key: cord-347547-makm0j09 authors: duran-frigola, miquel; bertoni, martino; blanco, roi; martínez, víctor; pauls, eduardo; alcalde, víctor; turon, gemma; villegas, núria; fernández-torras, adrià; pons, carles; mateo, lídia; guitart-pla, oriol; badia-i-mompel, pau; gimeno, aleix; soler, nicolas; brun-heath, isabelle; zaragoza, hugo; aloy, patrick title: bioactivity profile similarities to expand the repertoire of covid-19 drugs date: 2020-07-16 journal: j chem inf model doi: 10.1021/acs.jcim.0c00420 sha: doc_id: 347547 cord_uid: makm0j09 [image: see text] until a vaccine becomes available, the current repertoire of drugs is our only therapeutic asset to fight the sars-cov-2 outbreak. indeed, emergency clinical trials have been launched to assess the effectiveness of many marketed drugs, tackling the decrease of viral load through several mechanisms. here, we present an online resource, based on small-molecule bioactivity signatures and natural language processing, to expand the portfolio of compounds with potential to treat covid-19. by comparing the set of drugs reported to be potentially active against sars-cov-2 to a universe of 1 million bioactive molecules, we identify compounds that display analogous chemical and functional features to the current covid-19 candidates. searches can be filtered by level of evidence and mechanism of action, and results can be restricted to drug molecules or include the much broader space of bioactive compounds. moreover, we allow users to contribute covid-19 drug candidates, which are automatically incorporated to the pipeline once per day. the computational platform, as well as the source code, is available at https://sbnb.irbbarcelona.org/covid19. a new coronavirus, named sars-cov-2, is the responsible agent for the current 2019−2020 viral pneumonia (covid-19) outbreak, 1,2 which is already affecting millions of people worldwide and causing hundreds of thousands of deaths. the covid-19 pandemic has prompted an unprecedented effort by the scientific community to understand its molecular constituents and find an effective treatment to mitigate viral infectiveness and symptoms. this is reflected in the over 6000 covid-related publications that appeared in the past few weeks. 3 huge efforts are being invested in the discovery of an effective vaccine, but even the most optimistic scenarios suggest that it will not be available until 2021. other drug discovery projects have been launched to target specific viral proteins, particularly its main protease (mpro). 4 however, these initiatives, even if successful, could take even longer to deliver an approved drug. thus, the repurposing of existing drugs is our best chance to face the current outbreak therapeutically, since approved drugs have known safety profiles and are ready to be tested in humans. for instance, several compounds initially developed to treat hiv (e.g., lopinavir/ritonavir) 5 or ebola (e.g., remdesivir), 6 as well as antimalarial drugs (e.g., hydroxychloroquine), 7 are being tested against covid-19. indeed, we conducted a limited review of the most relevant scientific literature and identified over 200 compounds that are potentially active against covid-19 with different levels of experimental support, from purely computational predictions to preclinical and drugs already in clinical trials. we now exploit this literature mining effort to identify other compounds with the potential to be effective against covid-19. to this aim, we use the chemical checker (cc), a resource that provides processed, harmonized, and integrated bioactivity data for about 1 million small molecules. 8 in the cc, bioactivity data are expressed in a vector format, which naturally extends the notion of chemical similarity between compounds to similarities between bioactivity profiles. the cc organizes data into five levels of increasing complexity, ranging from drug binding profiles to clinical outcomes, and thus enables similarity searches that should be mechanistically and clinically relevant. in the current resource, we use cc signatures to identify similarities between bioactive compounds and the list of current covid-19 drug candidates (i.e., bait compounds). the similarity search is performed systematically across the large chemical space encompassed by the cc, thereby substantially expanding the portfolio of potential molecules effective against sars-cov-2. results are stratified between drug molecules and a broader medicinal chemistry space, thus offering ranked lists of compounds that should be of value for drug repurposing endeavors as well as preclinical screening campaigns. our resource capitalizes on an ongoing literature curation effort done by our group. additionally, we welcome contributions from the broader scientific community via web form, allowing users to include compounds under investigation in their laboratories, or to update the evidence level as new covid-19 experiments accumulate. the scientific evidence supporting covid-19 drug candidates is variable: some compounds come from computational predictions, some have proven their value in preclinical tests, others are approved drugs with a therapeutic indication unrelated to infectious diseases, and, finally, some are drugs currently used to fight sars-cov-2-related pathogens. the mechanisms of action (moa) suggested to confer efficacy are also variable, ranging from immunomodulators to protease inhibitors. during curation, we classify literature covid-19 candidates by their level of evidence and moa ( figure 1 ). by the 18th of april, 2020, we have found that 230 small molecules have been suggested as potential treatments for covid-19. starting from the smiles representation of a compound, we derive cc bioactivity signatures for each covid-19 literature bait compound. we then run bioactivity similarity searches against the ∼1 million bioactive molecules characterized in the cc and keep the top 10,000 most similar compounds for each search type. likewise, we conduct conventional similarity searches solely based on 2d representations of the compounds (2048-bit morgan fingerprints, radius 2). similarities are expressed as empirical p-values (−log 10 scale) derived from the expected similarity distribution across the full search space. a simple support measure is provided for each compound by adding up the number of similar covid-19 drugs (weighted by −log 10 p-value and level of evidence, as shown in figure 1 ). in addition, we complement our literature curation effort with a further level of evidence, namely, text-mining, based on the automatic detection of experiments (bioassays) that could be relevant to covid-19. more specifically, we process the text description of the ∼1.2 million bioassays catalogued in the chembl database and rank them according to their relevance to the current corpus of about 30,000 articles related to covid-19 and other coronavirus infections. 9 chembl bioassays 10 are ranked using two complementary approaches: (i) we construct a retrieval query from the bioassay descriptions and use it to score each of the paragraphs and abstracts contained in the articles collection. we then use statistics of the score distribution of top scoring documents to rank the bioassays. and (ii), we manually labeled a set of (seed) molecules that tested positive in ∼100 bioassays relevant to covid-19. we then automatically identify compounds from all the bioassay descriptions and compute their contextual embeddings. finally, we rank the bioassays according to their cosine similarity to the seed molecules. we then keep the 1000 most relevant covid-19 literature bioassays, as ranked by either text-mining approach and identify those bioactive molecules within the cc universe that tested positive (<10 μm) in at least one of them. finally, we cross these results with the 10,000 compounds obtained from the similarity searches described above and assign an extra literature-evidence level (text-mining) to those in common, which are then used as bait compounds. the pipeline runs automatically every day, so that we always provide the most updated results. searches are precomputed for each evidence strength and moa. results of the large-scale similarity search are made available as a web-resource at https://sbnb.irbbarcelona.org/covid19. the interface contains five tabs: figure 1 . methodological strategy. we use the list of covid-19 compounds extracted from the literature, with different levels of experimental evidence, as bait to search for compounds with similar bioactivity or chemical features among the 800,000 molecules contained in the cc. we also include compounds that are positive in relevant bioassays, identified through automatic mining of the covid-19 literature, and for which we find further bioactivity support in the cc. we keep and rank the top 10,000 most similar molecules to bait compounds and weight them to favor molecules with similar properties to those with higher levels of experimental evidence. candidates. we provide the 10,000 molecules, within the cc universe of 1 m bioactive compounds, that are more similar to the covid-19 bait compounds collected from the literature (figure 2 ). the precomputed similarity matrix can be queried to extract candidates that fulfill properties of interest by selecting among the levels of evidence for the bait compounds as well as their moa. in addition, the resulting list of molecules can be sorted following different criteria, including whether they are approved/experimental drugs, the cumulative level of support, or their similarity to specific covid-19 literature drugs. full and partial tables can be downloaded and exported to several formats, including the smiles string representation for all the compounds. literature. this tab lists the covid-19 bait compounds extracted from the literature, together with their level of experimental evidence and, if known, the moa that confers efficacy against sars-cov-2. documentation. here, we present a brief description of the methodological strategy, and more importantly, we offer updated statistics and benchmarks of the resource. in particular, we quantify the number of literature bait compounds available at each level of evidence and moa ( figure 3a ,b) and project cc signatures on a 2d plane to offer a global view of the chemical space explored by our resource (figure 3c,d) . we see that, while significantly diverse, covid-19 bait compounds cluster in certain regions of the chemical space, and we find new candidate molecules in their vicinity. reassuringly, when we analyze the therapeutic categories of the top-ranked candidates, as expected, we retrieve a significant number of anti-infective drugs ( figure 4a ). other therapeutic categories such as hormonal treatments are enriched after the highest-ranking compounds. note that, for this enrichment analysis, only drug molecules could be considered since atc annotations are not available for most of the compounds in the cc. finally, we perform a leave-one-out cross-validation to assess whether bait compounds can be retrieved by our similarity search. figure 4b shows that known covid-19 drugs are significantly up-ranked when using and evaluating all levels of evidence ( figure 4b ). contribute. through this form, users can contribute to the resource by including their molecules of interest. we require the name and smiles representation of the molecules as well as their level of experimental evidence, moa, and references, if available. after each submission, we manually check the data and incorporate it in the next daily update. code. this links to the gitlab repository containing the complete code to run the pipeline and analyze results. overall, we believe that the tool presented herein explores regions of the bioactive chemical space that could be relevant to covid-19 treatment. our web-based resource is updated daily and can be used to dynamically search for candidates related to covid-19 drugs with varying levels of evidence and moa. therefore, our resource will be useful to a broad range of covid-19 drug discovery approaches, ranging from those seeking a repurposing opportunity to those departing from the in vitro screening of compounds. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china structure of mpro from sars-cov-2 and discovery of its inhibitors compassionate use of remdesivir for patients with severe covid-19 quantifying treatment effects of hydroxychoroquine and azithromycin for covid-19: a secondary analysis of an open label non-randomized clinical trial extending the small molecule similarity principle to all levels of biology with the chemical checker chembl: towards direct deposition of bioassay data the authors declare no competing financial interest. this project has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no. 101003633 (ripcon). key: cord-328176-fck2ktxi authors: mahapatra, manojkumar; kulandaivelu, umasankar; saiko, philipp; graser, geraldine; szekeres, thomas; andrei, graciela; snoeck, robert; balzarini, jan; jayaprakash, venkatesan title: methyl-2-arylidene hydrazinecarbodithioates: synthesis and biological activity date: 2013-02-19 journal: chem zvesti doi: 10.2478/s11696-013-0346-4 sha: doc_id: 328176 cord_uid: fck2ktxi methyl-2-arylidene hydrazine-carbodithioate has not been of particular interest to researchers even though its metal complexes are extensively reported on due to their biological activity. this study examined the cytostatic and antiviral activity of twelve methyl-2-arylidene hydrazinecarbodithioates reported by many researchers as intermediates for the synthesis of thiosemicarbazides and the preparation of their metal complexes. compounds iic, iii, and iil with tridentate ligand features were found to have the lowest ic(50) value (6.5 μm, ≈ 1 μm, and 0.8 μm, respectively) against hl60 human promyelocytic leukemia cells. they were also most inhibitory to human embryonic lung (hel) fibroblast proliferation (5.3 μm, 17 μm, and 2.6 μm). compound iic and iil show antiviral activity against wild-type herpes simplex virus (hsv), varicella zoster virus (vzv), and acyclovirresistant hsv; however, these activities were observed at concentrations at which the compounds also markedly inhibit hl60 and hel cell proliferation. many n-substituted thiosemicarbazones were prepared by the condensation of schiff's bases of methyl-2-arylidene hydrazinecarbodithioate (mahcd) with amines (casero et al., 1980; collins et al., 1982; klayman et al., 1979 klayman et al., , 1991 scovill et al., 1982; shipman et al., 1981) . while thiosemicarbazones and their metal complexes were studied for their biological activity (beraldo & gambinob, 2004; ettari et al., 2010; jiang et al., 2006; katz, 1987; liberta & west, 1992; matesanz & souza, 2009; pandeya & dimmock, 1993; wnuk & robins, 2006; yu et al., 2009) , the intermediate mahcd was rarely studied for its metal complexation behaviour and the complexes for their biological activity (ali et al., 2002; kanwar et al., *corresponding author, e-mail: venkatesanj@bitmesra.ac.in 2008; neelam et al., 2000; saxena & tandon, 1983; singh et al., 1997) . very few investigators have reported biological activity of compounds with a dithiocarbamate side chain (cao et al., 2005; huang et al., 2009; kumar et al., 2010) . it is interesting to note that mahcd shares structural similarity with schiff's bases of n-hydroxy semicarbazides (snhs) and nhydroxy aminoguanidines (snhag) (fig. 1) . both snhs and snhag were rigorously documented for their anticancer activity and antiviral properties. it was proposed that these compounds act by inhibiting ribonucleotide reductase (rr) through metal chelation (das et al., 1999; ren et al., 2002; t'ang et al., 1985) . with this background, twelve mahcd derivatives were synthesised and evaluated for anticancer activity against hl60 human promyelocytic leukemia cells and antiviral activity against a panel of viral cell lines. synthesis of methyl hydrazinecarbodithioate (i) and substituted benzaldehyde hydrazones of methyl hydrazine carbodithioate (ii) to an ice cooled solution (< 10 • c) of 19.8 g (0.300 mol) of potassium hydroxide in 24 ml of water and 20 ml of propan-2-ol, 17.1 ml of 80 % pure hydrazine hydrate were added and constantly stirred. the amount of 18.2 ml (0.300 mol) of ice cooled carbon disulfide was added drop wise to the stirred solution, maintained at < 10 • c for over about 1-1.5 h. the bright yellow mixture formed was stirred for an additional 1 h after which ice cooled iodomethane (18.7 ml, 0.300 mol) was added drop wise over a 2 h period. stirring was continued for an additional 1 h, and the white precipitate obtained was filtered and washed with ice-cold water. the crude product was recrystallised from dichloromethane: yield of 43 %; audrieth et al. (1954) ; 81-83 • c, klayman et al. (1979) ). methyl hydrazinecarbodithioate (20.0 mmol) was dissolved in 20 ml of methanol and then, equimolar amount of aromatic/heteroaromatic aldehyde was added. the mixture was refluxed for 6-12 h and the reaction was monitored by tlc. the hot solution was poured onto crushed ice and the precipitate obtained was filtered (klayman et al., 1979; scovill et al., 1982) : yield of 60-80 %. the hl-60 human promyelocytic leukemia cell line was purchased from atcc (american type culture collection, manassas, va, usa). cells were grown in a rpmi 1640 medium supplemented with a 10 % heat inactivated fetal calf serum (fcs), 1 % l-glutamine and 1 % penicillin-streptomycin in a humidified atmosphere containing 5 % of co 2 . all media and supplements were obtained from life technologies (paisley, scotland, uk). cell counts were determined using a micro cell counter cc-108 (sysmex, kobe, japan). cells growing in the logarithmic phase of growth were used in all experiments described below. hl-60 cells (10 5 per ml) were seeded in 25 cm 2 nunc tissue culture flasks and incubated with increasing concentrations of drugs (iia-iil) at 37 • c under cell culture conditions. cell counts and ic 50 values were determined after 24 h, 48 h, and 72 h using the micro cell counter cc-108. viability of the cells was determined by trypan blue exclusion. results were calculated as the number of viable cells. antiviral assays (except anti-human immunodeficiency virus (hiv) assays) were based on the inhibition of virus-induced cytopathic effect in hel for the hcmv assays, confluent hel fibroblasts were grown in 96-well microtiter plates and infected with the human cytomegalovirus strains davis and ad-169 at 100 pfu per well. after a 2 h incubation period, the residual virus was removed and the infected cells were further incubated with a medium containing different concentrations of the test compounds (in duplicate). after incubation for 7-days at 37 • c, the virus-induced cytopathic effect was monitored microscopically after ethanol fixation and staining with giemsa dye. antiviral activity was expressed as the ec 50 or compound concentration required for the reduction of virus-induced cytopathogenicity by 50 %. the varicella zoster virus (vzv) drug susceptibility tests were performed on confluent hel cells in 96well microtiter plates by the plaque reduction assay. monolayers were infected with 20 plaque forming units (pfu) of the cell-associated virus per well. for each assay, virus controls (infected-untreated cells) were included. after a 2 h incubation period, the virus inoculum was removed and the media were replaced by different dilutions (in duplicate) of the tested molecules. serial dilutions of test compounds were incubated with the infected monolayers for five days. after a five day incubation period, the cells were fixed and stained with giemsa, and the level of the virus-induced cytopathic effect was determined by counting the number of plaques for each dilution. activity was expressed as ec 50 (effective compound concentration required to reduce virus plaque formation by 50 %) compared to the untreated control. the anti-hiv activity of the compounds was evaluated against the wild type hiv-1 strain iiib in mt-4 cell cultures using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (mtt) method. briefly, virus stocks were titrated in mt-4 cells and expressed as the 50 % cell culture infective dose (ccid50). mt-4 cells were suspended in culture medium at 10 5 cells per ml and infected with hiv at the multiplicity of infection of 0.02. immediately after viral infection, 100 µl of the cell suspension were placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. the test compounds were dissolved in dmso at 50 mm or more as stock solutions. then, serial dilutions of the test compounds were made. the highest concentration tested was 100 µm of the test compound containing 0.2 % of dmso, which is by itself not toxic to cell proliferation. after four days of incubation at 37 • c, the number of viable cells was determined using the mtt method. compounds iia-iil were synthesised following the reaction sequence outlined in fig. 2 . methyl hydrazinecarbodithioate (i ) was prepared by the reaction of hydrazine hydrate (85 mass %) with carbon disulfide in the presence of potassium hydroxide at a temperature below 10 • c; followed by the addition of methyl iodide (audrieth et al., 1954) . condensation of i with aromatic aldehydes/ketones in methanol provided compounds iia-iil (klayman et al., 1991; scovill etal., 1982) . all compounds were characterised by their 1 h-nmr and fab-ms data. physicochemical characterisation data are presented table 1 and spectral data of compounds iia-iil are presented in table 2 . all compounds were tested for their anticancer activity against the hl-60 human promyelocytic leukemia cell line and the results are presented in table 3 . the compounds were also tested for their antiviral activity against herpes simplex virus-1 (kos), herpes simplex virus-2 (g), herpes simplex virus-1 tk − (kos acv r ), varicella-zoster virus (oka and 07/1), cytomegalovirus (davis and ad169), vaccinia virus, vesicular stomatitis virus, feline corona virus, feline herpes virus, human immunodeficiency virus (hiv) type 1 (iii b ) and type 2 (rod), coxsackie virus b4, respiratory syncytial virus, parainfluenza 3 virus, retable 4 . compound iil, iii, and iic showed ic 50 values against hl60 cells at the concentration of 0.8 µm, ≈ 1 µm, and 6.5 µm, respectively, after 72 h. it is interesting to note that preferably compounds with tridentate ligand characteristics (iil, iii, and iic) showed potent anti-proliferative activity against the hl60 cells (table 3 ). the activity of compound iii (2 -hydroxy acetophenone derivative) was higher than that of iic (2-hydroxy benzaldehyde derivative) and almost equally potent to iil (pyridine 2carboxaldehyde derivative). all the other compounds (iia, iib, iid-iih, and iik) were inhibitory at concentrations between 55 µm and 63 µm after 72 h, except for iij, irrespective of the nature and position of the substitution. compound iij did not exert any inhibitory activity on the hl60 cells at the maximum concentration studied (100 µm, after 72 h). all the compounds exhibited a cytotoxic concentration (cc 50 ) of ≥ 100 µm in the hela cell cultures except for iil (36 µm), (table 3) . compounds iii and iil showed the best selectivity towards the hl60 cells and therefore, further investigations are required. compounds iic and iii resemble the iron chelator 311 (n -[(2-hydroxynaphthalen-1-yl)-methylidene]pyridine-4-carbohydrazide) and iil resembles triapine (3-aminopyridine-2-carbaldehyde thiosemicarbazone). compound iii having a ketonic methyl group showed activity comparable to that of iron chelator 311 at 48 h. compound iil also exhibited an anti-proliferative activity comparable to that of triapine. the analogue of iil with a ketonic methyl group has to be considered for further improvement of its activity. it is interesting to observe that only compounds with tridentate ligand characteristics (iic, iii, and iil) have shown antiviral activity against a few viruses and are presented in table 4 . compound iil displayed pronounced antiviral activity against hsv-1 (8-42 µm), hsv-2 (9-11 µm ), vzv (6.5-42 µm), and vaccinia virus (15-45 µm). it should be noted that iil inhibits the wild-type as well as the acyclovir-resistant hsv at comparable compound concentrations (8-20 µm, table 4). compound iic with a 2-hydroxy substitution and compound iii, an acetophenone analogue of iic, show antiviral activity at somewhat higher concentrations than iil. compound iid that differs from iic by a hydroxy functional group at the para position did not show any appreciable antiviral activity. additional analogues of iic and iil are currently under further investigation. it should, however, be noted that iic, iii, and iil are poorly cytotoxic but they show pronounced cytostatic activity in proliferating hel (and hl60) cell cultures. therefore, the observed antiviral activity might not be due to direct antiviral activity but rather due to indirect inhibitory action caused by the underlying cytostatic potential of these compounds. the present investigation revealed that: i) compounds with a metal chelating functional group (iic, iii, and iil ) at their aryl end were found to be active and ii) the methyl dithioate (-cssch 3 ) group is not an bioisosteric equivalent of hydroxamate (-conhoh) or amidoxime (-cnhnhoh) (fig 1.) . the compounds investigated are known as intermediates in the synthesis of many thiosemicarbazones but their usefulness as medicinally active agents has not yet been studied. the study suggests a possibility of intermediates with specific pharmacophoric features for the intended activities that can provide a new scaffold for these activities. hydrazine derivatives of the carbonic and thiocarbonic acids. i. the preparation and properties of thiocarbohydrazide preparation, characterization and antifungal properties of nickel(ii) complexes of tridentate ons ligands derived from n-methyl-s-methyldithiocarbazate and the x-ray crystal structure of the [ni(onmes)cn]·h 2 o complex the wide pharmacological versatility of semicarbazones, thiosemicarbazones, and their metal complexes. mini-reviews in medicinal chemistry synthesis and in vitro antitumor activity of 4(3h)-quinazolinone derivatives with dithiocarbamate side chains activity of 2-acetylpyridine thiosemicarbazones against trypanosoma rhodesiense in vitro activity of 2-acetylpyridine and 2-acetylquinoline thiosemicarbazones tested in vitro in combination with other antituberculous drugs inhibition of herpes simplex virus type 1 and adenovirus type 5 by heterocyclic schiff bases of aminohydroxyguanidine tosylate falcipain-2 inhibitors synthesis and antitumor activity of novel 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relationships approach. mini-reviews in medicinal chemistry palladium(ii) complexes of ns donor ligandsderived from s-methyl-dithiocarbazate, s-benzyldithiocarbazate and thiosemicarbazide as antiamoebic agents recent evaluations of thiosemicarbazones and semicarbazones and related compounds for antineoplastic and anticonvulsant activities synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new schiff bases of hydroxysemicarbazide as potential antitumor agents the potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents ii: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone antitumor activity of some diorganotin and tin(iv) complexes of schiff bases 2-acetylpyridine thiosemicarbazones. 4. complexes with transition metals as antimalarial and antileukemic agents antiviral activity of 2-acetylpyridine thiosemicarbazones against herpes simplex virus antitumor activity studies of newly synthesized n-salicyloyl-n -(p-hydroxybenzthioyl)hydrazine and its copper(ii) complex both in vivo and in vitro optimization of the schiff bases of n-hydroxy-n -aminoguanidine as anticancer and antiviral agents ribonucleotide reductase inhibitors as anti-herpes agents thiosemicarbazones from the old to new: iron chelators that are more than just ribonucleotide reductase inhibitors key: cord-293867-c4wnr5xe authors: gürsoy, elif; dincel, efe doğukan; naesens, lieve; ulusoy güzeldemirci, nuray title: design and synthesis of novel imidazo[2,1-b]thiazole derivatives as potent antiviral and antimycobacterial agents date: 2019-12-06 journal: bioorg chem doi: 10.1016/j.bioorg.2019.103496 sha: doc_id: 293867 cord_uid: c4wnr5xe a series of novel acyl-hydrazone (4a-d) and spirothiazolidinone (5a-d, 6a-d) derivatives of imidazo[2,1-b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. the antituberculosis activity was evaluated by using the microplate alamar blue assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. according to the biological activity studies of the compounds, 5a-c displayed hope promising antitubercular activity, 6d was found as potent for coxsackie b4 virus, 5d was found as effective against feline corona and feline herpes viruses. consequently, the obtained results displayed that, 5a-d and 6d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity. lately, much interest has been focused on the chemistry and the biological activity of fused heterocyclic systems because of their broad spectrum of physiological activities [1, 2] . among these heterocyclic compounds carrying nitrogen atom, imidazo [2,1-b] thiazole derivatives possess specific importance because of their diverse pharmacological activities. the imidazo [2,1-b] thiazole derivatives have been reported in the literature as antibacterial [3, 4] , antitubercular [5] , antifungal [6] , antitumoral [7] , antiviral [8, 9] , antihelmintic [10] , analgesic [11] , anti-inflammatory [12] , antihypertensive [13] , cardiotonic [14, 15] , diuretic [16] , herbicide [17] and insecticide [18] agents. levamisole [(6s)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole] (fig. 1) , which is the levogyre isomer of antihelminthic tetramisol and contains imidazo [2,1-b] thiazole moiety, is a drug that has significant immunomodulatory properties [19] in addition to its antihelminthic activity [20] . besides the wide biological activity spectrum of imidazo[2,1-b] thiazole derivatives, also the compounds bearing hydrazide, acyl-hydrazone and spirothiazolidinone moiety, have been reported in the literature with their various effects such as antibacterial [21] , antifungal [22] , antitubercular [23] , antiviral [24] , anticonvulsant [25] and antidepressant [26] . on the other hand, the two compounds bmy-27709 and bms-199945, which are known in the literature for their antiviral activity and especially for their effects on influenza virus, have attracted the attention of researchers as compounds, that has a specific chemical character by an amide structure which connects an aliphatic cyclic system with an aromatic system [27, 28] . in this study, we further explored the scaffold containing the imidazo [2,1-b] thiazole ring as the aromatic moiety, that is linked by an amide to a spirothiazolidinone ring system as the aliphatic cyclic moiety and from this point forward, novel derivatives were synthesized (table 1) , and broadly evaluated for their antiviral and antimycobacterial activity (fig. 2) . two hit compounds were found to inhibit feline coronavirus or coxsackie b4 virus. besides, we observed some activity against mycobacterium tuberculosis, suggesting the versatility and relevance of this compound class to achieved anti-infective agents. compound 1 was obtained according to the procedure described by robert et al [29] . compound 2 was obtained according to the procedure described by robert et al [29] . compound 3 was obtained according to the procedure described by harraga et al [30] . general procedure for the synthesis of 6-(4-bromophenyl)-n 2 -(substituted/non-substituted cycloalkylidene)imidazo[2,1-b]thiazole-3-acetohydrazides (4a-d) 0,005 mol of 3 was boiled in a water bath under reflux with 30 ml of ethanol until a clear solution was obtained. 0.01 mol of cyclic ketone was added and heated for 6 h. after cooling the mixture to room temperature, it was filtered and purified by crystallization with warm ethanol or washing. [2,1-b] to a suspension of 0.005 mol of 4a-d in 30 ml of anhydrous benzene is added 2 ml of mercaptoacetic acid / 2-mercaptopropionic acid. the reaction mixture is heated under a reflux condenser using a dean-stark trap in a water bath for 6 h. it is concentrated under reduced pressure and the excess acid is neutralized with nahco 3 solution. the resulting product is kept in the refrigerator until solidified. the crude product is filtered, washed with water, dried and purified by crystallization or elution with ethanol. white solid, mp 162-163°c, yield: 54.4%. anal. calcd. for c 21 white solid, mp, 178-180°c, yield: 66.4%. anal. calcd. for c 27 white [4.4] [4, 5] the antimycobacterial activity studies of the compounds were performed by the taacf (tuberculosis antimicrobial acquisition and coordinating facility by the national institute of health of the us government). primary screening was conducted at 6.25 µg/ml against mycobacterium tuberculosis h37rv in bactec 12b medium using a broth microdilution assay the microplate alamar blue assay (maba) [31] . compounds exhibiting fluorescence were tested in the bactec 460 radiometric system. compounds affecting less than 90% inhibition in the primary screen were not generally evaluated further. compounds demonstrating at least 90% inhibition in the primary screen were retested at lower concentrations against m. tuberculosis h37rv in order to determine the actual minimum inhibitory concentration (mic) using maba. rifampin was utilized as the standard compound in the assays and each assay was replicated four times. the mic was defined as the lowest concentration affecting a reduction in fluorescence of 90% relative to controls. concurrently with the determination of mics, compounds were tested for cytotoxicity (ic 50 ) in vero cells at concentrations ≤6.25 µg/ml or 10 times the mic for m. tuberculosis h37rv (solubility in media permitting). after 72 h exposure, viability was assessed on the basis of cellular conversion of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (mtt) into a formazan product using the promega celltiter 96 non-radioactive cell proliferation assay. compounds for which the selectivity index ic 50 :mic ratio) si > 10 were assumed to possess in vitro activity confirmed in the bactec 460 at 6.25 µg/ml. alamar blue susceptibility assay (maba). antimicrobial susceptibility testing was performed in black, clear-bottomed, 96-well microplates (black view plates; packard instrument, meriden, connecticut, usa) in order to minimize background fluorescence. outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. initial drug dilutions were prepared in either dmso or distilled deionized water, and subsequent twofold dilutions were performed in 0.1 cm 3 of 7h9gc (no tween 80) in the microplates. bactec 12b-passaged inocula were initially diluted 1:2 7h9gc, and 0.1 cm 3 was added to wells. subsequent determination of bacterial titers yielded 1 × 10 6 , 2.5 × 10 6 and 3.25 × 10 5 cfu cm −3 in plate wells for m. tuberculosis h37rv. frozen inocula were initially diluted 1:20 in bactec 12b medium followed by a 1:50 dilution in 7h9gc. addition of 0.1 cm 3 to wells resulted in final bacterial titers of 2.0x10 5 and 5x10 5 cfu cm −3 for h37rv. wells containing drugs only were used to detect autofluorescence of compounds. additional control wells consisted of bacteria only (b) and medium only (m). plates were incubated at 37°c. starting at day 4 of incubation, 20 mm 3 of 10x alamar blue solution (alamar biosciences/accumed, westlake, ohio, usa) and 12.5 mm 3 of 20% tween 80 were added to one b well and one m well, and plates were reincubated 37°c. wells were observed at 12 and 24 h for a color change from blue to pink and for a reading of ≥50,000 fluorescence units (fu). fluorescence was measured in a cytofluor ii microplate fluorometer (perseptive biosystems, framingham, massachusetts, usa) in bottom-reading mode with excitation at 530 nm and emission at 590 nm. if the b wells became pink by 24 h, the reagent was added to the entire plate. if the well remained blue or 50,000 ≤fu was twofold drug dilutions were prepared in either dmso (sigma) or distilled deionized water and delivered via a 0.5-cm 3 insulin syringe in a 50-mm 3 volume. drug-free control vials consisted of solvent with bacterial inoculum and solvent with a 1:100 dilution of bacterial inoculum (1:100 controls). vials were incubated at 37°c, and the gi was determined in a bactec 460 instrument (becton dickinson) until the gi of the 1:100 controls reach at least 30. all vials were read the following day, and the gi and daily changes in gi (δgi) were recorded for each drug dilution. the mic was defined as the lowest concentration for which the δgi was less than the δgi of the 1:100 control. if the gi of the test sample was greater than 100, the sample was scored as resistant even if the δgi was less than the δgi of the 1:100 control. the synthesized compounds (4a-d, 5a-d and 6a-d) were evaluated against diverse rna and dna viruses, using the following cellto perform the antiviral assays, the virus was added to subconfluent cell cultures in 96-well plates, and at the same time, the test compounds were added at serial dilutions. appropriate reference compounds were included such as the virus entry inhibitors urtica dioica agglutinin lectin and dextran sulfate; broad virus inhibitors ribavirin and mycophenolic acid; antiherpetic drugs ganciclovir and cidofovir; and hiv inhibitor azidothymidine and nevirapine. after 3-6 days incubation at 37°c (or the target compounds 4a-d, 5a-d and 6a-d were synthesized from 2-(6-(4-bromophenyl)imidazo [2,1-b] thiazol-3-yl)acetohydrazide by a four and five-step synthesis through the pathways shown in fig. 3 . 4-bromophenacyl bromide and ethyl 2-aminothiazole-4-acetate were dissolved in acetone and mixed in room temperature. after standing for 3 days, 2amino-3-[(4-bromobenzoyl)methyl]-4-(ethoxycarbonylmethyl)thiazolium bromide (1) was obtained [29] . thereafter, compound 1 was boiled under reflux in absolute ethanol and via the ring closure of compound 1, ethyl [6-(4-bromophenyl)imidazo[2,1-b]thiazole-3-yl]acetate hydrobromide (2) was obtained [29] . by heating the compound 2 and hydrazine hydrate in ethanol, 2-[6-(4-bromophenyl)imidazo[2,1-b]thiazole-3-yl]acetohydrazide (3) was obtained [30] . the compound 3 and cyclic ketones were heated under reflux in ethanol to yield 6-(4-bromophenyl)-n 2 -(substituted/non-substituted cycloalkylidene)imidazo [2,1-b]thiazole-3-acetohydrazides (4a-d). the reaction yields of compounds 4a-d were between the interval of 95%-72.5%. 4a-d were then reacted with mercaptoacetic acid / 2-mercaptopropionic acid in anhydrous benzene under reflux by using a dean-stark water separator. the obtained reaction mixture was eventually neutralized with nahco 3 solution and by that way 2-[6-(4-bromophenyl)imidazo[2,1-b]thiazol-3yl]-n-(2-nonsubstituted/methyl-6,7,8-nonsubstituted/alkyl/aryl-3-oxo-1-thia-4-azaspiro [4.4] non/ [4.5] dec-4-yl]acetamides (5a-d, 6a-d) were obtained. the reaction yields of the compounds 5a-d and 6a-d were between the interval of 87.8%-54.4% and 95%-54%, respectively. when the chemical structures of the synthesized compounds were examined, it was observed that the carbonyl group was not present in the compounds 4a-d whereas the same group was present in the spirothiazolidinone ring of the compounds 5a-d and 6a-d. also according to the ir spectroscopy results, the stretching band belonging to the carbonyl group was not visible in the spectrum of the compounds 4a-d whereas the mentioned band was able to be observed in the range of 1724-1707 cm −1 for 5a-d and 6a-d. the ir values belonging to the carbonyl groups of spirothiazolidinone ring of 5a-d and 6a-d were in agreement with the literature [33, 34] . in the 1 h nmr analysis, the nh 2 protons of compound 3, which were observed as a broad singlet peak (2h) at 4.38 ppm, was disappeared in compounds 4a-d, whereas the peaks of aliphatic ch 2 groups belonging to cyclic ketone arose as multiplet in the range of 3.15-1.44 ppm. when the results obtained from compounds 5a-d examined, it was determined that the peaks of s-ch 2 protons were added to the spectrum in the range of 3.68-3.60 ppm as a distinctive indicator related to the formation of the mentioned compound [33, 35, 36] . also from the 1 h nmr analysis data obtained from compounds 6a-d, it was determined that the s-ch and ch-ch 3 peaks of the spirothiazolidinone ring were in the range of 3.95-3.90 and 1.43-1.40 ppm, respectively and the values were in compliance with the literature [37, 38] . the ir, 1 h nmr, 13 c nmr and ms spectra of the novel compounds are in agreement with the assigned structures. no unacceptable side reactions were observed, and products were obtained in moderate to good yields. the broad antiviral evaluation demonstrated that compound 5d was quite effective against feline coronavirus in crfk cells (table 2) , with an antiviral ec 50 value of 4.8 µg/ml and a superior selectivity index (ratio of cytotoxic to antiviral concentration) higher than 20. 5d was four-fold less active against feline herpesvirus. similarly, two compounds (4c and 4d) had weak anti-dna virus activity in hel cells, with antiviral ec 50 values about 45 µg/ml ( table 3) . the most effective compound against feline coronavirus was 5d. when the sar analysis of 5d is evaluated by comparison with the other compounds, it can be understood that the existence of the structure of the spirothiazolidinone moiety is significant in the activity against the mentioned virus. it can also be observed that the spirothiazolidinone structure of 5d is separated from 5b-c by the 4-hydroxyphenyl substituent at the 8-position, and thus it can be concluded that the corresponding substituent is significant in the activity of the compound. additionally, the 2-position of the spirothiazolidinone structure separates 5d from 6b-d, indicating that the nonsubstitution of position 2 is crucial for the antiviral activity against the feline coronavirus. the results also displayed that, the compounds bearing spirothiazolidinone structure did not show activity against dna viruses, whereas compound 4c and 4d, which are carrying acyl-hydrazone moiety, showed activity against dna viruses. the obtained result displayed the importance of the existence of the acyl-hydrazone residue for the activity against dna viruses. also, 4-phenylcyclohexyl and 4-(4hydroxyphenyl)cyclohexyl derivatives of acyl-hydrazones have been found to further increase the activity against dna viruses. another hit compound, 6d, was somewhat active against coxsackie b4 virus (ec 50 : 10 µg/ml and selectivity index ≥2 (table 4) ). the antimycobacterial evaluation of compounds (4a-c, 5a-c) was performed. the results belonging in vitro primer analyses, that were performed against mycobacterium tuberculosis h37rv strain by using maba in bactec 12b media, were given in table 5 . according to the in vitro primer analyses, the compounds possessing any value where the mic value was equal to less than 10 µg/ml were considered as active for antimicrobial activity and further analysis of the mentioned compounds were conducted. from this point forward compounds 4a-c were not detected as active and no further antimycobacterial activity was performed for them. in contrast to compounds 4a-c, compounds 5a-c were detected as active and further antimycobacterial activity of these compounds was conducted and the results were displayed in table 6 . the obtained results displayed that, 4a-c, the acyl-hydrazone moiety containing imidazo[2,1-b]thiazole derivatives, showed no or weak antitubercular activity and the ring closure raised the antitubercular activity of the compounds as can be seen from the results related to the compounds 5a, 5b, and 5c. the fact that the conversion of acyl-hydrazone derivatives to spirothiazolidinone derivatives by ring closure increased the activity, indicates the importance of the existence of spirothiazolidinone structure for antitubercular activity. antibiotic resistance is rising to high levels sharply all over the world and new kinds of resistance mechanisms are emerging worldwide. infections such as pneumonia, tuberculosis, gonorrhea, blood poisoning are becoming harder to treat and current antibacterials are losing their effectivity day by day. designing of new effectual compounds to deal with these resistant bacterias has become one of the most important issues today. similarly, mycobacterium tuberculosis remains a leading infectious cause of death worldwide today. especially the evolution of multi-drug-resistant (mdr) strains of mycobacterium tuberculosis, is the main reason for the increased incidence of tuberculosis, therefore, the development of new antimycobacterial agents has become an obligation. another important is that needs new drug candidates are antiviral therapy and it is definitely necessary to design and develop new effective antiviral agents. in the present study, in an attempt to find novel antiviral and antitubercular agents, 12 diverse derivatives of imidazo[2,1-b]thiazole were designed and synthesized. the compounds were screened for their antiviral and antitubercular activity. the antimycobacterial activities of the compounds were tested against the mycobacterium tuberculosis h37rv strain and in the test method, each value, in which the mic value was equal to less than 10 µg/ml, was actively accepted for antimycobacterial activity. the mic values of the compounds 5a, 5b, and 5c were determined as 8.453 µg/ml, 1.566 µg/ml and 0.854 µg/ml and the molecules were found as active. also, compound 6d was found as effective for coxsackie b4 virus. the antiviral activity and cytotoxicity of the compounds against feline corona and feline herpes viruses were investigated in crfk cell cultures and in comparison with hha, uda and ganciclovir references, compound 5d was found as highly effective. the findings revealed the promising antiviral and antitubercular activity of acyl-hydrazone, spirothiazolidinone and 2-methyl-substituted spirothiazolidinone derivatives of imidazo [2,1-b] thiazole and these derivatives could be an interesting starting point for further structural optimization to obtain new promising and more potent antiviral and antitubercular agents. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the levamisole story the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discusses in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. the authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the declaration of helsinki for all human or animal experimental investigations. supplementary data to this article can be found online at https:// doi.org/10.1016/j.bioorg.2019.103496. key: cord-336759-cu1uprwm authors: cihan-üstündağ, gökçe; naesens, lieve; şatana, dilek; erköse-genç, gonca; mataracı-kara, emel; çapan, gültaze title: design, synthesis, antitubercular and antiviral properties of new spirocyclic indole derivatives date: 2019-07-17 journal: monatsh chem doi: 10.1007/s00706-019-02457-9 sha: doc_id: 336759 cord_uid: cu1uprwm abstract: a series of indole-based spirothiazolidinones have been designed, synthesized and evaluated, in vitro, for their antitubercular, antiviral, antibacterial, and antifungal activities. the structures of the new compounds were established by ir, (1)h nmr, (13)c nmr (proton decoupled, apt, and dept), electrospray ionization mass spectrometry, and microanalysis. compounds bearing a phenyl substituent at position 8 of the spiro ring, exhibited significant antitubercular activity against mycobacterium tuberculosis h37rv atcc 27294 at concentrations of 3.9 and 7.8 µm. still, some of the tested compounds displayed activity on mycobacteria with mic values of 16 and 31 µm. four of the indole-spirothiazolidinone derivatives were found to be moderately active against punta toro virus, yellow fever virus or sindbis virus in vero cells. the antiviral ec(50) values were in the range of 1.9–12 µm and the selectivity index (ratio of cytotoxic to antivirally effective concentration) was above 10 in some cases. the most potent effect was seen with the compound that is methylated at positions 2 and 8 of the spirothiazolidinone system. graphic abstract: [image: see text] electronic supplementary material: the online version of this article (10.1007/s00706-019-02457-9) contains supplementary material, which is available to authorized users. tuberculosis (tb) is a highly infectious disease caused by the bacillus mycobacterium tuberculosis. for the past 5 years, it has been the leading cause of mortality from a electronic supplementary material the online version of this article (https ://doi.org/10.1007/s0070 6-019-02457 -9) contains supplementary material, which is available to authorized users. 1 3 single infectious disease, ranking above hiv/aids [1] . major problems associated with the currently available tb treatment include long treatment duration, inadequate compliance, concurrent hiv infection, and increasing incidence of multidrug-resistant (mdr) and extensively drug-resistant (xdr) tuberculosis [2] [3] [4] [5] . this emergence of difficult to treat strains necessitates the discovery and development of novel antitubercular drugs. after this domain has been inactive for several decades, two new drugs became available, i.e., the mitochondrial atp synthase inhibitor bedaquiline and mycolic acid biosynthesis inhibitor delamanid, which received accelerated approval for the treatment of mdr tuberculosis in 2012 and 2014, respectively [6] . besides, diverse novel drug candidates are in preclinical or clinical development [6] [7] [8] . indole-2-carboxamides incorporating an alicyclic system ( fig. 1a ) have been extensively studied by different research groups [9] [10] [11] [12] [13] [14] . this type of compounds was found to be highly active against both drug-susceptible and drugresistant strains of mycobacterium tuberculosis by acting on the mmpl3 transporter protein. in previous investigations, we have identified the indole-spirothiazolidinone system (fig. 1b) as a promising scaffold against the mycobacterium tuberculosis h37rv strain [15, 16] . some of these analogues exhibited in vitro antitubercular activity with gi (growth inhibition) values of 91-95% at a mic (minimum inhibitory concentration) value of 6.25 µg/cm 3 . recently, we reported on the synthesis of novel 5-fluoro-3-phenyl-1h-indole derivatives containing a 4-thiazolidinone nucleus (instead of spirothiazolidinone system) [17] . two molecules (fig. 1c ) displayed notable antitubercular activity at concentrations tenfold lower than those that produced cytotoxicity in mammalian cell lines. furthermore, several spirothiazolidinone compounds synthesized in our laboratory were found to be efficient inhibitors of membrane fusion mediated by influenza virus hemagglutinin (ha) [18] [19] [20] . as demonstrated in fig. 1d , these compounds have a similar backbone structure, consisting of an aromatic/polycyclic ring linked to a non-aromatic spiro system via an amide bridge. some analogues displayed low micromolar activity against the influenza a/h3n2 subtype with a favorable selectivity index. based on these insights and our objective to optimize the antimicrobial activity of indolyl thiazolidinones and spirothiazolidinones, we here report the chemical synthesis, structural characterization and in vitro antitubercular, antiviral, antibacterial, and antifungal evaluation of new 5-chloro-3-phenyl-n(2,7,8,9substituted/nonsubstituted-3-oxo-1-thia-4-azaspiro [4.4] nonan/ [4.5] decan-4-yl)-1h-indole-2-carboxamides 4a-4i, 5a-5h (fig. 1e ). the synthetic pathways for the preparation of the spirothiazolidinones 4a-4i and 5a-5h are illustrated in scheme 1. thus, the diazonium salt, formed by the reaction of 4-chloroaniline with nano 2 and hcl, was reacted with ethyl 2-benzyl-3-oxobutanoate to obtain compound 1 [21] according to the japp-klingemann reaction. the fischer indole synthesis was carried out in acidic medium to cyclize 1 into ethyl 5-chloro-3-phenyl-1h-indole-2-carboxylate 2 [22] . subsequent exposure of 2 to an excess of hydrazine hydrate afforded compound 3 [23] . the target spirocyclic compounds 4a-4i, 5a-5h were synthesized by treatment of the key intermediate 3 with appropriate cyclic ketones and mercapto acids in a one-pot reaction [15] . the structures of the new compounds were characterized by ir, 1 h nmr, 13 c nmr (proton decoupled, apt, and dept), electrospray ionization mass spectrometry (esi-ms), and combustion analysis. the ir spectra of 4a-4i and 5a-5h exhibited the ring and the exocyclic c=o bands in the 1692-1713 cm −1 and 1651-1670 cm −1 , respectively. the shifts observed in the amide bands when compared to that of 3 (1636 cm −1 ) and the presence of additional lactam bands provided definite proof for the aimed cyclization. observation of nh signals assigned to the indole nh (δ = 12.11-12.18 ppm) and amide nh (9.97-0.21 ppm) together with the absence of the nh 2 resonance of the intermediate hydrazide 3 in the 1 h nmr spectra of 4 and 5, provided further evidence for the formation of new adducts. the s-ch 2 (4a-4i) and s-ch (5a-5h) protons of the newly formed spiroalkane residue resonated at about 3.50-3.65 and 3.87-3.93 ppm, respectively. the s-ch 2 protons of 4a-4i appeared as singlets except for the methylene hydrogens of compound 4g which were observed as separate doublets with large coupling constants (j = 16.1 hz) due to the geminal coupling resulting from the chiral centers of the spirothiazolidinone ring. the 1 h nmr spectra of 5a-5h displayed the thiazolidinone s-ch protons as quartets or broad/distorted singlets and doublets. assignment of the indole protons was achieved on the basis of the values and coupling constants reported for the 2,3,5-trisubstituted indole ring [15, 16, 24, 25] . the carbon resonances were assigned by chemical shifts and comparison with previously reported 13 c nmr data for compounds having a similar backbone structure [15, 19, 26] . ch 3 , ch 2 , ch, and c signals were assigned by the antitubercular activity of compounds 4a-4i and 5a-5h was tested in vitro against m. tuberculosis h37rv atcc 27294 using the microdilution method. the lowest concentration of compound that inhibited 100% of mycobacterial growth in the culture was defined as the mic. rifampicin was used as the reference drug. compounds were assayed using twofold dilutions starting at 1000 µm. as shown in table 1 , compounds 4 h and 5h, bearing a phenyl substituent at position 8 of the spiro ring, exhibited the highest anti-tb activity at concentrations of 3.9 and 7.8 µm, respectively. most of the compounds in series 4 (4b, 4c, 4d, 4f, and 4i) displayed some activity on mycobacteria with mic values of 16 and 31 µm. looking at the chemical structures of the active compounds, it can be observed that the presence of a methyl group at position 2 of the spirocyclic system (series 5) led to a significant reduction in antitubercular activity. introduction of a bulky aromatic substituent (c 6 h 5 ) at position 8 of the ring, as in 4h and 5h, enhanced the antitubercular activity. compounds 4a-4i and 5a-5h were evaluated against a variety of dna and rna viruses in cell culture, namely: herpes simplex virus type-1 (hsv-1) and type-2 (hsv-2), an acyclovir-resistant thymidine kinase-deficient (tk − ) mutant of hsv-1, vaccinia virus, human adenovirus-2, human coronavirus, vesicular stomatitis virus, coxsackie b4 virus, respiratory syncytial virus, parainfluenza-3 virus, reovirus, sindbis virus, punta toro virus, yellow fever virus, and influenza a and influenza b virus. the cytopathic effect reduction assays revealed that compounds 4b, 4c, 5b, and 5d were moderately active against punta toro virus, yellow fever virus or sindbis virus in vero cells ( table 2 ). the antiviral ec 50 values were in the range of 1.9-12 µm and the selectivity index (si: ratio of cytotoxic to antivirally effective concentration) was above 10 in some cases (see values between brackets in table 2 ). the most potent effect was seen with compound 5d that is methylated at positions 2 and 8 of the spirothiazolidinone system. of note, no antiviral activity was obtained for the analogues carrying a spiro-fused cyclopentane ring instead of cyclohexane (i.e., 4a and 5a) or a bulkier group than methyl on the cyclohexane residue. introduction of a methyl group at position 2 of the ring system (e.g., compare compounds 4b and 5b) seemed to have a slightly positive effect on antiviral activity. the test compounds did not exhibit activity against any of the other dna-or rna-viruses tested. nevertheless, this broad antiviral testing allowed to determine the compounds' cytotoxic activity in different mammalian cell lines (table 3 ). in general, the compounds endowed with antiviral activity (4b, 4c, 5b, and 5d) tended to be less cytotoxic than the inactive derivatives. the broad antibacterial and antifungal activity of compounds 4a-4i and 5a-5 h was further assessed using the experiment was performed twice and the same results were obtained a mic, the actual minimum inhibitory concentration required to inhibit the growth of 100% of organisms in the search for effective antimicrobial agents, we achieved the synthesis of novel spirothiazolidinone derivatives 4a-4i 5a-5h with the 5-chloro-3-phenyl-1h-indole scaffold. structures of the new compounds were characterized and confirmed by spectrometric methods (ir, 1 h nmr, 13 c nmr, and esi-ms) and elemental analysis. compounds 4a-4i and 5a-5h were evaluated for in vitro antitubercular, antiviral, antibacterial, and antifungal activity against various viral, bacterial, and fungal strains. compounds 4h and 5h, bearing a bulky phenyl group at position 8 of the spiro ring, displayed appreciable anti-tb activity against m. tuberculosis h37rv atcc 27294 with mic values of 3.9 and 7.8 µm, respectively. compounds 4b, 4c, 5b, and 5d exhibited inhibitory effect on the replication of punta toro virus, yellow fever virus or sindbis virus in vero cells. the antiviral ec 50 values were in the range of 1.9-12 µm and the selectivity index (si: ratio of cytotoxic to antivirally effective concentration) was above 10 in some cases. the most potent effect was seen with compound 5d that is methylated at positions 2 and 8 of the spirothiazolidinone system. neither of the indole-spirothiazolidinone compounds showed activity against any of the bacterial or fungal strains tested, at concentrations below 2500-156 µm. all purchased solvents and chemicals were of analytical grade and used as received. melting points were determined in open capillary tubes with a buchi b-540 melting point apparatus. microanalyses were performed on a thermo [22] 158-160 °c). a mixture of 3 (0.0025 mol), an appropriate cyclohexanone/cyclopentanone (0.003 mol), and mercaptoacetic acid or 2-mercaptopropionic acid (0.01 mol) in 20 cm 3 dry toluene was heated to reflux with a heating mantle for 5-6 h using a dean-stark water separator. excess toluene was evaporated in vacuo. the resulting residue was treated with saturated nahco 3 solution until co 2 evolution ceased and was allowed to stand overnight or in some cases refrigerated until solidification. the solid thus obtained was washed with water, dried, and recrystallized from ethanol or ethyl acetate. -n-(7,7,9-trimethyl-3-oxo-1-thia-4 the microdilution method was performed according to a standard protocol from the clinical and laboratory standard institute (clsi) [27, 28] . the resazurin microtitre assay (rema) has been developed as a colorimetric and standard method for drug susceptibility testing. the minimum inhibitory concentrations (mics) were determined according to color changes at the end of incubation [29] [30] [31] . the strain used, i.e., mycobacterium tuberculosis h37rv atcc 27294 is susceptible to all common antimycobacterial drugs. middlebrook 7h9 broth medium (becton and dickinson, usa) was used and the medium was adjusted to ph 7.0 at 25 °c. each bottle was controlled for sterility before it was used. resazurin purchased from sigma-aldrich (st louis, usa) was dissolved in sterile distilled water to a final concentration of 0.02% and sterilized by filtration, then stored at 4 °c until use. rifampicin purchased from becton-dickinson (bd, usa) was dissolved in sterile distilled water to a final concentration of 1 μg/cm 3 (critical concentration). the synthesized compounds were dissolved in 100% dimethyl sulfoxide according to clsi methods [27, 28] . stock solutions were obtained by 40-fold dilution in dmso followed by sterile filtration. from here, working stocks at 4000 μm were obtained by diluting 1/10 in mb7h9 medium. the final concentrations were 1000 μm to 0.49 μm for the synthesized compounds. for rifampicin, the critical concentration (1 μg/ cm 3 ) was used [27, 28] . inoculum suspensions of mycobacteria were prepared according to the clsi guidelines as described previously. the isolates were subcultured on löwenstein jensen medium and incubated at 37 °c for 20-25 days. a few colonies from freshly grown m. tuberculosis were suspended in middlebrook 7h9 broth medium to obtain 1.0 mcfarland turbidity, then diluted ten times with the same medium. the broth microdilution test was performed in sterile 96-well u-shaped microdilution plates (lp italiano spa, milano, italy). rows a-f contained 100 mm 3 of the compound dilutions, whereas rows g (positive control) and h (negative control) contained 100 mm 3 medium. one hundred mm 3 of the corresponding inoculum was added to all wells except for row h. the microplates were incubated at 35 °c for about 7-10 days, when mycobacterial growth was clearly visible as a white sediment in the positive control. microbial growth was confirmed by ehrlich-ziehl-neelsen acid-fast stain. resazurin solution (30 mm 3 ) was added to each well and the plates were incubated for one additional day. at that time, the first purple colored well in which no growth was visible, was defined as the compounds' mic value (table 1) . stock solutions of the test and reference compounds were prepared in 100% dmso at 5-25 mm. during incubation with the cells, the highest test concentration was 100 µm (or 250 µm for ribavirin). the antiviral reference compounds were: ganciclovir, brivudin, zanamivir, amantadine, ribavirin, dextran sulfate-10,000, and mycophenolic acid. antiviral evaluation was carried out with a broad panel of viruses using cytopathic effect (cpe) reduction assays. human influenza a (h1n1 and h3n2) and b viruses were examined on madin-darby canine kidney (mdck) cells. respiratory syncytial virus, vesicular stomatitis virus and coxsackie b4 virus were evaluated on human cervix carcinoma hela cells. african green monkey vero cells were used for parainfluenza-3 virus, reovirus-1, sindbis virus, coxsackie b4 virus, punta toro virus and yellow fever virus. human embryonic lung (hel) fibroblast cells were infected with herpes simplex virus types 1 and 2, vaccinia virus, human adenovirus-2, and human coronavirus 229e. semiconfluent cell cultures in 96-well plates were infected with virus at a multiplicity of infection of 100 ccid 50 (50% cell culture infective dose) per well. together with the virus, fourfold dilutions of the test or reference compounds were added. the plates were incubated at 37 °c (or 35 °c for influenza-and coronavirus) until far advanced cpe was visible, i.e., during 3-6 days or 10 days in the case of adenovirus-2. then, microscopy was performed to score the cpe and calculate the 50% antivirally effective concentration (ec 50 ). microscopy was also done to assess cytotoxicity, expressed as the compound concentration causing minimal changes in cell morphology (minimal cytotoxic concentration; mcc). [32, 33] . serial twofold dilutions ranging from 2500 µm to 1.22 µm were prepared in the test medium, i.e., mueller-hinton broth for bacteria and rpmi-1640 medium for yeast strains. the inoculum was prepared using a 4-6 h broth culture of each bacteria type and 24 h culture of yeast strains adjusted to a turbidity equivalent to 0.5 mcfarland standard, diluted in broth media to give a final concentration in the test tray of 5 × 10 5 cfu/cm 3 for bacteria and 5 × 10 3 cfu/cm 3 for yeast. the trays were covered and placed into plastic bags to prevent evaporation. the bacteria trays were incubated at 35 °c for 18-20 h while the yeastcontaining trays were incubated at 35 °c for 46-50 h. the mic was defined as the lowest concentration of compound giving complete inhibition of visible growth. amikacin and fluconazole were used as reference antibiotics for bacteria and yeast, respectively; their mic values were within the accuracy range of the clsi guidelines [34] . (2h, m, ch/ch 2 -sp.), 1.38 (3h, d, j = 6.8 hz, 2-ch 3 -sp.), 1.62-1.80 (4h, m, ch/ch 2 -sp.), 3.87 (1h, s*, s-ch-sp 27 (ch 2 -sp.), 36.84, 36.96 (c9-sp ms (esi +): m/z (%) = 497.1 decan-4-yl)-3-phenyl-1h-indole-2-carboxamide (5h, c 30 h 28 cln 3 o 2 s) beige powder ̄ = 3289 (n-h) 2-ch 3 -sp.), 1.58-2.00 (8h, m, ch 2 -sp 18 (1h, s, nh) ppm; 13 c nmr (apt, dmso-d 6 , 125 mhz): δ = 20.03 (2-ch 3 -sp.), 30.76, 31.24 (ch 2 -sp m-h) + 2] − , 33.8). references 1. world health organization (who tranquilizer benzodiazepine derivatives. south african patent za 6803041 a antibiotics in laboratory medicine, 5th edn. williams and wilkins, philadelphia 28 approved standard-7th edition m7-a7. clinical and laboratory standards institute, wayne 34. clsi document (2010) 7th informational supplement m100-s20 acknowledgements this work was supported in part by the research fund of istanbul university (project number byp-57695). ln acknowledges dedicated assistance from leentje persoons and her team members. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-296970-5yc6u5t3 authors: donmez, ataberk; rifaioglu, ahmet sureyya; acar, aybar; doğan, tunca; cetin-atalay, rengul; atalay, volkan title: ibioprovis: interactive visualization and analysis of compound bioactivity space date: 2020-08-15 journal: bioinformatics doi: 10.1093/bioinformatics/btaa496 sha: doc_id: 296970 cord_uid: 5yc6u5t3 summary: ibioprovis is an interactive tool for visual analysis of the compound bioactivity space in the context of target proteins, drugs and drug candidate compounds. ibioprovis tool takes target protein identifiers and, optionally, compound smiles as input, and uses the state-of-the-art non-linear dimensionality reduction method t-distributed stochastic neighbor embedding (t-sne) to plot the distribution of compounds embedded in a 2d map, based on the similarity of structural properties of compounds and in the context of compounds’ cognate targets. similar compounds, which are embedded to proximate points on the 2d map, may bind the same or similar target proteins. thus, ibioprovis can be used to easily observe the structural distribution of one or two target proteins’ known ligands on the 2d compound space, and to infer new binders to the same protein, or to infer new potential target(s) for a compound of interest, based on this distribution. principal component analysis (pca) projection of the input compounds is also provided, hence the user can interactively observe the same compound or a group of selected compounds which is projected by both pca and embedded by t-sne. ibioprovis also provides detailed information about drugs and drug candidate compounds through cross-references to widely used and well-known databases, in the form of linked table views. two use-case studies were demonstrated, one being on angiotensin-converting enzyme 2 (ace2) protein which is severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spike protein receptor. ace2 binding compounds and seven antiviral drugs were closely embedded in which two of them have been under clinical trial for coronavirus disease 19 (covid-19). availability and implementation: ibioprovis and its carefully filtered dataset are available at https://ibpv.kansil.org/ for public use. contact: vatalay@metu.edu.tr supplementary information: supplementary data are available at bioinformatics online. the chembl database (version 25) has 1 879 206 distinct small molecule compounds with 12 482 target proteins and 15 506 670 reported bioactivities (mendez et al., 2019) . even if only the data in chembl are considered, there are more than 11 billion possible compound-target protein pairs to be tested in vitro experimentally. unfortunately, public databases or datasets have limited coverage as only partial information is available regarding the compound-target interaction space, mainly due to high costs and labor requirements associated with large-scale screening experiments. therefore, prior knowledge about the eventual target proteins or cellular signaling events, in which a small molecule is involved, becomes crucial for novel drug-target discovery (rifaioglu et al., 2019) . furthermore, the representation of drugs and their targets in databases lack the comparative holistic view of the molecular action on multiple targets and structural similarity of the compounds. a small number of studies have recently become available to visualize the chemical space and the compound bioactivity space (awale and raymond, 2016; gaspar et al., 2015; gü tlein et al., 2012; janssen et al., 2019; karlov et al., 2019) . janssen et al. (2019) and karlov et al. (2019) we describe a tool called ibioprovis, which uses a map-based method to embed a given set of compounds, particularly active compounds in the context of their cognate target proteins, as points onto a real-coordinate 2d space, based on the structural descriptors of the compounds. ibioprovis allows the interactive visualization of these embeddings. the sources of the set of compounds are not restricted; the compounds may be coming from a list of user-defined compounds indicated as canonical smiles strings (e.g. the source of this list can be the output of a machine learning method, which predicts interacting compounds to the target of interest), drugs from drugbank or target proteins' active compounds that are extracted from a reliable compound-target bioactivity measurement dataset, which is a carefully processed and filtered subset of the chembl (v25) database. the output is the 2d embedding of the input set of compounds. by looking at the distribution of compounds as points in this embedding, the user can infer that the compounds that are close to each other may possess similar protein target characteristics. we use the extended connectivity fingerprint (rogers and hahn, 2010) with bond diameter four (ecfp4) as the compound descriptor and pca and t-stochastic neighbor embedding (t-sne) to generate the 2d embeddings. we also provide a reliable compound-target bioactivity measurement dataset, which is a carefully processed and filtered subset of chembl (v25) database, to be used with ibioprovis. ibioprovis is an interactive web-based visualization tool and it has advantages when compared with existing studies and tools. first of all, the computation is performed in real-time and visualization can be done for a variety of input set of compounds; i.e. ibioprovis is not restricted in terms of datasets. ibioprovis is a web-based tool and it does not require any installation. since the local neighborhood is essential for drug discovery and drug repurposing, ibioprovis employs t-sne. additionally, a pca projection of the compounds is provided as well, for comparison. furthermore, visual analysis of the compound bioactivity space is made possible in several contexts such as target proteins, drugs and drug candidate compounds. the user has the option to select the compounds from a reliable filtered compound-target bioactivity measurement dataset, which is a carefully processed and filtered subset of chembl (v25) database. ibioprovis has its own bioactivity dataset, processed and filtered from the chembl (v25) database, which originally contains a total of 15 506 670 data points (i.e. bioactivity measurements; mendez et al., 2019) . after the application of several filtering and preprocessing steps (which are outlined in the supplementary material and at https://ibpv.kansil.org/dataset) to generate the ibioprovis compound-target protein dataset, the number of bioactivity measurements was reduced to 890 886 which contains 3803 unique target proteins and 581 442 unique compounds. the whole dataset is available for download at https://ibpv.kansil.org/dataset. if the user desires, ibioprovis embedding operations are applied to this filtered dataset. upon a user submission of target protein identifier(s), ibioprovis first extracts ecfp4 for the compounds of the given target protein(s), to be used as compound feature vectors. the tool then generates a distance matrix for the given compounds, based on the tanimoto coefficient. the distance matrix becomes the input to the t-sne algorithm which produces the 2d embeddings of the compound feature vectors (van der maaten and hinton, 2008). finally, these 2d embeddings are plotted as a scatter plot and the point that corresponds to each compound is color-labeled based on the target protein that the compound is reported to bind to. it is also possible to give the representations of drugs or compounds of interest in canonical smiles notations during the input phase, to obtain their 2d embeddings along with the binders of the given target proteins. once the embedding process is completed and displayed, the user is able to select a set of compounds on the constructed plot and observe their chembl identifiers and the target proteins that they actively bind to. several cross-references to widely used and wellknown biological databases are also provided so that the user can easily relate the entities and navigate to those databases by clickable links. the cross-referenced databases are uniprot, intact, pubchem, drugbank and clinical trials. these steps taken to generate the t-sne embeddings are given in algorithm 1 in the supplementary material and its expected complexity is o(nlogn) where n is the total number of compounds. pca projection of the input compounds is provided as well, and the worst-case time complexity is o(n 2 ) when pca is used. the bokeh library is employed to generate interactive and user-friendly visualizations (bokeh development team, 2019). a sample web interface embedding is demonstrated in figure 1 . the active compounds are colored either in blue or in green. additional user-input compounds are shown in red and drugs (approved and experimental drugs found in the drugbank database) are represented by diamond shapes. when a user selects a set of compounds, the information about these compounds and their target proteins is shown in two different tables (side table: compounds, bottom table: drugs), where the compounds (rows) are grouped by their respective target proteins. an additional group is created for the user-input compounds since their target information is not presented. this information is shown under the 'target information' column. ibioprovis provides uniprot protein accessions, gene names and chembl identifiers for the target proteins. in addition to these, compound chembl id, molecular formulas and pubchem crossreferences are given under this table, for the selected compounds. the second (bottom) table is reserved to present only the approved or experimental drugs in the user's selection. here, ibioprovis provides drug names, and clinical trial cross-references in addition to the aforementioned information. at the top right side of the plot, there are buttons for easy navigation on the plot such as pan, box zoom, box select, wheel zoom, tap, reset and save. there is a bioactivity value filter at the bottom of the plot, which can be used interactively to remove the compounds that do not satisfy the selected bioactivity threshold [against the corresponding target protein(s)]. the first use-case example of ibioprovis is demonstrated on b-adrenergic receptors (b-ars) adrb2 (beta-2 adrenergic receptor) and adrb3 (beta-3 adrenergic receptor) (fig. 1) . recent studies have shown that isoform-specific activation of b-ars is associated with distinct cellular events in various tissues. hence, targeting b-ars selectively is important for their molecular pathology-specific actions. small molecules targeting adrb2 and adrb3 with affinities < 10 mm were embedded with ibioprovis' interactive web interface ( fig. 1a and b) . molecules with similar structures are located in close vicinity, although they are reported to act on different isoforms of b-ars (blue versus green nodes in fig. 1c ). molecules shared by both proteins are represented by magentacolored nodes. as seen in figure 1 , salmeterol (chembl1263) targets both isoforms of b-ars, whereas compound chembl1800935 targets the adrb2 protein and chembl3126381 targets the adrb3 protein. all three compounds possess very similar structure. this specific example clearly demonstrates that although chembl1800935 has been reported to act on ardb2, ibioprovis embedding indicates that this compound may also act on ardb3. hence, by examining the embedding by ibioprovis, one can hypothesize ardb3 as a new target of chembl1800935. the same argument is valid for compound together with 6516 small molecule drugs from the drugbank database ( fig. 1 , case study 2; wishart et al., 2018) . there are two significantly separated clusters of ace2 receptor binding compounds. although compounds of the cluster at the upper right side are defined as thiolbased ace2 inhibitors, the cluster of compounds in figure 1 , case study 2, panel b are from another study which designed ace2 peptidase activity inhibitors (deatonn et al., 2008; mores et al., 2008) . these experimental inhibitors were closely embedded with seven antiviral-protease inhibitors among which lopivanir and ritonavir are currently under clinical trials for use against sars-cov-2 (cao et al., 2020; harrison, 2020) . ibioprovis is an unprecedented tool that can be utilized for virtual screening and for chemical genomics. it can be used for several purposes, including the investigation and analysis of how active compounds of different target proteins are distributed on a 2d space, as well as the prediction of bioactivity profiles for new or uncharacterized compounds, based on the features of compounds with known bioactivity information. furthermore, it may provide insight to drug repurposing studies by identifying the compounds that are embedded close to an approved drug, especially when those compounds are known binders of a different target protein. web-based 3d-visualization of the drugbank chemical space bokeh: python library for interactive visualization a trial of lopinavir-ritonavir in adults hospitalized with severe coivd-19 thiol-based angiotensin-converting enzyme 2 inhibitors: p1 modifications for the exploration of the s1 subsite chemical data visualization and analysis with incremental generative topographic mapping: big data challenge ches-mapper-chemical space mapping and visualization in 3d coronavirus puts drug repurposing on the fast track sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor drug discovery maps, a machine learning model that visualizes and predicts kinome-inhibitor interaction landscapes chemical space exploration guided by deep neural networks chembl-towards direct deposition of bioassay data development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme recent applications of deep learning and machine intelligence on in-silico drug discovery extended-connectivity fingerprints visualizing data using t-sne drugbank 5.0: a major update to the drugbank database for 2018 this work was supported by scientific and technological council of turkey (tü b _ itak) grant number: eeeag-116e930.conflict of interest: none declared. chembl3126381 which acts on ardb2 but may also act on ardb3.we selected the angiotensin-converting enzyme 2 (ace2) receptor, which has been reported as the sars-cov-2 spike protein receptor for viral entry, as the second use-case demonstration (hoffmann et al., 2020) . ace2 receptor (chembl3736) is associated with 58 compounds which satisfy the ibioprovis bioactivity dataset criteria (supplementary material). the 58 compounds were embedded key: cord-345750-dk1exw9l authors: kulikov, a. s.; epishina, m. a.; batog, l. v.; rozhkov, v. yu.; makhova, n. n.; konyushkin, l. d.; semenova, m. n.; semenov, v. v. title: synthesis and antineoplastic properties of (1h-1,2,3-triazol-1-yl)furazans date: 2014-01-07 journal: russ chem bull doi: 10.1007/s11172-013-0113-2 sha: doc_id: 345750 cord_uid: dk1exw9l a method of 3-amino-4-[5-aryl(heteroaryl)-1h-1,2,3-triazol-1-yl)]furazan synthesis was optimized. condensation of these compounds with 2,5-dimethoxytetrahydrofuran resulted in a series of previously unknown 4-[5-aryl(heteroaryl)-1h-1,2,3-triazol-1-yl)]-3-(pyrrol-1-yl)furazans. all target compounds were evaluated for both antimitotic microtubule destabilizing effect in a phenotypic sea urchin embryo assay and cytotoxicity in a panel of 60 human cancer cell lines. pyrrolyl derivatives of triazolylfurazans were determined as antiproliferative compounds. the most potent microtubule targeting compounds 7a and 7e are of interest for further trials as antineoplastic agents. five membered heterocycles are frequently used in the synthesis of antimitotics that was studied in detail 1 for the analogs of natural compound combretastatin a 4 (ca 4). a water soluble phosphorylated prodrug of ca 4 is cur rently undergoes clinical trials in the usa as antitumor agent. 2, 3 an introduction of 1,2,3 triazole (1,2,3 tri azolocombretastatin) 4, 5 and furazan (combretafurazan) 6 rings into combretastatin framework was regarded as a non isomerizable and metabolically stable bioisosteric replacement of the double bond in cis stilbenes allowing the synthesis of new promising anticancer compounds. compared to combretastatin, combretafurazan is a more potent cytotoxic compound in vitro against neuro blastoma cells, yet maintaining similar pharmacokinetic properties. 6 1,2,3 triazole bridged combretastatin ana log 4,5 exhibits both strong cytotoxicity against ovarian can cer cells and vascular disrupting activity in tumors. 6 more over, this compound is more water soluble than combre tafurazan. water soluble biologically active compounds contain ing both cycles, e.g., (1,2,3 triazol 1 yl)furazans 1, ex hibiting other mechanisms of action were synthesized. thus, compounds with the (1,2,3 triazol 1 yl)furazan moieties inhibit glycogen synthase kinase (gsk 3), a tar get in the treatment of alzheimer's disease and type 2 diabetes. 7 other analogs of (1,2,3 triazol 1 yl)furazans inhibit the sars cov m pro cysteine protease, an impor tant enzyme responsible for the intracellular replication of severe acute respiratory syndrome coronavirus. 8a several (1,2,3 triazol 1 yl)furazan derivatives selectively stimu * dedicated to academician of the russian academy of sciences i. p. beletskaya on the occasion of her anniversary. late no dependent activation of soluble guanylate cy clase (sgc). 8b in the present work we aimed to study a series of (1,2,3 triazol 1 yl)furazans 1 as potential antineoplastic agents, since these compounds can be synthesized by well elabo rated methods. 9-15 synthesis of these compounds involved 1,3 dipolar cycloaddition of azidofurazans 2 to various dipolarophiles, e.g., acetylenes, morpholinonitroethylene, or compounds with the activated methylene group, e.g., activated nitriles and 1,3 dicarbonyl compounds. the dis advantage of majority of these methods is the formation of 1,2,3 triazole regioisomers. depending on the type of the substituents, dipolarophiles add differently to the azidofurazans yielding isomeric 4,5 or 5,4 derivatives (scheme 1). the regioselective cycloaddition of aroylacetic esters to azidofurazans was described; in the cycloaddition prod ucts, the aryl substituent and the ester group were located, respectively, at the positions 5 and 4 of the triazole ring. 10 however, only two compounds of this type with ar = ph and 4 clc 6 h 4 synthesized from the corresponding aroyl acetates are known. to provide feasible antineoplastic properties, it is necessary to introduce into the triazol ylfurazan structure either alkoxybenzene moieties or heterocyclic pharmacophores and to remove the es ter group. thereby, the aim of the present work was the optimi zation of the synthetic procedure for [5 aryl(hetaryl) 1h 1,2,3 triazol 1 yl]furazan derivatives and biological evaluation of the target compounds for antiprolifera tive properties. it was also necessary to clarify whether the regioselectivity of cycloaddition of azidofurazan to aroyl(hetaroyl)acetates 3 with other aromatic or hetero aromatic substituents will be retained. in addition, to extend the scope of triazolylfurazan derivatives with potential antineoplastic activity, we used the clauson-kaas pyrrole synthesis involving the reaction of primary amino group of the furazan ring with dimethoxytetra hydrofuran. for these purposes, aroylacetic esters 3a-g were in volved in the cyclocondensations with aminoazidofur azan 2a under conditions developed earlier. 10 the 1 h and 13 c nmr spectra of synthesized triazolylfurazans 4a-g indicated formation of single regioisomer in high yield; no signals for the second possible regioisomer were detected. the spectral characteristics also indicated high purity of the crude products. therefore, unpurified esters 4a-g were hydrolyzed to the corresponding acids 5a-g, which also without further purification were subsequently thermally decarboxylated to target 3 amino 4 [5 aryl(hetaryl) 1h 1,2,3 triazol 1 yl]furazans 6a-g in high yields. thus, we developed a preparative procedure for the synthesis of tri azolylfurazans 6a-g without purification of the interme diates (scheme 2). to transform the amino group of triazolylfurazans 6 into the pyrrole ring, compounds 6a-f and 6h 15 were involved in the clauson-kaas pyrrole synthesis with 2,5 dimethyltetrahydrofuran. the reaction was carried out in refluxing acetic acid. 14 pyrrole containing tri azolylfurazans 7a-f,h were obtained in 64-98% yields (scheme 3). the biological activity of seven triazolylfurazan deriv atives bearing amino groups 6a-d,g,i, ester 4c (a precur sor of compound 6c), and seven pyrrole derivatives 7a-f,h was studied. the initial trials were carried out on the sea urchin embryos widely used as a model in screening for compounds with antiproliferative effect. 16,17 re cently a simple and efficient pheno typic sea urchin embryo assay has been developed. the assay allows identification of compounds with antiproliferative properties and pro vides information about the mech anism of antimitotic activity. 18 specific changes of sea urchin embryo swimming pattern, namely, settlement to the bottom of the culture vessel and rapid spinning around the animal-vegetal axis, suggest a microtubule destabi lizing activity of a tested compound.* typical develop mental abnormalities caused by triazolylfurazan 7 are shown in fig. 1 . note that the compounds at effective concentrations leading to the alteration of sea urchin egg cleavage were comparable with the ic 50 for the cultured mammalian and human tumor cells. 18, 19 target compounds were further selected for cytotoxicity test in 60 human tumor cell lines (developmental therapeutics program at the national cancer institute of usa). the results are given in table 1 . triazolylfurazans 6a-d,g,i bearing alkoxybenzene moieties and the unsubstituted amino group, as well as ester 4c, did not affect cell division in both test systems. their analogs 7a-f,h bearing the pyrrole ring instead of the amino group exhibited moderate activity. the most potent compounds 7a and 7e altered cleavage of the sea urchin eggs at concentration of 50 nmol l -1 . compound 7e caused the sea urchin embryo spinning suggesting the antitubulin mechanism of action, namely, the ability to destabilize microtubules. apparently, compound 7a ex hibited similar mechanism of action. although, compound 7a failed to affect the sea urchin embryo swimming, the arrested eggs acquired tuberculate shape typical of micro tubule destabilizers. 18 the pyrrole ring was shown to be essential for antiproliferative effect, since the related struc tures 6 containing the amino group instead of the pyrrole ring were inactive. it is worth noting that the increase in the number of methoxy groups in the benzene ring (com pounds 7a-d) resulted in reduction of the antimitotic properties. in this respect, triazolylfurazans 7 is distin guished from the known analogs of plant antimitotics com bretastatin and podophyllotoxin interacting with the col chicine site of tubulin. specifically, trimethoxybenzene according to the data of the national cancer institute (nci) of usa, compounds 7a and 7e inhibited cancer cell growth at relatively low concentrations (gi 50 = 389 and 295 nmol l -1 , respectively). these compounds were referred to the biological expert committee of nci as promising for further studies. leukemia sr cells (7a), melanoma mda mb 435 cells (7a and 7e) , and the co lon cancer cells (7e) were the most sensitive to triazolyl furazans 7a and 7e. the "doze-effect" curves for seven colon cancer cell lines exposed to compound 7e are given in fig. 2 . in summary, the preparative synthesis of furazans 6 by 1,3 dipolar cycloaddition of azidoaminofurazan 2a to aroyl(hetaroyl)acetates 3a-g followed by further modifi cation was developed. the procedure does not require pu rification of the intermediates. subsequent clauson-kaas condensation of synthesized triazolylfurazans 6 with dimethoxytetrahydrofuran yielded a series of 4 [5 aryl (hetaryl) 1h 1,2,3 triazol 1 yl] (3 pyrrol 1 yl)furazans 7. the antiproliferative properties of both types of compounds were evaluated using the sea urchin embryo assay. it was found that the amino derivatives of triazolylfurazans 6 failed to affect cell division. however, their analogs 7 bear ing the pyrrole ring exhibited moderate antimitotic activi ty. two compounds, 7a and 7c, were referred to the nci biological expert committee as promising compounds for further trials. nmr spectra were recorded on bruker wm 250 ( 1 h, 250 mhz) and bruker am 300 ( 13 c, 75.5 mhz) spectrometers. chemical shifts are given in the  scale relative to me 4 si (inter nal standard). mass spectra were obtained on a varian mat ch 6 instrument (ei, 70 ev). thin layer chromatography was per formed on silufol uv 254 plate (elution with chcl 3 ), spots were visualized under uv light. elemental analyses were carried out on a perkin-elmer 2400 chn analyzer. ethyl aroylacetates with 4 methoxyphenyl (3a), 3,4 di methoxyphenyl (3b), 3,4,5 trimethoxyphenyl (3c), 4 fluoro phenyl substituents (3f) were commercially available (aldrich). synthesis of ethyl 1 (4 aminofurazan 3 yl) 5 r 1h 1,2,3 triazole 4 carboxylates 4a-g (general procedure). a mixture of 4 azidofurazan 3 amine 2a (0.88 g, 7 mol), aroylacetate 3a-g (7 mmol), and mgco 3 (0.34 g, 4 mmol) in ethanol (20 ml) was refluxed for 8-10 h (until complete consumption of 2a, tlc monitoring). the reaction mixture was filtered hot, the solvent was evaporated in vacuo. the precipitate was filtered off, washed with cold etoh, and dried in air. ethyl 1 (4 aminofurazan 3 yl) 5 (4 methoxyphenyl) 1h 1,2,3 triazole 4 carboxylate (4a). the yield was 96%, m.p. 13 synthesis of 1 (4 aminofurazan 3 yl) 5 r 1h 1,2,3 tri azole 4 carboxylic acids 5a-g (general procedure). a solution of naoh (0.5 g, 12.5 mol) in water (50 ml) was added to ethyl 1 (4 aminofurazan 3 yl) 5 r 1h 1,2,3 triazole 4 carboxylate 4a-g (7 mmol). the reaction mixture was refluxed for 1 h, the undissolved residue was filtered off, and the filtrate was acidified with dilute hcl to ph 2. a precipitate was filtered off, washed with water, and dried in air. when crude carboxylic acids 4a-g (unwashed with etoh) were used, the yields of products 5a-g were virtually the same. 82 (br.s, 4 h, 2 h in ar and 2 h, nh 2 ). 13 c nmr (dmso d 6 ), : 56.03 (ome) aminofurazan 3 yl) 5 (3,4 methylenedioxyphenyl) 1h 3 triazole 4 carboxylic acid (5d) aminofurazan 3 yl) 5 (4 ethoxyphenyl) 1h 1,2,3 tri azole 4 carboxylic acid (5e). the yield was 89%, m 13 c nmr (dmso d 6 ), : 13.72 (me) aminofurazan 3 yl) 5 (4 fluorophenyl) 1h 1,2,3 tri azole 4 carboxylic acid (5f). the yield was 79%, m aminofurazan 3 yl) 5 (2 thienyl) 1h 1,2,3 triazole 4 carboxylic acid (5g). the yield was 66%, m 28; n, 30.12. c 9 h 6 n 6 o 3 s 17; n, 30.20. ms, m/z (i rel (%) 2 hz); 7.73 (d, 1 h, c(5) thiophene ring, 3 j = 5.2 hz) the yield was 71%, m .81; n, 32.43. c 11 h 10 n 6 o 2 : 3.79 (s, 3 h, ome); 6.65 (s 13 c nmr (dmso d 6 ) ome) : 3.70 (s, 3 h, ome); 3.73 (s, 6 h, 2 ome); 6.74 (s, 2 h in ar) dmso d 6 ), : 56.04 (ome); 193 c .96; n, 30.87. ms, m/z (i rel : 6.08 (s, 2 h, ch 2 ) the yield was 63%, m the yield was 68%, m.p. 191-192 c : 6.63 (s, 2 h, nh 2 ); 7.22, 7.49 (both d, 2 h each the yield was 73%, m.p ) thiophene ring .79; n, 27.39. c 15 h 12 n 6 o 2 68 (br.s, 2 h, c(2) and c(5) of pyrrole ring) 13 c nmr (cdcl : 56.09 (ome)103 c ) and c(5) of pyrrole ring : 1.31 (t, 3 h, me, 3 j = 7.0 hz); 4.05 (q, 2 h, ch 2 , 3 j = 7.0 hz); 6.35 (s, 2 h, c(3) and c(4) of pyrrole ring); 6.83 (s, 2 h, c(2) and c(5) of pyrrole ring) 37 (s, 1 h, triazole ring) 98; n, 28.19. c 14 h 9 fn 6 o 35 (s, 2 h, c(3) and c(4) of pyrrole ring); 6.83 (s, 2 h, c(2) and c(5) of pyrrole ring) institute of developmental bi ology of ras in cyprus. adult sea urchins was carried out at the following devel opmental steps: (1) fertilized eggs, 8-15 min after fertilization 47, 3275; (b) pat. rf 2158265, byul. isobret modern analysis of antibiotics bioorganic marine chemistry biotech niques cytotoxicity in 60 human cancer cell lines was studied at the national cancer institute of usa according to the procedure described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.the authors are grateful to the national cancer insti tute (bethesda, maryland, usa) for the screening of com pounds in the framework of the developmental thera peutics program for the search of antineoplastic medici nal agents; http://dtp.cancer.gov). key: cord-328962-1c4vqaqr authors: benítez-cardoza, claudia guadalupe; vique-sánchez, josé luis title: potential inhibitors of the interaction between ace2 and sars-cov-2 (rbd), to develop a drug date: 2020-06-15 journal: life sci doi: 10.1016/j.lfs.2020.117970 sha: doc_id: 328962 cord_uid: 1c4vqaqr aims: the covid-19 disease caused by the sars-cov-2 has become a pandemic and there are no effective treatments that reduce the contagion. it is urgent to propose new treatment options, which are more effective in the interaction between viruses and cells. in this study was to develop a search for new pharmacological compounds against the angiotensin-converting enzyme 2 (ace2), to inhibit the interaction with sars-cov-2. materials and methods: docking, virtual screening using almost 500,000 compounds directed to interact in the region between the residues (gln24, asp30, his34, tyr41, gln42, met82, lys353, and arg357) in ace2. the average of δg(binding), the standard deviation value and the theoretical toxicity from compounds were analyzed. key findings: 20 best compounds directed to interact in ace2 with a high probability to be safe in humans, validated by web servers of prediction of adme and toxicity (protox-ii and preadmet), to difficult the interaction between ace2 and region binding domain (rbd) of sars-cov-2. significance: in this study, 20 compounds were determined by docking focused on the region of interaction between ace2 and rbd of sars-cov-2 was carried out. the compounds are publicly available to validate the effect in in vitro tests. currently, the pandemic that has developed has some antecedents related to the sars-cov 2002 outbreak, of which several works have been carried out to develop new drugs directed to specific regions of the coronavirus (sars-cov 2002). the disease caused by sars-cov-2 generates a wide range of signs and symptoms, causing respiratory, gastrointestinal and even death diseases [1] [2] . in the first reports on the sars-cov-2 (covid19) outbreak in china reported that the average age was 47 years, with an incubation period of 4 days, 41.9 % were women, with fever 88.7 % and cough the 67.8 % of the patients in the study, accompanied by lymphocytopenia in 83.2 %. it should be noted that there was no specific treatment for sars-cov-2, the treatment was based on antibiotics in 58.0 % and antivirals (oseltamivir) in 36.2 % [2] . recently, new antivirals have been developed, focusing on rna-dependent rna polymerase (rdrp), polyproteins (3clpro and plpro), spike protein (s-protein) [3] [4] and membrane fusion inhibitors (hr1 and hr2 of s-protein) [5] [6] [7] from sars-cov-2. without a treatment that demonstrating an advantage therapeutic, which demonstrates the urgent need for the development of specific drugs against a selective target that alters the evolution of this disease. there are works related to sars-cov, for the development of a specific drug, which have reported the development of peptides related to the key protein for the interaction between the sars-cov and the host cell; the angiotensin-converting enzyme 2 (ace2), reporting amino acids sequence that was essential for drug development (glu22, glu23, lys26, asp30, lys31, his34, glu35, glu37, asp38, glu56 and glu57) [8] . as well as another work that reported the important amino acids between the region binding domain (rbd) of the s-protein sars-cov with the ace2 (gln24, thr27, lys31, his34, glu37, asp38, tyr41, gln42, leu45, leu79, met82, tyr83, asn90, gln325, glu329, asn330, lys353 and gly354 in ace2) [9] . currently, has been reported the crystallographic structure of the interaction between sars-cov2-rbd and ace2 (gln24, asp30, his34, tyr41, gln42, met82, lys353, and arg357 in ace2) [10] , which we used in this study. there are reports of the development of pharmacological compounds that have an effect on the interaction of sars-cov and ace2 [11] [12] , as well as focused on important j o u r n a l p r e -p r o o f proteins in the sars-cov such as the main protease (mpro), which propose synthesized aromatic compounds [13] . although there is a great similarity between sars-cov and sars-cov-2 sequence (sequence identity of almost 80 %) [14] , the same results of compounds or antibodies that were tested in sars-cov are not presented in sars-cov-2; the differences in the residues found in sars-cov-2 explain the resistance generates against compounds and antibodies (s230) [15] [16] . the affinity of sars-cov-2 with ace2 has been determined to be up to 20 times higher than that reported in sars-cov in 2002, which may help explain why the complications that develop are more serious, and the probability of contagion is greater, having a great impact on the health of the population [17] . it was determined that in sars-cov, ace2 plays a very important role so that it can cross the cell membrane and be able to replicate the sars-cov; taking into account that sars-cov-2 also interacts with ace2. recently, an important protein for the interaction of ace2 with sars-cov2, tmprss2, was identified, where it is demonstrated that if it is limited to this protein, the interaction of the virus with the cell can be affected [18] . some works for the development of new drugs against sars-cov-2, propose epitopes as potential sites of interaction [19] , as well as using docking and compound libraries, as well as looking for a repositioning of drugs [5] , to search for compounds that interact with some sars-cov-2 region and thus be able to prevent interaction with ace2 [20] . a drug that was proposed to interact in ace2, is arbidol, which recently reported the crystallographic structure, demonstrating that arbidol interacts in s-protein (domain s2) from sars-cov-2 [21] , which demonstrates the low existence of drugs that are directly interacting with ace2. we use the amino acids reported in the crystallographic structure of the interaction between the s-protein-rbd of sars-cov-2 and ace2 (gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357 in ace2) [10] [22] , therefore, using the crystallographic structure of ace2 (pdb 1r42), we carried out a docking directed to these mentioned residues using a library of compounds (express-pick collection from chembridge corp.) to select the best compounds, and that these can affect the interaction between ace2 and sars-cov-2, making these results an important contribution to establishing the foundations that allow the development of a drug that optimizes the resolution of this pandemic. atomic coordinates of angiotensin converting enzyme 2 (ace2) were obtained from the protein data bank (pdb: 1r42). the structure was used as protein targets for docking procedures. the protonation and energy minimization of pdb file was performed using molecular operating environment (moe) software with the default parameters and the charmm27 force field [23] [24] . we select one region to interaction in ace2 (gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357) [10] . the express-pick collection stock of the small molecule screening library from chembridge copr. was used for docking [25] . this collection of small molecule screening compounds contains over 500,000 chemical compounds that fulfill the druggable properties of lipinski's rules [26] [27] and cover a broad area of chemical space. for docking, the receptors were kept rigid, while the ligand atoms were released to move to a maximal number of rotatable bonds. all crystallographic water molecules were deleted from the initial structures. high-throughput virtual molecular docking was carried out [25] [28] by means of the software autodock and moe, using default parameters (placement: triangle matcher, rescoring 1: london δg, refinement: forcefield, rescoring 2: london δg, for each ligand up to 20 conformations were generated and saved). the binding affinity of each complex (ligand-protein) was estimated with the ratio of general born vs volume integral (gb/vi), using parameters in moe [29] [30] . general born or nonbonded interaction energies comprise van der waals, coulomb electrostatic interactions and implied solvent interaction energies [30] . j o u r n a l p r e -p r o o f each compound was simulated with up to 50 conformations, to select the best compounds, the average of the δg binding interaction value of up to 20 conformers, the description of chemical properties by physchem -acd/labs [31], the theoretical toxicity [32] , carcinogenicity and mutagenicity [33] were considered. the calculated interactions between ace2 and compounds were visualized with ligand-interaction interactions implemented in moe. among the interactions in ace2 (gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357) with compounds ( figure 1 ), the selection criteria of the top poses, out of almost 500,000 compounds from chembridge library, were the frequency of the conformers of each compound and the δg binding values between -6.0 to -7.3 kcal/mol -1 . we made the choice of compounds based on the average of the score from up to 20 conformers per compound and better probability to be safe in humans. we selected 20 compounds depicted here as c1 to c20 (table 1) journal pre-proof j o u r n a l p r e -p r o o f for selection of the best compounds, the analysis from docking´s results was carried out, taking into account the average of the interaction δg binding (15 to 20 conformers) was determined, as well as the standard deviation for each compound. subsequently, the theoretical toxicity were evaluated with two website (protox-ii -prediction of toxicity and preadmet web server, prediction of carcinogenicity and mutagenicity, table 2 ). besides, we determined 30 compounds with good results, but with significant theoretical toxicity effects, we show them in table s21 . the description of the chemical properties of each compound (c1 -c20, table s22 ), adme (table s23 ) and theoretical toxicity (table s24) , are shown in the supplemental material. j o u r n a l p r e -p r o o f to propose the probable interaction sites between each compound (c1 -c20) with ace2 we analyzed up to 20 conformers of each compound that showed the better δg binding values of interaction in amino acids gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357 ( figure 1 ). from docking´s result (table s1 -s20), we determined the main amino acids in ace2 to interact with the 20 compounds, these are lys26, his34, glu37, asp38, tyr41, gln96, gln325, asn330, lys353, arg357, ala386, ala387, pro389 and arg393 ( table 2 ). the interaction of each compound and its conformers in ace2 are shown in the supplementary material ( figure s1 -s20). several arg393 are important for the majority of the compounds that we propose to interact in ace2 (table 1) . furthermore, we propose that the interaction site in ace2 presents little change in the structural conformation when the s-protein-rbd is present, since we perform an alignment and superposition of the three-dimensional structures, of the apo-ace2 (pdb: 1r42) and the ace2 with rbd (pdb: 6m17) and there is an rmsd between them of 2.4 å ( figure s21 ), which shows that the interaction of ace2 with rbd does not affect the three-dimensional conformation, moreover, the amino acids that we take into account to do the docking (amino [7] , in which they seek to prevent the hr1 region of s-protein ( figure s22 ) from interacting with hr2 and its ligand in the ace2 membrane, hindering the process of fusing the viral membrane and blocking the introduction of viral genetic material, there are currently very promising results of this type of drug, with evaluated doses with an ic50s between 1.3 and 15.8 nm against sars-cov-2 [7] , it shows that its development is viable. we determined that there are very few studies of new drugs against ace2, previously arbidol was thought to have ace2 as a selective target, but the description of the interaction of arbidol with s2-domain in s-protein of sars-cov-2 was made [21] . therefore, the development of antivirals against covid-19, still in development, shows that there are no specific drugs against sars-cov-2, since several of the drugs that are using, they have been developed against other diseases, such as ebola (remdisivir) [4] , influenza (arbidol) [21] , sars and mers [13] [14] , searching a drug repurposing [38] . besides, proposing combinations of drugs, with different mechanisms of action (such as those mentioned), will be used for the pandemic that is occurring, in addition, will be necessary to develop selective drugs against ace2 ( figure s23) , which may be able to prevent interaction with sars -cov-2. carrying out the selection of the compounds, taking into account the results of between 15 to 20 conformers of each compound, gives us a greater probability of choosing the compounds that could be selective in the amino acids sequence gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357 in ace2 (figure 1 ), subsequently validate them by two toxicity prediction web servers ( table 2 and table s22 ), obtaining important characteristics such as a lethal dose 50 (ld50) within acceptable values as well as a very low probability of toxicity; which must be fulfilled by each compound for its selection. thus, this could reduce the time that must be waited for to be used in humans, therefore we propose compounds (c1 -c20), with a high probability to be safe in humans. in addition, we show the next 30 compounds, which have some probability of generating side effects, such as carcinogenicity, hepatotoxicity and immunotoxicity mainly; these compounds are on the supplementary material (table s21 ) which could be tested in in vitro tests with ace2 -sars-cov-2 interaction. it will be necessary to evaluate by in vivo tests, the effect of these compounds could to generate when interacting with ace2 in humans, since the ace2 functions on angiotensin and its effects at the cardiovascular system level [39] [40] [41] [42] [43] , they would have to be considered to determine the therapeutic effect and the degree of impact that they could have on the health-disease process of covid-19 and/or some alteration in the functions of ace2. most of these proposed compounds do not have any specific use registered, nor a scientific article or registered patent, all the compounds are available to purchase or j o u r n a l p r e -p r o o f systematize them, to carry out in vitro assays for the interaction of sars-cov-2 with ace2, and in this way, be able to develop a new drug that helps combat this pandemic. furthermore, as already reported, sars-cov have an affinity for the same ace2 region, which could help in the future to prevent new viruses that have an affinity for this region of interaction in ace2. we propose 20 compounds that have a high probability of interacting in a specific region in ace2 (gln24, asp30, his34, tyr41, gln42, met82, lys353 and arg357), and thus hinder interaction with the rbd of sars-cov-2. furthermore, these 20 compounds have a high probability to be safe in humans, since they were validated by the protox-ii and preadmet server (adme and toxicity predictor). these 20 compounds are available from chembridge corp. (table 1) j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f table s23 : adme -preadmet | prediction of adme/tox of compounds c1 -c20. table s24 : toxicity -preadmet | prediction of adme/tox of compounds c1 -c20. declarations life sciences require that the corresponding author, signs on behalf of all authors, a declaration of conflicting interests. if you have nothing to declare in any of these categories then this should be stated. a conflicting interest exists when professional judgment concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). it may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. sars and mers: recent insights into emerging coronaviruses clinical characteristics of coronavirus disease 2019 in china molecular investigation of sars-cov-2 proteins and their interactions with antiviral drugs pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid-19 analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor structure of sars coronavirus spike receptor-binding domain complexed with receptor structural basis for the recognition of the sars-cov-2 by full-length human ace2, science (80-. ). (2020) eabb2762 antiviral drug discovery against sars-cov structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor discovery of unsymmetrical aromatic disulfides as novel inhibitors of sars-cov main protease: chemical synthesis, biological evaluation, molecular docking and 3d-qsar study genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding unexpected receptor functional mimicry elucidates activation of coronavirus fusion cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds arbidol: a potential antiviral drug for the treatment of sars-cov-2 by blocking trimerization of the spike glycoprotein characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine charmm: the biomolecular simulation program merck molecular force field. i. basis, form, scope, parameterization, and performance of mmff94 experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings dynamic structure-based pharmacophore model development: a new and effective addition in the histone deacetylase 8 (hdac8) inhibitor discovery use of amino acid composition to predict ligand-binding sites the generalized born/volume integral implicit solvent model: estimation of the free energy of hydration using london dispersion instead of atomic surface area in silico identification of promiscuous scaffolds as potential inhibitors of 1-deoxy-d -xylulose 5-phosphate reductoisomerase for treatment of falciparum malaria crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov therapeutic options for the 2019 novel coronavirus (2019-ncov) research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases synergistic effect of angiotensin-(1-7) on bradykinin arteriolar dilation in vivo angiotensin-(1-7) reduces smooth muscle growth after vascular injury angiotensin-converting enzyme 2 is an essential regulator of heart function a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 enzima conversiva de la angiotensina 2 y su papel emergente en la regulación del sistema renina-angiotensina the authors are very grateful for the financial support from sip-ipn méxico the authors declare that they have no conflict of interest. all sources of funding should also be acknowledged and you should declare any involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication. if the study sponsors had no such involvement, this should be stated. all authors listed on your paper must have made significant contributions to the study. to ensure clarity, you are required to enter the specific details of each author's contribution, which must substantiate the inclusion of each person on the manuscript. please detail this information below (submit additional sheets as necessary): key: cord-327946-mqakaisa authors: massari, serena; bertagnin, chiara; pismataro, maria chiara; donnadio, anna; nannetti, giulio; felicetti, tommaso; di bona, stefano; nizi, maria giulia; tensi, leonardo; manfroni, giuseppe; loza, maria isabel; sabatini, stefano; cecchetti, violetta; brea, jose; goracci, laura; loregian, arianna; tabarrini, oriana title: synthesis and characterization of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide-based compounds targeting the pa-pb1 interface of influenza a virus polymerase date: 2020-10-16 journal: eur j med chem doi: 10.1016/j.ejmech.2020.112944 sha: doc_id: 327946 cord_uid: mqakaisa influenza viruses (flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-flu armamentarium still remains inadequate. in search for new anti-flu drugs, our group has focused on viral rna-dependent rna polymerase (rdrp) developing disruptors of pa-pb1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. by merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. in particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important sar insight and identify new active compounds showing both the ability to inhibit pa-pb1 interaction and viral replication in the micromolar range and at non-toxic concentrations. for few compounds, the ability to efficiently inhibit pa-pb1 subunits interaction did not translate into anti-flu activity. chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, h-bonds enhanced its binding within the pa(c) cavity. influenza (flu) viruses are responsible for seasonal epidemics resulting in about 3 to 5 million cases of severe respiratory illness and 290,000 to 650,000 deaths each year all over the world [1] . moreover, they are also able to generate pandemic outbreaks that occur when new highly virulent flua subtypes generated by antigenic shift are transmitted from animals to humans and sustainably spread among people. in 1918, humanity experienced the "spanish flu" caused by flua(h1n1) [2] , an influenza pandemic that intensively and speedily struck world population infecting about 500 million people and killing from 20 to 40 million people globally [3] . other two pandemics occurred in 20 th century: the "asian flu" caused by flu a(h2n2) virus [4] , which started in china in 1957 and spread globally causing about one to four million deaths, and the "1968 flu pandemic" caused by flu a(h3n2) virus, which started in hong kong and spread to the united states causing one million deaths. in 2003, the avian flu a(h5n1) re-emerged passing the species barrier but fortunately not spreading sustainably from person to person, while, in 2009, the "swine flu" a(h1n1) virus was responsible for the first pandemic of the 21 st century [5] ; it started in mexico and spread rapidly around the world causing from 150,000 to 600,000 deaths [6] . to date, the avian flu a(h5n1) and flu a(h7n9) are of particular concern to public health due to their potential to cause an influenza pandemic [7] . [32] . computational studies performed on the positional isomers 3 and 4 within the pa c cavity suggested that the molecules have a different orientation and extents of hydrophobic and h-bond interactions within the cavity [32] . thus, while the elongated shape of 4 allows it to recognize all the three hydrophobic regions described by liu and yao [35] within the pb1 binding site, compound 3 is j o u r n a l p r e -p r o o f shifted toward the opposite side of the cavity and matches only the first hydrophobic region, generated by w706. nevertheless, compound 3 establishes a very favorable h-bond between its c-2 amidic carbonyl group and the q408 residue, which could be the reason for its efficient inhibition of pa-pb1 heterodimerization. in the present study, additional hybrid compounds were synthesized as analogues of compound 3 (compounds 5-18, figure 2 ) and compound 4 (compounds 19-26, figure 2 ). by mainly focusing on the chtc core, various structural modifications were undertaken investigating the cycloheptane, the thiophene and the 2-carboxamide moieties. from the antiviral activity, sar insights were obtained with the main indication entailing the favorable replacement of the chtc core by a simpler 2carbamoylphenyl moiety. in depth studies were pursued to determine the ability of the compounds to interfere with rdrp functions, their metabolic stability as well as to predict their binding mode within the pa c cavity. further studies were also performed on a couple of regioisomers to investigate the different behavior in inhibiting pa-pb1 interaction and viral growth. the synthesis of all the target compounds 5-13, 15-22 and 24-29 was accomplished, as reported in schemes 2-8, by coupling reaction of the appropriate reagent with 5-methyl-7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride 30 [32] or 7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbonyl chloride 31 [32] . as shown in scheme 1, derivatives 30 and 31 were obtained by chlorination of the corresponding carboxylic acids [32] , which were in turn prepared by cyclocondensation of ethyl 5-amino-1,2,4-triazole-3-carboxylate [36] and 1phenylbutane-1,3-dione in acetic acid at reflux furnishing ethyl 5-methyl-7-phenylinteraction. of note, derivative 8 emerged as the most active anti-flu compound herein reported, even more than hit compound 3. these results suggested that the presence of a cycloalkyl moiety fused to the thiophene ring is important to obtain anti-flu activity, although its aromatization is still tolerated but detrimental for anti-pa-pb1 activity. then, a set of four derivatives was synthesized in which the thiophene-based core was linked to the tzp moiety by the c-3 position. the thienopyridine derivative 12 was the most potent pa-pb1 inhibitor among the compounds herein reported (ic 50 of 3.3 μm), but at the expense of the antiviral activity (ec 50 > 100 μm), while derivative 13 exhibited a balanced biological profile (ic 50 of 31 μm and ec 50 of 43 μm). these results indicated that, in this series of compounds, the lack of a cycloalkyl ring fused to the thiophene does not impair the activity. no anti-pa-pb1 activity was shown by cycloheptathiophene compound 10 and benzothiophene compound 11, the strict analogs of compounds 3 and 9, respectively, although the latter showed all the compounds were non-toxic up to 250 μm concentration, with the exception of derivatives 10 and 18, which, however, showed a mild cytotoxic effect (cc 50 = 90 and 101 μm, respectively). in summary, the synthesis of novel hybrid compounds, aimed at clarifying the best substitution pattern for the c-2 position of the tzp ring, has led to the identification of some interesting compounds able to inhibit the pa-pb1 interaction and the viral growth. interesting sar insights have been outlined. in particular, the chtc portion can be replaced by a benzene ring (as in metabolic stability in human liver microsomes (hlm) was studied by monitoring the percentage of although the ms/ms fragmentation is not sufficient to identify the exact position of the site of metabolism in the aromatic ring, the most probable site for hydroxylation appears to be at c-4 position, according to metasite [64] predictions within the webmetabase analysis tool (table s3 and figure s3 , supporting information). regarding the human plasma protein binding, samples were analyzed by employing a rapid equilibrium system device and adding them in buffer and measuring the compound presence in the human plasma compartment after 4 h of incubation by means of uplc/ms/ms. it was observed that compound 23 showed an unbound fraction of 22.45%, which together with the good metabolic stability predicts a good bioavailability of the compound in human plasma. finally, with the aim to gain information on how two different moieties, such as the chtc and the 2-carbamoylphenyl, showed a favorable ability to disrupt pa-pb1 interaction, computational studies were performed to predict the binding mode of benzamide derivative 23 within the pa cavity with respect to hit compound 4. for comparative purpose, also its isomer 14, which was active in elisa-based assay but devoid of any antiviral activity, was studied in comparison to hit compound 3. the study was conducted using the same method applied for the evaluation of the binding mode of compounds 3 and 4 using the flap software [65] , and the most probable binding poses for the two isomers are illustrated in figure 4 . in agreement with the biological results, both compounds showed an efficient binding within the pa c , although through different orientations and interactions. in particular, both compounds were found to efficiently interact with w706 through hydrophobic interactions, as observed for all the inhibitors of the pa-pb1 interaction we have studied so far [24] . similarly to 4, compound 23 displayed a favorable hydrophobic interaction with all the three hydrophobic regions previously described by liu and yao [35] . indeed, according to the proposed j o u r n a l p r e -p r o o f description of the protein cavity, the first hydrophobic region is centered on residues w706 and f411, the second one is defined by f710 and l666, and the third one includes l640, v636, m595, and w619. however, differently from 4, 23 seems to be involved in the formation of a favorable hbond between the 2-carboxamide nh group and the hydroxyl group of t639. this amino acid was not found to be involved in such kind of interaction in other compounds studied so far by us. interaction. this data seems to confirm the importance of an efficient interaction with w706 for inhibition of pa-pb1 heterodimerization. in conclusion, the computational study confirmed the central interaction between the inhibitors and w706, and the 2-carboxyamide group of 23 and 14 was pivotal in the interaction with pa by establishing favorable h-bonds. this is also experimentally confirmed, since the analogues lacking the 2-carboxamide substituent showed a very weak activity in the elisa pa-pb1 interaction assays [32] . the flu rdrp is emerging as a privileged drug-target as demonstrated by the most recently approved compounds or in the pipeline such as favipiravir, baloxavir marboxil and pimodivir, which act by inhibiting each of the three rdrp subunits. by applying an alternative approach to inhibit flu rdrp functions, since many years we have been working on the development of compounds able to interfere with rdrp subunits interaction and in commercially available starting materials, reagents, and solvents were used as supplied. all after cooling, the reaction mixture was filtered over celite and the filtrate was evaporated to dryness, to give a residue that was stirred with saturated nahco 3 solution for 15 min. the solution was then extracted with ch 2 cl 2 and the organic layers were evaporated to dryness to give a grey solid, which was purified by flash chromatography eluting with meoh/ch 2 cl 2 (2%), to give 9 [37] (1.0 equiv) in well dry ch 2 cl 2 , oxalyl chloride (3 equiv) was added and after 30 min dry dmf (2 drops) was added. after 2h, the reaction mixture was evaporated to dryness to give 30 [37] or 31 [37] that was dissolved in well dry ch 2 cl 2 and added of the appropriate amine (1.0 equiv) and dipea (1.0 equiv). the reaction mixture was maintained at r.t. until no starting material was detected by tlc. then, it was worked up through two procedures: (procedure 1) the reaction mixture was evaporated to dryness to give a residue that was poured into ice/water providing a precipitate which was filtered and purified as reported in the description of the compounds; or (procedure 2) the precipitate formed in the reaction mixture was filtered and purified as reported in the description of the compounds. the title compound was prepared starting from 36 [38] and 30 [37] ( n-(3-carbamoyl-6-ethyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-7(9) . the title compound was prepared starting from 40 [41] and 30 [37] (11) . the title compound was prepared starting from 48 [48] and 30 [37] through . [49] the title compound was prepared starting from methyl 3-aminothiophene-2-carboxylate through method b providing 49 [47] in 100% yield, carboxamide (13). the title compound was prepared starting from 50 [49] and 30 [37] through carboxamide (22) . the title compound was prepared starting from 50 [49] and 31 [37] through the minireplicon assay was performed as described [27] , with some modifications. briefly, hek 293t cells (2 × 10 5 cells per well) were plated into 24-well plates and incubated overnight at 37 °c. the next day, cells were transfected using calcium phosphate co-precipitation method with pcdna the stock solutions (10-2 m) of the assayed compounds were diluted to decreased molarity, from 300 μm to 0.1μm, in 384 well transparent plate (greiner 781801) with 1% dmso: 99% pbs buffer. then, they were incubate at 37 ºc and read after 2 hours in a nephelostar plus (bmg labtech). the results were adjusted to a segmented regression to obtain the maximum concentration in which compounds are soluble. digossin, prazosin and progesterone were used as reference compounds (equilibrium solubility = 84.0, 62.8 and 6.5 μm, respectively) [71] . j o u r n a l p r e -p r o o f x-ray diffraction patterns of the crystals were recorded using a bruker d8 venture diffractometer equipped with an incoatec imus3.0 microfocus sealed-tube mo kα (λ = 0.71073 å) source and a ccd photon ii detector. the analyses were carried out at 120 (2) k using an oxford cryosystems 800 cooler. the data collected through generic φ and ω were integrated and reduced using the bruker axs v8 saint software. the structures were solved and all the thermal parameters were anisotropically refined using the shelxt and shelxl packages of the bruker apex3 software. the assay was carried out by employing rapid equilibrium dyalisis (red) from thermo flow: 0.6 ml/min. the chromatographic equipment employed was an uplc qsm waters acquity. compound concentrations were calculated from the ms peak areas. test compounds (10 μm www.moldiscovery.com). c 23 h 19 n 5 o 2 s 430.1338 (m+h) + , found 430.133652 (m+h) + . hplc, ret. time: 3.652, peak area: 97 the title compound was prepared starting from 2-aminobenzothiazole and 30 36% yield as yellow solid; 1 h nmr (dmsod 6 , 400 mhz): δ 2.70 (s, 3h, ch 3 ), 7.30 (t, j = 7.4 hz, 1h, aromatic ch), 7.40 (t, j = 7.3 hz, 1h, aromatic ch), 7.55-7.70 (m, 4h, aromatic ch and h-6), 7.80 (d, j = 7.9 hz, 1h, aromatic ch), 8.10 (d, j = 7.7 hz, 1h, aromatic ch) 102196 (2m+h) + . hplc, ret. time: 3.278 min, peak area: 98 the title compound was prepared starting from 2-aminobenzothiazole and 31 worked up through procedure 2, and purified by crystallization by dmf, in 57% yield as yellow solid; 1 h nmr (dmso-d 6 , 400 mhz): δ 2.85 (s, 1h 10 (s, 1h, h-6), 8.25-8.30 (m, 2h, aromatic ch) hrms: m/z calcd for c 20 h 11 n 6 os 387.1029 (m+h) + , found 387.1022 (m+h) + . hplc, ret. time: 2.898 min, peak area: organization who, influenza the 1918 influenza pandemic: 100 years of questions answered and unanswered centers for disease control and prevention h2 influenza viruses: designing vaccines against future h2 j o u r n a l p r e -p r o o f pandemics antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans influenza a virus recycling revisited in vivo characterization of avian influenza a (h5n1) and (h7n9) viruses isolated from canadian travelers antiviral treatments drugs for influenza treatment: is there significant news? next-generation direct-acting influenza therapeutics influenza virus polymerase inhibitors in clinical development antivirals targeting the polymerase complex of influenza viruses favipiravir (t-705), a novel viral rna polymerase inhibitor in vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase pa subunit discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 antiviral strategies against influenza virus: towards new therapeutic approaches focusing on the influenza virus polymerase complex: recent progress in drug discovery and assay development insights into rna-dependent rna polymerase inhibitors as anti-influenza virus agents structure and function of influenza polymerase, cold spring harb perspect med structure and function of the influenza virus transcription and replication machinery, cold spring harb crystal structure of the polymerase pac-pb1n complex from an avian influenza h5n1 virus the structural basis for an essential subunit interaction in influenza virus rna polymerase inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions polymerase acidic protein-basic protein 1 (pa-pb1) protein-protein interaction as a target for next-generation anti-influenza therapeutics inhibition of influenza virus polymerase by interfering with its protein-protein interactions human j o u r n a l p r e -p r o o f cytomegalovirus inhibitor al18 also possesses activity against influenza a and b viruses small molecule inhibitors of influenza a and b viruses that act by disrupting subunit interactions of the viral polymerase 6-diphenylpyridines as promising novel anti-influenza agents targeting the pa-pb1 protein-protein interaction: structure-activity relationships exploration with the aid of molecular modeling structural investigation of cycloheptathiophene-3-carboxamide derivatives targeting influenza virus polymerase assembly exploring the cycloheptathiophene-3-carboxamide scaffold to disrupt the interactions of the influenza polymerase subunits and obtain potent anti-influenza activity potent and broad-spectrum cycloheptathiophene-3-carboxamide compounds that target the pa-pb1 interaction of influenza virus rna polymerase and possess a high barrier j o u r n a l p r e -p r o o f to drug resistance a broad antiinfluenza hybrid small molecule that potently disrupts the interaction of polymerase acidic protein-basic protein 1 (pa-pb1) subunits optimization of small-molecule inhibitors of influenza virus polymerase: from thiophene-3-carboxamide to polyamido scaffolds synthesis and biological evaluation of a library of hybrid derivatives as inhibitors of influenza virus pa-pb1 interaction molecular basis of the interaction for an essential subunit pa-pb1 in influenza virus rna polymerase: insights from molecular dynamics simulation and free energy calculation synthesis of esters and amides of 5-amino-1,2,4-triazole-3-carboxylic and 5-amino-1,2,4-triazol-3-ylacetic acids efficient and regioselective one-step synthesis of 7-aryl-5-methyland 5-aryl-7-methyl-2-amino reactions of some anellated 2-aminothiophenes with electron poor acetylenes exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptaseassociated ribonuclease h discovery of novel fibroblast growth factor receptor 1 kinase inhibitors by structure-based virtual screening preparation of pyrido[3,4-b]indoles and pyrimido[4,5-b]benzo[d]thiophen-4-ones as serotonergic and dopaminergic agents evolution of the thienopyridine class of inhibitors of iκb kinase-β: part i: hit-to-lead strategies synthesis of substituted azulenes via pt(ii)-catalyzed ring-expanding cycloisomerization enantioselective synthesis of cyclic, quaternary oxonitriles from cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric hiv-1 synthesis and biological evaluation of 2-(alkoxycarbonyl)-3-anilinobenzo[b]thiophenes and thieno[2,3-b]pyridines as new potent anticancer agents synthesis and structure-activity relationship studies of 2-(1,3,4-oxadiazole-2(3h)-thione)-3-amino-5-arylthieno[2,3-b]pyridines as inhibitors of drak2 heteroaromatic carboxamide derivatives, particularly 3-aminothiophene-2-carboxamides, useful as protein kinase inhibitors, for the treatment of cancer, inflammation, and inflammation-related disorders synthesis of 3-amino-2-carbamoylthiophene and its reaction with cycloalkanones to form imines identification of 3-aminothieno[2,3-b]pyridine-2-carboxamides and 4-aminobenzothieno[3,2-d]pyrimidines as limk1 inhibitors rapid microwave-assisted solution phase synthesis of substituted 2-pyridone libraries the ecstasy and agony of assay interference compounds zinc 15 -ligand discovery for everyone swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules computational prediction of formulation strategies for beyond-rule-of-5 compounds molecular characteristics for solid-state limited solubility a critical account on π-π stacking in metal complexes with aromatic nitrogencontaining ligands † enhanced metabolite identification with ms e and a semi-automated software for structural elucidation high-throughput, computer assisted, specific metid. a revolution for drug discovery post-acquisition analysis of untargeted accurate mass quadrupole time-of-flight ms e data for multiple collision-induced neutral losses and fragment ions of glutathione conjugates webmetabase: cleavage sites analysis tool for natural and unnatural substrates from diverse data source understanding metabolism in human cytochromes from the perspective of the chemist a common reference framework for analyzing/comparing proteins and ligands. fingerprints for ligands and proteins (flap): theory and application exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptaseassociated ribonuclease h in silico p k a prediction and adme profiling tat-mediated delivery of heterologous proteins into cells residues of human cytomegalovirus dna polymerase catalytic subunit ul54 that are necessary and sufficient for interaction with the accessory protein ul44 selective anti-cytomegalovirus compounds discovered by screening for inhibitors of subunit interactions of the viral polymerase automated robotic liquid handling/laser-based nephelometry system for high throughput measurement of kinetic aqueous solubility key: cord-340832-412qre64 authors: liang, pi‐hui; cheng, wei‐chieh; lee, yi‐ling; yu, han‐pang; wu, ying‐ta; lin, yi‐ling; wong, chi‐huey title: novel five‐membered iminocyclitol derivatives as selective and potent glycosidase inhibitors: new structures for antivirals and osteoarthritis date: 2006-01-05 journal: chembiochem doi: 10.1002/cbic.200500321 sha: doc_id: 340832 cord_uid: 412qre64 a novel 5‐membered iminocyclitol derivative was found to be a potent and selective inhibitor of the glycoprotein‐processing α‐glucosidase with a k(i) value of 53 nm. this compound was further derivatized to antiviral agents against japanese encephalitis virus, dengue virus serotype 2 (den‐2), human sars coronavirus, and human β‐hexosaminidase (k(i) =2.6 nm), a new target for the development of osteoarthritis therapeutics. mannosidase (see supporting information). because core 4 had only moderate inhibitory activities against a-mannosidase and a-fucosidase, the library generated from 4 was screened against a-, b-glucosidase and n-acetyl-b-hexosaminidase in this study. a mixture of iminocyclitol 4, a carboxylic acid (1 equiv, scheme 2), diisopropyl ethylamine (diea, 2.2 equiv), and (1hbenzotriazole-1-yl)-1,1,3,3-(tetramethyl)uronium hexafluorophosphate (hbtu, 1.1 equiv) in dmso was shaken in 96-well microtiter plates for 5 h. crude reaction products were randomly selected (30 %) and analyzed by esi-ms to ensure the presence of the desired products. the reaction mixture was diluted with appropriate buffer solutions and transferred to another 96-well microtiter plate for screening directly without any purification. based on the ic 50 values of 4 (see supporting information), the concentrations of reaction products were set at 20 mm for a-, b-glucosidase and 30 mm for b-hexosaminidase. assuming that amide formation is complete, the percentage of inhibition relative to the control was calculated on the basis of absorbance at 405 nm for p-nitrophenol released from their corresponding substrates. of the 144 compounds generated from the amide-forming reaction, several inhibitors were found (see supporting information). good inhibitors for each enzyme were selected, resynthesized as pure compounds, and reevaluated for determination of ic 50 or k i values (table 1 , scheme 2). in the screening for 166 www.chembiochem.org a-glucosidase (bakers' yeast) inhibition, about two-thirds of reaction products were found to be more potent than 4 (see supporting information for overall library inhibitory activities). briefly, a-glucosidase was inhibited to a greater extent when the compound contained a fused-aromatic ring (e.g. 17 and 25) and a heteroaromatic component (e.g. [19] [20] [21] . it is worth noting that the enzyme was more interactive with a bicyclic than a tri-or mono-cyclic aglycon (e.g. 16 vs. 25 vs. 10). the aglucosidase from bacillus stearothermophilus was also investigated with the same library. the results were similar to those with bakers' yeast. the most potent one against yeast a-glucosidase was compound 24, with a k i value of 53 nm and a 600fold selectivity for aover b-glucosidase. interestingly, the ic 50 values of compounds 15-17 for a-glucosidase were about two-to-three orders of magnitude different. these results suggest that, while a lipophilic binding pocket exists that allows a bicyclic ring such as indole or naphthalene to fit, the specific orientation of these bicyclic rings is also very important. in the screening of b-glucosidase (almonds), about one-sixth of the reaction products were found to be comparable to or more potent than 4. c-1-modified a-iminocyclitols showed weaker effects for b-glucosidase than for a-glucosidase. a dimethylamino group attached to the aromatic ring could enhance the inhibitory activity (e.g. 11 and 29), as documented previously. [26] in particular, structures with a trans-cinnamic moiety were shown to significantly inhibit this enzyme, espe-cially those with meta-halogen substituents (e.g. 12, 13, and 26). because the trans-cinnamic moiety has a board range of biological properties, including hepatoprotective, [31] antimalarial, [32] and antioxidant, [33] our finding suggests a new application of trans-cinnamic acid and its derivatives. the most inhibitory was reaction product 28 (up to 67 % inhibition), with a k i value of 1.2 mm. in general, derivatives of 4 are more selective athan b-glucosidase inhibitors. inhibition of jack bean n-acetyl-b-hexosaminidase seems to be greater when there are hydrophilic substituents in the c-1 position (e.g. 6, 8, and 9). however, none of the substituents is more potent than the acetamido group; this indicates its crucial role in the enzyme active site. this study also showed that potent inhibitors of b-hexosaminidase can be found from derivatives of 4, though relatively weaker inhibitors of b-glucosidase were observed. overall, combinatorial synthesis followed by rapid screening in situ as described here provided a platform to identify selective glycosidase inhibitors through modification of a common transition-state core at the aglycon side chain. ruses. [34] previously, we reported the antivirus effects of an n-nnonyl dnj (nn-dnj) on flavivirus infections. [16] however, fivemembered iminocyclitols had not been tested for their antiviral activities. with the potent a-glucosidase inhibitors in hand, we tested their potential antiviral effect based on our previous assay system for jev and den-2. details of the antivirus assay are given in the experimental section. compounds 7, 17, 20, 22 , and 24 exhibited no inhibition at 50 or 100 mm (data not shown). the peracetylated compound 30, which is believed to increase the cellular uptake and is then converted to compound 24 by cellular esterases, [35] was subjected to the cell assay. however, it also showed no inhibition at 50 mm (data not shown). we then turned our attention to modifying the ring nitrogen on the most potent a-glucosidase inhibitor 24. side-chain modification by alkylation of dnj has previously been reported to enhance both the ability to inhibit glycan processing and virus production. [13, [36] [37] [38] to study the influence of the alkyl-chain length on the cellbased assay, a series of compounds with alkyl chains ranging from c 4 to c 12 attached to compound 24 was synthesized by using reductive amination with appropriate aldehydes to give rise to compounds 31-38 (scheme 3). the inhibition activities of n-alkylated derivatives against aand b-glucosidase were also investigated (see supporting information). the results showed that these molecules still have a-glucosidase selectivity, albeit with low activity compared to the parent compound 24. this result was also observed in the in vitro anti-a-glucosi-dase activity of dnj and nn-dnj. the cell-based sodium 3,3'-{1,[(phennylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (xtt) assay, which measured cell proliferation, indicated that most of the compounds were not toxic at 10 mm (figure 2 a) . the exception was compound 38, which showed a low level of cytotoxicity. in general, the alkylated iminocylitols were more active against den-2 than jev infection (figure 2 b ). relative to the virus titer derived from cells treated with nn-dnj, these newly synthesized molecules were less potent against den-2 infection (figure 2 c). however, in anti-jev infection, compounds 36-38 were more potent than nn-dnj. compound 37 ( figure 3 ) appeared to be less cytotoxic and more potent with ic 50 = 4.7 mm and ic 90 = 9.2 mm for den-2 ( table 2 ). in the present studies, compounds 36-38 with chain lengths of nine, ten, and twelve carbons, respectively, were found to be optimal for the antiviral activity. a similar effect was also noticed against varicella zoster virus. [39] longer alkyl chains, such as, the decyl chain in compound 38, provided a modest increase in potency in cell-based assays, but also resulted in an increase in cytotoxicity, presumably due to the disruption of the lipid bilayer. [36] from a structural perspective, the alkylated iminocyclitol can be viewed as consisting of two distinct molecular elements: 1) an imino sugar head group and 2) an n-alkyl side chain. the head group is recognized by the er-a-glucosidase. the role of the tail, as shown for nn-dnj, is unclear but it might be able to insert into the membrane to increase its local concentration near the membrane-associated er glucosidase. [40] in recent studies, n-nonyldeoxygalactonojirimycin (nn-dgj), a galactosetype iminocyclitol, was found to still have anti-hbv [12] and anti-bvdv [13] activities, although it lacks the ability to inhibit a-glucosidase. these observations suggest that nn-dnj might possess a different antiviral mechanism. nevertheless, we found that nn-dgj did not inhibit either jev or den-2 in our cellbased assay system. [16] the weaker a-glucosidase inhibitors 39-41, derived from core 4 with eight to ten carbons (scheme 3), were then evaluated, and we found that 39-41 at 10 or 50 mm did not inhibit either jev or den-2 infection (data not shown) in the cell-based assay. inhibitors 36-38 were also screened against the infection of sars-cov by following our previously established procedure. [41] the ic 50 for compound 37 was around 3.3-10 mm. the antiviral effects of compounds 36-38 on jev, den-2, and sars-cov shown in the present study are likely mediated by inhibition of the er a-glucosidase; however the possibility of other mechanisms besides a-glucosidase inhibition cannot be rigorously excluded. [16] discovery of potent human b-hexosaminidase inhibitors in our previous study, compound 5 and its n-methyl derivative 42 ((2r,3r,4r,5r)-n-methyl-2-(acetamidomethyl)-3,4-dihydroxy5-(hydroxymethyl)pyrrolidine) were found to be potent inhibitors of human n-acetyl-b-hexosaminidases, with k i % 24 nm. [30] in particular, incubation of human chondrosarcoma cells with iminocyclitol 42 resulted in an accumulation of glycosaminoglycans (gags) in the cell-associated fraction and a decrease in the release of gags into the culture supernatant. the discovery of iminocyclitols as potential chondroprotective agents suggests a new avenue for the development of drugs to treat osteoarthritis. in order to further improve the potency of 42, considerable effort has been directed toward modification of the ring nitrogen and the c1 nitrogen of iminocyclitol 4. [42] however, none of the synthesized inhibitors was more potent than compound 42. the structure-activity relationship of iminocyclitols revealed that the acetamido group at the c1 position is crucial, and that the active site pocket of b-hexosaminidase does not tolerate larger substituents. the methyl group at the ring nitrogen enhances the inhibition activity, whereas aromatic ring substituents cause a decrease in inhibition activity. our alternative approach to increasing potency is to probe a distant aglycon-binding site of b-hexosaminidase. we therefore decided to attach a long-chain alkyl group with a terminal amine to the ring nitrogen through reductive animation. the resulting primary amine is easy to diversify by amide-bond formation to generate libraries as mentioned above. our strategy began with reductive amination of compound 5 with aldehydes of different lengths to give compounds 43-49 (scheme 4). these intermediates were either deprotected under acidic conditions or hydrogenolyzed to give primary amines 50-56. in our first attempt to generate a library from compound 50 by amide-bond formation as mentioned above, the high-throughput screening showed no compounds with significantly enhanced inhibitory activities (data not shown). thus, inhibition studies of compounds 51-56 against human placenta n-acetyl-b-hexosaminidase were carried out. 4-methylumbelliferyl n-acetyl-b-d-glucosaminide (4-mu-gnac) was used as the substrate. the apparent k m and v max values for each substrate were calculated from the lineweaver-burke double reciprocal plot of [1/v]/[1/s]. the k i values were determined from a replot of the k m app versus the inhibitor concentration. compound 54 is the most potent competitive inhibitor with a k i value 2.6 nm ( figure 4) . interestingly, in varying the linkage from n-propyl to n-octyl of compounds 50-55, we observed a trend in human b-hexosaminidase inhibition. compound 54, with a heptamine moiety, was the strongest inhibitor. in an attempt to further optimize the inhibitor, the linker was replaced by an ethylene glycol chain (hydrophilic linker) to give compound 56 (k i = 60 nm) and by moderate n-alkyl chains to give compounds such as 57, 58, and 59 (k i = 180, 250, and 160 nm, respectively). however, these modifications had a negative effect; this indicated the important role of the amino group and the lipophilic chain in compound 54. as the crystal structure of human b-hexosaminidase b is now available, [43] its complex with 54 was modeled to reveal a narrow hydrophobic cleft in the active site, which is mainly enclosed by the sidechain groups of residues w424, y450, a447, and l453 and the backbone of residues k425 and d426 ( figure 5 ). the amino group at the terminal site is expected to be largely protonated when binding to the enzyme. it presumably forms a salt bridge with the secondary aglycon binding site of the enzyme. [44] it appears that the alkyl linkage is long enough to bring the amine end near enough to have hydrogen-bond interactions with the backbone carbonyl of k425 and a447 and a possible ionic interaction with the carboxyl group of d426. based on the structure of 1-amino-1,2,5-dideoxy-2,5-imnio-dmannitol (4), we applied a combinatorial synthesis in microtiter plates for in situ screening. this method is a powerful procedure to rapidly identify significant binding-site differences among similar enzymes and develop potent and selective inhibitors. for the inhibitors of a-glucosidase, structures with bicyclic rings such as indole and naphthalene gave the best inwww.chembiochem.org hibitory potency, as illustrated by 24 with a k i value of 53 nm and 100-fold increase in activity compared to parent core 4. the n-alkylated derivatives of compound 24 were also tested for antiviral activity, and 36-38, which contain lipophilic alkyl groups, were the most active, with an ic 50 of about 5-10 mm against jev, den-2, and sars-cov infection. given its important role in osteoarthritis, the inhibition of nacetyl-b-hexosaminidase was investigated with regard to the substituent effects on the c1 nitrogen and ring nitrogen of core 4. the results showed that the acetamido group at the c1 position was crucial, with modification at the ring nitrogen with aromatic groups causing loss of inhibition. however, extending the alkyl chain at the ring nitrogen gave the most potent human b-hexosaminidase inhibitor known to date, that is, compound 54, with a k i value of 2.6 nm. modeling indicated strong binding of compound 54 with b-hexosaminidase in its lipophilic cleft and an ionic interaction with the secondary binding site. altogether, this work clearly demonstrates the effectiveness of our simple combinatorial approach strategy for the rapid discovery of potent inhibitors as potential candidates for medical applications. materials: the sources of enzymes were as follows: general method for chemical synthesis: all nonaqueous reactions were run in oven-dried and vacuum-cooled glassware under nitrogen atmosphere. reactions were monitored by thin-layer chromatography (merck, silica gel 60f-254) by utilizing ninhydrin, p-anisaldehyde, or cerium molybdate as the stain reagent. silica gel used for flash column chromatography was mallinckrodt type 60 (230-400 mesh). unless otherwise noted, reagents and materials were obtained from commercial sources and used as provided without further purification. 1 h and 13 c nmr spectra were recorded on a bruker av-400 or av-500 spectrometer and referenced to residual solvent peaks (cdcl 3 : 1 h d = 7.24, 13 general procedure for coupling reactions and in situ screening: carboxylic acid (10 ml from a 10 mm stock solution in dmso, 0.1 mmol), hbtu (10 ml from a 11 mm stock solution in dmso, 1.1 equiv, 0.11 mmol), and diea (10 ml from a 22 mm stock solution in dmso, 2.2 equiv, 0.22 mmol) were added to dmso (10 ml) in each well of a 96-well microtiter plate. the reaction was initiated by adding compound 4 (10 ml from a 10 mm stock solution, 0.1 mmol) to each well. the reaction mixtures were shaken at room temperature for 5 h and were monitored for completion based on the disappearance of 4 by tlc with a mobile phase of chcl 3 / meoh/nh 4 oh (1:3:1, r f = 0.33). then an aliquot (5 ml) was withdrawn from the previous reaction mixture and mixed with phosphate buffer (95 ml, ph 7.0) to reach a 20-fold dilution. the same procedure was repeated to give the desired dilution (i.e. a final concentration of 20 mm product in each well of a microtiter plate). in each well of another plate, the a-glucosidase from baker's yeast (10 ml, 0.1 u ml à1 ) and p-nitrophenyl-a-glucopyranoside (50 ml, 2 mm) were mixed with an aliquot (50 ml) of the aforementioned mixture and 90 ml buffer (to give~20 mm inhibitor) for the enzyme inhibition assay. enzyme assay. general procedure for the assay with various glycosidases: the initial velocities of hydrolysis at 30 8c were measured spectrophotometrically at various concentrations of p-nitrophenylglycopyranoside (4 mm, 2 mm, 1 mm, 0.5 mm, 0.25 mm, 0.125 mm, 0.0625 mm) at 405 nm by using bio assay reader (perkin-elmer hts 7000 plus). the obtained data were fitted into the michaelis-menten equation by using the kaleida graph program to determine the apparent k m values. the substrate concentrations were used at three-to fivefold k m values for evaluation of the inhibitory effect against various glycosidases. to give an ideal progress curve, an appropriate enzyme concentration (0.01-0.2 units per ml) and inhibitor concentration (from 1 nm to 500 mm) were used. the 50 % inhibitory concentration (ic 50 ) was determined as the concentration at which the velocity of the hydrolysis was reduced to 50 % as compared to the untreated control. the assays performed in wells of the microtiter plate contained either sodium phosphate buffer (50 mm, ph 7, for aand b-glucosidase, aand bgalactosidase, aand b-mannosidase, a-fucosidase), or mcilvaine's buffer (25 mm, ph 6, n-acetyl-b-hexosaminidase, jack bean). the cell lines, viruses, and virus infection: bhk-21 cells were cultured in rpmi 1640 medium containing 5 % fetal bovine serum (fbs) and l-glutamine (2 mm). jev strain rp-9 [45] and the taiwanese den-2 strain pl046 [46] were used in this study. virus propagation was carried out in c6/36 cells by using rpmi 1640 medium containing 5 % fbs. inhibitors were dissolved in dmso. for infection with jev or den-2, monolayers of bhk-21 cells in six-or 12-well plates were adsorbed with virus for 1 h at 37 8c. after adsorption, unbound viruses were removed by gentle washing with serum-free medium, followed by addition of fresh medium containing various amounts of inhibitors for further incubation at 37 8c. to determine virus titers, culture media were harvested for plaque-forming assays. various virus dilutions were added to 80 % confluent bhk-21 cells and incubated at 37 8c for 1 h. after adsorption, cells were washed and overlaid with 1 % agarose (seaplaque; fmc bioproducts, cambrix bioscience, rockland, me, usa) containing rpmi 1640 with 1 % fbs. after incubation for 4 days for jev and 7 days for den-2, cells were fixed with 10 % formaldehyde and stained with 0.5 % crystal violet. the inhibitors concentrations required to inhibit virus production by 50 % (ic 50 ) and 90 % (ic 90 ) were determined. indirect immunofluorescence assay (ifa): cells were fixed in acetone/methanol (1:1) for 3 min and then treated with a monoclonal antibody (mab) against jev ns3 [47] or den-2 ns3 . [46] after being washed with phosphate-buffered saline (pbs), cells were further stained with a goat anti-mouse fluorescein isothiocyanate (fitc)conjugated secondary antibody (jackson immunoresearch, west grove, pa, usa), and the resulting cells were examined under a leica fluorescent microscope. the viral protein expressions by ifa were read by fluorescence microplate reader (molecular devices) with an excitation wavelength of 355 nm and an emission wavelength of 488 nm. data are shown as percentage infected bhk-21 cells versus those without inhibitor treatment (none, 100 %). cell nuclei were visualized by 4',6'-diamidino-2-phenylindole (dapi) staining in 0.9 % sodium chloride at room temperature for 5 min. xtt assay: to determine cell viability, a colorimetric xtt-based assay was performed (cell proliferation kit ii; roche). bhk-21 cells in a 96-well plate were incubated with various concentrations of inhibitors for 2 days before the xtt labeling reagent was added to the culture medium. cells were incubated at 37 8c for about 30 min and then read by an elisa reader at 450 nm (molecular devices). primary screening for anti-sars-cov activity: vero e6 cells (2 10 4 per well) were cultured in a 96-well plate in dulbecco's modified eagle medium (dmem) supplemented with 10 % fbs. the culture medium was removed after 1 day's incubation, when the cells reached 80-90 % confluence. a solution of dmem (100 ml), with 2 % fbs containing the compound to be tested was placed in three wells. cells were incubated in a co 2 incubator at 37 8c for 2 h and inoculated with sars-cov (h.k. strain) at a dose of 100 tcid 50 per well; the cytopathic morphology of the cells was examined by using an inverted microscope 72 h after infection. computer modeling. docking experiments were conducted by using autodock 3.0.5 with a lamarckian genetic algorithm (lga). [48] the crystal structure of a human b-hexosaminidase b complex with a transition-state-analogue inhibitor, 2-acetamido-2-deoxy-d-glucono-1,5-lactone (d-lactone), [44] was downloaded from the rcsb protein data bank (pdb code 1o7a). chain a of the structure was extracted and utilized in docking simulation. the structure models of inhibitors were built in cache (fujitsu, japan) and refined by performing an optimized geometry calculation in mechanics by using augmented mm3 parameters and stored in pdb format. mgltools (molecular graphics lab, scripps research institute) [49] was used for protein-structure preparation and parameter creation to meet the input requirements of autodock. briefly, essential hydrogen atoms were added to the structure model of hexosaminidase followed by assigning kollman united atom charges and solvation parameters. compound molecules were assigned gasteiger-marsili charges, nonpolar h atoms were merge, and torsions were defined. autogrid tool in autodock 3.0.5 was applied to produce energy grids (50 50 50 in xyz directions with 0.375 spacing) of various types of compound atoms. these grip maps were centered at the active site where the d-lactone bound. during docking experiments, each compound was kept flexible and the protein was kept rigid. solis & wets' local search method with lga was applied to generate available conformations of compound structures within the active site. the conformational search was conducted by utilizing 0.2 quaternion and 28 torsion steps. for each compound structure, a maximum of 5 10 6 energy units were evaluated, and 50 poses were selected from 2.7 10 5 generations per run. plausible docking modes were selected from the most abundant cluster (rmsd = 2.0 ), which had the strongest affinity energy. pictures of the final simulated complex were generated in mgltools. www.chembiochem.org proc. natl. acad. sci proc. natl. acad. sci iminosugars as glycosidase inhibitors-norjirimycin and beyond proc. natl. acad. sci proc. natl. acad. sci mgltools we thank dr. jia-tsrong jan at the national defense university (taipei, taiwan) for the anti-sars assay and the academia sinica for financial support.keywords: antiviral agents · glycoproteins · iminocyclitols · inhibitors · osteoarthritis key: cord-344598-5drr3fyt authors: khanna, leena; singhal, sugandha; jain, subhash c.; khanna, pankaj title: spiro‐indole‐coumarin hybrids: synthesis, adme, dft, nbo studies and in silico screening through molecular docking on dna g‐quadruplex date: 2020-03-19 journal: chemistryselect doi: 10.1002/slct.201904783 sha: doc_id: 344598 cord_uid: 5drr3fyt new series of hybrids were synthesized by combination of 4‐hydroxycoumarin with spiro[indol‐indazole‐thiazolidine]‐diones and spiro[indol‐pyrazole‐thiazolidine]‐diones, via hitherto unknown schiff bases. the effects of substituents, such as ‐f, ‐br and ‐ch(3), on the crucial characteristics pertaining to the hybrids were investigated through computational studies. in silico or virtual screening through molecular docking studies on the library of 22 compounds, including reference compounds, precursors, non‐hybrid and hybrid derivatives, was performed on dna g‐quadruplex of the human genome. all six freshly synthesized hybrids showed high binding energy as compared to non‐hybrids as well as reference compounds. the presence of substituents at 5‐position of indole enhanced the binding tendency of the ligand. adme studies indicated good oral bioavailability and absorption of these compounds. density functional theory (dft) calculations of hybrids were done at b3lyp/6‐311g++(d,p) level of computation. their homo and lumo energy plots reflected the presence of high charge transfer and chemical potential. natural bond order (nbo) calculations predicted hyperconjugative interactions. the molecular electrostatic potential (mep) surface plots showed possible electrophilic and nucleophilic attacking sites of the hybrids. compound 10 a (5‐fluoro‐spiro[indol‐indazole‐thiazolidine]‐dione‐coumarin hybrid), on the basis of global reactivity descriptors, was filtered to be chemically most reactive with the highest binding energy of −8.23 kcal/mol with dna g‐quadruplex. the synthesized hybrid coumarin derivatives in correlation with theoretical docking studies validate that hybrid derivatives are more reactive compared to their non‐hybrid counterparts. coumarins or 2h-chromen-2-ones constitute an important group of natural products and are known to possess varied activities viz. antibacterial, antiallergic, anti-inflammatory, antioxidant, anticoagulant. among the various coumarins known, o-alkylated coumarins constitute an important group of naturally occurring compounds e. g., 1 (figure 1 ) has been isolated from mutisia orbignyana. [1] the o-alkylated coumarins 2 ( figure 1 ), have shown antibacterial activity against bacillus subtilis and e. coli. [2] also, the anticancer activity of coumarin derivatives has been well screened. 6-brominated coumarin hydrazide-hydrazone derivatives (bchhd) were found to be more effective against resistant panc-1 cells than doxorubicin (dox). [3] whereas coumarin derivatives having 4,5-dihydropyrazole moiety exhibited as potential telomerase inhibition activity against human gastric cancer cell sgc-7901. [4] besides this, indole-2,3-diones when joined using alkyl bridges to different heterocyclic moieties have been known to act as sars coronavirus 3cl protease inhibitors. [5] bis-indolinone derivatives having 2,6-disubstituted pyridine core or 1,10disubstituted phenanthroline core showed high binding with g-quadruplexes as well as antitumor activity. [6] similarly, naturally occurring indole alkaloids, spirotryprostatins a and b obtained from the fermentation broth of aspergillus fumigates, inhibited the g2/m progression cell division in mammalian tsft210 cells. while synthetically prepared dispiro[3h-indole-3,2'-pyrrolidine-3',3''-piperidine]-2(1h),4''-diones displayed effective antitumor activities against the cervical cancer cell line (hela) more than that of cisplatin. [7] fascinatingly, hybrid molecules are generally designed to target simultaneously two different sites with synergistic effects or acting as dual drugs. [8] [9] [10] also, a single molecule carrying multiple pharmacophores acts as a hybrid multifunctional entity and is more useful as each pharmacophore displays diverse modes of action. therefore, considering the importance of coumarin and spiro-indoles, we thought of developing their hybrid molecules in anticipation of having better pharmacophoric features and biological profiles. hence, in vogue of combining more numbers of bioactive moieties in a single molecular framework and continuing our interest in spiro-indoles, [11] [12] [13] [14] [15] [16] we thought of synthesizing hybrids of spiro[indol-indazole/pyrazole-thiazolidine]-diones with coumarin. also, there has been no report of these types of potentially active indolyl compounds containing coumarin, thiazolidine and pyrazole moieties in one frame. however, as it was difficult to introduce the coumarin moiety into the spiro-indole already having two bioactive moieties, a literature survey revealed alkyl bridge between any two moieties as a possible route for the preparation of the desired compounds. all the six new hybrids were characterized using detailed spectroscopic analysis experimentally and theoretically. the computational studies were performed for these six spiro indole-coumarin hybrids, 10 a-c and 12 a-c in order to study their stability and biological potency. also, it is well known the fact that the formation of gquadruplex inside the human body at telomeres can change many cellular functions, inducing apoptosis or may cause cancer. however, the development of synthetic molecules that are able to bind and stabilize these telomeric g-quadruplexes is catching attention nowadays as there are proving as attractive therapy as antitumor agents. [17] [18] [19] as we have already discussed that literature revealed both spiro-indoles and coumarin derivatives possessed potent antitumor properties; hence, it can be evaluated in their hybrids also. thus, in the present study, the biological importance of 22 compounds including six new spiro indole-coumarin hybrids as dna quadruplex groove binders has been evaluated by performing molecular docking studies on dna g-quadruplexes of the human genome. finally, density functional theory (dft) calculations, nbo analysis, and mep plots are drawn for hybrids to prove their chemical reactivity and stability. the reaction of 4-hydroxy-2h-chromen-2-ones (3) with 1,6dibromohexane (4) in the presence of nah in dry dmf at à 10°c under inert atmosphere gave a mixture of two compounds 5 and 6 (scheme 1). these were separated by column chromatography. the compound 5 obtained as white solid, displayed a molecular ion peak at m/z 324 corresponding to the molecular formula c 15 h 17 o 3 br. the ir spectrum showed characteristic absorption at 1711 cm à 1 . the absence of absorption for -oh group indicated that alkylation has occurred. its 1 h nmr spectrum showed signals integrating for aromatic protons of coumarin at δ 7.81 (h-5), δ 7.55 (h-7) and δ 7.28 (h-6 and h-8). the h-3 proton appeared at δ 5.66 as a singlet. the protons of alkyl group appeared at δ 4.14 (t, och 2 ), δ 3.43 (t, ch 2 br) and as multiplets at δ 1.92 and δ 1.58 for the other four methylenes. 13 c nmr spectrum displayed the presence of carbonyl carbon at δ 165.3 in addition to carbons at δ 160.9 (c-4), δ 153.1-90.2 (aromatic carbons), δ 69.4 (och 2 ) and δ 34.1 (ch 2 br). the above spectral data confirmed the formation of 5 which was characterized as 4-(6-bromohexyloxy)-2h-chromen-2-ones. besides 5, another compound 6 formed in minor amount in the course of reaction was characterized as (2-oxo-2h-chromen-4-yloxy)-1,1'-(hexanediyl)bis. the bromide 5 on reaction with 5-fluoro-1h-indol-2,3-dione (7 a) in the presence of nah in dry dmf under nitrogen gave a red colored solid 8 a whose molecular ion peak appeared at m/z 409 which corresponded to the molecular formula c 23 h 20 no 5 f. its ir spectrum showed the presence of carbonyl peaks at 1735, 1727 and 1709 cm à 1 . the 1 h nmr spectrum showed aromatic protons of the indole nucleus at δ 7.30 (h-4'' & h-6'') and δ 6.86 (h-7'') besides the protons of coumarin moiety at usual values. the methylenes appeared at δ 4.12 (t, och 2 ), δ 3.74 (nch 2 ) as triplets besides the other four methylenes at δ 1.91, δ 1.75 and δ 1.54 as multiplets. the 13 c nmr spectrum showed characteristic carbonyl at δ 185.1 (c-3''), δ 163.2 (c-2'') and δ 165.9 (c-2), apart from the aromatic and alkyl carbons. the above spectral studies confirmed the formation of 1-[6-(2-oxo-2h-chromen-4-yloxy)hexyl]-5-fluoro-1h-indol-2,3-dione (8 a). the reaction of 8 a with 5-aminoindazole in absolute ethanol under refluxing conditions gave a reddish solid 9 a, which displayed molecular ion peak m + at m/z 524 corresponding to the molecular formula c 30 h 25 n 4 o 4 f. ir spectrum showed characteristic absorptions at 3259 cm à 1 (> nh, indazole) and 1644 cm à 1 (c=n) besides other carbonyl bands. 1 the schiff base 9 a was reacted with mercaptoacetic acid under refluxing conditions using dean-stark apparatus to obtain a new compound 10 a, whose molecular mass was found to be 598 corresponded to the molecular formula c 32 h 27 n 4 o 5 fs. the ir spectrum in this compound showed distinct characteristic absorption at 3321 cm à 1 (> nh, indazole), 1724 cm à 1 (thiazolidine carbonyl), 1718 cm à 1 (coumarin carbonyl) and 1681 cm à 1 (indole carbonyl), thus confirming the cycloaddition. 1 h nmr spectrum showed the presence of methylene of thiazolidine as two doublets at δ 4.37 and δ 4.05 besides the usual aromatic protons of indole, indazole and coumarin moieties. the 13 c nmr spectrum showed peaks at δ 175.2 (c-4''''), δ 173.3 (c-2''), δ 166.0 (c-2) and δ 33.2 (c-5'''') besides other aromatic and alkyl carbons. the above spectral data confirmed the formation of desired spiro compound 10 a characterized as 1the indol-2,3-dione (8 a) was also reacted with 4-aminoantipyrine in similar fashion to obtain a red colored solid 11 a, whose molecular ion peak appeared at m/z 594 corresponding to the molecular formula c 34 h 31 n 4 o 5 f (scheme 2). the ir spectrum showed absorptions at 1725, 1718 and 1641 cm à 1 . the 1 h nmr spectrum showed the peaks at δ 7.50 & δ 7.48 (n-c 6 h 5 ), δ 3.29 (n-ch 3 ) and δ 2.47 (3'''-ch 3 ) of pyrazoline moiety with corresponding integrations, besides the usual protons. the 13 c nmr spectrum showed characteristic peaks at δ 145.7 (c=n), δ 36.3 (n-ch 3 ) and δ 11.7 (3'''-ch 3 ). thus on the basis of the above spectral data 11 a was confirmed as 1-[6-(2-oxo-2hchromen-4-yloxy)hexyl]-3-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl) imino-5-fluoro-1h-indol-2-one. the schiff base 11 a was cyclocondensed with mercaptoacetic acid in dry toluene under refluxing conditions using dean stark apparatus to afford a new compound 12 a which gave a molecular ion peak at m/z 668 corresponding to molecular formula c 36 h 33 n 4 o 6 fs. the ir spectrum showed peak at 1722 cm à 1 . the 1 h nmr spectrum showed characteristic peaks of thiazolidine methylenes as two doublets at δ 4.39 and δ 3.83 with coupling constant of 15.0 hz integrating for one proton each. the 13 c nmr spectrum also showed peaks of thiazolidine moiety at δ 172.8 (c-4'''') and 32.7 (c-5'''') besides peaks of indole, pyrazoline, and coumarins moieties. thus, on the basis of above spectral data, the desired spiro compound 12 a was confirmed and characterized as 1the detailed spectroscopic data of all the compounds has been summarized in supplementary table 1 . the bioactivity scores and drug likeliness properties were determined using molinspiration virtual screening online software (www.molinspiration.com). the bioactivity scores for gpcr ligands, ion channel modulator, enzymes, and nuclear receptors were predicted. the physicochemical properties of compounds analyzed using lipinski's rule (logp, total polar surface area, molecular weight, number of hydrogen bond donors and acceptors, number of atoms, number of rotatable bonds, etc.). the preadmet online server was used to calculate pharmacokinetic parameters to determine the oral activity of the compounds. the parameters such as adsorption, distribution, metabolism and excretion were evaluated. density functional theory (dft) calculations of compounds were done using exchange functional by becke, [20] and correlational functional, [21] at b3lyp/6-311g + + (d,p) level of computation using gaussian 09 software. [22, 23] the optimized geometries of compounds 10 a-c and 12 a-c are depicted in figure 2 . these were further used as input for frontier orbital calculations. the chemical reactivity and chemical potential were predicted from the frontier orbital properties. the atomic charges and hyperconjugative interactions were predicted from natural bond order calculations in vapor phase. the molecular docking was carried out with autodock 4.2 program. [24] the crystal structure of g-quadruplex [d(tggggt)] 4 (pdb id: 1 s45) was retrieved from the protein data bank (www.rcsb.org). the dna was prepared by adding polar hydrogens, kollman united atom charges and solvation parameters. the ligand files were prepared using gasteiger charge assignment and merging non-polar hydrogens. the grid dimensions in x, y and z directions were kept at 60, 60 and 60 å respectively. lamarckian genetic algorithm (lga) method was used for the best conformer search. the docked poses were visualized using discovery studio visualizer 2017. nowadays, docking is proven to be a highly important technique, useful to predict the interaction of small ligands with biological macromolecules. therefore in the present work, (figure 3 ), [16] and three reference compounds (figure 4) , [18, 19, 25] were correlated with each other on the basis of their dna g quadruplex binding affinity via molecular docking as an in silico tool. all 22 compounds were docked with g-quadruplex [d (tggggt)] 4 (pdb id: 1s45) and their calculated binding energies are summarized in table 1 . in each case, the best favorable binding pose was selected from the docked structures, and binding energies were calculated. the data was interpreted by plotting graphs of various compounds versus binding energies. the hybrid compounds were compared with their non-hybrid counterparts to look insight into the influence of coumarin moiety on their activity ( figure 5) . similarly, the results also compare substitution at 5-position of isatin in spiro-indoles, it was noteworthy that both ch 3 and f were showing an increase in binding interactions, although fluorine was found to be the best ( figure 6 ). table 1 also shows all six newly synthesized spiro indolecoumarin hybrids 10 a-c and 12 a-c are having high binding energy as compared to intermediary schiff bases, isatin, their non-hybrid counterparts and the reference compounds. however, 10 a is found to be the best binding ligand. also, our motive behind using six carbon long chain for hooking up coumarin moiety with spiro-indoles was to enhance their lipophilicity and to provide flexibility to the molecule, which helped it to bind well with dna g quadruplex. the compound 10 a binds perfectly to the wide groove of g-quadruplex with binding energy à 8.23 kcal/mol. the nh hydrogen atom of pyrazole ring forms hydrogen bond with oxygen atom of dg403 residue (g3). the oxygen atom of thiazolidinone nucleus forms conventional hydrogen bond with hydrogen atoms of dg513 residue (g4) (figure 7) . the aromatic ring of coumarin forms pi-anion interaction with oxygen atom of dg843 residue (g7). the pyrazole ring forms pi-anion interaction with oxygen atom of dg403 residue (g3) ( figure 8 ). the indazole ring forms pi-lone pair interactions with dg515 and dg403 residue (g3). the aromatic ring of dt731 residue (g6) depicts pi-sulfur interaction with sulfur atom of thiazolidinone nucleus (figure 9 ). the compound is considered active if bioactivity score is > 0, moderately active if score between à 5.0-0.0, and inactive if score is < à 5.0. all the compounds depicted good bioactivity scores ( table 2 ). the adme parameters were within the permissible limits indicating good oral bioavailability of the compounds ( table 3 ). the hia (human intestinal absorption) values of > 90% indicated good oral absorption. the physicochemical properties values such as number of hydrogen bond donors, acceptors, rotatable bonds, and tpsa were also in well agreement (table 4) . thus, all the six coumaryl spiro-indoles have shown good overall bioactivity, oral availability despite 2 violations to lipinski's rule. the frontier orbitals homo (highest occupied molecular orbital) and lumo (lowest unoccupied molecular orbital) energies provide significant insight into reactivity and active site of the compounds. the negative chemical potential values indicate the spontaneous decomposition of compounds. the homo and lumo energies and global reactivity descriptors are given in table 5 . the lower energy gap value implies higher chemical reactivity. using the koopman's theorem the i and a values can be correlated with frontier orbitals by the relation: the global reactivity descriptors chemical hardness (η), chemical potential (μ), electronegativity (χ) and electrophilicity index (ω) described by parr, [27] and pearson, [28] are calculated using equations: [29] the hardness is given by h ¼ ði à aþ=2 (1) the chemical potential is given by m ¼ à ði þ aþ=2 the electronegativity is given by the electrophilicity index is given by w ¼ m 2 =2h (4) compound 10 a having fluoro substituent has the lowest energy gap value; therefore, it is more polarizable and has higher chemical reactivity, low kinetic stability and thus considered as soft molecule among 10a-c series. similarly, compound 12 a has the lowest energy gap value among series 12a-c rendering it more polarizable with higher chemical reactivity, and lower kinetic stability. the homo-lumo plots of compounds 10 a-c and 12 a-c are shown in figure 10 . nbo analysis provides information regarding atomic charges in molecular system including conjugative interactions or charge transfer. reed and weinhold performed the nbo calculation, [30] showing that hyperconjugation stabilizes due to the delocalization of electron density to neighboring electron deficient orbital (non-lewis type nbo) from filled lewis type nbo. also, for each donor nbo (i) and acceptor nbo (j), the stabilization energy can be described by means of second-order perturbation interaction energy e(2) which is given using following equation: where q i is the donor orbital occupancy, ei, ej are the diagonal elements, fij is the off diagonal nbo fock matrix element. [31] the nbo analysis was performed at b3lyp/6-311 + + g(d,p) basis set to estimate the nature of bonds. the second order perturbation theory analysis of compound 10 a (figure 11 ) is given in table 6 . the major interactions include lp(n17) to σ*(c22-h26), lp(o68) to σ*(c58-o67), and lp(o19) to σ* (c11-ν15) with stabilization energies 55.87, 41.06, 33.97 kj/mol respectively. the intramolecular hyperconjugative interactions are formed due to orbital overlap between π*(c20-c21) and π* (c22-c25), π*(c20-c21) and π*(c23-c27), and π*(c57-c62) and π*(c60-c63) with respective highest stabilization energies 188.00, 170.20, 182.00 kcal/mol. the high values of e2 indicate table 2 . similarly, the second order perturbation theory analysis for compound 12 a (figure 12 ) is given in table 7 . the intramolecular hyperconjugative interactions are formed due to anti bond orbital overlap between π*(c43-c48) and π*(c46-c49), π* figure 11 . labelled structure of 10a table 6 . second order perturbation analysis of the interaction between donor and acceptor orbitals of compound 10 a calculated at b3lyp/6-311 + + g(d,p) supplementary table 3 . the nhos, natural hybrid orbitals are a result of symmetrically orthogonalized hybrid orbital, which is derived from the natural atomic orbital (nao) centred on particular atom via unitary transformation. looking at the simple bond orbital picture, an nbo is defined as an orbital formed from nhos. the nbo for a localized σ-bond between atoms a and b, is defined as: where h a and h b are the natural hybrids centred on atoms a and b and c a and c b are the polarization coefficients for atoms a and b. the parameters spherical polar angles theta (θ) and phi (φ) from the nucleus and the deviation angle dev from the line of the centres between the bonded nuclei specify the direction of each hybrid. in general, for the sp λ d μ hybrids, the hybrid direction is numerically determined corresponding to maximum angular amplitude. further, it is compared with the direction of internuclear axis to determine the magnitude of bending in the bond as the deviation angle between them. table 4 ). similarly, for compound 12a the carbons are bent away from the line of c 1 -c 6 centres by 16.3°while c 11 -o 19 centre deviates by 6.9°(supplementary table 5 ). the 3d mep plots are a useful tool to establish relationship between molecular structure and its physicochemical property. it helps to display molecular size, positive, negative and neutral electrostatic potential regions with the help of color grading. therefore, 3d mep plots calculations for these six hybrids were done at b3lyp/6-311g (d,p) level of computation and plots are shown in figure 13 and 14 and supplementary figures s1-s4 . the positive electrostatic potential represents proton repulsion by atomic nuclei due to low electron density (blue shades). similarly, the negative potential represents the proton attraction due to excess electron density (red shades). [32] [33] the potential values are represented in varied colors and increase in the order red < orange < yellow < green < blue. the color code of these maps is in the range à 0.209 (deepest red) to 0.209 (deepest blue) for 10 a-c and à 0.169 to 0.169 for 12 a-c where red and blue represent strongest attraction and repulsion respectively. the esp plots clearly show that the maximum positive region (blue) is located on the alkyl chain due to hydrogen atoms. the maximum negative region (red) is localized on the spiro-indole ring system containing heteroatoms as well as the coumarin ring oxygen. it also indicates that electron donating ability is favorable from them. a contour plot is a 2d xy plot of a 3d xyz surface which displays lines at intersection point of surface and planes of constant elevation (z). the contour plots depict lines of constant density or brightness, such as electrostatic potentials and are drawn in the molecular plane. the electron rich red lines are around heteroatoms whereas the electron deficient region is shown by greenish-yellow lines. the calculations are done at the at 0.004 density values. the mep and contour plots are in correlation with the docking interactions which reveal that heteroatoms form pication, pi donor and classical h bonds with g-quadruplex. the coumarin ring with electron donating ability as suggested by mep and contour plots also forms h bond with g-quadruplex dna. six new spiro indole-coumarin hybrids 10 a-c and 12 a-c have been successfully synthesized using heterocyclic amines through six new schiff bases. the detailed spectroscopic analysis revealed the true structures of the compounds. binding interaction of about 22 compounds including these hybrids with dna g-quadruplex of the human genome, was screened using molecular docking studies. compound 10 a was evidenced to be the best ligand with the highest binding energy value of à 8.23 kcal/mol. also, all these hybrid compounds have shown good bioactivity scores and drug likeliness properties. the dft studies showed high chemical reactivity of all six hybrids through homo and lumo plots. the position of molecular orbitals reveals the charge transfer within the molecule. the detailed nbo analysis of 10 a and 12 a, their mep plots, hyperconjugative interactions and charge delocalization calculations verified the reactivity and stability of these compounds. however, hybrid 10 a on the basis of global reactivity descriptors filtered to be chemically more reactive than other compounds. the in vitro studies for the screened potential hybrid coumarin derivatives are underway in collaboration. experimental section, spectroscopic data, complete nbo and nho tables of 10 a and 12 a and additional molecular electrostatic potential and contour plots are available in supplementary information. figure 14. mep and contour plot for compound 12a proc. natl. acad. sci gaussian 09 density-functional theory of atoms and molecules the authors would like to thank faculty research grant scheme (frgs), guru gobind singh indraprastha university, dwarka, new delhi, and council of scientific industrial research new delhi, for financial support of this project. the authors declare no conflict of interest.keywords: coumarin hybrids · g-quadruplexes · molecular docking · nbo analysis · virtual screening key: cord-329078-gnnis7pl authors: musella, simona; di sarno, veronica; ciaglia, tania; sala, marina; spensiero, antonia; scala, maria carmina; ostacolo, carmine; andrei, graciela; balzarini, jan; snoeck, robert; novellino, ettore; campiglia, pietro; bertamino, alessia; gomez-monterrey, isabel m. title: identification of an indol-based derivative as potent and selective varicella zoster virus (vzv) inhibitor date: 2016-11-29 journal: eur j med chem doi: 10.1016/j.ejmech.2016.09.014 sha: doc_id: 329078 cord_uid: gnnis7pl we report the synthesis and antiviral activity of a new family of non-nucleoside antivirals, derived from the indole nucleus. modifications of this template through mannich and friedel-crafts reactions, coupled with nucleophilic displacement and reductive aminations led to 23 final derivatives, which were pharmacologically tested. tryptamine derivative 17a was found to have a selective inhibitory activity against human varicella zoster virus (vzv) replication in vitro, being inactive against a variety of other dna and rna viruses. a structure-activity relationship (sar) study showed that the presence of a biphenyl ethyl moiety and the acetylation at the amino group of tryptamine are a prerequisite for anti-vzv activity. the novel compound shows the same activity against thymidine kinase (tk)-competent (tk(+)) and tk-deficient (tk(−)) vzv strains, pointing to a novel mechanism of antiviral action. varicella zoster virus (vzv) is a ubiquitous and highly infectious human virus that belongs to the herpesviridae family. it is classified within the group of a-herpesviruses which also includes herpes simplex virus (hsv) [1] . a vzv primary infection leads to acute varicella or "chickenpox", while reactivation of latent virus, established in cranial nerve and dorsal root ganglia, causes herpes zoster (shingles). the course of varicella is generally benign in immune-competent children, but can cause severe morbidity and mortality in adults and in immune-compromised individuals [2] . complications of herpes zoster in immunecompetent hosts include post-herpetic neuralgia (phn), a persistent pain syndrome, which is the most challenging complication particularly in older individuals [2, 3] . central nervous system (cns) complications can follow both primary infection and reactivation of vzv [4, 5] . the most serious manifestations arise when vzv invades the spinal cord or cerebral arteries after reactivation of the virus, causing diseases such as myelitis and focal vasculopathies [2, 4, 5] . other neurological complications of herpes zoster include motor neuropathy, particularly in patients with zoster ophthalmicus [6, 7] . in patients with the acquired immune deficiency syndrome (aids), transplant recipients, and cancer patients, vzv infection can be associated with severe acute retinal necrosis (arn), a disease with poor prognosis [8, 9] . the outcomes of varicella and herpes zoster have been dramatically improved by the development of safe and effective antiviral drugs with potent activity against vzv [10] . three oral guanine-based antivirals are approved worldwide for the treatment of vzv-associated diseases: acyclovir, valacyclovir, and famciclovir [11] . the thymidine analog brivudin has been licensed for the therapy of herpes zoster in some european and central american countries [12] . these drugs are (a) nucleoside analogs that after predominant phosphorylation by the virus-encoded thymidine kinases (tks), act as competitive inhibitors of the viral dna polymerase or alternate substrates to the natural triphosphates, inhibiting dna replication [13] . other anti-vzv nucleoside inhibitors such as the stearyl/valyl diester valomaciclovir and the valyl-ester prodrug of the bicyclic nucleoside analog (bcna) fv100 are under clinical investigation [14e17]. one of the limitations of the use of nucleoside derivatives is the emergence of single and multiple drug resistance which could be partially avoided with the use of non nucleoside compounds [18, 19] . a drug of choice for treatment of acyclovir-resistant vzv disease is foscarnet, a direct inhibitor of viral dna polymerase that is not dependent on viral tk for activation [20e22] . a number of small molecules have been identified and reported as potent and selective vzv inhibitors with different mechanisms of action. some examples are the 4-oxo-dihydroquinoline [23] and 4-oxo-dihydrothieno [2,3-b] pyridine derivatives [24] as inhibitors of the viral dna polymerases, the oxadiazolephenyl derivative (asp2151) as a helicase-primase inhibitor [25] , and n-a-methylbenzyl-n 0 -arylthiourea derivatives that interfere with the function of the viral orf54 protein, impairing morphogenesis of the capsid [26, 27] . finally, a series of 4-benzyloxy-g-sultone derivatives has been also reported as non-selective vzv inhibitors with unknown mechanism of action [28] . given the difficulty of identifying initial hit compounds in this field, where the synthesized compounds are in primis subject to a cellular screening, we considered of interest to use a privileged scaffold as effective starting point in the search for anti-vzv ligands [29] . indole and its bioisosteres, as privileged scaffolds, represent one of the most important structural motifs in drug discovery [30e32], and it is widely used in antiviral research [32, 33] . arbidol [34] and delavirdine [35] , are examples of marketed indole-containing antiviral drugs, whereas panobinostat (lbh589) [36] , being a hdac (histone deacetylase) inhibitor, is actively undergoing clinical evaluation against human immunodeficiency virus (hiv) type 1 (see fig. 1 ). however the use of the indole-based structures in the research of anti-herpes virus agents is rather unusual. hence we explored the minimum structural requirements for anti-vzv activity starting from this easily derivatizable scaffold. two small libraries were synthesized based on substituted indoles (a, b) and tryptamines (c). their cytotoxic and antiviral activity was then evaluated using cellular assays. some interesting structure-activity relationships were evidenced regarding the n-1 and c-3 substituents. compounds 4a-d were prepared starting from indole 1 according to scheme 1. n-1 alkylation of 1 with propyl iodide or 4phenylbenzyl iodide in dcm/dmf using nah as base, gave the corresponding intermediates 2 and 3, respectively. the 3-acyl derivative 4a was obtained from 2 by friedel-crafts acylation, using 4chlorobenzoyl chloride and alcl3 in acetonitrile (32% yield). functionalization of 2 and 3 through a mannich reaction, using formaldehyde and piperidine/or biphenyl ethyl amine, and tfa as catalyst, led to 3-methylamine derivatives 4b-d (25e38% yield). the 5-substituted indole derivatives (8a-c) were synthesized using the indole-5-carboxyaldehyde (5) and the 5-aminoindole (9) as starting material and following the two-synthetic strategy indicated in scheme 2. 5 was first n-alkylated (6) and then subjected to a mannich reaction to obtain the corresponding aldehydes 7a and 7b, as described above. treatment of these intermediates with 4-chloro aniline and sodium triacetoxyborohydride in reductive amination conditions, led to final products 8a and 8b in 24% and 30% yield, respectively. on the other hand, reaction of 9 with benzoic acid using hobt/hbtu as coupling agents gave n-(1hindol-5-yl)benzamide 10 in 63% yield. n-alkylation of 10 with npropyl iodide followed by mannich reaction with formaldehyde and piperidine afforded final compound 8c. the tryptamine-based derivatives 15, 16a-i were prepared following scheme 3. n-alkylation of 3-(2-bromoethyl)-1h-indole (12) with methyl iodide or 4-phenylbenzyl iodide led to derivatives 13 and 14 in 67% and 61% yield, respectively. nucleophilic displacement of the bromine atom in these intermediates by different commercially available amines was performed under microwave conditions, using palladium acetate as catalyst, obtaining the compounds 15, 16a-i in 38e75% yield [37] . treatment of compounds 16a and 16d-i with acetyl chloride gave the corresponding acetyl derivatives 17a and 17d-i (42e58% yield). analogously, treatment of 16a with different aromatic and aliphatic acyl chlorides gave the corresponding acylated compounds 18a-c (48e57% yield). the carbamoyl derivative 18d was obtained in 58% yield by reaction of 16a with di-tert-butyl dicarbonate in dcm/tea (see scheme 4). the acyl derivatives 17, except 17d and 17e, and 18 were obtained as a mixture of rotamers due to the atropisomerism of the amide function, as confirmed by roesy experiments, which showed exchange cross-peaks between the hydrogen signals of the two conformers (as examples see nmr spectra of compounds 17f and 17 d in supporting information) [38, 39] . first, compounds 4, 8, and 15, 16 and 17a were evaluated for their ability to inhibit the replication of vzv in human embryonic lung (hel) cells, and the results were compared to those obtained for the reference compounds acyclovir and brivudin ( table 1 ). the antiviral activity was expressed as ec50, being the compound concentration required to reduce virus-plaque formation (vzv) by 50%. as shown in table 1 , the n-propyl-3-acyl (4a) and n-propyl-3piperidine methyl (4b) derivatives do not displayed antiviral or cytotoxic activities at 100 mm, while the substitution of piperidine with cc50 values of 1.7 and 6.9 mm, respectively. compounds 8 a-c, derivatized at the c-5 indole position, proved less cytotoxic, cc50 z 10e30 mm, but were also ineffective as inhibitors of vzvinduced plaque formation at similar concentrations. the most interesting results were obtained for the tryptamine derivatives. in this series, the presence of a methyl group at the nindole position lead to weaker cytotoxic derivatives (16a-d versus 15, cc50 20e41 versus 7.3 mm), while the acylation of the amine group (17a) also reduces cytotoxicity (cc50 ≈ 31e39 mm) but with an inhibitory effect on the anti-vzv activity. tryptamine derivative 17a was indeed able to inhibit replication of tkþ and tk-vzv strains with ec50 values in the range of 1.7e3.6 mm ( table 1 ). the potency of 17a against oka strain (ec50 ¼ 2.5e3.6 mm) was found comparable to that of the reference drug acyclovir (ec50 ¼ 1.9e2.1 mm) but two orders of magnitude lower than that of brivudin (ec50 ¼ 0.02e0.03 mm). however, the activity of this compound against thymidine kinase-deficient vzv strains, both 07-1 and ys-r, was maintained in the micromolar range with ec50 ¼ 1.7e3.1 mm being, at least, one order of magnitude more active than acyclovir (ec50 ¼ 33e103 mm) and brivudin (ec50 ¼ 26e165 mm). the activity of these compounds against other members of the herpesviridae family was tested in hel cell cultures. as shown in table 2 none of these compounds was found active either towards cytomegalovirus (hcmv, ad-169 strain and davis strain) or against herpes simplex virus-1 (hsv-1; kos and thymidine kinasedeficient acyclovir-resistant) (hsv-1/tk-kos acvr strains) or against herpes simplex virus-2 (hsv-2; g strain). the compounds were not active against other dna viruses such as vaccinia virus, adenovirus-2, and feline herpesvirus. all these compounds also lacked inhibitory activity against a broad variety of rna viruses, including hiv-1 and hiv-2, feline coronavirus, vesicular stomatitis virus, coxsackie virus b4 and respiratory syncytial virus, para-influenza-3 virus, reovirus-1, sindbis virus, coxsackie virus b4, punta toro virus, influenza a virus (h1n1 and h3n2 subtypes) and influenza b virus (data not shown). to further explore the sar within the tryptamine series, we next prepared a second set of 17a analogs featuring a) a modified aromatic moiety linking the acetamide group (compounds 17 d-i) and b) a modified acyl group (compounds 18a-d). as shown in table 3 , substitution of biphenyl ethyl moiety by the more rigid naphthyl (compounds 17d, 17e) or their superior analog naphthylmethyl groups (17f, 17g), as well as, the introduction of a biphenylmethyl or phenyloxybenzyl moieties (17h, 17i) led to a complete loss of the antiviral activity. however, compounds containing 1-naphtyl (17e) or 1-naphtyl methyl (17g) groups or the inferior analog biphenylmethyl group (17h) were less cytotoxic. on the other hand, substitution of the acylmethyl group by bulkier alkyl groups such as cyclohexyl, morpholinethyl and tert-butyloxy groups (compound 18a, 18c, 18d) led to an annihilation of antiviral activity. in this series of compounds, only the benzoyl derivative 18b somewhat maintains the antiviral activity at an ec 50 of 20 mm, displaying also a reduced cytotoxic activity (mmc >100 mm). the identification of a new family of non-nucleoside anti-vzv agents based upon the indole template has been reported. we have shown that the substitution on the tryptamine moiety strongly influences the activity/toxicity of these compounds. in particular, the concomitant presence of a biphenylethyl and a methyl(phenyl) carboxy group at the amino group of tryptamine is a requisite for antiviral activity. in fact, the n-biphenylethyl acetamide derivative 17a displayed an interesting inhibition against vzv and was endowed with a selectivity of 10e20 (ratio cc50/ec50). it must be also considered that 17a showed similar activity against tkþ and tk-vzv strains, thus indicating a mechanism of action independent from the virus-encoded thymidine kinase. additionally, this compound is able to exert antiviral activity exclusively on vzv, being totally inactive against other members of herpesviridae family as well as against a wide variety of rna virus strains suggesting a selective action on a specific antiviral target untargeted by the currently existing compounds. the possible mechanism of action of this novel class of compounds will be further investigated. reagents, starting materials, and solvents were purchased from sigma-aldrich (milan, italy) and used as received. reactions were carried out with magnetic stirring in round-bottomed flasks unless otherwise noted. moisture-sensitive reactions were conducted in oven-dried glassware under a positive pressure of dry nitrogen, using pre-dried, freshly distilled solvents. microwave assisted reactions were performed in a biotage initiator þ reactor. analytical thin layer chromatography (tlc) was performed on pre-coated glass silica gel plates 60 (f254, 0.25 mm, vwr international). purifications were performed by flash column chromatography on silica gel (230e400 mesh, merck millipore). nmr spectra were recorded on varian mercury-400 apparatus. 1hnmr and 13c nmr spectra were recorded with a varian-400 spectrometer, operating at 400 and 100 mhz, respectively. chemical shifts are reported in spectrometer. combustion microanalyses were performed on a carlo erba cnh 1106 analyzer, and were within 0.4% of calculated values and confirmed >95% purity for the final products. 4.1.1. general procedure for the synthesis of n-alkylated indole intermediates (2, 3, 6, 11, 13 and 14) indole (1, 1.0 eq) or 1h-indole-5-carbaldehyde (5, 1.0 eq), or n-(1h-indol-5-yl)benzamide (10, 1.0 eq), or 3-(2-bromoethyl)indole (13, 1.0 eq.) were dissolved in a mixture of anhydrous dcm/dmf (2/1 v/v) under magnetic stirring and the temperature was set to 0 c. to this solution, 1.5 equivalents of nah were added portionwise and the mixture was allowed to react for 30 min. then, 1.5 equivalents of alkyl iodide [methyl iodide, or n-propyl iodide, or 4-(phenyl)iodomethylbenzene] in dcm were added dropwise and the reaction was warmed to room temperature and maintained under stirring for further 12 h. the reaction was then quenched by a 10% aqueous solution of citric acid and washed with brine. the organic layer was separated, dried over anhydrous na2so4, filtered and evaporated in vacuo. crude products were purified by column chromatography using n-hexane/ethyl acetate (4:1 v:v) as mobile phase. n-alkylated compounds were obtained in 75% yield (derivative 2); 80% yield (derivatives 3 and 13); 67% yield (derivative 6); 55% yield (derivative 11) and 50% yield (derivative 14). to a solution of 1-propyl-1h-indole (2) in dry ch3cn was added aluminium trichloride (15 eq) and benzoyl chloride (10 eq), the reaction was stirred at room temperature overnight. the resulting mixture was dried in vacuo and reconstituted in dcm. the organic phase was washed with water (3 â 50 ml), dried over anhydrous na2so4, filtered, concentrated and purified by column chromatography using n-hexane/ethyl acetate (3/2) as mobile phase. compound 4a was obtained in 32% yield. a solution of formaldehyde (2 eq.), trifluoroacetic acid (2 eq.) and 2 equivalents of the proper amine (piperidine or bifenylethilamine) was added portionwise to a solution of the intermediate 2 or 3 (1 eq.) in dcm. the mixture was stirred for 3 h at room temperature, then was washed with water (3 â 50 ml), dried over anhydrous na2so4, filtered, concentrated and purified by column chromatography using dcm/meoh (9:1 v/v) as mobile phase. compounds 4b-d were obtained in 25%e38% yield. the intermediate 7a or 7b (1.0 eq) was dissolved in a solution of dcm:ch3cooh (5:1 v/v) at room temperature. to this solution 2.0 equivalents of 4-chloroaniline was added and the mixture was warmed to reflux for 1.5 h. then, 1.8 equivalents of sodium triacetoxyborohydride were added portionwise and the mixture was allowed to reflux for further 3e5 h. after cooling to room temperature, naoh 1 n was added. the organic phase was separated and extracted one more time with the alkaline solution. then it was dried over na2so4, filtered and concentrated in vacuo. the crude products were purified by column chromatography using mixtures of dcm/meoh as eluent giving desired compounds 8a and 8b in 24% and 30% yield respectively. to a solution of 5-amino indole (9, 1.0 eq) in dichloromethane/ dmf (20 ml/5 ml) were successively added benzoic acid (1.1 eq), hbtu (1.2 eq), hobt (1.2 eq), and diea (2.4 eq), and stirring was continued at room temperature for 24 h. afterward, the reaction mixture was diluted with dichloromethane (20 ml), and the resulting solution was washed successively with 10% citric acid (2 â 25 ml), 10% nahco3 (2 â 25 ml), and water (2 â 25 ml), dried over na2so4, and evaporated to dryness. intermediate 10 was obtained after flash chromatography using n-hexane/ethyl acetate 2/1 as ratio with 63% yield. substitution with propyl group of indolic n1 was carried out using the same conditions described elsewhere, to give compound 11 with 44% yield. mannich reaction performed on position 3 of intermediate 11 follows the procedure previously described yielded final compound 12 in 24% yield. 4.1.6. general procedure for the synthesis of derivatives 15, and 16a-i one equivalent of intermediate 14a or 14b was dissolved in thf and 1.5 eq of the proper amine, 1.5 eq of tea, 1.5 eq of nai and 0.3 eq of (ch3coo)2pd were added to this solution. the reaction was conducted under mw, at 100 c, for 20 min. the resulting mixture was filtered through celite, dried in vacuo and reconstituted in dcm. the organic phase was washed with water (3 â 50 ml), dried over anhydrous na2so4, filtered, concentrated and purified by column chromatography using dcm/meoh (9:1 v/v) as mobile phase giving derivative 15 in 38% yield and intermediates 16a-i in 55e75% yield. 4.1.7. general procedure for the synthesis of derivatives 17a-i and 18a-c one equivalent of intermediate 16a-i was dissolved in dcm and 1.5 eq of the proper acyl chloride and 1.5 eq of tea, were added to the solution. the reaction was conducted for 20 min at room temperature. the organic phase was washed with water (3 â 50 ml), dried over anhydrous na2so4, filtered, concentrated and purified by column chromatography using n-hexane/ethyl acetate (2:1 v/v) as mobile phase. final derivatives 17a-i and 18ad were obtained as an atropisomer mixtures in 42e58% yield. to a mixture of 16a (1.0 eq.) in dcm, (boc)2o (3.0 eq.) was added followed by tea (1.5 eq.). the mixture was allowed to stir at room temperature for 12 h. afterward, the reaction mixture was diluted with dichloromethane (20 ml), and the resulting solution was washed with 10% citric acid (2 â 25 ml). the organic phase was dried over na2so4, and evaporated to dryness. compound 17 k was obtained as atropisomer mixture after flash chromatography using n-hexane/ethyl acetate 2/1 in 58% yield. the compounds were evaluated against different herpes viruses, including varicella-zoster virus (vzv) strains oka and ys, tk à vzv strains 07-1 and ys-r, herpes simplex virus type 1 (hsv-1) strain kos, thymidine kinase-deficient (tk à ) hsv-1 kos strain resistant to acv (acv r ), herpes simplex virus type 2 (hsv-2) strain g, human cytotoxicity measurements were based on the inhibition of hel cell growth. hel cells were seeded at a rate of 5 â 103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. then, medium containing different concentrations of the test compounds was added. after 3 days of incubation at 37 c, the cell number was determined with a coulter counter. the 50% cytostatic concentration (cc50) was calculated as the compound concentration required to reduce cell growth by 50% relative to the number of cells in the untreated controls. cc50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. cytotoxicity was expressed as the minimum cytotoxic concentration (mcc) or the compound concentration that causes a microscopically detectable alteration of cell morphology. this work was supported by grant from the italian ministry of education (miur) (prin n 2010-11e61j12000210001) . the biological part of this work was supported by the ku leuven (goa 15/19 tba). morphological and biological characteristics of varicella-zoster virus varicella zoster virus infection: clinical features, molecular pathogenesis of disease, and latency herpes zoster and postherpetic neuralgia varicella zoster virus and central nervous system syndromes varicella-zoster virus latency in human ganglia varicella zoster virus vasculopathies: diverse clinical manifestations, laboratory features, pathogenesis, and treatment neurologic complications of the reactivation of varicella-zoster virus acute retinal necrosis: clinical features and therapy options zoster in patients infected with hiv: a review human herpesviruses: biology, therapy, and immunoprophylaxis current pharmacological approaches to the therapy of varicella zoster virus infections: a guide to treatment varicella zoster virus: natural history and current therapies of varicella and herpes zoster clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir in treatment of dna virus and retrovirus infections advances in the treatment of varicella-zoster virus infections chemotherapy of varicella-zoster virus by a novel class of highly specific anti-vzv bicyclic pyrimidine nucleosides pharmacokinetics and safety of fv-100, a novel oral anti-herpes zoster nucleoside analogue, administered in single and multiple doses to healthy young adult and elderly adult volunteers roscovitine, a cyclindependent kinase inhibitor, prevents replication of varicella-zoster virus resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms antiviral resistance: mechanisms, clinical significance, and future implications acyclovir-resistant herpes zoster in human immunodeficiency virus-infected patients: results of foscarnet therapy antivirals and antiviral strategies non-nucleoside inhibitors of herpesviruses inhibition of herpesvirus replication by a series of 4-oxo-dihydroquinolines with viral polymerase activity ]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases asp2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2 thiourea inhibitors of herpesviruses. part 3: inhibitors of varicella zoster virus identification of small molecule compounds that selectively inhibit varicella-zoster virus replication 4-benzyloxy-gamma-sultone derivatives: discovery of a novel family of non-nucleoside inhibitors of human cytomegalovirus and varicella zoster virus privileged scaffolds for library design and drug discovery homology modeling in tandem with 3d-qsar analyses: a computational approach to depict the agonist binding site of the human cb2 receptor comfa and comsia analyses on 1,2,3,4-tetrahydropyrrolo[3,4-b]indole and benzimidazole derivatives as selective cb2 receptor agonists biological importance of the indole nucleus in recent years: a comprehensive review antiviral activity of benzimidazole derivatives. iii. novel anti-cvb-5, anti-rsv and anti-sb-1 agents arbidol: a broad-spectrum antiviral compound that blocks viral fusion delavirdine mesylate, a potent non-nucleoside hiv-1 reverse transcriptase inhibitor comparison of hdac inhibitors in clinical development: effect on hiv production in latently infected cells and t-cell activation tryptamine-based derivatives as transient receptor potential melastatin type 8 (trpm8) channel modulators the nuclear overhauser effect in structural and conformational analysis synthesis, in vitro, and in cell studies of a new series of the authors wish to express their gratitude to mrs. leentje persoons, mrs. lies van den heurck and mrs. ellen de waegenaere for excellent technical assistance. supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2016.09. 014. these data include mol files and inchikeys of the most important compounds described in this article. key: cord-333675-vkk2frnf authors: hamada, manabu; roy, vincent; mcbrayer, tamara r.; whitaker, tony; urbina-blanco, cesar; nolan, steven p.; balzarini, jan; snoeck, robert; andrei, graciela; schinazi, raymond f.; agrofoglio, luigi a. title: synthesis and broad spectrum antiviral evaluation of bis(pom) prodrugs of novel acyclic nucleosides date: 2013-07-04 journal: eur j med chem doi: 10.1016/j.ejmech.2013.06.053 sha: doc_id: 333675 cord_uid: vkk2frnf a series of seventeen hitherto unknown anp analogs bearing the (e)-but-2-enyl aliphatic side chain and modified heterocyclic base such as cytosine and 5-fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide or 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as key synthetic step. all novel compounds were evaluated for their antiviral activities against a large number of dna and rna viruses including herpes simplex virus type 1 and 2, varicella zoster virus, feline herpes virus, human cytomegalovirus, hepatitis c virus (hcv), hiv-1 and hiv-2. among these molecules, only compound 31 showed activity against human cytomegalovirus in hel cell cultures with at ec(50) of ∼10 μm. compounds 8a, 13, 14, and 24 demonstrated pronounced anti-hcv activity without significant cytotoxicity at 100 μm. the discovery by a. holý and e. de clercq in 1986 of broadspectrum antiviral activity of (s)-hpmpa [9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine] and pmea [9-[2-(phosphonome thoxy)ethyl]adenine] led to a new family of nucleotides designated as acyclic nucleoside phosphonates (anp) [1e4],. anps are nucleotide analogs that are characterized by the presence of a phosphonate group linked to a pyrimidine or purine base through an aliphatic linker. three of these are approved drugs for the treatment of severe/fatal infectious diseases and represent three different types of anps: (i) hpmp derivatives such as (s)-1-[3hydroxy-2-(phosphonomethoxy)propyl]cytosine (hpmpc, cidofovir (vistide ò )] which is approved for the treatment of cytomegalovirus (cmv) retinitis in aids patients [5] ; (ii) pme derivatives such as pmea [adefovir (in its oral prodrug form, adefovir dipivoxil (hepsera ò )] for the treatment of hepatitis b virus infections [6] , and (iii) pmp derivatives such as pmpa [tenofovir (in its oral prodrug form, tenofovir disoproxil fumarate (viread ò )] is used for the treatment of hiv infections (aids) and hepatitis b virus [7] . from these data, it appears that small chemical alterations in the acyclic side-chain lead to marked differences in antiviral activity and the spectrum of activity of acyclic nucleoside phosphonates against various classes of viral agents [1] . thus, the synthesis and biological evaluation of a large panel of anps were systematically investigated as potential antiviral compounds [1] . in our search for antiviral compounds, we synthesized a new class of acyclic nucleoside phosphonates based on a 4-phosphono-but-2-en-1-yl base motif in which the oxygen heteroatom has been replaced with a double bond having trans stereochemistry [8] . we have shown that this modification allows mimicry of the three-dimensional geometry provided by the backbone of pmea, pmpa, and cdv while maintaining an electronic contribution similar to that brought by the oxygen atom [8] . several new derivatives are efficiently activated by human thymidylate kinase (htmpk), and the best substrates were converted to bis-(pivaloyloxymethyl)ester phosphonate prodrugs and found to be active against several herpes viruses in cell culture. on the basis of these findings, it was interesting to design and synthesize hitherto unknown anp analogs bearing the biolabile phosphonate (e)-but-2-enyl aliphatic side-chain and a series of modified heterobases selected from the literature as lead nucleobases with antiviral properties, such as cytosine and 5-fluoro cytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxamide or 4-substituted-1,2,3-triazoles (fig. 1) . from a chemical synthesis point of view, the strategy based on olefin cross metathesis we have developed to obtain a large library of (e)-4-phosphono-but-2 0 -en-1 0 -yl pyrimidine nucleosides [9e11]. first, we turned our attention to the synthesis of cytosine derivatives as cytosine modified nucleosides form a prolific family of antitumor and antiviral agents [12] . as it could be excepted, even if the ruthenium carbene complex 3 (grubbsenolan catalyst 2nd generation) is less affected by free amine and nitrogen-containing groups than the grubbs's 1st generation catalyst [13] , the crossmetathesis reaction between unprotected n 1 -crotylated cytosine 2 and bis-(pom) allylphosphonate 1 failed (scheme 1). the successful cross-metathesis occurred with protected n 1crotylated cytosines 6a, b. thus, cytosine 4a and 5-fluorocytosine 4b were converted to their n 4 -bis-boc cytosine derivatives 5a, b, respectively, through a n-peracylation followed by subsequent and regioselective n 1 deprotection by a saturated solution of nahco 3 in methanol [14, 15] . crotylation of the n 1 position of 5a, b using cs 2 co 3 and crotyl bromide afforded the desired compounds 6a (85%) and 6b (81%). compound 6a, b were then engaged in the olefin cross metathesis reaction with bis(pom)-allylphosphonate 1 using 5 mol% of the (nhc)ru]chr nolan's catalyst, cl 2 (pcy 3 ) (imes)ru (chph) (3), in dry ch 2 cl 2 (0.1 m) at reflux to afford (e)-n 1 -(4 0bis(pom)-phosphinyl-2 0 -butenyl)-bis-boc-cytosine 7a, b in moderate yields (43% (for r ¼ h) and 26% (for r ¼ f)). the removal of the boc group requires oftentimes harsh conditions (e.g. trifluoroacetic acid, trimethylsilyl iodide, hydrochloric acid in ethyl acetate, potassium carbonate, etc.) that are not compatible with the pom moiety. however, hwu et al. [16] have reported an efficient and milder selective boc deprotection under neutral conditions using ceric ammonium nitrate (can) that is consistent with the stability of the phosphonate biolabile group. thus, protected anp 7a, b were reacted with a catalytic amount of ceric ammonium nitrate (can) (20 mmol%) in ch 3 cnemeoh (1:1) to give the expected compounds 8a, b, respectively, in moderate yield, with no observed removal of the pom moiety (scheme 2). we turned then our attention to the synthesis of the pyrazinecarboxamide derivative 14 since, among the modified nucleobases, a series of pyrazinecarboxamide derivatives (including t-705, favipiravir) developed by furuta et al. [17, 18] have demonstrated good activity in various rna viral infections. in a first attempt, we struggled to introduce the pyrazine moiety through direct n-alkylation of 10 with the corresponding (e)-4-phosphonobut-2 0 -en-1 0 -yl bromide (9) , in the presence of k 2 co 3 in anhydrous dmf, (pathway a). unfortunately only the bis(pom)-but-1,3-dienyl phosphonate 9 0 resulting from undesired bromine elimination was obtained, (scheme 3). thus, we decided to reach the pyrazine phosphonate analogs 13, 14 following the same strategy developed for the cytosine derivatives, (pathway b). starting from the 3hydroxypyrazine-2-carboxamide 10, the n 1 -crotyl-3-oxo-pyrazine-n 3 -bis-boc-carboxamide 11 was obtained in 49% yield, via the two step crotylation/n-boc-protection. next, n 1 -crotyl-3-oxo-pyrazine-n 3 -bis-(boc)-carboxamide 11 in ch 2 cl 2 was treated in the presence of bis(pom)-allylphosphonate 1 and (nhc)ru]chr nolan's catalyst 3 to give the desired product 12 in 22% yield. to obtain the carboxamide 14, we first applied our previous described methods, using ceric ammonium nitrate and ch 3 cnemeoh (1:1) . surprisingly, the methylester 13 was isolated as the major compound in 47% yield. thinking that the presence of the undesired product 13 was due to the use of methanol as co-solvent, the preparation of the desired amide 14, was achieved in ch 3 cn using ceric ammonium nitrate in 25% yield. based on the above results, we extended this approach to the formation of a ribavirin analog bearing the 1,2,4-triazole derivative [19, 20] . starting from 15, the protection of both nitrogens provided compound 16, in quantitative yield, which is directly used in the next step. an attempt to selectively boc deprotect at the n 1 position alternatively, we decided to successively alkylate the n 1 position of the 1,2,4-triazole-3-carboxamide in the presence of crotyl bromide and cs 2 co 3 , followed by the protection of the free nitrogen in dry thf in the presence of boc 2 o and dmap at room temperature to give the desired triazole 19 in 34% in two steps (scheme 4). n 1crotyl-1,2,4-triazole-3-bis-boc-carboxamide 19 was then subjected to the olefin cross metathesis reaction, with bis(pom)-allylphosphonate in ch 2 cl 2 to obtain the desired compound 20 in 16% yield. the bis-boc groups were cleaved according to the previous procedure, by treatment with can in a mixture of ch 3 cnemeoh (1:1) to give the free anp analog 21 in 42% yield (scheme 4). to complete our investigation, we elaborated a small library of anps in their prodrug form bearing the substituted 1,2,3-triazolyl moiety 24e33. the triazole derivatives were obtained using cu(i)catalyzed azideealkyne cycloaddition (cuaac) using selected alkynes and phosphonate azide 22 [21e24] ,. the introduction of the azide group could be easily performed from the key intermediate previously described. the olefin cross metathesis of bis(pom)allylphosphonate with (e)-1,4-dibromobut-2-ene catalyzed by catalyst 3 afforded (e)-4-bromo-bis(pom)-allylphosphonate (9) in 88% yield. then, the introduction of azido on 9 with sodium azide in dmsoethfeh 2 o (5:2:1) afforded (e)-4-azide-bis(pom)-allylphosphonate 22 in 69% yield (scheme 5). among the modifications on the base moiety, carboxylic acid is an unavoidable function. in a first attempt, following our previously described method c, in the presence of propiolic acid under microwave irradiation, we observed the formation and isolation of the unexpected decarboxylated product 23 in 16% yield [25, 26] . to circumvent this decarboxylation, hall et al. reported boronic acid catalysis (bac) for the activation of carboxylic acids, to lead to a classic dipolar [3 þ 2] cycloaddition with several azides [27] . to obtain the desired (e)-4-(4-carboxylic acid-[1,2,3]-triazol-1-yl)methyl-bis(pom)-but-2enylphosphonate derivative 24, we selected the bac of the azideealkyne cycloaddition, which was converted in 58% yield in the presence of 2-nitrophenylboronic acid at room temperature. finally, the copper-catalyzed azide-alkynes 1,3-dipolar cycloaddition (cuaac) affording chemo-selectively and complete regioselectively the (1,4) substituted-1,2,3-triazoles [22] , that permitted the synthesis of a number of anps analogs, (table 1) . a first series of bis(pom)-(1,4-disubstituted-1,2,3-triazol)-but-2enyl-phosphonate congeners bearing a substituted 1,2,3-triazole by phenylacetylene moieties (chosen from apolar to bulky substituents) was obtained by cuaac reaction with cu(0)/cuso 4 $5h 2 o as catalyst in the presence of substituted phenylacetylenes, ethynylthiophene and non-aromatic alkynes, in moderate to good yields ranging from 35 to 93% (table 1 , entries 1e6) at room temperature (method a. the reaction between our synthon 22 and 2ethynylthiophene or prop-2-ynol at room temperature only led to trace amounts of the expected products 31 and 32. these were, however, isolated. the cycloaddition was then carried out at 60 c table 1 , entries 7 and 8, method b) to afford compounds 31 and 32 in 45 and 95% yield, respectively. however, the alkyne listed in table 1 , entry 9 did not allow the formation of the triazole product 33 under thermal conditions. microwave heating is known as a powerful tool that can produce a variety of nucleoside products [23] . following our previous work [24] , the microwave irradiation allowed to obtain in moderate yields the desired anps 33 and 34 in 44% and 34% yield respectively (table 1, entry 9 and 10, method c). the title bis(pom) (e)-4-phosphono-but-2-en-1-yl acyclic nucleosides were subjected to an in vitro antiviral screening using a wide spectrum of viruses, in mdkc cell cultures for anti-influenza virus activity, in vero cell cultures for an antiviral activity against para-influenza3 shown) only compound 31 showed activity at an ec 50 of w10 mm (ad-169 strain) with no observed cytotoxicity at 100 mm ( table 2) . the compounds were also evaluated for inhibition of hepatitis c virus (hcv) in the subgenomic hcv replicon system in huh 7 cells [28] , and cytotoxicity testing was performed in pbmc, human lymphoblastoid cem and vero cells [29, 30] . data for anti-hcv activity and anti-hiv activity, and the cytoxicity are shown in table 3 . many of the compounds were found to be moderately cytotoxic, which must be considered when interpreting the anti-hcv data. compounds 8a, 13, 14, and 24 demonstrated anti-hcv activity without significant cytotoxicity at 100 mm. the other molecules showed pronounced inhibition at 10 mm, but at compound concentrations that were close to their cytostatic activity. we have efficiently synthesized a series of seventeen hitherto unknown anp analogs bearing the e-but-2-enyl aliphatic side chain and a series of modified heterocyclic bases such as cytosine, 5fluorocytosine, 2-pyrazinecarboxamide, 1,2,4-triazole-3-carboxam ide and 4-substituted-1,2,3-triazoles and evaluated their antiviral activities. among the tested molecules only compound 31 showed activity against human cytomegalovirus at an ec 50 of w10 mm (ad-169 strain) at subtoxic concentrations with no observed cytotoxicity up to 100 mm. compounds 8a, 13, 14, and 24 demonstrated pronounced anti-hcv activity at 10 mm without significant cytotoxicity at 100 mm. further structural optimization of both the (e)-but-2-enyl aliphatic side chain and the heterocycle is well under way, alongside more detailed biological testing of the most active compounds, with the aim of improving their antiviral potency. commercially available chemicals were of reagent grade and used as received. solvents were dried following standard procedures. the reactions were monitored by thin layer chromatography (tlc) analysis using silica gel plates (kieselgel 60f 254 , e. merck). column chromatography was performed on silica gel 60m (0.040e0.063 mm, e. merck). the 1 h, 31 p and 13 c nmr spectra were recorded on a varian inova unity 400 spectrometer (400 mhz) in (d 4 ) methanol, cdcl 3 , shift values in parts per million relative to sime 4 as internal reference. high resolution mass spectra (hrms) were performed on a bruker maxis mass spectrometer by the "fédération de recherche icoa/cbm (fr 2708) platform". physico-chemical data are in agreement with reported information [9] . cas number: 1258789-63-7. to the stirred suspension of cytosine in an argon atmosphere (444.0 mg, 4.0 mmol, 1.0 equiv.) in dry thf (13 ml), dmap (44.0 mg, 0.4 mmol, 0.1 equiv.) boc 2 o (3.60 g, 16 mmol, 4.0 equiv.) were added. after 20 h stirring at room temperature, the mixture was diluted with etoac and then extracted with (2 â 30 ml) etoac. the combined organic layer was washed with brine, dried over mgso 4 , and concentrated in vacuo. the resulting tri-boc-cytosine was used for the next step without further purification. to a solution of tri-boccytosine in methanol (40 ml) was added saturated nahco 3 aq. solution (18 ml) at room temperature. after stirring 2 h at 60 c, confirming the complete consumption of substrate by examining a tlc developed with petroleum ethereetoac (1:1). after removal of methanol in vacuo, the mixture was diluted with etoac (30 ml), quenched with water (20 ml) and finally extracted with etoac (2 â 20 ml). the combined organic layer was washed with brine, dried over mgso 4 , and concentrated in vacuo. the residue was purified by silica gel column chromatography with petroleum ethere etoac (1:1 to 1:20) to give 5a (465 mg, 37%) as a white solid. 1 in an analogous manner to the preparation of 5a, 5b was prepared from 5-fluoro-cytosine (158.0 mg, 24%) as a white solid. 1 to a solution of 5a (90.0 mg, 0.29 mmol, 1.0 equiv.) in dry dmf (1 ml) was added cs 2 co 3 (104.3 mg, 0.32 mmol, 1.1 equiv.) and crotyl bromide (43.0 mg, 0.32 mmol, 1.1 equiv.) at room temperature and stirred under an argon atmosphere for 2 h. the resulting mixture was then diluted with etoac (2 â 20 ml), quenched with water and extracted with etoac. the combined organic layer was washed with brine, dried over mgso 4 , and concentrated in vacuo. the residue was purified by silica gel column chromatography with petroleum ethereetoac (1:1) to give a mixture of z (minor)/e (major) n 4 ,n 4 -bis-boc-n 1 -crotyl-cytosine 6a (90.0 mg, 85%) as a yellow oil. 1 in a similar manner as described for 8a, a solution of 7b (35.5 mg, 0.05 mmol) in 0.8 ml of ch 3 cnemeoh (1:1) was treated with ceric ammonium nitrate (5.5 mg, 0.01 mmol), to give (e)-n 1 -(4 0 -bis(pom)phosphinyl-2 0 -butenyl)-5-fluoro-cytosine 8b (8.2 mg, 35%) as a colorless oil. 1 to a solution of bis(pom)-allylphosphonate 1 (560 mg,1.60 mmol, 1.0 equiv.) in 24 ml of dry ch 2 cl 2 was added (e)-1,4-dibromobut-2ene (1.37 g, 6.40 mmol, 4.0 equiv.) and (nhc)ru]chr nolan's catalyst 3 (68.6 mg, 0.08 mmol, 0.05 equiv.). after 16 h of stirring at 45 c under an argon atmosphere, all volatiles were evaporated and the residue was purified by silica gel chromatography eluting with petroleum ethereetoac (4:1) to give (e)-4-bromo-bis(pom)-but-2enylphosphonate 9 (623 mg, 88%) as a brown oil. 1 in a similar manner as described for 6a, a solution of 3hydroxypyrazine-2-carboxamide 10 (139.1 mg, 1.0 mmol) in dry dmf (3 ml) was treated with cs 2 co 3 (358.4 mg, 1.1 mmol) and crotyl bromide (148.5 mg, 1.1 mmol) for 4 h at 70 c, to give a crude mixture of (e/z) n 1 -crotyl-3-oxo-pyrazine-2-carboxamide. after evaporation of all volatiles and rapid silica gel chromatography, the residue (134.0 mg) was treated with dmap (7.7 mg, 0.07 mmol) and boc 2 o (602.4 mg, 2.76 mmol) in dry thf (3 ml), to give n 1 -crotyl-3oxo-pyrazine-2-bis-boc-carboxamide 11 (134.0 mg, 49%) as a colorless oil. 1 in a similar manner as described for 7a, a solution of bis(pom)allylphosphonate 1 (273.5 mg, 0.78 mmol) and n 1 -crotyl-3-oxopyrazine-n,n-bis-boc-carboxamide 11 (338.2 mg, 0.86 mmol) in dry ch 2 cl 2 (8 ml) were treated with (nhc)ru]chr nolan's catalyst 3 (66.2 mg, 0.078 mmol), to give 12 (122.8 mg, 22%) as a yellow oil. 1 155.4, 135.1, 130.4, 130.3, 126.1, 126.0, 124.7, 83.4, 83.3, 53.3 to a solution of 1,2,4-triazole-3-carboxamide 15 (224.5 mg, 2.0 mmol, 1.0 equiv.) in dry dmf (6 ml) was added cs 2 co 3 (716.8 mg, 2.2 mmol, 1.1 equiv.) and crotyl bromide (270.0 mg, 2.2 mmol, 1.1 equiv.) and the reaction solution was stirred at room temperature under an argon atmosphere for 2 h and then warmed at 70 c for 12 h. after concentration to dryness in vacuo, the residue was subjected to silica gel chromatography with ch 2 cl 2 e meoh (5:1) and employed in the next step without further purification. the residue (330 mg) was suspended in thf (10 ml), dmap (44 mg, 0.2 mmol, 0.1 equiv.) and boc 2 o (1.31 g, 6.0 mmol, 3.0 equiv.) were added under an argon atmosphere. the solution was stirred for 20 h at room temperature and then the mixture was diluted with etoac and then extracted with etoac. the combined organic layer was washed with brine, dried over mgso 4 , and concentrated in vacuo. the residue was purified by silica gel column chromatography eluting with petroleum ethereetoac (4:1) to give a mixture of z (minor)/e (major) n 1 -crotyl-1,2,4-triazole-3-bis-boccarboxamide 19 (248.0 mg, 34%) as a colorless oil. 1 in a similar manner as described for 8a, a solution of (e)-n 1 -(4 0bis(pom)-phosphinyl-2 0 -butenyl)-1,2,4-triazole-3-bis-boc-carboxamide 20 (44.3 mg, 0.065 mmol) in 1 ml of ch 3 cnemeoh (1:1) was treated with ceric ammonium nitrate (7.2 mg, 0.013 mmol), to give (e)-n 1 -(4 0 -bis(pom)phosphinyl-2 0 -butenyl)-1,2,4-triazole-3carboxamide 21 (14.0 mg, 42%) as a colorless oil. to a solution of (e)-4-bromo-bis(pom)-but-2-enylphosphonate 9 (222.0 mg, 0.50 mmol, 1.0 equiv.) in mixture of dmso (5 ml), thf (2 ml) and h 2 o (1 ml) was added sodium azide (163.0 mg, 2.50 mmol, 5.0 equiv.). after 24 h stirring at room temperature, the mixture was diluted with etoac and water and then extracted with etoac. the combined organic layer was washed with brine, dried over mgso 4 , and concentrated in vacuo. the residue was purified by silica gel column chromatography, eluting with petroleum ethere etoac (4:1) to give (e)-4-azide-bis(pom)-but-2-enylphosphonate procedure a: to a solution of alkyne (1.3 equiv.) and (e)-4-azidobis(pom)-but-2-enylphosphonate 22 (0.11 mmol, 1.0 equiv.) in t-buoh/h 2 o (1:1 ratio, 400 ml) were added cu powder (11.6 mg, 0.40 mmol, 5.0 equiv.) and cuso 4 (5.0 mg, 0.020 mmol, 0.25 equiv.). the resulting suspension was stirred 8 h at room temperature, then the crude mixture was diluted in etoac (1 ml), and directly transferred on a preparative thin layer silica plate to give (e)-4 0 -(1,2,3-triazol-1-yl)-bis(pom)-but-2 0 -enylphosphonate. procedure b: following the procedure described above, the resulting suspension was stirred 16 h at 60 c. procedure c: in a similar procedure, the mixture was stirred 1 h under microwave conditions at 125 c. the title compound was prepared from 22 with procedure c to give 23 (16%) as a colorless oil. 1 the title compound was prepared from 22 with procedure a to give 25 (70%) as a yellow oil. 1 the title compound was prepared from 22 with typical procedure c to give 33 (44%) as a colorless oil. 1 the title compound was prepared from 22 with typical procedure c to give 34 (34%) as a yellow oil. 1 viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. antiviral activity was expressed as the ec 50 or compound concentration required reducing virus-induced cytopathogenicity or viral plaque (vzv) formation by 50%. the minimal cytotoxic concentration (mcc) of the compounds was defined as the compound concentration that caused a microscopically visible alteration of cell morphology. alternatively, the cytostatic activity of the test compounds was measured based on inhibition of cell growth. hel cells were seeded at a rate of 5 â 10 3 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. then, medium containing different concentrations of the test compounds was added. after 3 days of incubation at 37 c, the cell number was determined with a coulter counter. the cytostatic concentration was calculated as the cc 50 , or the compound concentration required to reduce cell proliferation by 50% relative to the number of cells in the untreated controls. the methodology of the anti-hiv assays was as follows: human cem (w3 â 10 5 cells/ cm 3 ) cells were infected with 100 ccid 50 of hiv(iii b ) or hiv-2(rod)/ml and seeded in 200 ml wells of a microtiter plate containing appropriate dilutions of the test compounds. after 4 days of incubation at 37 c, hiv-induced cem giant cell formation was examined microscopically. hepatitis c antiviral activity was evaluated as previously described [28] . huh 7 clone b cells containing hcv replicon rna were seeded in a 96-well plate at 3000 cells/well, and the compounds were tested at 10 mm in triplicate immediately after seeding. following five days incubation (37 c, 5% co 2 ), total cellular rna was isolated using the rneasy96 well extraction kit from qiagen. replicon rna and an internal control (taqman rrna control reagents, applied biosystems) were amplified in a single step multiplex real time rt-pcr assay. the antiviral effectiveness of the compounds was calculated by subtracting the threshold rt-pcr cycle of the test compound from the threshold rt-pcr cycle of the no-drug control (dcthcv). a dct of 3.3 equals a 1-log reduction (equal to 90% less starting material) in replicon rna levels. the cytotoxicity of the compounds was also calculated by using the dct rrna values. rs-446 (2 0 -c-me-c) was used as the control and tested at 10 mm. to determine ec50 and ec90 values [31] , dct values were first converted into fraction of starting material and then were used to calculate the % inhibition. nucleoside and nucleotide analogues for the treatment of herpesvirus infections: current stage and new prospects in the field of acyclic nucleoside phosphonates synthesis of acyclic nucleoside phosphonates acyclic nucleoside phosphonates: a key class of antiviral drugs acyclic nucleoside phosphonates: an unfinished story cidofovir, a new agent with potent anti-herpesvirus activity a novel selective broad-spectrum anti-dna virus agent differential antiherpesvirus and antiretrovirus effects of the (s) and (r) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (r)-9-(2-phosphonylme thoxypropyl)-2,6-diaminopurine novel antiviral c5-substituted pyrimidine acyclic nucleoside phosphonates selected as human thymidylate kinase substrates the shortest strategy for generating phosphonate prodrugs by olefin cross metathesis e application to acyclonucleoside phosphonates expeditious convergent procedure for the preparation of bis(poc) prodrugs of (e)-4-phosphono-but-2-en-1-yl nucleoside microwave assisted silylation-amination of uracil acyclonucleosides to 4-alkylamino-2(1h)-pyrimidone analogues antitumor activity of sugar-modified nucleosides ruthenium-catalyzed ring-closing metathesis: recent advances, limitations and opportunities efficient n-arylation and n-alkenylation of the five dna/rna nucleobases a practical and efficient approach to pna monomers compatible with fmoc-mediated solidphase synthesis protocols ceric ammonium nitrate in the deprotection of tert-butoxycarbonyl group mechanism of action of t-705 against influenza virus efficacy of orally administered t-705 on lethal avian influenza a (h5n1) virus infections in mice design, synthesis, and broad spectrum antiviral activity of 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides synthesis and biological evaluation of carbocyclic analogues of ribavirin synthesis of 1,2,3-triazolo-carbanucleoside analogues of ribavirin targeting an hcv in replicon cu(i)-catalyzed huisgen azideealkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide, and oligonucleotide chemistry microwave-assisted syntheses of nucleosides and their precursors preparation of ribavirin analogues by copper-and ruthenium-catalyzed azide-alkyne 1,3-dipolar cycloaddition ) analogs: synthesis and anti-hiv-1 activity a novel approach to 1-monosubstituted 1,2,3-triazoles by a click cycloaddition/decarboxylation process boronic acid catalysis for mild and selective [3þ2] dipolar cycloadditions to unsaturated carboxylic acids ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis c viruses in culture, antimicrob activities of 3 0 -azido-3 0 -deoxythymidine nucleotide dimers in primary lymphocytes infected with human immunodeficiency virus type 1 antiviral activities and cellular toxicities of modified 2 0 ,3 0 -dideoxy-2 0 ,3 0 -didehydrocytidine analogues a simple method of estimating fifty percent endpoints key: cord-330994-6nu7utu1 authors: abdelrheem, doaa a.; ahmed, shimaa a.; abd el-mageed, h. r.; mohamed, hussein s.; rahman, aziz a.; elsayed, khaled n. m.; ahmed, sayed a. title: the inhibitory effect of some natural bioactive compounds against sars-cov-2 main protease: insights from molecular docking analysis and molecular dynamic simulation date: 2020-10-01 journal: journal of environmental science and health. part a, toxic/hazardous substances & environmental engineering doi: 10.1080/10934529.2020.1826192 sha: doc_id: 330994 cord_uid: 6nu7utu1 this work aimed at evaluating the inhibitory effect of ten natural bioactive compounds (1–10) as potential inhibitors of sars-cov-2-3cl main protease (pdb id: 6lu7) and sars-cov main proteases (pdb ids: 2gtb and 3tnt) by molecular docking analysis. the inhibitory effect of all studied compounds was studied with compared to some proposed antiviral drugs which currently used in covid-19 treatment such as chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir. homology modeling and sequence alignment was computed to evaluate the similarity between the sars-cov-2-3cl main protease and other sars-cov receptors. admet properties of all studied compounds were computed and reported. also, molecular dynamic (md) simulation was performed on the compound which has the highest binding affinity inside 6lu7 obtained from molecular docking analysis to study it is stability inside receptor in explicit water solvent. based on molecular docking analysis, we found that caulerpin has the highest binding affinity inside all studied receptors compared to other bioactive compounds and studied drugs. our homology modeling and sequence alignment showed that sars-cov main protease (pdb id: 3tnt) shares high similarity with 3clpro (96.00%). also, admet properties confirmed that caulerpin obeys lipinski’s rule and passes admet property, which make it a promising compound to act as a new safe natural drug against sars-cov-2-3cl main protease. finally, md simulation confirmed that the complex formed between caulerpin and 3clpro is stable in water explicit and had no major effect on the flexibility of the protein throughout the simulations and provided a suitable basis for our study. also, binding free energy between caulerpin and 6lu7 confirmed the efficacy of the caulerpin molecule against sars-cov-2 main protease. so, this study suggested that caulerpin could be used as a potential candidate in covid-19 treatment. coronaviruses (cov) are a great family of viruses varying from the common cold virus which is the lowest serious disease in this family to the more serious diseases such as mers, cwid, and sars. also, their structure has a characteristic rna genome. however, cov are more familiar with animals; seven of them can induce the human respiratory system. [1] from december 2019, a new coronavirus called covid-19 that was first appeared in wuhan, hubei province of china, and now, is spreading rapidly across china and other parts of the world. covid-19 has become a serious threat to the world public health, causing 244,792 deaths from 3,484,640 cases as of 3 may 2020 from the entire world. the current covid-19 outbreak is caused by sars-cov-2 that has been known as the seventh member of the family of coronaviruses. [1] sars-cov-2 was established to have higher sequence homology toward sars-cov than that of mers-cov according to the whole genome sequence alignment analysis in different studies. [2] there are four structural proteins characterize coronaviruse genome, spike glycoprotein (s), matrix glycoprotein (m), nucleocapsid protein (n), and small envelope protein (e) were reported. [3] additionally, 3clpro is also known as nsp5 of sars-cov-2 required for the maturation of coronaviruses. 3clpro is first automatically cleaved from poly-proteins to produce mature enzymes, and then, further cleaves downstream nsps at 11 sites to release nsp4-nsp1631. 3clpro directly mediates the maturation of nsps. so, 3clpro is critical for the viral life cycle, making it an attractive target of anti-coronavirus drug evolution. [4, 5] there are different studies that had been performed on the main proteases of sars-coronavirus (2gtb) to develop antiviral treatments of covid-19 virus because it shares 96% similarity with 3clpro of covid-19. [6] the utilization of natural compounds alternatives or complementary therapies has received growing attention due to their low toxicity, fewer side effects and in some cases as the only effective treatment. chemical drugs are shown different side effects and ineffectiveness in some cases for longterm use. the world health organization (who) evaluated that about 80% of people use natural compounds in the treatment field. [7] actually, natural compounds have various and effective biological activities such as antimicrobial, anticancer, anti-inflammatory, and anti-diabetic. [8] medicinal plants are an important source of molecules with various pharmacological properties and effective biological activities like antimicrobial, anticancer, anti-inflammatory, antiviral, and antidiabetic. so, bioactive compounds isolated from medicinal plants have attracted significant attention because there are safe and effective against different pathogens since ancient times. [9, 10] caulerpin, the low toxic bisindole alkaloid is a more common compound of the genus caulerpa of green macroalgae and it was isolated from caulerpa racemosa, [11] red alga chondria armata, [12] and brown alga sargassum platycarpum. [13] caulerpin has various pharmacological properties and effective biological activities such as antitumor, anti-diabetic, anticancer, anti-larvicidal, anticorrosion, antitubercular, antimicrobial, spasmolytic, antinociceptive, plant growth regulatorory activity, and anti-inflammatory. [13, 14] caulerpin exhibited antiviral activities against chikungunya virus [15] and herpes simplex virus type 1. [16] also, caulerpin showed good inhibition activity against alzheimer's disease. [17] due to the low-toxicity and biological activity of all studied compounds especially caulerpin in different biological applications as summarized in table 1 . we expected that they could be used to develop formal inhibitors against covid-19. in this study, our homology modeling and sequence alignment of 2019-ncov main protease show that sars-cov main protease (pdb id: 3tnt) shares 96% similarity with 3clpro of covid-19. also, homology modeling and sequence alignment of 2019-ncov main protease show that sars-cov main protease (pdb id: 2gtb) shares 96.76% similarity with 3clpro of covid-19 as described in this study. [6] so, we study the inhibitory effect of some bioactive compounds obtained from natural sources against sars-cov-2-3clpro and sars-cov main proteases (pdb ids: 2gtb and 3tnt). due to their high similarity of 2gtb and 3tnt with 3clpro, inhibition of these receptors could be effective in 3clpro treatment. based on molecular docking analysis, we found that caulerpin has the highest binding affinity against 6lu7, 3tnt, and 2gtb receptors compared to other bioactive compounds and the proposed drugs. the pharmacogenic and toxicity properties of all studied compounds also are computed and confirmed that most of all studied bioactive compounds especially, caulerpin obey zaleya decandra [8] ceramium virgatum [18] ulva intestinalis [18] fucus sp. [19] chlorella vulgaris [20] torreya grandis [21] hazelnut [22] antimicrobial, [22] anticancer, [23] and antiviral activities [24] saringosterols (2,3) terrestrial plant red macroalgae green macroalgae brown macroalgae strychnos spinosa [25] acanthophora spicifera [26] cladophora fascicularis [27] sargassum muticum [28] antitrypanosomal [25] anti-obesity, [28] a novel selective lxrb agonist, [29] and anticancer activities [30] b-sitosterol (4) terrestrial plant red macroalgae green macroalgae brown macroalgae microalgae synadenium glaucescens [31] eucheuma cottonii [32] ulva fasciata [33] sargassum glaucescens [34] nannochloropsis [35] antimicrobial, [36] antiviral activity against hepatitis b virus, [37] antioxidant and anticancer activities [38] glycoglycerolipids (5, 9) terrestrial plant red macroalgae brown macroalgae cyanobacteria soybean [39] exophyllum wentii [40] sargassum horneri [41] phormidium sp. [42] accumulation inhibition, [39] antitumor, antiviral, anti-inflammatory and anticancr activities [30, 43] brown macroalgae sargassum naozhouense [44] antibacterial effect against e. coli and bacillus subtilis [45] loliolide (7) terrestrial plant brown macroalgae canscora decussata [46] sargassum naozhouense [44] antioxidant and a cell protective effect on a monkey kidney fibroblast cell line, [47] and anticancer, antibacterial and antifungal activities [48] hexadecanoic acid (8) terrestrial plant canthium parviflorum [49] antimicrobial, anti-inflammatory, antioxidant, hypocholesterolemia, pesticide, hemolytic and 5-alpha reductase inhibitor, [50] and antiviral activity [51] caulerpin (10) green macroalgae red macroalgae brown macroalgae caulerpa racemosa [11] chondria armata [12] sargassum platycarpum [13] antitumor, anti-diabetic, anticancer, antilarvicidal, anticorrosion, anti-herpes, antitubercular, antimicrobial, cytotoxic, antiviral, spasmolytic, antinociceptive, anti-alzheimer's disease, plant growth regulatorory activity and anti-inflammatory activities [13] [14] [15] [16] [17] lipinski's rule and pass adme property. finally, molecular dynamic (md) simulation confirmed that the complex formed between caulerpin and 6lu7 is stable in water explicit and had no major effect on the flexibility of the protein throughout the simulations and provided a suitable basis for our study. based on this work, we believe that caulerpin could be used to develop an antiviral molecule against covid-19. thus, this compound could be a potential candidate for in vitro and in vivo antiviral studies followed by clinical covid-19 treatment. it is worth mentioning that there is not any work in literature discusses the inhibition effect of all studied compounds against covid-19. also, most of all related work in this field studied the inhibition effect against only covid-19 receptors, but in this work we study the inhibition activity of all studied compounds against covid-19 main protease and two sars-cov main proteases receptors. finally, we also build homology modeling and sequence alignment on sars-cov-2-3cl main protease to show its similarity with other sars-cov receptors. while, most of all related work in this field did not study it. in our study, we choose ten public bioactive compounds that already are abundant and isolated from different species of plants or marine algae to study the inhibitory effect of them against main proteases of cov-2 (3clpro) with (pdb id: 6lu7) and main proteases of sars-cov (mpro) (pdb ids: 2gtb and 3tnt). according to literature survey, these compounds showed a broad spectrum of biological activities like antimicrobial, antioxidant, anticancer, antiviral, and anti-inflammatory that making them an attractive target to evaluate their potential to become potential candidate inhibitors against sars-cov-2-3clpro. all studied bioactive compounds in this work are shown in figure 1 . also, their sources, species names and biological activities of all studied compounds are illustrated in table 1 . the crystal structures of sars-cov-2-3clpro (pdb code: 6lu7) and main proteases of sars-coronavirus (mpro) with (pdb ids: 2gtb and 3tnt) were downloaded from the protein data bank (www.pdb.org), and any heteroatoms and water molecules were removed before molecular docking studies. the 3-dimensional (3d) structures of all studied compounds and some proposed antiviral drugs (bioactive compounds, chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir) were obtained from pubchem (https://pubchem.ncbi.nlm.nih.gov/), in .sdf format. pubchem is a chemical substance and biological activities repository consisting of three databases, including substance, compound, and bioassay databases. determination of the amino acids in the active site of a protein was determined using the biovia discovery studio software [52] to analyze the grid box and docking evaluation results. homology modeling is performed for the 2019-ncov main protease. the crystal structure of sars-cov-2-3clpro was used as a template (pdb id: 6lu7). therefore, sequence similarity searches were performed by using blastp analysis which showed suitable templates for the homology modeling. target sequences of the sars-cov-2-3clpro were created according to the build homology models implicit in the discovery studio software as described in this study. [53] molecular docking all ligands in this study (bioactive compounds, chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir) were optimized before docking by avogadro version 1.2 with force field type mmff94 and saved in .pdb format. discovery studio used for protein optimization, by removing water and other atoms to prepare protein for the docking process. molecular docking between ligands and 6lu7 was carried out using auto dock tools (adt) graphical user interface supported by mgl tools. polar hydrogen was added and atomic charges were computed by kollman and gasteiger method. we defined a grid size with 60 å â 60 å â 60 å for two receptors and the lamarckian genetic algorithm (lga) was assigned to carry out the molecular docking process, as described in this study. [54] the output of the auto dock was further analyzed with discovery studio program and pymol version 1.7.4.5 software package. the drug likeness prediction of all studied bioactive compounds was carried out by lipinski filter (http://www.scfbioiitd.res.in/software/drugdesign/lipinski.jsp), according to which an orally active drug should comply with a minimum of four of the five laid down criteria for drug likeness namely: molecular mass, clogp, hydrogen donor and acceptor, and molar refractive index. [55] furthermore, the pharmacokinetic properties like absorption, distribution, metabolism, excretion, and the toxicity of all studied compounds were predicted utilizing admetsar database (http:// lmmd.ecust.edu.cn/admetsar1/predict). [56] md simulation the structure of the highest binding complex obtained from molecular docking study between compound 10 (caulerpin) and 6lu7 was prepared for md simulation using slandered dynamic cascade implicit in discovery studio. the md simulation of sars-cov-2-3clpro-inhibitor complex was carried out at 30 ns using charmm force field for all atoms in the complex. the simulation started by solvating the complex in triclinic box using tip3p water model. the counter ions were added to the neutralized the system. in which caulerpin-6lu7 complex with solvated in 6506 water molecules and neutralized by 20 sodium and 17 chlorides as counter ions. periodic boundary conditions were used. throughout the simulation, the studied system is maintained at a temperature of 300 k with constant pressure. energy minimization was done for 50,000 steps. the trajectories were collected for every nanosecond to get insights into the interactions at the atomistic level. all md protocol was carried out according to this study. [3] the complexes result from md simulation was analyzed for root mean square deviation (rmsd), root mean square fluctuations (rmsf) and hydrogen bonds analysis. the binding free energy of protein and ligand complexes can be calculated by combining the molecular mechanic/ poisson-boltzmann surface area (mm-pbsa) with md. the md scripts were extracted to perform mm-pbsa calculations. the binding free energy provides an overview of the biomolecular interactions between protein and ligand. the binding energy constitutes of potential energy, polar and non-polar solvation energies. the mm-pbsa binding free energies were calculated by utilizing nanoscale molecular dynamics (namd) and visual molecular dynamics (vmd) software programs according to this study. [57] results and discussions homology modeling and sequence alignment of 2019-ncov main protease it is noticed that sars-cov and sars-cov-2-3clpro share remarkable 96.00% sequence alignment among all other human coronaviruses as shown in figure 2 , based on our homology modeling and sequence alignment of 2019-ncov main protease. the crystal structure of sars-cov-2-3clpro (pdb id: 6lu7) is highly similar to its sars-cov sister (pdb id: 3tnt) at high resolution with 1.59 å. multiple additional sequencing studies have been performed for sars-cov-2, including a landmark preprint, which suggested renaming 2019-ncov to sars-cov-2 on based on results similar to ours. [58] the highly conserved region of the structural elements was found, the least pdf total energy of 6lu7 and 3tnt are 2728.98 and 2881.43, respectively, that is, a reliable statistical potential to assess the quality of homology models in protein structure prediction. also, dope score of 6lu7 and 3tnt are 70754.34 and 70644.22, respectively. sars-cov-2 and sars-cov-3clpro have nine a-helices and 13 b-strands which make up three distinct domains, i.e., domain i, domain ii, and domain iii. all cov proteases family consist of three domain, in which domains i (residues 8-101) and ii (residues 102-184) have one antiparallel b-barrel, that resemble the trypsin-like serine proteases structure. domain iii (residues 201-306) consists of 5 a-helices (a5-a9), that are connected by a long loop (resi-dues185-200) with domain ii as reported in this study. [6] there are only four mutated residues (phe 13, asn 65, ala 94, and val 35) between sars-cov-2 and sars-cov-2-3clpro as shown in figure 3 . the ramachandran plot built also, by discovery studio shows that 100% of the residues in the allowed regions, 97.0% in the most favored region as shown in figure 4 . additionally, 88.2% of the residues have averaged 3d-1d score 0.3 based on the verify 3d software, while the overall quality factor of errat is 96.0%. our homology modeling and sequence alignment of 2019-ncov main protease is the best agreement with this study, [58, 59] which confirmed that the structure of sars-cov (pdb code 3tnt) and sars-cov-2-3clpro (pdb code 6lu7) share remarkable 96.75% sequence alignment. due to the high sequence alignment between 3tnt and 2gtb with 6lu7, 3tnt, and 2gtb could be a potential drug target for 2019-ncov and the inhibition of 3tnt and 2gtb protease would help to restrict the viral maturation thereby decreasing the sars-cov-2 infection in humans. the amino acids found in the active site pockets of 6lu7, 3tnt, and 2gtb and sars-cov-2, were elucidated from pdb files by discovery studio and summarized in table 2 . table 3 displays the molecular docking analysis of all studied bioactive compounds and some proposed antiviral drugs against 6lu7, including binding energy, ligand efficiency, inhibition constant, intermolecular energy, and van der waals (vdw)-h bond desolvation energy. figure 5 is shown the best docking poses of the studied bioactive compounds (1-10) inside 6lu7. also, figure 6 displays docking analysis visualization of 6lu7 binding with all studied compounds in which the yellow dots show h-bonds formed between bioactive compounds and 6lu7 residues. the number of h-bonds, h-bonding residues and h-bonding distance produced from docking for all studied bioactive compounds against 6lu7 shown in table 4 . chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir also docked inside 6lu7 using the same protocol applied on all studied compounds. an in silico analysis study showed that only compound 10 can inhibit 6lu7 which have the higher binding energies and inhibition constants and higher number of h-bonds with 6lu7 amino acid residues with compared to chloroquine, hydroxychloroquine, azithromycin, remdesivir, baloxvir, lopinavir, and favipiravir inhibitors as shown in tables 3 and 4 . compound 10 exhibits the highest binding energy (-9.28 k.cal/mol) with compared to other compounds and formed three hydrogen bond interactions with lys 137, glu 288, and lys 5 of 6lu7 amino acid residues with distance 2.25, 2.27, and 2.38 å, respectively. also, all studied bioactive compounds are docked inside 3tnt and 2gtb and the obtained binding energy are summarized in table 5 . also, compound 10 shows the highest binding energy compared to all studied bioactive compounds and antiviral drugs. so, compound 10 may act as a potential inhibitor of sars-cov-2-3clpro and sars-cov-2. lipinski's rule of five is commonly utilized in development and drug design to expect oral bioavailability of drug molecules. lipinski's rule was established based on five rules to compute the ability of the compound to act as an orally active drug was calculated and shown in table 6 . so, orally active drugs must have no more than one violation of the following standards: (i) octanol/water partition coefficient (log p) which measured the lipophilicity of a molecule must be not greater than five. (ii) a molecular weight (mw) less than 500 da. (iii) not more than five hydrogen bond donors (non). (iv) not more than 10 hydrogen bond acceptors (nohn). the topological polar surface area (tpsa) is measured the bioavailability of the drug molecule. tpsa is closely related to the hydrogen bonding potential of a compound. tpsa of studied compounds was noticed in the range of 25.87-153.50 å and is well below the limit of 160 å. it can be predicted that all studied bioactive compounds obeyed lipinski's rule of five and are likely to be orally active except compounds 5 and 9 as shown in table 6 . the database supports admet profiles which involve some features to study the ability of the studied compounds to act as drug leads such as blood-brain barrier (bbb) penetration, human intestinal absorption (hia), caco-2 cell permeability, cyp inhibitory promiscuity, ames toxicity, carcinogenicity, and rat acute toxicity ld50 are calculated and displayed in table 7 . as shown in table 6 , all studied compounds may cross blood brain barrier (bbb) and absorb in the human intestine (hia) along are permeable for cacoe2 cells, whereas, compound 5 showed a negative result for bbb, hia, and cacoe2 cell permeability. cytochrome p450 (cyp) is a group of isozymes containing the metabolism of drugs, steroids, fatty acids, bile acids, and carcinogens. the results indicate that these studied compounds are non-substrate and non-inhibitor of cyp enzymes. [13, 60] in terms of ames toxicity, all studied compounds were observed to be non-toxic. carcinogenicity model indicated non-carcinogenic nature of all studied compounds. rat acute toxicity ld50 of all studied compounds was found between 1.95 and 2.75 mol/kg. the finding strongly provides the ability of most of all studied compounds to act as a drug, except compound 5 as shown in table 7 . to confirm the docking results and get more insight into the stability of the ligand-protein complex, md simulations were carried out for the compound which has the highest binding affinity (compound 10) inside 6lu7 complex in the solvated states at 30 ns as shown in figure 7 . the results of md simulations have been examined based on rmsd, rmsf, and the number of hydrogen bonds as a function of time. to examine the change in the protein dynamics and the conformational stability of the protein-ligand complexes, the protein complexed with compound 10 was subjected to 30 ns md simulations. standard dynamics cascade module implicit in ds software was employed to measure the rmsd and rmsf. the rmsd measures the direct changes in the protein from the initial coordinates. the rmsd values of the protein backbone in complex with the potential inhibitors were computed concerning the initial structure as a frame reference (0 to 10 ns). the rmsd values steadily increased from 0 to 5 ns, and reached equilibration after that throughout the simulation period. the rmsd values for the studied complex showed oscillations between 3 to 5 ns indicating that the studied compound was adapting another conformation within the binding pocket as shown in figure 8 . the average rmsd values for the last 1 ns for the 6lu7 and caulerpin þ 6lu7 complex, were 1.13 ± 0.07 and 1. 22 ± 0.05, respectively. lower rmsd value of the caulerpin in the complex with 6lu7 indicates its stability and provided a suitable basis for our study. root mean square fluctuation rmsf was measured based on the backbone atom of each amino acid residues and the plot of rmsf was used to depict the fluctuations at the residue level. rmsf plot as shown in figure 9 of solvated 6lu7 and 6lu7 in complex with caulerpin during 30 ns mds simulations exhibited a similar trend of residue fluctuation profile for both free receptors (6lu7) and the complexes with a low average rmsf. this trend in the rmsf plot for the complex indicates that binding caulerpin to the receptor was stable and had no major effect on the flexibility of the protein throughout the simulations. to explore more insights on the local protein flexibility, the time average of rmsf values of the 300 amino acids of 6lu7 in the absence and presence of the caulerpin over simulation period were calculated. the rmsf values for the three complexes suggested that the following residues (ala206, val204, and leu205) showed less fluctuation in the presence and presence of the caulerpin. the average rmsf values of these residues were 0.33 ± 0.06, 0.40 ± 0.07, and 0.43 ± 0.08 å for 6lu7, and 0.37 ± 0.07, 0.48 ± 0.08, and 0.52 ± 0.09 å for caulerpin-6lu7 complex. hydrogen bonds are one of the essential elements answerable for the molecular interactions in biological systems. hydrogen bonds provide the basis for molecular recognition and selectivity by imparting directionality and explicitness to molecular interactions. the protein-ligand interactions were guided by the changes in the secondary structures, which in turn were regulated by the hydrogen bonds. md simulation provided different conformations in which a protein could be found in actual biological conditions. each conformation of a protein is supposed to have its interaction pattern with the ligand. we calculated the number of hydrogen bonds formed during the complete run of md simulations for selected complexes, as presented in figure 10 . as shown in figure 10 , in the caulerpin-6lu7 complex, the most number of conformations formed up to 10 hydrogen bonds during the simulation. a very few conformations showed less than 5 and greater than 10 hydrogen bonds. the bioactive caulerpin molecule was able to maintain strong interaction with the binding pocket of 6lu7 throughout the simulation period. we measured binding free energy at last 10 ns from md simulation. the final binding energy is a cumulative sum of van der wall, electrostatic, polar solvation, and sasa energy. except for the polar solvation energy, all other forms of energy contributed favorably to the interaction between caulerpin molecule and 6lu7. the caulerpin molecule showed the binding free energy equal to à256.44 ± 27.55 kj/mol, sasa equal to à28.58 ± 4.32 kj/mol, de polar solvation equal to à283.58 ± 44.53 kj/mol, de electrostatic equal to à127.76 ± 40.50 kj/mol, and de van der waal equal to à384.44 ± 34.40 kj/mol inside 6lu7. this confirmed the efficacy of the caulerpin molecule against sars-cov-2 main protease. recently, the rapidly spreading outbreak of covid-19 has challenged the healthcare sector of the world. numerous antiviral or other conventional drugs are being examined against covid-19, but not yet positive results have come. to contribute to this fight against covid-19, virtual screening-based molecular docking and md simulation were investigated to identify novel and potential to inhibit sars-cov-2-3clpro main protease. based on our molecular docking analysis we found that among all studied compounds, caulerpin has the highest binding affinity against all studied receptors 6lu7, 3tnt, and 2gtb with compared to some proposed antiviral drug currently used in covid-19 treatment. the admet and molecular descriptors properties strongly provide that most of all studied compounds obey lipinski's rule of five and exhibit significant biological activities especially, caulerpin compound. md simulation confirmed the stability of caulerpin-6lu7 receptor in the presence of explicit water solvent. rmsd of the receptor-ligands complex has maintained the stability at around 1.8 å and rmsd of caulerpin complexed with the protein are in the favorable range of within 1.2 å and has remained stable during the simulations. the backbone atoms of the complex and free receptor show similar rmsf, indicating the stability of the caulerpin inside 6lu7 receptor. also, the most number of conformations formed up to 10 hydrogen bonds during the simulation in caulerpin-6lu7 complex. a very few conformations showed less than 5 and greater than 10 hydrogen bonds. the bioactive caulerpin molecule was able to maintain strong interaction with the binding pocket of 6lu7 throughout the simulation period. finally, the caulerpin molecule showed the binding free energy inside 6lu7 equal to à256.44 ± 27.55 kj/mol which confirmed the efficacy of this molecule against sars-cov-2 main protease. hence, caulerpin is highly effective against sars-cov-2 main protease and can be explored further for drug repurposing against the successful inhibition of covid-19. hussein s. mohamed 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computational drug repurposing study key: cord-318854-xaus3bma authors: sun, yi; kurokawa, masahiko; miura, motofumi; kakegawa, tomohito; motohashi, shigeyasu; yasukawa, ken title: chapter 4 bioactivity and synthesis of diarylheptanoids from alpinia officinarum date: 2016-12-31 journal: studies in natural products chemistry doi: 10.1016/b978-0-444-63601-0.00004-1 sha: doc_id: 318854 cord_uid: xaus3bma abstract many diarylheptanoids were isolated from the galangal (rhizome of alpinia officinarum). we are reported the antitumor-promoting, antiinflammatory, cytotoxic, antiinfluenza virus, and antiherpes simplex-1 virus activities of diarylheptanoids. these components in our dietary and herbal supplements are considered to be relatively nontoxic to human. in addition, we synthesized the new compounds. in this review, we discuss the antitumor-promoting, antiinflammatory, cytotoxic, antimicrobial, antiinfluenza, and antiherpes simplex-1 virus activities of diarylheptanoids isolated from galangal resulting from our own and other research groups' investigation. alpinia officinarum hance (zingiberaceae), known as lesser galangal, is a wellknown medicinal herb distributed in east asia whose rhizomes are used for stomachic, analgesic, and antiemetic treatment [1] . the plant grows several feet tall and has long leaves and reddish-white flowers. alpinia officinarum is widely cultivated throughout china, thailand, india, sri lanka, malaysia, indonesia, saudi arabia, and egypt. the species is native to asia, australia, and the pacific islands, where it occurs in tropical and subtropical climates. alpinia is the largest genus of about 230 species in zingiberaceae. historically, its rhizomes possess stimulant and digestive effects owing to their spicy flavor and aromatic scent, and as a result, it is widely used in curries and perfumes throughout asia. although it was previously used throughout europe, its use has declined in recent years, and it is now mainly used in eastern europe. homoeopaths use it as a stimulant. the rhizomes of a. officinarum have been used in traditional japanese herbal prescriptions (kampo medicine) mainly for dyspepsia, vomiting, flatulence, stomach trouble, and diarrhea. the plant is also used in traditional chinese medicine as an aphrodisiac, abortifacient, carminative, antipyretic, antiinflammatory, and emmenagogue, as well as to treat disorders of the heart and kidneys, bronchitis, chronic enteritis, renal calculus, diabetes, and rheumatism. furthermore, its rhizomes are used in various asian cuisines (for example, in thai and lao tom yum and tom kha gai soups, vietnamese hu cuisine and throughout indonesian cuisine). the rhizomes contain numerous active constituents, including essential oils [2] , resin [3] , flavonoids [4] , diarylheptanoids [5] , and phenylpropanoids [6, 7] . as the main components distributed in this genus, diarylheptanoids possess cytotoxic, antiemetic, antiinflammatory, antivirus, and antiproliferative activities. the rhizomes of a. officinarum are an important medicinal herb recorded in chinese, korean, and japanese pharmacopoeias [8] [9] [10] . in this chapter, we discuss the biological activity of natural diarylheptanoids isolated from a. officinarum, as well as synthetic diarylheptanoids. diarylheptanoids are a major class of bioactive constituents in a. officinarum that are categorized into linear, cyclic, and dimeric diarylheptanoids, or diarylheptanoids bearing special moieties ( fig. 4 .1, table 4 .1). diarylheptanoids isolated from a. officinarum are mainly linear diarylheptanoids. diarylheptanoids 2-11 mostly possess a common structural moiety of 5-ene and 3-oxo or 3,5-dioxo groups on the heptane skeleton [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . the main types of linear diarylheptanoids (13-43) possess 5-hydroxy and 3-oxo groups in their structure [11] [12] [13] [14] [15] [16] [17] [18] [19] [22] [23] [24] [25] [26] [27] [28] [29] [30] . the other differences in structures lie in the pattern of substitution on aromatic rings. some diarylheptanoids (47) (48) (49) (50) (51) (52) (53) possess a common structural moiety of 3,5-dihydroxy on the heptane skeleton [12, 15, 17, 25, 29, 31 ]. in addition, compounds 1, 9, 12, and 44-46 are also linear diarylheptanoids that possess double bonds or ketone groups between c-1 and c-7 [15, 19, 32] . sun [14, [23] [24] [25] . several novel dimeric diarylheptanoids (58) (59) (60) (61) (62) (63) (64) were isolated from the rhizomes of a. officinarum [19, [32] [33] [34] . some dimeric diarylheptanoids were connected through cdc or cdodc bonds (58) (59) (60) (61) , and some were connected through the pyridine ring or six-numbered carbon ring (62) (63) (64) . both the pyridine and (5r)-5-(3,5,7-trihydroxyflavone)-7-(3-methoxy-4-hydroxyphenyl)-1-phenyl-3heptanone (officinin a) [35] six-numbered carbon rings are derived from the heptane unit of linear diarylheptanoids. liang et al. found that diarylheptanoid (65) has a novel skeleton bearing a flavonol moiety [35] . pharmacological research found that diarylheptanoids exhibited antiproliferative, cytotoxic, antiemetic, antiinflammatory, and antivirus activities. many related diarylheptanoids have been examined in order to study the structureactivity relationship (sar). several research groups have developed techniques for the detection, isolation, and identification of the components from a. officinarum. wong et al. isolated two diarylheptanoids (6 and 24) and two flavonoids using high-speed countercurrent chromatography (hsccc) from chloroform extracts [36] . ho et al. attempted to analyze the ethyl acetate extract directly by high-performance liquid chromatography (hplc) with photodiode array and electrochemical detection (hplc-ecd) techniques [37] . the hplc-edc method is also a very powerful tool for the detection of diarylheptanoid components at the nanogram level. furthermore, feng et al. discussed the separation and identification of diarylheptanoids in supercritical fluid extracts of a. officinarum using a uplc-q-tof-ms-ms system [38] . diarylheptanoids are widely distributed in nature. curcumin is the most common diarylheptanoid and possesses a range of bioactivities against human diseases, including antitumor, antiinflammatory, and antioxidant activities [39] . on the other hand, yashabushidiols were isolated from the male flowers of alnus sieboldiana (betulaceae) by hashimoto et al. [40] , and these compounds are also included in the diarylheptanoids from a. officinarum, such as compound (49) (fig. 4.2) . stereoselective synthesis of yashabushidiols and their derivatives has been reported by venkateswarlu's group, shinde's group, and yikang's group [41] [42] [43] . each group used a sugar or derivative as a starting material; for example, d-mannitol, d-glucose, and d-gluconolactone. after several steps, acetal compounds were obtained, and nucleophilic addition or wittig reactions then afforded yashabushidiols and their derivatives ( fig. 4.3) . we also synthesized yashabushidiols and their derivatives using kinetic resolution of sharpless asymmetric epoxidation reaction [44] (fig. 4.4) . first, 4-phenyl-1-butyne was treated with n-buli followed by addition of 3-phenylpropionaldehyde to a lithiated alkynyl solution. reduction of propargyl alcohol with red-al afforded the racemic allylic alcohol. sharpless epoxidation reaction of allylic alcohol gave enantio-enriched antiepoxy alcohol through kinetic resolution, followed by reduction using red-al to afford both natural and unnatural types of yashabushidiol b (49) . on the other hand, racemic allylic alcohol was treated with mcpba and preferentially generated syn-epoxy alcohol followed by reductions using cp 2 ticl 2 , zn, and zncl 2 to afford yashabushidiol a. compounds 35 and 32, and their enantiomers, were synthesized under almost the same conditions. compound 32 showed particularly strong antiviral activity against respiratory syncytial virus (rsv) in vitro and in vivo [44] . optically active compounds 35 and 32 were synthesized from racemic allylic alcohols according to the following procedure. first, kinetic resolution of optically active epoxy alcohol and chiral allylic alcohol was performed by sharpless asymmetric epoxidation (sharpless ae). chiral allylic alcohol was subjected to sharpless ae with l-dipt to afford the opposite configuration epoxy alcohol. these epoxy alcohols were then oxidized by dess-martin periodinane oxidation to afford optically active α-epoxy ketones. finally, compound 35 and its enantiomers were obtained by treating β-hydroxy ketone with meotf and 2,6-di-tert-butylpyridine; deprotection of β-hydroxy ketone by tbaf yielded compound 32 (fig. 4.5) . compound 50 was also isolated from a. officinarum. it resembled the structure of yashabushidiol b, which was synthesized by das et al. [45] . they also used kinetic resolution of sharpless ae reaction with the racemic allylic alcohol, followed by a ring opening reaction. the 3,5-dihydroxy compound was protected by 2,2-dimethoxypropane under acid conditions, followed by deprotection of the pmb group using ddq reagent. finally, the terminal hydroxyl group was oxidized by swern oxidation to afford aldehyde compound, and it was then subjected to wittig reaction followed by reduction using h 2 and pd on carbon to yield compound 50 ( fig. 4.6 ). alpinoids b (46) and c (45) were also isolated from a. officinarum by sun et al. [18] . both compounds have a unique moiety in the skeleton of the γ-hydroxy-α-enone carbon chain. generally, diarylheptanoids possess 3,5-diketo, 3-keto-5-hydroxy, or 3-keto-4-ene structures, but alpinoids b (46) and c (45) have a 3-keto-4-ene-5-hydroxy moiety. in addition, there is a chiral center at c-5, and its absolute configuration was determined as r by mosher's method. alpinoid c (45) and its analogues were synthesized by venkateswarlu's group and miura's group [46, 47] . the synthetic strategy of venkateswarlu's group is described below (fig. 4.7) . first, 4-phenyl-2-buten-1-ol was treated with (+) dipt, ti(oipr) 4 , and cumene hydroperoxide to afford chiral epoxy alcohol. after two further steps, epoxy alcohol formed chiral allylic alcohol followed by olefin metathesis coupling using grubb's second generation catalyst to afford alpinoid c (45) . on the other hand, miura et al. synthesized 45 using asymmetric 2,3-sigmatropic rearrangement of chiral α-sulfinyl enone ( fig. 4.8 ). chiral α-sulfinyl enone was readily synthesized from l-menthyl sulfinate [48] . chiral α-sulfinyl enone treated with catalytic amount of dbu and pph 3 , followed by oxidation with aqueous h 2 o 2 solution afforded target compound 45 in high enantiomeric excess. neuroblastoma is a common extracranial pediatric solid tumor, accounting for 10% of all tumors in the pediatric age group. the clinical presentation of neuroblastoma is variable and advanced cases are often found to be highly resistant to conventional treatment modalities based on surgery, chemotherapy, transplantation, and radiotherapy [49] . thus, development of new effective and safe therapeutic agents for the treatment of neuroblastoma is urgently needed. in a recent publication, we discussed the antitumor activity of naturally occurring compounds against neuroblastoma [50] . compounds 6, 24, and 30 exhibited the most potent activity against neuroblastoma imr-32 cells (table 4. 2), with ic 50 values of 0.11, 0.83, and 0.23 μm, respectively [17] , and were more potent than cisplatin (ic 50 : 0.85 μm). sun et al. found that diarylheptanoids containing the substituents of 3″-ome and 4″-oh on the benzene ring or only a carbonyl (c-3) and a double bond (c-4/5) at the aliphatic chain possessed potent cytotoxicity against the imr-32 cell line [17] . dose-dependent manner. they found that 5 induces s phase arrest and apoptosis via upregulation of activating transcription factor 3 (atf3) and stabilization of p53 in the shsy5y cell line [25] . compound 5 also exhibited potent cytotoxicity against hepg2, mcf-7, and sf-268 (atcc) human cancer cell lines (ic 50 : 6-10 μg/ml) [24] . furthermore, matsuda et al. also tested the inhibition of melanogenesis by 6, 21, 24, and 49 in theophylline-stimulated b16 melanoma 4a5 cells, and found ic 50 values of 10-48 μm. compound 6 showed the strongest activity among the four diarylheptanoids (ic 50 : 10 μm), and it also inhibited the mrna expression of tyrosinase, tyrosinase-related protein (trp)-1 and trp-2, as well as protein levels of microphthalmia-associated transcription factor (mitf) [52] . chronic inflammation may be a causative factor in a variety of cancers. the longer the inflammation persists, the higher the risk of cancer. in general, inflammatory leukocytes such as neutrophils, monocytes, macrophages, and eosinophils provide soluble factors that are thought to mediate the development of inflammation-associated cancer, including the cancer cells themselves, although other cells also participate. inflammatory mediators include metabolites of arachidonic acid, cytokines, chemokines, and free radicals. chronic exposure to these mediators leads to increased cell proliferation, mutagenesis, oncogene activation, and angiogenesis. emphasis will be placed on examining the role of the reactive oxygen (eg, o 2 − ) and nitrogen intermediates (eg, no), cytokines (eg, interferons, interleukins, tumor necrosis factor-α (tnf-α)), and prostaglandins (pgs). increased cancer incidence is associated with increased duration of inflammation. animal models have demonstrated experimentally that chronic inflammation can lead to the development of various forms of cancer, while providing further insights into possible mechanisms. skin tumors are induced by administration of carcinogens such as 7,12-dimethylbenz[a]anthracene (dmba), followed by repeated administration of tumor promoters such as 12-o-tetradecanoylphorbol-13-acetate (tpa) [53] . in recent publications, we discussed the inflammatory and tumor promotion, and its inhibitors from naturally occurring compounds [54, 55] . methanol extracts from the rhizomes of a. officinarum inhibited tumor promotion by tpa after initiation with dmba in icr mice [56] . fig. 4 .9a shows the percentage of tumor-bearing mice treated with dmba plus tpa was 80% at week 20, whereas that in the group treated with dmba plus tpa and methanol extract of a. officinarum was 20%. treatment with methanol extracts of the rhizomes of a. officinarum caused an 85% reduction in the average number of tumors per mouse at week 20 ( fig. 4 .9b). using bioassay-guided isolation, seven diarylheptanoids (2, 4, 6, 21, 24, 30, and 35) were isolated from active fractions of the methanol extracts of a. officinarum [56] . the inhibitory effects against tpa-induced inflammation closely paralleled those of the inhibition of tumor promotion in two-stage carcinogenesis initiated by dmba and tpa, a well-known tumor promoter, in a mouse skin model [57] . these diarylheptanoids inhibit tumor promotion in two-stage carcinogenesis in mouse skin. on the other hand, these diarylheptanoids inhibited tpa-induced inflammation in mice (table 4. 3). compounds 4 and 30 were similar in activity to indomethacin, an inflammatory drug [56] . the antiinflammatory mechanisms of diarylheptanoids have been reported by many researchers. matsuda et al. found that compounds 6, 21, 24, and 49 inhibited nitric oxide (no) production in lipopolysaccharide (lps)-activated mouse peritoneal macrophages on bioassay-guided isolation, and compound 6 showed particularly strong inhibitory activity with an ic 50 value of 33 μm [58] . bioassay-guided purification of ether extracts led to the isolation of a new diarylheptanoid (44) , as well as two known diarylheptanoids (2 and 17) . these compounds exhibited potent platelet-activating factor (paf) receptor binding inhibitory activity with ic 50 values of 1.3, 5.0, and 1.6 μm, respectively [16] . compound 15 also showed inhibitory and bactericidal activities against enteropathogenic escherichia coli (epec) clinical isolates and efficiently suppressed epec lps-induced inflammation in human peripheral blood mononuclear cells [59]. compound 6 exhibited antiinflammatory properties in a mouse macrophage cell line (raw 264.7) (6.25-25 μm) and suppressed lps-induced production of no, interleukin (il)-1β, and tnf-α by inhibiting nuclear factor-κb (nf-κb) activation and phosphorylation of p44/42 mitogen-activated protein kinases (mapks) [60] . compounds 2, 6, 15, 16, 47, and 48 were tested for their inhibitory effects on no production in the lps-activated macrophage cell line raw 264.7 [15] . compounds 6 and 2 showed potent inhibitory activities with ic 50 values of 0.6 and 6.8 μm, respectively. diarylheptanoid 3, isolated from the n-hexane extract, modulated nf-κb signaling involved in the inflammatory response, and inhibited lps-induced expression of tnf-α, il-1β, nitric oxide synthase (nos), and cyclooxygenase-2 (cox-2) at the gene level in raw 264.7 cells [21] . kiuchi et al. examined the effects of diarylheptanoids (6, 11, 13-15, 19, and 40) against pg and leukotriene. this suggested that compounds lacking the methoxy group adjacent to the phenol were less active than those possessing the methoxy group, presumably owing to the decrease in acidity from the phenol group [26, 28] . thus, the reports cited above suggest that diarylheptanoids possess inhibitory effects against inflammatory and tumor-promoting activities. diarylheptanoids exhibited antiviral activities against influenza virus [61] [62] [63] [64] [65] , rsv [44, 66] , poliovirus [44] , measles virus [44] , herpes simplex virus type 1 (hsv-1) [44] , human immunodeficiency virus (hiv) [67] , severe acute respiratory syndrome (sars) virus [68] , and epstein-barr virus in relation to carcinogenesis [69] . they are characterized as compounds possessing broad antiviral spectrum against dna and rna viruses. most diarylheptanoids possessing antiviral activities have been evaluated in vitro. antiviral activities in vivo have been documented for influenza virus and rsv using animal infection models [62, 65, 66] . sawamura et al. [61, 62] examined the antiinfluenza virus activity and cytotoxicity of 10 diarylheptanoids (2, 4, 6, 21, 24, 30, 32, 35, 36, and 49) isolated from a. officinarum by plaque reduction assay and mtt assay, respectively, using madin-darby canine kidney (mdck) cells (table 4 .4). in this study, influenza virus was more susceptible to 6 (ec 50 = 2.9 ± 0.3 μg/ml) and 32 (ec 50 = 0.7 ± 0.2 μg/ml) than to the others, and their therapeutic indexes (cc 50 / ed 50 ) were 11.7 and 114.3, respectively [61] . compound 32 has a 4-hydroxylphenyl moiety. platyphyllone and platyphyllone-5-xylopyranoside contain two 4-hydroxyphenyl moieties and have been reported to be active against influenza virus with therapeutic indexes of >10 [64] . thus, hydroxylation at the c-4 position of the phenyl moiety may be important for antiinfluenza virus activity in vitro. however, in a murine influenza virus infection model, 6 significantly reduced virus titers in bronchoalveolar lavage fluids of the lungs and prolonged survival times of the infected mice without toxicity, whereas 32 did not show this activity [62] . compound 6 possessed an unsaturated ketone and a methoxy group at the c-5 position of the 4-hydroxyphenyl moiety, but 32 did not. thus, the ketone and methoxy groups may be necessary for antiinfluenza activity in vivo. compound 6 exhibited antiviral activity against h1n1 virus, h3n2 virus, and b type virus, as well as oseltamivir-resistant h1n1 virus [62] . this indicates that the mode of antiinfluenza virus action of 6 was different from that of the known agents, such as oseltamivir, and suggests that it is a candidate antiviral compound against more virulent strains than the pandemic h1n1. in fact, 6 was shown to have no effect on virus adsorption or invasion into cells, instead suppressing the expression of viral messenger rna and antigens in infected mdck cells [62] . it is probable that 6 selectively suppressed influenza virus mrna synthesis in infected cells without cytotoxicity [62] . diarylheptanoids isolated from alpinia katsumadai such as katsumadain a and (e,e)-5-hydroxy-1,7-diphenyl-4,6-heptadien-3-one are reported to have inhibitory activity against the neuraminidase (na) of influenza virus (a/pr/8/34) in vitro [65] . some diarylheptanoids isolated from a. officinarum may therefore be effective in reducing na activity. konno et al. [34, 84] (table 4 .4). among these, 6 and 30 were not effective against the a2 strain of rsv [84] . the ec 50 values of 4 and 66 were 5.0 ± 0.0 and 7.0 ± 1.4 μg/ml (table 4 .4), respectively, and both compounds showed antiviral activity against the a2 strain of rsv with therapeutic indexes of 4.6 and 14.3, respectively, and were more potent than the other tested compounds [44, 66] . compound 4 was also active against influenza virus with an ec 50 value of <10 μg/ml [61] , which suggests that 4 is an effective diarylheptanoid against both rsv and influenza virus in vitro. compounds 22 and 22/32 were synthesized as an enantiomer and racemate, respectively, of 32, and compound 66 was synthesized as the enantiomer of 35 (fig. 4.10 ) [44] . the therapeutic indexes of stereoisomers (22, 32 , and 22/32) were similar (cc 50 /ec 50 : 1.3, 2.1, and 3.4, respectively) and their ec 50 values were 44.7 ± 1.5, 40.7 ± 3.5, and 24.3 ± 0.6 μg/ml (table 4 .4), respectively [44] . however, the therapeutic indexes of 35 and 66 (6.1 and 14.3) were higher than those of the three stereoisomers, with the ec 50 values of 16.3 ± 3.5 and 7.0 ± 1.4 μg/ml, respectively [44, 66] . the c-5 position of 35 and 66 is methoxylated with a saturated ketone at the c-3 position. compound 30 also contained a methoxy group with a saturated ketone and its ec 50 value was 13.3 ± 3.8 μg/ml [66] . thus, methoxylation in diarylheptanoids may contribute to anti-rsv activity in vitro. in a murine intranasal rsv infection model, compounds 22, 32, 22/32, and 66 were significantly effective in reducing virus titers, infiltration of lymphocytes and interferon-γ levels (marker of pneumonia severity) in the lungs of mice [44] . among these, 66 showed the strongest anti-rsv activity in mice, as shown by anti-rsv assay in vitro, and this may be a lead compound for the development of anti-rsv drugs in the future. six diarylheptanoids (2, 6, 21, 24, 30, and 35) were examined for their anti-rsv activity against poliovirus, measles virus, and hsv-1 by plaque reduction assay and trypan blue dye exclusion assay using vero cells (table 4 .4). among the six compounds, 6 and 30 did not exhibit any significant anti-rsv activity in hep-2 cells [84] . however, 30 exhibited relatively stronger antiviral activities against all three viruses (poliovirus, measles virus, and hsv-1), but 6 was only effective against measles virus. all the examined diarylheptanoids except 6 exhibited antipoliovirus activity with ec 50 values between 3.7 ± 0.6 and 44.3 ± 4.0 μg/ml, and all except 2 and 6 exhibited anti-hsv-1 activity with ec 50 values between 5.7 ± 0.6 and 58.7 ± 1.5 μg/ml (table 4 .4). all six examined compounds were significantly effective against measles virus with ec 50 values between 6.3 ± 0.6 and 47.0 ± 4.6 μg/ml. thus, diarylheptanoids appear to have a broad spectrum of antiviral activity. chareonkla et al. [67] reported that (3s,5s)-3,5-diacetoxy-1,7-bis(3,4,5-trimethoxyphenyl) heptane from zingiber mekongense exhibited anti-hiv activity in the antisyncytium assay using δtat/rev mc99 virus and 1a2 cell line system, but that it did not show activity on hiv-1 reverse transcriptase assay. hirsutenone isolated from alnus japonica has been shown to be a potent inhibitor of papain-like protease, which controls replication of the sars coronavirus. this compound is therefore thought to be a potential compound for the treatment of sars [68] . some cyclic diarylheptanoids isolated from acer nikoense and myrica rubra are reported to strongly inhibit epstein-barr virus early antigen activation in raji cells, and they also exhibit inhibitory activities on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test [69] . it is possible that some diarylheptanoids from a. officinarum possess antiviral activity against these viruses. obesity is a major health concern at present and is widely considered to be a global epidemic. conventional or allopathic medicines used to treat obesity have high abuse potential and frequently exhibit side effects. few botanicals included in natural weight loss products have been thoroughly researched on a basic or clinical level, and it is thus imperative to validate their widespread consumption for weight management. many natural weight loss products are sold and used globally with little or no proof of efficacy and quality, and concerns regarding safety have surfaced with good reason. orlistat inhibits pancreatic and gastric lipases that catalyze the hydrolysis of ingested fats, resulting in reduced dietary fat absorption and high fecal fat excretion [70] . it is certain that orlistat has a marked drug effect, as 35-45% of energy intake in western diets is attributed to dietary fat. however, malabsorption of fat leads to rather uncomfortable side effects, such as flatus with discharge, oil spotting from the rectum, fecal incontinence, fecal urgency, loose or liquid stools, and malabsorption of fat-soluble vitamins [70] . the water extract of rhizomes from a. officinarum showed the strongest inhibitory activity against pancreatic lipase in vitro. the extract was subsequently fractionated using organic solvents, and the ethyl acetate fraction had the strongest inhibitory activity. the most potent inhibitor in this fraction was identified as galangin-3-methyl ether (fig. 4.11) with an ic 50 value of 1.3 mg/ ml, as compared with the positive control (orlistat) with an ic 50 value of 0.8 mg/ml using triolein as the substrate. after oral administration of corn oil, triglyceride levels in mice decreased significantly to less than those in the control group by administration of h 2 o extract (1 g/kg) and the ethyl acetate fraction (0.5 g/kg). in triton wr-1339-induced hyperlipidemic mice, galanin-3-methyl ether significantly decreased triglyceride and cholesterol levels at a 20 mg/kg dose to 81.3% and 81.0%, respectively, while it increased high density lipoprotein (hdl) when compared with the control group. moreover, galanin-3-methyl ether did not show hypolipidemic activity in high cholesterol dietinduced hyperlipidemic mice [71] . compound 15 exhibited inhibitory activity against pancreatic lipase with an ic 50 value of 1.5 mg/ml using triolein as the substrate. the levels of serum triglyceride in corn oil fed mice were reduced significantly, while the levels of serum triglyceride and cholesterol were reduced in triton wr-1339-induced hyperlipidemic mice. hypolipidemic activity was not observed in the high-cholesterol diet-induced hyperlipidemic mice. inhibition of pancreatic lipase for both compounds when compared with the positive control (orlistat) was good in terms of other parameters, such as triglyceride, cholesterol and hdl levels. however, orlistat remained the most effective drug [72] . antiemetic drugs are effective against vomiting and nausea and may be used for severe cases of gastroenteritis, particularly if the patient is dehydrated. antiemetics can also be used for morning sickness, but there is little information about their effects on the fetus. some crude drugs inhibit vomiting among traditional japanese herbal prescriptions, while a. officinarum is used as an antiemetic in traditional chinese medicine. takahashi et al. reported that diarylheptanoids showed antiemetic activities in a copper sulfate (cuso 4 )-induced emesis assay in young chicks [12, 29] . the sar of diarylheptanoids was also investigated. among 14 diarylheptanoids, two types of essential functional structure showed inhibitory activities against emesis. diarylheptanoids 2, 4, 6, 21, 22, 24, 27 contained partial type a or b structures ( fig. 4.12) , and they therefore concluded that diarylheptanoids of types a and b might be the main antiemetic components of a. officinarum. 5α-reductase (reduced nicotinamide adenine dinucleotide phosphate: δ 4 -3-ketosteroid 5α-oxidoreductase), which is present as type 1 and type 2 isozymes in humans and rats, catalyze the reductive conversion of testosterone to 5α-dihydrotestosterone [73] . 5α-dihydroteststerone acts as a more active androgen than testosterone in many tissues, such as the prostate. therefore, inhibitors of this enzymatic conversion may be useful in the selective treatment of androgen-dependent diseases, such as benign prostate hyperplasia, male pattern baldness, and acne. most 5α-reductase inhibitors are steroids binding to the steroid receptors, and these steroids may produce various undesirable hormonal effects acting as agonists or antagonists. it has been reported that diarylheptanoids possess inhibitory activity against 5α-reductase. the sar of four diarylheptanoids (2, 22, 24, and 31) from a. officinarum was discussed [13] . it has been suggested that diarylheptanoids with unsaturated bonds, particularly the conjugated form in the alkyl structural moiety between the two aryl groups, had weak inhibitory activity, while diarylheptanoids with saturated counterpart bonds had potent inhibitory activity. thus, the alkyl parts of the diarylheptanoids appear to be important structural moieties for inhibitory activity against 5α-reductase. helicobacter pylori is a gram-negative, microaerophilic bacterium found in the stomach. it is also related to the development of duodenal ulcers and stomach cancer [74] . the eradication of h. pylori is effective in preventing duodenal ulcers and stomach cancer. lee et al. examined the inhibitory effects of compound 15 against h. pylori atcc 43504, atcc 700392, and atcc 700824 using the paper-disc diffusion and agar dilution methods [75] . rhizome-derived materials, particularly isolated diarylheptanoids, merit further study as potential antipylori functional food products or therapeutic products for preventing the diseases caused by h. pylori. zhang [19] . the ic 50 values of these compounds were 9-20 μg/ml and 25-47 μg/ml against hp-sydney and 1 hp-f44 strains, respectively. from bacteria to vertebrates, cells change their patterns of gene expression to respond to their microenvironment. in addition to classical dna microarray technology (transcriptome analysis), new analytical tools for high-throughput screening of whole transcriptome sequencing, polysome profiling (translatome analysis [76] ) or ribosome profiling (ribosome protection assay [77] ) have recently emerged and been applied to the analysis of gene expression (fig. 4 .13) [78] . in et al. reported that microarray global gene expression analysis showed 1′s-1′-acetoxyeugenol acetate (fig. 4.14) , a novel phenylpropanoid from alpinia conchigera, enhanced the apoptotic effects of paclitaxel in mcf-7 cells through nf-κb inactivation [79] . this suggests that the induction of tumor cell death through apoptosis is modulated through dysregulation of the nf-κb pathway. as genetic information transforms from dna to proteins, the cellular abundance of proteins is predominantly controlled at the translation level [80] . weak correlations between messenger rna (mrna) and protein levels [81] are observed because nontranslated mrnas may be present in rna granules, rna particles, processing bodies, stress granules, and mirna-risc (mirisc) complexes in cytosol (fig. 4.13) . analysis of the translatome can thus provide substantial and surprising new information [82] . whole free polysome and/or membrane-bound polysome analysis has also been applied. ribosome-associated mrnas (usually >3) are separated from mrnas associated with fewer ribosomes. these polysome-associated mrnas are applied to label probes on dna microarrays (translatome analysis) or are sequenced using next-generation sequencers (polysome profiling). these polysome-associated mrnas are then applied to ribosome-protection assay, and the resulting segments of rna are sequenced using next-generation sequencers (ribosome profiling). in these analyses, active and stalled ribosomes have been shown to cosediment during isolation of polysome complexes through sucrose gradients [83] , thus indicating that polysome profiling does not fully distinguish translationally active from repressed mrnas. diarylheptanoids 6, 24, and 30 inhibit proinflammatory mediators and exhibit cytotoxic and antiviral activities. however, the precise mechanisms of action and their effects on expression of specific genes are unknown. thus, we used translatome analysis to investigate the mechanisms and modes of action of these diarylheptanoids [84] . polysome-associated mrnas were prepared from diarylheptanoid-treated and control cells from a human b lymphoblastoid cell line; these mrna samples were then used for microarray analysis. the number of downregulated inflammatory-related transcripts was ranked as follows: 30 > 24 > 6. compound 6 showed greater influence on the translatome of bjab cells, while 24 showed less efficacy, except when upregulating the expression of genes related to rhodopsin-like gpcrs, mrna processing, and proteasomerelated proteins of wikipathways [85] . it is possible that the same host factors, such as splicing factors or hnrnps listed in mrna processing wp411 45374 (wp; wikipathways), affect virus structure and/or replication. sixteen transcripts were upregulated after treatment with 6, 24, or 30. among these, transcripts of heterogeneous nuclear ribonucleoprotein c (c1/c2), heterogeneous nuclear ribonucleoprotein k, non-pou domain containing, octamer-binding, and polypyrimidine tract-binding protein 1 were identified as internal ribosome entry site trans-acting factors. all of these studies have provided new insights into the mode of action of diarylheptanoids from a. officinarum with regard to its antiinflammatory, antitumor promotion, and antiviral effects. humans have used plants as foods and natural medicines since ancient times, and while they are crude drugs, are typically safer than synthetic drugs, and have been used as both spices and supplements. several active components have been isolated, and their chemical structures have been and continue to be determined. the diarylheptanoids of the rhizomes of a. officinarum are considered to be a particularly promising group of compounds. diarylheptanoids are minor but ubiquitous components in our diet and have the advantage of being nontoxic or relatively nontoxic to humans. natural diarylheptanoids have multiple physiological functions, including antiinflammatory, antitumor, cancer preventive, antiviral, antiemetic, and anti-pylori effects. challenges that must be overcome in order to find functionally useful compounds that can be applied clinically are further screening of natural diarylheptanoid compounds, examination of sars, elucidation of physiological action mechanisms, and the problems associated with supplying large quantities of compounds. in order to resolve these issues, collaboration between researchers in various fields will be necessary. activator protein-1 atf3 activating transcription factor 3 bjab the human b-lymphoma cell line encyclopedia of herbs & their uses studies in natural products chemistry chinese pharmacopoeia of the people's republic of china the korea food and drug administration notification, korean pharmacopoeia the japanese pharmacopoeia drug discovery research in pharmacognosy drug discovery and development -from molecules to medicine key: cord-328834-yetnlb2j authors: mohsin, noor ul amin; irfan, muhammad; hassan, shams ul; saleem, usman title: current strategies in development of new chromone derivatives with diversified pharmacological activities: a review date: 2020-06-15 journal: pharm chem j doi: 10.1007/s11094-020-02187-x sha: doc_id: 328834 cord_uid: yetnlb2j chromone derivatives possess a spectrum of biological activities. chromone has been recognized as a privileged structure for new drug invention and development. substitution pattern of chromone scaffold determines different type of biological activities. the type, number and position of substituents connected to the chromone core play a vital role in determining pharmacological activities. in the present review, we have discussed new chromone derivatives as anticancer, anti-diabetic, antimicrobial, anti-inflammatory, antioxidant and as anti-alzheimer agents. this review deals with the chromone derivatives prepared by combining chromone molecule with various natural and synthetic pharmacophores and pharmacological activities presented by them. the main aim is to highlight the diversified pharmacological activities exhibited by chromone hybrid molecules during the last eight to ten years. chromone is a heterocyclic compound containing oxygen as heteroatom and has a benzo-g-pyrone skeleton (fig. 1, compound 1 ). chromone is a natural molecule present in the diet of human and animals and shows less toxicity to mammalian cells [1] . chromone containing natural and synthetic molecules displayed interesting biological activities [2] . medicinal properties exhibited by chromone derivatives are antibacterial, antifungal, antioxidant, antimalarial, neuroprotective and hiv inhibitory potential [3 -8] . chromone derivatives have also shown promising anticancer and antiviral potential [9 -13] . anti-inflammatory, antiallergenic and antiulcer are other properties displayed by chromone derived molecules [14 -16] . chromone is treated as an attractive source for the synthesis of new drugs due to its valuable activities and low toxicity [17] . chromone is considered as a single molecule which can combine with different types of receptors [18] . modifications of chromone scaffold have been performed at benzene or pyrone ring by attachment of different substituents. 3-formylchromone (fig. 1, compound 2) is a frequently used precursor for the synthesis of chromone derivatives and can be prepared easily by the vilsmeir -haack reaction [19] . chromone derived molecules have been discussed with respect to chemical structures, pharmacological activities and structure -activity relationship (sar) in following sections. cancer is a serious problem all around the world due to high mortality and there is demand to discover new leads as anticancer agents [20] . a number of anticancer drugs have been discovered from natural sources [21] . chromone derived molecules have displayed excellent anticancer activities. amin and co-workers synthesized new molecules by merging benzofuran and 5h-furo [3,2g] chrome-5-one and attached different heterocyclic rings through sulfonamide group. these compounds displayed in vitro activity against breast cancer cell line (mcf-7) ranging from 0.004 -0.87 mm. compound 3 (fig. 2) presented prominent in vitro activity (ic 50 = 0.056 ± 0.0027 mm) against mcf-7 cell line in comparison to standard drug (doxorubicin, ic 50 = 0.62 ± 0.0316 mm) and proved less toxicity to normal cell line (ic 50 = 23 ± 1.02 mm). these derivatives also showed p38a mapk (mitogen-activated protein kinase) inhibition activity. mapk controls many biological functions such as cell growth, differentiation and inflammation [22] . molecular docking studies showed that compound 3 formed four hydrogen bonds with k-53, m-109 and g-170 amino acids of mapk [23] . singh, et al. attached indole, pyrimidine, pyrazole with chromone to produce new derivatives. it was observed that introduction of 2,6-dichlorophenyl, 2,6-dichlorobenzoyl group along with indolinone produce notable activities. compound 4 manifested prominent in vitro antitumor activity with 50 -90% growth inhibition of all tumor cell lines and showed an average gi 50 value of 3.2 mm. compound 4 was more potent against leukemia (rpmi-8226 gi 50 [24] . synthesis of chromone and sulfonamide comprising molecules was carried out by awadallah, et al. in which two molecules were linked to each other by a large heterocyclic ring or by small linker groups such as methine amine or alkyl amine. compound separated by small linker group dispensed greater activity. upon in vi-tro evaluation, compound 5 emerged as the most active against breast (mcf-7, ic 50 = 0.72 mm) and lung (a-549, ic 50 = 0.50 mm) cancer cell lines as compared to doxorubicin (mcf-7 ic 50 = 33.13 ± 2.90 mm, a-549 ic 50 = 26.81 ± 2.50 mm). compound 5 presented selectivity for isoforms ix and xii of the human carbonic anhydrase (hca). this compound induced apoptosis in both types of cancer cell. it was also observed that compounds having free sulfonamide group presented higher activity. when the sulfonamide group was attached with heterocyclic scaffold such as pyridine, pyrimidine, and isoxazole, less active derivatives were obtained [25] . chen, et al. attached chromone molecule to 1-alkyl-1h-imidazole-2-yl via dienone as linker group. the nitrogen-containing heterocycles were used as bioisostere for phenols in the natural compound while the dienone linker was used as substitute for dienone in curcumin. compound 6 presented excellent activity against prostate cancer (pc-3, ic 50 = 1.8 ± 0.3 mm and lncap ic 50 = 1.0 ± 0.2 mm) cell lines. the nitrogen atom of imidazole carries ethyl group. replacement of ethyl group by longer chain has no significant influence on anticancer activity. therefore they are excellent molecules for future investigations [26] . dolatkhah, et al. used the three-component reaction involving chromone-3-carboxaldehyde, alkyl acetoacetate, urea or thiourea to produce 4h-chromone-1,2,3,4tetrahydropyrimdine-5-carboxylates using mcm-41-so 3 h nanoparticles as catalyst. the catalyst can be recycled and reused. compound 7 presented prominent activity against leukemia cell line upon evaluation by microculture tetrazolium test (mtt) assay. this compound showed no toxicity to normal cell line human foreskin fibroblast (hu02). compound 5 showed high affinity (binding energy = -10.10 kcal/mol) with ab1-kinase enzyme by autodock-4 program [27] . nam, et al. developed chromone derived analogues of lavendustin. upon anticancer evaluation, compounds 8 (ic 50 = 6.01 ± 2.7 mm) and 9 (ic 50 = 9.92 ± 3.6 mm) showed prominent activities against a-549 cell line. compound 8 (ic 50 = 6.89 ± 2.6 mm) and 9 (ic 50 = 7.86 ± 2.2 mm) also showed activity against hct-15 cell lines. in compound 8, replacement of 4-methoxybenzyl with benzyl or phenethyl decreased the activity. in compound 9, replacement of 4-nitrobenzyl with 4-methoxybenzyl or benzyl group produced less active compounds against hct-15 cell line [28] . bhatia and co-workers synthesized chalcone-chromenone derived molecules. upon in vitro evaluation, compound 10 ( fig. 3) showed prominent activity (87% growth inhibition) against colon cancer cell line (hct-116) as compared to fluorouracil (67% inhibition). compound 10 carries two halogen atoms each on the chromone core and chalcone fragment [29] . ozen, et al. conjugated chromone molecule with 5-membered heterocyclic scaffolds thiazolidindiones, imidazolidindiones and thiohydantoins to produce new hybrid molecules. compound 11 showed prominent activity against liver (huh-7 ic 50 = 5.2 mm) and breast cancer (mcf-7, ic 50 = 4.9 mm) cell lines. this compound contains unsubstituted chromone while ethyl group is attached with thiohydantoin. when the chromone ring is substituted with methyl group, resultant compound showed less activity. compound 11 did not show apoptosis but only reduced the replication of cells [30] . singh and co-workers synthesized chromenopyridine derivatives based on the activity of chromones and pyridones. derivatives bearing electron withdrawing groups at positions # 5, # 6 and # 7 of chromone scaffold showed better activity. compound 12 appeared as the most potent against pc-3 (ic 50 = 2.4 ± 3.4 mm), mcf-7 (ic 50 = 10.7 ± 2.5 mm) and hela (ic 50 = 7.0 ± 3.5 mm) cancer cell lines. this compound contains allyl group attached to pyridone and showed better activity as compared to unsubstituted compounds [31] . obreque-balbua and coworkers synthesized chromone derivatives as abcc1 modulators. an amino or carboxamide group was introduced at position # 2. abcc1 overexpression is detected in different types of cancers. compound 13 was the most effective (ic 50 = 11.3 ± 1.8 mm) in reducing the abcc1 mediated resistance in comparison to reversan (ic 50 = 4.3 ± 0.2 mm). derivative 13 showed selectivity for abcc1 over abcg2 and abcb1. it consists of chromone molecule connected to benzo [1, 2, 5] oxadiazole via piperazine linker group. insertion of carbonyl group between chromone and piperazine decreased the activity [32] . abdelhafez at al. synthesized benzofuran derivatives as vascular endothelial growth factor (vegf) inhibitor. bromovisnagin (14) was an intermediate product and consist of chromone core in its structure. bromovisnagin (14) showed prominent anticancer (ic 50 = 3.67´10 -7 -7.65´10 -13 mm) potential as compared to other products in this series. compound 14 was the most potent agent against mcf-7 (ic 50 = 3.67´10 -7 ) as compared to standard drug epirubicin (ic 50 = 2.22´10 -9 ). docking studies of compound 14 with vegfr2 (vascular endothelial growth factor receptor) kinase enzyme showed hydrogen bonding between furan oxygen and amino group of d1046. methoxy group showed interaction with nh moiety of k868 [33] . chand and colleagues synthesized substituted chromone, 4-oxo-4h-1-benzopyran derivatives as anticancer agents. compounds having acrylate group at position # 3 demonstrated prominent activity. derivatives having amide and acid group at this position proved less active. compound 15 was the most active (50 -60% inhibition) agent against colon (ht-29), breast (sk-ov-3) and ovarian (mda-468) cell lines as compared to doxorubicin (80% inhibition). compound 15 did not show in vitro src kinase inhibitory activity (ic 50 < 300 mm) as compared to staurosporin (ic 50 = 0.6 mm) [34] . han and colleagues synthesized succinimide derivatives by c-h functionalization of chromone, naphthoquinone and xanthone scaffolds with maleimide scaffold. chromone derivatives (16a, b, c) showed only minute activity (ic 50 < 50 mm) versus mcf cell line [35] . el-garah, et al. synthesized chromone carboxamide derivatives. compound 17 presented prominent activity (ic 50 = 0.9 mm) against mcf-7 cancer cell line in comparison to tamoxifen (ic 50 = 0.39 ± 0.01 mm). structure activity relationship (sar) studies highlighted that attachment of fluorine atom at position # 6 of chromone core produced more active derivatives. replacement of fluorine atom by chloro and methyl groups produced less active derivatives. the presence of propyl side chain at the amide group also increased the activity. compound 18 also presented in-vitro anti-inflammatory activity (79.9 ± 6.6% inhibition) as lipoxygenase (lox) inhibitor. derivatives having hydrophilic group showed better anti-inflammatory activity [36] . ali, et al. synthesized chromone annulated phosphorous heterocycles as anticancer agents. phosphorous reagents used in the synthesis were phosphorous halides, phosphorous sulfides and phosphonic acid. anticancer activity was evaluated by using crystal violet blue assay. compounds 19 and 20 exhibited prominent activities against hepg-2 (ic 50 = 1.61 mg/ml, ic 50 = 2.49 mg/ml) and hct-116 (ic 50 = 1.72 mg/ml, ic 50 = 1.56 mg/ml) as compared to doxorubicin (hep-g-2 ic 50 = 0.467 mg/ml, hct-116 ic 50 = 0.468 mg/ml). the thiophosphoryl (p=s) bond was described as major cause of cytotoxicity of these compounds as compared to phosphoryl group (p=o) [37] . sun, et al. carried out synthesis of thiopyrano [4,3-d] pyrimidne derivatives having chromone molecules. compound 21 displayed significant inhibition of mtor (ic 50 = 1.10 ± 0.10 mm) and p13ka (ic 50 = 0.92 ± 0.12 mm) kinases. compound 21 (fig. 4 ) displayed marvelous in vitro activity against mcf-7 (ic 50 = 11.8 ± 6.9 mm), hela (ic 50 = 10.3 ± 0.58 mm) and hepg2 (ic 50 = 8.77 ± 0.83 mm) cancer cell lines as evaluated by mtt assay. docking investigation with mtor and p13ka kinases highlighted that morpholine, chromone and hydrazinyl groups are essential for antitumor activities. substitution on the chromone nucleus manifested significant influence on activity. introduction of carboxylic group on the chromone core proved more active as compared to methyl, ethyl and hydroxyl group [38] . abu-aisheh, et al. synthesized amidrazone derivatives having flavone scaffold. upon evaluation as anticancer agents by mtt assay, compounds 22 (ic 50 = 1.42 ± 0.13 mm) and 23 (ic 50 = 2.92 ± 0.94 mm) showed prominent in vitro activity versus breast cancer cell line (t47d) as compared to doxorubicin (ic 50 = 0.33 ± 0.05 mm). replacement of piperazine ring by piperidine produced less active agents against breast cancer. derivatives having phenyl group at position # 2 presented higher activity as compared to methyl group. these compounds can be good candidates as anticancer agents [39] . singh, et al. synthesized chromone and isoxazolidine based compounds by regioselective and stereoselective 1,3-dipolar cycloaddition reaction. compound 24 (ic 50 = 0.7 mm) exhibited prominent in vitro activity against a549 cell line in comparison to paclitaxel (ic 50 = 0.4 mm). this compound bears disubstituted isoxazolidine scaffold. this compound also induced apoptosis in hl-60 cancer cell line [40] . satyajit singh and co-workers synthesized chromano piperidine fused isoxazolidine molecules. derivatives having electron withdrawing groups at the chromone core presented superior activity. compounds having aromatic ring attached with isoxazolidine presented better activity. compound 25 showed prominent in vitro cytotoxicity against colo (ic 50 = 12.6 mm) as compared to fluorouracil (ic 50 = 21 mm). compound 25 also exhibited moderate activity against imr-32 (ic 50 = 55.2 mm) as compared to adriamycin (ic 50 = 1.7 mm). compound 26 (ic 50 = 10.7 mm) presented prominent activity against neuroblastoma (imr-32) cell line [41] . zwergel and co-workers synthesized chromone and coumarin based benzofuran derivatives. biological evaluation on human leukemia (k562) cell line was carried out. compounds 27 and 28 exhibited prominent activities having 72% and 63% cell survival respectively by using 50 mm concentrations. compound 27 showed 24% apoptosis. substitution on chromone nucleus does not have significant influence on activity. replacement of benzofuran by naphthofuranone produced more active compound towards apoptosis [42] . zhu, et al. designed and synthesized thienopyrimidine and chromone derived compounds as mtor/p13ka inhibitors [43] . compound 29 (97.2 ± 0.4% inhibition) presented prominent activity against mtor/p13ka kinase. derivative 29 also manifested notable in vitro activity versus h460 (ic 50 = 1.2 ± 0.3 mm) and pc-3 (ic 50 = 0.85 ± 0.04 mm) cell lines in comparison to positive control (ic 50 = 9.52 ± 0.29 mm, ic 50 = 16.27 ± 0.54 mm against h-460 and pc-3). chromone molecule having carboxylic acid or nitro group was found to be optimum for the activity in this series. docking studies revealed that chromone molecule is vital for activity. carboxylic group of chromone interacted via hydrogen bonding with trp-2239 and arg-2348. morpholine group formed hydrogen bonding with val-2240 [44] . diabetes mellitus is caused by the elevated level of glucose in the blood due to improper production of insulin. it is a very common problem all around the world [45] . currently available anti-diabetic drugs are linked with various side effects [46] . anti-diabetic properties of chromone derivatives are described in this section. ceylan-unlusoy, et al. attached chromone scaffold with 2,4-imidazolidindione and 2,4-thiazolidindione. attachment of heterocyclic rings at position # 3 produced more active derivatives as compared to position # 2. compounds 30 (142.7 ± 17.60%) and 31 (155.4 ± 35.16%) (fig. 5 ) showed prominent in vitro insulinotropic potential with dose of 1 mg/ml as compared to standard drug glibenclamide (138 ± 13.99%). incorporation of methyl and ethyl group in the heterocyclic ring showed comparable activity to unsubstituted derivatives [47] . later on, ceylan-unlusoy, et al. synthesized chromone derivatives having imidazolidindione, 2,4-thiazolidindione and 2-thioxoimidazolidine-4-one heterocyclic cores. derivatives having substituted 2,4-thiazolidinione ring demonstrated better activity and the most active agent carries methyl group at heterocyclic nitrogen. therefore lipophilic groups at the heterocyclic nitrogen were found significant for increasing the activity. compound 32 (dose = 0.001 mg/ml) was the most prominent which increased the release of insulin (120.6 ± 13.53%) as compared to glibenclamide (145.7 ± 7.74%) [48] . wang, et al. linked chromone to the benzene sulfonamide through hydrazone linkage to produce new a-glucosidase inhibitors. the a-glucosidase enzyme is involved in the hydrolysis of carbohydrates and releases glucose. compound 33 showed excellent in vitro inhibition (ic 50 = 20.1 ± 0.19 mm) of this enzyme in comparison to acarbose (ic 50 = 817.38 ± 6.27 mm). compound 33 carries a free sulfonamide group attached to the phenylhydrazone. replacement of phenylhydrazone by benzohydrazide and benzene ring by thiophine ring produced less active molecules. compound 33 displayed non-competitive inhibition as determined by lineweaver-burk plot. in molecular docking studies, hydrogen bonding was observed between the drug molecule and asp-214 and arg-439 of enzyme [49] . one year later, wang, et al. synthesized hybrid molecules comprising chromone-isatin as a-glucosidase inhibitors. these molecules exhibited excellent in vitro blockage of this enzyme and compound 34 (ic 50 = 3.18 ± 0.12 mm) appeared as the most potent in this series. substitution on the chromone core was very important where introduction of hydroxy group significantly enhances the activity. molecular docking studies also showed interaction (binding energy = -8.8 kcal/mole) of this compound with the glucosidase enzyme. 4-bromophenyl moiety showed hydrophobic interactions with phe-157 and phe-177, ala-278 and phe-300. benzopyrone formed ð-ð interaction with phe-300. hydroxyl group formed hydrogen bonding with gln-350 [50] . alpha-amylase is an enzyme involved in the hydrolysis of carbohydrates. salar, et al. connected chromone to hydrazinyl thiazole to produce the new hybrid molecules. upon in vitro evaluation as the a-amylase inhibitors, compound 35 showed prominent inhibition (ic 50 = 2.826 ± 0.06 mm) of this enzyme in comparison to acarbose (ic 50 = 1.9 ± 0.07 mm). compound 35 showed a docking score of -7.1717 and p-p interactions with trp-59 of a-amylase. compound 35 also exhibited dpph (2,2-diphenyl-1-picrylhydrazyl) (ic 50 = 1.13 ± 0.15 mm) and abts (2,2 / -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ic 50 = 1.083 ± 0.15 mm) radical scavenging activities. compound 35 bears unsubstituted phenyl ring with thiazole. replacement of phenyl group with biphenyl core also produced more active compounds. incorporation of hydroxyl group at the aromatic ring decreased the activity. dichloro-substituted derivatives at the aromatic ring produced more active compounds as compared to mono-substituted compounds. attachment of methyl group with chromone core also produced less active derivatives [51] . parthiban, et al. synthesized quercetin based chromone derivatives as alpha-amylase inhibitors. various modifications in the chromone core of quercitin were carried out such as introduction of furan and indole ring. compounds 36 (ic 50 = 13 ± 0.16 mm) and 37 (ic 50 = 12 ± 0.14 mm) presented prominent in vitro activities as alpha amylase inhibitor as compared to quercitin (ic 50 = 22 ± 0.01 mm). in these derivatives chromone core is attached with indole ring. derivatives having aliphatic side chains at the indole nitrogen presented higher activity as compared to aromatic rings. molecular docking studies showed that chromone core forms hydrophobic interaction with trp58, tyr62 and leu165. diethylamino group of compound 37 interacted via hydrogen bonding as well as hydrophobic interaction with glu233 and ile235. these derivatives have the potential for further investigations as anti-diabetic agents [52] . parasites such as bacteria, viruses, fungi, protozoa, trypanosomes, etc. cause various diseases in human beings and are responsible for high mortality and morbidity [53] . ate activity (10 -12 mg/ml) against sensitive h37rv as compared to ethambutol (2 mg/ml) and streptomycin (2 mg/ml). it was assumed that these compounds act on the lipoprotein part of the cell wall of mycobacterium tuberculosis [62] . cano, et al. synthesized fluorine-containing chromone and tetrazole hybrid molecules by ugi-azide reaction [63] these derivatives displayed moderate antimicrobial activity as is evident by compound 51 (mic = 20 mg/ml) activity versus pseudomonas aeruginosa (p. aeruginosa) as compared to cefotaxime (mic = 0.5 mg/ml). compound 52 also displayed low antiamoebic activity (ic 50 = 61.7 mg/ml) as compared to metronidazole (ic 50 = 1.5 mg/ml). compound 53 exhibited antifungal activity against sporothrix schenckii (s. schenckii) at the concentration of 6.25 mg/ml. introduction of halogen atoms at the chromone core produced more active derivatives as compared to nonhalogenated derivatives [64] . reddy, et al. produced the fused hybrid molecules by reacting 2-amino chromone, benzaldehyde, 1,4-dimedone to produce chromeno-tetrahydroquinoline. antibacterial activity was tested against p. aeruginosa, s. aureus, e. coli and bacillus subtilis (b. subtilis). in these derivatives, tetrahydropyridine is embed-ded in dimedone and chromone scaffolds. agar well diffusion method was used for assessing antibacterial activity. compounds 54, 55 and 56 were most active derivatives (zoi = 22 -37 mm) as compared to streptomycin (zoi = 25 -40 mm) against these bacteria. derivatives bearing small alkyl chains demonstrated better activity. introduction of cyclic rings e.g. cyclopropyl, cyclobutyl and cyclopentyl at this position produced less active derivatives [65] . pouramiri, et al. showed excellent activity (100%) against meliodogyne incognita by using concentrations 10 mg/ml and 25 mg/ml. standard drug tioxazafen exhibited 100% inhibition at 25 mg/ml and 92.9% at 10 mg/ml. sar studies revealed that incorporation of methyl, ethyl and phenyl groups in the linker chain produced less active derivatives. attachment of trifluoromethyl group at position # 3 of pyrazole scaffold produced more active derivatives as compared to bromide, cyanide and methyl groups [68] . siddiqui, et al. synthesized prazolyl pyranopyridines derivatives by the reaction of 3-acetoacetyl benzopyranopyridone with different hydrazine derivatives. benzopyranopyridones contain condensed chromone ring and are the intermediate products in this series. among these derivatives, compound 65 (fig. 8 ) manifested prominent activity against salmonella typhi (s. typhi, zoi = 12 mm), salmonella dysenteriae (s. dysenteriae, zoi = 19 mm) and klebsiella pneumoniae (k. pneumoniae, zoi = 18 mm) as compared to chloramphenicol (zoi = 16 mm, 23 mm, 23 mm against these bacteria) [69] . batula, et al. synthesized aryl isoxazole and 6-fluorochromone carboxylate derivatives in which both these scaffolds were linked by ester linkage. the introduction of aryl groups at the isoxazole core enhanced the antibacterial activity. compound 68 showed activity against a. niger (mic = 18.75 mg/ml) and c. albicans (mic = 18.75 mg/ml) as compared to amphotericin b (mic = 1.562, 6.25 mg/ml respectively against these microbes). introduction of halogen-substituted phenyl rings significantly increased antifungal activity. compounds 66 and 67 were most active against b. subtilis having mic value of 9.375 mg/ml for each compound as compared to streptomycin (mic = 6.25 mg/ml) [70] . hessein and co-workers synthesized furochromone, furocoumarin and benzofuran derivatives having sulfonamide group. upon in vitro evaluation, compound 69 exhibited antimicrobial potential against e. coli (zoi = 20 mm) and aspergillus ochraceus (zoi = 19 mm). compound 70 (zoi = 19 mm) was most prominent against e.coli as compared to chloramphenicol (zoi = 38 mm). attachment of aminophenyl and biphenyl ring to the sulfonamide group significantly improved the antibacterial activity. addition of benzotriazole or biphenyl acetamide group did not improve the antimicrobial activity [71] . aggarwal, et al. synthesized 1,2,4-dithiazolyl derivatives as antimicrobial agents. compound 71 showed prominent activity against b. subtilis (mic = 0.78 mg/ml), s. cerevisiae (mic = 6.25 mg/ml) and c. albicans (mic = 3.12 mg/ml) as compared to fluconazole (mic = 1.9 mg/ml, 3.9 mg/ml versus s. cerevisiae, c. albicans). compound 72 presented excellent activity against e. coli (mic = 1.56 mg/ml) and b. subtilis (mic = 1.56 mg/ml) as compared to gentamicin (mic = 0.9 mg/ml and 0.75 mg/ml against e. coli and b. subtilis). sar investigation declared that substitution of the phenyl ring at 1,2,4-dithiazolyl ring is very important for antimicrobial activity and addition of the halogen atom at para-position significantly improves the activity. addition of electron-withdrawing group e.g. chlorine and bromine at position # 6 and # 7 of chromone scaffold also enhances activity while attachment of electron-donating group e.g. methyl lowers the activity [72] . gadhave, et al. synthesized fluorine containing pyrazolone derivatives having chromone molecule by a knoevenagel condensation reaction [73] . compound 75 (fig. 9) showed prominent activity against streptococcus pyogenes (mic = 62.5 mg/ml) in comparison to ampicillin (mic = 100 mg/ml). compounds 76 was most active against s. aureus having mic value of 62.5 mg/ml. compound 77 was most active against p. aeruginosa (mic = 62.5 mg/ml) in comparison to ampicillin (mic = 250 mg/ml). it was found that addition of propyl and trifluoromethyl group at the pyrazole ring produced potent derivatives against gram-positive bacteria [74] . knoevenagel-cope condensation reaction was also used by bari, et al . different concentrations were used for testing antibacterial activity and no direct relationship was observed between concentration and antibacterial activity [77] . badhade, et al. synthesized chromone and isoxazole conjugates without any linker group. upon in vitro evaluation as antibacterial agents, these derivatives exhibited excellent activity. compounds 82 and 83 (fig. 9 ) exhibited notable activities against b. subtilis and s. aureus presenting mic values of 10 mg/ml equivalent to standard drug. incorporation of halogen and alkyl groups in the chromone ring produced more active compounds [78] . chromone scaffold was attached with 1,2,3-triazole by dofe and co-workers to produce new derivatives. compounds 84 and 85 exhibited prominent antifungal activities (mic = 12.5 mg/ml) against c. albicans for both compounds as compared to miconazole (mic = 12.5 mg/ml). derivatives 84 and 85 showed prominent antibacterial activity (mic = 25 mg/ml) against b. noor ul amin mohsin et al. the process of inflammation is related to various diseases. nonsteroidal anti-inflammatory agents are frequently used as remedy of inflammation but are associated with the major adverse effect of gastrointestinal ulceration [82] . therefore the development of new anti-inflammatory drugs having good safety profiles is necessary. chromone derivatives have also presented anti-inflammatory activities. tnfa (87% inhibition) as compared to standard drug dexamethasone (71 and 84% inhibition of tnfa and il-6 respectively). in compound 97, piperazine and chromone scaffolds are separated by methylene group and piperazine is further attached to a pyrimidine. replacement of pyrimidine ring by pyridine, p-methoxyphenyl ring produced equipotent derivatives. substitution of pyrimidine ring by methyl, ethyl and acetyl groups produced less active derivatives [85] . alzheimer's disease (ad) is a neurological ailment mostly among the elder people and is indicated by memory loss and dementia [86] . chromone core has been attached with various other pharmacophores to produce new multifunctional agents. natural compounds have potential to inhibit some toxicities of alzheimer disease [87] . liu, et al. attached chromone-2-carboxamide with alkylbenzylamines as anti-alzheimer agents. compound 98 (fig. 11 ) displayed excellent in vitro acetylcholinesterase (ache) inhibition (ic 50 molecular modeling investigation of this derivative also showed interaction with the active site of ache. the presence of amino group at position # 2 of chromone ring forms additional hydrogen bonding with tyr-510 and gly-523 of ache. benzene ring of chroman fragment showed p-cation interaction with arg-522. the increased activity was also linked to the extra amino functionality in the chromone ring [18] . hybrid molecules of 4-oxo-4h-chromene and tacrine were synthesized by fernandez-bachiller and colleagues in which cholinesterase inhibitory property of tacrine and antioxidant property of chromone were combined. alkylene diamine was used as linker between these two pharmacophores and a chain of ten carbon atoms was found optimal for best activity. hybrid proved more potent than parent drug tacrine but showed inhibition of ache and buche enzymes. 2-amino-4-methyl phenol group occupied the hydrophobic pocket formed by leu-171, ile-198 and ile-199 [90] . mackhaeva, et al. synthesized 2-vinyl chromone derivatives as selective butyryl cholinesterase (buche) inhibitors. derivatives having n-benzyl and n-vinyl methoxy carbonyl substituted compounds presented more selectivity for buche. in this series, compound 104 appeared as the most potent inhibitor (ic 50 = 2.27 ± 0.18 mm) of buche in comparison to tacrine (ic 50 = 0.0290 ± 0.0002 mm). compound 104 carry bromine atom at position # 6 of chromone core. introduction of ethoxy group at position # 8 of chromone core produced less active derivatives. molecular docking studies showed that methoxy carbonyl group interacted with anionic site formed by gly116, gly117 and ala199. the n-benzyl group interacted with tyr440 and trp430 and showed more affinity (binding free energy = 1.5 kcal/mol) as compared to n-methyl substituent at this position [91] . cagide and co-workers synthesized chromone-2-phenyl carboxamide derived compounds as adenosine a1 (ha1) receptor ligands. different substituents were attached at the exocyclic phenyl ring. compound 105 presented prominent activity at ha1 (ki=0.219 mm) receptor and this compound also exhibited selectivity for this receptor as compared to ha3. it bears nitro group at ortho position of phenyl ring. shifting of nitro group at meta or para position produced less active derivatives. authors explained that receptor sites of ha1 (lined by asn170, glu170, glu172 and thr270) and ha3 (ser73, gln167, val169, leu264) are different from each other. these derivatives also follow lipinski rule of five and possess drug like properties [92] . ali abid, et al. synthesized sulfonyl hydrazone derivatives of 3-formyl chromones as monoamino oxidase (mao) inhibitors. these compounds showed capacity to inhibit mao-a and mao-b enzymes simultaneously. compound 106 was the most active (ic 50 = 0.33 ± 0.01 mm) in vitro mao-a inhibitor in comparison to clorgyline (ic 50 = 0.004 ± 0.0003 mm). it bears chlorine atom at position # 6 of chromone skeleton. replacement of chlorine by methyl group produced less active derivatives. compound 107 was the most active (ic 50 = 1.12 ± 0.02 mm) mao-b inhibitor as compared to deprenyl (ic 50 = 0.0196 ± 0.001 mm). it carries fluorine atom at position # 6 and replacement of fluorine atom by chlorine or bromine atom produced less active derivatives. molecular docking studies of compound 107 with mao-b showed that sulfonamide oxygen interacted by hydrogen bonding with tyr60. oxygen atoms of chromone ring and carbonyl group interacted through hydrogen bonding with gln206 and tyr435. derivatives in this series exhibited good pharmacokinetic properties such as oral bioavailability [93] . reactive oxygen species produce oxidative damage to various biomolecules. naturally occurring compounds have also displayed antioxidant activities [94] . chromone derivatives also play significant role as antioxidants and free radical scavengers [95] . berczynski, et al. (2013) evaluated chromone conjugated derivatives of 2,4-thiazolidinedione, 2,4-imidazolidinedione, 2-thioxo-imidazolidine-4-one as antioxidant agents. derivatives having 2-thioxo-imidazolidine scaffold exhibited prominent activity against oh·, dpph· free radicals. compound 108 (fig. 12 ) appeared as the most active in vitro free radical scavenger (ic 50 = 0.353 ± 0.04 mmol/l) in comparison to standard drug tiron (ic 50 = 0.451 ± 0.065 mmol/l). in compound 108, 2, 3-double of chromone is in conjugation with the 4-carbonyl group and this is the reason for increased antioxidant activity [96] . berczynskai, et al. (2013) evaluated chromone molecules having 2,4-thiazolidinedione and 2,4-imidazolidinedione heterocyclic rings for antioxidant activities. heterocyclic rings were further attached with substituted aromatic rings. in dpph (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay, compounds 109 (ic 50 = 194 ± 3.5 mmol/l) and 110 (ic 50 = 364 ± 3.3 mmol/l) presented prominent activity as compared to vitamin c (ic 50 = 346 ± 28 mmol/l). by using the dmpo-oh (5,5-dimethyl-1-pyrroline-1-oxide) spin adduct method, 109 (41.7% inhibition) and 110 (35.1% inhibition) also exhibited free radical scavenging activity [97] . takao and colleagues synthesized 3-styryl chromone derivatives by konevenagel reaction as a-glucosidase inhibitor and as antioxidant agents. compounds 111 (ec 50 = 17 mm) and 112 (ec 50 = 23 mm) showed prominent in vitro activities as dpph scavenger as compared to ascorbic acid (ec 50 = 23 mm). compounds 111 (ic 50 = 16 mm) and 112 (ic 50 = 10 mm) also exhibited inhibition of a-glucosidase enzyme. both compounds contain catechol ring attached to the chromone scaffold. sar studies showed that incorporation of methyl group and halogen atoms on the ring b did not increase the activity but the incorporation of hydroxyl group produced active molecules. dihydroxy derivatives showed better activity as compared to monohydoxy derivatives. [98] . proenca, et al. attached chromone with 3,4-dihydroxy phenyl core via 1,3-diene system. compound 113 (ic 50 = 125 ± 13 mm) and 114 (ic 50 = 121 ± 9 mm) showed prominent activity as scavenger of hydrogen peroxide (h 2 o 2 ) as compared to standard drug quercetin (ic 50 = 1338 ± 42 mm). compound 114 also exhibited effective scavenging of hocl (ic 50 = 17 ± 3 mm), roo· and onoo -·(ic 50 = 0.29 ± 0.02 mm). incorporation of hydroxyl groups both at position # 5 and # 7 of chromone scaffold was important for the antioxidant activity. no prominent difference in the activity was observed for these hydroxyl groups [99] . soengas, et al. synthesized chromone-3-pyrazoly derivatives as free radical scavenger and alpha glucosidase inhibitor. compound 115 appeared as the most effective antioxidant agent (ic 50 = 23 mm) as compared to standard drug a-tocopherol (ic 50 = 23 mm). it possesses catechol group in its structure and it proved more active as compared to monohydroxyl derivatives. addition of hydroxyl group in the chromone ring had no significant influence on the activity [100] . rao, et al. synthesized styryl chromone derivatives in which styryl group was attached at position # 2 of chromone core. antioxidant activity was monitored as scavenger of superoxide free radical. antioxidant activity was dependent on the number of hydroxyl groups, and compound 116 emerged as the most active agent (ic 50 = 234 mm) in comparison to vitamin c (ic 50 = 852 mm). compound 116 also showed excellent antibacterial activity against xanthomonas campestris (zoi = 11.3 mm) and agarobacterium tumafaceins (zoi = 11 mm) as compared to streptomycin (zoi = 12 -15 mm). chromone derivatives having hydroxyl group also presented better antimicrobial activity. replacement of hydroxyl group with methoxy group decreased antibacterial activity and increased antifungal activity [101] . demetgül and co-workers linked chromone with chitosan to produce the chromone-chitosan schiff base. upon in vitro evaluation as antioxidant agent by dpph radical scavenging assay, compound 117 emerged as the most potent (ic 50 = 0.88 mg/ml) antioxidant agent as compared to chitosan alone. the increased antioxidant activity was related to the phenolic group present in chromone [102] . thus, chromone is a promising scaffold for the synthesis of new therapeutic agents, and hybrid molecules presented in this review can be used as lead for future drug discovery. actions of flavonoids and coumarins on lipoxygenase and cyclooxygenase el-edfawy, phosphorus, sulfur alzheimer's dis key: cord-292380-ulsejzqt authors: iwanejko, jakub; wojaczyńska, elżbieta; turlej, eliza; maciejewska, magdalena; wietrzyk, joanna title: octahydroquinoxalin-2(1h)-one-based aminophosphonic acids and their derivatives—biological activity towards cancer cells date: 2020-05-22 journal: materials (basel) doi: 10.3390/ma13102393 sha: doc_id: 292380 cord_uid: ulsejzqt in the search for new antitumor agents, aminophosphonic acids and their derivatives based on octahydroquinoxalin-2(1h)-one scaffold were obtained and their cytotoxic properties and a mechanism of action were evaluated. phosphonic acid and phosphonate moieties increased the antiproliferative activity in comparison to phenolic mannich bases previously reported. most of the obtained compounds revealed a strong antiproliferative effect against leukemia cell line (mv-4-11) with simultaneous low cytotoxicity against normal cell line (mouse fibroblasts-balb/3t3). the most active compound was diphenyl-[(1r,6r)-3-oxo-2,5-diazabicyclo[4.4.0]dec-4-yl]phosphonate. preliminary evaluation of the mechanism of action showed the proapoptotic effect associated with caspase 3/7 induction. at present, our attention is focused on the covid-19 pandemic and there is a tendency to neglect the civilization diseases which however remain the main reason for mortality worldwide. in particular, recently published data have shown that cancers are some of the leading causes of death in poland, with prostate cancer (almost 20% of patients) and lung cancer as the most common in the male population. the third cause of male mortality is colorectal (colon and rectum) cancer. in the female population, lung cancer dominates, followed by breast and colorectal cancer. among young poles, leukemias, lymphomas and brain cancers predominate and account for about 56% of cases [1] . anticancer drug design is a challenging field with a continuous demand for new, selective and non-toxic agents for treatment [2] . among various classes of compounds, aminophosphonic acids and their derivatives meet these requirements. due to their similarities to α-amino acids and a wide range of applications, α-aminophosphonic acids are continuously gaining importance in organic synthesis. so far, a number of applications such as antiviral [3, 4] or cytotoxic agents [5, 6] , enzyme inhibitors [7, 8] immune system activators [9] or antibacterial activities [10] have attracted considerable attention. the importance of these acids and their derivatives in the search for new pharmaceutical uses has been extensively discussed in numerous review articles [11] [12] [13] [14] . the α-aminophosphonic acid and its derivatives pose a crucial role in a variety of biological activities (figure 1 ), including cytotoxic properties. ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist [15] ; (b) antimicrobial aminophosphonate [16] ; (c) anti-hiv agent [17] ; (d) cytotoxic agent against human cervical carcinoma [18] . aminophosphonates bearing an n-heterocyclic fragment have received particular attention over the last years among other potential anticancer agents [19] [20] [21] [22] . α-amino acids and their esters are considered versatile pharmacophores, known for antitumor activity [23] . the group of nikalje successfully coupled indole-2,3-diones with α-aminophosphonates, which led to new selective, antiproliferative compounds, analogues of commercially available drugs, orantinib and sunitinib [24] . in another noteworthy example, huang and co-workers proved that the incorporation of an aminophosphonate moiety to irinotecan, a chemotherapeutic agent, increased the cytotoxicity against certain cancer cell lines [25] . the fact of their negligible mammalian toxicity [12] testifies to the usefulness of these compounds in drug discovery research. a convenient route to this class of compounds is the pudovik reaction-a nucleophilic addition of dialkyl phosphites to imines [26] . another approach, kabachnik-fields one-pot three-component protocol, requires an amine, a carbonyl compound and an alkyl phosphite, however, it fails with electron-deficient amines [27] . most of the reported reactions are base-or acid-catalyzed, and noncatalyzed procedures remain scarce [28] . a recent review covers the reactions and synthetic methods for aminophosphonates [29] . in our previous research, we reported on the synthesis and antiproliferative action of novel phenolic adducts of bicyclic imine 1 (scheme 1) [30] . two phenolic mannich bases were found to be comparatively active to cisplatin with a noticeable increase of selectivity against cancer cell lines (one of them shown on the scheme). the tested compounds exhibit a resemblance to bioactive diketopiperazines in their bicyclic fragments. our new goal was to synthesize phosphorus analogs of these structures and evaluate their cytotoxicity against cancer cell lines. among a variety of synthetic methods for heterocyclic aminophosphonates formation [31] , our group has focused on a convenient nucleophilic addition of dialkyl phosphites to a cyclic imine. bearing in mind the remarkable precedents of improving the efficacy of cytotoxicity against cancer cell lines by insertion of aminophosphonate moiety and the results of our previous research, we directed our examinations toward the evaluation of antiproliferative properties of the phosphonic derivatives of octahydroquinoxalin-2(1h)-one. [15] ; (b) antimicrobial aminophosphonate [16] ; (c) anti-hiv agent [17] ; (d) cytotoxic agent against human cervical carcinoma [18] . aminophosphonates bearing an n-heterocyclic fragment have received particular attention over the last years among other potential anticancer agents [19] [20] [21] [22] . α-amino acids and their esters are considered versatile pharmacophores, known for antitumor activity [23] . the group of nikalje successfully coupled indole-2,3-diones with α-aminophosphonates, which led to new selective, antiproliferative compounds, analogues of commercially available drugs, orantinib and sunitinib [24] . in another noteworthy example, huang and co-workers proved that the incorporation of an aminophosphonate moiety to irinotecan, a chemotherapeutic agent, increased the cytotoxicity against certain cancer cell lines [25] . the fact of their negligible mammalian toxicity [12] testifies to the usefulness of these compounds in drug discovery research. a convenient route to this class of compounds is the pudovik reaction-a nucleophilic addition of dialkyl phosphites to imines [26] . another approach, kabachnik-fields one-pot three-component protocol, requires an amine, a carbonyl compound and an alkyl phosphite, however, it fails with electron-deficient amines [27] . most of the reported reactions are base-or acid-catalyzed, and non-catalyzed procedures remain scarce [28] . a recent review covers the reactions and synthetic methods for aminophosphonates [29] . in our previous research, we reported on the synthesis and antiproliferative action of novel phenolic adducts of bicyclic imine 1 (scheme 1) [30] . two phenolic mannich bases were found to be comparatively active to cisplatin with a noticeable increase of selectivity against cancer cell lines (one of them shown on the scheme). the tested compounds exhibit a resemblance to bioactive diketopiperazines in their bicyclic fragments. our new goal was to synthesize phosphorus analogs of these structures and evaluate their cytotoxicity against cancer cell lines. among a variety of synthetic methods for heterocyclic aminophosphonates formation [31] , our group has focused on a convenient nucleophilic addition of dialkyl phosphites to a cyclic imine. bearing in mind the remarkable precedents of improving the efficacy of cytotoxicity against cancer cell lines by insertion of aminophosphonate moiety and the results of our previous research, we directed our examinations toward the evaluation of antiproliferative properties of the phosphonic derivatives of octahydroquinoxalin-2(1h)-one. in our studies of the aminophosphonic acids and aminophosphonates mv4-11 (b phenotypic myelomonocytic) cell line carrying translocation t(4;11) was used. mv4-11 corresponds to aml m5b (according to the previously used french-american-british (fab) classification based on the morphology features of the cells). such an aml subtype is characterized by a tendency to occupy the gums, lymph nodes and skin [32] . mv4-11 cell line growth in suspension has about 50 h doubling time that makes it a good and a sensitive model for searching for new synthesized compounds against leukemia cells. all the reagents and solvents were purchased from commercial suppliers and used without further purification. melting points were carried out on the apotec ® schmelzpunktbestimmer melting point apparatus and are uncorrected. 1 h, 13 c and 31 p nmr spectra were collected on jeol 400yh and bruker avance ii 600 instruments. nmr spectra were recorded in cdcl3, unless specified otherwise. the temperature of the samples was 298 k. fourier-transform infrared spectra were measured using the perkin elmer 2000 ftir spectrometer. the principle peaks and their assignments are listed in table 1 . the high-resolution mass spectra (hrms) measurements were performed using the waters lct premier xe tof instrument. optical rotations were measured at ambient temperature on optical activity ltd. model aa-5 automatic polarimeter; [α] d values are given in 10 −1 deg cm 2 g −1 . column chromatography was performed on silica gel 60 (particle size 0.063-0.200 mm). thin-layer chromatography was conducted with the merck silica gel 60 pre-coated plates (f254) and visualized with uv light and/or iodine vapors. (1r,6r)-3-oxo-2,5-diazabicyclo [4.4 .0]dec-4-ene (1). typical procedure (1r,2r)-trans-diaminocyclohexane (4.00 mmol, 456 mg, 2.00 equiv) was dissolved in 2-proh (8 ml). to the stirred solution ethyl glyoxylate solution (50% solution in toluene, 2.00 mmol, 0.420 ml, 1.00 equiv) was added and the mixture was stirred for 24 h at room temperature (293 k). the solvent was removed in vacuo and the product was purified by silica gel column chromatography (eluent: in our studies of the aminophosphonic acids and aminophosphonates mv4-11 (b phenotypic myelomonocytic) cell line carrying translocation t(4;11) was used. mv4-11 corresponds to aml m5b (according to the previously used french-american-british (fab) classification based on the morphology features of the cells). such an aml subtype is characterized by a tendency to occupy the gums, lymph nodes and skin [32] . mv4-11 cell line growth in suspension has about 50 h doubling time that makes it a good and a sensitive model for searching for new synthesized compounds against leukemia cells. all the reagents and solvents were purchased from commercial suppliers and used without further purification. melting points were carried out on the apotec ® schmelzpunktbestimmer melting point apparatus and are uncorrected. 1 h, 13 c and 31 p nmr spectra were collected on jeol 400yh and bruker avance ii 600 instruments. nmr spectra were recorded in cdcl 3, unless specified otherwise. the temperature of the samples was 298 k. fourier-transform infrared spectra were measured using the perkin elmer 2000 ftir spectrometer. the principle peaks and their assignments are listed in table 1 . the high-resolution mass spectra (hrms) measurements were performed using the waters lct premier xe tof instrument. optical rotations were measured at ambient temperature on optical activity ltd. model aa-5 automatic polarimeter; [α] d values are given in 10 −1 deg cm 2 g −1 . column chromatography was performed on silica gel 60 (particle size 0.063-0.200 mm). thin-layer chromatography was conducted with the merck silica gel 60 pre-coated plates (f 254 ) and visualized with uv light and/or iodine vapors. [(1r,6r)-3-oxo-2,5-diazabicyclo [4.4 .0]dec-4-yl]-phosphonic acid (3a). procedure: the imine 1 (1.00 mmol, 152 mg, 1.00 equiv) was dissolved in ch 2 cl 2 (15 ml) followed by the addition of the tris (trimethylsilyl) phosphite (1.00 mmol, 0.334 ml, 1.00 equiv). the reaction was kept at ambient temperature for 24 h, with magnetic stirring. the solvent was removed under reduced pressure and the residue dissolved in methanol (15 ml) followed by stirring overnight at ambient temperature. methanol was removed under reduced pressure and the acid was separated by crystallization (anhydrous etoh/et 2 o 1:5 v/v) which led to the product as a colorless solid. the ftir analysis was conducted to additionally confirm the structures of the products. in the spectrum of imine 1, the strong band at 1622 cm −1 has been assigned to the double-bonded imino group [33] . this stretching vibration was not present in the other products which confirmed the addition of phosphorus nucleophiles. moreover, these compounds exhibited signals in the range of 1416-1453 cm −1 (c-n), characteristic for secondary cyclic amines, which corresponds to the spectra of aminophosphonates known in the literature [34] . absorption in the region of 3100-3400 cm −1 has been attributed to the n-h stretching from the lactam group. the differences are observed for acids 3a and 3b, due to the possible intermolecular hydrogen bond formation. the p=o stretch was found at 1166-1349 cm −1 , in accordance to the literature [35] . the other essential signals between 909 and 1122 cm −1 were assigned to p-o and p-ar bonds as identified in tusek-bozic's work [36] . . balb/3t3 cell line was cultured in dmem (gibco, scotland, uk) supplemented with 2 mm l-glutamine and 5% fbs). all the culture media contained antibiotics: 100 u/ml penicillin (polfa tarchomin sa, warsaw, poland) and 100 µg/ml streptomycin (sigma-aldrich chemie gmbh, steinheim, germany)). all the cell lines were cultured in a humid atmosphere at 37 • c and in 5% co 2 . twenty four hours before adding the tested compounds, each of the cell lines was seeded in 96-well plastic plates (sarstedt, numbrecht, germany) in an appropriate medium at a density (10 4 cells/well), except a549 cell line (0.25 × 10 4 /well), and mcf7 cell line: (0.75 × 10 4 /well). the selected cell lines were exposed to each of the tested chemical compounds at four different concentrations in the range of 100 to 0.1 µg/ml for 72 h. as a reference, cisplatin (teva pharmaceuticals, poland) was used, and dmso (sigma-aldrich chemie gmbh, steinheim, germany) served as a solvent control at concentrations corresponding to these present in the dilutions of the tested compounds. for adherent cells, a sulforhodamine b assay (srb), and for leukemic-an mtt assay was performed. after 72 h of incubation, cells were fixed in situ by gently adding of 50 µl per well of ice-cold 50% tca (trichloroacetic acid, poch, gliwice, poland) and were incubated at 4 • c for one hour. afterwards, wells were washed five times with water and 50 µl of 0.4% solution of srb (sulforhodamine b, sigma-aldrich chemie gmbh, steinheim, germany) in 1% acetic acid (poch, gliwice, poland) was added to each well and plates were again incubated at rt for 30 min. the unbound dye was removed by washing plates five times with 1% acetic acid, while stained cells were treated with 10 mm tris (tris base, sigma-aldrich, chemie gmbh, steinheim, germany). the absorbance in each well was read using the elisa plate reader (biotek synergy h4, swindon, uk) equipped with gen5 software at the 540 nm wavelength [37] . the percentage of proliferation inhibition of leukemia cells by the tested compounds was determined by an mtt assay. briefly, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (sigma-aldrich, chemie gmbh, steinheim, germany) was added to each well and plates were left for 4 h at 37 • c. then, plates were centrifuged for 5 min, at 88× g, at 4 • c, the supernatant was thrown out and 200 µl of dmso per well (poch, gliwice, poland) was added. the plates were left in rt for 10 min and the absorbance in each well was read using the elisa plate reader (biotek synergy h4, swindon, uk), equipped with gen5 software at the 570 nm wavelength [38] . the results are presented as mean ic 50 values (the concentration of the compound, that inhibits cell proliferation by 50%) ± standard deviation. ic 50 values were assessed by the prolab-3 system based on cheburator 0.4, software developed by nevozhay [39] . at each concentration, chemical compounds were tested in triplicates in a single experiment. each experiment was repeated at least three times independently. for the cell cycle analysis, mv4-11 cell line was used. the cells were seeded in 24-well plastic plates (sarstedt, darmstadt, germany) at a density of 0.25 × 10 6 cells/1 ml. next, after 24 h, the tested compounds at the final concentration ic 50 and 2 × ic 50 , were added in a volume of 1 ml to the cells. the cells were exposed to the tested compounds for 48 h. next, the cells growing in suspension were collected, counted with the trypan blue solution (sigma-aldrich chemie gmbh, steinheim, germany), centrifuged for 5 min at +4 • c, 5 min, at 324× g, resuspended in 1 ml of 70% ice-cold ethanol (poch, gliwice, poland) and frozen at −20 • c for at least 24 h. after that, the cells were transferred to 5 ml propylene tubes (dedicated for flow cytometry analysis), washed in pbs (iiet, pas, wroclaw, poland) and centrifuged (+4 • c, 10 min, 324× g). then, the rnase solution (in pbs, 8 µg/ml) (life technologies, carlsbad, ca, usa) was added (500 µl for 0.5 × 10 6 cells) and the cells were incubated for 60 min at 37 • c with gentle mixing. after that, the cells were placed on ice, a propidium iodide (pi) solution (sigma-aldrich chemie gmbh, steinheim, germany) (in concentration 0.1 mg/ml) was added to the cells for 30 min. next, the flow cytometry analysis of the cell cycle was performed using bd lsr ii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva version 6.1. software (bd). the analysis of the obtained results was performed using flowing software version 2.5.1 developed by perttu terho. for each sample, the percentage of cells in each cell cycle phase was determined. each experiment was performed three times independently. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml (total 1 × 10 6 cells) in a culture medium on 24-well plastic plates (sarstedt, numbrecht, germany) and was exposed to the chemical compounds at the concentration of ic 50 and 2 × ic 50 for 48 h. as a solvent control, dmso (poch, gliwice, poland) was used at a concentration corresponding to the highest concentration of compounds. after 48 h of incubation, the cells were collected, washed in pbs (324 g, 10 min, 4 • c) and counted. the cells (0.5 × 10 6 /ml) were diluted in a 0.5 ml annexin binding buffer (10 mm hepes/naoh; 140 mm nacl, 2.5 mm cacl 2 : iiet pas, wrocław, poland), diluted in distilled water at a ratio of 1:4. 5 µl of annexin v conjugated with apc (bd bioscience, san jose, ca, usa) was added to each 195 µl of cell suspension. after 15 min of incubation at room temperature in the dark, and pbs washing, the pi solution at 0.5 mg/ml (sigma-aldrich gmbh chemie, steinheim, germany) was added to the samples. the data were processed using bd lsrii fortessa, equipped with diva 6.1 software. the data were analyzed using flowing software version 2.5.1., developed by perttu terho and described as: double negative (live), double-positive (late apoptotic), annexin v positive-propidine iodine negative (early apoptotic)-and annexin v negative-propidine iodine positive (necrotic). each experiment was repeated 4-5 times. the mv-4-11 cells were seeded at the density of 0.25 × 10 6 cells/ml in culture medium on 24-well plastic plates (sarstedt, numbrecht, germany). after a 48 h exposition to the compounds at the concentration of ic 50 and 2 × ic 50 and incubated for 48 h, cells were collected and washed in pbs. as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. camptothecin (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. the appropriate volume of lysis buffer ph 7.5 (50 mm hepes, 10% sucrose, 150 mm nacl, 1% triton x-100) (iiet, pas, wroclaw, poland) with the addition of 1% of dtt (dl-dithiotreithol, sigma-aldrich gmbh chemie steinheim, germany) was prepared. the reaction buffer ph 7.5 (20 mm hepes, 10% sucrose, 100 mm nacl) iiet pas, wrocław, poland with the dtt addition and with 10 µm of caspase-3 substrate (ac-devd-amc, cayman chemicals, ann arbor, mi, usa) was prepared and warmed to 37 • c before using. after incubation time the cells were centrifuged (324× g, 10 min, 4 • c), 50 µl of the lysis buffer was added to each sample and the probes were left at 4 • c for 30 min. then 40 µl of lysates were added to white 96-well plates (perkin-elmer, waltham, ma, usa) in triplicate, 160 µl of a pre-warmed reaction buffer was added to each well and the fluorescence was read out using a fluorescence plate reader (biotek synergy h4, swindon, uk) equipped with gen5 software the measurements were performed at 355 nm and 460 nm for 2 h every 10 min at 37 • c. at the same time, an mtt test was performed in order to normalize results. based on the results obtained, an mfi (mean fluorescence intensity) vs. reaction time curve was plotted and the vmax (reaction rate) values were determined. after normalization, the values obtained for the tested samples were compared to the control to assess how many times caspase-3 activity in tested probes is higher/lower than in control. each experiment was conducted at least 4-5 times. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml in a culture medium on 24-well plates (sarstedt, numbrecht, germany). the cells were exposed to the compounds at the concentration of ic 50 and 2 × ic 50 for 48 h. then, the cells were collected, washed in pbs and counted in a trypan blue solution. the collections of 0.2 × 10 6 cells/sample were centrifuged (300× g, 5 min, room temperature) and pellets were suspended in a jc-1 solution (cayman chemicals, ann arbor, mi, usa) in a warm culture medium (final concentration 2.5 µg/ml). after 10 min of incubation at 37 • c in the dark, cells were centrifuged (300× g, 5 min, room temperature) and pellets were suspended in 200 µl of pbs (iiet, pas, wrocław, poland). as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. valinomycin (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. the results were read using bd lsrii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva 6.1. software and were analyzed with flowing software 2.5.1, developed by perttu terho in dot plots presenting jc-1 monomers to aggregates. the mv-4-11 cells were seeded at a density of 0.25 × 10 6 cells/ml in culture medium on 24-well plates (sarstedt, numbrecht, germany). the cells were exposed to the compounds at the concentration of ic 50 and 2 × ic 50 and incubated for 48 h. as a solvent control, dmso was used at a concentration corresponding to the highest concentration of the compounds. tamoxifen (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. after 48 h of incubation, the cells were collected and washed in pbs (324 g, 10 min, 4 • c). for this purpose, 96-well black plates were used (perkin elmer, walthman, ma, usa). 100 µl of propidine iodine (pi) (final concentration: 10 µg/ml) was added to each sample, except the probes intended for background measurement. after 2 min of room temperature incubation, the cells were centrifuged (400× g, 5 min, room temperature), pellets were suspended in 100 µl of pbs (iiet pas, wrocław, poland) and again centrifuged. next 100 µl of 0.05 mmol/l dansyl cadaverine (sigma aldrich gmbh chemie, steinheim, germany) was added to each sample and the samples were incubated at 37 • c for 10 min. next, the cells were centrifuged and washed with pbs. finally, the obtained pellet was suspended in 300 µl of pbs and transferred into the appropriate wells. each sample was made in triplicate. autophagic vacuole staining intensity was detected at the excitation wavelength of 335 nm and emission of 512 nm, and the degree of cell death at excitation wavelength 536 nm and emission of 617 nm. each experiment was conducted at least 4-5 times. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml in a culture medium on 24-well plastic plates (sarstedt, germany) to the final volume of 2 ml. the cells were exposed to the compounds at the concentration of ic 50 and 2ic 50 and incubated for 48 h. as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. after 48 h of incubation, the cells were collected, washed in pbs (324 g, 5 min, room temperature) and counted. the collection of 0.2 × 10 6 cells were stained with 10 µg/ml of acridine orange (ao) (sigma aldrich gmbh chemie, steinheim, germany) for 20 min at 37 • c, washed two times with pbs and read using bd lsr ii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva 6.1. software and were analyzed with flowing software 2.5.1, developed by perttu terho. each experiment was performed at least 4 times. the presented compounds were synthesized using the previously published methods [40, 41] . a cyclic imine 1, derived from optically pure trans-(r,r)-1,2-diaminocyclohexane was reacted with h-phosphonates or phosphine oxides to give compounds 2a-f with good yields as mixtures of epimers (table 2) . a single epimer 2g of p-taddol derivative (taddol = α,α,α ,α -tetraaryl-2,2-disubstituted 1,3-dioxolane-4,5-dimethanol) was obtained by crystallization from mixture 2f. nmr spectra of products 1 and 2a-g are shown in the supplementary materials (figures s1-s8 ). the presented compounds were synthesized using the previously published methods [40, 41] . a cyclic imine 1, derived from optically pure trans-(r,r)-1,2-diaminocyclohexane was reacted with hphosphonates or phosphine oxides to give compounds 2a-f with good yields as mixtures of epimers (table 2) . a single epimer 2g of p-taddol derivative (taddol = α,α,α′,α′-tetraaryl-2,2disubstituted 1,3-dioxolane-4,5-dimethanol) was obtained by crystallization from mixture 2f. nmr spectra of products 1 and 2a-g are shown in the supplementary materials (figures s1-s8 ). in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of the accuracy of nmr technique (ca. 2%). in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of the accuracy of nmr technique (ca. 2%). all compounds were evaluated according to their antiproliferative activity towards human acute myeloid leukemia (aml-m5b) cell line (mv4-11) and three adenocarcinoma cell lines of different origin: lung (a549), colorectal (lovo) and breast (mcf-7). the results were compared with those obtained on the normal murine fibroblasts cell line (balb/3t3). among the modified compounds, only derivatives 2f and 2g (similarly to 1) exert antiproliferative activity against all the cancer cell lines tested, however in contrast to compound 1 their activity towards balb/3t3 cells was visibly lower as compared to the cancer cells (table 3) . on the other hand, 2b and 3a were most active towards leukemia cells, though they were not toxic neither for solid tumors cell lines nor murine fibroblasts. a preliminary sar (structure-activity relationship) analysis reveals a few conclusions about structural features that result in the desired activity. among the tested phosphonates, phenyl derivative 2b performed better than benzyl (2c), methyl (2a) and taddol esters (2f). however, the latter derivative bearing a substituent introducing a big steric hindrance was found to be more versatile and acted on all of the investigated cell lines. comparison of diastereomeric mixture (2f) and isolated single isomer (2g) reveals no significant differences between epimers with opposite configurations of the stereogenic center c-4. therefore, the separation of phosphonate diastereomers for biological tests seems unnecessary. remarkably, h-phosphinate 2d was practically inactive, while phosphine oxide 2e exhibited a moderate activity in comparison to the majority of the tested phosphonates. this indicates the directions of further modifications, which should be focused on esters of aminophosphonic acids. the acid itself (3a) exhibited high cytotoxicity, but only toward leukemia cells; comparison of compounds 3a and 3b suggests that complete substitution of a stereogenic center decreases the antiproliferative activity. compounds 2b, 2e and 2f were selected for further studies. for evaluation of the impact of the selected compounds on cell cycle distribution, we analyzed the percentage of cells in each of the cell divisions upon incubation with selected compounds. analyzing cell cycle distribution (figure 2a-d) , we could only observe a decrease of cells in the s phase after 48 h incubation with 2e. in parallel, the tendency to increase the percentage of cells in g0/g1 phase was observed. cisplatin used as a control of the test increased cells percentage in the g2m phase. analysis of dead cells (subg1) showed a significant increase in dead cells caused by 2f used in higher concentrations. next, we decided to analyze apoptotic and necrotic cells using annexin v/pi staining ( figure 2e-g) , as well as the activity of caspase 3/7 in the treated cells ( figure 2h ). compound 2b increased the level of early apoptotic cells with an increase of caspase 3/7 activity. compound 2f increased the percentage of necrotic cells (but also the tendency to increase the level of apoptotic cells: early and late, was observed) and also increased the activity of caspase 3/7. in the case of compound 2e, an increased percentage of late apoptotic cells was accompanied by an increase of caspase 3/7 activity. the drop of the percentage of cells with high mitochondrial membrane potential (∆ψ) was observed in mv4-11 cells incubated with compound 2f and 2e ( figure 2i ). for labeling autophagic vacuoles, two techniques with dansyl cadaverin and with acridine orange were used. in both methods, compound 2f increased the level of acidic autophagic vacuoles ( figure 2j,k) . increased the activity of caspase 3/7. in the case of compound 2e, an increased percentage of late apoptotic cells was accompanied by an increase of caspase 3/7 activity. the drop of the percentage of cells with high mitochondrial membrane potential (δψ) was observed in mv4-11 cells incubated with compound 2f and 2e ( figure 2i ). for labeling autophagic vacuoles, two techniques with dansyl cadaverin and with acridine orange were used. in both methods, compound 2f increased the level of acidic autophagic vacuoles ( figure 2j ,k). in this study, we prepared aminophosphonic acids and their derivatives based on octahydroquinoxalin-2(1h)-one scaffold via pudovik reaction. the syntheses proceeded efficiently giving stable products in case of all types of h-phosphonates, phosphine oxides and phosphite used. since no significant differences of antiproliferative activities for a diastereomeric mixture (2f) and a single epimer (2g) were observed, further studies were conducted for mixtures of both stereoisomers of each compound. compound 2b, which was found to be the most active in proliferation inhibition of the mv4-11 cells, induces apoptosis of these cells with an increased caspase 3/7 activity. compound 2f, which inhibited the proliferation of all neoplastic cell lines, tends to decrease the percentage of cells in the g2m phase and increases the percentage of dead cells, including cells undergoing necrosis. this compound increases the activity of caspases 3/7 and reduces the mitochondrial potential of cells. a long-lasting drop or rise of ∆ψ from control levels may induce a loss of cell viability. the higher is the level of intracellular atp, the more stable are the ∆ψ values, making atp a compound buffering mitochondrial ∆ψ [42] . however, compound 2f can also enhance autophagy. this phenomenon may not be beneficial from the point of view of cancer therapies, although there are different opinions, as well as it may depend on specific mechanisms of action of studied compounds [43] . derivative 2e reduces the percentage of cells in the s phase of the cell cycle, increases the percentage of cells in late apoptosis and necrotic cells, increases caspase 3/7 activity, and reduces mitochondrial potential, so it works as a pro-apoptotic agent. a similar mechanism of action was reported by huang's group in the paper on the synthesis of potential anticancer candidates for the use in the therapy of ovarian cancer cells [25] . the tested compounds, with the emphasis put on 2b, 2e and 2f, could be used as promising scaffolds in further antiproliferative drug design. supplementary materials: the following are available online at http://www.mdpi.com/1996-1944/13/10/2393/s1, figure s1 . (a) 1 h nmr and (b) 13 c nmr spectra of (1r,6r)-3-oxo-2,5-diazabicyclo [4.4 .0]dec-4-ene 1. figure s2 . cancer in poland in 2017 recent advances and perspectives in cancer drug design phosphonate inhibitors of west nile virus ns2b/ns3 protease synthesis and antiphytoviral activity of α-aminophosphonates containing 3, 5-diphenyl-2-isoxazoline as potential papaya ringspot virus inhibitors synthesis and cytotoxicity of o,o -dialkyl {[2-(substituted phenoxy)acetamido](substituted phenyl)methyl}phosphonates diversity-oriented synthesis of α-aminophosphonates: a new class of potential anticancer agents renin inhibitors. synthesis of transition-state analog inhibitors containing phosphorus acid derivatives at the scissile bond the synthesis and kinetic evaluation of aryl α-aminophosphonates as novel inhibitors of t. cruzi trans-sialidase aminobisphosphonates as new weapons for gammadelta t cell-based immunotherapy of cancer bioactive amide and α-aminophosphonate inhibitors for 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of some new heterocyclic α-aminophosphonates synthesis, antimicrobial and anticancer activities of a novel series of diphenyl 1-(pyridin-3-yl)ethylphosphonates -oxo-1,2-dihydroquinolin-3-yl)(arylamino)methyl)phosphonate as potential anticancer agents synthesis and anticancer activity of some novel diethyl {(chromonyl/pyrazolyl) [(4-oxo-2-phenyl-quinazolin-3(4h)-yl)amino]methyl}phosphonates. phosphorus sulfur silicon rel. elem new 2-oxoindolin phosphonates as novel agents to treat cancer: a green synthesis and molecular modeling synthesis, mechanisms of action, and toxicity of novel aminophosphonates derivatives conjugated irinotecan in vitro and in vivo as potent antitumor agents synthesis of quaternary α-aminophosphonic acids an extremely efficient three-component reaction of aldehydes/ketones, amines, and phosphites (kabachnik−fields reaction) for the synthesis of α-aminophosphonates catalyzed by magnesium perchlorate methods for the synthesis of 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an effective route to new bicyclic compounds: aminophosphonates, enamines and imines aminophosphonates and aminophosphonic acids with tetrasubstituted stereogenic center: diastereoselective synthesis from cyclic ketimines mitochondrial membrane potential the roles of autophagy in cancer funding: this research received no external funding. the authors declare no conflict of interest. key: cord-340331-51yq1rdo authors: tonelli, michele; naesens, lieve; gazzarrini, sabrina; santucci, matteo; cichero, elena; tasso, bruno; moroni, anna; costi, maria paola; loddo, roberta title: host dihydrofolate reductase (dhfr)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus date: 2017-07-28 journal: eur j med chem doi: 10.1016/j.ejmech.2017.04.070 sha: doc_id: 340331 cord_uid: 51yq1rdo we have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza a and b virus and respiratory syncytial virus (rsv) via targeting of the host dihydrofolate reductase (dhfr) enzyme. most analogues proved active against influenza b virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (ec(50) = 0.060 μm), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. activity was also observed for two influenza a strains, including a virus with the s31n mutant form of m2 proton channel, which is the most prevalent resistance mutation for amantadine. importantly, the compounds displayed nanomolar activity against rsv and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (ec(50) ∼0.008 μm), far surpassing the potency and safety profile of the licensed drug ribavirin (ec(50) = 5.8 μm, si > 43). the ortho-and paramyxoviridae families comprise important respiratory pathogens, i.e. influenza a and b viruses and respiratory syncytial virus (rsv), respectively. the acute respiratory illnesses caused by these viruses represent major medical problems, given their significant morbidity and potential mortality, particularly in vulnerable populations such as small infants, elderly people or patients with underlying medical conditions [1] . besides, the threat for new influenza a virus pandemics (such as that of 2009 [2] ) is a reason for global and constant concern. since the current arsenal of antiviral drugs to treat or prevent influenza or rsv infections is quite limited [1, 3] , new therapeutics are highly needed. according to a recommendation by the world health organization [4] , attention should be given to innovative agents with broad activity against diverse respiratory viruses. viruses, as obligate intracellular parasites, encode multiple virus-specific proteins essential for replication, which also depends on critical interactions with host cell proteins. most approved antiviral drugs target unique proteins encoded by one virus or a range of closely related viruses. this strategy is prone to selecting drug-resistance, particularly for viruses, which possess high mutability (such as influenza virus) or require long-term therapy. an alternative and relevant approach is to address host factors involved in the viral life cycle. this type of inhibitors is anticipated to possess a markedly higher barrier for selecting drug-resistant viruses and, furthermore, may display broad-spectrum antiviral activity when dealing with a cellular target that is recruited by different viruses. two host-directed antiviral drugs are maraviroc, a ccr5 receptor antagonist approved for hiv therapy, and alisporivir, a cyclophylin inhibitor that is undergoing phase iii tests for hepatitis c treatment [5] . specific host proteins were proven to be critical for the replication of diverse unrelated viruses [6] , yet the array of possible cellular targets (the 'virus-host interactome') is continuously growing, as recently reviewed for influenza [7] and rsv [8] . the first example of a broadly-acting antiviral drug is ribavirin, a nucleoside analogue that was proposed to act directly at the level of the viral polymerase, although an indirect effect via inhibition of the host-cell imp dehydrogenase and depletion of the gtp pool seems more plausible [9] . another enzyme of the purine and pyrimidine pathways is dihydrofolate reductase (dhfr) which catalyzes the reduction of dihydrofolate (dhf) to tetrahydrofolate (thf), a crucial cofactor for the biosynthesis of imp and thymidylate. folate antagonists interfering with dhfr can be applied in diverse pharmacological (i.e. antimalarial, antibacterial and antineoplastic) settings [10e13] . the licensed antifolates trimethoprim [14] , pyrimethamine [15] and cycloguanil are potent inhibitors of bacterial and protozoal dhfr, respectively, but only weak inhibitors of mammalian dhfr enzymes. on the other hand, the drug methotrexate (mtx) is a potent unselective dhfrs inhibitor (ki ¼ 0.01e0.2 nm) [16] , because of its close structural similarity with dihydrofolic acid, the natural substrate of the enzyme [17] . mtx shows a binding affinity to human dhfr (hdhfr) 1000-fold higher than that of folic acid [16] , explaining its clinical application as anticancer, anti-inflammatory and immunosuppressive agent. indeed, the mtx capability of affecting different intracellular pathways has been very recently described, highlighting a rather complex mechanism of action besides the most important therapeutic activity related to hdhfr inhibition [18] . ongoing research efforts to develop novel antifolates for cancer chemotherapy and microbial infections continue to be extensively reviewed [19] . cycloguanil is the active metabolite of the antimalarial drug proguanil (paludrine ® or malarone ® ), that is approved for prophylaxis and treatment of infections by plasmodium vivax or falciparum. the species-selective activity of cycloguanil (and pyrimethamine) has traditionally been attributed to higher affinity of the drug for plasmodium bifunctional dihydrofolate reductase-thymidylate synthetase (dhfr-ts) than for hdhfr [20] . since 1991, cycloguanil and related 1-aryl-4,6-diamino-1,2-dihydrotriazines were studied with the aim at treating pneumocystis carinii pneumonia [21] , searching for more selective inhibitors for p. carinii dhfr over host dhfr (especially human enzyme). indeed, trimethoprim, the antifolate most widely used for that kind of infection, was a poor inhibitor of p. carinii dhfr (ki ¼ 280 mm) and showed about 6-fold greater selectivity for hdhfr (ki ¼ 48 mm). some 1-aryl-4,6diamino-1,2-dihydrotriazines exhibited a selective p. carinii dhfr inhibition, while cycloguanil and some related analogues (two of them corresponding to our compounds 11 and 14) were disclosed to bind slightly stronger to hdhfr (cycloguanil, ki ¼ 43.0 mm) than to p. carinii enzyme (cycloguanil, ki ¼ 109.0 mm). finally, the author suggested that not only the expected selective fungal enzyme inhibitors, but even compounds with higher species-selectivity profile for hdhfr showed improvement over agents currently used to treat p. carinii infections. in our previous studies, we focused on the design of antiviral agents by exploring diverse and original chemotypes [22e26]. in the search of novel promising derivatives, in this manuscript we reported for the first time the intriguing antiviral profile of cycloguanil (1). based on this information, we deemed interesting to proceed our work directed to the design, synthesis and evaluation of the antiviral activity and cytotoxicity played by further compounds, against a wide range of rna and dna viruses. in particular, we explored the most relevant structure-activity relationship (sar) exhibited within a series of structural analogues of cycloguanil (1), including a number of 1-aryl-4,6-diamino-1,2-dihydrotriazines. these compounds proved to inhibit virus reproduction targeting the host cell dhfr. in particular, this new class of host-directed antiviral agents displayed promising dual activity against influenza and respiratory syncytial virus (rsv). selected compounds were also tested against hdhfr, in order to gain knowledge on their efficacy versus the recombinant protein. finally, docking studies were performed, in order to support and disclose the most probable binding mode for these derivatives as hdhfr ligands and foster lead optimization process. the compounds, object of the present study, are characterized by the 1-aryl-4,6-diamino-1,2-dihydrotriazine scaffold, such as the cited cycloguanil, which was identified by us as prototype (1) of a new class of antiviral agents exploiting a host dhfr inhibition mechanism. in order to better clarify the intriguing effectiveness of tuning an adequate host dhfr inhibition for the development of antiviral compounds endowed with optimized antiviral activity and safety profiles, we proceeded with the design, synthesis and evaluation of a series of cycloguanil (1) analogues. furthermore, several studies about compound (i) [27] , containing a pyridopyrimidine system bioisostere of the (1) triazine core, as hdhfr-targeting derivative, prompted us to deepen some sar requirements within the series of congeners (fig. 1) . thus, different functionalized 1-aryl-4,6-diamino-1,2dihydrotriazines (2e28) were designed (fig. 2) , by exploring the effect on biological activity as a result of the chemical variation of the para-cl substituent (r 3 ) on the phenyl ring and/or of the two methyl groups (r 1 , r 2 ) at c(2) of cycloguanil (1) with smaller/ bulkier alkyl groups. five compounds are newly synthesized, while the others have been re-synthesized, to be tested as new antiviral agents capable of inhibiting the host cell dhfr. we also included in our investigation the cycloguanil isomeric anilinodihydrotriazine 29; proguanil (30), the metabolic precursor of cycloguanil; 1-(4-chlorophenyl)biguanide (31), the second major metabolite of proguanil; and two 2,4-diaminopyrimidine planar analogues, pyrimethamine (32) and trimethoprim (33) which are potent inhibitors of protozoal and bacterial dhfr, respectively. all the compounds underwent cell culture evaluation for cytotoxicity and antiviral activity against a wide range of rna and dna viruses. with the aim of determining if the host dhfr was targeted by our compounds series along the virus replication pathway, the most promising compounds were tested against the recombinant protein of the hdhfr enzyme. successive docking studies allowed to find a molecular rationale for the mechanism by which our compounds could inhibit the hdhfr. we deemed interesting to synthesize and evaluate the antiviral activity of a series of 4,6-diamino-1,2-dihydrotriazines, bearing in position 1 an aromatic ring, variously substituted, and in position 2 one or two alkyl or aromatic moieties (fig. 2 ). most of the tested dihydrotriazines were already described and re-prepared according to cited references: syntheses of compounds 1e3, 15, 16, 18, 20, 23 and 28 [28] , 4 [29] , 5 [30] , 14 [31] , 19 [32] were achieved by a one-step acid-catalyzed cyclocondensation among an aromatic amine, dicyandiamide and a carbonyl compound (3component syntheses); for 26 and 27 was used the twocomponent syntheses which progresses in two steps by condensation of a preformed arylbiguanide and a carbonyl derivative [33] . the progress of the reaction was monitored by means of the negative result of colored copper complex formed characteristically by biguanide with a freshly cuprammonium sulphate solution. by the treatment of cycloguanil hydrochloride with an excess of alkali at reflux, it undergoes an intramolecular rearrangement to the isomeric anilinodihydrotriazine compound (29) [28] . compounds 12 and 17 were known as free base, while in our synthetic route they crystallize directly from the reaction mixture as pure hydrochlorides through the aforementioned three component synthesis, thus they were experimentally described together with the novel compounds 21, 22 and 25 (scheme 1). all new compounds showed excellent analytical and spectroscopic data, in good agreement with their structures (see experimental part). in the 1 h nmr spectra the protonated nitrogen of 4,6diamino-1,2-dihydrotriazine nucleus gives rise to a net singlet near d 9 to 10, while the other amino groups yield signals in the range d 7 to 8. the antiviral activities of compounds 1e33 against influenza a (h1n1 subtype) and b viruses are presented in table 1 . our procedure [34] uses exponentially growing mdck cells in which the virus-induced cytopathic effect (cpe) is monitored by either microscopy or cell viability testing. as shown in table 1 , there was overall correlation between the antiviral ec 50 values obtained by either method, indicating the reliability of the observed antiviral effect. on the other hand, microscopic evaluation revealed that several compounds produced cytostatic effect, yielding a minimum cytotoxic concentration (mcc) of, for instance, 4 mm for cycloguanil (1) . by comparison, its cc 50 value by cell viability assay was 52 mm, indicating that a true cytotoxic effect with manifest cell killing was only seen at higher concentrations of 1. it is worth noting that cycloguanil is the active metabolite of the prodrug proguanil, an antimalarial drug that is considered as safe even when used during pregnancy [35] . furthermore, most of the compounds were cytotoxic only in mdck cells while leaving unaffected (mcc > 100 mm) the human hela (table 2 ) and primate vero cell lines (table 3) used to grow the other viruses under investigation. in addition, some selected compounds (1, 11, 13, 14, 16, 25 and 32) were assayed for cytotoxicity against human airway epithelial calu-3 cells confirming mcc values higher than 100 mm (data not shown). table 1 revealed the following trends. twenty-four out of thirty-three compounds (73%) proved active against at least one influenza virus strain, and 30% displayed activity against all three influenza virus strains. cycloguanil (1), the prototype of this class of 4,6-diamino-1,2dihydrotriazine derivatives, exhibited modest activity against influenza a/h1n1 viruses, while being 15-fold more potent against influenza b virus (ec 50 ¼ 2.2 mm). based on this interesting observation, the study was oriented towards design and synthesis of more effective agents. firstly, replacement of the chlorine atom in position 4 of the aromatic ring was explored: the introduction of either electron-withdrawing group isosteres of chlorine (f, 5; br, 6) or electron-donor groups (ch 3 , 3; och 3 , 4) was permitted without changing the activity, whereas polar electron-withdrawing groups (no 2 , cooh and cooet: 7e9) were detrimental. having assumed that chlorine is a well-suited substituent, we investigated its influence on the mono-substituted cycloguanil-related 2-cl (10) and 3-cl (11) isomers, and on the di-substituted 2,4-dichloro (15) and 3,4-dichloro derivatives (16) . compounds 11 and 16, bearing a lipophilic electron-withdrawing group in position 3 of the aromatic ring, proved to be strong inhibitors with sub-micromolar potency against influenza b virus (ec 50 ¼ 0.10 and 0.080 mm, respectively) and only 2-fold lower activity against influenza a viruses. in contrast, compound 10 was only poorly active against influenza b (ec 50 ¼ 56 mm), while the 2,4-dichloro substitution abolished the activity (15) . since these data clearly indicated that a lipophilic electron-withdrawing atom in position 3 properly modulates the antiviral activity, we next synthesized the 3-f (12), 3-br (13) and 3-cf 3 (14) analogues. compound 14 showed an activity profile comparable to that of 11, whereas bromine derivative 13 was found to be the best influenza b inhibitor among the entire series, having an ec 50 of 0.016 mm. in the next step, we varied the two methyl groups on c(2) of the 4,6-diamino-1,2-dihydrotriazine scaffold, by introducing bulkier alkyl groups than the two ones derived from acetone. this was achieved by performing condensation with methyl ethyl ketone (for 18), methyl isopropyl ketone (for 19), cyclopentanone (for 20e22), or cyclohexanone (for 23e25). the progressive increase in the substituent's size led to a proportional decrease in the activity against influenza a virus, while having much less impact for influenza b, even when combined with 3-cl or 3-cf 3 substitutions. among the three compounds with mono-substitution at position c(2), the ch 3 group (26, ec 50 ¼ 17 mm) was tolerated to keep influenza b inhibition, whilst the n-propyl chain (27) and phenyl ring (28) were detrimental. since in vivo application of cycloguanil is through its prodrug proguanil (30), we checked whether also the prodrug and 1-(4chlorophenyl)biguanide (31) possess anti-influenza activity. the two main metabolites of the antimalarial drug proguanil are the active form cycloguanil and the inactive 1-(4-chlorophenyl)biguanide, which constitute about 30% and 23%, respectively, of the total drug concentration in plasma. 32 both compounds 30 and 31 were shown inactive, alike the anilinedihydrotriazine isomer 29 of cycloguanil. the 2,4-diaminopyrimidines trimethoprim and pyrimethamine were chosen as fully unsaturated cycloguanil analogues: only pyrimethamine (32) influenza b virus appears more sensitive to a reduction in the thf pool. this could point to a higher sensitivity of influenza b virus to nucleotide imbalances [perhaps related to the slower growth kinetics of influenza b compared to influenza a virus [36] , although this seems contradicted by the finding that the gtp-depleting agent ribavirin displayed similar ec 50 values for influenza a and b viruses (table 1) . recently, balgi et al. [37] used a high-throughput yeast growth restoration assay to screen 250,000 compounds. they identified three tetrahydrotriazines with antiviral activity in a plaque reduction assay and, with only one exception, efficacy (when assessed at 100 mm) against the wild type a/m2 proton channel in a twoelectrode voltage clamp (tevc) assay. based on the chemical resemblance, we deemed interesting to assess if our 4,6-diamino-1,2-dihydrotriazines (1, 14 and 16) were also able to inhibit the a/ m2 proton channel expressed in xenopus oocytes using the tevc assay. at 100 mm, neither of the three compounds significantly inhibited the wild type or s31n mutant m2 channel [38] , thus excluding m2 inhibition as the antiviral mechanism of action in virus-infected mdck cells (data not shown). several compounds produced promising antiviral effect in a similar cpe reduction assay for rsv in hela cells (table 2) . since most compounds were not cytotoxic at 100 mm (the highest concentration tested), the selectivity index, i.e. ratio of mcc to ec 50 , was calculated to be at least 10,000 for the most active analogues 11, 14 and 16. these compounds inhibited rsv replication at nanomolar concentrations, far surpassing the antiviral activity of ribavirin (ec 50 ¼ 5.8 mm, si > 43). the latter is the only small molecule drug to treat rsv infections, but, as a consequence of its limited efficacy, the need of prolonged aerosol administration and the risk of toxicity, its use is limited to children at high risk [39] . therefore, effective and safe drugs are strongly needed to treat severe rsv-linked respiratory pathologies which can also affect adults and, particularly, elderly. thus, the highly potent compounds 11, 14 and 16 could be interesting lead compounds for further chemical derivatization and development of improved rsv inhibitors. similar to what was observed for influenza virus, the drugs cycloguanil (1) and pyrimethamine (32) were found to be equipotent rsv-inhibitors with ec 50 values of 0.55 and 0.75 mm, respectively, which is nearly one order of magnitude superior to the value of ribavirin. the compounds with no activity against rsv (i.e. 8, 9, 15, 27e31, 33) were also inactive against influenza. the three most active rsv inhibitors, i.e. 11, 14 and 16 (ec 50~0 .008 mm; table 2 ) also had the best ec 50 values (table 1) for influenza. hence, it is clear that the same biochemical mechanism explains the inhibitory effect of this class of compounds towards rsv and influenza virus. as for the other rna viruses tested in either hela (table 2) or vero cells (table 3) , 11e14 and 16 also inhibited reovirus-1, a member of the rotaviridae. they were found active in the low micromolar range without visible cytotoxicity (mcc > 100 mm). compound 32 exhibited broader antiviral activity but only at concentrations quite close to those producing cytotoxicity (mcc !20 mm) (tables 2 and 3) . neither of the compounds inhibited the replication of yellow fever virus (table 3) , a member of the flaviviridae which was described in another report as sensitive to methotrexate, the prototype inhibitor of mammalian dhfr enzymes [40] . finally, we evaluated in human embryonic lung (hel) fibroblast cells, several dna viruses of the herpes-, adeno-or poxvirus families, plus coronavirus 229e (an rna virus), but neither of the compounds 1e33 proved active against any of these viruses (data not shown). since host cell dhfr inhibition seemed the plausible explanation for the observed antiviral effect, we performed a combination experiment in which rsv-infected hela cells were exposed to compound 14 in combination with different concentrations of the natural dhfr substrate dihydrofolic acid ( reaction has a much higher impact on virus replication than on cell growth, which concurs with the promising antiviral selectivity of our host-directed dhfr inhibitors. finally we assayed some selected compounds (1, 11, 13, 14, 16, 25 and 32) against hdhfr in order to confirm that the observed antiviral activity against rsv was clearly influenced by this enzyme inhibition. concerning the inhibition experimental data of the test compounds on hdhfr (table 5) , sars moved with the same trend compared to antiviral activity in cell-based assays: the ki values ranged between 0.07 and 0.13 mm for the best antiviral compounds (13, 11, 16, 14 in decreasing order) while the bulkier spirocompound 25 was less effective with ki value equal to 13.17 mm. cycloguanil (1) and pyrimethamine (32) displayed the same degree of potency, about 5-fold lower than that of the most potent compounds (11, 13, 14 and 16) . interestingly, for almost all the compounds tested (1, 11, 13, 14, 16, 25 and 32) a similar trend can be observed between the ki against hdhfr and the antiviral activity against influenza b virus and respiratory syncytial virus, thus supporting the concept that the observed antiviral effect is sustained also by the hdhfr inhibition (fig. 3) . moreover, kinetic inhibition studies of cycloguanil (1) were performed and a competitive inhibition pattern was observed towards the substrate for binding to the human enzyme (fig. 1s) . thus, these 1-aryl-4,6-diamino-1,2-dihydrotriazine derivatives proved to efficiently work weakening the virus replication machinery by a direct inhibitory effect versus the host (human) dhfr. molecular modelling studies were performed on the hdhfr inhibitors identified (1, 11, 13, 14, 16, 25 and 32) to explore the structural basis of the interaction between the mentioned compounds and the human enzyme. the docking studies were performed using the x-ray crystallographic structure of the hdhfr, in complex with a pyridopyrimidine-based inhibitor (i) (pdb code ¼ 4qhv; resolution ¼ 1.61 å) [24] . the human dhfr inhibitor, compound i, was considered as positive control (fig. 1) . the main issues to be addressed were to clarify, through docking studies of 1, 11, 13, 14, 16 and 25, the role played by the 4,6-diamino-1,2-dihydrotriazine nucleus and of the hydrophobic framework, including the phenyl ring and the r1-r3 substituents, with respect to the 2,4-diaminopyrimidine core and to the phenyl-substituted pyridine-3-amine moiety of i. in addition, pyrimethamine (32) was also submitted to docking calculations, having shown a similar antiviral behaviour to cycloguanil. as shown in fig. 4a , i was engaged in h-bonds between the two nh 2 substituents placed onto the pyrimidine core and the i7 and e30 carbonyl group and side-chain, respectively. the whole, planar bicyclic ring was involved in pàp stacking with y33 and f34, while the isopropyl phenyl ring was projected towards f31, i60, p61, displaying van der waals contacts. as consequence, these kinds of contacts efficiently stabilized i within the hdhfr binding site, leading the complex to a favourable value of the estimated binding affinity (hdhfr-i dg ¼ à28.0 kj/ mol), in harmony with the high compound potency profile (ki ¼ 11 nm, table 5 ). based on our docking calculations (table 6 ), when small bulky alkyl groups are chosen for r1 and r2, the presence of a meta substituent placed in r3, rather than at the ortho and para positions of the phenyl ring, proved to be preferred. accordingly, cycloguanil (1) was characterized by weak h-bonds with i7 and e30, while the 4-chlorophenyl ring moved towards t56 and y121, on the opposite side of the cavity occupied by the isopropyl phenyl ring of the reference inhibitor (fig. 4b) . a comparable docking mode was observed for pyrimethamine (32) . as shown in table 6 , the related complexes displayed quite adequate and comparable values of predicted binding affinity energies (hdhfr-1 dg ¼ à7 kj/mol; interestingly, the most promising derivatives 11, 13, 14, 16 shared a common docking mode, exhibiting the required h-bonds with i7 and e30, by means of the two nh 2 groups of 1,2-dihydrotriazine scaffold (as shown for compound 13 in fig. 5a ). in addition, the dimethyl substitution in r1 and r2 of 13, and also the presence of a 3-br-phenyl ring, proved to be particularly effective to properly mimic the same positioning displayed by i within the x-ray crystallographic structure of hdhfr. as consequence, all of them were efficiently characterized by the most important and crucial bonds with the biological target, as previously discussed for compound i, and therefore experienced the most favourable predicted binding affinity profiles, being in agreement with the experimental data. in particular, compound 13 (hdhfr-13 dg ¼ à19 kj/mol) proved to be the most promising (ki ¼ 0.07 mm) . conversely, the presence of bulky group or the introduction of a cycloalkyl ring in r1 and r2, moved the derivative toward a quite turned docking mode (if compared with i), as shown for compound 25 (fig. 5b) . indeed, only one of the two nh 2 groups onto the dihydrotriazine ring was able to mimic the role played by those of i, detecting only one h-bond with e30. in addition, the bioisosteric replacement of the pyridopyrimidine scaffold with the smaller dihydrotriazine one, inevitably impaired the ability of the compound to display the same pattern of hydrophobic contacts, previously mentioned for i. this compound was the only one of the series here proposed to exhibit this kind of docking mode, losing key-contacts such as one h-bond with i7 and several van on all these basis, it was expected that the 4,6-diamino-1,2dihydrotriazine nucleus, properly decorated with small, rigid and planar groups, could represent an interesting scaffold able to fulfil the minimal pharmacophore requirements to exert hdhfr inhibition. this report describes the discovery of a new class of hostdirected antiviral agents characterized by a 1-aryl-4,6-diamino-1,2-dihydrotriazine scaffold, responsible for a host (human) dhfr inhibition mechanism. host-targeting antivirals represent an alternative and emerging strategy to address host factors involved in virus life cycle. this type of inhibitors could show a markedly higher barrier for selecting drug-resistant viruses and, furthermore, display broad-spectrum antiviral activity when interacting with a common cellular target that is recruited by different viruses. the interesting dual activity of the 1-aryl-4,6-diamino-1,2dihydrotriazines against influenza and respiratory syncytial viruses, via inhibition of the cellular (human) dhfr enzyme, points to this host factor as a new therapeutic target for these two respiratory viruses. in fact, reversal effect on antiviral activity has been demonstrated in rsv-infected hela cells exposed to compound 14 in combination with different concentrations of dihydrofolic acid, such as natural dhfr substrate. the most promising compounds, tested against the recombinant protein of the hdhfr, also confirmed to bind this enzyme in the sub-micromolar range. kinetic inhibition studies of cycloguanil showed a competitive inhibition behavior, and docking studies disclosed the most probable binding mode for this class of compounds as hdhfr ligands. notably, the antiviral activity against rsv and influenza b viruses in cell-based assays, the ki values on the recombinant hdhfr enzyme and, also the estimated binding affinity (dg) shared an interesting comparable sar trend. the possibility to suppress influenza virus by interfering with the purine or pyrimidine pathway was proposed for a few other enzymes [9, 41] but our study is the first to identify the relevance of host (human) dhfr in antiviral therapy. most of the compounds proved effective inhibitors of influenza b virus, giving sub-micromolar activity in case of the most potent analogues 11, 13, 14 and 16, which compare favorably with the licensed antiviral drugs zanamivir, even exceeding the antiviral efficacy of ribavirin. compounds 11, 14 and 16 also possessed low micromolar activity against influenza a virus and, even more importantly, nanomolar activity against rsv with a selectivity index of at least 10,000, far surpassing the antiviral activity of the reference drug ribavirin. these novel host-directed 1-aryl-4,6-diamino-1,2dihydrotriazine derivatives combine promising antiviral activity with low cost and easily accessible chemical synthesis. therefore, our results provide the foundation to further explore the structureactivity relationships of this class of compounds versus dhfr (especially human enzyme), and the apparently important role of this host enzyme in influenza and rsv virus replication. 6.1.2. general method for the synthesis of 4,6-diamino-1,2dihydrotriazine derivatives a solution of the substituted aniline hydrochloride (4.36 mmol) in 40 ml of acetone and 5 ml of meoh was reacted at r.t. with dicyandiamide (4.58 mmol, 1.05 equiv.) with stirring for 24 h. compounds separated directly from reaction mixture, thus they were collected by filtration, washed with acetone and recrystallized from the same solvent. only in the case of compound 17, the solution was evaporated to dryness under vacuum and the residue was recrystallized from meoh. the compounds' antiviral activity in cell culture was determined with a broad panel of viruses and using cytopathic effect (cpe) reduction assays described in detail elsewhere [42] . human influenza a/h1n1 and b viruses were examined on madin-darby canine kidney (mdck) cells [34] . human cervix carcinoma hela cells were used to study respiratory syncytial virus (rsv; strain long); vesicular stomatitis virus (vsv); and coxsackie b4 virus. african green monkey vero cells were used for para-influenza-3 virus; reovirus-1; sindbis virus; coxsackie b4 virus; punta toro virus and yellow fever virus. the following viruses were investigated in human embryonic lung fibroblast cells: herpes simplex virus types 1 and 2; vaccinia virus; human adenovirus type 2; vsv; and human coronavirus 229e. finally, the activity against human immunodeficiency virus types 1 and 2 was assessed in human mt-4 lymphoblast cells. semiconfluent cell cultures in 96-well plates were infected with the virus at a multiplicity of infection of 100 ccid 50 (50% cell culture infective dose) or 20 pfu (plaque forming units) per well. simultaneously with the virus, serial dilutions of the test or reference compounds were added. the plates were incubated at 37 c (or 35 c in the case of influenza and coronavirus) until clear cpe was apparent, i.e. during 3e6 days, or 10 days in the case of ad2. then, microscopy was performed to score the cpe and determine the antiviral activity [expressed as 50% effective concentration (ec 50 )] and cytotoxicity [expressed as minimum cytotoxic concentration (mcc)]. in the case of influenza virus and hiv, virus-induced cpe was also monitored by a colorimetric formazan-based cell viability assay. the capability of synthesized chemical library to inhibit the hdhfr protein was evaluated by spectrophotometric assay performing the dhf substrate (dihydrofolate) time-reading enzymatic consumption at 340 nm for 180 s. each inhibitor compound was dissolved in dmso in order to have an initial concentration equal to 50 mm. first, an inhibition assay at one concentration point (50 mm) has been performed in order to determine the best concentrations range to calculate the ic 50 value. the inhibition assay was performed considering a final volume equal to 600 ml. in details, the several reagents were added in this following order: ddw, hdhfr enzyme at concentration of 0.36 mm, inhibitor compound at each single evaluated concentration, dhf substrate at concentration of 50 mm, tes buffer, and, at the end, nadph was added to the reaction mixture, at concentration value equal to 120 mm, for starting the kinetic enzyme reaction. based on the obtained inhibition data, the concentrations range for the ic 50 evaluation have been selected. each inhibitor compound was assayed at five concentration values equal to 1-2-4-8-16 mm, except for compound 25 which was assayed at 50-100-200-400-1000 mm. based on the resulting inhibition data, ic 50 values for each compound by michaelis-menten kinetic in steady-state conditions have been calculated. all the compounds were built, parameterized (gasteiger-huckel method) and energy minimized within moe using mmff94 forcefield [43] . all ligands were used in their protonated state. docking calculations within the x-ray structure of human dhfr (pdb code ¼ 4qhv) were performed using the leadit 2.1.8 software suite (www.biosolveit.com) including the flexx scoring algorithm which is based on calculation of the binding free energy by means of gibbs-helmholtz equation [44e47] . the software detects the binding site defining a radius of 10 å far from the co-crystallized ligand, in order to set up a spherical search space for the docking approach. the standard setting as docking strategy was followed, choosing the so-called hybrid approach (enthalpy and entropy criteria), the related scoring function evaluation is described in the literature [44] . the derived docking poses were prioritized by the score values of the lowest energy pose of the compounds docked to the protein structure. all ligands were refined and rescored by assessment with the algorithm hyde, included in the leadit 2.1.8 software. the hyde module considers dehydration enthalpy and hydrogen bonding [48, 49] . finally, the reliability of the selected docking poses was assessed using a short~1 ps run of molecular dynamics (md) at constant temperature, followed by an all-atom energy minimization (low-modemd implemented in moe software). this kind of module allowed to perform an exhaustive conformational analysis of the ligand-receptor binding site complex, as we previously discussed about other case studies [50e52]. -fluorophenyl)-1,2-dihydrotriazine hydrochloride (12) yield 68%. mp 209e210 c (acetone). 1 h nmr (200 mhz, dmsod 6 ): 9.45 (s, 1h, þ nh, exchanges with d 2 o), 7.92e7.17 (m, 4 arom h and 7.74 br s, 3h, amino groups, exchange with d 2 o, superimposed signals) 13 c nmr (50 mhz 5-ditrifluoromethylphenyl)-1,2-dihydrotriazine hydrochloride (17) yield 51%. mp 195e198 c (meoh). 1 h nmr (200 mhz, dmsod 6 ): 9.50 (s, 1h, þ nh, exchanges with d 2 o), 8.26 (s, 1 arom h) -chlorophenyl)-2,2-cyclotetramethylene-4,6-diamino-1,2-dihydrotriazine hydrochloride (21) yield 38%. mp 241 c (acetone). 1 h nmr exchange with d 2 o), 6.56 (br s, 1h, amino group, exchanges with d 2 o), 1.94e1.20 (m, 8 h, (ch 2 ) 4 ). 13 c nmr (50 mhz, dmso-d 6 ) mhz, dmsod 6 ): 9.44 (s, 1h, þ nh, exchanges with d 2 o), 7.98e7.23 (m, 4 arom h and 3h, amino groups, exchange with d 2 o), 6.61 (br s, 1h, amino group mhz, dmsod 6 ): 9.26 (s, 1h, þ nh, exchanges with d 2 o), 8.05e7.40 (m, 4 arom h and 3h, amino groups, exchange with d 2 o), 6.51 (br s, 1h, amino group overview of the 3rd isirv-antiviral group conferenceeadvances in clinical management, influenza other respir pandemic preparedness and responseelessons from the h1n1 influenza of fight against h1n1 influenza a virus: recent insights towards the development of druggable compounds brave research agenda, world health organization current progress in antiviral strategies human ddx3 protein is a valuable target to develop broad spectrum antiviral agents influenza virus-host interactomes as a basis for antiviral drug development novel insights into human respiratory syncytial virushost factor interactions through integrated proteomics and transcriptomics analysis the influenza virus polymerase complex: an update on its structure, functions, and significance for antiviral drug design dihydrofolate reductase as a therapeutic target for infectious diseases: opportunities and challenges the search for new dhfr inhibitors: a review of patents 3-disubstituted quinazoline-4(3h)-one molecules derived from amino acid linked sulphonamide as a potent malarial antifolates for dhfr inhibition dihydroorotate dehydrogenase: a drug target for the development of antimalarials sulfonamides, trimethoprim-sulfamethoxazole, quinolones, and agents for urinary tract infections chemotherapy of protozoal infections: malaria the pharmacological basis of therapeutics much more than you expected: the non-dhfr-mediated effects of methotrexate repositioning of dhfr inhibitors antimalarial chemotherapy: mechanisms of action, resistance and new directions in drug discovery inhibitors of pneumocystis carinii dihydrofolate reductase antiviral activity of benzimidazole derivatives. i. antiviral activity of 1-substituted-2-[(benzotriazol-1/2-yl)methyl]benzimidazoles antimicrobial and cytotoxic arylazoenamines. part iii: antiviral activity of selected classes of arylazoenamines acridine derivatives as anti-bvdv agents antiviral activity of benzimidazole derivatives. iii. novel anti-cvb-5, anti-rsv and anti-sb-1 agents antiviral activity of benzotriazole derivatives. 5-[4-(benzotriazol-2-yl)phenoxy]-2,2-dimethylpentanoic acids potently and selectively inhibit coxsackie virus b5 structure-activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and pneumocystis carinii dihydrofolate reductase chemical and biological studies on 1,2-dihydro-s-triazines. ii. three-component synthesis studies in dihydrotriazines: 1-aryl-2,4-diamino-1,6-dihydro-6,6-dialkyl-1,3,5-triazines inhibition of bovine and rat liver dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(4-substituted-phenyl)-s-triazines 1-m-trifluoromethylphenyl-4,5-diamino-1,2-dibydro-2,2-dimethyl-1,3,5-triazine, patent inhibitors of multiple mutants of plasmodium falciparum dihydrofolate reductase and their antimalarial activities chemical and biological studies on 1,2-dihydro-s-triazines. iii. two-component synthesis novel inhibitors of influenza virus fusion: structureactivity relationship and interaction with the viral hemagglutinin chemotherapy of protozoal infections: malaria replicationcompetent influenza a and b viruses expressing a fluorescent dynamic timer protein for in vitro and in vivo studies inhibitors of the influenza a virus m2 proton channel discovered using a high-throughput yeast growth restoration assay azapropellanes with anti-influenza a virus activity chemotherapy of respiratory syncytial virus infections: the final breakthrough flaviviruses are sensitive to inhibition of thymidine synthesis pathways host factors regulating the influenza virus replication machinery a versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents moe: chemical computing group inc. montreal. h3a 2r7 canada the computer program ludi: a new method for the de novo design of enzyme inhibitors the development of a simple empirical scoring function to estimate the binding constant for a proteineligand complex of known threedimensional structure a fast flexible docking method using an incremental construction algorithm docking assay of small molecule antivirals to p7 of hcv towards an integrated description of hydrogen bonding and dehydration: decreasing false positives in virtual screening with the hyde scoring function substantial improvements in large-scale redocking and screening using the novel hyde scoring function in silico evaluation of human small heat shock protein hsp27: homology modeling, mutation analyses and docking studies synthesis, biological evaluation and molecular modeling of 1-oxa-4-thiaspiroand 1,4-dithiaspiro[4.5]decane derivatives as potent and selective 5-ht1a receptor agonists tricyclic pyrazoles. part 8. synthesis, biological evaluation and modelling of tricyclic pyrazole carboxamides as potential cb2 receptor ligands with antagonist/inverse agonist properties supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2017.04.070. key: cord-330465-16j5vm7h authors: marciniec, krzysztof; chrobak, elwira; dąbrowska, aleksandra; bębenek, ewa; kadela-tomanek, monika; pęcak, paweł; boryczka, stanisław title: phosphate derivatives of 3-carboxyacylbetulin: synthesis, in vitro anti-hiv and molecular docking study date: 2020-08-05 journal: biomolecules doi: 10.3390/biom10081148 sha: doc_id: 330465 cord_uid: 16j5vm7h lupane-type pentacyclic triterpenes such as betulin and betulinic acid play an important role in the search for new therapies that would be effective in controlling viral infections. the aim of this study was the synthesis and evaluation of in vitro anti-hiv-1 activity for phosphate derivatives of 3-carboxyacylbetulin 3–5 as well as an in silico study of new compounds as potential ligands of the c-terminal domain of the hiv-1 capsid–spacer peptide 1 (ca-ctd-sp1) as a molecular target of hiv-1 maturation inhibitors. in vitro studies showed that 28-diethoxyphosphoryl-3-o-(3′,3′-dimethylsuccinyl)betulin (compound 3), the phosphate analog of bevirimat (betulinic acid derivative, hiv-1 maturation inhibitor), has ic(50) (half maximal inhibitory concentration) equal to 0.02 μm. compound 3 inhibits viral replication at a level comparable to bevirimat and is also more selective (selectivity indices = 1250 and 967, respectively). molecular docking was used to examine the probable interaction between the phosphate derivatives of 3-carboxyacylbetulin and c-terminal domain (ctd) of the hiv-1 capsid (ca)–spacer peptide 1 (sp1) fragment of gag protein, designated as ctd-sp1. compared with interactions between bevirimat (bvm) and the protein, an increased number of strong interactions between ligand 3 and the protein, generated by the phosphate group, were observed. these compounds might have the potential to also inhibit sars-cov2 proteins, in as far as the intrinsically imprecise docking scores suggest. despite significant advances in medicine and continuous work on new pharmacotherapy methods, in addition to common preventive vaccination programs, in the first two decades of the 21st century, the world health organization (who) recorded many epidemics of viral diseases. among the great epidemics of this period were severe acute respiratory syndrome (sars) in the year 2003, the influenza h1n1 pandemic in 2009, middle east respiratory syndrome (mers) in 2012, and the zika virus in 2005, as well as the hiv/aids pandemic, which peaked in 2005-2012 [1] . the beginning of 2020 brought a new pandemic produced by the rapidly spreading virus that causes covid-19 disease. in the case of each new pathogen, the science world faces the difficult challenge of finding an effective therapy. chemical modification of natural substances is an important method used to obtain promising new therapeutic agents. pentacyclic triterpenes and their semi-synthetic derivatives are a large group of compounds known to demonstrate biological activity, including antitumor, antiviral, antimalarial, melting points of obtained compounds were measured in open capillary tubes on an electrothermal melting point apparatus without correction. 1 h nmr (600 mhz), 13 c nmr (150 mhz) and 31 p nmr (243 mhz) spectra were recorded on a bruker avance iii hd 600 spectrometer (bruker, billerica, ma, usa) in deuterated cdcl 3 , using the residual solvent signal as an internal standard. chemical shifts values were reported in parts per million (ppm). multiplicity was designated as singlet (s), doublet (d), doublet of doublets (dd) and multiplet (m). infrared spectra (pellets, kbr, merck, darmstadt, germany) were obtained using an iraffnity-1 ftir spectrometer (shimadzu, kyoto, japan). the measurement was recorded in the range of 4000-1000 cm -1 at 295k. high-resolution mass spectra have been measured with bruker impact ii (bruker, billerica, ma, usa). calculation of the theoretical molecular mass for compounds was performed using "the exact mass calculator, single isotope version" (http://www.sisweb.com/referenc/tools/exactmass.htm; (ringoes, nj, usa)). synthesis of bvm [3-o-(3',3'-dimethylsuccinyl)betulinic acid], used in the study as a reference, was performed as was previously described [12] . the final product was obtained at a yield of 70%. the melting point and spectroscopic characteristics of the compound were consistent with the literature data [20] . to the solution of 1 mmol of betulin 1 in 3 ml tetrahydrofuran (thf), pyridine (2.6 mmol, 0.24 ml) and n,n-dimethylaminopyridine (dmap; 0.1 mmol, 12 mg) was added. the obtained mixture was cooled in an ice-water bath to 0 • c, then diethylchlorophosphate (2 mmol, 0.29 ml) was added dropwise. the reaction was carried out under argon atmosphere for 9 h. then, the volatile components were evaporated on a vacuum evaporator. dichloromethane (15 ml) was added to the residue and washed with saturated sodium bicarbonate solution and water. the organic layer was dried with anhydrous sodium sulphate (vi), then concentrated until dry. the product was purified by column chromatography (sio 2 ; hexane/ethyl acetate ratio: 3:2, v/v) yielding compound 2. to the solution of 1 mmol 28-diethoxyphosphorylbetulin 2 in 2 ml pyridine, dmap (1.5 mmol, 0.19 g) and 5 mmol of appropriate acid anhydride (2,2-dimethylsuccinic anhydride or 3,3-dimethylglutaric anhydride or 2,2-dimethylglutaric anhydride) was added. the reaction vessel was placed in a microwave reactor and the reaction was carried out for 1.5 h at a temperature of 130 • c at a maximum wave power (300 w). after cooling, the mixture was diluted with 25 ml of ethyl acetate, and then washed with a 20% hydrochloric acid solution and with water. the organic layer was dried with anhydrous sodium sulphate (vi) and concentrated until dry in a vacuum evaporator. the crude product was purified by column chromatography (sio 2 ; chloroform/ethanol ratio: 15:1, v/v) to obtain phosphorus derivatives of 3-carboxyacyl-28-diethoxyphosphorylbetulin 3-5. for the determination of compounds' cytotoxicity, cem-t4 cells were obtained from the nih aids reagent program (nih, us) and were cultured in rpmi supplemented with 10% fcs (biochrom) and antibiotics at 37 • c in 5% co 2 on 96-well culture plates. the experiments were carried out in media containing tested compounds in concentrations of the appropriate range. cultures in a neat medium (rpmi, 10% fcs) were used as a control. viability of cells was determined after 7 days using the mtt assay [21] in which 10 µl of mtt solution (5 mg/ml) was added to each culture plate well, and cultures were incubated for 3 h at a temperature of 37 • c. after the centrifugation, the supernatant was removed, and dmso was added for lysis of the cells and to dissolve crystals of formazan. color intensity was measured with a plate reader at 560 nm. cem-t4 cells were preincubated (culture plates with 96 flat bottom wells) for 24 h under standard conditions (37 • c, 5% co 2 ) and in a standard medium (rpmi, fcs 10%) enriched with tested compounds in the concentration range from 0.02 to 10 µm. in each well, 20,000 cells were suspended in the solution of a tested compound (200 µl). for each concentration, cultures were run in triplicate. a wild-type hiv-1 was isolated from the hiv-positive patient in the laboratory of virology of the national medicines institute (warsaw, poland) and was used as a reference. a culture of cem-t4 lines in a standard neat medium (rpmu, fcs 10%) was used to produce viruses. after 24 h of incubation in a medium enriched with a tested compound, cells were inoculated with a known amount of hiv, and after 7 days, hiv replication was evaluated through the measurement of secreted viral protein p24 carried out with the enzyme-linked immunosorbent assay (elisa) technique [22] . for each tested compound and for each concentration, the measurements of p24 antigen were done in triplicate using the genscreen ultra hiv ag-ab kit (biorad, warszawa, poland) and following manufacturer's instructions. the 3d structures of studied compounds were generated in their low-energy conformation using the gaussian 16 (revision a.03) computer code [23] with the density functional theory (dft, b3lyp) and 63-11 + g(d,p) basis sets. target macromolecules for molecular docking studies were obtained from the protein data bank (https://www.rcsb.org/; research collaboratory for structural bioinformatics, usa). we used crystal structure of the ca-ctd-sp1 fragment of hiv-1 gag, sars-cov-2 main protein, sars-cov-2 rna-dependent rna polymerase, and sars-cov-2 spike ectodomain (pdb ids: 5i4t, 5r7z, 6m71, and 6vyb respectively). incomplete or missing side chains of ca-ctd-sp1 protein were restored with the swiss-model workspace (https://swissmodel.expasy.org/; swiss institute of bioinformatics, basel, switzerland) [24] . geometry optimization of the model was achieved by the optimization protocol in yasara energy minimization server (http://www.yasara. org/-minimizationserver.htm; yasara biosciences gmbh, vienna, austria). the sars-cov-2 main protease (m pro ) structure was obtained from pdb (pdb id: 5r7z). the native ligand for 5r7z is n-[2-(5-fluoro-1h-indol-3-yl)ethyl]ethanamide (pdb id: hwh). the region of interest used for vina docking was defined as all m pro residues within 40 × 40 × 40 å of the reference ligand. the homology model of the sars-cov-2 e protein catalytic domain was also generated in the swiss-model workspace [24] . based on maximum sequence identity (95%), structure with pdb id 5x29 is detected as the best homologous structure of human sars-cov-2 e protein. geometry optimization of model was done by the optimization protocol in yasara. for toxic/side effect study we used crystal structure of the pyruvate kinase (pdb id: 6nu1), aconitase (pdb id: 1c96), cytochrome c (pdb id: 1hrc), carbamoyl phosphate synthetase 1 (pdb id: 5dou), hypoxanthine-guanine phosphoribosyltransferase (pdb id: 6ar9), glutamate dehydrogenase (pdb id: 1bvg). in this study, autodock vina [25] tool compiled in pyrx [26] was employed to perform molecular docking. autodock vina incorporates limited flexibility in the receptor, and it combines an empirical free-energy force field with a lamarckian genetic algorithm, providing fast prediction of bound conformations with predicted free energies of association. the volume was set as 40 × 40 × 40 å. after calculations, only the 9 highest-scored poses were returned as a docking result for ligand-cavity configuration. all the obtained results were ranked according to their score values and presented in kcal/mol. molecular docking details were visualized using the biovia discovery studio virtual environment [27] . appropriate autodock vina output complexes were prepared for simulations using qwikmd [28] built in visual molecular dynamics (vmd https://www.ks.uiuc.edu/research/vmd/; theoretical and computational biophysics group, university of illinois at urbana-champaign, illinois, usa) software ver 1.9.3 [29] . simulation was carried out with namd (https://www.ks.uiuc.edu/research/namd/; theoretical and computational biophysics group, university of illinois at urbana-champaign, illinois, usa) ver. 2.13 [30] using charmm27 force field. periodic boundary conditions with an explicit solvent were employed. parameterization of ligands was conducted using a cgenff server [31, 32] . all protein and protein-ligand systems have been solvated with tip3p cubic water box at 15 å thickness. to neutralize the system, 0.15 mol/l of nacl salt was added. simulation protocol consists of 2000 steps of minimization, 144,000 steps of annealing, 500,000 steps of equilibration and 5,000,000 steps (10ns) of production md simulation. the system was heated to 300 k at rate of one kelvin degree per 1 ps. langevin dynamics were used to control the temperature. minimization, annealing and equilibration were restrained to backbone atoms of the protein, while the production run was unrestrained. timestep was set to 2 fs, all runs were performed in npt ensemble. vmd was used to analyze the results. the above-mentioned literature data and results of the anti-hiv activity test obtained for phosphate and phosphonate derivatives of betulinic acid [12] have become the starting point for the synthesis of betulin phosphate derivatives. the obtained compounds have a carboxyacyl group in position c3, whose presence is important for action against hiv-1, and a diethyl phosphate group in position c28 (scheme 1). this work attempts to answer the question of how the introduction of a phosphate substituent affects the activity of synthesized compounds, compared to substances with known potential-in this case, bvm. betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3 ,3 -dimethylsuccinic, 4 ,4 -dimethylglutaric, and 3 ,3 -dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc 50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic 50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . betulin 1 obtained from birch bark harvested in southern poland by extraction with dichloromethane was used as the starting substrate. after concentrating the extracts, the produced substance was purified by crystallization from ethanol. in the first stage, a phosphorylation reaction of a hydroxyl group at the c28 position was carried out using diethyl chlorophosphate. after purification, 28-diethoxyphosphorylbetulin 2 was obtained at a yield of 86%. compound 2 was then reacted with 3′,3′-dimethylsuccinic, 4′,4′-dimethylglutaric, and 3′,3′-dimethylglutaric anhydride according to the method previously described [33] . derivatives 3-5 were obtained at a yield of 32−38%. newly synthesized compounds 2-5 were evaluated for their activity against hiv-1 in a cem-t4 cell line. first, a cytotoxicity assessment of the synthesized derivatives was performed using the mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. the results were expressed as cc50 (concentration of compound causing 50% cell death). anti-hiv activity was expressed as ic50 (concentration of compound causing 50% inhibition of replication) and consisted of measurements of p24 antigen made using the genscreen ultra hiv ag-ab kit. betulin 1 and bvm were used as reference compounds. the bioassay results are presented in table 1 . as can be seen, betulin 1 and betulin phosphate 2 did not demonstrate the ability to inhibit hiv replication in the tested range of concentrations. betulin derivatives containing a 3 ,3 -dimethylglutaric group that exhibit significant activity against hiv-1 are described in the literature [34, 35] . corresponding phosphorus derivative 4 demonstrated good activity and selectivity that were higher than obtained for 4 ,4 -dimethylglutaric derivative 5. on the other hand, compound 4 was more active than the previously described 3 ,3 -dimethylglutaric derivatives of betulinic acid, which in the c30 position contained a diethylphosphate or diethylphosphonate group (ic 50 = 0.6 µm and 0.9 µm, respectively) [12] . the results obtained for phosphate derivatives of 3-carboxyacylbetulin 3-5 indicate that the most active compound is derivative 3 with r = hoc(o)c(ch 3 ) 2 ch 2 c(o), which shows activity comparable to that of bvm (ic 50 = 0.02 and 0.03 µm, respectively). a chart of changes in hiv-1 inhibition of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range is attached to the supplementary materials ( figure s1 ). however, compound 3 has a higher selectivity for action (therapeutic index (ti) = 1250 and 967, respectively). among the tested phosphate derivatives, compound 3 showed the lowest cytotoxicity, but it was at a level comparable to bvm. other phosphate derivatives were more toxic and also less active against hiv. as results from the data presented in the table 1 , compounds with a longer carbon chain in the carboxyacylic substituent are more cytotoxic. dimethylglutaric derivatives have an ic 50 figure s2 ). an important issue in the study of new chemical substances considered as potential drug candidates is the analysis of their physicochemical parameters. the in silico pharmacokinetic study is being used in the development of new drugs with good oral bioavailability, low toxicity and the least side effects. for phosphate derivatives 2-5 the adme (adsorption-distribution-metabolism-excretion) properties (supplementary materials table s1 ) such as number of hydrogen bond acceptors (nhba), number of hydrogen bond donors (nhbd), lipophilicity (log p), molecular weight m, number of rotatable bonds (nrotb) and topological polar surface area (tpsa) were evaluated through the pkcsm online server (http://biosig.unimelb.edu.au/pkcsm/; university of melbourne, australia) [36] . the hydrophobicity of compounds was determined on the basis of log p values. the log p parameter determines the strength of the drug and its distribution in the body after absorption stage. the obtained log p values (9.20-10.65) of triterpene derivatives indicate poor permeability across the cell membrane. the compounds shown in the table s1 having a number of rotational bonds in the range 8-13 show high conformational flexibility and good binding affinity with the binding pocket. topological polar surface area (tpsa) is important criteria for the determination of oral bioavailability. compounds that meet the criterion that tpsa ≤ 140 å may show oral bioavailability [37, 38] . the values of the parameters log ps, log bb and tpsa for the tested derivatives are characteristic for the cns-nonactive drugs [36] . the tested compounds can exist as two chemical species, carboxylic acids or carboxylate salts, depending on the ph of the aqueous solution. calculations performed with acd/percepta software [39] for bvm and compounds 3-5 show that in all cases the content of the carboxylate states at physiological ph is 100%. therefore, the carboxylate states of betulin derivatives were used in docking calculations. the three-dimensional (3d) structures of ligands required for docking studies were generated in their low-energy conformation using gaussian 16 computer code [24] . the last step of hiv-1 replication occurs after release of immature virion outside host cell. during maturation process, viral protease cleaves gag precursor protein to individual matrix (ma), spacer peptide (sp1), capsid (ca) and nucleocapsid (nc) peptides. this step is essential to produce mature and infectious virions. recent studies report that the immature ca-ctd-sp1 assembles to create a hexameric structure which forms a cone-shaped core built up with multiple hexameric proteins. protease can access the cleavage site only after the six-helix bundle unfolds [40] . based on atomic coordinates of the ctd of ca and sp1 of hiv-1 gag deposited in the protein data bank (pbd id 5i4t), one active site cavity was predicted [41] . this cavity was located inside the pore formed by the six-helix sp1 bundle (figure 1 ). we would like to point out that we used autodock ver.4.2.6 in our previous research on the antiviral activity of betulinic acid derivatives [12] . in the present study, we decided to use the program autodock vina (referred to as vina) for in silico research. vina uses a sophisticated gradient optimization method in its local optimization procedure. the calculation of the gradient effectively gives the optimization algorithm a "sense of direction" from a single evaluation. evaluation of the speed and accuracy of vina during flexible docking showed an improvement in speed of approximately two orders of magnitude, and a significantly higher accuracy of the binding mode prediction compared to autodock [25] . we also decided to redock the bvm molecule to hiv-1 ca-ctd-sp1 with the use of vina software. the betulin derivatives ranked by autodock vina are shown in table 2 . the lowest scores for binding energy (kcal/mol) of protein−ligand complexes correspond to a strong binding affinity, and the most probable ligand−protein system in vivo. results obtained with vina indicate that compound 3 exhibits a lower binding energy compared to the reference bvm ( table 2) . analysis of the bvm complex ( figure 2 ) included calculations, distance measurements, and pose geometries that determined salt bridge interaction of the ligand pose with lys227 residue of chain g and hydrogen bonding with lys158 in chain l. in addition, numerous hydrophobic interactions influence the increased stability of the complex. analysis of the compound 3 complex, the most potent compound in vitro (figure 3 ), determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. analysis of the compound 3 complex, the most potent compound in vitro (figure 3) , determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. analysis of the compound 3 complex, the most potent compound in vitro (figure 3) , determined salt hydrogen bonding between the carboxylate group of the ligand and lys158 residue of chain i, and hydrogen bonding with lys158 in chain l and the phosphate group. in addition, numerous hydrophobic interactions, including carbon−hydrogen bonding, are also visible. in order to validate docking results a molecular dynamics simulation (md) has been performed. after a 10-ns run, a root mean square deviation (rmsd) of the atomic positions of ligand-protein complexes have been calculated. low value of ligand rmsd followed by relatively constant value of protein backbone rmsd indicate complex stability and validate docking protocol. md simulation has been performed for compound 3, possessing the lowest score for binding energy. both ligand and protein showed low rmsd values below 2 å, proving stability of the ligand-protein system ( figure 6 ). the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 [44] [45] [46] [47] [48] [49] . table 3 . toxicity and side effect-causing protein target of drugs. physiological function site of action effect of deficiency/inhibition ref. in order to validate docking results a molecular dynamics simulation (md) has been performed. after a 10-ns run, a root mean square deviation (rmsd) of the atomic positions of ligand-protein complexes have been calculated. low value of ligand rmsd followed by relatively constant value of protein backbone rmsd indicate complex stability and validate docking protocol. md simulation has been performed for compound 3, possessing the lowest score for binding energy. both ligand and protein showed low rmsd values below 2 å, proving stability of the ligand-protein system ( figure 6 ). the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 the docking results are in line with cytotoxicity binding assay findings, which means that the tested compounds can interact with the ca-ctd-sp1 protein. in vitro studies in human cells, as well as preclinical studies, suggest that bevirimat should have low potential for cytotoxicity. there is no evidence of reproductive or developmental toxicity [42] . in order to check the potential toxic properties of the compounds 3-5, docking study of phosphate betulin derivatives to cellular proteins was carried out. it is known that deficiency and inhibition of some proteins important in normal cellular function may result in toxicity or side effects. examples of these proteins are those involved in key cellular metabolism processes such as glycolytic pathway, amino acid and nucleotide metabolism, urea cycle, citric acid cycle and oxidative phosphorylation in mitochondria [43] . table 3 gives a list of selected cellular proteins that are known to be associated with potential toxicity and side effects. information about the physiological function, site of action and effect of deficiency/inhibition for these proteins is also given in table 3 [44] [45] [46] [47] [48] [49] . [45] cytochrome c oxidative phosphorylation mitochondria increased sensitivity to cell heath signals triggered by tnf-α [46] carbamoyl phosphate synthetase i urea cycle mitochondria hyperammonemia [47] hypoxanthine-guanine phosphoribosyltransferase nucleotide biosynthesis mitochondria hyperuricemia [48] glutamate dehydrogenase amino acid degradation mitochondria nephrotoxicity [49] the results of docking experiments between bvm, compounds 3-5 and selected cellular proteins are shown in table 4 . according to the results of docking for the target proteins, all tested compounds showed a lower degree of fit to tested proteins compared to bvm. these results may indicate that modification of the bevirimat molecule by substitution of the carboxyl group by a phosphate substituent probably does not increase toxicity of phosphate derivatives compared to bvm. due to 2020's covid-19 pandemic, another group of rna viruses-coronaviruses-have become interesting to scientists. the majority of its large genome is transcribed and translated into polypeptide encoding proteins. these proteins are important for gene expression. the main protease (m pro ), and rna-dependent rna polymerase (rdrp) are key enzymes for coronavirus replication [50] . the m pro mediates the maturation of non-structural proteins (nsps), essential in the life cycle of the virus [51] . in research on various coronaviruses inhibitors, nsps and its rdrp domains have been used as a promising target for new drug candidates [52] . the sars-cov e protein is an integral membrane. most of the phases of the virus life cycle, such as envelope pathogenesis, formation, budding, and assembly are dependent on this protein [53, 54] . it has been suggested by several studies that the absence of the sars-cov-2 e protein may result in an "attenuated virus" [54] . spike (s) is the fundamental protein of the coronavirus, and forms a characteristic corolla structure on the membrane of the virion [55] . structural integrity of spike and cleavage activation play a key role in virus invasion and virulence. therefore, blocking coronavirus form entering host cell by targeting specific receptors on the host surface, such as s protein, might be therapeutic strategy of great value for the development of the anti-viral agents [56] . no specific medicine or treatment is currently available for sars-cov-2-related diseases. studies of remdesivir, phosphoramidate of an adenosine c-nucleoside analog, have brought attention to the possible application of this molecule as an anti-sars-cov-2 agent (figure 7 ) [57] . this rdrp inhibitor [18] can inhibit the virus by inhibiting synthesis of viral nucleic acid, and has been recently authorized for emergency use in acute covid-19 patients. the new betulin phosphate derivatives 2−5 reported in this paper were obtained from easily available material, botulin 1, via a brief two-step synthesis. compounds 3−5, with different carboxyacylic substituents at the c3 position, exhibited in vitro anti-hiv activity with ic50 values in the range of 0.02−0.22 μm. derivative 3, which has a 3',3'-dimethylsuccinyl moiety at the c3 position, exhibits the highest anti-hiv-1 activity (ic50 = 0.02 μm). for bvm, a known maturation inhibitor, the ic50 value determined in this study was comparable and equal to 0.03 μm. derivative 3 was characterized by slightly higher selectivity (ti values of 1250 and 967 for 3 and bvm, respectively). considering the literature reports on various potential mechanisms of anti-hiv activity of triterpenes, molecular modeling with ca-ctd-sp1 has been carried out. the optimal fit was demonstrated by compound 3. this effect suggests a potential molecular target that determines anti-hiv activity of the studied compounds. supplementary materials: the following are available online at www.mdpi.com/xxx/s1, characteristics of synthesized compounds, figure s1 : anti-hiv-1 activity of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range, figure s2 : cytotoxicity of betulin phosphate 2-5 in the tested concentration range, table s1 : selected physicochemical properties of the compounds 2-5, table s2 : scoring functions of the tested compounds (sars-cov-2 proteins), figure s3 : the lowest-energy docking poses of sars-cov-2 mpro protein complexes with bvm (a) and betulinic acid (b), figure s4 : visualization of interaction between bvm (a) and compound 4 (b) with sars-cov-2 rdrp, figure s5 : docking pose of sars-cov-2 e protein complexes with bvm (a) and compound 6 (b), figure s6 : visualization of interaction between betulinic acid (a) and bvm (b) with sars-cov-2 spike protein monomer, figure s7 : rmsd for atoms of protein (mpro, rdrp, e) backbones (left) and ligands (right), figure s8 : rmsd for atoms of s protein backbones (left) and ligands (right), figure s9 : rmsd for atoms of e protein backbones without terminal residues, table s3 : interactions of tested compounds with sars-cov-2 proteins (references [59] [60] [61] are cited in the supplementary materials). on 31 january 2020, the new england journal of medicine reported the diagnosis and treatment of the first sars-cov-2 patient in the united states [58] , and remdesivir exhibited some potential in the treatment of this first patient. accordingly, we used remdesivir as a reference ligand. moreover, based on our previous study, we decided to dock to sars-cov-2 proteins betulinic acid and 30-diethoxyphosphoryl-3-o-(3 ,3 -dimethylsuccinyl)betulinic acid (compound 6, figure 7 ). compound 6 demonstrated in vitro anti-hiv-1 activity with an ic 50 value similar to that of bvm (ic 50 = 0.02 µm and 0.03 µm for 6 and bvm, respectively), and with a lower cytotoxicity (cc 50 = 68 µm and 29 µm for 6 and bvm, respectively) [12] . according to the results of docking (table s1 ) obtained from autodock vina, four potential sars-cov-2 inhibitors (bvm, betulinic acid, and compounds 4 and 6) were selected based on a lower negative dock energy value. detailed interactions between ligands and selected sars-cov-2 proteins are included in supplementary materials (figures s3-s6 and tables s2 and s3) . md simulation has been performed for compounds possessing the lowest score for binding energy (bvm, betulinic acid, and compounds 4 and 6). low fluctuations of rmsd of all proteins indicate that they reached stable conformation (supplementary materials figures s7-s9 ). the new betulin phosphate derivatives 2−5 reported in this paper were obtained from easily available material, botulin 1, via a brief two-step synthesis. compounds 3−5, with different carboxyacylic substituents at the c3 position, exhibited in vitro anti-hiv activity with ic 50 values in the range of 0.02−0.22 µm. derivative 3, which has a 3',3'-dimethylsuccinyl moiety at the c3 position, exhibits the highest anti-hiv-1 activity (ic 50 = 0.02 µm). for bvm, a known maturation inhibitor, the ic 50 value determined in this study was comparable and equal to 0.03 µm. derivative 3 was 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for anti-covid-19 drug design antiviral activity of nucleoside analogues against sars-coronavirus (sars-cov) insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates structural insights into coronavirus entry molecular docking study of receptor binding domain of sars-cov-2 spike glycoprotein with saikosaponin, a triterpenoid natural product remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the following are available online at http://www.mdpi.com/2218-273x/10/8/1148/s1, characteristics of synthesized compounds, figure s1 : anti-hiv-1 activity of 3-carboxyacylbetulin phosphate and bvm in the tested concentration range, figure s2 : cytotoxicity of betulin phosphate 2-5 in the tested concentration range, table s1 : selected physicochemical properties of the compounds 2-5, table s2 : scoring functions of the tested compounds (sars-cov-2 proteins), figure s3 : the lowest-energy docking poses of sars-cov-2 mpro protein complexes with bvm (a) and betulinic acid (b), figure s4 : visualization of interaction between bvm (a) and compound 4 (b) with sars-cov-2 rdrp, figure s5 : docking pose of sars-cov-2 e protein complexes with bvm (a) and compound 6 (b), figure s6 : visualization of interaction between betulinic acid (a) and bvm (b) with sars-cov-2 spike protein monomer, figure s7 : rmsd for atoms of protein (mpro, rdrp, e) backbones (left) and ligands (right), figure s8 : rmsd for atoms of s protein backbones (left) and ligands (right), figure s9 : rmsd for atoms of e protein backbones without terminal residues, table s3 : interactions of tested compounds with sars-cov-2 proteins (references [59, 60] are cited in the supplementary materials). key: cord-323093-u3ozc9ry authors: rathnayake, athri d.; zheng, jian; kim, yunjeong; perera, krishani dinali; mackin, samantha; meyerholz, david k.; kashipathy, maithri m.; battaile, kevin p.; lovell, scott; perlman, stanley; groutas, william c.; chang, kyeong-ok title: 3c-like protease inhibitors block coronavirus replication in vitro and improve survival in mers-cov–infected mice date: 2020-08-19 journal: sci transl med doi: 10.1126/scitranslmed.abc5332 sha: doc_id: 323093 cord_uid: u3ozc9ry pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and the newly emerged sars-cov-2, the causative agent of coronavirus disease 2019 (covid-19). we describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3c-like protease (3clpro), an enzyme essential for viral replication. the optimized compounds were effective against several human coronaviruses including mers-cov, sars-cov, and sars-cov-2 in an enzyme assay and in cell-based assays using huh-7 and vero e6 cell lines. two selected compounds showed antiviral effects against sars-cov-2 in cultured primary human airway epithelial cells. in a mouse model of mers-cov infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. these results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. coronaviruses are a large group of viruses that can cause a wide variety of diseases in humans and animals (1) . human coronaviruses generally cause the common cold, a mild upper respiratory illness. however, global outbreaks of new human coronavirus infections with severe respiratory disease have periodically emerged from animal reservoirs, including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and, most recently, sars-cov-2, the causative agent of coronavirus disease 2019 . sars-cov-2 emerged in china in december 2019 and subsequently rapidly spread throughout the world. genetic analysis of sars-cov-2 revealed that it is closely related to sars-like betacoronaviruses of bat origin, bat-sl-covzc45 and bat-sl-covzxc21 (2) . despite the periodic emergence of new coronaviruses capable of infecting humans, there are currently no licensed vaccines or antiviral drugs against any coronaviruses, underscoring the urgent need for the development of preventive and therapeutic measures against pathogenic coronaviruses. the coronavirus genome contains two overlapping open reading frames (orf1a and orf1b) at the 5′ end terminal, which encode polyproteins pp1a and pp1ab. the polyproteins are processed by a 3c-like protease [3clpro or main protease (mpro)] (11 cleavage sites) and a papain-like protease (plpro) (3 cleavage sites), resulting in 16 mature nonstructural proteins, including an rna-dependent rna polymerase (rdrp). both 3clpro and plpro are essential for viral replication, making them attractive targets for drug development (3) (4) (5) (6) (7) . coronavirus 3clpro is a cysteine protease that has two n-terminal domains containing two -barrel chymotrypsin-like folds (8) (9) (10) . the active site of 3clpro is located in the cleft between the two domains and is characterized by a catalytic cys-his dyad. we have developed broad-spectrum inhibitors of an array of viruses, including coronaviruses and noroviruses (11) (12) (13) (14) (15) (16) (17) (18) that use 3clpro for viral replication and picornaviruses that use 3c protease (19) . we have shown efficacy of the coronavirus 3clpro inhibitor gc376 (currently in clinical development) in animal models of coronavirus infection (20, 21) . specifically, administration of gc376 to cats with feline infectious peritonitis (fip), a coronavirus-induced systemic disease that is 100% fatal, reversed the progression of fip and resulted in clinical remission (20, 21) . we have recently reported the results of exploratory in vitro studies using a dipeptidyl series of mers-cov 3clpro inhibitors that embody a piperidine moiety as a new design element, as well as pertinent structural and biochemical studies (17) . here, we report the development of 3clpro inhibitors against multiple coronaviruses, including sars-cov-2, and demonstrate in vivo efficacy against mers-cov in a mouse model. the synthesis scheme for compound series 6a to 6k and 7a to 7k is shown in fig. 1 and described in supplementary materials and methods. the activity of compounds 6a to 6k and 7a to 7k against the 3clpro enzymes of mers-cov, sars-cov, and sars-cov-2 was evaluated in a fluorescence resonance energy transfer (fret) enzyme assay (table 1 and table s1 ). several compounds in this series (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) were also tested in cell-based assays ( table 2) . table 1 and table s1 show 50% inhibitory concentration (ic 50 ) values in a fret enzyme assay for select compounds (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) and gc376. fifty percent inhibitory concentration (ec 50 ) values and 50% cytotoxic concentration (cc 50 ) values for select compounds and gc376 were measured in cell culture assays (table 2) . cell culture assays included huh-7 cells infected with mers-cov, vero e6 cells infected with sars-cov-2, the crandell-rees feline kidney (crfk) cells infected with fip virus (fipv), and l929 cells infected with mouse hepatitis virus (mhv) ( table 2 ). inhibitors with a p 2 leucine (leu) residue were more potent than those with a cyclohexylalanine against mers-cov 3clpro (compounds 6h and 7h versus 6i and 7i), with submicromolar ic 50 values (table 1 and table s1 ). the compounds tested against mers-cov in cell culture (7a, 6c, 7e, 7h, and 6j) also displayed submicromolar ec 50 values. among these compounds, 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m. gc376 with a p 2 leu residue and a nonfluorinated benzyl cap exhibited 20-fold lower potency against mers-cov in cell culture compared to compound 6j ( table 2) . the compounds were also effective against sars-cov-2 with ec 50 values ranging from 0.15 to 0.9 m in vero e6 cells (table 2) . these compounds were also found to be potent against fipv and mhv, with ec 50 values ranging from 0.07 to 0.22 m. in the fret enzyme assay, these compounds were active against the 3clpro of sars-cov and sars-cov-2 ( table 1 ). the ic 50 values of these compounds against sars-cov-2 3clpro ranged from 0.17 to 0.82 m. among these compounds, 6e showed the most potent antiviral ac-tivity against sars-cov-2 in the enzyme assay (ic 50 , 0.17 m) and cell-based assay (ic 50 , 0.15 m) (tables 1 and 2) . gc376 also exhibited activity against the 3clpro of sars-cov-2 with an ic 50 value of 0.62 m in the enzyme assay ( table 1) . the antiviral effects of compounds 6j and 6e against sars-cov-2 were confirmed in cultured primary human airway epithelial cells from three donors. in the absence of a 3clpro inhibitor, viral titers in the infected cultured primary human airway epithelial cells reached 10 7.3 (donor 1), 10 7.1 (donor 2), and 10 8.4 (donor 3) plaque-forming units (pfu) per milliliter of culture medium. in the presence of compound concentrations that were about two-to threefold higher than the ec 50 values obtained in vero e6 cells (2 m, 6j or 0.5 m, 6e), viral titers were reduced to 10 6.4 (donor 1), 10 6.1 (donor 2), and 10 6.3 (donor 3) pfu/ml for compound 6j or 10 6.1 (donor 1), 10 6.5 (donor 2), and 10 8.1 (donor 3) pfu/ml for compound 6e (table 3) . although there was some variation in viral replication among infected cells from the three donors (especially donor 3), the antiviral effects of both 6j and 6e were evident at the tested concentrations. for infected cells from donors 1 and 2, both compounds inhibited viral replication about 10-fold at the tested concentrations. for infected cells from donor 3, 6e inhibited virus replication about 50%, whereas 6j inhibited virus replication 100-fold at the tested concentrations. we determined multiple high-resolution cocrystal structures of compounds 6b, 6d, 6g, 6h, 7i, or 7j with the 3clpro of mers-cov (fig. 2 , a to f, and figs. s1 to s3). these inhibitors bound to the active site of mers-cov 3clpro, demonstrating that the vicinity of the s 4 pocket is encompassed by an array of primarily hydrophobic residues, including phe 188 , val 193 , ala 171 , and leu 170 (fig. 2 , c and f, and fig. s3 ). hydrophobic and hydrogen-bonding functionalities were incorporated into the 3clpro inhibitors to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate congeners. the bisulfite adducts reverted to the corresponding aldehydes, which subsequently reacted with cys 148 to form nearly identical covalent complexes with a tetrahedral arrangement at the newly formed stereocenter (fig. 2 , a to f, and figs. s1 to s3). the backbone of compound 6h (table 1 and fig. 2 , a to c) engaged in h-bond interactions with amino acid residues gln 192 , gln 167 , and glu 169 . three additional side-chain h-bonds between the -lactam ring and his 166 , phe 143 , and glu 169 also were clearly evident (fig. 2b) fig. 1 . synthesis scheme for compound series 6a to 6k and 7a to 7k. stepwise compound synthesis with intermediate compounds is shown for 3c-like protease (3clpro) inhibitors of the 6a to 6k and 7a to 7k series. the alcohol inputs were reacted with (l) leucine isocyanate methyl ester or (l) cyclohexylalanine isocyanate methyl ester to yield products, which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. subsequent coupling of the acids to glutamine surrogate methyl ester "8" furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate. a amino acid methyl ester isocyanate/tea/ch 3 was ensconced in the hydrophobic s 2 pocket (fig. 2c) . the extra methylene group in compound 7j, which was converted to an aldehyde and thus became identical to 6j, resulted in the reorientation of the difluorocyclohexyl group and the formation of three h-bonds between gln 195 and ala 171 and the fluorine atoms, with concomitant loss of one of the gln 192 hydrogen bonds and the displacement of phe 143 (fig. 2e ). the substitution of the p 2 leu with p 2 cyclohexylalanine (compound 7i; table s1) resulted in the loss of an h-bond with gln 192 but otherwise adopted the same interactions as observed for compound 6h (fig. s2a ). the electron density, hydrogen bond interactions, and electrostatic surface representations for mers-cov 3clpro in complex with compounds 6b, 6g, and 6d are shown in figs. s1 to s3. next, compound 7j was cocrystallized with sars-cov or sars-cov-2 3clpro, and the structures were compared to that for mers-cov 3clpro and 7j. in the sars-cov 3clpro-7j complex (fig. 2 , g to i), the backbone of compound 7j formed direct h-bonds with cys 145 , his 163 , his 164 , glu 166 , and gln 189 . compound 7j also formed an additional h-bond with his 41 and a water-mediated contact with gly 143 (fig. 2h ). however, there was a loss of the three h-bonds between gln 195 and ala 171 and the fluorine atoms, compared to the cocrystal structure of 7j with mers-cov 3clpro. the electron density map was consistent with both possible enantiomers at the new stereocenter formed by covalent attachment of the s atom of cys 145 in the cocrystal structure of sars-cov 3clpro with 7j. the electron density map for compound 7j in complex with sars-cov-2 3clpro was most consistent with a single enantiomer, although it adopted a similar binding mode and hydrogen bond interactions as observed in the sars-cov 3clpro-7j cocrystal structure (fig. 2 , j to l). superposition of compound 7j with mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro ( fig. s4 ) revealed a very similar binding mode for 7j among all three viral proteases. the most potent compound of the series, 6j, was identified in a cellbased assay and had an ec 50 value of 0.04 m against mers-cov (fig. 3a) . we determined the efficacy of compounds 6j and 6h in transgenic hdpp4-ki mice expressing human dipeptidylpeptidase 4, a model of mers-cov infection. first, hdpp4-ki mice were infected with the mouse-adapted mers-cov (mers ma -cov) virus strain and then were treated with compounds 6h and 6j (50 mg/kg per day, once a day) or vehicle as a control starting 1 day post virus infection (1 dpi) and continuing until 10 dpi. all mice treated with vehicle control died by 8 dpi (fig. 3b ). in contrast, 40% of mice treated with compound 6h survived, and all mice treated with compound 6j were alive at the end of the study (15 dpi) (fig. 3b ). the survival of mice treated with compound 6j or 6h was increased compared to the vehicle control (p < 0.05), and the 6j-treated mice had an improved survival rate compared to 6h-treated mice (p < 0.05). all mice treated with compound 6j rapidly recovered from body weight loss starting at 3 dpi (fig. 3c ). the mice that survived after 6h treatment continued to lose body weight until 6 dpi but then started to gain weight from 9 dpi (fig. 3c ). after we observed that treatment with compound 6j resulted in the survival of mers ma -cov-infected hdpp4-ki mice, we conducted another study by delaying treatment initiation until 3 dpi. similar to the first study, no untreated mice or mice given vehicle (control) survived, and there was no statistical difference between these two groups (0% survival) (fig. 3d ). when 6j treatment was started on 1 dpi, four of the five mice survived (80% survival), and there was a statistically significant increased survival in mice treated starting at 1 dpi compared to untreated or vehicle control-treated mice (p < 0.05; fig. 3d ). when 6j treatment was delayed by one additional day (2 dpi), the survival of mice treated with 6j decreased to 40%, but this was still higher than the 0% survival for untreated or vehicle controltreated mice. however, there was no statistical difference between the 6j treatment group starting at 2 dpi and the untreated or vehicle control-treated groups (fig. 3d ). treatment with 6j starting at 3 dpi also failed to improve the survival of mice compared to the untreated or vehicle control-treated groups (fig. 3d ). all mice lost body weight after virus infection, but surviving mice treated with 6j regained the lost weight by 15 dpi (fig. 3e ). recovery of body weight was faster in mice treated with 6j starting at 1 dpi than table 3 . antiviral effects of compounds 6j and 6e against sars-cov-2 in cultured primary human airway epithelial cells. the study was a single experiment, and viral titers were measured in duplicate with a plaque-forming assay. vehicle control at 2 dpi (fig. 3e) . these results show that survival of mice markedly increased when 6j was given at 1 dpi. the lung pathology caused by mers ma -cov infection of hdpp4-ki mice resembles that seen in severe cases of human mers-cov infection, with diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . a group of hdpp4-ki mice were infected with mers ma -cov and treated with compound 6j or vehicle as a control starting at 1 dpi. mouse lungs were collected for determination of virus load at 3 and 5 dpi and for histopathology at 6 dpi. lung virus titers decreased in the 6j-treated mice compared to control mice at both 3 and 5 dpi (p < 0.01) (fig. 4a) . edema in the lungs of the treated mice was reduced compared to control mice (p < 0.01) (fig. 4b) . scores for hyaline membrane formation were reduced in 6j-treated mice but were not statistically different from control mice. mers ma -cov-infected hdpp4-ki mice treated with vehicle showed patches in lung tissue variably composed of cellular inflammation, vascular congestion, and atelectasis (fig. 4 , c and e). the airways of these animals were generally intact, with only scattered, uncommon sloughed cells (fig. 4 , c and e). in some lungs from these mice, lymphatic vessels were filled with degenerative cells and cellular debris (fig. 4 , c and e). alveolar edema was detected in some lung tissue sections (fig. 4 , c and e). in contrast, there were few observed lesions in the lungs of mers ma -cov-infected hdpp4-ki mice treated with compound 6j starting at 1 dpi (fig. 4 , d and f). there are currently no approved vaccines or small-molecule therapeutics for the treatment of mers-cov, sars-cov, or sars-cov-2 infection. however, numerous preventive and therapeutic options are under development (3) (4) (5) (6) (7) . the most clinically advanced antiviral compound with a broad spectrum of activity is remdesivir (gs-5734). this nucleoside analog was originally developed as an antiviral drug against ebola virus and has been shown to be effective against both mers-cov and sars-cov in cell culture assays and in animal models of coronavirus infection (23) (24) (25) (26) . prophylactic treatment or early therapeutic treatment of infected mice with remdesivir reduced mers-cov-or sars-cov-mediated weight loss and decreased lung virus titers and lung injury scores compared to those of vehicle-treated animals (23, 26) . remdesvir also showed potent activity against sars-cov-2 in cell culture assays and animal models (27) and was recently issued an emergency use authorization by the u.s. food and drug administration as an investigational antiviral drug for covid-19. another nucleoside analog, eidd-2801, which is a broad-spectrum inhibitor against multiple viruses including influenza viruses, was also shown to be effective against mers-cov and sars-cov in mouse models (28) . our group has been engaged in the discovery of broad-spectrum inhibitors targeting the 3clpro of multiple human and animal coronaviruses. we initially generated dipeptidyl and tripeptidyl series of compounds (29) and observed that the dipeptidyl compound series had superior pharmacokinetic (pk) profiles compared to the tripeptidyl compound series (20) . a representative compound of the dipeptidyl series is gc376, which is currently in clinical development for fip in cats and covid-19. the pk characteristics of multiple dipeptidyl compounds similar to compound 6j, including gc376, were examined through an intraperitoneal or subcutaneous administration in animals, including mice. it was determined that their maximum plasma concentration (c max ) values were >100-fold of the ec 50 for the target virus and that the elimination half-life (t 1/2 ) was 3 to 5 hours. the in vivo efficacy of gc376 against mice infected with mhv or murine norovirus has been demonstrated (11, 15) . inspection of previously obtained crystal structures of the dipeptidyl compounds in a complex with coronavirus 3clpro (17, 19) revealed the potential to achieve enhanced binding interactions with the s 4 subsite by introducing diverse functionalities at the cap position in the inhibitors. in the current study, a new dipeptidyl series focusing on the design of structural variants in the cap substructure were synthesized and evaluated for their activity against coronavirus 3clpro. the ec 50 of gc376 against mers-cov was determined to be ~1 m. one of our goals was to generate compounds with near or below 0.1 m potency against mers-cov and other target coronaviruses. all synthesized compounds displayed varying degrees of inhibitory activity against multiple coronaviruses in the fret enzyme assay and cell-based assays. among these compounds, 6e showed the most potent antiviral activity against sars-cov-2 3clpro in a fret enzyme assay (ic 50 , 0.17 m) and cell-based assays (ec 50 , 0.15 m), and 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m (table 2) . it was previously demonstrated that optimal potency is attained when the p 1 and p 2 residues are a glutamine surrogate and leu, respectively, and that replacement of the p 2 leu with a cyclohexylalanine is inimical to potency (17, 18) . this is clearly evident when comparing the relative potencies of 6h and 7h versus 6i and 7i (table 1 and table s1 ). furthermore, compounds with leu at the p 2 position showed higher cc 50 values compared to those with cyclohexylalanine at p 2 (table s1). x-ray crystallography confirmed the mechanism of action of the inhibitors, which involves formation of a covalent bond between the active site cysteine and the carbonyl carbon of the aldehyde. x-ray crystallography also identified the structural determinants associated with binding, accounting for the observed differences in potency. the high-resolution cocrystal structures of 3clpro inhibitors 7j and 7i with mers-cov 3clpro also confirmed that the difference in activity arose from the loss of a h-bond with gln 192 and the loss of two additional h-bonds from the displacement of gln 167 and phe 143 with 7i ( fig. s2a ). the nature of the interaction of 7j with the s 4 subsite is unique among the compounds examined and provides strong support for our approach, vis-à-vis our focus on the cap position for enhancing binding affinity and potency. compound 7j was cocrystallized with mers-cov, sars-cov, and sars-cov-2 3clpro. superposition of compound 7j with these 3clpro enzymes revealed a similar binding mode among all three proteases. however, a key difference lies with the different conformation adopted by the difluorocyclohexyl ring in the mers-cov 3clpro s 4 subsite, enabling it to engage in additional h-bond binding interactions (fig. 2e and fig. s4 ). compound 7j had a moderately lower potency against sars-cov 3clpro and sars-cov-2 3clpro compared to mers-cov 3clpro in the fret enzyme assay, suggesting that moieties forming h-bonds that were accommodated at the s 4 subsite had an important impact on potency. the barrier to the development of drug resistance increases when an inhibitor engages in h-bond interactions with the backbone of the 3clpro. we used a robust mouse model of mers-cov infection (30-32) to evaluate the efficacy of compounds 6j and 6h. hdpp4-ki mice expressing human dipeptidylpeptidase 4 were infected with a mouseadapted mers-cov virus (mers ma -cov). the infected mice develop fatal lung disease with severe inflammation and weight loss (32) . furthermore, the lung pathology caused by mers ma -cov infection of the hdpp4-ki mice closely resembles that of severe human mers-cov infection and is characterized by diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . in the current study, we demonstrated that survival rates in this mouse model were higher with 6j treatment compared to 6h treatment (fig. 3b) . compounds 6j and 6h share a near-identical structure except for the extra methylene group present in compound 6j. compound 6h showed potent anti-3clpro activity, whereas the antiviral activity of compound 6h in cell culture was lower than that of compound 6j (tables 1 and 2) , which may have been the reason for its diminished therapeutic efficacy in the mouse model (fig. 3b) . our findings indicate that therapeutic treatment of infected mice with compound 6j was associated with a reduction in lung viral load and lung pathology (fig. 4) . moreover, treatment of mice with 6j at 1 dpi resulted in the survival of infected mice, whereas delaying treatment initiation until 3 dpi resulted in decreased survival. overall, mouse survival was markedly increased only when 6j was given to mice at 1 dpi (p < 0.05; fig. 3 , d and e). treatment with compound 6j starting at 2 dpi resulted in moderately increased survival of infected mice, but this was not statistically significant (p > 0.05). these results emphasize the importance of early therapeutic intervention in attaining a positive clinical outcome. earlier studies from our group showed that gc376 can cure fatal feline coronavirus disease in cats (20, 21) , demonstrating that a specific coronavirus protease inhibitor can be effective therapeutically against coronavirus disease in a natural host. the mers-cov mouse model used here provides proof of principle regarding the therapeutic potential of our protease inhibitors for treating severe human respiratory coronavirus disease. limitations of the current study include differences in host receptor usage, mortality, and transmissibility between mers-cov and sars-cov-2. thus, further evaluation of our protease inhibitors in mice, hamsters, or nonhuman primates experimentally infected with sars-cov-2 will be crucial to assess these inhibitors as potential therapeutic options for covid-19. our study showed that these compounds were broadly active against the 3clpro of several coronaviruses, with compound 6j displaying the highest activity against mers-cov and compound 6e displaying the highest activity against sars-cov-2. clinical efficacy is influenced by many factors, including drug bioavailability, pk, metabolism, and the chemical stability of a compound. this poses a major challenge with respect to reliably predicting whether the difference in potency against different coronaviruses in assays in vitro can be translated to differences in clinical efficacy. therefore, further research is needed to establish whether one protease inhibitor can be an effective therapeutic for both mers-cov and sars-cov-2 infections in humans. the results from our previous and current studies suggest that the dipeptidyl compound series can serve as a platform suitable for the structure-guided design of one or more inhibitors against highly virulent human coronaviruses. we have generated potent inhibitors of the 3clpro of several coronaviruses, including sars-cov-2, and tested their efficacy in cultured cells and primary human airway epithelial cells. furthermore, we have demonstrated proof-of-concept therapeutic efficacy for one 3clpro inhibitor 6j in hdpp4-ki mice infected with mers ma -cov. our study lays the foundation for advancing this compound series further along the drug development pipeline. the goal of this study was to evaluate the efficacy of 3clpro inhibitors against human coronaviruses, including sars-cov-2, in a fret enzyme assay and cell culture assays, as well as in a mouse model of mers-cov infection. initial antiviral screening was performed with recombinant 3clpro from sars-cov, mers-cov, and sars-cov-2 in the fret enzyme assay. antiviral activity was then assessed in cultured huh-7 cells infected with mers-cov and vero e6 cells infected with sars-cov-2. selected 3clpro inhibitors were further examined using x-ray cocrystallization with mers-cov, sars-cov, and sars-cov-2 3clpro to elucidate the mechanism of action and identify the structural determinants of potency. last, two selected compounds were evaluated for in vivo efficacy in a mouse model of mers-cov infection (hdpp4-ki mice expressing human dipeptidylpeptidase 4 infected with a mouse-adapted mers-cov). age-and sex-matched mice were randomly assigned into various groups for virus infection and treatment studies. microscopic analysis of lung lesions was conducted in a blinded manner; other experiments were not blinded. no mice were excluded from analysis. in vivo studies were performed in animal biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional animal care and use committee at the university of iowa according to guidelines set by the association for the assessment and accreditation of laboratory animal care and the u.s. department of agriculture. the studies with mers-cov and sars-cov-2 were performed in biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional biosafety committee at the university of iowa according to guidelines set by the biosafety in microbiological and biomedical laboratories, the u.s. department of health and human services, the u.s. public health service, the u.s. centers for disease control and prevention, and the national institutes of health. compounds 6a to 6k and 7a to 7k were readily synthesized as illustrated in fig. 1 and are listed in table 1 and table s1 . briefly, the alcohol inputs were reacted with l-leucine isocyanate methyl ester or l-cyclohexylalanine isocyanate methyl ester to yield dipeptides "2", which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. the subsequent coupling of the acids to glutamine surrogate methyl ester "8" (33, 34) furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate (35) . the expression and purification of the 3clpro of mers-cov and sars-cov were performed by a standard method described previously by our lab (11, 19, 20) . we also cloned and expressed the 3clpro of sars-cov-2. the codon-optimized complementary dna (cdna) of the full length of 3clpro of sars-cov-2 (genbank accession number mn908947.3) fused with sequences encoding six histidine at the n terminus was synthesized by integrated dna (coralville, ia). the synthesized gene was subcloned into the pet-28a(+) vector. the expression and purification of sars-cov-2 3clpro were conducted after a standard procedure described by our lab (19) . briefly, stock solutions of compounds 6a to 6k and 7a to 7k were prepared in dimethyl sulfoxide (dmso) and diluted in assay buffer, which was composed of 20 mm hepes buffer (ph 8) containing nacl (200 mm), edta (0.4 mm), glycerol (60%), and 6 mm dithiothreitol (dtt). the protease (3clpro of mers-cov, sars-cov, or sars-cov-2) was mixed with serial dilutions of each compound or with dmso in 25 l of assay buffer and incubated at 37°c for 30 min (mers-cov) or at room temperature for 1 hour (sars-cov and sars-cov-2), followed by the addition of 25 l of assay buffer containing substrate (fam-savlq/sg-qxl 520, anaspec, fremont, ca). the substrate was derived from the cleavage sites on the viral polyproteins of sars-cov. fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (flx800, biotek, winooski, vt) 1 hour after the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously (19) . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) to determine the ic 50 values of the compounds. some compounds in the 6a to 6k and 7a to 7k series were also investigated for their antiviral activity against the replication of mers-cov, fipv, or mhv-1 in huh-7, crfk, or l929 cells, respectively (19) . briefly, medium containing dmso (<0.1%) or each compound (up to 100 m) was added to confluent cells, which were immediately infected with viruses at a multiplicity of infection (moi) of 0.01. after incubation of the cells at 37°c for 24 hours, viral titers were determined with the median tissure culture infectious dose (tcid 50 ) method (fipv or mhv) with the crfk or l929 cells or plaque assay with vero81 cells (mers-cov). for sars-cov-2, confluent vero e6 cells were inoculated with ~50 to 100 pfu per well, and medium containing various concentrations of each compound and agar was applied to the cells. after 48 to 72 hours, plaques in each well were counted. ec 50 values were determined by graph-pad prism software using a variable slope (graphpad, la jolla, ca) (19) . to confirm that these inhibitors also inhibit sars-cov-2 in primary human cells, differentiated human airway epithelial cells from three donors were used as previously described (36, 37) . two compounds (6j and 6e) were tested for their antiviral effects against sars-cov-2. briefly, airway epithelial cells were washed with phosphate-buffered saline (pbs), and sars-cov-2 was inoculated at a moi of 0.1 for 1 hour. after the inoculum was removed, media containing 6j (2 m) or 6e (0.5 m) was added. after 48 hours, cells were subjected to a freeze/thaw cycle, and virus titers were determined by plaque assay on vero e6 cells. the 50% cytotoxic concentration (cc 50 ) for compounds 6a to 6k and 7a to 7k was determined in huh-7, crfk, or l929 cells. confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 m) of each compound for 72 hours. cell cytotoxicity was measured by a cytotox 96 nonradioactive cytotoxicity assay kit (promega, madison, wi), and the cc 50 values were calculated using a variable slope by graphpad prism software. the in vitro therapeutic index was calculated by dividing the cc 50 by the ec 50. protein purification, crystallization, and data collection in x-ray crystallographic studies mers-cov 3clpro and sars-cov 3clpro were purified as described previously (17, 19) . an escherichia coli codon-optimized construct encoding residues ser 3264 to phe 3568 of the orf1ab polyprotein (sars-cov-2 3clpro, genebank accession number qhd43415.1) was cloned into a pet his6 sumo tev lic cloning vector (2s-t, addgene). expression and initial ni-column purification were performed as described for mers-cov 3clpro and sars-cov 3clpro. the small ubiquitin-like modifier protein (sumo) fusion elution fractions of sars-cov-2 were dialyzed against 20 mm tris (ph 8.0) and 100 mm nacl and treated with the tobacco etch virus (tev) protease (1:10, w/w) overnight. this mixture was loaded onto a 5-ml histrap hp column (ge healthcare) equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl and eluted with 20 mm tris (ph 8.0), 100 mm nacl, and 500 mm imidazole using an äkta pure fast protein liquid chromatography (fplc). the flow-through fractions containing sars-cov-2 3clpro without the sumo fusion were loaded onto a superdex 75 10/300 gl size exclusion column equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl. the fractions were pooled and concentrated to 9.6 mg/ml for crystallization screening. note that four residues from cloning (ser-asn-ile-gly) remain at the n terminus after treatment with tev protease. purified mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro in 100 mm nacl and 20 mm tris (ph 8.0) were concentrated to 10.6 mg/ml (0.3 mm), 22 mg/ml (0.64 mm), and 9.6 mg/ml (0.28 mm), respectively, for crystallization screening. all crystallization experiments were set up using an nt8 drop-setting robot (formulatrix inc.) and uvxpo mrc (molecular dimensions) sitting-drop vapor diffusion plates at 18°c. one hundred nanoliters of protein and 100 nl of crystallization solution were dispensed and equilibrated against 50 l of the latter. stock solutions (100 mm) of compounds 6b, 6d, 6g, 6h, 7i, and 7j were prepared in dmso, and complexes were generated by mixing 1 l of the ligand (2 mm) with 49 l (0.29 mm) of the protease and incubating on ice for 1 hour. crystals of the mers-cov 3clpro inhibitor complexes were obtained from the following conditions: compounds 6b, 6d, 6g intensities were integrated using xds (38, 39) using autoproc (40) , and the laue class analysis and data scaling were performed with aimless (41) . structure solution was conducted by molecular replacement with phaser (42) (46), respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity (47) , and figures were prepared using the ccp4mg package (48) . crystallographic data are provided in table s2. the two best compounds (6j and 6h) in the series were examined for their in vivo efficacy using 10-week-old male hdpp4-ki mice infected with mers ma -cov (30) . in the first study, animals were divided into three groups (n = 5 to 6) and were lightly anesthetized with ketamine/xylazine and infected with 50 l of 750 pfu mers ma -cov via intranasal inoculation. compound 6j or 6h were formulated in 10% ethanol and 90% peg400 and given to mice from 1 to 10 dpi at 50 mg/kg per day (once per day) via intraperitoneal administration. the control mice received vehicle. animals were weighed daily and monitored for 15 days. animals were euthanized when an animal lost 30% of initial weight or at 15 dpi. in the next study, treatment with compound 6j was delayed up to 3 dpi to determine the impact of delayed treatment on mouse survival. animals were divided into five groups (n = 5), and compound 6j (50 mg/kg per day, once per day) was administered to mice starting at 1, 2, or 3 days after virus challenge (1, 2, or 3 dpi, respectively) until 10 dpi. mice were monitored for weight loss and survival as described above for 15 days after virus challenge. as controls, vehicle (10% ethanol and 90% peg400) was administered equivalently to the experimental compound, or animals received no treatment (untreated). the third study was conducted to assess the effects of therapeutic treatment of compound 6j in the lungs. for lung harvest and virus titration, animals were divided into three groups (n = 4) of mice, and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi until euthanasia. animals were euthanized at 3 or 5 dpi, and lungs were removed aseptically, disassociated with a manual homogenizer in 1× pbs, briefly centrifuged, and supernatants removed. samples were titered on vero81 cells as reported elsewhere (49) . for lung histopathology analyses, animals were divided into two groups (n = 5), and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi for 5 days. mice were euthanized at 6 dpi, lungs were fixed with 10% formalin, and hematoxylin and eosinstained tissues were examined by a veterinary pathologist using the postexamination method of masking (50) . briefly, tissues were scored in an ordinal manner for edema and hyaline membrane formation using the following scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34 to 66% lung fields; and 4, >66% lung fields (30) . the analysis of survival curves in groups was performed using a log-rank (mantel-cox) test and a gehan-breslow-wilcoxon test using graphpad prism software (san diego, ca). log-transformed viral titers in the lungs and lung edema and hyaline membrane formation in groups of mice were analyzed with multiple t tests using graphpad prism software. stm.sciencemag.org/cgi/content/full/12/557/eabc5332/dc1 materials and methods table s1 . compound ic50 in the fret enzyme assay and cc50 in a cell culture assay for mers-cov 3clpro. table s2 . cocrystal structure data for compounds with the 3clpro of mers cov and sars cov. data file s1. individual-level data for all figures and tables. references (51) (52) (53) (54) (55) view/request a protocol for this paper from bio-protocol. genomic characterisation and 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respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software n y times web china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china towards automated crystallographic structure refinement with phenix.refine features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp4 molecular-graphics project rapid generation of a mouse model for middle east respiratory syndrome principles and approaches for reproducible scoring of tissue stains in research scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality resolving some old problems in protein crystallography linking crystallographic model and data quality we thank rudragouda channappanavar for performing the initial assays demonstrating drug efficacy against mers-cov. we thank d. george for technical assistance. key: cord-326922-bajpr5a2 authors: watson, c. james; whitledge, james d.; siani, alicia m.; burns, michele m. title: pharmaceutical compounding: a history, regulatory overview, and systematic review of compounding errors date: 2020-11-02 journal: j med toxicol doi: 10.1007/s13181-020-00814-3 sha: doc_id: 326922 cord_uid: bajpr5a2 introduction: medications are compounded when a formulation of a medication is needed but not commercially available. regulatory oversight of compounding is piecemeal and compounding errors have resulted in patient harm. we review compounding in the united states (us), including a history of compounding, a critique of current regulatory oversight, and a systematic review of compounding errors recorded in the literature. methods: we gathered reports of compounding errors occurring in the us from 1990 to 2020 from pubmed, embase, several relevant conference abstracts, and the us food and drug administration “drug alerts and statements” repository. we categorized reports into errors of “contamination,” suprapotency,” and “subpotency.” errors were also subdivided by whether they resulted in morbidity and mortality. we reported demographic, medication, and outcome data where available. results: we screened 2155 reports and identified 63 errors. twenty-one of 63 were errors of concentration, harming 36 patients. twenty-seven of 63 were contamination errors, harming 1119 patients. fifteen errors did not result in any identified harm. discussion: compounding errors are attributed to contamination or concentration. concentration errors predominantly result from compounding a prescription for a single patient, and disproportionately affect children. contamination errors largely occur during bulk distribution of compounded medications for parenteral use, and affect more patients. the burden falls on the government, pharmacy industry, and medical providers to reduce the risk of patient harm caused by compounding errors. conclusion: in the us, drug compounding is important in ensuring access to vital medications, but has the potential to cause patient harm without adequate safeguards. in the modern-day united states (us), medications are by-inlarge manufactured in commercial facilities, and this production is regulated and overseen by the us food and drug administration (fda). historically, however, medications were mixed-or compounded-by independent pharmacists for use by individual patients. while traditional compounding is becoming less prevalent, it still occurs in instances where a particular patient may require a formulation of a medication that is not otherwise available. furthermore, a new form of large-scale compounding has become commonplace, whereby pharmacies produce bulk volumes of medications which are not available commercially, and broadly distribute them to healthcare practices and individual patients. compounding does not traditionally fall under the purview of fda oversight, instead being regulated by individual states' boards of pharmacy. this approach has resulted in a patchwork and oftentimes underfunded regulatory framework, which has subsequently harmed patients [1] [2] [3] [4] . morbidity and mortality frequently result either from a compounded medication that is contaminated with bacteria, fungi, or another medication during production, or from an error whereby the concentration of the drug dispensed is not as intended, which can lead to inadvertent over-or underdosing. patient harm caused by compounded medications has been the focus of media, medical, and legislative attention in recent years, especially following a multistate, multi-fatality outbreak of fungal meningitis caused by contaminated steroid injections compounded at a pharmacy in framingham, ma [2, 3, 5, 6] . this article seeks to provide a comprehensive review of the state of outpatient compounding in the us. compounding performed by hospital pharmacies for inpatient use is beyond the scope of this paper. much has been written on compounding pharmaceuticals; this paper is an effort to succinctly address the history, purpose, and regulatory framework in a unified location, as well as to perform a systematic review of all us compounding errors over the past 30 years. to our knowledge, no systematic review of both contamination and non-contamination errors has to this point been undertaken. we will first explore the definition and modern role of compounding. then, we will briefly discuss the modern us history of compounding, with a particular focus on factors influencing the current state of compounding. next, we will examine compounding through a legislative and regulatory lens, to better decipher how governmental oversight-or a lack thereof-may contribute to errors in compounding resulting in patient harm. understanding the interventions being made on a federal level can help improve the safety of compounding. finally, we have performed a systematic review of documented compounding errors and categorized those errors by type and patient outcome. whereby, we elucidate just how and with what frequency patients are harmed by compounding errors, with the ultimate aim of identifying potential strategies for reducing these adverse events. compounding is defined by the fda as the combination, mixing, or alteration of drug ingredients to create medications tailored to individual patient needs [7] . the united states pharmacopeia (usp), which sets quality standards for drugs, describes compounding as "the preparation, mixing, assembling, altering, packaging, and labeling of a drug … in accordance with a licensed practitioner's prescription …" [8] put simply, it is the creation of a medication that is not commercially available. in the us, compounding is performed in both the inpatient hospital setting and in outpatient pharmacies, with a trend in recent decades towards larger scale outpatient production [9] . as will be discussed later in this paper, compounding may now occur in newly defined "outsourcing facilities," which are designed to compound in bulk; some examples of these facilities include quva pharma and leiters [10] . there are many indications for compounding. some patients may not tolerate pills and require a compounded liquid drug formulation; examples include young children taking antibiotics, feeding tube-dependent patients, or patients with dysphagia from neurologic compromise such as a stroke [11, 12] . patients may be allergic to binding agents, dyes, diluents, or other inactive ingredients in commercially available formulations. dietary restrictions, such as a ketogenic diet in pediatric epilepsy patients, may necessitate compounding of sugar-free medications [13] . refractory neuropathic pain may benefit from compounded analgesic topical creams containing multiple medications not commercially available in combination; examples include ketamine, baclofen, gabapentin, amitriptyline, bupivacaine, and clonidine [14] . painful oral lesions can be treated with "magic mouthwash" and dyspepsia can be treated with a "gastrointestinal (gi) cocktail"; these are terms that actually encompass a range of compounded preparations [15] . total parenteral nutrition (tpn) is needed for patients unable to take in sufficient oral nutrition, and numerous chemotherapy regimens must be compounded for cancer treatment [16, 17] . healthcare providers may need compounded medications to perform specialized procedures such as intraarticular or intravitreal injections. in some instances, commercial preparations may be available but expensive, and a compounded equivalent is more affordable [18] . drug shortages, a longstanding healthcare problem exacerbated by crises such as the covid-19 pandemic and the devastation of puerto rico by hurricane maria, may be addressed by compounding as well [19, 20] . the fda has responded to significant shortages during the covid-19 pandemic by temporarily relaxing restrictions on compounding of commercially available drugs [21, 22] . when a compounded medication is prescribed or administered, patient safety depends on adherence to current good manufacturing practices (cgmp), which are outlined in chapter 795 of the usp for non-sterile preparations and chapter 797 for sterile preparations. appropriateness of the prescription indication, safety, and dosing should be assessed by the pharmacist. ingredient quantities must be meticulously calculated, and the source quality of those ingredients assured. compounding facilities and equipment must be clean and monitored continuously. staff must routinely practice and be assessed for competency in proper hygienic measures. sterile preparations, by definition, require a higher level of care to prevent contamination than do non-sterile preparations, including differences in staff training and personal protective equipment (ppe), environment and air quality monitoring, and disinfection. compounded sterile preparations are further subdivided into low-, medium-, and high-risk depending upon the quantity of ingredients, number of manipulations required during compounding, and whether nonsterile ingredients requiring subsequent sterilization are incorporated. multiple medications must not be simultaneously compounded in the same workspace. the compounding process must be reproducible such that medication quality is consistent throughout many production cycles. finally, prescriptions must be correctly labeled and patients instructed in appropriate use [8, [23] [24] [25] . failure to adhere to these standards has the potential to result in patient harm through multiple mechanisms including medication suprapotency, subpotency, contamination, and consumer misuse. throughout pre-industrial history, pharmacists played the critical role of admixing various materials to produce a finished therapeutic substance. this role was, in essence, one of compounding [26, 27] . however, the industrial revolution and the resultant mass production of pharmaceuticalscoupled with the increasing presence of synthetic proprietary medications-led to a change in pharmacists' primary role. instead of compounding, community pharmacists in the early 1900s turned their focus to the dispensing of previously manufactured medications as well as to general retail, including operating the soda fountains which came into vogue with the prohibition of alcoholic beverages. in fact, by the 1930s, fewer than 1% of pharmacies in the us made a majority of their income from pharmaceutical sales [27] . the decline in community pharmacy compounding was precipitous through the mid-1900s. in the 1930s, 75% of prescriptions required some sort of in-pharmacy compounding. that number fell to 25% by the 1950s, less than 5% by 1960, and to 1% by 1970 [28] . interestingly, there was a concurrent increase in the need for hospital pharmacy compounding during the same period; largely due to the advent of chemotherapy, tpn, and cardiac surgery which necessitated the administration of complex cardioplegic regimens. by the 1980s, these advanced therapeutics began to spill into the outpatient setting, generating a novel home infusion industry for treatments such as tpn, antibiotics, and chemotherapeutics [29] . as a result, the 1990s and 2000s yielded further diversification within the compounding industry as pharmacies began to compound in bulk. this development was brought about by expanding home infusion programs, the more frequent outsourcing of hospital compounding to the outpatient setting, and the rise of hormone replacement therapy. large volume compounding blurs the line between traditional compounding which has state-based regulatory oversight, and the mass manufacture of pharmaceuticals which falls under wellestablished federal fda regulations [29] [30] [31] . the inspiration for this article is a well-documented history of medication errors attributable to pharmaceutical compounding, for which a lack of regulatory oversight persists as a common thread [3, 29, 32, 33] . the most lethal and infamous of these cases occurred in 2012, when an outbreak of fungal meningitis occurred amongst patients who had received epidural spinal injections. the outbreak affected 753 patients across 20 states, killing 64 [5, 6, 34, 35] . ultimately, the outbreak was linked to a compounding pharmacy, the new england compounding center (necc, located in framingham, ma). amongst other pharmaceuticals, necc produced injectable sterile methylprednisolone acetate for epidural injections, which it then distributed nationally. following the outbreak (hereafter "framingham"), the fda determined that the pharmacy had disregarded basic sanitary standards and had not taken corrective measures despite internal knowledge of potential contamination [2, 5, 6, [34] [35] [36] . as with many compounding pharmacies, necc operated in a historically murky regulatory space, producing medications in bulk as would a commercial pharmaceutical manufacturer, while only being subjected to reduced state oversight given to compounding pharmacies. in fact, in the years preceding the outbreak, the fda had thrice investigated necc and found sterility violations, but they were unable to enforce any interventions or penalties due to the fda's contested regulatory jurisdiction [4] . both preceding and following framingham, efforts have been made at the federal level to improve oversight of compounding; these are reviewed in depth later in this article. currently, there is incomplete tracking of compounded pharmaceuticals in the us, though they are estimated to comprise 1-3% of all prescriptions [30, 31, 37, 38] . ultimately, compounding is highly prevalent, and so clinicians must be familiar with the risks associated with compounded medications as they care for patients who may be suffering from a related adverse event. prior to framingham, modern compounding pharmacies evolved within a regulatory framework that lacked distinct federal or state oversight roles. in 1938, the federal food, drug, and cosmetic act (fdca) authorized fda oversight of pharmaceutical manufacturing [39] . however, because compounders traditionally produced drugs in response to individual prescriptions and on a much smaller scale than conventional drug manufacturers, pharmaceutical compounding developed and remained under the regulatory purview of individual state boards of pharmacy [32, 40] . towards the end of the twentieth century, pharmacies began bulk compounding in response to (1) the home infusion industry and (2) hospitals' financial interest in outsourcing compounding from their inpatient pharmacies to the outpatient setting [29] . concerned that bulk compounders were self-classifying as pharmacies to avoid the rigorous federal oversight required of drug manufacturers under the fdca, congress passed the 1997 food and drug administration modernization act (fdama) [40] . fdama addressed the changing nature of compounding pharmacies by creating a "safe harbor" exempting pharmacies from the more stringent fdca regulations so long as compounders refrained from advertising their product and abided by requirements designed to increase drug safety [40, 41] . despite congress's attempt to strengthen oversight of compounding pharmacies, litigation challenging fdama tempered the fda's authority to regulate compounders. in 2002, a narrowly divided us supreme court ruled in thompson v. western states medical center that the fdama advertising prohibition was unconstitutional on first amendment free speech grounds [42] . the ensuing regulatory confusion is well described in the literature, and the details are beyond the scope of this review [3, 28, 29, 31, 32, 40] . for reference, a summary of the relevant legislation and litigation is provided in fig. 1 . decades of regulatory uncertainty culminated in the 2012 framingham incident, which revived congressional efforts to address pharmaceutical compounding industry safety concerns. in response to framingham, congress passed and president barack obama signed into law the bipartisan-supported compounding quality act (cqa) as part of a broader legislative package (the 2013 drug quality and security act) [43] . the cqa delineated state and federal oversight authority by defining two distinct categories of compounding pharmacies. the first category is traditional compounding pharmacies, or "503a" pharmacies [44] . 503a pharmacies under the cqa may compound only in response to individual prescriptions. importantly, 503a pharmacies may not compound bulk medications either in anticipation of receiving prescriptions or with plans to distribute broadly to healthcare facilities [31, 45] . in exchange for complying with these limitations, 503a pharmacies largely avoid the more burdensome regulations required of drug manufacturers under the fdca, including adhering to cgmp [31, 37, [46] [47] [48] . accordingly, state boards of pharmacy continue to serve as the primary regulators of 503a pharmacies [45] . the cqa created a second category of compounding pharmacy, called an "outsourcing facility." [49] unlike 503a pharmacies, outsourcing facilities voluntarily opt-in to this category by paying the fda a user fee (approximately $18,298 in fy2020) [50] , and comply with stringent cgmp standards as well as reporting requirements [38, 46] . because they submit to more robust fda oversight, outsourcing facilities are permitted to compound in bulk in advance of receiving a prescription, and may distribute their products across state lines [31, 51] . though the fda enjoys primary regulatory authority over outsourcing facilities, states are not precluded from imposing additional requirements [51] . should a compounding pharmacy fail to comply with the 503a criteria or voluntarily register as an outsourcing facility, it is subject to the full breadth of regulations required of drug manufacturers under the fdca [37] . the distinctions between 503a pharmacies and outsourcing facilities are illustrated in fig. 2 . following enactment of the cqa, states have taken numerous steps to further develop their respective oversight structures under the new framework. a majority of states have strengthened regulations empowering state boards of pharmacy to hold 503a pharmacies accountable to higher safety practices, such as requiring conformation with recognized sterile compounding guidelines. however, state oversight of 503a pharmacies continues to vary, with fewer than half of all states reporting annual inspections of 503a pharmacies in 2018 [52] . the fda has similarly adjusted its enforcement priorities [53, 54] . for example, the cqa permits a 503a pharmacy to distribute no more than 5% of its total prescriptions out of state . the goal of this provision is to avoid another national outbreak by reducing the likelihood that contaminated drugs cross state lines. if a given state enters into a mou with the fda, 503a pharmacies in that state may distribute a higher percentage of prescriptions (now up to 50%) across state lines in exchange for that state's board of pharmacy agreeing to identify, investigate, and report associated adverse events [ 45] . importantly, the mou standardizes procedures for state boards of pharmacy to report concerns to the fda and other states; however, the agreement also grants states significant discretion in how states conduct investigations [54] . in short, states that participate in the mou, rather than the fda, will undertake primary responsibility for detecting poor quality or dangerous compounded medications distributed by 503a pharmacies from their state. the fda also announced an effort to entice more compounding pharmacies to register as outsourcing facilities by embracing a risk-based approach [45] . since enactment of the cqa, far fewer pharmacies have registered as outsourcing facilities than the fda had expected. in fact, the fda anticipated 50 pharmacies to register per year, but only 78 total were registered as of may 2020 (even fewer than the 91 registered in 2016) [38, 51, 56] . to attract compounding pharmacies-some of which have cited cost of compliance with cgmp as a prohibitively expensive barrier to registering as an outsourcing facility-the fda plans to reduce cgmp requirements for compounding pharmacies it deems as "lower risk." [45] though the fda published draft guidance in 2018 describing how the agency may tailor cgmp requirements for outsourcing facilities, the fda has yet to issue final guidance on this matter [53, 57] . critics warn that fda and state efforts to implement the cqa regulatory scheme excludes compounding pharmacies from the decision-making process and may limit patients' access to compounded medications. for example, the preserving patient access to compounded medications act (h.r. 1959) introduced in the us house of representatives attempts to address complaints expressed by compounders [58] . the proposed legislation seeks to ensure that compounders and other interested parties have an opportunity to comment on (and influence) fda compounding regulations. furthermore, the proposed legislation would explicitly allow physicians who engage in in-office sterile compounding, or who otherwise maintain a supply of compounded medications for "office use," to avoid complying (where state law permits) with outsourcing facility regulations [58] . meanwhile, the sterility practices of some compounding pharmacies continue to raise alarm: between 2013 and 2018, the fda issued more than 180 warning letters to compounding pharmacies, resulting in approximately 140 recalls. as acknowledged by the agency, the fda's transition to a risk-based approach may assist the agency in more efficiently targeting its limited resources, but it could also increase the likelihood of compounders engaging in unsafe practices that elude regulators [45] . in sum, the cqa and subsequent state and fda actions have somewhat clarified oversight roles after framingham, largely by defining separate 503a pharmacies and outsourcing facilities. seven years after its enactment, however, uncertainty regarding the relative strength and consistency of said regulatory framework remains. we performed a systematic review of compounding errors, including both errors that resulted in patient harm and those that did not, as reported in the academic literature. we searched the national center for biotechnology information (pubmed; u.s. national library of medicine: bethesda, m a r y l a n d ) a n d e m b a s e ( e l s e v i e r : amsterdam, the netherlands) using the following search criteria: "'compounding and pharmacy' and 'error,' 'overdos*,' 'toxicol*,' 'infect*,' 'death,' 'outbreak,' 'injur*,' or 'case report.'" this search was limited to january 1990 through march 2020. additionally, we reviewed abstracts for years 1990-2019 for the following conferences using keyword searches for "compound" and "compounding": american college of medical toxicology (acmt) annual scientific meeting, north american congress of clinical toxicology (nacct), american college of emergency physicians (acep) scientific assembly, society for academic emergency medicine (saem) annual meeting, american academy of pediatrics (aap) national conference & exhibition, and the pediatric academic societies (pas) meeting. we also reviewed the fda's online "drug alerts and statements" repository for alerts regarding compounding pharmacies' failures in sterility and potency standards. authors cjw and jdw screened reports by title and, when necessary for clarification, by abstract. under manual review, articles were excluded if they were obviously irrelevant, consisted of research comparing samples of compounded and commercial pharmaceuticals, were in a non-english language, regarded medications compounded outside of the us, were redundant with another included report, represented misuse of properly compounded medications, regarded veterinary patients, were compounded by an inpatient hospital pharmacy (including chemotherapeutics and parenteral nutrition), were published prior to 1990, or if the report lacked sufficient information to provide substantive value. redundant reports of the same error were included for analysis only once, but efforts were made to reference all identified reporting sources. for included reports, cjw and jdw extracted information including date, type of error, cause of error, number of patients affected, age of patients affected, and clinical course of patients affected. incomplete data was acknowledged and by-inlarge was not grounds for exclusion from the study. we categorized errors under the conceptual framework described by sarah sellers, pharmd, mph, former board member for the fda's advisory committee on pharmacy compounding, in testimony to the us senate committee on health, education, labor, and pensions, namely, that "suprapotency," "subpotency," and "contamination" are the primary risks associated with pharmaceutical compounding [59] . we further broke down "contamination" into subgroups of "microbiologic contamination" for cases of bacterial, viral, or fungal contamination and "toxic contamination" for noninfectious contaminants. when available, we documented patient age and outcome, route of administration, and medication-in-question, so as to better characterize the types of medications, errors, and patients most associated with adverse events. we referenced and applied the principles for authoring review articles delineated within the journal of medical toxicology when feasible and appropriate during the review process [60] . our search terms identified 1058 potential articles in pubmed and 721 potential articles in embase. the review of conference abstracts yielded additional potential cases as follows: [61, 62] . in total, a total of 2155 articles, statements, and reports were identified and underwent our manual review (performed by cjw and jdw). after the application of our exclusion criteria, a total of 63 errors were included. these 63 errors are documented as harming 1155 patients. when broken down by type, contamination accounted for 27 errors adversely affecting 1119 patients (appendix table 3 ) and errors in concentration accounted for 21 events adversely affecting 36 patients (appendix table 4 ). there were 15 reports of identified compounding errors which potentially exposed innumerable patients but did not end up causing any known harm; these were predominantly errors of contamination (appendix table 5 ). the number of patients exposed to potential harm cannot be calculated based on the available data, but reaches at least the several thousands (framingham alone exposed 13,534 patients with 753 documented instances of patient harm). table 1 is a summary of the 27 included contamination errors. with 1119 patients over 27 errors, the median number of patients affected per error is 8. the mean number of patients affected per error is 41; however, by excluding framingham, that number is 14. with 81 deaths over 27 errors, the mean number of fatalities per error is 3; however, excluding framingham drops that number to less than 1 (0.65). the median number of deaths per error is 0. five out of the 27 contamination errors were from intraarticular (including epidural) steroids, and eight of 27 were from medications injected intravitreally. a total of 25 of the 27 errors were from medications administered parenterally, in healthcare settings. three of 27 were from toxic contamination rather than microbiologic contamination. interestingly, six of 27 errors with documented adverse outcomes occurred following the cqa. table 2 is a summary of the 21 included sub-and supratherapeutic errors. one report describes a subtherapeutic error affecting 9 pediatric patients who were on posttransplant immunosuppression with tacrolimus. the remaining 20 reports involved errors of supratherapeutic drug concentrations; they affected a total of 27 patients, of which 14 (52%) were pediatric. of the 36 total patients affected by concentration errors, 23 (64%) were pediatric and 3 (8%) were over the age of 65 years. three patients died, all of whom received supratherapeutic intravenous colchicine at an alternative medicine infusion clinic for chronic back pain [91] . appended to this article are appendix tables 3, 4 , 5, which respectively catalog all contamination errors causing patient harm, all sub-and suprapotency errors causing patient harm, and all potential compounding errors identified and rectified before patient harm occurred. of the 15 potential errors identified before patient harm occurred, 13 came after the enactment of the cqa. in this study, we separated compounding errors into the categories of contamination, suprapotency, and subpotency. we found that medications with contamination errors are frequently (1) bulkproduced and distributed, (2) used parenterally, and (3) administered by physicians. because of their parenteral administration, medications contaminated with otherwise benign environmental flora are able to disseminate throughout the body and cause the devastating outcomes documented here. furthermore, because contamination errors are often associated with larger-even multistate-distribution networks, the reach of their impact is large. framingham was the archetypal contamination error. it woke much of the medical and lay communities to the potential dangers of compounding. it inspired the federal government to enact the cqa in 2013 and create an entirely new form of compounding facility-the outsourcing facility-to attempt to regulate the subsection of pharmacies who were bulk-compounding medications not available (or not available cheaply) through commercial channels, and who exported [45] certainly, contamination has persisted despite the cqa and the fda's efforts to oversee outsourcing facilities. given this ongoing concern, the medical community must bear some of the responsibility for reducing the number of medications manufactured in substandard environments. it should be the expected standard for healthcare practices to purchase exclusively from compounding pharmacies strictly adherent to cgmp standards and formally approved as outsourcing facilities by the fda. leading expert outterson referenced the potential for this approach in 2014 [46] , and it is unclear how purchasers have responded. while these policies may be more expensive; the physical, ethical, and even financial [92] consequences of purchasing compounded medications from organizations not sufficiently invested in safety are clearly documented here. subpotency and suprapotency can be considered as the single category of errors of concentration, as the sources and scope of concentration errors are largely similar. our findings demonstrate that subpotency is largely not a reportable issue, but that does not mean it is not a danger. as an example, beyond the cited series of subtherapeutic tacrolimus concentrations [93] , another case series (excluded for location outside the us) identifies dozens of patients who received subtherapeutic chemotherapy treatments [94] . these subtherapeutic errors are difficult to capture. identification must be done during routine serum testing, as occurred with the tacrolimus series; or on the supply side, as occurred with the chemotherapy series. when considering subtherapeutic and supratherapeutic errors together as errors of concentration, we found a somewhat different pattern than that which we identified amongst errors of contamination. the concentration errors we were able to identify, with a few notable exceptions [95, 96] , were caused by traditional compounding pharmacies. these pharmacies, labeled as 503a pharmacies under the cqa, are limited in their scope to producing compounded medications only after an individual prescription is in-hand. per the cqa, 503a pharmacies are still solely regulated by state boards of pharmacy, meaning that oversight is patchwork across the us. many concentration errors are of orders of magnitude, suggesting that simple mathematical and measurement mistakes are to blame. in addition to hoping that states will implement greater oversight of these 503a pharmacies, we call on the pharmacy industry to emphasize and standardize compounding training amongst its students and even consider a mandatory credential before allowing a pharmacist or pharmacy technician to compound a medication [26, [97] [98] [99] . it is worth noting that 4-aminopyridine and liothyronine are fairly uncommon medications, however they accounted for a large number of compounding concentration errors. there is nothing particularly special about these medications which make them prone to concentration errors, except for the fact that they are not readily commercially available, and so they must be compounded. the prescribers of these medications need to carefully consider the benefits and risks of prescribing a treatment which requires compounding; especially when the risks are so great (status epilepticus with 4-aminopyridine and thyrotoxicosis with liothyronine). liothyronine, in particular, has had its clinical utility recently questioned. for example, the national health service (uk) has recently called on general practitioners to stop prescribing liothyronine without specialist consultation, as most patients benefit equally from commercially prepared levothyroxine [100]. given the risks of inadvertent overdose due to compounding errors, providers must consider commercially available alternatives whenever able. in fact, it has been questioned w h e t h e r p r o v i d e r s w h o k n o w i n g l y p r e s c r i b e a compounded medication despite commercially available alternatives might be legally liable for any harm resulting from compounding errors [101] . at the very least, it is incumbent on prescribers as well as pharmacists to educate their patients on the risks of taking a compounded medication-both from errors in concentration and contamination-and to instruct them on when to present to a healthcare provider. additionally, practitioners must be aware of their patients' medication lists, and consider a possible compounding error as a cause of medical illness. notably, we found that the majority of concentration errors were made in pediatric and geriatric patients, vulnerable populations who are already at increased risk of providers failing to diagnose toxicity from prescription medications. in 2020 and beyond, we anticipate the demand for compounding to only increase. the number of novel therapeutics continues to rise rapidly, as do their approved routes of administration. the anti-angiogenesis medication bevacizumab is a classic example; it is commercially manufactured but is frequently compounded into smaller aliquots for intravitreal administration. as we have seen, this process has unfortunately resulted in multiple outbreaks of endophthalmitis [72] [73] [74] [75] [76] . furthermore, regional and global disasters have recently resulted in significant pharmaceutical supply chain issues. examples of this phenomenon include hurricane maria's impact on puerto rican manufacturers in 2017 and the covid-19 pandemic [120] [121] [122] [123] . these disruptions place increased demand on alternative means of supply, including via pharmaceutical compounding. in fact, the covid-19 pandemic and its associated drug shortages has already resulted in the loosening of fda restrictions, including allowing outsourcing facilities to compound copies of commercially available drugs for hospital use [21] . our study has its limitations. while we made every effort to capture published cases of compounding errors, it is possible that our search criteria missed some cases that would have impacted our analyses. while we also strove to review less-traditional sources, including conference abstracts and fda alerts, we are not free of publication bias and are at risk for having excluded compounding errors not associated with adverse events, or with very small numbers of patients affected. similarly, it must be noted that compounding errors can only be identified following adverse events, laboratory screening, or industry or governmental report. even once identified, we were dependent on the publication of the error in order to capture it here. as such, we are likely underreporting the frequency with which compounding errors occur. compounding is more relevant than ever. appreciating that the need for compounding is unlikely to diminish in the near future, we can only re-emphasize the critical nature of our recommendations for the federal and state governments to fully fund the oversight of outsourcing facilities, for healthcare practices to refuse medications compounded without strict adherence to cgmp and fda regulations, for pharmacy schools to expand compounding training and certification, and for physicians to think critically about the risks of prescribing medications that are not commercially produced. conversely, we must remain aware that compounding pharmacies frequently provide an essential service and poorly calibrated regulations may contribute to issues of access. ultimately, medical providers must remain vigilant, especially when caring for members of vulnerable 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presentation of iatrogenic phenytoin toxicity in a newborn fda alerts patients and health care providers not to use budesonide solution from the compounding shop. united states food and drug administration fda alerts health care professionals not to use sterile drugs from downing labs (aka nuvision pharmacy) fda announces medistat rx's nationwide voluntary recall of sterile drug products. united states food and drug administration fda alerts health care professionals not to use sterile drug products from qualgen. united states food and drug administration united states food and drug administration fda alerts health care professionals and patients not to use sterile drug products from vital rx, dba atlantic pharmacy and compounding. united states food and drug administration fda alerts health care professionals to voluntary nationwide recall of all sterile products from coastal meds. united states food and drug administration fda announces ranier's rx laboratory's voluntary recall of all sterile compounded drugs. united states food and drug administration fda alerts health care professionals and patients not to use sterile drug products from pharm d solutions. u.s. food & drug administration fda alerts health care professionals and patients not to use drug products intended to be sterile from promise pharmacy. united states food and drug administration fda alerts patients and healthcare professionals to infusion options' voluntary recall due to quality issues. united states food and drug administration dba amex pharmacy, voluntary recall of all sterile compounded drugs. united states food and drug administration publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-301349-m4nr3pqx authors: mirza, muhammad usman; saadabadi, atefeh; vanmeert, michiel; salo-ahen, outi m.h.; abdullah, iskandar; claes, sandra; de jonghe, steven; schols, dominique; ahmad, sarfraz; froeyen, matheus title: discovery of hiv entry inhibitors via a hybrid cxcr4 and ccr5 receptor pharmacophore‐based virtual screening approach date: 2020-09-02 journal: eur j pharm sci doi: 10.1016/j.ejps.2020.105537 sha: doc_id: 301349 cord_uid: m4nr3pqx chemokine receptors are key regulators of cell migration in terms of immunity and inflammation. among these, ccr5 and cxcr4 play pivotal roles in cancer metastasis and hiv-1 transmission and infection. they act as essential co-receptors for hiv and furnish a route to the cell entry. in particular, inhibition of either ccr5 or cxcr4 leads very often the virus to shift to a more virulent dual-tropic strain. therefore, dual receptor inhibition might improve the therapeutic strategies against hiv. in this study, we aimed to discover selective ccr5, cxcr4, and dual ccr5/cxcr4 antagonists using both receptorand ligand-based computational methods. we employed this approach to fully incorporate the interaction attributes of the binding pocket together with molecular dynamics (md) simulations and binding free energy calculations. the best hits were evaluated for their anti-hiv-1 activity against cxcr4and ccr5-specific nl4.3 and bal strains. moreover, the ca(2+) mobilization assay was used to evaluate their antagonistic activity. from the 27 tested compounds, three were identified as inhibitors: compounds 27 (ccr5), 6 (cxcr4) and 3 (dual) with ic(50) values ranging from 10.64 to 64.56 μm. the binding mode analysis suggests that the active compounds form a salt bridge with the glutamates and π-stacking interactions with the aromatic side chains binding site residues of the respective co-receptor. the presented hierarchical virtual screening approach provides essential aspects in identifying potential antagonists in terms of selectivity against a specific co-receptor. the compounds having multiple heterocyclic nitrogen atoms proved to be relatively more specific towards cxcr4 inhibition as compared to ccr5. the identified compounds serve as a starting point for further development of hiv entry inhibitors through synthesis and quantitative structure-activity relationship studies. the most effective treatment of patients infected with human immunodeficiency virus 1 (hiv-1) is currently highly active antiretroviral therapy (haart), which utilizes a combination of several drugs targeting viral proteins such as reverse transcriptase, integrase and protease [1, 2] . an alternative approach relies on the multi-targeted inhibition of viral cell entry [3] , which consists of a series of structural events arbitrated by viral and cellular membrane proteins. the interaction between the hiv-1 glycoprotein gp120 and the cellular receptor cd4 is an important initial step in viral entry. it induces a conformational change in gp120 and mediates further interaction with either one of the two co-receptors, chemokine receptor 5 (ccr5) and cxc-chemokine receptor 4 (cxcr4) during viral entry into the host cells [4, 5] . both chemokine receptors belong to the g-protein coupled receptor family and regulate hiv-1 cellular tropism. ccr5-tropic or r5 strains preferentially uses ccr5, whereas cxcr4-tropic or x4 strains preferentially use cxcr4. dual-tropic strains use both co-receptors for their entry in the cd4 + target cells [5] [6] [7] . the chemokine ligand 5 (ccl5) also known as rantes (regulated upon activation, normal t cell expressed, and secreted) is the natural ligand for ccr5 [8] , whereas the cxcchemokine ligand 12 (cxcl12), also known as sdf-1 (stromal cell-derived factor 1) is the natural ligand for cxcr4 [9] . inhibition of either ccr5 or cxcr4 leads hiv to shift to a more virulent dual-tropic strain. therefore dual inhibition of both co-receptors is of paramount importance to improve the therapeutic strategies against hiv infection [10, 11] . although numerous selective ccr5 and cxcr4 antagonists have been reported [12] [13] [14] [15] , only a few ccr5/cxcr4 dual antagonists have been identified so far [3] . ccr5 and cxcr4 share an overall similar architecture, and the overlapping regions of the binding pockets pave the way for the identification of dual co-receptor antagonists [16] . maraviroc received marketing approval by the food and drug administration (fda) as a very potent and selective ccr5 antagonist with a favourable hiv resistance profile. it inhibits rantes binding to a recombinant ccr5 expressed in the human embryonic kidney (hek-293) cell line with an ic 50 value of 5.2 nm [17] . plerixafor (or amd3100) is a selective cxcr4 antagonist that specifically inhibits sdf-1α binding to cxcr4 (ic 50 = 12.5 nm) [18] its close analogue amd3451 (a tetraazamacrocycle) was first reported as a dual cxcr4/ccr5 antagonist [11] . alongside, tetraazamacrocycle-based transition metal complexes were shown to act as dual cxcr4/ccr5 antagonist and were endowed with excellent in vitro activity against hiv [19] . other dual cxcr4/ccr5 antagonists include, peptide-based triazoles analogues [20] , diterpene derivatives (ec 50 0.02 and 0.09 µm against r5 and x4 hiv strains, respectively [21] ), and pyrazole derivatives (ic 50 3.8 and 0.8 µm against ccr5-and cxcr4-utilizing hiv-1 strains) [22] . an analogue of the antiparasitic drug suramin nf279 [23] , the natural penicillixanthone a [24] and a coumarin-based analogue gut-70 [25] are also known as dual ccr5/cxcr4 antagonists (figure 1 ). modern structure-based virtual screening approaches are indispensable in the discovery of novel hits targeting specific proteins and is nowadays a crucial part in the hit-to-leadoptimization phase of drug discovery [26] [27] [28] . this approach resulted in the discovery of various potent antiviral compounds against viruses such as ebola [29] , dengue [30] , zika [31, 32] , sars-coronavirus [33, 34] and influenza [35] . the aim of this study was to discover selective ccr5, cxcr4 and dual ccr5/cxcr4 antagonists based on both receptor-and ligand-based virtual screening methods together with molecular dynamics (md) simulations and binding free energy calculations. the most promising hits were evaluated for cxcr4 and ccr5 antagonism via a ca 2+ mobilization assay using u87.cd4.cxcr4 and u87.cd4.ccr5-transfected cell lines. in addition, their anti-hiv-1 activity was evaluated in tzm-bl cells, using nl4.3 (cxcr4 tropic) and bal (ccr5 tropic) hiv-1 strains. databases of commercially available compounds (molport and interbioscreen databases, ~1.2 million compounds) were used for the virtual screening (vs) and prepared using the ligprep module of maestro v.11 (schrödinger, llc, new york) software. possible protonation states were generated using the opls3 force field [36] at ph 7±2. at most, one stereoisomer was generated per ligand such that the specified chiralities were retained. for the pharmacophorebased screening, the database was converted to the phase database format with the generate phase database module of maestro [37, 38] . the vs was specifically performed on ccr5, based on the x-ray crystal structure of ccr5 complexed with maraviroc (pdb id: 4mbs; a chain) [39] was retrieved from the protein data bank (pdb; www.rcsb.org). the co-crystallized ligands, ions and water molecules were removed from the receptor and all hydrogen atoms, missing loops and side chains were added, and bond orders assigned using the protein preparation wizard of maestro [40] . the protonation states of histidine residues were inspected interactively; his88 and his231 were changed to hie (protonated at nɛ atom) and hip (double protonated), respectively, to optimize the h-bonding network. restrained minimization using the opls3 force field was first applied for hydrogen atoms only and then for heavy atoms until non-hydrogen atoms attained an average root-mean-square deviation (rmsd) of 0.3 å. the top hits from this virtual screening campaign against ccr5 were comparatively docked with cxcr4 in order to discover dual acting ccr5/cxcr4 antagonists. therefore, the x-ray crystal structure of cxcr4 complexed with an isothiourea derivative (it1t) (pdb id: 3odu) [41] was prepared for docking, similarly as described above. in cxcr4, the protonation state of his113 and his281 were changed to hie and hip, respectively. 2.3 receptor-based virtual screening and prime/mm-gbsa binding free energy vs by molecular docking was conducted with the glide module of maestro [42] . the docking site was defined with the receptor grid generation tool of maestro. the enclosing cubic grid was centered at coordinates (150.03, 108. 25, 22.41) and ligand diameter midpoint box was set to 10 å × 10 å × 10 å . the limitation length for ligands to be docked was set to 12 å . a maximum of five poses per ligand were considered. the glide high-throughput virtual screening (htvs) mode was used for the initial docking of the large database after which the top 10% of the hits based on the docking score were chosen and then re-docked using the standard precision (sp) algorithm. subsequently, 10% of the highest-ranked compounds from this step were redocked using the extra-precision (xp) mode and finally, the top 10% of the glide xp score-ranked ligands were selected for the binding free energy calculations with the prime/mm-gbsa module of maestro [43] . the approximate energies were calculated using the vsgb 2.0 solvation model [44] and opls3 force field, first keeping all binding site residues fixed and then allowing the residues within 5 å from the ligand to move. flexible sampling was done by minimization of the complex. for the best-ranked compounds, molecular property prediction was carried out with maestro's qikprop module. besides the lipinski's rule of five (≤ 2 violations), favorable cell permeability (qppcaco > 500 nm s -1 ; qppmdck > 500 nm s -1 ) and acceptable aqueous solubility (-6.5 < qplogs < 0.5), a series of pains filters were applied to eliminate compounds with potential toxicophores. as a second screening approach, pharmacophore modeling was carried out with the phase module of maestro [37, 38] against ccr5. after preparing the maraviroc-ccr5 crystal complex (pdb id: 4mbs) with the protein preparation wizard of maestro, this structure was used for generating the pharmacophore hypothesis. the nɛ nitrogen atom of the triazole ring and the amide nh of maraviroc form h-bonds with the hydroxyl groups of tyr37 and tyr251, respectively ( figure 2 ). in addition, the positively charged nitrogen in the bicyclic ring engages in an ionic interaction with glu283. therefore, a h-bond acceptor (a), a h-bond donor (d) and a positive ionic (p) feature were selected as the pharmacophoric features for the hypothesis (figure 2 ). this pharmacophore model was then used to screen through the commercial ligand database in search of matching ligands. the ligands were scored and ranked by the phase screen score according to how well they superimpose on the features associated with the hypothesis. the previously calculated qikprop properties were then used to filter the best-ranked 3000 hit compounds as described above. the 500 ligands that passed the filter were then docked into the maraviroc binding site with glide using the xp mode and the subsequent prime/mm-gbsa binding free energy calculation according to the abovedescribed protocol. phase generated pharmacophore model. pharmacophoric features: red sphere for hydrogen-bond acceptor (a); light blue sphere for hydrogen-bond donor (d); blue sphere for the positive ionic (p) feature. the arrows are pointing in the direction of the lone pair. ligand atom color code: carbongreen; nitrogenblue; oxygenred; fluorinelight green. all hydrogen atoms except for maraviroc's polar hydrogens have been omitted for clarity. after a careful analysis of the best-ranked virtual hits, the receptor-bound docked poses of the most promising compounds were subjected to md simulation to estimate the stability of the binding complexes. all simulations were performed with the amber 18 simulation package [45] using the same md simulation protocol as described previously [46, 47] . each solvated system was minimized stepwise, followed by the heating and equilibration stages. finally, a production run of 20 ns was performed at 300 k and 1 bar pressure. the time step was set to 2 fs and the trajectory snapshots were saved every 2 ps and analysed using the cpptraj program [48] of amber. moreover, h-bond occupancy was also examined for best compounds over simulation period. the binding free energies (δg total ) of the most promising hit compounds were calculated using the mm-gbsa (molecular mechanics-generalized born surface area) method, implemented in amber 18. the amber ff99sb molecular mechanics (mm) force field [49] was used to estimate energy contributions from the atomic coordinates of the ligand, receptor and the complex in a gaseous phase. the total binding free energy is calculated as a sum of the molecular mechanics binding energy (δe mm ) and solvation free energy (δg sol ) as given below: where, δe mm is further divided into internal energy (δe int ), electrostatic energy (δe ele ), and van der waals energy (δe vdw ), and the total solvation free energy (δg sol ) is contributed by the sum of polar (δg p ) and non-polar (δg np ) components. δg bind is the free energy of binding of the ligand evaluated after entropic calculations, which is (tδs). the mm-gbsa approach has been successfully used in binding free energy calculations [50] of antiviral inhibitors [51, 52] . the selected compounds were purchased from molport (riga lv1011, latvia) in 2 to 5 mg masses. the purity the compounds was greater than 95%. the most promising compounds resulting from vs were evaluated for antiviral activity by a luciferase assay in tzm-bl cells infected with wild type hiv-1 strains nl4.3 (cxcr4-tropic strain, x4) and bal (ccr5-tropic strain, r5). amd3100 (specific cxcr4 antagonist) and maraviroc (specific ccr5 antagonist) were included as best-in-class positive controls. the cellular toxicity of the compounds was also evaluated in tzm-bl cells. the anti-hiv replication assays in tzm-bl cells have been described previously [53] . the ability of the compounds to inhibit the ca 2+ flux induced by cxcl12 in u87.cd4.cxcr4 cells and by rantes/ccl5 in u87.cd4.ccr5 cells was investigated via calcium mobilization assays. intracellular calcium fluxes were measured with the flipr tetra system as described previously [54] . the ic 50 (i.e., the compound concentration that inhibits the cxcl12-induced cxcr4 signaling or ccl5-induced ccr5 signaling by 50%) was calculated for each compound. the well-known antagonists, amd3100 (cxcr4) and maraviroc (ccr5) were used as a positive control. to identify novel ccr5 antagonists, two parallel virtual screening campaigns were carried out using both receptor-and a ligand-based (pharmacophore) approaches. a total of ~1.2 million compounds were pre-filtered using a set of selected criteria: the lipinski rule of five (≤ 2 violations), predicted permeability in caco-2 and mdck cells (> 500 nm/s) [55] , and the predicted ic 50 value for the blockage of human ether-a-go-go related gene potassium ion (herg k + ) channel (qplogherg value set to -5). herg k + inhibition is a well-known safety issue encountered in the medicinal chemistry optimization of various chemokine receptor antagonists, including maraviroc [56] . as a result, the database was reduced to ~30%, which then underwent the subsequent glide docking protocol and the compounds were ranked based on xp docking and prime/mm-gbsa scores. in the pharmacophorebased screening, the top compounds were selected from the pool of the best-ranked 3000 compounds predefined by the pharmacophore hypothesis (as explained in methods). collectively, 137 compounds were selected and interactively visualized for molecular interactions. receptor-based virtual hits included the compounds with the lowest (best) binding affinity values and a good interaction profile with the binding site residues of ccr5. whereas, the main criteria in the pharmacophore-based screening included the potential to establish strong h-bond interaction (salt bridge) with glu283, π-π stacking interaction with trp86, and hydrophobic interactions with tyr108, ile198, and tyr198. furthermore, the visual inspection process contributed to the identification of compounds that revealed significant molecular interactions with the critical binding pocket residues of ccr5 and helped to exclude compounds that showed unrealistic docking conformations. among the top hits, 77 were found to follow the set selection criteria and showed strong interactions throughout the 20-ns md simulations with favourable binding free energies. although some of these compounds exhibited significantly different binding conformations, all top hits established an extensive interaction network with ccr5. these compounds were further investigated by mm-gbsa calculations and h-bond analysis. after a careful post-md inspection, 43 compounds were selected based on the following criteria; (1) overall backbone stability of the protein/ligand complex, (2) electrostatic (δe ele ) and van der waals (δe vdw ) interaction energy, (3) h-bonds occupancy, and (4) binding pocket residual contribution towards ligands. all these 43 hits were comparatively docked at cxcr4 receptor to investigate their potential for dual ccr5/cxcr4 antagonists [3] . as described earlier, both ccr5 and cxcr4 share marked similarities in their overall structures and in their active site geometry. these similarities include the presence of seven transmembrane domains with a high number of proline residues, a c-terminal threonine and serine-rich cytoplasmic region, four extracellular loops with a high number of cysteine residues and an n-terminal extracellular domain. the regions of the binding pockets contain critically important conserved residues which might allow ccr5 and cxcr4 to host the same ligand. based on these evidences, we specifically sorted out potential hits and 27 structurally diverse compounds were purchased and tested for their anti-hiv activity (figure s1) the ca 2+ mobilization assay allowed to evaluate the antagonistic activity (i.e., the potency to inhibit the rantes/ccl5 and sdf-1/cxcl12-induced calcium mobilization) of the selected compounds. both rantes (natural ccr5 ligand) and cxcl12 (natural cxcr4 ligand) produced a robust transient increase of cytosolic ca 2+ flux in u87.cd4.ccr5 and u87.cd4.cxcr4 cells, respectively. among the tested compounds, compounds 3, 6, and 27 showed antagonistic activity and dose-dependently inhibited the intracellular-induced ca 2+ flux ( table 1 ). the structures of these compounds are shown in the values of cc 50 , ec 50 and ic 50 are in micromolar if not otherwise stated. the results represent the mean of four readings. the ability of the compounds to inhibit the cytopathogenic effect induced by nl4.3 (cxcr4-tropic strain, x4) and bal (ccr5-tropic strain, r5) hiv-1 strains was further evaluated in tzm-bl cells ( the calcium mobilization clearly indicated that compounds 3, 6, and 27 bind to the host coreceptors. although compound 27 showed an ic 50 value in the low micromolar range as ccr5 antagonist, no antiviral activity was observed even at the highest tested concentration of 100 μm. the predicted binding modes of compounds 3 (ccr5/cxcr4 antagonist), 6 (cxcr4), and 27 (ccr5) were investigated to obtain an atomic understanding inside the binding pocket. for all three compounds, the md simulation time was further extended from 20 to 50 ns to enhance the investigations on the stability of the complexes and the compounds' binding energy contributions. the statistical correlation was also evaluated between the average amber/mm-gbsa total binding free energy (δg total ) at the co-receptors and the experimental ic 50 values from ca 2+ mobilization assay ( the stability analysis of the top-ranked compounds against ccr5 is diagrammatically illustrated in (figure 4, s2-s3) . overall, all 27 tested compounds occupied the bottom of the ccr5 binding cavity defined by the residues of the transmembrane (tm) helices (1, 2, 3, 4, 5 and 7), and these binding poses were supported by the co-crystallized conformation of maraviroc ( figure 4a) . moreover, the protonated nitrogen of the compounds identified through the pharmacophore-based hypothesis was found in the close vicinity of the negatively charged glu283, and most of these nitrogen atoms engaged in salt bridge interactions. all compounds exhibited excellent binding energies against ccr5 as indicated via the energy plot, where compounds 3, 6, and 27 ranked among the top, albeit no inhibition of ca 2+ flux evoked by rantes was observed for 6 ( figure 4b ). the stability of these compounds inside the deep binding cavity of the corresponding co-receptors revealed convergence in rmsd values ( figure s2) . compound 27 reached an equilibrium value of ~1.5 å (relative to the equilibrated position of the ligand) somewhat late than the others, at around 20 ns, due to its flexible fatty acid side-chain ( figure s2) . compound 6 adopted a favourable conformation inside the cxcr4 binding pocket at around 13 ns and the dual antagonist 3 displayed consistently stable binding at both co-receptors already from the early steps of the simulation (~ 2-3 ns) (figures s2) . the stable rmsd value in the last ~ 30 ns was evident from the stable molecular interactions governed by the ligand inside the respective binding site of the co-receptors. furthermore, the tm helices of cxcr4 and ccr5 also exhibited significant stability as observed from the root-mean-square fluctuations (rmsf < 1 å), although tm5, which is connected to tm4 by the largest extracellular loop ecl2, showed somewhat larger fluctuations (around 2 å) ( figure 4a and s3). the binding cavity of both co-receptors is mostly hydrophobic due to the presence of a relatively large number of nonpolar amino acids. the analysis of the binding interactions demonstrated that the stability of the compounds inside the binding pocket of cxcr4 and ccr5 is mostly governed by π-stacking and h-bond interactions ( figure 5a-d) . for further in-depth understanding of interactions, h-bond occupancy was also recorded (distance ≤ 3 å; angle ≥120°) throughout the 50 ns simulations ( figure 5e ). per-residue decomposition was also performed for the key residues involved binding the ligands. in 27/ccr5 complex, the interactions were mainly governed by multiple h-bond interactions with the binding site residues. compound 27 contains a short flexible fatty acid side-chain (9hydroxynonanoic acid) linked to monic acid by an ester linkage. as evident from the molecular interactions, compound 27 established a network of h-bonds over a period of 50 ns and adopted a more favourable conformation inside the binding cavity ( figure 5a) chain (figure 5a) . the per-residue decomposition analysis revealed favourable binding free energy contributions (< -1.5 kcal/mol) with trp86, tyr108, phe109, tyr148, whereas the interaction with glu283 revealed a positive value due to the desolvation effect. in the cxcr4/6 complex, the phenyl rings on either side of the molecule were fluctuating during the simulation, with especially the terminal phenyl moiety connected to the aliphatic carbon side chain, whereas the central piperazine ring remained stable in its position. as shown in figure 5b , the terminal side-chain benzene established a strong π-π stacking with trp94 and revealed the most favourable energy decomposition value of -4.6 kcal/mol as compared to the other π-π stacking interactions (with tyr108 and phe109). this π-π stacking interaction was found completely missing with trp86 in the 6/ccr5 complex because the molecule was flipped inside the binding cavity of ccr5 (figure s4) , which might explain the lack of activity against ccr5. moreover, the oxygen atom of the side-chain carboxylic acid of glu288 formed a strong and well populated (82.7%) salt-bridge (average distance, 2.47 å) with the protonated nitrogen atom of the piperazine ring of compound 6, which could stabilize the central piperazine ring. glu288 also showed a favourable binding free energy contribution of -3.37 kcal/mol. the dual ccr5/cxcr4 antagonist 3 contains a salicyl alcohol moiety, a chiral β-hydroxyl group, a secondary amine, and an ether-linked aryloxyalkyl sidechain. the nonpolar side chain facilitated binding with the hydrophobic binding cavity of the co-receptors. the protonated nitrogen of secondary amine in 3 retained similar spatial coordinates in both complexes and established well-populated salt-bridge (64.2 and 58.5%; average distance, 2.15 and 2.64 å) with the glutamate residue of ccr5 (glu283) and cxcr4 (glu288) at respective sites ( figure 5c-e) . the salt-bridge with glutamate provided an anchor point for a dual antagonist to extend in both directions. on one side, the aryloxyalkyl tail reached deep into the binding cavity of ccr5, where the terminal phenyl ring formed hydrophobic interactions with tyr37, tyr89, gln280, thr284 and met287 ( figure 5c) . whereas, the phenyl ring formed a π-π interaction with trp252 and established one h-bond with tyr255 (46.4%; average distance, 2.55 å) in cxcr4 ( figure 5d ). on the other side, the two hydroxyl groups of the salicyl alcohol moiety established h-bonds with thr195 of ccr5 (35.1%; average distance, 2.23 å), although no stable h-bond was observed by this group in cxcr4, except an additional stacking interaction with trp94. among the interacting residues, tyr108 (ccr5) and tyr116 (cxcr4) showed favourable interactions, while glu283 (ccr5) and glu288 (cxcr4) revealed a slightly negative value. the ec 50 values of the different compounds against ccr5 and cxcr4 were correlated quantitatively with the amber/mm-gbsa binding energy values derived from the md simulations in order to assess the performance of the calculations. for each md simulated coreceptor complex with the bound ligand, a total of 5000 snapshots (every 4 ps) were generated. the δg total values were calculated at 10 different time scales (0-2 to 0-20 ns) and compared with the experimental ec 50 values. table 3 tabulates average δg total values for ccr5 at different time scales (0-2 to 0-20 ns) of md simulation along with the pec 50 (-log of ec 50 ) values. the quantitative correlation between δg total and experimental pec 50 values at the shortest time scales (0-2 ns and 0-4 ns) was rather poor (r 2 = 0.347 and 0.285), albeit a gradual increase in correlation was observed with larger time scales (> 0-12 ns and above). the initial poor correlation was due to the event of a single conformation of the proteinligand complex, which did not incorporate the flexibility in the beginning of the simulation. this underlying phenomenon was also evident from the correlations obtained from the docking scores (glide xp) alone, which showed poor r 2 value (0.353) ( figure s5) . afterwards, improved correlations were observed with a longer time scales of 0-18 (r 2 = 0.622) and 0-20 ns (r 2 = 0.631) (table 3, figure 6 ). the overall trend of ligand rmsd trajectories of most complexes (except for few) showed stability after ~8 ns (8-20 ns), which is rationalized through the electrostatic and vdw interaction energies. consequently, the plot of δe mm components (δe elec and δe vdw ) with respect to md simulations at various time scales (8-10 to 8-20 ns) were correlated separately with experimental pic 50 values, as shown in figure 6 . ccr5 and cxcr4 are both important co-receptors involved in the hiv-1 entry process [3, 4, 11, 57] . the ccr5 binding cavity is much deeper and open, while the entrance to the active site of cxcr4 is relatively covered having a strong negative charge on the surface as compared to ccr5 [39] . a highly negative surface charge density across the binding site makes cxcr4 atypical among the chemokine receptors and this difference is evident form the structural attributes of the cxcr4-specific inhibitors. despite the substantial difference in the surface electrostatic potential, many compounds have been reported against both coreceptors [11, 19, 22, 58, 59] . comprehensive virtual screening experiments were carried out to investigate the multiple topographies of the ccr5 binding pocket using both receptor-and ligand-based approaches. the top hits from the ccr5 virtual screening campaign were afterwards docked in the binding cavity of cxcr4 to investigate the dual antagonism of these compounds. 27 compounds were selected for biological evaluation, from which three displayed promising activity. compound 3 is salmeterol, a β 2 -adrenergic receptor agonist acting as a dual cxcr4/ccr5 antagonists. compound 6 is the antihistamine drug cinnarizine with cxcr4 antagonistic potency and compound 27 is the antibiotic mupirocin displaying potent ccr5 antagonism. to date, no antiviral activity of these compounds has been reported, except for moderate activity of salmeterol against the dengue virus [60] . although, the pharmacological properties of these compounds have already been explained in the previous studies, very briefly, salmeterol is indicated to improve airflow in chronic obstructive pulmonary disease, and asthma. it falls under the category of long acting β-2 adrenergic receptor inhibitors with an elimination half-life of 5.5 hr when administered via pulmonary route. this long-acting feature of salmeterol is considered associated with its ability to bind with both active and exo-sites of the receptor. a blood maximum concentration of salmeterol is achieved within 15 min of pulmonary administration [61] . cyp3a4 is considered predominantly involved in the metabolism of salmeterol through α-hydroxylation. some frequently known adverse effects of salmeterol may include hyperlactatemia, metabolic acidosis, depression, anxiety, hypophosphatemia, and hypokalemia [62] . although, a 6-month long clinical trial of salmeterol have proved satisfactory in context of safety profile [63] . in context of these findings and keeping in view the serious side-effects of antiretroviral therapy, it could be most likely stated that further development and repurposing salmeterol for hiv treatment could serve a promising approach. cinnarizine is an antihistaminic compound used for motion sickness and vestibular disorders and is also considered as a nootropic agent [64, 65] . it is a long-known calcium channel blocker with slight potency towards certain dopamine receptors [66] . after oral administration, a maximum plasma concentration approaches in nearly 3 hours with elimination half-life of 3 to 4 hours [67] . its six metabolites are known, which are produces via different of cyps [68] . although, cinnarizine has long been used in clinical practice with considerable tolerance but concerns has been raised for its association with drug-induced acute and chronic parkinsonism, linked to its ability to interact with dopamine receptors [69] . on the other hand, long-term clinical trials up to 4-month have demonstrated reasonable safety [70] suggesting its beneficial application for further development as an anti-hiv agent. mupirocin, also known as pseudomonic acid a, is a natural produced by pseudomonas fluorescens that can inhibit bacterial isoleucyl-trna synthetase [71] . it is found effective against many gram-positive bacteria and some gram-negative bacteria. the presence of epoxide residue in mupirocin makes it prone to extensive metabolism quickly converting it into inactive monic acid, which makes its applications limited to topical antibacterial ointment [71] . its elimination half-life ranges between 20 and 40 minutes after an intravenous administration [72] . due to this short half-life and lack of detailed pharmacological data from parenteral or oral administration requires extensive further experimentation for the use of mupirocin against hiv. compounds 3, 6 and 27 showed highly significant amber/mm-gbsa (calculated) binding energy values during screening. through molecular modelling studies, the compounds were found well-fitted inside the binding site of both co-receptors and remained stable throughout the md simulation period. during md simulations, the compounds remained in a close proximity to a negatively charged glutamate residue in the respective co-receptors. molecular interactions of compounds 3 and 6 were in line with the pharmacophore hypothesis and the co-crystalized complexes of ccr5/maraviroc [39] and cxcr4/it1t [41] , respectively. the protonated nitrogen atoms of the tropane group of maraviroc engaged in a salt-bridge interaction (2.78 å) with glu283 of ccr5, while the protonated nitrogen of the imidazothiazole moiety (n1) of it1t showes a salt-bridge interaction (2.8 å) with glu288 of cxcr4. likewise, the piperazine nitrogen of compound 6 and the secondary amine of compound 3 established a consistent salt-bridge interaction over a period of 50 ns with the respective glutamates. the importance of the glutamate residue deep inside the active site in the stabilization of the complexes has been reported previously [73] [74] [75] [76] [77] [78] [79] and, therefore, glu288 of cxcr4 and glu283 of ccr5 are important residues for structure-based drug discovery of cxcr4 and ccr5 antagonists. in addition, both compounds engaged in significant nonpolar interactions with the corresponding hydrophobic residues of the respective co-receptors, and these interactions are supported with previous mutagenesis and modelling studies [39, 41, 80, 81] . compound 27, the most promising compound after receptor-based screening (ic 50 = 10.64 µm), showed a significant h-bond interaction profile with the residues lining the binding pocket of ccr5, as reported also for maraviroc [39, 80] . compound 6 did not inhibit the calcium flux induced by rantes in u87.cd4.ccr5 cells, despite the significant mm-gbsa binding energy with ccr5 (δg total = -56.48 kcal/mol). the difference in electrostatic potential of the entrance of the binding site may provide the reason to this discrepancy, which has long been recognized between r5 and x4 strains [82] , and is supported by numerous mutational studies [83] [84] [85] [86] . ccr5 bears a neutral to positive electrostatic surface at the binding site entrance, with a deep negatively charged binding pocket, which might hinder the cationic piperazine moiety of compound 6 [74] . cxcr4 has a strongly negative surface charge due to a highly negative n-terminus that can favourably accommodate compound 6 [87] . likewise, compounds 15 and 21, having relatively similar electronegative cyclic nitrogen atoms, showed selective anti-hiv activity against x4 hiv-1 strain (ic 50 of 41.13 and 64.33 µm, respectively). furthermore, the binding pocket of the h 2histamine receptor (pdb id: 6bqg), the primary target of cinnarizine [88] , has a similar, negatively charged surface ( figure s6) , which is in agreement with the selective binding of compound 6 with cxcr4. in contrast, compound 27 possesses an overall negative charge [89] and is a potent ccr5 antagonist, lacking activity against cxcr4. per-residue decomposition analysis revealed binding free energy contributions by important binding site residues involved in electrostatic and vdw interactions. among the residues that were found to stabilize the complex formation, glutamate residues produced positive decomposition values for compounds 3 and 27. this was due to the contribution of unfavourable solvation energy by blocking out a sufficient amount of solvent that might stabilize these negatively charged glutamates. however, due to the presence of long carbon chains in 3 and 27 reduced the solvent exposure of glutamate residues inside the binding cavity resulting in a more favourable value. compound 6 showed a fairly negative energy value for glutamate interaction due to the presence of two protonated nitrogen atoms of the piperazine ring, which counteracted the desolvation effect. this more negative per-residue decomposition value of glu288 in cxcr4 is supported by the evidence from taylor et al., [59] . they changed the protonated piperidine ring [22] to a doubly protonated piperazine ring aiming at increasing the electrostatic interactions, hence counteracting the desolvation effect. although the identified compounds were less active against ccr5 and cxcr4 as compared to the positive controls maraviroc and amd300, it should be underlined that these compounds are directly derived from virtual screening approaches without any further optimization. according to the literature, this is the first account of the activities of these compounds against ccr5 and cxcr4. moreover, the structural pharmacophores that are present in compounds 2, 3, 6, and 27 were not observed in the previously reported ccr5 and cxcr4 antagonists. pyrimidine, quinoline, tetrahydroquinoline, guanide, p-xylylenediamine, indole, and cyclic pentapeptide are commonly known pharmacophores as cxcr4 inhibitors [90] . the compounds 3, 6, and 27 possess relatively different structural architectures that do not know resemble with these moieties. this suggests that the discovery of 3, 6, and 27 as anit-hiv drugs could provide and new platform with a unique set of compounds that do not resemble with already known cxcr4 inhibitors. among potent ccr5 inhibitors, maraviroc has 8-azabicyclo[3.2.1]octane and triazole residues, vicriviroc has piprazine, trifluoromethyl, and pyrimidine residues, aplaviroc contain 1,4,9triazaspiro [5.5] undecane and cenicriviroc is composed of biphenyl with fused tetrahydroazocine and imidazole [91] . the only similarity of the compounds reported in this study was found in the form of piprazine in 6, which resembles vicriviroc. additionally, it is worth mentioning that the high selectivity of compound 27 towards ccr5 could serve as a promising starting point for further optimization towards novel and more potent ccr5 antagonists. moreover, it is notable that among the 27 screened compounds, 13 of them displayed antiviral activity against hiv-1 in low to medium micromolar range, which indicates an enrichment rate of approximately 48.14%. hence, this systematic study focuses on an additional argument emphasizing the advantages of virtual screening for discovering new drug candidates. the study outlines the identification of new selective ccr5 and cxcr4 antagonists, as well as the discovery of a dual ccr5/cxcr4 antagonist. the important pitfalls that must be addressed in virtual screening efforts against chemokine receptors is defining an effective scoring method using electrostatic surface potentials towards hit selection and prioritization. the pipeline of receptor-and ligand-based virtual screening, used in this study, proved as a promising tool to find antagonists for the selected targets. the results were validated through specific ca 2+ gpcr signaling assays 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activity of amd3100 cloning of a human seven-transmembrane domain receptor, lestr, that is highly expressed in leukocytes 5-ht2c receptor structures reveal the structural basis of gpcr polypharmacology the bioavailability of mupirocin in nasal secretions in vitro small molecule inhibitors of cxcr4 ccr5 inhibitors: emergence, success, and challenges key: cord-320591-re99v1qt authors: le, thanh ninh; chiu, chiu-hsia; hsieh, pao-chuan title: bioactive compounds and bioactivities of brassica oleracea l. var. italica sprouts and microgreens: an updated overview from a nutraceutical perspective date: 2020-07-27 journal: plants (basel) doi: 10.3390/plants9080946 sha: doc_id: 320591 cord_uid: re99v1qt sprouts and microgreens, the edible seedlings of vegetables and herbs, have received increasing attention in recent years and are considered as functional foods or superfoods owing to their valuable health-promoting properties. in particular, the seedlings of broccoli (brassica oleracea l. var. italica) have been highly prized for their substantial amount of bioactive constituents, including glucosinolates, phenolic compounds, vitamins, and essential minerals. these secondary metabolites are positively associated with potential health benefits. numerous in vitro and in vivo studies demonstrated that broccoli seedlings possess various biological properties, including antioxidant, anticancer, anticancer, antimicrobial, anti-inflammatory, anti-obesity and antidiabetic activities. the present review summarizes the updated knowledge about bioactive compounds and bioactivities of these broccoli products and discusses the relevant mechanisms of action. this review will serve as a potential reference for food selections of consumers and applications in functional food and nutraceutical industries. over the past twenty years, the heavy dependence of human nutrition on the sustainability of agricultural production has been highlighted owing to rapid population growth and environmental degradation [1] . increasing food and agricultural productivity has simultaneously imposed external expenses and consequences upon the financial resource, ecological system, and human health by excessive water use, soil occupation, fertilizers, pesticides, herbicides, and food waste treatment [2] . thus, to meet the united nations' sustainable development goals, the agriculture and food sectors have been confronted with a major challenge: to provide adequate nutrition for global food demand while minimizing the negative impacts on the environment [3] . furthermore, in recent years, improving public awareness about the healthy lifestyle framework has prompted the search for novel food sources, which are rich in essential nutrients and have positive effects on human health [4] . as a result, functional foods and nutraceuticals are gaining significant attention since these foods could provide both health benefits to reduce the risk of chronic diseases and basic nutrition [5, 6] . besides, many scientific projects and research groups are focusing on the recovery of food wastes and upgrading them into high-value byproducts to serve as functional ingredients in new product development [7] [8] [9] [10] . particularly, the issues of the food systems, including food safety, food security, and food sustainability, should be significantly addressed in the era of the coronavirus (covid19) pandemic crisis. the availability of bioactive constituent of food and functional foods may become bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing healthpromoting benefits [34] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [21] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure 2 and tables 1-3 ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [35] [36] [37] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [38, 39] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c18 analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables 1-3 ). it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure 2 ). bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing health-promoting benefits [34] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [21] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure 2 and tables 1-3 ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [35] [36] [37] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [38, 39] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c 18 analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables 1-3) . it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure 2 ). summary of the bioactive compounds analyzed in the last ten years of broccoli sprouts and microgreens. glucosinolates (glss), nitrogen−sulfur compounds (β-d-thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [40] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [19] . in broccoli sprouts and microgreens, 26 compounds of glss were identified (table 1) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [41] . in particular, glucoraphanin accounted for over 50% of the total glss content, which was quantified in the range of 605 to 1172 mg per 100 g of fresh weight (fw) [42] [43] [44] . glucosinolates (glss), nitrogen−sulfur compounds (β-d -thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [40] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [19] . in broccoli sprouts and microgreens, 26 compounds of glss were identified (table 1) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [41] . in particular, glucoraphanin accounted for over 50% of the total glss content, which was quantified in the range of 605 to 1172 mg per 100 g of fresh weight (fw) [42] [43] [44] . notably, glss are not the functional components in cruciferous vegetables, rather their hydrolysis products are the putative bioactive compounds [56] . when plant tissues are mechanically damaged, glss could be hydrolyzed by the enzyme myrosinase into a variety of degradation products, including isothiocyanates (itcs), thiocyanates, nitriles, epithionitriles, and oxazolidines. in particular, itcs have been proven to present strong anticarcinogenic activities [42] . as mentioned above, predominant glss in broccoli seedlings are glucoraphanin, glucoerucin, and glucobrassicin, which are enzymatically converted into sulforaphane (sfn), erucin (ern), and iberin, respectively [56] . sfn is a naturally occurring inducer of phase ii enzymes in human and animal bodies to detoxify cancer-causing chemicals. thus, it can reduce the risk of different cancers, especially those of the bladder, colon, and lung [59] . to date, sfn was analyzed as a major compound among the total of 17 itcs identified from broccoli sprouts and microgreens ( table 2 ). the total itc content of broccoli seedlings was reported to be around 11 mg per 100 g of fw. additionally, such seedlings were said to contain 90% sfn [19, 66] . uv/vis, ultraviolet-visible spectrophotometry; gc-ms, gas chromatography combined with mass spectrometry; gc−fid, gc with a flame ionization detector. besides glss and itcs, another important group of bioactive constituents present in cruciferous vegetables is the phenolic compounds. they are secondary metabolites produced in plants through the phenylpropanoid and shikimate pathways [74] . based on their structure, which comprises one or more aromatic rings with attached hydroxyl substituents, phenolic compounds can be categorized into various subgroups, such as phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. these compounds have been mainly reported for antioxidant activity. moreover, they are also associated with other health-promoting effects such as anticarcinogenic, antimicrobial, anti-inflammatory, and anti-aging properties [75] . hydroxycinnamic acids and flavonoid glycosides are among the main phenolic compounds found in broccoli seedlings [21, 76] . using quantitative and qualitative analysis, 37 phenolic compounds were characterized in broccoli sprouts and microgreens, including 29 hydroxycinnamic acids and derivatives, and 8 flavonoids and derivatives. their phenolic profile composed mostly of sinapic acid, gallic acid, flavonoids (quercetin and kaempferol), and other hydroxycinnamic acids (chlorogenic, caffeic acid, and ferulic acids) ( table 3 ). in general, total phenolic and flavonoid contents were determined to be in the ranges of 74-453 mg per 100 g of fw and 95-105 mg per 100 g of fw, respectively [43, 65, 77] . similar to other sprouted seeds, broccoli sprouts and microgreens are known for their high concentration of essential nutrient composition [17] . essential nutrients are compounds that must be supplied from foods, such as vitamins, minerals, fatty acids, and amino acids. they are required for normal body functions, including dna synthesis, energy production, and biosynthetic pathways [84] . nonetheless, the number of studies, which determined the nutritional composition of broccoli seedlings and the variations occurring during germination, is significantly smaller than that of glucosinolates and phenolic compounds. these studies indicated that broccoli sprouts are expressly rich in vitamins (a, c, k, and folic acid), minerals (potassium, calcium, magnesium, and selenium), pigments (carotenoids and chlorophylls) and some other important nutrients (amino acids, fatty acids, and dietary fiber) [20] [21] [22] 51, 57, 85, 86] . in general, the biochemical composition of broccoli sprouts and microgreens depends mainly on the germination time, and 8-day-old sprouts displayed the highest nutrient concentration [22] . among these compounds, carotenoids and chlorophylls were determined in several studies of broccoli sprouts [20, 50, 51, 73, 79] . carotenoids are a group of isoprenoid molecules synthesized as secondary metabolites by all photosynthetic plants, including broccoli [87] . they have lipophilic antioxidant and immunomodulatory activities owing to the conjugated double bonds of the long polyene chain, which are capable of inhibiting reactive oxygen species and reducing oxidative damage [21] . thus, carotenoids might prevent degenerative diseases, such as cardiovascular diseases, skin damage, diabetes, and several types of cancer [21] . the major carotenoids found in broccoli are β-carotene, α-xanthophyll (lutein), and β-xanthophylls (zeaxanthin, violaxanthin, and neoxanthin) [21] . in particular, β-carotene is the most typically studied carotenoids in broccoli sprouts and microgreens, due to its importance in medical science [50] . it supplies the human diet a substantial amount of vitamin a that is essential for organogenesis, tissue differentiation, immune function, and vision [87] . the total carotenoid (β-carotene) concentration of broccoli sprouts was reported in several studies with a range of 118 to 221 mg per 100 g of dry weight (dw) [20, 21] . chlorophylls are another group of light-absorbing pigments regularly present in broccoli sprout extract. they are primary metabolites that have a porphyrin structure [21] . similar to carotenoids, they also have antioxidant, anti-mutagenic, and anti-inflammatory potential [21] . the content of chlorophyll in broccoli sprouts was commonly determined along with total carotenoid content in previous studies, ranging from 738 to 850 mg per 100 g dw [21, 51] . additionally, a study showed individual concentrations of carotenoids (lutein and neoxanthin) and chlorophyll (chlorophyll a and b) in broccoli sprouts [21] . the human consumption of foods or nutraceuticals is not limited to bioactive compounds. in the human body, bioavailability is determined as the fraction of the dose administered that reaches the circulatory system and then distributes into target tissues so that the bioactive compounds are biologically available for exerting health-promoting benefits [88] . many factors could affect the bioavailability of the dietary phytochemicals, including plant cell wall compositions, chemical structures, processing conditions, environmental stress, as well as an individual's gastrointestinal system [56] . the effectiveness of a high consumption of broccoli sprouts in reducing the risk of cancer has been attributed mainly to their high content of glss. the daily intake of glss is difficult to estimate owing to the high variability of the constituent in cruciferous vegetables, particularly broccoli. a report for glucosinolate intake was indicated for european people ranging from 4.7-65 mg/day [32] . glss are broken down by both the plant enzyme myrosinase in the small intestine and the microbiota in the gastrointestinal tract into itcs, which are reported to have the prominent anticancer effect [89] . after intake 2-3 h, the itcs are absorbed from the small bowel and colon and metabolized by the mercapturic acid pathway. the metabolites are detectable in human urine and blood [53] . in broccoli sprouts, two of the most abundant glss are glucoraphanin and glucoerucin, and myrosinase hydrolysis of these glss form sfn and ern, respectively. sfn, distinguished itcs in broccoli sprouts, is one of the most potent naturally occurring inducers of phase 2 detoxification enzymes, boost antioxidant status, and protect animals against chemically induced cancer [40, 90] . its modes of action are involved with the repressor protein kelch-like ech associated protein 1 (keap1), nuclear factor erythroid 2 p45-related factor 2 (nrf2), and genes, which contain an antioxidant responsive element (are) [62] . thus, it is important for the inclusion of potential cancer chemopreventive agents in dairy foods. moreover, it has shown an inhibitory effect for urease from helicobacter pylori, a human pathogen [40] . the formation of other breakdown products from glss by intestinal microbiota is not well documented. for example, bifidobacterium strains belonging to the human intestinal microbiota can in vitro metabolize the glss to nitriles. it is also known that several microorganisms are able to convert nitriles into ammonia and organic acids [89] . phenolic compounds are substantial micronutrients in the human diet and the health benefits of polyphenols depend on the amount consumed and on their bioavailability [91] . the dietary intake of phenolic compounds has been associated with health-promoting effects, such as antioxidant, anti-aging, antiproliferative, and anti-inflammatory activities [19] . it has been reported that the average dietary intake of phenolic compounds for humans is about 780-1058 mg/day, including 50% of hydroxycinnamic acids, 20-25% of flavonoids, and 1% of anthocyanins [87] . bioavailability differs significantly among phenolic compounds so that the most abundant compounds in the human diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues [91] . there are two directions for the digestion of dietary phenolics, comprising digestion along the gastrointestinal tract and digestion inside the enterocytes. these digestions are based on hydrolase enzymes, which are available in the intestinal lumen, brush border, and enterocyte [88] . many studies showed that polyphenols are absorbed in both the small intestine and the large intestine by intestinal enzymes after microbial digestions [88] . in the human small intestine and stomach, gallic acid and caffeic acid are the most well-absorbed compounds (95%), followed by catechins (20%). the least well-absorbed polyphenols are proanthocyanidins and the anthocyanins. they are ph-sensitive, which could be broken down in the stomach and readily absorbable. flavonoid group is one of the group molecules with molecular weights > 500 da so that it has a low bioavailability level (1%) because molecules are improbable to be transported through passive diffusion pathways [76, 88, 91] . the dietary intake of carotenoids, particularly vitamin a, has been linked to the protection of dna, proteins, and lipids from oxidative damage [87] . digested carotenoids decrease oxidative dna damage so that they could protect colon cells against the stress of reactive oxygen species [87] . the dietary chlorophylls from fresh fruits and vegetables, which compose of chlorophyll a and b, showed distinct biological potentials, including wound healing, the control of calcium oxalate crystals, the modulation of xenobiotic metabolism, and the induction of apoptosis [21] . broccoli seedlings and their bioactive components exhibit many potential health-promoting roles. in the last decade, the beneficial effects, including antioxidant, anticarcinogenic, antimicrobial, anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [24, 25, 92] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables 4 and 5 ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure 3 ). anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [24, 25, 92] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables 4 and 5 ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure 3) . the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [93] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p450, lipoxygenases, and nitric oxide syntheses [94] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [95] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [93] . previous studies the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [93] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p450, lipoxygenases, and nitric oxide syntheses [94] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [95] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [93] . previous studies on broccoli sprouts and microgreens have demonstrated that they are recognized for their variety of naturally occurring antioxidants, comprising both nutritional antioxidants like vitamins (especially, ascorbic acid and α-tocopherol), and non-nutritional antioxidants such as carotenoids and phenolic compounds [96] . it was reported that these compounds are responsible for 80−95% of the total antioxidant capacity in broccoli sprouts [14] . the antioxidant activity of broccoli sprouts and microgreens has been determined in numerous studies using various methods. the capacity of broccoli sprouts and microgreens for chelating fe2 + and scavenging free radicals such as dpph (2,2-diphenyl-1-picrylhydrazyl) and abts (2,2 -azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) was demonstrated in numerous studies (table 4) . among the possible methods, dpph radical scavenging assay, abts radical cation decolonization assay, and ferric reducing antioxidant power (frap) assay were commonly employed in previous studies ( table 4 ). the dpph method is speedy, simple, and low cost in comparison to other test models. dpph, a dark crystalline molecule, is a stable chromogen radical formed by the delocalization of the spare electron over the molecule. the dpph scavenging assay is based on the electron donation of antioxidants to neutralize dpph radical. the reaction occurs with the loss of the violet color of dpph that is measured at 517 nm, and the discoloration acts as an indicator of the antioxidant effectiveness [31] . the abts decolonization assay is appropriate for both hydrophilic and lipophilic antioxidants. it uses a spectrophotometer to measure the ability of antioxidants to scavenge the stable radical cation abts + , a blue-green chromophore molecule with absorption at 734 nm. antioxidants can neutralize and decolorize the radical cation abts + by electron or hydrogen atom donations [97] . the frap assay was originally employed to measure reducing power in plasma, but it has been expanded for other biological fluids and plant extracts. thus, it is applicable for both in vitro and in vivo experiments. the frap mechanism is based on electron transfer rather than hydrogen atom transfer. the assay demonstrates the ability of antioxidants to reduce ferric iron. it measures the reduction of the ferric ion (fe 3+ )-ligand complex to the blue-colored ferrous (fe 2+ ) form at low ph by antioxidants (absorption at 593 nm) [98] . moreover, another mechanism of antioxidant activity of broccoli sprouts and microgreens have been confirmed in several in vitro and in vivo examinations by inhibiting the activity of prooxidant enzymes such as lipoxygenase (lox), and xanthine oxidase (xo), and activating antioxidant enzymes such as peroxidase (pod), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), nad(p)h-quinone acceptor oxidoreductase 1 (nqo1), and heme oxygenase-1 (ho-1) ( table 4) . prooxidant enzymes are considered as the main biological source of superoxide radicals, whereas antioxidant enzymes could eliminate the excess of reactive oxygen species and reduce oxidative damage during senescence [48, 83] . for example, enzyme activities of cytosolic glutathione peroxidase (gpx1), thioredoxin reductase(tr) in the thyroid, plasma glutathione peroxidase (gpx3), and ferric reducing ability of plasma (frap) significantly increased in response to broccoli sprouts ingestion in 4-week-old wistar rats [72] . in summary, previous studies showed that broccoli sprout extracts rich in vitamins, carotenoids, and phenolic compounds showed very high antioxidant activity in both in vitro and in vivo tests (table 4) . thus, they could be applied for antioxidant therapeutics to reduce the risk of chronic diseases caused by ros. interestingly, although the antioxidant capacity of broccoli seedlings has been reported comprehensively, it is still attracting considerable attention from investigators and researchers at present [20, 24, 80] . abts, 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulphonic acid decolonization activity; dpph, 2,2-diphenyl -1-picrylhydrazy radical scavenging activity; frap, ferric reducing-antioxidant power; teac, trolox equivalent antioxidant capacity; orac, oxygen radical absorbance capacity; pod, peroxidase activity; cat, catalase activity; sod, superoxide dismutase activity; gpx, glutathione peroxidase activity; chel, metal chelating activity; lpo, inhibition of lipid peroxidation; loxi, inhibition of lipoxygenase; xoi, inhibition of xanthine oxidase; nqo1, nad(p)h-quinone acceptor oxidoreductase 1; ho-1, heme oxygenase-1; ma/gc, malonaldehyde/gas chromatography. in the last decade, many studies and projects have supported the protective effects of natural products in cancer prevention. the protective role against cancer has been mostly attributed to the high content of glss, typically found in cruciferous vegetables [40] . broccoli sprouts and microgreens have been valued as a rich source of glss and their hydrolysis products (icts, particularly sfn), which are a well-known class of cancer chemotherapeutic agents that work by inducing apoptosis and arresting cell cycle progression (tables 1 and 2) . the potential mechanisms of action mainly involve the inhibition of proliferation and the induction of apoptosis in cancer (table 5) . hence, to demonstrate the anticancer activity of broccoli sprouts and microgreens, many in vitro tests have been carried out to determine the antiproliferative activity. their results indicated that broccoli seedlings exert strong cytotoxicity against different types of cancer cell lines, including hepatocellular carcinoma cells (hepg2), prostate carcinoma cells (pc-3, at-2, and sum159), lung carcinoma cells (a549), and colorectal adenocarcinoma cells (caco-2, and ht-29) ( table 5 ). in these studies, the highly effective ic 50 values were found to be from 29 to 190 µg/ml. on the another hand, the selectivity of broccoli seedlings was reported on normal skin fibroblasts (bj), normal colon fibroblasts (ccd18-co), and normal liver cells (fl83b) by displaying no toxic effects on their viability after the treatment of broccoli samples (table 5) . cancer is essentially a disease of uncontrolled cell division. it is caused by an imbalance between cell proliferation and apoptosis [101] . its development and progression are generally associated with the disorder of cell cycle regulators' activity. defects in the programmed cell death mechanism also make a key contribution to tumor pathogenesis [101] . thus, most chemotherapeutic agents exert their cytotoxic activity against cancer cells, since they cause dna damage and activate a complex signaling network resulting in cell cycle arrest and apoptosis induction [24] . targeting the cell cycle phase and the checkpoint signaling pathway, which leads to the arrests at g1/s or g2/m phases, would provide a promising opportunity for cancer treatment. besides, triggering cell apoptosis could be an effective strategy for potential chemotherapeutic agents [24, 25, 73, 101] . to confirm the antiproliferative activity of broccoli sprouts and microgreens, several in vitro studies revealed the mechanism of cell death based on inducing cell cycle arrest and apoptosis (table 5) . their results showed cell cycle arrests (obviously at g0/g1 and s phases) and significant increases in cell percentage with subg1 dna content, which is considered as a marker of apoptosis. besides, mitochondrial changes might activate the intrinsic apoptotic pathway. the loss of mitochondrial membrane potential (mmp) leads to the activation of several proteins linked to apoptosis, such as caspase-9 and cytochrome c [24] . hence, a few studies indicated the notable decrease in mmp levels of cancer cells after treatment by broccoli seedlings to prove the mechanism of programmed cell death [24, 73] . furthermore, some in vivo carcinogenesis models were employed to demonstrate the anticancer effects and related molecular mechanisms of broccoli sprouts and microgreens, such as c57bl/6 mice, balb/c mice, skh-1 hairless mice, and sprague-dawley rats (table 5 ). sfn extracted from broccoli sprouts showed the inhibition of breast cancer stem cells and downregulate the wnt/β-catenin self-renewal pathway in a nonobese diabetic/severe combined immunodeficient xenograft model [102] . sfn isolated from broccoli seeds and sprouts also exhibited anticancer effects in lung cancer cell lines and nude balb/c mice with lung cancer xenograft by inhibiting the pi3k-akt signaling pathway [25] . the transgenic adenocarcinoma of the mouse prostate model with broccoli sprout intake demonstrated a significant decline in prostate cancer occurrence and hdac3 protein expression in the epithelial cells of the prostate. hdac3 is one of the histone deacetylase enzymes that turn off tumor suppressor genes and promote the expression of oncogenes [103] . the broccoli sprout diet administered to adult her2/neu mice showed both preventive and suppressive effects on mammary cancer. these protective effects were associated with tumor-and epigenetic-related gene expression, and changed histone acetylation, dna methylation, and dna hydroxymethylation levels [104] . the dietary administration of broccoli sprout extract to the rat model inhibited bladder cancer development by inducing glutathione s-transferase and nad(p)h-quinone oxidoreductase 1 in the bladder, which are important enzymes against oxidants and carcinogens [105] . in vitro ns a549, h460, h446, hcc827, h1975, h1299 cells inhibiting cell proliferation; inducing apoptosis [25] in vitro ns caco-2, ccd18-co cells inducing cell cycle arrest and apoptosis; decreasing mmp level; increasing ros generation [73] in vivo ns female nod/scid mice eliminating breast cscs in vivo; downregulating wnt/β-catenin pathway [102] in vivo ns female nude balb/c mice inhibiting the pi3k-akt signaling pathway [25] in vivo 3 days female skh-1 hairless mice stabilizing p53; inducing phase 2 enzyme; inhibiting inos upregulation [106] in vivo ns male tramp mice in c57bl/6 background decreasing hdac3 protein expression [103] in vivo ns sv40 and her2/neu mice modulating epigenetic pathways; regulating epigenetic-controlled gene expression [107] in vivo ns female her2/neu mice regulating tumor-and epigenetic-related gene expression; increasing tumor suppressor gene expression [104] in vivo 3 days female sprague-dawley rats inducing gst and nqo1 [105] hepg2 the antimicrobial capacity of broccoli has been reported in several publications; however, most of these studies focused on broccoli florets. there are a few reports that displayed the detrimental effect of broccoli sprouts on pathogenic bacteria. a study investigated broccoli sprouts with high levels of gallic acid, esculetin, ferulic acid, and myricetin have the antibacterial activity against foodborne pathogens, including both gram-positive bacteria (staphylococcus aureus and bacillus subtilis) and gram-negative bacteria (salmonella typhimurium and escherichia coli), with minimum inhibition concentration (mic) values from 390 to 1560 µg/ml [24] . this study revealed that the gram-positive bacteria were more sensitive to broccoli sprout extract than the gram-negative bacteria, similar to the previously published results of other plant extracts [108, 109] . the possible reason might be the structural differences in the cell wall between these two classes of bacteria. gram-negative bacteria are surrounded by an additional outer membrane, which is a hydrophilic layer to prevent the access of many substances including natural compounds [108, 109] . thus, broccoli sprout extract was demonstrated to be more active on gram-positive bacteria. another in vitro study reported the powerful bactericidal activity of broccoli sprouts against helicobacter pylori by the presence of sulforaphane as the major anti-helicobacter active compound, with highly active inhibition zones (>5 cm) [26] . many reports showed that plant-derived bioactive compounds, such as phenolics, flavonoids, and glucosinolates, can inhibit the growth and activity of various microorganisms [110] . with the diversity in the molecular structure and chemical composition, these compounds can perform distinct antimicrobial effects, such as destabilization of the plasma membrane or inhibition of extracellular enzymes [110] . the antimicrobial activity against pathogenic bacteria, yeast, and phytopathogenic fungi was also proven by the presence of many antimicrobial peptides in broccoli floret extract [111] . moreover, the daily intake of sulforaphane-rich broccoli sprouts for 2 months was demonstrated to reduce gastric bacterial colonization and attenuate gastritis in helicobacter pylori-infected mice and patients [23] . in another clinical trial, the high-sulforaphane broccoli sprouts powder also showed a considerable effect on h. pylori eradication in type 2 diabetic patients with positive h. pylori [112] . collectively, these findings indicate that broccoli sprout extracts might serve as a potential source of antimicrobial agents in the food and pharmaceutical industry. several studies proved that broccoli sprouts and their bioactive components possess anti-inflammatory activity. hence, the application of broccoli sprouts and microgreens has potential in the prevention and treatment of inflammatory bowel diseases, such as ulcerative colitis and crohn's disease [27] . the anti-inflammatory mechanisms of broccoli sprouts are probably associated with the nuclear factor-kappa b (nf-κb) and nuclear factor erythroid 2-related factor 2 (nrf2) signaling pathways [27] . both in vitro and in vivo experiments, as well as clinical trials, mainly exhibit anti-inflammatory effects of broccoli sprouts, which showed high concentrations of sulforaphane and a group of phenolic compounds, including anthocyanins, isoquercetin, chlorogenic and cinnamic acids, via inhibiting inflammatory mediators such as a nitric oxide (no), decreasing the levels of proinflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin-6 (il-6), and il-1β, and increasing the levels of anti-inflammatory cytokines such as il-10 and il-22 [27, 28, 55, 72, 113, 114] . for example, a study showed that sulforaphane-enriched broccoli sprouts inhibited activation of the nf-κb signaling pathway and the secretions of inflammatory proteins (inducible nitric oxide synthase (inos), cyclooxygenase 2 (cox-2), tnf-α, il-6, il-1β, and prostaglandin e2 (pge2)) in microglial cells (bv2) and male icr (institute of cancer research) mice. in this study, broccoli samples also upregulated the expression of nrf2 and heme oxygenase-1 (ho-1) in normal bv2 cells and against scopolamine-induced amnesia in mouse brain tissue samples [27] . to analyze immunological parameters in the wistar rats with iodine deficiency, the significant decrease in il-6 level, along with no significant differences in il-10 concentration, were observed after adding broccoli sprouts to the diet [72] . high-sulforaphane broccoli sprouts also indicated favorable effects on inflammatory markers by reducing concentrations of serum high-sensitive c reactive protein (hs-crp), il-6, and tnf-α in type 2 diabetic patients [28] . broccoli is one of the few vegetables that is considered as a supplementary treatment for type 2 diabetes and for the prevention of its long-term complications [31] . a few studies investigated the potential benefits of broccoli sprouts and microgreens for patients with diabetes. a study determined the effects of broccoli sprout powder on insulin resistance in type 2 diabetic patients as a new approach by the use of its bioactive constituents. the results showed that broccoli sprout powder containing a high concentration of sulforaphane may significantly decrease in serum insulin concentration and lessen complications of diabetes [29] . in another report, the potential efficacy of sulforaphane extracted from young broccoli sprouts has been confirmed as an effective option for supplementary treatment in type 2 diabetes. it could induce some peroxisome proliferators-activated receptors, which contribute to glucose homeostasis in hyperglycemia and oxidative conditions [92] . obesity has become a worldwide health problem and leads to adverse metabolic disorders, such as cardiovascular disease and type 2 diabetes. a couple of studies documented positive actions of broccoli sprouts on obesity by regulating lipid metabolism. in a double-blind clinical trial, broccoli sprout powder as supplementary treatment in type 2 diabetic patients could have beneficial effects on lipid profiles and oxidized low-density lipoprotein ratio (ox-ldl/ldl), as risk factors for obesity and cardiovascular disease [30] . another mechanism to regulate lipid metabolism of broccoli sprouts may be associated with sulforaphane capacity to induce the nrf2 pathway [92] . a recent study revealed that glucoraphanin extracted from broccoli sprouts can decrease the lipid accumulation and increase the nrf2 activation. it leads to the downregulation of the expression of multiple genes involved in gluconeogenesis and lipogenesis, thereby alleviating obesity and diabetes [115] . several other health-promoting effects of broccoli sprouts and microgreens have also been explored by in vitro, in vivo, and clinical research. a couple of studies indicated that broccoli sprouts are a good source of bioactive compounds that act as xanthine oxidase (xo) inhibitors [76, 83, 99] . xo is the enzyme that catalyzes the metabolism of hypoxanthine and xanthine into uric acid, so inhibiting xo activity may be potentially useful in the treatment of gout or other xo-induced diseases [99] . broccoli sprout supplementation during pregnancy and the early newborn period could reduce brain injury following placental insufficiency in the newborn rats. the findings provide a new approach for the prevention of cerebral palsy and disabilities related to placental insufficiency [116] . the analgesic and antinociceptive effects of broccoli sprout extract were proven by the opioid mechanism using two in vivo experimental models of nociception. this study suggests the potential activity of broccoli sprouts in pain therapy [117] . in a double-blind study, the short-term ingestion of broccoli sprout homogenates showed the beneficial effects on nasal responses to the influenza virus in smokers by significantly decreasing virus-induced markers of inflammation and reducing the virus quantity. it may be a promising strategy for preventing influenza risk in smokers and other people exposed to airborne pollutants [118] . in recent years, broccoli sprouts and microgreens are one of the most consumed vegetable products. they have gained recognition as functional foods or nutraceutical foods by the increasing interest of consumers for diets that support health and longevity. consequently, the health-promoting compounds of broccoli seedlings have been extracted and isolated to integrate into food and pharmaceutical product formulations. over the past ten years, the extraction, isolation, and characterization of the functional properties of these broccoli products have been demonstrated by numerous scientific publications. in this review, we summarized for the first time the research findings of the last decade into bioactive constituents, bioactivities, and molecular mechanisms of broccoli sprouts and microgreens. they have been proven to present an abundant source of important natural compounds, including glucosinolates, phenolics, flavonoids, vitamins, minerals, and pigments. in particular, glucosinolates and their hydrolysis products are the main components analyzed, followed by phenolic compounds with a detailed profile. moreover, the previous studies have focused on several biological activities of broccoli seedlings, such as antioxidant, anticancer, antimicrobial, and anti-inflammatory, as well as the potentially beneficial effects for patients with cancers, diabetes, and obesity. in general, they are non-toxic or have low toxicity. hopefully, this updated review, in addition to being a reference for consumers' food selections, might attract more attention to broccoli sprouts and microgreens and their further applications in the food and nutraceutical industries, or even in clinical studies as cancer chemopreventive agents. in the future, more bioactive compounds in broccoli sprouts and microgreens will be isolated, identified, and evaluated. further investigations are required for exploring unrecognized biological functions and their underlying mode-of-action and molecular mechanisms in both in vitro and in vivo models, such as cardiovascular protective, hepatoprotective, neuroprotective, or enzyme inhibitory potentials. besides, well-designed clinical trials should be carried out to confirm these health-promoting benefits of broccoli seedlings on humans. food security and sustainable intensification broccoli microgreens: a mineral-rich crop that can diversify food systems how circular will you eat? the sustainability challenge in food and consumer reaction to either waste-to-value or yet underused novel ingredients in food small-seeded legumes as a novel food source. variation of nutritional, mineral and phytochemical profiles in the chain: raw seeds-sprouted seeds-microgreens relationships between bioactive food components and their health benefits the role of functional foods, nutraceuticals, and food supplements in intestinal health polyphenolic profile and varied bioactivities of processed taiwanese grown broccoli: a comparative study of edible and non-edible parts implementation of phenols recovered from olive mill wastewater as uv booster in cosmetics a facile water-induced complexation of lycopene and pectin from pink guava byproduct: extraction, characterization and kinetic studies phenols recovered from olive mill wastewater as additives in meat products the food systems in the era of the coronavirus (covid-19) pandemic crisis. foods recovery of high added-value components from food wastes: conventional, emerging technologies and commercialized applications separation of functional macromolecules and micromolecules: from ultrafiltration to the border of nanofiltration selecting sprouts of brassicaceae for optimum phytochemical composition microgreens: production, shelf life, and bioactive components micro-scale vegetable production and the rise of microgreens microgreen nutrition, food safety, and shelf life: a review bioactive compounds and bioactivities of germinated edible seeds and sprouts: an updated review broccoli and radish sprouts are safe and rich in bioactive phytochemicals evaluation of the bioaccessibility of antioxidant bioactive compounds and minerals of four genotypes of brassicaceae microgreens alone or combined, redirect the biosynthesis of glucosinolates, phenolics, carotenoids, and chlorophylls in broccoli sprouts biochemical composition of broccoli seeds and sprouts at different stages of seedling development dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in helicobacter pylori-infected mice and humans brassica oleracea l. var. italica) sprouts as the potential food source for bioactive properties: a comprehensive study on in vitro disease models the natural compound sulforaphene, as a novel anticancer reagent, targeting pi3k-akt signaling pathway in lung cancer analysis and anti-helicobacter activity of sulforaphane and related compounds present in broccoli (brassica oleracea l.) sprouts sulforaphane-enriched broccoli sprouts pretreated by pulsed electric fields reduces neuroinflammation and ameliorates scopolamine-induced amnesia in mouse brain through its antioxidant ability via nrf2-ho-1 activation effects of broccoli sprout with high sulforaphane concentration on inflammatory markers in type 2 diabetic patients: a randomized double-blind placebo-controlled clinical trial effect of broccoli sprouts on insulin resistance in type 2 diabetic patients: a randomized double-blind clinical trial broccoli sprouts powder could improve serum triglyceride and oxidized ldl/ldl-cholesterol ratio in type 2 diabetic patients: a randomized double-blind placebo-controlled clinical trial extraction, chemical characterization and biological activity determination of broccoli health promoting compounds health benefits and possible risks of broccoli-an overview physiological effects of broccoli consumption methods for determining bioavailability and bioaccessibility of bioactive compounds and nutrients pressurized hot water extraction (phwe) for the green recovery of bioactive compounds and steviol glycosides from stevia rebaudiana bertoni leaves evaluation of microwave-assisted extraction technology for separation of bioactive components of saffron (crocus sativus l.). ind. crops prod. 2020, 145, 111978 potential use of pulsed electric technologies and ultrasounds to improve the recovery of high-added value compounds from blackberries the effects of conventional and non-conventional processing on glucosinolates and its derived forms, isothiocyanates: extraction, degradation, and applications emerging technologies for the production of nutraceuticals from agricultural by-products: a viewpoint of opportunities and challenges glucosinolate metabolism, functionality and breeding for the improvement of brassicaceae vegetables glucosinolates in broccoli sprouts (brassica oleracea var. italica) as conditioned by sulphate supply during germination glucoraphanin, sulforaphane and myrosinase activity in germinating broccoli sprouts as affected by growth temperature and plant organs influence of light on health-promoting phytochemicals of broccoli sprouts effects of cacl2 on the metabolism of glucosinolates and the formation of isothiocyanates as well as the antioxidant capacity of broccoli sprouts glucosinolates fortification of cruciferous sprouts by sulphur supplementation during cultivation to enhance anti-cancer activity antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion establishing the occurrence of major and minor glucosinolates in brassicaceae by lc-esi-hybrid linear ion-trap and fourier-transform ion cyclotron resonance mass spectrometry calcium sulfate treatment enhances bioactive compounds and antioxidant capacity in broccoli sprouts during growth and storage uvb light doses and harvesting time differentially tailor glucosinolate and phenolic profiles in broccoli sprouts uv-b irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors photosynthetic pigments, phenolic, glucosinolates content and antioxidant capacity of broccoli sprouts in response to nanoselenium particles supply assessment of the anticancer compounds se-methylselenocysteine and glucosinolates in se-biofortified broccoli (brassica oleracea l. var. italica) sprouts and florets bioavailability of isothiocyanates from broccoli sprouts in protein, lipid, and fiber gels genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design effect of sucrose and mannitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts sucrose enhances the accumulation of anthocyanins and glucosinolates in broccoli sprouts metabolism and antiproliferative effects of sulforaphane and broccoli sprouts in human intestinal (caco-2) and hepatic (hepg2) cells characterization of products from the reaction of glucosinolate-derived isothiocyanates with cysteine and lysine derivatives formed in either model systems or broccoli sprouts physiological and biochemical metabolism of germinating broccoli seeds and sprouts sulforaphane bioavailability from glucoraphanin-rich broccoli: control by active endogenous myrosinase calcium mitigates the stress caused by znso 4 as a sulphur fertilizer and enhances the sulforaphane formation of broccoli sprouts effect of se treatment on glucosinolate metabolism and health-promoting compounds in the broccoli sprouts of three cultivars increasing antioxidant content of broccoli sprouts using essential oils during cold storage. agriculture (polnohospodárstvo) response surface optimization and identification of isothiocyanates produced from broccoli sprouts free radical scavenging, antiproliferative activities and profiling of variations in the level of phytochemicals in different parts of broccoli (brassica oleracea italica) absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days comparative study of predominant phytochemical compounds and proapoptotic potential of broccoli sprouts and florets selenium enrichment of broccoli sprout extract increases chemosensitivity and apoptosis of lncap prostate cancer cells effect of broccoli sprouts on thyroid function, haematological, biochemical, and immunological parameters in rats with thyroid imbalance antiproliferative effect of bioaccessible fractions of four brassicaceae microgreens on human colon cancer cells linked to their phytochemical composition sorting out the value of cruciferous sprouts as sources of bioactive compounds for nutrition and health phenolic acids: natural versatile molecules with promising therapeutic applications effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts influence of sodium and maturity stage on the antioxidant properties of cauliflower and broccoli sprouts phenolic profile and antioxidant activity in selected seeds and sprouts energy regulated nutritive and antioxidant properties during the germination and sprouting of broccoli sprouts (brassica oleracea var. italica) morphometric characteristics, polyphenols and ascorbic acid variation in brassica oleracea l. novel foods: sprouts, microgreens and baby leaves effect of sprouting and light cycle on antioxidant activity of brassica oleracea varieties antioxidant and antiproliferative activities in different maturation stages of broccoli (brassica oleracea italica) biofortified with selenium anticancer and antioxidant activity of bread enriched with broccoli sprouts health-affecting compounds in brassicaceae daily intake of broccoli sprouts normalizes bowel habits in human healthy subjects acylated anthocyanins in broccoli sprouts phytochemicals in daucus carota and their health benefits bioavailability of phytochemicals. in phytochemicals-a global perspective of their role in nutrition and health bioavailability of glucosinolates and their breakdown products: impact of processing bioavailability of sulforaphane following ingestion of glucoraphanin-rich broccoli sprout and seed extracts with active myrosinase: a pilot study of the effects of proton pump inhibitor administration chemical and biological characterisation of nutraceutical compounds of broccoli potential efficacy of broccoli sprouts as a unique supplement for management of type 2 diabetes and its complications oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options reactive oxygen species: the dual role in physiological and pathological conditions of the human body reactive oxygen species in health and disease analysis and antioxidant activity of extracts from broccoli (brassica oleracea l.) sprouts measurement of antioxidant activity review on in vivo and in vitro methods evaluation of antioxidant activity enhancement of antioxidant abilities and the lipoxygenase and xanthine oxidase inhibitory activity of broccoli sprouts by biotic elicitors induction of the phase 2 response in mouse and human skin by sulforaphane-containing broccoli sprout extracts pharmacological targeting of cell cycle, apoptotic and cell adhesion signaling pathways implicated in chemoresistance of cancer cells sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells broccoli sprouts delay prostate cancer formation and decrease prostate cancer severity with a concurrent decrease in hdac3 protein expression in transgenic adenocarcinoma of the mouse prostate (tramp) mice maternal epigenetic regulation contributes to prevention of estrogen receptor-negative mammary cancer with broccoli sprout consumption inhibition of urinary bladder carcinogenesis by broccoli sprouts protection against uv-light-induced skin carcinogenesis in skh-1 high-risk mice by sulforaphane-containing broccoli sprout extracts temporal efficacy of a sulforaphane-based broccoli sprout diet in prevention of breast cancer through modulation of epigenetic mechanisms antimicrobial activity and phytochemical analysis of organic extracts from cleome spinosa which approach is more effective in the selection of plants with antimicrobial activity? evid plant phenolics and phenolic-enriched extracts as antimicrobial agents against food-contaminating microorganisms antimicrobial activity of broccoli (brassica oleracea var. italica) cultivar avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast complementary and alternative medicinal effects of broccoli sprouts powder on helicobacter pylori eradication rate in type 2 diabetic patients: a randomized clinical trial nutraceutical improvement increases the protective activity of broccoli sprout juice in a aqueous extract of glucoraphanin-rich broccoli sprouts inhibits formation of advanced glycation end products and attenuates inflammatory reactions in endothelial cells glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency broccoli sprouts in analgesia-preclinical in vivo studies effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study funding: this research received no external funding. the authors declare no conflict of interest. key: cord-338436-0z828org authors: tzou, philip l.; tao, kaiming; nouhin, janin; rhee, soo-yon; hu, benjamin d.; pai, shruti; parkin, neil; shafer, robert w. title: coronavirus antiviral research database (cov-rdb): an online database designed to facilitate comparisons between candidate anti-coronavirus compounds date: 2020-09-09 journal: viruses doi: 10.3390/v12091006 sha: doc_id: 338436 cord_uid: 0z828org background: to prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. methods: we reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. results: as of august 2020, the coronavirus antiviral research database (cov-rdb; covdb.stanford.edu) contained over 2800 cell culture, entry assay, and biochemical experiments, 259 animal model studies, and 73 clinical studies from over 400 published papers. sars-cov-2, sars-cov, and mers-cov account for 85% of the data. approximately 75% of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors (21%), viral protease inhibitors (17%), miscellaneous host-acting inhibitors (10%), polymerase inhibitors (9%), interferons (7%), fusion inhibitors (5%), and host protease inhibitors (5%). of 975 compounds with known or likely mechanism, 135 (14%) are licensed in the u.s. for other indications, 197 (20%) are licensed outside the u.s. or are in human trials, and 595 (61%) are pre-clinical investigational compounds. conclusion: cov-rdb facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development. cov-rdb contains four main types of antiviral experimental data, six main lookup/explanation tables, and a registry of ongoing or planned clinical trials. the four main types of antiviral experimental data include (i) cell culture and entry assay experiments; (ii) biochemical experiments; (iii) animal model studies; and (iv) clinical studies. the six main lookup/explanation tables provide information on viruses, virus strains/isolates, tested compounds, compound targets, cell types, and animal models. cov-rdb data are stored in a postgresql relational database, but there is not necessarily a oneto-one relationship for the tables displayed on the web and their underlying database structure. indeed, several of the website tables contain information from more than one underlying database table. as of 14 august 2020, the cov-rdb contains data from more than 1800 virus cell culture experiments, 465 entry assay experiments, 519 biochemical experiments, 259 animal model experiments, and 71 clinical studies from more than 310 peer-reviewed publications and 90 preprints. research articles are identified through incremental daily searches of pubmed and biorxiv using the search term "coronavirus" and by citations identified through reading these papers. publications containing experimental data are imported into a staging zotero database and then annotated to extract the data described in the sections below. preprints that are subsequently published in a peer-reviewed journal are identfied using a computer script that parses datasets downloaded from the stephen b. thacker cdc (centers for disease control and prevention) library the cell culture experiments table contains 13 fields, including four fields present in each of the experiment tables: reference, compound, virus category, and virus isolate/strain. the nine fields unique to the cell culture experiments table include six that describe experimental conditions and the three experiment results are the half-maximal effective concentration (ec 50 ), percent inhibition, and the 50% cytotoxic concentration (cc 50 ). the ec 50 can only be determined using a series of compound dilutions. while the ec 50 is usually reported as µm, inhibitory activity for interferons is also often reported as international units (iu)/ml and inhibitory activity for monoclonal antibodies is often reported as ng/ml. the ec 50 is available for the vast majority of in vitro cell culture experiments. however, for a few experiments, the experimental setup involved a single compound concentration (rather than a dilution series). for these experiments, the percent virus inhibition with the single compound concentration is reported. there are two tables for entry assay experiments-one for pseudovirus entry assays and another for cell-cell fusion assays. the pseudovirus assay table contains the following six unique fields: (i) pseudovirus vector, (ii) pseudovirus number, (iii) target cell type, (iv) time to addition of drug, (v) indicator of virus replication, and (vi) ec 50 . in the pseudovirus experiments, the virus strain is a virus construct composed of a virus that does not require a biosafety level 3 (bsl-3) laboratory, such as vesicular stomatitis virus (vsv) or human immunodeficiency virus type 1 (hiv-1), into which the coronavirus spike (s) gene has been cloned. this construct also has a reporter gene such as luciferase or gfp. the cell-cell fusion assay table contains the following seven unique fields: (i) effector cell type, (ii) effector cell number, (iii) target cell type, (iv) target cell number, (v) time to addition of drug, (vi) indicator of virus replication, and (vii) ec 50 . the biochemical experiments table contains two unique fields: the biochemical target and the half maximal inhibitory concentration (ic 50 ). the biochemical target is usually one of the virus enzymes including rna-dependent rna polymerase (rdrp), main protease (also called 3c-like protease; 3cl pro ), papain-like protease (pl pro ), and helicase. however, cell-free assays that test inhibitors of the spike (s) protein binding to angiotensin converting enzyme 2 (ace2) are also included. the animal model experiments are characterized by comparisons between a group of animals receiving a treatment intervention either shortly before or after virus infection and a group of untreated virus-infected control animals. the animal model experiments table has two parts-experimental conditions and experimental results. the experimental conditions include the (i) animal model, (ii) size and route of virus challenge, (iii) treatment intervention, (iv) treatment dosage, (v) treatment timing in relation to the addition of virus, (vi) number of treated subjects, and (vii) number of control subjects. the experimental results, which often depend on the study, include endpoints such as mortality, weight loss, fever, respiratory rate, lung pathology, and virus load measurements. the reduction of endpoint severity is reported on an ordinal scale ranging from 0 to 3. there are more than 59 references containing more than 259 animal model experiments, nearly all involving sars-cov-2, sars-cov, or mers-cov. approximately 70% of the studies involve mice; 10% involve non-human primates (rhesus macaques, marmosets, and cynomolgus macaques); and 20% involve hamsters, ferrets, cats, dogs, or rabbits. the most commonly studied interventions have included monoclonal antibodies, fusion inhibitors, interferons, and the nucleoside analog polymerase inhibitors. the clinical studies are represented using several enumerated and free-text fields. the enumerated fields include the reference, virus category, and type of study (e.g., observational, randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not provide an assessment of study quality such as validity and risk of bias as there are other research groups providing this type of assessment. antiviral data on coronaviruses other than sars-cov-2 provide insight into the robustness of an antiviral compound, in that, compounds that are active against multiple viral species will be more likely to inhibit future pandemic coronaviruses and will be less vulnerable to the development of drug-resistance mutations. indeed, for many drug targets, such as the virus rdrp and 3cl pro enzymes, and for host processes upon which coronaviruses depend, inhibitory compounds will likely have broad-spectrum activity. cov-rdb contains antiviral data for six categories of coronaviruses: sars-cov-2, sars-cov, mers-cov, endemic human coronaviruses, bat coronaviruses, and non-bat mammalian coronaviruses. sars-cov-2 (37%), sars-cov (31%), and mers-cov (17%) account for 85% of the data. however, the proportion of data associated with sars-cov-2 is rapidly increasing. figure 2 shows the distribution of study types for sars-cov-2, sars-cov, and mers-cov. sars-cov and sars-cov-2 belong to the same betacoronavirus 2b (sarbecovirus) clade, and their amino acids are approximately 97% identical in the rdrp and 3cl pro enzymes and 84% identical in the spike protein. in contrast, mers, a clade 2c betacoronavirus, is approximately 75% identical to sars-cov and sars-cov-2 in the rdrp gene, 60% identical in the 3cl pro gene, and 40% identical in the s gene. within each of these three viruses, there is little diversity, with median pairwise distances ranging between 0% and 0.2%. the four endemic human coronaviruses include two clade 2a betacoronaviruses and two alphacoronaviruses. bat coronaviruses are distributed widely among different clades [2, 3] . indeed, 4 of the 9 betacoronavirus clades and 7 of 11 coronavirus clades are found only in bats. the mammalian coronaviruses include murine hepatitis virus (mhv), which is a longstanding experimental model for coronavirus infection, and several other coronaviruses that have been studied because they are important livestock diseases [4] . although infectious bronchitis virus is an avian gammacoronavirus, we have included it in the non-bat mammalian coronavirus category. cov-rdb uses the terms isolate and strains to describe the different viruses used in antiviral studies, although "strains" is usually reserved for describing isolates that have distinct phenotypic properties [5] . where possible, isolates are named according to the recommendations from the international committee on taxonomy of viruses [6] , i.e., virus/host/location/isolate/date. most sars-cov-2 isolates are nearly identical to one another with the upper limit for the pairwise amino acid distance being about 0.1%, although this number varies depending upon the gene [7] . therefore, the biological significance of the isolate used for a particular study is not known. however, for some treatments such as monoclonal antibodies, changes in the sequence encoding the relevant epitope, specifically in the s protein receptor binding domain may prove to have biological and clinical significance [8, 9] . although sars-cov resulted from at least two zoonotic introductions from civet cats [4] , and although mers-cov resulted from multiple zoonotic introductions from dromedary camels, these viruses also demonstrate little genetic variability. several of the most commonly used isolates have been cloned, either as intact viruses (e.g., by plaque purification or limiting dilution) or by constructing a cdna copy representing a single sequence variant. modification of these clones, such as selection of a resistant variant in vitro [10] or introduction of a reporter gene like gfp or luciferase, presumably retains the characteristics of the original parental virus isolate or strain [11] . commonly used isolates that have been cloned and manipulated in the laboratory include mers-cov/human/amsterdam/emc/2012 [12] , sars-cov/human/hanoi/urbani/2003 [13] , sars-cov-2/human/usa/wa1/2020 [14] , and sars-cov-2/human/munich/929/2020 [15] . the cell lines table provides descriptions for the cell lines used in cell culture and entry assay experiments. it contains four fields: (i) the cell line's commonly used name, (ii) the source of the cell line, (iii) closely related cell lines, and (iv) a description of the cell line and one or more of the closely related cell lines. the most commonly used cell lines for sars-cov and sars-cov-2 include a variety of different vero cell clones [16] [17] [18] [19] [20] , huh7 [16, 21] , caco-2 [22] , calu-3 [23] , and 293t/ace2 cells [16] [17] [18] ( table 1) . while each of these cell lines expresses ace2, only calu-3 cells were originally derived from lung epithelial cells. the 293t cells are typically used for cell-cell fusion and pseudovirus entry experiments. several studies have also used human alveolar epithelial cells or a variety of different respiratory system or kidney organoids [24, 25] . the cell lines used for mers-cov are similar, with the main exception that 293t/dpp4 (dipeptidyl peptidase 4) cells are used instead of 293t/ace2 cells because dpp4 (aka cd26) is the mers-cov receptor [18] . vero cells support the replication of many viruses often producing a visual cytopathic effect [16] [17] [18] . they express ace2, the receptor for sars-cov and sars-cov2, and dpp4, the receptor for mers-cov. although vero cells are ifn-deficient, they express the ifn-α/β receptor and thus retain the ability to respond to exogenous ifn [19] . vero e6 cells engineered to express greater amounts of tmprss2 produce higher sars-cov-2 titers of sars-cov-2 [20] . drugs that target tmprss2 are often inactive in vero cells. human lung epithelial cell line calu-3 cells form differentiated pseudostratified columnar epithelia highly permissible to coronavirus infection. they are polarized with an apical domain facing the airway lumen and a basolateral domain facing internally. they produce a visual cytopathic effect. the 2b4 clone has high ace2 expression. they are often used for the preclinical development of respiratory drugs [23] . heterogeneous human epithelial colorectal adenocarcinoma caco-2 cells are considered to be more pharmacologically relevant than vero cells for some studies because of their human origin [22] . human hepatoma huh-7 cells express ace2 and tmprss2, yet do not support levels of replication as high as vero cells [16, 21] . the 293t cells are derived from the human embryonic kidney 293 cell line. 293t cells contain the sv40 large t-antigen, which facilitates replication of transfected plasmids containing the sv40 origin of replication. the 293t/ace2 cells are transfected to express ace2 and have been used for many sars-cov cell-cell fusion and pseudovirus entry inhibitor studies [16] [17] [18] . human airway epithelial cells differentiated human airway cells have occasionally been used to study antiviral agents, although they are more commonly used to study viral pathogenesis [24, 25] . tmprss2-transmembrane serine protease 2; ace2-angiotensin converting enzyme 2; ifn-interferon; dpp4-dipeptidyl peptidase 4; sv40-simian virus 40. the over 10 different animal models used in experiments described in the cov-rdb include three non-human primate models (rhesus macaques, cynomolgus macaque, and marmosets), multiple transgenic and non-transgenic mouse models, and several additional rodent models including hamsters and ferrets [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . the transgenic mice have been modified in multiple ways, including to express hdpp4 so that they can be infected with mers-cov, to knock out the interferon (ifn)-α/β receptor to compromise innate immunity [44] ; to knock out recombination activating gene 1 (rag1) to compromise adaptive immunity [45] ; to knock out carboxylesterase 1c, which causes poor plasma stability of remdesivir; and to express human rather than mouse ace2 [39] [40] [41] . table 2 describes the utility of the most common non-human primate and mammalian models for studies of the pandemic coronaviruses. pathological changes observed in the aged mouse model infected with sars-cov more closely resemble those observed in humans [29] . rag −/− mice lack t and b cells and lack adaptive immunity and experience prolonged coronavirus shedding [45] . ifnar −/− mice are vulnerable to greater coronavirus disease severity [44] . there are many hace2 transgenic mouse models. these mice are more likely to experience weight loss, detectable virus loads, and interstitial pneumonia following challenge with sars-cov-2 than those with the murine ace2 receptor [39] [40] [41] . rhesus macaque infection causes a self-limiting disease associated with virus replication. radiographic and pathologic examination of sars-cov-2-infected animals display evidence of pneumonia [26, 30, 32, 42] . cynomolgus macaque infection results in a productive infection in respiratory epithelial cells. symptoms are minimal but virus shedding can last up to 2 weeks. chest radiographs reveal unifocal or multifocal pneumonia. autopsy reveals variable amounts of foci of diffuse alveolar damage [31, 33] . rag-recombination activating gene; ifnar-interferon-α/β receptor; hace2-human angiotensin-converting enzyme 2. the target table has two main fields: name and description. the target classification organizes drugs, treatments, and compounds according to the virus or host process targeted by a compound including virus enzymes, virus entry into cells, host immunological responses, and other host processes. there are three virus enzyme inhibitor classes: rdrp, protease (including 3cl pro , pl pro ), and helicase inhibitors. there are four inhibitor classes targeting virus entry: convalescent plasma and polyclonal antibody preparations, monoclonal antibodies, miscellaneous other compounds that inhibit virus receptor binding, and fusion inhibitors. there are two classes that target host immunological responses: interferons and other potential immunostimulatory compounds. although there are potentially many mechanisms by which targeting a host processes may interfere with virus replication, we have divided these into two broad categories: host protease enzymes used by coronaviruses to cleave the spike protein, thus, priming it for fusion and other host proteins or pathways utilized by coronaviruses. the classification of host-acting compounds is likely to continue to evolve as mechanistic pathways become better defined. table 3 describes each of the targets and compound classes described above. figure 3 shows the distribution of experimental data types according to target. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-nhydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-n-hydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. table 3 . antiviral coronavirus therapy targets. inhibitors of the coronavirus rna-directed rna polymerase (rdrp) enzymes include nucleoside analogs that cause immediate chain termination, delayed chain termination, or viral mutagenesis. coronaviruses contain two protease enzymes: 3 chymotrypsin-like cysteine protease (3cl pro or main (m)-pro) and papain-like (pl pro ). coronavirus helicases catalyze the unwinding of duplex rna molecules into single strands. entry convalescent plasma and polyclonal sera. convalescent plasma is one of the most widely studied treatments for covid-19. polyclonal sera and immunoglobuline preparations have also entered clinical trials. recovery during sars-cov, mers-cov, and sars-cov-2 is usually associated with the development of neutralizing antibodies. most sars-cov-2 neutralizing mabs target the part of the receptor binding domain (rbd) that binds ace2 while most mers-cov neutralizing mabs target the part of the rbd that binds dpp4. many highly potent neutralizing mabs targeting each of the pandemic coronaviruses have shown protection in vitro and in animal models. structural studies have defined specific rbd epitopes recognized by individual mabs and identified amino acid residues that are critical for mab binding. other receptor binding inhibitors sars-cov and sars-cov-2 spike s1 binds to the cellular angiotensin converting enzyme 2 (ace2) receptor. mers-cov binds to dipeptidyl peptidase 4 (dpp4). a variety of compounds including non-antibody proteins, peptides, and small molecules have been shown to prevent the binding of the coronavirus spike protein to its cellular receptor. following receptor binding and spike s1/s2 cleavage and s2 priming, heptad region 1 (hr1), which is close to the fusion peptide sequence, and hr2, which is close to the virus membrane, collapse on to one another to bring virus and cell membranes together. nearly all fusion inhibitors are hr2-mimicking peptides less than 70 kda that bind hr1, thus preventing hr1−hr2 binding. interferons have been extensively studied for their ability to inhibit each of the pandemic coronaviruses in cell culture, animal models, and/or clinical studies [47] . sars-cov-2 may be more susceptible to interferons than sars-cov is [48] . there are several clinical trials using immunostimulatory cytokines and compounds purported to induce interferon. cleavage of coronavirus spike proteins is necessary for the virus to transition from receptor attachment to cell fusion. for sars-cov-2, there is a poly-basic furin cleavage site at the s1/s2 boundary and another cleavage site within s2 believed to be cleaved at the cell surface by host tmprss2 enzymes [49] . multiple intracellular processes essential to virus replication are vulnerable to pharmacologic inhibitors including endosomal acidification, membrane formation, various signaling pathways, nucleotide biosynthesis, and autophagy [50] . many compounds with uncertain mechanisms of action have been found to inhibit coronaviruses in vitro. several of these are also being studied in clinical trials. mabs-monoclonal antibodies. figure 4 displays ec 50 values for many of the directly acting antiviral compounds currently in clinical trials for the treatment of covid-19 including six polymerase inhibitors (remdesivir, eidd-2801, favipiravir, ribavirin, galidesivir, and sofosbuvir), three hiv-1 protease inhibitors (lopinavir, atazanavir, and darunavir), and three entry inhibitors (receptor binding monoclonal antibodies, soluble recombinant human ace2, and umifenovir). figure 5 displays ec 50 values for many of the repurposed compounds that target host processes required for virus replication including two host pis that target the transmembrane serine protease 2 (tmprss2) enzyme (camostat and nafamostat), three chloroquine analogs that interfere with endosomal acidification (chloroquine, hydroxychloroquine, and mefloquine), three other compounds believed to interfere with membrane trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). figures 4 and 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec50s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 μm. however, there is also marked heterogeneity in the ec50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec50s consistently below 1 μm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec 50 s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 µm. however, there is also marked heterogeneity in the ec 50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec 50 s consistently below 1 µm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . viruses 2020, 12, x for peer review 11 of 22 table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display submicromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1′cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5′isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display sub-micromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1 -cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. regn10933 and regn10987 are mabs with subnanomolar inhibitory activity that bind to non-overlapping ace2-competing sars-cov-2 spike receptor binding domain epitopes [8, 63] . this mab combination also reduces virus replication and lung pathology in syrian hamsters and rhesus macaques [64] . the combination is being evaluated in phase iii trials for preventing and treating sars-cov-2 infection. ly3819253 is a sars-cov-2 mab in phase iii trials for preventing and treating covid-19. as of august 2020, there are no associated published preclinical data. mabs (phase i/ii trials) azd7442, brii-196, js016, scta01, sti-1499, and ty027 are mabs in phase i/ii trials. as of august 2020, there are no associated published preclinical data linked to mabs with these names. many research groups that have published preclinical data on one or more mabs (or mab variants such as nanobodies) including their in vitro inhibitory activity, genetic sequence data, three-dimensional structural data, and/or animal model data [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] . interferons ifn-α, ifn-β, and ifn-λ ifn-α, ifn-β, and ifn-λ each inhibit sars-cov-2 by 90%-99% at low concentrations of about 100 international units (iu)/ml [48, [81] [82] [83] [84] . inhalational ifn-α and parenteral ifn-β were associated with modest reductions in disease severity and/or virus loads in two small open-label clinical trials [84, 85] . an inhaled formulation of ifn-β was reported in the news to significantly reduce the odds of developing severe disease or death in a blinded randomized control trial (sng016) of 220 patients that has not yet been published (https://www.synairgen.com/covid-19/). there are currently four planned or ongoing placebo-controlled trials of parenteral or inhaled ifn-β (~1800 patients) and of parenteral ifn-λ (~400 patients). camostat and nafamostat are tmprss2 inhibitors with nanomolar coronavirus inhibitory activity in biochemical and cell culture assays [51, [53] [54] [55] [56] [86] [87] [88] . both drugs are used in japan for the treatment of pancreatitis, while nafamostat is also used as an anticoagulant and for the treatment of disseminated intravascular coagulation. although nafamostat has approximately 10-fold greater inhibitory activity than camostat, it may be associated with greater toxicity. camostat is being studied in two blinded and two open-label randomized controlled studies totaling about 900 patients. nafamostat is being studied in three small randomized open-label studies totaling about 200 patients. apilimod was found to inhibit sars-cov-2 at two-digit nanomolar levels with high selectivity indexes in multiple drug screens [89] [90] [91] [92] . it inhibits the membrane protein pi (3, 5) p2 by inhibiting the enzyme pi-3p-5-kinase (pikfyve) thus interfering with endosomal trafficking of sars-cov-2 and additional viruses utilizing the same endosomal pathway [93, 94] . it has been studied in humans in multiple clinical trials and been found to be safe and well tolerated. it is being studied for the treatment of mild sars-cov-2 infections in one randomized placebo-controlled phase ii trial. ptc299 is an inhibitor of dihydroorotate dehydrogenase (dhodh), a rate limiting enzyme in the pyrimidine biosynthesis pathway [95] . dhodh inhibitors are therapeutic targets for autoimmune disases and viral infections [96, 97] . ptc299 has been found to be safe and have favorable pharmacokinetics in more than 300 human subjects and has low nanomolar sars-cov-2 inhibitory activity and a high selectivity index [98] . as both viral replication and cytokine overproduction depend on pyrmidine synthesis, dhodh inhibition may have a dual role in covid-19 treatment. dhodh inhibition is synergistic with viral polymerase inhibition [97] . there is one phase ii/iii trial of ptc299 for patients with severe but not critical covid-19. leflunomide, another repurposed dhodh inhibitor was found to reduce virus load in an open-label pilot study of 27 patients [99] . soluble recombinant human ace2 (rhace2) rhace2 protects mice for sars-cov-1 ards and has been studied as a treatment for ards in humans [100, 101] . it inhibits sars-cov-2 spike binding at nanomolar concentrations in a wide variety of cell lines [102, 103] . there are two ongoing phase ii trials of an intravenous commercial rhace2 preparation. notes: some compounds with sub-micromolar in vitro activity are not included in this table either because (i) they have not been used in humans, (ii) they have been identified only in high-throughput drug screens, or (iii) they have unfavorable pharmacokinetics. several compounds that inhibit sars-cov-2 less potently but are being studied as inhalational and/or intranasal therapies are also not shown. this last category includes (i) compounds that inhibit the interaction of sars-cov-2 spike and cell surface heparin sulfate proteoglycans such as lactoferrin and heparin; and (ii) ciclesonide, an inhaled corticosteroid that may interfere with membrane trafficking by binding directly to nsp-3 or nsp-4 or indirectly through a host protein. the clinical trials registry table is a regularly updated, annotated list of ongoing, planned, or completed clinical trials obtained from the clinicaltrials.gov, who ictrp, and chinese clinical trial websites. it contains trials of compounds with potential antiviral activity but not studies of non-antiviral interventions, such as those designed to optimize intensive-care management or reduce the inflammatory response associated with severe covid-19. the clinical trials registry classifies trials according to the compound target, the type of trial (e.g., observational or randomized controlled study), the status of the trial (pending, active, or completed), and the population studied. as of 6 august 2020, it contains more than 700 trials of which about 73% are listed on clinicaltrials.gov and 27% are listed only on the who international clinical trials platform. figure 6a displays the distribution of planned, ongoing, and published studies according to the compound targets of the drugs studied. figure 6b displays the same distribution for those drugs in three or more studies. it is notable that many of the most commonly studied compounds have either little or no activity against sars-cov-2, including several drugs used for non-coronavirus infections such as the hiv protease inhibitors lopinavir and darunavir and the influenza inhibitors favipiravir, oseltamivir, and umifenovir. the chloroquine analogs, chloroquine and hydroxychloroquine, have weak in vitro activity but have failed to show clinical efficacy in multiple clinical trials [104] [105] [106] [107] [108] [109] [110] . the search function allows users to specify one or more of the following options from four drop-down lists: (i) compound target, (ii) compound, (iii) virus category, and (iv) study type. if the user selects "any" for one of these and leaves the others in their default position, the search function returns the database's complete set of cell culture experiments, biochemical experiments, entry assay experiments, animal model studies, and published clinical studies. by selecting one or more of the above options, the search function restricts the data returned to those meeting the search criteria. by using the "copy to clipboard" link, users can import the results of any query into a spreadsheet where they can further sort and filter query results. the search function also provides a link to the trials in the clinical trials registry for selected compounds and compound targets. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal model and clinical studies. compounds that are active in vitro, however, may also not be clinically useful if their associated in vitro data do not reflect physiologic conditions or if standard dosing with these compounds does not result in sufficient inhibitory concentrations at sites of infection. the creation of the cov-rdb was primarily motivated by the observation that many of the drugs being evaluated in covid-19 clinical trials demonstrate little or no in vitro anti-coronavirus activity. for example, as recently as july 2020, four of the most commonly studied drugs-chloroquine analogs, azithromycin, lopinavir/r, and favipiravir-demonstrated little if any in vitro activity. the creation of the cov-rdb was secondarily motivated by the observation that results for the same compound often vary across different laboratories as a result of experimental design such as cell line, inoculum size, drug-addition timing, duration of culture, and method for measuring virus replication. given sufficient data, a database makes it possible to eventually identify the experimental features responsible for the heterogeneity in published results, thus improving the ability to compare the antiviral activity of different compounds. indeed, we have already noted in this manuscript several compound classes for which viral inhibition is influenced by the cells used for virus culture. the cov-rdb is also designed to be educational as it provides multiple lookup tables for the viruses, drugs, cell lines, and animal models used in reported experiments. these tables contain descriptions of viruses, virus isolate/strains, cell lines, animal models, and more than 300 licensed and investigational compounds. work is underway to also add comprehensive, yet detailed, summaries of sars-cov-2 monoclonal antibodies and of pharmacokinetic data for those drugs with well-documented in vitro inhibitory activity. there are several additional web resources devoted to coronavirus drug development including sites devoted to high-throughput drug screening [111] , the genetics of monoclonal antibodies [112] , and meta-analyses of published clinical trials [113, 114] . the national institutes of health (nih) recognizes the importance of such resources and has recently announced a notice of special interest: national institute of allergy and infectious diseases (niaid) priorities for biomedical knowledgebases and repositories (not-ai-20-044). the cov-rdb database, user interface, and underlying computer code represent a framework for organizing a vast amount of data and for facilitating data curation. however, the value of this resource depends upon ongoing manual data curation and annotation. in conclusion, the cov-rdb provides a uniquely integrated interdisciplinary synthesis of in vitro, animal model, and clinical studies of compounds 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systematic review ongoing clinical trials for the management of the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-300574-nclkfw4h authors: donno, dario; mellano, maria gabriella; cerutti, alessandro kim; beccaro, gabriele loris title: chapter 9 nutraceuticals in alternative and underutilized fruits as functional food ingredients: ancient species for new health needs date: 2018-12-31 journal: alternative and replacement foods doi: 10.1016/b978-0-12-811446-9.00009-5 sha: doc_id: 300574 cord_uid: nclkfw4h abstract a diet containing high levels of fruit has been associated with a lowered risk of chronic diseases as, in addition to their vitamin and mineral content, they also contain various compounds with health-protective effects, in particular antioxidant and antiinflammatory compounds. wild plant species are of interest to the food industry because of their ability to replace synthetic chemicals and nutraceuticals; however, the nutritional, economical, and sociocultural values of some neglected and underutilized natural resources have not yet been fully exploited. some of these less well-known and underutilized fruits, which have the potential to provide novel sources of health-promoting agents, are presented in this chapter (i.e., asimina triloba (l.) dunal, crataegus azarolus l., lycium barbarum l., morus nigra l., and amelanchier canadensis (l.) medicus). underutilized fruits could represent an opportunity for growers to gain access to these special markets where consumers place emphasis on high contents of nutrients that are capable of preventing degenerative diseases. the development of specific horticultural models for nutraceutical fruit production could be an interesting opportunity to obtain a highly standardized raw material for fresh or derived products. the health benefits of different foods or diets. many are unwilling or unable to follow dietary guidelines, which is perhaps not surprising as dietary reference values take little or no account of today's busy lifestyles (hardy, 2000) . the concepts of nutraceuticals, dietary supplements, functional foods, medical foods, and dietary supplements are confusing and the terms are often used interchangeably. nutraceuticals are described as products extracted, purified, or produced from a plant, animal, or marine source (e.g., antioxidants from blueberries, elk velvet, or fish oils), or produced from dried, powdered, or pressed plant material. they have a demonstrable physiological benefit or provide protection against chronic diseases (prabu et al., 2012; srivastava et al., 2015) . dietary supplements have more defined health roles, as vitamins, minerals, herbs, botanicals, amino acids, and other dietary substances are intended to supplement the diet by increasing the total dietary intake of these ingredients (roberfroid, 2002) . dietary supplements are not intended to treat or cure disease (gibson and makrides, 2000) whereas nutraceuticals are used in the prevention or treatment of diseases. the concept of functional foods is different from that of nutraceuticals and they can be defined as food products that are used in the standard diet to provide beneficial health effects in addition to the traditional nutritional effects (whitman, 2001) . it is possible to link the key concepts (i.e., health, technology, and nutrition) with the main players in the functional food research and development process (i.e., food technologists, nutritionists, and specialists) ( fig. 9 .1). the combination of skills possessed by these different players is essential for the development of innovative nutraceutical products. these products must present higher quality standards (and organoleptic properties) compared to corresponding conventional products and aim to maintain well-being (bigliardi and galati, 2013) . a diet containing high levels of fruits and vegetables has been associated with a lower risk of chronic diseases because, in addition to their high vitamin and mineral content, these foods also contain compounds with health-protective effects, in particular antioxidant and antiinflammatory compounds (donno et al., 2013b) . in plant biochemistry these molecules are secondary metabolites (i.e., chemical compounds produced within the plants that are not directly involved in the normal growth, development, or reproduction of the organism). some are found to defend against diseases, predators, ultraviolet radiation, parasites, and oxidants; some facilitate the reproductive process (e.g., serving as attractive smells and coloring agents); and others are important for interspecies competition (kaur and das, 2011) . these same metabolites provide beneficial health and wellness effects in humans and animals. evidence from epidemiological, in vitro, in vivo, and clinical studies has consistently demonstrated that a diet rich in plant foods can reduce the risk of some degenerative diseases including diabetes, obesity, inflammatory disorders, cardiovascular complications, and cancer (cheung and mehta, 2015) . inflammation is a protective measure produced by an organism to eliminate an injurious stimuli. the use of antiinflammatory substances can be an effective tool in the therapeutic treatment of diseases; in this context, medicinal plants and their isolated compounds are used globally in folk medicine to treat different inflammatory conditions (de cassia da silveira et al., 2013) . in the continuous search for new natural bioactive antiinflammatory molecules, fruits are increasingly being seen as a rich source due to their essential oils and their isolated components, the monoterpenes (dell' agli et al., 2013) . moreover, polyphenols are the most abundant bioactive compounds reaching values of 1 g/day in the diet, approximately 10-times higher than vitamin c intake (arts and hollman, 2005; scalbert et al., 2005) . the quantity and quality of polyphenols in plant foods may vary significantly according to intrinsic and extrinsic factors such as plant genetics, soil composition, growth conditions, stage of maturity, and postharvest conditions (jeffery et al., 2003) . the dietary intake of phenolics is greatly affected by the eating habits and preferences of individuals (shahidi and ambigaipalan, 2015) . simple phenolics (i.e., hydroxycinnamic acid conjugates and flavonoids) are important constituents of fruits, vegetables, and beverages. these compounds show a wide range of antioxidant activities in vitro and are thought to exert protective effects against major diseases such as cancer and cardiovascular disease (rice-evans et al., 1997) . in the plant kingdom, antioxidant compounds can range from simple molecules (e.g., vitamin c and phenolic acids) to highly polymerized compounds (e.g., tannins). phenolic compounds can be divided into many different classes ( fig. 9 .2); however, bigliardi, b., galati, f., 2013. innovation flavonoids and phenolic acids are the most abundant in plant materials (tabart et al., 2011; tabart et al., 2012) . these low molecular weight secondary metabolites exhibit excellent antioxidant properties. moreover, organic acids are also considered as antioxidants with multiple uses in pharmacology (eyduran et al., 2015) ; however, their specific mechanisms of action can vary depending on both the structure and the chemical environment (komes et al., 2011) . nutraceuticals on the market today consist of both traditional and nontraditional foods. traditional nutraceuticals are natural whole foods with new information about their potential health qualities. many (if not most) fruits, vegetables, grains, fish, dairy, and meat products contain several natural components that deliver benefits beyond basic nutrition. even tea and chocolate have been noted to contain beneficial-health attributes in some studies. conversely, nontraditional nutraceuticals are foods resulting from agricultural breeding or with added nutrients or ingredients (prabu et al., 2012) . wild food plants have become very important for the food industry as they can be used to replace synthetic chemicals and nutraceuticals (sadia et al., 2014) ; however, the nutritional, economic, and sociocultural potential of neglected and underutilized natural food resources have yet to be fully exploited and are suffering from a lack of research interest (beccaro et al., 2015; donno et al., 2014) . for example, data on the antioxidant properties of several plant resources, in particular plants not used in nutrition and medicine, are still lacking. therefore studies on these plants may be of interest in the search for novel sources of natural antioxidants, functional foods, and nutraceuticals (donno et al., 2015c) . many alternative and underutilized fruits such as asimina triloba (l.) dunal, crataegus azarolus l., lycium barbarum l., morus nigra l., and amelanchier canadensis (l.) medicus are presented in this chapter as novel sources of health-promoting agents. the pawpaw (a. triloba l. dunal) is a tree fruit native to the temperate woodlands of the appalachian region. it belongs to the annonaceae family, which includes 120 genera and over 2100 species. a. triloba is the only temperate species as the rest of the family thrives in tropical or subtropical climates. during the growing season, the pawpaw has a whitish to light-green color that turns yellow-brown at maturity . the ripe fruit is highly aromatic and the banana-like flesh surrounds two rows of large bean-shaped brown seeds. pawpaw fruits were traditionally consumed by native americans, then by european explorers and settlers, and today they are consumed by local populations in rural areas. despite some consumer and professional interest, the high perishability of the fruit has been a major factor in slowing the development of this fruit in a larger market (mclaughlin, 2008) . pawpaws contain significant levels of phytochemicals and unique compounds that may promote health. the fruits are comparable to apples and oranges since they are rich in polyphenolic compounds, vitamin c (30-200 mg kg −1 ), magnesium (1000-1400 mg kg −1 ), iron (60-80 mg kg −1 ), copper (2-8 mg kg −1 ), and manganese (20-30 mg kg −1 ). their antioxidant activity may also be comparable to that of more extensively studied fruit. moreover, pawpaw fruits are also a good source of potassium (3000-3800 mg kg −1 ), essential amino acids (mean value of 40 mg kg −1 of protein), riboflavin (0.06-0.15 mg kg −1 ), niacin (10-12 mg kg −1 ), calcium (500-800 mg kg −1 ), phosphorus (400-500 mg kg −1 ), and zinc (10-12 mg kg −1 ). they are comparable to other important tropical fruits; for example, they contains levels of polyphenols that are similar (caffeic acid and vitamin c) or higher (ellagic acid, ferulic acid, and quercetin) than mango (mangifera indica l.). the levels of antioxidant compounds are also similar to other tropical fruits such as guava, papaya, banana, and ananas (kobayashi et al., 2008; kral, 1960) . some alternative food medicines and products that contain extracts of pawpaw (e.g., paw paw cell reg, graviola max, royal graviola, and graviola liquid extract) have been reported to exhibit antitumor efficacy in animal models and in a limited number of clinical studies (coothankandaswamy et al., 2010) . azarole fruits (c. azarolus l.) have attracted attention in the food, nutraceutical, and medical fields because of their widely reported health benefits (e.g., they reduce the risk of cardiovascular diseases). the systematics of the crataegus genus have been problematic because of hybridization, introgression, polyploidy, and apomixis. considerable revisionary work has led to a substantial reduction in the number of species that can plausibly be recognized as crataegus, with global estimates now suggesting there are 150-200 species, which is a much more manageable number to handle in studies of chemical variation within the genus (bahri-sahloul et al., 2009) . crataegus fruits and flowers are used for medicinal purposes; specifically, the fruits are not only consumed fresh and dried but are also used to produce jams, marmalades, and syrups. the weight of the fruit ranges from 2 to 8 g and they are variable in size (up to 25 mm diameter) and color (yellow to bright red to black). there are 1-3 large seeds in the center of each fruit and each contains on average 15 g of total sugar, 1.5 mg of total acidity, 30 mg of vitamin c, 11 mg of ca, 10 mg of p, 1 mg of fe, 160 mg of k, 7 mg of mg, 0.2 mg of cu, 0.25 mg of mn, 2 mg of na, 1 g of protein, and 13 g of carbohydrate per 100 g (bignami et al., 2003) . crataegus species. their high contents of flavonoids, proanthocyanidins, catechins, phenolic acids, essential oils, and terpenoids explains their use as natural therapeutics in the treatment of neurodegenerative diseases, some types of cancer, disorders of the immunological system, and cardiovascular diseases. extracts from crataegus spp. exert a wide range of pharmaceutical properties, especially on the cardiovascular system, including cardiotonic, antiarrhythmic, hypotensive, hypolipidemic, and antioxidant activities. the use of fruits in the treatment of heart ailments dates back to the late 1800s and numerous laboratory tests and clinical trials have demonstrated their efficacy in the treatment or prevention of cardiovascular diseases (belkhir et al., 2013) . goji is a solanaceous deciduous shrub native to china, tibet, and other parts of asia, and its bright orange-red ellipsoid berries are 1-2 cm long. the two closely related species, l. barbarum l. and lycium chinense miller, have been historically used as food and medicinal plants in china and other asian countries. their highly similar anatomy and tissue structure makes differentiation based on morphological and histological analyses very delicate. the most common name for the goji berry is "wolfberry," and it derives from the character "gou" as it is related to the one that means wolf. the fruits are collected from late summer to autumn, dried in the shade until the skin shrinks, and then exposed to the sun until the outer skin becomes dry and hard and the pulp remains soft (donno et al., 2015a (donno et al., , 2016a . lycium fruits are a rich source of nutrients and phytochemicals including organic acids, sugars and polysaccharides, carotenoids (zeaxanthin), vitamins (i.e., vitamin c, vitamin b complex, and vitamin e), flavonoids, phenolic acids, minerals, trace elements (i.e., zinc, iron, calcium, selenium, germanium, and phosphorus), 18 amino acids (including eight essential amino acids), alkaloids, and fats (amagase and farnsworth, 2011) . studies indicate that the goji fruit has positive effects on aging, neuroprotection, general well-being, metabolism and energy expenditure (i.e., glucose control in diabetics), glaucoma, immunomodulation, and cytoprotection, as well as having antitumor and antioxidant properties. l. barbarum and l. chinense fruits are widely used in traditional chinese medicine and they can also be sold as dietary supplements or classified as nutraceuticals for prolonged and safe use (donno et al., 2016b) . some authors report that the health-promoting agents in goji fruit might confer beneficial health effects by alleviating oxidative stress and counteracting free radicals (donno et al., 2016b; potterat, 2010) . lycium spp. fruits are most often incorporated into complex herbal preparations at 6-18 g/100 g of dried material. if decoction is used then scientific references indicate a dosage of 5-15 g/100 ml of goji, equivalent to 25-120 g of fresh berries. research recommends 30 ml of decoction four times daily (120 ml/day), which is equivalent to approximately 150 g of fresh berries. goji has been used as a food and herbal medicine for over 2500 years without any toxic effects; however, there have been two case reports of possible interactions between goji fruit tea and warfarin (coumadin) (amagase et al., 2009; larramendi et al., 2012) . the mulberry (morus spp., moraceae family) has become domesticated over thousands of years, adapting to tropical, subtropical, and temperate zones in the northern hemisphere, and growing in a wide range of climatic, topographic, and soil conditions. there are 24 species of morus (about 100 known cultivars) including the white mulberry (morus alba l.), black mulberry (m. nigra l.), and red mulberry (morus rubra l.). m. nigra has juicy fruits with a nice color and a unique, slightly acidic flavor (donno et al., 2015c) . mulberries are sweet fruits and they play an important role in the food industry due to their high levels of bioactive compounds (mulberry fruits can vary in terms of their chemical composition and antioxidant properties). for this reason, mulberries can be considered as a good source of nutrients and antioxidant compounds (especially anthocyanins and polyphenols) and they can also provide nutritionally useful amounts of minerals (liang et al., 2012) . mulberry trees have historically been cultivated for their leaves, which can be used as food for silkworms. mulberry fruits are currently consumed as both fresh and processed products (e.g., juices, fruit salads, and dried fruits) due to their nutritive value. the production and consumption of mulberry fruits is rapidly increasing, largely due to their aromatic taste, nutritional value, and biological activities. one of the most important bioactive constituents of mulberries are the anthocyanins. these pigments have dual value: first, they constitute an integral part of the sensory attributes since their levels and various forms pertain directly to the coloration of the final product; and second, they are thought to possess diverse biological properties and therefore are considered as secondary metabolites with potential nutritional value. several researchers have studied the contents of phenolics (i.e., flavonoids and anthocyanins) and carotenoids in mulberry extracts (sánchez-salcedo et al., 2015) . mulberry fruits are traditionally used as worming, hypoglycemic, emetic, or laxative agents. in chinese herbal medicine the mulberry fruit has been used as a folk remedy to treat several diseases including oral and dental diseases, diabetes, hypertension, arthritis, and anemia. several studies have confirmed that mulberries may have positive health effects in humans due to their phenolic composition, and this is especially true for people with type 2 diabetes mellitus. similarly, mulberry leaves have also shown promising biological effects (calin-sanchez et al., 2013). the genus amelanchier (family rosaceae) is subdivided into approximately 25 species (e.g., amelanchier alnifolia, amelanchier arborea, and a. canadensis) and is widespread in north america and in parts of europe and asia. a. canadensis, also known as the shadblow serviceberry, ripens in early june and is widely used in the landscape trade. the serviceberry is native to north america (western and north central united states, alaska, and western canada), but is less well-known and rarely grown in europe (except scandinavian countries) despite its edible fruits, frost resistance, and decorative value (stushnoff, 1991) . the maroon-purple fruits of a. canadensis are similar to those of a. arborea (i.e., glabrous, wax-coated pomes of 7-15 mm diameter). in the last two decades there has been growing interest in using the sweet serviceberry in the food industry in fresh fruits, pies, pastries, preserves, jams, jellies, spreads cereals, and snack foods, in particular in canada and the united states (michigan). the fruits have also been added to cider, wine, beer, and tea (cazares-franco et al., 2014) . the important health benefits of serviceberries, especially their ability to act as a good source of minerals (e.g., manganese, magnesium, iron, calcium, potassium, and copper) and carotenoids (e.g., lutein), has been emphasized in the literature. moreover, the fruit is also rich in nutraceutical compounds, especially phenolic compounds (e.g., anthocyanins, chlorogenic acid, catechins, and rutin). amelanchier spp. seed oil may serve as a potential dietary source of tocopherols, sterols, and unsaturated fatty acids (juríková et al., 2013; ozga et al., 2007) . indigenous north americans used different parts of the serviceberry plant for medicinal purposes; for example, in canada the fruits were used as a juice for treating stomach and intestinal ailments. eye drops and eardrops were also prepared from ripe serviceberries. the boiled bark can be used as a disinfectant while the root infusion can be used to prevent miscarriage after injury. native american communities also prepare a tea from the twigs to administer to women after birth, while a tonic from the bark is given to women after delivery to hasten discharge of the placenta. moreover, several cultivars of amelanchier spp. were found to possess free radical scavenging activity (due to their relatively high anthocyanin content) and antiviral activity against enteric coronavirus. finally, serviceberry fruits show antidiabetic properties (due to their aldose reductase inhibitor activity) and exhibit the ability to regulate lipid metabolism and energy expenditure (bakowskabarczak and kolodziejczyk, 2008; bakowska-barczak et al., 2007) . the type of sample determines the sampling protocol and how the samples are handled. the main considerations are the degree of homogeneity of the material and the possible variation in the compound content, not only between different materials but also between different samples of the same material. the cultivar, origin, season, and growth conditions will also affect the bioactive content (nedović et al., 2015) . it is essential that the sampling protocol and the number of collected samples reflects the composition of the whole sample. the time between sample collection and analysis should be as short as possible and the protocol should minimize any effects that may cause undesirable losses prior to analysis (mitra, 2004) . therefore identification is an important step in the collection of wild plants, even if it is often overlooked by the nutraceutical companies: in the european market only 20% of food product plants are collected in germplasm repositories (fowler, 2006) . moreover, an individual plant species may have different cultivars with different genetic characteristics, chemical compositions, and health effects on the efficacy of the final product and consumer health (kerslake and menary, 1985) . the fruit harvest period can vary across different sampling sites and years (dvaranauskaitė et al., 2009; lakusic et al., 2011) . the development of tree species is also influenced by natural factors (endogenous or exogenous) as well as by human factors. these factors can influence the chemical composition of plant organs and the respective derived products (tibaldi et al., 2011 ). food quality is not only dependent on intrinsic characteristics (i.e., genetic factors) but is also closely linked to the pedoclimatic conditions in the collecting area. indeed, plants growing in more suitable areas have greater potential in terms of product quantity and quality (vegvari et al., 2008) . knowledge of environmental characteristics, such as the best pedoclimatic conditions for plant development, can help identify the best raw material to be used for fresh plant material and derived functional foods (rates, 2001) . each species has its own requirement in terms of altitude, latitude, mean temperature, average annual rainfall, light availability, and physic-chemical soil properties. climatic conditions have direct effects on the physiological processes and phenology of the plant (e.g., growth, flowering, and fruit ripening) thus they can also affect the availability of essential metabolites for the biosynthesis of active compounds (vegvari et al., 2008) . a plant can almost completely lose the ability to synthesize specific bioactive molecules outside of its own habitat (vv.aa., 2012). therefore the use of advanced agrotechniques alongside dedicated genetic improvement allows tree species to fit better with the cultivation site and can also improve the final product quality (capasso et al., 2006) . assessing the chemical composition of fruit is very important because it allows the functional product to be standardized. each nutraceutical product should have the same qualitative and quantitative chemical composition in every production batch in order to maintain quality, safety, and efficacy (ong, 2004) . the relationship between these products and the different factors such as their origin, the pedoclimatic conditions (e.g., soil, latitude, altitude, mean temperature, light intensity, and rain frequency), and the agronomic techniques used means that standardization should first extend to the raw materials through the selection of plants with the best bioactive compound content. the subsequent transformation process and the use of standardized methods, together with analytical laboratory controls, contributes to the production of a final product with reproducible and consistent chemical and physical characteristics (i.e., bioactive compounds, appearance, texture, density, and solubility) (khan, 2006) . considering that food composition is affected by many factors, and that compounds are not uniformly distributed within and between samples of a given food, statistically valid sampling and sample preparation are of utmost importance to obtain representative and homogeneous samples (rodriguez-amaya, 2001) . the main causes of data inaccuracy are that only a single sample lot is analyzed per food and that errors are not observed between intralaboratory and interlaboratory evaluations in which the same homogenized samples are analyzed. the selection, number, handling, and preparation of samples prior to extraction are important factors that can affect data quality. sample preparation may consist of multiple steps such as drying, homogenization, sieving, extraction, preconcentration, derivatization, and hydrolysis (luthria, 2006) . qualitative and quantitative studies of bioactive compounds mostly rely on selecting the proper extraction method (smith, 2003) . extraction is the first step in the study of bioactive compounds and is crucial to the final result. the five main objectives of extraction are: (1) to extract targeted bioactive compounds from complex samples, (2) to increase the selectivity of analytical methods, (3) to increase the sensitivity of bioassays by increasing the concentration of targeted compounds, (4) to convert the bioactive compounds into a more suitable form for detection and separation, and (5) to provide a strong and reproducible method that is independent of variations in the sample matrix (nedović et al., 2015) . therefore extraction should be carefully optimized and performed. the polarities of the different bioactive compounds, which vary significantly due to their conjugation status and their association with the sample matrix, determines the solvents to be used in the extraction (sasidharan et al., 2011) . when selecting solvents for the extraction of bioactive compounds, the molecular affinity between the solvent and solute, mass transfer, use of a cosolvent, environmental safety, human toxicity, and financial feasibility should also be considered (nedović et al., 2015) . optimizing the extraction parameters not only increases the extraction efficiency of the analyte of interest but also reduces the solvent consumed and the waste generated during the extraction process, thus making it more environmental friendly (chemat et al., 2012) . specific pretreatments (e.g., storage under modified atmosphere, microbial and chemical acidification, pasteurization, and utilization of depolymerizing enzymes) are sometimes needed to preserve the raw materials and extract biomolecules; moreover, additional procedures (e.g., rotary shaking, stirring, and sonication) can also be used to improve the extraction (laufenberg et al., 2003) . during identification and characterization of bioactive substances, the critical point is usually the selection of the correct measuring method. spectroscopic methods usually permit crude identification of the bioactive compounds present in food, but in most cases the specific composition can remain obscure. however, spectrophotometry remains very useful for the evaluation of antioxidant activity (table 9 .1). detailed information about the composition of food requires additional methods, such as chromatography and mass spectrometry, to fully characterize the structures of the molecules in the samples (nedović et al., 2015) . technological innovation in analytical instruments has allowed increasingly sophisticated qualitative and quantitative assessments of the chemical composition of nutraceutical foods (cordell, 2011) . chromatographic techniques are mainly used to analyze fresh foods and derived products. chromatography offers a very powerful separation ability, such that the complex chemical components in the extracts can be separated into many relatively simple subfractions. furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as high-performance liquid chromatography-diode array detection (hplc-dad), gas chromatography-mass spectroscopy (gc-ms), capillary electrophoresis-dad, hplc-ms, and hplc-nuclear magnetic resonance (nmr), can provide additional spectral information. this is very helpful for qualitative analysis and for online structural elucidation (liang et al., 2004; steinmann and ganzera, 2011) . more specifically, hplc is currently the most widely used analytical method when combined with different detectors (e.g., diode arrays, mass spectrometry, and fluorescence) (donno et al., 2012) . it is regarded as the gold standard in the authentication of nutraceuticals, functional foods, pharmaceuticals, and herbal medicines due to its precision, sensitivity, and reproducibility despite its high instrumental cost, relatively long analytical time, and large organic solvent consumption (donno et al., 2015b) . identification and quality control of the food material can be performed using marker compounds: a chemical constituent of a raw material, drug substance, or food product that is used for identification or quality control purposes, especially when the active constituents are not known or identified. the active compounds are responsible for the intended pharmacological activity or synergistic therapeutic effects (ong, 2004) . it is necessary to determine most of the phytochemical constituents of analyzed products to ensure the reliability and repeatability of pharmacological and clinical research, as well as to understand the bioactivities and possible side effects of active compounds and to enhance quality control (liang et al., 2004) . chromatography combined with a suitable detection technique offers a powerful tool for separating the individual compounds and developing a characteristic profile of the sample, otherwise known as a fingerprint. combining a chromatographic separation system online with a spectroscopic detector has become the most important approach for identifying or confirming the identity of target and unknown chemical compounds (cuadros-rodríguez et al., 2016) . if hyphenated chromatography is further combined with chemometric approaches, clear pictures may be developed for chromatographic fingerprints (bertacchini et al., 2013) . the complexity of fresh foods and derived products means that it is necessary to consider many factors for their fingerprint evaluation. the quality of a functional food is closely related to its chemical constituents and their concentrations and may vary slightly according to differences in the climate, cultivation conditions, harvest time, drying, and storage (hibbert, 2012) . to analyze the large amount of generated data, a variety of analytical methods have proven to be useful and versatile tools for the extraction, visualization, and interpretation of the information. in particular, pattern recognition methods as a principal component analysis allows better visualization of the information included in the fingerprints. the original variables are transformed into latent variables to summarize the systemic patterns of variation between the samples (ieri et al., 2013) . exploratory data analysis is easier if represented as a multivariate data table as a low-dimensional plane. sustainability is a broad concept and is considered to be ambiguous because it means different things to different people at different periods of time. as a consequence there are many different definitions of sustainable agriculture; however, most of them are connected to the three pillars of sustainability: society, economy, and environment. for example, reganold et al. (2001) summarized this concept as "to be sustainable, a farm must produce adequate yields of high quality, be profitable, protect the environment, conserve resources, and be socially responsible in the long term." there are a number of indicators that can be used to assess the environmental performance of a production system. many studies claim that indicators that consider many aspects of the environmental impact at the same time are more useful to address the complexity of agricultural systems (bastianoni et al., 2007) . therefore some of the most important features of an indicator are its ability to summarize, focus, and condense extensive datasets (obtained from complex environmental parameters) to a manageable amount of meaningful information (godfrey and todd, 2001) . life cycle assessment (lca) is defined by the international organization for standardization (iso14040:2006) as the compilation and evaluation of the inputs, outputs, and potential environmental impacts of a product system throughout its life cycle. the origin of lca can be found in the late 1960s within the context of american industry (hunt et al., 1996) and numerous studies have been carried out to adapt this method to the agricultural sector (audsley et al., 2003) . lca is now considered a useful tool for comparing alternative food products, processes, or services, and as the background for environmental product declaration (schau and fet, 2008) . the results of lcas are commonly presented as impacts in a range of different categories such as global warming, acidification, nitrification, ozone depletion, and toxicity (pennington et al., 2004) . nevertheless, more importance and consideration is being given to the results in single impact categories. one example is the carbon footprint, which in the lca approach corresponds to the "global warming potential." this category expresses the number of co 2 equivalents (co 2 eq., a collective unit for all molecules with a global warming potential) that are associated, directly and indirectly, with all stages of development of the system under consideration. the term co 2 eq. is intended to express all greenhouse gases, with each being weighted according to their global warming potential. the advent of the carbon footprint has made lca results available to a wider audience (weidema et al., 2008) . food production systems have their own specific agronomic requirements. even different varieties of the same species may have unique requirements in terms of fertilizers, pesticides, water, plantation strategies, and field management, which may result in different environmental burdens (de gennaro et al., 2012) . one exemplary case is the growing of ancient cultivars or forgotten fruits with high nutraceutical values (donno et al., 2010 (donno et al., , 2012 . ancient cultivars are bound to the area they are grown through centuries of slow adaptation to the pedoclimatic conditions, thus resulting in lower agricultural inputs compared to introduced cultivars (bisht et al., 2007; jarvis et al., 2008) . a lower agricultural input theoretically produces a lower environmental impact; however, in order to be sure about this it is necessary to apply an environmental impact assessment method. a didactical case study was reported by cerutti et al. (2013) , in which the authors investigated the environmental burden of ancient varieties of apple in north west italy. hundreds of different cultivars of apple (malus domestica borkh.) were grown in italy until the 1950s. after this time the proliferation of commercial cultivars caused the local germplasm to lose importance and be forgotten by growers and consumers. many ancient cultivars were gradually replaced by commercial ones with low phytochemical and nutraceutical traits. the italian fruit-growing scene underwent significant change and golden delicious is now the most cultivated variety (more than 70% of total orchards); however, the genetic diversity has fortunately been largely preserved thanks to germplasm repositories (donno et al., 2012) . while golden delicious dominates, the ancient apple germplasm of the piedmont region (northern italy) is actually represented by ∼350 cultivars, almost all recently described by their nutraceutical, morphological, and agronomic traits (bounous et al., 2006) . the commercial appeal of traditional cultivars is related to their unique quality traits and to the claims of their lower environmental impacts due to being grown on the original agricultural land. ancient cultivars usually require fewer treatments and field operations per hectare of cultivation in comparison with foreign cultivars because they are more adapted to the pedoclimatic characteristics of the native region. cerutti et al. (2013) used lca methodology to calculate the environmental performance of grigia di torriana, magnana, and runsé cv., three ancient apple cultivars from torino and cuneo provinces. the environmental impacts of these cultivars were compared to that of golden delicious. the study was performed in accordance with the iso 14040:2006 standard series guidelines and with the cradle-to-gate approach as the basis for the life cycle inventory (lci) (international organization for standardization, 1997). data regarding orchard structure, agricultural inputs, resource consumption, and orchard management practices were obtained directly from the growers who filled in a questionnaire for the 2011 season. in order to consider minor geographical differences, the lci for each cultivar included the average of three orchards spread throughout the two provinces. environmental impacts were calculated according to several functional units. considering the impacts for growing 1 hectare of orchard, the golden delicious cultivar showed the lowest environmental performance emitting 6.5 t co 2 eq.; conversely, less emissions were observed in the life cycle of the three ancient cultivars (average 4.9 t co 2 eq.). this result confirms that if low-input agricultural systems are managed properly, and are producing high-value products (including high levels of nutraceutical components), they can be more sustainable than high-input systems (cerutti et al., 2013) . it could be important to apply the same methodology to some underutilized fruit species (i.e., those of nutraceutical value) in order to evaluate the sustainability of their production and consumption. worldwide interest in introducing the cultivation of these tree species to promote the differentiation of the cultivated agrobiodiversity and the consumption of new functional foods with excellent nutraceutical properties could also be encouraged by the specific agronomic traits of these species that could be managed with more environmentally friendly agrotechniques (if compared with the most commonly grown fruit species), and by the possible greater sustainability of their production. research carried out in the field of natural bioactive compounds is increasing our understanding of healthy compounds that are naturally available in food, in particular in fruit. this will permit their increased use in foods (instead of artificial drugs), which will in turn increase their quality and added value. new methodologies for the extraction, purification, identification, and quantification of biomolecules using ecofriendly analytical techniques will need to be developed to improve the extraction yields (chemat et al., 2012; oroian and escriche, 2015) . with the advent of high-throughput technologies and their applications in food science, the food and beverage industries have been taking steps to transform their traditional r&d and product development processes. the food and beverage industries are now faced with a situation in which they should quickly adapt to new market demands and regulatory updates and be prepared to make expensive and risky decisions as to whether they should proceed with significant investments in the development of functional products (younesi and ayseli, 2015) . alternative and underutilized fruits represent an opportunity for local growers to gain access to specialized markets where consumers lay emphasis on exotic character and the presence of nutrients that are capable of preventing degenerative diseases. the creation of specific horticultural models for nutraceutical fruit production could be an interesting opportunity to obtain a highly standardized raw material for fresh or derived products. in addition, phytochemicals from these fruits could have further applications in the food industry, such as increasing the stability and shelf life of food products. studies relating to the toxicological analysis of bioactive extracts; the metabolism of bioactive compounds, their bioavailability, and bioaccessibility; and the sensorial and nutritional traits of food products with added bioactive compounds from underutilized fruits should be undertaken (ayala-zavala et al., 2011) . the development of new functional products with intended health claims requires concrete scientific criteria. to meet the current stringent regulations for functional products, the food industry must approach this topic using integrative approaches. integration of complex biological knowledge relevant to human health and diseases into the functional product development process could not only increase the capacity of innovative product development, but could also support informed decision making (younesi and ayseli, 2015) . food science is a blend of many scientific and engineering disciplines, such as chemistry, biology, agricultural science, and chemical engineering, which is applied to provide a better understanding of food and its transformation and to deliver safe and desirable food products to the consumer (smithers, 2016) . in addition, economic analysis of the extraction processes and marketing of natural bioactive extracts should be contemplated. if food products are to be produced for use in the nutraceutical and pharmaceutical industries, there will be certain criteria that production system must meet: for example, it must clearly be economical, environmentally safe, and not have an impact on the environment (barnes and prasain, 2005; cerutti et al., 2014) . a source of bioactive substances should be identified and cultivars of the same species must be considered in order to choose the highest and the most stable concentration of natural compounds (fowler, 2006; joubert et al., 2008) . once this source has been identified, a constant and stable supply of plant material should be secured. this means that an agronomic system for biomass production should be developed in order to allow the full expression of the genetic capability of the cultivar (kerslake and menary, 1985; khan, 2006) . moreover, climate and soil conditions should fit with the species requirements. the impact of fertilization and irrigation on the biomass production and its chemical constituents should be understood and the economic growth and cultivation of the raw material should generally be explored (lakusic et al., 2011) . in the future, a plant production of secondary metabolites may be controlled with different growth regulators according to good agricultural practices used in this agronomic production (donno et al., 2013a) . the successful production of biomass should include the development of appropriate harvest techniques and storage processes and mechanization of the harvest process should be addressed in order to maintain an economic system of production (spinelli et al., 2012) . the integral exploitation of the entire food production supply chain could have economic benefits to producers and a beneficial impact on the environment, leading to a greater diversity of products for human consumption. these products represent a new class of functional foods that have not been completely exploited and that could also contribute health benefits to consumers. due to the safety and limitations surrounding the use of synthetic antioxidants, natural antioxidants and nutraceuticals obtained from edible sources (i.e., underutilized fruit species and derived products) are alternative sources of interest (shahidi and zhong, 2015) . in conclusion, further studies on the isolation of bioactive compounds using complementary methods and their effects on biological status in animal models and human subjects are needed to evaluate their potential benefits. moreover, it is necessary to further confirm lack of toxicity and bioavailability of these natural sources (moure et al., 2001) : the use of isolated nutraceuticals as dietary supplements or functional food ingredients may also be helpful in order to promote the human health, reduce the disease 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complementary and alternative nutraceuticals: lifestyle issues an integrated systems-based model for substantiation of health claims in functional food development key: cord-306934-29ljbl7g authors: tonelli, michele; vazzana, iana; tasso, bruno; boido, vito; sparatore, fabio; fermeglia, maurizio; paneni, maria silvia; posocco, paola; pricl, sabrina; colla, paolo la; ibba, cristina; secci, barbara; collu, gabriella; loddo, roberta title: antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives date: 2009-07-01 journal: bioorganic & medicinal chemistry doi: 10.1016/j.bmc.2009.05.020 sha: doc_id: 306934 cord_uid: 29ljbl7g abstract twelve aminoarylazocompounds (a–c) and 46 aryltriazene 7 derivatives (d–g) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 rna and dna viruses. eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. a marked activity against bvdv and yfv was prevailing among the former compounds, while the latter type of compounds affected mainly cvb-2 and rsv. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv. arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; bvdv: 1 > 7 > 8 > 4; yfv: 7 > 5; cvb-2: 25 > 56 > 18; rsv: 14 > 20 > 55 > 38 > 18 > 19; hsv-1: 2. for these compounds the ec50 ranged from 1.6μm (1) to 12μm (18), and the s. i. from 19.4 (1) to 4.2 (2). thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss rna viruses, particularly against rsv and bvdv, which are important human and veterinary pathogens. finally, molecular modeling investigations indicated that compounds of structure a–c, active against bvdv, could work targeting the viral rna-dependent rna-polymerase (rdrp), having been observed a good agreement between the trends of the estimated ic50 and the experimental ec50 values. we have recently shown that some arylazoenamines (fig. 1) are endowed with antiviral activity in vitro against rna viruses, particularly cvb-2, rsv, bvdv, yfv and sb-1, in many cases with ec 50 in the range from 0.8 to 10 lm. 1 arylazoenamines can be considered as substructures of ortho-(and para-) aminoazocompounds, many of which are endowed with antimicrobial and antiparasitic activities, but additional pharmacological activities have been also shown in particular cases (fig. 2) . 2,6-diamino-3-phenylazopyridine (pyridium) inhibits various cocci and coli bacilli, 5-(4-amino-1-naphthylazo)uracil is highly effective against schistosoma mansonii, 2 while 1-[3-(4-phenylazo-5,6,7,8-tetrahydronaphth-1-yl)amino]propylpiperidine 3 and 1-[3-(4-phenylazo-5,6,7,8-tetrahydro naphth-1-yl)amino]lupinane 4 are very active against mycobacterium tuberculosis. antifungal and anti-proliferative activities have been found in a peculiar class of cyclic aminoazocompounds (3,3-disubstituted-3,4-dihydro-1,2,4-benzo triazines), which, moreover, can display several other pharmacological activities depending on the substituents that are present on the bicyclic system. 5, 6 very potent relaxant activity on intestinal and uterine smooth muscles was displayed at nanomolar concentration by n-dialkylaminoalkyl derivatives of another cyclic aminoazo system, namely 11h-dibenzo-[1,2,5]-triazepine, which exhibited also local anesthetic and antithrombotic activities. 7 arylazoenamines could be also seen as vinylogues of aryltriazenes (fig. 2) , a class of compounds which hold an important position as carcinogens and/or anticancer agents. 3,3-dimethyl-1phenyltriazene and, even better, dacarbazine are able to methylate dna; thus the former resulted a powerful carcinogen, while the latter is in clinical use for the treatment of malignant melanoma and hodgkin's lymphoma. 8 temozolamide, 9 closely related to dacarbazine, is currently used to treat malignant glyomas. a recent development of this cyclic aryltriazene is represented by benzotriazepinones, 10 that are very promising agents against breast cancer. it is worth noting that these compounds are weak alkylating agents and may damage dna by a novel mechanism. the triazene pharmacophore has been linked to other bioactive moieties in order to obtain chymeric compounds which should be able to exert a double cytotoxic mode of action. thus, the triazene group was linked to 4-anilino-quinazoline backbone, 11 which is known to inhibit the epidermal growth factor receptor tyrosine kinase (egfr-tk), and also to a benzophenone residue, 12 that is present in an inhibitor of pharnesyl-transferase and of tubulinpolymerization. the introduction of a triazene group on the molecule of pyrimethamine, a dihydrofolate reductase inhibitor, generated a compound that combines antitumoral potential with inhibition of pneumocystis jirovecii 13 (p. carinii). the insertion of triazene group between two benzamidine units produced diminazene (berenil), a powerful agent against the african trypanosomiasis. to the best of our knowledge, no antiviral activity has been seen with either amino arylazocompounds or aryltriazenes, with the only exception for a patent claiming the activity of 1-phenyl-3,3dimethyltriazene against the tobacco mosaic virus. 14 figure 2 . some biologically active aminoarylazo compounds and aryltriazene derivatives. antiviral activity has been shown by several benzotriazole derivatives, which could be formally considered as cyclic triazenes, though the resonance stabilization of the heterocycle could imply even substantial differences in the chemical and hence the biological behavior of the two kinds of compounds. interestingly, some halogenated benzotriazoles have been shown to inhibit the ntpase/helicase activities of hepatitis c and related viruses. 15 aromatic and heteroaromatic esters of 1-hydroxybenzotriazole were found to strongly inhibit (k i = 7.5 nm) the 3cl-protease, which is essential for the replication of the coronavirus responsible of sars (severe acute respiratory syndrome). 16 more recently, a set of benzotriazole derivatives have been found endowed with potent activity against rsv and moderate activity against yfv, bvdv and cvb-2. 17 on this base we deemed interesting to investigate the possible antiviral activity of some assorted aminoarylazo compounds and aryltriazene derivatives, allotted in seven groups a-g (fig. 3) . on the whole 12 amino arylazo compounds (a-c) and 46 triazene derivatives (d-g) were evaluated in cell based assays for cytotoxicity and antiviral activity against a large panel of rna and dna viruses. thirty out of the 58 compounds, that were evaluated for antiviral activity, were already known and were prepared according to the literature. compounds 7-12 were already described by some of us. 4 (48) is reported as known, r but we failed to find out any data concerning its characterization. references, a-r concerning the previously described compounds, are available as supplementary data. the 28 novel compounds were prepared according to the following schemes 1 and 2. all new compounds were characterized by elemental analyses and 1 h nmr spectra. the n-(3'-dimethylaminopropyl)-3-trifluoromethylaniline and the n-(lupinyl)-3-trifluoromethylaniline, required by scheme 1, were already described by some of us. 18 it is observed that the presence of a 3-trifluoromethyl group prevented completely the coupling of diazonium salt on the para position of the n-substituted anilines giving place only to the triazene derivatives e. the prepared compounds (12 amino arylazocompounds a-c and 46 triazene derivatives d-g) were evaluated in vitro in parallel cell-based assays for cytotoxicity and antiviral activity (tables 3-5) against viruses representative of two of the three genera of the flaviviridae family, that is, flaviviruses (yfv) and pestiviruses (bvdv), as hepaciviruses can hardly be used in routine cell-based assays. title compounds were also tested against representatives of other virus families. among ssrna+ were a retrovirus (human immunodeficiency virus type 1, hiv-1), two picornaviruses (coxsackie azt (3 0 -azido-thymidine), nm 108 (2 0 -c-methyl-guanosine), nm 176 (2 0 -c-ethynyl-cytidine), ribavirin, nm 299 (6-azauridine), m 5255 (mycophenolic acid) and acg (acyclovir) were used as reference inhibitors of ssrna+, ssrnaà and dna viruses, respectively. interestingly, 35 (8 aminoazocompounds and 27 triazene derivatives) over 58 tested compounds exhibited antiviral activity against one or more viruses; in particular 16 compounds exhibited a selective activity against a single virus, while 13, 4 and 2 were, respectively, active against two, three and four viruses. on the other hand, 23 compounds (4 aminoarylazo and 19 triazene derivatives) were not able to inhibit the multiplication of any virus at concentrations up to 100 lm. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv, but an increasing number of them exhibited antiviral activity against, in the order, reo-1 (4), sb-1 (7), hsv-1 (8), bvdv (9), yfv (9), rsv (12) and cvb-2 (13) (tables 1 and 2 therefore, both the [(dialkylaminoalkyl)amino]azobenzene and the aryltriazene molecular patterns represent interesting pharmacophore for developing novel antiviral agents, particularly against ssrna viruses. cytotoxicity and antiviral activities of tested and reference compounds are reported in tables 3-5. in these tables, viruses for which no active compounds have been found are not indicated. test compounds showed different degrees of cytotoxicity against the confluent cell monolayers (in stationary growth) used to support the multiplication of the different viruses. the most susceptible to toxicity were the exponentially growing lymphoblastoid human cells (mt-4) used to grow hiv-1, while the non-human host cell lines exhibited a progressively reduced sensitivity in the order vero-76 > bhk > mdbk. on the other hand, it is observed that the toxicity is not evenly distributed among the different types of test compounds, being mainly shown by the triazene derivatives of structure e and by the amino azo compound a-c, which are characterized by the presence of the basic dimethylaminopropyl and lupinyl moieties. indeed the most toxic compounds were 29 and 12, with a mean of the cc 50 values for the host cells of 17.5 and 21.5 lm, respectively. the other groups of triazene derivatives of structure d, f and g are relatively non toxic, exhibiting in most cases cc 50 p 100 lm. as already seen, eight [(dialkylaminoalkyl)amino]azobenzenes (a-c) and 27 triazene derivatives (d-g) have shown antiviral activity, sometimes of high level against one or more viruses. from tables 1 and 3 appears that compounds of structure a-c have a marked activity against bvdv and yfv, with only occasional, though still of high level, activity against hsv-1, reo-1, cvb-2 and rsv. on the whole the triazene derivatives d-g are characterized by the most frequent activity against cvb-2 and rsv, to which are associated, with decreasing frequency, the activity on sb-1, hsv-1, yfv, reo-1 and bvdv (tables 2, 4 and 5). however, considering separately each subgroup of triazene derivatives, it is observed that the activity against sb-1 is mainly associated to the pyrrolidine-related triazenes f, while activity against hsv-1 is associated with the diaryltriazenes that bear a dimethylaminopropyl chain. the importance of a basic chain for the activity against hsv-1 is underlined by the high activity shown by compounds 2 and 4, bearing the same dimethylaminopropyl chain, even linked to the aminoazobenzene pharmacophore (a), but also by the moderate activity of triazene 53, which contains a basic n-methylpiperazine residue. conversely it is worth noting that the activity against bvdv, largely diffused among the amino azo compounds a-c, is very rarely present (2 over 46 compounds) among the triazene derivatives d-g, even among those (e) sharing the basic chain of the former groups. active and inactive compounds are found side by side in all groups a-g, thus their molecular patterns seem to address the activity against the different type of viruses, while the substituents on the aromatic nucleus are the determinants for activity or inactivity. however, attempts to define general structure-activity relationships resulted rather frustrating, since observations that hold for the activity against most viruses may not hold for some others. indeed the meta substitution with halogens, trifluoromethyl and nitro groups favors the display of activity in the groups a-e and g, but in compounds of group f is the para substitution that promotes the activity; however occasional exceptions are observed in both cases. an unsubstituted aromatic ring can be found in active (1, 7, 9, 21, 25, 52, 54-56) and inactive (13, 34, 45) compounds, while the presence of electron-releasing groups as ch 3 and och 3 (32, 42) or of a benzyl residue (15, 22) is always detrimental for the expression of activity. despite the high antiviral activity of many compounds, the corresponding selectivity index (s. i.) are generally low, due to the rather high cytotoxicity exerted on the host cells. only few compounds exhibited a s. i. higher than 10 for one or more viruses, while 13 compounds had a s. i. in the range from 9 to 5 and the remaining had s. i. below this value. taking into account the ec 50 and s. i. values, the best compounds were the following, which are arranged in the order of decreasing potency and selectivity; bvdv: 1 > 7 > 8 > 4; yfv: 7 > 5; cvb-2: 25 > 56 > 18; rsv: 14 > 20 > 55 > 38 >18 > 19 > 23; sb-1: 41; hsv-1: 2. for these compounds the ec 50 ranged from 1.6 lm (1) to 15 lm (23, 41) , while the corresponding s. i. values were in the range from 19.4 (1) to 4.2 (2) . though the activity shown by the above compounds may appear as not particularly impressive, it must be noted that, at the present, very few experimental agents are known to act against those viruses and that, moreover, they often display high cytotoxicity. a particular interest is deserved by compounds displaying good activity against rsv and bvdv. rsv, is responsible of serious respiratory tract infections in infants, elderly and immunocompromised patients with high mortality rate. very potent anti-rsv agents, interfering with the virus-host fusion process, have been developed in the last years, as the benzimidazole derivative bms-433771 which exhibited an ec 50 as low as 21 nm; however, attempts to demonstrate therapeutic efficacy were not successful, 19 probably due to the rapid emergence of resistance. quite recently a chiral 1,4-benzodiazepine derivative, acting through a different mechanism, though much less potent than bms-433771, showed very promising characteristics and is being examined in phase ii clinical studies. 20 nevertheless the chirality-associated developability issue cannot be underestimate, since the antiviral activity was found to reside mainly in the s-enantiomer (rsv-604), that exhibited an ec 50 = 0.9 lm, while racemate had ic 50 = 3.5 lm, a value comparable with that of compound 14. on the other hand, bvdv is the prototype of pestiviruses, which commonly affect cloven-hoofed animals (cattle, swine, sheep). bvdv infection is responsible of reduced dairy production and increased (even up to 30%) cattle mortality throughout the world. 21 thus, the availability of effective and inexpensive anti-pestivirus drugs is of importance to relieve such an heavy economic burden. the development of anti-bvdv drugs and the understanding of their mechanism of action is of further interest because it may provide valuable informations for the design of agents active against hepatitis c virus (hcv). 22 hcv, which like bvdv belongs to the flaviviridae family, infects about 3% of world population. since chronic hcv infection can progress to fatal cirrhosis and hepatocellular carcinoma, the development of effective and safe anti-hcv agents is urgently needed. the current combination therapy with pegylated interferon and ribavirin is effective in less than 60% of the treated patients and is associated with heavy side effects. for the understanding of some aspects of virus replication, bvdv has been considered even more advantageous than the currently used hcv subgenomic replicon system. 23 in the last years, several potent anti-bvdv agents have been developed and shown to target the rna-dependent rna-polymerase (rdrp), even if resulting almost inactive on the purified enzyme. compounds vp32947 (3-[(dipropylaminoethyl)thio]-5h-1,2,4-triazino [5,6-b] indole) 24 and bpip (523 exhibited ec 50 as low as 30-40 nm. other compounds (as the thiazolylurea dpc-a69280-29, 25 table 3 cytotoxicity against mt-4, mdbk, bhk and vero-76 cell lines and bvdv, yfv, reo-1, cvb-2, rsv and hsv-1 inhibitory activity of amino azocompounds of structure a-c the benzimidazolone 1453 26 and 7-amino-1,3-dihydroxy-10methyl-6-[4-(2-pyridinyl)-1-piperazinyl]-9(10h)acridone 27 ) have ec 50 in the range 0.6-1.5 lm, that are comparable with those of the presently described aminoaryl azocompounds 1 and 7 (1.6 and 2.5 lm, respectively). the structural simplicity of these compounds, which allows a wide range of easy and inexpensive molecular modifications, make them an attractive model to develop more active and less toxic anti-pestivirus agents. in view of these considerations, we deemed interesting to perform some molecular modeling investigations to study wether the bvdv rdrp could be the target of also our compounds 1, 2, 4 and 7-10. bvdv, the best-studied pestivirus, has a genome that consists of an approximately 12.6-kb positive sense ssrna. the bvdv genome is translated into a single polyprotein which is processed into at least four structural and six nonstructural (ns) proteins required for viral assembly and replication. among the nonstructural proteins, the ns5b is an rna-dependent rna-polymerase (rdrp) enzyme responsible for genome replication as a part of a larger, membrane associated replicase complex. the crystal structure of rdrp from several families of single-or double-strand rna viruses, including hcv [28] [29] [30] and bvdv, 31, 32 have been recently made available in the protein data bank (pdb) repository. despite the low sequence identity between different polymerases, the crystal structures of these proteins (from bvdv, hcv and other families of ss and dsrna viruses) all present the shape of a right hand with fingers, palm, and thumb domains. in particular, the bvdv rna-dependent rna-polymerase core domain (residues 139-679) has a dimension of approximately 74 â 60 â 58 å around a central cavity, 31 which serves as for rna template bind-ing, nucleotides recruitment, and polymerization reaction (see fig. 4a ). in addition, there is an n-terminal region (residues 71-138) of which residues 71-91 are disordered in the relevant crystal structure. a thorough search of a putative binding site for our molecules onto bvdv rdrp was conducted following our successful recipe developed for studying allosteric inhibitors of polio-virus helicase. 33 the portion of the enzyme making up the binding site interacting with the inhibitors is located in the fingers domain (residues 139-313 and 351-410), consisting of 12 a-helices and 11 b-strands (see fig. 4a ). in bvdv rdrp, as in other viral rdrps, the n terminus of the fingers domain, together with a long insert in the fingers domain (residues 260-288), form the fingertip region that associates with the thumb domain. this region is characterized by a three-strand conformation, and since the fingers and the thumb domains are associated through this fingertip region, the conformational change induced by the rna template binding into the central channel is somewhat limited. the remainder of the fingers domain is comprised of a b-strand rich region (b-fingers) and an a-helix rich region (a-fingers) close to the palm domain. according to the procedure adopted, all compounds were characterized by a similar docking mode in the putative binding site of the bvdv rdrp, as exemplified by compound 1 in figure 5 . in particular, the residues lining the pocket (see fig. 5d ) include the side chains of residues from n217 to f224 belonging to a loop between the two strands b3 and b4, and those of residues from k263 to e265 of a loop between strands b5 and b6. amino acids from i287 to e291 contribute to binding from the b-sheet portion b8, whilst another loop, connecting two a-helical motifs (a19 and a20, respectively), concurs with residues from r529 and l530. going into details, the aminoalkyl part of the molecule is engaged in stabilizing nonbonded interactions with the apolar side chains of residues i261, i287, a221, and a222, while the diarylazo moiety is nicely encased in a subsite lined by the side chains of n217, n264, e265, k266, r529, and l530. more importantly, however, this molecular scaffold is anchored in place by three persistent hydrogen bonds (hbs). a first hb bridge involves the nitrogen atom of the n(ch 3 ) 2 substituent group on 1 and the -oh group of y289 side chain, with an average dynamic length (adl) of 3.08 ± 0.3 å. the second hb interaction engages the terminal amino group of k263 and the n atom of the inhibitor secondary amino group (adl = 3.18 ± 0.2 å). finally, one of the nitrogen atoms belonging to the azo group characterizing compound 1 is involved in the third hb interaction with the backbone -nh group of e265 (adl = 2.92 ± 0.3 å). importantly, quantitative information about the affinities of our ligands towards bvdv rdrp can be inferred by applying the mm/ pbsa methodology to estimate the free energy of binding, dg bind , and its components. the calculated dg bind values for the dimethylaminopropyl derivatives 1, 2, 4, and the quinolizidinylmethyl derivatives 7-10 are listed in table 6 . generally speaking, and in harmony with our previous findings, 33,34 both the nonbonded mechanical energy components of dg bind , de vdw and de el , afford a substantial, favorable contribution to binding for both series of compounds. on the other hand, due the polar character of the azo group, the desolvation penalty paid by these molecules upon binding (dg pb ) is also quite substantial, so that the net, resulting electrostatic contribution to the affinity of these inhibitors to their enzyme receptor are notably unfavorable. specifically, for this series of compounds, the mean value of the electrostatic energy (de el + dg pb ) is 26 kcal/mol, whilst the corresponding mean values of the van der waals and hydrophobic overall interaction energies (de vdw + dg np ) are à46 kcal/mol. accordingly, it follows that the association between the ligands and the rdrp is mainly driven by more favorable nonpolar interactions in the complex than in the solution, in harmony with a proposed general scheme for noncovalent association. 33, 34 however, as indicated for instance by the energy components of compound 2, this driving force can be weakened when the polar groups on the molecule do not find an adequate bonding pattern in the protein compared to water. the free energy penalty for this (de el + dg pb ) term is least for compound 1 and this, together with its substantial van der waals contribution and moderate entropic unfavorable term, consequently leads to one of the highest binding affinity in this set of inhibitors. one of the most important benchmark in this study, however, is the correspondence between the estimated free energies of binding and the experimental measured ec 50 values. indeed, we can observe a good agreement between the trend exhibited by the ic 50 values reported in table 6 and the corresponding biological activity determined for these compounds in bvdv infected cell line (see table 3 ). although we obviously cannot directly compare the computed binding free energy (and hence the corresponding ic 50 ) with the ec 50 values deriving from experiment, we can observe that, in most cases, the rank of the inhibitors with respect to their activity towards their putative targets, the rdrps of bvdv, is maintained, although with some discrepancies. in fact, all estimated ic 50 values are systematically higher than those determined by in vitro experiments with infected cell lines. the overall results obtained from molecular modeling deserve some more comments. first of all, the binding site identified by our procedure is very close to the putative binding site proposed for two allosteric inhibitors of bvdv rdrp, vp32947 and bpip, 23 table 5 cytotoxicity against mt-4, mdbk, bhk and vero-76 cell lines and yfv, reo-1, cvb-2, rsv, hsv-1 and sb-1 inhibitory activity of triazene derivatives of structure f and g tables 3 and 4. and involves residue f224 (see fig. 5d ), a residue found mutated into s224 in bvdv bpip-resistant clones. the binding pocket is located in a turn of the fingers domain between two b-sheets which is believed to be involved in finger flexibility for rna template/product translocation, 35 dimerization of the rdrp in the replication complex, or protein/protein interactions, 36, 37 enabling the assembly of an active replication complex. according to our docking results, then, the hypothetical binding of our compounds to the polymerase could affect the finger flexibility or impair the capacity of the protein to translocate its template/product during rna polymerization. alternatively, the diarylazo moiety could possibly act by hampering the entrance of the template rna in the template binding cavity. although our in silico data clearly show the same trend of the corresponding experimental ec 50 values, at the same time they yield no evidence that binding of these inhibitors onto the bvdv rdrp result in a real allosteric inhibition of the enzyme or in a reduced template-binding ability. one of the explanations why we find ic 50 values higher than the corresponding ec 50 could be explained by the following rationale. recently an attempt of co-crystallize the bvdv rdrp with the inhibitor vp32947 failed do to a dimer interface near the putative binding site of vp32947 in the bvdv rdrp crystal. the authors of the work proposed that the for-mation of this dimer could constitute an important point in the replication complex. moreover, it was also hypothesized that the top of the fingers domain could be a protein-docking site pivotal to the interactions with other enzymes of the replicase complex. as the vp32947 binding site is part of the putative binding pocket for our molecular series, it could be that, upon binding, our compounds hinder the interactions among different proteins making up the replicase complex. last but not least, it is important to recall that rdrp functions in virus-infected cells in the context of membrane-bound replication complexes, that usually consist of several virus-encoded proteins, host proteins, and various forms of viral rna. the rna-dependent rna polymerase may then have a role in interacting with these other components and/or in the stability of the replication complexes. if one or more of these putative actions of the rdrp in infected cells is sensitive to our inhibitors, viral rna synthesis may be disrupted in a manner which is distinct from the one estimated by our in silico assays. now, given the similarity among the polymerases of pestiviruses, hepaciviruses, and flaviviruses, any knowledge gained from any one of these viral systems is likely to foster progress in the others. given the high importance of the hepatitis c pandemic and the stringent need for efficient therapeutics for this pathology, it is expected that new molecular entities such as those described in this work can provide meaningful insights towards the identification of possible anti-hcv agents. interestingly, the overall sequence identity between bvdv and hcv rdrp proteins is quite low, approximately 30%. if we consider the alignment of bvdv and hcv rdrp reported in figure 4c , and in particular focus on the highlighted fingers domain part, we can immediately speculate that: (i) the f224 residue in the bvdv protein has no counterpart in the corresponding hcv enzyme; accordingly, if the drug would bind in the same binding pocket with the same binding mode, there should in principle be no problem of resistant mutant at this position. (ii) the important region of k263-k266 in the bvdv rdrp is highly conserved in the corresponding hcv polymerase (knek in bvdv and knev in hcv, respectively, see fig. 4c ); importantly, both k263 and e265 are engaged in fundamental stabilizing hydrogen bonds with the inhibitors in the bvdv case but, being conserved, they are able to establish the same interactions with the compounds also in the case of hcv. (iii) the residue y289 of bvdv rdrp is not conserved in the corresponding hcv polymerase, being replaced by a phenylalanine. therefore, the anchoring hydrogen bond between the y289 -oh group and, for instance, the tertiary nitrogen of 1 can no longer take place. taken all together, these evidences can justify a minor potency of this molecular series towards the hcv rdrp as a target with respect to the bvdv counterpart. at the same time, in the case of both viruses, the binding pockets on the respective rdrps do share a sufficient degree of structural and sequence homology; thus, since these regions involve the fingers domains, which overhang the palm domains of the enzymes, they might be involved in rna substrate recognition and, hence, the the side chains of all residues that form the primary binding pocket interacting with compound 1 are shown as stick models, and the atom color-coding is as follows: n217, firebrick; a221, orange; a222, dark kaki; f224, red; e258, dim gray; t259, rosy brown; i261, green; k263, hot pink; n264, tan; e265, sienna; k266, dark magenta; i287, gold; q288, navy blue; y289, purple; p290, dark slate blue; e291, pink; r295, olive drab; r529, coral; l530, kaki. hydrogen bonds are highlighted as light gray broken lines. hydrogen atoms, counterions, and water molecules are omitted for clarity. free energy components and total binding free energies for compounds of the series a-c on bvdv rdrp inhibitor molecules may affect the rdrps interactions with the incoming rna molecule. twelve aminoarylazocompounds of structures a-c, and 46 aryltriazene derivatives d-g have been synthesized and assayed for antiviral activity against a panel of 10 rna and dna viruses. thirty-five (8 aminoazocompounds and 27 triazene derivatives) over 58 tested compounds exhibited antiviral activity against one or more viruses and many of them had shown an ec 50 6 10 lm against at least one virus. none of the active compounds inhibited the multiplication of hiv-1, vsv and vv. aminoazocompounds a-c have a marked activity against bvdv and yfv, while the triazene derivatives d-g are characterized by the most frequent activity against cvb-2 and rsv. despite the high antiviral activity of many compounds, the corresponding selectivity index (s. i.) are generally low, due the rather high cytotoxicity exerted on the host cell lines. taking into account the ec 50 a particular interest is deserved by compounds displaying good activities against rsv (as 14 and 20) and bvdv (as 1 and 7) . the former virus is responsible of serious respiratory tract infections in humans, with high mortality rate, while the latter is responsible of severe epidemic outbreaks in livestock, but is also a surrogate model for the evaluation of novel, urgently needed agents against hcv, an emerging threat to human health worldwide. the above compounds represent valuable starting models for developing better (more potent and less toxic) and inexpensive antiviral agents through the variation of the dialkylaminoalkyl chain and/or the substituents on the aromatic rings. in view of these considerations, molecular modeling investigations were performed to study wether the active compounds of structures a-c could target the bvdv rna-dependent rna-polymerase (rdrp), which shares some structural similarity with hcv rdrp. indeed a good agreement was observed between the trend exhibited by the ic 50 (calculated from the estimated free energies of binding) and the corresponding biological activities determined for these compounds in bvdv infected mdbk cell line. the overall sequence identity between bvdv and hcv rdrp proteins is approximately 30%, but the important region k263-k266 in the bvdv rdrp is highly conserved in the corresponding hcv polymerase, thus targeting of hcv rdrp by the a-c molecular series could be expected, even if with a minor potency in respect to the bvdv counterpart. a solution of arylamine (8 mmol) in 1.56 ml of conc. hydrochloridric acid and 1 ml of water was cooled to 0°c and slowly diazotized with a solution of sodium nitrite (8 mmol) in 2 ml of water. the diazonium salt solution was added dropwise, during about 10-20 min, to a cold water solution of the n-(3-dimethylaminopropyl/lupinyl)aniline monoacetate (8 mmol) , and maintaining carefully ph 7 with the simultaneous addition of a saturated solution of sodium acetate. after stirring the reaction mixture for 45 min at 0-5°c, the oil product was extracted with et 2 o, dried, then vacuum-distilled (0.1-0.2 torr) at 80-90°c to remove the unreacted n-substituted aniline. the residue was fractionated by cc, eluting firstly with et 2 o the triazene derivative (e) and then with et 2 o + 5%-meoh the isomeric azocompound (a) in quite lower yield. the isolated compounds were further purified either by crystallization or, when oily, by a second cc or distillation. the aromatic amine (25 mmol) was dissolved in 7.3 ml of conc. hydrochloridric acid and 10 ml of water, cooling in a ice bath. the solution was kept at 0-5°c, while adding drop by drop a solution of sodium nitrite (25 mmol) in 10 ml of water. the diazonium salt was then coupled with the relevant amine (25 mmol) dissolved in 45 ml of 1 n naoh (ph 7-8). after 20 min of stirring, the precipitate was collected by filtration. the solid was crystallized from the suitable solvent. in the case of compounds 14, 18 and 19 the proper oily aniline was added to the diazonium salt solution, followed by a solution of sodium acetate to reduce acidity. when the coupling-product separated as an oil (compounds 37, 43, 53) it was extracted with et 2 o and after removing the solvent the oil was purified by cc. the cytisine derivative 58 was extracted with chcl 3 , and the solid obtained was crystallized from acetone. cell lines were purchased from american type culture collection (atcc). the absence of mycoplasma contamination was checked periodically by the hoechst staining method. cell lines supporting the multiplication of rna and dna viruses were the following: cd4 + human t-cells containing an integrated htlv-1 genome (mt-4); madin darby bovine kidney (mdbk); baby hamster kidney (bhk-21) and monkey kidney (vero 76) cells. for cytotoxicity tests, run in parallel with antiviral assays, mdbk and bhk cells were resuspended in 96 multiwell plates at an initial density of 6 â 10 5 and 1 â 10 6 cells/ml, respectively, in maintenance medium, without or with serial dilutions of test compounds. cell viability was determined after 48-96 h at 37°c in a humidified co 2 (5%) atmosphere by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (mtt) method. 38 vero 76 cells were resuspended in 24 multiwell plates at an initial density of 4 â 10 5 cells/ml. the cell number of vero 76 monolayers was determined by staining with the crystal violet dye. for cytotoxicity evaluations, exponentially growing cells derived from human hematological tumors [cd4 + human t-cells containing an integrated htlv-1 genome (mt-4)] were seeded at an initial density of 1 â 10 5 cells/ml in 96 well plates in rpmi-1640 medium supplemented with 10% fetal calf serum (fcs), 100 units/ml penicillin g and 100 lg/ml streptomycin. cell cultures were then incubated at 37°c in a humidified, 5% co 2 atmosphere in the absence or presence of serial dilutions of test compounds. cell viability was determined after 96 h at 37°c by the mtt method. activity of compounds against human immunodeficiency activity of compounds against yellow fever virus (yfv) and reo virus type-1 (reo-1) was based on inhibition of virus-induced cytopathogenicity in acutely infected bhk-21 cells. activities against bovine viral diarrhoea virus (bvdv), in infected mdbk cells, were also based on inhibition of virus-induced cytopathogenicity. bhk and mdbk cells were seeded in 96-well plates at a density of 5 â 10 4 and 3 â 10 4 cells/well, respectively, and were allowed to form confluent monolayers by incubating overnight in growth medium at 37°c in a humidified co 2 (5%) atmosphere. cell monolayers were then infected with 50 ll of a proper virus dilution (in serum-free medium) to give a m.o.i = 0.01. one hour later, 50 ll of mem earle's medium, supplemented with inactivated fetal calf serum (fcs), 1% final concentration, without or with serial dilutions of test compounds, were added. after 3-4 days of incubation at 37°c, cell viability was determined by the mtt method. activity of compounds against coxsackie virus type b2 (cvb-2), polio virus type-1 sabin strain (sb-1), vesicular stomatitis virus (vsv), vaccinia virus (vv), herpes virus 1 (hsv-1) and respiratory syncytial virus (rsv), a-2 strain, in infected vero 76 cells, was determined by plaque reduction assays in vero 76 cell monolayers. to this end, vero 76 cells were seeded in 24-well plates at a density of 2 â 10 5 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium at 37°c in a humidified co 2 (5%) atmosphere. then, monolayers were infected with 250 ll of proper virus dilutions to give 50-100 pfu/well. following removal of unadsorbed virus, 500 ll of dulbecco's modified eagle's medium supplemented with 1% inactivated fcs and 0.75% methyl-cellulose, without or with serial dilutions of test compounds, were added. cultures were incubated at 37°c for 2 (sb-1 and vsv), 3 (cvb-2, vv and hsv-1) or 5 days (rsv) and then fixed with pbs containing 50% ethanol and 0.8% crystal violet, washed and air-dried. plaques were then counted. ec 50 (50% effective concentration) was calculated by linear regression technique. et al. coupling algorithm 47 with separate coupling of the solute and solvent to the heat, an integration time step of 2 fs, and the applications of the shake algorithm to constrain all bonds to their equilibrium values, thus removing high frequency vibrations. longrange nonbonded van der waals interactions were truncated by using a dual cutoff of 9 and 13 å, respectively, where energies and forces due to interactions between 9 and 13 å were updated every 20 time steps. the particle mesh ewald method was used to treat the long-range electrostatics. for the calculation of the binding free energy between the rdrp and each inhibitor in water, a total of 100 snapshots were saved during the md data collection period described above, one snapshot per each 0.1 ns of md simulation. the binding free energy dg bind of each rdrp/drug complex in water was calculated according to the procedure termed molecular mechanic/poisson-boltzmann surface area (mm/pbsa), and originally proposed by srinivasan et al. 48 in the mm/pbsa framework of theory, the binding free energy of a given ligand to its receptor protein can be evaluated as the individual terms of the mm/pbsa approach that contribute to the free energy of a molecule are where e mm denotes the sum of intra-and intermolecular mechanical (mm) energies of a molecule in the gas phase, g solv is its solvation free energy, and -ts solute represents an estimate of the solute entropy. e mm can be further divided into terms arising from electrostatic (e el ), van der waals (e vdw ), and internal (e int ) (i.e., bond, angle, and torsional energies): the solvation free energy: consists of a polar solvation energy component, g pb , which is calculated in a continuum solvent, usually a finite-difference poisson-boltzmann (pb) model, and a nonpolar term, g np , which is proportional to the solvent-accessible surface area (sa). the ensemble of structures for the uncomplexed reactants are generated either running separate md simulations for them, or by using the trajectory of the complex, simply removing the atoms of the protein or ligand. in this work we applied the latter variant. accordingly, the term e int in eq. 2 cancels out in the calculation of the free energy of binding. the calculations of the polar solvation term g pb were done with the delphi package, 49 with interior and exterior dielectric constants equal to 1 and 80, respectively. a grid spacing of 2/å, extending 20% beyond the dimensions of the solute, was employed. the non-polar component g np was obtained using the following relationship: g np = csa + b, in which c = 0.00542 kcal/(mol å 2 ), b = 0.92 kcal/mol, and the surface area was estimated by means of the msms software. 50 the last parameter in eq. 1, that is, the change in solute entropy upon association -ts solute , was calculated through normal-mode analysis. in the first step of this calculation, an 8-å sphere around the ligand was cut out from an md snapshot for each ligand-protein complex. this value was shown to be large enough to yield converged mean changes in solute entropy. on the basis of the size-reduced snapshots of the complex, we generated structures of the uncomplexed reactants by removing the atoms of the protein and ligand, respectively. each of those structures was minimized, using a distance-dependent dielectric constant e = 4r, to account for solvent screening, and its entropy was calculated using classical statistical formulas and normal mode-analysis. to minimize the effects due to different conformations adopted by individual snapshots we averaged the estimation of entropy over 50 snapshots. -tolylazo)phenylpropane-1,3-diamine (6) yield: 10%. mp 78-79°c (et 2 o) 96 (s, ch 3 ) bp (p = 0.1-0.2 torr) 100-105°c 3-trifluoromethylphenyl)-3-phenyltriazen-3-yl]-3-propanamine (26) yield: 13%. oil (cc: al 2 o 3 , et 2 o) et 2 o + 2%et 2 nh). 1 h nmr (cdcl 3 ): 1.83 (q, 2h, c(2)); 2.20 (s, n(ch 3 ) 2 ); 2.31 (t tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr anal. calcd for c 17 h 21 n 5 o 2 : c, 62 tlc: r f = 0.8 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 62 tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 64.30 n-dimethyl-3-propanamine (30) yield: 57%. oil (cc: al 2 o 3 , et 2 o) tlc: r f = 0.7 (al 2 o 3 , et 2 o + 2%et 2 nh). 1 h nmr found: c, 63.98 1-phenyl-3-(3 0 -trifluoromethylphenyl)triazene (14) yield: 92%. mp 95-96°c (hexane). 1 h nmr 5-difluorophenyl)-3-phenyltriazene (18) yield: 30%. mp 125-126°c (pentane). 1 h nmr 0 -trifluoromethylphenyl) triazene (19) yield: 61%. mp 103-104°c (hexane). 1 h nmr 5°c (hexane). 1 h nmr (cdcl 3 ): 1.96 (br s, 4h, c(3, 4)); 3.73 (br s, 4h, c(2, 5)) oil (cc: al 2 o 3 , et 2 o) 1 h nmr calcd for c 11 h 12 f 3 n 3 : c, 54.32 5-difluorophenyl)azopyrrolidine (43) yield: 89%. oil (cc: al 2 o 3 , et 2 o). 1 h nmr calcd for c 10 h 11 f 2 n 3 : c, 56 4-dichlorophenyl)azopyrrolidine (44) yield: 65%. mp 63-65°c (hexane). 1 h nmr anal. calcd for c 10 h 11 found: c, 49 -chlorophenyl)azopiperidine (46) yield: 30%. mp 32-33°c (hexane). 1 h nmr calcd for c 11 h 14 cln 3 : c, 59 -bromophenyl)azopiperidine (47) yield: 28%. mp 30°c (hexane). 1 h nmr mp 33-35°c (hexane). 1 h nmr 5-difluorophenylazo)piperazine (53) yield: 71%. oil (cc: al 2 o 3 , et 2 o). 1 h nmr mp 74-75°c (hexane). 1 h nmr found: c, 60 financial support from italian miur (firb rbne01j3sk01) and biomedicine project is gratefully acknowledged. the authors thanks o. gagliardo for performing the elemental analyses. all molecular dynamics simulations were carried out using the sander and pmemd module within the amber 9 suite of programs, 39 and the parm99 all-atom force field, 40 working in parallel on 32 processors of the tartaglia cluster at the university of trieste (trieste, italy). the crystallographic coordinates of the rna-dependent rna-polymerase (rdrp) of bvdv (pdb entry 1s48.pdb) 31 and hcv (pdb entry 1csj.pdb) 28 were employed as starting geometries for protein simulations in complex with the most active compounds (1, 2, 4, 7-10). missing hydrogen atoms were added to the protein backbone and side chains with leap module of amber 9. all ionizable residues were considered in the standard ionization state at neutral ph. the geometry of added hydrogens and ions was refined for 200 steps (steepest descent) in vacuum using the parm94 force field. 40 further protein geometry refinement was carried out using the sander module of amber 9 via a combined steepest descent-conjugate gradient algorithm, using as a convergence criterion for the energy gradient the root-mean-square of the cartesian elements of the gradient equal to 0.01 kcal/(mol å). the generalized born/surface area (gb/sa) continuum solvation model 41 was used to mimic a water environment. as expected, no relevant structural changes were observed between rdrp relaxed models and the original 3-d structure.the putative binding site for our compounds on the bvdv rdrp was determined using the activesite_search option of the binding site module of insightii (v. 2001, accelrys, san diego, usa). 33 active-site_search identifies protein active sites or binding sites by locating cavities in the protein structure. according to the site_search algorithm employed, the protein is first mapped onto a grid which covers the complete protein space. the grid points are then defined as free points and protein points. the protein points are grid points, within 2 å from a hydrogen atom or 2.5 å from a heavy atom. then, a cubic eraser moves from the outside of the protein toward the center to remove the free points until the opening is too small for it to move forward. those free points not reached by the eraser will be defined as site points. after a site is located, it can be modified by expanding or contracting the site. one layer of grid points at the cavity opening site will be added or removed by each expand or contract operation, respectively.the model structures of all ligand molecules were built and subjected to an initial energy minimization. the convergence criterion was set to 10 à4 kcal/(mol å). a conformational search was carried out using a well-validated, ad-hoc developed combined molecular mechanics/molecular dynamics simulated annealing (mdsa) protocol. 33, 34 accordingly, the relaxed structures were subjected to five repeated temperature cycles (from 310 k to 1000 k and back) using constant volume/constant temperature (nvt) md conditions. at the end of each annealing cycle, the structures were again energy minimized to converge below 10 à4 kcal/ (mol å), and only the structures corresponding to the minimum energy were used for further modeling. the atomic partial charges for the geometrically optimized compounds were obtained using the resp procedure, 42 and the electrostatic potentials were produced by single-point quantum mechanical calculations at the hartree-fock level with a 6-31g * basis set, using the merz-singh-kollman van der waals parameters. 43 eventual missing force field parameters for the compounds considered were generated as follows: am1 geometry optimization of the structure was followed by rhf/6-31g * single point calculation to obtain the electrostatic potentials. next, the resp method was used for charge fitting. the missing bond, angle torsion or van der waals parameters not included in the parm99 were generated using the antechamber module of amber 9.the optimized structures of the inhibitors were docked into the putative enzyme allosteric binding site by applying a consolidated procedure; 33, 34 accordingly, it will be briefly reported below. the software autodock 3.0 44 was employed to estimate the possible binding orientations of all compounds in the receptor. in order to encase a reasonable region of the protein surface and interior volume, centered on the binding site, the grids were 60 å on each side. grid spacing (0.375 å), and 120 grid points were applied in each cartesian direction so as to calculate mass-centered grid maps. am-ber 12-6 and 12-10 lennard-jones parameters were used in modeling van der waals interactions and hydrogen bonding (n-h, o-h and s-h), respectively. in the generation of the electrostatic grid maps, the distance dependent relative permittivity of mehler and solmajer was applied. 45 for the docking of each compound to the proteins, three hundred monte carlo/simulated annealing (mc/ sa) runs were performed, with 100 constant temperature cycles for simulated annealing. for these calculations, the gb/sa implicit water model 41 was used to mimic the solvated environment. the rotation of the angles u and /, and the angles of side chains were set free during the calculations. all other parameters of the mc/ sa algorithm were kept as default. following the docking procedure, the structure of all compounds were subjected to cluster analysis with a tolerance of 1 å for an all-atom root-mean-square deviation from a lower-energy structure representing each cluster family. in the absence of any relevant crystallographic information for our compounds, the structure of each resulting complex characterized by the lowest interaction energy in the prevailing cluster was selected for further evaluation.each best substrate/rdrp complex resulting from the automated docking procedure was further refined in the amber 9 suite using the quenched molecular dynamics (qmd) method. 33, 34 in this case, 1 ns md simulation at 310 k were employed to sample the conformational space of the substrate-enzyme complex in the gb/sa continuum solvation environment. the integration step was equal to 1 fs and the parm99 force field parameters were applied in the simulations. after each ps, the system was cooled to 0 k, the structure was extensively minimized, and stored. to prevent global conformational changes of the enzyme, the backbone of the protein binding site was constrained by a harmonic force constant of 100 kcal/å, whereas the amino acid side chains and the ligands were allowed moving without any constraint.the best energy configuration of each complex resulting from the previous step was allowed to relax in a 55-å radius sphere of tip3p water molecules. 46 the resulting system was minimized with a gradual decrease in the position restraints of the protein atoms. at the end of the relaxation process, all water molecules beyond the first hydration shell (i.e., at a distance > 3.5 å from any protein atom) were removed. finally, to achieve electroneutrality, a suitable number of counterions were added to neutralize the system using the leap module of amber 9, removing eventually overlapping water molecules. to reduce computational time to reasonable limits, all proteins residues with any atom closer than 20 å from the center of mass of each bounded ligand were chosen to be flexible in the dynamic simulations. subsequently, a spherical tip3p water cap of radius equal to 20 å was centered on each inhibitor in the corresponding rdrp complex, including the hydrating water molecules within the sphere resulting from the previous step. after energy minimization of the new water cap for 10 ps, keeping the protein, the ligand, and the pre-existing waters rigid, followed by a md equilibration of the entire water sphere with fixed solute for 1 ns, further unfavorable interactions within the structures were relieved by progressively smaller positional restraints on the solute (from 25 to 0 kcal/(mol å 2 ) for a total of 2 ns. each system was gradually heated to 310 k in three intervals, allowing a 1 ns interval per each 100 k, and then equilibrated for 5 ns at 310 k, followed by 10 ns of data collection runs, necessary for the estimation of the free energy of binding (vide infra). the md simulations were performed at constant t = 310 k using the berendsen supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmc.2009.05.020. key: cord-315193-z6v6s46n authors: adhikari, nilanjan; baidya, sandip k.; saha, achintya; jha, tarun title: structural insight into the viral 3c-like protease inhibitors: comparative sar/qsar approaches date: 2017-07-14 journal: viral proteases and their inhibitors doi: 10.1016/b978-0-12-809712-0.00011-3 sha: doc_id: 315193 cord_uid: z6v6s46n severe acute respiratory syndrome (sars), caused by sars-coronavirus (sars-cov), is a dreadful infection worldwide having economic and medical importance and a global threat for health. it was turned into an epidemic in south china followed by a chain of infections across three generations. a number of pathogeneses in human may occur due to the virus. this infection has not been taken into account before the sars outbreak, and still it is a neglected one. therefore, there is an urgent need to develop small molecule antivirals to combat the sars-cov. no vaccines are available till date though a number of sars-cov 3c-like and 3c protease inhibitors were reported. in this chapter, quantitative structure–activity relationship technique is used for development of anti-sars and anti-hrv drugs and outcome discussed in details. this approach may be a useful strategy to design novel and potential anti-sars drugs to combat these dreadful viral diseases. in early 2003, about 8500 people were diagnosed across the world with severe acute respiratory syndrome (sars). among them, almost 800 died due to its first outbreak. the disease was broken out and turned into an epidemic in guangdong of south china. two cases of sars infections were noticed in taiwan and singapore due to improper handling of the samples in the research laboratory. during april 2004, a "mini outbreak" of infections took place in a research laboratory of beijing that, in turn, led to a chain of infections across three generations. fortunately, the total number of sars-infected people was only nine that time. this incidence threatens us about the mini outbreaks of sars globally at any time (anand et al., 2005) . the sars is caused by the sars-coronavirus (sars-cov) that belongs to the family of coronaviridae. this family also includes viruses, such as feline infectious peritonitis virus, murine hepatitis virus, bovine coronavirus, transmissible gastroenteritis virus (tgev), as well as human coronavirus 229e . the sars is considered as a global threat to the health (khan, 2013; perlman and netland, 2009) . moreover, water and food-borne viral gastroenteritis may be caused by the noroviruses that belong to the family calciviridae (atmar, 2010; patel et al., 2009) . therefore, there is an urgent need to develop small molecule antiviral drugs to combat these viruses. the picornavirus belongs to the family of viruses namely picornaviridae, calciviridae, and coronaviridae (mandadapu et al., 2013a) . a number of pathogeneses in human may occur due to these viruses leading to economic and medical burden. for example, human rhinovirus (hrv) is the major reason 318 viral proteases and their inhibitors for upper respiratory tract infection (ren et al., 2012; turner and couch, 2007; winther, 2011) whereas nonpolio enteroviruses are responsible for symptomatic infections with 10-15 million cases per year in united states (mcminn, 2012; solomon et al., 2010) . depending on the similarity of the polycistronic organization of the genome, common and posttranscriptional strategies along with the conserved region of domain homology in viral proteins, the virus families are related phylogenetically though these families are not related morphologically (anand et al., 2005; cavanagh, 1997; cowley et al., 2000) . coronavirus is found to be responsible for causing a number of diseases not only in human but also in animals, though the human coronavirus has not been taken into account seriously before the sars outbreak (anand et al., 2005) . human coronavirus (hcov) oc43 and 229e may be responsible for illness in the upper portion of the respiratory tract along with common cold-like conditions (myint, 1995) . the hcov 229e is the only strain till date that can be cultured in cell culture technique efficiently. the symptoms of sars include rigor, malaise, high degree of fever, cough, headache, and dyspnoea. the symptoms may also lead to produce interstitial infiltrates in lungs that may be treated through ventilation and intubation . not only the lungs but also other organs may be affected by sars infection (such as liver, kidney, and gastrointestinal tract). therefore, the sars infection may be treated as a cause of systemic infection. face-to-face contacts may be supposed to be the reason for transmission of the pathogen though other routes are also possible. a number of inhibitors against sars-cov 3cl pro and hrv 3c pro were reported and the process of development of new antivirals against this class has been continued for a decade. in the present report, quantitative structure-activity relationships (qsars) techniques have been explored to understand the relation between the sars-cov 3cl pro and hrv 3c pro enzyme inhibitory activity with the physicochemical and structural properties of these inhibitors developed till now. this approach may be a useful strategy to design and develop novel and potential sars-cov 3cl pro and hrv 3c pro inhibitors to combat the dreadful viral infections. and the sequence of genomic structure (marra et al., 2003; rota et al., 2003) . three noncanonical m pro cleavage sites are observed in sars coronavirus polyproteins having val, met, or phe amino acid residues at p2 position whereas the same cleavage site is found dissimilar in other coronaviruses. therefore, the structural and functional criteria of m pro helps to identify it as an important target for developing anti-sars drugs or other anticoronaviral drugs (anand et al., 2005) . the structures of hcov 229e m pro , tgev m pro , and sars-cov m pro demonstrate that these enzymes have three distinct domains. the first two domains (domain i and ii) together possess similarity with chymotrypsin whereas the third one consists of an α-helical fold which is unique (anand et al., 2005) . the active site which is situated between the first two domains possesses a cys-his catalytic site. antiparallel β-barrels with six strands are composed of the domain i and ii (residues 8-99 of i and 100-183 of ii, respectively). the domain ii is connected to domain iii (residues 200-300) through a long loop (residues 184-199) (anand et al., 2005) . the hydrophobic amino acid residues are found to compose the domain i β-barrel. the α-helix (residues 53-58) helps to close the β-barrel like a lid. the domain i is bigger than domain ii, as well as the homologous domain ii of chymotrypsin and 3c pro of hav (allaire et al., 1994; bergmann et al., 1997; tsukada and blow, 1985) . moreover, a number of secondary structural elements are found to be missing in coronavirus m pro compared to hav 3c pro (such as strands b2ii and cii along with the linking loop). the gly135 to ser146 form a portion of the barrel though domain ii possesses maximum consecutive turns and loops. moreover, the structural alignment of coronavirus m pro domain ii with the picornavirus 3c pro domain ii is found to be different. superimposition of domain i of tgev m pro , with hav 3c pro domain i results in a root mean square deviation (rmsd) of 1.85 ǻ whereas superimposition of domain ii of both of these enzymes yields a rmsd of 3.25 ǻ. the overall rmsd for the c α atoms between their structures is >2 ǻ for all 300 c α positions and the three m pro structures possess similarity among themselves (anand et al., 2005) . the helical domain iii is the most variable domain that exhibits a better overlapping between hcov m pro and tgev m pro compared to the sars-cov m pro , with each other. moreover, tgev and hcov 229e (belongs to group i coronavirus) show 61% sequence similarity whereas sars-cov (belongs to group ii coronavirus) exhibits 40% and 44% sequence similarity with hcov 229e and tgev, respectively . a high degree of conserved region (42%-48%) between the domain i and ii is observed while comparing group i coronavirus m pro , and group ii sars-cov m pro . the domain iii comparatively exhibits a lower degree of sequence similarity (36%-40%) between these two groups coronaviral enzymes (anand et al., 2005) . the x-ray crystallography structures of sars-cov m pro , tgev m pro , and hcov 229e m pro show that these form dimers (anand et al., 2002 yang et al., 2003) . moreover, it was also confirmed that the dimer form is enzymatically active but the monomeric form is not active (anand et al., 2005; fan et al., 2004) . the dimerization process is found to be mandatory for enzyme activity and this process helps to discriminate the coronavirus m pro , and the picornavirus m pro distinctly. a catalytic dyad is formed by cys145 and his41 at the sars-cov active site, whereas other cysteine and serine protease are found to form a catalytic triad. a water molecule is found to have hydrogen bonding interaction with his41 and asp187. moreover, if the cysteine residue is replaced with serine at the enzyme active site, the enzymatic activity of sars-cov m pro is decreased. for coronaviral main protease, as well as the picornaviral protease, the cysteine residue is located at the same place of the active site of the his41 imidazole ring plane (distance 3.5-4 ǻ). for hydrogen bonding interaction between the side chains, the sulfur atom of cysteine residue should be along with the same plane of imidazole function (anand et al., 2005) . the substrate binding sites are found to be conserved in all coronavirus main proteases as suggested by the experimental observations (anand et al., , 2005 . the x-ray crystallographic study between the inhibitor-sars cov m pro suggests 322 viral proteases and their inhibitors that the imidazole function of his163 is located at the bottom of the s1 site of m pro to donate hydrogen bond to the backbone carbonyl function of glutamine. for interaction with glutamine at s1 site, the histidine amino acid residue has to be remained unaltered over a broad range of ph. this may be possible through two interactions involved in the imidazole ring. it may either stack to the phenyl ring of phe140 or may accept a hydrogen bond from the hydroxyl function of tyr161. replacement of the his163 is found to abolish the proteolytic activity (hegyi et al., 2002a; ziebuhr et al., 2000) . all these residues discussed are found to be conserved not only in sars-cov m pro but also in all other coronavirus main proteases. moreover, residues ile51, met151, glu166, and his172 of the s1 pocket take part in the conformation of sars-cov m pro (anand et al., 2005) . regarding the s2 specificity site, all the coronaviruses m pro consists of a leucine residue at the s2 cleavage site. this s2 site is hydrophobic in nature and is composed of side chain amino acid residues, such as his41, thr47, met49, tyr53, and met165. the longer methionine residue may restrict the s2 pocket and requires slight spatial orientation to accommodate the substrate leucine residue. due to the presence of ala46 residue and differences in amino acid sequences, the s2 pocket is bigger in sars-cov m pro compared to hcov 229e m pro and tgev m pro . in sars-cov m pro , a stretch of amino acid sequences is observed in 40-50 residues that help to enlarge the size by forming a helix which is not observed in other coronaviruses. this bigger size may be effective in the substrate binding (anand et al., 2005) . apart from the s1 and s2 pockets, some other substrate binding pockets should be taken into consideration. at the p4 position, small amino acid residues may be preferable (such as val, thr, ser, and pro) whereas no specificity at the p3 position is observed for coronavirus m pro . at the p4 position, some amino acid residues are found to be conserved in sars-cov m pro (such as met165 and thr190). moreover, p5 amino acid side chains are found to interact with the main chain at pro168, thr190, and gln192 in sars-cov m pro , and help like a linker between domain ii and iii. apart from antivirals to fight against these coronaviruses, rna interference (rnai) and vaccine development may be a useful strategy though it is a challenging task. the rnai is an important tool for gene silencing. apart from the use of rnai in cancer and genetic disorder , development of sirna inhibitors in sars infection may be a boon for the treatment of the disease (li et al., 2005) . the replication of the sars may be inhibited effectively through rnai in vero cells. therefore, sirna therapy may be effective to combat sars infection . short hairpin rna (shrna) may be useful to target the n gene sequence of sars coronavirus and to inhibit shrna of sars-cov antigen expression (tao et al., 2005; zhai et al., 2007) . these results suggest that gene silencing through rnai may effectively inhibit the sars-cov antigen expression, and, therefore, rnai approach may be effectively utilized as possible therapy for inhibiting sars-cov infection. moreover, the rnai is used to target the replicase enzyme of human sars virus. it not only targets the hsars gene but also produces inhibitory effects on the sars rna virus expression (zhai et al., 2007) . as far as the development of sars vaccines is concerned, the inactivated sars-cov along with the full-length s protein and an attenuated weak virus, and recombinant sars protein may be used (jiang et al., 2005; zhai et al., 2007) . the s protein and the inactivated virus were reported to be used to neutralize antibodies. the attenuated or the weak form of the virus might be used to induce immunity, as well as to neutralize antibodies (finlay et al., 2004) . the development of recombinant vaccines may be a useful strategy to prevent sars infections. it mainly depends on the best antigen identification, as well as the choice of expression system. the s glycoprotein of sars-cov along with its truncated form may be targeted for development of recombinant vaccine as the best candidate (babcock et al., 2004; bisht et al., 2004; buchholz et al., 2004; yang et al., 2004; zhai et al., 2007) . a number of reports were published regarding recombinant s protein vaccine against different sars-cov through aryl delivery system (pogrebnyak et al., 2005; tuboly et al., 2000) . qsar is a useful tool to understand the relation between the structural and physicochemical properties of the drug molecules, and their biological activity which may be useful for predicting the activity or toxicity profile of drugs (gupta, 2007; verma and hansch, 2009) . the data required for developing the qsar models are collected from the literature (see individual qsar for corresponding references). the ic 50 (molar concentration required to produce 50% inhibition of the enzyme), ec 50 (effective concentration), or k i (binding affinity) data are obviously considered as the biological activity term or dependent variable. the independent variables include physicochemical parameters [such as hydrophobicity, molar refractivity, dipole moment along with different axes, molecular weight (mw), polar surface area (psa), and polar volume, as well as surface area (sa) and volume] and many topological parameters, such as kier's molecular connectivity indices, balaban indices, etc. regarding the statistics of qsar models, n is used to indicate the number of compounds in the set, r to indicate the correlation coefficient of the qsar model obtained, r 2 refers to the squared correlation coefficient exhibiting the goodness of fit, q 2 indicates square of the leave-one-out cross-validated correlation coefficient (represents the internal validation of the model), r a 2 refers to the adjusted r 2 , f value represents the fischer statistics (fischer ratio) that actually means the ratio between the explained and unexplained variance for a particular degree of freedom, p stands for the probability factor related to f-ratio, see means the standard error of estimate, q is the quality factor that can be a measure of chance correlation. a high q represents the high predictivity, as well as the lack of over-fitting of the model. compounds that misfit in the correlation are considered as outliers and are usually removed from the regression. we discuss here the qsar models obtained for different categories of sars-cov 3cl pro and hrv 3c pro inhibitors. this model suggested that the increasing value of the psa may contribute positively to the binding enzyme. compounds with a higher psa (compounds 4-6, table 11 .2) have higher activity than compounds with a lower psa (compounds 2, 7, table 11 .2). compound 1 has a lower psa but higher activity whereas compound 3 having the higher psa has lower activity. these molecules are not explained properly by this model. therefore, these molecules (compounds 1, 3, table 11 .2) were considered as outliers. they may have different mechanism(s) of action(s). blanchard et al. (2004) reported some sars-cov 3cl pro inhibitors (fig. 11.3; table 11 .3). the qsar model for these compounds was as shown by eq. (11.2). this model also exhibited that the psa of the molecule might be conducive to the enzyme inhibitory activity of the compounds. as obvious from table 11 .3, compounds 2 and 4 with a higher psa have higher activity than compounds with the lower psa. the sulfone and amino functions of compound 2 and the disubstituted amino acid function of compound 4 may produce higher psa as compared to the trichloro-substituted compound 5 and the monohydroxy trifluoro substituted ester analog (compound 2). it also suggested that the enzyme-drug interaction might be taking place in a nonhydrophobic space at the enzyme active site. compound 1 has the lower psa but possesses comparatively higher activity than other compounds. it may be assumed that compound 1 may behave differently. probably, the ester function and the chloro group may have some electronic interaction with the enzyme responsible for the higher inhibitory activity. therefore, compound 1 may be considered as an outlier. jain et al. (2004) synthesized and evaluated some keto-glutamine analogs as potent sars-cov 3cl pro inhibitors (table 11 .4). for this series of compounds, the qsar model obtained was as shown by eq. (11.3). in this equation, "i" is an indicator parameter that was used with a value of 1 for the presence of the conme 2 . for the absence of this group, its value was zero. the negative coefficient of i suggests that compounds with conme 2 function (compounds 1-4, table 11 .4) are less active than compounds with 3-pyrrolidinone function (compounds 5-8, table 11 .4). therefore, compounds with 3-pyrrolidinone functions (compounds 5-8) are preferable for the higher inhibitory activity. shie et al. (2005a) reported a series of potent anilide inhibitors against sars-3cl pro (fig. 11.4; table 11 .6), for which the qsar model obtained was as shown by eq. (11.5). this model showed the importance of dipole moment along the x-axis (d x ), mw, psa, and volume (vol) for controlling the enzyme inhibition. the positive coefficient of the dipole moment along x-axis suggested that the bulky substitutions along x-axis may favor the activity. moreover, the mw was also shown to have a positive impact on the activity, whereas psa was shown to have the negative effect. therefore, it may be suggested that molecules with the bigger size along with bulky substituents may be conducive to the inhibition. moreover, the volume is found to have a parabolic relation with the enzyme inhibition. the optimum value of the volume is 6500. compounds 1, 7, 9, and 14 were considered as outliers as these molecules may work with a different mechanism(s). shie et al. (2005b) reported a series of peptidomimetic α,β unsaturated esters as promising sars-3cl pro inhibitors (table 11 .7). the qsar model obtained for them was as shown by eq. (11.6). it was observed from eq. (11.6) that increase in the value of both the mw, as well as the psa may be detrimental to the activity. thus, the model suggested that the smaller molecules with less steric bulk might favor the activity. moreover, the enzyme-drug interaction would be more favored in nonhydrophobic space. it was observed that compounds having unsaturation at the r' position (compounds 11-16, table 11 .7) possess lower mw compared to the other molecules in the dataset and possess higher inhibitory activity. in compound 13, both the phenyl rings might be accommodated in the s2 and s3 pockets. moreover, the (dimethylamino) cinnamyl function adopts a coplanar rigid structure at the end terminal, which may help it in forming hydrogen bonding with amino acids residues glu166, glu189, and glu192 at the enzyme active site. compound 8 may behave differently and hence, this was considered as an outlier. wu et al. (2006) reported some benzotriazole esters as promising sars-3cl pro mechanism-based inhibitors (table 11 .8). the model obtained for this series is as shown by eq. (11.7). it was observed from eq. (11.7) that increasing value of the dipole moment along y-axis (d y ) may lead to a decrease in the activity, whereas increasing value of the mw may be conducive to the activity. it also suggests that compounds with the higher molecular bulk with lower steric effect may be favorable for the higher inhibitory activity. compounds with ester functions (compounds 1-8, table 11 .8) is better active than compounds with acetyl function (compounds 9-11) as these molecules (compounds 1-8) possess higher bulkiness. therefore, it may be assumed that the ester analogs impart less steric effect with the enzyme and hence, produce higher activity. chen et al. (2006a) reported some diverse chemical entities through virtual screening, surface plasmon resonance and fluorescence resonance energy transfer based assays as promising against sars-cov 3cl pro (fig. 11 .5; table 11 .9). the qsar model obtained was as shown by eq. (11.8): it is observed from eq. (11.8) that increasing the value of the psa may be detrimental to the activity. thus it suggested that less polar molecules may have better inhibitory activity. due to the presence of electronegative function (such as carboxyl, chloro, etc.), the molecule may have larger psa. compounds 5 and 7 (table 11 .9), though possess lower psa, have higher activity and this could not be explained by this model. thus these compounds might be supposed to involve the different mechanism of action for producing the higher activity. therefore, these compounds are considered as outliers. zhou et al. (2006) reported some isatin analogs as sars-cov 3cl pro inhibitors (table 11 .10), for which the correlation obtained was as in eq. (11.9): it was observed from eq. (11.9) that increasing the sa of these molecules may impart higher inhibitory activity. bulky substitution at the r 1 position, such as β-napthylmethyl (compounds 3 and 8, table 11 .10) may impart higher sa and hence, produce higher activity. thus substitution with -conh 2 function at the r 2 position in place of iodo function may have a better effect (compound 8 vs. 3, compound 6 vs. 1, and compound 7 vs. 2, table 11 .10). similarly, bulky aryl function may be more favorable than the alkyl function. the larger sa may help the molecule to occupy more space in the enzyme active site to have better binding interaction as evidenced by the molecular docking analysis (zhou et al., 2006) . compound 8 having maximum sa exhibits hydrogen bonding with his41 and cys145 through the keto functions of the isatin moiety. moreover, the carboxamide function at the r 2 position makes hydrogen bonding with phe140 and his163. the β-napthyl moiety (compound 8) fits well into the hydrophobic s2 pocket whereas smaller and less bulky substituents, such as methyl (compound 4), n-propyl (compound 5), n-butyl (compound 6), and benzyl (compound 7) do not accommodate well into the s2 pocket. it is not clear why the compound 4 behaves aberrantly though possessing a comparable good sa. therefore, compound 4 may be considered as an outlier. tsai et al. (2006) reported a series of sars-cov 3cl pro inhibitors through pharmacophore mapping and virtual screening approach (fig. 11 .6; table 11 .11). for this, the qsar model obtained was as shown by eq. (11.10). .080, p < 0.00000, see = 0.282, q 2 = 0.754, q = 3.245, outlier = compounds 9, 13, 24 it was observed from eq. (11.10) that increasing the value of the molar refractivity (cmr) and decreasing the value of the dipole moment along y-axis (d y ), as well as the volume (vol) may contribute positively to the enzyme inhibitory activity. it was, therefore, suggested that increasing the total molecular bulk may increase the activity whereas bulky substituent along y-axis may be detrimental to the activity. bulky substitution along y-axis may produce some unfavorable steric interaction with the enzyme. therefore, the bulky molecule with less steric effect may be favorable for the activity. compounds 9, 13, and 24 (table 11 .11) may act through different mechanism(s) of action and hence, they were considered as outliers. table 11 .12) through structure-based drug design approach, for which the qsar model obtained was as shown by eq. (11.11). it was observed from this equation that the increase in the value of the sa and the polar volume (pol vol) may be conducive to the activity, whereas the increasing in the value of volume and dipole moment along x-axis (d x ) might be detrimental to the activity. thus it could be suggested that bulky substitutions along x-axis might produce unfavorable steric hindrance that may lower the activity. moreover, this model also revealed that compounds having higher polar volume may favor the activity compared to compounds with lower polar volume. in compound 1 (table 11 .12), one of the nitro groups is closer to the imidazole function of his41 and thus there may be some electrostatic interaction between them leading to better activity. moreover, the phenyl ring may form π-π interactions with his237 at the enzyme active site leading to potent activity. compounds 1 and 8 (table 11 .12) might act in a different manner and hence, they were considered as outliers. chen et al. (2006b) reported some quercetin-3-β-galactoside and its analogs as promising sars-cov 3cl pro inhibitors (table 11 .13), for which the qsar model obtained was as in eq. (11.12): it was observed from eq. (11.12) that decreasing the value of the psa would have the positive effect on the biological activity. it meant that less polar molecules would be preferred to the high polar molecules. due to the presence of a number of hydroxyl groups, these molecules may interact with the enzyme as hydrogen bond acceptors. the molecular modeling study revealed that the side chain of gln189 forms four hydrogen bonds with compound 5 (table 11 .13), whereas two hydrogen bonding interactions are observed with the nitrogen atom of glu166. it was, however, observed that compound 4 having the highest psa value due to the presence of two galactose rings was less active. probably, compound 4 might behave in an aberrant fashion and hence, it was considered as an outlier. zhang et al. (2007) synthesized and evaluated some phthalhydrazide ketones (table 11 .14) and heteroatomic ester as potential sars-3cl pro inhibitors. the qsar model developed for this set of compounds was as shown by eq. (11.13): eq. (11.13) suggested that high polar volume of the compound would not favor the activity. a molecular modeling study had revealed that the halopyridine moiety of the compounds was well accommodated in the s1 binding pocket where it could have van der waals interactions. moreover, it was observed that the halogen atom does not interact with the enzyme and is directed toward the solvent exposed area. the furyl group of compound 3 is located near the catalytic cys145 residue where it can have hydrophobic interaction. compounds 2 and 7 being a misfit in the correlation were excluded. ghosh et al. (2007) reported some peptidomimetic sars-cov 3cl pro inhibitors (table 11 .15), for which a qsar model obtained was as: 5.009 ( 0.174) 0.504 ( 0.067) z 50 = ± − ± (11.14) it was observed from eq. (11.14) that increasing the value of the dipole moment along z-axis (d z ) will lead to decrease the enzyme inhibitory activity. it thus suggested that the bulky substituent along z-axis will not be conducive to the activity. the long chain linear aminobutoxy derivatives (compounds 4 and 5, table 11 .15) are better than the isoxazole analogs (compounds 1, 2, 6, and 7, table 11 .15) as the isoxazole moiety may produce more bulkiness that may impart unfavorable steric effect with the enzyme. zhang et al. (2008) reported some arylmethylene ketones and fluorinated methylene ketones as sars-cov 3cl pro inhibitors (table 11 .16). the qsar model for them was as shown by eq. (11.15), where the indicator variable "i" stands for a value of unity for the ester group. a positive coefficient of it suggested that the ester group may be favorable for imparting the higher inhibitory activity. compounds bearing ester functions (compounds 2-4, table 11 .16) are highly active compared to the nonester derivatives (compounds 5-8, table 11 .16). compounds 5-7 are found to be oriented from s1 to s4 pocket and the furan oxygen atom forms hydrogen bonds with the amino function of glu166. moreover, it is observed that compound 1 though having ester function, may behave in an aberrant fashion. therefore, it was considered as an outlier. the qsar model obtained for a series of chloropyridine esters reported by niu et al. (2008) as potent sars-cov 3cl pro inhibitors (table 11 .17) was as shown by eq. (11.16), which suggested that increasing the molar refractivity and decreasing the total dipole moment may favor 3cl protease inhibitory activity this also suggested that the smaller molecules with less steric effect may be conducive to the inhibitory activity. the α-naphthyl (compound 11, table 11.17) and the 2-oxochromene function (compound 12, table 11 .17) at ar position yield less dipole moment and better molar refractivity compared to the nitrophenyl (compounds 9 and 10), the chlorophenyl (compound 8) or the 350 viral proteases and their inhibitors pyridyl analog (compound 7) and thus compounds 11 and 12 are more potent than compounds 7-10. furyl derivatives (compounds 1-4) are better inhibitors as compared to the other aryl ester analogs (compounds 7-12) as they have higher molar refractivity despite having comparatively moderate bulky p-chlorophenyl or the p-nitrophenyl groups at r position. a slight reduction in the activity is noticed for the disubstituted aryl function (compound 3) and the alteration of the nitro function at the 2nd position of the phenyl ring (compound 4) in contrast to the 4th position (compound 2), which increases the bulkiness or total bulk, and reduces the activity slightly. it is observed from the molecular modeling study that increasing the length of the side chain may increase the interaction between s2 and s4 pocket and the inhibitor that can be reflected by the qsar model. compound 5 possesses the higher molar refractivity while compound 6 possesses the lowest value of total dipole moment but it is not reflected in their activity. probably, these compounds behave differently from other compounds in the dataset and hence were outliers. yang et al. (2008) reported some cinanserin analogs as sars-cov 3cl pro inhibitors (table 11 .18), for which the qsar model obtained was as shown by eq. (11.17). this equation clearly exhibited that high molecular volume will not be favorable to the activity. thus compounds having aryl (compound 4), the heteroaryl (compounds 6, 7), or the long chain amide function (compounds 1, 2) at y position have the lower activity than compounds having at this position the unsaturation (compounds 8, 9) or the ester function (compound 5). compound 8 enters into the deep s1 pocket and has hydrophobic interactions. however, compound 3 has the lowest volume but it does not show the highest activity. probably, this compound (compound 3) may behave in a different manner with the enzyme and hence it is considered as an outlier. the qsar model derived for some trifluoromethyl, benzothiazolyl, and thiazolyl ketone compounds with peptide side chain reported by regnier et al. (2009) eq. (11.18) showed that the enzyme inhibitory activity was correlated with the polar volume of the molecules through a parabolic relation. it, therefore, suggests that the activity would decrease upto an optimum value of polar volume (pol vol opt = 135) and beyond that will start increasing. compound with the 2-oxo-pyrrolidin-3-yl function (compound 10, table 11 .19) possesses the higher polar volume and hence, possess the maximum inhibition. comparing the activity of this compound with those of compounds 8, 10-13, it may be suggested that the 2-oxo-pyrrolidin-3-yl function in compound 10 is favorable than the diethylamino function in compounds 11-13 and morpholino function in compound 8 at the x position. moreover, the benzothiazole-2-yl function in compounds 14, 15 is favorable than the thiazole function in compounds 8, 12, 13. the bulky group, such as the morpholino in compound 4 and the benzylmethylamino function in compound 5 are favorable than the smaller substituents, such as the hydroxyl in compound 1, the amino group in compound 2, and the diethylamino group in compounds 3, 6, and 7 at x position. comparing compound 14 with 15, it may be inferred that the bulky amino acid moiety (compound 14) is favorable than smaller amino acid functions (compound 15), as bulky functions may produce the higher polar volume. the molecular modeling study revealed that the benzyloxycarbonyl moiety of compound 10 did not make any hydrophobic interaction rather had hydrogen bonding interactions with glu166 through its adjacent amino function. ramajayam et al. (2010) reported some pyrazolone analogs as promising sars-cov 3cl pro inhibitors (table 11 .20), for which the qsar model obtained was as: eq. (11.19) thus suggested that increasing value of the hydrophobicity of these molecules may be detrimental to the activity. compounds with the smaller halogen substitution, such as fluorine at r position (compound 8, table 11 .20) are better than compounds with the bigger halogen substituents, such as the chloro (compounds 2 and 6, table 11 .20). further, a dihalo substituted compound, such as compound 7 was shown to be less active as compared to monohalo-substituted analogs (compounds 2, 6, and 8) . the cyano (compound 4) and the nitro (compound 9) substitutions also produced the higher activity as compared to the methoxy substitution (compound 3) . the docking study suggested that the n1-phenyl group was located near to the s1' pocket. one of the oxygen atoms of the nitro group forms a hydrogen bond with gly143. the keto function of the pyrazolone ring was also found to form another hydrogen bond with glu166. the c-3 phenyl ring was found to be well-accommodated in the s2 pocket. the benzylidene ring without any carboxyl functions lost the activity. therefore, it may be assumed that hydrogen bonding interaction is more important than the hydrophobic interaction. the oxygen atom of the carboxyl group forms a hydrogen bond with gln192. therefore, apart from s2 pocket, none of the aryl functions has exhibited hydrophobic interactions, whereas three hydrogen bonding interactions were observed. compounds 1 and 5 considered as outliers. (11.20) . it was observed from eq. (11.20) that the increasing value of the dipole moment along x-axis may be conducive to the activity. thus, the bulky substitution at x-axis of these molecules may be favorable for activity. compounds 10-12 (table 11 .21) possess higher dipole moment due to much bulky aryl groups as compared to the compounds 1-2, 4-8 and, therefore, have higher activity. compounds 3 and 9 exhibited the aberrant behavior and thus were considered as outliers. nguyen et al. (2011) reported some promising sars-cov 3cl pro inhibitors through virtual screening (fig. 11.9; table 11 .22). the qsar model obtained for these compounds was as shown by eq. (11.21), which exhibited that the activity is well correlated with the hydrophobicity of the molecules. the docking study had revealed that compound 7 (table 11 .22) had good hydrophobic interactions with his41, phe140, leu141, cys145, his163, glu166, gly170, and his172 apart from a number of hydrogen bonding interactions (the nitro group with gly143, methacrylamide group with phe140, one of the oxygen atoms of the nitro group with cys145). the nitrophenyl group was found to be the most crucial moiety to enter into the s1 pocket for imparting potent inhibition. compounds 2 and 4 though possessed a higher value of hydrophobicity but less activity than expected, hence, they were considered as outliers. some peptidomimetic sars-cov 3cl pro inhibitors (table 11 .23) were synthesized and evaluated by akaji et al. (2011) and the qsar model obtained for this [eq. (11.22) ] indicated that the activity is controlled by a single indicator parameter "i" used for an imidazolyl-4-yl methyl substituent at the r 1 position. the positive coefficient of this indicated that such a substituent would conducive to the activity. the reason of this may be that this substituent might have better steric fitting in the s1 pocket of the enzyme formed by phe140, leu141, and glu166. compounds 4 and 8 (table 11 .23) were considered as outliers. nguyen et al. (2012) reported some flavonoids from pichia pastoris (fig. 11 .10; table 11 .24) having sars-cov 3cl pro inhibitory activity. for these compounds, the inhibition activity was shown to be correlated with the psa of the molecule [eq. (11.23)], suggesting that highly polar molecules may have better activity. substituents like hydroxy might give better psa, leading to better figure 11.10 general structure of some flavonoids as sars-cov 3cl pro inhibitors. activity and also such substituents might form the hydrogen bonds. a molecular docking study showed that the galloyl group forms hydrogen bonds with leu141, gly143, ser144, and his163 at the enzyme active site. compounds without any b ring (compounds 3 and 4) are less active. compound 1 with no 2, 3 double bond in the c ring is less active than the compound 2 though possessing the higher psa. compound 1 was found to act as an outlier. it was also observed that the rigid aryl substitution with the hydroxyl group (compound 6) was better than the flexible cycloalkyl substitution with the hydroxyl group (compound 5). mandadapu et al. (2013b) reported some dipeptidyl aldehydes and α-keto amides as potent norovirus 3cl pro inhibitors (table 11 .25). the qsar model obtained for these compounds was as shown by eq. (11.24) that again exhibited that the hydrophobicity of the compounds may be beneficial to sars-cov 3cl pro inhibitory activity of the compounds. compounds with cyclohexylmethyl group appeared to be more potent than other compounds. this might be due to the bulkiness of this group providing the higher c log p value and due to its better fitting in the active site of the enzyme. compounds 1 and 7, however, showed aberrant behaviors and thus were considered as outliers. thanigaimalai et al. (2013a) reported a series of dipeptide-type sars-cov 3cl pro inhibitors (table 11 .26), for which the qsar model obtained was as shown by eq. (11.25). it was suggested from eq. (11.25) that decreasing value of the sa may be conducive to the enzyme inhibition. however, the volume of the molecules was found to exhibit a parabolic relation with the enzyme inhibitory activity. it, therefore, suggested that increase in volume may be responsible for enhancing the activity only up to an optimum value of 7250. beyond this value, the activity would decrease. thus it indicated that molecules with limited bulk or with substituents with limited bulk might be favorable to the activity. thus indole derivatives with less bulky substitution (compounds 3-11, table 11 .26) resulted in higher activity than those with a greater bulk (compounds 12, 13) . compared to the indole analogs, the oxopyrrolidine (compound 1), the pyrrole (compound 2), the benzothiazole (compound 15), and the benzofuran (compound 17) analogs were comparatively less active. however, it could not be explained by the model why benzimidazole analog (compound 14) had higher activity as compared to the benzothiazole (compound 15) and benzofuran analogs (compound 17). probably, this compound may behave differently as compared to the other compounds in the dataset. therefore, this compound is considered as an outlier. in the subsequent study, thanigaimalai et al. (2013b) reported a series of dipeptide-type sars-cov 3cl protease inhibitors (table 11 .27) whose activity was shown to be controlled by the molar refractivity (cmr) and the polar volume (pol vol) of the compounds [eq. (11.26)]. since the correlation was quadratic with respect to both cmr and pol vol, it suggested that compounds with limited bulk and polarity may have a better binding affinity. several compounds, however, were treated as outliers. 1, 6, 7, 15, 21, 24, 25 turlington et al. (2013) reported a series of n-(benzo [1,2,3]triazol-1-yl)-n-(benzyl)acetamido) phenyl) carboxamides as promising sars-cov 3cl pro inhibitors (table 11 .28). the qsar model obtained for these compounds [eq. (11.27)] suggested that highly hydrophobic (c log p > 4.1) molecule with high molar refractivity but the less mw will be conducive to the activity. with the structural insight into the viral 3c-like protease inhibitors chapter | 11 365 n-(benzo[1,2,3]triazol-1-yl)-n-( (table 11 .29) reported by prior et al. (2013) suggested that the psa of the molecules will control their activity and that a -cho group at their x-position, for which an indicator parameter "i" was used, will have an added advantage. while the psa may affect the activity due to a polar interaction of the molecule, the -cho group might be involved in some hydrogen bond interactions with some residue of the active site. (table 11 .30), for which the qsar model was as shown by eq. (11.29). it was observed from eq. (11.29) that increasing value of hydrophobicity may be favorable for the activity and that the moderate dipole moment of the compound will also be conducive to the inhibition of the enzyme. however, a negative value of the indicator variable "i", which was used with a value of 1 for compounds having a β-napthylmethyl function at r 1 -position, indicated that such a substituent would not be preferred, probably such a substituent might create steric hindrance in the interaction of the compounds with the receptor. mohamed et al. (2015) recently reported some substituted pyrazoles and substituted pyrimidines as promising sars-cov 3cl pro inhibitors (fig. 11.11 ; table 11 .31). the qsar model [eq. (11.30) ] obtained for these compounds simply suggested that highly polar fraction of the molecule with the high value of its x-component of dipole moment (d x ) will not be conducive to the activity. galasiti et al. (2015) recently reported a series of dipeptidyl norovirus 3cl pro inhibitors having potent inhibitory activity (table 11 .32). the qsar model obtained for these inhibitors was as shown by eq. (11.31). this equation simply suggested that while the z-component of the dipole moment will be favorable to the activity, its moderate psa will have an adverse effect. (table 11 .33), for which the qsar model obtained [eq. (11.32 )] suggested that molecules should have an optimum lipophilicity value for imparting the higher activity. the activity may be further supported by the x-component of the dipole moment and the psa of the molecule. notwithstanding, the high polar volume of the molecule will be delirious to the activity. where the activity is shown to be correlated with two indicator variables, i 1 and i 2 . i 1 and i 2 , with a value of 1 each, indicate the presence of the ipropyl and the t-butyl moiety at r 3 -position, respectively. the positive coefficients of both these parameters suggested that the i-propyl and the t-butyl functions at r 3 -position will be favorable for inhibitory activity. compound 6 behaved aberrantly and therefore it was considered as an outlier. it may be observed that the t-butyl substitution at r 3 -position in compounds 5 and 10 gave better activity than the i-butyl substitution at the same position in compounds 4 and 9. dragovich et al. (1998a) reported a series of michael acceptor type potent hrv 3c pro inhibitors (fig. 11 .12; table 11 .35), for which the qsar model [eq. (11.34) ] exhibited that the positive effect on the activity of the compounds will be produced by the z-component of the dipole moment and the sa of the compounds till it attains an optimum value. these two properties indicate the same kind of electronic interactions of the molecule with the receptor. dragovich et al. (1998b) reported a series of peptide-derived potent hrv 3c pro inhibitors (table 11 .36), the qsar model [eq. (11.35) ] suggested that the activity would be primarily controlled by the hydrophobicity of the molecule. the polar volume and the total dipole moment of the compounds would also help to increase the activity of the compounds. "i" is an indicator parameter indicating the presence of i-butyl moiety at r 3 position. its negative coefficient suggested that i-butyl function is not favorable at r 3 -position. this might be creating some steric problem. dragovich et al. (1999a) reported some ketomethylene group containing peptide-based hrv 3c pro inhibitors (table 11. it is observed from eq. (11.36) that decreasing value of the dipole moment along y-axis may increase the activity. this model also showed the importance of two indicator variables, i 1 and i 2 , each of which was used with a value of unity i-butyl and the i-propyl substituent at r 2 position, respectively. the negative coefficient of i 1 suggested that i-butyl group will not be conducive to the activity, while the positive coefficient of i 2 suggested that i-propyl group would be favorable to the activity. these facts are observed from the i-butyl substituted compounds 1-3 and 5-7and the i-propyl substituted compounds 4, 8-11. dragovich et al. (1999b) reported some hrv 3c pro inhibitors (table 11 .38). the qsar model is shown in eq. (11.37) obtained for them suggested that the high polarity of the compound in the x-direction will be detrimental to the inhibition potency of the compound. the simple structure-activity relationship study of (continued ) dragovich et al. (1999c) had also reported some tripeptidyl n-methyl amino acids as hrv 3c pro inhibitors (table 11 .39), for which the qsar model obtained [eq. (11.38) ] had suggested that the activity will be totally governed by the total dipole moment of the compound. this indicated the strong electronic interaction between the molecule and the receptor. however, for some ketone containing tripeptidyl hrv 3c pro inhibitors (table 11 .40) reported by dragovich et al. (2000) , the qsar model [eq. (11.39) ] exhibited the importance of only z-component of the dipole moment of the compound. in the z-direction, the polarity of the compound may be more favorable for electronic interaction with the receptor. webber et al. (2001) reported some depsipeptidyl hrv 3c pro inhibitors ( table 11 .41), for which the qsar model obtained was as is shown by eq. (11.40). this model suggested that in this case, the polar volume of the molecule will not be conducive to the activity. = ± + ± − ± + ± − ± p c p d d i = ± + ± − ± + ± − ± + ± − ± p c p d d for some 2-pyridone containing peptidomimetics as promising hrv 3c pro inhibitors (table 11 .42) reported by dragovich et al. (2002) , the qsar model [eq. (11.41) ] had, however, suggested that the sa of the molecule will be favorable to the activity, probably because of dispersion interaction between the active surface of the molecule and that of the receptor. however, a very high sa was shown to be detrimental to the activity, probably because of the steric problem. (table 11 .43). for these inhibitors, the qsar model [eq. (11.42) ] indicated that the activity of compounds will simply depend upon the presence or absence of β-naphthyl group or chromene ring at the r-position of the compound. of the two indicator variables, i 1 and i 2 , the former with a value of 1 indicated the presence of β-naphthyl group and the latter with a value of unity indicated the presence of chromene ring at the r-position. the positive coefficients of both the variables indicated the favorable contribution of either of the substituent. both might have steric interactions with the active site of the enzyme. dragovich et al. (2003) reported a series of 2-pyridone containing peptidomimetics as hrv 3c pro inhibitors (table 11 .44). the qsar model obtained for these compounds was as shown by eq. (11.43). this model suggested that have less bulky molecules with small x-component of their dipole moment will be favored. simultaneously, the high value of the psa of these molecules may be conducive to activity. further, the positive coefficient of the indicator variable "i" that was defined with a value of unity for a benzyl substituent at r 1 -position indicated that such a substituent should be desired for the better activity of the compound. this benzyl group might have steric interaction with the receptor. a total of 43 qsar models (33 for sars-cov 3cl pro inhibitors and 10 for hrv 3c pro inhibitors) have been reported here to get an insight into the relation between the enzyme inhibitory activities of the antiviral compounds and their physicochemical and structural properties. qsar models exhibited that the physicochemical parameters, such as dipole moment, psa, polar volume, hydrophobicity, molar refractivity, sa, and molecular volume of the compounds play a crucial role in controlling both sars-cov 3cl pro and hrv 3c pro inhibitory activities. moreover, some structural indicator variables were found to play an important role for inhibition of these enzymes. in many cases, the dipole moment and the psa were found to be dominant factors. the bulk of the inhibitors and their flexibility and polarity also appeared to play crucial roles in the inhibition of the enzyme. most of the qsar models exhibited a direct correlation of dipole moment with the 3cl pro or 3c pro inhibitory activity, where a majority of them showed the positive effect of dipole moment on activity but few showed the negative effect, too. these positive and negative effects may be attributed to the orientation of the inhibitor molecules in the active site of the enzyme. the psa and the polarity of the inhibitors were some other important factors that were found in some cases to influence the activity. with their positive coefficients in the correlation, they indicated the attractive electronic interactions of the molecules with the enzyme, and with negative coefficient, they indicated the repulsive interaction. in many cases, the polar volume was found to govern the activity. the polar volume also indicated the attractive or repulsive dispersion interaction between the molecule and the receptor. among all, the hydrophobicity of the molecules had its own role. in any drug-receptor interaction, hydrophobicity is found to play an important role because in most of the enzymes the active site has a hydrophobic pocket which plays an important role in the activity of the site. in most of the cases, the bulky portion of the molecule tries to occupy this hydrophobic pocket where it may have hydrophobic interaction. the molecular volume, mw, or molar refractivity all sometimes become synonymous to the hydrophobic property of the molecules. if they are not found related to hydrophobicity and are crucial for the activity, then it means that the inhibitor-enzyme interaction involves dispersion interaction. the qsar models discussed here may be a great help to design some new, more potent compounds in any given category of sars-cov 3cl pro or hrv 3c pro inhibitors. structure-based design, synthesis, and evaluation of peptide-mimetic sars 3cl protease inhibitors picornaviral 3c cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases structural insight into the viral 3c-like protease inhibitors chapter structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alphahelical domain coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs coronavirus main proteinase: target for antiviral drug therapy noroviruses-state of the art amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor the refined crystal structure of the 3c gene product from hepatitis a virus: specific proteinase activity and rna recognition severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice high-throughput screening identifies inhibitors of the sars coronavirus main proteinase contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity nidovirales: a new order comprising coronaviridae and arteriviridae discovering severe acute respiratory syndrome coronavirus 3cl protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with sars-cov 3cl (pro): structure-activity relationship studies reveal salient pharmacophore features gill-associated virus of penaeus monodon prawns: an invertebrate virus with orf1a and orf1b genes related to arteri-and coronaviruses structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 1. michael acceptor structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 2. peptide structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 3. structure-activity studies of ketomethylene-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 4. incorporation of p1 lactam moieties as l-glutamine replacements structure-based design of irreversible, tripeptidyl human rhinovirus 3c protease inhibitors containing n-methyl amino acids structure-based design of ketone-containing, tripeptidyl human rhinovirus 3c protease inhibitors structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase rapid response research to emerging infectious diseases: lessons from sars structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3cl protease.structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors structural insight into the viral 3c-like protease inhibitors chapter evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery quantitative structure-activity relationship studies on zinc-containing metalloproteinase inhibitors mutational analysis of the active centre of coronavirus 3c-like proteases conservation of substrate specificities among coronavirus main proteases nucleotide sequence of the human coronavirus 229e rna polymerase locus sars coronavirus: a new challenge for prevention and therapy evaluation of metal-conjugated compounds as inhibitors of 3cl protease of sars-cov synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome 3clpro sars vaccine development structure-based design of a parallel synthetic array directed toward the discovery of irreversible inhibitors of human rhinovirus 3c protease a novel coronavirus capable of lethal human infections: an emerging picture broad-spectrum inhibitors against 3c-like proteases of feline coronaviruses and feline caliciviruses a major outbreak of severe acute respiratory syndrome in hong kong using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque synthesis, modification and docking studies of 5-sulfonyl 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the s1 pocket of sars-cov m pro noroviruses: a comprehensive review coronaviruses post-sars: update on replication and pathogenesis severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine design, synthesis, and bioevaluation of viral 3c and 3c-like protease inhibitors synthesis and evaluation of pyrazolone compounds as sars-coronavirus 3c-like protease inhibitors new developments for the design, synthesis and biological evaluation of potent sars-cov 3cl (pro) inhibitors structural insight into the viral 3c-like protease inhibitors chapter viral infections of the lower respiratory tract biflavonoids from torreya nucifera displaying sars-cov 3cl (pro) inhibition discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3cl protease inhibition of the severe acute respiratory syndrome 3cl protease by peptidomimetic alpha, beta-unsaturated esters current status of anti-sars agents virology, epidemiology, pathogenesis, and control of enterovirus 71 targeting zoonotic viruses: structurebased inhibition of the 3c-like protease from bat coronavirus hku4-the likely reservoir host to the human coronavirus that causes middle east respiratory syndrome (mers) potent and specific inhibition of sars-cov antigen expression by rna interference development of potent dipeptide-type sars-cov 3cl protease inhibitors with novel p3 scaffolds: design, synthesis, biological evaluation, and docking studies design, synthesis, and biological evaluation of novel dipeptide-type sars-cov 3cl protease inhibitors: structure-activity relationship study viral replicase gene products suffice for coronavirus discontinuous transcription discovery of a novel family of sars-cov protease inhibitors by virtual screening and 3d-qsar studies structure of alpha-chymotrypsin refined at 1.68 å resolution immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants discovery of n-(benzo [1,2,3]triazol-1-yl)-n-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (sars-cov) 3clpro inhibitors: identification of ml300 and noncovalent nanomolar inhibitors with an induced-fit binding rhiniviruses camptothecins: a sar/qsar study inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells design and synthesis of irreversible depsipeptidyl human rhinovirus 3c protease inhibitors rhinovirus infections in the upper airway small molecules targeting severe acute respiratory syndrome human coronavirus stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3cl protease the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor design and synthesis of cinanserin analogs as severe acute respiratory syndrome coronavirus 3cl protease inhibitors a dna vaccine induces sars coronavirus neutralization and protective immunity in mice recent patents on treatment of severe acute respiratory syndrome (sars) design, synthesis, and evaluation of inhibitors for severe acute respiratory syndrome 3c-like protease based on phthalhydrazide ketones or heteroaromatic esters aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (sars) 3c-like proteinase isatin compounds as noncovalent sars coronavirus 3c-like protease inhibitors virus-encoded proteinases and proteolytic processing in the nidovirales structural insight into the viral 3c-like protease inhibitors chapter further reading structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand x-ray structure and biological evaluation papain-like protease (plpro) inhibitory effects of cinnamic amides from tribulus terrestris fruits key: cord-011251-rjyipcfv authors: chernyshov, vladimir v.; yarovaya, olga i.; fadeev, dmitry s.; gatilov, yuriy v.; esaulkova, yana l.; muryleva, anna s.; sinegubova, katherina o.; zarubaev, vladimir v.; salakhutdinov, nariman f. title: single-stage synthesis of heterocyclic alkaloid-like compounds from (+)-camphoric acid and their antiviral activity date: 2019-02-28 journal: mol divers doi: 10.1007/s11030-019-09932-9 sha: doc_id: 11251 cord_uid: rjyipcfv abstract: an effective technique for one-stage synthesis of new polycyclic nitrogen-containing compounds has been developed. the procedure involves refluxing mixtures of camphoric acid with aliphatic or aromatic diamine without catalysts. in cases where the starting amine has a low boiling point (less than 200 °c), phenol is used as a solvent, as it is the most optimal one for obtaining products with good yields. it has been shown that the use of lewis acids as catalysts reduces the yield of the reaction products. a set of compounds have been synthesized, which can be attributed to synthetic analogues of alkaloids. in vitro screening for activity influenza virus a was carried out for the obtained compounds. the synthesized quinazoline-like agent 14 has inhibitory activity against different strains of influenza viruses. graphical abstract: [image: see text] electronic supplementary material: the online version of this article (10.1007/s11030-019-09932-9) contains supplementary material, which is available to authorized users. influenza represents one of the most serious challenges to medical science and health care all over the world. it causes annual epidemics and from time to time pandemics, both resulting in significant increase in morbidity and mortality [1] . due to fast replicative cycle, ability to reassort the fragments of segmented genome and lack of correcting activity of viral polymerase influenza virus can quickly select mutants that do not match the virus-inhibiting antibodies and can therefore escape from the immune response [2] . several classes of chemically distinct compounds are currently used for treatment of influenza: amantadine and rimantadine (two blockers of m2 proton channel) [3, 4] , four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir and laninamivir) prevent budding of viral progeny [5] , umifenovir (arbidol) is used against influenza in russia and china [6, 7] , pyrazinecarboxamide derivative favipiravir (t-705) was approved for stockpiling for potential treatment of pandemic influenza [8, 9] , and baloxavir marboxil (xofluza ® ) that has been recently approved by fda interferes with endonuclease activity of viral pa subunit of polymerase complex [10] . the same features of influenza virus that cause the emergence of antibody-escape mutants lead also to the selection of drug resistance to direct-acting antivirals. indeed, all current isolates of influenza virus are resistant to adamantane derivatives so that who does not recommend their use for treatment of influenza anymore [10] [11] [12] . in 2007-2009 almost total resistance of seasonal influenza a(h1n1) viruses to oseltamivir was achieved. these strains were further replaced with oseltamivir-susceptible pandemic influenza viruses a(h1n1)pdm09 [13, 14] . taken together, these facts suggest that novel anti-influenza drugs of alternative mechanism(s) of activity and viral target(s) are therefore of high priority for medicinal science and health care. the abundance, crystallinity and variety of transformations of (+)-camphor were interesting throughout the history of organic chemistry. our work is devoted to the investigation of chemical properties of (+)-camphoric acid (product of oxidation of (+)-camphor). camphoric acid is a cyclopentane derivative containing two carboxylic acid functional groups on the first and third carbon atoms. the c-1 atom has an additional methyl group, thereby converting camphoric acid into an enantiomeric ditopic organic linker with different coordination regimes. moreover, the coordination chemistry of camphoric acid specifically gives rise to many interesting chiral features applicable to both materials and life sciences, such as asymmetrical synthesis or crystallization, homochiral structural design, chiral induction, absolute helical control and ligand handedness [15] . there are examples of the use of camphoric acid derivatives as ligands [16] [17] [18] . the condensation of carboxylic acids with amines and anilines is known to be a classical way of preparing amides. this interaction is shown for a wide spectrum of various substrates [19] . the methods for preparing 2-substituted benzimidazoles based on direct cyclocondensations of carboxylic acids with benzene-1,2-diamine are also well known, but almost all of them involve rigid conditions, high temperatures or acidic conditions [20] [21] [22] . for example, many carboxylic acids are condensed with benzene-1,2-diamine at temperatures of about 200° [23] [24] [25] . thus, we consider it relevant to study the interaction of camphoric acid with various aliphatic and aromatic diamines. this study is important, because all nitrogen-containing heterocyclic compounds play a great role in medicinal and organic chemistry in whole. the present work is devoted to the synthesis of new polycyclic nitrogen-containing compounds from (+)-camphoric acid and aliphatic or aromatic diamines. the first investigated reaction was interaction between ethylenediamine 2 and (+)-camphoric acid 1. as a result of refluxing 1 eq. camphoric acid with 2 eq. ethylenediamine in phenol, product 3 was obtained (scheme 1). this technique led to an almost quantitative conversion to 3 after 2 h with 80% yield. product 3 was not observed, if refluxing the starting reagents was carried out without a solvent, apparently due to the low boiling point of the starting amine 2. also we used toluene, dmso and o-xylene as a solvent for this interaction. it should be noted that using toluene or xylene significantly increased the conversion time to 24 h. the use of dmso as a solvent resulted in a double decrease in the desired product. thus, using phenol as a solvent proved to be the most optimal for the production of the tricyclic product 3. refluxing a tenfold excess of camphoric acid with diamine 2 in i-proh gives us a mixture of cyclic amide of symmetrical structure 4 and product 3 (1:1). product 4 was purified by column chromatography and isolated with 9% yield. further, the interaction of 1,3-diaminopropane 5 with camphoric acid was investigated. refluxing 5 with 1 in phenol for 3 h led to compound 6 with 90% yield (scheme 1). unfortunately, we were unable to isolate the cyclic amide of a symmetric structure based on 1,3-diaminopropane. product 8 was obtained by refluxing 1 with 7 in an i-proh with 25% yield. this interaction allows us to obtain compound 8 and presumably mixture of polycyclic compounds with a similar structure to the compound 6, which, unfortunately, are not received in a pure form. for compounds 3, 8 and perchlorate of the compound 6 (6·hclo 4 ) x-ray crystallographic analysis was carried out (fig. 1) . then, we decided to investigate the possibility of obtaining compounds of a similar structure from aromatic amines. thus, o-phenylenediamine 9 was chosen as the next object of our research. we show that refluxing of mixture of 9 with 1 without a solvent for 3 h leads to the formation of a mixture of two benzimidazole derivatives 10a and 10b with a total yield of 70% (scheme 2). in this case, we carried out a study of the conversion rate and the ratio of the final products, by replacing camphoric acid 1 with its anhydride, and also by adding 5 mol% of lewis acid to the reaction mixture, for example, anhydrous zinc chloride. it is shown that the use of the lewis acid increases the conversion time from 3 to 5 h and decreases the total yield of the reaction products to 55% and the quantity of the major isomer 10a in the final mixture. using camphoric acid anhydride as a starting reagent slightly reduces the overall yield of the reaction products. compounds 10a and 10b have been isolated after the column chromatography with a yield of 40% and 2%, scheme 1 interaction of camphoric acid with aliphatic diamines respectively. the structure of compound 10a has been confirmed by x-ray crystallographic analysis (fig. 1) . the benzimidazole derivative 10a was previously described [19] . the procedure involved refluxing the mixture of reagents with 10 mol% of boric acid in toluene for 48 h, and a dean-stark trap was used for the azeotropic removal of h 2 o. we have also shown the possibility of synthesizing compound 10a by single-stage synthesis without solvent and catalysts, which significantly reduced the reaction time. then, we chose naphthalene-1,8-diamine 11 as parent aromatic diamine. refluxing double excess of 11 with 1 without a solvent for 6 h led to the mixture of compounds 12a and 12b with a total yield of 50% (scheme 2). compounds 12a and 12b have been isolated by column chromatography with a yield of 30% and 2%, respectively. we consider that the minor product in all the transformations above is obtained by the primary nucleophilic attack to a more sterically hindered carboxylic group. this assumption is confirmed by 1 h, 13 c and 2d nmr spectra (see supporting information). also, the interaction of o-aminobenzylamine 13 with 1 was studied. refluxing the mixture of 1 and excess of 13 without a solvent for 4 h resulted in compound 14 with 45% yield (scheme 2). x-ray crystallographic analysis shows that, in this case, the main product is the compound formed as a result of the primary nucleophilic attack to a more sterically hindered carboxyl group (fig. 1 ). synthesized polycyclic nitrogen-containing compounds 3, 6, 10a, 10b, 12a, 12b, 14 can be referred to as synthetic analogues of natural quinazoline alkaloids. nowadays, natural and synthetic quinazolines attract considerable attention due to their diverse and sometimes very high biological activity [26] . for example, the most common active metabolites of plants of the genus peganum are quinazoline alkaloids (such as vasicine 15a, desoxyvasicine 15b, vasicinone 16a, deoxyvasicinone 16b [27] ). these natural compounds have a broad spectrum of native biological activity, in particular: anti-ad for 15a-b [28] , anti-parasitic for 16a-b [29] , insecticidal for 15a [30] . at the same time, synthetic derivative of quinazoline diproqualone 17 was previously used as an analgesic for osteoarthritis and rheumatoid arthritis [31] (fig. 2) . along with the pronounced biological activity, the compounds of the quinazoline series can be successfully used as chiral catalysts [32, 33] . thus, the use of vasicine 15a as an organic catalyst for direct c-h arylation of unactivated arenes with aryl iodides/bromides without assistance of any transition metal catalyst has been described [34] . vasicine 15a, a quinazoline alkaloid, from the leaves of adhatoda vasica, has been utilized as an efficient catalyst for metal-and base-free henry reaction of various aldehydes with nitro alkanes [35] . the quinazoline structure possibly imparts rigidity to the ligand and hence consistently high scheme 2 interaction of camphoric acid with aromatic diamines enantioselectivity [36] . at present time, attention of numerous groups is being paid to the synthesis of analogues of natural alkaloids. synthetic and natural quinazoline alkaloids can exhibit pronounced antiviral properties [37, 38] . the compounds synthesized in this work contain both the monoterpenic fragment and the n-heterocycle. we have previously shown that various derivatives of monoterpenoids, in particular compounds including a 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold and n-heterocyclic fragment, exhibit antiviral properties against the influenza virus [39, 40] . in this regard, the obtained derivatives were screened for their inhibitory activity against influenza virus a h1n1. for each compound, the values of 50% cytotoxic dose (cc 50 ), 50% virus-inhibiting dose (ic 50 ) and selectivity index (si) were calculated. the results are shown in table 1 . adamantane-and norbornanebased derivatives were used as reference compounds due to their close similarity to the compounds under investigation in having rigid cage fragments in their structures. it is worth noting that compounds 3, 6, 10b, 12b, 14 are less cytotoxic than reference compounds. compounds 3, 6, 10b, 14 are most effective in inhibiting the influenza virus a (h1n1) and can be used for further studies this type of activity. we believe that aliphatic polycyclic compounds (with a similar structure to compounds 3, 6) or compounds containing an additional aromatic cycle may exhibit potentially high antiviral activity, but in this case, we are particularly interested in the isomers with the hem-dimethyl bridge directed upwards. for compound 14, which showed the highest activity, we studied the antiviral activity against different strains of influenza virus (table 2) . it has been shown that compound 14 has inhibitory activity against different strains of influenza virus a. the compound synthesized has inhibitory activity against strain h5n2 (comparable to reference compounds) and strain h1n1 (exceeding that of reference compounds). unfortunately, the inhibitory activity of compound 14 against strain h3n2 is lower than that of the reference compounds. activity of compounds 3, 4, 6, 10a, 12a in conclusion, a simple and effective method of singlestage synthesis of polycyclic nitrogen-containing heterocyclic compounds, synthetic analogues of natural alkaloids, is suggested for the first time. it was shown that the optimal technique of synthesis is refluxing mixtures of parent compounds in phenol or without solvent and catalysts. using this method, we synthesized a number of polycyclic amides containing in their structure both a heterocyclic fragment and a bicyclic fragment (3, 6, 10a, 10b, 12a, 12b, 14) and two cyclic symmetrical amides (4, 8) . the structures of all synthesized compounds were confirmed by a complete set of spectral data, including x-ray crystallographic analyses of crystalline products 3, 6, 8, 10a and 14. in vitro screening for inhibitory activity against influenza virus was carried out for the obtained compounds and compound 14, was shown, exhibits inhibitory activity against different strains of influenza virus a (h1n1, h3n2, h5n2). compounds 3, 6, 10b and their derivatives, in turn, can be used in the further study of this type of activity. media centre influenza (seasonal) fact sheet constraints, drivers, and implications of influenza a virus reassortment multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza a m2 proton channel structural basis for proton conduction and inhibition by the influenza m2 protein antiviral treatments arbidol as a broad-spectrum antiviral: an update arbidol: a broad-spectrum antiviral compound that blocks viral fusion favipiravir as a potential countermeasure against neglected and emerging rna viruses favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase baloxavir marboxil investigators group baloxavir marboxil for uncomplicated influenza in adults and adolescents adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states the origin and global emergence of adamantane resistant a/h3n2 influenza viruses comparison of antiviral resistance across acute and chronic viral infections drug resistance in influenza a virus: the epidemiology and management chiral chemistry of metalcamphorate frameworks novel tridentate ligands derived from (+)-camphoric acid for enantioselective ethylation of aromatic aldehydes enantioselective alkylation of aromatic aldehydes with (+)-camphoric acid derived chiral 1,3-diamine ligands synthesis of some new chiral bifunctional o-hydroxyarylphosphonodiamides and their application as ligands in ti(iv) complex catalyzed asymmetric silylcyanation of aromatic aldehydes boric acid-catalyzed direct condensation of carboxylic acids with benzene-1,2-diamine into benzimidazoles synthesis, reactivity and biological activity of benzimidazoles synthesis and biological evaluation of 4′-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfonamide derivatives as dual angiotensin ii/endothelin a receptor antagonists novel pyrazolo[3,4-d]pyrimidine with 4-(1h-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer agents: synthesis, cell based assay, topoisomerase inhibition, dna intercalation and bovine serum albumin studies efficient propylphosphonic anhydride ( ® t3p) mediated synthesis of benzothiazoles, benzoxazoles and benzimidazoles simulating microwave chemistry in a resistance-heated autoclave made of semiconducting silicon carbide ceramic synthesis and tuberculostatic activity evaluation of novel benzazoles with alkyl quinazoline derivatives: synthesis and bioactivities chemistry, pharmacology and medicinal properties of peganum harmala l rapid and sensitive detection of the inhibitive activities of acetyl-and butyryl-cholinesterases inhibitors by uplc-esi-ms/ms alkaloids from the seeds of peganum harmala showing antiplasmodial and vasorelaxant activities toxicity and growth inhibitory activities of methanol extract and the β-carboline alkaloids of peganum harmala l. against two coleopteran stored-grain pests quinazolinone: an overview evidence for involvement of cationic intermediate in epoxidation of chiral allylic alcohols and unfunctionalised alkenes catalysed by mn iii (quinazolinone) complexes 3-aminoquinazolinones as chiral ligands in catalytic enantioselective diethylzinc and phenylacetylene addition to aldehydes vasicine catalyzed direct c-h arylation of unactivated arenes: organocatalytic application of an abundant alkaloid vasicine from adhatoda vasica as an organocatalyst for metal-free henry reaction and reductive heterocyclization of o-nitroacylbenzenes vasicine as tridentate ligand for enantioselective addition of diethylzinc to aldehydes synthesis, antiviral activity and cytotoxicity evaluation of schiff bases of some 2-phenyl quinazoline-4(3)h-ones antiviral alkaloids produced by the mangrove-derived fungus cladosporium sp. pjx-41 synthesis and in vitro study of novel borneol derivatives as potent inhibitors of the influenza a virus synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity acknowledgements this work was supported by the russian science foundation (18-03-00271 a). the authors confirm that this article content has no conflict of interest. key: cord-296560-ehrww6uu authors: bender, andreas; jenkins, jeremy l.; li, qingliang; adams, sam e.; cannon, edward o.; glen, robert c. title: chapter 9 molecular similarity: advances in methods, applications and validations in virtual screening and qsar date: 2006-11-07 journal: annu rep comput chem doi: 10.1016/s1574-1400(06)02009-3 sha: doc_id: 296560 cord_uid: ehrww6uu this chapter discusses recent developments in some of the areas that exploit the molecular similarity principle, novel approaches to capture molecular properties by the use of novel descriptors, focuses on a crucial aspect of computational models—their validity, and discusses additional ways to examine data available, such as those from high-throughput screening (hts) campaigns and to gain more knowledge from this data. the chapter also presents some of the recent applications of methods discussed focusing on the successes of virtual screening applications, database clustering and comparisons (such as drugand in-house-likeness), and the recent large-scale validations of docking and scoring programs. while a great number of descriptors and modeling methods has been proposed until today, the recent trend toward proper model validation is very much appreciated. although some of their limitations are surely because of underlying principles and limitations of fundamental concepts, others will certainly be eliminated in the future. molecular similarity [1] [2] [3] [4] follows, in principle, a simple idea: molecules which are similar to each other exhibit similar properties more often than dissimilar pairs of molecules. this is often written as the relationship which leaves open two major questions: 1. how to represent molecular structure (the connectivity table or the coordinates of atoms are not per se suitable choices)? 2. what is the functional form between structure (or rather structural representation) and the property under consideration so that we can derive an empirical measure of similarity? in order to explicitly include both challenges mentioned one can reformulate to give mðpropertyþ ¼ f ðgðstructureþþ where m is the measurement outcome of a molecular property concept (such as log p as a surrogate measure of 'lipophilicity'), g represents the transformation of a molecular structure into a 'descriptor' which is amenable to a statistical analysis or machine-learning treatment and f connects experimental measurement and structural representation. both steps are generally independent of each other, although some combinations of molecular representation and model generation technique are more sensible than others. the problem in establishing a suitable function g, which translates a molecular structure into a descriptor representation, is that it is usually not known a priori which molecular features contribute to a certain property. for example, some functional groups in ligand-receptor binding will establish ligand-receptor interactions, while others simply point into bulk solvent. often a large number of descriptors need to be calculated in order to (hopefully) capture the relevant factors for a certain molecular property, since often no direct experimental observation is known. the problem in establishing a function f, which correlates descriptor representation and property is that its functional form is also usually not known. again, no underlying theory exists and its character can vary between two extremes. linear regression, for example, represents a simple functional form between input and output variables with the advantage of a very small number of free parameters -and following occam's razor it should be applied in cases where there is a sound physical reason to believe in an underlying linear relationship between input and output variables. at the other end, neural networks are able to model any (also nonlinear) relationships between input and output variables. however, they depend on a large number of variables, which may lead to spurious correlations. often the choice of a functional form, in the absence of physical laws, is governed simply by trial-and-error. the problems in establishing the optimal choice of f and g are increased by the fact that the relationship between structure and measured property (the only relationship available from experimental data!) is rarely given over a large region of chemical space. data are sparse -estimations of the size of the chemical space for typical drug molecules [5] (up to 30 heavy atoms) are in the region of 10 60 , experimental datasets on a property of interest are rarely available for more than 10 6 compounds and are often considerably smaller. a solution to the problem of identifying the 'best' molecular descriptor will never be fully established -for both practical reasons (the limited size of datasets) and theoretical reasons. a wide variety of different features are important for each property and the functional forms between descriptor representation and property can usually not be established from physical laws (and thus cannot be optimized analytically). still, we can establish empirical measures of molecular similarity to predict some particular properties better than others, tested on some of the more or less restricted datasets available. this review deals with both novel molecular representations, function g from above, as well as novel model generation and machine learning methods, function f from above. as soon as a relationship between molecular representation and a particular property's values is established a crucial question arises: how good are predictions for novel molecules? ideally, all of chemical space would be covered with zero error. limits in descriptor generation as well as in experimentally available data clearly prevent us from reaching this goal. still, in order to establish confidence in models in practical settings, this requirement can be replaced by the question: which area of chemical space is covered with acceptable error? different methods (best known among them are approaches like crossvalidation), attempt to provide empirical answers to this question. intuitively one might guess that for the question which region is covered by a given model, the distance of compounds from the training set to the novel compounds whose properties are to be predicted is relevant. this is indeed the case, as has been established in recent articles (see section 4) . the question of how good predictions for novel compounds are is often established by cross-validation, where portions of the available datasets are, in turn, taken as an external test set, while the remainder of the dataset is used for training purposes. the test set thus attempts to simulate a novel set of molecules, unknown to the training phase of the model and root-mean-square errors (rmse) or cross-validated correlation coefficients (q 2 ) on the test set are often reported as a measure of the generalizability of models. recently, it has emerged that cross-validation actually shows merely that a model is internally consistent, but not necessarily predictive for new compounds. the question of how reliability of models can be assured is also discussed in section 4, and indeed several recent publications propose approaches to determine the 'domain' of models (the area in which they are applicable, see section 4 for details). conventionally, enrichment over random selection is often cited, giving an estimate of how many more active compounds are retrieved from a database than by pure chance. while this measure is correct in the way it is calculated, more recently the performance of 'sophisticated' fingerprints has been compared to trivial features, namely counts of atoms by element, without any structural information [6] . the performance ratio of 'state-of-the-art' methods (i.e., circular fingerprints and unity fingerprints) to those 'dumb' descriptors can then be interpreted as the 'added value' of more sophisticated methods. soberingly, on many datasets of actives 'real' fingerprints do not perform significantly better than atom counts (see fig. 1 ). this also relates to the suitability of current databases employed for retrospective virtual screening runs, which are often derived from the mddr [7, 8] . while on the one hand, multiple activity classes are present, those datasets still possess two major disadvantages; first, no information about definite inactivity of compounds is contained in the database. still, if experimental data for retrieved hits are subsequently obtained, many of the 'false-positive' predictions may well be active. second, following bioisosteric considerations in combination with 'fast follower' approaches to synthesis, it should be noted that this database contains a large number of close analogues. the hit rates obtained on this dataset may thus be overly optimistic compared to real-world libraries employed for virtual screening. still, the two databases referenced above, which are both subsets of the mddr, were very important as they enabled comparison of similarity searching approaches on multiple, identical datasets. we would also like to emphasize that more suitable datasets are too often -unfortunatelyunavailable from the pharmaceutical and biotechnology companies. in the following sections, we will also cover other recent developments in some of the areas, which exploit the 'molecular similarity principle'. section 3 will present novel approaches to capture molecular properties by the use of novel 'descriptors'. since molecular descriptors and the methods used to analyze the data they represent cannot be separated easily, the second part of this section also covers novel data analysis methods. section 4 focuses on a crucial aspect of computational models -their validity. in the previous few years, about two dozen publications that focused on 'model validation' have appeared, an area which shall be summarized in this review. finally, sections 5 and 6 turn to the application of the methods described earlier. in section 5, we discuss additional ways to examine data available such as those from high-throughput screening (hts) campaigns and to gain more knowledge from this data. section 6 describes some of the recent applications of methods described in the preceding sections, focusing on successes of virtual screening applications, database clustering and comparisons (such as drug-and in-house-likeness) and recent large-scale validations of docking and scoring programs. we will now describe some of the recent developments in the calculation of molecular descriptors. pot-dmc [9] (short for potency-scaled dynamic mapping of consensus positions) takes not only the (binary) activity of a compound into consideration for virtual screening applications, but also the quantitative activity of a structure. accordingly, each bit of the descriptor vector (which consists of a combination of one-, two-and three-dimensional (1d, 2d and 3d) features) is multiplied depending on the ic 50 value of the compound. scaled bits are summed and normalized at each position. afterward, the descriptor can be used for virtual screening. when applied to a database of ccr5 chemokine receptor antagonists, serotonin receptor agonists and gonadotropin-releasing hormone agonists, the method overall did not retrieve a larger number of structures -but those which were retrieved were, as intended, of higher activity than in cases where no scaling according to activity was applied. the fepops [10] (feature points of pharmacophores) descriptor aims to exploit a (relative) advantage of 3d descriptors, the ability to discover novel scaffolds against a given target, based on active sample structures. after generation of tautomers and conformers, k-means clustering of atomic coordinates is performed. thus, no knowledge about the active conformation of a structure is necessary. interaction types are assigned to characteristic 'feature points' in a subsequent step, and are again subject to k-medoids clustering to reduce redundant conformer coverage. cluster representatives can now be used for similarity searching. validations are presented using both mddr (cox-2, hiv-rt and 5ht3a inhibitors and ligands, respectively) and in-house datasets. in addition, it was shown that inhibitors can be identified from a database, based simply on endogenous ligands (for dopamine and retinoic acid). a completely different path is followed by the lingo [11] approach, which is based on a textual representation of molecules. based on the smiles string of a structure, and without time-consuming conversion and descriptor generation, a molecule is represented by a set of overlapping 'lingos', each of which represents a substring of the complete smiles structure. while being a straightforward concept (in the best possible sense), favorable performance is presented on log p and solubility datasets, where cross-validated rms errors are 0.61 and 0.89 log units, respectively. the descriptor also shows applicability to bioactivity, where significant discrimination between bioisosteres and random functional groups can be observed. reduced graph descriptors have been the subject of interest for a considerable time, and recently further work was performed in this area with success. earlier comparison algorithms of reduced graphs represent the graph as a binary fingerprint, sometimes leading to molecules perceived as similar by the algorithm, which are not similar to the eyes of most chemists. this problem was recently addressed [12] by applying 'edit distance' measures to the similarity of compounds -the number of operations needed to transform one reduced graph structure of a molecule into another. through this emphasis of not only the fragments present in reduced graphs, but also the way in which they are connected, better agreement with the human perception of 'molecular similarity' could be achieved. molecular binding can be thought of as being mediated by complementary shapes and matching properties -where, due to solvation and other effects, 'matching' does not only mean complementarity. accordingly, a 'shape fingerprint' method has recently been presented [13] which implements shape similarity measures akin to volume overlap methods, but which, due to the employment of database-derived reference shapes, is several orders of magnitude faster. (note of course that shape also plays an important role in other areas of science [14] .) employing gaussian descriptions of molecular shape, about 500 shape comparisons can be performed per second and the resulting shape similarity was shown to be useful in virtual screening applications. only some parts of a ligand bound to its target will actually interact with the target, other parts will just be pointing into the bulk solvent. by analyzing the variability of ligands' regions, features which correspond to each of the regions can be inferred -molecular features which are involved in ligand-target interactions will be more highly conserved than those which point into the solvent, due to the stricter requirements imposed on them. the 'weighted probe interaction energy (wep) method' [15] exploits exactly this principle, and can be used to derive ligand-based receptor models. this was applied to the steroid dataset (which is well known from comfa studies) a set of dihydrofolate reductase (dhfr) inhibitors as well as hydrophobic chlorinated dibenzofurans. in particular, the dhfr model was able to elucidate interactions relevant to binding which very closely resemble the target-derived model complex. previously applied to the calculation of inter-substituent similarities, which might be exploited for the identification of bioisosteric groups [16] , the r-group descriptor (rgd) was more recently also the subject of qsar investigations [17] . the rgd describes the distribution of atomic properties at a distance of n bonds (n ¼ 1, 2, 3 y) away from a core that is common to a series of compounds. in combination with partial least squares the descriptor was applied to several datasets for qsar studies, comprising of benzodiazepin-2-ones active at gaba a , triazines exhibiting anticoccidal activity and a set of tropanes active at serine, dopamine and norepinephrine transporters. rgdss in combination with pls showed comparable performance overall to hqsar and eva models in a cross-validation study, in some cases outperforming the other qsar approaches. another alignment-free method for the time-efficient generation of qsar models is fingal [18] (a short and straightforward acronym for 'fingerprint algorithm'). unlike rgds, a hashed fingerprint is generated which encodes structural features of the molecule, where distances may be measured either topologically or by employing spatial information between atoms. applied to d2 ligands, the 2d version of fingal, in particular, was able to outperform comfa-and comsia-based approaches. for estrogen ligands, performance was highly dependent on the structural class of compounds, not only for fingal but also for models based on comfa, hqsar, fred/skeys (fast random elimination of descriptors/substructure keys) and dragon descriptors. in subsets such as a pesticide subset, no model was obtained via comfa (correlation coefficient of zero), whereas fingal gave correlation coefficients as high as 0.85 in a cross-validation study. the grid force field [19] has been the basis of a number of descriptors developed recently, among the best-known ones being the grind descriptor [20] . some extensions of the descriptor have been presented recently, which include the incorporation of shape [21] into the descriptor. it was recognized that molecular shape is a major factor determining ligand-receptor binding, a property that was previously not emphasized enough by the original grind descriptors. this was due to the fact that only maximum products of interactions are incorporated into the descriptor, omitting large lipophilic features which do not contribute significantly to calculated interaction energies with probes, but might still have profound influence on binding through steric effects. introducing the new 'tip' probe (which is not a probe in the traditional sense but a measure of curvature of the molecular surface) led to significant improvements in qsar studies of adenosine receptor antagonists (of the xanthine structural class) and plasmodium falciparum plasmepsin inhibitors being observed. interestingly, tip-tip correlations were also found to be the most significant descriptors in case of a 1 antagonists, showing the importance of the shape descriptor on this class. the second development was the 'anchor-grind' approach [22] , which focuses on user-defined features to calculate a distribution of interaction points relative to it, thereby incorporating pre-existing biological knowledge about a target. models are found to be both of better quality and easier to interpret on congeneric series of hepatitis c virus ns3 protease and of acetylcholinesterase inhibitors, as well as more discriminatory between factor xa inhibitors of both high and low affinity. a virtual screening methodology also based on the grid force field was developed recently [23] . this method was validated on a large dataset containing thrombin inhibitors and also showed potential to select suitable replacements for scaffolds typically encountered in the lead optimization stage. a molecular 'descriptor' which actually does not employ an explicit transformation of the molecular structure into descriptor space was recently presented [24] . it employs a graph kernel description of the structure in combination with support vector machines (svms) for regression analysis. the computational burden is alleviated through employing a morgan index process as well as the definition of a second-order markov model for random walks on 2d structures. the method was then validated on two mutagenicity datasets. while already exhibiting the ability to capture molecular features responsible for bioactivity (here mutagenicity) in its current form, future developments might include more abstract representations of the molecular scaffold such as some form of reduced graph representation. while the bioinformatics area has a multitude of methods which can be applied to the analysis on 1d representations of protein sequences and dna, due to branching and cyclization the case is far more difficult for small molecules. one of the few 1d representations of molecules [25] , based on multidimensional scaling of the structure from 3d into 1d space, has more recently been extended to allow for the alignment of multiple structures [26] . applied to skc kinase ligands as well as herg channel blockers, significant improvement in retrieval rates could be observed in a retrospective study if multiple (in this case 10) ligands were used for screening. the concept of feature trees was also recently extended to allow for the incorporation of knowledge derived from multiple ligands into a single query [27] , and retrospective screening results on ace inhibitors as well as adrenergic a 1a receptor ligands showed considerable improvements over searches using single queries, both in terms of enrichments as well as the diversity of structures identified. when structures are encoded in a discrete fashion, 'binning' is often employed in order to convert real-valued distance ranges into binary presence/absence features. this approach is followed in, for example, the cats autocorrelation descriptor in its 3d version (cats3d) [28] . however, binning borders may introduce artifacts such that feature distances close to each other but on opposites sides of bin borders being perceived to be as different from each other (simply since features do not match) as much more distant features. accordingly, a related descriptor termed 'squid' was recently introduced which incorporates a variable degree of fuzziness [29] . applied to cox-2 ligands considerable retrieval improvement was observed, with best performance at intermediate degrees of fuzziness. using cox-2 ligands as well as thrombin inhibitors in combination with graph-based potential pharmacophore point triangles, typed according to interaction types, features responsible for ligand-target binding could be identified [30] . in addition, prospective screening was performed and a benzimidazole identified as a potent cox-2 inhibitor was experimentally found to be active in a cellular assay with high affinity (ic 50 ¼ 200 nm). the ultimate descriptor, in the realm of virtual screening, is the response of the biological system. while structure-derived descriptors are quick and (usually) easy to calculate, they are not the final goal -it is the effect that the compound has in a 'real world' setting. using those biological effects as descriptors, namely percent inhibition values across a range of 92 targets for a number of 1567 molecules, the 'biospectra similarity' (the similarity of effects on the respective targets) was established via hierarchical clustering [31] . it was found that biospectra similarity provides a solid descriptor for forecasting activities of novel compounds and this was validated by removal of some important target classes after which clustering of compounds was overall still very stable. while the response of single targets is already a step toward biology, protein readouts of cell cultures [32] also incorporate cell signaling networks, thus stepping even closer to whole organism systems (of course at the price of increased complexity and cost involved). also based on biological response data (phenotypic screening) a 'class scoring' technique was recently developed [33] , which does not assign binary (hit/non-hit) activities to individual compounds but to classes of compounds instead. this way, more robust assignments are achieved as well as a lower number of false-positive predictions. svms have been previously used for distinguishing, for example, between drug-and non-drug-like structures [34] and recently have been applied in virtual screening [35, 36] . using dragon descriptors and a modification of the traditional svm to rank molecules (instead of just classifying them), performance was in this study [35] validated on inhibitors (or ligands) of cyclin-dependent kinase 2, cyclooxygenase 2, factor xa, phosphodiesterase-5 and of the a 1a adrenoceptor. compared to methods such as binary kernel discrimination in combination with jchem fingerprints the new approach was found to be superior. the ability of lead hopping was also demonstrated recently through the combination of svms with 3d pharmacophore fingerprints (defined as smarts queries) [36] . there is a trend in the recent cheminformatics literature toward ensemble methods, i.e., methods where multiple models (instead of a single model) are generated and used together (as an ensemble) to make either qualitative or quantitative predictions about new instances. random forests [37] are an ensemble of unpruned classification or regression trees created by bootstrapping of the training data and random feature selection during tree induction. prediction is then made by majority vote or averaging the predictions of the ensemble. on a set of diverse datasets (blood-brain-barrier penetration, estrogen-binding, p-glycoprotein-activity, multidrug-resistance reversal-activity and activity against cox-2 and dopamine receptors) superior results to methods such as decision trees and pls were reported. more recently, 'boosting' was applied to the same (and additional) datasets [38] , and as a general rule this new method seems to be slightly superior in large regression tasks, whereas random forests are claimed to excel in classification problems. additionally, employing k-nearest neighbor classifiers, svms and ridge regression in an ensemble approach [39] gave significant improvement over single classifiers on a 'frequent hitter' dataset. most models derived in qsar studies, for example, ordinary and partial least-squares regression or principal components regression, employ a linear parametric part and a random error part, the latter of which is assumed to show independent random distributions for each descriptor. however, since molecular descriptors never capture 'complete' information about a molecule, this independence assumption is often not valid. kriging [40] has replaced the independent errors by, for example, gaussian processes. applied to a boiling point dataset and compared to other regression methods (ordinary and partial least-squares and principal component regression) improved performance could be observed. alongside model generation, feature selection is also an important step in many studies. since no perfect descriptors of the molecular system are known, often a multitude of descriptors (often several thousands) are calculated and it is hoped that they capture information, which is relevant to the respective classification or regression task. a comparative study of feature selection methods in drug design appeared recently [41] , which compares information gain, mutual information, w 2 -test, odds ratio and the gss coefficient (named after the authors, galavotti, sebastiani and simi; a simplified version of the w 2 -test) in combination with the naı¨ve bayes classifier as well as svms. while svms were found overall to perform favorably in higher-dimensional feature spaces (and do not benefit much from feature selection), feature selection is found to be a crucial step for the bayes classifier. (note that this has at the same time been shown empirically in virtual screening experiments [42, 43] .) some of the methods, namely mutual information and genetic programing, have also been evaluated separately for their use in qsar studies [44] with respect to a dataset which showed some (typical) problems present in the area, such as a very different sizes of 'active' vs. 'inactive' data subsets. the problem that structure-activity relationships are rarely linear has been addressed previously through the application of nonlinear methods [45, 46] such as k-nearest neighbor approaches [47, 48] . more recently, k-nn has also been combined with a comfa-like approach, termed k-nn mfa, to predict bioactivity of a compound based on its k-nearest neighbors in 'field space' [49] . as discussed by the authors, some of the disadvantages of comfa such as alignment problems are retained; nonetheless, multiple models are produced in each run, giving more room for appropriate model selection. removing limitations of the statistical model is possible using non-parametric models which have recently been used in qsar studies [50] and were shown to improve results over more conventional regressiontype models. also bayesian regularized networks have been found to be of interest in recent qsar studies [51] [52] [53] . those networks possess inherent advantages including that they run less risk of being overtrained than non-bayesian networks (since more complex models are punished by default). the effect of binary representations of fingerprints has been known for some time, such as combinatorial preferences [54] and size effects [55] (depending on the similarity coefficient used). more recently, another aspect of the binary representation of features in a fingerprint has been analyzed [56] . integer or real-valued representations of feature vectors were calculated for 12 activity classes and employed cats2d and cats3d autocorrelation descriptors as well as ghose and crippen fragment descriptors. afterward, retrospective virtual screening calculations were performed for both the original (quantitative) representations and the binary (presence/absence) fingerprints. surprisingly, in only 2 out of the 12 cases did significantly different numbers of actives get retrieved (defined as more than 20% difference). in addition, the retrieved actives showed, depending on the activity class, very different overlap, between 0% and 90%, indicating some orthogonality of the same descriptor, differing by its representation (integer/real-values vs. binary format). exploiting the 'molecular similarity principle' by not only looking for neighbors of an active compound and assuming they are active (as is usually done in virtual screening) but also using this knowledge further to improve the model, has recently been exploited in a method called 'turbo similarity searching' [57] . by feeding back information about the nearest neighbors of an active compound into the model generation step, an increased number of active compounds can be retrieved in a subsequent step. this is analogous to the re-use of hot air in turbo chargers in cars, where the output (hot gas, nearest neighbor in this case) is fed back into the loop to improve performance. a number of publications have appeared recently focusing on the validation of qsar models. a wealth of parameters exist here, such as training/ test/validation set splits, the dimensionality of descriptors used in relation to the number of degrees of freedom of a model, or the way selection of features is performed. while it has been recognized for some time that a larger number of descriptors increases the likelihood of chance correlations [58] , more recently a discussion of the validity of statistical significance tests, such as the f test, has appeared [59] which puts the number of features considered into relation to the significance of a model. this study cautions in agreement with earlier work that one needs to be very careful when judging the statistical significance of correlation models if feature selection is applied -and that statistically 'significant' models can hardly be 'avoided' if too large a variable pool is chosen to select features in the first place. since datasets are generally limited in size, a suitable split into training and test set(s) is crucial in order to achieve sufficient training examples on the one hand, and as high as possible a predictivity of the model on the other. often, leave-one-out cross-validation has been used to judge model performance -where the compound 'left out' was supposed to be a novel compound found for which property predictions had to be made. unfortunately this is, according to recent studies, not a suitable validation method [60, 61] . in the case of leave-one-out cross-validation, where features are selected from a wider range, the tendency exists in every case to select those features which perform best on a particular compound -thus decreasing generalizability of the model. results were summarized in a simple statement: 'beware of q 2 !', where specifically the cross-validated correlation coefficient of a leave-one-out cross-validation is alluded to. in addition, general guidelines for developing robust qsar models were developed, namely a high cross-validated correlation coefficient and a regression, which shows slope close to 1 and no significant bias. using theoretical considerations as well as empirical evaluations the question of leave-one-out vs. separate test sets was recently considered in detail [62] . performing repeated cross-validations of both types on a large qsar dataset, the conclusion was drawn that in the case of smaller datasets, separate test sets are wasteful, but in case of larger datasets (at least large three-digit numbers of data points) it is recommended. this partly contradicts the above conclusion, that separate test sets should always be used. the discrepancy was explained by the fact that in the earlier work only small separate test sets were used (containing 10 compounds), which was not able to provide a sufficiently reliable performance measure. the finding that cross-validation often overestimates model performance was corroborated in a recent related study [63] , in particular, in cases where strong model selection such as variable selection is applied. the main influence on quality overestimation was found to be a (small) dataset size; other factors are the size of the variable pool considered, the objectto-variable-ratio, the variable selection method, and the correlation structure of the underlying data matrix. while in case of conventional stepwise variable selection overconfidence is commonly encountered, as a remedy lasso (least absolute shrinking and selection operator) selection is proposed, as well as the utilization of ensemble averaging. both techniques give more reliable estimates of the quality of the developed model. given that the latter was shown to improve performance in many cases on its own the generation of reliable performance measures is an additional advantage of ensemble techniques. overfitting is a problem which describes good model performance on a training set but much worse performance on subsequent data, and thus, mediocre generalizability of the model (the model is not robust). a recent discussion of this problem, with many accessible examples, gives similar guidelines to those above, such as that leave-one-out cross-validation is not sufficient [64] . it also emphasizes the recommendation of multiple training/test set splits even in the case of very large dataset sizes and of performing cross-validation across classes of compounds in the case of close analogues (instead of molecule-by-molecule splits). in order to have some measure of overfitting, the use of 'benchmark models' such as partial least squares is recommended (depending on the particular problem) in order to determine whether there might be simpler models appropriate to the task (indicating that the more complex model overfits the data). using a toxicity dataset of phenols against tetrahymena pyriformis [65] the conclusion that q 2 is not a sufficient predictor for the applicability of a qsar model to unseen compounds is corroborated, and suggests using the rms error of prediction (rmsep) instead. this guideline is presented along with additional important points: that outliers should not necessarily be deleted since this step reduces the chemical space covered by the model, that the number of descriptors in a multivariate model needs to be chosen carefully and finally that an 'appropriate' number of dimensions is required for pls modeling. in addition, the influence of the number of variables on predictive performance for training and test sets is investigated. several recent publications have attempted to investigate what the actual scope of a qsar model is -and attempted to develop guidelines to assess the applicability of a model to a novel compound whose properties are to be predicted [66, 67] . two measures for applicability are proposed: the similarity of the novel molecule to the nearest molecule in the training set and the number of neighbors of the novel compound within the training set with a similarity greater than a certain cutoff. as expected, molecules with the highest similarity are best predicted, and this was found to be true across datasets as well as across methods. the applicability measures described above can also be used numerically to derive error bars for estimations of how likely the prediction of a specific model is within a certain error threshold. the issue of model validity was also briefly reviewed from a regulatory viewpoint [68] . in a similar vein, a 'classification approach' has been presented for determining the validity of a qsar model for predicting properties of a novel compound [69] . focusing on linear models (though the underlying concept is more generally applicable), the predictions made for compounds within the initial training set are differentiated between 'good residuals' and 'bad residuals'. using three different datasets (an artemisinin dataset as well as two boiling point datasets) machine-learning methods were employed to predict whether a novel compound belongs to the 'good' or 'bad' class of residuals, thereby making predictions as to whether its properties can be predicted -with a success rate of between 73% and 94%. a stepwise approach for determining model applicability [70] considers physicochemical properties, structural properties, a mechanistic understanding of the phenomenon and, if applicable, the reliability of simulated metabolism in a step-by-step manner. with several qsar datasets, it could be shown that for substances that are well covered by the training set improved predictions can be made for novel compounds, in agreement with the conclusions stated above. the performance of similarity searching methods varies widely, comprising both target-and ligand-based approaches. while large enrichment factors (often in the hundreds) are reported, the question arises of how much 'added value' more sophisticated methods actually provide, compared to very simple approaches, and where the gain-to-cost ratio actually shows an optimum. a recent study illustrated that simple 'atom count descriptors' (which do not capture any structural knowledge but represent a molecule by a set of integers which represent the number of atoms of each element) are able to have comparable performance to state-of-the-art fingerprints [6] . thus, when averaged over multiple target classes, the added value of virtual screening approaches is probably closer to two (compared to trivial descriptors) than in the region of often published double-digit numbers (compared to random selection). it should be added that performance of 'dumb' and more sophisticated descriptors varied widely, between virtually no difference in performance up to high single-digit performance improvements of state-of-the-art fingerprints (which are, with respect to retrieval rate and on a mddr-dataset, circular fingerprint descriptors). hts results are notorious for the amount of noise they contain and methods such as multiple screening runs are routinely applied to alleviate the problem. still, additional experiments are required. an alternative method was recently presented [71] which, applying purely computational methods, is able to predict truly active compounds with improved reliability in screenings where multiple compounds are screened per well. using scitegic circular fingerprints [72] , similarities between molecules in wells containing compounds predicted as being active (which may be true positives or, often, just noise) are calculated. the compounds most similar to active compounds are more likely to be active themselves; by predicting (across wells) those compounds which are similar to each other and at the same time are located in wells showing activity, the active compounds out of the mixtures can be estimated. this way, between 29% and 41% of the active compounds could be retrieved in the top 10% of the sorted compounds. another approach which attempts to improve knowledge derived from hts campaigns was recently proposed [73] ; the conventional selection of a fixed number of compounds showing activity in a primary screen is replaced for secondary screens ('top x approach'). alternatively, methods based on partitioning are frequently employed. in the approach presented here, an ontology-based pattern identification method is employed, which originated from bioinformatics methods (the prediction of gene function based on microarray data). taking scaffold diversity into account and also applying the 'molecular similarity principle', the overall probability of selecting active compounds from different clusters is maximized. based on earlier hts data, significant improvement of hit confirmation rates was demonstrated, compared to a conventional 'top x' approach. related work was recently also performed with a focus on scaffold clustering [74] . as discussed below, scoring functions are not yet able to predict binding affinities sufficiently well across the board of target proteins. still, the identification of active ligands was shown to be improved by a second data post-processing step. first, ligands are docked to the target. subsequently, predicted active and inactive compounds are subject to model generation via a naı¨ve bayesian model [75] based on circular fingerprints. applied to protein kinase b and protein-tyrosine phosphatase 1b, significant performance improvements could be observed in combination with dock, flexx as well as glide scores on protein-tyrosine phosphatase 1b. on the other hand, results on protein kinase b results were not improved, which was attributed to the fact that the predicted actives used to train the model were 100% false positives. understandably, performance cannot be improved if the initial enrichments are not able to identify true positive binders. more recently, another step was introduced between scoring and selecting active and inactive compounds for training the bayes classifier [76] , which is one of the available consensus scoring methods. since consensus scoring is often able to rescue docking results in cases where a specific scoring function fails, rank-by-median consensus scoring was shown to improve results for protein kinase b considerably. other consensus approaches (rank-by-mean, and rank-by-vote) did not perform as well. this was attributed to their sensitivity to cases where one of the scoring functions performs badly. (the median of a set of numbers is less sensitive to outliers than its mean.) an alternative method for post-processing docking scores is the post-dock approach whose final goal is the elimination of false-positive predictions and their discrimination from artifacts [77] . based on a ligandtarget database, derived descriptors (dock score, empirical scoring and buried solvent accessible surface area) and models from machine-learning methods were derived to identify false-positive predictions. validating the method on 44 structurally diverse targets (plus the same number of decoy complexes), 39 of 44 binding and only 2 of 44 complexes were predicted to be of true-positive nature. compared to purely docking-based methods, dock and chemscore achieve enrichments on the order of five to seven, depending upon the database used, while the method presented here claims to obtain about 19-fold enrichment. consensus prediction of docking scores is often able to improve results over single functions and multiple ways have been proposed to combine scores from different functions such as rank-by-rank, rank-by-vote or rankby-number [78] . performance improvement could not be observed in every case and a theoretical study [79] to elucidate the way in which consensus scoring improves results, concluded that this was due to the simple reason that multiple samplings of a distribution are closer to its true mean than single samplings. assumptions made by the study, such as the performance of each individual scoring function is comparable, have led to the work later being criticized [80] , and it has been concluded that consensus scoring can improve results but that it is not true in every case (as observed in practice). more recently, it was demonstrated [81] that two criteria are important if consensus scoring is to be successful: first, each individual scoring function has to be of high quality, and second, the scoring functions need to be distinctive. even if no training data are available to judge those points, rank-vs.-score plots were proposed to gauge the success of target-based virtual screening against a particular target. while consensus predictions for ligand-based virtual screening have been known for some time, a more recent study extended the descriptors employed to include structural, 2d pharmacophore and property-based fingerprints as well as bcut descriptors and 3d pharmacophores [82] . logistic regression and rank-by-sum consensus approaches were found to be most advantageous due to repeated samplings, better clustering of actives (since multiple sampling will recover more actives than inactives) and agreement of methods to predict actives but less so inactives. in addition, more stable performance across a range of targets was observed. if multiple active compounds are known in a virtual screening setting, the question arises of how to combine the retrieved lists of individual compounds. applied to different activity classes from the mdl drug data report as well as the natural products database [83] it was recently found that the rank-by-max method generally outperforms the rank-by-sum method, while concluding that the tanimoto coefficient is superior to 10 other similarity coefficients considered. as to the applicability of consensus approaches, it is found that more dissimilar activity classes profit more than more homogeneous classes, where best retrieval performance is already obtained using lower numbers of query structures (which are then already able to cover the 'activity island' inhabited by the particular class of compounds). while many applications of virtual screening tools have appeared in the literature, only some examples can be given here. a phosphodiesterase-4 (pde4) inhibitor recently has been optimized through the application of small combinatorial libraries [84] . affinity was increased by three orders of magnitude by screening only 320 compounds after prioritization by flexx docking. following the recent sars scare, a virtual screening procedure via docking (dock program) was able to find inhibitors of sars coronavirus 3c-like proteinase with binding affinities of k i ¼ 61 mm out of 40 compounds tested [85] . virtual screening based on a homology model of the neurokinin-1 (nk1) receptor led to the discovery of submicromolar ligands [86] , while even nanomolar binding compounds against checkpoint kinase 1 (chk1) could be discovered [87] by applying successive filtering for physicochemical properties, pharmacophore filters and docking stages. ligand-based pharmacophore models generated by catalyst [88] were used to discover nanomolar ligands of erg2, emopamil-binding protein (ebp), and the sigma-1 receptor (s 1 ) [89] . out of 11 compounds tested, 3 exhibited affinities of less than 60 nm. high levels of biliary elimination of a cck2 antagonist led to the quest for novel compounds, which retained activity and selectivity while improving half-life. using field points derived from xed charges [90] , novel heterocycles were proposed [91] (switching from an indole to pyrrole and imidazole series), which decreased molecular weight and polarity and achieved the desired scaffold hop. apart from this list of applications against particular targets, only two further applications shall be described here (since the field is simply too large to capture it in its entirety). first, ligand-and target-based approaches were recently compared in their abilities to identify ligands for g-protein coupled receptors [92] . evaluating docking into homology models, ligand-based pharmacophore models and feature trees, 3d similarity searches as well as models built on 2d descriptors, all ligand-based techniques were shown to outperform the docking-based approaches. however, docking also provided significant enrichment. second, the 'hts data mining and docking competition' presented its results recently [93] [94] [95] . duplicate residual activities of 50,000 compounds against escherichia coli dhfr in primary screening were released in late 2003 [96] , upon which 42 groups submitted activity predictions for a test set of the same size (but with unknown activity). approaches employed ranged from docking [97, 98] to purely ligandbased methods [99, 100] . overall, none of them was able to predict actives from the test set reliably. while this was partly due to difference in chemical composition of the training and test sets, an additional problem was posed by the test set which did not contain real 'actives' (showing proper dose-response curves in secondary assays), thus making predictions difficult. several novel clustering algorithms have been presented recently, each of which extends previous approaches in its own way. a combination of fingerprint and maximum common substructure (mcs) descriptors [101] speed up clustering (compared to purely mcs methods) enabling its application to large datasets, and the method was shown to be able to identify the most frequent scaffolds in databases, to select analogues of screening hits and to prioritize chemical vendor libraries. a modification of k-means clustering also showed a considerable speed increase to be possible when processing large libraries [102] , as demonstrated on a dataset containing about 60,000 compounds derived from the mddr. the desired speed-up was observed along with favorable enrichment of activity classes within the clusters. by introducing fuzziness into the clustering process [103] , superior results can be obtained compared to the original (non-fuzzy or 'crisp') approaches to k-means and ward clustering, depending on the particular dataset and the property one attempts to predict. fuzzy clustering assigns partial memberships to multiple classes (instead of binary values); with a log p dataset the best fuzzy parameterization was shown to clearly outperform the best crisp clustering. in addition, partial class memberships were shown to capture the 'chemical character' of a compound more satisfyingly than conventional (crisp) class assignments. while the concept of 'drug-likeness' has to be applied with care (and one needs to be aware of its limitations) it has nonetheless received considerable attention in recent years, based on datasets derived from the available chemicals directory (acd) and the world drug index (wdi). first applications employed ghose/crippen descriptors in combination with neural networks for classification, and correct classification was achieved for 83% of the acd and 77% of the wdi, respectively [104] . later, the application of svms was not able to improve overall performance significantly, but the new method was able to correctly classify compounds that were misclassified by the ann-based technique [34] . very recently a further analysis of the drug/non-drug dataset appeared, which analyzed svm performance (as well as that of other machine-learning methods) in more detail [105] . it was found that, in spite of problems with the dataset (some descriptor representations of compounds were, for example, identical in the drug and non-drug dataset) performance could be improved considerably to about 7% misclassified compounds by optimizing the kernel dimensions employed. an application using 'humanunderstandable' descriptors of drug-vs. non-drug-like properties has also been presented [106] recently, and was able to distinguish between both datasets with the most important descriptors being proper saturation level and the heteroatom-to-carbon ratio of the molecule. the concept of database comparison is also more generally applicable, as was shown recently when the question of how 'in-house like' external databases are was addressed in order to help to decide whether they should be acquired or not [107] . a number of validations of docking programs have appeared recently, and it is interesting to observe that they grow in size in every respect -including the number of docking and scoring functions considered as well as the number and diversity of ligand-target complexes employed for their evaluation. using dock, gold and glide in order to evaluate the performance of docking programs in target-based virtual screening on five targets (hiv protease, protein tyrosine phosphatase 1b, thrombin, urokinase plasminogen activator and the human homologue of the mouse double minute 2 oncoprotein), it was concluded that performance is both target-and method-dependent [108] . performance varied widely, between near-perfect behavior (for example, gold in combination with protein tyrosine phosphatase 1b) to negative enrichment (for example, gold with hiv protease). employing fred, dock and surflex, and adopting the algorithm to the particular binding pocket, it was found that target-based virtual screening is successful in some cases [109] , with surflex probably performing the best overall. investigating phosphodiesterase 4b [110] and a set of 19 known inhibitors with 1980 decoys, the scoring functions pmf, jain, plp2, ligscore2 and dockscore were compared with respect to their ability to enrich known ligands. it was found that pmf and jain showed high-enrichment factors (greater than four-fold) alone, while a rank-based consensusscoring scheme employing pmf and jain in combination with either dockscore or plp2 showed more robust results. in what is probably one of the most extensive studies yet, 14 scoring functions in combination with 800 protein-ligand complexes from the pdbbind database have been compared for evaluation [111] . the scoring functions compared were x-score and drugscore, five scoring functions implemented in sybyl (chemscore, d-, f-and g-score and pmf-score), four implemented in cerius2 (ligscore, ludi, plp and pmf) as well as two scoring functions implemented in gold (goldscore and chemscore) as well as the hint function. performance was assessed by their ability predicting affinity (k i /k d values). overall, x-score, drugscore, sybyl with chemscore and cerius2 with plp performed better than the other combinations, giving standard deviations in the range of 1.8-2.0 log units. another very comprehensive evaluation [112] employed 10 docking programs in combination with 37 scoring functions against eight proteins of seven types. three criteria were used for assessment, namely the ability to predict binding modes, to predict ligands with high affinity and to correctly rank-order ligands by affinity. while nearly all programs were able to generate crystallographic ligand-target complexes, the identification of the correct structure by the scoring function was found to be considerably more error-prone. averaged over all targets, none of the programs was able to predict more than 35% of the ligands within an rmsd of equal to or less than 2 å . while active compounds were correctly identified, activity prediction was more difficult -to the extent that 'for the eight proteins of seven evolutionarily diverse target types studied in this evaluation, no statistically significant relationship existed between docking scores and ligand affinity' [112] . similar results were obtained on five datasets (serine, aspartic and metalloproteinases, sugar-binding proteins and a 'miscellaneous' set) using the scoring functions bleep, pmf, gold and chemscore [113] , where across all complexes on average no function returned a better correlation than r 2 ¼ 0.32. interestingly, another recent study drew quite different conclusions from similar observations [114] . docking endogenous ligands into a panel of proteins it was concluded that proteins are often very promiscuous and do not interact with only a single clearly defined small molecule. while this is surely possible, given the limitations of today's scoring functions it might well be the case that predictions are just not yet good enough. while a great number of descriptors and modeling methods has been proposed until today, the recent trend toward proper model validation is very much appreciated. applications of the 'molecular similarity principle' do not yet show the power one would like them to have -and although some of their limitations are surely due to underlying principles and limitations of fundamental concepts, others will certainly be eliminated in the future. concepts and applications of molecular similarity chemical similarity searching approaches to measure chemical similarity -a review molecular similarity: a key technique in molecular informatics the art and practice of structurebased drug design: a molecular modeling perspective a discussion of measures of enrichment in virtual screening: comparing the information content of descriptors with increasing levels of sophistication in vitro and in silico affinity fingerprints: finding similarities beyond structural classes comparison of fingerprint-based methods for virtual screening using multiple bioactive reference structures pot-dmc: a virtual screening method for the identification of potent hits a 3d similarity method for scaffold hopping from the known drugs or natural ligands to new chemotypes lingo, an efficient holographic text based method to calculate biophysical properties and intermolecular similarities the reduced graph descriptor in virtual screening and data-driven clustering of high-throughput screening data small molecule shapefingerprints extracting the three-dimensional shape of live pigs using stereo photogrammetry novel receptor surface approach for 3d-qsar: the weighted probe interaction energy method calculation of intersubstituent similarity using r-group descriptors use of the r-group descriptor for alignment-free qsar fingal: a novel approach to geometric fingerprinting and a comparative study of its application to 3d-qsar modelling a computational procedure for determining energetically favorable binding sites on biologically important macromolecules grid-independent descriptors (grind): a novel class of alignment-independent three-dimensional molecular descriptors incorporating molecular shape into the alignment-free grid-independent descriptors anchor-grind: filling the gap between standard 3d qsar and the grid-independent descriptors virtual screening and scaffold hopping based on grid molecular interaction fields graph kernels for molecular structure-activity relationship analysis with support vector machines one-dimensional molecular representations and similarity calculations: methodology and validation fast small molecule similarity searching with multiple alignment profiles of molecules represented in one-dimension multiple-ligand-based virtual screening: methods and applications of the mtree approach comparison of correlation vector methods for ligand-based similarity searching fuzzy pharmacophore models from molecular alignments for correlation-vector-based virtual screening extraction and visualization of potential pharmacophore points using support vector machines: application to ligand-based virtual screening for cox-2 inhibitors biospectra analysis: model proteome characterizations for linking molecular structure and biological response can cell systems biology rescue drug discovery? identifying biologically active compound classes using phenotypic screening data and sampling statistics comparison of support vector machine and artificial neural network systems for drug/nondrug classification virtual screening of molecular databases using a support vector machine lead hopping using svm and 3d pharmacophore fingerprints random forest: a classification and regression tool for compound classification and qsar modeling boosting: an ensemble learning tool for compound classification and qsar modeling ensemble methods for classification in cheminformatics new approach by kriging models to problems in qsar a comparative study on feature selection methods for drug discovery molecular similarity searching using atom environments, information-based feature selection, and a naive bayesian classifier similarity searching of chemical databases using atom environment descriptors (molprint 2d): evaluation of performance evaluation of mutual information and genetic programming for feature selection in qsar nonlinear prediction of quantitative structure-activity relationships nonlinear quantitative structure-activity relationship for the inhibition of dihydrofolate reductase by pyrimidines novel variable selection quantitative structure-property relationship approach based on the k-nearest-neighbor principle development and validation of k-nearest-neighbor qspr models of metabolic stability of drug candidates three-dimensional qsar using the knearest neighbor method and its interpretation application of non-parametric regression to quantitative structure-activity relationships robust qsar models using bayesian regularized neural networks predictive bayesian neural network models of mhc class ii peptide binding an in silico approach for screening flavonoids as p-glycoprotein inhibitors based on a bayesian-regularized neural network combinatorial preferences affect molecular similarity/diversity calculations using binary fingerprints and tanimoto coefficients analysis and display of the size dependence of chemical similarity coefficients comparison of three holographic fingerprint descriptors and their binary counterparts enhancing the effectiveness of similarity-based virtual screening using nearest-neighbor information chance factors in studies of quantitative structureactivity relationships judging the significance of multiple linear regression models rational selection of training and test sets for the development of validated qsar models beware of q2! assessing model fit by cross-validation chance correlation in variable subset regression: influence of the objective function, the selection mechanism, and ensemble averaging the problem of overfitting the better predictive model: high q(2) for the training set or low root mean square error of prediction for the test set? assessing the reliability of a qsar model's predictions similarity to molecules in the training set is a good discriminator for prediction accuracy in qsar improving opportunities for regulatory acceptance of qsars: the importance of model domain, uncertainty, validity and predictability determining the validity of a qsar model -a classification approach a stepwise approach for defining the applicability domain of sar and qsar models prioritization of high throughput screening data of compound mixtures using molecular similarity novel statistical approach for primary high-throughput screening hit selection hiers: hierarchical scaffold clustering using topological chemical graphs finding more needles in the haystack: a simple and efficient method for improving high-throughput docking results combination of a naive bayes classifier with consensus scoring improves enrichment of high-throughput docking results postdock: a structural, empirical approach to scoring protein ligand complexes consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins how does consensus scoring work for virtual library screening? an idealized computer experiment virtual screening using protein-ligand docking: avoiding artificial enrichment consensus scoring criteria for improving enrichment in virtual screening the use of consensus scoring in ligand-based virtual screening enhancing the effectiveness of virtual screening by fusing nearest neighbor lists: a comparison of similarity coefficients design of small-sized libraries by combinatorial assembly of linkers and functional groups to a given scaffold: application to the structure-based optimization of a phosphodiesterase 4 inhibitor virtual screening of novel noncovalent inhibitors for sars-cov 3c-like proteinase successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model identification of compounds with nanomolar binding affinity for checkpoint kinase-1 using knowledge-based virtual screening pharmacophore modeling and three-dimensional database searching for drug design using catalyst discovery of high-affinity ligands of sigma1 receptor, erg2, and emopamil binding protein by pharmacophore modeling and virtual screening scaffold hopping with molecular field points: identification of a cholecystokinin-2 (cck(2)) receptor pharmacophore and its use in the design of a prototypical series of pyrrole-and imidazole-based cck(2) antagonists virtual screening of biogenic amine-binding g-protein coupled receptors: comparative evaluation of protein-and ligand-based virtual screening protocols mcmaster university data-mining and docking competition -computational models on the catwalk experimental screening of dihydrofolate reductase yields a ''test set'' of 50,000 small molecules for a computational data-mining and docking competition evaluating the highthroughput screening computations high throughput screening identifies novel inhibitors of e. coli dihydrofolate reductase that are competitive with dihydrofolate here be dragons: docking and screening in an uncharted region of chemical space virtual ligand screening against e. coli dihydrofolate reductase: improving docking enrichment using physics-based methods using extended-connectivity fingerprints with laplacian-modified bayesian analysis in high-throughput screening follow-up screening for dihydrofolate reductase inhibitors using molprint2d, a fast fragment-based method employing the naive bayesian classifier: limitations of the descriptor and the importance of balanced chemistry in training and test sets database clustering with a combination of fingerprint and maximum common substructure methods a hierarchical clustering approach for large compound libraries clustering files of chemical structures using the fuzzy k-means clustering method a scoring scheme for discriminating between drugs and nondrugs classifying 'drug-likeness' with kernel-based learning methods a new rapid and effective chemistry space filter in recognizing a druglike database in-house likeness'': comparison of large compound collections using artificial neural networks comparison of automated docking programs as virtual screening tools fast structure-based virtual ligand screening combining fred, dock, and surflex retrospective docking study of pde4b ligands and an analysis of the behavior of selected scoring functions an extensive test of 14 scoring functions using the pdb bind refined set of 800 protein-ligand complexes a critical assessment of docking programs and scoring functions predicting protein-ligand binding affinities: a low scoring game? ligand selectivity and competition between enzymes in silico key: cord-317628-1inxq7t5 authors: cuccarese, michael f.; earnshaw, berton a.; heiser, katie; fogelson, ben; davis, chadwick t.; mclean, peter f.; gordon, hannah b.; skelly, kathleen-rose; weathersby, fiona l.; rodic, vlad; quigley, ian k.; pastuzyn, elissa d.; mendivil, brandon m.; lazar, nathan h.; brooks, carl a.; carpenter, joseph; probst, brandon l.; jacobson, pamela; glazier, seth w.; ford, jes; jensen, james d.; campbell, nicholas d.; statnick, michael a.; low, adeline s.; thomas, kirk r.; carpenter, anne e.; hegde, sharath s.; alfa, ronald w.; victors, mason l.; haque, imran s.; chong, yolanda t.; gibson, christopher c. title: functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and covid-19 drug discovery date: 2020-08-14 journal: biorxiv doi: 10.1101/2020.08.02.233064 sha: doc_id: 317628 cord_uid: 1inxq7t5 development of accurate disease models and discovery of immune-modulating drugs is challenged by the immune system’s highly interconnected and context-dependent nature. here we apply deep-learning-driven analysis of cellular morphology to develop a scalable “phenomics” platform and demonstrate its ability to identify dose-dependent, high-dimensional relationships among and between immunomodulators, toxins, pathogens, genetic perturbations, and small and large molecules at scale. high-throughput screening on this platform demonstrates rapid identification and triage of hits for tgf-βand tnf-α-driven phenotypes. we deploy the platform to develop phenotypic models of active sars-cov-2 infection and of covid-19-associated cytokine storm, surfacing compounds with demonstrated clinical benefit and identifying several new candidates for drug repurposing. the presented library of images, deep learning features, and compound screening data from immune profiling and covid-19 screens serves as a deep resource for immune biology and cellular-model drug discovery with immediate impact on the covid-19 pandemic. acting through autocrine, paracrine, and endocrine mechanisms, endogenous immune stimuli maintain homeostasis and signal response to invasion, injury, or malignancy. immune dysregulation underlies a broad set of human diseases including inflammation 1 , autoimmune disease 2 , neuroinflammation 3 , neurodegenerative disease 4 , secondary effects of traumatic brain injury 5 , cancer 6, 7 , infection [8] [9] [10] , and cytokine storm 11, 12 . improvements in the understanding of how immune stimuli amplify or suppress the immune system, trigger new cell fate differentiation, and remodel tissue have resulted in the discovery of a wide range of successful therapeutics 13 , as demonstrated by the anti-tnf antibody adalimumab (humira), noted both for its discovery 14 and its application in rheumatic disease 15 . however, the immune system is vastly complex and dependent on cell type and context; reliably intervening in such a highly interdependent process is a formidable drug discovery challenge. with few exceptions 16 , intercellular immune signaling has been explored by studying specific factors in isolation. although cellular response to individual immune stimuli can be effectively profiled by high-dimensional molecular methods such as rnaseq 17 , proteomics 18 , and chipseq 19 , these technologies lack the speed and cost-effectiveness for systems-level analysis of large panels of immune stimuli and screening libraries across myriad inflammatory models. by contrast, image-based methods have demonstrated utility at many stages of drug discovery 20 , including compound profiling 21 , prediction of assay performance 22 , clustering by mechanism of action 23, 24 , and toxicology predictions 25 . here we present phenomics, the analysis of fluorescence microscopy images as a scalable approach to examine cellular response to a wide range of perturbations. deep-learning algorithms extract high-dimensional and dose-dependent fingerprints of cellular morphological changes, or 'phenoprints', from images to support a variety of downstream applications. these phenoprints detect subtle morphological changes far beyond human ability, and a standardized assay pipeline allows the phenoprints of millions of cellular samples to be related across time and experimental conditions. in this work, we first demonstrate the ability to use images alone to accurately quantify and relate hundreds of immune stimuli. we then show how profiles resulting from these perturbations can be employed in drug screening, particularly highlighting the utility of a relatable dataset to accurately predict the mechanism of action for unknown compounds. we used these capabilities to rapidly develop high-throughput-ready disease models for both sars-cov-2 viral infection and the resulting cytokine storm, and immediately launched large-scale drug screens that recapitulated known effective and ineffective therapies and, more importantly, identified several new potential treatments for both sars-cov-2 infection and covid-19-associated cytokine storm. in order to discern the breadth of immune response that might be captured in a single profiling assay for various applications (fig. 1) , we treated endothelial cells, fibroblasts, peripheral blood mononuclear cells (pbmc), and macrophages with diverse immune stimuli (table s1 ), crispr gene editing reagents, antibodies, and small molecules. we used a single fully automated experimental workflow in which cells are plated, treated, labeled with a panel of fluorescent stains, and imaged with high-throughput fluorescence microscopy 26 . vector representations of more than one million multi-channel fluorescence microscopy images analyzed in this manuscript were generated using a proprietary analytics workflow based on an extension of a densenet-161 neural network 27 various cell types (top left) are treated with a range of biological perturbants and treatments (bottom left), including recombinant proteins, antibodies, crispr-based genetic modifications, and small molecules. high-throughput fluorescence microscopy (middle-top) and deep learningenabled image featurization generates high-dimensional phenoprints that are used for interrogating a range of experimental questions (middle-top and middle bottom). this approach suits a suite of applications (right) with a single workflow and on a single platform. specific applications demonstrated in this paper: systems biology exploration of immune relationships, adaptation across diverse disease models, compound screening, and mechanism prediction. phenoprints are high-dimensional vector representations (embeddings) of cellular morphology derived from fluorescent images resulting from treatment with a biological perturbation. to provide landmark phenoprints across a diverse range of immune function, 446 stimuli were added to four different primary human cell types at a range of biologically relevant concentrations and compared to untreated cells and adjacent concentrations. for example, cells treated with tumor necrosis factor alpha (tnf-ɑ), the anti-pd-1 antibody nivolumab, transforming growth factor beta (tgf-β), or interferon alpha (ifn-ɑ) each displayed concentration-dependent phenotype strength measured as intra-replicate consistency as well as increasing convergence of the phenotype ( fig. 2a) . in total, 131 of 446 stimuli produced highly consistent, dose-dependent phenoprints in at least one cell type (fig. 2b) , with representation from cytokines, growth factors, chemokines, antibodies, microbial toxins, and others (fig. s2) . a subset of factors, such as innate stimulants like lps, interferons, and microbial toxins (e.g. enterotoxins) produced strong dose-dependent phenoprints in all cell types tested. by contrast, other stimuli produced phenoprints only in the expected cell type: vascular endothelial growth factor (vegf) in endothelial cells, fibroblast growth factor (fgf) family in fibroblasts, innate stimulants and interferons in macrophages and pbmcs, and tgf-β family in both macrophages and fibroblasts 28, 29 . example phenoprints for indicated immune stimuli and cells. the intra-dose cosine similarity among well replicates (n=6) for each perturbation dose, demonstrating perturbation consistency(red), and average pairwise cosine similarity between each perturbation dose (blue) and the highest dose, demonstrating phenotypic convergence. b. statistically significant phenoprints induced by immune stimuli (rectangles, colored by class as in fig. 2c ) are connected by edges to the cell type (circles) in which the phenoprint was observed. thicker edges reflect stronger interactions. c. classes of all immune stimuli in immune perturbant library. to test whether phenoprints could capture known functional relationships, we applied hierarchical clustering and confirmed appropriate grouping for factors with similar function and/or structural homology, often in a cell-type-specific manner (fig. 3a , table s2 ). for example, related factors il-4 and il-13, and all type-i ifns clustered in unique groups in any cell type in which a phenoprint was observed. chemokines (such as ccl-4 and mcp-2) and innate stimulants (such as lps and flagellin) are also readily grouped with other stimulants, but only in their appropriate cell types: macrophages and pbmcs (fig. 3a , green/purple edges). growth factors cluster in fibroblasts, consistent with their sensitivity to environmental cues for matrix remodeling and organ function, and ability to differentiate into other states such as myofibroblasts under inflammatory pressure (fig. 3a , blue edges) 30, 31 . phenoprints also captured associations between initial stimuli and overlapping secondary effects, as in the case of clusters for activators of nfkb (tnf-ɑ, il1β), interferons induced by irf3, and tlr3 ligands (poly (i:c) variants) in endothelial cells (fig. s1c ). double-stranded rna motifs such as poly (i:c) are recognized by tlr3, which signals through both nfkb and irf3 pathways 32 . fine-grained distinctions were visible in clustered phenoprints. for example, the tgf-β superfamily (tgf-β proteins, growth differentiation factors, activins, and müllerian inhibiting substance) formed a distinct cluster from other growth factor families, which included the epidermal growth factor (egf) family (egfs, tgf-ɑ, betacellulin, heparin-binding egf), platelet-derived growth factor (pdgf) family, fgf family and the insulin-like growth factor (igf) family (fig. s1d ). within these larger groupings, nearly structurally-identical tgf-β isoforms tgf-β1, tgf-β2, and tgf-β3 clustered more tightly than do other members of the tgf-β superfamily 33 . pathogen-derived toxins also revealed expected relationships. c. difficile toxins a and b produced cell-type-specific similarities with members of the human immune stimulant panel (fig. s1e ). these toxin phenoprints were similar to those of interferon proteins in macrophages, as well as to il-6 superfamily members and nfkb-mediated stimulants in huvec. this aligns with the known dual pathology of c. diff. infection, involving both inflammation through macrophage activation 34 and direct gut permeabilization effects 35 . high-dimensional morphological relationships in four cell contexts. immune stimuli are arranged based on hierarchical clustering of all factors in all cell types; only factors with a statistically strong phenoprint in at least one cell type are shown. lines between nodes are filtered for similarity and colored based on the cell type in which the association was observed. b. hierarchical clustering of phenoprints resulting from huvec treated with an annotated compound library. highlighted clusters pertain to small molecule inhibitors of agc family kinases: akt (blue), rock (orange), mek (orange)/erk (blue), and mtor inhibitors: rapalogues (blue), pi3k inhibitors (orange) and mtorc1/2 (green). c. process for reduction of high-dimensional data into two dimensions. d. projections of compound response in the context of on-and off-perturbation vectors and logistical regression for on-perturbation values by drug concentration for tnf-ɑ, il-6 (receptor chimera) ligands, and socs3 knockout in huvec (mean, n=6). contour lines depict perturbation state (orange) and target state (blue) replicates at the 50, 90, and 99th percentiles, respectively. confidence in phenotypes resulting not only from the immune perturbation but those resulting from compounds in a screening library is critical for evaluation of high-throughput screens. to this end, we generated individual compound phenoprints from an annotated bioactive library, which indeed clustered by mechanism of action ( fig. 3b ). for example, inhibitors of agc kinases akt and rock clustered within a super-group as did inhibitors of mek and erk, which is expected based on protein homology and sequential signaling, respectively. more granular sub-clusters were also observed; for example, a diverse group of inhibitors of mtor can be sub-classified into rapalogues, pi3k inhibitors and mtorc1/2 inhibitors. we next tested whether phenomics could uncover compounds that rescue the complex high-dimensional effect of various immune perturbants. we applied several disease-related phenoprints identified above and defined a corresponding perturbation vector in high-dimensional embedding space. compounds that rescued on-perturbation morphology with minimal deviation in off-perturbation morphology were selected as screen hits, as they offer a potential combination of efficacy and specificity (fig. s3a, b) . we first validated the strategy using approved anti-tnf antibodies in the context of a tnf-ɑ phenoprint (fig. 3d ). all tested antibodies rescued the phenoprint induced by tnf-ɑ (perturbed state) back towards the unperturbed phenoprint (target state), but infliximab was less potent relative to adalimumab and golimumab. it is known that infliximab, adalimumab, and golimumab all have similar affinities for tnfɑ 36 , but only adalimumab and golimumab are effective in the absence of concomitant immunosuppression 37 . although many factors can affect antibody performance, this finding suggests phenomics can differentiate subtleties of antibody efficacy beyond affinity for the ligand. next, we selected a set of clinical-stage and approved jak inhibitors to test the ability of phenomics to model compound rescue of il-6 signaling when cells are activated by the ligand or when disrupted by genetic modification. tofacitinib, baricitinib, ruxolitinib, and oclacitinib reverse the phenoprint of the il-6 receptor chimera in huvec in a dose-dependent manner. in addition to identifying compounds that block receptor-mediated signaling, we demonstrated compound-induced rescue of a phenotype resulting from knockout of an intracellular mediator. here, knockout of socs3 using crispr/cas9 leads to hyperactivation of jak signalling 38 , resulting in a phenoprint that is rescued by the same set of compounds ( fig. 3d ; comparison to inactives: fig. s3c ). well-designed phenotypic screening efforts benefit from being a more proximal model of the disease, and since they are not limited to pre-defined targets, offer the ability to uncover novel therapeutic pathways 39 . however, this carries the risk of investing time and resources into compounds that are later discovered to interact with known or disadvantageous pathways. to address this challenge, we leveraged the relatability of our nce screening and annotated compound datasets to identify novel therapeutic opportunities while rapidly deprioritizing high-risk mechanistic space, as demonstrated in a screen against the tgf-β-induced phenoprint. tgf-β, signaling through the receptor alk5, is recognized as a primary driver of fibrosis in debilitating diseases such as idiopathic pulmonary fibrosis and renal fibrosis, as well as a significant contributor to immune exclusion in the tumor microenvironment 29 . the diversity and consistency of phenoprints recapitulating known biology suggested phenomics might discover new chemical entities (nce) and predict mechanism of action. we therefore screened 90,000 diverse chemical starting points (fig. s4a ) against the tgf-β phenoprint (fig. 4a) . a novel compound of interest (rec-0104937) completely reversed the phenoprint at low micromolar concentrations. further, this same compound rescued an orthogonal functional validation assay, mitigating tgf-βinduced collagen deposition with an ec50 of 0.763 µm (fig. 4b) . we then compared the rec-0104937 phenoprint to reference phenoprints derived from a set of 6,000 diverse, well-annotated small molecules; several known alk5 inhibitors were highly similar (fig. 4c) . we experimentally validated the accuracy of these predictions in gold standard assays of alk5 activity: rec-0104937 inhibited cellular p-smad activity and cell-free biochemical alk5 activity at 0.585 µm and 0.725 µm respectively (fig. s4d, fig. 4d ) 40, 41 . because the advancement of tgf-β receptor inhibitors has been hampered by cardiac toxicity 42 , and because our research goals are to identify compounds acting against novel pathways, we rapidly deprioritized this compound in favor of others based on the primary screening data. overactive tnf-ɑ signaling is a major driver of inflammation in inflammatory and autoimmune diseases including alzheimer's disease 43 , multiple sclerosis 44 , and traumatic brain injury 45 , and significant benefit has been achieved with monoclonal antibody intervention 46 . we therefore sought to rescue the tnf-ɑ phenoprint with an intervention that not only targets a novel mechanism of action, but also benefits from advantages of small molecules over existing antibodies, such as oral availability and increased central nervous system penetration. we found 2,073 compounds that statistically altered the tnf-ɑ phenoprint at a single dose in a 90,000-compound primary screen. we tested these for dose response and selected a subset for validation. although suppression of tnf-ɑ signaling through nfkb blockade is a plausible anti-inflammatory strategy, reduction of tnf-ɑ signaling via global inhibition of nfkb leads to a challenging safety profile 47 . two molecules (rec-0150357 and rec-0082469) rescued the tnf-ɑ-induced phenoprint (fig. 4e ) and prevented secretion of il-6, a marker of tnf-ɑ stimulation (fig. 4f ) while preserving nfkb activation (fig. s4e) . by comparing the phenoprint of rec-0150357 to phenomics data from annotated compounds in prior experiments, we found strong similarity between the phenoprints of rec-0150357 and rho kinase inhibitors (fig. 4g ). this finding was corroborated by kinase profiling, revealing rock1 and rock2 inhibition at 0.097 and 0.066 µm, respectively (fig. s4f ). during intellectual property exploration, the target was further confirmed; the scaffold was previously evaluated as a rock inhibitor 48 . kinases that were inhibited to a lesser degree were cdk7 and dyrk1b (fig. s4f ). the effect of rock2 inhibition on tnf-ɑ-induced inflammation is documented 51 and similar compounds are in active development for autoimmune disease 49, 50 . therefore, we deprioritized the compound using high-dimensional primary screening data in favor of another compound, rec-0082469. the alternative scaffold also reduced tnf-ɑ-induced il-6 release ( fig. 4f ) but did not phenotypically cluster with any of the mechanistic classes annotated in our libraries, thus enabling informed prioritization of the compound for further study. in a >400 kinase biochemical screen, the compound showed no significant inhibition of any kinase at 1 µm. to investigate the potential benefit of rec-0082469 in neuroinflammation, we explored the role of the molecule to suppress microglial activation in vitro. using an immunofluorescence stain for the microglial activation marker iba-1 51 we confirmed that rec-0082469 reduced the activation of bv-2 mouse microglia ( fig. 4h ; example images fig. s4g ). given the potential of rec-0082469 to operate via a novel mechanism of action (moa) we initiated a hit optimization effort initially focused on enhancing the series potency (rec-0082469 ec50 > 1 um). we used phenomics as to assess our efforts to optimize potency while maintaining or improving efficacy against an unknown target; we succeeded in improving potency by more than ten-fold (rec-0648455 ec50 = 71 nm) and improving the on-perturbation score to 0.05 (fig. 4i ). the ongoing covid-19 pandemic presents an urgent need for quick and adaptable drug discovery in the context of a complex and poorly understood disease. we leveraged our phenomics platform to screen for approved and reference (e.g., development stage antiviral) compounds that could address two key components of covid-19 disease progression: direct effects of viral infection and the damaging effects of an unresolved inflammatory response, or cytokine storm. 1,670 and 2,913 compounds were applied to cells in the infection and cytokine storm modes, respectively, and compound rescue was evaluated with the same processing pipeline described above. many features of terminal covid-19 are the result of inflammatory pressure on endothelial cells, manifesting as barrier disruption, lymphocyte recruitment, induction of blood coagulation, and acute respiratory distress syndrome (ards) 52 . we modeled the cytokine storm associated with late-stage covid-19 in endothelial cells by applying cocktails of circulating proteins that mirror those from severe covid-19 53 patients (perturbed state) as well as healthy control patients (target state) (table s3 , fig. s5a ). we hypothesized that rescue of the perturbed state toward the target state would reveal anti-inflammatory compounds specifically relevant to the covid-19-associated cytokine storm. following identification of hit compounds, electric cell-substrate impedance sensing (ecis) was employed to confirm the activity of the aforementioned compounds in an orthogonal functional model of vascular integrity challenged with the same cytokine cocktails. presently, jak inhibitors have shown benefit in one nonrandomized trial 54 and represent one of the most common mechanisms being evaluated among hundreds of clinical trials active for covid-19. in our screen against the cytokine storm phenotype, jak inhibitors were capable of potent rescue of the severe cytokine storm phenoprint, confirming strong potential for this mechanism's efficacy in the context of a complex immune cascade (fig. 5a) . we also identified rescue by compounds in three classes of inhibitors outside of the jak/stat pathway that have been less deeply explored in the context of covid-19, including inhibitors of syk, pi3k and c-met. compounds were then applied to cells with the same inflammatory cocktail and evaluated with ecis ( fig. s5a ) to inform on benefit to vascular integrity. rescue of this orthogonal functional assay was observed for each mechanism identified in the high-dimensional assay (fig. 4g, fig. s5b -c). we next developed a model of sars-cov-2 infection to screen for repurposable compounds acting directly against viral targets or on host pathways. to define the model, we evaluated the effect of sars-cov-2 infection in multiple cell types, of which three resulted in robust phenoprints as compared to either mock infected or inactivated virus control populations: calu3 (a lung adenocarcinoma line), vero (an immortalized interferondeficient african green monkey kidney line 55 ), and primary human renal cortical epithelium (hrce) (fig. 5c, fig. s6d ). we confirmed active infection with sars-cov-2 nucleocapsid antibody staining and quantification of productive viral replication ( fig. s6a-c) . we reasoned that a primary human cell type would be most directly translatable 56 to human pathology, especially from tissues demonstrated to be directly infected by sars-cov-2 57 , and thus conducted a screen of 1660 compounds against the hrce phenotype, while testing a limited subset of those compounds in vero and calu3 cells. the majority of compounds currently under evaluation 58 in human clinical trials for covid-19 showed no or weak efficacy in the hrce model 56 . however, in these screens remdesivir and its metabolite, gs-441524, demonstrated strong efficacy and aligned with potency described in the literature (ec50 of 100 nm and 2 μm, respectively) ( fig. 5d) 59, 60 . remdesivir is a nucleoside analog that directly interferes with the viral-rna-dependent rna polymerase to inhibit viral replication and, importantly, successfully reduced recovery time for treated patients in clinical trials 61 announced after our data and analysis was publicly released. further illustrating the predictive capacity of the model, two other antivirals, lopinavir and ritonavir were not found to be efficacious and were later discontinued in clinical testing for covid-19 62 . additionally, aloxistatin (e64d), an irreversible cysteine protease inhibitor initially developed for muscular dystrophy 63 , also demonstrated suppression of the viral phenoprint in hrces (ec50 of 40 nm). recent studies have confirmed that cathepsin l, a cysteine protease, is required for sars-cov-2 entry in some cell types, and aloxistatin treatment significantly reduced entry of sars-cov-2 pseudovirions 64,65 . we then tested a subset of these antiviral compounds in additional cell types, vero and calu3, and found aloxistatin did not rescue in these models. however, another protease inhibitor, camostat mesilate, was efficacious in the calu3 model (ec50 of 260 nm), but not the vero or hrce models (fig. 5d-f, fig. s6e ). camostat inhibits tmprss2, which was recently shown to be required for sars-cov-2 entry in human airway cells 66 . similar to findings in recent clinical trials 67,68 , we found chloroquine and hydroxychloroquine to have no benefit in the hrce or calu3 models; however, they showed modest benefit in vero cells (ec50 of 730 nm and 1.65 µm, respectively) with very high offperturbation activity (fig. 5d-f) . overall, compound efficacy in human cell types was poorly recapitulated in vero cells (fig. 5d-f, fig. s6f ). taken together these findings suggest that sars-cov-2 entry protease inhibitor activity varies across cell type and species; however, remdesivir and gs-441524 show strong rescue of the viral phenoprint in all cell types tested. we identified jak inhibitors ruxolitinib and baricitinib as efficacious in both viral and cytokine storm models (fig. 5a, 5i, fig. s6g ). however, we found that high concentrations of these compounds led to increased infection in hrce cells (fig. s6h) . suppression of interferon production is a known component of sars-cov-2 infection at a low multiplicity of infection 69 . it is unclear however, what effect additional interferon suppression would have in vivo, especially at higher viral loads, warranting investigation into alternative mechanisms of cytokine storm suppression, such as pi3k or c-met inhibition. notably, bortezomib exhibited poor performance in both assay modes, is reported to impair endothelial cells in inflammatory contexts 70 , and also enhances susceptibility to viral infection 71 , particularly coronaviruses 72 . biology is massively complex and highly networked, but the tools to explore and discover novel biology and develop medicines have until recently relied on simple, univariate measurement. the genomic revolution yielded a taste of what is possible if high-dimensional biology can be scaled by massively increasing the rate of understanding of the role of thousands of genes in human biology and disease. following this advancement, several new high-dimensional approaches have been developed to add clarity to complex functional relationships and discover new therapeutics, but these are hindered from highthroughput screening application by engineering and cost bottlenecks. we present here early data from our experience using phenomics (fig. 1) as one strategy to accelerate drug discovery. we first established that diverse immune biology and pharmacology can be detected and discriminated using phenomics (fig. 2) . these data also reveal that phenomics is not simply a classification technology: deep quantification of rich, multi-parametric signal and assessment of dose response is achievable, enabling comparisons and clusterings of diverse biological perturbants alone, or in combination across diverse cell types (fig. 3) . diving more deeply on just two of the 131 immune phenoprints we uncovered that are suitable for drug screening, we explored 90,000 new chemical entity starting points in the context of tgf-β-and tnf-ɑ-induced phenoprints (fig. 4) . among hits in each context, prediction of alk5 and rock inhibition allowed us to rapidly shift resources to higher priority hits. in particular, we focused our efforts on a suppressor of the tnf-ɑ phenoprint with a high potential to potentially be active against a novel, but as of yet unknown, target. further, we drove medicinal chemistry work against this unknown target(s) using phenomics, demonstrating a 10-fold increase in potency while also increasing the magnitude of rescue. the application of phenomics can be extended to more complex disease-causing perturbations as well: the platform was rapidly adapted for the characterization and exploration of actionable therapies in the context of a novel and poorly understood disease, covid-19. within 28 days of initiating the project, we identified hits through high throughput chemical screens against covid-19 cytokine storm and sars-cov-2 infection in the relevant tissue types, without the need to develop cytokine-or virus-specific reagents and assays. we demonstrated that a handful of drugs currently in clinical trials strongly modulate the infection model (e.g. remdesivir), the cytokine storm model (pi3k inhibitors) or both (jak inhibitors), prior to their clinical trial results becoming available. conventional antiviral research relies heavily on univariate assays that measure attributes like cell death or expression level of one protein. using a single platform, we found not only conventional antivirals, but also compounds with unconventional effects on disease-associated host pathways such as inflammation. in the sars-cov-2 model remdesivir and its analog, gs-441524 demonstrated efficacy in all cell models tested. unfortunately, remdesivir is dosed via an intravenous route, typically in an inpatient setting and a time at which cytokine storm may be primarily responsible for the pathology (wherein remdesivir had no unexpected efficacy in our cytokine storm model). sars-cov-2 is able to use a variety of receptors to facilitate cell entry, with receptor specificity by cell type apparent in our data: aloxistatin (e64d), inhibiting the cathespinmediated entry pathway, and camostat, inhibiting the tmprss2-mediated pathway, each demonstrated strong response in hrce and calu3 cells respectively. nevertheless, pseudovirus entry assays 66 have shown that even in cells with both pathways active, modulating a single pathway still quantitatively reduces viral infection load. further study of the proportional activity of each pathway in relevant human tissues may be warranted. as aloxistatin is orally bioavailable, simply and inexpensively synthesized, and has a relatively strong safety profile based on chronic treatment of muscular dystrophy patients in a phase 3 trial, it deserves further study for covid-19, with the expectation that early treatment in the course of infection may be most efficacious. in our model of more advanced covid-19 symptoms driven by cytokine storm, jak inhibitors were noteworthy rescuers of the inflammatory phenoprint and moderate rescuers of the viral phenoprint at low concentrations. due to oral bioavailability and the safety profile of acute treatment they are excellent candidates for repurposing. however, we observed that jak inhibitors enhanced cellular infection of sars-cov-2 at higher concentrations, suggesting an effect on interferon signaling, a possible clinical liability that should be closely monitored during trials. supporting this finding and underlining the importance of identifying diverse options to address cytokine storm, jak inhibitors are known to increase the prevalence or severity of other viral infections including herpes zoster, jc virus, and hepatitis b [73] [74] [75] . this study also identified alternative mechanisms of action which have been much less deeply considered in the context of covid-19, such as certain syk inhibitors, c-met inhibitors and pi3k inhibitors. such molecules could be critical additions to remdesivir therapy in severe patients. this work and the recent success of dexamethasone in clinical trials for covid-19 also identified a key limitation of our current phenomics approach: when studying a cell type in isolation, phenomics surfaces compounds that act via cellautonomous mechanisms 76 . compounds that intervene in multicellular processes might be revealed by development of coculture models. taken together, our results demonstrate that systems-level modeling and drug discovery is achievable using a single phenomics platform. first, this approach simplifies and extends the ability to work across many disease models rapidly because assay development work for any new model is minimized. second, this work partially overcomes a historical limitation of phenotypic screening, predicting mechanism of action, by relating the high-dimensional phenoprint of hit compounds to those of reference molecules. finally, we show the potential of this platform in optimizing nce compounds through medicinal chemistry in a high-dimensional, target-agnostic manner. unlike other high-dimensional approaches, the relatively inexpensive nature of these image-based assays allows them to be scaled to levels of throughput comparable to more traditional lowdimensional screening modalities. in the hopes that it will be valuable to others, we have made images and embeddings from huvec treated with the immune perturbant library, and from our covid-19 primary screens (both infection and cytokine storm) available online (including raw image data, metadata, and deep learning embeddings from images) at rxrx.ai/rxrx2 and rxrx.ai/rxrx19, respectively. beyond the applications described here, the modular nature of this phenomics platform enables rapid adaptation to different libraries of immune stimulants, antibodies, or other large molecules, and incorporation of additional cellular contexts like co-culture models. in future work, comparisons of hits to phenoprints associated with knockout of each gene in the genome (achieved by arrayed whole-genome crispr knockout) may further expand our ability to predict mechanisms beyond those represented within our annotated small molecule library, bridging a key gap in phenotypic screening. critically, these data can be related over time and across disparate research programs-supporting the creation of large biological image datasets for deep-learning applications 77,78 that will accelerate drug discovery and yield functional maps of human cellular biology. human umbilical vein endothelial cells (lonza, c2519a) were cultured according to manufacturer's recommendations in egm2 (lonza, cc-3162). nhlf: normal human lung fibroblasts (lonza, cc-2512) were cultured according to manufacturer's recommendations in fgm2 (lonza: cc-3131, 4126) and used at passage four for all assays. pbmc: peripheral blood mononuclear cells (pbmcs), from healthy donors, were prepared from fresh (< 24 hours old) leukopaks (stemcell technologies inc., catalog # 70500). following 3 120 rcf washes (brake off) for platelet removal, the samples were processed by easyseptm rbc depletion reagent in accordance to manufacturer's instructions (stemcell technologies inc., catalog #18170). following isolation, pbmcs were pelleted (300 rcf) and resuspended in cryopreservation medium (cryostor® cs10 freeze media, biolifesolutions inc., part #: 210102) for long term storage. macrophage: macrophages were derived from either cryopreserved or freshly isolated pbmcs. monocytes were enriched by plastic adherence, first seeding pbmcs in serum free rpmi-1640 medium followed by 1.5 hours of incubation and washed 2x with pbs. cells were incubated for 3 d in complete medium (rpmi+ 10% heat inactivated fbs, 25 ng/ml m-csf, 10 ng/ml il-10). media was replenished after 3 d with one third of the conditioned medium and ⅔ fresh complete medium. monocyte derived macrophages (mdms) were harvested from each vessel after an additional 3 d using accutase following manufacturer's instructions (thermo -a1110501). mdms were pelleted (300 rcf) and resuspended in cryo-preservation medium. hrce: primary human renal cortical epithelial cells lonza (cc-2554) were propagated at 37°c with 5% co2 in epicm, (sciencell # 4101) supplemented with epithelial cell growth supplement (epicgs, sciencell #4152). vero: an immortalized african green monkey kidney (atcc ccl-81) were propagated at 37°c with 5% co2 in eagle's minimum essential medium (emem) supplemented with 10% fbs. calu3: human lung adenocarcinoma line (atcc htb-55) were propagated at 37°c with 5% co2 in emem supplemented with with 10% fbs. bv-2: murine microglial cells (iclc atl03001, ospedale policlinico san martino) were propagated at 37°c with 5% co2 in rpmi media + 10% heat inactivated fbs. immune stimuli (table s1 ) were solubilized in sterile phosphate buffered saline (pbs) containing 0.1% bsa (sigma cat.# a1595-50ml) to make stock solutions of .04 mg/ml in echoqualified 384w low-dead volume source plates. source plates were stored at -80°c until use. cells were seeded into 1536-well microplates (greiner, 789866) via multidrop (thermo fisher) and incubated at 37c in 5% co2 for the duration of the experiment. immune stimuli or virus were added 24 hours post-seeding (huvec, macrophage, fibroblast) or 1 h (pbmc). treatments were randomized across treatment plates with a 6-log range of immune stimuli (typically 0.001-100 ng/ml) at 6 replicates each with acoustic transfer (echo 555, labcyte) and incubated 37°c for 24 or (complete immune stimuli panel) or 48 h (for pbmc with pembrolizumab or nivolumab). active sars-cov-2 was added via multidrop 24 hours post seeding of the specified cell type. plates were stained using a modified cell painting protocol 29 . cells were treated with mitotracker deep red (thermo, m22426) for 35m, fixed in 3-5% paraformaldehyde, permeabilized with 0.25% triton x100, and stained with hoechst 33342 (thermo), alexa fluor 568 phalloidin (thermo), alexa fluor 555 wheat germ agglutinin (thermo), alexa fluor 488 concanavalin a (thermo), and syto 14 (thermo) for 35 minutes at room temperature and then washed and stored in hbss+0.02% sodium azide. live-virus experiments omitted the mitochondrial stain due to operational constraints of the biosafety level environment. one hour prior to addition of the immune stimulant or 18 hours prior to the addition of virus, cells were treated with compound via acoustic transfer (echo 555, labcyte). primary screening of new chemical entity libraries was performed at 10 or 30 µm with concentration-response confirmation spanning 100 nm to 30 µm in half-log steps. sars-cov-2 screening was completed in dose response in half-log steps between 10 nm to 3µm. after 24 h incubation (or 96 hours post viral infection), plates were imaged using image express micro confocal high-content imaging system (molecular devices) microscopes in widefield mode with 20x objectives. four sites per well were acquired with 6 channels per site. the following bandpass filters were used to visualize the channels: ff409/493/573/652, ff459/526/596, ff01-432/515/595/730-25, ff01-475/543/702, and ff01-600/37/25. kinase profiling was performed using a kinomescan tm panel of 97 or >400 kinases at eurofins-discoverx (san diego, ca). targets exhibiting > 50% inhibition were followed by kdelect ⓡ analyses for dyrk1b, cdk7, rock2 and rock1 to determine ic50's. data presented as mean and standard deviation. alt-r crispr-cas9 reagents were purchased from integrated dna technologies, inc. (idt) and prepared following the manufacturer's guidelines and protocols (alt-r crispr-cas9 crrna, alt-r crispr-cas9 tracrrna cat #1072534, alt-r s.p. cas9 nuclease v3, cat #1081059, and alt-r cas9 electroporation enhancer, cat #1075916). alt-r crispr-cas9 crrna was duplexed to alt-r crispr-cas9 tracrrna and then combined with alt-r s.p. cas9 nuclease v3, following idt guidelines, to form a functional crispr-rnp complex. this crispr-rnp complex was transfected into cells using the lonza 4d nucleofection system and standard protocols with proprietary modifications, or with a proprietary lipofectionbased process for high-throughput application. alt-r cas9 electroporation enhancer was included into the nucleofection reactions to enhance transfection efficiency following standard guidelines from idt. all images were uploaded to cloud storage and featurized by embedding them with a trained neural network using google cloud platform. this network is based on the convolutional neural network densenet-161 30 . we adapt this network in the following ways. first, we change the first convolutional layer to accept image input of size 512 x 512 x 6. like densenet-161, we use global average pooling to contract the final feature maps, which in our case are tensors of dimensions 16 x 16 x 2,208, to a vector of length 2,208. however, instead of following immediately with a classification layer, we add two fullyconnected layers of dimension 1,024 and 128, respectively, and use the 128-dimensional layer as the embedding of the image. the weights of this network were learned by adding two separate classification layers to the embedding layer, one using softmax activation and the other using arcface 78 activation, which were simultaneously optimized by training the network to recognize perturbations in the public dataset rxrx1 79 and in a proprietary dataset of immune stimuli in various cell types. due to operational constraints of the bsl-3 assay conditions, a modified assay protocol lacking one image channel was used for the live-virus experiments. to accommodate this change, we trained a separate network of the same basic architecture that used only five input channels and one fully-connected final layer of dimension 1,024. immune stimuli phenoprints were observed by calculating the mean embedding of all but one biological replicate, finding the angle between that average and the held-out replicate well, and repeating this process for every replicate to find the average cross-validated angle for that perturbation. statistical significance of these phenotypes was determined by comparing their similarity at high dose against a distribution of similarities between embeddings of images of untreated cells. we used the benjamini-hochberg multiple tests correction with a 5% false discovery rate and considered phenotypes acceptable if they had a corrected p-value<0.05 in two independent experimental batches. the similarity between a pair of immune stimuli was determined by calculating the cosine similarity between all pairs of embeddings of one immune stimulant at high concentration with the embeddings of another immune stimulant at high concentration, and testing whether the mean of this pairwisesimilarity distribution was significantly different from zero using a one-sample t-test and employing the benjamini-hochberg multiple tests correction with a 5% false discovery rate. only significant pairs are used in this paper, and the means of their pairwise-similarity distributions are the values reported in the figures. for small molecule screens, post-processing of the embedded images included normalization to remove inter-plate variance, pca to reduce the feature space, and anomaly detection to remove outliers from the control populations. the vector pointing between the barycenters of the untreated and perturbed conditions was computed, and the embedded image vectors were decomposed into the signed scalar projection (the onperturbation score) and the scalar rejection (the off-perturbation score) with respect to this vector. these scores were normalized so that the mean on-perturbation score was 0 for the untreated condition and 1 for the perturbed condition. separation of the untreated and perturbed conditions along the on-perturbation axis was assessed by z-factor. for compound moa inference, cosine similarities were computed between the embeddings of an nce compound and the set of embeddings of a compound library annotated for moa, and significantly large similarities (relative to the distribution of similarities of pairings of annotated compounds with the nce compound) were reported. for the cocktail representing severely affected patients, top concentration of the most abundant protein, cxcl-10 was selected to be 200 ng/ml based on a practical screen concentration and previously identified phenotypes for this factor. all other proteins were prepared at appropriate concentrations relative to cxcl-10. cocktails representing healthy patients and those with moderate disease severity were prepared with each concentration relative to the severe cocktail. iba1 immunofluorescence assay bv-2 microglia were thawed from liquid nitrogen and plated at 2500 cells per well in 384-well pdl/collagen-coated plates (greiner #781866). the next day, the cells were treated with compound first, followed an hour later by stimulant (recombinant murine tnf-ɑ+ifn-γ (peprotech), 200 μg/ml in 0.1% low endotoxin bsa/pbs). twenty-four hours after treatment, bv-2 microglia were fixed and stained for the microglial activation marker iba1. briefly, cells were fixed with 4% pfa for 15 min at room temperature. primary antibody solution was added to a 1:100 final dilution (iba1 antibody abcam cat #ab5076) incubated overnight at 4°c. after overnight incubation with primary antibody cells were washed with pbs, and secondary antibody solution was added (alexafluor 488 donkey anti-goat igg, 1:1000 final dilution. invitrogen cat# a11055). cells were then incubated for 1 hr at room temperature, protected from light. following secondary antibody incubation cells were washed and the plate was sealed for imaging, which was performed on an image express micro confocal high-content imaging system (molecular devices). data and error presented as mean and standard deviation. quantitative measurement of cytokines in supernatants obtained from cultured cells was performed by using a homogenous timeresolved fluorescence assay (htrf, cisbio). il-6 htrf assays were performed in accordance with the manufacturer's protocol. briefly, cells were seeded and after 24 h treated with compound over 8 concentrations and 5 replicates each. after an additional 24 h, supernatant was collected from assay plate and appropriate sample dilutions and standards were made and dispensed into barcoded labeled 384-perkinelmer proxiplates (cat# 6008280). after the recommended incubation time, the plate was read using an envision ⓡ 2105 microplate reader (perkinelmer). data and error presented as mean and standard deviation. nfkb reporter cells (tr860a-1, system biosciences) were seeded at 4000 cells per well in 384 well imaging plates (781948, greiner). compounds were added at at least 4 replicates per concentration followed by 1 ng/ml tnf-ɑ after 1 h. plates were imaged (gfp channel) once every 3 h via incucyte (sartorius). data for integrated intensity over at the 16 h time point is presented as mean and standard deviation, significance analyzed with 2-way anova. normal human lung fibroblasts (nhlf, lonza) were plated in 1536-well plates (greiner) at 0.25 x 10^6 cells/ml in fgm-2 (lonza). after 24 hours, the media was replaced with fbm media (lonza) and incubated for 24 hours. cells were treated with compounds of interest in a 11-point dose response curve at 32 replicates per concentration using an acoustic liquid handler, and incubated for 1 hour at 37°c, 5% co2. cells were then treated with 1ul of 11ng/ml tgf-β1 (r&d systems), for a final concentration of 1ng/ml. cells were incubated for 30 minutes at 37°c and 5% co2. cells were fixed with 4% pfa, blocked for 1 hour with 1% bsa/0.1% triton x-100/pbs, and then stained for psmad (cell signaling, 1:800). after an overnight incubation at 4°c, cells were stained with alexafluor 647 (thermo, 1:1000) and hoescht (thermo, 1:5000) for two hours at 25°c, washed with pbs twice, and imaged on an image express micro confocal high-content imaging system (molecular devices). images were analyzed with cellprofiler to observe nuclear translocation of psmad2. data and error presented as mean and standard deviation. normal human lung fibroblasts (nhlf, lonza) were plated in 1536-well plates (greiner) at 0.25 x 10^6 cells/ml in fgm-2 (lonza). cells were treated with compounds of interest in a 11point dose response curve at 32 replicates per concentration using acoustic transfer (echo 555, labcyte), and incubated for 1 hour at 37°c, 5% co2. cells were then treated with 1ul of 11ng/ml tgf-β1 (r&d systems), for a final concentration of 1ng/ml using multidrop (thermo fisher). cells were incubated for 96 hours at 37°c and 5% co2. cells were fixed with 4% pfa, blocked for 1 hour with 5% bsa/0.2% triton x-100/pbs, and then stained for collagen i (cell signaling, 1:500). after an overnight incubation at 4°c, cells were stained with alexafluor 750 (thermo, 1:1000), cellmask orange (thermo, 1:5000) and hoescht (thermo, 1:5000) for two hours at 25°c, washed with pbs twice, and imaged on an image express micro confocal high-content imaging system (molecular devices). images were analyzed with cellprofiler. data and error presented as mean and standard deviation electric cell-substrate impedance sensing (ecis) prior to use, 96-well ecis plates (applied biophysics, 96w20idf pet) were pre-treated with 10mm l-cysteine (sigma-aldrich, c7352-25g) and then coated with fibronectin (gibco, phe0023). human umbilical venous endothelial cells were plated in the fibronectin coated 96-well ecis plates at 55,000 cells/well in ebm-2 (lonza cc-3156) +egm-2 (lonza, cc -4176). cells were allowed to settle at room-temperature for 1 hour and then incubated for 24 hours at 37 o c, 5% co2. following incubation, the plates were placed on the ecis readers for 1 hour to establish baseline resistance. cells were then treated in a 9point dose response curve at 4 replicates per concentration using acoustic transfer (echo 555, labcyte) and returned to the incubator for 1 hour. following this, cells were treated with the cytokine storm cocktail (table s3) . resistance was measured for 24 hours following this. the assay window was defined as the time range with the greatest observable difference in membrane resistance between empty and disease control (approximately 6 hours following cocktail addition). resistance was normalized to each plate and graphed as a dose-response curve where 1 and 0 correspond to health and disease controls, respectively. after staining and imaging to establish high dimensional phenotypes, plates were rinsed once with wash buffer (1xhbss + 0.02% sodium azide) before incubating with primary antibody raised against sars-cov-2 nucleocapsid protein for 60 mins at rt (sino biological catno. 40588-t62, 1:1000 dilution). media was evacuated from wells by inverted centrifugation, and secondary antibody was added and incubated another 60 minutes at rt (thermo scientific catno. a31573, 1:2000 dilution). primary and secondary antibodies were diluted in stain base media (1xhbss, 1% bsa, 0.3% triton-x 100). plates were washed one final time using inverted centrifugation and wash buffer before imaging as described above. data presented as mean value. cell-level image segmentations and per-cell log-mean nucleocapsid staining intensities were calculated using standard image segmentation techniques (cellprofiler 80 ). these intensities were normalized by plate with respect to log-mean-intensity in the mock control cells. to adjust for optical effects that changed the background fluorescence level in infected wells, a gaussian mixture model was used to align the lowest peak in log-meanintensity across well conditions. cells were estimated to be infected if they exhibited an adjusted log-mean-intensity above the 99th percentile of intensity for the control cell population. this estimate of number of infected cells was used to compute a fraction of cells infected, which was adjusted to account for the 1% of uninfected cells expected to be above the 99% threshold. the usa-wa1/2020 strain of sars-cov-2 was propagated in vero cells. cells were grown in standard tissue culture flasks (60% confluence) and were infected at a multiplicity of infection (moi) of 0.001, in emem + 2% fbs and 50 g/ml gentamicin, incubated at 37°c with 5% co2 for 5 days. supernatants containing virus were removed from these cultures, spun down to remove cellular debris and stored at -20°c until use. viral titers were determined through standard tissue culture infectious dose 50% (tcid50) methods, where cytopathic effect (cpe) on vero 76 cells was measured by visual observation under a light microscope. to create a suitable control with inactivated virus, sars-cov-2 was irradiated with a uv lamp for 10 or 20 minutes. viral inactivation in this sample was verified using visual cpe on vero cells, where undetectable level of active virus was observed. an additional "mock" control was created using conditioned media preparations generated from uninfected vero 76 cells grown in 2% fbs in emem for five days. cellular debris were removed through centrifugation and the supernatants were frozen at -80°c until use. all experiments using sars-cov-2 were performed using biosafety level 3 (bsl-3) containment procedures at partner facilities including one at utah state university. data and error presented as mean and standard deviation. a 1536-well plate was pre-treated with compounds, at 13 concentrations (ranging from 0 to 100 ng/ml) with at least 2 replicates of each concentration, and a reaction mix containing 15 ng alk5 (thermofisher), using poly 4:1 as substrate was added to the plate. the reaction was started by the addition of 20 µm atp, the plate was mixed on a plate shaker (500 rpm for 2 min) and the reaction allowed to incubate at room temp for 60 min. the reaction was terminated by the addition of adp-glo reagent (promega v9102) (40 min incubation) and kinase detection reagent (30 min incubation). luminescence was captured using an envision xcite plate reader. data presented as mean and standard deviation. all assessments of compound diversity and similarity were performed in datawarrior (openmolecules.org). neighbors were determined to be at 83% or greater similarity as determined with the skelspheres descriptor. tables containing metrics for immune stimulant phenotypes in each cell type are provided in the supplementary materials. underlying images, metadata, and deep learning embeddings for soluble factor perturbations in huvec and all primary screens in primary cell types for covid-19 virus and cytokine storm screening have been made available at rxrx.ai. an interactive server containing drug response projections, hit scores, and structures for covid-19 screening data has been made available for custom search at covid19.rxrx.ai. the code underlying this report leverages proprietary algorithms for image processing, data standardization, outlier detection, and compound efficacy scoring. as such the code underlying this report will not be made available. instead, much of the output of these algorithms is provided in the provided supplemental tables. virus graphic in fig. 1 schematic for interpreting projection of drug response in 2-dimensional plot. contours show the 99%, 90%, and 50% distributions of on-and off-perturbation scores for the perturbation (orange) and target (blue) states. ideal rescues are compounds that rescue along the on-perturbation xaxis toward the target state with minimal increase to the off-perturbation score on the y-axis. b. potential hits are prioritized by the proximity of any dose to the target state, illustrated here with dashed lines and increasing red highlight intensity for higher-ranked dose-curve trajectories. c. projections of treatments along the on-perturbation vector: rescue of the tnf-ɑ phenoprint with clinically approved monoclonal antibodies, reversal il-6-il-6r receptor chimera, and reversal of socs3 crispr gene knockout. ec50 for high-dimensional compound rescue are indicated in parentheses representative images of hrce, calu3 and vero cells immunostained with sars-cov-2 nucleocapsid protein (pink) and modified cell paint dyes c. infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. of note, hrce donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. antibody stains were performed after the principal analysis concluded and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. d. infection of hrce yielded a phenoprint against the mock-infected target population with an assay z-factor of 0. 43 cytokines in rheumatoid arthritis -shaping the immunological landscape cytokine networks in neuroinflammation immune signaling in neurodegeneration inflammatory cytokine and chemokine profiles are associated with patient outcome and the hyperadrenergic state following acute brain injury cytokines in cancer immunotherapy cytokines in the treatment of cytokines in the balance of protection and pathology during mycobacterial infections the temporal role of cytokines in flavivirus protection and pathogenesis insights into the hiv latency and the role of cytokines cytokine storm and sepsis disease pathogenesis immunotherapeutic implications of il-6 blockade for cytokine storm biologics for targeting inflammatory cytokines, clinical uses, and limitations 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golimumab, a human monoclonal antibody specific for human tumor necrosis factor α infliximab vs. adalimumab in crohn's disease: results from 327 patients in an australian and new zealand observational cohort study socs3 binds specific receptor-jak complexes to control cytokine signaling by direct kinase inhibition opportunities and challenges in phenotypic drug discovery: an industry perspective the alk-5 inhibitor a-83-01 inhibits smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta preclinical assessment of galunisertib (ly2157299 monohydrate), a first-in-class transforming growth factor-β receptor type i inhibitor selective inhibition of tgfβ1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape targeting tumor necrosis factor alpha for alzheimer's disease selective modulation of tnf-tnfrs signaling: insights for multiple sclerosis treatment the far-reaching scope of neuroinflammation after traumatic brain injury anti-tnf-α therapies: the next generation nf-kb signaling in inflammation rho kinase (rock) inhibitors and their therapeutic potential cutting edge: selective oral rock2 inhibitor reduces clinical scores in patients with psoriasis vulgaris and normalizes skin pathology via concurrent regulation of il-17 and il-10 nct03919799 ibrutinib suppresses lps-induced neuroinflammatory responses in bv2 microglial cells and wild-type mice the trinity of covid-19: immunity, inflammation and intervention 2019-novel coronavirus (2019-ncov) infections trigger an exaggerated cytokine response aggravating lung injury baricitinib restrains the immune dysregulation in covid-19 patients regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production identification of potential treatments for covid-19 through artificial intelligence-enabled phenomic analysis of human cells infected with sars-cov-2 human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. infectious diseases (except hiv/aids covid-19 clinical trial dashboard mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio remdesivir for the treatment of covid-19-preliminary report who discontinues hydroxychloroquine and lopinavir/ritonavir treatment arms for covid-19 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov cell entry mechanisms of sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor covid-19) update: fda revokes emergency use authorization for chloroquine and hydroxychloroquine effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial imbalanced host response to sars-cov-2 drives development of covid-19 hypoxia-inducible transcription factor-1 alpha determines sensitivity of endothelial cells to the proteosome inhibitor bortezomib the proteasome inhibitor bortezomib enhances the susceptibility to viral infection the proteasome inhibitor velcade enhances rather than reduces disease in mouse hepatitis coronavirus-infected mice a systematic review and meta-analysis of infection risk with small molecule jak inhibitors in rheumatoid arthritis fatal encephalopathy with wild-type jc virus and ruxolitinib therapy evaluation of hepatitis b virus in clinical trials of baricitinib in rheumatoid arthritis low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid-19 rxrx1 additive angular margin loss for deep face recognition rxrx1: an image set for cellular morphological variation across many experimental batches cellprofiler: image analysis software for identifying and quantifying cell phenotypes we declare competing interests. all authors were employees of or advisors to recursion during the course of this work. all authors have real or potential ownership interest in recursion. key: cord-309052-3h0g7s9v authors: alam, fiaz; khan, gul nawaz; asad, muhammad hassham hassan bin title: psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review date: 2017-12-15 journal: phytother res doi: 10.1002/ptr.6006 sha: doc_id: 309052 cord_uid: 3h0g7s9v psoralea corylifolia l. (leguminosae) is a well‐known traditional medicinal plant used from ancient times for treatment of various ailments. it is widely distributed and an important part of therapeutics in ayurveda and in chinese medicines. the aim of this review is to present comprehensive and most up to date report on its ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects. studies on the ethnobotanical, ethnopharmacological, clinical, phytochemical, and side effects of p. corylifolia were published until year 2017 and were searched using various scientific databases. the scientific literature searched revealed that these plant species has been extensively investigated in vivo and in vitro for various biological and phytochemical studies. it has cardiotonic, vasodilator, pigmentor, antitumor, antibacterial, cytotoxic, and anti‐helminthic properties and locally used for alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema. so far, about a hundred bioactive compounds have been isolated from seeds and fruits, and most important compounds identified belongs to coumarins, flavonoids, and meroterpenes groups. this review article summarized the most updated scientific literature on bioactive phytochemical and biological activities of p. corylifolia. this article will be a useful addition to providing information for future research, and more standard clinical trials are needed for the plant to be used as therapeutic agent. the family leguminosae contains about 500 genera and is among the largest families of flowering plants. the plants of leguminosae are widely distributed, and in terms of number of specie, it is one of the largest terrestrial family of plants after orchidaceae and asteraceae (stevens, 2015) . the genera that belong to this family are important medicinally and contain a variety of biologically important molecules; p. corylifolia is an important part of therapeutics in ayurveda and in chinese medicines. the plant is cardio active and showed antimicrobial and cytotoxic properties. it is used as a pigmentor. the plant showed cytotoxicity against tumors and worms (baquar, 1989; rizvi, saeed, & zubairy, 2007; sharma, 2004) . in chinese medicines, the psoralea plant is considered warm by nature, and therefore shows many healing actions on kidney and spleen meridians (daiquan, 2000 (daiquan, -2006 . the seeds of p. corylifolia are used in indigenous medicine systems for healing of various ailments. the seeds are diuretic, aphrodisiac, laxative, anti-helminthic, and are used in febrile conditions. in ayurveda, the seeds are used in the form of paste and as an ointment for external as well as internal use for treatment of different conditions such as alopecia, inflammation, leukoderma, leprosy, psoriasis, and eczema (huang, 1998; judd, campbell, kellogg, stevens, & donoghue, 1999; sjc, 2015) . in india, the powder of seeds is mixed with haratalabhasma (yellow arsenic) and converted to paste with the urine of cow. this paste is used to treat leukoderma lesions. in another formulation, the mixture of powdered seeds with buttermilk have been used externally for treating ringworm and scabies. the seed oil is taken orally with betel nut leaf for treatment of leprosy. another skin condition, dermatosis, has been treated with adjuvants therapy of bakuchi (p. corylifolia) with other local plants such as amalaki and khadira. similarly, the oils of bakuchi and karanja are mixed with vaseline to treat chronic skin diseases (khare, 2008) . the seeds of the plant are also considered useful in bilious disorder, snakebite, and in scorpion sting. the seeds are also used in the production of perfumes (deshaprabhu, 1966; maisch, 1889; sharma et al., 2000) . the root of p. corylifolia has shown its effectiveness in dental caries. the fruits are aphrodisiac and have laxative effect, and the leaves are antidiarrheal (baquar, 1989; sjc, 2015) . the plant is also valuable in treating alopecia areata (dua, kumar, pandey, & kumar, 2013b) . in combination with haritka and gokshura, this plant is used for urinary frequency, and with ashwagandha and bala, it is used for treatment of reproductive diseases and cough. in chronic diarrhea and for treating cold symptoms, this plant is used in combination with nutmeg and haritaki (anderson & voorhees, 1980) . the seeds of bakuchi in powder form mixed with the decoction of bibhitaka (terminalia bellirica bark) and kaakodumbara (ficushispida) was effective to treat vitiligo. the infection of ringworm is treated with a combination of tila (sesame seeds) and bakuchi (gupta, dhar, & atal, 1978) . a combination of bakuchi with haratalabhasma provides relief against leukoderma when applied externally (khatune et al., 2002) . in japan, the plant extracted with alcohol has been used as an additive in processed foods and pickles (nadkarni, 1976) . when the fixed oils are removed, what is left after the seed cake are used as feed or manure due to the presence of nitrogen (6.7%) and the minerals (7.8%). volatile oils obtained from fruits have an irritant effect on the skin and mucous membranes and stimulate the voluntary muscles in high concentrations (chaudhuri, 2015; gidwani et al., 2010; huang, 1998) . p. corylifolia in the form of extracts have been used in various herbal formulations when combined with other herbs and used in handling psoriasis and many other skin disorders . this plant is also reported to be used in cardiac problems, asthma, and urinary discharge (kr, 1975) . it also has anti-leishmaniasis activity (ali, akhtar, sultana, baboota, & ahuja, 2008) . it is used to control watery stool, urinary frequency, and reproductive imbalances (pole, 2006) . in china, it is specially used against vitiligo disease (anderson & voorhees, 1980; khare, 2004) . p. corylifolia is available in the form of various ayurvedic marketed formulations in india and across the world, the most famous brands are safuf bars®, zimad kibrit®, svitrakaravati®, khadirarista®, algushadi yoga®, sarvangasundarigutika®, bhallatakawaleha®, maheshwa-raghrita®, ayorajodilepa®, brihatsomarajitaila®, somarajighrita®, bawchichurna®, etc. (ali et al., 2008; pole, 2006; qiao et al., 2007) . the investigation showed that p. corylifolia possessed a wide range of phytochemicals including flavones, coumarins, monoterpenes, chalcones, lipids, resins, stigmasteroids, and flavonoids. the volatile oils are also reported from this plant (zhang, zhao, wang, lu, & chen, 2016) . it is revealed that seasonal variation also effects the phytochemistry of p. corylifolia. mostly, the bioactive compounds are found to be concentrated in the seeds. the phytochemistry of this plant is discussed as follows. the whole plant of p. corylifolia was extracted with organic solvents such as petroleum ether and chloroform. the subsequent isolation methods lead to the purification of bioactive compounds, for example, psoralen, isopsoralen, corylifolin, corylin, and psoralidin (gupta, gupta, & gupta, 2013) . peng and colleague obtained a new compound identified as neo-psoralen from the whole plant of p. corylifolia in 1996 and elucidated its structure on the grounds of chemical indications and spectroscopic analysis (chaudhuri, 2015; gupta et al., 2013; table 1 and figure 2 ). it was already discussed that most of the active constituents isolated so far from p. corylifolia are part of the seed. sen and colleague extracted seed oil from p. corylifolia and found that the oil is unsaponifiable with boiling points 180-190°c. during the experiment, another compound was also identified as methyl glucoside with melting points of 105-127°c (chopra & chopra, 1933) . chopra and chaterjee, in 1927 , identified essential oil, a fixed oil and resin of dark brown color with some traces of alkaloids in p. corylifolia (chopra et al., 2013) . dymock studied the sugar contents, extractive matter, albumin concentraton, and ash value, and also found some traces of manganese in seeds of p. corylifolia. in continuation of research for compounds from the seeds of p. corylifolia, three more important components were fractionated and identified as bakuchiol a monoterpene phenol and the two novels dimeric monoterpenoids, bisbakuchiols a and b (panda, 1999) . a very important pharmacological compound known as bakuchiol has also been biosynthesized in 1983, and it was concluded that it is a derivative of phenylpropane pathway (banerji & chintalwar, 1983) . similarly, bisbakuchiols a and b structures were evaluated, and it was noted that dimeric monoterpenoid skeleton contains two monoterpenes, which are connected through a dioxane bridge (wu et al., 2007) . the low polarity ether extract of p. corylifolia seeds was investigated, and it revealed the presence of various ketones and aldehydes containing compounds such as corylinal, c-formylated chalcone, and isoneobayachalcone. a new isoflavone compound known as psorlenal was also identified in the seeds (gupta et al., 1978) . the same compounds, psoralen and isopsoralen, were also isolated by applying other chromatographic techniques such as high-speed counter-current chromatography (liu, li, sun, & kong, 2004) . the seed's sample has been the main focus for the search of bioactive compounds, and such an isolation procedure involving spectroscopic methods and crystal x-ray diffraction resulted in isolation of five novel compounds. these compounds are named as psoracorylifols a-e and chalcone and bavachromanol (yin, fan, dong, & yue, 2006) . similarly, three new flavonoid compounds named as corylifols a, b, and c and bavachalcone were also fractionated from p. corylifolia seeds. another compound known as bakuchicin was also identified from the seeds (yin, fan, wang, dong, & yue, 2004) . the seeds are also reported to contain some other flavonoids such as bavachinin (bcn), bavachin, isobavachin, and isobavachalcone (ibc). the glycosides identified in seeds of p. corylifolia were psoralenoside and isopsoralenoside, which were of the benzofuran type. p. corylifolia also reported to contain some polar compounds, namely, neobavachalcone, 7-methyl bavachin, and bavachromene. these compounds were isolated and identified from the insoluble portion of the ethanolic extract. these compounds identified as (khatune et al., 2004) 2 aryl coumarin coumarin seeds anticancer (limper et al., 2013) 3 astragalin flavonoid seeds antioxidant 4 bakuchiol meroterpene seeds/fruit anti-acne antibacterial (katsura et al., 2001; newton et al., 2002) antifungal (newton et al., 2002) , (hosamani et al., 2012; lau et al., 2010; lau et al., 2014; prasad et al., 2004; savoia, 2012; srinivasan & sarada, 2012; yang et al., 2006) retinal regeneration (seo et al., 2013) anti-aging (seo et al., 2013) estrogen receptor agonist, postmenopausal symptoms (lim et al., 2011) anti-diabetic (behloul & wu, 2013) lymphangiogenesis inhibition (jeong et al., 2013) anticancer (chen et al., 2010; li et al., 2016) 5 bavachinin flavone seeds antibacterial (khatune et al., 2004) estrogen receptor agonist (lim et al., 2011) lymphangiogenesis inhibition (jeong et al., 2013) osteoporosis (liu et al., 2014) anti-alzheimer (chen et al., 2013) carboxylesterase inhibitors 6 bakuisoflavone flavone fruit antibacterial (siva et al., 2015) 7 bakuflavanone flavone fruit antibacterial (siva et al., 2015) 9 bavachin flavnonoid seeds/fruit osteoblast 10 bakuchicin coumarin seeds topoisomerase inhibitor (sun et al., 2003) 11 bavachalcone chalcone seeds anticancer (shan et al., 2014) cvs protective effect (dang et al., 2015) 12 bavachinone a flavonoid fruit antibacterial (won et al., 2015) 13 bavachinone b flavonoid fruit antibacterial (won et al., 2015) 14 bavacoumestan c flavonoid fruit antibacterial (won et al., 2015) 15 corylifolinin chalcone seeds antibacterial (khatune et al., 2004) carboxylesterase inhibitors (sun et al., 2016) 16 corylifols prenyl flavonoid seeds antibacterial (yin et al., 2004) 17 corylifol a flavonoid seeds/fruit carboxylesterase inhibitors 18 corylifol b flavonoid seeds carboxylesterase inhibitors 19 corylifol c flavonoid seeds protein kinase inhibition (limper et al., 2013) anticancer (limper et al., 2013) 20 corylifol d flavonoid seeds anticancer (stomach; yang et al., 1996; teschke et al., 2014) 21 corylifol e flavonoid seeds anticancer (stomach; yang et al., 1996; teschke et al., 2014) 22 coryfolin flavonoid whole plant antioxidant, anti-diabetic (behloul & wu, 2013) 23 corylin flavonoid whole plant osteoblast wang et al., 2001) anticancer (shan et al., 2014) carboxylesterase inhibitors (sun et al., 2016) 24 coryaurone a flavonoid fruit antibacterial (won et al., 2015) 25 dadzin isoflavnoid fruit antioxidant (shinde et al., 2010) 26 dadzein isoflavnoid fruit antioxidant (shinde et al., 2010) antidiabetic (behloul & wu, 2013) topoisomerase inhibitor (sun et al., 2003) 27 dihydroxy coumestan essential oil component seeds insecticidal, genotoxic (khatune et al., 2002; dua et al., 2013b) 28 genistein isoflavone fruit ani-diabetic, anti-obesity (behloul & wu, 2013) , antioxidant (shinde et al., 2010) 29 hydroxy bukuchiol meroterpene seeds lymphangiogenisis inhibition (jeong et al., 2013) 30 hydroxypsoralenol a flavonoid fruit antibacterial (won et al., 2015) 31 hydroxypsoralenol b flavonoid fruit antibacterial (won et al., 2015) 32 types of esters were also studied in p. corylifolia, and psoralester and psorachromene were identified as two new metabolites. the lymphangiogenesis inhibition (jeong et al., 2013) anti-alzheimer (chen et al., 2013) carboxylesterase inhibitors 33 isobavachin flavonoid seed/fruit osteoblast (li et al., 2014) 34 isopsoralen furanocoumarin whole plant antiprotozoal 35 neobavaisoflavone seeds antibacterial (khatune et al., 2004) 36 psoralen furanocoumarin whole plant/root leucoderma, psoriasis anticancer (hao et al., 2014) , antioxidant , anti-alzheimer (somani et al., 2015) , collagengenesis 37 psoralidin coumarin whole plant/seed estrogen receptor modulator (liu et al., 2014; lim et al., 2011) antioxidant (wang, yin, zhang, peng, & kang, 2013b) , antibacterial (khatune et al., 2004) anti-diabetic (behloul & wu, 2013) , antiprotozoal anticancer (hao et al., 2014; limper et al., 2013; yang et al., 1996) , anti-depressent (farahani et al., 2015) 38 psoracorylifol d flavonoid seed lymphangiogenesis inhibition (jeong et al., 2013) psoracoumestan coumestans seeds essential oil anti-cancer (limper et al., 2013) 39 xanthoangelol chalcone seeds anticancer (limper et al., 2013) figure 2 structures of important compounds isolated from psoralea corylifolia psoralester is a 10-membered lactone compound and the latter is an isomer of already known compound bayachromene (tewari & bhakuni, 2010) . khatune and colleague isolated new coumestan derivative 6-(-3-methylbut-2-enyl)-6 n-7-dihydroxycoumestan while working on the chloroform soluble portion of the seeds of p. corylifolia (khatune et al., 2002) . coumestans-3, 5′-dihydroxy-6′, 6′-dimethyldihydropyrano (2′, 3′: 8,9) coumestan, 3-hydroxy-5′-(1hydroxy-1-methylethyl) 4′, 5′-dihydrofuro (2′, 3′: 8,9) coumestan, and sophoracoumestan a have been fractionated from the seeds of p. corylifolia. lin and kuo isolated two new benzofuran derivatives; corylifonol and isocorylifonol along with astragalin, p-hydroxy benzoic acid from p. corylifolia seed extract (lin & kuo, 1992) . in 1989, zaka and colleague investigated the lipid and fatty acid composition of p. corylifolia. the lipids recognized were triacylglycerols, diacylglycerols, free fatty acids, monoacylglycerols, hydrocarbons, wax esters, and polar lipids. the purified crude lipids of p. corylifolia seeds were analyzed by thin layer and gas chromatographic techniques. among the components, the major polar lipid was c18: the sticky and oily pericarp constitute the fruit of p. corylifolia, and chemical investigation revealed some similar compounds as already isolated from seeds. a new isoflavone, corylinin, (7,4′-dihydroxy-3′in another experiment, hplc protocol was applied to identify some isoflavonoids such as daidzein, genistein, and biochanin a (sehrawat, sangwan, & yadav, 2014) . further work on the fruit extracted with hexane showed the presence of a phenolic monoterpene known as bakuchiol (cui, taniguchi, kuroda, & hatano, 2015) . dried p. corylifolia fruit powder extracted with methanol and analyzed on hplc reverse column showed the presence of isoflavonoids known as genistein, daidzein, and biochanin a (hsu, wu, chen, yang, & wang, 2001) . raun and colleagues have isolated seven compounds from the fruit of p. corylifolia and established the structure of compounds after the spectroscopic analysis. the components identified were corylinin (new compound), psoralen, neobavaisoflavone, sophoracoumestan a, uracil, and daidzin (ruan et al., 2007) . in 2015, two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corlylifolia with antimicrobial activities (lee, kim, baik, ryu, & lee, 2015) . in one study, six new flavonoid compounds and a meroterpenoid were isolated and identified by spectroscopic methods from p. corylifolia fruits and displayed medium activity against staph. mutans (won et al., 2015) . the dried fruits of the plant was investigated, and n-hexane the roots of p. corylifolia has been investigated for bioactive compounds. it was found that furanocoumarins psoralen and isopsoralen isolated from a petroleum ether extract were responsible for the anti-feedant activity against instar spodoptera litura larvae (sah et al., 2006 ; figure 2 ). the above discussions about phytochemical investigation of different parts of p. corylifolia clearly indicates that this plant is a very useful source of variety of bioactive constituents including flavonoids, terpenes, glycosides, alkaloids, coumarins, and others. p. corylifolia is a widely used herb and have many diverse ethnopharmacological and medicinal applications. the numerous chemical and pharmacological research that have been carried out have resulted in the isolation of the diverse bioactive compounds that are summarized below. p. corylifolia proved a promising agent in anti-acne formulations due to the presence of phenolic compounds bakuchiol. it proved to be safe and non-irritant and can be used for longer periods of the day because it showed no irritation and is non-sensitized . one of the bioactive isolated compound "soralen" found to have the ability to stimulate the development of melanin, and therefore it is employed for leucoderma treatment (kim, shim, ahn, & jung, 2013) . the plant is also used against the skin disease known as psoriasis (chen, yang, & zhang, 2013) . in one experiment, seeds of p. corylifolia was extracted with hexane and oil in water, cream was prepared with stearic acid as a base. in the next step, an open clinical trial was conducted on 30 patients suffering from eczema for a period of 30 days. after 2 weeks of cream application, symptoms score reduced. final day observation revealed that the symptom scores for eczema reduced from 6.367 ± 1.098 to 0.333 ± 0.279 for length of the lesion, from 1.333 ± 0.994 to 0.165 ± 0.087 for exudation rate, and from 2.567 ± 0.504 to 0.165 ± 0.132 for the rate of itching. this formulation was compared with the placebo preparation, in which the formulated cream contained all the ingredients except for the hexane extract of p. corylifolia. this study concluded that this plant could be effectively used for the treatment of eczema (gidwani et al., 2010) . p. corylifolia has been tested for antibacterial activity. wang et al. nine compounds exhibited significant antibacterial activity against sa and s. epidermidis (yin et al., 2004 that showed 11.5 mm zone of inhibition (acharya et al., 2015) . p. corylifolia in the form of the mouth rinsing solution has prevented the growth of s. mutans bacteria in a very low concentration. along with mouth rinsing ability, the ethanol extract of p. corylifolia was also reported to inhibit the human gingival fibroblast. in the same series of experiments, it was concluded that the extract is safe to use and has no toxic effect in normal doses . in 2015, two more new flavonoids, bakuisoflavone, and bakuflavanone were isolated from the fruit of p. corylifolia and were tested against the strains of mrsa (mrsa481 and mrsa 584) and it was found that two compounds showed some good antibacterial effects with mic values of (>32 (>9.4) and >32 (>9.4)) and (>32 (>9.5) and >32 (>9.5 μg/ml)), respectively . tissue culture studies showed that jasmonic acid treated plants of p. corylifolia enhanced psoralen content in leaves and roots of p. corylifolia. the methanol extract of root sample displayed effective antimicrobial activity against tested bacterial and fungal pathogens in range 25, 50, and 75 μg/ml. most effective activity 28 mm zone of inhibition was observed against p. aerugenosa at 50 μg/ml (siva et al., 2015) . p. corylifolia was also tested against the microbes sa, pseudomonas aeruginosa, and candida albicans, which are known to contaminate the cosmetics products. in the 96-well microplate assay, it was found to have antimicrobial activity . the a phenolic compound bakuchiol extracted from p. corylifolia (seeds) exhibited antifungal activity against many strains of pathogenic fungi, (hosamani, lakshman, & sandeepkumar, 2012; lau et al., 2010; lau et al., 2014; newton et al., 2002; prasad, anandi, balasubramanian, & pugalendi, 2004; savoia, 2012; srinivasan & sarada, 2012; yang et al., 2006) . in another study, activity was found against other fungi such as alternari brassicae, aspergillus niger, fusarium oxysporum, and rhizoctonia cerealis, in which mycelial growth was inhibited (satish, raghavendra, & raveesha, 2009; vonshak et al., 2003; yang et al., 2006) . in one study, p. corylifolia significantly reduced the incidents of the anti-worm property of the seeds of p. corylifolia is clinically proven on roundworms and flatworms (gidwani et al., 2010) . the seeds and the leaves of p. corylifolia was extracted with water and alcohol, and were tested on the spontaneous movements of setaria cervi whole worm and again on the isolated nerve muscle preparations. the survival of the microfilariae was tested in vitro. the dose required to inhibit the movements of whole worm and nerve muscle preparations for alcohol extracts of leaves were 160, 30, and for seeds were 50, 20 μg/ml (maurya, singh, & seth, 2014; mendam, kavitha, & naik, 2015; qamaruddin, parveen, khan, & singhal, 2002) . volatile oil extracted from the seeds of p. corylifolia displayed strong toxicity against both larvae and adult of the southern house mosquito, the bioactivity guided isolation from chloroform extract of seeds leads to the identification of pure compound, 6-(-3-methylbut-2enyl)-6 n-7-dihydroxycoumestan and found active against larvae and adult cx. quinquefasciatus. the same compound has also showed activity against the red flour beetle, tribolium casteneum hebrst. in this detailed study, both the male and female adults were exposed to the compound for 24-72 hr period and a range of ld 50 were calculated for dose starting from 400 to 1600 ppm and doses were used in five replications. it was concluded that the isolated coumestan compound can be a useful botanical insecticide (dua, kumar, pandey, & kumar, 2013a; khatune et al., 2002) . an external protozoan parasite ichthyophthirius multifiliis (also called "ich") has been reported to infest freshwater fish species. the p. corylifolia extracted with methanol showed excellent activity against i. multifiliis theronts in concentration of 1.25 mg/l or more when was exposed for a period of 4 hr. the p. corylifolia extract at 5.00 mg/l concentration has caused 100% mortality of protomonts and 88.9% of encysted tomonts. it was found that longer period (24 hr) and higher concentration (5.00 mg/l) caused the significant reduction of the survival rate and reproduction of tomont of i. multifiliis, which were exited from the fish after in-bath handling in situ (ling et al., 2013) . p. corylifolia has been found to be an alternative to malachite green to control i. multifiliis, an external protozoan parasite. the screening showed that p. corylifolia extract have the maximum activity against i. multifiliis theronts. when the experiments were conducted in vivo, at 1.25 mg/l or more concentrations of methanol extract of p. corylifolia, it caused 00% mortality of theronts during the 4 hr of exposure (ling et al., 2013) . this study showed the damaging effect of p. corylifolia against i. multifiliis trophont in situ. the same study leads to the evaluation of the activity of antiprotozoal compounds extracted form p. corylifolia against i. multifiliis. the two bioactivity guided isolated compounds, isopsoralen and psoralidin, were assessed. in comparison, study of psoralidin and isopsoralen, the inhibitory activity of psoralidin, was found more efficient. further, when experimented in vivo, the compound psoralidin at 2.5 mg/l, efficiently reduced the theronts numbers in infected fish over an exposure period of 5 hr. this study showed that psoralidin could be a very good candidate for the formulation as a commercial drug against i. multifiliis . the compounds from p. corylifolia were found to have a protective effect when tested against retinal damage caused by oxidative stress. to evaluate the effects of bakuchiol on the cell viability of dif(park et al., 2005) . a recent and very useful research on the same subject of hepatotoxicity shows that the isolated compound bakuchiol when administered at a dose of 52.5 and 262.5 mg/kg for 6 weeks in rats, many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. the mrna expression of cyp7a1, hmg-coa reductase, pparα, and srebp-2 decreased in bakuchiol-treated group, the expression of bsep increased in bakuchiol-treated low dosage, and the expression of bsep decreased in bakuchiol-treated high dosage. it was concluded that bakuchiol could induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. the mechanism may be related to effects on liver lipid metabolism (li et al., 2017) . in one study, six compounds isolated from p. corylifolia were studied for their binding affinities for estrogen receptors, erα and erβ, using the yeast transactivation test. among the compounds, bakuchiol was in another study, psoralidin activity as er (α and β) agonist was evaluated in endometrial and human breast cell lines. psoralidin at 10 μm was able to get the highest reporter gene expression conforming to that of e2-treated cells and such an activation of the ere-reporter gene by psoralidin was totally stopped by the treatment of a pure er antagonist, indicating that the biological actions of psoralidin are intermediated by er. psoralidin was also able to stimulate the endogenous estrogen-responsive gene (ps2) in mcf-7, the human breast cancer cells. it was observed that activation of the classical er-signaling pathway by psoralidin is mediated via induction of er conformation by psoralidin and direct binding of the psoralidin-er complex of the eres present in the promoter region of estrogenresponsive genes. lastly, molecular docking of psoralidin to the ligand-binding pocket of the erα exposed that psoralidin is able to mimic the binding interactions of e 2 , and therefore, in the cellular environment it could act as an er agonist (liu et al., 2014) . psoralen isolated from p. corylifolia fruits were investigated as an inhibitor of ache enzyme in an attempt to explore its potential for the management of alzheimer's disease. the concentration of psoralen used was 25-400 μg/ml. it inhibited the ache in a dose-dependent way in animal models. adult male wistar rats, weighing 180-250 g, were used in the study. while a molecular docking study was also carried out, which showed that psoralen binds well within the binding site of the enzyme showing interactions such as π-π stacking and hydrogen bonding (somani et al., 2015) . although the activity measured in this study was moderate when compared with the standard compound used, despite that, the compound could serve as lead for synthetic analog preparation to improve the inhibitory activity. p. corylifoia also found to possess antidepressant activity. marzieh sarbandi farahani and colleague mentioned the mechanism of action of the plants with antidepressant action and the chemical components isolated from them. they mentioned that psoralidin isolated from seeds of p. corylifolia modify the hypothalamic-pituitary-adrenal axis (farahani, bahramsoltani, farzaei, abdollahi, & rahimi, 2015) . a similar study was conducted on psoralidin by yi and colleague on the icr strain of male mice. the dose was administered orally in forced swimming test and they observed the increased levels of 5hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain and an altered dopamine level. the mechanism for antidepressant activity was proposed to be through involvement of monoamine neurotransmitter and the hypothalamic pituitary adrenal axis systems (yi et al., 2008) . some previous studies are also available, for example, one study was conducted on mice models and it was concluded that furocoumarins were actually responsible for antidepressant activity. in this study, the well-established antidepressants were used as standards for comparison with the seed extract of p. corylifolia. the dose range used was 7.5 to 100 mg/kg in comparison with amitriptyline (10 and 20 mg/kg) and fluoxetine (13 mg/kg). this study was well designed and results indicate the potential of the seed extract as competitive antidepressant when compared to the conventional therapeutic agents (chopra et al., 2013) . p. corylifolia also has a wide range of antioxidant activity. different compounds isolated from p. corylifolia were tested for their antioxiin the understanding of the reputation of this plant species in medicines now, attention has been given to produce callus culture. in one study, relationship between isoflavone and antioxidant activity of p. corylifolia cultures were experimented, and it was found that root-derived callus cultures produced more daidzein, whereas leafdervied callus produced more genistein, and this enhanced production was related to enhanced antioxidant activities (shinde, malpathak, & fulzele, 2010) . one of the isolated compound, psoralen showed the promising antioxidant activity (ic 50 value = 1.10 ± 0.60 μg/ml) against the superoxide anion production by human neutrophils in response to formyl-lmethionyl-l-leucyl-l-phenylalanine/cytochalasin b (fmlp/cb; chen et al., 2011). p. corylifolia extract possesses anti-diabetic activity by its action as the protective effects on pancreatic β cells (behloul & wu, 2013) . a more detailed biochemical study was conducted on the aqueous extract of seed of p. corylifolia that caused a significant recovery in the activities of hexokinase, glucose-6-phosphatase, and glucose-6-phosphate dehydrogenase and antioxidant enzymes such as peroxidase, catalase, and superoxide dismutase, along with the lipid peroxidation level in liver tissue and serum transaminase, and corrected the fasting blood glucose level in streptozotocin-induced diabetic rats at a dose of 20 mg/0.5 ml water/100gm body weight (ghosh, bera, chatterjee, ali, & debasis, 2009 ). p. corylifolia has been the part of many ayurvedic formulation that are used for the treatment of various central nervous system conditions such as for neurotropic activity and as central nervous system protective agent (goel & ojha, 2015) . based on such report, a study was conin another study, it was revealed that ibc, a flavonoid from p. corylifolia, has the ability to ameliorate the neuronal injury in brain diseases related to inflammation, and this was accomplished through inhibition of lipopolysaccharide induced intercellular adhesion molecule-1 expression and leukocyte adhesion to brain endothelial cell by blocking toll-like receptor 4 signaling . various studies on animals showed that genistein has the ability to decrease body weight by decreasing food intake. it also reduced the fat pad weight and enhanced the apoptosis of adipose tissues. for example, one such study was conducted on ovariectomised mice. this well-known trihydroxyflavone, genistein, has also been isolated from p. corylifolia, exhibited a potential anti-obesity and obesity related low grade inflammation activities through multiple mechanisms and cell signaling pathways. p. corylifolia extract possesses anti-obesity and antdiabetic activity by its action on adipocyte life cycle, obesity-related low-grade inflammation, and oxidative stress (behloul & wu, 2013) . the well-known chinese herb p. corylifolia l. (scurfpea fruit) has been employed for the treatment of bone fractures and also for joint diseases for thousands of years. p. corylifolia also improved the pathological bone condition, hyperosteoidosis, by increasing the serum inorganic phosphate level at a dose of 30 mg/kg. it was observed previously that the extract markedly decreased osteoid volume and there has been improvement in bone calcification (miura, nishida, & linuma, 1996) . the flavonoids of corylin and bavachin has the osteoblastic proliferation stimulating activity in umr106 cell line cultured in vitro (cho et al., 2001; wang, li, & jiang, 2001) . p. corylifolia extract when administered orally to ovx rats, it was noted that there was a decrease in urinary calcium excretion and serum osteocalcin at a dose of 25-50 mg/kg body weight. the experiments in this study showed that the extract also increased the bone mineral density and bone formation at 50 mg/kg bw (tsai et al., 2007) . such experiment, which were extended to 3 months, it can be concluded that p. corylifolia extract can be used at postmenopause state to prevent the osteoporosis. a further in depth study is still needed to make a therapeutic candidate. this study leads to the isolation of the compound responsible for the mentioned activity by weng and colleagues. the isolated compounds from p. corylifolia, known as bakuchiol and bavachin, showed to prevent the estrogen deficiency by inducing the upregulation in primary human osteoblast differentiation (weng et al., 2015) . an advanced herbal formula containing psoraleae fructus has previously showed promising bone protecting effect when tested in rats, and later on showed excellent results in women with osteoporosis. this herbal formula could efficiently have promoted the osteogenesis and suppress the adipogenesis in mesenchymal stem cells (siu et al., 2013 ). an hplc analysis was carried out to study the important constituents in scurfpea fruit. in the method employed, the compounds were identified by comparing their retention indexes with standard substances and it resulted in the identification of 11 compounds. the biological methods such as mtt and alp were employed to study the osteoblasts proliferation and differentiation activity. bavachin and isobavachin showed significant cell proliferation by stimulation, the compound bakuchiol displayed higher effect to boost osteoblasts differentiation. from the results, it was hypothesized that prenyl group as a side chain might be responsible for the activity mentioned, because this structural component was found as a common entity among the compounds tested for activity (li et al., 2014) . previous surveys revealed bakuchiol and bavachin, the two important components of p. corylifolia l., showed osteoblastic property. both the compounds displayed the prevention of bone loss caused by deficiency of estrogen in overiectomized animal models such as rats. one of in vitro study proposed that bavachin and bakuchiol caused the induction of primary human osteoblast differentiation by up regulating the wnt signaling pathway. this research proposes that boneprotective role makes these two compounds a promising and safe estrogen supplement for the estrogen replacement therapy (weng et al., 2015) . in one investigation, the compound psoralen with different concentrations (1, 10, and 100 μm) was tested on chondrocytes isolated from rats at 3-and 9-day intervals. it was found that at the low dose concentration psoralen was safe toward chondrocytes; however, at higher dose suppression of chondrocytes, proliferation was observed. the compound psoralen also increased the synthesis of type ii collagen at 100 μm, by 0.48-fold on day 3 and 0.56-fold on day 9. this was a detailed study, including mts assay, alcian blue colorimetry, western blotting, and qrt-pcr. the compound psoralen was also tested for cytotoxicity and exhibited low cytotoxicity toward chondrocytes at a dose range of 1-10 μm. a much higher dose of 100 μm suppression of chondrocytes proliferation was observed. the compound psoralen caused the inhibition of the type i collagen in gene expression and also in protein synthesis. it was concluded that psoralen could be an important biological compound for triggering the cartilaginous cellular functions of chondrocytes . the (jeong et al., 2013) . this was the first kind of study on p. corylifolia. the plant p. corylifolia extract neutralized the coagulation of caused by naja naja karachiensis snakebite when compared with the antidote used as a standard. the snake venom was experimented on human plasma (citrated) to evaluate its effect on activated partial thromboplastin time (aptt), prothrombin time (pt), and thrombin time (tt). snake venom (200 μg/ml) was found to delay pt (13 ± 0.57 to 23 ± 0.57 sec), aptt (35 ± 1.52 to 48 ± 2.0 sec), and tt (13 ± 0.57 to 33 ± 0.57 sec). pt and tt were prolonged, and it suggested the occurrence of thrombin-like or plasminogen activating enzymes (asad et al., 2013; asad et al., 2014) . a further in depth study of this activity is still underway in the author's laboratory. the extract of seeds of p. corylifolia have been reported to have stimulant activity against natural killer cells when tested in mice. this study report that the extract also modulates the antibody dependent cellular toxicity. during tumor development, the seed extract also inhibited the antibody complement mediated cytotoxicity. the study was conducted on balb/c male mice. the dose of 100 mg/kg was administered intraperitoneally. blood collected from punctured heart and serum was separated to study the antibody complement-mediated cytotoxicity. the natural killer cells were removed from spleen, and antibodydependent cellular cytotoxicity was assessed (latha, evans, panikkar, & jayavardhanan, 2000) . the isolated compounds from p. corylifolia including arylcoumarin and psoracoumestan showed strong anticancer potential by strongly hepatocarcinoma cells, it showed its inhibitory activity by inducing the mechanism of apoptosis (guo, liu, ye, & han, 2011; jiang & xiong, 2014; khan, iqbal, ahmed, & jamil, 2015; mohammadparast, rustaiee, rasouli, zardari, & agrawal, 2014; nehybova, smarda, & benes, 2014; rajan, tripathi, variyar, & pandey, 2014; tang et al., 2011; wong & rabie, 2011; yang et al., 2012) . similarly, two more compounds from the same specie identified as ibc and bcn attenuate aβ42-induced cell toxicity. the investigation was carried out on yeast two-hybrid system (chen et al., 2013) . during this study, eight compounds were tested, and among them ibc (3 μm) and bcn (30 μm) were proved to be active at non-toxic concentrations. the results were confirmed by a standard tht fluorescence method. the compound ibc also exhibited strong inhibitory effect on tht fluorescence, and bcn showed more efficient activity, which was comparable with the reference. moreover, the determination of ic 50 of ibc and bcn in the tht assay were about 25 and 45 μm, respectively. psoralidin in another study, caused the generation of reactive oxygen and it also thought to cause the inhibition of a549 cell proliferation. the method adopted was mtt assay. this study was designed to study the relationship of time and concentration used. the ic 50 values obtained after 24-, 48-and 72-hr treatment were 19.2, 15.4, and 11.8 μm, respectively. (hao, zhang, zhao, & chen, 2014) . another evidence of anticancer activity came from the compound bakuchiol that suppressed the testosterone induced cell proliferation and gene expression in lncap cells. this study was designed to explore the bakuchiol action in the androgen-dependent pca cell line (lncap). mtt assay and real-time pcr method were employed. the ic 50 of bakuchiol to androgen receptor was 8.87 × 10 4 , which was similar to the standard flutamide (10.00 × 10 4 ; miao et al., 2013) . these experiments showed that bakuchiol is a useful agent for drug development for androgen dependent pca. the same compound, bakuchiol, has also showed a strong anticancer action against human lung adenocarcinoma cell line a549 and showed much better results than its analogue resveratrol. ic 50 of bakuchiol at 72 hr was 9.58 ± 1.12 μmol/l, much lower than that of resveratrol (33.02 ± 2.35 μmol/l). bakuchiol triggered the process of apoptosis to a higher level, compared with resveratrol. it was also noted that oxygen species related apoptosis also contribute the cytotoxic properties of bakuchiol, and therefore, it also supports the use of bakuchiol against non-small-cell lung cancer. (chen et al., 2010) . bakuchiol also showed very selective clearance activity in hepatic stellate cells by a mechanism involving apoptosis. this study showed that p. corylifolia also has anticancer activity in liver cancer (chen et al., 2010; yang, paik, cho, cho, & kim, 2008) . in search of cytotoxic compounds from p. corylifolia, two isoflavnoids were isolated, named as corylifols d and e from the ethyl acetate extract. similarly, psoralidin was also found active against stomach carcinoma cell lines (teschke, wolff, frenzel, & schulze, 2014; yang et al., 1996) . bakuchiol is also an active ingredient of the dried ripe fruit of p. corylifolia that and corylin from p. corylifolia has been investigated against udp-glucuronosyltransferases and showed strong inhibition (shan et al., 2014) . it also inhibits lipopolysaccharide-induced endothelial-mesenchymal transition via down regulation of the nf-κb-snail signaling pathway (jung et al., 2015) . in one investigation against dna polymerase enzyme, p. corylifolia extract showed strong inhibitory activity. importance of isolated components from p. corylifolia is obvious from the fact that compounds such as psoralen is being produced from callus derived from various plant portions. in one experiment, it was found that in the in vitro conditions, cinnamic acid proved a very strong precursor of psoralen pathway that induced a maximum amount of psoralen (mohammadparast, rustaiee, rasouli, zardari, & agrawal, 2014 succeeding experiments with many kinase inhibitors propose that pf-mediated degradation of cyclin d1 and cdk4 is dependent on erk1/2 and/or gsk3β (park, sung, song, & jeong, 2016) . the crude extract of p. corylifolia with solvent ethanol was found a strong dna polymerase inhibitor of dna replication enzyme in an activity directed isolation assay that resulted in the purification of novel compound corylifolin, bakuchiol, neobavaisoflavone, and resveratrol. in a similar enzyme assay, some topoisomerase ii inhibitors were also isolated, namely, daidzein and bakuchicin (sun, woo, cassady, & snapka, 2003) . many studies have been designed so far to study the behavior of p. corylifolia extract and isolated compounds on important enzyme. additionally, the inhibition kinetics were calculated by dixon and lineweaver-burk plots for the inhibitory activities toward ces1. the inhibition kinetic parameters (k i ) were calculated to be 5.3, 9.4, 1.9, 0.7, and 0.5 μm for neobavaisoflavone, corylifolinin, coryfolin, corylin and bcn, respectively. it was concluded that this inhibition by the constituents might be responsible for the possible adverse effects of fp through the disrupting ces1-catalyzed metabolism of endogenous substances and xenobiotics (sun et al., 2016) . cytochrome p450 (cyp) is an assembly of heme-containing enzymes fixed essentially in the lipid bilayer of the endoplasmic reticulum, and helps metabolize several drugs and carcinogens. the plant p. corylifolia has been reported to be used in cardiac problems in traditional medicine (kr, 1975) . bavachalcone, a compound from p. corylifolia, has reported to have increased the luciferase activity of the manganese superoxide dismutase (mnsod) promoter and enhanced mnsod mrna and protein expressions. further, it was found that bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. on the other hand, bavachalcone (in concentration range of 1, 2.5, and 5 μmol/l for 24 hr) stimulated liver kinase b1 and ampkα phosphorylation in a converse manner. mrna interfering by using short hairpin rna (shrna) of amp-activated protein kinase (ampk) inhibited bavachalcone-induced mnsod expression. moreover, ampk reduced by shrna-ampk reversed the inhibition of bavachalcone on mitochondrial superoxide production in endothelial cells. these results showed that bavachalcone could shield the endothelial function by enhancing the ampk activity and mnsod expression and lowering the mitochondrial oxidative stress, which is considered to be the key etiological reason in cardiovascular diseases (dang et al., 2015) . many herbal remedies, including essential oils, contain psoralen, and methoxy psoralen are reported to cause photosensitivity in the form of erythema and blisters (koh & ong, 1999) . p. corylifolia has been the part of many herbal remedy used to treat vitiligo and patients using such treatments in the form of creams are also reported to experienced erythema when exposed to sunlight (maurice & cream, 1989) . p. corylifolia is frequently indicated for vitiligo in india, and it contains psoralen, isopsoralen, and psoralidin. its extracts when tested on guinea pig skin have been stated to possess potent sensitizing action (pathak, daniels, & fitzpatrick, 1963) . another study reported the gonadal toxicity. although the ethanol extract of p. corylifolia seeds are proposed to use in processed food preservation, but it showed toxicity when tested on male and female rats. the period of the experiment was 90 days, and various concentrations were administered were 0%, 0.375%, 0.75%, 1.5% or 3.0%. histopathological investigation showed atrophy of seminiferous tubules, leydig cells, seminal vesicles, and prostate cells were observed in male rats administered with the 1.5% and 3.0%. with the same concentrations, the female rats showed a reduced number of corpora lutea in the ovaries and less frequent endometrial glands in the uterus. it was suggested that the extract caused the hypothalamus-pituitary-gonadal axis (takizawa et al., 2002) . in one investigation, p. corylifolia and its natural compounds (bavachin, corylifol a, neobavaisoflavone, ibc, and bcn) were evaluated for its potential toxicity, and results showed it had a potent inhibitory effect against human udp-glucuronosyltransferase 1a1 (ugt1a1), and it is considered as main stimulant for p. corylifolia related toxicity, including hepatic injury and raised bilirubin levels . in another study p. corylifolia extract and fractionated compounds such as psoralen and isopsoralen were incubated with the recombinant cyp3a4 enzyme or differentiated huh-7 and heparg cells. p. corylifolia extract, psoralen, and isopsoralen caused the inhibition of concentration cyp3a4 activity in a dose dependent manner with different potency in vitro. it was also noted that none of the sample tested showed any toxicity (liu & flynn, 2015) . several compounds isolated from p. corylifolia have many industrial applications and are available commercially. some of the examples are the following: the compound angelicin (cas 523-50-2) is available as antifungal compound (sardari, amin, sekhon, micetich, & daneshtalab, 2000) . the compound psoralen has been used as photochemical probe in studies of dna mutation and repair mechanisms (zarmouh, eyunni, & soliman, 2017) . methoxsalen (8-methoxypsoralen; cas 298-81-7) has been known as a potent suicide inhibitor of cytochrome p-450 (toyooka & ibuki, 2009 ). bakuchiol (cas 10309-37-2) is available as a ptp1b and dna polymerase inhibitor (choi et al., 2015) . bavachin (cas 19879-32-4 ) is available as a weak antioxidant that stimulates bone formation (jing et al., 2017) . corylifol c and, to a lesser extent, xanthoangelol are potent protein kinase inhibitors (inhibitory concentration 50% values for epidermal growth factor receptor; limper et al., 2013) . a well-known compound daidzin (cas 552-66-9) isolated from many plant species is a potent inhibitor of human mitochondrial aldehye dehydrogenase that demonstrates chemopreventitive activities (keung & vallee, 1993) . the compound genistein (cas 446-72-0) is available as a highly specific inhibitor of protein tyrosine kinase (zarmouh, messeha, elshami, & soliman, 2016) . these examples clearly show the importance of bioactive compounds isolated from p. corylifolia, an important traditional medicinal plant. the above-mentioned summary about p. corylifolia clearly showed that it is a very important plant from ethnobotanical, pharmacological, and chemical point of view. to date, more or less hundreds compounds have been separated from p. corylifolia, which was also mentioned by zhang et al., (2016) . we here presented the latest version of scientific literature on p. corylifolia, including the research work carried out in the recent years. p. corylifolia contains a wide variety of chemical constituents belonging to various groups, including flavonoids, coumarins, and meroterpenes, which are more dominant. p. corylifolia l. (fabacese) is a fortified source of biological active compounds, which gives the plant with great value for its use in pharmaceuticals, health, and body-care products. sehrawat and colleagues reported that p. corylifolia is a rare and endangered herbaceous medicinal plant. because most of the plant materials are collected from naturally occurring stands and are therefore being depleted rapidly and there is a possibility of extinction. therefore, it is suggested that there is a need for in vitro propagation of this species (sehrawat et al., 2014) . authors declare that there is no conflict of interest. anti microbial activity of different dosage forms of bakuchi (psoralea corylifolia linn.) taila, an ayurvedic formulation medicinal plants of bombay presidency efficacy of seed hydropriming with phytoextracts on plant growth promotion and antifungal activity in maize antipsoriatic microemulsion gel formulations for topical drug delivery of babchi oil (psoralea corylifolia) phytochemical studies and antimicrobial screening of non/less-polar fraction of psoralea corylifolia by using gc-ms psoralen photochemotherapy of cutaneous disorders compensatory effects of medicinal plants of pakistan upon prolongation of coagulation assays induced by naja naja karachiensis bite anti-venom potential of pakistani medicinal plants: inhibition of anticoagulation activity of naja naja karachiensis toxin biosynthesis of bakuchiol, a meroterpene from psoralea corylifolia medicinal and poisonous plants of pakistan genistein: a promising therapeutic agent for obesity and diabetes treatment systema naturae antibacterial activity of psoralea corylifolia l. seed and aerial parts with various extraction methods bakuchiol: a retinol-like functional compound, modulating multiple retinol and non-retinol targets new isoflavones and bioactive constituents from the fruits of psoralea corylifolia isobavachalcone and bavachinin from psoraleae fructus modulate aβ42 aggregation process through different mechanisms in vitro anti-tumor effects of bakuchiol, an analogue of resveratrol, on human lung adenocarcinoma a549 cell line bakuchiol: a hepatoprotective compound of psoralea corylifolia on tacrine-induced cytotoxicity in hep g2 cells prediction of drug-induced liver injury in hepg2 cells cultured with human liver microsomes psoralea corylifolia l. 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of psoralea corylifolia (somraji) and seed of trigonella foenum-graecum l.,(methi) in separate and composite manner in streptozotocin-induced diabetic male albino rat evaluation of a novel herbal formulation in the treatment of eczema with psoralea corylifolia ashtang ghrita: a noble ayurveda drug for central nervous system inhibitory effects of osthole, psoralen and aconitine on invasive activities of breast cancer mda-mb-231bo cell line and the mechanisms. zhong xi yi jie he xue bao= quality standards of indian medicinal plants corylinal: a new isoflavone from seeds of psoralea corylifolia phytochemical analysis of methanol extracts of psoralea corylifolia psoralidin induces autophagy through ros generation which inhibits the proliferation of human lung cancer a549 cells antimicrobial activity of leaf extract of psoralea corylifolia l the presence of three isoflavonoid compounds in psoralea corylifolia the pharmacology of chinese herbs neuroprotective effects of psoralea corylifolia linn seed extracts on mitochondrial dysfunction induced by 3-nitropropionic acid cytotoxicity of corylifoliae fructus. ii. cytotoxicity of bakuchiol and the analogues studies on lymphangiogenesis inhibitors from korean and japanese crude drugs induction of apoptosis in human hepatocarcinoma smmc-7721 cells in vitro by psoralen from psoralea corylifolia isobavachalcone attenuates mptp-induced parkinson's disease in mice by inhibition of microglial activation through nf-κb pathway medicinal plants. oxford and ibh publishing medicinal plants. tropical horticulture plant systematics: a phylogenetic approach aqueous extract of psoralea corylifolia l. inhibits lipopolysaccharideinduced endothelial-mesenchymal transition via downregulation of the nf-κb-snail signaling pathway handbook of ayurvedic medicinal plants: herbal reference library in vitro antimicrobial activities of bakuchiol against oral microorganisms. antimicrobial agents and chemotherapy daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase antioxidant, hemolytic and mutagenic potential of psoralea corylifolia encyclopedia of indian medicinal plants indian medicinal plants: an illustrated dictionary pesticidal activity of a novel coumestan derivative isolated from psoralea corylifolia linn. against tribolium casteneum herbst. adults and larvae (coleptera: tenebrionidae) antibacterial compounds from the seeds of psoralea corylifolia phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia protective effects of the compounds isolated from the seed of psoralea corylifolia on oxidative stress-induced retinal damage antimicrobial effect of commercially available mouth rinsing solutions and natural herbal extracts on streptococcus mutans selective inhibition of bakuchicin isolated from psoralea corylifolia on cyp1a in human liver microsomes. evidence-based complementary and alternative medicine phytophotodermatitis due to the application of citrus hystrix as a folk remedy indian medicinal plants the wealth of india: raw materials: vol. viii. ph-re. the wealth of india: raw materials cytotoxicity of corylifoliae fructus. i. isolation of the effective compound and the cytotoxicity immunomodulatory and antitumour properties of psoralea corylifolia seeds two antifungal components isolated from fructus psoraleae and folium eucalypti globuli by bioassay-guided purification anti-dermatophytic activity of bakuchiol: in vitro mechanistic studies and in vivo tinea pedis-inhibiting activity in a guinea pig model isobavachalcone attenuates lipopolysaccharide-induced icam-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways phytoestrogen bakuchiol exhibits in vitro and in vivo anti-breast cancer effects by inducing s phase arrest and apoptosis osteoblasts proliferation and differentiation stimulating activities of the main components of fructus psoraleae corylifoliae fructus psoraleae contains natural compounds with potent inhibitory effects towards human carboxylesterase 2 bakuchiol contributes to the hepatotoxicity of psoralea corylifolia in rats estrogenic activities of psoralea corylifolia l. seed extracts and main constituents compounds isolated from psoralea corylifolia seeds inhibit protein kinase activity and induce apoptotic cell death in mammalian cells analysis of bakuchiol, psoralen and angelicin in crude drugs and commercial concentrated products of fructus psoraleae two new benzofuran derivatives, corylifonol and isocorylifonol from the seeds of psoralea corylifolia antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of psoralea corylifolia against ichthyophthirius multifiliis in goldfish antimalarial activity of artemisia annua flavonoids from whole plants and cell cultures preparative isolation and purification of psoralen and isopsoralen from psoralea corylifolia by high-speed counter-current chromatography psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from psoralea corylifolia cyp3a4 inhibition by psoralea corylifolia and its major components in human recombinant enzyme, differentiated human hepatoma huh-7 and heparg cells useful plants of the genus psoralea granzyme a cleaves a mitochondrial complex i protein to initiate caspaseindependent cell death the dangers of herbalism potential medicinal plants for lymphatic filariasis: a review natural sources used for treatment and prevention of filariasis bakuchiol inhibits the androgen induced-proliferation of prostate cancer cell line lncap through suppression of ar transcription activity effect of crude fractions of psoralea corylifolia seed extract on bone calcification effect of crude fractions of psoralea corylifolia seed extract on bone calcification in vitro enhancement of psoralen as an important anticancer compound in psoralea corylifolia through precursor feeding indian materia medica with ayurvedic plant coumestans: recent advances and future perspectives in cancer therapy. anti-cancer agents in medicinal chemistry the evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from psoralea corylifolia and sanguinaria canadensis phytochemical and pharmacognostic investigation of antidiabetic scoparia dulcis linn scrophulariaceae whole plant grown in nigeria herbs cultivation and medicinal uses protective effect of (s)-bakuchiol from psoralea corylifolia on rat liver injury in vitro and in vivo anti-cancer activity of psoralea fructus through the downregulation of cyclin d1 and cdk4 in human colorectal cancer cells the presently known distribution of furocoumarins (psoralens) in plants larvicidal property of essential oils against culex quinquefasciatus say (diptera: culicidae). industrial crops and products ayurvedic medicine: the principles of traditional practice antidermatophytic activity of extracts from psoralea corylifolia (fabaceae) correlated with the presence of a flavonoid compound potential antifilarial activity of the leaves and seeds extracts of psoralea corylifolia on cattle filarial parasite setaria cervi chemical fingerprint and quantitative analysis of fructus psoraleae by high-performance liquid chromatography mechanism of cytotoxicity by psoralea corylifolia extract in human breast carcinoma cells medicinal plants history, cultivation and uses studies on the chemical constituents of psoralea corylifolia l screening for inhibition activity of plant extracts on microorganism contaminating in cosmetics isolation and identification of furocoumarins from the seeds of psoralea corylifolia linn antifungal activity of diplotaenia damavandica antifungal potentiality of some plant extracts against fusarium sp plant-derived antimicrobial compounds: alternatives to antibiotics psoralea corylifolia l. an endangered medicinal plant with broad spectrum properties protective role of psoralea corylifolia l. seed extract against hepatic mitochondrial dysfunction induced by oxidative stress or aging comparison of the inhibitory potential of bavachalcone and corylin against udp-glucuronosyltransferases database on medicinal plants used in ayurveda agro-techniques of medicinal plants determination of isoflavone content and antioxidant activity in psoralea corylifolia l. callus cultures the effects of an antiosteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus on density and structure of rat long bones under tail-suspension, and its mechanisms of action optimization of elicitation condition with jasmonic acid, characterization and antimicrobial activity of psoralen from direct regenerated plants of psoralea corylifolia l systema naturae the netherlands in vitro acetylcholinesterase inhibition by psoralen using molecular docking and enzymatic studies in vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from psoralea corylifolia against ichthyophthirius multifiliis in fish antifungal activity of phenyl derivative of pyranocoumarin from psoralea corylifolia l. seeds by inhibition of acetylation activity of trichothecene 3-o-acetyltransferase (tri101) pharmacognostic evaluation of the root of berberis asiatica fabaceae" angiosperm phylogeny website, 13 ed. missouri botanical garden and university of inhibition behavior of fructus psoraleae's ingredients towards human carboxylesterase 1 (hces1) dna polymerase and topoisomerase ii inhibitors from psoralea c orylifolia gonadal toxicity of an ethanol extract of psoralea corylifolia in a rat 90-day repeated dose study psoralen stimulates osteoblast differentiation through activation of bmp signaling review article: herbal hepatotoxicity-an update on traditional chinese medicine preparations new constituents from psoralea corylifolia histone deacetylase inhibitor sodium butyrate enhances the cell killing effect of psoralen plus uva by attenuating nucleotide excision repair psoralea corylifolia extract ameliorates experimental osteoporosis in ovariectomized rats screening south indian medicinal plants for antifungal activity against cutaneous pathogens osteoblastic proliferation stimulating activity of psoralea corylifolia extracts and two of its flavonoids chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= chemical constituents from psoralea corylifolia and their antioxidant alphaglucosidase inhibitory and antimicrobial activities identification and characterization of naturally occurring inhibitors against udp-glucuronosyltransferase 1a1 in fructus psoraleae (bugu-zhi) indian medicinal plants. a compendium of 500 species positive skeletal effect of two ingredients of psoralea corylifolia l. on estrogen deficiency-induced osteoporosis and the possible mechanisms of action bioactive metabolites from the fruits of psoralea corylifolia effect of psoralen on bone formation bisbakuchiols a and b, novel dimeric meroterpenoids from psoralea corylifolia effects of gaultheria yunnanensis on adjuvant arthritis in rats. zhongguo zhong yao za zhi= zhongguo zhongyao zazhi= psoralen activates cartilaginous cellular functions of rat chondrocytes in vitro psc-afp, an antifungal protein with trypsin inhibitor activity from psoralea corylifolia seeds resveratrol induces the suppression of tumor-derived cd4+ cd25+ regulatory t cells the cytotoxicity of psoralidin from psoralea corylifolia the osteoprotective effect of psoralen in ovariectomy-induced osteoporotic rats via stimulating the osteoblastic differentiation from bone mesenchymal stem cells antidepressant-like effects of psoralidin isolated from the seeds of psoralea corylifolia in the forced swimming test in mice psoracorylifols a-e, five novel compounds with activity against helicobacter pylori from seeds of psoralea corylifolia antibacterial prenylflavone derivatives from psoralea corylifolia, and their structure-activity relationship study lipid class and fatty acid composition of psoralia corylifolia seed the benzopyrone biochanin-a as a reversible, competitive, and selective monoamine oxidase b inhibitor evaluation of the isoflavone genistein as reversible human monoamine oxidase-a and-b inhibitor. evidence-based complementary and alternative medicine the chemical constituents and bioactivities of psoralea corylifolia linn.: a review psoralea corylifolia l: ethnobotanical, biological, and chemical aspects: a review key: cord-003341-z5w56zeq authors: nguyen, thi thanh hanh; lee, sun; wang, hsi-kai; chen, hsin-yen; wu, ying-ta; lin, simon c.; kim, do-won; kim, doman title: in vitro evaluation of novel inhibitors against the ns2b-ns3 protease of dengue fever virus type 4 date: 2013-12-13 journal: molecules doi: 10.3390/molecules181215600 sha: doc_id: 3341 cord_uid: z5w56zeq the discovery of potent therapeutic compounds against dengue virus is urgently needed. the ns2b-ns3 protease (ns2b-ns3(pro)) of dengue fever virus carries out all enzymatic activities needed for polyprotein processing and is considered to be amenable to antiviral inhibition by analogy. virtual screening of 300,000 compounds using autodock 3 on the gvss platform was conducted to identify novel inhibitors against the ns2b-ns3(pro). thirty-six compounds were selected for in vitro assay against ns2b-ns3(pro) expressed in pichia pastoris. seven novel compounds were identified as inhibitors with ic(50) values of 3.9 ± 0.6–86.7 ± 3.6 μm. three strong ns2b-ns3(pro) inhibitors were further confirmed as competitive inhibitors with k(i) values of 4.0 ± 0.4, 4.9 ± 0.3, and 3.4 ± 0.1 μm, respectively. hydrophobic and hydrogen bond interactions between amino acid residues in the ns3(pro) active site with inhibition compounds were also identified. among members of the flavivirus genus dengue virus (denv) is responsible for the highest rate of disease and mortality and consists of a group of four serologically related viruses referred to as denv types 1-4. infection with these viruses results in a range of clinical diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome [1] [2] [3] . global epidemics of denv have occurred over the past few years. there were 390 million (95% credible interval 284-528 million infections) dengue infections per year, of which 96 million (67-136 million infections) apparently manifested some level of disease severity [4] . denv are transmitted to humans by the bite of infective female mosquitoes of the genus aedes. denv has a single-stranded rna genome that is packaged by the virus capsid protein in a hostderived lipid bilayer and is surrounded by 180 copies of two glycoproteins. the complete virion is approximately 50 nm in diameter and contains an approximate 10.7 kb positive-sensed rna genome that has one open reading frame encoding a single polyprotein [2] . the 5'-end encodes three structural proteins: the capsid (c); membrane precursor protein (prm) proteolytically cleaved by the host protease furin to form the membrane protein in mature virions; and the envelope (e), constituting the enveloped virus particle [2, 5] . seven non-structural (ns) proteins essential for viral replication are encoded by the remainder of the genome. the order of proteins encoded is 5'-c-prm-e-ns1-ns2a-ns2b-ns3-ns4a-ns4b-ns5-3' [5, 6] . ns3 pro is responsible for cleaving both in the cis and trans directions to generate viral proteins that are essential for viral replication and maturation of infectious dengue virions. a number of different strategies have been employed to search for denv ns2b-ns3 protease inhibitors, including high-throughput screening [7] [8] [9] , synthesizing rationally designed substrate-based peptidomimetics [10, 11] , cyclopeptide [12] , and structure-based virtual screening (sbvs) [13] [14] [15] as well as screening natural products [16, 17] . however, only a few peptide or small molecule inhibitors of the denv ns2b-ns3 protease with moderate activity have so far been reported [18, 19] . there are currently no effective antiviral agents to treat dengue infection [20] as well as no licensed vaccine against dengue infection is available, and the most advanced dengue vaccine candidate did not meet expectation in a recent large trial [4, 21, 22] . over the past decade, high-throughput virtual screening (vs), and particularly substrate-based virtual screening (sbvs) has emerged as a reliable, cost-effective and time-saving technique for discovering lead compounds as an alternative to high-throughput screening [23] . vs, as applied to new enzyme inhibitor discovery, involves docking, computational fitting of compound structures to the enzyme active site, and scoring and ranking of each compound [24, 25] . grid application platform virtual screening service (gvss) has been developed with the autodock 3.0.5 docking engine [26] . thus, a user friendly grid service, gvss, was developed to conduct large-scale molecular docking easily [26] . in this study, ns2b-ns3 pro was expressed in pichia pastoris culture. this enzyme was used for in vitro enzyme inhibition assays against 36 compounds selected from vs of ns3 pro using the gvss. the novel compound inhibitors were tested for their ic 50 values and used for an enzyme kinetic study. 2.1. expression of active ns2b-ns3 pro in p. pastoris denv4 ns2b-ns3 pro , fused to the α-factor secretion signal sequence and placed under the control of the methanol inducible alcohol oxidase promoter, was constructed to express the secreted protein in p. pastoris. the expressed ns2b-ns3 pro was 45 kda based on western blot using the anti-his antibody ( figure 1 ). the ns2b-ns3 pro proteolytic activity was monitored with fluorogenic tetrapeptide substrate containing the 7-amino-4-methylcoumarin (amc) leaving group (benzoyl-norleucine-lysinearginine-arginine-7-amino-4-methylcoumarin, bachem, bubendorf, switzerland) for an increase in fluorescence at a λ ex = 380 nm and λ em = 460 nm at 37 °c. no enzyme activity was detected before induction or in the p. pastoris negative control without ns2b-ns3 pro gene (data not shown). ns2b-ns3 pro activity increased steadily with increasing culture time (and induction time) and reached a final activity of 480 u·l −1 after 96 h of induction with methanol. ns2b-ns3 pro was purified from p. pastoris culture supernatants. after dialysis to remove the potassium phosphate buffer, 77.6 mg of proteins remained in the culture medium supernatant. a total of 9.7 mg of ns2b-ns3 pro was isolated by ammonium sulfate precipitation. the final preparation of ns2b-ns3 pro was 1.4 mg following ion-exchange chromatography using a deae-sepharose column, which resulted in a 13.5-fold purification ( table 1 ). the recombinant protein was expressed in approximately 0.8 mg·l −1 of the induced culture medium and specific activity of the purified recombinant ns2b-ns3 pro was 59.3 u·mg −1 . the michaelis-menten constant (k m ) derived with amc peptide substrates from the lineweaver-burk plot was 1.60 ± 0.1 μm ( figure s1 ). gvss was developed with the autodock 3.0.5 docking engine [26] . a java-based graphical user interface and grid application platform (gap) allows end-users to specify target and compound libraries, set up docking parameters, monitor docking jobs and computing resources, visualize and refine docking results, and download the final results. gvss was designed for conducting large-scale molecular docking more easily by providing a user-friendly grid service [26] . a total of 300,000 compounds were used for the large-scale screening with gvss against ns3 pro protein (pdb accession number 2vbc [27] ) by using 4,167 cpu per day, and the results were completed in 60 days. a top scoring of 10% was selected from the first post-docking filtering strategy based on the free binding energy of the lowest energy conformation. thirty-six compounds were selected for in vitro assay based on their free energy binding (table s1 ) and h-bonds interactions with amino acid residues at the ns3 pro active site. each compound obtained after vs was tested in duplicate at 100 μm for its ability to inhibit ns2b-ns3 pro activity. the primary inhibition assay of the 36 selected compounds is shown in table s2 . among them, seven compounds that showed higher inhibition activities against ns2b-ns3 pro were selected for determining their ic 50 values ( table 2 ). their physicochemical properties were shown in table s2 . the chemical structure of each compound is depicted in table 2 . the seven compounds displayed ns2b-ns3 pro inhibitory activities with ic 50 values of 3.9 ± 0.6-86.7 ± 3.6 μm ( table 2) . compounds 2, 14, and 22, which displayed over 90% inhibition activity at 100 μm, were subjected to kinetic characterization. inhibitory kinetic experiments were performed at different constant inhibitor concentrations and different substrate concentrations. lineweaver-burk and dixon plots were used to analyze the inhibition modes of these compounds. compounds 2, 14, and 22 were analyzed and compared by molecular docking as potent binders to the ns3 pro active site pocket ( figure 3a) . figure 3b provides the details of the specific interactions between compound 2 and ns3 pro ; carbon atoms of compound 2 formed hydrophobic interactions with met49, trp50, val72, arg73, asp75, pro132, gly151, asn152, ala164, and thr166 of ns3 pro . the the ns2b-ns3 pro gene was synthesized for p. pastoris after codon optimalization (dna2.0, menlo park, ca, usa) based on the amino acid sequence of ns2b-ns3 pro (ambl aaw30973.1) [28] . the protein encoding the ns2b-ns3 pro comprises 49 ns2b amino acid residues (amino acid residues 1393-1441), which are linked by a flexible ggggsgggg linker with the 186 ns3 pro amino acid residues (amino acid residues 1475-1660) ( figure s3 ). the ns2b-ns3pro gene (730 bp) was isolated from the vector (pj201) with the ns2b-ns3 pro gene by cutting with ecori/noti (takara, shiga, japan) and was ligated into the ecori/noti-digested ppiczαa vector (invitrogen, carlsbad, ca, usa). this construct allowed secretion of ns3 pro into the culture medium because of the presence of an in frame n-terminal α-factor secretion signal peptide. the novel construct was named ppiczαa-ns2b-ns3 pro and transformed into e. coli dh5α (invitrogen) using a standard heat shock method. transformants harboring ppiczαa-ns2b-ns3 pro were selected from low salt medium lb agar [1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) nacl, ph 7.5] containing 25 μg·ml −1 zeocintm (invitrogen). the ppiczαa-ns2b-ns3pro plasmid was amplified in e. coli dh5α, linearized by saci digestion, and transformed into p. pastoris gs115 using a modified lithium chloride method. the ppiczαa vector was also linearized by saci digestion and transformed into p. pastoris gs115 as a negative control strain. after transformation, cells were plated on ypd agar [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose] containing 100 µg zeocin·ml −1 and incubated for 3 days at 30 °c. positives were screened by polymerase chain reaction (pcr) with two sets of primers (bioneer, deajeon, korea): set one contained the ns2b-ns3pro primers [dv4-f (5'-gaattcgctgatctta gtttag-3') and dv4-r (5'-gcggccgctttctttctaaa-3')] and set two contained the α-factor and 3aox1 primers. the selected clone was inoculated for gene expression into 100 ml bmgy [1% (w/v) yeast extract, 2% (w/v) peptone, 100 mm potassium phosphate, ph 6.0, 1.34% (w/v) yeast nitrogen base, 4 × 10 −5 % (w/v) biotin, and 1% glycerol] in a 500 ml flask and incubated at 30 °c with shaking at 250 rpm for 16 h. the cells were harvested and resuspended in 500 ml bmmy [1% (w/v) yeast extract, 2% (w/v) peptone, 100 mm potassium phosphate, ph 6.0, 1.34% (v/v) yeast nitrogen base, 4 × 10 −5 % (w/v) biotin, and 0.5% (v/v) methanol] medium in a 2 l flask to an optical density at 600 nm of 1.0 and then incubated at 30 °c with shaking at 250 rpm. to maintain methanol induction by the aox1 promoter, 0.5% (v/v) methanol was fed every 24 h during the fermentation period [29] . culture aliquots (1 ml each) were collected every 24 h for analyses of ns2b-ns3 pro expression and enzyme activity. ppiczαa-transformed p. pastoris x-33 proteins were expressed as a negative control. the yeast cells were then separated from the broth by centrifugation at 8,000 ×g for 15 min at 4 °c. the pellet was discarded, and the supernatant was exchanged with 20 mm tris buffer (ph 7.5) using a 10 kda membrane (millipore, billerica, ma, usa). the supernatant was used for ammonium sulfate fractionation (from 0%-90%). the proteins obtained were dissolved in 20 mm tris buffer (ph 7.5) and dialyzed against 20 mm tris buffer (ph 7.5). concentrated protein sample was loaded onto a deae-sepharose column equilibrated with a low-salt buffer containing 50 mm nacl and 40 mm tris-hcl (ph 7.5). the recombinant protein was eluted with a salt gradient of 50-1,000 mm nacl in a buffer containing 40 mm tris-hcl (ph 7.5). one ml fractions were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and checked for enzyme activity. the fractions containing recombinant ns2b-ns3pro were pooled and concentrated using a 9,000 da molecular weight cut-off pierce ® concentrator. ns2b-ns3 pro proteolytic activity was measured in a spectramax gemini xps apparatus (molecular devices, sunnyvale, ca, usa) (λex = 380 nm, λem = 460 nm) performed in a final volume of 100 µl containing 40 mm tris-hcl (ph 7.5) and 25 µm of fluorogenic tetrapeptide substrate, benzoyl-norleucine-lysine-arginine-arginine-7-amino-4-methyl coumarin (bachem, bubendorf, switzerland), at 37 °c using a fluorescence plate reader. the proteolytic reaction was monitored by an increase in fluorescence (relative fluorescence units min-1), which was subsequently converted to µm·min −1 from a standard 7-amino-4-methyl coumarin (amc) calibration curve [30] . one unit of ns2b-ns3 pro activity was defined as the quantity of enzyme required to produce 1 μm of amc per min at 37 °c and ph 7.5 in 40 mm tris-hcl. ns2b-ns3 pro kinetic parameters were obtained using 1-20 μm amc peptide substrates in the fluorescent assay with a 20 min measurement period. reaction responses were linear within this time period. the michaelis-menten constant (k m ) was calculated from a lineweaver-burk using the sigmaplot program (systat software, san diego, ca, usa). the vs process for denv4 has been described previously [26] . the three-dimensional coordinates in the x-ray crystal structure of ns3 pro (pdb accession number 2vbc) [27] obtained from the protein data bank [26, 31] were used as the receptor model for the structural-based vs docking simulations. the ns3 pro docking library comprised 300,000 compounds from chemdiv inc. (san diego, ca, usa), a commercially available compound library, was used for vs. autodock version 3.0.5 was used for the computational molecular docking simulation of flexible small molecules to rigid proteins using ligand and rigid proteins [32, 33] . large-scale computations were conducted between 2vbc and 300,000 compounds using the gvss [26] . important docking parameters for the lamarckian genetic algorithm were a population size of 100 individuals, maximum of 1.5 million energy evaluations, maximum of 27,000 generations, mutation rate of 0.02, crossover rate of 0.8, and 50 docking runs (each docking job produced 50 docked conformations). the probability of performing a local search on an individual in the population was set to 0.06 and the maximum number of iterations per local search was set to 300. one percentage top scoring function was extracted and the potential hydrogen bond among ligands and key residues in the active site pocket of hma was identified using chimera software [34] , and selected compounds for next step were analyzed for their hydrophobic and h-bond interactions using the ligplot program [35] . among 300,000 compounds, 36 were selected for testing in vitro inhibitory activity against ns2b-ns3 pro based on hydrogen bond interactions [25, 36] . each compound was tested in duplicate at a concentration of 100 μm for its ability to inhibit ns2b-ns3 pro activity during initial screening. each compound was dissolved in dimethyl sulfoxide (dmso) as a 5 mm stock solution. assays were performed in a reaction mixture (final volume 100 μl) containing 0.04 u enzymes, 1.65 μm amc peptide substrate, 100 μm of inhibitor, and 40 mm tris buffer, ph 7.5. reactions were run for 20 min at 37 °c with continuous fluorescence monitoring using a spectramax gemini xps apparatus (molecular devices, eugene, or, usa) with excitation and fluorescence emission wavelengths of 380 nm and 460 nm, respectively. inhibitor kinetic studies were performed for compounds 2, 14, and 22 against ns2b-ns3 pro . the ns2b-ns3 pro kinetic parameters were obtained using 0.75, 1, 1.25, 1.5, and 1.65 μm amc peptide substrate in the fluorescent assay with a 20 min measurement period. reaction responses were linear within this time period. the inhibitor concentration used was 0-20 μm for compound 2, 0-25 μm for compound 14, and 0-6 μm for compound 22. the type of inhibition was determined using lineweaver-burk plots, and k i values were determined with a dixon plot (1/v as a function of [i], inhibitor concentration) from different inhibitor concentrations [36, 37] . thirty-six compounds from vs were tested for their inhibitory activities towards ns2b-ns3 pro expressed from p. pastoris. the ic 50 values of seven were 3.9 ± 0.6-86.7 ± 3.6 μm, and three of the compounds (2, 14, and 22) were competitive inhibitors of ns2b-ns3 pro with k i values of 4.0 ± 0.4, 4.9 ± 0.3, and 3.4 ± 0.1 μm, respectively. detailed docking simulation binding mode analyses showed that the inhibitors were stabilized by formation of h-bonds with catalytic residues and the establishment of hydrophobic interactions with amino acids in the ns3 pro active site pocket. more detailed inhibition studies are underway to elucidate the inhibitory mechanism of compounds 2, 14, and 22. supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/12/15600/s1. activity of recombinant dengue 2 virus ns3 protease in the presence of a truncated ns2b co-factor, small peptide substrates, and inhibitors structure of dengue virus: implications for flavivirus organization, maturation, and fusion expression of dengue virus e glycoprotein domain iii in non-nicotine transgenic tobacco plants the global distribution and burden of dengue the dengue viruses biological characteristics of dengue virus and potential targets for drug design identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen a novel, potential dengue virus ns2b/ns3 protease inhibitor, bp13944, was discovered through high-throughput screening with dengue replicon cells use of parallel validation high-throughput screens to reduce false positives and identify novel dengue ns2b-ns3 protease inhibitors peptide inhibitors of dengue virus ns3 protease. part 1: warhead peptide inhibitors of dengue virus ns3 protease. part 2: sar study of tetrapeptide aldehyde inhibitors synthesis and disulfide bond connectivity-activity studies of a kalata b1-inspired cyclopeptide against dengue ns2b-ns3 protease novel dengue virus-specific ns2b/ns3 protease inhibitor, bp2109, discovered by a high-throughput screening assay structure-guided fragment-based in silico drug design of dengue protease inhibitors discovery of novel small molecule inhibitors of dengue viral ns2b-ns3 protease using virtual screening and scaffold hopping inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue-2 virus ns3 protease structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional ns3 protein from dengue virus as a target strategies for development of dengue virus inhibitors dengue: guidelines for diagnosis, treatment, prevention and control; world health organization protective efficacy of the recombinant, live-attenuated, cyd tetravalent dengue vaccine in thai schoolchildren: a randomised, controlled phase 2b trial dengue vaccine development: a 75% solution? recent development and application of virtual screening in drug discovery: an overview virtual screening and its integration with modern drug design technologies virtual screening identification of novel severe acute respiratory syndrome 3c-like protease inhibitors and in vitro confirmation gvss: a high throughput drug discovery service of avian flu and dengue fever for egee and euasiagrid crystal structure of the ns3 protease-helicase from dengue virus recombination in the nonstructural gene region in type 2 dengue viruses efficient expression and purification of recombinant alcohol oxidase in pichia pastoris a fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease the protein data bank automated docking using a lamarckian genetic algorithm and an empirical binding free energy function inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition ucsf chimera-a visualization system for exploratory research and analysis ligplot: a program to generate schematic diagrams of protein-ligand interactions discovery of novel inhibitors for human intestinal maltase: virtual screening in a wisdom environment and in vitro evaluation flavonoid-mediated inhibition of sars coronavirus 3c-like protease expressed in pichia pastoris sample availability: all tested compounds are commercially available from chemdiv inc this work was partially supported by the national research foundation of korea (nrf) grant funded by the korea government (mest) (no. 2013056129) . the authors declare no conflict of interest. key: cord-014863-jyti99xq authors: karaküçük-i̇yidoğan, a.; aydınöz, b.; taşkın-tok, t.; oruç-emre, e. e.; balzarini, j. title: synthesis, biological evaluation and ligand based pharmacophore modeling of new aromatic thiosemicarbazones as potential anticancer agents date: 2019-05-15 journal: pharm chem j doi: 10.1007/s11094-019-01968-3 sha: doc_id: 14863 cord_uid: jyti99xq two series of new aromatic thiosemicarbazone derivatives were synthesized by condensation of n-(4-cyanophenyl)hydrazine carbothioamide (i) and n-(4-methylsulfanylphenyl)hydrazine carbothioamide (ii) with appropriate aromatic aldehydes in order to investigate their antiviral and cytostatic potency. the chemical structures of all compounds were fully characterized by elemental analysis and spectroscopic techniques. the results of the bioassays indicated that compounds id, ie, if and iif proved inhibitory against influenza virus a (ec(50) = 13 – 27 μg/ml for strain h1n1 and 9.3 – 18 μg/ml for strain h3n2). compounds ig and iig were the most cytostatic compounds with inhibition of hela cell proliferation at an ic(50) = 0.3 μg/ml for ig and 1.9 μg/ml for iig. especially, compound ig showed the highest cytostatic activity with ic(50) of 0.30, 0.70 and 2.50 μg/ml against hela, cem and l1210 cell lines, respectively. this inhibition range was within the same order of magnitude as that for cisplatin. furthermore, molecular modeling was carried out to examine the cytostatic activity and determine the best pharmacophore model as a guide for the design and development of potential prodrugs in future studies. thiosemicarbazones are important class of synthetic products and potential biologically active compounds. domagk, et al. [1] first reported that thiosemicarbazone pharmacophore had antituberculosis activity. then, antiviral activity of benzaldehyde thiosemicarbazone derivatives against vaccina virus in mice was found by hamre, et al. [2] . the antiviral activity of isatin-b-thiosemicarbazone (ibt) and n-methyl-isatin-b-thiosemicarbazone (commercially known as methisazone or marboran) was widely investigated against orthopoxviruses in the 1960s [3] . after these discoveries, thiosemicarbazones draw considerable interest due to their application in the pharmaceutical chemistry and proved to be chemotherapeutic agents potentially useful for inhibiting cancer cells [4] . for example, 3-aminopyridine-2carboxaldehyde thiosemicarbazone (commercially known as triapine) inhibited the biosynthesis of dna in murine leukemia l1210 cells by blocking the activity of ribonucleotide reductase [5] . in recent years, it has been commonly accepted that agents containing more than one pharmacophore can have superior efficacy as compared to single-pharmacophore drugs [6] . pharmacophore hybridization is a method of rational drug design, and a single molecule containing different modes of action can be beneficial for the treatment of diseases. as an important pharmacophore, bis-alkylating nitrogen mustard such as chlormethine, melphalan, chlorambucil and many more have anticancer activities against hematologic tumors, myeloma, ovarian cancer and solid tumors [7] . alkylating agents are electrophilic entities that interact with nucleophilic moieties of dna resulting in the covalent transfer of an alkyl group. another pharmacophore para-substituted aryl nitrile, which is bioisostere of a ketone group, has a variety of anticancer activities, e.g., against hormonally-responsive breast cancer (letrozole), breast cancer (neratinib), prostate cancer (bicalutamide), pancreatic cancer, non-small cell lung cancer, head and neck cancer (l-778,123, phase i), and chronic myelogenous leukemia (bosutinib, ski-606, phase iii) [8] . previously, we reported in vitro cytostatic and broadspectrum antiviral activity of the thiosemicarbazones derived from 5-substitutedthiophene-2-carboxaldehydes and their platinum(ii) and palladium(ii) complexes [9] . encouraged by these results and aimed at developing effective anticancer agents, we designed two series of thiosemicarbazones by the pharmacophore hybridization method using two or more different pharmacophores in view of prospecting their cytostatic and antiviral activity (fig. 1) . in order to gain detailed information about this issue and to develop effective anticancer agents for future studies, we have performed virtual screening of the aforementioned compounds by using gaussian09 and discovery studio 3.5 software [10, 11] . the intermediates n-(4-cyanophenyl)hydrazinecarbothioamide (i) and n-(4-methylthiophenyl)hydrazinecarbothioamide (ii) were prepared from 4-cyanophenyl isothiocyanate and 4-methylthiophenyl isothiocyanate by reaction with hydrazine monohydrate, respectively [12] . n-(4-cyanophenyl)hydrazinecarbothioamide (i) was reported previously [13] and n-(4-methylthiophenyl)hydrazinecarbothioamide (ii) was commercially available. the novel thiosemicarbazones (ia -ig and iia -iig) as well as the known analog of ib [14] were synthesized in high yield (71 -92%) from the corresponding 4-substitutedbenzaldehydes by treatment with thiosemicarbazides in methanol, according to the general procedure of a previously described method [15] . the synthetic route of target compounds ia -ig and iia -iig is shown in scheme 1. the chemical structures of new compounds were throughly eludicated by elemental analysis and spectral data (uv-vis, ir, 1 h nmr, 13 c nmr, and esi-ms). assignments of selected diagnostic ir bands provided significant indication for the formation of the thiosemicarbazone deriva-tives. all thiosemicarbazone derivatives (ia-g and iia-g) exhibited two intense bands in the region of 3396 -3266 cm -1 and 3162 -3120 cm -1 due to n(n-h) stretching. the strong band at 839 -818 cm -1 was present due to n(c=s) stretching. it was suggested that thiosemicarbazones in the solid phase remain in the thione form. in addition, the characteristic azomethine stretching vibrations n(c=n) at 1587 -1543 cm -1 were observed, which also confirmed the formation of thiosemicarbazones [16] . results of the 1 h nmr integrations and signal multiplicities were in line with the proposed structures and other spectral data. the 1 h nmr spectra of ia -ig and iia -iig showed a singlet peak attributable to the =n-nh proton in arange of d = 12.60 -11.62 ppm as well as a singlet peak attributable to the phnh proton in the range d = 10.80 -9.92 ppm. the signal of the azomethine proton (hc=n) appeared as a singlet at d = 8.63 -8.04 ppm [16] . all aromatic protons were observed with the expected chemical shift (d = 8.48 -6.80 ppm) and coupling constant in the nmr spectra of all thiosemicarbazone derivatives. the 13 c nmr spectra of the thiosemicarbazones exhibited two important signals at d = 176.92 -174.23 and at d = 143.89 -142.57 ppm assigned to thioamide (c=s) and imine (c=n) carbon atoms, respectively. the signals at d = 164.01 -112.06 ppm in the spectra were assumed to be due to the aromatic carbons. the ms spectra of all thiosemicarbazones were in line with the proposed structures. the progress of all reactions were monitored by thin layer chromatography (tlc). tlc was performed on silica gel plates (merck silica gel 60, f 254 , 0.2 mm) with visualization by exposure to iodine vapor and uv light using etoac/hexane (v/v 1:1 and 1:3) as solvent system. melting points were determined on a ez-melt mpa120 automated melting point apparatus and were uncorrected. the ir spectra were recorded on a perkin elmer 100 ft-ir spectrometer with universal atr sampling accessory. the 1 h nmr and 13 c nmr spectra were obtained at room temperature with a bruker avance-dpx-400 nmr spectrometer in dmso-d 6 using tms as the internal standard. the mass spectra were obtained using an lc/ms agilent 1100 msd series spectrometer in the electrospray mode. elemental (chns) analyses were performed using a variomicro elemental analyzer. general procedure: to a hot solution of thiosemicarbazide (1.04 mmol) in methanol (25 ml) was added dropwise a solution of the appropriate aldehyde (1.04 mmol) in methanol (10 ml) with continuous stirring. after the addition of a catalytic amount of glacial acetic acid, the reaction mixture was refluxed for 6 -24 h. the progress of the reaction was monitored by tlc. the reaction mixture was cooled and the precipitate was filtered. the crude product was washed with cold diethylether or ethanol and recrystallized from appropriate solvent. table 1 orange solid (acetone). yield: 0.30 g (82%); m.p. the antiviral assays were based on the inhibition of virus-induced cytopathicity in confluent cell cultures, and the cytostatic assays on inhibition of tumor cell proliferation in exponentially growing tumor cell cultures according to previously described methods [17] . the synthesized compounds were evaluated against the following viruses: herpes simplex virus type 1 (hsv-1) strain kos, thymidine kinase-deficient (tk-) hsv-1 kos strain resistant to acv (acv r ), herpes simplex virus type 2 (hsv-2) strain g, cytomegalovirus strains ad-169 and david, varicella zoster virus (vzv) strains oka and ys, vaccinia virus lederle strain, respiratory syncytial virus (rsv) strain long, vesicular stomatitis virus (vsv), coxsackie b4 virus, parainfluenza 3 virus, influenza virus a (subtypes h1n1, h3n2), influenza virus b, reovirus-1, sindbis and punta toro virus. the antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation (for vzv) in human embryonic lung (hel) fibroblasts, african green monkey cells (vero), human epithelial cervix carcinoma cells (hela) or madin-darby canine kidney cells (mdck). confluent cell cultures in microtiter 96-well plates were inoculated with 100 ccid 50 of virus (1 ccid 50 being the virus dose to infect 50% of the cell cultures) or 20 or 100 plaque forming units (pfu) (for vzv and cmv, respectively) in the presence of varying concentrations of the test compounds. viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. antiviral activity was expressed as ec 50 (50% effective concentration) defined as the compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%). inhibition of hiv-1(iii b )-and hiv-2(rod)-induced cytopathicity in cem cell cultures was measured in microtiter 96-well plates containing~3´10 5 cem cells/ml infected with 100 ccid 50 of hiv per milliliter and containing appropriate dilutions of the test compounds. after 4 -5 days of incubation at 37°c in a co 2 -controlled humidified atmosphere, cem giant (syncytium) cell formation was examined microscopically and characterized by the ec 50 value (50% effective concentration inhibiting hiv-induced giant cell formation by 50%). all cytostatic/toxic activity assays were performed in 96-well microliter plates. to each well were added (5 -7.5)´10 4 tumor cells and a given amount of the test compound. the cells were allowed to proliferate for 48 h (murine leukemia l1210 cells) or 72 h (human cd 4 + t-lymphocytic cem and human cervix carcinoma hela cells) at 37°c in a humidified co 2 -controlled atmosphere. at the end of the incubation period, the cells were counted in a coulter counter. the ic 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%. cisplatin was purchased from sigma and used as a reference drug. compound concentrations of 100, 20, 4, 0.8, 0.16 and 0.032 mg/ml have been tested (5-fold dilutions) and the data represent the mean ± sd (standard deviation) of at least two to three independent experiments. all values are significantly different from cisplatin (less active), except for ig (no statistical difference, p > 0.05). the biological activity depended on only on the chemical structure of substituents, but also on the three dimensional (3d) configuration of compound ig. for this reason, we again applied computational methods and ligand-based pharmacophore modeling to evaluate this state. the new derivatives of thiosemicarbazones (ia -ig and iia -iig) were drawn and converted from 2d to 3d, and the pm3mm basis set was assigned and then minimized using a semi-empirical method with the aid of gaussian09 program package. a conformational search of the ligands was carried out using best algorithm for 3d pharmacophore generation. the auto pharmacophore generation protocol in discovery studio 3.5 was used based on a bioactive conformation. in this study, we used compound ig as the bioactive conformation with ic 50 values between 0.30 and 2.50 mg/ml. the pharmacophore models were generated based on the main features of compound ig. these features were hb-acceptor, hb-donor, hydrophobic and ring aromatic in the pharmacophore models. besides, the genetic function approximation (gfa) model for the selectivity of pharmacophores was used. the best model was determined with the help of clusters pharmacophores subprotocol. the model contains six features, representing two hydrophobic (cyan), two ring aromatic (orange), one hydrogen bond donor (pink) and one hydrogen acceptor (green). all newly synthesized compounds were evaluated for their antiviral, cytostatic, and cytotoxic properties. none of the compounds effectively inhibited dna virus replication (i.e. hsv-1, hsv-2, vv), except i and ii that showed slight activity against vaccinia virus replication in hel cell cultures (ec 50 = 45 -50 mg/ml) and compounds ib, ic and iic bearing an electron-donating group (-oh and -och 3 ) against feline herpes virus (ec 50 = 17.8 -54.3 mg/ml). all compounds were also not significantly inhibitory against a wide variety of rna viruses, including vesicular stomatitis virus (vsv), respiratory syncytial virus and coxsackie virus in hela cell cultures at subtoxic concentrations (table 2) . however, in vero cell cultures compound ig consistently showed an antiviral activity of 3 mg/ml (ec 50 ) against coxsackie virus b4 but there was no inhibitory activity observed for parainfluenza virus-3, reovirus-1, sindbis virus and punta toro virus ( table 2 ). the fact that the activity of ig against coxsackie virus b4 was only observed in one cell line (monkey kidney vero), but not in another cell line (human cervix carcinoma hela) led us to conclude that this compound should not be considered as a consistent new antiviral lead agent. a marginal activity was observed for ib, ic and iic against feline herpes virus (ec 50 = 17.8 -54.3 mg/ml) and for ib, ic and iia against feline corona (fipv) virus (ec 50 = 7.0 -31.5 mg/ml) in crfk cell cultures (table 3) . whereas no activity was found in mdck cell cultures against influenza b virus, compounds id, ie, if and iif which have an electron-withdrawing group (-cf 3 , -f and -no 2 ) proved inhibitory against influenza a virus (ec 50 : 12.7 -26.7 mg/ml for strain h1n1 and 9.3 -18.5 mg/ml for strain h3n2). these compounds were only 5-to 20-fold less effective than the clinically used drug oseltamivir and were neither cytotoxic (mcc) nor cytostatic (cc 50 ) at 100 mg/ml against mdck cells (table 3) . thus, a certain degree of selectivity was present for these compounds against influenza a virus in cell culture and should be further considered for optimization as potential anti-influenza virus lead compounds. the synthesized compounds were also evaluated for their cytostatic activity against murine leukemia l1210, human cd 4 + t-lymphocyte cem and cervix carcinoma hela cells (table 4 ). most compounds in the iia -iig series did not show pronounced cytostatic activity (ic 50 ³ 100 mg/ml). however, iib was moderately cytostatic (ic 50 = 13 -15 mg/ml), whereas iig seemed to be poorly inhibitory to l1210 and cem cell proliferation, but had a pronounced antiproliferative activity against hela cells (ic 50 = 1.9 mg/ml). this unusual selectivity between tumor cell lines should be further investigated. in fact, the i series of compounds were often somewhat more cytostatic than their corresponding ii series of compounds. it may not be a coincidence that ig (ic 50 = 0.3 mg/ml against hela cells) was also most cytostatic among series i and also showed the most pronounced antiproliferative potency against hela cells. the structure -activity relationship (sar) studies were performed to determine how the substituent on the benzene ring affected the cytostatic activity. compounds ia and iia without any substituent group on the benzene ring did not show cytostatic activity. then, we examined the effect of introducing various substituent groups in the benzene ring of compounds ia and iia. the -oh, -och 3 , -cf 3 , -f, -no 2 and -n(ch 2 ch 2 cl) 2 groups at the para position of the benzene ring of compounds ia and iia were used to form compounds punta toro virus i >100 >100 >100 50 >100 >100 ³100 >100 >100 >100 100 >20 >20 >20 >20 >20 ia >100 >100 >100 >100 >100 >100 ³100 >100 >100 >100 >100 >100 >100 >100 >100 >100 ib 100 >20 >20 >20 >20 >20 100 >20 >20 >20 100 >20 >20 >20 >20 >20 ic >100 >100 >100 >100 >100 >100 100 >20 >20 >20 100 >20 >20 >20 >20 >20 id >100 >100 >100 >100 >100 >100 100 >20 >20 >20 100 >20 >20 >20 >20 >20 ie >100 >100 >100 >100 >100 >100 ³100 >100 >100 >100 100 >20 >20 >20 >20 >20 if >100 >100 >100 >100 >100 >100 ³100 >100 >100 >100 >100 >100 >100 >100 >100 >100 ig >100 >100 >100 >100 >100 >100 ³20 >20 >20 >20 >20 >4 >4 >4 3 >4 ii >100 >100 >100 45 >100 100 100 >20 >20 >20 100 >20 >20 >20 >20 >20 iia 100 >20 >20 >20 >20 >20 100 >20 >20 >20 100 >20 >20 >20 >20 >20 iib >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 iic >100 >100 >100 >100 >100 >100 100 >20 >20 >20 100 >20 >20 >20 >20 >20 iid 100 >20 >20 >20 >20 >20 >100 >100 >100 >100 100 >20 >20 >20 >20 >20 iie 100 >20 >20 >20 >20 >20 >100 >100 >100 >100 100 >20 >20 >20 >20 >20 iif >100 >100 >100 >100 >100 >100 100 >20 >20 >20 100 >20 >20 >20 >20 >20 iig 100 >20 >20 >20 >20 >20 ³100 >100 >100 >100 100 >20 >20 >20 >20 >20 ds-5000: dextran sulfate (molecular weight, 5.000). d) (s)-dhpa: (s)-9-(2,3-dihydroxypropyl)adenine. (ib -ig and iib -iig). especially ig with a bis(2-chloroethyl)amino moiety, the ic 50 values (0.30 -2.50 mg/ml) were the lowest (most effective) among all compounds and compared to cisplatin as a reference compound. a two-tailed, unpaired t-test (graphpad prism) revealed no significant difference between the cytostatic inhibition values of ig and cisplatin obtained against the three tumor cell lines (p = 0.647). the results showed that compounds ia -ig containing electron-withdrawing group, such as -cn located at the para position of the benzene ring, increased the cytostatic activity, according to the electron-donating group (-sch 3 ) at the same position of the structure (like compounds synthesis, biological evaluation and ligand 147 iia -iig). furthermore, the introduction of electron-donating groups (-oh, -och 3 ) at the para position of the benzene ring weakened the cytostatic effects. while the same situation for electron-withdrawing groups (-f, -no 2, -cf 3 ) was also observed, compound iib exhibited moderate activity against all tumor cell types. besides, compound 1g displayed the better cytostatic activity than its analog compound iig, which has an electron-donating group (-sch 3 ) at the para position. these results indicated that the introduction of the bulky alkylating group (bis(2-chloroethyl)amino) and a cyano group reinforced the cytostatic activity. the pharmacophore model ( fig. 2a ) was hypothetically superimposed with compounds ig (fit value 4.6216) and iig (fit value 3.0032). figures 2b and 2c summarize the results of model fitting obtained for compounds ig and iig. the other compounds were not superimposed to fit with the best model. in addition to comparing the results of modeling with experimental data, they show compatible and significant trends. it is established that hb-acceptor (-cn; -sch 3 ), hydrophobic groups (-n(ch 2 ch 2 cl) 2 ), and 3d-conformations are responsible for the cytostatic activity of compounds such as ig and iig, according to ligand-based pharmacophore modeling. especially, 3d-conformation of the investigated compounds showed the major effect in this study. unfortunately, compounds ia -if and iia -iif did not show any activity in comparison to compounds ig and iig. the proposed methods were able to provide valuable information about key features and interactions that are important for the biological activity of compound ig. thus, ligand-based pharmacophore modeling was increasingly successful in delineating why compound ig displayed better cytostatic activity than compound iig. gaussian 09, revision a.1 gaussian the present work was supported by the scientific re key: cord-002237-200ondzx authors: dashtdar, mehrab; dashtdar, mohammad reza; dashtdar, babak; khan, gazala afreen; kardi, karima title: phenol-rich compounds sweet gel: a statistically more effective antibiotic than cloxacillin against pseudomonas aeruginosa date: 2016-09-17 journal: j pharmacopuncture doi: 10.3831/kpi.2016.19.026 sha: doc_id: 2237 cord_uid: 200ondzx objectives: the purpose of this study was to obtain a natural antibiotic from phenol-rich compounds; for the dressing and the treatment of chronic wounds. methods: the phenol-rich compound sweet gel was prepared by blending four natural herbal extracts, acacia catechu (l.f.), momia (shilajit), castanea sativa, and ephedra sinica stapf, with combination of a sweet gel medium, including honey, maple saps, phoenix dactylifera l. (date), pomegranate extract and azadirachta indica gum as a stabilizer. the combinations were screened by using a well-diffusion assay with cloxacillin as a control. pseudomonas spp. was tested with our novel antimicrobial compound. the zones of inhibition in agar culture were measured for each individual component and for the compound, and the results were compared with those of the control group which had been treated with cloxacillin. data were expressed as means ± standard deviations. quantitative analyses were performed using the paired t-test. results: the antibiotic effect of the phenol-rich compound sweet gel was statistically shown to be more significant than that of cloxacillin against pseudomonas aeruginosa (p < 0.05). conclusion: our novel approach to fighting the antibiotic resistance of pseudomonas proved to be successful. the phenol-rich compound sweet gel was found to be suitable for use as an alternative medicine and bioactive dressing material, for the treatment of patients with various types of wounds, including burns, venous leg ulcers, ulcers of various etiologies, leg ulcers on the feet of diabetic, unhealed graft sampling sites, abscesses, boils, surgical wounds, necrotic process, post-operative and neonatal wound infection, and should be considered as an alternative to the usual methods of cure. pseudomonas aeruginosa (p. aeruginosa) is an aerobic gram-negative bacterium; that is ubiquitous, exist in aqueous habitats, and take advantage of humid en-vironments. it is also saprophytic and naturally resistant to antibiotics (beta-lactams, hydrophilic), and may become an opportunistic pathogen responsible for serious infections when favorable circumstances exist. because it is environmentally present in soils, plants (including fruits and vegetables), aqueous habitats and humid environments [1], it has an ability to acquire resistance to antibiotics, and has multiplicity of virulence factors (diffusible or constituent) that baffle the host's defenses and allow the development of infections in susceptible patients, such as malnourished patients; burn victims, trauma patients, patients suffering from diabetes, cystic fibrosis, cancer, human immunodeficiency virus (hiv), and blood disorders, patients undergoing mechanical ventilation or long-term corticosteroid therapy; patients in whom carrier surveyed peripheral and central catheters are being used [2] . p. aeruginosa infections are most often acquired in a hospital, but are sometimes community acquired; factors limiting p. aeruginosa proliferation on the skin are dryness of the skin's surface and normal flora (gram + cocci). the bacterium p. aeruginosa can grow or become permanent on the skin in case of the traumatic rupture of the skin barrier; chronic wounds such as leg ulcers, extensive burns, diabetes include ulcers, oozing dermatitis, and wet or macerated lesions the disappearance of gram + cocci normal flora caused by the use of systemic or topical antimicrobials. the first signs of infection of a chronic wound or ulcer are the production of greenish pus and the appearance of a characteristic aromatic odor. the appearance of the ulcer or wound becomes inflammatory or necrotic. these are vesicular or bullous lesions with a content of serum are hemorrhagic based on an erythematous and edematous base and can initially have the clinical appearance of multiform erythema. these lesions can change very quickly in a few days to ulceration, and they then take the appearance of a so-called ecthyma gangrenosum [3] [4] [5] [6] . one can also have abscesses or single or multiple subcutaneous nodules that can change very quickly to necrosis and ulceration. biofilm formation seems to facilitate the survival of p. aeruginosa in the environment and in their hosts. bacterial biofilms are structured masses of bacterial cells coated with a hydrated polymeric matrix of their own synthesis. the biofilm protects the bacteria and allows them to survive in hostile environmental conditions. the biofilm bacteria can withstand the immune response of the host and are much more resistant to antibiotics and disinfectants than planktonic bacterial cells. the ability to form a biofilm is now recognized as a characteristic of many microorganisms [7] [8] [9] . the presence of biofilms during infections therefore requires new methods of prevention, diagnosis and treatment. the existence of biofilms and antibiotic resistance has emerged as one of the greatest threats to global health and, brings an additional level of complexity to the control of chronic ulcers. the "black beast" of burn centers, pseudomonas, is responsible for 10% of bloodstream infections and 28% of deaths. therefore, alternative antimicrobial strategies are urgently needed, and this situation has led to a re-evaluation of the therapeutic use of ancient remedies, such as herbal extracts and herbal-based products. the medicinal properties of plants are due to its chemicals. plants synthesize many compounds called primary metabolites that are essential to their existence. these include proteins, lipids and carbohydrates that are used for subsistence and reproduction; in not only the plant itself but also the animals that feed on them. in addition, plants synthesize an extraordinary range of other compounds, called secondary metabolites, whose functions are far from being unanimously accepted. many secondary metabolites can be broadly considered to be "antibiotics" as they protect plants against fungi, bacteria, animals and even other plants. secondary metabolites include two types of compounds: phenol and flavonoids compounds. phenol compound are involved in plant-plant interactions (allelopathy, and inhibitions of both germination and growth). these compounds include lignin, flavonoids, phenylpropanoids, anthocyanins and "nitrogen compounds including alkaloids and glycosides. recently, phenol compounds have drawn much attention due to their antioxidants properties and their potential beneficial implications for human health, such as the treatment and the prevention of cancer, cardiovascular disease and other inflammatory diseases. polyphenol are composed of various phenol compounds such as phenolic acid, catechins, flavonoids, and tannins. phenols, which are aromatic organic compounds, are present in many plants. they usually have antiseptic, antibacterial actions. the simplest is phenol (an antimicrobial) present in thyme oil. the phenolic acids have antioxidant and protective effects against cardiovascular disease and cancer. other phenols have analgesics, anti-inflammatory, cholesterol-lowering, hypotensive, anticoagulant, anti-allergenic, hypotensive, and hepato-protective effects [10] . flavonoids are pigments giving color to flowers and are present in some leaves. these substances can be yellow (origin of the word flav), red, blue or purple. the main properties of flavonoids are their veinotonic, protective (vessels), anti-cholesterol or antioxidants effects [11, 12] . tannins are phenol compounds that precipitate proteins. these complex compounds may be soluble in water or alcohol, are the flavonoid family. tannins are widely distributed in the plant kingdom. they are common both in gymnosperms and angiosperms. within angiosperms, tannins are more common in dicotyledons than they are in monocotyledons. tannins can be found mainly in the cortex, roots, fruits and leaves. they have especially astringent properties. tannins are mainly used externally, particularly to treat patients with injuries, wounds or hemorrhoids. internally they are also used to treat patients with diarrhea and gastroenteritis. tannins also exhibit antioxidant and antibacterial properties [13] . catechin is a molecule of the flavonoid family; the name comes from the fact that this molecular compounds found in the fruits of acacia catechu. catechins are astringent juices from various sources and involve decoction of the fruit of areca catechu l. (palms), and the wood of the acacia catechu. catechins are present in significant quantities in green tea. the fermentation of the leaves being stopped soon after harvest, they are barely oxidized, which is the reason that green tea retains most of its original catechins , which gives them their slightly bitter and astringent taste. green tea con-tains flavonoids 70% of which are catechins. interesting amounts of catechins also exist in chocolate, red wine, apples and grapes. five catechins molecules are: catechins, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate molecules. the latter is the most abundant and most active of all the catechins. according to numerous studies, catechins have, in addition to their antioxidant properties, a protective role in the prevention of certain chronic diseases such as diabetes and osteoporosis [14] . several studies that have been conducted in recent years have manifested the antibiotic activities of some phenol compounds in natural plants extracts, including acacia catechu (l.f.) willd, castanea sativa, momia (shilajit) and ephedra sinica stapf [15] . honey, maple saps, phoenix dactylifera l. (dates) extract and pomegranate which are well known for their high levels of antioxidants and polyphenols have also shown promise as novel antimicrobial agents. phenol compounds have recently attracted much interest due to their antioxidants properties and their potential beneficial implications for human health, such as their use in the treatment and the prevention of cancer, cardiovascular diseases and inflammatory diseases. the effectiveness of these above substances; has been demonstrated in several studies [16] [17] [18] [19] . however, no effort has been made to evaluate the synergic effects of combined natural sweeteners on enhancing the antibiotic activities of natural plants extracts. the topical formulations that on certain vitamins and nutrients, are rich in phenol compounds, and are applied directly to wounds are more effective for reducing the risk of infection and for stimulating healing. meanwhile, phenol-rich compounds; provides a number of essential minerals such as zinc, potassium, iron, magnesium and calcium (table 1 ). in order to obtain a natural antibiotic for the dressing and the treatment of chronic wounds, we conducted this study to find an innovative strategy for using natural medicine derived from phenol-rich compounds that have been tested on bacterial strains of a reference such p. aeruginosa. this research was conducted through 2015 at the laboratories of dubai pharmacy college united arab emirates (uae). the processing of the plants performed in this study was the same as the traditional method used by the people in the iranian bakhtiari tribe, as mentioned in ref [15] . the phenol-rich compound sweet gel was prepared by blending four natural herbal extract acacia catechu (l.f.) willd, castanea sativa, ephedra sinica stapf, and momia in to combination of sweet gel medium, including honey, maple saps, date syrup, pomegranate extract and azadirachta indica gum as a stabilizer. the combinations were screened by using a well-diffusion assay with cloxacillin as a control. suspension assays were used to determine the antimicrobial activities of the medium gel alone and of the medium gel in combination with the four natural herbal extract. the test organism was p. aeruginosa. in this study, eight plant species were used as shown in table 2 and 3. the ingredients of the sweet gel compound are presented in table 4 , bacterial strains and growth conditions. a bacterial strain used in this study was pseudomonas spp. the inhibition of bacterial growth was studied by using the well-diffusion method with nutrient agar, as commonly practiced in medical bacteriology, and was purchased from himedia laboratories, india. plates were inoculated with 100 µl of each pathogenic microorganism adjusted to standardized inoculum (1.5 × 10 8 cfu/ml) in triplicates and were spread with sterile swabs. eight mm wells were drilled into the agar by using a sterile stainless steel borer. for the preparation of phenol-rich compound herbal extracts, one gram of aslan crude extract (table 3) was added to 10 ml of distilled water to form 10% aslan (w/v), which was heated and stirred until all ingredients had dissolved. it was then mixed with an equal amount of sweet gel, which included honey, date, maple syrup and pomegranate in specific percentages, as shown in table 3 , after which neem gum as much as 5 percent of the total weight, was added as stabilizer. one drop of each sample solution (50 ul) was applied to each well on the plate by using a pasteur pipette. the petri dishes thus prepared were left at room temperature for ten minutes to allow diffusion of the extract into the agar. after incubation for 24 hours at 37°c, the plates were observed. anti-bacterial activity was indicated by an inhibition zone surrounding the well (including the well diameter) containing the plant extract. the diameters of the zones of inhibition were measured in millimeters and interpreted based on published standards [20] . anti-bacterial activity was recorded if the zone of inhibition was greater than 8 mm. the interpretation of the anti-bacterial activity results was done according to the diameter of the zone of inhibition as follows: zones with diameters < 9 mm zone were considered inactive, zones with diameters from 9 to 12 mm were considered partially active, zones with diameters from 13 to 18 mm were considered active, and zones with diameters from > 18 mm were considered very active. the means and standard deviations of the diameters of the inhibition zones were calculated. the standard anti-bacterial agent was cloxacillin. data were evaluated using the ibm spss software program (version 19; ibm spss inc., il, usa). the herbal extract groups and the control groups were compared at the 95% confidence interval, and the results were expressed as means ± standard deviations. differences between the control group and the herbal extract groups were the criteria for the anti-bacterial activities. the t-test (paired tests) was used to detect differences between the treatment groups and the control group. a value of p < 0.05 was considered significant. the table 5 shows the zones of inhibition for the individual ingredients in the phenol-rich compound that were used against p. aeruginosa and that produced a greater inhibition of pseudomonas spp. than distilled water did. table 6 shows that the phenol-rich compound sweet gel produced a greater inhibition of pseudomonas spp. than cloxacillin did. also, the table shows that phenol-rich compound sweet gel had a greater inhabitation had a greater inhabitation on pseudomonas spp. than the individual ingredients did an indication of synergic effects. ® table 5 antibiotic effects of individual ingredients in phenol-rich compound against p. aeruginosa in this study based on the results for the antibacterial effects, we can state that phenol-rich compound sweet gel is a good candidate for the prevention and the treatment of chronic ulcers. our findings showed that the phenol-rich compound sweet gel had a significant antiseptic effective against pseudomonas spp. and were in agreement with those of previous studies [15] [16] [17] [18] [19] that found that phenol compound exhibited fairly good antimicrobial activities against both gram-negative and gram-positive bacteria and that remarkable activity was exhibited by p. aeruginosa. also, the greater inhibition due to the compound compared to the inhabitation due to the individual ingredients on pseudomonas spp. indicates significant synergic effects. however, no effort has been made so far to evaluate the synergic effects of combined natural sweeteners on enhancing the antibiotic activities of natural plants extracts. phenol compounds rich in minerals and trace elements are involved in many biological processes in wound healing: several studies revealed a higher postoperative morbidity, delayed healing, and more frequent secondary infections in malnourished patients with amputations than in non-malnourished patients. malnutrition encourages dropping sutures. an earlier acute or chronic malnutrition trauma slows healing. zinc as a trace element is quantitatively the most important one and is involved in the construction of over 200 enzymes with effects as follow: improving cell growth and differentiation, fibroblast proliferation, collagen synthesis, strengthening the immune system and increasing steroid receptors [21, 22] . iron; also plays a key role in the healing process [23] . it preventing necrosis and accelerating the repair of radiation-induced wounds. it also promotes collagen synthesis, improves oxygen delivery to tissue, and is a component of many enzyme systems. chronic iron deficiency or anemia is associated with an extended period of healing and othprolonged supplementation may in turn affect the absorption of zinc, copper and iron. this is because copper, iron and zinc use the same routes to cross the intestinal barrier and reach the bloodstream. therefore excessive inputs of a given mineral salt can interfere with the absorption of others. no significant benefit for wound healing is seen with nutritional supplements such as vitamins c, a, e, and zinc in a non-deficient individuals. [25] [26] [27] [28] in contrast to oral administration, topical administration of zinc appears to be superior due to its action in reducing super infections and necrotic material via enhanced local defense systems and collagenolytic activity; and to the sustained release of zinc ions, which stimulates epithelialization of wounds in normozincemic individuals. zinc oxide in paste bandages (unna boot) protects and soothes inflamed peri-wound skin. zinc from these formulations is transported through the skin although the systemic effects seem insignificant. the topical administration formula that focuses on certain vitamins and nutrients applied directly on the wounds is more effective as a supplements to reduce the risk of infection and to stimulate healing. furthermore, phenolrich compounds, provides a number of essential minerals, such as zinc, potassium, iron, magnesium and calcium (table 1 ). in previous studies [29-31] on phenol compounds, no contraindications and side effects have been reported to date for any of the ingredient used in our study, even though all of the benefits seem to have been reported. the topical use of phenol-rich compound sweet gel should be safer than that of the other formulations, but to be on the safe side, more clinical studies and toxicology studies need to be conducted. the antibiotic effect of phenol-rich compounds is due to the presence of hydrophilic components, such as polyphenols, polysaccharides, flavonoids and tannins in one or more parts of the plant. thus is an innovative approach to fighting the antibiotic-resistant pseudomonas. our results and those of previous studies provide pharmacology and microbiology information to explain the advantages of the phenol-rich compound sweet gel and its mechanisms of action as a bioactive dressing material in treating chronic ulcer: √ sterilization of wounds √ rapid autolytic debridement √ inhibition of potential pathogens of wounds and enzymes that destroy tissue, √ stimulations of tissues growth to speed healing, √ protection against cross-contamination, √ reduction of scars, √ anti-inflammatory effect: reductions of infections, deodorization of wounds, reducing edema and against maceration due reduced of exudate , √ provision of moist healing environment without risk of surrounding skin maceration and preventions of adhesion of the dressings to the wound, thus preventing pain and tissue damage when dressing is changed. √ low enough ph to slow or prevent the growth of many pathogenic species. the increase in the number of bacterial infections resistant to current antibiotics is an extremely worrying phenomenon, especially in the hospital setting (nosocomial infections). therefore new strategies and innovative antibiotics for use against these particularly virulent microorganisms need to be developed if a therapeutic impasse is to be avoided. this work evaluated the synergic effects of combined natural sweeteners on enhancing the antibiotic sensitivities of natural plants extracts and found the phenol-rich compounds sweet gel to be an alternative medicine and bioactive dressing material, for the treatment of patients with various types of wounds, including burns, venous leg ulcers, ulcers of various etiologies, diabetes induced ulcers on the feet, unhealed sampling sites grafts, abscesses, boils, surgical wounds, necrosis process, post-operative and neonatal wound infection. based on these results, that compound should be considered an alternative to the usual methods of cure. on burns its antibacterial and anti-inflammatory properties allow a moist healing environment that protects the wounds from deterioration and fibrosis to be maintained. isolation of pseudomonas aeruginosa from open ocean and comparison with freshwater, clinical, and animal isolates antimicrobial resistance profile of pseudomonas aeruginosa producing metallo-beta-lactamases pseudomonas aeruginosa o-11 folliculitis. development into ecthyma gangrenosum in immunosuppressed patients bilateral periorbital ecthyma gangrenosum drug-induced pseudomonas aeruginosa ecthyma gangrenosum twenty five years review of pseudomonas aeruginosa bacteremia in a burn center bacterial biofilms: a common cause of persistent infections. science biofilm formation as microbial development a genetic basis for pseudomonas aeruginosa biofilm antibiotic resistance chemistry and biochemistry of dietary polyphenols antioxidant activity of plant extracts containing phenolic compounds flavonoids and cardiovascular disease antioxidant properties and bioactive components of norton (vitis aestivalis) and cabernet franc (vitis vinifera) wine grapes food biotechnology -plant biotechnology in vitro, anti-bacterial activities of aqueous extracts of acacia catechu (l.f.) willd, castanea sativa, ephedra sinica stapf and shilajita mumiyo against gram positive and gram negative bacteria in vitro anti-bacterial activity of ethanolic bark extract of acacia catechu willd against enteric pathogens medical honey for wound care -still the 'latest resort'? the antibacterial nature of honey: 1. the nature of the antibacterial activity phenolic profile and antioxidant activity of the algerian ripe date palm fruit (phoenix dactylifera) performance standards for antimicrobial disk susceptibility tests; approved standard-eleventh edition. clsi document m02-a11 zinc in human health: effect of zinc on immune cells iron and zinc interactions in humans the role of iron in the skin and cutaneous wound healing human coronavirus 229e remains infectious on common touch surface materials zinc in wound healing: theoretical, experimental and clinical aspects current concepts in laboratory testing to guide antimicrobial therapy drug and nutrient aspects of wound healing randomized clinical trial of ascorbic acid in the treatment of pressure ulcers potential of honey in the treatment of wounds and burns honey and sugar as a dressing for wound and ulcers effects of topical honey on post-operative wound infections due to gram positive and gram negative bacteria following caesarean sections and hysterectomies we would like to express our gratitude to professor saeed ahmad khan dean of dubai pharmacy college, dubai, united arab emirates, for his great support. this project would not have been possible without his collaboration and support. in addition, we would like to express our sincere thanks and appreciation to havva dashtdar, phd, biotechnology professional and embryologist; for her review of and suggestions concerning this article. we also would like to thanks joyce velasquez, laboratory technologist, dubai specialized medical center & medical research lab, who assisted us in this research. the authors declare that there is no conflict of interest. mehrab dashtdar. http://orcid.org/0000-0001-5931-4715. key: cord-003427-0dddrh4e authors: el-faham, ayman; farooq, muhammad; khattab, sherine n.; abutaha, nael; wadaan, mohammad a.; ghabbour, hazem a.; fun, hoong-kun title: synthesis, characterization, and anti-cancer activity of some new n′-(2-oxoindolin-3-ylidene)-2-propylpentane hydrazide-hydrazones derivatives date: 2015-08-13 journal: molecules doi: 10.3390/molecules200814638 sha: doc_id: 3427 cord_uid: 0dddrh4e eight novel n′-(2-oxoindolin-3-ylidene)-2-propylpentane hydrazide-hydrazone derivatives 4a–h were synthesized and fully characterized by ir, nmr ((1)h-nmr and (13)c-nmr), elemental analysis, and x-ray crystallography. the cyto-toxicity and in vitro anti-cancer evaluation of the prepared compounds have been assessed against two different human tumour cell lines including human liver (hepg2) and leukaemia (jurkat), as well as in normal cell lines derived from human embryonic kidney (hek293) using mtt assay. the compounds 3e, 3f, 4a, 4c, and 4e revealed promising anti-cancer activities in tested human tumour cells lines (ic(50) values between 3 and 7 μm) as compared to the known anti-cancer drug 5-fluorouracil (ic(50) 32–50 μm). among the tested compounds, 4a showed specificity against leukaemia (jurkat) cells, with an ic(50) value of 3.14 μm, but this compound was inactive in liver cancer and normal cell lines. hydrazide-hydrazone derivatives are molecules containing a highly reactive group (co-nh-n=ch) and considered to be a good candidate for development of a new drug [1] . recently, hydrazide-hydrazones have been considered to be of great interest in medicinal chemistry due to their diverse biological properties, including anti-microbial [2] [3] [4] , anti-mycobacterial [5, 6] , anti-convulsant [7] , analgesic [8] , anti-inflammatory [9] , anti-platelet [10] , anti-tubercular [10] [11] [12] [13] , and anti-tumoral activities [14] [15] [16] [17] [18] [19] . in addition, hydrazide-hydrazones have been reported to elicit anti-cancer [18] [19] [20] [21] and anti-hiv properties [22] and they are therefore increasingly considered to be of great value in medicinal chemistry [16, [23] [24] [25] [26] [27] . because of the remarkable biological value of isatin as an important constituent of bioactive compounds exhibiting caspase inhibitory [28, 29] , antibacterial, and antiproliferative activity [30] , schiff bases of isatin derivatives have antismallpox [31] and gal3 receptor antagonist capabilities [32] . in addition, the analogous of isatin derivatives displayed inhibitory activity against elf2 kinase activator [33] , tnf-α, cdk2 [34] and sars protease [35] . isatin also displays antiviral [36] , anti-inflammatory, analgesic [37] , and anticonvulsant activities [38] . valproic acid (vpa, 1) has an important biological value as a potent antiepileptic molecule [39] [40] [41] , as well as showing inhibition of angiogenesis both in vitro and in vivo [42] [43] [44] [45] . vpa also acts as a powerful histone deacetylase inhibitor [46, 47] and induces differentiation and apoptosis in a variety of malignant cells in vitro [48] . clinical trials with vpa have focused on acute myeloid leukaemia and the myelodysplastic syndromes. when it was used as mono therapy or in combination with all-trans retinoic acid, which synergizes in vitro, vpa achieved hematologic improvement in a subset of patients [48] . as a continuation to our previously reported data [49] [50] [51] , herein we designed eight isatin hydrazide-hydrazone derivatives, considering some of the factors responsible for such activity, including (i) the presence of isatin moiety; (ii) the presence of the hydrazide-hydrazone functionality; and (iii) valproic acid moiety ( figure 1 ). all the prepared compounds were assessed against two different human tumour cell lines, including human liver (hepg2) and leukaemia (jurkat), as well as in normal cell lines derived from human embryonic kidney (hek293) using mtt assay. compound 2 was prepared following the reported method [49] and condensed with isatin derivatives 3a-h in the presence of 2-3 drops of glacial hoac and ethanol as the solvent to afford products 4a-h (scheme 1). the structures of all synthesized compounds were in a good agreement with their spectral data. the ir spectra of the compounds 4a-g reveal absorption bands in the region 3195-3217 cm −1 corresponding to the (nh), a band at 1720 and 1688 cm −1 corresponding to the c=o group, and a peak around 1596 cm −1 related to the c=n bond. the nmr of all the products 4a-g are in good agreement with their structures (figures s1-s7). as a prototype 1 h-nmr of 4g showed multiple peaks at δ 0.86-0.89, 1.27-1.29, 1.40-1.44, 1.56-1.60 ppm, and a broad singlet at δ 2.51 ppm corresponding to the valporic acid moiety (2 ch3, 4 ch2, and ch, respectively). also, two triplet peaks were observed at δ 3.77 and 4.20 ppm corresponding to the two methylene group (ch2-ch2br), respectively. the observed peaks in the aromatic region at δ 7.18 (t, 1h, ar-h), 7.32 (d, 1h, ar-h), 7.53 (t, 1h, ar-h), and 7.88 (brs, 1h, arh) are related to the isatin moiety, while the broad singlet peak at δ 12.28 ppm is related to the nh. the 13 c-nmr of 4g showed peaks at δ 14.5, 20.6, 29.9, 35.3, 39.7, 42.2, and 46.5 ppm, corresponding to the valporic acid moiety and the two methylene groups, in addition to seven peaks related to the aromatic carbons and imino function group. the two peaks at δ 169.8 and 176.0 ppm corresponded to the carbonyl groups of the isatin moiety and the hydrazide group, respectively. it is expected that compound 4g could adopt two different geometrical isomers (z and e) as shown in figure 2a . therefore, it is considered worthwhile to model the compounds using molecular mechanics mm2 calculations. in addition, quantum chemical calculations were carried out with the gaussian 98 suite of programs. geometry optimizations were carried out using the dft level (b3lyp/6-31g **) of theory to assess the relative stability of the z and e isomeric species. calculated relative energies of 4g z and e isomers are −3585.4860873 au and 3585.4635100 au, respectively. computed energies indicate the stability of the z isomer over the e one by 0.0225773 au (14. 1675 kcal/mol) ( figure 2b ). the x-ray single crystal structure determination of compound 4g (ccdc: 996592) confirmed that the structure exists in the z-conformer rather than the e-conformer ( figure 3 ). the details of data collection and structure refinement are listed in table 1 , and the x-ray structures are shown in figure 3 . compound 4g crystallized from ethanol in the triclinic space group p-1. all bond lengths and angles are in normal ranges [52] . in the crystal packing (figure 4 ), the molecules are linked by intermolecular c17a-h17b···br1b, c17b-h17d···o2b and c18a-h18b···o2a hydrogen bonds ( table 2) into two-dimensional networks parallel to the bc plane ( figure 4 and table 2 ). the asymmetric unit contains two molecules of the compound with disorder in one of the ethylene arms. the molecular structure of compound 4g is composed of a 2-oxoindoline ring (c1/n1/c2-c8) which is linked with two side chains at n1 and c8. the two molecules in the asymmetric unit are with z configuration about the c8=n2 double bond ( figure 3 ). the z configuration of 4a is stabilized with intramolecular hydrogen n3-h3n···o2 ( table 2 ). the single bond n2-n3 is clearly characterized by the distances of 1.355 (3) å and 1.361 (4) å, respectively, for molecules a and b. the double bond of c8=n2 is characterized by the distances of 1.298 (3) å and 1.283 (4) å, for molecules a and b, respectively. a b figure 3 . ortep diagram of the asymmetric unit consisting of two symmetry-independent molecules. the ellipsoids are drawn at the 50% probability level with hydrogen atoms being shown as spheres of arbitrary radii. one of the molecules has disorder in the side chain c11b-c13b. (3) 149.00 c17b−h17d···o2b ii 0.9700 2.4100 3.139 (4) 131.00 c18a−h18b···o2a iii 0.9700 2.2900 3.096 (3) 140.00 symmetry codes: reaction of valporic acid hydrazide 2 with n-acetylisatin 3h proceeds in a different way, where the ring opening occurred to afford the product 4h. the ir and nmr data proved its structure in the open form, due to the attack of the hydrazide group on c2 instead of c3 [53] . the ir spectrum of 4h reveals absorption bands in the region 3310 and 3223 cm −1 corresponding to the nh, a band at 1720, 1709, 1688, and 1608 cm −1 corresponding to the c=o and c=n group, respectively. 1 h-nmr of 4h showed multiple peaks at δ 0.86-0.89, 1.27-1.29, 1.40-1.44, 1.56-1.60, and a broad singlet peak at δ 2.21 ppm corresponding to the valporic acid moiety (2 ch3, 2 ch2, and 2ch2, ch, respectively). the observed peak at δ 2.16 ppm is corresponding to the n-acetyl group. the observed peaks in the aromatic region at δ 7.28 (t, 1h), 7.68 (t, 1h), 7.99 (d, 1h), and 8.17(d, 1h) ppm, are related to the phenyl ring, while the two singlet peaks at δ 10.03 and 10.72 ppm corresponded to the two nh. the 13 c-nmr of 4h showed peaks at δ 14.6, 20.6, 29.9, 35.3, 43.8, and 46.5 ppm corresponding to the valporic acid moiety group, while the acetyl group was observed at δ 25.0 ppm, in addition to six peaks related to the aromatic carbons at δ 121.5, 121.8, 123.8, 133.6, 135.9, and 140.3 ppm. the three peaks observed at δ 164.1, 169.6, and 174.7 ppm related to the three conh groups, while the peak observed at δ 193.0 ppm is related to the α-ketoamide group. the cyto-toxicity activity was measured in vitro in hepg2, jurkat, and hek 293 cells using the mtt 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan colorimetric assay. for comparison, 5-fluorouracil (5-fu) was used as a standard anti-cancer drug. treatment with dimethylsulfoxide (dmso) was used as a control for the cancer cells. the anti-cancer activity of the new prepared compounds against the hepg2 cell line revealed that compounds 3a, 3h, 4a, 4b, 4f, 4g, and 4h showed no anti-cancer activity. similarly, the starting compounds 1 and 2 did not show promising anti-cancer activity in hepg2 cells. compound 3e exhibited higher potency against hepg2 cell line with ic50 = 2.82 ± 40.7 μm, which is much lower than the ic50 value of 5-fu (32.15 ± 48.1 μm). moreover, the results showed that compounds 3f, 4c, and 4e were also found to be potent and selective against hepg2 with ic50 values of 3.13 ± 42.0, 4.39 ± 43.2, and 7.61 ± 44.6 μm, respectively, which are also much lower than 5-fu ( figure 5 and table 3 ). the anti-cancer profile of newly synthesized compounds was also tested against leukaemia cells (jurkat cells, originated from human t lymphocytes). the compound 4c was most active, with an ic50 value of just 2.90 ± 36.3 μm, which was much lower than the positive control (5-fu ic50 = 51.16 ± 47.4 μm). similarly, the compounds 3f, 4a, and 4c showed strong activity and ic50 values were much lower as compared to 5-fu (table 3 and figure 5 ). the order of activity was 3f, 4a, 4e, and 3e in ascending order (table 3 and figure 5 ). the compound 1 showed a weaker level of activity against jurkat cells (ic50 79.35 ± 40.7 μm) and, indeed, this was much weaker than 5-fu (ic50 51.16 ± 47.4 μm). finally the cyto-toxicities of these compounds were screened in the normal human embryonic kidney (hek293) cell line, in order to assess whether these compounds are active only in cancer cells or whether they possess toxicity against normal cells as well. the results revealed that the compound 4c turned out to be most toxic by disrupting the cell survival of normal cells (hek 293) with ic50 4.77 ± 42.7 μm. the compounds 3a, 4b, and 4g also possessed some level of cyto-toxicity against hek 293 cells with ic50 values 16.73 ± 50.0, 26.08 ± 31.3, and 45.43 ± 48.9 μm, respectively. the ic50 values of these compounds in hek cells, however, are much higher compared to their activity in cancer cells (table 3 and figure 5 ). the compounds 3e, 3f, 4a, and 4e showed no activity in hek 293 cells, but strong activity in liver (hepg2) and leukaemia (jurkat) cancer cells, suggesting that these compounds are potent and potentially viable anti-cancer molecules. the solvents used were of hplc reagent grade. melting points were determined with melting points were obtained in open capillary tubes using a mel-temp melting point apparatus (sigma-aldrich chemie gmbh, 82024 taufkirchen, germany) and are uncorrected and are uncorrected. infrared (ir) spectra were recorded on a perkin-elmer 1600 series fourier transform instrument (perkinelmer life and analytical sciences, shelton, ct, usa) as kbr pellets. nuclear magnetic resonance spectra ( 1 h-nmr and 13 c-nmr spectra) were recorded on 400 mhz jeol spectrometer (jeol ltd., tokyo, japan) at room temperature. chemical shifts are reported in parts per million (ppm) and are referenced relative to residual solvent (e.g., chcl3 at δ 7.26 ppm for cdcl3, dmso at δ 2.50 ppm for dmso-d6). spin multiplicities are represented by the following signals: singlet (s), broad singlet (br s), doublet (d), broad doublet (br d), doublet of doublets (dd), triplet (t), doublet of triplets (dt), and multiplet (m). elemental analyses were performed on a perkin-elmer 2400 elemental analyzer (perkinelmer inc., waltham, ma usa), and the values found were within ±0.3% of the theoretical values. follow-up of the reactions and checks of the purity of the compounds was done by tlc on silica gel-protected aluminum sheets (type 60 gf254, merck millipore, billerica, ma, usa) and the spots were detected by exposure to uv-lamp (company seven, montpelier, md, usa) at λ 254 nm for a few seconds. the compounds were named using chem. draw ultra version 11, cambridge soft corporation. crystallographic data for the structure reported in this paper have been deposited at the cambridge crystallographic data center and allocated with the deposition numbers: ccdc 996592 for compound 4g. ccdc 996592 contains the supplementary crystallographic data for this paper. these data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the ccdc, 12 union road, cambridge cb2 1ez, uk; fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk). the product was prepared according to the reported procedure [50] compounds 3e-h were prepared following the reported procedure [54] . the entire prepared compounds were in a good agreement with the reported data. a solution of valproic hydrazide 2 (316 mg, 2 mmol) in ethanol (20 ml) was added to a solution of substituted isatin 3a-h (1 mmol) in ethanol (20 ml), and glacial acetic acid (2 drops); the reaction mixture was refluxed for 3-4 h. the product was separated out on cooling, filtered, and recrystallized from ethanol or ethylacetate to afford n′-(2-oxoindolin-3-ylidene)-2-propylpentanehydrazide derivatives 4a-h. the product was obtained as yellow solid, in 89% yield; single crystals were obtained by slow evaporation from ethanol. a good crystal with a suitable size was selected for x-ray diffraction analysis. data were collected on a d8 venture area diffractometer equipped with graphite monochromatic mok/α radiation (λ = 0.71073 å) at 100 k. cell refinement and data reduction were done by bruker saint (bruker, madison, wi usa); the program used to solve structure and refine structure is shelxs-97 [55] . the final refinement was performed by full-matrix least-squares techniques with anisotropic thermal data for non-hydrogen atoms on f 2 . all the hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. multi-scan absorption correction was applied by use of sadabs software [56] . the stock concentration of the entire compound in dmso was 10 mm and this concentration was used to prepare the working dilution. the final dmso concentration used in the experiments was ≤0.5% as the working concentration. human liver cancer cell lines (hepg2), human leukemia cell line (jurkat), and human embryonic kidney cell line (hek293) cells were cultured in high glucose dulbecco's modified eagle medium (dmem: life technologies cat #11995073) supplemented with 10% fetal bovine serum (fbs: life technologies cat #16000044) in a humidified incubator with 5% co2 at 37 °c. around 2 ×10 3 cells were seeded in each well of a 24-well cell culture plate and were allowed to adhere and grow for 24 h. the jurkat cells grow in suspension so they were cultured and allowed to grow overnight before the addition of compounds. the compounds in serial dilutions (1, 3, 15, 45, 100, and 300 μm) were added after 24 h of culture and the cells were cultured for another 24 h at 37 °c. the cell viability was determined in each experiment using mtt 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan colorimetric assay. briefly, the treated or untreated cells were trypsenized and centrifuged, and the resulting pellet was re-suspended in 100 μl of dmem serum-free medium and incubated at 37 °c for 2 h. after incubation, 20 μl of mtt solution (5 mg/ml in pbs: sigma aldrich cat #m2003) was added to each well and further incubated for 2 h. the plate was centrifuged at 40,000 rpm for 10 min then the medium was removed from each well and 2-propanol containing 0.04 m hcl was added to dissolve the formazan produced in the cells. the optical density of the formazan product in solution was measured with a microplate reader at 540 nm. the experiment was conducted in triplicate. data were calculated as percent of cell viability by the following formula: % cell viability = (mean absorbance in test wells/mean absorbance in control wells) × 100 (1) the ic50 values were calculated from the means of six different concentrations by bio data fit 1.02 using the software bio tool kit (version 300; chang bioscience inc., castro valley, ca, usa) and online program http://ic50.tk/index.html. all data were analyzed using origin (version 6.1052; origin lab corp northampton, ma, usa). one-way anova analysis of variance and student t test was used to compare different experimental groups, and data were considered statistically significant for p values less than 0.05. in conclusion, the screening results from human cancer and normal cell lines suggested that isatin derivatives were remarkably influenced by various substituents on the isatin ring and at the n-terminal of the hydrazide-hydrazone moiety. the data revealed that replacement of n-hydrogen at position 1 of the isatin moiety 3a by methyl and benzyl of the targeted compound (3e and 3f) noticeably enhanced the activity against the selected two cell lines, hepg2 and jurkat, for compound 3f. the presence of the valproic acid moiety as hydrazide-hydrazone derivatives 4a make the compound more targeted towards jurakt cells in vitro. introducing the chlorine atom to the molecule 4c, meanwhile, increased the reactivity more significantly than with 4a and 3a towards the three cell lines hepg2, jurkat, and hek293. furthermore, the methyl group in the same analogous compound 4e increased the reactivity towards the two cell lines hepg2 and jurkat more than the benzyl group 4f. the rest of the compounds had no effect on the three cell lines. further studies to assess the effect of more derivatives on various cancer cell biomarkers are currently underway in our lab. supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/08/14638/s1. microwave assisted synthesis and antimicrobial activity of 2-quinoxalinone-3-hydrazone derivatives synthesis and antimicrobial activity of some new hydrazones of 4-fluorobenzoic acid hydrazide and 3-acetyl-2,5-disubstituted-1,3,4-oxadiazolines synthesis, antibacterial and antifungal activity of some new thiazolylhydrazone derivatives containing 3-substituted cyclobutane ring efficacy of voriconazole in treatment of systemic scedosporiosis in neutropenic mice synthesis and antimycobacterial activity of (3,4-diaryl-3h-thiazol-2-ylidene)-hydrazide derivatives synthesis, characterization and antibacterial activity of biologically important vanillin related hydrazone derivatives anticonvulsant properties of various acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and unsaturated carbonyl compounds synthesis and analgesic activity of novel n-acylarylhydrazones and isosters, derived from natural safrole 1-acylthiose-micarbazides, 1,2,4-triazole-5(4h)-thiones, 1,3,4-thia-diazoles and hydrazones containing 5-methyl-2-benzoxazolinones: synthesis, analgesic-anti-inflammatory and antimicrobial activities new class of potent antinociceptive and antiplatelet 10h-phenothiazine-1-acylhydrazone derivatives a new modification of anti-tubercular active molecules antituberculosis drugs: ten years of research tuberculosis chemotherapy: current drug delivery approaches new α-(n)-heterocyclichydrazones: evaluation of anti-cancer, anti-hiv and antimicrobial activity cyanoacetic acid hydrazones of 3-(and 4-) acetyl-pyridine and some derived ring systems as potential antitumor and anti-hcv agents synthesis and anti-tumor evaluation of novel hydrazide and hydrazide-hydrazone derivatives novel synthesis of hydrazide-hydrazone derivatives and their utilization in the synthesis of coumarin, pyridine, thiazole and thiophene derivatives with antitumor activity synthesis and anti-cancer evaluation of some new hydrazone derivatives of 2,6-dimethylimidazo characterization of the conjugate of the (6-maleimidocaproyl) hydrazone derivative of doxorubicin with lactosaminated human albumin by 13 c-nmr spectroscopy synthesis of new acridines and hydrazones derived from cyclic α-diketone for cytotoxic and antiviral evaluation discovery and sar of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based hts assay anti hiv evaluation of benzo[d]isothiazole hydrazones synthesis, stereochemistry and biological activity of some novel long alkyl chain substituted thiazolidin-4-ones and thiazan-4-one from 10-undecenoic acid hydrazide -chloro-3-methyl-1-phenyl-1h-pyrazol-4-yl)methylene] 2/4-substituted hydrazides: synthesis and anticonvulsant activity synthesis and structure-activity relationships of novel 1-arylmethyl-3-aryl-1h-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against a549 lung cancer cells chemical synthesis and biological evaluation of triazole derivatives as inhibitors of inha and antituberculosis agents in silico screening, synthesis and in vitro evaluation of some quinazolinone and pyridine derivatives as dihydrofolate reductase inhibitors for anti-cancer activity n-benzylisation sulfonamide analogues as potent caspase-3 inhibitors: synthesis, in vitro activity, and molecular modeling studies synthesis and in vitro evaluation of sulfonamide isatin micheal acceptors as small molecule inhibitors of caspase-6 isatin-derived antibacterial and antifungal compounds and their transition metal complexes combinatorial optimization of isatin-β-thiosemicarbazones as anti-poxvirus agents arylimino-2-indolones are potent and selective galanin gal3 receptor antagonists 3-diaryl-1,3-dihydroindol-2-ones as antiproliferatives mediated by translation initiation inhibition isatins inhibit cyclooxygenase-2-and inducible nitric oxide synthesis in a mouse macrophage cell line isatin compounds as noncovalent sars coronavirus 3c-like protease inhibitors synthesis, antibacterial, antifungal and antiviral activity evaluation of some new bis-schiff bases of isatin and their derivatives some pharmacological activities of new substituted pyrolloindoles, indolothiazepine and azole derivatives anticonvulsant activity of schiff bases of isatin derivatives valnoctamide as a valproate substitute with low teratogenic potential in mania: a double-blind, controlled, add-on clinical trial new antiepileptic drugs that are second generation to existing antiepileptic drugs valproic acid: second generation valproic acid inhibits angiogenesis in vitro and in vivo valproic acid inhibits angiogenesis in vitro and glioma angiogenesis in vivo in the brain modulation of angiogenesis-related protein synthesis by valproic acid valproic acid exerts anti-tumor as well as anti-angiogenic effects on myeloma histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish valproic acid defines a novel class of hdac inhibitors inducing differentiation of transformed cells valproic acid for the treatment of myeloid malignancies teratogenic profile of valproic acid and newly synthesized derivatives in zebrafish embryos synthesis and biological activity of schiff base series of valproyl, n-valproyl glycinyl, and n-valproyl-4-aminobenzoyl hydrazide derivatives biological screening of novel derivatives of valproic acid for anti-cancer and antiangiogenic properties. asian pac tables of bond lengths determined by x-ray and neutron diffraction. part 1. bond lengths in organic compounds microwave irradiation: synthesis and characterization of α-ketoamide and bis (α-ketoamide) derivatives via the ring opening of n-acetylisatin facile method for the synthesis of silver nanoparticles using 3-hydrazino-isatin derivatives in aqueous methanol and their antibacterial activity a short history of shelx the authors extend their sincere appreciation to the deanship of scientific research at king saud university for funding this work through research group project no. rgp-234 (saudi arabia). ayman el-faham and sherine khattab carried out the synthesis and designed the proposed methods and analyzed the data statistically together. hazem ghabbour and hoong-kun fun carried out x-ray method and the characterization; muhammad farooq, nael abutaha, and mohammad wadaan carried out all the biological activities studies. all authors read and approved the final manuscript. the authors declare no conflict of interest.molecules 2015, 20 key: cord-025119-201ac32t authors: salman, saad; shah, fahad h; idrees, jawaria; idrees, fariha; velagala, shreya; ali, johar; khan, abid a title: virtual screening of immunomodulatory medicinal compounds as promising anti-sars-cov-2 inhibitors date: 2020-05-21 journal: nan doi: 10.2217/fvl-2020-0079 sha: doc_id: 25119 cord_uid: 201ac32t aim: severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a pernicious viral disease, causes acute respiratory distress responsible for mortality and morbidity worldwide. to screen different immunomodulatory medicinal compounds to unravel their interaction with sars-cov-2 viral proteins. materials & methods: a library of immunomodulatory medicinal compounds with antiviral capability were analyzed against sars proteases, spike protein and nonstructural proteins (nsp-9, 15) using autodock vina. results: out of more than 300 medicinal compounds, only six compounds: arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone showed significant interaction with the sars viral proteins by forming hydrogen bonds with the active site residues with low binding energy. further admet (absorption, distribution, metabolism, excretion and toxicity) analysis showed good pharmacokinetic properties and low acute toxicity of these compounds. conclusion: the current study provides convincing evidence that these medicinal compounds exert antiviral activity against the sars-cov-2 virus and could be further exploited for the treatment of this disease. severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a novel pathogen from the class of coronaviruses, is taking over the world at an unprecedented rate. it is one of the most debilitating and deadly viral respiratory diseases, and has raised many concerns among healthcare professionals and the general public. the dilemma is the severity profile, enhanced surged capacity of diagnostic and laboratory tools, the spectrum of illness and that there is no specific therapeutic intervention, while the lack of sufficient understanding of viral pathogenesis makes it even more enigmatic [1, 2] . recent studies are currently underway to decipher the molecular mechanism adopted by these viruses to understand the nature of the disease and to identify potential therapeutic targets for vaccine and drug development. coronaviruses, by taxonomical hierarchy, belong to order nidovirales and family coronaviridae. the family is further genetically classified into four different genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronvirus. the first two genera (alpha and beta-) of coronaviridae cause diseases in mammals, whereas the other two affect birds. both sars-cov-1 (middle east respiratory syndrome) and sars-cov-2 (novel coronavirus19) belong to genus betacoronavirus that are highly contagious viruses and notorious for causing a multitude of diseases with neurological, respiratory, enteric and hepatic manifestations [3] [4] [5] . they are assumed to be transmitted from bats to different organisms, which ultimately lead to transmission to humans. sars-cov-2, previously known as novel coronaviruses , are enveloped positive-sense singlestranded rna viruses with a genome size of 27-30 kb and are equipped with 5 -cap structure and 3 poly-a tails that act as typical messenger rna upon transduction [6, 7] . the genomic organization of sars-cov-2 is comprised of 14 annotated open-reading frames (orfs) translating 27 proteins. at the 5 -terminus, lies orf1a and orf1b, which encode for polyproteins that after proteolytic processing produce 16 nonstructural proteins (nsp), involved in direct rna replication and transcription. the structural region is congregated adjacent to the 3 -end and encodes for four structural proteins (spike, envelope, membrane and nucleocapsid) and other accessory proteins through ribosomal frameshifting. the function of sars spike glycoprotein is to recognize angiotensin converting enzyme-2 receptor on human epithelial cells to attach and transduce its viral rna, to commence their proliferation [8] , whereas membrane and envelope proteins are responsible for viral assembly and packaging. nucleocapsid protein encapsulates the viral genetic material inside the virion [9] . among nsps, nsp-5 protease [10] and peptidase [11] facilitate polyprotein cleavage, nsp-9 replicase [12] encourages replication of viral rna and nsp-15 endoribonuclease, [13] (a subunit of sars-cov-2 rna-dependent rna polymerase) catalyzes viral rna replication. the role of other nsps is still elusive. all these protein culprits together cause serious respiratory distress sometimes leading to death. inhibiting some of these viral proteins might impede viral replication and could be considered invaluable therapeutic targets for this disease. to recognize potential inhibitors of these viral proteins, initial screening of various pharmacological agents is essential. among these agents, medicinal compounds are promising drug candidates for sars-cov-2, as they are known for their antiviral and immunomodulatory activity since medieval times and some of them are well studied [14] [15] [16] . other abilities of these compounds include immune response amelioration, rejuvenation of damaged tissues [16, 17] and reduction of viral load [15, 18] . here, we analyzed different medicinal compounds using a virtual screening method to obtain promising inhibitors for these viral proteins that could be further utilized for sars-cov-2 treatment. in this study, essential sars-cov-2 proteins facilitating viral-host interaction, polyprotein processing and vital replication proteins were selected from the rcsb protein databank. these proteins are sars coronavirus peptidase (2gtb) [11] , sars-cov-2 protease (6lu7) [10] , spike glycoprotein (6vsb) [8] , nsp-9 replicase protein (6w4b) [12] and nsp-15 endoribonuclease (6vww) [13] . modrefiner was used for structure refinement and energy minimization of these protein receptors [19] . the refined protein structures were used to predict their active site residues using metapocket 2.0 [20] and subsequently exploited for docking analysis. medicinal compounds, known for antiviral and the immunomodulatory activity [14] [15] [16] 18] , were selected as ligands and prepared with the prodrg server [21] prior to docking. the entire docking study was facilitated by autodock vina equipped with raccoon2 (a virtual screening plugin), and the ligands were focused at the predicted active site of the viral protein receptors to perform site-specific docking to procure potential inhibitors. the successful ligands obtained from screening analysis were further investigated for admet (absorption, distribution, metabolism, excretion and toxicity) properties facilitated by gusar [22] and swissadme database [23] . molecular docking is a computer-assisted drug design and development method utilized to predict the interaction and binding mode of different compounds with a target protein. these compounds (ligands) when focused at the active site of a particular protein (receptor) establish different types of chemical bonds with their active residues. these chemical bonds include hydrogen, vanderwaal, salt bridges, π-π interaction, π-sigma bond, π-sulfur and many other hydrophobic interactions. among these bonds, hydrogen bonds play a critical role in protein-ligand interaction and lower the binding energy in order to stabilize the docked complex [24] . it is already established that when a protein active site is blocked by a chemical substrate (ligand) this abrogates its functional enzymatic activity. this approach was adopted to find novel inhibitors for sars-cov-2 by inhibiting critical proteins responsible for virus attachment and replication. more than 300 medicinal compounds with immunomodulatory and antiviral activity were added to the raccoon2 plugin of autodock vina to perform virtual screening to obtain promising inhibitors for sars-cov-2 proteins. both target proteins and compounds were prepared for docking studies by subjecting them for energy minimization and structure refinement. molecular docking was performed by exposing these compounds to the predicted active site residues of these viral proteins. the results obtained from this analysis were filtered based on compounds interaction with the active site residues, low binding energy and hydrogen and hydrophobic interaction. it was observed that, among these medicinal compounds, only six compounds showed promising interaction with the active site of sars proteins as summarized in table 1 and molecularly represented in figure 1 and supplementary figures 1, 3 , 5 and 7, respectively. the majority of these compounds exploited oxygen (o-, o=) and hydroxyl groups (-oh) to form hydrogen bonds with the sars proteins whereas carbon rings participated in establishing hydrophobic interactions. some compounds showed similar binding affinity for the active residues of these viral proteins to establish hydrogen bonds, such as in the case of peptidases (2gtb), threonine-111 and asparagine-151 were favored by arzanol, ferulic acid and thymohydroquinone while threonine-199 by genistein, and resveratrol as shown in figure 2 . mentary figure 8 ). the binding energy observed for these compounds was approximately 5-7.0 kcal/mol and they were further subjected for ligand conformational analysis, which was predicted by the ligrmsd database [25] . the prime purpose of root-mean-square deviation (rmsd) analysis is to evaluate experimentally solved ligand structure against predicted docked ligand conformations. the predicted value of the rmsd for these compounds was approximately 0.9-1.5å, which is considered successful and stable. a value beyond 2å indicates instability and aberrancy in ligand conformation and docking parameters. the principal aim of acute toxicity prediction is to determine unwanted side effects of a compound after single or multiple exposures to an organism by a known administration route (subcutaneous, oral, inhalation, intravenous or intraperitoneal). the successful compounds were further used to evaluate their acute toxicity using gusar. gusar analyses compounds based on the quantitative neighborhoods of atoms descriptors and prediction of activity spectra for substances algorithm and correlate the obtained results with symyx mdl toxicity database and further classify them on the organisation for economic co-operation and development (oecd) chemical classification manual [22] . it has been observed that these compounds elicit toxicity when the compound dose surpasses more than 500,000 mg/kg in case of the oral route, more than 7000 mg/kg for intravenous route and more than 20,000 mg/kg for subcutaneous and intraperitoneal route database as shown in table 2 . the results of the oecd chemical classification of these compounds revealed that arzanol, genistein, rosmanol and thymohydroquinone are class 4 chemicals, whereas ferulic acid and resveratrol are class 5 chemicals. to ascertain the behavior of these compounds inside an organism in terms of absorption, distribution, metabolism, and excretion (adme), it is imperative to discover their pharmacokinetic properties prior to animal and clinical studies. for this reason, the swissadme database [23] was exploited to elucidate the pharmacokinetics and drug likeness of these compounds. the lipophilicity of arzanol had a logp o/w value of 3.42 that indicates high sublingual absorption, whereas for other compounds the value remained in between 1.3 and 2.8 suggesting high oral, intestinal and central nervous system absorption. all these compounds possess high gastrointestinal absorption and watersoluble capability and ferulic acid, resveratrol and thymohydroquinone are permeable to the blood-brain barrier. out of six compounds, two of them; ferulic acid and resveratrol are cyp1a2 inhibitors, which increases the drug half-life of these compounds and also avert serious drug interactions. the drug-likeness criteria are qualified by all these compounds with no violations and possess an appreciable bioavailability score. the results are summarized in table 3 . with a growing number of infected cases with thousands of mortalities worldwide, it is imperative to discover potential therapies in order to curb this devastating situation. who approved chloroquine on an emergency basis and hydroxychloroquine based on promising in vitro and clinical results [26] . but in hindsight, these drugs are highly toxic, capable of jeopardizing patient's health by disrupting essential heart and neurological functions [27, 28] . on the other hand, hundreds of different anti-hiv protease inhibitors have been reported that are capable of terminating sars-cov-2 viral replication and rna extension. however, these viruses possess proof-reading and other coping mechanisms to attenuate the interaction, thus making these antiprotease drugs inefficient as evident 10 .2217/fvl-2020-0079 future virol. (epub ahead of print) future science group from recent clinical trials [9, 28, 29] . to address these problems, we utilized a novel approach by gathering medicinal compounds with antiviral and immunomodulatory activity to check their inhibitory interaction against critical sars-cov-2 proteins. the viral proteins include structural proteins such as spike glycoprotein that allow these viral particles to attach themselves to ace-2 receptor of the host cell, whereas nonstructural proteins (nsp-9, nsp-15) facilitate viral replication and proteases modulate the production of different proteins through proteolytic cleavage of sars-cov-2 polyproteins. this study aimed to obtain novel drug candidates that have the capability to interact with the active site of all of these viral proteins and should possess efficient pharmacokinetic profile with low toxicity to ensure safety during administration. both proteins and ligands were prepared with modrefiner and prodrg server to eradicate any bad contacts, unwanted potential energy and structural anomaly that might result in false interaction. these structurally refined molecules were used and added to raccoon2 virtual screening plugin of autodock vina to perform virtual screening of these ligands and to filter out possible drug candidates establishing hydrogen bonds, other than vanderwaal interaction, with the active site residues of these viral protein receptors. only six medicinal compounds: arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone formed hydrogen bonds with these viral proteins. these compounds were further analyzed for ligand rmsd to evaluate their interaction stability through ligrmsd [25] . the predicted value of rmsd of these compounds was in between 0.9-1.5å, which is considered adequate and stable [30] . determining the acute toxicity and pharmacokinetic properties of these compounds was facilitated by gusar software [22] and swissadme [23] . the toxicity profile of these compounds is relatively low and require high doses to elicit toxic response. the majority of the compounds are class 4 chemicals that have mild toxic effects (piloerection and diarrhea), whereas ferulic acid and resveratrol are class 5 chemicals with low toxic effects [31] . hence, the dosage of these compounds must be calibrated to exploit its full benefits and avert adverse effects. the pharmacokinetic attributes are in favor of these compounds to be exploited as promising drug candidate for sars-cov-2 treatment. apart from their antiviral activity, these medicinal compounds rejuvenate immunological response, and prevent the onset of cytokine storm that is classical hallmark of this disease. medicinal compounds combined with standard antiviral medications, synergistically ameliorate the inhibitory action on antiviral proteins [32] , reduce toxic effects [33] promote tissue repair and alleviate patients' symptoms [16] . incorporating these compounds with temporary approved anti-sars drugs as adjuvants might develop and sustain good immunological response against this lethal infection. another probable action of these compounds is to encourage phagocytotic functions, regulate the proliferation of macrophages and neutrophils and expedite the development of adaptive immunity by promoting t-cells cytokine production, natural killer cells activity and dendritic cells stimulation, which in reality takes 4-7 days for activation [14, 16] . such intervention might be able to build immunity and ameliorate the deplorable condition of sars-cov-2-affected individuals. virtual screening of different immunomodulatory compounds successfully filtered promising drug candidates for the treatment of sars-cov-2. these compounds have low toxicity, efficient pharmacokinetic profile and an excellent binding affinity for sars-cov-2 proteins. they may be further subjected to in vitro and in vivo experimentations to determine their therapeutic efficacy to be further exploited as lead compounds for clinical trials. this study elucidated the chemical affinity and mechanism of these medicinal compounds against sars-cov-2 protein targets. the results obtained from this in silico study could be used as a vehicle by other researchers and clinicians looking for promising anti-sars therapies. • docking interaction of immunomodulatory medicinal compounds library filtered six promising medicinal compounds against severe acute respiratory syndrome coronavirus-2 (sars-cov-2) viral proteins. • these six compounds, arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone, possess strong binding affinity for sars-cov-2 proteins by forming hydrogen bonds within active site residues of these proteins. • these compounds have low acute toxicity profile, excellent pharmacokinetic properties and show nominal deviation from the viral protein backbone, as evident from their root-mean-square deviation values. • this study suggests that these compounds are promising anti-sars-cov-2 inhibitors and can also serve as immunomodulatory agents to enhance immunogenicity. • the findings of this study might serve as a guide for clinicians, pharmaceutical experts and other researchers to further validate these insights in vitro and in large clinical settings to confirm their therapeutic efficacy in this disease. to view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/sup pl/10.2217/fvl-2020-0079 all the authors equally contributed for this study. financial & competing interests disclosure pathological findings of covid-19 associated with acute respiratory distress syndrome characteristics of covid-19 infection in beijing covid-19: gastrointestinal manifestations and potential fecal-oral transmission the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients liver injury in covid-19: management and challenges genome composition and divergence of the novel coronavirus (2019-ncov) originating in china covid-19 (novel coronavirus 2019)-recent trends cryo-em structure of the 2019-ncov spike in the prefusion conformation emerging coronaviruses: genome structure, replication, and pathogenesis structure of mpro from covid-19 virus and discovery of its inhibitors nelfinavir was predicted to be a potential inhibitor of 2019-ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation epub ahead of print) future science group crystal structure of the sars-cov-2 non-structural protein 9 crystal structure of nsp15 endoribonuclease nendou from sars-cov-2 plant-derived immunomodulators: an insight on their preclinical evaluation and clinical trials antiviral effect of phytochemicals from medicinal plants: applications and drug delivery strategies a review of immunomodulators in the indian traditional health care system medicinal plants in tissue engineering and regenerative medicine in the african continent antiviral potential of medicinal plants against hiv, hsv, influenza, hepatitis, and coxsackievirus: a systematic review improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization identification of cavities on protein surface using multiple computational approaches for drug binding site prediction prodrg: a tool for high-throughput crystallography of protein-ligand complexes qsar modelling of rat acute toxicity on the basis of pass prediction swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing ligrmsd: a web server for automatic structure matching and rmsd calculations among identical and similar compounds in protein-ligand docking world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? prolonged neuropsychiatric effects following management of chloroquine intoxication with psychotropic polypharmacy a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 is it reliable to take the molecular docking top scoring position as the best solution without considering available structural data? biometrical evaluation of the performance of the revised oecd test guideline 402 for assessing acute dermal toxicity medicinal plants used by traditional medicine practitioners to boost the immune system in people living with hiv/aids in uganda synergy and antagonism in natural product extracts: when 1+ 1 does not equal 2 the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord-252108-04xr5xdl authors: havrylyuk, dmytro; zimenkovsky, borys; vasylenko, olexandr; day, craig w.; smee, donald f.; grellier, philippe; lesyk, roman title: synthesis and biological activity evaluation of 5-pyrazoline substituted 4-thiazolidinones date: 2013-06-06 journal: eur j med chem doi: 10.1016/j.ejmech.2013.05.044 sha: doc_id: 252108 cord_uid: 04xr5xdl a series of novel 5-pyrazoline substituted 4-thiazolidinones have been synthesized. target compounds were evaluated for their anticancer activity in vitro within dtp nci protocol. among the tested compounds, the derivatives 4d and 4f were found to be the most active, which demonstrated certain sensitivity profile toward the leukemia subpanel cell lines with gi(50) value ranges of 2.12–4.58 μm (4d) and 1.64–3.20 μm (4f). the screening of antitrypanosomal and antiviral activities of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-diones was carried out with the promising influence of the mentioned compounds on trypanosoma brucei, but minimal effect on sars coronavirus and influenza types a and b viruses. the non-condensed heterocyclic systems with thiazolidinone [1] and pyrazoline [2, 3] moieties have emerged as powerful scaffolds in drug design. among diazole-substituted 4-thiazolidinones highly active anticancer agents have been identified including inhibitors of necroptosis [4] , tumor necrosis factor a [5] and tyrosine phosphatase [6] . our previous study, based on a hybrid pharmacophore approach, allowed to establish a number of patterns in the structureeactivity relationship (sar) context for 4-thiazolidinones with a pyrazoline fragment in 2, 3 and 4 positions of the thiazolidone cycle, which possessed antitumor activity [7, 8] . on the other hand, thiazolidinones and pyrazolines have occupied a unique position in the design and synthesis of novel biologically active agents that exert trypanocidal activity [9e13]. the 2-thioxo-4thiazolidinone-3-acetic acid derivatives were identified as inhibitors of trypanosoma brucei dolicholphosphate mannose synthase [11] . the 2-hydrazolyl-4-thiazolidinone-5-carboxylic acid derivatives have shown promising activity on the cruzipain protease [12] . the most promising compound in series of aryl-4-oxothiazolylhydrazones was shown to be very active at non-cytotoxic concentrations in in vitro assays against trypanosoma cruzi cell cultures and exhibited potency comparable with the reference compounds (ic 50 (y strain) ¼ 0.3 mm) [9] . among pyrazoline derivatives, some novel compounds have been identified as inhibitors of the trypanosomal cysteine protease cruzain with ic 50 of 40e230 nm [13] . the antiviral activity of heteryl substituted 4-thiazolidinones is promising. among thiazoleethiazolidine conjugates [14] and noncondensed derivatives with thiazolidinone and pyridine [15e17] or pyrimidine [18e20] cycles, anti-hiv agents were identified. in addition, this group of compounds was active against hepatitis c virus [21] , tobacco mosaic virus [22] and vesicular stomatitis virus [23] . previously we also demonstrated the efficiency of certain pyrazolineethiazolidinone conjugates on influenza viruses and sars coronavirus [24] . the present work is an extension of our ongoing efforts toward developing promising biologically active agents using a hybrid pharmacophore approach. we made the design (fig. 1 ) and synthesized hybrid compounds by linking the main structural unit of the 4-thiazolidinone ring system with the pyrazoline, and examined their antitumor, trypanocidal and antiviral activities in vitro. we have found two compounds from 5-pyrazoline substituted 4-thiazolidinones, which possessed a commensurate antitumor activity compared to the pyrazolineethiazolidinone analogous compounds reported previously [7, 8] and evaluated anti-trypanosomal activity and antiviral activity of the synthesized compounds. the general methods for synthesis of target thiazolidinonee pyrazoline conjugates are depicted in schemes 1 and 2. the starting 3,5-diaryl-4,5-dihydropyrazoles synthesized using known methods from appropriate chalcones [25] easily reacted with 5-bromothiazolidine-2,4-dione [26] yielding 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1a and 1b. it is known that chemical modification of the n3 position of thiazolidinone cycle has an essential influence on the antitumor activity [27, 28] . relying on these observations we utilized potassium salt of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione, generated in situ, in the reactions with 2-chloro-n-arylacetamides. following the mentioned reaction the new n3substituted non-condensed thiazolidinoneepyrazoline conjugates 2ae2c were synthesized. based on the mannich reaction of 1a and 1b with secondary amines the thiazolidinone analogs 3ae3g were obtained (scheme 1). aiming at the detailed elaboration of sar, especially the influence of the linking group of thiazolidinoneepyrazoline conjugates on the anticancer activity, 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2oxoethylidene]-thiazolidine-2,4-diones (4ae4f) were synthesized by the method, described previously [29] . reaction of 3,5-diaryl-4,5-dihydropyrazoles with (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] afforded in excellent yield and purity the compounds 4ae4f. following the reaction of generated in situ potassium salts of 4b and 4e with 2-chloro-n-arylacetamides the group of n3substituted 4-thiazolidinones 5ae5e were synthesized (scheme 2). the data characterization of synthesized novel heterocyclic substituted thiazolidones are presented in experimental part. analytical and spectral data ( 1 h nmr, 13 c nmr) confirmed the structure of the synthesized compounds. protons ch 2 ech of pyrazoline fragment in the 1 h nmr spectra of synthesized compounds showed characteristic patterns of an amx system. the proton (ch) of thiazolidinone core of 1ae1b, 2ae2c and 3ae3g showed the broad singlet at d w5.59e5.99 and the protons of the methylene group (ch 2 co) of 2ae2c and 5ae5e appeared as a broad singlet at d w4.44e4.49 ppm. in the 1 h nmr spectra of the 1ae1b and 4ae4f nh proton of thiazolidinone cycle the broad singlet at dw12.20e12.72 was found. synthesized derivatives 1a, 1b, 2a, 3ae3d, 3f, 4a, 4d, 4e, 4f and 5d were selected by national cancer institute (nci, bethesda usa) developmental therapeutic program (dtp) and evaluated at the concentration of 10 à5 m toward a panel of approximately sixty cancer cell lines (http://dtp.nci.nih.gov). the human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, central nervous system, ovarian, renal, prostate and breast cancers. primary anticancer assays were performed according to the nci protocol as described elsewhere [30e33]. the compounds were added at a single concentration and the cell cultures were incubated for 48 h. the end point determinations were made with a protein binding dye, sulforhodamine b (srb). the results for each compound are reported as the percent growth (gp%) of treated cells when compared to untreated control cells ( table 1 ). the range of percent growth shows the lowest and the highest percent growth found among the different cancer cell lines. the most active compounds 4d and 4f were found to be effective against 12 and 18 cell lines, respectively, compound 4a was found to be moderately effective against few cell lines, while the other compounds (1a, 1b, 2a, 3ae3d, 3f, 4e, 5d) did not show any activity (table 1) . finally, compounds 4d and 4f were selected for an advanced assay against a panel of approximately sixty tumor cell lines at 10fold dilutions of five concentrations (100 mm, 10 mm, 1 mm, 0.1 mm and 0.01 mm) [30e33] . the percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48-h exposure and using srb protein assay to estimate cell viability or growth. doseeresponse parameters were calculated for each cell line: gi 50 e molar concentration of the compound that inhibits 50% net cell growth; and tgi e molar concentration of the compound leading to the total inhibition. furthermore, a mean graph midpoints (mg_mid) were calculated for each of the parameters, giving an average activity parameter over all cell lines for the tested compound. for the mg_mid calculation, insensitive cell lines were included with the highest concentration tested ( table 2 ). the tested compounds showed inhibition activity (gi 50 < 10 mm) against 47 from 55 (4d) and 56 from 59 (4f) human tumor cells with average gi 50 /tgi values of 7.02 mm/38.07 mm (4d) and 4.38 mm/ 50.99 mm (4f) ( table 2) . with regard to the sensitivity against some individual cell lines among several subpanel, the compounds 4d and 4f demonstrated a certain sensitivity profile toward the leukemia subpanel tumor cell lines with gi 50 values range of 2.12e4.58 mm (4d) and 1.64e3.20 mm (4f) ( table 3 ). the sar study revealed that: (1) the level of antitumor activity of active thiazolidinones with pyrazoline fragment in 5 position (4d and 4f) is compatible with effectivity levels of heteryl substituted thiazolidones, described previously [34e42]; (2) conjugation of pyrazoline and thiazolidinone cycles using oxomethylidene linking group (4f) allowed us to increase the activity, in comparison with the structurally related conjugate representative 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1b; (3) introduction of the substituents in 3n-position of thiazolidine fragment did not have significant influence on the antitumor activity. nci's compare algorithm [30e33] allows to assume biochemical mechanisms of action of the novel compounds on the basis of their in vitro activity profiles when comparing with those of standard agents. we performed compare computations for the compounds 4d and 4f against the nci "standard agents" database at the gi 50 and tgi levels (table 4 ). however, obtained pearson correlation coefficients (pcc) did not allow to distinguish cytotoxicity mechanism of tested compounds with high probability. the compound 4d showed the highest correlation at the gi 50 level with dihydroorotate dehydrogenase inhibitor brequinar (pcc ¼ 0.651) and compound 4f e with maytansine (rna/dna antimetabolite, pcc ¼ 0.636). antiviral activity of 1a, 1b, 2ae2c, 3d and 3e was determined against sars coronavirus (sars cov) and influenza types a and b viruses (flu a, flu b). the obtained results are summarized in table 5 . although antiviral activity was evident, virus inhibition occurred at or near the cytotoxic concentration. the compounds showed insignificant activities against the four strains of influenza virus with the range levels of selectivity index from 1.0 to 2.1. compound 2a had moderate activity against the duck strain of influenza a with a 50% effective concentration (ec 50 ) of 21.78 mm and selective index (si) of >16.3; but did not have significant activity against other influenza strains. the majority of the compounds showed no activity against sars cov. the positive control compounds ribavirin, oseltamivir carboxylate, and m128533 were active as expected in the assays. the compounds 1a, 2b and 2c were selected in advanced in vitro assay against trypanosoma brucei brucei (tbb) and trypanosoma brucei gambiense (tbg). the doseeresponse curves with drug concentrations ranging from 10 mg/ml to 0.625 mg/ml are depicted on the results showed a moderated activity of compounds (table 6) on both parasite strains, namely ic 50 (tbb) ¼ 5.43e13.87 mm and ic 50 (tbg) ¼ 2.53e6.66 mm. in the present paper new 4-thiazolidinone based conjugates with pyrazoline moiety at 5 position are described. antitumor activity assay of thirteen synthesized compounds allowed us to identify highly active thiazolidinoneepyrazoline hybrids 4d and 4f, which demonstrated certain sensitivity profile toward the leukemia subpanel tumor cell lines with gi 50 values range of 2.12e4.58 mm (4d) the starting 3,5-diaryl-4,5-dihydro-1h-pyrazole [25] , 5-bromothiazolidine-2,4-dione [26] , and (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] were obtained according to the table 1 anticancer screening data at the concentration of 10 mm. methods described previously. preparation of compounds 4ae4d and 4f was described in our previous report [29] . a suspension of compound 1a or 1b (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-n-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in etoh (10 ml). obtained powders were filtered off, washed with ethanol and recrystallized with dmf:ethanol (1:2) mixtures. (2b). yield 68%, mp 220e222 c. 1 anti-trypanosomal activity of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1yl)-thiazolidine-2,4-dione derivatives (1a, 2b, 2c). 137.9, 133.9, 133.3, 133.2, 130.1, 129.3, 128.9, 128.7, 128.6, 128.2, 127.6, 127.2, 127.1, 123.6 a solution of (2,4-dioxothiazolidin-5-ylidene)-acetyl chloride (3 mmol) in 5 ml of dioxane was added to a mixture of appropriate 3,5-diaryl-4,5-dihydro-1h-pyrazole (3 mmol) and triethylamine (3 mmol) in 5 ml of dioxane and later was heated to 70e80 c during 15 min, cooled and poured water (50 ml). obtained powder was filtered off, washed with water and recrystallized with dmf:ethanol (1:2) mixtures. results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. the percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. the cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of human tumor cell lines at concentrations ranging from 10 à4 to 10 à8 m. 48-h continuous drug exposure protocol was followed and an srb protein assay was used to estimate cell viability or growth. using absorbance measurements [time zero (tz), control growth in the absence of drug (c), and test growth in the presence of drug (ti)], the percentage growth was calculated for each drug concentration. percentage growth inhibition was calculated as: ½ðti à tzþ=ðc à tzþ â 100 for concentrations for which ti ! tz; ½ðti à tzþ=tz â 100 for concentrations for which ti < tz: dose response parameters (gi 50 , tgi) were calculated for each compound. growth inhibition of 50% (gi 50 ) was calculated from [(ti à tz)/(c à tz)] â 100 ¼ 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by srb staining) as compared to the net protein increase seen in the control cells. the drug concentration resulting in total growth inhibition (tgi) was calculated from ti ¼ tz. values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was excessive, the value for that parameter was expressed as more or less than the maximum or minimum concentration tested. the lowest values were obtained with the most sensitive cell lines. compounds having gi 50 values 100 mm were declared to be active. primary antiviral assay was performed on a respiratory viruses panel (flu a (h1n1), flu a (h3n2), flu a (h5n1), flu b, sars cov) [43] . compounds were diluted to 20 mg/ml in dmso then eight half-log dilutions were prepared in mem solution with 50 mg/ml gentamicin. each dilution was added to 5 wells of a 96-well plate with 80e100% confluent cells, and three wells of each dilution were then infected with the test virus using a multiplicity of infection of <0.006 ccid50 per cell for each virus. two wells remained uninfected as toxicity controls. a known active compound was run in parallel as a control. after cytopathic effect (cpe) was observed microscopically, plates were stained with neutral red dye for approximately 2 h, then supernatant dye was removed from the wells and the incorporated dye was extracted in 50:50 sorensen citrate buffer/ethanol and read on a spectrophotometer at 540 nm. the optical density of test wells was converted to percent of cell and virus controls, then the concentration of test compound required to inhibit viral cpe by 50% (ec 50 ) was calculated by regression analysis. the concentration of compound that would cause 50% cytotoxicity in the uninfected cells was similarly calculated (cc 50 ). ec 50 and cc 50 were presented in mm. the selective index (si) is the cc 50 divided by ec 50 . bloodstream forms of t. brucei brucei strain 90-13 and t. brucei gambiense feo strain were cultured in hmi9 medium supplemented with 10% fcs at 37 c under an atmosphere of 5% co 2 [44] . in all experiments, log-phase parasite cultures were harvested by centrifugation at 3000âg and immediately used. drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [45] . drug stock solutions were prepared in pure dmso. t. brucei bloodstream forms (10 5 cells/ml) were cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 ml. after a 72-h incubation, resazurin solution was added in each well at the final concentration of 45 mm and fluorescence was measured at 530 nm and 590 nm absorbance after a further 4-h incubation. the percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. dmso was used as control. concentration inhibiting 50% of parasite growth (ic 50 ) was determined from the doseeresponse curve with a drug concentrations ranging from 10 mg/ml to 0.625 mg/ml and presented in mm. ic 50 value is the mean þ/à the standard deviation of three independent experiments. yield 82%, mp 268e270 c. 1 h nmr (400 mhz, dmso-d 6 þ ccl 4 ): d 12.71 (br. s, 1h, nh), 8.27 (s, 1h, arom), 7.95e8.11 (m, 4h, arom), 7.88 (s, 1h, ch), 7.56e7.60 (m, 2h, arom) 5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene]-2,4-dioxothiazolidin-3-yl}-n-arylacetamides (5ae5e) a suspension of compound 4b or 4f (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-n-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in etoh (10 ml). obtained powders were filtered off 3-phenyl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-n-p-tolylacetamide (5a) 04 (d, 2h, j ¼ 7.9 hz, arom), 5.70 (dd, 1h, ch 2 ch dmso-d 6 þ ccl 4 ): d 10.04 (s, 1h, nh), 8.02 (s 4 -d ioxoth iazolidin-3-yl )-n-(4-chlorophenyl)-acetamide (5c). yield 79%, mp 289e290 c. 1 h nmr (400 mhz, dmso-d 6 þ ccl 4 ): d 10.51 (s, 1h, nh), 7.98 (s, 1h, coch), 7.83e7.86 (m, 2h, arom), 7.48e7.58 (m, 5h, arom), 7.34e7.42 (m, 3h, arom) -{2-[5-(4-methoxyphenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-acetamide (5d). yield 86%, mp 268e269 c 46 (d, 2h, j ¼ 6.4 hz, arom), 7.21 (d, 2h, j ¼ 6.4 hz, arom), 6.90 (br. s, 3h, arom), 5.68 (dd, 1h, ch 2 ch 4-thiazolidones: centenarian history, current status and perspectives for modern organic and medicinal chemistry biological activities of pyrazoline derivatives e a recent development recent advances in the therapeutic applications of pyrazolines structureeactivity relationship study of a novel necroptosis inhibitor photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of tnf-a 2-thiazolylimino/heteroarylimino-5-arylidene-4-thiazolidinones as new agents with shp-2 inhibitory action synthesis of novel thiazolone-based compounds containing pyrazoline moiety and evaluation of their anticancer activity synthesis of new 4-azolidinones with 3,5-diaryl-4,5-dihydropyrazole moiety and evaluation of their antitumor activity in vitro synthesis, cruzain docking, and in vitro studies of aryl-4-oxothiazolylhydrazones against trypanosoma cruzi synthesis and structureeactivity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against plasmodium falciparum, trypanosoma brucei, and trypanosoma cruzi first small molecular inhibitors of t. brucei dolicholphosphate mannose synthase (dpms), a validated drug target in african sleeping sickness synthesis of 2-hydrazolyl-4-thiazolidinones based on multicomponent reactions and biological evaluation against t. cruzi synthesis and structureeactivity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain design, synthesis, and evaluation of 2-aryl-3-heteroaryl-1,3-thiazolidin-4-ones as anti-hiv agents synthesis and anti-hiv studies of 2-adamantyl-substituted thiazolidin-4-ones discovery of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-hiv-1 agents design and synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones as anti-hiv agents synthesis and evaluation of 2-(2,6-dihalophenyl)-3-pyrimidinyl-1,3-thiazolidin-4-one analogues as anti-hiv-1 agents predicting anti-hiv activity of 1,3,4-thiazolidinone derivatives: 3d-qsar approach design, microwave-assisted synthesis and hiv-rt inhibitory activity of 2-(2,6-dihalophenyl)-3-(4,6-dimethyl-5-(un)substituted-pyrimidin-2-yl)thiazolidin-4-ones non-nucleoside inhibitors of the hepatitis c virus ns5b rna-dependant rna polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives synthesis and biological activity of 4-(3-aryl-4-oxo-2-thioxothiazolidin-5-ylimino)-3-methyl-1-(n,n-disubstituted aminomethyl) pyrazolin-5-ones synthesis and antiviral activity of new pyrazole and thiazole derivatives synthesis and anticancer and antiviral activities of new 2-pyrazoline-substituted 4-thiazolidinones synthesis and antidepressant activities of some 3,5-diphenyl-2-pyrazolines synthesis and antihyperglycemic activity of novel 5-(naphthalenylsulfonyl)-2,4-thiazolidinediones synthesis and in vitro anticancer activity of 2,4-azolidinedione-acetic acids derivatives structureeanticancer activity relationships among 4-azolidinone-3-carboxylic acids derivatives synthesis and evaluation of antitumor activity of 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen cancer drug discovery and development the nci60 human tumour cell line anticancer drug screen synthesis and anticancer activity of novel nonfused bicyclic thiazolidinone derivatives, phosphorus new 5-arylidene-4-thiazolidinones and their anticancer activity a facile synthesis and anticancer activity evaluation of spiro thiazolidinone motif in anticancer drug discovery. experience of dh lnmu medicinal chemistry scientific group synthesis and anticancer activity evaluation of 4-thiazolidinones containing benzothiazole moiety synthesis and anticancer activity of isatin-based pyrazolines and thiazolidines conjugates synthesis and antitumor activity of novel 2-thioxo-4-thiazolidinones with benzothiazole moieties synthesis of new 4ethiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity study of molecular mechanisms of proapoptotic action of novel heterocyclic 4-thiazolidone derivatives in vitro and in vivo assay systems for study of influenza virus inhibitors prolyl oligopeptidase of trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice new protein farnesyltransferase inhibitors in the 3-arylthiophene 2-carboxylic acid series: diversification of the aryl moiety by solid-phase synthesis we are grateful to dr. v.l. narayanan from drug synthesis and chemistry branch, national cancer institute, bethesda, md, usa, for in vitro evaluation of anticancer activity. evaluations of compounds for antiviral activity were supported by funds from contract n01-ai-15435 from the virology branch, division of microbiology and infectious diseases, national institute of allergy and infectious diseases, national institutes of health, bethesda, md, usa. key: cord-006139-9063uhox authors: egan, timothy j; kuter, david title: dual-functioning antimalarials that inhibit the chloroquine-resistance transporter date: 2013-03-27 journal: future microbiol doi: 10.2217/fmb.13.18 sha: doc_id: 6139 cord_uid: 9063uhox malaria remains a major international health challenge. resistance to a number of existing drugs and evidence of the emergence of artemisinin resistance has emphasized the need for new antimalarials. a new approach has been the preparation of dual-function compounds that include a chloroquine-like antimalarial group and a group that resembles a chloroquine chemosensitizer. this article reviews the recent discovery of such dual-function antimalarials that are proposed to target both hemozoin formation and the chloroquine resistance transporter, pfcrt. these are discussed in relation to the mechanism of action of 4-aminoquinolines, chloroquine resistance and resistance reversal. according to the who world malaria report of 2011, there were 216 million cases of malaria in 2010, with 655,000 deaths, 91% of which occurred in africa, with 86% of the victims being children under the age of 5 years [1] . despite a 25% decrease in mortality since 2000, one child still dies of malaria every minute. the disease is caused by four species of protozoan parasite of the genus plasmodium that are specific to humans, namely plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale. a fifth species, plasmodium knowlesi, which primarily infects monkeys, has also been recognized as a zoonotic cause of human malaria in indonesia [2] . of these species, p. falciparum is the most deadly. all are transmitted by mosquitoes of the genus anopheles. control and eradication of malaria relies on a multifaceted strategy. this involves prevention of mosquito bites using screening methods, elimination of mosquitoes and treatment of infected individuals to eliminate the parasite from the host. large-scale deployment of bed nets [3] , reintroduction of ddt in some areas, such as mozambique [4] , and the introduction of artemisinin combination therapy (act) have all contributed to the significant progress made in the last decade in fighting malaria [5] . drug resistance: an ominous threat the first highly active synthetic antimalarials introduced after world war ii were used either as monotherapies (e.g., chloroquine) or combinations targeting a single pathway (antifolates; e.g., sulfadoxine with pyrimethamine). this strategy eventually led to the emergence and widespread dissemination of drug-resistant parasites. consequently, starting in the 1990s, new combination therapies in the form of acts were introduced. these consist of an artemisinin derivative, such as artesunate, artemether or dihydroartemisinin on the one hand, combined in a fixed dose with a 4-aminoquinoline, aryl methanol or a related derivative such as amodiaquine, lumefantrine (coartem ® , norvatis, switzerland), mefloquine, piperaquine (euartesim ® , mmv, switzerland) or pyronaridine (pyramax ® , mmv) on the other. currently, these acts are highly effective. however, ominous first signs of possible future resistance have begun to appear. patients have been identified with prolonged parasite clearance times, resulting in artemisinin treatment failures in western cambodia and western thailand [6] [7] [8] . this appears to have occurred as a result of the use of artemisinin monotherapy [6] . at the same time, resistance to mefloquine is widespread [9] , while some strains of chloroquine-resistant parasite are also cross-resistant to amodiaquine [10] . in addition, evidence suggestive of the possible development of lumefantrine tolerance, which is the first step towards resistance, has begun to emerge in east africa, where coartem is widely used [11] . while resistance to act may still be some time away, it is prudent that new antimalarials be developed against this possibility. thus, considerable work continues to find new and improved antimalarials. future science group important. prior to the emergence of resistance, it was highly effective, generally well tolerated at appropriate doses, safe for use in pregnancy and inexpensive. unfortunately, chloroquineresistant p. falciparum is now very widespread. nonetheless, this has not led to the complete loss of this class of compound, because resistance is not coupled to the drug target itself. indeed, the 4-aminoquinoline piperaquine has recently entered the clinic (in combination with dihydroartemisinin) [12] , while another 4-aminoquinoline, ferroquine, is in phase iib clinical trials [13] . in addition, the 4-aminoquinoline, amodiaquine, and the quinoline methanol, mefloquine, as well as the aryl methanol, lumefantrine, are all currently crucial components of act. these antimalarials are widely believed to act by inhibiting heme detoxification in the malaria parasite, a hypothesis that is best established in the case of chloroquine. the process of hemoglobin digestion and heme detoxification is summarized in figure 1 . hemozoin formation is the dominant fate of heme released into the digestive vacuole (dv), with at least 95% of the iron present in late trophozoites (32 h into the 48-h blood cycle) present as hemozoin [14] . the parasite also possesses an endogenous cytosolic fe-superoxide dismutase and imports a host peroxiredoxin from the red blood cell into the parasite cytosol [15, 16] . these enzymes remove o 2 and h 2 o 2 , both of which are probably produced in part during the oxidation of fe(ii)heme. while it has long been known that antimalarials such as chloroquine and quinine interact directly with fe(iii)heme [17, 18] , the first crystal structure of a drug-fe(iii)heme complex (that of halofantrine-fe[iii]heme), was only reported in 2008 (figure 2a) figure 1 . a schematic representation of the process of hemoglobin degradation and hemozoin formation in plasmodium falciparum. rbc cytoplasm is taken up into the malaria parasite via transport vesicles in an endocytotic process and delivered to the acidic digestive vacuole (~ph5) [86] . hemoglobin is digested by a series of proteases: plasmepsins, hap, falcipains and falcilysin [87] [88] [89] . the resulting peptides are ultimately hydrolyzed to amino acids. currently, agreement about the details of this step has not been reached [90] [91] [92] . the toxic heme released during hemoglobin digestion is oxidized to the fe(iii) state and then incorporated in less-toxic hemozoin in a process associated with neutral lipids [93, 94] , represented in the figure by the elliptical structure enclosing the hemozoin crystals. hap: histoaspartic protease; rbc: red blood cell. future science group p. falciparum could be accounted for on the basis of an additional interaction: a chargeassisted hydrogen bond between a protonated amine nitrogen atom in the side chain of the drug and one of the heme propionate groups. this interaction has recently been confirmed in crystal structures of quinine-fe(iii)heme and quinidine-fe(iii)heme ( figure 2b & 2c) [20]. by contrast, the structures of 4-aminoquinoline-fe(iii)heme complexes remain less well understood, despite various efforts to use nuclear magnetic resonance techniques to elucidate them [21] [22] [23] [24] [25] [26] [27] [28] [29] . in addition, the precise relationship between heme binding and hemozoin inhibition remains unclear. early postulates involved stoichiometric solution complexes of antimalarials with fe(iii)heme [30] and included suggestions that drugs act by increasing fe(iii)heme solubility, thus preventing aggregation [30, 31] . more recently, however, an alternative suggestion by buller et al. has enjoyed considerable attention [32] . these authors, among others, have suggested that this class of drug can dock into the fastest-growing face of the hemozoin crystal, as well as inhibit growth of some of the other faces [32] [33] [34] [35] . this hypothesis has been able to mathematically account for the effects of chloroquine and quinidine on the kinetics of synthetic hemozoin (b-hematin) formation [36] . in addition, it can also explain how substoichiometric quantities of a drug can inhibit hemozoin formation and provides a well-defined binding site for these compounds. to date, however, this model does not appear to have been directly used to try to design new inhibitors using a rational approach, probably because conventional drug-docking programs are not able to handle crystal surfaces well. despite ample evidence that quinoline and related antimalarials inhibit b-hematin formation under abiotic and biomimetic conditions, direct evidence of inhibition of hemozoin formation in the parasite itself is much more sparse. chloroquine and, more recently, ruthenoquine, an analog of the 4-aminoquinoline ferroquine in which the ferrocene moiety is replaced by ruthenocene, have been shown to accumulate in the parasite dv in close proximity to hemozoin [37, 38] . smaller hemozoin crystals and a premature halt in their growth within the parasite dv has been observed in the presence of chloroquine [39] . finally, at 120 nm and 12-h incubation times, chloroquine causes a build-up of transport vesicles in the parasite, which contain undigested hb [40] , indicating that the endocytotic feeding process is inhibited. recently, a fractionation strategy has been applied together with electron spectroscopic imaging to chloroquine-treated p. falciparum [41] . this has clearly shown a dose-dependent increase in free fe(iii)heme occurring together with the decrease in hemozoin. undigested hb only seems to appear at higher doses of chloroquine, and the levels of free fe(iii)heme are closely correlated with the parasite survival curve (figure 3 ). electron spectroscopic imaging using electron energy loss spectroscopy clearly showed a translocation of iron to the parasite cytoplasm ( figure 4a & 4b) . since virtually all of the iron present is heme iron and there is little undigested hb at the dose used, this is likely to be the location of the free fe(iii)heme. there the aromatic ring lies parallel to the porphyrin ring in a p-stacking arrangement. in the cases of the quinidine and quinine complexes, a charge-assisted hydrogen bond (salt bridge) occurs between one of the heme propionate groups and the protonated quinuclidine tertiary amino group. the structural models shown here were created using data taken from [19, 20] . future science group is also evidence that chloroquine causes a disruption in the growth of the hemozoin crystals, which show evidence of a mosaic structure with grain boundaries ( figure 4c ). thus, in combination with earlier work, this study provided strong evidence that chloroquine does indeed act by inhibiting hemozoin formation in the parasite. preliminary evidence also showed an increase in free fe(iii)heme and a decrease in hemozoin at 2.5× ic 50 for a number of other drugs, including amodiaquine, mefloquine and lumefantrine, as well as artesunate. however, it must be emphasized that further work would be required to substantiate whether the observations made with these other drugs are causal or the effect of inhibiting other targets. despite ongoing efforts to understand the mechanism of action of 4-aminoquinolines and the structures of their complexes with fe(iii)heme, the existence of structure-activity relationship data has substantially aided the task of designing new compounds in this class. four studies in the late 1990s and early 2000s provided a detailed structure-activity model for the 4-aminoquinolines [42] [43] [44] [45] . this is summarized in figure 5 . further studies have revealed that alterations to the alkyl side chain of the quinoline can abolish cross-resistance with chloroquine [46] . indeed, the structure of the side chain appears to be the primary determinant of resistance, with the quinoline ring itself having, at most, a minor role in cross-resistance [47] [48] [49] . this has encouraged the design of new quinoline antimalarials with the aim of overcoming resistance. as mentioned above, p. falciparum that are resistant to chloroquine or other quinolines exhibit no known changes in the process of heme detoxification. rather, resistance arises from mutations and changes in expression levels of membrane proteins located in the dv membrane. the principal determinants of chloroquine resistance are mutant forms of a protein known as pfcrt [50, 51] . a second protein, pfmdr1, has also been implicated in resistance to quinoline antimalarials [52, 53] . mutations in this protein are associated future science group with mefloquine resistance in field isolates, and an increased copy number has previously been associated with decreased sensitivity to quinine. pfmdr1 is not thought to be directly responsible for chloroquine resistance, but mutant forms of this protein can affect chloroquine sensitivity in the presence of mutant forms of pfcrt [54] , and there is evidence of a complex interaction between pfcrt and pfmdr1 [55] . in addition, pfmdr1 has recently been implicated in the transport of chloroquine and other quinolines into the parasite dv [56] and has subsequently also been shown to bind to a selection of these drugs [57] . furthermore, evidence from a cross of two different drug-resistant strains (gb4 and 7g8) has indicated that quinolines actually inhibit transport of the natural substrates of pfcrt and pgh-1 (the protein encoded by pfmdr1) [58] . a third protein, pfmrp1, located in the parasite plasma membrane, has also been suggested to play a role in chloroquine resistance, but in this case, the evidence remains uncertain [59, 60] . notwithstanding the role of these other membrane proteins, a pfcrt mutation is accepted to be the major factor involved in chloroquine resistance. pfcrt is predicted to be an integral membrane protein localized to the dv membrane. it is believed to be a member of the drug metabolite transporter family of proteins [61] . chloroquineresistant strains of the parasite exhibit several mutations in this protein ( figure 6 ) [62] , but all naturally occurring chloroquine-resistant mutants exhibit one crucial mutation, that of lys-76 to thr-76 (k76t). the additional mutations to k76t are thought to counteract the loss of function that would occur in the case of the k76t mutation alone [63] . studies conducted using xenopus laevis oocytes injected with mrna encoding pfcrt have convincingly shown that this protein transports chloroquine. these findings support the hypothesis that decreased activity of chloroquine stems from its extrusion by pfcrt from the dv. this effectively lowers the concentration of chloroquine in this organelle, thus permitting hemozoin formation to resume unhindered [64] . chloroquine is also thought to bind to pfcrt, and a possible site of interaction has been proposed based on photoaffinity labeling [65] . several recent and comprehensive reviews covering pfcrt and chloroquine resistance are available, and readers are encouraged to consult them for in-depth discussion [62, [66] [67] [68] . it has been known for two and a half decades that verapamil, a calcium channel blocker, can restore the activity of chloroquine in several resistant laboratory strains of p. falciparum [69, 70] . subsequent to this initial discovery, many other compounds have been found to have similar chemosensitizing properties. these include other calcium channel blockers, including analogs of verapamil and nifedipine; dibenzazepines and their analogs, which include imipramine; phenothiazines; dihydroanthracenes; dibenzylmethylamines (dibemethins); plant-derived alkaloids; and others, including primaquine [71] . a common feature of nearly all of these compounds is a basic amino group that is expected to be protonated at the ph of the parasite dv. these compounds are believed to act by inhibiting chloroquine transport by pfcrt, and this has been directly demonstrated in the case of verapamil, primaquine and several dibemethins in the xenopus oocyte system [64, 72] . it is not known whether these compounds act as competitive or noncompetitive inhibitors of chloroquine transport. two significant quantitative structure-activity relationship studies have been carried out on chloroquine chemosensitizers. one exclusively investigated a series of dihydroanthracene derivatives with rigid bicyclic structures [73] . based on this study, the authors suggested that these molecules interact with a serine (or threonine) and an aspartate (or glutamate) side chain in pfcrt, which are separated by 9.2 å. in a second study, which has turned out to be more influential, a the 4-aminoquinoline group is the smallest fe(iii)heme-binding fragment [43] . replacement of the quinoline ring n atom with a ch group abolishes interaction with fe(iii)heme, as well as hemozoin inhibitory activity and biological activity [45] . replacement of the 4-amino nh group with a ch 2 group weakens fe(iii)heme binding and also abolishes hemozoin inhibition and biological activity [45] . the cl atom is necessary for the inhibition of hemozoin formation. it can be replaced with other electron-withdrawing hydrophobic groups [44] . the tertiary n atom in the side chain is important for accumulation in the parasite digestive vacuole through ph trapping, and it usually improves activity [43] . analogs are known in which this n atom is replaced with a ch, retaining biological activity [42] . future science group series of imipramine analogs were investigated, leading to a pharmacophore model consisting of two suitably positioned aromatic groups and a weak base amino group (figure 7 ) [74] . the latter study has underpinned the design of a new type of antimalarial that incorporates the features required for both an active anti malarial and a resistance-reversing chemosensitizer. these dual-function compounds, consisting of so-called 'reversed chloroquines' and related compounds, are the subject of the remainder of this review. dual-function antimalarials with both chloroquine-like activity (hemozoin inhibition) and resistance-reversing activity form a relatively new class of compounds. the first example, a so-called reversed chloroquine (compound 1), was reported in 2006 [75] . this consisted of a 4-amino-7-chloroquinoline moiety linked to the n atom of imipramine via a three-carbon alkyl linker group ( figure 8a) . compound 1 was shown to be active in vitro against both the chloroquine-sensitive d6 and chloroquine-resistant dd2 strains of p. falciparum. it was also shown to associate with fe(iii)heme, both at ph 5.7 and 7, with log association constant (logk ) values of 5.48 (comparable to chloroquine, with values of 5.48 and 6.00, respectively, at these two ph values). however, compound 1 itself was not considered suitable for further drug development owing to its high lipophilicity (log distribution constant [clogp] = 8.9). in a follow-up study, peyton and coworkers investigated the effects of the linker and head groups in the activity of a range of reversed chloroquines [76] . in one study, the head group was altered to a diphenylamine or dibenzylamine, while the aliphatic portion of the side chain and linker chain were varied in length in an effort to reduce lipophilicity [76] . all of these compounds demonstrated strong activity against parasites cultured in vitro (ic 50 <120 nm), with only minor differences between chloroquinesensitive and -resistant d6 and dd2 strains. larger differences were seen in cytotoxicity, with molecules possessing amide or piperazine linkers exhibiting the lowest toxicity. nonetheless, all of the compounds tested exhibited well over 100-fold selectivity against malaria parasites. closer scrutiny of the ic 50 values showed that a dibenzylamine head group usually gave rise to a more active compound than the corresponding compound with a diphenylamine head group, and that replacement of the linear diamine linker with a piperazine group also generally decreased activity. on the other hand, introduction of an amide head group was tolerated and permitted a decrease in clogp to values comparable to chloroquine. in a subsequent and more comprehensive study, this group made further variations to this chemotype, replacing the diphenyl-or dibenzylamine head group with others such as benzhydryl, the reliability of this structural model remains untested. side chains of residues at sites of mutations implicated in chloroquine resistance are shown as space-filling spheres, and k76 is marked by an arrow. the structural model of the protein shown here was created using data taken from [95] . future science group adamantyl, triphenylmethyl and pyridine-2-yl methyl groups [77] . benzhydryl head groups with substituents were also investigated. interestingly, the variations in head group had a relatively small influence on activity against either the d6 chloroquine-sensitive or dd2 chloroquineresistant strains of the parasite, but considerably more variation was observed in activity against the 7g8 chloroquine-resistant strain. indeed, in many of the derivatives, activity against the 7g8 strain was four-to five-fold weaker than against the d6 or dd2 strains. this is notable in view of the fact that the 7g8 strain, which originates from south america, differs from the old world d6 and dd2 strains in as much as verapamil has only weak chemosensitizing activity on it. this perhaps supports the proposal that the activities of these compounds relates to the presence of a resistance-reversing pharmacophore. of particular note, however, is a derivative, compound 2, in which the phenyl rings in the head group were replaced with ortho-pyridyl groups ( figure 8b ) [77] . this compound was equipotent against all three of the tested strains and is substantially less lipophilic (clogp = 3.6) than the other compounds, a factor that is important in improving solubility and potentially lowering systemic toxicity. this compound represents a potential lead compound for further development. indeed, it was found to possess good oral activity in the plasmodium berghei mouse model of malaria, with four doses at 30 mg/kg reducing parasitemia by more than 99% and curing two out of three treated mice. a selection of these reversed chloroquines were investigated for their fe(iii)heme binding and b-hematin inhibiting activities. they were found to exhibit dissociation constant values (k d ) ranging from 8.6 to 1.0 µm (logk = 5.1-6.0), similar to that of chloroquine with a k d of 4.0 µm (logk = 5.4). the ic 50 values for b-hematin inhibition (1.6-14 µm) were lower than that of chloroquine (24 µm), and a significant (r 2 = 0.66) correlation between b-hematin inhibitory ic 50 and in vitro antimalarial activity against p. falciparum was observed. finally, it has also been demonstrated that these compounds could inhibit b-hematin formation and decrease hemozoin formation within the parasite cell. the most active compound was also a more potent hemozoin inhibitor than compound 1 [77] . consideration of the structures of these reversed chloroquines in light of known structure-activity relationships for active hemozoininhibiting quinolines, as well as resistance-reversing chemo sensitizers, permits rationalization of their structure-activity relationships (figure 9 ). this approach was subsequently used in the design of other resistance-reversing antimalarials. other studies have built on the concept of reversed chloroquines. dihydropyrimidinones are a well-known class of calcium channel blockers. similar to verapamil, they are capable of chemosensitizing multidrug-resistant cancer cells. these molecules have been attached to the 4-amino-7-chloroquinoline structure to produce a series of compounds (e.g., compound 3 in figure 10a ) with strong activity against chloroquine-resistant and -sensitive parasites [78] . interestingly, these compounds do not have basic n atoms in the side chain and therefore do not conform to the more commonly observed structure-activity relationship model. tricyclic heteroaromatics, especially phenothiazines such as chlorpromazine (compound 4), are well-known chloroquine chemosensitizers. furthermore, it has been demonstrated that chlorpromazine ( figure 10b) can be modified to produce an analog (compound 5) with antimalarial activity with improved water solubility and improved oral bioavailability from the same group [76] . future science group by the introduction of an additional basic amino group ( figure 10c ) [49] . kelly et al. later showed that the related tricyclic aromatic acridones (e.g., compound 6) also exhibit chloroquine chemosensitizing activity ( figure 10d ) [79] . subsequently, these authors went on to introduce a further weak base-containing side chain onto this scaffold to produce a dual-function compound (compound 7) that exhibited antimalarial activity ( figure 10e ) [80] . this compound was shown to accumulate in the parasite dv by confocal fluorescence microscopy and to inhibit hemozoin formation. isobolograms were used to demonstrate that it exhibited an additive relationship when mixed with chloroquine in a chloroquine-sensitive p. falciparum strain (d6), but was synergistic in the chloroquine-resistant dd2 strain. this observation is consistent with that expected if the compound acts as a chemosensitizer in chloroquine-resistant parasites and also acts as an antimalarial in a manner similar to chloroquine. this compound exhibited excellent activity against chloroquine-sensitive and -resistant parasites and was active in vivo. a recent addition to the class of dual-function quinolines that have resistance-reversing activity is the dibemequines [81] , consisting of a 4-amino-7-chloroquinoline with a dibemethin (dibenzylmethylamine) side chain. these were designed to fulfill the structure-activity relationship criteria of both an active hemozoin-inhibiting quinoline antimalarial and a chloroquine n n cl hn n figure 9 . rationalization of the structure-activity relationships in the reversed chloroquines. the parts of the molecule highlighted are the 4-aminoquinoline moiety required for fe(iii)heme binding, the cl atom required for hemozoin inhibition, the basic tertiary n atom that aids accumulation in the digestive vacuole (see figure 5 ) and the two aromatic rings and hydrogen bond acceptor required for a chloroquine chemosensitizer (see figure 7) . [79] . (e) a dual-function acridone (compound 7) that both reverses chloroquine resistance and is an active antimalarial [80] . future science group chemosensitizer ( figure 11 ). the dibemethin side chains themselves were found to reverse chloroquine resistance and to inhibit chloroquine transport by pfcrt [72] . the crystal structure of the prototype dibemequine (compound 8) demonstrated the structural requirements for a resistance future science group reverser, with the quinoline group folded around to establish an approximately triangular relationship between the quinoline n atom and the two phenyl groups of the dibemethin side chain. this compound was shown to directly inhibit chloroquine transport by pfcrt in the xenopus oocyte model ( figure 11b ). two analogs (compounds 9 & 10) were also shown to exhibit such activity ( figure 11c ). these represent the first blood-stage antimalarially active compounds for which direct evidence of the inhibition of chloroquine transport by pfcrt has been provided. isobologram ana lysis of compound 10 revealed an additive relationship with chloroquine in the chloroquine-sensitive d10 strain of parasite, but a synergistic relationship in the chloroquineresistant dd2 strain. this strongly supported the hypothesis that this class of compound inhibits pfcrt in the parasite under conditions in which it inhibits parasite growth. these compounds were also shown to inhibit b-hematin formation, thus also supporting the hypothesis that their activity against malaria parasites is linked to inhibition of hemozoin formation. indeed, their biological activity against p. falciparum cultured in vitro was found to be correlated with b-hematin inhibitory activity, albeit in combination with the predicted dv accumulation ratio and substitution pattern in the dibemethin side chain, with f = 9.70 > f 0.95 = 8.45 ( figure 11d) . the dibemequines tested for cytotoxicity, which included the three most active derivatives, were found to have low cytotoxicity, with selectivity indices well above 1000. the whole series was found to show little cross-resistance with chloroquine in the k1 strain of chloroquine-resistant parasite. compound 10 was also highly active against the k1, dd2, w2 and rsa11 strains of chloroquine-resistant parasites, with resistance indices below 2. the three prototype compounds (compounds 8, 9 & 10) also had good in vivo antimalarial activity in the p. berghei mouse malaria model. two of the compounds were curative when using three or four oral doses at 100 mg/kg, with parasites being undetectable 30 days after treatment and with 100% of the mice surviving. this series of compounds again illustrates the potential of the dual-function approach to chloroquine resistance-reversing antimalarially active compounds. in comparison with the initially reported reversed chloroquine compounds, the clogp values of this series were considerably lower (5.42-6.19 ) and were comparable to chloroquine (clogp = 5.1). however, less hydrophobic compounds would still be more desirable and represent a priority for any further development of this series. several dual-function quinolines designed to exhibit both antimalarial and chloroquine resistance-reversal properties have been made that exhibit good in vivo activity, have low cytotoxicity and have been shown by isobologram ana lysis to work synergistically with chloroquine, supporting the hypothesis that they do indeed inhibit chloroquine transport by pfcrt. in addition, in at least one group, the dibemequines, inhibition of the transport of chloroquine by pfcrt in the xenopus oocyte has been directly demonstrated. in a review, peyton has also reported that similar activity has been observed, but not yet published in the case of the reversed chloroquines [82] . these compounds have been shown to exhibit strong in vitro antimalarial activity against a substantial number of chloroquine-sensitive and -resistant parasite strains, with no significant cross-resistance with chloroquine. thus, in many respects, these compounds have excellent properties for further development. however, there are some serious obstacles that will need to be overcome. weak base compounds, particularly hydrophobic weak bases, frequently exhibit inhibitory activity against herg, a potassium channel found in cardiac muscle. this can lead to prolonged qt intervals and potentially fatal heart arrhythmias [83] . indeed, in the case of at least one antimalarial drug, halofantrine, this problem has actually been encountered clinically [84] . unfortunately, herg toxicity is difficult to predict with certainty. nonetheless, gleeson has carried out a principal components ana lysis of approximately 30,000 compounds for which absorption, distribution, metabolism, excretion and toxicity data had been collected at glaxo-smithkline and found that while there is a greater potential for herg liability in basic molecules than in acidic or neutral ones, the liability is generally much reduced if the compound has a molecular weight below 400 and a calculated clogp below 4 [85] . indeed, this also improves druggability in a number of other ways. for example, it improves solubility, permeability and volume of distribution, decreases protein binding and lowers the potential of the compound to inhibit cytochrome p450. as noted above, peyton and coworkers have already produced reversed chloroquines with much improved clogp values [77] . a dibemequine analog has also been produced with a clogp well below 4 future science group and a molecular weight below 400 [egan tj et al., unpublished data] . while it has not yet been demonstrated that any of these compounds actually have reduced herg liability, it does at least suggest that improvement is possible with this class of compound. a second challenge relates to the mechanism of inhibition in the mutant pfcrt. it is not currently known whether these compounds inhibit chloroquine transport competitively or noncompetitively. they could bring about inhibition either by themselves being transported in preference to chloroquine or by binding to pfcrt and blocking chloroquine transport. the latter seems more likely, since one would expect resistant parasites to be resistant to the dualfunction molecule if it were transported out of the dv more efficiently than chloroquine. if used as monotherapies, these compounds might have an increased risk of rapid development of resistance because of their ability to bind to chloroquine-resistant mutants of pfcrt. it is possible that point mutations may then lead from binding to transport. this potential problem would likely be lessened by use of combination therapy. in addition, with the exception of one group of reversed chloroquines, the activities of these compounds against south american strains such as 7g8, with reduced sensitivity to verapamil resistance reversal, have not been n malaria is responsible for the deaths of over 600,000 people a year, approximately 90% of them in africa. n resistance to existing drugs and evidence of delayed parasite clearance with artemisinins, which is currently the most important class of antimalarial, is a major cause for concern. n continued discovery of new antimalarials is an important strategy. n historically, the quinolines have been one of the most important classes of antimalarial drug. n they are known to interact with fe(iii)heme. recently, crystal structures of the fe(iii)heme complexes of the quinoline methanols, quinine and quinidine and the related aryl methanol, halofantrine, have been reported. n chloroquine is believed to act by inhibiting the incorporation of fe(iii)heme, released when the parasite digests host hemoglobin, into hemozoin, an insoluble crystalline form of fe(iii)heme. n recent work has shown that chloroquine causes a dose-dependent increase in free fe(iii)heme and a decrease in hemozoin in treated parasites that is correlated with parasite survival. in addition, chloroquine treatment has been shown to redistribute heme into the parasite cytoplasm and disrupt the hemozoin crystal lattice. n chloroquine resistance is now widespread. n such resistance is largely attributable to mutations in a protein, pfcrt, found in the membrane of the digestive vacuole where hemoglobin digestion takes place. n a mutant form of pfcrt from chloroquine-resistant parasites has been shown to directly transport chloroquine in xenopus laevis oocytes. n a variety of compounds have been discovered that chemosensitize chloroquine-resistant parasites to chloroquine. these are often referred to as resistance reversers. n combination of a 4-amino-7-chloroquinoline with an imipramine-like group led to the first example of a putative dual-function 'reversed chloroquine'. n subsequent work has led to improvements in water solubility and oral bioavailability of this class of compound. n reversed chloroquines have been shown to interact with fe(iii)heme and inhibit parasite hemozoin formation. n new reversed chloroquine-like molecules have been discovered, including dual-function acridones, reversed chloroquines containing a dihydropyrimidinone group and dibemequines. n dibemequines inhibit b-hematin formation and have been shown to directly inhibit chloroquine transport by pfcrt. n the dibemequines have been shown to maintain activity against a range of chloroquine-resistant parasite strains, to exhibit little cytotoxicity and to be curative for mouse malaria. n dual-function antimalarials that inhibit chloroquine transport by pfcrt are innovative compounds that have been shown to have good activity, including oral activity in mice. n obstacles to future development do, however, exist. these include potential for herg toxicity, the possibility of increased potential for the development of resistance resulting from interaction with pfcrt and the need to compete with numerous other quinoline antimalarials. dual-functioning antimalarials that inhibit the chloroquine-resistance transporter review future science group explored. lack of activity against such strains is potentially a risk factor for these compounds. the final and probably most difficult hurdle is not a technical one. currently, as noted earlier, a number of new quinolines and related compounds are in development. in addition, there are several such compounds in current use. obtaining the necessary support to develop yet another set of quinoline compounds under these circumstances is likely to be a major challenge and probably represents the single biggest hurdle to further development of this class of compound. despite the challenges noted above, the dualfunction antimalarials that act against both hemozoin formation in the parasite and chloroquineresistant mutants of pfcrt represent a unique series of molecules with considerable potential as antimalarials. combinations with existing quinolines and related compounds, including chloroquine, amodiaquine and quinine, which 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parasites efflux of chloroquine from plasmodium falciparum: mechanism of chloroquine resistance reversal of chloroquine resistance in plasmodium falciparum by verapamil quinolineresistance reversing agents for the malaria parasite plasmodium falciparum. drug resist a series of structurally simple chloroquine chemosensitizing dibemethin derivatives that inhibit chloroquine transport by pfcrt synthesis and effects on chloroquine susceptibility in plasmodium falciparum of a series of new dihydroanthracene derivatives a 3d qsar pharmacophore model and quantum chemical structureactivity analysis of chloroquine(cq)-resistance reversal a chloroquine-like molecule designed to reverse resistance in plasmodium falciparum reversal agent and linker variants of reversed chloroquines: activities against plasmodium falciparum synthesis, structure-activity relationship, and mode-of-action studies of antimalarial reversed chloroquine compounds reversed chloroquines based on the 3,4-dihydropyrimidin-2( 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generation of a set of simple, interpretable admet rules of thumb the ph of the digestive vacuole of plasmodium falciparum is not associated with chloroquine resistance four plasmepsins are active in the plasmodium falciparum food vacuole, including a protease with an activesite histidine falcipains and other cysteine proteases of malaria parasites identification and characterization of falcilysin, a metallopeptidase involved in haemoglobin catabolism within the malaria parasite plasmodium falciparum characterization of the plasmodium falciparum m17 leucyl aminopeptidase roles for two aminopeptidases in vacuolar hemoglobin catabolism in plasmodium falciparum distribution and biochemical properties of an m1-family aminopeptidase in plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism haemozoin (b-haematin) biomineralization occurs by self-assembly near the lipid/water interface the role of neutral lipid nanospheres in plasmodium falciparum haem crystallization on the mechanism of chloroquine resistance in plasmodium falciparum key: cord-253276-mqcwk2ow authors: desai, n. c.; bhatt, nayan; somani, hardik; trivedi, amit title: synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-oxadiazoles date: 2013-09-30 journal: european journal of medicinal chemistry doi: 10.1016/j.ejmech.2013.06.029 sha: doc_id: 253276 cord_uid: mqcwk2ow abstract a series of thiazole clubbed 1,3,4-oxadiazole derivatives (5a–l) have been synthesized and characterized by ir, 1h nmr, 13c nmr and mass spectral analysis. synthesized compounds were evaluated for their antimicrobial and cytotoxic activities. the results indicated that, compounds 5c and 5i exhibited the most potent antibacterial activity. compound 5f was found to be the most potent antifungal agent. the structure activity relationship revealed that the presence of electron withdrawing groups at para position of phenyl ring remarkably enhanced the antibacterial activity of synthesized compounds. further, the results of preliminary mtt cytotoxicity studies on hela cells suggested that potent antimicrobial activity of 5b, 5c, 5f, 5h and 5i is accompanied by low cytotoxicity. the treatment of infectious diseases still remain a challenging task because of combination of factors such as an alarming increase in number of multi-drug-resistant microbial pathogens and advent of newer infectious diseases such as severe acute respiratory syndrome (sars), and avian influenza. despite the availability of a large number of antibiotics and chemotherapeutics, the increasing clinical importance of drug-resistant microbial pathogens have lent additional urgency in microbiological and antifungal research [1e4] . a potential solution to the antibiotic resistance is to design and explore innovative heterocyclic agents with novel mode of actions. in this context, thiazole derivatives have been playing a crucial role in medicinal chemistry. thiazole nucleus constitutes an integral part of all the available penicillins, which have transformed the bacterial diseases therapy [5] . they display quite a broad spectrum of biological activities [6] , which have found applications in the treatment of allergies [7] , hypertension [8] , inflammation [9] , schizophrenia [10] , microbial infections [11, 12] , hiv infections [13] , hypnotics [14] and pain [15] . they are also used as new inhibitors of bacterial dna gyrase b [16] . further, thiazoles have emerged as new class of potent antimicrobial agents, which are reported to inhibit bacteria by blocking the biosynthesis of certain bacterial lipids and/ or by additional mechanisms [17, 18] . on the other hand, 1,3,4-oxadiazole heterocycles are very good bioisosteres of amides and esters, which can contribute substantially in enhancing pharmacological activity by participating in hydrogen bonding interactions with the receptors [19] . potent pharmacological activity of 1,3,4-oxadiazoles can be attributed to the presence of toxophoric en]ceoe linkage which may react with the nucleophilic centers of the microbial cell [20] . further, the widespread use of 1,3,4-oxadiazoles as a scaffold in medicinal chemistry established this moiety as a member of the privileged structures class and its derivatives have exhibited a wide range of biological activities such as antibacterial [21] , antitubercular [22] , vasodialatory [23] , antifungal [24] , cytotoxic [25] , anti-inflammatory and analgesic [26, 27] , hypolipidemic [28] , anticancer [29] and ulcerogenic [30] . oxadiazole derivatives have been found to possess broad spectrum antimicrobial activity and therefore are useful substructures for further molecular exploration [30] . the most prominent examples of prescribed agents featuring the 1,3,4-oxadiazoles nucleus include the antiretroviral raltegravir [31] , antihypertensive nesapidil [32] and the antibiotic furamizole [33] (fig. 1) . prompted by above-mentioned observations and in continuation of our search for new, potent, selective, and less toxic antimicrobial agents [34e39], we report herein the synthesis of some novel structural hybrids by combining thiazole and 1,3,4oxadiazole pharmacophores in single molecular framework in order to investigate their in vitro antimicrobial activity. in addition, cytotoxicity studies were also conducted in hela cell lines to evaluate the ability of these compounds to inhibit the cell growth. the substitution pattern of 1,3,4-oxadiazole ring was carefully selected in order to confer different electronic environment to the molecules. the reaction sequences employed for synthesis of the target compounds 5ael are outlined in scheme 1. ethyl 2-amino-4methylthiazole-5-carboxylate (1) was taken as starting material and reacted with acetic anhydride to afford ethyl 2-acetamido-4methylthiazole-5-carboxylate (2) , which on further reaction with hydrazine hydrate in absolute ethanol yielded intermediate n-(5-(hydrazine carbonyl)-4-methylthiazol-2-yl)acetamide (3) . the intermediate obtained thus, was refluxed with carbon disulfide in the presence of potassium hydroxide in ethanol (99.5%) to yield intermediate n-(4-methyl-5-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2yl)thiazol-2-yl)acetamide (4) . mannich condensation of intermediate 4 with 36% formaldehyde and an appropriately substituted aniline derivative in ethanol (99.5%) furnished the desired compounds 5ael. both analytical and spectral data of the synthesized compounds 5ael were fully in agreement with proposed structures. formation of titled compounds 5ael was confirmed by characteristic ir spectrum absorption bands in the range of 3330e 3350 cm à1 , 3200e3250 cm à1 , 2810e2840 cm à1 , 2750e2800 cm à1 and 1660e1690 cm à1 corresponding to enh stretching of secondary amine, enh stretching of amide, ech 3, ech 2 e and >c]o of amide respectively. singlets at around d 2.03e2.10, 2.40e2.50, 4.15e4.25, 4.35e4. 45 and 9 .10e9.20 ppm in 1 h nmr were due to e ch 3 in anilide group, hetech 3 , secondary amine, ech 2 e and enh amide group respectively. the aromatic ring protons were observed at d 6.20e8.05 ppm and j value were found to be in accordance with substitution pattern on phenyl ring. characteristic peaks at around d 168.5e168. 9 and 177.0e177.3 ppm in 13 c nmr confirmed the presence of >c]o and >c]s groups respectively. the mass spectrum of 5ael revealed that observed molecular ion peaks were in agreement with molecular weight of respective compound. all the synthesized compounds 5ael were evaluated for their in vitro antibacterial activity against staphylococcus aureus mtcc 96, streptococcus pyogenes mtcc 442 (gram-positive), escherichia coli mtcc 443 and pseudomonas aeruginosa mtcc 1688 (gramnegative) by conventional broth microdilution method using chloramphenicol as a control drug for antibacterial activity [40] . the results of the antimicrobial studies are presented in table 1 . in general, compounds 5ael demonstrated better antibacterial activity than antifungal activity. compounds 5c and 5i emerged as the most effective antibacterial agents with 2-to 4-fold higher mic (12.5e25 mg/ml) than the reference drug chloramphenicol. while, compounds 5b and 5h exhibited comparable antibacterial activity with the standard drug. from the results of the antimicrobial activity of the synthesized compounds 5ael, the following structure activity relationships can be derived: the antibacterial activity was considerably affected by substitution pattern on the phenyl ring and the most active compounds contain electron withdrawing substituent at para and meta positions of the phenyl ring (p > m > o). in contrast, the presence of electron releasing groups on the phenyl ring witnessed a substantial decrease in antibacterial activity for compounds 5def and 5kel. the role of electron withdrawing group in improving antimicrobial activity is very well supported by previous studies [41, 42] . compounds 5c and 5i, substituted with inductively electron withdrawing fluoro and nitro groups, respectively at para position showed the highest antibacterial activity (f > no 2 ). it is a very well-known fact that electron withdrawing substitution such as fluoro/nitro at the para position of the aromatic ring increases the lipophilicity of molecules. this property is directly related to antimicrobial activity as it facilitates a compound to diffuse through the biological membranes and reach to its site of action. the presence of lipophilic substituent at para position of phenyl ring provided a positive influence on antibacterial activity. on the other hand, presence of the same functional groups at meta position resulted in slight decrease in the antibacterial activity (5b and 5h) but, still produced significant inhibitory action compared to the standard drug chloramphenicol (f > no 2 ). while, substituting the phenyl ring with fluoro and nitro group at ortho position resulted in noticeable decrease in the antibacterial activity of compounds 5a and 5g respectively. on the basis of mic values, it may be concluded that electron withdrawing atom/group such as fluoro and nitro at the para position of phenyl ring induced a positive effect while electron donating groups such as methyl and methoxy induced a negative effect on antibacterial activity of compounds 5ael. the in vitro antifungal activity of synthesized compounds 5ael were determined against candida albicans mtcc 227, aspergillus niger mtcc 282 and aspergillus clavatus mtcc 1323 by conventional broth microdilution method. the results indicated that compound 5f substituted with methoxy group at para position of the phenyl ring was found to be the most promising agent against both c. albicans and a. niger having 2-fold higher mic (25 mg/ml) in comparison with control drug ketoconazole. the enhanced activity of compound 5f may be attributed to the presence of electron releasing group at para position. the contrasting nature of substitution pattern at para position of the phenyl ring of most active antibacterial and antifungal agents indicate that the structural requirements are different for binding of drug to bacterial or fungal targets, respectively [43] . all other compounds showed less inhibition against the tested microorganisms as compared to the standard drug. in vitro cytotoxicity of compounds 5b, 5c, 5f, 5h and 5i were evaluated against human cervical cancer cell line (hela) by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (mtt) colorimetric assay [44] , which measures the reduction of tetrazolium bromide salt into a formazan dye by mitochondrial dehydrogenases in treated versus untreated cells. the results are summarized in table 2 . it was observed that none of the tested compounds exhibited any significant cytotoxic effect on hela cells, suggesting a great potential for their in vivo use as antimicrobial agents. in conclusion, some new structural hybrids of thiazole and 1,3,4oxadiazole were synthesized and investigated for their antimicrobial property with an anticipation of generating new structural leads serving as potent antimicrobial agents. many of the synthesized motifs (5b, 5c, 5h and 5i), possessing electron withdrawing atom/group such as fluoro and nitro at para and meta positions were identified as the most potent antibacterial agents. albeit, it was observed that para position was more favorable for enhancing the antibacterial activity. while, compound 5f with electron releasing group at para position came out as the most promising antifungal agent. the potent antimicrobial activity of most active compounds 5b, 5c, 5f, 5h and 5i were accompanied with relatively low level of cytotoxicity. the results described here, merits further investigations in our laboratories using a forward chemical genetic approach for finding lead molecules as antimicrobial agents. all reactions except those in aqueous media were carried out by standard techniques for the exclusion of moisture. melting points were determined on an electrothermal melting point apparatus and were reported uncorrected. tlc on silica gel plates (merck, 60, f 254 ) was used for purity checking and reaction monitoring. column chromatography on silica gel (merck, 70e230 mesh and 230e400 mesh asth for flash chromatography) was applied when necessary to isolate and purify the reaction products. elemental analysis (% c, h, n) was carried out by a perkineelmer 2400 chn analyzer. ir spectra of all compounds were recorded on a perkine elmer ft-ir spectrophotometer in kbr. 1 h nmr spectra were recorded on varian gemini 300 mhz and 13 c nmr spectra on varian mercury-400 100 mhz in dmso-d 6 as a solvent and tetramethylsilane (tms) as an internal standard. mass spectra were scanned on a shimadzu lc-ms 2010 spectrometer. all reactions requiring anhydrous conditions were carried out under nitrogen atmosphere using oven-dried glassware. ethyl 2-amino-4-methylthiazole-5-carboxylate (0.01 mol) was taken in a round bottom flask and acetic anhydride (0.02 mol) was added. the reaction mixture was refluxed at 140e150 c for 1 h and then poured into cold water under constant stirring to get solid product. the mixture was heated to boiling to decompose excess of acetic anhydride and cooled. the solid obtained was filtered, washed with water and dried. the ethyl 2-(acetyl amino)-4methyl-1,3-thiazole-5-carboxylate was collected and recrystallized from ethanol to get pure white crystals. yield: 74%; m.p. 158 c; anal. obs. c, 47.08%; h, 5.48%; n, 12.44%. calcd. for c 9 h 12 n 2 o 3 s: c, 47.35%; h, 5.30%; n, 12.27%. intermediate (2) (0.01 mol) and 99% hydrazine hydrate (0.015 mol) were taken in a round bottom flask and mixture was refluxed for 10 min. alcohol was added till both the layers were miscible and refluxing was continued for 5 h. excess of alcohol and unreacted hydrazine hydrate was distilled out and the contents were poured into a beaker. the solid was recrystallized from ethanol to get pure crystalline product. dmso was used as diluents to get desired concentration of compounds to test upon standard bacterial strains. serial dilutions were prepared in primary and secondary screening. the control tube containing no antibiotic was immediately subcultured (before inoculation) by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 c overnight. the tubes were then incubated overnight. the mic of the control organism was read to check the accuracy of the compound concentrations. the mic was defined as the lowest concentration of the antibiotic or test sample allowing no visible growth. all the tubes showing no visible growth (same as control tube) were subcultured and incubated overnight at 37 c. the amount of growth from the control tube before incubation (which represents the original inoculum) was compared. subcultures might show: similar number of colonies indicating bacteriostatic; a reduced number of colonies indicating a partial or slow bactericidal activity and no growth if the whole inoculum has been killed. the test must include a second set of the same dilutions inoculated with an organism of known sensitivity. each synthesized compound was diluted obtaining 2000 mg/ml concentration as a stock solution. in primary screening 500, 250 and 200 mg/ml concentrations of the synthesized compounds were taken. the active synthesized compounds found in this primary screening were further tested in a second set of dilution against all microorganisms. the compounds found active in primary screening were similarly diluted to obtain 100, 62.5, 50 and 25 mg/ml concentrations. the highest dilution showing at least 99% inhibition is taken as mic. in vitro cytotoxicity was determined using a standard mtt assay with protocol appropriate for the individual test system. test compounds were prepared prior to the experiment by dissolving in 0.1% dmso and diluted with medium. the cells were then exposed to different concentrations of the drugs (1e100 mm) in the volume of 100 mm/well. cells in the control wells received the same volume of medium containing 0.1% dmso. after 24 h, the medium was removed and cell cultures were incubated with 100 ml mtt reagent (1 mg/ml) for 5 h at 37 c. the suspension was placed on micro vibrator for 10 min and absorbance was recorded by the elisa reader. the experiment was performed in triplicate. human cancer cell lines, hela cells were cultured in mem medium supplemented with 10% fbs, 1% glutamine and 50 mm/ml gentamicin sulphate in a co 2 incubator in a humidified atmosphere of 5% co 2 and 95% air. ir (kbr): v/cm à1 3329 (secondary amine nh), 3225 (secondary amide nh), 3010 (aromatic ring ch -nitrophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5i) yield: 60%, m.p. 159e161 c; ir (kbr): v/cm à1 3331 (secondary amine nh), 3227 (secondary amide nh) (o-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5j) yield: 62%, m.p. 142e144 c; ir (kbr): v/cm à1 3331 (secondary amine nh), 3220 (secondary amide nh (m-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5k) yield: 57%, m.p. 151e153 c; ir (kbr): v/cm à1 3330 (secondary amine nh), 3229 (secondary amide nh anal. calcd. for c 16 h 17 n 5 o 2 s 2 c-51 p-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5l) yield: 61%, m.p. 163e165 c; ir (kbr): v/cm à1 3327 (secondary amine nh) anal. calcd. for c 16 h 17 n 5 o 2 s 2 c-51 we would like to express our sincere gratitude to the department of chemistry, mahatma gandhi campus, maharaja krishnakumarsinhji bhavnagar university, bhavnagar for providing research and library facilities. key: cord-323479-vlgv3nwq authors: speranza, jasmine; miceli, natalizia; taviano, maria fernanda; ragusa, salvatore; kwiecień, inga; szopa, agnieszka; ekiert, halina title: isatis tinctoria l. (woad): a review of its botany, ethnobotanical uses, phytochemistry, biological activities, and biotechnological studies date: 2020-03-01 journal: plants (basel) doi: 10.3390/plants9030298 sha: doc_id: 323479 cord_uid: vlgv3nwq isatis tinctoria l. (brassicaceae), which is commonly known as woad, is a species with an ancient and well-documented history as an indigo dye and medicinal plant. currently, i. tinctoria is utilized more often as medicinal remedy and also as a cosmetic ingredient. in 2011, i. tinctoria root was accepted in the official european phytotherapy by introducing its monograph in the european pharmacopoeia. the biological properties of raw material have been known from traditional chinese medicine (tcm). over recent decades, i. tinctoria has been investigated both from a phytochemical and a biological point of view. the modern in vitro and in vivo scientific studies proved anti-inflammatory, anti-tumour, antimicrobial, antiviral, analgesic, and antioxidant activities. the phytochemical composition of i. tinctoria has been thoroughly investigated and the plant was proven to contain many valuable biologically active compounds, including several alkaloids, among which tryptanthrin, indirubin, indolinone, phenolic compounds, and polysaccharides as well as glucosinolates, carotenoids, volatile constituents, and fatty acids. this article provides a general botanical and ethnobotanical overview that summarizes the up-to-date knowledge on the phytochemistry and biological properties of this valuable plant in order to support its therapeutic potential. moreover, the biotechnological studies on i. tinctoria, which mainly focused on hairy root cultures for the enhanced production of flavonoids and alkaloids as well as on the establishment of shoot cultures and micropropagation protocols, were reviewed. they provide input for future research prospects. the genus isatis l., which belongs to the brassicaceae family, comprises about 80 herbaceous annual to perennial species diffused in the middle east and central asia and extending to the mediterranean region [1] [2] [3] . i. tinctoria has a long and well documented history as a medicinal plant in both eastern and western cultures. its therapeutic properties have been esteemed in europe and in traditional chinese medicine (tcm) for centuries. the first written records in europe about the medicinal uses of this plant were by hippocrates (460 b.c.) who supported its use for treating wounds, ulcers, and haemorrhoids. galen (129-216 b.c.) and pliny (23-79 b .c.) recommended it as well [6, 28] . due to the huge cultivation in europe for the production of indigo dye, from the 12th century until the 17th century, i. tinctoria was also extensively used as a medicinal plant to treat snake bites, wounds, and other inflammatory ailments [6, 29] . these medicinal properties have been widely described in a number of renaissance and baroque herbal texts, which recommended woad for treating haemorrhoids, ulcers, and tumours [6] . various authors have mentioned different medicinal uses for i. tinctoria leaves and roots. the leaves have been utilized in the treatment of typhoid, measles, and the flu [30] . garland [31] reported their use against the tremendous inflammation of the skin due to st. anthony's fire, which is a dreaded illness that was common in the middle ages. other uses such as treating iposideremic the root is cylindrical, slightly tortuous, externally greyish-yellow, or brownish-yellow, wrinkled longitudinally and lenticellate transversally, with rootlets or rootlet scars. root stock slightly expanded, which exhibited dark green or dark brown petiole bases arranged in whorls, and dense tubercles. basal leaves are oblong-lanceolate, entire to toothed, and long-petioled. cauline leaves, which are narrower than basal and gradually reduced upwards, are simple, entire, sagittate, usually amplexicaul, with acute auricles. the flowers are gathered in a racemose inflorescence, with yellow petals, tetradynamous androecium, consisting of six stamens with two filaments shorter than the others. fruits are pendulous siliques, oblong-obovate, or elliptic-obovate hairless or shortly hairy. this species is very variable, particularly in the size, shape, and hairiness of the silique [1, 2, 13, 24] . there are conflicting views on the taxonomy of i. tinctoria (european woad) and i. indigotica (chinese woad). the latter was described for the first time by fortune in 1846, and initially considered plants 2020, 9, 298 4 of 40 as a distinct species. afterward, some taxonomists have classified it as a variety of i. tinctoria [17] . angelini et al. [25] reported that i. indigotica presents morphological, genetic, and physiological differences with respect to the european i. tinctoria, even though it is closely related to this one. the seedlings of both species develop a rosette in the first year of their cycle. the leaves i. indigotica show a glaucous instead of a shiny surface, which is rarely pubescent with a greater thickness and a more upright habit. the high degree of genetic diversity between the two species was confirmed by various studies [26, 27] . despite this, i. indigotica is currently considered a synonym of i. tinctoria, and not a separate species, as confirmed by the consulted taxonomic databases known as the plant list, international plant names index (ipni), and tropicos. on the other hand, the euro+med plantbase does not mention i. indigotica in the list of synonyms. i. tinctoria has a long and well documented history as a medicinal plant in both eastern and western cultures. its therapeutic properties have been esteemed in europe and in traditional chinese medicine (tcm) for centuries. the first written records in europe about the medicinal uses of this plant were by hippocrates (460 b.c.) who supported its use for treating wounds, ulcers, and haemorrhoids. galen (129-216 b.c.) and pliny (23-79 b.c.) recommended it as well [6, 28] . due to the huge cultivation in europe for the production of indigo dye, from the 12th century until the 17th century, i. tinctoria was also extensively used as a medicinal plant to treat snake bites, wounds, and other inflammatory ailments [6, 29] . these medicinal properties have been widely described in a number of renaissance and baroque herbal texts, which recommended woad for treating haemorrhoids, ulcers, and tumours [6] . various authors have mentioned different medicinal uses for i. tinctoria leaves and roots. the leaves have been utilized in the treatment of typhoid, measles, and the flu [30] . garland [31] reported their use against the tremendous inflammation of the skin due to st. anthony's fire, which is a dreaded illness that was common in the middle ages. other uses such as treating iposideremic anaemic diseases (due to iron deficiency), scurvy, and, in general, hypovitaminosis have been correlated to the relevant levels of iron, and of vitamins a and c present in the leaves. moreover, the leaf extracts have been used for their anabolic, astringent, and detergent properties. according to these skills, the extracts were utilized in treating acrodermatitis, haemorrhagic diathesis, eczema from scrofulous, furunculosis, intoxications, hypercholia, jaundice, gastric neurosis, hives, torpid sores, scrofulous, heartburn, and seborrhoea [32] . the bitter and refreshing roots have been utilized to reduce scarlet fever. the root extracts have been used to treat patients with solid tumours and leukaemia, which is a traditional usage that led to purification of indirubin [9] . after i. tinctoria lost its importance as a source of indigo for dying, its curative properties in europe fell into oblivion. it has only just recently emerged into western awareness as having medicinal properties, which is mostly due to its antiviral properties [28] . the european pharmacopoeia currently reports the monograph of i. tinctoria root (isatidis radix) for use in official european phytotherapy [24] . in china, woad has an equally rich history as dye and a medicinal plant. the chinese pharmacopoeia reports three monographs for i. tinctoria: băn lán gēn (isatidis radix), dà qīng yè (isatidis folium), and qing dai (indigo naturalis) [9, 33] . these herbal preparations are considered as somewhat differing medicines. băn lán gēn (isatidis radix) is the dried root of i. tinctoria harvested in some provinces of r.p. china such as hebei, jiangsu, and anhui during the autumn, dried in the sun, and processed into granules (banlangen keli). the product is very popular throughout china and it is most commonly consumed when dissolved in hot water or tea [34] . it reduces the body temperature and soothes the sore throat. it is indicated for eruptive epidemic diseases, pharyngitis, laryngitis, scarlet fever, erysipelas, and carbuncles. it is utilized for treating hepatitis, mumps, the flu, mononucleosis, viral skin plants 2020, 9, 298 5 of 40 diseases such as herpes simplex, herpes zoster, and pityriasis rosea, epidemic cerebrospinal meningitis, and diphtheria [34] [35] [36] [37] . băn lán gēn has been one of the eight major medicines recommended by the chinese government for preventing and controlling the deadly severe acute respiratory syndrome (sars) [38] . dà qīng yè (isatidis folium) consists of i. tinctoria dried leaves [39] collected in the summer and the autumn from different provinces of r.p. china such as hebei, jiangsu, anhui, and henan and dried in the sun. dà qīng yè is used against the virus of b-encephalitis, mumps, and influenza and for treating the leptospirosis [36] . qing dai (indigo naturalis) is a dark blue powder prepared from the leaves of various plants including i. tinctoria, baphicacanthus cusia (nees) bremek., indigofera suffruticosa mill., indigofera tinctoria l., or polygonum tinctorium ait. it is used in tcm as a haemostatic, antipyretic, anti-inflammatory, and in the treatment of bacterial and viral infections [40, 41] . another preparation, called dang gui long hui wan, consisting of 11 herbal medicines including i. tinctoria, has been used in tcm as a remedy for various chronic diseases including chronic myelocytic leukaemia [14] . clinical trials have not assessed the safety of i. tinctoria leaf or root preparations. traditional chinese herbal texts do not list adverse effects, except gastro-intestinal disorders such as nausea and vomiting in some patients [36, 42] . i. tinctoria, like other species belonging to the brassicaceae family, shows an interesting chemical profile characterized by a large variety of compounds. i. tinctoria represents a valuable source of bioactive compounds such as alkaloids, phenolic compounds, polysaccharides, glucosinolates, carotenoids, volatile constituents, and fatty acids (table 1) . i. tinctoria is an important source of two known indolic alkaloids called indigo and indirubin. the first one has a blue colour instead of the second one that has a red colour and both of them are extensively used to dye textiles, cosmetics, foods, and pharmaceutical preparations. the plant is not able to synthetize directly into the indigoid pigments, but it produces several precursors: indican, isatan a, isatan b, and isatan c ( figure 2 ). when the leaves are damaged and exposed to air, precursors are submitted to enzymatic hydrolysis by β-d-glucosidase and β-glucuronidase. after cleavage of the o-glycosidic linkage, the indoxyl is released giving, in turn, indigo (blue indigoid pigments) through the oxidation process, and producing isatin as a side reaction thanks to an oxygen-rich environment. eventually, the condensation of indoxyl with isatin produces indirubin (red indigoid pigment), whereas the condensation with dioxindole, coming from isatan c, produces isoindirubin (red indigoid pigment) [44] . the indigo precursors indicant as well as isatans b and c were detected by maugard et al. [44] in the acetone/acetic acid 1% v/v extracts of i. tinctoria young and old fresh leaves. moreover, the number of indigo precursors in these samples was quantified by the same team, who established that isatan b was the major precursor. the concentration of indigo precursors was higher in the young, fresh leaves than in the oldest ones. a later study of oberthur et al. [53] . about i. tinctoria liophylized or air-dried leaves identified isatan a as the major indoxyl glycoside and gave the correct structure of isatan b, which was previously assigned by epstein et al. [54] . furthermore, the same team showed how the content of indigo precursors strictly depended on the post-harvest treatments. a mix of i. tinctoria young and older rosette leaves were submitted to three different post-harvest treatments: immediate shock freezing with liquid n 2 followed by freeze drying, air drying at room temperature, and drying in a thermostatic oven at 40 • c. the highest concentrations of isatans a and b were found in freeze dried leaf samples whereas the indican had the lowest amount. on the other hand, the other two treatments lead to high concentrations of indican and complete disappearance of isatans a and b. in all samples, the isatan b concentrations were lower than those of isatan a. these data and the enzymatic conversion of indican to isatan b underlined the biosynthetic pathway where isatan b derived from indican is, at the same time, a biogenetic precursor and degradation product of isatan a. eventually, the production and amount of indigo precursors depend on the cultivars and harvest period, which also influenced the indigo dyes' composition [55] . (7z, 10z, 13z)-hexadecatrienoic acid corchorifatty acid b 9-hydroxy-(10e, 12e, 14e)-octadecatrienoic acid 9-oxo-(10e, 12z, 15z)-octadecatrienoic acid polysaccharides root [34] 4.1. alkaloids i. tinctoria is an important source of two known indolic alkaloids called indigo and indirubin. the first one has a blue colour instead of the second one that has a red colour and both of them are extensively used to dye textiles, cosmetics, foods, and pharmaceutical preparations. the plant is not able to synthetize directly into the indigoid pigments, but it produces several precursors: indican, isatan a, isatan b, and isatan c ( figure 2 ). when the leaves are damaged and exposed to air, precursors are submitted to enzymatic hydrolysis by β-d-glucosidase and β-glucuronidase. after cleavage of the o-glycosidic linkage, the indoxyl is released giving, in turn, indigo (blue indigoid pigments) through the oxidation process, and producing isatin as a side reaction thanks to an oxygenrich environment. eventually, the condensation of indoxyl with isatin produces indirubin (red indigoid pigment), whereas the condensation with dioxindole, coming from isatan c, produces isoindirubin (red indigoid pigment) [44] . the indigo precursors indicant as well as isatans b and c were detected by maugard et al. [44] . in the acetone/acetic acid 1% v/v extracts of i. tinctoria young and old fresh leaves. moreover, the number of indigo precursors in these samples was quantified by the same team, who established that isatan b was the major precursor. the concentration of indigo precursors was higher in the young, fresh leaves than in the oldest ones. a later study of oberthur et al. [53] . about i. tinctoria liophylized or air-dried leaves identified isatan a as the major indoxyl glycoside and gave the correct structure of isatan b, which was previously assigned by epstein et al. [54] . furthermore, the same team showed how the content of indigo precursors strictly depended on the post-harvest treatments. a mix of i. tinctoria young and in addition to the indigo precursors, indigoid pigments named trans-indigo (indigotin, blue), trans-indirubin (isoindigotin, red), cis-indirubin (isoindirubin, red), iso-indigo (indigo brown), and cis-indigo were detected by hplc in the acetone/acetic acid 1% v/v extracts of i. tinctoria young and old fresh leaves ( figure 3 ) [44] . it is also important to point out that the indigo dyes indigo and indirubin appear under the fermentative conditions, used in the old natural indigo production, or after a drying process at 40 • c [45] . beyond the study of i. tinctoria fresh leaves, one unique analysis on dried rosette leaf extracts were carried out by mohn et al. [33] . thanks to the extractions performed through two different solvents, known as dichloromethane and methanol, it was possible to obtain a broad-based characterisation of the i. tinctoria profile. the lc-ms analysis of dichloromethane extracts showed various indolic alkaloids such as isatin, isoindigo, indoxyl indigo, and indirubin. instead, in the methanolic extracts, only indican was detected [33] . recently, lc-ms analysis of i. tinctoria frozen and lyophilized and dried leaf extracts were performed and the obtained metabolite profiles were compared. in the lyophilized extracts' analysis, beyond the characterization and quantification of 122 compounds previously described, the following indole derivatives were described for the first time: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside (isatan c), 6-hydroxyindole-3-carboxylic acid 6-o glucoside, and 6-hydroxyindole-3-carboxylic acid glucose ester [45] . several research studies carried out on i. tinctoria dried roots showed two new and five known indole alkaloid glycosides. the structure of the two new indole alkaloid glycosides isatindigoside a and isatindigoside b were determined by extensive spectroscopic data analysis including 1 d, 2 d nmr, ir, and hr-esi-ms data analysis. the known glycosides isatindosulfonicacid a 3-o-b-d-glucopyranoside, indole-3-acetonitrile 6-o-b-d-glucopyranoside, isatindigobisindoloside a, isatindigobisindoloside b, and isatindigobisindoloside f, were identified by comparing spectroscopic and optical rotation data [37] . furthermore, a new indole alkaloid with an unusual carbon skeleton named isatisindigoticanine a were detected by the same team [56] . leaf samples whereas the indican had the lowest amount. on the other hand, the other two treatments lead to high concentrations of indican and complete disappearance of isatans a and b. in all samples, the isatan b concentrations were lower than those of isatan a. these data and the enzymatic conversion of indican to isatan b underlined the biosynthetic pathway where isatan b derived from indican is, at the same time, a biogenetic precursor and degradation product of isatan a. eventually, the production and amount of indigo precursors depend on the cultivars and harvest period, which also influenced the indigo dyes' composition [55] . in addition to the indigo precursors, indigoid pigments named trans-indigo (indigotin, blue), trans-indirubin (isoindigotin, red), cis-indirubin (isoindirubin, red), iso-indigo (indigo brown), and cis-indigo were detected by hplc in the acetone/acetic acid 1% v/v extracts of i. tinctoria young and old fresh leaves ( figure 3 ) [44] . it is also important to point out that the indigo dyes indigo and indirubin appear under the fermentative conditions, used in the old natural indigo production, or after a drying process at 40 °c [45] . beyond the study of i. tinctoria fresh leaves, one unique analysis on dried rosette leaf extracts were carried out by mohn et al. [33] . thanks to the extractions performed through two different solvents, known as dichloromethane and methanol, it was possible to obtain a broad-based characterisation of the i. tinctoria profile. the lc-ms analysis of dichloromethane extracts showed various indolic alkaloids such as isatin, isoindigo, indoxyl indigo, and indirubin. instead, in the methanolic extracts, only indican was detected [33] . recently, lc-ms analysis of i. tinctoria frozen and lyophilized and dried leaf extracts were performed and the obtained metabolite profiles were compared. in the lyophilized extracts' analysis, beyond the characterization and quantification of 122 compounds previously described, the following indole derivatives were described for the first time: acetylindican, malonylindican, two another important compound of this class is tryptanthrin, indolo-[2,1-b]-quinazoline alkaloid ( figure 4 ), which is also responsible for some biological activities of i. tinctoria. honda et al. [43] isolated and identified tryptanthrin from i. tinctoria dried rosette leaf chloroform extracts for the first time. moreover, more than 70 isatis samples of different origin were analysed and the tryptanthrin content in the leaves varied from 0.56 to 16.74 × 10 −3 % [57] . the tryptanthrin synthetic pathway is not known, but it is a product of the post-harvest process. its formation is apparently favoured by the drying process and elevated temperature (40 • c) and, on the contrary, the lyophilization and fermentative conditions decreased its concentration [58] . plants 2020, 9, x for peer review 19 of 43 dioxindole glucosides, dioxindole malonylglucoside (isatan c), 6-hydroxyindole-3-carboxylic acid 6-o glucoside, and 6-hydroxyindole-3-carboxylic acid glucose ester [45] . several research studies carried out on i. tinctoria dried roots showed two new and five known indole alkaloid glycosides. the structure of the two new indole alkaloid glycosides isatindigoside a and isatindigoside b were determined by extensive spectroscopic data analysis including 1 d, 2 d nmr, ir, and hr-esi-ms data analysis. the known glycosides isatindosulfonicacid a 3-o-b-dglucopyranoside, indole-3-acetonitrile 6-o-b-d-glucopyranoside, isatindigobisindoloside a, isatindigobisindoloside b, and isatindigobisindoloside f, were identified by comparing spectroscopic and optical rotation data [37] . furthermore, a new indole alkaloid with an unusual carbon skeleton named isatisindigoticanine a were detected by the same team [56] . another important compound of this class is tryptanthrin, indolo-[2,1-b]-quinazoline alkaloid ( figure 4 ), which is also responsible for some biological activities of i. tinctoria. honda et al. [43] isolated and identified tryptanthrin from i. tinctoria dried rosette leaf chloroform extracts for the first time. moreover, more than 70 isatis samples of different origin were analysed and the tryptanthrin content in the leaves varied from 0.56 to 16.74 × 10 −3 % [57] . the tryptanthrin synthetic pathway is not known, but it is a product of the post-harvest process. its formation is apparently favoured by the drying process and elevated temperature (40 °c) and, on the contrary, the lyophilization and fermentative conditions decreased its concentration [58] . the main class of secondary metabolites in i. tinctoria polar extracts is represented by phenolic compounds [16, 47] . phenolics can be divided into three main categories: flavonoids, phenolic acid, and their conjugates [16] . the identified flavonoids represent the major class of the phenolic the main class of secondary metabolites in i. tinctoria polar extracts is represented by phenolic compounds [16, 47] . phenolics can be divided into three main categories: flavonoids, phenolic acid, and their conjugates [16] . the identified flavonoids represent the major class of the phenolic constituent and are derivatives of flavones and of flavonols [46] . in methanolic extract from dried leaves, the presence of p-hydroxybenzoic, o-methoxybenzoic, p-methoxybenzoic, dihydrocaffeic, and 4-hydroxy-3-methoxyphenylpropanoic acid has been reported [48] . they found methyl esters of several phenolic acids, like ferulic, sinapic, salicylic, vanillic, and 4-hydroxyphenylacetic acids as a result of acidic hydrolysis. the same kind of extract was analysed by mohn et al. [33] . in dried rosette leaves, they found vicenin-2, stellarin-2, isoorientin, isovitexin, isoscoparin, and some of their glucosides ( figure 5 ). simultaneously, the isocoparin, sinapic, and ferulic acids turned out from the analysis of dichloromethane extract from the same plant material. simultaneously, the isocoparin, sinapic, and ferulic acids turned out from the analysis of dichloromethane extract from the same plant material. the drying process may cause changes in the metabolite profile even among phenolic compounds. on this account, lyophilization of freshly harvested plant material was introduced to prevent biochemical decomposition, e.g., deglycosylation. as a result of this procedure, a large group of conjugates between hydroxycinnamic acids and a hexose or dihexose (10 compounds), glycerate (two compounds), malate (one compound), or glucaric acid (45 compounds) was detected in the rosette leaf methanol extract. all of the conjugates showed one or two moieties of p-coumaric, ferulic, or sinapic acid. furthermore, two glucaric acid conjugates containing a higher molecular weight dilignol-like esterified moiety in which coniferyl alcohol was linked to ferulic acid via a typical lignin 8-o-4 linkage were detected. in addition, hydroxycinnamic acids conjugated with flavone glucosides were identified. among benzoic acid derivatives, only two compounds were confirmed, which include hexoside and hexose ester of protocatechuic acid [16] . many flavonoids showed carbohydrate and acid moieties and, according to it, many derivatives of the flavonoid aglycones were detected. nguyen et al. [16] was found in the polar extract from rosette leaves' flavone compounds mono or diglicosylated on the c-6 position with a rare 1→3 glycosidic linkage. flavone compounds with additional glycosylation of the o-7 position and esterification with a hydroxycinnamic acid derivative such as p-coumaric, ferulic, or sinapic acid were identified. moreover, the same authors reported the presence of six flavone glucosides conjugated with hydroxycinnamic acid. they identified isoscoparin with its eight derivatives including: 3"-osinapoyl glucoside, 3"-o-feruloyl glucoside and 3"-o-p-coumaroyl glucosides of isoscoparin, in the same extract, the luteolin-6-c-glucoside-7-o-glucoside was detected for the first time and it has not been described in other plants before. miceli et al. the drying process may cause changes in the metabolite profile even among phenolic compounds. on this account, lyophilization of freshly harvested plant material was introduced to prevent biochemical decomposition, e.g., deglycosylation. as a result of this procedure, a large group of conjugates between hydroxycinnamic acids and a hexose or dihexose (10 compounds), glycerate (two compounds), malate (one compound), or glucaric acid (45 compounds) was detected in the rosette leaf methanol extract. all of the conjugates showed one or two moieties of p-coumaric, ferulic, or sinapic acid. furthermore, two glucaric acid conjugates containing a higher molecular weight dilignol-like esterified moiety in which coniferyl alcohol was linked to ferulic acid via a typical lignin 8-o-4 linkage were detected. in addition, hydroxycinnamic acids conjugated with flavone glucosides were identified. among benzoic acid derivatives, only two compounds were confirmed, which include hexoside and hexose ester of protocatechuic acid [16] . many flavonoids showed carbohydrate and acid moieties and, according to it, many derivatives of the flavonoid aglycones were detected. nguyen et al. [16] was found in the polar extract from rosette leaves' flavone compounds mono or diglicosylated on the c-6 position with a rare 1→3 glycosidic linkage. flavone compounds with additional glycosylation of the o-7 position and esterification with a hydroxycinnamic acid derivative such as p-coumaric, ferulic, or sinapic acid were identified. moreover, the same authors reported the presence of six flavone glucosides conjugated with hydroxycinnamic acid. they identified isoscoparin with its eight derivatives including: 3"-o-sinapoyl glucoside, 3"-o-feruloyl glucoside and 3"-o-p-coumaroyl in the same extract, the luteolin-6-c-glucoside-7-o-glucoside was detected for the first time and it has not been described in other plants before. miceli et al. [46] characterised, for the first time, the phenolic compounds of the hydroalcoholic extract (70% methanol) from i. tinctoria lyophilized cauline leaves. among 13 identified flavonoids, derivatives of flavones and flavonols, which were quantitatively dominant, were vicenin-2, isovitexin, and apigenin glucosides. some of them like kaempferol, buddleoside, and quercetin have not been detected in isatis before. moreover, the authors reported the presence of phenolic acids like neochlorogenic, chlorogenic, caffeic, ferulic, sinapic, p-coumaric, and coumarylquinic acids ( figure 6 ). neochlorogenic and sinapic acids were dominant compounds. the same authors also performed a comparative analysis of hydroalcoholic extracts from rosette leaves, cauline leaves, and flowers of i. tinctoria [47] . according to the results, the phenolic profile of both types of leaves was very similar, but cauline leaves showed more than double the phenolic content than rosette leaves. a lower number of phenolic compounds was detected in the flower extract. nonetheless, they were found to be quantitatively close to those of cauline leaves. the main flavonoids detected in leaves and flowers from i. tinctoria were vicenin-2, stellarin-2, isovitexin, luteolin-glucuronide, and quercetin. among them, vicenin-2 turned out to be the most abundant flavonoid detected in the cauline leaf extract, whereas luteolin-glucuronide and stellarin-2 were the main compounds for rosette leaves and flowers, respectively [47] . i. tinctoria is a notable source of glucosinolates, which are synthesis and storage products of isothiocyanates. when the plant tissue is damaged, glucosinolates are released and converted by myrosinase (β-thioglucosidase). this is an enzyme that coexists with these compounds in plants even though it is physically separated [19] . the enzyme hydrolyzes the glucose moiety into the main skeleton by giving isothiocyanates and, under certain conditions, this reaction may lead to other types of products like thiocyanates, indoles, epithionitriles, and nitriles [59] . these compounds are responsible for the bitter and pungent taste and for most of the biological activities of the glucosinolates such as anticancer, antioxidant, and antibacterial factors. moreover, glucosinolates are known as goitrogens because they prevent the absorption of iodine, which causes swelling of the thyroid. from the same authors also performed a comparative analysis of hydroalcoholic extracts from rosette leaves, cauline leaves, and flowers of i. tinctoria [47] . according to the results, the phenolic profile of both types of leaves was very similar, but cauline leaves showed more than double the phenolic content than rosette leaves. a lower number of phenolic compounds was detected in the flower extract. nonetheless, they were found to be quantitatively close to those of cauline leaves. the main flavonoids detected in leaves and flowers from i. tinctoria were vicenin-2, stellarin-2, isovitexin, luteolin-glucuronide, and quercetin. among them, vicenin-2 turned out to be the most abundant flavonoid detected in the cauline leaf extract, whereas luteolin-glucuronide and stellarin-2 were the main compounds for rosette leaves and flowers, respectively [47] . i. tinctoria is a notable source of glucosinolates, which are synthesis and storage products of isothiocyanates. when the plant tissue is damaged, glucosinolates are released and converted by myrosinase (β-thioglucosidase). this is an enzyme that coexists with these compounds in plants even though it is physically separated [19] . the enzyme hydrolyzes the glucose moiety into the main skeleton by giving isothiocyanates and, under certain conditions, this reaction may lead to other types of products like thiocyanates, indoles, epithionitriles, and nitriles [59] . these compounds are responsible for the bitter and pungent taste and for most of the biological activities of the glucosinolates such as anticancer, antioxidant, and antibacterial factors. moreover, glucosinolates are known as goitrogens because they prevent the absorption of iodine, which causes swelling of the thyroid. from the chemical point of view, glucosinolates are (z)-n-hydroxyminosulfate esters characterized by sulphur-linked β-d-glucopyranose moiety and an amino acid-derived side chain. they are divided into three groups according to their amino acid side chain: aliphatic glucosinolates, aromatic glucosinolates, and indole glucosinolates [60] . in i. tinctoria seeds, aliphatic glucosinolates as gluconapin, progoitrin, and epipogoitrin, which were mainly detected, but indolic glucosinolates like glucoisatisin/epiglucoisatisin (inseparable epimeric mixture present only in seeds), glucobrassicin and neoglucobrassicin were also identified. the glucosinolate pattern of the seeds extract showed differences when compared to the extract obtained from the rosette leaves. in fact, the extracts obtained from i. tinctoria frozen and liophylized rosette leaves, mainly contained indolic glucosinolates including glucobrassicin, neoglucobrassicin, sulfoglucobrassicin, and glucotropaeolin ( figure 7 ) [49, 50] . the analysis carried out by the same authors also showed that the glucosinolate content in the leaf samples was higher than that of the seed samples. taviano et al. [47] showed that a very low amount of glucoiberin, glucobrassicin, and 4-methoxyglucobrassicin are contained in the liophylized leaf (rosette and cauline) and flower of 70% methanol extracts. furthermore, recent studies reported, for the first time, the presence of gluconapoleiferin in the the analysis carried out by the same authors also showed that the glucosinolate content in the leaf samples was higher than that of the seed samples. taviano et al. [47] showed that a very low amount of glucoiberin, glucobrassicin, and 4-methoxyglucobrassicin are contained in the liophylized leaf (rosette and cauline) and flower of 70% methanol extracts. furthermore, recent studies reported, for the first time, the presence of gluconapoleiferin in the frozen and lyophilized rosette leaf methanol extracts [16] . carotenoids are highly lipophilic metabolites and, for this reason, the extraction and separation conditions were optimized for the analysis. on one hand, the dichloromethane extract of i. tinctoria dried rosette leaves showed several known carotenoids like (all-e)-β-carotene and various lutein isomers [33] . on the other hand, in the hexane/acetone extract (1:1 v/v) obtained from the dried rosette leaves, it was possible to detect more and other unknown carotenoids like (all-e)-lutein, (z)-neochrome, (15z)-violaxanthin [33] . moreover, in the same extract, the lipophilic chlorophyll degradation products phaeophytin a, b, and pyrophaeophytin a, b were identified. phenolic derived compounds with higher molecular weight belonging to monolignols and oligolignols were described by nguyen et al. [16] . in methanol extracts of i. tinctoria frozen and lyophilized rosette leaves, monolignols and oligolignols, derived from coniferyl and synapyl alcohol, were characterized by lc-ms for the first time. some of these compounds were previously identified in other plants such as arabidopsis thaliana and linum usitatissimum. furthermore, several compounds containing hexose, guaiacyl, and syringyl units linked by 8-5 and 8-o-4 bonds were detected for the first time and all compounds were found in a hexosylated form. authors underlined that they were accumulated in the vacuole of mesophyll and epidermal cells rather than in the cell walls of the leaf nervature. this finding suggested that oligolignols may play some physiological role in i. tinctoria leaves. the volatile constituents of i. tinctoria were characterized in leaves and roots. the first extraction from fresh leaves was carried out by condurso et al. [19] through solid-phase microextraction and gas chromatography-mass spectrometry (spme/gc-ms). this analysis allowed them to identify several compounds such as acids, alcohols, aldehydes, esters, ethers, furans, hydrocarbons, isothiocyanates, thiocyanates, ketones, nitriles, sulfurated compounds, monoterpenoids, and sesquiterpenoids. a high portion of volatile isothiocyanates was detected in the leaves (40% of the total volatile fraction), especially 3-butenyl isothiocyanate that is the aglicone of gluconapin. the 2-hydroxy-3-butenyl isothiocyanate, that is, the aglicone of progoitrin and epiprogoitrin, was also identified. after the isothiocyanates, aldehydes were the most represented class and both aliphatic and aromatic types were identified, especially the (z)-2-esenal (a typical aldehyde from leaves). among the sulfurated compounds, the 2-ethylthiophene was the most abundant one. moreover, saturated and unsaturated alcohols were identified with tetradecanol as the main component. almost 7% of the volatile fraction was represented by monoterpenoids and sesquiterpenoids. ten monoterpenoids and two sesquiterpenoids were detected and the monocyclic monoterpenoid limonene was the major one. furthermore, the nitriles that derive from isothiocyanates due to the loss of sulphur were detected, which were 4-pentenenitrile, 3-hydroxy-4-pentenenitrile, heptanenitrile, octanenitrile, and 2-phenylacetonitrile as the most abundant ones. verzera et al. [51] carried out the first extraction of i. tinctoria dried root volatile constituents by spme/gc-ms and the same main classes of compounds were identified. the volatile content of roots was also characterized by a high portion of 3-butenyl isothiocyanate that represents 82% of the total plants 2020, 9, 298 20 of 40 volatiles. after isothiocyanates, the alcohols and nitriles were the most abundant classes and they, respectively, constituted 4.9% and 3.6% of the total volatile content. the other classes of compounds are even less represented. different studies have shown that polysaccharides from roots are important bioactive components of i. tinctoria. in a study carried out by han et al. [34] , the conditions that gave the maximum extraction yield of the polysaccharides from i. tinctoria root (11.19% ± 0.04) have been established. the seeds of i. tinctoria, like those of other plants belonging to family brassicaceae, show an interesting fatty acid composition, which is the highest among all isatis spp. (10%). according to kizil et al. [52] , seeds contain erucic acid (26.48% of total fatty acid fraction), oleic, linoleic, linolenic acids as the major components, and other fatty acids like palmitic, stearic, arachidic, and tetracosanoic. moreover, mohn et al. [33] reported the presence of palmitoleic and α-lysolecithin in dichloromethane dried rosette leaf extracts. additionally, the presence of the triterpenoid ursolic acid was confirmed by these authors. the microelement analysis of the i. tinctoria seeds carried out by kizil et al. [52] showed a number of studies have been conducted to evaluate different biological activities of i. tinctoria in both in vitro and in vivo experimental models. biological investigations are mainly based on the ethnopharmacological uses of the plant as an effective anti-inflammatory, anti-tumour, and antiviral herbal remedy. in addition, other biological activities such as analgesic, antimicrobial, and antioxidant have been highlighted (table 2 ). i. tinctoria has been appreciated for centuries in europe and, in tcm, for its anti-inflammatory properties. for this reason, several in vivo and in vitro research studies were carried out in order to assess its anti-inflammatory potential. solvents with different polarity and diverse extraction procedures (maceration, reflux extraction, co 2 supercritical fluid extraction (sfe), and accelerated solvent extraction (ase)) were utilized in order to establish the best condition to extract the bioactive compounds contained in the leaves and roots of i. tinctoria. one of the earliest studies investigated the effects of the treatment with an aqueous extract of i. tinctoria in a model of chronic pseudomonas aeruginosa lung infection mimicking cystic fibrosis in rat. i. tinctoria (400 mg/kg b.w., s.c. once a day for 10 days) was able to decrease the frequency of the lung abscess and the severity of the macroscopic pathological changes in lungs. the i. tinctoria extract was also able to modulate and decrease the inflammatory response in the lungs by enhancing the shift in the inflammatory response from an acute type inflammation dominated by polymorphonuclear leukocytes to a chronic type inflammation dominated by mononuclear leukocytes [61] . the anti-inflammatory activity of lipophilic i. tinctoria leaf extracts was demonstrated. recio et al. [18] showed the efficacy of sfe and dichloromethane (dcm) extracts obtained from dried rosette leaves of i. tinctoria, using two experimental models of acute inflammation including carrageenan-induced mouse paw oedema and 12-o-tetradecanoylphorbol 13-acetate (tpa)-induced mouse ear oedema. the sfe (os, 75-125 mg/kg) and dcm (os, 125-175 mg/kg) extracts showed dose-dependent anti-inflammatory activity in the carrageenan-induced mouse paw oedema. particularly, the potency of the sfe extract was found higher than that of dcm. in the second in vivo model of acute inflammation, the topical application of the dcm and sfe extracts (0.5 mg/ear) significantly inhibited the tpa-induced ear oedema and the topical administration was more effective than the systemic one. in sub-chronic inflammation induced by repeated application of tpa in the mouse ear, both oral (150 mg/kg) and topical (1 mg/kg) administration of dcm extract inhibited oedema formation and reduced the neutrophil infiltration, which decreased the various parameters of the inflammatory response such as the hypertrophy of fibroblasts, papillomatosis, acanthosis, hyperkeratosis, and spongiosis [18] . the anti-inflammatory properties of these extracts have been explained with multi-faceted effects including, among others, a marked cox inhibitory activity, with a preferential inhibition of cox-2, inhibition of 5-lox, inos, histamine and serotonine release, and leukocytic elastase, as previously demonstrated by danz et al. [17, 92] . in a model of delayed-type hypersensitivity (dht) induced by dinitrofluorobenzene (dnfb) in mice, the lipophilic dcm extract, topically administered (1 mg/ear), inhibited the response both during the induction phase and the inflammatory phase. instead, the oral administration had no effect [18] . adjuvant-induced arthritis in rats is an experimental in vivo model mimicking the rheumatoid arthritis and it was utilized with the aim to evaluate the effect of a dcm extract on chronic inflammatory diseases [64] . in this model, the dmc extract (os, 150 mg/ml once/day, on days 17-23 by injection of the adjuvant) led to a significant reduction of paw oedema induced by injection of mycobacterium butyricum. this effect did not result in a dose-dependency. a decreased joint damage, a reduction of tissular and articular inflammatory markers (oedema, cell infiltration, articular damage, pannus, cysts etc.), bone, and cartilage erosion was observed. interestingly, the treatment over two weeks with a high dose of i. tinctoria extract did not determine macroscopic lesions of the rat gastric mucosa. the treatment did not cause a significant difference in tissue levels of cox-1 and cox-2, immunohistochemically determined. these results appear to disagree with those reported by orberthür et al. [29] where a preferential inhibition of cox-2 and a reduced expression of inos was observed. in vitro studies demonstrated that the dcm extract is able to inhibit both tumour necrosis factor-α (tnf-α) and il-β production in raw 264,7 macrophages [64] . thus, the anti-inflammatory and anti-allergic activity of i. tinctoria dcm extract highlighted in the arthritis and dth models was related to the reduction of the production of these cytokines. brattström et al. [65] demonstrated the anti-inflammatory effects of sfe extract from the i. tinctoria leaves in a murine experimental model of allergic airway disease (asthma). the extract, administered intranasally (10-100 µg/mouse) to ovalbumin immunised balb/c mice, determined a dose-dependent inhibition of the allergic reaction, which decreases the methacholine-induced airway hyperresponsiveness (ahr) and eosinophil recruitment, as determined both in the bronchoalveolar lavage (bal) fluid and in the lung homogenates. the observed effect was related to the reduced production of the typical mediators of the th2 immune response il-4, il-5, and rantes (regulated on activation normal t cell expressed and secreted) highlighted after treatment with i. tinctoria extract. the anti-inflammatory activity of i. tinctoria was also evaluated in a clinical pilot study. the efficacy of the topical administration of different lipophilic extracts of i. tinctoria leaves was confirmed on healthy human volunteers using two experimental models, called sodium lauryl sulfate-induced irritant contact dermatitis and uvb-induced erythema. i. tinctoria extracts are effective when administered during the induction phase of dermatitis whereas they did not show any anti-inflammatory activity in the uvb-induced erythema model [63] . various lipophilic constituents belonging to different chemical classes play an important role in the anti-inflammatory activity of i. tinctoria extracts such as the alkaloidis tryptanthrin, indirubin, indolinone, and fatty acids as linolenic acid. tryptanthrin possesses a unique pharmacological profile. it has been shown to be highly selective toward the cox-2 isoenzyme [17, 92] . nonetheless, other studies reported tryptanthrin as a non-selective cox inhibitor [93] . it inhibited 5-lipooxygenase (5-lox) catalysed leukotriene synthesis in vitro and in vivo [18, 92, 94] , and inos catalysed nitric oxide (no) production [93] . in particular, tryptanthrin was found to be a potent natural inhibitor of cellular leukotriene biosynthesis in human whole blood and it is effective in vivo after oral administration in several experimental models such as in a murine model of inflammatory bowel disease (100 mg/kg p.o. for 3 days), in carrageenan-induced paw oedema in mice (50 mg/k p.o.), and in the rat pleurisy model (10 mg/kg p.o.) [18, 94, 95] . on the contrary, this compound, when topically administered, showed no significant anti-inflammatory effect in an animal model (tpa-induced ear oedema) [18] as well as in a clinical pilot study. in this study, carried out on healthy human volunteers, tryptanthrin, topically administered, did not manifest any protective effect both in the uvb-induced erythema model and in sodium lauryl sulfate-induced dermatitis [63] . thus, the pure compound is not effective when administered by topical application. this observation was also confirmed by oberthür et al. [62] through an experimental model of cutaneous micro-dialysis. it has been shown that the tryptanthrin contained in the extracts penetrates to a greater extent than that of pure solutions. this is because, in the first case, the alkaloid remains in molecular dispersion while, in the second case, it tends to crystallize on the surface of the skin. one of the active compounds isolated from i. tinctoria leaves responsible for the anti-allergic properties is the alkaloid indolin-2-one. it has been demonstrated that indolinone inhibits compound 48/80-induced histamine release from rat peritoneal mast cells [96] . moreover, indolinone was found to block ige-mediated degranulation of sensitized mast cells at nm concentrations without directly interfering with signalling upstream of the histamine-containing granules [97] . several studies demonstrated that indirubin is a potent inhibitor of cyclin-dependent kinase 5 (cdk5/p25) and glycogen synthase kinase-3-β (gsk-3-β) [98] as well as interferon-γ and interleukin (il)-6 [99] . in the experimental model of lps-induced pulmonary oedema in mice indirubin was shown to diminish oxidative stress and inflammation by reducing mda production as well as il-1β and tnf-α expression [100] . the analgesic potential of the lipophilic extract of i. tinctoria dried rosette leaves (dcm extract) was shown in vivo by an acetic acid-induced abdominal writhing test. the oral administration of the extract (200 mg/kg), before the intraperitoneal injection of acetic acid, reduced the number of writhing and stretching movements in mice while tryptanthrin (40 mg/kg) had no such effect [18] . i. tinctoria has been used in china to treat patients with solid tumours and leukaemia [9] . phase i/ii clinical trials highlighted very encouraging therapeutic effects of i. tinctoria in treating chronic myelocytic leukaemia [101] . experimental studies with transplantable tumours demonstrated that i. tinctoria significantly prolonged the life span of walker carcinosarcoma 256 bearing rats [101] . the anti-cancer properties of this species have been mainly attributed to the alkaloids' indirubin and tryptanthrin, whose activity has been demonstrated by in vivo and in vitro experimental models. numerous studies showed the potential efficacy of these compounds against a broad range of tumour types and the mechanisms of action underlying their activity have been elucidated. the bisindole indirubin has been described about 40 years ago as being active in treating human chronic myelocytic leukaemia. several trials published in china indicated that indirubin orally administered at the dose of 150-200 mg of per day led to remission in 60% of patients [9] . it was reported to induce complete remission in 26% and partial remission in 33% of 314 patients suffering from chronic myelocytic and chronic granulocytic leukaemia, which exhibited low toxicity and had limited adverse effects [66] . in animal leukaemia and lung carcinoma models, indirubin showed a good inhibitory effect and low toxicity [6] . it was found to inhibit dna synthesis in rats bearing walker-256 sarcoma [102] . a weak binding of indirubin to dna has been demonstrated in vitro by wu et al. [103] using the isotope labelling method, the spectrophotometric method, and thermal denaturation measurements, which shows that the binding between indirubin and dna might be of a hydrogen bond rather than ionic. in an experimental study carried out by zhang and colleagues [75] , the treatment for 25 days with indirubin (10 mg/kg per day) has proven to be effective in inhibiting prostate tumour growth in a xenograft mouse model (balb/c nude mice) by inhibiting tumour angiogenesis, as confirmed through a chick chorioallantois membrane (cam) assay and a mouse corneal model (c57bl/6 mice). western blot analysis highlighted that indirubin suppressed endothelial cell viability by blocking a vascular endothelial growth factor receptor 2-(vegfr2) mediated janus kinase (jak)/stat3 signalling pathway in endothelial cells. in particular, the phosphorylation and activation of jak2 and stat3 was suppressed, and the stat3 downstream genes including bcl-2, bcl-xl, cyclin d1, cyclin a, and survivin were down-regulated. the same authors also showed that indirubin effectively inhibited proliferation and induced apoptosis in human umbilical vein endothelial cells (huvec), while also inhibiting migration and capillary-structure formation. the core proteins involved in apoptosis including caspase-3 and parp were found to be activated. the anti-tumour properties of indirubin and its derivatives appear to correlate with their antimitotic potential and inhibition of cyclin-dependent kinases (cdks) and cell cycle regulatory molecules, which play an essential role in the regulation of cell proliferation, transcription, and apoptosis [70, 104] . it was found that indirubin and its analogues inhibited the proliferation of the human large cell lung carcinoma lxfl529l and human mammary carcinoma mcf-7 cells. the more soluble analogue indirubin-3 -monoxime induced g2/m arrest in mcf-7 cells synchronized in the g2/m phase by a transient exposure to nocodazole and this effect was mediated by the inhibition of cdk1 and cdk1/cyclin b activity. this was suggested as a major mechanism by which indirubin derivatives exert their potent anti-tumour efficacy [70] . indirubins are also efficient inhibitors of cdk2 and cdk5/p35. they act by competing with atp for binding to the catalytic subunit of the kinase [102, 105] . the crystal structure of the complex cdk2/indirubin derivatives showed that indirubin interacts with the kinase's atp-binding site through van der waals interactions and three hydrogen bonds [102] . another study published by damiens et al. [106] reported that, after transient treatment with indirubin-3'-monoxime of human breast epithelial hbl-100 cells synchronized in g2/m by nocodazole, cells underwent an endoreplication. after the compound has been removed, the polyploid cells became aneuploid and, later, died from necrosis. this mechanism of endoreplication followed by cell death may contribute to the anti-tumour properties of indirubins. the indole quinazolinone alkaloid tryptanthrin showed cancer chemo-preventive efficacy in intestinal tumour formation induced by azoxymethane in f344 rats [69] . it was found to be able to inhibit the growth of the murine myeloid leukaemia wehi-3b jcs cells in the tumour-bearing balb/c mice [73] . recently, tryptanthrin has also been shown to be an effective suppressor of non-melanoma skin cancer (nmsc), as demonstrated by using dimethylbenz[a]anthracene/phorbol 12myristate 13acetate (dmba/pma) induced skin carcinogenesis model in swiss albino mice [79] . it was found that the compound disrupted dmba/pma-induced expansion of hair follicle cells by suppressing the activation of β-catenin, which is a major driver of hair follicle cell proliferation. additionally, tryptanthrin suppressed the activation of erk1/2 and p38, in which both promote β-catenin activation and lowers the expression of c-myc and cyclin-d1. these are the transcriptional targets of β-catenin, which are widely implicated in carcinogenesis. increasing evidence demonstrated that tryptanthrin is highly cytotoxic to a variety of a solid tumour and leukaemia cell lines and has a broad range of downstream targets that regulate tumour-associated cell processes including cell growth, cell cycle progression, and survival. tryptanthrin was found to inhibit the growth of human gastric cancer (hgc), lung cancer (hlc), and promyelocytic leukemia hl-60 cells with ic 50 values of 1.5-4.2 µg/ml [67] . it showed significant cytotoxicity against non-small cell lung cancer nci-h460, human glioblastoma sf-268, and human breast cancer mcf-7 cell lines with ic 50 values of 8.5-22.6 µm [78] . tryptanthrin also exhibited a multi-drug resistance reversing effect in doxorubicin-resistant breast cancer mcf-7 cells (mcf-7/adr), which is higher than that of the multi-drug resistance (mdr)-reversing agent verapamil through down-regulation of mdr1 gene expression, and partly by modulating the gstpi-related pathway, which is a non-transporter pathway [71, 72] . liao and leung [76] reported that tryptanthrin is able to inhibit the growth of the n-myc amplified human neuroblastoma la-n-1 cells (ic 50 = 15.8 ± 1.41 µm) as well as two other human neuroblastoma cell lines sh-sy5y and sk-n-dz, which induced cell cycle arrest at the g0/g1 phase. western blot analysis showed a marked down-regulation of cyclin d1, cyclin d3, cdk4, and cdk6, which are known to be associated with the cell-cycle progression from the g1 to the s phase. la-n-1 cells treated with tryptanthrin underwent marked morphologic differentiation, enhancement of acetylcholine esterase activity, and up-regulation of various differentiation markers including growth-associated protein 43 (gap43), microtubule-associated protein tau (mapt), calcitonin gene related peptide (cgrp), and somatostatin, with an effect superimposable to that of 5 µm 9-cis-ra used as a positive control. moreover, tryptanthrin treatment led to a significant reduction of the n-myc proto-oncogene expression. the anti-angiogenic activity has been proposed as an important mechanism underlying the anti-cancer properties of tryptanthrin against various solid tumours. a study published by liao and colleagues [77] demonstrated that tryptanthrin inhibited angiogenesis both in vitro and in vivo. in particular, it inhibited the proliferation of human microvascular endothelial hmec-1 cells and interrupted the migration and capillary-like structure formation. the anti-angiogenic effect was confirmed in vivo by a matrigel plug assay. the underlying molecular mechanisms of action were explained by a reduced expression of pro-angiogenic factors such as ang-1, pdgfb, and mmp2, suppression of the phosphorylation of vegfr2, and blockade of the vegfr2-mediated erk1/2 signalling pathway. molecular docking studies indicated that tryptanthrin could bind to the atp-binding site of vegfr2. concerning the antileukemic activity, at low concentrations (0.5 µg/ml), tryptanthrin was found to be able to enhance the expression of cell differentiation markers in human monocytic u-937 and promyelocytic hl-60 leukaemia cells, which is indicative of their differentiation into monocytes and macrophages. higher concentrations inhibited cell proliferation and induced cell death by apoptosis in the cultures by damaging the mitochondria that induces the apoptotic cascade through a caspase-3/fas antigen pathway [68] . chan et al. [73] reported the potent anti-proliferative efficacy of tryptanthrin against murine myeloid leukaemia wehi-3b jcs cells, with an ic 50 value of 1.5 µm at 48 h of treatment. flow cytometric analysis showed the cell cycle arrest of the cells at the g0/g1 phase in a dose-dependent manner. the expression of the cell cycle-related genes including cyclin d2, d3, cdk 2, 4, 6 was found to be significantly down-regulated at 24 h as measured by real time pcr, which led us to propose the prevention of the cell cycle progression from the g1-phase into the s-phase. this causes cell cycle arrest as one of the mechanisms for leukemic cell growth inhibition. furthermore, it has been suggested that tryptanthrin exerts its anti-tumor effect on wehi-3b jcs cells by triggering cell differentiation, as demonstrated by morphological and functional studies. miao et al. [74] had proved that tryptanthrin significantly inhibited human chronic myeloid leukaemia k562 cell proliferation with an ic 50 value of 8.8 µg/ml after 48 h of treatment. cell cycle distribution analysis showed that it inhibited proliferation by blocking the cell cycle progression at the g0/g1 phase and, subsequently, progressing into apoptosis. the underlying mechanism has been attributed to the damage of mitochondrial membrane and the cyt-c-caspase-3 dependent mechanisms. besides the lipophilic compounds indirubin and tryptanthrin, the polar phenolic constituents have also been shown to contribute to the anti-cancer properties of i. tinctoria. recently, the anti-proliferative properties of polar extracts obtained from i. tinctoria rosette leaves together with those from the cauline leaves and the flowers have been evaluated [47] . to achieve the separation of the polar compounds from the lipophilic ones, frozen and lyophilized plant material was sequentially extracted with dichloromethane and 70% methanol. i. tinctoria polar extracts showed anti-proliferative effects against the human anaplastic thyroid cancer atc cell lines cal-62, 8505c and c-643. particularly, after 48 h of treatment, the basal leaf extract markedly inhibited the growth of cal-62 cells, which caused nearly 85% reduction of viability at the highest tested dose (1 mg/ml). the significant anti-proliferative effects against atc cell lines have been related to the flavonoids and the phenolic acids contained in the extracts, characterized by hplc-pda/esi-ms. another study published by the same authors confirmed that phenolics were mainly responsible for the observed activity. the anti-proliferative properties of the phenolic-rich fraction obtained from i. tinctoria cauline leaf polar extract has been demonstrated, which caused 80% and 65% growth inhibition in cal-62 and 8505c cells, respectively, after 48 h of exposure at the maximum tested dose (0.1 mg/ml). the activity of the phenolic-rich fraction against these cell lines was similar to that of sunitinib used as a positive control, when tested at the dose of 10 µm for 72 h [46] . a few research studies investigated i. tinctoria as a potential source of valuable compounds with anti-microbial efficacy against bacterial and fungal species. more thorough studies have been conducted on components with antiviral activity, which have led to the identification of possible mechanisms of action ( table 2) . the results of a study carried out by dornberger and lich [81] on the anti-microbial activity of a hydroalcoholic (water/ethanol) extract of the fresh whole plant evaluated by an agar diffusion test indicated that, among the 23 tested strains (gram-positive, gram-negative, yeast, and fungi), only the gram-positive tetracycline-resistant bacillus mycoides sg 756 tf, b. subtilis atcc 6633, and micrococcus luteus sg 125 a, and the yeast saccharomyces cerevisiae jh 3 showed weak sensitivity to the extract. by contrast, ullah and colleagues [85] recently reported the good anti-microbial properties of different extracts from i. tinctoria. in particular, the study concerned the evaluation of anti-microbial activity of fractions obtained from different parts of the plant (branches, flowers, leaves, and roots) by extraction with 14 different solvents, which was performed using a micro-titer plate method against seven bacterial and four fungal strains. the obtained results showed that the extracts displayed antibacterial activity, more elevated against gram-positive strains (b. subtilis, m. luteus and staphylococcus aureus). in many cases, it was higher than the antibiotic cefotaxime used as a positive control as well as antifungal activity (except for branches). in general, the leaves were found to be the best plant part and ethyl acetate, n-hexane, chloroform, and acetone as the most efficient solvents for the extraction of antimicrobial compounds. the possible utilization of i. tinctoria in combination therapy with commonly used antibiotics for treating infections caused by meticillin-resistant s. aureus (mrsa), which is a widespread cause of both community-acquired and hospital-acquired infections associated with significant mortality and morbidity, has been suggested. the dried leaf hydroalcoholic (75% ethanol) extract of i. tinctoria was found to be able to potentiate the effects of four antibiotics (penicillin g, gentamicin, ciprofloxacin, and ceftriaxone) against both mrsa (isolated from hospital patients) and standard (atcc25923) s. aureus strains [82] . among the phytochemicals contained in i. tinctoria, the alkaloid tryptanthrin has been proven to have an antimicrobial effect. nonetheless, there are discrepancies between the results of various research studies. one of the earliest published works is that of honda and colleagues, who evaluated the activity of tryptanthrin against a wide variety of microorganisms including bacteria, yeasts, dermatophytes by the agar dilution streak method, and phytopathogens by the paper disc method. the results of these investigations demonstrated that it displays strong activity against bacillus subtilis (mic: 25 µg/ml) and b. polymyxa (mic: 50 µg/ml), and it is a highly specific antimicrobial agent against dermatophytes, which are causative of athlete's foot, namely trichophyton mentagrophytes, t. rubrum ifo 5808, t. tonsurans var. sulfureum ifo 5945, microsporum canis ifo 9182, m. gypseum ifo 8307, and epidermophyton floccosum ifo 9045 (mics: 3.1-6.3 µg/ml) [43, 80] . by contrast, the results of the agar diffusion assay carried out by chiang et al. [83] revealed that tryptanthrin showed no effect on tested fungi including the dermatophytic pathogens e. floccosum atcc 18397, m. gypseum atcc 14683, and t. rubrum atcc 10218. the same authors reported that tryptanthrin significantly (≥12.5 µg/disc) inhibited the growth of s. epidermis atcc 12228 and s. aureus atcc 6538, and only weakly suppressed mrsa atcc 43300 even at 100 µg/disc. this last result is in disagreement with that of a more recent study, which highlighted a promising anti-bacterial activity of tryptanthrin against mrsa, as evaluated by the agar diffusion method (mic 62.5 µg/ml) [84] . the traditional chinese medicine bǎn lán gēn (isatidis radix) is widely employed in the prevention and treatment of a wide range of viral infections including seasonal flu, the deadly severe acute respiratory syndrome (sars), viral pneumonia, mumps, pharyngitis, and hepatitis [87, 107] . different types of compounds effective against influenza viruses have been isolated from i. radix. indirubin has been shown to have powerful activity vs. the influenza virus a/nws/33-and b/lee/40-infected bronchial epithelial cells h292 by reducing both the expression and production of the chemokine rantes [86] . the polysaccharide compounds contained in i. radix also have anti-influenza virus activity, as demonstrated by in vitro investigations. it has been reported that a polysaccharide isolated from i. radix inhibited the attachment of the influenza virus to the red blood cells, which promoted the generation of anti-influenza viral igg antibodies [88] . in a study carried out by li et al. [87] , i. radix polysaccharides showed potent anti-influenza a virus activity against human seasonal influenza viruses (h1n1 and h3n2) and avian influenza viruses (h6n2 and h9n2). they were found to effectively inhibit the expression of pro-inflammatory cytokines (il-6) and chemokines (ip-10, mig, and ccl-5) induced by the human influenza virus pr8/h1n1 and the avian influenza virus h9n2. furthermore, the polysaccharides reduced the expression of the toll-like receptor (tlr)-3 induced by the pr8/h1n1 virus. these results led to hypothesize that the underlying mechanism of action of the polysaccharides is to impair the upregulated expression of pro-inflammatory cytokines/chemokines induced by the influenza virus by inhibiting the expression of host tlr-3. the in vitro antiviral efficacy of the i. radix polysaccharide against type ii herpes simplex virus (hsv-2) has been recently demonstrated [88] . using the 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay, this effect was shown to occur mainly by inhibiting the viral duplication and adsorption. in the last decade, some research has focused on the study of i. tinctoria as a source of natural antioxidants. the first published articles concerning the antioxidant properties of i. tinctoria extracts were those of fialova et al. and of yang et al. [89, 90] . in the first work carried out on a hydroalcoholic extract of the leaves, good radical scavenging activity in the 1,1-diphenyl-2-picrylhydrazyl (dpph) test was found (sc 50 = 103.9 µg/ml) whereas the second one reported very weak activity in the same test as well as in the trolox equivalent antioxidant capacity (teac) and in reducing power assays for an ethanol extract obtained from dried chinese i. tinctoria. the results of subsequent investigations on i. tinctoria leaves indicated that phytochemicals belonging to different chemical classes act as antioxidants, such as alkaloids and phenolic compounds. in a study carried out by zhao and collegues [91] , the antioxidant properties of the indole alkaloids indigo and indirubin were evaluated by means of different in vitro assays. indigo and indirubin were found to be more effective for superoxide anion scavenging (ec 50 = 0.61 mg/ml and 0.74 mg/ml, respectively). this effect has been attributed to the presence of some electrophilic groups, such as keto or aldehyde, which facilitate the release of hydrogen from the o-h bond and stabilize the superoxide anion. both compounds exhibited scavenging activities on a dpph-free radical, whereas they did not show hydroxyl radical (oh•) scavenging activity and reducing power. it is well known that polyphenols represent the main class of natural antioxidants, which act in different ways such as radical scavengers because phenolic groups are excellent nucleophiles and are also able to inhibit lipid peroxidation, as breakers of the oxidation reaction by binding with free radicals generated through lipid peroxidation, and as chelators of metal ions that induce oxidation [108] . aimed at establishing the involvement of phenolic compounds in the antioxidant properties of i. tinctoria, recently, the polar extract obtained from the rosette leaves and also those from the cauline leaves and the flowers have been investigated by in vitro assays based on different mechanisms [47] . lyophilized plant material was sequentially extracted with dichloromethane and 70% methanol in order to separate the polar compounds from the lipophilic ones. the polar extracts were found to possess good radical scavenging (dpph test) and ferrous ions' chelating activities and moderate reducing power, and those from cauline leaves and flowers displayed better antioxidant efficacy than the rosette leaf extract. the calculated coefficient of determination (r 2 ) indicated that flavonoids were the phenolic compounds mainly involved in the observed antioxidant properties. another study carried out by the same authors on the phenolic-rich fraction obtained from i. tinctoria cauline leaf polar extract confirmed that phenolics were mainly responsible for the good radical scavenging properties [46] . in particular, the activity has been mainly related to the flavonols detected in the fraction, which have all the essential structural elements for potent radical scavenging activity. compared to flavone compounds, the flavonols contain more hydroxyl groups (one to six) among which the oh group at the c-3 position has a very high ability to scavenge the dpph radical, such as quercetin and rutin, which are well-known potent antioxidants. interestingly, these works have highlighted the antioxidant activity not only of the rosette leaves, which are commonly utilized for their medicinal properties, but also of the cauline leaves and the flowers, which indicates a broader range of usage possibilities for this relevant species. even the roots of i. tinctoria are a source of antioxidant compounds. indeed, the polysaccharides contained in the roots were found to possess antioxidant properties in vitro. in particular, in a study conducted by han et al. [34] , the polysaccharides extracted from the dried roots exhibited an appreciable radical scavenging activity in the 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (abts) assay, reaching, at the maximum tested concentration (0.3 mg/ml), a percentage of inhibition (64.3%) close to that of vitamin c used as a reference compound. it has been reported that the phytochemical profile of i. tinctoria field-cultivated is highly affected by various factors including cultivar, geographical regions, climatic fluctuations, and soil conditions [109] . the techniques concerning plant in vitro cultures and the possibilities of increasing the accumulation of secondary metabolites established by plant biotechnology are a known, alternative source of many valuable biologically active secondary metabolites used in medicine as well as in cosmetology and the food industry [110, 111] . the many strategies used in plant biotechnology aimed at increasing the in vitro culture biomass and/or bioactive metabolites' production. the elaboration of micropropagation protocols creates the possibilities of increasing and facilitating the specific, often very demanding, plant multiplication [112] . for increasing the secondary metabolites production in in vitro cultured biomass, different, specific strategies have been introduced [113] . the most important ones are: the selection of high productivity cell lines, elaboration of the optimal culture conditions (e.g., selection of basic formulation of the culture medium, type of plant growth regulators, and their concentrations in the media, types of cultures, and optimal lighting and temperature conditions), establishment of cultures with a high degree of organogenesis (shoot, root cultures), application of biotic or abiotic elicitors, and genetic transformation (e.g., hairy root cultures). increasing the biomass production and the scale-up of cultures' production in the special plant bioreactors are also important aspects of biotechnology studies [111, 114, 115] . the growing interest in the beneficial values of i. tinctoria has been concentrated mainly on the roots of this plant as the best source of demanding metabolites. as apparent from the analysis of the latest scientific literature, the biotechnological studies on i. tinctoria are limited mainly to hairy root cultures. interestingly, within the last four years, the teams of two researchers: gai and jiao from northeast forestry university, harbin in china, have published a series of research publications on i. tinctoria hairy root cultures. the cultures were successfully initiated from in vitro petiole explants co-cultured with agrobacterium rhizogenes with the addition of acetosyringone and arginine. in the obtained hairy root cultures, the high production of alkaloids [116] and flavonoids [117] was confirmed. within the alkaloids epigoitrin, isatin, indole-3-carboxaldehyde, tryptanthrin, indigo, and indirubin were qualitatively and quantitatively determined by lc-ms/ms. the total alkaloid content (521.77 µg/g dw (dry weight)) was 1.12 times higher than in two-year-old roots of field grown plants (464.69 µg/g dw) [116] . under the study on flavonoid composition, eight bioactive constituents were estimated by the lc-ms/ms method: rutoside, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol, and isoliquiritigenin [117] . under optimal conditions, the total flavonoid content (438.10 µg/g dw) was found to be 1.28 times higher than in two-year-old field grown roots (341.73 µg/g dw) analyzed for comparison. additionally, in vitro antioxidant assays demonstrated that hairy root extracts exhibited higher activities than those from roots of field grown plants [117] . such promising results prompted the chinese team to perform the elicitation experiments on these cultures in order to boost the bioactive metabolites' production. the elicitation experiment with chitosan was proven to increase the flavonoid yield in i. tinctoria hairy root biomass [118] . in comparison with the control (2.31 mg/g dw), a 7.08-fold enhancement of total flavonoids (16.35 mg/g dw) was achieved in 24-day-old hairy root cultures elicited by 150 mg/l chitosan for 36 h [118] . under another study, the i. tinctoria hairy root cultures were exposed to ultraviolet radiation (uv-a, uv-b, and uv-c) in an attempt to promote the production flavonoids. results showed that the maximum flavonoid accumulation (7259.12 µg/g dw) in hairy root cultures treated by 108 kj/m 2 dose of uv-b radiation increased 16.51-fold as compared to the control (439.68 µg/g dw) [119] . moreover, the same teams proved the increase of flavonoid production after i. tinctoria hairy root cultures co-cultivation with two immobilized live gras (generally recognized as safe) fungi: aspergillus niger and aspergillus oryzae. a. niger strains were exhibited as the better biotic elicitor in the plant-fungus co-cultivation system. the highest flavonoid production (3018.31 µg/g dw) was achieved after treatment with spores at the concentration of ca.104 spores/ml, temperature of 30 • c, ph 7.0, and time of 72 h. the total amount of flavonoids increased 6.83 times in comparison to control hairy roots (441.91 µg/g dw) [120] . a recent work on elicitation of i. tinctoria hairy root cultures proved enhanced production of flavonoids as well as alkaloids after abiotic elicitation protocol applied with salicylic acid and methyl jasmonate [121] . as determined by central composite design based on a mathematical model, the maximum accumulation of alkaloids was found in biomass elicited by 142.61 µm salicylic acid for 28.18 h and flavonoids in biomass elicited by 179.54 µm methyl jasmonate for 41.87 h. these amounts increased 5.89-times and 11.21-times, respectively, in comparison with the control. the above described biotechnological studies on i. tinctoria hairy roots are a good example of the importance of optimization culture conditions, aimed at high secondary metabolites' production. authors claim that their research studies are a promising and effective approach for the enhanced production of flavonoids and alkaloids in i. tinctoria in vitro cultures, which allows for the improved applicability of these valuable compounds in pharmaceutical fields, and that their studies pave the way toward the successful commercialization of this culture system in the future. in the available scientific literature, there is only one publication on the development of the micropropagation protocol for i. tinctoria, based on the adventitious shoot regeneration method. under the work of saglam and ciftci [122] , the effective shoot regeneration system was developed using 'isubgol' as a cheap alternative gelling substance. the leaf and hypocotyl explants were cultured on murashige and skoog (ms) [123] medium solidified with 6.5 g/l agar or 15 g/l isubgol containing different concentrations of ba (6-benzyladenine) and naa (1-naphthaleneacetic acid). maximum regeneration of 12.65 and 17.80 shoots per leaf explant was observed on ms medium solidified with agar containing 1 mg/l ba with 0.25 mg/l naa and on ms medium solidified with isubgol containing 0.50 mg/l ba, respectively. maximum regeneration of 19.87 and 20.55 shoots per hypocotyl explant on agar and isubgol gelled media was recorded on ms medium containing 0.50 mg/l ba with 0.25 mg/l naa, and 1 mg/l ba, respectively. the gelling agent isubgol gave better shoot regeneration as compared to agar. in the existing literature, only one study on shoot cultures of i. tinctoria is available. in particular, reference [124] obtained the establishment of i. tinctoria shoot cultures starting from the nodal segments of young plants cultured on ms and gamborg (b5) [125] media supplemented with ba or kin (kinetin) with or without naa. the best multiplication rates were obtained on b5 medium supplemented with 1 mg/l ba (3.4 shoots per explant). the addition of naa to the tested media did not increase multiplication. currently, under the cooperation of our teams, from the department of chemical, biological, pharmaceutical and environmental sciences, university of messina (messina, italy), and the department of pharmaceutical botany, jagiellonian university, collegium medicum (kraków, poland), in vitro shoot cultures of i. tinctoria have been successfully initiated, which aimed to investigate whether they could represent a potential source of bioactive compounds (figure 8 ) [unpublished] . the cultures were successfully initiated from the seeds, and the preliminary media composition optimization was performed. the ms media variants with ba and kin as cytokinins and naa and iba (indole-3-butyric acid) as auxins in different concentrations (range from 0 to 2 mg/l) were tested. the best conditions for agar shoot cultures were proven to be ms medium containing 1 mg/l ba and 1 mg/l naa on which the biomass increment was equal to 3.58-fold (fresh weight). not increase multiplication. currently, under the cooperation of our teams, from the department of chemical, biological, pharmaceutical and environmental sciences, university of messina (messina, italy), and the department of pharmaceutical botany, jagiellonian university, collegium medicum (kraków, poland), in vitro shoot cultures of i. tinctoria have been successfully initiated, which aimed to investigate whether they could represent a potential source of bioactive compounds (figure 8 ) [unpublished] . the cultures were successfully initiated from the seeds, and the preliminary media composition optimization was performed. the ms media variants with ba and kin as cytokinins and naa and iba (indole-3-butyric acid) as auxins in different concentrations (range from 0 to 2 mg/l) were tested. the best conditions for agar shoot cultures were proven to be ms medium containing 1 mg/l ba and 1 mg/l naa on which the biomass increment was equal to 3.58-fold (fresh weight). intensive biotechnological studies on i. tinctoria shoot culture are in progress and are aimed at obtaining high production of secondary metabolites in the biomass (as well as in the growth media) such as phenolic acids or glucosinolates, which were never studied before. i. tinctoria has an ancient and well-documented history as an indigo dye and medicinal plant. it has been long utilized as an ornamental crop and an animal feeding plant, and, currently, its importance increased in cosmetic industry. due to the relevance of this species, a large number of in vitro and in vivo investigations carried out on i. tinctoria mostly in the last 20 years have provided reasonable support for its various traditional uses, which shows that the diverse extracts or compounds isolated from different parts of this species have a broad spectrum of biological activities. as far as we know, there is only one previous review article by hamburger (2002) , which reported studies published from 2000 to 2002 focusing only on the anti-inflammatory activity of i. tinctoria extract and its active component tryptanthrin. this article review provides an up-to-date and comprehensive overview mainly on the phytochemistry and the biological properties demonstrated for this valuable species in order to support its therapeutic potential and to offer input for future research prospects. the article also focuses on all attempts in plant biotechnology studies for the enhanced production of bioactive compounds from i. tinctoria hairy root and shoot cultures as an alternative to intensive biotechnological studies on i. tinctoria shoot culture are in progress and are aimed at obtaining high production of secondary metabolites in the biomass (as well as in the growth media) such as phenolic acids or glucosinolates, which were never studied before. i. tinctoria has an ancient and well-documented history as an indigo dye and medicinal plant. it has been long utilized as an ornamental crop and an animal feeding plant, and, currently, its importance increased in cosmetic industry. due to the relevance of this species, a large number of in vitro and in vivo investigations carried out on i. tinctoria mostly in the last 20 years have provided reasonable support for its various traditional uses, which shows that the diverse extracts or compounds isolated from different parts of this species have a broad spectrum of biological activities. as far as we know, there is only one previous review article by hamburger (2002) , which reported studies published from 2000 to 2002 focusing only on the anti-inflammatory activity of i. tinctoria extract and its active component tryptanthrin. this article review provides an up-to-date and comprehensive overview mainly on the phytochemistry and the biological properties demonstrated for this valuable species in order to support its therapeutic potential and to offer input for future research prospects. the article also focuses on all attempts in plant biotechnology studies for the enhanced production of bioactive compounds from i. tinctoria hairy root and shoot cultures as an alternative to plant raw materials. plant in vitro culture technology has emerged as an attractive alternative for the field cultivation of i. tinctoria. due to the high therapeutic value of i. tinctoria, the biotechnological approach represents a promising way to develop safe and more effective extracts than traditional ones. the large-scale production of biotechnological extracts, enriched in desired metabolites and obtained by different in vitro systems, should become the goal of further scientific studies. flora europaea the european garden flora systematics and phylogeny of the brassicaceae (cruciferae): an overview genetic variation and population structure in a eurasian collection of isatis tinctoria l adaptability and variation in isatis tinctoria l.: a new crop for europe isatis tinctorial-from the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical an ancient dye plant of interest as a multifunctional crop new archaeobotanical finds of isatis tinctoria l. (woad) from iron age gaul and a discussion of the importance of woad in ancient time spectral and photophysical studies of substituted indigo derivatives in their keto forms cultivation and use of isatis tinctoria l. (brassicaceae) in southern italy diversity of the intracellular mechanisms underlying the anti-tumor properties of indirubins geographic population structure in an outcrossing plant invasion after centuries of cultivation and recent founding events phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in isatis tinctoria leaves identification and isolation of the cyclooxygenase-2 inhibitory principle in isatis tinctoria anti-inflammatory and antiallergic activity in vivo of lipophilic isatis tinctoria extracts and tryptanthrin the leaf volatile constituents of isatis tinctoria by solid-phase microextraction and gas chromatography/mass spectrometry european directorate for the quality of medicines cosmetic ingredientdatabase (cosing) sowing date, transplanting, plant density and nitrogen fertilization affect indigo production from isatis species in a mediterranean region of spain nutraceutical value of woad (isatis tinctoria) flower buds of ecotypes from sicily european directorate for the quality of medicines differences in leaf yield and indigo precursors production in woad (isatis tinctoria l.) and chinese woad a high degree of genetic diversity is revealed in isatis spp. (dyer's woad) by amplified fragment length polymorphism (aflp) discussion on the botanical origin of isatidis radix and isatidis folium based on dna barcoding herbal antivirals: natural remedies for emerging, resistant and epidemic viral infections hplc based activity profiling for 5-lipoxygenase inhibitory activity in isatis tinctoria leaf extracts medicinal plants of east and southeast asia: attributed properties and uses a comprehensive metabolite profiling of isatis tinctoria leaf extracts optimisation of extraction conditions for polysaccharides from the roots of isatis tinctoria l. by response surface methodology and their in vitro free radicals scavenging activities and effects on il-4 and ifn-γ mrna expression in chicken lymphocytes medicinal plants of china indole alkaloid glycosides from isatis tinctoria roots antiviral decoction of isatidis radix (bǎn lán gēn) inhibited influenza virus adsorption on mdck cells by cytoprotective activity the pharmacology of chinese herbs in chinese drugs of plant origin assessment of indigo (polygonum tinctorium ait.) water extracts' bioactive compounds, and their antioxidant and antiproliferative activities handbook of chinese herbs and formulas; institute of chinese medicine isolation of an antidermatophytic, tryptanthrin, from indigo plants, polygonum tinctorium and isatis tinctoria identification of an indigo precursor from leaves of isatis tinctoria (woad) metabolic markers for the yield of lipophilic indole alkaloids in dried woad leaves (isatis tinctoria l.) chemical characterization and biological activities of phenolic-rich fraction from cauline leaves of isatis tinctoria l. (brassicaceae) growing in sicily phenolic profile, antioxidant and cytotoxic properties of polar extracts from leaves and flowers of isatis tinctoria l. (brassicaceae) growing in sicily a novel anthranilic acid derivate from isatis tinctoria extraction and analysis of intact glucosinolates-a validated pressurized liquid extraction/liquid chromatography-mass spectrometry protocol for isatis tinctoria, and qualitative analysis of other cruciferous plants glucosinolate pattern in isatis tinctoria and i. indigotica seeds volatile constituents in dried roots of isatis tinctoria l. (brassicaceae) microelement contents and fatty acid compositions of some isatis species seeds the elusive indigo precursors in woad (isatis tinctoria l.)-identification of the major indigo precursor, isatan a and a structure revision of isatan b origin of indigo of woad the content of indigo precursors in isatis tinctoria leaves-a comparative study of selected accessions and post-harvest treatments isatisindigoticanine a, a novel indole alkaloid with an unpresented carbon skeleton from the roots of isatis tinctoria quantitative determination of the dual cox-2/5-lox inhibitor tryptanthrin in isatis tinctoria by esi-lc-ms tryptanthrin content in isatis tinctoria leaves-a comparative study of selected strains and post-harvest treatments thermal degradation of glucosinolates phytochemical characterization and biological activities of a hydroalcoholic extract obtained from the aerial parts of matthiola incana (l.) r. br. subsp. incana (brassicaceae) growing wild in effects of chinese medicinal herbs on a rat model of chronic pseudomonas aeruginosa lung infection a comparative study on the skin penetration of pure tryptanthrin and tryptanthrin in isatis tinctoria extract by dermal microdialysis coupled with isotope dilution esi-lc-ms prevention of experimentally induced irritant contact dermatitis by extracts of isatis tinctoria compared to pure tryptanthrin and its impact on uvb-induced erythema anti-arthritic activity of a lipophilic woad (isatis tinctoria) extract the plant extract isatis tinctoria l. extract (ite) inhibits allergen-induced airway inflammation and hyperreactivity in mice clinical studies of 314 cases of cml treated with indirubin cytotoxic effects of substances in indigo plant (polygonum tinctorium lour.) on maligant tumour cells cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (u-937 and hl-60) by tryptanthrin, an active ingredient of polygonum tinctorium lour prevention of azoxymethane-induced intestinal tumors by a crude ethyl acetate-extract and tryptanthrin extracted from polygonum tinctorium lour inhibition of cyclin-dependent kinase 1 (cdk1) by indirubin derivatives in human tumour cells tryptanthrin inhibits mdr1 and reverses doxorubicin resistance in breast cancer cells downregulation of gstpi expression by tryptanthrin contributing to sensitization of doxorubicin-resistant mcf-7 cells through c-jun nh2-terminal kinase-mediated apoptosis modulatory effects and action mechanisms of tryptanthrin on murine myeloid leukemia cells proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on human chronic myeloid leukemia k562 cell line in vitro indirubin inhibits tumor growth by antitumor angiogenesis via blocking vegfr2-mediated jak/stat3 signaling in endothelial cell tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma la-n-1 cells tryptanthrin inhibits angiogenesis by targeting the vegfr2-mediated erk1/2 signalling pathway isolation and cytotoxicity evaluation of the chemical constituents from cephalantheropsis gracilis pre-clinical evidences for the efficacy of tryptanthrin as a potent suppressor of skin cancer the antimicrobial specificity of tryptanthrin screening for antimicrobial and presumed cancerostatic plant metabolites the synergistic activity of antibiotics combined with eight traditional chinese medicines against two different strains of staphylococcus aureus an in vitro study of the antimicrobial effects of indigo naturalis prepared from strobilanthes formosanus moore in vitro anti-mrsa activity of couroupita guianensis extract and its component tryptanthrin antibacterial and antifungal activity of isatis tinctoria l. (brassicaceae) using the micro-plate method inhibition of rantes expression by indirubin in influenza virus-infected human bronchial epithelial cells radix isatidis polysaccharides inhibit influenza a virus and influenza a virus-induced inflammation via suppression of host tlr3 signaling in vitro antiviral activities of radix isatidis polysaccharide against type ii herpes simplex virus in vitro isatis tinctoria l. (dyer's woad) from the standpoint of phenolic compound content and scavenging activity evaluation of antioxidant and antimicrobial activities from 28 chinese herbal medicines optimization of ultrasound-assisted extraction of indigo and indirubin from isatis indigotica fort and their antioxidant capacities inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis tryptanthrin inhibits nitric oxide and prostaglandin e2 synthesis by murine macrophages on the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo the natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice inhibitory activity of indolin-2-one derivatives on compound 48/80-induced histamine release from mast cells e,z)-3-(3',5'-dimethoxy-4'-hydroxybenzylidene)-2-indolinone blocks mast cell degranulation structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases indirubin inhibits inflammatory reactions in delayed-type hypersensitivity indirubin improves antioxidant and anti-inflammatory functions in lipopolysaccharidechallenged mice indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in china indirubin, the active constituent of a chinese antileukemia medicine, inhibits cyclin-dependent kinases studies on the mechanism of indirubin action in the treatment of chronic granulocytic leukemia. v. binding between indirubin and dna and identification of the type of binding molecular mechanisms of indirubin and its derivatives: novel anticancer molecules with their origin in traditional chinese phytomedicine indirubins inhibit glycogen synthase kinase-3β and cdk5/p25, two kinases involved in abnormal tau phosphorylation in alzheimer's disease. a property common to most cdk inhibitors? anti-mitotic properties of indirubin-3 -monoxime, a cdk/gsk-3 inhibitor: induction of endoreplication following prophase arrest anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds phenolic profile and biological properties of the leaves of ficus vasta forssk. (moraceae) growing in egypt environmental factors affecting growth and development of banlangen (radix isatidis) in china biotechnology for the production of plant secondary metabolites factors influencing somatic embryogenesis induction and plant regeneration with particular reference to arabidopsis thaliana (l.) heynh. plant growth regul factors affecting the production of secondary metabolites elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories schisandra lignans production regulated by different bioreactor type establishment of high-productive isatis tinctoria l. hairy root cultures: a promising approach for efficient production of bioactive alkaloids establishment of hairy root cultures by agrobacterium rhizogenes mediated transformation of isatis tinctoria l. for the efficient production of flavonoids and evaluation of antioxidant activities chitosan elicitation of isatis tinctoria l. hairy root cultures for enhancing flavonoid productivity and gene expression and related antioxidant activity ultraviolet radiation for flavonoid augmentation in isatis tinctoria l. hairy root cultures mediated by oxidative stress and biosynthetic gene expression remarkable enhancement of flavonoid production in a co-cultivation system of isatis tinctoria l. hairy root cultures and immobilized aspergillus niger elicitation of isatis tinctoria l. hairy root cultures by salicylic acid and methyl jasmonate for the enhanced production of pharmacologically active alkaloids and flavonoids effects of agar and ısubgol on adventitous shoot regeneration of woad (isatis tinctoria) a revised medium for rapid growth and bio assays with tobacco tissue cultures acknowledgments: jasmine speranza thanks the foundation "prof. antonio imbesi" for the fellowship. the authors declare no conflict of interest. key: cord-033493-kslzdy8q authors: hebishy, ali m. s.; salama, hagar t.; elgemeie, galal h. title: new route to the synthesis of benzamide-based 5-aminopyrazoles and their fused heterocycles showing remarkable antiavian influenza virus activity date: 2020-09-21 journal: acs omega doi: 10.1021/acsomega.0c02675 sha: doc_id: 33493 cord_uid: kslzdy8q [image: see text] this study describes a new route to the synthesis of novel benzamide-based 5-aminopyrazoles and their corresponding pyrazolo[1,5-a]pyrimidine and pyrazolo[5,1-c][1,2,4]triazine derivatives. benzamide-based 5-aminopyrazoles were prepared through a reaction of benzoyl isothiocyanate with malononitrile in koh–etoh followed by alkylation with alkyl halides and then a reaction with hydrazine. in an attempt to react benzoyl isothiocyanate with ethyl cyanoacetate in koh–etoh followed by alkylation with methyl iodide at room temperature and then a reaction with hydrazine has resulted in the formation of 3-ethoxy-5-phenyl-1h-1,2,4-triazole. the structures of the new compounds were characterized by mass spectroscopy, (1)h nuclear magnetic resonance ((1)h nmr) spectroscopy, infrared spectroscopy (ir), and x-ray analysis. the new compounds were tested in vitro for their anti-influenza a virus (subtype h5n1) activity. among the synthesized compounds, eight compounds 3b, 4, 10b, 10c, 12a, 19, 21a, and 21b were found to possess significant antiviral activities against bird flu influenza (h5n1) with viral reduction in the range of 85–65%. the emergence of bird flu virus (h5n1) 23 years ago has caused several diseases in mankind over the past few years, and now, it is spreading in the world a very deadly influenza pandemic, which is covid-19 and has caused the death of many people. we had to put in place several measures to prevent the spread of these influenza viruses by developing pharmaceutical strategies to design drugs for combating influenza viruses of all kinds. 5-aminopyrazoles and their fused triazine and pyrimidine ring systems are analogs of purines and thioguanines, which are the mostly used antimetabolic agents. pyrazoles share their structural design with several drugs, and biologically dynamic compounds exhibit activities such as anti-inflammatory, anticancer, and antihypertensive. 1−3 our group has actively embarked on a program for the development of new methods for the synthesis of 5-aminopyrazoles as potential bioactive agents. 4,5 5-aminoyrazoles have been used to synthesize numerous other fused heterocycles. pyrazolo [1,5-a] pyrimidines, in particular, have shown valuable pharmaceutical uses including various antiviral, antimicrobial, and antitumor activities. 6−11 some compounds comprising this scaffold are official and commercialized drugs, for example, zaleplon (a), lorediplon (b), indiplon (c), anagliptin (d), and ocinaplon (e) (figure 1 ). we have recently reported different successful synthetic methods to prepare pyrazolo [1,5-a] pyrimidines, which found application and appear to constitute new classes of antimetabolic agents. 12−15 pyrazolo [5,1-c] triazines synthesis of 3-ethoxy-5-phenyl include a large number of compounds with broad biological activities. the pyrazolo [5,1-c] triazines are well known as the most potent scaffold for synthesizing potential therapeutics. the applications of these ring systems as kinase inhibitors are growing and are very successful in the last few years. 16−19 we have recently reported different successful synthetic methods to prepare pyrazolotriazines, which found application and appear to constitute new classes of antimetabolic agents. 20, 21 one of our previously reported series of novel 5-aminopyrazoles f and g ( figure 2 ) was used by others as a starting material for the synthesis of pyrazolopyrimidines. 22−27 studies revealed that 5-aminopyrazoles act as a building block for various functionalized pyrazolopyrimidines as purine analogs. 28−37 these interesting results have promoted our research group to explore other synthetic methods for the preparation of 5-aminopyrazoles for synthesizing pyrazolotriazines and pyrazolopyrimidines and investigating their use as antiviral agents in chemotherapy. in light of these results and as part of our program directed toward the preparation of potential antiviral antibiotics, the present paper deals with a novel synthesis of novel benzamide-based 5-aminopyrazoles and their use in synthesizing pyrazolo[5,1-c] triazine and pyrazolo[1,5-a]pyrimidine ring systems using innovative synthetic approaches. chemistry. it has been found that benzoyl isothiocyanate reacted with malononitrile in potassium hydroxide−ethanol with heating to give the corresponding stable potassium 2cyano-ethylene-1-thiolate salt 2. 5-aminopyrazole 4 was prepared by alkylation of the potassium 2-cyano-ethylene-1thiolate salt 2 with an alkyl halide at room temperature to offer n-(2,2-dicyano-1-(alkylthio)vinyl)benzamide 3 followed by a reaction with hydrazine hydrate by refluxing ethanol containing a catalytic amount of piperidine (scheme 1). the structures of 3 and 4 were established on the basis of their elemental analysis and spectral data (ir, 13 c nmr, 1 h nmr, and ms). structure 4 was confirmed by its mass (m/z 227), which agrees with its molecular formula c 11 h 9 n 5 o. the 1 h nmr spectrum of compound 4 revealed a broad singlet at 5.37 ppm for the nh 2 group, a multiplet at a range of 7.50−8.09 ppm for the phenyl moiety, and two broad singlets at 11.73 and 12.33 ppm assigned to two nh groups. the reactivity of 5-aminopyrazole derivative 4 to form the diazonium salt 5 was studied, and the latter was coupled with malononitrile or ethyl cyanoacetate in sodium acetate/etoh to give the corresponding pyrazolotriazines 6a,b (scheme 2). the structures of the resultant n-(pyrazolo [5,1-c] [1, 2, 4 ]triazin-7-yl)benzamides 6a,b were confirmed according to their spectral data. thus, the 1 h nmr spectrum for compound 6a revealed a multiplet at a range of 7.46−8.19 ppm for the presence of the phenyl group, a broad singlet at 3.60 ppm for the nh 2 group, and a broad singlet at 10.47 ppm for an nh group. it was found that the 5-aminopyrazole 4 reacts with the sodium salts of (hydroxymethylene)cycloalkanones 7a, b in the presence of piperidine acetate−acetic acid to give an adduct, where the structure of 8a, b was established. the reaction begins with an initial nucleophilic attack from the external amino group to the formyl group followed by cyclization and then removal of one molecule of water to produce the angular tricyclic compounds 8a,b. in the presence of an acid medium, first, the protonation of the ring nitrogen occurs, which is the most nucleophilic center 38−43 in compound 4, which directs the exocyclic amino group to attack the unhindered formyl group of 7 to yield compounds 8a,b (scheme 3). the 1 h nmr spectrum of 8a showed the existence of a signal at δ 8.80 ppm assigned to a pyrimidine-h proton. the 5-aminopyrazole 4 reacted with arylmethylene malononitriles 9 with piperidine as a catalyst to give the pyrazolo[1,5-a]pyrimidines 10a−c. the reaction proceeded by michael addition of the exocyclic amino group of 4 to the double bond of 9 followed by cyclization through the addition of the ring nh to the cyano group to give the pyrazolo[1,5a]pyrimidines 10 (scheme 3). the structures of 10a−c were confirmed by 1 h nmr, which revealed for compound 10b a singlet at 3.88 ppm assigned to the och 3 group, a singlet at 9.18 ppm assigned to the nh 2 group, a multiplet at a range of 7.15−8.23 ppm for aromatic protons, and a broad singlet at 12.19 ppm to indicate the presence of the nh group. the reactivity of diketones and keto esters such as acetyl acetone 11a and ethyl acetoacetate 11b with 5-aminopyrazole 4 was studied through a reaction with piperidine and boiling ethanol to afford the corresponding pyrazolo[1,5-a]pyrimidines 12a,b. an attempt to react benzoyl isothiocyanate with ethyl cyanoacetate in koh−etoh with heating followed by alkylation with methyl iodides at room temperature has resulted in the formation of the corresponding (e)-ethyl 3benzamido-2-cyano-3-(methylthio)acrylate 14. the reaction of (e)-ethyl 3-benzamido-2-cyano-3-(methylthio)acrylate 14 with hydrazine was investigated. the reaction between 14 and hydrazine gave a product whose mass spectra were not consistent with the proposed pyrazole structure 16, and other spectroscopic measurements did not allow us to unambiguously identify the product, and thus, the x-ray crystal structure was determined as shown in figure 3 , 44 confirming the exclusive presence of the triazole derivative 19 as the sole product in the solid state. the formation of 19 from the reaction of 14 and hydrazine is suggested to proceed via initial addition of basic nitrogen in hydrazine to the double bond of 14 followed by the formation of the adduct 15 and elimination of ethyl cyanoacetate. the adduct 15 leads to the formation of the favored, kinetically and thermodynamically controlled 3ethoxy-5-phenyl-1h-1,2,4-triazole product 19 (scheme 4). the 1 h nmr spectra of the product 19 revealed the presence of an ethoxy group as a broad singlet at δ1.37 ppm assigned to the ch 3 group and a broad singlet at δ 4.34 ppm assigned to the ch 2 group, a multiplet at δ 7.47−7.93 ppm assigned to the phenyl group, and a triazole ring nh at δ 13.72 ppm. the potassium ethylenethiolate salts 2 and 13 reacted with tetra-o-acetyl-glucopyranosyl bromide 20 at room temperature and in ethanol to give with a high yield the corresponding sglucosides 21a,b, respectively. the chemical structures of the prepared compounds 21a,b were confirmed by elemental analyses and spectroscopy ( 1 h nmr and ir) studies. for example, the 1 h nmr spectrum of compound 21b showed an anomeric proton at δ 6.10−6.12 ppm as a doublet, and the coupling constant (j 1′,2′ = 9.2 hz) proved h-1′to be transdiaxial to h-2′. 45−50 the signals resonating at 3.92, 3.99, 4.04, 4.89, 5.03, and 5.61 ppm are assigned to six glucose protons, and the four singlets appearing from δ 1.59 ppm to 2.00 ppm are assigned to four acetyl groups. the signal for the c-1′ atom in the 13 c nmr spectrum of 21b appeared at δ 80.31, and the signals for c-6′, c-4′, c-2′, c-3′, and c-5′ appeared at δ 61.50, 68.76, 69.46, 73.47, and 79.82 ppm, respectively. an attempt to remove the protection groups at 21 by methanol−ammonia did not result in the formation of the corresponding free glycosides. the structure 21 was suggested to be present in the e form and not in the z form, which was demonstrated by the reaction of compounds 21 with hydrazine at room temperature in piperidine−ethanol to give the corresponding 5-aminoprazole 4. the structure of 4 was confirmed on the basis of elemental analysis and spectral data. antiviral activity. the antiviral activity was measured for the synthesized compounds with respect to the h5n1 influenza virus strain a/egypt/m7217b/2013 using mtt 50 ) and plaque reduction assays 52 exploring the cytotoxicity and inhibition percentage values, respectively. the anti-influenza drug zanamivir was used as a positive control. 53 antiviral bioassay data (see table 1 and figure 4 ) indicated that most of the compounds demonstrated a dosedependent inhibition behavior. based on the u.s. national cancer institute (nci) and geran protocol, 54,55 the ld 50 values indicate that most tested compounds, especially compound 3b, are safe for healthy cells, while the plaque reduction assay showed that compounds 3b, 4, and 10b have higher therapeutic indices compared to the other synthesized compounds. in particular, compound 3b exhibited the highest antiviral activity at the given concentrations with percentage of virus reduction reaching 85% at a concentration of 0.25 μmol/ ml. on the other hand, compounds 3c, 10c, 12a, 19, 21a, and 21b demonstrated a moderate anti-h5n1 activity with viral inhibition over 65 percent. in general, it has been observed that compound 3b with an ethylthio group at a concentration of 0.25 μmol/ml was more active compared to 3a and 3c with methylthio and benzylthio groups, respectively. additionally, conversion of n-(2,2-dicyano-1-(alkylthio)vinyl)benzamides 3a and 3c to the 5-aminopyrazole 4 relatively enhanced the activity at the same concentration. the presence of pyrazolotriazine moieties in compounds 6a, b decreases the activity compared to the 5-aminopyrazole 4. compound 10b with a pyrazolo[1,5-a]pyrimidine scaffold and amino-, cyano-, and methoxy phenyl groups was found to have the highest potency in the series at a concentration of 0.125 μmol/ml (83%). unexpectedly, the synthesis of derivatives (21a and 21b) with a sugar moiety had no significant effect on the antiviral activity (scheme 5), where the study showed that n-(2,2-dicyano-1-(alkylthio)vinyl)benzamide 3b with s-ethyl seemed to be more active than the corresponding derivatives of acyclic thioglucosides 21a,b. synthesis of n-(5-amino-3,6-dicyano-7-arylpyrazolo[1,5a]pyrimidin-2-yl)benzamides 10a−c. method: a mixture of compound 4 (2.27 g, 0.01 mol) and benzylidene propane-dinitrile 9a (1.54 g, 0.01 mol), (4-methoxy benzylidene) propanedinitrile 9b (1.84 g, 0.01 mol), or (4-chlorobenzylidene) propanedinitrile 9c (1.88 g, 0.01 mol) was refluxed for 5 h in ethanol (20 ml) containing drops of piperidine. the reaction mixture was allowed to cool to room temperature. the precipitate was collected by filtration and crystallized from dmf. n-(5-amino-3,6-dicyano-7-phenylpyrazolo[1,5-a]pyrimidin-2-yl)benzamide 10a. yellow powder; (dmf); yield 55%; mp>300°c; 1 synthesis of (e)-ethyl 3-benzamido-2-cyano-3-(methylthio)acrylate 14. method: a mixture of ethyl cyanoacetate (1.13 g, 0.01 mol) and benzoyl isothiocyanate (1.63 g, 0.01 mol) was heated for 30 min in ethanol (25 ml) in the presence of potassium hydroxide (0.56 g, 0.01 mol). after cooling, methyl iodide (1 mmol) was added, and the mixture was stirred overnight. the resulting solid product was collected by filtration and recrystallized from ethanol. mtt cytotoxicity assay (ld 50 ). the samples were diluted to 10-fold serially using the dulbecco's modified eagle's medium (dmem). 51−56 then, a preparation of test compound stock solutions was carried out in diluted dmso with distilled water (10%). cytotoxicity testing of the extracts was performed with madin−darby canine kidney (mdck) cells using the method of mtt (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) 51 with some minor modifications. cells were placed in 96-well plates and incubated for 1 day at room temperature in 5% co 2 . after 1 day, the cells were treated with different concentrations of the tested compounds. the supernatant was discarded after further 24 h, and the cell monolayers were washed with a solution of phosphate saline (pbs). an mtt solution was added to each well and incubated at room temperature for 4 h, and then, a medium suction was applied. the formazine crystals formed were then dissolved in 200 μl of acidified isopropanol. the absorbance measurement of the formazine solutions at λ max of 540 nm with 620 nm as the reference wavelength was performed using a multiplate reader. then, the determination of the cytotoxicity compared to the untreated cells was performed using the following equation. the plot of cytotoxicity percentage versus concentration of sample was used to calculate the concentration, which showed 50% cytotoxicity (ld 50 ). plaque reduction assay. the plaque reduction assay was performed according to the method described by hayden et al. 52 in a plate of six wells in which the mdck cells (105 cells/ ml) were cultivated for 1 day at room temperature. 51−56 the virus a/chicken/7217b/1/2013 (h5n1) was diluted to give 105 pfu/well and mixed with the tested samples and incubated for half hour at room temperature before being added to the cells. the growth medium was removed from the cell culture plates, and mixtures of virus−cpd or virus−extract and virus−oseltamivir (100 μl/well) were inoculated (100 μl/well). after 1 hour of contact time for viral uptake, 3 ml of dmem was added with 2% agarose on the monolayer cell, and platelets were left to harden and were incubated at room temperature until viral plaques form (3−4 days). formalin (10%) was added for 2 h, and then, the plates were stained with 0.1% crystal violet in h 2 o. control wells were included where untreated virus was incubated with mdck cells, and at the end, plaque was counted and the percentage decrease in plaque formation compared to control wells was calculated as follows: the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02675. all spectral analysis data such as ir, 1 h nmr, and 13 c nmr spectra for the newly synthesized compounds (pdf) yellow powder; (ethanol); yield 53%; mp 64°c; 1 h nmr (400 mhz, dmso-d 6 ): δ 1.36−1.41 (t, 3h, ch 3 ), 2.39 (s, 3h method: hydrazine hydrate (0,50 g, 0.01 mol) was added to a solution of (e)-ethyl 3-benzamido-2-cyano-3-(methylthio) acrylate (14) (2.90 g, 0.01 mol) in ethanol (20 ml) in the presence of drops of piperidine. the mixture was heated under reflux for 2 h and then poured onto ice. the solid product was filtered off, dried synthesis of acyclic thioglucosides 21a,b. method: a mixture of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and benzoyl isothiocyanate (1.63 g, 0.01 mol) was heated for 10−20 min in ethanol 11 g, 0.01 mol) was added. the reaction mixture was stirred overnight at room temperature. the resulting solid was collected by filtration and was separated from traces of the reactant by preparative thinlayer chromatography using dcm as an eluent where compounds 21a yellow powder; yield 84%; mp>300°c; 1 h nmr (400 mhz, dmso-d 6 ): δ 1.18−1.27 (t, 3h, ch2-ch 3 ), 1.59−2.00 (4 s, 12h synthesis of new pyrazole derivatives and their anticancer evaluation antimicrobial screening and one-pot synthesis of 4-(substituted-anilinomethyl)-3-(2-naphthyl)-1-phenyl-1h-pyrazole derivatives fused pyrazole derivatives as kinase inhibitors, wo patent novel synthesis and biological evaluation of the first pyrazole thioglycosides as pyrazofurin analogues novel synthesis of new pyrazole thioglycosides as pyrazomycin analogues pyrimidine ttk inhibitors: cfi-402257 is a potent, selective, bioavailable anticancer agent design and synthesis of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety and evaluation of their antitumor activity in vitro and in vivo regioselective synthesis of 1-and 4-substituted 7-oxopyrazolo[1,5-a]pyrimidine-3-carboxamides ,5-a]pyrimidine-based inhibitors of hcv polymerase discovery of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives as c-src kinase inhibitors for the treatment of acute ischemic stroke reactions with 3,5-diaminopyrazoles: new routes to pyrazolo[1,5-α]pyrimidines novel synthesis of mercaptopurine and pentaaza-as-indacene analogues: reaction of [bis(methylthio)methylene]malononitrile and ethyl-2-cyano-3,3-bis(methylthio)acrylate with 5-aminopyrazoles design, synthesis, docking, and antimicrobial evaluation of some novel pyrazolo[1,5-a] pyrimidines and their corresponding cycloalkane ring-fused derivatives as purine analogs potential purine analogue antagonists: synthesis of novel cycloalkane ring-fused pyrazolo[1,5-a]pyrimidines 1,3,5-triazine-based analogues of purine: from isosteres to privileged scaffolds in medicinal chemistry synthesis and biological activity yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of tyrosine threonine kinase ttk reactions of chlorocarbonyl isocyanate with 5-aminopyrazoles and active methylene nitriles: a novel synthesis of pyrazolo[1,5-a]-1,3,5-triazines and barbiturates the reaction of dimethyl ncyanodithioiminocarbonate with amino-and oxo-azoles: a new general synthesis of methylsulfanylazoloazines 5-amino-3-anilino-n-(chlorophenyl)-1h-pyrazole-4-carboxamide ethanol solvate novel synthesis of fluorinated cyanoketene n,s-acetals and their conversions to fluorinated pyrazole derivatives. phosphor. sulfur silicon relat novel cyanoketene n,s-acetals and pyrazole derivatives using potassium 2-cyanoethylene-1-thiolates direct route to a new class of acrylamide thioglycosides and their conversions to pyrazole derivatives synthesis of some novel α-cyanoketene s,s-acetals and their use in heterocyclic synthesis potassium 2-cyanoethylene-1-thiolate derivatives: a new preparative route to 2-cyanoketene s,n-acetals and pyrazole derivatives synthesis of some pyrazolopyrimidines as purine analogues synthesis and anti-tumor activities of some new pyridines and pyrazolo[1,5-a]pyrimidines cyanoacetanilides intermediates in heterocyclic synthesis. part 5: preparation of hitherto unknown 5-aminopyrazole and pyrazolo[1,5-a]pyrimidine derivatives containing sulfamoyl moiety synthesis of some new purine-related compounds: regioselective one-pot synthesis of new tetrazolo[1,5-a]pyrimidine, pyrazolo[1,5-a]pyrimidine and pyrimido[1,6-a]-pyrimidine derivatives novel synthesis of some new pyrimido[1,6-a]pyrimidine and pyrazolo[1,5-a]pyrimidine derivatives synthesis and in vitro cytotoxic activity of novel pyrazolo[1,5-a]pyrimidines and related schiff bases synthesis, characterization, and cytotoxicity of some new 5-aminopyrazole and pyrazolo[1,5-a]pyrimidine derivatives synthesis and antitumor activity of some new pyrazolo[1,5-a]pyrimidines synthesis, structural elucidation, and in vitro antitumor activities of some pyrazolopyrimidines and schiff bases derived from 5-amino-3-(arylamino)-1h-pyrazole-4-carboxamides synthesis and in vitro anticancer activity of pyrazolo synthesis of structurally related purines: benzimidazo[1,2-a]pyridines, benzimidazo-[1,2-c]-pyrimidines, and pyrazolo-[1,5-a]pyrimidines 1,2,3,4-tetrahydrobenzimidazo[2,1-b]-quinazoline 1,2-dimethylpyrido[1,2-a]benzimidazole-4-carbonitrile antimetabolites : a novel synthesis of nonclassical condensed carbocyclic purine analogues. egypt novel purine thioglycoside analogs: synthesis, nanoformulation and biological evaluation in in vitro human liver and breast cancer models antimetabolites: design, synthesis, and cytotoxic evaluation of novel dihydropyridine thioglycosides and pyridine thioglycosides crystal structure of 1-amino-2-oxo-2,5,6,7,8,9-hexahydro-1h-cyclohepta new synthetic strategies for acyclic and cyclic pyrimidinethione nucleosides and their analogues design, synthesis, molecular docking and anti-hepatocellular carcinoma evaluation of novel acyclic pyridine thioglycosides a direct route to a new class of acrylamide thioglycosides rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays plaque inhibition assay for drug susceptibility testing of influenza viruses synthesis and screening of some novel fused thiophene and thienopyrimidine derivatives for anti-avian influenza virus (h5n1) activity synthesis and anti-proliferative activity of novel oxepin-annulated coumarins protocols for screening chemical agents and natural products against animal tumors and other biological systems toward developing therapies against corona virus: synthesis and anti-avian influenza virus activity of novel cytosine thioglycoside analogues key: cord-255862-84u3c33m authors: kim, ji won; ha, thi-kim-quy; cho, hyomoon; kim, eunhee; shim, sang hee; yang, jun-li; oh, won keun title: antiviral escin derivatives from the seeds of aesculus turbinata blume (japanese horse chestnut) date: 2017-07-01 journal: bioorganic & medicinal chemistry letters doi: 10.1016/j.bmcl.2017.05.022 sha: doc_id: 255862 cord_uid: 84u3c33m abstract porcine epidemic diarrhea virus (pedv) causes severe diarrhea and high fatality of piglets, influencing the swine industry. japanese horse chestnut (seed of aesculus turbinata) contains many saponin mixtures, called escins, and has been used for a long time as a traditional medicinal plant. structure-activity relationship (sar) studies on escins have revealed that acylations at c-21 and c-22 with angeloyl or tigloyl groups were important for their cytotoxic effects. however, the strong cytotoxicity of escins makes them hard to utilize for other diseases and to develop as nutraceuticals. in this research, we investigated whether escin derivatives 1–7 (including new compounds 2, 3, 5 and 6), without the angeloyl or tigloyl groups and with modified glycosidic linkages by hydrolysis, have pedv inhibitory effects with less cytotoxicity. compounds 1–7 had no cytotoxicity at 20μm on vero cells, while compounds 8–10 showed strong cytotoxicity at similar concentrations on pedv. our results suggest that escin derivatives showed strong inhibitory activities on pedv replication with lowered cytotoxicity. these studies propose a method to utilize japanese horse chestnut for treating pedv and to increase the diversity of its bioactive compounds. from the middle east. until the patient was diagnosed as infected with mers-cov, 29 secondary infections occurred by visiting different clinics, resulting in 186 confirmed cases. 5 coronaviruses at a molecular level have important features such as high rates of rna recombination, extraordinarily large rna genomes and rapid stability after transmission to other species, and leading to genetic diversity, unlike other enveloped rna viruses. 6 pedv of family coronaviridae shares phylogenetically common features with other coronaviruses. pedv causes severe diarrhea, dehydration, vomiting in pigs of all ages, and high mortality of piglets, resulting in tremendous financial loss. 7 thus, these results imply the necessity of studying the characteristics of coronaviruses and discovering active drugs to prevent the fast and extensive spread of coronaviruses. aesculus l (hippocastanaceae) contains 12 species of deciduous trees and has been cultivated as pharmaceutical crops for the production of standardized therapeutics extracts of escins in china. the common name ''horse chestnut" came from the uses of seeds for horses to treat overexertion or coughs, and it has been used as therapeutics purposes for anti-fever. 8 japanese horse chestnut (aesculus turbinata) is a medicinal plant widely distributed in japan and also has a small amount of cultivation in korea and china. 9 the seeds, which a large amount of escins were reported as its constituents, 9 have been used for diverse biological activities including anti-inflammatory, anti-obesity, hypoglycemic, and anti-cancer effects. [10] [11] [12] [13] [14] escins were also reported to possess strong antiviral effects against sars-cov with an ec 50 of 6.0 lm (si value of 2.5) 15 and against anti-hiv-1 protease. 9 however, the industrial utilization of escins for application to diseases and development as nutraceuticals has been limited to date due to their strong nonspecific cytotoxic effects. these reports prompted us towards the development of safer escin derivatives with anti-cov activities. previous studies on structure-activity relationship with escins suggested that acylation at c-21 and c-22 was necessary for the cytotoxic effects. [16] [17] [18] [19] [20] the cytotoxicity can be enhanced with methylation at c-24 and a free hydroxyl at c-16 at oleanane-type structure and altered by the site of glycosides. 14, 21 thus, alkaline and acid hydrolysis of escins was applied to detach acyl moieties at c-21 and c-22, and provide varieties of sugar moieties at c-3. in this research, we reported ten compounds (1-10), including four new compounds 2, 3, 5 and 6, from the extract of a. turbinate after the two-step hydrolysis. we also measured their antiviral activities using the pedv assay with isolated compounds and each fraction for safer utilization of japanese horse chestnut. the air-dried seeds of a. turbinata were extracted and separated through column chromatography using silica gel, rp-c 18 and preparative hplc to afford ten compounds, including four new (2, 3 and 5, 6) and six known (1, 4 and 7-10). 22 11 . the ir spectrum showed absorption due to hydroxyl table 1 1 h nmr and 13 c nmr spectroscopic data of compounds 2, 3, 5, and 6 in pyridine-d 5 . table 1) . the 1 h and 13 c nmr data of 2 were consistent with those of escinidin (1), except for the chemical shift of c-3 and the presence of one b-d-glucopyranosiduronic acid moiety in 2 (fig. 1) . these results indicated the attachment of b-d-glucopyranosiduronic acid to c-3 (d c 89.0). the linkage position of this b-dglucopyranosiduronic acid was confirmed by the hmbc experiment from the correlation from h-1 0 (d h 5.03) to c-3 (d c 89.1) ( fig. 2a) . therefore, the structure of 2 was elucidated as (3b,16a,21b,22a)16 . the 13 c nmr spectrum of 3 was very similar to that of 2, apart from the presence of one b-d-glycopyranosyl moiety in 3 (table 1) table 1 ). the hmbc correlation between h-3 (d h 3.63) and c-1 0 (d c 106.7) confirmed the position of b-d-glucopyranosiduronic acid. the relative configuration of 5 was investigated by analysis of its roesy spectrum (fig. 2b) . correlations 4 .64) were observed in the roesy data, implying that all these protons were on the same side of the molecule. the relative configuration of compound 5 remained unaltered even after the two-step reaction except for the deacylation and the cleavage of the glucose linkage. therefore, the structure of 5 was identified as (3b,16a,21b,22a)-16,21,22,24,28-pentahydroxyolean-12-en-3-o-b-d-glucopyranosiduronic acid. 23 compound 6 by analysing the roesy data of compound 5, we also confirmed that the overall skeleton and relative configurations of new compounds 2, 3, 5, and 6 were identical with the escin series, after a two-step hydrolysis. six known compounds 1, 4, and 7-10 were determined as protoaescigenin (1), 24 escinidin (4), 25 aesculuside b (7), escin ia (8), escin ib (9), 13 and isoescin ia (10) 26 by comparison with literature data. the cytotoxicity assay 27 was done at a concentration of 10 lg/ ml to compare the cytotoxic effects of the total extract and partitioned fractions before and after a two-step hydrolysis (fig. 3a) . the n-buoh fraction, containing a large amount of escins, showed strong cytotoxicity compared to fractions obtained after the twostep hydrolysis. interestingly, compounds 1-7 isolated from the fraction with the two-step hydrolysis were evaluated to have much lower cytotoxic effects than compounds 8-10 from the n-buoh part at concentration of 20 lm (fig. s22) . additionally, dosedependent cytotoxic effects of compounds 8-10 were ascertained at concentrations of 2, 5 and 10 lm (fig. s23) . the n-buoh and the other fractions from a two-step hydrolysis were evaluated for their pedv inhibitory activities with 6-azauridine as positive control at 1, 2, 5, and 10 lg/ml (fig. 3b) . 28 up to 2 lg/ml, both fractions showed similar and mild inhibitory effects on pedv replication, proving the original horse chestnut's antiviral activities. the fraction after a two-step hydrolysis inhibited pedv replication in a dose-dependent manner without cytotoxicity. the n-buoh fractions above 5 lg/ml, which are expected to contain many escins, exhibited poor cell viability because of strong cytotoxic effects, even if it could show better pedv inhibitory effects than the fraction from a two-step hydrolysis. based on these data, the ten purified oleanane triterpenoids (1-10) were evaluated for their pedv inhibitory effects with the same methods (fig. s24) . as compounds 8-10 showed strong cytotoxic effects on vero cells at 20 lm, their pedv inhibitory activities were evaluated at a concentration of 2 lm. compounds 1-7 were tested at a concentration of 20 lm to compare their inhibitory effects on pedv replication, providing less cytotoxicity in relatively high concentrations. compound 4 showed the strongest inhibitory activity among the ten compounds 1-10. additionally, compounds 4-6 exhibited concentration-dependent inhibition of pedv replication at concentrations of 10, 20 and 40 lm, indicating improved cell viability from the two-step hydrolysis (fig. s25 ). based on cytotoxicity and cpe assays, structure-activity relationships (sars) were studied. isolated compounds 1-10 after the two-step hydrolysis suggested the presence of three important groups: (1) acylation at c-21, c-22 or c-28 (1-7 and 8-10), (2) methylation at c-24 (1-3 and 4-6), group 1 (1-7 and 8-10) indicated that deacylation at c-21 could improve the cell viability (figs.3a and s22) . the pedv inhibitory effects of group 2 (1-3 and 4-6) demonstrated that methylation at c-24 could reduce antiviral activity. and (4 and 5-7) ] showed that the absence of glycosidic linkage also improved the antiviral effects (fig. s24) . during the pedv replication, two key structural proteins, spike and nucleocapsid proteins, take part in important roles. 29 the spike protein regulates the entry stage of the virus 30 and binding of nucleocapsid protein to viral rna is crucial for viral transcription. 31 following the data of the cytotoxicity and cpe assays (figs. s22-24), the five compounds 1 and 4-7 were selected for further evaluation. the inhibitory effects of compounds 1 and 4-7 on nucleocapsid protein synthesis at 20 lm were measured using western blot (fig. 4a ). 32 the five compounds showed moderate inhibitory effects on nucleocapsid protein synthesis, and compound 4 significantly inhibited nucleocapsid protein synthesis. thus, compound 4 was further analyzed for its effects in nucleocapsid and spike protein synthesis with western blot at concentrations of 10, 20 and 40 lm, and it was found to inhibit pedv replication in a concentration-dependent manner (fig. 4b) . on the basis of the above findings, compounds 4 and 6 were also measured with key genes and proteins crucial for pedv replication by real time qpcr (qpcr). 33 to measure the expression level of viral rna encoding nucleocapsid and spike proteins, compounds 4 and 6 were treated in vero cells at a concentration of 40 lm and total rna was extracted for reverse transcription followed by polymerase chain reaction using the primers for pedv (stable 1 ). fig. 5a shows the rna expression levels of two kinds of proteins with compounds 4, 6 and positive control. when the inhibitory effect of compound 4 was analyzed in detail at the concentrations (b) cpe inhibition assay of the n-buoh fraction and the reaction fraction at concentrations of 1, 2, 5, and 10 lg/ml. up to 2 lg/ml, the n-buoh fraction and the reaction fraction from a two-step hydrolysis showed similar activities, but at high concentrations, the n-buoh fraction showed cytotoxic effects and the reaction fraction had pedv inhibitory effects in dose-dependent manner. of 10, 20 and 40 lm, compound 4 inhibited the rna expression of nucleocapsid and spike proteins in a dose-dependent manner (fig. 5b) . on the basis of inhibition of pedv rna expression, compound 4 was further studied for its inhibitory effects on pedv replication, by performing an immunocytochemistry assay (fig. 5c ). 34 we observed green fluorescence in virus-infected cells but no signals in mock-treated cells. this result revealed that compound 4 had noticeable inhibitory effects on pedv replication in a dose-dependent manner at concentrations of 10, 20 and 40 lm. 3c-chymotrypsin-like protease (3cl protease) is vital for proteolytic processing of viral replication in coronaviruses. as escin was reported as a sars-cov 3cl protease inhibitor, 15 we performed docking modelling of compound 4 into the active site of sars-cov 3cl pro (pdb id code 3v3m). 35 the binding site was predicted by the 2d program of ds 4.0. as shown in fig. 5d , the hydroxyl group of c22 and c16 of 4 formed hydrogen bonds with the oxygen atom of the carbonyl group of glu166. additionally, the methyl group of c23 and the b ring of 4 showed hydrophobic interactions with cys145 and leu27 through their side chains. the cdocker interaction energy was calculated to be à38.63 kcal/mol. the 3cl pro binding energy value of compound 4 was unstable and weaker than that of the reference ligand 0em. however, clear key amino acid interactions of compound 4 with 3cl pro , proposed the mode of action as inhibition of 3cl protease and explained inhibitory possibility of the sars-cov of escin derivatives. this research demonstrated that including the four new compounds (2, 3, 5, and 6), ten oleanane-type triterpenoids (1-10) were isolated from the seeds of aesculus turbinata (japanese horse chestnut). the cytotoxicity of the n-buoh fraction was decreased with compounds 1-7 isolated from two-step hydrolysis. especially, two compounds 4 and 6 showed strong inhibitory activities against pedv in a dose-dependent manner. the present study proposed a way to utilize japanese horse chestnut for treating pedv with lowered cytotoxic effects and to increase the diversity of bioactive compounds. which is funded by the korean government. references 1 since the n-buoh-soluble fraction (70.8 g) contains a large amount of mixed triterpenoidal saponins called escins, this fraction was directly applied to hydrolysis reactions of two steps. acyl group hydrolysis was done at 90°c with 0.5 n naoh in 50% etoh aqueous solution for 2 h. further partial hydrolysis of the glucose moieties was also followed with 1.0 n hcl in 50% etoh aqueous solution at 90°c for 2 h. this reaction mixture was directly placed on an hp-20 cc (10 â 60 cm) to discard salt, washed with 10% etoh (3 l), and finally eluted with etoh (3 l) for the saponin fraction. the partial saponin fraction (5.59 g) was then chromatographed over an rp-c 18 cc (40-63 lm particle size) and eluted with a gradient solvent system of meoh:h 2 o (from 4:6 to 1:0), to yield five fractions (f1-f5). fraction f1 was further applied to semi-preparative hplc :40) over 25 min) resulted in the isolation of compound 4. fraction f5 was purified by preparative hplc (mobile phase mecn/h 2 o (30:70-60:40) over 25 min) to provide compound 1. compounds 8-10 were isolated from the n-buoh fraction of the dried seeds of a. turbinata extract by semi-preparative hplc using an isocratic solvent of 40% mecn 1023 cm à1 ; see table 1 for 1 h (500 mhz) and 13 c nmr 3901 (calcd for c 36 h 57 o 11 [màh] à , 665.3906). (3b,16a,21b,22a)-16,21 1028 cm à1 ; see table 1 for 1 h (500 mhz) and 13 c nmr 4434 (calcd for c 42 h 67 o 16 [màh] à , 827.4435). (3b,16a,21b,22a)-16,21 3852 (calcd for c 36 h 57 o 12 [màh] à , 681.3856). (3b,16a,21b,22a)-16,21,22,24,28-pentahydroxyolean-12-en-3-yl-o ir (kbr) mmax 3405, 2942, 1604, 1051, 1033 cm à1 ; see table 1 for 1 h (800 mhz) and 13 c nmr hresims m/z 843.4384 (calcd for c 42 h 67 o 17 vero cells (african green monkey kidney cell line cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with virus stock was maintained at à80°c before use. to assess the cell viability, a mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) assay was carried out. vero cells were seeded for 24 h in 96-well plates at 1 â 10 5 cells per well. then, the cells were exposed to different concentrations of fractions and compounds for 48 h. the final concentration of dmso was maintained at 0.05% (v/v) to avoid solvent toxicity. twenty microliters of mtt solution (2 mg/ml) was then added to cultures and incubated for 4 h pedv (0.01 moi) were inoculated onto confluent monolayers of vero cells for 2 h. the media were replaced by dmem with different concentrations of compounds aliquots of lysates were separated by 10-12% sds-page and electrophoretically transferred to pvdf membranes (pvdf 0.45 lm. immobilon-p, usa) after 24 h, total rna from the cells was isolated by the trizol method and reverse transcribed using random primer (intron biotechnology, seongman, korea) according to the manufacturer' protocol. amplifications were carried out using selective primers for pedv, which are listed in s-table 1 (supporting information), using 2 ll of cdna and maxima sybr green qpcr master mix 2x ) for 1 h. after washing three times with pbs (ph 7.4), the slides were stained with 500 nm dapi solution for 10 min at room temperature and washed with pbs (ph 8.0) three times. mounting reagent (vectashield this work was supported in part by grants from the marine biotechnology program of the ministry of oceans and fisheries (pjt200669) and the korea bioactive natural material bank supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05. 022. key: cord-300872-blycbi4u authors: saadeh, haythem a.; sweidan, kamal a.; mubarak, mohammad s. title: recent advances in the synthesis and biological activity of 8-hydroxyquinolines date: 2020-09-21 journal: molecules doi: 10.3390/molecules25184321 sha: doc_id: 300872 cord_uid: blycbi4u compounds containing the 8-hydroxyquinoline (8-hq) 1 nucleus exhibit a wide range of biological activities, including antimicrobial, anticancer, and antifungal effects. the chemistry and biology of this group have attracted the attention of chemists, medicinal chemists, and professionals in health sciences. a number of prescribed drugs incorporate this group, and numerous 8-hqbased molecules can be used to develop potent lead compounds with good efficacy and low toxicity. this review focusses on the recent advances in the synthesis of 8-hq derivatives with different pharmacological properties, including anticancer, antiviral, and antibacterial activities. for this purpose, recent relevant references were searched in different known databases and search engines, such as medline (pubmed), google scholar, science direct, scopus, cochrane, scientific information database (sid), scifinder, and institute for scientific information (isi) web of knowledge. this review article provides a literature overview of the various synthetic strategies and biological activities of 8-hq derivatives and covers the recent related literature. taken together, compounds containing the 8-hq moiety have huge therapeutic value and can act as potential building blocks for various pharmacologically active scaffolds. in addition, several described compounds in this review could act leads for the development of drugs against numerous diseases including cancer. 8-hydroxyquinoline derivatives are an important group of compounds with rich and diverse biological activities. these compounds incorporate the 8-hydroxyquinoline (8-hq) moiety, which is a bicyclic compound that consists of a pyridine ring fused to phenol, in which the hydroxyl group is attached to position 8 [1] . in this respect, the pyridine ring maintains its properties as an electron-deficient entity with a basic nitrogen. in addition, and due to the presence of the phenolic group, 8-hq displays typical phenolic properties that make it susceptible to numerous chemical reactions and structural modifications, such as electrophilic aromatic substitution, diazonium coupling, and molecular rearrangements. the close proximity of the hydroxyl group to the heterocyclic nitrogen makes 8-hydroxyquinolines good monoprotic bidentate chelating agents, which form four-and six-covalent complexes with a wide range of metal ions, including cu 2+ , zn 2+ , bi 2+ , mn 2+ , mg 2+ , cd 2+ , ni 2+ , fe 3+ , and al 3+ [2] . figure 1 is a schematic representation of the different sites of reactions of 8-hq. all of the aforementioned properties make 8-hq a privileged structure with a variety of structural modifications, possessing a rich diversity of physical, chemical and biological properties. 8-hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [3] . 8-hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of 2og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [4] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [5, 6] . on the basis of the preceding discussion, and owing to the importance of 8-hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of 8-hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of 8-hq and its derivatives, covering the period ranging from 2017 to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel 8-hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of 8-hq and its derivatives, as well as their biological activities against different diseases and disorders. due to the coronavirus 2019 (covid-19) pandemic and its devastating economic, social, and health effects, we decided to start this study with the antiviral activities of some recent 8-hq derivatives. a few publications have dealt with the antiviral activities of 8-hq and its derivatives. de la guardia et al. described the synthesis of novel 8-hydroxyquinoline derivatives (scheme 1) and investigated their activity against dengue virus [7] . these researchers prepared a number of compounds from 8-hydroxyquinoline n-oxide 2, which upon treatment with copper-catalyzed grignard reagents (rmgx; r = i-pr and i-bu) gave 2-alkyl-8-hydroxyquinoline 3. subsequent chlorination using n-chlorosuccinimide (ncs) under acidic conditions afforded the corresponding 2-alkyl-5,7-dichloro-8-hydroxyquinoline 4 in a good yield. scheme 1. synthesis of 2-isopropy, 2-isobutyl-5,7-dichloro-8-hydroxyquinoline 4. 8-hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [3] . 8-hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of 2og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [4] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [5, 6] . on the basis of the preceding discussion, and owing to the importance of 8-hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of 8-hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of 8-hq and its derivatives, covering the period ranging from 2017 to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel 8-hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of 8-hq and its derivatives, as well as their biological activities against different diseases and disorders. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted 8-hydroxyquinoline-2-carboxanilides, as shown in scheme 2 [8] . condensation of activated 8-hydroxyquinoline-2-carboxylic acid (5) with substituted aniline 6 in the presence of by phosphorus trichloride yielded the desired target compound 7 in good amounts (61-79%) . molecules 2020, 25, x for peer review 3 of 26 the antiviral activities of the two novel quinoline derivatives (r = i-pr and i-bu) 4 were evaluated in vitro against the dengue virus serotype 2 (denv2). both exhibited significant inhibitory activities against this virus. the results indicated that the iso-pr-substituted derivative exhibits a half-maximal inhibitory concentration (ic50) of 3.03 µm and a half-maximal cytotoxic concentration (cc50) of 16.06 µm, for an estimated selectivity index (si) of 5.30. on the other hand, the iso-bu derivative was also active, showing a higher si value of 39.5, with an ic50 of 0.49 µm and cc50 of 19.39 µm. the mechanism of action was also investigated and the results showed that these two derivatives are not virucidal, but appear to act at an early stage of the virus lifecycle, reducing the intracellular production of the envelope glycoprotein and the yield of infectious virions in treated and infected cells. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted 8-hydroxyquinoline-2-carboxanilides, as shown in scheme 2 [8] . condensation of activated 8-hydroxyquinoline-2-carboxylic acid (5) with substituted aniline 6 in the presence of by phosphorus trichloride yielded the desired target compound 7 in good amounts (61-79%). these prepared compounds were subjected to bioactivity screening against the highly pathogenic h5n1 avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a549 cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase highperformance liquid chromatography (rp-hplc). based on the presented results, most monosubstituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, 8-hydroxy-n-(3-nitrophenyl)quinoline-2-carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of 85.0% and 4%, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = 3-no2 and log k = ca. 0.41 showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = 3,4-cl, 3,4,5-cl, 3-cl-2-f, and 2,4-no2) showed higher inhibition of h5n1 growth and simultaneous low cytotoxicity. for example, 3-cl-2-f and 3,4,5-cl exhibited virus growth inhibition activity values of 91.2 and 9.7% and cytotoxicity values of 79.3 and 2.4%, respectively; the log k values for these derivatives were 1.44 and 1.26, respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid-19 patients and ultimately for a covid-19 vaccine, the 8-hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; these prepared compounds were subjected to bioactivity screening against the highly pathogenic h5n1 avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a549 cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase high-performance liquid chromatography (rp-hplc). based on the presented results, most mono-substituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, 8-hydroxy-n-(3-nitrophenyl)quinoline-2-carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of 85.0% and 4%, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = 3-no 2 and log k = ca. 0.41 showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = 3,4-cl, 3,4,5-cl, 3-cl-2-f, and 2,4-no 2 ) showed higher inhibition of h5n1 growth and simultaneous low cytotoxicity. for example, 3-cl-2-f and 3,4,5-cl exhibited virus growth inhibition activity values of 91.2 and 9.7% and cytotoxicity values of 79.3 and 2.4%, respectively; the log k values for these derivatives were 1.44 and 1.26, respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid-19 patients and ultimately for a covid-19 vaccine, the 8-hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent 2,6-difluoro-3-hydroxybenzamide (dfmba) [9] . the substituted 8-hq 8 was alkylated with 1,3-dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate 9. target compound 10 was obtained via alkylation of dfmba with the corresponding 8-hq mono bromide (scheme 3). molecules 2020, 25, x for peer review 4 of 26 hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent 2,6-difluoro-3-hydroxybenzamide (dfmba) [9] . the substituted 8-hq 8 was alkylated with 1,3-dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate 9. target compound 10 was obtained via alkylation of dfmba with the corresponding 8-hq mono bromide (scheme 3). scheme 3. synthesis of 2,6-difluoro-3-hydroxybenzamide-8-hydroxyquinoline derivatives. the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc190723 (11) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc190723. in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of 0.25 and 0.18 against s. aureus were observed for derivatives where r = 5-cl and 5,7-dicl, respectively. moreover, when comparing the activity of 8-hq derivatives with 8-hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new 5-amino-7-bromoquinolin-8-yl sulfonates 15 in good yield using a multistep synthesis method [10] . bromination of 8-hydroxquinoline 1 with nbromosuccinimide (nbs) in chloroform afforded 7-bromoquinolin-8-ol, which upon treatment with nano2/hcl followed by reduction with na2s2o4 in 1:1 tetrahydrofuran (thf) and water gave 5amino-7-bromoquinolin-8-ol 14. the target sulfonate derivatives 15 were obtained from 5-amino-7bromoquinolin-8-ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme 4). the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc190723 (11) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc190723. in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of 0.25 and 0.18 against s. aureus were observed for derivatives where r = 5-cl and 5,7-dicl, respectively. moreover, when comparing the activity of 8-hq derivatives with 8-hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new 5-amino-7-bromoquinolin-8-yl sulfonates 15 in good yield using a multistep synthesis method [10] . bromination of 8-hydroxquinoline 1 with n-bromosuccinimide (nbs) in chloroform afforded 7-bromoquinolin-8-ol, which upon treatment with nano 2 /hcl followed by reduction with na 2 s 2 o 4 in 1:1 tetrahydrofuran (thf) and water gave 5-amino-7-bromoquinolin-8-ol 14. the target sulfonate derivatives 15 were obtained from 5-amino-7-bromoquinolin-8-ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme 4). the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no2ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch3ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no2, r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibiotic-resistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no 2 ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch 3 ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm) . a study published by rbaa the newly synthesized sulfonate 15 derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of 22 mm compared to the reference drug (24 mm). on the other hand, derivatives with ar = 4-fph and 2-oh-5-no2ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of 22 mm and 23 mm, respectively, compared to the standard drug (24 mm), whereas the derivative with r = 4-ch3ph was potent against klebsiella pneumonia, with an inhibition zone of 25 mm compared to the standard drug (27 mm) . a study published by rbaa these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no2, r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gram-negative strains compared to the standard antibiotics used. one derivative (r = no 2 , r = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, a. baumanii, e. coli, e. cloacae, and e. cloaca, with minimum inhibitory concentration (mic) values (mg/ml) of 1 × 10 −6 , 1 × 10 −5 , 1 × 10 −5 , 1 × 10 −4 , and 1 × 10 −4 , respectively, while the mic for the standard drug was 1 × 10 −4 . derivatives with r = r = h and r = h, r = cl displayed similar activities against s. aureus, with mic values of 1 × 10 −4 . bioinformatic petra, osiris, and molinspiration (pom) analyses indicated that all compounds exhibit good bioavailability and less toxic profiles when compared with penicillin g. interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . interestingly, two research groups have reported on the hybridization of 8-hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of 5-chloro-8-hydroxyquinoline-ciprofloxacin 19 via the mannich reaction (scheme 6). thus, 5-chloro-8-hydroxyquinoline 18 reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at 75% yield [12] . the hybrid 19 was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of 4-16 µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of 0.125-0.5 µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound 21 at 96% yield through coupling of 8-hydroxyquinoline-2-carboxylic acid 20 and ciprofloxacin by using 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme 7) [13] . the hybrid was screened for cell toxicity against hela cells, a tumor cell line, primary cell cultures of mouse fibroblasts, and against non-cancerous mammalian cell line. it showed no cell toxicity at the concentrations tested (0-200 µm). then, the antibacterial activity was tested against gram-negative and gram-positive bacteria, including pathogenic bacteria present in the hospital environment that are difficult to treat (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species). the results indicated that that hybrid 21 exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of 0.0625 (mic of the standard drug = 0.125). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that 8-hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of 7-hydroxycoumarin (umbelliferone) with several heterocylic rings, including 4-and 8-hydroxyquinolines [14] . 7-hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch 2 ) n -br (n = 2,4) in refluxing acetone in the presence of k 2 co 3 -naoh (2:1). bromoalkylated coumarins were then reacted with 4-and 8-hydroxyquinoline in the presence of 1 equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at 55-80% yields (scheme 8). molecules 2020, 25, x for peer review 7 of 26 indicated that that hybrid 21 exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of 0.0625 (mic of the standard drug = 0.125). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that 8-hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of 7hydroxycoumarin (umbelliferone) with several heterocylic rings, including 4-and 8hydroxyquinolines [14] . 7-hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch2)n-br (n = 2,4) in refluxing acetone in the presence of k2co3-naoh (2:1). bromoalkylated coumarins were then reacted with 4-and 8-hydroxyquinoline in the presence of 1 equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at 55-80% yields (scheme 8). the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone-8hydroxyquinoline (n = 2), umbelliferone-8-hydroxyquinoline (n = 4), and umbelliferone-4hydroxyquinoline (n = 4) were 49-63, 57-90, and 18-41%, respectively, compared to carbendazim (95-99%) at 200 µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone-8hydroxyquinoline (n = 4) was the most active compared to the reference drug at 10.6 and 2.3 µg/ml, respectively. the rest of the 8-hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of 4 carbon atoms exhibit better antifungal the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone-8-hydroxyquinoline (n = 2), umbelliferone-8-hydroxyquinoline (n = 4), and umbelliferone-4-hydroxyquinoline (n = 4) were 49-63, 57-90, and 18-41%, respectively, compared to carbendazim (95-99%) at 200 µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone-8-hydroxyquinoline (n = 4) was the most active compared to the reference drug at 10.6 and 2.3 µg/ml, respectively. the rest of the 8-hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of 4 carbon atoms exhibit better antifungal activity than those with a spacer of 2 carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position 8 were more active than those with oh at position 4. the structure-activity relationship suggests that modification of the umbelliferone-8-hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at 200 mg/l with l. sativa, with the results showing that umbelliferone-8-hysroxyquinoline (n = 4) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel 3,4,5-trisubstituted triazole derivatives bearing 8-hydroxyquinoline 31, evaluating their antimicrobial activity [15] . synthesis was achieved by first reacting 8-hydroxyquinoline 1 with ethyl 2-chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester 26 with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea 28. ring closure using koh in refluxing ethanol led to the formation of 3-mercapto-1,2,4-triazole derivative 29. reaction of 3-mercapto-1,2,4-triazole with 2-chloro-n-(substituted (benzo)/thiazole)acetamide 30 afforded the target compounds, as depicted in scheme 9. molecules 2020, 25, x for peer review 8 of 26 activity than those with a spacer of 2 carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position 8 were more active than those with oh at position 4. the structure-activity relationship suggests that modification of the umbelliferone-8hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at 200 mg/l with l. sativa, with the results showing that umbelliferone-8-hysroxyquinoline (n = 4) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel 3,4,5-trisubstituted triazole derivatives bearing 8-hydroxyquinoline 31, evaluating their antimicrobial activity [15] . synthesis was achieved by first reacting 8-hydroxyquinoline 1 with ethyl 2-chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester 26 with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea 28. ring closure using koh in refluxing ethanol led to the formation of 3-mercapto-1,2,4-triazole derivative 29. reaction of 3mercapto-1,2,4-triazole with 2-chloro-n-(substituted (benzo)/thiazole)acetamide 30 afforded the target compounds, as depicted in scheme 9. some sulfonates reported by krishna (scheme 4) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = 2-nitro-5-hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of 10 and 13 mm, respectively, compared to fluconazole (15 mm); and against penicillium spinulosum, with inhibition zones of 12 and 10 mm, respectively, compared to fluconazole (12 mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [16] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [17] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme 10 [18] . 6-bromo-8-methoxyquinoline (1) was prepared according to a published procedure [19] . afterwards, compound 32 was subjected to a suzuki cross-coupling reaction with p-formphenylboronic (33) acid to afford synthon 34 in excellent yield. all target products (35) were synthesized via a simple and effective method using a one-pot mannich-type reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. molecules 2020, 25, x for peer review 9 of 26 some sulfonates reported by krishna (scheme 4) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = 2-nitro-5hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of 10 and 13 mm, respectively, compared to fluconazole (15 mm); and against penicillium spinulosum, with inhibition zones of 12 and 10 mm, respectively, compared to fluconazole (12 mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [16] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [17] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme 10 [18] . 6-bromo-8-methoxyquinoline (1) was prepared according to a published procedure [19] . afterwards, compound 32 was subjected to a suzuki crosscoupling reaction with p-formphenylboronic (33) acid to afford synthon 34 in excellent yield. all target products (35) were synthesized via a simple and effective method using a one-pot mannichtype reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. the cytotoxicity of these prepared compounds against esophageal (eca109) and hepatocellular (huh7) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic50 values ranging from 2.26 to 7.46 µmol/l for the two most promising derivatives containing 2-methylphenyl and 4-methylphenyl groups for r 2 and iso-propyl for r 1 ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic50 values of 16.54 and 5.27 µmol/l towards eca109 and huh7, respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. the cytotoxicity of these prepared compounds against esophageal (eca109) and hepatocellular (huh7) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic 50 values ranging from 2.26 to 7.46 µmol/l for the two most promising derivatives containing 2-methylphenyl and 4-methylphenyl groups for r 2 and iso-propyl for r 1 ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic 50 values of 16.54 and 5.27 µmol/l towards eca109 and huh7, respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic 50 values of 0.8-2.49 mg/ml, whereas the ic 50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. molecules 2020, 25, x for peer review 10 of 26 faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic50 values of 0.8-2.49 mg/ml, whereas the ic50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 the prepared compounds were then subjected to cell viability evaluation against hela, mcf-7, a-549, and mda-mb-231 (triple-negative breast cancer cell line) cell lines using the mtt assay [26] [27] [28] at a concentration of 20 mm. the results revealed that only 4 compounds designated with r = phenyl, 3,5-dimethylphenyl, 4-fluorophenyl, and 4-trifluoromethylphenyl exhibited significantly reduced cell viability percentages towards the tested cancer cell lines. the ic50 values for these compounds were between 26.30 and 63.75 mm against selected cancer cell lines, whereas the ic50 of docetaxel (positive control) was in the range of 3.37-4.46 mm. a new series of glycoconjugates composed of various sugar units (40, 41) (d-glucose or dgalactose) and 8-hydroxyquinolines (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. (figure 3 ) in moderate to excellent yields [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 c nmr, ir, and hrms. molecules 2020, 25, x for peer review 10 of 26 faydy and coworkers described the synthesis of new derivatives of 8-hydroxyquinoline (36-38) ( figure 2 ) in a multistep approach [20] according to published procedures [21] [22] [23] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the 2,2-diphenyl-1-picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic50 values of 0.8-2.49 mg/ml, whereas the ic50 value of 1-ascorbic acid was 0.1 mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. (figure 3 ) in moderate to excellent yields [24] following a published procedure [25] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including 1 h nmr, 13 (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. (42, 43) was prepared in an effective and simple method [29] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene 1,2,3-triazole linker, as shown in scheme 11. sugar derivatives of 44 and 45 were prepared according to published procedures [30] [31] [32] ; sugar was used to enhance the bioavailability and solubility of potential drugs. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r 1 = h and r 2 = oac, was the most active, with ic 50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r1 = h and r2 = oac, was the most active, with ic50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated 2-amino-4-aryl-4-pyrano[3,2-h]quinolone-3-carbonitrile (48) derivatives (scheme 12) [33] . these derivatives were prepared through interactions of various 8hydroxyquinolines (46) with α-cyanocinnamonitriles (47) . these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic50 values (mg/ml) of these compounds were in the ranges of 0.9-38. all prepared derivatives were tested against different cancer cell lines, including hela, hct 116, and mcf-7, in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound 2, designated with r = r1 = h and r2 = oac, was the most active, with ic50 values of 30.98, 22.7, and 4.12 mm against hela, hct 116, and mcf-7, respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the 1,2,3-triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated 2-amino-4-aryl-4-pyrano[3,2-h]quinolone-3-carbonitrile (48) derivatives (scheme 12) [33] . these derivatives were prepared through interactions of various 8hydroxyquinolines (46) with α-cyanocinnamonitriles (47) . these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic50 values (mg/ml) of these compounds were in the ranges of 0.9-38.2, 1.3-45.5, 0.7-44.5, and 1.23-36.7 against mcf-7, hct 116, hepg2, and a549, respectively; while the colchicine reference showed ic50 values of 6.1, 2.6, 4.6, and 3.78 mg/ml, respectively. in a similar fashion, chhabra et al. (2017) described an amberlite ira 402(oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [34] . a synthetic procedure (scheme 13) involved a condensation reaction between the amino group (nh2) in 49 and the carbonyl group in salicylic aldehyde (50), followed by intramolecular cyclization under a these prepared compounds were screened for potential anticancer activity against mcf-7, hct 116, hepg-2, and a549 using the mmt assay. structure-activity relationship (sar) results revealed that 6-chloroanalogues were the most active, whereas the 9-methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions 4, 6, and 9. the ic 50 values (mg/ml) of these compounds were in the ranges of 0.9-38.2, 1.3-45.5, 0.7-44.5, and 1.23-36.7 against mcf-7, hct 116, hepg2, and a549, respectively; while the colchicine reference showed ic 50 values of 6.1, 2.6, 4.6, and 3.78 mg/ml, respectively. in a similar fashion, chhabra et al. (2017) described an amberlite ira 402(oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [34] . a synthetic procedure (scheme 13) involved a condensation reaction between the amino group (nh 2 ) in 49 and the carbonyl group in salicylic aldehyde (50), followed by intramolecular cyclization under a microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8-hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2-benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic 50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. molecules 2020, 25, x for peer review 12 of 26 microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. scheme 13. synthesis of novel benzothiazole-quinoline derivatives. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and single-crystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell lines using mmt assay, giving ic 50 values of 21.02 and 27.73 mm, respectively [35] . molecules 2020, 25, x for peer review 12 of 26 microwave approach to form the benzothiazole (51) . the reaction between 51 and 5,7-dialkyl-8hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product 52; dibromoalkanes were employed as linkers between the two fragments, 8-hydroxyquinoline and 2benzothiazol-2-ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf-7, a549, and human ovarian carcinoma (a2780), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic50 values in the ranges of 5-19, 7-49, 10-30, and 10-38 mm against mcf-7, hela cells, a2780, and a549, respectively. in addition, the target products were 3-25-fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure 4 ) bearing three quinolinone moieties in a simple procedure that involved a reaction of 3,6-bis(bromomethyl)-2-chloroquinoline and 8-hydroxyquinoline at a ratio of 1:2 in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf-7 and hela cancer cell lines using mmt assay, giving ic50 values of 21.02 and 27.73 mm, respectively in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. in addition, shamsi and coworkers prepared 16 quinoline-based 1,3,4-oxadiazole-triazole derivatives (scheme 14) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [36] . these compounds were considered to be high-impact motifs with a wide range of biological activities [37, 38] . the synthetic strategy was based on the treatment of 1 with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under s n 2 conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced 54, which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound 55 (1,3,4-oxadiazole) after acidification. the thiol group (ar-sh) in 55 is acidic and gives the ar-s − anion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound 56. finally, reaction of various azide derivatives of 56 yielded the target derivative 57 through a (3 + 2) cycloaddition mechanism. the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic 50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic 50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8-hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a-549), hepatocellular carcinoma (hepg2), human cervical carcinoma-hpv18 (hela), and human cervical carcinoma-hpv16 (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic50 value of 5.6 mm against the a-549 cell line, which is higher than that of doxorubicin (ic50 = 1.83 mm). interestingly, this compound was not toxic towards normal cells (up to 200 mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme 15 [39] . in the first step of the synthesis, the hydroxyl group in 58 was protected by conversion into the acetyl analogue 59. then, a condensation reaction between a methyl group at position 2 of protected 8hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [40] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products 60. all prepared compounds were screened for anticancer activity against the wild-type hct 116 p53 +/+ and hct 116 p53 −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position 8 of the quinolone moiety exhibit moderate activities (4.60-25.00 mm) and (2.61-25.00 mm) towards p53 +/+ and p53 −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p53 +/+ and p53 −/− (0. 28-13.85 [41] . their synthetic procedure involved a knoevenagel condensation between malonitrile (61) and the respective aryl aldehyde 62 to afford the corresponding arylidene-malonitrile intermediates 63, as shown in scheme 16. then, the phenolate anion attacks the β-carbon via c-7 to produce an acyclic michael adduct, which undergoes cyclization reaction (6-exo-dig) and tautomerization to afford the target product 64. all prepared derivatives in the study (along with compound ly290181 used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma 518a, colon carcinoma cells ht-29, dld-1, hct 116, cervix carcinoma kb-v1 vbl , and mcf-7 tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic 50 values in nanomolar concentrations, meaning these results are even better than the activity of ly290181. two compounds designated with r = ch 3 , r 1 = r 3 = h, r 2 = r 4 = f and r = ch 3 , r 1 = r 3 = r 4 = h, r 2 = no 2 were the most active among all examined molecules. the first one had ic 50 values of 20.1 and 14 nm against mcf-7 and kb-v1 vbl , respectively. on the other hand, the second compound had an ic 50 value of 20 nm against kb-v1 vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. all prepared compounds were screened for anticancer activity against the wild-type hct 116 p53 +/+ and hct 116 p53 −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position 8 of the quinolone moiety exhibit moderate activities (4.60-25.00 mm) and (2.61-25.00 mm) towards p53 +/+ and p53 −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p53 +/+ and p53 −/− (0. 28-13.85 [41] . their synthetic procedure involved a knoevenagel condensation between malonitrile (61) and the respective aryl aldehyde 62 to afford the corresponding arylidene-malonitrile intermediates 63, as shown in scheme 16. then, the phenolate anion attacks the β-carbon via c-7 to produce an acyclic michael adduct, which undergoes cyclization reaction (6-exo-dig) and tautomerization to afford the target product 64. all prepared derivatives in the study (along with compound ly290181 used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma 518a, colon carcinoma cells ht-29, dld-1, hct 116, cervix carcinoma kb-v1 vbl , and mcf-7 tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic50 values in nanomolar concentrations, meaning these results are even better than the activity of ly290181. two compounds designated with r = ch3, r1 = r3 = h, r2 = r4 = f and r = ch3, r1 = r3 = r4 = h, r2 = no2 were the most active among all examined molecules. the first one had ic50 values of 20.1 and 14 nm against mcf-7 and kb-v1 vbl , respectively. on the other hand, the second compound had an ic50 value of 20 nm against kb-v1 vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp-2 and mmp-9 being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [42, 43] . along this line, chen and colleagues described the synthesis of two series of 8-hydroxyquinolines, as shown in schemes 17 and 18 [44] . in scheme 17, the first step involved protecting the amino group in 65 with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in 66 reacted with the amino groups in various substrates leading, to the formation of carboxamide 67. other steps involved deprotection to liberate the free primary amine 68 [45] , which matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp-2 and mmp-9 being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [42, 43] . along this line, chen and colleagues described the synthesis of two series of 8-hydroxyquinolines, as shown in schemes 17 and 18 [44] . in scheme 17, the first step involved protecting the amino group in 65 with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in 66 reacted with the amino groups in various substrates leading, to the formation of carboxamide 67. other steps involved deprotection to liberate the free primary amine 68 [45] , which reacts with 5-chloro-8-hydroxyquinoline-7-carboaldehyde or 8-hydroxycarbaldehyde through reduction amination to yield target product 69. furthermore, in scheme 18, the phenolic hydroxyl group in 70 was protected and reaction of the primary amino group in 70 with substituted carboxylic acids afforded derivative 71. finally, deprotection of the hydroxyl group yielded the target derivative 72. molecules 2020, 25, x for peer review 15 of 26 reacts with 5-chloro-8-hydroxyquinoline-7-carboaldehyde or 8-hydroxycarbaldehyde through reduction amination to yield target product 69. furthermore, in scheme 18, the phenolic hydroxyl group in 70 was protected and reaction of the primary amino group in 70 with substituted carboxylic acids afforded derivative 71. finally, deprotection of the hydroxyl group yielded the target derivative 72. ho all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic50 values (mm) were in the ranges of 1.3-10, 5.1-10, and ˃10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic50 values in the range of 0.69-22 mm. finally, the positive control, all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic50 values (mm) were in the ranges of 1.3-10, 5.1-10, and ˃10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic50 values in the range of 0.69-22 mm. finally, the positive control, all of these derivatives were screened as potential mmp-2/9 inhibitors. the results revealed that compounds (of series 1) that have substituents at c-7 on the quinolone moiety showed ic 50 values (mm) in the range of 0.81-10, while for those with substituents at c-5, the ic 50 values ranged from 5.7 to 10. on the other hand, derivatives belonging to series 2 showed ic 50 values in the range of 6.5-10 against mmp-2. as for mmp-9, using the same sequence, the ic 50 values (mm) were in the ranges of 1.3-10, 5.1-10, and >10. some selected compounds (those with substituents at c-7) were further screened against a panel of cancer cell lines (hl60, k562, kg1, a549, pc-3, and mcf-7), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic 50 values in the range of 0.69-22 mm. finally, the positive control, the hydroxamate-based mmp inhibitor nngh [46] , showed ic 50 values of 29-187 mm for antiproliferation activities against various cancer cell lines, and against mmp-2 and mmp-9 showed values of 0.0091 and 0.0088 mm, respectively. ökten and coworkers described the synthesis of 5,7-dibromo-8-hydroxyquinoline [47] in excellent yield via reaction of 8-hydroxyquinoline with two equivalents of bromine in chloroform. the target molecule exhibited ic 50 values (mg/ml) of 5.8, 17.6, 18.7, 5.4, 16.5, and >1000 against a549, fl, hela, ht29, mcf7, and hep3b, respectively. from all of these studies, one can see the importance of the 8-hq moiety in potential anticancer drugs. in addition, some of the prepared compounds could be leads towards the development of potent and safe drugs. however, more work is required in this category, which could involve the use of animals and possibly human subjects to evaluate the efficacy and safety profiles of the prepared compounds. alzheimer's disease (ad), characterized by a loss of cognitive ability and severe behavioral irregularities, is a chronic neurodegenerative disorder. it is most common among the elderly, and can be described as an irreversible brain disorder that breaks down memory and reduces the ability of a patient to carry out simple mental and cognitive functions, such as comprehension, solving simple problems, and trivial calculations. this disease is becoming a universal health problem, and it can eventually lead to death [48] . statistics indicate the presence of approximately 2.5 to 4.0 million alzheimer's disease patients in the united states, and 17 and 25 million worldwide [49] . published research findings indicate that cholinergic dysfunction could be associated with selective and irreversible deficiency of the neurotransmitter acetylcholine, which is controlled by hydrolysis of acetylcholine via acetylcholinestrase (ache) and butyrylcholinestrase (bche). additionally, it was suggested that ache predominates in a healthy brain, whereas bche is considered to play a minor role in regulating the brain's ach levels [49] . the approved prescribed commercial drugs for the treatment of ad, which provide slight improvements in memory, include donepezil, rivastigmine, and others; their action is based on the inhibition of acetylcholinesterase. it is also worth mentioning that other factors contribute to ad, such as β-amyloid (a β) deposits and oxidative stress. in this respect, several studies have shown that levels of redox-active metal ions, including cu 2+ and zn 2+ , are observed in the brains of ad patients [50] . these metal ions can interact with β-amyloid peptides to form insoluble plaques [51, 52] . in the search for potent buche and ache inhibitors, hirbod et al. (2017) designed and prepared eight novel compounds incorporating coumarin and 8-hydroxyquinoline moieties (73), as shown in the [53] . these researchers used various dibromoalkanes (n = 3-5) as cross-linkers between 8-hydroxyquinoline and coumarin rings in the presence of an aprotic solvent (n,n-dimethylformamide). in addition, the activity levels of these prepared compounds were evaluated against buche and ache using ellman's method. the results demonstrated that some of the prepared compounds exhibited potent ache and buche inhibition activities, with half-maximal inhibitory concentration (ic 50 (74 and 75) , as shown in figure 5 [54] . the target compounds were prepared by chloromethylation of 8-hq to give 5-chloromethyl-8-hydroxyquinoline. then, the former compound was reacted with tert-butylpiperazine-1-carboxylate followed by trifluoroacetic acid to remove the protected boc group. finally, the resulting compound was reacted with the appropriate cinnamic or hydroxycinnamic acids, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc). in this context, β-amyloid (aβ) significantly contributed to the progression of alzheimer's disease (ad), where elevated levels of aβ have been detected in the brains of ad patients. moreover, aβ a aggregation can be clearly observed in the presence of cu 2+ , zn 2+ , and fe 2+ ions, since these ions can readily bind to aβ through some of its specific residues. in addition, aβ can aggregate by itself. th prepared target compounds were tested for their inhibition of aβ aggregation using the thioflavon t-binding assay [55, 56] . furthermore, chelating studies of cu 2+ , zn 2+ , fe 3+ , and fe 2+ were conducted using the former prepared derivatives by means of a uv-vis spectrophotometer. the results revealed that the compound designated with r 1 = h, r 2 , r 3 = och 3 showed the maximum percentage inhibition of aβ 1→42 aggregation of 65.82% with an ic 50 of 5.64 mm compared to resveratrol, which showed corresponding values of 51.74% and 12.43 mm. in addition, the previous compound was selected as a representative to examine its metal chelation behavior; it exhibited significant activity in this context, producing even better results than clioquinol. molecules 2020, 25 [57] . synthesis of these compounds involved protection of the phenolic group in 76 using boc group to allow the reaction of the primary aromatic amine in 77 with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group 78. in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt 79. regarding scheme 20, the phenoxide anion in 80 was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in 81 was then subjected to pinnick oxidation using seo2 to afford the corresponding acid 82. reaction of the carboxylic acid group in 82 with thionyl chloride and then with 3-(cyclopentyloxy)-4-methoxyaniline or 3,4-dimethoxyaniline afforded 83 upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product 84. on the other hand, synthesis of compound 87 was achieved by converting 1 into tert-butyldimethylsilyl (tbs)-protected 2-aminoquinolin-8-ol (85). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh4oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative 86 was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative 87 (scheme 21). compounds 79, 84, and 87 are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase 4d (pde4d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde4d is involved in the process of longterm potentiation and memory consolidation. one of the derivatives of 79, for which x = h, r 1 = cf2h, [57] . synthesis of these compounds involved protection of the phenolic group in 76 using boc group to allow the reaction of the primary aromatic amine in 77 with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group 78. in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt 79. regarding scheme 20, the phenoxide anion in 80 was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in 81 was then subjected to pinnick oxidation using seo 2 to afford the corresponding acid 82. reaction of the carboxylic acid group in 82 with thionyl chloride and then with 3-(cyclopentyloxy)-4-methoxyaniline or 3,4-dimethoxyaniline afforded 83 upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product 84. on the other hand, synthesis of compound 87 was achieved by converting 1 into tert-butyldimethylsilyl (tbs)-protected 2-aminoquinolin-8-ol (85). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh 4 oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative 86 was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative 87 (scheme 21). during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (± 0.44) × 10 6 cm s -1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s -1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) x 10 6 cm s -1 , respectively. during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (± 0.44) × 10 6 cm s -1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s -1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) x 10 6 cm s -1 , respectively. figure 6 [61] . compounds 88 and 89 were prepared from 8-hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate [56] . in this reaction, the phenolic group was protected via reaction with chloro(methoxy)methane to yield diethyl ((8-(methoxymethoxy)quinolin-5-yl)methyl)-phosphonate. subsequent reaction of the last compound with various 2-nitrobenzaldehydes in the presence of sodium hydride yielded derivatives of 8-(methoxymethoxy)-5-(2-nitrostyryl)quinolone. reductive cyclization of 2-nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound 89, diethyl ((7-chloro-8compounds 79, 84, and 87 are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase 4d (pde4d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde4d is involved in the process of long-term potentiation and memory consolidation. one of the derivatives of 79, for which x = h, r 1 = cf 2 h, r 2 = cyclopentylethyl, exhibited an ic 50 of 0.399 mm against pde4d compared to rolipram as the reference compound (ic 50 0.621 mm), whereas the derivative showing x = h, r 1 = cyclopentylmethyl of family 84 exhibited the highest value among all tested compounds in the orac assay (its value is 1.98 expressed as trolox equivalents). in this respect, the orac value of antioxidant activities increases the ability of a given compound as the antioxidant becomes better [58] . clioquinol, rolipram, and roflumilast showed values of 0.60, 0.070, and 0.067, respectively. oxidative stress has a major effect in the production of excess free radicals, which lead to cell death and cytosceletal damage in ad [59] . finally, the bbb has a major role in the generation of chronic brain inflammation during ad [60] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of 87 with r 1 = ch 3 and r 2 = cyclopentylmethyl exhibited a maximum value of pe 16.41 (±0.44) × 10 6 cm s −1 , whereas other tested compounds showed values higher than 4.7 × 10 6 cm s −1 , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as (18.87 ± 0.57), (9.22 ± 0.62), and (5.20 ± 0.33) × 10 6 cm s −1 , respectively. an earlier paper by wang et al. (2018) described the synthesis of new 8-hydroxyquinoline derivatives, as shown in figure 6 [61] . compounds 88 and 89 were prepared from 8-hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate [56] . in this reaction, the phenolic group was protected via reaction with chloro (methoxy)methane to yield diethyl ((8-(methoxymethoxy) quinolin-5-yl)methyl)-phosphonate. subsequent reaction of the last compound with various 2-nitrob enzaldehydes in the presence of sodium hydride yielded derivatives of 8-(methoxymethoxy)-5-(2-nitro styryl)quinolone. reductive cyclization of 2-nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound 89, diethyl ((7-chloro-8-hydroxyquinolin-5-yl)methyl)-phosphonate was prepared by reacting diethyl ((8-hydroxyquinolin-5-yl)methyl)phosphonate with sodium hypochlorite, followed by the same previous steps. on the other hand, compound 91 was prepared through a series of steps that involved reacting 2-methyl-8-hydroxyqunolines 90 with acetic anhydride, then with 2-nitrobenzaldehydes, followed by reductive cyclization of 2-nitrostyerenes in the presence of the palladium (ii) acetate catalyst under carbon monoxide gas, and finally with a base (carbonate or methoxide ions, depending on the nature of x). compounds 88, 89, and 91 were tested against the orac assay, bbb permeability assay, and inhibition activity towards amyloid beta (aβ) self-induced aggregation. the results revealed that compounds 88 (r = oh), 89 (x = h, r 1 = oh, r 2 = h), and 91 (x = h, r 1 = oh, r 2 = ch 3 ) exhibited the highest activities among all prepared compounds (6.6, 5.3, 5.9, and 5.4, respectively). the presence of the phenolic hydroxyl group at c-5 of the indole moiety enhances the activity. on the other hand, the presence of a chlorine atom in compound 89 lowers the activity compared to a hydrogen. the reference standards, including clioquinol, melatonin, and a mixture of clioquinol and melatonin, exhibit the values of 0.5, 2.4, and 2.9, respectively. this test was performed based on a fluorescein (orac-fl) method with a trolox as the internal standard. similarly, permeation of the bbb is considered an important parameter for potential central nervous system (cns) candidates; this assay was accomplished using the parma method. the results showed that most of the target products could effectively permeate the bbb through passive diffusion. two compounds of series 91, designated as x = h, r 1 = oh, r 2 = h and x = h, r 1 = oh, r 2 = och 3 , showed higher bbb permeability activity (pe values 14.8 and 12.1, respectively) compared to other derivatives that have a hydrogen atom instead of the phenolic hydroxyl group. in contrast, compounds 88 and 89, bearing the indole moiety at position 5 of the quinolone scaffold, gave pe values of 9.1 and 6.8, respectively, even in the presence of a phenolic hydroxyl group. aβ represents the major component of amyloid plaques found in the brains of alzheimer's patients [62] . inhibition of aβ self-induced aggregation for the prepared 8-hydroxyquinoline-indole derivatives was examined using thioflavin fluorescence. one of the derivatives of 91, for which x = h, r 1 = oh, r 2 = h, caused 51.2% percent inhibition, which was the highest percentage among the prepared compounds. on the other hand, compounds 88 and 89 caused 57.2% and 68.7% inhibition, respectively; whereas clioquinol, curcumin, and resveratrol showed 1.9%, 36.7%, and 42.1% inhibition, respectively. in a recent publication, prati and coworkers [63] described the synthesis of a new series of 8-hydroxyquinoline derivatives (94) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme 22 are the steps involved in the synthesis of these compounds. in a recent publication, prati and coworkers [63] this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8-hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references donepezil, tacrine, and galantamine exhibited inhibition towards hbche, with values of 84.3, >90, and 65.8%, respectively. on the other hand, the inhibition activity of compound 10 and its derivatives towards the aβ 42 antiaggregating property was evaluated. the obtained results demonstrated that the inhibition potencies of all derivatives ranged from 19.1 to 65.0% at the concentration of 50 mm; both series (x = h, cl) had close activity rates. in addition, the metal chelating ability of the selected compounds was examined using cu 2+ and zn 2+ in phosphate buffer solution (ph 7.4). spectroscopic data showed that there is a bathochromic shift of about 18 nm from the original band (243 nm) upon complex formation; as the metal ion concentration increases (1.56 to 50 mm), the intensity of the absorption band also increases. finally, all derivatives showed antioxidant activity, whereby some prepared compounds exhibited higher activity than trolox. raj and padhi reported that the condensation of 8-hydroxyquinoline-2-carbaldehyde (95) with aromatic diamines (96) afforded quinoline-based benzimidazole (97) followed by intracyclization reaction to produce derivatives of 100. with aliphatic diamines (98), compound 95 produced bis-imines (99) without undergoing intracyclization reaction, as shown in scheme 23 [64] . products of these reactions have been well-characterized using various techniques, including ft-ir, nmr, ms, and single-crystal x-ray diffraction method. in the first step of both reactions, one equivalent of 8-hydroxyquinoline-2-carbaldehyde underwent a condensation reaction with one equivalent of primary amine to form the mono-imine product (schiff bases). however, in the second step and with the presence of aromatic amine, an intramolecular ring cyclization occurred, where the resulting precursor reacts further with the second equivalent of 8-hydroxyquinoline-2-carbaldehyde, followed by migration of hydride to afford compound 100. in the case of an aliphatic amine, the second step represents a second condensation reaction with another molecule of 8-hydroxyquinoline-2-carbaldehyde to give the final product 99. in a recent publication, prati and coworkers [63] described the synthesis of a new series of 8hydroxyquinoline derivatives (94) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme 22 are the steps involved in the synthesis of these compounds. this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine (92), paraformaldehyde, and 8-hq or its 5-chloroanalogue under a microwave-assisted procedure to afford 7-(piperazin-1-ylmethyl)-8-hydroxyquinolines (93). this synthon was then reacted with various benzyl chlorides in dmf. compound 94 and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of 40 mm, all derivatives exhibited inhibition, with values ranging from 9.0 to 63.8% and 49.2 to 89.1% for 5-chloro-8-hydroxyquinoline and 8-hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position 5 of the 8hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references scheme 23. synthesis of new derivatives of 8-hydroxyquinolie-based benzimidazole. on the other hand, kong et al. (2017) prepared novel barbituroquinoline derivatives 106 and 107 in a one-pot procedure by combining three components in water, namely 1, 2-thiobarbituric acid (104), and aldehyde (isatin) 105 [67] , as shown in scheme 25. this reaction was conducted under mild experimental conditions and without a catalyst. nitriles or acetonitrile in aqueous potassium hydroxide solution yielded 1,2,4-triazole-based quinolone (103 on the other hand, kong et al. (2017) prepared novel barbituroquinoline derivatives 106 and 107 in a one-pot procedure by combining three components in water, namely 1, 2-thiobarbituric acid (104), and aldehyde (isatin) 105 [67] , as shown in scheme 25. this reaction was conducted under mild experimental conditions and without a catalyst. the 8-hydroxyquinoline moiety can act as a building block for various pharmacologically active scaffolds. in the present work, we have reviewed the recent literature pertaining to the synthesis and bioactivity of numerous 8-hq derivatives as anticancer, antiviral, antimicrobial, antibacterial, antifungal, and anticancer agents. the results obtained from this review highlight the importance of numerous derivatives of 8-hq as possible chemotherapeutic agents and as possible leads towards the development of new drugs to treat various diseases, including cancer. we hope that data presented in this review could help researchers in the fields of medicinal chemistry and pharmacology in designing new active compounds and in the modification of existing compounds in the search for new drug leads. the development of drugs, either natural or synthetic, is gaining popularity in the fight against diseases, such as cardiovascular disorders, cancer insurgence, and immune dysfunction. there are certain nuclei such as 8-hq that are important building blocks in the medicinal arena. therefore, new synthetic methods for bioactive 8-hq derivatives should be pursued. in this respect, more biological testing, including in vivo studies, should accompany these syntheses. studies should also involve different pharmacokinetic parameters related to the safety profiles of potent derivatives. in this review, we have shown different synthetic strategies for pharmaceutically important chemicals that incorporate the 8-hq moiety. these compounds exhibited a wide range of biological activities and could be used as therapeutic agents against different diseases, including cancer. some of these compounds could be envisioned as leads in the development of drugs. compound 101 then reacted with alkenes or alkynes containing electron-withdrawing substituents under the mechanism of 1,3-dipolarcycloaddition and in the presence of a base (k 2 co 3 ) to afford 102. on the other hand, the reaction of 101 with aromatic nitriles or acetonitrile in aqueous potassium hydroxide solution yielded 1,2,4-triazole-based quinolone (103) 8-hydroxyquinoline and its derivatives: synthesis and applications immiscible polymers in double spin-coated electroluminescent devices containing phenyl-substituted tris (8-hydroxyquinoline) aluminum derivatives soluble in a host polymer 8-hydroxyquinolines in medicinal chemistry: a structural perspective 8-hydroxyquinoline: a privileged structure with a broad-ranging pharmacological potential synthesis, characterization, and anti-corrosion properties of an 8-hydroxyquinoline derivative synthesis and characterization of 8-hydroxyquinoline complexes of tin (iv) and their application in organic light emitting diode antiviral activity of novel quinoline derivatives against dengue virus serotype 2 8-hydroxyquinoline-2-carboxanilides as antiviral agents against avian influenza virus synthesis and antibacterial activity of 3-benzylamide derivatives as ftsz inhibitors chemoselective synthesis of 5-amino-7-bromoquinolin-8-yl sulfonate derivatives and their antimicrobial evaluation synthesis, antibacterial properties and bioinformatics computational analyses of novel 8-hydroxyquinoline derivatives synthesis and biological evaluation of quinoline derivatives as a novel class of broad-spectrum antibacterial agents in vitro activities of a new fluoroquinolone derivative highly active against chlamydia trachomatis antifungal activity of umbelliferone derivatives: synthesis and structure-activity relationships synthesis of some novel 3, 4, 5-trisubstituted triazole derivatives bearing quinoline ring and evaluation of their antimicrobial activity a comprehensive review on the chemotherapeutic potential of piceatannol for cancer treatment, with mechanistic insights a novel potent nicotinamide phosphoribosyltransferase inhibitor synthesized via click chemistry synthesis and structure-activity relationships study of a-aminophosphonate derivatives containing a quinoline moiety an extremely stable and orthogonal dna base pair with a simplified three-carbon backbone synthesis and investigation of antibacterial and antioxidants properties of some new 5-subsituted-8-hydroxyquinoline derivatives synthesis, spectroscopic characterization, x-ray analysis, and dft-hf calculations of 5-ethoxymethyl-8-hydroxyquinoline synthesis and antimicrobial activities of sulfonohydrazide-substituted 8-hydroxyquinoline derivative and its oxinates bioconjugation via azide-staudinger ligation: an overview synthesis and biological evaluation of 8-hydroxyquinoline-hydrazones for anti-hiv-1 and anticancer potential design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents design, synthesis and biological evaluation of 1, 3, 6-trisubstituted b-carboline derivatives for cytotoxic and anti-leishmanial potential co-delivery of docetaxel and gemcitabine by anacardic acid modified self-assembled albumin nanoparticles for effective breast cancer management novel gemcitabine conjugated albumin nanoparticles: a potential strategy to enhance drug efficacy in pancreatic cancer treatment synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their b1,4-galt inhibitory and anti-cancer properties synthesis and characterization of a new cationic galactolipid with carbamate for gene delivery click'assembly of glycoclusters and discovery of a trehalose analogue that retards aβ40 aggregation and inhibits aβ40-induced neurotoxicity chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein halogenated 2-amino-4h-pyrano [3,2-h] quinoline-3-carbonitriles as antitumor agents and structure-activity relationships of the 4-, 6-, and 9-positions amberlite ira 402 (oh) mediated green synthesis of novel benzothiazole-quinoline conjugates as cancer theranostics comparative theoretical and experimental study on novel tri-quinoline system and its anticancer studies synthesis, anticancer evaluation and dna-binding spectroscopic insights of quinoline-based 1, 3, 4-oxadiazole-1, 2, 3-triazole conjugates synthesis and anti-breast cancer activities of substituted quinolines design, synthesis and biological evaluation of new 4-(4-substituted-anilino) quinoline derivatives as anticancer agents the synthesis and anticancer activity of 2-styrylquinoline derivatives. a p53 independent mechanism of action an efficient microwave-assisted synthesis of structurally diverse styrylquinolines new pyranoquinoline derivatives as vascular-disrupting anticancer agents matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability review on epidermal growth factor receptor (egfr) structure, signaling pathways, interactions, and recent updates of egfr inhibitors design, synthesis and preliminary bioactivity evaluations of 8-hydroxyquinoline derivatives as matrix metalloproteinase (mmp) inhibitors synthesis of tetracyclic pyrrolidine/isoxazolidine fused pyrano [3,2-h] quinolines via intramolecular 1,3-dipolar cycloaddition in ionic liquid discovery of cgs 27023a, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors acetylcholinesterase inhibition by flavonoids from agrimonia pilosa coumarin derivatives as acetyl-and butyrylcholinestrase inhibitors: an in vitro, molecular docking, and molecular dynamics simulations study cholinesterase inhibitors and beyond pet imaging of copper trafficking in a mouse model of alzheimer disease nanoprobing of the effect of cu 2+ cations on misfolding, interaction and aggregation of amyloid b peptide coumarin derivatives bearing benzoheterocycle moiety: synthesis, cholinesterase inhibitory, and docking simulation study novel 8-hydroxyquinoline derivatives targeting b-amyloid aggregation, metal chelation and oxidative stress against alzheimer's disease design, synthesis, and evaluation of multitarget-directed resveratrol derivatives for the treatment of alzheimer's disease design, synthesis, and evaluation of orally available clioquinol-moracin m hybrids as multitarget-directed ligands for cognitive improvement in a rat model of neurodegeneration in alzheimer's disease synthesis and evaluation of clioquinol-rolipram/roflumilast hybrids as multitarget-directed ligands for the treatment of alzheimer's disease profiling donepezil template into multipotent hybrids with antioxidant properties antioxidant capacity in the lipophilic fraction of alzheimer's brain tissues inflammatory events at blood-brain barrier in neuroinflammatory and neurodegenerative disorders: implications for clinical disease design, synthesis, and evaluation of orally bioavailable quinoline-indole derivatives as innovative multitarget-directed ligands: promotion of cell proliferation in the adult murine hippocampus for the treatment of alzheimer's disease the amyloid beta peptide: a chemist's perspective. role in alzheimer's and fibrillization novel 8-hydroxyquinoline derivatives as multitarget compounds for the treatment of alzheimer's disease synthesis, characterization, and structure of quinoline-based benzimidazole derivatives synthesis of pyrazolo-and [1,2,4] triazolo-[1,5-a] quinolin-9-ols by cycloaddition to 8-hydroxyquinoline n-imide o-mesitylenesulfonylhydroxylamine and related compounds-powerful aminating reagents convenient one-pot synthesis of thiobarbituro-quinoline derivatives via catalyst-free multicomponent reactions in water this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord-000445-2x7dfl1q authors: paliwal, sarvesh k.; verma, ankita narayan; paliwal, shailendra title: neglected disease – african sleeping sickness: recent synthetic and modeling advances date: 2011-05-10 journal: sci pharm doi: 10.3797/scipharm.1012-08 sha: doc_id: 445 cord_uid: 2x7dfl1q human african trypanosomiasis (hat) also called sleeping sickness is caused by subspecies of the parasitic hemoflagellate trypanosoma brucei that mostly occurs in sub-saharan africa. the current chemotherapy of the human trypanosomiases relies on only six drugs, five of which have been developed more than 30 years ago, have undesirable toxic side effects and most of them show drug-resistance. though development of new anti-trypanosomal drugs seems to be a priority area research in this area has lagged far behind. the given review mainly focus upon the recent synthetic and computer based approaches made by various research groups for the development of newer anti-trypanosomal analogues which may have improved efficacy and oral bioavailability than the present ones. the given paper also attempts to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness. african sleeping sickness remains one of the most neglected life threatening diseases that have been left untreated till date. two forms of human african trypanosomiasis (hat) have been identified that are parasite dependent. the first one trypanosoma brucei gambiense (t. b. gambiense) causes a human chronic infection, endemic in western and central africa while the other trypanosoma brucei rhodesiense (t. b. rhodesiense) has a vast animal reservoir and causes acute illness in people in eastern and southern african countries. hat has high occurance in the remote rural areas, where the surveillance is weak or nonexistent, with 50 to 70 thousand estimated cases. according to world health organization (who) estimation there are half-million cases of hat or "sleeping sickness" resulting from infections with t. brucei rhodesiense and t. brucei gambiense. who has attributed 50.000 deaths annually to the disease [1] . by more recent estimates, up to 25.000 new cases occur per year, and 50 million people are at risk [2, 3] . out of six clinically approved drugs for the treatment of hat, five (suramin, pentamidine, melarsoprol, eflornithine and nifurtimox) have had been discovered more than 30 years ago. because suramin and pentamidine are ionized at physiological ph, they are unable to cross the blood brain barrier in therapeutic concentrations and are thus used for the treatment of hemolymphatic early stage hat, caused by t. b. rhodesiense and t. b. gambiense infections, respectively. the treatment of the second or neurological stage, when the parasites invade the central nervous system (cns), relies on the organoarsenical drug melarsoprol and the more recently registered eflornithine. the latter is ineffective against t. b. rhodesiense sleeping sickness and is used primarily to control cns-involved hat caused by t. b. gambiense. all the existing anti-trypanosomal therapies suffer from unacceptable toxicity, poor efficacy, difficulties of administration, and increasing treatment failures due to the development of parasite resistance [4] [5] [6] [7] [8] [9] . in the last couple of decades, no new drug has been developed for treatment of early-stage hat, and only one drug has been developed for late-stage hat [1, 3, 5] . the need is great for new orally active drugs for the control and eradication of this disease. novel medicines are typically developed using a trial-and-error approach, which is timeconsuming and costly but yet has the potential to yield new drugs. the application of computer-assisted drug design (cadd) methodologies to this problem has the potential to greatly decrease the time and effort required to discover new medicines or improve current ones in term of their efficacy. this review focuses on the synthetic and computer-assisted drug design (cadd) approaches made by various research groups for the development of newer antitrypanosomal agents having improved efficacy and oral bioavailability. the current research also endeavors [10] to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity that may be helpful in development of potent anti-trypanosomal agents against sleeping sickness. safe, effective and affordable orally active therapies for trypanosomiasis capable of overcoming resistance are required, so the identification of new anti-trypanosomal drug candidates is an urgent priority. compared to the last 15 years there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing [11] . in view of this, the researchers have endeavored to compile in the present section of the review, diverse series of compounds that have been recently synthesized by various research groups targeting dna minor groove and many other new targets. based on the mechanism of action this section has been divided into two sub-sections. the first sub-section includes the series of compounds acting as dna minor groove binders while in the second sub-section are included all the other anti-trypanosomal compounds and their respective targets. also are described herein prodrug approaches to provide oral bioavailability for the dication class. the wide range of antifungal and antiparasitic activities related to aromatic diamidines and its excellent results in preclinical and clinical phase of drug development has made aromatic diamidines an interesting class for the development of newer anti-trypanosomal drug therapy. the aromatic diamidines i.e. pentamidine ( figure 1) show their anti-parasitic action by binding strongly to at-rich sequences in the minor groove of dna. therefore dna minor groove has evolved as a productive target for designing new ant-parasitic drugs. in order to design newer analogues belonging to diamidine series, studies of the dna complexes with diamidine compounds have been conducted and a number of diamidines have been crystallized with the dna duplex d(cgcgaattcgcg)2 which provide valuable models for drug development in the diamidine series. structures of dna complexes of furamidine, berenil, and pentamidine, for example, reveal that they all bind in the dna minor groove at the central aatt sequence. these drugs penetrate deeply into the groove and fit snugly between the walls of the groove. their amidines form h-bonds with thymine-o2 and/or adenine-n3 acceptor groups on the edges of the bases at the floor of the groove. the amino group of g protrudes into the minor groove and prevents the compounds from assuming their preferred orientation deep in the minor groove. this binding to dna eventually leads to inhibition of one or more of the several dna-dependent enzymes (e.g., topoisomerases and nucleases) or direct inhibition of transcription. for more than 50 years aromatic diamidines and related dicationic molecules have been extensively used but so far only pentamidine [12] [13] [14] [15] has been widely employed as a drug in humans despite several adverse effects, such as hypotension, abdominal pain, vertigo, hypersalivation, hypoglycemia, nausea, and mild nephrotoxocity [5] [6] [7] [8] . being a highly flexible molecule that can assume an array of linked conformations related through torsional rotation, changes can be made in the nucleus to produce improved analogous against hat. in line to this with an aim to increase the efficacy and decrease the side effect related to pentamidine various research groups have made several changes in the pentamidine molecule [16] [17] [18] [19] [20] [21] [22] [23] large numbers of structurally related congeners of pentamidine were synthesized by tidwell rr et al [24] by introducing substitutions on the cationic groups, changing the position of dications from 4,4' to 3,3' position, changing the length of the aliphatic chain between the two aromatic rings, adding substituent on the aromatic rings at 2,2' and 3,3' position and replacing oxygen atoms in the alkyl linker with isosteric sulfur or nitrogen atoms (figure 2a-g) . on comparing the activities of the synthesized compounds with pentamidine and melarsoprol, it was found that few compounds showed activity in the range or better than that of pentamidine and/or melarsoprol. the unsubstituted diamidine compound 64 ( figure 3a ) with secondary amino group in place of oxygen atom exhibited subnanomolar activity (<0.001 µm) against t. b. rhodesiense and was nearly twice as selective against the pathogen as pentamidine. at the same time the diimidazoline compound 66 (figure 3b ) exhibited the second highest anti-trypanosomal activity in the series with the ic 50 value of 0.001 µm and also had the highest parasite selectivity (si t = 34500), being 63 times more selective against t. b. rhodesiense than pentamidine. the replacement of oxygen atom with sulphur atom as in compound 62 ( figure 3c ) also generated active congeners having ic 50 (0.005 µm) value better than that of pentamidine (0.007 µm). the compounds 20-31 ( figure 2c ) in which the cationic groups were present in the 3,3′-position of the aromatic rings and the length of the carbon linker was varied from 3-6 exhibited lower anti-trypanosomal activity compared to pentamidine whereas among the compounds 12-19 ( figure 2b ) of the series, in which the amidine groups were present at 4,4' position and the length of aliphatic chain was varied from three carbon atoms to six carbon atoms, compound 12 ( figure 3d , ic 50 =0.007 µm) with three methylene linker between the aromatic rings had same in vitro activity as that of pentamidine (0.007 µm). further introduction of 2,2′-dichloro substituent in compound 12 improved the antitrypanosomal properties as evident from the in vitro activity of compound 32 (figure 3e , ic 50 =0.004 µm). these compounds having excellent in vitro activity were evaluated in vivo in the stib900 mouse model of african trypanosomiasis. the screening was conducted using intraperitoneal dosing at 20 mg/kg daily for four days. the compounds 12, 62 and 64 showed very poor in vivo activity, whereas compound 32 and 66 exhibited excellent in vivo efficacies in the acute mouse model of trypanosomiasis, providing cures of all infected animals. thus, because of high selectivity, excellent in vitro and in vivo activity compounds 66 and 32 can serve as a novel lead for further pre-clinical and clinical trials, but its cytotoxcity profile needs to be monitored during its evaluation. also compound 64 which showed excellent in vitro, in vivo activity and high selectivity index against the t. b. rhodesiense merits further sar optimization in order to synthesized newer lead compounds with reduced cytotoxicity compared to pentamidine. pentamidine analogues with activity better than that of melarsoprol and/or pentamidine increased efficacies of pyridyl analogues of 2,5-bis(4-amidinophenoxy)furan (furamidine) [25] [26] [27] encouraged tidwell rr et al [28] to synthesize pentamidine analogues in which the phenyl rings of pentamidine were replaced with pyridyl fragments. this replacement resulted in series of 18 compounds (figure 4a ), most of which had lower cytotoxicity than pentamidine. sar study pointed out that the antiprotozoal properties of these compounds depended on the placement of cationic moieties on the pyridine rings as well as the nature of substituents on the amidine groups. the n-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the n-alkylation of cationic fragments reduces the activity of compounds against t. brucei rhodesiense compared to pentamidine. a same trend was observed in pentamidine analogues series. the 2, 6-substituted dications (compounds 11-13, figure 4a ) and 2,4-substituted dications (compounds 14-18, figure 4a ) displayed lower potencies against t. brucei rhodesiense than the corresponding 2,5-substituted isomers (compounds 1-10, figure 4a), while among the 2, 5-substituted dications, the compounds possessing cationic substituents adjacent to nitrogen atoms in pyridine rings displayed superior activities against parasites compared to pentamidine as evident from compound 6 (figure 4b) which showed promising anti-trypanosomal activity (0.001µm) and lower cytotoxicity (4.90µm) than pentamidine and melarsoprol, but had poor in vivo activity giving only 1/4 cures in the stib900 mouse model. however diamidoxime compound 9 (figure 4c ), an oral prodrug of diamidine compound 6 ( figure 4b ), exhibited excellent in vivo activity curing four out of four animals upon oral administration in stib900 mouse model. although compound 9 did not provide cure in the cns mouse model of infection but its bbb permeability could potentially be improved by developing prodrug using lipophillic substitutions in place of hydrophilic substitution as used in compound 9. this could provide higher concentration of compound 6 in cns. thus excellent in vitro activity of diamidine 6 and high in vivo efficacy of its prodrug diamidoxime 9 warrant further investigation of these dications as potential antitrypanosomal drug candidates with improved oral efficacies. the structure activity relationship of the two series indicates that in both the cases the unsubstituted diamidines were more active than the n-substituted diamidines. the replacement of oxygen atoms in alkyl chain with secondary amino group improves the activity against t. b. rhodesiense of pentamidine analogues while in case of pyridyl analogue the compounds with oxygen atoms in the alkyl chain have been the active one. the most potent compound of these two series are compound 6 ( figure 4b ) and compound 66 (figure 3b ), which have excellent in vitro activity (0.001µm) better than pentamidine and melarsoprol and also have high selective against the t. b. rhodesiense parasite as evident from their selectivity index (si of compound 6 = 4900 and compound 66 = 34500 µm). initially it has been believed that the oxygen atom in the aliphatic linker and the amidine groups are important for anti-trypanosomal activity of pentamidine as these groups were part of recognition motif for p2 amino purine transporter in trypanosoma species. thus the activity of compound 6 can be explained on this basis, but in case of compound 66 the amidine groups have been replaced by imidazoline and the oxygen atom has been replaced by secondary amino groups. despite these replacements, the compound has shown excellent in vitro and in vivo activity. thus this is in accordance to recently reported work [29] according to which there is no direct connection between the affinity for p2 carrier and anti-trypanosomal activity. in view of this, these two compounds can serve as a lead structure for the development of newer analogues of pentamidine with reduced side effect and improved pharmacokinetic profile. in the early 1970s dann o et al [30] [31] [32] fig. 6 . structure of two phenylbenzofuran dications with promising anti-trypanosomal activity tidwell rr et al found that the in vitro anti-trypanosomal activities of bisbenzofuran derivatives against trypanosoma brucei rhodesiense, and cytotoxicity against mammalian cells depended on the position and the type of cationic substituents as well as the length of the carbon linker between aromatic moieties. as observed in most of the dicationic molecules, the n-substituted congeners were lesser cytotoxic than the unsubstituted diamidines whereas the n-alkylation of cationic fragments reduced the activity of compounds against t. brucei rhodesiense compared to pentamidine. at same time the 5-substituted bisbenzofurans were generally less cytotoxic than compounds bearing substituents in the 4-or 6-positions where as the 4-substituted bisbenzofurans were significantly less active against t. b. rhodesiense than corresponding 5-and 6-substituted isomers. the in vitro activity of the bisbenzofuran series was not so promising and only lead compound 8, (figure 5b ) exhibited in vitro anti-trypanosomal activity (0.008 µm) comparable to that of pentamidine and melarsoprol. however some compounds from tidwell series (figure 7b ) showed reduced cytotoxicity profile compared to pentamidine (for example compound 43 has cytotoxicity of 1.85 µm), but had very poor selectivity profile. thus was proved that the selectivity of bisbenzofurans against t. b. rhodesiense decreases as number of methylene groups in the alkyl bridge increases. however, how the substitution on the amidine groups affects the anti-trypanosomal properties of bisbenzofurans has not been explained. thus in light of their reduced cytotoxicity compared to pentamidine these molecules require further investigation to study the influence of the type of cationic substituents and the distance between aromatic moieties on uptake and intracellular distribution of dicationic bisbenzofurans in order to understand better the mode of action and thus improve the efficacy of aromatic diamidines. thus promising in vitro, anti-trypanosomal activity, excellent potency in the acute mouse model of trypanosomiasis and the reduced cytotoxicity (1.9 µm) of compound 1 compared to pentamidine warrants further pre-clinical and clinical trials of this molecule. while compound 32 and 20 can serve as lead compounds for further sar optimization to derive congener with enhanced activity and lesser cytotoxicity. to combine the rigidity of 2-phenylbenzofurans with the flexibility of pentamidine congeners, tidwell rr et al [35] incorporated the benzofuran ring into molecules of pentamidine-related analogues. a series of 48 pentamidine congeners containing benzofuran fragments (figure 10a , b) were synthesized and tested in vitro against t. b. rhodesiense. most of the compounds in the series showed cytotoxicity less than that of pentamidine, but had lesser potency and selectivity index as compared to pentamidine. the properties and cytotoxicities of these dications depended on the nature of the cationic substituents, the placement of the benzofuran motif, and the length of the carbon linker. within the series cytotoxicity of the compounds decreased with the substitution on cationic groups and increased with the elongation of the carbon linker. dications with the benzofuran motif in the 4'-position (compounds 25-48, figure 10b ) and connected by the propylene linker are the most potent and more selective against t. b. rhodesiense among the series. thus on the basis of sar study further improvements can be made in order to produce newer anti-trypanosomal agents with improved pharmacological activity. the sar of the three series clearly indicates that the nature of cationic substituents and their position is important in deciding the anti-trypanosomal activity of benzofuran derivatives. the introduction of phenoxy fragment into a benzofuran structural motif may result into retention of affinity for the aminopurine p2 transporter, which could be the reason for anti-trypanospomal activity of benzofuran derivatives. however the precise mechanism by which these compounds show their anti-trypanosomal activity in not known. among the three series the 2-phenylbenzofuran derivatives are attractive molecules because of their high activity, low cytotoxicity and high selectivity against the hat parasite. the introduction of phenoxy fragment into the aromatic ring protects the phenylbenzofuran dervatives from metabolic deactivation. this structural modification retains affinity for the aminopurine p2 transporter. the diamidine and the hydroxy or methoxy group may be involved in binding to the dna minor groove. thus multiple modes of action may be the reason for promising anti-trypanosomal propertries of 2-phenylbenzofuran derivatives. thus these compounds require further assessment in order to develop as newer antitrypanosomal agents with promising pharmacokinetic profile. since a prodrug of furamidine, 2,5-bis[4-(n-methoxy)amidinophenyl]furan (pafuramidine), has shown promising results in clinical trials against hat [36] , therefore furamidine ( figure 11a ) has gained attention for further evaluation to produce newer antitrypanosomal analogous. two other analogues of furamidine (figure 11b &c) have been evaluated against t. b. rhodesiense mouse model and have shown promising results. one approach to enhance the activity of furamidine has been the replacement of the central furan ring with other heterocyclic systems, including thiophene, pyrrole, oxazole, oxadiazole, thiadiazole, pyridazine, methylpyrimidine, and triazine [37] [38] [39] [40] [41] . such structural modification in past has resulted into compound with good anti-parasitic activity. for example in 1977 boykin dw and das pb have reported the synthesis and antiprotozoal activity of eighteen substituted 2,5-bis(4-guanylphenyl)furans and related analogues, including "masked" amidines in which the guanyl function was incorporated into a heterocyclic ring. among these, six compounds have produced cures in mice at submilligram dosage levels and have been somewhat more active in this screen than stilbamidine, hydroxystilbamidine, and pentamidine. . 11 . structure of furamidine and its analogue that have shown promising antitrypanosomal properties tidwell rr et al [43] has recently synthesized a series of 3,5-diphenylisoxazole analogues ( figure 12 ), in which the central ring of furamidine was replaced by isoxazole, and their activities with furamidine and melarsoprol were compared. among 43 synthesized dications, the compounds with at least one p-amidine moiety (compound 22, figure 13a and compound 3, figure 13b ), displayed good ic 50 values (3.5 nm and 5.1 nm respectively) comparable to that of furamidine (4.3 nm) while loss in potency was observed in compounds with substituted diamidine at m-position (compound 32, ic 50 =29 nm, figure 13c ). in general, the introduction of nitro, chloro, or methoxy substituents on either aromatic ring resulted in decreased anti-trypanosomal activity. however, the introduction of methoxy group on the aromatic rings of compound 32 resulted into compound 41 ( figure 13b ) with comparable anti-trypanosomal activity (4.2 nm) as that of furamidine. hence these compounds of the isoxazole series that showed good in vitro antitrypanosomal activity and less cytotoxcity profile relative to furamidine, could be a candidate for further evaluation against animal models of the diseases. however in vivo evaluation of the present 3,5-diphenylisoxazole series has not been reported yet. [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] and also because 1,4-diphenyl-1,2,3-triazoles showed geometrical resemblance to furamidine. it was observed that the cytotoxicities of triazoles were as lower compared to that of pentamidine and were not affected by the alkylation on the amidine groups or the substitution on the aromatic rings. however, the placement of the cationic moiety in the 4-position increased the cytotoxicities of diamidines 46 ( figure 15 ) with respect to the pentamidine. except few, majority of unsubstituted diamidines exhibited higher in vitro activities against t. brucei rhodesiense than bis(n-isopropyl)amidines and diimidazolines. the compounds with cationic fragments in the 5,5'-position (compounds 1-15, figure 14a the replacement of central furan ring of furamidine either by isoxazole or 1,2,3-triazole resulted into compounds with better activity than that of furamidine. position and the nature of the cationic substituent governed the anti-trypanosomal activity of the two series. in general the para substituted diamidines in both the series were the most promising compounds with good activity. thus these compounds should be further evaluated to produce better analogue of furamidine the recent report on n,n'-bis(4-amidinophenyl)piperazine (figure 16) , which was shown to be very effective in vivo anti-trypanosomal agent has attracted the interest in this compound [56] . hence in search for new hat chemotherapy dardonville c et al [57] decided to carry out an in vitro screening of a total of 62 compounds against the parasite t. brucei rhodesiense taken from their in-house library. based on this they showed that bisguanidine and especially bis(2-aminoimidazoline)diphenyl compounds displayed potent anti-trypanosomal activity in vitro and vivo against t. b. rhodesiense, the causative agent of acute hat [58, 59] . among the 62 compounds screened, compounds1c, 28b, 32b and 41b ( figure 17 ) showed excellent in vitro activity (49nm, 69 nm, 22 nm and 118 nm respectively) as well as high selectivity (>5294, 3072, 29.5 and 881respectively) for the parasite. these studies revealed that compounds bearing 2-aminoimidazoline cations had higher selectivity for the parasite and similar activities with respect to their guanidine counterparts. in addition, a correlation between anti-trypanosomal activity and dna binding affinity was observed, suggesting a possible mechanism of action for these compounds. those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite represent new anti-trypanosomal lead compounds. fig. 16. n,n'-bis(4-amidinophenyl) piperazine in light of these promising results, bis(2-aminoimidazoline) derivatives deserve more investigation as anti-trypanosomal agents and dna minor groove binders. the synthesis and study of new derivatives and prodrugs of these lead compounds is ongoing. progress has also been made on targets other than dna minor grove. in addition to newer targets some new lead compounds have also been identified with proper antitrypanosomal activity but uncertain mechanism of action. the enzyme trypanothione reductase (tr) which restores the oxidized trypanothione to reduced state has evolved as an effective drug target. many inhibitors of this enzyme have been developed but most of them combat with problems related to bioavailability, pharmacokinetics and metabolism. despite this quinolines have evolved as most important class of compounds against tr due to their broad spectrum of activity, excellent pharmacological and pharmacokinetic properties such as high plasma levels, high clearance, oral and parenteral applicability, chemical stability and rare side effect. in view of this gilbert ih [60] reported the screening of 62000 compound libraries against t. brucei. high through put screening (hts) which resulted into identification of two novel compound series active against the enzyme trypanothione reductase. series 1 was based on the quinoline scaffold (figure 18a ) in which compound 2 (figure 19a) with methylfuran group at r 3 position, br at 6 th position of quinoline ring and n-methylethanamine group at r 1 position was the most potent and significant tr inhibitor with tr inhibitory activity of 1.1 µm. according to sar study the replacement of methylfuran with other groups like furan, phenyl, pyridinyl, thiophene led to decrease in activity. also the replacement of br with h or f reduced the activity while cl group retained the activity. at 4 th position the nh and nme groups were equally active. the alkylamines at r 1 were active whereas the simple alkyl or aryl groups led to inactive compounds. the second series was based on the pyrimidopyridazine scaffold (figure 18b) , in which the compound 49 ( figure 19b ) showed promising inhibitory activity of 2.6 µm. in case of series 2 replacement of methyl group at r 1 position by h led to the decrease in activity. at r 2 position methyl and ethyl group showed activity while the h and chain extensions to propyl, butyl and cyclopentane led to decrease in activity. substituted phenyl and alkenylphenyl group at r 3 position gave the most active compound of the series. thus these quinoline compounds with promising activity could serve as lead compounds for further development of targeted drugs against african trypanosomiasis. in addition to this synthetic optimization study based on the lead anti-trypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate ( figure 20 ) was undertaken by werbovetz ka et al [61] in an attempt to discover new trypanocides with potent in vivo activity targeting tr enzyme. in course of this a total of 53 compounds were evaluated in vitro for their anti-trypanosomal activity and cytotoxicity. the compounds with oxygen atom at 6 th position were the most active compounds in the series, for example compound 9 ( figure 21 ) showed better anti-trypanosomal activity (0.007 µm) and low cytotoxicity (6.8 µm) than melarsoprol (ic 50 = 0.008µm and cytotoxicity = 7.9µm). whereas compounds lacking the 6-oxygen atom or bearing an oxygen atom at the 7-position rather than the 6-position were far less potent than those containing an alcohol or acyloxy group at the 6-position. compounds carrying aliphatic or a 2-phenylacetyl ester side chains were as potent and selective as their benzoylated or acetylated counterparts. however compound 9a was unstable due to auto-oxidation, so the unstable alcohols were esterified to generate prodrug 10a ( figure 21 ) having promising activity of 0.014µm against these parasites and a selectivity index of 1700. the in vivo evaluation of compound 10a in a murine model of african trypanosomiasis showed good results as the prodrug extended the lifespan of mice infected with t. b. brucei. thus compound 10a can serve as lead compound for further investigation of this class of molecules as potential candidates against hat. efforts are also be undertaken to further elucidate the metabolism, pharmacokinetics, and the anti-trypanosomal mechanism of action of this novel and promising class of compounds. dna topoisomerases have evolved as an effective drug target in prokaryotic and eukaryotic systems as these enzyme mediate mechanistic interactions such as supercoiling, relaxation, knotting or catenating of dna double helices. based on their mechanism of action, topoisomerases can be classified as type i enzymes, which break a single strand of the dna helix during the catalytic cycle, and type ii enzymes, which make double-stranded breaks. on the basis of primary sequence and reaction mechanism, type i topoisomerases are further subdivided into type ia and type ib. recently shapiro ta et al [62] evaluated the activity of indenoisoquinolines (figure 22) , originally known to have anti-cancer activity, against t. brucei and found that most of the compounds showed in vitro activity at submicromolar concentrations. the compound 12 ( figure 23 ) with propylamino group at r 6 position and methoxy group at r 2 , r 3 and r 9 was the most active one with in vitro anti-trypanosomal activity of 0.05 µm. the compound also showed good in vivo activity as it delayed parasitemia and extended survival in infected mice. according to structure-activity analysis the compounds with enhanced potency included alkylamino substitutions on n-6, methoxy groups on c-2 and c-3, and a methylenedioxy bridge between c-8 and c-9. testing of indenoisoquinolines with promising activity on l1210 mouse leukemia cells revealed all the compounds were more effective against trypanosomes than against mammalian cells. the indenoisoquinolines also showed appreciable water solubility indicating that these compounds have good quality for drug development. these compounds showed their anti-trypanosomal action by multiple mechanisms. the study indicated that they stabilize topoisomerase-dna complexes in situ and may also impede topoisomerase binding to dna. these agents markedly inhibited dna synthesis by interfering with topoisomerase and possibly other dna-metabolizing enzymes. concentrations in the range of 300 ng/ml up to 900 ng/ml. some of the dupont compounds, developed as anti-tumour drugs, were highly active but also showed high cytotoxicity on ht-29 cells. it was observed that the position r 1 and position r 2 in the quinolone core nucleus was not prerequisite for anti-trypanosomal activity and also substitution at r 8 position was not necessary for trypanocidal activity. thus, based on the result obtained from sar analysis special attention should be given to r 7 position and the tetracyclic derivatives. the in vivo results of these compounds were very poor, as none of the compounds evaluated produced cure of mice in dose escalation experiment up to 100mg/kg i.p. however no signs of toxicity were observed during the experiments. the in vivo ineffectiveness had not been explained and no drug level determination in the plasma of the treated mice was performed. polyamines are generally involved in growth and differentiation [64] [65] [66] [67] within the cell and their analogs are also used as anticancer agents, antiparasitic agents, antidiarrhoeals, anti-hiv agents, metal chelators, and gene delivery agents. since the inhibition of the initial polyamine biosynthesis enzyme, ornithine decarboxylase, by dl-α-difluoromethylornithine (dfmo) is toxic to african trypanosomes cells, [68, 69] polyamines can become a promising anti-trypanosomal compound. dfmo [70] [71] [72] is the most recently developed agent for late stage t. b. gambiense and t. b. brucei sleeping sickness, but has not been active against all strains of t. b. rhodesiense. the major drawbacks of dfmo are its cost, the duration of treatment and its availability. recent clinical studies have investigated that dmfo can be used in combination with clinically used trypanocides including suramin, nifurtimox and melarsoprol [73, 74] . these combinations result in significant reduction in dfmo dosage and time of administration. initial clinical study has shown that dfmo + nifurtimox are superior to dfmo + melarsoprol and melarsoprol + nifurtimox. the dfmo + nifurtimox regimen (nect regimen) allowed reduction in dfmo regimen from 14 to 7 days (56 versus 28 infusions) with a 94% cure rate and are associated with significantly reduced adverse side effects as compared to melarsoprol-based therapy. the success of the combined regimen has been most likely due to ability of dmfo to reduce trypanothione levels and resistance to oxidative stress and the ability of nifurtimox to generate oxidative stress. recently gilbert et al [76] designed, synthesized, and evaluated substituted polyamines, carrying 1,3,5-triazine units, as potential anti-trypanosomal drugs. preliminary results indicated that this route might be successful, and lead structure a (figure 26 ) was used as a starting point for the synthesis of two series of analogues. in the first series, the influence of structural changes of the central core unit was investigated while in the second series, the effect of additional methyl substituents on the 1,3,5-triazine was studied. the compounds were designed with the intention to selectively target the interior of t. brucei via the p2 amino-purine transporter. in the first series the compound containing the ndodecyl chain as core unit, showed weak activity against t. b. rhodesiense. the compound with n-nonyl chain was the most promising compound and its various analogues were designed by replacing nh 2 groups on the triazine ring with nhme and nme 2 groups. introduction of one or two methyl groups per triazine unit resulted in a 10fold increase in anti-trypanosomal activity. when four methyl groups per triazine unit were introduced, an 80-fold increase in activity was observed. similarly, replacement of nh 2 group in n-dodecyl chain led to 2-20-fold higher anti-trypanosomal activity for the methylated derivatives. monosubstituted compounds showed a slight increase in activity against t. b. rhodesiense as compared to the disubstituted compounds. the methylamino substituted triazines attached to the c9-(compound 8c, figure 27 ) or c12-(compound 8f, figure 27 ) polyamine precursor via an additional ch 2 linker resulted in most active trypanocidal compounds(ic 50 of 8c = 0.27µm and 8f = 0.18 µm). beside good activity, the compounds showed poor in vivo activity producing no cure to the infected mice and concentrations greater than 10 mg kg −1 induced severe acute toxicity. the actual mode of action for the reported triazine substituted polyamines remains unclear. so to understand and improve the activities of these compounds, further research has to verify intracellular drug targets and possible metabolic pathways. stanislaw fw et al [77] have reported the anti-trypanosomal activity of 5'-deoxy-5'-(e)-(iodomethylene)adenosine, which is a known inhibitor of adohcy hydrolase, [78, 79] the 5'-deoxy-5'-(e)-(iodomethylene)adenosine (eiddha) and its 6-n cyclopropyl analogue (figure 28a , b) have shown promising in vitro inhibitory activity (ic50 at 9 and 12 μg/ml) against t. brucei. the utilization of adenosine analogues as anti-parasitics should be explored as a therapeutic paradigm, as it has been shown previously that inhibitors of adohcy hydrolase are also very potent inhibitors against the growth of plasmodium falciparum [79] . this class of 6-n-cyclopropyl adenosine analogues modified at carbon 5', does not exhibit an inhibitory effect on human or parasite forms of the enzyme and displays only marginal antiviral activity in comparison to analogues which have been unmodified at 6-amino position (that are potent inhibitor of adohcy hydrolase). therefore these compounds require further structural modification in order to develop newer analogues with improved activity against t. brucei. the synthesis of 4-[5-(4-phenoxyphenyl)-2h-pyrazol-3-yl] morpholine derivatives by perozzo r et al [80] resulted in to the discovery of newer class of anti-trypanosomal compounds having stage specific action, as these compounds have shown moderate to very good activity against the blood stage of t. b. rhodesiense. the two compounds, 4-[3-(4-phenoxyphenyl)-1h-pyrazol-5-yl]morpholine (1.0µm) ( figure 29a ) and 1-[3-(4-phenoxyphenyl)-1h-pyrazol-5-yl]piperazine (1.1 µm) (figure 29b ) with a pyrazol ring, are the most potent anti-trypanosomals of the series and have same cytotoxicity (61.6 µm), indicating that the pyrazol ring is very important for anti-trypanosomal activity. the substitution of pyrazol ring with isoxazole derivative leads to a six fold reduction in activity as compared to the most potent compound. in addition, substitution with nitrophenyl or aminophenyl also results in strong reduction in activity. the phenoxy ring in the compounds is also important for activity as replacing it by an ethylene group results in nine fold reduction in efficacy. further substitution with a nitro group or an amino group reduces potency up to 4-fold or 18-fold respectively. the stage specific action of these compounds is unknown. further optimization of this class of compounds is required in order to lower the cytotoxicity profile of the compound. in vitro evaluation of a series of n-, s-, and cooh-blocked glutathione derivatives have been carried out by d'silva and daunes [81] against bloodstream form trypanosoma brucei trypomastigotes, to identify the determinants necessary for activity and for further development into an active lead structure. the results shows that n, s-blocked glutathione diesters are the most active inhibitors of t. brucei parasites and that n-acetyl-s-benzyloxycarbonylglutathione dimethyl ester (compound 5) and the n,s-benzyloxycarbonyl-s-2,4dinitrophenylglutathione diester derivatives (compounds 17-19 & 21) (figure 30 ) represent lead structures possessing minimal toxicity which potentially could be developed further to yield a therapeutically active agent for the treatment of trypanosomiasis. pentamidine, furamidine and its analogues lack oral bioavailability [82, 83] .in addition several analogues of furamidine show excellent activity on intravenous dosing but are ineffective on oral administration [83, 84] . generally, oral administration is the preferred dosing regime, and hence, prodrug strategies for diamidines that have the potential to overcome their limited oral bioavailability merit attention. the following works have been performed by boykin dw et al to develop prodrugs. boykin dw et al [85] syhthesized and evaluated five o-alkoxyamidine analogues of the prodrug 2,5-bis [4-methoxyamidinophenyl] furan against trypanosoma brucei rhodesiense in the stib900 mouse model by oral administration. it was observed that the size of the o-alkyl side-chain determined the metabolic stability of the prodrugs. the prodrugs with the o-methyl analogue were most susceptible to metabolism while the larger o-n-butyl and o-n-hexyl groups were least susceptible to metabolism. the in vivo studies in the stib900 mouse model for t. b. rhodesiense showed that compounds with an o-methoxy-amidine or o-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. therefore the o-alkoxyamidine prodrugs, where the alkyl chain is less than three carbons, could effectively be used as prodrugs for amidines. in addition to above mentioned prodrug synthesis boykin dw et al [86] also reported that bis-amidoximes and bis-o-alkylamidoximes of a number of diamidine systems are effective prodrugs. in order to develop orally effective anti-trypanosomal agents, they synthesized these two types of potential prodrugs in the terphenyl series. it was found that compound 10b and 10d ( figure 32 ) showed good activity in the range of 2 nm. among these compounds 10b had lower cytotoxicity (6.4μm) profile but had very poor in vivo activity (cured none of the infected animal in stib900 mouse model). whereas compound 10d showed excellent in vivo activity, by curing all the infected animals upon oral administration in stib900 mouse model. to capitalize on the efficacy of these potent dications, other prodrugs that rely on different bioconversion pathways need to be developed. boykin dw et al [87] showed that some of the dicationic guanidine, n-alkylguanidine, and reversed amidine derivatives of fused ring systems have good in vitro activity against trypanosoma brucei rhodesiense [87, 88] . the dicationic n-isopropylguanidino-9hfluorene (12c, figure 33 ) showed promising in vivo biological results by giving 4/4 cures of the treated animals in the stib900 animal model for african trypanosomiasis. in addition the n-methyl analogue (12a, figure 33 ) also showed high activity giving 3/4 cures of the treated animals in the stib900 animal model for african trypanosomiasis. in order to enhance the oral bioavailability, two novel classes of potential guanidine prodrugs were prepared. the n-alkoxyguanidine derivatives 12d and 12e ( figure 33) were not effective as prodrugs. whereas the carbamate prodrugs (11c, figure 33 ), gave promising results with 4/4 cures on oral administration in the stib900 mouse model. the result showed that these compounds bind strongly to the dna minor groove, but despite strong bonding these compounds do not have high antiparasitic activity. as compared to the last 15 years, there has been a revival of drug research and development regarding neglected parasitic diseases, and a number of drug development projects are currently ongoing. however discovering lead compounds with anti-trypanosomal activity remains a crucial step to sustain the progress achieved till date. the use of computer-assisted drug design (cadd), since their start, has become increasingly helpful in understanding many aspects of chemical-biological interactions in drug and other scientific research. the latest technological advances (qsar, structure-based design, ligand-based design, cheminformatics & bioinformatics) are providing a much improved basis for the design of ligands and inhibitors with desired specificity. recently, computerassisted drug design approaches based on ligand-based and structure-based drug design have been successfully employed to develop new drugs for the treatment of cancer, aids and other diseases [89] [90] [91] [92] [93] [94] [95] [96] . qsar [97] has been widely used for years to provide quantitative analysis of structure and activity relationships of compounds. our core research group has also performed qsar analysis of dicationic diphenylisoxazoles [10] . in this study, attempt has been to investigate the relationship between the various physiochemical parameters and anti-trypanosomal activity of dicationic 3,5-diphenylisoxazoles that may be helpful in development of potent antitrypanosomal agents against sleeping sickness. several statistical expressions have been developed using stepwise multiple linear regression analysis (mlr) and partial least squares (pls). the best mlr model showed good correlative and predictive ability as shown in following equation 84 has also been obtained. the developed model has been validated by leave-one-out method of cross-validation and prediction of test set. the study indicates that the anti-trypanosomal activity is largely explained by cosmic energy, log p and total lipole descriptors. the qsar study has reported in present study provides important structural insights, related to anti-trypanosomal activity. authors have developed a validated and highly predictive model sharing important structural requirement for effective binding of anti-trypanosomal compounds to minor groove of t. b. rhodesiense dna. the model reported in the study should be helpful in development of new compounds with improved efficacy and oral bioavailability. in line to the developed model authors have also designed some molecules which showed good activity in silico. the further study of these compounds is in progress. the poor pharmacokinetic profile and toxicity produced by the drugs currently used to treat trypanosomiasis requires an immediate attention for the development of safe, economical and high affinity chemotherapeutic agents to meet the need for this class of drugs. the unacceptable truth is the lack of attention of the government and less interest of pharmaceutical companies in light of less monitory gain in the area of protozoal infection since most of the affected people belong to poorer country. surprisingly the endeavors of different research group discussed in this review belong to academic institutions. it is felt by the researchers that the treatment for hat requires a combined approach by government organization, pharmaceutical companies and academic institutions. the most promising fact is that in the last decade several synthetic approaches have been made in the field of development of anti-trypanosomal therapies. several compounds are being synthesized that yield new compounds for the treatment of hat, as a result of these synthetic approaches newer leads have been identified, which are under different phase of drug development. the promising in vitro and in vivo activities of dicationic molecules clearly indicate that aromatic diamidines are the most promising class of compounds for the development of newer drugs against hat. the most important one is the newer analogous of existing drugs pentamidine (compounds 32, figure 3e and 66, figure 3b ) which shows excellent in vivo and in vitro activity (0.004 and 0.001 μm respectively) and also has very good selectivity index against the parasite but is cytotoxic. in an attempt to synthesise newer compounds with reduced cytotoxicity than pentamidine molecules, several benzofuran derivatives have been synthesized. however the replacement of phenoxy fragement of pentamidine with a benzofuran motif has resulted in poor analogues with lower antitrypanosomal activity but improved cytotoxic profile. however the strong activity against t. b. rhodesiense isolates indicates that steps should be taken to initiate further studies of compound 66 and compound 32 which can be further evolved as new lead compound. the newer analogues of furamidine also show promising anti-trypanosomal activity along with lower cytotoxicity than furamidine. thus strong activity against t. b. rhodesiense and lower cytotoxicity of compound 3 (figure 13b ) indicates that these compounds should be further evaluated. thus the dna minor grove binders are still the most interesting and potential target for the development of newer anti-trypanosomal agents. apart from diamidines, polyamines and guanidine also have the potential to give antitrypanosomal compounds. the success of dmfo as polyamine inhibitors has attracted researchers for synthesis and development of agents targeting polyamine metabolism. recently polyamines carrying 1,3,5-triazine units have been synthesized and evaluated against t. brucei. these compound exhibited good activity but are cytotoxic. morpholine and dihydroquinolines have also shown promising in vivo anti-trypanosomal activity. in addition to this development of prodrugs, the existing compounds also promise to address the pharmacokinetic related problems, in near future. thus excellent in vitro and in vivo activities and high selectivity of aforementioned compounds merit further investigation in order to reduce the cytotoxicity that may result in development of newer anti-trypanosomal drug with reduced toxicity, improved efficacy and pharmacokinetic profile. as the drug discovery and development process is expensive in terms of time and money, the cross application of existing series of compounds with selective trypanocidal activity may be the best prospect to new anti-trypanosomal drugs in the short term. more emphasis has to be put in the field of cadd approaches for development of anti-trypanosomal agents as cadd study reduces the time and cost required for development of newer analogues. the trypanosomiases african trypanosomiasis (sleeping sickness) the fall and rise of sleeping sickness treatment of human african trypanosomiasis-present situation and needs for research and development chemotherapy of human african trypanosomiasis: current and future prospects human african trypanosomiasis. cook gc, zumla a, editors. in manson's tropical diseases chemotherapy of human african trypanosomiasis current chemotherapy of human african trypanosomiasis quantitative structure activity relationship analysis of dicationic diphenylisoxazole as potent anti-trypanosomal agents new approaches to the development of anti-protozoan drug candidates: a review of patents small molecule dna and rna binders; from synthesis to nucleic acid complexes treatment perspectives for human african trypanosomiasis challenges and new discoveries in the treatment of leishmaniasis treatment and control of human african trypanosomiasis the biochemical basis of arsenicaldiamidine crossresistance in african trypanosomes transporters in african trypanosomes: role in drug action and resistance adenosine transporters in bloodstream forms of trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs a drug resistance determinant in trypanosoma brucei synthesis and trypanocidal activity of the bis-carba analogue of pentamidine structure-activity relationships of analogs of pentamidine against plasmodium falciparum and leishmania mexicana amazonensis analogues of 1,5-bis(4-amidinophenoxy)pentane (pentamidine) in the treatment of experimental pneumocystis carinii pneumonia trypanocidal activity of conformationally restricted pentamidine congeners structure-activity study of pentamidine analogues as antiprotozoal agents synthesis and antiprotozoal activity of 2,5-bis(4-guanylphenyl)furans synthesis and antiprotozoal activity of aza-analogues of furamidine accumulation and intracellular distribution of anti-trypanosomal diamidine compounds db75 and db820 in african trypanosomes synthesis and antiprotozoal activity of pyridyl analogues of pentamidine design and synthesis of a series of melamine-based nitroheterocycles with activity against trypanosomatid parasites trypanocide diamidine mit drei ringen in zwei isolierten ringsystemen trypanocide diamidine mit vier ringen in einem oder zwei ringsystemen synthesen biskationischer, trypanocider 1-benzofuran-verbindungen synthesis and in vitro antiprotozoal activity of bisbenzofuran cations synthesis and antiprotozoal activity of cationic 2-phenylbenzofurans synthesis and antiprotozoal properties of pentamidine congeners bearing the benzofuran motif db-289 immtech international synthesis and antiprotozoal activity of 2, 5-bis(4-guanylphenyl)furans synthesis and anti-trypanosomal activity of some bis(4-guanylphenyl) five-and six-membered ring heterocycles anti-pneumocystis carinii pneumonia activity of dicationic diaryl methylpyrimidines synthesis of dicationic diaryltriazines nucleic acid binding agents 4-diphenylfurandiamidines as novel anti-pneumocystis carinii pneumonia agents trypanocidal diamidines with three isolated ring systems synthesis and in vitro antiprotozoal activities of dicationic 3,5-diphenylisoxazoles superoxide dismutase-like activity of 1, 2, 3-triazole derivatives synthesis and muscarinic activities of quinuclidin-3-yltriazole and -tetrazole derivatives 3-triazoles: synthesis and evaluation of antiinflammatory and analgesic properties i 3-triazoles: synthesis and evaluation of antiinflammatory and analgesic properties ii bioisosteres of arecoline: 1,2,3,6-tetrahydro-5-pyridyl-substituted and 3-piperidyl-substituted derivatives of tetrazoles and 1,2,3-triazoles. synthesis and muscarinic activity synthesis and biological evaluation f novel 2-pyridinyl-[1,2,3]triazoles as inhibitors of transforming rowth factor beta 1 type 1 receptor rapid discovery and structure-activity profiling of novel inhibitors of human immunodeficiency irus type 1 protease enabled by the copper(i)-catalyzed synthesis of 1,2,3-triazoles and their further functionalization synthesis and biological evaluation of 4-aryl-5-cyano-2h-1, 2, 3-triazoles as inhibitor of her2 tyrosine kinase synthesis, hiv-rt inhibitory activity and sar of 1-benzyl-1h-1, 2, 3-triazole derivatives of carbohydrates 3-triazole derivatives as new cannabinoid cb1 receptor antagonists synthesis and cb1 cannabinoid receptor affinity of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles trypanocidal activity of conformationally restricted pentamidine congeners new bis(2-aminoimidazoline) and bisguanidine dna minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity dna binding affinity of bisguanidine and bis(2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity and polyamine derivatives as potent and selective chemotherapeutic agents against trypanosoma brucei rhodesiense. synthesis and in vitro evaluation investigation of tryoanothione reductase as a target in trypanosoma brucei activity of 1,2-dihydroquinolin-6-ols and their ester derivatives activity of indenoisoquinolines against african trypanosomes evaluation of quinolone derivatives for anti-trypanosomal activity biological activity and synthesis of polyamine analogues and conjugates synthesis of polyamines, their derivatives, analogues and conjugates polyamines as targets for therapeutic intervention trypanosoma brucei ornithine decarboxylase -enzyme purification, characterization, and expression in escherichia-coli polyamine metabolism: a potential therapeutic target in trypanosomes advances in sleeping sickness therapy safety and effectiveness of first line eflornithine for trypanosoma brucei gambiense sleeping sickness in sudan: cohort study three drug combinations for late-stage trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in uganda nifurtimox eflornithine combination therapy for second-stage trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in congo cure of trypanosoma brucei brucei and trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of s-adenosylmethionine synthesis and biological evaluation of s-triazine substituted polyamines as potential new anti-trypanosomal drugs anti-trypanosomal activity of 5'-deoxy-5'-(iodomethylene) adenosine and related 6-n-cyclopropyladenosine analogues synthesis of 6' (e and z)-halohomovinyl derivatives of adenosine, inactivation of s-adenosyl-lhomocysteine hydrolase, and correlation of anticancer and antiviral potencies with enzyme inhibition structure, evolution, and inhibitor interaction of s-adenosyl-l-homocysteine hydrolase from plasmodium falciparum synthesis and evaluation of antiparasitic activities of new 4 structure-activity study on the in vitro antiprotozoal activity of glutathione derivatives dicationic diaryl furans as anti-pneumocystis carinii agents anti-pneumocystis activity of aromatic diamidoxime prodrugs 5-bis[4-(n-alkylamidino)phenyl]furans as pneumocystis carinii agents prodrugs of furamidine: in vitro transport and microsomal metabolism as indicators of in vivo efficacy in a mouse model of trypanosoma brucei rhodesiense infection dna affinity, and antiprotozoal activity of linear dications: terphenyl diamidines and analogues synthesis, dna affinity, and antiprotozoal activity of fused ring dicationic compounds and their prodrugs prodrugs for amidines: synthesis and anti-pneumocystis carinii activity of carbamates of 2,5-bis rational design of potent sialidase-based inhibitors of influenza virus replication bis tertiary amide inhibitors of the hiv-1 protease generated via protein structure-based iterative design structure-based inhibitor design by using protein models for the development of antiparasitic agents conformation-activity relationship study of 5-ht3 receptor antagonists and a definition of a model for this receptor site 3-hydroxy-3-methylglutaryl-coenzyme. a reductase: molecular modeling, three-dimensional structureactivity relationships, inhibitor design a 3d model of sars_cov 3cl proteinase and its inhibitors design by virtual screening a novel strategy for improving ligand selectivity in receptor-based drug design corona virus main proteinase (3clpro) structure: basis for design of anti-sars drugs correlation of biological activity of phenoxyacetic acids with hammett substituent constants and partition coefficients 3d qsar on a library of heterocyclic diamidine derivatives with antiparasitic activity activity of bisphosphonates against trypanosoma brucei rhodesiense qsar study on the contribution of log p and es to the in vitro antiprotozoal activity of glutathione derivatives authors pay sincere thanks to prof. aditya shastri, vice chancellor, banasthali university and dr. monali bhattacharga, dept. of english, banasthali university, rajasthan, india. the author declares no conflict of interest key: cord-000536-0mn1gbll authors: hu, le-le; chen, chen; huang, tao; cai, yu-dong; chou, kuo-chen title: predicting biological functions of compounds based on chemical-chemical interactions date: 2011-12-29 journal: plos one doi: 10.1371/journal.pone.0029491 sha: doc_id: 536 cord_uid: 0mn1gbll given a compound, how can we effectively predict its biological function? it is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. in this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) carbohydrate metabolism, (2) energy metabolism, (3) lipid metabolism, (4) nucleotide metabolism, (5) amino acid metabolism, (6) metabolism of other amino acids, (7) glycan biosynthesis and metabolism, (8) metabolism of cofactors and vitamins, (9) metabolism of terpenoids and polyketides, (10) biosynthesis of other secondary metabolites, (11) xenobiotics biodegradation and metabolism. it was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. it is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions. metabolism refers to a collection of chemical reactions in vivo, which keep an unceasing supply of matter and energy for living organisms to maintain life (e.g., growth and reproduction) [1] . these energy-using and energy-releasing chemical reactions catalyzed by enzymes are organized into many metabolic pathways. some compounds/small molecules play major roles in these pathways and are vital for many activities essential for life. for example, during the digestion, the energy rich molecules (i.e. carbohydrate) are broken apart to provide energy, which is then used by cells to build up complex molecules from simple molecules, such as utilizing amino acids to synthesize new proteins that the body needs. identifying the biological functions of compounds is an effective way to study the mechanisms of many basic biological processes [2] . on the other hand, small molecules are the cause, and the cure, for many diseases. for example, diabetes mellitus is a metabolic disease caused by insufficient or inefficient insulin secretary response and elevated blood glucose level [3] . compounds such as sulfonylureas [4] , acarbose [5] , biguanides, thiazolidinediones [5] , and sitagliptin [3] have been used as effective drugs for diabetic therapy. therefore, it is essential to annotate the bioactivities of compounds, which will benefit drug design and disease treatment. besides the conventional biochemical experiments, computational methods are alternative ways to annotate the biological functions of compounds. in recent years, various bioinformatics and structural bioinformatics [6] tools were developed to address this issue, such as quantitative structure activity relationship (qsar) [7, 8] , pharmacophore modeling [9] , molecular docking [10] , and monte carlo simulated annealing approach [11, 12] . different from these methods, lu et al. [1] and cai et al. [2] analyzed the biological functions of compounds by mapping them to the corresponding metabolic pathway classes, which are strongly associated with the biological functions of compounds. the functional group composition was used to represent the compounds, and the nearest neighbor algorithm and adaboost learner [13] were used to construct the prediction models by cai et al. [2] and lu et al. [1] , respectively. both the two prediction methods achieved quite promising results on their own datasets. however, none of their datasets contained the ''multi-function'' compounds that belong to two or more metabolic pathway classes. since these authors were only focused on addressing the singlelabel classification problem, their methods could not be used to deal with the ''multi-function'' compounds. actually, according to kegg [14] , among all the compounds with functional annotations, the ''multi-function'' compounds occupy about 8%. particularly, these multi-function compounds may play some unique role intriguing to both basic research and drug development and hence are worthy of our special attention. recently, the systems biology methods based on protein-protein interactions have been widely applied for predicting protein attributes [15, 16, 17, 18, 19] . these algorithms suggest that interactive proteins are likely to share the common biological functions [16, 17, 18, 19] , also more likely tending to have the same biological function than non-interactive ones [20, 21] . likewise, we can assume that the interactive compounds may tend to share the common biological functions. in this study, the chemical-chemical interactions were retrieved from stitch [22] (search tool for interactions of chemicals), where the interaction unit consists of two chemicals and their interaction weight. the interaction weight (confidence score) represents the probability that the interaction occurs between the two chemicals concerned. the interactive compounds can be classified into the following three categories: (i) ones that participate in the same reactions; (ii) ones that share the similar structures or activities; (iii) ones with the literature associations [22] . in a metabolism system, chemical reactions are organized into many metabolic pathways, thus the compounds involved in the same reactions are in the same metabolic pathways. similar structures or activity means that they share the similar functions, and hence they are likely to be in the same metabolic pathways. the co-occurrence of two compounds in many literatures suggests some kinds of direct or indirect relationships, indicating they have the potential to be in the same metabolic pathways. accordingly, it is rational to suppose that the interactive compounds tend to participate in the same metabolic pathways. in this study, we proposed a multi-target model based on chemical-chemical interactions for predicting the metabolic pathways where compounds participate in. our method sorts the possible metabolic pathways that are associated with the query chemical, providing a more comprehensive view of the biological effects of the compound. according to a recent comprehensive review [23] , to establish a really useful statistical predictor for a biological system, we need to consider the following procedures: (1) construct or select a valid benchmark dataset to train and test the predictor; (2) formulate the statistical samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the attribute to be predicted; (3) introduce or develop a powerful algorithm (or engine) to operate the prediction; (4) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor. below, let us describe how to deal with these steps. the compounds were retrieved from public available database kegg [14] (kyoto encyclopedia of genes and genomes) compound [ftp://ftp.genome.jp/pub/kegg/release/archive/ kegg/42/ligand.tar.gz] (release 42.0). subsequently, these compounds were mapped to the following 11 major metabolic pathway classes that are strongly associated with the biological functions of compounds (http://www.genome.jp/kegg/pathway. table 1 under the title of group-i). from the 4,366 compounds of group-i, 3,137 compounds were retrieved that can interact with any of the others as annotated by stitch database [22] (see table 1 under the title of group-ii). of the 4,366 compounds of group-i, 4,027 are involved in only one metabolic pathway class, 246 in two metabolic pathway classes, 54 in three metabolic pathway classes, 24 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes. of the 3,137 compounds of group-ii, 2,820 are involved in only one metabolic pathway class, 226 in two metabolic pathway classes, 53 in three metabolic pathway classes, 23 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes. note that since one compound may occur in more than one pathway class, the sum of the compounds over the 11 pathway classes in group-i turns out to be 4,860, which is greater than 4,366. likewise, the sum of the compounds over the 11 pathway classes in group-ii is 3,606, which is greater than 3,137. this is quite similar to the case of proteins with multiple location sites, as elaborated in [24, 25] . the chemicals interactions were retrieved from stitch [22] , a large database of known and predicted interactions of chemicals and proteins derived from experiments, literature, databases, and so on. as mentioned in introduction, there are three types of associations between two compounds in stitch: (i) cooccurrence in reactions, (ii) similar structures or activities, and (iii) literature associations. in the downloaded stitch chemicals interactions file: chemical_chemical.links.detailed.v2.0.tsv from http://stitch.embl.de/cgi/show_download_page.pl, there are 337,482 pairs of interactive compounds belonging solely to type i, 73,598 pairs solely in type ii, 2,152,508 pairs solely in type iii, 384 pairs in both type i and ii, 120,936 pairs in both type i and iii, 10,372 pairs in both type ii and iii, and 1,990 pairs in the three types, in total of 2,697,270 interactions. each of the interaction is quantified by the interaction confidence score, which represents the likelihood that the interaction occurs. in this study, the interactions with both interactive compounds occurring in the 4,366 compounds of group-i were extracted. as a result, 3,137 compounds with 75,949 interactions were collected to constitute the benchmark dataset of the current study (see table 1 under the title of group-ii). besides the 4,366 compounds (cf. table 1 under the title of group-i) with known metabolic pathway classes, there are 11,661 compounds without known metabolic pathway classes in kegg. among these compounds, 5,549 compounds that have annotated interactions with the compounds of the 4,366 compounds in stitch were collected. such 5,549 compounds are to form an independent dataset, being used to test our prediction method in hopes to acquire useful information for further investigation. as mentioned in introduction, the interactive compounds tend to participate in the same metabolic pathways. accordingly, for a query compound, the higher interaction confidence score with its interactive compound, the more likely they are to participate in the same metabolic pathway. the more its interactive compounds involving in a certain metabolic pathway, the more likely it is to participate in such metabolic pathway. based on these points, we should count not only the number of compounds interacting with the query compound, but also the corresponding interaction scores. thus, the desired predictor can be formulated via the following procedures. suppose the training dataset contains n compounds, which are denoted as fc 1 ,c 2 ,:::,c n g. the 11 metabolic pathway classes (cf. table 1 ) are expressed as fp 1 ,p 2 ,:::,p 11 g, where p 1 represents the 1 st metabolic pathway class (''carbohydrate metabolism''), p 2 the 2 nd metabolic pathway class (''energy metabolism''), p 3 the 3 rd metabolic pathway class (''lipid metabolism''), and so forth. thus, the descriptor of metabolic pathway classes to which the compound c i belongs to can be formulated as p(c i )~½p i,1 ,p i,2 ,:::,p i,j ,:::,p i,11 t (i~1,2,:::,n; j~1,2,:: where given a query compound c q , its interaction with the compounds in the training dataset can be defined as w (c q )~½w q,1 ,w q,2 ,:::,w q,i ,:: where w q,i represents the interaction confidence score between c q and c i . t is the transpose operator, and w q,i~0 if no interaction exists between them. here, we did not consider the selfinteraction, therefore w q,i~0 when q~i. accordingly, the likelihood that the query compound c q is involved in the j-th metabolic pathway class can be formulated by the following score which is the sum of the interaction confidence scores of c q with its interactive compounds in the training dataset by counting both the number of interactive compounds and the interaction confidence scores. obviously, the higher the score of eq. 4, the more likely c q is to be involved in the j-th metabolic pathway c j . thus, for a given query compound c q , we can use eq. 4 to calculate its 11 scores, with each associated with one of the 11 metabolic pathway classes. the class to which the compound c q most likely belongs should be the one with the highest score. in other words, the query compound c q will be predicted to belong to the mth metabolic pathway class if m~arg max j s(c q [j)jj~1,2,:::,11 where m is the argument of j that maximize the value of s(c q [j). since the problem in this study is of multi-label classification, we intend to provide flexible information by predicting some candidate metabolic pathway classes for the query compounds, rather than just the most likely metabolic pathway class. therefore, instead of eq. 5, let us consider the following equation containing 11 scores in a one-column vector: where d ; is a descending operator that sorts the 11 scores of eq. 4 for s(c q [j) according to the descending order (s 1 §s 2 § á á á §s j § á á á §s 11 ). if there is a tie among these scores, a random order will be made among those with a tie. consequently, the predicted metabolic pathway classes for the query compound can be derived according to the descending order of eq. 6; i.e., if s 1~s (p k [6), s 2~s (p k [1), s 3~s (p k [10) , then it follows that the query compound c q is involved in the 6 th metabolic pathway class (''metabolism of other amino acids'') will be ranked as the highest in the likelihood, that c q in the 1 st metabolic pathway class (''carbohydrate metabolism'') as the 2 nd , and that c q in the 10 th metabolic pathway class (''biosynthesis of other secondary metabolites'') as the 3 rd . the corresponding results thus obtained are, respectively, called the 1 st -order, 2 nd -order, and 3 rd -order predicted metabolic pathway classes. and so forth. in statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (such as 5-fold, 7-fold, or 10-fold cross-validation) test, and jackknife test [26] . in this study, the 5-fold cross-validation was employed to examine the performance of our method. the concrete procedures were that the training dataset were divided into five groups by splitting each of its subsets into five approximately equal-sized subgroups. each of these five groups was in turn used as a testing dataset and the rest used as training dataset, thereby generating five different success rates, with their average representing the success rate by the 5-fold cross-validation. for the j-th order prediction, the accuracy w j was calculated by where m j is the number of the compounds whose j-th order predicted metabolic pathway class is one of the true pathway classes that the compounds are involved with, and n is the total number of compounds in the dataset. such 11-order accuracies were used to evaluate our prediction method. it is obvious according to the definition of eq. 7 that, the higher the value of w j with a smaller value of j, or the lower the value of w j with a larger value of j, the better the prediction quality will be by our method. in the dataset, the average number of metabolic pathway class that each compound is involved in is calculated as where e i is the number of metabolic pathway classes that the compound c i is involved with. hence, another measurement -the likelihood that the first k order predicted metabolic pathway classes cover all the true metabolic pathway classes that the compound is involved in -can be formulated as usually, k is the smallest integer equal or greater than the average number of metabolic pathway classes (h). it is obvious from eq. 9 that the larger the value of l k , the better the prediction quality will be by our method. given a query compound, according to the information of its interactions with the 4,366 compounds in group-i ( table 1 ) whose metabolic pathway classes are known, the likelihood of its belonging to each of the 11 metabolic pathway classes can be easily calculated according to eq. 4. and the scores thus obtained were sorted according to a descending order (eq. 6) to yield the predicted metabolic pathway classes according to their different ranks or orders. in this study, our method was evaluated by the 5-fold crossvalidation on the benchmark dataset that contains 3,137 compounds in group-ii of table 1 . the 11-order prediction accuracies are shown in figure 1 . the first order (most likely) prediction accuracy is 77.97%, and the last order (least likely) prediction accuracy is 0.38%, which indicates a quite good performance of our method. the average number of metabolic pathway classes with which each compound is involved is 1.15 (cf. eq. 8), meaning that the average success rate by a random guess would be 1.15/ 11 = 10.45%, which is much lower than that by our method. accordingly, the parameter k in eq. 9 was set to (1.15+1) = 2; i.e., we may select the results of the first two orders of the predicted metabolic pathway classes for the query compounds. as we can see from figure 1 , the accuracies of both the 1 st and 2 nd order predictions are higher than that of the random guess. according to eq. 9 the metabolic pathway classes predicted by the 1 st and 2 nd orders have actually covered more than 80% of all the true metabolic pathway classes, suggesting that, of the results predicted by the 11 orders, more attention should be paid to those by the first two orders. listed in table 2 are the accuracies by each of the 11 prediction orders for the 3,137 compounds about their involvement in the 11 metabolic pathway classes using the 5-fold crossvalidation test. the highest accuracy achieved by the 1 st -order prediction was 80.96% for the 1 st metabolic pathway class (''carbohydrate metabolism''). and the results obtained by the 1 st and 2 nd prediction orders have covered 89.00% of the true metabolic pathway classes. the second highest accuracy by the 1 storder prediction was 78.77% for the 11 th metabolic pathway class (xenobiotics biodegradation and metabolism), while the results obtained by the 1 st and 2 nd prediction orders have covered 87.00% of the true metabolic pathway classes. both the two 1 st -order accuracies are higher than the overall 1 st -order prediction accuracy of 77.97%, and each of their combinations with the 2 nd -order predictions is also higher than the overall likelihood of 80.00%. as for the metabolic pathway classes with less compounds, such as ''glycan biosynthesis and metabolism'' class that contains only 68 compounds in group-i and 43 in group-ii (cf . table 1) , the predicted accuracies were relatively not as good as the others. it is anticipated that with more experimental data are available in future for the compounds in these classes, the corresponding prediction success rates will be improved. overall speaking, the aforementioned results are quite encouraging, indicating that our approach may become a useful tool to deal with this kind of very complicated systems. as stated in the method section, the interactive compounds derived from stitch tend to participate in the same metabolic pathways. for example, table 3 lists the interactions of dihydrouracil with other compounds. among the 32 interactive compounds, most of them appear in ''metabolism of cofactors and vitamins'' or ''metabolism of other amino acids'' or ''nucleotide metabolism'' pathway class (cf. table 1 ) just like dihydrouracil. dihydrouracil and uracil participate in pyrimidine metabolism pathway (belong to ''nucleotide metabolism''), where 5,6-dihydrouracil and nadp+ are catalyzed by dihydropyrimidine dehydrogenase (dpd) to form uracil and nadph+h+ [14, 27] . they are also co-mentioned in many pubmed abstracts such as [28, 29, 30, 31, 32, 33, 34, 35, 36, 37] . another two interactive compounds -dihydrouracil and dihydrothymine share a very similar structure, the only difference is that dihydrothymine has a methyl at the 5th position of the hexatomic ring while dihydrouracil has not [38] . according to the prediction criteria, when dihydrouracil was treated as a query compound, the first three order predicted metabolic pathways that it participates in are ''nucleotide metabolism'', ''metabolism of cofactors and vitamins'' and ''metabolism of other amino acids'', respectively, which are consistent with the true metabolic pathways that it is involved in. predicted results for the compounds with unknown metabolic pathway encouraged by the quite promising results obtained by the 5fold cross-validation test on the benchmark dataset of the 3,137 compounds, we applied the method to the 5,549 compounds whose metabolic pathways are unknown as mentioned in the materials and methods section. the predicted results thus obtained are given in table s1 . as discussed above, we selected the metabolic pathway classes obtained by the 1 st and 2 nd order predictions for these compounds, in hoping that the information thus obtained may provide useful clues for further investigations. actually, it is interesting to see that many of our predicted results have proved to be reasonable according to the reports from other investigators. for example, n-acetylgalactosamine 4-sulfate and its interactive compounds with pathway information are shown in table 4 . n-acetylgalactosamine 4-sulfate can bind to sulfate, glucuronic acid, galactose, xylose, fucose, na(+), glycerol, and phosphate to form complex to perform the biological function [39] . in pubmed abstracts, n-acetylgalactosamine 4-sulfate is comentioned with sulfate [40] , glucuronic acid [41] , galactose [42] , 39-phospho.pho. [43] , sugar-1-phosph. [44] , udp-glcnac [45] , indole-3-glyce. [46] , n-acetyl-d-glucosamine [47] , and gdpmannose [44] . besides, n-acetylgalactosamine 4-sulfate and nacetyl-d-glucosamine share a similar structure and the difference is that n-acetylgalactosamine 4-sulfate has a sulfate at the position 4 of the ring while n-acetyl-d-glucosamine has not [38] . from these evidences, n-acetylgalactosamine 4-sulfate is supposed to participate in the same metabolic pathways as its interactive compounds. it can be seen from table 4 that most of the interactive compounds of n-acetylgalactosamine 4-sulfate belong to the 1 st and 2 nd metabolic pathway classes. by considering all the interactions and the interaction confidence scores, it was predicted that carbohydrate metabolism (the 1 st class) and energy metabolism (the 2 nd class) would be the possible metabolic pathway classes that n-acetylgalactosamine 4-sulfate belongs to. actually, as a carbohydrate, n-acetylgalactosamine 4-sulfate reacts with chondroitin 4-sulfate to form hydrogen oxide and g12336 (i.e. (galnac) 2 (glca) 1 (s) 2 ), one kind of glycan which can participate in carbohydrate and energy metabolism. therefore, n-acetylgalactosamine 4-sulfate may also participate in carbohydrate and energy metabolism. another example is that cyclopropylamine in table 4 has 23 interactive compounds with known pathway information. cyclopropylamine, cyanuric acid, ammonia, n-cyclopropylammelide, c0761, hydroxyl radicals are in the same pathway -n-cyclopropylmelamine degradation [48, 49] , where n-cyclopropylmelamine first reacts with hydrogen oxide to form n-cyclopropylammeline and ammonia, and then n-cyclopropylammeline also reacts with hydrogen oxide to form ncyclopropylammelide and ammonia. after that, n-cyclopropylammelide reacts with hydrogen oxide to form cyanuric acid, cyclopropylamine and hydroxyl radicals. finally, cyanuric acid is transformed into hydrogen oxide and ammonia through cyanurate degradation. cyanuric acid, n-cyclopropylammelide, and c0761 are all in the 11 th pathway class. therefore, cyclopropylamine may also belong to the 11 th pathway class (xenobiotics biodegradation and metabolism). for other interactive compounds, they are comentioned with cyclopropylamine in pubmed abstracts, such as polyethylene [50] , 1-aminocyclopropane-1-carboxylic acid [51] , cyclopropanecarboxylic acid [52] , 3-hydroxyphenylacetic acid [53] , and acetophenone [54] . in table 4 , most of the interactive compounds of cyclopropylamine belong to the 11 th metabolic pathway classes. according to above analysis, cyclopropylamine is suggested to participate in the xenobiotics biodegradation metabolism, which was the 1 st -order predicted class for cyclopropylamine by our method. accordingly, it is quite reasonable to expect that our method may provide useful information for further investigating into biological functions of compounds from the viewpoint of system biology. as indicated by the above discussion and analysis, the results derived from the 1 st and 2 nd order predictions should be considered as the candidates for the metabolic pathway classes with which the query compound may be involved. in view of this, biochemical experiments should be conducted by mainly focusing on the targets predicted by the 1 st and 2 nd order predictions. the results obtained by the last five order predictions can be ignored due to their very low likelihood (,2%). consequently, the current prediction method can provide useful clues for further validation by experiments and expedite the research progress by prioritizing the targets concerned. it is instructive to note that for the 4,366 compounds in group-i of table 1 , there are still 1,229 compounds that can not be processed by the current method due to lack of the interaction information with other compounds within the dataset. it is expected that the problem can be solved by collecting as much chemical-chemical interaction information as possible from stitch, which is a large-scale and well-maintained resource in chemical biology, including the interactions information for over 2.5 million proteins and over 74,000 small molecules in 630 organisms. with the continuous increase of the interactions information, the performance of our method will be further improved. based on the chemical-chemical interactions information, a multi-target model was proposed for identifying the metabolic pathway classes with which a query compound is involved. since some compounds may be involved with more than one metabolic pathway class, our method is featured by the capacity able to provide a series of potential metabolic pathway classes for each of the query compounds investigated, instead of only one metabolic pathway class. it is anticipated that our method may become a useful tool in helping annotate the compound for their biological functions. table s1 each order predicted metabolic pathway class for the collected 5,549 compounds without known metabolic pathway classes. the predicted metabolic pathway class code corresponds to the code in table 1 . among the 11 predicted pathway classes, the first 2 order predicted metabolic pathway classes should be paid more attention to. 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oxidase by n-cyclopropylbenzylamine the authors are very much indebted to the two anonymous reviewers for their constructive comments, which were very helpful for strengthening the presentation of this paper. many thanks are also to kegg and stitch for providing data to support the current study. key: cord-016425-8yd2bkf1 authors: strobel, gary; daisy, bryn; castillo, uvidelio title: novel natural products from rainforest endophytes date: 2005 journal: natural products doi: 10.1007/978-1-59259-976-9_15 sha: doc_id: 16425 cord_uid: 8yd2bkf1 endophytic microorganisms are found in virtually every higher plant on earth. these organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to pathogenic. endophytes may contribute to their host plant by producing a plethora of substances that provide protection and survival value to the plant. ultimately, these compounds, once isolated and characterized, may also have potential for use in modern medicine. novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation and culturing of individual endophytes followed by purification and characterization of some of their natural products. the potential of finding new drugs that may be effective candidates for treating newly developing diseases in humans is great. the need for new and useful compounds to provide assistance and relief in all aspects of the human condition is ever growing. drug resistance in bacteria, the appearance of new life-threatening viruses, the recurrent problems of diseases in persons with organ transplants, and the tremendous increase in the incidence of fungal infections in the world's population all underscore our inadequacy to cope with these medical problems. environmental degradation, loss of biodiversity, and spoilage of land and water also add to problems facing humanity, and each of these in turn can have health-related consequences. endophytes, microorganisms that reside in the tissues of living plants, are relatively unstudied as potential sources of novel natural products for exploitation in medicine. however, some of the most extensive and comprehensive work on natural products produced by endophytes has been done on the neotyphodium sp. found on grasses (1) . alkaloids synthesized by this fungus in its grass hosts have been implicated in fescue toxicosis in rangeland animals (1) . the chemistry and biology of this and other grass endophytes are reviewed elsewhere (2) . unfortunately, because this work is so comprehensive, one may be led to the conclusion that endophytes produce toxic compounds only in their respective hosts and hold no promise for any medicinal applications whatsoever (2) . it turns out that this is simply not the case. as endophytes are examined from a plethora of sources, an overwhelming number have been found to produce natural products with promising potential for medicinal applications. of the approx 300,000 higher plant species that exist on the earth, each individual plant, of the billions that exist here, is host to one or more endophytes. only a handful of these plants (grass species) have ever been completely studied relative to their endophytic biology (2) . consequently, the opportunity to find new and interesting endophytic microorganisms among myriads of plants in different settings and ecosystems is very great. the intent of this review is to provide insights into their occurrence in nature, the products that they make, and indicate how some of these organisms are beginning to show some potential for human use. the majority of the report discusses rationale for study, methods used, and examples of a number of endophytes isolated and studied in the authors' laboratories over the course of many years. this review, however, also includes some specific examples that illustrate the work of others in this emerging field of bioprospecting the microbes of the world's rainforests. there is a general call for new antibiotics, and for chemotherapeutic agents that are highly effective and possess low toxicity. this search is driven by the development of resistance in infectious microorganisms (e.g., staphylococcus, mycobacterium, streptococcus) to existing drugs and by the menacing presence of naturally resistant organisms. the ingress to the human population of new disease-causing agents such as acquired immunodeficiency syndrome (aids), ebola, and severe acute respiratory syndrome (sars) requires the discovery and development of new drugs to combat them. not only do diseases such as aids require drugs that target them specifically, but new therapies are needed for treating ancillary infections, which are a consequence of a weakened immune system. furthermore, others who are immunocompromised (e.g., cancer and organ transplant patients) are at risk of infection by opportunistic pathogens, such as aspergillus, cryptococcus, and candida, which normally are not major problems in the human population. in addition, more drugs are needed to efficiently treat parasitic protozoan and nematodal infections such as malaria, leishmaniasis, trypanomiasis, and filariasis. malaria by itself is more effective in claiming lives each year than any other single infectious agent with the exception of aids and tuberculosis (tb) (3) . however, the enteric diseases claim the most lives each year of any disease complex, and unfortunately, the victims are mostly children (3) . novel natural products and the organisms that make them offer opportunities for innovation in drug discovery. exciting possibilities exist for those who are willing to venture into the wild and unexplored territories of the world to experience the thrill of engaging in the discovery of endophytes, their biology, and potential usefulness. it may also be true that a reduction in interest in natural products for use in drug development has happened as a result of people growing weary of dealing with the traditional sources of bioactive compounds, including plants of the temperate zones and microbes from a plethora of soil samples gathered in different parts of the world by armies of collectors. in other words, why continue to do the same thing when robots, combinatorial chemistry, and molecular biology have arrived on the scene? furthermore, the logic and rationale for time and effort spent on drug discovery using a targetsite-directed approach has been overwhelming. while combinatorial synthesis produces compounds at random, secondary metabolites, defined as low-molecular-weight compounds not required for growth in pure culture, are produced as an adaptation for specific functions in nature (4) . shutz notes that certain microbial metabolites seem to be characteristic of certain biotopes, both on an environmental as well as organismal level (5) . accordingly, it appears that the search for novel secondary metabolites should center on organisms that inhabit unique biotopes. thus, it behooves the investigator to carefully study and select the biological source before proceeding, rather than to take a totally random approach in selecting the source material. careful study also indicates that organisms and their biotopes that are subjected to constant metabolic and environmental interactions should produce even more secondary metabolites (5) . endophytes are microbes that inhabit such biotopes, namely higher plants, which is why they are currently considered as a wellspring of novel secondary metabolites offering the potential for exploitation of their medical benefits. in addition, it also is extremely helpful for the investigator interested in exploiting endophytes to have access to, or have some expertise in, microbial taxonomy, and this includes modern molecular techniques involving sequence analyses of 16s and 18s rdna. currently, endophytes are viewed as an outstanding source of bioactive natural products because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. thus, it would appear that a myriad of biotypical factors associated with plants can be important in the selection of a plant for study. it may be the case that these factors may govern which microbes are present in the plant as well as the biological activity of the products associated with these organisms. since the discovery of endophytes in darnel, germany, in 1904, various investigators have defined endophytes in different ways, which usually depended on the perspective from which the endophytes were being isolated and subsequently examined (6) . bacon et al. give an inclusive and widely accepted definition of endophytes: "microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects" (2) . while the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests they can also be aggressive saprophytes or opportunistic pathogens. both fungi and bacteria are the most common microbes existing as endophytes. it would seem that other microbial forms most certainly exist in plants as endophytes, such as mycoplasmas, rickettsia, and archebacteria; however, no evidence for them has yet been presented. the most frequently isolated endophytes are the fungi (7) . it turns out that the vast majority of plants have not been studied for their endophytes. thus, enormous opportunities exist for the recovery of novel fungal forms, including genera, biotypes, as well as species in the myriad of plants yet to be studied. hawksworth and rossman estimated there may be as many as 1 million different fungal species, yet only approx 100,000 have been described (8) . as more evidence accumulates, estimates keep rising as to the actual number of fungal species. for instance, dreyfuss and chapela estimate there may be at least 1 million species of endophytic fungi alone (9) . it seems obvious that endophytes are a rich and reliable source of genetic diversity and may represent previously undescribed species. finally, in our experience, novel microbes (as defined at the morphological and/or molecular levels) often have novel natural products associated with them. this fact alone helps eliminate the problems of dereplication in compound discovery. it is important to understand the methods and rationale used seem to provide the best opportunities to isolate novel endophytic microorganisms at the genus, species, or biotype level. thus, since the number of plant species in the world is so great, creative and imaginative strategies must be used to quickly narrow the search for endophytes displaying bioactivity (10) . a specific rationale for the collection of each plant for endophyte isolation and natural product discovery is used. several hypotheses govern this plant selection strategy, and these are as follows: 1. plants from unique environmental settings, especially those with an unusual biology, and possessing novel strategies for survival, are seriously considered for study. 2. plants that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. these plants are chosen either by direct contact with local peoples or via local literature. ultimately, it may be learned that the healing powers of the botanical source, in fact, may have nothing to do with the natural products of the plant, but of the endophyte inhabiting the plant. 3. plants that are endemic, having an unusual longevity, or that have occupied a certain ancient land mass, such as gondwanaland, are also more likely to lodge endophytes with active natural products than other plants. 4 . plants growing in areas of great biodiversity, it follows, also have the prospect of housing endophytes with great biodiversity. just as plants from a distinct environmental setting are considered to be a promising source of novel endophytes and their compounds, so too are plants with an unconventional biology. for example, an aquatic plant, rhyncholacis penicillata, was collected from a river system in southwest venezuela where the harsh aquatic environment subjected the plant to constant beating by virtue of rushing waters, debris, and tumbling rocks and pebbles (11) . these environmental insults created many portals through which common phytopathogenic oomycetes could enter the plant. still, the plant population appeared to be healthy, possibly owing to protection by an endophytic product. this was the environmental biological clue used to pick this plant for a comprehensive study of its endophytes. eventually, an unusual and potent antifungal strain of serratia marcescens, living both as an epiphyte and an endophyte, was recovered from r. penicillata. this bacterium was shown to produce oocydin a, a novel antioomycetous compound having the properties of a chlorinated macrocyclic lactone ( fig. 1) (11) . it is conceivable that the production of oocydin a by s. marcescens is directly related to the endophyte's relationship with its higher-plant host. currently, oocydin a is being considered for agricultural use to control the ever-threatening presence of oomyceteous fungi such as pythium spp. and phytophthora spp. oocydin a also has activity against a number of rapidly dividing cancer cell lines (11) . plants with ethnobotanical history, as mentioned above, also are likely candidates for study, since the medical uses for which the plant was selected may relate more to its population of endophytes than to the plant biochemistry itself. for example, a sample of the snakevine, kennedia nigriscans, from the northern territory of australia, was selected for study since its sap has traditionally been used as bush medicine for many millenia. in fact, this area was selected for plant sampling because it has been home to the world's longest standing civilization-the australian aborigines. the snakevine is harvested, crushed, and heated in an aqueous brew by local aborigines in southwest arnhemland to treat cuts, wounds, and infections. as it turned out, the plant contained a streptomycete that possessed unique partial 16s rdna sequences when compared to those in genbank. the organism was designated streptomyces nrrl 30562, and it produces broad-spectrum novel peptide antibiotics called munumbicins, which are discussed below (12) . it seems likely that some of the healing properties in plants, as discovered by indigenous peoples, might be facilitated by compounds produced by one or more specific plant-associated endophytes as well as the plant products themselves. in addition, it is worthy to note that some plants generating bioactive natural products have associated endophytes that produce the same natural products. such is the case with taxol, a highly functionalized diterpenoid and famed anticancer agent that is found in each of the world's yew tree species (taxus spp.) (11, 12) . in 1993, a novel taxol-producing fungus, taxomyces andreanae, from the yew taxus brevifolia, was isolated and characterized (13). of the myriad of ecosystems on earth, those having the greatest general biodiversity seem to be the ones also having the greatest number and most diverse endophytes. tropical and temperate rainforests are the most biologically diverse terrestrial ecosystems on earth. the most threatened of these spots cover only 1.44% of the land's surface, yet they harbor over 60% of the world's terrestrial biodiversity (10) . in addition, each of the 20-25 areas identified as supporting the world's greatest biodiversity support unusually high levels of plant endemism (10) . as such, one would expect, with high plant endemism, there also should exist specific endophytes that may have evolved with the endemic plant species. biological diversity implies chemical diversity, because of the constant chemical innovation that is required to survive in ecosystems where the evolutionary race to survive is most active. tropical rainforests are a remarkable example of this type of environment. competition is great, resources are limited, and selection pressure is at its peak. this gives rise to a high probability that rainforests are a source of novel molecular structures and biologically active compounds (14) . bills et al. describe a metabolic distinction between tropical and temperate endophytes through statistical data that compare the number of bioactive natural products isolated from endophytes of tropical regions to the number of those isolated from endophytes of temperate origin (15) . not only did they find that tropical endophytes provide more active natural products than temperate endophytes, but they also noted that a significantly higher number of tropical endophytes produced a larger number of active secondary metabolites than did fungi from other substrata. this observation suggests the importance of the host plant as well as the ecosystem in influencing the general metabolism of endophytic microbes. tan and zou believe the reason why some endophytes produce certain phytochemicals, originally characteristic of the host, might be related to a genetic recombination of the endophyte with the host that occurred in evolutionary time (6) . this is a concept that was originally proposed as a mechanism to explain why t. andreanae may be producing taxol (16) . thus, if endophytes can produce the same rare and important bioactive compounds as their host plants, this would not only reduce the need to harvest slow-growing and possibly rare plants, but also help to preserve the world's everdiminishing biodiversity. furthermore, it is recognized that a microbial source of a high-value product may be easier and more economical to produce effectively, thereby reducing its market price. all aspects of the biology and interrelatedness of endophytes with their respective hosts is a vastly under-investigated and exciting field (17, 18) . thus, more background information on a given plant species and its microorganismal biology would be exceedingly helpful in directing the search for bioactive products. presently, no one is quite certain of the role of endophytes in nature and their relationship to various host plant species. although some endophytic fungi appear to be ubiquitous (e.g., fusarium spp., pestalotiopsis spp., and xylaria spp.), one cannot definitively state that endophytes are truly host-specific or even systemic within plants, any more than one can assume that their associations are chance encounters. frequently, many endophytes of the same species are isolated from the same plant, and only one or a few biotypes of a given fungus will produce a highly biologically active compound in culture (19) . a great deal of uncertainty also exists between what an endophyte produces in culture and what it may produce in nature. it does seem possible that the production of certain bioactive compounds by the endophyte in situ may facilitate the domination of its biological niche within the plant or even provide protection to the plant from harmful invading pathogens. furthermore, little information exists relative to the biochemistry and physiology of the interactions of the endophyte with its host plant. it would seem that many factors changing in the host, related to the season, age, environment, and location, may influence the biology of the endophyte. indeed, further research at the molecular level must be conducted in the field to study endophyte interactions and ecology. all of these interactions are probably chemically mediated for some purpose in nature. an ecological awareness of the role these organisms play in nature will provide the best clues for targeting particular types of endophytic bioactivity with the greatest potential for bioprospecting. after a plant is selected for study, it is identified, and its location is plotted using a global positioning device. small stem pieces are cut from the plant and placed in sealed plastic bags after excess moisture is removed. every attempt is made to store the materials at 4â°c until isolation procedures can begin (20, 21) . in the laboratory, the surfaces of plant materials are thoroughly treated with 70% ethanol, sometimes flamed, and ultimately they are air dried under a laminar-flow hood. this is done in order to eliminate surface-contaminating microbes (20) . then, with a sterile knife blade, outer tissues are removed from the samples and the inner tissues carefully excised and placed on water agar plates. after several days of incubation, hyphal tips of the fungi are removed and transferred to potato dextrose or other suitable agar. bacterial forms also emerge from the plant tissues, including, on rare occasions, certain streptomyces spp. the endophytes are encouraged to sporulate on specific plant materials and are eventually identified via standard morphological and molecular biological techniques and methods. eventually, when an endophyte is acquired in pure culture, it is tested for its ability to be grown in shake or still culture using various media and growth conditions (21) . it is also immediately placed in storage under various conditions including 15% glycerol at -70â°c. ultimately, once appropriate growth conditions are found, the microbe is subjected to fermentation, extraction, and the bioactive compounds are isolated and characterized. virtually all of the common and advanced procedures for product isolation and characterization are utilized in order to acquire the product(s) of interest. central to the processes of isolation is the establishment of one or more bioassays that will guide the compound purification processes. one cannot put too much emphasis on this point, since the ultimate success of any natural-product isolation activity is directly related to the development or selection of appropriate bioassay procedures. these can involve target organisms, enzymes, tissues, or model chemical systems that relate to the purpose for which the new compound is needed. the following section shows some examples of natural products obtained from endophytic microbes and their potential in the pharmaceutical and agrochemical arenas. many of the examples are taken from our work, and thus, this review is by no means inclusive of all natural-product work in endophytes. fungi are the most commonly isolated endophytic microbes. they usually appear as fine filaments growing from the plant material on the agar surface. generally, the most commonly isolated fungi are in the group fungi imperfecti or deuteromycetes. basi-cally, they produce asexual spores in or on various fruiting structures. also, it is quite common to isolate endophytes that are producing no fruiting structures whatsoever, such as mycelia sterilia. quite commonly endophytes do produce secondary metabolites when placed in culture. however, the temperature, the composition of the medium, and the degree of aeration will affect the amount and kind of compounds that are produced. sometimes endophytic fungi produce antibiotics. natural products from endophytic fungi have been observed to inhibit or kill a wide variety of harmful microorganisms including, but not limited to, phytopathogens, as well as bacteria, fungi, viruses, and protozoans that affect humans and animals. described below are some examples of bioactive products from endophytic fungi. cryptosporiopsis cf. quercina is the imperfect stage of pezicula cinnamomea, a fungus commonly associated with hardwood species in europe. it was isolated as an endophyte from tripterigeum wilfordii, a medicinal plant native to eurasia (22) . on petri plates, c. quercina demonstrated excellent antifungal activity against some important human fungal pathogens, including candida albicans and trichophyton spp. a unique peptide antimycotic, termed "cryptocandin," was isolated and characterized (22) . this compound contains a number of peculiar hydoxylated amino acids and a novel amino acid, 3-hydroxy-4-hydroxy methyl proline (fig. 2) . the bioactive compound is related to known antimycotics-the echinocandins and the pneumocandins (23) . as is generally true, not one but several bioactive and related compounds are produced by an endophytic microbe. thus, other antifungal agents related to cryptocandin are also produced by c. quercina. cryptocandin is also active against a number of plant pathogenic fungi, including sclerotinia sclerotiorum and botrytis cinerea. cryptocandin and its related compounds are currently being considered for use against a number of fungi causing diseases of the skin and nails. cryptocin, a unique tetramic acid, is also produced by c. quercina (discussed previously) (fig. 3)(24) . this unusual compound possesses potent activity against pyricularia oryzae, the causal organism of one of the worst plant diseases in the world, as well as a number of other plant pathogenic fungi (24) . the compound was generally ineffective against a general array of human pathogenic fungi. nevertheless, with minimum inhibitory concentrations against p. oryzae at 0.39 î¼g/ml, this compound is being examined as a natural chemical control agent for rice blast and is being used as a platform for the synthesis of other antifungal compounds. as mentioned earlier, p. microspora is a common rainforest endophyte (17) (18) (19) (20) . it turns out that enormous biochemical diversity does exist in this endophytic fungus, and many secondary metabolites are produced by various strains of this widely dispersed organism. one such secondary metabolite is ambuic acid, an antifungal agent, which has been recently described from several isolates of p. microspora found as representative isolates in many of the world's rainforests (fig. 4) (25) . this compound as well as another endophyte product, terrein, have been used as models to develop new solidstate nuclear magnetic resonance (nmr) tensor methods to assist in the characterization of the molecular stereochemistry of organic molecules. a strain of p. microspora was also isolated from the endangered tree torreya taxifolia and produced several compounds having antifungal activity, including pestaloside, an aromatic î²-glucoside (fig. 5) , and two pyrones-pestalopyrone and hydroxypestalopyrone (26) . these products also possess phytotoxic properties. other fig. 4 . ambuic acid, a highly functionalized cyclohexenone produced by a number of isolates of pestalotiopsis microspora found in rainforests around the world. this compound possesses antifungal activity and has been used as a model compound for the development of solid-state nuclear magnetic resonance methods for the structural determination of natural products. newly isolated secondary products obtained from p. microspora (endophytic on taxus brevifolia) include two new caryophyllene sesquiterpenes-pestalotiopsins a and b (27) . additional new sesquiterpenes produced by this fungus are 2î±-hydroxydimeninol and a highly functionalized humulane (28, 29) . variation in the amount and kinds of products found with this fungus depends on both the cultural conditions as well as the original plant source from which it was isolated. pestalotiopsis jesteri is a newly described endophytic fungal species from the sepik river area of papua new guinea, and it produces jesterone and hydroxyjesterone, which exhibit antifungal activity against a variety of plant pathogenic fungi (30) . these compounds are highly functionalized cyclohexenone epoxides. jesterone, subsequently, has been prepared by organic synthesis with complete retention of biological activity (fig. 6) (31) . jesterone is one of only a few products from endophytic microbes in which total synthesis of a bioactive product has been successfully accomplished. phomopsichalasin, a metabolite from an endophytic phomopsis sp., represents the first cytochalasin-type compound with a three-ring system replacing the cytochalasin macrolide ring. this metabolite exhibits antibacterial activity in disk diffusion assays (at a concentration of 4 î¼g/disk) against bacillus subtilis, salmonella gallinarum, and staphylococcus aureus. it also displays moderate activity against the yeast candida tropicalis (32) . an endophytic fusarium sp. from the plant selaginella pallescens, collected in the guanacaste conservation area of costa rica, was screened for antifungal activity. a new pentaketide antifungal agent, cr377, was isolated from the culture broth of the fungus and showed potent activity against c. albicans in agar diffusion assays (33) . colletotric acid, a metabolite of colletotrichum gloeosporioides, an endophytic fungus isolated from artemisia mongolica, displays activity against bacteria as well as against the fungus helminthsporium sativum (34) . another colletotrichum sp., isolated from artemisia annua, produces antimicrobial metabolites as well. a. annua is a traditional chinese herb that is well recognized for its synthesis of artemisinin (an antimalarial drug) and its ability to inhabit many geographically different areas. not only did the colletotrichum sp. found in a. annua produce metabolites with activity against human pathogenic fungi and bacteria, but also metabolites that were fungistatic to plant pathogenic fungi (35). there are only a limited number of bacterial species known to be associated with plants, and one of the most common is pseudomonas spp. pseudomonas has representative biotypes and species that are epiphytic, endophytic, and pathogenic. they have been reported from every continent including the antarctic. some of these species produce phytotoxic compounds as well as antibiotics. the ecomycins are produced by pseudomonas viridiflava (36) . this bacterium is generally associated with the leaves of many grass species and is located on and within the tissues (36) . the ecomycins represent a family of novel lipopeptides and have masses of 1153 and 1181. besides common amino acids such as alanine, serine, threonine, and glycine, some nonprotein amino acids are incorporated into the structure of the ecomycins, including homoserine and î²-hydroxyaspartic acid (36) . the ecomycins are active against such human pathogenic fungi as cryptococcus neoformans and c. albicans. the pseudomycins produced by a plant-associated pseudomonad are another group of antifungal peptides (37, 38) . they are active against a variety of plant and human pathogenic fungi, including candida albicans, cryptococcus neoformans, and a variety of plant pathogenic fungi, including ceratocystis ulmi (the dutch elm disease pathogen) and mycosphaerella fijiensis (causal agent of black sigatoka disease in bananas). the pseudomycins are cyclic depsipeptides formed by acylation of the oh group of the n-terminal serine with the terminal carboxyl group of l-chlorothreonine. variety in this family of compounds is imparted via n-acetylation by one of a series of fatty acids, including 3,4-dihydroxydecanoate, 3-hydroxy-tetradecanoate (38) . the pseudomycins contain several nontraditional amino acids, including l-chlorothreonine, l-hydroxyaspartic acid, and both d-and l-diaminobutryic acid. the molecules are candidates for use in human medicine, especially after structural modification by chemical synthesis has successfully eliminated mammalian toxicity (39) the pseudomycins are also effective against a number of ascomycetous fungi, and are being considered for agricultural use for the control of the black sigatoka disease in bananas (strobel, unpublished). streptomyces spp. are filamentous bacteria, belonging to the order actinomycetales, that live in widely diverse ecological settings. generally, this group is gram positive, has a high g+c content, and does not have an organized nucleus. to date, actinomycetes have been the world's greatest source of natural antibiotics (40) . in fact, just one genus, streptomyces, is the source of 80% of these compounds. the majority of the antibiotic producers are from soil sources, and until recently it was not realized that these organisms can exist as endophytes. one of the first endophytic streptomyces spp. isolated was that from lolium perenne, a grass species (41) . this isolate produces a diketopiperazine that is a weak antibiotic and has been designated "methylalbonoursin" (41) . using the ethnobotanical approach to plant selection, the snakevine plant, k. nigriscans, was chosen as a possible source of endophytic microbes because of its long-held traditional use by australian aborigines to treat cuts and open wounds, resulting in reduced infection and rapid healing. this plant was collected near the aboriginal community of manyallaluk in northern territory, australia, and consistently yielded an endophytic actinomycete designated streptomyces nrrl 30562 (12) . the organism was not found in several tree species supporting the vine, suggesting a host-selective or -specific association of the endophyte with a specific plant genus. this streptomycete produces a family of extremely potent peptide antibiotics, and these compounds may not only protect the plant from fungal and bacterial infections, but also have unknowingly served the aborigines as a source of bush medicine. the antibiotics produced by streptomyces nrrl 30562, called "munumbicins," possess widely differing biological activities, depending on the target organism. in general, the munumbicins demonstrate activity against gram-positive bacteria such as bacillus anthracis and multidrug-resistant mycobacterium tuberculosis, as well as a number of other drug-resistant bacteria. however, the most impressive biological activity of any of the munumbicins is that of munumbicin d against the malarial parasite plasmodium falciparum, having an ic 50 of 4.5 â± 0.07 ng/ml (12) . the munumbicins are highly functionalized peptides, each containing threonine, aspartic acid (or asparagine), and glutamic acid (or glutamine). since the peptides are yellowish orange, they also contain one or more chromophoric groups, whose structures have not been determined. their masses range from 1269 to 1326 da. the isolation of this endophytic streptomycete represents an important finding, providing one of the first examples of plants serving as reservoirs of actinomycetes. more than 40 of these endophytic streptomycetes, now in hand in our laboratory, possess antibiotic activity (castillo, u., strobel, g.a., unpublished data). endophytic actinomycetes are now being tested and considered for use in controlling plant diseases (42) . another endophytic streptomyces sp. (nrrl 30566), from a fern-leaved grevillea (grevillea pteridifolia) tree growing in the northern territory of australia, produces novel antibiotics called "kakadumycins," which are related to the echinomycins (43) . each of these antibiotics contains alanine, serine, and an unknown amino acid. kakadumycin a has wide-spectrum antibiotic activity similar to that of munumbicin d, especially against gram-positive bacteria, and it generally displays better bioactivity than echinomycin. for instance, against b. anthracis strains, kakadumycin a has mics of 0.2-0.3 î¼g/ ml, in contrast to echinomycin at 1.0-1.2 î¼g/ml. both echinomycin and kakadumycin a have impressive activity against p. falciparum, with ld 50 s in the range of 7-10 ng/ml (43) . kakadumycin a and echinomycin are related by virtue of their very similar structures (amino acid content and quinoxoline rings), but differ slightly with respect to their elemental compositions, aspects of their spectral qualities, chromatographic retention times, and biological activities (53) . echinomycin and kakadymycin a were studied as inhibitors of macromolecular synthesis, with control substances such as ciprofloxacin, rifampin, chloramphenicol, and vancomycin used as standards with well-established modes of action. tests were done for dna, rna, protein, and cell-wall synthesis inhibition activities, respectively. kakadumycin a significantly inhibited rna synthesis in b. subtilis (43) . kakadumycin a also inhibited protein synthesis and cell-wall synthesis substantially, but had a lower effect on dna synthesis. kakadumycin a shares a very similar inhibitory profile with echinomycin in four macromolecular synthesis assays. kakadumycin a preferentially inhibits rna synthesis, and may have the same mode of action as echinomycin, which inhibits rna synthesis by binding to a dna template (53) . more recently, endophytic streptomycetes have been discovered in an area of the world claimed to be one of the most biologically diverse-the upper amazon of peru. the inner tissues of the follow me vine, monstera sp., commonly yielded a verticillated streptomycete with outstanding inhibitory activities against pythiaceous fungi as well as the malarial parasite plasmodium falciparum. the bioactive component is a mixture of lipopeptides named "coronamycins" (44). another fascinating use of products from endophytic fungi is the inhibition of viruses. two novel human cytomegalovirus (hcmv) protease inhibitors, cytonic acids a and b, have been isolated from solid-state fermentation of the endophytic fungus cytonaema sp. their structures were elucidated as p-tridepside isomers by ms and nmr methods (45) . it is apparent that the potential for the discovery of compounds having antiviral activity from endophytes is in its infancy. the main limitation to com-pound discovery to date is probably related to the absence of common antiviral screening systems in most compound-discovery programs. muscodor albus is a newly described endophytic fungus obtained from small limbs of cinnamomum zeylanicum (cinnamon tree) (46) . this xylariaceaous (non-spore producing) fungus effectively inhibits and kills certain fungi and bacteria by producing a mixture of volatile compounds (47) . the majority of these compounds have been identified by gas chromatography (gc)/ms, synthesized or acquired, and then formulated into an artificial mixture. this mixture not only mimicked the antibiotic effects of the volatile compounds produced by the fungus, but also was used to confirm the identity of the majority of the volatiles emitted by this organism (47) . each of the five classes of volatile compounds produced by the fungus had some microbial effects against the test fungi and bacteria, but none was lethal. however, they acted synergistically to cause death in a broad range of plant and human pathogenic fungi and bacteria. the most effective class of inhibitory compounds was the esters, of which isoamyl acetate was the most biologically active. the composition of the medium on which m. albus grows dramatically influences the kind of volatile compounds that are produced (48) . the ecological implications and potential practical benefits of the "mycofumigation" effects of m. albus are very promising, given the fact that soil fumigation utilizing methyl bromide will soon be illegal in the united states. methyl bromide is not only a hazard to human health, but it has been implicated in causing destruction of the ozone layer. the potential use of mycofumigation to treat soil, seeds, and plants may soon be a reality. the artificial mixture of volatile compounds may also have usefulness in treating seeds, fruits, and plant parts in storage and while being transported. muscodor albus already has a limited market for the treatment of human wastes. its gases have both inhibitory and lethal effects on such fecal-inhabiting organisms as escherichia coli and vibrio cholera. using m. albus as a screening tool, it has now been possible to isolate other endophytic fungi producing volatile antibiotics. the newly described m. roseus was obtained twice from tree species growing in the northern territory of australia. this fungus is just as effective in causing inhibition and death of test microbes in the laboratory as m. albus (49) . in addition, for the first time, a nonmuscodor species (gliocladium sp.) was discovered as a producer of volatile antibiotics. the volatile components of this organism are totally different from those of either m. albus or m. roseus. in fact, the most abundant volatile inhibitor is [8] -annulene, formerly used as a rocket fuel and discovered for the first time as a natural product. however, the bioactivity of the volatiles of this gliocladium sp. is not as good or comprehensive as those from muscodor spp. (21, 47) . taxol and some of its derivatives represent the first major group of anticancer agents that are produced by endophytes (fig. 6) . taxol (fig. 7) , a highly functionalized diterpenoid, is found in each of the world's yew (taxus) species, but was originally isolated from taxus brevifolia (50) . the original targets for this compound were ovarian and breast cancers, but now it is used to treat a number of other human tissue proliferating diseases as well. the presence of taxol in yew species prompted the study of their endophytes. by the early 1990s, however, no endophytic fungi had been isolated from any of the world's representative yew species. after several years of effort, a novel taxol-producing endophytic fungus, taxomyces andreanae, was discovered in taxus brevifolia (13) . the most critical line of evidence for the presence of taxol in the culture fluids of this fungus was the electrospray mass spectrum of the putative taxol isolated from t. andreanae. in electrospray mass spectroscopy, taxol usually gives two peaks-one at mass 854, which is m+h + , and the other at 876, which is m+na + . fungal taxol had a mass spectrum identical to authentic taxol (16) . then, 14 c labeling studies showed the presence of fungal-derived taxol in the culture medium (26) . this early work set the stage for a more comprehensive examination of the ability of other taxus species and many other plants to yield endophytes producing taxol. some of the most commonly found endophytes of the world's yews and many other plants are pestalotiopsis spp. (17) (18) (19) (20) . one of the most frequently isolated endophytic species is pestalotiopsis microspora (17) . an examination of the endophytes of taxus wallichiana yielded p. microspora, and a preliminary monoclonal antibody test indicated that it might produce taxol (20) . after preparative tlc, a compound was isolated and shown by spectroscopic techniques to be taxol. labeled ( 14 c) taxol was produced by this organism from several 14 c precursors (20) . furthermore, several other p. microspora isolates that produce taxol were obtained from a bald cypress tree in south carolina (19) . this was the first indication that endophytes, residing in plants other than taxus spp., produce taxol. therefore, a specific search was conducted for taxolproducing endophytes on continents not known for any indigenous taxus spp., e.g., south america and australia. from the extremely rare, and previously thought to be fig. 7 . taxol, the world's first billion-dollar anticancer drug, is produced by many endophytic fungi. it too, possesses outstanding anti-oomycete activity. extinct, wollemi pine (wollemia nobilis), pestalotiopsis guepini was isolated, which was shown to produce taxol (51) . also, quite surprisingly, a rubiaceous plant, maguireothamnus speciosus, yielded a novel fungus, seimatoantlerium tepuiense, that produces taxol. this endemic plant grows on the top of the tepuis in the venzuelan-guyana border in southwest venezuela (52) . furthermore, fungal taxol production has also been noted in periconia sp. (53) and seimatoantlerium nepalense, another novel endophytic fungal species (54) . simply, it appears that the distribution of taxol-making fungi is worldwide and is not confined to endophytes of yews. the ecological and physiological explanation for fungi making taxol seems to be related to the fact that taxol is a fungicide, and the organisms most sensitive to it are plant pathogens such as pythium spp. and phytophthora spp. (55) . these pythiaceous organisms are some of the world's most important plant pathogens and are strong competitors with endophytic fungi for niches within plants. in fact, their sensitivity to taxol is based on their interaction with tubulin, in an identical manner as in rapidly dividing human cancer cells (55) . thus, bona fide endophytes may be producing taxol and related taxanes to protect their respective host plant from degradation and disease caused by these pathogens. other investigators have also made observations on taxol production by endophytes, including the discovery of taxol production by tubercularia sp. isolated from the chinese yew (taxus mairei) in the fujian province of southeastern mainland china (56) . at least three endophytes of taxus wallichiana produce taxol, including sporormia minima and trichothecium sp. (57) . using hplc and esims, taxol has been discovered in corylus avellana cv. gasaway (58) . several fungal endophytes of this plant (filbert) produce taxol in culture (58) . it is important to note, however, that taxol production by all endophytes in culture is in the range of sub-micrograms to micrograms per liter. also, commonly, the fungi will attenuate taxol production in culture, with some possibility for recovery, if certain activator compounds are added to the medium (53) . efforts are being made to determine the feasibility of making microbial taxol a commercial possibility, e.g., the discovery of endophytes that make large quantities of one or more taxanes that could then be used as intermediates for the organic synthesis of taxol or one of its anticancer relatives. torreyanic acid, a selectively cytotoxic quinone dimer and potential anticancer agent, was isolated from a p. microspora strain (fig. 8) . this strain was originally obtained as an endophyte associated with the endangered tree torreya taxifolia (florida torreya) (59) . torreyanic acid was tested in several cancer cell lines, and it demonstrated 5 to 10 times more potent cytotoxicity in lines that are sensitive to protein kinase c agonists; it causes cell death by apoptosis. recently, torreyanic acid has been successfully synthesized by a biomimetic oxidation/dimerization cascade (60) . alkaloids are also commonly found in endophytic fungi. fungal genera such as xylaria, phoma, hypoxylon, and chalara are representative producers of a relatively large group of substances known as the cytochalasins, of which more than 20 are now known. many of these compounds possess antitumor and antibiotic activities, but because of their cellular toxicity they have not been developed into pharmaceuticals. three novel cytochalasins have recently been reported from rhinocladiella sp., as an endophyte on tripterygium wilfordii. these compounds have antitumor activity and have been identified as 22-oxa[12] -cytochalasins (61) . thus, it is not uncommon to find one or more cytochalasins in endophytic fungi, and this provides an example of the fact that redundancy in discovery does occur, making dereplication an issue even for these under-investigated sources. two compounds, pestacin and isopestacin, have been obtained from culture fluids of pestalotiopsis microspora, an endophyte isolated from a combretaceaous plant, terminalia morobensis, growing in the sepik river drainage system of papua new guinea (62, 63) . both pestacin and isopestacin display antimicrobial as well as antioxidant activity. isopestacin was attributed with antioxidant activity based on its structural similarity to the flavonoids (fig. 9) . electron spin resonance spectroscopy confirmed this antioxidant activity; the compound is able to scavenge superoxide and hydroxyl free radicals in solution (62) . pestacin was later described from the same culture fluid, occurring naturally as a racemic mixture and also possessing potent antioxidant activity (fig. 10) (63) . the proposed antioxidant activity of pestacin arises primarily via cleavage of an unusually reactive c-h bond and, to a lesser extent, through o-h abstraction (63) . the antioxidant activity of pestacin is at least one order of magnitude more potent than that of trolox, a vitamin e derivative (63). a nonpeptidal fungal metabolite (l-783,281) was isolated from an endophytic fungus (pseudomassaria sp.) collected from an african rainforest near kinshasa in the democratic republic of the congo (64). this compound acts as an insulin mimetic but, unlike insulin, is not destroyed in the digestive tract and may be given orally. oral administration of l-783,281 in two mouse models of diabetes resulted in significant lowering of blood glucose levels. these results may lead to new therapies for diabetes. immunosuppressive drugs are used today to prevent allograft rejection in transplant patients, and in the future they could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes. the endophytic fungus fusarium subglutinans, isolated from t. wilfordii, produces the immunosuppressive but noncytotoxic diterpene pyrones subglutinols a and b (fig. 11) (65) . subglutinol a and b are equipotent in the mixed lymphocyte reaction (mlr) assay and thymocyte proliferation (tp) assay, with an ic f f 50 of 0.1 î¼m. in the same assay systems, the famed immunosuppressant drug cyclosporin a, also a fungal metabolite, was roughly as potent in the mlr assay and 10 4 more potent in the tp assay. still, the lack of toxicity associated with subglutinols a and b suggests that they should be explored in greater detail as potential immunosuppressants (65). of some compelling interest is an explanation as to how the genes for taxol production may have been acquired by p. microspora (66) . although the complete answer to this question is not at hand, relevant genetic studies have been performed on this organism. p. microspora ne 32 is one of the most easily genetically transformable fungi that have been studied to date. in vivo addition of telomeric repeats to foreign dna generates extrachromosomal dnas in this fungus (66) . repeats of the telomeric sequence 5'-ttaggg-3' were appended to nontelomeric transforming dna termini. the new dnas, carrying foreign genes and the telomeric repeats, replicated independently of the chromosome and expressed the information carried by the foreign genes. the addition of telomeric repeats to foreign dna is unusual among fungi. this finding may have important implications in the biology of p. microspora ne 32, because it explains at least one mechanism as to how new dna can be captured by this organism and eventually expressed and replicated. such a mechanism may begin to explain how the enormous biochemical variation may have arisen in this fungus (19) . also, this initial work represents a framework to aid in the understanding of how this fungus may adapt itself to the environment of its plant hosts and suggests that the uptake of plant dna into its own genome may occur. in addition, the telomeric repeats have the same sequence as human telomeres, and this points to the possibility that p. microspora may serve as a means to make artificial human chromosomes, a totally unexpected result. endophytes are a poorly investigated group of microorganisms that represent an abundant and dependable source of bioactive and chemically novel compounds with potential for exploitation in a wide variety of medical applications. the mechanisms through which endophytes exist and respond to their surroundings must be better understood in order to be more predictive about which higher plants to seek, study, and employ in isolating microfloral components. this may facilitate the natural-product discovery process. although work on the utilization of this vast resource of poorly understood microorganisms has just begun, it has already become obvious that an enormous potential for organism, product, and utilitarian discovery in this field holds exciting promise. this is evidenced by the discovery of a wide range of products, and microorganisms that present potential. it is important for all involved in this work to realize the importance of acquiring the necessary permits from governmental, local, and other sources to pick and transport plant materials (especially from abroad) from which endophytes are to be eventually isolated. in addition to this aspect of the work is the added activity of producing the necessary agreements and financial sharing arrangements with indigenous peoples or governments in case a product does develop an income stream. certainly, one of the major problems facing the future of endophyte biology and natural-product discovery is the rapidly diminishing rainforests, which hold the greatest possible resource for acquiring novel microorganisms and their products. the total land mass of the world that currently supports rainforests is about equal to the area of the united states (10) . each year, an area the size of vermont or greater is lost to clearing, harvesting, fire, agricultural development, mining, or other human-oriented activities (10) . presently, it is estimated that only a small fraction (10-20%) of what were the original rainforests existing 1000-2000 yr ago, are currently present on the earth (10). the advent of major negative pressures on them from these human-related activities appears to be eliminating entire mega-life forms at an alarming rate. few have ever expressed information or opinions about what is happening to the potential loss of microbial diversity as entire plant species disappear. it can only be guessed that this loss is also happening, perhaps at the same frequency as the loss of mega-life forms, especially because certain microorganisms may have developed unique specific symbiotic relationships with their plant hosts. thus, when a plant species disappears, so too does its entire suite of associated endophytes and consequently all of the capabilities that they might possess to make natural products with medicinal potential. multistep processes are needed now to secure information and life forms before they are lost. areas of the planet that represent unique places housing biodiversity need immediate preservation. countries need to establish information bases of their biodiversity and at the same time begin to make national collections of microorganisms that live in these areas. endophytes are only one example of a life-form source that holds enormous promise to impact many aspects of human existence. the problem of the loss of biodiversity should be one of concern to the entire world. coevolution of fungal endophytes with grasses: the significance of secondary metabolites microbial endophytes niaid global health research plan for hiv/aids, malaria and tuberculosis industrial microbiology bioactive fungal metabolites-impact and exploitation. british mycological society endophytes: a rich source of functional metabolites endophytic fungi in grasses and woody plants where are the undescribed fungi? potential of fungi in the discovery of novel, low-molecular weight pharmaceuticals hotspots: earth's biologically richest and most endangered ecoregions. washington dc. cemex conservation international oocydin a, a chlorinated macrocyclic lactone with potent anti-oomycete activity from serratia marcescens munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl 30562, endophytic on kennedia nigriscans taxomyces andreanae a proposed new taxon for a bulbilliferous hyphomycete associated with pacific yew lessons from nature: can ecology provide new leads in the search for novel bioactive chemicals from rainforests? recent and future discoveries of pharmacologically active metabolites from tropical fungi taxol and taxane production by taxomyces andreanae microbial gifts from rain forests rainforest endophytes and bioactive products endophytic taxol producing fungi from bald cypress taxodium distichum taxol from pestalotiopsis microspora, an endophytic fungus of taxus wallichiana an endophytic gliocladium sp. of eucryphia cordifolia producing selective volatile antimicrobial compounds cryptocandin, a potent antimycotic from the endophytic fungus cryptosporiopsis cf. quercina inhibitors of î²-glucan synthesis cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina ambuic acid, a highly functionalized cyclohexenone with antifungal activity from pestalotiopsis spp. and monochaetia sp the relationship between an endangered north americn tree and an endophytic fungus pestalotiopsin-a and pestalotiopsin-b-new caryophyllenes from an endophytic fungus of taxus brevifolia a new isodrimeninol from pestalotiopsis sp metabolites of endophytic fungi of taxus brevifoliathe first highly functionalized humulane of fungal origin jesterone and hydroxy-jesterone antioomycete cyclohexenenone epoxides from the endophytic fungus pestalotiopsis jesteri exploring chemical diversity of epoxyquinoid natural products: synthesis and biological activity of jesterone and related molecules phomopsichalasin, a novel antimicrobial agent from an endophytic phomopsis sp cr377, a new pentaketide antifungal agent isolated from an endophytic fungus metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua ecomycins, unique antimycotics from pseudomonas viridiflava pseudomycins, a family of novel peptides from pseudomonas syringae, possessing broad spectrum antifungal activity structure of the pseudomycins, new lipodepsipeptides produced by pseudomonas syringae msu 16h synthesis and antifungal activities of novel 3-amido bearing pseudomycin analogs practical streptomycetes genetics. the john innes foundation biosynthesis of 1-n-methylalbonoursin by an endophytic streptomyces sp endophytic actinomycetes: attractive biocontrol agents kakadumycins, novel antibiotics from streptomyces sp. nrrl 30566, an endophyte of grevillea pteridifolia coronamycins, peptide antibiotics produced by a verticillated streptomyces sp. (msu-2110) endophytic on monstera sp cytonic acids a & b: novel tridepside inhibitors of hcmv protease from the endophytic fungus cytonaema species muscodor albus gen. et sp. nov., an endophyte from cinnamomum zeylanicum volatile antimicrobials from a novel endophytic fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus muscodor roseus anna. nov. an endophyte from grevillea pteridifolia plant antitumor agents,v1. the isolation of taxol, a novel antitumor agent from taxus brevifolia pestalotiopsis guepinii, a taxol producing endophyte of the wollemi pine, wollemia nobilis seimatoantlerium tepuiense gen. nov. a unique epiphytic fungus producing taxol from the venezuelan guyana the induction of taxol production in the endophytic fungus periconia sp. from torreya grandifolia seimatoantlerium nepalense, an endophytic taxol producing coelomycete from himalayan yew (taxus wallichiana) antifungal properties of taxol and various analogues taxol from tubercularia sp. strain tf5, an endophytic fungus of taxus mairei evidence for paclitaxel from three new endophytic fungi of himalayan yew of nepal bioprospecting for taxol in angiosperm plant extracts torreyanic acid: a selectively cytotoxic quinone dimer from the endophytic fungus pestalotiopsis microspora total synthesis of the quinine epoxide dimer (+) torreyanic acid: application of a biomimetic oxidation/ electrocyclization/diels-alder dimerization cascade three new chytochalasins produced by an endophytic fungus in the genus rhinocladiella ispoestacin, an isobenzofuranone from pestalotiopsis microspora, possessing antifungal and antioxidant activities pestacin: a 1,3 -dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities discovery of small molecule insulin mimetic with antidiabetic activity in mice subglutinols a & b: immunosuppressive compounds from the endophytic fungus fusarium subglutinans in vivo addition of telomeric repeats to foreign dna generates chromosomal dnas in the taxol-producing fungus pestalotiopsis microspora we thank dr. gene ford and dr. david ezra for helpful discussions. the authors express appreciation to the nsf, usda, novozymes biotech, nih, the bard foundation of israel, the r&c board of the state of montana, and the montana agricultural experiment station for providing financial support for some of the work reviewed in this chapter. key: cord-003539-tazd6dvm authors: yang, kun; jin, ming-ji; quan, zhe-shan; piao, hu-ri title: design and synthesis of novel anti-proliferative emodin derivatives and studies on their cell cycle arrest, apoptosis pathway and migration date: 2019-03-02 journal: molecules doi: 10.3390/molecules24050884 sha: doc_id: 3539 cord_uid: tazd6dvm emodin is a cell arrest and apoptosis-inducing compound that is widely distributed in different plants (rhubarb, aloe), lichens and terrestrial fungi, and also isolated from marine-derived fungi and marine sponge-associated fungi. in this study, we designed and synthesized a novel series of emodin derivatives by binding emodin to an amino acid using linkers of varying lengths and composition, and evaluated their anti-proliferative activities using hepg2 cells (human hepatic carcinoma), mcf-7 cells (human breast cancer) and human normal liver l02 cells. most of these derivatives showed moderate to potent anti-proliferative activities. notably, compound 7a exhibited potent anti-proliferative activity against hepg2 cells with the half maximal inhibitory concentration (ic(50)) value of 4.95 µm, which was enhanced 8.8-fold compared to the parent compound emodin (ic(50) = 43.87 µm), and it also exhibited better selective anti-proliferative activity and specificity than emodin. moreover, further experiments demonstrated that compound 7a displayed a significant efficacy of inducing apoptosis through mitochondrial pathway via release of cytochrome c from mitochondria and subsequent activation of caspase-9 and caspase-3, inducing cell arrest at g0/g1 phase, as well as suppression of cell migration of tumor cells. the preliminary results suggested that compound 7a could be a promising lead compound for the discovery of novel anti-tumor drugs and has the potential for further investigations as an anti-cancer drug. emodin (3-methyl-1,6,8-trihydroxyanthraquinone, figure 1 ) is a naturally occurring anthraquinone derivative that is widely distributed in different plants (rhubarb, aloe), lichens and terristrial fungi, that has also been isolated from marine-derived fungi and marine sponge-associated fungi [1] [2] [3] [4] . it is also an active ingredient in chinese medicinal herbs used in the treatment of constipation, jaundice, gastro-intestinal hemorrhage, and ulcers. emodin has the same tricyclic planar chromophore skeleton as certain anti-tumor antibiotics such as daunorubicin, doxorubicin, epirubicin, and several synthetic analogs distributed such as mitoxantrone and pixantrone ( figure 1) ; all of which are in used for clinical treatment of various cancers. many reports have demonstrated that emodin possesses a wide spectrum of pharmacological effects such as anti-tumour [5] [6] [7] , anti-inflammatory [8, 9] , antiviral [10] , antibacterial [11] , anti-allergic [12] , anti-osteoporotic [13] , anti-diabetic [14, 15] , immunosuppressive [16] , neuroprotective [17] , and hepatoprotective [18, 19] activities, etc. among these effects, anti-tumor anti-osteoporotic [13] , anti-diabetic [14, 15] , immunosuppressive [16] , neuroprotective [17] , and hepatoprotective [18, 19] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [20] , inducing of cell differentiation [21] and apoptosis [22] , and activating of caspase cascade pathway [23, 24] and mitochondrial death pathway [25, 26] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [27] [28] [29] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [30] , and tan et al. reported dna-binding pyrazole emodin derivatives [31] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [32] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [33] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure 2 ). among these derivatives, compound 3b derived from (r)-2-aminopropanoic acid exhibited the most anti-proliferative activity against both hepg2 cells and mcf-7 cells. this prompted us to screen the different length of diol linker between 2-aminopropanoic acid and emodin. our results also suggest that compared to compound 3a, compound 7a exhibited a slightly more potent chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [27] [28] [29] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [30] , and tan et al. reported dna-binding pyrazole emodin derivatives [31] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [32] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [33] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure 2 ). anti-osteoporotic [13] , anti-diabetic [14, 15] , immunosuppressive [16] , neuroprotective [17] , and hepatoprotective [18, 19] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [20] , inducing of cell differentiation [21] and apoptosis [22] , and activating of caspase cascade pathway [23, 24] and mitochondrial death pathway [25, 26] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [27] [28] [29] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [30] , and tan et al. reported dna-binding pyrazole emodin derivatives [31] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [32] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [33] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure 2 ). among these derivatives, compound 3b derived from (r)-2-aminopropanoic acid exhibited the most anti-proliferative activity against both hepg2 cells and mcf-7 cells. this prompted us to screen the different length of diol linker between 2-aminopropanoic acid and emodin. our results also suggest that compared to compound 3a, compound 7a exhibited a slightly more potent among these derivatives, compound 3b derived from (r)-2-aminopropanoic acid exhibited the most anti-proliferative activity against both hepg2 cells and mcf-7 cells. this prompted us to screen the different length of diol linker between 2-aminopropanoic acid and emodin. our results also suggest that compared to compound 3a, compound 7a exhibited a slightly more potent anti-proliferative activity against hepg2 cells and mcf-7 cells. following this, the anti-cancer mechanism of compound 7a was evaluated against hepg2 cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in schemes 1-3. for the synthesis of compounds 3a-3z, compound 1 was subjected to an alkylation reaction with 2-iodoethanol and cs 2 co 3 in dmf that resulted in compound 2. then coupling reactions under the conventional coupling condition (dcc/dmap) between compound 2 and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng 20% tfa in dcm, the target compounds 3a-3z were obtained as tfa salts. molecules 2019, 24, x for peer review 3 of 23 anti-proliferative activity against hepg2 cells and mcf-7 cells. following this, the anti-cancer mechanism of compound 7a was evaluated against hepg2 cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in scheme 1-3. for the synthesis of compounds 3a-3z, compound 1 was subjected to an alkylation reaction with 2-iodoethanol and cs2co3 in dmf that resulted in compound 2. then coupling reactions under the conventional coupling condition (dcc/dmap) between compound 2 and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng 20% tfa in dcm, the target compounds 3a-3z were obtained as tfa salts. following the dcc/dmap coupling condition and deprotection under 20% tfa in dcm for the synthesis of compounds 3a-3z, the target compounds 4a and 4b were obtained as tfa salts (scheme 2). scheme 1. synthesis of compounds 3a-3z. reagents and conditions: (a) 2-iodoethanol, cs 2 co 3 , dmf, 60 • c, 65%; (b) (i) various n-boc amino acids, dcc, dcm, 0 • c; (ii) 20% tfa in dcm, r.t., 20%-50% over two steps. following the dcc/dmap coupling condition and deprotection under 20% tfa in dcm for the synthesis of compounds 3a-3z, the target compounds 4a and 4b were obtained as tfa salts (scheme 2). these products were then coupled with formaldehyde (37% aqueous solution) and reduced utilizing nabh3cn to provide 5a and 5b as tfa salt in 85% and 89% yields, respectively. as shown in scheme 3, by employing the route for the synthesis of compounds 3a-3z, compounds 7a-7l were obtained as tfa salts. scheme 3. synthesis of compounds 7a-7l. reagents and conditions: (a) various hydroxyalkyl bromides or iodides, cs2co3, dmf, 60 °c, 30%-55%; (b) (i) r/s-n-boc-ala-oh, dcc, dcm, 0 °c; (ii) 20% tfa in dcm, r.t., 65%-70% over two steps. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg2 cells and mcf-7 cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin (1) and compound 2 were also included for comparison. as shown in table 1 , emodin (1) showed weak inhibitory activity against hepg2 cells and mcf-7 cells with ic50 values of 43.87 ± 1.28 µm and 52.72 ± 2.22 µm, respectively. compared to emodin (1), compound 2 showed reduced activity due to the introduction of the hydroxyethyl group at the 3-position of emodin. among compounds 3a-3z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin (1) . compound 3a with the gly group displayed near double the activity of emodin (1) against hepg2 cells, but had slightly decreased activity against mcf-7 cells. compound 3b with the d-ala group displayed the most potent anti-proliferative activity against hepg2 cells and mcf-7 cells with ic50 values of 8.22 ± 0.13 µm and 8.99 ± 0.64 µm, respectively. compound 3c with the l-ala group displayed slightly weaker activity than compound 3b, but still displayed stronger activity than emodin (1). compounds 3d-3j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin (1) . overall, with the exception of compound 3n, compounds 3k-3m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin (1); especially compound 3m containing a heterocycle. interestingly, compound 3n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg2 cells and mcf-7 cells with ic50 values of 12.48 ± 0.59 µm scheme 2. synthesis of compounds 4a, 4b, 5a and 5b. reagents and conditions: (a) (i) boc-n-me-r/ s-ala-oh, dcc, dcm, 0 • c; (ii) 20% tfa in dcm, r.t., 40%-50% over two steps; (b) formaldehyde (37% aqueous solution), nabh 3 cn, meoh, r.t., 85%-89%. these products were then coupled with formaldehyde (37% aqueous solution) and reduced utilizing nabh 3 cn to provide 5a and 5b as tfa salt in 85% and 89% yields, respectively. as shown in scheme 3, by employing the route for the synthesis of compounds 3a-3z, compounds 7a-7l were obtained as tfa salts. these products were then coupled with formaldehyde (37% aqueous solution) and reduced utilizing nabh3cn to provide 5a and 5b as tfa salt in 85% and 89% yields, respectively. as shown in scheme 3, by employing the route for the synthesis of compounds 3a-3z, compounds 7a-7l were obtained as tfa salts. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg2 cells and mcf-7 cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin (1) and compound 2 were also included for comparison. as shown in table 1 , emodin (1) showed weak inhibitory activity against hepg2 cells and mcf-7 cells with ic50 values of 43.87 ± 1.28 µm and 52.72 ± 2.22 µm, respectively. compared to emodin (1), compound 2 showed reduced activity due to the introduction of the hydroxyethyl group at the 3-position of emodin. among compounds 3a-3z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin (1) . compound 3a with the gly group displayed near double the activity of emodin (1) against hepg2 cells, but had slightly decreased activity against mcf-7 cells. compound 3b with the d-ala group displayed the most potent anti-proliferative activity against hepg2 cells and mcf-7 cells with ic50 values of 8.22 ± 0.13 µm and 8.99 ± 0.64 µm, respectively. compound 3c with the l-ala group displayed slightly weaker activity than compound 3b, but still displayed stronger activity than emodin (1). compounds 3d-3j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin (1) . overall, with the exception of compound 3n, compounds 3k-3m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin (1); especially compound 3m containing a heterocycle. interestingly, compound 3n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg2 cells and mcf-7 cells with ic50 values of 12.48 ± 0.59 µm the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg2 cells and mcf-7 cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin (1) and compound 2 were also included for comparison. as shown in table 1 , emodin (1) showed weak inhibitory activity against hepg2 cells and mcf-7 cells with ic 50 values of 43.87 ± 1.28 µm and 52.72 ± 2.22 µm, respectively. compared to emodin (1), compound 2 showed reduced activity due to the introduction of the hydroxyethyl group at the 3-position of emodin. among compounds 3a-3z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin (1) . compound 3a with the gly group displayed near double the activity of emodin (1) against hepg2 cells, but had slightly decreased activity against mcf-7 cells. compound 3b with the d-ala group displayed the most potent anti-proliferative activity against hepg2 cells and mcf-7 cells with ic 50 values of 8.22 ± 0.13 µm and 8.99 ± 0.64 µm, respectively. compound 3c with the l-ala group displayed slightly weaker activity than compound 3b, but still displayed stronger activity than emodin (1). compounds 3d-3j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin (1) . overall, with the exception of compound 3n, compounds 3k-3m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin (1); especially compound 3m containing a heterocycle. interestingly, compound 3n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg2 cells and mcf-7 cells with ic 50 values of 12.48 ± 0.59 µm and 23.51 ± 1.93 µm, respectively. compounds 3o and 3p with d-and l-ala groups exhibited similar inhibitory activity to compound 3n. compared to emodin (1), compounds 3q-3z with aryl (phenyl, substituted phenyl, benzyl, substituted benzyl, etc.) amino acid groups displayed enhanced activity in varying degrees when tested against two cancer cell lines, with ic 50 values ranging from 11.41 ± 0.77 to 42.44 ± 1.16 µm. following our initial results, we performed further modification of compounds 3b and 3c to improve the anti-proliferative activity, thus mono-and dimethylation of the amino moiety in the amino acid afforded compounds 4a, 4b, 5a and 5b. unfortunately, this modification led to decreased activity to varying degrees against both the hepg2 and mcf-7 cell lines, with ic 50 values ranging from 13.69 ± 0.89 to 68.81 ± 2.25 µm, as shown in table 2 . notably, compound 4b displayed a 6-fold activity reduction against mcf-7 cell lines compared to emodin (1). the results revealed that "nh 2 " was the moiety that favors improving the anti-proliferative activity. the preliminary explanation was that amino group can interact with other biomolecules to improve the anti-proliferative activity via formation of hydrogen bond. mono-or dimethylation of amino group might weaken the trend of hydrogen bonding and decrease the anti-proliferative activity. next, as shown in table 3 , the linking group of compounds 3b and 3c was further optimized by increasing the linker length in compounds 3b and 3c by one additional methylene group. the resulting compounds, 7a and 7b, displayed further enhanced potency at inhibiting cell growth against both hepg2 cells and mcf-7 cells. however, compounds 7c-7f initially failed to increase the potency of cell growth inhibition against both tested cancer cell lines; this was successfully addressed by increasing the linker length using additional methylene bridge. on the other hand, elongation of the linker by two to four ethanediol groups resulted in compounds 7g-7l, which showed decreased activity compared to compounds 3b and 3c. three pairs of compounds possessing stronger anti-proliferative activities were screened out and their cytotoxic activities were preformed against human normal liver cells (l02) in vitro. as shown in table 4 , the parent compound emodin exhibited stronger cytotoxic activity against l02 cells with an ic 50 value of 22.52 ± 0.18 µm than against cancer cell lines (ic 50 = 43.87 ± 1.28 µm against hepg2 cells and ic 50 = 52.72 ± 2.22 µm against mcf-7 cells). however, all the six compounds exhibited weaker cytotoxic activities against l02 cells compared with their corresponding cancer cell lines. above all, we finally chose to investigate the mechanism of action for compound 7a because it had the strongest anti-proliferative activity against hepg2 cells. lack of selective cytotoxicity is the main factor that hinders conventional chemotherapeutic agents. thus, to evaluate the selective anti-proliferative activity of the compound 7a, the selectivity index (si) between cancer and normal cells was calculated and the results are summarized in table 5 . the si was calculated by dividing the ic 50 values in normal cells by the ic 50 values in cancer cells. emodin displayed stronger cytotoxic activity, with si values of 0.51 (hepg2) and 0.43 (mcf-7), respectively, indicating that both normal cells and cancer cells would be killed. however, compound 7a exhibited weaker cytotoxic activity with si value of 2.82 (hepg2) and 2.36 (mcf-7), respectively. it means that 7a exhibited a better selective anti-proliferative activity and specificity than emodin. in order to verify whether compound 7a is able to induce apoptosis in hepg2 cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound 7a for 48 h at concentrations 2.5, 5, and 10 µm. as shown in figure 3a , few (5.5%) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to 22.2% after treatment with compound 7a at 5 µm for 48 h and further increased to 50.7% after treatment with 7a at the concentration of 10 µm. as illustrated in figure 3b , the quantitative analysis of apoptosis strongly suggests that treatment with compound 7a effectively induced apoptosis in hepg2 cells in a concentration-dependent manner in comparison to the control. in order to verify whether compound 7a is able to induce apoptosis in hepg2 cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound 7a for 48 h at concentrations 2.5, 5, and 10 µm. as shown in figure 3a , few (5.5%) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to 22.2% after treatment with compound 7a at 5 µm for 48 h and further increased to 50.7% after treatment with 7a at the concentration of 10 µm. as illustrated in figure 3b , the quantitative analysis of apoptosis strongly suggests that treatment with compound 7a effectively induced apoptosis in hepg2 cells in a concentration-dependent manner in comparison to the control. statistical significance is determined by two-tailed student t-test: "***" denote p < 0.001, "**" denote p < 0.01, respectively (supplementary table s1 ). (c, e) western blot analysis effect of compound 7a on the levels of bax, bcl-2, cytochrome c, procaspase-3, caspase-3 and procaspase-9 expression in hepg2 cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < 0.001, "**" denote p < 0.01, "*" denote p < 0.05, respectively (supplementary table s2 ). to verify the molecular mechanisms of apoptosis induction of compound 7a, we performed a western blot assay. it is well known that the bcl-2 family of pro-apoptotic and anti-apoptotic the quantitative analysis of apoptosis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < 0.001, "**" denote p < 0.01, respectively (supplementary table s1 ). (c, e) western blot analysis effect of compound 7a on the levels of bax, bcl-2, cytochrome c, procaspase-3, caspase-3 and procaspase-9 expression in hepg2 cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < 0.001, "**" denote p < 0.01, "*" denote p < 0.05, respectively (supplementary table s2 ). to verify the molecular mechanisms of apoptosis induction of compound 7a, we performed a western blot assay. it is well known that the bcl-2 family of pro-apoptotic and anti-apoptotic proteins regulates the mitochondrial pathway of apoptosis. these bcl-2 family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure 3c and 3d, in comparison with the control cells, compound 7a induced an increase in the levels of bax and a decrease in the expression of bcl-2 in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound 7a, while procaspase 9 and procaspase 3 decreased after treatment with 7a, indicating that the caspase 9 and caspase 3 were activated. as shown in figure 3e ,f, the increased expression of cleaved caspase-3 after treatment with 7a provided a further evidence that compound 7a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg2 cells was formed by pro-apoptotic protein bax after the treatment of compound 7a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor-1 (apaf-1) and atp, which then binded to procaspase-9 to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase-9 to its active form of initiator caspase-9, which in turn activated procaspase-3 and then the effector caspase-3 and finally resulted in cell apoptosis. to further examine how compound 7a suppressed the growth of hepg2 cells, the effect of compound 7a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure 4a . as determined by flow cytometry, the exposure of hepg2 cells to compound 7a for 48 h resulted in an obvious increase in the percentage of cells in g0/g1 phase in comparison with the control. treatment with compound 7a resulted in an increase of g1 phase cells (63.37%) compared to the control (33.16%). inversely, s phase cell population decreased to 14.60% compared to the control (48.13%). therefore, compound 7a resulted in a significant g0/g1 phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. proteins regulates the mitochondrial pathway of apoptosis. these bcl-2 family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure 3c and 3d, in comparison with the control cells, compound 7a induced an increase in the levels of bax and a decrease in the expression of bcl-2 in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound 7a, while procaspase 9 and procaspase 3 decreased after treatment with 7a, indicating that the caspase 9 and caspase 3 were activated. as shown in figure 3e ,f, the increased expression of cleaved caspase-3 after treatment with 7a provided a further evidence that compound 7a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg2 cells was formed by pro-apoptotic protein bax after the treatment of compound 7a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor-1 (apaf-1) and atp, which then binded to procaspase-9 to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase-9 to its active form of initiator caspase-9, which in turn activated procaspase-3 and then the effector caspase-3 and finally resulted in cell apoptosis. to further examine how compound 7a suppressed the growth of hepg2 cells, the effect of compound 7a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure 4a . as determined by flow cytometry, the exposure of hepg2 cells to compound 7a for 48 h resulted in an obvious increase in the percentage of cells in g0/g1 phase in comparison with the control. treatment with compound 7a resulted in an increase of g1 phase cells (63.37%) compared to the control (33.16%). inversely, s phase cell population decreased to 14.60% compared to the control (48.13%). therefore, compound 7a resulted in a significant g0/g1 phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. table s3 ). to evaluate the effect of compound 7a on cancer migration, a wound healing assay was conducted to determine whether compound 7a could prevent hepg2 cell migration. after culturing hepg2 cells for 48 h in the presence and absence of compound 7a at 0, 0.25, 0.5, and 1 µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg2 cells was used to observe the migration inhibition capability of compound 7a. the empty space reduced significantly in size in the absence of compound 7a because of cell proliferation and migration and results indicate compound 7a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure 5 ). cell cycle distribution after treatment of compound 7a. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < 0.001, "*" denote p < 0.05 and ns means no significance, respectively (supplementary table s3 ). to evaluate the effect of compound 7a on cancer migration, a wound healing assay was conducted to determine whether compound 7a could prevent hepg2 cell migration. after culturing hepg2 cells for 48 h in the presence and absence of compound 7a at 0, 0.25, 0.5, and 1 µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg2 cells was used to observe the migration inhibition capability of compound 7a. the empty space reduced significantly in size in the absence of compound 7a because of cell proliferation and migration and results indicate compound 7a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure 5 ). table s4 ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel 60-f254, e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel (200-300 mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d6 on a bruker-250 spectrometer (bruker biospin, fällanden, switzerlahd), at 400 mhz for 1 h-nmr, 101 mhz for 13 c-nmr and 376 mhz for 19 f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent 1100 series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). table s4 ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel 60-f254, e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel (200-300 mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d 6 on a bruker-250 spectrometer (bruker biospin, fällanden, switzerlahd), at 400 mhz for 1 h-nmr, 101 mhz for 13 c-nmr and 376 mhz for 19 f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent 1100 series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). 1,8-dihydroxy-3-(2-hydroxyethoxy)-6-methylanthracene-9,10-dione (2) to a mixture of emodin (10.0 g, 37.0 mmol) in dry dmf (150 ml) were added cs 2 co 3 (13.2 g, 40.5 mmol) and 2-iodoethanol (19.1 g, 111 mmol) at room temperature. after stirring for 36 h at 60 • c, the resulting mixture was evaporated under reduced pressure and then mixed with water (500 ml). the ph value of aqueous phase was adjusted to around 5 with 10% hydrochloric acid solution. the yellow precipitate was collected and washed with water to give the crude product, which was in further purification by triturating twice with ethyl acetate (100 ml) and filtered to afford compound 2 (7.6 g, 65%) as a brown solid; 1 general procedure a for preparation of compounds 3a-3z, 4a-4b, and 7a-7l to a mixture of compound 2 (1.00 mmol) in dry dichloromethane (20 ml) were added various n-boc amino acids (1.20 mmol), dicyclohexyl carbodiimide (dcc) (4.50 mmol) and 4-(n,n-dimethlyamino) pyridine (dmap) (1.00 mmol) at 0 • c. after stirring about 30 min to 5 h at 0 • c, tlc analysis showed the complete consumption of compound 2, and then the resulting mixture was added dropwise tfa (5 ml) at the same temperature and kept stirring for anther about 2 h. the insoluble side product was filtered out and the filtrate was evaporated to give the residue, which was purified by reverse phase flash chromatography with the following conditions: column: spherical c18, 20-40 µm, 330 g; mobile phase a: water (plus 5 mm tfa); mobile phase b: acn; flow rate: 80 ml/min; gradient: 5% b gradient in 10 min, 25% b-45% b gradient in 25 min; detector: 254 nm. the fractions containing the desired product were collected at around 40% b and concentrated under reduced pressure to afford compounds 3a-3z in 20% to 50% yields. 2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-2-oxoethanaminium 2,2,2-trifluoroacetate (3a). according to the general procedure a, compound 2 was treated with n-boc-glycine and then purified by reverse phase flash chromatography to give compound 3a: yellow solid; yield, 45%; 1 (s)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (3c). according to the general procedure a, compound 2 was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 3c: yellow solid; yield, 48%; 1 h-nmr (s)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-3-hydroxy-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (3d). according to the general procedure a, compound 2 was treated with n-boc-o-tert-butyl-l-serine and then purified by reverse phase flash chromatography to give compound 3d: yellow solid; yield, 20%; 1 (r)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-3-methyl-1-oxobutan-2-aminium 2,2,2-trifluoroacetate (3e). according to the general procedure a, compound 2 was treated with n-boc-d-valine and then purified by reverse phase flash chromatography to give compound 3e: yellow solid; yield, 46%; 1 189.89, 181.04, 168.97, 164.68, 164.26, 161.46, 148.57, 134.85, 132.73, 124.23, 120.57, 113.37, 110.09, 107.74, 107.05, 66.65, 63.45, 57. 161.48, 148.62, 134.89, 132.78, 124.26, 120.61, 113.42, 110.13, 107.79, 107.11, 66.63, 63.71 1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-2-methyl-1-oxopropan-2aminium 2,2,2-trifluoroacetate (3j). according to the general procedure a, compound 2 was treated with n-boc-aib-oh and then purified by reverse phase flash chromatography to give compound 3j: yellow solid; yield, 38%; 1 h-nmr δ 11.96 (br, 2h), 8.51 (br, 3h), 7.54 (s, 1h), 7.34-7.14 (m, 2h), 6 1-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)cyclopropan-aminium 2,2,2-trifluoroacetate (3k). according to the general procedure a, compound 2 was treated with 1-(boc-amino)cyclopropanecarboxylic acid and then purified by reverse phase flash chromatography to give compound 3k: yellow solid; yield, 28%; 1 h-nmr δ 11.96 (brs, 1h), 8 189.28, 180.10, 169.83, 164.69, 164.22, 161.39, 148.39, 134.15, 132.07, 123.98, 120.37, 112.73, 109.54, 107.67, 106.74, 66.57, 63.78 1-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)cyclobutan-aminium 2,2,2-trifluoroacetate (3l). according to the general procedure a, compound 2 was treated with 1-(boc-amino)cyclobutanecarboxylic acid and then purified by reverse phase flash chromatography to give compound 3l: yellow solid; yield, 28%; 1 h-nmr δ 11.95 (brs, 1h), 8 3-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)oxetan-3-aminium 2,2,2-trifluoroacetate (3m). according to the general procedure a, compound 2 was treated with 3-boc-amino-3-oxetanecarboxylic acid and then purified by reverse phase flash chromatography to give compound 3m: yellow solid; yield, 37%; 1 h-nmr δ 11.97 (brs, 2h), 8 1-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)cyclopentan-aminium 2,2,2-trifluoroacetate (3n). according to the general procedure a, compound 2 was treated with n-boc-aminocyclopentanecarboxylic acid and then purified by reverse phase flash chromatography to give compound 3n: yellow solid; yield, 31%; 1 h-nmr δ 11.98 (brs, 2h), 8 (r)-2-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)-pyrrolidinium 2,2,2-trifluoroacetate (3o). according to the general procedure a, compound 2 was treated with n-boc-d-proline and then purified by reverse phase flash chromatography to give compound 3o: yellow solid; yield, 44%; 1 h-nmr δ 7.52 (d, j = 1. 7 (s)-2-((2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)carbonyl)-pyrrolidinium 2,2,2-trifluoroacetate (3p). according to the general procedure a, compound 2 was treated with n-boc-l-proline and then purified by reverse phase flash chromatography to give compound 3p: (r)-2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-2-oxo-1-phenylethanaminium 2,2,2-trifluoroacetate (3q). according to the general procedure a, compound 2 was treated with n-boc-d-phenylglycine and then purified by reverse phase flash chromatography to give compound 3q: yellow solid; yield, 39%; 1 h-nmr δ 11.97 (brs, 2h), 8 (r)-2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-(4-hydroxyphenyl)-2-oxoethanaminium 2,2,2-trifluoroacetate (3s). according to the general procedure a, compound 2 was treated with n-boc-d-4-hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound 3s: yellow solid; yield, 42%; 1 h-nmr δ 11.99 (brs, 2h), 9. (s)-2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-(4-hydroxyphenyl)-2-oxoethanaminium 2,2,2-trifluoroacetate (3t). according to the general procedure a, compound 2 was treated with n-boc-l-4-hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound 3t: yellow solid; yield, 40%; 1 h-nmr δ 11.96 (brs, 1h), 9. (r)-2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-(4-fluorophenyl)-2-oxoethanaminium 2,2,2-trifluoroacetate (3u). according to the general procedure a, compound 2 was treated with (r)-n-boc-4-fluorophenylglycine and then purified by reverse phase flash chromatography to give compound 3u: yellow solid; yield, 45%; 1 h-nmr δ 11.97 (brs, 2h), 8 (s)-2-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-(4-fluorophenyl)-2oxoethanaminium 2,2,2-trifluoroacetate (3v). according to the general procedure a, compound 2 was treated with (s)-n-boc-4-fluorophenylglycine and then purified by reverse phase flash chromatography to give compound 3v: yellow solid; yield, 43%; 1 h-nmr (400 mhz, dmso-d 6 ) δ 11.95 (brs, 2h), 8 (r)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-oxo-3-phenyl-propan-2-aminium 2,2,2-trifluoroacetate (3w). according to the general procedure a, compound 2 was treated with n-boc-d-phenylalanine and then purified by reverse phase flash chromatography to give compound 3w: yellow solid; yield, 48%; 1 h-nmr δ 11.90 brs, 1h), 8 (s)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-1-oxo-3-phenyl-propan-2-aminium 2,2,2-trifluoroacetate (3x). according to the general procedure a, compound 2 was treated with n-boc-l-phenylalanine and then purified by reverse phase flash chromatography to give compound 3x: yellow solid; yield, 45%; 1 h-nmr δ 11.90 (brs, 1h), 8 (r)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (3y). according to the general procedure a, compound 2 was treated with n-boc-o-tert-butyl-l-tyrosine and then purified by reverse phase flash chromatography to give compound 3y: yellow solid; yield, 26%; 1 h-nmr δ 9.33 (s, 1h), 7.54 (d, j = 1. 6 (s)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-3-(4-hydroxyphenyl)-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (3z). according to the general procedure a, compound 2 was treated with n-boc-l-tyrosine and then purified by reverse phase flash chromatography to give compound 3z: yellow solid; yield, 23%; 1 h-nmr δ 11.04 (s, 1h), 7.50 (d, j = 5. 7 (r)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-n-methyl-1-oxo-propan-2-aminium 2,2,2-trifluoroacetate (4a). according to the general procedure a, compound 2 was treated with boc-n-methyl-d-alanine and then purified by reverse phase flash chromatography to give compound 4a: yellow solid; yield, 48%; 1 h-nmr δ 11.93 (brs, 2h), 9.18 (s)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-n-methyl-1-oxo-propan-2-aminium 2,2,2-trifluoroacetate (4b). according to the general procedure a, compound 2 was treated with boc-n-methyl-l-alanine and then purified by reverse phase flash chromatography to give compound 4b: yellow solid; yield, 45%; 1 h-nmr δ 12.15 (brs, 1h), 11.96 (brs, 1h), 9.11 general procedure b for preparation of compounds 5a and 5b to a solution of compound 4a or 4b (100 mg, 0.25 mmol) and paraformaldehyde (37% aqueous solution, 62 mg, 0.76 mmol) in meoh (15 ml) was added nabh 3 cn (24 mg, 0.38 mmol) at 0 • c. after stirring 2 h at room temperature, the reaction was quenched by tfa (0.1 ml). the resulting reaction solution was used directly in purification by reverse phase flash chromatography with the following conditions: column: spherical c18, 20-40 µm, 330 g; mobile phase a: water (plus 5 mm tfa); mobile phase b: acn; flow rate: 80 ml/min; gradient: 5% b gradient in 10 min, 25% b-45% b gradient in 25 min; detector: 254 nm. the fractions containing the desired product were collected at around 40% b and concentrated under reduced pressure to afford compounds 5a or 5b in 85% or 89% yield, respectively. (r)-1-(2-(4,5-dihydroxy-7-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yloxy)ethoxy)-n,n-dimethyl-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (5a). yellow solid; yield, 85%; 1 general procedure c for preparation of compounds 6a-6f to a mixture of emodin (10 mmol) in dry dmf (50 ml) were added cs 2 co 3 (12 mmol) and hydroxybromides or iodides (30 mmol) at room temperature. after stirring for 36 h at 60 • c, the resulting mixture was evaporated under reduced pressure and then mixed with water (100 ml). the ph value of aqueous phase was adjusted to around 5 with 10% hydrochloric acid solution, extracted with dichloromethane (2 × 100 ml). the combined organic layer was washed with brine (200 ml), dried over anhydrous sodium sulfate and evaporated to dryness. the crude product was purified by silica gel column chromatography with 1%-10% ethyl acetate in petroleum to afford compounds 6a-6f. total cell lysates from cultured hepg2 cells treated with different concentrations of compound 7a for 48 h were obtained by lysing the cells in ice-cold ripa buffer (1 pbs, 1% np-40, 0.5% sodium deoxycholate and 0.1% sds) containing 100 mg/ml pmsf, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 5 mg/ml pepstatin and 100 mg/ml naf. after centrifugation at 12,000 rpm for 10 min, the protein in the supernatant was quantified by the bradford method (bio-rad, hercules, ca, usa) using a multimode varioscan instrument (thermo fischer scientific, waltham, ma, usa). twenty micrograms of protein per lane was applied in 12% sds polyacrylamide gel. after electrophoresis, the protein was transferred to a polyvinylidine difluoride membrane (amersham biosciences, marlborough, ma, usa). the membrane was blocked at room temperature for 2 h in tbst containing 5% blocking powder (santa cruz, dallas, tx, usa). the membrane was washed with tbst for 5 min, and the primary antibody was added and incubated at 4 • c overnight (o/n). bax (5023, cst, danvers, ma, usa), bcl-2 (15071, cst), cytochrome c (4280, cst), procaspase-9 (ab138412, abcam, cambridge, ma, usa), procaspase-3 (ab32150, abcam), caspase-3 (66470-2-lg, proteintech group, rosemont, il, usa) and gapdh (ab8245, abcam) antibodies were employed. after three tbst washes, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) (santa cruz) at room temperature for 2 h. membranes were washed with tbst for 15 min five times and the protein blots were visualized with chemiluminescence reagent (thermo fischer scientific ltd.). the x-ray films were developed with a developer and fixed with fixer solution. the grey levels were analyzed using imagequant las 4000 system (ge, marlborough, ma, usa). the hepg2 cells were treated with indicated concentrations of compound 7a. after incubation for 48 h, cells were washed twice with ice-cold pbs, fixed and permeabilized with ice cold 70% ethanol at −20 • c overnight. the cells were treated with 100 µg/ml rnase a at 37 • c for 30min, then washed with ice-cold pbs and finally stained with 1mg/ml pi in the dark at 4 • c for 30 min. the cellular dna content for the cell cycle distribution analysis was performed with the system software (cellquest; bd biosciences), plotting at least 30,000 events per sample. the percentage of cells in the g1, s and g2 phases of the cell cycle were determined using the modfit lt version 5.0 software package (verity software, topsham, me, usa). hepg2 cells were grown in dmem medium containing growth factors at a cell density of 1 × 10 5 cells/ml for 24 h. a disposable 200 ml plastic pipette tip was used to scratch the monolayer of cells in a streaking motion. compounds were added to the streaked cell culture at the indicated concentrations. the streaked cells were then cultured in serum-free medium for an additional 48 h and photographed. to quantify the experimental results, the % cell inhibitory rate was calculated by the equation: cell inhibitory rate (%) = (1 − d drug /d control ) × 100%, where d drug is the mean distance of cell migration in drug group and d control is the mean distance of cell migration in control group. pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation, and data were presented as mean ± sd from three independent experiments. in conclusion, a novel series of emodin derivatives via the introduction of an amino acid were designed and synthesized. their in vitro anti-proliferation tests revealed that these derivatives exhibited moderate to potent anti-proliferative activity against hepg2 cells and mcf-7 cells. among these compounds, the most potent compound, 7a, exhibited better selective anti-proliferative activity and specificity than emodin, displayed a significant effect in inducing cell cycle arrest at g0/g1 phase and inducing cell apoptosis in hepg2 cells via release of cytochrome c and subsequent activation of caspase-9 and caspase-3, which revealed that the possible molecular mechanism of 7a apoptosis induction may mainly through the mitochondrial death pathway. moreover, compound 7a also resulted in inhibition of hepg2 cells migration in the wound healing assay. these preliminary molecular mechanism results suggest that compound 7a could be a promising lead compound for the development of novel antitumor drugs and has the potential for further investigations as an anti-cancer drug. supplementary materials: the following are available online at http://www.mdpi.com/1420-3049/24/5/884/s1, tables s1-s4: biochemistry analytical data from three independent experiments; figures s1-s140: 1 h-nmr, 13 c-nmr and 19 f-nmr spectra of these compounds. according to the general procedure c, emodin was treated with 3-iodopropan-1-ol and then purified by silica gel column chromatography to give compound 6a: brown solid; yield, 55%; 1 h-nmr δ 12.14 (s, 1h), 11.96 (s, 1h), 7.51 (s, 1h according to the general procedure c, emodin was treated with 4-bromobutan-1-ol and then purified by silica gel column chromatography to give compound 6b: brown solid; yield, 47%; 1 h-nmr δ 12.16 (s, 1h), 11.98 (s, 1h), 7.53 (s, 1h) according to the general procedure c, emodin was treated with 5-bromopentan-1-ol and then purified by silica gel column chromatography to give compound 6c: brown solid; yield, 52%; 1 h-nmr δ 12.15 (s, 1h), 11.96 (s, 1h) according to the general procedure c, emodin was treated with 2-(2-bromoethoxy)ethanol and then purified by silica gel column chromatography to give compound 6d: brown solid; yield, 38%; 1 h-nmr δ 12.14 (s, 1h), 11.95 (s, 1h), 7.50 (d, j = 1 -bromoethoxy)ethoxy)ethanol and then purified by silica gel column chromatography to give compound 6e: brown solid; yield, 42%; 1 h-nmr δ 12.01 (s, 1h) -bromoethoxy)ethoxy)ethoxy) ethanol and then purified by silica gel column chromatography to give compound 6f: brown solid h-nmr δ 12.13 (s, 1h), 11.94 (s, 1h), 7.49 (d, j = 1.6 hz, 1h), 7.18 (t, j = 1.3 hz, 1h), 7.15 (d, j = 2.5 hz, 1h) according to the general procedure a, compound 6a was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7a: yellow solid; yield, 70%; 1 h-nmr δ 7.52 (d, j = 1 according to the general procedure a, compound 6a was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7b: yellow solid according to the general procedure a, compound 6b was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7c: yellow solid according to the general procedure a, compound 6b was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7d: yellow solid hz, 1h), 2.44 (s, 3h), 1.92-1.76 (m, 4h), 1.41 (d, j = 7.2 hz, 3h) according to the general procedure a, compound 6c was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7e: yellow solid; yield, 70%; 1 h-nmr δ 12.00 (brs, 2h), 8.32 (brs, 3h), 7.54 (d, j = 1 according to the general procedure a, compound 6c was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7f: yellow solid 43 (s, 3h), 1.83-1.76 (m, 2h), 1.75-1.68 (m, 2h), 1.56-1.46 (m, 2h), 1.40 (d, j = 7.1 hz, 3h) according to the general procedure a, compound 6d was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7g: yellow solid; yield, 65%; 1 h-nmr δ 9 according to the general procedure a, compound 6d was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7h: yellow solid according to the general procedure a, compound 6e was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7i: yellow solid; yield, 66%; 1 h-nmr δ 9 according to the general procedure a, compound 6e was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7j: yellow solid; yield, 65%; 1 h-nmr δ 9 according to the general procedure a, compound 6f was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound 7k: yellow solid; yield, 66%; 1 h-nmr δ 9.76 (brs, 3h), 7.46 (d, j = 1.6 hz, 1h) according to the general procedure a, compound 6f was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound 7l: yellow solid; yield, 65%; 1 h-nmr δ 9 ) containing 10% fetal bovine serum (fbs) and 1% penicillin (100 units/ml)-streptomycin (100 µg/ml) in a humidified incubator at 37 • c under 5% co 2 and 95% relative humidity (rh) atmosphere. the cells were harvested for metabolomics experiments using cell scrapers cell viability assay hepg2 cells and mcf-7 cells in 40 µl dmem supplemented with 10% fbs were seeded at a density of 500 cells/well in 384-well cell culture plate (3570, corning), respectively. after 24 h, the medium was substituted with fresh medium containing various sample solutions in dmso usa) was added to each well containing cells and the contents were mixed for 2 min on an orbital shaker to induce cell lysis. the plates were then incubated at room temperature for 20 min to stabilize the luminescent signal. the luminescence was recorded on an envision cells were seeded at 2 × 10 5 /well in 10% fbs-dmem into 6-well plates and treated with compounds 7a for 48 h. the cells were washed twice with cold phosphate buffered saline (pbs) and then analysis is as follows: lower left quadrant, viable cells (annexin v−/pi−); lower right quadrant, early apoptotic cells (annexin v+/pi−); upper right quadrant, late apoptotic cells (annexin v+/pi+); upper left quadrant, necrotic cells a new diphenyl ether from marine-derived fungus aspergillus sp b-f-2 a: a new antimicrobial anthraquinone from a sea urchin-derived fungus monodictys sp a new ergosterol analog, a new bis-anthraquinone and anti-obesity activity of anthraquinones from the marine sponge-associated fungus talaromyces stipitatus kufa 0207. mar. drugs bis-indolyl benzenoids, hydroxypyrrolidine derivatives and other constituents from cultures of the marine sponge-associated fungus aspergillus candidus kufa0062 emodin and aloe-emodin suppress breast cancer cell proliferation through er alpha inhibition. evid.-based complement emodin suppresses wnt signaling in human colorectal cancer cells sw480 and sw620 emodin suppresses hyperglycemia-induced proliferation and fibronectin expression in mesangial cells via inhibiting cflip emodin suppresses inflammatory responses and joint destruction in collagen-induced arthritic mice emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction effect of emodin on the cariogenic properties of streptococcus mutans and the development of caries in rats emodin, a naturally occurring anthraquinone derivative, suppresses ige-mediated anaphylactic reaction and mast cell activation ct imaging biomarker for evaluation of emodin as a potential drug on lps-mediated osteoporosis mice formula optimization of the jiashitang scar removal ointment and antiinflammatory compounds screening by nf-kappa b bioactivity-guided dual-luciferase reporter assay system emodin protects against diabetic cardiomyopathy by regulating the akt/gsk-3 beta signaling pathway in the rat model emodin prolongs recipient survival time after orthotopic liver transplantation in rats by polarizing the th1/th2 paradigm to th2 emodin induces neurite outgrowth through pi3k/akt/gsk-3 beta-mediated signaling pathways in neuro2a cells emodin ameliorates ethanol-induced fatty liver injury in mice emodin protects against concanavalin a-induced hepatitis in mice through inhibiting activation of the p38 mapk-nf-kappa b signaling pathway emodin suppresses cell proliferation and fibronectin expression via p38mapk pathway in rat mesangial cells cultured under high glucose emodin accelerates osteoblast differentiation through phosphatidylinositol 3-kinase activation and bone morphogenetic protein-2 gene expression emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase-8-mediated bid cleavage aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway emodin inhibits tnf-alpha-induced human aortic smooth-muscle cell proliferation via caspase-and mitochondrial-dependent apoptosis emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway synthesis and antitumor activity of emodin quaternary ammonium salt derivatives synthesis and biological activity, evaluation of emodin quaternary ammonium salt derivatives as potential anticancer agents synthesis, sar and pharmacological characterization of novel anthraquinone cation compounds as potential anticancer agents antitumor effects and mechanism of novel emodin rhamnoside derivatives against human cancer cells in vitro synthesis, dna binding and cytotoxicity of new pyrazole emodin derivatives synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates preparation of amino acid conjugates of betulinic acid with activity against human melanoma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-103271-l9n27ocf authors: carozza, jacqueline a; brown, jenifer a.; böhnert, volker; fernandez, daniel; alsaif, yasmeen; mardjuki, rachel e.; smith, mark; li, lingyin title: structure-aided development of small molecule inhibitors of enpp1, the extracellular phosphodiesterase of the immunotransmitter cgamp date: 2020-05-31 journal: biorxiv doi: 10.1101/2020.05.30.125534 sha: doc_id: 103271 cord_uid: l9n27ocf cancer cells initiate an innate immune response by synthesizing and exporting the small molecule immunotransmitter cgamp, which activates the anti-cancer stimulator of interferon genes (sting) pathway in the host. an extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1), hydrolyzes cgamp and negatively regulates this anti-cancer immune response. small molecule enpp1 inhibitors are much needed as tools to study basic biology of extracellular cgamp and as investigational cancer immunotherapy drugs. here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of enpp1. additionally, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. this study yielded several best-in-class compounds with ki < 2 nm and excellent physicochemical and pharmacokinetic properties. finally, we demonstrate that an enpp1 inhibitor delays tumor growth in a breast cancer mouse model. together, we have developed enpp1 inhibitors that are excellent tool compounds and potential therapeutics. adaptive immune checkpoint inhibitors such as anti-pd-1, anti-pd-l1, and anti-ctla-4 are now curing cancer patients who were previously considered terminally ill. 1 these inhibitors work by removing the immunological brakes that cancer cells place on tumor-infiltrating lymphocytes (tils), therefore increasing the cancer-killing efficacy of tils. however, only "hot" tumors -those that already have high numbers of tils -respond to checkpoint inhibitor therapy. most tumors do not exhibit this til inflammation and thus are immunologically "cold." [2] [3] [4] turning "cold" tumors "hot" by activating the innate immune detection of cancer, which is upstream of the recruitment of adaptive immune tils, could revolutionize cancer immunotherapy. the cytosolic double-stranded dna (dsdna) sensing-stimulator of interferon genes (sting) pathway is the key innate immune pathway that responds to cancerous cells. chromosomal instability and extrachromosomal dna are hallmarks of cancer that can lead to leakage of dsdna into the cytosol. [5] [6] [7] [8] [9] the cytosolic dsdna is detected by the enzyme cyclic-gmp-amp synthase (cgas), 10 which synthesizes the cyclic dinucleotide 2',3'-cyclic gmp-amp (cgamp). 11, 12 cgamp then binds and activates sting, which leads to production of type i interferons (ifns) and downstream til infiltration. we recently discovered that cancer cell lines basally synthesize cgamp and export it to the extracellular space. 13 extracellular cgamp is then internalized by host cells to trigger anti-cancer innate immune responses. [13] [14] [15] [16] [17] [18] however, we also discovered that the ubiquitously expressed extracellular enzyme enpp1, which was previously known only as an atp hydrolase, is the dominant hydrolase for extracellular cgamp and dampens innate responses to cancer. 13, 19 we previously developed phosphorothioate cgamp analogs that are resistant to enpp1 hydrolysis. 19 since then, other stable cgamp analogs have entered clinical trials in combination with anti-pd-1 checkpoint blockers (trial ids nct03172936 and nct03010176, respectively). however, these cgamp analogs need to be injected directly into the tumors to achieve efficacy and to avoid systemic interferon response. alternatively, we propose to inhibit enpp1 to boost the efficacy of the endogenous extracellular cgamp that is only locally exported by cancer cells. indeed, genetic knockout and pharmacological inhibition of enpp1 both increased tumor-infiltrating dendritic cells and controlled tumor growth, without any safety concerns. 13 this work suggests that enpp1 inhibitors could turn "cold" tumors "hot" and render them more sensitive to adaptive immune checkpoint inhibitors. outside of its role in cancer immunotherapy, cgamp has shown excellent adjuvant activities in vaccination against influenza 20 and may aid in the current urgent demand to develop vaccines against sars-cov-2. we, therefore, propose that enpp1 inhibitors may also stabilize cgamp when administered as an adjuvant. enpp1 is a single-pass transmembrane protein that is anchored with the catalytic domain outside of the cell. it can also be exported extracellularly in a soluble form that is abundant in the circulation. [21] [22] [23] was known to convert nucleotide triphosphates (preferentially atp) to nucleotide monophosphates (cite something). we reported that enpp1 also converts cgamp to amp and gmp by hydrolyzing first the 2'-5' phosphodiester bond, and then the 3'-5'. 19 similar to the other npp family members, its catalytic site coordinates two zinc ions that hold the substrate phosphate in place for threonine-mediated nucleophilic attack. the adenosine base of enpp1 substrates stacks with aromatic residues that form a tight pocket in the active site. 24 later co-crystal structures of enpp1 with papg, the degradation intermediate of 2'3'-cgamp, and with 3'3'-cgamp provided mechanistic insight into why enpp1 degrades 2'3'-cgamp, but not 3'3'-cgamp. 25 however, all natural enpp1 substrates have km values higher than 20 μm. 19, 24 despite enpp1 being a highly sought-after target, [26] [27] [28] [29] [30] [31] [32] [33] [34] developing drug-like enpp1 inhibitors has proved difficult. here, we report the development of the most potent enpp1 inhibitors to date and the co-crystal structure of an exemplary phosphonate inhibitor with enpp1. inspired by the molecular scaffold of a previous inhibitor, qs1, 26, 28 which lacks potency at physiological conditions, we build structure-activity relationships (sar) around the three sections of the molecule -the zinc-binding head, the core, and the tail -and develop several inhibitors with nanomolar ki values. our crystal structure revealed extensive interactions between the inhibitor and enpp1 and explains its 1000-10,000-fold improvement in affinity over the natural substrates. finally, these inhibitors have desirable physicochemical and pharmacokinetic properties, which enables their systemic use in mouse studies. indeed, we demonstrate that treatment with one of our top enpp1 inhibitors delays tumor growth in a breast cancer mouse model. given that a soluble form of enpp1 is present in the circulation, we first asked how much enpp1 activity is present in freshly drawn mouse and human plasma by measuring the half-life of radiolabeled cgamp. cgamp was degraded rapidly (t1/2 = 16 minutes for mouse; t1/2 = 30-60 minutes for human, based on 5 healthy donors) (fig 1a-c) . detectable hydrolysis of cgamp ex vivo occurs only in wt, and not enpp1 -/mouse plasma 19 , and inhibiting enpp1 activity using edta to chelate the catalytic zinc ions abrogated cgamp degradation in both mouse and human plasma (fig 1a-c) . rapid degradation of extracellular cgamp by circulating enpp1 underscores the need to develop potent and systemic enpp1 inhibitors. assays used to assess the potency of previously attempted enpp1 inhibitors are inconsistent 26-34 ; however, developing an appropriate assay is key to determining the utility of the molecules in inhibiting cgamp degradation under physiological conditions. the most common model substrate is p-nitrophenyl-5'-tmp (p-nptmp), but ic50 values measured using this substrate have been shown to deviate significantly from ic50 values measured using a natural substrate such as atp. 30 in addition, perhaps to increase the speed of the assay, most reported assays are conducted at ph 9, where enpp1 is most active. however, since enpp1 is active in serum at physiological ph ( fig. 1a-c) , an effective enpp1 inhibitor needs to be potent at ph 7.4 or even lower, as can occur in the tumor microenvironment. 35, 36 therefore, we evaluated inhibitors with an assay using cgamp as a substrate at ph 7.4 (fig. 1d) . 37 there were only two non-nucleotide inhibitor hits from the literature which had the potential to be developed into a lead compound for inhibiting enpp1. first, a thioacetamide inhibitor (compound 1, fig. 1e ) was reported to have a ki of 5 nm against human enpp1 using p-nitrophenyl-5'-tmp (p-nptmp) as the substrate, but the ki increased to 5 µm when using atp as the substrate, both assays conducted at ph 9. 27 when we tested the potency of compound 1 using mouse enpp1 and cgamp as a substrate at both ph 7.4 and ph 9, we detected no activity at concentrations below 10 µm (fig. 1e) , suggesting that it cannot block cgamp degradation activity specifically compared to other substrates, or that it may be specific to human enpp1. regardless, lack of efficacy against cgamp and/or mouse enpp1 disqualifies this molecule as a scaffold to use for further development. second, patel et al reported a quinazoline-piperidine-sulfamide inhibitor (qs1, compound 2) with an ic50 of 36 nm against enpp1 using atp as a substrate. 26 however, we found that the potency of qs1 dropped 100-fold when adjusted the ph down to 7.4 instead of ph 9 (ki = 1.6 μm using cgamp as a substrate, fig. 1e ), making it unsuitable for experiments at physiological ph and in vivo. in addition, we have previously shown that qs1 non-specifically blocks cgamp export, limiting its use as a tool for studying extracellular cgamp biology. 13 we, therefore, sought to develop more potent and specific enpp1 inhibitors. we hypothesized that qs1 lacked potency at ph 7.4 because deprotonation of the sulfamide group (pka around 7-8) is important for efficient binding to the zinc atoms at the catalytic site of enpp1. therefore, we tested several other classes of zinc-binding head groups on the qs1 scaffold ( fig. 2a-b) . we also tried methylene, ethyl, and/or propyl linkage between the head groups and the piperidine core. ureas (compounds 4 and 5) and carboxylic acid (compound 6) were less potent compared to sulfamides (compounds 2 and 3), but we observed improvement in potency with boronic acid (compound 7), hydroxamic acids (compounds 8 and 9), and especially phosphates (compounds 10 and 11), the natural zinc binding group of the enpp1 substrates cgamp and atp. to increase compound stability while preserving the core properties of phosphates, we tested thiophosphates (compounds 12 and 13), phosphonates (compounds 14-16), and a thiophosphonate (compound 17), all of which are known to be more stable against enzymatic degradation compared to phosphates. the phosphonates showed ki values less than 50 nm independent of the ph (fig. 2a-b) . we chose to proceed with phosphonates because they are potent, stable, and synthetically tractable. in addition, there are several phosphonate drugs on the market including tenofovir and pradefovir as antivirals, fosfomycin as an antibiotic, and bisphosphonates (pamidronic aicd, zoledronic acid, alendronic acid) for osteoporosis and bone disease. finally, they are negatively charged at all physiological ph values, which is crucial for zinc binding and keeping them cell impermeable to act on the extracellular target enpp1. we also chose the ethylene linker (compound 15, ki = 33 nm, previously reported as stf-1084 13 ) because it has been shown previously that for the sulfamides, analogs with shorter linker lengths have less affinity for the cardiac potassium channel herg, a detrimental off target. 28, 32 co-crystal structure of enpp1 and compound 15 reveals molecular determinants of potency enpp1 is a >100 kda multidomain glycoprotein with three glycosylation chains. it has a catalytic domain, a nuclease-like domain which provides structural support important for catalysis, and two disordered smb domains. we used a mouse enpp1 construct where the transmembrane anchor was truncated and replaced by a signal peptide, and expressed the construct in hek293s gnt1cells to generate secreted soluble enpp1 with simplified glycosylation chains as previously described 24, 25, 38 . we then determined the structure of enpp1 in complex with compound 15 to 3.2 å resolution using x-ray crystallography (fig. 3, fig. s1 , tables s1-3). compound 15 occupies the substrate-binding pocket ( fig. 3a-d) and forms extensive interactions with zinc ions and residues in the catalytic site, demonstrating that compound 15 is a competitive inhibitor. the phosphonate oxygens bind both zinc ions, and the third phosphonate oxygen forms a hydrogen bond with n259, a residue previously determined to be important for catalytic activity. the piperidine group is engaged in hydrophobic interactions with l272, and the quinazoline group sits between y322 and f239 to form p-p interactions with both ( fig. 3b-d) . although compound 15 adopts a similar binding conformation as the reaction product amp 24 (pdb 4gtw, fig. 3e -f), there are two significant differences between the ligands that suggest why compound 15 has a much higher affinity than the substrates atp or cgamp. first, the quinazoline ring of compound 15 sits ~2.5 a further back in the catalytic pocket compared to the purine ring of amp, perhaps facilitating hydrophobic interactions with residues in the back of the pocket, and possibly a polar interaction between n3 of the quinazoline and y353. second, the 7-methoxy group of compound 15 makes a direct hydrogen bond with d308, whereas amp makes a water-mediated hydrogen bond (fig. 3e ). in addition, residue k277 forms a hydrogen bond with the 7-methoxy oxygen, further strengthening this direct hydrogen bond network. together, the potency of compound 15 is driven by its zinc ion binding, hydrophobic interactions with the back of the binding pocket, and direct hydrogen bonds with active side resides of enpp1. in addition, this structure guided our sar for the core and tail parts of the inhibitor. since our crystal structure suggests that the zinc-binding phosphonate head and quinazoline tail form the most important interactions with enpp1, we next sought to explore the core region to achieve optimal geometry between these two functional groups ( fig. 4a-b) . inverting the nitrogen in the piperidine from aryl to alkyl (compound 18) resulted in more than a 200-fold loss in potency, as did installing a piperazine using the phosphonate zinc-binding head and the piperidine core as a scaffold, we then sought to determine optimal substitution of the quinazoline tail ( fig. 5a-b) . based on our co-crystal structure of enpp1 with compound 15, we predicted that the 7-methoxy would be critical for binding to enpp1, while the 6-methoxy would be dispensable since it is solvent-exposed. indeed, when we deleted the methoxy groups individually, we found that the 6-methoxy alone (compound 25) was 100-fold less potent, while the we then moved to substituents at the 8-position of the quinazoline ring. the 8-methoxy (compound 32) and 8-ethoxy (compound 33) quinazolines displayed impressive potency with a ki values less than 2 nm, which is the limit of quantification of the assay when using a 3 nm concentration of enpp1. we reasoned that combining other methoxy groups with the 8-methoxy could make additional interactions with the binding pocket, since the 7-methoxy (compound 27) alone was also potent. however, combinations of 8-methoxy with substituents in either the 5, 6, or 7 positions (compounds 36-38 and 40-43) all decreased potency. from these data and the crystal structure, we hypothesize that the 8-methoxy (compound 32) and 8-ethoxy (compound 33) quinazolines make hydrogen bonds with both d208 and/or k277 similar to the 7-methoxy in compound 15. this would suggest that compounds 32 and 33 shift in the pocket to accommodate these interactions. adding more substituents to the quinazoline does not necessarily lower potency further, as the inhibitor-interacting residues may already be occupied. in addition, similar to previous sar, 26,32 the 2-substituted vinyl-3-pyridyl combined with the 6,7-dimethoxy (compound 44) showed identical potency to its parent 6,7-dimethoxy (compound 15). this is surprising, since the crystal structure shows limited space in this area of the pocket, suggesting that the pyridine may make a specific interaction with a protein residue. however, this trend did not hold for the 8-methoxy (compound 45), where the addition of the 2-substituted vinyl-3-pyridyl decreased potency by more than 100-fold. this further supports the hypothesis that different substitutions cause shifts in the inhibitor binding to the pocket, possibly excluding space that was previously available. from the crystal structure of compound 15 bound to enpp1, we noted that the quinazoline tail of compound 15 is close to residues near the back of the binding pocket. we perturbed the tail aromatic ring structure itself to investigate these possible interactions (fig. 5a,c) . working from the quinazoline compound 15, we deleted the nitrogens on the ring on by one. deleting n3 (quinoline, compound 46) led to merely a 3-fold decrease in potency, but deleting n1 (isoquinoline, compound 51) led to a more than 100-fold decrease in potency. we tested whether we could replace the inhibitor-protein interaction that involved the n3 or n1 with a nitrile, a more electronegative group and possible hydrogen bond acceptor, as has been previously suggested. 39 indeed, the 6,7-dimethoxy, 3-nitrile quinoline (compound 47) was more potent than the quinazoline itself (compound 46), and this pattern repeated with all of the other methoxy substituents (compounds 48-50). compound 50 was the most potent molecule with a ki less than our limit of quantification of 2 nm. when we attempted to substitute back the missing nitrogen with a nitrile group on the isoquinoline scaffold (compound 52), we lost potency, possibly due to molecular clashes between inhibitor and protein. in summary, the n1 nitrogen is critical for potency, possibly due to polar interactions with the protein backbone ~4 å away. it cannot be replaced by a nitrile since space is limited. the n3 nitrogen can be replaced by a more electronegative nitrile, leading to much more potent compounds. taking into account of all of our sar data, we then synthesized hybrid molecules composed of the most potent heads, cores, and tails (8-methoxy quinazoline, e.g., compound 32, and 8-methoxy quinoline 3nitrile, e.g., compound 50) (fig. 6 ). for all of these molecules, ki values fell below 100 nm. combining the benzyl amine core with the 8-methoxy quinoline 3-nitrile tail (compound 57) yielded another inhibitor with a ki less than our limit of quantification of 2 nm. attaching other zinc binding head groups, including thiophosphate (compound 53), boronic acid (compounds 60 and 61), and hydroxamic acid (compound 62), to the most potent core/tail combinations also yielded potent inhibitors. we further evaluated the potency and in vitro adme properties of the seven most potent inhibitors. first, we evaluated the protein-shifted ic50, which can help predict what the efficacy will be in vivo, as this value is generally higher than that observed in vitro due to protein binding. we observed that all of the inhibitors displayed a shift in potency when we did the assay in the presence of human serum albumin, although several still stayed below 50 nm (fig. 7a, table 1, fig. s2 ). we then tested their potency in both mouse and human plasma, and obtained similar values, confirming that mid-nanomolar concentrations are sufficient in biological fluids to prevent degradation of cgamp (fig. 7b, table 1 ). although enpp1 protein is present in our circulation, these data demonstrate that we can completely block serum enpp1 activity with as little as 100 nm of our most potent inhibitors, showing that protein binding does not negate the inhibition, and suggesting that serum enpp1 will not sequester all the systemically administered enpp1 inhibitor. finally, all of our inhibitors are stable and cell impermeable. although we expect these phosphonate inhibitors to have few intracellular off-targets due to their impermeability, we also confirmed that the inhibitors are non-toxic to primary human peripheral blood mononuclear cells (pbmcs) ( table 1 , fig. s3 ). since qs1 has an off-target herg liability, 26 we tested two phosphonate inhibitors (compounds 15 and 32) and observed no inhibition at 25 μm (table 1) . we then assessed the pharmacokinetic (pk) profile of one of the top inhibitors, compound 32, in mice. since compound 32 has an ic50 value of 4 nm in mouse plasma, we aim to achieve serum concentrations of 40 nm (ic95 value). first, we administered intravenous (iv) and subcutaneous (sc) doses of compound 32 at 10 mg/kg to mice and analyzed the concentration of compound 32 in the serum, kidney, and liver for the next 8 hours. we found that serum concentrations declined to 10 nm or less within 8 hours for both iv and sc administration, and the half-life was only 10-15 minutes. concurrent with the rapid decline in serum concentrations, we saw concentration plateaus in the kidney and liver in the micromolar range, suggesting that compound 32 is rapidly excreted through these organs. this is in agreement with our previously reported pk study of compound 32 performed at 300 mg/kg via sc administration where we observed that the serum concentration drops to ~100 nm at the end of 24 hours. 13 therefore, we were only able to test the efficacy of compound 32 in that study dosing via an osmotic pump to maintain serum concentration of compound 32 above its ic95. to achieve sustained serum concentrations without surgically implanting osmotic pumps, we repeated the 300 mg/kg sc dosing daily for 6 days. we collected serum 24 hours after the previous dose, which corresponds to the expected lowest point in the trough. we observed on average ~100 nm of compound 32 across several days, and the lowest concentration measured was 40 nm. we also observed no adverse effects and the mice maintained healthy weights, suggesting that this dosage amount and timing is tolerated well. together, we succeeded in achieving convenient, systemic, once daily dosing of compound 32 that results in sustained serum concentrations above the ic95. after optimization of compound 32 pk, we then asked if compound 32 has anti-tumor efficacy in the orthotopic and syngeneic e0771 triple negative breast cancer model. we chose this model because e0771 cells basally export cgamp in cell culture. in addition, we previously saw that e0771 cells implanted into mice grow more slowly in enpp1 -/mice than in wild type mice. 13 we treated mice with established e0771 tumors (~100 mm 3 ) with compound 32 using the optimized dosing schedule for seven consecutive days. remarkably, we observed a delay in tumor growth in mice treated with compound 32 relative to the controls, which also led to prolonged survival. the tumor growth delay observed with pharmacological inhibition of enpp1 mirrors that from genetic ablation of enpp1 we previously reported, 13 demonstrating that our enpp1 inhibitors are therapeutically beneficial. here we report the development of highly potent phosphonate enpp1 inhibitors. we demonstrate that they are active against the physiological substrate cgamp under physiological conditions, including an in vitro assay at ph 7.4 and an ex vivo assay against enpp1 in mouse and human plasma. our compounds are by far the most potent inhibitors reported to date. although some of the boronic acids (compounds 60 and 61) and hydroxamic acids (compound 62) are also potent, further investigation of their cell permeability and possible off-target effects would be needed. the lead phosphonate compounds, however, are cell impermeable through passive diffusion, avoiding all potential intracellular off-targets. we, therefore, nominate them as specific tool compounds to study enpp1 and extracellular cgamp biology. to understand the potency of our compounds, we solved the crystal structure of enpp1 with compound 15. we found that compound 15 adopts a similar binding pose as amp, the product of cgamp and atp hydrolysis, but there are extra interactions that explain its enhanced potency including a direct hydrogen bond between the 7-methoxy and d308 (instead of water-mediated), extra hydrogen bond interactions with k277, and other hydrophobic and polar interactions with l272 and y353. our sar of other phosphonate inhibitors allowed us to build a model of the drivers of potency. the p-p stacking interactions are key, as only a couple of the cores we tried resulted in potent inhibitors and the core region could be important for positioning of the zinc-binding phosphonate with respect to the p-p stacking tail. we hypothesize that moving the methoxy groups to different positions on the ring would also engage d308 and/or k277 in hydrogen bonding. even though some of the positions on the ring are solvent-exposed in the compound 15 structure in complex with enpp1, combinations of more substituents are detrimental to potency, suggesting that these compounds could be shifted in the binding pocket leading to steric hinderance when extra substituents are added. it is interesting to note that enpp1 makes better interactions with the methoxy group, in contrast to the hydroxy or amine (e.g., compare compound 27 to compounds 29 and 30; compare compound 32 to compound 34), suggesting that aliphatic carbons are important in addition to hydrogen bonding. in addition, we can speculate about the importance of the 3-nitrile group off the quinoline, e.g. compound 47 and related analogs. compared to the n3 on the quinazoline, it is possible that the nitrile can make stronger polar interactions with the protein backbone or surrounding residues (e.g., d200, y353, or e355), act as a hydrogen bond acceptor, or polarize the quinoline ring for better p-p stacking. 40 previous modeling showed that nitriles can replace water-mediated azomethine-protein interactions 39 . this could extend to our scenario in replacing a weak polar interaction with a stronger one. in addition, it is surprising that the 2-vinyl-3-pyridyl substituent (compound 44) is potent, given that the 2 position faces the protein backbone. it is possible that this large substituent can slide into the narrow pocket between h242 and y353 or could make a specific interaction with one of the residues. it also suggests that this space is not as available with the 8-methoxy substituent, as compound 45 (with 2-vinyl-3-pyridyl) was more than one hundred-fold less potent than compound 32 (without 2-vinyl-3-pyridyl). in addition to unmatched potency, our enpp1 inhibitors also have desirable adme profiles in vitro, and we have measured the pharmacokinetics of one of the top inhibitors to demonstrate efficacious concentrations in mice with once daily systemic dosing. compared to jump starting the anti-cancer innate immune response by treating with direct sting agonists, our enpp1 inhibitors provide two advantages. first, although endogenous extracellular cgamp enhances the anti-tumor immune response, 13 the same may not be true for cgamp analogs. because cgamp is specifically transported into different cell types through different transporters, 14, 15 it is difficult to design cgamp analogs that match the cell-targeting profile of cgamp itself. it is important to target specific cells because sting activation in cancer cells promotes metastasis, 8 and sting activation in t cells leads to t cell death, [41] [42] [43] both of which would be detrimental to cancer patients. in contrast, enpp1 inhibitors should increase the half-life of endogenous cgamp and thus enhance the natural anti-tumor response. indeed, treatment of mice with compound 32 delayed tumor growth in the e0771 breast cancer mouse model, which is a promising result for enpp1 inhibitors as cancer therapeutics. second, sting agonists can only be introduced intratumorally to achieve efficacy and to avoid systemic inflammation, which limits treatment to palpable and injectable tumors. since our enpp1 inhibitors can be administered systemically, we hypothesize that enpp1 inhibitors could treat a wide variety of cancers and may even be effective against undetectable micrometastasis. they could also avoid causing widespread toxic interferon production, since they only enhance endogenous cgamp. this hypothesis is supported by the fact that enpp1-inactivating mutations in both humans and mice do not cause interferonopathy. our enpp1 inhibitors are biological tools as well as candidate investigational drugs that have shown efficacy in mouse tumor models, and they hold the promise to potentiate the efficacy of radiation and other immune checkpoint inhibitors. finally, since cgamp has shown stunning results as an adjuvant for influenza vaccination, 20 our lead enpp1 inhibitor that prevents its extracellular degradation may have the potential to maximize cgamp's adjuvancy effect in developing vaccines for pandemic threats. all procedures to obtain plasma after blood draw were performed at 4 ºc. blood from c57b6/j mice was obtained by cardiac puncture and deposited in heparin-coated tubes (bd microtainer with pst additive). mouse enpp1 (3 nm; expressed and purified as described previously 24 ) was incubated with 5 µm cgamp (synthesized as described previously 13 the extracellular region (residues 92-905) of mouse enpp1 was expressed, purified, and crystallized as multiple needle-like crystals were tested for x-ray diffraction which, when x-ray exposed, showed weak diffracting power. one needle crystal was isolated and used to collect a data set to a minimum d-spacing of around 3.2 å. data was collected at cryogenic temperature (100 k) at beamline 5.0.1 of the advanced light source (als) synchrotron (berkeley, ca, usa) at a single 0.97741 å wavelength. data was reduced with mosflm 44 and scaled with scala 45 within the ccp4 suite. 46 the crystal belonged to the trigonal space group p 31 and contained two polypeptide chains per asymmetry unit. data collection and refinement statistics are listed in table 3 . the structure was solved by the molecular replacement method with phaser 47 using mouse enpp1 (pdb code: 4gtw) polypeptide chain stripped from ligands, ions, and glycan chains, as the search model. structural refinement was done using refmac 48 iteratively with visual inspection of electron density maps and manual adjustment of atomic coordinates in coot 49 until progression to convergence. the final refined structure shows an excellent agreement with reference protein data as shown by ramachandran statistics (table 4 ). data collection statistics are derived from scala. 45 to calculate rfree, 5% of the reflections were excluded from the refinement. rsym is defined as rsym = σhklσi|ii(hkl) -<i(hkl)>| / σhklσiii(hkl). data refinement statistics are derived from refmac. 48 the final quality check was done with procheck. 50 graphic renderings were prepared with pymol. 51 as previously observed, 24 ligands are shown as sticks/spheres, protein residue d308 is shown as sticks, and water is shown as sticks/spheres. density for water molecule is present only in amp-enpp1 crystal structure. (f) chemical structure of amp. dotted line represents the minimum ic50 value (2 nm) measurable with the given enzyme concentration. chemical structures of inhibitors are displayed below. dots represent the mean of two independent replicates, and shaded areas around the fitted curves represent the 95% confidence interval of the fit. cell viability of primary human peripheral blood mononuclear cells (pbmcs) after incubation with indicated compounds for 16 hours measured by celltiterglo. data is normalized to no compound (100% cell viability). two cell culture replicates are plotted. 45 to calculate rfree, 5% of the reflections were excluded from the refinement. rsym is defined as rsym = σhklσi|ii(hkl) -<i(hkl)>| / σhklσiii(hkl). data refinement statistics are derived from refmac. the final quality check was done with procheck. safety, activity, and immune correlates of anti-pd-1 antibody in cancer innate immune recognition of cancer innate immune signaling and regulation in cancer immunotherapy host type i ifn signals are required for antitumor cd8 + t cell responses through cd8α + dendritic cells the multifaceted role of chromosomal instability in cancer and its microenvironment cgas surveillance of micronuclei links genome instability to innate immunity mitotic progression following dna damage enables pattern recognition within micronuclei chromosomal instability drives metastasis through a cytosolic dna response extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna. science (80-. ) cgas produces a 2′-5′-linked cyclic dinucleotide second messenger that activates sting extracellular cgamp is a cancer-cell-produced immunotransmitter involved in radiation-induced anticancer immunity slc19a1 is an importer of the immunotransmitter cgamp the lrrc8a:c heteromeric channel is a cgamp transporter and the dominant cgamp importer in human vasculature cells tumor-derived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response blockade of the phagocytic receptor mertk on tumor-associated macrophages enhances p2x7r-dependent sting activation by tumor-derived cgamp hydrolysis of 2'3'-cgamp by enpp1 and design of nonhydrolyzable analogs pulmonary surfactant-biomimetic nanoparticles potentiate heterosubtypic influenza immunity. science (80-. ) identification and characterization of a soluble form of the plasma cell membrane glycoprotein pc-1 variants of enpp1 are associated with childhood and adult obesity and increase the risk of glucose intolerance and type 2 diabetes structure of npp1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification crystal structure of enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling structural insights into cgamp degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1 quinazolin-4-piperidin-4-methyl sulfamide pc-1 inhibitors: alleviating herg interactions through structure based design imidazopyridine-and purine-thioacetamide derivatives: potent inhibitors of nucleotide pyrophosphatase/phosphodiesterase 1 (npp1) quinazoline-4-piperidine sulfamides are specific inhibitors of human npp1 and prevent pathological mineralization of valve interstitial cells 2-a]benzimidazol-3(2h)-one derivatives: structure-activity relationships of selective nucleotide pyrophosphatase/phosphodiesterase1 (npp1) inhibitors substrate-dependence of competitive nucleotide pyrophosphatase / phosphodiesterase1 (npp1) inhibitors synthesis of novel substituted pyrimidine derivatives bearing a sulfamide group and their in vitro cancer growth inhibition activity synthesis and biological evaluation of novel quinazoline-4-piperidinesulfamide derivatives as inhibitors of npp1 highly selective and potent ectonucleotide pyrophosphatase-1 (npp1) inhibitors based on uridine 5'-pa,a-dithiophosphate analogues deazapurine analogues bearing a 1h-pyrazolo[3,4-b]pyridin-3(2h)-one core: synthesis and biological activity blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review the acidic tumor microenvironment as a driver of cancer development of cgamp-luc, a sensitive and precise coupled enzyme assay to measure cgamp in complex biological samples expression, purification, crystallization and preliminary x-ray crystallographic analysis of enpp1 optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity nitrile-containing pharmaceuticals: efficacious roles of the nitrile pharmacophore intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes cutting edge: activation of sting in t cells induces type i ifn responses and cell death signalling strength determines proapoptotic functions of sting evolving methods for macromolecular crystallography: processing diffraction data with mosflm an introduction to data reduction: space-group determination, scaling and intensity statistics d concentration); the maximum is 10 µm. (e) chemical structures and ki values of compounds 1 and 2 (mean of at least 2 independent replicates residues in...* number of residues (%) most favored regions 962 (83.3%) additional allowed regions 185 (16.0%) generously allowed regions 6 (0.5%) disallowed regions 2 (0.2%) *according to procheck for non-proline and non-glycine residues (1155 residues). key: cord-254036-0karwgz2 authors: wang, runming; li, hongyan; sun, hongzhe title: bismuth: environmental pollution and health effects date: 2019-09-12 journal: encyclopedia of environmental health doi: 10.1016/b978-0-12-409548-9.11870-6 sha: doc_id: 254036 cord_uid: 0karwgz2 although it has been used for centuries, bismuth remains one of the least understood elements in the periodic table. metallic bismuth and bismuth compounds have been widely used in the manufacture of alloys, pigments, cosmetics, and pharmaceuticals. as a “green” heavy metal, the substitution of lead with bismuth in some industries may partially resolve the environmental problems related to heavy metal pollution. in health care, as bismuth has low toxicity to humans, bismuth-based drugs such as colloidal bismuth subcitrate (cbs), ranitidine bismuth citrate (rbc), bismuth subsalicylate (bss), bismuth iodoform and radioactive bismuth ((212)bi/(213)bi) complexes have been developed and used in clinics to treat various diseases. in most cases, bismuth therapies exhibit high therapeutic efficacies and little side effects; nevertheless, there are still reported cases of bismuth toxicity caused by bismuth over-dosage. bismuth is a metallic chemical element, symbolled as bi, with atomic number of 83. although it has been known since ancient times, it became familiar to the greeks and romans during the middle ages, before that bismuth was often confused with other metals, such as lead and tin. it is considered to be a metal in the periodic table but has more similarity to semimetals. bismuth is categorized among the group of elements conventionally known as "poor elements" due to its rarity. it has been used quite extensively for various purposes including cosmetic, industrial, laboratory, and pharmaceutical. bismuth serves as a leading nontoxic replacement for lead in brass plumbing fixtures, fishing sinkers, free machining steels, and solders, as well as a metallurgical additive in the foundry. the manufacture applications of bismuth also include ceramic glazes, pearlescent pigments, lubricating greases, and crystal ware. major applications of bismuth in medicine and health care are related to its high effectiveness in treating burns, intestinal disorders, and stomach ulcers as well as its potential activities against microorganisms, viruses and malignant tumors. with the development of new therapies and better understanding of the mechanism of action of bismuth, the applications of bismuth-based agents in health care will be further extended. bismuth is a relatively rare element in the earth and ranked 69th in terms of its natural abundance in the earth's crust, estimated at about 2 ppb by atoms. the world reserves of bismuth is usually estimated based on the content of lead resources since bismuth is always a by-product of the processing of other metal ores such as lead, silver, tin, copper, and zinc. only the tasna mine in bolivia and a mine in china produce bismuth as a major product. world reserves of bismuth are estimated at around 320,000 tons. bismuth has been consumed rapidly over the last decade. in 2018 approximately 16,000 tons of refinery bismuth were produced worldwide. among all the countries, china is the leading producer of refined bismuth with ca. 79.9% of the world's total production, followed by laos, japan, and mexico with ca. 12.3%, 3.6%, and 2.1% respectively. the estimated global reserve of bismuth is shown in fig. 1 . comparison to majority of other heavy metals, bismuth has low toxicity, which marks it as a "green metal" for the environment. bismuth and its alloys have widespread commercial applications such as in the production of lubricating grease, chemicals, catalysts, shot bullets, cosmetics, fire sprinkler systems, solders, thermoelectric materials, pigments, fishing sinkers, 235 u/ 233 u carriers, medicines, malleable steels, etc. the semimetal crystal structure of bismuth along with its other physical-chemical properties such as expansion on solidification, the widest range between melting and boiling points among all metals, and the lowest thermal and heat conductivity make bismuth an ideal substitute for lead analogs in extreme-pressure additives (ep additives). substitution of the formerly widely used lead-based ep additives by bismuth-sulfur additives not only provides a higher lubricant efficacy, but also reduces the amount of heavy metal used in extreme-pressure lubricants by a half, in accordance with the new ecological and environmental philosophy of the world. in recent decades, the substitution of lead with bismuth in glass production may lead to potential application for bismuth in the manufacturing of automobile glass, the finest tableware, and art objects, with the purpose of environmental protection. in north america, biesn has been used to replace lead in shotshells for the hunting of wetland birds. although the bismuth-containing shotshells cannot be approved as "nontoxic," in comparison to the high toxicity of lead (e.g. a toxic intake level of 1 mg for a 70 kg human), the higher tolerance to bismuth among humans (e.g. a toxic intake level of 15 â 10 3 mg for a 70 kg human) renders it a "relatively" safe substitute for preventing environmental problems caused by lead accumulation. owing to their satiny luster and low absorption properties, some bismuth compounds such as bismuth oxychloride and bismuth vanadate have also been used in cosmetics including nail polishes, lipsticks, and eye shadows. recently in europe, the restriction of hazardous substances directive (rohs-2002/95/ec) has stated that lead must be eliminated in the manufacturing of various types of electronic and electrical equipment. this restriction, which has been supported by commercial and research organizations in japan and the north america, may further increase the demand for bismuth in industry in future, and accelerate the replacement of lead and other heavy metals with bismuth in the manufacturing industry. the medical use of bismuth can be traced back to 200 years ago, and bismuth-related agents have been involved in the treatment of a wide range of diseases including syphilis (sodium/potassium bismuth tartrate, bismuth quinine iodide, iododbismitol, bismuth chloride, etc.), colitis (bismuth subnitrate and bismuth citrate), wound infection (bismuth oxide) and parasite infections (sodium bismuth thioglycolate). the massive usage of bismuth compounds for the treatment of syphilis and wound infections in early 20th century was dramatically dropped owing to the discovery of antibiotics, thus, contemporary use of bismuth in medicine principally lies in the treatment of gastrointestinal disorders such as gastric and duodenal ulcers (e.g. colloidal bismuth subcitrate (cbs), bismuth subsalicylate, and bismuth subnitrate), dyspepsia (bismuth subsalicylate, bismuth subnitrate, etc.), and diarrhea (bismuth subsalicylate, bismuth nitrate, etc.). bismuth subsalicylate (bss, peptobismols; the procter & gamble company, cincinnati, ohio, united states) is one of the most commercially successful bismuth medicines with annual sales of 82.6 million usd in united states in 2013 (statistics from the statistics portal). it has been widely used for rapid relief of heartburn, nausea, indigestion and diarrhea. on the basis of crystallographic analysis, bss possesses a complex cluster including bi(iii) ions, mono-deprotonated salicylate, deprotonated h 2 o and other oxo ligands. upon interaction with gastric acid in stomach, this medicine releases bi(iii) ions, which is known to inhibit the growth of helicobacter pylori (h. pylori, fig. 2 ), and salicylate, which ameliorates gastritis and antagonizes inflammation. bismuth citrate-based compounds, represented by cbs (de-nol; gist brocades and yamanouchi), are the most typical anti-h. pylori bismuth drugs and have been widely utilized in "cocktail" therapy to treat a variety of h. pylori-associated infections. the therapeutic efficacy of bismuth citrate-based drugs against the h. pylori-associated infections may be attributed to (1) preferential formation of a "protective coating" on the ulcer craters owing to the fact that cbs can form polymeric structures (fig. 3); (2) induction of the secretion of increasing mucosal protective factors; (3) antimicrobial activity of bi(iii), which is attributable to its ability to disrupt multiple biological pathways via binding to key proteins andenzymes. ranitidine bismuth citrate (rbc, tritec and pylorid, glax-osmithkline plc.) was later found to be a more efficient antiulcer agent, since it incorporates an h 2 -receptor antagonist, ranitidine, to suppress secretion of excess stomach acid. the newly developed bismuth-based triple or quadruple regimens, which combine the bismuth citrate-based drugs with antibiotics such as amoxicillin, tetracycline, clarithromycin, or nitroimidazole, and proton-pump inhibitors (ppis) have been often recommended in clinics for treating h. pylori-associated infections. in comparison to the non-bismuth therapies, bismuth-based regimens significantly increase the eradication rate of antibiotic resistant h. pylori (table 1 ) even for antibiotic resistant strains. ) or bi(cit)) polymeric structure by the dimeric unit ([bi 2 (cit) 2 ] 2 2n à ). the polymeric framework probably coats on the ulcer craters to prevent the erosion of gastric acid. the negatively charged ([bi 2 (cit) 2 ] 2 2n à ) framework is balanced by cations. color code: bi, yellow; c, gray; o, red; cation, blue. a new drug of bismuth with d-poly galacturonic acid, colloidal bismuth pectin, was approved for clinical use in china in the treatment of peptic ulcers and is as effective as cbs. apart from over-the-counter bismuth drugs, numbers of novel bismuth compounds with remarkable anti-h. pylori activity have been synthesized in recent decades. bi(iii) complexes with hydroxamic acids, indole-carboxylic acids, a-amino acids, nonsteroidal antiinflammatories, acetylsalicylic acid, glycosaminoglycan, polysaccharide, and some other organic ligands exhibit higher or comparable anti-h. pylori activity to bss and cbs. since nanotechnology has been extensively explored, bismuth-based nanomaterials have also received more attention in pharmaceuticals. for example, bismuth subcarbonate ((bio) 2 co 3 ) nanotubes exhibit slightly higher activities against h. pylori than the clinically used colloidal bismuth subcitrate under similar conditions, and importantly, bismuth nanotubes could be used as "capsules" in bismuth-based triple or quadruple therapies for the treatment of h. pylori infection, or as drug "carriers" for sustained-release of other ingredients in the human body, when used in combination with other drugs for the treatment of other diseases. bismuth is an effective anti-h. pylori agent though it appears more bacteriostatic than bactericidal. what differentiates bismuth from organic antibiotics is that bismuth drugs have multiple cellular targets. thus, the anti-h. pylori actions of bismuth drugs are resulted from comprehensive factors including but not limited to, hindrance of synthesis of bacterial cell wall, impediment of bacterial adhesion, inhibition of atp synthesis, abolishment of defense of oxidative stress and ph buffering ability, interference of metal homeostasis (e.g. ni 2 þ homeostasis), inhibition of activity of key enzymes such as alcohol dehydrogenase, urease, dnak. a recent report showed that bismuth inhibits urease activity by disruption of the cellular maturation of urease via functional perturbation of one of its accessory proteins, ureg, rather than directly targets the metal center of urease. despite that extensive studies have been made at molecular levels, the exact mode of action of bismuth drug in h. pylori still remains to be unclear. bismuth also exhibits potential activities against microorganisms beyond h. pylori. bismuth thiolates are one of the most representative antimicrobial agents among those newly developed bismuth compounds. the antimicrobial activities of bismuth are greatly enhanced when coordinated with lipophilic thiol-containing ligands such as 1,3-propanedithiol, 3-mer-capto-2butanol, b-mercaptoethanol, dimercaprol, and dithiothreitol. these complexes show antimicrobial activity against some gram-negative bacterial strains such as escherichia coli, burkholderia cepacia complex, klebsiella pneumoniae, as well as some gram-positive bacterial strains such as staphylococcus aureus (s. aureus) and streptococcus pyogenes, with minimum inhibitory concentration (mic) at micromolar levels. subsequent studies show that bi(iii) thiolates exhibit remarkable anti-biofilm activity against pseudomonas aeruginosa and methicillin-resistant s. aureus biofilms. recent research indicates that the antimicrobial spectrum of bismuth thiolates could be further broadened when prepared in nanoparticle form (exemplified as bisbal nps). the enhanced antimicrobial potency of bismuth thiolates is possibly in part due to the chelation effect of thiols that significantly improves the solubility and lipophilicity of bi(iii) compounds, as well as the rapid exchange of those kinetically labile thiolate ligands inside bacterial cells. the antimicrobial activity of bismuth can be synergistically enhanced by coordination with other antimicrobial agents. for example, bismuth conjugates with fluoroquinolone, i.e., norfloxacin and ciprofloxacin, show notable activity with mic at micromolar levels against both gram-positive and-negative bacterial strains such as e. coli, s. aureus, s. epidermideis, e. facecalis, b. cereus and b. pumilus. recent research reveals that bismuth compounds or drugs could be potentially used as a novel class of antibiotic adjuvant in the treatment of infections caused by metallo-b-lactamase (mbl) producing superbugs (fig. 4) . bismuth drugs, i.e., colloidal bismuth subcitrate (cbs), exhibit potent inhibition on mbl activity through displacement of zinc ions (as essential cofactors) from enzyme's active site. bismuth-based antibiotic combination therapy greatly boosted the antimicrobial effectiveness of existing antibiotic, by which the survival of mice infected by mbl superbugs was significantly increased. it shows that bismuth-based compounds and related metallodrugs could become a novel class of mbl inhibitors, and they have great potential to be used as effective antibiotic adjuvants to treat infections caused by mbl superbugs in a wider clinical context. bismuth compounds have been widely found to exhibit potential antineoplastic activity against malignant tumor cell lines. the most promising candidates with potent chemotherapeutic activity are those bismuth compounds with bismuth coordinated to the ligands including thiosemicarbazone/thiocarbonohydrazone, hydrazine, dithiocarbamate and halide, as well as some radiosensitive bismuth-based nanomaterials. for example, a 9-coordinated bi(iii) complex of [bi(h 2 l)(no 3 ) 2 ]no 3 (h 2 l ¼ 2,6diacetylpyridine bis((4)n-methylthiosemicarbazone)) has an ic 50 value of 26.8 mm against k562 leukemia cells and an inhibitory rate of 61.6% in a hepatocarcinoma (h22 cell line) xenograft mouse model. bi(iii) dithiocarbamate complexes with a general formula of bi(s 2 cnr2) 3 exhibit powerful anticancer activity against seven cancer cell lines including breast cancer cell line (mcf-7) and ovarian cancer cell line (igrov) with ic 50 values of no more than 0.22 mm. a water-soluble bismuth cyclen based compound, abbreviated as bi-tpc, exhibits great anticancer activity that is 100-time more potent than cisplatin, probably via interactions with dna under physiologically relevant conditions. it has been shown that radioactive bismuth ( 212 bi and 213 bi) exhibits promising potential as a novel therapeutic agent for mall volume tumors. both 212 bi and 213 bi have a series of branched decays, resulting in the emission of aà/b-particles. although the half-life of 212 bi is short (t 1/2 1 h), and radioactive lead ( 212 pb, t 1/2 10.6 h) can be used as an in vivo generator of 212 bi. with their short-ranged penetration (50-80 mm), bismuth radionuclides can reduce the nonspecific irradiation to normal tissues around the target cells. furthermore, in order to deliver bismuth to the site of action effectively, a chelate ligand can be used together with bismuth radionuclides. in this type of combination treatment, the strong chelate ligand is conjugated to a monoclonal antibody or a fusion protein, a standard treatment for tumors, via modification of the ligand to eventually produce a bismuthradiolabeled "complex." once the metal radiolabeled "complex" is introduced into the host, it targets specific cell types or sites of diseases and releases the a-particles only at or near the tumor tissues, thereby minimizing damage to the surrounding normal tissues. recently, a chemical conjugation, abbreviated as tam-bi 2 s 3 @mps nps, incorporating bismuth sulfide@mesoporous silica coreshell nanoparticles and trastuzumab, an antibody targeting her-2 overexpressed breast cancer cells, is rationally designed to exert good anticancer activity with good biocompatibility and drug loading ability. this conjugate reaches certain degree of directed targeting with 16-fold higher bismuth content in targeted tumor group compared with non-targeted group. this type of bi-based nanomaterial may pioneer a new direction for the development of target-directed therapeutic agents for cancer treatment. the antitumor mechanism of action of bi(iii) compounds is principally correlated to the generation of ros, reduction in mitochondrial membrane potential, induction of apoptosis via caspase-3 mediated or g2/m arrest mechanisms, rather than dna binding as what platinum compounds carry out, though the bismuth-dna binding was sporadically reported. these unique modes of action of bi(iii)-based anticancer agents display a great potential to circumvent the therapeutic stress placed upward by cisplatin resistance. but the detailed mechanism of action of bi-based anticancer agents still remains a mystery. further exploration at the mechanistic and therapeutic levels on bismuth compounds as anticancer agents might serve as a new direction in future. there have not been reports about antiviral applications of bismuth compounds with the exception of bismuth sodium triglycollamate, historically used to treat warts. recently, bismuth was discovered to be effective in inhibiting severe acute respiratory syndrome coronavirus (sars-cov) with an ic 50 less than 1 mm. the potential target of bismuth is the scv ntpase/helicase, a zinc-containing enzyme that has rna capping activity and controls the virus reproduction. binding of bismuth may induce conformational changes in the enzyme, which subsequently affects the helicase rna/dna unwinding activity, thereby inhibiting the virus proliferation (fig. 5) . another potential application of bismuth compounds is in the amelioration of side effects induced by the anticancer drug, cisplatin. cisplatin and its analogs (e.g. carboplatin and oxaliplatin) have been used in clinics worldwide to treat various solid cancers. however, a major obstacle to the more widespread use of cisplatin-based drugs resides in their severe side effects. the major dose-limiting factor is nephrotoxicity, including tubular degeneration, loss of brush border, necrosis, and mineralization of the tubular epithelial cells. these renal damages are caused by platinum (pt). the mechanism of platinum nephrotoxicity may be similar to that of mercury (hg) and likely relates to the depletion of thiolate groups in the renal tubes. bismuth compounds, such as bismuth (sub)nitrate, have been reported to alleviate cisplatin-induced nephrotoxicity without interfering its antitumor response. recent studies show that a bismuth-citrate based complex, bizn, significantly preserved the renal function of mice receiving overdose of cisplatin treatment. the compound could potentially simulate the production of antioxidative biomolecules, metallothionein and glutathione from kidney cells and minimize cisplatin-induced apoptosis. in vivo experiments displayed that when compared with the cisplatin-alone group, the group of mice receiving pretreatment of bismuth compounds can greatly reduce the blood urea nitrogen (bun) and creatinine level, defined as an indicator of renal damage and the survival rate was significantly increased (fig. 6) . unlike other heavy metals, e.g. lead and mercury, bismuth and its related drugs have been involved in clinic treatment of h. pyloriassociated infections for decades, and seldom reported to have acute toxicity in humans even being administered at extreme dosages in some cases. over a long period of time, it remains a mystery that why bismuth(iii) is selectively toxic to certain human pathogens rather than human beings. one possible reason is the limited gastrointestinal tract (git) absorption of bismuth after its oral administration. recent studies indicated that glutathione (gsh) may play a vital role for bismuth metabolism and detoxification in mammalian cells. bismuth ions were found to be passively absorbed, conjugated to gsh and then transported into vesicles via mrp transporter; the sequestration of absorbed bismuth consumed cytosolic glutathione and activated was in de novo biosynthesis, which in turn facilitated passive uptake of bismuth (fig. 7) . the self-propelled positive feedback cycle actively eliminated bismuth from both intra-and extracellular space, thus protecting critical systems of human body from acute toxicity. considering gsh is ubiquitous in most living cells, but only absent in certain anaerobic or microanaerobic bacteria such as h. pylori, the gsh and mrp mediated self-propelled disposal of bismuth in host cells might be accountable for the selective toxicity of bismuth drugs. in biological systems, proteins and enzymes have been regarded as potential targets for bismuth. investigations of bismuthprotein interactions will not only improve understanding of the mechanism of action of bismuth but will also provide a basis for the design of more effective bismuth agents. bismuth was found to be binding to transferrin (an iron transport protein) preferentially to the c-lobe with carbonate (co 3 2 à ) as a synergistic anion. transferrin is likely to act as a bismuth transport "vehicle," as it is only 30% saturated with iron in blood plasma. importantly, the metal-bound transferrin can be rapidly recognized by the transferrin receptor because of the higher affinity of the receptor to metal-bound transferrin than to its apo-form (fig. 8) . although human serum albumin is the most abundant protein in serum (0.63 mm) and has been hypothesized to be the target of bismuth in plasma. around 70% bismuth associates to transferrin and the rest to increasing bismuth concentration serum albumin, indicating a higher selectivity of bismuth to transferrin. lactoferrin is another type of major iron binding protein in the transferrin family, which can also strongly bind to bismuth ions. the bismuth-bound lactoferrin is able to compete with the iron-bound lactoferrin in both the membrane and intracellular, providing another route for bismuth transport. histidine-rich proteins, such as hpn and hpn-like (46.7% and 25% histidine residues, respectively) in h. pylori are also potential targets of bismuth ions. mutagenesis experiments have shown that h. pylori with hpn gene knock-out are fourfold susceptible to bismuth antiulcer drugs than that of the wild-type, indicating a protective role of hpn in h. pylori responses to bismuth-based therapies. the interaction of bismuth with enzymes is related to the high affinity to cysteine residues. for example, bismuth inhibits urease activity probably by blocking the enzyme active site by coordinating to a cysteine residue at the entrance of the active site. although bismuth is considered to be nontoxic as stated previously, the long-term use of bismuth may result in certain degree of side effects on human subjects. besides the few cases caused by occupational exposure to bismuth in the manufacturing industry, most of the poisoning incidents occur in the form of accidental or deliberate over-dosage of bismuth drugs. the extent of bismuth toxicity depends on individual cases, i.e., the types of bismuth compounds and the amounts absorbed. it is still not clear why only selected individuals develop bismuth toxicity. patients suffer toxicity at different bismuth levels in blood but the syndrome is rare when bismuth levels are below 50 mg/l. among the bismuth-based regimens, the use of insoluble bismuth compounds such as bismuth oxychloride and bismuth subcarbonate are related to low toxicity, whereas the use of soluble bismuth organic compounds such as bismuth sodium tartrate and tripotassium dicitratobismuthate, or the combined use of bismuth with thiolate-containing ligands, are associated with high toxicity, such as neurotoxicity and nephrotoxicity. this is probably due to the enhanced uptake of soluble bismuth salts in human bodies. it has also been suggested that the oral bismuth drugs need to undergo methylation by intestinal microbes to enable them to be absorbed. absorbed bismuth will accumulate in the kidneys, lungs, spleen, liver, brain, and muscles, and will be eliminated in urine and feces via bile and intestinal secretions. in the clinic, depending on the administration time of bismuth, its toxicity can be roughly divided into acute and chronic exposures. both exposure doses can cause neurotoxicity, gastrointestinal toxicity, nephrotoxicity, hepatotoxicity, and increased bismuth concentration in blood. in spite of the toxicity, most of these side effects can be alleviated after the discontinuation of bismuth therapies. bismuth iodoform paraffin paste (bipp), which reduces the risk of bacterial infection, renders a deep necrotic wound cavity clean and promotes the development of granulation tissue, and is widely used in oral, maxillofacial, and ent surgery (ear, nose, and throat surgery) as an antiseptic dressing. however, a few examples of serious adverse effects of bipp were observed in the clinic when some patients were treated with bipp. in one case, the patient became acutely confused and the gait became unsteady, indicative of an encephalopathy caused by over-dosage of bismuth. this was confirmed by the observation of a toxic level of bismuth in the patient's serum. another case involved using bipp to cover the dura mater in a wound after removal of a large basal cell carcinoma. the patient became confused and then comatose. an encephalopathy was confirmed by the observation of diffuse cerebral edema in a tomographic scan. however, upon removal of the bipp, the patient recovered, and deteriorated if the pack was applied again. the mechanism of intoxication has not been well understood till now. it was probably caused by the interference of bismuth with the oxidative metabolism of the central nervous system by binding to essential enzymes and reducing cerebral blood flow. there are only few cases on nephrotoxicity after over-dosage of cbs. in one case, even the gastric lavage was performed initially, a 2-year-old boy suffered from an acute renal failure (arf) associated with uremia and oliguria after ingestion of 28 de-nol tablets (cbs, 8.4 g) for 2 days. after ingestion of cbs for 10 days, bismuth levels in both the blood and urine were 739 and 693 mg/l respectively. with the treatment of peritoneal dialysis, his urine volumes increased and plasma bun and creatinine levels decreased gradually. after 100 days of admission, the patient recovered and his bismuth level in blood went back to normal. to cure some serious side effects caused by bismuth over-dosage, some antidotes such as d-penicillamine, 2,3-dimercapto-1-propane-sulfonic acid (dmps), dimercapto-succinic acid (dmsa), and dimercaprol have been tested in animals and limited clinical trials. it was shown that dmps and dmsa effectively alleviate bismuth poisoning due to their strong chelating ability to bismuth ions. further reading efficacy of helicobacter pylori eradication therapies: a single centre observational study bismuth compounds and preparations with biological or medicinal relevance a novel synthetic compound, bismuth zinc citrate, could potentially reduce cisplatin-induced toxicity without compromising the anticancer effect through enhanced expression of antioxidant protein bioinorganic chemistry of bismuth and antimony: target sites of metallodrugs a proteomic approach for the identification of bismuth-binding proteins in helicobacter pylori metals in medicine glutathione and multidrug resistance protein transporter mediate a self-propelled disposal of bismuth in human cells reversible nephrotoxicity after overdose of colloidal bismuth subcitrate determining the background levels of bismuth in tissues of wild game birds: a first step in addressing the environmental consequences of using bismuth shotshells systems approaches for unveiling the mechanism of action of bismuth drugs: new medicinal applications beyond helicobacter pylori infection recent advances in bioinorganic chemistry of bismuth unexpectedly strong binding of a large metal ion (bi 3 þ ) to human serum transferrin the determination of metals (antimony, bismuth, lead, cadmium, mercury, palladium, platinum, tellurium, thallium, tin and tungsten) in urine samples by inductively coupled plasma-mass spectrometry helicobacter pylori eradication: a new, single-capsule bismuth-containing quadruple therapy bismuth antimicrobial drugs serve as broad-spectrum metallo-b-lactamase inhibitors integrative approach for the analysis of the proteome-wide response to bismuth drugs in helicobacter pylori metallochaperone ureg serves as a new target for design of urease inhibitor: a novel strategy for development of antimicrobials biocoordination chemistry of bismuth: recent advances bismuth complexes inhibit the sars coronavirus bipp madness; an iatrogenic cause of acute confusion key: cord-023584-yaxawqhj authors: bucknall, r.a. title: the continuing search for antiviral drugs date: 2008-04-10 journal: adv pharmacol doi: 10.1016/s1054-3589(08)60460-3 sha: doc_id: 23584 cord_uid: yaxawqhj this chapter discusses the continuing search for antiviral drugs. many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, at present it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. one may cite smallpox, yellow fever, polio, and recently measles among human diseases, newcastle disease, marek's disease, and infectious bronchitis among poultry diseases—an area of veterinary disease control where vaccines have been particularly important. research into the treatment of virus diseases by drugs is at present directed toward three general areas: (1) attempts to stimulate the defense mechanism of the host animal, (2) large screening programs to find drugs which directly block some virus-specific process, and (3) alleviation of the symptoms of the disease. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs and in cases where symptoms have already appeared. although research into the mechanisms of virus infection is carried out by many sections of the scientific community, the search for antiviral drugs is almost exclusively the province of pharmaceutical manufacturers. the reasons for this are partly historical, but chiefly it is because the facilities for running large-scale screening programs are expensive and can only be met by commercial and, occasionally, governmental resources. because of the need to protect their discoveries from unauthorized exploitation, a good deal of secrecy inevitably surrounds the work being carried out in commercial organizations. this secrecy is regrettable but necessary, since the survival of such organizations depends largely then on getting a fair financial return on the money invested by them in research. the security aspects of commercial research naturally restrict frank and constructive discussions between competitors as well as third parties, and this has been particularly true in antiviral research. perhaps it was a revolt against this enforced isolationism which led, at least in part, to the first conference on antiviral substance's held by the new york academy of sciences in 1965, in which manufacturers disclosed many of the details of their antiviral research and had a chance to discuss their failures and comparative successes (whipple, 1965) . 295 since then there have been a number of reviews of progress in the field of antiviral chemothrrapy, and in most of these there have appeared comments and recommendations ivhich indicate that a radical rethinking is taking place of the prospects for antiviral drugs (osdene, 1967; mcfadzean, 1969 ; goz and prusoff, 1970; swallow, 1971 ). it was natural in the early 1950s to espect that antiviral drugs would be discoverrd which would be analogous to the antibacterial antibiotics, the darlings of the prrvious decade. the experience up to date has proved that this was not to br the case, and despite prodigious efforts by the drug houses, only three clinically useful antiviral agents, which are far from perfect, have emerged. i believe thcre are lessons to be learned from this disappointing record, and i hope that the following remarks may help to continue further the critical reappraisal of this field, so that future progress may be faster. before embarking on a search for antiviral drugs, a number of points must be considered in order to assess thr technical feasibility of treating or preventing a virus disrase u-ith a drug. too often in the past massive screens have bccn set up against many viruses in the hope that a drug would turn up, and it would then find a natural place in human or veterinary medicine. although it is true that random screening still offers the best chance of discovering new drugs, unless a realistic appraisal of the whole project is made a t the outset, the products of random screens could well be useless as potential medicines. somr of the more important considcrations are listed below. a. diseases of economic importance i n ordcr to he commercially viable a drug must sell in sufficient quantities to pay for its devclopmcnt and manufacture as well as for future research. because of this, and because antiviral chemicals tend to inhibit specific viruses. diseases of low incidence or loneconomic importance are ruled out as primary targets for drug devclopment. this may seem inhumane, particularly in the field of human virus diseases, but the fact remains that it is no easier to find a drug against rabies than against influenza, and the sales from a specific antirabies drug would never cover its own developmrnt costs. diseases of loiv incidence or low cconomic importance, no matter how serious the outcome may be for the infected individual, inevitably must remain the subjects of sponsored research. nevertheless, treatments for some of these diseases may emerge as drugs are developed against major diseases. b. immunological control many virus diseases, both of humans and animals, have been successfully controlled by vaccines. these successes have naturally led to improvements in the spectrum and duration of protection offered by vaccines until, today, it is difficult to see how antiviral drugs could compete with vaccines in the control of many virus diseases. as examples one may cite smallpox, yellow fever, polio, and, more recently, measles among human diseases, and newcastle disease, marek's disease, and infectious bronchitis among poultry diseases-an area of veterinary disease control where vaccines have been particularly important. despite these triumphs, vaccines are not, and probably never will be, the complete answer to the control of certain virus diseases. it is in these areas where drugs would be useful, and it is on these diseases that efforts should be concentrated. the two most important human diseases in this class are influenza and the common cold. when a novel strain of influenza appears among the human population, as happened in 1933, 1947, and 1956, existing immunity to the previous current influenza strain is not effective, and widespread epidemics of disease occur. the disease spreads so rapidly after it first appears that it is not possible to develop, distribute, and administer a vaccine based on the new strain soon enough to protect useful numbers of the population. even after the initial overwhelming pandemic, successive epidemics will occur as the disease penetrates into pockets of the community that 'had escaped infection. the disease is then maintained partly by the continuing appearance of susceptible juveniles, partly by spontaneous antigenic modifications in the virus enabling it to overcome previous immunity, and partly by the general decline in immunity of other individuals with the passage of time. after the initial pandemic, it is theoretically possible to control the disease with widespread vaccination, but in practice this is not done. consequently the disease smoulders on in the community, appearing as isolated cases and occasional outbreaks and epidemics. these are the conditions under which the disease normally exists, and it is this situation, rather than the much publicized pandemics, which causes the greatest economic loss to industrialized countries. the overall loss in 1956-1957 in great britain due to the asian influenza pandemic was estimated at x100 million, but the continuing loss, from that time on has been at least x30 million each year. losses in europe, north america, japan, and similar industrialized communities must be romparablr, and if only a fraction of the disease could be prevented by a drug, the economic benefits would be enormous. losses due to the common cold are comparable. the careful study of lidwell and williams (1961) showed that approximately 11 x lo6 working days arc lost cach year in great britain alone from the common cold. scvertheless, the prospects for a common cold vaccine are poor because of the large number of viruses which are known to cause the disease. there are 89 known rhinoviruses (kapikian, 1971) , probably a comparable number of as yct unclassified rhinoviruses, a growing catalog of coronaviruses (kapikian, 1969) , and a selection of other viruses including myxoviruses, adenoviruses, and herpcsviruses (tyrrell, 1965) , all of which have been isolated from clinical colds. it is the serological diversity of these etiological agents which makes the prospects for a vaccine so poor, and the common cold must, therefore, be considered as a target, even though a difficult target, for antiviral drugs. besides the problems of antigenic variation, exemplified by influenza, and thc multiplicity of serotypes, exemplified by the common cold, there are two further problems associated with the control of respiratory diseases by parmterally administered vaccines. the first is that circulating antibodies appear in only small amounts in respiratory mucus, and, consequently, the degree of protection afforded to the respiratory tract is less and of shorter duration than might be expected. the second problem has been brought to light by the use of experimental vaccines against respiratory syncytial virus disease in infants. when infants who had been vaccinated parenterally against this disease contracted the natural disease, they were more ill than infants who had not received the vaccine. the reason seems to be that the circulating antibodies resulting from parenteral vaccinations not only offer little protection to the respiratory tract, but when a natural infection occurs, these antibodies combine with the virus antigens at the surface of the respiratory epithelial cclls causing an inflammatory response with a corresponding increase in the severity of clinical symptoms kim et al., 1969; kapikian et al., 1969) . chanock et a2. (1970) have pointed out that if an immunopathological process involving serum antibodies occurs during respiratory syncytial virus infection, then stimulation of local, respiratory tract, secretory antibody by intranasal instillation of live or inactivated virus may give adequate protection without unwanted hyperreactivity. a similar allergic reaction between virus antigen and preexisting antibody is a factor, possibly a major factor, in the pathological processes initiated by herpesviruses (jones and patterson, 1967) and marks the herpes diseases as possible candidates for antiviral drug development. one factor that deserves more careful consideration than it usually receives is the way in which a potential antiviral drug would be administered to the animal or patient requiring protection. clearly a common cold treatment would be unacceptable if it had to be given intravenously 3 times a day. but there are less obvious, but no less real, difficulties in dosing large herds of cattle or sheep so that effective protection is maintained. with freeranging animals the duration of protection from a single dose would need to be prolonged to offset the labor of administering the dose. also, it is easier to dose herds by injection than by mouth, so that certain veterinary antiviral drugs may not be required in an orally active formulation. conversely, oral dosing, preferably by an addition to food or drinking water, is the most convenient way of dosing poultry. the onset of symptoms in most virus diseases is acute and may be the first indication that the host has contracted an infection. because of this, it is usually assumed that antiviral drugs will only be of value in preventing and not in curing virus diseases. nevertheless, although the periods of virus growth in infected individuals may be short, they may well be long enough to allow useful therapy. for example, pate1 et al. (1964) have shown in volunteers infected intranasally with coxsackie a21 virus that maximum virus growth precedes the onset of symptoms by 24 hours. but from their work it may be seen that a considerable amount of virus growth is concurrent with the period of overt symptoms, and application of antiviral drugs during this period of 2-3 days might well prevent the full development of the disease. the work of douglas et al. (1966) with volunteers shows that a similar period exists in acute rhinovirus infections, and dawkins et al. (1968) and wingfield et al. (1969) have shown that the course of influenza in humans can be modified, even after the onset of symptoms, by treatment with 1-aminoadamantane. it hardly seems necessary to point out that before undertaking a search for a drug against a particular disease, the etiological agent of the disease should be unequivocally identified. there are a number of important virus diseases for which the causative agent is either unknown or is in doubt, for example, bovine pneumonia and human epidemic viral gasteroenteritis. it would be a mistake to set up screens against agents that were only suspected of being implicated in these diseases in case subsequent work should demonstrate that these were not in fact the causative agents. although it is difficult to predict changes in governmental legislation toward public or animal health, nevertheless, the existing and prospective legal position in various countries should be considered since these may affect the prospects for potential drugs. for example, in great britain, foot and mouth disease is controlled by the policy of slaughter and compensation, but in other countries the disease is controlled by vaccination. in the lattrr countries, a drug may find a ready market, but if legislation should change, then that market would be lost. in summary, we may say that before embarking on a search for antiviral drugs, the target disease must be carefully selected by a consideration of all the relevant factors. the more important of thesc a r t : 1. there must be an adequate market for the drug. 2. there should be no effective immunological control, and no prospects 3. due regard should be given to the practicability and the timing of 4. the etiology of the disease should be clearly established. other relevant factors, e.g., medical or veterinary legislation, should be takcn into consideration. in table i , a number of virus diseases of economic importance are listed together with some comments to illustrate how many seemingly attractive drug-target diseases are in fact precluded by other factors. for such control. dosing the patients or animals at risk. it is easy to list the properties of the ideal antiviral drug-wide spectrum of activity, nontoxic, accessible to the target organ, etc. what is not so easy is to predict what sort of properties one might expect from antiviral leads detected by screening programs. all the same, it is only by intelligent attempts to do just this that screens may be designed to detect compounds that one day may lead to the development of useful medicines. the scientific literature abounds with reports of antiviral chemicals discovered by screening random compounds in tissue culture systems, but all too frequently these compounds turn out to be false positives or active only against some relatively unimportant virus. for the products of a tissue culture screen to be of potential value, the screen must meet the requirements outlined below. first, the screen should be able to process large numbers of chemical compounds, or fermentation products, since the more that are tested, the greater the chance of success in finding active leads. perhaps the time will come when new drugs can be designed and synthesized on entirely rational grounds, but at present most active leads are chance discoveries. buthala (1965) quotes 6% of all compounds tested in a tissue screen as showing some antiviral activity, but in my experience the rate varies from 1 to o.ol$!&, the higher rate referring to influenza a viruses, and the lower rate to picornaviruses. bauer (1967) has suggested that the larger the virus, the more susceptible it is to inhibition, since, for any given intracellular concentration of drug, a large virus will encompass, both physically and in terms of synthetic requirements, more drug molecules than a small one. undoubtedly, this principle will contribute to our observed high rate of inhibition of influenza virus, but another important factor seems to be that the adsorption of influenza viruses to cellular receptors is particularly vulnerable to interference by extraneous substances. given that only a fraction of a percent of all compounds tested in tissue culture will show activity and that of these only a small proportion will show activity in animal models, a realistic screening rate would be not less than 5000 compounds a year. at rates less than this, the chances of finding useful compounds become so small as to make the whole project not worthwhile. in many of the published screening procedures, the virus with which the screens are run seem to have been chosen more for the ease with which they can be handled rather than for their relevance to any virus disease target. for example, ehrlich et al. (1965) describes a screen where the primary test viruses include parainfluenza type 3, measles, and poliovirus, and johnson (1965) describes a screen that includes pseudorabies, adenovirus 111, and mouse hepatitis virus. of course, if wide-spectrum leads appear, the choice of test virus may be irrelevant, but the antiviral compounds (as distinct from interferon inducers) known at present are characterized by their relatively limited spectrum of activity, e.g., methisazone is active only against poxviruses (bauer and sadler, 1960) and possibly adenoviruses (bauer and apostolov, 1966) ; l-aminoadamantane is active only against influenza a1 and as and not against other myxo-or paramyxoviruses (davies et al., 1964) ; guanidine and a-hydroxybenzyl benzimidazole are active only against picornaviruses and not against other small ribonucleic acid (rna) viruses (eggers and tamm, 1961) . thus, whenever possible, the viruses used in routine screens should be those that are responsible for the clinical large-scale screens use large numbers of tissue culture cells, and there has been an inevitable trend toward the use of continuous cell lines in screening procedures. such cells have many attractive features. they grow rapidly, they can easily be obtained in large quantities, they remain "the same" year after year, they can be madr to prrform useful technical tricks such as rapidly changing the ph of their medium and surviving for long periods under agar, and, perhaps most important, they will support the growth of a wide range of viruses. continuous cell lines seem to be the natural choice for running routine screens. xevertheless, it is worthwhile remembering that many of the desirable technical properties exhibited by these cells may be a direct result of their neoplastic nature, and to use a continuous rather than a primary or diploid cell in a tissue culture system is to take yet another step away from the natural disease. it is truc that antiviral agents, such as l-aminoadamantane and methisazone, which can be shown to protect humans against virus diseascs, also exert their antiviral action in neoplastic cells in tissue culture, for example hela and kb cells, but we have striking evidence that this may not always follow. we have recently discovered a family of chemical compounds that have high activity against rhinoviruses when grown in human diploid lung cells, but virtually no activity against the same viruscs growing in monkey kidney cells, hela cells, or kb cells (bucknall, unpublished results) . these compounds and any others that may exhibit this property would have been missed in tests carried out in continuous cell lines. in summary, a tissue culture screen should be able to proccss large numbers of tcst compounds, using viruses as relevant as possible to the diseases for which a drug is required, and should employ normal rather than neoplastic cells. unfortunately, in most of the published screening procedures the last two requirements have been sacrificed to technical considerations designed to increase the number of compounds tested, as the following descriptions will show. in its simplest form, a test for antiviral activity involves treating cultures of cells with a range of concentrations of a test compound. first the maximum concentration tolerated by the cclls is assessed; then, second, the growth of virus at lowcr concentrations of compound that are not cytotoxic is measured. several ingenious methods have been devised for measuring these two responses-cytotoxicity and virus growth-all designed to facilitate the screening of large numbers of compounds. for example, herrmann et al. (1960) devised a zone-inhibition test in which large flat dishes of chicken cells were infected with test virus, overlaid with agar containing a vital stain, and paper discs impregnated with test compounds placed on the surface of the agar. compounds with antiviral activity showed two concentric zones around the paper disc, the innermost being pale in color due to the destruction of host cells by cytotoxic concentrations of compound diffusing from the disc. outside this was a deeply staining zone where cells were exposed to nontoxic concentrations of compound which also protected them from the destructive effects of the virus with which they had been infected. beyond this, where the concentration of compound was too low to protect the cells, the cell sheet was destroyed by virus and stained poorly. thus, by visual inspection of the dishes after 3 to 4 days, active compounds could be quickly detected. rada et al. (1960) devised a similar agar diffusion test in which test compounds were applied to the virus-infected cell sheets in circular wells in the agar overlay. although agar diffusion tests are capable of processing large numbers of test compounds, they suffer from two drawbacks. first, they are of comparatively low .sensitivity in detecting both the cytotoxic and antiviral levels of compounds, and second, they are limited to viruses that produce plaques under agar. rightsel et al. (1956) devised a system based on the fact that if cells were damaged either by the toxic effects of a chemical compound or by virus growth, they would not swing the ph of their medium. thus, by incubating virus-infected cells in a series of concentrations of a compound and then looking for the cultures that had changed the color of the phenol red indicator in their medium from pink to yellow, active compounds could be detected. this technique has also been used to assay neutralizing antibody and interferon action (pauker, 1965). finter (1970) described a system whereby the cytotoxic effects of test compounds could be assayed by the reduction in the amount of neutral red taken up by treated cells, and, similarly, the cytopathic effects of virus growth could be quantitated by measuring the reduction in the uptake of neutral red by infected cells. the system can readily be adapted to the screening of test compounds for antiviral activity. if myxoviruses are used in this system, because their cytopathic effects may not be pronounced, their growth is best monitored, not by a reduction in neutral red uptake, but by a quantitative hemadsorption method which matches the neutral red uptake method in its accuracy and sensitivity (finter, 1964) . in contrast to the eone-inhibition and ph-swing tests, the neutral red uptake test is precise in operation and may be used to demonstrate fine differences in the relative toxicity and activity of test compounds; it is probably no more timeconsuming than the former tests. a system of testing for antiviral agents based on the inhibition of nucleic acid synthesis was dcscribcd by lliller et al. (1970) and has been used to screen compounds and mold metabolites for antiviral activity (miller et ul., 1968) . for the test, hela cells were suspended in a medium containing uridine-3h. if a test compound has toxic effects on the hela cells, then the cellular r s a synthesis, as measured by u r i d i n~-~h fixation, will be reduced. similarly, if cells are infected with an r s a virus and treated with sctinomycin d. then r s a synthesis will be due to virus growth only. thus, if test compounds reduce this virus-directed rxa synthesis at concentrations that do not affect cellular r s a synthesis, then the compound is exerting a specific effect on virus growth. the test can also be used for deoxyribonucleic acid (dsa) viruses, the cellular and virus dxa synthesis being monitored by including th~midine-~h in the medium. virus dsa synthesis is distinguished from cellular dsa synthesis by disrupting the cells at the end of the test and treating them with dcoxyribonuclease when encapsulated virus dsa is resistant to digestion and the unprotected host cell dsa is not. thus, the selective effect of test compounds on the synthesis of virus dsa can be measured. the authors claim that the system operates satisfactorily ivith a range of viruses-some of them important disease organisms-and is simple, reliable, and rapid. the chief criticism of this method is that, because nucleic acid synthesis is used as the sole measure of virus growth, test compounds that might act on subsequent stages in the virus replicative cycle may not be detected. for example, any disturbances in the sequencing of virus nucleic acid, inhibition of structural protein synthesis, or failure of assembly or release of mature virions, would provide a sound basis for a useful drug, but these phenomena may not be detected in this type of test. also, since high infecting doses of virus are used to give satisfactory operation of this test (up to 15 virus particles per cell), the test may be rather insensitive in detecting antiviral activity. all antiviral activity is, in the broadest sense, competitive, either a t the level of cellular membrane receptors or at an cnzymic or template level. thus, the more virus is used to initiate infection, the less effective an antiviral compound is likely to be. although this factor will not turn a highly active compound into an inactive one, it may well obscure low levels of activity which might be useful starting points for chemical exploitation. the real value of miller's test is the use of the important biochemical system of nucleic acid synthesis to monitor the toxic manifestations of test compounds. this subject is discussed furthrr in the following section. reference has already been made to four methods for measuring the toxicity of chemical compounds in tissue culture cells: direct cytopathic ef-fects, vital dye uptake, metabolic activity (ph-swing), and nucleic acid inhibition, and there is no shortage of other methods. nevertheless, the inadequate assessment of compound toxicity in antiviral testing probably gives rise to more false leads than any other single cause, before discussing how compound toxicity might be measured, it must first be defined. strictly, any interference with cellular metabolism by an extraneous compound is a toxic effect, and the most stringent tissue culture test of lack of toxicity is the continued normal division and growth of cells in the presence of an extraneous compound. however, this test is too cumbersome for use in rapid screening procedures, and simpler, but less critical, tests are invariably used in primary screens. undoubtedly, the simplest method of assessing compound toxicity is by direct microscopic examination of cells for cytopathic effects or more subtle morphological changes. it is necessary for the observer to be trained to detect such changes, and an arbitrary scale must be devised to record the observations, but if these simple requirements are met, the method is generally successful. it may be objected that this system would be unworkable where large numbers of compounds are being screened because of the correspondingly large numbers of microscopic examinations required; but given a good low-power microscope, an experienced reader, and the fact that most random compounds tested will show no antiviral activity and will, therefore, not require more than a cursory examination, the system is reliable, fast, and economical. in the author's laboratory a system of this kind has been in use for over 5 years, and it is possible for one worker to screen a hundred compounds against three viruses each week. we have found that with this system, compounds appear to be toxic a t lower concentrations than with either the zone diffusion or the dye uptake method. we conclude, therefore, that our method is more sensitive than the others mentioned in detecting the toxic effects of compounds. the direct microscopic assessment of toxicity is not without its deficiencies, but if the method is seen only as a preliminary determination of toxicity, these deficiencies are not serious. chief among these (and this applies even more to indirect methods) is the occasional failure to detect certain types of toxicity. for example, from time to time we have had compounds which appeared to prevent virus growth and to show no toxicity to confluent sheets of tissue culture cells, these cultures looked normal for several days in the presence of the compound, but viruses would not grow in these cells. nevertheless, further studies (see below) have shown that the compounds were exerting an inhibitory effect on some aspect of the cellular metabolism and it was this which prevented virus growth. we have investigated this effect with (a) inhibitors of nucleic acid synthe-sis and ( b ) uncouplers of oxidative phosphorylation, two classes of compound that are particularly prone to giving misleading results. nucleic acid inhibitors are often slow to produce cytopathic effects in confluent monolayers of cultured cells. the d s a synthrsis of such cells is low, and sufficient r s a synthesis is often maintained in the presence of partially effective concentrations of an inhibitor, enabling the cellular structural integrity to be sustained. all the same, an invading virus is unable to replicate in a cell under these reduced circumstances, and this will lead to an apparent antiviral specificity. this is the mechanism by which the chlorinated ribofuranosylbenzimidazoles exert their antiviral effects (bucknall, 1967) . these compounds were extensively studied as antiviral agents before their "activity" was found not to be specific for the virus (tamm et al., 1954; tamm and srmes, 1957; tamm and overman, 1957) . uncouplers of oxidative phosphorylation also often appear to be antiviral agents becausc concentrations that greatly reduce the energy-generating systems of cells in confluent monolayers are often slow to produce morphological changes. in this half-poisoned state, the cultures appear normal, but do not support virus growth, and thus another false "lead compound" is generated. as mentioned earlier, these remarks apply to all tissue culture systems to a greater or lesser extent, and tissue culture tests for antiviral activity must always be regarded as strictly preliminary. active leads from such tests must always be subjected to the closest scrutiny to determine whether the activity is truly specific for a virus-coded process or simply results from a subtle toxic effect on the host cell. the margin betwen the maximum nontoxic concentration and the minimum antiviral concentration of a test compound is conveniently expressed as thrrapcutie ratio = max. nontoxic concentration/min. antiviral concentration and will vary according to how these two concentrations are determined. the simplest and most stringent test of the maximum nontoxic concentration of a compound in zdtro is to grow cells in the presence of the compound and determine the maximum concentration a t which division and growth will proceed normally. if this concentration, and lower ones, protect the cells from virus attack, then this is an unequivocal demonstration that the compound is exerting a specific effect on some aspect of virus replicat ion. like miller et al. (1970) , we have found the inhibition of cellular nucleic acid synthesis, particularly r s a synthesis, to be a useful system for detecting the toxic effects of test compounds. cells are treated with a range of concentrations of a compound, then the uptake of ~r i d i n e -~h into acidinsoluble material is measured and compared with that of normal cells (bucknall, 1967) . the test is simple to run, and since the nucleic acid metabolism is a cardinal area in the cellular metabolism, even if a compound has no direct effect on the nucleic acid synthesis, disturbances of other synthetic or homeostatic mechanisms are quickly reflected in changes in the synthesis of rna or dna or both. in fig. 1 , the dose-response curves of one experimental compound are determined in human diploid lung cells by the three methods outlined above-direct cytopathic effect in confluent monolayers, inhibition of rna synthesis, and inhibition of cell growth in newly seeded cultures. although this compound is not a specific inhibitor of rna synthesis, the inhibition of rna synthesis and the production of cytopathic effects run close together. at concentrations that indirectly affect the nucleic acid synthesis, sufficient disturbance is caused in other areas of the cellular metabolism to lead to a general cytopathic effect. cell division is affected a t lower concentrations and in this particular case, inhibitory (and therefore toxic) effects can be detected with concentrations 10 times lower than those that cause cell destruction, nevertheless, even with cell growth as a measure of toxicity, and effects on the cellular growth rate (&-a) are higher than those that suppress virus growth, indicating that ici 65,709 is exerting a specific effect on virus growth. (50% end points: cpe, 10 pg/ml; rna synthesis, 10 pglml; cell growth, 1.5 pg/ml; virus growth, 0.05 pg/ml.) there is a clear margin between the toxic and antiviral effects, as may be seen from the virus yield curve. fusidic acid, which has been reported to show specific antiviral activity in tissue culture (acornley et al., 1967) , was also tested in the same way (fig. 2) . in this case, the concentration causing 50% cytopathic effect after 48 hours was 100 pg/ml, whereas virus yield was depressed to 50% by only 3 pg/lml, giving an apparent therapeutic ratio of 33. but when the effects of fusidic acid on cellular synthesis were studied, it was clear that cellular rka synthesis was drastically reduced by 3 pg,!ml. thus, fusidic acid probably reduces virus growth by inhibiting cellular, rather than virus, synthetic processes, and probably accounts for the fact that this compound showed no clinically useful effects in virus-infected volunteers, despite good levels of drug in blood and nasal secretions (acornley et al., 1967) . since the toxic effects of compounds in vitro usually increase with time, it is important when comparing toxicity and antiviral activity, to ensure that the compound has been in contact xvith cells for the same length of time in each case. in the above experiment, the cytopathic effect, rna synthesis, cell growth, and virus inhibition were all measured in cells that had been exposed to the compound for 48 hours. when a virus disease is limited to a particular target organ, such as the respiratory tract, it is of great value to be able to culture a portion of the organ for in vitro studies. the culture of portibns of trachea has been extensively used to study the growth of respiratory viruses (hoorn and tyrell, 1965, 1966; mcintosh et al., 1967; craighead and brennan, 1968; herbst-laier, 1970) , and recently organ cultures of human embryonic gut have been used to study the agents of human "virus" gastroenteritis ( d o h et al., 1970) . the technique could presumably be extended to the study of viruses, such as polio, rabies, smallpox, and herpes, which localize in specific organs of infected individuals. we have found the use of human embryo and animal tracheal pieces of value in studying the toxicity and the antiviral activity of leads produced by tissue culture screening programs. our technique is to excise a trachea and cut it transversely into rings 1-2 mm thick. these are placed in 3 x 3 in. tubes with 1 ml of eagle's medium and rolled exactly as conventional tissue cultures. with a low-power microscope the ciliary activity of the respiratory epithelium is assessed on an arbitrary scale of 0 to 4. in the presence of a test compound, the reduction of ciliary action is a highly sensitive measure of compound toxicity, and it is easy to determine the concentration at which full ciliary activity can be maintained (fig. 3) . at lower concentrations the effects on virus growth may be measured by harvesting the culture fluid at intervals and titrating for infectious virus. by using this technique, we have found that almost 70% of the so-called active compounds produced by a tissue culture screen against influenza a appear negative when tested in ferret tracheal cultures. in almost all cases, compounds were toxic at lower concentrations in tracheal cultures than in conventional tissue culture monolayers, as judged by a cessation of ciliary activity. in a proportion of these compounds, some data concerning their biochemical action were available, and in most cases these drugs were uncouplers of oxidative phosphorylation, nucleic acid inhibitors, or general antimetabolites. the inhibition of ciliary action in tracheal cultures is, therefore; a much more sensitive index of toxic effects than morphological changes in monolayers. in fig. 3 , the 50% cilia-inhibitory concentration of ici 65,709 is 2.0 pg/ ml. this effect is detected at a concentration 5 times lower than is necessary to cause morphological changes in conventional tissue culture cells (fig. l) , presumably because the metabolic patterns of the ciliated cells are more complex than those of static cells in culture and are, therefore, more readily disturbed. in general, the concentrations of compounds that suppress ciliary activity arc comparable to those that prevent cell growth, except in the case of specific inhibitors of dsa synthesis for which cell division and growth are usually more sensitive than ciliary activity. any conventional technique may be used to measure virus growth in antiviral tests-cytopathic effect, plaque reduction, yield of infectious virus, and ht.madsorption, bring thc most common. because of thcir simplicity, cytopathic effect and hcmadsorption are widdy used, hut these techniques must be used with some precautions if certain types of antiviral activity are not to be missed. in order to speed up the rate of antiviral testing, the quantity of challenge virus is often increased to a theoretical maximum of 1 virus particle per cell. the whole cell culture then behaves synchronously, and results are obtained in whatever time the virus takes to complete its replicative cycle-usually between 10 and 20 hours. with this procedure, however. a test compound that prevented the formation of infectious virus but was unable to protect the infected cell from destruction would not be detected. for example, in a relatively complex virus, such as influenza, it is not difficult to imagine that rna synthesis could be interrupted while hemagglutinin production continued, much as it does in the van magnus effect. the result would be a monolayer showing full hemadsorption and yet no transmissible virus would have been formed. test compounds that produce this effect would be of great interest as potential drugs but could be missed in tests where the dose of challenge virus is too high. wherever possible, tests should permit several cycles of virus growth to occur so that compounds that interrupt any part of the cycle may be detected. there is a school of thought that tissue culture testing is so artificial as to be of little value in detecting useful antiviral substances. it is argued that by testing for antiviral effects directly in animals, the activity that is detected is likely to be more valid and more useful than that detected in tissue culture. it is true that the majority of active compounds detected in tissue culture screens are not active in animals, even after full authentication of the antiviral activity by tests such as those discussed above. the reason for this is usually that the compounds do not reach the target organs in sufficient amounts to show activity rather than because of some intrinsic defects in the antiviral activity of the compounds. also, apart from interferon inducers, unless a compound manifests some activity in vitro, it is highly unlikely to do so in vivo. on the one hand, it is argued that a virus growing in a tissue culture cell is of little relevance to the processes by which that virus causes disease in the whole animal. on the other hand, it can be said that, since virus growth is the basis of the pathological processes, if virus growth can be halted, then the disease can be stopped. furthermore, the literature discloses that some highly irrelevant viruses are being used in animal test systems and that, even when human pathogens are used, e.g., influenza, they require extensive adaption to their animal host and the course of the disease is usually very different from that in humans. finally, there are no convenient animal models for studying the growth of human rhino-and coronaviruses and, unless tissue culture tests are used, there could be no screening for antiviral compounds against these important pathogens. there is, therefore, no convincing theoretical advantage in using animals for routine antiviral screens. this, together with the cumbersome nature of animal tests and the difficulties of "scaling-up" to test large numbers of compounds makes tissue culture testing a more attractive proposition for the initial screening program. the foregoing remarks apply, of course, only to the detection of compounds that have a direct effect on specific virus processes, e.g., absorption, penetration, and replication. in the field of interferon inducers, immune enhancers, and other stimulators of thr host defense mechanisms, obviously one has no choice but to use test animals; but here one is concerned to detect any overall rffcct on the course of a disease rathrr than accurately t o model a particular human or veterinary infection. accordingly, the choice of test system is less critical provided it fulfills certain requirements. for instancc, (a) thr virus and test compound should be administered a t separate sites to avoid any possible local destruction of the challenge virus by test compound; (6) the test compound should be given parenterally to give the best chance of absorption, and (c) the dose of challenge virus should be sufficiently small to allow a useful incubation period before symptoms develop. the following system has been used successfully by us. groups of 5 mice are dosed intraperitoneally with test compounds a t 50, 12.5, and 3.1 mg/ kg on four successive days. twenty-four hours after the first dose, they are challenged intramuscularly with 100 jild,, of semliki forest virus, and after the last dose they are observed for symptoms twice daily. the mcan rcciprocal day of death (mrdd) for each test group is calculated after 14 days and comparcd with that of a similar undosed group as w l l as with that of a group givcn four daily injections of a protective agent such as polyinsinie-polyrytidilir acid (poly ic) . figure 4 shows the typical response of mice treated nith poly ic and untreated controls. under these particular conditions, the t~7 0 response curves overlap. by selecting an mrdd of 0.133 as the criterion of "active" or '5nactive," then theoretically 10% of all inactive compounds tested will appear as spurious actives and would require to be retested to establish their true status. some caution is needed in interpreting the converse overlap. given 100 known active compounds, or a single active compound tested 100 times, then 6 tests in every 100 would miss such a compound. but in practice, the vast majority of compounds passing through the test will be inactive, and the chance that the occasional true active compound will by chance fall into the "6% missed" category is correspondingly reduced. even with an in vivo test reduced to such a minimum as this, it still requires a large effort in terms of manpower and facilities to test realistic numbers of compounds. although many human viruses will grow in animal hosts it is often difficult to assess the potential value of an antiviral compound for human use by using animal models. there are five main reasons for this. first, a relatively benign human virus infection will often follow a very different, and often severe course in an animal. for example, influenza virus, herpes simplex, and coxsackie viruses may cause much more serious diseases in laboratory animals than they do in man. second, human viruses often must be adapted by multiple passaging before they will grow satisfactorily in animal hosts, and, therefore, the challenge virus in the animal model may be a very different creature from the original human pathogen. third, the quantity of virus administered to an animal in order to produce some measurable effect, e.g., symptoms, virus isolation, and seroconversion, is usually vastly greater than would ordinarily be encountered by the natural host, and this can have a profound bearing on the efficacy of any curative agent. for example, finter (1967) has shown that the protection offered to mice by doses of interferon is greatly increased as the quantity of challenge virus is reduced. fourth, the fate of a drug when administered t o an animal may be very different from that seen in man. and last, a compound may show toxio effects in man which it did not show in animals. the last two points are probably the most important in determining how far the results obtained with animal models are relevant to man. in theory the above considerations should operate in both directions; that is, a compound that shows a positive result in an animal model may be positive or negative in man, and a compound that is negative in an animal may be negative or positive in man. but, since many animal models offer a greater challenge to the therapeutic potential of a drug than the natural disease in man, a positive result in an animal model always gives great hopes that a positive result might be achieved in man. this consideration often encourages the evaluation of potential antiviral drugs in man on the very slimmest of grounds. an example of the dilemma that an animal model may pose is afforded by the �ork of boyle and his colleagues (1970) on the compound skf 30097. this compound was shown to be active against a number of viruses, including a wide range of human rhinoviruses, in tissue culture. the problem arose of how to evaluate this compound in vivo. there are no smallanimal modrls of human rhinovirus infections, and the authors, therefore, decided to test the compound in chimpanzees, which are one of the few primates susceptible to human rhinoviruses. because of lack of knowledge on infectivity of human rhinoviruses for chimpanzees, the authors gave up to 10, ooo tcd,, of challenge virus to each animal to ensurc infcction. the animals were given the drug orally 3 times a day, and the course of the disease was monitored by virus shedding from the nose. the rate of antibody rise was also measured. the numbers of animals in each experiment were necessarily small-usually 2 or 3 treated with drug and 2 or 3 controls. the final results were tantalizingly inconclusive: not clearly negative, nor convincingly positive that the drug had produced a curative effect. the investigators admit that this system is far from satisfactory but conclude from their results that the compound is n-orthwhile studying further in human subjects. the same conclusion, however, would probably have been reached if the compound had been clearly inactive in the chimpanzees, on the grounds that the excessive doses of challenge virus and unknown factors in the chimpanzee metabolism could have led to this result. animal models of human virus disease must at best be regarded as poor imitations of the natural condition, and results obtained with animal models, whether they are positive or negative, encouraging or discouraging, should be interprcted cautiously and never be used as the sole basis for predicting the outcome in man. research into the treatment of virus diseases by drugs is a t present directed toward three general areas: ( 1 ) attempts to stimulate the defense mechanism of the host animal; (2) large screening programs to find drugs that directly block some virus-specific process; and (3) alleviation of the symptoms of the disease. the first approach is exemplified by the variety of interferon inducers which are a t present under intensive study. a disappointing feature of these is their uniformly low activity in man, despite highly promising results in laboratory animals, such as mice, rats, and rabbits. perhaps the interferon response in man and primates is less important in defense against virus disease than it is in other taxonomic groups. searches are being made for more general stimulants of host defense mechanisms, for example, stimulators of phagocytosis and the immune response, but very little progress has been reported so far. the search for drugs that will directly inhibit virus replication, by stopping a virus-coded synthetic process, or the absorption, penetration, uncoating, assembly, or release of virions, has been intensive and is still continuing. nevertheless, the products of this effort will find application only in a relatively small number of virus diseases for the reasons outlined above. only for those diseases of high economic importance, and for which no effective vaccines are available, will specific antiviral drugs ever be a commercial reality. the present paucity of such drugs is undoubtedly due largely to the intimate association of viruses at the molecular level with their host cells. for this reason, disease targets must be carefully defined, screening procedures made as meaningful as possible, and the limitations of animal models be clearly recognized. only by attention to these details can the maximum effort be brought to this difficult problem. again, the lack of clinically useful drugs allows no more than speculation on the possibilities of drug-resistant viruses emerging when antiviral drugs are eventually in widespread use. the phenomenon of drug resistance in viruses is well established in the laboratory (melnick et al., 1961; tamm and eggers, 1962; renis and buthala, 1965) , and, unless potential antiviral drugs are free of this serious defect, their commercial life will be embarrassingly short. the treatment of the symptoms, rather than the cause of a disease, has been the mainstay of medical practice from time immemorial, and this is still the case with most virus disease. the short incubation period of many virus diseases will inevitably restrict the therapeutic use of antiviral drugs, and in cases where symptoms have already appeared, the physician and layman alike will have recourse to the extensive armamentanurn of palliatives available for alleviating the symptoms of virus disease. all the same, this is clearly an unsatisfactory state of affairs, and the ultimate goal of antiviral research is the prevention of virus disease. as the understanding of viruses increases, so a more rational approach to the chemotherapy of virus diseases will become feasible. also, random discoveries of antiviral activity in novel chemical compounds will shed further light on those areas of virus metabolism that are susceptible to chemical attack. with increasing con-tributions from both these approaches, virus chemotherapy should soon emerge from a theoretical possibility to a practical reality, and antiviral drugs ivill make their long awaited contributions to clinical medicine. modern trends in medical virology zn "virus-induced immunopathology arch. gesanlfe virusforsch. 30 zn "diagnostic procedures for virus and rickettsia1 infections amer zn "topics in medicinal chemistry ezperientia 16, 487 virology 4, 483. tamm, j., and overman antiviral substances key: cord-273372-69rlh9or authors: litterman, nadia; lipinski, christopher; ekins, sean title: small molecules with antiviral activity against the ebola virus date: 2015-02-09 journal: f1000res doi: 10.12688/f1000research.6120.1 sha: doc_id: 273372 cord_uid: 69rlh9or the recent outbreak of the ebola virus in west africa has highlighted the clear shortage of broad-spectrum antiviral drugs for emerging viruses. there are numerous fda approved drugs and other small molecules described in the literature that could be further evaluated for their potential as antiviral compounds. these molecules are in addition to the few new antivirals that have been tested in ebola patients but were not originally developed against the ebola virus, and may play an important role as we await an effective vaccine. the balance between using fda approved drugs versus novel antivirals with minimal safety and no efficacy data in humans should be considered. we have evaluated 55 molecules from the perspective of an experienced medicinal chemist as well as using simple molecular properties and have highlighted 16 compounds that have desirable qualities as well as those that may be less desirable. in addition we propose that a collaborative database for sharing such published and novel information on small molecules is needed for the research community studying the ebola virus. viruses remain a constant threat to global health, with new infections from human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) killing more than 3 million people annually 1, 2 . the flavivirus that causes dengue fever infects up to 100 million people each year, leading to death in 2.5% of cases 3, 4 . other viral outbreaks including severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory coronavirus (mers-cov), affect far fewer people but have high mortality rates and the potential to spread to epidemic size 1,5,6 . thus, even with the development of vaccines and other treatments, viruses lead to a large burden on human health. more than 30 small molecule drugs have been developed that have activity against individual viruses, including hiv, influenza, hbv, and more recently hcv 7,8 . however, a large number of virus types remain without any effective therapeutics, and there are few broadspectrum anti-virals available. thus, when viruses emerge that cause life-threatening infections, such as the recent ebola virus epidemic in west africa, there are no treatment options. since it is likely that these and other types of infectious agents will emerge in the future, an important goal is to identify inhibitors to be available to contain such outbreaks. a large array of drug discovery efforts have proven that despite their small size, viral genomes represent suitable targets for drugs. direct-acting antivirals, which target viral proteins rather than the host's, have been the subject of extensive investigation 3 . targets in this class fall into multiple categories: virus adsorption inhibitors, inhibitors of viral dna or rna synthesis, viral protease inhibitors required for virus maturation, and viral neuraminidase inhibitors required for virus elution 7 . in addition, cellular targets exist that are required for viral replication, including inosine monophosphate (imp) dehydrogenase, which is required to supply the pool of guanosine triphosphate (gtp) that serves as a substrate for rna and dna, and s-adenosylhomocysteine (sah) hydrolase, which is required for the methylation and hence maturation of viral dna. while finding specific inhibitors to target each viral threat individually may be ideal, the cost-savings and feasibility of finding drugs that act as broad-spectrum antivirals, or those that target specific viral genus or family, is an important goal given the expense associated with developing any one drug for one disease 3 . there are important individual aspects of each virus, and viral family to consider, but nonetheless, many mechanisms of the viral life cycle are mirrored across families, and thus represent opportunities to learn from the many experimental studies that have already been performed. like many around the world, we have been watching the devastating effects of the ebola virus in west africa. we have been impressed by the important contributions of the health organizations and the personal sacrifices the medical workers are making to serve patients and hamper the spread of disease. and yet we began to wonder, why has there been relatively little focus on small molecules apart from a recent review of ebola virus therapeutic strategies in general 9 ? small molecules have several advantages over other therapeutic approaches including the ability to be produced at a large scale and stability necessary for broad distribution. we felt it was time to therefore focus more on small molecules while we await a vaccine. we have found that indeed there is much prior knowledge regarding small molecules that have been shown to be active against the ebola virus in vitro or in animal models 10-13 , including a number of fda-approved drugs 14-16 . a thorough literature search of pubmed, and cas scifinder tm (cas, columbus oh) using terms including "ebola" identified 55 molecules suggested to have activity against ebola virus in vitro and/or in vivo (supplemental table 1 ). fda approved small molecules with activity against the ebola virus recently, a pharmacophore 17 was generated from four fda approved compounds for other diseases (non-antivirals) resulting from two high throughput screens against the ebola virus 14,15 and closely matched the receptor-ligand pharmacophores for the ebola viral protein 35 (vp35) 10 . follow-up docking studies suggested that these compounds may have favorable inhibitory interactions with this receptor. vp35 is a cofactor in the rna polymerase transcription complex, and helps the virus evade the immune response by blocking activation of the interferon regulatory factor 3, which is required for the induction of interferons alpha and beta. thus, blocking vp35 should allow for an enhancement of the host immune response to the ebola virus. it is proposed that similar compounds may be acting via a closely related mechanism, though there has been no experimental evidence to directly prove this yet. another recent study 16 has highlighted the ability of three clinically approved ion channel blockers to inhibit the ebola virus cellular entry. the drugs amiodarone, dronedarone, and verapamil, were given at concentrations that are possible in human serum, and were effective against a number of filoviruses. the authors hypothesized that these drugs may act by disrupting late endosomal processing or by disrupting calcium signaling that is required for viral entry. of course, none of these aforementioned fda approved drugs were designed to target the ebola virus. amodiaquine and chloroquine are antimalarials, clomiphene and toremifene are selective estrogen receptor modulators. amiodarone, dronedarone, and verapamil are anti-arrhythmics. interestingly, all of these compounds have a common tertiary amine feature, which may suggest they could act through similar mechanism 18, 19 . however, they are all orally bioavailable and generally safe for humans. thus these repurposed drugs may represent a fast track to potential evaluation and approval as a feasible option for preventing the spread and mortality associated with the ebola virus in a large population. small molecules tested in humans with the ebola virus several small molecules have actually been tested in very small numbers of humans for activity against the ebola virus. for example there has been some press on favipiravir, which is undergoing phase 3 clinical trials in the us for influenza and is approved in japan, as it has shown promising efficacy against the ebola virus in mice 20 . faviparavir is thought to act by inhibiting the viral rna-dependent rna polymerase selectively and has demonstrated activity against a number of other viruses. at least one ebola patient, who has since recovered, was given favipiravir 21 , and japan offered to supply it to the world health organization. a second experimental drug, brincidofovir 22 , in phase 3 clinical trials for treatment of cytomegalovirus and other dna viruses has shown efficacy against the ebola virus in vitro and animal studies are ongoing 23 . brincidofovir is thought to mimic cytidine, a building block of dna, and thereby inhibit viral dna polymerases, and its mechanism of action against the ebola virus, an rna virus, is yet unknown. brincidofovir, which has demonstrated safety in humans, has been given to at least two ebola virus patients, one in dallas and one in nebraska. while unfortunately the dallas patient died, the nebraska patient survived 24 . it is of course too early to know the effect of this molecule on the progression of the disease. this compound is a pro-drug that is converted into the active antiviral, cidofovir diphosphate. brincidofovir has higher oral bioavailability, intracellular concentrations of drug and increased antiviral potency 22 . this compound only appeared in the literature in 2014 and there is very little published information. beyond these early stage drugs, there are a number of other compounds that have been identified as active against the ebola virus as summarized by erik de clercq 9 . while many are not ready for in human use, they may present an attractive starting point to be refined in a future drug discovery effort. for example, a novel nucleoside analog, bcx4430 demonstrated efficacy in mice and nonhuman primates against the ebola virus 25 . this compound targets viral rna polymerase activity by inducing early termination of transcription and thus blocking replication. bcx4430 is not only active against ebola virus, but also targets other members of the filovirus family as well as 8 other rna virus families. because of the potent and efficacious effects, bcx4430 is being fast-tracked for clinical trials in humans. medicinal chemistry analysis of small molecules active against the ebola virus we have recently described an expert's medicinal chemistry 26 analysis of the over 320 nih probe compounds using public and commercial sources of chemical structures and the issues related to doing this type of analysis 27 . the likely chemistry quality of these probes was scored based on a number of criteria including literature related to the probe and potential chemical reactivity. through a series of machine learning models, we also computationally predicted the scores which were being identified through a painstaking manual process 26 . external validation and comparison with other measures of drug-likeness and filtering rules suggested a comparable level of accuracy 26 . we have now carefully analyzed in a similar manner the 55 small molecules with activity against the ebola virus identified from our literature search. the chemist's (c.a.l.) decisions on compound quality are summarized in supplemental table 1 . in contrast to the previous work in scoring compounds as chemical probes, the current aim is very specific -to treat a very serious viral disease for which there is a phenotypic readout. all the known drugs in clinical use were rejected as the chemist looked for compounds that looked more interesting in his opinion. only 16 out of 55 were selected as desirable in this analysis with no potential problems based on medicinal chemistry experience. in addition to this manual approach, we applied computational filters such as pan assay interference compounds (pains) to identify potentially problematic compounds from structures 28,29 (supplemental table 1 ). pains analysis was enabled using the mmds mobile app 30 . based on this approach we have identified several molecules that appear problematic and agreed with the medicinal chemistry analysis. for example 4 molecules appear to fail the pains filters, including the rhodanine compound lj-001 which was found to be active against numerous enveloped viruses and was found through a screen of inhibitors of nipah virus entry (ic 50 1μm) 31 . in vivo a steady state plasma concentration could not be maintained at a therapeutic level. rhodanines are known to be problematic pains compounds 28, 29 . interestingly amodiaquine was not scored favorably by pains or the medicinal chemist, yet this is a successful antimalarial drug. in addition we analyzed the simple chemical properties (calculated in the collaborative drug discovery (cdd) vault (collaborative drug discovery, inc) using the chemaxon toolkit (chemaxon, budapest, hungary)) of the molecules and compared these to their medicinal chemistry classification ( table 1 ). the mean calculated molecular property values for compounds were compared using the t-test and anova with jmp v. 8.0.1 (sas institute, cary, nc). significant differences were noted in logp and lipinski rule of 5 violations between desirable and undesirable compounds although one molecule sara-133 skewed the data with a molecular weight over 3000 ( table 1) . removal of this compound leads to significant differences for molecular weight, number of hydrogen bond table 1 . mean ± sd molecular properties calculated in cdd vault using chemaxon software for the 55 molecules with activity against the ebola virus. * statistically significant p < 0.05 using the t-test and anova. ** statistically significant p < 0.0001 using the t-test and anova. note data are skewed by sara-133. when this molecule is removed the mean values and significance data are shown in parentheses. collaboration for the ebola virus drug discovery the research described for small molecule inhibitors against the ebola virus has occurred in a disconnected manner and there have been few efforts to summarize the total medicinal chemistry efforts to date. we think this research can learn from other areas in which there are currently efforts to improve collaboration and screening. in the area of tuberculosis research, funding from the bill and melinda gates foundation and the european commission have enabled the tb drug accelerator and the more medicines for tuberculosis, as large-scale collaborations between academia, research institutes and industry. these collaborations have promoted the selective sharing of related data in a secure environment between collaborators using the cdd vault 32 (figure 1 ). the centralized availability of publicly available data on compounds screened for activity against mycobacterium tuberculosis enables researchers to leverage the existing literature alongside their private data. this knowledge can be used for building of validated computational models that can help in selecting additional compounds or lead optimization 33,34 , saving time and effort. by organizing the data on small molecules tested against the ebola virus similarly in a central database and using machine learning models based on public data may help identify additional compounds for testing. such an effort may also prevent duplication of efforts as we have seen with the screening of multiple libraries of fda approved compounds against the ebola virus 1416 . in order to catalyze this we have made the 55 compounds (supplemental table 1 ) freely available as a dataset in cdd public (https://app.collaborativedrug. com/register). the compound representation in cdd vault can be accessed via the text compound descriptors (as in the supplemental table 1 ) or in a batch connection table format as an mdl format structure data file (*.sdf) that is universally read by all chemistry aware software. being able to easily retrieve the chemical structures in machine retrievable form has considerable value. identifying compounds by name or company code number does not per se allow direct access to chemical structure and often fails entirely to link compound identifier with the chemistry structure of the compound. inchikey has the great advantage of being searchable on the web (e.g. via google) but requires a lookup table (e.g. embl's unichem) to get back to the chemical structure. smiles and inchi representations allow direct access to chemical structure and are compatible with structure searching in most public chemistry databases as well as the proprietary chemistry acs cas scifinder database. the iupac name is universally used in naming chemical compounds in patents and software exists for going from iupac name to chemical structure. the compounds which are fda approved drugs for other diseases 14-16 but with activity against ebola virus in vitro or in vivo may represent useful starting points with the advantage that much is known regarding their adme and tox properties. however they may not be ideal in the opinion of a medicinal chemist. by bringing together all 55 compounds described in the literature with activity against the ebola virus, this body of evidence may be more convincing than each study in isolation. in addition it allows us to consider structural features and molecular properties, which may provide insights into the target or mechanism of action. it is unclear whether any of the 55 compounds might also have activity when used as combination therapy as is the current standard of care for hiv, in order to overcome drug resistance, and this needs experimental evaluation. clearly, we are a considerable distance from having an fda approved drug for the ebola virus, but this analysis illustrates that there are already drugs on the shelf that can be potentially repurposed in a shorter time period and at a lower cost and yet they do not appear to have been tested in patients with the ebola virus. in addition there are at least 16 molecules which in the opinion of an experienced medicinal chemist are possibilities for further optimization. while novel compounds are likely more commercially viable they also will require considerable effort to assess safety. resources will need to be allocated to this effort and administrators and scientists should perhaps consider some of the medicinal chemistry insights we have provided as well as using a collaborative database to share molecules that are active amongst all scientists. it is hoped these efforts could inspire further drug discovery efforts around small molecules. to illustrate the level of interest in repurposing efforts for the ebola virus, the following studies were identified upon submission that describe additional compounds as well as those already described herein 35-37 . all authors contributed to the collaborative writing of this project. antiviral drug discovery: broadspectrum drugs from nature the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry a common feature pharmacophore for fdaapproved drugs inhibiting the ebola virus publisher full text lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (fa2n-4 cells) pubmed abstract | publisher full text | free full text a high content screening assay for identifying lysosomotropic compounds successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model french nurse cured of ebola contracted in liberia development of cmx001 (brincidofovir) for the treatment of serious diseases or conditions caused by dsdna viruses chimerix's brincidofovir has in vitro activity against ebola potential and emerging treatment options for ebola virus disease protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 computational prediction and validation of an expert's evaluation of chemical probes parallel worlds of public or commercial bioactive chemistry data new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays chemistry: chemical con artists foil drug discovery cheminformatics workflows using mobile apps a broad-spectrum antiviral targeting entry of enveloped viruses acknowledgments se acknowledges several discussions with dr. megan coffee, dr. joel s. freundlich, dr. nancy connell and dr. peter madrid. the author(s) declared that no grants were involved in supporting this work. the authors have collected 55 small molecules reported to have activity against the ebola virus from the literature and organized them into a dataset that is easily searchable for those who may be interested in pursuing further research on the design of inhibitors of this virus. no competing interests were disclosed. the authors have searched a collection of compounds for a common pharmacophore directed against ebola proteins, and detected 55 hits. these were filtered by an experienced medicinal chemist using well-established techniques and intuition to provide 16 legitimate compounds that may serve the ebola research community as a publicly available resource. this is an excellent example of the power of shared, collaborative research databases providing valuable resources to the research community. no competing interests were disclosed. competing interests: key: cord-034363-6uscua0y authors: cerda-cavieres, christopher; quiroz, gabriel; iturriaga-vásquez, patricio; rodríguez-lavado, julio; alarcón-espósito, jazmín; saitz, claudio; pessoa-mahana, carlos d.; chung, hery; araya-maturana, ramiro; mella-raipán, jaime; cabezas, david; ojeda-gómez, claudia; reyes-parada, miguel; pessoa-mahana, hernán title: synthesis, docking, 3-d-qsar, and biological assays of novel indole derivatives targeting serotonin transporter, dopamine d2 receptor, and mao-a enzyme: in the pursuit for potential multitarget directed ligands date: 2020-10-10 journal: molecules doi: 10.3390/molecules25204614 sha: doc_id: 34363 cord_uid: 6uscua0y a series of 27 compounds of general structure 2,3-dihydro-benzo[1,4]oxazin-4-yl)-2-{4-[3-(1h-3indolyl)-propyl]-1-piperazinyl}-ethanamides, series i: 7(a–o) and (2-{4-[3-(1h-3-indolyl)-propyl]-1-piperazinyl}-acetylamine)-n-(2-morfolin-4-yl-ethyl)-fluorinated benzamides series ii: 13(a–l) were synthesized and evaluated as novel multitarget ligands towards dopamine d(2) receptor, serotonin transporter (sert), and monoamine oxidase-a (mao-a) directed to the management of major depressive disorder (mdd). all the assayed compounds showed affinity for sert in the nanomolar range, with five of them displaying ki values from 5 to 10 nm. compounds 7k, ki = 5.63 ± 0.82 nm, and 13c, ki = 6.85 ± 0.19 nm, showed the highest potencies. the affinities for d(2) ranged from micro to nanomolar, while mao-a inhibition was more discrete. nevertheless, compounds 7m and 7n showed affinities for the d(2) receptor in the nanomolar range (7n: ki = 307 ± 6 nm and 7m: ki = 593 ± 62 nm). compound 7n was the only derivative displaying comparable affinities for sert and d(2) receptor (d(2)/sert ratio = 3.6) and could be considered as a multitarget lead for further optimization. in addition, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds in the most relevant protein targets were carried out. furthermore, in order to obtain information on the structure–activity relationship of the synthesized series, a 3-d-qsar comfa and comsia study was conducted and validated internally and externally (q(2) = 0.625, 0.523 for comfa and comsia and r(2)(ncv) = 0.967, 0.959 for comfa and comsia, respectively). the fluorinated benzamides derivatives 12a-d were finally connected to indolylpropylpiperazines 9a-c to achieve the expected compounds 13a-l, with yields ranging from 47% to 85% (scheme 5). in summary, 15 compounds were synthesized for series i in yields ranging from 42% to 91%. the synthetic pathway of this series involved the fluorinated benzamide derivatives 12a-d, which were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence with good to excellent yields as shown in scheme 4. in summary, 15 compounds were synthesized for series i in yields ranging from 42% to 91%. the synthetic pathway of this series involved the fluorinated benzamide derivatives 12a-d, which were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence with good to excellent yields as shown in scheme 4. the fluorinated benzamides derivatives 12a-d were finally connected to indolylpropylpiperazines 9a-c to achieve the expected compounds 13a-l, with yields ranging from 47% to 85% (scheme 5). the fluorinated benzamides derivatives 12a-d were finally connected to indolylpropylpiperazines 9a-c to achieve the expected compounds 13a-l, with yields ranging from 47% to 85% (scheme 5). molecules 2020, 25 table 1 summarizes the affinity of compounds 7a-o for sert, d2 receptor, and mao-a. most compounds were potent and clearly selective as sert ligands, showing in all cases affinities in the nanomolar range, whereas the affinities for d2 and mao-a ranged from micromolar to much higher values, respectively. a detailed analysis of sert activities indicates that a c-5 substitution of the indole ring with halogens (fluorine or bromine; compounds 7g or 7m) leads to more potent compounds than the unsubstituted derivative (7a). on the other hand, the presence of a halogen atom at c-6 of the benzoxazine ring increased the affinity (e.g., 7c, 7d, and 7e vs. 7a). accordingly, the most potent compounds were those exhibiting a dual halogen substitution pattern (7i, 7j, and 7k), with ki values below 10 nm. the c-7 halogen substitution on the benzoxazine ring gave no consistent effects, slight increases (7b and 7h) or decreases (7n) of affinity were observed, as compared with the corresponding c-7 unsubstituted compounds (7a, 7g, and 7m, respectively). the d2 receptor affinity for this series indicates that no conclusive structure-activity relationships can be extracted for these compounds. nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen atom at the c-5 of the indole ring and the other at either the c-6 or c-7 of the benzoxazine moiety (7h-7k, 7m-7o), resulted in more potent compounds than their corresponding monohalogenated or unsubstituted counterparts (7a-7e, 7g). moreover, the presence of a methoxyl group at the c-6 of the benzoxazine ring has almost no effect on the affinity of the compounds for d2 receptor. it is worth mentioning that the dihalogenated compound 7n was the only derivative displaying comparable affinities for sert and d2 receptor (d2/sert ratio = 3.6) and could be considered as a potential leader in the search of more potent multitarget compounds. scheme 5. synthesis of series ii derivatives 13a-l. reagents and conditions: k 2 co 3 , ch 3 cn, 80 • c, yield (47-85%). table 1 summarizes the affinity of compounds 7a-o for sert, d 2 receptor, and mao-a. most compounds were potent and clearly selective as sert ligands, showing in all cases affinities in the nanomolar range, whereas the affinities for d 2 and mao-a ranged from micromolar to much higher values, respectively. a detailed analysis of sert activities indicates that a c-5 substitution of the indole ring with halogens (fluorine or bromine; compounds 7g or 7m) leads to more potent compounds than the unsubstituted derivative (7a). on the other hand, the presence of a halogen atom at c-6 of the benzoxazine ring increased the affinity (e.g., 7c, 7d, and 7e vs. 7a). accordingly, the most potent compounds were those exhibiting a dual halogen substitution pattern (7i, 7j, and 7k), with ki values below 10 nm. the c-7 halogen substitution on the benzoxazine ring gave no consistent effects, slight increases (7b and 7h) or decreases (7n) of affinity were observed, as compared with the corresponding c-7 unsubstituted compounds (7a, 7g, and 7m, respectively). the d 2 receptor affinity for this series indicates that no conclusive structure-activity relationships can be extracted for these compounds. nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen atom at the c-5 of the indole ring and the other at either the c-6 or c-7 of the benzoxazine moiety (7h-7k, 7m-7o), resulted in more potent compounds than their corresponding monohalogenated or unsubstituted counterparts (7a-7e, 7g). moreover, the presence of a methoxyl group at the c-6 of the benzoxazine ring has almost no effect on the affinity of the compounds for d 2 receptor. it is worth mentioning that the dihalogenated compound 7n was the only derivative displaying comparable affinities for sert and d 2 receptor (d 2 /sert ratio = 3.6) and could be considered as a potential leader in the search of more potent multitarget compounds. table 1 . affinities, measured as ki values at the serotonin transporter (sert), d 2 receptor, and percent of monoamine oxidase-a (mao-a) inhibition (at 100 µm) of indolepiperazinyl benzoxazine derivatives (series i). molecules 2020, 25, x for peer review 6 of 30 considering the pharmacological results, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds were carried out only in the human sert (hsert) and in selected cases at the d2 receptor. the most potent compounds 7g, 7h, 7i, and 7k showed a common docking pose (figure 1 ), which favors the following stabilizing interactions: a π-π interaction between the indole ring and the πdonor aromatic residue tyr176, a coulombic interaction between the protonated piperazine n-1 with the asp98 residue, and a π-cation interaction for the protonated piperazine with tyr95. furthermore, aromatic interactions were also observed for the benzoxazine ring with the residues phe341 and considering the pharmacological results, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds were carried out only in the human sert (hsert) and in selected cases at the d 2 receptor. the most potent compounds 7g, 7h, 7i, and 7k showed a common docking pose (figure 1 ), which favors the following stabilizing interactions: a π-π interaction between the indole ring and the π-donor aromatic residue tyr176, a coulombic interaction between the protonated piperazine n-1 with the asp98 residue, and a π-cation interaction for the protonated piperazine with tyr95. furthermore, aromatic interactions were also observed for the benzoxazine ring with the residues phe341 and phe335. these drug-target interactions are in agreement with those described in the crystal structure of the hsert in complex with the inhibitor (s)-citalopram [47, 48] . the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds 7f and 7l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for 7f. thus, the least potent compound of the series (7f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c-5 fluorinated counterpart ( figure 2 ). compounds showing intermediate affinities (7a-7e and 7m-7o) exhibited docking poses between the most and least favorable binding modes (not shown). the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds 7f and 7l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for 7f. thus, the least potent compound of the series (7f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c-5 fluorinated counterpart ( figure 2 ). compounds showing intermediate affinities (7a-7e and 7m-7o) exhibited docking poses between the most and least favorable binding modes (not shown). phe335. these drug-target interactions are in agreement with those described in the crystal structure of the hsert in complex with the inhibitor (s)-citalopram [47, 48] . the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds 7f and 7l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for 7f. thus, the least potent compound of the series (7f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c-5 fluorinated counterpart ( figure 2 ). compounds showing intermediate affinities (7a-7e and 7m-7o) exhibited docking poses between the most and least favorable binding modes (not shown). docking simulations showed that compounds of this series adopt, at the d 2 receptor, a binding mode similar to that experimentally determined for the atypical antipsychotic risperidone [49] . thus, the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined by cys118, thr119, ser197, phe198, and trp386, while the protonated piperazine n-1 locates in a favorable position to establish a coulombic interaction with asp114 ( figure 3) . furthermore, the benzoxazine portion extends to the additional hydrophobic sub-pocket lined by val91, trp100, phe110, and tyr408, in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety of risperidone. interestingly, this general binding mode was observed for both the most and the least potent compounds of this series (7a, 7b, and 7m, 7n), respectively ( figure 3a ,b). therefore, it is tempting to speculate that the higher affinity showed by brominated derivatives (7m and 7n) is due to the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue (e.g., ser197). as observed (figure 3 ), this could also change the position of the benzoxazine moiety, favoring its interactions at the more external hydrophobic sub-pocket. docking simulations showed that compounds of this series adopt, at the d2 receptor, a binding mode similar to that experimentally determined for the atypical antipsychotic risperidone [49] . thus, the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined by cys118, thr119, ser197, phe198, and trp386, while the protonated piperazine n-1 locates in a favorable position to establish a coulombic interaction with asp114 ( figure 3) . furthermore, the benzoxazine portion extends to the additional hydrophobic sub-pocket lined by val91, trp100, phe110, and tyr408, in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety of risperidone. interestingly, this general binding mode was observed for both the most and the least potent compounds of this series (7a, 7b, and 7m, 7n), respectively ( figures 3a and 3b) . therefore, it is tempting to speculate that the higher affinity showed by brominated derivatives (7m and 7n) is due to the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue (e.g., ser197). as observed (figure 3 ), this could also change the position of the benzoxazine moiety, favoring its interactions at the more external hydrophobic sub-pocket. table 2 summarizes the affinity of series ii compounds 13a-13l for sert, d2 receptor, and mao-a. as in the case of indole benzoxazine derivatives (series i), most indole morpholine ethylbenzamides (series ii) were potent sert ligands, showing much lower affinities for d2 receptor and virtually no effect upon mao-a activity. regarding sert activity, and in agreement with our previous studies, halogen substitution at c-5 of the indole ring with fluorine or bromine (compounds 13a-g) conducted an increase in affinity as compared with the unsubstituted analogues 13i-l, with the fluoro derivatives 13a-d being the most potent of the series. on the other hand, when the acetanilide portion, connected to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at c-2) and a morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was located at meta position (compounds 13c, 13g, and 13k). table 2 summarizes the affinity of series ii compounds 13a-13l for sert, d 2 receptor, and mao-a. as in the case of indole benzoxazine derivatives (series i), most indole morpholine ethylbenzamides (series ii) were potent sert ligands, showing much lower affinities for d 2 receptor and virtually no effect upon mao-a activity. regarding sert activity, and in agreement with our previous studies, halogen substitution at c-5 of the indole ring with fluorine or bromine (compounds 13a-g) conducted an increase in affinity as compared with the unsubstituted analogues 13i-l, with the fluoro derivatives 13a-d being the most potent of the series. on the other hand, when the acetanilide portion, connected to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at c-2) and a morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was located at meta position (compounds 13c, 13g, and 13k). similar to the analysis of series i and considering the pharmacological results, docking studies were carried out only in hsert. in this series, seven compounds exhibited ki values between 7 and 60 nm (13a, 13b, 13c, 13f, 13g, 13k, and 13l) . docking simulations showed that compounds with the lowest ki values (13c and 13g) share a common binding mode into the s1 site of the sert, which is similar to that described for compounds of series i ( figure 4a ). thus, the piperazine n-1 can establish ionic and π-cation interactions with asp98 and tyr176, respectively, while the indole moiety can participate in aromatic interactions with tyr176 and phe341. interestingly, the ethylmorpholinic chain extends towards the extracellular vestibule (also known as the s2 site). on the other hand, for the compounds with the lowest affinities (13e and 13i), docking simulations showed that the piperazine n-1 was located farther away from asp98 and tyr95, making the possible ionic interactions with these residues unlikely or much weaker ( figure 4b ). the analysis of the docking poses indicates that the most potent compounds, i.e., those having a 5,2-substitution pattern (13c, 13g, and 13k) exhibited an extended conformation at the binding site, while the least potent similar to the analysis of series i and considering the pharmacological results, docking studies were carried out only in hsert. in this series, seven compounds exhibited ki values between 7 and 60 nm (13a, 13b, 13c, 13f, 13g, 13k, and 13l). docking simulations showed that compounds with the lowest ki values (13c and 13g) share a common binding mode into the s1 site of the sert, which is similar to that described for compounds of series i ( figure 4a ). thus, the piperazine n-1 can establish ionic and π-cation interactions with asp98 and tyr176, respectively, while the indole moiety can participate in aromatic interactions with tyr176 and phe341. interestingly, the ethylmorpholinic chain extends towards the extracellular vestibule (also known as the s2 site). on the other hand, for the compounds with the lowest affinities (13e and 13i), docking simulations showed that the piperazine n-1 was located farther away from asp98 and tyr95, making the possible ionic interactions with these residues unlikely or much weaker ( figure 4b ). the analysis of the docking poses indicates that the most potent compounds, i.e., those having a 5,2-substitution pattern (13c, 13g, and 13k) exhibited an extended conformation at the binding site, while the least potent compounds (13e and 13i, showing a 2,4-substitution pattern) adopted a more constrained binding mode, impairing the most relevant interactions. molecules 2020, 25, x for peer review 10 of 30 compounds (13e and 13i, showing a 2,4-substitution pattern) adopted a more constrained binding mode, impairing the most relevant interactions. to systematize the structure-activity relationship of the synthesized molecules, we carried out a 3-d-qsar study of the comfa and comsia type. the complete series of 27 molecules was divided into training (19 compounds) and test sets (8 compounds) in a ratio of 70:30, selecting the test set compounds at random to avoid bias. the q 2 values for the best models were 0.625 and 0.523 for comfa and comsia, respectively while the r 2 ncv values were 0.967 and 0.959 for comfa and comsia, respectively. the statistical summary, as well as the tables of affinities for both models and their respective graphs, are incorporated in the supplementary material. the steric contour map of comfa ( figure 5a ) shows a green polyhedron on the bromine atom of compound 7k, the most active of the series. this means that the insertion of bulky atoms or groups in this position is favorable for biological activity. this is consistent with docking studies showing that compounds of series i place halogen into the void space close to lipophilic residues like trp100 and tyr408. in the case of compounds of series ii, the meta-substituted benzamides placed the chain towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the meta-position. this is confirmed in the docking of these compounds, in which better accommodation is observed in the sert binding site. on the other hand, the electrostatic contour map ( figure 5b ) shows three blue polyhedra of significant size. this means that the presence of positively charged atoms in these positions would be favorable for affinity. such polyhedra are located on the carbon atom bonded to the halogen in the case of series i, suggesting that the presence of electronegative atoms bonded to the aforementioned carbon is favorable. the second blue polyhedron is localized on the oxygen atom of the carbonyl group belonging to the ortho-substituted series ii amidecompounds. therefore, oxygen atom remotion would be favorable for affinity. finally, the third polyhedron is observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series ii, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better affinities. furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted in less favorable affinities compared to meta substitutions as was experimentally corroborated. the hydrophobic contour map of comsia ( figure 5c ) showed a gray polyhedron at position c5 of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. in fact, the c5 fluorine-substituted indoles displayed the best affinities of the series. other polar groups like a b figure 4 . docking poses in sert obtained for compounds 13c in cyan and 13g in purple (a), and 13e in light blue, and 13i in orange (b). nearby residues < 5 å (grey sticks) and na + atoms (pink spheres) are shown. dotted lines represent ionic interactions, orange lines represent π-cation interactions, and aromatic interactions are shown with green lines. to systematize the structure-activity relationship of the synthesized molecules, we carried out a 3-d-qsar study of the comfa and comsia type. the complete series of 27 molecules was divided into training (19 compounds) and test sets (8 compounds) in a ratio of 70:30, selecting the test set compounds at random to avoid bias. the q 2 values for the best models were 0.625 and 0.523 for comfa and comsia, respectively while the r 2 ncv values were 0.967 and 0.959 for comfa and comsia, respectively. the statistical summary, as well as the tables of affinities for both models and their respective graphs, are incorporated in the supplementary material. the steric contour map of comfa ( figure 5a ) shows a green polyhedron on the bromine atom of compound 7k, the most active of the series. this means that the insertion of bulky atoms or groups in this position is favorable for biological activity. this is consistent with docking studies showing that compounds of series i place halogen into the void space close to lipophilic residues like trp100 and tyr408. in the case of compounds of series ii, the meta-substituted benzamides placed the chain towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the meta-position. this is confirmed in the docking of these compounds, in which better accommodation is observed in the sert binding site. on the other hand, the electrostatic contour map ( figure 5b ) shows three blue polyhedra of significant size. this means that the presence of positively charged atoms in these positions would be favorable for affinity. such polyhedra are located on the carbon atom bonded to the halogen in the case of series i, suggesting that the presence of electronegative atoms bonded to the aforementioned carbon is favorable. the second blue polyhedron is localized on the oxygen atom of the carbonyl group belonging to the ortho-substituted series ii amide-compounds. therefore, oxygen atom remotion would be favorable for affinity. finally, the third polyhedron is observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series ii, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better affinities. furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted in less favorable affinities compared to meta substitutions as was experimentally corroborated. red polyhedron on the halogen atom at position 5 of the indole ring means that the presence of electron-rich atoms is favorable for affinity. it is interesting to note that the blue polyhedron intersecting the carbon atom of indole at position 5 is complementary to the red polyhedron. in consequence, the presence of a positive charge on the indole ring is favorable for activity. other potential electron-withdrawing groups to be explored are cn, no2, and cor. docking studies showed π-stacking interaction between the π-deficient indole ring with tyr176, phe341, and trp386 residues. the hydrophobic contour map of comsia ( figure 5c ) showed a gray polyhedron at position c5 of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. in fact, the c5 fluorine-substituted indoles displayed the best affinities of the series. other polar groups like oh, nh 2 , or nr 2 would also be interesting to evaluate at this position. similarly, a yellow polyhedron located on the bromine atom of the benzoxazine framework (compound 7k) means that the presence of lipophilic groups is favorable for activity. in concordance, halogens like cl, br, and i would be the most appropriated substituents and groups, such as aromatic rings, alkyl, and/or alkoxy chains, could also be explored. in the case of compounds of series ii, a yellow polyhedron is located on the amide group of the meta-substituted compounds; therefore, the replacement of the amide by a less-polar function, such as a ketone or ester, would be an interesting option to explore. on the other hand, the electrostatic contour map of comsia ( figure 5d ) showed two polyhedra around compound 7k. a red polyhedron on the halogen atom at position 5 of the indole ring means that the presence of electron-rich atoms is favorable for affinity. it is interesting to note that the blue polyhedron intersecting the carbon atom of indole at position 5 is complementary to the red polyhedron. in consequence, the presence of a positive charge on the indole ring is favorable for activity. other potential electron-withdrawing groups to be explored are cn, no 2 , and cor. docking studies showed π-stacking interaction between the π-deficient indole ring with tyr176, phe341, and trp386 residues. melting points were determined on a hot-stage apparatus and were uncorrected. the 1 h and 13 c-nmr spectra were obtained on a bruker drx-300 spectrometer (300 and 75 mhz, respectively) in cdcl 3 , dmso-d 6 , and cd 3 cocd 3 -d 6 . chemical shifts were recorded in ppm (δ) relative to tms as an internal standard. j values are given in hz. micro-analyses were carried out on a fisons ea 1108 analyzer. high-resolution mass spectra were recorded on a dsa-tofaxion 2 tof ms (perkin elmer, shelton, ct, usa), positive mode. silica gel merck 60 (70-230 mesh) and aluminum sheets coated with silica gel 60 f254 were used for column and tlc chromatography, respectively. to a solution containing 2-chloro-1-(2,3-dihydrobenzo[b] [1, 4] oxazin-4-yl) ethanamide 4a (1.5 g; 7.09 mmol) in dry ch 3 cn (60 ml), n-boc-piperazine (1321 mg; 7.09 mmol) and anhydrous k 2 co 3 (980 mg; 7.09 mmol) were added. the mixture was stirred at 80 • c for 24 h. after this time, the mixture was diluted with water (100 ml) and the solution extracted with etoac (100 ml × 3), dried over anhydrous na 2 so 4 , and concentrated under reduced pressure. the organic crude was purified by silica gel column chromatography with etoac as eluent, to provide 5a (2152 mg; 84% yield) as a white solid. m. [1, 4] oxazin-4-yl)-ethanamide 4c (1.5 g; 6.53 mmol), n-boc-piperazine (1216 mg; 6.53 mmol), and anhydrous k 2 co 3 (902 mg; 6.53 mmol), to afford 5c (2033 mg; 82% yield) as a white solid. m. [1, 4] oxazin-4-yl)-2-oxo-ethyl]-1-piperazinyl] tert-butylcarbamate 5a (2 g; 5.53 mmol) in dry ch 2 cl 2 (20 ml) and trifluoroacetic acid (12 ml) was stirred at 0 • c, for 4 h. after this time, dry ch 2 cl 2 (200 ml) was added and neutralized with solid nahco 3 (10 g) to later filter on celite. the mixture was finally diluted with a saturated solution of nahco 3 (200 ml), extracted with etoac (8 × 50 ml), dried over anhydrous na 2 so 4 , and concentrated under vacuum to obtain pure 6a (867 mg; 82% yield) as an unstable yellow light solid, highly hygroscopic; 1 to a solution of 3-(5-fluoro-1h-3-indolyl)-propyl-4-methylbencensulfonate 1b (268 mg; 0.72 mmol) in ch 3 cn (50 ml), 1-(7-fluoro-2,3-dihydro-benzo[b] [1, 4] [1, 4] oxazin-4-yl) ethanamide 4e (227 mg; 0.92 mmol), and anhydrous k 2 co 3 (127 mg; 0.92 mmol) were added. the mixture was heated at 80 • c for 24 h. after this time, the resulting mixture was poured into water (100 ml) and extracted with etoac (4 × 50 ml), dried over anhydrous na 2 so 4 , and concentrated under reduced pressure. the organic crude was purified by column chromatography etoac/meoh 111.1, 111.8, 114.8, 118.5, 118.7, 119.1, 121.2, 122.6, 125.4, 126.3, 127.6, 128.1, 136.7, 146.0 to a mixture containing water-acetic acid-ethanol (1:1:1), 4-fluoro-n-(2-morpholin-4-yl-ethyl)-2nitro-benzamide 10a (1 g; 3.36 mmol) and iron powder (734 mg; 13.1 mmol) were added. the resulting mixture was heated and stirred for 3 h at 70 • c. after this time, the mixture was filtered to remove excess metallic iron, transferred to a flask containing a mixture of etoac/h 2 o (400 ml, 1:1), and neutralized with nahco 3 (10 gr). the aqueous phase was extracted with etoac (50 ml × 3). the organic layer was dried over anhydrous na 2 so 4 and concentrated under vacuum to give a crude, which was purified by column chromatography with etoac/meoh (6:1) to give 11a (891 mg; 98% yield) as a yellow light solid. m.p.: 120.3-121. to determine the binding of all compounds at sert, competitive binding assays were performed according to previously reported procedures with some modifications [36] . briefly, assays were carried out in a total volume of 0.5 ml containing 9 µg protein of membrane from a clonal cell line hek-293 that overexpresses sert, 50 mm tris buffer, ph 7.4, 120 mm nacl, 5 mm kcl, 2 nm [ 3 h]-paroxetine (specific activity 20.8 ci/mmol, perkinelmer), and the compounds to be tested at different concentrations (10 −9 -10 −4 m). after 1 h at 27 • c, incubations were stopped by rapid filtration through whatman gf/c filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 ml of ice-cold buffer, dried, and put in eppendorf tubes with scintillation liquid. radioactivity was counted by a liquid scintillation counter (microbeta 2450 microplate counter, perkinelmer). control curve was performed with fluoxetine in the same experimental conditions. non-specific binding was determined with 10 µm fluoxetine. to determine the binding of all compounds at d 2 receptor, competitive binding assays were performed according to provider indications with some modifications. briefly, assays were carried out in a total volume of 0.5 ml containing 3 µg protein of membrane from a cho-k1 clonal cell line that overexpresses d 2 receptor, 50 mm tris buffer, ph 7.4, 120 mm nacl, 5 mm kcl, 5 mm mgcl 2 , 1 mm edta, 0.5 nm [ 3 h]-methylspiperone (specific activity 64.1 ci/mmol, perkinelmer), and the compounds to be tested at different concentrations (10 −9 -10 −4 m). after 2 h at 27 • c, incubations were stopped by rapid filtration through whatman gf/c filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 ml of ice-cold wash buffer (50 mm tris buffer, ph 7.4, 154 mm nacl), dried, and put in eppendorf tubes with scintillation liquid. radioactivity was counted as described before. control curve was performed with haloperidol in the same conditions. non-specific binding was determined with 10 µm haloperidol. analysis of data: all curves were fitted using the sigmoidal dose-response inhibition curve (variable slope) equation built into graphpad prism 5.01 (graphpad software inc., san diego, ca, usa). the analysis gives the ic 50 value (i.e., the drug concentration inhibiting specific binding by 50%) to calculate ki (affinity constant) by the cheng-prussof equation (ki = ic 50 /(1 + ([radioligand]/kd (radioligand))). the kd values used correspond to 0.13 nm to [ 3 h] paroxetine on sert [50] , and 0.1 nm to [ 3 h]-methylspiperone on d 2 (provided by the manufacturer). the ic 50 and ki values correspond to the results of three independent experiments, each in triplicate. all data are expressed as the mean ± sem. all experimental procedures were approved by the ethics committee of the university of santiago de chile and the science council (fondecyt) of chile and followed internationally accepted guidelines (nih guide for the care and use of laboratory animals). the effects of the compounds on rat mao-a activity were studied following a previously reported methodology [50, 51] , using a crude rat brain mitochondrial suspension as a source of enzyme. serotonin (100 µm) was used as the selective substrate for mao-a. this compound and its metabolite were detected by hplc with electrochemical detection. as an exploratory evaluation, the percentage of mao-a inhibition in the presence of 100 µm of the different compounds was determined, with the idea of evaluating in detail those compounds showing an inhibitory activity in the range of 70-100%. molecular docking studies for the two families of compounds were performed on two different protein targets (sert and d 2 receptor). all dockings were carried out at ph 7.4 in the crystal structures of human sert (hsert pdb: 5i73) [47] and human d 2 receptor [hd 2 pdb: 6cm4) [49] . all compounds were modelled using the spartan'14 software (wavefunction, inc. irvine, ca) and geometry optimization calculations were carried out using the software package at the hartree-fock level using the 6-31g* basis set. docking studies were performed using autodockv4.2 [52] software suite with autodock tools adt 1.5.6 [52, 53] following the standard docking procedure for rigid proteins. grid maps were calculated using the autogrid option with a grid volume of 70 × 70 × 70 points with a grid spacing of 0.375 å and centered on the coordinates x, y, z: 33.3 184.5; 0.565 −9.397; and 37.019 28.136 for the sert and d 2 receptor respectively. docking simulations were performed with a lamarckian genetic algorithm (lga) and binding energies were estimated according to the internal scoring function implemented by the program; 250 independent runs per ligand were carried out with an initial population of 300 individuals. default settings were used for all other parameters. the lowest free-energy resulting complexes were selected and further analyzed using the visual molecular dynamic (vmd) visualization program [54] . validation of the docking protocol was performed using the co-crystallized ligands (s)-citalopram and risperidone for sert and d 2 , respectively. comfa and comsia studies were performed with sybyl x-1.2 software [55] installed in a windows 10 environment on a pc with an intel core i7 cpu. the geometric optimization, field calculation, and charges calculation were performed as previously reported [56] ( figure s1 and table s3 in supplementary material) [57] . the internal validation of the models was done by calculating the cross-validation coefficient q 2 [58] . the models with the highest value of q 2 were selected and then subjected to external validation [59] [60] [61] (table s2 ). in all cases, the best models passed the validation limits [59] (table s2 ). the regression graphs of each model and the tables of experimental versus calculated values are in the supplementary material (table s3, figure s2 ). according to these results, the design of hybrid or bifunctional compounds, i.e., molecules that incorporate two pharmacophores known to act at different receptors into a single chemical entity, is an attractive approach for the development of agents having a targeted polypharmacological profile [19, 62, 63] . in the present work, we attempted to combine sert effects previously demonstrated for indolylalkylpiperazine derivatives, functionalizing the parent scaffold with structural fragments of drugs with known activity upon d 2 receptor or mao-a. unexpectedly, the synthesized compounds did not show, in most cases, a multitarget profile, since they exhibited a high affinity for sert while showing almost no effect at d 2 receptor or mao-a. this indicates that this strategy, although plausible, requires a very fine design of the fragments to be connected and how these are going to be linked. beyond these considerations, our results highlight the remarkable stability of the indolylpropylpiperazine skeleton as sert ligand, which exhibits a high affinity by this target, apparently regardless of the type of the associated moiety [34] [35] [36] . we think that this represents an important feature for the design of polypharmacological molecules, in which an effect upon sert is pursued. docking and qsar results allowed us to rationalize the high sert affinity observed for compounds in both studied families. thus, the presence of a halogen at the c-5 position of the indole ring and fluorine atoms at the benzoxazine (series i) or acetanilide (series ii) moieties probably induces electronic deprotection of the corresponding aromatic rings, favoring stronger π-π interactions of these frameworks with donor aromatic residues at the binding site. interestingly, one of the compounds (7n) showed a promissory multitarget profile, being the only derivative showing a relatively high and comparable affinity for sert and d 2 receptor (ki = 84.4 and 307 nm, respectively). even though at this time it is difficult to determine the molecular aspects underlying this pharmacological promiscuity, it is clear that for polypharmacological drugs, a similar affinity for different receptors is the most relevant characteristic, and therefore 7n stands as a very attractive lead for further optimization. figure s1 . the superimposed structures of all compounds used in the comfa/comsia models. figure s2 . plots of experimental versus predicted pki values for the training and test set molecules for comfa (a, b) and comsia (c, d) models. figure s3 . hsert affinity curves for compounds of series i (7a, 7b, 7c, 7d, 7e, 7f, 7g, 7h, 7i, 7j, 7k, 7l, 7m, 7n, 7o , and fluoxetine), displaying ic 50 values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s4 . d2 affinity curves for compounds of series i (7a, 7b, 7c, 7d, 7e, 7f, 7g, 7h, 7i, 7j, 7k, 7l, 7m, 7n, 7o , and haloperidol), displaying ic 50 values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s5 . hsert affinity curves for compounds of series ii (13a, 13b, 13c, 13d, 13e, 13f, 13g, 13h, 13i, 13j, 13k, 13l , and fluoxetine), displaying ic 50 values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s6 . d2 affinity curves for compounds of series ii (13a, 13b, 13c, 13d, 13e, 13f, 13g, 13h, 13i, 13j, 13k, 13l , and haloperidol), displaying ic 50 values. each determination was made in triplicate and the data expressed as the mean ± sd. hrms: (ei) calculated for c 30 h 38 brfn 6 o 3 (m + ) = 629 -morpholin-4-ylethyl) benzamide (13f) 5-bromo-3-(3-piperazin-1-yl-propyl)-1h-indole 9c (187 mg; 0.58 mmol), 3-(2-chloro-acetylamino)-4-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12b (199 mg; 0.58 mmol), and anhydrous k 2 co 3 (80 mg; 0.58 mmol), to afford 13f (193 mg mmol), 5-(2-chloro-acetylamino)-2-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12d (199 mg; 0.58 mmol), and anhydrous k 2 co 3 (80 mg; 0.58 mmol), to afford 13g (249 mg 53 (dd, 1h, h-3 , j o = 9 -morpholin-4-ylethyl) benzamide (13h) 5-bromo-3-(3-piperazin-1-yl-propyl)-1h-indole 9c (187 mg; 0.58 mmol), 2-(2-chloro-acetylamino)-5-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12c 14 (s, 2h, h-6 ), 3.40-3.43 (m, 2h, h-8 ), 3.57 (t, 4h, h-2 hrms: (ei) calculated for c 30 h 38 brfn 6 o 3 (m + ) = 629 piperazin-1-yl-propyl)-1h-indole 9a (150 mg; 0.62 mmol), 2-(2-chloro-acetylamino)-4-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12a (213 mg; 0.62 mmol), and anhydrous k 2 co 3 dmso-d 6 ): δ 1.86 (m, 2h, h-2 ), 2.25-2.49 (m, 12h, h-3 , h-4 , h-9 and h-1 ), 2.58 (m, 4h, h-5 ) 5 hz), 141.4, 163.8 (d, 1 j c-f = 245 hz), 167.3, and 170.3 ppm. hrms: (ei) calculated for c 30 h piperazin-1-yl-propyl)-1h-indole 9a (150 mg; 0.62 mmol), 3-(2-chloro-acetylamino)-4-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12b (213 mg; 0.62 mmol), and anhydrous k 2 co 3 19 (s, 2h, h-6 ), 3.38-3.40 (m, 2h, h-8 ), 3.57 (t, 4h, h-2 , j = 4.5 hz), 6.96 (td, 1h, h-5 or h-6 3 hz), 136.3, 154.7 (d, 1 j c-f = 249 hz), 165.1, and 168.5 ppm. hrms: (ei) calculated for c 30 h piperazin-1-yl-propyl)-1h-indole 9a (150 mg; 0.62 mmol), 5-(2-chloro-acetylamino)-2-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12d (213 mg; 0.62 mmol), and anhydrous k 2 co 3 13 (s, 2h, h-6 ), 3.39 (q, 2h, h-8 , j = 6.4 hz), 3.58 (t, 4h, h-2 , j = 4.2 hz), 6.96 (t, 1h, h-5 or h-6, j = 7.2 hz), 7.06 (t, 1h, h-6 or h-5 piperazin-1-yl-propyl)-1h-indole 9a (200 mg; 0.82 mmol), 2-(2-chloro-acetylamino)-5-fluoro-n-(2-morpholin-4-yl-ethyl)-benzamide 12c (281 mg (m, 2h, h-8 ), 3.56 (t, 4h, h-2 , j = 4.5 hz), 6.96 (td, 1h, h-5 or h-6 ci/mmol; code net869) ci/mmol; net856), membrane from clonal cell line hek-293 that overexpresses sert (code: rbhstm400ua), and membrane from cho-k1 clonal cell line that overexpresses d 2 receptor (code: rbhd2cm400ua) were purchased from perkin-elmer depression fact sheet depression, mania and self-reported creativity in bipolar disorder characterizing neurocognitive markers of familial risk for depression 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potential acetylcholinesterase inhibitors based on a benzoxazine core x-ray structures and mechanism of the human serotonin transporter structural basis for action by diverse antidepressants on biogenic amine transporters structure of the d2 dopamine receptor bound to the atypical antipsychotic drug risperidone monoamine oxidase inhibitory properties of some methoxylated and alkylthio amphetamine derivatives: structure-activity relationships pharmacological profile of antidepressants and related compounds at human monoamine transporters autodock4 and autodocktools4: automated docking with selective receptor flexibility python: a programming language for software integration and development vmd: visual molecular dynamics 1.2; tripos international three-dimensional quantitative structure-activity relationships (3d-qsar) on a series of piperazine-carboxamides fatty acid amide hydrolase (faah) inhibitors as a useful tool for the design of new cannabinoid ligands molecular similarity indices in a comparative analysis (comsia) of drug molecules to correlate and predict their biological activity validation of the general purpose tripos 5.2 force field beware of q2! best practices for qsar model development, validation, and exploitation some case studies on application of "r(m)2" metrics for judging quality of quantitative structure-activity relationship predictions: emphasis on scaling of response data polypharmacology of dopamine receptor ligands multitarget opioid ligands in pain relief: new players in an old game this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest. key: cord-260014-q5sug7uu authors: szűcs, zsolt; naesens, lieve; stevaert, annelies; ostorházi, eszter; batta, gyula; herczegh, pál; borbás, anikó title: reprogramming of the antibacterial drug vancomycin results in potent antiviral agents devoid of antibacterial activity date: 2020-06-29 journal: pharmaceuticals (basel) doi: 10.3390/ph13070139 sha: doc_id: 260014 cord_uid: q5sug7uu influenza a and b viruses are a global threat to human health and increasing resistance to the existing antiviral drugs necessitates new concepts to expand the therapeutic options. glycopeptide derivatives have emerged as a promising new class of antiviral agents. to avoid potential antibiotic resistance, these antiviral glycopeptides are preferably devoid of antibiotic activity. we prepared six vancomycin aglycone hexapeptide derivatives with the aim of obtaining compounds having anti-influenza virus but no antibacterial activity. two of them exerted strong and selective inhibition of influenza a and b virus replication, while antibacterial activity was successfully eliminated by removing the critical n-terminal moiety. in addition, these two molecules offered protection against several other viruses, such as herpes simplex virus, yellow fever virus, zika virus, and human coronavirus, classifying these glycopeptides as broad antiviral molecules with a favorable therapeutic index. seasonal infections by influenza a and b viruses are each year responsible for significant morbidity and mortality [1] . besides, zoonotic influenza a viruses occasionally enter the human population to cause serious pandemics with a high number of fatalities [2] . antiviral drugs are essential for influenza treatment and prevention, including in the context of pandemic preparedness. at the moment, four drug classes are available: the m2 ion channel blockers and neuraminidase inhibitors, approved in all countries [3] , and two polymerase inhibitors, recently approved in a few countries [4, 5] . for each of these drugs, emergence of resistant mutants is possible; this is particularly problematic when the mutant viruses are fit and human-to-human transmissible [6] [7] [8] [9] . hence, additional drug classes with a distinct mechanism of action remain essential [10] [11] [12] . several recent investigations demonstrated that some antibiotics from bacterial origin display interesting antiviral properties [13] representing a unique example of drug repurposing [14] . to synthesize the first variant, we used literature procedures to prepare vancomycin aglycone (1) and its hexapeptide derivative (2) by the edman degradation (scheme 1) [35, 36] . then, by the copper-catalyzed diazotransfer reaction, the n-terminal azido derivative (3) was successfully prepared in analogy to our previous work [24, 30] . pharmaceuticals 2020, 13, 139 3 of 15 to synthesize the first variant, we used literature procedures to prepare vancomycin aglycone (1) and its hexapeptide derivative (2) by the edman degradation (scheme 1) [35, 36] . then, by the copper-catalyzed diazotransfer reaction, the n-terminal azido derivative (3) was successfully prepared in analogy to our previous work [24, 30] . the subsequent copper-catalyzed azide-alkyne click reaction using alkyne compound 4 (scheme 2) yielded triazole derivative 6. the second compound modified on the n-terminus (7) was prepared by the same method using the already described maleimide derivative 5 [30] as the alkyne compound in the final step. the third derivative (8) in this group was synthesized using derivative 2 and hexanesulfonyl chloride by sulfonamide formation, similar to what we previously published for teicoplanin congeners [30] . the subsequent copper-catalyzed azide-alkyne click reaction using alkyne compound 4 (scheme 2) yielded triazole derivative 6. the second compound modified on the n-terminus (7) was prepared by the same method using the already described maleimide derivative 5 [30] as the alkyne compound in the final step. the third derivative (8) in this group was synthesized using derivative 2 and hexanesulfonyl chloride by sulfonamide formation, similar to what we previously published for teicoplanin congeners [30] . structures of previously prepared alkynes 4 and 5 used for the synthesis of derivatives 6 and 7. as for the c-terminal modifications, we decided to prepare two derivatives carrying a 1,2,3-triazole ring, since this moiety often generates bioactive analogues [37] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used 2-azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme 3). after preparation of the amines, the reaction of compound 2 with amine 9 using the pybop reagent gave amide derivative 11 (scheme 4). using the same conditions, the reaction between compound 2 and maleimide 10 as the alkyne compound yielded compound 12. in the case of the cterminal sulfonamide derivative 13 we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound 2 using pybop yielded compound 13. structures of previously prepared alkynes 4 and 5 used for the synthesis of derivatives 6 and 7. as for the c-terminal modifications, we decided to prepare two derivatives carrying a 1,2,3-triazole ring, since this moiety often generates bioactive analogues [37] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used 2-azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme 3). as for the c-terminal modifications, we decided to prepare two derivatives carrying a 1,2,3-triazole ring, since this moiety often generates bioactive analogues [37] . in order to minimize the structural difference between the side chain in the n-versus c-terminal position, we used 2-azidoethylamine as a small linker for the synthesis of the appropriate c-terminal analogues (scheme 3). after preparation of the amines, the reaction of compound 2 with amine 9 using the pybop reagent gave amide derivative 11 (scheme 4). using the same conditions, the reaction between compound 2 and maleimide 10 as the alkyne compound yielded compound 12. in the case of the cterminal sulfonamide derivative 13 we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound 2 using pybop yielded compound 13. scheme 3. synthesis of amines 9-10 for the c-terminal modifications. after preparation of the amines, the reaction of compound 2 with amine 9 using the pybop reagent gave amide derivative 11 (scheme 4). using the same conditions, the reaction between compound 2 and maleimide 10 as the alkyne compound yielded compound 12. in the case of the c-terminal sulfonamide derivative 13 we also used the similar, small linker moiety. in this approach, an amine functionalized sulfonamide was first synthesized from n-hexanesulfonyl chloride and ethylenediamine, then the peptide coupling reaction between this compound and compound 2 using pybop yielded compound 13. modified on the c-terminus. antibacterial tests were carried out by the broth microdilution method on a panel of eight grampositive bacterial strains, using vancomycin and teicoplanin as reference compounds. neither of the new compounds exhibited significant activity against any bacterium, proving successful elimination of antibacterial activity, as anticipated (table 1) . antibacterial tests were carried out by the broth microdilution method on a panel of eight gram-positive bacterial strains, using vancomycin and teicoplanin as reference compounds. neither of the new compounds exhibited significant activity against any bacterium, proving successful elimination of antibacterial activity, as anticipated (table 1) . with regard to antiviral activity, the two n-terminal triazole derivatives 6 and 7 displayed robust activity against the three influenza a or b viruses tested. upon microscopic inspection, no virus-induced cytopathic effect (cpe) was observed in virus-infected cells treated with 6.25 µm of compound 6 or compound 7 ( figure 1a ). the quantitative antiviral efficacy (ec 50 ) and cytotoxicity (cc 50 ) values, both determined by the mts cell viability assay, are summarized in table 2 . with ec 50 values of~3 µm and a cc 50 value of 41 µm (compound 6) and 18 µm (compound 7), the molecules had a selectivity index (ratio of cc 50 to ec 50 ) of 14 and 6, respectively. both molecules exhibited clear inhibition of influenza virus replication, since they strongly reduced the virus yield in the supernatant ( figure 1b ), giving ec 99 values of 3.5 µm (compound 6) and 4.6 µm (compound 7), which is 5-to 6-fold lower than the ec 99 for ribavirin (23 µm) . at these concentrations, the compounds were devoid of cytotoxicity, as assessed by mts cell viability assay in mock-infected cells ( figure 1c ). the n-terminal n-hexanesulfonyl derivative 8 and c-terminally modified compound 11 proved inactive. for compound 8, this was somewhat surprising since the analogous teicoplanin pseudoaglycone derivative showed high activity [30] . on the other hand, this result is in line with our previous findings on ristocetin and teicoplanin aglycone derivatives, indicating that even minor structural differences in the peptide core can lead to significantly different anti-influenza virus activity [27] . the c-terminally modified compounds 12 and 13 were only slightly active against one or both influenza a virus strains. compared to compounds 6 and 7, compound 13 displayed an 8-fold higher antiviral ec 50 value by mts assay ( table 2 ); its lower potency was also evident in the virus yield reduction assay ( figure 1b ). this points to the importance of the modification site, since the c-terminally modified compounds were clearly inferior to the n-terminal analogues. pharmaceuticals 2020, 13, 139 6 of 15 with regard to antiviral activity, the two n-terminal triazole derivatives 6 and 7 displayed robust activity against the three influenza a or b viruses tested. upon microscopic inspection, no virusinduced cytopathic effect (cpe) was observed in virus-infected cells treated with 6.25 µm of compound 6 or compound 7 ( figure 1a ). the quantitative antiviral efficacy (ec50) and cytotoxicity (cc50) values, both determined by the mts cell viability assay, are summarized in table 2 . with ec50 values of ~3 µm and a cc50 value of 41 µm (compound 6) and 18 µm (compound 7), the molecules had a selectivity index (ratio of cc50 to ec50) of 14 and 6, respectively. both molecules exhibited clear inhibition of influenza virus replication, since they strongly reduced the virus yield in the supernatant ( figure 1b) , giving ec99 values of 3.5 µm (compound 6) and 4.6 µm (compound 7), which is 5-to 6fold lower than the ec99 for ribavirin (23 µm) . at these concentrations, the compounds were devoid of cytotoxicity, as assessed by mts cell viability assay in mock-infected cells ( figure 1c ). the nterminal n-hexanesulfonyl derivative 8 and c-terminally modified compound 11 proved inactive. for compound 8, this was somewhat surprising since the analogous teicoplanin pseudoaglycone derivative showed high activity [30] . on the other hand, this result is in line with our previous findings on ristocetin and teicoplanin aglycone derivatives, indicating that even minor structural differences in the peptide core can lead to significantly different anti-influenza virus activity [27] . the c-terminally modified compounds 12 and 13 were only slightly active against one or both influenza a virus strains. compared to compounds 6 and 7, compound 13 displayed an 8-fold higher antiviral ec50 value by mts assay ( table 2 ); its lower potency was also evident in the virus yield reduction assay ( figure 1b ). this points to the importance of the modification site, since the c-terminally modified compounds were clearly inferior to the n-terminal analogues. encouraged by the promising anti-influenza virus activity of compounds 6 and 7, we tested the two compounds against a range of dna-and rna-viruses evaluated in human embryonic lung (hel) fibroblast, hela or vero cells. for each virus, appropriate reference compounds were included. protection against virus-induced cytopathicity as well as compound cytotoxicity were determined by the mts cell viability assay. as shown in table 3 , the two compounds exhibited broad protection against a large variety of viruses, including herpesvirus types 1 and 2 and vaccinia virus. they retained full effectivity against a thymidine kinase deficient form of hsv-1, which was 61-fold (acyclovir) and 89-fold (ganciclovir) resistant to antiherpetic drugs. moreover, the compounds proved effective against two emerging pathogens for which no therapy is currently approved, i.e., coronavirus (inhibited by compounds 6 and 7) and zika virus (inhibited by compound 7) . at a non-toxic concentration of 25 µm of compound 6, no coronavirus 229e-induced cytopathicity could be observed microscopically (figure 2a ), which agrees with an ec 50 value of 11 µm as determined by mts cell viability assay (table 3 ). in addition, treatment of infected cells with 25 µm of compound 6 resulted in a 1000-fold reduction of the viral rna copy number in the supernatant, yielding an ec 99 value of 20 µm for reduction of virus yield ( figure 2b ). hence, we established, by virus yield assays, that compound 6 suppresses the replication of influenza virus and coronavirus, and for the other viruses, activity was indicated by the protection against viral cpe. this broad activity against distinct viruses fits with our hypothesis that these molecules may act by disrupting the viral endocytosis process, similarly to what we reported for a glycopeptide active against influenza virus [23] and what was described for ebola virus and mers and sars coronaviruses [15, 16] . this should become clear from mechanistic work ongoing in our laboratory. vancomycin hydrochloride was a gift from teva pharmaceutical industries ltd. (debrecen, hungary). vancomycin aglycone hexapeptide, trifluoromethanesulfonyl azide, compounds 4 and 5 were prepared as described elsewhere [24, 30] . tlc was performed on kieselgel 60 f254 (merck) with detection either by immersing into ammonium molybdate-sulfuric acid solution followed by heating vancomycin hydrochloride was a gift from teva pharmaceutical industries ltd. (debrecen, hungary). vancomycin aglycone hexapeptide, trifluoromethanesulfonyl azide, compounds 4 and 5 were prepared as described elsewhere [24, 30] . tlc was performed on kieselgel 60 f 254 (merck) with detection either by immersing into ammonium molybdate-sulfuric acid solution followed by heating or by using pauly's reagent for detection. flash column chromatography was performed using silica gel 60 (merck 0.040-0.063 mm) and silica gel 60 silanized (0.063-0.200 mm). the 1 h nmr (500mhz, 400 mhz) 13 c nmr (125 mhz, 100 mhz) and 2d nmr spectra were recorded with a bruker drx-400 and bruker avance ii 500 spectrometers at 300k. chemical shifts are referenced to me 4 si and to the solvent residual signals. maldi-tof ms analysis of the compounds was carried out in the positive reflectron mode using a biflex iii mass spectrometer (bruker, bremen, germany) equipped with delayed-ion extraction. 2,5-dihydroxybenzoic acid (dhb) was used as matrix and cf 3 coona as cationizing agent in dmf. elemental analysis (c, h, n, s) was performed on an elementar vario microcube instrument. synthesis of azido vancomycin aglycone hexapeptide (3): 350 mg (0.34 mmol) vancomycin aglycone hexapeptide (2) was obtained from 750 mg (0.5 mmol) vancomycin hydrochloride (1) by edman degradation as described in the literature [35, 36] . sodium azide (65 mg, 1.0 mmol) was added to dry pyridine (1.5 ml) cooled to 0-5 • c. tf 2 o (0.8 mmol, 134 µl) was added dropwise over the course of about 30 min. the reaction mixture was stirred for another 2 h at 0-5 • c. then, 350 mg (0.34 mmol) of 2 was dissolved in 15 ml pyridine, then 95 µl (2.0 equiv., 0.68 mmol) et 3 n was added followed by the solution (1.5 ml) of the freshly prepared trifluoromethanesulfonyl azide, and finally 800 µl of a 10 mg/ml cuso 4 · 5h 2 o solution. the solution was stirred at room temperature overnight, then the solvents were evaporated. the crude product was dissolved in dilute nh 4 oh, then the ph was set to 1-2 with 1n hcl, the resulting cloudy mixture was extracted with n-buoh three times, the butanolic phase was washed with water, then evaporated and purified by flash column chromatography using step gradient elution (mecn:h 2 o = 100:0→97:3→94:6→92:8). the title compound was obtained in 250 mg yield (70%) as an off-white powder. nmr: see synthesis of compound 6: 132 mg (0.127 mmol) of 2 was dissolved in 2.0 ml of dmf:h 2 o 3:1 mixture. next, 72 mg (1.25 equiv., 0.16 mmol) of alkyne 4 was added, followed by 6 mg cuso 4 · 5h 2 o. the reaction mixture was stirred at room temperature for 12 h. by this time tlc (cellulose, n-proh: cc. nh 4 oh = 6:4) indicated good conversion. the solvents were evaporated until a syrupy residue was obtained. ether was added, and the product was filtered off after precipitation and washed with additional ether to remove the excess alkyne. purification was carried out by c 18 reverse phase column chromatography (h 2 o:mecn = 70:30→60:40→55:45) followed by gel chromatography using sephadex lh-20 in meoh. the title compound was obtained as a white powder in 69 mg yield (37%). nmr: see table s2 in supporting information. elemental analysis: see table s8 after 12 h stirring at room temperature, tlc indicated good conversion. the reaction mixture was worked up and purified by c 18 reverse phase column chromatography as described above. the title compound was obtained as a yellow powder in 52 mg yield (38%). nmr: see table s3 in supporting information. elemental analysis: see table s8 in supporting information. maldi-ms m/z calcd. for c 69 h 74 cl 2 n 10 o 22 s 2 + na + [m + na] + : 1551.369. found: 1551.367. synthesis of compound 8: 78 mg (0.077 mmol) of 2 was dissolved in 3 ml dry pyridine and 0.5 ml dry dmf, then 19 µl (1.5 equiv., 0.115 mmol) of n-hexanesulfonyl chloride was added. after stirring 3 h at room temperature, ethyl acetate and ether was added, the resulting precipitate was filtered and washed with ether. the crude product was purified by flash column chromatography using toluene: meoh = 7:3→6:4 as eluent, followed by gel chromatography using sephadex lh-20 with meoh: h 2 o = 6:4 as eluent. the yield was 20 mg (22%). nmr: see table s4 in supporting information. elemental analysis: see table s8 synthesis of 2-(4-(13-(4-((decyloxy)methyl)-1h-1,2,3-triazol-1-yl)-2,5,8,11-tetraoxatridecyl)-1h-1,2, 3-triazol-1-yl)ethanamine (9): 207 mg of 4 [24, 30] (0.46 mmol) and 39 mg (0.46 mmol) of 2-azidoethylamine were dissolved in 2 ml dry dmf under argon. 70 µl (1.08 equiv., 0.5 mmol) et 3 n was added, then 17 mg (20 mol%, 0.09 mmol) cu(i)i. the reaction mixture was stirred at room temperature for an hour. after evaporation of the solvents, the crude product was purified by flash column chromatography using dcm: meoh = 95:5 (+0.1% v/v nh 4 oh) as eluent. the title compound was obtained as an off white solid in 68% yield (171 mg). addition of ethyl acetate, filtered out and washed with diethyl ether. the product was purified by flash column chromatography using step gradient elution (toluene: meoh = 7:3→1:1) then by gel column chromatography (sephadex lh-20, acetone:h 2 o = 1:1). the title compound was obtained as an off-white powder in 36 mg yield (34%). nmr: see table s7 in supporting information. elemental analysis: see table s8 in supporting information. maldi-ms m/z calcd. for c 54 h 57 cl 2 n 9 o 17 s + na + [m + na] + : 1228.29. found: 1228.27. for antiviral testing, 25 mm compound stocks were prepared in 100% dmso and stored at 4 • c. the compounds were fully soluble under these conditions. in the antiviral tests, the highest concentration tested was 100 µm, corresponding to a non-toxic dmso content of 0.4%. the virus strains (a/h1n1: a/ned/378/05 and a/pr/8/34; a/h3n2: a/victoria/361/11; and b/ned/537/05) were propagated in embryonated hen eggs. the antiviral procedure was published elsewhere [38, 39] . madin-darby canine kidney (mdck) cells were seeded at 7500 cells per well into 96-well plates, using infection medium (ultramdck medium (lonza) with 225 mg/l sodium bicarbonate, 2 mm l-glutamine, and 2 µg/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-treated trypsin). one day later, virus (moi: 0.001) was added together with 1:5 serial compound dilutions, to reach a total volume of 200 µl per well. besides the test compounds, two references were included, i.e., zanamivir and ribavirin (positive controls) plus a condition receiving medium instead of compound (negative control). in parallel, the compound dilutions were also added to a mock-infected plate (in which medium was added instead of virus), to determine compound cytotoxicity. each plate contained two wells in which all reagents yet no cells were added, to serve as blanks in the mts calculations. after three days incubation at 35 • c, viral cpe was first monitored by microscopy. then, the supernatants were replaced by mts reagent (celltiter 96 ® aq ueous mts reagent from promega) diluted 1:10 in pbs, and 4 h later, absorbance at 490 nm was measured in a plate reader. to monitor the inhibitory effect of the compounds on virus replication, mdck cells were seeded, infected (with a/pr/8/34 virus; moi: 0.001) and treated with 1:2 serial compound dilutions. the plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). at day 3 p.i., supernatants were collected and frozen at -80 • c, to quantify the virus yield by one-step qrt-pcr analysis of viral copy number [40] . two µl supernatant was mixed with 10 µl resuspension buffer and 1 µl lysis reagent (cellsdirect one-step rt-qpcr kit; invitrogen) and heated during 10 min at 75 • c. next, 10 µl lysate was transferred to a qpcr plate containing the qrt-pcr enzymes and buffer (cellsdirect one-step rt-qpcr kit; invitrogen), and influenza virus m1-specific primers and probe [40] . the program consisted of: 15 min at 50 • c; 2 min at 95 • c; and 45 cycles of 15 s at 95 • c followed by 90 s at 60 • c. absolute quantification of vrna copies was performed by including an m1-plasmid standard. the ec 99 values were calculated by interpolation and defined as the compound concentration causing 100-fold reduction in vrna copy number, as compared to the virus control receiving no compound. it was ascertained that the cell controls showed no detectable qpcr signal. the viruses were propagated and evaluated in the following cell lines: human embryonic lung (hel) fibroblast cells, used for human coronavirus 229e [41] , herpes simplex virus type 1 (hsv-1 strain kos, including a thymidine kinase deficient hsv-1/tkmutant), herpes simplex virus type 2 (hsv-2, strain g) and vaccinia virus (strain lederle); human cervixcarcinoma hela cells, used for respiratory syncytial virus (rsv, strain long), and african green monkey kidney vero cells, used for yellow fever virus (vaccine strain 17d) and zika virus (strain mr766). the medium used for virus infection was dulbecco's modified eagle's medium supplemented with 2% fetal calf serum. to prepare virus stocks, confluent cell cultures in 75-cm 2 flasks were infected with the virus and frozen after 3 to 5 days incubation at 37 • c (or 35 • c in case of human coronavirus 229e), when full-blown cpe was visible. after freeze-thawing and centrifugation, the clarified lysates were stored at −80 • c. for the antiviral experiments, the cells were grown in 96-well plates until confluent. virus was added (moi: 0.001) together with 1:5 serial dilutions of the compounds. for each virus, appropriate reference compounds were included. the compound dilutions were also added to a mock-infected plate, to determine compound cytotoxicity. when manifest cpe was reached, i.e., after 3 to 5 days incubation at 37 • c (or 35 • c in case of human coronavirus 229e), cpe and compound cytotoxicity were quantified by the mts assay, and ec 50 and cc 50 values were calculated as explained above for influenza virus. to assess inhibition of human coronavirus 229e replication, hel cells were infected and treated with 1:2 serial compound dilutions. the plate contained three virus controls (receiving no compound) and two cell controls (receiving no virus and no compound). at day 4 p.i., supernatants were frozen at −80 • c to determine the virus yield by one-step rt-qpcr assay. two microliters supernatant was mixed with 11 µl lysis mix containing lysis enhancer and resuspension buffer at a 1:10 ratio (cellsdirect one-step rt-qpcr kit; invitrogen), and heated for 10 min at 75 • c. five microliters of lysate was transferred to a pcr plate containing 9.75 µl rt-qpcr mix (cellsdirect one-step rt-qpcr) and 0.25 µl superscript iii rt/platinum taq enzyme, and coronavirus-229e n-gene specific primers and probe (forward primer 5 -ttagagagcgtgttgaaggtg-3 ; reverse primer 5 -gttctgaattcttgcgcctaac-3 ; probe 5 -fam-tctgggttg-zen-ctgttgatggtgcta-ibfq-3 ). the rt-qpcr program consisted of 15 min at 50 • c, 2 min at 95 • c, and 50 cycles of 15 s at 95 • c and 45 s at 60 • c. an n-gene plasmid standard was included for absolute quantification. compound activity was expressed as the ec 99 value, i.e., the concentration causing 100-fold reduction in vrna copy number, as compared to the virus control receiving no compound. it was ascertained that the cell controls showed no detectable qpcr signal. starting from vancomycin, we have successfully prepared two derivatives with strong activity against influenza virus. the modifications that we introduced were based on our previous work on the glycopeptide antibiotic teicoplanin. interestingly, some of these modifications yielded compounds 6 and 7 having the same antiviral potency as the analogous teicoplanin pseudoaglycon derivatives [24, 30] but lacking antibacterial activity. the short work described here validates that the glycopeptide scaffold is an underexplored entity to conceive new antivirals with a broad activity spectrum, that besides influenza virus includes emerging pathogens like coronavirus and zika virus. supplementary materials: the following are available online at http://www.mdpi.com/1424-8247/13/7/139/s1, table s1 : nmr data for compound 3, table s2 : nmr data for compound 6, table s3 : nmr data for compound 7, table s4 : nmr data for compound 8, table s5 : nmr data for compound 11, table s6 : nmr data for compound 12, table s7 : nmr data for compound 13, table s8 mg (0.43 mmol) of compound 5 [30] and 44 mg (1.2 equiv., 0.52 mmol) of azidoethylamine were dissolved in 2 ml dry dmf under argon. 66 µl (1.1 equiv., 0.47 mmol) et 3 n was added followed by 16 mg (20 mol%) cu(i)i. the reaction mixture was stirred for 2 h at room temperature x ch 2 ), 32.32, 31.36, 21.48 (6c, 6 × ch 2 ), 13.43 (2c, 2 × ch 3 ). maldi-ms m/z calcd. for c 25 h 43 n 5 o 6 s 2 + na + [m + na] + : 596.25. found: 596.238. synthesis of compound 11: vancomycin aglycone hexapeptide 2 (90 mg, 0.089 mmol) was dissolved in 1.0 ml dry dmf. 95 mg (2.0 equiv., 0.177 mmol) of compound 9 was added followed by 25 µl (2.0 equiv., 0.177 mmol) et 3 n and 55 mg (1.2 equiv., 0.107 mmol) of pybop. the reaction mixture was stirred for 2 h at room temperature, after which tlc (n-proh:nh 4 oh = 7:3, cellulose) indicated complete conversion. the product was precipitated by the addition of 100 ml of cold etoac:et 2 o = 1:1 mixture, filtered off and washed thoroughly with diethyl ether. the crude product was purified by gel column chromatography using sephadex lh-20 in mecn:h 2 o = 8:2 mixture, followed by flash column chromatography in mecn:h 2 o = 9:1 mixture. the product was obtained as a white powder in 52 mg yield (38%) 18 mmol) was added followed by 70 mg n-(2-aminoethyl)hexane-1-sulfonamide (~4 equiv., 0.34 mmol) obtained in step 1 risk factors for serious outcomes associated with influenza illness in high-versus low-and middle-income countries: systematic literature review and meta-analysis influenza a virus transmission via respiratory aerosols or droplets as it relates to pandemic 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drugs to fight against a long-lived enemy teicoplanin inhibits ebola pseudovirus infection in cell culture glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of hiv entry inhibition of feline (fipv) and human (sars) coronavirus by semisynthetic derivatives of glycopeptide antibiotics inhibition of hepatitis c virus replication by semi-synthetic derivatives of glycopeptide antibiotics an analogue of the antibiotic teicoplanin prevents flavivirus entry in vitro anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study synthesis of fluorescent ristocetin aglycone derivatives with remarkable antibacterial and antiviral activities synthesis of isoindole and benzoisoindole derivatives of teicoplanin pseudoaglycone with remarkable antibacterial and antiviral activities a few atoms make the difference: synthetic, cd, nmr and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycone derivatives semisynthetic teicoplanin derivatives as new influenza virus binding inhibitors: synthesis and antiviral studies synthesis and biological evaluation of lipophilic teicoplanin pseudoaglycone derivatives containing a substituted triazole function structure-activity relationship studies of lipophilic teicoplanin pseudoaglycon derivatives as new anti-influenza virus agents structure-activity relationship studies of a series of antiviral and antibacterial aglycon derivatives of the glycopeptide antibiotics vancomycin, eremomycin, and dechloroeremomycin structural modifications of the active site in teicoplanin and related glycopeptides. 1. reductive hydrolysis of the 1,2-and 2,3-peptide bonds teicoplanin, antibiotics from actinoplanes teichomyceticus nov. sp. viii. opening of the polypeptide chain of teicoplanin aglycone under hydrolytic conditions structural modifications of the active site in teicoplanin and related glycopeptides. 2. deglucoteicoplanin-derived tetrapeptide the edman degradation of vancomycin -preparation of vancomycin hexapeptide synthesis and evaluation of vancomycin aglycon analogues that bear modifications in the n-terminal d-leucyl amino acid click chemistry: 1,2,3-triazoles as pharmacophores superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones influenza virus entry via the gm3 ganglioside-mediated platelet-derived growth factor receptor beta signalling pathway mutational analysis of the binding pockets of the diketo acid inhibitor l-742,001 in the influenza virus pa endonuclease synthesis and anti-coronavirus activity of a series of 1-thia-4-azaspiro we acknowledge kind assistance of l. persoons and her team, and of ria van berwaer and julie vandeput. the authors declare no conflict of interest. key: cord-012773-wtgk2d68 authors: xu, ming-ming; ryan, philip; rudrawar, santosh; quinn, ronald j; zhang, hai-yan; mellick, george d title: advances in the development of imaging probes and aggregation inhibitors for alpha-synuclein date: 2019-10-04 journal: acta pharmacol sin doi: 10.1038/s41401-019-0304-y sha: doc_id: 12773 cord_uid: wtgk2d68 abnormal protein aggregation has been linked to many neurodegenerative diseases, including parkinson’s disease (pd). the main pathological hallmark of pd is the formation of lewy bodies (lbs) and lewy neurites, both of which contain the presynaptic protein alpha-synuclein (α-syn). under normal conditions, native α-syn exists in a soluble unfolded state but undergoes misfolding and aggregation into toxic aggregates under pathological conditions. toxic α-syn species, especially oligomers, can cause oxidative stress, membrane penetration, synaptic and mitochondrial dysfunction, as well as other damage, leading to neuronal death and eventually neurodegeneration. early diagnosis and treatments targeting pd pathogenesis are urgently needed. given its critical role in pd, α-syn is an attractive target for the development of both diagnostic tools and effective therapeutics. this review summarizes the progress toward discovering imaging probes and aggregation inhibitors for α-syn. relevant strategies and techniques in the discovery of α-syn-targeted drugs are also discussed. parkinson's disease (pd) is a progressive neurodegenerative disorder that causes severe motor deficits. common symptoms include rigidity, bradykinesia, tremor and postural instability [1] . with disease progression, nonmotor symptoms, such as depression, psychosis, falls, and sleep disturbance, also emerge [2] . globally, 1.5% of the population over 65 years of age [3] and more than 5 million people [4] are affected by this devastating disease. pd is pathologically characterized by the substantial loss of dopamine (da)-containing neurons in the midbrain [5] and the presence of intraneuronal cytoplasmic inclusions [6] , known as lewy bodies and lewy neurites [7] , both of which comprise alphasynuclein (α-syn) aggregates. a definitive diagnosis of pd has to rely on histopathological postmortem analysis and requires the detection of dopaminergic cell loss and the presence of lewy bodies and lewy neurites. for living patients, several clinical diagnostic criteria have been formulated, including the uk parkinson's disease society brain bank (ukpdsbb) [8] , the national institute of neurological disorders and stroke (ninds) criteria [9] and the movement disorder society clinical diagnostic criteria for pd [10] . however, the diagnostic accuracy obtained by using these criteria is only between 75% and 85% [11, 12] . compared with the observation of clinical symptoms, neuroimaging may help to increase diagnostic precision. clinically available imaging approaches include positron emission tomography (pet), single-photon emission computed tomography (spect) and magnetic resonance imaging (mri). pet and spect use a variety of radiotracers to quantitatively measure the metabolic and neurochemical changes in the brains. for example, fluorine-18-l-dihydroxyphenylalanine ( 18 f-dopa) pet can mark dopaminergic deficiencies in the brains of patients with pd [13] . mri uses different sequences and contrasts to study brain structure and function [14] . although substantial progress has been made in pd neuroimaging, currently, it can only be used as a supplementary tool to clinical examination [15] . increasing evidence suggests that prior to the motor phase of classical pd there is a prodromal period that lasts for several years [16] . after the appearance of typical motor features, the disease can continue to progress for many years or even decades, although it is impossible to predict the trajectory of this progression at diagnosis [17] . the pathological changes in the central nervous system during the prodromal phase, such as the formation of α-syn aggregates, appear to mirror the occurrence of motor and nonmotor symptoms. a timeline of pd from onset to death has been proposed [17] , which not only promotes a comprehensive understanding of the overall disease process but is also a reminder that before a patient shows any pd-related clinical symptoms, the disease may have progressed severely and possibly irreversibly as a result of neuronal dysfunction and cell loss [18, 19] . in the future, the traditional symptom-based clinical examination may be pre-empted by the use of biomarkers that may assist in identifying early disease-specific changes by early prodromal diagnosis, namely detecting specific changes in biomarkers during the early development of pd [20] . since diagnosis informs treatment, novel therapeutics targeting the potential pathological culprits need to be developed. until now, no neuroprotective or neurorestorative therapy has been found for the treatment of pd. existing treatments predominantly target dopamine-related symptoms. few, if any, target nondopaminergic symptoms, which cause a severe burden for patients in the advanced stages of the disease. in addition, current pd treatments provide little or no relief for disease progression because they do not alter the rate or extent of neuronal cell loss [21, 22] . therefore, for the future direction of pd medications, the priority is the development of neuroprotective or neurorestorative drugs that can stop or at least relieve disease progression and the relevant nonmotor symptoms [23] . to realize this goal, it is necessary to identify and target the key culprit underlying the pathogenesis of pd. native α-syn is a small protein encoded by the gene snca, with a molecular mass of 14.5 kda (140 amino acids) [24] . its primary structure ( fig. 1) shows three different regions: the n-terminal region (residues 1−60) that is characterized by repetitions of a highly conserved lysine-rich motif ktk(e/q)gv, the hydrophobic central part (residues 61−95, known as the non-amyloidogenic component (nac) region), and the acidic c-terminal region (residues 96−140) . the amphipathic n-terminal region has a structural alpha-helix propensity similar to apolipoprotein-binding domains, suggesting that α-syn is a membrane-bound protein [25] . the nac region is believed to be involved in protein aggregation by mediating the conformational changes of α-syn from random coil to a beta-sheet structure [24] . the c-terminal region, having no distinct secondary structure, has been reported to interact with the n-terminal region or the nac region to maintain the natively unfolded state of α-syn [26] . although it is widely believed that α-syn exists mainly as monomer, studies have also shown that endogenous α-syn can form tetramers [27, 28] , which comprise an alpha-helical secondary structure and show little tendency to form aggregates [29] . all these findings suggest that there may be a dynamic equilibrium between different α-syn conformational states [30] . the exact normal functions of α-syn are not well understood. although some animal studies suggest that an α-syn-knockout is not fatal, deficiencies in synaptic transmissions were observed in some knockout animal lines [31, 32] . in neurons α-syn is mainly localized at the presynaptic terminals [33, 34] and is associated with the reserve pool of synaptic vesicles [35] [36] [37] . α-syn can promote the assembly of the presynaptic soluble n-ethylmaleimide-sensitive factor activating protein receptor (snare) protein complex, that mediates vesicle fusion, which is a vital step for the release of neurotransmitters, including dopamine [38] . in addition to its role in synaptic transmission, α-syn is also involved in the suppression of apoptosis [39] , antioxidation [40] and even regulation of dopamine biosynthesis [41, 42] . the first connection between α-syn and pd was made in 1997 when point mutations in the snca gene were identified in familial pd cases [43] . to date, six missense mutations in snca are known to lead to autosomal dominant pd: a53t [43] , a30p [44] , e46k [45] , h50q [46] , g51d [47] , and a53e [48] . meanwhile, the link between α-syn and pd was further elucidated with the discovery of duplications and triplications of the snca gene in pd families, which also suggested that increased expression of α-syn was a causative factor of pd [49, 50] . moreover, certain polymorphisms in snca, such as the dinucleotide repeat rep1 located in the snca promoter (snca-rep1), a −770 and −116 base-pair (bp) singlenucleotide polymorphism, are major risk factors for sporadic pd, and are thought to result from altered expression of the gene product [51, 52] . genome-wide association studies have also consistently revealed highly significant regions of genetic variation around the snca gene as contributors to the risk of pd. a meta-analysis of genome-wide association studies has identified 17 new pd risk loci [53] . as mentioned earlier, the nac region of monomeric α-syn mediates the misfolding from a random coil to a beta-sheet structure, leading to the formation of oligomers, protofibrils and eventually mature fibrils [54] . this pathway is also shared by other amyloid-forming proteins such as beta-amyloid (aβ), tau and human islet amyloid polypeptide (hiapp). the fluorescent dye thioflavin-t (tht) is a widely used "gold standard" for staining and identifying fibrils of these amyloid proteins [55] . kinetics studies of α-syn aggregation using tht [56, 57] revealed a sigmoidal curve of fibril growth, which is composed of the lag phase representing the nucleation stage, an exponential phase representing the elongation stage and a plateau phase corresponding to the completion of fibril formation [58] . it is natural to deduce that α-syn can cause toxicity to neurons, as cell death is a major hallmark of pd and α-syn plays a causal role in the disease. given that all known clinical mutations are thought to be linked to increased α-syn aggregation and that the main components of lewy bodies and lewy neurites are aggregated α-syn fibrils, it was initially believed that α-syn fibrils were the toxic species. later, increasing evidence from both in vitro and in vivo studies has supported the proposal that oligomeric species are the most pathogenically relevant [59] [60] [61] [62] [63] . as a result, the oligomers may be a better therapeutic target, as the aggregated lewy bodies themselves might be protective and represent a form of an aggresome [64, 65] . α-syn oligomers are believed to exert toxicity both intracellularly and extracellularly. within the cytoplasm, α-syn oligomers can inhibit the hsp 70 chaperone system which is important for protein refolding [66] . α-syn oligomers can also bind to and inhibit the activities of proteasomes [67] , thus affecting protein degradation. consequently, proteostasis is impaired, leading to endoplasmic reticulum (er) stress and finally cell death. in vivo, α-syn oligomers have been detected within the er lumen, and treatment with an inhibitor of er stress, dramatically delayed the onset of motoric symptoms and decreased the accumulation of α-syn oligomers [68] , suggesting that er stress is involved in the toxicity of α-syn oligomers. in addition, α-syn plays an important role in the assembly of the presynaptic snare protein complex. the formation of oligomeric α-syn can inhibit snare-mediated vesicle docking and consequently reduce exocytosis, suggesting that inhibition of dopamine release is a potential mechanism in pd [63] . α-syn oligomers can also induce the dysfunction of synapses by compromising the axonal transport of critical presynaptic proteins [69] . other potential intracellular targets of α-syn oligomers include mitochondria [70] , lysosomes [71] and microtubules [72] . extracellular sources of α-syn oligomers can form pores in cell membranes, causing an increase in intracellular calcium levels that leads to cell death [59] . exposure of neurons to exogenous α-syn oligomers can activate glutamatergic receptors, resulting in longterm potentiation (ltp) and excitotoxicity that leads to neuronal death [73, 74] . moreover, there is significant evidence that α-syn oligomers can transfer from neuron to neuron or to glial cells via a prion-like process [75, 76] , which may explain the spread of lewy pathology throughout the brain in pd [77] . overall, the accumulation of α-syn aggregates is a major pathological hallmark of pd and a priority target for drug development given its hypothesized contribution to neurodegeneration. in vivo imaging of α-syn pathology could be useful as a biomarker of the presence of the disease, disease progression and as a pharmacodynamic tool for drug development. therefore, αsyn imaging is a critical need for pd research [78] . moreover, since α-syn aggregation is regarded as a major pathogenic process in pd, several strategies exist for the prevention of α-syn toxicity [79] . among them, inhibition of α-syn aggregation remains an extremely attractive target for drug development [30] . the representative progress in the development of imaging probes and aggregation inhibitors over the past decade will be discussed further. although several pet/spect tracers targeting the dopamine system have been used clinically, as reviewed by politis [15] , braak staging of the pd brain suggests that the severe loss of dopaminergic neurons occurs at stage 4, while lewy body pathology appears as early as stage 1. as such, imaging α-syn pathology, rather than dopaminergic changes, would be more suitable for early diagnosis in the prodromal period of pd. while great progress has been made toward developing imaging probes for other amyloid-forming proteins [80] (and notably three aβ imaging probes have gained fda approval), the development of α-syn imaging probes is still at an early stage. despite the fact that there are no selective α-syn pet or spect probes for clinical pd diagnosis, several α-syn imaging probes have been developed and tested over the past decade (fig. 2 , table 1 ). amyloid proteins, such as aβ and α-syn, tend to form similar beta-sheet structures upon aggregation. as a result, probes that can interact with aβ aggregates also have the potential to bind to aggregated α-syn. following this rationale, some established aβ pet probes have been evaluated against α-syn. one well-known example is 11 c-pittsburgh compound-b (pib, 1), a derivative of thioflavin-t (tht) which is the gold standard for staining all types of amyloid proteins. probe 1 exhibited a similar binding affinity (k d = 4 nm) for in vitro generated α-syn fibrils compared to aβ [81] , but failed to bind to brain homogenates containing lewy bodies [82] . additionally, probe 1 displayed poor selectivity for α-syn in the staining of brain sections where aβ was also present [82] . benzoxazole bf227 is another established aβ-binding probe. 18 f-bf227 (2) was proven to bind to α-syn fibrils at an equimolar concentration compared to aβ 1-42 fibrils, but the binding affinity for α-syn fibrils (k d = 9.63 nm) was approximately sevenfold lower [83] . however, like 11 c-pib, probe 2 also failed to bind to brain homogenates containing lewy bodies [83] . a follow-up in vivo study using an accelerated mouse model of synucleinopathy failed to observe a significant difference in the brains of α-syn transgenic mice compared with α-syn-ko mice after treatment with probe 2 [84] . these results suggest that it is necessary to develop specific α-syn pet probes. to discover selective ligands for α-syn aggregates, yu et al. synthesized a series of phenothiazine derivatives and evaluated their binding affinities for α-syn fibrils with a tht competition assay. compounds 11b, 16a and 16b exhibited k i values of less than 60 nm (32.10 nm for 11b, 48.96 nm for 16a and 57.94 nm for 16b) and were considered for further study [85] . later, 125 i labeled 16b (3), denoted 125 i-sil23, was synthesized and tested by the same research group. probe 3 can bind to recombinant α-syn fibrils and brain homogenates from pd patients and α-syn transgenic mice. probe 3 exhibited a relatively high binding affinity for α-syn fibrils compared with aβ 1−42 fibrils (fivefold lower than α-syn) and tau fibrils (twofold lower than α-syn) but the selectivity was not high enough for in vivo imaging [86] . using a [87] . in addition, 11 c-2a (4) and 18 f-2b (5) were synthesized and proven to cross the blood−brain barrier (bbb) in sprague−dawley (sd) rats. both radiotracers exhibited high initial uptake (0.953% id/g for 4, 0.758% id/g for 5), homogeneous distribution and rapid washout kinetics. the authors believed that these two radiotracers could be good leads for the discovery of selective imaging probes for α-syn [87] . identifying new chemical scaffolds is necessary for developing specific radiotracers with high selectivity for α-syn fibrils versus other amyloid proteins such as aβ and tau. recently, a series of compounds based on the 3-(benzylidene)-indolin-2-one scaffold were synthesized and the most potent compound was 46a, having a high affinity (k i = 2 nm) and very good selectivity for α-syn versus aβ (k i = 142.4 nm) and tau fibrils (k i = 80.1 nm). the binding affinity of 18 f-labeled 46a (6) was determined from a saturation binding assay. the k d of probe 6 for α-syn fibrils was 8.9 nm while the values for aβ and tau fibrils were 271 and 50 nm, respectively. however, probe 6 is not believed to be a suitable pet tracer for in vivo imaging due to poor druggability [88] . flavonoids are a common source for the discovery of inhibitors of aβ aggregation as well as α-syn. chalcone is a good example and ono et al. synthesized a series of chalcone derivatives in the hope of discovering new scaffolds for α-syn imaging. of all four synthesized compounds, idp-4 showed the most selective binding to α-syn aggregates (k d = 5.4 nm, k d = 16.24 nm for aβ). fluorescent staining of pd brain sections confirmed the affinity of idp-4 for lewy bodies. unfortunately, 125 i-idp-4 (7) displayed the lowest initial uptake of the four compounds synthesized (0.45% id/g) in normal mice, making it difficult to use for in vivo imaging [89] . more recently, the same group also synthesized three novel radioiodinated benzoimidazole (bi) derivatives based on the previous findings: (1) the benzoimidazole scaffold could bind to α-syn aggregates; (2) the diene moiety helps to increase the affinity for α-syn aggregates; and (3) introduction of large substituents increases the selectivity for α-syn. compound bi-2 showed the highest selectivity and binding affinity for α-syn aggregates (k d = 99.5 nm, k d = 727 nm for aβ). fluorescent staining of pd and ad brain sections using bi-2 was conducted and bi-2 clearly stained lewy bodies but failed to label aβ aggregates, further confirming its selectivity for α-syn. however, the promising compound 125 i-bi-2 (8) exhibited low initial uptake (0.56% id/g) and poor washout kinetics in normal mouse brains, suggesting that the introduction of bulky substitution groups may affect penetration into the bbb [90] . the authors also mentioned that criteria exist for ideal aβ and tau imaging probes according to previous reports [91, 92] : (1) an initial brain uptake up to 4% id/g at 2-min postinjection in mice; and (2) a remaining amount of less than 1% id/g at 30-min postinjection in normal mouse brains. clearly, none of the reported probes for α-syn met these criteria, including probe 8. in the past decade, significant progress toward discovering useful α-syn radiotracers has been made; however, an ideal, druggable probe possessing high affinity and selectivity for α-syn has yet to be identified [93] . fluorescence imaging presents a promising alternative technique to radiotracers. in contrast to pet/spect techniques, fluorescence imaging is monitored in real time, is inexpensive, is nonradioactive and is of high-resolution when using near-infrared fluorescence (nirf) imaging probes. as a technique, fluorescence imaging is progressively being explored for the neuroimaging of aβ aggregates [94] . the ideal fluorescent probe will not only possess high selectivity and binding affinity, penetrate rapidly into the bbb and be cleared quickly from normal brain regions, but will also have low background fluorescence (ideal emission wavelength of greater than 650 nm) in addition to producing a significant increase in fluorescence upon binding to target proteins. given that aggregated α-syn is far less abundant in the brain than aβ, αsyn fluorescent probes will require very high selectivity for α-syn over aβ and tau. since the majority of α-syn is found intracellularly, in addition to penetrating the bbb, such a probe has to cross the cell membrane [78] . tht is a widely used fluorescent dye for the nonselective staining of protein aggregates. its in vivo utility is limited due to its positive charge which affects its penetration through the bbb and its emission wavelength is not suitable for in vivo imaging. to search for improved fluorescent probes for α-syn, celej et al. evaluated the abilities of several n-arylaminonaphthalene sulfonate (nas) derivatives to be used as fluorescent markers for α-syn aggregates since the nas scaffold has a long history of being applied to study the molecular microenvironment and conformation of proteins. compounds 2,6-ans (fig. 3, 9) , 2,6-tns (10), bis-ans (11), and bis-tns (12) exhibited slightly improved binding affinity (k d values: 8.8, 11.7, 8.6 and 11.6 μm, respectively) for αsyn fibrils compared with tht (k d = 14.9 μm) [95] . although these molecules also displayed advantages over tht in terms of providing structural information during α-syn fibrillation, they are still not useful as imaging probes as a result of their charged properties and unideal emission wavelengths. in an endeavor to search for novel specific fluorescent probes for α-syn, volkova and coworkers studied the potential of a series of monomethine and trimethinecyanines to detect the formation of α-syn fibrils. the most potent dyes, t-284 (13) and sh-516 (14), had similar binding affinities to tht but showed large increases in fluorescence (~9.5-fold for 13 and~7.6-fold for 14) upon binding to α-syn fibrils relative to tht (2.9-fold). a notable improvement was that the emission wavelengths of probes 13 and 14 after binding to α-syn fibrils were determined to be 570 and 580 nm, respectively, compared with tht (478 nm) [96] . the specificity of these two dyes was not evaluated by assays against other amyloid proteins. later, carbocyanine compound jc-1 (15) was introduced to conduct real-time analyses of α-syn fibril formation. probe 15 could bind to α-syn monomers as well as fibrils. interestingly, the maximum emission wavelengths of probe 15 after binding to monomers and fibrils were different (590 nm for monomers, 538 nm for fibrils), indicating that probe 15 is able to distinguish between monomeric and fibrillar α-syn. furthermore, probe 15 did not interact with either monomeric or fibrillary aβ. the high selectivity of probe 15 might be explained by its interaction with the acidic c-terminal region of α-syn [97] . the results also suggested that promising selective probes for α-syn may be discovered by searching for compounds that can interact with the monomeric form of α-syn, since different amyloid proteins have different primary structures but share similar beta-sheet structures once aggregated. some fluorescent probes previously used as sensors for other biomolecules or microenvironments were tested to monitor α-syn aggregation. one of the compounds, coumarin 6 (16), displayed a similar sigmoidal curve as tht when monitoring the aggregation process of α-syn. the lag phase (58 h) was shorter than that of tht (74.9 h) and the concentration required (50 nm) was much lower than that of tht (10 μm), suggesting that probe 16 is more sensitive than tht. the other compound 1,6-diphenyl-1,3,5hexatriene (dph, 17), failed to show a sigmoidal curve and was believed to interact with hydrophobic environments without selectivity, while probe 16 could bind to the beta-sheet structure more specifically [98] . emerging evidence indicates that α-syn oligomers are more toxic and more pathologically connected to pd than fibrils. consequently, recent research has focused on developing [99] . the emission wavelength of probe 18 after binding to α-syn oligomers was determined to be 664 nm, showing its potential to be used in vivo as a nirf imaging probe. however, it remains questionable whether the bulky probe 18 could penetrate the bbb. another novel probe aimed at detecting α-syn oligomers is tetraphenylethene tethered with triphenylphosphonium (tpe-tpp, 19) . unlike sl-631, probe 19 is able to detect the monomeric, oligomeric and also fibrillar forms of α-syn. at the same concentration, probe 19 emitted over four times more fluorescence than tht after incubation with α-syn fibrils. the calculated k d for probe 19 was 4.36 μm, lower than that of tht (8.48 μm) [100] . compound selectivity among different amyloids has yet to be determined. huge challenges still exist in the discovery of probes targeting α-syn oligomers and major breakthroughs in terms of the 3d structure of oligomeric α-syn and the synthesis of high contrasting and selective imaging probes are urgently needed. a known modulator of α-syn aggregation, anle138b (20) , was found to exhibit a significant increase in fluorescence upon binding to α-syn fibrils with high affinity (k d = 190 nm) [101] . the study of probe 20 indicates not only the feasibility of discovering fluorescent probes from known inhibitors of α-syn aggregation but also the possibility of developing drugs with both therapeutic and diagnostic functions. until now there have not been any useful α-syn fluorescent probe reported for in vivo imaging (table 2 ). in fact, the great progress made in the development of aβ fluorescent probes might negatively affect the discovery of a specific imaging probe for α-syn because naturally the same strategies to design aβ probes have been applied to the development of α-syn probes [102] . since most of the existing aβ fluorescent probes share the classical push−pull structure [94] , which shows little selectivity for the different amyloid protein aggregates, it has been recommended to work on novel strategies to search for or design specific ligands for α-syn. the idea of inhibiting α-syn aggregation, especially oligomerization, using small molecules to combat α-syn toxicity and prevent neurodegeneration has been gaining increasing attention. it has been proposed that the identification of aggregation inhibitors from screening compound libraries should be a priority [30] . until now, a variety of small molecules have been discovered to inhibit α-syn aggregation (table 3 ). in many cases, however, the inhibitory effects have also been translated to other amyloid proteins. in addition to their roles as antimicrobial agents to fight infectious diseases, antibiotics have demonstrated other properties, such as neuroprotective activity. rifampicin (fig. 4, 21) stabilized α-syn as a monomer and blocked the fibrillation process. moreover, rifampicin was also able to disaggregate existing fibrils [103] . another study confirmed the efficacy of compound 21 by discovering that rifampicin could prevent 1-methyl-4-phenylpyridinium (mpp + )induced toxicity in pc12 cells, increase their survival and reduce α-syn oligomer formation [104] . another molecule tetracycline (22) is also able to inhibit the formation of fibrils and destabilize preformed fibrils. compound 22 exhibited moderate effective concentrations (ec 50 values) for the formation (6.06 μm) and destabilization (18.78 μm) of α-syn fibrils while the values for aβ 1−40 and aβ 1−42 were higher (10 and 10 μm respectively for fibril formation; 23 and 45 μm respectively, for fibril destabilization), indicating a slight selectivity for α-syn over aβ [105] . dopamine and its analogs dopamine (23) is believed to react with α-syn covalently to form αsyn-quinone adducts which are primarily large molecular mass oligomers. these oligomeric intermediates can cause cytotoxicity, implying a potential role for the interaction of compound 23 with αsyn in pd pathogenesis [106] . however, other evidence suggests that compound 23 inhibits α-syn fibrillation via binding noncovalently to α-syn [107] . based on this fact, latawiec et al. screened 70 analogs of compound 23 and selected five potent compounds (24 −28) by molecular dynamics (md) simulations. atomic force microscopy (afm) and transmission electron microscopy (tem) analysis confirmed the in silico simulation predictions that the selected compounds may affect the aggregation process of α-syn [108] . the combination of in silico approaches with in vitro assays to discover ligands that interact with target proteins may emerge as a novel and efficient way to identify aggregation inhibitors for α-syn. some common dyes, such as lacmoid (29) , congo red (30) and phthalocyanine tetrasulfonate (31) , have been reported to inhibit α-syn fibrillation [109] . compounds 29 and 30 were found to be nonspecific inhibitors; however, their effects were mediated by the formation of aggregates of these compounds which can interact with different regions of α-syn monomer [110] . unlike these two nonspecific inhibitors, the inhibition of α-syn fibrillation by compound 31 is believed to be mediated by specific interactions with the n-terminus of α-syn [111] . this molecule has also been tested in animal models for the treatment of scrapie disease and exhibited anti-prion activity and showed low toxicity [112] . therefore, compound 31 could be a potential therapeutic candidate for amyloid protein-related diseases. recently, another dye, coomassie brilliant blue r (32) not only exhibited significant inhibition of α-syn fibrillation but also prevented the formation of oligomers that caused notable neurotoxicity in cells, making it a potentially useful candidate for future in vivo studies [113] . the red blood pigment hemin (33) is a well-known inhibitor of aβ aggregation [114] . until recently, compound 33 was also thought to interfere with α-syn aggregation [115, 116] . the effects of compound 33 on α-syn remain controversial; however, it has not been confirmed whether compound 33 inhibits tht fluorescence via inhibition of α-syn aggregation or by obstructing the interaction between tht and the proteins [117] . polyphenols are the largest group of inhibitors of α-syn aggregation and many of them can also inhibit the aggregation of other amyloid proteins such as aβ. we have categorized representative polyphenols into the following groups. curcuminoids. the interaction of curcumin with α-syn seems complicated in that different mechanisms of anti-aggregation by curcumin (34) have been reported. early studies from the tht assay and tem showed that compound 34 not only inhibited the formation of α-syn fibrils but also destabilized the preformed fibrils [105] . later, the anti-aggregation ability of compound 34 was confirmed by western blot analysis and in a cell model of αsyn aggregation [118] . interestingly, another report stated that compound 34 does not interact with α-syn monomers but binds to oligomers and fibrils, causing morphological changes and consequently reducing their toxicity. moreover, the interaction of compound 34 with early oligomers could impact the toxicity by converting the preformed oligomers into the less toxic fibrils [119] . although compound 34 has been widely studied, its instability and poor bioavailability limits its medical use. with the aim of discovering more druggable molecules with a scaffold similar to that of compound 34, ahsan et al. 13 μm) . surprisingly, unlike compound 37, compound 36 failed to reduce the cytotoxicity caused by α-syn oligomers [120] . while compound 37 is a promising therapeutic agent for the potential treatment of pd, the case of compound 36 strengthens the importance of evaluating the anti-oligomerization ability of any molecules that are expected to show neuroprotection in cells and/ or animals. another group focused on modifications of the two aromatic rings of curcumin to increase the hydrophobicity. of the nine reported analogs, only two (c2 and c4) showed lower stability than curcumin, and one (c3) exhibited significant cytotoxicity. among the remaining compounds, c6 (38) , which was synthesized by replacing all the hydroxyl groups of compound 34 with −och 2 ph groups, showed the highest reduction in cytotoxicity caused by the preformed oligomers and fibrils. consistent with previous study [119] , curcumin and its analog compound 38 were found to accelerate the process of α-syn aggregation into less toxic fibrils [121] . flavanols. the green tea polyphenol egcg (fig. 5, 39) is perhaps the most studied inhibitor of aggregation of different amyloid proteins. it is also a common positive control in many studies aiming to develop new anti-amyloidogenic molecules. ehrnhoefer et al. demonstrated that compound 39 can directly bind to monomeric α-syn and promote the formation of unstructured, nontoxic α-syn oligomers. nmr results suggested that the compound 39 binds randomly to the backbone of α-syn [122] , which, along with another report using ms to study the binding of compound 39 with α-syn [123] , may explain why the effects of compound 39 on α-syn can also be seen on aβ. additionally, compound 39 is able to bind to preformed α-syn fibrils (k d = 100 nm) and transform them into smaller amorphous aggregates that are less toxic [124] . however, the mechanism by which compound 39 reduces the toxicity of α-syn oligomers is not the same as in the case of fibrils. one group found that treatment with compound 39 does not cause notable changes in the structure or size of α-syn oligomers. instead, the binding of compound 39 to α-syn oligomers prevents permeabilization of the α-syn oligomers with cell membranes, thus reducing the toxicity [125] . in animal studies, tea polyphenols mainly containing compound 39 were reported to reduce the level of α-syn oligomers in a pd monkey model [126] . theaflavins, which are abundant in black tea, exert antiamyloidogenic effects in the same way as compound 39, promoting the assembly of monomeric α-syn and aβ into nontoxic aggregates and converting the preformed fibrils into nontoxic aggregates [127] . nevertheless, the tendency of compound 39 and theaflavins to be oxidized could compromise their ability to inhibit amyloidogenesis. although preoxidized compound 39 is still able to significantly inhibit the aggregation process, a reduction in efficacy can be seen after long-term preoxidation in the case of compound 39 but not in the case of another theaflavin, tf3 (40) , which is less rapidly oxidized than egcg [127] . these results suggest that the anti-amyloidogenic abilities of common polyphenols may partially depend on the antioxidant properties. developing polyphenols that are resistant to oxidation, such as compound 40, may help to discover promising therapeutic agents for amyloid protein-related disease. stilbenes. previous evidence has demonstrated that some stilbenes can inhibit aβ aggregation [128] . to explore the potential application of stilbenes in the discovery of inhibitors for α-syn aggregation, temsamani et al. studied three wine stilbenes, piceatannol (41) , ampelopsin a (42) and isoheopeaphenol (43) . although all three stilbenes inhibited fibrillation, only compound 41 showed notable protection of cells treated with α-syn aggregates [129] . an explanation for this may be that compound 41 is also able to disaggregate preformed fibrils, and its relatively small size could enable compound 41 to easily penetrate the cell membrane to exert its protective effects. other phenolic compounds. using confocal single-particle fluorescence techniques, caruana et al. investigated the effects of different polyphenolic compounds. in addition to the abovementioned theaflavins and compound 39, they also studied flavones (apigenin, baicalein and scutellarein), flavonols (myricetin and quercetin), phenolic acids (rosmarinic acid and tannic acid), a stilbene (resveratrol) and others (ginkgolide b, nordihydroguaiaretic acid). the most potent compounds, baicalein (44) , scutellarein (45) , myricetin (46) , compound 39, nordihydroguaiaretic acid (47) and black tea extract (mainly theaflavins), were selected because they exhibited significant inhibition and disaggregation of α-syn at low concentrations (ic 50 < 4 μm). the structure activity relationship (sar) study suggested that aggregation inhibitors should contain aromatic rings for interactions with monomeric and oligomeric α-syn and adjacent hydroxyl groups on the same ring [130] . moreover, the authors proposed that the selected compound would be promising for tests in pd animal models. in addition to compound 39, compound 44 has also been studied in vivo and should be considered a potential drug candidate for clinical trials. in mpp + -treated sd rats, compound 44 reduced the increase of α-syn aggregates and protected the nigrostriatal dopaminergic system in the brain [131] . similar effects from compound 44 were observed in the rotenone mouse model, where α-syn oligomers greatly decreased the striatal neurotransmitters, including dopamine, and rotenone-associated behavioral dysfunction was improved [132] . as a main metabolite of green tea polyphenols, protocatechuic acid (48) is able to inhibit α-syn aggregation, disaggregate preformed α-syn fibrils and protect pc12 cells from toxicity caused by α-syn aggregates [133] . however, this phenolic acid can also exert similar effects on aβ. oleuropein aglycone (49) , which is commonly found in olive oil, was found to protect sh-sy5y cell viability via stabilization of α-syn in the monomeric state, directing α-syn to form nontoxic aggregates and obstructing the binding of α-syn to cell membranes [134] . another α-syn aggregation inhibitor discovered from olives is hydroxytyrosol (50) . similar to compound 48, compound 50 can also inhibit α-syn aggregation and destabilize preformed α-syn fibrils. notably, compound 50 at a concentration of 25 μm almost completely reversed the toxicity caused by α-syn aggregates [135] while compound 48 only rescued the cell viability to approximately 80% at the same concentration [133] . the discovery of active anti-amyloidogenic compounds from olive indicates another good dietary source that may help to prevent neurodegenerative diseases, in addition to green tea. glucosylation of common organic compounds may help to increase their bioavailability [136] . curcumin-glucoside (51), which was synthesized by replacing the two hydroxyl groups on the aromatic rings with glucose moieties, showed similar effects to inhibit α-syn oligomerization as well as to destabilize preformed fibrils compared with compound 34 but was believed to be more potent. importantly, the introduction of glucose increased the solubility [137] . there are also natural glycosides that have been reported to be anti-amyloidogenic agents. one common group is ginsenosides, the main biologically active extract from ginseng. rb1 (52) is one of the most studied ginsenosides and can inhibit oligomerization and fibrillation as well as disaggregate preformed fibrils. administration of compound 52 in cells can significantly attenuate the toxicity induced by α-syn aggregates, thus making it worthwhile for compound 52 to be further tested in animal models [138] . another glucoside, liquiritin (53), comes from glycyrrhizauralensis, a common traditional chinese medicine for the treatment of pd. in vitro studies have demonstrated that compound 53 can inhibit both oligomerization and fibrillation of α-syn. in a transgenic c. elegans expressing human α-syn, compound 53 significantly inhibited α-syn aggregation and extended the life span of the treated worms [139] . it has been reported that several vitamins k, unlike many promiscuous inhibitors that bind to α-syn via nonspecific hydrophobic interactions, can inhibit α-syn fibrillation and disassemble preformed fibrils by specifically binding to the nterminal region of α-syn. these vitamins (fig. 6 , phylloquinone, 54, menaquinone, 55 and menadione, 56) are actually derivatives of 1,4-naphthoquinone (1, 57) , indicating a potential role for the scaffold of compound 57 in the design of novel specific inhibitors for α-syn [140] . interactions of these vitamins k with other amyloid proteins have yet to be explored. tanshinone i (58) and tanshinone ii a (59) are the main active components in the traditional chinese medicine danshen. similar to egcg, these two phenanthrenequinones have been demonstrated to reduce the formation of α-syn oligomers and fibrils as well as destabilize the preformed fibrils. the benefits of these two molecules were further confirmed in a dye leakage assay where they managed to prevent the membrane damage caused by α-syn aggregates. the good performance in vitro paved the way for later in vivo assays conducted in a c. elegans model expressing α-syn. compounds 58 and 59 both significantly prevented α-syn aggregation and extended the life span of treated c. elegans [141] . since other neuroprotective effects of these compounds have already been studied in different rodent models, compounds 58 and 59 are expected to target α-syn aggregation in pd rodent models. hybrid molecules in light of the complex pathogenesis of pd, designing molecules that are capable of targeting different factors related with the disease is an attractive approach. compounds 18 (60) and 24 (61) , which are 3-arylcoumarin-tetracyclic tacrine derivatives, were synthesized and selected as promising leads for the further development of potential pd therapeutics. consistent with the design strategy, these two compounds exhibited multitargeted properties, including inhibition of α-syn aggregation, antioxidation and enhancement of the content of dopamine [142] . the concept of designing hybrid compounds for multitargeted functions may contribute to discovering novel therapeutics for future pd treatment. it has been found that lipid vesicles can assist the nucleation of monomeric α-syn, which is a key step in α-syn aggregation [143] . the potential effects of the aminosterol compound squalamine (62) on α-syn aggregation was studied as this compound is able to translocate proteins from the cell membrane to the cytoplasm [144] . in an α-syn aggregation system containing lipid vesicles, compound 62 intervened in the interaction between α-syn and the surface of the vesicles, thereby inhibiting α-syn aggregation [145] . this mechanism of compound 62 also counteracted the toxicity of oligomeric α-syn in sh-sy5y cells, reduced α-syn aggregation and alleviated the immobility caused by α-syn aggregates in c. elegans overexpressing α-syn [145] . later, the same group studied another structurally similar compound, trodusquemine (63) . the side chain of compound 63 is spermine rather than spermidine as in compound 62. the introduction of additional positive charges was attributed to the increased ability of compound 63 to displace α-syn from both lipid vesicles and preformed α-syn fibrils, thus making this aminosterol able to inhibit not only the nucleation of α-syn monomers but also fibrilinduced aggregation [146] . like compound 62, compound 63 also protected sh-sy5y cells from the toxicity of oligomeric α-syn and improved the fitness of c. elegans overexpressing α-syn [146] . future investigations of these two aminosterols in rodent pd models are anticipated. pyrimido pyrazine targeting the interaction between α-syn and membranes has become an important therapeutic strategy for synucleinopathies [147] . while the aforementioned aminosterols are molecules isolated from nature, neuropore therapies, inc. along with its collaborators developed a promising compound, npt100-18a (64), a de novo synthesized molecule with a pyrimido pyrazine scaffold. compound 64 was designed to target the 96−102 domain of α-syn that is believed to mediate the dimerization of α-syn on membranes [148] . by inhibiting the interaction between α-synuclein imaging probes and aggregation inhibitors mm xu et al. α-syn and lipid membranes, compound 64 reduced the formation of toxic α-syn oligomers in primary rat neurons. the long-term effects of compound 64 in a wild-type α-syn transgenic mouse model improved the performance of treated mice in motor behavioral assessments [148] . in the brains of these treated mice, the accumulation of α-syn was significantly reduced in different regions (neocortex, hippocampus, and striatum). a reduction in α-syn was also seen in the substantia nigra but this result was not significant. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) demonstrated that compound 64 decreased the amount of α-syn dimers and oligomers, a phenomenon that was also seen in an oligomer-prone e57k α-syn transgenic mouse model [148] . pathologically, neuronal death, synapto-dendritic damage and astrogliosis were notably improved, indicating an association between inhibition of α-syn accumulation, recovered neurodegeneration, and ameliorated motor function [148] . the short-term effects of compound 64 were evaluated in real time in α-syn-gfp transgenic mice. the reduction of the α-syn-gfp signal at synapses could be seen between 30 and 60 min after administration, indicating the efficiency of compound 64 to inhibit α-syn accumulation at synaptic terminals [148] . compound 64 suffered from poor oral bioavailability and poor brain penetration; therefore, this compound was not advanced further for clinical studies [149] . based on the structure of compound 64, a new compound, npt200-11 (65) , with improved pharmacokinetic properties, was introduced. compound 65 maintained similar efficacies compared to compound 64, including a decrease in α-syn accumulation, amelioration of neurodegenerative pathology and improvements in behavioral performance [149] . according to the authoritative database of clinical trials (clinicaltrials.gov), the phase 1 study of compound 65 in healthy subjects was successfully completed in 2016. a phase 1b clinical study in both healthy subjects and pd patients is expected to be conducted in europe. without a doubt, compound 65 is the most promising small molecule drug candidate targeting α-syn aggregation. there are still many other molecules that have been reported to be inhibitors of α-syn aggregation at the molecular level, such as aldehyde 4-hydroxy-2-nonenal (hne, 66) [150] and scyllo-inositol (67) [151] . compound 66 has perhaps the simplest structures among all the reported inhibitors since it has no aromatic ring and the molecular mass is only 156 da. compound 67 is already well known for its efficacy to inhibit aβ aggregation in vitro and in vivo. in cell models, compounds such as dieckol (68) [152] , melatonin (69) [153] , selegiline (70) [154] , and synuclean-d (71) [155] can exert neuroprotection against toxicity induced by α-syn aggregates. the discovery of compound 71 resulted from drug screening based on the tht assay [156] . the ameliorative effects of compound 71 were also observed in pd c. elegans models. treatment with compound 71 remarkably reduced the formation of α-syn aggregates and protected dopaminergic cells from death in c. elegans, attributing to the improved motility [155] . the following three molecules are promising compounds to be further developed into useful pd disease-modifying drugs for clinical applications. the first one, clr01 (72), also termed molecular tweezer, was first studied systematically by prabhudesai et al. in vitro assays including tht assays and tem demonstrated that compound 72 can inhibit α-syn aggregation as well as disassemble preformed fibrils. compound 72 also exhibited significant protection in both hek293 cells expressing α-syn endogenously and pc12 cells treated with aggregated α-syn. excitingly, compound 72 managed to reduce apoptosis induced by α-syn aggregation in zebra fish embryos expressing α-syn, which contributed to increased survival of the treated embryos [157] . both intracerebroventricular and subcutaneous administration of compound 72 to mice overexpressing α-syn can notably alleviate the motor deficits caused by α-syn pathology and is accompanied by a decrease in the soluble α-syn fraction. the long-term efficacy of compound 72 could be explained by its stable kinetics in vivo [158] . modifications of this molecule to increase its ability to penetrate the bbb would be required before moving to human trials. anle138b (20) was discovered by a high-throughput screening system against the aggregation of the prion protein. interestingly, compound 20 is also able to reduce the formation of α-syn oligomers. administration of compound 20 in the rotenone mouse model successfully ameliorated motor dysfunction. in another transgenic mouse model expressing human a30p mutated α-syn, compound 20 was shown to improve motor performance, prevent the spread of deposited α-syn in the brain and reduce the level of α-syn oligomers [159] . since compound 20 possesses ideal pharmacokinetic properties, it would not take long for it to be tested clinically to treat pd and prion diseases. the identification of fasudil (73) as a potent inhibitor of α-syn aggregation is quite exciting, as it is already an approved drug for cerebral vasospasm. therefore, it would not be difficult for compound 73 to be used clinically in the future for treatment of pd given that this molecule can effectively inhibit α-syn aggregation via specific binding to the c-terminal region and more importantly, treatment with compound 73 can improve motor performance and recognition memory in α-syn a53t mutated mice [160] . lewy bodies were first identified over 100 years ago and their main component, α-syn, though seemingly discovered much later, has been investigated for over 20 years. it is believed that the aggregation process of α-syn plays a central role in pd pathogenesis and as a result, the past decade has seen a large number of studies focusing on α-syn aggregation and its role as a biomarker. a variety of small molecule probes and inhibitors of α-syn have been developed over the past decade. some of these inhibitors have been tested in vivo and therefore are promising candidates for clinical trials. many of the discovered molecules are also able to affect the aggregation of other amyloid proteins, indicating their roles as nonspecific amyloid inhibitors. the reason for this may be explained by discussing the common techniques for the discovery of molecules capable of interacting with α-syn. similar to other biophysical techniques, tht assays are not able to sensitively detect and individually characterize unique protein subspecies with which a small molecule ligand interacts [161] . a tht assay can also be compromised if the small molecule can competitively bind to the dye-binding site on the protein or quench the emission of fluorescence via interaction with the dye [162] . the advantages of mass spectrometry (ms) over other screening assays include the rapid discovery of candidate inhibitors and, more importantly, the ability to identify binding modes and the particular protein conformations responsible for the interactions with the small molecules [116, 117, 163] . based on previous studies, we have recently established an automated screening system for screening scaffolds that can bind to monomeric α-syn. by screening over 2500 pure compounds, we identified a new αsyn aggregation inhibitor (74) with a pyrazolo [1, 5-a] pyrimidine-5-carboxylic acid scaffold. this molecule exhibited similar effects in the tht assay and circular dichroism (cd) and tem experiments with the positive controls egcg and hemin. coincubation of α-syn with compound 74 protected sh-sy5y cells from the toxicity of α-syn aggregates. ms techniques also allow the electron capture dissociation (ecd) fragmentation of the protein−ligand complexes, which can offer information about the binding regions of the ligands [164, 165] . compound 74 displayed a more specific binding region on the primary sequence of monomeric α-syn and a clear binding preference for low-charge states, suggesting its higher selectivity for monomeric α-syn than egcg and hemin [166] . in an endeavor to discover specific α-syn aggregation inhibitors, research has started to focus on developing molecules that target specific regions of monomeric α-syn, such as vitamins (54, 55 and 56) [140] and the two de novo synthesized pyrimido pyrazine derivatives (64 and 65) [148, 149] . the ability to detect the interactions between small molecules and monomeric α-syn α-synuclein imaging probes and aggregation inhibitors mm xu et al. makes ms a powerful technique to discover specific α-syn binding compounds and identify the specific regions to which the compounds bind. we also propose that the α-syn binding scaffolds screened by ms have the potential to be further developed into both fluorescent and pet imaging probes. this means that a single molecule may have both therapeutic and diagnostic functions. these types of 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alpha-synuclein accumulation in a rotenone mouse model of parkinson's disease protocatechuic acid: inhibition of fibril formation, destabilization of preformed fibrils of amyloid-beta and alpha-synuclein, and neuroprotection oleuropein aglycone stabilizes the monomeric alpha-synuclein and favours the growth of non-toxic aggregates protective effects of hydroxytyrosol against alpha-synuclein toxicity on pc12cells and fibril formation bioavailabilities of quercetin-3-glucoside and quercetin-4′-glucoside do not differ in humans curcuminglucoside, a novel synthetic derivative of curcumin, inhibits α-synuclein oligomer formation relevance to parkinson's disease ginsenoside rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils isoliquiritigenin and liquiritin from glycyrrhiza uralensis inhibit α-synuclein amyloid formation vitamins k interact with n-terminus alpha-synuclein and modulate the protein fibrillization in vitro. exploring the interaction between quinones and alphasynuclein inhibition effects of tanshinone on the aggregation of alpha-synuclein discovery of 3-arylcoumarin-tetracyclic tacrine hybrids as multifunctional agents against parkinson's disease lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation synuclein imaging probes and aggregation inhibitors mm membrane phosphatidylserine regulates surface charge and protein localization a natural product inhibits the initiation of alpha-synuclein aggregation and suppresses its toxicity multistep inhibition of alpha-synuclein aggregation and toxicity in vitro and in vivo by trodusquemine modulating membrane binding of α-synuclein as a therapeutic strategy a de novo compound targeting alpha-synuclein improves deficits in models of parkinson's disease the small molecule alpha-synuclein misfolding inhibitor, npt200-11, produces multiple benefits in an animal model of parkinson's disease effect of 4-hydroxy-2-nonenal modification on alpha-synuclein aggregation alpha-synuclein aggregation, seeding and inhibition by scyllo-inositol dieckol, an edible seaweed polyphenol, retards rotenone-induced neurotoxicity and α-synuclein aggregation in human dopaminergic neuronal cells effect of melatonin on α-synuclein self-assembly and cytotoxicity the anti-parkinsonian drug selegiline delays the nucleation phase of alpha-synuclein aggregation leading to the formation of nontoxic species small molecule inhibits alpha-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons high-throughput screening methodology to identify alpha-synuclein aggregation inhibitors a novel "molecular tweezer" inhibitor of alpha-synuclein neurotoxicity in vitro and in vivo a molecular tweezer ameliorates motor deficits in mice overexpressing alpha-synuclein anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and parkinson's disease fasudil attenuates aggregation of alpha-synuclein in models of parkinson's disease proteins behaving badly: emerging technologies in profiling biopharmaceutical aggregation dye-binding assays for evaluation of the effects of small molecule inhibitors on amyloid (aβ) self-assembly mass spectrometry-based screening for inhibitors of β-amyloid protein aggregation top-down esi-ecd-ft-icr mass spectrometry localizes noncovalent protein-ligand binding sites molecular basis for preventing α-synuclein aggregation by a molecular tweezer identification of a new α-synuclein aggregation inhibitor via mass spectrometry based screening in vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics a bifunctional curcumin analogue for two-photon imaging and inhibiting crosslinking of amyloid beta in alzheimer's disease this work was supported by the griffith university international postgraduate research scholarship (guiprs) and the griffith university postgraduate research scholarship (guprs). the authors also want to thank dr. stephen wood and dr. alex sykes for their helpful discussions and proofreading of this paper. author contributions mmx wrote the paper; pr, sr, rjq, hyz and gdm read and revised the paper. competing interests: the authors declare no competing interests. key: cord-270123-m8utyd1m authors: enmozhi, sukanth kumar; raja, kavitha; sebastine, irudhayasamy; joseph, jerrine title: andrographolide as a potential inhibitor of sars-cov-2 main protease: an in silico approach date: 2020-05-05 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1760136 sha: doc_id: 270123 cord_uid: m8utyd1m sars-cov-2 virus which caused the global pandemic the coronavirus disease2019 (covid-2019) has infected about 1,203,959 patients and brought forth death rate about 64,788 among 206 countries as mentioned by who in the month of april 2020. the clinical trials are underway for remdesivir, an investigational anti-viral drug from gilead sciences. antimalarial drugs such as chloroquine and hydroxychloroquine derivatives are being used in emergency cases; however, they are not suitable for patients with conditions like diabetes, hypertension and cardiac issues. the lack of availability of approved treatment for this disease calls forth the scientific community to find novel compounds with the ability to treat it. this paper evaluates the compound andrographolide from andrographis paniculata as a potential inhibitor of the main protease of sars-cov-2 (mpro) through in silico studies such as molecular docking, target analysis, toxicity prediction and adme prediction. andrographolide was docked successfully in the binding site of sars-cov-2 mpro. computational approaches also predicts this molecule to have good solubility, pharmacodynamics property and target accuracy. this molecule also obeys lipinski’s rule, which makes it a promising compound to pursue further biochemical and cell based assays to explore its potential for use against covid-19. communicated by ramaswamy h. sarma the onset of symptoms such as fever, cough, fatigue, production of sputum, shortness of breath, sore throat, headache along with some with reports of diarrhoea and vomiting began to rise as the group of pneumonia cases from december 2019 and later they were identified as bcoronavirus in wuhan, hubei province, china (guan et al., 2020; the bcoronavirus was firstly named as 2019-novel coronavirus (2019-ncov) on 12 january 2020 by who and formally named the disease as coronavirus 2019 and as a world emergency disease of cause and concern globally, international committee of coronavirus study group (csg) recommended the use of the name as sars-cov-2 which was published on 11 february 2020 . through analyzing the viral sequence and evolutionary analysis, bat was suspected to be the natural host of the virus. and the virus might have been transferred to humans as their intermediate host by binding to ace-2 receptor (angiotensin converting enzyme-2 receptor) (zhou et al., 2020) . the first lethal case was reported on 11 january 2020. the infection from patients to healthcare workers was first verified on 20 january 2020. it was further reported that during chinese new year people migrated from wuhan to various countries of the world. new cases evolved to other countries especially to various patients with no travel history to china which notified scientific and medical communities that local human to human transmission were seen in those countries (rothe et al., 2020) . recently, the total number of cases around the world was recorded to be 1,203,959 confirmed cases with more than 64,788 deaths (https://www.worldometers.info/coronavirus/). various types of treatments have been proposed which are mainly antiviral drugs, sars-cov and mers-cov antibodies are being used by clinicians and recent recommended combination therapy of hydroxychloroquine and azithromycin was studied and its results of open labelled non randomized clinical trial was published gautret et al., 2020) . meanwhile, food and drug administration (fda) has stated that both chloroquine phosphate and hydroxychloroquine sulphate are not approved of treating covid-19. and upon certain in vitro and some clinical data chloroquine phosphate and hydroxychloroquine sulphate was advised to be the treatment for covid-19 and enough randomized trials on these compounds to be provided and allowed the administration of the above drugs to be used for emergency (https://www.fda.gov/emergency-use-authori-zation#covidtherapeutics). hydroxychloroquine may have inhibitory mechanism over the viral processes and metabolisms. they may be involved in other mechanisms as inhibition of ace2 cellular receptor, acidification of the cell membrane preventing the entry of virus and modulation of immune response through respective cytokine release (covid-19 drug therapy-elsevier, 09 march 2020). but recent studies have shown that the hydroxychloroquine can also cause drug poisoning and severe or moderate adverse effects in individuals who are already taking treatments for diabetic and hypersensitive patients, the same patient group who are found to be affected severely by covid-19. administration of hydroxychloroquine has found to inhibit pro-inflammatory cytokines which finally leads to acute respiratory distress syndrome (ards) (guastalegname & vallone, 2020) . it has been found out that adverse neuropsychiatric condition was seen in post treatment of hydroxychloroquine which is hypothesized that it specifies the lysosomal dysfunction leading to psychiatric symptoms, which initiated the normal state of the patient who has been administered with the drug (ali & jones et al., 2018) . lethal adverse effect of retinal toxicity was seen in patient with acute renal impairment when administered with hydroxychloroquine (tailor et al., 2012) . a study of high doses of hydroxychloroquine along with atorvastatin in diabetic patients showed highest decline of blood glucose in patients (wondafrash et al., 2020) . when antimalarial drug, hydroxychloroquine when administered to patients with dermatomyosis, non-life threatening cutaneous reactions are seen most in dermatomyosis patients than cutaneous lupus erythrematosus (pelle & callen, 2002) and many side effects has been reported. and according to the website, (https://www. guidetopharmacology.org/coronavirus.jsp) many ligands which are synthetic in nature have been proposed for the treatment of covid-19 and are in clinical trials and are in process of peer review. due to these high adverse effects and the site of target through which hydroxychloroquine acts on the viral proteasome, spike proteins and proteins involved in the life cycle of the virus are unknown (covid-19 drug therapy-elsevier, 09 march 2020). a potential natural, non-synthetic drug compound has to be found with minimal side effects. the drugs which are necessary to act on the targets such as ace-2 receptors, tmprss2, sars-cov-2 and cd147 (https://www. guidetopharmacology.org/coronavirus.jsp) are in the process of being found in order to decrease the prognosis of the disease and life cycle of the virus. in silico studies of chemically synthetic drugs such as paritaprevir and raltegravir, doultegravir and bictegravir for the targets 3clpro and 2 0 -omtase (khan, jha, et al., 2020) , theophylline and pyrimidone derivatives as possible inhibitors of rna bound n terminal domain (sarma et al., 2020) and remdesivir, saquinavir and darunavir also with two natural compounds, flavone and coumarine derivatives for the inhibition of 3cl pro (khan, zia, et al., 2020) have been published. though there are many targets are found for the treatment of covid-19, the main protease (m pro ) of sars-cov-2 was chosen due to interest of treating infected patients, to stop the multiplication of virus within the cells, through which m pro was involved in the release of polypeptides which are functional extensive proteolysis and cleavage of the enzyme itself from the sites of genome, pp1a and ppa1ab . plant compounds are an ideal of finding drug components of interest and most economical one to produce quickly as possible. this is known as the concept of repurposing the natural phytomolecules which will hasten the drug discovery process. during a search for such potent plant compounds we found a recent study on potential plant compounds which are able to inhibit the m pro is in a process of publication (khaerunnisa et al., 2020) , while discussing the findings on the paper with group of siddha and ayurvedic doctors, they have advised us to examine the properties of the traditional available plant, several plant molecules obtained from medicinal plants were explored. the outcome was that andrographis paniculata which was found to have anti-viral property and already reported in ancient texts could be investigated as a good bet. we examined the main compound in andrographis paniculata and found that the main compound in the plant was andrographolide. the plant compound has been evidenced to have anti-inflammatory, anti-cancer, anti-obesity and anti-diabetes (dai et al., 2019) . andrographolide was found to have antiviral properties over many types of viral infections (gupta et al., 2017) and was found to have activity against chikungunya (wintachai et al., 2015) potential inhibitor of herpes simplex virus type-1 (seubsasana et al., 2011) . also, during the outbreak of dengue in india at 2006, aqueous extracts of andgrographis paniculata were given through the advice of ministry of ayush (ayurveda, unani, siddha and homeopathy) department of india which led to decrease in cases and infection of the disease as a preventive measure even to normal people acting as a immune booster. the study of anti-dengue activity was found and published through quantification of dengue viral inhibition and showed most antiviral inhibitory effects when denv 1-4 infected vero cells with maximum nontoxic dose (krishnasamy et al., 2018) . due to these antiviral activities the compound andrographolide was chosen. due to these interests, this paper involves the in silico analysis of andrographolide against crystal structure of sars-cov-2 main protease which is provided with an inhibition site (pdb id: 6lu7) , prediction of adme (https://www.swissadme.ch), target prediction (https://www. swisstargetprediction.ch) were done by using swiss-bioinformatics online tools. the prediction of toxicity of andrographolide was seen using pkcsm online web tool (https://biosig.unimelb.edu.au/pkcsm/). 2.1. docking of andrographolide on sars-cov-2 main protease the sars-cov-2 main protease (figure 1a and 1b) (pdb id: 6lu7) was used as the receptor. the inhibitor was selected and removed. the receptor preparation was done using the dock prep tool of ucsf-chimera. hydrogens were added and optimized by a hydrogen bonding network and allowed the method to determine the histidine protonation state. the receptor was saved in mol2 format (rec_charged.mol2) using untransformed coordinates and sybyl-style hydrogen naming. hydrogen present in the structure were removed from the protein and saved in pdb format (rec_noh.pdb). the 3 d structure of andrographolide (figure 2a and 2b) (pubchem cid:5318517) was downloaded from pubchem (https://pubchem.ncbi.nlm.nih.gov/compound/ andrographolide). hydrogen was added to the ligands using the addh function in structure editing tools in ucsf-chimera. charges were added to the ligands using am1-bcc method. the ligand file was saved in mol2 format (lig_charged.mol2) using untransformed coordinates. the molecular surface of the receptor (rec_noh.pdb) was prepared using the write dms tool in ucsf-chimera. spheres outside the surface were generated using sphgen function having 4.0 in angstroms as the maximum sphere radius and 1.4 in angstroms as the minimum sphere radius. the cluster present in the binding site of the inhibitor was chosen using showsphere function. a box was created around the chosen cluster, having extra margins enclosed of 5.0 in angstroms in all the 6 directions. a grid was generated using the program grid of dock6. flexible docking parameters were employed for andrographolide. is important to analyze the pharmacodynamics of the proposed molecule which could be used as a drug. swiss-adme is a website (https://www.swissadme.ch) which allows the user to draw their respective ligand or drug molecule or include smiles data from pubchem and provides the parameters such as lipophilicity (ilogp, xlogp3, wlogp, mlogp, silicos-it, log p 0 /w), water solubility-log s (esol, ali, silicos-it), drug likeness rules (lipinski, ghose, veber, egan and muegge) and medicinal chemistry (pains, brenk, leadlikeness, synthetic accessibility) methods are analyzed (daina et al., 2017) . the data from pubchem which consists of smiles of andrographolide (https://pubchem.ncbi.nlm.nih. gov/compound/andrographolide) was entered into the search bar and was analyzed. molecular target studies are important to find the phenotypical side effects or potential cross reactivity caused by the action of small biomolecules (keiser et al., 2007; gfeller et al., 2014) . swiss target prediction website (https://www.swisstargetprediction.ch) was logged on and the zinc number for andrographolide (zinc3881797) was entered on to the search bar and was analyzed. toxicology prediction of small molecules is important to predict amount of tolerability of the small molecule before being ingested into the human and animal models. pkcsm is an online database in which the small molecule can be drawn virtually or can be analyzed by submitting the smiles of the same. the website can provide details of toxicology effects in the fields of ames toxicity, human maximum tolerance dose, herg-i inhibitor, herg-ii inhibitor, ld50, loael, hepatotoxicity, skin toxicity, t. pyriformis toxicity and minnow toxicity. the website was logged on and the smiles of the andrographolide data from pubchem was searched and submitted into the website and toxicity mode was selected (pires et al., 2015) . the docking analysis of the compound with sars-cov-2 protease generated negative values for free energy -3.094357 kj/ mol in the grid box, suggesting high affinity for the binding pocket. all the binding conformations of the compound in the active binding pocket involved both h-bond and salt bridge interaction. the compound did bind to the protease with 4 hydrogen bonds with 3 residues namely gly143, cys145 and glu166 as shown (figure 3a and 3b ). all details of the atoms involved in bonding with ligands, bond lengths, docking energies and salt bridges are given in table 1 . the adme prediction which was done using swissadme database came with the results following after submission of the small biomolecule, andrographolide. water solubility properties calculated are esol -3.18, solubility of 2.36e à02 mg/ml and of soluble class; ali -3.62, solubility of 8.42e à02 mg/ml and of soluble class, silicos-it -2.69, solubility of 7.22e à01 mg/ml and of soluble class. pharmacokinetic data predicted was found to be of high gastrointestinal absorption (gi), not blood brain barrier permeant, acts as a p-gp substrate, does not inhibit cyp1a2, cyp2c19, cyp2c9, cyp2d6 and cyp3a4 cytochromes. skin permeation kinetics (log k p ) was found to be -6.90 cm/s. druglikeness factors was found to be of drug like compound which obeys lipinski's rules with no violation, also obeys druglikeness score rules such as ghose, veber, egan, muegge and with 0.55 bioavailability score. medicinal chemistry parameters were found to be of no pains alert, violates brenk's laws with two alerts of being an isolated alkene, one michael acceptor, no lead likeness with molecular weight of greater than 350 and synthetic accessibility of 5.06 rate. the target prediction analysis was displayed in the web page with the following observations the top 25 of the results were given as a pie-chart ( figure 4) . the pie chart predicts 32% of kinase, 12% of protease, 4% of fatty acid binding protein family, 4% of transferases, 8% of enzymes, 4% of ligases, 8% of nuclear receptors, 8% of electrochemical transporters, 4% of secreated protein, 4% of family ag protein coupled receptor, 4% of unclassified protein and 8% of lyase. the output table consistting of target, common name, uniprot id, chembl-id, target class, probability and known actives in 2 d/3d are given in the supplementary-1. the possible sites of target which the compound may bind to are mostly the targets which are predicted by the software and the probability score are very less that is from 0.10560 to 0.0972. this makes an inference that the small compound may have high target attraction towards the specific binding site it is directed to. the toxicity predicted was displayed in the website and the results is as follows, the andrographolide does not have ames toxicity, maximum tolerated dose for human is about 0.128 log mg/kg/day, it does not inhibit herg-i and herg-ii, acute oral rat toxicity (ld 50 ) was found to be 2.162 mol/kg, chronic oral rat toxicity (loael) was found to be 1 log mg/ kg_bw/day, does not produce hepatotoxicity, it does not cause skin sensitivity, 0.491 log mg/l causes t. pyriformis toxicity and 1.37 log mm causes minnow toxicity. the need of the hour is a therapy for sars-cov-2 virus, many small molecules like remdesivir are in trial to provide cure for this dreadful viral outbreak. hydroxychloroquine and azithromycin complex is being advised to be given for the affected in case of emergency, though it has been studied to increase the ph of the protease and is published as a potent inhibitor of sars-cov-2 infection and spread heald-sargent & gallagher, 2012; vincent et al., 2005) . the hydroxychloroquine and azithromycin complex mentioned above may be potent, but the adverse side effects they bring to the patients are very alarming as shared already, with these drastic side effects, the need to bring equally or more potent alternatives in the form of plant derived drug is very essential as they are much safe and have no known side effects. as we are interested only in finding pure potent plant compounds without adding any analogs or derivatives we found that the plant compound, andrographolide intriguing due to its awesome properties. the drug compound, andrographolide can be isolated and produced easily by extracting from the plant andrographis paniculata. when the compound was analyzed by in silico computational docking tools it successfully docked against the inhibitor region of the main protease of sars-cov-2 virus with docking score of -3.094357 kcal/mol, the docking score showed great binding when compared to synthetic compounds when they are docked against m pro such as disulfiram, tideglusib and shikonin which are -46.16 kcal/mol, à61.79 kcal/ mol and -17.35 kcal/mol . and it also shows great binding score when compared against recently proposed combination of three drugs namely, lopinavir, ostelmivir and ritonavir whose binding scores are -4.1kcal/mol,-4.65 kcal/mol and -5.11 kcal/mol (muralidharan et al., 2020) . even some plant molecules which are studied to inhibit the main protease of sars-cov-2 failed to prove their binding score when compared with the andrographolide. they are compounds such as kaempferol -9.41 kcal/mol, quercetin -8.58 kcal/mol, demethoxycurcumin -8.17 kcal/mol, curcumin -7.31 kcal/mol, catechin -7.05 kcal/mol, epichatechin gallate-7.24 kcal/mol, zingerol -6.67 kcal/mol and gingerol -5.40 kcal/mol respectively (khaerunnisa et al., 2020) . even proposed inhibitor of m pro such as prd_002214 has a docking score of -10.466 kcal/mol (bouchentof & missoum, 2020) which proclaims that andrographolide has better properties than other proposed inhibitors. the compound possesses excellent properties of druggability as well small biomolecule. the molar refractivity of the compound confirms that the drug compound is permeable through particular membranes and can remain constant even in the midst of strong or weak solute-solvent, solventsolvent interactions. and exemplary tpsa says that it has great transport properties. through lipophilicity of the drug compound we can know that the compound has ideal property for oral and intestinal absorption and is able to be absorbed sub-lingual as well. through water solubility properties predicted the drug is free soluble. the pharmacokinetic data predicted about absorption and permeability echoes the predicted values of lipophilicity and solubility, they also predict that is able to release phosphate from atp and simultaneous binding of adp to the glycoprotein, thus the compound acts as a p-gp substrate. the compound, andrographolide does not inhibit liver metabolism by inhibiting cyp1a2, it does not stop the metabolism of several therapeutic drugs especially, anti-ulcer, anti-malarial, anti-convulsant, anesthetic and sedative drugs via inhibiting cyp2c19, it does not stop metabolism of anti-hypersensitive drugs, b blockers, anti-arrhythmic drugs and anti-depressants via inhibition of cyp2d6, it does not stop the metabolism of anti-clotting agents, anti-seizure, management of type-ii diabetes, anti-hypertensive, nonsteroidal anti-inflammatory drugs (nsaids) via cyp2d9 and it does not stop oxidation of steroids, fatty acids and xenobiotics as well as for hormone synthesis and breakdown through cyp3a4. druglikeness factor rules were obeyed accordingly without any violation to this compound which describes the compound can act as a drug in the biological systems. medical chemistry parameters exclaims that, zero pan-assay interference compounds (pains) alert for the compound meaning that it is a progressive compound worthy of testing for biochemical assays. the toxicity prediction says that the compound, andrographolide is safe and can be given as a drug with the value of tolerance prescribed for human consumption as predicted by the website. though these properties are appreciable in silico, due to extensive lockdown and work from home command through the government the findings was not continued in a wet lab. so, further studies of in vitro and clinical studies dealing with sars-cov-2 should be considered for further studies. 23. an adverse neuropsychiatric reaction following treatment with hydroxychloroquine: a case report identification of compounds from nigella sativa as new potential inhibitors of 2019 novel coronasvirus (covid-19): molecular docking study overview of pharmacological activities of andrographis paniculata and its major compound andrographolide swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial swisstargetprediction: a web server for target prediction of bioactive small 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2'-o-ribosemethyltransferase identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach anti-dengue activity of andrographis paniculata extracts and quantification of dengue viral inhibition by sybr green reverse transcription polymerase chain reaction. ayu (an computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 adverse cutaneous reactions to hydroxychloroquine are more common in patients with dermatomyositis than in patients with cutaneous lupus erythematosus pkcsm: predicting small-molecule pharmacokinetic and toxicity properties using graphbased signatures transmission of 2019-ncov infection from an asymptomatic contact in germany in-silico homology assisted identification of inhibitor of rna binding against 2019-ncov n-protein (n terminal domain) a potential andrographolide analogue against the replication of herpes simplex virus type 1 in vero cells a case of severe hydroxychloroquine-induced retinal toxicity in a patient with recent onset of renal impairment: a review of the literature on the use of hydroxychloroquine in renal impairment chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan activity of andrographolide against chikungunya virus infection potential effect of hydroxychloroquine in diabetes mellitus: a systematic review on preclinical and clinical trial studies crystal structure of sars-cov-2 main protease provides a basis for design of improved a-ketoamide inhibitors a pneumonia outbreak associated with a new coronavirus of probable bat origin i would like to express my sincere gratitude to derek and sharon for their daily updates on sars-cov-2, dr. kaleeswaran for his advice on this compound and mr. rahul vivek for his work on docking. no potential conflict of interest was reported by the author(s). key: cord-261366-mtcalbo5 authors: da rosa guimarães, tatiana; quiroz, carlos guillermo; rigotto, caroline; de oliveira, simone quintana; rojo de almeida, maria tereza; bianco, éverson miguel; moritz, maria izabel goulart; carraro, joão luís; palermo, jorge alejandro; cabrera, gabriela; schenkel, eloir paulo; reginatto, flávio henrique; oliveira simões, cláudia maria title: anti hsv-1 activity of halistanol sulfate and halistanol sulfate c isolated from brazilian marine sponge petromica citrina (demospongiae) date: 2013-10-29 journal: mar drugs doi: 10.3390/md11114176 sha: doc_id: 261366 cord_uid: mtcalbo5 the n-butanol fraction (bf) obtained from the crude extract of the marine sponge petromica citrina, the halistanol-enriched fraction (tsh fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate c (2), were evaluated for their inhibitory effects on the replication of the herpes simplex virus type 1 (hsv-1, kos strain) by the viral plaque number reduction assay. the tsh fraction was the most effective against hsv-1 replication (si = 15.33), whereas compounds 1 (si = 2.46) and 2 (si = 1.95) were less active. the most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. the anti-hsv-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into vero cells, the virucidal effect on virus particles, and by the impairment in levels of icp27 and gd proteins of hsv-1. in summary, these results suggest that the anti-hsv-1 activity of tsh fraction detected is possibly related to the synergic effects of compounds 1 and 2. the drug of choice for the prophylaxis and treatment of herpex simplex virus (hsv) infections is acyclovir (acv), which selectively inhibits hsv dna replication with low host-cell toxicity. however, the intensive use of antiviral drugs has led to the emergence of resistant viruses [1] [2] [3] . recently, de clercq [4] described the evolution of antiviral agents against some viral infections, including hsv, confirming that the search for new antiviral agents is still relevant. pharmaceutical interest in marine organisms has provided thousands of new and novel compounds that have shown important biological properties, such as anticancer, antiviral, antiprotozoal, and antibacterial activities [2, [5] [6] [7] [8] . in this context, marine sponges have been a prolific source of diverse secondary metabolites with complex and unique structures [2, [9] [10] [11] [12] [13] . some of them were used as lead compounds to obtain new drugs that are currently used in clinics, such as acyclovir, vidarabine, cytarabine, eribulin mesylate, and others, that are now in clinical stages of evaluation such hemiasterlin [14] [15] [16] . in addition, several highly active compounds from marine sponges have been reported as new biologically active structures [17] [18] [19] [20] [21] [22] [23] [24] . petromica citrina (porifera, demospongie) belongs to a marine sponge genus that occurs only on the brazilian coast [25] . there are few studies with this species, and most of them describe the evaluation of different pharmacological properties such as antibacterial and antiviral activities for its aqueous extracts [26, 27] and n-butanol fraction [28] . moreover, a restricted number of chemical investigations and a few bioactive constituents have been reported, in particular, a sulfated steroidal compound, identified as halistanol sulfate [29, 30] . recently, our research group described the anti-herpes activity of the n-butanol fraction of p. citrina [28] . thus, the aim of this investigation was to determine, through a bioguided study, the active compounds responsible for the anti-hsv-1 activity detected. in a previous screening of the anti-infective potential of marine invertebrates and seaweeds [28] , we observed a promising activity for the n-butanol fraction (bf) obtained from the ethanolic crude extract of this sponge that led us to perform this study. our goal was to isolate, through a bioguided study, the anti-herpes bioactive metabolites present in this fraction. first, the bf fraction was submitted to several sephadex lh-20 chromatography procedures yielding five fractions (sep-1 to sep-5), which were pooled based on thin-layer chromatography (tlc) similarity. among these fractions, only fraction sep-5 showed anti hsv-1 activity and was submitted to nmr analysis. the 1 h nmr spectrum of sep-5 displayed characteristic signals of the presence of halistanol sulfates as the major compounds. these major compounds were isolated by c18 column chromatography, yielding compounds 1 and 2 ( figure 1 ). the complete structure of compound 1 was determined based on hsqc, hmbc, and cosy spectra, as well as by esi mass spectrometry and by comparison with literature data [29] [30] [31] [32] [33] . the presence of three sulfate groups in the structure could be clearly defined by esi mass spectrometry these sulfate groups was also supported by the ir band (1230 cm −1 ). in addition, the 1 h nmr spectrum of compound 1 showed carbinol signals at δ h 4.83 (sl), δ h 4.76 (sl; j = 1.8 hz), and δ h 4.20 (dt; j = 11.0; 4.4 hz), corresponding in the hsqc spectrum to the signals at δ c 75.6 (ch-2 and ch-3), and δ c 78.8 (ch-6), respectively. these data, together with characteristic signals of two methyl singlets at δ 0.70 (ch 3 -18) and δ 1.07 (ch 3 -19) , suggested a sulfated sterol nucleus. the structure of the side chain of compound 1 was elucidated by analysis of 2d nmr data. the nmr spectra showed the presence of a side chain containing two secondary methyls at δ 0.95 (d; j = 6.4 hz) and δ 0.84 (d; j = 6.8 hz) attributed to positions c 21 and c 28 , also based on hmbc data. the 1 h nmr spectra revealed a singlet at δ 0.86 (9h), which was connected to carbon at δ 27.9, suggesting a t-butyl group on the side chain. hmbc correlations of carbons at δ 27.9 (c 26 , c 27 and c 29 ), δ 34.2 (c 25 ), and δ 45.5 (c 24 ) to the proton at δ 0.86 confirmed that c 26 , c 27 , and c 29 were connected to c 25 . therefore, compound 1 was identified as halistanol sulfate, a steroid previously reported for marine sponges such as halichondria cf. [31] , epipolasis sp. [32] , petromica ciocalyptoides [29] , haliclona sp. [33] , and petromica citrina [30] . halistanol sulfate (hs) was first reported in 1981 by fusetani et al. [31] and, in that work, the authors only showed the 13 c nmr data of hs. new compounds of the halistanol sulfate series (halistanol sulfates a to h) were isolated in the subsequent years [32, 34] , but the nomenclature and the chemical shift values in the 1 h nmr spectra of the side chain are still not completely defined [29, 32] . therefore, it is important that the details of the structural elucidation of compounds 1 and 2 are also presented. compound 2 also showed the same halistanol steroidal nucleus signals, but with a shorter side chain, which was inferred by nmr data together with the information of the esi mass spectrum. ) in the structure. as well as for compound 1 the presence of sulfate groups in the structure was also supported by the ir band (1226 cm −1 ). although the 1 h-nmr spectra of compound 1 displayed two methyl doublets at δ 0.95 and δ 0.84 on the side chain, corresponding to c 21 and c 28 , respectively, the 1 h nmr of compound 2 only one doublet signal at δ 0.94 (d; j = 6.6 hz), corresponding to the c 21 methyl group. in addition, the 1 h nmr data did not show a t-butyl group at the end side of the chain. furthermore, two new methyl signals at δ 0.87 (d; j = 6.6 hz) and δ 0.89 (d; j = 6.6 hz) were identified. considering the j values of these protons, we could suggest the presence of an isopropyl on the side chain. thus, based on the data obtained, compound 2 was identified as halistanol sulfate c, a steroid previously reported for pseudoaxinissa digitata [34] and epilopasis sp. [32] . as far as we are aware, this is the first report of halistanol sulfate c for petromica citrina. sulfated sterols have been described from a wide variety of marine organisms, such as sponges and echinoderms. several of these sterols have a great structural diversity and broad spectrum of biological activities [35] [36] [37] [38] [39] . the first reported compound of the halistanol family was halistanol sulfate, isolated from the marine sponge halichondria cf. moorei bergquist [31] . important biological activities have been reported for this steroid sulfate, such as anti-hiv effects [38] , cytotoxic activity against human hepatoma cells (qgy-7701), and chronic myelogenous leukemia cells (k562) [40] . afterwards, the same compound was isolated from petromica ciocalyptoides and topsentia ophiraphidites, showing inhibitory activity of leishmania tarentola [29] and a wide spectrum of activity against resistant bacteria such as staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, mycobacterium fortuitum, and neisseria gonorrheae [30] . thus far, eight sulfated sterols have been described with this fundamental nucleus and named as halistanol sulfates a to h ( figure 2 ). all of them are characterized by the same 2β, 3α, 6α-trisulfoxy functionalities, differing only in their side chains [32, 34, 35] . the most promising pharmacological activities described for these compounds were the anti-hiv-1 and anti-hiv-2 effects for halistanol sulfates f and g [32] . in addition, there are many other reports about different members of the halistanol series that have shown important pharmacological properties. one of the first reported members of this series was ibisterol sulfate, isolated from topsentia sp., which showed anti-hiv activity [41] . other examples of halistanol-type compounds with antiviral activity are weinbersterol disulfates a and b isolated from the sponge petrosia weinbergi which exhibited activity against leukemia virus (felv), mouse influenza virus (pr8), and mouse coronavirus (a59) replication [42] . as compounds with sulfated groups are described to have antiviral properties [34, 38, [43] [44] [45] [46] , and due to the anti-herpetic activity shown by the bf fraction, we decided to verify the anti-hsv-1 activity of compounds 1 and 2 and the tsh fraction and to elucidate their mode of action. the evaluation of potential antiviral activity of p. citrina fractions [sep-1, sep-2, sep-3, sep-4, and sep-5 (tsh fraction)] as well as the isolated compounds (1 and 2) was performed against hsv-1 (kos strain) using the viral plaque number reduction assay. according to the results obtained (table 1) , the isolated compounds 1 (si = 2.46) and 2 (si = 1.45) showed weak activity. on the other hand, the tsh fraction that contains these compounds as the major constituents showed the most promising activity (si = 15.33). as it is important to understand the targets and the mode of action of a potential useful new antiviral agent, a set of experiments was carried out to determine the stages at which the most active samples (tsh fraction and compounds 1 and 2) affect the viral replication cycle. pretreatment of vero cells with tsh fraction and compounds 1 and 2 for three hours before viral infection showed that these samples did not affect viral infectivity suggesting that they did not exert protective effects against the hsv-1 infection process (data not shown). the direct virus inactivating activity of the tested samples, in the absence of cells, was also evaluated. it was also observed that the tsh fraction and compounds 1 and 2 were able to reduce hsv-1 infectivity at concentrations 8×, 12× and 6× lower than their ic values ( table 2 ). this is in accordance with previous studies that have reported the virucidal activity of halistanol sulfates f and h against hiv replication [34, 38] . in order to determine whether these samples were able to interfere with early events of hsv infection, their effects on hsv-1 attachment and penetration were investigated separately. all the tested samples inhibited virus attachment and penetration, as shown in table 2 . therefore, the inactivation of hsv-1 could be related to virions binding to heparan sulfate receptors, inhibiting these two early stages of viral replication. other natural sulfated molecules, such as sulfated polysaccharides, were also active against hiv, hsv-1, and hsv-2 replication [43] [44] [45] [46] , inhibiting these same early events of viral replication. it is well documented that the antiviral potency of sulfated compounds depends on their degree of sulfation [4, 47] . moreover, it has become clear that the antiviral properties of sulfated compounds are not only a simple function of their detailed structural features, but also of their charge density. for instance, a highly charged molecule is more likely to interfere with electrostatic interactions between the positively charged region of a viral glycoprotein and the negatively charged hs chains of the cell-surface glycoprotein receptor, which could explain the blockade of viral attachment and penetration by competitive inhibition [48] . additionally, we also tested the anti hsv-1 activity of halistanol disulfate (ds) and halistanol monosulfate (ms) (data not shown). it was observed that ds was less active than halistanol sulfate and halistanol sulfate c (compounds 1 and 2, respectively; both trisulfated derivatives) as well as the ms being inactive against hsv-1. in view of the fact that our results suggest that tsh fraction and compounds 1 and 2 affect the early stages of hsv replication, we also investigated the effects of these samples on protein expression during hsv-1 replication by western blotting (figure 3) . the results showed that the tsh fraction was the only sample that reduced the expression of all the tested proteins, in a concentration-dependent manner. nevertheless, the (α) immediate icp27 protein expression of hsv-1 (kos strain) was reduced by all the tested samples, confirming that they interfere with the early events of hsv-1 replication. in addition to this event, a concentration-dependent inhibition of gb glycoprotein synthesized in the late phase (γ) of hsv-1 replication was also observed. only tsh fraction reduced gd expression. these results suggest that an alteration in immediate early protein expression could affect the expression of late proteins. this statement is supported by the findings of fontaine-rodrigues and knipe [49] , who demonstrated that icp27 is required for the efficient expression of hsv late proteins. given the fact that tsh fraction, compounds 1 and 2 seemed to act in a different way than acv, the potential synergistic effects between them were tested at different concentrations ( table 3 ). the results obtained suggest a strong synergism between tsh fraction, compound 2 and compounds 1 + 2 and acv, and a moderate synergism when compound 1 was tested with this drug, at the higher concentration (2 × ic 50 ). when compounds 1 and 2 were tested in association, a strong synergism was also detected, at the three tested concentrations. in relation to the other combinations, a slight or a moderate antagonism was detected, exception to the association of acv and tsh fraction, at the intermediate concentration (1 × ic 50 ), when an additive effect was detected. the observed synergism between these samples and acv could be explained by the fact that the samples act in different steps of hsv-1 replication than those affected by this anti-herpes drug, which could be considered an interesting result. therefore, the most important result obtained was when compounds 1 and 2 were tested in association showing that the detected anti-hsv activity could be explained by the strong synergic effects of these major compounds present in the tsh fraction. other natural compounds with anti-herpes activity, such as sulfated polysaccharides [44, 45] , docosanol [51] , and oxiresveratrol [52] have already been reported to present synergistic effects with acv, which corroborate our results. general 1d and 2d nmr experiments were performed on a bruker avance 2 (500 mhz) instrument at 500 mhz for 1 h and 125 mhz for 13 c. all spectra were recorded in cd 3 od using the signals of residual non-deuterated solvent as internal reference. mass spectrometric analyses were performed using a bruker microtof-q ii mass spectrometer (bruker ® daltonics, billerica, ma, usa), equipped with esi. multi-point mass calibration was carried out using a mixture of sodium formate from m/z 50 to 900. data acquisition and processing were carried out using the bruker compass data analysis version 4.0 software supplied with the instrument. all the analytical solutions (0.5 mg/ml) were prepared using methanol lcms grade. compounds were infused into the source using a kds 100 syringe pump ( the frozen sponge (1700 g, wet) was exhaustively extracted with ethanol for three days at room temperature. the crude ethanolic extract (che) was filtered, the ethanol was eliminated under reduced pressure, and the gummy residue was suspended in h 2 o before being extracted successively with ethyl acetate (etoac) and n-butanol (n-buoh) (3 × 500 ml) yielding three fractions: etoac (eaf), n-buoh (bf) and aqueous residue (ar), respectively. next, the bf fraction (2.0 g) was subjected to sephadex lh-20 column chromatography (790 mm × 25 mm) using methanol (meoh) as eluent. a total of 180 tubes (20 ml) were collected and combined into five fractions (sep-1, 650 mg; sep-2, 450 mg; sep-3, 350 mg; sep-4, 250 mg; and sep-5, 300 mg) based on silica gel thin-layer chromatography (tlc) similarity. because the fraction sep-5 (named tsh fraction) showed only one spot by tlc analysis, this fraction was forwarded to 1 hnmr analysis and proved to be a mixture of halistanol sulfate (compound 1) and halistanol sulfate c (compound 2) as the major compounds. the tsh fraction (200 mg) was then dissolved in methanol and submitted to a reversed-phase column chromatography (300 mm × 20 mm) packed with rp18 as stationary phase and meoh:h 2 o (1:1 v/v) as mobile phase. this procedure resulted in two isolated compounds: halistanol sulfate (30 mg, compound 1) and halistanol sulfate c (12 mg, compound 2). figure 4 shows the steps of purification. hsv-1 (kos strain, faculty of pharmacy, university of rennes, france) was propagated in vero cells. viral stocks were stored at −80 °c and titrated based on plaque forming units (pfu) counted by plaque assay as previously described [53] . vero (atcc: ccl 81) cells were grown in eagle's minimum essential medium (mem; cultilab ® , campinas, brazil) supplemented with 10% fetal bovine serum (fbs; gibco ® , carlsbad, ca, usa), 100 u/ml penicillin g, 100 µg/ml streptomycin, and 25 µg/ml amphotericin b (cultilab ® ), and maintained at 37 °c in humidified 5% co 2 . vero cell viability was measured by the mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-sigma-aldrich ® , st. louis, mo, usa) [54] . briefly, confluent vero cells were exposed to different concentrations of samples for 72 h, and after incubation, the 50% cytotoxic concentration (cc 50 ) of each one was calculated as the concentration that reduces cell viability by 50%, when compared to untreated controls. viral plaque number reduction assay: to evaluate the anti-herpes activity, a plaque reduction assay was performed following the general procedures described by silva et al. [55] . vero cell monolayers were infected with approximately 100 pfu of the virus for 1 h at 37 °c, then overlaid with mem containing 1.5% carboxymethylcellulose (cmc; sigma-aldrich ® , st. louis, mo, usa) either in the presence or absence of different concentrations of the samples. after 72 h of incubation at 37 °c, cells were fixed and stained with naphtol blue-black (sigma-aldrich ® , st. louis, mo, usa ), and the plaques were counted. the ic 50 of each sample was calculated as the concentration that reduced the number of viral plaques in 50%, when compared to the untreated controls. acv was used as a positive control. the selectivity index (si = cc 50 /ic 50 ) was calculated for each sample tested. virucidal assay: mixtures of serial two-fold dilutions of samples and 4 × 10 4 pfu of hsv-1 in serum free mem were co-incubated for 15 min at 37 °c prior to the dilution of these mixtures to non-inhibitory concentrations (1:100) [56] . the residual infectivity was determined by the viral plaque number reduction assay, as described above. pretreatment: this assay was performed as described by bettega et al. [57] . briefly, vero cell monolayers were pretreated with different concentrations of samples for 3 h at 37 °c prior virus infection. after washing, cells were infected with 100 pfu of hsv-1 for 1 h at 37 °c. the infected cells were washed, overlaid with mem containing 1.5% cmc, incubated for 72 h, and treated as described earlier for the viral plaque number reduction assay. simultaneous treatment: this assay was performed as described by silva et al. [55] . briefly, 100 pfu of hsv-1 and different concentrations of samples were added concomitantly to vero cells for 1 h at 37 °c. after washing, cells were overlaid with mem containing 1.5% cmc, incubated for 72 h, and treated as described earlier for the viral plaque number reduction assay. adsorption and penetration assays: these assays followed the procedures described by silva et al. [55] , with minor modifications. briefly, for the adsorption assay, confluent vero cells, pre-chilled at 4 °c for 1 h, were infected with 100 pfu of hsv-1 and treated with different concentrations of samples, then incubated at 4 °c for 2 h. the unabsorbed viruses were removed by washing with cold pbs, the cells were covered with overlay medium, the temperature was raised to 37 °c, and treated as described earlier for viral plaque number reduction assay. dextran sulfate (sigma) was used as a positive control. for the penetration assay, 100 pfu of hsv-1 was adsorbed for 2 h at 4 °c on confluent vero cells, after that incubated at 37 °c for 5 min to maximize virus penetration. the cells were then treated with different concentrations of samples. after 1 h at 37 °c, unpenetrated viruses were inactivated with warm citrate-buffer (ph 3.0) for 1 min. the cells were washed with pbs and treated as described above for the viral plaque number reduction assay. western blotting analysis: procedures were performed as described by bertol et al. [58] . briefly, vero cell monolayers were infected with hsv-1 at moi 0.2 for 1 h. next, residual viruses were removed with pbs and the cells were submitted to the different treatments for 18 h. the proteins were then extracted from the cells, separated on 12% sds-polyacrylamide gel electrophoresis (sds-page), transferred to polyvinylidene difluoride (pvdf) membranes (millipore, billerica, ma, usa) and blocked with 5% non-fat milk in blotting buffer [25 mm tris-hcl (ph 7.4), 150 mm nacl, 0.1% tween 20] . the membranes were incubated for 90 min with the following primary antibodies: goat monoclonal antibody against icp27 protein (1:700 dilution) (santa cruz biotechnology, santa cruz, ca, usa); mouse monoclonal antibody against ul42 protein (1:5000 dilution) (millipore ® , st charles, mo, usa); mouse monoclonal antibody against gd (1:5000 dilution) (santa cruz ® biotechnology, santa cruz, ca, usa); mouse monoclonal antibody against gb (1:5000 dilution) (millipore ® , st charles, mo, usa); and rabbit monoclonal antibody against beta-actin (1:5000 dilution) (millipore ® , st charles, mo, usa). after washing, the membranes were incubated with the respective secondary antibodies for 1 h. the immunoblots were developed and detected using the pierce ecl western blotting substrate (thermo ® scientific, rockford, il, usa), according to the manufacturer's instructions. the effects of tsh fraction, compounds 1 and 2 in combination with acv, and compounds 1 more 2 were evaluated by plaque reduction assay as described above and according to the experimental design proposed by chou et al. [50] . briefly, each sample alone or in combination was tested at a fixed ratio of its corresponding ic 50 value (i.e., at ic 50 × 0.5 × 1 and × 2). the interaction degree between samples, based on the median-effect principle of the mass-action law, using calcusyn software (version 2.1, biosoft ® , cambridge, uk). according to the ci theorem, ci values <1, =1, and >1 indicate synergism, additive effect, and antagonism, respectively. in summary, these results suggest that the tsh fraction and compounds 1 and 2 present antiherpes activity through the reduction of viral infectivity, inhibition of virus entry into the cells, and by the impairment of levels of icp27 and gd proteins of hsv-1. the relevant selectivity index of 15.33 of tsh fraction and its content (compounds 1 and 2) as well as the strong synergism effects observed suggest that the detected anti-hsv activity could be explained by the synergic effects of these major compounds present in the tsh fraction. the management of 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oxyresveratrol derived from thai medicinal plant: mechanism of action and therapeutic efficacy on cutaneous hsv-1 infection in mice virology: a laboratory manual rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays in vitro antiherpes effects of a c-glycosylflavonoid enriched fraction of cecropia glaziovii sneth anti-herpes simplex virus activities of two novel disulphated cyclitols evaluation of the antiherpetic activity of standardized extracts of achyrocline satureioides antiherpes activity of glucoevatromonoside, a cardenolide isolated from a brazilian cultivar of digitalis lanata we would like to thank conselho nacional de desenvolvimento cientí fico e tecnológico the authors declare no conflict of interest.mar. drugs 2013, 11 key: cord-259744-r9j5yzfc authors: mcdonagh, phillip; sheehy, paul a; norris, jacqueline m title: identification and characterisation of small molecule inhibitors of feline coronavirus replication date: 2014-12-05 journal: vet microbiol doi: 10.1016/j.vetmic.2014.10.030 sha: doc_id: 259744 cord_uid: r9j5yzfc feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were 217, 24, and 20 for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. feline infectious peritonitis (fip), a feline coronavirus (fcov) induced disease, is almost invariably fatal with median life expectancy measured in days. current treatment options are, at best, palliative. the objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against fcov in vitro to determine viable candidates for therapy. a resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against fcov. plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and cpe inhibition and ifa-based time of addition assays. three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced cpe at low micromolar concentrations. orthogonal assays confirmed inhibition of cpe was associated with significant reductions in viral replication. selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were 217, 24, and 20 for chloroquine, mefloquine, and hexamethylene amiloride respectively. preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. these results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with fip. ß 2014 elsevier b.v. all rights reserved. a number of compounds have demonstrated an inhibitory effect on the virus in vitro (barlough and shacklett, 1994; hsieh et al., 2010; keyaerts et al., 2007) , but there is little or no published data regarding their use in treating fip. the broad spectrum antiviral ribavirin demonstrated in vitro efficacy but provided limited clinical benefit and produced toxicity in cats (weiss et al., 1993) . more recently in a small study involving experimentally infected cats treatment with chloroquine, a drug with demonstrated in vitro antiviral efficacy, was associated with mild improvements in clinical signs, however there was no statistically significant difference in survival time compared to untreated cats (takano et al., 2013) . efficacious and safe antiviral therapeutics are desperately needed for fip treatment. modern antiviral drug discovery often involves high throughput screening of vast chemical libraries. these large scale unfocused screens are expensive and beyond the reach of companion animal medicine. an alternative approach is to utilise a more focused screening strategy, enriching the screening library with compounds considered likely to have an antiviral effect based on a prior knowledge of their pharmacodynamics and the viral life cycle. focused screening panels may consist of compounds related to those demonstrated effective against the challenge virus or those demonstrated effective against closely related viruses. in the current study we screened 19 compounds with previously demonstrated antiviral activity against coronaviruses or other rna viruses, for antiviral activity against fcov using an optimised resazurin-based cpe inhibition assay. cytotoxicity of compounds was determined prior to screening using sequential resazurin-and srb-based assays to determine the optimal minimally toxic test concentration and to enable calculation of selectivity indices. the antiviral effects of compounds identified during screening were confirmed with plaque reduction and virus yield reduction assays. virucidal suspension assays and time of addition assays provided initial information on the stage of viral replication targeted and the potential mechanism of action. crandell rees feline kidney (crfk) cell line was propagated in dulbecco's modified eagle's medium (dmem; sigma-aldrich, castle hill, nsw, australia) supplemented with 10% fbs (sigma-aldrich) (dmem-10) in a humidified incubator at 37 8c in 5% co 2 in air. two strains of fcov, fipv wsu 79-1146 (fipv1146) and fecv wsu 79-1683 (fecv1683), acquired from the american type culture collection (virginia, usa), were used. fcov fecv1683 was originally isolated from mesenteric lymph nodes and intestinal washes of a 1.5 year old female domestic shorthaired cat that died of acute haemorrhagic gastroenteritis while fcov fipv1146 was originally isolated from the liver, spleen, and lungs from a case of neonatal death in a 4-day-old male persian kitten (mckeirnan et al., 1981) . pathogenicity studies of these two isolates have shown that fipv1146 is highly virulent and reliably causes signs of classic fip following oronasal inoculation, while fecv1683 causes a low grade fever and mild enteritis, but no signs of fip (pedersen, 2009) . despite the dissimilar in vivo biological properties of the two isolates, the two have similar in vitro properties in immortalised cell lines. compounds were selected for the test panel based on their reported in vitro antiviral properties against coronaviruses or other rna viruses (see supplementary material for details). the compounds tested and their screening concentrations are shown in table 1 . stock solutions were prepared by dissolving compounds in ultrapure water or dmso (sigma-aldrich). compounds were sterile filtered with a 0.22 mm regenerated cellulose filter (corning inc., corning, ny, usa), aliquoted into sterile single use microtubes (sarstedt, numbrecht, germany), and stored for a maximum of 6 months at à80 8c until use. to determine an appropriate screening concentration, cytotoxicity of test compounds was determined using sequential resazurin and sulforhodamine b assays. the resazurin-based assay was performed as for the antiviral screening assay except compounds were added in 50 ml volume and there was no infection step. to perform the srb assay, cells were immediately fixed post fluorescent data acquisition by decanting culture media by inverting plates and adding 10% trichloroacetic acid for 1 h at 4 8c. srb staining was as previously described by (vichai and kirtikara, 2006) except that 0.2% srb was used for staining. following solubilisation of bound dye, od510 was measured using the fluostar omega microplate reader (bmg labtech, mornington, australia). viability was compared to untreated controls. test compound concentrations selected for subsequent antiviral screening were those resulting in cell viability of 80% or greater. compounds showing marked, moderate, or mild antiviral effects were defined as those showing 75-100%, 50-74%, and 25-49% inhibition of cpe respectively. compounds demonstrating marked cpe inhibition were classified as candidate compounds and were selected for further characterisation. using the resazurin-based cpe inhibition assay a concentration-response experiment was conducted with serial dilutions of identified candidate compounds (nine concentrations per compound). to enable calculation of the selectivity index, a repeat cytotoxicity screen was performed concurrently. each treatment was performed in triplicate and repeated in three independent experiments. data were exported to microsoft excel for calculation of cell viability and cpe inhibition according to the formulae described above. data analysis were conducted in graphpad prism, with the 50% inhibitory concentration (ic50) and 50% cytotoxic concentration (cc50) values calculated using the inbuilt non-linear curve fitting functions following log 10 transformation of compound concentrations. the selectivity index (si) for each compound was calculated according to the following formula: 2.5. confirmatory assays plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified using the cpe inhibition assay. virus yield reduction assays were performed in 24-well plates (sarstedt). wells were seeded with 4.0 â 10 4 cells well à1 in 400 ml dmem-10. plates were kept at room temperature for 30 min and then at 37 8c in 5% co 2 in air for 5 h prior to the addition of test compounds. compounds were diluted in dmem to the required concentrations with 75 ml added to each well. cells were incubated at 37 8c in 5% co 2 in air for an additional 1 h prior to infection with fcov fipv1146 at moi 0.1 in 25 ml dmem. cells were incubated for a further 48 h at 37 8c in 5% co 2 . at 24 and 48 h post-infection (hpi) cell monolayers were visually assessed for cpe using an olympus ckx41 inverted phase-contrast microscope (olympus, melville, ny, usa) and culture media was collected and stored at à80 8c for virus titration. untreated infected cells, untreated uninfected cells, and treated uninfected cells were included as controls. this latter control was included to allow assessment of morphological changes to cells due to compound treatment. titration of extracellular virus harvested at 24 and 48 hpi was performed using the tcid50 method as described by mcdonagh et al. (2011) . each treatment and time point was performed in triplicate and repeated in two independent experiments, with results representing mean ae se. plaque reduction assays were performed in 12-well plates (corning). cells seeded at a density of 6 â 10 4 cells well à1 in 1 ml dmem-10 were held at room temperature for 30 min prior to incubation at 37 8c in 5% co 2 in air for 60 h, by which time monolayers were approximately 90% confluent. culture media was discarded and replaced with 400 ml dmem supplemented with 2% fbs plus 75 ml of various concentrations of test compounds in dmem (or 75 ml dmem only for control wells) using five or six concentrations per compound. after exposure to the compound for 1 h, cells were infected with 30 pfu well à1 fcov fipv1146 in 25 ml dmem. virus was allowed to adsorb for 90 min with plates rocked every 15 min to ensure an even distribution of inoculum. culture media was discarded after 90 min and cells overlaid with 1 ml 0.9% carboxymethylcellulose, 2% fbs in dmem containing the same concentration of compound as present prior to and during infection. cells were fixed and stained with 0.1% (w/v) crystal violet 48 hpi prior to manual plaque counting. the relative plaque number was calculated for each treatment, with the value of untreated control defined as 100%. each treatment was performed in duplicate, and repeated in three independent experiments, with data representing mean ae se. a virucidal suspension assay was performed to assess virucidal effects of test compounds. the assay was performed as above with the exception that virus was mixed and incubated with test compounds prior to infection. stock fcov fipv1146, diluted in dmem to 2 â 10 6 pfu ml à1 , was mixed with an equal volume of test compound diluted in dmem to 2â the test concentration used during screening. the control virus suspension was mixed with dmem containing an equal concentration of dmso as the test samples. virus suspensions were incubated for 1 h at room temperature before serial dilution in dmem to infect cells with 25 pfu well à1 in 100 ml. following serial dilution of the virus, cells were exposed to test compounds at concentrations greater than 4 log 10 lower than concentrations previously shown to have no antiviral effect. the experiment was performed in triplicate and repeated in two independent experiments. data represent mean ae se. a modification of the resazurin-based cpe inhibition assay was performed to assess the effect of time of compound addition on the antiviral efficacy of identified compounds. the cpe inhibition assay was performed as previously described with the exception that test compounds were added at various time points before and after infection. the selected time points were 1 h prior to infection, concurrent with infection, and 1, 3, or 6 h postinfection. treatments were performed in triplicate and repeated in three independent experiments. data represent mean ae se. to further elucidate the stage of viral replication affected by each compound the effect of time of addition on viral antigen expression was examined. cells were seeded at a density of 5.0 â 10 3 cells well à1 in 100 ml dmem-10 in 96-well plates (mclear 1, greiner bio-one). after seeding plates were kept at room temperature for 30 min and then incubated at 37 8c in 5% co 2 in air for 5 h prior to the first time-point of compound addition. compounds were added in 30 ml to duplicate wells at different time points prior to, concurrent with, or postinfection. cells were infected with fcov fipv1146 at moi 0.5 in 20 ml or mock infected with 20 ml dmem for an infection period of 1 h. an infection period of 12 h was selected based on the reported one step growth curve of fcov (rottier et al., 2005) . at 12 hpi (measured from the end of the infection period) cells were fixed in 20% formaldehyde in pbs and permeabilised in ice cold methanol. viral antigen was detected with a biotinylated anti-fcov antibody (ccv2-2; custom monoclonals international, sacramento, ca, usa) and visualised with streptavidin-conjugated alexafluor 555 (life technologies, mulgrave, vic, australia). to enable accurate segmentation, cells were stained with the whole cell stain hcs cell mask blue (life technologies) and dapi (life technologies) to enhance nuclear visualisation. fluorescent imaging was performed using the bd pathway 855 bioimager (bd bioscience, franklin lakes, nj, usa). images of wells were acquired using a 10â objective (na 0.4) using a 3 â 3 montage with laser autofocus performed for each montage frame. hcs cell mask blue/dapi, images were acquired with ex 380/10 bp and em 435 lp filters, and alexa fluor 555 images acquired with ex 548/20 bp and em 570 lp filters. image analysis was performed using the free opensource image analysis software cellprofiler (r11710, www.cellprofiler.org) with data exported to fcs express image cytometry (version 4.07.0005, de novo software, los angeles, ca, usa) for analysis. each treatment was performed in duplicate, and data represents mean ae sd. to assess efficacy against different fcov strains, identified candidate compounds were tested against fcov fecv1683 using the resazurin-based cpe inhibition assay. the assay was performed as described, except that cells were infected with either fcov fipv1146 or fecv1683 at moi 0.01. each treatment was performed in triplicate and repeated in three independent experiments, with data representing mean ae se. three of nineteen tested compounds showed marked inhibition of virus induced cpe (fig. 1) and were selected for further characterisation. pre-treatment with chloroquine at 25 mm, mefloquine at 10 mm, and hexamethylene amiloride at 10 mm resulted in 93.3%, 89.8%, and 77.6% inhibition of cpe respectively. a further two compounds, glycyrrhizic acid at 25 mm and cinanserin at 20 mm displayed a mild antiviral effect with a 26.7% and 34.0% reduction in cpe respectively. all other compounds demonstrated limited or no inhibitory effect on cpe. included among these ineffective compounds was ribavirin, a broad spectrum antiviral compound that had previously shown in vitro (barlough and scott, 1990; weiss and oostrom-ram, 1989) , and to a limited extent in vivo efficacy against fcov (weiss et al., 1993) , as well as rfeifn-v which had previously shown in vitro efficacy against fcov (mochizuki et al., 1994; truyen et al., 2002) . a concentration-response study was conducted with chloroquine, mefloquine, and hexamethylene amiloride. a repeat cytotoxicity screen was concurrently performed for these compounds to allow calculation of selectivity indices. all compounds demonstrated a clear concentration-response effect over the tested range (fig. 2) . calculated ic50, cc50, and si values for the compounds are shown in table 2 . virus yield reduction assays confirmed the cpe inhibition identified during screening was associated with a marked reduction in extracellular viral titre. determination of extracellular virus titre was performed at 24 and 48 hpi with results shown in fig. 3 . for chloroquine and mefloquine there was a considerable difference in the resulting concentration-response curves at 24 and 48 hpi, while for hexamethylene amiloride the shape of the curve was similar at both time points. differences in concentration-response curves between the two time points is reflected in the ic50 values, with increased ic50 values for chloroquine and mefloquine at 48 hpi compared to 24 hpi, while for hexamethylene amiloride ic50 values were similar at both time points (table 3) . plaque reduction assays confirmed the findings of the cpe inhibition and virus yield reduction assays. pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride resulted in a concentration-dependent decrease in plaque number, with high concentrations completely inhibiting macroscopic plaque formation. for all compounds plaque morphology was similar between treated and untreated wells however plaque size was smaller in treated versus untreated wells. during the virus yield reduction assay cells were monitored for the development of cpe using phase contrast microscopy. it was noted that infected and uninfected cells treated with chloroquine, mefloquine, or hexamethylene amiloride displayed characteristic morphological changes. these changes consisted of a large number of variably sized cytoplasmic (predominantly perinuclear) inclusions in addition to the presence, in some cells, of an increased number of cytoplasmic vacuoles. to investigate the nature of these inclusions, separate wells were stained with 33 mg ml à1 neutral red in dmem for 2 h. these inclusions appeared to accumulate the vital dye neutral red following suggesting they were likely dilated endosomes/lysosomes (fig. 4) . using a virucidal suspension assay no virucidal effects were seen for chloroquine, mefloquine, or hexamethylene amiloride, with the infectivity of virus suspensions exposed to the compounds not significantly different from virus incubated with media alone. the effect of time of addition on the antiviral activity of selected compounds was assessed using a modification of the resazurin-based cpe inhibition assay and through ifa of viral protein expression. based on the cpe inhibition assay maximum antiviral effect was seen when compounds were added prior to or concurrent with infection, following which there was a time-dependent reduction in cpe inhibition (fig. 5) . for all tested compounds cpe inhibition remained greater than 50% when compounds were added at the latest tested time point of 6 h postinfection. the cpe inhibition assay encompasses multiple rounds of viral replication. to further elucidate the stage of viral replication affected by test compounds a single replication cycle ifa-based assay was conducted which confirmed that, based on viral antigen expression, all three compounds possess antiviral properties when added prior to, or at the time of infection. furthermore all compounds displayed a time of addition dependent reduction in antiviral effect; however the extent and timing of this reduction varied. the inhibitory effect of chloroquine was reduced, based on an increase in the percentage of fcov antigen positive cells, when added at any time postinfection (fig. 6) . a similar result was seen for hexamethylene amiloride, although in this case a significant increase in the number of infected cells was not seen until compound addition was delayed until 1 hpi. in contrast, mefloquine remained effective when added up to 5 hpi suggesting it may act at a later stage of viral replication than chloroquine and hexamethylene amiloride. the efficacy of the three identified candidate compounds was tested against fcov fecv1683, a serotype ii enteric biotype fcov. comparison of the virus control (no treatment) wells showed fcov fipv1146 infection resulted in more pronounced cpe over the 72 h infection period compared to fcov fecv1683. pre-treatment with chloroquine, mefloquine, or hexamethylene amiloride provided a degree of protection against strain fcov fecv1683. pretreatment with hexamethylene amiloride provided protection against virus induced cpe that was similar for the two strains, with a reduction in cpe of 89.5% and 86.0% for fcov fipv1146 and fecv1683 respectively. both chloroquine and mefloquine however were more effective against fcov fipv1146 than fecv1683, with cpe inhibition for chloroquine of 76.9% for versus 63.8%, and for mefloquine 79.0% versus 67.5% for strains fipv1146 and fecv1683 respectively. this study identifies three compounds (chloroquine, mefloquine, and hexamethylene amiloride) demonstrating a marked inhibitory effect on fcov replication in vitro by significant reductions in virus induced cpe and viral titres at low micromolar concentrations when present during the early stages of viral replication. an antiviral effect of chloroquine had previously been demonstrated against fcov, and hexamethylene amiloride had previously demonstrated efficacy against other coronaviruses, however this is the first demonstration of antiviral efficacy of mefloquine against a coronavirus. initial compound screening was performed using a cpe inhibition assay, with subsequent virus yield reduction assays and plaque reduction assays used for confirmatory testing. for the effective compounds the ic50 values, and corresponding selectivity index, varied with the assay method utilised. this is not unexpected given the assays measure different endpoints, and has been reported for other antiviral drugs such as the retroviral protease inhibitor saquinavir where the reported ic50 calculated based on production of viral p24 antigen is approximately 30-fold lower than that based on production of mature virions (buss and cammack, 2001) . similarly variation in assay conditions may result in the calculation of significantly different ic50 values. the concentration-response curve of chloroquine against sars-cov determined using a pcr based virus yield reduction assay was shown to shift considerably to the right when viral genome copies were assayed 3 days post-infection compared to 1 day postinfection (keyaerts et al., 2004) . a similar finding was noted in the current study for both chloroquine and mefloquine, with differences in potency reported with the tcid50 based virus yield reduction assay performed at 24 and 48 hpi, however this was not seen for hexamethylene amiloride. two compounds, ribavirin and rfeifn-v, which had previously demonstrated in vitro efficacy against fcov, failed to demonstrate significant inhibition of cpe during screening. for both compounds these discordant results are likely attributable to testing at concentrations below their useful therapeutic range and variations in assay conditions and sensitivity compared with previous work. the screening concentration of compounds used in this study was determined based on cytotoxicity testing to achieve cell viability greater than 80%. previous studies with ribavirin demonstrated ic50 values of 41.7 mg ml à1 (170 mm) (barlough and scott, 1990 ) based on a visual assessment of protection from cytopathic effect and 2.5 mg ml à1 (10.2 mm), based on the reduction of extracellular viral titre (weiss and oostrom-ram, 1989) . the concentration used for screening (2.5 mm) was therefore more than 60 times lower than the ic50 previously calculated based on a similar assay endpoint. from the results of the current study, virus yield reduction assays appear to provide a more sensitive assessment of antiviral efficacy than cpe inhibition assays, with the ic50 values calculated based on viral titre reduction significantly lower than those calculated based on cpe inhibition for all compounds. a small antiviral effect of ribavirin cannot therefore be ruled out based on the current findings, as although the tested concentrations did not provide protection against virus induced cpe, it may have been associated with a reduction in extracellular viral titre. the practical relevance of such a small antiviral effect is questionable, particularly given the known toxicity profile of this compound in cats. for rfeifn-v reductions in viral titres of 0.2-1.2 logs have been reported when crfk cells were treated with 50,000 u ml à1 1 h post-infection (truyen et al., 2002) and 0.5-0.6 logs when fcwf cells were pre-treated with 100-100,000 u rfeinf-v (mochizuki et al., 1994) . protection from cpe was not seen in the current study when cells were pre-treated with rfeinf-v at 100 u ml à1 , a concentration significantly lower than that previously shown to be effective using the same virus strain and cell line (truyen et al., 2002) . the tested concentration was however similar to that used by mochizuki et al. (1994) . this apparent lack of efficacy in this case may reflect differences in the drug exposure and infection conditions, the viral isolate tested, or an intrinsic enhanced susceptibility to the antiviral effects of interferon in fcwf cells compared to crfk cells as used in this study (weiss and toivio-kinnucan, 1988) . alternatively it may be that, as suggested for ribavirin, virus yield reduction assays provide a more sensitive assessment of antiviral effects than cpe inhibition assays, and that the screening method utilised failed to identify mild antiviral effects. a number of different mechanisms of action have been suggested to account for the antiviral properties of the compounds identified in this study against other viruses. for chloroquine antiviral effects have been ascribed to inhibition of glycosylation of viral proteins (savarino et al., 2004) or cellular receptors for viral attachment (vincent et al., 2005) , inhibition of glycoprotein expression (dille and johnson, 1982) , or inhibition of endosome mediated viral entry (savarino et al., 2003) . the antiviral effect of mefloquine against jc virus has been postulated to be due to its action as an adenosine mimetic (brickelmaier et al., 2009) , while for hexamethylene amiloride it has been suggested antiviral properties against different viruses may arise through competitive inhibition of viral rna polymerase (gazina et al., 2011) , an indirect mutagenic effect (levi et al., 2010) , or inhibition of viroporins (wilson et al., 2006) . interestingly all three compounds showing marked antiviral efficacy against fcov in this study resulted in similar morphological changes in cells exposed to sub-toxic concentrations. increased numbers of variably size cytoplasmic inclusions that accumulate the viral dye neutral red suggests these compounds result in a perturbation of the normal endocytic pathway in crfk cells. alterations in the endocytic pathway have previously been reported for chloroquine (dean et al., 1984) , mefloquine (labro and babin-chevaye, 1988) , and for amiloride and some of its derivatives (dutta and donaldson, 2012 ). this suggests a common physiological effect on treated cells for all three candidate antivirals and possibly a shared mechanism of action. viruses are known to usurp a variety of host endocytic pathways for cell entry and intracellular movement and inhibition of these pathways may be a useful therapeutic approach. although targeting a cellular pathway may be associated with an increased risk of toxicity, if that pathway is critical for viral replication this approach may slow or limit the development of resistance. time of addition studies demonstrated all compounds were most effective when added prior to infection, suggesting a mechanism of action involving early stages of viral replication. the cpe inhibition based time of addition assay involved infection at low moi with a 72 h infection period, allowing for multiple rounds of viral replication. as a result of this, even with the delayed addition of compounds, cells uninfected by the original inoculum are effectively pre-treated prior to challenge with progeny virions produced during the primary replication cycle. using an ifa-based time of addition study involving a single replication cycle we were able to further clarify of the effect of time of addition, and refine the possible stage of the viral life cycle targeted by each compound. based on the ifa results chloroquine was effective only if present at the time of infection, supporting the hypothesis that chloroquine acts during cell entry for fcov fipv1146, possibly through inhibition of endosomal ph (takano et al., 2008) . hexamethylene amiloride and mefloquine provided significant antiviral effects when compound addition was delayed for up to 1 and 5 hpi respectively, suggesting that if the antiviral effects of these compounds arise through perturbation of endosomal function, the effects occur at different stages of the viral life cycle. alternatively distinct mechanisms of action may account for the observed effects of these compounds, as suggested for other viruses. there is limited published pharmacokinetic or safety data to inform the potential therapeutic application of the identified compounds in cats and given the relatively low si of all three compounds consideration must be given to their in vivo safety in this species. the human approved pharmaceuticals chloroquine and mefloquine are generally considered well-tolerated drugs, albeit with a narrow therapeutic index, while the clinical use of hexamethylene amiloride has not been reported. pharmacokinetic data available for chloroquine and mefloquine in humans would suggest that effective plasma concentrations could be achieved at standard therapeutic doses (pussard and verdier, 1994; simpson et al., 1999) . chloroquine has been shown to accumulate in leukocytes, where the concentration may be two orders of magnitude greater than that of plasma (mackenzie, 1983) , with the highest concentration reported in monocytes (french et al., 1987) . thus therapeutic concentrations may be attained in the target cells of virulent biotype fcovs at relatively low plasma concentrations, minimising the risk of dose-dependent adverse effects. mefloquine is known to accumulate within brain parenchyma at concentrations approximately 10-30 times higher than found in serum, with tissue concentrations of up to 50 mm reported (nevin, 2009; pham et al., 1999) . mefloquine may therefore be useful in the treatment of dry (non-effusive) fip, where cns lesions are common (pedersen, 2009 ) although the potential for neurotoxicity must be considered. although the concentration of mefloquine achieved in the cns is greater than the cc50 of this compound in immortalised feline kidney cells, in humans this tissue concentration is achievable at therapeutic doses, despite in vitro data in human cells showing a cc50 approximately equal to that determined in the current study (brickelmaier et al., 2009) . it may be therefore that the more static cell population of the cns is more refractory to the toxic effects of mefloquine than mitotically active immortalised cells. this study has identified three compounds demonstrating marked in vitro inhibition of fcov in an immortalised cell line at low micromolar concentrations, including the first demonstration of antiviral effects of mefloquine against a coronavirus. although the low si of the three compounds may limit their therapeutic utility, these preliminary studies open the way for further investigation and potential optimisation of these compounds as antiviral agents. effectiveness of three antiviral agents against fip virus in vitro antiviral studies of feline infectious peritonitis virus in vitro identification and characterization of mefloquine efficacy against jc virus in vitro. antimicrob measuring the effectiveness of antiretroviral agents effects of exogenous amines on mammalian cells, 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inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication this study was supported by donations from the rex cat club (especially sharon barton and tracey gleeson), participants of the national annual feline health seminars, christine atkins, ruth thurling, cat fanciers association of nsw, and the cat protection society of nsw. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2014.10.030. key: cord-013387-q91052qw authors: leão, rozires p.; cruz, josiane v.; da costa, glauber v.; cruz, jorddy n.; ferreira, elenilze f. b.; silva, raí c.; de lima, lúcio r.; borges, rosivaldo s.; dos santos, gabriela b.; santos, cleydson b. r. title: identification of new rofecoxib-based cyclooxygenase-2 inhibitors: a bioinformatics approach date: 2020-08-26 journal: pharmaceuticals (basel) doi: 10.3390/ph13090209 sha: doc_id: 13387 cord_uid: q91052qw the cyclooxygenase-2 receptor is a therapeutic target for planning potential drugs with anti-inflammatory activity. the selective cyclooxygenase-2 (cox-2) inhibitor rofecoxib was selected as a pivot molecule to perform virtual ligand-based screening from six commercial databases. we performed the search for similarly shaped rapid overlay of chemical structures (rocs) and electrostatic (eon) compounds. after, we used pharmacokinetic and toxicological parameters to determine the best potential compounds, obtained through the softwares qikprop and derek, respectively. then, the compounds proceeded to the molecular anchorage study, which showed promising results of binding affinity with the hcox-2 receptor: lmqc72 (∆g = −11.0 kcal/mol), lmqc36 (∆g = −10.6 kcal/mol), and lmqc50 (∆g = −10.2 kcal/mol). lmqc72 and lmqc36 showed higher binding affinity compared to rofecoxib (∆g = −10.4 kcal/mol). finally, molecular dynamics (md) simulations were used to evaluate the interaction of the compounds with the target hcox-2 during 150 ns. in all md simulation trajectories, the ligands remained interacting with the protein until the end of the simulation. the compounds were also complexing with hcox-2 favorably. the compounds obtained the following affinity energy values: rofecoxib: δgbind = −45.31 kcal/mol; lmqc72: δgbind = −38.58 kcal/mol; lmqc36: δgbind = −36.10 kcal/mol; and lmqc50: δgbind = −39.40 kcal/mol. the selected lmqc72, lmqc50, and lmqc36 structures showed satisfactory pharmacokinetic results related to absorption and distribution. the toxicological predictions of these compounds did not display alerts for possible toxic groups and lower risk of cardiotoxicity compared to rofecoxib. therefore, future in vitro and in vivo studies are needed to confirm the anti-inflammatory potential of the compounds selected here with bioinformatics approaches based on rofecoxib ligand. cyclooxygenases are an important and thoroughly studied group of enzymes present in two isoforms in mammals: constitutive form cyclooxygenase-1 (cox-1) and an inducible form cyclooxygenase-2 (cox-2) [1] . the cox-1 enzyme is expressed in most tissues and is responsible for maintaining homeostasis and production of prostaglandins (pgs) [2] . cox-2 is found predominantly in the brain, renal, and endothelial cells and is significantly increased through various acute and chronic inflammatory infections [3, 4] . the inhibition of the cox-2 enzyme through selective anti-inflammatory drugs has been an important strategy to contain the inflammation process. many selective cox-2 inhibitors achieve the market as anti-inflammatory drugs, such as celecoxib (celebrex ® ), valdecoxib (bextra ® ), and rofecoxib (vioxx ® ) [5] , once it was thought that more selective drugs leads to less gastric side-affects-see figure 1 . nevertheless, some of these selective inhibitors of cox-2 also depress prostacyclin (pgi2), an atheroprotective agent, which might predispose patients to heart attack and stroke [6] . pharmaceuticals 2020, 13, x for peer review 2 of 27 cyclooxygenases are an important and thoroughly studied group of enzymes present in two isoforms in mammals: constitutive form cyclooxygenase-1 (cox-1) and an inducible form cyclooxygenase-2 (cox-2) [1] . the cox-1 enzyme is expressed in most tissues and is responsible for maintaining homeostasis and production of prostaglandins (pgs) [2] . cox-2 is found predominantly in the brain, renal, and endothelial cells and is significantly increased through various acute and chronic inflammatory infections [3, 4] . the inhibition of the cox-2 enzyme through selective anti-inflammatory drugs has been an important strategy to contain the inflammation process. many selective cox-2 inhibitors achieve the market as anti-inflammatory drugs, such as celecoxib (celebrex ® ), valdecoxib (bextra ® ), and rofecoxib (vioxx ® ) [5] , once it was thought that more selective drugs leads to less gastric side-affects-see figure 1 . nevertheless, some of these selective inhibitors of cox-2 also depress prostacyclin (pgi2), an atheroprotective agent, which might predispose patients to heart attack and stroke [6] . thus, the side effects promoted by these therapeutic agents directed the search for new compounds, whose anti-inflammatory potential is accompanied by greater selectivity and specificity, minimal side effects, and lower cost [7] . rofecoxib (vioxx ® ) was approved by the food and drug administration (fda) for human use in may 1999, and withdrawn from the market on september, 2004 [8] . this drug, from the coxibs family, presents risks of cardiovascular events; however, it presents anti-inflammatory effects and properties similar to traditional non-steroidal anti-inflammatory drugs (nsaids) with reduced gastrointestinal toxicity, which would have the potential [5] [6] [7] [8] [9] without side-effects, such as ulcers and gastrointestinal problems [10] . since then, rofecoxib became an important prototype for the design of new promising nsaids for the cox-2 target and with possible minor side effects in humans [11] . in this work, we used a virtual screening ligand-based methodology to identify new potential cox-2 inhibitors based on the rofecoxib structure [12] [13] [14] . the virtual screening strategy was chosen once it has been widely applied in the early phase of drug discovery, being able to accelerate hit discovery and reducing drug development costs. thus, the similarity and electrostatic potential of the selected structures were performed using computer programs and commercial databases of compounds [15] and then we performed the filtering of the results considering the pharmacokinetic and toxicological properties [16] [17] [18] [19] [20] . furthermore, the docking simulation evaluated the binding affinity of compounds to cox-2 in comparison with rofecoxib [20, 21] . biological target prediction was used as a screening step through the web server swiss [22] and the bioactivity was determined on the molinspiration web server [23] . finally, we used molecular dynamics to investigate interaction over time in cox-2 of the promising compounds. general scheme of the methodological steps in this article is presented in figure 2 (see more details in the materials and methods section). thus, the side effects promoted by these therapeutic agents directed the search for new compounds, whose anti-inflammatory potential is accompanied by greater selectivity and specificity, minimal side effects, and lower cost [7] . rofecoxib (vioxx ® ) was approved by the food and drug administration (fda) for human use in may 1999, and withdrawn from the market on september, 2004 [8] . this drug, from the coxibs family, presents risks of cardiovascular events; however, it presents anti-inflammatory effects and properties similar to traditional non-steroidal anti-inflammatory drugs (nsaids) with reduced gastrointestinal toxicity, which would have the potential [5] [6] [7] [8] [9] without side-effects, such as ulcers and gastrointestinal problems [10] . since then, rofecoxib became an important prototype for the design of new promising nsaids for the cox-2 target and with possible minor side effects in humans [11] . in this work, we used a virtual screening ligand-based methodology to identify new potential cox-2 inhibitors based on the rofecoxib structure [12] [13] [14] . the virtual screening strategy was chosen once it has been widely applied in the early phase of drug discovery, being able to accelerate hit discovery and reducing drug development costs. thus, the similarity and electrostatic potential of the selected structures were performed using computer programs and commercial databases of compounds [15] and then we performed the filtering of the results considering the pharmacokinetic and toxicological properties [16] [17] [18] [19] [20] . furthermore, the docking simulation evaluated the binding affinity of compounds to cox-2 in comparison with rofecoxib [20, 21] . biological target prediction was used as a screening step through the web server swiss [22] and the bioactivity was determined on the molinspiration web server [23] . finally, we used molecular dynamics to investigate interaction over time in cox-2 of the promising compounds. general scheme of the methodological steps in this article is presented in figure 2 (see more details in the materials and methods section). in this initial stage, the pivot molecule rofecoxib was used as a research model for the virtual screening in six commercial molecule databases: chembridge diversetexp, diverset core library (https://www.chembridge.com) [24] , maybridge collections (www.maybridge.com) [25, 26] , zinc drug database, zinc natural stock (http://zinc.docking.org) [27] , and drug fda bindingdb (http://www.bindingdb.org) [27] using the programs rapid overlay of chemical structures (rocs) and electrostatic similarity (eon). in the rocs software [28] [29] [30] , we used a virtual screening tool for searching three-dimensional (3d) structures with chemical similarity and shape with the pivot molecule rofecoxib [16, 28] . the rofecoxib molecule was used as a comparison model with each of the molecules in the databases looking for chemical similarity [16, 31] , according to the structural characteristics and molecular volume fractions of the pivot molecule, observing the maximum overlap in relation to the shape (chemical structure), using as a parameter the gaussian functions [32] implemented in the rocs software. the compounds were selected and classified by means of an algorithm that generated relative scores for the overlapping of forms in the databases according to the pharmacophoric characteristics of rofecoxib [15, 33] . this stage of virtual screening identified the most similar two thousand (2000) molecules in each database (top_2000), resulting in twelve thousand (12,000) tracked structures, which exhibited highest scores of chemical similarities. in the sequence, the selected compounds were submitted to electrostatic correlations of aligned molecules based on the tanimoto electrostatic score in eon software [34, 35] . this electrostatic potential is calculated using openeye's poisson-boltzmann (pb) electrostatic calculation [33, 36] . the top 100 molecules by database (top_100), led to six hundred structures (600) hits with best alignment based on the electrostatic potential [15] . the remaining six hundred structures (600) were then evaluated for their pharmacokinetic properties (the absorption, distribution, metabolism) using the qikprop software [37] [38] [39] . structures submitted for pharmacokinetic study, resulted in two hundred and thirty-three (233) hits that presented satisfactory pharmacokinetic properties, especially electronic affinity, lipinski's rule, and the central nervous system (cns) parameter, when compared with the properties of rofecoxib. in this initial stage, the pivot molecule rofecoxib was used as a research model for the virtual screening in six commercial molecule databases: chembridge diversetexp, diverset core library (https://www.chembridge.com) [24] , maybridge collections (www.maybridge.com) [25, 26] , zinc drug database, zinc natural stock (http://zinc.docking.org) [27] , and drug fda bindingdb (http://www. bindingdb.org) [27] using the programs rapid overlay of chemical structures (rocs) and electrostatic similarity (eon). in the rocs software [28] [29] [30] , we used a virtual screening tool for searching three-dimensional (3d) structures with chemical similarity and shape with the pivot molecule rofecoxib [16, 28] . the rofecoxib molecule was used as a comparison model with each of the molecules in the databases looking for chemical similarity [16, 31] , according to the structural characteristics and molecular volume fractions of the pivot molecule, observing the maximum overlap in relation to the shape (chemical structure), using as a parameter the gaussian functions [32] implemented in the rocs software. the compounds were selected and classified by means of an algorithm that generated relative scores for the overlapping of forms in the databases according to the pharmacophoric characteristics of rofecoxib [15, 33] . this stage of virtual screening identified the most similar two thousand (2000) molecules in each database (top_2000), resulting in twelve thousand (12,000) tracked structures, which exhibited highest scores of chemical similarities. in the sequence, the selected compounds were submitted to electrostatic correlations of aligned molecules based on the tanimoto electrostatic score in eon software [34, 35] . this electrostatic potential is calculated using openeye's poisson-boltzmann (pb) electrostatic calculation [33, 36] . the top 100 molecules by database (top_100), led to six hundred structures (600) hits with best alignment based on the electrostatic potential [15] . the remaining six hundred structures (600) were then evaluated for their pharmacokinetic properties (the absorption, distribution, metabolism) using the qikprop software [37] [38] [39] . structures submitted for pharmacokinetic study, resulted in two hundred and thirty-three (233) hits that presented satisfactory pharmacokinetic properties, especially electronic affinity, lipinski's rule, and the central nervous system (cns) parameter, when compared with the properties of rofecoxib. the "surviving structures" were submitted to derek software [40] to evaluate toxicological properties, having as reference the properties of the commercial drug rofecoxib. thus, only seventy-nine structures were selected because they did not present toxicity alerts and toxicophoric groups [21] . subsequently, these structures were subjected to a molecular study to assess binding mode and affinity with hcox-2 receptor. at the end of this process, only three structures (lmqc72, lmqc36, and lmqc50) were selected, for having binding affinity with the cox-2 molecular target and good pharmacokinetic and toxicological profile. therefore, this study discusses the main selected structures (lmqc72, lmqc36, and lmqc50) that offer promising results with the therapeutic ligand of interest. in silico prediction of absorption, distribution, metabolism, excretion, and toxicity (admet) properties are fundamental for the selection of the most promising molecules for further development. the selected structures were subjected to predictions of pharmacokinetic properties absorption, distribution, metabolism, and elimination using the qikprop software. to evaluate these properties, nine parameters (see table 1 ) were used, related to the inflammatory process, and based on the compound rofecoxib. the #star parameter compares results obtained with properties of drugs present in database of the qikprop software [37] . an alert is given when a result is outside the 95% range of values similar to commercially available drugs. this parameter takes into account a set of properties and descriptors such as: molecular weight (mw), dipole moment, electron affinity (ea), total solvent accessible surface area (sasa), hydrophobic component of the sasa (fosa), hydrophilic component of the sasa (fisa), π (carbon and attached hydrogen) component of the sasa (pisa), weakly polar component of the sasa (halogens, p, and s) (wpsa), polar surface area (psa), molecular volume, number of rotatable bonds (#rotor), number of hydrogen bond donor groups (donorhb), number of hydrogen bond acceptor groups (accpthb), predicted polarizability in cubic angstroms (qppolrz), predicted hexadecane/gas partition coefficient (qplogpc16), predicted octanol/gas partition coefficient (qplogpoct), predicted water/gas partition coefficient (qplogpw), predicted octanol/water partition coefficient (qplogpo/w), predicted aqueous solubility (logs), prediction of binding to human serum albumin (qplogkhsa), predicted brain/blood partition coefficient (qplogbb), number of likely metabolic reactions (#metabol) [38] . these results for the three selected compounds are shown in table 1 . the pharmacokinetic predictions for lmqc72, lmqc35, and lmqc50 show no violations in the descriptors and properties analyzed, which indicates that its properties are similar to commercial drugs (#star = 0). however, rofecoxib has an alert (#star = 1) in the molecular descriptor electronic affinity (ea), which is out of range (−0.9 to 1.7), with a value of 1.99 ev. ea is an essential characteristic for intermolecular interactions and charge transfer complex [41] [42] [43] [44] . lipinski's (ro5) investigation are based on molecular weight (mw), lipophilicity (represented by the partition coefficient, logp) and hydrophilicity (represented by the number of hydrogen bond donors and acceptors groups) descriptors. ro5 represents a well-established form of limits for the absorption and permeability of a drug [45] . in this study, lmqc72, lmqc36, and lmqc50 showed no violations to ro5, indicating that these compounds would make it a likely orally active drug in humans. rofecoxib is an orally administered drug and in consonance, its properties did not violate the rule of lipinski (ro5). thus, this result predicts similarity to biological activity designed for oral administration [37, 46] . the percentage of human oral absorption (%hoa) was evaluated through a set of properties based on number of metabolites (#metab), number of rotating bonds (#rotor), solubility and cell permeability in comparison within the standards [38] . the prediction %hoa of the selected compounds showed excellent results, once lmqc72, lmqc36, and lmqc50 exhibited values of 100% hoa. moreover, rofecoxib showed a value of 82.40% hoa, which indicates a better oral absorption of the novel compounds. ; e apparent permeability of compound between octanol/water (qplogpo/w); f permeability of the differentiated cells of intestinal epithelium caco-2 (qppcaco); g madin-darby canine kidney (qppmdck); h activity in the central nervous system; i apparent permeability of compound in the blood-brain barrier [38] . pharmaceuticals 2020, 13, 209 6 of 26 the apparent permeability between octanol/water (qplogpo/w) is a parameter used in drug design processes to estimate solubility, membrane permeability, and bioavailability [47] [48] [49] [50] . the calculated values regarding qlogpo/w for lmqc72, lmqc36, and lmqc50 are higher than the value found for rofecoxib (qplogpo/w = 1.45). lmqc72, lmqc36, and lmqc50 values ranged from 2.18 ≥ qlogpo/w ≥ 4.21, considered more lipophilic compounds (logpo/w ≥ 0). thus, this means that the novel compounds are mainly absorbed by passive transcellular processes in the intestine. lmqc72, lmqc36, and lmqc50 are within the limits indicated in ranges 2 to 5, favoring better absorption, that is, easily overcome the lipid bilayer of biological membranes [51] . models predictive of intestinal drug absorption are important in drug development to identify compounds with promising biopharmaceutical properties [52] . in this study, the intestinal absorption was estimated by caco-2 and madin-darby canine kidney (mdck) cell values [53] . predictions values of these cells make it possible to evaluate the cell permeability of potential drug candidates and routes of drug transport (e.g., passive versus carrier mediated) [54, 55] . descriptors used for the prediction of passive transport should have values above 500 nm/s to be considered good, whereas values less than 25 nm/s are considered poor. lmqc72, lmqc36, and lmqc50 showed values between 1470.77 and 1751.71 nm/s for caco-2 cells and between 900.66 and 3415.52 nm/s for mdck cells. thus, the compounds showed good results, indicating a promising intestinal absorption and even better in comparison with rofecoxib. the blood-brain barrier (bbb) is a critical factor in drug design. high penetration is needed for cns-active drugs, while negligible penetration may be desirable in order to minimize cns-related side-effects of drugs with a peripheral site of action [56, 57] it is a selective barrier formed by narrow junctions between endothelial cells, to limit the penetration of different blood substances in the brain [56, 58] . in our study, compounds lmqc71, lmqc36, and lmqc50 were evaluated by the brain-blood partition coefficient (qplogbb). the parameter established to indicate inactivity for penetration into the blood-brain barrier and consequent cns activity includes values below 1 (c brain /c blood < 1) and, for values greater than 1, it suggests activity in the central nervous system [20, 37] . evaluation of the penetration capacity (qplogbb) of the lmqc72, lmqc36, and lmqc50 exhibited negative values (<1) which reveals low penetrability to cns [57] . then, the prediction of the central nervous system activity of the selected compounds was performed. the established cns activity parameter ranges from −2 (inactive) to +2 (active). in our study, lmqc72, lmqc36, and lmqc50 exhibited values equal to zero (0), which indicates that they are inactive and do not produce cns side effects in humans [38] . therefore, these results are similar to the pivot compound rofecoxib, which has values of below 1 (inactive) for the parameters: qplogbb and cns. in terms of pharmacokinetic properties, one may evaluate that the new hcox-2 inhibitors show better pharmacokinetic performance without violations in their descriptors and molecular properties when compared to rofecoxib. the seventy-nine compounds selected here by the toxicological studies followed the study of molecular docking to assess the binding mode and affinity with the hcox-2 receptor. to validate the molecular docking protocol, the crystallographic ligand was re-docked in the hcox-2 with the protein data bank (pdb) id 5kir structure with resolution 2.69 å [14] . the root mean square deviation (rmsd) obtained by re-docking, and the bonding pose found in the complex was 0.98 å [15] . the comparison between the crystallographic ligand and the pose predicted by docking overlap of the ligand can be visualized in figure 3 . according to literature, the binding mode prediction using docking should present rmsd value <2.0 å when superimposed to the crystallographic pose of the ligand [20, 59, 60] . we also evaluated the interaction affinity of rofecoxib to hcox-2. the binding affinity value obtained in re-docking was ∆g = −10.4 kcal/mol. it was considered close to the experimental value (∆g = −9.2 kcal/mol). thus, our protocol showed satisfactory performance in predicting the interaction conformation once the interaction affinity value was close to the observed experimentally, see table 2 . the seventy-nine compounds selected here by the toxicological studies followed the study of molecular docking to assess the binding mode and affinity with the hcox-2 receptor. to validate the molecular docking protocol, the crystallographic ligand was re-docked in the hcox-2 with the protein data bank (pdb) id 5kir structure with resolution 2.69 å [14] . the root mean square deviation (rmsd) obtained by re-docking, and the bonding pose found in the complex was 0.98 å [15] . the comparison between the crystallographic ligand and the pose predicted by docking overlap of the ligand can be visualized in figure 3 . according to literature, the binding mode prediction using docking should present rmsd value <2.0 å when superimposed to the crystallographic pose of the ligand [20, 59, 60] . we also evaluated the interaction affinity of rofecoxib to hcox-2. the binding affinity value obtained in re-docking was ∆g = −10.4 kcal/mol. it was considered close to the experimental value (∆g = −9.2 kcal/mol). thus, our protocol showed satisfactory performance in predicting the interaction conformation once the interaction affinity value was close to the observed experimentally, see table 2 . protein-ligand binding affinity is essential for biological processes, as these physical and chemical interactions determine biological recognition at the molecular level. in this way, it is possible to look for a ligand capable of inhibiting or activating a specific target protein through its interaction. in such a way, it is important to find a ligand that binds to a target protein with high affinity [61] . all 79 compounds that showed good pharmacokinetic and toxicological profiles were subjected to the molecular docking simulations in order to verify the binding affinity at the target receptor binding site (hcox-2, pdb 5kir). binding affinity values of the compounds with higher affinity to hcox-2 compounds are shown in figure 4 . protein-ligand binding affinity is essential for biological processes, as these physical and chemical interactions determine biological recognition at the molecular level. in this way, it is possible to look for a ligand capable of inhibiting or activating a specific target protein through its interaction. in such a way, it is important to find a ligand that binds to a target protein with high affinity [61] . all 79 compounds that showed good pharmacokinetic and toxicological profiles were subjected to the molecular docking simulations in order to verify the binding affinity at the target receptor binding site (hcox-2, pdb 5kir). binding affinity values of the compounds with higher affinity to hcox-2 compounds are shown in figure 4 . tables 3 and 4 . molecular docking studies (autodock/vina) [62] also allowed us to determine the types of interactions between the target receptor's binding site with the promising compounds lmqc72, lmqc36 and lmqc50. table 3 shows the interactions between the hcox-2 inhibitor rofecoxib (pdb id 5kir). in comparison with the pivot, table 4 shows the types of interactions and amino acid residues between hcox-2 and lmqc72, lmqc36, and lmqc50. crystallographic complex of rofecoxib with hcox-2 deposited in the pdb under the code 5kir exhibits the main interactions in the region of monomer b of the protein. rofecoxib methyl sulfone group binds to the active site of the enzyme, specifically with residues: his90 and arg513 in the α helix of the hydrophilic part of hcox-2 (chain b) [14, 63] . figure 5a shows the amino acid residues phe518, leu352, ala527, ser530, val349, and val523 of hcox-2 interacting with rofecoxib [14] . experimental data shows that the selected compounds share the following interactions with hcox-2: lmqc72 makes a hydrophobic and pi-alkyl type interaction with residues val523 and ala527, respectively in the α-helix and β-leaf regions of the protein ( figure 5b ). lmqc36 makes a hydrophobic and pi-alkyl interactions with ala527 residue located in the α-helix region of the protein ( figure 5c ). lmqc50 interacts with three amino acid residues that are present in the hcox-2 interaction with rofecoxib, which are val349 (pi-alkyl), phe518 (pi-pi stacked) and arg513 (hydrogen bond), in the α-helix region of the protein ( figure 5d ). therefore, the evaluation carried out through the autodock vina program enables us to affirm that the selected compounds are close to the interactions made with the rofecoxib (rcx) ligand (5kir) at the active site of hcox-2, as the molecules share the main interactions in the hydrophobic part with the and amino acid residues val523, val349, ala527, phe518, and arg513 linked by b chain. table 5 summarizes the chemical information from the selected structures resulting from the molecular docking study. the three remaining compounds were subjected to the bioactivity prediction, through the molinspiration server (https://www.molinspiration.com/). in this prediction, table 5 summarizes the chemical information from the selected structures resulting from the molecular docking study. the three remaining compounds were subjected to the bioactivity prediction, through the molinspiration server (https://www.molinspiration.com/). in this prediction, biological activity measured by the bioactivity score for enzyme inhibitor was evaluated enzyme (see table 6 ), which are classified into three different ranges: molecule having bioactivity score more than 0.00 is most likely to possess considerable biological activities, while values −0.50 to 0.00 are expected to be moderately active, and if score is less than −0.50, it is presumed to be inactive [21] . the bioactivity scores of the lmqc72, lmqc36, and lmqc50 structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [23] . a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [64] . the bioactivity scores of the lmqc72, lmqc36, and lmqc50 structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [23] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc72, lmqc50 and rofecoxib structures with values greater than 0.00. meanwhile, the lmqc36 has a moderately active score between −0.5 to 0.00. the score values of the lmqc 72, lmqc36, and lmqc50 structures are considered good because they are close to the pivot compound with probable biological activity (see table 6 ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [65] . the results of the ion channel modulators' scores for the lmqc36, lmqc50, and rofecoxib structures are estimated score values between −0.50 to 0.00 considered moderately active and the lmqc72 structure with a score value above 0.00 considered biologically active. these ionic pharmaceuticals 2020, 13, x for peer review 10 of 27 table 5 . selected structures resulting from the molecular docking. a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [64] . the bioactivity scores of the lmqc72, lmqc36, and lmqc50 structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [23] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc72, lmqc50 and rofecoxib structures with values greater than 0.00. meanwhile, the lmqc36 has a moderately active score between −0.5 to 0.00. the score values of the lmqc 72, lmqc36, and lmqc50 structures are considered good because they are close to the pivot compound with probable biological activity (see table 6 ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [65] . the results of the ion channel modulators' scores for the lmqc36, lmqc50, and rofecoxib structures are estimated score values between −0.50 to 0.00 considered moderately active and the lmqc72 structure with a score value above 0.00 considered biologically active. these ionic a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [64] . the bioactivity scores of the lmqc72, lmqc36, and lmqc50 structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [23] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc72, lmqc50 and rofecoxib structures with values greater than 0.00. meanwhile, the lmqc36 has a moderately active score between −0.5 to 0.00. the score values of the lmqc 72, lmqc36, and lmqc50 structures are considered good because they are close to the pivot compound with probable biological activity (see table 6 ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [65] . the results of the ion channel modulators' scores for the lmqc36, lmqc50, and rofecoxib structures are estimated score values between −0.50 to 0.00 considered moderately active and the lmqc72 structure with a score value above 0.00 considered biologically active. these ionic the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc72, lmqc50 and rofecoxib structures with values greater than 0.00. meanwhile, the lmqc36 has a moderately active score between −0.5 to 0.00. the score values of the lmqc 72, lmqc36, and lmqc50 structures are considered good because they are close to the pivot compound with probable biological activity (see table 6 ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [65] . the results of the ion channel modulators' scores for the lmqc36, lmqc50, and rofecoxib structures are estimated score values between −0.50 to 0.00 considered moderately active and the lmqc72 structure with a score value above 0.00 considered biologically active. these ionic modulators are important for planning potential anti-inflammatory drugs because they participate in the protection of tissues against lesions induced by the inflammatory process, they carry charged particles across cell membranes and their activity can be directed towards the discovery of new potential drugs for the regulation of the depolarization of ionic charges [66, 67] . the lmqc72 and lmqc50 structures have score values for kinase inhibitors greater than 0.00 considered biologically active. meanwhile, the compound rofecoxib and lmqc36 have moderately active score values (see table 5 ) for protein kinase inhibitors, as cyclooxygenase-2 is induced by various extracellular signals including pro-inflammatory stimuli and growth promoters. a cyclooxygenase-2 is induced by several extracellular signals, including pro-inflammatory and stimulating growth promoters. thus, all of the signals converge for the activation of mitogen-activated protein kinases (mapk) that regulate cyclooxygenase-2 mrna and contribute to the infection treatment process [68] . moreover, the nuclear receptor score values (nrs), in the lmqc72 and lmqc36 structures, are considered moderately active, as they have score values between −0.5 to 0.00. lmqc50 and rofecoxib are considered biologically active, with a score value above 0.00, according to the classification ranges of smant and chowdhary. the bioactivity of nuclear receptors (nrs) is important because they are involved in several physiological processes, including homeostasis, an important process that regulates inflammation [69] . the lmqc72, lmqc36, lmqc50 structures have moderately active score values between −0.5 and 0.00 for protease inhibitors. already, the dynamic compound of rofecoxib has an estimated value greater than 0.00 considered active. therefore, the results of the lmqc72 and lmqc50 structures are considered to have biological activity (active), enzyme inhibitor, since they had score values greater than zero, such as the compound rofecoxib. while lmqc36 is expected to be moderately active with a score between −0.50 to 0.00. the activity score profile of the selected structures demonstrates the probability that they are biologically active and that they have the necessary properties to act with potential enzyme inhibitors of cyclooxygenase-2 (cox-2) [70] . compounds lmqc72, lmqc36, and lmqc50 were also submitted to web server swiss target prediction (http://www.swisstargetprediction.ch) [22] . to identify the likelihood of bioactivity through similarity based on chemical structure and molecular form (electroshape) [71] . the server uses a database of molecules: chembl [72, 73] , drugbank [74] , pubchem [75] , and zinc [76] to track sets of molecules and identify proteins with ligands similar to bioactive molecules and also uses species selection for virtual screening (top_25 homo sapiens). the results of the virtual bioactivity screening for the enzymatic target performed by the swiss targetprediction [77] server issued a summary displayed in percentages with the probability of being the enzymatic target [78] . the table 5 shows the percentage probability values for enzyme inhibition. prediction analysis of enzymatic inhibition for rofecoxib was 32%; while the selected compounds exhibited the following probability of reaching the enzyme: lmqc72 16%, lmqc36 8%, and lmqc50 4%. thus, it is observed that rofecoxib did not reach 100% of the enzyme and the selected compounds had lower enzyme values. however, the results were assessed as likely for possible bioactivity and the structures proceeded with analysis taking into account the pharmacokinetic and toxicological profiles in which they presented favorable results. to evaluate the conformational changes in the receptor-ligand complexes along the time, the md simulations were applied in 150 ns simulation nodes, for each complex hcox and ligand: rofecoxib, lmqc72, lmqc36, and lmqc50. the simulations also allowed the evaluation of the conformational changes in the structure of the ligand and the protein backbone. these conformational changes in the backbone and ligand were evaluated from the root mean square deviation plot (rmsd). to plot the rmsd of the backbone, cα atoms were used, while to plot the rmsd of the ligand, all heavy atoms were used. in addition, the fluctuation of the residues from the protein backbone was evaluated, for this, the cα atoms were also used. this analysis was performed to evaluate the difference in the structural fluctuation of the protein during the interaction with the different ligands, throughout the 150 ns md simulation (see figure 6 ) [25, [79] [80] [81] [82] [83] . the rmsd plot reveals that the ligands showed small conformational variations when interacting with the protein along the time. their rmsd graphs show slight variations, which suggests that the ligands remained interacting with the active site of the protein undergoing minor conformational changes. this conformational stability over 150 ns of md simulations demonstrates a good interaction of the ligands with the molecular target, thus, remained in a favorable conformation to inhibit the biological receptor. conformational changes. this conformational stability over 150 ns of md simulations demonstrates a good interaction of the ligands with the molecular target, thus, remained in a favorable conformation to inhibit the biological receptor. the low rmsd fluctuation of the ligands is also related to the interactions established in the binding pocket. all ligands showed interactions with residues observed in the results of molecular docking, which are summarized in tables 2 and 3. these interactions were able to keep the ligands interacting with the active site throughout the entire trajectory, allowing the maintenance of the receptor-ligand. the different ligands were able to impact the flotation of the atoms of the hcox-2 backbone in different ways, as can be seen from the differences of the root-mean-square fluctuation (rmsf) plot. the greatest differences in fluctuations in protein residues are observed at residues 34-107. this region of the protein corresponds to the n-terminal portion; in addition, it is initially composed of a small alpha-helice, followed by two beta-leaves that will connect to another alpha-helice through a region's relatively large loop. finally, the residue gap is formed by three more alpha helices that are connected by loop regions (see figure 7 ). tables 2 and 3 . these interactions were able to keep the ligands interacting with the active site throughout the entire trajectory, allowing the maintenance of the receptor-ligand. the different ligands were able to impact the flotation of the atoms of the hcox-2 backbone in different ways, as can be seen from the differences of the root-mean-square fluctuation (rmsf) plot. the greatest differences in fluctuations in protein residues are observed at residues 34-107. this region of the protein corresponds to the n-terminal portion; in addition, it is initially composed of a small alpha-helice, followed by two beta-leaves that will connect to another alpha-helice through a region's relatively large loop. finally, the residue gap is formed by three more alpha helices that are connected by loop regions (see figure 7) . colors. (a) general view of the protein structure with emphasis on the binding pocket occupied by the ligands. the protein was represented in cyan color and ligand protein was represented in spheres (red color). (b) rmsds of hcox-2-rofecoxib-system, (c) rmsds of hcox-2-lmqc72 system, (d) rmsds of hcox-2-lmqc36 system, (e) rmsds of hcox-2-lmqc50 system. ligands and residues were represented in sticks. the greatest fluctuation of residues 34-107 was observed in the complex established with the lmqc36 ligand. apparently, this greater fluctuation should impair the stability of the ligand at the active site, since residues 34-107 correspond to a region of the protein that is close to the active site. however, this behavior was not observed, since the rmsd plot of the ligand shows that the maintenance of the molecule in the binding pocket with conformational stability along the 150 ns trajectory. additionally, the affinity energy value (δgbind = −36.10 kcal/mol) demonstrates that the ligand was able to interact favorably with the protein. this result demonstrates that this region of the protein, despite showing high fluctuation, was not able to impair the interaction of the ligand with the active site. this suggests that the residues around the active site are sufficient to keep the ligands complexed to the protein, despite fluctuations conformations observed in the region of the protein formed by the residues 34-107. to evaluate the interaction energy of the selected compounds with hcox-2, the molecular mechanics/generalized born surface area (mm/gbsa) method was applied and the obtained results are summarized in table 7 . the greatest fluctuation of residues 34-107 was observed in the complex established with the lmqc36 ligand. apparently, this greater fluctuation should impair the stability of the ligand at the active site, since residues 34-107 correspond to a region of the protein that is close to the active site. however, this behavior was not observed, since the rmsd plot of the ligand shows that the maintenance of the molecule in the binding pocket with conformational stability along the 150 ns trajectory. additionally, the affinity energy value (∆gbind = −36.10 kcal/mol) demonstrates that the ligand was able to interact favorably with the protein. this result demonstrates that this region of the protein, despite showing high fluctuation, was not able to impair the interaction of the ligand with the active site. this suggests that the residues around the active site are sufficient to keep the ligands complexed to the protein, despite fluctuations conformations observed in the region of the protein formed by the residues 34-107. to evaluate the interaction energy of the selected compounds with hcox-2, the molecular mechanics/generalized born surface area (mm/gbsa) method was applied and the obtained results are summarized in table 7 . according to the values of affinity energy (∆gbind), all ligands selected by molecular docking are able to establish stable complexes with hcox-2. rofecoxib achieved the free energy value of ∆gbind= −45.31 kcal/mol. the other compounds reached the following affinity energy values: lmqc72: ∆gbind = −38.58 kcal/mol; lmqc36: ∆gbind = −36.10 kcal/mol; and lmqc50: ∆gbind = −39.40 kcal/mol. the compounds lmqc72, lmqc36, and lmqc50 showed favorable values of affinity energy for formation of the complexes. van der waals (∆evdw) interactions showed the greatest contributions to the formation of the different systems of this study. in addition, electrostatic (∆eele) and non-polar (∆gnp) interactions also contributed to complexes being formed spontaneously. the values of affinity energy for the three selected compounds were promising, as the values were relatively close to the obtained for rofecoxib. this demonstrates that the selected substances can be considered as putative hcox-2 inhibitors, being promising leads for new anti-inflammatory drugs project. the chemical structures of cox-2 inhibitors are heterogenic and can be classified into tricyclics and non-tricyclics compounds. contrary to the classic nsaids, this new class of enzyme inhibitors is lacking a carboxylic group, thus effecting cox-2 affinity by a different orientation within the enzyme without formation of a salt bridge in the hydrophobic channel of the enzyme [6] . celecoxib, rofecoxib, valdecoxib share in common the same structural features of the selected compounds lmqc36, lmqc50, and lmqc72, which exhibit a tricyclic scaffold, and a 1,2-diarylsubstitution on a central hetero ring system. in addition, these compounds show characteristic groups on one of the aryl rings that plays a crucial role on cox-2 selectivity. all selected compounds present five membered core heterocycles, even though all different from rofecoxib, which shows a furanone ring (see figure 1) . compound lmqc72 present a pharmacophore-based 1,2,4 triazole group, which increases a certain degree of conformational rigidity to compound, which can be seen in the binding free energy essay. lmqc50 shows a pyrazole moiety, the same core as celecoxib (figures 1 and 8) , which favors a hydrogen bond interaction with hcox-2 ( figure 5b ). moreover, lmqc50 presents a 4-sulfonylmethylphenyl substitution at 1 position on the pyrazole ring which increases the inhibitory effects against cox-2 enzyme [84] . according to the values of affinity energy (δgbind), all ligands selected by molecular docking are able to establish stable complexes with hcox-2. rofecoxib achieved the free energy value of δgbind= −45.31 kcal/mol. the other compounds reached the following affinity energy values: lmqc72: δgbind = −38.58 kcal/mol; lmqc36: δgbind = −36.10 kcal/mol; and lmqc50: δgbind = −39.40 kcal/mol. the compounds lmqc72, lmqc36, and lmqc50 showed favorable values of affinity energy for formation of the complexes. van der waals (δevdw) interactions showed the greatest contributions to the formation of the different systems of this study. in addition, electrostatic (δeele) and non-polar (δgnp) interactions also contributed to complexes being formed spontaneously. the values of affinity energy for the three selected compounds were promising, as the values were relatively close to the obtained for rofecoxib. this demonstrates that the selected substances can be considered as putative hcox-2 inhibitors, being promising leads for new anti-inflammatory drugs project. the chemical structures of cox-2 inhibitors are heterogenic and can be classified into tricyclics and non-tricyclics compounds. contrary to the classic nsaids, this new class of enzyme inhibitors is lacking a carboxylic group, thus effecting cox-2 affinity by a different orientation within the enzyme without formation of a salt bridge in the hydrophobic channel of the enzyme [6] . celecoxib, rofecoxib, valdecoxib share in common the same structural features of the selected compounds lmqc36, lmqc50, and lmqc72, which exhibit a tricyclic scaffold, and a 1,2-diarylsubstitution on a central hetero ring system. in addition, these compounds show characteristic groups on one of the aryl rings that plays a crucial role on cox-2 selectivity. all selected compounds present five membered core heterocycles, even though all different from rofecoxib, which shows a furanone ring (see figure 1) . compound lmqc72 present a pharmacophore-based 1,2,4 triazole group, which increases a certain degree of conformational rigidity to compound, which can be seen in the binding free energy essay. lmqc50 shows a pyrazole moiety, the same core as celecoxib (figure 1 and figure 8 ), which favors a hydrogen bond interaction with hcox-2 ( figure 5b ). moreover, lmqc50 presents a 4-sulfonylmethylphenyl substitution at 1 position on the pyrazole ring which increases the inhibitory effects against cox-2 enzyme [84] . lmqc36 shows an isoxazole ring such as valdecoxib ( figure 1 and figure 8 ), linked to the aryl ring by an amide group, which also confers rigidity to the structure and favors an additional pi-sigma interaction with hcox ( figure 5c ). compound lmqc72 present an imidazole ring, favoring a pi-pi stacking interaction with hcox-2 ( figure 5d ) [85] . according to previous studies, imidazole, triazole, ozaxol, benzene sulfonamide, and pyrazole favors the formation of hydrogen bonds capable of introducing a certain degree of conformational rigidity, indicating a wide range of pharmacological activity as a desired, anti-inflammatory activity ( figure 8 ) [86, 87] . lmqc36 shows an isoxazole ring such as valdecoxib (figures 1 and 8) , linked to the aryl ring by an amide group, which also confers rigidity to the structure and favors an additional pi-sigma interaction with hcox ( figure 5c ). compound lmqc72 present an imidazole ring, favoring a pi-pi stacking interaction with hcox-2 ( figure 5d ) [85] . according to previous studies, imidazole, triazole, ozaxol, benzene sulfonamide, and pyrazole favors the formation of hydrogen bonds capable of introducing a certain degree of conformational rigidity, indicating a wide range of pharmacological activity as a desired, anti-inflammatory activity ( figure 8 ) [86, 87] . due to the aforementioned facts, the compounds with the most promising results (figure 8) , were submitted to an investigation in scifinder ® , available on the internet, and linked to the chemical abstract service (cas) (https://scifinder.cas.org/), in order to verify additional information about structures and/or experiments with biological activities (patents). no additional information on the promising structures was found in the search. this demonstrates that the molecules mentioned above, with great potential for inhibition in cox-2, still do not have in vitro or in vivo studies that evaluate this activity. therefore, these are important findings for future research and development studies of cox-2 selective anti-inflammatory drugs. lmqc72, lmqc36, and lmqc5 were also submitted for evaluation of their toxicological properties using derek software. this assessment was carried out to investigate whether these compounds had a profile of adverse toxicological effects on humans, mice, and rats. according to the results (table 8) , the selected compounds did not present any toxicity alert. results of the pivot compound rofecoxib, on the other hand, were flagged as "plausible", since it presented a warning of hepatotoxicity (humans, mice and rats) for derivatives of the furanone group [63] . information on the promising structures was found in the search. this demonstrates that the molecules mentioned above, with great potential for inhibition in cox-2, still do not have in vitro or in vivo studies that evaluate this activity. therefore, these are important findings for future research and development studies of cox-2 selective anti-inflammatory drugs. lmqc72, lmqc36, and lmqc5 were also submitted for evaluation of their toxicological properties using derek software. this assessment was carried out to investigate whether these compounds had a profile of adverse toxicological effects on humans, mice, and rats. according to the results (table 8) , the selected compounds did not present any toxicity alert. results of the pivot compound rofecoxib, on the other hand, were flagged as "plausible", since it presented a warning of hepatotoxicity (humans, mice and rats) for derivatives of the furanone group [63] . [88] . table 8 shows also the oral lethal dose prediction (ld50) based on mg/kg body weight and toxicity class ranging from i to vi, performed on the protox-ii web server (http://tox.charite.de/protox_ii). ld50 of the lmqc72 structure was 674 mg/kg and of the lmqc50 1400 mg/kg both with iv classification was considered harmful if ingested (300 < ld50 ≤ 2000), however, they showed higher lethal dose when compared to rofecoxib. lmqc36 presented a ld50 value of 6500 mg/kg and classification vi, which is non-toxic if ingested, estimated as the best result of an oral lethal dose. therefore, the results for ld50 of the investigated compounds are better than the commercial compound and may present greater safety in use [21] . the compounds were also submitted to the preadmet [18, 19] software to assess the cardiotoxicity. drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (herg). the blockage leads to long qt syndrome (lqts), which is a severe life-threatening cardiac side effect [89] . the evaluation of this parameter was by means of herg ( [17] takes into account the electro-affinity calculation (ea) of the compounds. the results of the evaluation of the cardiotoxicity capacity for lmqc72, lmqc36, lmqc50, and rofecoxib can be seen in table 9 . [88] . table 8 shows also the oral lethal dose prediction (ld 50 ) based on mg/kg body weight and toxicity class ranging from i to vi, performed on the protox-ii web server (http://tox.charite.de/protox_ii). ld 50 of the lmqc72 structure was 674 mg/kg and of the lmqc50 1400 mg/kg both with iv classification was considered harmful if ingested (300 < ld 50 ≤ 2000), however, they showed higher lethal dose when compared to rofecoxib. lmqc36 presented a ld 50 value of 6500 mg/kg and classification vi, which is non-toxic if ingested, estimated as the best result of an oral lethal dose. therefore, the results for ld 50 of the investigated compounds are better than the commercial compound and may present greater safety in use [21] . the compounds were also submitted to the preadmet [18, 19] software to assess the cardiotoxicity. drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (herg). the blockage leads to long qt syndrome (lqts), which is a severe life-threatening cardiac side effect [89] . the evaluation of this parameter was by means of herg ( [17] takes into account the electro-affinity calculation (ea) of the compounds. the results of the evaluation of the cardiotoxicity capacity for lmqc72, lmqc36, lmqc50, and rofecoxib can be seen in table 9 . lmqc72, lmqc36, lmqc50, and rofecoxib showed a medium risk of cardiotoxicity in the electro-affinity calculation. this pharmacokinetic property that is related to drug-receptor interaction and electron transfers, we consider an aspect of paramount importance for therapeutic activity and in determining toxicity [90] . the human ether-a-go-go-related gene (herg) is codified for a protein that forms a voltage-dependent potassium ion channel found in heart and nervous system [91] [92] [93] ; a myocardial conduction disorder (electrical conduction) can alter ventricular repolarization and, consequently, increase the vulnerability for the development of a cardiac action [91] . therefore, lmqc72, lmqc36, and lmqc50 have a lower risk of cardiotoxicity when compared to rofecoxib, since they do not present violation in the electro-affinity parameter. crystallographic structure (pdb 5kir at 2.7 å resolution) of human cyclooxygenase-2 (hcox-2) was obtained as pdb file from the protein data bank (pdb) (https://www.rcsb.org/pdb) complexed with the pivot rofecoxib [12, 14] . in this step, we used six commercial databases for virtual screening based of rofecoxib ligand: chembridge diverset™-express-pick™ collection (diverset™-exp), diverset core library (diverset™-cl), zinc drug database, zinc natural stock e zinc drug@fda bindingdb, and maybridge. for each molecule in the database, we obtained 300 conformers using the mmff94 molecular force fields were generated [94] , running on omega v3.3.1.2 software (open eye scientific software, santa fe, nm, http://www.eyesopen.com) for windows 7 operating system and intel core i7 machine of 2.4 ghz. initially, for each molecule in the database, the fast conformer generation method was used with a maximum energy tolerance of 9 kcal.mol −1 and mean square deviation (rmsd) of 0.6 å [15, 16, 25] . in this study, rapid overlay of chemical structures (rocs) v3.3.2.2 (openeye) software was used as a tool for three-dimensional (3d) molecular similarity research. we used six databases to select chemical compounds through the rocs software (https://www.eyesopen.com/rocs) [30] , with gaussian function algorithm located in atoms that proposes the best overlap between molecules in a characteristic set that can be a steric volume or the molecular interaction, called comboscore. this was done to generate and score three-dimensional (3d) overlays of the database with the pivot compound (rofecoxib) in order to seek better compounds for the cox-2 receptor, to get the highest rated structures (top_200) of each base, totaling 12,000 compounds [15, 16, 25, 34] this software generates input files for the eon program. eon v2.3.2.2 (openeye) software is an electrostatics comparison program (https://www.eyesopen. com/eon) [95] -it compared the electrostatic potential maps of pre-aligned molecules and determined the tanimoto measures for the comparison of the six databases. moreover, it calculated the new partial load to minimize energy using the mmff94 force field [94] . electrostatic classification was based on tanimoto's electrostatic scores; the electrostatic arrangement was obtained from the overlapping of positive and negative charges when completing the variation of an identical to negative values. in this study, a lower energy of rofecoxib conformer was used to perform electrostatic comparisons (more rigid conformation, based on the available crystallographic structure). the output files were grouped according to the scores and the results were classified based on "et combo" analogous to "tanimoto combo". in the end, only the "100 best compounds/base" were selected, affording 600 molecules [15, 16, 25, 34, 35, 96, 97] . the assessment of a number of key physicochemical properties, pharmacokinetic parameters, and toxicity endpoints was carried out for the compounds that passed the virtual screening step-the top 100 of each database. pharmacokinetic (#star, "rule of five", human intestinal absorption, qppcaco, qppmdck, qplogpo/w, cns, and qplogbb) properties were predicted using the schrodinger's suite qikprop v.3.5, and derek nexus software 2.0 [25, 40] . the toxicity of the compounds with the best pharmacokinetic profiles was assessed using (derek) 10.0.2 nexus program [25, 40] . deductive estimation of risk from existing knowledge (derek) predicts potential toxicity and toxicophoric groups and also includes the following toxicological parameters: carcinogenicity, mutagenicity, genotoxicity, skin sensitization, teratogenicity, irritation, respiratory sensitization, reproductive toxicity [37, 63] . this software analyses qualitative predictions and, in this way, generates alerts about the possible toxic action of the chemical compounds analyzed. in this step, the compounds were evaluated in aspects involving the types of toxicity and possible toxicophoric effects [37, 98] . we have considered derek toxicity alerts involving the human species and also classified as plausible in mammals, but compounds containing any toxicophoric groups were also discarded [20] , through visual inspection using the maestro 9.9 program. the selected compounds were submitted to the protox web server (http://tox.charite.de/protox_ ii) [99] , which identifies lethal oral doses (ld 50 ) [88] . the prediction method is based on the analysis of the two-dimensional (2d) similarity to compounds with known ld 50 values and the identification of fragments over-represented in toxic compounds. the results are generated instantly on the server page, showing the predicted average lethal dose (ld 50 ) in mg/kg of weight and the toxicity classes (i, ii, iii, iv, v and vi) [21, 98] . the prediction of cardiotoxicity was determined using the online server preadmet (https: //preadmet.bmdrc.kr/) [18] and qikprop [100] software. the prediction method for the risk of cardiac toxicity is based on the inhibition property of the human ether-to-go-go (herg) gene based on the electron affinity of the compounds. preadmet instantly generates alerts on the server page classified as: low risk, medium risk, and high risk for the herg property [17] . this screening step for the prediction of the biological target was performed via web servers: molinspiration and swisstargetprediction. the bioactivity score of selected compounds was evaluated using the molinspiration server cheminformatics tool (http://www.molinspiration.com) [101] . the prediction made was based on the enzyme inhibition score, taking into account the pivot molecule. the results are analyzed according to roy; samant; chowdhary [102] . therefore, it is recommended that if the value is equal to or greater than 0.00, the more active it will be, while if the values are between −0.50 and 0.00, it is moderately active, and, if the score is less than −0.50, it will be considered inactive [21, 103] . then, the query structures were submitted to the swisstargetprediction web server (http://www. swisstargetprediction.ch), to predict small molecule protein targets in homo sapiens (top_25). targets are classified according to their percentage probability on the assumption that if the molecule is active, it is likely to bind to some protein. the investigation of the bioactivity target was based on the value of the enzymatic target of the pivot molecule rofecoxib with known bioactivity. the results of the server prediction via swisstargetprediction web server are presented as a percentage in a pie chart [22, 64] . molecular docking simulations were based on fitting the ligand to the active site of an enzyme. this simulation is called re-docking which aims to recover, from computer simulation, the original position of a ligand present in a crystallographic structure of a protein-ligand complex [59] . for the determination of this protocol an approach called validation is used, where we used as reference a crystallographic structure already determined [62, 83] . for this study, the crystallographic structure of hcox-2 complexed with rofecoxib ligand deposited in the pdb was used with code 5kir (homo sapiens) and a resolution of 2.7 å [11, 50] . the enzyme structure was prepared by removing water and binders, and adding hydrogen atoms, using discovery studio 4.1 software. then, the autodock/vina software was subjected to molecular coupling [62, 104, 105] autodock is a set of tools that allow the interaction between ligand and macromolecule and provides combinations with algorithm options: simulated annealing (sa), genetic algorithm (ga), and lamarckian genetic algorithm (lga). in this work, the search algorithm used was lga (lamarckian genetic algorithm), that presents the best results in the search for the global minimum [62, 83] . the interactions between inhibitors and the receptor were visualized using the discovery studio 4.1 software with standard parameters. the evaluation of the molecular coupling was determined by means of the ligand obtained experimentally and the theoretical conformation performed with the molecular coupling in the pdb (5kir), and were validated by the rmsd value. table 10 shows the x, y and z coordinates according to the interaction between cox-2 and the standard ligand. the x, y and z coordinates of the receivers were determined according to the average region of the active site. moreover, ten solutions were calculated for each ligand and minimum conformations of binding energy were analyzed [15, 16, 20, 25, 59] . the energy scoring function was used to assess the free binding energy (∆g) of interactions between cox and ligands in pyrx 0.8.30. the analysis of the poses (conformation + orientation) of the binders was also taken into account in the selection of the best binding free energy and binding affinity calculations in autodock 4.2/vina 1.1.2 in order to assess selectivity towards homo sapiens as a function of binding affinity at the cox-2 receptor. the initial structure for the system was obtained from molecular docking methods, as described in the previous section. the restrained electrostatic potential (resp) protocol with the hf/6-31g* basis sets was applied to obtain the charges of the atoms of each ligand [106] [107] [108] atomic charge calculated using gaussian [81, 109, 110] the parameters of the ligand were constructed with the antechamber module, available in the amber16 package [111] [112] [113] . the protonation state of ionizable residues of protein structure was analyzed using the propka [114] server in the neutral ph before performing the md simulations. the ligand was treated with the general amber force field (gaff) and protein was treated with the ff14sb [10] . the force field parameters developed by giammona were used for the heme group [115] . the system was constructed for the simulation using the tutorial for the leap (tleap) of amber 16 package. the system was solvated in an octahedron periodic box containing water molecules in the tip3p model [116] . the partial charges of the systems were neutralized by adding counterions. we used the sander.mpi for the four stages of energy minimization. in each of these stages, it took 3000 cycles using the steepest descent method and 5000 cycles using the conjugate gradient algorithm. in the first stage the hydrogen atoms of the water molecules were optimized; then, the ions and the water molecules were minimized; in the third stage, the hydrogen atoms of the protein, and in the last step, the solute and the solvent, underwent the process of energy minimization. three heating steps were used for a total time of 800 picoseconds to raise the system temperature to 300 k. first, the solute was restricted with a constant harmonic force of 25 kcal mol −1 . å −2 , so only the solvent and the counter ions moved. in the next step, the constant harmonic force was removed. to equilibrate the systems, we performed 2 ns simulations with no restriction at constant temperature. finally, for each system, we performed 150 ns of molecular dynamics of production. particle mesh ewald method [117] was used for the calculation of electrostatic interactions and the bonds involving hydrogen atoms were restricted with the shake algorithm [118] . temperature control was performed with the langevin thermostat [119] within collision frequency of 2 ps-1. to estimate the binding affinity (∆g bind ), we used the molecular mechanics/generalized born surface area (mm-gbsa) method [80, 120, 121] the affinity energy (∆gbind) is the summation of the interaction energy of the gas phase between protein-ligand (∆emm), desolvation free energy (∆gsolv) and system entropy (-t∆s). ∆emm is the result of the sum of internal energy (∆einternal, sum of the energies of connection, angles, and dihedral) electrostatic contributions (∆eele) and the van der waals term (∆evdw). ∆gsolv is the sum of the polar (∆ggb) and non-polar (∆gnp) contributions. ∆gsa was determined from the solvent accessible surface area (sasa) estimated by the linear combination of pairwise overlaps (lcpo) algorithm. the mm/gbsa method was used to determine the energy contribution of each protein residue, thus, rendering it possible to determine which residues are most important for the ligand interaction with the active site. the interaction energy of residues with the inhibitor can be described from four terms: van der waals contribution (∆e vdw ), electrostatic contribution (∆e ele ), polar solvation contribution (∆g pol ), and nonpolar solvation contribution (∆g nonpol ), according to the equation [25, 122, 123] : ∆g ligand-residue = ∆e vdw + ∆e ele + ∆g pol + ∆g nonpol (5) in this study, a computational strategy was applied to identify new potential selective hcox-2 inhibitors base conclusions d on the known drug rofecoxib. compounds from six databases were filtered by a ligand-based virtual screening study, followed by pharmacokinetic, toxicological, and molecular dynamic studies. the selected structures lmqc72, lmqc36, and lmqc50 have aspects strictly related to physical-chemical properties and biological activity. therefore, such selected structures reproduce values within the limits established in the pharmacokinetic predictions: absorption and distribution in the human body. moreover, in the prediction of toxicity the structures lmqc72, lmqc36, and lmqc50 did not present alerts for possible toxic groups. through the study of molecular dynamics, lmqc72, lmqc36, and lmqc50 were identified as promising due to values of affinity energy relatively close to those obtained for rofecoxib. along the trajectories of molecular dynamics simulations, the selected compounds showed conformational stability, as well as the pivot compound. lmqc72, lmqc50, and lmqc36 showed satisfactory pharmacokinetic results related to absorption and distribution. the toxicological predictions of these compounds did not display alerts for possible toxic groups and lower risk of cardiotoxicity compared to rofecoxib. this demonstrates that lmqc72, lmqc36, and lmqc50 can be considered as putative hcox-2 inhibitors, in addition to serving as the basis for the new anti-inflammatory drug project. cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition distinct functions of cox-1 and cox-2 molecular inflammatory mediators in peripheral nerve degeneration and regeneration regulation of cyclo-oxygenase-2 optimization and validation of a docking-scoring protocol; application to virtual screening for cox-2 inhibitors selective cox-2 inhibitors: a review of their structure-activity relationships cyclooxygenase-2: a therapeutic target rofecoxib (vioxx) voluntarily withdrawn from market selective cox-2 inhibitors development and testing of a general amber force field the protein data bank protein data bank (pdb): the single global macromolecular structure archive crystal structure of rofecoxib bound to human cyclooxygenase-2 ligand-and structure-based virtual screening of 16-((diiso-butylamino)methyl)-6α-hydroxyvouacapane-7β,17β-lactone, a compound with potential anti-prostate cancer activity preadmet | prediction of adme/tox-just another bmdrc sites preadmet version 2.0; bioinformatics and molecular design research center identification of novel protein kinase receptor type 2 inhibitors using pharmacophore and structure-based virtual screening silico evaluation of ibuprofen and two benzoylpropionic acid derivatives with potential anti-inflammatory activity andrographolide as a potential inhibitor of sars-cov-2 main protease: an in silico approach determination of molecular property, bioactivity score and binding energy of the phytochemical compounds present in cassia auriculata by molinspiration and dft method identification of novel parasitic cysteine protease inhibitors using virtual screening. 1. the chembridge database potential inhibitors of the enzyme acetylcholinesterase and juvenile hormone with insecticidal activity: study of the binding mode via docking and molecular dynamics simulations virtual screening, identification and experimental testing of novel inhibitors of pbef1/visfatin/nmprtase for glioma therapy a web-accessible database of experimentally determined protein-ligand binding affinities rocs-derived features for virtual screening rocs openeye | rocs software | virtual screening|lead hopping comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis gaussian shape methods integration of ligand-and target-based virtual screening for the discovery of cruzain inhibitors hppd: ligand-and target-based virtual screening on a herbicide target discovery of novel ppar ligands by a virtual screening approach based on pharmacophore modeling, 3d shape, and electrostatic similarity screening hierarchical virtual screening of potential insectides inhibitors of acetylcholinesterase and juvenile hormone from temephos identification of new inhibitors with potential antitumor activity from polypeptide structures via hierarchical virtual screening qikprop 3.5 user manual qikprop user manual web services as applications' integration tool: qikprop case study in silico tools for sharing data and knowledge on toxicity and metabolism: derek for windows, meteor, and vitic free energy calculations to estimate ligand-binding affinities in structure-based drug design the calculation of the electron affinity of atoms and molecules dft study on ground state electronic structures of simple to complex molecular specimens calculation of absolute binding free energies between the herg channel and structurally diverse drugs experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings drug metabolites and their effects on the development of adverse reactions: revisiting lipinski's rule of five effect of atomic charges on octanol-water partition coefficient using alchemical free energy calculation medicinal chemical properties of successful central nervous system drugs the log p parameter as a molecular descriptor in the computer-aided drug design-an overview octanol water partition coefficients of simple organic compounds predicting a drug's membrane permeability: a computational model validated with in vitro permeability assay data predicting apparent passive permeability of caco-2 and mdck cell-monolayers: a mechanistic model correlation between human ether-a-go-go-related gene channel inhibition and action potential prolongation the human intestinal epithelial cell line caco-2; pharmacological and pharmacokinetic applications variability in caco-2 and mdck cell-based intestinal permeability assays computational approaches to the prediction of the blood-brain distribution admet in silico modelling: towards prediction paradise? blood-brain barrier: from physiology to disease and back an in silico study of the antioxidant ability for two caffeine analogs using molecular docking and quantum chemical methods molecular modeling approaches of selective adenosine receptor type 2a agonists as potential anti-inflammatory drugs calculation of protein-ligand binding affinities unit using autodock for ligand-receptor docking toward of safer phenylbutazone derivatives by exploration of toxicity mechanism swisstargetprediction: a web server for target prediction of bioactive small molecules role of g protein-coupled receptors in inflammation ion channels in inflammatory processes: what is known and what is next? mediat. inflamm potassium channel modulators as anti-inflammatory agents kinase inhibitors for the treatment of inflammatory and autoimmune disorders structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics signalling networks regulating cyclooxygenase-2 improving the accuracy of ultrafast ligand-based screening: incorporating lipophilicity into electroshape as an extra dimension chembl: a large-scale bioactivity database for drug discovery using chembl web services for building applications and data processing workflows relevant to drug discovery drugbank 3.0: a comprehensive resource for 'omics' research on drugs nuclear receptors and the adaptive response of the heart zinc: a free tool to discover chemistry for biology swisstargetprediction: updated data and new features for efficient prediction of protein targets of small molecules systems biology shaping the interaction landscape of bioactive molecules identification of novel chemical entities for adenosine receptor type 2a using molecular modeling approaches in silico study to identify new antituberculosis molecules from natural sources by hierarchical virtual screening and molecular dynamics simulations molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of mycobacterium tuberculosis polyketide synthase 13 computational design of new protein kinase 2 inhibitors for the treatment of inflammatory diseases using qsar, pharmacophore-structure-based virtual screening, and molecular dynamics oil from the fruits of pterodon emarginatus vog.: a traditional anti-inflammatory. study combining in vivo and in silico recent advancement in the discovery and development of cox-2 inhibitors: insight into biological activities and sar studies synthesis of some thiophene, imidazole and pyridine derivatives exhibiting good anti-inflammatory and analgesic activities pharmacological significance of triazole scaffold pharmacological significance of triazole scaffold evolution of nonsteroidal anti-inflammatory cyclooxygenase (cox) inhibition and beyond drugs (nsaids) protox-ii: a webserver for the prediction of toxicity of chemicals computational determination of herg-related cardiotoxicity of drug candidates benzodiazepines: electron affinity, receptors and cell signaling-a multifaceted approach the human ether-a-go-go-related gene (herg) potassium channel represents an unusual target for protease-mediated damage +) channels: structure, function, and clinical significance hidden cardiotoxicity of rofecoxib can be merck molecular force field. ii. mmff94 van der waals and electrostatic parameters for intermolecular interactions similarity for lead-hopping akritopoulou-zanze, i. the use of three-dimensional shape and electrostatic similarity searching in the identification of a melanin-concentrating hormone receptor 1 antagonist bringing the mmff force field to the rdkit: implementation and validation computer prediction of possible toxic action from chemical structure protox: a web server for the in silico prediction of rodent oral toxicity release 2017-2: maestro calculation of molecular properties and bioactivity score research article in silico pharmacokinetics analysis and admet of phytochemicals of datura in silico pharmacokinetic, bioactivity and toxicity evaluation of some selected anti-ulcer agents designing quorum sensing inhibitors of pseudomonas aeruginosa utilizing fabi: an enzymic drug target from fatty acid synthesis pathway application of resp charges to calculate conformational energies, hydrogen bond energies, and free energies of solvation exploring the potentiality of natural products from essential oils as inhibitors of odorant-binding proteins: a structure-and ligand-based virtual screening approach to find novel mosquito repellents spectroscopic methods and in silico analyses using density functional theory to characterize and identify piperine alkaloid crystals isolated from pepper (piper nigrum l.) chemical profile of lippia thymoides, evaluation of the acetylcholinesterase inhibitory activity of its essential oil, and molecular docking and molecular dynamics simulations the amber biomolecular simulation programs naphthoquinones isolated from eleutherine plicata herb: in vitro antimalarial activity and molecular modeling to investigate their binding modes phytochemical profile, antioxidant activity, inhibition of acetylcholinesterase and interaction mechanism of the major components of the piper divaricatum essential oil obtained by supercritical co2 pdb2pqr: an automated pipeline for the setup of poisson-boltzmann electrostatics calculations improving the accuracy of protein side chain and backbone parameters from ff99sb comparison of simple potential functions for simulating liquid water particle mesh ewald: an n·log(n) method for ewald sums in large systems numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes langevin stabilization of molecular dynamics calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models insight into the interaction mechanism of nicotine, nnk, and nnn with cytochrome p450 2a13 based on molecular dynamics simulation insights into protein-protein binding by binding free energy calculation and free energy decomposition for the ras-raf and ras-ralgds complexes measuring the structural impact of mutations on cytochrome p450 21a2, the major steroid 21-hydroxylase related to congenital adrenal hyperplasia the authors declare no conflict of interest. key: cord-283128-9mbi75de authors: singh, ramendra k.; rai, diwakar; yadav, dipti; bhargava, a.; balzarini, j.; de clercq, e. title: synthesis, antibacterial and antiviral properties of curcumin bioconjugates bearing dipeptide, fatty acids and folic acid date: 2009-12-09 journal: eur j med chem doi: 10.1016/j.ejmech.2009.12.002 sha: doc_id: 283128 cord_uid: 9mbi75de curcumin bioconjugates, viz. di-o-tryptophanylphenylalanine curcumin (2), di-o-decanoyl curcumin (3), di-o-pamitoyl curcumin (4), di-o-bis-(γ,γ)folyl curcumin (6), c(4)-ethyl-o-γ-folyl curcumin (8) and 4-o-ethyl-o-γ-folyl curcumin (10) have been synthesized and tested for their antibacterial and antiviral activities. the conjugates 2, 3, 4, 6 and 8 have shown very promising antibacterial activity with mic ranging between 0.09 and 0.67 μm against gram-positive cocci and gram-negative bacilli. further, the conjugates 2, 3, 6, 8 and 10 have been screened for their antiviral activities against hsv, vsv, fipv, piv-3, rsv and fhv and the molecules 2 and 3 have shown good results with ec(50) 0.011 μm and 0.029 μm against vsv and fipv/fhv, respectively. however, the molecules did not show expected results against hiv-1 iii(b) and rod strains in mtt assay. curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, commonly known as diferuloyl methane, is a natural yellow pigment derived from rhizomes of the plant curcuma longa (zingiberaceae) -a plant grown in tropical southeast asian and indian subcontinent, and has been proved as potent antioxidant, anti-inflammatory, antiviral and anticancer agent through modulation of multiple cellular machinery [1] [2] [3] [4] [5] [6] . turmeric, a spice used to provide specific flavor and yellow color to curry, has been used for many centuries as an indian folklore medicine in ayurveda -an ancient traditional system of medicine for treatment of wide range of illnesses. current traditional indian medicine uses it for biliary disorders, anorexia, cough, diabetic wounds, hepatic disorder, rheumatism, blood purification and rheumatoid arthritis [7] [8] [9] . recent studies have shown curcumin as a potential molecule in the treatment of different forms of cancer, e.g., cervical cancer caused by hpv [10] [11] [12] . it has been observed that both curcumin and the oil fraction, suppress the growth of several bacteria like streptococcus, staphylococcus, lactobacillus, etc [12, 13] and human pathogenic fungi. turmeric oil is also active against aspergillus flavus, aspergillus parasiticus, fusarium moniliforme and penicilium digitatum [14, 15] . results have shown that curcumin treatment effectively reduced coxsackie virus b3 replication through desregulation of ubiquitin-proteosome system (ups) and cop9 signalosome (csn) performing a critical role in their life cycle, i.e., it strongly reduced viral rna expression and further protein synthesis [16, 17] . curcumin has been identified as inhibitor of hiv-1 ltr directed gene expression and viral replication. a previous study has shown that curcumin, a pharmacologically safe compound, is able to block hiv replication by inhibiting hiv-integrase and protease [18, 19] . the double bonds in curcumin provide definite conformational flexibility to the molecule, which accounts for its various properties. further, blocking of phenolic groups decreases its antioxidant activity since these groups play critical role in enzymatic activity at receptor sites [20, 21] . studies have revealed that curcumin has very low bioavailability due to its poor absorption and rapid metabolism in the liver and intestinal wall [22, 23] . curcumin is highly hydrophobic and cannot be administered systemically. on intravenous administration, it disappears rapidly from the blood and quickly appears as metabolites in the bile [10, 24, 25] . therefore, one of the most appreciable approaches is to make biodegradable conjugates of curcumin molecule with suitable ligands to enhance its cellular uptake. for preparing bioconjugate of curcumin, amino acids and fatty acids -natural components of bacterial cell wall, and folic acid -a cofactor in the synthesis of thymidine and other nucleotides, were selected [26] [27] [28] [29] . these bioconjugates are supposed to enhance cellular uptake, lipophilicity of the molecule and sustained release of drug molecule to improve the half-life and reduce the rate of metabolism of curcumin molecule inside the cell. we have previously reported a series of nucleosidic molecules [30] [31] [32] [33] of significant therapeutic applications. in the present pursuance, we are focusing on naturally occurring moleculescurcumin and its bioconjugates, e.g., di-o-tryptophanylphenylalanine curcumin (2), di-o-decanoyl curcumin (3), di-o-pamitoyl curcumin (4), di-o-bis-(g,g)folyl curcumin (6), c 4 -ethyl-o-g-folyl curcumin (8) and 4-o-ethyl-o-g-folyl curcumin (10) . these bioconjugates have been screened for their antibacterial and antiviral activities. curcumin, 1, has two phenolic groups and one active methylene group which can be utilized as potential sites for chemical variations and covalent linkage with biomolecules. we have synthesized curcumin bioconjugates 2, 3, 4, 6, 8 and 10, wherein the phenolic hydroxyls and active methylene group on curcumin have been utilized. for the synthesis of compound 2 (scheme 1), curcumin 1 was reacted with t-boc-n-trp-phe-cooh (indigenously synthesized) in 1:2 molar proportion using dicyclohexylcarbodiimide (dcc) coupling procedure in the presence of dmap in anhydrous dichloromethane (dcm) to yield di-o-tryptophanylphenylalanine curcumin 2 in 53% yield [34] . further, curcumin was reacted with decanoyl chloride and palmitoyl chloride in 1:2 molar proportion in the presence of dmap in anhydrous pyridine to get the molecules 3 and 4 (scheme 1) in 56% and 44% yield, respectively [35] . for the syntheses of conjugates 6, 8 and 10, folic acid was activated to p-nitrophenyl folate 5 (fa-pnp) (scheme 2) using pnitrophenol in the presence of dcc in pyridine and triethylamine (tea). the completion of the reaction was assessed by precipitation of dicyclohexylurea (dcu) [36] . the activated folate 5 was reacted with curcumin 1 in 2:1 molar proportion in the presence of dcc and dmap to get the compound 6 (scheme 3) in 48% yield. for synthesizing the molecule 8, both phenolic groups on curcumin were protected using benzoyl chloride. this di-o-benzoyl curcumin was conferred on a carbanionic character at the active methylene site by using a strong base, like naoet and reacted with 2-chloroethanol -a linker unit, followed by reaction with activated folate 5 in 1:1 molar proportion, using dcc and dmap to yield the compound 8 in 45% yield (scheme 3). the compound 10 was synthesized using only one phenolic group on curcumin molecule. curcumin was taken up in 5% aqueous naoh and reacted with 2-chloroethanol as linker unit. the resulting product, dissolved in anhydrous pyridine, was added dropwise to the activated folate 5 in 1:1 molar proportion in the presence of dcc and dmap to yield the compound 10 in 26% (scheme 4). all the newly synthesized bioconjugates were characterized by uv, 1 h nmr, 13 c nmr, mass spectra and elemental analyses. the molecules 2, 3, 4, 6 and 8 have been screened for their antibacterial activities against gram-positive cocci and gram-negative bacilli and the molecules 2, 3, 6, 8 and 10 for their antiviral activities against a range of viruses using standard protocols. the molecules 2, 3, 4, 6 and 8 have shown remarkable antibacterial activities with mic ranging between 0.09 and 0.67 mm (in vitro) against gram-positive cocci (streptococcus virudans) as well as gram-negative bacilli (escherichia coli, klebsiella pneumoniae, proteus mirabeilis) bacterial strains. results are given in table 1 . the most encouraging results were found against streptococcus viridans, e. coli, k. pneumoniae and p. mirabeilis with molecules 6 and 8 having mic 0.27 mm (0.54 mm against p. mirabeilis) and 0.09 mm, respectively, for each bacterial strain. the molecules 2, 3 and 4 have shown highly satisfactory results as antibacterials with mic 0.43, 0.369 and 0.591 mm, respectively, against e. coli and k. pneumoniae (curcumin shows mic value 1.23-2.47 mm). these results were compared with known antibiotics as standards as shown in table 1 . the antibacterial activity of the bioconjugates was 3.7-27 times higher than that of curcumin itself. the better results obtained with these bioconjugates may be because of their structural similarity with the bacterial cell wall that possesses amino acids and lipids as its integral part and folic acid is a requirement as a cofactor in the synthesis of thymidine and other nucleotides. this ensured high cellular uptake and thus enhanced bioavailability [26] [27] [28] [29] . the ester bonds in these molecules are biodegradable, i.e., they get hydrolysed by carboesterases present in the cells and this results in the enhanced effective concentration of the drug at the target site and sustained release of curcumin further ensures low toxicity, if any. the molecules 2, 3, 6, 8 and 10 were examined for their cytotoxicity and antiviral activity against a variety of dna and rna viruses using different cell cultures. the results have been shown in table 2 . the compound 2 has shown good result against vesicular stomatitis virus and compound 3 against feline corona and feline herpes viruses. the ec 50 value (0.011 mm) of compound 2 was almost nine times lower than its cc 50 value (0.096 mm). similarly, the ec 50 (0.029 mm) values of compound 3 against feline corona and feline herpes viruses were almost five times lower than its cc 50 (0.146 mm) value. the ec 50 (0.029 mm) of compound 3 has been observed as almost ten times better than that of curcumin (0.271 mm) against fipv and fhv. further, the cc 50 values of these bioconjugates were much higher than that of curcumin. thus, these results confirmed the better acceptability of curcumin bioconjugates by the cells under study than curcumin itself. all these compounds were also tested against influenza a h1n1/h3n2 subtype and influenza b in mdck cell lines. however, none of the test compounds was able to inhibit cytopathic effects of influenza a or b virus at subtoxic concentrations or the highest concentration tested (100 mg/ml). the positive results with compounds 2 and 3 might be due to their higher lipophilicity, which enhances their cellular uptake. the lipophilicities of compounds 2 and 3 are expressed by log p values determined experimentally (table 3) using the standard stir-shake flask method in an octanol-water system [37] . these values are also compared with values obtained by a computer programme -molinspiration method. the log p values of the compounds 2 and 3 were higher than that of curcumin. as expected, the lipophilicity of the compound 3 was much higher than that of the compound 2. this reaffirmed that the bioconjugates have better biocompatibility when we are concerned about bioavailability of curcumin molecule. since ester bonds are biodegradable by carboesterases, these molecules ensure higher and sustained bioavailability. the compound 6 which did not show positive result against the viruses under study has been found to be very active against hpv causing cervical cancer in indian women [singh et al. unpublished results]. the bioconjugates, however, did not show any activity against hiv-1 (iii b and rod strains) as shown in table 4 . we have prepared a number of curcumin bioconjugates bearing covalent linkage with suitable ligands, viz. dipeptide, fatty acids and folic acid to enhance cellular uptake of curcumin [26] [27] [28] [29] . these bioconjugates were screened for their antibacterial activity against gram-positive and gram-negative bacteria using microdilution broth susceptibility test method [38] . the rod strains) in mt 4 cells using mtt method but no encouraging results were obtained [39] . however, the present work has shown promise in developing a natural edible substance-curcumin, into a potential antibacterial and antiviral agent and preparing the bioconjugates of curcumin has proved beneficial in order to enhance its cellular delivery. further mechanistic studies on compounds 2 and 3 are in progress against vesicular stomatitis, feline corona and feline herpes viruses. silica gel g for tlc and silica gel (60-120) for column chromatography were obtained from e. merck india ltd. curcumin, folic acid, dicyclohexylcarbodiimide (dcc), p-nitrophenol (pnp), trifluoro acetic acid (tfa) and 2-chloroethanol were purchased from aldrich chemical co. usa. the dipeptide, t-boc-n-trp-phe-cooh, was indigenously synthesized. peptone (std.) was purchased from hi media laboratory ltd., mumbai, india. melting points were determined by electrothermal apparatus and were uncorrected. uv measurements were carried out on hitachi 220s spectrophotometer. 1 h nmr spectra were recorded using drx 300 instrument with dmso as solvent using tms as an internal standard. 13 c nmr spectra were recorded on varian xl-300 and bruker am-200 spectrometers operating at 75 and 50 mhz, respectively. all solvents were dried and distilled prior to use. curcumin (100 mg, 0.27 mmol) and dipeptide (266 mg, 0.59 mmol) were dissolved in dry dcm (10 ml), and stirred for a few minutes to get a clear solution. further, dcc (302 mg, 1.47 mmol) and dmap (66.35 mg) dissolved in dcm (15 ml) were added to the reaction vessel and stirred the reaction mixture overnight at room temperature. the completion of the reaction was assessed by precipitation of dicyclohexylurea (dcu). dcu was filtered off and the filtrate evaporated to a small volume and partitioned between water and ethyl acetate. the organic layer was evaporated and treated with tfa in dcm (1:1, v/v) to remove t-boc group from -nh 2 function. a positive ninhydrin test indicated the presence of free amino group. the residue was dissolved in dcm (30 ml) and this organic fraction was washed consecutively with 5% nahco 3 solution (20 ml), nacl (20 ml), h 2 o (20 ml), dried over anhydrous na 2 so 4 , filtered and reduced under vacuum. the title compound was purified by silica gel column chromatography using 1-2% ch 3 oh in ch 2 cl 2 as a light yellow solid which was recrystallized from ethanol/water. yield: 53%; m.p. ¼ 204 c, to curcumin (100 mg, 0.27 mmol) dissolved in dry pyridine (10 ml), added decanoyl chloride (0.12 ml, 0.61 mmol) and dmap (66.35 mg) under cooled condition and stirred the reaction mixture overnight at room temperature. after completion of the reaction, as indicated by tlc, added chilled water (5 ml) and stirred for 10 min. further, the volume of reaction mixture was reduced, the residue dissolved in etoac (20 ml), washed with water and dried up. the title compound was purified by column chromatography using ethyl acetate (1.5-2%) in hexane as a dark red solid which was recrystallized from etoac and hexane. yield: 56%; m.p. ¼ 225 c, to a stirred solution of folic acid (2 g, 5 mmol) in anhydrous ethyl acetate (10 ml), added dropwise p-nitrophenol (0.834 g, 6 mmol) dissolved in ethyl acetate (10 ml). after 15 min, pyridine and triethylamine (1 ml each) were added to make it more basic, stirred for 15 min and further added dcc (2.579 g, 12.5 mmol). the reaction mixture was stirred for 2.5 h and monitored on tlc. the completion of the reaction was assessed by total consumption of the starting material. curcumin (94 mg, 0.25 mmol) dissolved in dry pyridine (5 ml) was added dropwise to activated folate ester 5 (323 mg, 0.57 mmol), stirred for 15 min and tea (0.5 ml), dcc (292 mg, 1.42 mmol) and dmap (61 mg) were further added. the reaction mixture was stirred for 5 h and monitored on tlc. the precipitate of dcu started to appear after 30 min. at the end of the reaction, dcu was filtered off, the filtrate evaporated and the residue dissolved in etoac (20 ml). this organic fraction was washed consecutively with 5% nahco 3 solution (20 ml) (to neutralize the residual acid and to separate excess amount of pnp), nacl (20 ml), h 2 o (20 ml), dried over anhydrous na 2 so 4 , filtered and reduced under vacuum. the title compound was purified by silica gel column chromatography using ethyl acetate (1.5-2%) in hexane as eluant and recrystallized from ethanol/water. yield: 48%; m.p. ¼ 238 c, r f ¼ 0.6 (etoac:hexane, 3.5:6.5), uv(etoac) l max 280, 300 nm. 1 to curcumin (184 mg, 0.5 mmol) dissolved in dry pyridine (10 ml), added koh (67.2 mg, 1.2 mmol), cooled the solution and further added benzoyl chloride (1.4 ml, 1.2 mmol) dropwise with constant stirring which continued at ambient temperature for another 2 h. the completion of the reaction was assessed on tlc. the volume of the reaction mixture was reduced to half and poured on to crushed ice and the product was extracted with etoac. the organic phase was dried over anhydrous na 2 so 4 , concentrated and the product was purified by silica gel column chromatography using 2% etoac/hexane. this di-o-benzoyl curcumin (200 mg, 0.34 mmol) was dissolved in ethanol (7 ml) and naoet was added dropwise at room temperature over 20 min and the reaction mixture was stirred for another 30 min. the sodium salt was concentrated under vacuum, washed thoroughly with ethanol, dissolved in pyridine (5 ml) and further mixed with 2-chloroethanol (0.067 ml, 1 mmol). the reaction mixture was stirred at room temperature for 6 h. after completion of the reaction, the mixture was poured on to crushed ice and extracted thoroughly with etoac, dried over anhydrous na 2 so 4 and concentrated to get the intermediate compound 7 (183 mg, 0.29 mmol). the compound 7, dissolved in dry pyridine (5 ml), was added dropwise to the activated folate ester 5 (286 mg, 0.51 mmol) and stirred the reaction mixture for 15 min. tea (0.5 ml), dcc (257 mg, 1.25 mmol) and dmap (61 mg) were further added. the reaction mixture was stirred for 5 h and the reaction was monitored on tlc. the precipitate of dcu started to appear after 30 min. after the completion of the reaction, dcu was filtered off and the filtrate was evaporated to dryness. after deprotection of both phenolic hydroxyl functions on curcumin with nh 3 -pyridine (9:1v/v) at 55 c, ammonia was removed using water pump, the solvent evaporated and the residue dissolved in etoac (20 ml). to curcumin (184 mg, 0.5 mmol) dissolved in aq naoh (5%, 10 ml), added 2-chloroethanol (0.05 ml, 0.75 mmol) and stirred the reaction mixture for 7 h. the reaction mixture was extracted with etoac. the organic extract was concentrated under vacuum, washed with 5% nahco 3 , dried over anhydrous na 2 so 4 , evaporated and crystallized with ethanol and water to get the intermediate compound 9. yield (100 mg, 0.24 mmol, 48%). compound 9 (100 mg, 0.24 mmol), dissolved in dry pyridine (6 ml), was added dropwise to the activated ester 5 (157 mg, 0.28 mmol), stirred the reaction mixture for 20 min and tea (0.5 ml), dcc (123 mg, 0.6 mmol) and dmap (32 mg) were further added. the reaction mixture was stirred for 5 h and the reaction was monitored on tlc. dcu was filtered off and the product was obtained following the usual work up procedure and purification by silica gel column chromatography using ethyl acetate (2%) in hexane as eluant as a red solid which was recrystallized from etoac and hexane. yield: 26%; m.p. 13 the in vitro antibacterial activity of each curcumin bioconjugate against gram-positive cocci (s. viridans) and gram-negative bacilli (e. coli, k. pneumoniae, p. mirabeilis) was evaluated by microdilution broth susceptibility test method. the bacterial strains were isolated from clinical patients following the standard protocols [38] . the antibacterial activities of curcumin bioconjugates, 2, 3, 4, 6 and 8 were compared with that of curcumin. the stock solutions of the conjugates along with curcumin prepared in water/dmso to ensure complete solubilization were doubly diluted to the concentrations 1000 mg; 500 mg, 250 mg, 125 mg and 65.5 mg to which peptone-water (1 ml) was added. the fresh culture of aforementioned strains was prepared and a standardized bacterial suspension (100 ml) of 0.5 mcfarland turbidity was added to each dilution. suitable solvent control (dmso), positive growth control and standard control were also run simultaneously [39] . the tubes bearing different concentrations of the compounds and control tubes were incubated at 37 c for 24 h. after incubation, antibacterial activity of molecules in the tube was detected by lack of turbidity which indicated the inhibition of bacterial growth. the concentration in the tube with highest dilution showing no turbidity has been reported as mic. each test was performed in triplicate and the mics reported represent the result of at least two repetitions. the antiviral assay was based on inhibition of virus-induced cytopathogenicity in various cell cultures as described previously [39] [40] [41] [42] [43] . the assays were performed against various viruses, viz. herpes simplex virus type 1 (strain kos), herpes simplex virus type 2 (strain g), cytomegalovirus (cmv) from sindbis virus (sv), parainfluenza virus type 3 (piv-3) and reovirus type-3, vesicular stromatitis virus (vsv), coxsackie virus (coxs v), respiratory syncytial virus (rsv) using crfk, hel, hela and vero cell lines and hiv-1 (iii b and rod strains) in mt-4 cells using mtt method. the confluent cell cultures were prepared in microdilution trays and inoculated with 100 ccid50 (1 ccid50 corresponding to the virus stock dilution that proved infective for 50% of cell cultures). after 1 h of virus adsorption to the cells at 37 c, the residual virus was replaced by cell culture medium (eagle minimum essential medium supplemented with 3% fetal calf serum and antibiotics) and various concentrations of the test compounds 2, 3, 6, 8 and 10. virus cytopathogenicity was recorded as it reached completion in the untreated virus-infected cell cultures, i.e., at 1-2 days for vesicular stomatitis virus; 2 days for coxasackie; 2-3 days for vaccinia, herpes simplex type 1 and 2 and sindbis, 4 days for respiratory syncytial virus and 6-7 days for reo and parainfluenza viruses [44, 45] . the antiviral activity of the compound is expressed as the ec 50 or the concentration (mm) required to inhibit virus-induced cytopathogenicity by 50%. the viruses used are either dna or (þ)/(à) stranded rna viruses. the cytotoxicity of the test compounds 2, 3, 6, 8 and 10 was assessed on the basis of two parameters: (i) alteration of normal cell morphology, and (ii) inhibition of macromolecule (dna, rna and protein) synthesis. cytotoxicity (cc 50 ) of the compounds was examined by trypan blue exclusion test. to evaluate cytotoxicity, uninfected confluent cell cultures treated with various concentrations of the test compounds, were incubated in parallel with virus-infected cell cultures prepared in plastic trays containing 24 wells (16 mm diameter; falcon plastics). after 2 days of incubation at 37 c in a co 2 incubator, when the cell cultures were confluent, culture medium was removed from each well and 1 ml of maintenance medium containing serial concentrations of the test compounds was added. for the cell control, 1 ml of maintenance medium without compound was added. all cultures were incubated at 37 c, and after 2 and 7 days of incubation, compounds were withdrawn and the viability of the cells was determined by the trypan blue exclusion method. antiviral screening against hiv -1 (iii b and rod strains) was monitored by the efficiency of drug compounds to inhibit syncytia formation after hiv infection of mt -4 cells following the mtt method [45] [46] [47] . the activity of the compounds against hiv-1 was monitored by inhibition of hiv-1-induced cytopathogenicity in mt-4 cells. briefly, mt-4 cells (3 â 10 4 cells per well in 96 well plate) were cultured in microdilution trays in the presence of various concentrations of the test compounds added immediately after infection with 50% cell culture infective doses of hiv-1. after 5 days of incubation at 37 c, the number of viable cells was determined by the mtt (3 0 -[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) method. the partition coefficients of bioconjugates 2 and 3 along with curcumin were determined by the stir-flask method [37] . octanol and 0.1 naoh solution were mutually saturated and both phases were separated by centrifugation (5000 rpm, 10 min). a stock solution of each molecule (4 â 10 à3 m) was prepared using octanol-saturated naoh solution. a part of these solutions were kept aside for uv measurement. further, naoh-saturated octanol was added at various volume ratios to other part of the stock solution. then the two-phase mixtures were intensively stirred for 4 h at constant temperature (25 c) . the absorbance of the aqueous solutions was measured by uv-vis spectrophotometer at l max 258 nm for compound 2 and l max 250 nm for compound 3 and 223 nm for curcumin. the p value was calculated as follows: where a 0 and a 1 represent the absorbance of the molecule in the aqueous phase before and after partitioning, v w and v 0 are the water and octanol volumes, respectively. log p values are an average of three measurements. biochemistry of the amino acids, vols. ii and i. compressive review of amino acid biosynthesis and precursor functions, particularly in vol. ii. academic lipid biochemistry: an introduction oligonucleotide synthesis a practical approach methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically drugs, microbes, host-the elements of chemotherapy the authors thank the department of biotechnology (dbt) and indian council of medical research (icmr), new delhi, government of india for financial support. key: cord-261170-arnwk287 authors: gallimore, w. title: chapter 18 marine metabolites oceans of opportunity date: 2017-12-31 journal: pharmacognosy doi: 10.1016/b978-0-12-802104-0.00018-4 sha: doc_id: 261170 cord_uid: arnwk287 abstract the marine environment provides an array of compounds often with unique molecular architectures boasting an equally wide array of bioactivities including anticancer, antiinflammatory, and antimicrobial activity. typically without the benefit of folklore therapeutic knowledge, marine organisms are collected, extracted, and fractionated to afford compounds that undergo evaluation with in vivo and in vitro assays en route to clinical applications. the pharmaceutical industry has benefited from research into marine metabolites with the development of marine-derived drugs including cytarabine, vidarabine, and ziconotide along with the more recently developed formulation carragelose, an antiviral spray. cosmetic applications incorporating marine extracts include abyssine and refirmar. research with macroinvertebrates, macroalgae, and microorganisms continue in the hope that drugs of the future will be culled from the oceans of the world. while obtaining a consistent and adequate supply of the bioactive compounds remains a challenge to be overcome, synthetic methods are being employed along with the application of biotechnological techniques to ensure that the drugs, when developed, will be in sufficient quantities for distribution to those who are in need. to gain an understanding of the importance of marine natural products chemistry in drug development g to be able to map the process involved in drug development from marine natural products g to gain an appreciation of the range of biological activities associated with compounds isolated from micro-and macroorganisms g to identify the marine-derived drugs which are undergoing clinical evaluation over 75% of the earth's surface is covered by vast expanses of ocean. its inhabitants are diverse with 15 of the 34 phyla occurring exclusively in the oceans with only one phylum (onychophora) being reported as present on land only [1] . the marine environment provides an array of structurally unique and diverse constituents produced by an equally diverse consortium of marine organisms living on our coral reefs and in benthic communities. the marine organisms are highly variable in species, color, and morphology and belong to several phyla including porifera (sponges), ascidiacea (sea squirts), and octacorallia (soft corals). the metabolites of marine origin emanate from a variety of parts of the plants and animals and are thought to be produced as a form of chemical communication, defense, or to ward off potential predators [2à12] (figs. 18.1à18.3). the potential for a range of applications including anticancer, antibacterial, antiviral, antiinflammatory, antimalarial, antituberculosis activity, as well as pharmacological and industrial applications. the classes of compounds manufactured by marine organisms include alkaloids, terpenoids, shikimates, peptides, and polyketides [2à15] . temperature, salinity, ph, and dissolved oxygen concentrations in the water, thereby providing useful information to facilitate environmental studies [20, 21] . caution should always be exercised in the collection of marine species. gloves should be worn in the collection and subsequent handling of specimens. scuba divers should be clad in wet suits to protect against the possible deleterious effects of chemicals being exuded into the water by the organisms being collected. the personal unfortunate experience (author's) of hours of severe discomfort and rashes as a result of collecting the sponge neofibularia nolitangere from a reef in discovery bay, jamaica, provides clear evidence regarding the level of respect which should be accorded to figure 18 .5 student snorkeling to collect marine specimens. marine organisms whose chemistry is yet to be investigated. records are made of the depth, habitat, global positioning system coordinates (latitude and longitude), color, morphology, and associated organisms. an appropriate coding system should be employed to distinguish specimens. where possible, the specimens are photographed in situ as well as by the dockside (figs. 18.7 and 18.8) . a voucher specimen of each organism is usually preserved in 70% aqueous ethanol for the purpose of taxonomic identification. ascidians are usually preserved in seawater containing menthol crystals with more long-term storage in 10% formalin solution [22] . it should be noted that the recollection of organisms has proved to be a challenge in some instances. an ascidian species, e.g., found to be thriving on the mangrove in the summer of one year could all but disappear from the ecological landscape 6 or 12 months later, while a healthy bed of algae may be short-lived if there are dynamic factors involved in their growth. for example, the occasional nutrient runoff or groundwater seepage event could provide the ideal environment for the growth of selected algal species. environmental factors are key in the marine landscape and often provide a source of frustration to the specimen collector. prior to extraction of the collected organism, the specimens may be frozen, air-dried, freeze-dried, or could be retained in the fresh state. the majority of the marine organisms are extracted fresh or frozen while the remaining specimens are lyophilized or dried in air before extraction [22] . in some instances, dried algal species are ground to a powder prior to extraction as described by sansom and coworkers who isolated an antiproliferative bis-prenylated quinone from the alga perithalia capillaris [23] . the extraction of marine organisms may be carried out using a range of organic solvents including hexanes, dichloromethane, acetone, ethyl acetate, as well as more polar solvents such as ethanol and methanol. in many instances, a mixture of polar and medium polarity or nonpolar solvents is utilized in the extraction protocol. for example, the extraction of the madagascar sponge monanchora dianchora was achieved in ch 3 cl:meoh (1:1) to yield two polycyclic guanidine alkaloids [24] . extractions are usually exhaustively performed over several days with at least three aliquots of the solvent being used. the solvent is then removed in vacuo by rotary evaporation. solvent partitioning is another strategy employed in the extraction of the organisms. this involves single one-step or two-step partitioning systems usually involving an aqueous phase portioned with a solvent immiscible with that phase. the kupchan and modified kupchan procedures are often employed in natural products as was described in the isolation of a diterpene from an axinella species [25] . in this procedure, the concentration of the aqueous layer is progressively adjusted to afford three or four different fractions. complex partitioning procedures are also employed, albeit rarely so. simple partitioning has been most commonly employed with kupchan schemes being utilized with less frequency [22] . chromatographic methods of separation include gravity column chromatography, flash column chromatography, and vacuum liquid column chromatography utilizing silica gel as the packing material. with silica gel, the components of the marine extract are separated on the basis of polarity of the compounds. as the polarity of the eluting solvent increases compounds of increasing polarity are eluted from the column with hydrocarbons, e.g., eluting before alcohols. the elution of the components of a column is monitored by using thin layer chromatography (tlc) plates which are spotted to show the sequence of elution of the compounds (figs. 18.9 and 18.10). bonded reverse phase silica is employed in instances where the constituents of the marine extract include polar metabolites. bonded phases include ods (c 18 ), c 8 , cyano, and diol columns. separation of constituents may also be effected using gel permeation chromatography which effects separation of constituents on the basis of the size of the compounds. in this regard, sephadex lh-20 is commonly utilized in marine natural products isolation work [26] . resins such as biobeads, amberlite, xad-2, and xad-4 are also utilized in separating components of relatively high polarity. the use of xad-2 in the separation of antiviral trisulfated triterpene glycosides from the sea cucumber staurocucumis liouvillei is one such example in marine natural products isolation work [27] . the use of hplc employing a reversed phase stationary phase system is commonplace in marine natural products isolation work with c 18 and c 8 semipreparative and preparative columns being used. mplc and recycling hplc techniques are related techniques for purification of a range of metabolites including alkaloids, peptides, and terpenoids. tandem systems such as liquid chromatography-mass spectrometry systems are also employed to assist with dereplication efforts. unusual ms peaks in the profile suggest that novel components are present in the fraction or extract being evaluated. those fractions with unusual constituents may then become the focus of the research efforts. solidphase extraction methods are also employed in separating compounds. the structural identification of compounds isolated from the range of marine sources is facilitated by the use of spectroscopic techniques such as 1d and 2d nuclear magnetic resonance (nmr) spectroscopy and infrared (ir) spectroscopy. x-ray crystallographic techniques are also important in aiding in the determination of the stereochemistry of the compound. the identification of nanogram quantities of a novel compound is becoming increasingly more facile with the use of the cryoprobe, capillary probe, and mans probe [15] . in vitro activities of marine metabolites have been investigated for a diverse range of cell systems including antiinflammatory, antimicrobial, and anticancer activities. crude extracts, fractions from crude extracts, as well as pure compounds are typically evaluated for biological activity. the in vitro biological evaluation of the isolated compound may be performed using cell lines from human subjects or animals. brine shrimp, fish, and sea urchin are among the organisms employed in the evaluation of compounds or extracts for ecological and therapeutic importance (figs. 18.11 and 18.12) . a summary of the biological activity of some of the organisms discussed in this section is presented in preclinical trials are an essential component of the process of evaluation of the therapeutic potential of a compound. these trials often include animal models such as rats, dogs and monkeys. the major sources of biologically relevant compounds have been found to be from sponges, coelenterates, algae, echinoderms, ascidians, molluscs and microorganisms [14] . macroinvertebrates include sponges, ascidians, and soft coral. it has been found that the vast majority (75%) of novel compounds obtained from the marine environment have been sourced from the porifera and coelenterata (cnidaria) phyla [15] . scheme 18.1 shows representative structures of compounds isolated from macroinvertebrates. macroinvertebrates include: 1. sponges 2. ascidians 3. soft coral sponges (porifera) are sedentary, filter feeding metazoans which utilize a single layer of flagellated cells (choanocytes) to pump water current through their bodies in a unidirectional manner. there are over 5000 species of sponges accounting for much of the epifaunal biomass. extracted fresh or freeze-dried, sponge extracts are an important source of biologically active compounds. these isolates exhibit an impressive array of biological activities, some of which are described here. one sponge which has gained a place in history due to the promising biological activity being displayed is halichondria okadai, the producer of halichondrin b, which underwent evaluation as an anticancer agent. okadaic acid, also from h. okadai, exhibited inhibitory activity against phosphatase-1 and phosphatase-2a [28] (fig. 18 .13). agelaspin, an antitumor glycosphingolipid obtained from the marine sponge agelas mauritianus, demonstrated antitumor activity in vivo against murine b16 melanoma. this compound was also found to stimulate the immune system. a derivative of agelaspin, krn-7000, underwent clinical investigations for cancer immunotherapy [28] . more recently, the extracts of another agelas sp., a. nakamurai, contained the compound agelasine d which exhibited high antibacterial activity [29] . the deep water sponge discodermia dissoluta produced discodermolide, a polyhydroxylated lactone which exhibited anticancer activity, as well as immunosuppressive activity. it was found to stabilize microtubules in a manner similar to the drug taxol and underwent evaluation for use in tumors resistant to taxol [30, 31] . dysidea arenaria was found to contain arenastatin a which showed potent activity against kb cell lines (ic 50 5 5 pg/ml) [32] . girolline is a substituted imidazole isolated from the sponge pseudaxinyssa cantharella which functions by inhibiting the termination step in eukaryotic protein synthesis. having entered phase 1 clinical trials, it was withdrawn due to its adverse hypertensive effects seen in treated patients [28] . mycalamides a and b are protein synthesis inhibitors isolated from the new zealand sponge mycale sp. in vivo activity against a59 coronavirus was observed in mice when treated with a 2% mycalamide mixture at a dosage of 0.2 μg/kg daily with 100% survival over a two-week period. pure mycalamide a inhibited the herpes simplex virus 1 and polio virus type 1 at a concentration of 0.005 μg/disk. mycalamide b was found to exhibit more potent antiviral activity and cytotoxicity than mycalamide a [28] . the baculiferins i, j, l, and m from the marine sponge iotrochota baculifera have been found to inhibit human immunodeficiency virus-1 (hiv-1) with ic 50 values between 0.2 and 7.0 μm [33] . jasplakinolide, the first example of a cyclodepsipeptide isolated from a sponge, is a 19-membered macrocyclic depsipeptide from the jaspis sp. exhibiting in vitro antimicrobial activity at a minimum inhibitory concentration of melinacidins and gencidin antibacterial marinospora sp. lynamicins a-e antibacterial 25 μg/ml against candida albicans. with a topical administration of 2% jasplakinolide solution, an effect similar to that of miconazole nitrate was achieved in vivo [28] . discorhabdin r is a novel pyrroloiminoquinone isolated from the southern australian sponge negombata sp. and antarctic latruncula sp. which was found to display antibacterial activity against both gram-positive (staphylococcus aureus and micrococcus luteus) and gram-negative bacteria (serratia marcescens and escherichia coli), respectively [34] . antibacterial activity against a strain of the bacterial parasite plasmodium falciparum was reportedly identified in monanchora arbuscula with the active agents being the batzellidine alkaloids (ic 50 5 0.2à0.9 μm) [35] . an important isolate from a spongia sp. is the polyhydroxylated steroid, agosterol a, which functions by reversing multidrug resistance caused by the overexpression of two kinds of membrane glycoprotein in cancer cells [36] . from the phylum cnidaria the genera sinularia and briareum have proven to be prolific sources of novel compounds. cembranoids, 5,8-epidoxysteroids, sinulaflexiolides, and africanenes have been isolated from sinularia species [1] (fig. 18.14) . examples of other species of soft corals include the taiwanese soft coral cespitularia taeniata which was extracted with ethanol to yield a group of verticillene diterpenoids including cespitulactam k. the compounds were evaluated against human epidermal carcinoma and murine l1210 leukemia cell lines. cespitulactam k exhibited activity against the cancer cell lines (3.7à5.1 μg/ml) and also showed marked antimicrobial activity against m. luteus and cryptococcus neoformans [37] . the methanol extract of the octocoral muricea austera showed in vitro activity against chloroquine-resistant p. falciparum and was found to contain a range of different classes of compounds including tyramine derivatives, steroidal pregnane glycosides, and sesquiterpenoids [38] . cytotoxic dolabellane diterpenes were isolated from the formosan soft coral clavularia inflata var luzoniana and bioactivity against p388 cell lines with ed 50 values between 0.5 and 3.6 μg/ml was observed [39] . tunicates, sea squirts, or ascidians belong to the subphylum of tunicata (urochordata). they are so named because of their cellulose-containing protective tunic surrounding the organism. tunicates attach to a substratum, usually a marine solid surface such as a mangrove root, rocks, jetties, or even algal species (fig. 18.15 ). much like sponges and soft corals, ascidians have also been found to be a good source of bioactive agents. didemnin b, isolated from the tunicate trididemnum solidum, is one such bioactive compound, showing remarkable antiviral and cytotoxic activity. didemnin b demonstrated activity against p388 and l1210 murine leukemia cell lines. it was advanced into preclinical and clinical trials 1 and 2, but had to be withdrawn due to its harsh toxicity [30] . aplidine, formally known as dehydrodidemnin, an isolate from the mediterranean tunicate aplidium albicans, is one such bioactive compound. being structurally related to didemnin b, aplidine was found to be up to 10 3 more active and less toxic than didemnin b. it entered into phase 1 clinical trials in 1999 under investigation for the treatment of solid tumors and non-hodgkin's lymphoma. broad spectrum activity was displayed in vitro and in vivo against leukemia, melanoma, breast, ovarian, colon, and lung (nonsmall cell) cancer. having advanced to phase 2 clinical trials, aplidine affects protein synthesis through gtp-dependent inhibition of elongation factor 1-α [30] . the extract of the palauan ascidian didemnum guttatum afforded the sulfonated serinolipid cyclodidemniserinol trisulfate which exhibits an antiviral effect by inhibiting hiv-1 integrase, an attractive target for antiretroviral chemotherapy [30] . macroalgae belong to three main phyla: rhodophyta (red algae), chlorophyta (green algae), and phaeophyta (brown algae). biological activities identified in extracts and metabolites of algal origin include anticancer, antiobesity, neuroprotective, and antioxidant activity and scheme 18.2 shows chemical structures of representative bioactive compounds isolated from the macroalgae. a wide range of algal species are utilized in fresh or dried forms as food particularly in asian countries where folklore traditions govern their industrial and medicinal usage [40] . macroalgae are the source of agar, carrageenan, and alginate, which are all of importance in the food industry. the range of compounds isolated from algal sources has been variable. representative examples of bioactive constituents from macroalgae are mentioned below. cytotoxic activity has been identified in 8α,11-dihydroxypachydictyol a, a diterpenoid compound from a dictyota sp. collected on bangsaen beach in thailand. antimalarial activity was also found in the diterpene isolated from this extract when the compound was tested with malarial parasites [41] . stypolactone, an isolate from the brown alga stypopodium zonale, was found to exhibit weak cytotoxic activity in vitro when evaluated with a-549 and h-116 cell lines [42] . zonaquinone acetate, obtained from jamaican populations of s. zonale, displayed in vitro activity against breast and colon cancer cell lines [43] . specimens of taonia atomaria produced atomarianones a and b which were reportedly found to be cytotoxic against nsclc-n6 and a-549 cell lines [44] (fig. 18.16) . crude extracts of algal species have been found to exhibit a range of biological activities. for example, aqueous extracts of gracilaria corticata and sargassum oligocystum exhibit bioactivity against cancerous human leukemia cells [45, 46] while a methanol extract of plocamium telfairiae was observed to display bioactivity against ht-29 colon cancer cells [47] . antiinflammatory activity was found in the green alga from which 2-(2,4 0 -dibromophenoxy)-4,6-dibromoanisol was isolated. this activity was identified using a snake toxin-induced mouse limb model [48] . also exhibiting antiinflammatory activity is a mixture of phytosterols obtained from dunaliella tertiolecta. when administered in a sheep model of inflammation-induced cytokine production, an inhibitory effect was observed [49] . polyphenolic extracts from the red alga laurencia undulatea displayed antiinflammatory activity in vivo. these extracts served to inhibit asthmatic reactions in mice sensitized and challenged with ovalbumin which was used to induce murine allergic reactions in test subjects [50] . antiinflamatory agents floridoside and d-isofloridoside from the south korean alga l. undulatea were found to inhibit free radical oxidative stress at ic 50 values between 22 and 43 μm [51] . biologically active compounds have been isolated from the brown seaweed dictyota cervicornis from which was obtained sulfated polysaccharides with powerful anticoagulant activity [52] . antioxidant activity, evaluated using the dpph method, was reported in phenolic isolates of halimeda monile when liver injury was induced in a rat model. the phenolic fraction was administered over a 20-day period and led to protective effects against chemicals harmful to the liver [53] . with ic 50 values between 0.5 and 2.9 μm, potent antimalarial activity against the human malarial parasite p. falciparum was identified in new macrolides bromophycolides j, m, n, o, p, and q from the red algae callophycus serratus [54] collected in fiji. the marine alga halimeda tuna was studied by koehn and coworkers, leading to the isolation of halitunal, a diterpene displaying in vitro antiviral activity against murine coronavirus a59 [55] . ecologically important roles are played by some compounds from alga sources. for example, halimedatrial, a diterpene isolated from halimeda lamouroux, exhibited toxicity toward reef fishes and appeared to be a feeding deterrent. antimicrobial activity was also reported from this compound [56] . almost 20% of all bioactive marine compounds currently being studied are obtained from marine microorganisms [15] . these microbes are found in swabs from the surfaces of marine plants and animals, suspended in the water from geothermal vents and deep water environments, or on sediment surfaces. they thrive in a variety of environments including locales characterized by high pressures of up to 600 atmospheres, high temperatures, and high salinities. efforts at culturing some of the microorganisms have met with varying degrees of success. the ability to propagate these microorganisms in an economically feasible way will be of great significance as potent bioactive metabolites are discovered [57] (figs. 18.17à18.19 ). marine microorganisms are found 1. on the surface of marine plants and animals 2. suspended in water 3. on sediment surfaces historically, terrestrial microbes have been a potent source of pharmaceutical agents with the seminal discovery of penicillin. the discovery of new antibacterial agents is a serious priority because of the development of potent resistance to current antibiotics on the market. marine bacteria produce a wide variety of secondary metabolites for the purpose of defending themselves against other microbes. scheme 18.3 shows structures of representative compounds from microorganisms associated with marine specimens. marine bacteria which produce compounds of biological significance include pseudoalteromonas species which was found to produce 3,3 0 , 5,5 0 -tetrabromo-2,2 0 -diphenyl diol, an inhibitor of methicillin-resistant s. aureus. the class of 4-methoxypyrrole-containing compounds, the tambjamines, isolated from p. tunicata, was found to be active antifungal, immunosuppressive, and antimicrobial agents. biologically active compounds from marine bacteria also include streptomyces species from sediment and fish gut from which anticancer (e.g., halichomycin and δ-indomycinone) and antibacterial agents (e.g., phenazines) have been obtained [58à60]. vibrio species obtained from sponge specimens have produced phenolic and trisindole compounds with antibacterial activity [61, 62] . a micromonospora sp. obtained from a soft coral produced thiocoraline, a compound exhibiting anticancer activity [63] . marine fungi have also been known to produce compounds with a range of bioactivities including antiviral, antifungal, enzyme inhibition, and anticancer and antibacterial activities. the isolation and cultivation of fungi from the marine environment is of critical importance for propagation of the microbes from which biologically relevant compounds may be obtained. protocols have been established for this work [64] . fungal species which have produced antibacterial compounds include corallospora pulchella isolated from sand. this species produced melinacidins and gencidin [65] . anticancer activity has been reported from metabolites of aspergillus sp. (including the aspergillamides and fumiquinazolines) and penicillium sp. sourced from a marine alga which was found to contain pentostatins and communesins among other compounds [66, 67] . antiviral activity, attributable to the presence of halovirs, was identified in a scytalidium sp. collected from a seagrass species. potent antiviral activity against h. simplex virus (type 1) was observed and may be acting by binding directly to the virus [68] . actinomycetes have been the source of a wide range of antimicrobial agents, the most common of which include tetracycline and streptomycin. other bioactive compounds originating from actinomycetes include antitumor and antimicrobial agents. a marinospora sp. produced a group of bisindole pyrroles, lynamicins aàe, which exhibited biological activity against gram-positive and gram-negative species. importantly, activity was also shown against drug-resistant pathogens including methicillin-resistant s. aureus [69] . anticancer activity against lung, colon, and breast cancer cell lines was exhibited by isolates from the fermentation of a streptomyces sp. (mbg-04-17-069). tartrolon d was found to be the bioactive agent [70] . microalgae are found in seven phyla. these include chlorophyta, phaeophyta, rhodophyta, crystophyta, cryptophyta, eugelophyta, and pyrrhophyta. the blue-green algae, cyanophyta, are cyanobacteria which have been found to share characteristics with eukaryotic algae. these microalgae produce compounds with a high degree of structural diversity and species, such as lyngbya majuscula, have produced a vast array of biologically active compounds [71] . curacin a, e.g., isolated by gerwick and coworkers in 1994 [72] , was found to function by disturbing microtubule assembly, thereby functioning as a lead compound in chemotherapy. microcystis aeruginosa is the source of potent protein phosphatase-1 and phosphatase-2a inhibitors identified in microcystins [73] . other microalgal species under examination include dinoflagellates which produce an array of bioactive toxins including saxitoxin and maitotoxin which function by blocking or activating sodium/calcium channels. challenges exist with respect to the culturing of these organisms due to relatively low proliferation rates and the large quantities of culture required to obtain small amounts of bioactive compounds. diatoms, microscopic unicellular colonial algae, grow at a faster rate and are amenable to culturing but few bioactive metabolites have been identified from these microalgae [28] . some marine compounds sourced from microbes are of clinical significance, undergoing evaluation as potential pharmaceutical agents. the marine-derived drug pipeline, almost nonexistent in decades gone by, now has a range of candidates at various stages of development as shown in table 18 drugs in phase three clinical trials include tetrodotoxin, a guanidinium alkaloid under the trademark name tectin obtained from the pufferfish [34] . affecting the sodium channels, this drug is being investigated for the treatment of chronic pains (scheme 18.5). a depsipeptide from a tunicate, plitidepsin, is being tested by pharmamar in the treatment of a variety of cancers, namely leukemia, multiple myeloma, and lymphoma. another drug under evaluation by pharmamar for cytotoxic activity is zalypsis (pm00104) sourced from a mollusc which targets the dna-binding capacity of diseased uterine, lymphoma, cervical, and endometrial cancer cells. the alkaloid-derived compound pm01183 is another drug candidate from pharmamar being evaluated for its efficacy against a range of cancers including ovarian, breast, lung, acute leukemia, and endometrial cancer [74, 75] . bryostatin i, from the bryozoan bugula neritina has been involved in a battery of clinical trials being investigated for its potency against cancer. it is currently under phase i evaluation as a treatment for alzheimer's [74] . in the early years, the challenge associated with the supply of the drug was underscored by the fact that, in order to obtain 18 g of a cgmp quality bryostatin i, 13 tonnes of b. neritina had to be collected in californian waters [13, 76] . the gene cluster of the uncultivated microbial symbiont of b. neritina, candidatus endobugula sertula has been successfully identified, thereby opening the potential for the supply of the compounds [77] . kahalalide f, a cyclic depsipeptide, was found in the mollusc elysia rufescens as well as the green algae bryopsis sp. on which it feeds. this compound is currently in phase i/ii trials as a treatment against prostate cancer [74] (fig. 18.20) . dmxba [(3-(2,4-dimethylxybenzylidene)]-anabaseine is a derivative of anabeseine, an alkaloid found in marine worms. found to improve cognition in animal models, dmxba and other related compounds have demonstrated neuroprotective activity in both in vitro and in vivo screens. thought to have an effect on macrophage 7 receptors, antiinflamatory activity was also observed in animal models. phase i evaluation of healthy males and schizophrenics have shown that dmxba has led to marked improvements in cognitive function [74] . there are several marine compounds sourced from microbes which are of clinical significance. clinical trials are being conducted on plinabulin (npi-2358), a vascular disrupting agent obtained from a marine fungal extract with potential for activity against multidrug resistant tumor cells. marizomib (salinosporamide a, npi-0052), an isolate from a marine bacterium salinospora tropica, is a novel proteasome inhibitor which is currently under investigation for its efficacy against solid tumor models. the compound exhibits low cytotoxicity to normal cells and has significant potential for oral and intravenous administration [74] . the ultimate goal of many marine natural products and synthetic chemists is that the isolated or synthesized molecule possesses therapeutic applications. there are several food and drug association (fda)-approved drugs of marine origin obtained from sponges, a fish, a cone snail, a mollusc, and cyanobacterium species, while yondelis (trabectidin) obtained from the ascidian ecteinascidia turbinata, has been approved in the european union. the antitumor effects of aqueous ethanol extracts of e. turbinata were observed from 1969. in vitro trials had been carried out on a 60 human cancer cell panel by the company developing the drug, pharmamar, and the national cancer institute. aquaculture of the ascidian proved to be the initial strategy used to obtain sufficient quantities for evaluation of the efficacy of the compound. semisynthetic procedures involving the fermentation of pseudomonas florescens are now currently employed in the pharmaceutical preparation of the drug which is sold in over 80 countries, including south korea and russia, under the trade name yondelis. yondelis is also used in patients with relapsed platinum-sensitive ovarian cancer. this drug is currently under evaluation in phase ii for breast, prostate, lung, and pediatric cancers. the sponge tethya crypta (cryptotethia crypta) was the original source from which the drug cytarabine was developed. cytarabine is a synthetic analogue of the nucleoside which was originally isolated from the sponge. sold under the trade name cytosar-u, this cytotoxic agent inhibits deoxyribonucleic acid (dna) polymerase and dna synthesis. acute lymphocytic leukemia, non-hodgkin's lymphoma, and acute myelocytic leukemia are among the conditions being treated by this drug approved by the fda in 1969 [74] . produced by fermentation of streptomyces griseus, cytarabine has limited bioavailability but improvements in the delivery system have been made [78] . a slow-release liposomal form of cytarabine (depo cyle) has been approved in the united states and europe for the prolonged administration/exposure in cerebrospinal fluid. a related drug, vidarabine (vira-a), was developed from spongouridine and found use as an antiviral treatment for epithelial and superficial keratitis caused by the h. simplex virus types 1 and 2. viral dna polymerase and dna synthesis of herpes are inhibited by this drug which was discontinued over 10 years ago. this drug is still in use in europe for ophthalmological challenges. prialt (ziconotide) was obtained from a peptide ω-conotoxin mviia isolated from the cone snail conus magus. with a unique mode of action, this drug acts by reversibly blocking n-type calcium channels in some specific nerves in superficial layers of the spinal cord. this drug is used for the management of severe and chronic pains in patients suffering from cancer and acquired immunodeficiency syndrome who are unable to use or are unresponsive to other drugs such as morphine. ziconotide had to be synthesized using solid-phase peptide synthesis due to the insufficient quantities supplied by the cone snail, c. magus [79] . the blockage of the spinal cord induced by this drug prevents the release of neurotransmitters responsible for pain from specific neurons. related conus peptides are undergoing evaluation in human clinical trials [80] . brentuximab vedotin (sgn-35) is being marketed under the trade name adcetris by seattle genetics and has gained repute for the treatment of hodgkin and systemic anaplastic large cell lymphoma [81] . this drug is an analogue of dolastatin 10, a compound isolated from the sea hare dolobella auricularia, which was later found to be produced by diet-associated cyanobacteria symploca hydnoides and l. majuscula. preliminary phase i and ii clinical trials of dolastatin 10 and a related analogue were largely unsuccessful. antibody-drug conjugates function by selectively delivering the drug to the cancer cell by linking the dolastatin 10, e.g., to an antibody that targets a cell membrane protein on the surface of hodgkin's lymphoma cells. this technology has proven to be a seminal development. omega-3 fatty acids from fish oils are being marketed under the trade name lovaza by glaxosmithkline. used in the treatment of hypotriglyceridemia, the drug controls ethyl esters of eicosapentaenoic acid and docosahexaenoic acid and functions by lowering triglyceride levels. [81] . eribulin mesylate (e 7389), with the trade name halaven was formulated from the macrolide halichondrin b sourced from the sponge h. okadai. studies related to the anticancer activity of simpler analogues of halichondrin b showed that the efficiency is retained leading to the development of eribulin mesylate which is more water soluble than the parent macrolide. now approved for use, potent and irreversible inhibition in cancer cells medicated by this drug resulted in the death of the cells by apoptosis. in the absence of tubulin, cell growth grinds to a halt. related compounds are currently being evaluated in phase ii trials [81] . one of the more recent formulations on the market is carragelose, an antiviral nasal spray which functions by creating a physical antiviral barrier in the nasal cavity. the company marinomed biotechnologie gmbh, utilized iotacarrageenan, sulfated polysaccharides found in the rhodophyceae seaweed as well as other seaweeds. the product is effective against the early symptoms of the common cold [81] . it should be noted that, in addition to the pharmaceutical applications of marine-sourced therapies, a range of cosmetic applications also exist and are thriving industries. the foray into cosmetic applications was led by estee lauder with the antiaging skin care remedy resilience which contains an extract from the caribbean sea whip pseudopterogorgia elisabethae. the active antiinflammatory and analgesic agents are the pseudopterosins, tricyclic diterpene glycosides, which have been found to inhibit pla2 and 5-lipoxygenase. derivatives of the pseudopterosins underwent phase i and ii trials to examine wound healing efficiency but the lipophilic and insoluble nature of the compounds have served to limit its potential as an effective drug. compounds from this group of tricyclic diterpene glycosides also underwent preclinical evaluation as antiinflammatory drugs [81] . abyssine is marketed as a product used to soothe and reduce irritation in skin sensitive to ultraviolet b light as well as chemical and mechanical attack. it consists of an extract from an alteromonas species and contains a high molecular weight polymer with two different oligosaccharides (exopolysaccharide), while seacode represents another exopolysaccharide which occurs as a mixture of extracellular glycoproteins and other glucidic exopolymers produced by fermentation of a pseudoalteromonas sp. this product has been found to improve skin roughness after up to four weeks of administration. refirmar, a recent product to be introduced, was obtained from an intracellular extract from a fermentation of a new pseudoalteromonas sp. isolated from a deep (2300 m) hydrothermal vent in portugal's exclusive economic zone, extraction of the cultured biomass afforded a mixture of macromolecules which inhibit muscle contraction. the hydrating and antiaging potential of the product has been evaluated in vivo and in topically applied formulations [81] . the area of marine natural products chemistry has clearly developed leaps and bounds as evidenced by the relatively large number of marine-derived drugs undergoing evaluation as potential therapeutic agents. buoyed by the potential for the development of natural products from the sea, research work continues to advance with the discovery of new bioactive compounds and new applications for previously isolated molecules [2à15]. the supply issue, however, remains one of critical importance as it relates to the development of drugs from a marine organism. for example, (1) spongistatin 1 has been reported to be highly cytotoxic. it has been deemed to be the most active of all natural and synthetic compounds investigated by the national institute of cancer (usa). three tonnes of the sponge yielded 0.8 mg of the compound. another collection and processing of 400 kg of the sponge afforded 10 mg of the compound. this isolation work facilitated structure elucidation work. the ic 50 value for this compound was evaluated at 10 26 m in colon cancer cells and 10 212 m for breast cancer cell lines [82] . synthetic approaches to the compound have been presented by research groups including petit and coworkers [83, 84] . total synthesis of biologically active marine compounds is often fraught with its attendant challenges due to the length of multistep synthetic procedures and the general complexity of the structural motifs which must take into account stereochemical considerations. propagation through mariculture and aquaculture are also being studied to determine the viability of using these approaches to deal with the challenges associated with procuring sufficient quantities for clinical trials and subsequent formulation into drugs [85] . the timeline from discovering the drug, leading to the entry into the market typically spans a 20-to 30-year period during which time the capital injection is considerable, often necessitating support from the large pharmaceutical entities which are sometimes hesitant about making investments which may not yield significant financial rewards [86] . the caribbean region, being an important source of marine species with which much research work has been carried out, is not likely to become the recipient of the potential benefits to be derived from the development unless more research work in this area is undertaken in the region with support from the appropriate collaborators. in the future, it is expected that new strategies will be employed to ensure the supply of large quantities of the target compounds. these include optimization or fermentation techniques for propagation of microbes, including mixed fermentation methods. biotechnological approaches are likely to include whole genome sequencing, genome mining, genetic engineering, chemoenzymatic synthesis, and in vitro enzymatic synthesis in the hope that new therapeutic drugs will come from our seas [87] . 1. if you were required to evaluate an extract for its potential as a drug, what approach would you adopt? 2. silica gel chromatography is essential for the purification of organic compounds. identify three methods of chromatography. 3. design a form which could be used to document information when collecting a specimen. trends in the discovery of new marine natural products from invertebrates over the last two decades à where and what are we bioprospecting? marine natural products: metabolites of marine algae and herbivorous marine molluscs marine natural products: metabolites of marine invertebrates marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products marine natural products drugs and cosmetics from the sea marine natural products and their potential applications as anti-infective agents biogeography of sponge chemical ecology: comparisons of tropical and temperate defenses temperature and spatiotemporal variability of salicylihalamide a in the sponge haliclona sp sources of secondary metabolite variation in dysidea avara (porifera: demospongiae): the importance of having good neighbors chemical mediation of interactions among marine organisms marine advanced technology education-marinetech.org. date accessed a survey of deep-water coral and sponge habitats along the west coast of the us using a remotely operated vehicle. noaa technical memorandum nos nccos 138 isolation of marine natural products an antiproliferative bis-prenylated quinone from the new zealand brown alga perithalia capillaris ptilomycalin d, a polycyclic guanidine alkaloid from the marine sponge monanchora dianchora a new cycloamphilectene metabolite from the vanuatu sponge axinella sp bastadin 20 and bastadin o-sulfate esters from ianthella basta: novel modulators of the ry 1 r fkbp12 receptor complex two new cytotoxic and virucidal trisulfated triterpene glycosides from the antarctic sea cucumber staurocucumis liouvillei drugs from the sea from anti-fouling to biofilm inhibition: new cytotoxic secondary metabolites from two indonesian agelas sponges marine natural products and related compounds in clinical and advanced pre-clinical trials discodermolide, a new bioactive polyhydroxylated lactone from discodermia dissolute arenastatin a, a potent cytotoxic depsipeptide from the okinawan marine sponge dysidea arenaria baculiferins aào, o-sulfated pyrrole alkaloids with anti-hiv-1 activity, from the chinese marine sponge iotrochota baculifera marine pharmocolgy in 2000: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiplatelet, antitubercolosis, and antiviral activities; affecting the cardiovascular, immune, and nervous systems and other miscellaneous mechanisms of action bioactive guanidine alkaloids from two caribbean marine sponges agosterol a, a novel polyhydroxylated sterol acetate reversing multidrug resistance from a marine sponge of spongia sp nitrogen-containing verticillene diterpenoids from the taiwanese soft coral cespitularia taeniata antiplasmodial metabolites isolated from the marine octocoral muricea austera cytotoxic constituents from the formosan soft coral clavularia inflata var. luzoniana a guide to the common edible and medicinal sea plants of the pacific islands novel diterpenes with cytotoxic, anti-malarial and anti-tuberculosis activities from a brown alga dictyota sp an interesting diterpenoid from the brown alga stypopodium zonale antiproliferative activity and absolute configuration of zonaquinone acetate from the jamaican alga stypopodium zonale atomarianones a and b: two cytotoxic meroditerpenes from the brown alga taonia atomaria in vitro antitumor activity of gracilaria corticata (a red alga) against jurkat and molt-4 human cancer cell lines anticancer activity of sargassum oligocystum water extract against human cancer cell lines methanolic extracts of plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in ht-29 human colon carcinoma cells biological importance of marine algae a mixture of phytosterols from dunaliella tertiolecta affects proliferation of peripheral blood mononuclear cells and cytokine production in sheep anti-asthmatic effect of marine red alga (laurencia undulata) polyphenolic extracts in a murine model of asthma inhibitors of oxidation and matrix metalloproteinases, floridoside, and d-isofloridoside from marine red alga laurencia undulata biological activities of sulfated polysaccharides from tropical seaweeds free phenolic acids from the seaweed halimeda monile with antioxidant effect protecting against liver injury antimalarial bromophycolides jàq from the fijian red alga callophycus serratus halitunal, an unusual diterpene aldehyde from the marine alga halimeda tuna isolation of halimedatrial: chemical defense adaptation in the calcareous reef-building alga halimeda screening for new metabolites from marine microorganisms δ-indomycinone: a new member of pluramycin class of antibiotics isolated from marine streptomyces sp rare phenazine l-quinovose esters from a marine actinomycete a novel antimicrobial substance from a strain of the bacterium vibrio sp marine natural products. 34. trisindoline, a new antibiotic indole trimer, produced by a bacterium of vibrio sp. separated from the marine sponge hyrtios altum thiocoraline, a new dipsipeptide with antitumor activity produced by a marine micromonospora. 1. taxonomy, fermentation, isolation, and biological activities methods for isolation of marine-derived endophytic fungi and their bioactive secondary products corollospora pulchella, a marine fungus producing antibiotics, melinachidins iii, iv and gancidin w new cytotoxic sequiterpenoid nitrobenzoyl esters from a marine isolate of the fungus aspergillus halovirs a-e, new antiviral agents from a marine-derived fungus of the genus scytalidium lynamicins a-e, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete tartrolon d, a cytotoxic macrodiolide from the marine-derived actinomycete streptomyces sp. mdg-04-17-069 continuing studies on the cyanobacterium lyngbya sp.: isolation and structure determination of 15-norlyngbyapeptin a and lyngbyabellin d structure of curacin a, a novel antimitotic, antiproliferative, and brine shrimp toxic natural products from the marine cyanobacterium lyngbya majuscula structure and biosynthesis of toxins from blue-green algae (cyanobacteria) the odyssey of marine pharmaceuticals: a current pipeline perspective the bryostatins identification of the putative bryostatin polyketide synthase gene cluster from "candidatus endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan bugula neritina development of cytarabine prodrugs and delivery systems for leukemia treatment industrial natural product chemistry for drug discovery and development conus peptides: biodiversity-based discovery and exogenomics marketed marine natural products in the pharmaceutical and cosmeceutical industries: tips for success marine natural products: a way to new drugs towards a more step-economical and scalable synthesis of spongistatin 1 to facilitate cancer drug development efforts antineoplastic agents. 257 isolation and structure of spongistatin 1 aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process development and economics mariculture trials with mediterranean sponge species. the exploitation of an old natural resource with sustainable and novel methods marine natural products: a new wave of drugs? key: cord-290539-8ak2tths authors: cagno, valeria; tintori, cristina; civra, andrea; cavalli, roberta; tiberi, marika; botta, lorenzo; brai, annalaura; poli, giulio; tapparel, caroline; lembo, david; botta, maurizio title: novel broad spectrum virucidal molecules against enveloped viruses date: 2018-12-07 journal: plos one doi: 10.1371/journal.pone.0208333 sha: doc_id: 290539 cord_uid: 8ak2tths viral infections are an important cause of death worldwide. unfortunately, there is still a lack of antiviral drugs or vaccines for a large number of viruses, and this represents a remarkable challenge particularly for emerging and re-emerging viruses. for this reason, the identification of broad spectrum antiviral compounds provides a valuable opportunity for developing efficient antiviral therapies. here we report on a class of rhodanine and thiobarbituric derivatives displaying a broad spectrum antiviral activity against seven different enveloped viruses including an hsv-2 acyclovir resistant strain with favorable selectivity indexes. due to their selective action on enveloped viruses and to their lipid oxidation ability, we hypothesize a mechanism on the viral envelope that affects the fluidity of the lipid bilayer, thus compromising the efficiency of virus-cell fusion and preventing viral entry. viral infections are one of the ten leading causes of death worldwide [1] . nowadays, although effective antiviral strategies have been successfully developed for some important pathogens such as hiv and hcv, antiviral drugs or vaccines are still missing for the majority of viruses. as an example, no effective antiviral strategies are yet available for viruses causing chronic infections such as hbv [2] , as well as for tropical viruses like dengue virus that is causing 390 million infections per year [3] . a particular interest is addressed to the recent epidemics of ebola in africa and zika (zikv) in south america, for which, despite the huge effort to find antivirals, the research community was not able to execute in time an efficient antiviral plan. the majority of emerging or re-emerging viruses are zoonoses, and it has been demonstrated that the passage among different species is easier for enveloped viruses in comparison a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 with non-enveloped viruses [4, 5] . it is estimated that there are approximately 500,000 unknown mammalian viruses in animal reservoirs [6] . in this scenario, the possibility to find antiviral molecules directed against viral envelopes is of particular interest in order to identify broad spectrum antiviral drugs [7] . in our previous papers we reported some rhodanine and aminothiazolone derivatives endowed with nanomolar activities against hiv-1 infected cells [8] [9] [10] . in a recent work, we showed that our compounds were also active against hsv-1/2, while they were completely ineffective against hpv, a nonenveloped virus [11] . these results suggested that the mode of action of our molecules could involve the viral envelope. compounds acting on viral envelopes have been previously described: rafi compounds inhibit a broad range of enveloped viruses by inserting into viral envelopes and altering the fusion kinetics in the hemifusion stalk due to their inverted cone structure [12] . liposomes extracting cholesterol from viral envelopes have been reported to inhibit hiv, hcv and hbv [13] . virolytic antiviral peptides derived from mastoparan were shown to inhibit different enveloped viruses acting on the envelope and causing its detachment from the viral core [14] . the selectivity of these strategies is based on the fact that viral envelopes are static and characterized by an absence of repair mechanisms, in contrast with the biogenic membranes of the cells that are endowed with plenty of tools to repair membrane damage or alteration [15] . furthermore, since envelope lipids are derived from host cells, it is more complicated to the virus to develop resistance to these compounds if compared to those targeting the classical viral components; for this reason they are extremely promising targets also for viruses with a high mutation rate. therefore, we investigated the activity on hsv-2 of a new series of compounds. after having selected the most potent derivative of the series, we verified its broad spectrum activity against an hsv-2 acyclovir-resistant strain and other six important enveloped pathogens such as zikv, influenza a virus (iav) and respiratory syncytial virus (rsv), amongst the others, and we confirmed its complete inactivity against three non-enveloped viruses. finally, we investigated its mechanism of action, identifying a lipid oxidizing activity and the impairment of viral entry of hsv-2. the compounds were synthesized using the one pot two step approach previously generated by us for the synthesis of rhodanine derivatives. according to fig 1, aldehydes 4-6 were obtained through suzuki reaction between commercially available iodides 1 or 2 and 5-formyl-2-furanylboronic acid 3. basic hydrolysis of ester 5 provided the corresponding acid 6. nucleophilic substitution between trithiocarbonate and the opportune primary amine led to the rhodanine intermediates 8a-e, which were converted into the final compounds 9a-e by knoevenagel condensation with aldehyde 4 or 6. thiobarbituric derivatives were synthesized as shown in fig 2. reaction between benzoyl chloride 10, ammonium thiocyanate and the opportune amine led to intermediates 11a and 11b; consequent hydrazinolisis and coupling reaction with diethylmalonate 13 in basic conditions furnished monoalkylated thiobarbituric derivatives 14a and 14b. finally knoevenagel condensation with aldehyde 6 in refluxing acidic etoh led to compounds 15a and 15b. having proved that rhodanine derivatives belonging to this series of compounds are potential microbicide active against hiv-1 and hsv-1/2 [11] , we further investigated the inhibitory activity against hsv-2 of the novel derivatives bearing both rhodanine and thiobarbituric scaffolds. the compounds were tested with a plaquing efficiency assay, preincubating with the virus for 1 h at 37˚c and subsequently adding the mixtures on cells. all the compounds exhibited potent inhibitory activities at non-cytotoxic doses, as demonstrated by the high selectivity indexes (fig 3) . compound 9d, which showed the most promising antiviral activity, was subjected to further investigations. the toxicity of compound 9d was evaluated at different time points on vero cells, and also after 96h exposure the cc 50 was evaluated to be 2.09 μm confirming the favorable selectivity index (s1 fig) . due to the lipophilic nature of the compound and the results obtained in previous works, in which derivatives of the same series proved to be effective against hiv and hsv [11] , we investigated the spectrum of antiviral activity of 9d against different enveloped viruses (i.e. hsv-1, hcmv, rsv, zikv, iav, vsv) and non-enveloped viruses (i.e. ad5, hpv and hrov). as reported in table 1 , the compound showed a potent antiviral activity against all enveloped viruses tested at not cytotoxic doses, while it was found to be inactive against nonenveloped viruses, suggesting a possible action on the viral envelope. interestingly, compound 9d retained its antiviral activity also against hsv-2 acyclovir resistant strain, suggesting a different mechanism of action. we performed assays aimed at better understanding the mechanism of action. in the pretreatment assay compound 9d was added on cells before the virus in order to determine if the antiviral activity was due to an interaction with the cells. moreover, an assay was conducted adding virus and compound on cells without the preincubation performed in previous assays. the results showed in fig 4 demonstrate that the antiviral activity of 9d is not due to an interaction with cells, since the compound was not active in pre-treatment assays. on the contrary, we observed a significant reduction of activity in the during-infection assay (ec 50 = 0.147 μm), if compared to the pre-incubation assay (ec 50 = 6.55 nm) suggesting that the loss of activity could be related to a competition between the viral envelope and the cell membrane for the insertion and activity of the compound. we then investigated whether compound 9d was able to inhibit multiple cycles of infection, in viral yield reduction assays, when added post infection. as shown in fig 5, in this condition the compound retained a good antiviral activity, suggesting possible therapeutic uses. to further elucidate the mechanism of action we performed a virucidal assay in which 9d was incubated with the virus at 10 μm 5μm or 1μm concentration for different times (fig 6a) or for 1h with serial dilutions of compound ( fig 6b) ; subsequently, the mixture was titrated on cells and the viral titer was evaluated at dilutions at which the compound concentration was known not to be active in plaquing efficiency assays. in these conditions it was possible to observe that the viral infectivity in presence of the compound was decreased at 15' post treatment and completely abrogated from 30' on when the virus was exposed to 10 μm of compound, and at lower doses ( fig 6a) . while after 1h of exposure, the ec50 was 37.3 nm ( fig 6b) . the irreversibility of the mechanism was also tested with an assay in which the compound was incubated with the virus for 1h and subsequently the mixture has been diluted in drug free medium for additional 1, 2, 3 or 4 hours before the addition on cells (s2 fig) . also in this condition the infectivity was not regained further verifying the permanent virucidal activity. since the observed irreversible effect can be exerted at different stages of viral infection, we investigated more in detail if compound 9d produced a physical disruption of the virus, thus preventing its attachment to the cells or a successive irreversible modification. we performed entry and binding assays, evaluating the amount of bound virus through immunostaining or qpcr. the results shown in fig 7a, 7b and 7c, demonstrate that the compound is not altering viral binding capability, differently from heparin, a known attachment inhibitor [16] . on the contrary, when entry assays were performed, it was possible to observe a reduction in the amount of virus associated to the cells ( fig 7d) . with this assay, the signal from the virus could be related to virus in the cytoplasm or blocked in the endosomes, since the treatment with acidic glycine is affecting only the virus at the surface of the cell. for this reason we performed also an in immunofluorescence (if) in which it was possible to visualize that there is virus signal in discrete dots, possibly endosomes, while in the control the viral signal is diffused in the cytoplasm (fig 7e) , supporting the hypothesis that part of the virus could be blocked in endosomes due to an impaired fusion. due to the chemical structure of compound 9d and its affinity for lipids, we envisioned that its mechanism of action might involve the peroxidation of viral phospholipids, which would lead to altered fusion properties of enveloped viruses. to assess this hypothesis, we carried out an in vitro lipoperoxidation assay based on the oxidation of linoleic acid as model lipid substrate, incorporated in a dppc liposomes. the lipid peroxidation was evaluated by monitoring the production of the lipid degradation end-product malondialdehyde (mda). after the exposure to compound 9d, the linoleic acid underwent a remarkable lipid peroxidation in the liposome system (fig 8) . the results of the tba assay suggest the peroxidant activity of the compound. the peroxidation capability of 9d was further confirmed by the reduction of linoleic acid peroxidation extent in a control sample in which in the preparation of liposomes, an antioxidant such as (±)-α-tocopherol, was added. interestingly the addition of 0.05% w/w (±)-α in this work we demonstrated that the series of compounds herein reported, and in particular compound 9d, are endowed with a broad-spectrum activity against a panel of seven different enveloped viruses including a hsv-2 acyclovir resistant strain, due to an irreversible mechanism that impairs viral entry into the host cell. the selective activity on enveloped viruses and the lipid peroxidation capability of the compound suggest a mechanism of action on the viral envelope that is affecting viral and cell membrane fusion. these results open interesting possible applications for these molecules as antivirals or as components of microbicidal preparations for topical use. this is of particular interest if we consider that all the recent epidemics of emerging and re-emerging viruses are caused by enveloped viruses such as ebola virus, lassa virus, zika virus, sars and mers coronavirus and avian flu [3] , without considering the threatening infections of hendra and nipah viruses and the emergence of novel mutant strains. finally, the activity of the reported compounds against hiv [6] , hsv-1, hsv-2 and zikv, which can be all transmitted through sexual activity, suggest a possible use of this class of compounds as vaginal microbicide components. general information. all commercially available chemicals were used as purchased. anhydrous reactions were run under a positive pressure of dry n 2 . thin-layer chromatography (tlc) was carried out using merck tlc plates: silica gel 60 f254. chromatographic purifications were performed on columns packed with merck 60 silica gel, 23-400 mesh, for the flash technique. 1 h and 13 c nmr spectra were recorded at 400 mhz on a bruker avance dpx400 spectrometer. melting points were measured using a gallenkamp melting point apparatus and are uncorrected. microwave irradiation experiments were conducted using a cem discover synthesis unit (cem corp., matthews, nc, usa). the instrument consists of a continuous focused microwave power delivery system with operator-selectable power output from 0 to 300 w. the temperature of the contents of the vessel was monitored with a calibrated ir temperature control mounted under the reaction vessel. all experiments were performed using a stirring option, whereby the contents of the vessel were stirred by a rotating magnetic plate located below the floor of the microwave cavity and a teflon-coated magnetic stir bar in the vessel. general procedure for the synthesis of aldehydes 4 and 5. methyl-4-iodosalycilate 1 or 4-chloro-3-iodobenzotrifluoride 2 (1.00 mmol) and 5-formyl-2-furan boronic acid 3 were dissolved in 10 ml of dmf and 15 ml of etoh. the reaction mixture was stirred for 10 min under n 2 , then pd(pph 3 ) 2 cl 2 (0.10 mmol) was added and finally na 2 co 3 2m (6.00 mmol). the reaction mixture (light-orange) was stirred under n 2 at room temperature. after 1h the reaction went to completion and was quenched with h 2 o and 2n hcl; then etoac was added and the mixture was stirred until the two layers became clear. the aqueous layer was extracted three times with etoac, then the organic phase was washed several times with h 2 o and brine, dried over na 2 so 4 , filtered and evaporated under reduced pressure. 4-(5-formylfuran-2-yl)-2-hydroxybenzoic acid (6). compound 5 was dissolved in 25 ml of ch 3 oh, then a solution of naoh 1m (5.00 mmol) was added dropwise, after the reaction mixture was heated at reflux. the reaction mixture was stirred overnight until completion (tlc). organic solvent was removed under reduced pressure, then some water was added and the aqueous layer was extracted three times with et 2 o; the aqueous layer was then acidified to ph 1 with hcl 6n and a precipitated appeared. (6) 13 ( 13 ( general procedure for the synthesis of compounds 11a and 11b. benzoylchloride 10 (4.31 mmol, 1.00 eq) was dissolved in 3 ml of acetone. to this, nh 4 scn (5,17 mmol, 1.2 eq) was added in one portion and the mixture irradiated at 60˚c for 15 min at the microwave. after this time, the opportune amine (1.00 eq) respectively benzylamine for 8a, and phenethylamine for 8b, was added, and the mixture was irradiated for further 15 min at the microwave. the resulting suspension was filtered, and the precipitate washed with h 2 o and ch 3 oh, to furnish pure 8a and 8b as white solids. general procedure for the synthesis of compounds 12a and 12b. the opportune thioureido derivative (11a or 11b) (1.61 mmol) was dissolved in 10 ml of hydrazine monohydrate. the solution was stirred at rt for 3 h. then 5 ml of fuming hydrochloric acid were added and the mixture was extracted with ch 2 cl 2 (3 x 15 ml). the organic phase was washed with na 2 co 3 (aq. sol.) (2 x 50 ml), and dried over na 2 so 4 . the solvent was removed at reduced pressure and the corresponding residue purified by flash chromatography (acoet/ch 2 cl 2 1:1). general procedure for the synthesis of compounds 14a and 14b. sodium metal (3,61 mmol, 3 eq) was dissolved in anhydrous etoh (10 ml), then the opportune thiourea compound (12a or 12b) (1.20 mmol) and diethylmalonate 13 (2.41 mmol) were added subsequently, and the mixture was refluxed under argon atmosphere. the solvent was removed at reduced pressure and the corresponding residue was solubilized in water. the mixture was acidified at ph 2 with hcl 1n and filtered. the residue was then purified by flash chromatography (ch 2 cl 2 /ch 3 oh 8:2). 1-benzyl-2-thioxodihydropyrimidine-4,6(1h,5h)-dione (14a). yield 80% red solid. general procedure for the synthesis of compounds 15a and 15b. compound 6 (0,22 mmol, 1 eq) and compound 13a or 13b (0.22 mmol, 1 eq) were suspended in etoh (6 ml); to this, 3 drops of hcl (conc.) were added and the mixture was stirred at 70˚c overnight. after this time 3 ml of hcl were added and the resulting precipitate was filtered at reduced pressure to give a solid that was washed with h 2 o, ch 3 oh and hexane. (z)-4(5-((1-benzyl-4,6179.42, 171.68, 162.05, 161.72, 160.30, 159.71, 158.73, 151.34, 138.28, 136.86, 134.45, 131.61, 130.83, 128.57, 127.31, 116.43, 114.46, 113.68, 113.38, 113. clinical isolates of hsv-1 and hsv-2 were kindly provided by prof. m. pistello, university of pisa, italy. hsv-1 and hsv-2 strains were propagated and titrated by plaque assay on vero cells. a hsv-2 strain with phenotypic resistance to acyclovir was generated by serial passage in the presence of increasing concentrations of acyclovir and tested for acyclovir resistance with dose-response inhibition assay with an ec50 of 319 μm, as previously described [17] . hcmv strain towne was kindly provided by prof. w. brune, heinrich pette institut, hamburg, germany; it was propagated and titrated by plaque assay on helf cells. rsv strain a2 (atcc vr-1540) was propagated in hep-2 and titrated by the indirect immunoperoxidase staining procedure using an rsv monoclonal antibody (ab35958; abcam, cambridge, united kingdom), as described previously [15] . human rotavirus strain wa (atcc vr-2018) was activated with 5 mg/ml porcine pancreatic trypsin type ix (sigma, st. louis, mo.) for 30 min at 37˚c and propagated in ma104 cells using mem containing 0.5 mg trypsin per ml, as described previously [18] . vsv (atcc vr-1238) was propagated on vero cells and titrated by plaque assay. zika virus (prvabc59) was kindly provided by marco alves and was propagated and titrated by plaque assay on vero cells. iav h1n1 isolated from a clinical specimen was propagated and titrated on mdck. adenovirus 5 encoding gfp (gfp-ad5), with a e1/e3 deletion, was purchased from vector biolabs (philadelphia, pa, usa). hpv-16 pseudovirions were produced with 293tt as previously described [19] and their concentration was assessed on hela cells. virus stocks were maintained at -80˚c. cell viability was measured using the mts [3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxy methoxy phenyl)-2-(4-sulfophenyl)-2h-tetrazolium] assay. cell cultures were seeded in 96-well plates and were incubated with different concentrations of compounds in duplicate under the same experimental conditions described for the antiviral assays, (i.e. if the incubation time of the compound on cells was carried for 2 hours and then the evaluation of the antiviral activity was done 72h later, the same timing was used for the cytotoxicity assays, in order to exclude any toxicity effect in the antiviral evaluation). cell viability was determined using the celltiter 96 proliferation assay kit (promega, madison, wi, usa) according to the manufacturer's instructions. absorbances were measured using a microplate reader (model 680, biorad) at 490 nm. the effect on cell viability at different concentrations of the compound was expressed as a percentage, by comparing absorbances of treated cells with those of cells incubated with culture medium and equal volumes of vehicle. the 50% cytotoxic concentrations (cc 50 ) and 95% confidence intervals (cis) were determined using prism software (graph-pad software, san diego, ca). for compound 9d a continuous incubation of 1, 2, 3 and 4 days on vero cells was carried in order to evaluate the toxicity after long exposure the results are shown in s1 fig and fig 3. the antiviral effect on hsv and vsv and zikv infection was evaluated by plaquing efficiency assay. vero cells were pre-plated 24 h in advance in 24-well plates at a density of 10 5 cells. increasing concentrations of compounds were mixed with hsv-2 (moi 0.001 pfu/cell) or hsv-2 acyclovir resistant (moi 0.001) or hsv-1 (moi 0.0005) or vsv (moi 0.005) or zikv (moi 0.005) and incubated for 1 hour at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 2 h. the virus inoculum was then removed and the cells washed and overlaid with a medium containing 1.2% methylcellulose (sigma). after further incubation at 37˚c for 24 h (hsv-2 and vsv) or 48 h (hsv-1) or 72h (zikv), cells were fixed and stained with 0.1% crystal violet in 20% ethanol and viral plaques counted. the effective concentration producing 50% reduction in plaque formation (ec 50 ) was determined using prism software by comparing drug-treated with wells treated with medium and solvent. the selectivity index (si) was calculated by dividing the cc 50 by the ec 50 value. hcmv inhibition assays. helf cells were pre-plated in a 96-well plate. the following day increasing concentrations of compounds were mixed with hcmv (moi 0.005) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 3 h; monolayers were then washed and overlaid with 1.2% methylcellulose medium supplemented with 3% fcs and 1mm sodium pyruvate. after five days incubation, cells were observed under an inverted zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and the percentages of infection were calculated by comparing gfp positive cells in treated and untreated wells. hep-2 cells were pre-plated in a 96-well plate. the following day increasing concentrations of compounds were mixed with rsv (moi 0.005) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 3 h; monolayers were then washed and overlaid with 1.2% methylcellulose medium. 72 h later cells were fixed and subjected to specific immunostaining using an rsv monoclonal antibody (ab35958; abcam, cambridge, united kingdom) in order to visualize syncytia. percentages of infection were calculated by comparing numbers of syncytia in treated and untreated wells. hrov inhibition assays. ma104 cell were plated in 96-well trays. the following day virus infectivity was activated by adding 5 μg porcine trypsin (sigma)/ml for 30 min at 37˚c. increasing concentrations of compounds were mixed with hrov (moi 0.02) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 1 h; monolayers were then washed and overlaid with medium. after 16 h, cells were fixed with cold acetone-methanol and viral titers were determined by indirect immunostaining using the monoclonal antibody mab-0036 (specific for human 41 kda inner capsid protein-vp6 -of rotavirus) purchased from covalab (villeurbanne, france) and the ultratech hrp streptavidin-biotin detection system (beckman colter). hpv-16 and ad5 inhibition assays. hela cells were plated in 96-well plates. the following day, increasing concentrations of compounds were mixed with hpv-16 (approximately 1 ng/ml l1) or ad5 (moi 0.02) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 72 h. the gfp-expressing infected cells were observed under an inverted zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and the percentages of infection were calculated by comparing gfp positive cells in treated and untreated wells. iav h1n1 inhibition assays. mdck cells were pre-plated in 96-well plates. the following day, increasing concentrations of compounds were mixed with iav (moi 0.05) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells for 1 h at 37˚c, after a washout cells were overlaid with medium for 16 h at 37˚c. cells were then fixed and subjected to specific immunostaining using a flu a monoclonal antibody (merck 5001) in order to visualize infected cells. percentages of infection were calculated by comparing numbers of infected cells in treated and untreated wells. vero cells were subjected to different assays: pretreatment: cells were pretreated for 2 h at 37˚c with increasing concentrations of compounds, the inocula were then removed, cells washed and hsv-2 was added on cells for 2 h at 37˚c. subsequently the same protocol described above was followed. during infection: cells were subjected to the same experiment described above without the pre-incubation between compounds and virus. post treatment: cells were infected with hsv-2 (moi 0.01) for 2 h at 37˚c, the viral inoculum was removed and cultures were exposed to different compound concentrations and incubated until control cultures displayed extensive cytopathology. supernatants and cells were harvested and cell-free virus infectivity titers were determined in duplicate by plaque assay in vero cell monolayers. percent inhibition was determined by comparing the titer measured in the presence of the compounds to that measured in untreated wells. binding: cells were plated in 96 well plates. the following day 10 μm of compound and hsv-2 (moi 10) were incubated for 1 h at 37˚c and subsequently added on cells for 2 h at 4˚c. cells were then fixed with 4% paraformaldehyde, air dried, and blocked with 5% bovine serum albumin (bsa) in phosphate-buffered saline (pbs)-tween. bound virus was detected using the polyclonal hsv-2 antibody (dako, denmark) (diluted 1:250) incubated for 1 h at room temperature, washed three times with pbs-tween, and incubated for 1 h at 37˚c with anti-rabbit conjugated to horseradish peroxidase (1:500). at the end of incubation, plates were washed three times with pbs-tween, abts [2,2-azinobis(3-ethyl benz thiazoline sulfonic acid)] substrate was added for 30 min (thermo scientific, rockford, il) and the absorbance at 405 nm was read. entry: cells were plated in 96 well plates. the following day 10 μm of compound and hsv-2 (moi 10) were incubated for 1 h at 37˚c and subsequently added on cells for 1 h at 4˚c. cells were then washed and shifted at 37˚c for 1.5 h or 4 h, after they were subjected to a wash with acidic glycine to inactivate unentered virus. cells were then fixed with 4% paraformaldehyde, air dried and permeabilized with pbs-triton (0.5%) and then the same protocol described for binding was followed. virucidal assay: approximately 10 5 pfu of hsv2 plus 10 μm or different concentration of compound were added to mem and mixed in a total volume of 100 μl. the virus-compound mixtures were incubated for different times at 37˚c then diluted serially to the non-inhibitory concentration of test compound on cells or in tubes for additional 1, 2, 3 or 4 hours; the residual viral infectivity was determined by viral plaque assay. cells were plated on coverslips and the following day the binding or entry protocol described above was applied. cells were than fixed with 4% paraformaldehyde, air dried and for entry protocol permeabilized with pbs-triton. blocking was performed with pbs-bsa 1% for 1 h at room temperature and subsequently hsv-2 polyclonal antibody (1:250) was added on cells, after 1 h at 37˚c the inoculum was removed and coverslips were washed with pbs-tween for 3 times. rhodamine conjugated anti-rabbit (santa cruz) was then added on cells for 1 h at 37˚c following 3 washes coverslips were mounted on microscope glasses and observed with zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and images were acquired. qpcr cells subjected to binding assays were then lysed and subjected to total dna/rna extraction and subsequently subjected to qpcr using as primers 5'-ccgtcagcaccttcatcga -3' and 5'-cgctggacctccgtgtagtc -3' and as probe 5'-fam ccacgagatcaagga cagcggcc-tamra for 40 cycles at 94˚c for 15" followed by 60˚c for 60". the results were normalized according to values of rnasep and the % of bound virus were measured comparing the δct of treated wells to wells treated with equal volume of solvent. to evaluate the oxidation capability of 9d an in vitro lipoperoxidation test was carried out. in lipid peroxidation, free radicals attack double bonds of polyunsaturated fatty acids forming lipid hydroperoxides, which undergo homolytic scission to form a variety of cytotoxic compounds, such as aldehydes. this oxidative stress plays an important role in damaging membrane lipids. according to previous literature, the potential oxidative effect of 9d was evaluated toward the oxidation of linoleic acid, a carboxylic acid with two double bonds as a model lipid substrate. the lipoperoxidation of linoleic acid, incorporated in dipalmitoylphosphatidylcholine (dppc) liposomes was determined using the tba assay [20, 21] . dppc liposomes were prepared with the thin film evaporation method and used to mimic a phospholipid bilayer, and to dissolve linoleic acid. tba assay, commonly used as an index of lipid peroxidation, is based on the reactivity of malondialdehyde (mda), a colourless end-product of degradation, with 2-thiobarbituric acid (tba) to produce a pink adduct (tba-mda-tba) that absorbs at 535 nm. mda is indeed one of the final products of polyunsaturated fatty acids peroxidation in the cells and is generally considered an indicator of lipid peroxidation. mda was detected spectrophotometrically according to the method described by bay et al. with the following modifications [22] . for the tba assay,9d compound was dissolved in dmso at the concentration of 10 μm. then, 100 μl of the 9d dmso solution were incubated at 37˚c for 30 minutes with a 0.5% linoleic acid containing liposome aqueous dispersion. blank samples were prepared incubating the liposome aqueous dispersion either with saline solution (0.9% nacl aqueous solution) or dmso in the absence of 9d. after incubation, a volume of the blank and 9d samples (0.2 ml) were withdrawn and introduced in a glass tube closed with a screw cap and added with 0.1 ml of water, 0.2 ml of 4% w/w sds, 1.5 ml of 1.0% w/w phosphoric acid and 1.0 ml of 0.6% w/w tba. the mixture was stirred and heated in water bath at 95-100˚c for 45 min to favor the formation of the complex. after cooling in an ice bath, 4.0 ml of 1-butanol were added to each tube and the tba-mda-tba complex was extracted upon stirring and centrifugation. the organic supernatant was evaluated by spectrophotometry. the calibration curve of tba-mda-tba complex was obtained using a mda precursor 1,1,3,3-tetraethoxypropane. mda can be obtained by acid hydrolysis from 1,1,3,3-tetraethoxypropane in an equimolecular reaction. for this purpose, standard solutions of 1,1,3,3-tetraethoxypropane in sds (4% w/w) within the concentration range 5-250 μm were prepared. the solutions were subjected to tba assay and analyzed at spectrophotometer. the final concentration of mda derived from the reaction of linoleic acid, calculated exploiting the calibration curve, was expressed as micromoles of mda per mg of lipid substrate. to further confirm the oxidant activity of 9d the peroxidation experiment was also carried out using a control sample containing 0.05% w/w of (±)-α-tocopherol, molecule with antioxidant properties, added during the preparation of liposomes. mda formation was monitored in the same conditions above reported. the results are mean and sd of 3 independent experiments. results are presented as the mean values from 2 to 6 independent experiments, in case of 2 independent experiments results are expressed ± square root of the sum of squares, in other cases as ± sd. the ec 50 values for inhibition curves were calculated by regression analysis using the software graphpad prism (graphpad software, san diego, california, u.s.a.) by fitting a variable slope-sigmoidal dose-response curve. for binding and entry assays the significance was analyzed with a one-way anova followed by a bonferroni. results are mean and sd. n = 3. (docx) chronic hepatitis b infection: a review dengue and severe dengue new pharmacological strategies to fight enveloped viruses reservoirs and vectors of emerging viruses a strategy to estimate unknown viral diversity in mammals human ddx3 protein is a valuable target to develop broad spectrum antiviral agents a versatile and practical synthesis toward the development of novel hiv-1 integrase inhibitors 2-aminothiazolones as anti-hiv agents that act as gp120-cd4 inhibitors antimicrob one drug for two targets: biological evaluation of antiretroviral agents endowed with antiproliferative activity rhodanine derivatives as potent anti-hiv and anti-hsv microbicide antivirals acting on viral envelopes via biophysical mechanisms of action polyunsaturated liposomes are antiviral against hepatitis b and c viruses and hiv by decreasing cholesterol levels in infected cells membrane repair: mechanisms and highly sulfated k5 escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures in vitro anti-herpes simplex virus activity of crude extract of the roots of nauclea latifolia smith (rubiaceae) identification of equine lactadherin-derived peptides that inhibit rotavirus infection via integrin receptor competition the agmatine-containing poly (amidoamine) polymer agma1 binds cell surface heparan sulfates and prevents attachment of mucosal human papillomaviruses influence of hydroxypropyl-b-cyclodextrin on the photostability and antiradical activity of trolox lipid peroxidative stress and antioxidative enzymes in brains of milk-supplemented rats conceptualization: valeria cagno, maurizio botta. key: cord-017041-0zxoq68m authors: volochnyuk, dmitriy m.; grygorenko, oleksandr o.; gorlova, alina o. title: fluorine-containing diazines in medicinal chemistry and agrochemistry date: 2014-06-13 journal: fluorine in heterocyclic chemistry volume 2 doi: 10.1007/978-3-319-04435-4_7 sha: doc_id: 17041 cord_uid: 0zxoq68m the combination of a fluorine atom and a diazine ring, which both possess unique structural and chemical features, can generate new relevant building blocks for the discovery of efficient fluorinated biologically active agents. herein we give a comprehensive review on the biological activity and synthesis of fluorine containing, pyrimidine, pyrazine and pyridazine derivatives with relevance to medicinal and agrochemistry. abstract the combination of a fl uorine atom and a diazine ring, which both possess unique structural and chemical features, can generate new relevant building blocks for the discovery of effi cient fl uorinated biologically active agents. herein we give a comprehensive review on the biological activity and synthesis of fl uorine containing, pyrimidine, pyrazine and pyridazine derivatives with relevance to medicinal and agrochemistry. although being present in very small amounts, they are highly odiferous and can be detected at extremely low concentrations. unlike other heterocycles found in many important natural products, pyridazines were discovered only after 1970, and relatively few pyridazines have thus far been isolated from natural sources. as synthetic compounds, all diazines constitute an important pharmacophoric moiety present in many drugs acting on various pharmacological targets as well as agrochemicals. inspite of organofl uorine compounds are almost absent as natural products, ~25 % of drugs in the pharmaceutical pipeline and ~15 % of agrochemicals contain at least one fl uorine atom. one of the earliest synthetic fl uorinated drugs is the antineoplastic agent 5-fl uorouracil, derivative of pyrimidine, an antimetabolite fi rst synthesised in 1957. since the advent of 5-fl uorouracil, fl uorine substitution is commonly used in contemporary medicinal and agrochemistry to improve metabolic stability, bioavailability and protein-ligand interactions. in this review only compound bearing fl uoro or fl uoroalkyl substituent in diazine ring are discussed. among fl uorine containing diazines now 12 drugs and 10 agrochemicals are presented on the market. this review provides an information about fl uorinated diazines as drugs or agrochemicals and their mode of action as well as synthesis. the review is divided in two parts. first part dedicated to the medicinal and synthetic chemistry of fl uorinated diazines that have reached at least clinical development phase. the second one dedicated to the biological role and the chemistry of the marketed agrochemicals based on fl uorinated diazines. it is widely accepted that compounds containing fl uorine atoms have a remarkable record in medicinal chemistry and play a continuing role in providing lead compounds for potential therapeutic applications. the reasons for that have been discussed extensively in a number of books and reviews [ 1 , 2 ] . in this view, fl uorine-containing diazines are not the exception; they have attracted attention of medicinal chemists since 1950s when fluorouracil ( 1 ) was introduces as anti-cancer drug. analysis of mddr (mdl drug data report) data retrieved 1,150 hits derived from fl uorine-containing diazines [ 3 ] . nearly a third part of them is represented by anti-cancer agents (fig. 1 ) ; other important classes (more than 100 examples) include compounds with antiviral (mainly anti-hiv) and antiarthritic activity. according to mddr, 106 compounds containing a fl uorinated diazine moiety have entered pre-clinical studies, 40 of them have reached clinical phase, and 12 of these have become drug substances (fig. 2 ) . in the following sections, fl uorinecontaining diazine derivatives that have reached at least clinical development phase will be discussed, focusing on their aspects related to medicinal and synthetic organic chemistry. the use of fl uorinated diazines as anti-cancer agents is the major fi eld of their application in medicinal chemistry. the fi rst representative of this class, fluorouracil ( 1 ) was developed by charles heidelberger and co-workers in 1957 [ 4 ] . it was approved by u.s. fda [ 5 ] in 1962 as antineoplastic agent in the treatment of advanced colorectal cancer. fluorouracil represents a class of rationally designed anticancer agents which act as antimetabolites. the observation that rat hepatomas utilized radiolabeled uracil more avidly than normal tissues [ 6 ] implied that the enzymatic pathways for utilization of uracil or its close analogs differed between malignant and normal cells -a feature which might provide a target for antimetabolite chemotherapy. a minimal modifi cation of uracil by introducing a single fl uorine atom allowed for implementation of cellular uptake and metabolic activation of 1 via the same transport processes and enzymes involved in the case of uracil. however, in the case of essential biological targets, remarkable differences are observed due to unique properties of the fl uorine atoms, which result in inhibition of the metabolic and signal pathways involved. although all the details of the mechanism by which fluorouracil gives its biological effect are not elucidated, a remarkable progress has been made over the past half a century in elucidating its cellular and clinical pharmacology [ 7 , 8 ] . the key steps in fluorouracil metabolism are shown in scheme 1 . up to 80 % of 1 administered as injection is transformed to dihydrofl uorouracil (dhfu, 13 ) by dihydropyrimidine dihydrogenase (mostly in liver tissues). however, this metabolite is not involved into antineoplastic activity; instead, 13 itself and its further metabolites are responsible for most of the toxic effects of 1 . the main mechanism of activation of fluorouracil is conversion to fl uorouridine monophosphate (fump, 14 ), either directly by orotate phosphoribosyltransferase, or via fl uorouridine (fur, 15 ) through the sequential action of uridine phosphorylase and uridine kinase. 14 is then phosphorylated to give fl uorouridine diphosphate (fudp, 16 ), which can be either phosphorylated again to the active metabolite fl uorouridine triphosphate (futp, 19 ) , or reduced to fl uorodeoxyuridine diphosphate (fdudp, 18 ) by ribonucleotide reductase. in turn, 18 can either be dephosphorylated or phosphorylated to generate an alternative activation pathway involves the thymidine phosphorylase catalysed conversion of 1 to floxuridine (fudr, 4 ), which is then phosphorylated by thymidine kinase to give 19 . the metabolite of 1 -floxuridine -is itself used as an anti-cancer agent [ 9 ] . it was launched in 1970 by hospira inc [ 5 ] . upon rapid injection, most of floxuridine is catabolized to fluorouracil; hence similar effects on the organism are obtained in this case. on the contrary, when 4 is slowly administered into the arterial blood, it is mostly transformed to 19 ; thus toxic effects are diminished comparing to 1 [ 10 ] . it has long been recognized that one of the main mechanisms underlying fluorouracil action is inhibition of thymidylate synthase by fl uorodeoxyuridine monophosphate ( 19 ) [ 11 ] . thymidylate synthase belongs to a class of enzymes required for dna replication, and its activity is higher in rapidly proliferating cells. in particular, thymidylate synthase is responsible for methylation of deoxyuridine monophosphate (dump, 21 ) to deoxythymidine monophosphate (dtmp, 22 ) with the use of 5,10-methylenetetrahydrofolate ( 23 ) as a cofactor (scheme 2 ) [ 12 ] . with fl uorodeoxyuridine monophosphate, a slowly-reversible ternary complex 24 is formed instead. inhibition of thymidylate synthase leads to deoxyribonucleotide imbalance, and hence to interference with dna synthesis and repair. alternative mechanism of dna-directed fluorouracil effect is misincorporation of fl uorodeoxyuridine triphosphate ( 20 ) into dna. analogously, fl uorouridine triphosphate ( 17 ) is extensively incorporated into different rna species, disrupting their normal processing and function [ 7 , 8 , 11 ] . two principal approaches were used for the preparation of fluorouracil (scheme 3 ). one of the fi rst methods [ 13 , 14 ] commenced from ethyl fl uoroacetate which was subjected to claisen condensation with ethyl formate to give 25 . the salt 25 was introduced into reaction with s -alkylisothiourea to give fl uoropyrimidines 26 , which were hydrolysed to give 1 . several variations of this method were also described; their common drawback was the use of highly toxic fl uoroacetic acid derivatives. in an alternative approach, fluorouracil was prepared by direct fl uorination of different pyrimidine derivatives, including uracil [ 15 ] , cytosine [ 16 ] , and orotic acid [ 17 ] . in the latter method, the initially obtained fl uoroorotic acid 27 was subjected to decarboxylation. the use of two-step reaction sequence was claimed to be advantageous due to simplifi ed product isolation and purifi cation. early synthesis of floxuridine commenced from fluorouracil ( 1 ) which was transformed into its mercury salt 28 and then allowed to react with 2-deoxy-dribofuranosyl chloride derivative 29 (scheme 4 ) [ 18 ] . the product 30 was subjected to alkaline hydrolysis to give floxuridine ( 4 ). as in the case of fluorouracil, newer syntheses of floxuridine relied on direct fl uorination of uracil derivatives. fluorination of uridine 31 was done using fl uorine [ 19 ] , acetyl fl uoride [ 20 ] , and cf 3 of [ 21 ] . the latter reagent gave good but still moderate yield of the product 4 (47 %). the use of a two-step reaction sequence, i.e. fl uorination of diacetoxy derivative 32 and hydrolysis, improved the yield of 4 to 82 % over two steps [ 21 , 22 ] . despite fluorouracil remains the main agent for the treatment of certain cancer types ( i.e. colorectal) [ 23 ] , it displays various side effects due to its nonspecifi c cytotoxicity, poor distribution to tumor sites, and serious limitations in effectiveness due to drug resistance. apart from modulation of fluorouracil biological action through combination therapies [ 7 , 24 ] , a number of drugs and clinical candidates acting as prodrugs of 1 and/or 4 were developed (table 1 ) . the fi rst example of fluorouracil prodrug is tegafur ( 3 ) developed in 1960s in latvia [ 25 , 26 ] . tegafur is an oral slow-release prodrug formulation of fluorouracil which is readily absorbed through the gastrointestinal tract. the major pathway of metabolic activation of 3 includes hydroxylation by hepatic cytochrome p450 enzymes, mostly cyp2a6 (scheme 5 ) [ 27 ] . apart from fluorouracil, 4-hydrohybutyraldehyde and succinic dialdehyde are also formed, which are further transformed into γ-butyrolactone and 4-hydrohybutyric acid [ 28 ] . tegafur was shown to be 2-5 times more potent and less toxic than 1 ; hence lower doses of 3 can be utilized, resulting in decreased neurotoxicity without compromising the antitumor effects. another prodrug of fluorouracil -doxifl uridine ( 5 ), which also implies the idea of attachment of sugar-like moiety to the molecule of 1 , was launched in japan in 1987 [ 29 ] . the mechanism of metabolic activation of 5 is rather simple and includes hydrolysis to fluorouracil by thymidine phosphorylase [ 299 ] . since the level of thymidine phosphorylase is signifi cantly higher in several types of solid tumours (in particular, colorectal, breast, and kidney cancers) as compared with normal tissues, doxifl uridine possesses a higher therapeutic index for these types of cancers. the use of 5 is somewhat limited by gastrointestinal toxicity after oral administration due to release of 1 by intestinal pyrimidine nucleoside phosphorylase [ 30 ] . yet another sugar-modifi ed fluorouracil derivative -ogt 719 ( 33 ), in which galactose is incorporated onto the fl uoropyrimidine moiety, was developed by oxford glycosciences and had reached phase i clinical study [ 31 ] . in 1999, the company decided to discontinue development of 33 as the results of phase i/ii clinical study were not suffi ciently strong to justify large scale phase ii studies. ogt 719 was rationally designed to reduce the systemic toxicity normally associated with fluorouracil while retaining activity against tumors localized in the liver, in which it may be preferentially localized through the asialoglycoprotein receptors [ 32 ] . these receptors are present on the surface of hepatocytes and recognise various sugar-containing biomolecules through terminal galactose and n -acetylgalactosamine residues. the metabolic activation of ogt 719 occurs once the compound enters hepatocytes, where the galactose molecule is cleaved from the fluorouracil residue. two derivatives of floxuridine -tt-62 ( 34 ) and t-506 ( 35 ) have reached phase ii clinical trials in japan [ 3 ] . the compounds showed signifi cant antitumor activity by oral administration; moreover, they slowly released floxuridine, and the effective level of 4 was prolonged [ 33 , 34 ] . the gastro-intestinal disturbances and loss of body weight were serious side effects of 34 and 35 . several prodrugs of flourouracil were obtained by acylation or carbamoylation of n-1 and/or n-3 atoms of the pyrimidine ring of 1 . in particular, an oral drug carmofur ( 2 ) which is 1-hexylcarbamoyl derivative of 1 was launched in japan in 1981 and later -in other countries [ 35 ] . the carbamate moiety in 2 decomposes gradually in neutral water or in basic conditions, but it is strongly resistant to acidic hydrolysis and hence can survive acid in the stomach. the 1-hexylcarbamoyl moiety also facilitates the rapid uptake of 2 through the cell membrane [ 36 ] . the metabolic activation of carmofur involves oxidation and scission of the side-chain with slow release of 1 [ 37 ] . two main routes of the side chain transformation are ω-oxidation and (ω-1)-oxidation: metabolites 40 -43 were detected after administration of carmofur (fig. 3 ) [ 38 ] . non-enzymatic hydrolytic decomposition of 2 and its metabolites also contributes to release of 1 . another oral prodrug of fluorouracil, atofl uding ( 36 ) is a diacyl derivative of 1 . atofl uding has reached phase iii clinical trials in china [ 39 ] . the activation of 36 includes its fast non-enzymatic hydrolysis to 3o -toluyl-5-fluorouracil ( 44 ) following oral administration; 44 is then slowly metabolized to 1 (scheme 6 ) [ 40 ] . since the acetyl group of atofl uding is not stable and prone to decompose, impairing quality control for the preparation, a possibility of direct application of 44 was also considered [ 41 ] . an interesting idea was behind design of emitefur ( 37 ), a prodrug of fluorouracil which was developed by otsuka pharmaceutical and has reached phase iii clinical trials in japan [ 3 , 42 , 43 ] . the structure of 37 contains the fragments of two biologically active components: fluorouracil ( 1 ) and 3-cyano-2,6-dihydroxypyridine ( 45 ), which is a potent inhibitor of dihydropyrimidine dehydrogenase. therefore, 37 is a double prodrug which not only delivers fluorouracil but also prevents its enzymatic biotransformation to the dihydropyrimidine derivative 12 . metabolic activation of 37 occurs via rapid cleavage of the ester bonds by esterase to give 45 and 1-ethoxymethyl-5-fl uorouracil ( 46 ) (scheme 7 ). the intermediate 46 is further metabolized to 1 by microsomal enzymes in the liver [ 44 ] . all the prodrugs of fluorouracil discussed above contained the fragment of 1 in their structure; their transformation to 1 included hydrolysis reaction as the key step. on the contrary, 5-fl uoro-2-pyrimidinone (5-fp, 38 ) which has been studied in phase i clinical trials [ 45 ] is activated through oxidative process. in particular, pyrimidine 38 is transformed to 1 by aldehyde oxidase, which is present in high concentrations in the human livers but not in the gastrointestinal tract [ 46 ] . two prodrugs of 1 , capecitabine ( 6 ) and galocitabine ( 39 ), are 5-fl uorocytidine derivatives. both the compounds were developed by hoffman la roche; whereas capecitabine was launched in 1998, galocitabine was terminated at phase ii clinical trials [ 47 ] . both the compounds are close analogues as well as prodrugs of doxifl uridine ( 5 ), which was used as the lead compound in their design. the main goals of such design were to minimize the mielotoxicity and to increase the tumor selectivity of 5 . in fact, capecitabine ( 6 ) indeed demonstrated minimal mielotoxicity in clinical studies. although the therapeutic indices of 39 were much higher in mice tumor models than in the case of 5 , it was not effi ciently metabolised to the active species in humans. the metabolic activation of 6 and 39 includes their hydrolysis by carboxylesterase or acylamidase in liver to give 5′-deoxy-5-fl uorocytidine ( 47 ), which is then transformed to 5 by cytidine deaminase (scheme 8 ) [ 48 ] . syntheses of fluorouracil prodrugs relied on either chemical modifi cation of 1 or direct fl uorination of the corresponding pyrimidine derivatives. in particular, tegafur ( 3 ) was obtained from 1 by reaction with 2,3-dihydrofuran [ 49 -54 ] , 2-chloro[ 55 , 56 ] , 2-alkoxy[ 57 ] , 2-acetoxytetrahydrofuran [ 58 , 59 , 300 ] , and 4-trimethylsilyloxybutyraldehyde dimethyl acetal ( 48 ) (scheme 9 ) [ 60 ] . alternatively, 3 was prepared via fl uorination of compound 49 [ 61 ] or ester 50 [ 62 ] . one of the early syntheses of doxifl uridine ( 5 ) [ 63 , 64 ] commenced from floxuridine ( 4 ) which reacted with thionyl chloride to give cyclic sulphite 51 (scheme 10 ). methanolysis of 51 upon treatment with sodium methylate gave 52 , which was reduced with tributyltin to give 5 . in an analogous approach, the compound 5 was prepared via iodide 53 , in turn obtained from 4 in two steps (scheme 11 ) [ 65 ] . it should be noted that direct transformation of 4 into the corresponding iodide was done with low yield of the product, hence the protection strategy was necessary to use. bromide 54 was a key intermediate in one more analogous scheme [ 66 ] . several syntheses of doxifl uridine relied on glycosylation of fluorouracil derivative 55 . in particular, 5′-deoxyrybose derivatives 56 , 57 , and 58 were used for that purpose (scheme 11 ) [ 67 , 68 ] . finally, direct fl uorination of 5′-deoxyuridine derivatives with f 2 /n 2 [ 69 ] or acof [ 70 ] was also described. syntheses of ogt 719 ( 33 ) relied on glycosylation of the compound 55 (scheme 12 ). reaction of 55 with bromide 59 [ 71 , 72 ] or acetate 60 [ 73 ] gave tetraacetyl derivative 61 , which was transformed to 33 upon deprotection. with 60 as the glycosylating reagent, in situ generation of 55 from fluorouracil was also described [ 74 ] . synthesis of carmofur ( 2 ) and atofl uding ( 36 ) was performed in obvious and straightforward manner. carmofur ( 2 ) was prepared by reaction of fluorouracil ( 1 ) and n -hexylisocyanate (scheme 15 ) [ 77 , 78 ] . alternative approach included reaction of 1 with phosgene and then -with n -hexylamine. early syntheses of 5-fl uoro-2-pyrimidinone ( 38 ) relied on desulfurization of fluorouracil thio-derivatives. in particular, reaction of pyrimidine derivatives 68 with p 2 s 5 followed by treatment with raney nickel and gave alkoxy derivative 69 , which was transformed to 38 upon acidic hydrolysis (scheme 18 ) [ 83 ] . a more straightforward transformation sequence was also described; including reaction of fluorouracil ( 1 ) with p 2 s 5 and reduction of thione 70 with raney nickel [ 84 , 85 ] . alternatively, the thione 70 was alkylated to give derivative 71 , which was either oxidated and then hydrolyzed [ 86 ] or subjected to reaction with hydrazine and then -silver oxide [ 301 ] ; in both cases, 38 was obtained. a completely different synthetic scheme commenced from fl uoroacetic acid which was subjected to vilsmeiertype formylation to give 2-fl uoro-3-dimethylamino-acrolein ( 72 ) [ 87 ] . reaction of 72 with triethyloxonium tetrafl uoroborate and dimethylamine gave the salt 73 , which led to 38 upon reaction with urea. finally, 38 was also obtained by direct fl uorination of 2-pyrimidinone [ 88 , 89 ] . syntheses of capecitabine ( 6 ) started from 5-fl uorocytosine ( 9 ) (see further sections for the preparation of 9 , which is used as antifungal drug). in particular, compound 70 reacted with 1,2,3-tri-o -acetyl-5-deoxy-β-d -ribofuranose ( 58 ) to give diacetyl derivative 72 , which was acylated with n -pentylchloroformate and then hydrolyzed, resulting in the formation of 6 (scheme 19 ) [ 90 -95 ] . variations of this method using a silyl derivative of 70 instead of 70 itself [ 68 , 96 ] , as well as 1-o -acetyl-2,3-o-isopropylidene-5-deoxy-d -ribofuranose ( 73 ) (scheme 20 ) [ 96 ] or 1,2,3-tri-o -methoxycarbonyl-5-deoxy-d -ribofuranose [ 97 ] as the sugar sources were also reported. syntheses of galocitabine ( 39 ) were performed analogously to that of capecitabine, 3,4,5-trimethoxybenzoyl chloride being used instead of npentylchloroformate at the corresponding steps [ 68 , 89 , 90 , 98 ] . [ 3 ] . the active principle of both tas-102 and ftc-092 with anti-cancer effect is trifl uridine ( 7 ). as in the case of fluorouracil, one of the mechanisms by which compound 7 exhibits its antitumor activity is inhibition of thymidylate synthase [ 100 ] . more precisely, trifl uridine is transformed into α,α,α-trifl uorothymidine monophosphate ( 76 ) by thymidine kinase (scheme 21 ); similarly to the fluorouracil derivatives discussed in the previous sections, compound 76 is true inhibitor of thymidylate synthase. however, compound 7 exhibits an anticancer effect on colorectal cancer cells that have acquired fluorouracil resistance as a result of the overexpression of thymidylate synthase. therefore, an alternative mechanism of action is also in operation, namely, incorporation of α,α,α-trifl uorothymidine triphosphate ( 77 ) into dna, which results in single-strand breaks, followed by double-strand breaks when the cells progress to a subsequent dna replication phase [ 101 ] the major drawback of trifl uridine ( 7 ) is its high susceptibility to biodegradation, which is catalysed by thymidine phosphorylase and gives α,α,α-trifl uorothymine ( 78 ) and 2-deoxy-α-dribose 1-phosphate ( 79 ) [ 102 ] . in the case of tas-102, this issue is overcome by co-administration of thymidine phosphorylase inhibitor tipiracil ( 75 ) [ 103 ] , whereas improved biological effect of ftc-092 upon oral administration is achieved by its gradual biotransformation, mainly through the action of liver microsomes, releasing 7 over a long period [ 104 ] . the fi rst synthesis of trifl uridine commenced from trifl uoromethylacrylonitrile ( 80 ) which reacted with hbr and then with urea to give amide 81 in moderate yield. hydrolysis of 81 was accompanied by cyclization and led to dihydropyrimidine 82 (scheme 22 ). two-step aromatization of 81 gave α,α,α-trifl uorothymine ( 78 ). compound 78 was transformed to 7 in low yield (8 %) by enzymatic glycosylation [ 105 ] . the yield of the last step in this sequence was signifi cantly improved when 78 was preliminarily transformed to bis-silyl derivative 83 , and chloride 84 was used for glycosylation [ 106 , 107 ] an alternative approach to 7 was based on direct trifl uoromethylation of the corresponding deoxyuridine derivatives 32 or 84 , using cf 3 cooh-xef 2 [ 108 ] and cf 3 i-cu-hmpa [ 109 ] as the reagents, respectively (scheme 23 ). ftc-092 ( 74 ) was prepared by regioselective benzylation of trifl uridine ( 7 ) (scheme 24 ) [ 110 ] . as in the case of 7 , direct trifl uoromethylation was also used for synthesis of 74 . the following sequence was established as the most practical: tritylation of 2′-deoxy-5-iodouridine ( 85 ), 3′-o -benzylation, n 3 -benzoylation, crosscoupling reaction with cf 3 cu reagent, and acidic deprotection (scheme 25 ) [ 111 ] . alternatively, 74 was prepared in low yield by glycosylation of α,α,α-trifl uorothymine using the bis-silyl derivative 83 (scheme 26 ) [ 112 ] . an approach to cancer treatment which relies on using fl uorinated uracil analogues as antimetabolites is the most recognised in the fi eld of fl uorinated diazines relevant to medicinal chemistry. however, other strategies are also gaining momentum; in particular, several compounds which act as kinase inhibitors ( i.e. 87 -92 ) have reached clinical development phase (table 2 ) . compound ly-2835219 ( 87 ) is currently being developed by eli lilly and co.; monomesylate salt of 87 has entered phase i clinical trials in patients with advanced cancer in 2011 [ 113 ] . it acts as a potent oral inhibitor of the cyclin-dependent kinases 4 and 6 (cdk4/6), playing a key role in regulating cellular proliferation [ 114 ] . in particular, these cyclin d-dependent kinases facilitate progression of gap 1 cell cycle phase (g 1 ) by phosphorylating retinoblastoma susceptibility protein (rb), which prevents association of rb with e2f transcription factor, and thus relieves transcriptional repression by the rb-e2f complex. in addition, these fluorine-containing diazines in medicinal chemistry and agrochemistry kinases also sequester cdk interacting and kinase inhibitory proteins (cip/kip) from their complexes with cyclin-dependent kinase 2 (cdk2), facilitating activation of cdk2 with cyclin e [ 115 ] monomesylate salt of 87 inhibits cdk4 and cdk6 with ic50 values of 2 and 10 nm, respectively; moreover, it is able to cross blood-brain barrier and therefore has the potential for the treatment of brain tumors and metastases [ 114 ] . fostamatinib disodium (tamatinib fosdium, 88 ), which is prodrug of tamatinib ( 92 ) (scheme 27 ), was discovered by rigel; it is currently studied in phase ii clinical trials by rigel and astra zeneca plc. for treatment of b-cell lymphoma [ 113 ] . apart from that, compound 88 is also investigated as agent for treatment of autoimmune thrombocytopenia and rheumatoid arthritis. because of its poor pharmaceutical properties, tamatinib ( 92 ) is orally administered as the methylene phosphate ( 88 ) is quickly cleaved to 92 by alkaline phosphatases that are present on the apical brush-border membranes of the intestinal enterocytes, after which the more hydrophobic 92 can be readily absorbed [ 116 ] . tamatinib ( 92 ) acts as an atp-competitive inhibitor of spleen tyrosine kinase (syk) -a non-receptor tyrosine kinase which is a key component of the b-cell receptor (bcr) signaling pathway [ 117 ] . it is shown that bcr-mediated signaling through syk occurs to a greater degree and for a longer duration in neoplastic cells than in nonmalignant b-cells. inhibition of the syk pathway prevents chronic lymphocytic leukemia (cll) cells from interacting with the microenvironment, and promotes proapoptotic signals. r-763 ( 89 ), also known as as-703569, is another kinase inhibitor discovered by rigel. it was investigated in phase i clinical trials for several types of tumors by rigel and merck serono; the latest study was terminated in 2012, concerning a review of the available clinical data and low probability of completing the trial based on the observed recruitment rate [ 113 ] . compound 89 inhibits aurora kinases -serine/threonine kinases which are essential for cell proliferation, mainly due to regulation of gap 2 and mitotic cell cycle phases (g 2 /m). over-expression of aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. although the biological functions of two types of aurora kinases (a and b) are different, in both cases their inhibition induces apoptosis of the cell, leading to similar phenotypes. some other kinases are also inhibited by 89 , in particular fms-like tyrosine kinase 3 (flt3) [ 118 ] . one more aurora kinase inhibitor -pf-03814735 ( 90 ) -was developed by pfi zer; it has been investigated in phase i clinical trials for treatment of solid tumors (the study completed in 2012) [ 113 ] . pf-03814735 was generally well tolerated with manageable toxicities, and a recommended phase ii dose could be established; however, clinical or metabolic antitumour activity was limited [ 119 ] . similarly to r-763 ( 89 ), compound 90 inhibits both aurora a and b kinases; other kinases are affected to a lesser extent [ 120 ] . therefore, pf-03814735 ( 90 ) produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. azd-1480 ( 91 ) was developed by astrazeneca and studied in phase i clinical trials for treatment of advanced solid malignancies (the study terminated in 2012) [ 113 ] . azd-1480 is an atp-competitive inhibitor of janus kinase 2 (jak2) -an intracellular non-receptor tyrosine kinase that transduce cytokine-mediated signals via the janus kinase -signal transducer and activator of transcription (jak-stat) signaling pathway. in particular, inhibition of jak2 blocks stat3 signaling, associated with chronic cytokine stimulation in some tumors [ 121 ] . x-ray diffraction study of complex formed by 91 and jak2 shows that the donor-acceptor-donor hydrogen-bonding motif provided by aminopyrazole fragment forms three hydrogen bonds with an adenine binding pocket, whereas the fl uoropyrimidine ring occupies a nearby hydrophobic pocket [ 122 ] . synthesis of ly-2835219 ( 87 ) relied on selective functionalization of 2,4-dichloro-5-fl uoropyrimidine ( 93 ), which can be easily obtained from fluorouracil ( 1 ) (scheme 28 ) [ 123 ] . first, boronic ester 94 was prepared from aniline 95 in three steps, including benzimidazole ring construction and palladiumcatalyzed coupling with pinacol diborane. suzuki-type reaction of 93 and 94 resulted in selective functionalization at c-4 of the pyrimidine ring and gave chloride 96 . buchwald-hartwig coupling of 96 with amine 97 (prepared in two steps from 1-ethylpiperazine ( 98 ) and ( 99 )) gave the fi nal product 87 . analogously, selective functionalization of 93 was used for the preparation of fostamatinib disodium ( 88 ) (scheme 29 ). in particular, reaction of 93 with equimolar amount of amine 100 and then -with 3,4,5-trimethoxyaniline ( 101 ) gave tamatinib ( 92 ) [ 124 ] . it should be noted that no detailed procedures of performing these transformations were given in the initial patent; moreover, synthesis of the starting compound (amine 100 ) is not documented to date. to obtain fostamatinib disodium ( 88 ), compound 92 was treated with chloride 102 and cs 2 co 3 ; further deprotection subsequent and salt formation gave the target product 88 [ 125 ] . similar approach was used for the synthesis of r-763 ( 89 ) (scheme 30 ) [ 126 ] . in this case, lactam 102 , which was obtained from norbornadiene ( 103 ) and graf isocyanate (clso 2 nco), was protected with boc 2 o and then subjected to ringopening with aqueous ammonia to give amide 104 . deprotection of 104 followed by arylation with 93 gave an intermediate 105 , which was then treated with n -arylpiperazine derivative 106 (prepared in two steps from 4-fl uoro-3-methylnitrobenzene ( 107 )) to give racemic 89 . optically pure 89 was obtained either by chiral stationary phase hplc applied at different steps of the synthesis, or via enzymatic resolution of boc-protected lactam 102 . it is not surprising that synthesis of pf-03814735 ( 90 ) also followed analogous strategy, 2,4-dichloro-5-trifl uoromethylpyrimidine ( 111 ) being used as a key fl uorinated diazine building block instead of 93 (scheme 31 ) [ 302 ] . the synthetic scheme commenced from amine 108 which was n -trifl uoroacetylated, then nitrated, and subjected to a change of the protecting group to give boc derivative 109 . two alternative pathways were developed for further transformations. in the fi rst one, compound 109 was reduced into fused aniline derivative 110 which reacted with 111 to give compound 112 . deprotection of 112 followed by coupling with n -acetylglycine led to the formation of chloride 113 . alternatively, compound 109 was deprotected, coupled with n -acetylglycine, reduced catalytically and then arylated with 111 to give 113 . finally, compound 113 reacted with cyclobutyl amine to give the fi nal product 90 as racemate. both enantiomers of 90 were also obtained using this scheme if boc derivative 109 was subjected to chiral stationary phase hplc prior further transformations. although a similar strategy was used for the preparation azd-1480 ( 91 ), in this case the fl uorinated diazine moiety is not in a central part of the molecule; hence a different approach was used for the construction of the fl uorinated the fi ght against hiv infection is another important fi eld where fl uorinated diazines have remarkable record, including approved drug emricitabine ( 8 ) and 7 compounds that have reached clinical development phase (compounds 125 -131 ) ( table 3 ) . all these compounds act as hiv reverse transcriptase inhibitors and fall into two categories: fl uorocytidine analogues ( 8 and 125-127 ) and trifl uoromethyl-substituted quinazolone derivatives ( 128 -131 ). emtricitabine ( 8 ) was discovered in emory university (atlanta, usa); development of the drug was completed by gilead sciences, and the compound was approved by fda under trade name emtriva ® in 2003. it is also marketed in combinations with other anti-hiv agents, i.e. tenofovir ( 132 , used as a prodrug) (truvada ® ), efavirenz ( 133 ) and tenofovir (atripla ® ), rilpivirine ( 134 ) and tenofovir (complera ® ), and elvitegravir ( 135 ), cobicistat ( 136 ), and tenofovir (stribild ® ) [ 5 ] emricitabine is a close analogue of lamivudine ( 137 ), which is an example of nucleoside analogs -an important class of reverse transcriptase inhibitors, which has gained much attention since the initial success of the fi rst representative, zidovudine ( 138 ) [ 128 ] (fig. 5 ) . emtricitabine ( 8 ) was discovered in emory university (atlanta, usa); development of the drug was completed by gilead sciences, and the compound was approved by fda under trade name emtriva ® in 2003. it is also marketed in combinations with other anti-hiv agents, i.e. tenofovir ( 132 , used as a prodrug) (truvada ® ), efavirenz ( 133 ) and tenofovir (atripla ® ), rilpivirine ( 134 ) and tenofovir (complera ® ), and elvitegravir ( 135 ), cobicistat ( 136 ), and tenofovir (stribild ® ) [ 5 ] emricitabine is a close analogue of lamivudine ( 137 ), which is an example of nucleoside analogs -an important class of reverse transcriptase inhibitors, which has gained much attention since the initial success of the fi rst representative, zidovudine ( 138 ) [ 128 ] . emtricitabine ( 8 ) is very similar to lamivudine ( 137 ) with respect to its activity, convenience, safety and resistance profi le; the only remarkable difference is longer intracellular half-life of 8 . analogously to 137 , the biologically active form of 8 is triphosphate 139 , which is formed by a stepwise phosphorylation of 8 (scheme 33 ). compound 139 can be considered as 2,3-dideoxycytidine trifosphate analogue and acts as a competitive inhibitor and alternate substrate of the normal deoxycytidine triphosphate ( 140 ). as a competitive inhibitor of the normal substrate, 139 inhibits incorporation of 140 into the growing dna chain by viral reverse transcriptase; as an alternate substrate, it is incorporated into this chain (as 141 ) and acts as a chain terminator (since 141 is missing the 3′-hydroxyl group required for further chain elongation) [ 128 , 129 ] . although emtricitabine might have the potential for toxicity caused by interaction with human mitochondrial dna enzymes, both in vitro and in vivo testing results show that this is not a serious issue. low toxicity of 8 as compared to other nucleoside reverse transcriptase inhibitors is a remarkable advantage of this drug. as with all representatives of this class, the major drawback of 8 is rapid development of drug resistance by a single point mutation of viral reverse transcriptase [ 129 ] . the main route of elimination of 8 is renal excretion, mostly unchanged (86 % of the dose). the metabolic transformations of emtricitabine include oxidation of the sulphur atom to form the 3′-sulfoxide diastereomers (9 %) and conjugation with glucuronic acid to give 2′-o -glucuronide (4 %) [ 130 ] . a racemic form of emtricitabine, racivir, was also studied in clinics by pharmasset and has reached phase ii trials [ 113 ], designed to measure its effi cacy in patients harbouring virus resistant to lamivudine. it was shown that d (+)enantiomer 125 is less potent and more toxic than emtricitabine itself. one of the reasons behind lower potency of 125 is that 8 is phosphorylated by deoxycitidine kinase to a greater extent; therefore, the active form ( 139 ) is formed more readily for (-)-enantiomer [ 131 , 132 ] . elvucitabine ( 126 ) and its enantiomer dexelvucitabine ( 127 ) were discovered in yale university (new haven, usa) and emory university (atlanta, usa), respectively. both compounds were further developed by commercial companies (achillion pharmaceuticals and incyte co., respectively), and have reached phase ii clinical trials [ 113 ] . development of 127 was terminated due to inability to pair with other cytidine analogues and higher risk of hyperlipasemia. phase ii studies of 126 were suspended because of bone marrow suppression in several patients [ 133 ] . the mode of action of elvucitabine is quite similar to that of emtricitabine; the major advantages of 126 include long plasma half-life (up to ten times greater than that of 8 ) and superior potency against common resistance mutations [ 134 ] . four compounds dpc-961 ( 128 ), dpc-961 ( 129 ), dpc-083 ( 130 ), and dpc-082 ( 131 ) were developed by dupont pharmaceuticals as non-nucleoside reverse transcriptase inhibitors. al the compounds have reached phase i clinical trials; dpc-083 ( 130 ) was further progressed into phase ii trials by bristol-myers squibb after the company had acquired dupont pharmaceuticals; however, the development was stopped in 2003 due to poor pharmacokinetics [ 135 ] . the compounds are close analogues of efavirenz ( 133 ) -a non-nucleoside reverse transcriptase inhibitor approved by fda in 1998 [ 5 ] . all the compounds 128 -131 showed similar to efavirenz activity towards wild-type virus in vitro ; however, they were more effective towards singlemutation variants and showed lower plasma serum protein binding [ 136 , 137 ] . it might be assumed that mechanism of action of 128 -131 is similar to that of efavirenz, which is known to bind within the non-nucleoside inhibitor binding pocket of reverse transcriptase [ 138 ] , both spatially and also functionally associated with the substrate-binding site. metabolism of dpc-961 ( 128 ) was studied in rats. analogously to efavirenz, the main metabolite is glucuronide conjugate 142 (more than 90 % of excreted dose in the bile) (scheme 34 ). however, a glutatione conjugate 143 was also isolated, which is presumably formed via oxirene intermediate 144 ; in this view, metabolism of 128 was different from that of 133 [ 139 ] . approach starting from d-mannose or d-galactose was used for the preparation of d-enantiomer 125 [ 141 ] . most of the methods describing the preparation of emtricitabine (and racivir) rely on the construction of 1,3-oxathiolane ring by reaction of glycolaldehyde or glyoxalic acid derivatives with mercaptoacetic acid or mercaptoacetic aldehyde (which exists as 1,4-ditiane 154 ). for example, one of the fi rst of syntheses of this type commenced from allyl alcohol which was silylated and then subjected to ozonolysis to give glycolaldehyde derivative 155 (scheme 36 ) [ 142 ] . reaction of 155 with mercaptoacetic acid afforded 1,3-oxathiolane 156 , which was reduced with lialh(o t bu) 3 or dibal and then acetylated to form 157 . finally, reaction of 157 with silylated fl uorocytosine derivative 158 followed by deprotection led to the formation of racemic 8 (racivir). more than 15 preparations described in patents are variations of the above synthetic scheme. in particular, to obtain optically pure emtricitabine, lipase-catalyzed enzymatic resolution, as well as chiral stationary phase hplc was used [ 143 ] . however, the most effective procedure included separation of menthyl derivatives. this method evolved signifi cantly since the fi rst publication (which in fact relied on separation of all the 4 possible diastereomers) [ 144 ] ; one of the recent multigram preparations is shown in the scheme 37 [ 145 ] . the fi rst step of the synthesis included formation of methyl ester 159 from glyoxalic acid and l -menthol. reaction of 159 with 1,4-ditiane 154 gave 1,3-oxathiolane 160 as a mixture of cis diastereomers. compound 160 was transformed to chloride 161 by treatment with thionyl chloride and methanesulfonic acid. reaction of 161 and 158 led to the formation of 162 , which was separated as a single diastereomer by transformation to oxalate and subsequent crystallization. finally, reduction of 162 with nabh 4 gave emtricitabine ( 8 ) which was isolated as hydrochloride. an interesting variation of the method was patented by glaxo wellcome inc [ 146 ] . their synthesis was started from 2,4-dichloro-5-fl uoropyrimidine ( 93 ) (scheme 38 ). reaction of 93 with naoet and then -with anion of 2,2-dimethoxyethanol gave pyrimidine derivative 163 , which upon detection formed aldehyde 164 . reaction of 164 and 154 led to the formation of 1,3-oxathiolane 165 , which was acetylated to give 166 . treatment of 166 with tmsotf resulted in rearrangement leading to 167 , which was transformed to racemic 8 (racivir) by reaction with ammonia. a number of methods for the preparation of elvucitabine ( 126 ) were reported in the literature. in the fi rst synthetic scheme developed in yale university [ 147 ] , 2′-deoxy-5-fl uoro-β-l-uridine ( 168 ), which is enantiomer of floxuridine ( synthesis of elvucitabine ( 126 ) developed by chemists from vion pharmaceuticals commenced from lactone 171 (scheme 40 ), which can be obtained in 4 steps from d-glutamic acid [ 148 ] . phenylselenation of enolate generated from 171 proceeded highly diastereoselectively and led to 172 . phenylselenide 172 was reduced with dibal and then acetylated to give acetate 173 as a mixture of anomers. reaction of 173 with 158 was also diastereoselective due to the steric effect of bulky phenylselenyl substituent and gave β anomer 174 in almost quantitative yield. oxidative elimination of the selenide substituent from 174 and subsequent deprotection gave elvucitabine ( 126 ) as a single enantiomer. an analogous synthesis was described by chemists from emory university [ 149 ] . syntheses of dexelvucitabine ( 127 ) [ 150 ] and later -elvucitabine ( 126 ) [ 151 ] were described, starting from d-and l-xylose, respectively, both using almost the same methodology. in particular, d-xylose was transformed into the dibenzoyl derivative [ 152 ] . compound 184 was subjected to bromoacylation with excess of 2-acetoxy-2methylpropionyl bromide ( 185 ) to give a mixture of esters 186 and 187 . this mixture was subjected to reductive elimination to give 188 , which was transformed to 127 upon alcoholysis. another synthesis of 127 relied on palladium mediated ferrier rearrangementtype glycosidation of a furanoid glycal (scheme 43 ) [ 153 ] . the initial steps of fluorine-containing diazines in medicinal chemistry and agrochemistry the synthesis were quite similar to those shown in scheme 41 . the major difference was the use of polymer-supported pph 3 at the glycal generation step, which allowed for isolation of unstable glycal 189 with more than 90 % purity. palladium-catalyzed reaction of 189 with 5-fl uorocytosine ( 9 ) was accompanied by ferrier-type rearrangement and led to derivative 190 , which was transformed to 127 upon deprotection. all the reported syntheses of dpc-961 ( 128 ) and dpc-963 ( 129 ) commenced from the corresponding o -amino-α,α,α-trifl uoroacetophenones 191 (scheme 44 ). in the fi rst preparations of 128 and 129 , 191 reacted with tmsnco to give adducts 192 , which were transformed to cyclic imines 193 upon dehydratation. reaction of 193 with lithium cyclopropylacetylenide gave racemic 128 and 129 , which were subjected to chiral stationary phase hplc to isolate 128 and 129 as pure enantiomers [ 136 , 137 ] . several improvements were reported for this synthetic scheme. in particular, diastereoselective additions of lithium cyclopropyl acetylenide to the derivatives of 193 containing residues of α-phenylethyl amine or campheic acid were developed [ 154 , 155 ] . moreover, an enantioselective modifi cation of this method employing amino alcohol 194 as an asymmetric catalyst was discovered [ 156 , 157 ] . another enantioselective method involved reaction of the derivatives of 193 and cyclopropyl acetylene itself, catalysed by amino alcohol derivatives ( e.g. 195 ) and zn(otf) 2 [ 158 ] . dpc-083 ( 130 ) and dpc-082 ( 131 ) were obtained by reduction of 128 and 129 , respectively, with lialh 4 [ 136 , 137 ] . recently, an alternative approach to the synthesis of 130 was reported, which relied on enantioselective organocatalytic mannich-type reaction of imine derivative 196 and cyclopropyl methyl ketone (scheme 45 ) [ 159 ] . although enantioselectivity of the key step was moderate ( ee 75 %), it could be easily enhanced to >99 % by a single recrystallization of intermediate 197 . apart from anti-hiv drugs discussed in the previous section, two additional antiviral agents can be mentioned: trifl uridine ( 7 ) and favipiravir ( 198 ) . trifl uridine ( 7 ) was mentioned above as a component of phase iii investigational drug tas-102. it is however more known as an ophthalmic anti-herpes agent launched by glaxo wellcome (now merged into glaxosmithkline) in 1980 [ 5 ] . it is effective against herpetic keratitis, and seems to be especially useful in 'diffi cult' cases [ 160 ] . high susceptibility to biodegradation of trifl uridine is advantageous for its use as ophthalmic drug, as its action in other tissues is thus prevented. as in the case of anti-tumor activity, the mechanism of antiviral action of 7 involves the inhibition of viral replication. trifl uridine does this by incorporating into viral dna during replication, which leads to the formation of defective proteins and an increased mutation rate [ 161 ] . inhibition of thymidylate synthetase also seems to contribute into antiviral effect of 7 . the details of these processes, as well as synthesis of 7 were discussed in the above sections. favipiravir ( 198 ) has been discovered by toyama chemicals; it is currently in phase iii (japan) and phase ii (usa) clinical trials [ 113 , 162 ] . favipiravir is under development as an agent against infl uenza virus, however, it was also tested against other rna viruses, including arenaviruses, bunyaviruses, west nile virus (wnv), yellow fever virus (yfv), and foot-and-mouth disease virus (fmdv) [ 163 ] . a proposed mechanism of action of 198 includes its biotransformation into ribofuranosyltriphosphate derivative 199 (scheme 46 ), which inhibits infl uenza virus rna polymerase in the host cells [ 164 ] . [ 166 , 167 ] . acidic hydrolysis of 205 gave amide 206 , which upon mild alkaline hydrolysis led to 198 . alternatively, compound 198 was obtained by mild alkaline hydrolysis of 205 followed by reaction with h 2 o 2 -naoh, or by reaction of 205 with allyl or benzyl alcohol, removal of the protection, and hydrolysis. recently, an improved version of this method was patented, which allowed authors to claim its industrial applicability [ 168 ] . one more method for the preparation of 198 commenced from pyrazine derivative 207 , which was transformed to dichloride 208 using sandmeyer reaction (scheme 49 ) [ 166 ] . hydrolysis of the ester moiety in 208 followed by one-pot chloroanhydride formation, introduction of fl uorine atom and amination gave derivative 209 , which was transformed into 198 by diazotization and subsequent hydrolysis. several other approaches to the synthesis of favipiravir were also described, most of them relying on direct fl uorination of pyrazine derivatives with molecular fl uorine [ 166 ] all they were low-yielding and allowed for the preparation of milligram quantities of the fi nal product. a single compound is discussed in this category, namely gsk-1322322 ( 210 ), which was developed by glaxosmithkline and has reached phase ii clinical trials in bacterial skin infections [ 113 ] and phase iii -in community-acquired bacterial pneumonia [ 169 ] . compound 210 acts as an inhibitor of peptide deformylase -an enzyme that removes the formyl group during eubacterial peptide elongation. bacterial protein synthesis initiates with formyl-methionyl-trna and, consequently, all polypeptides newly synthesized in bacteria contain an n -formylmethionine terminus. this residue is further removed in two steps catalyzed by peptide deformylase and methionine aminopeptidase, respectively. inhibition of peptide deformylase increase production of bacterial n -formylated polypeptide, which prevents bacteria growth and possibly triggers an enhanced immune response [ 170 ] . peptide deformylase is a metalloprotease, which mostly utilizes fe 2+ in its active site. it was shown for analogs of 210 that n -formyl-n -hydroxylamine function coordinated to metal ion when the inhibitor was bound to the enzyme [ 171 ] . synthesis of 210 was started from preparation of chiral diamine 211 (scheme 50 ) [ 172 ] . in particular, d -serine methyl ester was converted to n -benzyl derivative 212 , which was transformed into carboxylic acid 212 using reaction with chloroacetyl chloride and subsequent hydrolysis. carboxylic acid 212 was subjected to coupling with benzyl amine, reduction, reaction with ethyl oxalyl chloride and reductive cyclization to give bicyclic compound 213 . finally, 211 two-step reduction of 213 led to the formation of diamine 211 , which was isolated as dihydrochloride. reaction of 211 with dichloro derivative 215 and then -hydrazine hydrate gave the product 216 , which was coupled with carboxylic acid 217 and subjected to catalytic hydrogenation to give 210 . two drugs were launched as anti-fungal agents to date: flucytosine ( 9 ) (valeant, 1971) and voriconazole ( 10 ) (pfi zer, 2002) (fig. 6 ) [ 5 ] . flucytosine itself has no antifungal activity; its activity results from the rapid conversion into fluorouracil ( 1 ) within susceptible fungal cells [ 173 ] . the mechanism of cytotoxic effect of fluorouracil has been discussed in the previous sections. flucytosine is taken up by fungal cells by cytosine permease, which is the transport system for cytosine and adenine. inside the fungal cells, 9 is deaminated to 1 by cytosine deaminase. the specifi city of this step is crucial for the narrow antifungal spectrum of 9 : mammalian cells as well as fungi lacking cytosine deaminase are not sensitive to 9 . on the other hand, fluorouracil itself cannot be used as an antifungal drug, since it is only poorly taken up by fungal cells and is too toxic to human cells. the major drawback of flucytosine is rapid development of resistance in fungi, either by mutations or by increased synthesis of pyrimidines; this limits the use of 9 as a single antifungal agent. monotherapy with flucytosine is currently only used in some cases of chromoblastomycosis and in uncomplicated candidosis; in all other cases, 9 is used together with other agents, usually amphotericin b [ 173 ] . the effect of voriconazole ( 10 ) is exerted within the fungal cell membrane. in particular, cytochrome p450-dependent 14-α-lanosterol demethylase is inhibited, which prevents the conversion of lanosterol ( 217 ) to ergosterol ( 218 ) -an important component of yeast and fungal cell membranes which does not occur in mammalians (scheme 51 ). this mechanism results in the accumulation of toxic methylsterols and inhibition of fungal cell growth and replication [ 174 ] . voriconazole is active against many fungal infections, including invasive aspergillosis, pseudallescheria , scedosporium , fusarium infections [ 175 ] . it is also proposed for empirical antifungal therapy [ 176 ] . an important advantage of voriconazole is high oral bioavailability (96 %). the most common side effect, which is unique for voriconazole among other azole antifungals, is a reversible disturbance of vision (photopsia): it occurs in nearly a third of patients but rarely leads to discontinuation of the drug [ 174 ] . resistance to voriconazole still remains uncommon, although an increase of resistance and continued surveillance with greater use of the drug has been reported [ 177 ] . the fi rst synthesis of flucytosine ( 9 ) has been reported in 1957 [ 13 , 14 ] . the synthetic scheme is quite similar to that for fluorouracil ( 1 ); in the case of 9 , compound 27 was subjected to reaction with pcl 5 and then -liquid ammonia to give 219 , which was transformed to 9 upon hydrolysis (scheme 52 ). in an alternative method, compound 70 (prepared from fluorouracil) reacted with socl 2 to give 220 , which was transformed to 9 upon reaction with ammonia in methanol [ 84 ] . another synthesis commenced from 2,5-difl uoro-4-chloropyrimidine, which, however, is not readily accessible [ 178 ] . flucytosine was also obtained by direct fl uorination of cytosine using cf 3 of (85 % yield) [ 179 , 180 ] , fl uorine [ 181 , 182 ] , and acof [ 20 ] . despite numerous syntheses of voriconazole ( 10 ) were documented, they all followed the same synthetic strategy, namely, addition of anion 221 to ketone 222 , followed by isolation of necessary diastereomeric pair and its resolution with 10-camphorsulphonic acid (scheme 53 ). three different approaches were used for the generation of anion 221 or the corresponding organometallic species. first of them relied on deprotonation of the pyrimidine derivative 222 (prepared from the fl uorinated keto ester 223 or dichloro derivative 93 ) by strong bases such as lda (scheme 54 ) [ 183 -189 ] . the main drawback of this method was low diastereoselectivity of the key step; therefore tedious separation of diastereomers was necessary. another approach to generation of 221 relied on zncl 2 -catalyzed decarboxylation of salts 224 , prepared from 225 (scheme 55 ) [ 190 ] . in this case, the desired diastereomeric pair was obtained with much better selectivity (6.5: 1). the last approach relied on reformatsky-type reaction involving 222 and bromides 226 (prepared from 223 [ 191 , 192 ] or its thio analogues [ 193 -195 ] ) or sulfonates 227 (prepared from 93 ) (scheme 56 ) [ 196 , 197 ] . in this case, good diastereoselectivities were obtained. seven compounds designed as agents acting at central and/or peripheral nervous system have reached at least phase ii clinical trials, and only one of them was launched (table 4 ) [ 3 , 113 ] . these compounds address different biological targets and act as skeletal muscle relaxants (afl oqualone ( 11 )), antipsychotics a representative of fl uorinated diazines, afl oqualone ( 11 ), was launched in 1983 in japan as a central acting muscle relaxant [ 198 ] . it is an analogue of methaqualone ( 234 ) (fig. 7 ) -a drug widely used as a hypnotic, for the treatment of insomnia, and as a sedative and muscle relaxant in 1970s, but reclassifi ed as a schedule i controlled substance in usa in 1984 [ 199 ] . the mechanism of action of afl oqualone is not well studied. it was shown that its site of action is different from that of other central acting muscle relaxants, i.e. mephenesin, chlormesazone or diazepam [ 200 ] . gaba-enhancing effect was also demonstrated [ 303 ] . the main routes of metabolism of 11 in human include n -acetylation, followed by hydroxylation at the 2′-methyl and acetyl methyl carbons, as well as glucuronidation of the aromatic amino group. this pattern of metabolism is similar to that observed in monkeys and rats, but drastically different from that in dogs [ 304 ] . synthesis of afl oqualone commenced from 5-nitroanthranilic acid ( 235 ) which was transformed to amide 236 via the corresponding chloroanhydride (scheme 57 ) [ 201 ] . catalytic reduction of 236 followed by acetylation gave 237 , which reacted with chloroacetyl chloride to form quinazoline 238 . nucleophilic substitution of chlorine atom in 238 with fl uorine led to the formation of 239 , which upon deprotection gave afl oqualone ( 11 ). alternatively, compound 236 was subjected to acylation with fl uoroacetyl chloride or anhydride to give amide 240 [ 202 ] . refl uxing of 240 with acetic anhydride gave quinazoline 241 , which was reduced to afl oqualone either by catalytic hydrogenation or using sncl 2 . all three compounds discussed in this section ( i.e. 228 -230 ) have reached phase ii clinical trials as agents for treatment schizophrenia. development of bmy-14802 ( 228 ) was discontinued more than 10 years ago. fluorine-containing diazines in medicinal chemistry and agrochemistry noted that relative role of these two targets in biological effect of 228 was debated in the literature. whereas in pigeons, the effect was serotonergically mediated primarily through 5-ht 1a receptors [ 203 ] , in other model systems, these interactions did not seem to contribute signifi cantly to the potential antipsychotic action of the compound [ 204 ] . although studies in animal models supported for the suggestion that bmy-14802 ( 228 ) may possess antipsychotic properties [ 205 ] , clinical trials showed lack of effi cacy in schizophrenia treatment [ 206 ] . recently, bmy-14802 was proposed as a promising candidate for clinical trials of l -dopa-induced dyskinesia -a common side effect observed during prolonged use of l -dopa in parkinson disease patients [ 207 ] . it was shown that the compound suppresses abnormal involuntary movements related to l -dopa-induced dyskinesia via its 5-ht 1a agonistic effect. abt-925 ( 229 ) developed by abbott is a selective d3 receptor antagonist [ 208 ] . it was suggested that selective antagonists of d3 receptor might be promising antipsychotic agents lacking the presumed d2 receptor-mediated side effects, although d3 antagonists may express their effect via mechanisms that cannot be refl ected by the commonly used animal models [ 209 ] . it was shown that abt-925 produced cognitive signals but did not achieve suffi cient d3 receptor occupancy at the doses used in clinical studies [ 210 ] . nevertheless, these studies allowed for the assumption that the development and clinical testing of newer d3 receptor antagonists with higher potency at d3 receptors, enabling suffi cient receptor occupancy, is highly warranted [ 211 ] . on the contrary, jnj-37822681 ( 230 ) is a d2 highly selective receptor antagonist and hence acts in a mode analogous to that of most marketed antipsychotics [ 212 ] . jnj-37822681 is characterized by a rapid dissociation rate from the dopamine d2 receptor, which was hypothesized to confer antipsychotic effi cacy and improved tolerability [ 213 ] . clinical studies in patients with an acute exacerbation of schizophrenia showed that jnj-37822681 had similar biological activity but lesser tendency to induce weight gain compared to a known antipsychotic drug, olanzapine ( 242 ) [ 214 ] (fig. 8 ) . synthesis of bmy-14802 ( 228 ) commenced from pyrimidine derivative 243 which reacted with piperazine 244 to give derivative 245 (scheme 58 ) [ 215 , 216 ] . reduction of the compound 245 followed by deprotection gave amine 246 , which was alkylated with chloride 247 and then subjected to acidic hydrolysis to form ketone 248 . reduction of 248 allowed bmy-14802 ( 228 ) to be obtained. pure enantiomers of 228 were also obtained. to achieve this, the following methods were used: resolution of 228 with using reaction with α-phenylethyl isocyanate [ 217 ] or lipase-catalyzed acetylation or hydrolysis [ 218 ] , alkylation of 245 with enantiopure alcohols 249 [ 219 ] ; and microbial reduction [ 305 ] or ru-catalyzed enantioselective hydrogenation [ 220 ] of 248 . abt-925 ( 229 ) was obtained starting from amidine 250 and ethyl trifl uoroacetoacetate to give pyrimidine 251 (scheme 59 ) [ 221 ] . reaction of 251 with socl 2 and then -piperazine led to the formation of amine 252 . selective alkylation of 252 with 1-bromo-3-chloropropane gave chloride 253 , which reacted with thiouracil anion to form abt-925 ( 229 ). bmy-21502 ( 231 ) was developed by bristol-myers squibb as nootropic agent ( i.e. for cognition disorders) and has reached phase ii clinical studies. the compound was effective in vitro [ 223 ] as well as in animal models [ 223 -227 , 306 ] that may predict cognitive enhancement. the mode of action of bmy-21502 is poorly understood. it was shown that the compound has an anti-anoxic action, and activation of the cns cholinergic system is involved as one of the causative mechanisms for this effect [ 228 ] . clinical trials showed that bmy-21502 was not significantly superior to placebo in alzheimer's disease; moreover, although generally well tolerated, 231 also had a higher rate of discontinuations [ 229 , 230 ] . synthesis of bmy-21502 ( 231 ) optimized for large scale preparations commenced from malonodiamide and ethyl trifl uoroacetate, which reacted to give pyrimidine 255 (scheme 61 ) [ 231 ] . compound 255 was transformed into dichloro derivative 256 upon treatment with pocl 3 . reaction of 256 with piperidine 257 (prepared from 4-pyridinylmethyl chloride in two steps) gave 258 , which was reduced catalytically to form bmy-21502 ( 231 ). alternatively, bmy-21502 was obtained by arylation of 257 with 4-chloro-2-trifl uoromethylpyrimidine ( 259 ) [ 232 ] . synthesis of jnj-37822681 ( 230 ) was quite trivial and relied on selective functionalization of 4-aminopiperidine core, fi rst with 3-chloro-6-trifl uoromethylpyridazine ( 254 ) and then -with 3,4-difl uorobenzaldehyde (scheme 60 ) [ 222 ] . . bw-4030w92 ( 232 ) was developed as a cns-acting antihyperalgesic agent ( i.e. for treatment of increased sensitivity to pain). it is an analogue of anticonvulsant drug lamotrigine ( 260 ) (fig. 9 ) , used n the treatment of epilepsy and bipolar disorder [ 233 ] . like lamotrigine, bw-4030w92 binds to the transmembrane segment s6 in domain iv of α subunit of voltage-gated sodium channels (na v ), thus acting as a pore blocker [ 234 ] . it is assumed that neuropathic pain is partially mediated by an increase in the density of na v channels in injured axons and their dorsal root ganglions. clinical studies in patients with chronic neuropathic pain showed that although bw-4030w92 signifi cantly lowered allodynia severity at the fi rst day, the effect did not maintain in further treatment [ 235 ] . gw-842166x ( 233 ) is a selective cb2 receptor full antagonist which has potent analgesic, anti-infl ammatory and anti-hyperalgesic actions. it was selected as a clinical candidate after lead optimization of a pyrimidine ester 261 (gk02076, fig. 9 ) , identifi ed in a focused screen as a partial agonist at the cb2 receptor with micromolar potency [ 236 ] . the compound was evaluated as an analgesic for treatment of infl ammatory pain (phase i trials) and dental pain (phase ii trials) [ 113 ] . in the latter study, single doses of gw842166 failed to demonstrate clinically meaningful analgesia in the setting of acute dental pain [ 237 ] . fluorine-containing diazines in medicinal chemistry and agrochemistry trifl uoride (dast) to give racemic 232 . alternatively, nitrile 263 reacted with ethyl fl uoroacetatet -buok and then -ethyl iodide to give enol ether 267 , which was transformed to racemic 232 by reaction with guanidine. resolution of enantiomers of 232 was achieved by crystallization of dibenzoyl-l -tartaric acid salt; the more active r -enantiomer was isolated. in the synthesis of gw-842166x ( 233 ), commercially available pyrimidine 268 reacted with 2,4-dichloroaniline to give ester 269 , which was subjected to hydrolysis followed by amide coupling with 4-aminomethyltetrahydropyran ( 270 ) to afford 233 (scheme 63 ) [ 236 , 239 , 240 ] . in the previous sections, compounds targeting cancer cells or nervous system, as well as those fi ghting foreign organisms were discussed. three compounds do not fall into any of these categories. fostamatinib disodium ( 88 ) was mentioned above as an anti-cancer investigational drug, but it was also studied as agent for autoimmune diseases, i.e. rheumatoid arthritis (currently in phase iii) and autoimmune thrombocytopenia (in phase ii). gemigliptin ( 12 ) was approved as an anti-diabetic drug in south korea in 2012. pf-04634817 ( 271 ) was discontinued after phase i studies as an agent for liver fi brosis; nevertheless, it is currently investigated in diabetic nephropathy (fig. 10 ) (phase ii, october 2012) [ 113 ] . as it was mentioned in the previous sections, the active principle of fostamatinib disodium ( 88 ) is tamatinib ( 92 ), which is formed by enzymatic hydrolysis of 88 in the intestine. as in the case of lymphoma, the effect of 88 in autoimmune diseases is related to inhibition of spleen tyrosine kinase (syk) by 92 [ 241 , 242 ] . as syk has the central role in transmission of activating signals within b cells, inhibition of this enzyme lowers expression of a number of proinfl ammatory cytokines and hence leads to immunosuppression [ 243 ] . fostamatinib has shown signifi cant effi cacy in the treatment of patients with rheumatoid arthritis not responding to methotrexate ( 272 ) (a drug which is used conventionally in therapy), although a number of adverse events were observed [ 244 ] . if these results are confi rmed once phase iii studies are completed, it may fi nd a place in the treatment of patients with rheumatoid arthritis with poor response to conventional therapy (fig. 11 ) . gemigliptin ( 12 ) was developed by lg life sciences as an inhibitor of dipeptidyl peptidase 4 (dpp-4) -a target of oral drugs used to treat used to treat type 2 diabetes (characterized by high blood glucose in the context of insulin resistance and relative insulin defi ciency) [ 245 ] . the fi rst representative of this class, sitagliptin ( 273 ) was launched in 2006. in human body, gemigliptin is metabolized to lc15-0636, which is a major active metabolite, by cytochrome p450 3a4 isozyme [ 246 ] . inhibition of dpp-4 results in increase of incretin levels (which is normally inactivated by dpp-4), in particular glucagon-like peptide-1 (glp-1) and gastric inhibitory peptide (gip) [ 247 ] . incretins inhibit glucagons release and stimulate insulin secretion, which leads to decrease in glucose blood levels. clinical trials showed effi cacy and safety of gemigliptin administered once daily as a monotherapy, [ 248 ] as well as in addition to metformin ( 274 ) [ 249 ] for type 2 diabetes patients. pf-04634817 ( 271 ) is a phizer's investigational drug, initially developed as agent for liver fi brosis -formation of excess fi brous connective tissue in liver [ 250 ] . the development of the compound was discontinued since february 2012 after phase i trials. recently, a phase ii study of pf-04634817 in diabetic nephropathya progressive kidney disease caused by angiopathy of capillaries in the kidney glomeruli [ 251 ] -was registered [ 113 ] . pf-04634817 is an antagonist of chemokine receptors ( i.e. ccr2 and ccr5) [ 252 ] . these chemokine receptors are important players in the traffi cking of monocytes/macrophages and in the functions of other cell types relevant to pathogenesis of many diseases [ 253 ] , including liver fi brosis [ 307 ] and diabetic nephropathy [ 254 ] . gemigliptin ( synthesis of optically active pf-04634817 ( 271 ) based on commercially available (-)-vince lactam as chirality source. starting from (-)-vince lactam the chiral key 4-amino-2-cyclopentene-1-carboxylic acid derivative 286 was synthesized. the compound 286 is dimethyl pyrrole protected form of corresponding aminoacid, which was subjected to amide coupling with boc-protected diamine 287 to give amine 288 (scheme 65 ) [ 252 ] . removing of the pyrrole function followed by catalytic hydrogenation gave amine 289 , which was subjected to reductive amination of ketone 290 , separation of diastereomers, deprotection and then -arylation with pyrimidine derivative 291 to afford the fi nal product, 271 . agrochemistry is one of more important fi eld of application of the fl uorinated compounds which is widely recognized [ 256 , 257 ] (fig. 12 ). uracil derivatives butafenacil ( 292, inspire®, rebin®) and benzfendizone ( 293 ) were introduced as herbicides in 1998, whereas their pyridazine-derived analogue flufenpyr-ethyl ( 295 ) -in 2000 [ 258 ] . butafenacil (developed by syngenta ag) is used for weed control in grapes, nut crops, pome and stone fruits and also as a cotton defoliant [ 259 ] . it was registered in australia and approved by u. s. environmental protection agency. benzfendizone (developed by fmc corporation) is a post-emergence herbicide that provides good control of grass and broadleaf weeds in tree fruits and vines, as a cotton defoliant, and in total vegetation control [ 256 ] . flufenpyr-ethyl (developed by sumitomo chemical company) was registered in usa for use on corn, soybeans and sugarcane [ 259 ] . the most recent example is safl ufenacil ( 294 , kixor®), introduced by basf in 2009 for preplant burndown and selective pre dicot weed control in multiple crops, including corn. [ 260 ] . compounds 292 -295 act as inhibitors of protoporphyrinogen oxidase (protox) -an enzyme in the chloroplasts of the plant cells that oxidizes protoporphyrinogen ix ( 303 ) to produce protoporphyrin ix ( 304 ) (scheme 66 ) [ 261 ] . in turn, 304 is a precursor molecule for both chlorophyll and heme. when protoporphyrinogen oxidase is inhibited, protoporphyrinogen ix is accumulated and transferred from chloroplasts into the cytoplasm, where non-enzymatic conversion of 303 to 304 occurs. when present in cytoplasm, 304 is cytotoxic due to interaction with oxygen upon action of light, which results in formation of singlet o 2 molecules. 1 o 2 causes lipid peroxidation, membrane disruption and plant cell death. butafenacil is known to be eye, skin and respiratory tract irritant in humans [ 262 ] . it also demonstrated very high toxic effect to algae, and moderate toxicity to fi sh, aquatic invertebrates and honeybees. for benzfendizone and flufenpyr-ethyl, no reports on toxic effects are available. acute mammalian toxicology studies of safl ufenacil indicate that herbicide has low toxicity for mammals after ingestion, dermal exposure or inhalation. it is not an irritant for eyes and skin and does not act as a sensitizer. studies of the structure-activity relationship (sar) of uracile derivatives as protox inhibitor showed that presence of a polyfl uorinated alkyl group at position 6 of the uracil ring critical. alkyl groups such as methyl at position 6 of the uracil ring resulted in compounds with low or no biological activity [ 263 ] . limited data are available on the synthesis of butafenacil ( 292 ). in particular, it was prepared by esterifi cation of carboxylic acid 305 , [ 264 ] as well as by reaction of isocyanate 306 with ester 307 (scheme 67 ) [ 265 ] . preparation of neither 305 nor 306 was disclosed in the corresponding patents, although synthesis of carboxylic acid 305 was partially described elsewhere [ 266 ] . benzfendizone ( 293 ) was obtained from ethyl trifl uoromethylaminocrotonate ( 308 ) which reacted with isocyanate 309 in the presence of nah and then directly methylated to give 310 (scheme 68 ) [ 267 ] . demethylation of phenol moiety in 310 followed by alkylation with benzyl chloride 311 gave benzfendizone. in the preparation of flufenpyr-ethyl ( 295 ), hydrazones 318 or 319 were the key synthetic intermediates (scheme 70 ) [ 269 -271 ] . both compounds 318 and 319 were prepared by reaction of dibromoketone 320 and the corresponding hydrazines 321 and 322 , in turn obtained by reduction of diazonium salts 323 and 324 . alternatively, hydrazone 319 was prepared by reaction of 324 and ethyl trifl uoroacetoacetate, followed by hydrolysis and decarboxylation. further transformations of 319 included reaction with (carbethoxylidene)triphenylphosphorane resulting in the formation of pyridazine derivative 327 . acidic hydrolysis of 327 led to 328 , which was alkylated with ethyl bromoacetate to give 295 (scheme 71 ). alternatively, either 318 or 319 reacted with methylmalonic acid to give adducts 329 or 330 , which underwent cyclization upon heating with carboxylic acid and a base to give 331 and 327 , respectively. both 331 and 327 were transformed to flufenpyr-ethyl ( 295 ) as described above (scheme 72 ). compounds discussed in this section are derivatives or analogues of sulfonylurea herbicides -agrochemicals which began the present low-dose era of herbicide chemistry in 1970s [ 257 ] . primisulfuron-methyl ( 299 ) (from ciba-geigy corporation and syngenta ag) is a sulfonylurea derivative introduced in 1990 [ 262 ]. it is used for post-emergence control of actively growing weeds in corn and in non-cropland areas [ 272 ] . cloransulam-methyl ( 296 ), florasulam ( 298 ), and diclosulam ( 297 ), all developed by dow agrosciences, are examples of the triazolopyrimidine sulfonanilide herbicides; they were introduced in 1998, 1999, and 2000, respectively. cloransulam-methyl is used for soil-applied and post-emergence control of broadleaf weeds in soybeans [ 273 ] . florasulam is a highly-selective broadleaf herbicide which is registered for use in cereals in many countries around the world. diclosulam-based products are registered for use to control annual and certain perennial broadleaf weeds; they can be can be applied as soil, foliar, or burndown treatments in crops such as sugar cane, peanuts and soybeans and in forestry applications. compounds 296 -299 inhibit acetohydroxy acid synthase (ahas), formerly known as acetolactate synthase. its activity is not present in animals, but it has been found in all plants where measurements have been attempted. acetohydroxy acid synthase catalyses the fi rst step in production of branched amino acids (leucine, valine and isoleucine) (scheme 73 ), which are obviously needed for the protein synthesis and cell growth. the compounds 296 -299 seem to bind within the substrate-access channel of the enzyme, thus blocking α-ketocarboxylate access to the active site. while these herbicides are undoubtedly highly successful, resistance developed due to mutations within ahas is becoming a serious problem [ 274 , 275 ] . primisulfuron-methyl is a slightly toxic for skin, inhalation and eye exposure, with little metabolic activity in mammalian. it is slightly toxic to freshwater fi sh, aquatic organisms and to marine shrimp and has no toxic effect on birds and honeybees [ 276 ] . cloransulam-methyl can be highly toxic to certain aquatic plants and algae on an acute basis; it is practically nontoxic to other non-plant organisms. florasulam is highly toxic to aquatic organisms and slightly toxic to birds, and diclosulam is very highly toxic to aquatic organisms [ 272 ] . in contrast to uracile herbecides in which cf 3 -group is critical for activity in fl uorinated triazolopyrimidine series fl uorine atom responsible for the methabolitic transformation of the herbecides. the different metabolic pathway of the triazolopyrimidine herbicide diclosulam and cloransulam-methyl are guided by the fl uorine atom at the 7-position on the triazolopyrimidine ring system (scheme 74 ). the predominance of one pathway is very crop specifi c. in cotton, 296 and 297 are metabolized by the displacement of the 7-fl ouro substituent on the triazolopyrimidine ring by a hydroxy group, forming 332 . its soybean selectivity is attributed to facile conjugation with homo-glutathion (homogsh), which displaces the 7-fl uoro substituent ( 333 ). this mechanism was found to only occur in soybeans for these herbecides. in maize and wheat, 296 and 297 are detoxifi ed by hydroxylation at the 4-th position on the aniline moiety ( 334 ) followed by subsequent glycosidation [ 277 ] . cloransulam-methyl ( 296 ) and diclosulam ( 297 ) were obtained by reaction of sulfonyl chloride 340 with the corresponding aniline derivatives (scheme 75 ). synthesis of 340 commenced from dichloropyrimidine 335 [ 278 ] , which reacted with kf and then -hydrazine hydrate to give 337 . reaction of 337 with cs 2 /et 3 n and then -benzyl chloride was accompanied by dimroth rearrangement and gave the synthesis of primisulfuron-methyl ( 299 ) started from reaction of diethyl malonate and thiourea (scheme 77 ) [ 284 ] . the resulting pyrimidine derivative 348 was methylated, difl uoromethylated and then oxidized to give sulfone 351 . reaction of 351 with aqueous ammonia gave heteroaromatic amine 352 , which was transformed to primisulfuron-methyl ( 299 ) upon treatment with isocyanate 353 . fluoxastrobin ( 300 ) is a pesticide from bayer cropscience for the control of fungal diseases, which was registered by u. s. environmental protection agency (epa) in 2005 [ 276 ] . fluoxastrobin is used on peanuts, tuberous and corm vegetables, leaf petiole vegetables, fruiting vegetables and turf. fluacrypyrim ( 301 ) was discovered by basf ag and introduced in 2002 by nippon soda co., shows acaricidal effect against all stages of tetranychid [ 285 ] . both 299 and 300 are representative of strobilurin family with parent compound strobilurin a ( 354 ) (fig. 13 ) , discovered in late 1970s [ 286 ] . interestingly, fluacrypyrim ( 301 ) is the fi rst representative of strobilurin family which is not used as a fungicide. strobilurins are the part of the larger group of the so-called quinone outside inhibitors (qoi) -compounds which act at the quinol outer binding site of the cytochrome bc 1 complex. this enzyme, also referred to as ubiquinol: ferricytochrome c reductase, or complex iii, is the third complex in the electron transport chain -a cascade of enzymes which couples electron transfer between nadh and o 2 with the transfer of h + ions across a membrane to generate chemical energy in the form of adenosine triphosphate (atp) [ 287 ] . the overall result of the reaction catalyzed by cytochrome bc 1 complex is reduction of ferricytochrome c by oxidation of ubiquinol ( 355 ) and the concomitant pumping of 4 protons from the mitochondrial matrix to the intermembrane space. the mechanism of this process is too sophisticated to be discussed herein. it is important that the enzyme has two binding sites for the substrate 355 or its oxidized form 356 (fig. 14 ) , i.e. outer (q 0 ) and inner (q 1 ), and the quinone outside inhibitors bind to the outer site. this leads to inhibition of mitochondrial respiration -a process which is essential to all living organisms. the selective biological effect of quinone outside inhibitors on certain organisms ( i.e. fungi or mites) is achieved by differential penetration and degradation between various species, leading to a combination of high fungicidal (or acaricidal, in the case of 301 ) potency and good crop safety [ 288 ] . unfortunately, resistance has already evolved to this class of pesticides in some plant pathogens in certain geographical areas [ 289 ] . although the in vitro fungicidal activity of the natural strobilurin a was discovered soon, its agrobiological testing in vivo was diffi cult because of its volatility and the inherent lability of the ( e,z,e )-triene system, which resulted in rapid photolytic or metabolic degradation. the unusual structural simplicity of this natural product soon made it a target for chemical derivatization. below a set of isosterical replacement in a course of lead optimization of natural strobirulin a leading to commercial synthetic products shown on the fig. 15 . the fi rst sequence leads to fi rst commercialized strobilurin azoxystrobin (1996, amistar®, syngenta) and than to fl uoxastrobin ( 300 ), which structure combines a methoximino 5,6-dihydro-1,4,2-dioxazin-2-yl toxophore (bayer toxofore) with an optimally adjusted side-chain bearing a 6-(2-chlorophenoxy)-5-fl uoro-pyrimidin-4yl-oxy moiety as an essential element. fluoxastrobin ( 300 ) has an advantage as no reorientation of the toxophore is necessary for binding to the target. the sars studies indicate that the fl uorine atom has a benefi cial effect on the phytotoxicity and leaf systemicity. another sequence leads to picoxystrobin (2002, acanto®, syngenta), which has a 6-cf 3 -pyridin-2-yl moiety in its arylalkyl ether side-chain. an indication switch from the fungicidally to acaricidally active strobilurin type with β-methoxyacrylate pharmacophore is achieved by exchange of the 6-cf 3pyridin-2-yl moiety in the arylalkyl ether side-chain of picoxystrobin with a 2-i pro-6-cf 3 -pyrimidin-4-yl moiety to give fl uacrypyrim ( 301 ). fluoxastrobin ( 300 ) was obtained by reaction of compounds 359 and 360 in the presence of k 2 co 3 (scheme 78 ) [ 290 ] . compound 359 was prepared by reaction of 4,5,6-trifl uoropyrimidine ( 358 ) with potassium o -chlorophenolate. in turn, 358 was obtained from 5-chloro-4,6-difl uoropyrimidine ( 357 ) by reaction with kf. preparation of fluacrypyrim ( 301 ) started with reaction of o -isopropylisourea hydrochloride and ethyl trifl uoroacetoacetate to give pyrimidine 361 (scheme 80 ) [ 292 ] . alkylation of 361 with bromide 362 (or the corresponding chloride 363 [ 293 , 294 ] ) in the presence of alkali or k 2 co 3 gave fluacrypyrim. cu 2 o-catalyzed alkylation of 361 was also developed for the synthesis of 300 [ 295 ] . compounds 362 and 363 were obtained using several closely related methods. in particular, ticl 4 -mediated reaction of chloride 366 and methyl orthoformate was used to obtain 363 (scheme 81 ) [ 293 , 294 ] . alternatively, 366 reacted with methyl formate in the presence of ticl 4 -et 3 n to give 367 , which was treated with p -toluenesulfonic acid in methanol to give 363 . yet another method included reaction of 367 with methyl orthoformate to give 368 , which was transformed to 363 upon treatment with methanesulfonic acid. another approach to fluacrypyrim ( 301 ) commenced from pyrimidine derivative 364 , which reacted with methyl formate in the presence of ticl 4 -et 3 [ 293 , 294 ] . methylation of 365 using methyl orthoformate or dimethyl sulphate and alkali led to the formation of 301 . the last pesticide from this section is flufenerim (flumfen® 302 ), which is under development by ube industries as an insecticide. it is reported to control aphids, whitefl ies, and cotton leafworm, but has no activity against thrips [ 296 ] . since flufenerim is chemically related to pyrimidifen (miteclean® 369 ) (fig. 16 ) , it was initially believed to have similar mechanism of action, i.e. inhibition of the mitochondrial electron transport of nadh dehydrogenase (nadh: ubiquinone oxidoreductase, complex i) -an enzyme which transfers electrons from nadh to ubiquinone and hence opens the electron transport chain cascade. nevertheless, it was shown that 302 reduced activity of acetylcholinesterase -an effect which possibly can be addressed to interaction with other systems [ 297 ] . flufenerim ( 302 ) was prepared from 4,5-dichloro-6-ethylpyrimidine ( 347 ) (scheme 82 ) [ 298 ] . compound 370 was chlorinated with chlorine gas; the product 371 thus obtained was subjected to nucleophilic substitution with acok to give acetate 372 , which upon hydrolysis and subsequent reaction with diethylaminosulphur trifl uoride (dast) gave fl uoride 374 . finally, reaction of 374 with amine 375 led to the formation of flufenerim ( 302 ). since discovery of the fi rst fl uorinated diazine -antineoplastic agent 5-fl uorouracil more than 20 compounds from the class were introduced into the market. undoubtedly the success was achieved due to joint progress of medicinal chemistry, agrochemistry as well as synthetic methods of heterocyclic and fl uoroorganic chemistry. the continued progresses in these fi elds of science allow us to predict that the number of fl uorine containing diazines as drugs or agrochemicals on the market will be increased. recent trends in using of perfl uorinated diazines as core scaffold for the synthesis of a diverse array of polysubstituted fl uorinated diazines for hts increases probability of these compounds as potential hits and leads. also the new methodologies of direct introduction of fl uorinated substituent, like baran approach, continue to appear facilitating further investigation. moreover in the chemical space covered by fl uorinated diazines remains "white spots". thus diazine scaffold decorated by important in med and agrochem fl uorinated fragments such as -chf 2 , -ch 2 cf 3 , -ocf 3 , -scf 3 , -sf 5 not investigated because the synthetic chemistry of these compounds on development phase or not developed at all. therefore the comprehensive investigations in the fi eld of fl uorinated diazines still are interesting both for academic and industrial scientists. fluorine in medicinal chemistry fluorine in medicinal chemistry: a century of progress and a 60-year retrospective of selected highlights elsevier mdl, version 2012.1 fluorinated pyrimidines, a new class of tumourinhibitory compounds united states food and drug administration www.fda.gov studies in 2-acetylaminofl uorene carcinogenesis. 3. the utilization of uracil-2-c-14 by preneoplastic rat liver and rat hepatoma -fl uorouracil: mechanisms of action and clinical strategies 5-fl uorouracil: forty-plus and still ticking. a review of its preclinical and clinical development medicinal chemistry of anticancer drugs cancer drug discovery and development: combination cancer therapy: modulators and potentiators fluorouracil: biochemistry and pharmacology the catalytic mechanism and structure of thymidylate synthase the synthesis of 5-fl uoropyrimidines process for fl uorinating uracil and derivatives thereof process for production of 5-fl uorouracil and its derivatives process for producing 5-fl uorouracil preparation of thymidine and deoxyfl uorouridine, and intermediates therefor über die reaktion von uracil und seinen nucleosiden mit elementarem fluor reaction of acetyl hypofl uorite with pyrimidines. part 3. synthesis, stereochemistry, and properties of 5-fl uoro-5,6-dihydropyrimidine nucleosides nucleic acid related compounds. 21. direct fl uorination of uracil and cytosine bases and nucleosides using trifl uoromethyl hypofl uorite. mechanism, stereochemistry, and synthetic applications nucleic acid related compounds. iii. facile synthesis of 5-fl uorouracil bases and nucleosides by direct fl uorination clinical colorectal cancer: ode to 5-fl uorouracil 5-fl uorouracil derivatives: a patent review verfahren zur herstellung von n1-(2′tetrahydrofuryl)-und n1-(2′tetrahydropyranyl)-derivaten 5-substituierter urazile und deren alkalimetallsalzen ussr 2218 n1-(2′-furanidyl)-derivatives of 5-substituted uracils involvement of microsomal cytochrome p450 and cytosolic thymidine phosphorylase in 5-fl uorouracil formation from tegafur in human liver formation pathways of γ-butyrolactone from the furan ring of tegafur during its conversion to 5-fl uorouracil comparison of gastrointestinal toxicity of 5-fu derivatives a phase i safety and pharmacokinetic study of ogt 719 in patients with liver cancerю a novel, orally administered nucleoside analogue, ogt 719, inhibits the liver invasive growth of a human colorectal tumor, c170hm2 antitumor effect and tumor level of 5-fl uoro-2′-deoxyuridylate following oral administration of tetradecyl 2′-deoxy-5-fl uoro-5′-uridylate antitumor activity of t-506, a novel synthetic fudr derivative, on murine colon cancer and its hepatic metastasis synthesis and antitumor activity of 5-fl uorouracil derivatives sensitivity to six antitumor drugs differs between primary and metastatic liver cancers metabolism of 1-hexylcarbamoyl-5-fl uorouracil (hcfu), a new antitumour agent, in rats, rabbits and dogs low dose 1-hyxylcarbamoyl-5-fl uorouracil (hcfu) recommended for cirrhotic patients with hepatocellular carcinoma phase iii clinical study of a new anticancer drug atofl uding atofl uding n(3)-o-toluyl-fl uorouracil inhibits human hepatocellular carcinoma cell growth via sustained release of 5-fu a phase ii trial of a new 5-fl uorouracil derivative, bof-a2 (emitefur), for patients with advanced gastric cancer effi cacy of a new 5-fl uorouracil derivative, bof-a2, in advanced non-small cell lung cancer. a multi-center phase ii study phase i assessment of the pharmacokinetics, metabolism, and safety of emitefur in patients with refractory solid tumors phase i clinical trial of 5-fl uoro-pyrimidinone (5fp), an oral prodrug of 5-fl uorouracil (5fu) 5-fl uoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fl uorouracil capecitabine preclinical studies: from discovery to translational research metabolism of capecitabine, an oral fl uorouracil prodrug: 19 f nmr studies in animal models and human urine process for producing 5-fl uorouracil derivative with a calcium chloride catalyst process for the preparation of 1-(2-tetrahydrofuryl)-5-fl uorouracil method for the preparation of derivatives of uracil process for the preparation of n.sup.1-(2′-furanidyl)-5-fl uoro-uracil synthesis of tetrahydro-2-furyl derivatives of 5-substituted uracils liepin'sh e (1981) nitrogen-containing organosilicon compounds c. reaction of 5-fl uoro-2,4-bis-o-(trimethylsilyl)uracil with 2,3-dihydrofuran analogs of pyrimidine nucleosides i. n,(atetrahydrofuryl) derivatives of natural pyrimidine bases and their antimetabolites the synthesis of 1 (tetrahydro-2-furanyl)-5-fl uorouracil (ftorafur) via direct fl uorination synthetic studies on chemotherapeutics. ii. synthesis of phenyl-substituted 1,4-dihydro-4-oxonicotinic acid derivatives. [studies on the syntheses of heterocyclic compounds. part 704 analogs of pyrimidine nucleosides facile synthesis of tetrahydro-2-furylated pyrimidines and purines using a new catalyst of cesium chloride a novel synthesis of 5-fl uorouracil derivatives having oxacycloalkane moieties synthesis of 1-(tetrahydro-2-furanyl)-5-fl uorouracil (ftorafur) via direct fl uorination studies on fl uorinated pyrimidines. i. a new method of synthesizing 5-fl uorouracil and its derivatives 5′-halogeno-2′,3′-cyclic sulphite isomers in the preparation of 5′-halogeno nucleosides. synthesis of 5′-deoxyuridine and 5′-deoxy-5-fl uorouridine 5′-halogeno-2′,3′-sulphites in the synthesis of 2′,5′-dideoxy-5-fl uorouridine and related analogues fluorinated pyrimidine nucleosides. 3. synthesis and antitumor activity of a series of 5′-deoxy-5-fl uoropyrimidine nucleosides 149. stereospezifi sche synthese des cancerostatikums 5'-desoxy-5-fl uor-uridin (5-dfur) und seiner 5′-deuterierten derivate rapid continuous synthesis of 5-deoxyribonucleosides in fl ow via brønsted acid catalyzed glycosylation preparation, antibacterial effects and enzymatic degradation of 5-fl uorouracil nucleosides reaction of acetylhypofl uorite with pyrimidines. part 3. synthesis, stereochemistry and properties of 5-fl uor-5,6-dihydropyrimidine-nucleosides gb 080491 chem abstr synthesis and antitumor activity of phagocytic conjugates of 5-fl uorouracil with albumin synthesis of f-18 labeled nucleoside analogues therapeutic compounds with pyrimidine base 5-fl uoro-2′-deoxyuridine derivatives and a process for the preparation thereof novel 5-fl uoro-2-deoxyuridine derivatives and salts thereof, process for producing the same, and antitumor agents containing the same 5-fl uorouracil derivatives. i. the synthesis of 1-carbamoyl-5-fl uorouracils 1-carbamoyl-5-fl uorouracil derivatives studies on the syntheses of heterocyclic compounds. 845. studies on the synthesis of chemotherapeutics. 10. synthesis and antitumor activity of n -acyl-and n -(alkoxycarbonyl)-5-fl uorouracil derivatives synthesis of 5-fl uorouracil derivatives containing an inhibitor of 5-fl uorouracil degradation 5-fl uorouracil derivatives 5-fl uorouracil derivatives derivatives of pyrimidine some derivatives of 5-fl uoropyrimidine antitumor properties of 2(1 h )-pyrimidinone riboside (zebularine) and its fl uorinated analogs synthesen mit substituierten malondialdehyden, xix. darstellung fl uorsubstituierter carbo-und heterocyclen verfahren zur herstellung von 5-fl uorpyrimidin-2-on und seinen n -1-substituierten derivaten a simple synthesis of 5-fl uoro-2-pyrimidinone and its n 1 -substituted derivatives n4-(substituted-oxycarbonyl)-5′-deoxy-5-fl uorocytidine compounds, compositions and methods of using same the design and synthesis of a new tumor-selective fl uoropyrimidine carbamate, capecitabine processes related to making capecitabine process for the preparation of capecitabine combination therapy for the treatment of cancer using cox-2 inhibitors and dual inhibitors of egfr synthesis and biological activity evaluation of cytidine-5′-deoxy-5-fl uoro-n-[(alkoxy/aryloxy)] carbonyl-cyclic 2′,3′-carbonates process for preparing capecitabine methods for preparing capecitabine and beta-anomer-rich trialkyl carbonate compound used therein a novel combination antimetabolite, tas-102, exhibits antitumor activity in fu-resistant human cancer cells through a mechanism involving ftd incorporation in dna trifl uorothymidine exhibits potent antitumor activity via the induction of dna double-strand breaks thymidine phosphorylase. substrate specifi city for 5-substituted 2′-deoxyuridines potentiation of the antitumor activity of α, α, α-trifl uorothymidine by the co-administration of an inhibitor of thymidine phosphorylase at a suitable molar ratio in vivo antitumor activity of ftc-092, a masked 5-trifl uoromethyl-2′-deoxyuridine derivative syntheses of 5-trifl uoromethyluracil and 5-trifl uoromethyl-2′-deoxyuridine alternative synthesis of 2′-deoxy-5-(trifl uoromethyl)-uridine and the α-anomer thereof the synthesis of 2′-deoxy-5-trifl uoromethyluridine utilizing a coupling reaction direct perfl uoroalkylation including trifl uoromethylation of aromatics with perfl uoro carboxylic acids mediated by xenon difl uoride studies on organic fl uorine compounds. part 35. trifl uoromethylation of pyrimidine and purine nucleosides with trifl uoromethyl-copper complex studies on antitumor agents. 8. antitumor activities of o -alkyl derivatives of 2′-deoxy-5-(trifl uoromethyl)uridine and 2′-deoxy-5-fl uorouridine studies on antitumor agents. ix. synthesis of 3′-o -benzyl-2′-deoxy-5-trifl uoromethyluridine us 4898936 113. clinicaltrials.gov: a service of the u.s. national institutes of health abstract b234: ly2835219, a potent oral inhibitor of the cyclindependent kinases 4 and 6 (cdk4/6) that crosses the blood-brain barrier and demonstrates in vivo activity against intracranial human brain tumor xenografts cyclin-dependent kinase pathways as targets for cancer treatment metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor r406 in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation tyrosine kinase inhibitors as potential drugs for b-cell lymphoid malignancies and autoimmune disorders preclinical characterization of aurora kinase inhibitor r763/as703569 identifi ed through an imagebased phenotypic screen phase i, open-label, multicentre, dose-escalation, pharmacokinetic and pharmacodynamic trial of the oral aurora kinase inhibitor pf-03814735 in advanced solid tumours pf-03814735, an orally bioavailable small molecule aurora kinase inhibitor for cancer therapy the jak2 inhibitor azd1480 potently blocks stat3 signaling and oncogenesis in solid tumors discovery of 5-chloro-n 2-[(1 s )-1-(5-fluoropyrimidin-2-yl)ethyl]-n 4-(5-methyl-1 h -pyrazol-3-yl)pyrimidine-2,4-diamine (azd1480) as a novel inhibitor of the jak/stat pathway pyrimidinediamine compounds for use in the treatment or prevention of autoimmune diseases -aminopyrazole)pyrimidine derivatives for use as tyrosine kinase inhibitors in the treatment of cancer anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv emtricitabine, a new antiretroviral agent with activity against hiv and hepatitis b virus pharmacokinetics of antiretrovirals in pregnant women the 5′-triphosphates of the (-) and (+) enantiomers of cis-5-fl uoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine equally inhibit human immunodefi ciency virus type 1 reverse transcriptase the antihepatitis b virus activities, cytotoxicities, and anabolic profi les of the (-) and (+) enantiomers of cis-5-fl uoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine new nucleoside reverse transcriptase inhibitors for the treatment of hiv infections overview of antiretroviral agents expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodefi ciency virus type 1 inhibition of clinically relevant mutant variants of hiv-1 by quinazolinone nonnucleoside reverse transcriptase inhibitors structural basis for the resilience of efavirenz (dmp-266) to drug resistance mutations in hiv-1 reverse transcriptase characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (s)-6-chloro-4-(cyclopropylethynyl)-4-(trifl uoromethyl)-3,4-dihydro-2(1h)-quinazolinone (dpc 961), in rats. possible formation of an oxirene metabolic intermediate from a disubstituted alkyne asymmetric synthesis and biological evaluation of β-l-(2 r ,5 s )-and α-l-(2 r ,5 r )-1,3-oxathiolane-pyrimidine and -purine nucleosides as potential anti-hiv agents structure-activity relationships of β-d-(2s,5r)-and α-d-(2s,5s)-1,3-oxathiolanyl nucleosides as potential anti-hiv agents method for the synthesis, compositions and use of 2′-deoxy-5-fl uoro-3′-thiacytidine and related compounds intermediates in the synthesis of 1,3-oxathiolane nucleoside enantiomers processes for the diastereoselective synthesis of nucleoside analogues novel process for the preparation of cis -nucleoside derivative design and synthesis of 2′,3′-dideoxy-2′,3′-didehydro-beta-l-cytidine (beta-l-d4c) and 2′,3′-dideoxy 2′,3′-didehydro-beta-l-5-fl uorocytidine (beta-l-fd4c), two exceptionally potent inhibitors of human hepatitis b virus (hbv) and potent inhibitors of human immunodefi ciency virus (hiv) in vitro stereoselective syntheses of β-l-fd4c and β-l-fddc synthesis and biological evaluation of 2′,3′-didehydro-2′,3′-dideoxy-5-fl uorocytidine (d4fc) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against hiv-1 reverse transcriptase synthesis and comparative evaluation of two antiviral agents: β-l-fd4c and β-d-fd4c method for synthesizing beta-l-fl uoro-2′,3′didehydcytidine (β-l-fd4c) method for the synthesis of 2′,3′-dideoxy-2′,3′-didehydronucleosides synthesis of d-d4fc, a biologically active nucleoside via an unprecedented palladium mediated ferrier rearrangement-type glycosidation with an aromatization prone xylo-furanoid glycal a new asymmetric 1,4-addition method: application to the synthesis of the hiv non-nucleoside reverse transcriptase inhibitor dpc 961 general scope of 1,4-diastereoselective additions to a 2(3h)-quinazolinone: practical preparation of hiv therapeutics an effi cient chiral moderator prepared from inexpensive (+)-3-carene: synthesis of the hiv-1 non-nucleoside reverse transcriptase inhibitor dpc 963 nmr spectroscopic investigations of mixed aggregates underlying highly enantioselective 1,2-additions of lithium cyclopropylacetylide to quinazolinones highly enantioselective construction of a chiral tertiary carbon center by alkynylation of a cyclic n-acyl ketimine: an effi cient preparation of hiv therapeutics highly enantioselective construction of a quaternary carbon center of dihydroquinazoline by asymmetric mannich reaction and chiral recognition trifl uridine: a review of its antiviral activity and therapeutic use in the topical treatment of viral eye infections physical and biological consequences of incorporation of antiviral agents into virus dna fluorine-containing diazines in medicinal chemistry and agrochemistry t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections mechanism of action of t-705 against infl uenza virus nitrogen-containing heterocyclic carboxamide derivatives or salts thereof and antiviral agents comprising the same novel pyrazine derivatives or salts thereof, pharmaceutical composition containing the same, and production intermediates thereof organic amine salt of 6-fl uoro-3-hydroxy-2-pyrazinecarbonitrile and method for producing the same method for producing dichloropyrazine derivative antibiotic r&d gets a dose of funding method for producing dichloropyrazine derivative antibiotic activity and characterization of bb-3497, a novel peptide deformylase inhibitor peptide deformylase inhibitors flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions ii pharmacology and clinical use of voriconazole voriconazole: a new triazole antifungal agent voriconazole compared with liposomal amphotericin b for empirical antifungal therapy in patients with neutropenia and persistent fever multiple-triazole-resistant aspergillosis process for the preparation of 5-fl uorocytosine nucleic acid related compounds. 21. direct fl uorination of uracil and cytosine bases and nucleosides using trifl uoromethyl hypofl uorite. mechanism, stereochemistry, and synthetic applications a direct synthesis of 5-fl uorocytosine and its nucleosides using trifl uoromethyl hypofl uorite über synthesen von difl uoraminopyrimidinen process for preparing 5-fl uorocytosine salt triazole antifungal agents novel antifungal 2-aryl-1-(1 h -1,2,4-triazol-1-yl)butan-2-ol derivatives with high activity against process for the preparation of voriconazole process for preparing voriconazole improved process for the preparation of (2 r ,3 s )-2-(2,4-difl uorophenyl)-3-(5-fl uoropyrimidin-4-yl)-1-(1 h -1,2,4-triazol-1-yl)butan-2-ol (voriconazole) improved process for the preparation of (2 r ,3 s )-2-(2,4-difl uorophenyl)-3-(5-fl uoropyrimidin-4-yl)-1-(1 h -1,2,4-triazol-1-yl)butan-2-ol process for the preparation of voriconazole a process for making voriconazole preparation of triazoles by organometallic addition to ketones and intermediates therefor an improved process for the preparation of voriconazole and intermediates thereof process for the production of voriconazole intermediates of voriconazole and preparation method of voriconazole using the same process for preparing voriconazole by using new intermediates a novel process to manufacture (2r,3s)-2-(2,4-difl uorophenyl)-3-(5-fl uoropyrimidin-4-yl)-1-(1h-1,2,4-triazol-1-yl)butan-2-ol section vii. worldwide market introductions the encyclopedia of addictive drugs pharmacological studies on 6-amino-2-fl uoromethyl-3-( o -tolyl)-4(3 h )-quinazolinone (afl oqualone), a new centrally acting muscle relaxant (i) studies on biologically active halogenated compounds. 1. synthesis and central nervous system depressant activity of 2-(fl uoromethyl)-3-aryl-4(3 h )-quinazolinone derivatives discriminative stimulus characteristics of bmy 14802 in the pigeon a role for sigma binding in the antipsychotic profi le of bmy 14802 the sigma ligand bmy-14802 as a potential antipsychotic: evidence from the latent inhibition model in rats bmy 14802, a sigma receptor ligand for the treatment of schizophrenia the sigma-1 antagonist bmy-14802 inhibits l -dopa-induced abnormal involuntary movements by a way-100635-sensitive mechanism in vitro characterization of the selective dopamine d3 receptor antagonist a-437203. 32th annual meeting, society of neuroscience effect of dopamine d3 antagonists on ppi in dba/2j mice or ppi defi cit induced by neonatal ventral hippocampal lesions in rats a double-blind, randomized, placebocontrolled study of the dopamine d3 receptor antagonist abt-925 in patients with acute schizophrenia dopamine d3 receptor antagonism -still a therapeutic option for the treatment of schizophrenia third generation antipsychotic drugs: partial agonism or receptor functional selectivity pharmacology of jnj-37822681, a specifi c and fast-dissociating d2 antagonist for the treatment of schizophrenia a double-blind, randomized, placebo-controlled study with jnj-37822681, a novel, highly selective, fast dissociating d2 receptor antagonist in the treatment of acute exacerbation of schizophrenia agents for treatment of brain ischemia antipsychotic 1-fl uorophenylbutyl-4-(2-pyrimidinyl)piperazine derivatives synthesis and biological characterization of α-(4-fl uorophenyl)-4-(5-fl uoro-2-pyrimidinyl)-1-piperazinebutanol and analogues as potential atypical antipsychotic agents resolution of α-(4-fl uorophenyl)-4-(5-fl uoro-2-pyrimidinyl)-1-piperazinebutanol (bms 181100) and α-(3-chloropropyl)-4-fl uorobenzenemethanol using lipase-catalyzed acetylation or hydrolysis evaluation of the effects of the enantiomers of reduced haloperidol, azaperol, and related 4-amino-1-arylbutanols on dopamine and σ receptors asymmetric hydrogenation of amino ketones using chiral rucl 2 (diphosphine)(1,2-diamine) complexes piperidin-4-yl-pyridazin-3-ylamine derivatives as fast dissociating dopamine 2 receptor antagonists a 1-heteroaryl-4-piperidinyl-methyl pyrrolidinone, bmy 21502, delays the decay of hippocampal synaptic potentiation in vitro effect of bmy 21502 on acquisition of shape discrimination and memory retention in monkey effect of bmy 21502 on classical conditioning of the eyeblink response in young and older rabbits bmy 21502 and piracetam facilitate performance of two-choice win-stay water-escape in normal rats effects of oral bmy 21502 on morris water task performance in 16-18 month old f-344 rats effects of bmy-21502 on anoxia in mice effi cacy and safety of bmy 21502 in alzheimer disease the use of the computerized neuropsychological test battery (cntb) in an effi cacy and safety trial of bmy 21,502 in alzheimer's disease process for large-scale production of bmy 21502 cerebral function enhancing diazinylpiperidine derivatives small molecule blockers of voltage-gates sodium channels molecular determinants of voltage-dependent gating and binding of pore-blocking drugs in transmembrane segment iiis6 of the na + channel α subunit a multicenter, double-blind, randomized, placebo-controlled crossover evaluation of a short course of bw-4030w92 in patients with chronic neuropathic pain discovery of 2-[(2,4-dichlorophenyl)amino]-n -[(tetrahydro-2 h -pyran-4-yl)methyl]-4-(trifl uoromethyl)-5-pyrimidinecarboxamide, a selective cb2 receptor agonist for the treatment of infl ammatory pain a randomized, controlled study to investigate the analgesic effi cacy of single doses of the cannabinoid receptor-2 agonist gw842166, ibuprofen or placebo in patients with acute pain following third molar tooth extraction optically active phenyl pyrimidine derivatives as analgesic agents pyrimidine derivatives and their use as cb2 modulators combination of cb2 modulators and pde4 inhibitors for use in medicine role of spleen tyrosine kinase inhibitors in the management of rheumatoid arthritis of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of syk fostamatinib, a syk inhibitor prodrug for the treatment of infl ammatory diseases the status of fostamatinib in the treatment of rheumatoid arthritis recent advances in non-peptidomimetic dipeptidyl peptidase 4 inhibitors: medicinal chemistry and preclinical aspects effects of ketoconazole and rifampicin on the pharmacokinetics of gemigliptin, a dipeptidyl peptidase-fluorine-containing diazines in medicinal chemistry and agrochemistry iv inhibitor: a crossover drug-drug interaction study in healthy male korean volunteers an update in incretin-based therapy: a focus on dipeptidyl peptidase 4 inhibitors a multicentre, multinational, randomized, placebo-controlled, double-blind, phase 3 trial to evaluate the effi cacy and safety of gemigliptin (lc15-0444) in patients with type 2 diabetes effi cacy and safety of the dipeptidyl peptidase-4 inhibitor gemigliptin compared with sitagliptin added to ongoing metformin therapy in patients with type 2 diabetes inadequately controlled with metformin alone liver fi brosis infl ammation and the pathogenesis of diabetic nephropathy -aminocyclopentanecarboxamides as chemokine receptor modulators dual targeting of ccr2 and ccr5: therapeutic potential for immunologic and cardiovascular diseases chemokine receptor genotype is associated with diabetic nephropathy in japanese with type 2 diabetes dipeptidyl peptidase-iv inhibiting compounds, methods of preparing the same, and pharmaceutical compositions containing the same as an active agent fluorine-containing agrochemicals: an overview of recent developments. in: tressaud a (ed) fluorine and the environment: agrochemicals, archaeology, green chemistry & water agricultural products based on fl uorinated heterocyclic compounds. in: petrov va (ed) fluorinated heterocyclic compounds: synthesis, chemistry, and applications a history of weed control in the united states and canada -a sequel the pesticide book, 6th edn. meisterpro information resources advanced technologies for parasitic weed control protoporphyrinogen oxidase inhibitor: an ideal target for herbicide discovery synthesis and chemistry of agrochemicals v process for the production of 3-aryl-uracils synthesis and chemistry of agrochemicals vi method for producing sulfonic acid diamides pyridazin-3-one derivatives, their use, and intermediates for their production production of pyridazine herbicides herbicidal composition. us 6218338 272 structure and mechanism of inhibition of plant acetohydroxyacid synthase acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids environmental protection agency offi cial site www.epa.gov weed management in peanut ( arachis hypogaea ) with diclosulam preemergence n -arylsulfi limine compounds and their use as catalysts in the preparation of n -arylarylsulfonamide compounds preparation of n -arylarylsulfonamide compounds process for heterocyclic sulfonyl chloride compounds preparation of n-arylarylsulfonamide compounds pyrimidinyl)amino)carbonyl)amino)sulfonyl)benzoic acid methyl ester (primisulfuron) acaricides -biological profi les, effects and uses in modern crop protection review of strobilurin fungicide chemicals lehninger principles of biochemistry recent developments in the mode of action of fungicides mechanisms of resistance to qoi fungicides in phytopathogenic fungi halogen pyrimidines and its use thereof as parasite abatement means -alkoxy-6-trifl uoromethylpyrimidin-4-yl)oxymethylene]phenylacetic acid derivatives, their preparation and intermediate therefor, and use thereof processes for producing acrylic acid derivative processes for producing acrylic acid derivative methods for highly selectively o -alkylating amide compounds with the use of copper salts flufenerim, a novel insecticide acting on diverse insect pests: biological mode of action and biochemical aspects inhibitors of mitochondrial electron transport: acaricides and insecticides 4-phenethylaminopyrimidine derivative, and agricultural and horticultural chemical for controlling noxious organisms containing the same kinetics and metabolism of a new fl uoropyrimidine, 5′-deoxy-5-fl uorouridine studies on tetrahydrofuryl-5-fl uorouracils. iv. mode of reaction of 5-fl uorouracil with 2-acetoxytetrahydrofuran preparation of 2-pyrimidinone and derivatives pyrimidine derivatives for the treatment of abnormal cell growth effects of afl oqualone on vestibular nystagmus and the lateral vestibular nucleus identifi cation and measurement of urinary metabolites of afl oqualone in man biocatalytic synthesis of some chiral drug intermediates by oxidoreductases the nootropic compound bmy-21502 improves spatial learning ability in brain injured rats modifi cation of chemokine pathways and immune cell infi ltration as a novel therapeutic approach in liver infl ammation and fi brosis key: cord-021013-xvc791wx authors: wink, michael title: chapter 1 allelochemical properties or the raison d'être of alkaloids date: 2008-05-30 journal: nan doi: 10.1016/s0099-9598(08)60134-0 sha: doc_id: 21013 cord_uid: xvc791wx this chapter provides evidence that alkaloids are not waste products or functionless molecules as formerly assumed, but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells, which are seen in molecules that mimic endogenous neurotransmitters. the chapter discusses that microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. many herbivores have potent mechanisms to detoxify xenobiotics, which allow the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. it needs more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. organisms. we must also consider that plants compete with other plants (of the same or different species) for light, water, and nutrients. how do plants defend themselves against microorganisms (including bacteria, fungi, and viruses), herbivores, and plants? because plants do rather well in nature, this question has often been overlooked. we are well aware of the defensive strategies of higher animals against microbes and predators (1,2,4,15,17,28,494) . the complex immune system with its cellular and humoral components is a well-studied area in the context of vertebrate-microbe interactions. against predating animals, nature evolved weapons, armor, crypsis, thanatosis, deimatic behavior, aposematism, flight, or defense chemicals (usually called "poisons") (1). it is evident that most of these possibilities are not available for plants with their sessile and "passive" life-style. what then is their evolutionary solution? we can distinguish the following defense mechanisms in plants (3, 4, 7, 15, 17) ; the mechanisms are not independent and may act cooperatively and synergistically. we should be aware that many species have additionally evolved specialized traits in this context. 1 . mechanical protection is provided by thorns, spikes, trichomes, glandular hairs, and stinging hairs (which are often supported by defense chemicals). 2. formation of a thick bark on roots and stems can be considered as a sort of armor, and the presence of hydrophobic cuticular layers as a penetration barrier directed against microbes. b. cell walls are biochemically rather inert with reduced digestibility to many organisms because of their complex cellulose, pectin, and lignin molecules. callose and lignin are often accumulated at the site of infection or wounding (6,7) and form a penetration barrier. c. synthesis of inhibitory proteins (e.g., lectins, protease inhibitors) or enzymes (e.g., chitinase, lysozyme, hydrolases, nucleases) that could degrade microbial cell walls or other microbial constituents would be protective, as well as synthesis of peroxidase and phenolase, which could help inactivate phytotoxins produced by many bacteria and fungi. these proteins are either stored in the vacuole 1. allelochemical properties of alkaloids 3 or are secreted as exoenzymes into the cell wall or the extracellular space (8, 9) . these compounds are thus positioned at an "advanced and strategically important defense position." in addition, storage proteins (of cereals and legumes) are often deficient in particular essential amino acids, such as lysine or methionine. d. as a widely distributed and important trait, secondary metabolites with deterrenthepellent or toxic properties against microorganisms, viruses, and/or herbivores may be produced (2-4, 10-21) . these allelochemicals can be constitutively expressed, they may be activated by wounding (e .g., cyanogenic glycosides, glucosinolates, coumaryl glycosides, alliin, ranunculin), or their de ~o u o synthesis may be induced by elicitors (so-called phytoalexins), infection, or herbivory (4,7, [22] [23] [24] . these products are often synthesized and stored at strategically important sites [epidermal tissues or in cells adjacent to an infection (25,26)] or in plant parts that are especially important for reproduction and survival [flowers, fruits, seeds, bark, roots (2, 3, 15) ]. in animals, we can observe the analogous situation in that many insects and other invertebrates (especially those which are sessile and unprotected by armor), but also some vertebrates, store secondary metabolites for their defense which are often similar in structure to plant allelochemicals (1,4,12,16,17,28-30, [494] [495] [496] 503) . in many instances, the animals have obtained the toxins from their host plants (4, 12,15,17,27-33). hardly any zoologist or ecologist doubts that the principal function of these secondary metabolites (which are often termed ''toxins" in this context) in animals is that of defense against predators or microorganisms (1, 17, 28, [494] [495] [496] . these defense compounds are better known as natural products or secondary metabolites. the latter expression originally meant compounds which are not essential for life, and thus distinct from primary metabolites (34, 35, 38) . unfortunately the term "secondary" has also a pejorative meaning, indicating perhaps that the compounds have no importance for the plant. as discussed in this chapter, just the opposite is true. more than 30,000 natural products have been reported from plants so far (2, 4, 17) . owing to the sophistication in phytochemical methods, such as chromatography (hplc, glc) and spectroscopy (nmr, ms) , new products are reported at rapid intervals. because only 5-10% of all higher plants, which consist of over 300,000 species, have been analyzed phytochemically in some detail, the overall real number of secondary products is certainly very large. it is a common theme that an individual plant does not produce a single natural product, but usually a moderate number of major metabolites and a larger number of minor derivatives. within a taxon secondary metabolites often share a common distribution pattern and are therefore of some importance for phytochemical systematics. classic taxonomy, however, has taken little account of alkaloid distribution: if the same alkaloid is present in two plants of the same taxon, this is interpreted as evidence for a relationship, but its occurrence in two plants of nonrelated taxa is taken as evidence of independent evolution. because secondary metabolites are also derived characters that were selected during evolution, their general value for taxonomy and systematics is certainly smaller than formerly anticipated (233). for many years, secondary metabolites were considered as waste products or otherwise functionless molecules, merely illustrating the biochemical virtuosity of nature (34, 35) . in 1887 and 1888, errera and stahl (92, 308, 504) published the idea that natural products are used by plants for chemical defense against herbivores. since the leading plant physiologists of that time were mostly anti-darwinian, they were not willing to accept the defense argument, which was too much in line with the darwinian concept. therefore, this early defense concept was negated and remained forgotten for nearly 60 years. in 1959, fraenkel(10) reopened the debate in a review article and presented new data supporting the view that secondary metabolites serve as chemical defense compounds against herbivores. during the next three decades this concept was improved experimentally, and we can summarize the present situation as follows although the biological function of many plant-derived secondary metabolites has not been studied experimentally, it is now generally assumed that these compounds are important for the survival and fitness of a plant and that they are not useless waste products, as was suggested earlier in the twentieth century (34, 35) . in many instances, there remains a need to analyze whether a given compound is active against microorganisms (viruses, bacteria, fungi), against herbivores (molluscs, arthropods, vertebrates), or against competing plants (so-called allelopathy). in some instances, additional functions are the attraction of pollinating or seed-dispersing animals, for example, by colored compounds such as betalains (within the centrospermae), anthocyanins, carotenoids, and flavonoids or by fragrances such as terpenes, amines, and aldehydes (15, 17) . physiological roles, such as uv protection [by flavonoids or coumarins (4,17)], nitrogen transport or storage (14, 36, 37) , or photosynthesis (carotenoids), may be an additional function. allelochemicals are often not directed against a single organism, but generally against a variety of potential enemies, or they may combine the roles of both deterrents and attractants (e.g., anthocyanins and many essential oils can be attractants in flowers but are also insecticidal and antimicrobial). thus, many natural products have multiple functions, a fact which is easily overlooked since most scientists usually specialize on a narrow range of organisms (i.e., a microbiologist will usually not check whether an antibiotic alkaloid also deters the feeding of caterpillars). to understand all the interactions we need to adopt a holistic, that is, interdisciplinary, approach. it might be argued that the defense hypothesis cannot be valid since most plants, even those with extremely poisonous metabolites (from the human point of view), are nevertheless attacked by pathogens and herbivores. however, we have to understand and accept that chemical defense is not an absolute process. rather, it constitutes a general barrier which will be effective in most circumstances, that is, most potential enemies are repelled or deterred. plants with allelochemicals at the same time represent an ecological niche for potential pathogens and herbivores. during evolution a few organisms have generally been successful in specializing toward that niche (i.e., in a particular toxic plant) in that they found a way to sequester the toxins or become immune to them (14, 15, 32) . this is especially apparent in the largest class of animals, the insects (probably with several million species on earth), which are often highly host plant specific. the number of these "specialists" is exceedingly small for a given plant species as compared to the number of potential enemies that are present in the ecosystem. we can compare this situation with our immune system: it works against the majority of microorganisms but fails toward a few viruses, bacteria, fungi, and protozoa, which have overcome this defense barrier by clever strategies. nobody would call the immune system and antibodies useless because of these few adapted specialists! we should adopt the same argument when we consider plants' defenses by secondary metabolites (2) . since secondary metabolites have evolved in nature as biologically active compounds with particular properties in other organisms, many of them are useful to mankind as pharmaceuticals, fragrances, flavors, colors, stimulants, or pesticides. in addition, many allelochemicals provide interesting lead structures that organic medicinal chemists can develop into new and more active compounds. about 20-30% of higher plants accumulate alkaloids (505,506). the incidence of alkaloid production varies between taxa to some degree; for example, about 60-70% of species of the solanaceae and apocynaceae are 6 michael wink alkaloidal, whereas other families contain few alkaloid-producing species. some alkaloids have a wide distribution in nature: caffeine occurs in the largest number of families, lycorine in the largest number of genera and berberine in the largest number of species. alkaloids are not restricted to higher plants (although they are here most numerous); they are also present in club mosses (lycopodium), horsetails (equisetum), fungi, and animals such as marine worms (e.g., nereidae), bryozoans, insects (e.g., coccinellidae, solenopsidae), amphibians (toads, frogs, salamanders), and fishes. alkaloids thus represent one of the largest groups of natural products, with over 10,000 known compounds at present, and they display an enormous variety of structures, which is due to the fact that several different precursors find their way into alkaloid skeletons, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan (38) (39) (40) . in addition, part of the alkaloid molecule can be derived from other pathways, such as the terpenoid pathway, or from carbohydrates ( [38] [39] [40] . whereas the structure elucidation of alkaloids and the exploration of alkaloid biosynthetic pathways have always commanded much attention, there are relatively few experimental data on the ecological function of alkaloids. this is the more surprising since alkaloids are known for their toxic and pharmacological properties and many are potent pharmaceuticals. alkaloids were long considered to be waste products [even by eminent alkaloid researchers such as w. 0. james and kurt mothes ( 3 4 3 , 505,526)l. because nitrogen is a limiting nutrient for most plants, a nitrogenous waste product would be a priori unlikely. the waste product argument probably came from animal physiology: carnivorous animals take up relative large amounts of proteins and nucleic acids, containing more nitrogen than needed for metabolism, which is consequently eliminated as uric acid or urea. a similar situation or need, however, is not applicable for plants. in fact, many plants remobilize their nitrogenous natural products (including alkaloids) from senescing organs such as old leaves (2,37,506). if alkaloids were waste products, we would expect the opposite, namely, accumulation in old organs which are shed. on the other hand, the alkaloids produced by animals were never considered to be waste products by zoologists, but rather regarded as defense chemicals (16,28,494496) . thus, the more plausible hypothesis is that alkaloids of plants, microorganisms, and animals, like other allelochemicals, serve as defense compounds. this idea is intuitively straightforward, because many alkaloids are known as strong poisons for animals and homo sapiens. as a prerequisite for an alkaloid to serve as a chemical defense compound we should demand the following criteria. (1) the alkaloid should have significant effects against microbes and/or animals in bioassays. 7 (2) the compounds should be present in the plant at concentrations that are of the same order (or, better, even higher) as those determined in the bioassays. (3) the compound should be present in the plant at the right time and the right place. (4) evidence should be provided that a particular compound is indeed important for the fitness of a plant. although more than 10,000 alkaloids are known, only few (-2-5%) have been analyzed for biochemical properties, and even fewer for their ecophysiological roles. in most phytochemical studies only the structures of alkaloids have been elucidated, so that often no information is available on their concentrations in the different parts and through the ontogenetic development of a plant, or on their biological activities. furthermore, the corresponding studies were usually designed to find useful medicinal or sometimes agricultural applications of alkaloids, not to elucidate their evolutionary or ecological functions. these objections have to be kept in mind, because an alkaloid is sometimes termed "inactive" in the literature, which usually means less active than a standard compound already established as a medicinal compound (such as penicillins in antimicrobial screenings). in many medicinal experiments relatively low doses are applied because of the toxic properties of many alkaloids. if the same compound would have been tested at relevant (which normally means elevated) concentrations that are present in the plant, an ecologically relevant activity might have been detected. another restriction is that the activities of alkaloids have been tested with organisms that are sometimes irrelevant for plants but medicinally important. however, if a compound is active against escherichia coli, it is likely that is is also active against other gram-negative and plant-relevant bacteria. nevertheless, most of the data obtained in these studies (tables i-viii ) provide important information which at present permits extrapolation to the function of alkaloids in plants. in this chapter the focus is on the biological activity of alkaloids (the information available on the pharmacological properties of alkaloids is mostly excluded), and we try to discuss these data from an ecological perspective. in the following, the possible functions of alkaloids in plant-animal, plant-plant, and plant-microbe interactions are discussed in more detail. it is nearly impossible to cover the literature exhaustively. therefore, an overview of the allelochemical properties of alkaloids is presented. because of the large amount of data (literature up to 1990 is included), the selection of examples must remain subjective to some degree. nevertheless, the author would be grateful to receive information or publications about relevant omissions. because homo supiens and domestic animals are to some degree herbivores, a large body of empirical knowledge has accumulated on the toxic properties of alkaloids (tables i through v) and alkaloid-containing plants. previously, the toxic properties of alkaloids in vertebrates was part of the definition (as a common denominator) for this group of natural products (38, 39) . in the following, the toxic or adverse effects of alkaloids are separately discussed for invertebrates (mainly insects) and vertebrates. among the invertebrates, insects have been extremely successful from the evolutionary point of view, and they form the largest class of organisms on our planet as far as the number of both individuals and species is concerned. entomologists estimate that the number of insects is at least 1 million, but tropical rain forests may harbor up to 20-30 million species, many of which are still unknown and, owing to the fast extinction of this ecosystem, will probably also disappear without having been discovered and studied by scientists. most insects are herbivores, and adaptation to host plants and their chemistry is often very close and complex ( i ,4,10,14,15, 28-33, 494496,503) . whereas insects rely on plants for food, many plants need insects for pollination and seed dispersal. in the latter context we often find that plants attract insects by chemical means (colors, fragrances, sugars, amino acids). at the same time, other secondary metabolites are employed to discourage the feeding on flowers and seeds. the close association between plants, especially the angiosperms, and insects evolved during the last 200 million years. some scientists have called this phenomenon a "coevolutionary" process, but it has to be recalled that the associations seen today are not necessarily those in which the chemical interactions originally evolved (18,505,506). applications of synthetic insecticides have shown that resistance to these new compounds can occur rapidly, sometimes encompassing only a dozen generations. times can also be much longer. if plant species are introduced to a new continent or island, it usually takes a long time before new pathogens or herbivores become adapted and specialized to this new species. for example, lupinus polyphyllus from north america has a number of specialized herbivores, but is rarely attacked by herbivores in europe. this lupine left its enemies behind when it was transferred to europe three centuries ago. about 10 years ago, however, the north american lupine aphid (macrosiphum albifrons) was introduced to europe accidentally. this aphid is specialized to alkaloid-rich lupines with lupanine as a major alkaloid. at present, this aphid has spread over most of europe and is now colonizing its former host, l. polyphyllus (2, 503) . insect herbivores can be divided into two large groups whose strategies with respect to the plant's defense chemistry differ substantially (15). the polyphagous species can exploit a wide range of host plants, whereas the mono-/oligophagous insects are often specialized on one or a small number of (often systematically related) hosts. polyphagous insects, namely, species which feed on a wide variety of food plants, are usually endowed with fantastic and powerful olfactory receptors (501) that allow the distinction between plants with high or low amounts of "toxins." the receptors also allow insects to ascertain the quality of the essential products present, such as lipids, proteins, or carbohydrates (507). these "generalists," as we can also call this subgroup of herbivores, are usually deterred from feeding on plants which store especially noxious metabolites and select those with less active ones (such as our crop species, where man has bred away many of the secondary metabolites that were originally present; see table xi ). alternatively, they change host plants rapidly and thus avoid intoxication. in addition, most polyphagous species have evolved active detoxification mechanisms, such as microsomal oxidases and glutathione peroxidase, which lead to the rapid detoxification and elimination of dietary secondary products (4,15,17,508). in contrast, mono-and oligophagous species often select their host plants with respect to the composition of the nutrients and secondary metabolites present. for these "specialists" the originally noxious defense compounds are often attractive feeding and oviposition stimulants. these insects either tolerate the natural products or, more often, actively sequester and exploit them for their own defense against predators or for other purposes (1,4,10-12,144 7,28,31,33,494-496). these observations seem to contradict the first statement, that secondary metabolites are primarily defense compounds, and a number of renowned authors have fallen into this logical pit, such as mothes (35) and robinson (505). however, these specialized insects are exceptions to the general rule. for these specialists, the defense chemistry of the host plant is usually not toxic, but they are susceptible to the toxicity of natural toxins from non-host plants (32) . as compared to the enormous number of potential herbivores, the number of adapted monophagous species is usually very small for a particular plant species. quite a number of alkaloids have been tested toward herbivorous insects (table i ). in general it is observed that many alkaloids can act as feeding deterrents at higher concentrations (>i%, w/w). given the choice, insects tend to select a diet with no or only a small dose of alkaloids. also, specialists avoid most "toxins" except those of their host plants. these data indicate that under natural conditions plants with a high content of alkaloids should be safe from most herbivorous insects, with the exception of particular monophagous species or a few very potent polyphagous ones. if insects have no choice or if they are very hungry, the deterrency threshold value is much reduced, and they often feed on a diet with alkaloids that they would normally avoid (15,32). in this case we have the chance to test the toxicity of an ingested alkaloid. if insects do not take up alkaloid-containing food, alkaloid toxicity can be assessed to some degree by topical application or by injection ( table i) . as can be seen from table i a substantial number of alkaloids display significant insect toxicity, including nicotine, piperine, lupine alkaloids, caffeine, gramine, strychnine, berberine, ephedrine, and steroidal alkaloids. only the specialists can tolerate the respective alkaloids. the tobacco hornworm (manduca sexta), for example, can grow on a diet with more than 1% nicotine without any adverse effects. most of the nicotine is either degraded or directly eliminated via the malpighian tubules and in feces (182). because nicotine binds to the acetylcholine (ach) receptor, it is likely that in manduca this receptor has been modified in such a way that ach can still bind, but not nicotine (so-called target site modification). the toxic effects of alkaloids in insects (table i) can be caused by their interference with diverse cellular and intracellular targets. since most mechanisms have not yet been elucidated for insects, this issue is discussed below in the section on vertebrate toxicity (see table iv ). with some caution we can extrapolate to insect toxicity. because homo sapiens and domestic animals are largely herbivores, a voluminous body of information on the adverse effects of secondary metabolites has accumulated over the centuries. many allelochemicals and alkaloids are feeding deterrents for vertebrates, owing to their bitter or pungent taste or bad smell, and instinctively a foul-smelling, bitter, or pungent diet is normally avoided. examples of bitter alkaloids (at least for man) are quinine, strychnine, brucine, and sparteine, and for pungent alkaloids are capsaicin, and piperine. it should be recalled that these taste properties are not identical for all animals. for example, geese, which are obligate herbivores, hardly avoid food with alkaloids or smelly compounds (amines, mercaptoethanol) that man would hardly touch (185). conversely, fragrances that are attractive to us are highly repellent to geese (185). even within a given population taste can differ significantly. it has been observed that a substantial proportion of homo sapiens cannot detect the smell of hcn, whereas others are highly sensitive. furthermore, olfactory sensitivity can differ with age, sex, and hormonal cycles. bitterness varies with the chemical structure of an alkaloid. with the quinolizidine alkaloids (qas) the following scale was assessed for man: mean detection levels are 0.00085% for sparteine, 0.0021% for lupanine, and 0.017% for hydroxylupanine (503). whereas we know a few parameters of olfactory qualities in homo sapiens, often much less or hardly anything is known for most other vertebrates. alkaloids are famous for their toxic properties in vertebrates, and plants that produce alkaloids are often classified by man as poisonous or toxic plants. for a number of alkaloids the respective ld,, values have been determined with laboratory animals, especially mice, but also rats, guinea pigs, cats, rabbits, dogs, or pigeons. table i1 presents an overview for 132 alkaloids, including the very poisonous alkaloids aconitine, coniine, atropine, brucine, curarine, ergocornine, physostigmine, strychnine, colchicine, germerine, veratridine, cytisine, delphinidine, and nicotine. toxicity is usually highest if the alkaloids are applied parenterally [intravenously (i.v.), intraperitoneally (i.p.), and subcutaneously (s.c.)] as compared to oral application [per 0s (p.o.)]. also, some of the alkaloids which are made or stored by animals are strong vertebrate poisons, including batrachotoxin, batrachotoxinin a, anabasine, glomerine, maitotoxin, nereistoxin, palytoxin, saxitoxin, and tetrodotoxin (1, 28, 29, 259) . although the general toxicity of alkaloids differs from species to species, the data in table i1 generally show that many alkaloids are more or less toxic to vertebrates. the toxic effects observed with intact animals has its counterpart in the cytotoxic effect, which has been recorded for nearly 180 alkaloids (table 111 ). these data have been obtained by screening many natural products for anticancer activity. however, an alkaloid that can kill a cancer cell is usually also toxic for "normal" cells. therefore, the data shown in table i11 are another indication of the general toxicity of alkaloids toward animals. because this toxicity applies also for herbivores, the production of alkaloids by plants can certainly be interpreted as a potent antiherbivore mechanism. for a number of alkaloids the mechanisms underlying the toxic effects have already been elucidated in some detail. we can distinguish molecular targets and processes that are important for all cells, such as synthesis of dna, rna, and proteins, replication, transcription, translation, membrane assembly and stability, electron chains, or metabolically important enzymes or proteins including receptors, hormones, and signal compounds (table iv ). in the following we discuss some of these toxic effects. a. cellular targets nucleic acids. dna, the macromolecule which holds all the genetic information for the life and development of an organism, is a highly vulnerable target. it is not surprising that a number of secondary metabolites have been selected during evolution which interact with dna or dnaprocessing enzymes. some alkaloids bind to or intercalate with dna/rna (table iv) and thus affect replication or transcription, or cause mutations, leading to malformations or cancer (table v) : 9-methoxyellipticine, dictamnine, ellipticine, harmane alkaloids, melinone f, quinine and related alkaloids, skimmianine, avicine, berberine, chelerythrine, coptisine, coralyne, fagaronine, nitidine, sanguinarine, pyrrolizidine alkaloids (pas), cycasin, olivacine, etc. many of the intercalating molecules are planar, hydrophobic molecules that fit within the stacks of at and gc base pairs. other alkaloids act at the level of dna and rna polymerases, such as vincristine, vinblastine, avicine, chelilutine, coralyne, fagaronine, nitidine, amanitine, hippeastrine, and lycorine, thus impairing the processes of replication and transcription. whereas these toxins usually cause a rapid reaction, some alkaloids cause long-term effects in vertebrates in that they are mutagenic or carcinogenic (table v) . besides basic data obtained in salmonella or drosophila, there are a few reports which illustrate the potent mutagenic effect of alkaloids on vertebrates. anagyrine, anabasine, and coniine cause "crooked calf disease" if pregnant cows or sheep feed on these alkaloids during the first period of gestation (329,341,348,349,351,352) . the offspring born show strong malformation of the legs. some of the steroid alkaloids (e.g., cyclopamine, jervine, and veratrosine), which are produced by veratrum species, cause the formation of a central large cyclopean eye (329-330, an observation that was probably made by the ancient greeks and thus led to the mythical figure of the cyclops. it is likely that any herbivore which regularly feeds on plants containing these alkaloids will suffer from reduced productivity and reduced fitness in the long term. in effect, the plants which contain these alkaloids are usually avoided by vertebrate herbivores. another long-term effect caused by alkaloids with carcinogenic properties has been discovered only recently (tables iv and v) . the alkaloid aristolochic acid, which is produced by plants of the genus aristolochia, is carcinogenic. the mechanism of action of this alkaloid is believed to be similar to the well-known carcinogen nitrosamine (344,345) , because of its no, group. pyrrolizidine alkaloids and their n-oxides, which are abundantly produced by members of the asteraceae and boraginaceae but also occur in the families apocynaceae, celestraceae, elaeocarpaceae, euphorbiaceae, fabaceae, orchidaceae, poaceae, ranunculaceae, rhizoesterified (425,426) . after oral intake, the n-oxides are reduced by bacteria in the gut. the lipophilic alkaloid base is resorbed and transported to the liver, where it is "detoxified" by microsomal enzymes. as a result, a reactive alkylating agent is generated, which can be considered as a pyrrolopyrrolidine. the alkaloid can then cross-link dna and rna and thus cause mutagenic or carcinogenic effects (especially in the liver) (502). thus, pyrrolizidine alkaloids represent highly evolved and sophisticated antiherbivore compounds, which utilize the widespread and active detoxification system of the vertebrate liver. the pa story is very intriguing, since it shows how ingenious nature was in the "arms race." the herbivores invented detoxifying enzymes, and nature the compound which is activated by this process. a herbivore feeding on pa-containing plants will eventually die, usually without reproducing properly. only those individuals which carefully avoid the respective bitter-tasting plants maintain their fitnes and thus survive. the protection due to pa can easily be seen on meadows, where senecio and other pa-containing plants are usually not taken by cows and sheep, at least as long other food is available. protein biosynthesis is essential for all cells and thus another important target. indeed, a number of alkaloids have already been detected (although few have been studied in this context) that inhibit protein biosynthesis in uitro (table iv) , such as vincristine, vinblastine, emetine, tubulosine, tyramine, sparteine, lupanine and other quinolizidine alkaloids, cryptopleurine, haningtonine, homohamngtonine, haemanthamine, isohamngtonine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrebrine, tylophorine, and tylocrepine. for lupine alkaloids, it was determined that the steps which are inhibited are the loading of acyl-trna with amino acids, as well as the elongation step. the inhibitory activity was strongly expressed in heterologous systems, that is, protein biosynthesis in the producing plants, such as lupines, was not affected (503). electron chains. the respiratory chain and atp synthesis in mitochondria demand the controlled flux of electrons. this target seems to be attacked by ellipticine, pseudane, pseudene, alpinigenine, sanguinarine, tetrahydropalmatine, ch,-(ch2),,-2,6-methyl-piperidines, capsaicin, the hydroxamic acid dimboa, and solenopsine. as mentioned before, however, only a few alkaloids have been evaluated in this context (table v) . biomembranes and transport processes. a cell can operate only when it is enclosed by an intact biomembrane and by a complex compartmentation that provides separated reaction chambers. because biomembranes are impermeable for ions and polar molecules, cells can prevent the uncontrolled efflux of essential metabolites. the controlled flux of these compounds across biomembranes is achieved by specific transport proteins, which can be ion channels, pores, or carrier systems. these complex systems are also targets of many natural products (table iv) . disturbance of membrane stability is achieved by 9-methoxyellipticine, ellipticine, berbamine, cepharanthine, tetrandrine, steroidal alkaloids, irehdiamine, and malouetine. steroidal alkaloids, such as solanine and tomatine, which are present in many members of the solanaceae, can complex with cholesterol and other lipids of biomembranes; cells are thus rendered leaky. cells carefully control the homeostasis of their ion concentrations by the action of ion channels (na+,k+, ca2+ channels) and through na+,k+-atpase and ca2+-atpase. these channels and pumps are involved in signal transduction, active transport processes, and neuronal and neuromuscular signaling. inhibition of transport processes (ion channels, carriers) is achieved by (table iv) acronycine, ervatamine, harmaline, quinine, reserpine, colchicine, nitidine, salsolinol, sanguinarine, stepholidine, caffeine, sparteine, monocrotaline, steroidal alkaloids, aconitine, capsaicine, cassaine, maitoxin, ochratoxin, palytoxin, pumiliotoxin, saxitoxin, solenopsine, and tetrodotoxin. a special class of ion channels in the central nervous system and involved in neuromuscular signal transfer are coupled with receptors of neurotransmitters such as noradrenaline (na), serotonin, dopamine, glycine, and acetylcholine (ach). we can distinguish two types. type 1 is a ligand-gated channel (i.e., a receptor), which is part of an ion-channel complex, such as the nicotinergic ach-receptor. in type 2 the receptor is an integral protein. when a neurotransmitter binds, the receptor changes its conformation and induces a conformational change in an adjacent gprotein molecule, which consists of three subunits. the a subunit then activates the enzyme adenylate cyclase, which in turn produces camp from atp. the camp molecule is a second messenger which activates protein kinases or ion channels directly, which in turn open for milliseconds (e.g., the muscarinergic ach receptor). a number of alkaloids are known whose structures are more or less similar to those of endogenous neurotransmitters. targets can be the receptor itself, the enzymes which deactivate neurotransmitters, or transport processes, which are important for the storage of the neurotransmitters in synaptic vesicles. alkaloids relevant here include (table iv) brucine, ergot alkaloids, eseridine, serotonin, physostigmine, gelsemine, p-carboline alkaloids, strychnine, yohimbine, berberine, bicuculline, bulbocapnine, columbamine, coptisine, coralyne, corlumine, ephedrine, galanthamine, laudanosine, nuciferine, palmatine, papaverine, thebaine, cytisine and other quinolizidine alkaloids, heliotrine, chaconine and other steroidal alkaloids, cocaine, atropine, scopolamine, anabaseine, arecoline, dendrobine, gephyrotoxin, histrionicotoxin, methyllycaconitine, muscarine, nicotine, pilocarpine, psilocin, psilocybin, morphine, mescaline, and reserpine. a number of these alkaloids are known hallucinogens, which certainly decrease the fitness of an herbivore feeding on them regularly. cytoskeleton. many cellular activities, such as motility, endocytosis, exocytosis, and cell division, rely on microfilaments and microtubules. a number of alkaloids have been detected which can interfere with the assembly or disassembly of microtubules (table iv) , namely, vincristine, vinblastine, colchicine, maytansine, maytansinine, and taxol. colchicine, the major alkaloid of colchicum autumnale (liliaceae), inhibits the assembly of microtubules and the mitotic spindle apparatus. as a consequence, chromosomes are no longer separated, leading to polyploidy . whereas animal cells die under these conditions, plant cells maintain their polyploidy, a trait often used in plant breeding because polyploidy leads to bigger plants. because of this antimitotic activity, colchicine has been tested as an anticancer drug; however, it was abandoned because of its general toxicity. the derivative colcemide is less toxic and can be employed in the treatment of certain cancers (312). also, cellular motility is impaired by colchicine; this property is exploited in medicine in the treatment of acute gout, in order to prevent the migration of macrophages to the joints. for normal cells, and thus for herbivores, the negative effects can easily be anticipated, and colchicine is indeed a very toxic alkaloid which is easily resorbed because of its lipophilicity . another group of alkaloids with antimitotic properties are the bisindole alkaloids, such as vinblastine and vincristine, which have been isolated from catharanthus roseus (apocynaceae). these alkaloids also bind to tubulin (312). both alkaloids are very toxic, but are nevertheless important drugs for the treatment of some leukemias. from taxus baccata (taxaceae) the alkaloid taxol has been isolated. taxol also affects the architecture of microtubules in inhibiting their disassembly (322). nonalkaloidal compounds to be mentioned in this context include the lignan podophyllotoxin (312). in conclusion, any alkaloid which impairs the function of microtubules is likely to be toxic, because of their importance for a cell, and, from the point of view of defense, a wellworking and well-shaped molecule. enzyme inhibition. the inhibition of metabolically important enzymes is a wide field that cannot be discussed in full here (see table iv ). briefly, inhibition of camp metabolism (which is important for signal transduction and amplifications in cells), namely, inhibition of adenylate cyclase by anonaine, isoboldine, tetrahydroberberine and inhibition of phosphodiesterase by 1-ethyl-p-carboline, p-carboline-1-propionic acid, papaverine, caffeine, theophylline, and theobromine are some examples. inhibition of hydrolases, such as glucosidase, mannosidase, trehalase, and amylase, is specifically achieved by some alkaloids (table iv) b. action at organ level. whereas the activities mentioned before are more or less directed to molecular targets present in or on cells, there are also some activities that function at the level of organ systems or complete organisms, although, ultimately, they have molecular targets, too. central nervous system and neuromuscular junction. a remarkable number of alkaloids interfere with the metabolism and activity of neurotransmitters in the brain and nerve cells, a fact known to man for a thousand years (table iv) . the cellular interactions have been discussed above. disturbance of neurotransmitter metabolism impairs sensory faculties, smell, vision, or hearing, or they may produce euphoric or hallucinogenic effects. a herbivore that is no longer able to control its movements and senses properly has only a small chance of survival in nature, because it will have accidents (falling from trees, or rocks, or into water) and be killed by predators. thus euphoric and hallucinogenic compounds, which are present in a number of plants, and also in fungi and the skin of certain toads, can be regarded as defense compounds. some individuals of homo sapiens use these drugs just because of their hallucinogenic properties, but here also it is evident that long-term use reduces survival and fitness dramatically. the activity of muscles is controlled by ach and na. it is plausible that an inhibition or activation of neurotransmitter-regulated ion channels will severely influence muscular reactivity and thus the mobility or organ function (heart, blood vessels, lungs, gut) of an animal. in the case of inhibition, muscles will relax; in the case of overstimulation, muscles will be tense or in tetanus, leading to a general paralysis. alkaloids which activate neuromuscular action (so-called parasympathomimetics) include nicotine, arecoline, physostigmine, coniine, cytisine, and sparteine. inhibitory (or parasympatholytic) alkaloids include hyoscyamine and scopolamine, (see above) (312) . skeletal muscles as well as muscle-containing organs, such as lungs, heart, circulatory system, and gut, and the nervous system are certainly very critical targets. the compounds are usually considered to be strong poisons, and it is obvious that they serve as chemical defense compounds against herbivores, since a paralyzed animal is easy prey for predators or, if higher doses are ingested, will die directly (compare ld,, values in table 11 ). inhibition of digestive processes. food uptake can be reduced by a pungent or bitter taste in the first instance, as mentioned earlier. the next step may be the induction of vomiting, diarrhea, or the opposite, constipation, which negatively influences digestion in animals. the ingestion of a number of allelochemicals such as emetine, lobeline, morphine, and many other alkaloids causes these symptoms (312). another mode of interference would be the inhibition of carriers for amino acids, sugars, or lipids, or of digestive enzymes. relevant alkaloids are the polyhydroxyalkaloids, such as swainsonine, deoxynojirimycin, and castanospermine, that inhibit hydrolytic enzymes, such as glucosidase, galactosidase, trehalase (trehalose is a sugar in insects which is hydrolyzed by trehalase), and mannosidase selectively (table iv) . nutrients and xenobiotics (such as secondary metabolites) are transported to the liver after resorption in the intestine. in the liver, the metabolism of carbohydrates, amino acids, and lipids takes place with the subsequent synthesis of proteins and glycogen. the liver is also the main site for detoxification of xenobiotics. lipophilic compounds, which are easily resorbed from the diet, are often hydroxylated and then conjugated with a polar, hydrophilic molecule, such as glucuronic acid, sulfate, or amino acids (312). these conjugates, which are more water soluble, are exported via the blood to the kidney, where they are transported into the urine for elimination. both liver and kidney systems are affected by a variety of secondary metabolites, and the pyrrolizidine alkaloids have been discussed earlier (tables iv and v) . the alkaloids are activated during the detoxification process, and this can lead to liver cancer. also, many other enzyme or metabolic inhibitors (e.g., amanitine), discussed previously, are liver toxins. many alkaloids and other allelochemicals are known for their diuretic activity (312). for an herbivore, an increased diuresis would also mean an augmented elimination of water and essential ions. since na' is already limited in plant food (an antiherbivore device?), long-term exposure to diuretic compounds would reduce the fitness of an herbivore substantially. disturbance of reproduction. quite a number of allelochemicals are known to influence the reproductive system of animals, which ultimately reduces their fitness and numbers. antihormonal effects could be achieved by mimicking the structure of sexual hormones. these effects are not known for alkaloids yet, but have been confirmed for other natural products. estrogenic properties have been reported for coumarins, which di-merize to dicoumarols, and isoflavones (4,17) . insect molting hormones, such as ecdysone, are mimicked by many plant sterols, which include ecdysone itself, such as in the fern polypodium uulgare, or azadirachtin from the neem tree (4,17) . juvenile hormone is mimicked by a number of terpenes, present in some coniferae. spermatogenesis is reduced by gossypol from cottonseed oil (17) . the next target is the gestation process itself. as outlined above, a number of alkaloids are mutagenic and lead to malformation of the offspring or directly to the death of the embryo ( table v) . the last step would be the premature abortion of the embryo. this dramatic activity has been reported for a number of allelochemicals, such as mono-and sesquiterpenes and alkaloids. some alkaloids achieve this by the induction of uterine contraction, such as the ergot and lupine alkaloids (312) . the antireproductive effects are certainly widely distributed, but they often remain unnoticed under natural conditions. nevertheless, they are defense strategies with long-term consequences. blood and circulatory system. all animals need to transport nutrients, hormones, ions, signal compounds, and gas between the different organs of the body, which is achieved by higher animals through blood in the circulatory system. inhibitors of the driving force for this process, the heart muscle, have already been discussed. however, the synthesis of red blood cells is also vulnerable and can be inhibited by antimitotic alkaloids such as vinblastine or colchicine (312) . some allelochemicals have hemolytic properties, such as saponins. if resorbed, these compounds complex membrane sterols and make the cells leaky. steroidal alkaloids from solanum or veratrum species display this sort of activity as well as influencing ion channels (table iv) . allergenic effects. a number of secondary metabolites influence the immune system of animals, such as coumarins, furanocoumarins, hypericin, and helenalin. common to these compounds is a strong allergenic effect on those parts of the skin or mucosa that have come into contact with the compounds (4,17,312) . activation or repression of the immune response is certainly a target that was selected during evolution as an antiherbivore strategy. the function of alkaloids in this context is hardly known. this selection of alkaloid activities, though far from complete, clearly shows that many alkaloids inhibit central processes at the cellular, organ, or organismal level, an important requisite for a chemical defense compound. however, most of the potential targets for the 10,000 alkaloids known at present remain to be established. if no activity has been reported, it often means that nobody looked into this question scientifically, and not that a particular alkaloid is without a certain biological property. summarizing this section, it is safe to assume that most alkaloids can affect animals and thus herbivores significantly. dead plants easily rot due to the action of bacteria and fungi, whereas metabolically active, intact plants are usually healthy and do not decay (7) . how is this achieved? the aerial organs of terrestrial plants have epidermal cells that are covered by a more or less thick cuticle, which consists of waxes, alkanes, and other lipophilic natural products (4,7) . this cuticle layer is water repellent and chemically rather inert, and it thus constitutes an important penetration barrier for most bacteria and fungi. in perennial plants and in roots we find another variation of this principle in that plants often form resistant bark tissues. the only way for microbes to enter a healthy plant is via the stomata or at sites of injury, inflicted by herbivory, wind, or other accidents. at the site of wounding, plants often accumulate suberin, lignin, callose, gums, or other resinous substances which close off the respective areas (4,17) . in addition, antimicrobial agents are produced such as lysozyme and chitinase, lytic enzymes stored in the vacuole which can degrade bacterial and fungal cell walls, protease inhibitors which can inhibit microbial proteases, or secondary metabolites with antimicrobial activity. secondary metabolites have been routinely screened for antimicrobial activities by many researchers, since the corresponding assays are relatively easy to perform. these studies have usually been directed toward a pharmaceutical application, and they often employ the routine methods for screening microbial or fungal antibiotics. it may happen that these tests do not detect an antibacterial activity of a compound because the wrong test species or a nonrelevant concentration was assayed. in the pharmaceutical context we search for very active compounds which can be employed at low concentrations. therefore, the higher concentrations, which would be more meaningful ecologically, are often not tested. these precautions have to be kept in mind when screening the literature for data on the antimicrobial activity of alkaloids. secondary compounds known for their antimicrobial activity include many phenolics (e.g., flavonoids, isoflavones, and simple phenolics), glucosinolates, nonproteinogenic amino acids, cyanogenic glycosides, acids, aldehydes, saponins, triterpenes, mono-and disesquiterpenes, and last but not least, alkaloids (4,17,42,149,322) . in table vi 183 alkaloids are tabulated for which antibacterial activities have been detected. the alkaloids usually affect more gram-positive than gram-negative bacteria. especially well represented are alkaloids which 3'-hydroxytabernamine 16hydroxytetrahydrosecamine 10 tetrandrine thalicarpine thalicerbine thalidasine thalidezine thaliglucinone thalistine thalistyline thalmelatine thalmirabine thalphenine thalrugosaminine thalrugosidine thalrugosine tubocurarine 3 ( i +. active; -, no activity observed in the concentration range tested (many alkaloids were only assayed in low concentrations as microbial antibiotics); ad, agar diffusion, al, agar dilution; bg, biogram; ld, liquid culture; mic, minimal inhibitory concentration; pd, paper disk; sp, suspension; tlc, tlc disk test according to wolters and eilert (95) . if more than one value is given, the data refer to different bacterial species tested. derive from tryptophan (indole alkaloids) and phenylalaninehyrosine, which may be due to the fact that these alkaloids have obtained considerable scientific attention since the discovery of many medicinally important compounds within these groups (42,50,59,60,63,68,75-84) . some of these alkaloids are highly antibiotic, with similar activities as fungal antibiotics, namely, cinchophylline (69), dictamnine ( 9 3 , fagarine (95), stemmadine (70), yuehchukene (71), liriodenine ( 8 3 , lysicamine (82), oxonantenine (82), sanguinarine (87), solacasine (50,92) , rutacridone epoxide (95), tryptanthrine (i@#), and tuberin (107,108) (table vi) . in many instances, when alkaloids are assessed for their antibacterial activity, they are often also tested for antifungal properties. usually yeasts and candidu are used as test organisms (table vii) . table vii lists ( i 19,121), thaliglucinone (79), demissidine (126,127), solacasine (92), soladulcidine (126,127), solasodine (26,127)tidine (126,127), tomatine (42,126) , verazine (124), cryptopleurine (133) hydroxyrutacridone epoxide ( 9 3 , tryptanthrine (104), and tuberin (107) . whereas the mode of action and targets of antibiotics of fungal and bacterial origin have been elucidated in many instances (see table iv ), relevant information for plant-derived compounds is scant. however, the molecular targets of some alkaloids have been determined at the general level, but not specifically for bacterial or fungal systems (table iv) that may be responsible for the antibiotic effects observed. the following interactions of alkaloids having antimicrobial properties with molecular targets of bacterial or fungal cells are likely (compare tables vi and vii with tables iv and v) . protein biosynthesis in ribosomes is affected by sparteine (56,423, lupanine, angustifoline, 13-tigloyloxylupanine, and 13hydroxylupanine (56,98,99,417,421,422) . intercalation or binding to dna is influenced by fagaronine, dictamnine (367), harman alkaloids (376,378) [binding to dna is light dependent (66)], berberine (396-3981, chelerythrine (400), and sanguinarine (400,409) ; these compounds may thus inhibit important processes such as dna replication and rna transcription that are also vital for microorganisms. the stability of biomembranes may be disturbed by cepharanthine, tetrandrine, and steroidal alkaloids such as solamargine ( 4 3 3 , solanine (430,432,433), and solasonine (435) , thus leading to an uncontrolled flux of metabolites and ions into microbial cells. inhibition of metabolically important enzymes is affected by berberine (399), chelerythrine (259,401), chelidonine (402), palmatine (399), sanguinarine (143,259), solacongestidine (434) , and papaverine. in contrast to antibiotics of microbial origin that could be classified as alkaloids from a chemical point of view in many instances, and which often interfere with the biosynthesis or maintenance of the cell wall (murein) (table iv) , such an interaction has not been described for plantderived compounds. since this topic has not been studied in detail it remains open whether this complex is another target for alkaloids. we can distinguish between secondary metabolites that are already present prior to an attack or wounding, so-called constitutive compounds, and others that are induced by these processes and made de now. inducing agents, which have been termed "elicitors" by phytopathologists, can be cell wall fragments of microbes, the plant itself, or many other chemical constituents (4,17,22-24) . the induced compounds are called "phytoalexins," which is merely a functional term, since these compounds often do not differ in structure from constitutive natural products. in another way this term is misleading, since it implies that the induced compound is only active in plant-microbe interactions, whereas in reality it often has multiple functions that include antimicrobial and antiherbivoral properties (see below). many of the antimicrobial alkaloids found are constitutively expressed and accumulated, that is, they are already present before an infection. using plant cell cultures, it was observed that some cultures start to produce new secondary metabolites when challenged with bacterial or fungal cell walls, culture fluids, or other chemical factors (4,17,22-24) . among the compounds found to be inducible are alkaloids such as sanguinarine and hydroxyrutacridone epoxide (see table xi ). quinolizidine alkaloids display some antimicrobial properties, besides their main role in antiherbivore defense (503) (see table i ). on wounding, qa production is enhanced, thus increasing the already high alkaloid concentration in the plant; in other words, the antimicrobial and herbivoral effect is further amplified (table xi) (2,184,503) . the reactions leading to the induction and accumulation of phytoalexins with phenolic structures have been studied in molecular detail (4,17,22-24) . these studies revealed that plants can detect and react rapidly to environmental problems, such as wounding or infection: within 20 min of elicitation, mrnas coding for enzymes that catalyze the reactions leading to the respective defense compounds are increasingly generated, leading to the accumulation of the respective enzymes and consequently the production of the secondary metabolites (4,17,22-24) . similar processes are likely for alkaloids, but so far the mechanisms have not been elucidated. we assume that a substantial number of the 10,000alkaloids have antimicrobial properties (which remain to be tested in most cases) that are directed against the ubiquitous and generalist microbes which have not table vi . if a range is given, the first value gives a 10% inhibition, the second value a 100% inhibition. specialized on a particular host plant. however, alkaloid production does not necessarily have to be involved with antimicrobial defense. for example, phytophthora or fusarium will attack alkaloid-rich plants of nicotiana, solanum esculentum, and s . tuberosum. cladosporium and fusarium can develop in nutrient-containing media enriched with alkaloids, and aspergillus niger can utilize alkaloids as a nitrogen source (506). in addition, most plant species are known to be parasitized or infected by at least a few specialized bacteria or fungi which form close, often symbiotic, associations. in these circumstances an antimicrobial effect expected from the secondary metabolites present in the plant can often no longer be observed. we suggest that these specialists have adapted to the chemistry of their host plants. mechanisms may include inhibition of biosynthesis of the respective compounds, degradation of the products, or alteration of the target sites, which are then no longer sensitive toward a given compound (so-called target site modification). these mechanisms need to be established for most of the microbial specialists living on alkaloid-producing plants. some associations between plants and fungi are symbiotic in nature, such as rhizobia in root nodules of legumes or microrhizal fungi in many species. in lupines, nitrogen-fixing rhizobia are present both in alkaloid-rich and alkaloid-free plants. they must therefore be able to tolerate the alkaloids, which are also present in the root. alkaloid production in lupines is more or less unaffected whether or not the plants harbor rhizobia (185,506) . an ecologically important symbiosis between plants and fungi can be observed in fungal species that produce ergot alkaloids. graminaceous species that are infected by ergot suffer much less from herbivory because of the strong antiherbivoral alkaloids produced by the fungi (4). a similar relationship may occur for other fungal species of plants, many of which produce secondary metabolites possessing animal toxicity. from the pharmaceutical point of view, few alkaloids are interesting as antibiotics, because many are highly toxic to vertebrates (tables i1 and 111 ). since many alkaloids are antibacterial and antifungal (tables vi and vii) and are present in plants at relatively high concentrations (section iila), it seems likely that from an ecological perspective alkaloids, besides their prominant role in antiherbivore strategies, may play an important role also in the defense against microbial infections. it should be recalled that even alkaloid-producing plants synthesize antimicrobial proteins, such as chitinase and lysozyme, and other antimicrobial secondary products, such as simple phenolics, flavonoids, anthocyanins, saponins, and terpenes (2-4,7) . a cooperative, or even synergistic, process could thus be operating. c. antiviral properties plants, like animals, are hosts for a substantial number of viruses, which are often transmitted by sucking insects such as aphids and bugs (heteroptera). resistance to viral infection can be achieved either by biochemical mechanisms that inhibit viral development and multiplication or by warding off vectors such as aphids in the first place. the assessment of antiviral activity is relatively difficult. as a result, only a few investigators have studied the influence of alkaloids on virus multiplication. nevertheless, at least 45 alkaloids have been reported with antiviral properties (table viii) . only sparteine (527) and cinchonidine (142) have been tested for antiviral activities against a plant virus, the potato x virus. all other evidence for antiviral activities (table viii) table viii are difficult to interpret at present. polyhydroxy alkaloids, such as swainsonine, can block the action of endoplasmic reticulum-and golgi-localized glucosidases and mannosidases, which are important for the posttranslational trimming of viral envelope proteins. because alkaloids often deter the feeding of insects, such as aphids and bugs (table i ), viral infection rates may be reduced in alkaloid-rich plants. such a correlation exists for alkaloid-rich lupines (so-called bitter 141 109 109 147 148 147 147 148 146 147 109 147 147 148 147 148 150 150 150 150 lupines) and low-alkaloid varieties (the so-called sweet lupines) (see table xii) . plants often compete with other plants, of either the same or different species, for space, light, water, and nutrients. this phenomenon can be intuitively understood when the flora of deserts or semideserts is analyzed, where resources are limited and thus competition intense (4,17,498-500) . a number of biological mechanisms have been described, such as temporal spacing of the vegetation period in which some species flower at an earlier season, when others are still dormant or ungerminated. it was observed by molisch in 1937 (497) that plants can also influence each other by their constituent natural products, and he coined the term "allelopathy" for this process. secondary products are often excreted by the root or rhizosphere to the surrounding soil, or they are leached from the surface of intact leaves or from decaying dead leaves by rain (4,17) . both processes will increase the concentration of allelochemicals in the soil surrounding a plant, where the germination of a potential competitor may occur. allelopathy, namely, the inhibition of germination or of the growth of a seedling or plant by natural products, is well documented at the level of controlled in v i m experiments (4,17,19,497-500) , but how it operates in ecosystems is still often a matter of controversy. it is argued, for example, that soil contains a wide variety of microorganisms which can degrade most organic compounds. thus allelochemicals might never reach concentrations high enough to be allelopathic. allelopathic natural products have been recorded in all classes of secondary metabolites. few research groups have studied the effect of alkaloids in this context, but at least 50 alkaloids have been reported with allelopathic properties (table ix) . as can be seen from table ix , allelopathic activities can be found within nearly all structural types of alkaloids. at higher alkaloid concentrations, a marked reduction in the germination rate can be recorded regularly. more sensitive, however, is the growth of the radicle and hypocotyl. they respond to alkaloids at a much lower level, and usually a reduction in growth can be observed but sometimes also the opposite, either of which reduces the fitness of a seedling. in species which produce the compounds, the inhibitory effects can be absent, as was reported for quinolizidine alkaloids in lupines and colchicine in colchicum autumnale (503, 506) . it is likely that autotoxicity is prevented either by a special modification of cellular target sites or by other mechanisms. alkaloids (56,166,378), berberine (396-398), sanguinarine (400,409) and veratrum alkaloids]; inhibition of protein biosynthesis [e.g., emetine (404) and quinolizidine alkaloids (56,99,416-418,422) tables iv and ix) . the inhibitory action of quinolizidine alkaloids should be explained in this context (184, 503) . they are very abundant in lupine seeds (up to 3-8% dry weight). during germination, 13-hydroxylupanine is converted to ester alkaloids, such as 13-tigloyloxylupanine. the latter compound is predominantly excreted via the roots of young seedlings and in germination assays proved to be the most allelopathic qa. these alkaloids influence only heterologous systems, not the germination of lupine seeds themselves. when lupine and lepidium seeds were grown together in the same pot, growth of the lepidium seedlings was much reduced and inhibited, indicating that qas may also be relevant in the ecological context (184) . although the number of alkaloids with known allelopathic properties is not large, owing to the limited number of studies conducted, it is clear from table ix that alkaloids can be toxic to plants, probably by interfering with basic metabolic or molecular processes. although comparably few alkaloids have been studied for their biological activities in detail, and considering that our data collection (tables i-ix) is far from complete, we can safely state that alkaloids have potent deterrent or poisonous properties in herbivorous animals, and also affect bacteria, fungi, viruses, and plants. the next question will be whether all the adverse activities of alkaloids, which are often assayed in in uitro systems only, are meaningful in nature. because most of the allelochemical activities are dose dependent (others may be synergistic, additive, etc.), the question is whether the amounts of alkaloids produced and stored in plants are high enough to be ecologically meaningful. it is difficult, and also dangerous, to make a general statement concerning alkaloid levels in plants. we must remember that alkaloid composition and levels are often tissue or organ specific (4,25,38) . they may vary during the day [a diurnal cycle has been observed for qas and tropane alkaloids (185,503,506)l or during the vegetation period (39. 505,506) . furthermore, as in all biological systems, there are differences at the level of individual plants and between populations and subspecies. unfortunately, many phytochemical reports do not contain any quantitative information, or these data are given for the whole plant without realizing the above-mentioned variables. in addition, concentrations are usually given on a dry weight basis, which is appropriate in the chemical or pharmaceutical context. however, herbivores or pathogens do not feed on the dry plant in general, but on the "wet" fresh material. in the context of chemical ecology we urgently need data on a fresh weight basis. as an approximation, in this chapter we use a conversion factor of 10 to convert dry weight to fresh weight data if only the dry weight data are available. summarizing the relevant phytochemical literature, we find that alkaloid levels are between 0.1 and 15% (dry weight), which is equivalent to o.oi-ls%fresh weight, or 0.1-15 mg/gfresh weight. for plantscontaining quinolizidine alkaloids, actual alkaloid contents are given for a number organs or parts (table x ) , which fall in the range deduced before. we have evaluated the situation for quinolizidine alkaloids and found that the actual concentrations of alkaloids in the plant are usually much higher than the concentrations needed to inhibit, deter, or poison a microorganism or herbivore (2, 184, 503, 527) . this means that plants obviously play safe and have stored more defense chemicals than actually needed. if we look at the ed,, and ld,, values given in tables 1 through ix, it is likely that the situation is similar for other alkaloid-producing plants, but these correlations need to be experimentally established in most instances. it seems trivial that plants not only synthesize but also store their secondary products, which makes sense only in view of their ecological functions as defense compounds, since they can fulfil these functions only if the amounts stored are appropriate. achieving and maintaining the high levels of a defense compound are very demanding from the point of view of physiology and biochemistry. most allelochemicals would probably interfere with the metabolism of the producing plant if they would accumulate in the compartments where they are made (25). whereas biosynthesis takes place in the cytoplasm, or in vesicles (berberine) or organelles such as chloroplasts (qas, coniine), the site of accumulation of water-soluble alkaloids is the central vacuole, and that of lipophilic compounds includes latex, resin ducts, or glandular hairs (e.g., nicotine) (4,25) . in this context it should be recalled that many alkaloids are charged molecules at cellular ph and do not diffuse across biomembranes easily. during recent years, evidence has been obtained that at least some alkaloids pass the tonoplast with the aid of a carrier system. the next problem is determining how the uphill transport, that is, the accumulation against a concentration gradient, is achieved. proton-alkaloid antiport mechanisms and ion trap and chemical trap mechanisms have been postulated and partially proved experimentally (503,510,512) . thus, the sequestration of high amounts of alkaloids in the vacuole is a complex and energy-requiring task, which would certainly have been lost during evolution were it not important for fitness. as a rule of thumb, we can assume that all parts of an alkaloidal plant contain alkaloids, although the site of synthesis is often restricted to a particular organ, such as the roots or leaves. translocation via the phloem, xylem, or apoplastically must have therefore occurred. phloem transport has been demonstrated for quinolizidine, pyrrolizidine, and indolizidine alkaloids, and xylem transport for nicotine and tropane alkaloids (36,39,511) . if the plant relies on alkaloids as a defense compound, these molecules have to be present at the right place and at the right time. alkaloids are often stored in specific cell layers, which can differ from the site of biosynthesis (25, 38, 39) . in lupines, but also in other species (486,4891, alkaloids are preferentially accumulated in epidermal and subepidermal cell layers, reaching local concentrations between 20 and 200 mm (table x) , which seems advantageous from the point of view of chemical ecology, since a pathogen or small herbivore encounters a high alkaloid barrier when trying to invade a lupine. the accumulation of many alkaloids in the root or stem bark, such as berberine, cinchonine, and quinine, can be interpreted in a similar way. a number of plants produce laticifers filled with latex. for example, isoquinoline alkaloids in the family papaveraceae are abundant in the latex (39), where they are sequestered in many small latex vesicles. in latex vesicles of chelidonium mujus the concentration of protoberberine and benzophenanthridine alkaloids can be in the range of 0.6-1.2 m, which is achieved by their complexation with equal amounts of chelidonic acid (512). if a herbivore wounds such a plant, the latex spills out immediately. besides gluing the mandibles of an insect, the high concentration of deterrent and toxic alkaloids will usually do the rest, and, indeed, chelidonium plants are hardly attacked by herbivores. in addition, as these alkaloids are also highly antimicrobial (table iv) , the site of wounding is quickly sealed and impregnated with natural antibiotics. other well-known plants that have biologically active alkaloids in their latex belong to the families papaveraceae (genera papauer, macleya, and sanguinaria) and campanulaceae (genus lobelia) (39) . it is intuitively plausible that a valuable plant organ must be more protected than others. alkaloid levels are usually highest during the time of flowering and fruit/seed formation. in annual species actively growing young tissue, leaves, flowers, and seeds are often alkaloid-rich, whereas in perennial ones, like shrubs and trees, we find alkaloid-rich stem and root barks in addition. all these plant parts and organs have in common that they are important for the actual fitness or for the reproduction and thus the long-term survival of the species. spiny species, which invest in mechanical defense, accumulate fewer alkaloids than soft-bodied ones (15); examples are isoquinoline alkaloids in cacti or qas in legumes (184) . if a plant produces few and large seeds, their alkaloid levels tend to be higher than in species with many and small seeds (15,184); thus. a plant with few and big seeds is generally a rich source of alkaloids, which makes sense in view of the defense hypothesis. these few examples show that accumulation and storage of alkaloids have been optimized in such a way that they are present at strategically important sites where they can ward off an intruder at the first instance of attack. thus, specialized locations must be regarded as adaptive. alkaloid concentrations can fluctuate during the vegetation period, or even during a day (36,506). but in biochemical terms their biosynthesis and accumulation are constitutive processes. this ensures that a certain level of defensive compounds is present at any time. furthermore, continuous turnover is a common theme for molecules of the cells whose integrity is important, such as proteins, nucleic acids, and signal molecules. the same seems to be true for a defense compound. an alkaloid which mimics a neurotransmitter, such as hyoscyamine, nicotine, or sparteine, could be oxidized or hydrolyzed in the cell by chance, and thus would be automatically inactivated. only by replacing these molecules continuously can the presence of the active compounds be guaranteed. for example, it was suggested that nicotine has a half-life of 24 hr in nicotiana plants, and that more than 10% of the co, fixed passes through this alkaloid (505). in other groups of natural products it was possible to show that plants can react to infection by microbes or to wounding by herbivores by inducing the production of new defense compounds. these compounds are termed "phytoalexins" in phytopathology (22) (23) (24) . classic examples of phytoalexins include isoflavones, phenolics, terpenes. protease inhibitors, coumarins, and furanocoumarins. using plant cell cultures it could be shown that a similar process can be observed with some alkaloidal plants, which start to produce alkaloids with antimicrobial properties (e.g., sanguinarine, canthin-6-one, rutacridone alkaloids) when challenged with elicitors from bacterial or fungal cell walls (table xi) . but what is the situation after herbivory? when plants are eaten by large herbivores, a de nouo synthesis would be almost useless for a plant (except maybe trees), since this would not be quick enough. the situation is different, however for small herbivores such as insects or worms, which may feed on a particular plant for days or weeks. here the de nouo production of an allelochemical would be worthwhile. there are indeed some preliminary experimental data that support this view. in liriodendron rirlipifera several aporphine alkaloids accumulate after wounding, which are otherwise not present (506). in tobacco the produc" cc, cell culture; pl, plant tion of nicotine, in lupines that of qas, and in atropci belleidonnu that of hyoscyamine are induced by wounding, thus increasing the already high levels of alkaloids by up to a factor of 5. whereas the response was seen after 2-4 hr in lupines, it took days in nicotiunu and in atropei (table xi) . we suggest that the wound-induced stimulation of alkaloid formation is not an isolated phenomenon, but rather an integral part of the chemical defense system. the induced antimicrobial and antiherbivoral responses show that plants can detect environmental stress and that secondary metabolism is flexible and incorporated in the overall defense reactions. many details on how a plant perceives and transmits information remain to be disclosed, but this will surely be a stimulating area of research in the future. although the physiology and metabolism of most alkaloids are extremely intricate (3839) and often not known, the available data suggest that they are organized and regulated in such a way that alkaloids can fulfill their ecological defense function. in other words, the alkaloids are present at the right time, the right place, and the right concentration. the aforementioned arguments strongly support the hypothesis that alkaloids serve as defense compounds for plants. besides circumstantial evidence, we would welcome critical experiments which clearly prove that alkaloids are indeed important for the fitness and survival of the plants producing them. we suggest that if a plant species which normally produces alkaloids is rendered alkaloid-free, it should have a reduced fitness because it is much more molested by microorganims and herbivores than its alkaloid-producing counterpart. for one group of alkaloids, the quinolizidine alkaloids, these experiments have already been performed (2,184,484,503,527) . as mentioned before, qas constitute the main secondary products of many members of the leguminosae, especially in the genera lirpinus, genistu, cyfisiis, bccptisiu, thrrmopsis, sophoru, ormosici, and others (503). lupines have relatively large seeds which contain up to 40-50% protein, up to 20% lipids, and 2-8% alkaloids. to use lupine seed for animal or human nutrition, homo scipiens, for several thousand years, used to cook the seeds and leach out the alkaloids in running water. this habit has been reported for the egyptians and greeks in the old world, and for the indians and incas of the new world. the resulting seeds taste sweet, in contrast to the alkaloid-rich ones which are very bitter. in mediterranean countries people still process lupines in the old way, and sometimes the seeds are salted afterward and served as an appetizer, comparable to peanuts. at the turn of the twentieth century, german plant breeders set out to grow alkaloid-free lupines, the so-called sweet lupines. although sweet lupines are extremely rare in nature ( 1 in >100.000), the efforts were largely successful, and at present, sweet varieties with an alkaloid content lower than 0.01% exist for lupinus albus, l. mutabilis, l . luteus, l. angustifolius, and l . polyphyllus. as far as we know, the sweet varieties differ from the original bitter wild forms only in the degree of alkaloid accumulation. this offers the chance to test experimentally whether bitter lupines have a higher fitness than sweet ones with regard to microorganisms and herbivores. the results of these experiments were clearcut (2,184503,506,527 ) (table xu). in the greenhouse, where plants are protected from herbivores or pathogens, no clear advantage was seen. when lupines were planted in the field, without being fenced in and without man-made chemical protection, however, a dramatic effect was regularly encountered, especially with regard to herbivores (2,184,503,527) . rabbits (cuniculus europaeus) and hares (lepus europaeus) clearly prefer the sweet plants and leave the bitter plants almost untouched, at least as long as there was an alternative food source. before dying rabbits will certainly try to eat bitter lupines. a similar picture was seen for a number of insect species, such as aphids, beetles, thrips, and leaf-mining flies (table xii) , namely, the sweet forms were attacked, whereas the alkaloid-rich ones were largely protected. the alkaloid-poor variety of l . luteus also became a host of acyrthosiphon pisii (506). in poland, where the sweet yellow lupine is one of the more important fodder plants, the invasion of the aphids became a serious problem not only because the aphid enfeebles the plants by sucking its phloem sap, but also because it transfers a viral disease. the disease, known as lupine narrow leafness, decreases seed production in infected plants, and the infection takes place early, that is, prior to the plants' blossoming. thus, a mixed population of sweet and bitter lupines can, after a few generations, lose all sweet forms. infestation by the aphid and the following viral infection accelerate the elimination of alkaloid-poor plants, which, even without infection, are already inferior in seed production (506). this observation again stresses the importance of alkaloids for the fitness of lupines. plant breeders have also observed that bacterial, fungal, and viral diseases are more abundant in the sweet forms, but this effect has not been documented in necessary detail. these experiments and observations clearly prove the importance of qas for lupines, but it should not be forgotten that other secondary metabolites, such as phenolics, isoflavones, terpenes, saponins, stachyose, erucic acid, and phytic acid, are also present in lupines and may exert additional or even synergistic effects. the lupine example also tells us about the standard philosophy and problems of plant breeding. with our present knowledge on the ecological importance of qas for the fitness of lupines, it seems doubtful whether the selection of sweet lupines was a wise decision. in order to grow them we have had to build fences and, worse, to employ man-made chemical pesticides, which have a number of well-documented disadvantages. it can be assumed that similar strategies, namely, breeding away unwanted chemical traits, have been followed with our other agricultural crops, with the consequence that the overall fitness was much reduced (2). we can easily observe the reduced fitness by trying to leave crop species to themselves in the wild: they will quickly disappear and not colonize new habitats. there are, however, alternatives. taking lupines as an example, we could devise large-scale technological procedures to remove alkaloids from the seeds after harvest (similar to sugar raffination from sugar beets). at present a few companies are actively exploring these possibilities. one idea is to produce pure protein, lipids, dietary fibers from bitter seeds. a spin-off product would be alkaloids, which could be used either in medicine (sparteine is exploited as a drug to treat heart arrhythmia) or in agriculture as a natural plant protective, that is, as an insecticide (185,503) . it is evident, however, that each plant has developed its own strategy for survival. if all plants would follow the same strategy, it would be an easy life for herbivores and pathogens, since being adapted to one species would mean adapted to all species. this specialization becomes evident if we analyze the qualitative patterns of secondary metabolite profiles present in the plant. we regularly see one to five main alkaloids in a plant, but also several (up to 80) minor alkaloids. this qualitative pattern is not constant, but differs among organs, developmental stages, individuals, populations, and species. normally, we classify the compounds as belonging to one or two chemical groups. this does not mean, however, that their biological activities are identical. on the contrary, the addition of a lipophilic side chain to a molecule seems to be a small and insignificant variation from the chemical point of view, but this may render the compound more lipophilic, and thus more resorbable. in consequence, its toxicity may be higher (see qas in table i ). thus, a herbivore or pathogen has to adapt not only to one group of chemicals but to the individual compounds present. as the composition of these chemicals changes, it is even more difficult for them to cope. therefore, we suggest that structural diversity and continuous variation are means by which nature counteracts the adaptation of specialists. in medicine, we do a similar thing if we want to control microbial diseases. to overcome or to prevent resistance of bacteria toward a particular antibiotic, very often mixtures of structurally different antibiotics are applied, whose molecular targets often differ. if only one antibiotic were given to all patients, the development of resistance would be much favored. it has been argued that alkaloids cannot have a significant role in plants because not all plant species produce alkaloids (only 30% of all plants do). these authors, such as robinson ( 5 0 3 , have overlooked the fact that if all plants would produce one single alkaloid, even a very toxic alkaloid such as colchicine, it could be certain that nearly all herbivores would have developed a resistance toward this alkaloid. only the variation of secondary metabolites, and thus of the targets which they affect, provides a means to develop efficient defense compounds. the arguments of robinson would be correct if there were higher plants without any secondary metabolites, which, nevertheless, would thrive in nature; however, these plants are not known. from an evolutionary perspective it is not important whether the defense chemical is an alkaloid or a terpene; it is only essential that it affect certain and important targets in herbivores or pathogens. although the biological activities of many alkaloids have not yet been studied and their ecological functions remain to be elucidated or proved, we can nevertheless safely say that alkaloids are neither waste nor functionless molecules, but rather they are important fitness factors, probably mostly antiherbivore compounds. since nature obviously favored multitasking, additional activities, such as allelopathic or antimicrobial activities, are plausible. for quinolizidine and pyrrolizidine alkaloids, these multiple functions are already well documented (tables i-x) . plants that defend themselves effectively constitute an ecological niche almost devoid of herbivores and pathogens. it is not surprising that during evolution a number of organisms evolved which have specialized on a particular host plant species and found ways to tolerate, or even to exploit, the defense chemistry of their hosts (4, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . as compared to the huge number of potential enemies, the number of adapted specialists is usually small, and in general a "status quo" or equilibrium can be observed between the specialists (or parasites) and their hosts. a specialist is not well advised to kill its host, since this would destroy its own resources; a mutualism is more productive for survival. host plant-specific specialists occur within bacteria, fungi, and herbivores. the interaction of the former two groups is a central topic for plant pathologists. they often find that susceptible and nonsusceptible microbe strains exist. in most cases, it is not known how these microbial specialists achieved a relationship with the host plant chemistry, for example, whether they degrade secondary metabolites or whether they simply toler-ate them. many phytopathogenic bacteria and fungi produce their own secondary metabolites, which are often toxic to plants. it is assumed that these phytotoxins serve to weaken the host plants' defense, but may be this is not the whole story. many grasses are infected with fungi that produce ergot alkaloids. it has been assumed that these fungi (e.g., clauiceps) are proper parasites. in recent years, however, experimental evidence suggests that the relationship between grasses and ergot may be of a symbiotic nature (513). ergot alkaloids are strong vertebrate toxins (tables i-iv) ; they mimic the activity of several neurotransmitters, such as dopamine, serotonin, and noradrenaline (table iv) . in fact, the impact of herbivores on populations which were highly infected by fungi was more reduced than those without. this means that the fungi exploit the nutrients of their host plants and supply them with strong poisons, which are not produced by the plants themselves. since the fungi do not kill their hosts, this close interrelationship seems to be of mutual interest. we expect that similar relationships are likely to be detected in the future. as mentioned earlier, a large number of mono-and oligophagous insects exist which have adapted to their host plants and the respective defense chemistry in complex fashions. in general, we can see the following main schemes (4,15, 17, 32, 507, 508) . in type 1 adaptations, a species "learns" (or, as we should say, during evolution variants have been selected by natural selection which can tolerate a noxious defense compound) (a) by finding a way to avoid its resorption in the gut; (b) if resorption cannot prevented, by eliminating the toxin quickly via the malpighian tubules or degrading it by detoxifying microsomal and other enzymes; and (c) by developing a target site that is resistant to the toxin, such as a receptor which no longer bind the exogenous ligand. alternatively, in type 2 strategies a species not only tolerates a plants' defense compound, but exploits it for its own defense or for other purposes, such as pheromones i7,494496,506) . examples of type 1 include manduca sexra, whose larvae live on nicoriana and other solanaceous plants. the alkaloids present in these plants, such as nicotine or hyoscyamine, are not stored but are degraded or directly eliminated with the feces (182). in addition, it has been postulated that nicotine may either not diffuse into nerve cells or that the acetylcholine recpetor no longer binds nicotine as in "normal" animals (17). the potato beetle (leptinotarsa decernlineata) lives on solanurn species containing steroid alkaloids, which are tolerated, but not stored, by this species, the bruchid beetle callosohruchus fasciarus predates seeds of qa-rich plants, such as laburnum anagyroides; this beetle eliminates most of the dietary cytisine with the feces (492). examples of type 2 are to some degree more interesting. in a number of plants alkaloids are translocated via the phloem (511). when aphids live on these plants they are in direct contact with the alkaloids present. a number of examples are known at present which show that adapted aphids can store the dietary alkaloids. examples are the quinolizidines in aphis cytisorum, a. genistae, and macrosiphum albifrons, the pyrrolizidines in aphis jacobaea, a . cacaliaster, and aconitine in aphis aconiti (185, 511) . for alkaloid-storing m . albifrons it was shown experimentally that the qas stored provide protection against carnivorous beetles, such as carabus problematicus or coccinella septempunctata (465, 503) . acyrthosiphon spartii prefers sparteine-rich cytisus scoparius plants (506); although it is likely that this species also stores qas, it has not been demonstrated to do so. larvae of the pyralid moth uresiphita reversalis live on qa-producing plants, such as teline monspessulana. the larvae store some of the dietary alkaloids, especially in the integument and also the silk glands. the uptake is both specific and selective and is achieved by a carrier mechanism. whereas alkaloids of the 10-oxosparteine type dominate in the plant, it is the more toxic cytisine that is accumulated by the larvae, with the 10oxosparteines being eliminated with the feces (503,514) . the larvae gain some protection from storing qas, as was shown in experiments with predatory ants and wasps. when the larvae pupate, most of the alkaloids stored are used to impregnate the silk of the cocoon, thereby providing defense for this critical developmental stage (503,514). the emerging moth lives cryptically, has no aposematic coloring, and does not contain alkaloids. in contrast the alkaloid-rich larvae are aposematically colored and live openly on the plants (503,514) . the larvae of the blue butterfly (plebejus icaroides) feed only on lupines, rich in alkaloids. as far as we know, the larvae do not sequester or store the dietary alkaloids (506). helopeltis feeds on cinchona bark, which is rich in cinchonine-like alkaloids; it stores and uses them for its own defense (506). larvae of the butterflies pachlioptera aristolochiae, zerynthia polyxena, ornithoptera priamus, and battus philenor live on arisrolochia plants and were shown to take up and sequester aristolochic acid, a carcinogenic alkaloid discussed earlier, as an effective defense compound (4,28,236) . the best-studied group of acquired alkaloids are the pyrrolizidines, which are produced by plants, especially in the families asteraceae and boraginaceae (502). some arctiid larvae of tyria jacobaea, cycnia mendica, amphicallia bellafrix, arginia cribaria, and arctia caja were shown to store the dietary pas and exploit them for their own defense (4,17,28,31,222-224,237) . in tyria jacobaea, arctia caja, diacrisia sannio, phragmatobia fuligonosa, and callimorpha dominula pas are taken up and stored in the integument (523). monarch butterflies (e.g., danaus plexipus) combine two sets of natural compounds. larvae feed on plants rich in cardiac glycosides and use them as chemical defense compounds. adult butterflies visit plants with pas, where they collect pas that are converted to pheromones or transferred to their eggs (4,f 7,31,33,36f,515) . a similar pa utilization scheme was observed with larvae of the moth utetheisa ornatrix (367, 516) , where the compounds were shown to be deterrent for spiders and birds (225, 525) . the chrysomelid beetle oreina feeds on pa-containing plants, such as adenostyles, and stores the dietary pas in the defense fluid (463,524). in the arctiid creatonotos transiens was observed an advanced exploitation of pas (31,33,429,517-521) . the alkaloids are phagostimulants for larvae, which are endowed with specific alkaloid receptors. dietary pyrrolizidine n-oxides are resorbed by carrier-mediated transport. after resorption, free pas are converted to the respective n-oxides and (7s)-heliotrine to (7r)-heliotrine. the latter form is later converted to a male pheromone, (7r)-hydroxydanaidal. pas are stored in the integument, where they serve as defense compounds and are not lost during metamorphosis. in the adult moth, however, the pas are mobilized. in the female adult, pas are translocated into the ovary and subsequently into the eggs. in the male, pas are necessary for the induction of abdominal scent organs and concomitantly for the biosynthesis of pa-derived pheromones, which are dissipated from these coremata. in addition, pas are transferred into the spermatophore and thus donated to the female. a significant amount of pas is further transferred to the eggs, which thus obtain chemical protection from the pas previously acquired by both male and female larvae. marine dinoflagellates produce a number of toxins, such as saxitoxin, surugatoxin, tetrodotoxin, and gonyautoxin, that affect ion channels (table iv). these algae are eaten by some copepods, fish, and molluscs that also store these neurotoxins (4,17,28,29,494,495) . as a consequence, these animals have acquired chemical defense compounds, which they can use against predators. this discussion is not meant to be complete, but should illustrate that a number of insect herbivores exploit the chemistry of their food plants. these insects are adapted and have evolved a number of molecular and biochemical traits that can be considered as prerequisites. however, many of the respective plant-insect interactions have not yet been studied, and it is therefore likely that the acquisition of dietary defense compounds is even more widely distributed in nature than anticipated. whereas insect herbivores are often highly host plant specific, vertebrate herbivores tend to be more of the polyphagous type, although some specialization may occur. for example, grouse (lagopus lagopus) or capercaillies (tetra0 urogallus) prefer plants of the families of ericaceae or coniferae, and crossbills seeds of picea and abies species, which are rich in terpenes. the australian koala is oligophagous and prefers terpene-rich species of the genus eucalyptus. for approximately 65 million years, the only true herbivorous vertebrates have been the mammals. the mesozoic reptiles disappeared following the mesophytic flora. birds, though a few species feed on seeds and berries, seldom eat leaves (except geese and grouse), and they frequently use insects, in addition to plant parts, as a food source (18). although a single plant can be a host for hundreds of insect larvae, hundreds of plants comprise a daily menu for a larger mammal. the strategies of the polyphagous species include the following. avoidance of plants with very toxic vertebrate poisons (these species are usually labeled toxic or poisonous by man) by olfaction or taste discrimination. often such compounds may be described as bitter, pungent, bad smelling, or in some other way repellent. 2. sampling of food from a wide variety of sources and thus minimizing the ingestion of high amounts of a single toxin. 3. detoxification of dietary alleochemicals, which can be achieved by symbiotic bacteria or protozoa living in the rumen or intestines, or by liver enzymes which are specialized for the chemical modification of xenobiotics. this evolutionary trait is very helpful for homo sapiens, since it endowed us with a means to cope with our man-made chemicals which pollute the environment. carnivorous animals, such as cats, are known to be much more sensitive toward plant poisons (505). it was suggested that these animals, which d o not face the problem of toxic food normally, are thus not adapted to the handling of allelochemicals. some animals, such as monkeys, parrots, or geese, ingest soil. for geese (185) it was shown that the ingested soil binds dietary allelochemicals, especially alkaloids (185). this procedure would reduce the allelochemical content available for resorption. 5. animals are intelligent and can learn. the role of learning in food and toxin avoidance should not be underestimated, but it has not been studied in most species. for most vertebrate herbivores, the ways they manage to avoid, tolerate, or detoxify their dietary allelochemicals have not been explored. sometimes, only domesticated animals were used in experiments, but they tend to make more mistakes in food choice than the wild animals. more evidence on this subject is available for homo sapiens, who has evolved a number of "tricks," some of them obviously not anticipated by evolution. first, man tends to avoid food with bitter, pungent, or strongly scented ingredients. as a prerequisite he needs corresponding receptors in the nose or on the tongue which evolved during the long run of evolution as a means to avoid intoxication. second, our liver still contains a set of detoxifying enzymes which can handle most xenobiotics. furthermore, some of these enzymes, such as cytochrome p.450 oxidase, is inducible by dietary xenobiotics. third, besides these biological adaptations, man has also used his brain to avoid plant allelochemicals. (a) many fruits or vegetables are peeled. as many alkaloids and other compounds are stored in the epidermis, for example, steroid alkaloids in potato tubers or cucurbitacins in cucurbits, peeling eliminates some of these compounds from consumption. (b) most food is boiled in water. this leads to the thermal destruction of a number of toxic allelochemicals, such as phytohaemagglutinins, protease inhibitors, and some esters and glycosides. many watersoluble compounds are leached out into the cooking water and are discarded after cooking (e.g., lupines or potatoes). (c) south american indians ingest clay when alkaloid-rich potato tubers are on the menu. since clay binds steroidal alkaloids, geophagy is thus an ingenious way to detoxify potential toxins in the diet (522) . (d) man has modified the composition of allelochemicals in his crop plants, in that unpleasant taste components have been reduced by plant breeding. from the point of view of avoidance, this strategy is plausible, but, as was discussed earlier, it is deleterious from the point of view of chemical ecology. these plants often lose their resistance against herbivores and pathogens, which then has to be replaced by man-made pesticides. in general, only a few plants are exploited by man as food, as compared to the 300,000 species present on our planet. this means that even homo sapiens with all his ingenuity has achieved only a rather small success, indicating the importance and power of chemical plant defenses. in this context, it is worth recalling that a number of animals are able to synthesize their own defense compounds, among them several alkaloids (4,17,28,494-496) . these animals have the common feature that they are usually slow-moving, soft-bodied organisms. marine animals, such as mol-luscs, sponges, zooanthids, and fishes, have been shown to contain a variety of alkaloids, such as acrylcholine, neosaxitoxin, murexin, pahutoxin, palytoxin, petrosine, and tetramine, that are toxic to other animals (4.17,28,29,221,226,229,232,233,234,495) . a number of nemertine worms, such as amphiporus or nereis, produce alkaloids such as 2,3-bipyridyl, anabaseine, nemertelline, or nereistoxin, which are toxic to predators such as crayfish ( 4 1 7,28,230,226,) . arthropod-made alkaloids include glomerine and homoglomerine in glomerus (215) , adaline in adalia (227), coccinelline, euphococcinine, and derivatives in coccinella, epilachna, and other coccinellid beetles (28,226,227,235) , and stenusine in stenus (215) , which are considered to be antipredatory compounds (4,17,28,494-496) . solenopsis ants produce piperidine alkaloids which resemble the plant alkaloid coniine. these alkaloids are strong deterrents and inhibit several cellular processes, such as electron transport chains (table iv) (28, 494) . many insects indicate the content of toxic natural products by warning colors (aposematism) or by the production of malodorous pyrazines (4,17,231,494) . not only are lower animals able to synthesize alkaloids, but also vertebrates, especially in the class amphibia. tree frogs of the genus dendrobates accumulate steroidal alkaloids, such as batrachotoxin, pumiliotoxins a-c, gephyrotoxin, and histrionicotoxin, in their skin, which are strong neurotoxins (table iv) (4,17,28) . natives have used the alkaloids as arrow poisons. similar alkaloids (i.e., homobatrachotoxin) have recently been detected in passerine birds of the genus pitohui (528) . salamanders, salamandra maculosa, which are aposematically colored, produce the toxic salamandrine and derivatives, alkaloids of the steroidal group (4,17,28). salamandrine is both an animal toxic (paralytic) and an antibiotic. toads (bufonidae) produce in their skin cardiac glycosides of the bufadienolide type, but also a set of alkaloids, such adrenaline, noradrenaline, adenine, bufotenine, or bufotoxin (4,17,28). except for bufotoxin, the other chemicals are, or mimic, neurotransmitters. these examples show that alkaloids found in animals can either be derived from dietary sources (see section 111,d,2) or be made endogenously. common to both origins is their use as chemical defense compounds, analogous to the situation found in plants. in animals we can observe the trend that sessile species, such as sponges and bryozoans, or slow-moving species without armor, such as worms, nudibranchs, frogs, toads, and salamanders, produce active allelochemicals (28,29,494,495) , but not so those with weapons, armor, or the possibility for an immediate flight. plants merely developed a similar strategy as these "unprotected" animal species. in this context it seems amazing that hardly anybody has doubted the defensive role of alkaloids in animals, whereas people did, and still do, where alkaloids in plants are concerned. evidence is presented in this overview that alkaloids are not waste products or functionless molecules as formerly assumed (34,35), but rather defense compounds employed by plants for survival against herbivores and against microorganisms and competing plants. these molecules were obviously developed during evolution through natural selection in that they fit many important molecular targets, often receptors, of cells (i.e. they are specific inhibitors or modulators), which can clearly be seen in molecules that mimic endogenous neurotransmitters (table iv; section ii,a,3,a). on the other hand, microorganisms and herbivores rely on plants as a food source. since both have survived, there must be mechanisms of adaptations toward the defensive chemistry of plants. many herbivores have evolved strategies to avoid the extremely toxic plants and prefer the less toxic ones. in addition, many herbivores have potent mechanisms to detoxify xenobiotics, which allows the exploitation of at least the less toxic plants. in insects, many specialists evolved that are adapted to the defense chemicals of their host plant, in that they accumulate these compounds and exploit them for their own defense. alkaloids obviously function as defense molecules against insect predators in the examples studied, and this is further support for the hypothesis that the same compound also serves for chemical defense in the host plant. the overall picture of alkaloids and their function in plants and animals seems to be clear, but we need substantially more experimental data to understand fully the intricate interconnections between plants, their alkaloids, and herbivores, microorganisms, and other plants. defense in animals introduction to ecological biochemistry defense mechanisms in plants herbivores: their interaction with secondary 16 okologische biochemie allelochemicals: role in agriculture and forestry cell culture and somatic cell genetics of plants" (f. constabel and 25. m. wink gifttiere und ihre waften perspectives in chemoreception behavior biochemie und physiologie der sekundaren pflanzenstoffe plant metabolites biosynthese der alkaloide biochemistry of alkaloids the alkaloids: the fundamental chemistry antiseptika baerheim svendsen lloydia 51. n. m. rojas hernandez vallejos and 0. a. roveri the merck index proc. 3rd inf. lupin conf insect biology in the future micromolecular evolution, systematics and ecology phytochernical ecology: allelochernicals, mycotoxins and insect pheromones phytochem biogene gifte rozniki nauk rolnikzych die gift-und arzneipflanzen in mitteleuropa antibiotics: mechanism of action of antimicrobial and antitumour agents antimicrob. agents chemo pharmazeutische biologie 11, biogene arzneistoffe pharmazeutische biologie drug use in pregnancy the alkaloids handbook of enzyme inhibitions cold spring harbor conf primary and secondary metabolism of plant cell cultures chemical defenses of arthropods der einfluss einer pflanze auf die andere-allelopathie the science of allelopathy allelopathy lectures on insect olfaction focus on insect-plant interactions alkaloid biology and metabolism in plants molecular aspects of insect plant associations methods of plant biochemistry secondary products in plant tissue culture proc. natl the alkaloids the work of the author was supported by the deutsche forschungsgemeinschaft. i thank dr. th. twardowski for reading an earlier draft of the manuscript. key: cord-021419-nypnib0h authors: olsufyeva, evgenia n.; yankovskaya, valentina s. title: main trends in the design of semi-synthetic antibiotics of a new generation date: 2020-03-17 journal: nan doi: 10.1070/rcr4892 sha: doc_id: 21419 cord_uid: nypnib0h this review summarizes main advances achieved by russian researchers in the synthesis and characterization of semi-synthetic antibiotics of a new generation in the period from 2004 to 2019. the following classes of compounds are considered as the basis for modification: polycyclic antibacterial glycopeptides of the vancomycin group, classical macrolides, antifungal polyene macrolides, the antitumour antibiotic olivomycin a, antitumour anthracyclines and broad-spectrum antibiotics, in particular, oligomycin a, heliomycin and some other. main trends in the design of modern anti-infective and antitumour agents over this period are considered in relation to original natural antibiotics, which have been independently discovered by russian researchers. it is shown that a new type of hybrid structures can, in principle, be synthesized based on glycopeptides, macrolides and other antibiotics, including heterodimers containing a new benzoxaborole pharmacophore. the review addresses the influence of the length of the spacer between two antibiotic molecules on the biological activity of hybrid structures. a combination of genetic engineering techniques and methods of organic synthesis is shown to be useful for the design of new potent antifungal antibiotics based on polyenes of the amphotericin b group. many new semi-synthetic analogues exhibit important biological properties, such as a broad spectrum of activity and low toxicity. emphasis is given to certain aspects related to investigation of a broad range of biological activity and mechanisms of action of new derivatives. the bibliography includes 101 references. the review addresses the influence of the length of the spacer between two antibiotic molecules on the biological activity of hybrid structures. a combination of genetic engineering techniques and methods of organic synthesis is shown to be useful for the design of new potent antifungal antibiotics based on polyenes of the amphotericin b group. many new semi-synthetic analogues exhibit important biological properties, such as a broad spectrum of activity and low toxicity. emphasis is given to certain aspects related to investigation of a broad range of biological activity and mechanisms of action of new derivatives. the bibliography includes 101 references. antibiotics are commonly used in the treatment and prevention of various infectious diseases. one of the major problems of modern chemotherapy is the disappointing efficacy when using available drugs against resistant bacterial strains. natural antibiotics, i.e., antibiotics produced by various microorganisms, have been and continue to be an important source of new highly active antimicrobial and antitumour agents. one of the most relevant approaches to the design of new drugs relies on targeted chemical transformations of natural antibiotics. 1 in the world science, considerable efforts are currently underway to combat the problem of resistance of microorganisms to available drugs. however, in comparison with other drugs, the development of new antibiotics is not carried out sufficiently. the design of anti-infective drugs and the creation of marketable products are still a challenge. 2 since the development of medicines for the treatment of chronic diseases is much more profitable and because of high requirements for safety, large cap pharmaceutical companies (big pharma) shut down their antibiotic research projects. currently, small-and medium-sized enterprises are developing a majority of new drugs through investments, venture capital, etc. because of high demands for new anti-infective drugs and extremely high cost of these works, cross-country collaborations are needed for research in this field. only a few new compounds were approved for therapeutic use in human medicine or have completed phase-iii clinical trials. 3 the problems are compounded by the fact that the increasing percentage of the population, particularly in developed countries and russia, suffer from infections that were not earlier dangerous, i.e., from opportunistic infections. this is due to a significant decrease in the immune status of the population caused by natural or man-made factors. the translation of semi-synthetic antitumour antibiotics (e.g., doxorubicin) into clinics resulted in the development of gold standard chemotherapeutic agents. many antibiotics have made a great contribution to understanding of mechanisms of development of resistance in bacterial and tumour cells. due to high innate or acquired drug resistance of cancer cells, chemotherapy of malignant tumours is often ineffective. the international research community has focused its attention on the search for new, more effective and less toxic antitumour agents. one of the most rational approaches to the targeted therapy is based on the search for inhibitors of important tumour cell targets among natural products, primarily antibiotics. 4 even the repurposing of known antitumour antibiotics is considered in order to address the problem of antibiotic resistance of antiinfective agents. 5 researchers of the gause institute of new antibiotics (gina) headed by academician of the ussr academy of medical sciences g.f.gause in the 1960 ± 1990s made considerable contribution to the discovery of a series of original antibacterial and antitumour agents (a total of 15 compounds), their characterization and introduction to medical practice. major achievements of the institute during this period are considered in the review. 6 antibiotics comprise an important class of natural products with unique structural diversity. chemical transformations of natural antibiotics imply a change of particular functional groups of the starting molecules with preservation of structural elements responsible for biological activity. the structure determines the possibility of chemical transformation of the antibiotic and reaction conditions. for example, many antibiotics contain nitrogenous and(or) nitrogen-free sugars, which are easily eliminated in acidic or alkaline media. many antibiotics are sensitive to oxidants, are poorly soluble in organic media or, on the contrary, in aqueous solutions, etc. on the other hand, in the case of similar structures of certain moieties [e.g., the presence of nh 2 , co 2 h, oh, c(o), etc. groups)], methods developed for one class of antibiotics can be applied to antibiotics of another class. the goal of synthetic chemists is to transform natural products with preservation of the sites responsible for biological activity. besides, targeted modification can be performed to gain better understanding of the mechanisms of action, in particular in order to investigate the interaction between the antibiotic and the target. in this review, the following classes of compounds are considered as scaffolds for the synthesis of new antibiotics: polycyclic glycopeptides of the vancomycin ± teicoplanin group, classical macrolides, macrolides of the amphotericin b ± oligomycin group, anthracyclines, aureolic acid derivatives, heliomycin, synthetic benzoxaboroles and some other antibiotics. such representatives as eremomycin, carminomycin, olivomycin a, oligomycin a and heliomycin 6 . olivomycin a 364 6.1. modification of olivomycin a at the aromatic ring of the aglycone 364 6.2. modification of olivomycin a at the side-chain 2 h -keto group of the aglycone 366 6.3. modification of the side chain at the c(2 h )7c (3 h the discovery of vancomycin (1) and teicoplanin (2) (fig. 1 ) has given impetus to research on polycyclic glycopeptide antibiotics. 7 natural antibiotics 1 and 2 are still used in medical practice and are considered as reserve antibiotics. they are commonly applied for the treatment of infections caused by gram-positive cocci, particularly, methicillinresistant staphylococcus aureus (mrsa) strains. glycopeptide antibiotics bind with high affinity to the terminal d-ala-d-ala group of the growing peptidoglycan chain on the outer bacterial cell wall, thereby inhibiting the enzymes transpeptidase and transglycosylase. the vancomycin resistance in enterococcus strains (vre) (for vana and vanb phenotypes) arises due to replacement of the d-ala-d-ala group by d-ala-d-lactate, which weakly interacts with the antibiotic. semi-synthetic glycopeptide analogues, such as oritavancin, telavancin and dalbavancin, have recently been used worldwide in medicine. these drugs only partially solve the problem of the treatment of infectious diseases caused by vancomycinresistant enterococci. 7, 8 the search for more effective glycopeptide analogues is an ongoing process. 7 ± 9 eremomycin 3 (see fig. 1 ), the natural antibiotic of this group, was discovered in the gause institute of new antibiotics. 17 this compound differs from vancomycin (1) by the absence of a chlorine atom and the presence of the additional amino sugar eremosamine in the side group of amino acid 6 (aa6), as well as by the structure of the amino sugar (4 h -epivancosamine or eremosamine) at the d-glucopyranose moiety attached to aa4. eremomycin (3) is 3 ± 5 times more active against gram-positive bacteria than antibiotic 1; however, drug 3 is also ineffective against vre and vancomycin-intermediate resistant staphylococcus aureus (visa). in recent years, series of new semi-synthetic derivatives of eremomycin, vancomycin and teicoplanin active against resistant vre and visa strains were prepared. 8 ± 10 figure 1 presents main possible directions of modification of the cand n-terminal groups of the peptide core (a and f ), 3 h -amino sugar (b), the amide group of asparagine (asn) (c), sugar elimination (d ) and edman degradation (e) for antibiotics 1 ± 3. the presence of (benzotriazol-1-yl)oxytripyrrolidinophosphonium hexafluorophosphate (pybop) as the peptide coupling reagent afforded a series of new carboxamide derivatives of eremomycin 4 ± 6 (scheme 1, fig. 1 a3) . 11 ± 14 after the purification, these compounds were isolated in *50% ± 80% yields. eremomycin pyrrolidide (4) has high in vitro antibacterial activity against sensitive and resistant gram-positive bacterial strains, including mrsa, visa and vre isolates. 13, 14 besides, compound 4 is much more effective in the treatment of induced sepsis in mice compared to vancomycin (1) and does not cause a pseudoallergic reaction typical of many antibiotics of this group. compound 4 was successful in preclinical evaluation (in collaboration with the limited liability company`medicine technology') and was recommended for further clinical trials. 13 eremomycin n-adamantan-2-ylamide (5) was synthesized in a similar way as amide 4 (see scheme 1). 15 in in vitro assays, compound 5 exhibits activity against mrsa, visa, vre and bacillus anthracis strains. this compound is also effective against ciprofloxacin-resistant strains of bacillus anthracis. model in vivo assays in mice infected with s. aureus or bacillus anthracis showed that compound 5 provides a higher survival rate of animals compared to ciprofloxacin and has pharmacologically relevant properties, exhibiting an excellent distribution in tissues. the synthesis of eremomycin carboxamides containing bulky substituents, such as 2-aminoadamantane (2-ad) (compound 5), in the presence of pybop at ph * 8.5 afforded the previously characterized unsubstituted eremomycin amide (6) as a by-product. 16 compound 6 is produced by the competitive amidation reaction of the antibiotic with ammonia, which is eliminated through transpeptidation of asparagine-containing peptides in an alkaline medium. an original method was developed for the selective introduction of different amino acids containing a hydrophobic substituent into glycopeptide antibiotics 1 or 2 via selective aminoacylation of the 3 h -amino group of the amino sugar moiety of the disaccharide branch. 17 for instance, the reaction of vancomycin 1 with n-fmoc-(n-n-octyl-o-4benzyl)-l-alanine n-hydroxysuccinimide (osu) ester gave figure 1 . structures of vancomycin (1), teicoplanin a2-2 (2) and eremomycin (3) and directions of their chemical modifications: amidation (a), acylation (b), alkaline hydrolysis of the c(o)nh2 group to co2h followed by amidation (c), sugar elimination (d ), edman degradation (e) and modification of the n-terminal amino group of the peptide core ( f ). fig. 1 b1) . in this reaction, the n-terminal group of the peptide core of the antibiotic remains intact. the n-fmoc protecting group can easily be removed by the treatment with a 5% secondary amine solution. compound 8 exhibits high activity against sensitive and resistant clinical strains of gram-positive bacteria, including vre. 18 in order to study in detail the interaction between the antibiotic and the target in the intact bacterial cell by solid-state nmr spectroscopy using the rotational-echo double resonance (redor) technique, 15 nh 2 -or f-labelled substituents were introduced into amino acid residues 3 (aa3) and(or) 7 (aa7) of the peptide chain of the antibiotic eremomycin (3). 19 the 15 n label was introduced in the vicinity of the binding pocket of the antibiotic. this was accomplished using carboxyeremomycin (9) , which was synthesized previously by the selective alkaline hydrolysis of compound 3 in a saturated aqueous solution of ba(oh) 2 . under these conditions, vancomycin (1) decomposes. carboxyeremomycin [ 15 n]-bisamide 10 was synthesized by the reaction of compound 9 with appropriate amines in the presence of pybop (scheme 3, fig. 1 a3,c3) . 19 eremomycin 4-fluorophenyl-n-piperazide (11) was synthesized by the conventional amidation method in the presence of pybop. the redor experiments were performed using intact staphylococcus aureus cells, which were grown in a culture medium containing bioprecursors with isotope-labelled atoms (e.g., 13 c-amino acid). the 15 n-or f-containing antibiotic that was added to the medium inhibits bacterial growth by forming a stable complex with 13 c-labelled peptidoglycan moieties (see fig. 2 , hydrogen bonds are indicated by dashed lines). 19, 20 the study of the complex with compound 10 provides an estimate of the distance from the distance between the c-terminal [ 15 n]-amide of eremomycin (10) and l-[ 13 c(3)]ala 1 of the peptidoglycan stem is 3.5 # a (see fig. 2 , a solid arrow). 20 consequently, higher activity of eremomycin amide 10 (compared to antibiotics 1 or 3) against resistant visa staphylococci can be attributed to the fact that this compound interacts with the peptidoglycan not only via a classical model (i.e., with the d-ala-d-ala target) but also with the l-[ 13 c(3)]ala group of its stem. besides, there is an additional binding site of derivative 11 to the target, which can also account for its high antibacterial activity against vre and visa. 21 previously, it was shown that the elimination of sugars (see fig. 1 d ) and the introduction of a hydrophobic residue into the aglycone can give rise to aglycone derivatives of antibiotics exhibiting activity against different types of enveloped viruses. 22 the modification of the eremomycin aglycone (12a), its de-d-meleu analogues (hexapeptide, 13a), which was produced by the cleavage of amino acid 1 (aa1) using the edman method (see fig. 1 e), and the teicoplanin aglycone (14) gave a series of new hydrophobic derivatives. (1-adamantylmethyl)amide of the eremomycin aglycone (12b) and its hexapeptide analogue (13b) are derived by the reaction of 1-adamantylmethylamine with 12a or 13a in the presence of diphenylphosphoryl azide (dppa) (scheme 4). diphenylphosphoryl azide rather than pybop is the reagent of choice for the amidation of aglycones, because the reactions in the presence of pybop often afford by-products containing the pybop moiety in the phenol group of the aglycone at aa4. 23 the acylation of compound 14 with di-tert-butyl dicarbonate (boc 2 o) followed by amidation under standard conditions in the presence of pybop gives the disubstituted derivative ð (2-adamantyl)amide of the n-boc-teicoplanin aglycone (15) . the acylation of 14 with 1-adamantylmethyloxy carbonate (adoc 2 o) affords the n-adoc-teicoplanin aglycone (16) (see fig. 1 f, scheme 4). compounds 12b, 13b and 14 ± 16 exhibit high in vitro activity against different corona-and flaviviruses, in partic-ular feline infectious peritonitis virus (fipv) and the coronavirus (sars-cov). 24 the most interesting data were obtained when studying antiviral activity of (1-adamantylmethyl)amide of the eremomycin aglycone (12b) and its de-(d-meleu) analogue (13b) against human immunodeficiency viruses (hiv): ic 50 = 1.6 and 5.5 mmol l 71 for hiv-1, 7.0 and 3.5 mmol l 71 for hiv-2, respectively. compounds of type 13b are promising selective anti-hiv agents because they cannot bind to bacterial targets. 23 apparently, they cannot induce resistance of bacteria during long-term application and can be used in the future for the prevention of hiv infections. the doubly modified teicoplanin derivative ð n-bocprotected 2-adamantylamide of the teicoplanin aglycone 15 ð exhibits high in vitro activity against a series of flaviviruses: hepatitis c virus (hcv), 25 yellow fever virus (yfv), japanese encephalitis virus (jev), tick-borne encephalitis virus tbev) and dengue virus (denv). 25, 26 compound 15 is unique in that it can inhibit replication of the closely related denv and hcv viruses by different mechanisms: in the former case, the inhibition occurs in the stage of virus entry in the host cell; in the latter case, after virus entry. protein kinases play an essential role in the virus entry in the cell and virus replication. hence, 15 analogues of the eremomycin and teicoplanin aglycones (including compounds 12a,b, 13a,b and 14 ± 16) were tested on a panel of 12 recombinant human protein kinases (pks) and two rat liver pks (ck1 and ck2). 27 these compounds were shown to inhibit pk activity by 50% at a concentration of <10 mmol l 71 and by 90% at a concentration of 10 mmol l 71 . teicoplanin aglycone derivatives 15 and 16 exhibit higher activity against many pks compared to eremomycin derivatives 12b and 13b, which also correlates with their higher activity against many types of enveloped viruses. the kinetic analysis of the inhibition of protein kinase ck2a demonstrated that teicoplanin n-adoc-aglycone 16 does not compete with atp and peptide substrates. 27 on the available data, it was suggested that one of the mechanisms of antiviral activity of glycopeptide derivatives can be based on the inhibition of serine/threonine protein kinases. the synthesis of hybrid analogues containing covalently bonded compounds of different classes (dual-acting antibiotics) with different spectra of antibacterial activity is a promising approach to the search for new antibacterial agents to combat antibiotic resistance of bacteria. 28, 29 boronic acids and benzoxaboroles are compounds capable of interacting with various biologically important components of the living cell, such as alcohols, amino alcohols, carbohydrates, rna and some peptides. a new class of synthetic antibiotics possessing antifungal, antimicrobial and antiparasitic activity was designed and synthesized based on benzoxaboroles and is currently developed by anacor pharmaceuticals (usa). 30 certain starting benzoxaboroles used in the synthesis also exhibit biological activity (see below). series of hybrid analogues 17 ± 20 linked to the borole or benzoxaborole moiety either directly or through a spacer were synthesized for the first time from glycopeptides 1 and 3. to introduce a substituent containing a boronic acid moiety into molecule 1 or 3, it is necessary to employ picolinic acid as a protecting group, which is easily removed in a weakly acidic medium (scheme 5). 31 the amidation of the carboxyl group of antibiotics 1 and 3 with 4-or 3-aminomethylphenylboronic acid picolinate esters in the presence of pybop gave new carboxamides of these antibiotics (17a ± 20a). the hydrolysis of the picolinic group under mild conditions in a weakly acidic aqueous medium affords derivatives 17b ± 20b containing the unprotected boronic acid moiety. borole-containing derivatives 17 ± 20 were found to be as effective as the starting antibiotics 1 and 3. eremomycin derivative 19b exhibits the highest activity against gram-positive bacteria and is more effective against resistant staphylococcus strains (visa) compared to compounds 1 and 3. a series of vancomycin conjugates containing different types of benzoxaborole substituents were synthesized: amido derivatives 21a,b, n-acyl derivatives (22) n-alkyl derivatives (23) (scheme 6). similar schemes were applied to synthesize benzoxaborole derivatives of eremomycin (7) (see scheme 1) and the teicoplanin aglycone (scheme 7). 32 carboxamides of eremomycin (7) (see scheme 1), vancomycin (21a) (see scheme 6) and the teicoplanin aglycone (24a) (see scheme 7) were synthesized by a standard procedure based on the treatment of compounds 1, 3 and 14, respectively, with 3-(aminomethyl)benzo[c] [1, 2] oxaborol-1(3h)-ol in the presence of pybop. the reaction of compound 1 or 14 with o-amino-n-alkylamines affords the corresponding amides 21b and 24b,c (n = 2, 3 and 5) containing a longer spacer. the reactions with osu-activated esters of the same in situ generated compounds were used to synthesize n-[3-(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborol-7-yl)propanoyl] derivatives of vancomycin (22) and the teicoplanin aglycone (25a). the alkylation of vancomycin (1) with appropriate aldehyde in the presence of nabh 3 cn affords n,n hdi(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole-6-methyl)vancomycin (23) in quantitative yield (see scheme 6) . 32 the amidation of n-acyl-substituted teicoplanin aglycone 25a with appropriate amine (in a similar way as the synthesis of amides 21 and 24a) gave disubstituted teicoplanin aglycone derivative 25b. the five-membered oxaborole ring cleavage is not observed in various reactions of benzoxaboroles (see scheme 7) . the presence of peaks at m/z 1619. 49 [m7oh], 1474.39 and lower (1312. 34 and 1284.34) in the tandem mass spectrometry (esi-ms/ms-mrm) spectrum of compound 22 indicates that the antibiotic molecule contains a substituent in the n-terminal amino group of the peptide core rather than in the amino sugar n(3 h ) group of vancosamine (fig. 3) . 32 vancomycin derivatives 21a, 22 and 23 proved to be less effective against gram-positive bacteria than the starting compound 1, except for amide 21a, which exhibits activity comparable with that of vancomycin 1. 32 hybrid derivatives, in which benzoxaborole and the teicoplanin aglycone are linked by a spacer with a particular length, exhibited the highest antibacterial activity against clinical isolates of gram-positive bacteria. 3-amino-n-(1hydroxy-1,3-dihydro[c] [1, 2] oxaborol-6-yl)propylamide of the teicoplanin aglycone (24c, n = 2) possesses particularly high activity, in particular against vancomycin-resistant strains. 32 besides, this compound exhibits moderate activity against vancomycin-resistant enterococci (vre); the minimum inhibitory concentration (mic) is 4 ± 8 mg ml 71 . an increase or a decrease in the spacer length and the introduction of two benzoxaborole substituents into the n-and c-terminal groups of the peptide were found to decrease antibacterial activity. the broad-spectrum antibacterial drug kanamycin a (26a) is an important antibiotic of the aminoglycoside (aminocyclitol) class, which is still used in medicine for the treatment of many infectious diseases and also in agriculture. 7 aminoglycosides are active against gram-positive and gram-negative bacteria. the mechanism of their action is related to the interaction with the decoding site (a site) of the 16s subunit of ribosomal ribonucleic acid (rrna), which leads to disturbance of translation, i.e., protein biosynthesis. according to the literature data, a number of semi-synthetic derivatives of a new generation were synthesized based on aminoglycosides. 33 various heterodimeric aminoglycoside conjugates with other antibiotics were reported, and some of them are used in medicine. 28, 29 the synthesis of hybrid kanamycin a conjugates with glycopeptides 1 and 3 was described for the first time in our publication. 34 kanamycin a (26a) was conjugated with compounds 1 or 3 via an amino group of the antibiotic at the 1 position of 2-deoxy-d-streptamine. the acylation of this amino group is known to reduce the risk of the development of resistance, because it prevents deactivation of the antibiotic by enzymes. the amino group at the 3 position of 2-deoxy-dstreptamine and the 6 h -amino group of 6 h -deoxy-6 h -amino-d-glucopyranose of aminoglycoside 26a were protected by the benzyloxycarbonyl group (cbz). 3,6 h -bis-(cbz)-kanamycin a (26b) was synthesized by the reaction of the zinc complex of compound 26a with cbzcl in the presence of a base (et 3 n) using a modified method 35 (scheme 8). (mic *2 ± 4 mg ml 71 for compounds 27, 28a,b) and vre stains (mic = 8 mg ml 71 for compound 28a). 34 based on the results of these studies, methods were developed for the selective introduction of functional groups at the amino sugar amino group of vancomycin (1) or eremomycin (3), with the terminal methylamino group of the peptide core of the antibiotic remaining intact. 17 the amidation of the terminal carboxyl group of these antibiotics with various amines, including amines with bulky substituents, was studied in detail. 12, 13, 15 an unusual byproduct of amidation (previously unknown for these classes of antibiotics) was isolated, and the optimal conditions were found for the amidation providing the target products in high yields. 15, 16 the introduction of various groups into glycopeptides at certain positions of the molecule can give new derivatives active against bacteria that are resistant to the initial antibiotics ð glycopeptide-resistant enterococci. 5, 10 this resulted in the discovery of compounds exhibiting high activity against glycopeptide-resistant enterococci (mic = 2 ± 8 mg ml 71 ) and staphylococcus aureus with intermediate resistance to glycopeptide antibiotics (mic = 1 ± 2 mg ml 71 ). generally, eremomycin derivatives possess higher in vitro antibacterial activity than analogous vancomycin derivatives and show significant advantages over vancomycin in the treatment of animals in a mouse model of staphylococcal sepsis. 10, 13 conditions were found for the selective introduction of isotopic labels at both the terminal carboxyl group and the asparagine residue (aa3) of the peptide core of eremomycin, with carbohydrate moieties and other labile functional groups remaining intact. 19 the investigation of interactions of these compounds with native cells of gram-positive bacteria by the redor technique confirmed the mechanisms of action of this group of antibiotics proposed in our previous studies. 20, 21 modifications of glycopeptide aglycones at the carboxyl and(or) amino group were performed in a series of studies. 22 ± 25 this resulted in the discovery and characterization of a new class of polycyclic peptides exhibiting antiviral activity at micromolar concentrations against hiv-1 and hiv-2, as well as against the enveloped viruses hcv, denv and many other. 22 ± 26 the correlation between antiviral and pk inhibitory activities was established for a class of hydrophobic derivatives of glycopeptide aglycones. 27 the first heterodimeric conjugates of glycopeptide antibiotics with boroles, 31, 32 and with kanamycin a 34 were synthesized. the conjugates exhibit activity against resistant vre and(or) visa strains. the synthesis of chimeric (heterodimeric) macrolide-based antibiotics is a promising area of research to search for new antimicrobial agents. 28 these antibiotics are among the most effective broad-spectrum antibacterial agents. the semi-synthetic antibiotics clarithromycin (29) and azithromycin (30) are commonly used in medicine for the treatment of various infectious diseases caused by many gram-positive and gram-negative bacteria (fig. 4) . azithromycin (30) has the best pharmacological profile among macrolide antibiotics. the mechanism of action of macrolides is based on the inhibition of protein synthesis. the target of macrolides is the peptidyl transferase centre on the large 50s subunit of bacterial ribosome. however, clarithromycinand azithromycin-resistant clinical isolates of bacteria were isolated. 36 researchers at the gause institute of new antibiotics have developed methods for the conjugation of macrolide antibiotics with benzoxaboroles or polycyclic glycopeptide antibiotics and synthesized series of new chimeric antibiotics. conjugates based on clarithromycin (29), azithromycin (30) and various substituted benzoxaboroles were synthesized by the targeted modification of macrolides at the c(9), c(2 h ) and c(4 hh ) atoms and the c(11) ± c(12) bond (see fig. 4 a ± d ). according to the literature data, the introduction of arylalkyl groups at the 4 hh position of the cladinose moiety may help antibiotics overcome resistance caused by methylation of the macrolide-binding site of 23s rrna of the large ribosomal subunit. 37 a method was developed for the introduction of aminobenzoxaboroles at the c(4 hh ) atom of the cladinose moiety of the antibiotic through the carbamoyl group. 38 hybrid structures containing hydroxamic acid-derived benzoxaborole at the c(9) keto group of the aglycone were prepared, because modification of this group does not lead to the loss of antibiotic activity. scheme 10 shows the synthesis of conjugates based on clarithromycin 29 via the introduction of benzoxaborole groups (a and b) into the antibiotic molecule at the c(9) atom of the aglycone or at c(4 hh )7o-cladinose with acetyl protection of the c(2 h )7oh group of desosamine. 38 the former approach is based on the treatment of clarithromycin (29) with aminoacetic acid giving intermediate clarithromycin 9-syn(anti)-(o-carboxymethyl)oxime (31a). the reaction of 31a with aminobenzoxaboroles ha figure 4 . structures of the macrolide antibiotics clarithromycin (29) and azithromycin (30) . arrows indicate the directions of modification: at the 9 position of the aglycone (a), at the c(2 h )7oh group of desosamine (b), at the c(4 hh ) atom of cladinose (c), the formation of c(11),c(12)-cyclic carbonate and modification of the c(11) atom (d ). or hb gives the corresponding amides of clarithromycin (e/z)-9-carboxymethoxime 31b,c (see scheme 10). another approach was accomplished by a modified procedure 39 involving the following four steps: the protection of the c(2 h )7oh group of desosamine by the acetyl group giving 2 h -oac-clarithromycin (32), the transformation of the latter into activated 2 h -oac-clarithromycin 4 hh -o-1h-imidazole-1-carboxylate (33) by the treatment with carbonyldiimidazole (cdi), the amidation of 33 with amines ha or hb in the presence of the peptide coupling reagent 1,8-diazobicyclo [5.4 .0]undec-7-ene (dbu) giving 2 h -oac-substituted carbamoyl derivatives of clarithromycin 34a,b, and the deacetylation of 34a,b by heating in methanol to form target unprotected carbamoyl derivatives of clarithromycin 35a,b. compounds 31b,c and 35a,b exhibit the inhibitory effect against staphylococci and streptococci comparable with the activity of starting compound 29. in these assays, derivatives at the c(4 hh )-cladinose position were found to be more effective than the derivatives at the c(9) position of the aglycone of antibiotics 31b and 31c. compound 35b is the most effective against the strains staphylococcus epidermidis atcc 12228 and streptococcus pneumoniae atcc 49619. clarithromycin analogues 31c and 35b possess an opposite activity against gram-negative bacteria, such as sensitive strains of e. coli and resistant strains of e. coli (tolc and tolc puc erm42). thus, compound 31c is more active than 35b. in the former case, e. coli tolc and tolc puc erm42 are bacterial strains containing the outer membrane protein tolc, which is responsible for antibiotic efflux from the cell. in the latter cases, the strain contains, apart from tolc, the puc plasmid cloning vector and methylase erm42. 38 attempts were made to extend the method developed for the synthesis of c(4 hh )-substituted clarithromycin ± benzoxaborole conjugates to the synthesis of related azithromycin conjugates (scheme 11). 39 it appeared that the success of introducing the aminobenzoxaborole moiety into a macrolide antibiotic depends on the structure of antibiotics 29 and 30, as well as on the structure of aminobenzoxaborole. the acetylation of azithromycin (30) giving the 2 h -oac derivative (36) followed by the treatment of the latter with n,n h -carbonyldiimidazole in the presence of et 3 n produces the desired activated imidazole derivative ð azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate (37) (see scheme 11) . however, unlike the synthesis of clarithromycin analogue 34a, the amidation of compound 37 with 7-(hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole)methylamine in the presence of dbu does not give the desired outcome. the introduction of the latter amine was accomplished using the trisubstituted derivative, the 11,12-cyclic carbonate azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate 38 as the substrate. compound the same conditions as those used for 37 was not successful (see scheme 11) . 40 meanwhile, this reaction with another amine containing an aminoethyl spacer gives the corresponding azithromycin carbamoyl derivative 39 (see scheme 11) . 40 however, the elimination of the 2 h -oac group finally results in the decomposition of the deacetyl derivative during its purification on silica gel. an alternative procedure for the introduction of benzoxaboroles into molecule 30 using carboxy derivatives and diaminoalkane spacers (scheme 12) proved to be more successful. 40 the treatment of imidazole derivative 38 with stronger bases, such as diaminoethane or 1,3-diaminopropane, in the presence of dbu resulted in the formation of aminoalkylcarbamoyl derivatives 40 (n = 2) and 41 (n = 3). the subsequent acylation of the latter with various benzoxaborole acids under standard conditions (dcc, hobt) gives a series of acylaminoalkylbenzoxaborole-containing carbamoyl derivatives of 11,12-cyclic carbonate, 2 h -oacazithromycin 42a ± 44a (see scheme 12) . the deacetylation of these compounds affords the corresponding cyclic carbo-nates 42b ± 44b containing the free c(2 h )7oh group of the desosamine moiety (r 1 = h) in quantitative yields. compound 46b and its 2 h -oac analogue 46a were synthesized from azithromycin 2 h -oac-4 hh -o-1h-imidazole-1-carboxylate 37 through intermediate 2 h -oac analogues 45 (n = 2, 3) (scheme 13). 40 therefore, the synthesis of compound 46a and its 2 h -oac analogue 46b showed that the introduction of the acylaminoalkylbenzoxaborole moiety can be accomplished using a scheme described above without protection of the c(11)7oh and c(12)7oh groups by cyclic carbonate. the evaluation of antibacterial activity of 4 hh -o-substituted derivatives 39, 42a ± 44a (n = 2), 42b ± 44b (n = 2, 3) and 46b (n = 2, 3) compared with that of compound 30 showed that the activity of compounds 39, 42 ± 44 and 46 against gram-negative bacteria (5 isolates) is lower than that against gram-positive bacteria (8 isolates). for example, the activity of compounds 39, 42a (n = 2), 43a (n = 3) and 42b (n = 3) against the gram-positive strains streptococcus pyogenes atcc 19615 and propionibacterium acnes atcc 6919 is comparable with that of the starting compound 30, while conjugates 42b (n = 3), 43a (n = 2), 43b (n = 2) and 44b (n = 2) are more effective than compound 30 against the strains streptococcus pneumoniae atcc 49619 or enterococcus faecium. the presence of the 2 h -oac group or 11,12-cyclic carbonate in analogous hybrid antibiotics was found to have almost no effect on antibacterial activity. for compounds 42a (n = 2, 3), 43a (n = 2, 3), 44a (n = 3), 42b ± 44b (n = 3) and 46b (n = 2, 3), the mechanism of antibacterial activity was studied using the prfpcer-trpl2a reporter construct, which responds to inhibitors of translocation of the ribosome along the matrix nucleic acid (mrna). 40 all compounds were found to inhibit the peptide chain growth at the exit from the ribosome tunnel like typical macrolide antibiotics. it is worth noting that one of the starting benzoxaborole acids, 3-(1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborol-7-yl)propanoic acid (see schemes 12 and 13, marked with an asterisk), also exhibited activity in this assay. 3.3. azithromycin ± benzoxaborole conjugates at the 11-oh group of the aglycone 11,12-cyclic carbonate can be used not only as the protecting group for two hydroxyl groups but also as the activated group to introduce benzoxaborole substituents at the 11 position of antibiotics. 41 the reaction of 11,12-cyclic carbonate azithromycin 2 h -oac derivative 47, which was generated from azithromycin (30) in two steps (treatment with ethylene carbonate followed by acylation of the 2 h -o group of the desosamine moiety), with diaminoalkane gave 2 h -oacetylazithromycin 11-aminoalkylcarbamates 48 (n = 3, 5) (scheme 14). the acylation of compound 48 with benzoxaborole acids under standard conditions (dcc, hobt) produced a series of azithromycin 2 h -oac derivatives 49a ± 51a. the deacetylation of these compounds afforded target benzoxaborole derivatives 49b ± 51b. new hybrid antibiotics 49b ± 51b exhibit broader-spectrum antibacterial activity against gram-positive and gram-negative bacteria compared to azithromycin (30) and tobramycin. compound 50b proved to be the most effective compound in this series, but its activity is lower than that of compound 30. the modified antibiotics do not overcome the antibiotic resistance in mrsa strains (strain atcc 33591). higher activity of these three compounds against the sensitive strain s. pneumonia atcc 6301 compared to tobramycin (mic is 40.06 ± 0.25 versus 4 mg ml 71 ) is a particularly valuable property. 41 , or , or , dcc, hobt; dcc is n,n h -dicyclohexyl carbodiimide, hobt is 1-hydroxybenzotriazole; in the former case, the reaction of azithromycin 11-aminoalkylcarbamates 48 (n = 3, 5) with antibiotics 1, 3 or 13 in the presence of pybop affords the corresponding derivatives of vancomycin (52, 53) , eremomycin (54) or the teicoplanin aglycone (55, 56) (scheme 15). 42 in the latter case, 11,12-cyclic carbonate azithromycin 4 hh -o-alkylaminocarbamoyl derivatives 40 and 41 are amidated with antibiotics 1, 3 or 13 in the presence of pybop (scheme 16). after the removal of the 2 h -o-acetyl protecting group from compounds 57a ± 61a and the column chromatographic separation on silanized silica gel followed by sephadex lh-20 chromatography, five new conjugates 57b ± 61b were isolated in 35% ± 45% yields based on the corresponding starting antibiotic. 42 antibacterial activity of derivatives 52 ± 55 modified at the c(11)7oh group of the aglycone was evaluated compared to the starting antibiotics vancomycin (1) and azithromycin (30) on a panel of gram-positive and gramnegative bacterial strains (8 and 3 strains, respectively). 42 none of the conjugates exhibited activity against gramnegative bacteria, which attests to the absence of the effect of the azithromycin moiety active against gram-negative bacteria. generally, compounds 52 ± 55 display similar or somewhat lower activity against gram-positive bacterial strains compared to compounds 1 and(or) 30, the activity against staphylococci being higher than that against the streptococci s. pneumoniae 49619 atcc and s. agalactis 52-2. azithromycin ± teicoplanin aglycone conjugate 56 containing a long spacer (n = 5) exhibits higher activity against all tested gram-positive bacterial strains (staphylococci and 42 derivatives at the 4 hh -cladinose position show a similar tendency. thus, they are inactive against gram-negative bacteria. the fact that the hybrid structures are ineffective against e. coli 25922 atcc and other gram-negative bacterial strains indicates that they cannot penetrate the outer phospholipid layer of the bacterial cell. in all the tested gram-positive bacterial strains, compounds 52 ± 55 exhibited activity comparable to or higher than that of azithromycin (30) and vancomycin (1) . their activity is provided by the presence of the glycopeptide moiety. unlike hybrid vancomycin analogue 58b, hybrid eremomycin analogue 59b displays significant activity against the vancomycin-resistant enterococci (vre) strains enterococcus faecium 569 and enterococcus faecalis 560 (mic = 3.2 and 6.5 mmol l 71 ), which can be attributed to the effect of the azithromycin moiety attached to the c-terminal group of the peptide core of antibiotic 3. 42, 43 the mechanism of action against gram-positive bacteria was confirmed by quantum chemical calculations of the energy of interaction dg 298 in relation to hybrid antibiotics 58b and 59b with the model d-ala-d-ala ligand typical of glycopeptides. 43 these studies resulted in the development of methods for the synthesis of macrolide-based hybrid antibiotics containing benzoxaborole as a new pharmacophore. 38, 40, 41 the behaviour of the macrolide antibiotic aglycone in chemical reactions was found to be affected by its structure, in particular it depends on the presence of an additional methylamino group in azithromycin (30) . 38, 40 the position of the benzoxaborole substituent was shown to influence the antibacterial activity of antibiotics 29 and 30. it was established that the c(11)-substituted analogues are less effective inhibitors of gram-positive and gram-negative bacteria compared to 4 hh -substituted analogues. 40, 41 the presence of 11,12-cyclic carbonate or the 2 h -o-acetyl group in the azithromycin molecule was shown to have no significant effect on the antibacterial activity of the conjugates, while an increase in the spacer length generally leads to an increase in activity of the final compounds. a method was developed for the synthesis of a series of chimeric antibiotics based on glycopeptides and azithromycin (30) . 42 the activity of almost all the synthesized compounds against the tested gram-positive bacterial strains, including vancomycin-resistant strains, is similar to or higher than that of the starting antibiotic. the range of antibacterial activity of the resulting hybrid derivatives and quantum chemical calculations suggest that the antibacterial activity is determined by the presence of the glycopep carbohydrate-containing polyene macrolides are commonly used in medicine for the treatment of both superficial and systemic mycoses due to their high activity and a broad spectrum of action. 44, 45 the mechanism of action of polyene macrolides is related to their ability to interact with sterol-containing cytoplasmic membranes and form pores (channels) in these membaranes, by which ions leave the cell causing its death. the efficacy of polyenes against fungal pathogens is due to their stronger binding to fungal membrane ergosterols compared to cholesterol present in human and animal cell membranes. nystatin (62a), partricin and pimaricin are administered locally, whereas amphotericin b (amb, 63a) is the only polyene that is applied for the treatment of systemic mycoses. unfortunately, polyenes are rather toxic agents because of their low selectivity for fungal versus mammalian cell. poor solubility of polyenes in water, their high hematotoxicity and nephrotoxicity and a number of other adverse effects have stimulated an extensive search for new, less toxic and more effective agents. previously, it was shown that toxicity of compound 63a and other polyenes can be reduced by chemical modification, which leads to a decrease in side effects. 44, 45 the toxic effect of compound 63a on blood cells (haemolysis) is particularly dangerous. in order to improve antifungal properties, cytotoxic and therapeutic characteristics and to study the mechanisms of action, series of new semi-synthetic derivatives based on amb (63a) and bioengineered analogues s44hp (64a), bsg005 (65a), bsg022 (66a), bsg019 (67), bsg003 (68a) and bsg018 (69) were synthesized (in collaboration with the company biosergen, norway) (scheme 17). 46 ± 49 the structural diversity of the above-mentioned polyenes 64a ± 66a, 67, 68a and 69 was provided by using methods of genetic engineering to alter genes encoding the nystatin-producing strain streptomyces noursei. 50 new analogues compare favourably with nystatin (62a), primarily due to the presence of a double bond (instead of a single one) at c(28) ± c(29) characteristic of 63a and other polyenes 64 ± 69. the heptaene group of the aglycone imparts rigidity to the antibiotic structure and improves antifungal activity. new monosubstituted polyene macrolides at the terminal 16-co 2 h group of the aglycone were synthesized based on compounds 63a and 64a; monosubstituted polyene macrolides at the mycosamine 3 h -amino group were prepared based on 63a, 64a and 65a. 47, 48 the synthesis of doubly modified analogues of antibiotics 63a and 64a was reported. 48 the related amido derivatives (64b ± 64h) substituted in a similar way at the c(16)-carboxamide group of s44hp were prepared. 46 ± 49 the yields of c(16)-amido derivatives were *50% ± 90% depending on the structure of the starting amine and the antibiotic. the antibiotic structure was found to have almost no effect on antifungal activity against the fungal strains candida albicans (atcc 14053), cryptococcus humicolus (atcc 9949), aspergillus niger (atcc 16404) and fusarium oxysporum (vkm f-140). the activity in each pair of the derivatives containing the same substituents is almost the same in magnitude. carboxamides 63b,d,g and 64b,e,i were found to be the most effective against the above-mentioned strains (mic 50 & 0.5 ± 2 mg ml 71 ). the major directions of chemical modification of s44hp (64a) and bsg005 (65a) at the mycosamine 3 h -amino group in alkylation and aminoacylation reactions, the aminocontaining reagents are protected by the 9-fluorenylmethoxycarbonyl (fmoc) group. conventional 3 h -n-alkyl derivatives of polyenes, such as the 3 h -n-(4-dimethylaminobenzyl)-substituted compounds s44hp (64l) and bsg005 (65c) and the n,n-di(aminopropyl)-substituted compounds s44hp (64m) and bsg005 (65d), were synthesized by reductive alkylation of compounds 64a and 65a with appropriate aldehydes in the presence of nacnbh 3 (see scheme 19, conditions b and c). 47 the yields of compounds 64m and 65d with respect to the starting antibiotics are *12% ± 20%. 3 h -n-acyl derivatives 64n and 65e were synthesized by the reaction of antibiotics 64a and 65a with n a ,n e -(fmoc) 2 -l-lysine in the presence of pybop in *71% ± 74% yields (see scheme 19, conditions d ). the removal of the fmoc group from intermediate derivatives 64o,p and 65f,g with a 5% piperidine solution in dmso 47 affords the corresponding 3 h -n-aminoacyl derivatives 64m,n and 65d,e in *11% ± 20% yields. the evaluation of the influence of substituents in the terminal group at the c(16) atom of the aglycone and the mycosamine amino group on the antifungal activity of polyenes showed that the replacement of the co 2 h group by me has no significant effect on antifungal activity against the tested strains. 48 the activity of s44hp (64a) and its analogue 64k is similar to that of bsg005 (65c) and its analogue 65b. thus, the effect of the same modifications on the activity of the initial antibiotics s44hp and bsg005 with very close values of antifungal activity can be multidirectional. the mic 50 values are changed in the following series: 64a = 65a, 64k = 65b, 64l > 65c, 64m > 65d, 64n < 65e. (77) . the removal of the fmoc protecting group from compound 72a gave 3 h -n-(l-lysyl)-s44hp n-(3-dimethylaminopropyl)amide (72b). 49 an alternative scheme involves the initial synthesis of fmoc-protected 3 h -n-aminoacyl derivatives of s44hp followed by their transformation into the corresponding c(16)-carboxamides (scheme 21). the reaction of compound 64a with n-fmoc-4-aminomethylbenzoic acid in the presence of pybop affords 3 h -n-(n-fmoc-4-aminomethylbenzoyl)-s44hp (78) . the amidation of the latter with appropriate amines in the presence of pybop gives 3 h -n-(n-fmoc-4-aminomethylbenzoyl)-s44hp dmae-amide (79a) and 3-hydroxypropylamide (80a). known compound 64p, which was prepared by the reaction of 64a with n a ,n e -(fmoc) 2 -l-lys in the presence of dcc and hobt, was used to synthesize n-(2-dimethylaminoethyl)amide (81a) and 3-hydroxypropylamide (82a) of n a ,n e -(fmoc) 2 -l-lysyl-s44hp. the removal of the fmoc group from compounds 79a ± 82a under mild conditions gave target products 79b ± 82b containing free amino groups (see scheme 21) . 49 the evaluation of antifungal activity of doubly modified s44hp derivatives 70 ± 82 against the above-mentioned four fungal strains compared to the corresponding monomodified s44hp c(16)-carboxamides 64b,c demonstrated that the additional modification of the mycosamine 3 h -amino group of carboxamide 64b has no significant effect on antifungal activity against these fungal and yeast strains (mic *0.5 ± 2 mg ml 71 ). meanwhile, the corresponding modifications of s44hp 3-hydroxypropylamide lead to a considerable decrease in antifungal activity. in the series of doubly modified derivatives, s44hp 2-n,n-dimethylethylamides (73 and 74, respectively), prepared via the amadori rearrangement with d-glucose or d-galactose, exhibit the highest activity, similar to that of the starting antibiotics 63a and 64a. 49 experiments in animals play a significant role in the selection of lead antifungal agents. since only rather toxic amb (63a) is used for the treatment of systemic fungal infections, compounds exhibiting the highest in vitro activity are currently tested for the haemolysis and(or) acute toxicity. new genetically engineered polyene macrolides 64a, 65a, 66a and 68a, the semi-synthetic derivatives dmae-s44hp (64b), 3 h -n-lys-bsg005 (65e) and doubly modified 3 h -n-(1deoxy-d-fructos-1-yl)-s44hp dmae (73) were evaluated for antifungal activity in the treatment of candida albicansinduced sepsis in mice and tested for toxicity. 47, 49 the largest margin between the therapeutic and toxic doses was observed for compounds 64b and 73. compounds, the effective dose was 2% and 6% of the maximum tolerated dose (mtd), respectively, whereas amb (63a) is effective only at a dose of 62% of mtd. the antifungal activity of c(16)-methyl-c(16)-decarboxypolyenes against four fungal strains changes in the following order: bsg005 [c(7), c(10)] (65a) > bsg019 [c(7)] (67) > bsg018 [c(7), c(9)] (69) . 48 this confirms the pattern of changes in the activity against c. albicans observed previously in the series of c(16)-carboxy-containing antibiotics with a similar arrangement of hydroxyl groups at c(7)7c(10): s44hp (64a) > bsg022 (66) [c(7)] > bsg003 (68a) [c(7), c(9)]. 50 the following amides were synthesized from polyenes 64a, 66a and 68a and 2-(n,n-dimethylamino)ethylamine according to the conventional amidation method in the presence of pybop: dmae-s44hp (64b), dmae-bsg022 (66b) and dmae-bsg003 (68b) (see scheme 17) . 47, 48 the activity of these compounds, like that of the starting antibiotics, changes in a similar series: dmae-s44hp (64b) > dmae-bsg022 (66b) > dmpe-bsg003 (68b). it is worth noting that low antifungal activity of compounds 66b and 68b was confirmed also by animal experiments related to the treatment of murine candida sepsis. these studies clearly demonstrated that the c(7)7c(10) group of the polyol moiety plays a critical role in antifungal activity, although this group has not previously been considered of importance in the model of antibiotic binding to the target. compounds containing a single hydroxyl group at the 7 position of c(7)7c(10) (66a and 67) exhibit low activity against the tested fungal strains. polyenes containing two hydroxyl groups at the 7 and 9 positions (68a and 69) are inactive. 48 antibiotics and semi-synthetic derivatives containing hydroxyl groups at the 8 and 9 positions (amb, 63a,b) and at the c(7) and c(10) atoms [s44hp (64a,b) and bsg005 (65a,b)] displayed the best results. the design of hybrid analogues of antibiotics containing pharmacophore moieties, which affect targets different from those used by the starting antibiotics, is a promising line of research. 28, 29 some benzoxaborole-containing compounds exhibit pronounced antifungal activity. 51 methods were developed for the synthesis of dual-action antibiotics based on amb (63a) and different types of benzoxaboroles. the following five types of conjugates were synthesized depending on the nature of the functional group in benzoxaborole, which can be used to attach the latter to the amb molecule: c (16) 3 h -n-sulfo derivative 87 and 3 h -n-mono-and 3 h -n,n-dialkyl derivatives 88a and 89 (scheme 22). 52 amide 83 was produced by the standard procedure that was applied to prepare the above-described amides; however, the yield of 83 was low (10%). apparently, the amidation interferes with the formation of the aminoborole complex with amb. as mentioned above, the yields of amb carboxamides in this reaction using other amines are higher than 50%. 3 h -n-derivatives 84a ± 86a were prepared from amb (63a) using in situ generated osu-activated esters. 3 h -n-sulfo derivative 87 was synthesized by the reaction of 63a with the appropriate sulfochloride in the presence of pyridine (py); 3 h -n-alkyl analogues 88a and 89 were prepared by the reaction of 63a with the appropriate aldehyde in the presence of nabh 3 cn. 52 the amidation of derivatives 84a and 87a with n,n-dimethylethylenediamine in the presence of pybop gave 84b and 87b, respectively (scheme 23). an attempt to synthesize disubstituted 3 h -n-alkyl-dmae analogue 88b according to a similar scheme from 3 h -n-alkylamino derivative 88a failed (see scheme 22) . 50 nevertheless, compound 88b was prepared using an alternative approach by the alkylation of c(16)-amide derivative 63b with 1-hydroxy-1,3-dihydrobenzo[c] [1, 2] oxaborole-6-carbaldehyde in the presence of nabh 3 cn (see scheme 23). 52 tandem mass spectrometry (esi-ms/ms-mrm) studies showed that different fragmentation patterns are possible depending on the modification of the starting polyene (scheme 24). 52 in many cases, the introduction of the benzoxaborole substituent leads to a decrease in cytotoxicity and haemolytic activity with retention of high antifungal activity. these facts were confirmed by membrane activity assays. 53 the results are given in table 1 . semi-synthetic amb derivatives 63b,d,e, 84a,b, 85a, 87b and 88a,b were shown to have a significant pore-forming ability in artificially formed sterol-containing membranes. 53 compounds with high antifungal activity and low haemolysis have higher selectivity for ergosterol-containing fungal membranes (c chol /c erg ) versus cholesterol-containing human cell membranes compared to compound 63a. for example, the high selectivity (5.4 ae 1.0) of compound 84b correlates with low haemolysis of human erythrocytes (6%). on the contrary, high haemolysis (47%) is determined by low or even reverse selectivity (c chol /c erg = 0.5 ae 0.2). for amb (63a) and its derivative 63b, the corresponding parameters have intermediate values (c chol /c erg = 2.3 ± 2.4, haemolysis 15% ± 23%). 53 amphotericin b (63a) was selectively modified at the mycosamine 3 h -amino group or the c(16) carboxyl group. the developed approaches were extended to the related polyenes s44hp (64a), bsg005 (65) and other genetically engineered polyenes, which differ from the starting amb by the substituent at the c(16) atom and the positions of hydroxyl groups at c(7) ± c(10) of the aglycone moiety. the structure ± activity relationship analysis of new semisynthetic derivatives of polyene macrocycles revealed several general features of antifungal activity. in particular, antibiotics and, consequently, their semi-synthetic analogues containing two hydroxyl groups in this region at the 8 and 9 positions (amb, 63a) or the 7 and 10 positions (s44hp and bsg005) exhibit high activity. a series of new semi-synthetic derivatives were shown to have pore-forming ability in artificially formed sterol-containing membranes. it is worth noting that they have selective activity against ergosterol-containing fungal membranes and lower haemolysis compared to amphotericin b. the aim of chemical modifications of the antibiotic oligomycin a (90a) (fig. 6 ) acting as the f 0 f 1 -atp synthase inhibitor is to prepare new analogues possessing selective antitumour or anti-infective activity and to elucidate the mechanisms of sensitivity of microorganisms to this agent. oligomycin a (90a) (hereinafter oligomycin) belongs to macrolides ð 26-membered a,b-unsubstituted macrolactones. it is a highly specific inhibitor of oxidative phosphorylation in the mitochondria of eukaryotes. oligomycin inhibits adenosine triphosphate (atp) synthesis and causes cell death. note. cerg is the minimum concentration of the compound that causes the pore formation in the dphpc/erg bilayer; cchol/cerg is the ratio of the minimum concentrations of the polyene in the dphpc/chol and dphpc/erg bilayers (dphpc is 1,2-diphytanoyl-sn-glycero-3-phosphocholine, chol is cholesterol, erg is ergosterol). the oligomycin molecule (90a) was found to contain a functional group, the chemical modification of which can produce the largest number of semi-synthetic analogues with various biological activities. the docking study of the interaction between the antibiotic and the target of the enzyme f 0 f 1 -atp synthase showed that the c(32)7c (34) side chain is not directly involved in the formation of this complex. 54 a series of 33-substituted oligomycin derivatives were synthesized using 33-deoxy-o-mesyl oligomycin (91) as the key compound, which was prepared by the selective treatment of 90a with methanesulfonyl chloride in a dmap ± py mixture (scheme 25). 55 the nucleophilic substitution of the group r via the s n 2 or s n 1 mechanism using various reagents gave the following 33-substituted derivatives: 33-(s)-oligomycin a (90b), 56 33-deoxy-33-(s)-thiocyanooligomycin (92), 57 33-deoxy-33-(r,s)-bromooligomycin (93) 58 and 33-deoxy-33-(s)-azidooligomycin (94) 55 (see scheme 25) . the racemization is observed only for 33bromo derivative 93. the 33-epimer of this antibiotic, 33-(s)-oligomycin a (90b), was synthesized by the solvolysis of 33-(r)-deoxy-omesyl oligomycin (91) with an aqueous mixture of tiourea and methyl cellosolve on heating. the reaction involves the walden inversion of configuration at the c(33) atom through a plausible mechanism presented in scheme 25. the biological activity assay of compound 90b revealed that the inversion of the hydroxyl group decreases activity against the actinobacteria streptomyces fradiae, while the antifungal activity remains at the same level. 33-(s)-oligomycin a (90b) exhibits somewhat higher activity against tumour cells compared to the starting analogue 90a. both antibiotics are able to overcome different drug resistance phenotypes and have low toxicity to non-malignant cells. 56 the treatment of oligomycin 91 with 98% formic acid afforded c(33)-o-formyloligomycin (95) 59 (see scheme 25) . formylated derivative 95 retains the ability to inhibit tumour cell growth, whereas the activity against most other test cultures, including non-malignant cells, decreases. due to selectivity against tumour cells, c(33)-o-formyloligomycin (95) holds promise for further investigation. 33-deoxy-o-mesyl oligomycin (91) can be quantitatively converted into 33-dehydrooligomycin (96) by the kornblum oxidation in a dmso ± et 3 n mixture at 105 8c (see scheme 25) . 54 attempts to oxidize the oh group at the c(33) atom of oligomycin (90a) to the keto group using different oxidizing agents failed. the cited study is interesting because this structure was previously presented as a new natural compound. however, the structure of this compound was not described in detail and its biological activity was not evaluated. derivative 96 exhibits twice lower activity against s. fradiae atcc-19609 compared to com pound 90a; its activity against candida spp. and other filamentous fungi is very similar to that of compound 90a. docking studies of the binding of derivative 96 to f 0 f 1 -atp synthase also showed that the affinity for the enzyme decreases compared to the starting antibiotic 90a. 54 a method for the synthesis of 1,4-disubstituted 1,2,3triazoles of oligomycin was developed based on 33-azido-33-deoxyoligomycin 94. the method involves the regioselective [3+2]-dipolar cycloaddition of the 33-azido group to monosubstituted alkynes (scheme 26). 55 the reaction of azide 94 with alkynes (phenylacetylene, propiolic acid and methyl propiolate) in a tert-butanol ± water mixture (1 : 1) can be performed both in the presence of a catalyst (cu i ) or without catalysts. this approach was applied to synthesize 33-deoxy-33-(4-phenyltriazol-1-yl)oligomycin (97), 33-deoxy-33-(4-methoxycarbonyltriazol-1-yl)oligomycin (98) and 33-deoxy-33-(4-carboxytriazol-1-yl)oligomycin (99) in 53%, 52% and 50% yields, respectively. compound 99 served as the starting compound for the synthesis of water-soluble 33-deoxy-33-[(4-dmae-carbonyl)triazol-1yl]oligomycin amide (100) exhibiting selective antitumour activity. 55 oligomycin 90a undergoes a retro-aldol rearrangement accompanied by dehydration in the presence of bases (scheme 27). 60 the structure of alkaline degradation product 101a was established by the detailed 1 h and 13 c nmr study, including heteronuclear correlation, combined with tandem mass spectrometry (esi-ms/ms-mrm). the structure of the carbon skeleton of the starting antibiotic 90a undergoes a significant transformation at c(7)7c(12) (the cleavage pathways a, b and c) giving open-ring compound 101a. the mechanism of alkaline degradation of oligomycin 90a presented in scheme 27 accounts for the formation of derivative 101a (through intermediates 90c,d) but not for the formation of 101b (through intermediates 90e,f). 61 as opposed to compound 90a, compound 101a does not exhibit activity against proteasomal f 0 f 1 -atp synthase at a concentration of 1 mmol l 71 because of the loss of conformational rigidity. the hydrogenation of oligomycin (90a) on a palladium catalyst occurs both at the 2,3-unsaturated bond of lactone and the diene system at c (16) the hydrogenation of compound 90a under other conditions leads to the sequential selective reduction of keto groups ð first at the c(7) atom and then at the c(11) atom. thus, the use of nabh(oac) 3 latter with nabh 4 in ethanol afforded (7s,11r)-tetrahydrooligomycin (104) (see scheme 28) . 59 the hydrogenation of double bonds of the macrolactone ring causes a decrease in the activity of compound 90a both against actinobacteria and fungal and mammalian cells. 62 this may be attributed to the loss of conformational rigidity and the fact that the geometry of the macrocycle favourable for the interaction with the target (f 0 f 1 -atp synthase) is changed because of destruction of the diene system of the starting antibiotic 90a. the retention of activity of the starting compound 102 against some strains of candida spp. supports the previous suggestion that there are additional targets in yeast cells of this genus, the binding to which is apparently independent of the geometry of the macrocycle. the reaction of compound 90a with m-chloroperoxybenzoic acid (m-cpba) in dichloromethane on decreasing the reaction temperature to 717 8c allows the selective epoxidation of oligomycin at one of c = c bonds to form unstable intermediate epoxide 105 (scheme 29). 59 in the presence of formic acid, the latter compound gives a stable disubstituted oligomycin derivative ð 16,17-dihydro-16s,17s-dihydroxy-16,33-diformyloligomycin (106). previously, 16-bromo derivative 107 of this antibiotic was synthesized using n-bromosuccinimide as the brominating agent (see scheme 29). 63 the addition of hydroxylamine and related compounds to 90a was studied. 64 the comprehensive study of the resulting compound by 1 h and 13 c nmr spectroscopy, including heteronuclear correlation, made it possible to establish the structure of nitrone 108a and exclude the possible existence of isomer 108b. it was found that an additional ring involving the c(3) ± c(7) atoms is formed, the activity of the antibiotic against f 0 f 1 -atp synthase being reduced. the reaction of antibiotic 90a with aminopyridinium and 4-dimethylaminopyridinium iodides in pyridine affords cyclic derivatives of pyrazolo [1,5-a] pyridine and 4-methylpyrazolo[1,5-a]pyridine 109a,b, respectively, annulated to the macrocycle (see scheme 30) . 64 their structures were established by 1 h and 13 c nmr spectroscopy, including heteronuclear correlation, and structure 109c was excluded. biological assays of new derivatives of oligomycin (90a) demonstrated that, in most cases, modifications reduce biological activity of the starting antibiotic against s. fradiae. 54, 65 ± 67 the growth inhibition assay of the strain s. fradiae atcc 19609 sensitive to very low concentrations of oligomycin (<0.001 nmol ml 71 or 0.0005 nmol per disc surface) and hypersensitive to most of the known antibiotics was used to study the mechanism of resistance of microorganisms. 66 mutant strains of this microorganism sensitive to analogues of compounds 92 (ref. 67 ) and 108a, as opposed to the starting antibiotic 90a and other derivatives, were produced under experimental conditions. the results of assays demonstrated that antibiotic 90a has several biotargets. these data confirm the conclusion that both the diene system of the macrocycle and its hydroxypropyl side chain play an important role in biological activity. 65 therefore, the chemical modification of the antibiotic oligomycin (90a), a highly active f 0 f 1 -atp synthase inhibitor, was performed for the first time. more than 20 new semi-synthetic oligomycin derivatives were prepared and the mechanism of their action was studied. analogues with selective antitumour or anti-infective activity were synthesized. the chemical modification of antibiotic 90a, which enables the efficient modulation of its biological activity due to a decrease in the binding to f 0 f 1 -atp synthase, was found. this provides the possibility to optimize pharmacological properties. olivomycin a (110) (hereinafter olivomycin) is a highly effective antibiotic with a unique mechanism of antitumour activity discovered in the gause institute of new antibiotics 6, 68, 69 (fig. 7) . its structure consists of the aglycone olivin and two carbohydrate chains attached to the aglycone at the 2 and 6 positions. the antibiotic is a dna duplex minor groove ligand, which binds to the guanine-cytosine (gc)-rich region. compared to other antibiotics of the aureolic acid group (mithramycin and chromomycin a), olivomycin 110 has better therapeutic efficacy. in recent years, antibiotics of this group have attracted increasing interest. 70, 71 antibiotics of this group were shown to be able to prevent the development of resistance of tumour cells to other antitumour agents, in particular via a mechanism involving the inhibition of transcription of the mdr1 gene, overexpression of which is responsible for multidrug resistance of tumour cells. mithramycin is used to treat paget's disease and testicular carcinoma. chromomycin is a drug of limited use in japan for the treatment of gastrointestinal cancer. olivomycin (110) was applied in the ussr for the treatment of ovarian cancer, reticulosarcoma and some other tumours. however, serious adverse effects limit the therapeutic potential of these drugs. various aspects of antitumour activity of antibiotics of the aureolic acid group were addressed in detail; however, chemical modifications of this class of antibiotics are poorly known. selective modifications of olivomycin (110) at the c(5) atom (a), the c(8)7oh (b) and c(2 h ) = o groups (c), the c(2 h )7c(3 h ) bond (d ) and the residues a4 and e4 (e) (see fig. 7 ) were developed in the gause institute of new antibiotics. the selective acylation of compound 110 at the phenolic hydroxyl group at the c(8) atom with a ac 2 o7py mixture affords 8-o-acetylolivomycin (111) (scheme 31). 72 an investigation of the reaction of olivomycin (110) with aryldiazonium salts showed that the azo coupling gives aryldiazenes monosubstituted at the 5 position of the aglycone accompanied by the elimination of the disaccharide branch at the c(6) atom: 5-(phenyldiazenyl)olivomycin (112), 5-(4-sulfamidophenyldiazenyl)olivomycin (113), 5-(4methoxyphenyldiazenyl)olivomycin (114), 5-(3,4-dichlorophenyldiazenyl)olivomycin (115) and 5-(4-methylphenyldiazenyl)olivomycin (116) (see scheme 31) . 72 to explain the outcome of this reaction, the frontier electron density in the highest occupied molecular orbital (homo) was calculated by the semiempirical quantum chemical am1 method (fukui indices f). alternative directions of the nucleophilic attack by the phenyldiazonium cation were chosen by considering possible anionic forms of olivomycin (110a ± 110c) (scheme 32 a). it was found that the 5 position in anion 110a is the most favourable for electrophilic attack giving compound 112 (the fukui index f homo is 0.617). meanwhile, the nucleophilicities of the c(7) and c(10) atoms are lower (0.356 and 0.185, respectively). the formation of anion 110a in an alkaline medium is confirmed by the above-mentioned selective acylation of compound 110 giving acetate 111 at the same hydroxyl group at the c(8) atom in high yield (see scheme 32) . 72 the calculated energy parameters of alkaline hydrolysis of the disaccharide branch are in agreement with the experimental data. thus, the hydrolysis proceeds very rapidly in the presence of the diazenyl substituent at the 5 position, while the storage of 110 in an alkaline medium (used for the azo coupling) on cooling to 0 ± 5 8c for 2 h in the absence of diazonium salt does not lead to hydrolysis. the azo coupling of aryldiazonium salts with the aglycone of olivomycin ð olivin (117), which was synthesized by quantitative acid hydrolysis of compound 110, was investigated. aryldiazenyl derivatives of olivin 117a ± c containing the same substituents as compounds 112, 114 and 115 (scheme 33) were synthesized. 70 the geometric configurations of the most probable tautomeric structures a ± d of derivative 117a were determined (scheme 34) and the total energies of each tautomeric form were calculated by the density functional theory at the b3lyp/6-31g(d) level of theory. the 1 h nmr spectroscopic data also indicate that compounds 117a ± c exist as equilibrium mixtures of isomeric forms a ± d. the reaction of olivomycin (110) with (o-carboxymethyl)hydroxylamine affords 2 h -(carboxymethoxime)olivomycin (cm) 118 (scheme 35). 74 the introduction of the carboxyl group into molecule 110 makes it possible to subject this compound to further modification. the reaction of compound 118 with appro75 the introduction of the carboxyl group into molecule 110, as in the case of long acid 118, provides a route to its further modification. the reaction of short osa 124 with appropriate amines in the presence of pybop or diphenylphosphoryl azide (dppa) affords osa n-methylamide it is worth noting that the method developed for the synthesis of intermediate osa (124) can be applied to prepare the related derivative of mithramycin bearing a similar side chain at the c(3) atom of the aglycone. hence, short' mithramycin acid becomes more available compared to the combinatorial biosynthesis and can be used for further chemical modification of mithramycin. 76 olivomycin a (110) has two carbohydrate chains bearing the acetyl group at the a4 position of the oliose moiety and the isobutyryl group at the e4 position of the olivomycose moiety. a series of analogues of compound 110, which differ in the set of functional groups, were synthesized in order to elucidate the influence of acyl substituents in carbohydrate chains on biological activity. 77 two analogues, de-e4-isobutyrylolivomycin a (133) and de-e4-isobutyryl-de-a4-acetylolivomycin a (or de-e4isobutyrylolivomycin c) (134), were synthesized by selective alkaline hydrolysis of the e4-isobutyryl group of olivomycin a (110) or c (135), respectively (scheme 37). it is worth noting that the hydrolysis of the e4-isobutyryl group in olivomycin (110) antibiotics 135 (de-e4-isobutyrylolivomycin a) and 136 (de-e4-isobutyryl-e4-acetyl-olivomycin a or olivomycin b) were isolated from the natural olivomycin complex produced by fermentation of the streptoverticillum cinnamoneum strain. after the purification by silica gel column chromatography and semipreparative hplc, compounds 135 and 136 were isolated in 40% and 45% yields, respectively. 70, 77 these compounds are identical to the previously characterized natural olivomycins c and b, respectively. 78 the modification of antibiotic 110 at the c(8) phenol group of the aromatic ring (compound 111) was found to have no effect on antiproliferative activity against cancer cell lines and does not alter its ability to inhibit topoisomerase i. 72 the transformation of 110 giving diazenyl derivatives accompanied by elimination of the disaccharide branch from the aglycone (compounds 112 ± 116) leads to a sharp decrease in antiproliferative activity. compared to the starting olivomycin 110, compounds 112, 115 and 116 acquire considerable selective activity against human immunodeficiency viruses hiv-1 and hiv-2 in assays in the human t-lymphocyte cell line (cem). 72 the evaluation of antiproliferative activity of olivomycin (110) and its analogues modified at the side-chain 2 h -keto group of the aglycone (cm amides 119 ± 121 and 123) against the leukaemia cell lines k562 and l1210 showed that these compounds are more effective than the starting acid 118 but are less active than compound 110. 74 for derivatives of antibiotic 110 with a shorter aglycone side chain (osa, 124) and its amides 125 ± 132, the evaluation of antiproliferative activity in the human chronic myeloid leukaemia cell line k562 and the human colon 75 the evaluation of antiproliferative activity of olivomycin analogues 133 ± 136 against hct116 cells showed that acyl groups in carbohydrate chains of antibiotic 110 play a significant role. the activity decreases with elimination of acyl groups from molecule 110; ic 50 is 0.02 (for 110), 0.064 (for 136), 0.28 (for 133) and >50 mmol l 71 (for compounds 117, 134 and 135). 77 these results correlate with the data on the ability of these compounds to inhibit dna-dependent topoisomerase i. 79 in the absence of antibiotics, the relaxation of supercoiled (sc) dna leads to the disappearance of inhibitory activity and the formation of a set of topoisomers. the inhibition of topoisomerase i is detected from the presence of residual amounts of sc-dna and a decrease in the amount of rapidly migrating topoisomers. a decrease in the inhibitory activity of the enzyme is consistent with a decrease in the antiproliferative activity in the series of compounds 110 > 136 > 133. the removal of the e4-isobutyryl group from the trisaccharide branch has a lower effect than the removal of the a4-acetyl group from the disaccharide branch. a similar pattern of activity is observed in another group of olivomycin derivatives. compound 118 displays low antiproliferative activity and is inactive against topoisomerase i. 2 h -(carboxymethoxime)olivomycin n-(2-adamantyl)amide (121) is active in both assays. 2 h -(carboxymethoxime)olivomycin n-(tert-butyl)amide (122) and 2 h -(carboxymethoxime)olivomycin n-(1,3-dihydroxy-2-methylpropan-2-yl)amide (123) are even more effective inhibitors of the enzyme, capable of inhibiting sc-dna relaxation even at concentrations of 0.1 mmol l 71 . 74 the data on antiproliferative activity of some analogues of antibiotic 110 do not correlate with the activity against topoisomerase i. for example, the analogue of olivomycin osa (124), which exhibits weak antiproliferative activity, displays inhibitory activity against topoisomerase i similar to that of olivomycin a (110), whereas dmae-osa (127) possessing high antiproliferative activity is a weaker inhibitor of this enzyme. 74, 79 recently, it was shown that highly effective analogue 127 can act as a dna duplex minor groove ligand in another assay. it can disrupt the key epigenetic dna methylation process with the dnmt3a enzyme on an equal basis with antibiotic 110. both compounds inhibit the formation of the dna ± enzyme7intermediate covalent bond, required for the methylation, at nearly equal micromolar concentrations. 80 olivomycin complexes with model oligonucleotide ± dna duplexes were studied by circular dichroism (cd) and fluorescence titration (ft). according to hartree ± fock 3-21g calculations of the 3d structure of the dimer [110] 2 mg 2+ , the presence of the dmae group in compound 127 leads to an increase in the binding constant (k a ) of the mg 2+ complex with the dna duplex by an order of magnitude compared to the acid osa (124) (k a = 1.35610 5 versus 2.1610 4 mol l 71 , as evaluated by ft). however, the presence of the bulky adamantyl substituent in compound 121 results in a decrease in the binding constant of the dimer [121] 2 mg 2+ with the dna duplex by an order of magnitude (k a = 1.32610 4 mol l 71 ). 81 the elimination of one acyl substituent also leads to a decrease in the antiproliferative activity compared to that of antibiotic 110 due probably to a decrease in its affinity for dna. 77 the molecular docking of complex of 110 with dna shows that the antibiotic can bind only to gc-rich regions in the minor groove of the dna duplex. 82 carbohydrate chains of olivomycin interact with the sugar ± phosphate backbone of dna, and the aglycone interacts with nucleic acid bases. the sites of 110 responsible for the interaction with dna (an additional hydrogen bond with the nh 2 group of the g base) and the complexation of the antibiotic with dna were identified. the structural fragment, which is not directly involved in the interaction with dna but models the affinity of the antibiotic to the target ð the minor groove of the dna duplex, was determined. based on these data, a schematic model of the interaction between olivomycin a and dna was proposed (scheme 38). 73 two semi-synthetic olivomycin derivatives (121 and 127) with the modified aglycone side chain were chosen based on the evaluation of antiproliferative activity in animal assays. compound 121 is more effective in the treatment of p388 murine leukaemia than the starting antibiotic 110 due to a decrease in toxicity and the absence of the cumulative effect. 83, 84 compound 127 (olivamide) is also more effective in this assay compared to the starting antibiotic 110. 85 detailed preclinical assays in transplanted syngeneic tumours confirmed the efficacy of the agent based on compound 127 for further clinical trials. 85 ± 88 these studies enabled the development of methods for selective chemical modification of the antibiotic olivomycin a, resulting in the synthesis of a series of semi-synthetic derivatives of this antibiotic; besides, structure ± activity relationship analysis was performed. 70, 73 many of these compounds exhibited high antiproliferative activity in different tumour cell lines. some aspects of the mechanism of action of olivomycin a and its natural and semi-synthetic analogues were considered. 74, 75, 77, 79 ± 82 it was concluded that high antitumour activity of these compounds is related to their high affinity to the dna duplex. based on the results of in vitro assays, compounds were chosen for the evaluation of antitumour activity in in vivo assays. after the preclinical evaluation of antitumour activity and toxicity in laboratory animals, semi-synthetic olivomycin analogue 127 ð olivamide ð was recommended for further clinical trials. 85 ± 89 anthracycline antibiotics are important chemotherapeutic agents commonly used in the treatment of malignant tumours. daunorubicin (137a), which was independently discovered under the name of rubomycin in the gause institute of new antibiotics (gina), 6 is applied mainly for the treatment of leukaemia in children and adults. doxorubicin (14-hydroxydaunorubicin, dox) (138a) is used in combination chemotherapy of breast cancer, smallcell lung cancer, sarcoma, tumours in children and haemoblastosis. an original method for the one-pot preparation of semi-synthetic doxorubicin from rubomycin was developed in the gina. 6 carminomycin (139a), which was also discovered in the gina, has lower cardiotoxicity than other anthracyclines and can inhibit the growth of doxorubicin-resistant tumours (138a). 90 the structures of anthracycline antibiotics 137a, 138a and 139a are shown in fig. 8 . the mechanism of cytotoxic activity of anthracycline antibiotics is related mainly to the inhibition of nucleic acid synthesis via intercalation between nitrogeneous base pairs, dna damage and inhibition of dna topoisomerases. an adverse effect of anthracyclines is the potentially irreversible and cumulative dose-related cardiotoxicity, apparently associated with the free-radical damage of myocardial cell membranes. the chemical modification of anthracycline antibiotics was extensively studied in the gina for a long period of time. in recent years, efforts were focused on the synthesis of new semi-synthetic analogues of drugs with improved anticancer properties. 91 daunorubicim (137a) and carminomycin (139a) were modified at the c(14) atom (b), the c(13) = o bond (c) and the daunosamine 3 h -amino group (d, e) (see fig. 8 ). 3 h -n-alkyl or 3 h -n-aminoacyl derivatives exhibit high antitumour activity, because they retain the amine function at the daunosamine moiety. 90 this function is required for the primary interaction of the antibiotic with the sugar ± phosphate backbone of dna. however, the drawback of reductive alkylation of anthracyclines 137a and 139a with aldehydes in the presence of nabh 3 cn is that it is accompanied by the reduction of the c(13) = o group as the side reaction giving n-alkyl-13-dihydro derivatives of daunorubicin (137b) and carminomycin (139b), respectively (scheme 39). these analogues are less active than the related compounds containing the c(13) = o group. 92 ± 94 13-dimethyl ketals of 14-bromo derivatives of daunorubicin (137c) and carminomycin (139c) were used to avoid this side reaction (see scheme 39) . the reductive alkylation of compounds 137c and 139c in the presence of nabh 3 cn can be accomplished using d,lglyceraldehyde and aldoses (the monosaccharide arabinose and the disaccharide melibiose) to form the following 14bromo-substituted 13-dimethyl ketals in quantitative yields: the antiproliferative activity of compounds 139e and 143 against leukaemia cells k562 is virtually as high as that of derivative 137e, but is lower than that of carminomycin (139a). the activity of 14-hydroxycarminomycin derivative 143 evaluated in the same assay is an order of magnitude lower than that of the starting anthracycline 139a, and the activity of doxorubicin 137e is two orders of magnitude lower than that of compound 138a. it should be emphasized that 14-hydroxycarminomycin derivatives 142 and 143 are equally active against the wildtype human breast adenocarcinoma cell lines mcf-7 and k562 and resistant pgp-expressing cell lines (mcf-7dox, k562i/s9). the antitumour activity of compound 139e evaluated in the murine leukaemia cell line p388 compares favourably with that of analogue 138a. thus, an increase in life span (ils) of mice bearing p388 leukaemia after a single admin-istration of 10 mg kg 71 of compound 139e is the same as that observed after administration of analogue 138a at a dose of 8 mg kg 71 (ils 108%). 92 it was shown that anthracycline antibiotics 137a, 138a and 139a, which are known to inhibit dna topoisomerase ii at micromolar concentrations, can inhibit dna topoisomerase i at the same concentrations. 75 new derivatives 137e, 139e, 142 and 143 have higher inhibitory activity against topoisomerase i compared to compounds 138a and 139a. the introduction of polyhydroxylated substituents at the 3 h -amino group of the daunosamine moiety of anthracycline antibiotics increases the inhibitory activity of anthracyclines against topoisomerase a method was developed for the preparation of new watersoluble depot forms of doxorubicin (138a) ð conjugates with high-molecular-mass polysaccharides. 95, 96 galactomannan davanat (144) was used as the polysaccharide. the latter was prepared by controlled partial hydrolysis of water-insoluble high-molecular-mass 1,4-b-d-galactomannan isolated from seeds of cyamopsis tetragonoloba or guar gum. the molecular mass of compound 144, determined by gel chromatography on a sephadex g-200 column calibrated with pullulans, is *92 kda. to conjugate compound 144 with 138a, the former is activated by the oxidation with periodate using a deficient amount of the oxidizing agent (0.07 ± 0.11 equiv. with respect to the total amount of sugar residues). this gives rise to schiff bases between the aldehyde groups of the oxidized polysaccharide (145a ± 145c) and the 3 h -amino group of the antibiotic (scheme 40). different structures of the final products are possible (146a ± 146c). the content of the starting antibiotic 138a in conjugate 146 is 5 mass % (determined from the content of the chromophore per unit mass of the dried powder by uv spectroscopy at 490 nm). in order to increase the doxorubicin content in the conjugate with davanat 145, the antibiotic molecule was attached to the polysaccharide through a spacer giving 3 h -n-l-lysyldoxorubicin (147) (scheme 41). 96 compound 147 contains two amino groups (while doxorubicin contains one amino group), which may facilitate the formation of the schiff base. besides, it is known that a series of n-acyl conjugates of daunorubicin or doxorubicin with amino acids possess high antitumour activity. 90 3 h -n-l-lysyldoxorubicin (147) was synthesized by the acylation of the amino group of doxorubicin (138a) with n a ,n e -(fmoc) 2 -l-lysine osu-ester followed by the deprotection of the fmoc-protected intermediate with a morpholine solution. 96 the schiff base in the resulting conjugate can have either a linear (148a) or cyclic structure (148b). scheme 41 presents the possible structures for one of the oxidized subunits of activated davanat 145. it should be noted that 3 h -n-l-lysyldoxorubicin can be attached to conjugate 145 through either the a-amino-or e-amino group of 3 h -n-l-lysyldoxorubicin (is not shown in scheme 41). the introduction of the lysine spacer between compounds 138a and 145 allows an increase in the antibiotic content in the conjugate to 10 mass % with retention of water solubility. the conjugates were purified by gel chromatography on a sephadex g-200 column and dialysis against deionized water using a membrane with molecular weight cut-off (mwco) m w > 15 kda. the molecular masses of conjugates 146 and 148 evaluated by gel chromatography on a sephadex g-200 column calibrated with pullulan standards are * 95 and * 98 kda, respectively. the antiproliferative activity of the doxorubicin conjugates was tested in three tumour cell lines: the murine melanoma cell line b16-f1, the breast cancer cell line mcf-7 and the colon cancer cell line ht-29 (htb-38). 96 the ic 50 values for conjugate 146 (taking into account the percentage of antibiotic 138a in the conjugate) are 0.025 ± 0.04, 0.15 ± 0.22 and 0.65 ± 1 mg ml 71 , respectively; for antibiotic 138a, 0.01 ± 0.02, 0.08 ± 0.12 and 0.2 ± 0.3 mg ml 71 . despite the fact that the cytotoxicity of conjugate 146 is *1 ± 2 orders of magnitude lower (data were not reported) than that of doxorubicin (138a), these results indicate that conjugate 146 is an active depot form of doxorubicin (138a). the antiproliferative activity of the 3 h -n-l-lysyldoxorubicin ± davanat conjugate (148) (ic 50 > 50 mg ml 71 ) against these tumour cell lines is much lower compared to cytotoxicity of conjugate 146. this can be due to the fact that the imine bonds in the 3 h -n-l-lysyldoxorubicin ± da-vanat conjugate (148) are more stable than those in conjugate 146. it should be noted that 3 h -n-l-lysyldoxorubicin is not released in in vitro assays. therefore, a biological model, which would provide release of 147, is apparently required to evaluate the therapeutic potential of conjugate 148. based on these studies, a method was developed for the introduction of polyhydroxylated substituents of different types and different length into anthracycline antibiotics, which was used to synthesize a series of new hydrophilic 3 h -n-alkyl derivatives of doxorubicin and 14-hydroxycarminomycin, including those containing mono-and disaccharide residues. 92, 93 this modification was found to enhance the inhibitory activity of anthracyclines against topoisomerase i with retention of antitumour activity of the antibiotics. the new 14-hydroxycarminomycin derivatives, unlike doxorubicin derivatives, were shown to suppress the tumour cell growth insensitive to doxorubicin. 93 a new water-soluble depot form of doxorubicin ð a conjugate with the highmolecular-mass polysaccharide galactomannan dava-nat ð was prepared. 94, 95 this depot form exhibits antiproliferative activity against the above-mentioned three tumour cell lines. 91 the antibiotic heliomycin (resistomycin, 3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione) (149) with a broad spectrum of biological activity (scheme 42) was discovered in the gause institute of new antibiotics. 6 first of all, this antibiotic is highly effective against grampositive and some gram-negative microorganisms, including drug-resistant strains. more recently, heliomycin was found to exhibit antifungal 97 and antiviral (anti-hiv) activity. 98 besides, compound 149 can block proliferation of some tumour cells in in vitro assays. 99 in the soviet union, heliomycin (149) was produced on a commercial scale and was used as a gel for topical treatment of skin infections and healing of burn wounds. the chemical modification of heliomycin is poorly known. hence, structure ± activity relationship studies require the development of method for the synthesis of new semi-synthetic derivatives and evaluation of their biological properties. the main goal is to prepare series of derivatives with improved water solubility in order to expand their practical application. a method was developed for the synthesis of aminomethyl derivatives 149 at the 4 position of compounds 150 (where x is an amine moiety or a nitrogen-containing heterocycle). typical synthetic procedures are presented in relation to compounds 150a ± c (see scheme 42) . 100, 101 the mannich aminomethylation of compound 149 can be performed using amines and formaldehyde in dmf (see scheme 42 b ). an alternative procedure for the synthesis of derivatives 150 is based on the use of pre-prepared stable iminium salts (see scheme 42, conditions a). in the case of aminomethylation of compounds 149 with polyfunctional amines, the amino group is protected with boc (see scheme 42 c) and then deprotected with acid (see scheme 42 d ). the reaction of compound 149 with n,n-dimethylamino(methylene)ammonium chloride afforded 4-[(n,n-dimethylamino)methyl]-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1-h-benzo[cd ]pyrene-2,6-dione hydrochloride (150a). 100 4-[(tert-butylamino)methyl]-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione hydrochloride (150b) is produced by the treatment of compound 149 with tertbutylmethylamine in the presence of formaldehyde. the treatment of compound 149 with tert-butylpiperidin-4-ylcarbamate in the presence of a formaldehyde solution gives 4-(4-boc-aminopiperidinomethyl)-3,5,7,10-tetrahydroxy-1,1,9-trimethyl-1h-benzo[cd ]pyrene-2,6-dione, which is quantitatively converted to the corresponding amine dihydrochloride (150c) upon the treatment with hcl7meoh. the antiproliferative activity (ic 50 ) was evaluated by the colorimetric determination of cell metabolic activity (mtt assay) using a standard procedure in eight tumour cell lines, including both drug-sensitive and drug-resistant cell lines. 100 the resulting compounds inhibit tumour cell proliferation in a low submicromolar to micromolar concentration range, similar to that of doxorubicin (138a). however, most of these compounds significantly outperform doxorubicin in terms of the drug-resistance index. these compounds block the growth of the wild-type cell lines k562 and hct116 and the following multidrugresistant cell lines: the k562/4 subline isogenic to p-glycoprotein (pgp)-positive multidrug resistance and the hct116p53ko subline with p53 gene deletion. the development of multidrug resistance in these tumour cell lines is the crucial factor responsible for a decrease in activity of antitumour drugs. as opposed to compound 149, derivatives 150a ± c show a high level of induced apoptosis in the t24 bladder cancer cell line model. the introduction of the 4-aminomethyl moiety enhances the dna-binding affinity and the inhibitory activity against dna topoisomerase i. hence, compound 150c is the most promising candidate for preclinical trials. based on these studies, methods were developed for chemical modification of heliomycin (149). series of new analogues, water-soluble salts of amino-containing derivatives, were synthesized and they were shown to exhibit high antiproliferative activity against many tumour cell lines and inhibitory activity against various targets. 100 the value of these studies is that the majority of aminomethyl derivatives of heliomycin are active against both wild-type and drugresistant cancer cells at micromolar or submicromolar concentrations. the development of new-generation drugs is a challenging problem because of the increasing risk of the development of drug resistance in microorganisms. different approaches to the search for new compounds were developed in recent years. particular attention is given to semi-synthetic derivatives based on available natural antibiotics, since there are numerous examples of the successful application of this approach. some semi-synthetic derivatives based on macrocyclic glycopeptides have advantages over the gold standard chemotherapeutic agent ð vancomycin. 3, 7, 10 the distinguishing features of new representatives of this class are selectivity against multidrug-resistant pathogenic bacteria and higher bioavailability. the new semi-synthetic vancomycin analogue telavancin (vibativ) (manufactured by theravance and astellas pharma, us) was approved for use in the united states and europe. two semi-synthetic derivatives of glycopeptide antibiotics have completed phase 3 clinical trials and were approved by the united state food and drug administration (fda): chloroeremomycin (discovered by eli lilly, acquired by the medicine co in 2009) and dalbavancin (discovered by lepetit; acquired by pfizer in 2005). 8, 10 the drawbacks of vancomycin are the poor accumulation within tissues, because of which it is not used in the treatment of, for example, pneumonia, and a pronounced pseudoallergic reaction typical of glycopeptides. research in international cooperation showed the promise of chemical transformations of the antibiotic eremomycin belonging to this group of glycopeptide antibiotics. eremomycin is a highly active domestic antibiotic that suppresses the growth of gram-positive organisms; it is 3 ± 5 times more effective than vancomycin but is inactive against drug-resistant strains of staphylococci and enterococci. 6 original approaches and methods were developed under the supervision of professor m.n.preobrazhenskaya in the gause institute of new antibiotics. these methods can be applied to prepare derivatives of antibiotics of this group with desired properties. in 2006, the method for the synthesis of glycopeptide analogues was protected by an international patent. 18 promising analogues, various n h -derivatives and carboxamides of eremomycin, were synthesized. these derivatives compare favourably in efficacy against drug-sensitive and drug-resistant strains of staphylococci and enterococci with the related vancomycin derivatives and they are even more effective in a number of assays. 12 ± 15 some carboxamides show no allergenicity. 13 other derivatives (e.g., the adamantane derivative of eremomycin) are promising as a protection against biological risks because they were found to be active against the bacterium bacillus anthracis, including fluoroquinolone-resistant strains. 15 high activity of the resulting hydrophobic glycopeptide derivatives can be attributed to the dual mechanism of action on gram-positive bacteria. 20, 21 modifications of aglycones of glycopeptide antibiotics with hydrophobic substituents resulted in the discovery of a new class of polycyclic peptides active against a large group of enveloped viruses, such as hiv 23 or hepatitis c virus. 25 studies on the mechanisms of antiviral activity are currently ongoing. it was suggested that protein kinase is one of possible targets for this agluco analogue, because antiviral activity was shown to correlate with the inhibition of serine/ threonine protein kinases. 27 comprehensive research on the synthesis and characterization of heterodimeric conjugates based on different classes of antibiotics was initiated in the gause institute of new antibiotics under the supervision of professor m.n.preobrazhenskaya. the following hybrid antibiotics were synthesized: glycopeptide ± macrolide, glycopeptide ± aminoglycoside and hybrid compounds containing the benzoxaborole chromophore. the literature data provide evidence that this line of research holds promise. 28, 29 macrolides modified through the carbamoyl group at the 4 hh position can acquire activity against drug-resistant bacterial cell lines. generally, conjugates containing a long spacer exhibit higher activity than the related compounds with a shorter spacer. 40, 41 investigations of interactions between different benzoxaboroles and antibiotics made a significant contribution to the chemistry of not only antibiotics with complex structures but also the relatively poorly studied borole compounds. benzoxaboroles were found to be quite stable under different reaction conditions. in particular, the acylation, amidation and reductive alkylation with reactive agents and on heating are virtually not accompanied by oxaborole ring opening. 32 the introduction of the benzoxaborole substituent into the polyene macrolide amphotericin b (amb) also enhances biological activity. the resulting compounds possess valuable properties, such as lower cytotoxic and haemolytic activity compared to amb combined with high antifungal activity. 52 the efficiency of the approach to the design of antibiotics of a new generation was demonstrated in relation to antifungal polyene macrolides. it is based on a combination of genetic engineering techniques and methods of organic synthesis 48 ± 50 and is protected by an international patent. 46 the chemical modification of polyene antibiotics, which were obtained via the genetic engineering of nystatin a1 biosynthesis at the norwegian university, gave a series of agents exhibiting lower toxicity and higher activity compared to amphotericin b in animal assays. the dependence of antifungal activity on the structure of the polyol region [c(7) ± c(10)] of these antibiotics was revealed for the first time by preobrazhenskaya et al. 49 pioneering studies on the development of methods for selective chemical modification of the unique macrolactone oligomycin a, a specific f 0 f 1 -atp synthase inhibitor, and the original antitumour antibiotic olivomycin a of the aureolic acid group were performed. in both cases, examples of chemical modifications of related compounds for these antibiotics are absent in the literature. these results are of great scientific value. investigations of f 0 f 1 -atp synthase inhibitors are of considerable interest because this enzyme is involved in the development of resistance in microorganisms. chemical transformations of oligomycin a using different reagents were studied in detail. alkaline hydrolysis reactions were performed and conditions for the selective reduction of double bonds and keto groups, cyclization, etc. were found. the replacement of the c(33)-hydroxyl group by the activated mesyl group has proved to be particularly successful. 55 this modification has no significant effect on biological activity of the antibiotic but provides a possibility to optimize its pharmacological properties. 65, 67 only transformations via the biosynthesis were described for the aureolic acid group antibiotics mithramycin and chromomycin. 70, 76 these natural antibiotics, which have a limited use in the treatment of some malignant tumours, have attracted increasing attention of researchers in different fields. 71, 73 the synthesized compounds were tested for antiproliferative activity. based on the results of in vitro studies of a series of new olivomycin a analogues of different types, compounds were selected for the in vivo evaluation. after preclinical trials in laboratory animals (evaluatrion of antitumour activity and toxicity), one olivomycin a analogue was recommended for further clinical trials. 87 ± 89 a systemic approach applied to series of new semisynthetic analogues of olivomycin a allows a detailed study of the molecular mechanism of antitumour action of this antibiotic. 79 ± 81 in particular, it was shown that olivomycin and its analogues, acting as dna duplex minor groove ligands, can inhibit topoisomerase i and the dnmt3a enzyme responsible for the disruption of the key epigenetic dna methylation process. 79, 80 the structural fragments of olivomycin a critical for antitumour activity were identified. a model of the interaction of the antibiotic and some its analogues with the dna duplex was proposed. 82 new analogues of anthracycline antibiotics with substituents of different types and different length, including those containing mono-and disaccharide residues, were prepared. the evaluation of the effect of polyhydroxylated moieties of doxorubicin and 14-hydroxycarminomycin on antitumour activity showed that this modification does not lead to the loss of activity of anthracyclines 93 ± 95 and enhances inhibitory activity against topoisomerase i. 92 14-hydroxycarminomycin derivatives proved to be particularly valuable because these compounds, as opposed to related doxorubicin derivatives, inhibit the proliferation of both doxorubicin-sensitive and doxorubicin-resistant tumour cell lines. 96 a new water-soluble depot form of doxorubicin with the high-molecular-mass polysaccharide galactomannan davanat was constructed and it was shown that it exhibits antiproliferative activity against three tumour cell lines. 94, 95 series of new heliomycin analogues ð water-soluble salts of amino derivatives ð were synthesized and these compounds were shown to have high antiproliferative activity against many tumour cell lines and inhibitory activity against different targets. 100 the value of these studies is that the majority of aminomethyl derivatives of heliomycin are active against both wild-type and drugresistant cancer cells at micromolar or submicromolar concentrations. the method is protected by a patent. 101 targeted chemical modifications of antibiotics are not only performed in order to prepare new potential drugs of a new generation but are used as an efficient tool for investigation of the mechanisms of action on bacterial or tumour cells. it should be emphasized that many potent antibiotics are indispensable tools for molecular biology research of the living world. such comprehensive studies can lead to the design and synthesis of new-generation drugs, which would be more effective than the starting antibiotics and which are of theoretical interest as new compounds with high biological activity. figures 2, 3 , 5 and 6 were composed by the authors based on the cited publications (the references are given in the figure captions). new approaches to natural anticancer drugs. springer briefs in pharmaceutical science and drug development antibiotics: challenges, mechanisms, opportunities markovnikov congress on organic chemistry (book of abstracts) handbook of experimental pharmacology doctoral thesis in chemical sciences, m.v.lomonosov institute of fine chemical technology glycobiology and drug design. (acs symp. ser. 1102) carbohydrates and drug design. (asc symp. ser. 932) key: cord-264316-do0px1gq authors: mucha, artur; drag, marcin; dalton, john p.; kafarski, paweł title: metallo-aminopeptidase inhibitors date: 2010-05-10 journal: biochimie doi: 10.1016/j.biochi.2010.04.026 sha: doc_id: 264316 cord_uid: do0px1gq aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the n-terminus of proteins and peptides. they are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. the largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. the optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases. amino-terminal modifications of nascent polypeptides are the most common processing events, occurring on nearly all proteins. aminopeptidases are a class of enzymes that play a pivotal role in this processing. aminopeptidases (ec 3.4.11 e hydrolases/peptidases/aminopeptidases, according to the classification of the international union of biochemistry and molecular biology) are proteolytic enzymes that hydrolyze peptide bonds from the amino termini of polypeptide chains. they may hydrolyze the first peptide bond in a polypeptide chain with the release of a single amino acid residue (aminopeptidases in a strict sense) or may remove dipeptides or tripeptides (dipeptidyl-and tripeptidylpeptidases) from polypeptide substrates. regarding catalytic mechanism, most of the aminopeptidases are metallo-enzymes but cysteine and serine peptidases are also included in this group. this review focuses on the strict metallo-aminopeptidases because they constitute the largest and the most homogenous class of these enzymes and use one or two metal ions in their active sites to specifically release the n-terminal amino acid residues of polypeptides and proteins. since 1990 over 5000 papers dealing with aminopeptidases have been published (medline database). aminopeptidases are ubiquitous enzymes widely distributed throughout the biological kingdoms and are found in many subcellular organelles, in cytoplasm, and as membrane components where they perform essential cellular functions. aminopeptidases act in concert with other peptidases to complete diverse proteolytic pathways. they play a vital role in a range of biological processes and disease situations. processes as distinct as angiogenesis, antigen presentation, neuropeptide and hormone processing, pregnancy and reproduction, protein turnover, memory, inflammation, tumor growth, cancer and metastasis, blood pressure and hypertension all involve one or more critical aminopeptidases. these enzymes can efficiently retrieve amino acids from dietary proteins and endogenous proteins degraded during protein turnover, thereby also covering a nutritional role. in addition to the book of hooper and lendeckel [1] , which describes role of aminopeptidases in biology and medicine, several excellent reviews on various aspects of their biology and the application of their inhibitors have been published [2e4]. we therefore limit our review to selected recent achievements in this field, although we present this in a certain historical context. the classification of aminopeptidases has often been based on mechanism of catalysis, the structure of the active site, substrate specificity (broad or narrow) and molecular properties. the nomenclature of many peptidases has been determined by their preferences or requirements for a particular n-terminal amino acid. the rapidly accumulating data covering new representatives of proteolytic enzymes required the development of an integrated source of information [5] . such a role was fulfilled by the merops database (http://merops.sanger.ac.uk/), which uses hierarchical, structure-based classification of these enzymes. this database relies on the fact that enzymes performing the same (or similar) chemical functions in different organisms generally turn out to have similar overall three-dimensional structures, and also show significant conservation of their amino acid sequences, polypeptide chain lengths and domain organization. in particular, in the regions of their active sites, a high degree of conservation of residue identities and structural positions is observed. in the merops peptidase information database each protease is assigned to a certain family on the basis of statistically significant similarities in amino acid sequence, and families that are thought to be homologous are grouped together into clans. clans consist of families of peptidases that are believed to share a single evolutionary origin, evidenced by similarities in their tertiary structures and/or their active site architectures. fifteen clans of metalloproteases have been identified, with metallo-aminopeptidases found in six which are designated as, ma (the largest one, containing over 35 families), mf, mg, mh, mn and mq. the families in clan ma are united by the presence of an hexxh motif in which the two his residues are zinc ligands and the glu has a catalytic function. clans mf (two zinc ions in the active site), mg (with the pita-bread fold and containing two cobalt or two manganese ions in their active centers) and mq (typically with two zinc ions) consists of only one family of peptidases each (m17, m24 and m29, respectively). the mh clan forms the most heterogeneous group and contains a variety of zinc-dependent exopeptidases. their structures show similar protein folds and are co-catalytic zinc peptidases containing two atoms of zinc per molecule, which have five amino acid ligands. clanmn contains only one enzyme e damino acid-specific aminopeptidase from bacillus subtilis. although metallo-aminopeptidases occur in all types of organisms, the mammalian enzymes were amongst the first proteases discovered in tissues and therefore they have been most intensively studied. human enzymes are particularly increasing in interest since the alterations in their function and regulation underline many human diseases. for example, leucine aminopeptidases (laps, ec 3.4.11.1), belonging to m17 family, have been the most extensively studied because they play a key role in the metabolism of proteins and biologically active peptides. these enzymes, perhaps the first that were isolated, are cytosolic exopeptidases of broad substrate specificity that are ubiquitous in nature being present not only in animals, but also in plants and bacteria [6] . they have medical and biological importance because of their functions in the metabolism of hormones and neurotransmission, cell maturation, and turnover of proteins, including utilization of exogenous proteins as nutrient substances and elimination of nonfunctional proteins. human lap is important in processing of antigenic peptides and in determination of immunodominance of various peptides [7e9] as well as in development of human eye lens cataract [10] . the protease plays a vital role in progression of cancers [11] . it may also have an important function in early events of hiv infection and thus serum activity of this enzyme may be useful as a surrogate marker for hiv infection and response to chemotherapy [12] . another example of medically important enzyme is microsomal aminopeptidase (belonging to m1 family), known also as aminopeptidase n, cd13 or alanyl aminopeptidase (ec 3.4.11.2) [13] . in biological systems, their primary peptide substrates include a wide variety of neuropeptides and hormones [14, 15] . it has been established as a myelomonocytic marker in leukemia typing [16] , as a receptor for human coronavirus 229e and cytomegalovirus [17, 18] , as a mediator of both inflammation and cell invasion [16] , as a regulator of blood pressure and the pathogenesis of hypertension [19] , and as a regulator of analgesia via metabolism of endorphins and enkephalins [20] . furthermore, it also regulates il-8 bioavailability in the endometrium and therefore may contribute to the process of angiogenesis [21] . it also plays key roles in physiological and pathological processes, such as embryogenesis, immune responses, angiogenesis, tumor cell invasion, and metastasis [22] . methionine aminopeptidases (aminopeptidase m, metaps, ec 3.4.11.18), belonging to m24 family, are an example of peptidases that exhibit narrow specificity [23] . generally they are responsible for the removal of methionine from the amino-terminus of newly synthesized proteins. they maintain stringent specificity for the n-terminal methionine and accept no other natural amino acid residues. they also have a strong preference for small and uncharged second residues in peptide chains. since the mammalian enzymes play a critical role in the regulation of post-translational processing and protein synthesis they play an important role in the development and malignancy of different types of cancer [24e28]. human aminopeptidase m is also involved in neurofibromatosis, one of the most common tumor predisposition syndromes [29] . although scarce, there are also reports on aminopeptidase isolation and characterization from other vertebrate species, as exemplified by recent findings in fishes (carp and red sea bream) [30, 31] and birds (chicken) [32] . far more information is known about insect aminopeptidase n, which is one of the membrane proteins identified as a receptor to cry proteins in various species [33e36] . cry proteins produced by bacillus thuringiensis are toxic to insects and thus this strain is exploited commercially as a bioinsecticide. aminopeptidases involved in the degradation of insect neuropeptides have been also studied in some respects [37] . the other groups of metallo-aminopeptidases explored intensively are of bacterial origin. the first studies on these enzymes were carried out over 40 years ago, and since then a large number of aminopeptidases of microbial origin have been characterized. they may be localized in cytoplasm, on membranes associated with the cell envelope or secreted into the extracellular media [4] . the interest in these enzymes stems from their potential to act as targets to combat bacterial diseases. in this respect, a wide variety of structurally diverse aminopeptidases have been recently isolated and characterized from a range of bacterial species. these include: aminopeptidase p isolated from common strain of escherichia coli [38] , aminopeptidase m from pathogenic mycobacterium tuberculosis [39] and leucine aminopeptidase from helicobacter pylori [40] , cold-active aminopeptidase from psychrotropic colwellia psychrerythraea [41] , a thermophilic enzyme from geobacillus thermoleovorans [42] , an extracellular zinc metalloprotease and putative virulence factor involved in pathogenicity of the fish pathogen vibrio anguillarum [43] , and a lysine-specific enzyme from unusually resistant, hyperthermophilic archaeon pyrococcus furiosus [44] . this clearly indicates that bacterial aminopeptidases are widely distributed and are of vital importance. in recent years there has been a considerable interest in aminopeptidases of parasitic protozoans, which cause important diseases in humans, animals and birds. the alanine and leucine aminopeptidases from plasmodium falciparum, a causative agent of malaria, the most significant parasitic disease of humans, are the most comprehensively studied [45, 46] . these aminopeptidases are promising targets for designing anti-malarial drugs (for a recent review see [47] ). another important enzyme of plasmodium e aspartyl aminopeptidase is being considered as an additional target for drug design [48, 49] . intensive studies on the role and biochemistry of aminopeptidases isolated from other parasitic organisms, including legionella pneumophila (causative agent of legionnaires' disease) [50] , eimeria tenella (causes hemorrhagic cecal coccidiosis in young poultry) [51] , babesia gibsoni (parasite found in red blood cells and transmitted by ticks) [52] and microsporidia (causing diseases in immunosuppressed patients) [53] are ongoing. diseases caused also by trematodes, commonly known as bloodflukes, affect hundreds of million people in impoverished areas of africa, central and south america and east asia, with those caused by schistosoma spp. are considered by the world health organization as second in importance only to malaria. leucine aminopeptidase is thought to play a central role in hatching of the miracidium from the schistosome egg and therefore is intensively studied as candidate for drug design [54, 55] . similar motivation has driven studies on the leucine aminopeptidase from paragonimus westermani, a tissue-invading trematode that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals [56] . specific parasite aminopeptidases might also be considered as targets for vaccine design as shown for fasciola hepatica [57] , a vector of an important freshwater snail-borne helminthiasis that produces a chronic liver infection of cattle and sheep. understanding the mechanism of action for each family of metallo-aminopeptidases is of a key importance to rational design of more potent and more specific inhibitors of these enzymes and, consequently, to obtain drugs of improved properties. therefore, a substantial effort has been made in studying the mode of binding of their substrates and transition state inhibitors in enzymatic binding sites, as well as in the elucidation of the three-dimensional structure of active sites and the detailed mechanisms of catalyzed reactions. a feature common to all metallo-aminopeptidase active sites is that the metal ion (in most cases zinc) is surrounded by a shell of hydrophilic groups that is embedded within a larger environment of hydrophobic groups. in addition, amino acid side chains serving as ligands usually form hydrogen bond contacts with neighboring residues, perhaps to preorder the metal ion binding site and to decrease its entropic cost of binding. the structures of active sites suggest that a number of reaction paths are possible. two catalytic roles of metal ions might be considered. first, they might stabilize a highly reactive hydroxide ion, thereby ensuring that an activated nucleophile is available for catalysis at physiological ph (mechanism a in fig. 1) . second, the positively charged metal ion may serve as an electrophilic catalyst complexing an oxygen atom of the scissile peptide bond and facilitating the nucleophilic attack of water molecule (mechanism b in fig. 1 ). there is also a possibility that the active site glutamate (especially in the case of m17 family of peptidases) acts as a nucleophile resulting in formation of a covalent enzymeeinhibitor complex followed by its fast hydrolysis by water (mechanism c in fig. 2 ). the glutamate assisted process is being considered as a less probable mechanism than a and b. in the case of the enzymes containing binuclear metal centers the substrate carbonyl oxygen is coordinated to one of them and a hydroxide ion bridging two metal ions acts as the nucleophilic agent (mechanism d in fig. 2 ). this may explain the fact that several dinuclear metallopeptidases retain some catalytic activity when converted into mononuclear 1 . catalytic roles considered for the metal (zinc) ions in the mechanism of metalloaminopeptidases action: stabilization of a highly reactive hydroxide ion (mechanism a), complexation of the oxygen atom of the scissile peptide bond to facilitate the nucleophilic attack of a water molecule (mechanism b). fig. 2 . alternatives of catalytic mechanisms considered for metallo-aminopeptidases: glutamate acting as a nucleophile e formation of a covalent enzymeeinhibitor complex and its fast hydrolysis (mechanism c), bridging of a hydroxide ion in binuclear metal centers (mechanism d). ones but typically exhibit faster rates with dinuclear active sites. finally, in all cases the additional role of a metal ion is to stabilize developing negative charge(s) in the transition state of the catalytic mechanism. there is no single method for the elucidation of the mechanism of certain enzymatic reaction. thus, a combination of several methods is required. enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. however, some kinetic data can suggest possibilities to be examined by other techniques. the evaluation of the role of metal ion is usually studied by metal exchange technique. in particular, the role of each metal ion in the dinuclear sites of aminopeptidases from the m17 family towards peptide hydrolysis was studied by kinetic and spectroscopic methods after replacement of one of active site zinc ions by mn(ii), co(ii), ni(ii), zn(ii), and cd(ii) [58e60] . important data about detailed mechanisms of action of aminopeptidases are also gained by production of altered enzymes by means of site-directed mutagenesis [61e66] . good examples are the studies on the role of active site glutamic acid in various aminopeptidases of m17 family [66, 67] . this was achieved by replacing glutamic acid either by structurally related aspartic acid (possessing an acidic side group), glutamine (lacking an acidic side group), or structurally different alanine, and studies of kinetic properties of the mutated enzyme. the method that provides the most important insights into domains organization and architecture of active sites of aminopeptidases, and thus into mechanisms of their action, is crystallography. in that respect crystal structures of native enzymes and those complexed with small ligands are extremely useful (for representative recent examples see [68e73]), especially those determined for enzymes bound with inhibitors being considered as transition state analogues [70,72,74e76] . there is also an emerging power in the application of computeraided methods as a mean to study mechanisms of enzymatic reactions. the calculations enable the researcher to choose one of the several possible reaction pathways (and thus to determine reaction mechanism) and to establish the structures of transition states. these methods are based on the knowledge of enzyme threedimensional structure available either by crystallographic methods or obtained by computations using homology approach techniques (for representative examples considering metallo-aminopeptidases see [77e80]). besides the elucidation of the functional roles of active-site residues, an estimation of the environment effects are also possible. in the case of metallo-aminopeptidases such popular techniques such as studies employing modified substrates [81] , studies on isotopic effects [67] or construction of the chemical models of enzyme active sites [82] are quite scarce. due to their association in several medical disorders, metalloaminopeptidases are considered important targets for the design of inhibitors, which could potentially enter clinical trials as candidates for drugs. the presence of the metal ion(s) in the active center has determined a general strategy applied for the development of such synthetic ligands. these possess two fundamental structural features, a specific war-head portion dedicated to recognize the active site of the enzymes and specific functional groups that create appropriate complexes with the metal ions. a metal ligand fragment can be incorporated into a backbone containing optimized side chain(s) that are able to interact with the enzyme binding pocket(s). as the result, non-covalent inhibitors of the amino acid/ peptide structure (natural substrate, transition state or product analogues) have appeared the most suitable for this purpose. indeed, the majority of compounds designed to date have such characteristics. bidentate tetrahedral phosphonates and aldehydes (hydrated in the gem-diolate form), bidentate planar carboxylates and hydroxamates, as well as monodentate thiols, are classical examples. recently, a variety of promising heteroaromatic or miscellaneous (heteroaromatic based sulfonamides/carboxylates/amides) inhibitors of metallo-aminopeptidases have being identified from random screening of compound libraries. their structure usually involve a bidentate n,n, n,o or o,o donor system incorporated into a rigid hydrophobic environment, and thus such compounds also act in a non-covalent mode. finally, natural products are a also a source of the appropriate effectors of this group of peptidases, with bestatin being a prototypical representative. its peptide-like framework offers a choice of heteroatom groups that get involved in an active site metal ion complexation. other natural non-covalent heteroatom-rich systems are based on terpene or polyphenol scaffolds. fumagillin and ovalicin are quite unique examples of very specific inhibitors that possess an electrophilic moiety (an epoxide) capable of reacting with the nucleophilic his side chain in the active site. the proven medicinal importance of aminopeptidase n (apn, cd13), leucine aminopeptidase (lap) and methionine aminopeptidases (metaps) has resulted in the focus on design of inhibitors primarily for these three enzymes and extensive thematic reviews have been recently published [6,83e86] . here, we present a selection of several recent and historic approaches for design and optimization of the inhibitors of metallo-aminopeptidases, which could serve as lead compounds in the future studies of this group of proteases. among de novo constructed targeted molecules, organophosphorus compounds, namely a-aminoalkanephosphonates (general formula 1, fig. 3 ) and phosphorus containing pseudodipeptides (predominantly phosphinic, 2), have probably contributed the most to the inhibition studies on the neutral aminopeptidases: apn (alanyl m1) and lap (leucine m17). although the phosphonate/phosphinate group is a rather weak zinc complexing moiety, it offers other advantageous structural and electronic features. similar to other amino acid and peptide mimetics used as protease inhibitors, this is the effect of the incorporation of a covalent or non-covalent binding group (here involved in coordination of a catalytic metal ion(s) in the enzyme active site) into a substrate structure. for the phosphorus modified compounds it is also uniquely combined with its tetrahedral shape that is considered to mimic the high energy transition state of the peptide bond hydrolysis. additionally, the p1 side chain of the aminophosphonic acid analogues (or more effectively, both p1 and p1 0 residues of the pseudopeptides phosphoryl moiety) gives further possibility of structural optimization of substituents interacting with the s1 and s1 0 binding pockets of the enzyme (fig. 3) fig. 4 ), appeared to be efficient inhibitors of lap with a k i ¼ 0.15 [90] and 0.23 mm [87] for the r (l) enantiomers. the d (s) antipodes were strongly discriminated, by 2e3 orders in magnitude for the given examples. non-coded arylalkyl derivatives, exemplified by phosphonic homophenylalanine (5) and homotyrosine (6), were bound preferentially to an even slightly greater extent (k i ¼ 0.14 and 0.12 mm, respectively, for the racemic mixture, fig. 4 ) [91] . promising affinity (k i 1.0 mm) was also found for extended linear homologues, namely 1-amino-nhexanephosphonic [87] and 1-amino-n-octanephosphonic acid [91] . these results indicate that the s1 binding pocket of the leucine aminopeptidase can accommodate hydrophobic ligands even bulkier than indicated by its substrate preferences (the hydrolase cleaves substrates with the broad band specificity, however, those of the hydrophobic character at the n-termini, like leucine, methionine, isoleucine, valine, etc., are processed noticeably faster [79, 84] ). somewhat similar situation is observed for the alanyl aminopeptidase apn, both the mammalian one [91] and the orthologue from a lower organism, the protozoan p. falciparum [93] . for example, phosphonic amino acid analogues of strong hydrophobic character, such as phenylalkyl or (cycloalkyl)alkyl (compounds 7 and 8, fig. 4 ), inhibited the porcine kidney enzyme with the k i values at low micromolar range [91] . an interesting attempt of mapping of the apn s1 binding pocket and rationalisation of these data in the context of the substrate-inhibitor structural relationship has been recently undertaken [94] . the specificity of apn was determined using an extensive collection of fluorogenic substrates bearing both natural and non-natural p1 residues. then, the obtained kinetic parameters were correlated with the activity of the corresponding a-aminophosphonic inhibitors. surprisingly, not the turnover velocity (expressed by the k cat/ k m ) but only the strength of substrate binding (described by the k m value) predicted the most reliably structural features responsible for the inhibitory potency. thus, the appropriate p1 residue incorporated into the a-aminophosphonate core was proved to be indispensable for the tight binding of a ligand to apn. because of the resemblance in the substrate specificity, the two aminopeptidases (lap and apn) are frequently studied together to refer the selectivity of newly developed ligands [89e91]. in general, a-aminoalkanephosphinic acids are much more potent inhibitors of the leucine aminopeptidase. for example, hydrophobic aliphatic compounds, such as 3 and 4 ( fig. 4) , expressed an affinity of more than two orders of magnitude higher for lap compared to apn as indicated by the appropriate k i values [90] . the reason for this observation seems to be the presence of the two zinc ions in the binding site of the cytosolic aminopeptidase (lap contain two zinc ions while apn contains one). involvement of the necepo 3 portion in interactions with both metal ions definitely results with a tighter binding. contrarily, the leucine aminopeptidase does not readily accept p1 substituents containing a nitrogen atom. thus, to achieve a high differentiating factor in favor of apn, bulky hydrophobic residues should be appropriately modified with a heteroatom. compounds 9 and 10 ( fig. 4) , bearing an additional amino moiety, was obtained by aziridinephosphonate ring opening with an amine and are low micromolar inhibitors of apn but do not effect lap [90] . it is worth noting that among the phosphorus analogues of amino acids studied so far, n 0 -cyclhexyl-1,2-diaminoethanephosphonic acid (9) appeared the most active towards apn. the availability of a broad collection of the applied a-aminophosphonates allowed a systematic structureeactivity relationship in this context of the s1 binding pocket preferences and the specificity of lap and apn. however, to achieve more significant inhibition these compounds needed to be extended to interact with the s1 0 pocket as well. three kinds of phosphonate elongation/modification were envisaged to provide such dipeptidic transition state mimetics. formally, their structure is the result of the replacement of the scissile amide bond by the phosphonodepsi, phosphonamidate or phosphinate moiety. the potential of all three variations for aminopeptidase inhibition was evaluated in detail for the leu-leu mimetics and lap as the target [87, 95] . the synthesis of the phosphonate analogue (compound 11, x ¼ o, fig. 5 ) was described in the early work of bartlett, however, the compound showed only moderate potency towards the enzyme studied [87] . despite this, a systematic computer-aided approach was undertaken by grembecka et al. to design new generations of more active inhibitors. the methodology was preliminary validated to rationalize the structureeactivity relationship obtained for various phosphorus containing amino acid analogues [96, 97] . then, when applied for pseudodipeptides, it positively confirmed the idea of addition of the p1 0 portion, albeit only via pen or pec bonding [95] . pseudodepsidipeptide bond (peo) was found to be unfavourable because of entropic effects upon inhibitor binding and oxygeneoxygen electrostatic repulsions with the carbonyl of ala113. the two other types of analogues (12, x ¼ nh and 13, x ¼ ch 2 , fig. 5 ) were synthesized and evaluated [95] . disappointedly, the fully deprotected phosphonamidate 12 appeared to be unstable at ph below 11. the hydrolysis of the pen bond that occurred, which effected two component amino acids, was clearly correlated with the presence of free neighboring amino group (from the other side, crucial for the effective binding) [98] . thus, despite being the most promising compounds according to the computed predictions (compound 12 was theoretically calculated to bind with the affinity of k i ¼ 5 nm), phosphonamidates were excluded for practical reasons. in turn, phosphinic pseudodipeptides exhibited perfect hydrolytic stability and inhibition constants in nanomolar range, and were ranked among the most effective ligands of lap reported to date. the mixture of two diastereoisomers of the leu-leu analogue 13 showed k i ¼ 65 nm, similarly to the phosphinate hphe-phe and hphe-tyr mimetics, both containing arylalkyl p1 and p1 0 residues (compounds 14 and 15, respectively, fig. 5 , tested as the mixture of four diastereoisomers) [95] . chiral chromatography performed for compound 14 allowed separation and assignment of the activity of its individual stereoisomers. the final k i value for r,s-14, counterpart of the l,l natural peptide configuration, was determined as 45 nm [99] . interestingly, compounds 14 and 15, particularly the latter one, appeared also effective inhibitors of apn (k i ¼ 276 nm and 36 nm, respectively, for the mixture of four diastereoisomers) [95] . clear preference for the tyr residue at the p1 0 position indicated the significance of the terminal phenolic oh group. this observation was explained by formation of a very specific hydrogen bond (to the carboxylate of glu 413 of apn) with the use of a model homologous to the leukotriene a 4 hydrolase structure. further exploration of the p1 0 structure and its termini, in the context of lap versus apn selectivity, was undertaken via a parallel n-alkylation strategy of appropriate amino acid building blocks. unfortunately, the final products appeared only moderate and poorly selective inhibitors (k i for both enzymes varied at 0.5e20 mm range) [100] . importantly, phosphinic pseudodipeptides were found to be excellent inhibitors of parasite counterparts of lap and apn and useful tools for their validation as potential targets in a novel treatment of malaria [47] . p. falciparum m1 and m17 aminopeptidases are responsible for the cleavage of the neutral residues in the terminal stages of the host haemoglobin degradation. thus, being a limiting stage in generating amino acids essential to parasite growth and development, they represent an attractive target for the development of novel anti-malarial drugs. compounds 14 and 15 inhibited recombinant m17 enzyme with the potency superior to that observed for the mammalian peptidase (k i ¼ 13.2 and 10.4 nm, respectively) [101] . the affinity of 14 measured for the pfm1 was also elevated (k i ¼ 78.4 nm) when compared to porcine apn (k i ¼ 276 nm). in addition, the phosphinates efficiently controlled the growth of p. falciparum in culture, including malaria cells lines that were resistant to the well-known anti-malarial chloroquine. finally, in vivo studies using a non-lethal plasmodium chabaudi murine malaria model demonstrated that treatment of mice with compound 14 reduced infection by 92% compared with controls [101] . recently, this compound was co-crystallized with pfm1 [75] and pfm17 [76] to give an insight into the active site architecture and the mechanism of action of both aminopeptidases which will greatly facilitate the design of new optimized ligands with potential as anti-parasite agents. appropriate phosphinic pseudodipeptide building blocks can be further elongated by means of solution or solid-phase peptide synthesis to produce tripeptide analogues that possess an additional p2 0 substituent. such optimized compounds were reported to be the most potent organophosphorus inhibitors of the alanyl (apn) and glutamyl (aminopeptidase a, apa, ec 3.4.11.7) metalloaminopeptidases reported to date. chen et al. described a series of nanomolar ligands of mammalian apn, exemplified by the ala-phe-phe analogue (16, fig. 6 anti-nociceptive activity thanks to the dual action against apn and neprilysin which caused an analgesic response after administration in mice [103] . compound 16 inhibited equipotently both the mammalian alanyl aminopeptidase as well as its bacterial orthologue. it also served as a ligand to resolve the three-dimensional structure of the latter enzyme [70] . related phosphinate tripeptidic analogues were used for co-crystallization with the leukotriene a 4 hydrolase/aminopeptidase, a prototypic m1 family member. as the result, substrate and transition state binding details together with a presumed catalytic mechanism, scarce data for the m1 enzymes, were reported [104] . a phosphinic pseudotripeptide, the glu-leu-ala analogue (17, fig. 6 , tested as mixture of four diastereoisomers), showed high affinity towards the zinc glutamyl aminopeptidase (mice recombinant) expressed by its k i equal to 0.8 nm [105] . within a series of compounds, n-terminal pseudoglutamyl residue was found to be crucial for the high potency and selectivity. for example, the differentiating factor between apa and apn (k i ¼ 31 mm) exceeded four orders of magnitude in favor of the apa. modifications of the phosphinate metal binding group in order to increase the number of coordination to the ion present in the active site can represent another approach to improve the potency of aminopeptidases inhibitors. this effect can be achieved by the introduction of a neighboring group containing a heteroatom that is additionally involved in metal complexation. four types of such modifications have been proposed and evaluated recently for the alanyl aminopeptidase apn [106, 107] . they involved the application of a-aminoalkane-a 0 -hydroxyalkanephosphinic acids (general formula 18, fig. 7 ), bis-a-aminoalkanephosphinic acids (19) , carbamoylated and thiocarbamoylated a-aminoalkanephosphinic acids (20 and 21). the structural variant 18 was previously described by bartlett for lap but the inhibition achieved for the leu-leu analogue (r ¼ i[87] . more interesting results were reported for apn. combination of the hydrophobic residues p1 (r ¼ i-pr, i-bu, n-bu, ph or (ch 2 ) 2 ph) and p1 0 (r ¼ ph, (ch 2 ) 2 ph or ch 2 (p-ome-c 6 h 4 )) allowed for regulation of the enzyme activity with the ic 50 value to 0.25 mm (this result corresponded to k i ¼ 143 nm) [106, 107] . interestingly, all four structural variations 18e21 produced comparable, low micromolar or sub-micromolar ic 50 values of inhibition. originally isolated from streptomyces olivoreticuli (md976-c7) more than 30 years ago by umezawa and co-workers, bestatin ((2s,3r)-3-amino-2-hydroxy-4-phenylbutanoyl-l-leucine e ubenimex d , 22, fig. 8 ) was one of the first potent inhibitors of metalloaminopeptidases with broad spectrum of action [108] . bestatin has been extensively investigated in biological systems both in vitro and in vivo, which resulted in discovery of several interesting properties of this compound such as ability to induce apoptosis in cancer cells, anti-angiogenic, anti-malarial or immunomodulatory effects [109] . presently, bestatin is on the market in japan where it is applied for treatment of cancer and bacterial infections. examples of successful inhibition of aminopeptidases by bestatin include aminopeptidase n (cd13), leucine aminopeptidase (lap), aminopeptidase b (ec 3.4.11.6) or lta 4 hydrolase [110e112]. bestatin can act as slow (lap) or fast (apn) binding, competitive inhibitor of aminopeptidases [113] . it resembles a phe-leu dipeptide substrate, however its phe residue is b-amino-a-hydroxy amino acid [114] . this a-hydroxy group together with the neighboring carbonyl group coordinate a zinc ion, which results in a competitive active site-directed inhibition. bestatin is weaker inhibitor of aminopeptidases containing one metal ion in the active center (apn, apb) and much stronger of enzymes with two metal ions (lap). this feature is explained by larger amount of interactions made by inhibitor with both enzyme metal ions in the active center along with additional contacts made by side chains of the inhibitor in s1 and s1 0 pockets of the enzyme. to date several stereoselective, synthetic methods leading to desired bestatin diastereomer have been described [115e119] (see recent reviews presenting available synthetic methods for bestatin and some modifications [120, 121] ). absolute configuration is a key issue responsible for good inhibitory effect and discrimination of the appropriate binding partners (substrates and inhibitors) for most of the proteases. diastereomer of bestatin with inverse configuration at carbon atom with hydroxy group ((2r,3r)-3-amino-2-hydroxy-4phenylbutanoyl-l-leucine) is known as epibestatin and is frequently used as negative control in biological experiments [122] . the structure of bestatin has also been a subject of several modifications in hope to improve its inhibitory and pharmacological properties. examples of such derivatives are bestatin thioamide (compound 23, fig. 9 ) [123] , para-hydroxybestatin (24) [124] or 2-thiolbestatin (25) [125] . the scaffold of bestatin was also used for the design of activity [113, 129] . these compounds are tri-and tetrapeptides and are better inhibitors of aminopeptidase n when compared to bestatin. this is due to an increased amount of contacts made with s1, s1 0 , s2 0 and s3 0 pockets of the enzyme and side chains of the inhibitors, which in most cases have very hydrophobic character (phe, leu, val). hydroxamic acids (n-hydroxyamides) can be considered as analogues of carboxylic acids and amides that uniquely combine the features of both of these groups. simultaneously, the hydroxamate moiety is an effective planar bidentate o,o metal chelating system. this close structural similarity to the products/substrates of the peptide bond hydrolysis and metal-complexing properties make it an attractive war-head for constructions of inhibitors targeted towards metallo-dependent proteases. the synthesis is not problematic and usually involves a one-step transformation of an acid or an ester with the use of an appropriate hydroxylamine derivative. accordingly, one-side optimized hydroxamate analogues of acids, amino acids and peptides have found biomedical relevance. the most promising applications have been associated with inhibition of matrix metalloproteinases as perspective targets for anti-cancer therapy. matrix metalloproteinases, involved in normal and abnormal tissue remodeling, angiogenesis and tumor metastasis, have been potently regulated by hydroxamates (e.g. batimastat, marimastat) that reached advanced phases of the clinical trials. these finally failed because of side effects associated with cross interactions with other metal containing proteins [130, 131] . fundamental work on the metallo-aminopeptidases inhibition by a-aminohydroxamates was performed by chan et al. and concerned the leucine aminopeptidase [132] . the derivatives of hydrophobic amino acids (exemplified by l-leu-nhoh, compound 30, fig. 12 ) regulated the enzyme activity with the k i values of micromolar range. other c-terminally modified compounds, such as the l-leu amide, alcohol, or hydrazide, together with the amino acid alone, were much less potent, typically at least by a 100-fold ratio. these observations were consistent with a subsequent study by a series of phenylalanine derivatives targeted towards apn. compound 31 (fig. 12 ) appeared a moderate inhibitor of the enzyme [133] . both l-leu-nhoh and l-phe-nhoh were overpowered by the corresponding thiols which indicated a much tighter individual sulfurezinc interaction than the bidentate oxygenezinc binding. since then, a choice of hydroxamic acid inhibitors (not necessarily based on the amino acid skeleton) of different metallo-aminopeptidases have been reported in the literature. a representative selection from recent examples is presented in fig. 13 . compound 32, bearing a hydrophobic substituent, was identified by screening of a 3000 natural and synthetic compound library and appeared equipotent to bestatin for apn [134] . compound 32 controlled the basic fibroblast growth-factorinduced invasion of endothelial cells at low micromolar range. the hydroxamate was highly selective as it exerted no action towards members of the matrix metalloprotease family. interesting hydroxamate based inhibitors were also developed for the methionine aminopeptidases from different organisms. hu et al. described the synthesis of compounds possessing an additional substitution at the hydroxamate nitrogen atom [135] . these n-hydroxydipeptides, derivatives of met-gly (compound 33, fig. 13 ), allowed for structural optimization of both sides of the molecule. the products inhibited the bacterial as well as both forms of human metap, with a slight preference to e. coli enzyme. a cooperative action of the n-terminal amino group and two hydroxamate oxygen atoms towards two catalytic metal ions in the active center was suggested as the binding pattern. the bacterial form of metap1 served also as a model for comparison of the activity of 5-aryl-furane-2-carboxylic acids with other variants of the c-termini [136] . such heteroaromatic hydroxamic acids (exemplified by 34, fig. 13 ) were found to be superior inhibitors to corresponding acids, esters, amides, hydrazides, alcohols and nitriles towards distinct metalloforms of the ecmetap1. certain metallo-aminopeptidases are promising targets in anticancer therapy in humans, but they can also be exploited in the development of antibacterial, antifungal and antiparasitic agents. the zinc p. falciparum m1 aminopeptidase is being actively explored in this context (as described in the section devoted to organophosphorus compounds). the dual function amide-hydroxamate template was also used to target the malaria enzyme [137] . the compounds developed consisted of three portions: hydroxamic acid termini dedicated for zn(ii) complexation, a hydrophobic a-substituent and the amide function that served for extensive structural diversification. the use of bulky hydrophobic amines for the amide formation (as exemplified by 35, fig. 14) yielded potent inhibitors of the parasite enzyme, with the ic 50 values in low nanomolar range [138] . importantly, consecutive iterative optimization gave derivatives that were characterized by spectacular selectivity versus the mammalian orthologue. compounds were active in parasite growth inhibition and displayed good pharmacokinetic properties [138] . tosedostat (chr-2797, 36, fig. 15 ) [139] seems to be the most attractive novel pharmacologically active product among hydroxamic acid metallopeptidase inhibitors. this orally available cyclopentyl ester prodrug is converted in vivo into the intracellularly active acid metabolite. the latter is a potent inhibitor of a number of aminopeptidases, including leucine aminopeptidase, aminopeptidase n, puromycin sensitive aminopeptidase and leukotriene a4 hydrolase/aminopeptidase (with the ic 50 in nanomolar range) [140] . chr-2797 exerted anti-proliferative effects against tumor cell lines in vitro and in vivo, and exhibited 300 times more potency than bestatin. the proposed mechanism of tumor cell killing involves depletion of amino acids by blocking protein processing/recycling [140] . chr-2797 is well tolerated and can be safely administered at doses that result in the metabolite activity as evidenced in preclinical models [141] . recently, tosedostat demonstrated promising efficacy in patients with acute myeloid leukemia and myelodysplastic syndrome in the phase ii of clinical trials [142] . activation of the latter and its subsequent substitution with a sulfur containing nucleophile [143e146]. alternatively, the mitsunobu reaction is utilized as the oh replacement procedure [145, 146] . as the optically active substrates are easily available and there is no risk of racemization, final products are obtained in the enantiomerically pure form. when the mode of binding to metalloaminopeptidases is considered, 2-aminothiols are typical analogues of the n-terminal portion of the peptide substrates that act in reversible competitive manner. the thiol group is a termini designed to interact non-covalently with the catalytic metal ion(s). except for the functional groups essential for the polar contacts, 2aminothiols contain a side chain that occupies the s1 pocket. despite a structural simplicity and low molecular weight, the potency of these compounds against metallo-aminopeptidases is very high. the reason for this is a strong affinity of the sulfur atom for divalent soft acid metal ions, such as zn(ii). consequently, 2aminothiols exhibit much higher activity than amino acid analogues of the corresponding complexity, but containing other metal chelating moieties, such as phosphonate or hydroxamate ones. frequently, these compounds are discriminated with an affinity more than 1000-fold ratio lower in comparison to the analogous thiols. in general, 2-aminothiols are one-handed inhibitors directed specifically towards the s1 subsite part. the availability of extended ligands that possess an additional pn 0 portion is limited because of a complex synthesis and diastereomeric purity. nevertheless, 2-aminothiols have found some fundamental and practical applications connected with metallo-aminopeptidase inhibition. the most spectacular achievements are associated with the regulation of action of aminopeptidases a and n, enzymes involved in the brain renineangiotensin cascade that represent perspective targets in a hypertension therapy. a series of representative 2-aminothiols, derived from the natural amino acids, is given in fig. 16 . l-lysine thiol (the absolute configuration s, compound 37) was shown to be an extremely potent inhibitor of arginyl aminopeptidase (aminopeptidase b, apb) with a subnanomolar k i value [143] . l-leucine thiol (compound 38) binds to the same target but with a lower activity. more interestingly, it exhibited a 700-fold ratio stereochemical preference towards the enzyme for the natural configuration (k i ¼ 980 mm for the d (r) enantiomer). unexpectedly, compound 38 poorly inhibited leucine aminopeptidase (lap), what was explained by the use of the zinc-magnesium hybrid enzyme in these experiments [143] . according to an earlier study, l-leucine thiol (38) was shown to be a potent competitive inhibitor of the microsomal aminopeptidase from porcine kidney (apn, k i ¼ 22 nm) [144] . this compound was then used as a lead structure to optimize the size of a neutral hydrophobic p1 residue, in the context of construction of potentially analgesic dual inhibitors of neprilysin (nep) and apn [133] . a broad series of inhibitors was synthesized that showed high equipotent efficacy for the aminopeptidase n (k i ¼ 11e50 nm). among them, the methionine thiol (compound 39, fig. 16 ) appeared the most active in vitro. 2-aminothiols were at least four orders in magnitude more effective than carboxylates, phosphonates and hydroxamates of the corresponding structure [145] . despite such potency, the use of selected compounds in intravenous administration did not induce anti-nociceptive responses in mice on the hot plate test, evidently because of difficulties to cross the bloodebrain barrier. indeed, the oxidation to the disulfide form to increase the lipophilicity made them efficient prodrugs. much more significant response was stimulated by application of a mixed prodrug which structure was disulfide based combination of two thiol inhibitors, each directed towards a certain enzyme, either apn or nep [147] . elucidation of an intrinsic function in the brain and, therefore, the medical potential of selected aminopeptidases in the renineangiotensin system stimulated continuation of studies on 2aminothiols. these two metallo-aminopeptidases, namely apn and glutamyl aminopeptidase (aminopeptidase a, apa), are supposed to participate in metabolism of brain angiotensin ii and iii (angii and angiii). glu-thiol (compound 40, fig. 17 ) [148] was described as first efficient inhibitor of apa, however it was equipotent to apn (k i ¼ 140 versus 120 nm, respectively). to study the physiological importance of these enzymes, specific agents, discriminating between the two targets to a high degree are indispensable. in a quest for a selective inhibition, a number of 2-aminothiol derivatives were synthesized and evaluated [145, 146] . it appeared to be a relatively simple task to identify the p1 residue responsible for high nanomolar potency of an apn ligand. an extended p1 hydrophobic portion, favorably terminated with a heteroatom group (as exemplified by compounds 41 and 42, fig. 17 ), represented such an option. the affinity of these compounds towards apa was of 100-fold ratio lower. anyway, according to recent data l-methionine thiol developed earlier [149] gave even more for apa, respectively [149] ). such a convenient modification was not found for the opposite case. a compromise was achieved for short side chains with an acid function other than carboxyl at the terminus (such as 43, fig. 17 ) [145, 146] . the affinity of the sulfonic acid 43 for apa was not elevated in comparison to the lead, but a drop in potency measured for apn was more significant and finally resulted in a 100-fold discrimination ratio. these parameters were furthermore improved by exploration of the sn 0 subsite of the aminopeptidase a. the synthesis of tripeptidomimetics that contained an optimum p1 substituent, attached as the 3-amino-2-mercaptocarboxyl to the n-termini of dipeptide libraries, allowed determination of the advantageous p1 0 and p2 0 side chains [150] . the s1 0 binding pocket accommodated hydrophobic residues whereas the s2 0 pocket preferred negatively charged residues. as a result, an exceptionally potent compound (44, fig. 17 ) characterized by a striking 20,000fold selectivity ratio, was identified. interestingly, the most active diastereoisomer exhibited non-natural configuration r at the p1 portion which was explained by sterical constrains of the whole molecule [150] . despite a remarkable activity (a first subnanomolar thiol inhibitor of apa), compound 44 was poorly bioavailable. a limited p1 0 fragment optimization starting from the lead 43 gave a much simpler molecule: (3s,4s)-3-amino-4-mercapto-6-phenylhexane-1-sulfonic acid [151] . the substitution of the c3 atom with a hydrophobic arylalkyl fragment resulted with a satisfactory potent apa inhibition (k i ¼ 30 nm) (however, the kinetic data for apn were not reported). over-activity of the renineangiotensin system in the brain has been implicated in the development and maintenance of hypertension. using specific agents described above, it was demonstrated that apa and apn are involved in the metabolism of angiotensin ii and iii, respectively, within this system. angiii (2e8) is generated from angii by apa assisted cleavage of the n-terminal asp-arg bond, whereas apn functions in subsequent inactivation of angiii. apa specific sulfonate 43 increased the half-life of angii in mice, blocked the angii to angiii conversion which in turn controlled vasopressin release [152, 153] . the dose-dependent decrease of blood pressure was achieved only by intra-cerebroventricular injection. contrarily, selective inhibitors of apn (39 and 41), administered by the same route, caused elevated blood pressure. thus, the level of angiotensin ii and iii in the brain, in particular that controlled by the aminopeptidases, was suggested to be a potential target of hypertension treatment [153, 154] . to this end, the disulfide dimer of the lead 43 was reported as a first orally available candidate for the therapy based on aminopeptidase a inhibition [154, 155] . the majority of inhibitors discovered for metallo-aminopeptidases are compounds which interact with enzyme in a non-covalent way. however, for some enzymes covalent inhibitors have also been found. fumagillin ((2z,4e,6e,8e) 10-oxodeca-2,4,6,8-tetraenoic acid, 45, fig. 18 ) and ovalicin ((1s,2r,3s,4r,5s)-7-hydroxy-8-methoxy-7-[2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-4-oxaspiro [2.5] octan-2-one, 46), which are fungal metabolites inhibit methionine aminopeptidase type 2 (metap2) by the formation of a covalent, irreversible bond between a reactive epoxide and his-79 present in the active center of the enzyme [156, 157] . these compounds are not effective towards other aminopeptidases. selectivity towards metap2 and complete lack of selectivity towards metap1 is explained by difference in only one amino acid (ala-202 in metap2 and threonine in metap1) around the active center of these enzymes as determined by comparison of crystal structures [158, 159] . this approach revealed also that tight binding of fumagillin and derivatives to metap2 is also a combination of several other interactions made by the inhibitor components with the surface around the active center of the enzyme. in biological studies fumagillin and ovalicin have been found as potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. this resulted in extensive sar studies and synthesis of several derivatives of these compounds. compound tnp-470 (47, fig. 18 ) has entered clinical trials and was evaluated for application in anti-angiogenic therapy [156, 160] . numerous competitive inhibitors of metallo-aminopeptidases that have been recently developed, frequently starting from leads identified by random screening (e.g. high throughput screening utilizing fluorogenic [161, 162] and chromogenic assays [163, 164] or virtual screening [165, 166] ), are based on a heteroatom-rich fragment. such an appropriate heteroaromatic (or rarely heterocyclic) scaffold contains a set of the nitrogen, oxygen and/or sulfur atoms involved in coordination of the catalytic metal ion(s). structural rigidness and constraint of this portion of a ligand is often privileged for formation of hydrophobic interactions, specifically pep stacking, with the neighboring residues of the active site. the synthetic procedures used for obtaining such compounds should allow the combination with reliable methods of the substituent(s) diversification. this can be performed prior to a ring formation/ aromatization or by parallel decoration of the target scaffold with the use of a simple chemistry, such as the amide bond formation. an extensive structural optimization of certain scaffolds has led to very potent inhibition of selected aminopeptidases by surprisingly low molecular weight compounds (even below 200). methionine aminopeptidases (particularly human metap2), are representative examples of successful application of this strategy. 1,2,4-triazoles were originally described in the patent literature as potent reversible non-peptidic inhibitors of the methionine aminopeptidase type 2 [167, 168] . variously substituted compounds bound to the cobalt form of metap2 with the affinity expressed by fig. 19 ) and their biological relevance was also subsequently presented [169] . iterative refinement of the inhibitor lead structure allowed the identification of compounds with the potency against metap2 in the picomolar range. systematic modifications of the key structural fragments revealed certain tendencies responsible for elevation or decrease in their activity. the 3 0 and 4 0 substitution of the aniline phenyl ring with small residues was the most favorable (compounds 49 and 50, fig. 19 ). contrarily, any changes in the optimal benzylthio s1 residue structure were found deleterious, similarly to methylation of any of the triazole nitrogen atoms (to a relatively smaller extent for the n4). selected derivatives inhibited human endothelial cell proliferation and the growth of new blood vessels in a model of angiogenesis. low molecular weight 4-aryl substituted 1,2,3-triazoles were found to act as almost equipotent reversible inhibitors of the human metap2 [170] as described for 1,2,4 derivatives. diversification of the position 4 was performed by the use of various aromatic aldehydes that were transformed into the appropriate alkyne substrates for the cycloaddition. several effective ligands of the cobalt-activated metap2 were identified, showing the k i,app values down to 1 nm. for example, k i,app ¼ 15 nm for 51 given in fig. 19 corresponded to k i ¼ 4.2 nm and ic 50 ¼ 10 nm in full kinetic analysis [170] . sar studies performed for the phenyl ring excluded bulky 3 0 and 4 0 substitution showed that only small alkyl chains were tolerated whereas the halogens were favorable in these positions (see for example compound 52 in fig. 19 ). contrarily, a 2 0 addition of a bulky residue, even a heteroaromatic one, was well accepted and indicated some additional room in the active site. replacement of the whole phenyl ring by a substituted pyridyl was also profitable. the use of the pyrazole based scaffolds instead of the triazole one, revealed the key role of n1 and n2 atoms in high affinity. regarding their biological activity, the compounds inhibited human and mouse endothelial cell growth. enzyme-ligand crystal structures accompanied studies on the triazoles [169, 170] . they revealed, among others, the significance of the p1 aromatic ring of a certain size (substituted or not, present in potent metap2 inhibitors) that should occupy the hydrophobic pocket formed by phe219, his331, tyr444 and his231, and form the appropriate pep stacking. the size of the pocket (narrower for metap1) was postulated to be a discrimination factor between the two forms of the methionine aminopeptidase. the difference in the binding affinity is usually 3e4 orders in magnitude in favor for metap2 for the triazoles studied (for example, k i,app ¼ 0.5 nm versus 3900 nm for 48, ic 50 ¼ 10 nm versus 7100 nm for 51, fig. 19 ). the structural data provided a rationale for sar discussion, but first of all confirmed the significance of the key interactions between n1 and n2 nitrogen atoms of the inhibitors and the cobalt ions. the molecular mechanism of triazole action was elucidated originally using the crystal structures of the staphylococcus aureus orthologue of the metap1 enzyme complexed with 3,5-disubstituted-1,2,4triazoles (such as compound 53, fig. 19 ) [171] . according to these results, each of the triazole n1 and n2 nitrogen atoms interacts with one of the two cobalt ions such that the nen bond is nearly co-linear with the co to co. the distances between the nitrogen and the coordinated cobalt are very similar to each other and tight (close to 2 å) which indicates strong interactions [171] . the triazole ring forms a stacking contact with the hydrophobic face of the imidazole side chain of h76, a residue that is believed to be involved in the catalytic process. somewhat surprisingly, the substituent of the substrate-like methionine structure of 53 docks to the s1 0 subsite, and not to the s1 site. in turn, its natural position is occupied by the thiobenzyl residue as described above. metap is believed to be a dinuclear cobalt dependent enzyme as co(ii) activates all its known forms, however, the identity of the metal cofactor is still ambiguous. triazoles that inhibited exclusively the cobalt-activated human metap2 even with high nanomolar potency, although failed in cell proliferation assays, were used to shed light on this matter [172, 173] . such metal dependent action indicates that other divalent ions must be considered as the relevant cofactors, particularly in respect to the fact that the architecture of the enzyme active site does not depend on the identity of the bound metal. in this context, manganese was postulated as the physiologically relevant metal [172] . indeed, the triazoles investigated were not effective towards such activated metap form. contrary to triazoles, 1,3-thiazoles appeared selective inhibitors of the methionine aminopeptidase type 1 of both bacterial and human origin. the essential function of metap1 in bacteria suggested that the enzyme could be a promising target for the development of novel broad spectrum antibiotic agents. accordingly, thiazole based active compounds were identified by screening of libraries towards the e. coli methionine aminopeptidase. it is worth noting that selected leads did not represent typical heteroaromatic scaffolds. these were not the central core of the molecule, but rather a proximity group linked with another hydrophobic portion (frequently heteroaromatic as well) by a carbonecarbon, amide or carbonyl based bonding. thus, the complexing mode of the enzyme catalytic ion(s) was not as characteristic as for triazoles and usually involved participation of two neighboring heteroatom groups. the random screening of 4500 compounds by nan and coworkers led to the amide of pyridine-2-carboxylic acid and thiazol-2-ylamine (54, fig. 20 ) that inhibited the e. coli metap1 with the ic 50 ¼ 5 mm and served as a perspective lead compound [174] . all primary chemical mutation in the basic structure, such as change in position of pyridine substitution or heteroatom replacement by an isosteric group, appeared deleterious for the potency [174, 175] . most probably, the combination of three electronegative groups that are involved in the metal ions coordination, namely the aromatic nitrogen of the pyridine ring, the carbonyl moiety in position 2 and a heteroatom of the thiazole, forms a system essential for the tight binding. nevertheless, substitution of the position 3 in pyridine with an amide or ester group appeared profitable in terms of optimization of interactions with the binding pocket. the affinity was elevated to nanomolar range, as for example for compound 55, fig. 20 [174] . importantly, introduction of a bulky cyclic or aromatic residue at the proximity of the amide allowed for efficient discrimination between the bacterial enzymes from different sources. for x ¼ benzoyloamino or cyclohexanecarbamino, by substituting the structure 54 the selectivity factor between e. coli and the saccharomyces cerevisiae exceeded 100-fold ratio (ic 50 ¼ 0.89 and 0.87 mm for ecmetap1, respectively and ic 50 > 100 mm for scmetap1 for both compounds). wide optimization of the x moiety demonstrated that introduction of an additional heteroatom to the extended amide structure was also well tolerated (56, fig. 20) [175, 176] . finally, replacement of the whole pyridine ring by another thiazole system gave compound 58 (fig. 20) with low nanomolar potency [177] . interestingly, 3-substituted pyridine-2-carboxylic acid thiazol-2-ylamides selectively inhibited human metap1 versus metap2, as exemplified by a 200-fold differentiating factor for compound 57 (fig. 20 ) [178] . despite their moderate potency in vitro, the treatment of tumor cell lines with such specific agents suggested a role of metap1 in g 2 /m phase transition. furthermore, this study confirmed the previously reported results on variations in the metal binding in the active site of the co(ii)-ecmetap in the presence of the thiazole based ligands [165] . according to the appropriate crystal structures, these compounds seemed to be responsible for driving a third metal ion into the enzyme active site. the auxiliary cobalt atom was not present in the native enzyme, even when crystallized in the presence of high concentration of the metal salt. the additional cobalt interacts with a single histidine residue of the enzyme, three water molecules at the entrance of the active center and two nitrogen atoms of the ligand (those of the pyridine and the amide). inhibitor binding is stabilized by several contacts, mostly of the hydrophobic character. obviously, such an unusual situation must be taken into consideration when sar of novel metap effectors is discussed and the in vitro versus in vivo experiments data are correlated (see below for another example). closely related thiazole inhibitors of the bacterial methionine aminopeptidase were discovered by the high throughput screening of a ten times larger library [161] . the main goal of the study was identification of potent but predominantly selective inhibitors that could distinguish between various metalloforms of e. coli metap. indeed, thiazole based compounds exemplified by compound 59 (fig. 20) strongly inhibited the cobalt dependent enzyme and only moderately the manganese, iron or nickel ones [161] . the opposite pattern of activity was found for other heteroaromatic compounds. 5-arylfuran-2-carboxylic acids and their derivatives exhibited preferences towards mn(ii)-ecmetap [136, 161] , whereas the cathecholcontaining thiazoles preferred fe(ii)-ecmetap [179] . however, these two groups of compounds did not follow the mode of binding typical for heteroaromatic inhibitors of the cobalted form of metap. they did not employ the heteroatom(s) of the ring but instead binding involved the two proximity oxygen atoms, either of the carboxylate [161, 162] or the phenol moieties [179] , respectively, for complexing to the metal ion(s). direct combinations of two heteroaromatic rings represent another approach to developing methionine aminopeptidase inhibitors. these compounds bind the driven active site auxiliary cobalt ion by employing nitrogen atoms from each of the rings that are placed in co-planar manner. thiabendazoles (benzimidazoles substituted in position 2 with the thiazole ring) and their derivatives (compounds 60e62, fig. 21 ) regulated the co(ii)-ecmetap activity with k i value in sub-micromolar range, although they appeared ineffective in vivo [166] . this was explained with the use of the metap-ligand crystal structures that revealed variations in the metal binding in the active site [165, 166] as described above for pyridine-2-carboxylic acid thiazol-2-ylamides. a family of related 2-(2-pipyridinyl)pyrimidines was identified upon screening of a 175,000 member library for inhibitors of human and p. falciparum methionine aminopeptidases [164, 180] . interestingly, the compounds were virtually equipotent towards both mammalian types of the enzyme (for example see 63, fig. 21 ). similarly as before, they complexed to the auxiliary co(ii) that is normally not involved in the catalytic process [164] . nevertheless, they appeared to be novel potential anti-malarial agents. three active isoforms of pfmetap1 were obtained after cloning, expression and purification, and tested in vitro. the inhibitors were remarkably selective towards the 1b type, with the highest obtained potency with ic 50 ¼ 112 nm. the most active compound 63 inhibited also the p. falciparum proliferation of both chloroquine sensitive and dependent erythrocyte cultures with ic 50 ¼ 0.9 and 3.1 mm, respectively [180] . consequently, this compound was used in murine malaria models and positively confirmed pfmetap1b as a promising target for development of new anti-malarials. boronic acids as inhibitors of aminopeptidases were described first by baker et al. for aeromonas aminopeptidase (ec 3.4.11.10) [181, 182] . in this report simple aliphatic derivatives were used as competitive, transition state analogues that bound to the active center of enzyme with good efficiency. among five tested derivatives 1-butaneboronic acid (64, fig. 22 ) was the best inhibitor of the enzyme. in another approach, shenvi described a series of a-aminoboronic acids as effective inhibitors of human enkephalin degrading aminopeptidase (heda), microsomal leucine aminopeptidase and cytosolic leucine aminopeptidase [183] . the advantage of these inhibitors over simple aliphatic derivatives was the presence of the free amine group at carbon a, a feature that is known to improve binding of the ligand to aminopeptidases. detailed analysis of kinetic data for cytosolic leucine aminopeptidase revealed biphasic slow-binding inhibition mechanism of a-aminoboronic acids. this suggested that slow-binding step is responsible for formation of tetrahedral boronate molecule from trigonal boronic acid. the inhibitory activity of the tested derivatives (65e68) also strongly correlated with the side chain type used in the study (fig. 23 ). aldehyde derivatives of amino acids have been also described as inhibitors of aminopeptidases. andersson et al. first reported this group of compounds as very effective transition states analogue inhibitors (upon hydratation they formed a gem-diolate involved in zinc complexation) of porcine cytosolic and microsomal leucine aminopeptidases [184] . the most effective inhibitor described in this report was l-leucinal (compound 70, fig. 24 , k i ¼ 6 nm for lap and k i ¼ 76 nm for apn). importance of the aldehyde war-head was demonstrated in this report by comparison of the value of l-leucinal inhibitory constant with those found for simple amino acid l-leucine as well as its hydroxy derivative l-leucinol. these compounds were around 4e5 orders of magnitude less effective towards both aminopeptidases tested. additionally, glycine aldehyde derivative (glycinal, compound 69, fig. 24 ) investigated in this study was also much less effective (k i ¼ 0.68 mm for lap). this result confirmed the importance of the side chain in binding efficiency of the designed inhibitors, but also proved that information from the substrate activity screening can be used for the design of the inhibitors. l-leucine p-nitroanilide substrate is much more efficiently processed by lap than analogous glycine derivative. another group of inhibitors for aminopeptidases with aldehyde scaffold were proposed by tarnus et al., 3-amino-2-hydroxy-propionaldehyde and 3-amino-1-hydroxypropan-2-one derivatives (71 and 72, respectively, fig. 25 ) [185] . these compounds designed as general inhibitors of metallo-aminopeptidases were micromolar competitive inhibitors of lap and apn. for some derivatives selective inhibition of apn over lap was observed. unfortunately, due to their susceptibility to oligomerization as well as very high reactivity aldehyde derivatives are not the best candidates for in vivo studies. however, they are an interesting alternative for design of inhibitors, which can be used for investigation of aminopeptidases in vitro. sulfonamides belong to a group of the most recognized compounds with biomedical relevance. easily obtained in the reaction of appropriate sulfonyl chlorides with amines, they offer a great potential of structural variations of both substrates. indeed, since the discovery of the antimicrobial properties, sulfonamides have found a vast number of other biological applications connected with regulation of enzymatic activity. in the context of proteases inhibition, a rationale for the sulfonamide moiety application is the isosteric and isoelectronic resemblance to the high energy tetrahedral transition state that is present in the amide bond hydrolysis (similarly to the phosphorus containing peptide analogues). surprisingly, there are not many recent examples of the use of this strategy towards metallo-aminopeptidase targets. what is more significant, the mode of sulfonamides action in those rare cases is miscellaneous and does not follow the theoretically considered pattern of transition state interactions. the title functional group plays more of a role as a linker between hydrophobic portions of the inhibitor, with another metal binding entity incorporated into one of them. alternatively, a cooperative action of two heteroatom-rich moieties (including the sulfonamide one) is observed. the most advanced studies on this topic seemed to be performed in abbott laboratories and concerned anthranilic acid sulfonamides as inhibitors of the human recombinant methionine aminopeptidase type 2 (as a target for orally available drugs in an anti-cancer therapy) [186e189]. a series of sulfonamide compounds, such as 73 (fig. 26) , was identified using affinity selection by a mass spectroscopy screening method [186] . they exhibited micromolar activity for the manganese form of the human metap2 and were moderately potent in a cell proliferation assay. thanks to promising pharmacokinetics and synthetic viability they were pointed out as novel attractive leads. consecutive x-ray studies allowed for the rational design of a new generation of ligands and tracking of the efficiency of structural optimizations. first iteration 73, ic 50 enlargement of the aromatic portion of the anthranilic acid to a naphthalene or tetrahydronaphthalene system (compound 74, fig. 26 ) allowed 1000-fold improvement in activity of the starting molecule [186] . as revealed by the crystal structure, tetrahydronaphthyl ring fitted tightly into a hydrophobic pocket of the active site. the carboxylate was an actual metal chelating group, whereas the sulfonamide moiety simply ensured the proper twist of the molecule to point the aromatic rings into a lipophilic environment. unfortunately, these inhibitors exhibited a limited cellular activity and extensive binding to human serum albumin. a positively charged ortho substitution in the arylsulfonamide ring was predicted to obey these drawbacks, in particular to disrupt interactions with albumin [187] . a set of substituents to the structure based on complex amines and diamines was introduced at this position and positively validated the approach. the modified products (exemplified by 75, fig. 26 ) showed potent activity for metap2, associated with high selectivity versus related aminopeptidases (3000-fold ratio less active for metap1, for example) [187] . importantly, their efficiency in cell proliferation assays was improved by a greater than 100-fold gain in potency and ranked in low nanomolar range. similar parameters were achieved for 5,6 disubstituted anthranilic acids containing and additional heteroatom group that was presumed to tighten interactions with manganese ions [188] . since they exhibit strong anti-cancer activity, enhanced accessibility and oral available such sulfonamides could be considered as optimized for therapeutic use [189] . a cooperative binding mode was observed for quinoline-based sulfonamides potent towards the e. coli metap1. these inhibitors were discovered by screening of a 100,000 member small organic compounds library [190] . selected hits were screened for different metalloforms of the enzyme, with the highest affinity towards the cobalted form displayed by compound 76 (fig. 27) . the sulfonamides behaved as typical competitive inhibitors; however, as disclosed by the x-ray structural studies, their mode of interactions was not typical. consistent with data described for the heteroaromatic ligands, the enzyme active site was loaded with three metal ions. the inhibitor bound as a bidentate sulfonamide/ quinoline n,n donor to the auxiliary manganese or cobalt atom [190] . although the methanesufonate fragment was deeply buried, the molecule had no direct interactions with the catalytic metal ions. 14. tetralone derivatives first described in 1994 by schalk et al. derivatives of 3-amino-2-tetralone were found to be nanomolar inhibitors of porcine kidney aminopeptidase n [191] . these compounds (77 and 78, fig. 28 ) have non-peptidic character and are competitive inhibitors of the enzyme. the possible mechanism of their coordination with enzyme zinc ion is via carbonyl and amine groups located on the neighboring carbons in the cyclohexyl scaffold. interestingly, these compounds were almost completely inactive towards aspartate and arginine aminopeptidases and only slightly active towards lap. in another approach albrecht et al. synthesized various new derivatives of 3-amino-2-tetralone [192] . several methyl ketone, substituted oximes or hydroxamic acids, phosphinic acids and hydrazides derivatives (exemplified by compounds 79e81, fig. 29 ) were obtained and tested towards leucine aminopeptidase, aminopeptidase n, aeromonas proteolytica aminopeptidase, and leukotriene a 4 hydrolase. even if theoretically equipped with better zinc chelating groups, these compounds were rather weak (up to low micromolar k i values) inhibitors of aminopeptidases with one active site zinc and very weak inhibitors of aminopeptidases with two zinc ions. inhibitors bearing gallic acid in the structure were designed based on the previously known biological properties of this compound as well as by assumption that methoxy (natural gallic acid has three free hydroxy groups) substituted hydrophobic ring of this compound will tightly bind in the s1 pocket of aminopeptidase n. several amino acids derivatives of this compound like 4-amino-l-proline, l-iso-glutamine and cyclo-l-iso-glutamine have been obtained [193e195] . among them gallolylamide derivatives based on proline scaffold (compounds 82 and 83, fig. 30) were extremely good inhibitors of aminopeptidase n. the best compounds had ic 50 values in low nanomolar range [194] . in another study l-iso-glutamine and cyclo-l-iso-glutamine derivatives (compounds 84 and 85, fig. 31 ) have been synthesized. these compounds were not as good inhibitors as proline derivatives and had ic 50 [195] . betulinic acid (86, fig. 32 ) is a triterpene isolated from bark of several different plants (birch bark is rich source of this compound) [196] . this compound has been found to be potent inducer of apoptosis in cancer cells and is currently in clinical trials [197] . it is proposed that betulinic acid acts by increasing mitochondrial membrane permeability, which subsequently facilitates release of apoptosis stimulating proteins (cytochrome c). however, several others biological targets for this compound have been proposed and one of them is membrane aminopeptidase n (cd13). melzig et al. determined the ic 50 for betulinic acid against aminopeptidase to be 7.3 mm [198] . naturally occurring in plants polyphenolecurcumin (87, fig. 33 ) has been also described as inhibitor of aminopeptidase n. curcumin is known as potent antitumor agent and currently its mechanism of action as well as biological targets are extensively investigated [199, 200] . this compound interacted with cd13 in irreversible and non-competitive mode and strongly inhibited apn-positive tumor cell invasion as well as induced angiogenesis by basic fibroblast growth factor [201] . interestingly, curcumin did not influence invasion of apn-negative cells, what further strengthened hypothesis that anti-invasive activity of this compound is a result of cd13 inhibition. aminopeptidases play pivotal roles in the turnover of proteins and the regulation of intracellular amino acids pools. they are essential to the metabolism, growth and development of all cells and tissues, and are part of the processes that regulate our immune and neurological systems. they also perform a broad spectrum of functions outside the cell, on the surface or even in the surrounding milieu (receptors, hormone processing and regulation etc). their involvement in the cause or maintenance of certain pathological diseases, particularly cancer, has focused our attention on their structure and function in the hope of developing new treatments. more recently, we have learned that aminopeptidases are also central to the cellular physiology of many parasites, including malaria, which has opened new avenues for development of antiinfectious disease drugs. crucial to the development of new drugs, however, is our detailed understanding of the mechanism of binding and interaction of inhibitory compounds to the active site of their targets. the present review highlights how this has been progressing well for several aminopeptidases, including leucine, alanine and methionine aminopeptidases, but also exposes our lack of information on a large number of aminopeptidase families. clearly, these gaps will be filled in time due to the improvements in inhibitor discovery and design (e.g. screening of chemical libraries followed by medicinal chemistry) and in methods for threedimensional structure determination. the challenge will be to discover inhibitors with selectivity not only for specific enzyme types so as to avoid off-target effects on other systems, but can be delivered to block specific functions or physiological roles of a particular aminopeptidase since each enzyme often performs a variety of defined and ancillary roles. aminopeptidases in biology and disease, proteases in biology and disease human aminopeptidases: a review of the literature industrial enzymes the properties and functions of bacterial aminopeptidases merops: the peptidase database leucine aminopeptidase as a target for inhibitor design. mini rev proteolysis and class i major histocompatibility complex antigen presentation leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for mhc class i antigen presentation interferon-gamma can stimulate postproteasomal trimming of the n terminus of an antigenic peptide by inducing leucine aminopeptidase identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens lap to cleave bovine crystallins expression of adipocyte-derived leucine aminopeptidase in endometrial cancer. association with tumor grade and ca-125 bestatinmediated inhibition of leucine aminopeptidase may hinder hiv infection the moonlighting enzyme cd13: old and new functions to target proteomic analysis of microglia-derived exosomes: metabolic role of the aminopeptidase cd13 in neuropeptide catabolism physiology of local renineangiotensin systems cd13-not just a marker in leukemia typing cd13 (human aminopeptidase n) mediates human cytomegalovirus infection development of a transgenic mouse model susceptible to human coronavirus 229e aminopeptidase n in arterial hypertension enkephalin metabolism by microglial aminopeptidase n (cd13) impaired angiogenesis in aminopeptidase n-null mice cd13 is a novel mediator of monocytic/endothelial cell adhesion maps and poep of the roads from prokaryotic to eukaryotic kingdoms methionine aminopeptidase 2 and cancer immunomodulatory activity of a methionine aminopeptidase-2 inhibitor on b cell differentiation ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation map1d, a novel methionine aminopeptidase family member is overexpressed in colon cancer roles of p67/metap2 as a tumor suppressor cerebrospinal fluid proteomic analysis reveals dysregulation of methionine aminopeptidase-2 expression in human and mouse neurofibromatosis 1-associated glioma purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (cyprinus carpio) leucine aminopeptidase from red sea bream (pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution purification and properties of aminopeptidase h from chicken skeletal muscle purification and properties of major midgut leucyl aminopeptidase of morimus funereus (coleoptera, cerambycidae) larvae purification and characterization of aminopeptidase n from spodoptera litura expressed in sf21 insect cells mutation of an aminopeptidase n gene is associated with helicoverpa armigera resistance to bacillus thuringiensis cry1ac toxin identification and characterization of aedes aegypti aminopeptidase n as a putative receptor of bacillus thuringiensis cry11a toxin neuropeptidases and the metabolic inactivation of insect neuropeptides complexes of mutants of escherichia coli aminopeptidase p and the tripeptide substrate valproleu expression and characterization of two functional methionine aminopeptidases from mycobacterium tuberculosis h37rv the leucyl aminopeptidase from helicobacter pylori is an allosteric enzyme crystal structure of the cold-active aminopeptidase from colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene a4 hydrolase purification and characterization of hyperthermotolerant leucine aminopeptidase from geobacillus thermoleovorans 47b mutational analysis of the zinc metalloprotease empa of vibrio anguillarum characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon pyrococcus furiosus characterization of the plasmodium falciparum m17 leucyl aminopeptidase. a protease involved in amino acid regulation with potential for antimalarial drug development the m17 leucine aminopeptidase of the malaria parasite plasmodium falciparum: importance of active site metal ions in the binding of substrates and inhibitors plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials the m18 aspartyl aminopeptidase of plasmodium falciparum binds to human erythrocyte spectrin in vitro the m18 aspartyl aminopeptidase of the human malaria parasite plasmodium falciparum the type ii secretion system of legionella pneumophila elaborates two aminopeptidases, as well as a metalloprotease that contributes to differential infection among protozoan hosts partial purification and characterization of an aminopeptidase from eimeria tenella characterization of a leucine aminopeptidase of babesia gibsoni investigations into microsporidian methionine aminopeptidase type 2: a therapeutic target for microsporidiosis leucine aminopeptidases of the human blood flukes schistosoma mansoni and schistosoma japonicum rna interference targeting leucine aminopeptidase blocks hatching of schistosoma mansoni eggs identification and characterization of paragonimus westermani leucine aminopeptidase fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis mechanistic role of each metal ion in streptomyces dinuclear aminopeptidase: peptide hydrolysis and 7 â 10(10)-fold rate enhancement of phosphodiester hydrolysis metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase analyzing the binding of co(ii)-specific inhibitors to the methionyl aminopeptidases from escherichia coli and pyrococcus furiosus role of the invariant asn345 and asn435 residues in a leucine aminopeptidase from bacillus kaustophilus as evaluated by site-directed mutagenesis a functional comparison of the tet aminopeptidases of p. furiosus and b. subtilis with a proteinengineered variant recombining the former's structure with the latter's active site histidines 345 and 378 of bacillus stearothermophilus leucine aminopeptidase ii are essential for the catalytic activity of the enzyme analyzing the catalytic role of asp97 in the methionine aminopeptidase from escherichia coli kinetic and spectroscopic analysis of the catalytic role of h79 in the methionine aminopeptidase from escherichia coli kinetic, spectroscopic, and x-ray crystallographic characterization of the functional e151h aminopeptidase from aeromonas proteolytica the catalytic role of glutamate 151 in the leucine aminopeptidase from aeromonas proteolytica structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and tnp-470 crystal structure of isoaspartyl aminopeptidase in complex with l-aspartate structure of aminopeptidase n from escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor pl250 structural basis of catalysis by monometalated methionine aminopeptidase zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from vibrio proteolyticus crystal structure of aminopeptidase n from human pathogen neisseria meningitidis crystal structures of staphylococcus aureus methionine aminopeptidase complexed with keto heterocycle and aminoketone inhibitors reveal the formation of a tetrahedral intermediate structural basis for the inhibition of the essential plasmodium falciparum m1 neutral aminopeptidase structure of the plasmodium falciparum m17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases peptide hydrolysis by the binuclear zinc enzyme aminopeptidase from aeromonas proteolytica: a density functional theory study the reaction mechanism of bovine lens leucine aminopeptidase on the origin of the broad-band selectivity of bovine-lens-leucine-aminopeptidase development of a working model of the active site in bovine lens leucine aminopeptidase: a density functional investigation hydrolysis of thionopeptides by the aminopeptidase from aeromonas proteolytica: insight into substrate binding iron substitution for sodium in a carboxylate-bridged, heterodinuclear sodiumeiron complex aminopeptidase n (apn/cd13) as a target for anti-cancer agent design metalloaminopeptidases: common functional themes in disparate structural surroundings aminopeptidase-n/cd13 (ec 3.4.11.2) inhibitors: chemistry, biological evaluations, and therapeutic prospects the structure and main functions of aminopeptidase n phosphorus amino acid analogues as inhibitors of leucine aminopeptidase biological activity of aminophosphonic acids and their short peptides inhibition of aminopeptidases by phosphonic acid and phosphinic acid analogues of aspartic and glutamic acids inhibition of aminopeptidases by aminophosphonates alpha-aminoalkylphosphonates as a tool in experimental optimisation of p1 side chain shape of potential inhibitors in s1 pocket of leucine-and neutral aminopeptidases stereoselective synthesis of 1-aminoalkanephosphonic acids with two chiral centers and their activity towards leucine aminopeptidase chemical target validation studies of aminopeptidase in malaria parasites using alpha-aminoalkylphosphonate and phosphonopeptide inhibitors aminopeptidase fingerprints. an integrated approach for identification of good substrates and optimal inhibitors the most potent organophosphorus inhibitors of leucine aminopeptidase. structure-based design, chemistry, and activity computer-aided design and activity prediction of leucine aminopeptidase inhibitors quantum chemical analysis of the interactions of transition state analogs with leucine 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aminopeptidase a (ec 3.4.11.7, apa) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the p-1 position novel hydroxamic acid-related phosphinates: inhibition of neutral aminopeptidase n (apn) first synthesis of alpha-aminoalkyl-(n-substituted) thiocarbamoyl-phosphinates: inhibitors of aminopeptidase n (apn/cd13) with the new zinc-binding group bestatin, an inhibitor of aminopeptidase b, produced by actinomycetes bestatin as an experimental tool in mammals leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis leukotriene a4 hydrolase. inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin inhibition of aminopeptidases by amastatin and bestatin derivatives the structure of bestatin a stereospecific synthesis of (à)-bestatin from l-malic acid application of acyl cyanophosphorane methodology to the synthesis of protease inhibitors: poststatin, eurystatin, phebestin, probestin and bestatin acylnitrene route to vicinal amino alcohols. application to the synthesis of (à)-bestatin and analogues a new one-pot method for the synthesis of alpha-siloxyamides from aldehydes or ketones and its application to the synthesis of (à)-bestatin new stereoselective synthesis of the peptidic aminopeptidase inhibitors bestatin, phebestin and probestin bestatin: three decades of synthetic strategies the review of the synthesis of bestatin, an effective inhibitor of aminopeptidase n bestatin inhibits covalent coupling of [3h]lta4 to human leukocyte lta4 hydrolase design of novel inhibitors of aminopeptidases. synthesis of peptide-derived diamino thiols and sulfur replacement analogues of bestatin synthesis of p-hydroxyubenimex synthesis of sulfur-containing analogues of bestatin. inhibition of aminopeptidases by alpha-thiolbestatin analogues development of bestatin-based activity-based probes for metallo-aminopeptidases phebestin, a new inhibitor of aminopeptidase n, produced by streptomyces sp. mj716-m3 probestin, a new inhibitor of aminopeptidase m, produced by streptomyces azureus mh663-2f6. i. taxonomy, production, isolation, physico-chemical properties and biological activities the metabolism of neuropeptides. phase separation of synaptic membrane preparations with triton x-114 reveals the presence of aminopeptidase n matrix metalloproteinase inhibitors and cancer: trials and tribulations matrix metalloproteinase inhibitors for cancer therapy: the current situation and future prospects inhibition of leucine aminopeptidase by amino acid hydroxamates potent and systemically active aminopeptidase n inhibitors designed from active-site investigation n-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide, a novel hydroxamic acid-based inhibitor of aminopeptidase n and its anti-angiogenic activity peptidyl hydroxamic acids as methionine aminopeptidase inhibitors metalloform-selective inhibition: synthesis and structureeactivity analysis of mn(ii)-form-selective inhibitors of escherichia coli methionine aminopeptidase deprez-poulain, design, synthesis and antimalarial activity of novel, quinoline-based, zinc metalloaminopeptidase inhibitors novel selective inhibitors of the zinc plasmodial aminopeptidase pfa-m1 as potential antimalarial agents cytostatic agents chr-2797: an antiproliferative aminopeptidase inhibitor that leads to amino acid deprivation in human leukemic cells a first-in-man phase i and pharmacokinetic study on chr-2797 (tosedostat), an inhibitor of m1 aminopeptidases, in patients with advanced solid tumors aminopeptidase inhibitor, oncolytic l-lysinethiol: a subnanomolar inhibitor of aminopeptidase b l-leucinthiol e a potent inhibitor of leucine aminopeptidase investigation of the active site of aminopeptidase a using a series of new thiol-containing inhibitors differential inhibition of aminopeptidase a and aminopeptidase n by new beta-amino thiols mixed inhibitor-prodrug" as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes inhibition of angiotensin iii formation by thiol derivatives of acidic amino acids llorens-cortes, pc18, a specific aminopeptidase n inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin iii to the design of the first highly potent and selective inhibitors of this enzyme synthesis and in vitro activities of new non-peptidic apa inhibitors identification of metabolic pathways of brain angiotensin ii and iii using specific aminopeptidase inhibitors: predominant role of angiotensin iii in the control of vasopressin release aminopeptidase a inhibitors as potential central antihypertensive agents brain renineangiotensin system blockade by systemically active aminopeptidase a inhibitors: a potential treatment of salt-dependent hypertension orally active aminopeptidase a inhibitors reduce blood pressure: a new strategy for treating hypertension methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors agm-1470 and ovalicin the antiangiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, metap-2 the anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the escherichia coli methionine aminopeptidase structure of human methionine aminopeptidase-2 complexed with fumagillin synthetic analogues of tnp-470 and ovalicin reveal a common molecular basis for inhibition of angiogenesis and immunosuppression metalloform-selective inhibitors of escherichia coli methionine aminopeptidase and x-ray structure of a mn(ii)-form enzyme complexed with an inhibitor inhibition of monometalated methionine aminopeptidase: inhibitor discovery and crystallographic analysis two continuous spectrophotometric assays for methionine aminopeptidase identification of pyridinylpyrimidines as inhibitors of human methionine aminopeptidases metal ions as cofactors for the binding of inhibitors to methionine aminopeptidase: a critical view of the relevance of in vitro metalloenzyme assays metal-mediated inhibition of escherichia coli methionine aminopeptidase: structureeactivity relationships and development of a novel scoring function for metal-ligand interactions substituted 1,2,4-triazole methionine aminopeptidase type 2 inhibitors, their preparation, and their therapeutic use substituted triazole methionine aminopeptidase type 2 inhibitors, their preparation, and their therapeutic use highly potent inhibitors of methionine aminopeptidase-2 based on a 1,2,4-triazole pharmacophore 3-triazole: a novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo the 1.15a crystal structure of the staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese small molecule inhibitors of methionine aminopeptidase type 2 (metap-2) discovery and structural modification of inhibitors of methionine aminopeptidases from escherichia coli and saccharomyces cerevisiae inhibitors of type i metaps containing pyridine-2-carboxylic acid thiazol-2-ylamide. part 2: sar studies on the pyridine ring 3-substituent identification of potent type i metap inhibitors by simple bioisosteric replacement. part 1: synthesis and preliminary sar studies of thiazole-4-carboxylic acid thiazol-2-ylamide derivatives identification of potent type i metaps inhibitors by simple bioisosteric replacement. part 2: sar studies of 5-heteroalkyl substituted tcat derivatives elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression discovery of inhibitors of escherichia coli methionine aminopeptidase with the fe(ii)-form selectivity and antibacterial activity inhibitors of plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity hydroxamates and aliphatic boronic acids: marker inhibitors for aminopeptidase a transition-state-analog inhibitor influences zincbinding by aeromonas aminopeptidase shenvi, alpha-aminoboronic acid derivatives: effective inhibitors of aminopeptidases orchymont, 3-amino-2-hydroxy-propionaldehyde and 3-amino-1-hydroxy-propan-2-one derivatives: new classes of aminopeptidase inhibitors development of sulfonamide compounds as potent methionine aminopeptidase type ii inhibitors with antiproliferative properties discovery and optimization of anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2: a structural basis for the reduction of albumin binding lead optimization of methionine aminopeptidase-2 (metap2) inhibitors containing sulfonamides of 5,6-disubstituted anthranilic acids correlation of tumor growth suppression and methionine aminopetidase-2 activity blockade using an orally active inhibitor metal mediated inhibition of methionine aminopeptidase by quinolinyl sulfonamides 3-amino-2-tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-m (ec 3.4.11.2) synthesis and structure activity relationships of novel non-peptidic metalloaminopeptidase inhibitors the preparation of novel 1-iso-glutamine derivatives as potential antitumor agents 226th acs national meeting novel 3-galloylamido-n 0 -substituted-2,6-piperidinedione-n-acetamide peptidomimetics as metalloproteinase inhibitors comparison of the cytotoxic effects of birch bark extract, betulin and betulinic acid towards human gastric carcinoma and pancreatic carcinoma drug-sensitive and drug-resistant cell lines activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid betulinic acid inhibits aminopeptidase n activity multiple molecular targets in cancer chemoprevention by curcumin antitumor, antiinvasion, and antimetastatic effects of curcumin irreversible inhibition of cd13/aminopeptidase n by the antiangiogenic agent curcumin key: cord-023284-i0ecxgus authors: nan title: abstracts of publications related to qasr date: 2006-09-19 journal: nan doi: 10.1002/qsar.19900090309 sha: doc_id: 23284 cord_uid: i0ecxgus nan tive mechanisms p.2-25. edited by magee, p.s., henry, d.r., block, j.h., american chemical society, washington, 1989. results: an overview is given on the approaches for the discovery and design concepts of bioactive molecules: a) natural products derived from plant extracts and their chemically modified derivatives (cardiac glycosides, atropine, cocaine, penicillins, cephalosporins, tetracyclines and actinomycins, pyrethrins and cyclosporin; b) biochemically active molecules and their synthetic derivatives: acetylcholine, histamine, cortisonelhydrocortisone, indole-3-acetic acid (phenoxyacetic acid herbicides); c) principles of selective toxicity is discussed exemplified by trimethoprimlmethotrexate, tetracyclines, acylovir, azidothymidine, antifungal agents; d) metabolism of xenobiotics; e) exploitation of secondary effects (serendipity); f) receptor mapping; g) quantitative structure-activity relationship studies; h) empirical screening (shotgun approach). results: past and present of qsar is overviewed: a) historical roots; b) the role of qsar models in rational drug design, together with a simplified diagram of the steps involved in drug development, including the place of qsar investigations; c) classification of qsar models: structure-cryptic (property-activity) models, structure-implicit (quantum chemical) models, structure-explicit (structure-activity) and structure-graphics (computer graphics) models; d) a non-empirical qsar model based on quantities introduced for identification of chemical structures, using szymansk's and randic's identification (id) numbers, including applications for alkyltriazines. bioessays, 1989, 11(5) , 136-141. results: a review is given on recent observations about receptor structure and the dynamic nature of drug receptors and the significance of receptor dynamics for drug design: a) receptors are classified according to structure and function (i) ion channels (nicotinic acetylcholine, gaba, glycine); (ii) g protein linked [adrenergic ( c x ,~) , muscarinic acetylcholine, angiotensin, substance k, rhodopsin]; (iii) tyrosine kinase (insulin, igf, egf, pdgf); (iv) guanylate cyclase (atrial natriuretic peptide, speractin); b) protein conformational changes can be best studied on allosteric proteins whose crystal structure is available (e.g. hemoglobin, aspartate transcarbamylase, tryptophan repressor) (no high resolution of a receptor structure is known); c) receptor conformational changes can be studied by several indirect approaches (i) spectral properties of covalent or reversibly bound fluorescent reporter groups; (ii) the sensitivity of the receptor to various enzymes; (iii) the sedimentation of chromatographic properties of the receptor; the affinity of binding of radioligands; (iv) the functional state of the receptor; d) there are many unanswered questions: e.g. (i) are there relatively few conformational states for receptors with fluctuations around them or many stable conformational states; (ii) how can static structural information be used in drug design when multiple receptor conformations exist. title: designing molecules and crystals by computer. (review) author: koide, a. ibm japan limited, tokyo scientific center, tokyo research laboratory 5-19 sanban-cho, chiyoda-ku, tokyo 102, japan. source: ibm systems journal 1989, 28(4), 613 -627. results: an overview is given on three computer aided design (cad) systems developed by ibm tokyo scientific center: a) molecular design support system providing a strategic combination of simulation programs for industrial research and development optimizing computational time involved and the depth of the resulting information; b) molworld on ibm personal systems intended to create an intelligent visual environment for rapidly building energetically stable 3d molecular geometries for further simulation study; c) molecular orbital graphics system designed to run on ibm mainframe computers offering highly interactive visualization environment for molecular electronic structures; d) the systems allow interactive data communication among the simulation programs for their strategically combined use; e) the structure and functions of molworld is illustrated on modeling the alanine molecule: (i) data model of molecular structures; (ii) chemical formula input; (iii) generation of 3d molecular structure; (iv) formulation of bonding model; (v) interactive molecular orbital graphics; (vi) methods of visualizing electronic structures; (vii) use of molecular orbital graphics for chemical reactions. title: interfacing statistics, quantum chemistry, and molecular modeling. (review) author: magee, p.s. biosar research project vallejo ca 94591, usa. source: acs symposium series 1989, no.413 . in: probing bioactive mechanisms p.37-56. edited by magee, p.s., henry, d.r., block, j.h., american chemical society, washington, 1989. results: a review is given on the application and overlap of quantum chemical, classical modeling and statistical approaches for the quant. struct.-act. relat. 9, 234 -293 (1990) abstr. 225-228 235 understanding of binding events at the molecular level. a new com-a) qsar of cns drugs has been systematically discussed according plementary method called statistical docking experiment is also to the following classes: (i) general (nonspecific) cns depressants: presented: general anesthetics; hypnotics and sedatives; (ii) general insights obtained using energy-minimized structures; activation in the bound state, types and energies of interactions at the receptor site and in crystal; four successful examples (significant regression equations) are given for the modeling of binding events using physico-chemical descriptors and correlation analysis: (i) binding of a diverse set of pyridines to silica gel during thin-layer chromatography; (ii) binding of meta-substituted n-methyl-arylcarbamates to bovine erythrocyte ache; (iii) binding of meta-substituted n-methyl-arylcarbamates to ache obtained from susceptible and resistant green rice leafhoppers; (iv) activity of phenols inhibiting oxidative phosphorylation of adp to atp in yeast; a new statistical method for mapping of binding sites has been developed based on the hypermolecule approach, identifying key positions of binding and nature of the energy exchange between the hypermolecule atoms and the receptor site; two examples are given on the successful application of statistical modeling (statistical docking experiment) based on the hypermolecule approach: (i) inhibition of housefly head ache by metasubstituted n-methyl-arylcarbamates (n = 36, r = 0.841, s = 0.390, f = 25.82); (ii) inhibition of housefly head ache by orthosubstituted n-methyl-arylcarbamates (n = 46, r = 0.829, s = 0.485, f = 14.24). (nonspecific) cns stimulants; (iii) selective modifiers of cns functions: anticonvulsants, antiparkinsonism drugs, analgetics and psychopharmacological agents; (iv) miscellaneous: drugs interacting with central a-adrenoreceptors, drugs interacting with histamine receptors, cholinergic and anticholinergic drugs; b) the review indicates that the fundamental property of the molecules which mostly influence the activity of cns drugs is hydrophobicity (they have to pass the cell membrane and the bloodbrain barrier); c) electronic parameters, indicative of dipole-dipole or charge-dipole interactions, charge-transfer phenomena, hydrogen-bond formation, are another important factor governing the activity of most cns agents; d) topographical, lipophylic and electronic structures of cns pharmacophores are reviewed; e) 191 qsar equations, 24 tables and 3 figures from 294 references are shown and discussed. the relevant template for each atom in the molecule is mapped into a bit array and the appropriate atomic position is marked; volume comparisons (e.g. common volume or excluded volume) are made by bit-wise boolean operations; the algorithm for the visualization of the molecular surface comprising the calculated van der walls volume is given; comparisons of cpu times required for the calculation of the van der waals molecular volumes of various compounds using the methods of stouch and jurs, pearlman, gavazotti and the new method showed that similar or better results can be achieved using the new algorithm with vax-class computers on molecules containing up to several hundred atoms. abtstr. 229-232 quant. struct.-act. relat. 9, 234-293 (1990) one of the important goal of protein engineering is the design of isosteric analogues of proteins; major software packages are available for molecular modeling are among others developed by (i) biodesign, inc., pasadena, california; (ii) biosym technologies, san diego, california; (iii) tripos, st. louis, missouri; (iv) polygen, waltham, massachusetts; (v) chemical design ltd. oxford; the molecular modelling packages use three basic parameters: (i) descriptive energy field; (ii)algorithm for performing molecular mechanics calculations; (iii) algorithm for performing molecular dynamics calculations; modelling study of the binding events occurring between the envelop protein (gp120) of the aids (hiv) virus and its cellular receptor (cd4) protein supported the hypothesis that this domain was directly involved in binding the gp120 envelop protein leading to the design of conformationally restricted synthetic peptides binding to cd4. 19901229 title: finding washington, 1989. results: a new technique called "homology graphing" has been developed for the analysis of sequence-function relationships in proteins which can be used for sequence based drug design and the search for lead structures: a) as target protein is inhibited by the ligands of other proteins having sequence similarity, computer programs have been developed for the search of the similarity of proteins; b) proteins are organized into hierarchical groups of families and superfamilies based on their global sequence similarities; c) global sequence similarities were used to find inhibitors of acetolactate synthase (als) and resulted in a quinone derivative as a lead structure of new als inhibitors; d) local sequence similarities of bacterial and mammal glutathione synthase (gsh) were used to find inhibitors of gsh; e) it was shown that the sequence segment of gsh was similar to dihydrofolate reductase (dhfr) is part of the atp-binding site; f) biological bases of local similarity between sequences of different proteins were indicated: molecular evolution of proteins and functionally important local regions; g) homology graph, as a measure of sequence similarity was defined; h) sequence-chemical structure relationship based on homology graph and the procedure to find lead structures was illustrated by an example resulting in a list of 33 potential inhibitors selected by the procedure based on the sequence segment from residue 150 to 210 of the sequence of tobacco als. source: acs symposium series 1989, no.413. in: probing bioactive mechanisms p.198-214. edited by magee, p.s.. henry, d.r., block, j.h., american chemical society, washington. 1989. results: a review is given on the molecular design of the following major types of antifungal compound in relation to biochemistry, molecular modeling and target site fit: a) squalene epoxidase inhibitors (allilamines and thiocarbanilates) blocking conversion of squalene 2,3-oxidosqualene; b) inhibitors of sterol c-14 demethylation by cytochrome p-450 (piperazines pyridines, pyrimidines, imidazoles and triazoles); c) inhibitors of sterol a' -t a' isornerization andlor sterol reductase inhibitors (morpholines); d) benzimidazoles specifically interfering with the formation of microtubules and the activity phenylcarbamates on benzimidazole resistant strains; e) carboxamides specifically blocking the membrane bound succinate ubiquinone oxidoreductase activity in the mitochondria1 electron transport chain in basidiomycetes; f) melanin biosynthesis inhibitors selectively interfering with the polyketide pathway to melanin in pyricularia oryzae by blocking nadph dependent reductase reactions of the pathway (fthalide, pcba, chlobentiazone, tricyclazole, pyroquilon, pp389). title: quantitative modeling of soil sorption for xenobiotic chemicals. (review) author: sabljic, a. theoretical chemistry group, department of physical chemistry, institute rudjer boskovic hpob 1016, yu-41001 zagreb, croatia, yugoslavia. source: environ. health perspect. 1989, 83(2), 179 -190. results: the environmental fate of organic pollutants depends strongly on their distribution between different environmental compartments. a review is given on modeling the soil sorption behavior of xenobiotic chemicals: a) distribution of xenobiotic chemicals in the environment and principles of its statistical modeling; b) quantitative structure-activity relationship (qsar) models relating chemical, biological or environmental activity of the pollutants to their structural descriptors or physico-chemical properties such as logp values and water solubilities; c) analysis of the qsar existing models showed (i) low precision of water solubility and logp data; (ii) violations of some basic statistical laws; d) molecular connectivity model has proved to be the most successful structural parameter modeling soil sorption; e) highly significant linear regression equations are cited between k , values and the first order molecular connectivity index ( ' x ) of a wide range of organic pollutants such as polycyclic aromatic hydrocarbons (pahs) and pesticides (organic phosphates, triazines, acetanilides, uracils, carbamates, etc.) with r values ranging from 0.976 to 0.986 and s values ranging from 0.202 to 0.300; f) the molecular connectivity model was extended by the addition of a single semiempirical variable (polarity correction factor) resulting in a highly significant linear regression equations between the calculated and measured ko, values of the total set of compounds (n = 215, r = 0.969, s = 0.279, f = 3291); g) molecular surface areas and the polarity of the compounds were found to be responsible for the majority of the variance in the soil sorption data of a set of structurally diverse compounds. title: strategies for the use of computational sar methods in assessing genotoxicity. (review) results: a review is given on the overall strategy and computational sar methods for the evaluation of the potential health effects of chemicals. the main features of this strategy are discussed as follows: a) generalized sar model outlining the strategy of developing information for the structure-activity assessment of the potential biological effects of a chemical or a class of chemicals; b) models for predicting health effects taking into account a multitude of possible mechanisms: c) theoretical models for the mechanism of the key steps of differential activity at the molecular level; d) sar strategies using linear-free energy methods such as the hansch approach; e) correlative sar methods using multivariate techniques for descriptor generation and an empirical analysis of data sets with large number of variables (simca, adapt, topkat, case, etc.); f) data base considerations describing three major peer-reviewed genetic toxicology data bases (i) national toxicology program (ntp) containing short term in vitro and in vivo genetic tests; (ii) data base developed by the epa gene-tox program containing 73 different short term bioassays for more than 4000 compounds, used in conjunction with adapt, case and topkat; (iii) genetic activity profile (gap) in form of bar graphs displaying information on various tests using a given chemical. title: quantitative structure-activity relationships. principles, and authors: benigni,, r.; andreoli, c.; giuliani, a. applications to mutagenicity and carcinogenicity. (review) laboratory of toxicology and ecotoxicology, istituto superiore di sanita rome, italy. source: mutat. res. 1989, 221(3), 197 -216. results: methods developed for the investigation for the relationships between structure and toxic effects of compounds are summarized: a) the extra-thermodynamic approach: the hansch paradigm, physical chemical properties that influence biological activity and their parametrization, originality of the hansch approach, receptors and pharmacophores: the natural content of the hansch approach, predictive value of qsars, a statistifa1 tool: multiple linear regression analysis, the problem of correlations among molecular descriptors, other mathematical utilizations of extrathermodynamic parameters; b) the substructural approach: when topological (substructural) descriptors are needed, how to use topological decriptors; c) qsar in mutagenicity and carcinogenicity: general problems, specific versions of the substructural approach used for mutagenicity and carcinogenicity, applications to mutagenicity and carcinogenicity. title: linking structure and data. (review) author: bawden, d. source: chem. britain 1989, 25(nov) , i107 -1108. address not given. results: the integration of information from different sources, particularly linking structural with non-structural information is an important consideration in chemical information technology. a review is given on integrated systems: a) socrates chemicallbiological data system for chemical structure and substructure searching combined with the retrieval of biological and physicochemical data, compound availability, testing history, etc.; b) psidom suite of pc based structure handling routines combining chemical structure with the retrieval of text and data; c) cambridge crystal structure databank on x-ray data of organic compounds integrating information on chemical structure, crystal conformation, numerical information on structure determination, bibliographic reference and keywording; d) computer aided organic synthesis for structure and substructure search, reaction retrieval, synthetic analysis and planning, stereochemical analysis, product prediction and thermal hazard analysis. title: determination of three-dimensional structures of proteins and nucleic acids in solution by nuclear magnetic resonance spectroscopy. source: critical rev. biochem. mol. biol. 1989, 24(5) , 479 -564. results: a comprehensive review is given on the use of nmr spectroscopy for the determination of 3d structures of proteins and nucleic acids in solution discussing the following subjects: a) theoretical basis of two-dimensional (2d) nmr and the nuclear overhauser effect (noe) measurements for the determination of 3d structures is given; b) sequential resonance assignment for identifying spin systems of protein nmr spectra and nucleic acid spectra, selective isotope labeling for extension to larger systems and the use of site specific mutagenesis; c) measurement and calculation of structural restraints of the molecules (i) interproton distances; (ii) torsion angle restrains; (iii) 4 backbone torsion angle restraints; (iv) side chain torsion angle restraints; (v) stereospecific assignments; (vi) dihedral angle restraints in nucleic acids; d) determination of secondary structure in proteins; e) determination of tertiary structure in proteins using (i) metric matrix distance geometry (ii) minimization in torsion angle space; (iii) restrained molecular dynamics; (iv) dynamical simulated annealing; (v) folding an extended strand by dynamical simulated annealing; (vi) hybrid metric matrix distance geometry-dynamical simulated annealing method; (vii) dynamical simulated annealing starting from a random array of atoms; f) evaluation of the quality of structures generated from nmr data illustrated by studies for the structure determination of proteins and oligonucleotides using various algorithms and computer programs; g) comparisons of solution and x-ray structures of (i) globular proteins; (ii) related proteins; (iii) nonglobular proteins and polypeptides; h) evaluation of attainable precision of the determination of solution structures of proteins for which no x-ray structures exist (i) bds-i (small 43-residue protein from the sea anemone sulcata; (ii) hirudin (small 65-residue protein from leech which is a potent natural inhibitor of coagulation); i) structure determination by nmr is the starting point for the investigation of the dynamics of conformational changes upon ligand abtstr. 236-239 quant. struct.-act. relat. 9, 234 -293 (1990) binding, unfolding kinetics, conformational equilibria between different conformational states, fast and slow internal dynamics and other phenomena. title: aladdin. an integrated tool for computer-assisted molecular design and pharmacophore recognition from geometric, steric, and substructure searching of three-dimensional molecular structures. ( aladdin has the ability to (i) objectively describe receptor map hypothesis; (ii) scan a database to retrieve untested compounds which is predicted to be active by a receptor map hypothesis; (iii) quantitatively compare receptor map hypotheses for the same biological activity; (iv) design compounds that probe the bioactive conformation of a flexible ligand; (v) design new compounds that a receptor map hypothesis predicts to be active; (vi) design compounds based on structures from protein x-ray crystallography; a search made by aladdin in a database for molecules that should have d2 dopaminergic activity recognized unexpected d2 dopamine agonist activity of existing molecules; a comparison of two superposition rules for d2 agonists was performed by aladdin resulted in a clear discrimination between active and inactive compounds; a compound set was designed that match each of the three lowenergy conformations of dopamine resulting in novel active analogues of known compounds; mimics of some sidc ~1 1 . 1 1 1 1 . 111 p.piide beta turns were designed, in order to demonstrate that aladdin can find small molecules that match a portion of a peptide chain and/or backbone; results: lately a number of chemical information systems based on three-dimensional (3-d) molecular structures have been developed and used in many laboratories: a) concord uses empirical rules and simplified energy minimization to rapidly generate approximate but usually highly accurate 3-d molecular structures from chemical notation or molecular connection table input; b) chemical abstracts service (cas) has added 3-d coordinates for some 4 million organic substances to the cas registry file; c) cambridge structural database system contains x-ray and neutron diffraction crystal structures for tens of thousands of compounds; d) maccs3d developed by molecular design ltd., contains the standard maccs-i structures to which additional 3-d data, such as cartesian coordinates, partial atomic charges and molecular mechanics energy are added; maccs3d allows exact match, geometric, submodel and substructure searching of 3-d models with geometric constrains specified to certain degree of tolerance; two 3-d databases are also available from molecular design that can be searched using maccs3d [drug data report (10,000 models) and fine chemicals directory (90,000 models)]; e) aladdin (daylight chemical information systems) is also searches databases of 3-d structures to find compounds that meet biological, substructural and geometric criteria such as ranges of distances, angles defined by three points (dihedral angles) and plane angles that the geometric object must match. aladdin is one of a number of menus working within the framework provided by daylight's chemical information system. title: improved access to supercomputers boosts chemical applica-author: borman, s. c&en 1155 sixteenth st., n.w., washington dc 20036, usa. source: c&en 1989, 67(29) , 29-37. results: supercomputers have been much more accessible by scientists and engineers in the past few years in part as a result of the establishment of national science foundation (nsf) supercomputer centers. the most powerful class of supercomputers have program execution rates of 100 million to 1 billion floating-point operations per second, memory storage capacities of some ten million to 100 miltion computer words and a standard digital word size of 64 bits, the equivalent of about 15 decimal digits. the following examples are given for the use of supercomputer resources for chemical calculations and modeling: a) modeling of key chromophores in the photosynthetic reaction center of rhodopseudomonas viridis showing the heme group, the iron atom and the chlorophyll which absorbs light and causes rapid transfer of electron to pheophtin and then to the quinone; modeling includes a significant part of the protein having about 2000 atoms out of a total of some 12,000; quant. struct.-act. relat. 9, 234-293 (1990) abstr. 240-242 239 b) modeling of transition state of reaction between chloride and methyl chloride including electron clouds and water molecules surrounding the reaction site; c) analysis of nucleic acid and protein sequences to evaluate the secondary structure of these biopolymers; d) construction of a graphical image of hexafluoropropylene oxide dimer, a model for dupont krytox high performance lubricant; e) calculation of the heats of formation of diaminobenzene isomers indicated that the target para isomer was 3 kcal/mol less stable then the meta isomer byproduct therefore the development for its large scale catalytic synthesis was not undertaken (saving was estimated to be $1 to $2 million). , b) comparison of the newly defined eo, parameter with the taft-kutter-hansch e, (tkh e,) parameter showed characteristic steric effects of ortho-alkoxy and n-bonded planar type substituents (e.g. no,, ph); c) in various correlation analyses using retrospective data eo, satisfactorily represented the steric effects of ortho-substituents on reactivity and biological activity of various organic compounds; d) semi-empirical am1 calculations using a hydrocarbon model to study the steric effects of a number of ortho-substituents resulted in the calculation of the es value (difference in the heat of formation between ortho-substituted toluene and t-butylbenzene) which linearly correlated with the eo, and the tkh e, parameters; e) effects of di-ortho substitution on lipophilicity could be mostly expressed by the summed effect of the 2-and 6-position substituents; t) highly significant regression equations were calculated for the pk, values of di-ortho-substituted benzoic acids using various substituent parameters; g) quantitative analysis of the effect of ortho-substitution is difficult because it is a result of overlapping steric and electronic effects. title: calculation of partition coefficient of n-bridgehead com( i i ) is more lipophilic than propanolol-4-sulphate (iv)]. fig. 1 shows the relationship between lipophilicity and ph for the compounds (circle represents (i), triangle (ii), rhomboid (m) and square gv 234 -293 (1990) abstr. 245-246 241 f (rekker's constant, characterizing hydrophobicity). results: a good agreement was found between the observed and calculated logp values of i1 (3.98 and 3.65, respectively) and for iii. the hydrophobicity of i was found to be significantly lower than that of i1 (2.78 and 4.72, respectively) . the large deviation was attributed to the surface reduction as a result of condensed ring formation in i. since interesting pharmacological activities have been reported for several derivatives of this type of compounds, the hydrophobicity of the unsubstituted 1 lh-indolo[3,2-c]quinoline has been calculated to be 2.22: (1) [interaction energy between a molecule and the binding site model was assumed to be the sum of its atomic contributions according to the expres-e , . , , ,~~(~) was the interaction energy parameter between the site region rand the atom-type of atom a and ag(b) was the total interaction energy for the binding mode b (binding mode was regarded as feasible when the molecule was in its energetically most favorable conformation)]. sion ag(b) = erelion reatomi a in r er,typc(a). where results: for development of the binding site model, first a simple geometry was proposed and agm-5 agm,calc i agm+) was calculated for the whole set of compounds. if the calculated binding energy of any of the compounds was outside of the above boundary, the proposed site geometry was rejected and a more complex one was considered. this procedure had been repeated until all molecules in the set could be fitted within the experimental data range. as a result a 3d, five-region voronoi binding site model has been developed for the pahs containing a trigonal pyramid (rl) in the center and portions r2rs having infinite volumes and indicated by boundary planes. region r, represented access to the solvent and regions r3rs were blocked for binding ( fig. 1) : pyrene is shown in its optimal binding mode with its atom barely touching the boundary surfaces and edges: calculations showed that benzene and other monoaromatic ring compounds should be very weak competitors for the b[a]p site. the model correctly predicted the binding energy of nine competitors outside of the training set. '% (wiener index calculated as the sum of all unique shortest distances between atoms in the hydrogen suppressed graph of the compound); (wiener index calculated as the sum of all geometric distances between atoms in the hydrogen suppressed molecule of the compound). results: the traditional 2d wiener number is defined as the sum of the lengths of all possible routes in the molecular graph. here the length is proposed to be calculated as the real three-dimensional length between atoms: this is the 3d wiener number. this number has many of the advantageous features of the related and very much studied 2d wiener number. additionally, it is highly discriminative and its use in quantitative structure-property relation studies (qspr) appears to be encouraging, according to the preliminary calculations. of these the most convincing is the set of statistical parameters for the linear correlation between the experimental and calculated enthalpy functions of 3dw the lower alkanes not shown here. three different models have been tried and in all cases the 3d wiener number seemed to be superior to the 2d one as it is reflected in (eqs.1-6). a) gaba receptors in human mouse, rat and bovine brain tissues, membrane preparations and cellular uptake systems; b) gaba receptors in cat and rat spinal cord preparations; c) cultured astrocytes. as in the equations nearly all indicator variables had negative regression coefficients it was concluded that instead of searching for better analogs, the research should be directed toward degradable pro-gaba or pro-muscimol derivatives that are efficiently taken up into the central nervous system (cns). 0.585(+2.22) irng + 4.16 (3) title: synthesis and qsar of 1-aryl-4-(~-2-quinolyi/l-isoqui-noly1ethyl)piperazines and some related compounds as hypotensive agents. authors (1) based on eq. 1, an optimal logp is predicted (logpo = 4.23). the highest activity was produced by the 1-(3-methylphenyl)-4-(~-2-qui-data determined: chemical descriptors: abtstr. 251-252 quant. struct.-act. relat. 9, 234 -293 (1990) nolylethyl) piperazine, its logp value being near to the optimal value (4.52). l.og(bph) values calculated by eq. 1 agree well with the observed ones. source: toxicology 1989, 58(2), 197 -210. compounds: 3,5-dimethoxyphenol, 4-chlorophenol, 2.6-dichlorophenol, 4-methyl-2-nitropheno1, 2,4dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,5-tetrachlorophenol, 2,4,6-triiodophenol, pentachlorophenol. biological material: chinese hamster ovary (cho) cells. data taken from the literature: ezoc; ecsoc; eczoa; ec~oa [concentration (mmol1l) of the compound leading to a 20 or 50 % inhibition of the cell growth or adenosine uptake, respectively]. data determined: ego; ecso [concentration (mmol1l) of the compound leading to a 20 or 50 % inhibition of the na+/k+-atpase activity, respectively]. chemical descriptors: logp (logarithm of the partition coefficient in i-octanollwater); u (hammett's constant, characterizing the electron-withdrawing power of the substituent); e, (taft's constant, characterizing steric effects of the substituent); x (molecular connectivity index, calculated by koch's method). results: highly significant linear relationships were calculated between log (eczo) and logp (r = -0.963). the relationship between log(ec,,) and u being less good (r = -0.767). combining the two parameters the relationship has improved (eq. i): (1) (logarithm of the partition coefficient in i-octanollwater); (hansch-fujita's substituent constant characterizing hydrophobicity); (hammett's constant, characterizing the electron-withdrawing power of the substituent); (sterimol steric parameter, characterizing the steric effect of the meta substituents); (rplc derived hydrophobic substituent constant, defined by chen and horv6th, and extrapolated to 0 x methanol); (indicator variable 1 for the present 0 for the absence of hydrogen bonding substituents). results: logk' values were determined for the benzenesulfonamides and correlated with chemical descriptors. a highly significant linear relationship between logk' and logp was calculated (eq. 1): ( pk, (negative logarithm of the acidic dissociation constant); logp (logarithm of the partition coefficient in i-octanol/water). results: relationships between ki values and the chemical descriptors were investigated for cpz and its listed metabolites. relationship between log(l/ki) and logp was calculated (eq. 1) no numerical intercept (c) is given: ( in spite of the complexity of the full mechanism of inhibition involving at least six transition states and five distinct intermediates, a significant linear regression equation was calculated for ki (eq. 3): since the crystal structure of the acyl-enzyme complex, the acylation and deacylation rate were available, it was concluded that the inhibition begins with the histidine 57 catalyzed attack of serine 195 0, at the benzoxazinone c4, while the carbonyl oxygen occupies the oxyanion hole formed by glycine 194 and serine 195. title: antifolate and antibacterial activities of 5-substituted authors: harris, n.v.; smith, c.; bowden, k. rhone results: it was shown earlier that binding of diaminoquinazolines to dhfr correlated with the torsional angle of the 4-amino group of the quinazoline nucleus. it was postulated that the interaction between the adjacent 5-substituent and the 4-amino group was very important in determining dhfr binding of the compounds possibly, because of the influence on the hydrogen-bond formed between the 4-amino group and a residue at the active site. the existence of such interaction in 5-substituted 2,4-diaminoquinazolines were shown by measuring a , , and 6~~1 values. the ui and uor electronic parameters correlated well with chemical shifts of the 2-nh, groups (eq. 1) but showed poor correlation for the 4-nh, group (eq. 2), respectively: (1) the equations suggest that the through-ring resonance interactions between the 5-substituent and the adjacent 4-amino group are disrupted by some other effects which might have significance for binding. a) an extensive set of compounds based on the nalidixic acid structure of type i. where r', r3, r6 and r7 are various substituents; x6 and x* = c, n (for nalidixic acid: x6 = c, x8 = n, r' = et, r' = cooh. r6 = h, r7 = me); b) subset of (i) (set a) containing fifty two 6,7-disubstituted 1 -alky l-1,4-dihydro-4-oxoquinoline-3-carboxylic acids; compounds: abtstr. 260-261 quant. struct.-act. relat. 9, 234-293 (1990) c) subset of (i) (set b) containing one hundred and sixty two xylic acids; d) subset of (i) (set c) containing eighty five 1,4-dihydr0-4-oxo-1,8-naphthyridine-3-carboxylic acids with substituted azetidinyl, pyrrolidinyl and piperidinyl rings at position 7, fluorine at position 6 and ethyl, vinyl or 2-fluoroethyl substituent at position 1. biological material: ps. aeruginosa v-1, e. coli nihj jc-2, s. the study showed that the most active compounds have fluorine in position 6, r7 can be a wide variety of nitrogen containing substituent and the best predictor for r7 is its lipophilicity. compounds: 17 phytoalexins: pisatin, 3,6a-dihydroxy-8,9-(methylenedioxy)pterocarpan, 6a, 1 la-dehydropisatin, 3-hydroxy-8,9-(methylenedioxy)-6a, 1 1 a-dehydropterocarpan, (*)-3-hydroxy-9-methoxypterocarpan, (+)-3-hydroxy-9-zmethoxypterocarpan, (-)-3-zhydroxy-9-methoxypterocarpan, vestitol, sativan, formonenetin, coumestrol, 4'-o-methylcoumestro1, phaseoilin, phaseollinisoflavan, 2'-methoxyphaseollin-isoflavan, glyceollin, 6a-11 a-dehydroglyceollin, tuberosin, 6a, 1 ladehydrotuberosin. (capacity factor determined rp-hplc). calculated for logp of six reference compounds using their k' values (eq. 1): (1) n = 6 r = 0.993 s not given f not given the lipophilicity of the phytoalexins were within the range of log p = 1.5 -4.2. it was found that the antifungal activity of similar compounds positively correlated with antifungal activity but no equation could be calculated for the whole set of compounds. it was suggested, however, that compounds with logp values higher than 3.5 were retained in the membranes, therefore phytoalexins with slightly lower lipophilicity, as well as greater fungitoxicity and systemic activity should be searched. certain structural features seemed to correlate with antifungal activity such as the presence of phenolic oh and benzylic hydrogen. it was suggested that the ability of the ortho oh group to form fairly stable intramolecular hydrogen bond may contribute to the greater stability of the shiff base hnctional group and the higher biological activity of the substances (various subsets required different equations). results showed that compounds with increasing lipophilicity and electron donating substituents at the 3-and 5-positions have high inhibitory activity. i-[(3'-allyl-2'-hydroxybenzilidene)amino]-3-hydroxyguanidine was found to be the most active compound. the use of parameter focusing of the substituent hydrophobic constant and electronic constants was suggested for the selection of further substituents to design effective compounds. biological material: a) rabbits; b) rats; c) guinea pig. data taken from the literature: analogue results: prp, ecjoh, ecsob, ecsot values were measured and presented for the c,, paf analogue and compared with that of other analogues. c,, paf analogue was less potent than the c 1 6 or cis paf analogues and equivalent to c,, paf analogue, showing that the activity decreased with lipophilicity. a highly significant parabolic relationship was calculated between log(rps) and cf (eq. 1): the maximum activity was calculated cf = 6.78, this corresponds to the cl6 paf. (energy minimizatipn of the compounds were calculated using the free valence geometry energy minimization method); (molecular shape analysis according to hopfinger was used to quantitatively compare the shape similarity of analogs in their minimum energy conformer states (within 8 kcal/mol of their global minimum energy ( fig. 1 shows the superposition of the reference conformations of the phenylalanine and tryptophane analogues). quant. struct.-act. relat. 9, 234-293 (1990) chemical descriptors: logp (logarithm of the partition coefficient in 1 -octanol/water); (hansch-fujita's substituent constant characterizing hydrophobicity of a substituent on the aromatic ring and the hydrophobicity of the aromatic ring itself, respectively) ; [common overlap steric volumes (a3) between pairs of superimposed molecules in a common low energy conformation]; [dipole moment (debeyes) of the whole molecule and of the aromatic ring, respectively, calculated using the cndoi2 method] ; quantum chemical indices (partial atomic charges calculated by the cndoi2 method); 0 1 -0 4 [torsion angles (deg) (fig. 1 ) rotated during the conformational analysis of the compounds]. results: significant parabolic regression equations were calculated for the antigelling activity of the phenylalanine and tryptophan analogues (eq. 1 and eq. 2, respectively): the different qsar for the phenylalanine and tryptophan analogues indicated that they interact with hemoglobin in different ways or at different sites. for the phenylalanine analogues the hydrophobicity of the side chain, the aromatic dipole moment and the steric overlap volume explained about 50 %, 20 % and 10 % of the variance in antigelling activity, respectively. for the tryptophan analogues the square of the dipole moment or the steric overlap volume explained 70% or 60% of the variance in ra, respectively, being the two descriptors highly correlated. the results show that the tryptophan analogs have a relatively tight fit with the receptor site. title: s-aryl (tetramethyl) isothiouronium salts as possible antimicrobial agents, iv. in both eq. 3 and eq. 4, log(l/c) depended primarily on electronic factors (eu' ) and only secondarily on hydrophobicity (ctobsd). a threshold logp value for the active isothiuronium salts was indicated, as the compounds with logp values between -0.70 and -1.58 were found to be totally inactive with the exception of the nitro-derivatives. title: comparative qsar study of the chitin synthesis inhibitory activity of benzoyl-ureas versus benzoyl-biurets. source: tagungsbericht 1989, no.274 \ r* ponents explaining 69.61 %, 19.02 % and 9.30 % of the variance. fig. 1 shows the minimum energy conformation of a highly active representative of the urea analogs (dimilin) with 5.6 a distance between the 1 and 15 carbon atoms. fig. 2 shows the low energy conformation of the corresponding biuret analog with the two benzene rings in appro:imately the same plane and with the same c1-c18 distance (5.6 a) allowing to fit a hypothetical benzoylurea phamacophore. the similarity of the regression equations and the modelling study supported the hypothesis that the benzoylbiurets act by the same mechanism as the benzoylureas. biological material: 8 insect species: aedes aegypti, musca domestica, chilo suppressalis, hylemya platura, oncopeltus suppressalis, oncopeltus fasciatus, pieris brassicae, leptinotarsa decemlineata. [concentration of the benzoylurea derivative (various dimensions) required to kill 50% of insect larvae (a. aegypti, m. domestica, c. suppressalis, h. platura, 0. suppressalis, 0. fasciatus, p. brassicae or l. decemlineata]. data determined: lcso [concentration of the biuret analogue (ppm) required to kill 50% of insect larvae (a. aegypti or m. domestica]; molecular modeling (models of the compounds were built using molidea); conformational analysis (minimum energy conformations of the compounds were calculated using molecular mechanics method). chemical descriptors: the thesis is devoted to the quantitative analysis of the uncoupling activity of substituted phenols using chemical descriptors in order to obtain further information on the mode of action of phenol uncouplers: the study of the partition coefficient of substituted phenols in liposomelwater system [p(l/w)] showed that (i) p(l/w) depended primarily on the logp value; (ii) influence of steric and electronic parameters depended on the type of the lipid involved; qsar analysis of uncoupling phenols in rat-liver mitochondria identified the relevant physicochemical parameters required for phenols being protonophore in inner mitochondrial membrane and quantitatively separated the potency as the protonophore in the inner mitochondrial membrane and the incorporation factor (iogp); protonophoric potency of substituted phenols was linearly related to uncoupling activity when certain critical physicochemical parameters of the experiment were taken into account; linear relationship was calculated between uncoupling activities of substituted phenols and related uncouplers in the mitochondria from the flight muscles of house flies and in spinach chloroplasts; the results indicated a shuttle type mechanism for the uncoupling action of substituted phenols. title: uncoupling properties of a chlorophenol series on acer cell 234 -293 (1990) compounds: 22 chlorinated phenols substituted with 2-c1, 3-c1, 2,4,5-cl, 2,4,6-ci, pentachlorophenol, 4-ci-2-me. 4-c1-3-me, 4-c1-2,3-me, 4-c1-3,5-me, 4-ci-2-ally1, 4-c1-2-pr-5-me, 4z1, 2,3-c1, 2,443, 2,5-c1, 2,6-cl, 3,4-c1, 3,5-ci, 2,3,6-c1, 2-cl-6-no2, 2,4-c1-6-no,, 2-ci-4,6-no,. biological material: acer pseudoplatanus l. cell suspensions. data determined: dso [concentration of the compound (pmolll) required for 50 % uncoupling effect registered by measuring the oxygen consumption rate by polarography]; [minimal concentration of the compound (pnol/l) required for giving a full uncoupling effect]. chemical descriptors: logp (logarithm of the partition coefficient in 1-octanol/water); mr (molar refractivity); ed (steric parameter representing the perimeter of coplanary molecules projected onto the aromatic plane); a (angular parameter expressing the hindrance in the neighborhood of the hydroxyl group in positions 2 and 6, respectively) ; ui, 02 (hammett's constants, characterizing the electron-withdrawing power of the para-substituent and the ortho-or 4-nitro substituents, respectively). results: highly significant linear regression equations were calculated for the uncoupling effects of chlorophenols in acer cell suspensions: the equations for the uncoupling effects in the whole cells and those calculated previously for isolated mitochondria or chloroplasts possess similar structures. 0.202(*2.049) a, -4.250 (2) title: effects of 3' substituents on diphenyl ether compounds. results: sar suggested that the space for the n' and nz substituents in the psi1 binding site is relatively large. the variation of the number of the carbon atoms of r2 on the photosynthetic inhibitory activity is shown in fig. 1 (hansch-fujita's substituent constant characterizing hydrophobicity); chemical descriptors: 0.32(*0.29) ior + 0.41(&0.43) hb + 5.62 the biological activity of three out of the 30 (dpe-16, 19 and 28) substituted diphenyl esters were measured and listed. igr values were measured for the three compounds and compared with that of a-23 and methoprene. it was found that the position of acetamido group in the phenol moiety when it is in the ortho position abtstr. 272-273 234 -293 (1990) increases the lipophilicity of the compound with a logp value of 2.54. if the same group is in mr para position, the logp values are 2.16 and 1.99, respectively and they are comparatively ineffective. when both the ortho positions are substituted with tertiary butyl groups (dpe-28) the logp value is relatively higher (3.30) which increases the lipophilicity of the compound and explains the pronounced idr activity at relatively low concentrations. abstr. results: a highly significant linear regression equation was calculated for the descriptors of r' (r' = i-pro was eliminated as an outlier) (eq. 1): the compound with r' = eto, r2 = me and z = 0 was found to be an effective, broad spectrum insecticide. the replacement of the quaternary carbon with a silicon atom cansimplify the synthesis of test compounds and thus can be advantageously utilized for the preparation of large compound sets for qsar studies. the data suggest that the initial electron loss from the given compounds is the preeminent factor effecting the reaction rate. a single mechanism is suggested over the entire range of reactivities, where a transition state with a considerable positive charge is involved. title: connection models of structure and activity: ii. estimation of electronoacceptor and electronodonor functions of active centers in the molecules of physiologically active materials. research institute of physiology active materials chernogolovka, moskow district, ussr. engl. summary). authors chemical descriptors: logp (logarithm of hydrophobicity). results: calculations for electronoacceptor and electronodonor entharpic and free energy factors on the base of functional groups were made according to the principle of independence of active centers: data determined: linear correlation was found between the calculated and measured characteristics: the accuracy of the fitting was the same as the measurement error of ah,,, and agm.the entropy might be calculated from enthalpy, gibbs energy and temperature: the good linear correlations between the measured and calculated data show that the functional group approaches might be used for these compound types. the substituent effects for the a-acceptorlr-donor substituents (f, c1, br, i) were found to be very much larger for the c6fsr relative to the nitrobenzenes. these results indicate that the extra electron enters a o*-orbital, which is localized on the c-r atoms. for the structure-solubility relationship of aliphatic alcohols. the study indicated that solubility of aliphatic alcohols depends primarily on molecular connectivity ('x), the number of carbon atoms in the alkyl chain (n'), the number of hydrogens on the a-carbon atom (normal, iso, secondary, ternary) and the degree of branching (vg): (1) n not given r not given s not given f not given eq. 1 was found to be a highly significant predictor of s (eq. 2): the result support kier's, furthermore kier and hall's earlier models on the structural dependence of water solubility of alcohols. -log(s) = 113 'x + (113)* sg -2.5075 title: linear free energy relationships for peroxy radical-phenol reactions. influence of the para-substituent, the orthodi-tert-butyl groups and the peroxy radical. k (reaction rate constant (m -'s -i ) of the reaction between the reaction of cumyl-, 1 -phenylethyl-and t-butyl-peroxy radicals and ortho-para-substituted phenol inhibitors). data taken from the literature: chemical descriptors: u+ r. ui, ur (charton's electronic substituent constant and its decomposition to inductive and resonance components, respectively for the characterization of the para substituent); (indicator variable 1 for the presence or absence of the t-bu groups in 2,6-position of the phenols). results: highly significant linear regression equations were calculated by stepwise regression analysis for logk in spite of the diverse data set originating from different laboratories using different peroxy radicals (eq. 1, eq. 2): quant. struct.-act. relat. 9, 234 -293 (1990) (2) n = 32 r = 0.848 s = 0.432 f = 37.2 i c~" was not selected by stepwise regression indicating that the orthodi-t-bu substitution had no significant effect on the rate of hydrogen abstraction from phenols by the radicals. the form of the equations for different subsets of the phenols and radicals indicated that the reaction mechanism was the same for the different peroxy radicals. title: a fractal study of aliphatic compounds. a quantitative structure-property correlation through topological indices and bulk parameters. the following descriptors are considered as 'bulk parameters': vw (van der waals volume, calculated from the van der waals radii of the atoms); mw (molecular weight); sd (steric density of the functional group). results: highly significant equations are presented for calculating vw, sd and mw r values ranging from 0.92 to 1.00, other statistics and the number of investigations are not given. q and 0 values calculated by these equations were introduced to the equation given above and the physicochemical properties were calculated. the observed and calculated iogv,, d and p values are presented and compared for the alkanes, alcohols, acids and nitriles. the observed and calculated physicochemical parameters agreed well. fractal nature of the alkyl chain length was discussed and a relationship was presented between the fractal-dimensioned alkyl chain length and a generalized topological index. title: application of micellar liquid chromatography to modeling of organic compounds by quantitative structure-activity relationships. chemical descriptors: logp (logarithm of the partition coefficient in 1-octanol/water). results: in a series of experiment with the listed compounds micellar liquid chromatography has been applied to model hydrophobicity of organic compounds in a biological system. the measured logk' values of the substituted benzenes were found to be superior predictors of logp. fig. 1 shows the plot of logp versus logk' of the substituted benzenes. highly significant correlation was calculated for the logk' values of phenols (open squares) (n = 6, r = 0.985), for the rest of the compounds (full squares) (n = 16, r = 0.990) and for the entire set (n = 22, r = 0.922). further experiments using various surfactant types in the mobil phase suggested that logk' values generated on a lamellar phase may be better predictors of hydrophilicity than logp obtained from binary solvent systems. title: isoxazolinyldioxepins. 2. the partitioning characteristics and the complexing ability of some oxazolinyldioxepin diastereoisomers. authors quant. struct.-act. relat. 9, 234 -293 (1990) source: j. chem. soc. perkin trans. i1 1989 . no. 11, 1935 -1937 compounds: 10 oxazolinyldioxepin derivatives of type i and 11, where x = h, f, ci, cf3 or ch3. data determined: logk' [logarithm of the capacity factor, measured by reversed-phase liquid chromatography (rplc)]; mep (molecular electrostatic computed by geesner-prettre and pullman's vsspot procedure). chemical descriptor: logp (logarithm of the partition coefficient in 1-octanollwater). results: the logk' and logp values were measured for the two type of diastereomers and a highly significant linear relationship between logk' and logp was presented (r = 0.995): the meps of i and 11's f-derivatives were determined and presented, "a" for type i, "b" for type ii: the complex forming ability of the diastereoisomers with mono-cations was investigated and explained in terms of the structures and electronic properties of the compounds. results: linear relationships are presented plotting y versus n for the 9 hydrophobic sorbents (fig. 1 ) and the slopes of these straight lines are suggested for experimental determination of . q, values. ~0 values determined by the suggested method are listed. while no linear relationships were found between kd and n, y depend linearly on n for the test compounds [alkanols (i). alkane diols (2) results: three linear models were fittedwith independent variabies of log(p), mr and o x . the best fitting parameters (independent of composition) were obtained from the following models (no statistical characteristics is presented): (1) (2) the two types of correlations (with structural and with moving phase parameters) together might be used for the optimization of chromatographic separation of complex mixtures of sulphur-containing substances. (zero order molecular bonding type connectivity index); ig(k) = a0 + a1 p' + a2 logp + a3 p' logp ig(k) = a0 + a1 tg(cm) + a, logp + a3 tg(cm) logp -3-1 19901285 the kd values derived by the suggested method were compared by kd values calculated by martin's rule and a good agreement was found. title: mathematical description of the chromatographic behaviour of isosorbide esters separated by thin layer chromatography. compounds: 9 isosorbide esters: isosorbide (l), 1-5-monoacetate, 1-2-monoacetate, 1-5-mononitrate, 1-2-mononitrate, l-diacetate, 1-5-nitro-2-acetate, 1-2-nitro-5-acetate, l-dinitrate. rn, r~i [retention factors obtained by thin-layer chromatography in benzene/ethylacetate/isopropanol/ (7:3: 1.5) and in dichloromethane/diisopropylether/isopropanol (20:4:2: 1) eluent systems, respectively]. data determined: chemical descriptors: (information index, based on the distribution of the elements in the topological distance matrix); (the geometrical analogue); (randic connectivity index); (maximum geometric distance in the molecule); compounds: 46 highly diverse chemicals. grouped according to the following properties: contains (ester or amide or anhydride) or (heterocyclic n) or (0 bound to c) or (unbranched alkyl group with greater than 4 carbons). data determined: aerud chemical descriptors: (aerobic ultimate degradation in receiving waters). 2 v x 4x, nci m, (molecular weight). results: the paper has aimed at developing a model for predicting aerud. the data sets were collected from 22 biodegradation experts. the experts estimated the biodegradationtime that might be required for aerud on the time scales of days, weeks, months and longer. 46 (valence second order molecular connectivity index); (fourth order path/cluster connectivity index); (number of covalently bound chlorine atoms); highly diverse chemicals but typical in wastewater treatment systems were examined. zero to six order molecular and cluster connectivity indexes were calculated using computer programs wrinen in for-tran for ibm pc/xt. the best fitted linear regression model is: [first order rate constant: transport or transformation parameter (mol/pa. h)]. results: the qwasi fugacity model describes the fate of a (contaminating) chemical, such as organo-chlorine compounds, pesticides or metals. the lake model consists of water, bottom and suspended sediments, and air. the model includes the following processes: advective flow, volatilization, sediment deposition, resuspension and burial, sediment-water diffusion, wet and dry atmospheric deposition, and degrading reactions. the steady state solution of the model is illustrated by application to pcbs in lake ontario using the equilibrium criterion of fugacity as the variable controlling environmental fate of the chemical. the applications are based upon inaccurate data. use of fugacity is inappropriate for involatile chemicals, such as metals, or ionic species, because fugacities are calculated from a basis of vapor phase concentrations. for these materials the use of the equilibrium concentration activity is more appropriate since activities are calculated from a water phase base. thus, a new equilibrium criterion, termed the "aquivalent" concentration (or equivalent aqueous concentration) is suggested as being preferable. this concentration has the advantage of being applicable in all phases, such as water, air and sediments. the formalism developed in the qwasi approach can also be applied, making possible a ready comparison of the relative rates (and thus, the importance) of diverse environmental fate processes. all these are illustrated by applying the model on a steady state basis to quant. struct.-act. relat. 9, 234-293 (1990) abstr. 288-289 261 the pcb example and to the fate of lead in lake ontario. the estimated and observed concentrations of pcbs and lead in lake ontario agree well: the largest difference in the case of pcbs in rain amounts to a factor of three. in other phases, and especially in the case of lead, the difference is usually less than 30 per cent. although in order to judge the biological effects of a contaminant it is of fundamental importance to know its transport and transformations, and the present model has been proven to useful to describe this; direct biological implications are not deduced at the present stage. the similar slopes of the equations show that these compounds exert their cytotoxicity primarily by alkylation. while the majority of the tested compounds showed no hypoxia-selective cytotoxicity (ratio awa 1 .o), the 4-n02 and 3-no2 substituted compounds were more toxic to uv4 cells under hypoxic conditions (ratio = 3.2 for the compound with r = 4-n02), indicating cellular reduction of the nitro-group. the measured hypoxic selectivity of the 3-no2 and 4-n0, substituted compounds was a fraction of the calculated ratio (measured 220 fold and calculated 3500 fold by eq. 2 between the 4-n02 and 4-nh, substituted compounds). the main reason for the difference between the calculated and measured hypoxic selectivity is suggested to be the low reduction potential of the 4-n02 and 3-no, groups (e = -500 mv and e = -470 mv, respectively). 19901288 title: quantitative structure-activity relationships for the cytotoxici(hammett's constant, characterizing the electron-withdrawing power of the substituent); (hammett's polar electronic constant characterizing the electron withdrawing power of the substituent for anilines). results: significant linear regression equations were calculated for the halflife (t1/2), growth inhibition (150) and clonogenicity data (ctlo) using hammett constants (eq. 1, eq. 2, eq. 3): (1) n = 11 r = 0.96 s = 0.24 f not given 234 -293 (1990) type, test animals, the mean level of toxicity and the form of the equation. e.g. analysis of the toxicity of phenols showed a transition between simple dependence from logp to exclusive dependence to reactivity factors indicating two separate classes of phenol toxicity (eq. 1 for mouse i.p. toxicity, and eq. 2 for rat oral toxicity): results: an additivity model, plc50 = cni a ti 4-to, where ni is the number of ith substituents in a benzene derivative, at; is the toxicity contribution of the ilh substituent and to is the toxicity of the parent compound (benzene), was used for predicting toxicity of lo similar correlation was found between mutagenicity and u (fig. 2) indicating that both biochemical and chemical processes involve a ph dependent nucleophilic ring opening (including the protonation of the aziridin nitrogen as rate controlling step) and in.,uenced by electronic and steric factors (equation not given). resonance effect). (electron density on n1 in the homo calculated by the mndo). results: highly significant linear relationships between log( 1 ic) and logp, &homo (eq. 1); iogp, qhomo (eq. 2) are presented indicating that the more hydrophobic and more electron-rich triazines are more active according to the ames test: substructures [a total of 32355 fragments were generated from the 189 compounds using the program case (computer-automated structure evaluation) system]. results: a comparative classification of the compounds were performed using case for identifying molecular fragments associated with cancerogenic activity (biophores) as well as deactivating fragments (biophobes). case identified 2 1 biophores and 2 biophobes from the 32355 fragments of the 189 compounds with a less than 12.5% probability of being associated with carcinogenicity as a chance. the sensitivity and specificity of the analysis was unexpectedly high: 1.00 and 0.86, respectively. the predictive power of case biological material: chemical descriptors: was tested using the identified biophores and biophobes on a group of chemicals not present in the data base. the ability of case to correctly predict carcinogens and presumed non-carcinogens was found to be very good. it was suggested that non-genotoxic carcinogens may act by a broader mechanism rather than being chemical specific. compounds: 9 compounds of type i where r = h, ch3, c,h5, czh7, c3h7, czh9, c4h9, c3h5, c4h7, sc4h7; 3 compounds of t y p i1 where r = h, c~heoh, sc4h80h. data determined: p t pi" (a priori probability of appearance of the i-th active compound); (a priori probability of appearance of the i-th nonactive compound). (the first order molecular connectivity index); (the second order molecular connectivity index); (information-theoretic index on graph distances calculated by the wiener index according to gutmann and platt); chemical descriptors: (rank of smell, where the rank is defined to equal with one for the most active compound). results: the authors' previously proposed structure-activity relationship approach was applied for structure-odor relationship. 13 different compounds of groups i and i1 were examined using the topological indices w, r, i, x as independent variables and v as the dependent variable. the best correlation was obtained between r and v ( fig. i) results: logp and iogp, values were determined for the nitroimidazole derivatives. significant linear equations were calculated, the best one related for logp and logp,r (eq. 1): (1) logp = 1.14 logp,i + 0.37 n = 9 r = 0.92 s not given f not given chemical descriptors: logp descriptors (logarithm of the partition coefficient in i-octanoll water); (12 indicator variables taking the value of 1 for the presence of cr/p-hydroxy , 6a-fluoro, 6a-methy1, 9afluoro, 17-hydroxy, i6a-fluoro. 16,17-acetonide, 21-deoxy, 21-acetate, 21-propionate, 2 i-butyrate or 2 1-isobutyrate, respectively). results: a data set of 43 steroids were compiled after removing those ones containing unique substituents. the set was divided into two categories of approximately equal membership by defining a threshold logp value of 1.45. a descriptor set was created and the non-significant ones were eliminated using the weight-sign change feature selection technique. linear leaning machine was applied to calculate the weight vectors and complete convergence was achieved in the training procedure. the predictive ability of the linear pattern classifier thus obtained was tested using the leave one out procedure. the predictive ability was found to be 8 i .4 %. the predictive ability of the approach was found to be good and improvement was expected with larger data set. steroids, however, containing new substituents would have to be subjected to a repeated pattern-recognition calculation. lengthlbreadth descriptors (2 descriptors)]. results: for modeling the shape of the compounds, simca was used: the approach was to generate disjoint principal models of clustered points in a multidimensional space. the number of clusters for each structure was determined by using hierarchical cluster analysis. fig. 1 shows the orthogonal views of a schematic representation of the sim-ca models for the atom clusters in senecionine: each compound in turn was used as a reference structure. every other structure was superimposed on the reference using the ends of the corresponding binding moment vector plus the ring nitrogen atom. canonical correlation analysis was used for calculating the correlation between the five biological activity data and shape descriptors of 21 structures. the best correlation was observed for jurs' shadow descriptors. the msa and simca descriptors were comparable. the model was able to express both the amount and direction of shape the differences, and also for encoding relevant information for correlation with the biological activity. compounds: 6 n-substituted 3-methyl-4-nitropyrazole-5-carboxamides (11), 6 n-substituted 4-amino-3-methylpyrazole-5-carboxamides (iii), 14 n-substituted 3-methyl-4-diazopyrazole-5-carboxamides and n-piperidiny 1-n-( 1,3-dimethyl-4-nitrosopyrazol-5-yl)-urea (vii) . title: structure-activity correlations for psychotomimetics. 1. phenylalkylamines: electronic, volume, and hydrophobicity parameters. abtstr. 300 quant. struct.-act. relat. 9, 234-293 (1990) data determined: edso conformational analysis g [dose of the compound (mg/kg) which causes 50% of the rats which were trained on 1 rngfkg reference compound to respond as they would to the training drug]; (geometries of the compounds were calculated using mmf2 from starting geometries determined by the program euclid). discriminant analysis resulted in a function containing six variables which misclassified only one compound in the training set. when the data was repeatedly split randomty into a training and a test sb, the misclassification rate was 9% (15 out of 161 classifications). fig. 2 shows the plot of the two canonical varieties from discriminant analysis visualizing the separation of hallucinogenic and nonhallucinogenic derivatives (meaning of symbols are the same as in fig. 1 ). multiple regression analysis (mra) was found to be the most useful for identifying relevant and discarding redundant variables. highly significant parabolic regression equations were calculated for the human activity data (a) (n = 50, r ranging from 0.9004 to 0.9563, f not given) and for animal data (edso) (n = 16, r = 0.8679 and r = 0.9825, f not given). eight descriptors were found to be highly significant. among these the importance of directional hydrophobicity and volume effects indicated that steric and hydrophobic interactions participate in the interaction with the receptor.mra indicated a strong interaction between the meta-and para-substituents and the presence of the formation of charge transfer complex by accepting charge. data did not support the hypothesis that the human activity data and animal ( 1 ) data taken from the literature: sweet(n) (sweet taste of the compound, where n represent the number of times a sample has to be diluted to match the taste of 3% sucrose solution). [class fit distances of a compound to sweet and nonsweet class (dimension not given) calculated by principal component analysis]. chemical descriptors: mr (molar refractivity); bi, l (sterimol steric parameters, characterizing the steric effect of the substituent); r (hansch-fujita's substituent constant characterizing hydrophobicity); urn, up (hammett's constants, characterizing the electron-withdrawing power of the substituent in meta-and para-position, respectively). results: no statistically significant regression equation was obtained by the hansch-fujita approach using the chemical descriptors listed. d', d2 quant. stact.-act. relat. 9, 234 -293 (1990) abstr. 301-302 267 principal component analysis of the data set extracted 2 principal components, explaining 64 % of the variance of the sweet compounds. the sweet compounds clustered in a relatively confined region of the 15d space whereas the tasteless and bitter compounds were scattered around the sweet compounds. a coomans plot, however., indicated, when plotting d' versus d2, that sweet and nonsweet compounds could be well separated along the d' axis ( fig. 1, title: conformation of cyclopeptides. factor analysis. a convenient tool for simplifying conformational studies of condensed poly-ring systems. prolyl-type cyclopeptides. authors conformations of the six-membered dop-ring family may be reproduced by means of a superposition of the canonical twist (t), boat (b) and chair (c) forms. physically, the coefficients have the meaning of relative contributions (amplitudes) of the t, b and c forms into the total conformation of the ring. here factor analysis (fa) and principal component analysis was used in conformational studies of 30 various x-ray conformers of dop/pyr. a correspondence was found between factors identified and rpt, when the rings are considered separately. this fact allows a physical interpretation of the fa results: two or three puckering variables were found for the dop and pyr rings expressing the absolute amplitudes of the basic pucker modes. subse-quent fa treatment of the condensed system revealed five conformational variables necessary and sufficicnt to describe the twolring puckering completely. each of the basic pucker modes defines a unique pattern of conformational variation of the whole two-ring system. the results demonstrate that fa is a powerful technique in analysing condensed poly-ring systems, not amenable to the rpt treatment. title: preprocessing, variable selection, and classification rules in the application of simca pattern recognition to mass spectral data. authors: dunn m*, w.j.; emery, s.l.; glen, g.w; scott, d.r. college of pharmacy, the university of illinois at chicago 833 south wood, chicago il 60612, usa. source: environ. sci. technol. 1989, 23(12) , 1499-1505. compounds: a diverse set of 121 compounds observed in ambient air classified as (1) nonhalogenated benzenes; (2) chlorine containing compounds; (3) bromo-and bromochloro compounds; (4) aliphatic hydrocarbons; (5) miscellaneous oxygen-containing hyhocarbon-like compounds (aliphatic alcohols, aldehydes and ketones). pattern recognition was applied to autocorrelation-transformed mass spectra of the compounds using providing chemical class assignment for an unknown]; m/z;, mlz,, m/zl (first three principal components scores of simca). results: simca pattern recognition method was applied on a training set of 78 toxic compounds targeted for routine monitoring in ambient air. the analysis resulted in very good classification and identification of the compounds (87 % and 84 %, respectively). however, the training procedure proved to be inadequate as a number hydrocarbons from field samples (gcims analysis) were incorrectly classified as chlorocarbons. a new approaches for the preprocessing (scaling the ms data by taking the square root of the intensitiesfollowed by autocorrelation transform), variable selection (only the 16 most intense ions in the ms spectrum were taken), and for the classification rules of simca has been introduced to improve results on real data. fig, 1 and as a result of the revised rules the classification performance has been greatly improved for field data (97 -94 %). title: a qsar model for the estimation of carcinogenicity. 234-293 (1990) it was suggested that the mechanism of mutagenicity of the cimeb[a]ps measured in the ames test is probably more complex than the simple reactivity of carbocation intermediates. [dipole interaction potential (dimension not given)]; [molecular electrostatic potential (kcallmol) in a plane]; [molecular electrostatic field map (kcall mol), mapping the 1e(r)j values of a molecule surface in a plane, predicting the directions and energies of the interactions with small polar molecules at distances greater than the van der waals sphere]; (construction of surfaces corresponding to a given value of potential); 3d mep and mef maps (3d maps weregenerated by superimposing the equipotential curves corresponding to a value of 20 kcallmol in the case of mep. and 1.5 kcallmol in the case of mef, computed in several planes perpendicular to the mean plane of the analogues in low energy conformations, stacking over each other in 1 a distance). results: the three vasopressin analogues differ significantly in their biological activities. both mep and mef maps of the of the biologically active (mpa')-avp and (cpp')-avp are similar, but they are different from that of the inactive (ths')-avp. fig. 1, fig. 2 abtstr. 306-307 quant. struct.-act. relat. 9, 234-293 (1990) a new method for calculating the points of the equipotential curves was also presented. crystal structure (crystal coordinates of the molecules were determined by x-ray diffraction methods). data taken from the literature: [electrostatic molecular potential (ev) were calculated using am-1 type semiempirical mo calculations]; conformational analysis [minimum energy conformations were calculated using x-ray structures as input geometries followed by am 1-method (fletcher-powell algorithm)]. chemical descriptors: ui, u2 [rotational angles of the n-ally1 group (deg)]. results: four similar energy minima were located by am-i calculations for both namh+ and nlph+. the energy minima for the protonated nam + and nlph + were the most populated ones with conformational enantiomers relative to the involved n-allyl-piperidine moiety (37 % and 44 %, respectively). it was shown that the isopotential curve localization of emp contour maps were very similar for the corresponding conformations of both nlph * and namh + indicating that both molecules should interact a the same anionic sites of the opioid receptor, ( p morphine receptor). fig. 2 and fig. 3 shows the emp contour maps of namh' and nlph + , respectively, in their preferred conformations: compounds: esfenvalerate (ss and sr isomers) of type i, 3-phenoxybenzyl 2-(4-ethoxyphenyl)-3,3,3-trifluoropropyl ether (r quant. struct.-act. relat. 9, 234-293 (1990) abstr. 308 271 isomer) (11). a-cyano-3-phenoxybenzyl 2-(4-chlorophenyl)-2-methylpropionate (s isomer) (iii) and deltamethrin (iv). " cn data determined: conformational analysis (minimum energy conformations of the compounds in vacuum were calculated using am1 molecular orbital method and broyden-fletcher-goldfarb-shanno method integrated into mopac); (root mean square, indicating the goodness of fit between two conformers in 3d); (logarithm of the partition coefficient in i-octanol/water estimated using clogp program); [heat of formation of the most stable conformer (kcallmol)]. rms logp e chemical descriptors: 0 1 -0 6 results: it was assumed that the 3d positions of the benzene rings of the pyrethroids are decisive for good insecticidal activity. the lower energy conformers of (i) (ss and sr isomers), (11) (r isomer), (111) (s isomer) and deltamethrin (iv) were compared by superimposition. inspite of their opposite configuration, esfenvalerate (i) (ss isomer) and the new type pyrethroid i1 (r isomer) were reasonably superimposed, indicating that the positions of the benzene rings in space are important and the bonds between them are not directly determinant (fig. 1) crystal structure (x-ray crystal coordinates of penicillopepsin was obtained from the protein data bank). data determined: electrostatic potential [electrostatic potential of the protein atoms (kcallmol) is calculated using the partial charges in the amber united atom force field); docking (the dock program was used to find molecules that have a good geometric fit to the receptor). results: a second generation computer-assisted drug design method has been developed utilizing a rapid and automatic algorithm of locating sterically reasonable orientations of small molecules in a receptor site of known 3d structure. it includes also a scoring scheme ranking the orientations by how well the compounds fit the receptor site. in the first step a large database (cambridge crystallographic database) is searched for small molecules with shapes complementary to the receptor structure. the second step is a docking procedure investigating the electrostatic and hydrogen bonding properties of the receptor displayed by the midas graphics package. the steps of the design procedure is given. the algorithm includes a simple scoring function approximating a soft van der waals potential summing up the interaction between the receptor and ligand atoms. directional hydrogen bonding is localized using electrostatic potential of the receptor at contact points with the substrate. the shape search of (i) was described in detail. a new method has been developed for the construction of a hypothetical active site (hasl), and the estimation of the binding of potential inhibitors to this site. the molecules were quantitatively compared to one another through the use of their hasl representations. after repeated fitting one molecule lattice to another, they were merged to form a composite lattice reflecting spatial and atomic requirements of all the molecules simultaneously. the total pki value of an inhibitor was divided to additive values among its lattice points presumed to account for the binding of every part of the molecule. using an iterative method, a self consistent mathematical model was produced distributing the partial pki values of the training set in a predicting manner in the lattice. the hasl model could be used quantitatively and predictively model enzyme-inhibitor interaction. a lattice resolution of 2 -3 a was found to be optimal. a learning set of 37 e. coli dhfr inhibitors were chosen to test the predictive power of the hasl model at various resolutions. binding predictions (pki values) were calculated for the entire inhibitor set at each resolution and plotted separately for the learning and test set members at 2.8 a resolution (fig. i) : . ala-). data determined: molecular models (3d structure of the molecules have been constructed and displayed using the program geom communicating with cambridge x-ray data bank, brookhaven protein data bank, sandoz x-ray data bank sybyl and disman; quant. struct.-act. relat. 9, 234-293 (1990) abstr. 311-312 273 distance geometry [nuclear overhauser enhancements (noe) and spin-spin coupling constants were measured by 2d nmr methods, semiempirically calibrated as proton-proton distance (a) and dihedral angle (deg) constrains and used in distance geometry calculations (disman) andlor in restrained molecular dynamics calculations to determine 3d structure of molecules in solution]; crystal structure (atomic coordinates of the compounds were determined by x-ray crystallography); rms [root mean square deviation (a) of the corresponding atoms of two superimposed molecular structures]. chemical descriptors: results: distance geometry calculations were carried out using geom and disman, to identify all conformations of the compounds in solution which were consistent with experimental data obtained by noe measurements. the application of geom was demonstrated by modelling cycbsporin a with and without a limited set of h-bond constrains and with a full nmr data set. in case of cyclosporin a, 100 randomly generated linear analogues of the cyclic structure were formed from the monomers. geometric cyclization was achieved using disman, resulting in many different but stereochemically correct conformations of cyclosporin a. superposition of the backbones of the 10 best cyclic conformers showed rms deviations between 1.8 a and 3.1 a. fig. 1 shows the superposition of a disman generated ring conformation (thick line) with its x-ray structure of cyclosporin a (thin line) with h-bond constraints (rms = 1.25 a): fig.1 37 distance and 4 dihedrl-angle constraints have been extracted from noe and vicinal coupling data and used to generate the conformation and the cyclization conditions of the hexapeptide (fig. 2) (position of residual distance violations and their direction is shown by arrows): although the described method is not exhaustive, it explores a much greater variety of initial structures than had been previously possible. title: a new model parameter set for @-lactams. authors: durkin, k.a.; sherrod, m.j.; liotta*, d. department of chemistry, emory university atlanta gl 30322, usa. source: j. org. chem. 1989, 54(25) , 5839-5841. compounds: 22 @lactam antibiotics of diverse structure. data taken from the literature: crystal structures (crystal coordinates of the p-lactams were determined using x-ray diffraction method). results: superposition of the x-ray structures and the calculated geometries of 8-lactams using the original parameter set in the mm2 force field in model gave satisfactory rms values. a lack of planarity of the 8-lactam ring and significant differences in the calculated bond lengths and anglesaround the &lactam nitrogen were detected, however. = 0, s, so, so,]. in order to improve fit, a new atom type with new parameters has been developed for the p-lactam nitrogen (wild atom type 6 0 coded with symbol 2 2 in model). the new parameters were evaluated by comparison of the calculated and x-ray geometries of the 22 0-lactams. using the new parameter set, the x-ray data were satisfactorily reproduced except for the sulfone 8-lactams. it was indicated that the ampac data were not suitable for the sulfones as the hypervalent sulfur compounds are not well described in the am1 hamiltonian. an additional parameters was, however, derived giving good structural data unrelated to the ampac information. it is not known which the new parameter sets is the best for the sulfone /3-lactams. title: a molecular modelling study of the interaction of compounds noradrenalin. biological material: a) cdna of the hamster lung 0,-adrenergic receptor and &-adrenergic receptor kinase; b) bacterio-ovine-and bovine-rhodopsin and rhodopsin kinase. protein primary sequence (amino acid sequence of the hamster lung p,-adrenergic receptor has been deduced by cloning the gene and the cdna of the hamster lung &adrenergic receptor);, (the cosmic molecular modeling program was used for modeling a-helices in a hydrophobic environment using p and w torsion angles of -59" and -44", respectively, according to blundell et al.); (the two highest lying occupied and the two lowest lying unoccupied orbitals, respectively, calculated using indo molecular orbital calculation); crystal structure (crystal coordinates of noradrenaline has been determined by x-ray diffractometry); conformation analysis (minimum energy conformation of the &-adrenergic receptor model has been calculated using molecular mechanics method). results: strong experimental evidences suggested that rhodopsin and 8,-adrenergic receptor had similar secondary structure. thus, it was assumed, that similarly to bacterio-ovine-and bovine-rhodopsins, d2-adr ener gic receptor -. . b2-adrenergic receptor possesses a structure consisting of seven ahelices traversing the cell membrane. fig. 1 shows the postulated arrangements of the a-helices of rhodopsin and the &-receptor. using the experimental data, a model of the &-adrenergic receptor has been generated for the study of its interaction with noradrenaline. a possible binding site was created. successful docking indicated that homo and lumo orbitals contributed to the binding in a chargetransfer interaction between trp-109 and noradrenaline. a hydrogen bond was detected between the threonine residue of the model receptor and the noradrenaline side chain hydroxyl explaining why chirality was found to be important for the activity of adrenergic substances. title: three-dimensional steric molecular modeling of the [binding affinity (nm) of the compounds to the 5-ht3 receptor]. data determined: molecular modeling (3d molecular models of each 19 compound were made using camseqlm molecular modeling system); [distance (a) from the center of the aromatic ring to the ring-embedded nitrogen, when the nitrogen is placed in the same plane as the aromatic ring]. results: in order to derive rules for the 5-ht3 pharmacophore, a molecular graphics-based analysis was made using six core structures. the structures were aligned so as to overlay the aromatic rings and to place the ring embedded nitrogen atom in the same plane as the aromatic ring. nine steric rules were derived from the analysis common to all 19 potent 5-ht3 agents. fig. 1 shows the 3d representation of the six overlaid 5-ht3 core structures using camseqim: the 5-ht3 inactivity of atropine could be explained because its steric properties differed from those the active ics 205-930 only by a single atom and failed to meet two of the nine hypothetical criteria. uv-visible spectra [spectrophotometric studies of mixtures of the dyes and nicotine in 10% (vlv) aqueous ethanol mixture at 29"ci. results: cyanine dyes demonstrate a multitude of biological activities which may be due to the interference of the adsorbed dye molecule on active sites of the living cell. it was shown by uv-and visible spectrophotometry that the hydroxy styryl cyanine dyes and nicotine formed 1 : 1 charge-transfer complexes. the absorption band of the complex formed between nicotine and dye was detected at wavelengths longer than those of the individual pure substances having identical concentrations to those in mixture. fig. 1 shows that the two partially positive centres of the dye (2-(2-hydroxystyryl)-pyridinium-i-ethyliodide) were located at a similar distance than the two nitrogen atoms of pyridine or pyrrolidinyl moieties of nicotine allowing the suggested 1: i parallel stucking interaction between the two molecule: molecular modeling (200 conformations were calculated using a distance geometry algorithm and energy minimized by a modified mm2 force field in moledit). results: all conformers within 5 kcallmol of the lowest energy conformer were superposed on the x-ray structure of mk-329. crystal structure (crystal coordinates of the proteins were determined using x-ray diffraction method). data taken from the literature: was less than 1 a); (probability that a given tetrapeptide sequence is superimposable on the ribonuclease a structure); [probability that the ith residue (amino acid) will occur in the jth conformational state of the tetrapeptide which is superimposable to ribonuclease a]. results: it was suggested that the five tetrapeptides were essential components of larger peptides and might be responsible for their biological activity (binding to the cd4 receptor). earlier it was hypothesized that the critical tetrapeptide located in a segment of ribonuclease a, would assume low energy conformations (residues 22 -25, a @-bend, having a segment homologous to the sequence of peptide t). low energy conformers of the tetrapeptides could be superimposed to the native structure of segment 22-25 of ribonuclease a. fig. shows the superimposition of peptide t (full square): many low energy conformers could be calculated for the tetrapeptides but for the polio sequence. the p, value for most tetrapeptides were 5 -10 times higher that the value of the less active polio sequence. the results supported the hypothesis that the active peptide t adopts the native ribonuclease @-bend. title: potential cardiotonics. 4. synthesis, cardiovascular activity, molecule-and crystal structure of 5-phenyl-and 5-(pyrid-4-yl) data determined: [dose of the compound (mollkg) required for 30 4% increase of the heart beat frequency of guinea pig or dog heart]; [dose of the compound (mollkg) required for 10 % decrease of systolic or diastolic blood pressure of dog]; crystal structure (atomic coordinates of the compounds were determined by x-ray diffraction); molecular modeling (molecule models were built using molpac); mep [molecular electrostatic potential (mep) (dimension not given) was calculated using cndoiz]. results: milrinon and its oxygen containing bioisoster possess highly similar crystal structure and mep isopotential maps ( fig. 1 and fig. 2) both compounds show strong positive inotropic and vasodilatoric activity. it was suggested that the negative potential region around the thiocarbonyl group such as the carbonyl group in milrinon imitates the negative potential field around the phosphate group of camp. title: molecular mechanics calculations of cyclosporin a analogues. effect of chirality and degree of substitution on the side chain conformations of (2s,3r,4r,6e)-3-hydroxy-4-methyl-2-(meth~lamino)-6octenoic acid and related derivatives. [solution conformation of csa in cdch has been elucidated via molecular dynamics simulation incorporating 58 distance constrains obtained from ir spectroscopy and nuclear overhauser effect (noe) data]; (conformational analysis was performed using the search subroutine within sybyl); energy minimization (low energy conformers were calculated using molecular mechanics withinmacromodel ver. 1.5 applying an all-atom version of the amber force field). results a total of 12 conformations of csa have been identified within 4 kcalfmol of the minimum energy conformer. population analysis showed that one conformer dominates in solution. fig. 1 shows the superposition of the peptide backbone of the crystal and solution structures of csa (crystal structure is drawn with thick line and the solution structure with thin line). it was shown that the boltzmann distribution between active and inactive conformers correlated with the order of the immunosuppressive activity. a common bioactive conformer serving as a standard for further design has been proposed for csa and its analogs. abtstr. 320 quant. struct.-act. relat. 9, 234 -293 (1990) 4 data determined: molecular modeling (models of (i), (11) and (111) were built using sybyl based on x-ray coordinates of the compounds); conformational analysis (minimum energy conformations of the compounds were calculated using the search option of sybyl and mndo method; [interaction energy of the molecules (kcall mol) with a hypothetical receptor probe (negatively charged oxygen atom) calculated by grid]. results: the specific receptor area of the sodium channel was modeled with a negatively charged oxygen probe (carboxyl group), interacting with the positively charged (protonated) ligand. fig. 1 shows areas for energetically favorable interaction (areas i, 11 oh 0 ho2c *. . quant. struct.-act. relat. 9, 234 -293 (1990) abstr. 321-322 279 biological material: a) aspergillus ochraceus; b) carbopeptidase a. data determined kobr [first order rate coefficient (io-6/sec) of the hydrochloric acid hydrolysis of ochratoxin a and b]; x-ray crystallography (coordinates of the crystal structure of ochratoxin a and b was obtained using x-ray diffraction); (models of ochratoxin a and b was built using alchemy); ["c nmr chemical shifts (ppm) of the amide and ester carbonyls of the ochratoxins]. chemical descriptors: pka (negative logarithm of the acidic dissociation constant). results: a reversal of the hydrolysis rate between ochratoxin a and b was observed comparing the hydrolysis rates obtained in vitro (carbopeptidase a) and in vivo (hydrochloric acid). the difference in hydrolysis rates cannot be due to conformation since the two toxins have the same conformation in both crystal and in solution. fig. 1 shows the fit of ochratoxin a and b based on superimposing the phenolic carbon atoms. it is suggested that the relative large steric bulk of the chloro atom hinders the fit between ochratoxin a and the receptor site of carbopeptidase a. thus, probably the slower metabolism is the reason, why ochratoxin a is more toxic than ochratoxin b. title: inhibitors of cholesterol biosynthesis. 1. trans-6-(2-pyrrolcharge distribution studies showed that compactin had two distinct regions of relatively large partial charges corresponding to the pyrrol ring and the isobutyric acid side chain. experiments for more closely mimicking the polar regions associated with the high activity of compactin indicated that potency of the new compounds was relatively insensitive to the polarity of the r' group. it was also suggested that an electron deficient pyrrole ring was required for high potency. title: synthesis and biological activity of new hmg-coa reductase inhibitors. 1. lactones of pyridine-and pyrimidine-substituted 3.5dihydroxy-6-heptenoicf-heptanoic) acids. chemical descriptors: results: an attempt was made to correlate electrophysiological activity with the effect of the position of the aryl group on the conformation of the side chain using molecular modeling. the study suggested that the compounds with class 111 activity prefer a gauche (a in fig. 1 ) and compounds in which class i activity prefer trans relationship of the nitrogens (b in fig. 1) : the study indicated that the point of attachment of the aryl moiety had an effect on the side chain conformation which appeared to be a controlling factor of the electrophysiological profile of these compounds. title: a molecular mechanics analysis of molecular recognition by cyclodextrin mimics of a-chymotrypsin. authors ( 1 ) quant. struct.-act. relat. 9. 234 -293 ( 1990) biological material: chymotrypsin. data taken from the literature: crystal structure (crystal coordinates of the macrocycles determined using x-ray diffraction analysis). data determined: molecular modeling structure superposition (models of b-cd and in chains by nmethylformamide and n-dimethyl-formamide substituted (capped) b-cd were built using the amber program and the coordinates for building the n-methylformamide substituent were calculated using mndo in the mopac program); (energy minimization of the molecules were calculated in vacuo using molecular mechanics program with the amber force field); (energy minimized structures of b-cd and capped b-cd were separately fit to the xray structure of the b-cd complex); [molecular electrostatic potential (kcallmol) of b-cd and capped b-cd were approximated by the coulombic interaction between a positive point charge and the static charge distribution of the molecule, modeled by the potential derived atomic point charges at the nuclei and visualized as 2d mep map]. results: b-cd and capped b-cd were analyzed as biomimetic models of the active site of chymotrypsin. capped b-cd was shown to be the more effective biomimetic catalyst. capping also altered certain structural features of molecular recognition. the orientation of the secondary hydroxyls were altereddue to twisting of some of the glucose units. secondary hydroxyl oxygen mimics the ser-195 of chymotrypsin in initiating the acyl transfer event through nucleophilic attack on the substrate. fig. 1 shows the energy minimized structures of b-cd (a) and capped b-cd (b) (fragment number is given in parenthesis). the mep maps of b-cd and capped b-cd showed that the qualitative features of the electrostatic recognition were practically the same in the two mimics. biologicai material: four monocotyledonous (johnson grass, yellow foxtail, barnyard grass, yellow millet) and four dicotyledonous weed species (velvetleaf, morning glory, prickly sida, sicklepod). data determined: [pre-emergence and postemergence herbicidal activities of the compounds were measured and rated using a scale ranging from 0 (no activity) to 9 (complete kill]; [measure of the compound's ability (dimension not given) to translocate upwards in plants through xylem vessels); [soil sorption coefficient calculated by the formula k, = c,/c,, where c, is the concentration of the compound (pg compoundlg soil) and c. is the concentration of the compound (pg compoundlml) in water solution in equilibrium with the soil]; (models of the compounds were built using maccs and prxbld programs); tscf kd molecular modeling quant. struct.-act. relat. 9, 234 -293 (1990) abstr. 327-328 283 conformational analysis (minimum energy conformations of the compounds were calculated using mm2 molecular mechanics method); (molecules were visualized using program mogli on an evans and sutherland picture system 33); [total energies, orbital eigenvalues, atomic charges and dipole moments of simple model analogs of type i were calculated using prddo (partial retention of diatomic overlap) level of approximation]. electronic structure chemical descriptors: logp (logarithm of the partition coefficient in 1-octanollwater). results: conformational analyses and high level quantum mechanical calculations of the conformational preferences showed that the compounds with r = 4-c1 and 5-ci substituents adopt a coplanar structure stabilized by intramolecular hydrogen bond, whereas the 3-c analogue does not (fig. 1 ): higher logp values (0.6 -1.0 logarithmic unit difference), higher kd and tscf values of the 4-ci and 5-ci substituted compounds relative to the 3-ci analog were interpreted as the result of the intramolecular hydrogen bond and were consistent with the observation that the 4-ci and 5-ci analogs were active as post-emergence but not pre-emergence herbicides while the 3-ci derivative was active in both modes. title: application of molecular modeling techniques to pheromones of the marine brown algae cutleria multifida and ectocarpus siliculosus (phaeophyceae). metalloproteins as chemoreceptors? (geometrical models of the compounds were constructed using information from the cambridge structural data base (csd) and calculated using molecular mechanics methods in sybyl); (minimum energy conformations of the compounds were calculated using molecular mechanics method within sybyl). chemical descriptors: kfcq [partition coefficient in fc72/water (fc72 = fluorocarbon results: as both ectocarpene (i) and multifidene (11) trigger mutual cross reactions between male gametes of ectocarpus siliculosus and cutleria multifida males it was supposed that a common mode of binding should exist for the two structurally different pheromones. the active analogue approach was applied to model the pheromone receptor by superposing the minimum energy conformations of active structural analogues (hi, iv, v, vi) on ectocarpene and multifidene. the common active conformation of (i) and (11) was extracted by systematic superimposition of the analogues. to explain the function of the double bonds in the pheromones, the presence of a receptor bound metal cation was assumed. simultaneous optimization,of both structures without and with a receptor bound metal cation resulted in virtually the same conformations. fig. 1 shows the mapping of multifidene onto ectocarpene in their biologically relevant conformations. solvent)]. title: critical differences in the binding of aryl phosphate and carbamate inhibitors of acetylcholinesterases. conformational analysis [minimum energy conformations of (asn-ala-asn-pro)9 was calculated using charmm (chemistry at harvard macromolecular mechanics), amber (assisted model building with energy refinement) and ecepp (empirical conformational energy program for peptides) potential energy functions]; [root mean square deviation (a) of the position of the corresponding atoms of two superimposed molecular sructures], results: 24 low energy conformations of (asn-ala-asn-pro)9 has been determined using charmm, ecepp and amber in order to determine their final conformations and relative energies. the final conformations were compared calculating the rms values of their c" atoms and matching the parameters of the energy minimized (asn-ala-asn-pro), peptide to that of the ideal helix or coiled coil. the similarity of the final conformations obtained by using any two different potentials starting from the same conformation varied from the satisfactory to highly unacceptable. the extent of difference between any pairs of the final conformations generated by two different potential energy functions were not significantly different. the lowestenergy conformation calculated by each of the energy potentials for any starting conformation was a left handed helix and pair-wise superposition of the c" atoms in the final conformations showed small rms values (1 .o -1.3 a) . it was suggested that the native conformation of (asn-ala-asn-pro), in the cs protein may be a left-handed helix, since all three potential energy functions generated such conformation. 234 -293 (1990) source: proteins 1989 proteins , 6(2), 193 -209, 1989 . biological material: crambin. data determined phi-psi probability plot (probabilities of the occurrences of phi-psi dihedral angle pairs for each amino acid were determined and plotted using the data of approximately 100 proteins from the brookhaven protein data bank); (optimization technique for the reproduction the folding process converging to the native minimum energy structure by dynamically sampling many different conformations of the simplified protein backbone). chemical descriptors: phi-psi values [dihedral angles (deg) defined by the bonds on either side of the a-carbon atom of the amino acid residue in a protein]. results: a simplified model has been developed for the representation of protein structures. protein folding was simulated assuming a freely rotating rigid chain where the effect of each side chain approximated by a single atom. phi-psi probabilities were used to determine the potentials representing the attraction or repulsion between the different amino acid residues. many characteristics of native proteins have been successfully reproduced by the model: (i) the optimization was started from protein models with random conformations and led to protein models with secondary structural features (a-helices and 0strands) similar by nature and site to that of the native protein; (ii) the formation of secondary structure was found to be sequence specific influenced by long-range interactions; (iii) the association of certain pairs of cysteine residues were preferred compared to other cysteine pairs depending on folding; (iv) the empirical potentials obtained from phi-ps probabilities led to the formation of a hydrophobic core of the model peptide. x [dihedral angle (deg) ]. results: four kinds of monte carlo simulations of about 20,000 steps of the conformations of crambin were carried out by using the second derivative matrix of energy functions (starting from native and unfolded conformations both in two kinds of systems, in vacuo and in solution). fig. 1 shows the native (a) and theunfolded (b) conformation of crambin. starting from native conformation, the differences between the mean properties of the simulated crambin conformations obtained from in vacuo and solution calculations were not very large. the fluctuations around the mean conformation during simulation were smaller in solution than in vacuo, however. simulation starting from the unfolded conformation resulted in a more intensive fluctuation of the structure in solution than in vacuo indicating the importance of the hydration energy term in the model. the conformations generated in the simulations starting from the native conformation deviate slightly from the xray conformation (rms = 0.70 a and i . 10 a for in vacuo and solution simulations, respectively). the results indicate that the simulations of the protein with hydration energyare more realistic that the simulations without hydration energy. fig.1 results: earlier studies overestimated the catalytic rate decrease of the hypothetical d102a point mutant of thrombin (20 orders of magnitude decrease calculated instead of the 4 order of magnitude measured). the source of error was due to an overestimation of v and neglecting the effects of the surrounding water molecules and induced dipoles. to compensate for these errors, a scale factor of 0.12 was introduced into the calculations. as aresult of the rescaling, one magnitude increase of tat for the d121 mutant and two magnitudes decrease of k,, of the k41 mutant of ribonuclease a was predicted. it was shown that the effect of the mutations on the catalytic rate depended almost entirely on steric factors. it was suggested that in mutants of serine proteases where the buried asp is replaced by ala or asp, the kcat value will decrease between 4 -6 orders of magnitude. title: high-resolution structure of an hiv zinc fingerlike domain via a new nmr-based distance geometry approach. authors: summers*, m.f.; south, t.l.; kim [root mean square deviation (a) of the corresponding atoms of two superimposed molecular sructures]. results: the atomic resolution structure of an hiv zinc fingerlike domain has been generated by a new nmr-based dg method using 2d noesy backcalculation. the quality of the structures thus obtained were evaluated on the basis of the consistence with the experimental data (comparison of measured and back-calculated nmr spectra) rather than comparing it tostructural informations from other sources (e.g. x-ray data). the method provided a quantitative measure of consistence between experimental and calculated data which allowed for the use of tighter interproton distance constraints. the folding of the c( l)-f(2)-n(3)-c(4)-g(s)-k(6) residues were found to be virtually identical with the folding of the related residues in the x-ray structure of the iron domain of rubredoxin (rms values 0.46 and 0.35 a). the backbone folding of the peptide was found to be. significantly different from that of the "classical" dna-binding zn-finger. fig. 1 shows the wire frame model of all the back%ne atoms and certain side chain atoms of the peptide (dg struciure) (dashed lines indicate hydrogen atoms): active site of the protease dimer in an extended conformation with extensive van der waals and hydrogen bonding and was more than 80 % excluded from contact with the surrounding water (fig. i , where the inhibitor is shown in thicker lines and the hydrogen bonds in dashed lines): data determined: ago,, ago, [standard free energy (callmol) of transfer of a molecule from an apolar phase to an aqueous phase, observed or calculated by eq. 1: ago, = c aui ai, where aoi is the atomic solvation parameter of atomic group i, ai is the accessible surface area of atom i, respectively]. results: atomic solvation parameters (asps) characterizing the free energy change per unit area for transfer of a chemical group from the protein interior to aqueous surroundings were determined. ago, and ago, were determined and compared, and a highly significant linear relationship is presented (fig. 1) . one letter symbols indicate amino acid side chains: fig .1 the binding of the inhibitor induced substantial movement in the en-'2 zyme around the residues 77 to 82 in both subunits at places exceeding i the structure of glutamine synthetase is discussed. it was established that hydrophobic interactions are important for the intersubunit interactions, and the hydrophobic interactions between the two rings of subunits are stronger than between the subunits within a ring. the cterminal helix contribute strongly to the inter-ring hydrophobic interaction. asps are suggested to estimate the contribution of the hydrophobic energy to protein folding and subunit assembly and the binding of small molecules to proteins. title: determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing. authors: holak*, t.a.; gondol, d.; otlewski, j.; wilusz, t. max-planck-hstitut fiir biocbemie d-8033 martinsried bei miinchen, federal republic of germany. title: interpretation of protein folding and binding with atomic crystal structure (atomic coordinates of cmti-i was determined by solvation parameters. x-ray diffraction method). results: in order to obtain information of the 3d structure of the free cmti-i in solution, a total of 34 inhibitor structures were calculated by a combination of distance-geometry and dynamical simulated annealing methods, resulting in well defined 3d positions for the backbone and side-chain atoms. fig. 1 shows the superposition of the backbone (n, c", c, 0) atoms of the structures best fitted to residues 2 to 29 (binding loop): the average rms difference between the individual structures and the minimized mean stfucture was 0.35(*0.08) a for the backbone atoms and 0.89(+0.17) a for all heavy atoms. title: electron transport in sulfate reducing bacteria. molecular modeling and nmr studies of the rubredoxin-tetraheme-cytochrome-c3 complex. biological material: a) sulfate reducing bacterium (desulfovibrio vulgaris); b) rubredoxin (iron-sulfur protein); c) tetraheme cytochrome c3 from d. vulgaris; e) flavodoxin. detected in the segments from the residues 16 to 18 and 25 -25. fig. 1 shows the best superposition (residues 2 to 29) of the nmr and crystal structure of cmti-i indicating the backbone c, c", n, 0, as well as the disulfide c8 and s atoms: fig.1 it was demonstrated that uncertainty in nmr structure determination can be eliminated by including stereospecific assignments and precise distance constraints in the definition of the structure. crystal structure (coordinates of the crystal structure of the compounds were determined by x-ray crystallography). results: the speed of the homolysis of the organometallic bond is 10" times higher in the apoenzyme bound coenzyme biz than in a homogenous solution. structural changes occurring during the co-c bond homolysis of the coenzyme biz leading from cobalt(ii1) corrin to cobalt(i1) corrin were investigated. fig. 1 shows the superposition of structures of the cobalt corrin part of the biz (dotted line) and of cob(ii)alamin (solid line): biological material: apoenzyme, binding the coenzyme biz and the data determined: . 1 shows that the crystal structure of biz and cob(i1)alamin are strikingly similar and offers no explanation for the mechanism of the protein-induced activation of homolysis. it was suggested that the co-c bond may be labiiized by the apoenzyme itself and in addition to a substrate-induced separation of the homolysis fragments (which mights be supported by a strong binding of the separated fragments to the protein). 'h nmr (complete stereospecific assignments were carried out and proton-proton distance constrains were determined by the analyses of dqf-cosy, hohaha and noesy spectra); nh, ah, oh [chemical shifts (ppm) of proton resonances of human eti ; 3d structure (3d structure of et was calculated using the distance geometry program dadas based upon the noesy proton-proton distance constrains determined by nmr spectroscopy); [root mean square distance (a) between 5 et conformers calculated by distance geometry (dadas)]. results: the solution conformation of et has been determined by the combined use of 2d 'h nmr spectroscopy and distance geometry calculations. five structures of et have been calculated from different initial conformations. the superposition of the backbone atoms the calculated structures is shown in fig. 1 . the average rms value in the core region for the main-cahin atoms was 0.46 a. quant. struct.-act. relat. 9, 234-293 (1990) the lack of specific interactions between the core and tail portions of et and a characteristic helix-like conformation in the region from lys' to cys" was shown. literature data indicated that neither the eti -1 5 nor the etl6-21 truncated derivatives of et showed constricting or receptor binding activity suggesting that the et receptor recognizes an active conformation consisting of both the tail and core portion. the present study, however, suggested that the receptor bound conformation of et is probably different from that in solution because the lack of interaction between tail and core. the hydrophobic nature of the tail suggested the importance of a hydrophobic interaction with the receptor. compounds: triphenyl-methyl-phosphit cation (tpmp+). biological material: nicotinic acetylcholine receptor (achr), a prototype of the type i of membrane receptor protein from the electric tissue of torpedo and electrophorus. results: a computer model of the achr ion channel has been proposed. fig. 1 shows the side view of the ion channel model with five pore-forming mz-helices and the channel blocking photoaffinity label (tpmp +) represented by the dotted sphere: fig.1 it was supported by electronmicroscopy, electrophysiological-and biochemical experiments that the mz-helices were formed by homologous amino acid sequences containing negatively charged amino acid side chains which act as the selectivity filter. the amino acid side chains may undergo conformational changes during the permeation of the cation. the predicted transmembrane folding of four transmembrane a-helices of type i receptors is shown in fig. 2: fig. 2 energy profile calculations indicate that other transmembrane sequences of the receptor protein besides m2 may affect the ion channel. source: cabios 1989, 5 (3), 219 -226. results: an interactive computer program tefoojj2 has been developed for drug design on ibm/pc and compatible computers. the program contains the following modules and performs the following calculations: a) series design for selecting an optimal starting set of compounds using a modified version of austel's method; b) regression analysis calculating the hansch's equation; c) hansch searching method using the equation calculated by the regression analysis routine or the use of an input equation for the identification of the 10 most active compounds; d) geometrical searching methods for finding the optimum substituents in the parameter space using the sphere, ellipse, quadratic or polyhedric cross algorithms with or without directionality factors; e) space contraction for reducing the dimension of the parameter space by eliminating non-significant parameters; f) an example is given for the lead optimization of an aliphatic lead compound correctly predicting the n-pentane to be the optimum substituent. results: a new expert system sparc is being developed at epa and at the university of georgia to develop quantitative structure-activity relationships for broad compound classes: a) classical qsar approaches predict therapeutic response, environmental fate or toxicity from structure/property descriptors quantifying hydrophobicity, topological descriptors, electronic descriptors and steric effects; b) sparc (sparc performs automated reasoning in chemistry), an expert system written in prolog, models chemistry at the level of physical organic chemistry in terms of mechanism of interaction that contribute to the phenomena of interest; c) sparc uses algorithms based on fundamental chemical structure theory to estimate parameters such as acid dissociation constants (pk,s), hydrolysis rate constants, uv, visible and ir absorption spectra, and other properties; d) the information required to predict input data for broad classes of compounds is dispersed throughout the entire ir spectrum and can be extracted using fourier transforms; e ) the accuracy of sparc algorithm was demonstrated on the close match of calculated and experimental pk, values of 20 carboxylic acid derivatives near to the noise level of measurement. abtstr. 347-351 quant. struct.-act. relat. 9, 234-293 (1990) results: a new stand-alone molecular simulation program, nmrgraf integrating molecular modeling and nmr techniques has been introduced by biodesign inc. a) nmrgraf is a molecular modeling program utilizing force fields which incorporate empirical properties such as bond lengths and angles, dihedral, inversion and nonbonded interactions, electrostatic charges and van der waals interactions; b) the molecular structural properties are combined with nuclear overhouser effect (noe) and j-coupling nmr data (experimental interproton distance constrains and bond angle data); c) the nmr proton-proton distance data are accurate only at relatively short distances (5 to 10 a) which restricts the use of nmr noe approaches only for the analysis of molecules with known x-ray structure; d) the combination of nmr and molecular modeling approaches, however, makes it possible to model virtually any molecule even if its structure does not exist in the databases. title: electronic structure calculations on workstation computers. results: the main features of the program system turbomole for large-scale calculation of scf molecular electronic structure on workstation computers is described: a) the program system allows for scf level treatments of energy, first-and second-order derivatives with respect to nuclear coordinates, and an evaluation of the me? correlation energy approximation; b) the most important modules of turbomole are (i) dscf performing closed and open shell rhf calculations; (ii) egrad used for analytical scf gradient evaluations; (iii) kora calculating direct two-electron integral transformation (iv) force for the computation and processing of integral derivatives and the solution of cphf equations; c) comparison and evaluation of timings of representative applications of turbomole on various workstations showed that apollo ds 1o.ooo and iris 4d1210 were the fastest and comparable to the convex c210 in scalar mode. results: a new algorithm has been developed for the calculating and visualizing space filling models of molecules.the algorithm is about 25 times faster than a conventional one and has an interesting transparency effect when using a stereo viewer. a) the algorithm is briefly described and and the result is visualized on modeling a ribonucleotide unit; b) in the order of increasing atomnumbers, the (x,y) sections of the hemispherical disks of the atoms are projected on the screen with decreasing value of the azimuthal angle (p) of the van der wads radius and as the value of p decreases the projection is increasingly whitened to obtain shading effect on the surfaces of the spheres; c) the transparency of the van der waals' surfaces of atoms of a molecule makes possible to perceive almost the whole space filling structure and not only the surface, hiding the underlying atoms. title: supercomputers and biological sequence comparison algo-authors: core*, n.g. ; edmiston, e.w. ; saltz, j.h. ; smith, r.m. rithms. yale university school of medicine new haven ct 06520-2158, usa. source: computers biomed. res. 1989, 22(6) , 497 -515. compounds: dna and protein fragments. chemical descriptors: sequences of monomers. results: a dynamic programming algorithm to determine best matches betweenpairs of sequences or pairs of subsequences has been used on the intel ipsc/l hypercube and ,the connection machine (cm-i). parallel processing of the comparison on cm-i results in run times which are 65 to 230 times as fast as the vax 8650, with this factor increasing as the problem size increases. the cm-i and the intel ipsc hypercube are comparable for smaller sequences, but the cm-i is several times quicker for larger sequences. a fast algorithm by karlin and his coworkers designed to determine all exact repeats greater than a given length among a set of strings has been tried out on the encore multimax/32o. the dynamic programming algorithms are normally used to compare two sequences, but are very expensive for multiple sequences. the karlin algorithm is well suited to comparing multiple sequences. calculating a multiple comparison of 11 dna sequences each 300-400 nucleotides long results in a speedup roughly equal to the number of the processors used. source: cabios 1989, 5(4), 323. results: a program has been developed for the prediction and display of the secondary structure of proteins using the primary amino acid sequence as database. a) the program calculates and graphically demonstrates four predictive profiles of the proteins allowing interpretation and comparison with the results of other programs; b) as a demonstration the sliding averages of n sequential amino acids were calculated and plotted for four properties of human interleukin 6: (i) plot of the probabilities of a-helix, p-structure and p-turns according to chou and fasman; (ii) @-turn index of chou and fasman; (iii) plot of the hydrophobicity index of hopp and woods; (iv) flexibility index of karplus and schulz; c) the regions of primary structure having properties which usually go together agreed reasonably well with each other, i.e. loops and turns with bend probability and hydrophilicity with flexibility. title: 3dsearch. a system for three-dimensional substructure searching. quant. struct.-act. relat. 9, 234-293 (1990) sci. 1989, 29(4) , 255 -260. results: the search for threedimensional substructures is becoming widely used in 3d modeling and for the construction of pharmacophores for a variety of biological activities. a system (3dsearch) for the definition and search of three-dimensional substructures is described: a) representation of atom types consists of five fields (i) element (he-u); (ii) number of non hydrogen neighbors (bonded atoms); (iii) number of 'k electrons; (iv) expected number of attached hydrogens; (v) formal charge; (vi) four type of dummy atoms are also used to define geometric points in space (e.g. centroid of a b) definition of queries (i) definition of spatial relationship between atoms; (ii) matches in atom type (iii) preparation of keys (constituent descriptors); (iv) execution of key search; (v) geometric search including the handling of angleldihedral constrains and takes into account "excluded volume"; c) time tests showed that a search of 3d structures with 3 to 5 atoms in large databases with more than 200,000 entries took only a few minutes (22 -492 s). results: a database containing about 265,000 compounds in connection tables and 30,000 experimentally determined structures from the cambridge structural database has been transformed into a database of low energy 3d molecular structures using the program concord. the strategy for building the 3d database consisted of the following four steps: a) generation of approximate 3d coordinates from connection tables (hydrogens were omitted); b) assignment of atom types from connection table information characterized by five descriptors: (i) element type (he-u); (ii) number of attached non-hydrogen neighbors (0 -8); (iii) number of 'k electrons (0 -2); (iv) calculated number of attached hydrogens (0-4); (v) formal charge ( -1, 0, 1); c) addition of three types of chemically meaningful dummy atoms for purposes of 3d substructure searching: (i) centroids of planar 5and 6-membered rings; (ii) dummy atoms representing the lone electron pairs; (iii) ring perpendiculars positioned orthogonal to and 0.5 a above and below each planar ring; d) efficient storage of the resultant coordinate database indexing the compounds with identification number; e) the database can be used among others to deduce pharmacophores essential for biological activity and to search for compounds containing a given pharmacophore. title source: c&en 1989, 67(43) , 18-20. results: a complex carbohydrate structure database (ccsd) has been developed by the complex carbohydrate research center at the university of georgia, having more than 2000 structures and related text files, with about 3000 more records to be added over the next two years. the following are the most important features of ccsd: a) in ccsd, database records include full primary structures for each complex carbohydrate, citations to papers in which sequences were published, and suplementary information such as spectroscopic analysis, biological activity, information about binding studies, etc; b) structural display format visualize branching, points of attachment between glycosyl residues and substituents, anomeric configuration of glycosyl linkages, absolute configuration of glycosyl residues, ring size, identity of proteins or lipids to which carbohydrates are attached and other data; c) it is planned that ccsd will provide threedimensional coordinates, to visualize and rotate the structures in stereo and study their interaction with proteins or other biopolimers. probing bioactive mechanisms p commercial carbamate insecticides of type ii, where r' = h, s-bu biological material: acetylcholinesterase. data determined: d [distance (a) between the serine oxygen of acetylcholinesterase and the methyl substituents of the carbamate and phosphate inhibitors molecular modeling (models and minimum energy conformations of acetylcholine, aryl carbamate and phosphate ester inhibitors were created using the draw mode of a maccs database and the prxbld modeling program) results: transition state modeling of the reaction of the serine hydroxyl ion of acetylcholinesterase with the methylcarbamoyl and dimethyl phosphoryl derivatives of 3,4-dimethyl-phenol showed that the active site binding for these two classes of acetylcholinesterase inhibitors should be different. the model shows that the distances between the serine oxygen and the ring substituents (meta-and para-me-thy1 groups) are different in both spacing and direction. fig. 1 shows the transition state models of serine hydroxyl d values for the meta-and para-methyl substituents of n-methylcarbamate and dimethylphosphate were meta = 6.20, para = 8.10 and meta = 5.48, para = 7.03 a, respectively title: a comparison of the charmm, amber and ecepp potentials for peptides. 1. conformational predictions for the tandemly repeated peptide (asn-ala-asn-pro)9 biological material: tandemly repeated peptide (asn-ala-asn-pro) which is a major immunogenic epitope in the circumsporozoite (cs) protein of plasmodium falciparum conformational analysis dream or reality? a authors: nbray-szab6*, g.; nagy, j.; bcrces, a. priori predictions for thrombin and ribonuclease mutants molecular modeling (geometric model of subtilisin and trypsin was built using protein data bank coordinates and model of thrombin was built using the theoretical coordinate set of graphic representations of the triad of the tetrahedral intermediate for the enzymes formed on the active side chain and residues (ser-221, his-64 and asp32 in subtilisin; ser-195, his-64 and asp-i02 in trypsin and thrombin; his-119, lys-41 and his-i2 in ribonuclease a) were created using pcgeom electrostatic properties (electrostatic surfaces and fields of the molecules were calculated and displayed using amber and associated programs); [chemical shift (ppm) measured by nmr results: a hypothetical model between rubredoxin and cytochrome c3 was built as a model for the study of electron transfer between different redox centers, as observed in other systems. fig. i shows the main chains atoms of the proposed complex where the hemes of the cytochromes are shown along with the center of rubredoxin and stabilized by hydrogen bonds and charge-pair interactions (the nonheme iron of the rubredoxin is in close proximity to heme 1 of cytochrome c3):6 spectroscopy].the model was consistent with the requirements of steric factors, complementary electrostatic interactions and nmr data of the complex. comparison of the new model and the nmr data of the previously proposed flavodoxin-cytochrome c3 complex showed that both proteins interacted with the same heme-group of cytochrome c3. title: nuclear magnetic resonance solution and x-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.authors: holak*, t.a.; bode, w.; huber, r.; otlewski, j.; wilusz, t. max-planck-institut fiir biochemie d-8033 martinsried bei munchen, federal republic of germany.source: j. mol. biol. 1989, no.210, 649-654. biological material: a) trypsin inhibitor from the seeds of the squash cucurbita maxima; b) p-trypsin and trypsin inhibitor complex.title: retention prediction of analytes in reversed-phase high-performance liquid chromatography based on molecular structure. 5. quant. struct.-act. relat. 9, 234-293 (1990) results: an expert system (cripes) has been developed for the prediction of rp-hplc retention indices from molecular structure by combining a set of rules with retention coefficients stored in a database. the method underlying the system is based on the "alkyl aryl retention index scale" and aims to improve the reproducibility of prediction and compatibility between various instruments and column materials. the vp-expert system shell from microsoft was used for the development. the performance of cripes was demonstrated on several subtypes of substituted benzenes (phenacyl halides, substituted arylamines, arylamides and other types). in general the calculated and measured retention indices agreed well but relatively large deviations were observed between the ie and ic values for phenacyl bromides and chlorides, o-and p-bromo anilines, n-methylbenzamide and n,n-dimethylbenzamide and phthalate esters. the extension of the database with further interaction values was regarded as necessary for a consistently high accuracy at prediction. [out-of-plane bending energy (kcal/mol) given by the formula e, = kd', where d is the distance from the atom to the plane defined by its three attached atoms and k is a force constant]; eb e, [torsional energy (kcal/mol .deg2) associated with four consecutive bonded atoms i,j,k,l given by the formula e, = ki,j.k,l(l fs/lslcos(lslbi,j,k,~)), where b is the torsion angle between atoms i j , k and 1, s and k are constants]; e, [potential energy (kcallmol) (nonbonded energy term) associated with any pair of atoms which are neither directly bonded to a common atom or belong to substructures more than a specified cutoff distance away given by the formula e, = kij(l.0la" -2.0/a6), where a is the distance between the two atoms divided by the sum of their radii, and k is the geometric mean of the k constants associated with each atom]. results: model geometries produced by the tripos 5.2 force field have been assessed by minimizing the crystall structures of three cyclic hexapeptides, crambin and 76 diverse complex organic compounds. comparative force field studies of the tripos 5.2, amber and amberlopls force fields were carried out by energy minimization of three cyclic hexapeptides starting from the crystal structures showed the tripos 5.2 force field superior to the others with the exception of amber, ecep2 and levb force fields as published by other workers. a direct comparison between the performance of tripos 5.2 and . amber using isolated crambin showed that the bond and torsion angles of tripos 5.2 averaged closer to the crystal structure than the angles calculated by amber (rms = 0.025 a, 2.97 deg and 13.0 deg for bond lengths, angles, and torsions, respectively, and rms = 0.42 a for heavy atoms). fig. 1 shows the superimposed structures of crambin before and after energy minimization:fi 9.1 tripos 5.2 was assessed for general purpose applications by minimizing 76 organic compounds starting from their crystal structures. the test showed that tripos 5.2 had a systematic error in overestimating the bond lengths of atoms in small rings. statistical analysis of the results showed that t r i p s 5.2 had an acceptable overall performance with both peptides and various organic molecules, however, its performance was not equal to the best specialized force fields. title: new software weds molecular modeling, nmr. author: krieger. j. c&en 1155 sixteenth st., n.w., washington dc 20036, usa. source: c&en 1990, 68(13) , 16. key: cord-024652-4i6kktl0 authors: santra, hiran kanti; banerjee, debdulal title: natural products as fungicide and their role in crop protection date: 2020-05-12 journal: natural bioactive products in sustainable agriculture doi: 10.1007/978-981-15-3024-1_9 sha: doc_id: 24652 cord_uid: 4i6kktl0 seeking solutions from nature for solving one and all problems is the age-old practice for mankind, and natural products are proved to be the most effective one for keeping up the balance of development as well as the “healthy, wealthy, and well” condition of mother nature. fungal pathogens are proved to be a common and popular contaminant of agroecosystem that approximately causes 70–80% of total microbial crop loss. to meet the proper global increasing need of food products as a result of population explosion, managing agricultural system in an eco-friendly and profitable manner is the prime target; thus the word “sustainable agriculture” plays it part, and this package is highly effective when coupled with nature-derived fungicidal products that can minimize the event of fungal infections in agrarian ecosystem. present study enlists the most common and effective natural products that might be of plant or microbial origin, their mode of action, day-by-day development of phytopathogenic resistance against the prevailing fungicides, and also their role in maintenance of sustainability of agricultural practices with special emphasis on their acceptance over the synthetic or chemical one. a large number of bioactive compounds ranging from direct plant (both cryptogams algae and moss and phanerogams)-derived natural extracts, essential oil of aromatic plants, and low-molecular-weight antimicrobial compounds known as phytoalexins to secondary metabolites that are both volatile and nonvolatile organic compounds of microbes (fungal and actinobacterial members) residing inside the host tissue, called endophyte, are widely used as agricultural bioweapons. the rhizospheric partners of plant, mycorrhizae, are also a prime agent of this chemical warfare and protect their green partners from fungal invaders and emphasize the concept of “sustainable agriculture.” natural products are the best weapon for the survival of any type of problems regarding infection, pathogenesis, or protection from diseases. due to their degradability in nature, they are the first options to be used by agriculturalists and plant biologist for combating fungal pathogenesis. the eukaryotic organism fungi have a separate kingdom in whittaker's five kingdom classification and are prime member of this ecosystem as a potent decomposer. in spite of their heavy and multidimensional applications in agricultural, medicinal, and industrial field ranging from the production of life-saving medicines to food supplements, they are the cause of huge global crop loss each year and lead to economic exhaustion. macro-and microscopic fungi-producing fruit bodies on different portions of a plant body (stem, leaf, fruit, root) lead to the death and decline of the crop species. several methods have already been tried since the start of civilization for crop protection but because of plant and fungal coevolution, fungi have dominated on the green eukaryotes and caused significant reduction in the crop yield. particularly in a country like india where the central gdp largely depends on agricultural output, the fungal pathogenesis has been a matter of grave concern for the agriculture department and policy makers. huge amount of money and manpower is invested to fight against the fungal diseases for ensuring higher and qualitative yield, but still it has been a burning problem of today's conditions. the problem with chemical and synthetic tools for combating the parasitic infections includes their toxicity leading to quality deterioration and environmental pollution accompanied with side effects on human health. in a case study, it has been reported that the extreme use of antibiotics in agricultural field and their direct consumption by humans through their daily meal have led to resistance of those antibiotics in human fungal pathogens. so we are in search of bioactive agents that will be of biological origin, selective to their host, and produce no secondary symptoms with least negative impact. the problem with fungi is their secretion of various types of mycotoxins (aflatoxins, ochratoxins, patulin, fumonisin, zearalenone, deoxynivalenol, etc.) in the stored food products, causing postharvest loss of cereals, pulses, dry fruits, and spices. mycotoxins are not only food spoilers but also potent disease-causing agents in humans leading to cancer, liver damage, kidney failure, and paralysis (miller 1995) . the severe effects of fungal crop loss are visible largely in tropical or subtropical regions where the temperature is moderately higher than the other parts of the world. fungal devastation occurs in two phases: firstly, when the crops are growing on the field and, secondly, when they are stored for further transportation, postharvest loss. the third type of contamination occurs when the microscopic airborne pathogens like molds grow on cooked foods and lead to food spoilage. at each and every level, scientists have developed techniques to minimize fungal food loss. on a gross annual estimate, almost 25% of agricultural food items are of no use due to fungal contamination (pittet 1998) . the major issues with fungi-related crop loss are deterioration as a result of increase of fatty acid conditions, change of color and texture of food items, poor nutritional conditions, and poor germinability of stored seeds (dhingra et al. 2001) . reports from asia and africa include death of humans and animals due to consumption of mycotoxin-contaminated foods (reddy and raghavender 2007) . fungal pathogens are sometimes dependent on more than one host for their successful completion of life cycle and disease development (puccinia graminis var. tritici, causal agent of black stem rust of wheat that requires berberis aristata for successful infection other than there main target wheat plant). so physical controls like eradication of secondary or collateral host and burning of the old livestocks and remnants of the field are the primary measures adopted by the farmers for disease-free crop production. so maintaining the sustainability along with less pathogenic infection is the deep ecological movement for crop maintenance. there are reports of resistance developed against the common and widely used antibiotics of agricultural importance. blasticidin s, an antibiotic obtained from streptomyces sp. (a type of actinobacteria predominantly present in soil samples), interacts with the protein synthesis and causes the death of the rice blast pathogens. development of resistance of this antibiotic is reported to be present in some fungal pathogens that detoxify it by deamination (dayan et al. 2009 ). compounds of bacterial and fungal origin from both soil and endophytic sources are used as an alternative source over the chemical ones. plant extracts especially essential oils from plant taxa of lamiaceae family are of immense importance and are used as fungicidal or fungistatic. most of the active ingredients act upon the fungal cell wall by either blocking the cellular processes like respiration, cell wall and cell membrane synthesis, ergosterol biosynthesis, protein synthesis, or dna replication. not only the secondary metabolites of plant and microbial origin but also the direct application of microorganisms in terms of biocontrol agent could be used as potent antifungals. other than these, plants' own defense molecules, known as the phytoalexins, could provide a strong line of defense against mycorrhizae; the root symbionts of higher plants can physically, biologically, and biochemically protect the plant root from pathogenic invasion and provide an enhanced resistance conditions to their hosts. this study includes the role of these compounds as natural agents of antifungal property and their role in disease prevention. mycorrhizae as a biocontrol agent mycorrhiza being the perfect example of symbiosis is known to be the oldest association between higher plant (both angiosperm and gymnosperm, monocot and dicot plants) and fungi and is an astonishing phenomenon of nature. the mycorrhizal association is one of nature's privileges for maintaining the sustainability of agriculture. in present day's changing environment, haphazard use of pesticides (fungicides) and chemicals poses a great risk to the existence and survival of mycorrhizal species in its complete biologically active form. there is a need to increase awareness in order to save mycorrhizal fungi from extinction. plants form beneficial association with other variants of life forms (animals, bacteria, or fungi) to complete their life processes, to fight against pathogenic microorganisms, and most importantly to thrive in adverse environmental situations. the plant root and its associated living microbial flora are together called "rhizosphere," particularly the area of mycorrhizal occurrence. the term mycorrhiza is derived from two greek words: mycos which means fungus and rhiza which means roots. in nature, more than 80% of angiosperms and almost all of gymnosperms are known to have mycorrhizal associations. the common two types of mycorrhizal associations that exist in nature are endomycorrhizae, also called arbuscular mycorrhizae (am), for example, endogone sp. and rhizophagus sp., and ectomycorrhizae (em), for instance amanita muscaria and laccaria bicolor. mycorrhizal associations support its host plants to survive in untimely soil conditions and drought situations by increasing the surface area of root and efficiency of mineral uptake. environmental threats including problems of temperature increase, climate changing, drought, and infertility of soil are some of the major challenges in agriculture and have to be mitigated to ensure global food security. in this context, mycorrhiza-based crop production is one of the key components of sustainable agriculture practices. in most of the cases, am fungi-mediated suppression of root pathogenic fungi is achieved by either morphological, physiological, or biochemical alterations of the host. several experiments on fungistatic activity of the mycorrhizal species have been done, and fruitful results are found against pathogenic fungi such as aphanomyces spp., botrytis fabae, chalara (thielaviopsis) basicola, dothiorella gregaria, fusarium oxysporum, gaeumannomyces graminis var. tritici, ganoderma pseudoferreum, pythium ultimum, p. splendens, phytophthora parasitica, p. cactorum, p. vignae, rhizoctonia solani, r. bataticola, and sclerotium rolfsii (lioussanne et al. 2009; bagyaraj 2006; bagyaraj and chawla 2012) . the most common outcome of am fungal colonization is seen as an increase in number of branches, resulting in a relatively larger proportion of higher-order roots in the root system. thickening of the cell walls due to lignification and production of polysaccharides in mycorrhizal plants are the common mode of prevention of penetration and growth of pathogens like fusarium oxysporum and phoma terrestris. a huge percentage of am-root pathogen interaction studies have been conducted in crop plants of agricultural and horticultural importance. but the information available on forest tree species is scanty. mycorrhizal technology can thus play an important role in production of low-cost quality seedlings and provide plant protection. like other methods of biological control, am fungi are not able to offer complete immunity against the infection caused by plant pathogens. they could only impart a degree of resistance against soilborne plant pathogens. however, the possibility of biologically controlling soilborne plant pathogens looks promising. am fungi play a protective role for plants by activating the defense mechanisms for the better resistance of crop plants and thus may protect the host plant from further fungal pathogenic attack, thus working as a potent biocontrol agent. researchers have proved that am symbiosis triggers the activation of several defense-related genes and also expression of pathogenesis-related proteins. evidences are drawn from modern techniques like molecular biology methods and immunological and histochemical analysis that strongly supports this concept. am fungi first act as a biotrophic agent, and before entering the host plant's root cell, they cause a sharp change in endogenous salicylic acid that is reflected in quick accumulation of reactive oxygen species (ros), a wide range of hydrolytic enzymes, and also the activation of phenylpropanoid biosynthetic pathway (güimil et al. 2005 , paszkowski 2006 , roman et al. 2011 . research findings have proved that the amount of defense-related compounds (essential enzymes like pal, phenylalanine ammonialyase, a product of phenylpropanoid pathway, enzymes needed for flavonoid or isoflavonoid biosynthesis like chalcone isomerase) that act for the protection of plant from fungal and bacterial pathogen is higher in the case of mycorrhiza-inoculated plant than in the uninoculated ones (volpin et al. 1994 (volpin et al. , 1995 . host's physiological and biochemical processes are greatly influenced by the mycorrhizal association in terms of decreased root exudation, higher concentration of phenylalanine and serine contents, ortho-dihydroxy phenols, increased membrane phospholipid content, etc. (smith et al. 1994) . when the phospholipid contents are high, it reduces the chances of root pathogenic attack, and higher concentrations of ortho-dihydroxy phenols show inhibitory activity against root rot pathogen sclerotium rolfsii (causal agent of southern blight), whereas the non-mycorrhizal plants are affected by the southern blight disease. tomato plants when inoculated with g. fasciculatum show inhibitory activity against root knot nematodes. host-am association leads to the formation of defense-related compounds like phytoalexins, chitinases (chi), β-1,3-glucanase (glu) (enzyme related to hydrolysis of fungal cell wall), peroxidases (pox), hydroxyproline-rich glycoproteins, and phenolics (st arnaud and vujanovic 2007) . synergistic effect of pgprs along with am fungi is proved to be a system of better protection than the use of am fungi alone (linderman 1994; bagyaraj and chawla 2012) . fungal wilt of common medicinal plant indian coleus (coleus forskohlii) caused by fusarium chlamydosporium could be minimized by the joint action of am fungus and trichoderma viride and cause a sharp increase in root yield and root forskolin concentration and may also reduce the severe disease conditions (singh et al. 2012 ). am causes a drastic change in the rhizospheric microbiota and intentionally either removes directly the pathogenic microorganisms or stimulates the accumulation of potent microbial partners especially fungus that are heavily antagonistic to the plant pathogenic ones. plants with mycorrhizal association harbor higher population of rhizospheric microorganisms, thus making it impossible for the pathogen to compete and invade the root. in the case of phytophthora cinnamomi, the numbers of sporangia and zoospores are found to be reduced when rhizospheric soil extracts of am plants are applied; it means the am fungi are able to alter the microbial population and particular functional groups of rhizospheric microorganisms (meyer and linderman 1986; larsen and bodker 2003) . they cause qualitative and quantitative changes in the fungal community by several factors like changed exudation patterns; altered root size and architecture; different physiological and biochemical parameters like sugar, organic acids, and amino acids; and also putative direct am fungal effects (toljander et al. 2007; ahmed et al. 2013; vigo et al. 2000) . fungistatic siderophore (low-molecular-weight chelating agents having higher affinity for ferric ion)-producing microorganisms are found to be crowded in mycorrhiza-infected roots and rhizospheric regions. mycorrhizal plants are to be reported with more actinomycetes and bacterial (gram-positive paenibacillus sp. against phytophthora parasitica) flora antagonistic to soilborne root pathogens (azcon-aguilar and barea 1996; budi et al. 1999 ). apart from providing biochemical and physiological defense strategies, arbuscular mycorrhizal species also exhibit physical barrier of defense by changing the root anatomy, morphology, and even architecture in terms of increased nutrient uptake, meristematic and nuclear activities of root cell, higher rate of growths, and branching patterns (atkinson et al. 1994; gamalero et al. 2010; gutjahr and paszkowski 2013) . thus responses of root morphology as a result from afm colonization seem to depend on plant characters, tap root system, etc. more benefits are seen in tap root system than fibrous root system in terms of gained biomass and nutrient acquisition. though there is a gap of knowledge in how increased root branching caused by mycorrhizal infection help the plant to defend fungal pathogenesis, synergism is seen as something that can balance the suppressed root growth caused by several root pathogens and restore the root health. mycorrhiza-mediated strengthening of the vascular system allows the higher rate of flow of nutrients, increased mechanical strength, and also inactivation of vascular pathogens. in conditions of limited resource such as carbon requirement and space for inoculation, a competition between the symbiotic partner (mycorrhizae) and pathogenic fungi is very common and expected (vos et al. 2014 ). in the direct warfare, mycorrhizae win over the pathogenic one and thus obtain higher amount of nutrients (almost 4-20% of total assimilated carbon by host plant) and occupy large areas of available root cortical cells (jung et al. 2012; vierheilig et al. 2008) . defeating the pathogenic fungi in terms of nutrient uptake and providing a little or no room for infection are probably the mightiest cause of biocontrol ability of am fungus (hammer et al. 2011) . output of am and phytophthora interaction indicates that the pathogen does not penetrate cortical arbuscular cells, suggesting that localized competition for infection site does occur between the pathogenic fungi and the am fungus. not only fungi but also plant-invading nematodes are in the queue for colonization and nutrient uptake (smith 1988) . the infection of southern root-knot nematode (meloidogyne incognita, m. exigua) is reduced when the roots are priorly inoculated with symbiotic partners like in the case of coffee plants also (alban et al. 2013; dos et al. 2010) . reports have suggested that the number of infected sites is reduced within mycorrhizal root system than in the uninoculated one and thus strongly supports the mycorrhizal role as a biofungicide (vigo et al. 2000) . am fungi can help the plant uptake of nutrients like phosphate, nitrogen, minerals, microelements (zinc), and water at a higher rate than the uninfected one (baum et al. 2015; parniske 2008) , and as a result, they are provided with photosynthetic carbon (smith and smith 2011) . the plants capable of absorbing higher amount of nutrients in terms of am fungal association have the potential to tolerate pathogenic infections (karagiannidis et al. 2002) . though the improved nutrition and increased tolerance are not involved in a cause-effect relationship, proofs are there that higher uptake of phosphate results in remarkable reduction in pathogenic infection in mycorrhizal plant but not in non-mycorrhizal plant (bodker et al. 1998) . tomato plants already infected with rhizophagus irregularis are not colonized by the pathogen a. solani, whereas non-mycorrhizal plant is affected by the pathogen (fritz et al. 2006) . mixed action of arbuscular mycorrhizal fungi (amf) glomus intraradices and trichoderma harzianum as a biocontrol agent significantly reduces the damping off disease caused by rhizoctonia solani in the case of tomato seedlings (amer and seud 2008) . in order to combat parasitic (fungal, bacterial, viral, nematoidal, and insectal) infection like mammalian cells, plant cells also develop defense systems that mediate the release of low-molecular-weight and short-lived (generally 72-96 h of existence) antimicrobial compounds or molecules known as the phytoalexins (braga 1991; echiverri et al. 2010; paxton 1980) . these secondary metabolites help the plant to withstand biotic and abiotic stress (grayer and kukubun 2001) . most of them being lipophilic compounds can cross the plasma membrane and act inside the fungal cell causing cytoplasmic granulation of the infecting fungal cells, disorganization of the cellular components, rupture of the plasma membrane, and inhibition of the fungal enzymes and mycelial growth (cavalcanti 2005) . mode of action of phytoalexins against fungal pathogenesis varies from species to species (table 9 .1). metabolism of phytoalexin mediated by fungus involves the tendency for its increased polarity by addition of hydroxyl group (oxygenation), removal of methyl group (demethylation), etc. (jeandet et al. 2014) . muller and borger first enlightened the concept of phytoalexins almost 70 year ago (muller and borger 1940). the first reported case analyzed with the concept of phytoalexin was potato tuber infection by the different strains of causal organism of "late blight of potato," phytophthora infestans. this pathogenic fungus initiated the hypersensitive reactions that lead to the formation of some "plant secondary metabolite" that inhibited further infection of the same plant when infected with another strain of the same genus of phytophthora. muller and his coworker named this "principle" as "phytoalexins" that have protected the plant from secondary infection (deverall 1982) . accumulation of phytoalexins in the green plant tissue clearly indicates the presence of remarkable amount of fungal and bacterial infections in the host tissue (stoessl 1980) . phytoalexins are naturally occurring products secreted and accumulated temporarily by plants in response to pathogenic attack or abiotic stress and agents like heavy metal toxicity, uv radiation, and wounds on tissue (naoumkina et al. 2007 ). the inducer agent may be of two types, elicitor and elicitin. the elicitors are commonly the oligosaccharides from fungal cell origin (like hepatosaccharide from soja cell wall) (sharp et al. pezet and pont (1990) , adrian et al. (1997) , adrian and jeandet (2012) camalexin induction of the fungal programmed cell death (pcd) by apoptotic mechanisms et al. (2011) 1984). the elicitin types of molecules are generally a type of glycoproteins secreted by the fungal cells (cordelier et al. 2003) . reports on detailed investigations about phytoalexins have covered a very few families (leguminosae and solanaceae) of the green world (ingham 1982; kuc 1982) . though investigations on some selected number of species and genera are made from plant families including both monocotyledonous (amaryllidaceae, orchidaceae, poaceae) and dicotyledonous plants (apiaceae, asteraceae, convolvulaceae, chenopodiaceae, euphorbiaceae, linaceae, moraceae, piperaceae, rosaceae, rutaceae) and even gymnospermic taxa (ginkgoaceae) (coxon 1982) , cash crops like members of poaceae (focusing on maize and rice), vitaceae, and malvaceae (cotton) have been studied for their phytoalexin production (schmelz et al. 2014; langcake and pryce 1976; jeandet et al. 2010; sunilkumar et al. 2006) . though till date a lot of researches have already been performed regarding phytoalexins, a natural weapon against mycopathogens, but still to increase the fungitoxic effectivity of these stress metabolites, further advancement in design and genetic control is needed ). phytoalexin synthesis not only is dependent on pathogenic attack but also could be influenced by various abiotic factors such as temperature, humidity, and water availability ( fig. 9.1 ). there are evidences that different parts of the plant like leaves, flowers, stems, seeds, and root tubers are site of phytoalexin biosynthesis (mikkelsen et al. 2003) . different biochemical pathways are used for producing various types of phytoalexins. the three most common pathways include (i) the phenylpropanoic-polymalonic acid pathway, (ii) the methylerythritol phosphate (mep) and geranylgeranyl diphosphate (ggdp) route, and (iii) the indole phytoalexin (ip) pathway (jeandet et al. 2014 ). it is not always obvious that phytoalexins could be categorized not only by their chemical structure or biosynthetic pathway but also by their function and tissue specificity. examples include the occurrence of momilactone a on different plant parts of rice plant (lee et al. 1999; cartwright 1981) . momilactone a is known to be residing in rice husks and rice stems constitutively, but they are also a phytoalexin of rice leaves. further studies by toyomasu and his coworkers conclude that momilactone a is constitutively synthesized and oozed out from root of rice plants. still there is no sufficient data available to consider phytoalexins as ubiquitous throughout the whole plant kingdom. a lot of studies have revealed their complex biochemical synthetic machinery that involves their de novo synthesis, regulation, and mode of action (jeandet et al. 2013 ahuja et al. 2012 . regulatory mechanisms involve defense-related marker genes, calcium sensors, hormone signaling, phosphorylation cascades, and also their antipathogenic activity. there are reports on genetic engineering-mediated manipulation of phytoalexin production and increased disease resistance in the case of plants (delaunois et al. 2009; jeandet et al. 2012 jeandet et al. , 2013 . phytoalexins are secondary or stress metabolites that are produced when the host plant is infected with pathogenic fungus. phytoalexin-mediated defense response includes the expression of lytic enzymes such as chitinases and glucanases and a number of pathogenesis-related (pr) proteins, oxidizing agents, and lignification of cell walls (dixon and lamb 1999) . mode of action of phytoalexin involves the coordinated synergism between several defense factors for the effective inhibition of the fungal pathogen (purkayastha 2017; mansfield 1999) . in the case of sorghum plant, significant infection caused by fusarium proliferatum and fusarium thapsinum stimulates the production of 3-deoxyanthocyanidin, apigeninidin, and luteolinidin and also the concentration of defense-related proteins like peroxidases, β-1,3 glucanases, and chitinases that help to fight the pathogenic infection (huang and backhouse 2004 (koga et al. 1997; fukuta et al. 2007 ). there are several ways of blocking the fungal infection in host plant tissues by phytoalexin-mediated response. that includes inhibition of fungal spore on the leaf surface and inhibition during and after penetration to host cell (usman et al. 2018) . the occurrence of fungal germ tube on the leaf surface and diffusion of fungal metabolites through the leaf cells cause the accumulation of phytoalexins by the underlying cells and provide the first line of induced chemical defense (vanwees et al. 2003) . phytoalexins may be located on papillae or cell walls, thereby producing a localized, fungitoxic barrier to penetration (friend 2016) . examples include occurrence of fungitoxic (against erysiphe graminis) flavonoid (thought to be phytoalexin) on papillae of resistant barley leaves. phytoalexins are known to be solely produced as a result of induction or stimulus by external agents. fruitful evidences could be drawn regarding this fact. induction of disease resistance in plants is developed through the direct and indirect involvement of elicitors. extracts of fungal basidiocarp, essential oils of aromatic plants (walters et al. 2013) , and also synthetic chemicals like aminobutyric acid, salicylic acid, jasmonic acid, and acibenzolar-s-methyl ( (matiello and bonaldo 2013) , hymenolobium petraeum, qualea albiflora, and corymbia citriodora (matiello et al. 2016 ) that act as the elicitors of deoxyanthocyanidins and glyceolin production. homeopathic preparations of species of calcarea (c. citriodora and calcarea carbonica), essential oils of eucalyptus globulus (telaxka et al. 2014; oliveira et al. 2014) , and mild concentrations of salicylic acid (durango et al. 2013 ) are major elicitins of pistain production and accumulation in cotyledons of common bean (phaseolus vulgaris). silicon-mediated enhancement of disease resistance by peroxidase (pox), polyphenol oxidase (ppo), chitinases (chi), β-1,3-glucanases (glu), and phenylalanine ammonia-lyase (pal) is found in the case of leaf spot of cotton plant caused by ramularia areola (curvêlo et al. 2013 ). southern amazonian amphibian family bufonidae represents the true toads, and their cutaneous secretions are of diverse source of bioactive compounds which can be fruitful as new chemical weapons for agrochemical development. use of elicitors in the case of crop protection nowadays is becoming a very popular method of inducing response which are proved to be durable and broad-spectrum disease control mechanism where the plant's own resistance is used. a group of seven brazilian scientists (deice et al. 2019) evaluated the possibilities of methanolic extracts of cutaneous secretions of two species of bufonidae, rhaebo guttatus and rhinella marina, on synthesis of phytoalexins named glyceolin (soybean plant), deoxyanthocyanidins (sorghum plants), and phaseolin (mung plant) in soybean cotyledons, sorghum mesocotyls, and bean hypocotyls, respectively. there is a direct relationship between the phytoalexins production and defense ability of the host plant against the fungal pathogenesis. studies reveal that when the phytoalexin glyceolin is produced in higher amount in the soybean plant (cultivar tmg 132 rr) as a result of methanolic extracts of amphibian's (r. guttatus) cutaneous secretion (at a concentration of 0.2 mg/ml), stimulates the enzyme β-1,3-glucanase that can cause the hydrolysis of the fungal cell wall along with other defense-related enzymes (chitinase) is also produced in higher amount, but when suppression of glyceolin occurs, that particular enzyme is also not produced. there are evidences in the case of glycine max that the effectivity of phytoalexins varies from cultivar to cultivar. application of r. marina (amphibian) methanolic extracts induced glyceolin production in tmg 132 rr and monsoy 8372 cultivars ipro but did not induce tmg 132 rr cultivars to synthesize these defense-related compounds. less toxicity of phytoalexins than chemical fungicides is the reason for their universal acceptance. for over 75 years, phytoalexins have been a detailed area of study for its antimicrobial activity, especially antifungal properties. several investigations include the in vivo bioeffectivity of the phytoalexins against serious plant pathogenic fungi (table 9 .2). phytoalexin synthesizing genes have also been genetically modified to cope up with the pathogenic evolution. still reports are there that include examples of cruciferous phytoalexins detoxification by fungal enzymes (pedras and abdoli 2017) . modification of pathogen to overcome phytoalexinmediated damage includes curved germ tubes as a result of asymmetric growth of the germ tube. phytoalexins are natural products of diverse chemical nature, for example, alkaloids, coumarins, isoflavonoid (coumestans, isoflavans, isoflavones, isoflavanones, pterocarpans, pterocarpenes), lignans, polyacetylenes, pterocarpons (pisatin, phaseolin, glyceollin, medicarpin, and maackiain), terpenes, and non-isoflavonoid compounds (furanoacetylenes and stilbenes) ( fig. 9 .2) (grayer and kokubun 2001) . both in vitro and in vivo fungicidal activity are shown by sakuranetin (rice phytoalexins) against the blast fungus (hasegawa et al. 2014 ). reduction of green mold (caused by penicillium digitatum) infections is achieved by the action of coumarin type of phytoalexin (scopoletin) of orange (sanzani et al. 2014 ). the loss of apples production caused by penicillium expansum and accumulation of patulin is minimized by the action of phenolic phytoalexins like resveratrol, scopoletin, scoparone, and umbelliferone (sanzani et al. 2009 ). in the case of medicago sativa (alfalfa), the isoflavonoid 7-o-methyltransferase provides increased resistance against phoma medicaginis by synthesizing maiackiain (he and dixon 2000) . for soybean plants, transformation of resveratrol to pterostilbene 7, langcake and pryce (1976) , coxon (1980) , and harding and heale (1981) (continued) (zernova et al. 2014) . scientists have proven that not only fungal infection acts as the stimulus for phytoalexin synthesis but also the hormone levels; phosphorylation cascades play a major role in this purpose. cytokinin overexpression in nicotiana tabacum is directly associated with its resistance against p. syringe by higher concentration of capsidiol and scopoletin (grosskinsky et al. 2011 .) the fungitoxicity of the phytoalexin could be enhanced by methylation or presence of electron-attracting groups on aromatic rings that is directly involved in affinity with membrane proteins being an uncoupler of ets system. endophytes are a type of hidden beneficial microorganisms that reside within the host plant causing no visible disease symptoms and syndrome and promote the plant to maintain its existence in typical harsh conditions. sometimes they could be latent pathogens at a very distant path of the host's life cycle but are simply a unique area of research where plant science and their microbial association get new definition. endophytes have been a constant and reliable source of exploration of bioactive compounds, but extensive search has not been performed till date, and that has given the endophyte biologists a great opportunity to search endophytic fungal and actinobacterial flora for the establishment of novel bioactive compounds. selection of plant for endophytic isolation is the most vital part of this study. exploitation of the proper isolates accelerates this search and opens up new angle of research. the search for uncommon products of agrochemical importance is a common demand of todays' world. the safer the antifungal agents become, the more it is well accepted in the scientific community as well as agricultural market. in general, the screening of thousands of natural products ends up giving only one commercial product. so indeed it's a tough job to end the search of new antibiotics with a hopeful result. a total of 6 out of 20 of the popular prescribed medicines are of fungal origin, and it is a fact that 5% of the fungi have been described till date (hawksworth 1991 (hawksworth , 2001 . so fungi serve as a continuous dependable source of new natural products. the intelligent screening procedure includes the selection of fungal flora of endophytic sources to open up the untapped potential of secondary metabolites synthesized by fungi. microorganisms grown in the petri plates or culture broth constitute minimal growth medium needed for their survival. any kind of stress or transfer of microorganisms on selective media acts as a stimulation for production of their secondary bioactive compounds. these secondary metabolites are produced for their survival in odd environments and strictly act as the selection force for the expression of their antimicrobial-producing genes. these crude by-products of microbial cultures are filtered and purified for their industrial, medicinal, and agricultural exploitation. soil microorganisms have been exploited for a long time for production of antibiotics, but microorganisms inhabiting plants are a new source in that respect. plants are selected usually with potent medicinal applications. here the knowledge of ethnobotany and folk taxonomy contributes a lot in this selection procedure. a strong literature survey supports the plant selection. the plants are surface sterilized and plated in nutrient-less solid plates. the fungi emerge out from different explants using the decaying plant parts as their primary growth substance. the isolates are identified by microscopic structures focusing on their conidial morphology, spore sculpturing, and colony characters. confirmatory identification includes 18s rrna analysis. endophytic fungi are tested for their antifungal activity against phytopathogenic fungi by one-to-one inhibition assay or antagonistic test ( fig. 9 .3). two agar portions containing fungal hypha of endophyte and pathogen are placed on opposite sides of the plate. if the growth of pathogenic one is arrested partially or completely, that endophytic isolate is marked as antifungal agent and selected for further studies. another way of screening includes separating the agar plate into two equal halves, and two fungi are placed on two separate sides of the discontinuous plate. this test aims to screen the endophytes that produce volatile antifungals. if the isolate is potent enough to produce volatile organic compounds with fungi static or cidal activity, this will cease the growth of the pathogenic strain. then that isolate would be qualitatively and quantitatively measured for their volatile emissions using gc-ms as the master equipment ( fig. 9 .4). liquid extracts of endophytic fungi are also tested for antifungal potentials by agar well-diffusion method. the fungal extract having antibiotic property shows clear zone of inhibition of growth of the pathogenic fungus surrounding the area of application of that fungal liquid. the potent isolate will be mass cultured, and the bioactive molecules will be extracted using organic solvents like ethyl acetate, ethyl ether, and n-hexane. steps include purification of that fungal extract by column chromatography, detection of the compound by thin-layer chromatography, and analysis of compounds by hplc and mass chromatography. field experiment includes the synergistic effect of a pure compound with mixture of natural compounds, and the effectivity of a newly applied antifungal agent strictly depends on the host plant and pathogenic microorganism's interaction, environmental condition, and development of drug resistance by that organism. application of pellets soaked in fungal extracts also is a method of determination of antifungal activity. secondary metabolites are itself diverse in nature. a variety of bioactive secondary metabolites are produced at significant concentrations by the endophytic microbial flora. the major components include quinones, phenols, phenolic esters, steroids, terpenoids, cytochalasins, benzopyranone, alkaloids, isocoumarins, and chromones. till date, a large number of plants have been studied for their endophytic flora as antifungal agents (table 9 .3). alkaloid was the first ever reported insecticidal bioactive product. cryptocin was isolated from endophyte of tripterygium wilfordii, a plant of celastraceae family. the inner barks of the stem were used as explant, and cryptosporiopsis cf. quercina was isolated as a potent endophyte active against pyricularia oryzae and some other phytopathogenic fungi (li et al. 2000) . colletotrichum sp. produces 6-isoprenylindole-3-corboxylic acid having inhibitory action against phytophthora capsici, a pathogen of cucurbitaceae, fabaceae, and solanaceae, and also other phytopathogens rhizoctonia cerealis and gaeumannomyces graminis var. tritici, a common pathogen of poaceae family . epoxycytochalasin h and cytochalasins n and h were isolated as chloroform and methanolic extracts of (fu et al. 2011) . a lot of endophytes have been explored for their antifungal production, but only a few of them were positive for antifungal metabolites categorizing in alkaloids. the common alkaloids acting as the antifungal agents of endophytic fungal origin are gliotoxin, cryptocanadin, tyrocidine a, fumigaclavine c, fumitremorgin c, 1-n-methyl albonoursin, and phomapsichalasin. the terpenoids, usually called isoprenoids, are large and diverse group of naturally occurring organic compounds derived from terpenes that are multicyclic. sixty percent of all the known natural products are terpenoids in nature. some endophytic fungicidal products are of terpenes by their native chemical structure. endophytic isolates (hormonema sp.) of gymnospermous plant juniperus communis were reported to be antifungal producers of a triterpene glycoside enfumafungin (pelaez et al. 2000) . known antifungal sterols of endophytic origin are 3β-hydroxyergosta-5-ene, 3-oxoergosta-4,6,8,22-tetraene, etc. the sterols are strong inhibitors of helminthosporium sativum (present name: bipolaris sorokiniana), the asexual stage of cochliobolus sativus, a common root rot pathogen of wheat and barley crops which also infects leaf and stems of poaceae plants . sesquiterpenes are reported to be the growth inhibitors of cladosporium phlei (causal organism of leaf spot disease of timothy grass, phleum pratense). this is a unique example where the host plant (phleum pratense) itself harbors the endophyte (epichloe typhina) that inhibits the growth of its leaf spot pathogen (cladosporium phlei). from the point of view of organic chemistry, isocoumarins are defined as the isomer of coumarin where the orientation of the lactone is reversely arranged. zhang and his coworkers in the year 2008 isolated an endophytic fungus named microdochium bolleyi from fagonia cretica (also known as virgin's mantle of zygophyllaceae family), a herb of semiarid regions of gomera. isocoumarins were identified as the active compounds having antifungal activity against microbotryum violaceum (previously known as ustilago violacea), an obligate parasite of basidiomycete group and a common infectant of members of caryophyllaceae causing smut of anther. the four isolated and identified isocoumarins are monocerin, 12-oxo epimers of monocerin, and open-ring derivative compounds of monocerin. the compounds are obtained as mixtures by column chromatography followed by sephadex lh-20 chromatography techniques. preparative tlc further differentiated the four compounds. monocerin and its analogues were previously reported as antifungal compounds from fungal sources of drechslera monoceras, exserohilum monoceras, helminthosporium monoceras, exserohilum turcum, and fusarium larvarum (aldridge and turner 1970; robeson and strobel 1982; grove and pople 1979; claydon et al. 1979) . these secondary metabolites act on pathogens by interfering stages of divisional phases of cell cycle. the second isocoumarin was colorless oil. the third and fourth one are represented by the empirical formula of c 16 h 20 o 7 and c 16 h 22 o 7 . the fourth one is structurally correlated to fusarentin 6,7-dimethyl ether. both the compounds are of heptaketide in their origin, and it is revealed that fusarentins are the probable precursors of the active compounds monocerins (scott et al. 1984; axford et al. 2004 ). dihydroisocoumarins, mellein (an isocoumarin derivative), (r)-7-hydroxymellein, and fonsecinone were reported from species of xylaria (endophyte of piper aduncum), pezicula, penicillium (alibertia macrophylla), and aspergillus (cynodon dactylon), respectively (oliveira et al. 2011; schulz et al. 1995; song et al. 2004 ). phenols (popularly known as phenolics) represent a class of chemical compounds characterized with a hydroxyl group attached to an aromatic hydrocarbon group. phenol (or carbolic acid) is a colorless crystalline solid, aromatic compound having benzene rings. they are predominantly found in the plant kingdom as a response to stress and are of utmost importance. endophytic culture extracts are also known to be rich sources of phenolics; usually they are directly proportional to the antioxidative property of any fungal isolate, but in some particular cases, they are characterized with their antifungal potentials against phytopathogenic fungus. usually the liquid culture extracts of the endophytic isolates are subjected to solvent extraction using ethyl acetate, n-hexane, ethyl ether, etc. those organic solvents are believed to extract the phenolics from the water-based culture broth. those extracted compounds are further screened for their antifungal efficiency. ethyl acetate extracts of endophytic phoma sp. are reported to contain tetralone metabolites (derivatives of α-tetralone, 3,6,7-trihydroxy-α-tetralone) inhibiting the growth of two common broad phytopathogenic fungus fusarium oxysporum and rhizoctonia solani. griseofulvin is known to be the first antifungal compound isolated from penicillium griseofulvum. later it is isolated from several species of fungi including endophytic penicillium canescens and xylaria sp. (member of xylariaceae family). griseofulvin from endophytic p. canescens of popular chinese medicinal plant polygonatum cyrtonema (polygonaceae) showed strong inhibitory effectivity against phytopathogenic botrytis cinerea, sclerotinia sclerotiorum, colletotrichum orbiculare, and didymella bryoniae . other than penicillium, endophytic xylaria sp. isolated as an endophyte of abies holophylla yields griseofulvin and dechlorogriseofulvin for in vitro and in vivo effectivity against pathogenic magnaporthe grisea, corticium sasakii, blumeria graminis (park et al. 2005) . the ascomycete fungus pestalotiopsis is known to be a common plant pathogen but also has been reported many times because of their endophytic existence in the host plants. the two common species pestalotiopsis microspora (host: tropical plant terminalia morobensis) and p. fici are reported to be producing antifungal metabolites isopestacin and pestalofones d-e (harper et al. 2003; liu et al. 2009a, b) . chlorogenic acid and colletotric acids are antifungal phenolics of colletotrichum gloeosporioides and sordariomycetes sp., respectively zou et al. 2000) . they were isolated from medicinal plants of china (artemisia mongolica and eucommia ulmoides) and effective against fungi imperfecti helminthosporium sativum. orcinol is used for the production of a dye called orcein used randomly for the staining of cells and chromosomes. orcinol is popularly known for its antifungal activity too and has been isolated as a product of endophytic origin of penicillium sp. from alibertia macrophylla (a plant of rubiaceae) showing bioactivity against cladosporium cladosporioides and cladosporium sphaerospermum (oliveira et al. 2014) . endophytic phomopsis sp., dothiorella sp., and diaporthe sp. have also been tested for their antifungal production and antifungal compounds that were detected (brady et al. 2000; xu et al. 2004; huang et al. 2008 ). volatile organic compounds (vocs) are said to be a type of organic low-molecularweight carbon-containing small compounds (up to c20) that have a high vapor pressure with low molecular mass (100-500 daltons) at room temperature. the high vapor pressure results from a low boiling point of that chemical compound, which causes a huge quantity of molecules to evaporate from the liquid, solid, or semisolid form of the compound and gets released into the surrounding environment. the endophytes are unique in their volatile emissions. the term mycofumigation that is very much popular with the treatment of agricultural phytopathogens is actually the output of vocs that originated from endophytic isolates. the first ever reported volatile antibiotic producer was muscodor albus (xylariaceae family), an endophyte of guazuma ulmifolia (a plant of sterculiaceae family collected from tropical forest of sw ecuador), isolated by gary strobel and his co-workers ). the major compounds isolated by gcms are known to be involved in antifungal, antibacterial activity. compounds include butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, n-(1-methylpropyl); azulene and naphthalene derivatives; caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl-derivatives. these compounds were tested against a number of phytopathogenic fungi (botrytis cinerea, mycosphaerella fijiensis, pythium ultimum, phytophthora cinnamomi) showing partial or complete death or growth inhibition of those pathogens after 2 or 4 days of incubation. muscodor albus was reported from a diverse type of host plants, i.e., myristica fragrans, terminalia prostrata, cinnamomum zeylanicum, and ginkgo biloba, by several workers (worapong et al. 2001; sopalun et al. 2003; ezra and strobel 2003; mercier et al. 2004; ezra et al. 2004a, b; atmosukarto et al. 2005; lacey and naven 2006; lacey et al. 2009; strobel et al. 2007; banerjee et al. 2010a, b; corcuff et al. 2011; alpha et al. 2015) . the mycofumigants are effective against pathogen fusarium culmorum, causal agent of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals. sexual stage (teleomorph) of glomerella cingulata, a fungus of glomerellaceae, is a potent pathogen causing anthracnose-like symptoms of water-soaked, sunken spots and necrotic lesions on fruits of forest trees. this phytopathogen is strictly inhibited by the volatile emissions of this novel endophyte. banerjee et al. (2010a, b) first reported muscodor albus strain gba from the usa as an isolate of ginkgo biloba (first isolate of m. albus from g. biloba) and tested the biological efficacy of its volatile mixtures against agricultural pathogens and also evaluated its promises to be used as a commercial mycofumigant agent for controlling the fungal diseases in storage fruits and vegetables, that is, agricultural productions and during food transportation. the strain gba in comparison to other strains of muscodor e6 and cz620 completely inhibits and potentially kills the member of phycomycetes, pythium ultimum after 2 days of exposure of the mixture of volatiles. the organic compounds include alcohols, acids, esters, ketones, and lipids as their active components. 1-butanol, 3-methyl-, acetate was found in significant quantities. vitrine, a terpenoid, was first isolated from muscodor albus strain gab. the volatile mixture is artificially produced by the mixture of the pure compounds, and that mixture is again checked for antifungal activity. a positive mycocidal or mycostatic effect similar to the effect of endophyte's volatile emission will confirm establishment of the endophyte and its mixture as the biocontrol or antifungal agent. myrothecium inundatum, an endophyte of herbaceous acalypha indica (euphorbiacea member collected from northeastern part of india), produces unique mixture of volatile components having 3-octanone, 3-octanol, 7-octen-4-ol, sesquiterpenes, organic acids, methyl esters, naphthalene, 2-octanoic acid, heptanoic acid, etc. this endophyte produces foam in its liquid culture predominant with long-chain carbon compounds like octane, 1,4-cyclohexadiene, 1-methyl-and cyclohexane, and 1-ethylpropyl. (meshram et al. 2013 (meshram et al. , 2017 suwannarach et al. 2015; saxena et al. 2015; suwannarach et al. 2010 suwannarach et al. , 2012 suwannarach et al. , 2013 kudalkar et al. 2012; mitchell et al. 2010; worapong et al. 2002; daisy et al. 2002; siri-udom et al. 2016 postharvest fungal disease is one of the prime causes of agricultural loss of crops. use of biological agent to minimize this loss is one of the vital targets of agriculturalists, horticulturalists, and plant biologists. several chemical agents have been already tested for practical applications, but endophytes are less explored organisms in this arena. volatiles from endophytic source open up new scope of utilization of unique mixtures of chemicals to be used as mycofumigant agents. a large number of endophytes have already been screened for their postharvest disease management ability (table 9 .4). the volatiles of the endophyte could be considered as the natural fungicides. muscodor vitigenus, an endophyte of hevea brasiliensis, was analyzed in gc-ms for their volatile production. the isolates produce a unique mixture of myroxylon balsamum 1, 4-cyclohexadiene, 1-methyl-; 1,4-pentadiene and cyclohexene, 1-methyl-4-(1methylethenyl)-; alkyl alcohols starting with 1-butanol-3-methyl, 1-propanol-2-methyl, cinnamomum bejolghota. this isolate was known to produce azulene, a new compound detected first from any muscodor species. this species was tested in vitro and in vivo for antifungal activity against a common worldwide devastating pathogen rhizoctonia solani (causal agent of damping off). the vocs produced by this fungi include (s)-(+)-5-methyl-1-heptanol; ethyl acetate; propanoic acid, 2-methyl-, methyl ester; cis-2,4-dimethylthiane; s,s-dioxide; cyclopentane; butanoic acid, 2-methyl-, methyl ester; 1-butanol, 3-methyl-, acetate; β-humulene; azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl)-; 1s-(1.α., 7.α., 8a.β); and eudosma-4(14),11-diene 1,1,1,5,7,7,7-heptamethyl-3,3-bis(trimethylsiloxy) tetrasiloxane. rhizoctonia solani-infected seedlings were treated with volatile mixtures to assess the mycofumigation property. in vivo experiment was conducted on four seedlings of bird pepper, bush bean, garden pea, and tomato. it was concluded that 30 gm of muscodor cinnamomi prepared on rye grain solid media is the minimum dose required for inhibition of rhizoctonia infection and total control and elimination of damping off symptoms. muscodor cinnamomi-infected soil does not show any seed germination inhibition in comparison to rhizoctonia solani-infected soil. so it is a type of pioneer study of using endophytic species as potent agents of fumigation and biocontrol. candida intermedia strain c410 (saccharomycetaceae) was isolated as an endophyte of strawberry (fragaria ananassa), and the volatile emission was known to be a mixture of 49 organic compounds including esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, and aldehydes of which 1, 3, 5, 7-cyclooctatetraene and 3-methyl-1-butanol were the most dominant (huang et al. 2011 ). volatiles of strawberry endophyte were itself useful as postharvest control agent for the host plant against botrytis fruit rot. other compounds include 1,3,5,7-cyclooctatetraene; 3-methyl-1-butanol; 2-nonanone; pentanoic acid, 4-methyl-, ethyl ester; 3-methyl-1-butanol, acetate; acetic acid, pentyl ester; and hexanoic acid, ethyl ester that were found to be extremely inhibitory to conidial germination (reproductive growth) and also vegetative (mycelial) proliferation of b. cinerea. when the fruits are exposed to c. intermedia synthetic volatiles or itself to the fungus, the incidence of botrytis fruit rot reduces significantly. strawberry fruits inoculated directly with the endophyte also remain disease-free. so mixtures of candida intermedia c410, the unique natural products, are useful as mycofumigation technique or for postharvest disease management by biological control policies. tangerine fruit (citrus tangerine), the commercial citrus crop of northern thailand, faces huge postharvest losses due to pathogenesis of green mold (penicillium digitatum). the pathogen is the prime cause of worldwide deterioration of tangerine fruits by mycopathogenesis. out of 32 detected compounds, the most predominant were 2-methylpropanoic acid and 3-methylbutan-1-ol. other compounds include carbitol, octanoyl chloride, azulene, 3-methylhexane, 2-methylpropan-1-ol, 2,3-butanediol, caryophyllene, 2-methylbutyric acid, ethyl 2-hydroxyproponate, etc. the pathogen was treated both in vitro and in vivo for their inhibition by endophytic volatile components. in both cases, pathogen growth was restricted. during transportation of the fruits, fungus causes huge crop loss by infecting the fruits; when fruits were inoculated with 30 gm of rye grain culture of m. suthepensis (1 month old), the disease development is ceased. so it is a classic example of mycofumigation by the biocontrol agent of tangerine fruit for the control of rot lesions caused by p. digitatum infection. the in vivo application requires the proper surface sterilization (using sodium hypochlorite) of the targeted parts where the inoculation is going to be done, for example, fruit, stem, root, and leaf. usually infection on fruits for assessing the biocontrol potential is the most common and popular method. the seeds will be washed in distilled water, and using sterile needle, uniformly the whole area would be done, and the whole area would be infected or inoculated with the endophytic liquid extracts containing spore suspensions. muscodor albus vocs are potent enough to cause a significant reduction of in vitro spore germination of the tilletia species t. horrida, t. indica, and t. tritici. endophytic nodulisporium spp., trichoderma spp., phomopsis spp., and oxyporus latemarginatus are reported to produce vocs that inhibit mycelial growth of phytopathogenic fungi (lee et al. 2009; park et al. 2010; ajith and lakhsmidevi 2010; amin et al. 2010) . black sigatoka disease (also known as leaf spot or black leaf streak disease) of banana (musa paradisiaca) is caused by mycosphaerella fijiensis (ascomycete fungus). this phytopathogen is inhibited by the volatile emissions of muscodor sutura, an endophytic isolate of prestonia trifidi. the volatiles are effective also against ceratocystis ulmi, the causal agent of dutch elm disease of american elm (ulmus americana). the volatile mixtures include thujopsene, chamigrene, isocaryophyllene, and butanoic acid, 2-methyl-that are potent inhibitors of the common anthracnose pathogen of cucumber, muskmelon, and watermelon (members of cucurbits), colletotrichum lagenarium. so this unique endophyte and its chemical mixtures are potent mycofumigants and ensure crop protections against destructive pathogens like c. ulmi and c. lagenarium, sclerotinia sclerotiorum (causing white mold, cottony rot, water soft rot, stem rot, drop, crown rot, and blossom blight diseases of the host), and also phytophthora palmivora (oomycete fungi), the causal agent of bud root of palms and areca nut predominantly occurring in regions of south india (kudalkar et al. 2012) . liarzi and his coworkers tested the biological control efficacy of the endophytic daldinia cf. concentrica, isolated from olive tree (olea europaea l.) of israel against 18 phytopathogens, and the unique mixtures of 27 volatile were effective against the phytopathogenic mycelial growths. the mixtures include a variety of organic compounds: 3-methyl-1-butanol, 2-methyl-1-butanol, 1-methyl-1,3-cyclohexadiene, 1-methyl-1,4-cyclohexadiene, 4-heptanone, isoamyl acetate, 4-heptyn-2-ol, 2-octenal, octanal, β-elemene, α-guaiene, β-selinene, α-selinene, α-bulnesene, germacrene a, etc. the unique mixtures having broad-spectrum antifungal property could be used for fumigation for eliminating the pathogenic infections of aspergillus niger (mold-causing organism on fruits of economic importance). so the endophytic d. cf. concentrica opens up opportunities for fungal disease control in food and agricultural industries (liarzi et al. 2016) . nodulisporium sp. strain gs4d2ii1 (hypoxylon anthochroum) and hypoxylon anthochroum strain blaci are potent enough to be used as biopesticide against fusarium oxysporum, a common contaminant of solanum lycopersicum var. cerasiforme (cherry tomato) causing a great percentage of crop loss globally. six vocs of alcohols' mixture, phenylethyl alcohol, 2-methyl-1-butanol, 3-methyl-1-butanol, eucalyptol, ocimene, and terpinolene, were detected and applied together with synergistic effect and individually both in vitro and in vivo. inoculation of pathogen on the cherry tomato fruits yields significant reduction in fusarium contamination. both agar dilution techniques and gas test were done to assess the in vitro antifungal activity, and the endophytic volatile mixtures were effective in both the cases. volatiles kill the pathogens probably by interfering cell membrane permeability, hyphal morphology, and respiratory activity of the pathogenic fusarium oxysporum. so it is a great opportunity to use the unique mixture of volatile organic compounds of the endophytic isolate to reduce the crop loss caused by the pathogenic infection on the commercially valuable plant of cherry tomato worldwide. endophytic phoma sp. (didymellaceae) and phomopsis sp. (valsaceae) were isolated from larrea tridentata and odontoglossum sp. singh et al. 2011) . the volatiles detected are effective against phytopathogens verticillum sp., ceratocystis sp., cercospora sp., sclerotinia sp. sclerotinia sp., and botrytis sp. algae are diverse group of autotrophs and the leading producers of o 2 in the ecosystem. they range from prokaryotic unicellular to eukaryotic complex multicellular forms involved in the marine and terrestrial food chain. antifungal activity of the seaweed (members of phaeophyceae and rhodophyceae) is a major weapon for natural fungicides along with their antibacterial, anti-protozoan, and antiviral activities. algal seaweeds are potent holders of large number of secondary metabolites including phenolics, terpenes, alkaloids, and lectins which are not directly involved in photosynthesis and reproduction and thus fall under the category of secondary metabolites. they are common antimicrobial of algal origin that act on the target organisms by altering the microbial cell permeability accompanied with the loss of internal macromolecules or sometimes interfere with the membrane function causing cellular disintegrity ultimately leading to cell death (abu-ghannam and rajauria 2013). several studies include antifungal activity of algal members against human pathogens; a very few studies include their efficacy against plant pathogens (cheung et al. 2014; singh et al. 2007; stirk et al. 2007; padmakumar and ayyakkannu 1997; ismail et al. 2014; genovese et al. 2013; lopes et al. 2015 ). padmakumar and ayyakkannu tested 80 species of algae against a variety of bacterial and fungal pathogens. out of the all screened organisms, 70% exhibited antibacterial efficiency, and only 27.5% inhibited fungal growth. polysaccharides found in the cell wall and deposited in terms of storage food from red and brown algal sources include ulvans (obtained from ulva sp.), alginates and fucans (from fucus sp.), laminarin (laminaria sp.), and carrageenans that can induce defense responses in plants against phytopathogens by pathogen-associated molecular patterns (maps) and are capable of inducing plant resistance (vera et al. 2011) . polysaccharides stimulate regular cellular changes associated with pathogen perception and defense activation by change in ca 2+ concentration and burst due to oxidative stress activation of salicylate, ethylene, and jasmonate biosynthetic pathways and by activating pathogenesis-related proteins (prps) (jaulneau et al. 2010; zhao et al. 2012) . as a result of the depolymerization of the polysaccharides, the obtained oligosaccharides induce protection against a variety of fungal, viral, and bacterial diseases by accumulation of the antimicrobial compounds in the cell. algal polysaccharides as an alternative weapon over the synthetic agricultural drugs for controlling plant disease have been widely studied (stadnik and freitas 2014; hahn et al. 2008 ). brown algae laminaria digitata, a genus of phaeophyceae, is commonly called seaweeds and known to be the potent producers of kelp, an iodine-rich substance needed for the normal functioning of thyroid gland. laminaria produces laminarin, glucan polysaccharide-containing 1,3-linked β-d-glucose moiety, a reserve food material found on the vacuoles of the vegetative cells of this genus. β-glucans are involved as a major part of daily diet and obtained from the brands of common cereals. they are involved in the defense responses of agricultural crops like tomato (lycopersicon esculentum), eggplant (solanum melongena), pepper (piper nigrum), watermelon (citrullus lanatus), grape (vitis vinifera), apple (malus sp.), and pear (pyrus communis). elicitation of defense response by laminarin against causal agents of gray mold (botrytis cinerea) and downy mildew (plasmopara viticola) in grapevine plants remarkably suppresses their infection up to 55% and 75%, respectively (copping et al. 2004 ). so, natural product from brown algae laminaria sp. known as laminarin or laminaran can act as the biofungicide or biocontrol compounds. use of laminarin significantly reduces the mycelial growth and aflatoxin production in aspergillus flavus and ensures its use as a fungicide (liangbin et al. 2012) . the advantage of using laminarin over other products is that as it breaks down finally to glucose molecules, it has no maximum residue limit (mrl) on the plant treated with this product. so, there is no need of preharvest interval constraint. this has been a prime cause why laminarin has substituted five popular fungicides involved in the treatment of apple scab (venturia inaequalis) in france (mery et al. 2013 ). this phyto-pharmaceutical is used widely in france and some countries of europe in the name of vacciplant (major active constituent is laminarin). laminarin has broad-spectrum applicability on fire blight of apples and pears in greece, france, belgium, switzerland, portugal, and also morocco. it is effective for apple scab disease in france and belgium and for curing storage diseases of apples caused by gloeosporium sp. in belgium. laminarin comes out as a fungicide of natural origin after being eligible in 33 tests between 2001 and 2011 in several parts of europe, for example, france, belgium, italy, and poland, on natural contamination of orchards on several sensitive strains of scab fungus including golden delicious, golden smoothie, read cheaf, galaxy, gala, and pink lady. laminarin is applied widely against secondary scab (to minimize secondary scab during summer and up to harvest) as a result of its uniqueness in its mode of action. it does not involve cell death of the host plant or hypersensitivity induction in the host organism but rather stimulates plants' natural resistance (klarzynski et al. 2000) . aziz et al. (2003) reported its effectiveness in tobacco plants, wheat, strawberries, apples, and vines. the application of laminarin and alginate reduced the development of wilt symptoms caused by verticillium dahliae on olive twigs, stimulating its phenolic metabolism (salah et al. 2018) . moreover, alginates reduced pathogen growth in vitro. laminarin induces the release of h 2 o 2 in cells of tobacco plants and leads to the increase in pal activity (phenylalanine ammonia-lyase) and causes the accumulation of pr-1, pr-2 (glucanase), pr-3 (chitinase), and pr-5. concerning red algae polysaccharides, carrageenans induced protection against a broad range of pathogens such as tobacco mosaic virus (tmv), b. cinerea, and e. carotovora on tobacco (vera et al. 2011) . again on tobacco, mercier et al. (2001) showed that carrageenan infiltrated the leaves and increased the expression of genes coding for a sesquiterpene cyclase involved in the synthesis of the antimicrobial terpenoid capsidiol, pr-3 proteins (basic chitinases), and proteinase inhibitor with antipathogenic activity. an adequate percentage of growth and spore germination inhibition of botrytis cinerea was mediated by the hexane extracts of laminaria digitata and undaria pinnatifida. porphyra umbilicalis, laverbread, is an edible seaweed (corato et al. 2017) . other than b-glucan polysaccharides (laminarin) of laminaria, other secondary metabolites (phenols, terpenes) of phaeophycean algae (sargassum sp.) showed effectivity against common pathogens fusarium solani, rhizoctonia solani, aspergillus spp., fusarium oxysporum, penicillium spp., and botrytis cinerea (khallil et al. 2015; ibraheem et al. 2017; mabrouk et al. 1985; liu et al. 2014 ). cyanophycean blue-green algae are abundant all over the world and ranging from pond ecosystem to oceanic system. though they have been reported to produce a large number of toxins and involved in death and disease of cattle and human being, they are of serious interest from the point of view of natural fungicidal products. drawing the similarities with bacteria, they are characterized with a mucilaginous or gelatinous sheath composed of polysaccharides which are the weapon against fungal pathogenesis. cyanobacterial polysaccharides (pol) show higher disease resistance against b. cinerea when they are applied on the intact fruit (preharvest conditions when fruit is attached to the plant) rather than the fruit detached (postharvest conditions) from the plant (zheng et al. 2011; feliziani et al. 2015; yao and tian 2005) . polysaccharides are involved in elicitation as elicitors for development of local and systemic disease resistance and expression of defense enzyme synthesis, for example, chitinases and glucanases that are involved directly in antifungal responses (paulert et al. 2009; reymond and farmer 1998; sharma et al. 2014) . water extracts of common bga anabaena sp., ecklonia sp. (common edible marine algae of japan and korea), and corallina sp. (hard seaweed of corallinaceae family) exhibit antifungal activity against podosphaera xanthii (causal agent of powdery mildew of cucurbits) on zucchini plant, cucurbita pepo, of cucurbitaceae (roberti et al. 2015 (roberti et al. , 2016 . in the recent past, fungi inhibitory ability of algal members has been reported by several workers (righini et al. 2018; corato et al. 2017; khallil et al. 2015; ibraheem et al. 2017) . in vitro growth inhibition of aspergillus oryzae and penicillium notatum has been seen by cyanophycean anabaena laxa (frankmölle et al. 1992) . devastating plant pathogens pythium sp., fusarium sp., and rhizoctonia sp. were restricted by extracts of anabaena sp. (moon et al. 1992; manjunath et al. 2010) . the use of bga extract as the growth inhibitor of pathogenic chaetomium globosum, cunninghamella blakesleeana, aspergillus oryzae, rhizoctonia solani, fusarium sp., pythium sp., and sclerotinia sclerotiorum is reported. the extracts of phormidium fragile and nostoc muscorum (rizk 2006 bryophytes, the simplest member of the broad umbrella of embryophyta, are situated between algae and pteridophytes, are known to be plant amphibians growing in the marshy or shady habitat, and require water for their fertilization and for the perfect swimming motility of their sperms. they have been evaluated for their antimicrobial activity for a long time. it has been proved that these cryptograms are rich source of bioactive secondary metabolites and can easily be exploited as an alternative source of fungicidal compounds. as they grow in marshy habitats and can protect themselves from biotic (ultraviolet rays, heat stress, and predation) and abiotic stress (fungal or bacterial attack), they are store house of diverse bioactive chemicals (xie and lou 2008) . members of hepaticopsida and mosses (the evolved members of bryophytes) are known to possess antifungal activity and are rich source of flavonoids, terpenoids, bibenzyls, and fatty acids of therapeutic importance (krzaczkowski et al. 2008) . bryophytes are known to possess antibiotic property (banerjee and sen 1979; banerjee 2000; singh et al. 2007; shirzadian et al. 2009; savaroglu et al. 2011) . their antibiosis has been evaluated against a large number of plant and human pathogenic fungus (mekuria et al. 2005) . antimicrobial compounds from bryophyte can cure the problems of conventional antibiotic resistance (vanden bossche et al. 1998) . the antifungal efficacy is tested by disc diffusion assay and microdilution method ( fig. 9 .5). different concentrations of the extracts are prepared and checked for their antifungal efficacy against phytopathogenic fungi. they may be fungicidal or fungistatic in nature, interfering at cellular, genetic level and creating blockage at metabolic pathways. extracts are made on several organic solvents or water extractions and also mixture of one or two organic solvents. the solvents popularly used are ethanol, methanol, chloroform, ether, dimethyl sulfoxide (dmso), acetone, chloroform, and hexane (table 9 .5). sporophytes and gametophytes of different bryophytes at different stages of growth and at a different amount are first surface sterilized and then crushed on the organic solvents and used as antifungals in vitro against the fungal pathogens (wolters 1964 (sabovljevic et al. 2011; pejin et al. 2012; veljic et al. 2009; gahotri and chaturvedi 2011; alam et al. 2011; deora and jain 2008; dey and de 2011; deora and suhalka 2017) . actinobacteria are a group of gram-positive filamentous bacteria that are called as the branched bacteria or ray fungi (from greek actis, ray beam, and mykes, fungus) and are characterized with the high g + c content occurring in mostly aerobic conditions but occasionally being anaerobes (ludwig and klenk 2005; olanrewaju and babalola 2019). their morphology varies from forming branching filaments or mycelial growth to external spores. they are ubiquitous in nature ranging their distribution from soil and human microbiota to plant and even animal kingdom. they are predominant in aquatic as well as terrestrial ecosystem playing a major part in mineralization and recycling of organic matters leading to soil formation (sharma et al. 2014 ). they are not only free-living members of the ecosystem but also a plant symbiont or endophyte, contributing to the plants' survival in extreme conditions and pursuing several bioactivities in vivo and in vitro. actinomycetes produce a diverse range of secondary metabolites, for example, antibiotics, antitumor, insectrepellent, and immunosuppressive agents, and plant growth-promoting regulators (pgprs) that are of immense pharmaceutical and agricultural importance. they are the prime producers of diverse antibiotics after the landmark discovery of penicillin in the year 1928. the single genus of streptomyces sp. itself produces 76% of the total known bioactive (10,000 are produced by actinobacteria out of 23,000 produced by microorganisms, almost 45%) compounds from actinobacterial and riccia gangetica curvularia lunata deora and suhalka (2017) , guhil (2015, 2016) bacterial source (berdy 2012) and is known to be the prime organism in the pharmaceutical world. they are equally profitable when isolated from plant source and designated as endophytic actinomycetes. so exploitation of the actinobacterial novel bioactive compounds both from endophytic and non-endophytic source is the ultimate way to fight against human and plant diseases. here we focus only on actinobacterial compounds' antifungal activity and role in plant protection from deadly diseases caused by severe phytopathogens leading to irreparable crop loss and economic breakdown of agricultural sectors. actinomycetes from soil source are selected based on the enrichment culture technique and are plated on selective media for isolation. antifungal agents, for example, nystatin and cycloheximide, are supplemented for the inhibition of fungal contamination. for isolation of endophytic actinobacteria from plant source, plants are first selected and surface sterilized for the elimination of the epiphytic contaminants and finally plated on the selective growth media like starch casein nitrate agar (scna), chitin-vitamin b, tap water yeast extract agar (twya), soybean, humic acid-vitamin b (hv), yeast extract casamino acid (yeca), modified gausse, and glycine-glycerol (ivantiskaya et al. 1978; küster 1959; küster and williams 1964; williams and davies 1965; hayakawa and nonomura 1987; crawford et al. 1993) . international streptomyces project (isp) medium is also popular media used for isolation, and they are supplemented with amino acids (l-asparagine for isp 5, tryptone for isp 1), inorganic trace salts, starch or carbohydrate sources (malt extract for isp 4), and agar as solidifying agent. ph set at near to optimum or slightly basic is mandatory for proper isolation techniques using isp medium. the actinomycete isolates are grown in solid or liquid medium for their antifungal bioactivity detection. antagonistic activities of the potent isolates are tested by growing them on both sides of the fungal hyphae, and isolate having anti-phytopathogenic activity will inhibit the growth of the pathogens. actinobacterial aqueous-or solvent-based extracts will be evaluated for either fungistatic or fungicidal activity by agar welldiffusion techniques. soluble bioactive compounds of antifungal importance will be extracted using wide range of organic solvents followed by purification by column and thin-layer chromatographic techniques. hplc analysis will be the most useful method for the detection of the purity of the compound, and further nmr studies are needed for the proper identification of the bioactive compound. cell line studies are made with the coupling of bioinformatics tools for the proper knowledge about their mode of action. actinobacteria can be a part of plant as endophyte, rhizospheric soil as symbiont for plant growth-promoting substance producer, and organisms' normal microbial flora as gut microorganism. so they are ubiquitous in their distribution. out of several biologically potent compound produced from the actinobacterial source, antibiotics are the major contribution of these microorganisms toward human civilization. all the known antibiotics (blasticidin, mildiomycin, natamycin, validamycin, kasugamycin) are of actinobacterial (most of them are the streptomyces sp.) source showing protective activity for the plants against agricultural fungal pathogens (tables 9.6 and 9.7). as human are dependent completely on nature and more particularly natural components of agricultural origin and importance, dependence on agricultural crops is of a known fact. but the problem arises when the crops are affected most by the fungal pathogens leading to huge crop loss, and thus the search for novel antibiotics is on, and the search has shifted to actinobacterial source, and endophyte plays an important role in this respect. there are significant reports of antifungal compounds from bacterial origin, but now the focus has shifted to microbes of endophytic origin (table 9 .8). till date, a huge number of antibiotics are already reported and have minimized the crop loss to a notable amount ( fig. 9.6 ). antibiotics and other antifungal compounds include munumbicins a, b, c, d, e-4, and e-5, vanillin, saadamycin, 5,7-dimethoxy-4-p-methoxyphenyl coumarin, coronamycin, and fistupyrone isolated from different strains of streptomyces (shan et al. 2018; costa et al. 2013; igarashi et al. 2002; tian et al. 2004; zin et al. 2007 ) and are protecting a large number of cereals and other important cash crops from being affected by these common contaminants. endophytic actinobacteria directly counteract with fungal plant pathogens not only by producing bioactive compounds but also by enhancing the plant's growth through the production of plant growth promoters and making the plant less susceptible to pathogenic invasion. they are efficient agent of reducing the symptoms that arise due to exposure to environmental stress (shimizu 2011) . enhanced production of indole acetic acid (iaa) was mediated by streptomyces sp. (isolated from centella asiatica) and nocardiopsis sp. (dochhil et al. 2013; shutsrirung et al. 2014; gangwar et al. 2014) . experimental trials on cucumber indicate positive result as the isolates actinoplanes campanulatus, micromonospora chalcea, and streptomyces spiralis enhanced plant growth and improved yield conditions (el-tarabily et al. 2010) . other than auxin, auxin-like similarly functioning molecules named as pteridic acids a and b are found to be inducers of adventitious root proliferation in kidney bean plants at very minute concentrations of 1 mm (igarashi et al. 2002) . chitin is a major fungal cell wall polysaccharide (the second most abundant polysaccharide in nature after cellulose) component and is the first line of defense of fungal cells. actinobacteria antagonize the fungal cell by producing chitinases (an enzyme capable of hydrolyzing fungal cell wall) and break the glycosidic bonds in chitin and lead to the death of the pathogenic cell. endophytic kitasatosporia sp. (isolate of catharanthus roseus) and kibdelosporangium sp. (isolate of achillea fragrantissima) are reported to be chitinase producers (el-shatoury et al. 2009; mini priya 2012) . actinoplanes missouriensis isolated from lupinus sp., a member of fabaceae family, produces chitinase causing hyphal cell lysis and reducing the conidial germination rate and protects the plant from pathogenic attack of plectosporium tabacinum, the causal agent of lupin root rot in egypt (el-tarabily 2003; el-tarabily and sivasithamparam 2006) . siderophores are soluble, small, high-affinity iron carriers produced by bacterial or fungal members and are involved in the transportation of iron (fe 3+ ) across the cell membrane. they have caught sudden attention due to their involvement in plant growth promotion as well as antagonistic ability against phytopathogens (cao et al. 2005; tan et al. 2006; rungin et al. 2012) . endophytic actinobacteria from aloe vera, mentha arvensis, and ocimum sanctum are known to be producers of hydroxymate type and catechol type of siderophores, and the isolate saccharopolyspora o9 is known to be the potent inhibitor howell and stipanovic (1980) , homma et al. (1989) , thomashow et al. (2002) and smith et al. (1993) harpin proteins (erwinia amylovora), trade name: harpin αβ (proact) induction of systemic acquired resistance (sar) and less susceptibility to fungal and bacterial disease wei et al. (1992) strobilurin and oudemansin (members of basidiomycete grows on dead wood) commercial synthetic analogues: azoxystrobin and kresoxim-methyl of alternaria brassicicola, botrytis cinerea, and fusarium oxysporum (gangwar et al. 2014; el-shatoury et al. 2009 ). endophytic isolates of cucumis sativus (cucumber), identified as actinoplanes campanulatus, micromonospora chalcae, and streptomyces spiralis, are reported to control the growth and development of damping off, crown rot, and root rot pathogen pythium aphanidermatum. they are known to promote plant growth and to protect seedlings and mature plants. a novel bioactive compound identified as 6-prenylindole was isolated from endophytic streptomyces sp. showing strong antifungal activity against a broad range of phytopathogens: alternaria brassicicola and fusarium oxysporum (igarashi 2004) . another new prenylated indole derivative from endophytic actinobacterial source inhibited the growth of colletotrichum orbiculare, phytophthora capsici, corynespora cassiicola, and fusarium oxysporum (zhang et al. 2014) . naphthomycins a and k isolated from streptomyces sp. cs have antifungal activity against penicillium avellaneum shen 2003, 2007) . biocontrol ability of fistupyrone has made it a useful tool to minimize the crop loss of brassica due to black leaf spot disease caused by alternaria brassicicola (igarashi 2004) . interest on actinomycetes of endophytic origin as an alternative tool for antifungal agent is increasing day by day (table 9 .9). since the beginning of human civilization, whenever human race has faced any turbulence in its path of existence, they have rushed to their green friends, trees, for the ultimate solution. search for bioactive products of medical importance has been a thirst area from time immemorial. whether it is a concern of human or plant health, trees have given answers in all aspects. in the recent past, phytopathogenic infection has pushed the agricultural productive parameters to a real challenge, and plant extracts in its crude and purified form are applied as biocontrol methods (table 9 .10). the existing synthetic chemicals are facing problem of immediate or delayed drug resistance and also issues of nephrotoxicity (the gold standard; amphotericin b), biomagnification, or quality assurance of the food products and thus are inconsistent in their business (goa and barradell 1995; cuenca-estrella et al. 2000) . so green plant extracts are the novel, safest, and the best effective treatment tool in this arena. plants are mysterious in their chemical nature and in respect to their secondary metabolite production. the faith is consistent on green plants due to the fact that plants protect themselves from fungal or bacterial diseases specially for the taxa that occur in marshy shady or water-logged or stress conditions (gurgel et al. 2005) . so the search is primarily made on the wild native taxa or invasive species that have higher potential of antimicrobial production. the knowledge of ethnobotany comes in this context, and tribal people are imitated for the gathering of crude knowledge. the problem of fungal pathogenesis is mainly faced by plants of economic importance, that is, cash crops. a single event of pathogenic attack can affect seriously the demand and supply ratio; thus the sustainability is lost, and restoring the good health of crops is a basic need of agricultural sectors but in an efficient way not hampering the soil health, ecosystem characters, and human health and also should be budget friendly. the search is strictly focused on plants of ethnomedicinal importance as history indicates the ability of medicinal plant extracts in human and animal mycoses and antifungal ability (mathias-mundy and mccorkle secondary metabolites are plants' best weapon against phytopathogenic invasion. several plant extracts have been assessed for their antifungal activity against a variety of phytopathogens of serious agricultural threats (table 9 .11). the metabolites are divided into terpenoids, saponins, phenolic compounds, flavones, flavonoids, flavonols, alkaloids, and coumarins (table 9 .12). plant extracts are primarily tested for antifungal efficacy and further are purified by solvent extraction and chromatographic procedures leading to discovery of new antifungal agents. terpenoids, also called as isoprenoids (under the chemical subclass of prenyllipids), are known to be the oldest group of widespread molecular compounds produced by plants. scher et al. (2004) reported a variety of six sesquiterpenes of antifungal importance against the causal organisms of bunch rot (botrytis cinerea) on grapes, scab of cucurbits (cladosporium cucumerinum), potato blight (phytophthora infestans), rice blast (pyricularia oryzae), and blotch of wheat (septoria tritici). sesquiterpene isolated from polygonum punctatum (dotted knotweed of knotweed family polygonaceae) named after the chemical polygodial is an effective control agent of zygosaccharomyces bailii (a common food spoilage yeast). scab of cucurbits is a common and devastating fungal pathogenic disease in agricultural fields, and this disease is to some extent prevented by the use of clerodane diterpenes extracted from detarium microcarpum, a plant of leguminosae family (cavin et al. 2006) . skaltsa (2000) reported fungi inhibitory (cunninghamella echinulata) activity of costunolide and eudesmane derivatives isolated from centaurea plants. other than terpenes, saponins (triterpene ad steroidal saponins) are also effective antifungals reported from plant sources. tea is one of the most vital cash crops in terms of foreign money earning and the most popular beverage having antioxidative properties. pathogenic infection by pestalotia longiseta causes a huge loss of tea production. nagata et al. in the year 1985 isolated triterpenoid saponins camelids i and ii from the leaves of camellia japonica (japanese camellia) that inhibited the tea pathogen p. longiseta. cucurbitacins i, a, b, q, and e isolated from cucurbitacins (ecballium elaterium) have antifungal activity against botrytis cinerea (har-nun and meyer 1990) . phenolics are odorous compounds having antifungal compounds and are also responsible for the plant pigment production. phenolics cover a large number of chemical compounds, for example, alkylated phenols, anthraquinones, coumarins, phenolic acid, phenols, phenylpropanoids, quinines, xanthones, hydroxycinnamic acid, p-coumaric acid, ferulic acid, and chlorogenic acid. phenol derivatives like crassinervic acid (p. crassinervium), aduncumene (p. aduncum), hostmaniane (p. hostamannianum), and gaudichaudanic acid (p. gaudichaudianum) are effective against strawberry blossom blight pathogen cladosporium cladosporioides (lago et al. 2004 ). 3-acetyl-4-acetoxyacetophenone showed antifungal activity against spendley et al. (1982) , potterat et al. (1987) , martson et al. (1988) , marston et al. (1993) , viturro et al. (2004) , cavin et al. (2006a) , and dhatwalia et al. (2009) pinocembrin from leaves of populus deltoides (salicaceae) shain and miller (1982) , hoof et al. (2008) cladosporium fruit and leaf rot and bitter root (cladosporium gloeosporioides) long-chain alcohol from peels of young fruit of persea americana from lauraceae, methylripariochromene a from roots of eupatorium riparium (asteraceae) prusky et al. (1983) , ratnayake bandara et al. (1992) pine needle pathogen (dothistroma pini) stearic acid from needles of pinus radiata (pinaceae) franich et al. (1983) pathogen of corn, sorghum, apple (helminthosporium carbonum) luteone and wighteone from leaf surface of lupinus albus (leguminosae) ingham et al. (1983) black and brown spot of banana (colletotrichum musae) dopamine from unripe banana fruit (musa sp.) muirhead and deverall (1984) powdery mildew of grains (erysiphe graminis) gramine from leaves of hordeum vulgare (poaceae) wippich and wink (1985) leaf spot, rots, and blights (alternaria alternata), disease of cereal (penicillium verrucosum) alizarin and emodin from root of rubia tinctorum of rubiaceae, alkylated phenols of peel and pulp of mangifera indica (anacardiaceae) cojocaru et al. (1986) , manojlovic et al. (2005) maize rot (fusarium moniliforme), epidemic outbreak of glume and kernel discoloration (curvularia lunata) flavan-4-ols of root bark of sorghum cultivars of poaceae jambunathan et al. (1986) (continued) kobayashi et al. (1987) , endo et al. (1990) , cho et al. (1998) blue mold of tobacco (peronospora tabacina) diterpenoids from nicotiana tabacum of solanaceae reuveni et al. (1987) leaf and fruit pathogen (cladosporium cladosporioides) canaliculatol from bark of stemonoporus canaliculatus and long-chain alcohol from persea americana, phenylethanone from euodia lunuankenda, sinharine and methylsinharine from glycosmis cyanocarpa, illukumbin from glycosmis mauritiana (rutaceae), phenylethanone from euodia lunuankenda (lauraceae), benzoquinone from croton lacciferus (euphorbiaceae) bokel et al. (1988) , ratnayake bandara and wimalasiri (1988) , kumar et al. (1990) , greger et al. (1992) , pacher et al. (2001) , springob and kutchan (2009) miles et al. (1991) , miles et al. (1993) , lee et al. (2003) , deng and nicholson (2005) and yoganandam et al. (2009) (continued) sclerotinia sp. phenolic structures when contain a carbonyl group are known to be flavones, and the addition of an extra 3-hydroxyl group indicates flavonol. flavonoids are also known to hydroxylated phenolics but occurring as a c6-c3 unit linked to aromatic ring. not only plant samples directly but also plant derivatives like porpolis (galangin isolated from the bee glue or resinous mixture produced as a result of the mixture of tree buds, sap, botanical extracts, and bee exudates) are shown to be antifungal against green rot or mold of tangerine (pathogens penicillium digitatum, p. italicum) and also control postharvest disease of cereal grains, legumes, and tree nuts caused by a. flavus (afolayan and meyer 1997) . flavones (6,7,4′-trihydroxy-3,5′dimethoxyflavone, 5,5′-dihydroxy-8,2′,4′-trimethoxyflavone) from artemisia giraldi are effective against a. flavus infections (cowan 1999) . leaf wax of arrabidaea brachypoda (brazilian medicinal plant from bignoniaceae) contains herger et al. (1988) and abdu-allah and elyousr (2017) cassia tora (dealcoholized extract of leaves) mukherjee et al. (1996) thymol, carvacrol, citronellol, geraniol, citral, perillyl, menthol, eugenol, 1,8cirsiliol, cirsimaritin, and hispidulin and is showed to be effective against cladosporium sphaerospermum (alcerito et al. 2002) . galeotti et al. (2008) against fusarium oxysporum f. sp. dianthi (galeotti et al. 2008) . fusarium culmorum, a serous pathogen of seedling blight, foot rot, ear blight, stalk rot, and common rot of cereals and grasses, is found to be inhibited by six commercial coumarins: bergapten, herniarin, umbelliferone, xanthotoxin, and scopoletin. tithonia diversifolia, the source of tithoniamarin, is effective against the anther smut fungus microbotryum violaceum, earlier known as ustilago violacea (yemele-bouberte et al. 2006) . berberine and jatrorrhizine (alkaloids) are isolated from mahonia aquifolium (a plant of berberidaceae family commonly called as oregon grape and native to western north america) and are effective against human pathogenic candida species. pathogens of mango (c. gloeosporioides), anthracnose of lupin species, postbloom fruit drop of citrus, valencia and navel oranges in florida (caused by c. acutatum), and strawberry (caused by colletotrichum fragariae) are inhibited by findersine, anhydroevoxine, and haplamine (cantrell et al. 2005) . roots of cyathobasis fruticulosa are source of beta-carboline, tryptamine, and phenylethylamine-derived alkaloids and are antifungal in nature (bahceevli et al. 2005 ). essential oils (eos) of aromatic and medicinal plant origin are reported to possess antifungal properties and are of wide spectrum in their application for the control of agricultural pathogen (table 9 .13). eos are mainly categorized under the plants' secondary metabolites and may fall under the category of terpenes, ketones, esters, aromatic phenols, ethers, alcohols, oxides, etc. (fig. 9.7) . they act by inhibiting the fungal hyphal growth either by accumulating in the fungal cell membrane or by crossing the cell membrane and entering into the eukaryotic cell. being lipophilic in their chemical nature, they can easily cross the cell and interrupt in sterol biosynthesis leading to growth retardation and finally cell death. as sterols are the maintenance, compounds of cellular integrity treatment with eo cause fungal cell death. metabolic processes like respiration, replication, transcription, and translation are inhibited. membrane permeability is drastically changed as they cause swelling and disruption of protein-lipid-protein membrane. leakage of useful ions like ca 2+ and k + causes cell death. thymol, carvacrol, eugenol, and related phenolic compounds cause h + and k + leakage and water imbalance and deplete intracellular high-energy molecule (atp). essential oils are extracted from almost every parts of a plant, for example, roots, fruits, barks, twigs, leaves, seeds, and flowers, by several extraction procedures that include hydro and steam distillation, cold pressing, and zataria multiflora lamiaceae fermentation. the antifungal efficacy is checked by direct contact of the essential oil components and fungal hypha and poison food method, following micro or broth dilution techniques, or in vivo fumigation assay is also performed in case of field trials. essential oils from leaves of chenopodium ambrosioides, a member of amaranthaceae family, are effective against storage fungi aspergillus flavus, a. glaucus, a. niger, a. oryzae, colletotrichum gloeosporioides, c. musae, fusarium oxysporum, and fusarium semitectum (jardim et al. 2008) . lemongrass oil from cymbopogon citratus and cymbopogon martini are potent inhibitors of botrytis cinerea, rhizoctonia solani, aspergillus tamari, a. fumigatus, and a. conicus (tzortzakis and economakis 2007; mishra et al. 2015) . the members of lamiaceae family are well known for their pungent odor and are tested for their antifungal activity by agar and broth dilution methods (roby et al. 2013; omidbeygi et al. 2007 ). essential oils extracted from laurus nobilis, syzygium aromaticum, and origanum vulgare are effective antifungal compounds against two pathogens of rice, fusarium culmorum and fusarium verticillioides (rosello et al. 2015) . essential oils from cymbopogon exhibited antifungal activities against rot molds (soundharrajan et al. 2003) . antifungal activities of peppermint and sweet basil were tested against plant pathogenic fungi s. sclerotiorum, rhizopus stolonifer, and mucor sp. (edris and farrag 2003) . antifungal activity of β-dolabrin, γ-thujaplicin, and 4-acetyltropolone was tested against pythium aphanidermatum ifo 32440 (morita et al. 2004) . boyraz and ozcan (2006) tested the antifungal activity of the essential oils isolated from wild turkish summer savory (satureja hortensis). essential oils (carvacrol, thymol, p-cymene) extracted from origanum acutidens are effective against phytopathogens. growth of a. humicola, colletotrichum gloeosporioides, rhizoctonia solani, and phytophthora cactorum was inhibited by the essential oil of asarum heterotropoides var. mandshuricum (dan et al. 2010) . though there are several reports of essential oils being potent anti-phytopathogenic (penicillium purpurogenum, rhizopus stolonifer, spondylocladium austral, penicillium digitatum, penicillium luteum, monilinia laxa, curvularia lunata, etc.) in nature, still there are some problems regarding their maximum use and optimum effectivity. that includes their volatile natures, requirement of close systems, and degradation of eos by oxidation due to presence of extreme amount of hydrogenated compounds (kim et al. 2003 ). we are nourished by mother nature. so it is our prime duty to keep up the normal equilibrium of natural parameters. but in a way to seek solutions, some steps taken toward success may have negative impact on our environment. to fight against the fungal pathogens for the ensuring of better crop productivity, use of chemical fungicide is just another example of that fact. but we must emphasize on products from direct natural origin over the chemically synthesized one. natural products are the best weapon to fight fungal pathogenic diseases on economically important crop species. they are less toxic, stable, and of no side effects when used in crop fields. the crying need of modern era is obtaining pathogen-free crop species in one hand and assurance of environmental sustainability on the other. fungal and bacterial products are already used in large scales followed by the plants' secondary metabolites. phytoalexins as internal molecules are the plants' own defense system. the detailed biochemical analysis of the phytoalexins and study of their regulatory mechanisms are opening up new horizons for universal use of phytoalexin inducing elicitors as plant defense enhancers. mycorrhizae provide the basic line of physical barrier against pathogenic invasion, and reports include their ability to enhance plant growth, thus making the plant nonsusceptible to fungal attack. endophyte on the other hand can enhance the plants' defense system by direct incorporation and open up popular angles of green immunization or plant vaccination. researches on these fields are still scanty, but in the near future, they could lead to the ultimate solution of fungal pathogenic crop loss. effect of certain plant extracts and fungicides against powdery mildew disease of grapevines in upper egypt antimicrobial activity of compounds isolated from algae flavonoid and other constituents of bauhinia manca effects of resveratrol on the ultrastructure of botrytis cinerea conidia and biological significance in plant/pathogen interactions biological activity of resveratrol, a stilbenic compound from grapevines, against botrytis cinerea, the causal agent for gray mold the antimicrobial activity of 3, 5, 7-trihydroxyflavone isolated from the shoots of helichrysum aureonitens extraction and identification of bioactive compounds (eicosane and dibutyl phthalate) produced by streptomyces strain kx852460 for the biological control of rhizoctonia solani ag-3 strain kx852461 to control target spot disease in tobacco leaf 2-9 phytoalexins in defense against pathogens effect of volatile and non-volatile compounds from trichoderma spp. against colletotrichum capsici incitant of anthracnose on bell peppers in vitro antifungal efficacies of aqueous extract of dumortiera hirsuta (swaegr.) nees against sporulation and growth of postharvest phytopathogenic fungi evaluation of streptomyces griseorubens e44g for the biocontrol of fusarium oxysporum f. sp. lycopersici: ultrastructural and cytochemical investigations interactions between a root-knot nematode (meloidogyne exigua) and arbuscular mycorrhizae in coffee plant development (coffea arabica) foliar epicuticular wax of arrabidaea brachypoda: flavonoids and antifungal activity metabolites of helminthosporium monoceras: structures of monocerin and related benzopyrans trans-trans-3, 11-tridecadiene5, 7, 9-triyne-1,2-diol, an antifungal polyacetylene from diseased safflower (carthamus tinctorius) mycofumigation by the volatile organic compound-producing fungus muscodor albus induces bacterial cell death through dna damage mycorrhizal fungi and trichoderma harzianum as biocontrol agents for suppression of rhizoctonia solani damping off disease of tomato effect of volatile metabolites of trichoderma species against seven fungal plant pathogens in vitro production of gliotoxin on natural substrates by trichoderma virens studies on antagonistic effect against plant pathogenic fungi from endophytic fungi isolated from houttuynia cordata thunb. and screening for siderophore and indole-3-acetic acid production effect of mushroom extracts in the induction of phytoalexins and in the control of soy oidium in a greenhouse impact of mycorrhizal colonisation on root architecture, root longevity and the formation of growth regulators isolation and characterization of muscodor albus i-41.3s, a volatile antibiotic producing fungus influence of seed priming on the development of pearl millet downy mildew (sclerospora graminicola) synthesis and incorporation of the first polyketide synthase free intermediate in monocerin biosynthesis laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and plasmopara viticola streptomyces sanglieri which colonised and enhanced the growth of elaeis guineensis jacq. seedlings was antagonistic to ganoderma boninense in in vitro studies current status of biological control of plant diseases using antagonistic organisms in india status and prospects for enhancing the uptake of antagonistic organisms for nematode management in india alkaloids and aromatics of cyathobasis fruticulosa (bunge) aellen antimicrobial activities of bryophytes a review antibiotic activity of bryophytes an endophytic myrothecium inundatum producing volatile organic compounds muscodor albus strain gba, an endophytic fungus of ginkgo biloba from united states of america, produces volatile antimicrobials increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: a review seaweed polysaccharides as bio-elicitors of natural defenses in olive trees against verticillium wilt of olive thoughts and facts about antibiotics: where we are now and where we are heading in vitro screening of bryophytes for antimicrobial activity effect of phosphate and the arbuscular mycorrhizal fungus glomus intraradices on disease severity of root rot of peas (pisum sativum) caused by aphanomyces euteiches canaliculatol, an antifungal resveratrol trimer from stemonoporous canaliculatus induction and identification of sativan and vestitol as two phytoalexins from lotus corniculatus the cytosporones, new octaketide antibiotics isolated from an endophytic fungus phytoalexins induction in rubiaceae the camalexins: new phytoalexins produced in the leaves of camelina sativa (cruciferae) anti-fungal effects of cocoa tannin on the witches' broom pathogen crinipellis pernicious applied and environmental microbiology the chemical composition, antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with thai orchids isolation and identification of antifungal and antialgal alkaloids from haplophyllum sieversii isolation and characterization of endophytic streptomyces antagonists of fusarium wilt pathogen from surface sterilized banana roots isolation and characterization of two phytoalexins from rice as momilactones a and b munumbicins, wide-spectrum antibiotics produced by streptomyces nrrl 30562, endophytic on kennedia nigriscans munumbicins e-4 and e-5: novel broad-spectrum antibiotics from streptomyces nrrl 3052 aspectos bioquímicos e moleculares da resistência induzida bioactive diterpenes from the fruits of detarium microcarpum in vitro evaluation of fungicides, plant extracts and biocontrol agents against brown leaf spot of paddy bioactive diterpenes from the fruits of detarium microcarpum crop diseases and their management. phi learning private limited antifungal activity and action mechanism of ginger oleoresin against pestalotiopsis microspora isolated from chinese olive fruits studies on a chlorogenic acid-producing endophytic fungi isolated from eucommia ulmoides oliver antifungal activity of cinnamaldehyde and eugenol congeners against wood-rot fungi antifungal and antiviral products of marine organisms cytotoxic and antifungal triterpene glycosides from the patagonian sea cucumber hemoiedema spectabilis antimicrobial activity of 4-hydroxybenzoic acid and trans 4-hydroxycinnamic acid isolated and identified from rice hull diversity and antifungal activity of fungal endophytes of asparagus racemosus willd insecticidal secondary metabolic products from the entomogenous fungus fusarium larvarum 12-heptadecenyl)-resorcinol, the major component of the antifungal activity in the peel of mango fruit antifungal activity of neo-clerodane diterpenoids from scutellaria the manual of biocontrol agents effect of water activity on the production of volatile organic compounds by muscodor albus and their effect on three pathogens in stored potato biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by phytophthora megasperma glycoprotein elicitin biological control of phytopathogenic fungi by endophytic actinomycetes isolated from maize (zea mays l) plant products as antimicrobial agents identification of three hydroxyflavan phytoalexins from daffodil bulbs phytoalexins from other plant families isolation and characterization of actinomycete antagonists of a fungal root pathogen susceptibility of fluconazole-resistant clinical isolates of candida spp. to echinocandin ly303366, itraconazole and amphotericin b biochemical defense mechanisms in cotton plants against ramularia leaf spot mediated by silicon in vitro antifungal activity of 2-(3,4-dimethyl-2,5-dihydro-1h-pyrrol-2-yl)-1-methylethyl pentanoate, a dihydro -pyrrole derivative phytoalexin accumulation in tissues of brassica napus inoculated with leptosphaeria maculans naphthalene, an insect repellent, is produced by muscodor vitigenus, a novel endophytic fungus activities of essential oils from asarum heterotropoides var. mandshuricum against five phytopathogens antagonist actinomycetes metabolites against plant pathogens fungi of agricultural importance induction of phytoalexins and proteins related to pathogenesis in plants treated with extracts of cutaneous secretions of southern amazonian bufonidae amphibians natural products in crop protection bioassay-guided isolation of allelochemicals from avena sativa l.: allelopathic potential of flavone c-glycosides antifungal activity of crude extracts from brown and red seaweeds by a supercritical carbon dioxide technique against fruit postharvest fungal diseases rhizospheric streptomycetes as potential biocontrol agents of fusarium and armillaria pine rot and as pgpr for pinus taeda geldanamycin, a new antibiotic induction of fusarium solani mutants insensitive to tomatine, their pathogenicity and aggressiveness to tomato fruits and pea plants molecular engineering of resveratrol in plants antifungal properties of surangin b, a coumarin from mammea longifolia phytochemical analysis and antifungal activity of moss bryum cellulare against some phytopathological fungi studies on antifungal potential of bryum cellulare against spore germination of fungus curvularia lunata in vitro antifungal activity of plagiochasma appendiculatum against alternaria solani evaluation of bryophyte for green fungicides as alternative treatment to control plant pathogen oxidative ring contraction of the phytoalexin cyclobrassinin: a way to brassilexin brassilexin, a novel sulphur-containing phytoalexin from brassica juncea l., (cruciferae) secondary metabolites production by actinomycetes and their antifungal activity antifungal bryophytes: a possible role against human pathogens and in plant protection isolation, characterization and antimicrobial activity at diverse dilution of wheat puroindoline protein free fatty acid accumulation and quality loss of stored soybean seeds invaded by aspergillus ruber molecular communication in interactions between plants and microbial pathogens seed germination enhancing activity of endophytic streptomyces isolated from indigenous ethno-medicinal plant centella asiatica interactions between an arbuscular mycorrhizal fungus (scutellospora heterogama) and the root-knot nematode (meloidogyne incognita) on sweet passion fruit (passiflora alata) application of plant extracts as inducers to challenge leaf rust of wheat inhibitory effect and mechanism of tagetes erecta l. fungicide on fusarium oxysporum f evaluating novel microbe amended composts as biocontrol agents in tomato biological activity summary for cocoa (theobroma cacao l.) effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean phenylphenalenone phytoalexins, will they be a new type of fungicide? antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plant pathogenic fungi from the vapor phase production and genetic improvement of a novel antimycotic agent, saadamycin, against dermatophytes and other clinical fungi from endophytic streptomyces sp. hedaya48 antimicrobial activities of actinomycetes inhabiting achillea fragrantissima (family: compositae) an endophytic chitinase-producing isolate of actinoplanes missouriensis, with potential for biological control of root rot of lupine caused by plectosporium tabacinum performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of pythium aphanidermatum, a pathogen of cucumber under commercial field production conditions in the united arab emirates nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters structures of antifungal diarylheptenones, gingerenones a, b, c and isogingerenone b, isolated from the rhizomes of zingiber officinale coronamycins, peptide antibiotics produced by a verticillate streptomyces sp. (msu-2110) endophytic on monstera sp antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus xylaria sp. strain pb3f3 isolated from haematoxylum brasiletto new endophytic isolates of muscodor albus, a volatileantibiotic-producing fungus effect of substrate on the bioactivity of volatile antimicrobials produced by muscodor albus antifungal volatile organic compounds from the endophyte nodulisporium sp. strain gs4d2ii1a: a qualitative change in the intraspecific and interspecific interactions with pythium aphanidermatum preharvest treatments with chitosan and other alternatives to conventional fungicides to control postharvest decay of strawberry fungistatic effects of pinus radiata needle epicuticular fatty and resin acids on dothistroma pini blue-green alga anabaena laxa. i isolation and biological properties plant phenolics, lignification arbuscular mycorrhiza reduces susceptibility of tomato to alternaria solani antifungal metabolites from phomopsis sp. by254, an endophytic fungus in gossypium hirsutum comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilactones a and b antifungal and antibacterial potential of methanol and chloroform extracts of marchantia polymorpha l flavonoids from carnation (dianthus caryophyllus) and their antifungal activity interactions between a fluorescent pseudomonad, an arbuscular mycorrhizal fungus and a hypo virulent isolate of rhizoctonia solani affect plant growth and root architecture of tomato plants diversity and biopotential of endophytic actinomycetes from three medicinal plants in india the diversity, plant growth promoting and antimicrobial activities of endophytic actinomycetes isolated from emblica officinalis gaertn agriculture and bioactives: achieving both crop yield and phytochemicals isolation and characterization of endophytic actinomycetes from mangrove plant for antimicrobial activity two phytoalexins from sugar beet (beta vulgaris) leaves lass-florl c (2013) the mediterranean red alga asparagopsis taxiformis has antifungal activity against aspergillus species introduction of some new endophytic bacteria from bacillus and streptomyces genera as successful biocontrol agents against sclerotium rolfsii fluconazole: an update of its pharmacodynamic and pharmacokinetic properties and therapeutic use in major superficial and systemic mycoses in immunocompromised patients plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants sulfur containing cinnamides with antifungal activity from glycosmis cyanocarpa phytoalexin emit indolstruktur aus kohlrabi (brassica oleracea var. gongylodes) cytokinins mediate resistance against pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling metabolic products of fusarium larvarum fuckel. the fusarentins and the absolute configuration of monocerin potential of horsetail (equisetum sp.) preparations in the synthesis of defense metabolites in soy (glycine max l.) cotyledons and the effect on the growth of rhizoctonia solani kuhn, in vitro comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization chemical composition, antifungal and antitumor properties of ether extracts of scapania verrucosa heeg. and its endophytic fungus chaetomium fusiformis in vitro antifungal activity of dragon's blood from croton urucurana against dermatophytes multiple control levels of root system remodelling in arbuscular mycorrhizal symbiosis antifungal effect of five aqueous plant extracts on mycelial growth of penicillium expansum isolated from rotted yam tubers in storage alterations in root exudation of intercropped tomato mediated by the arbuscular mycorrhizal fungus glomus mosseae and the soil borne pathogen fusarium oxysporum f.sp. lycopersici host-pathogen interactions: xix. the endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans tit for tat? a mycorrhizal fungus accumulates phosphorus under low plant carbon availability the accumulation of inhibitory compounds in the induced resistance response of carrot root slices to botrytis cinerea cucurbitacins protect cucumber tissue against infection by botrytis cinerea pestacin: a 1,3-dihydro isobenzofuran from pestalotiopsis microspora possessing antioxidant and antimycotic activities the effect of post-infectional potato tuber metabolites and surfactants on zoospores of oomycetes the isolation of xanthoxylin from the bark of phytophthora and hendersonula-infected citrus lemon and its fungitoxic effect analysis on blast fungus-responsive characters of a flavonoid phytoalexin sakuranetin; accumulation in infected rice leaves, antifungal activity and detoxification by fungus the fungal dimension of biodiversity: magnitude, significance, and conservation the magnitude of fungal diversity: the 1±5 million species estimate revisited efficacy of artificial humic acid is a selective nutrient in hv agar used for the isolation of actinomycetes genetic manipulation of isoflavone 7-o-methyltransferase enhances biosynthesis of 4′-o-methylated isoflavonoid phytoalexins and disease resistance in alfalfa die wirkung von auszigen aus dem sachalin-staudenknoterich reynoutria sachalinensis (f. schmidt) nakai gegen plizkrankheiten, insbesondere echte mehltauplize production of antibiotics by pseudomonas cepacia as an agent for biological control of soilborne plant pathogens screening of poplar trees for antibacterial, antifungal and antiviral activity suppression of pythium ultimum induced damping -off of cotton seedlings by pseudomonas fluorescens and its antibiotic, pyoluterin effects of fusarium species on defence mechanisms in sorghum seedlings control of post harvest botrytis fruit rot of strawberry by volatile organic compounds of candida intermedia biodiversity of endophytic fungi associated with 29 traditional chinese medicinal plants novel acidic sesquiterpenoids constitute a dominant class of pathogeninduced phytoalexins in maize antimicrobial activities of some brown macroalgae against some soil borne plant pathogens and in vivo management of solanum melongena root diseases antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of markhamia tomentosa screening of novel bioactive compounds from plant-associated actinomycetes isolation of actinomycetes from live plants and evaluation of anti phytopathogenic activity of their metabolites isolation and identification of endophytic actinomycetes and their antifungal activity phytoalexins from the leguminosae fungitoxic isoflavones from lupinus albus and other lupinus species the polyoxins: pyrimidine nucleoside peptide antibiotics inhibiting fungal cell wall biosynthesis validoxylamines as trehalase inhibitors suppression of damping-off disease in host plants by the rhizoplane bacterium lysobacter sp. strain sb-k88 is linked to plant colonization and antibiosis against soilborne peronosporomycetes antibacterial and antifungal activities of brown alga zonaria tournefortii (jv lamouroux) direct isolation of micromonospora on selective media with gentamicin polyphenol concentrations in grain, leaf and callus tissues of mold-susceptible and mold-resistant sorghum cultivars composition and antifungal activity of the essential oil of the brazilian chenopodium ambrosioides l ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway modulation of phytoalexin biosynthesis in engineered plants for disease resistance metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol biosynthesis, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants deciphering the role of phytoalexins in plant-microorganism interactions and human health phytoalexins produced in the leaves of capsella bursapastoris (shepherd's purse) xanthotoxin: a phytoalexin of pastinaca sativa root mycorrhiza-induced resistance and priming of plant defenses isolation of endophytic actinomycetes from catharanthus roseus (l.) g. don leaves and their antimicrobial activity. iranian effect of verticillium wilt (verticillium dahliae kleb.) and mycorrhiza (glomus mosseae) on root colonization, growth and nutrient uptake in tomato and eggplant seedlings the possible association of phytoalexins with resistant gene expression in flax to melampsora lini antifungal potential in crude extracts of five selected brown seaweeds collected from the western libya coast in vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of cinnamomum-, syzygium-and cymbopogon-species against aspergillus fumigatus and trichophyton rubrum insecticidal activities of aromatic plant extracts and essential oils against sitophilus oryzae and callosobruchus chinensis anthraquinones isolated from cassia tora (leguminosae) seed show an antifungal property against phytopathogenic fungi recent development in the use of blasticidin s, a microbial fungicide, as a useful reagent in molecular biology linear b-1,3 glucans are elicitors of defense responses in tobacco-france induce systemic resistance and promotion of plant growth by bacillus spp antifungal activity of pisiferic acid derivatives against the rice blast fungus functional moiety for the antifungal activity of phytocassane e, a diterpene phytoalexin from rice phytoalexin induction in the sapwood of plants of the maloideae (rosaceae): biphenyls or dibenzofurans structural and functional characterization of gene clusters directing non-ribosomal synthesis of bioactive lipopeptides in bacillus amyloliquefaciens strain fzb42 evaluation of essential oils and their components for broad-spectrum antifungal activity and control of late leaf spot and crown rot diseases in peanut bryophytes, a potent source of drugs for tomorrow's medicine? a plant extract acts both as a resistance inducer and an oomycide against grapevine downy mildew outline of a comparative study of criteria used in characterization of the actinomycetes selection of media for isolation of streptomycetes phytoalexins from the solanaceae muscodor sutura, a novel endophytic fungus with volatile antibiotic activities endophytic fungi isolated from oil-seed crop jatropha curcas produces oil and exhibit antifungal activity identification of antifungal principle in the solvent extract of an endophytic fungus chaetomium globosum from withania somnifera isolation, characterization, and bioactivity of endophytic fungi of tylophora indica an endophytic nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential two fungicidal phenylethanones from euodia lunu-ankenda root bark antifungal and insect antifeedant 2-phenylethanol esters from the liverwort balantiopsis cancellata from chile efficacy of the biofumigant fungus muscodor albus (ascomycota: xylariales) for control of codling moth (lepidoptera: tortricidae) in simulated storage conditions the potential of the fungus, muscodor albus, as a microbial control agent of potato tuber moth (lepidoptera: gelechiidae) in stored potatoes benzoic acid derivatives from piper species and their fungitoxic activity against cladosporium cladosporioides and c. sphaerospermum the production of resveratrol by vitis vinifera and other members of the vitaceae as a response to infection or injury interactions between pea root-inhabiting fungi examined using signature fatty acids mycosubtilin overproduction by bacillus subtilis bbg100 enhances the organism's antagonistic and biocontrol activities momilactones a and b in rice straw harvested at different growth stages antibacterial activity of oriental medicinal plant extracts toward helicobacter pylori mycofumigation with oxyporus latemarginatus ef069 for control of postharvest apple decay and rhizoctonia root rot on moth orchid screening for endophytic fungi with antitumour and antifungal activities from chinese medicinal plants cryptocin, a potent tetramic acid antimycotic from the endophytic fungus cryptosporiopsis cf. quercina antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata effect of laminarin on aspergillus flavus growth and aflatoxin production use of the endophytic fungus daldinia cf. concentrica and its volatiles as bio-control agents mycorrhizae and plan health isoquinoline alkaloids from macleaya cordata active against plant microbial pathogens pestalofones a-e, bioactive cyclohexanone derivatives from the plant endophytic fungus pestalotiopsis fici bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, a marine algae derived bromophenol, inhibits the growth of botrytis cinerea and interacts with dna molecules screening of a marine algal extract for antifungal activities a new macrolide antibiotic with antitumor activity produced by streptomyces sp. cs, a commensal microbe of maytenus hookeri a novel ansamycin, naphthomycin k from streptomyces sp new bioactive metabolites produced by colletotrichum sp., an endophytic fungus in artemisia annua overview: a phylogenetic backbone and taxonomic framework for prokaryotic systematics inhibitory activities of some marine algae on aflatoxin accumulation naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus edenia gomezpompae plant disease control: understanding the roles of toxins and phytoalexins in host-pathogen interaction plant extracts, bau-biofungicide and fungicides in controlling some important diseases of rice cv. brri dhan40 biocontrol potential of cyanobacterial metabolites against damping off disease caused by pythium aphanidermatum in solanaceous vegetables bioprospecting for endophytes from australian flora with mycofumigation potential antifungal activity of rubia tinctorum, rhamnus frangula and caloplaca cerina antimicrobial compounds and resistance: the role of phytoalexins and antianticipins fungicidal and molluscicidal saponins from dolichos kilimandscharicus xanthones from polygala nyikensis ethnoveterinary medicine and development: a review of the literature elicitor activity of phytoalexins in soy and sorghum by extracts and tinctures of medicinal plant species synthesis of phytoalexins in soy and sorghum by extracts and tinctures from three forest species structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort herberta adunca endophytic fungi associated with monarda citriodora, an aromatic and medicinal plant and their biocontrol potential bioactivity of bryophyte extracts against botrytis cinerea, alternaria solani and phytophthora infestans control of fungal decay of apples and peaches by the biofumigant fungus muscodor albus the algal polysaccharide carrageenans can act as an elicitor of plant defense laboratoires goëmar. parc technopolitain atalante muscodor ghoomensis and muscodor indica: new endophytic species based on morphological features, molecular and volatile organic analysis from northeast india muscodor camphora, a new record from cinnamomum camphora muscodor kashayum sp. nov. -a new volatile antimicrobial producing endophytic fungus muscodor strobelii, a new endophytic species from south india response of subterranean clover to dual inoculation with vesicular-arbuscular mycorrhizal fungi and a plant growth-promoting bacterium modulation oh cyp79 genes and glucosilate profiles in arabidopsis by defense pathways potential agrochemicals from leaves of wedelia biflora 60-trihydroxydihydrochalcone from psidium acutangulum fungi and mycotoxins in grain: implications for stored product research endophytic actinomycetes from indian medicinal plants as antagonists to some phytopathogenic fungi chemically characterized cymbopogon martinii essential oil for shelf life enhancer of herbal raw materials based on antifungal, antiaflatoxigenic, antioxidant activity and favorable safety profile volatile antimicrobials from muscodor crispans, a novel endophytic fungus volatile plant metabolites for postharvest crop protection 4-methoxybrassinin, a sulphur-containing phytoalexin from brassica oleracea brassicanal c and two dioxindoles from cabbage brassicanal a and b, novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp pekinensis dehydro-4-methoxycyclobrassinin, a sulfur-containing phytoalexin isolated from turnip brassica campestris l. ssp. rapa calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga calothrix fusca biological activity of β-dolabrin, γ-thujaplicin, and 4-acetyltropolone, hinokitiol-related compounds bacillomycin d: an iturin with antifungal activity against aspergillus flavus chemical composition and fungitoxic properties to phytopathogenic fungi of essential oils of selected aromatic plants growing wild in turkey evaluation of 3, 4-dihydroxybenzaldehyde, dopamine and its oxidation products as inhibitors of colletotrichum musae (berk. and curt.) arx in green banana fruits antifungal activities of the leaf extract of cassia tora linn experimentelle untersuchungen über die phytophthora resistenz der kartoffel camellidins, antifungal saponins isolated from camellia japonica different mechanisms for phytoalexin induction by pathogen and wound signals in medicago truncatula glyceollin, a soybean phytoalexin with medicinal properties an endophytic actinomycete, streptomyces sp. aok-30, isolated from mountain laurel and its antifungal activity antimicrobial activities of vernonia tenoreana streptomyces: implications and interactions in plant growth promotion dihydro isocoumarins produced by xylaria sp. and penicillium sp., endophytic fungi associated with piper aduncum and alibertia macrophylla activation of biochemical defense mechanisms in bean plants for homeopathic preparations inhibition of protein biosynthesis by mildiomycin, an antimildew substance antifungal activity of thyme, summer savory and clove essential oils against aspergillus flavus in liquid medium and tomato paste activity of fungal endophytes against four maize wilt pathogens efficacy of some agricultural wastes in controlling root rot of glycine max l. induced by rhizoctonia solani effect of seed inoculation with bacillus subtilis and streptomyces griseus on the growth of cereals and carrots stress induced carbazole phytoalexins in glycosmis species seasonal variation of antibacterial and antifungal activities of the extracts of marine algae from southern coasts of india griseofulvin from xylaria sp. strain f0010, and endophytic fungus of abies holophylla and its antifungal activity against plant pathogenic fungi arbuscular mycorrhiza: the mother of plant root endosymbiosis potential of the volatile producing fungus nodulisporium sp. cf016 for the control of postharvest diseases of apple isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential distribution and identification of endophytic streptomyces species from schima wallichii as potential biocontrol agents against fungal plant pathogens mutualism and parasitism: the yin and yang of plant symbioses effects of sulfated polysaccharide and alcoholic extracts from green seaweed ulva fasciata on anthracnose severity and growth of common bean (phaseolus vulgaris l.) biological control in greenhouse systems a new working definition of the term "phytoalexin biotransformation of the brassica phytoalexin brassicanal a by blackleg fungus phytoalexins from brassicas: overcoming plants' defenses phytoalexin accumulation and antifungal compounds from the crucifer wasabi pathogen inactivation of cruciferous phytoalexins: detoxification reactions, enzymes and inhibitors antimicrobial activity of rhodobryum ontariense. hemijska industrija the discovery of enfumafungin, a novel antifungal compound produced by an endophytic hormonema species biological activity and taxonomy of the producing organisms ultrastructural observations of pterostilbene fungitoxicity in dormant conidia of botrytis cinerea pers natural occurrence of mycotoxins in foods and feeds -an update review relation between the chemical structure and biological activity of hydroxystilbenes against botrytis cinerea two new antifungal naphthoxirene derivatives and their glucosides from sesamum angolense welw potential of plant extracts and fungicides for managing fusarium oxysporum f. sp lycopersici further evidence for the involvement of a pre-formed antifungal compound in the latency of colletotrichum gloeosporioides on unripe avocado fruits progress in phytoalexin research during the past 50 years antifungal potential and defense gene induction in maize against rhizoctonia root rot by seed extract of ammi visnaga (l.) lam diterpene alcohols from croton lacciferus an antifungal chromene from eupatorium riparium outbreaks of aflatoxicoses in india removal of duvatrienediols from the surface of tobacco leaves increases their susceptibility to blue mold jasmonate and salicylate as global signals for defense gene expression use of algae in strawberry management antimicrobial activity of essential oils and ethanol natural products from plants and fungi as fungicides 227 extract of phlomis fruticosa l. (lamiaceae) growth activities of the sugar beet pathogens sclerotium rolfsii sacc. rhizoctonia solani kühn. and fusarium verticillioides sacc. under cyanobacterial filtrates stress induction of defense responses in zucchini (cucurbita pepo) by anabaena sp. water extract activity of seaweed and cyanobacteria water extracts against podosphaera xanthii on zucchini antioxidant and antimicrobial activities of essential oil and extracts of fennel (foeniculum vulgare l.) and chamomile elicitation of foliar resistance mechanisms transiently impairs root association with arbuscular mycorrhizal fungi antifungal activity and potential use of essential oils against fusarium culmorum and fusarium verticillioides plant growth enhancing effects by a siderophore producing endophytic streptomycete isolated from a thai jasmine rice plant (oryza sativa l. cv. kdml105) bioactivities of extracts from some axenically farmed and naturally grown bryophytes antimicrobial activity of bryum argenteum screening of antimicrobial and antioxidant secondary metabolites from endophytic fungi isolated from wheat (triticum durum) structure biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi pantoea agglomerans strain eh318 produces two antibiotics that inhibit erwinia amylovora in vitro effectiveness of phenolic compounds against citrus green mould control of penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone determination of antimicrobial and antiproliferative activities of the aquatic moss fontinalis antipyretica hedw muscodor tigerii sp. nov.-volatile antibiotic producing endophytic fungus from the northeastern himalayas muscodor darjeelingensis, a new endophytic fungus of cinnamomum camphora collected from northeastern himalayas bioactivity guided isolation of antifungal compounds from the liverwort bazzania trilobata biosynthesis, elicitation and roles of monocot terpenoid phytoalexins biologically active secondary metabolites of endophytic pezicula sp pinocembrin: an antifungal compound secreted by leaf glands of eastern cottonwood endophytic actinomycetes from tea plants (camellia sinensis): isolation, abundance, antimicrobial, and plant-growth-promoting activities isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production plant bio-stimulants: a review on the processing of macroalgae and use of extracts for crop management to reduce abiotic and biotic stresses purification and partial characterization of a b-glucan fragment that elicits phytoalexin accumulation in soybean diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds in: maheshwari dk (ed) bacteria in agrobiology: plant growth responses studies on endophytic actinomycetes (i) streptomyces sp. isolated from rhododendron and its antifungal activity introduction study of antifungal activities of bryophyte extracts anti-apoptotic machinery protects the necrotrophic fungus botrytis cinerea from host-induced apoptotic-like cell death during plant infection diversity of endophytic actinomycetes in mandarin grown in northern thailand, their phytohormone production potential and plant growth promoting activity diversity and antifungal activity of the endophytic fungi associated with the native medicinal cactus opuntia humifusa (cactaceae) from the united states antifungal activity of securinine against some plant pathogenic fungi antimicrobial activity of some indian mosses an endophytic phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds existence of muscodor vitigenus, m. equiseti and m. heveae sp. nov. in leaves of the rubber tree (hevea brasiliensis müll. arg.), and their biocontrol potential casbene: an antifungal diterpene produced in cell-free extracts of ricinus communis seedlings sesquiterpene lactones from centaurea thessala and centaurea attica: antifungal activity mechanisms of resistance to plant diseases what is the significance of the arbuscular mycorrhizal colonisation of many economically important crop plants? the role of phosphorous nutrition in interactions of vesicular arbuscular mycorrhizal fungi with soil borne nematodes and fungi suppression of cottony leak of cucumber with bacillus cereus strain uw85 management of mycorrhiza in agriculture, horticulture and forestry endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, sw1116 cell and some microbial growths a record of muscodor albus, an endophyte from myristica fragrans in thailand antifungal activity of some essential oils two novel antifungal alka-2,4-dienals from triticum aestivum plant-derived natural products synthesis, function, and application streptomyces sp. 9p as effective biocontrol against chilli soilborne fungal phytopathogens algal polysaccharides as source of plant resistance inducers mycorrhizae in crop production an endophytic gliocladium sp. of eucryphia cordifolia producing selective volatile antimicrobial compounds seasonal variation in antifungal, antibacterial and acetyl cholinesterase activity in seven south african seaweeds phytoalexins -a biogenetic perspective the role of phytoalexins in the seedling resistance to leptosphaeria maculans in some crucifers synergism among volatile organic compounds resulting in increased antibiosis in oidium sp an endophytic/ pathogenic phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential an endophytic nodulisporium sp. from central america producing volatile organic compounds with both biological and fuel potential the production of mycodiesel hydrocarbons and their derivatives by the endophytic fungus gliocladium roseum (nrrl 50072) muscodor albus e-6, an endophyte of guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant antifungal activities of a steroid from pallavicinia lyellii, a liverwort engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol muscodor cinnamomi, a new endophytic species from cinnamomum bejolghota evaluation of muscodor suthepensis strain cmu-cib462 as a postharvest biofumigant for tangerine fruit rot caused by penicillium digitatum molecular and morphological evidence support four new species in the genus muscodor from northern thailand biocontrol of rhizoctonia solani ag-2, the causal agent of damping-off by muscodor cinnamomi cmu-cib 461 antitumor activity of 4-arylcoumarins from endophytic streptomyces aureofaciens cmuac130 identification of streptomyces sp. tc022, an endophyte in alpinia galanga, and the isolation of actinomycin d structures of moracins e, f, g and h, new phytoalexins from diseased mulberry isolation of three novel sulphur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) novel sulfur-containing phytoalexins from the chinese cabbage brassica campestris l. ssp. pekinensis (cruciferae) mechanism of kasugamycin action on polypeptide synthesis isolation of endophytic actinomycetes from different cultivars of tomato and their activities against ralstonia solanacearum in vitro isolation, purification and characterization of trichothecinol-a produced by endophytic fungus trichothecium sp. and its antifungal, anticancer and antimetastatic activities antibacterial activity and induction of phytoalexins in bean plants by homeopathic preparations of essential oil of eucalyptus globulus antibiotic production by soil and rhizosphere microbes in situ study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro identification of volatile metabolites from fungal endophytes with biocontrol potential towards fusarium oxysporum f. sp. cubense race 4 influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure antifungal activity of lipopeptides from bacillus xt1 cect 8661 against botrytis cinerea hypoxylon sp., an endophyte of persea indica, producing 1,8-cineole and other bioactive volatiles with fuel potential diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings antifungal activity of lemongrass (cymbopogon citratus l.) essential oil against key postharvest pathogens antifungal drug resistance in pathogenic fungi phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte hypoxylon anthochroum strain blaci isolated from bursera lancifolia (burseraceae) volatile organic compounds from endophytic fungi as innovative postharvest control of fusarium oxysporum in cherry tomato fruits studies on the mode of action of the phytoalexin phaseolin characterization of the early response of arabidopsis to alternaria brassicicola infection using expression profiling antimicrobial activity of methanol extracts of fontinalis antipyretica, hypnum cupressiforme and ctenidium molluscum sea weed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants endophytic actinomycetes from azadirachta indica a. juss.: isolation, diversity, and anti-microbial activity the biocontrol effect of mycorrhization on soil borne fungal pathogens and the autoregulation of the am symbiosis: one mechanism, two effects? biocontrol of the pathogen phytophthora parasitica by arbuscular mycorrhizal fungi is a consequence of effects on infection loci 5-methylcoumaranones from mutisia friesiana and their bioactivity a vesicular arbuscular mycorrhizal fungus (glomus intraradix) induces a defense response in alfalfa roots suppression of an isoflavonoid phytoalexin defense response in mycorrhizal alfalfa roots fungal (−like) biocontrol organisms in tomato disease control controlling crop diseases using induced resistance: challenges for the future antifungal activities of essential oils and their constituents from indigenous cinnamon (cinnamomum osmophloeum) leaves against wood decay fungi antifungal activity screening of soil actinobacteria isolated from inner mongolia isolation and identification of an endophytic fungus of polygonatum cyrtonema and its antifungal metabolites loroglossol: an orchid phytoalexin evaluation of fungicides, bio-agents and plant extracts against pyricularia oryzae temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoalexins and the anthocyanin, cyanidin 3-dimalonyl glucoside peptide synthetase gene in trichoderma virens use of antibiotics for selective isolation and enumeration of actinomycetes in soil biological properties of alkaloids. influence of quinolizidine alkaloids and gramine on the germination and development of powderly mildew, erysiphe graminis f. sp. hordei die verbreitung antifungaler eigenschaften bei moosen the role of stilbenes in resistance of sitka spruce (picea sitchensis (bong) carr) to entry of fungal pathogens muscodor roseus anam. sp. nov., an endophyte from grevillea pteridifolia muscodor albus anam. sp. nov., an endophyte from cinnamomum zeylanicum chemical constituents from the chinese bryophytes and their reversal of fungal resistance metabolites from mangrove endophytic fungus dothiorella sp tetran or triterpenoids from chisocheton paniculatus effects of pre-and post-harvest application of salicylic acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage endophytic fungi harbored in the root of sophora tonkinensis gapnep: diversity and biocontrol potential against phytopathogens tithoniamarin and tithoniamide: a structurally unique isocoumarin dimer and a new ceramide from tithonia diversifolia evaluation of wedelia biflora (linn) d.c for anthelmintic and antimicrobial activity recent trends in studies on botanical fungicides in agriculture effect of polyacetylenic acids from prunella vulgaris on various plant pathogens potent in vivo antifungal activity against powdery mildews of pregnane glycosides from the roots of cynanchum wilfordii fungitoxic non-glycosidic iridoids from alibertia macrophylla diversity and antifungal activity of endophytic fungi associated with camellia oleifera potential of endophytic fungi isolated from cotton roots for biological control against verticillium wilt disease natural plant products as eco-friendly fungicides for plant diseases control-a review regulation of plant immunity through modulation of phytoalexin synthesis a new prenylated indole derivative from endophytic actinobacteria streptomyces sp. neau-d50 studies on chemical constituents in root tuber of cynanchum auriculatum muscodor fengyangensis sp. nov. from southeast china: morphology, physiology and production of volatile compounds bioactive isocoumarins isolated from the endophytic fungus microdochium bolleyi effects of yeast polysaccharide on growth and flavonoid accumulation in fagopyrum tataricum sprout cultures actinobacteria associated with chinaberry tree are diverse and show antimicrobial activity the diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in panxi plateau china preharvest l -arginine treatment induced postharvest disease resistance to botrytis cinerea in tomato fruits neoverataline a and b, two antifungal alkaloids with a novel carbon skeleton from veratrum taliense bioactive endophytic streptomycetes from the malay peninsula camalexin accumulation in arabis lyrata metabolites of colletotrichum gloeosporioides, an endophytic fungus in artemisia mongolica key: cord-283301-adjjkqt2 authors: awolade, paul; cele, nosipho; kerru, nagaraju; gummidi, lalitha; oluwakemi, ebenezer; singh, parvesh title: therapeutic significance of β-glucuronidase activity and its inhibitors: a review date: 2020-02-01 journal: eur j med chem doi: 10.1016/j.ejmech.2019.111921 sha: doc_id: 283301 cord_uid: adjjkqt2 the emergence of disease and dearth of effective pharmacological agents on most therapeutic fronts, constitutes a major threat to global public health and man’s existence. consequently, this has created an exigency in the search for new drugs with improved clinical utility or means of potentiating available ones. to this end, accumulating empirical evidence supports molecular target therapy as a plausible egress and, β-glucuronidase (βglu) – a lysosomal acid hydrolase responsible for the catalytic deconjugation of β-d-glucuronides has emerged as a viable molecular target for several therapeutic applications. the enzyme’s activity level in body fluids is also deemed a potential biomarker for the diagnosis of some pathological conditions. moreover, due to its role in colon carcinogenesis and certain drug-induced dose-limiting toxicities, the development of potent inhibitors of βglu in human intestinal microbiota has aroused increased attention over the years. nevertheless, although our literature survey revealed both natural products and synthetic scaffolds as potential inhibitors of the enzyme, only few of these have found clinical utility, albeit with moderate to poor pharmacokinetic profile. hence, in this review we present a compendium of exploits in the present millennium directed towards the inhibition of βglu. the aim is to proffer a platform on which new scaffolds can be modelled for improved βglu inhibitory potency and the development of new therapeutic agents in consequential. the world today is embattled with an increasing paucity of effective therapeutic agents or regimen for many pathological conditions, as well as the menace of drug resistance and adverse effects of available drugs [1] . as a result, smooth and efficient clinical practice is rigidly stymied, while global public health, social security and man's life expectancy are seriously threatened and trickles to a disquieting edge [2] . likewise, the burdens of developing new therapeutic agents to ameliorate the status quo has become heavier on all stakeholders in drug research. in this regard, molecular target therapy is fast becoming a spearhead in the search for new drugs with improved therapeutic effects. amongst many targets explored, glycosyl hydrolases (ghs) are notable due to their role in many important biological processes. their principal function is to catalytically cleave the glycosidic bond of glycans thereby eliciting different physiological responses. therefore, inhibitors of this class of enzymes have enjoyed intense research and development owing to their potentials as antiviral, anticancer and antidiabetic agents as well as therapeutic agents for some genetic disorders [3e5] . ghs have been classified using different indices [6] . for example, based on substrate specificity, those cleaving o-or sglycosides are grouped into ec 3.2.1 class, while hydrolases of nglycosides belong to ec 3.2.2 class. advancements in genomic science have also enabled classification into gh families based on their amino acid sequence similarities [7] . this system further groups gh families into clans, given the improved conservation of protein fold than the sequence [8] . accordingly, the reviewed enzyme, b-glucuronidase (ec 3.2.1.31) is classified into gh family 1, 2, 30, 79, 154 and gh-a clan. b-glucuronidase (bglu) is mainly a lysosomal hydrolase widely distributed in mammalian tissues, body fluids and microbiota; but significantly retained in the endoplasmic reticulum [9] . the enzyme is also found in plants, fishes, insects and molluscs. specifically, human bglu belongs to gh family 2. it is a 332 kda ellipsoidal and homotetrameric glycoprotein with each 75e78 kda monomer containing 651 amino acid residues (fig. 1a) . the monomer precursor is synthesized initially on membrane-bound ribosomes and suffers c-terminal proteolytic processing of 18 amino acid propeptide en route or after their transport to the lysosomes [10e13]. x-ray crystallography of the protein structure reveals a dihedral symmetry for the tetramer with two identical monomers in the asymmetric unit arising from disulphide-linked dimers. each monomer contains three structural domains (fig. 1b) . the first domain has a barrel-like structure with a jelly roll motif; the second domain exhibits a geometry identical to immunoglobulin constant domains; while the third c-terminal domain forms a tim barrel motif (b/a) 8 [14] . the active sites of human bglu ( fig. 1c) viz. catalytic acid glu451 (proton donor), catalytic nucleophile glu540 (carbonium ion stabilizer), asp207 (plausible role as glu540) and tyr504 (unclear catalytic role), are all housed in the third domain and in each of the four catalytic centres of the tetramer [14, 15] . moreover, the enzyme has an optimal activity at acidic ph~4.5, corresponding to its lysosomal environment and thermally stable up to 70 c [10] ; although hyperthermophilic variants exists in other media [16] . bglu is encoded by the gus gene. a deficiency arising from mutations in this encoding gene is associated with atherosclerosis [17] and lysosomal storage disease e sly syndrome or mucopolysaccharidosis type vii [18] . on the other hand, bacterial bglu, which is expressed in human gut microbiota and most strains of escherichia coli shows 45% sequence similarity with human bglu. also, it has a bacterial loop containing 17-amino acid residues not found in human bglu, an optimal activity at neutral ph and active site catalytic residues as glu413 (catalytic acid) and glu504 (catalytic nucleophile) [19] . consistent with the activities of lysosomal ghs, bglu deconjugates b-d-glucuronides to their corresponding aglycone and b-dglucuronic acid via an s n 2 reaction and "configuration retaining" mechanism ( fig. 2) . the catalytic mechanism is conceived to proceed as follows; catalytic glutamic acid residue glu451 (or glu413 in bacterial ortholog) protonates exocyclic glycosidic oxygen of glucuronide (1) hence releasing the aglycone via a putative oxocarbenium ion-like transition state (2) . 'back-side' nucleophilic attack by glutamate ion glu540 (or glu504 in bacterial ortholog) e the catalytic nucleophile, stabilizes the transition state and results in glucuronyl ester intermediate (3) with an inverted configuration. finally, hydrolysis through an inverting attack of water molecule on the anomeric centre releases glu540 to form b-d-glucuronic acid (4) and a concurrent overall retention of substrate configuration [14,15,19e21 ]. due to the increased expression of bglu in necrotic areas and other body fluids of patients with different forms of cancer such as breast [22] , cervical [23] , colon [24] , lung [25] , renal carcinoma and leukaemia [26] , compared to healthy controls, the enzyme is proffered as a reliable biomarker for tumour diagnosis and clinical therapy assessment [27] . this overexpression is also a potential diagnostic tool for other disease states such as urinary tract infection [28] , hiv [29] , diabetes [30] , neuropathy [31] and rheumatoid arthritis [32] . in this vein, empirical data update on clinical applications of bglu for these and other disorders is provided on brenda database [33] . bglu activity is also harnessed in prodrug monotherapy. in normal body systems, drugs and other xenobiotics are detoxified via glucuronidation, an s n 2 conjugation reaction and important pathway in phase ii metabolism, catalysed by udpglucuronosyltransferases (ugts). the resulting usually less active glucuronide metabolite is readily excreted by renal clearance due to increased polarity or sometimes via biliary clearance [34] . however, elevated levels of bglu activity reverts this process through deglucuronidation, which hydrolyses the phase ii metabolites to their active forms (fig. 2) . hence, glycosidation of a drug to give its glucuronide enhances selective release of the active form at necrotic sites via bglu-mediated deglucuronidation thus improving the drug's therapeutic potential [35] . bglu's postulated ability to increase t regulator cells (treg) is also applied in low-dose immunotherapy (ldi) for managing allergic diseases [36, 37] , lyme disease [38] and other chronic conditions. while it's hydrolytic activity on glucuronide conjugates is harnessed in forensic analysis [39] and assessment of microbial water quality [40] . nonetheless, enterobacterial bglu deconjugation of drug and xenobiotic glucuronides in the gastrointestinal (gi) tract has been implicated in colonic genotoxicity [41] and certain drug-induceddose-limiting toxicities. for example, the gi toxicity of anticancer drug irinotecan (cpt-11) [42] , enteropathy of non-steroidal antiinflammatory drug (nsaid) diclofenac [43] , tissue inflammation and hepatoxicity. furthermore, bglu is deemed a potential molecular target for; (1) anticancer chemotherapy considering its role in tumour growth and metastasis [44, 45] . (2) neonatal jaundice treatment due to its high expression in breast milk and role in enterohepatic bilirubin circulation (hyperbilirubinemia) [46, 47] . (3) diabetes mellitus management consequent to the positive correlations between the disease state and enzyme activity level as well as associated periodontitis [48, 49] . (4) anti-inflammatory agents development owing to its pro-inflammatory role following significant release from degranulated mast cells and neutrophils [50, 51] . expectedly, inhibition of bglu markedly alleviated these pathological conditions and their adverse effects hence improving regimens' efficacy. based on the foregoing, we extrapolate that the development of potent, specific and non-cytotoxic inhibitors of bglu is imperative to improving the clinical efficacy of therapeutic agents and effective disease management while bearing in mind the physiological significance of both human and bacterial orthologs of the glycosyl hydrolase. however, the fate of these inhibitors rests on their inhibition constants (k i ), since ghs are generally characterized by high rate enhancement (k cat /k uncat > 10 17 -fold). also, accumulating evidence suggests the dependence of inhibitory potency on the ability to mimic the highly enzyme-stabilized transition state of an enzymatic reaction (k i z 10 à20 m) en route to catalytic product [21, 52, 53] . considering the proven and encouraging potentials of enzyme inhibition and molecular target therapy in drug development, and in continuation of our exploits and expositions thereon [54e58], herein we present a comprehensive review of research undertakings in the present millennium (2000e2019) directed towards the development of potent inhibitors of bglu that are either natural products or synthetic scaffolds. apropos, before discussing the different inhibitors, this article will first highlight the potentials of bglu activity as a diagnostic tool within the defined period. however, therapeutic application in prodrug monotherapy and enzyme replacement therapy (ert) will not be covered as these have been excellently treated in other reviews [59e61]. hitherto, our search of extant literature revealed that, although there exists a plethora of scholarly research on potential inhibitors of bglu activity, no review article is exclusively devoted to the subject matter. the aim of this review is therefore to bring to light those bioactive frameworks bestowed with promising bglu inhibitory potency. our principal goal is to intimate the reader on key structural features of reviewed molecules crucial to their inhibitory activities and toxicity profiles, while establishing the comprehensive relationships existing between reported molecules. the availability of safe, easy to use, consistent, less-invasive and cost-effective tool for early diagnosis of diseases or appraisal of therapeutic interventions is of uttermost importance in clinical medicine. since most disease states are accompanied by elevated levels of specific enzymes in the diseased milieu (tissues, plasma and other body fluids), quantification of enzymes' activity levels is seen as a reliable biomarker of either disease status, severity, effects, susceptibility or exposure [62e64]. moreover, the substrate specificity and selective quantification of enzymes in the presence of other biomolecules makes them a tool of choice thereto [65] . a review of bglu activity as a biomarker of some physiologically important conditions is hereby presented together with a concise summary in table 1 . periodontal disease is a group of inflammatory disorders triggered by host's immune response to the actions of virulent subgingival plaque bacteria biofilms which activates the release of polymorphonuclear leukocytes and macrophages into the gingival crevice. this leads to gingivitis e an inflammation of periodontal tissues and distortion of periodontal histology that is reversible with improved oral hygiene; or, subsequent tissue destruction, alveolar bone resorption and tooth loss if left unattended i.e. periodontitis [66, 67] . therefore, a reliable tool to ascertain disease status, severity, risk or efficacy of administered therapy is highly desirous to clinicians. however, conventional diagnosis involving the measurement of periodontal clinical parameters such as probing depth (pd), clinical attachment level (cal), gingival index (gg-i), bleeding on probing (bop) and alveolar bone loss (abl), suffers from intrinsic limitations. they only define the status of patient's periodontium at the time of examination and not periodontal disease susceptibility or risk [68] . thus, since periodontitis is characterized by an influx of inflammatory mediators and corresponding enzymes into the gingival sulcus, the quantification of neutrophil-derived bglu activity in gingival crevicular fluid (gcf) or gcf's outflow into the oral cavity and subsequent less invasive estimation of bglu activity in saliva is considered a reliable biomarker for periodontal disease diagnosis. to this effect, the relationship between salivary bglu activity and periodontal clinical parameters (pd, cal and gg-i) was investigated in subjects with different stages of periodontal disease [69] . the mean pd and gg-i, number of sites with pd ! 5 mm and total number of white blood cells, blood neutrophils and monocytes all showed highly significant correlations with enzyme's activity, while cal had a weaker correlation. using logistic regression modelling and the presence of at least 1 or 4 sites with pd ! 5 mm as disease criterion, bglu activity showed promising potentials as a tool for periodontal disease screening or assessment of therapeutic intervention. the study also observed smoking status to be insignificant on enzyme activity. however, in a similar study, only pd, cal and lymphocyte count exhibited positive correlation with salivary enzyme activity while no significant relationship was observed for gg-i [70] . recently, subjects with chronic generalized periodontitis were also found to have significant increase in bglu activity (8-fold) compared to normal ones. although, a reduction in enzyme activity persisted in smokers regardless of periodontal status [71] . table 1 reported potential applications of bglu activity as a biomarker. [112] 284** ache activity chronic exposure (w) acute exposure (n) elevated in 16.5% and 60% of subjects with chronic and acute exposure respectively [113] ns: not specified; w: weak correlation; n: no correlation; pi: russell periodontal index; *threshold to distinguish culture positive from culture negative balf; ** acute exposure (5), chronic exposure (230) . the efficacy of therapeutic intervention using amoxicillin and metronidazole to downregulate amplified neutrophil activity was studied in 14 patients with aggressive periodontitis [72] . treatment involved seven consecutive days of antibiotic administration with concurrent scaling, root planning and surgical therapy and a total of 36 months posttreatment evaluation period. subsequently, a markedly downregulated neutrophil activity with approximately 50% inhibition of bglu activity in gcf was observed. periodontal health was also restored and maintained during posttreatment evaluations. in another study, bglu activity was posited as a better biomarker compared to alkaline phosphatase for evaluating the response to non-surgical periodontal therapy in patients with different stages of periodontal disease [73] . taken together, these results articulate the potentials of bglu activity as an indication of pd, tissue inflammation or destruction as well as a biomarker of neutrophil influx, disease risk, susceptibility, status, or severity for timely diagnosis of the inflammatory disorder. however, administration of doxycycline hyclate in 16 subjects with aggressive periodontitis was inefficient on salivary bglu activity [74] . surprisingly, an increase in enzyme activity was found even after 2 months of treatment in 12 patients and a decrease in 4. although, the authors concluded bglu concentration only facilitated the detection of periodontal inflammation and not worthy as biomarker of susceptibility, their contrasting result is linkable to periodontal pretreatment of subjects prior to examination and short treatment time using doxycycline. empirical evidence affirms the role of inflammatory mediators and signalling pathways in the pathogenesis of insulin resistance and b-cell dysfunction in diabetes mellitus [75e77]. in parallel, these inflammatory mediators e.g. cytokines and mmps are also produced in periodontal tissues [78e82]; hence, leading to compromised glycaemic control after accessing systemic circulation. the susceptibility to periodontitis is therefore heightened in persons with diabetes or a history of the hormonal imbalance and vice versa. putatively, an effective therapy for one affords an improved management of the other [83e85]. accordingly, bglu activity was found to be significantly elevated in the saliva of patients with chronic periodontitis and diabetes compared to nondiabetic ones [86] . a significant correlation to bglu activity was observed for pd and cal and not gg-i in nondiabetic subjects with periodontitis; whereas, these periodontal parameters were similar in diabetics. the increased disease burden was also established when bglu activity was measured in the sera of patients with both diabetes and periodontitis [87] . compared to controls, enzyme activity was 9-fold higher in diabetic subjects with periodontitis and only 2-fold higher in diabetic subjects without periodontitis. this difference was attributed to damaged lysosomal membrane and consequent enzyme leakage into the cytosol. the quantification of bglu activity in neutrophil leukocytes exposed to bacteria stimuli has shown that diabetic patients with chronic periodontitis have strikingly higher enzyme activity compared to nondiabetics burdened with periodontitis and healthy subjects [88] . using discriminant analysis, the study established that bglu activity has a diagnostic potential with great accuracy in distinguishing healthy subjects from diseased ones. bglu activity stimulated by nonopsonized staphylococcus aureus showed strongest correlation with the intensity of periodontal parameters compared to opsonized zymosan and prodigiosan, while the highest enzyme activity was stimulated by opsonized prodigiosan. the strength of association between salivary bglu activity, periodontitis and type 2 diabetes mellitus has been examined in dentate patients with different stages of periodontal disease, diabetic patients and edentulous patients [89] . in all subjects, diabetic status contributed significantly to bglu activity, while periodontal status had greater influence on enzyme activity. higher enzyme activity was also found in nondiabetic dentate patients with periodontitis compared to edentulous controls. overall, compared to il-1b, bglu activity level was more reliable as biomarker of disease severity for periodontitis than it was for the presence of diabetes. in a predating study [90] , gcf bglu activity also correlated strongly with pd, cal and bop regardless of diabetic status. lower enzyme activity was seen in diabetic subjects compared to those with periodontitis. the results suggested a lower release of bglu in response to systemic inflammation (diabetes) due to reduced deficiency in neutrophil activity, in contrast to amplified activity in response to local inflammation (periodontitis). despite landmark developments in oncology, the high rates of morbidity and mortality and increased medical costs associated with all forms of cancer coupled with patients' psychological trauma on disease diagnosis has remained a major threat to global public health. the successful disease management and sustained wellbeing of affected individuals is however subject to early disease diagnosis and constant evaluation of administered therapy. this in turn relies on efficient tumour markers to ascertain disease risk or status i.e. early stage or metastatic cancer [91] . although a vast number of biomarkers have been identified for cancer diagnosis, only few have gained clinical approval due to inconsistencies and false positives in their utility [92] . a successful biomarker is that which will not only be specific and selective but will also predict treatment response, while differentiating lethargic and aggressive tumours. the aetiology of cancer is known to be closely associated with inflammatory pathways and oxidative stress, which jointly create microenvironments favouring neoplasia [93, 94] . hence, in the tumour milieu, an increase in extracellular activity of lysosomal exoglycosidases responsible for the catalytic degradation of glycoconjugates occur, due to malignancy-mediated cell-death and/or lysosomal damage. in this vein, available practical data supports the overexpression of bglu in extracellular fluids and tissues around tumour sites as a prime factor in cancer aetiology [24] ; thus, suggesting the enzyme's viability as cancer biomarker [95] . for example, bglu activity was 2-folds higher in the blood samples of 21 patients with colorectal adenocarcinoma compared to healthy controls [96] . based on cell maturity and clinical grading, enzyme activity was highest in subjects with low or moderately differentiated cells and in subjects with tumours infiltrating surrounding tissues and organs or visceral peritoneum respectively. estimation of serum bglu activity in this case proved to be 80% sensitive and 82% specific in distinguishing diseased and healthy subjects. likewise, enzyme activity was elevated by 6-fold in the peritoneal fluids of patients with ovarian or endometrial cancer compared to women with infertility used as controls [97] . the stronger correlation between cancer stage and bglu activity, b-galactosidase and a-mannosidase, reiterated the improved clinical viability of bglu activity as biomarker of gynaecologic tumour status and severity. however, bglu activity was equally elevated in the peritoneal fluid of patients with pelvic inflammation hence compromising its application for differential diagnosis of gynaecologic cancer and pelvic inflammatory disease. bacterial peritonitis is a life-threatening inflammation of the peritoneum e the tissues lining of the inner abdominal walls. as with other inflammatory conditions, cellular damage and polymorphonuclear leukocytes activity increases the level of lysosomal enzymes in the extracellular space via enzyme leakage through the cellular membrane. thus, quantification of these enzymes provides a potential diagnostic platform. in fact, bglu activity in the peritoneal fluid of patients with culture positive bacterial peritonitis was 9 and 33-fold greater compared to that of patients with acute mesenteric lymphadenitis and controls respectively [98] . peritoneal fluid-bglu activity measurement in this case holds greater clinical potential compared to b-galactosidase and a-mannosidase for early disease diagnosis and evaluating patient's response to treatment. similarly, considering the current global prevalence of antimicrobial resistance, timely diagnosis of bacteria-mediated meningeal inflammation (bacterial meningitis) is crucial to the outcome of therapeutic intervention. it has been shown that bglu activity is elevated in cell-free cerebrospinal fluid (csf) of bacterial meningitis patients early in the disease pathogenesis, even when traditional laboratory parameters such as number of csf cells, csf-blood glucose ratio and protein concentration indicated normal status [99] . csf-bglu as biomarker was superior to these traditional parameters for early and sensitive prediction of patient's response to antibiotic treatment. csf-bglu activity in neonates and infants has also been studied as biomarker for differential diagnosis of sterile csf pleocytosis due to urinary tract infection (uti) or meningitis [100] . median bglu activity in csf of patients showed significant difference without overlapping in each disease state i.e., bacterial meningitis (168), viral meningitis (26.5) and uti with sterile csf pleocytosis (44.1); median enzyme activity was lowest (19.1) in febrile subjects without csf pleocytosis used as controls. this proffers an unambiguous diagnosis of each condition with 100% sensitivity and specificity. in contrast, broad overlapping was found with classic csf laboratory parameters viz., csf cell number, neutrophil number, protein concentration and csf-blood glucose ratio. recently, a case-control study concluded that bglu activity in bronchoalveolar lavage fluid (balf) is clinically useful as biomarker of bacterial lung infection [101] . in balf samples from 92 children, enzyme activity was significantly higher in patients with positive balf bacterial culture (cþ) compared to those with culture negative balf (ce). 43 nmol 4-methylumbelliferone (4mu)/ml/h was identified as optimum activity value, which allowed differential sample screening (i.e. cþ from ce) with 84.8% sensitivity and 78.3% specificity. moreover, receiver operating characteristics (roc) curve analysis established the superiority and higher prognostic value of bglu activity for bacterial lung infection compared to % polymorphonuclear cell count, human leukocyte elastase, il-8 and tnfa. repeated exposure to organophosphorus compounds (op) in pesticides is responsible for the lethal poisonings seen in agricultural and veterinary workers; particularly, those in developing countries. op exerts their fatal effects by inhibiting acetylcholinesterase (ache) in nervous tissues, leading to muscarinic and nicotinic effects with central nervous disturbance [102] . on the other hand, bglu is retained in liver microsomal endoplasmic reticulum by forming non-covalent binding complexes with its accessory 64 kda glycoprotein, egasyn, a carboxylesterase isozyme [103, 104] . liver intake of op however cleaves microsomal bgluegasyn complex thus elevating the level of bglu in plasma to consequently making it an alternative biomarker for op pesticide poisoning diagnosis [105e107]. a cross-sectional study of pest control workers [108] and plastic greenhouse workers [109] , established that plasma bglu activity was more sensitive as biomarker of op poisoning compared to butyrylcholinesterase (buche) and acid phosphatase (acp). the enzyme's activity was higher in subjects with increased exposure than those with low exposure and controls. bglu activity correlated significantly with buche and acp activities. likewise, in a case-control study, serum bglu activity was significantly increased in patients with severe poisoning compared to mildly affected patients and controls [110] . the difference in enzyme activity between the latter groups was insignificant. nonetheless, mildly exposed subjects have been reported to show elevated bglu activity than severely exposed and control subjects, which suggests the enzyme's utility for diagnosing lowlevels of op exposure. for instance, a study [111] , observed the order of serum bglu activity based on poisoning severity as mild > severe > moderate poisoning, while the order after 12 and 24 h of admission was mild > severe z moderate poisoning; strong correlation also persisted between serum bglu and buche activities. similar results were also obtained in a recent cross-sectional study [112] . therein, moderately exposed subjects showed higher enzyme activity than the highly exposed, while non-significant statistical difference persisted between control and highly exposed groups. notably, bglu activity correlated well with diabetes propensity, lipid profile, liver function and buche but not ache. in another cross-sectional study, significant difference in plasma bglu activity only exists between controls and subjects with chronic exposure (1e45 years) to op [113] . activity level was similar in controls and patients with acute poisoning; although 3 out of the 5 examined acutely poisoned patients showed increased level of plasma bglu activity. however, a case report of an acute op self-poisoned patient reached contrasting conclusion [114] . the opposing result can be linked to sample size and limited data on patient's medical history. moreover, the reduced susceptibility of bglu-egasyn complex to op in humans is another primal factor [115] . in contrast to murine bglu-egasyn interaction, binding in humans is independent of the c-terminal 18 amino acids propeptide in bglu and esterase active site of egasyn. rather, it involves the 51 amino residues in bglu internal segment i.e. residues 228 to 279. the progress and momentous achievements in drug discovery cannot be isolated from the wealth of chemical entities i.e. natural products, gifted to man by nature. many successful molecular candidates of pharmaceutical drug discovery programmes are indebted to the presence of natural product-derived/inspired fragments in their scaffolds [116] . therefore, the chemical space of natural products (both plants and microbes) has continued to be a much-researched depository for therapeutically significant molecules. in this section, we review some application of natural products including isolated compounds, whole plant extracts and natural product-inspired molecules as potent inhibitors of bglu. the structures of selected inhibitors (ic 50 5 mm) are presented in table 2 at the end of the section. the clarification [117, 118] that d-saccharic acid-1,4-lactone (5, fig. 3 ) (saccharolactone or d-glucaric acid-1,4-lactone) is the active and non-toxic bglu inhibitor responsible for the strong inhibitory potency found with saccharate solutions has precipitated an increased interest in the compound. despite the poor stability at physiological ph, d-saccharic acid-1,4-lactone (d-sal) has been explored extensively for its therapeutic significance. although an ic 50 value of 3.6 mm was reported in the pioneering work [117] , a value ca. 40 mm is recurrent in literature. nevertheless, at a concentration of 1 mm, d-sal completely inhibited the hydrolytic action of human liver-derived bglu on quercetin glucuronides [119] , while over 90% inhibition of bglu in breast milk was recorded at 10 mm [120] . also, using urine samples of male sprague-dawley rats, d-sal was identified via metabolomics strategy as one of the therapeutic constituents in liuweidihuang pills, a famous traditional chinese prescription for cancer treatment and prevention [121] . whereas, in a 6-days cumulative study, intraperitoneal pre or cotreatment of female wistar rats with 3 mg/ml, 10 mg/ml or 10 mg/0.5 ml d-sal reduced the severity of cpt-11 induced smallintestine mucosal damage assessed by the number of apoptotic cells or mitotic figures, compared to cpt-11 treated controls [122] . damage reduction was independent of treatment schedule. the synthetic and natural precursors of d-sal i.e., 2,5-di-oacetyl-d-glucaro-1,4:6,3-dilactone (d-sdl, 6, fig. 3 ) and d-glucurono-g-lactone (d-gl, 7) respectively, have also potently inhibited bglu activity in male fischer rats thereby providing chemopreventive effects against azoxymethane-induced colon carcinogenesis [123] . diet supplementation with 0.5 or 2% d-sdl for 5 weeks significantly reduced aberrant crypt foci formation (i.e. preneoplastic lesions) by over 48.6 and 55.3% respectively, compared table 2 most active natural product-derived bglu inhibitors with ic 50 to azoxymethane-controls. d-gl did not afford any significant reduction in colonic tumour incidence during this initiation phase. in addition, 32 weeks treatment with d-sdl or high dose (2%) of d-gl, after 3-weeks subcutaneous injections of 15 mg/kg azoxymethane, provided over 70% inhibition of colon carcinogenesis during the post-initiation phase. it was suggested that d-sdl is a blocking agent which may inhibit pre-neoplasia during the initiation phase, while d-gl is only active during post-initiation of colon carcinogenesis. the increased hydrolytic stability of d-sdl to d-sal in vivo might also be responsible for these observed phenomena [124] . in another study, d-sal was more therapeutically efficient compared to its natural precursor (d-gl), for reducing epidermal hyperplasia (lethargic tumour promoter) and inflammation in 7,12dimethylbenz(a)anthracene (dmba)-induced complete skin carcinogenesis of sencar mice [125] . pre and cotreatment of murine models with d-sal for 4-weeks (twice weekly) by topical administration (0.5e4 mg) or dietary treatment (0.5 and 1%), both significantly reduced epidermal hyperplasia and inflammation by up to 57% of dmba-controls in a dose-dependent manner. d-sal also inhibited the initiation of carcinogenesis by reducing dmbainduced oxidative dna damage (c-8 hydroxylation of guanine) and mutations in codon 61 of ha-ras gene, by up to 78%. in contrast, d-gl only inhibited epidermal hyperplasia with topical treatment and inflammation by dietary treatment (5% in diet); albeit with inferior potency compared to d-sal. interestingly, another lactone-based bglu inhibitor (8, fig. 3 ), exhibited 8-fold superior potency compared to d-sal [126] . isolated from the ethyl acetate extracts of aspergillus terreus, an endophytic fungus initially isolated from marine alga laurencia ceylanica, butyrolactone (8) with ic 50 ¼ 6.2 mm possesses 16-fold stronger potency than its prenylated isomer (9) . bglu in breast milk is believed to be involved in neonatal jaundice and hyperbilirubinemia, by increasing serum bilirubin levels via enterohepatic bilirubin circulation in breastfed newborns, in contrast to those fed with infant formulas [47] . therefore, the suppression of bglu-mediated deglucuronidation has been proffered as a practicable regimen. l-aspartic acid (10, fig. 3 ) in casein hydrolysate formulas was identified as the active bglu inhibitor responsible for the lower levels of neonatal jaundice observed in newborns receiving such formulae [127] . at 100 mm, the natural amino acid showed~86% inhibition of bglu that is, 100-fold more potent than d-isomer. moreover, in a randomized and double-blind clinical trial involving 64-newborns, supplementing breastfeeding in the first week of life with 6 doses of l-aspartic acid (180 mg/5 ml of water/day) was more potent against bglu activity than higher concentrations of enzymatically hydrolysed casein (infant formula containing the inhibitor) or whey/casein (routine formula lacking the inhibitor) [128] . l-aspartic acid supplementation at minimal aliquot concentration significantly lowered transcutaneous bilirubin levels (25% lower than control), leading to higher faecal bilirubin excretion and reducing neonatal jaundice, with no adverse effects. flavonoids are evidently an indispensable class of natural products due to their ubiquity in the vegetal domain and their therapeutic significance. found in a variety of plant parts (leaves, flowers, stems, nuts, seeds etc.), they perform important functions especially plant's growth and protection against pathogenic invasion. this intrinsic property encourages their utility as a major constituent of different local medicinal formulations and diets, while their acceptable toxicity profile and physiologic tolerance further presents them as druggable subjects. flavonoids are typified by the c6ec3ec6 ring system that is, their basic structural skeleton consists of two benzene rings (a and b) linked by heterocyclic pyran ring c (fig. 4) . variations on the chromane core (ring a and c) and attachment position of ring b due to biosynthetic origins lead to classification into flavones, flavanols, flavanones, flavonols, isoflavones, neoflavonoids, anthocyanins and chalcones [129] . this ring system and the presence of hydroxyl units has availed flavonoids with different pharmacological properties such as antidiabetic, antioxidant, antimicrobial, antiplasmodial, antiproliferative and particularly enzyme inhibition [130, 131] . luteolin (11, fig. 4 ), a dietary 3 0 , 4', 5, 7-tetrahydroxyflavone has been reported as a viable chemopreventive and anticarcinogenic agent plausibly by inhibiting bacterial bglu-mediated enterohepatic circulation of colonic carcinogens [132] . the 30-weeks cumulative study using male wister rats showed that pre or cotreatment with luteolin by intragastric gavage per os (p.o.) at 0.1, 0.2 or 0.3 mg/kg body weight/day, significantly reduced bacterial bglu activity thereby suppressing 1,2-dimethylhydrazine (dmh)induced colon adenocarcinomas in a dose-dependent manner compared to dmh-controls. although 0.2 and 0.3 mg kg à1 day à1 produced similar result. luteolin supplementation also reduced tumour size from 2 cm to 0.25 and 0.50 cm during the initiation and post-initiation stages respectively, as well up to 90% reduction in tumour incidence. in addition, luteolin (11), its 7-o-glycoside (12), apigenin-7-o-glycoside (13) and catechin (14) were identified as key constituents in the leaf extracts of pistacia terebinthus responsible for the high e. coli bglu inhibitory potency [133] . at the highest test concentration (8.2 mg/ml), the leaf extracts exhibited 97.2% inhibition of bglu activity, corresponding to an ic 50 value of 2.11 mg/ml. in another exploit, 32 natural flavonoids were evaluated for their inhibitory strengths against e. coli bglu [134] . it was established that luteolin (11) , similar flavones e baicalein (15), scutellarein (16) and its glucuronidated analogue scutellarin (17) , as well as dietary and ubiquitously occurring flavonol e quercetin (18) , are superior inhibitors (ic 50 ¼ 5.76e29.64 mm) compared to reference inhibitor d-sal (ic 50 ¼ 36.07 mm); isoflavones and dihydroflavones displayed weaker inhibition. overall, luteolin (11) and scutellarein (16) emerged the most potent flavone-based e. coli bglu inhibitors with ic 50 ¼ 8.68 and 5.76 mm respectively. sar analysis (fig. 4 ) revealed the importance of 5,6,7-trihydroxy (pyrogallol) unit to bacterial bglu inhibition, as unsubstitution or replacement of a hydroxy unit with methoxy or glycosyl unit led to significant loss of inhibitory activity. o-methylation at positions-6, 7 and 4 0 and installing oh unit at position-3 was also detrimental to potency, whereas the presence of hydroxy unit at c-4' favoured potency. in addition, molecular docking studies of luteolin and scutellarein in the active site of e. coli bglu showed hydrogen bonding (h-b) interactions of phenolic oh units at c-5 and c-7 with catalytic acid glu413 and arg562 as well as hydrophobic contacts with ser360 and leu361 in the bacterial loop. furthermore, subjecting the methanolic extracts of edible flower and pedicel of aquatic rhizomatous herb e nymphaea pubescens (water lily) to bioactivity-guided fractionation established the superior (3-fold) bacterial bglu inhibitory potency of crude flower extracts compared to pedicel extracts and silymarin e a marketed natural bglu inhibitor [135] . kaempferol (19) was subsequently identified as one of the active metabolites with promising activity; ic 50 ¼ 36.47 mm and 76-fold superior to silymarin. interestingly, the lower activity of kaempferol (19) compared to flavonoids (11, 15e18) affirmed the sar results described earlier. amidst 21 constituents found in commonly used chinese herbal medicines, two prenylflavonoids, sanggenon c (20, fig. 5 ) and kuwanon g (21) emerged as the most potent broad-spectrum inhibitors (>70% inhibition; ic 50 ¼ 12.5 and 7.4 mm respectively) of whole human gut bacterial bglu (consisting of eight bacterial isolates), compared to reference compound amoxapine (7.3% inhibition) [136] . compound 20 exhibited improved potency against recombinant bglu from e. coli (ecogus) and s. pasteuri (spasgus), ic 50 ¼ 0.40 and 0.33 mm respectively, compared to compound 21 (ic 50 ¼ 1.6 and 0.98 mm respectively). however, compound 21 was a stronger inhibitor of bglu from three representative bacterial isolates (e. coli, e. fergusonni and s. pasteuri) compared to compound 20. additionally, molecular docking studies of 20 and 21 in the binding pockets of ecogus and spasgus revealed both flavonoids bind (via their hydroxy groups) in the allosteric site and not the active site of the recombinant enzymes. the higher number of h-b interactions formed by compound 21 supported the overall increased potency compared to compound 20. the realization that pro-inflammatory mediators such as bglu, released from degranulated mast cells or neutrophils, play significant roles in inflammatory disorders has sparked increased interest in the search for potent inhibitors of such processes as antiinflammatory drug candidates. in this regard, dihydroxychalcones 22 and 23 (fig. 6 ) isolated from the roots of hypericum geminiflorum holds therapeutic promise [137] . compounds 22 and 23 potently inhibited the release of bglu from degranulated rat neutrophils with ic 50 values of 5.80 and 6.60 mm respectively, better than the reference inhibitor trifluoperazine. however, only compound 23 showed moderate activity against the enzyme's release from degranulated rat peritoneal mast cells with ic 50 ¼ 70.0 mm. conversely, an isomer of luteolin e norartocarpetin 24 (fig. 6) and flavonoid-like mornigrol d 25 (fig. 6) , both isolated from the barks of morus nigra (black mulberry), were moderately potent bglu inhibitors [138] . at 100 mm, compounds 24 and 25 showed 67.7% and 65.9% inhibition respectively against the release of bglu from activated rat neutrophils. however, chiral flavonoid alkaloids isolated from the ethanolic extracts of scutellaria moniliorrhiza and subsequently separated using chiral hplc, e scumonilines 26e29 (fig. 6) , are better drug candidates compared to compounds 24 and 25, but similar to compounds 22 and 23 [139] . scumonilines 26e29 displayed~63% inhibition at 10 mm against bglu release from activated rat neutrophils with ic 50 values of 5.21, 5.85, 5.47 and 5.16 mm respectively; better than reference inhibitor ginkgolide b (ic 50 ¼ 6.63 mm). compounds 26e29 were also more potent lead molecules compared to both glucuronate esters 30e34 (fig. 6 ) isolated from similar scutellaria regeliana [140] and chiral egeninebased alkaloids 35e37 isolated from the rhizomes of corydalis decumbens [141] . compounds 30e34 showed 43.7e47.1% inhibition at 10 mm, whereas compounds 35e37 were more inferior inhibitors at the same concentration with 32.4e41.3% inhibition. evidently due to its phycocyanin-rich content and potent reduction of zymosan-induced damage to knee joint histological architecture, edible blue-green microalgae e spirulina is deemed a therapeutically viable anti-arthritic agent [142] . an analysis of the synovial fluid of female of1 mice knee joints after 4-days intraarticular injection of zymosan, followed by 8-days oral administration of spirulina water-suspension at 100 mg/kg and 400 mg/kg, showed 78.7% and 89.2% inhibition of bglu activity respectively. subsequently, arthritic parameters such as tibial articular cartilage destruction, erosion of bone structure, articular tissue inflammation, loss of general joint architecture and pannus formation, were all markedly reduced in spirulina fed mice compared to those receiving zymosan only. these anti-inflammatory and anti-arthritic effects were comparable to reference drug triamcinolone, a tuber extracts of arisaema tortuosum (whipcord cobra lily) has shown moderate anti-inflammatory effect via bglu inhibition in a dose-dependent manner [143] . conceivably, the presence of quercetin, rutin and lectin, identified through chromatographic profiling, facilitated maximum inhibition of 92.9% at 100 mg/ml, superior to reference inhibitor salicylic acid. employing a similar approach with ginger rhizomes [144] , only 6-gingerol (38, fig. 7) was identified with 85% inhibition of bglu at 1 mm, comparable to salicylic acid (82% inhibition). it is also noteworthy that constituents profiling of essential oils extracted from the leaves of seven local varieties of piper betle l. identified eugenol (39) with an ic 50 value of 616.68 mg/ml similar to 794.62 mg/ml of silymarin; although the essential oils were only moderate bglu inhibitors with ic 50 ! 5.26 mg/ml [145] . however, metabolomics profiling of the leafy shoots-methanolic extracts of three swertia species viz. s. chirayita, s. decussata and s. bimaculate, presented s. chirayita as the strongest inhibitor of bglu and xanthones as the most active metabolites responsible for the swertia species' potency [146] . the c-2-glycoside of norathyriol, mangiferin (40) emerged as the most active and therapeutically promising xanthone with ic 50 ¼ 38 mm and 50-fold more potent than silymarin. shikunshito-kamiho (sktk), a traditional oriental formulation and fenugreek seeds (fgs), an indian spice, are natural ethnotherapeutic agents with potentials identical to luteolin (11) i.e., attenuation of colon carcinogenesis via potent inhibition of bacterial bglu activity [147, 148] . 5-weeks oral administration of spf male icr mice's diet supplemented with 20 mg/kg or 60 mg/kg sktk water extracts, after 10 weeks subcutaneous injection of dmh (weekly), equipotently reduced bglu activity during and after treatment by 14.9% and 21.3% of controls (dmh alone) respectively. tumour incidence in colon, measured by the number of aberrant crypt foci was also significantly reduced from 21.6 (distributed in sigmoid and descending colons) in controls, to 6.9 and 6.4 (distributed in rectum) at 20 mg/kg and 60 mg/kg doses respectively. similarly, diet supplementation with fgs significantly reduced intestinal bglu activity, leading to a marked reduction in tumour incidence from 93.3% in male wister rats receiving dmh alone for 15 weeks to 16.6% in rats receiving dmh þ 2 g/kg fgs water extracts for 30-weeks cumulative period. flavonoid, saponin and fibre rich content of fgs were presumed to be responsible for its anticarcinogenic activity. the hepatoprotective activity of chinese medicinal formulation e reduohanxiao-tang, used in the treatment of stroke and liver diseases has also been attributed to its ability to inhibit bacterial bglu activity in vivo [149] . water extracts of this local formulation and two of its ingredients viz. rhizomes of pueraria thunbergiana and scutellaria baicalensis potently inhibited the activities of e. coli and rat liver-derived bglu with ic 50 values ranging from 0.35 to 1.42 mg/ml. subsequently, oral administration of these extracts at 100 mg/kg alleviated ccl 4 -induced liver injury measured by significant reduction in serum aspartate aminotransferase (ast), alanine aminotransferase (alt), and lactic acid dehydrogenase (ldh) levels, compared to controls. the superior hepatoprotective effect of pueraria thunbergiana was accredited to isoflavone daidzein (41, fig. 7) , an aglycone metabolite of main components puerarin and daidzin by intestinal bacteria. daidzein inhibited e. coli and rat liver bglu with ic 50 ¼ 0.41 and 0.50 mg/ml respectively whereas puerarin and daidzin were inactive. fascinatingly, structurally similar tectorigenin (42), an aglycone metabolite of 7-o-glycoside e tectoridin (43), isolated from the flowers of pueraria thunbergiana, exhibited better inhibitory potency (ic 50 ¼ 0.30 mg/ml) than its congeners [150] . 50 mg/kg intraperitoneal pre-treatment of male icr mice with tectorigenin or 100 mg/kg oral administration of tectoridin provided better hepatoprotection than dimethyl diphenyl bicarboxylate (ddb) and daidzein, by significantly lowering serum ast, alt and ldh levels relative to ccl 4 -treated controls. the prodrug behaviour of tectoridin, identical to puerarin and daidzin above, was also established by its absence in the serum after 250 mg/kg oral administration (only tectorigenin was detected) and its failure to provide desired hepatoprotection after 100 mg/kg intraperitoneal administration. the triterpenoid 18b-glycyrrhetinic acid (44, fig. 8 ) also known as enoxolone, is the aglycone metabolite by human intestinal bacteria responsible for the hepatoprotective activity exhibited by saponin e glycyrrhizinic acid 45 [151] . glycyrrhizinic acid (45) is isolated from the rhizomes of glycyrrhiza uralensis e the sweetening agent in sktk. it exists as a natural glucuronide conjugate of 18b-glycyrrhetinic acid (44) hence making it (45) a substrate for bglu-mediated hydrolysis for consequent release of active inhibitor 44. in vitro evaluation against e. coli and rat liver bglu revealed stronger potency of glycyrrhizinic acid (ic 50 ¼ 12.15 and 97.21 mm respectively) compared to 18b-glycyrrhetinic acid (ic 50 ¼ 509.90 fig. 7 . natural bglu inhibitors profiled from plant extracts and ethnomedicinal preparations. and 42.49 mm respectively). moreover, at 100 mg/kg doses, oral treatment with glycyrrhizinic acid or intraperitoneal treatment with 18b-glycyrrhetinic acid, protected the hepatocytes of male wistar rats against ccl 4 -induced liver injury evident by the significant reduction in ast, alt and ldh levels compared to ccl 4controls. intraperitoneal administration of glycyrrhizinic acid also did not provide hepatoprotection akin to tectoridin (43) . in addition, structurally similar emodinol (46) , isolated from the roots of perennial herb paeonia emodi showed stronger e. coli bglu inhibition in vitro with ic 50 ¼ 63 mm compared to 18b-glycyrrhetinic acid [152] . microbial biotransformation of drugs and/or their precursors to new molecules with modulated pharmacological profile is considered an invaluable tool in drug design and development [153] . the natural product class e steroids, are key substrates in this regard [154] . accordingly, naturally occurring androstane steroid hormone, 5-dehydroepiandrosterone (47, fig. 8 ) and its macrophomina phaseolina metabolites were examined for their bglu inhibitory potentials [155] . the inferior potency/inactivity of metabolites compared to parent 5-dehydroepiandrosterone (ic 50 ¼ 77.9 mm) suggests that having an alkene unit at position-5 and cyclopentanone unit in the molecular architecture is crucial to inhibitory potency. conversely, the synthetic anabolic steroid, dianabol and its metabolites by filamentous fungus cunninghamella elegans or macrophomina phaseolina were inactive against bglu; except metabolite (48) with ic 50 ¼ 60.7 mm [156] . the e-oxime derivative (49) of an inactive metabolite however showed decent inhibitory potency with ic 50 ¼ 49.0 mm, equipotent with d-sal and 2-fold superior than its z-isomer. nonetheless, the therapeutic safety of these dianabol derived bglu inhibitors is questionable considering the increased risk of hepatotoxicity associated with oral administration of 17a-alkylated anabolic steroids [157, 158] . the fascinating ability of prebiotics and probiotics to positively influence the composition and behaviour of human intestinal microbiota, has been continuously explored with considerable success to improve human health [159e161]. precisely, lactic acid bacteria (lab) probiotics can selectively utilize non-digestible oligosaccharide prebiotics as carbon source to produce active metabolites, which modulates or stimulates the activities of certain intestinal bacteria hence, eliciting important physiological response and conferring health benefit [162] . consequently, lab e lactobacillus acidophilus csg afforded stronger inhibition of intestinal bacteria (including e. coli) producing bglu thus providing hepatoprotection to male icr mice, compared to lactobacillus brevis hy7401 and bifidobacterium iongum hy8001 [163] . when anaerobically cocultured with e. coli (hgu-3), l. acidophilus csg also potently inhibited bglu productivity of e. coli compared to other labs. as a result, oral treatment of the murine models with 500 mg/kg of l. acidophilus csg alleviated ccl 4 -induced hepatotoxicity by lowering ast and alt levels to 66% and 57% respectively, of ccl 4 control group. whereas, for t-bhpinduced hepatotoxicity, ast and alt levels were lowered to 62% and 48% respectively, better than reference hepatoprotective agent ddb. similarly, lactobacillus plantarum cfr 2194 was the most effective strain of lactobacilli metabolizing fructooligosaccharide prebiotics to short chain fatty acids, thereby altering bglu productivity of e. coli [164] . lactic acid (50, fig. 9 ) and especially nbutyric acid (51) were identified as the major short chain fatty acid metabolites in the culture filtrate of lactobacillus plantarum cfr 2194 and fructooligosaccharides responsible for the observed decrease in bglu activity. the most abundant and relatively stable thiosulfinate, s-methyl methanethiosulfinate (52, fig. 9 ), found in chinese chive (allium tuberosum rottier), has exhibited strong inhibitory potency (ic 50 ¼ 3.60 mm) against e. coli bglu [165] . compared to other disulphides viz., dimethyl, allyl methyl, and diallyl disulphides, compound 52 is more useful for alleviating drug induced toxicities or providing hepatoprotection. in parallel, a naturally occurring acetal isolated from red macroalga neodilsea yendoana, isogloiosiphone b (53), possesses similar potential, although with weaker potency (ic 50 ¼ 57.0 mm) compared to compound 52 [166] . the adverse role of inflammatory pathways and corresponding mediators including lysosomal enzymes during the pathogenesis of myocardial infarction again implicates bglu in the cardiovascular disease. in a study which examined the protective effects of thymol (54, fig. 9 ) on inflammation in isoproterenol-induced myocardial infarction using male albino wistar rats [167] , the therapeutic benefit of inhibiting the release of bglu, other lysosomal enzymes and pro-inflammatory cytokines was evident in the restoration of near-normal myocardial histology and function, compared to diseased controls. rats pre and cotreated with thymol by intragastric gavage at 7.5 mg/kg body weight/day for 7 days and subcutaneous injection of 100 mg/kg isoproterenol for 2 days (day 6 and 7), showed significantly reduced levels of cardiac troponin-t (a cardiotoxicity biomarker) and high-sensitive c-reactive protein in their sera, compared to those treated with isoproterenol only. most importantly, the activities of lysosomal enzymes i.e. bglu, bgalactosidase, cathepsin b and d in serum and heart were also potently inhibited to near-normal. these anti-inflammatory effects of thymol afforded a well-protected myocardium with stable histological architecture. polyhydroxylated piperidine and pyrrolidine alkaloids commonly referred to as iminosugars (fig. 10a) , are a noble class of sugar mimics in which the pyrano or furano endocyclic oxygen has been replaced with a basic nitrogen atom [168] . this class also include isoiminosugars (i.e. 1-azacarbasugars or 1-n-iminosugars) having a methylene unit in place of endocyclic oxygen and the anomeric carbon replaced with nitrogen. although these ring alterations render iminosugars metabolically inert, their protonated forms mimic the pyranosyl or furanosyl unit in gh substrates, especially the putative oxocarbenium ion-like transition state (2, fig. 2 ) in gh catalysed hydrolysis [21, 169] . this in turn facilitates their recognizability by ghs and other carbohydrate-recognizing proteins for corresponding enzyme inhibition. since the isolation of nojirimycin (55, first iminosugar) and siastatin b (56, first isoiminosugar) from streptomyces cultures in 1966 and 1974 respectively [170, 171] , these sugar mimics have been a recurring subject of intensive research owing to their extensive biological activities and therapeutic applications elicited majorly through potent inhibition of ghs [172] . the strongest iminosugar-based inhibitors of bovine liver-derived bglu reported so far (fig. 10b) , with promising antitumor potentials are siastatin b derivatives 57, 58 and 59 with ic 50 ¼ 65, 62 and 68 nm respectively [173] . therefore, this section reviews iminosugars and their analogues as natural productinspired bglu inhibitors, without undue repetitions of other monographs [4, 21, 168, 169] covering them. uronic analogues of nojirimycin bearing glycaro-1,5-lactams or the bicyclic analogues with imidazole and tetrazole units (i.e. tetrahydrotetrazolopyridine-5-carboxylates and imidazopyridine-5carboxylates respectively), were prepared to examine the effect of sugar-acid configuration and presence of lactam, tetrazole or imidazole moieties on inhibitory potency against bovine liver bglu [174, 175] . evidenced by the most potent inhibitor 60 ( fig. 11 ) with k i ¼ 12 nm, gluco-configured units, are stronger bglu inhibitors compared to galacto-and manno-analogues, due to their better mimicry of glucuronic acid (4). the imidazole ring also conferred stronger inhibitory potency than tetrazole or lactam units whereas glycarolactams and tetrazoles shared similar potencies; except galacto-tetrazole (64) with~200-fold inferior potency to galactolactam (62) . further, although sugar configuration at c-4 was found to be ineffective on bglu inhibition for glycaro-1,5-lactams 61 and 62, it was very significant for tetrazole and imidazole derivatives. gluco-tetrazole (63) was 300-fold better than galacto-tetrazole (64), while gluco-imidazole (60) was 600-fold more potent than galacto-imidazole (65) and 1200 superior to manno-imidazopyridine (66) , which differs at c-2. moreover, sugar configuration of acid moiety at c-5 also had significant influence on potency. lconfigured units 67, 68 and 69 were 20-50-fold weaker than their d-isomers 63, 62 and 61 respectively. more importantly, the 2-fold increased potency of gluco-imidazole (60) compared to gluco-tetrazole (63) was attributed to stronger interaction of gluco-imidazole (60) with catalytic nucleophile glu540 compared to glucotetrazole (63) . interaction with catalytic acid glu451 was compromised due to protonation of the imidazole ring in the zwitterionic form. conversely, similar bicyclic molecules of nojirimycin with cyclic carbamate, urea or guanidine pharmacophores are weaker bglu inhibitors [176] . in all, only cyclic carbamates 70e73 (fig. 12) showed moderate inhibitory activity against bovine liver bglu. cyclic carbamates with carboxylic acid unit at c-5 of nojirimycin ring were also better inhibitors than their hydroxymethyl analogues. thus, compounds 71 and 73 were the most potent overall with ic 50 ¼ 218 and 259 mm respectively. it is noteworthy that bicyclic indolizidine iminosugar 74, with hydroxymethyl unit is also a weak inhibitor (<50% inhibition at 1 mm), although it exhibits desirable selectivity (7-fold) for e. coli bglu [177] . surprisingly, carbasugar 75 shared a similar fate with ic 50 ¼ 170 mm against e. coli bglu, even though it is not an iminosugar [178] . taken together, these results partly suggest that ability to mimic d-glucuronic acid favours bglu inhibitory activity. accordingly, glucuronic acid analogue of naturally occurring hemiaminal calystegine b 2 (76, fig. 13 ), uronic-noeurostegine (77) , was synthesized in 27-steps from levoglucosan [179] . compound conferred with improved selectivity for e. coli bglu. this is partly due to their improved lipophilic balance for bacterial cell penetration afforded by their c-alkyl unit and improved chemical stability of cec bond at the pseudoanomeric c-1 centre [180] . liminosugar c-glycoside, (à)-adenophorine (80, fig. 14a ), an enantiomer of natural iminosugar c-glycoside (þ)-adenophorine (81), was prepared together with it analogues via skeletal rearrangement of corresponding azepanes [181] . however, compound 80 and analogues were found to be weak but selective inhibitors of e. coli bglu. the c-propyl analogue, compound 82 with ic 50 ¼ 586 mm showed total selectivity for the bacterial ortholog. it was also superior to compound 80, its azepane precursor 83, and other iminosugars in the study which showed moderate selectivity with 3.1e18.1% inhibition at 1 mm. however, increasing the lipophilicity of azepane scaffold 84 via c-2 or n-alkylation, while tuning the configuration of alkyl and hydroxy substituents at c-2 and c-6 respectively (fig. 14b) , birthed e. coli bglu inhibitors with markedly increased potency and highly conserved selectivity [182] . sar analysis revealed that a combination of (6s)eoh unit and n-alkylation with hexyl, nonyl or dodecyl produced stronger inhibitors and their potency increased with increasing chain length. in contrast, (2r, 6r)-c-butyl and (2s, 6r)-cnonyl derivatives, compounds 85 and 86 respectively, were the most active c-alkylated analogues with ic 50 ¼ 261 and 27 mm respectively. compound 85 was stronger than its n-alkylated analogue, while compound 86 showed a compromised selectivity (ic 50 ¼ 97 mm against bovine liver bglu). overall, highly lipophilic compound 87, having (6s)en-dodecyl framework was the strongest inhibitor with ic 50 ¼ 3.30 mm, 3-fold superior potency to (6s)e n-nonyl analogue 88 (ic 50 ¼ 10.0 mm) and absolute selectivity for e. coli bglu. furthermore, substituent type and configuration at c-6 also influenced the inhibitory potencies of noeuromycin azepane analogues [183] . (6s)-configured compound 89 and 90 were weakly potent, compared to their inactive (6r)-configured analogues. compound 89 (ic 50 ¼ 139 mm) was 4-fold more potent than its 6hydroxymethylated analogue compound 90. incorporating the acetamido unit in siastatin b (56), while retaining the iminosugar c-glycoside scaffold via similar skeletal rearrangement of azepanes, afforded l-configured c-glycosides of 1-deoxynojirimycin (91) with weak inhibitory potencies [184] . sugar configuration at c-1 and c-2 (fig. 14d) , significantly influenced selectivity for ghs. hence, (2r, 3r)-configured molecules were selective inhibitors of e. coli bglu but inactive against bovine liver bglu, a-n-acetylgalactosaminidase and b-n-acetylgalactosaminidase. whereas, (2s, 3s)-configuration infused selectivity for b-n-acetylgalactosaminidase. consequently, (2r, 3r)-configured compound 92 with a rigid benzyl substituent emerged as the best e. coli bglu inhibitor in the series with ic 50 ¼ 90.7 mm compared to 37.2% inhibition at 1 mm of compound 93 with (2s, 3s)-configuration and butyl unit. the authors attributed these inferior activities to stereochemical mismatch between the l-configured units and dconfigured substrates of ghs. we also conceive that the presence of hydroxymethyl and not carboxylic unit (to mimic glucuronic acid) might also be responsible for the weak inhibitory potencies. this was indeed the case for 3,4,5-trihydroxypipecolic acids, uronic analogues of 1-deoxynojirimycin [185] . d-isomers were stronger than corresponding l-isomers, while the most active bglu inhibitors were those with better mimicry of glucuronic acid viz. dgluco and d-galacto configured units; compounds 94 and 95 respectively (fig. 15 ). compound 94 showed 3-fold selectivity for bovine liver bglu (ic 50 ¼ 70 mm), whereas compound 95 displayed an exclusive inhibition (ic 50 ¼ 86 mm). azoles are a prominent class of heterocycles with at least one nitrogen atom in their 5-membered aromatic ring. due to their structural rigidity and amazing physicochemical properties, which confers highly coveted and broad pharmacological activity spectrum and therapeutic potentials, they have remained a targeted scaffold of many synthetic protocols. members of this class include pyrazoles, imidazoles, thiazoles, triazoles, oxadiazoles, thiadiazoles and tetrazoles together with their benzo analogues i.e., indoles, benzimidazoles, benzothiazoles and benzotriazoles. these compounds also enjoy increased hydrogen bonding (h-b) capability furnished by ring n and/or o atoms for biomolecular targets binding. consequently, this section is an overview of reported bglu inhibitors bearing one or more azole nuclei. the critical focus is on the most potent inhibitor(s) in a given series, the pharmacological profile, structure-activity relationship (sar) and molecular docking analysis. bglu inhibitory potency (ic 50 ¼ 1.20e44.16 mm) on a set of imidazolylethylaryl carboxylates compared to reference inhibitor d-sal (ic 50 ¼ 48.38 mm) [186] . compound 96 (fig. 16 ) emerged as the most potent bglu inhibitor (ic 50 ¼ 1.20 mm) with 40-fold superior potency to d-sal. sar analysis supported by in silico studies articulated that compounds with electron-donating groups (edgs) displayed inferior inhibitory activities compared to those with electron-withdrawing groups (ewgs). moreover, molecular hybridization with indole nucleus resulted in a potent bglu inhibitor compound 97 with ic 50 ¼ 2.10 mm and 23-fold increased potency than d-sal. compound 96 and 97 displayed strong h-b interaction of indole nh unit and p-p interaction with active site residues of bglu. in vitro screening of imidazolopyridines ( fig. 17 ) for their bglu inhibitory potentials presented dihydroxy-substituted derivatives as promising inhibitors of enzyme activity [187] . (fig. 19) . however, replacing oh unit with more lipophilic meo or eto groups led to significant loss of activity. molecular docking analysis also disclosed the importance of iminic nitrogen and proximity of thiazole nitrogen to catalytic acid glu451 for strong inhibitory potency. compound 102 with adjacent oh units had a favourable fit into bglu catalytic pocket for stronger interactions with amino acid residues glu540, glu451 and tyr508. however, installing pyren-1-ylmethylenehydrazinyl moiety on thiazole skeleton improved bglu inhibitory potency [190] . comwith br, me and meo substituents were weaker inhibitors [191] . as a result, compound 108 with ic 50 ¼ 2.16 mm (fig. 21) , was the most potent inhibitor. it was 2 and 22-fold stronger than compound 109 and d-sal respectively. docking studies revealed that the free amino nh unit in compound 108 formed strong h-b interaction with oh unit of catalytic acid glu451. the dichloro substituents also substituted phenyl units at position-2 of benzimidazole core [192] . activity was dependent on the presence and position of cl and oh substituents hence 3,4-dichlorophenyl analogue 110 (fig. 22 ) was found with strongest inhibitory potency (ic 50 ¼ 6.33 mm). interestingly, introducing 5,7-dichloro unit on benzimidazole nucleus gave remarkable results as previously inactive molecules gained significant activity [193] . compound 111 emerged as the most potent inhibitor overall with ic 50 ¼ 4.48 mm and 11-fold stronger than d-sal. moreover, replacing an oh unit of the dihydroxy substituent with meo led to significant or total loss of activity. f, me, no 2 , naphthyl and anthracenyl units also gave inactive compounds. [194] . molecules containing ewgs no 2 , cl and f groups were superior in the series (fig. 23) . thus, the best activity was found with orthoakin to imidazole-indole hybrid compound 96 with ic 50 ¼ 2.10 mm (fig. 16) , molecular hybridization of benzimidazole with amide bond bioisostere, 1,3,4-oxdiazole, resulted in an isopotent hybrid 113 ( fig. 24 ) with ic 50 ¼ 2.14 mm [195] . again, ohsubstituted derivatives were stronger inhibitors compared to those containing f, cl and no 2 , me and meo substituents. docking studies revealed that the molecular hybrids adopted a linear configuration in the active site of bglu. the oxadiazole nucleus and central-phenyl ring of compound 113 formed strong h-b interaction with nh 2 unit of asn484 and phenolic oh of tyr508 respectively. compound 114 (ic 50 ¼ 3.14 mm) on the other hand interacted with catalytic acid glu451 and tyr508 via its central phenyl and phenol rings respectively. in another comparative study using 113 as lead but replacing benzimidazole with benzohydrazone unit (fig. 25) , the importance of benzimidazole nucleus to bglu inhibitory activity was established by the pronounced loss of activity [196] . nevertheless, oh-substituted derivatives and benzenetriol compound 115 emerged as the strongest inhibitor with ic 50 ¼ 7.14 mm. inhibitory potency was dependent on substituent's position in the order; para ˃ meta ˃ ortho for oh, no 2 and cl groups, while the presence of me or meo groups gave inactive compounds. molecular docking studies showed that inhibitory activity correlated strongly with the strength of h-b interaction hence the improved potency seen with oh-substituted derivatives. notably, the benzenetriol oh unit on compound 115 formed h-b interactions with glu540, asp207, tyr508, his385, and asn450, while the benzohydrazide unit interacted similarly with tyr508, glu540 and tyr504. in like manner, replacing the pharmacophores in compound 115 i.e. hydroxyl for methoxy and imine for more polar sulfonamide unit (fig. 26) , led to stronger bglu inhibitory potency for the resulting oxadiazole-benzenesulfonamides [197] . the most potent inhibitors were compounds 116 and 117 with ic 50 ¼ 2.40 and 6.34 mm respectively; compound 116 showed similar potency as compound 113. inhibitory activity was improved for parasubstituted derivatives, while ewgs produced stronger inhibitors than edgs. binding interactions in the active site of bglu especially h-b interaction of benzamide nh unit with glu451 established the improved activity of the sulfonamides compared to benzohydrazones. compound 116 formed h-b interaction via sulfonyl oxygens with asp207 and asn450, while the benzamide oxygen interacted in similar fashion with tyr508. additional ionic bonding between no 2 groups in compound 117 and glu451 also accounted for the improved potency. molecular hybridization (fig. 27) inhibitor d-sal respectively (fig. 28) . in silico studies of these benzothiazoles revealed that they adopted a linear conformation which allowed appropriate fit into the binding groove of bglu. ortho-oh unit on compound 120 formed strong h-b interactions with glu451 while the para-oh unit provided h-b interaction with asp207. the enzyme-inhibitor complex was stabilized in the active site through hydrophobic interactions of benzenetriol ring with indole nucleus of trp587, his385, asn484, tyr504, his509, arg600, and lys606. most importantly, all the active inhibitors were noncytotoxic in a cytotoxicity assay using mouse embryo fibroblasts (3t3-l1) and wistar rat hepatocytes (cc-1). benzothiazole and benzimidazole are bioisosteres due to their structural and pharmacological similarities; therefore, bioisosteric replacement of one for the other has been consistently explored in drug development. accordingly, bioisosteric replacement of the benzimidazole moiety in compound 113, to give new benzothiazole-oxadiazole hybrids resulted in equally potent bglu inhibitors [200] . the most potent compound 122 (fig. 29 ) with ic 50 ¼ 2.16 mm, was equipotent with compound 113. fascinatingly, compound 123 (ic 50 ¼ 4.38 mm) was also isopotent with similarly substituted compounds 121 and 111. further, docking studies of compounds 122 and 123 showed that h-b interactions of phenolic oh units with active site residues viz., glu451, tyr508, and asp207, favoured their strong inhibitory potency. moreover, compound 122 formed hydrophobic interactions with tyr504 and lys606 via its thiazole core and benzene ring respectively. the reduced potency of compound 123 was established in the strength of these interactions as well as the absence of additional van der waal contacts of benzothiazole ring in compound 122 with catalytic residues glu451 and glu540. in silico pharmacokinetic properties modelling of these inhibitors predicted good cell permeability and solubility. the compounds were also non-cytotoxic to 3t3-l1 and cc-1 cell lines. following a similar molecular development strategy leading to compound 122, new benzothiazole-hydrazone hybrids were assembled by deleting the 2-oxadiazolylphenol fragment in compounds 122 and 115 (fig. 30) [201] . compared to the parent compound series, the new hybrids had marked reduction in potency. the recurring pattern of reduced or abolished activity also persisted with me and meo substituents. nevertheless, compound 124 was 3.2.1.7. triazole. the triazole ring also affords potent bglu inhibitors. in vitro evaluations of 1,2,4-triazole-schiff bases [202] presented isatin-containing compound 125 (fig. 31) as the strongest inhibitor with ic 50 ¼ 2.50 mm and 19-fold superior activity than d-sal. cl, oh and no 2 substituents at ortho-positions also gave appreciable activities whereas me, meo, cumyl and other aryl derivatives were inactive. docking simulations of compound 125 in the active site of bglu revealed strong h-b interaction between isatin nh and catalytic residues glu540 and tyr504. the phenyl ring also provided p-anion and p-p interactions with glu451 and tyr504 respectively while van der waal contacts with lys606, tyr508, val410 and asn450 stabilized the inhibitor-bglu complex. however, amide bond bioisostere, 1,2,3-triazole, is a stronger bglu inhibitor compared to its 1,2,4-triazole isomer. molecular hybridization with carbazole [203] delivered eight compounds having ic 50 < 3 mm (fig. 32) . compound 126 (ic 50 ¼ 0.55 mm) was the most potent hybrid in the series and 83-fold stronger than d-sal. all the carbazole-1,2,3-triazole tethers were non-cytotoxic to 3t3 cells. 3.2.1.8. indole-based hybrids. a library of indole analogues containing the hydrazone unit in compound 115 have been examined for bglu inhibition [204] . evinced by the increased potency, the indole pharmacophore delivers stronger bglu inhibitors compared to benzothiazole and oxadiazole. for instance, the most potent inhibitor 127 with ic 50 ¼ 0.50 mm (fig. 33) , was 100-fold more potent than d-sal and 42-fold stronger than similarly substituted oxadiazole variant from compound 115 library. compounds 128 (ic 50 ¼ 2.40 mm) and 129 (ic 50 ¼ 2.50 mm) were also 3-fold and 43fold stronger respectively than their corresponding oxadiazole variants. moreover, substitution at ortho and para-positions gave stronger inhibitors compared to meta-position, while potency in monosubstituted analogues followed the order; f ˃ oh ˃ cl ˃ pyridyl ˃ no 2 ˃ meo. the excellent potency of compound 127 was afforded by strong h-b interaction of position-5-oh with glu451 and glu540 as well as position-4-oh with tyr508. indole and benzohydrazone nh units formed h-b interactions with asn502 whereas the ligandreceptor complex was stabilized by p-alkyl interaction of indole ring with trp528. expectedly, the reduced activity of compound 128 was seen in its weaker interactions in the active site of bglu. based on the pharmacological significance of thiadiazole pharmacophore and the promising inhibitory activity of indole hybrid compound 127, hybrids of indole-thiadiazole were assembled as new class of bglu inhibitors [205] . generally, thiadiazole nucleus infused stronger inhibitory potencies on the resulting hybrids compared to hydrazone unit. dihydroxy substituted compound 130 emerged as the strongest inhibitor with ic 50 ¼ 0.50 mm and equipotent as compound 127. ortho-substitution was more beneficial to potency than para and meta-substitutions, while inhibitors' strength followed the order; oh ˃ f ˃ me ˃ pyridyl ˃ cl ˃ no 2 (fig. 34) . however, bioisosteric replacement attempt with oxadiazole proved that thiadiazole unit is preferred for stronger inhibition [206] . the strongest potency remained with 2,3-dihrdoxy substituted derivative 131 (ic 50 ¼ 0.90 mm), although 2-cl derivative had an outlying potency. bazedoxifene (fig. 35) is a novel indole-based inhibitor of il-6/ gp130 for the management of triple negative breast cancer [207] . it is also a selective estrogen receptor modulator administered as co-drug with conjugated estrogens (duavee) in estrogen replacement therapy for the prevention of postmenopausal osteoporosis. albeit, the high ratio of circulating estrogen metabolites and parent estrogen aided by bglu deglucuronidation activity in the gut is considered a risk factor for postmenopausal estrogen receptor positive (erþ) breast cancer. accordingly, a study on the therapeutic utility of this combination drug in an ovariectomized mouse model showed significant reduction in faecal bglu activity without altering faecal microbiota diversity [208] . the study articulated the significance of bacterial bglu inhibition to improving the outcome of long-term administered estrogens for postmenopausal women or breast cancer patients. consistent with the excellent inhibitory potencies of indolebased compounds, a library of bis-indoles (fig. 36) have been reported as strong bglu inhibitors [209] . once more, the importance of (di)hydroxy substitution to potency was prominent. compound 132 emerged as the most active molecule with ic 50 ¼ 1.62 mm and 30-fold superior to d-sal. in silico studies suggested that h-b donor-acceptor potential of oh units on compound 132 significantly influenced ligand-receptor interaction. ortho-oh formed strong h-b with amino acid residues asp207 and tyr508, while para-oh interacted similarly with his385. arene-arene interaction of indole ring with tyr504 also aided the favourable binding to bglu. in another study [210] , both compound 132 and its 2,3dihydroxy compound 133 (ic 50 ¼ 1.2 mm) were the strongest inhibitors of bacterial bglu; albeit with moderate cytotoxicity against 3t3 mouse fibroblasts. the presence of n-phenyl substituted thiosemicarbazide unit however introduced marked increase in bglu inhibition of 1hbisindoles [211] . inhibitory potency for monosubstituted analogues followed the order f z cf 3 ˃ cl ˃ br ˃ me ˃ meo and ortho-substitution produced the best result (fig. 37) . accordingly, 2-f to glu540, asn450, lys606, trp528. tyr508 also formed h-b interaction with amide nh and arene-arene interaction with phenyl ring of tolyl unit. chalcones are both a class of naturally occurring flavonoid family and synthetically obtainable compounds with extensive therapeutic applications [213] . the presence of a highly reactive a,b-unsaturated ketone unit and delocalized electrons makes them coveted precursors for many synthetic manipulations and pharmacological explorations. the pro-inflammatory role of bglu in inflammatory disorders motivated the design of a series of (di)hydroxychalcones (fig. 39) , to inhibit the release of bglu from stimulated rat mast cells and neutrophils [214e216]. it was established that hydroxychalcones were stronger and selective inhibitors of neutrophil-derived bglu over mast cell-derived bglu. the best inhibitors were compounds 139 (ic 50 ¼ 0.6 mm; ˃ 160-fold selectivity) and 140 (ic 50 ¼ 1.3 mm; 7fold selectivity). sar analysis showcased the importance of a,bcompound 141 (fig. 40 ) has emerged as the most potent inhibitor with 75% inhibition at 10 mm and only 3-fold stronger than standard salicylic acid, amongst a series of chalcone-mannich adducts [217] . adopting a similar molecular design [218] , a library of chalcone-benzamides were found with slightly improved potency and sar analysis established the preference of ewgs over edgs for better enzyme inhibition. compound 142 containing similar 3-br substituent as compound 141, emerged as the most potent molecule (84.68% inhibition at 1 mm); 3-fold stronger potency than salicylic acid (24.77% at 1 mm). it was also non-cytotoxic in cck-8 assay in contrast to 2-fold increased cytotoxicity of equally potent 3-cf 3 analogue. notably, activity modulation by substituting trimethoxy unit for indole was unsuccessful [219] , leading to prominent reduction in percentage inhibitory activity of the chalcones (˃ 8-fold). structural development of a coumarin (chromen-2-one) compound 143 (fig. 41 ) recovered from virtual screening delivered a set of moderately potent inhibitors of bglu activity [220] . superior activity compared to d-sal (ic 50 ¼ 48.40 mm) was recorded compounds 144e147 with ic 50 values of 9.90, 11.70, 21.40 and 34.20 mm respectively. in another attempt [221] , despite different molecular tunings including polar oh and cl groups, the coumarin pharmacophore remained inactive against bacterial bglu. conversely, structurally isomeric flavenone (chromen-4-one) bearing a pyranose appendage, showed stronger inhibition of bacterial bglu [210] . compound 148 (fig. 42) , the most active inhibitor thereof with ic 50 ¼ 4.50 mm and 10-fold superior potency to d-sal, was totally non-cytotoxic against 3t3 mouse fibroblasts. in silico studies further disclosed the importance of pyranose ring in h-b interactions with active site residues glu503, asp161 and glu413. fascinatingly, introducing 1,3,4-oxadiazole pharmacophore to a chromen-4-one scaffold (fig. 43) , led to significant increase in potency [222] . the new hybrids exhibited consistent trend in their potency based on substituent type and position in the order; f > cl > me > no 2 > pyridyl > meo and ortho > para > meta respectively. thus on the other hand, azacoumarins (1h-quinolin-2-ones) having a basic nitrogen atom in place of endocyclic coumarin oxygen, are conferred with stronger and selective inhibitory potencies against bacterial bglu. plausibly, their admirable activity is provided by the ability to mimic or interfere the oxocarbenium ion-like transition state akin to iminosugars; thus, allowing the alleviation of druginduced toxicities and prevention of colon carcinomas. renowned examples in this class of bglu inhibitors are the strongly potent azacoumarins 151 and 152 (fig. 44) , identified from high-throughput screening [19, 223] . the compounds exhibited in vitro ic 50 values of 0.28 and 0.37 mm respectively, over 1000-fold selectivity for e. coli bglu and 100-fold increased potency than reference inhibitor glucaro-d-lactam 61. the inhibitors also maintained their potency in living bacterial cells with ec 50 ¼ 0.018 and 0.028 mm. other similarly active compounds (ic 50 (once daily), alleviated irinotecan (cpt-11) induced diarrhoea evident by a protected gi epithelium of balb/cj mice compared to bloody diarrhoea due to damaged tissues and glandular structure in those receiving cpt-11 alone [19] . pre-treating c57bl/6j mice with 10 mg of inhibitor 151 before intraperitoneal administration of ulcerogenic dose of nsaids e diclofenac (60 mg/kg), indomethacin (10 mg/kg) and ketoprofen (100 mg/kg), also successfully protected the mice models against nsaid-induced small intestine mucosal damage [224, 225] . moreover, compound 151 reduced all enteropathy parameters by inhibiting gi bacterial bglu-mediated hydrolysis of nsaid-acyl glucuronide in a concentration-dependent manner (ic 50 z 0.164 mm). interestingly, the compound did not alter drug's systemic exposure, hepatobiliary excretion of glucuronides and gi microbiota diversity. although it possesses a short half-life (˂1 h) and pan-cytochrome p450-mediated poor bioavailability (21%). however, an attempt to reduce the inhibitor's serum exposure for increased gi residence by replacing ethoxy unit with hydroxyl or morpholinyl groups led to inferior pharmacological profile compared to parent compound 151 [226] . further, compound 152 exerted 2-fold reduction in the severity of diclofenacinduced anastomotic leakage, without affecting drug's plasma concentration in wistar rats receiving 0.8 mg of the inhibitor and 3 mg/kg of diclofenac [227] . in contrast, rats receiving diclofenac only suffered severe leakage thus suggesting the inhibitor's potential clinical utility to improve anastomotic healing. emerging from the screening protocol which birthed compound 151, three piperazine-containing compounds 153, 154 and 155 ( fig. 45) showed promising inhibitory potentials against bacterial bglu [228] . though with slightly weaker inhibitory strengths (ic 50 the therapeutic significance of fused pyridinone-furans as antiinflammatory drug candidates via inhibition of neutrophil-derived bglu has been reported [234] . compounds bearing meo and cf 3 substituents on ring b (fig. 47) showed over 70% inhibition at 1 mm with no cytotoxic effects (<32% at 10 mm) in cck-8 assay as compared to reference salicylic acid (25% inhibition). replacing ring b with furan, thiophene or pyridine however gave poor inhibitors (<45% inhibition). compound 158 was the most potent with 75% inhibition. the inhibitory potencies of dihydropyrimidinone carboxylates ( fig. 48) , against bovine liver-derived bglu has reiterated that small polar substituents are necessary for strong enzyme inhibition [235] . appreciable potency (ic 50 ˂ 20 mm) was exclusive to cf 3 ˃ f ˃ oh ˃ thienyl ˃ cl substituted compounds in that order. whereas, meo, br, benzyloxy, furanyl, aliphatic alkyl or aryl units either reduced potency when co-substituted with polar f or oh groups, gave inferior activity with monosubstitution or rendered the molecule totally inactive. therefore, the most active compound 159 with ic 50 ¼ 9.38 mm, found favourable fit in the active site of bglu to form strong h-b interactions with key residues. nh of asp207 was both a h-b donor and acceptor to pyrimidinone carbonyl oxygen and free nh at position-1 respectively. carboxylate carbonyl oxygen interacted similarly with arg600 whereas position-3 nh interacted with catalytic residues glu451 and tyr508. however, pyrimidinetriones (barbituric acid) [236, 237] , exhibited inferior potencies compared to standard d-sal (ic 50 ¼ 45.75 mm), despite their similar binding interactions as compound 159 with active site residues and non-cytotoxicity to 3t3 cells. in a predating study, the structural homolog of pyrimidinone, quinazolinone [238] , exhibited a unique trend in inhibitory potency against e.coli bglu with significant tolerance for lipophilic alkoxy groups, although poor activity with methyl substituent persisted. the most active compound 160 (fig. 49) intriguingly, replacing alkoxy units with more polar oh, no 2 and cl substituents led to significant reduction in potency. inhibitory potency therefore followed the order; meo ˃ eto ˃ n(me) 2 ˃ no 2 ˃ oh ˃ cl and ortho ˃ para ˃ meta. importantly, the quinazolinone-based inhibitors were all non-cytotoxic to 3t3 cells. it is also noteworthy that a quinazolinone compound (table si) from ref. [223] , also showed strong inhibitory potency (ic 50 ¼ 1.90 mm) against bacterial bglu. the quinoline nucleus can be regarded as a household name in medicinal chemistry, due to its inexhaustible pharmacological application and occurrence in many clinically important natural products and synthetic molecules. the desirable pharmacokinetic profile and ease of accessibility with wide substrate tolerance for different synthetic protocols, also encourages its utility in countless synthetic explorations. research endeavours aimed at quinolinebased bglu inhibitors are therefore presented in this section. quinoline-3-carbohydrazides [239] , prepared analogously as hydrazone tethers above also holds promising inhibitory activities. similarly, oh unit at ortho-position yielded stronger inhibitors than f > cl > no 2 > pyridyl > thiophenyl > furanyl z me > meo, in that order. moreover, ortho-substitution consistently gave superior inhibitors compared to para-position, while meta-substitution conferred the weakest potency. 2 to 3-fold reduction in potency persisted when oh unit is substituted for meo unit (fig. 50) . hence, compound 162 (ic 50 ¼ 2.11 mm) and 163 (ic 50 ¼ 2.60 mm) emerged as the most potent inhibitors overall. docking studies revealed that oh unit at position-4 of quinoline ring fostered important h-b interaction with catalytic acid glu451, while carbonyl unit of hydrazone moiety formed h-b interaction with nh of tyr504. in another quest for new anti-inflammatory agents, quinolinefuran hybrids (fig. 51) were designed as inhibitors of bglu release from activated neutrophils [240] . sar analysis, established that 2-(furan-2-yl)quinolines (type a) were stronger inhibitors of inflammatory mediators than tricyclic furo[2,3-b]quinolines (type b). whereas, formyl unit favoured potency compared to acetyl, oxime, methyl oxime as well as both a,b-unsaturated butan-2-one and its saturated variant. therefore, non-cytotoxic compound 164 (ic 50 ¼ 5.0 mm) was the strongest inhibitor of bglu release with 3fold superiority to reference trifluoperazine. acetyl isomer compound 165, also had similar non-cytotoxic and strong inhibitory potency (ic 50 ¼ 7.5 mm). akin to azacoumarins (1h-quinolin-2-ones) 151 and 152, new quinoline-pyrazole conjugates were prepared via hit development protocol of compound 166 (fig. 52) identified from highthroughput screening, for the inhibition of intestinal bacterial bglu's hydrolytic activity on sn-38g fo alleviate cpt-11 druginduced toxicity [241, 242] . interestingly, all the resulting derivatives showed good selectivity for e. coli bglu except 2-cl, 3-oh, 3-me and 4-nh 2 substituted derivatives. the trend in inhibitory potency, based on substituent's position varied in the order; meta < ortho < para, while the presence of strongly hydrophilic oh, no 2 and nh 2 units at para-position was detrimental to potency. following oral administration to balb/cj mice for 5 consecutive days compared to compound 168 (40%). whereas, pharmacokinetic profiling of compound 168 revealed its lower plasma concentration and increased gi residence for improved intestinal bglu inhibition compared to azacoumarin compound 151. most importantly, oral co-administration of compounds 167 or 168 at 3 mg kg à1 day à1 for 10 consecutive days with 50 mg kg à1 day à1 intraperitoneal injection of cpt-11, protected balb/cj mice models against cpt-11 drug-induced intestinal mucosal injury, without altering drug's therapeutic efficacy. furthermore, mechanistic studies established that the presence of electronegative and electroneutral surface charges on e. coli active site pocket and the protonation of n-1 and n-5 atoms avails a ph-dependent and selective inhibition for the compounds (fig. 52) . consequently, compounds 167 and 168 are clinically viable agents for protecting against gi e. coli bglumediated mucosal damage and preventing the release of chemical carcinogens crucial to the development of precancerous lesions for colon carcinogenesis. the 200-fold increased inhibitory potency of benzohydrazideen-cyanoethyl tethers [243] , compared to parent compounds articulates the significance of a balanced molecular lipophilicity to bglu inhibition. o-alkylation with benzyl, benzoyl or tosyl groups slightly influenced potency for oh-substituted units; benzylation produced the best result (fig. 53) . hence, 169 (ic 50 ¼ 1.60 mm) and 170 (ic 50 ¼ 2.20 mm) were the most active conjugates in the series. similarly, phenoxyacetohydrazones [244] have displayed 2 to 5-fold improved potency compared to d-sal (ic 50 ¼ 48.40 mm). compounds 171 and 172 (fig. 54 ) emerged as the the desirable therapeutic significance of fused thienothiophenes has stimulated the synthetic exploration of a thienothiophene skeleton 173 (fig. 55) for bacterial bglu inhibition [245] . from the resulting thienothiophene library with interesting structural diversity, only compound 174 showed inhibitory activity against bacterial bglu with ic 50 value of 0.9 mm; 51-fold superior potency to d-sal. notably, the predicted pharmacokinetic properties using molinspiration and osiris calculations suggested that the inhibitor possesses good bioavailability and no toxicity risk as bacterial bglu inhibitor. in vitro study of phenyl disulphideebenzenesulfonamide tethers [246] , disclosed compounds 175 and 176 (fig. 56 ) as potent inhibitors of bglu (ic 50 ¼ 2.20 and 3.34 mm respectively), analogous to similarly substituted oxadiazole-benzenesulfonamide compounds 116 and 117 (fig. 28) . interestingly, bulkier phenyl disulfides 175 and 176 shared similar potency with lower molecular mass thiosulfinate 52, fig. 9 (ic 50 ¼ 3.60 mm). however, substitution at ortho-position gave stronger inhibitors than para-and meta-positions, in contrast to compounds 116 and 117 series. docking studies further revealed that the strong inhibitory potency of compound 175 is aided by the h-b interactions of sulfonamide nh and oxygen with tyr205 and asp207 respectively. the efficacy of urea unit for bglu inhibition was also examined using a library of no 2 substituted n-phenyl ureas [247] . therefrom, appropriate positioning of polar no 2 unit was important to inhibitory potency of examined molecules. compound 177 (fig. 57) , emerged as the only inhibitor with promising activity with ic 50 value of 3.38 mm. however, thiourea analogues bearing meta-cl substituent on n-phenyl ring [248] , were stronger inhibitors compared to those with unsubstituted n-phenyl ring or no 2 substituted ureas (fig. 58 ). akin to compound 141, thioureas containing piperazine unit also exhibited improved potency compared to their morpholine and piperidine analogues, while n-alkylation of piperazine with n-(o or p-methoxyphenyl) or n-(2-pyridyl), extant empirical data identified bacterial bglu as a chelating agent, since enzyme activity was totally lost in the presence of cu 2þ , hg 2þ and ag þ metal ions and restored in the presence of other chelating agents e versene and cysteine [249] . therefore, metal complexes with coordinatable metal centres exhibit significant bacterial bglu inhibitory activities. to this end, pd(ii) complexes containing aniline and triphenylphosphine ligands were synthesized [250] . para-chloroaniline derived metal complex 182 (fig. 59) , displayed the strongest activity against bacterial bglu with 98.5% inhibition and ic 50 ¼ 15.40 mm. it was also 2-and 5-fold superior to meta-chloroaniline derived and n-methyl substituted metal complexes respectively. similarly, n-heterocyclic carbene (nhc) complexes of au(i) [251] , as well as bis (nhc) complexes of au(i) and au(iii) [252] , containing hydroxy, chloride, acetoxy and acetonyl ligands were equally potent bacterial bglu inhibitors with ic 50 ranging from 0.14 to 2.60 mm. the strongest au(i) nhc complex 183 (ic 50 ¼ 0.14 mm) was 327-fold more potent than reference d-sal and 7-fold superior to strongest bis (nhc) complexes 184. nonetheless, undesirable cytotoxicity against 3t3 cells discomfits the therapeutic significance of these metal complexes. parallel to iminosugars 60e74, the inhibitory potencies of structurally similar glycopolymers (fig. 60 ) was dependent on the presence of carboxyl unit, type and configuration of sugar unit. hence, glycopolymers with d-glucaric pendants linked to the polymer frame at c-1 (185), were stronger inhibitors compared to glycopolymers linked at c-5 (186) or those bearing d-gluconic (187) [253], d-mannaric (188) [254] and shorter chain d,l-xylaric (189) and l-tartaric (190) [255] pendants. however, these glycopolymers only show >60% inhibition at millimolar concentrations. patents and patent applications disclosing potent inhibition of bglu particularly bacterial bglu and the therapeutic significance thereof, have been documented. we therefore review in this section, patents applications filed in the present millennium relevant to bglu inhibition. we have also included the only biomarker application of the enzyme filed within the period [256] . however, some of the patents and applications [257e263] emanated from published papers [19, 210, 228, 229, 241, 262] , reviewed in previous sections; therefore, only their summary is presented in table 3 . considering the limitations associated with traditional clinical assessments for periodontal diseases diagnosis and the improved reliability of bglu as a biomarker, a simple method requiring less expertise has been claimed to quantify elevated levels of the enzyme in the saliva of diseased patients relative to healthy ones, as a biomarker of disease risk and status [256] . the invention involves adding known b-d-glucuronide substrates (4-methylumbelliferone or phenolphthalein b-d-glucuronides) to a sample of patient's saliva in bglu activity assay and thereafter quantifying the amount of aglycone produced via fluorometry, colorimetry or spectrophotometry. the addition of a labelled polyclonal or monoclonal antibody specific for bglu to saliva sample and subsequent estimation of the amount of labelled antibody forming bglu-antibody complex was also claimed, as well as a test-kit for claimed methods. bacterial bglu-mediated hydrolysis of steroid glucuronides on the skin leads to body odours hence inhibitors of the process were developed and applied in deodorant and antiperspirant formulations [264] . the invention claimed that deodorant formulations with described compounds only inhibits bglu activity without altering the natural composition of skin microbiota; in contrast to bacteriostatic or bactericidal mode of action of prior arts. amongst the claimed inhibitors of different class, the strongest inhibition of e. coli bglu was found with galactaric acid (99% inhibition at 0.1% test concentration in water), as well as zinc glycinate and zinc gluconate; 99% and 97% inhibition respectively at 0.25% test concentrations. in the same vein, potent inhibitors of bacterial bglu activity on urine have found non-therapeutic application in hygiene and sanitary products for suppressing the generation of urine odour [265] . at 0.1 wt% in dipropylene glycol, all the claimed inhibitors viz. macrocyclic ketones, ketones, macrocyclic lactones, macrocyclic oxalactones, esters, aldehydes, alcohols, ethers and terpenes, showed over 60% inhibition of e. coli bglu. macrocyclic ketones were stronger inhibitors than others. consequently, the invention's most active inhibitor was compound 191 with 100%, 99.9% and 95.8% inhibition at 0.1, 0.01 and 0.001 wt% respectively. it was also effective after 48 h in suppressing the increase in urine odour when incorporated into different hygiene and sanitary products. phenoxy thiophene sulfonamides [266] were designed as adjuvants to eliminate the dose-limiting gi toxicity (diarrhoea) of cpt-11, through potent inhibition of bacterial bglu-mediated deconjugation of active metabolite's glucuronide (sn-38g) in the intestine; thus, improving the drug's therapeutic efficacy. the synthesis of 76 bglu inhibitors and 18 analogues of brite-355252 (compound 192) , the most active compound (ic 50 ¼ 20 nm), together with their therapeutic application was claimed. sar study of compound 192 analogues revealed that inhibitory potency was markedly dependent on n-piperazinyl pendant at meta-position of phenoxy ring. over 500-fold reduced inhibitory potency occurred with n-methylation (akin to compound 156), 15-fold reduction when appended at para-position and total loss of activity when removed. uninstalling the chloro unit on thiophene ring also slightly diminished potency by 5-fold. whereas, replacing naphthyl with substituted phenyl units afforded inhibitors with similar potencies i.e. ic 50 < 50 nm (table si) ; as a result, 193 was equipotent with 192. bglu is a physiologically important lysosomal glycosyl hydrolase with appreciable therapeutic potentials such as biomarker for disease diagnosis, endogenous bioactivator in prodrug monotherapy and enzyme replacement therapy. the enzyme's role in cell proliferation and inflammation also renders it a potential target for anticancer and anti-inflammatory drugs development respectively. moreover, since a significant number of commercially available drugs are metabolised by glucuronidation, hydrolytic activity (i.e. deglucuronidation) by bglu in human intestinal microbiota has been linked to colon carcinogenesis and genotoxicity as well as drug-induced dose-limiting toxicities of anticancer agents and nsaids, which thwarts their therapeutic potential and clinical utility. therefore, potent inhibition of bglu holds enormous clinical importance. generally, our literature survey found that a significant number of natural products-derived inhibitors display moderate inhibition of bglu, and increased potency is found with inhibitors containing a flavonoid skeleton. the physiological tolerance, acceptable toxicity and favourable pharmacodynamic profiles of these natural inhibitors, posits them as worthy scaffolds warranting witty molecular developments. in addition, iminosugars distinguish themselves as a unique class of natural product-inspired molecules with promising potentials regardless of their usually multiple synthetic steps. notable amongst these are the nojirimycin analogues and iminosugar c-glycosides, both conferred with highly selective inhibition of bacterial bglu. however, the inhibitory potency of these carbohydrate mimics relies on several factors such as sugar configuration, mimicry of glucuronic acid substrate and correct lipophilic balance. hence, to harness their remarkable potential, ingenious manipulations of the synthetic medicinal chemist interested in exploring their chemical space is expressly required. on the other hand, purely synthetic inhibitors show fascinating diversity in their inhibitory potencies and pattern, due to the plethora of pharmacophoric enrichments possible for a single molecular design. based on the foregoing, a comprehensive list of potent synthetic inhibitors (ic 50 5 mm) in this review is provided in table si of supporting information. evinced by their nanomolar ic 50 values, quinoline-pyrazoles (167 and 168), piperazinecontaining compounds (156 and 157; 192 and analogues) as well as the indole nucleus, are conferred with the strongest inhibition of bglu activity overall. the order of potency for hydrazone tethers was found as; bisindole ˃ indole > quinoline > thiazole ˃ benzothiazole z oxadiazole. whereas, no 2 , cl, particularly f and oh substituents at ortho-or para-positions usually produce stronger bglu inhibitors compared to their meta-substitutions. taken together, these suggests that the presence of polar groups with strong hydrogen bonding potential is crucial to inhibitory potency, while an indiscriminate increase in molecular lipophilicity is detrimental to strong bglu inhibition. consequently, we envisage that the model bglu inhibitor will be that which is appositely tuned in its lipophilicity and polarity for easy cell penetration, favourable fit into the binding/catalytic pocket of bglu for energetically stable binding and strong enzyme inhibition. we also perceive that rationale molecular hybridization of those active class of natural products and synthetic molecules will furnish stronger inhibitors of bglu with improved selectivity and acceptable toxicity profile. furthermore, we found that due to the mixture of electroneutral and electronegative spots on the surface of bacterial bglu binding pocket at physiological ph, in contrast to the electropositive spots for human bglu, the presence of a protonatable n-atom confers improved selectivity for bacterial bglu inhibition. this was particularly true for piperazine based inhibitors. therefore, we believe further probing of this phenomenon will allow structureactivity guided molecular tuning to afford stronger bglu inhibitors. to the best of our knowledge, mechanistic studies on the behaviour of these inhibitors is scanty in 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treating adverse intestinal effects of nonsterodal anti-inflammatory drugs compounds, compositions, and methods for selectively inhibiting b-glucuronidases and alleviating side effects associated with drug treatment induced diarrhea selective b-glucuronidase inhibitors as a treatment for side effects of camptothecin antineoplasticagents inhibitors of microbial b-glucuronidase enzymes and uses thereof c]quinoline derivatives for inhibition of b-glucuronidase quinazolines as b-glucuronidase novel inhibitors in-silico based techniques in the identification of potent b-glucoronidase inhibitors glucuronidase inhibitors for use in deodorants and antiperspirants. us 7 dmh: 1,2-dimethylhydrazine d-sal: d-saccharic acid-1,4-lactone d-sdl: 2,5-di-o-acetyl-d-glucaro-1,4:6,3-dilactone d-gl: d-glucurono-g-lactone gcf: gingival crevicular fluid gh: glycosyl hydrolase gi: gastrointestinal gg-i: gingival index gpcr: g protein-coupled receptors h-b: hydrogen bonding ic 50 : half maximal inhibitory concentration il-1b: interleukin-1 beta il-8: interleukin-8 k cat : rate constant of catalysed reaction kda: kilodalton k i : inhibition constant k uncat : rate constant of uncatalyzed reaction lab glucuronide conjugate of sn-38 tnf-a: tumour necrosis factor-a udp: uridine-5 0 -diphosphate ugt: udp-glucuronosyltransferase the national research foundation (nrf) of south africa is appreciated for kic grant and other financial support. supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111921. key: cord-352844-wggg3ynb authors: annunziata, francesca; pinna, cecilia; dallavalle, sabrina; tamborini, lucia; pinto, andrea title: an overview of coumarin as a versatile and readily accessible scaffold with broad-ranging biological activities date: 2020-06-29 journal: int j mol sci doi: 10.3390/ijms21134618 sha: doc_id: 352844 cord_uid: wggg3ynb privileged structures have been widely used as an effective template for the research and discovery of high value chemicals. coumarin is a simple scaffold widespread in nature and it can be found in a considerable number of plants as well as in some fungi and bacteria. in the last years, these natural compounds have been gaining an increasing attention from the scientific community for their wide range of biological activities, mainly due to their ability to interact with diverse enzymes and receptors in living organisms. in addition, coumarin nucleus has proved to be easily synthetized and decorated, giving the possibility of designing new coumarin-based compounds and investigating their potential in the treatment of various diseases. the versatility of coumarin scaffold finds applications not only in medicinal chemistry but also in the agrochemical field as well as in the cosmetic and fragrances industry. this review is intended to be a critical overview on coumarins, comprehensive of natural sources, metabolites, biological evaluations and synthetic approaches. coumarins are a wide family of secondary metabolites found in various species of plants (more than 1300 coumarins have been identified from natural sources, especially green plants) but also fungi and microorganisms [1, 2] . the main pathway of coumarin biosynthesis occurs by shikimic acid route, via cinnamic acid, through phenylalanine metabolism [3] . the history of these natural products began 200 years ago-the name of the class derived from the plant coumarouna odorata (dipteryx odorata) from which the simplest member of this family, coumarin itself ( figure 1) , was isolated by vogel in 1820 [3, 4] . chemically speaking, coumarins are organic heterocycles and their nucleus is represented by benzo-α-pyrone (2h-1-benzopiran-2-one), whose systematic nomenclature was established by international union of pure and applied chemistry (iupac) [5] . natural coumarins are subdivided in different classes based on their chemical diversity and complexity-simple coumarins, isocoumarins, furanocoumarins and pyranocoumarins (both angular and linear), biscoumarins and other coumarins such as phenylcoumarins (table 1 ) [6] . coumarins have several attractive features, such as low molecular weight, simple structure, high in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric thunberginols (antidiabetic) [19] phenylcoumarins int. j. mol. sci. 2020, 21, x for peer review 3 of 83 thunberginols (antidiabetic) [19] phenylcoumarins isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and ohydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7-hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and o-hydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . int thunberginols (antidiabetic) [19] phenylcoumarins isodispar b (anti-inflammatory) [20] this privileged scaffold serves as an important platform for the design of compound libraries in the search for new drug candidates. here, we critically review the actual state of the art on natural and synthetic coumarins, focusing on the biological activity, structure-activity relationships (sars), pharmacokinetics (pks) and their potential applications in the pharma and agri-food sectors. furthermore, an overall overview of the most common synthetic routes applied to obtain simple coumarins are provided, together with a further discussion on alternative, innovative and green synthetic methodologies. coumarin is metabolized by cytochrome p450-linked mono-oxygenase enzyme (cyp2a6) system in liver microsomes, which leads to hydroxylation; subsequently, the hydroxylated metabolite follows phase ii conjugation reactions. although coumarin could potentially be hydroxylated at all six possible positions (i.e., carbon atoms 3, 4, 5, 6, 7 and 8) (figure 1 ), 7hydroxycoumarin and 3-hydroxycoumarin are the main metabolites. the former one faces phase ii conjugation reaction resulting in the glucuronide derivative, whereas 3-hydroxycoumarin can be further metabolized by ring splitting to form two products, o-hydroxyphenyllactic acid and ohydroxyphenylacetic acid ( figure 2 ) [4, 21] . since the expression of cyp2a6 varies between individuals, due to genetic and environmental factors, an inter-individual variation in the metabolism of coumarin drugs is possible [4] . in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric in a recent work, eight coumarin metabolites, which had not been identified previously, were detected by means of uplc/quadrupole-tof tandem mass spectrometry in human urine [22] . among them, positional isomers of 7-hydroxycoumarin glucuronide and 7-hydroxycoumarin sulphate were found. it was proposed that such isomers should bear the substituent in position 5, 6 or 8. metabolites coming from a double hydroxylation and subsequent conjugation with glucuronic acid or sulphate group (and their isomers) were detected as well. another metabolite was the one obtained by a double hydroxylation of the coumarin ring, followed by methylation and glucuronidation at the two newly generated hydroxyl groups. finally, the n-acetylcysteine conjugated metabolite was identified. in this work, o-hydroxyphenylacetic acid was also detected in the samples, whereas free coumarin and o-coumaric acid were not found, indicating that coumarin was completely metabolized before excretion, after oral administration. this meant that o-coumaric acid found in human plasma [23] underwent a biotransformation process before being eliminated, probably leading to o-hydroxyphenylacetic acid. in a healthy human body, normal metabolic processes produce free radicals and other highly reactive species such as ions, molecules with unpaired electrons, reactive oxygen, carbon, nitrogen or sulfur species (ros, rcs, rns or rss). when these species are overproduced, oxidative processes might cause cellular damage, affecting cellular components and causing ionic imbalance or mitochondrial disfunction [24] . the role of oxidative stress in different pathologies is well know: inflammation, cardiovascular diseases, cancer, diabetes and even neurodegenerative disorders often count oxidative damage among their pathological features [25] [26] [27] . therefore, exogenous antioxidants might be useful in order to maintain the right concentration of radicals, reducing the amounts of free radicals and avoiding oxidative stress [28] . the antioxidant potential of natural and synthetic coumarins has been deeply investigated in the last years and it became clear that poly-hydroxy or phenolic coumarins are efficient antioxidants in biologicals systems [29] . here below the most recent updates in this field are reported. in 2019, couttolenc and collaborators studied the radical scavenging activity of three hydroxy-4-methylcoumarins (1-3, figure 3 ) by means of experimental and theoretical methods [30] . int acid found in human plasma [23] underwent a biotransformation process before being eliminated, probably leading to o-hydroxyphenylacetic acid. in a healthy human body, normal metabolic processes produce free radicals and other highly reactive species such as ions, molecules with unpaired electrons, reactive oxygen, carbon, nitrogen or sulfur species (ros, rcs, rns or rss). when these species are overproduced, oxidative processes might cause cellular damage, affecting cellular components and causing ionic imbalance or mitochondrial disfunction [24] . the role of oxidative stress in different pathologies is well know: inflammation, cardiovascular diseases, cancer, diabetes and even neurodegenerative disorders often count oxidative damage among their pathological features [25] [26] [27] . therefore, exogenous antioxidants might be useful in order to maintain the right concentration of radicals, reducing the amounts of free radicals and avoiding oxidative stress [28] . the antioxidant potential of natural and synthetic coumarins has been deeply investigated in the last years and it became clear that poly-hydroxy or phenolic coumarins are efficient antioxidants in biologicals systems [29] . here below the most recent updates in this field are reported. in 2019, couttolenc and collaborators studied the radical scavenging activity of three hydroxy-4-methylcoumarins (1-3, figure 3 ) by means of experimental and theoretical methods [30] . firstly, the scavenging activity of the compounds was evaluated on abts (2,2′-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt), dpph (2,2-diphenyl-1-picrylhydrazyl) and galvinoxyl radicals as trolox (a vitamin e analogue) equivalent antioxidant capability (teac). the results showed that, whereas 1 did not exhibit radical scavenging activity, 2 resulted more active than trolox against the abts•+ radical (ec50 30.83 μm) and 3 displayed better antioxidant activity than trolox against abts•+, dpph and galvinoxyl radicals (ec50 values of 39.98, 150.99 and 13.19 μm, respectively). it is likely that such differences in antioxidant activity may rely on the differences in the relative positions of hydroxy groups [31] . then, compound 3, which showed the best scavenging activity, was evaluated for its primary antioxidant capacity. in this step, three reaction mechanisms were considered: single electron transfer (set), hydrogen transfer (ht) and radical adduct formation (raf), involving •ooh, •ooch3 and •ch(oh)ch3 as radicals. these experiments were carried out in lipidic and aqueous media, in order to mimic membrane and intra-cellular environment [32] , [33] . the results indicated that different mechanisms are involved depending by the medium and that positions 4, 7 and 8 of compound 3 are probably involved in ht mechanism, whereas positions 3, 4, 5 and 7 could be involved in raf mechanism. finally, the secondary antioxidant activity of compound 3 in aqueous media at physiological ph was evaluated. in the haber-weiss reaction (i.e., the reduction of copper in aqueous media and subsequent copper-catalyzed hydroxy radical formation), 3 was able to inhibit copper (ii) reduction avoiding oxidative stress. it was found that hydroxy groups in position 7 and 8 are fundamental for the primary and secondary antioxidant activity in both lipid and aqueous media. recently, wang and co-workers investigated the antioxidant activity and the mechanism of the antiradical action of six coumarin-fused coumarins (4-9, figure 4 ) previously synthesized by xi and liu [34, 35] . firstly, the scavenging activity of the compounds was evaluated on abts (2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), dpph (2,2-diphenyl-1-picrylhydrazyl) and galvinoxyl radicals as trolox (a vitamin e analogue) equivalent antioxidant capability (teac). the results showed that, whereas 1 did not exhibit radical scavenging activity, 2 resulted more active than trolox against the abts•+ radical (ec 50 30 .83 µm) and 3 displayed better antioxidant activity than trolox against abts•+, dpph and galvinoxyl radicals (ec 50 values of 39.98, 150.99 and 13.19 µm, respectively). it is likely that such differences in antioxidant activity may rely on the differences in the relative positions of hydroxy groups [31] . then, compound 3, which showed the best scavenging activity, was evaluated for its primary antioxidant capacity. in this step, three reaction mechanisms were considered: single electron transfer (set), hydrogen transfer (ht) and radical adduct formation (raf), involving •ooh, •ooch 3 and •ch(oh)ch 3 as radicals. these experiments were carried out in lipidic and aqueous media, in order to mimic membrane and intra-cellular environment [32] , [33] . the results indicated that different mechanisms are involved depending by the medium and that positions 4, 7 and 8 of compound 3 are probably involved in ht mechanism, whereas positions 3, 4, 5 and 7 could be involved in raf mechanism. finally, the secondary antioxidant activity of compound 3 in aqueous media at physiological ph was evaluated. in the haber-weiss reaction (i.e., the reduction of copper in aqueous media and subsequent copper-catalyzed hydroxy radical formation), 3 was able to inhibit copper (ii) reduction avoiding oxidative stress. it was found that hydroxy groups in position 7 and 8 are fundamental for the primary and secondary antioxidant activity in both lipid and aqueous media. recently, wang and co-workers investigated the antioxidant activity and the mechanism of the density functional theory (dft) calculations were performed, followed by the examination of the primary mechanisms, including ht, electron transfer-proton transfer (set-pt) and sequential proton loss transfer (splet). the most stable conformation of all the compounds was a non-planar structure, due to the steric repulsion of the groups in positions 5 and 5′. such conformation was retained by the correspondent anions and cation radicals (aro − , aroh +• ). ht process resulted the most significant in gas or non-polar phase, where compound 9 showed the highest activity. the ht path was possible for compounds 7, 8 and 9, having two or three oh groups, whereas 4 resulted inactive due to the absence of oh groups; compound 5, with only the 6-oh group, was less active than other compounds and 6 could merely trap dpph radical with a small rate constant. a second hat process was possible only for compound 9 and this finding could explain the higher activity of this molecule. in polar media, splet mechanism was favored-in this case the studied compounds were more efficient than trolox in the deprotonation step and, among them, compound 7 resulted to be the most promising, being more prone than the other compounds to deprotonate in a polar environment. the antioxidant potential of coumarin nucleus can be exploited in the production of new hybrid compound with enhanced antioxidant activity. a recent example of this strategy is the synthesis of a new series of chitosan derivatives (10a-d, figure 5 ) containing the coumarin nucleus, achieved by li and collaborators [36] . the antioxidant potential of compounds 10a-d was investigated by evaluating the inhibition of lipid peroxidation, metal ion chelation and free-radical scavenging activity. since both chitosan and coumarins have antioxidant properties themselves, the synthesized compounds were expected to show a stronger activity. lipid peroxidation inhibition was determined by quantifying thiobarbituric acid-reactive substance (tbars), using linoleic acid as a reference compound [37] . the results displayed the ability of the synthesized molecules to inhibit tabrs in a concentration-dependent manner; compound 10d emerged as the most active (ic50 = 0.11 mg/ml), showing a more efficient scavenging activity than chitosan alone (ic50 = 0.38 mg/ml). then, the radical-scavenging activity was the antioxidant potential of compounds 10a-d was investigated by evaluating the inhibition of lipid peroxidation, metal ion chelation and free-radical scavenging activity. since both chitosan and coumarins have antioxidant properties themselves, the synthesized compounds were expected to show a stronger activity. lipid peroxidation inhibition was determined by quantifying thiobarbituric evaluated. for this purpose, free radicals •oh, dpph and o2• − were used. compounds 10a-d showed a stronger •oh scavenging activity (ic50 = 0.09-0.12 mg/ml) compared to that of chitosan. these results suggested that the coumarin moiety strongly enhances chitosan antioxidant properties. since the chelating ability of antioxidants prevents oxidative stress by avoiding •oh production, iron-chelating properties of the new compounds were also evaluated through measurements of inhibition of ferrozine-fe 2+ complex formation. again, the obtained results (ic50 = 0.02-0.04 mg/ml) were better than those of chitosan alone. finally, cytotoxicity of the synthesized compounds was tested: no toxic effects were detected on 3t3-l1 and hhl-5 cells. interestingly, 3t3-l1 cells showed an increase in their viability, probably because of the antioxidant activity of the tested compounds. a similar approach was followed by popova and co-workers, who designed and synthesized a series of 4-methylcoumarins with tert-butyl, isobornyl and isocamphyl substituents (11, 12, 13-17, figure 6 ) [38] . the synthesized compounds were evaluated in terms of antioxidant, membrane-protective (mpa) and radical-scavenging (rsa) activities in vitro. all the tested compounds, at a concentration of 100 μm, exhibited good inhibitory activity against lipid peroxidation products (lpo) formation (ic50 = 3.33 -7.12 nm), whereas 7-hydroxy-4-methylcoumarin, used as reference compound, showed no activity. the scavenging activity, evaluated using dpph, strictly depended on the structure: only isobornyl derivatives showed moderate activity in the dpph assay (compound 15 showed rsa% = 57.48 ± 0.60 at 100 μm; rsa% = 87.95 ± 0.22 at 500 μm). moreover, the protective activity towards cell membrane was assessed, measuring the inhibitory activity against h2o2-induced hemolysis of red blood cells (rbcs). in all the experiments, the most promising compound was 15, having two isobornyl moieties. from these studies, it appears clear that coumarin derivatives show a great potential as antioxidant, membrane-protective and radical-scavenging compounds and that their activity depends mainly on the number and the position of the hydroxy groups. the term "cancer" defines a wide range of diseases caused by the accumulation of mutations and characterized by a multi-step process, involving many different factors which may not directly cause cancer themselves but can increase the chances of genetic mutations [39, 40] . recently, maleki et al. have synthesized eighteen o-prenylated coumarin derivatives and tested them on hela cervical cancer and hdf normal cells by mtt assay [41] . the most promising compounds are reported in figure 7 . the synthesized compounds were evaluated in terms of antioxidant, membrane-protective (mpa) and radical-scavenging (rsa) activities in vitro. all the tested compounds, at a concentration of 100 µm, exhibited good inhibitory activity against lipid peroxidation products (lpo) formation (ic 50 = 3.33-7.12 nm), whereas 7-hydroxy-4-methylcoumarin, used as reference compound, showed no activity. the scavenging activity, evaluated using dpph, strictly depended on the structure: only isobornyl derivatives showed moderate activity in the dpph assay (compound 15 showed rsa% = 57.48 ± 0.60 at 100 µm; rsa% = 87.95 ± 0.22 at 500 µm). moreover, the protective activity towards cell membrane was assessed, measuring the inhibitory activity against h 2 o 2 -induced hemolysis of red blood cells (rbcs). in all the experiments, the most promising compound was 15, having two isobornyl moieties. from these studies, it appears clear that coumarin derivatives show a great potential as antioxidant, membrane-protective and radical-scavenging compounds and that their activity depends mainly on the number and the position of the hydroxy groups. the term "cancer" defines a wide range of diseases caused by the accumulation of mutations and characterized by a multi-step process, involving many different factors which may not directly cause cancer themselves but can increase the chances of genetic mutations [39, 40] . recently, maleki et al. have synthesized eighteen o-prenylated coumarin derivatives and tested them on hela cervical cancer and hdf normal cells by mtt assay [41] . the most promising compounds are reported in figure 7 . the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic50 values between 136.4 ± 1.90 μm and 172.2 ± 1.80 μm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] benzothiazine) as reference compound. when pc3 and du145 human pca cells were treated with different concentrations of both 4-mmpb and 18 for 24, 48 and 72 h, a dose-dependent and time-dependent decrease in the survival of the cells was exhibited. pc3 cells resulted to be more sensitive than du145 cells to both inhibitors (ic50 in μg/ml for compound 18 on pc3 cells: 24 h, 40 19.52 ± 4.92) . moreover, compound 18 had no significant anti-proliferative activity on normal cells. concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g0/g1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic 50 values between 136.4 ± 1.90 µm and 172.2 ± 1.80 µm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] . the results represent a good starting point for the design of novel derivatives, because most of the examined compounds exhibited selective toxicity on hela cells (ic50 values between 136.4 ± 1.90 μm and 172.2 ± 1.80 μm after 24 h), whereas no negative effects on hdf normal cell's growth was detected. sar studies proved that the substitution on c6 position of coumarin nucleus provided the best anticancer activity, followed by substitution on c8. in addition, it was found that the cytotoxic properties of o-prenylated coumarins depends on the length of the prenyl chain, which increases the lipophilicity of the molecule, thus facilitating its penetration into the cells. the cytotoxic activity of prenylated-coumarin derivatives had been evaluated also by other groups. recent studies have shown a functional role of lipoxygenases (loxs) in carcinogenesis, precisely in prostatic cancer and the capability of 5-farnesyloxycoumarin (18, figure 8 ) to inhibit 15-lox-1 [42] [43] [44] starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] benzothiazine) as reference compound. when pc3 and du145 human pca cells were treated with different concentrations of both 4-mmpb and 18 for 24, 48 and 72 h, a dose-dependent and time-dependent decrease in the survival of the cells was exhibited. pc3 cells resulted to be more sensitive than du145 cells to both inhibitors (ic50 in μg/ml for compound 18 on pc3 cells: 24 h, 40 19.52 ± 4.92) . moreover, compound 18 had no significant anti-proliferative activity on normal cells. concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g0/g1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of starting from these observations, orafaie and collaborators investigated the inhibitory activity of compound 18 on 15-lox-1 [45] . the cytotoxic effects of 18 were evaluated by means of mtt assay on two carcinoma cell lines (pc-3 and du145) and a normal cell line (hff3), using 4-mmpb (4-methyl-2-(4-methylpiperazinyl) pyrimido [4,5-b] concerning the mechanism of action, it was found that 5-farnesyloxycoumarin acts as a cytotoxic agent causing chromatin condensation and dna damage and induces the arrest of the cell cycle in g 0 /g 1 phase. a similar study was carried out on 8-farnesyloxycoumarin (19, figure 8 ) by hosseinymehr and collaborators [46] . again, the coumarin derivative showed inhibitory activity on 15-lox-1 in pc3 and du145 cell lines, thus inducing apoptosis of the cancer cell, with the same mechanism of compound 18. pc3 cells resulted to be more sensitive to 19 inhibition than du145 cells; ic 50 halawa et al. synthesized and characterized a new series of 4-arylamino-3-nitrocoumarin analogues from 4-hydroxycoumarin and tested them on the human cervix carcinoma cell line kb-3-1 [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic 50 value of 21 µm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5 -thio-2 deoxyguanosine phosphonic acid of the dna backbone. [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic50 value of 21 μm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5′-thio-2′ deoxyguanosine phosphonic acid of the dna backbone. herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed antiproliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic50 = 1.01 μm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed anti-proliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic 50 = 1.01 µm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . [47] . these compounds were found to target the dna-topo i (human topoisomerase i) complex, thus blocking cell replication and leading to cell death. among this series, thiazolidinylidene derivative 20 ( figure 9 ) containing a malononitrile fragment exhibited the best cytotoxic activity with an ic50 value of 21 μm. the cytotoxicity of 20 was explained also by docking studies which highlighted that it forms important h-bonds with arg364, asp533, gln633 and 5′-thio-2′ deoxyguanosine phosphonic acid of the dna backbone. herrera et al. synthesized a series of 3-and 7-styrylcoumarins, some of which showed antiproliferative activity on sw480 human colon adenocarcinoma cells [48] . among them, 7-(4-hydroxy-3,5-dimethoxystyryl)-2h-chromen-2-one (21, figure 10 ) showed the highest activity (ic50 = 1.01 μm) as it was capable of inducing apoptosis in sw480 cells, probably by modulating the tumor-suppressor protein p53. the new compound was also tested in vivo, demonstrating to be able to inhibit the early progression of colon adenocarcinoma [49] . as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) as coumarin nucleus can be widely decorated, it can be used in the synthesis of hybrid compounds, targeting different proteins involved not only in tumor growth but also in metastatic and angiogenetic processes. in this context, diao and collaborators synthesized a series of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives (22a-l, figure 11 ) [50] . several isatin-based or 1,2,3-triazole or coumarin-based compounds (semaxanib, carboxyamidotriazole and stx64) are involved in clinical trials or have already been used the treatment of various cancers (as colon-rectal, prostatic, endometrial and breast cancer) [51, 52] , whereas isatin-1,2,3-triazole-coumarin derivatives showed activity against different cancer types. in addition, sar studies demonstrated that the linker between isatin and 1,2,3-triazole influences the activity [52, 53] and that hydrogen bonds are fundamental for the biological activity [54] . these evidences guided diao and collaborators in the choice of diethylene glycol as a linker [47] . ten compounds were tested on hepg2 (liver carcinoma), hela (cervical cancer), a549 (lung adenocarcinoma), du145 (prostatic cancer), skov3 (ovarian carcinoma), mcf-7 (breast cancer) and drug-resistant mcf-7/dox (doxorubicin-resistant mcf-7) human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic50 14.62 μm for mcf-10a cells and ic50 35.60 μm for l02 cells), indicating a higher selectivity than stattic. carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic 50 human cancer cell lines. all the synthesized compounds showed weak to moderate in vitro activity against all the cell lines, therefore further sar studies are necessary for the obtainment of new, more active compounds. figure 11 . general structure of diethylene glycol tethered isatin-1,2,3-triazole-coumarin derivatives 22a-l. cai et al. developed a series of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates [55] . these compounds act on stat3, which is involved in the regulation of mitochondrial apoptotic pathway [56] . in particular, they hypothesized that the inhibition of phosphorylation of tyr705 and ser727 might prevent stat3 activation. four stat3 over-activated human cancer cell lines (i.e., human breast carcinoma mda-mb-231 and mcf-7 cells, human colonic carcinoma hct-116 cells and human hepatocellular carcinoma hepg2 cells) were selected to assess the biological activity of the newly-synthesized compounds. compound 23 ( figure 12 ) showed high potency in inducing cancer cell apoptosis and ros generation, inhibiting stat3 phosphorylation on tyr705, affecting mitochondrial membrane potential and preventing stat3 dna-binding activity. in addition, 23 inhibited implanted 4t1 breast cancer growth in vivo. the antiproliferative effects of compound 23 on normal cells were investigated by mtt assays comparing it to the stat3 inhibitor stattic, which showed growth inhibition activity in both tumor and normal cells, whereas compound 23 exhibited selective inhibitory activity against cancer cells (ic50 14.62 μm for mcf-10a cells and ic50 35.60 μm for l02 cells), indicating a higher selectivity than stattic. carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential carbonic anhydrases (cas) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, which is an essential physiological reaction [57] . thus, this enzyme is involved in a wide range of physiological and pathological processes (ph regulation, co 2 homeostasis, respiration, bone resorption and tumorigenesis) [58] and its deregulation by means of carbonic anhydrases inhibitors (cais) may be useful in the treatment of many disorders [59] [60] [61] . the ideal ca inhibitor would selectively act against those isoforms (hca ix, xii, for instance) related to a certain disease [62, 63] . in this context, coumarins emerged as potential atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7-hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (k i = 5.8 nm) against ca xii and highly selective over isoforms i and ii. atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (ki = 5.8 nm) against ca xii and highly selective over isoforms i and ii. umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic50 =75 μm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 μm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (ki (hca xii) = 26.4 nm), they all had ki values comparable to those of the reference drug acetazolamide (ki (hca xii) = 5.7 nm). it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic 50 = 75 µm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 µm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (k i (hca xii) = 26.4 nm), they all had k i values comparable to those of the reference drug acetazolamide (k i (hca xii) = 5.7 nm). atypical hca ligands that, unlike classical hcais, do not need to chelate the prosthetic zinc ion but, after binding the catalytic site, are hydrolyzed to the corresponding 2-hydroxy cinnamic acid derivatives, the actual inhibitors [64, 65] . some studies highlighted that coumarins are capable of binding at the entrance of hca catalytic site, blocking the enzyme activity. furthermore, 7hydroxycoumarins derivatives showed good selectivity toward ix and xii isoforms over hca i and ii and, in some cases, they exhibited cytotoxic activity on cancer cells [66] [67] [68] [69] . many efforts have been made in this direction and the most recent results are here reported. fois and collaborators have recently investigated the selective inhibitory activity towards ca ix and ca xii of extracts of magydaris pastiacea seeds, from which they isolated ten linear furocoumarins, four simple coumarins and a new angular dihydrofurocoumarin [70] all the mentioned compounds were tested against four isoforms of hca: both extracts showed selective activity towards ca ix and xii whereas no effect was seen on isoforms i and ii at a concentration of 100 ng/ml. the most promising compound was umbelliprenin (24, figure 13 ), highly active (ki = 5.8 nm) against ca xii and highly selective over isoforms i and ii. umbelliprenin (24) was then tested on hela cancer cells in order to evaluate its cytotoxic activity and resulted to possess a moderate cytotoxicity (ic50 =75 μm) proving to be able to inhibit tumor growth, angiogenesis and metastasis formation in mice (after i.p. administration). it is noteworthy that ca ix and xii are overexpressed in tumor cells under hypoxic conditions, whereas the mentioned tests were carried out under normoxic conditions, which could explain the moderate cytotoxic activity of the isolated compound. in 2019, starting from 4-methylumbelliferone (25, figure 14 ), buran and co-workers synthesized a series of 8-substituted coumarin-based compounds bearing alkylpiperazine and arylpiperazine chains (26-37, figure 14 ), and evaluated their inhibitory activity against hca i, ii, ix and xii [71] . all the tested compounds were able to inhibit hca isoforms ix and xii, without showing any inhibitory activity towards the cytosolic isoforms i and ii up to a 10 μm concentration. the test pointed out that these compounds had higher affinity for hca xii over ix and, except for compound 36 (ki (hca xii) = 26.4 nm), they all had ki values comparable to those of the reference drug acetazolamide (ki (hca xii) = 5.7 nm). it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) it is remarkable that the substitution of c8 position of 4-methylumbelliferone (25) did not have any influence on inhibition of hca xii, suggesting that no significant interaction may be achieved between the side chains of compounds 26-37 and the catalytic site of isoform xii. on the other hand, alkylpiperazine derivatives showed better activity on hca ix when compared with the other synthesized compounds, being compound 30 the one with the highest activity among them (ic 50 = 27.1 nm). similar results were obtained by many other groups that have recently synthesized coumarin-based compounds and evaluated them as hca ix and xii inhibitors. sulphocumarins, bis-coumarins and coumarins 1,3,4-oxadiazole derivatives are some examples [72] [73] [74] . the battle against infections and multidrug resistant bacteria is almost certainly one of the most challenging issue that the scientific community and the whole mankind will face in the near future. multidrug resistant (mdr) bacteria are defined as non-susceptible strains to one or more antimicrobials on three or more antimicrobial classes, whereas strains that are non-susceptible to all antimicrobials are classified as extremely drug-resistant strains [75, 76] . the plant kingdom provides a virtually endless source of novel chemicals and scaffolds, such as polyphenols and coumarins [77] . in 2005, the antibacterial potency of nearly 50 coumarin derivatives, natural and synthetic, was evaluated and then correlated by a sar study. bacterial susceptibility to coumarins was evaluated by determining the minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc), considering active compounds exhibiting mic values ranging from 62.5 to 2000 µg/ml. among the active compounds, osthenole (a natural coumarin having also the anti-inflammatory activity) showed the most potent activity with a mic of 62.5 µg/ml against s. aureus and b. cereus [78] . in 2015, nagamallu and co-workers exploited the vilsmeier-haack reaction to obtain a series of novel pyrazole-containing coumarins and then evaluated their antioxidant and antibacterial activities [79] . among the series, two compounds (38 and 39, figure 15 ) showed a good antibacterial and antifungal activity, with mic values comparable with the ones of ciprofloxacin (positive control against bacteria species) and fluconazole (positive control against fungi strains). the battle against infections and multidrug resistant bacteria is almost certainly one of the most challenging issue that the scientific community and the whole mankind will face in the near future. multidrug resistant (mdr) bacteria are defined as non-susceptible strains to one or more antimicrobials on three or more antimicrobial classes, whereas strains that are non-susceptible to all antimicrobials are classified as extremely drug-resistant strains [75, 76] . the plant kingdom provides a virtually endless source of novel chemicals and scaffolds, such as polyphenols and coumarins [77] . in 2005, the antibacterial potency of nearly 50 coumarin derivatives, natural and synthetic, was evaluated and then correlated by a sar study. bacterial susceptibility to coumarins was evaluated by determining the minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc), considering active compounds exhibiting mic values ranging from 62.5 to 2000 μg/ml. among the active compounds, osthenole (a natural coumarin having also the anti-inflammatory activity) showed the most potent activity with a mic of 62.5 μg/ml against s. aureus and b. cereus [78] . in 2015, nagamallu and co-workers exploited the vilsmeier-haack reaction to obtain a series of novel pyrazole-containing coumarins and then evaluated their antioxidant and antibacterial activities [79] . among the series, two compounds (38 and 39, figure 15 ) showed a good antibacterial and antifungal activity, with mic values comparable with the ones of ciprofloxacin (positive control against bacteria species) and fluconazole (positive control against fungi strains). following the idea that the introduction of an additional coumarin nucleus on a parent coumarin molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017, chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80] . figure 16 shows the common scaffolds of the bis-coumarins synthesized using l-proline as catalyst in a multi-component reaction approach. following the idea that the introduction of an additional coumarin nucleus on a parent coumarin molecule could improve the pharmacological activity (i.e., dicumarol as anticoagulant), in 2017, chougala and colleagues designed and synthesized a series of bis-coumarin derivatives [80] . figure 16 shows the common scaffolds of the bis-coumarins synthesized using l-proline as catalyst in a multi-component reaction approach. the antibacterial potency of the compounds was evaluated against gram-positive and gramnegative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch3 > 7,8-benzo > -cl > -och3. furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized the antibacterial potency of the compounds was evaluated against gram-positive and gram-negative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch 3 > 7,8-benzo > -cl > -och 3 . furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. the antibacterial potency of the compounds was evaluated against gram-positive and gramnegative strains, comparing the mic with ciprofloxacin and most of the newly synthesized compounds showed modest to good inhibiting activity against the tested microorganisms. compounds 40a-e ( figure 16 ) were highly active and more potent than ciprofloxacin against s. aureus and e. faecalis, whereas 41c and 41d were active only against gram-positive e. faecalis. actually, compounds 41a-e were the most promising against e. coli. once established the broad spectrum of action of the coumarin nucleus, various researchers focused their attention on the activity against multidrug resistant strains, in particular on the eskape pathogens, the coterie that escape the lethal action of antibiotics: enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa and enterobacter species [81] . in 2017, naik et al. synthesized and evaluated against s. aureus and other bacterial strains a series of 3,4-dihydropyrimidinone-coumarin analogues, measuring the mic values and comparing them to ciprofloxacin [82] . the structures and the mic values for compounds 42-49, the most promising derivatives, are reported in figure 17 : the substitution at c6 position seemed to be excellent for the activity and able to modulate the potency, decreasing the efficiency in the order of -ch3 > 7,8-benzo > -cl > -och3. furthermore, it was revealed from docking studies that that one hydrogen atom and two oxygen atoms of 3,4-dihydropyrimidinone substituted coumarins form interactions with the active site residues of s. aureus gyrase, indicating that the presence of hydrogen bond acceptor and donor groups may enhance antimicrobial activity. in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized in 2018, chavan and hosamani proposed a facile method for the microwave assisted synthesis of a series of pyrazole-containing coumarins and tested their antibacterial and anti-inflammatory activities [83] . the researchers evaluated the in vitro antibacterial activity of the newly synthesized compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 µg/ml and mic 1.562 µg/ml, respectively). docking studies were in good agreement with the biological results. compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. 23857) and staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 μg/ml and mic 1.562 μg/ml, respectively). docking studies were in good agreement with the biological results. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6′,7′-dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. compounds through agar-well diffusion method against two gram-positive (bacillus subtilis (atcc no. 23857) and staphylococcus aureus (atcc-29213)) and two gram-negative (escherichia coli (atcc-25922) and pseudomonas aeruginosa (atcc no.25619)) bacterial strains [84] . the mic values were compared to those of ciprofloxacin and all the compounds showed significant antibacterial activity. in particular, compounds 50 and 51 ( figure 18 ), showed an excellent activity against s. aureus (mic 0.78 μg/ml and mic 1.562 μg/ml, respectively). docking studies were in good agreement with the biological results. in 2017, madeiro and co-workers focalized their interest towards the antibiotic activity of coumarins isolated from rutacea species ( figure 19 ) [85] . bergapten, xantoxin, isopimpinellin and imperatorin did not exhibit any antibacterial activity, even at the highest concentration, against s. aureus strains resistant to tetracycline, erythromycin and norfloxacin. however, their role as modulator of other antibiotics seemed quite promising, because isopimpinellin and imperatorin reduced the mic of erythromycin, tetracycline and norfloxacin. nevertheless, more detailed research is necessary in order to enable the use of these natural products as adjuvants to antimicrobial agents. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6′,7′-dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. in 2018, widelsky and co-workers isolated some similar linear furanocoumarins from the fruits of peucedanum luxurians tamamsch, with more encouraging results. plants of the peucedanum genus have been used for centuries as antibacterial agents and, for some of them, the activity was confirmed by biological and pharmacological studies on plant extracts and on a few isolated compounds [86] . all the six isolated compounds showed a broad growth-inhibitor activity against several bacteria strains but three of them ( figure 20 ) resulted particularly interesting. 6 ,7 -dihydroxybergamottin was the most active against all the bacteria strains tested, probably because of the aliphatic chain in c5 position; similar activity was noticed for peucedanin and officinalin isobutyrate. liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic50 values in the range 9.4-25 mg/l. fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic 50 values in the range 9.4-25 mg/l. liu and colleagues made a huge effort to synthesize and identify coumarin-pyrazole carboxamide derivatives as potential topoisomerase-ii inhibitors: 70 novel compounds were obtained and evaluated [87] . three of them (52-54, figure 21 ) were endowed with promising antimicrobial activities. in particular, compound 52 showed a considerable inhibitory activity compared with ciprofloxacin against escherichia coli and compound 53 exhibited excellent antibacterial activity against salmonella typhi. the selected compounds exhibited also potent inhibition against topo ii and topo iv with ic50 values in the range 9.4-25 mg/l. fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this fungal diseases are a well-known plague for animal and plant worlds but a more hidden menace for human health. more than 90% of all reported fungal-related deaths results from species that belong to one of four genera: cryptococcus, candida, aspergillus and pneumocystis [88] [89] [90] . coumarin derivatives are endowed with antifungal activity, potentially useful in both pharma and agri-food sectors. in this paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in section 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in paragraph 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et3nh][hso4] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et 3 nh][hso 4 ] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. paragraph, we will focus on the recent progresses in the development of novel antifungal drugs for human use whereas agri-food applications can be found in paragraph 3.2, food systems. diseases by candida species, a family of fungi that normally live on the host epithelial species but can initiate a fatal infection in particular cases like immunodeficiency, are the fourth most common cause of nosocomial blood-stream infections. despite several species of candida can cause disease, candida albicans prevails in term of incidence [88, 91, 92] . therefore, in 2016, shaik and colleagues designed a novel series of coumarin derivatives conjugated with 1,2,3-triazole moieties, on the basis of a previous work by shi and zhou and of the common use of azoles as antifungal drugs [93, 94] . the antifungal potency of the novel compounds was tested against candida albicans and other four fungal pathogens (i.e., fusarium oxysporum, aspergillus flavus, aspergillus niger and cryptococcus neoformans); mic values were evaluated and compared to mic of the reference compounds miconazole and fluconazole. compounds 55-57 and 59 ( figure 22 ) were found to be equipotent to miconazole against c. albicans and compound 58 was found to be two-fold more active compared with miconazole and equipotent to fluconazole. furthermore, molecular docking studies showed that these compounds have a high affinity toward the active site of enzyme p450 cytochrome lanosterol 14α-demethylase and analysis of adme parameters confirmed their drug-like properties [94] . in 2017, fifteen novel coumarin derivatives were synthesized by tiwari et al., under solvent free conditions and exploiting the ionic liquid [et3nh][hso4] as a catalyst [95] . the compounds were tested both for their antifungal and antibacterial activities and, among the series, compounds 60 and 61 resulted to be the most potent as fungicides ( figure 23 ). the mic values observed for compound 60 and 61 were comparable to the standard drug miconazole. further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. furthermore, it should not be forgotten the contribution of natural coumarins to the fight against candida infections. ferulago trachycarpa boiss. is one of the species of ferulago w. koch., common in anatolian region, exploited in traditional medicine but also in culinary field. previous studies showed that coumarins are the main compounds found in ferulago but only in 2018, dikpinar and colleagues conducted the first antimicrobial activity guided isolation of the molecular constituent of this particular species of ferulago [97] . four coumarin compounds ( figure 25 ) were isolated and purified and then three of them were tested against bacterial and fungal strains; in particular, marmesin senesioate, suberosin and crenulatin showed antifungal activity with 625 mg/l mic against candida albicans. the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . further studies demonstrated that compound 60 acts by inhibiting ergosterol biosynthesis in c. albicans. molecular docking studies revealed, as for previous study on compounds 55-59, a highly spontaneous binding ability of the tested compounds to the active site of lanosterol 14α-demethylase, which suggests that the tested compounds inhibit the synthesis of this enzyme. moreover, in silico admet properties were evaluated and demonstrated that all the compounds exhibited a good percent absorption (% abs) ranging from 84.9% to 100%. moreover, these compounds had proven to be safe after in vitro toxicity, in vivo acute oral toxicity and behavioral studies. coumarin-based antifungal azoles had been further investigated by elias and co-workers, who, in 2019, developed a series of 11 coumarins conjugated with 1,2,4-triazole and imidazole motifs [96] . the analysis of the biological effects of these novel chemical entities highlighted that imidazole-based derivatives ( figure 24 ) were more efficient against several candida strains compared to 1,2,4-triazole derivatives. moreover, the mode of action of the two classes of compounds were different. whereas the antifungal activity of the triazole-based azoles was dependent on expression of cyp51, the target of the azole antifungals, imidazole-based compounds displayed antifungal activity against a mutant lacking cyp51, indicating that imidazole-based azole antifungals have additional modes of action. this peculiarity could be favorable in the struggle against drugs-resistant fungal strains. furthermore, it should not be forgotten the contribution of natural coumarins to the fight against candida infections. ferulago trachycarpa boiss. is one of the species of ferulago w. koch., common in anatolian region, exploited in traditional medicine but also in culinary field. previous studies showed that coumarins are the main compounds found in ferulago but only in 2018, dikpinar and colleagues conducted the first antimicrobial activity guided isolation of the molecular constituent of this particular species of ferulago [97] . four coumarin compounds ( figure 25 ) were isolated and purified and then three of them were tested against bacterial and fungal strains; in particular, marmesin senesioate, suberosin and crenulatin showed antifungal activity with 625 mg/l mic against candida albicans. the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . the antifungal potency of coumarin itself against candida albicans had been previously evaluated, in particular its antibiofilm activity [98, 99] . recently, xu and co-workers focused their attention on the possible way to prevent the adhesion and formation of biofilm by candida albicans on implanted medical devices. the research group observed that coumarin not only suppresses biofilm formation but also affects the structure of the mature biofilm; moreover the adhesion, the cell surface hydrophobicity (csh) and the filamentous growth of c. albicans significantly decreased after coumarin treatment, indicating that coumarin inhibits biofilm formation by suppressing adhesion [100] . the antifungal potency of coumarin-based triazoles against other fungal strains in addition to candida species had been evaluated by dharavath and colleagues in 2020. coumarin-based 1,4-disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly protocol via a copper(i)-catalyzed click reaction between various substituted arylazides and terminal alkynes. the in vitro antifungal activity was tested against aspergillus niger, aspergillus flavus and fusarium oxysporum by using the disc diffusion method and the results were compared with those of clotrimazole, the reference drug. compounds 63-68 ( figure 26 ), characterized by the presence of a para-substituted phenyl moiety, directly linked to the triazole ring, showed comparable activity in respect to the reference compound clotrimazol [101, 102] . the antifungal potency of coumarin-based triazoles against other fungal strains in addition to candida species had been evaluated by dharavath and colleagues in 2020. coumarin-based 1,4disubstituted 1,2,3-triazole derivatives were synthesized using a highly efficient, eco-friendly protocol via a copper(i)-catalyzed click reaction between various substituted arylazides and terminal alkynes. the in vitro antifungal activity was tested against aspergillus niger, aspergillus flavus and fusarium oxysporum by using the disc diffusion method and the results were compared with those of clotrimazole, the reference drug. compounds 63-68 ( figure 26 ), characterized by the presence of a para-substituted phenyl moiety, directly linked to the triazole ring, showed comparable activity in respect to the reference compound clotrimazol [101, 102] . 2020 has been a crucial year for the timeless war between human and viruses: world health organization (who) declared the outbreak of sars-covid-19 a public health emergency of international concern on 30 january 2020 and on 11 march who characterized covid-19 as a pandemic [103] . whole developed countries have been quarantined and generations that never faced medical crisis are now struggling with the consequences of the viral diffusion. human history is afflicted by the cyclic succession of pandemic events and the research of new antivirals is still ongoing, due to the ability of viruses to mutate and the continuous appearance of new viruses on the medical scenario [104] . the natural world is a priceless source of valuable medical compounds and also in the fight against viral diseases there are several natural molecules which exhibit antiviral activity [105] [106] [107] [108] [109] . coumarins, likewise other polyphenolic compounds, exert a remarkable antiviral activity [110, 111] . the antiviral activity of coumarins explicates through different mechanisms which affect the life cycle of viruses and their biological activities could be changed depending upon the combination of various substituents and conjugates [104, 112] . coumarins appears to be active against several viruses, like hiv, influenza, hepatitis, dengue and chikunguya [104] . liu and co-workers after a phytochemical study on the stem of clausena lenis isolated three new and nine known prenylated coumarins [113] . all the prenylated coumarins were evaluated both for their anti-inflammatory and anti-hiv reverse transcriptase (rt) activities. in this last case, the inhibition assay for the cytopathic activities of hiv-1 (ec50) as well as cytotoxic activity assay against c8166 cell line (cc50) according to mtt methods were applied [114, 115] . the three new isolated compounds (69-71, figure 27 ) showed the best inhibitory activity with an ec50 of 0.29, 0.68 and 0.17 μm, respectively. furthermore, no cytotoxicity was observed against c8166 cell line (cc50 > 200.00 μm). 2.6. antiviral activity 2020 has been a crucial year for the timeless war between human and viruses: world health organization (who) declared the outbreak of sars-covid-19 a public health emergency of international concern on 30 january 2020 and on 11 march who characterized covid-19 as a pandemic [103] . whole developed countries have been quarantined and generations that never faced medical crisis are now struggling with the consequences of the viral diffusion. human history is afflicted by the cyclic succession of pandemic events and the research of new antivirals is still ongoing, due to the ability of viruses to mutate and the continuous appearance of new viruses on the medical scenario [104] . the natural world is a priceless source of valuable medical compounds and also in the fight against viral diseases there are several natural molecules which exhibit antiviral activity [105] [106] [107] [108] [109] . coumarins, likewise other polyphenolic compounds, exert a remarkable antiviral activity [110, 111] . the antiviral activity of coumarins explicates through different mechanisms which affect the life cycle of viruses and their biological activities could be changed depending upon the combination of various substituents and conjugates [104, 112] . coumarins appears to be active against several viruses, like hiv, influenza, hepatitis, dengue and chikunguya [104] . liu and co-workers after a phytochemical study on the stem of clausena lenis isolated three new and nine known prenylated coumarins [113] . all the prenylated coumarins were evaluated both for their anti-inflammatory and anti-hiv reverse transcriptase (rt) activities. in this last case, the inhibition assay for the cytopathic activities of hiv-1 (ec 50 ) as well as cytotoxic activity assay against c8166 cell line (cc 50 ) according to mtt methods were applied [114, 115] . the three new isolated compounds (69-71, figure 27 ) showed the best inhibitory activity with an ec 50 prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec50 value of 0.12 μm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic50 between 13 nm (compound 76) and 31 nm (ca ic50=10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec 50 value of 0.12 µm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic 50 between 13 nm (compound 76) and 31 nm (ca ic 50 = 10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. prenylated coumarins came to the fore in 2019 thanks to a work by liu et al., who revealed for the first time the presence of these type of derivatives in the fruits of manilkara zapota, an ever-green tropical tree. also in this case, three new derivatives were identified (72-74, figure 28 ) together with seven known compounds and the team evaluated their anti-inflammatory and anti-hiv activities, exploiting the methods described above [116] . the new prenylated coumarins showed the highest anti-hiv rt effect among the prenylated coumarins derived from the fruit of manilkara zapota; in particular, compound 72 displayed the most powerful effect with an ec50 value of 0.12 μm. comparative studies between compounds 72-74 and the other coumarin derivatives isolated from the fruits highlighted the importance of the isopentenyl group as a substituent at c6 and 2-methylbut-3-en-2-yl group as a substituent at c3 for the anti-hiv rt effect. in the same year, jesumoroti and co-workers approached the problem from another point of view, both regarding the target and the origin of the coumarin derivatives [117] . first of all, a different viral target was chosen, hiv-1 in, which is essential for a stable infection. moreover, there are no homologous enzymes in the host cell [118] . secondarily, the coumarin derivatives were obtained decorating the coumarin nucleus with an hydrazide group, in order to achieve a synergistic effect against-hiv-1-in and reducing the toxicity correlated to the salicylhydrazide, which was however essential for the activity [119] [120] [121] . the synthesized compounds were evaluated for their in vitro hiv-1 in inhibitory activity using chicoric acid (ca) as a reference compound, according to the method described by mccoll et al. [122] . compounds 75-78 ( figure 29 ) appeared to be the most active among the whole series showing an inhibition of extracellular in (evaluated in vitro according to the method described by mccoll et al. [122] ) in a range from 95% to 86 % and ic50 between 13 nm (compound 76) and 31 nm (ca ic50=10 nm). furthermore, the cytotoxicity of all the obtained compounds was tested and derivatives 75-78 showed low or negligible cytotoxicity. seasonal influenza claims around 0.29-0.65 million victims annually, even if several drugs are commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic 50 values of 4.84, 19.72, 6.12 and 9.13 µg/ml, respectively, against the h1n1 virus. commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic50 values of 4.84, 19.72, 6.12 and 9.13 μg/ml, respectively, against the h1n1 virus. bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic50 = 69.9 μg/ml) and 89 (ic50 = 47.9 μg/ml) turned out to be more active than catechol derivatives 84 (ic50 = 106.5 μg/ml) and 85 (ic50 = 91.5 μg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic50 110 μg/ml for both the parent compounds). commercially available [123] . the flu is a contagious respiratory disease caused by influenza viruses, manifesting major epidemics with no predictable periodicity or pattern and all different from one to another [124] . for these reasons, a constant effort in the development of new drugs for the treatment of this disease is of greatest importance. in 2019 osman and co-workers combined two bioactive moieties into a single molecule in order to obtain new bioactive compounds with antibacterial and antiviral effects [125] . in particular, based on a previous study of the same research team, in which coumarin scaffolds and thiazole moiety were combined leading to compounds endowed with both antibacterial and antiviral activities, the potentiality of this combination was further explored [126] , [127] . four new molecules of the series showed a remarkable antiviral activity against h1n1 virus. compounds 79-82 ( figure 30 ) seemed to be promising agents, having ic50 values of 4.84, 19.72, 6.12 and 9.13 μg/ml, respectively, against the h1n1 virus. bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic50 = 69.9 μg/ml) and 89 (ic50 = 47.9 μg/ml) turned out to be more active than catechol derivatives 84 (ic50 = 106.5 μg/ml) and 85 (ic50 = 91.5 μg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic50 110 μg/ml for both the parent compounds). bizzarri et al. exploited the regioselective oxidation of coumarin derivatives with 2-iodoxybenzoic acid (ibx) in order to obtain catechol and pyrogallol moieties [129] . after cytotoxicity studies that confirmed the safety of the series of derivatives, the antiviral activity against influenza viruses a/pr8/h1n1 was evaluated and compounds 84-89 ( figure 32 ) resulted able to inhibit the viral replication. interestingly, pyrogallol derivatives 88 (ic 50 = 69.9 µg/ml) and 89 (ic 50 = 47.9 µg/ml) turned out to be more active than catechol derivatives 84 (ic 50 = 106.5 µg/ml) and 85 (ic 50 = 91.5 µg/ml). moreover, pyrogallol and catechol derivatives were more active than the monohydroxycoumarins 6-hydroxycoumarin and 7-hydroxycoumarin (ic 50 110 µg/ml for both the parent compounds). one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as antihepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec50 values ranging from 5.1 to 8.4 μm and selective indices (si = cc50/ec50, which is a measure for the therapeutic window of the compound in an assay system) higher than 20. huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as anti-hepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec 50 values ranging from 5.1 to 8.4 µm and selective indices (si = cc 50 /ec 50 , which is a measure for the therapeutic window of the compound in an assay system) higher than 20. one of the possible advantages of oxidized coumarins could be their mode of action against viruses. indeed, due to their antioxidant activity, coumarins derivatives could affect intracellular redox-sensitive pathways useful for viral replication, independently from the variability of the strains [129] . as already mentioned, coumarins have been studied also for their potential application as antihepatitis agents. tsay and co-workers studied the activity against hepatitis c virus (hcv) of some unnatural imidazole-coumarin conjugates [130] . above all, three compounds (90-92, figure 33 ) showed a noteworthy antiviral activity against hcv with ec50 values ranging from 5.1 to 8.4 μm and selective indices (si = cc50/ec50, which is a measure for the therapeutic window of the compound in an assay system) higher than 20. huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . huang and co-workers focused their attention on the investigation of the potentiality expressed by esculetin (or 6,7-dihydroxycoumarin) against hepatitis b virus (hbv) [131] . the results suggested that esculetin efficiently inhibits hbv replication both in vitro and in vivo, which provides an opportunity for further development of the compound as antiviral agent. inflammation is a general physiological response that aims, firstly, to the circumscription of the harmful factors, which could be both endogenous (e.g., cancer, ischemia, autoimmune diseases) and exogenous (e.g., viral or bacterial infections, trauma), secondarily, to the removal of the causes of the damage and finally to the reparation of the tissues and restoration of the functions. nevertheless, the inflammation and the consequent restorative process could become harmful for the organism itself when there is a persistent stimulation and the phases of inflammation and reconstruction are contemporary activated, causing tissue injuries and fibrosis [132] . during an inflammatory process, many inflammatory effectors and mediators are produced and involved, often with a common effect on vascular system and on the recruitment of leukocytes [133] . frequently, inflammatory mediators are the target of anti-inflammatory drugs [134] [135] [136] [137] . among the numerous biological activities shown by coumarin derivatives, the anti-inflammatory effect could not certainly miss. dawood et al., in 2015 , developed a new series of coumarin derivatives hybridizing two pharmacophoric moieties-the coumarin scaffold itself and thiazoline or thiazolidinone groups, both showing cyclooxygenase 2 (cox-2) inhibitor effect [127] , [138] [139] [140] [141] [142] [143] . the compounds were evaluated in vivo for their systemic effect, in vitro for their ability to inhibit human cox-1 and cox-2 and also to evaluate the ulcerogenic potential compared to the reference drug, indomethacin, always following standard methods reported in literature. most of the new compounds showed significant in vivo anti-inflammatory activity with a superior gastro-intestinal safety profiles (0-7% ulceration) as compared to indomethacin. the ic 50 among the numerous biological activities shown by coumarin derivatives, the antiinflammatory effect could not certainly miss. dawood et al., in 2015 , developed a new series of coumarin derivatives hybridizing two pharmacophoric moieties-the coumarin scaffold itself and thiazoline or thiazolidinone groups, both showing cyclooxygenase 2 (cox-2) inhibitor effect [127] , [138] [139] [140] [141] [142] [143] . the compounds were evaluated in vivo for their systemic effect, in vitro for their ability to inhibit human cox-1 and cox-2 and also to evaluate the ulcerogenic potential compared to the reference drug, indomethacin, always following standard methods reported in literature. most of the new compounds showed significant in vivo anti-inflammatory activity with a superior gastrointestinal safety profiles (0-7% ulceration) as compared to indomethacin. the ic50 values of all the bioactive compounds ranged between 0.31 and 0.78 mm, showing an in vitro high affinity and selectivity toward the cox-2 isoenzyme, compared to the reference celecoxib. ethyl thiosemicarbazone 93, thiazoline derivatives 94-98 and the thiazolidinone compounds 99-100 exhibited the highest in vivo and in vitro anti-inflammatory activities with good cox-2 selectivity ( figure 34 ) [143] . nevertheless, cyclooxygenases are not the only enzymes involved in the inflammatory process. actually, 5-lipoxygenase (5-lox) catalyzes two different steps in the arachidonic acid metabolism that brings to the production of leukotriene a4, which is successively metabolized into leukotriene b4 [144] . molecular inhibitors of leukotrienes competitively bind the active site of 5-lox and are divided in three category: redox-active compounds (i.e., coumarins), iron-ligand inhibitors with weak redoxactive properties and non-redox-type inhibitors [145] . in 2016, srivastava and colleagues evaluated the anti-inflammatory and analgesic effect of a series of synthesized 7-substituted coumarins and, consequently, the most active compounds were assessed in vitro for 5-lox inhibition [146] . compounds 101 and 102 ( figure 35 ) resulted the most promising derivatives, also in the ulcerogenic risk evaluation when compared to acetylsalicylic acid. in vitro kinetic study of compound 102 (ic50 = 2.09 nm) showed mixed or non-competitive type of inhibition of 5-lox. the presence of a substituent on c6 position of benzothiazole ring was found very important for increasing the activity. nevertheless, cyclooxygenases are not the only enzymes involved in the inflammatory process. actually, 5-lipoxygenase (5-lox) catalyzes two different steps in the arachidonic acid metabolism that brings to the production of leukotriene a 4 , which is successively metabolized into leukotriene b 4 [144] . molecular inhibitors of leukotrienes competitively bind the active site of 5-lox and are divided in three category: redox-active compounds (i.e., coumarins), iron-ligand inhibitors with weak redox-active properties and non-redox-type inhibitors [145] . in 2016, srivastava and colleagues evaluated the anti-inflammatory and analgesic effect of a series of synthesized 7-substituted coumarins and, consequently, the most active compounds were assessed in vitro for 5-lox inhibition [146] . compounds 101 and 102 ( figure 35 ) resulted the most promising derivatives, also in the ulcerogenic risk evaluation when compared to acetylsalicylic acid. in vitro kinetic study of compound 102 (ic 50 = 2.09 nm) showed mixed or non-competitive type of inhibition of 5-lox. the presence of a substituent on c6 position of benzothiazole ring was found very important for increasing the activity. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic50 value of 8.6 μm. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the antiinflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic 50 value of 8.6 µm. in 2018, liu et al. identified ten new coumarin derivatives (3 monomers and 7 dimers) isolated in a phytochemical study on murraya exotica, a dwarf tree that is native of the tropical and subtropical areas of asia and traditionally used in the treatment of inflammatory-related diseases [147] . previous studies had shown that the main active components in m. exotica were coumarins; also bis-coumarins were isolated from the plant. a further investigation on the 95% aqueous etoh extract of the roots allowed the obtainment of new coumarins, together with other bioactive molecules. the compounds were tested for their inhibitory effect on no production and compound 103 ( figure 36 ) stood out among the coumarins in the inhibition against lps-induced no production in bv-2 microglial cells with ic50 value of 8.6 μm. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the antiinflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. the nuclear factor-kb (nf-kb) family of transcription factors is composed by a set of related, evolutionarily conserved dna-binding proteins. in response to multiple stimuli such as inflammatory cytokines, bacterial lipopolysaccharide (lps), viral infection or stress, a series of cascade reactions bring towards the entry on nf-kb into the nucleus and to the activation of several genes participating in immune and inflammatory responses, cell adhesion, growth control and regulation of apoptosis [148, 149] . in 2019, fan and co-workers evaluated in vivo and in vitro the anti-inflammatory activity of osthole ( figure 37 ) [150] , a natural prenylated coumarin from cnidium monnieri (l.) cuss. but also found in other medicinal plants, which has already shown different biological and pharmacological properties such as neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective and antimicrobial activities [13] . the team established that osthole inhibited the production of no, pge2, tnf-a and il-6 in lps-induced macrophages and suppressed the activity of inos and cox-2, probably by blocking the activation of nf-kb and p38/mapk signaling pathways. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 μg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 μg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. in the same year, mu and colleagues synthesized a series of eleven 7-substituted coumarins and evaluated their anti-inflammatory activities and their ability to exploit the nf-kb pathway [151] . all the screened compounds showed a relevant anti-inflammatory activity. in the series, compound 104 ( figure 38 ) was identified as a hit and further studies of molecular docking were conducted confirming that 104 could bind to the active site (nls polypeptide) of nf-κb p65. its binding affinity was further confirmed by surface plasmon resonance (spr) analysis. moreover, western blot assay showed that 104 remarkably blocked the nf-κb signaling pathway in vitro. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 μg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 μg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. alzheimer's disease (ad) is the most common form of dementia (60-70% of cases of dementia are cause by ad) and consists in a neurodegenerative disorder characterized by a slow, progressive and irreversible loss of cognitive function and memory [152] [153] [154] [155] [156] [157] . the current therapeutic approach, mainly based on the use of acetylcholinesterase (ache) inhibitors (rivastigmine, donepezil, galantamine) or n-methy-d-aspartic acid (nmda) receptor inhibitors (memantine), is merely symptomatic and does not counteract degeneration progression. new innovative approaches, such as multi-targeted strategies, are urgently required in order to cure cognition and motor dysfunctions, neurodegeneration and depression. among their biological activities, coumarins showed the ability to inhibit some biological targets involved in ad. with this in mind, some recent works aimed at investigating coumarins potential in the treatment of ad are discussed below. in 2019, karakaya and collaborators investigated the antioxidant and ache/buche inhibitory activity of aerial parts, fruits, flowers and root extracts from ferulago cassia boiss [158] . root's and fruit's dichloromethane extracts showed the highest antioxidant potential in tba assay. the evaluation of anticholinesterase activity was carried out by means of the ellman's method [159] : dichloromethane extracts showed significant inhibition against buche (96.56% ± 2.98 and 82.33% ± 2.69, respectively) at 20 µg/ml and appreciable inhibition against ache (53.24 ± 1.22 and 31.38 ± 5.41%, respectively) at 20 µg/ml. four coumarins were isolated from ferulago cassia-peucedanol, suberosin, grandivitinol and umbelliferone ( figure 39 ). therefore, f. cassia can be considered a starting point for the development of new compounds with antioxidant and anticholinesterase activity. thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and coworkers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic5 0= 0.027 ± 0.009 μm; tacrine ic50 = 0.048 ± 0.011 μm, donepezil ic50 = 0.039 ± 0.097 μm) and compound 106 was the most promising buche inhibitor (ic50 = 0.006 ± 0.002 μm; tacrine ic50 = 0.010 ± 0.004 μm, donepezil ic50 = 8.416 ± 0.628 μm) ( figure 40 ). thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and co-workers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic 50 thanks to their simple structural architecture and chemical stability, coumarins can be easily synthesized and modified in order to produce more active and selective compounds. najafi and coworkers synthesized a series of tacrine-coumarin derivatives linked to a 1,2,3-triazole moiety and evaluated their activity in terms of ache and butyrylcholinesterase (buche) inhibition, keeping donepezil and tacrine as reference drugs [160] . in addition, their beta-secretase 1 (bace1) inhibitory activity and neuroprotective potential were evaluated. since tacrine is a well-known inhibitor of the catalytic site of ache, whereas coumarins showed affinity for the peripheral anionic site (pas) [161] , this new compounds may be potential dual-and therefore more powerful -inhibitors of ches. the in vitro ache and buche inhibitory activity was evaluated using the ellman's method [159] ; among all the tested molecules, compound 105 resulted the best in ache inhibition (ic5 0= 0.027 ± 0.009 μm; tacrine ic50 = 0.048 ± 0.011 μm, donepezil ic50 = 0.039 ± 0.097 μm) and compound 106 was the most promising buche inhibitor (ic50 = 0.006 ± 0.002 μm; tacrine ic50 = 0.010 ± 0.004 μm, donepezil ic50 = 8.416 ± 0.628 μm) ( figure 40 ). concerning the anti-buche activity, structure-activity relationship studies showed that cl and me substituents, as well as the methylene linker, play a complex and not completely understood role in the enzyme inhibition. from the evaluation of inhibitory activity of the synthesized compounds on bace1, a moderate inhibitory activity of compound 105 was observed (inhibition of 28.69% and 13.97% at 50 and 10 µm, respectively). nevertheless, compound 105 did not show neuroprotective action on pc12 cells exposed to aβ [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] . then, in vivo evaluation of compound 105 using the morrison water maze method [162] was made and valuable results based on memory improvement in scopolamine-induced impairment were observed. similarly, rastegari and collaborators synthesized a series of 1,2,3-triazole-chromenone carboxamide derivatives and investigated their potential as anti-alzheimer's agents, in terms of inhibitory activity against ache, buche and bace1, besides their neuroprotective and metal-chelating properties [163] . the anti-ache activity of coumarin-3-carboxamide was already known [164] , as well as the ability of the 1,2,3-triazole moiety to enhance achei activity, if linked with tacrine or phenanthridinium derivatives and its bace1 inhibitory potential [165] . in vitro achei and buchei activities of the new compounds were evaluated, keeping donepezil as reference. higher activities were shown by compound 107, bearing 3,4-dimethylbenzyl connected to 1,2,3-triazole moiety and by compound 108, having 3-morpholinopropyl and 2-bromobenzyl moieties ( figure 41 ), even if they are much less active than donepezil (ic 50 = 0.027 µm). anti-bche activity was also modest and resulted to be affected by the type of amine connected to the amide moiety, that is, morpholine or piperidine and by the position and electronic properties of substituents on the benzyl group connected to 1,2,3-triazole ring. concerning the anti-buche activity, structure-activity relationship studies showed that cl and me substituents, as well as the methylene linker, play a complex and not completely understood role in the enzyme inhibition. from the evaluation of inhibitory activity of the synthesized compounds on bace1, a moderate inhibitory activity of compound 105 was observed (inhibition of 28.69% and 13.97% at 50 and 10 μm, respectively). nevertheless, compound 105 did not show neuroprotective action on pc12 cells exposed to aβ25-35. then, in vivo evaluation of compound 105 using the morrison water maze method [162] was made and valuable results based on memory improvement in scopolamine-induced impairment were observed. similarly, rastegari and collaborators synthesized a series of 1,2,3-triazole-chromenone carboxamide derivatives and investigated their potential as anti-alzheimer's agents, in terms of inhibitory activity against ache, buche and bace1, besides their neuroprotective and metal-chelating properties [163] . the anti-ache activity of coumarin-3carboxamide was already known [164] , as well as the ability of the 1,2,3-triazole moiety to enhance achei activity, if linked with tacrine or phenanthridinium derivatives and its bace1 inhibitory potential [165] . in vitro achei and buchei activities of the new compounds were evaluated, keeping donepezil as reference. higher activities were shown by compound 107, bearing 3,4-dimethylbenzyl connected to 1,2,3-triazole moiety and by compound 108, having 3-morpholinopropyl and 2bromobenzyl moieties ( figure 41 ), even if they are much less active than donepezil (ic50=0.027 μm). anti-bche activity was also modest and resulted to be affected by the type of amine connected to the amide moiety, that is, morpholine or piperidine and by the position and electronic properties of substituents on the benzyl group connected to 1,2,3-triazole ring. compound 107 was chosen for bace1 inhibitory activity evaluation, showing only modest results (ic50 = 21.13 μm compared to the reference om99-2 having ic50 = 0.014μm). also, neuroprotective potential of 107 was investigated by comparing 107-treated cells with intact (no intervention), quercetin+h2o2-treated (positive control) and h2o2-treated (negative control) cells. the mentioned compound showed moderate to good neuroprotective activity, not stronger than quercetin. finally, since ros, which are potentially involved in the neurodegenerative process of ad, may be generated from unregulated reaction of molecular oxygen with the redox active metals such as fe, cu and zn, the metal-chelating properties of 107 toward cu 2+, fe 2+ and zn 2+ were tested in methanol by means of uv-vis spectrophotometry. interaction between 107 and cu 2+ and zn 2+ was detected, whereas no interaction was observed between 107 and fe 2+ . compound 107 was chosen for bace1 inhibitory activity evaluation, showing only modest results (ic50 = 21.13 µm compared to the reference om99-2 having ic50 = 0.014 µm). also, neuroprotective potential of 107 was investigated by comparing 107-treated cells with intact (no intervention), quercetin+h 2 o 2 -treated (positive control) and h 2 o 2 -treated (negative control) cells. the mentioned compound showed moderate to good neuroprotective activity, not stronger than quercetin. finally, since ros, which are potentially involved in the neurodegenerative process of ad, may be generated from unregulated reaction of molecular oxygen with the redox active metals such as fe, cu and zn, the metal-chelating properties of 107 toward cu 2+, fe 2+ and zn 2+ were tested in methanol by means of uv-vis spectrophotometry. interaction between 107 and cu 2+ and zn 2+ was detected, whereas no interaction was observed between 107 and fe 2+ . de souza and co-workers designed and synthesized a series of 3-substituted-7-aminoalcoxycoumarin derivatives (109a-d, 110a-c, 111a-c, figure 42 ) whose achei/buchei activities and antioxidant properties were evaluated [166] . all the compounds showed good inhibitory activity on ache, with potencies in the nanomolar range, whereas their activity against buche was lower (ic 50 between 0.90 to 15.85 µm); 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin (111a) was considered a hit, showing an excellent inhibitory activity (ic 50 = 20 nm) and selectivity towards ache (ic 50 buche/ache = 354) compared to the reference drug donepezil (ic 50 = 6 nm, ic 50 buche/ache = 365). investigation of antioxidant properties showed that only compounds 111a-c presented activity in ferric ion reduction method (frap), whereas series 109a-d and 110a-c did not show significant results, suggesting that the dimethylaminophenyl moiety may be responsible for the antioxidant activity. de souza and co-workers designed and synthesized a series of 3-substituted-7-aminoalcoxycoumarin derivatives (109a-d, 110a-c, 111a-c, figure 42 ) whose achei/buchei activities and antioxidant properties were evaluated [166] . all the compounds showed good inhibitory activity on ache, with potencies in the nanomolar range, whereas their activity against buche was lower (ic50 between 0.90 to 15.85 μm); 3-(4-(dimethylamino)phenyl)-7-aminoethoxycoumarin (111a) was considered a hit, showing an excellent inhibitory activity (ic50 = 20 nm) and selectivity towards ache (ic50 buche/ache = 354) compared to the reference drug donepezil (ic50 = 6 nm, ic50 buche/ache = 365). investigation of antioxidant properties showed that only compounds 111a-c presented activity in ferric ion reduction method (frap), whereas series 109a-d and 110a-c did not show significant results, suggesting that the dimethylaminophenyl moiety may be responsible for the antioxidant activity. docking simulations showed that all the compounds were able to bind simultaneously the pas and cationic active site (cas) of the electric eel ache (eeache): the interaction with the cas took place by means of cation-π interaction between piperidinyl group and trp86. compounds 111a and 111c showed the ability to give a π-stacking interaction in the pas, with trp286. the smaller spacer of 111a allowed the coumarin moiety to be located properly in the gorge, achieving h-bonds with tyr337 and phe295. this is probably the reason for the most efficient binding mode (and consequently the best activity) to eeache of compounds with a short spacer. yang and his group synthesized a series of twenty-four 3-aryl coumarin derivatives and tested their cholinesterase inhibitory activity, monoamine oxidase (mao) inhibitory activity and antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic50 = 3.04 ± 0.32 μm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic50 = 2.76 ± 0.57 μm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r5 and r6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. docking simulations showed that all the compounds were able to bind simultaneously the pas and cationic active site (cas) of the electric eel ache (eeache): the interaction with the cas took place by means of cation-π interaction between piperidinyl group and trp86. compounds 111a and 111c showed the ability to give a π-stacking interaction in the pas, with trp286. the smaller spacer of 111a allowed the coumarin moiety to be located properly in the gorge, achieving h-bonds with tyr337 and phe295. this is probably the reason for the most efficient binding mode (and consequently the best activity) to eeache of compounds with a short spacer. yang and his group synthesized a series of twenty-four 3-aryl coumarin derivatives and tested their cholinesterase inhibitory activity, monoamine oxidase (mao) inhibitory activity and antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic 50 = 3.04 ± 0.32 µm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic 50 = 2.76 ± 0.57 µm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r 5 and r 6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. antioxidant activity in vitro [167] . as far as cholinesterase inhibition is concerned, most of the mentioned compounds showed moderate activity. compound 117 ( figure 43 ) exhibited a strong activity (ic50 = 3.04 ± 0.32 μm), whereas compound 116 showed selectivity towards ache. compound 114 was more active against buche (ic50 = 2.76 ± 0.57 μm) than donepezil, whereas compounds 112, 113 and 115 showed selectivity towards buche. 3-arylcoumarins bearing hydroxy group both at r5 and r6 positions displayed higher activity respect to the mono-substituted counterparts, especially towards ache, whereas no significant impact on buche inhibition was observed. as mentioned, mao-inhibitory activity was evaluated as well. in fact, mao is one of the enzymes responsible for oxidative stress, which is another factor involved in the neurodegenerative process characterizing ad and different studies identified various che/mao inhibitors as tools for ad treatment, showing positive results in clinical trials [168] . in yang's work, 3-arylcoumarins anti-mao activity was compared to that of rasagiline (a selective mao-b inhibitor): almost half of the compounds showed relevant activity [167] . among them, compound 117 was the most promising, with an ic 50 = 27.03 ± 0.50 µm, though weaker than rasagiline (ic 50 = 0.125 ± 0.005 µm). again, the presence of r 5 , r 6 hydroxy groups provided better mao inhibitory activity. finally, the antioxidant power of 3-arylcoumarins was studied by means of ferric ion reduction method (frap), using vitamin c as a reference. compound 117 resulted again the most active (frap value = 41.42 ± 0.35), also showing a certain activity in vivo, when tested on zebrafish, leading to an increase of total distance of zebrafish movement proportional to the concentration of compound used. in 2017, joubert and collaborators designed and synthesized a series of 7-substituted coumarins (118-146, figure 44 ), consisting in a coumarin motif (mao inhibitor) condensed with a benzyl-, piperidinyl-, n-benzylpiperidinylor p-bromo-n-benzylpiperizinyl moiety, which resemble the n-benzylpiperidine function present in donepezil (ache inhibitor), connected via an alkyl ether linkage at position 7 [169] . their biological activities were evaluated in terms of mao and che inhibitory potential. inhibition of hmao was achieved from all the designed compounds, which also showed selectivity towards mao-b. the benzyloxy series showed higher activity, in some cases even better than the reference selegiline (ic 50 = 0.008 µm), with ic 50 values in the nanomolar range (0.5-73 nm). piperidinyl-, n-benzylpiperidinyl-or p-bromo-n-benzylpiperizinyl moiety, which resemble the nbenzylpiperidine function present in donepezil (ache inhibitor), connected via an alkyl ether linkage at position 7 [169] . their biological activities were evaluated in terms of mao and che inhibitory potential. inhibition of hmao was achieved from all the designed compounds, which also showed selectivity towards mao-b. the benzyloxy series showed higher activity, in some cases even better than the reference selegiline (ic50 = 0.008 μm), with ic50 values in the nanomolar range (0.5-73 nm). . docking studies showed that 136 binds the substrate cavity through the coumarin ring, whereas the benzyl moiety occupies the entrance cavity. in addition, 136 was able to bind both the cas and pas of cholinesterase, which suggested that it could interfere with pas-induced β-amyloid aggregation. a different approach was followed by shi and collaborators, who designed four derivatives obtained by coupling two pharmacophores (carbazole and coumarin) to obtain potential multitarget agents for the treatment of ad [170] . the aim was to exploit the biological activities of both the mentioned moieties: on one hand coumarins are known to have inhibitory activity on ache, buche and mao, besides antioxidant properties; on the other hand, carbazole exhibits antioxidant activity [171] and the ability to inhibit aβ aggregation [172] . in addition, carbazole derivatives are reported to have inhibitory effect on ches, thus providing neuroprotection from β-amyloid toxicity [173] . thus, compounds 147a-d ( figure 45 ) were synthesized, characterized and their biological activities were evaluated: anticholinesterase activity was tested on eeache and on hache, using ellman's method [159] , keeping tacrine as reference drug. whereas all derivatives showed dose-dependent inhibitory activity on eeache (compound 147d resulted the best with an ic 50 of 3.75 µm), on hache they exhibited weak effects (147b ic 50 = 76.63 µm, 147d ic 50 = 70.51 µm). none of the tested compounds inhibited eqbuche or hbuche more than 30%, which means that compounds 147b and 147d showed selectivity for ache over buche. synthesized, characterized and their biological activities were evaluated: anticholinesterase activity was tested on eeache and on hache, using ellman's method [159] , keeping tacrine as reference drug. whereas all derivatives showed dose-dependent inhibitory activity on eeache (compound 147d resulted the best with an ic50 of 3.75 μm), on hache they exhibited weak effects (147b ic50 = 76.63 μm, 147d ic50 = 70.51 μm). none of the tested compounds inhibited eqbuche or hbuche more than 30%, which means that compounds 147b and 147d showed selectivity for ache over buche. as the alkyl linker length increased, the anti-cholinesterase activity improved, probably because a 5-methylene chain allowed the carbazole and coumarin moieties to bind both the cas and the pas, respectively. antioxidant properties were evaluated through the orac-fl method (oxygen radical absorbance capacity by fluorescein) [174] , using trolox (vitamin e analogue) as a standard; none of the tested compounds showed significant activity. among the tested derivatives, compound 147d resulted a promising lead candidate for the treatment of ad. epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable attacks, involving seizures and/or transient behavioral changes. its pathogenesis has not been completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory neurotransmission is involved [175] [176] [177] [178] [179] here, we report some recent advances in the use of coumarins as anticonvulsant compounds. abd-allah and collaborators recently studied the anticonvulsant activity of a series of coumarin derivatives, achieved by merging two or more pharmacophoric scaffolds in order to create new chemical entities with an improved biological activity [180] . the compounds here described possess all the necessary elements to exert anticonvulsant activity: a lipophilic aryl ring, a hydrogen-bonding domain and an electron-donor moiety [178, 179, 181, 182] . all the compounds were initially screened (phase i) using two standard animal seizure models, subcutaneous pentylenetetrazole (scptz) and maximal electric shock (mes) seizure tests using ethosuximide as reference drug. an assessment of the potential neurotoxicity was also done by means of rotarod test. phase ii consisted in the determination of ed50 value for compounds that conferred 100% protection in one or both tests. in the end, gaba level measurements were carried out in whole mouse brain for the most active compounds, using gabapentin as a reference drug. the results of phase i tests showed that all the tested compounds had protective activity against scptz-induced absence epilepsy (variable results in the range of 17-100% protection). among them, 148, 149 and 150 ( figure 46 ) were the most active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg, meaning that the compounds are 1.49, 1.48, 1.25 folds more potent than ethosuximide, respectively. as the alkyl linker length increased, the anti-cholinesterase activity improved, probably because a 5-methylene chain allowed the carbazole and coumarin moieties to bind both the cas and the pas, respectively. antioxidant properties were evaluated through the orac-fl method (oxygen radical absorbance capacity by fluorescein) [174] , using trolox (vitamin e analogue) as a standard; none of the tested compounds showed significant activity. among the tested derivatives, compound 147d resulted a promising lead candidate for the treatment of ad. epilepsy is a widespread neurological disorder, characterized by periodic and unpredictable attacks, involving seizures and/or transient behavioral changes. its pathogenesis has not been completely clarified yet; it is known, however, that an impairment between excitatory and inhibitory neurotransmission is involved [175] [176] [177] [178] [179] here, we report some recent advances in the use of coumarins as anticonvulsant compounds. abd-allah and collaborators recently studied the anticonvulsant activity of a series of coumarin derivatives, achieved by merging two or more pharmacophoric scaffolds in order to create new chemical entities with an improved biological activity [180] . the compounds here described possess all the necessary elements to exert anti-convulsant activity: a lipophilic aryl ring, a hydrogen-bonding domain and an electron-donor moiety [178, 179, 181, 182] . all the compounds were initially screened (phase i) using two standard animal seizure models, subcutaneous pentylenetetrazole (scptz) and maximal electric shock (mes) seizure tests using ethosuximide as reference drug. an assessment of the potential neurotoxicity was also done by means of rotarod test. phase ii consisted in the determination of ed 50 value for compounds that conferred 100% protection in one or both tests. in the end, gaba level measurements were carried out in whole mouse brain for the most active compounds, using gabapentin as a reference drug. the results of phase i tests showed that all the tested compounds had protective activity against scptz-induced absence epilepsy (variable results in the range of 17-100% protection). among them, 148, 149 and 150 ( figure 46 ) were the most active (100% protection) at 0.238, 0.239 and 0.283 mmol/kg, meaning that the compounds are 1.49, 1.48, 1.25 folds more potent than ethosuximide, respectively. in the mes-induced seizures though, all the compounds failed in completely protecting the animals. the best profile was exhibited by compound 151 (figure 46 ) which was capable of a 50% protection at 2.1 mmol/kg. a quantitative determination of ed 50 values was carried out for compounds 148, 149 and 150, which were able to fully protect animals in the scptz test. compound 148 was found to be the most active with an ed 50 of 54.86 mg/kg (0.131 mmol/kg). consequently, it was chosen for further investigation to elucidate the mechanism of action, which was assessed through an evaluation of gaba levels in mice brain. as a result, the proposed mechanism for compound 148 is a gaba-mediated one, perhaps non-vesicular release of gaba, gaba a receptor activation or gaba b receptor inhibition; probably an enhanced synthesis or reduced metabolism of gaba are also involved. the most active with an ed50 of 54.86 mg/kg (0.131 mmol/kg). consequently, it was chosen for further investigation to elucidate the mechanism of action, which was assessed through an evaluation of gaba levels in mice brain. as a result, the proposed mechanism for compound 148 is a gabamediated one, perhaps non-vesicular release of gaba, gabaa receptor activation or gabab receptor inhibition; probably an enhanced synthesis or reduced metabolism of gaba are also involved. a similar bivalent drug approach was followed by mohammadi-khanaposhtani and collaborators, who synthesized a series of coumarin-1,2,4-oxadiazole derivatives in order to create a new chemical entity with better anticonvulsant profile than coumarin and oxadiazole alone [183] . in fact, different 5-member heterocyclic rings-containing compounds such as oxadiazoles, triazoles and thiadiazoles were reported to have good anticonvulsant activity [184] [185] [186] through benzodiazepine (bdz) receptor [187] . the activity of the new derivatives was tested using ptz-and mes-induced seizures in mice, keeping diazepam as a reference drug. most of the new compounds did not show activity against ptz-induced seizures, except for three of them, 152a, 152b and 152c (figure 47 ), being 152b the most active (25% of protection using 10 mg/kg). compounds 152d, 152e and 152f ( figure 47 ) showed a 100% protection against mes-induced seizures at the doses of 7, 40 and 20 mg/kg, respectively (it should be considered that diazepam shows 100% protection at 2 mg/ml) [188] . compound 152d, which showed the best activity, was characterized by the absence of substituents on position 4 of the coumarin ring and by a 4-chloroaryl group connected to the 1, 2, 4-oxadiazole ring. the most active compounds 152d and 152e were used to investigate the mechanism of action; to do so, the effect of flumazenil (a bdz receptor antagonist) on their activity was evaluated. flumazenil antagonized both 152d and 152e, confirming that these compounds act as bzs receptor agonists. finally, in vivo neurotoxicity of compounds 152d and 152e was assessed and the tested compounds gave less neurological deficits that the reference drug diazepam. a similar bivalent drug approach was followed by mohammadi-khanaposhtani and collaborators, who synthesized a series of coumarin-1,2,4-oxadiazole derivatives in order to create a new chemical entity with better anticonvulsant profile than coumarin and oxadiazole alone [183] . in fact, different 5-member heterocyclic rings-containing compounds such as oxadiazoles, triazoles and thiadiazoles were reported to have good anticonvulsant activity [184] [185] [186] through benzodiazepine (bdz) receptor [187] . the activity of the new derivatives was tested using ptz-and mes-induced seizures in mice, keeping diazepam as a reference drug. most of the new compounds did not show activity against ptz-induced seizures, except for three of them, 152a, 152b and 152c (figure 47 ), being 152b the most active (25% of protection using 10 mg/kg). compounds 152d, 152e and 152f ( figure 47 ) showed a 100% protection against mes-induced seizures at the doses of 7, 40 and 20 mg/kg, respectively (it should be considered that diazepam shows 100% protection at 2 mg/ml) [188] . compound 152d, which showed the best activity, was characterized by the absence of substituents on position 4 of the coumarin ring and by a 4-chloroaryl group connected to the 1, 2, 4-oxadiazole ring. the most active compounds 152d and 152e were used to investigate the mechanism of action; to do so, the effect of flumazenil (a bdz receptor antagonist) on their activity was evaluated. flumazenil antagonized both 152d and 152e, confirming that these compounds act as bzs receptor agonists. finally, in vivo neurotoxicity of compounds 152d and 152e was assessed and the tested compounds gave less neurological deficits that the reference drug diazepam. a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920′s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. a series of 4-amino-3-nitrocoumarins (153a-e, figure 48 ) was synthesized and biologically evaluated by mokrov and co-workers [189] . the anticonvulsant activity was investigated using the mes mice model (grand mal seizures) and the corazole-antagonism test (modelling the so-called petit mal seizure). the only statistically significant result was given by compound 153a at doses in the range of 60-80 mg/kg, as it could increase the animal survival in mes test up to 60% (in the control group survival was 10%). corazole-induced seizures and animals' death were not prevented by compounds 153a, 153c and 153d. compound 153e exhibited good protection potential at 10-40 mg/kg and was able to prevent death in 50-63% of animals. the most active compound in the mes test was 153a, containing a coumarin ring with a 3-nitro group and a γ-aminobutirric acid fragment. the corresponding methyl-ester (153c) did not show any activity, as well as compound 153d, with an additional nitro group. compound 153e, bearing the methyl-ester of gaba pharmacophore and two nitro groups on the coumarin ring, showed the best profile in corazole-antagonism test. the corresponding free carboxylic acid 153d was found to be inactive, as well as compound 153c, having one less nitro group. compound 153a, bearing one nitro group and no substituent on gaba moiety, did not show any corazole-antagonist activity. therefore, it can be speculated that compounds 153a and 153e possess different mechanisms responsible for their anticonvulsant activity and they may be a starting point for further studies on coumarin derivatives as anticonvulsant agents. the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920′s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4 the recent medical history of anticoagulant drugs has been largely dominated by a simple class of molecules that has been discovered thanks to a mysterious livestock mortality and, later, employed as a powerful rodenticide: coumarins. the anticoagulant activity of coumarins was identified when in 1920 s seemingly healthy cattle of canada and north america died inexplicably for internal hemorrhages. the main cause of this decimation was attributed to a mold infestation of the damp hay, later known as the "sweet clover disease." however, it was not before 1940 that the responsible molecule was identified by karl link and his student harold campbell: it was 3,3'-methylenebis(4-hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. hydroxycoumarin), later known as dicoumarol. further studies by link's team brought to the discovery of warfarin in 1948 (so named from warf, wisconsin alumni research foundation, that financed the project), approved as a rodenticide in the usa in 1952 and for anticoagulation therapy in human in 1954, under the name of coumadin. currently, warfarin is one of the most widely used anticoagulation drug (in the uk it has been estimated that at least 1% of the population and 8% of people over 80 years are taking warfarin), together with other coumarin derivatives like dicumarol and acenocoumarol ( figure 49 ) [18, [190] [191] [192] . warfarin and other anticoagulant coumarins are vitamin-k antagonists (vkas). indeed, due to the structural similarity with vitamin-k, the compounds block the vitamin-k dependent pathways of coagulation, involving several factors (ii, vii, ix and x). despite the efficacy and the advantages of an oral therapy, warfarin is not devoid of side effects, mainly associated with bleeding and complications, like the narrow therapeutic range and interindividual genetic difference in pharmacokinetics, which requires a continuous monitoring of the patient [193, 194] . for this reason, the research of new safer and efficient compounds lead to the discovery of a novel vka, tecarfarin (ati-5923, figure 50 ), currently under development [195] . tecarfarin is active after oral administration and acts as a vitamin-k epoxide reductase (vkor) inhibitor; unlike warfarin, is not metabolized by the cytochrome p450 system but by human carboxylesterase-2 (hce-2) in hepatic microsomes. consequently, drug-drug or food-drug interactions are avoided, as well as genetic variability of cyp-450 system, providing a more stable anticoagulation effect compared to warfarin [196] . a detailed study on pharmacokinetics and pharmacodynamics of tecarfarin had been conducted on healthy patients by albrecht et al., together with the recent phase i study on its tolerability among patients with severe kidney disease [197, 198] . tecarfarin has the potentiality to be a valid substitute of warfarin in the oral therapy of thromboembolic disease. another approach reported in literature is the chemical modification of the coumarin scaffold by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives of salicylic acid. among the compounds evaluated by bang and co-workers in 2019, derivatives 154 and 155 ( figure 51 ) showed high anticoagulant activity, with an increased prothrombin time (pt) of 10.88 ± 0.56 sec and 13.10 ± 3.56 sec, respectively. both compounds resulted 1.5 times more active than warfarin (pt 7.97 ± 1.93) [199] . structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . another approach reported in literature is the chemical modification of the coumarin scaffold by conjugation of 7-hydroxylcoumarin and 7-hydroxy-4-methylcoumarin with some derivatives of salicylic acid. among the compounds evaluated by bang and co-workers in 2019, derivatives 154 and 155 ( figure 51 ) showed high anticoagulant activity, with an increased prothrombin time (pt) of 10.88 ± 0.56 sec and 13.10 ± 3.56 sec, respectively. both compounds resulted 1.5 times more active than warfarin (pt 7.97 ± 1.93) [199] . structural modifications of the 4-hydroxycoumarin core was also the rational of montagut-romans et al., who in 2017 explored the potentiality given by modifications performed on c3 position by introducing a side chain (with at most one unsaturation) structurally related to vitamin-k cofactor [200] . the underlying premise was the sar study performed by gebaur in 2007, which pointed out that the activity of 4-hydroxycoumarin was enhanced only by structural modification for c3 position by isoprenyl motifs [201] . in this contest, 14 functionalized 4-hydroxycoumarins with alkyl chains of different length, both linear and branched, were synthesized and their activity was evaluated in vitro and ex vivo (phenprocoumon was included in the test as internal standard). the ability to inhibit vkorc1 in rat liver microsomes was evaluated in vitro and, except for two compounds, the c3-alkyl derivatives showed a sub-micromolar activity (from 20 nm to 200 nm) overcoming the benchmark compound phenprocoumon. further ex vivo studies assessed the ability to increase in vivo the prothrombin time (pt) and compounds 156a and 156b ( figure 52 ) showed a promising anticoagulant activity after 24 h. it is possible that the presence of the halogen atom protects the drug from liver metabolism. despite the interesting anticoagulant activity, follow-up studies on the liver metabolism are necessary to determine if these molecules are substrate of cyp2c9, to which is to attribute the variability in the dosage of oral vitamin-k antagonists, due to its polymorphism [202] . figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. figure 53 ) presented a remarkable anticoagulant activity (pt = 41.2s and tt 128.5s) and no significant hepatic or renal toxicity when compared to warfarin (pt = 55.7s and tt 80.6s) [203] . although further studies are necessary to understand the mode of action of compound 157, it could be a promising anticoagulant agent for preclinical studies. diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] menteşe et al. synthesized a novel series of n -(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8 substituted-2-oxo-2h-chromen-3-carbohydrazides (158a-e, 159a-e, figure 54 ) [207] , merging the 1,2,4-triazole and the coumarin moieties, both characterized by a wide range of biological activities (including inhibition of α-glucosidases) and low toxicity profiles [208] [209] [210] [211] [212] [213] . then, their activity on α-glucosidases was studied, evaluating the enzyme inhibition in the presence of pnpg (p-nitrophenyl-α-d-glucopyranoside) as a substrate in the buffer (ph 6.8). among the new compounds, four molecules showed high inhibition activity, compared to acarbose (ic 50 = 8.85 ± 0.23 µg/ml): 158d (ic 50 = 4.28 ± 0.10 µg/ml), 158e (ic 50 = 0.96 ± 0.02 µg/ml), 159d (ic 50 = 6.75 ± 0.10 µg/ml) and 159e (ic 50 = 1.44 ± 0.06 µg/ml). diabetes is a chronic metabolic disease characterized by high blood sugar levels and is generally caused by an insufficient production of insulin by β-pancreatic cells or by the inability of human body to use this hormone. the consequences of diabetes might be very serious: blindness, kidney failure, stroke, heart attacks, lower limb amputation [204] [205] [206] menteşe et al. synthesized a novel series of n'-(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8 substituted-2-oxo-2h-chromen-3-carbohydrazides (158a-e, 159a-e, figure 54 ) [207] , merging the 1,2,4-triazole and the coumarin moieties, both characterized by a wide range of biological activities (including inhibition of α-glucosidases) and low toxicity profiles [208] [209] [210] [211] [212] [213] . then, their activity on α-glucosidases was studied, evaluating the enzyme inhibition in the presence of pnpg (p-nitrophenyl-α-d-glucopyranoside) as a substrate in the buffer (ph 6.8). among the new compounds, four molecules showed high inhibition activity, compared to acarbose (ic50 = 8.85 ± 0.23 μg/ml): 158d (ic50 = 4.28 ± 0.10 μg/ml), 158e (ic50 = 0.96 ± 0.02 μg/ml), 159d (ic50 = 6.75 ± 0.10 μg/ml) and 159e (ic50 = 1.44 ± 0.06 μg/ml). compounds 158e and 159e resulted the most active, probably because of the metoxy-group at position 8 of the coumarin ring. derivatives without substituents on positions 3 and 5 of the phenyl ring linked to the triazole nucleus resulted more active than compounds bearing a chlorine atom or a phenyl moiety on such positions. according to kinetic studies, the tested compounds inhibit αglucosidases in a competitive way. other studies focused on coumarins-mediated inhibition of αglucosidases were carried out by different groups. hu and collaborators synthesized through microwave radiation heating a new series of more than forty 3-arylcoumarins which were screened for antioxidant activity, α-glucosidases inhibition and advanced glycation end-products (ages) formation inhibition [214] . only eight of the synthesized compounds (160-167, figure 55 ) exhibited moderate to high inhibitory activity on α-glucosidase. compounds 158e and 159e resulted the most active, probably because of the metoxy-group at position 8 of the coumarin ring. derivatives without substituents on positions 3 and 5 of the phenyl ring linked to the triazole nucleus resulted more active than compounds bearing a chlorine atom or a phenyl moiety on such positions. according to kinetic studies, the tested compounds inhibit α-glucosidases in a competitive way. other studies focused on coumarins-mediated inhibition of α-glucosidases were carried out by different groups. hu and collaborators synthesized through microwave radiation heating a new series of more than forty 3-arylcoumarins which were screened for antioxidant activity, α-glucosidases inhibition and advanced glycation end-products (ages) formation inhibition [214] . only eight of the synthesized compounds (160-167, figure 55 ) exhibited moderate to high inhibitory activity on α-glucosidase. compound 165 was the most promising (ic50 = 1.37 ± 0.67 μm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic50 = 0.050 ± 0.003 μm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163,164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 ( compound 165 was the most promising (ic 50 = 1.37 ± 0.67 µm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic 50 = 0.050 ± 0.003 µm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163, 164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld 50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 (30 mg/kg/day) restored glucose blood levels near normal values, showing an effect similar to that of the oral antidiabetic glibenclamide. asgari and co-workers synthesized a new series of biscoumarin-1,2,3-triazole derivatives ( figure 56 ) and evaluated their α-glucosidase inhibitory potential, using acarbose as a reference drug [215] . compound 165 was the most promising (ic50 = 1.37 ± 0.67 μm), with a α-glucosidase inhibitory activity slightly weaker than acarbose (ic50 = 0.050 ± 0.003 μm). from an extensive sar study, it emerged that the 7-hydroxy group is important for the inhibition of α-glucosidase. compounds 160, 163,164, 165 and 166 were then tested in vivo: none of them showed toxicity on mice (ld50 > 5000 mg/kg). the mentioned compounds were also tested on streptozocin-induced diabetic mice. compound 166 showed the best profile, exhibiting a strong reduction of glucose blood levels. oral daily administration of 166 (30 mg/kg/day) restored glucose blood levels near normal values, showing an effect similar to that of the oral antidiabetic glibenclamide. asgari and co-workers synthesized a new series of biscoumarin-1,2,3-triazole derivatives ( figure 56 ) and evaluated their α-glucosidase inhibitory potential, using acarbose as a reference drug [215] . here, again, two active moieties, both characterized by a wide range of biological activities, were merged together: the bis-coumarin and the 1,2,3-triazole moieties [216] . all the synthesized compounds showed excellent activities (ic50 between 13.0 ± 1.5 and 75.5 ± 7.0 μm) compared to acarbose (ic50 750.0 ± 12.0). compound 168c, bearing 2-chloro phenyl moiety, resulted the most active. the substitution of the chlorine atom with a methyl group or its shift on the c4 position caused a decrease in activity. moreover, the inhibitory activity seemed to depend importantly on the electron properties of the substituents. from further kinetic studies it emerged that compound 168c inhibits α-glucosidases in a competitive mode (ki = 11 μm). a different therapeutic approach may be the stimulation of insulin secretion. in this perspective, ahmed and collaborators extracted from the aerial parts of clutia lanceolata (a medicinal plant native to sub-saharan africa and the arabian peninsula) twenty-one coumarins, including methyltio-and methylsulfinil-coumarins, thirteen of which were reported for the first time [217] . the structures of these natural compounds were elucidated from 2d-nmr and ms spectra, whereas their anti-diabetic activity was tested measuring the glucose-triggered insulin secretion of freshly isolated murine islets. here, again, two active moieties, both characterized by a wide range of biological activities, were merged together: the bis-coumarin and the 1,2,3-triazole moieties [216] . all the synthesized compounds showed excellent activities (ic 50 between 13.0 ± 1.5 and 75.5 ± 7.0 µm) compared to acarbose (ic 50 750.0 ± 12.0). compound 168c, bearing 2-chloro phenyl moiety, resulted the most active. the substitution of the chlorine atom with a methyl group or its shift on the c4 position caused a decrease in activity. moreover, the inhibitory activity seemed to depend importantly on the electron properties of the substituents. from further kinetic studies it emerged that compound 168c inhibits α-glucosidases in a competitive mode (k i = 11 µm). a different therapeutic approach may be the stimulation of insulin secretion. in this perspective, ahmed and collaborators extracted from the aerial parts of clutia lanceolata (a medicinal plant native to sub-saharan africa and the arabian peninsula) twenty-one coumarins, including methyltio-and methylsulfinil-coumarins, thirteen of which were reported for the first time [217] . the structures of these natural compounds were elucidated from 2d-nmr and ms spectra, whereas their anti-diabetic activity was tested measuring the glucose-triggered insulin secretion of freshly isolated murine islets. the applications and properties of coumarin scaffold have remarkably wide boundaries. coumarin-based compounds have been exploited in numerous research and industrial sectors, as active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser the applications and properties of coumarin scaffold have remarkably wide boundaries. coumarin-based compounds have been exploited in numerous research and industrial sectors, as active pharmaceutical ingredients, pesticides, fragrances, dyes for several purposes from laser technology to organic photoredox catalysis, cell imaging, photocleavable protecting groups and fluorescent biological probes [6, [218] [219] [220] [221] [222] [223] [224] [225] . in the following paragraphs, the most recent applications associated with the photophysical properties of coumarins have been reviewed. the development of a suitable formulation is a crucial step in order to achieve new functional therapeutics. the design of novel strategies aimed at selectively release the bioactive in a specific district at a determinate time to maximize efficacy and reduce off-target adverse effects represents an extremely active research frontline. so far, various stimuli-responsive systems have been considered in therapeutic approaches to regulate the release of the therapeutic cargo, including endogenous stimuli (e.g., ph, enzymes, redox reactions, etc.) and exogenous stimuli (e.g., light, magnetic field, ionizing radiations, etc.) [226, 227] . light-mediated therapies have shown excellent results in achieving on-demand therapeutics and optical tools for studying and controlling complex chemical and biological processes in localized areas, owing to their superior non-invasiveness and spatiotemporal precision upon applying a specific light-irradiation wavelength [227] [228] [229] . one method for the regulation of molecular processes with light is the use of photolabile "protecting" groups in key locations. ideally, this modification completely blocks the activity of any molecule and restores it only with light [230] . coumarins, particularly 4-hydroxymethyl derivatives, are known to undergo photolysis. keeping this concept in mind, several biomolecules of interest have been linked to the coumarin nucleus, mostly as acyl derivatives. then, under uv irradiation, the biomolecules can be released in biological systems. the photophysical parameters of the formed derivatives are determined by different factors as the mode of fusion, the chemical nature of additional rings and the presence of electron-donating and electron-withdrawing substituents [12] . fournier and co-workers in 2013 proposed a series of methyl-coumarins with redshifted absorption. in particular, three compounds (172-174, figure 58 ), were synthetically easily accessible and exhibited a significant action cross section for uncaging with blue-cyan light, whereas their uncaging ability in the uv spectral domain remained low in order to avoid their photoactivation when a properly tuned uv illumination is applied [231] . in the same year, fournier and co-workers further proved that compound 172 was a good blue-absorbing caging group, owing to its strongly donating substituent conjugated to the thiocarbonyl group. moreover, the research team demonstrated that this particular caging group could be used in zebrafish embryos in the context of development biology to perform chromatic orthogonal photoactivation of two biologically active species [232] . in 2017, gandioso and colleagues reported the development of green/red-absorbing chromophores based on coumarin scaffolds that could be useful as photocleavable protecting groups [224] . a series of coumarin derivatives in which the carbonyl of the lactone was replaced by a cyano(4-nitrophenyl)methylene moiety, by condensation of a thiocoumarin precursor with the corresponding arylacetonitrile derivatives, was synthesized and subsequently refined with the insertion of electro-withdrawing groups at the phenyl ring, leading to absorption in the green to red region (175, figure 58 ) [224] . the insertion of more than one electro-withdrawing group (such as -no 2 and -cn) decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a strong emission in the red region upon excitation with green light, as denoted by their significantly large stokes shifts. in order to demonstrate the utility of these new compounds as ppgs, a small collection of coumarin-based photocages of benzoic acid was prepared. thanks to photolysis studies with green light, it was demonstrated that the structure of the coumarin chromophore influenced the rate of the uncaging process. this observation gave the opportunity to exploit these new coumarin scaffolds as caging groups removable with visible light. on the other hand, bojtar and colleagues proposed water soluble red-shifted coumarin caging groups (176-178, figure 58 ), activated with green-light [233] . 58) [224] . the insertion of more than one electro-withdrawing group (such as -no2 and -cn) decreased the fluorescence emission, whereas the mononitro-containing coumarin derivatives had a strong emission in the red region upon excitation with green light, as denoted by their significantly large stokes shifts. in order to demonstrate the utility of these new compounds as ppgs, a small collection of coumarin-based photocages of benzoic acid was prepared. thanks to photolysis studies with green light, it was demonstrated that the structure of the coumarin chromophore influenced the rate of the uncaging process. this observation gave the opportunity to exploit these new coumarin scaffolds as caging groups removable with visible light. on the other hand, bojtar and colleagues proposed water soluble red-shifted coumarin caging groups (176-178, figure 58 ), activated with green-light [233] . the optical properties of coumarins as photo-responsive unites could be also applied to polymers that after a photochemical activation rapidly degrade into small molecules. in 2018, iturmendi and co-workers proposed that, through functionalization of polyphosphazenes with a coumarin-caged amino acid as a pendant group along the backbone, the sensitivity of the polymers to hydrolysis would be accelerated upon irradiation and effectively catalyze its own degradation [234] . coumarins possess a large electron-rich π-π conjugated system with charge transfer properties, reason why coumarin-based fluorophores are widely used for monitoring a variety of biologically important species and biochemical process in living cells, for example as diagnostic agent for detection of biothiols, enzymes, mitochondrial ph values, glucose and ions [3, 222, 235] . in particular, several coumarin scaffolds have been proposed and evaluated for the detection of ions in different fields, from cellular imaging to environmental waters. gong and co-workers based their work on an easily synthesized coumarin-based fluorescent probe (179, figure 59 ) that already was effective in the detection of glutathione (ghs) in the presence of cu 2+ ions, expanding its potentiality to the detection of hypochlorite ions with high selectivity and sensitivity. the probe showed a remarkable fluorescent intensity change in response to hypochlorite ions; moreover, this probe could be applied to detect cloin cells via intracellular fluorescent imaging [236, 237] . given the importance of hypochlorite the optical properties of coumarins as photo-responsive unites could be also applied to polymers that after a photochemical activation rapidly degrade into small molecules. in 2018, iturmendi and co-workers proposed that, through functionalization of polyphosphazenes with a coumarin-caged amino acid as a pendant group along the backbone, the sensitivity of the polymers to hydrolysis would be accelerated upon irradiation and effectively catalyze its own degradation [234] . coumarins possess a large electron-rich π-π conjugated system with charge transfer properties, reason why coumarin-based fluorophores are widely used for monitoring a variety of biologically important species and biochemical process in living cells, for example as diagnostic agent for detection of biothiols, enzymes, mitochondrial ph values, glucose and ions [3, 222, 235] . in particular, several coumarin scaffolds have been proposed and evaluated for the detection of ions in different fields, from cellular imaging to environmental waters. gong and co-workers based their work on an easily synthesized coumarin-based fluorescent probe (179, figure 59 ) that already was effective in the detection of glutathione (ghs) in the presence of cu 2+ ions, expanding its potentiality to the detection of hypochlorite ions with high selectivity and sensitivity. the probe showed a remarkable fluorescent intensity change in response to hypochlorite ions; moreover, this probe could be applied to detect clo − in cells via intracellular fluorescent imaging [236, 237] . given the importance of hypochlorite ions both in living systems (being one of the biologically most important reactive oxygens species) and in the environment (owing to its use as disinfectant), in 2019, shangguan and colleagues proposed another probe for this particular ion based on coumarin dye and malononitrile (180, figure 59 ). the fluorescence response of probe 180 at 459 nm towards hypochlorite ions gradually enhanced with the increase of clo − concentrations, resulting in 45-fold fluorescence enhancement. furthermore, probe 180 exhibited high accuracy for quantitative measurement of hypochlorite ions in real water samples and it can be used as a potential chemosensor for the detection of clo − in chemical environmental and biological systems [238] . in the same year, tang and co-workers worked on a coumarin based fluorescent probe (181, figure 59 ) able of rapidly discern hypochlorite and copper(ii) ions in water sample and biological systems. upon the reaction with clo − , the fluorescence wavelength of 181 displayed a strong blue shift along with the naked-eye visible changes from yellow to colorless. moreover, it exhibited an obvious fluorescence quenching behavior to copper(ii) with colorimetric analysis from yellow to luminous yellow [239] . because of the presence of the 7-diethylamino group and the 3-substituted lactone ring that are wellknown structural pattern accountable for the fluorescence properties. it was observed a different reactivity profile depending on the ph levels, probably due to the different reactivity of hypochlorite ions ascribable to the variation of the dissociation of the salt at different ph values. afterwards, a deeply investigation on the possible formation of chlorinated derivatives was conducted: hplc-pda-esi-ms analyses highlighted the presence of chlorinated derivatives and proved that the chlorination reaction was responsible for the linear fluorescence decays. the results suggest the possibility to exploit these coumarin ionic probes for the detection and quantitative determination of hypochlorite species in vivo. a coumarin fluorescent probe based on a nitro-3-carboxamide derivative for selective copper (ii) ions detection was reported by bekhradnia and colleagues. compound 185 (figure 60 ) showed the highest fluorescence intensity in presence of cu 2+ compared to a variety of other common heavy and toxic metal ions (for example pb(ii), co(ii), hg(ii)) and in aqueous solution at 320 nm [241] . another approach was attempted for the selective detection of copper (ii) ions by he et al. in 2018, which based the fluorescent probe on a coumarin-schiff base derivative (186, figure 60 ). this probe resulted to be particularly selective for cu 2+ even in the presence of several other ions [242] . saravana a noteworthy research on the mechanism of interaction between coumarin based ionic probes and hypochlorite ions had been conducted by starzak and collaborators [240] . first, the research team confirmed the linear decrease in the fluorescence emissions together with the increase in clo − concentration of three different coumarin derivatives, 182-184 (figure 59 ), which were selected because of the presence of the 7-diethylamino group and the 3-substituted lactone ring that are well-known structural pattern accountable for the fluorescence properties. it was observed a different reactivity profile depending on the ph levels, probably due to the different reactivity of hypochlorite ions ascribable to the variation of the dissociation of the salt at different ph values. afterwards, a deeply investigation on the possible formation of chlorinated derivatives was conducted: hplc-pda-esi-ms analyses highlighted the presence of chlorinated derivatives and proved that the chlorination reaction was responsible for the linear fluorescence decays. the results suggest the possibility to exploit these coumarin ionic probes for the detection and quantitative determination of hypochlorite species in vivo. a coumarin fluorescent probe based on a nitro-3-carboxamide derivative for selective copper (ii) ions detection was reported by bekhradnia and colleagues. compound 185 ( figure 60 ) showed the highest fluorescence intensity in presence of cu 2+ compared to a variety of other common heavy and toxic metal ions (for example pb(ii), co(ii), hg(ii)) and in aqueous solution at 320 nm [241] . another approach was attempted for the selective detection of copper (ii) ions by he et al. in 2018, which based the fluorescent probe on a coumarin-schiff base derivative (186, figure 60 ). this probe resulted to be particularly selective for cu 2+ even in the presence of several other ions [242] . saravana mani and colleagues designed in 2019 a coumarin hydrazine-based fluorescent probe for the detection of copper(ii), called benzepyr (187, figure 60 ), exploiting a reaction of condensation between 2-hydrazino benzothiazole and n,n -diethylamino-3-acetyl coumarin [243] . this particular fluorescent chemosensor could selectively detect cu 2+ among other disturbing metal ions, resulting particularly specific and highly responsive, with a visible colorimetric change of the solution, which turned from yellow to wine red. moreover, the limit of detection (lod) had been estimated to be 40 nm. benzepyr 187 was also tested for the fluorescence bioimaging of cu 2+ ions in hela cells using fluorescence microscopic analysis, resulting suitable for the exploitation as an ion marker in living cells. but also al ions and amino acids lys and arg [244] . in particular, the detection of lys and arg took place with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum absorption at 335 nm to 429 nm). at the same time the probe detected either way the presence of cu 2+ ions but could be used only for the fluorescent sensing of al 3+ . a further interesting exploitation of probe 188 (figure 60 ) was the fluorescent and colorimetric identification of cys, hcy and gsh when it was complexed with copper ions. despite the key role of chemical species like copper and hypochlorite, they are not the only ions valuable for detection. for this reason, a dual coumarin probe, fluorescent and colorimetric, was designed by chen and colleagues for the detection of palladium (ii) ions that can be used in living cells. this oxime-ether coumarin probe (189, figure 61 ) exhibited a strong green fluorescence with an emission peak at 500 nm. when palladium(ii) was added to the solution with compound 189, the fluorescence intensity at 500 nm decreased consequently, until 2 equivalents of pd 2+ were reached; at that point the fluorescence was almost completely quenched, which could clearly be observed with the naked eye. a linear fit between fluorescence and palladium (ii) concentration was observed in the range 0.0-8.0 μm, while the detection limit was measured to be 40 nm, which is far lower than the threshold for palladium content in drugs (5.0 ppm to 10.0 ppm -47.0 mm to 94.0 mm) specified by the world health organization [245] . it is also noteworthy the development of thioacetalised coumarin based fluorescent probes for the detection of mercury (ii), a hazardous ion both for human health and reproduction. cheng and co-workers exploited the known hg 2+ promoted deprotection reaction of dithioacetals to design two novel reactive fluorescent probes (190,191, figure 61 ) that showed a different behavior due to the different chemical structures: 190 displayed remarkable fluorescence quenching with the addition of another remarkable case of coumarin-based chemosensor had been presented by li and co-workers, who synthesized a multifunctional probe able to selectively detect not only copper (ii) ions but also al 3+ ions and amino acids lys and arg [244] . in particular, the detection of lys and arg took place with a colorimetric (from yellow to colorless) and a fluorescent response (from a maximum absorption at 335 nm to 429 nm). at the same time the probe detected either way the presence of cu 2+ ions but could be used only for the fluorescent sensing of al 3+ . a further interesting exploitation of probe 188 (figure 60 ) was the fluorescent and colorimetric identification of cys, hcy and gsh when it was complexed with copper ions. despite the key role of chemical species like copper and hypochlorite, they are not the only ions valuable for detection. for this reason, a dual coumarin probe, fluorescent and colorimetric, was designed by chen and colleagues for the detection of palladium (ii) ions that can be used in living cells. this oxime-ether coumarin probe (189, figure 61 ) exhibited a strong green fluorescence with an emission peak at 500 nm. when palladium(ii) was added to the solution with compound 189, the fluorescence intensity at 500 nm decreased consequently, until 2 equivalents of pd 2+ were reached; at that point the fluorescence was almost completely quenched, which could clearly be observed with the naked eye. a linear fit between fluorescence and palladium (ii) concentration was observed in the range 0.0-8.0 µm, while the detection limit was measured to be 40 nm, which is far lower than the threshold for palladium content in drugs (5.0 ppm to 10.0 ppm -47.0 mm to 94.0 mm) specified by the world health organization [245] . it is also noteworthy the development of thioacetalised coumarin based fluorescent probes for the detection of mercury (ii), a hazardous ion both for human health and reproduction. cheng and co-workers exploited the known hg 2+ promoted deprotection reaction of dithioacetals to design two novel reactive fluorescent probes (190, 191, figure 61 ) that showed a different behavior due to the different chemical structures: 190 displayed remarkable fluorescence quenching with the addition of hg 2+ ions while, in the presence of mercury ions, 191 displayed ratiometric fluorogenic and chromogenic response [246] . it should not be forgotten an on-off fluorescent probe for the tracking of iron (iii) ions based on 7-hydroxy-2-oxo-n-(pyridin-2-ylmethyl)chromene-3-carboxamide. in their work, warrier and kharkar demonstrated that compound 192 (figure 61 ) was selective towards fe 3+ ions and exhibited high fluorescence emission profile at 447 nm. the presence of other ions did not interfere with the detection of iron (iii) ions and the limit of detection was found to be 0.76 µm. moreover, cell imaging and mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential utility of probe 192 as cell-permeable chemosensor of fe 3+ in living cells [247] . the approach of jiao and colleagues for the detection of fluoride ions was based on the linkage between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive fluorescent probe. the mechanism of f − ions detection by compound 193 (figure 61 ) was explained and involved a desilylation reaction in the presence of fluoride ions. moreover, a linear relationship between the ratio of emission intensities at 532 and 465 nm and f − concentration over the range of 0-20 µm with a limit of detection of 0.025 µm was found [248] . differently, yao and co-workers exploited the capability of fluoride ions to form stable complex with ca 2+ to design a novel fluorescent sensor (194, figure 61) , synthesized from the combination of mandelic acid with 7-hydroxy-8-formylcoumarin through a hydrazine hydrate bridge, in order to selectively identify these two ionic species over other metal ions [249] . the fluorescence spectrum of compound 194 clearly increased when calcium ions were added to the solution with a limit of detection of 5.81 × 10 −7 m, while, once the complex between the probe and ca 2+ ions was obtained, the addition of fluoride ions to the solution lead to the turn-off of the fluorescence response with a limit of detection 4.28 × 10 −7 m. moreover, bio-imaging studies were performed in order to assure the possibility to exploit this novel chemosensor for the identification of ca 2+ and f − ions in vivo, with positive outcome. finally, reddy and choi designed and synthesized three dicyanovinylcoumarin probes as turn-on fluorescent sensor for the detection of cn − ions among other anions [250] . within the different synthesized probes only compound 195 ( figure 61 ) showed a remarkable increase of the fluorescence in presence of fluoride and cyanide ions with an interesting sensibility towards cn − ions: the limit of detection was up to 11.4 nm, lower than the maximum level in drinkable water according to who guidelines. high fluorescence emission profile at 447 nm. the presence of other ions did not interfere with the detection of iron (iii) ions and the limit of detection was found to be 0.76 μm. moreover, cell imaging and mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay proved the potential utility of probe 192 as cell-permeable chemosensor of fe 3+ in living cells [247] . the approach of jiao and colleagues for the detection of fluoride ions was based on the linkage between the coumarin scaffold and fluorescein in order to obtain a highly selective and sensitive fluorescent probe. the mechanism of fions detection by compound 193 (figure 61 ) was explained and involved a desilylation reaction in the presence of fluoride ions. moreover, a linear relationship between the ratio of emission intensities at 532 and 465 nm and f − concentration over the range of 0-20 μμ with a limit of detection of 0.025 μμ was found [248] . differently, yao and co-workers exploited the capability of fluoride ions to form stable complex with ca 2+ to design a novel fluorescent sensor (194, figure 61) , synthesized from the combination of mandelic acid with 7-hydroxy-8formylcoumarin through a hydrazine hydrate bridge, in order to selectively identify these two ionic species over other metal ions [249] . the fluorescence spectrum of compound 194 clearly increased when calcium ions were added to the solution with a limit of detection of 5.81 × 10 -7 m, while, once the complex between the probe and ca 2+ ions was obtained, the addition of fluoride ions to the solution lead to the turn-off of the fluorescence response with a limit of detection 4.28 × 10 -7 m. moreover, bio-imaging studies were performed in order to assure the possibility to exploit this novel chemosensor for the identification of ca 2+ and fions in vivo, with positive outcome. finally, reddy and choi designed and synthesized three dicyanovinylcoumarin probes as turnon fluorescent sensor for the detection of cnions among other anions [250] . within the different synthesized probes only compound 195 ( figure 61 ) showed a remarkable increase of the fluorescence in presence of fluoride and cyanide ions with an interesting sensibility towards cnions: the limit of detection was up to 11.4 nm, lower than the maximum level in drinkable water according to who guidelines. coumarins have found important applications also in the agri-food sector. in fact, the antimicrobial activity characterizing these natural compounds could be exploited for food coumarins have found important applications also in the agri-food sector. in fact, the antimicrobial activity characterizing these natural compounds could be exploited for food preservation or for the treatment of plant pathogens, infections in aquaculture or biofouling caused by eukaryotic organisms. in addition, because coumarin scaffold has been used in fluorescent probing, natural coumarins might be used in the detection of some substances in food samples. for instance, zhang and collaborators developed a new near-infrared probe constituted by a conjugated coumarin-indolium system, for rapid, colorimetric and ratiometric fluorescent detection of bisulfite and sulfite anions [251] . (bi)sulfite anions (hso 3 − /so 3 2− ) are widely used as preservative for foods and beverages in order to prevent oxidation, browning and microbial reaction during products' life cycle [252, 253] . unfortunately, high doses of (bi)sulfites can cause asthma or other allergic reactions. some individuals are very sensitive even to low levels of these anions [254] . in addition, sulfur dioxide (so 2 ) is one of the most distributed pollutants and it has a relevant impact on human health [252] , [253, 255] . an efficient tool to detect such molecules is provided by fluorescent probes; to date, different probes for hso 3 − /so 3 2− have been designed but many of them are intensity-based, which means that the signal output can be conditioned by different factors such as instrumental efficiency, probe concentration, environmental conditions. in addition, many of these probes show emissions only in the visible region and some of them need ultraviolet excitation, having limited biological applications. thus, new, more efficient probes are required. in zhang's work, a conjugated coumarin-indolium system with intramolecular charge transfer (ict) effect was developed, merging an electron-donating 7-diethylamino coumarin moiety and an electron-withdrawing positively charged indolium derivative, connected through an ethylene linker (figure 62 ). the whole system resulted in a typical 'push-pull' large conjugation dye system with ict properties. the so-designed probe showed nir fluorescence (667 nm) and a rapid, highly selective and sensitive detection process for hso 3 − /so 3 2− in aqueous solution under mild conditions; in addition, this probe exhibited significant colorimetric, nir fluorescent and ratiometric signal responses upon one excitation wave and a detection lower limit of ∼27 nm. this probe can be applied to detect hso 3 − /so 3 2− in real food samples, serum samples and living cells. when tested on real food samples (sugar, soft sugar, crystal sugar and wine in aqueous solution) this probe proved to be able to determine hso 3 − with good recovery. in addition, a paper test strip was developed, simply by wetting a strip of neutral filter paper with a solution of the probe in methanol (200 µm figure 62 ) can be used to develop a cheap, easy-to-prepare and easy-to-use paper test strip system for the detection of (bi)sulfites. and sulfite anions [251] . (bi)sulfite anions (hso3 − /so3 2− ) are widely used as preservative for foods and beverages in order to prevent oxidation, browning and microbial reaction during products' life cycle [252, 253] . unfortunately, high doses of (bi)sulfites can cause asthma or other allergic reactions. some individuals are very sensitive even to low levels of these anions [254] . in addition, sulfur dioxide (so2) is one of the most distributed pollutants and it has a relevant impact on human health [252] , [253, 255] . an efficient tool to detect such molecules is provided by fluorescent probes; to date, different probes for hso3 − /so3 2− have been designed but many of them are intensity-based, which means that the signal output can be conditioned by different factors such as instrumental efficiency, probe concentration, environmental conditions. in addition, many of these probes show emissions only in the visible region and some of them need ultraviolet excitation, having limited biological applications. thus, new, more efficient probes are required. in zhang's work, a conjugated coumarinindolium system with intramolecular charge transfer (ict) effect was developed, merging an electron-donating 7-diethylamino coumarin moiety and an electron-withdrawing positively charged indolium derivative, connected through an ethylene linker ( figure 62 ). the whole system resulted in a typical 'push-pull' large conjugation dye system with ict properties. the so-designed probe showed nir fluorescence (667 nm) and a rapid, highly selective and sensitive detection process for hso3 − /so3 2-in aqueous solution under mild conditions; in addition, this probe exhibited significant colorimetric, nir fluorescent and ratiometric signal responses upon one excitation wave and a detection lower limit of ∼27 nm. this probe can be applied to detect hso3 − /so3 2-in real food samples, serum samples and living cells. when tested on real food samples (sugar, soft sugar, crystal sugar and wine in aqueous solution) this probe proved to be able to determine hso3 -with good recovery. in addition, a paper test strip was developed, simply by wetting a strip of neutral filter paper with a solution of the probe in methanol (200 μm) . the result was a deep blue test paper ready for use: when a hso3 − solution is spotted on the test paper, rapid color changes can be observed, even if many other ions are present in the sample. different colors correspond to different concentrations of hso3 − . thus, probe 196 ( figure 62 ) can be used to develop a cheap, easy-to-prepare and easy-to-use paper test strip system for the detection of (bi)sulfites. a more recent example is a probe developed by nair and collaborators, able to selectively detect the amphiphilic bisulfate ion (hso4 − ) in edible plant foods, dog urine and drugs [256] . bisulfate consumption normally takes place through the ingestion of different edible plants, such as cabbage, broccoli, brussels sprouts, horseradish or seeds (black and white mustard, for instance). these plants contain glucosinolates [257, 258] that are hydrolyzed in our organism by the enzyme myrosinase, thus producing bisulfate ion [259] . in addition, bisulfate salts of many apis are currently on market, constituting another source of bisulfate ions [260] . when trying to evaluate the actual concentration of hso4 − , it is important to take account of the deprotonation equilibrium between hso4 − and so4 2-, a more recent example is a probe developed by nair and collaborators, able to selectively detect the amphiphilic bisulfate ion (hso 4 − ) in edible plant foods, dog urine and drugs [256] . bisulfate consumption normally takes place through the ingestion of different edible plants, such as cabbage, broccoli, brussels sprouts, horseradish or seeds (black and white mustard, for instance). these plants contain glucosinolates [257, 258] that are hydrolyzed in our organism by the enzyme myrosinase, thus producing bisulfate ion [259] . in addition, bisulfate salts of many apis are currently on market, constituting another source of bisulfate ions [260] . when trying to evaluate the actual concentration of hso 4 − , it is important to take account of the deprotonation equilibrium between hso 4 − and so 4 2− , using a highly selective probe able to discriminate between these two ions. nair and co-workers developed two fully water-soluble probes, coumarin-integrated glycine (cg) and coumarin-integrated alanine (ca) zwitterions, for the selective detection of hso 4 − at picomolar level (from 50 to 325 pm) ( figure 63 ). int. j. mol. sci. 2020, 21, x for peer review 41 of 83 using a highly selective probe able to discriminate between these two ions. nair and co-workers developed two fully water-soluble probes, coumarin-integrated glycine (cg) and coumarinintegrated alanine (ca) zwitterions, for the selective detection of hso4 − at picomolar level (from 50 to 325 pm) ( figure 63 ). glycine and alanine can interact selectively with the target through h-bond, due to their zwitterionic nature at physiological ph, whereas the 7-hydroxycoumarin moiety constitutes a good fluorophore probe, thanks to its biocompatibility, non-toxicity and water solubility. the cg/ca probes proved to be able to penetrate and stain living cell. when different unknown concentrations of clopidogrel bisulfate were added to a water solution of cg/ca, it was possible to precisely measure such concentration by measuring the emission intensity of each sample. confirmation of the experimentally observed values with the theoretically calculated ones supported the accuracy of the presented method. cg/ca probes were also tested on food samples: water extracts of cruciferous plant foods (cabbage, broccoli, mustard seeds, carrots) were added to aqueous solutions of the probes. again, titration of bisulfate ions was performed by emission measurements-addition of increasing volumes of aqueous food saps caused a growing reduction of probe's emission. cucumber and fenugreek were used as controls, as they do not contain bisulfates. eventually, bisulfate content was measured in pet urine samples (bisulfate is one of the common components of pet foods): urine samples were collected from two adult dogs and were treated with aqueous solution of cg/ca; a reduction of probe emission was noticed, revealing the presence of hso4 − (quenching was proportional to bisulfate quantity). in conclusion, cg and ca probes demonstrated the ability to detect bisulfate ions in aqueous solution at ph = 7.4, wherein hso4 − concentration was 10 5.4 lower than that of so4 2-, at concentration as low as 50 pm, even in presence of other ions. with regards to the antimicrobial activity of coumarin scaffold, some studies have been recently carried out, showing the potential of natural coumarins as food preservatives. yang and co-workers have studied the antimicrobial activity of eighteen natural compounds against r. solanacearum [261] , a bacterium responsible for the wilting of different plants such as tobacco, tomato, potato in (sub)tropical regions, causing significant economic losses [262, 263] . among them, four coumarins ( figure 64 ) showed an antibacterial activity stronger than that of thiodiazole copper treatment (antibacterial rate (mbc/mic) of 63.3%). daphnetin showed the highest activity, followed by xanthotol and esculetin (antibacterial rate 97.43%, 80.12% and 71.44%, respectively). antibacterial activity seemed to be enhanced by c6, c7 or c8 substitution, so hydroxycoumarins umbelliferone, esculetin and daphnetin were selected for further investigation of the mechanism of action. hydroxycoumarins were tested from 10 to 100 mg/l concentrations and from results it was clear that the good activity of umbelliferone can be enhanced by the additional hydroxylation of c6 position (esculetin), whereas even better results can be achieved by the dihydroxylation of c7 and c8 positions (daphnetin). tem images of r. solanacearum showed that daphnetin and esculetin caused irreversible damages to the cell membrane, whereas umbelliferone must follow a different path in inducing cell damage. it is worth noting that hydroxycoumarins showed very low cytotoxicity on human cells and have no effects on tobacco seeds' germination. furthermore, because r. solanacearum forms biofilm-like aggregations on host's roots, facilitating bacterial infection [264] , yang and his group speculated that hydroxycoumarins may interfere with biofilm formation. in fact, daphnetin, umbelliferone and esculetin (100 mg/l) reduced biofilm formation by 99.22%, 85.20% and 93.90%, respectively, probably influencing the swimming motility of the bacterium. thus, the glycine and alanine can interact selectively with the target through h-bond, due to their zwitterionic nature at physiological ph, whereas the 7-hydroxycoumarin moiety constitutes a good fluorophore probe, thanks to its biocompatibility, non-toxicity and water solubility. the cg/ca probes proved to be able to penetrate and stain living cell. when different unknown concentrations of clopidogrel bisulfate were added to a water solution of cg/ca, it was possible to precisely measure such concentration by measuring the emission intensity of each sample. confirmation of the experimentally observed values with the theoretically calculated ones supported the accuracy of the presented method. cg/ca probes were also tested on food samples: water extracts of cruciferous plant foods (cabbage, broccoli, mustard seeds, carrots) were added to aqueous solutions of the probes. again, titration of bisulfate ions was performed by emission measurements-addition of increasing volumes of aqueous food saps caused a growing reduction of probe's emission. cucumber and fenugreek were used as controls, as they do not contain bisulfates. eventually, bisulfate content was measured in pet urine samples (bisulfate is one of the common components of pet foods): urine samples were collected from two adult dogs and were treated with aqueous solution of cg/ca; a reduction of probe emission was noticed, revealing the presence of hso 4 − (quenching was proportional to bisulfate quantity). in [262, 263] . among them, four coumarins ( figure 64 ) showed an antibacterial activity stronger than that of thiodiazole copper treatment (antibacterial rate (mbc/mic) of 63.3%). daphnetin showed the highest activity, followed by xanthotol and esculetin (antibacterial rate 97.43%, 80.12% and 71.44%, respectively). antibacterial activity seemed to be enhanced by c6, c7 or c8 substitution, so hydroxycoumarins umbelliferone, esculetin and daphnetin were selected for further investigation of the mechanism of action. hydroxycoumarins were tested from 10 to 100 mg/l concentrations and from results it was clear that the good activity of umbelliferone can be enhanced by the additional hydroxylation of c6 position (esculetin), whereas even better results can be achieved by the dihydroxylation of c7 and c8 positions (daphnetin). tem images of r. solanacearum showed that daphnetin and esculetin caused irreversible damages to the cell membrane, whereas umbelliferone must follow a different path in inducing cell damage. it is worth noting that hydroxycoumarins showed very low cytotoxicity on human cells and have no effects on tobacco seeds' germination. furthermore, because r. solanacearum forms biofilm-like aggregations on host's roots, facilitating bacterial infection [264] , yang and his group speculated that hydroxycoumarins may interfere with biofilm formation. in fact, daphnetin, umbelliferone and esculetin (100 mg/l) reduced biofilm formation by 99.22%, 85.20% and 93.90%, respectively, probably influencing the swimming motility of the bacterium. thus, the expression of the regulating and structural flagellar genes flia, flhc and flhd in presence of daphnetin, umbelliferone and esculetin was evaluated and it turned out that the expression of flia and flhc was significantly repressed by the mentioned compounds. expression of the regulating and structural flagellar genes flia, flhc and flhd in presence of daphnetin, umbelliferone and esculetin was evaluated and it turned out that the expression of flia and flhc was significantly repressed by the mentioned compounds. another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . another interesting biological activity ascribed to the coumarin nucleus is its antifungal activity, which can be useful not only in the medicinal field but also in the agricultural one. as far as the latter is concerned, there has been an increasing interest in the exploitation of coumarin scaffold for the design and synthesis of novel fungicides useful in the treatment of many plant diseases. many pathogenic fungi still cause massive death of crops, limiting production and causing significant financial losses. to date, farmers deal with this problem by using chemical fungicides but such solution comes with some drawbacks: the long and extensive use of these chemicals may cause environmental pollution, accumulation of pesticides residues in the plants and drug resistance by fungi. thus, there is an urgent need for alternative (and green) solutions. in 2019, ramìrez-pelayo and his collaborators studied the coumarin-based compounds contained in the peel of citrus grown in colombia [265] . six coumarins were isolated (197-202, figure 65 ) and their antifungal activity, in terms of mycelial growth and spore germination inhibition, was evaluated against the fungus causing anthracnose (colletotrichum sp.). the production of oranges, mandarins, grapefruit, lemons, limes is limited by the action of microorganisms able to proliferate in such fruits because of peculiar condition such as high nutrient content, humidity and low ph values. nevertheless, these plants can defend themselves from pathogens thanks to the production of antimicrobial compounds which can be constitutive (phytoanticipins) or induced (phytoalexins) [266, 267] . as already mentioned, from the methanol extracts of c. latifolia and c. aurantifolia peels six compounds were isolated-5-geranyloxy-7-methoxycoumarin (197) , bergamottin (198) , bergapten (199) , isopimpinellin (200) , limettin (201) and oxypeucedanin hydrate (202) and their activity was compared to that of umbelliferone, scoparone and scopoletin. compounds 197-201 were tested at 0.25, 0.50 and 1.00 mm, whereas 202 was tested at 1.00 mm concentration. carbendazin and thymol were used as reference compounds (0.5 mm). the results showed that compounds 197-202 significantly inhibited mycelial growth of colletotrichum sp., the activity being proportional to the dose used. the (201)) was investigated, each of them showing enhanced activity respect to both the individual compounds. furthermore, the activity of 199, 201 and their mixtures was compared to that of the phytolaxins scoparone, scopoletin and umbelliferone; the results showed that phytolaxins were just slightly more active than the isolated compounds, although the highest fungistatic activity was shown by the mixture of 201 (0.75 mm) and 199 (0.25 mm). this mixture, along with compounds 197, 199 and 201, was selected for the evaluation of the inhibitory effect on spore germination in comparison with scopoletin, scoparone and umbelliferone. compounds 201 and 199 exhibited very good activity, with 96.7% and 95.3% inhibition, respectively but these percentages rapidly decrease, being less than 5% after 24 h. again, the 199/201 mixture showed the highest activity causing a complete, long-lasting inhibition of spore germination, thus suggesting that there may be an additive or synergistic effect between these compounds. therefore, coumarins and furanocoumarins may be useful scaffolds in the design of new antifungal agents. in 2018, yu and co-workers designed a series of coumarin-3-carboxamide derivatives and evaluated their antifungal activity against botrytis cinerea, alternaria solani, gibberella zeae, rhizoctonia solani, cucumber anthrax and alternaria leaf spot [268] . these phytopathogenic fungi are all relevant in agriculture, because they can cause significant losses in crops and investments. in this work, two different scaffolds were merged in order to design a new category of more effective fungicides. in fact, the use of carboxylic acid amides (caa) in this context is well-established, because amide fungicides have been used for over 50 years [269] . more recently, new amide fungicides with high activity and broad spectrum of action were developed (e.g., fluopyram, bixafen, sedaxane, isopyrazam, penthiopirad and boscalid) [1, 270] . in addition, hydrazide scaffolds have been found to have interesting biological and pharmacological activities [271, 272] . similarly, the antifungal activity of coumarin nucleus is well known (paragraph 0). considering these elements, the authors decided to combine the coumarin and the carboxamide/hydrazide scaffolds with the aim of synthesizing high-performance fungicides. all the synthesized compounds were tested and their antifungal activities against the mentioned phytopathogenic fungi were evaluated. at 100 µg/ml concentration, most of the tested compounds showed poor antifungal activity. only compounds 203 and 204 ( figure 66 ) showed ec 50 values of 1.57 µg/ml and 1.65 µg/ml, respectively, against botrytis cinerea, proving to be equivalent to the reference boscalid (0.51 µg/ml), whereas 203 (ec 50 = 1.80 µg/ml) resulted more active than boscalid (ec 50 = 2.98 µg/ml) against rhizoctonia solani. as already mentioned, from the methanol extracts of c. latifolia and c. aurantifolia peels six compounds were isolated-5-geranyloxy-7-methoxycoumarin (197) , bergamottin (198) , bergapten (199) , isopimpinellin (200) , limettin (201) and oxypeucedanin hydrate (202) and their activity was compared to that of umbelliferone, scoparone and scopoletin. compounds 197-201 were tested at 0.25, 0.50 and 1.00 mm, whereas 202 was tested at 1.00 mm concentration. carbendazin and thymol were used as reference compounds (0.5 mm). the results showed that compounds 197-202 significantly inhibited mycelial growth of colletotrichum sp., the activity being proportional to the dose used. the highest inhibition was exhibited by compounds 199 and 201 (32% and 25%, respectively); therefore, the fungistatic (201)) was investigated, each of them showing enhanced activity respect to both the individual compounds. furthermore, the activity of 199, 201 and their mixtures was compared to that of the phytolaxins scoparone, scopoletin and umbelliferone; the results showed that phytolaxins were just slightly more active than the isolated compounds, although the highest fungistatic activity was shown by the mixture of 201 (0.75 mm) and 199 (0.25 mm). this mixture, along with compounds 197, 199 and 201, was selected for the evaluation of the inhibitory effect on spore germination in comparison with scopoletin, scoparone and umbelliferone. compounds 201 and 199 exhibited very good activity, with 96.7% and 95.3% inhibition, respectively but these percentages rapidly decrease, being less than 5% after 24 hours. again, the 199/201 mixture showed the highest activity causing a complete, long-lasting inhibition of spore germination, thus suggesting that there may be an additive or synergistic effect between these compounds. therefore, coumarins and furanocoumarins may be useful scaffolds in the design of new antifungal agents. in 2018, yu and co-workers designed a series of coumarin-3-carboxamide derivatives and evaluated their antifungal activity against botrytis cinerea, alternaria solani, gibberella zeae, rhizoctonia solani, cucumber anthrax and alternaria leaf spot [268] . these phytopathogenic fungi are all relevant in agriculture, because they can cause significant losses in crops and investments. in this work, two different scaffolds were merged in order to design a new category of more effective fungicides. in fact, the use of carboxylic acid amides (caa) in this context is well-established, because amide fungicides have been used for over 50 years [269] . more recently, new amide fungicides with high activity and broad spectrum of action were developed (e.g., fluopyram, bixafen, sedaxane, isopyrazam, penthiopirad and boscalid) [1, 270] . in addition, hydrazide scaffolds have been found to have interesting biological and pharmacological activities [271, 272] . similarly, the antifungal activity of coumarin nucleus is well known (paragraph 0). considering these elements, the authors decided to combine the coumarin and the carboxamide/hydrazide scaffolds with the aim of synthesizing highperformance fungicides. all the synthesized compounds were tested and their antifungal activities against the mentioned phytopathogenic fungi were evaluated. at 100 μg/ml concentration, most of the tested compounds showed poor antifungal activity. only compounds 203 and 204 ( figure 66 ) showed ec50 values of 1.57 μg/ml and 1.65 μg/ml, respectively, against botrytis cinerea, proving to be equivalent to the reference boscalid (0.51 μg/ml), whereas 203 (ec50 = 1.80 μg/ml) resulted more active than boscalid (ec50 = 2.98 μg/ml) against rhizoctonia solani. starting from these data it was possible to suggest some structure-activity relationships: the replacement of amide bond with a hydrazide bond led to an improvement in antifungal activity spectrum. furthermore, coumarins bearing amide group resulted to be remarkably more active against botrytis cinerea and rhizoctonia solani, whereas against alternaria solani, gibberella zeae, starting from these data it was possible to suggest some structure-activity relationships: the replacement of amide bond with a hydrazide bond led to an improvement in antifungal activity spectrum. furthermore, coumarins bearing amide group resulted to be remarkably more active against botrytis cinerea and rhizoctonia solani, whereas against alternaria solani, gibberella zeae, cucumber anthrax and alternaria leaf spot they showed less potency. it is noteworthy that compounds bearing an electron-withdrawing group did not show any activity, whereas compounds with an electro-donating group were active against alternaria leaf spot. the addition to the coumarin nucleus of cl/f-substituted phenylidrazine gave compounds active on cucumber anthrax. a similar approach is the one followed by yang and collaborators, who decided to exploit coumarin nucleus for the functionalization of chitosan, in order to create more effective chitosan-based fungicides [273] . in fact, chitosan is becoming more and more appreciated due to its antimicrobial activity, low toxicity, biodegradability, biocompatibility and film forming activity. however, its application as fungicide is limited by its low solubility and weaker activity respect to the on-market fungicides. in this work, four coumarin-functionalized chitosan derivatives (205a-d, figure 67 ), were synthesized and tested against the phytopathogens alternaria solani sorauer, fusarium oxysporum f.sp. vasinfectum and fusarium moniliforme by evaluating the mycelial growth rate in vitro. at 1.0 mg/ml, compounds 205a-d showed inhibitory activity against a. solani, following the sequence 205d > 205b > 205c > 205a (38.25%, 51.23%, 44.01% and 52, 78%, respectively). cucumber anthrax and alternaria leaf spot they showed less potency. it is noteworthy that compounds bearing an electron-withdrawing group did not show any activity, whereas compounds with an electro-donating group were active against alternaria leaf spot. the addition to the coumarin nucleus of cl/f-substituted phenylidrazine gave compounds active on cucumber anthrax. a similar approach is the one followed by yang and collaborators, who decided to exploit coumarin nucleus for the functionalization of chitosan, in order to create more effective chitosan-based fungicides [273] . in fact, chitosan is becoming more and more appreciated due to its antimicrobial activity, low toxicity, biodegradability, biocompatibility and film forming activity. however, its application as fungicide is limited by its low solubility and weaker activity respect to the on-market fungicides. in this work, four coumarin-functionalized chitosan derivatives (205a-d, figure 67 ), were synthesized and tested against the phytopathogens alternaria solani sorauer, fusarium oxysporum f.sp. vasinfectum and fusarium moniliforme by evaluating the mycelial growth rate in vitro. at 1.0 mg/ml, compounds 205ad showed inhibitory activity against a. solani, following the sequence 205d > 205b > 205c > 205a (38.25%, 51.23%, 44.01% and 52, 78%, respectively). these results suggested that the introduction of halogens caused an increase in activity, depending on the number and the type of halogens. for instance, the 3,5-dichloro derivatives were more active against a. solani than the correspondent 5-cl or 5-br derivatives, probably because of an increase of hydrophobicity. the four coumarin-chitosan derivatives showed higher activity than chitosan alone also against f. oxysporum and f. moniliforme. compound 205d showed an inhibitory index of 57.09% against a. solani, 77.24% against f. oxysporum and 66.12% against f. moniliforme. the great biological potential of coumarins might be even increased by considering the possibility to complex coumarin scaffold with other substances as, for example, some metals. in fact, it has been proved that it is possible to enhance the activity of a certain drug simply binding it to a metallic-element [274] ; moreover, by combining known active moieties with metals, it would be possible to improve the parent compound with a certain selectivity or even with a new mechanism of action. therefore, many groups have already started to explore this strategy, aiming to produce more active compounds, exploiting metals as copper, platinum, zinc or silver. in some cases, due to its intrinsic potential, coumarin scaffold was exploited in this approach. some examples are discussed below. the nature of coumarin-copper complexes had already been evaluated in 2001 by karaliota and co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper (ii) complex (206) [275] . in this work, thanks to multiple instrumental characterization by means of ir, raman and nmr spectroscopy, the authors were able to identify the structure of the complex ( figure 68 ): a binuclear molecule [cu2(cca)4(h2o)2], where copper is coordinated by four carboxylic oxygens, one from each cca molecule and two water molecules. these results suggested that the introduction of halogens caused an increase in activity, depending on the number and the type of halogens. for instance, the 3,5-dichloro derivatives were more active against a. solani than the correspondent 5-cl or 5-br derivatives, probably because of an increase of hydrophobicity. the four coumarin-chitosan derivatives showed higher activity than chitosan alone also against f. oxysporum and f. moniliforme. compound 205d showed an inhibitory index of 57.09% against a. solani, 77.24% against f. oxysporum and 66.12% against f. moniliforme. the great biological potential of coumarins might be even increased by considering the possibility to complex coumarin scaffold with other substances as, for example, some metals. in fact, it has been proved that it is possible to enhance the activity of a certain drug simply binding it to a metallic-element [274] ; moreover, by combining known active moieties with metals, it would be possible to improve the parent compound with a certain selectivity or even with a new mechanism of action. therefore, many groups have already started to explore this strategy, aiming to produce more active compounds, exploiting metals as copper, platinum, zinc or silver. in some cases, due to its intrinsic potential, coumarin scaffold was exploited in this approach. some examples are discussed below. the nature of coumarin-copper complexes had already been evaluated in 2001 by karaliota and co-workers, who synthesized and characterized a binuclear coumarin-3-carboxilate-copper (ii) complex (206) [275] . in this work, thanks to multiple instrumental characterization by means of ir, raman and nmr spectroscopy, the authors were able to identify the structure of the complex ( figure 68 ): a binuclear molecule [cu 2 (cca) 4 (h 2 o) 2 ], where copper is coordinated by four carboxylic oxygens, one from each cca molecule and two water molecules. figure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stressinduced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. in the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cisfigure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stress-induced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. figure 69 ) which were tested on mcf-7 human breast cancer cells to evaluate their cytotoxicity [276] . in fact, the increase of ros levels might promote oxidative stressinduced cancer cell death, thus representing an alternative strategy for the treatment of some forms of tumors [277] [278] [279] . interestingly, it was found that the activity of such compounds depended also on their speciation; for example, 207, monomeric at neutral ph, showed valuable activity, whereas 212 -a dinuclear specie-resulted inactive. moreover, their cytotoxicity was not related to their ability to induce ros generation. such activity was showed only by complex 207, whereas 210 and 212 exhibited limited ros induction. 211 and 212 proved to be able to bind dna but none of the tested compounds showed significant nuclease activity. this means that imine-copper(ii) complexes showing cytotoxicity towards mcf-7 cells do not act by means of ros generation or nuclease activity, being these two the mechanisms of action previously ascribed to copper (ii) complexes [280] . in fact, complexes 208-210 acted as superoxide dismutase (sod) and catalase mimics. this is noteworthy, if we consider that in some cancer cells hypoxia induces an adaptive mechanism providing protection against damage and oxidative stress. in this context, copper (ii)-imine complexes acting as sod mimics may constitute a valid tool for the treatment of hypoxic tumors. in the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cisin the same year, qin and collaborators investigated the potential of platinum (ii) complexes against cisplatin resistant human lung adenocarcinoma (a549/ddp) and hela cells [281] . cisplatin was used as reference. in this work, eleven complexes between platinum (ii) and quinoline-coumarin derivatives (214-224, figure 70) were designed, synthesized and tested. all the new complexes showed an improved antineoplastic activity on a549/ddp and hela cells when compared to cis-pt(dmso) 2 cl 2 and the parent quinoline-coumarin derivatives, with ic 50 values ranging between 100 nm and 10. however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electronwithdrawing substituents. finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electron-withdrawing substituents. 100nm and 10.33 μm (75.02 ± 1.18 μm for a549/ddp or 12.09 ± 0.24 μm for hela). in addition, compounds 214-224 displayed selectivity towards the mentioned cancer cells over other tumoral cell lines and hl-7002 non-tumoral cell line. however, metal-coumarin complexes constitute an interesting tool not only in the pharmacological field but also in the chemical one. as an example of such application, we report here a work from aslkhademi and collaborators, who designed, synthesized ad characterized a new zn(ii) complex with a coumarin-hydrazone ligand (225, figure 71 ) [282] . in this case, the complex was evaluated as a catalyst for the synthesis of tetrazoles by means of 3 + 2 cycloaddition between a nitrile and an azide. to this purpose, many experiments were carried out using different nitriles and reaction conditions. in the end, the zn(ii)-coumarin-hydrazone complex resulted to be a suitable catalyst for the synthesis of tetrazoles; the most effective amount of catalyst to employ was 0.05 mmol and substrates bearing electron-donating groups reacted better than the ones with electronwithdrawing substituents. finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity finally, it is worth mentioning the application of metal-coumarin complexes as constituents of sol-gel coatings. lately, sol-gels are gaining attention due to their chemical stability, homogeneity and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3-carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using ag-coumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. and technological applications (e.g., reservoir for the prolonged release of antimicrobial substances). in 2013, jaiswal and co-workers proposed the synthesis of silver-coumarin complexes useful as antibacterial agents in sol-gel coatings [283] . these complexes were based on coumarin-3carboxylatosilver and its hydroxylated derivatives in 6, 7 and 8. antimicrobial activity was displayed even at concentration of 0.3% (w/w). the most active compound showed significative antibiofilm activity at 0.5% and 0.7% (w/w). these results are encouraging in the perspective of using agcoumarin complexes as biomedical coatings. table 2 reports some of the coumarins mentioned above, summarizing the biological activity, the molecular target and the origin. knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative knoevenagel condensation, wittig reaction, claisen rearrangement, heck reaction, pechmann condensation and perkin reaction are widely used methods for the obtainment of coumarin derivatives and have been widely reviewed over the years [284, 285] . the increasing interest in coumarins as potential biologically active compounds led to the development of innovative procedures and approaches aimed at the production of coumarins in high yield and in a sustainable manner. in the next paragraphs, we will focus on the most recent advances in coumarin synthesis. in the past two decades, flow chemistry emerged has a promising synthetic technology, due to the many advantages it offers compared to the traditional batch method. in fact, by using continuous flow reactors it is possible to control reaction parameters, such as temperature, stoichiometry, reaction time and others, very precisely, thus having the possibility to achieve better and more reproducible reactions. furthermore, the great surface area/volume ratio in continuous flow reactors often leads to better reaction yields. this new technology gives also the possibility to work with hazardous reagents and to perform superheated or pressurized reactions in safer conditions [286] [287] [288] . the applications of this technology are numerous and the improvement of chemical synthesis of natural compounds is one of them. in 2015, li and collaborators developed a two-stage synthesis of coumarins via o-acetylation of salicylaldehyde [289] . the reaction occurred via o-acetylation and intramolecular aldol-type condensation, followed by dehydration and it was performed using two heated coiled reactors (scheme 1). using this system, it was possible to reach a 120 g scale production. procedures and approaches aimed at the production of coumarins in high yield and in a sustainable manner. in the next paragraphs, we will focus on the most recent advances in coumarin synthesis. in the past two decades, flow chemistry emerged has a promising synthetic technology, due to the many advantages it offers compared to the traditional batch method. in fact, by using continuous flow reactors it is possible to control reaction parameters, such as temperature, stoichiometry, reaction time and others, very precisely, thus having the possibility to achieve better and more reproducible reactions. furthermore, the great surface area/volume ratio in continuous flow reactors often leads to better reaction yields. this new technology gives also the possibility to work with hazardous reagents and to perform superheated or pressurized reactions in safer conditions [286] [287] [288] . the applications of this technology are numerous and the improvement of chemical synthesis of natural compounds is one of them. in 2015, li and collaborators developed a two-stage synthesis of coumarins via o-acetylation of salicylaldehyde [289] . the reaction occurred via o-acetylation and intramolecular aldol-type condensation, followed by dehydration and it was performed using two heated coiled reactors (scheme 1). using this system, it was possible to reach a 120 g scale production. the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). scheme 1. set-up for the synthesis of coumarins via o-acetylation of salicylaldehyde in a continuous flow reactor. the use of immobilized reagents in organic synthesis is gaining ever more attention, due to the advantages offered by such type of reagents (for example, the simplification of product isolation and purification). this synthetic strategy has seen some applications in the production of natural scaffolds too. in 2018, mhiri and co-workers proposed a synthetic pathway for coumarin derivatives involving polymer supported reagents [290] . in the mentioned study, coumarin derivative a was prepared from 3-metoxy salicylaldheyde using reagents supported in a macroporous ion exchange resin, through the hydrolysis of the two intermediates iminocoumarin b and unsaturated nitrile c (scheme 2). coumarin synthesis with polymer-supported reagent. coumarin synthesis with polymer-supported reagent. the first step provided the preparation of polymer supported phenate ions that then reacted with phenylacetonitrile, leading to the formation of iminocoumarin and unsaturated nitrile, which were finally deblocked from the resin with chloroform. the acid hydrolysis of the obtained mixture of compounds b and c gave coumarin derivative a in very good yield (95%). it is worth speaking about another emerging strategy in organic synthesis: photocatalysis. in 2015, metternich and gilmour utilized this approach to emulate coumarins biosynthesis, using (−)-riboflavin as a photocatalyst [291] . in this work, two discrete activation modes of (−)-riboflavin were sequentially exploited to induce the isomerization and cyclisation of e-cinnamic acids used as starting material (scheme 3), thus overcoming the use of phenol-derived and pre-functionalized aryl rings as starting materials. the first step provided the preparation of polymer supported phenate ions that then reacted with phenylacetonitrile, leading to the formation of iminocoumarin and unsaturated nitrile, which were finally deblocked from the resin with chloroform. the acid hydrolysis of the obtained mixture of compounds b and c gave coumarin derivative a in very good yield (95%). it is worth speaking about another emerging strategy in organic synthesis: photocatalysis. in 2015, metternich and gilmour utilized this approach to emulate coumarins biosynthesis, using (−)riboflavin as a photocatalyst [291] . in this work, two discrete activation modes of (−)-riboflavin were sequentially exploited to induce the isomerization and cyclisation of e-cinnamic acids used as starting material (scheme 3), thus overcoming the use of phenol-derived and pre-functionalized aryl rings as starting materials. similarly, in 2019, song and co-workers reported a one-pot photoredox-catalyzed protocol to achieve a series of 3-fluoroalkylated coumarins starting from ortho-hydroxycinnamic esters (scheme 4) [292] . ortho-hydroxycinnamic esters and brcf2cor' (or other commercially available perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy)3 was found to be the best among the tested catalysts, whereas ch3cn, dmf, dmso and thf were all suitable solvents for this reaction. k2co3, et3n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. similarly, in 2019, song and co-workers reported a one-pot photoredox-catalyzed protocol to achieve a series of 3-fluoroalkylated coumarins starting from ortho-hydroxycinnamic esters (scheme 4) [292] . ortho-hydroxycinnamic esters and brcf 2 cor' (or other commercially available perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy) 3 was found to be the best among the tested catalysts, whereas ch 3 cn, dmf, dmso and thf were all suitable solvents for this reaction. k 2 co 3 , et 3 n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. perfluoroalkylated radical resources) were exposed to 30w blue leds irradiation under argon atmosphere for 12 h.; fac-ir(ppy)3 was found to be the best among the tested catalysts, whereas ch3cn, dmf, dmso and thf were all suitable solvents for this reaction. k2co3, et3n and dipea were all able to neutralize hbr produced during the process. this procedure gave 3-fluoroalkylated coumarins in good yields on milligram and gram scales. another example is the bichromatic synthesis of coumarins proposed by eivgi and collaborators in 2017 [293] . in this work, 2-nitrobenzyl-protected 2-hydroxystyrenes and acrylates underwent a uv-a (380 nm) photoinduced cross metathesis (cm) reaction, in the presence of ruthenium as a catalyst. in this step a 1-pyrenecarboxaldheyde solution was used as a uv filter, without whom the catalyst would be permanently inactivated due to phenol deprotection and phenolate chelation to the ruthenium. after cm, the uv filter was removed and irradiation with uv-c light (254 nm) led to more complex structures, such as coumarins. in fact, irradiation with uv-c light started a threereactions chain-photodeprotection of the intermediate, e/z isomerization and cyclization to form the desired coumarins (scheme 5). another example is the bichromatic synthesis of coumarins proposed by eivgi and collaborators in 2017 [293] . in this work, 2-nitrobenzyl-protected 2-hydroxystyrenes and acrylates underwent a uv-a (380 nm) photoinduced cross metathesis (cm) reaction, in the presence of ruthenium as a catalyst. in this step a 1-pyrenecarboxaldheyde solution was used as a uv filter, without whom the catalyst would be permanently inactivated due to phenol deprotection and phenolate chelation to the ruthenium. after cm, the uv filter was removed and irradiation with uv-c light (254 nm) led to more complex structures, such as coumarins. in fact, irradiation with uv-c light started a three-reactions chain-photodeprotection of the intermediate, e/z isomerization and cyclization to form the desired coumarins (scheme 5). chemists usually carry out reactions in solution, following the aristotelian principle "corpora non agunt nisi fluida seu soluta" or in other and more contemporary words "compounds do not react unless fluid or if dissolved" [294] . however, this is not always completely accurate, because many solventless reactions proceed efficiently. an enhancement in kinetics, owed to the different concentrations of reactants given the lack of solvents, bring actually to a higher reactivity and, where necessary, milder experimental conditions. at the same time, the absence of solvents provides the chance to heat over the boiling point of the most common solvents and to exploit the benefits of microwave assisted irradiation. the occurrence of efficient solid-state reactions shows that the reacting molecules are able to move freely in the solid state, whereas monitoring of the reaction is possible thanks ir and uv spectra in the solid state [295, 296] . the most remarkable advantage of solvent-free reaction, in all probability, is the noteworthy cut in the waste production associated with solvents handling, resulting in a more effective and ecological chemical process. there are three solvent-free techniques: (1) reactions conducted on mineral supports, (2) reaction without any solvent, support or catalyst and (3) solid-liquid phase transfer catalysis [297] . the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 °c for 10 min. without solvent, chemists usually carry out reactions in solution, following the aristotelian principle "corpora non agunt nisi fluida seu soluta" or in other and more contemporary words "compounds do not react unless fluid or if dissolved" [294] . however, this is not always completely accurate, because many solventless reactions proceed efficiently. an enhancement in kinetics, owed to the different concentrations of reactants given the lack of solvents, bring actually to a higher reactivity and, where necessary, milder experimental conditions. at the same time, the absence of solvents provides the chance to heat over the boiling point of the most common solvents and to exploit the benefits of microwave assisted irradiation. the occurrence of efficient solid-state reactions shows that the reacting molecules are able to move freely in the solid state, whereas monitoring of the reaction is possible thanks ir and uv spectra in the solid state [295, 296] . the most remarkable advantage of solvent-free reaction, in all probability, is the noteworthy cut in the waste production associated with solvents handling, resulting in a more effective and ecological chemical process. there are three solvent-free techniques: (1) reactions conducted on mineral supports, (2) reaction without any solvent, support or catalyst and (3) solid-liquid phase transfer catalysis [297] . the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 • c for 10 min. without solvent, giving after the work-up 7-hydroxy-4-methylcoumarin in 98% yield (scheme 6). the experimental conditions required for the classical synthesis of coumarin derivatives are, in some cases, drastic, as in the production of hymecromone via pechmann condensation where the final compound is obtain by stirring a mixture of resorcinol and ethyl acetoacetate in concentrated h2so4 for 12-24 h [295] . solvent-free reactions seemed to be a more ecological option. consequently, in 2001, sugino and tanaka proposed the synthesis of several coumarin derivatives both via pechmann and knoevenagel condensation reactions [298] . for example, p-toluensolfonic acid was employed as a catalyst and the reaction mixture was heated at 60 °c for 10 min. without solvent, giving after the work-up 7-hydroxy-4-methylcoumarin in 98% yield (scheme 6). scheme 6. solvent-free synthesis of 7-hydroxy-4-methylcoumarin. scheme 6. solvent-free synthesis of 7-hydroxy-4-methylcoumarin. knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . scheme 8. solvent-free synthesis of hetero-annulated coumarins. another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . knoevenagel condensation was also studied under solventless conditions and efficiently brought to the production of both coumarins and benzocoumarin derivatives [298] . another methodology was proposed in 2012 by kalita and kumar, that exploited the pechmann reaction using trifluoromethanesulfonic acid functionalized zr-tms (zr-tms, zirconia-based transition metal oxide mesoporous molecular sieves) catalysts, under solvent-free heterogeneous catalytic conditions. the product selectivity was always found to be about 100% (scheme 7) [296] . more recently, a novel series of hetero-annulated coumarins was obtained by ebrahimi and colleagues through a one-pot reaction under solvent-free conditions and evaluated as ache/buche inhibitors. as acidic catalyst nano-silica sulfuric acid was employed and the versatility of the system was assayed on appropriated benzaldehydes, dimedone and 4-hydroxycoumarin (scheme 8) [299] . also noteworthy is the application, once again, of a reusable heterogeneous catalyst, which contributed to the general environmental sustainability of the process thanks to the possible recovery and reuse [300] . another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team for the synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions, so the one-pot synthesis of coumarin derivatives was the natural next step (scheme 9) [301, 302] . because the catalyst reusability contributes to reduce the cost of the practical application processes, the reusability of the catalyst was investigated in the pechmann condensation. the catalyst, another solventless procedure worth noting is the one presented by kour and paul, which exploited lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. this particular kind of catalyst and its efficacy had been already studied by the team for the synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions, so the one-pot synthesis of coumarin derivatives was the natural next step (scheme 9) [301, 302] . because the catalyst reusability contributes to reduce the cost of the practical application processes, the reusability of the catalyst was investigated in the pechmann condensation. the catalyst, recovered by filtration, after five catalytic runs had a mild decrease in the catalytic activity and exhibited almost the same tg curve as the fresh one, depicting that the present heterogeneous system showed high thermal stability after successive reaction cycles [302] . in order to obtain a different and ecological procedure for the synthesis of 3-substituted coumarins via knoevenagel condensation, ghomi and akbarzadeh developed a procedure starting from various salicylaldehydes and 1,3-dicarbonyl compounds. they used mgfe2o4 nanoparticles as an efficient catalyst under solvent-free condition and ultrasound irradiation (scheme 10). basically, ultrasound irradiation increased the dimension of the single bubbles together with the number of active cavitation bubbles. the resulting effect was expected to be a higher maximum collapse temperature and a faster synthesis of coumarin compounds by knoevenagel reaction. comparing ultrasound irradiation to conventional heating the research team demonstrated that the first method allowed to obtain higher yields in shorter times, probably because of the shock wave and microjet generated by the cavitation. furthermore, with this method, mgfe2o4 nanoparticles were dispersed in the reaction and provided more sites for the construction of cavity over their surface. the catalyst was recovered via magnetic separation and reused up to 6 cycles without relevant loss of activity [303] . the convenience given by the removal of the catalyst by means of an external magnetic field is a significant improvement in the work-up of a reaction mixture. consequently, another magnetic nano structured catalyst was employed for the synthesis of coumarin nucleus, this time through pechmann condensation. in 2019, pakdel and co-workers proposed the synthesis of coumarin derivatives by leveraging fe3o4@boehmite-nh2-coii nps as a catalyst, under solvent-free conditions that resulted the best choice comparing the results obtained with classic solvents. the catalyst could be recovered easily and reused up to six times. however, no desired coumarin derivatives were observed in the case of phenols bearing electron-withdrawing substituents (such as -cl and -no2), behavior attributed to the proposed mechanism of the reaction [304] . in the field of solvent-free reactions, the application of ionic liquids as catalyst for the synthesis of coumarin scaffolds deserves a stand-alone status. ionic liquids (ils) have become increasingly popular in the last decades as a safer alternative to classic solvent systems, in a perspective of more sustainable chemical processes. most ils possess a number of properties that made them appealing, proving to be non-flammable, to possess a negligible vapor-pressure, that means that solvent evaporation is eliminated and to dissolve a wide range of organic and inorganic compounds [305] . furthermore, since ionic liquids are low-melting salts, they are made at least from two components which can be varied (the anion and the cation), thus the solvents could be designed with a particular end use in mind or possessing a particular set of properties [306] . however, their use in large quantities is restricted by some limitations like biodegradability, toxicity and high costs [307] [308] [309] . mahato and colleagues in 2017 used ionic liquids as an acidic catalyst in the regioselective synthesis of pyrano [3,2-c] coumarins under solvent-free conditions. in particular, given the fact that brønsted acidic ion liquids (bails) acquired recognition in the field of catalysis, the research team reported the catalytic effect of 1-butane sulfonic acid-3-methylimidazolium tosylate, [bsmim]ots (bail-1), on the tandem reaction between 4-hydroxycoumarin and α,β-unsaturated carbonyl compounds for the formation of pyrano[3,2-c]coumarins (scheme 10) [310, 311] . in addition to the high yields and the regioselectivity, this procedure allowed to avoid column chromatography for further purification. lewis acid grafted sulfonated carbon@titania composite as catalyst for the pechmann condensation. in order to obtain a different and ecological procedure for the synthesis of 3-substituted coumarins via knoevenagel condensation, ghomi and akbarzadeh developed a procedure starting from various salicylaldehydes and 1,3-dicarbonyl compounds. they used mgfe 2 o 4 nanoparticles as an efficient catalyst under solvent-free condition and ultrasound irradiation (scheme 10). noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of byproducts. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph2) for the synthesis of e-cinnamates and coumarin derivatives via basically, ultrasound irradiation increased the dimension of the single bubbles together with the number of active cavitation bubbles. the resulting effect was expected to be a higher maximum collapse temperature and a faster synthesis of coumarin compounds by knoevenagel reaction. comparing ultrasound irradiation to conventional heating the research team demonstrated that the first method allowed to obtain higher yields in shorter times, probably because of the shock wave and microjet generated by the cavitation. furthermore, with this method, mgfe2o4 nanoparticles were dispersed in the reaction and provided more sites for the construction of cavity over their surface. the catalyst was recovered via magnetic separation and reused up to 6 cycles without relevant loss of activity [303] . the convenience given by the removal of the catalyst by means of an external magnetic field is a significant improvement in the work-up of a reaction mixture. consequently, another magnetic nano structured catalyst was employed for the synthesis of coumarin nucleus, this time through pechmann condensation. in 2019, pakdel and co-workers proposed the synthesis of coumarin derivatives by leveraging fe 3 o 4 @boehmite-nh 2 -coii nps as a catalyst, under solvent-free conditions that resulted the best choice comparing the results obtained with classic solvents. the catalyst could be recovered easily and reused up to six times. however, no desired coumarin derivatives were observed in the case of phenols bearing electron-withdrawing substituents (such as -cl and -no2), behavior attributed to the proposed mechanism of the reaction [304] . in the field of solvent-free reactions, the application of ionic liquids as catalyst for the synthesis of coumarin scaffolds deserves a stand-alone status. ionic liquids (ils) have become increasingly popular in the last decades as a safer alternative to classic solvent systems, in a perspective of more sustainable chemical processes. most ils possess a number of properties that made them appealing, proving to be non-flammable, to possess a negligible vapor-pressure, that means that solvent evaporation is eliminated and to dissolve a wide range of organic and inorganic compounds [305] . furthermore, since ionic liquids are low-melting salts, they are made at least from two components which can be varied (the anion and the cation), thus the solvents could be designed with a particular end use in mind or possessing a particular set of properties [306] . however, their use in large quantities is restricted by some limitations like biodegradability, toxicity and high costs [307] [308] [309] . mahato and colleagues in 2017 used ionic liquids as an acidic catalyst in the regioselective synthesis of pyrano [3,2-c] coumarins under solvent-free conditions. in particular, given the fact that brønsted acidic ion liquids (bails) acquired recognition in the field of catalysis, the research team reported the catalytic effect of 1-butane sulfonic acid-3-methylimidazolium tosylate, [bsmim]ots (bail-1), on the tandem reaction between 4-hydroxycoumarin and α,β-unsaturated carbonyl compounds for the formation of pyrano[3,2-c]coumarins (scheme 10) [310, 311] . in addition to the high yields and the regioselectivity, this procedure allowed to avoid column chromatography for further purification. moreover, given the mild experimental conditions, there were no by-products attributable to decomposition or polymerization. noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of by-products. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph 2 ) for the synthesis of e-cinnamates and coumarin derivatives via the one-pot horner-wadsworth-emmons-type reaction (scheme 11). moreover, given the mild experimental conditions, there were no by-products attributable to decomposition or polymerization. noroozizadeh and co-workers focused their efforts on the synthesis of bis-coumarins thanks to a different catalyst, acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb), in continuous of a previous work of the research team on the preparation of brønsted acidic ionic liquids and solid salts [312, 313] . the aim was the development of a novel and efficient procedure for the synthesis of bis-coumarins which lacked the classical drawbacks as the use of solvents, high costs and low yields [314] . temperature is a key parameter in almost the totality of chemical reactions involving both inorganic and organic compounds. generally, most organic reactions are performed heating by traditional heat transfer equipment like oil baths, sand baths and heating jackets. the drawbacks associated with these heating techniques are the slowness, the possible development of temperature gradient within the sample and consequent local overheating that could lead to the formation of byproducts. microwave technology started to emerge in inorganic chemistry since the late 1970s and almost a decade after attracted the attention of organic chemists [315] . compared to the traditional heating, microwave dielectric heating has several advantages: (1) higher heating rates, (2) no direct contact between the source of energy and the reacting chemicals, (3) possible selective heating, because reacting chemicals interact differently with the commonly used microwave radiations and (4) rapid increase of the temperature also above boiling point in pressurized systems [316] . microwave irradiation is often associated with other technologies, such as ionic liquids and neat reactions (already mentioned in the previous paragraph) [295, 317] . in the late 1990s progresses in the classic coumarins synthesis via microwave irradiation were reported by singh and co-workers, who confirmed a reduction in reaction time of the pechmann condensation and by saidi and colleagues, who demonstrated the improvement in the synthesis of pyranocoumarins and furanocoumarins through claisen rearrangement [317, 318] . ten years later, valizadeh and shockravi pushed further the opportunities given by microwave heating combining it with a task-specific imidazolium-based phosphinite ionic liquid (il-opph2) for the synthesis of e-cinnamates and coumarin derivatives via the one-pot horner-wadsworth-emmons-type reaction (scheme 11). scheme 11. imidazolium-based phosphinite ionic liquid (il-opph2) for the microwave assisted synthesis of coumarins. the same reactions were in parallel conducted with traditional heating and microwave irradiation: the former technique showed considerably longer reaction times (13) (14) (15) (16) the same reactions were in parallel conducted with traditional heating and microwave irradiation: the former technique showed considerably longer reaction times (13-16 h vs. 10-12 min) and lower yields (65-60% vs. 79-83%). furthermore, il-opph 2 worked at the same time as reaction medium and reagent as the research team already demonstrated in a previous study focused on the synthesis of coumarin nucleus via knoevenagel and witting reactions in ionic liquids [319, 320] . in 2016, fiorito and colleagues merged the advantages of solvent-free reactions with microwave heating for the synthesis of several coumarin derivatives. they started from variously substituted phenols and propiolic acids in the presence of ytterbium triflate hydrate 10% mol as catalyst [321] . during the last thirty years, triflates of metals belonging to the lanthanide series have been regarded as effective water tolerant recyclable lewis acids. these metals have been discovered to catalyze different carbon-carbon and carbon-heteroatom bond formation reactions, providing the desired products in excellent yields by means of a green chemical approach [322, 323] . furthermore, wang and colleagues already employed this particular catalyst for the synthesis of 4-methylcoumarins via pechmann condensation [324] . fiorito and co-workers developed for the first time a synthetic procedure to obtain different substituted coumarins in 2 min. under microwave irradiation in 91-98% yields [321] . moreover, the research team recovered the ytterbium triflate catalyst and demonstrated that no loss of activity occurred during several cycles. in the same year, bouasla et al. compared the efficacy of different heterogeneous acidic catalysts in the pechmann synthesis of 4-methylcoumarin and 7-hydroxy-4-methylcoumarin when coupled by microwave heating. the efficacy of different amberylst-type catalyst in the pechmann synthesis of 7-hydroxy-4-methylcoumarin was already pointed out by sabou and colleagues in 2005 [325] . therefore, the catalytic performance of amberlyst-15 was compared with zeolite h-β and sulfonic acid functionalized hybrid material ts-os-so 3 h, performing the reaction under solvent-free conditions, at 130 • c for 20 min in a microwave reaction tube. amberlyst-15 showed a higher catalytic activity (97% yield against 21-44% obtained with two others catalysts) due to its high density of acid centers [326] . it is also worth mentioning the work of konrádová et al., who in 2017 developed an interesting protecting-group-free method starting from commercially available aromatic aldehydes and using a microwave promoted wittig reaction of a stabilized ylide for the synthesis of different natural products, including coumarins. after the optimization of the experimental parameters and the development of a general synthetic methodology for the synthesis of different substitute coumarin derivatives (scheme 12a), the method was further developed to incorporate a claisen rearrangement step. in order to demonstrate the synthetic utility and versatility of the developed method, the research team performed the total synthesis of two coumarins, one of which being osthole, a natural coumarin derivative already mentioned in the anti-inflammatory paragraph (scheme 12b) [327] . and lower yields (65-60% vs 79-83%). furthermore, il-opph2 worked at the same time as reaction medium and reagent as the research team already demonstrated in a previous study focused on the synthesis of coumarin nucleus via knoevenagel and witting reactions in ionic liquids [319, 320] . in 2016, fiorito and colleagues merged the advantages of solvent-free reactions with microwave heating for the synthesis of several coumarin derivatives. they started from variously substituted phenols and propiolic acids in the presence of ytterbium triflate hydrate 10% mol as catalyst [321] . during the last thirty years, triflates of metals belonging to the lanthanide series have been regarded as effective water tolerant recyclable lewis acids. these metals have been discovered to catalyze different carbon-carbon and carbon-heteroatom bond formation reactions, providing the desired products in excellent yields by means of a green chemical approach [322, 323] . furthermore, wang and colleagues already employed this particular catalyst for the synthesis of 4-methylcoumarins via pechmann condensation [324] . fiorito and co-workers developed for the first time a synthetic procedure to obtain different substituted coumarins in 2 min. under microwave irradiation in 91-98% yields [321] . moreover, the research team recovered the ytterbium triflate catalyst and demonstrated that no loss of activity occurred during several cycles. in the same year, bouasla et al. compared the efficacy of different heterogeneous acidic catalysts in the pechmann synthesis of 4-methylcoumarin and 7-hydroxy-4-methylcoumarin when coupled by microwave heating. the efficacy of different amberylst-type catalyst in the pechmann synthesis of 7hydroxy-4-methylcoumarin was already pointed out by sabou and colleagues in 2005 [325] . therefore, the catalytic performance of amberlyst-15 was compared with zeolite h-β and sulfonic acid functionalized hybrid material ts-os-so3h, performing the reaction under solvent-free conditions, at 130 °c for 20 min in a microwave reaction tube. amberlyst-15 showed a higher catalytic activity (97% yield against 21-44% obtained with two others catalysts) due to its high density of acid centers [326] . it is also worth mentioning the work of konrádová et al., who in 2017 developed an interesting protecting-group-free method starting from commercially available aromatic aldehydes and using a microwave promoted wittig reaction of a stabilized ylide for the synthesis of different natural products, including coumarins. after the optimization of the experimental parameters and the development of a general synthetic methodology for the synthesis of different substitute coumarin derivatives (scheme 12a), the method was further developed to incorporate a claisen rearrangement step. in order to demonstrate the synthetic utility and versatility of the developed method, the research team performed the total synthesis of two coumarins, one of which being osthole, a natural coumarin derivative already mentioned in the anti-inflammatory paragraph (scheme 12b) [327] . a broad range of indolo [2,3-c] coumarins have been obtained in good yields (46-88%) by gu and co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative coupling using palladium catalyst. the advantages of this methodologies compared to classic a broad range of indolo [2,3-c] coumarins have been obtained in good yields (46-88%) by gu and co-workers in 2019 under microwave-assisted base-free intramolecular cross dehydrogenative coupling using palladium catalyst. the advantages of this methodologies compared to classic synthesis of coumarin lactone ring were both atom economy and step efficiency, combined with c-h/c-h coupling without pre-functionalization [328] . the coumarin nucleus has been gaining increasing attention in the last years because of the variety of its applications both in medicinal chemistry and in agri-food sectors. in this review, we presented a collection of recent works that highlight the wide range of pharmacological activities ascribable to coumarins (antioxidant, antibacterial, antifungal, antiviral, anti-proliferative, anti-inflammatory, antidiabetics, anticoagulant and anti-neurodegenerative) and the possibility to easily modify and decorate this natural scaffold to perform structure activity relationships studies. the coumarin nucleus possesses some fundamental properties that ensure it an advisable role in the design of new biologically active derivatives, mainly due to its stability, low molecular weight and to the easiness to decorate it for increasing pharmacodynamic and pharmacokinetic properties. coumarin scaffold has been a valuable source of inspiration in the design of novel biologically active compounds and its role as a starting point is very attractive. this natural core is present in a number of currently available drugs used in the treatment of various diseases. for instance, the use of warfarin, acenocumarol and phenprocoumon in the treatment and prevention of thromboembolic diseases is now well established. other examples include the use of hymecromone as choleretic agent and that of the antihelminthic haloxon in veterinary medicine. even though the coumarin nucleus presents an impressive number of biological activities, its presence among marketed drugs is not widespread yet. further efforts are needed in order to achieve coumarin-based compounds with appreciable pharmacokinetic properties, along with high efficacy and a low toxicity profile. moreover, in this review we discussed coumarin photoproperties and their applications as photocleavable protecting groups and fluorescent probes. finally, we analyzed the latest updates in coumarin synthesis from both chemical and technological points of view. one of the most interesting approaches that emerged from this study is the design and use of coumarin-containing hybrid compounds. it is our hope that this review will contribute to further development in the investigation and exploitation of coumarins' potential. author contributions: conceptualization, a.p.; original draft preparation, f.a. and c.p.; writing-review and editing, s.d., a.p. and l.t.; supervision, a.p. and l.t. all authors have read and agreed to the published version of the manuscript. simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity pharmacological and biochemical actions of simple coumarins: natural products with therapeutic potential coumarin compounds in medicinal chemistry: some important examples from the last year recent advances and therapeutic journey of coumarins: current status and perspectives nomenclature of organic chemistry pharmacological and nutritional effects of natural coumarins and their structure-activity relationships coumarin: a natural, privileged and versatile scaffold for bioactive compounds natural products as fungicide and their role in crop protection sustainable production 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on the antioxidant property of coumarin-fused coumarins coumarin-fused coumarin: antioxidant story from n,n -dimethylamino and hydroxyl groups significantly enhanced antioxidant activity of chitosan through chemical modification with coumarins the evaluation of antioxidant and antifungal properties of 6-amino-6-deoxychitosan in vitro synthesis and biological evaluation of novel coumarins with tert-butyl and terpene substituents the hallmarks of cancer discovering the structure-activity relationships of different o-prenylated coumarin derivatives as effective anticancer agents in human cervical cancer cells primers on molecular pathways lipoxygenases: their role as an oncogenic pathway in pancreatic cancer overexpression of 12/15-lipoxygenase, an ortholog of human 15-lipoxygenase-1, in the prostate tumors of tramp mice synthesis and sar studies of mono o-prenylated coumarins as potent 15-lipoxygenase inhibitors 5-farnesyloxycoumarin: a potent 15-lox-1 inhibitor, prevents prostate cancer cell growth kaseb-mojaver, n. 8-farnesyloxycoumarin induces apoptosis in pc-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity synthesis, in vitro cytotoxicity activity against the human cervix carcinoma cell line and in silico computational predictions of new 4-arylamino-3-nitrocoumarin analogues synthesis and antiproliferative activity of 3-and 7-styrylcoumarins styrylcoumarin 7-sc2 induces apoptosis in sw480 human colon adenocarcinoma cells and inhibits azoxymethane-induced aberrant crypt foci formation in balb/c mice synthesis and in vitro anticancer activities of diethylene glycol tethered isatin-1,2,3-triazole-coumarin hybrids pyrimidine derivatives as novel lsd1 inhibitors design, synthesis, cytotoxicity and mechanism of novel dihydroartemisinin-coumarin hybrids as potential anti-cancer agents triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: design, synthesis, biological investigation and docking studies azide-alkyne cycloaddition towards 1h-1,2,3-triazole-tethered gatifloxacin and isatin conjugates: design, synthesis and in vitro anti-mycobacterial evaluation discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as mitochondria-targeting antitumor stat3 inhibitors signal transducer and activator of transcription-3, inflammation and cancer: how intimate is the relationship? carbonic anhydrases: novel therapeutic applications for inhibitors and activators multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? anhydrase inhibition and the management of glaucoma: a literature inhibition and the management of glaucoma: a literature antiglaucoma carbonic anhydrase inhibitors: a patent review anticonvulsant/antiepileptic carbonic anhydrase inhibitors: a patent review targeting tumour hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase ix inhibitors interfering with ph 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nosocomial pathogens: no eskape 4-dihydropyrimidinone-coumarin analogues as a new class of selective agent against s. aureus: synthesis, biological evaluation and molecular modelling study microwave-assisted synthesis, computational studies and antibacterial/ anti-inflammatory activities of compounds based on coumarin-pyrazole hybrid bioassay techniques for drug develompent modulation of the antibiotic activity against multidrug resistant strains of coumarins isolated from rutaceae species isolation and antimicrobial activity of coumarin derivatives from fruits of peucedanum luxurians tamamsch novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase ii inhibitors: design, synthesis and antibacterial activity hidden killers: human fungal infections gaffi fungal disease frequency|gaffi-global action fund for fungal infections how common are fungal diseases?-fungal infection trust nosocomial bloodstream infections in us hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study epidemiology of invasive candidiasis: a persistent public health problem synthesis and evaluation of a class of new coumarin triazole derivatives as potential antimicrobial agents 1,2,3-triazole incorporated coumarin derivatives as potential antifungal and antioxidant agents facile synthesis of novel coumarin derivatives, antimicrobial analysis, enzyme assay, docking study, admet prediction and toxicity study antifungal activity, mode of action variability and subcellular distribution of coumarin-based antifungal azoles antimicrobial activity of rhizomes of ferulago trachycarpa boiss. and bioguided isolation of active coumarin constituents coumarins reduce biofilm formation and the virulence of escherichia coli o157:h7 coumarin: a novel player in microbial quorum sensing and biofilm formation inhibition activity of coumarin against candida albicans biofilms susceptibility tests: diffusion test procedures microwave-assisted synthesis, biological evaluation and molecular docking studies of new coumarin-based 1,2,3-triazoles covid-19) events as they happen coumarin: an emerging antiviral agent. heliyon 2020, 6, e03217 plant derived antivirals: a potential source of drug development anti-viral activity of compounds from agrimonia pilosa and galla rhois extract mixture evaluation of antiviral effect and toxicity of total flavonoids extracted from robinia pseudoacacia cv. idaho in vitro and in silico study to evaluate the effectiveness of quercitrin as antiviral drug to dengue virus dehydrojuncusol, a natural phenanthrene compound extracted from juncus maritimus, is a new inhibitor of hepatitis c virus rna replication antiviral activity of resveratrol therapeutic potential of coumarins as antiviral agents bioactive prenylated coumarins as potential anti-inflammatory and anti-hiv agents from clausena lenis bioactive monoterpene indole alkaloids from nauclea officinalis novel tetrahydrofuran derivatives from trigonostemon howii with their potential anti-hiv-1 activities prenylated coumarins from the fruits of manilkara zapota with potential anti-inflammatory effects and anti-hiv activities evaluation of novel n -(3-hydroxybenzoyl)-2-oxo-2h-chromene-3-carbohydrazide derivatives as potential hiv-1 integrase inhibitors three new structures of the core domain of hiv-1 integrase: an active site that binds magnesium design and discovery of hiv-1 integrase inhibitors hydrazide-containing inhibitors of hiv-1 integrase chromone and chromanone derivatives as strand transfer inhibitors of hiv-1 integrase strand transfer inhibitors of hiv-1 integrase: bringing in a new era of antiretroviral therapy who -up to 650 000 people die of respiratory diseases linked to seasonal flu each year influenza pandemics of the 20th century new thiazolyl-coumarin hybrids: design, synthesis, characterization, x-ray crystal structure, antibacterial and antiviral evaluation synthesis and antimicrobial properties of some new thiazolyl coumarin derivatives synthesis, antimicrobial activity and advances in structure-activity relationships (sars) of novel tri-substituted thiazole derivatives bis coumarinyl bis triazolothiadiazinyl ethane derivatives: synthesis, antiviral activity evaluation and molecular docking studies regioselective ibx-mediated synthesis of coumarin derivatives with antioxidant and anti-influenza activities synthesis and structure-activity relationships of imidazole-coumarin conjugates against hepatitis c virus anti-hepatitis b virus activity of esculetin from microsorium fortunei in vitro and in vivo risposta del tessuto al danno origin and physiological roles of inflammation inflammation and the mechanism of action of anti-inflammatory drugs clinical perspectives of anti-inflammatory therapy in the elderly: the lipoxigenase (lox)/cycloxigenase (cox) inhibition concept cyclooxygenase-2 inhibitors: a painful lesson duration of treatment with nonsteroidal anti-inflammatory drugs and impact on risk of death and recurrent myocardial infarction in patients with prior myocardial infarction: a nationwide cohort study thiazole analogues of the nsaid indomethacin as selective cox-2 inhibitors synthesis, in vitro antiproliferative activity and in silico studies of fused tricyclic coumarin sulfonate derivatives molecular modeling, synthesis and screening of some new 4-thiazolidinone derivatives with promising selective cox-2 inhibitory activity synthesis and pharmacological evaluation of n-substituted 2-(2-oxo-2h-chromen-4-yloxy)propanamide as cyclooxygenase inhibitors synthesis and antiinflammatory activity of coumarin derivatives new coumarin derivatives as potent selective cox-2 inhibitors: synthesis, anti-inflammatory, qsar and molecular modeling studies 5-lipoxygenase development of 5-lipoxygenase inhibitors-lessons from cellular enzyme regulation synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-lox) inhibition activities and molecular docking study of 7-substituted coumarin derivatives anti-inflammatory prenylated phenylpropenols and coumarin derivatives from murraya exotica the nf-κb signaling pathway in human genetic diseases phosphorilation meets ubiquitination: the control of nf-kb activity the in vitro and in vivo anti-inflammatory effect of osthole, the major natural coumarin from cnidium monnieri (l.) cuss, via the blocking of the activation of the nf-κb and mapk/p38 pathways anti-inflammatory effect of novel 7-substituted coumarin derivatives through inhibition of nf-κb signaling pathway alzheimer's disease: overview the cholinergic hypothesis of geriatric memory dysfunction butyrylcholinesterase inhibitors ameliorate cognitive dysfunction induced by amyloid-β peptide in mice mechanisms of disease: alzheimer's disease the b-secretase enzyme bace1 as a therapeutic target for alzheimer's disease antioxidant and anticholinesterase potential of ferulago cassia with farther bio-guided isolation of active coumarin constituents a new and rapid colorimetric determination of acetylcholinesterase activity novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-alzheimer's compounds: in vitro and in vivo biological evaluation and docking study coumarins as cholinesterase inhibitors: a review novel tacrine-1,2,3-triazole hybrids: in vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors design, synthesis and anti-alzheimer's activity of novel 1,2,3-triazole-chromenone carboxamide derivatives novel coumarin-3-carboxamides bearing n-benzylpiperidine moiety as potent acetylcholinesterase inhibitors multifunctional iminochromene-2h-carboxamide derivatives containing different aminomethylene triazole with bace1 inhibitory, neuroprotective and metal chelating properties targeting alzheimer's disease discovery of novel dual-active 3-(4-(dimethylamino)phenyl)-7-aminoalcoxy-coumarin as potent and selective acetylcholinesterase inhibitor and antioxidant synthesis and biological evaluation of 3-arylcoumarins as potential anti-alzheimer's disease agents dual inhibitors of cholinesterases and monoamine oxidases for alzheimer's disease synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-b and cholinesterase inhibitors for the treatment of alzheimer's disease synthesis, characterization, crystal structure and evaluation of four carbazole-coumarin hybrids as multifunctional agents for the treatment of alzheimer's disease free-radical-scavenging effect of carbazole derivatives on aaph-induced hemolysis of human erythrocytes inhibition of beta-amyloid peptide aggregation by multifunctional carbazole-based fluorophores design, synthesis and biological evaluation of d-ring opened galantamine analogs as multifunctional anti-alzheimer agents aurone mannich base derivatives as promising multifunctional agents with acetylcholinesterase inhibition, anti-β-amyloid aggragation and neuroprotective properties for the treatment of alzheimer's disease cellular and molecular basis of epilepsy anticonvulsant profiles of certain new 6-aryl-9-substituted-6 design, synthesis and biological evaluation of new hybrid anticonvulsants derived from n-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives synthesis, molecular modeling studies and anticonvulsant activity of certain (1-(benzyl (aryl) amino) cyclohexyl) methyl esters synthesis and docking study of pyrimidine-triazine hybrids for gaba estimation in animal epilepsy models design, synthesis and docking studies of novel benzopyrone derivatives as anticonvulsants design and synthesis of novel parabanic acid derivatives as anticonvulsants synthesis and pharmacological screening of pyridazinone hybrids as anticonvulsant agents design, synthesis, in vivo and in silico evaluation of new coumarin-1,2,4-oxadiazole hybrids as anticonvulsant agents -arylthiazol-4-yl)methyl]azoles as a new class of anticonvulsants: design, synthesis, in vivo screening and in silico drug-like properties new gaba-modulating 1,2,4-oxadiazole derivatives and their anticonvulsant activity design, synthesis and pharmacological evaluation of novel 2-[2-(2-chlorophenoxy) phenyl]-1,3,4-oxadiazole derivatives as benzodiazepine receptor agonists design, synthesis, pharmacological evaluation and docking study of new acridone-based 1,2,4-oxadiazoles as potential anticonvulsant agents anticonvulsant effect of satureja hortensis aerial parts extracts in mice synthesis and anticonvulsant activity of n-substituted 4-amino-3-nitrocoumarins pharmacogenetics of warfarin: current status and future challenges the future prospects of pharmacogenetics in oral anticoagulation therapy almost 60 years old and still causing problems acenocoumarol in thromoembolic disorders current and emerging direct oral anticoagulants: state-of-the-art current and emerging pharmacotherapy for ischemic stroke prevention in patients with atrial fibrillation tecarfarin, a novel vitamin k reductase antagonist, is not affected by cyp2c9 and cyp3a4 inhibition following concomitant administration of fluconazole in healthy participants pharmacokinetics and pharmacodynamics of tecarfarin, a novel vitamin k antagonist oral anticoagulant pharmacokinetics of tecarfarin and warfarin in patients with severe chronic kidney disease synthesis and in vivo evaluation of new coumarin conjugates as potential indirect-action anticoagulants synthesis and biological evaluation of c-3 aliphatic coumarins as vitamin k antagonists synthesis and structure-activity relationships of novel warfarin derivatives pharmacogenomics of 4-hydroxycoumarin anticoagulants coumarin derivatives from ainsliaea fragrans and their anticoagulant activity world health organization (who) a new oral therapy for diabetes management: alpha-glucosidase inhibition with acarbose use of the alpha-glucosidase inhibitor acarbose in patients with 'middleton syndrome': normal gastric anatomy but with accelerated gastric emptying causing postprandial reactive hypoglycemia and diarrhea synthesis and kinetics studies of n -(2-(3,5-disubstituted-4h-1,2,4-triazol-4-yl)acetyl)-6/7/8-substituted-2-oxo-2h-chromen-3-carbohydrazide derivatives as potent antidiabetic agents pharmacological significance of triazole scaffold 1,2,4-triazole: a review of pharmacological activities synthesis, spectroscopic characterization, reactive properties by dft calculations, molecular dynamics simulations and biological evaluation of schiff bases tethered 1,2,4-triazole and pyrazole rings synthetic heterocyclic candidates as promising α-glucosidase inhibitors: an overview synthesis, α-glucosidase inhibition and molecular docking study of coumarin based derivatives syntheses of new 3-thiazolyl coumarin derivatives, in vitro α-glucosidase inhibitory activity and molecular modeling studies synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-diabetic agents biscoumarin-1,2,3-triazole hybrids as novel anti-diabetic agents: design, synthesis, in vitro α-glucosidase inhibition, kinetic and docking studies synthesis, in vitro evaluation and molecular docking studies of novel triazine-triazole derivatives as potential α-glucosidase inhibitors stimulation of insulin secretion by 5-methylcoumarins and its sulfur analogues isolated from clutia lanceolata forssk the in vivo dermal absorption and metabolism of [4-14c]coumarin by rats and by human volunteers under simulated conditions of use in fragrances a new coumarin laser dye 3-(benzothiazol-2-yl)-7-hydroxycoumarin lack of evidence for allergenic properties of coumarin in a fragrance allergy mouse model two novel aggregation-induced emission active coumarin-based schiff bases and their applications in cell imaging an injectable peg-bsa-coumarin-gox hydrogel for fluorescence turn-on glucose detection evaluation of antifungal activities and structure-activity relationships of coumarin derivatives development of green/red-absorbing chromophores based on a coumarin scaffold that are useful as caging groups application of coumarin dyes for organic photoredox catalysis the smart drug delivery system and its clinical potential light-triggered release of photocaged therapeutics-where are we now? photoactivated drug delivery and bioimaging beyond proof of principle light-controlled tools coumarinylmethyl caging groups with redshifted absorption a blue-absorbing photolabile protecting group for in vivo chromatically orthogonal photoactivation water-soluble red-shifted coumarin photocages coumarin-caged polyphosphazenes with a visible-light driven on-demand degradation a ratiometric two-photon probe for quantitative imaging of mitochondrial ph values a sensitive and selective fluorescent coumarin-based probe for detection of hypochlorite ion and its application to cellular imaging a sensitive and selective fluorescent probe for detection of glutathione in the presence of cu 2+ and its application to biological imaging a coumarin-based fluorescent probe for hypochlorite ion detection in environmental water samples and living cells a coumarin based fluorescent probe for rapidly distinguishing of hypochlorite and copper (ii) ion in organisms fluorescence quenching-based mechanism for determination of hypochlorite by coumarin-derived sensors novel coumarin-based fluorescent probe for selective detection of cu(ii) synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group coumarin based hydrazone as an ict-based fluorescence chemosensor for the detection of cu 2+ ions and the application in hela cells coumarin-based multifunctional chemosensor for arginine/lysine and cu 2+ /al 3+ ions and its cu 2+ complex as colorimetric and fluorescent sensor for biothiols a coumarin-based colorimetric and fluorescent dual probe for palladium(ii) ions that can be used in live cells thioacetalized coumarin-based fluorescent probe for mercury(ii): ratiometric response, high selectivity and successful bioimaging application highly selective on-off fluorescence recognition of fe 3+ based on a coumarin derivative and its application in live-cell imaging a novel fluorescein-coumarin-based fluorescent probe for fluoride ions and its applications in imaging of living cells and zebrafish in vivo a novel coumarin-based fluorescent sensor for ca 2+ and sequential detection of f − and its live cell imaging dicyanovinylcoumarin as a turn-on fluorescent sensor for cyanide ion a near-infrared fluorescent probe for rapid, colorimetric and ratiometric detection of bisulfite in food, serum and living cells a review of sulphites in foods: analytical methodologyand reported findings sulfites in foods: uses, analytical methods, residues, fate, exposure assessment, metabolism, toxicity and hypersensitivity clinical effects of sulphite additives health hazards from volcanic gases: a systematicliterature review estimation of bisulfate in edible plant foods, dog urine and drugs: picomolar level detection and bio-imaging in living organisms research on cruciferous vegetables, indole-3-carbinol and cancer prevention: a tribute to lee w. wattenberg crouciferous vegetables: cancer protective mechanism of glcisunolate hydrolisi products and selenium studies on the mechanism of myrosinase: investigation of the effect of glycosil acceptors on enzyme activity rosen's emergency medicines new insights into the antibacterial activity of hydroxycoumarins against ralstonia solanacearum molecular traits controlling host range and adaptation to plants in ralstonia solanacearum environmental cues controlling the pathogenicity of the plant pathogen ralstonia solanacearum needs aerotaxis for normal biofilm formation and interactions with its tomato host coumarins from the peel of citrus grown in colombia: composition, elicitation and antifungal activity secondary metabolites in plant innate immunity: conserved function of divergent chemicals two classes of plant antibiotics: phytoalexins versus phytoanticipins design, synthesis and antifungal activity evaluation of coumarin-3-carboxamide derivatives synthesis of new coumarin 3-(n-aryl) sulfonamides and their anticancer activity explosive properties of 1-hydroxybenzotriazoles synthesis and antitumor activities of a series of novel quinoxalinhydrazides synthesis, characterization and antibacterial activities of n-[1-(substituted phenyl) ethyl]-2-hydroxybenzohydrazide synthesis, characterization and antifungal activity of coumarin-functionalized chitosan derivatives principles of bioinorganic chemistry synthesis and characterization of a binuclear coumarin-3-carboxylate copper(ii) complex copper(ii) complexes of coumarin-derived schiff base ligands: pro-or antioxidant activity in mcf-7 cells? role of reactive oxygen species (ros) in therapeutics and drug resistance in cancer and bacteria the two faces of reactive oxygen species in cancer oxidative stress and lipid peroxidation products in cancer progression and therapy inhibition of landschutz ascites tumour growth by metal chelates derived from 3,4,7,8-tetramethyl-1,1 0-phenanthroline strong in vitro and vivo cytotoxicity of novel organoplatinum(ii) complexes with quinoline-coumarin derivatives synthesis, crystal structure and investigation of the catalytic and spectroscopic properties of a zn(ii) complex with coumarin-hydrazone ligand non-cytotoxic antibacterial silver-coumarin complex doped sol-gel coatings recent advances in the synthesis of coumarin derivatives via knoevenagel condensation: a review an overview on synthetic strategies to coumarins deciding wether to go with the flow: evaluating the merits of flow reactors for synthesis microreaction technology as a novel approach to drug design, process development and reliability continuous-flow technology-a tool fo the safe manifacturing of active pharmaceutical ingredients two-stage flow synthesis of coumarin via o-acetylation of salicylaldehyde synthesis of coumarin derivative using polymer supported reagents one photocatalyst, n activation modes strategy for cascade catalysis: emulating coumarin biosynthesis with (-)-riboflavin visible-light-driven, photoredox-catalyzed cascade of ortho-hydroxycinnamic esters to access 3-fluoroalkylated coumarins bichromatic photosynthesis of coumarins by uv filter-enabled olefin metathesis solvents and solvent effects in organic chemistry solvent-free coumarin synthesis via pechmann reaction using solid catalysts solvent-free reactions solvent-free coumarin synthesis hetero-annulated coumarins as new ache/buche inhibitors: synthesis and biological evaluation nano-silica sulfuric acid as an efficient catalyst for the synthesis of substituted pyrazoles preparation and characterization of lewis acid grafted sulfonated carbon@titania composites for the multicomponent synthesis of 4h-pyrimido[2,1-b]benzothiazoles and benzoxanthenones under solvent-free conditions a green and convenient approach for the one-pot solvent-free synthesis of coumarins and β-amino carbonyl compounds using lewis acid grafted sulfonated carbon@titania composite ultrasonic accelerated knoevenagel condensation by magnetically recoverable mgfe 2 o 4 nanocatalyst: a rapid and green synthesis of coumarins under solvent-free conditions fe 3 o 4 @boehmite-nh 2 -coii nps: an environment friendly nanocatalyst for solvent free synthesis of coumarin derivatives through pechmann condensation reaction ionic liquids-an overview ionic liquids. green solvents for the future when can ionic liquids be considered readily biodegradable? biodegradation pathways of pyridinium, pyrrolidinium and ammonium-based ionic liquids biodegradation studies of ionic liquids toxicity of ionic liquids: eco(cyto)activity as complicated but unavoidable parameter for task-specific optimization brønsted acidic ionic liquid-catalyzed tandem reaction: an efficient approach towards regioselective synthesis of pyrano[3,2-: c] coumarins under solvent-free conditions bearing lower e-factors novel brønsted acidic ionic liquids and their use as dual solvent-catalysts condensation of 2-naphtol with arylaldehydes using acetic acid functionalized ionic liquids as highly efficient and reusable catalysts tandem knoevenagel-michael-cyclocondensation reactions of malononitrile, various aldehydes and dimedone using acetic acid functionalized ionic liquid synthesis of bis-coumarins over acetic acid functionalized poly(4-vinylpyridinum) bromide (apvpb) as a green and efficient catalyst under solvent-free conditions and their biological activity microwave-assisted organic synthesis-a review dielectric parameters relevant to microwave dielectric heating acceleration of the pechmann reaction by microwave irradiation: application to the preparation of coumarins microwave promoted and improved thermal synthesis of pyranocoumarins and furocoumarins task-specific ionic liquid as reagent and reaction medium for the one-pot horner-wadsworth-emmons-type reaction under microwave irradiation one-pot wittig and knoevenagel reactions in ionic liquid as convenient methods for the synthesis of coumarin derivatives ytterbium triflate promoted coupling of phenols and propiolic acids: synthesis of coumarins microwave-assisted synthesis of xanthones promoted by ytterbium triflate ytterbium triflate catalysed meerwein-ponndorf-verley (mpv) reduction synthesis of coumarin by yb(otf) 3 catalyzed pechmann reaction under the solvent-free conditions synthesis of 7-hydroxy-4-methylcoumarin via the pechmann reaction with amberlyst ion-exchange resins as catalysts coumarin derivatives solvent-free synthesis under microwave irradiation over heterogeneous solid catalysts microwave-assisted synthesis of phenylpropanoids and coumarins: total synthesis of osthol synthesis of indolo[2,3-c]coumarins and indolo[2,3-c]quinolinones via microwave-assisted base-free intramolecular cross dehydrogenative coupling this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord-020010-q58x6xb0 authors: nan title: 19th icar abstracts: date: 2006-03-13 journal: antiviral res doi: 10.1016/j.antiviral.2006.02.001 sha: doc_id: 20010 cord_uid: q58x6xb0 nan the society was organized in 1987 as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its 20 th year of existence, and has about 600 members representing 30 countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, 10777 science center drive, san diego, ca 92121; phone +1 858 622 3117; fax +1 858 678 8182; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the 3 -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv-1teki (nsi phenotype) and hiv-1nl4-3 (si phenotype), and in a magi-x4 assay against hiv-1nl4-3 with hela-cd4-ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch 1 , imma clotet-codina 1 , julia blanco 1 , eduard pauls 1 , gemma coma 1 , samandhy cedeño 1 , francesc mitjans 2 , anuska llano 1 , margarita bofill 1 , bonaventura clotet 1 , jaume piulats 2 , jose este 1 1 retrovirology laboratory, fundacio irsicaixa, badalona, spain; 2 laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, 17e6, inhibited hiv-1 infection of primary macrophages. the effect of 17e6 on r5 or x4 hiv-1 replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (ec50) of 17 ± 2 g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb6 integrin (14d9.f8) also inhibited hiv-1 infection in this cell type. 17e6 reduced the detection of hiv-1 bal proviral dna in acutely infected macrophages but was completely ineffective against hiv-1 bal production in chronically infected macrophages, suggesting that 17e6 inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb6 integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant 1 , hong lu 2 , shuwen liu 2 , carol lackman-smith 3 , roger ptak 3 , jean-marc juteau 1 , shibo jiang 2 1 replicor inc., laval, que., canada; 2 f. lindsay kimball research institute, new york blood center, new york, ny, usa; 3 southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv-1 by blocking interactions between the v3 loop and cd4 has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully 2 -o-methylated derivatives) are also potent inhibitors of hiv-1-mediated membrane fusion and hiv-1 replication in a sequence-independent, sizedependent (optimal size ∼50-60 bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp41 and the hiv-1 fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp41 six-helix bundle formation, a critical step during the process of hiv-1 fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v3 loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥40 base) ps-ons may contribute to their inhibitory activity against hiv-1 fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp41 sixhelix bundle formation than shorter ps-ons (<30 bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv-1 and other enveloped viruses with type i fusion proteins. chris meier 1 , soenke jessel 1 , bastian reichardt 1 , olaf ludek 1 , jan balzarini 2 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the 3 -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv-1. additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita 1 , candace pert 2 , maria-teresa polianova 2 , alessandro ranazzi 1 , michael ruff 2 , carlo-federico perno 1 , stefano aquaro 1 1 university of rome tor vergata, italy; 2 georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv-1 commonly infected by ccr5-using (r5) strains of hiv-1. ccr5 is an attractive target for inhibition of ccr5 mediated hiv-1 entry. thus, ccr5 antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv-1 infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp120 v2 region of hiv-1, able to bind ccr5. dapta acts as selective viral entry inhibitor, displacing the binding of gp120 with ccr5. dapta anti-hiv-1 activity was evaluated in m/m infected with two different hiv-1 r5 strains, bal and 81a, in presence of several doses of the compound. dapta inhibited hiv-1 replication in m/m (>80% compared to control), measured by the p24 gag ag released in the cell culture supernatants, at concentration of 10-3 nm. pcr analysis of integrated hiv-1 proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv-1 r5 strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr5. in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of 10-3 and 10-4 nm, strongly inhibited apoptosis in sk-n-sh of 58 and 56%, respectively, compared to control. unexpectedly, tak-779 (a nonpeptidic ccr5 antagonist with potent anti-hiv-1 activity) inhibited apoptosis only of 30% compared to control. our results suggest that the development of new anti-hiv-1 compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv-1 r5 strains in the brain as direct inhibition of hiv-1 replication in m/m and gp120 related damage by ccr5 binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv-1 clades in pbmcs, in primary macrophages and several hiv-2 and siv strains in laboratory cell lines (range of 50% effective concentrations: 0.69-11 g/ml; 50% cytostatic concentration: >50 g/ml). prm-a also inhibits syncytium formation between persistently hiv-1-infected hut-78 cells and uninfected sup t1 cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp120 of hiv-1/mn in the presence of ca 2+ . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv-1. a variety of multiple mutations at n-glycosylation sites in hiv-1 gp120 are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv-1 gp120 glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/05/15), and the european commission (empro). jan muench 1 , ludger ständker 2 , knut adermann 2 , axel schulz 2 , michael schindler 2 , raghavan chinnadurai 2 , wolf-georg forssmann 2 , frank kirchhoff 2 1 department of virology university of ulm, albert-einstein allee 11, 89081 ulm, germany; 2 ipf pharmaceuticals gmbh, feodor-lynen-strasse 31, 30625 hannover, germany a variety of components in human blood might influence hiv-1 replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below 30 kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv-1 replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a 20-residue peptide corresponding to a c-terminal fragment of ␣1-antitrypsin (␣1-at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv-1 variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv-1 variants and primary isolates. virip specifically inhibited hiv-1 env function, and did not affect infection by virions containing hiv-2, siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the 20-residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv-1 gp41 fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp41 fp. our observations are evidence that a naturally occurring human substance inhibits hiv-1 infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than 350 virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv-1 entry inhibitors. stefano aquaro 1 , valentina svicher 1 , roberta d'arrigo 2 , ubaldo visco-comandini 2 , andrea antinori 2 , mario santoro 1 , giovanni di perri 3 , sergio lo caputo 4 , pasquale narciso 2 , carlo-federico perno 1,2 1 university of rome tor vergata, italy; 2 inmi l. spallanzani, italy; 3 university of turin, italy; 4 sm annunziata hospital, florence, italy to investigate gp41-variability and correlation with viroimmunological parameters in 54 hiv-infected patients (pts) receiving t20 added as a single active drug to a failing regimen. two hundred and ten hiv-gp41 sequences and clinical follow-up from 54 t20-treated patients were analyzed from baseline up to 48 weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd4 count (c/ul) was assessed by mann-whitney test. the addition of t20 to the failing antiretroviral regimen induced at 4 weeks a significant vl decrease from 5.1 log (stable in the last 12 weeks prior t20) to 4.3 log (p = .0002) and a significant cd4 increase from 48 c/ul (decreasing in the last 12 weeks prior t20) to 106 c/ul (p = .008). while vl rebounded to 4.7-5 log at 12-48 weeks, respectively, cd4 increased to 129 c/ul at 48 weeks. t20 resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v38a was the most common sign of t20 failure (27.8% of pts). the viroimmunological outcome of t20-treated pts varied according to gp41-mutations. v38a/e (38.5% of pts) was associated with a cd4 increase from bl (48 c/ul) of 4.5-fold (142 c/ul) at 24 weeks and 6.0-fold (196 c/ul) at 36 weeks (p = .004 and .02 compared without v38a/e, respectively). no significant correlation with vl was observed (from 4.9 log at bl to 4.4-4.8 at 24-36 weeks). by contrast, q40h + l45m (11.1% of pts) was associated with cd4 loss from 71 c/ul at bl to 26 c/ul at 36 weeks (p = .02), without significant changes in vl (from 5 log at bl to 5 log at 36 weeks). mutation n126k (observed in 6 pts, but never found at bl) abrogates the 4th gp41-glycosylation site and correlated with 1.7-fold cd4 increase at 24 weeks. conformational changes induced by v38a/e in the highly conserved giv motif of gp41-hr1, are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd4-recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h5n1 influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h5n1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as 10 2 eid 50 of a/vietnam/1203/04 (h5n1) influenza virus caused high fever (39.8-41.8 • c), weight loss (25.4% of initial), anorexia, extreme lethargy and death of animals on days 7-10 post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of 5 mg/kg/day for 5 days twice daily initiated 4 h p.i. inhibited the febrile response, reduced weight changes (14.6% of initial) and, most importantly, completely protected ferrets from lethal h5n1 infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of 2.8-6.5 log 10 eid 50 /ml on days 3, 5 and 7 p.i. systemic spread of the h5n1 virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day 5 p.i. in the brains of treated animals virus was detected in one of the two animals tested with >100-fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha1 subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h5n1 mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn 38105, usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/1203/04-like (h5n1) viruses. one virus possessed asparagine at position 31 of the m2 protein that was found in the naturally circulating virus (rgvn-1203) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn-1203sens) . balb/c mice were administered oseltamivir (1 or 10 mg/kg/day) and amantadine (1.5 or 15 mg/kg/day) twice daily for 5 days by oral gavage; the first doses were given 24 h before inoculation with 10 mld50 of h5n1 virus. combination treatment with 10 mg/kg/day oseltamivir and 15 mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h5n1 viruses (p < 0.01). treatment with oseltamivir at dosage of 10 mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days 3, 6, and 9 after inoculation (p < 0.05), but resulted in low survival rate (20%). single-drug amantadine showed dose-dependent effect only against rgvn-1203sens strain. notably, risk of death for mice that received 15 mg/kg/day of amantadine or 10 mg/kg/day of oseltamivir was similar (p < 0.05). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn-1203sens (p < 0.05) compared to singledrug usage and protected 90% of animals. importantly combination chemotherapy completely protected h5n1 virus spread to the brain of the mice: virus was not detected in brain of treated animals on days 3, 6, and 9 after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean 1 , reid asbury 2 , meera raj 1 , david lawrence 3 , martin petric 3 1 the university of british columbia, vancouver, bc, canada v6t 1z3; 2 ge healthcare bio-sciences, piscataway, nj 08855, usa; 3 british columbia center for disease control, vancouver, bc, canada v5z 4r4 in late 2002, severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the 21st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with 772 deaths reported from 29 countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh7: human liver] and primate (veroe6: monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over 50 host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard 1 , craig day 1 , robert montgomery 1 , kevin bailey 1 , matt heiner 1 , larry lauridsen 1 , robert sidwell 1 , kurt berg 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, 2005. 3, 251-262) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., 2004. apmis 112, 605-611) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning 12 h pre virus exposure at doses of 100,000 and 10,000 iu and the hydroxyethylrutoside mix (100 and 10 mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at 1.32 mg/ml (likely equivalent to an i.p. dose of 100 mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n01-ai-15435 from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk-21) cells that express the essential viral replication proteins (n, p, l and m2-1). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded 596 compounds (hit rate = 0.75%) with ec50 values ≤10 m and with selectivity index (si) values ≥10. seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a2) in a cytopathic effects inhibition assay (ec50 < 10 m; si > 10). these anti-rsv compounds represent nine different chemical classes. two compounds, a 4-aminoquinoline (ec50 = 0.25 m) and a thienopyrimidine (ec50 = 0.82 m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r44 ai047552-02. we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf1743(cf-1743) (1) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers (2) as new analogues in their own right, and 5 -phosphates (3) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides (4) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei 1 , joos van den oord 2 , pierre fiten 1 , ghislain opdenakker 1 , erik de clercq 1 , robert snoeck 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf29 gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after 4 days of differentiation and treated with serial dilutions of the test compounds. at 12 days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at 4 and 0.4 g/ml, foscarnet (pfa) at 40 g/ml and cidofovir (cdv) at 4, 0.4 and 0.04 g/ml inhibited viral dna content by more than 95%. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) 07-1 strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk-07-1 strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang 1 , oliver kregler 1 , john c. drach 2,3 , leroy b. townsend 3 , elke bogner 1 1 institut für klinische und molekulare virologie, erlangen, germany; 2 department of biologic and materials sciences, school of dentistry; 3 interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul56 and the small pul89, where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul89 is only required for duplex nicking. inhibitors targeting pul56 and/or pul89 are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul56. analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [2-bromo-4,5,6-trichloro-1-(2,3,5-tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl4rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic 50 in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv-1 infection. we report that indomethacin and aspirin inhibited hsv-1 replication at non-cytotoxic doses. in vero cells, 500 um indomethacin and 20 mm aspirin reduced hsv-1 titers 99.999 and 99.5%, respectively. electromobility shift assays revealed that hsv-1 activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv-1 titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p65, a component of the dimeric nf-kb complex, translocated to the nucleus of hsv-1 infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp4, an essential hsv immediate-early gene, 2.9and 2.5-fold, respectively, resulting in significant decreases of icp4 protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv-1 replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul97 phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp97 locus, and carrying the ul97 gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul97 by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul97-specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul97-specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic 50 of 15 m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a 3-log reduction (compared to wild-type virus) in the presence of 50 m mbv, and an ic 50 of 5 m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment (25 mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul97 in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against hsv-2 infection of human cervical cells and in a vaginal murine model. rep 9 has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). at a concentration of 100 m, rep 9 inhibited 6-logs of hsv-2 infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep 9 acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep 9 or pbs control at various times prior to vaginal challenge with a lethal dose of hsv-2 strain 186 (10 log 4 pfu). rep 9 prophylaxis provided protection to mice from hsv-2 infection and disease. protection was significant when challenged 30 min after treatment (p < 0.01). additionally, treatment with an analog of rep 9, which cannot activate tlr-9 mediated immune stimulation, was at least as active as rep 9, suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged 30 min after treatment (p < 0.001) with a trend toward protection when administered 60 min prior to challenge (p = 0.07). in summary, treatment with the rep 9 analog which has superior resistance to low ph and nuclease degradation was more effective than rep 9, in some experiments protecting 100% of mice from viral infection and disease. the testing of this ph resistant rep 9 analog in a gel formulation is currently underway. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi-4065, has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi-4065 inhibits translation initiated at the aug start site with ec50 of 308 nm (2.1 ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh-7 cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi-4065 produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi-4065 in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately 90% of mice with undetectable viral titer and the remaining mice show 1 log reduction in viral titer with 0.1 mg/mouse/day for 7 consecutive days. the fractional bioavailability of avi-4065 from a sq dose is approximately 1. the apparent elimination half life in rat, nonhuman primate and humans was 2.3, 4.5 and 11.4 h, respectively. the volume of distribution ranged from 0.6 to 1.0 l/kg and the cmax is linearly related to the dose in mg/m 2 . a phase i study in healthy volunteers in which 14 daily sq doses of 50 and 100 mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately 40-60% of the genotype-1 (gt-1) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns5b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of 4 -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a 4 -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is 4 -azido-cytidine, which shows good activity in the genotype 1b sub-genomic replicon (ic50 = 1.28 m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of 4 -azido-cytidine is a potent and highly selective inhibitor of ns5b (ic50 = 0.32 m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, 100 technology square, cambridge, ma 02139, usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns5b polymerase and ns3-4a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns5b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell 2005) . nim811, a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim811 exhibited potent anti-hcv activities in vitro. moreover, the combination of nim811 with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim811 than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim811 to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim811, with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r1479 was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r1479-tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns5b. in a transient replicon system r1479 inhibited hcv rna replication driven by genotype 1b polymerase with similar potency as compared to that driven by genotype 1a polymerase. r1479-tp inhibited native hcv replicase and recombinant ns5b from genotype 1a and 1b with similar potency. in contrast, r1479-tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and 3tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r1479-tp at concentrations up to 1 mm, suggesting high selectivity for the hcv rdrp. thus, r1479 was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype 2a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh7 cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati 1 , haitao guo 2 , gael westby 1 , anand mehta 2 , timothy block 1,2 1 institute for hepatitis and virus research; 2 drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of 80,288 drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg2.2.15, we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in 1758 initially positive hits, a hit rate of 2.2%. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to 77, currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec50 measurements below 5.0 m and cc50 measurements >50.0 m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: (1) an isobutyl-acetamide with an ec50 of 87.0 nanomolar, and a cc50 of >50 m, and (2) a carbothiamide with an ec50 of 1.6 micromolar and a cc50 of >50 m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m204v/i ± l180m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t184, s202, or m250. these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg2 cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a 3d homology model based on the hiv-1 rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m250 changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t184, s202 or m250 along with pre-existing lvdr substitutions m204v/i ± l180m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose (10 8 pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at 0, 24, or 48 h postinfection (pi). later treatment times were not evaluated. treatment at 24 or 48 h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than 10-fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by >90%; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing 10 9 -10 10 genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at 24 h pi, viral replication in spleen exceeded 10 9 genomes/g while liver and bone marrow yielded 10 8 genomes/ml. in comparison, titers in other tissues ranged between 10 5 and 10 6 genomes/g and blood yielded 10 4 genomes/ml at 24 h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately 10 10 genomes/g at day 5, then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days 7-9 at 10 8 genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv 24 h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl-4 laboratory. earl kern 1 , kathy keith 2 , robert jordan 2 , dennis hruby 2 , debra quenelle 2 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st-246, a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st-246 for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about 0.1-0.5 um of st-246. for in vivo studies, st-246 was administered once daily by oral gavage to mice using 100 mg/kg for 5, 7, 10, or 12 days beginning 4 or 24 h after intranasal inoculation with vv or cv. st-246 was highly effective (p < 0.001) in preventing mortality due to vv or cv even when treatment was delayed up to 24 h post-infection. a dosing duration of 5 days was adequate for vv infected mice, but duration of 7 days or longer was required for efficacy in cv infected mice. when st-246 was given once daily for 14 days at 100, 30, or 10 mg/kg daily at 24, 48, or 72 h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with 50 mg/kg of st-246. on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st-246 treatment successfully reduced virus titers from 3 to 5 logs 10 in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st-246 has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he16, in a mouse model when administered later in infection (oliphant et al., 2005. nat. med. 11, 522) . in this study, virus was demonstrated to be in the brains of hamsters at 5 days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. 5 dpi with 100 mg/kg of humanized monoclonal antibody, he16, survived wnv disease, whereas, 37% of placebo-treated hamsters survived ( *** p < 0.001). if administered at 2 dpi, 100% survived. we tested the hypothesis that he16 is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain 5 dpi using convectionenhanced delivery through a cannula implanted into the brain. the he16 was detected in the cns, but none was detected in the kidney. the survival of he16-treated hamsters was 88% as compared to 22% of placebo-treated animals ( *** p < 0.001). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he16 administered i.p. at 5 dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih, and grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with 50% effective concentrations (ec50s) of less than 100 nm against lassa fever virus and with ec50s of less than 2 nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein 1 , pramila walpita 1 , allison groseth 2 , heinz feldmann 2 , ramon flick 1 1 university of texas medical branch, department of pathology, galveston, tx, usa; 2 national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to 70%. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level 4 (bsl-4) agent. to avoid bsl-4 containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t7 minigenome rescue system (without the need for recombinant vaccinia virus mva-t7) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl4 agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t7) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl4 conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level 4 (bsl-4) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp30. anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire '76 strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec50 values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and 4-aminoquinoline compounds. acknowledgement: supported by nih r44 ai052917-02 and r01 ai066502-01. human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv31 episomal dna content. two of the 19 compounds caused a dose-dependent reduction in hpv31 episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼1000 copies of hpv31 per cell, compounds 2-ta and 4-ta both reduced hpv31 dna levels to below 50 copies per cell after 48 h incubation with 10 m compound. an alternative taqman tm amplicon within the hpv31 e7 gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv31 dna to the human apoe gene also demonstrated dramatic loss of hpv31 dna copies, further confirming our initial observations. finally, cells were treated with polyamides for 48 h, polyamide-containing media was removed, and episome levels were followed for 8 days. at day 6, 4 days after removal of polyamide and 3 days after sub-culturing of the cells, viral episome levels remained approximately 60% lower than control samples. by day 8, 6 days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh3 groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t7 rna polymerase from ribonucleoside 5 -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: (1) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; (2) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by 0.5-0.8 • c per modification; and (3) sirnas with borano-modifications were shown to be at least 10-fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: (1) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; (2) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; (3) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and (4) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy-2 -fluoro-2 ,3dideoxydidehydroadenosine (fd4ap, gs9148), a novel ntrti, with an excellent resistance profile toward hiv-1 variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d4) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt-2 cells. mitochondrial toxicity was assessed in hepg2 cells by measuring mitochondrial dna content. results: the d4 scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy-2 ,3dideoxydidehydroadenosine (d4ap) inhibited hiv-1 replication with a mean ec50 of 2.1 m and an 0.8-, 2.9-, and 2.9-fold change in potency against viruses containing m184v, k65r, and 6 thymidine analog mutations (tams), respectively. further exploration of d4ap was limited by its mitochondrial toxicity, which was then addressed in 2 ways: (i) preparation of l-d4ap or (ii) 2 fluorine substitution. l-d4ap exhibited an ec50 of 5.9 m but had substantially reduced potency (14-fold) toward m184v mutant viruses. fd4ap exhibited an ec50 of 12.3 m, with 0.8-, 1.2-, and 3.5-fold change in potency against viruses containing m184v, k65r, and 6 tams, respectively. no cytotoxic effects were measured up to 1 mm in mt-2 cells and no effects on mitochondrial dna were detected up to 300 m in hepg2 cells for both fd4ap and l-d4ap. conclusion: fd4ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv-1 strains. compared to d4ap, the 2 -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd4ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr-5 antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr-5 antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr-5 antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr-818, prevents the reactivation of dormant virus by inhibiting both the hiv-1 p24 and viral mrna production from latently hiv-1-infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr-818 was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr-818 was able to inhibit the ser5 phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk9 subunit of p-tefb, which is recruited to the viral promoter by the hiv-1 tat protein. since we did not find an inhibition at the level of cdk9 activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr-818 must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález 1 , sandra franco 2 , miguel a. martínez 2 , luis menéndez-arias 1 1 centro de biología molecular "severo ochoa", csic-uam, madrid, spain; 2 fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons 69-70 together with substitutions t69s and t215y in the reverse-transcriptase (rt)-coding region of hiv-1 are known to confer resistance to zidovudine (azt) and stavudine (d4t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and 10 additional mutations related to drug resistance (including t215y) showed >10-fold increased unblocking activity on azt-and d4t-terminated primers, when compared with an rt containing the insertion together with mutations t69s and t215y, in an otherwise wild-type bh10 sequence. these results suggested that other mutations associated with the complex t69sss/t215y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d4t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues 1-135 and wild-type bh10 rt residues 136-560 (l1 rt), or residues 1-135 of the bh10 rt and 136-560 of the ss rt (l3 rt), as well as an l1 rt variant with the substitution t215y (l2 rt) and an l3 rt derivative with t69sss (l4 rt). additional rts containing mutations m41l, a62v, or k70r together with the combination t69sss/t215y in the bh10 background were also obtained. atp-mediated excision activities on azt-and d4tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv-1. the l2 rt containing mutations t69sss/t215y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l1, l3, and l4 rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m41l, a62v and k70r) were responsible for the increased excision activity shown by rts bearing mutations t69sss and t215y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the 6-trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi-033, was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the 50% effective concentration (ec50) was 0.30 ± 0.18 g/ml, while the cc50 (50% cytotoxic concentration) of chi-033 for vero cells was more than 100 g/ml, thus resulting in a selectivity index of >500. detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi-033resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses 42 hiromichi tanaka 1 , kazuhiro haraguchi 1 , hiroki kumamoto 1 , takao nitanda 2 , masanori baba 2 , ginger e. dutschman 3 , yung-chi cheng 3 1 school of pharmaceutical sciences, showa university, tokyo, japan; 2 center for chronic viral diseases, kagoshima university, kagoshima, japan; 3 school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for 4 -carbon-substituted nucleosides has led to a new strategy, ring opening of 4 ,5 -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the 4 -position. as a result of this study, 4 -ethynylstavudine (4 -ed4t) was found to be more anti-hiv active than the parent compound stavudine (d4t). this compound (4 -ed4t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d4t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k103n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of 4 -ed4t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. 46 (1), 44]. in focus of the anti-hiv potency the ivs provide a 10-100-fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist 20-500-fold amplifying the anti-hiv-1 selectivity up to is∼10000. augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least 10-fold amplification of anti-hiv-1 potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as 2,6,-diamino-(9-[2-phosphonomethoxyethyl]purine (pme-dap) and 2-amino-6-cyclopropylamino-(9-[2phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv-1), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic 2,6diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv-1. the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p24 reduction assays in mt-2 cells infected with hiv-1. for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were >3 logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt-2 cells, the selectivity indexes are more than 10-fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv-1 in vitro. victor kuz'min 1 , eugene muratov 1 , anatoly artemenko 1 , ludmila koroleva 2 , vladimir silnikov 2 , v. lozitsky 3 , a. fedchuk 3 1 a.v. bogatsky physical-chemical institute, odessa, ukraine; 2 institute of chemical biology and fundamental medicine, novosibirsk, russian federation; 3 ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv-1, % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r 2 = 0.836, q 2 = 0.788). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd4 receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv-7 (vermeire et al., 2002. virology 302, 342-353) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd4 down-modulating and antiviral potencies of more than 25 cada analogs have been described (vermeire et al., 2003. mol. pharmacol. 63, 203-210) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. 1) . the anti-hiv and cd4 down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa05), an isomer of cada, was synthesized by cyclization of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (tbd) (fig. 1 ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. 1) . the anti-hiv and cd4 down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga 30602, usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code 1bl3. building of molecules and primary modeling was done with sybyl 7.1 on a silicon graphics onyx3 (r14000) workstation. the program gold 3.0 (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver 1.1, a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga 30602, usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein 1 , alexander serbin 2 , alissa bukrinskaya 1 d.i. ivanovsky institute of virology, moscow, russia; 2 health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd50). the antiviral effect of the compounds was analyzed in lymphoblastoid mt-4 cells and in hela cd4+/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv-1 replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed 1 h after infection. the anti hiv-1 effect of the compounds was gradually decreased if they were added 1 and 2 h after infection, no inhibition was observed when the compounds were added 4 h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt-2 cells or stimulated primary cd4+ t-cells were incubated with graded concentrations of tfv or cbv for 14 h (h); then extracellular drug was removed by washing. cells were further incubated without drug for 0-24 h and then infected with hiv-1 (iiib or bal). p24 was quantified on day 2; inhibition of hiv-1 replication due to intracellular drug persistence (pc50) was determined relative to a standard ec50. decay of intracellular dp/tps in cd4+ t-cells was measured using lc/ms/ms. results: in mt-2 cells, the pc50 value for tfv 12 h after drug removal remained unchanged relative to the ec50 (<3-fold shift) whereas the pc50 for cbv shifted >65-fold, indicating less persistence of cbv. in cd4+ t-cells, the pc50 value for tfv also showed a minimal shift relative to the ec50 (2.4-fold) 24 h after drug removal. cbv showed a much larger relative shift (>243-fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd4+ t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, 21 h versus cbv-tp, 5 h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt-2 cells and primary activated cd4+ t-cells, tfv had the longest persistence of antiviral activity. in cd4+ t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis 5320 is a phosphorothioate oligonucleotide with a molecular structure of t2g4t2. the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v3 loop of gp120. mapping studies with monoclonal antibodies targeting epitopes in and around the v3 loop have been used to define the binding site of isis 5320. in vitro, isis 5320 inhibits all laboratory and clinical strains of hiv-1 and hiv-2 tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr4 and ccr5 chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis 5320 resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis 5320 suggest that it may be an excellent anti-hiv topical microbicide. isis 5320 was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr5-tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd4-dependent and cd4-independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis 5320 used in combination with other microbicidal compounds. the results of these studies suggest that isis 5320 may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only 60% effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv-1 drugs, protease inhibitors (pi) are the unique to inhibit the hiv-1 production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv-1 production when pi have been removed in hiv-1 chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p24 gag protein was measured starting from 12 h after drug removal yet virus infectivity remained 1 log lower than control up to 1 week. inhibition of hiv-1 replication was still 48% and 18% upon amprenavir 20 and 4 m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd4/cd8 ratio above the levels of untreated controls until the 5th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt-2 cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt-2 cells. results: capravirine resistance selections were initiated at 1 nm (ec 50 of 1.5 nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l100i mutation emerged in rt and additional passaging led to v179d and f227c mutations at higher concentrations (100-300 nm). further increases in capravirine concentrations led to the emergence of a l100i + v179d + f227c triple mutant, which confers >1000-fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l100i, v179d and f227c were present on the same genome, with l100i as the primary mutation, and f227c and v179d were acquired sequentially at later passages. through sgs analysis, a l100i + k166r + v179d + f227c quadruple mutation on the same genome was also observed at higher capravirine concentrations (>1000 nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k103n nor y181c mutations in rt were observed. instead, the l100i mutation was initially acquired, followed by mutations f227c and v179d. addition of the k166r mutation to the triple mutant genome, l100i + v179d + f227c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi 1 , adeyemi falegan 2 1 medical microbiology, university college hospital, ibadan, nigeria; 2 dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = 0.87] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, 50,000-sample size, selected through simple random sampling technique. the mean age is 25.5 years old. relative risk [rr] calculated is 3.1, i.e. rr >1, indicating that the factor is the risk factor, and the confidential interval [ci] for rr at 95% significant level is 2.61 < 3.1 < 3.90 from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which 20% had the unprotected intercourse once or more, 25% had protected sex while 25% were not sure of using protection means. while, 20% have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu 2,3 , jacob barnor 1,6 , norio yamamoto 4 , kahoko hashimoto 1,2 , naoko miyano-kurosaki 1,2 , koichi ishikawa 5 , naoki yamamoto 5 , david ofori-adjei 6 , hiroshi takaku 1,2,7 1 department of life and environmental sciences, chiba institute of technology, chiba, japan; 2 high technology research center, chiba institute of technology, chiba, japan; 3 japan foundation of aids prevention; 4 department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; 5 aids research center, national institute of infectious disease, tokyo, japan; 6 department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; 7 bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv-1 replication in a long-term transduced pbmcs, h9, and jurkat cell culture assays (9 weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv-1 replication was similarly suppressed in the control cells expressing only vif shrna for about 3 weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of 6 avian and 12 porcine influenza a viruses isolated in germany between 1981 and 2002. virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only 3 of the 12 porzine isolates but all 6 avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between 1981 and 1987. all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g16e, s31n, and r77q in the matrix protein 2 (m2). additionally, l27a was detected in two h1n1 strains. s31n and/or l27a are well known amino acid substitutions in m2 that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin (2,3-dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin-3-thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h5n1) in mdck cells. antiviral activity was studied by virus yield assay (ec90), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h5n1) virus replication in mdck cells and compounds spiii-5h and spiii-5cl were most active (ec90 5.5 g/ml, cc50 >100 g/ml and si > 18). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai-15433 and noi-ai-30048 from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/8/34 (h1n1) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb1 segment mrnas, or the 3 -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by 1-3 orders of magnitude in a dose-dependent and sequence-specific manner over a period of 2 days. two of the p-pmo, targeting the pb1 translation start site region (pb1-aug) and the 3 terminus of np vrna (np v3 ), were evaluated by endpoint dilution (tcid50) or elisa assays against another h1n1 strain (a/wsn/33), as well as a/memphis/8/88 (h3n2) and a/thailand/1(kan-1)/04 (h5n1). the pb1-aug arp-pmo generated over 85% specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of 10-20 m. the np v3 p-pmo yielded similar results, with the exception of considerably lower efficacy against the h3n2 strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h1n1 and h5n1 fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep-2 cells were infected with hsv-1 strain us in dose 5 ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of 10 −4 m in experimental samples, or without them in control samples. then cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/1/68 (h3n2) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of 10 −3 m during the study of their anti-influenza action. diaza-18crown-6 and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza-18crown-6 derivatives that contain 2-oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv-1 by 13 and 29%, respectively. both of these compounds inhibited replication of influenza virus on 1.7 log 10 tid 50 aza-15crown-5 did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza-15crown-5 derivatives that contain 2-amino-3-phenyl-propanoyl-or 5-benzyloxy-3-oxapentyl-fragments decreased amount of cells infected by hsv-1 with virus-specific intranuclear inclusions by 41 and 47%, respectively. first compound inhibited replication of influenza virus on 1.5 log 10 tid 50 and the second one decreased virus amount on 2.0 log 10 tid 50 . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # 3147) . robert w. sidwell 1 , kevin w. bailey 1 , min-hui wong 1 , donald f. smee 1 , dale l. barnard 1 , shanta bantia 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx-1812, rwj-270201) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h1n1, h3n2) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to 6 h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n9 neuraminidase for over 24 h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to 48 h after virus exposure was protective to mice infected with influenza a (h1n1) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection 1 h pre-virus exposure in separate experiments to mice infected with an influenza a (h5n1) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of 20 and 10 mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao 2 ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to 72 h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h5n1) virus infections. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hiroshi saitoh 1 , naoko miyano-kurosaki 1,2 , hiroshi takaku 1,2 1 department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; 2 department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv2neo-u6 plasmid vectors and pvl1393based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u6 pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and 24 h later the cells were infected with either a/pr8 or b/ibaraki virus at a moi of 0.01. at 72 h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, 1992) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., 2004) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with 5-10 ld 50 of a/aichi/2/68 (h3n2) influenza virus. pre was administered by i.n. instillation 3 h before infection in the dose of 10 mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days 6 and 9 after infection their levels reached 140-160% of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h 2 o 2 , o 2 •− and no production by alveolar macrophages; the generation of ros and rni peaked on day 9. pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s11, a natural herb extract, ji-sun kwon 1 , hyun-jeong lee 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea the antiviral activity of s11, one of the traditional korean medical herb extract, against influenza virus was investigated. the 50% effective concentration (ec50) using plaque reduction assay was 31.25 ug/ml and the mean 50% cytotoxic concentration (cc50) using wst-1 assay in the mdck cells was 334 ug/ml. oral gavage treatment of the s11 to balb/c mice infected with a/pr/8/34 (h1n1) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s11 was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s11 is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku 1,2,4 , takayuki abe 3 , hitoshi takahashi 1 , naoko miyano-kurosaki 1,2 1 department life environ. sci., chiba inst. tech., chiba, japan; 2 high tech. res. center, chiba inst. tech., chiba, japan; 3 res. inst. microbial dis., osaka university, osaka, japan; 4 bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il-6, in murine raw 264.7 macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/8/34 (h1n1) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, 2 weeks apart, at a dose of 1.1 × 10 8 pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with 10 8 pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant 1 , annie lebel 2 , nathalie goyette 2 , guy boivin 2 , jean-marc juteau 1 , phil wyde 3 1 replicor inc., laval, que., canada; 2 chuq-chul and laval university, st. foy, que., canada; 3 baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥40 bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep 9 (a 40 mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep 9 was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep 9 aerosol has excellent characteristics for lung deposition and aerosol treatment with rep 9 was well tolerated and highly effective against infections with influenza a both in prophylaxis and 24 h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no1-ai-15437. irina v. alymova 1 , y. sudhakara babu 2 , allen portner 1 1 virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn 38105, usa; 2 biocryst pharmaceutical, inc., birmingham, al 35244, usa bcx 2798 is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 2798's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type 1 (rsev(hpiv-1 hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk 2 cells at moi 0.1 in the presence of increasing (from 100 to 3200 m) concentrations of compound; infected 129×1/svj mice were treated with 10 mg/kg/day of bcx 2798 twice for five days. treatment started 4 h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e527k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv-1 hn) . several mutations in hn gene of rsev (hpiv-1 hn) were selected in an animal model after the second passage of virus from mice treated with bcx 2798. two mutations, n23s and p29q, were located in the cytoplasmic domain of hn protein; mutations n173s and t553a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk 2 cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx 2798 both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv-1 hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx 2798 may not lead to appearance of clinically significant variant of viruses. kie-hoon jung 1 , michelle mendenhall 1 , lawrence m. blatt 2 , robert w. sidwell 1 , brian b. gowen 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates (30-50%) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon-1. both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level 4 containment facilities. acknowledgement: supported by contract no1-ai-30048 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding 5mono-, 5 -di-, and 5 -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers (2004) has been successfully applied to a number of different nucleosides (azt, d4t, dda, d4a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe 1b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of 4 -substituted nucleosides, including 4azido-cytidine, 4 -azido-uridine, and 2 ,3 -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (>1000-fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., 2004. mini-review. med. chem. 4, 371-382 . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns5b polymerase, an essential enzyme for viral replication. the micromolar ns5b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns5b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns5b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale 1 , dimitrios vlachakis 1 , maria chiara barbera 1 , romano silvestri 2 , colin berry 3 , johan neyts 4 1 cardiff university, the welsh school of pharmacy, cardiff cf10 3 xf, uk; 2 universita' degli studi "la sapienza", dipartimento di studi farmaceutici, 00185 roma, italy; 3 cardiff university, cardiff school of biosciences, cardiff cf10 3us, uk; 4 rega institute for medical research, k.u. leuven, b 300 leuven, belgium hepatitis c is a viral infection that affects 170 million people worldwide, including 4 million in the united states and 8 million in europe. the virus establishes a chronic infection in 55-85% of cases and 20% of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound 1 is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., 3650 wesbrook mall, vancouver, bc, canada v6s 2l2 hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns5b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin-4(3h)-one derivatives a series of novel 6-bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)-benzenesulphonamides were synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). 6-bromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)benzenesulphonamide (sps-ii) and 4-(4-oxo-2-phenyl-4hquinazolin-3y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv-1 in acutely infected mt-4 cells at a concentration of approximately 10 g/ml, while not being toxic to the host cell at a concentration of 60 or >125 g/ml (selectivity index: 8 and >12), respectively. in huh 5-2 cells sps-i inhibited hcv rna synthesis at ec50 of 8 g/ml, while at cc50 for cell growth 32 g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv-1, hsv-2 or vaccinia (vv) at a concentration of 50 g/ml, while not being toxic to the cells up to a concentration of 400 g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct 06511, usa hcv nonstructural protein ns5b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns5b have been used to investigate ns5b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns5b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns5b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns5b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns5b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns5b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than 170 million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than 100,000 cases of liver cancer annually worldwide and accounts for at least 40% of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p7 protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes 1a strain h77c and genotype 1b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype 2a strain jhf-1 10 um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells 1 h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after 72 h incubation (p < 0.05 and <0.01, respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype 1a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣-2b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣2b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼3-fold), or the ribavirin-interferon ␣2b combination (∼2-fold). similarly, the castanospermine-interferon ␣2b double combination was more synergistic than the castanospermine-ribavirin combination (∼5-fold), or the ribavirin-interferon ␣2b combination (∼3.3-fold). the combinations of celgosivir-interferon ␣2b or castanospermine-interferon ␣2b led to significant decreases in the ec50s of celgosivir (up to >20-fold) and castanospermine (up to >50-fold). the effective ec50s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣2b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣2b or with interferon ␣2b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about 50% of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm-107, amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (>100 m). celgosivir and castanospermine were synergistic with both amantadine and nm-107, with volumes of synergy between 60 and 150 m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm-107. mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul 120-749, korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately 9.6 kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately 3010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. the structural proteins include c, e1, e2, and p7 and the nonstructural (ns) proteins include ns2, ns3, ns4a, ns4b, ns5a, and ns5b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns5b protein, the viral rna-dependent rna polymerase (rdrp), and ns3 protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like 2 (prk2) regulates virus rna replication. hcv rna replication was inhibited when prk2 expression level was down-regulated by using a prk2-specific sirna. in this study, we investigated the anti-hcv effect of prk2 inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk2 inhibitors suppressing the endogenous prk2 activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk2 inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk2 inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk2 can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk2 activity will provide an opportunity to interfere with hcv rna replication. haitao guo 1 , tianlun zhou 2 , ju-tao guo 1 , andrea cuconati 3 , anand mehta 1 , timothy block 1 1 drexel university college of medicine, doylestown, pa, usa; 2 nucleonic inc., irvine, ca, usa; 3 hepatitis b foundation, doylestown, pa, usa more than 400 million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated 500,000 deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad38 cells, a hepg2-derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the 5 terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the 3 terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad38 produced and accumulated cccdna, which became detectable between 7 and 8 days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec50 >3 logs less than unmodified hpmpa in mt-2 cells by p24 reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc-5 human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for 6, 24 and 48 h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc-5 cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after 24 h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every 2 days for 0-10 days. levels of the diphosphates declined slowly with a t 1/2 of 5-10 days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of 15-me-hdp-cdv (1) was tested in liver s9 fractions from various species. in mouse and human liver s9 fractions, compound 1 was completely stable for 90 min while 15-20% of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound 1 retained full activity compared to hdp-cdv and compound 2 showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([1-(s)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (<5%), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs 1 that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs 2ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., 1994) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs 2 were converted to drug by cellular (caco-2, hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of 1 in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with 50% inhibitory concentrations ranging from 0.1 to 0.5 and 10 m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than 30-fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r43ai056864 and u01ai061457. lawrence trost 1 , bernhard lampert 1 , lloyd frick 2 , merrick almond 1 , george painter 1 1 chimerix, inc., durham, nc, usa; 2 dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv-1. however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx012) decreases the apparent ec 50 value against hiv-1 by up to 40-fold. cmx012 was esterified to produce cmx052 in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx052). rats were given oral doses of 10, 30 and 100 mg/kg cmx052 daily for 7 days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx012 (c max > 4 g/ml), the biologically active deesterified form of cmx052. in conclusion, cmx052 is absorbed after oral administration, converted to cmx012, and has a good preliminary toxicity profile. these results support the development of cmx052 for the treatment of drug resistant hiv infection. zhiqian wu 1 , julie breitenbach 2 , ulrika erickson 1 , john hilfinger 3 , john drach 2 , gordon amidon 1 1 department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; 2 school of dentistry, the university of michigan, ann arbor, mi, usa; 3 tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. 5 -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco-2 cell homogenate (t1/2: 2-40 min). 5 -l-isoleucyl and 5 -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept1mediated uptake as well as caco-2 permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada1. preliminary animal study shows 5 -lisoleucyl vidarabine results in >10-fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [9-␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types 1 and 2. it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼5-fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about 10-fold by combination with 1 m 2 -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on 5 -substituted vidarabine analogs (lipper et al., 1978. mol. pharmacol. 14, 366-369), we determined that minimizing metabolism of vidarabine by synthesizing 5 -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a 10-fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig 1 , anne baum 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active 3,5-bis-tertbutyl-6-fluoro-cyclosal-d4t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d4t with the antiviral active nucleoside analogues like d4a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen 1 , wolfgang fendrich 1 , tilmann schulz 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany, 2 rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d4a. for the pyrimidine-type series the fluorescent nucleoside m 5 k was synthesized as well as the dideoxy-compound dm 5 k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of 5000 compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv-1 and hiv-2 strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar 1 , richard mackman 1 , adrian ray 1 , dean boojamra 1 , lijun zhang 1 , deborah grant 1 , hon hui 1 , jennifer vela 1 , neil parkin 2 , yolanda lie 2 , kirsten white 1 , michael miller 1 , gerry rhodes 1 , manoj desai 1 1 gilead sciences, foster city, ca, usa; 2 monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine (gs9148) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs9148, resulting in the identification of gs9131 (ethylalaninyl phenyl ester of gs9148). results: gs9131 exhibits potent anti-hiv-1 activity in primary lymphocytes and t-cell lines (ec 50 < 150 nm). low cytotoxicity (cc 50 > 100 m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs9148 was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv-1 reverse transcriptase (rt) (k i = 0.8 m). unlike ddi, d4t, or d4fc, neither gs9148 nor its prodrugs inhibited mitochondrial dna replication in hepg2 cells. in a phenosense assay, gs9148 retained its full activity against hiv-1 variants with k65r, m184v or l74v mutations in rt. viruses with ≥4 thymidine analog mutations showed ≤2fold reduced susceptibility to gs9148, a shift that was smaller than that of any other tested nrti. following an oral dose of 3 mg/kg gs9131 in dogs, the bioavailability of prodrug exceeded 20%, resulting in high intracellular levels (9.0 ± 2.3 m) and prolonged retention (t 1/2 > 24 h) of gs9148 diphosphate in blood lymphocytes. conclusions: both gs9148 and its prodrug gs9131 exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs9131 possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs-7340 and gs-9131 are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; 9-[(2-phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd4ap (phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine), respectively. both gs-7340 and gs-9131 exhibit potent in vitro anti-hiv-1 activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver >10-fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs-7340 and gs-9131 is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs-7340 and gs-9131 was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was 3500-fold. sds-page analysis showed the presence of a prominent protein band at 29 kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec 3.4.16.5, cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ 14 c]gs-7340 and [ 3 h]difluorophosphonate resulted in the labeling of an identical 29 kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs-7340, approximately 6-9-fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside 5 -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to 30% by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of 0.1-0.2 m. the rate of passive drug release in physiological solution was from 33 to 50% of loaded drug during the 24-h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only 1-2 h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. 5 -triphosphate of 1-␤-d-ribofuranosyl-1h-1,2,4-triazole 3-carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin 5 -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong 1 , ebrima gibbs 2 , joel oger 2 1 department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; 2 neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the 2 (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately 60-70-folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky 1 , i. kravchenko 2 , v. larionov 2 1 research anti-plague institute, odessa, ukraine; 2 national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: (1) prolongation of the drug's action; (2) drug's concentration is maintained in therapeutic range; (3) there is no trauma to patient's skin while using td; (4) removing tts from the skin immediately stops drug's entering to the organism; (5) first-pass effect in the liver is reduced; and (6) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and 1.2-propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr (1 mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: 10 mg/mouse of dmso or 10 mg/mouse of octanol or 1 mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from 1 day before infection to 10th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/8/34 (h1n1), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of 10 −1 to 10 −7 . deaths of animals were recorded for 14 days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on 1 log 10 tid 50 . dmso and octanol did not demonstrate such activity. mikhail dobrikov 1 , serguei vinogradov 2 , barbara ramsay shaw 1 1 department of chemistry, duke university, durham, nc, usa; 2 center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv-1 reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano-2 ,3 -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r01 al52061 to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a 384 microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic50 stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, 08916 badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type 1 (hiv-1) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv-1 protease, p17-p24, was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv-1 protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv-1 repressor and hiv-1 protease, lambda phage replicated up to 1000-fold more efficiently than in cells that did not express the hiv-1 protease. this assay responds appropriately to well-known hiv-1 proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns3 protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) 3c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t20) and reverse transcription (sustiva, uc781 and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé 1 , kerstin angner 1 , anna overby 1 , david stein 2 , patrick iversen 2 , ramon flick 1 1 utmb, department of pathology, galveston, tx, usa; 2 avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about 20 subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. 1b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. 1a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic50 on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us-1 interacting with the hep-2 sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant #3147. we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve 10,000× to 100,000×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding 25 mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant #3147. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., 2002) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with 5-iodo-2 -deoxyuridine, to give intermediate 5-alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: (1) the side chain; (2) the bicyclic base; and (3) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec50 1 nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the 3 hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new 3 substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., 2001) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with 5-iodo-2 -deoxyuridine, to give intermediate 5alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis-5-nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc 50 , g/ml); (b) antiherpetic activity (hsv-1 strain kupka, ic 50 , g/ml); and (c) selectivity index (ratio of cc 50 to ic 50 ) and the structure of 48 n,n -bis-5-nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r 2 = 0.816-0.991, q 2 = 0.637-0.868). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of 5-arylethynyl-2deoxyuridines (1a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv-1. unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel 2 -deoxy (1a) and 2 -arabino (1b) uridine derivatives possessing different 5-arylethynyl substituents. dinucleosides 2 containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides 1a. these compounds as well as some of 1a and 1b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; 3,4dihydroxybenzohydroxamic acid) and trimidox (tx; 3,4,5trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a 43-and 39-fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by 50% in the resistant strain (cdv wt: 0.03 m, r: 1.28 m/gcv wt: 3.17 m, r: 122.85 m). this compared to only a moderate increase in drug dose required for dx and tx to achieve 50% inhibition in the resistant strain (dx wt: 20.71 m, r: 28.88 m/tx wt: 7.12 m, r: 11.59 m), corresponding to a 1.4-and 1.6-fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from 1-(phosphonomethoxyethyl)-cytosine (pme-c), 1-(phosphonomethoxyethyl)-5-bromo-cytosine (pme-5brc), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine (pme-5fc), 9-(phosphonomethoxyethyl)-2,4-diaminopurine (pme-dap) and 9-(phosphonomethoxyethyl)-2-amino-4cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec 50 values of 0.2, 0.3, and 0.03 m in hff cells infected with hsv-1, hsv-2 or hcmv, representing a 12-26-fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec 50 values of 0.7, 0.2, and 0.6 um in hff cells infected with hsv-1, hsv-2, and hcmv, representing a 16-43-fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type 6 and 8, is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach 1 , katherine z. borysko 1 , jiri zemlicka 2 , john c. drach 1 1 biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; 2 karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., 1998. j. med. chem.) and second generation (zhou et al., 2004. j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were 10-20-fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 50 's in plaque assays = 0.35 and 21 m, respectively for wild-type (wt) and synguanol-resistant (1982r) virus]. likewise a ul97 deletion mutant (prichard et al., 1996. j. virol.) was resistant to both first and second-generation compounds (ic 50 's = 2.1 and 0.25 m in wt; 100 and 15 m in ul97 del , respectively for synguanol and cyclopropavir). ul97 from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m460i and c603y. because hcmv with either m460i or the related c607y mutation alone was sensitive to synadenol and synguanol (baldanti et al., 2002 . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad169 bac with one, the other, or both mutations in ul97. as expected, the two strains with the single mutations were 3-to 6-fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol (0.5-to 1-fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir (6to 8-fold) but less so than observed in the 1982r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul97 is required for activity against hcmv and that is likely that two mutations in ul97 are required for significant resistance. acknowledgement: supported by grants p01-ai46390 and r44-ai054135 from nih and funds from the university of michigan. svitlana zagorodnya 1 , nadiya nesterova 1 , inna alexeeva 2 , larisa palchikovskaya 2 , galina baranova 1 , alexander kobko 2 , anna golovan 1 1 zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; 2 institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. 1 and 2, as well as n-glycosidic derivatives no. 3 against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no 1, no 2, and no 3 was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc-100 r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of 1000, 500, 250, 125, 64, 32, 16, 4 , 1, 0.5 and 0.1 g/ml. the concentrations that inhibited the quantity of live cells on 50% (id50) were equal to substances no. 1-750 g/ml, no. 2-625 g/ml and no. 3-125. the minimal inhibiting concentration (mic) of nos. 1, 2, and 3 was equal to 1 g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with 28.0 up to 14. hence, the index of selectivity (is) was equal to 750 and 625 for triazine bearing tricyclic bases nos. 1 and 2, for n-glycosidic derivatives-125. in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti 1 , l. nencioni 1 , g. macrì 2 , c. nucci 2 , u. benatti 3 , m. magnani 4 , e. garaci 5 , a.t. palamara 1 1 department public health sciences, university rome "la sapienza," rome, italy; 2 department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; 3 department exp. medicine biochemistry section, university genova, genoa, italy; 4 inst. biochemistry, university urbino, urbino, italy; 5 department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c4), with increased hydrophobic properties, inhibited in vitro parainfluenza-1 and hsv-1 replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c4 administration in hsv-1-induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with 2 × 10 5 pfu/ml of hsv-1. gsh-c4, dissolved in a saline solution (150 mm, 100 ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using 0.5% fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c4 treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c4 treated animals, conjunctival hsv-1 titre, assayed by tcid50 on day 4 post-infection, was significantly reduced as compared to that of control animals (mean = 1.4 × 10 3 units/ml versus 1.1 × 10 5 units/ml, n = 10 for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c4-treated animals versus placebo animals (mean = 3.5 × 10 3 units/ml versus 8.5 × 10 5 units/ml, n = 5 per group). such results highlight the antiviral activity of gsh-c4 in vivo and suggest that topical gsh-c4 treatment could be considered as complementary therapy of hsv-1-induced keratitis. debra quenelle 1 , deborah collins 1 , latisha pettway 1 , caroll hartline 1 , james beadle 2 , w. wan 2 , karl hostetler 2 , earl kern 1 1 university of alabama school of medicine, birmingham, al, usa; 2 department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated 16-20 weeks later with 4700 pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through 21-28 days and then gradually decreased to undetectable levels by 8 weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or 10 mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated 24 h after infection and continued for 28 days. cidofovir (cdv) at 20 mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at 30 or 10 mg/kg beginning 24 h post-viral inoculation and 30 mg/kg when treatment was delayed until 48 h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep 9 has potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep 9 and compared to saline-treated infected control mice. compound was administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting at 2 days prior to infection. mice were infected with 5 × 10 5 pfu mcmv on day 0, at 3 h post-treatment. sera were collected at −22 h, at 36 hpi, and at 3 dpi for elisa analysis of ifn␥ production. spleens and livers were collected at 3 dpi for determination of virus titers. at 3 dpi, virus titers in the spleens and livers were significantly reduced by rep 9 treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep 9 but not in saline treated, infected mice or in rep 9 treated, uninfected mice. ifn␥ levels in mice treated with rep 9 peaked at 36 hpi compared to 72 hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep 9 with two oligonucleotide analogs that do not activate tlr-9 mediated immune stimulation suggests that direct antiviral activity of rep 9 and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep 9 analog exhibited even greater antiviral activity than rep 9 while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. kathy keith 1 , joseph maddry 2 , namita bansal 2 , kochurani jacob 2 , secrist john 2 , earl kern 1 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: (1) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and (2) to investigate new substituents at the purine 2-and 6-positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over 100 analogs of pmea substituted at the purine 2-or at the adenine n-6 site have been synthesized and evaluated for activity against orthopoxvirus infections. many n-6 substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine 2-position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) 2-amino-n(6)-cyclopropyl pmea had ec 50 values of 0.03-0.07 m and ode 2-hydroxylamino-n(6)-cyclopropyl pmea had ec 50 values of 0.3-1.6 m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc 50 values of 30-70 m giving selective indices of 200-1000. these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin (2,3-dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin-3thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel 4-[(1,2dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested,4-[(5-methyl-1,2-dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2-pyrimidiny)-benzene sulphonamide(spiii-5me), was the most active compound with an ec50 value of 18 mol, compared with cdv, which had an ec50 of 20 mol against vaccinia virus. all the compounds were non-toxic (>300 lm)using a neutral red uptake assay. substitution of a halogen atom in 5th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no1-ai-30049 from virology branch, niaid, nih, usa. evgeny belanov 1 , svetlana kotovskaya 2 , nikolay bormotov 1 , sergey balakhnin 1 , olga serova 1 , nataliya perova 2 , zina baskakova 2 , galina dzhumbaeva 2 , valerii charushin 3 , oleg chupakhin 2 1 state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; 2 ural state technical university, yekaterinburg, russia; 3 institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of 1,2,4-benzotriazine ( fig. 1) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk-2 cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c-3 and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project #1989. yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., 4575 sw research way-suite 230, corvallis, or 97333, usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st-246) via high throughput screening (yang et al., 2005. j. virol. 79, 13139-13149) . marker rescue of st-246 resistant variants localized compound resistance to the f13l gene that encodes a major orthopoxvirus envelope protein (p37), which is required for extracellular viral particle formation. p37 participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st-246, we examined the effects of st-246 on the production of the extracellular viral particles in bsc40 cells infected with recombinant vaccinia virus containing a gfp-tagged p37 protein. in the presence of st-246, iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st-246 prevented the intracellular localization of p37 to the late endosome compartment as measured by confocal microscopy. in contrast, st-246 did not affect localization of p37 expressed from a st-246 resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p37 in transfected cells. these results suggest that st-246 inhibits an unknown virusspecific activity that requires f13l. this work underscores the exquisite specificity of st-246 and supports continued development of st-246 as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. 4575 sw research way, suite 230 corvallis, or 97333, usa st-246 is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st-246 resistant variants was measured by fluctuation analysis and found to be 3.58 × 10 −6 . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f13l gene. the spectrum of mutations that confer st-246 resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by 100-fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f13l coding sequence by error-prone pcr and the mutated f13l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f13l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st-246 resistant, f13l proteins. sequence analysis of the compound resistant f13l alleles identified numerous silent mutations scattered throughout the f13l coding sequence and 27 point mutations leading to amino acid changes that clustered around amino acid positions 258-302 within the f13l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: (1) mutations in at least 7 positions within f13l can confer resistance to st-246 and (2) st-246 resistant mutations cluster to a 58 amino acid domain in a region of the protein of unknown function. several 5-substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec 50 values below 10 m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the 5 position had ec 50 values below 15 m against both hsv-1 and hsv-2, whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec 50 values below 10 m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the 5 position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al 35233, usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec 50 values below 1 g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec 50 values of 0.55 and 1.5 g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec 50 values 14-fold greater than a tk positive isolate. similarly, a tk negative strain of hsv-1 was 90-fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus 8 (hhv-8), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated 24 h after infection and at doses as low as 17 mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a314t and a684v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a314t and the a684v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a314t and the a684v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a314t and the a684v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a684v mutant that showed only a two to three-fold decrease in susceptibility towards the 3-hydroxy-2-phosphonomethoxypropyl (hpmp) purine derivatives, the a314t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a314t mutation appeared before the a684v substitution, and the latter mutation only occurred in conjunction with a314t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of 50 mg/kg/day for 5 days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a314t + a684v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg 1 defence science and technology laboratories (dstl), salisbury, wiltshire, uk; 6 coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor 9. there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna (7909) could provide protection from lethality against vacv in balb/c mice when administered up to 7 days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of 7909 ranging between 15 and 100 ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, 1, 2, 3, or 4 days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was 60% when treated with 30 ug 1 day post-challenge (70 mld50 challenge). a survival rate of 80% was observed when treated with 50 ug 2 days post-challenge (40mld50 challenge). the delay of treatment to either 3 or 4 days post-challenge was ineffective, indicating that the window of opportunity for delivery of 7909 is within 2 days. multiple doses of 7909 were used to attempt to extend this window of opportunity, delivering 7909 twice within a 3-day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl 2005. department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of 5 × 10 5 plaque forming units (pfu), was lethal for 50% of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c-1 cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c-1 cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of 10 7 pfu, or an intracranial injection of 5 × 10 4 pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (n)-mct (2x/day for 7 days) or cidofovir (1x/day for 2 days). (n)-mct treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival (placebo). statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the 30 and 100 mg/kg/day treatments significantly reduced day 5 virus titers and lung weights, and the 100 mg/kg/day treatment reduced lung consolidation. cidofovir (100 mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no-1-ai-15435 from the virology branch, niaid, nih. chelsea byrd 1 , elena sbrana 2 , shu-yuan xiao 2 , marina siirin 2 , robert tesh 2 , dennis hruby 1 , robert jordan 1 1 siga technologies, inc., corvallis, or, usa; 2 university of texas medical branch, galveston, tx, usa st-246 is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st-246 against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than 1 pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st-246 at 100 mg/kg once per day for 14 days protected ground squirrels from a lethal challenge with 100 and 1000 pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day 12 post-infection. when treatment with 48, 72, and 96 h, 100% protection was observed in the 24, 48, and 72 h groups, and 66% protection in the 96 h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st-246 at 0, 24, 48, and 72 h postinfection appeared grossly and microscopically normal. thus, st-246 appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen 1 , guang yang 2 , dennis hruby 2 , erik de clercq 1 , robert jordan 2 , johan neyts 1 1 rega institute for medical research, k.u. leuven, belgium; 2 siga technologies, inc. corvallis, or, usa st-246 is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st-246 maps to a mutation in v061, which is homologous to vaccinia virus f13l (yang et al., 2005. j. virol.) . the latter encodes the envelope protein p37 required for production of extracellular virus. deleting f13l resulted in a virus ( f13l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with 2 × 10 5 pfu of wr-vac resulted in 59 ± 11 pox tail lesions per mouse, the same inoculum of f13l-vac caused no lesions (p < 0.001). athymic nude (nu/nu) or scid mice inoculated iv with 2 × 10 5 pfu f13l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f13l-vac was 22 ± 1 days as compared to 9 ± 1 days for wr-vac-infected mice (p < 0.001); scid mice survived the infection. we next studied whether f13l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with 5 × 10 5 pfu f13l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with 5 × 10 5 pfu of wr-vac. all placebo animals, but none of the f13l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f13l-vac and were infected one week later with 2 × 10 4 pfu of wr-vac by the iv route. placebo animals developed an average of 14 ± 4 pox tail lesions; no lesions developed in the f13l-vac animals (p < 0.001). in a third set of experiments, nmri mice were inoculated iv with either 2 × 10 4 pfu of f13l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with 2 × 10 4 pfu wr-vac. the placebo group developed an average of 11 ± 8 lesions as compared to 1.2 ± 0.7 lesions in the f13l-vac mice (p < 0.05). f13l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan 1 , travis lim 1 , shuan coughlin 1 , dexin qiu 1 , zhen liu 1 , dave stein 2 , decheng yang 1 1 the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; 2 avi biopharma, inc., corvallis, or, usa background: coxsackievirus b3 (cvb3) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p007) is conjugated to the 5 end of the oligomer to improve its delivery into cells. these features make p007-conjugated pmos (p-pmos) promising candidates for the inhibition of cvb3 infection. methods: total 8 p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp1, viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the 8 p-pmos, p-pmo-6, targeting the internal ribosomal entry site in the 5 utr, showed the most potent anti-cvb3 ability in a dose-dependent manner. both infected hela and cardiomyocytes hl-1 cells treated with p-pmo-6 showed drastically reduced vp1 production and 3.0 log decreases in viral titres as compared to the controls. cell viability assay revealed that 83 and 89% of treated hela and hl-1 cells were still alive as compared to 11 and 10% of control-treated cells and 47% antiviral activity still existed after 5 days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb3 replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb3 isolate 97-927 (ic 50 , g/ml); (b) cytotoxicity in hela cells (cc 50 , g/ml); and (c) selectivity index (si = ratio of cc 50 to ic 50 ), and structure of 21 [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r 2 = 0.831-0.931, q 2 = 0.674-0.896) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb3 activity. ivanka nikolova 1 , roumena petkova 2 , boris atanassov 3 , stoyan chakarov 2 , angel s. galabov 1 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 scientific technological service, ltd., sofia, bulgaria; 3 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b1 was carried out. the wild-type disoxaril-sensitive strain (connecticut 5) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b1 and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by 9 passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b1 genome coding for the capsid protein vp1 was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp1 gene of coxsackievirus b1 was performed with the blast alignment tool. distinct alterations in the vp1 locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. 2749-2751 and an insertion of uuu at nt.2769) were observed. high-degree similarity (97%) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only 91%. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative 3-d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu-23 (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b1 infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic50 values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of 5methylthio-3-aryl-isoxazole-4-carbonitrile derivatives whose in vitro anti-coxsackievirus b1 activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds 5-methylthio-3-[4-(3-phenyl-1-propoxy)phenyl]isoxazol-4-carbonitrile (on-7) and 5-methylthio-3-[4-(4-phenoxy-1-buthoxy)phenyl]isoxazol-4-carbonitrile (on-10) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on-7 was effective when added within 1 h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on-10 when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time 0). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on-10 primarily interferes with coxsackie b1 cellular attachment. at a concentration 100 times the id 50 , inhibition of adsorption of coxsackievirus by on-10 was complete, while similar concentration of on-7 had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤1 (tgf-␤1) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in 30 non-infected c57/bl6 mice. in subsequent experiments, any animal expressing a permeability value greater than 2 sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice 8 days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of 3000, 1000, or 300 ng/kg/day of tgf-␤1 or with drug vehicle once daily via the i.p. route on 7 and 8 days post-virus inoculation (dpi), and then assayed for bbb permeability on 8 dpi. sixty-two percent (8/13) of placebo-treated animals had abnormally high permeability values, while animals treated with 3000 and 1000 ng/kg/day of tgf-␤1 had 29% (2/7) and 57% (4/7) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with 300 ng/kg of tgf-␤1 (0/8) expressed abnormally high permeability values, which was significantly lower (p < 0.01) than placebo-treated animals. these results suggest that tgf-␤1 may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. and contract no1-ai-15435 from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents 3-5 days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for 10 days beginning 4 h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine (7 mg/kg) and mk-801 (1 mg/kg), and one ampa-receptor antagonist, gyki 52466 (10 mg/kg), were administered twice daily (b.i.d.) on 4 though 9 days post-virus inoculation (dpi). gyki 52466 slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki 52466, slightly improved hamster survival (p ≤ 0.05) when treatment began on 5 dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning 5 dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using 105.3 tissue culture infectious doses of wnv. tissues of infected and control animals from 3 to 11 days post-viral injection (dpi) were fixed by cardiac perfusion using 4% paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc 1020). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as 3 and 5 dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of 7-day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly 50 million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type 2 and 3 in vero (monkey kidney cells) and hepg2 (human hepatoma cells), showing inhibitory concentration 50% (ic 50 ) values in the range 0.14-1.1 g/ml and selectivity indexes (cc 50 /ic 50 ) in the range 1000-10000. in the present work we studied the mode of action of these polysulfates against denv-2 in vero and hepg2 cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at 1 h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv-2 adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( 35 s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv-2 rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv-2 was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. 2 -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus 17d (ec 50 = 3.1 g/ml in cpe reduction assays and >98% reduction at 5 g/ml as assessed by qpcr) and dengue fever virus type 2 (ec 50 = 16.5 g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (>98% at 5 g/ml as assessed by qpcr and >98% by plaque reduction neutralization test at 50 g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin 1 , andrew shiryaev 1 , igor moiseev 1 , v sabynin 2 , larisa rustamova 2 , alexandr petkevich 2 1 state technical university, samara, russia; 2 institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an-3739 strain) on the vero cells culture. different level of antiviral activity was shown by 15 compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen 1 , donald smee 1 , min-hui wong 1 , anne pace 1 , kie-hoon jung 1 , kevin bailey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon 1) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., 2005 . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon-1 for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for 7 days, and ifn alfacon-1 was administered intraperitoneally, once per day for 10 days. treatments were initiated 1-5 days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin (5-10 mg/kg/day) with ifn alfacon-1 (5-10 mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no1-ai-15435 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler 1 , laure deflubé 1 , andrew vaillant 2 , jean-marc juteau 2 , ramon flick 1 1 department of pathology, university of texas medical branch, galveston, texas, usa; 2 replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep 9 is a 40 mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp12) as well as the wild-type rvfv (zh501), to test the ability of rep 9 to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep 9 at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep 9 analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep 9 is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep 9. adriana garozzo 1 , rossella timpanaro 1 , aldo stivala 1 , gianna tempera 1 , christian c.c. cutrì 2 , angelo castro 1 1 department of microbiological sciences, university of catania, via androne 8, 95124 catania, italy; 2 department of pharmaceutical sciences, university of catania, viale a. doria 6, 95125 catania, italy our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (is-2) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ 3 h]uridine-labeled poliovirus to hep-2 cells in the presence or absence of is-2. no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is-2 action. justin julander 1 , aaron olsen 1 , john morrey 1 , lawrence blatt 2 , kristiina shafer 1 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c3h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc-83. virus was detected in the brain 2 days post-viral injection (dpi). brain titers increased to a peak titer of 10 9.5 50% cell culture infectious doses per gram tissue (ccid 50 /g) on 4 dpi, maintained a titer of 10 9 ccid 50 /g through 7 dpi, and dropped slightly to 10 8.6 ccid 50 /g by 8 dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in 100% survival, whereas all placebotreated animals died by 9 dpi. syrian golden hamsters were infected with 10 3 ccid 50 wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as 4 dpi. the majority of deaths occurred on 5 dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring 4 dpi. interferon alfacon 1 (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < 0.001 as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer 10 4.8 ccid 50 /g) as compared with placebo (mean titer 10 9.5 ccid 50 /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. justin julander 1 , kristiina shafer 1 , john morrey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. 183, 1431 -1436 . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a 10 −4 dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken 3 days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was 66% mortality in animals challenged with the virus up to 9 dpi. virus titers in the liver peaked 6 dpi as determined by qrt-pcr. a significant increase in serum levels of alt (6 dpi), alkaline phosphotase (6 dpi) and bilirubin (6 dpi), and a significant decrease in amylase (8 dpi), albumin (6 dpi), and glucose (6 dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon 1, a consensus interferon. ribavirin and ifn alfacon 1 both significantly (p < 0.001) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < 0.05) and ifn alfacon 1 (p < 0.01) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon 1. viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv40). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv40 has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv40 in vitro has already been demonstrated (andrei et al., 1998. antimicrob. agents chemother. 41, 587-593) . here, the activity of a new class of anp's, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4diaminopyrimidines, against polyomaviruses was assessed. confluent uc1-b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, 2pta2 or lid-1, while bsc-1 cells were infected with either the primate sv40 strain a2895, the sv40 pml-1 strain ek or the sv40 pml-2 strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after 4-6 days (murine polyoma virus) or 5-7 days (sv40). hpmpo-dapy (2,4-diamino-6-(r)-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy (2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv40 strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara 1 , sylvester mosley 1 , brian bakke 1 , joshua sadler 1 , katherine seley(radtke) 1 , sunny zhou 2 1 university of maryland-baltimore county, baltimore, md, usa; 2 washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional 5 -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv-1, thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc 27708-0346, usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv-1 reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of 2 3 -dideoxycytidine triphosphate (ddctp) resulted in a 28-fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox-2 transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr5 as a co-receptor for hiv-1 infections revealed a novel approach to hiv-1 treatments and preventions. ccr5, a member from the family of 7tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr5 antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv-1 gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p55 cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt-4 cells were labeled with [3h]-leucine or myristic acid, and 2 h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. 18-20 h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt-4 cells before infection or 1-2 h later and at concentration 2-6 ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire 1 , thomas bell 2 , sreenivasa anugu 2 , noah duffy 2 , roger le grand 3 , erik de clercq 1 , dominique schols 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 department of chemistry, university of nevada, reno, usa; 3 service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec 50 : 0.3-3.2 m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv-1, irrespective of chemokine receptor preference (r5, x4, r5/x4). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd4 receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd4 receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv-1 subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv-2 strains examined. in addition, cada potently inhibited sivmac251 infection of pbmcs isolated from macaques (ec 50 : 1.6 m). comparable results were obtained in human cells infected with sivmac251. flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd4 receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate 1,2benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus 3) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within 48 h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi (0.01) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for 48 h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole-3-carbanol [i3c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin (833), purvalanol a (570), and i3c (1000) were greater than the positive controls acv (250) and paa (60). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a 2,6,9-trisubstituted purine, primarily inhibits cdk1; and i3c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: (1) polyelectrolyte-specific interferon induction and immunomodulation; (2) electrostatic-selective prevention of viruses absorption on plasma membranes; (3) membrane-targeted blocking of post-absorption steps (fusion); (4) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as (5) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the (1) and (2) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a1/a2) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv-1 absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr5/cxcr4. as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = 20,000 (against hiv-1 strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova 1 , alexander ivanov 1,2 , georgii galegov 3 , valeria andronova 3 , maxim jasko 1 1 engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; 2 centre for medical studies, university of oslo, moscow, russia; 3 ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines, were synthesized, and their efficacies against hiv-1 and hsv-1 were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines were evaluated as substrates towards hiv-1 reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the 3 -end of the growing dna chain. available to this virus. jev genome is an approximately 11-kb single-stranded positive-sense rna that has a cap structure at its 5 terminus but lacks a poly(a) tail at its 3 -terminus. the coding region of the genome is flanked by 5 -and 3 -untranslated region (utr). the 3 -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-(2-aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the 3 -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns5 protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns5 protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand 3 -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the 3 -end 83-nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev 3 -utr 83-nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva 1 , reneta toshkova 2 , milena nikolova 3 , reneta tsvetkova 3 , stefka antonova 4 , ivana roeva 1 , munnever sokmen 5 , bektas tepe 5 , medine gulluce 6 , fikrettin sahin 6 , atalay sokmen 5 1 institute of microbiology; 2 institute of experimental pathology and parasitology; 3 institute of botany, bulgarian academy of sciences; 4 faculty of biology, department of microbiology, sofia university, sofia, bulgaria; 5 faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; 6 faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of 134 plant products, obtained from 67 bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples (11.2%) inhibited flu reproduction, and twelve samples (7.5%) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in 55.5%; the antiviral effect was connected with antioxidant activity in 100%; the anti-flu effect was associated with immunogenic properties in 100%; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy 2,6-di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose 10-3 m was studied in vitro on the model of a/hong kong/1/68 (h3n2) reproduction in tissue cultures of chorioallantoic membranes of 12 days old chick embryos. anti-hsv activity in doses 10-4 m was studied on models of reproduction of hsv-1 on cell culture hep-2. compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity (39.4 and 61% of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). 3,5-dihydrazine-carbonyl-2,6-dimethylpyridine showed high anti-hsv (52%) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # 3147 and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb 1 , angelina trifonova 1 , stephan filipov 2 , angel s. galabov 1* 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus 1 replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's 50% inhibitory concentration for poliovirus 1 (mahoney) was found to be 0.188 g/ml in the cpeinhibition test and 0.041 g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type 1 strain, lsc-2ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva-9, the six coxsackie b viruses and 6 echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by 1 lg ranged from 0.01 to 1.0 g/ml. selectivity index was greater than 100 and even greater than 1000 for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva 1 , angel s. galabov 2 1 department of medical physics and biophysics, medical university, sofia, bulgaria; 2 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/2/68 (h3n2) (2.0 of ld50) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary (5 days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee 1 , ji-sun kwon 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s22 was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s22 showed potent antiviral activities against a/pr/8/34 (h1n1) influenza virus with the 50% effective concentration (ec50) values of 62.5 g/ml and the 50% cytotoxic concentration (cc50) values of 673.86 g/ml in mdck cells. treatment with the s22 appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s22 treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s22 was extracted with chcl3, etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or 2 h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by 50, 61, 72, and 94%, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by 25, 100, 72, and 100%, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by 92, 6, 67, and 100%, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by 94, 78, and 94%, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the 20th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april 29, 2007 and will end on thursday afternoon, may 3, 2007. all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the 20 th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs 05-04-49500 and russian ministry of sciences (lot 11). key: cord-277802-f8pyn3rx authors: roman, gheorghe title: mannich bases in medicinal chemistry and drug design date: 2015-01-07 journal: eur j med chem doi: 10.1016/j.ejmech.2014.10.076 sha: doc_id: 277802 cord_uid: f8pyn3rx the biological activity of mannich bases, a structurally heterogeneous class of chemical compounds that are generated from various substrates through the introduction of an aminomethyl function by means of the mannich reaction, is surveyed, with emphasis on the relationship between structure and biological activity. the review covers extensively the literature reports that have disclosed mannich bases as anticancer and cytotoxic agents, or compounds with potential antibacterial and antifungal activity in the last decade. the most relevant studies on the activity of mannich bases as antimycobacterial agents, antimalarials, or antiviral candidates have been included as well. the review contains also a thorough coverage of anticonvulsant, anti-inflammatory, analgesic and antioxidant activities of mannich bases. in addition, several minor biological activities of mannich bases, such as their ability to regulate blood pressure or inhibit platelet aggregation, their antiparasitic and anti-ulcer effects, as well as their use as agents for the treatment of mental disorders have been presented. the review gives in the end a brief overview of the potential of mannich bases as inhibitors of various enzymes or ligands for several receptors. the classical mannich reaction, a three-component condensation between structurally diverse substrates (xeh) containing at least one active hydrogen atom, an aldehyde component (generally r 1 -cho) and an amine reagent leads to a class of compounds generally known as mannich bases 1 (scheme 1). because mannich bases may be regarded as derivatives of the substrate obtained through substitution by an aminoalkyl moiety, mannich reactions are also known as aminoalkylation reactions. in the particular instance when formaldehyde is employed as aldehyde component, the substrate is converted into the corresponding mannich base through an aminomethylation process. although primary amines and even ammonia (in the form of an ammonium salt) may be employed as amine reagents in aminomethylations or aminoalkylations, secondary aliphatic amines (r 2 nh) are the most commonly encountered as amine reagents in the mannich reaction. as formaldehyde is used to a great extent as aldehyde component in the mannich reaction, the structural diversity of mannich bases stems primarily from the miscellaneous types of the substrates that can be subjected to aminomethylation, and secondarily from the variety of amine reagents that can be potentially employed in the mannich reaction. regardless of their structural diversity, the substrates should all have an activating functional group as a crucial structural feature that is required to render the substrate active in the mannich reaction. the carbonyl function in ketones, the phenolic hydroxyl in phenols, the terminal carbonecarbon triple bond in alkynes, the heteroatom in heterocycles, or electronwithdrawing groups that substitute the carbon atom a to the carboxylate group in esters of aliphatic carboxylic acids are common examples of pairs of activating groups and corresponding substrates, but the list is far from being exhaustive. a general classification of the most common types of mannich bases with respect of the substrates from which they derive and the nature of the atom substituted by the aminomethyl function is given in fig. 1 . under normal reaction conditions, substitution of a substrate with a single aminomethyl function results in mono-mannich bases, but two aminomethyl groups may also be grafted onto a substrate containing more than one active hydrogen atom, leading to double mannich bases such as 2e5 derived from dialkyl ketones, alkyl aryl ketones, 4-substituted phenols and pyrrole, respectively (fig. 2) . also, the aminomethylation of substrate xeh with amine reagents other than secondary amines (such as ammonia, having three reactive hydrogen atoms at nitrogen, or primary amines renh 2 , having two reactive hydrogen atoms at nitrogen) may lead to tris-mannich bases 6 and bis-mannich bases 7, respectively (fig. 2 ). in addition, the capability of some polyfunctional substrates to aminomethylate chemoselectively at a single potential reaction site under the appropriate reaction condition, or aminomethylate indiscriminately at multiple reaction sites, or even undergo aminomethylation simultaneously with ring closure, contributes considerably to the structural variety of the resulting mannich bases. two excellent, albeit rather old reviews provide more details on the synthesis and reactions of mannich bases to the interested reader [1, 2] . mannich bases have found numerous practical applications in the treatment of natural macromolecular materials such as leather, paper and textiles, the production of synthetic polymers, as additives used by the petroleum industry, as products used in water treatment, analytical reagents, cosmetics, dyes, etc. [3] . nonetheless, the most important application of the mannich reaction lies in the field of medicinal chemistry, and this claim is supported by the substantial number of papers published on this topic every year. first of all, mannich bases could present interesting biological activities, many of these having yet to be discovered through a diligent screening process. second, aminomethylation of drugs could be used to improve their delivery into the human body. aminomethylation may increase the hydrophilic properties of drugs through the introduction of a polar function in their structure, the long-known rolicycline being one of the most common examples [4] . the solubility in water of a drug could be further enhanced through the quaternization of the nitrogen atom in its aminomethyl derivative and conversion into an ammonium salt. alternatively, the lipophilic properties of a drug could be tailored through a mannich reaction if the appropriate amine reagent is employed [5] . in addition, the aminomethylated drugs could act as prodrugs, releasing the active substance under controlled hydrolytic conditions via deaminomethylation [6] or deamination [7] . in spite of the tremendous potential of mannich bases in medicinal chemistry, the wealth of information from studies concerning the structureeactivity relationship (sar) involving mannich bases or the use of aminomethylated drugs as prodrugs does not appear to have inspired many recent literature reviews of consequence, to the best of our knowledge. the present review fills this void by providing a comprehensive coverage of the most relevant developments in the medicinal chemistry of mannich bases generated exclusively through aminomethylation, the information being ordered according to the reported biological activity. due the large number of articles published on this topic, the coverage of this review is limited to the last decade. anticancer properties and cytotoxicity of ketonic mannich bases (with an emphasis on mannich bases of type 8 derived from acetophenones [8] ) and of structurally related a,b-unsaturated ketones [9] were reviewed 15 years ago. these two groups of compounds were shown to exert their cytotoxic action through the alkylation of cellular thiols such as glutathione or cysteine, and may be useful in sensitizing tumor cells to antineoplastic agents, and even reverse drug resistance [10] . it is therefore no surprise that compounds having both a ketonic mannich base moiety and an activated unsaturated carbonecarbon double bond in their structure (for example, mannich bases of chalcones such as 9) have been considered as candidates for the evaluation of the sequential cytotoxicity theory [11] . this theory hypothesizes that the successive release of two or more cytotoxic agents will result in increased toxicity to malignant tissue rather than to normal cells [12] . in addition, mannich bases 10 of enones, which are easily accessible from alkyl aryl ketones in one synthetic step, also demonstrated marked toxicity towards numerous cancer cell lines [13] . furthermore, as ortho-phenolic mannich bases undergo deamination easily to yield ortho-quinone methides, mannich bases of chalcones derived from either phenolic aldehydes or ketones, a class of compounds for which structure 11 is prototypical, have been examined also as cytotoxic agents [14] . in the last decade, the quest for more potent anticancer agents amongst these four general types of cytotoxic mannich bases 8e11 (fig. 3 ) has steadily continued. cytotoxicity of ketonic mannich bases of type 8 with various substitution patterns in the aromatic ring and of a few types of their derivatives has been studied in detail. gul et al. have shown that the structural modification of single mannich bases 8 (r 1 ¼ r 2 ¼ h) derived from acetophenone and secondary aliphatic amines into double mannich bases 12 (fig. 4) generally results in increased cytotoxicity against mouse renal carcinoma (renca) and transformed human t-lymphocyte (jurkat) cell lines [15] to the extent that double mannich bases were more cytotoxic than reference drugs 5-fluorouracil or melphalan. the cytotoxicity of these single and double mannich bases 8 and 12, respectively, was reversed when the compounds were used in a brine shrimp bioassay, presumably due to the fast deamination of the double mannich bases before they could reach their target [16] . also, ketonic mannich bases 8 with dimethylamino, 1-piperidinyl, 4-morpholinyl as amine moiety and featuring either an unsubstituted, variously monosubstituted phenyl rings, or a thiophene ring were evaluated with respect of their cytotoxicity towards jurkat cells [17, 18] or androgen-independent prostate cancer (pc-3) cells [19] , and the cytotoxicity for some of these compounds was 2.5-to 5.2-fold higher than that of the standard 5-fluorouracil. in addition, mannich bases of type 8 derived from 4-aryloxyacetophenones were shown to display moderate cytotoxic properties towards murine l1210 cells as well as human molt 4/c8 and cem t-lymphocytes, and a number of these compounds possessed remarkable potencies towards seven human colon cancer cell lines [20] . the use of primary aliphatic amines in the mannich reaction leads to reaction products with diverse structures (fig. 4) . aminomethylation of acetophenones using methylamine as amine reagent afforded both bis-mannich bases 13 (r ¼ ch 3 ) and piperidinols 14 (r ¼ ch 3 ) , and the evaluation of their cytotoxicity towards jurkat cells showed that bis-mannich bases 13 were generally more potent than the corresponding piperidinols 14 or mono-mannich bases 8 [21] . besides their ability to alkylate cellular glutathione, compounds 13 (r ¼ ch 3 ) may exert their cytotoxic action through the inhibition of dna topoisomerase i; as the corresponding piperidinols 14 were generally devoid of dna topoisomerase i inhibitory action, the authors tentatively attribute the activity of bis-mannich bases 13 to their linear structure and the possibility of formation of hydrogen bonds with dna nucleotides [22] . on the other hand, aminomethylation of acetophenones using isopropylamine [23] and n-butylamine [24] as amine reagent yielded only secondary mono-mannich bases 15 (r ¼ ch(ch 3 ) 2 , (ch 2 ) 3 ch 3 ), whose cytotoxicity was evaluated against huh-7 hepatoma cells, human jurkat and rat skeletal muscle derived myoblasts (l6) cells. compared to reference drug 5-fluorouracil, these compounds were 2.1-to 2.8-fold more cytotoxic towards huh-7 hepatoma cells, 2.6-to 4.2-fold more cytotoxic towards human jurkat cells, and 1.2-to 2.2-fold more cytotoxic towards l6 cells. aminomethylation of acetophenones using phenethylamine as amine reagent could led under carefully controlled reaction conditions either to mono-mannich bases 15 (r ¼ ch 2 ch 2 c 6 h 5 ) [25] or to the corresponding piperidinols 14 (r ¼ ch 2 ch 2 c 6 h 5 ) [26] ; the cytotoxicity of these compounds towards androgen-independent prostate cancer (pc-3) cells ranged from 8.2 to 32.1 mm, whereas the best compounds from each series had an average value for dna topoisomerase i interference of approximately 40%. anticancer activity of ketonic mannich bases has been compared with that of derivatives of the carbonyl function (fig. 4) . a series of azines 16 of ketonic mannich bases 8 were designed as bifunctional cytotoxic agents, but their activity towards jurkat cells [19] or pc-3 cells [17] was less potent or, in the best of cases, equipotent to the parent ketonic mannich bases. on the other hand, hydrazones 17 were consistently more cytotoxic than the corresponding ketonic mannich bases [18] . noteworthy is the contribution of gul et al. to the understanding of the mechanism of the cytotoxic action of these compounds. his group has provided evidence that connects the anticancer activity of ketonic mannich bases of various structures or piperidinols 14 with their ability to alkylate glutathione [21,27e29] , whereas a few of the same compounds had mixed effects on thioredoxin, glutaredoxin, or heat shock proteins hsc70 and grp75 [30] . only a limited number of examples of mannich bases of a,bunsaturated ketones of type 9 with cytotoxic action are available in recent publications. a small series of mannich bases 18 of 1arylidene-2-tetralones ( fig. 5) were evaluated as cytotoxic agents using human molt 4/c8 and cem t-lymphocytes, as well as murine p388 and l1210 leukemic cells [31] . compared to the parent a,bunsaturated ketones, mannich bases 18 were consistently more potent, with half maximal inhibitory concentration (ic 50 ) values in the 0.2e10 mm range. furthermore, mannich bases 18 derived from aromatic aldehydes substituted with chlorine, carboxyl, methoxy or cinnamoyloxy groups exhibited significant potencies towards human tumor cell lines, with an emphasis on their antileukemic effect. in most instances, the compounds prepared in this study demonstrated selective toxicity to different cells, which further enhances their potential utility. in addition to compounds 18, simpler mannich bases 19 derived from 2benzylidenecyclohexanones were synthesized, and their evaluation against the same cell lines proved once more that mannich bases were more cytotoxic than the corresponding 2arylidenecyclohexanones, some of them showing growthinhibiting properties (ic 50 of approximately 2 mm) more potent than reference drug melphalan [32] . because n-myristoyltransferase is expressed in larger quantities in tumors than it is in normal cells, this enzyme has been under consideration as a molecular target for cancer [33, 34] . however, the substantially high ic 50 value of 500 mm towards n-myristoyltransferase for a representative compound of the series of candidates 19 suggests that the inhibition of this enzyme does not play an important role in the mechanism of the cytotoxic activity of these compounds. several compounds 19 were also tested against murine cancer cells mac13 (sensitive to most cytotoxic agents) and mac16 (resistant to most cytotoxic agents), and they demonstrated high cytotoxicity against the latter, but also against normal murine cells c2c12 and 3t3 [35] . on the other hand, mannich bases 20 showed no activity against mac16, which points to the importance of the double bond conjugated to the carbonyl function. the exploration of possible mechanisms of cytotoxic action of these compounds revealed that compounds 19 may interfere with a number of essential cellular mechanisms by alkylation of thiols on enzyme or proteins, by disrupting mitochondrial electron transport, or by creating holes in the cell membrane, and thus promoting atp leakage [35] . finally, mannich bases 21 had high cytotoxic activity against two human breast cancer cell lines (mcf-7 and mcf-7/adr) cells and human leukemia hl-60 cells, showed glutathione binding ability, and exhibited inhibitory action on glutathione-s-transferase p, whereas their analogues obtained through the hydrogenation of the double carbonecarbon bond were slightly less active [36] . the nature of the dialkylamino group did not seem to affect the cytotoxic activity of these compounds, while the substitution of the aromatic rings with a methyl selectively increased the cytotoxic effect on breast cancer cells, but not on immortalized mammary epithelial (184b5) cells. the literature reporting the anticancer activity of mannich bases of type 10 is even scarcer. given the significant antineoplastic properties of compounds 10 [13] , a novel series having substituents r 1 in the phenyl ring that were carefully selected with a view to impart a variety of physicochemical properties has been designed and synthesized [37] . since a gradual release of mannich base 10 from niosomes had improved its bioactivity in vivo, amino alcohols 22 (fig. 5) , which may slowly undergo dehydration to yield the desired mannich base 10, were also examined as cytotoxic agents. finally, in order to explore the hypothesis that cytotoxicity would be retained even when a thiol is liberated, the synthesis of adducts 23 was carried out. compounds 10, 22 and 23 ( fig. 5 ) were evaluated against both human widr colon cancer cells and human crl-2522 foreskin fibroblasts. ic 50 values lesser than 10 mm were obtained when compounds 10 were evaluated towards human widr colon cancer cells, and the corresponding candidates 22 also had ic 50 values in the low micromolar range. on the other hand, conversion of mannich bases 10 into the corresponding adducts 23 led to a 37-fold reduction in potency. in addition, compounds 10 and 22 demonstrated a preferential cytotoxicity to cancer cells compared to normal fibroblasts [37] . further studies showed that compounds 10 and 22 are cytotoxic towards a large number of human tumor cell lines, two important features of many of these compounds being their lethal effects toward promyelocytic leukemic hl-60 cells and their selective toxicity for the aforementioned cancer cell line (selectivity index of 10 or more) [38] . because divergence in the mechanism of action is required for drug candidates that are developed to be tumor-specific and spare normal tissues, it is noteworthy that a representative mannich base 10 caused apoptosis and activated caspase-3, caspase-8, and caspase-9 in hl-60 cells, but not in hsc-2 cells. cytotoxic phenolic mannich bases of chalcone analogues (type 11 in fig. 3 ) are well represented in the recent literature. one of the strategies that are available for the synthesis of this type of mannich bases consists in the aminomethylation of chalcone analogues derived from at least either a phenolic aldehyde or a phenolic ketone. in line with this strategy, a series of five mono-mannich bases 24 (fig. 6 ) were obtained through a mannich reaction of chalcone analogues derived from 4-hydroxyacetophenone, employing piperidine as amine reagent [39] . despite the use of an excess of both paraformaldehyde and piperidine, no double mannich bases analogous to 11 were isolated. the evaluation of compounds 24 against androgene-independent prostate cancer (pc-3) cell line showed that although three of them were more potent than the parent chalcone analogues, the most cytotoxic mannich base 24 was 2.5-fold less potent than the reference drug 5fluorouracil. based on correlations between cytotoxicity and hammet constant on one hand and cytotoxicity and partition coefficient on the other hand, the authors hypothesized that an increase in the general hydrophobicity of the molecule would result in enhanced cytotoxicity. therefore, a novel series of mannich bases 24 was designed to incorporate a dibenzylaminomethyl residue as a replacement for the piperidinomethyl group [40] . again, every attempt to obtain double mannich bases by varying the reaction conditions failed, and only mono-mannich bases could be isolated. when evaluated against androgene-independent prostate cancer (pc-3) cell line, these compounds consistently displayed lower cytotoxicity than that of the parent chalcone analogues. unexpectedly, cytotoxicity in the series of mannich bases with a dibenzylaminomethyl residue was also much lower than that of the corresponding mannich bases in the series containing a piperidinomethyl motif, thus invalidating the hypothesis put forth by the authors in the previous study. furthermore, no adduct between ethanethiol and a representative mannich base 24 could be detected after 48 h, whereas the incubation of ethanethiol with the corresponding parent chalcone analogue under the same conditions resulted in formation of small amounts of adduct. the authors concluded that no active cyclohexadienone species are formed following a potential deamination of mannich base 24, and that the introduction of the dibenzylaminomethyl group further reduces the ability of the chalcone moiety to undergo thiol addition. with a view to explore the effect of variation of dialkylamino moiety on the cytotoxicity of phenolic mannich bases of chalcone analogues, 27 candidates were synthesized through aminomethylation of three chalcone analogues derived from 4hydroxyacetophenone and diverse secondary aliphatic amines [41] . equimolar ratio of reactants afforded mono-mannich bases of type 24, which were evaluated against hepatocellular carcinoma (hepg2), human lung carcinoma (sk-lu-1), and human breast cancer (mcf-7) cell lines. mannich bases with 4-phenylpiperazine residue exhibited reduced cytotoxicity towards all three lines of cancer cells, whereas the candidates with 4-methylpiperazine or 4ethylpiperazine residues were the most active in each series, but less cytotoxic than reference drug ellipticine. with respect to the substitution pattern in the b phenyl ring, mannich bases 25 ( fig. 6 ) derived from 4-chlorobenzaldehyde (r 1 ¼ h, r 2 ¼ cl) or 2methoxybenzaldehyde (r 1 ¼ och 3 , r 2 ¼ h) were consistently more active than those derived from 4-methoxybenzaldehyde (r 1 ¼ h, r 2 ¼ och 3 ). the screening identified five compounds whose ic 50 values against mcf-7 cell line were lower than 2 mg/ml, whereas the most cytotoxic compound 25 (r 1 ¼ h, r 2 ¼ cl, nr 2 ¼ 4ethylpiperazinyl) had ic 50 values lower than 2 mg/ml against all three cancer cell lines used in this study. a larger library of phenolic mannich bases of chalcone analogues featuring the dialkylaminomethyl moiety either in ring a or ring b of the chalcone system was synthesized through the clai-seneschmidt condensation of the appropriately substituted mannich bases of phenolic aldehydes or ketones with heterocyclic ketones or aldehydes, respectively [42] . the use of 4-alkoxy-2hydroxyacetophenones as substrates in the mannich reaction yielded a mixture of 5-aminomethylated derivative with the isomeric 3-aminomethylated derivative, the former being the major reaction product in all cases. the adept tailoring of the ratio between the substrate, formaldehyde and morpholine in the mannich reaction of 4-hydroxyacetophenone resulted in the selective preparation of either single or double mannich base from this substrate. on the other hand, 3-hydroxyacetophenone afforded a mixture of 4-morpholinylmethyl derivative with 2morpholinylmethyl derivative and 2,4-bis(morpholinylmethyl) derivative, even when an equimolar ratio between the reagents was used. isovanillin, which underwent aminomethylation only at position 2 with morpholine and piperidine as amine reagents, was employed as an example of a phenolic aldehyde substrate in the mannich reaction. with the help of these intermediates, several small series of mannich bases of heterocyclic chalcone analogues 26e33 (fig. 6 ) were synthesized and evaluated for cytotoxic activity against four human cancer cell lines, namely pc-3, mcf-7, nasopharyngeal carcinoma (kb), and resistant nasopharyngeal carcinoma (kb-vin). the rich diversity within this library comprised of structurally related entities allowed interesting insight on the cytotoxicityestructure relationship. first, the presence of two phenolic groups in ring a seems to enhance the cytotoxic activity, as proven by a candidate of type 26 (r 1 ¼ h, het ¼ 2-pyridinyl), which was the most potent in the entire library against all four types of cancer cell lines. then, the presence of a methoxy group in series 27 (r 1 ¼ ch 3 ) appears to be generally preferable to ethoxy and isopropoxy, whereas a comparison of the cytotoxicity of similar compounds of type 26 and type 27 usually favors the candidates in the latter series, which have the aminomethyl group para to the phenolic hydroxyl. an analysis of the cytotoxicity within series of candidates of type 28 demonstrated that six-membered heterocycles (particularly a 2-pyridinyl residue) are preferred to five membered heterocycles as ring b moieties, and this observation was validated by the inspection of anticancer activity of compounds in series 29. however, the presence of a second morpholinylmethyl group appears to be detrimental to the cytotoxic activity of candidates 29. shuffling of hydroxy and morpholinylmethyl groups in mannich bases 28 and 29 led to compounds in series 30e32, which are generally less cytotoxic than their counterparts derived from chalcone analogues having a 4-hydroxy substituent in ring a. selective high cytotoxicity against mcf-7 cell line was displayed by the compounds in series 33 featuring the morpholinylmethyl moiety in ring b of the chalcone system. overall, more than 80% of the mannich bases in this library of mannich bases of heterocyclic chalcone analogues 26e33 are cytotoxic (ic 50 < 4 mg/ml), while four members of the library are highly cytotoxic (ic 50 < 1 mg/ml) against all four cell lines, and other four against at least three cell lines, with mcf-7 and pc-3 being usually more sensitive than kb and kb-vin cell lines. later, novel mannich bases of type 24 (ar ¼ 3pyridinyl) were evaluated against several cancer cell lines, and the candidates showed cytotoxicity in the low micromolar range only towards promyelocytic leukemic cells (hl-60) and oral squamosa cell carcinomas (hsc-2, hsc-3 and hsc-4), whereas the ic 50 values against non-malignant gingival fibroblasts, pulp cells and periodontal ligament fibroblasts were higher [43] . the tumor selectivity of these mannich bases may be the result of their proven ability to cleave poly[adp-ribose]polymerase-1 in hsc-2 cells, but not in gingival fibroblasts cells. in addition, the cytotoxic activity of mannich bases of chalcone analogues with an aminomethyl moiety in b ring structurally similar to 33 against a panel of breast cancer (mcf7), melanoma (uacc62) and renal cancer (tk10) cell lines was described in a patent [44] . most compounds were active, and some were potent mostly towards the first two cancer cell lines. a series of mannich bases 34 of bichalcone analogues (fig. 6) , in which the two chalcone units are linked through a bis(aminomethyl) function generated by the use of a bifunctional amine reagent such as piperazine, has also been synthesized and evaluated against 25 cancer cell lines [45] . aminomethylation of acetovanillone with piperazine afforded a bis-mannich base, which subsequently led to compounds 34 through a claiseneschmidt condensation with various aldehydes. surprisingly, compound 34 (ar ¼ 2-pyridinyl) was selectively cytotoxic to human tongue squamous carcinoma (cal-27) and human pharyngeal squamous carcinoma (fadu) cell lines, whereas 3-pyridinyl and phenyl analogues were the most cytotoxic compounds towards all cell lines. substitution of the phenyl ring (ar ¼ c 6 h 5 ) with methoxy groups stripped mannich bases 34 of their cytotoxicity towards most cell lines, whereas the decrease in cytotoxic activity induced by the presence of chlorine as substituent was not so drastic. replacement of phenyl with 2-furanyl or 2-thiophenyl led to compounds that are selectively cytotoxic to one or more cell lines, but further substitution with methyl of these five-membered heterocycle renders them devoid of cytotoxicity against all lines. despite a few notable examples of selectivity, the results obtained for this collection of compounds are not very encouraging, and they suggest that the incorporation of a second chalcone unit does not enhance the cytotoxicity of mannich bases derived from chalcone analogues. cytotoxic activity of mannich bases of bichalcone analogues was further explored using candidates with a modified design. the synthetic strategy comprised the synthesis of mono-phenolic mannich bases starting from 4-hydroxyacetophenone or acetovanillone as substrates and employing 1-(4-(piperazin-1-yl)phenyl) ethanone as amine reagent, then the bichalcone unit was generated through a claiseneschmidt condensation of both acetyl functions with aromatic aldehydes [46] . only mannich bases from bichalcone analogues featuring a pyridinyl moiety (such as candidate 35, fig. 6 ) were active towards prostate cancer (du145), non-small cell lung cancer (a549), ileocecal (hct) and nasopharyngeal carcinoma (kb) cell lines with ic 50 values between 0.7 and 4 mm; all other compounds had ic 50 values greater than 20 mm. compared to compound 35, mannich base 36 having a single chalcone unit was less active (ic 50 between 10 and 13 mm). an exploration of the mechanism of action for these compounds suggested that compound 36 most likely acted via the fas/cd95 apoptosis signaling pathway. ferulic acid and its derivatives provide another example of a type of substrate containing an a,b-unsaturated system activated by an electron-withdrawing group, similar to that in phenolic chalcone analogues, from which phenolic mannich bases can be synthesized. the growing body of evidence suggesting that 3 0 -azido-2 0 -deoxythymidine (azt), a known antiviral, also possesses anticancer activity [47e49] has sparked a study aiming at cytotoxic evaluation of a series of conjugates of azt and ferulic acid derivatives [50] . thus, propargyl ester and n-propargylamide of ferulic acid chemoselectively underwent aminomethylation ortho to the phenolic hydroxyl with various secondary aliphatic amines, and the resulting phenolic mannich bases 37 (x ¼ o, nh) reacted through the terminal alkyne moiety with azt via a cu(i)-catalyzed click chemistry process to afford the corresponding 1,2,3-triazoles. evaluation of cytotoxicity for both mannich bases 37 (fig. 7 ) and the related 1,2,3-triazoles against human breast adenocarcinoma (mda-mb-231), lung adenocarcinoma (sk-lu-1) and colon adenocarcinoma (sw480) cell lines showed that only some of compounds 37 were cytotoxic. despite the presence in their structure of an identical scaffold comprising a phenolic mannich bases moiety and an activated carbonecarbon double bond motif that has been deemed responsible for the cytotoxic effect of mannich bases 37, all of the corresponding 1,2,3-triazoles were inactive. out of eight mannich bases of propargyl ester of ferulic acid reported in this study, three were inactive against all three lines, while the rest had weak to moderate cytotoxicity, and the most active candidate (ic 50 ¼ 20e44 mg/ml) was mannich base 37 (x ¼ o) having a pyrrolidinylmethyl moiety. with the exception of mannich base 37 (x ¼ nh) with a piperidinylmethyl moiety, all others candidates derived from n-propargylamide of ferulic acid were inactive. other aminomethylated phenols with cytotoxic activity have been reported besides phenolic mannich bases of chalcone analogues. unfortunately, the wide structural diversity of phenolic substrates from which these phenolic mannich bases originate and the lack of a systematic and comprehensive search for structureecytotoxic activity relationships within a particular type of phenolic substrate undermine any efforts to discover good lead compounds for further development as drugs. phenolic substrates subjected to the mannich reaction with a view to obtain novel cytotoxic agents include both simple and very complex structures. examples that illustrate structurally simple phenolic substrates are 1-naphthol and 8-hydroquinoline, whose mannich bases with piperidine and 4-arylsulfonylpiperazines exhibited growthinhibitory effects towards a panel of carcinoma cell lines, including hela (cervical epithelioid carcinoma cell), bt483 (mammary gland adenocarcinoma cell), skhep (hepatocellular carcinoma cell), and ce81t (esophageal carcinoma cell) [51] . although the cytotoxic effect of aminomethylated naphthols and 8hydroxyquinoline derivatives has been known for some time [52, 53] , a mechanistic study presented in the aforementioned report showed that these phenolic mannich bases induce apoptosis by activation of caspase-dependent pathways. furthermore, upon addition of copper ions, these compounds dramatically stimulate production of reactive oxygen species and activate various kinases, a group of enzymes that are known to be important in the oxidative stress-mediated cell death. candidate 38 (fig. 7) was the most potent in this small series, with a concentration for 50% cell growth inhibition of 0.71 mm; this value decreased to 0.06 mm in the presence of 50 mm copper. a more detailed study [54] of the structureecytotoxic activity relationship within this class of compounds showed that either replacement of sulfonyl function in 38 with a methylene group, or replacement of piperazine ring with an ethylenediamino moiety (as in compound 39, fig. 7 ) led to a significant increase in cytotoxic activity. the cell lines used in this study exhibited selective sensitivity towards different mannich bases, which appeared to be modulated by the nature of the arylsulfonyl group. as for the 8-hydroxyquinoline part of the molecule, substitution at position 5 of the quinoline motif (especially with a nitro group) had a beneficial effect, whereas its replacement with phenol, 3-hydroxypyridine or 1-naphthol led to a decrease of cytotoxic activity [54] . cytotoxicity of enantiomerically-enriched mannich bases 40 (fig. 7) derived from 2-naphthol, aromatic aldehydes and either 4-piperidinol (r ¼ h) or its acetylated counterpart (r ¼ coch 3 ) against murine leukemic l1210 and human lymphoblast molt 4/c8 and cem cell lines has been examined by another study [55] . all of the compounds were only moderately cytotoxic, with ic 50 values in the middle micromolar range, and were also 10e70-fold less potent than that reference drug melphalan. substitution of the aryl group in 40 with r 1 ¼ 4dialkylaminoethoxy resulted in a series of compounds whose cytotoxicity potential against estrogen-responsive human mcf-7 breast cancer cells was found to be comparable to that of tamoxifen. however, removal of the 4-piperidinol moiety from mannich bases 40 led to benzylnaphthols with enhanced cytotoxicity against mcf-7 cells, which is most likely due to their binding and antagonistic effects against human estrogen receptor alpha [55] . flavones represent another type of phenolic substrate from which cytotoxic mannich bases have been synthesized. because flavones such as chrysin bear structural resemblances to androgens, mannich bases 41 fig. 7) have been designed as inhibitors of human aromatase, an enzyme which converts androgens to estrogens, and therefore represents a key target in the treatment of hormone-dependent tumors, including breast cancer [56] . several single and double mannich bases of chrysin were found to inhibit human aromatase more effectively than reference drug aminoglutethimide [57] . in addition, mannich bases 41 (r ¼ oh, r 1 ¼ h, r 2 ¼ aminomethyl) of apigenin ( fig. 7 ) have been prepared from aliphatic primary and secondary amines via chemoselective aminomethylation at c-8 in the benzopyran ring system [58] . antiproliferative activity of these mannich bases against four human cancer cell lines, namely human cervical (hela), human liver (hepg2), human lung (a549), and human breast (mcf-7) cancer cells, was determined using the standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium) bromide (mtt) assay. pyrrolidine mannich base of apigenin was the most promising compound in this series, its inhibition of cell proliferation being greater than 90% against all four cell lines at a concentration of 1 mg/ml. many natural or synthetic carbazoles, either simple or condensed with other heterocycles (such as pyridocarbazoles, indolocarbazoles, pyranocarbazoles, pyrrolocarbazoles, etc), have been reported as anticancer agents. a recent study examines the cytotoxicity of a series of oxazinocarbazoles, which were the major products arising from the mannich reaction of n-substituted 2-or 4-hydroxycarbazoles with primary amines (fig. 8 ) [59] . under the appropriate reaction condition, 4-hydroxycarbazoles yielded 2,3,4,7-tetrahydro [1, 3] oxazino [5,6c]carbazoles 42, whereas 2hydroxy-9-methylcarbazole led to a mixture of regioisomeric 2,3,4,7-tetrahydro [1, 3] oxazino [6,5-b] carbazoles 43 and 2,3,4,7tetrahydro [1, 3] oxazino[5,6-a]carbazoles 44. use of allylamine, 3,3-dimethylallylamine or benzylamine as amine reagents afforded 44 as the major product, while isomer 43 was the major component of the mixture when 2-pyridinylmethylamine was employed. in addition, small to moderate yields of bis-mannich bases 45 or 46 were isolated from n-substituted 4-hydroxycarbazoles and 2hydroxy-9-methylcarbazole, respectively, but only when 2pyridinylmethylamine was employed as amine reagent. evaluation of the antiproliferative action of these compounds against cem (t cell leukemia), jurkat (acute t cell leukemia), raji (burkitt's lymphoma), mcf-7 (breast cancer cells) and caco-2 (colorectal cancer) using the wst-1 colorimetric assay showed, after the primary screening at 100 mm, that bis-mannich bases 45 and 46 were less active than the oxazinocarbazoles. in the series of compounds 42, the best antiproliferative effect was observed for candidates having either an allyl or a prenyl group at the carbazole nitrogen atom and an allyl group at the oxazine nitrogen atom. thus, jurkat and raji cell lines. the majority of candidates 43 and 44 exhibited significant antiproliferative action at 100 mm, and compound 43 (r ¼ 2-pyridinylmethyl) was the most active towards jurkat and raji cell lines (ic 50 ¼ 12 mm) [59] . aminomethylated derivatives of hydroxycarbazoles have been also mentioned in a different study [60] , which described the synthesis of a small series of phenolic mannich bases 47 (fig. 8 ) obtained from 5-substituted 2-hydroxy-5h-benzo[b]carbazole-6,11-diones along with their in vitro anticancer evaluation at national cancer institute (nci) using an in-house developed screening panel of approximately 60 cell lines derived from nine different types of cancer. only one mannich base 47 (r ¼ 4-h 3 coc 6 h 4 ) was more active than the parent benzocarbazoledione, and a compare analysis [61] revealed that its mechanism of action is novel and does not resemble the known mechanisms of action for standard anticancer drugs, but this candidate was not selected for further studies concerning its interaction with dna. in a related report, phenolic mannich bases 48 of 5-hydroxy-1h-naphtho [2,3-g] indoles were found to be inactive in a similar screening [62] . quinones are a class of compounds that have been widely investigated as anticancer agents [63] . besides anthracycline antibiotics, other natural hydroxyquinones such as plumbagin [64, 65] , juglone [66, 67] or lapachol [68] and their derivatives have been reported to exhibit significant cytotoxicity. the mannich bases of another natural phenolic quinone, namely lawsone, and their pt(ii) complexes 49 ( fig. 9) have been synthesized and shown to be highly cytotoxic towards six cancer cell lines: mda-mb-435 (melanoma), hl-60 (promyelocytic leukemia), hct-8 (colon), sf-295 (brain), ovcar-8 (ovary) and pc-3 (prostate) [69] . the ligands and the complexes that have long alkyl chains (r ¼ n-heptyl or ndecyl) were the most active (the complexes were actually more cytotoxic than cisplatin), whereas the neutral complexes (x ¼ cl) were generally more cytotoxic than the corresponding charged complexes (x ¼ h 2 o or nh 3 ). examination of the mechanism of action for some of these complexes has shown that aqua complexes 49 were more efficient inhibitors of ethidium bromide intercalation into dna than amino complexes, and candidate 49 (r ¼ (ch 2 ) 3 ch 3 , x ¼ h 2 o) was more efficient than cisplatin [70] . the same aqua complex also induced dna strand breaks, while the corresponding amino complex was ineffective. the ability of these complexes to inhibit topoisomerase i was also examined. most chlorido and amino complexes were as active as reference drug camptothecin in the dna relaxation assay, and did not cause major unwinding of dna, with the exception of complex 49 (r ¼ n-decyl, x ¼ cl). in addition, cellular platinum accumulation was shown to increase with the increase of the length of the alkyl chain of the amino moiety in the mannich base ligand. the chlorido pt(ii) complexes were oxidized to the corresponding chlorido pt(iv) complexes, but the cytotoxicity of these new complexes was comparable to the cytotoxicity of the parent pt(ii) complexes, presumably owing to the rapid reduction of pt(iv) complexes before entering the cancer cells [71] . furthermore, miscellaneous aminomethylated phenols from structurally diverse substrates have been reported as anticancer agents (fig. 10) . thus, a small series of seven phenolic mannich bases 50 were synthesized through aminomethylation of naturally occurring antibiotic lasalocid, with simultaneous elimination of the carboxyl group neighboring the phenolic hydroxyl [72] . antiproliferative effect of mannich bases 50 was evaluated against mcf-7 (human breast adenocarcinoma), a549 (human lung adenocarcinoma), ht-29 (human colon carcinoma) and p388 (murine leukemia) using either mtt or sulforhodamine b (srb) assay, and four candidates were more cytotoxic towards a549, ht-29 and mcf-7 cell lines that anticancer drug cisplatin. these candidates also presented higher selectivity towards cancer cells than towards balb/3t3 (normal murine embryonic fibroblast) or hlmec (human lung microvascular endothelial) cell lines. the lack of activity of the other three mannich bases was attributed to the aralkyl or long alkyl chains in the amine moiety of these compounds [72] . camptothecin, another naturally occurring phenolic substrate and lead compound for a plethora of cytotoxic substances, was converted into the oxazino derivatives 51 (fig. 10 ) by means of the mannich reaction using primary aliphatic and aromatic amines [73] . evaluation of these novel hexacyclic camptothecin derivatives towards nine human cancer cell lines (bxpc-3, nci-446, mcf-7, hepg-2, a549, a2780, bel7402, ht-29, and kb), using mtt assay and camptothecin and topotecan as reference compounds, showed that most of them exhibit cytotoxicity towards several cell lines that is superior or comparable to topotecan, while only a few of the candidates presented cytotoxicity comparable to camptothecin. because candidates 51 (r ¼ c 2 h 5 or n-c 3 h 7 ) were the most potent antiproliferative agents in this series, the presence of a small alkyl group at the nitrogen in the oxazine moiety seems to be preferable for a high cytotoxicity [73] . a series of oxazino derivatives 52 (r 1 ¼ ch 3 ) (fig. 10) were also prepared from g-tocotrienol and primary amines via the mannich reaction, whereas d-tocotrienol afforded under the same conditions a mixture of oxazino derivatives 52 (r 1 ¼ h) and 53, in which the former is the major component [74] . no reaction occurred when secondary amines were used instead, but two phenolic mannich bases 54 were obtained indirectly from the corresponding oxazino derivatives 52. out of 42 candidates in this library, thirty compounds had greater antiproliferative activity against the highly metastatic þ sa mouse mammary epithelial cancer cells than that of the parent tocotrienols (ic 50 ¼ 3 mm), and seven candidates had ic 50 values in the nanomolar range. mannich bases 54 were less active than the corresponding oxazino derivatives 52 against nci's standard panel of 60 cell lines, which suggests that the oxazine ring is an essential pharmacophore for the cytotoxic activity of these tocotrienol derivatives. generally, the oxazino derivatives 52 of dtocotrienol were more active than the corresponding isomers 53 derived from g-tocotrienol, and a long alkyl chain at the nitrogen atom (preferably with a terminal hydroxyl group) proved beneficial for the antiproliferative activity. the same conclusions were drawn after the evaluation of structureeantimigratory activity relationship using the highly metastatic mdamb-231 breast cancer cell line [74] . novel g-quadruplex ligand/alkylating hybrid structures 55 ( fig. 10) were obtained by tethering a naphthalene diimide core having g-quadruplex recognizing properties to phenolic mannich bases using flexible spacer [75] . the assessment of cytotoxic effects of these compounds 55 (n ¼ 1, 2, or 3) and their corresponding methiodides against human embryonic kidney 293t cell line by mtt assay suggests that the length of the spacer between the core and the phenolic mannich bases moiety modulates the cytotoxicity: candidates 55 having two-carbon atoms and one-carbon atom spacers were the most active (ic 50 4.5 and 10.5 mm, respectively), whereas the mannich base 55 with a three-carbon atoms spacer was less active. cytotoxicity of these compounds parallels their ability to alkylate dna, and the grafting of the alkylating mannich base moiety to the central core contributes to the enhancement of the g-quadruplex folding induction and stabilization. a similar g-quadruplex ligand/alkylating hybrid structure was shown to significantly slow the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression [76] , which suggests that these hybrids could be possible candidates for the development of novel targeted anticancer therapies. in connection to this, the methiodide of double mannich base 56 (fig. 10 ) with a quinazoline core was also shown to cross-link linear dna at concentrations as low as 1 mm, and to inhibit dna transcription almost completely at 10 mm [77] . phenolic mannich bases 57 of norvisnagin ( fig. 10 ) were prepared through direct aminomethylation, and their ability to interact with dna was evaluated using both a qualitative binding assay and a colorimetric microassay based on the displacement of methyl green from dna [78] . three of the candidates 57 (r ¼ pyridinyl-2-amino, diethylamino, and methylamino) showed moderate dna binding affinity, and could be potentially cytotoxic. also, mannich bases 58 (r 1 ¼ h) of 1,3-dihydroxyxanthone ( fig. 10 ) displayed moderate to good cytotoxicity against lung cancer (nci-h460), tongue squamosa cell carcinoma (tca-8113), liver cancer (bel-7402), hepatocarcinoma (hepg2), gastric carcinoma (sgc-7901) and urinary bladder carcinoma (t24) in an mtt assay [79] . several reports of mannich bases of indoles as cytotoxic agents are also available in recent literature. cytotoxicity of a few indole mannich bases 59 derived from 4-substituted piperazines (fig. 11 ) was evaluated against liver (huh7), breast (mcf7) and colon (hct116) cancer cell lines using srb assay, and some of these compounds had ic 50 values in the lower micromolar range. compound 59 (r ¼ 3,4-dichlorobenzyl) was cytotoxic towards all three cancer cell lines, and fared better than reference drug 5-fluorouracil (5-fu) [80] . on the other hand, n-mannich bases 60 of 3methylindole did not inhibit the growth of cancer cells or had high ic 50 values. in spite of this fact, a subsequent study [81] was dedicated exclusively to the investigation of cytotoxicity of a novel series of n-mannich bases of type 60 against the same cancer cell lines. the broadening of the nature of substituent at position 4 of piperazine proved favorable, as several compounds reported in this later study presented cytotoxic activity comparable to reference drug 5-fu. a comparison between the morphological features of cancer cells for which apoptosis was induced either by a selected 3metylindole n-mannich base 60 or by paclitaxel suggests that compounds 60 (r 1 ¼ ch 3 ) and paclitaxel share the same mechanism of action. design of novel cytotoxic mannich bases in which indole itself was the substrate for aminomethylation was also revisited using an extended panel of 4-substituted piperazines as amine reagents in aminomethylation [82] . the novel candidates 59 proved to be cytotoxic towards the same cancer cell lines (huh7, mcf7, and hct116); however, no cytotoxicity was observed for the candidates with an electron-withdrawing group (such as 4nitrophenyl, benzoyl or acetyl) as substituent at position 4 of piperazine. other c-mannich bases of indole derivatives have been claimed as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (imct), an enzyme that plays an important role in the posttranslational modification of proteins that are involved in the regulation of cell growth, and therefore represents a potential therapeutic target in oncogenesis. among the few small molecules inhibitors of imct that were discovered so far, the most promising appears to be cysmethynil, an indol-3-ylacetamide derivative which impairs growth factor signaling and induces cell cycle arrest and autophagy. because cysmethynil suffers from poor water solubility and strong binding to plasma proteins, rational modification of this hit compound has been expected to yield more potent imct inhibitors with improved bioavailability. replacement of the acetamide function in cysmethynil with various aminomethyl moieties led to compounds 61 (r ¼ dialkylamino) with an inhibitory effect on icmt that was generally 2e3-fold more potent than that of the parent inhibitor [83] . evaluation of the effects of these candidates on viability of mda-mb-231 human breast cancer cells using the colorimetric tetrazolium assay confirmed the results for icmt inhibition. thus, compounds 61 ( fig. 11 ) were found to be more cytotoxic (ic 50 3e13 mm) than cysmethynil (ic 50 22 mm). other modifications of the lead compound 61 (r ¼ diethylamino) either preserved the potency of the candidates (e.g., shuffling of the methyl group on the phenyl ring), or resulted in a potency decrease (e.g., replacement of n-octyl with prenyl). however, the replacement of m-tolyl moiety in 61 with more polar heteroaromatic rings led to submicromolar ic 50 values in the icmt inhibition assay, and to ic 50 values in the antiproliferative assay on breast mda-mb-231 and prostate pc3 cell lines that are 2e3-fold lower than that of fig. 11 . cytotoxic indole and azaindole mannich bases. the lead 61 (r ¼ diethylamino) [84] . compound 62 (fig. 11 ) was the most potent compound in this series, and presented a series of improvements of the drug-like profile over cysmethynil, such as good solubility in water, acceptable permeability through an artificial membrane, and limited tendency to form light scattering aggregates. using naphtho[2,3-f]indole-5,10-dione as scaffold, a series of 3aminomethylated derivatives was synthesized, and four candidates were evaluated as antiproliferative agents against the standard panel of 60 human cancer cell lines at nci [85] . compounds 63 (r ¼ primary or secondary aliphatic amine residue, r 1 ¼ oh) (fig. 11 ) were less potent than doxorubicin against any of the cell lines, but multidrug resistant breast cancer cells were found to be more sensitive to 63 than to doxorubicin. in addition, mannich bases 63 showed potency for cancer cell lines that are otherwise resistant to anticancer drugs, such as the p-glycoprotein-positive subline of k562 leukemia cells or the p53-null subline of hct116 colon carcinoma cell line. replacement of phenolic hydroxyl groups r 1 in 63 (r ¼ dimethylamino) by 2-aminoethyleneamino moieties led to mixed results, as the potency improved for some of the cell lines and declined for others, but the sensitivity of multidrug resistant breast cancer cells to these modified candidates was completely lost [86] . furthermore, candidate 63 (r ¼ quinuclidin-3ylamino, r 1 ¼ oh) was shown to inhibit topoisomerase i-mediated relaxation of dna, but the suppression of the topoisomerase i activity is presumably the leading although probably not the only factor contributing to cytotoxicity of mannich bases of naphtho [2,3-f]indole-5,10-diones [87] . preobrazhenskaya et al. have also shown that a series of single and double mannich bases 64 (r 1 ¼ h or dialkylaminomethyl) of 3,4-bis(indol-1-yl)maleimides (fig. 11) , structurally related to rebeccamycin or staurosporine, were highly cytotoxic towards к562 and hcт116 cell lines, but their cytotoxicity does not correlate well with their ability to either inhibit protein kinase c-a or constrain activation of multiple drug resistance [88] . mannich bases of an indole isostere, namely 5h-pyrrolo[3,2-d] pyrimidine, have been designed as inhibitors of phosphatidylinositol-3-kinase a (pi3ka), a lipid kinase that modulates activity of the pi3k downstream effectors akt and mtor. since the consequences of biological activation of akt include tumor progression, proliferation, survival, growth, invasion, angiogenesis, and metastasis, pi3ka represents an attractive target for development of anticancer drugs. although aminomethylated pyrrolopyrimidine 65 (fig. 11 ) was an efficient inhibitors of pi3ka (ic 50 ¼ 20 nm) and showed good selectivity for pi3ka over mtor (170-fold) , this compound exhibited low cytotoxicity towards pc3 cancer cells [89] . in addition, all the other analogues of mannich base 65 were found to be even weaker inhibitors of pi3ka than the lead compound. isatin is nowadays a well recognized and privileged scaffold in the design of cytotoxic and anticancer compounds [90] . several isatin-containing substrates, namely isatin and its 5-halogenated analogues, the corresponding imine derivatives obtained from sulfadiazine, sulfadoxine and trimethoprim, and a hydrazone derived from isoniazid, were aminomethylated using gatifloxacin as amine reagent [91] . the resulting mannich bases were tested against nci's standard panel of 60 cell lines using srb assay, and compound 66 (fig. 12 ) emerged as an efficient anticancer agent that was generally more potent than reference drug etoposide against most cell lines in the panel. another library of mannich bases derived from isatin, 4-halogenated isatins and their schiff bases with 2-amino-6-methylbenzothiazole was evaluated for cytotoxic effects on three breast cancer cell lines (mda-mb468, mdamb231 and mcf7) using srb assay [92] . the introduction of a halogen as substituent at position 4 of isatin mannich bases led to an increase in cytotoxicity, and modification of isatins into schiff bases followed by aminomethylation resulted in mannich bases further enhanced the cytotoxicity of these candidates. compounds 67 and 68 ( fig. 12) were the most potent in each series (ic 50 < 20 mm), and more potent than reference drug cisplatin, while their cytotoxicity against normal cells was low. as far as their mechanism of action is concerned, compound 67 induced cell cycle arrest in g2/m phase at concentrations similar to those observed for cell growth inhibition, whereas compound 68 did not [92] . furthermore, another collection of mannich bases of isatin imines generated either from 2aminobenzimidazole or 2-amino-4,5-dihydrothiazole was evaluated against mcf-7 human breast adenocarcinoma cell line using srb assay, but the cytotoxicity of these compounds was moderate (ic 50 > 20 mm) and inferior to that of doxorubicin [93] . generally, aminomethylated schiff bases of isatin derived from 2aminobenzimidazole were more cytotoxic than their counterparts derived from 2-amino-4,5-dihydrothiazole, compound 69 ( fig. 11) being the most potent in this collection (ic 50 ¼ 22.6 mm). cytotoxic mannich bases of isatins were also designed employing hybridization of isatin with a 4-aminoquinoline scaffold to generate the substrate subjected to aminomethylation [94] . the cytotoxicity of these compounds towards breast cancer cell lines mda-mb468 and mcf7 was moderate (ic 50 values between 15 and 65 mm), but the activity improved slightly in the series of the corresponding thiosemicarbazones (ic 50 in the range of 10e55 mm). mannich bases 70 and 71 (fig. 12) were the most potent candidates in every series (2e3-fold more cytotoxic than cisplatin), and they preferentially inhibited the growth of cancer cell over normal cells. studies using flow cytometry also suggest that these compounds induce cancer cell death by apoptosis. beside isatin derivatives, several other classes of nh-azoles have been aminomethylated with a view to synthesize cytotoxic mannich bases. 2,3-dihydro-1,3,4-oxadiazole-2-thiones appear to be the preferred substrate within this category, most likely owing to their straightforward preparation. starting from methyl salicylate, good yields of the corresponding oxadiazolethione mannich bases 72 ( fig. 13) were obtained in three steps [95] . most compounds in this collection arise from primary aromatic amines diversely substituted in the aromatic ring, whereas mannich bases 72 derived from secondary aliphatic amines are poorly represented. selected candidates from this series have been initially evaluated by nci against a panel consisting of nci-h460 (lung), mcf7 (breast), and sf-268 (glioblastoma) cancer cell lines using srb assay. seven of these thirteen mannich bases 72, most of them having either chlorine or carboxy group as substituent in the aromatic ring of the amine moiety, reduced the growth of nci-h460 cell line to 30% or less, and they were further selected for the standard 60-cell lines panel assay. compounds 72 (r ¼ h, r 1 ¼ 3-clc 6 h 4 or 4-clc 6 h 4 ) presented higher cytotoxicity than reference drugs 5-fu or cyclophosphamide against most cancer cell lines in this panel [95] . the ability of several oxadiazolethione mannich bases 73 featuring variously substituted aromatic ring at position 5 of the oxadiazolethione ring (r 1 ¼ h and r ¼ no 2 , oh, ch 3 , or r ¼ r 1 ¼ cl) to inhibit the growth of tumors in vivo has been also investigated [96] . tumor volume and tumor weight in mice injected with ehrlich ascites carcinoma cells were reduced by 52e74% at a dose of 50 mg candidates 73 (fig. 13 ) per kg body weight, whereas similar dose of reference drug 5-fu inhibited tumor formation by 93%. mannich bases 73, especially those having hydroxyl, methyl or chloro substituents on the phenyl ring at position 5, were the most potent. also, the counts of red blood cells and leukocytes, as well as hemoglobin levels, have been restored almost to the normal values in mice treated with mannich bases 73 [96] . aminomethylated oxadiazolethiones 74 (fig. 13 ) were generally more cytotoxic against colon carcinoma (ht29) and less cytotoxic against breast cancer (mcf7) cells using srb assay, but the most potent candidate 74 (nr 2 ¼ nhc 6 h 4 ch 3 -4) was 5-fold less cytotoxic than reference drug doxycycline [97] . cytotoxicity of a series of mannich bases 75 (fig. 13 ) of a norharmaneoxadiazolethione structural hybrids was evaluated against a panel comprising melanoma (uacc-62), breast (mcf7), ovarian resistant (nci/adr), renal (786-0), lung (nci-460), prostate (pco-3), ovarian (ovcar) and colon (ht-29) cell lines using srb assay [98] . several of candidates 75 (r ¼ h or n(ch 3 ) 2 , r 1 ¼ isopropylamino or benzylamino) exhibited a broad spectrum cytotoxic activity, and aminomethylation of the parent oxadiazolethiones significantly enhanced the cytotoxicity of each resulting mannich bases 75 compared to that of the corresponding substrate. furthermore, mannich bases 76 of a fluoroquinoloneeoxadiazolethione hybrid were prepared using either secondary aliphatic amines or substituted arylamines as amine reagents [99] . the in vitro evaluation of cytotoxicity against hep3b cancer cells using mtt assay showed that compounds 76 ( fig. 13 ) were more potent than the parent fluoroquinolone pefloxacin. also, mannich bases in this series derived from aliphatic amines were generally more cytotoxic than those derived from arylamines, and candidate 76 (r ¼ n(ch 3 ) 2 ) was even more potent than reference drug bisantrene. 2,3-dihydro-1,2,4-triazole-3-thiones could also act as substrates for the preparation of cytotoxic n-mannich bases. schiff bases obtained from 5-aryloxymethyl-4-amino-3-mercapto-1,2,4-triazoles and 3(5)-substituted pyrazole-4-carboxaldehydes were aminomethylated using either morpholine or diphenylamine to afford mannich bases 77 (fig. 13) , whose cytotoxicity against hepg2 cell line was evaluated using mtt assay [100] . out of five tested candidates, mannich bases 77 having a morpholine moiety were more potent than those with a diphenylamine moiety, but they were still 2e3-fold less cytotoxic than reference drug doxorubicin. eleven dimethylamine mannich bases 78, that were obtained through aminomethylation of schiff bases derived from a 4-amino-1,2,4triazole-3-thione having at position 5 a moiety originating from fluoroquinolone ofloxacin, were screened for cytotoxicity against murine leukemia cell line (l1210) and human leukocytoma cell line (hl60) [101] . mannich bases 78 (fig. 13) were generally more cytotoxic than the corresponding parent schiff bases, and candidates 78 with a hydroxyl group in the aromatic ring of the azomethine function (r ¼ oh) were the most cytotoxic compounds in the series (ic 50 values in the range of 0.14e0.83 mm). mannich bases of thiazolidinone derivatives have also been investigated as cytotoxic agents. c-aminomethylation of two 4thiazolidinones using secondary aliphatic amines afforded mannich bases 79 (x ¼ o, ch 2 ) (fig. 14) , which were evaluated against colon (hct116) and breast (t47d) cancer cell lines by srb assay, but their cytotoxicity was generally moderate to low (ic 50 values between 13 and 50 mm) [102] . in addition, n-aminomethylation of two thiazolidine-2,4-diones (r ¼ cl, och 3 ) with morpholine, piperidine and variously 1-substituted piperazines yielded mannich bases 80 (fig. 14) , which were investigated at nci against the standard 60-cell lines panel using srb assay, and proved to be virtually inactive (growth inhibition for the most sensitive cell line between 18 and 33%) [103] . several examples of p-mannich bases derived from organic esters of phosphorous acid that were disclosed as cytotoxic agents are available in the literature. thus, a-aminophosphonates 81 were synthesized from alkyl phosphites (r 3 ¼ ch 3 , c 2 h 5 , n-c 3 h 7 , i-c 3 h 7 , n-c 4 h 9 ), fluorine-substituted benzaldehydes and 2aminobenzothiazoles, and their cytotoxicity was evaluated against pc3 (prostate), a375 (melanoma), a431 (epidermoid carcinoma), and bcap-37 (breast) cancer cells using mtt assay [104] . most mannich bases 81 ( fig. 15 ) exhibited low to moderate growth inhibition of a375 and bcap-37 cells, but their cytotoxicity towards pc3 and a431 cells was generally greater. the nature of the fluorine substituent appears to influence cytotoxicity, as candidates 81 having fluorine directly attached to the aromatic ring are more potent than those with a trifluoromethyl substituent. an improvement of cytotoxicity with the increase of the length of the alkyl residue r 3 from the initial phosphite was also noted. p-mannich bases 82 (r 1 ¼ ch 3 , c 2 h 5 , i-c 3 h 7 ) (fig. 15 ) were prepared using thieno [3,2-c] pyridine-2-carboxaldehyde as aldehyde component in the mannich reaction of dialkyl phosphites with variously substituted arylamines [105] . the majority of candidates 82 showed good cytotoxicity against esophageal cancer cells (ec109), but they were generally more potent against hepatocellular liver carcinoma cells (hepg2) at a concentration of 50 mg/ml. a few aaminophosphonates 83 (fig. 15 ) were obtained through a mannichtype process from diphenyl phosphite, 3-acetylpyridine and variously substituted anilines, and proved to be cytotoxic to hepg liver carcinoma cell line (ic 50~1 5 mm) and mcf7 breast adenocarcinoma cell line (ic 50~2 0 mm) using mtt assay [106] . although these candidates were approximately 7-fold less cytotoxic than reference drug doxorubicin, their ld 50 values were greater than the corresponding ic 50 values, making them safe to use. aminomethylation of diethyl phosphite with either aromatic aldehydes and aromatic diamines or terephthaldehyde and various amines led to bis(aaminophosphonates) 84 or 85 (fig. 15 ), respectively, whose cytotoxicity against jurkat (t-cell lymphoma), raji (burkit's lymphoma) and mcf-7 (breast cancer) cell lines was determined using mtt assay [107] . in this structurally diverse series of compounds, a few were devoid of cytotoxicity while most of them were moderately cytotoxic. compound 85, derived from tryptamine as amine reagent in the mannich reaction, emerged as the most potent in this series, its cytotoxicity being comparable to that of doxorubicin. furan belongs to the category of electron-rich heterocycles known to undergo electrophilic substitutions such as the mannich reaction with great ease. for example, aminomethylation of synthetic lactone of natural furanditerpene 6a,7b-dihydroxyvouacapan-17b-oic acid as substrate and using various secondary aliphatic amines led to the furan mannich bases 86 (fig. 16 ) [108] . antiproliferative effect of these compounds was more potent than that of the parent lactone against a panel of nine cancer cell lines (melanoma (uacc-62), breast (mcf7), ovarian expressing the resistance phenotype for adryamycin (nci-adr/res), kidney (786-0), lung, non-small cells (nci-h460), prostate (pc3), ovarian (ovcar-03), colon (ht-29), and k562 erythromyeloblastoid leukemia), as determined by srb assay. mannich bases 86 were also equipotent to reference drug doxorubicin against at least one, if not many, of these cancer cell lines, often with ic 50 values as low as 1 mg/ml. in addition, 1,2,4-triazolo[1,5-a]pyrimidine-7-amines having at position 2 a side chain capped with a furan ring were aminomethylated with various secondary aliphatic amines to yield a large series of furan mannich bases 87 (fig. 16 ) [109, 110] . cytotoxicity of compounds 87 against liver cancer (bel-7402) and fibro sarcoma (ht-1080) cells was established using mtt assay, and the results suggest that both the substitution of the arylamine moiety and the nature of the aliphatic amino residue in the aminomethyl function have a significant influence on the potency of these candidates. in particular, the presence of a 4-trifluoromethyl group or a 4-fluoro-3-trifluoromethyl substitution pattern in the arylamine moiety led to high cytotoxicity against both cell lines at levels comparable to that of reference drug cisplatin. also, the presence of dimethylamino, 1-piperidinyl or 1-pyrrolidinyl moieties as aliphatic amino residues in the aminomethyl group of mannich bases 87 appears to result in significant antiproliferative effects, while 4-morpholinyl or 4-methylpiperazinyl moieties drastically decrease or even abolish cytotoxicity. titanium-based chemical entities enjoy the reputation of having a tremendous potential against solid tumors. recently, a number of studies presented the synthesis and the cytotoxicity for a series of titanocenes, some of them featuring various aminomethylated fivemembered heterocycles as substituent of either one or both cyclopentadiene moieties [111e114]. thus, titanocenes 88 and 89 ( fig. 17) containing mono-and bis-aminomethylated pyrroles, respectively, or titanocenes 90 and 91 (fig. 17 ) having a dimethylaminomethyl group at position 1 and 3 of indole, respectively, as well as titanocene 92 (fig. 17 ) presenting an aminomethylated imidazole ring, have been screened against pig kidney epithelial cells (llc-pk1) or human renal cancer cells (caki-1) and found to have ic 50 values quite similar to that of cisplatin (in the range of 5e10 mm). the presence of at least one aminomethyl function in their structure is claimed to be crucial for the high cytotoxicity of these titanocenes. the aminomethyl groups are able to coordinate the titanium center, and could therefore stabilize the mono-or dication formed through hydrolysis of either one or both chlorine atoms inside the cell. this results in enhanced interactions between titanocene and dna, leading to cell death at low concentrations. aminomethylation of terminal alkynes has also been employed for the generation of mannich bases with potential cytotoxic effect. 10-(prop-2-ynil)phenothiazines underwent aminomethylation with secondary aliphatic amines to give propargylamines 93 (r 1 ¼ h, cl, cf 3 ) (fig. 18 ), which were first evaluated for cytotoxic activity using two hematological tumor cell lines, namely hl60 (promyelocytic leukemia) and the ccrf/cem (lymphocytic leukemia), and were afterwards tested, in combination with doxorubicin, for ability to revert activity in the corresponding multidrug resistant variants, hl60r and cem/vbl300 [115] . although most compounds 93 were devoid of significant antiproliferative effect on the sensitive cell lines, a few of them were highly cytotoxic for the resistant cell lines, and appear to arrest cells in g1 phase of the cell cycle, unlike classic anticancer agents. furthermore, several mannich bases 93 were able to restore sensitivity to doxorubicin of the resistant cell lines, an effect that was concentration-dependent and reached maximum at 10 mm. propargylamines 93 seem to induce apoptosis by activating the caspase cascade, although neither the extrinsic nor the intrinsic pathways appear to be involved in apoptosis [115] . betulin derivatives bearing an ethynyl function have also served as starting materials for acetylenic mannich bases with cytotoxic potential. for example, propargylamines 94 ( fig. 18 ) have been obtained from alkynes prepared through addition of an organometallic derivative of acetylene to the carbonyl function in betulonic acid esters [116] , and alkynes synthesized from betulin through a sequence comprising the oxidation of the primary alcohol function to aldehyde, followed by addition of an organometallic derivative of acetylene to aldehyde carbonyl, afforded propargylamines 95 (fig. 18 ) [117] . cytotoxicity of these mannich bases was evaluated on a panel of nine human cancer cell lines using srb assay, and the results prove that some of these compounds show considerable toxicity (ic 50 values as low as 4 mm). introduction of the aminomethyl group significantly improved the cytotoxicity of propargylamines 94 and 95 compared to that of the parent alkynes, presumably by enhancing their solubility and bioavailability. highly hydrophobic and sterically hindered amino moieties, such as dicyclohexylamino or dibenzylamino, led to a decrease in cytotoxicity of the corresponding aminomethylated alkynes. mannich bases of this type were shown to act by triggering apoptosis, although a complementary process of autophagy could also be involved. in an attempt to circumvent the resistance mechanism developed by cancer cells after prolonged administration of doxorubicin and address the issues of poor solubility, short lifetime and high toxicity of prodrug doxoform [118] , a second-generation, watersoluble prodrug of doxorubicin was developed by conjugation of the active drug with salicylamide by means of a mannich reaction [119] . doxorubicinesalicylamide conjugate doxaliform 96 (fig. 19 ) has a half-life of approximately one hour, and was more cytotoxic than doxorubicin against mcf-7 sensitive (4-fold) and mcf-7/adr resistant (10-fold) breast cancer cells. furthermore, doxaliform is amenable to functionalization with a view to provide a site for attachment of a releasable targeting group that might direct the conjugate to a specific receptor that is overexpressed by cancer cells. because many breast cancer cells overexpress estrogen receptor a, this receptor was chosen for targeting by doxasaliform having tethered a hydroxytamoxifen moiety, as in prototype 97 ( fig. 19 ) [120] . cytotoxicity of these candidates as a function of the length of the tether showed that a triethylene glycol unit provides a lead compound whose growth inhibition of four selected breast cancer lines (mcf-7, mcf-7/adr, mda-mb-231 and mda-mb-435) was enhanced up to 140-fold relative to doxorubicin. later work confirmed that uptake of hydroxytamoxifen-targeted doxorubicinesalicylamide conjugate is mediated by both the antiestrogen binding site and estrogen receptor [121] . also, several doxorubicineformaldehyde conjugates tethered to the nonsteroidal antiandrogen cyanonilutamide were designed, synthesized and evaluated as androgen receptor-targeted ligands for specific delivery of the conjugate to prostate cancer cells [122] . such a construct was later used in studies intended to evidence binding to androgen receptor in live pc3 prostate cancer cells and the subsequent translocation of the construct bound to the receptor to the nucleus, but the results were not very promising [123] . other efforts [124] were directed towards the conjugates of doxasaliform with the cyclic peptide neme-vrgdf (known as cilengitide), which is a potent antagonist of a v b 3 integrin involved in many cell-matrix recognition and cell adhesion phenomena, and plays an important role in angiogenesis and tumor metastasis. although the complete construct maintained a high affinity for a v b 3 integrin, the ic 50 for growth inhibition of mda-mb-435 cells was 2-fold greater than that of doxasaliform; the poor results have been tentatively blamed on limitation of drug delivery caused by the specific reduced abundance of receptors in this type of cell. eventually, because doxasaliform is not as active as prodrugs doxoform or doxazolidine, this line of research was terminated. however, the topic was later revisited by other authors, who reported the synthesis of constructs derived from either doxorubicinesalicylamide, daunorubicinesalicylamide or their 2-acyloxymethyl derivative, and amino-terminated poly(ethylene glycol), and their use for in vitro and in vivo studies [125] . these constructs presented cytotoxicities comparable to those of the parent drugs, and the lifetime of one of these constructs was determined to be longer than that of doxorubicin. also, the construct was more efficient than doxorubicin at reducing the weight of s-180 xenografted tumors [125] . cytotoxic mannich bases derived from miscellaneous, structurally unrelated substrates are grouped in the last paragraph of this section. 2-aminomethylated 9-alkyl-1,2,3,4-tetrahydrocarbazole1-ones 98 (fig. 20) show moderate to potent cytotoxicity towards a549 (human lung adenocarcinoma), sgc (human gastric cancer), k562 (human myelogenous leukemia), hct116 (human colorectal carcinoma), and kb-vcr (human oral cancer) cells using mtt assay [126] . one of candidates 98 (r ¼ c 2 h 5 , r 1 ¼ ch 3 ) was more cytotoxic than reference drug taxol against a549 cell line (ic 50 ¼ 70 nm), and at least one of its mechanism of action appears to be the inhibition of tubulin polymerization. a series of 3aminomethyl imidazo[2,1-b]benzothiazoles 99 (fig. 20) were evaluated for antiproliferative activity against hepatocellular carcinoma (hepg2), human breast (mcf-7) and human cervical (hela) cancer cell lines using mtt assay [127] . mannich bases 99 inhibited the proliferation of cancer cells at concentrations lower than 10 mm, arrested the cell cycle at g2/m phase while downregulating cyclin b and upregulating chk2 protein, and appeared to induce apoptosis based on the elevated levels of caspase-3. 6-cinnamoyl-benzoxazol-2-ones (r 1 ¼ h, ch 3 o) were the substrates used for the synthesis of mannich bases 100 (fig. 20) , which had high to moderate cytotoxicity (ic 50 between 5 and 40 mm) towards human pre-b-cell leukemia cell line bv-173 and chronic myeloid leukemia k-562, and appear to exert their cytotoxic action at least in part through induction of apoptosis [128] . replacement of the methoxy substituent in compounds 100 with chlorine led to a marginal improvement of ic 50 values [129] . a series of mannich bases 101 (r ¼ h, ch 3 ; x ¼ ch 2 , o, n-r 1 ) (fig. 20 ) of fused pyridazinone derivatives were synthesized and tested in vitro using srb assay to determine their growth inhibitory properties at a single 10 mm dose against sixty different human tumor cell lines. most of them showed no activity of all, but two candidates moderately inhibited the growth of non-small cell lung cancer (ekvx and hop-92) and glioblastoma (snb-75) cell lines [130] . an impressive number of articles dealing with the antibacterial activity of mannich bases have been published in the last decade. from a structural point of view, mannich bases reported in these studies are derived from nearly all of the major types of substrates capable of undergoing aminomethylation. these mannich bases have been evaluated against both gram-positive and gramnegative bacteria belonging to various families. the evaluation of the antibacterial activity was performed using different screening methods, and not always the standardized versions, and this complicates the interpretation and the comparison of results obtained in different studies. owing to the particular relevance of tuberculosis, the reports concerning the screening of mannich bases against mycobacterium species are presented in a separate section. ketonic mannich bases with antibacterial potential are at the heart of several studies within the period of time covered by this review. aminomethylation of a,b-unsaturated cyclic ketones with variable ring sizes afforded a library of compounds 102 (n ¼ 1e4) (fig. 21 ) derived from secondary cyclic aliphatic amines and having on the aromatic ring either no substituent at all, or a methyl group, or a variable number of methoxy groups [131] . evaluation of the antibacterial activity of these mannich bases against a panel of both gram-positive and gram-negative bacteria using the serial dilution method showed that the candidates derived from seven-or eightmembered a,b-unsaturated cyclic ketones were the least active of all, and that the nature of the amine moiety, or the number and the position of the methoxy groups on the aromatic ring, did not influence the antibacterial activity. none of the compounds were active against pseudomonas aeruginosa, and only a few were moderately active against escherichia coli. mannich bases 102 affected gram-positive bacteria (minimum inhibitory concentration (mic) < 12.5 mg/ml in most cases), but the candidates were less potent than reference antibiotics. another set of mannich bases 103 ( fig. 21 ) obtained from benzo-fused cyclic ketones (indanone, 1tetralone, benzosuberone) were tested under the same conditions against an extended panel of gram-positive and gram-negative bacteria [132] . in this collection, both the nature of the amine moiety and the alkoxy substituent r 1 influence the antibacterial activity, to a greater extent for gram-positive bacteria and to a lesser extent for gram-negative bacteria. indanone derivatives were active against both classes of bacteria (mic between 1.56 and 25 mg/ml), whereas tetralone and benzosuberone derivatives were mostly active against gram-positive bacteria (presumably due to the higher outer membrane permeability of these bacteria). also, a statistical comparison between the antibacterial activity of mannich bases 102 and mannich bases 103 showed that the latter were more active than the former [131, 132] . based on oxazolidinone antibacterials linezolid and eperezolid, a small series of mannich bases 104 (r ¼ nhcoch 3 or nhcsch 3 ) (fig. 21) derived from cyclohexanone or benzo-fused cyclic ketones was synthesized and evaluated against three selected gram-positive microorganisms using the standard serial dilution method [133] . all of the compounds having an acetamido group had low antibacterial activity, but the replacement of this group with thioacetamido restored the activity up to the level exhibited by the parent oxazolidinone antibacterial. further modification (r ¼ nhcsnh 2 ) produced a candidate whose antibacterial activity was superior to that of linezolid. 4,6-dimethoxybenzofuran-3(2h)-one double mannich bases 105 (fig. 21 ) had moderate to high antibacterial activity, as determined using disc diffusion method at a concentration of 0.5% in dmso [134] . the majority of compounds 105 were active against e. coli, and those derived from pyrrolidine, diethylamine and di-nbutylamine as amine reagents showed significant antibacterial activity (zone of inhibition 17e22 mm) towards most bacteria, comparable to that of reference drug gentamycin (zone of inhibition 18e24 mm). on the other hand, compound 105 derived from n-ethylmethylamine was inactive (no inhibition at concentrations higher than 100 mg/ml). also, structurally diverse types of mannich bases derived from acetophenones or 2-acetylthiophene, such as mono-mannich bases of type 8 (r ¼ ch 3 ) (fig. 3) , bis-mannich bases of type 13 (r ¼ ch 3 ) (fig. 4) , and piperidinols of type 14 (r ¼ ch 3 ) (fig. 4) , as well as their azine derivatives 16 (fig. 4) , are devoid of antibacterial activity against a wide range of human-and plant-pathogenic bacteria [135] . methiodide 106 (fig. 21 ) of dimethylamine mannich base of acetophenone was the sole active compound in this series, and only against staphylococcus aureus, but the diameter of the inhibition zone measured 9 mm (compared to 25 cm in the case of reference drug ofloxacin), and its mic was 500 mg/ml. the antibacterial activity of ketonic mannich bases generated from arylamines and aromatic aldehydes was also investigated by several groups. starting from cyclohexanone, a set of eleven compounds 107 was obtained, and screening against four common bacteria using serial dilution method showed that many of these candidates have excellent antibacterial activity, comparable to that of ceftriaxone [136] . however, the lack of rational design and systematic use of the aldehyde and amine components used to generate mannich bases 107 ( fig. 21 ) preclude any logical conclusions regarding sar in this collection. use of four differently substituted acetophenones, 4-fluorobenzaldehyde and several para-substituted anilines led to mannich bases 108 (fig. 21) , which were screened against four common bacteria [137] . most of these compounds were inactive, but candidate 108 (r 1 ¼ 2-br, r 2 ¼ f) was more potent than reference drug ampicillin against all four microorganisms, whereas another candidate 108 (r 1 ¼ 2-br, r 2 ¼ h) was more potent than ampicillin against e. coli and s. aureus. arylamine mannich bases 109 and 110 ( fig. 21 ) were obtained from 3-acetylcoumarine and 2-acetylbenzofuran, respectively, and evaluated against e. coli, s. aureus and p. aeruginosa using disc diffusion method [138] . since most compounds in this series are derivatives of 4-aminobenzoic acid, it has been expected that at least a few of these compounds have good antibacterial activity. three candidates 109 (r ¼ h, cl or f) had antibacterial activity comparable to that of reference drug streptomycin, but the rest were almost inactive. the series of candidates 110 provided only one remarkably active mannich base (r ¼ n(ch 3 ) 2 ), but the rest of the compounds with a benzofuran moiety are generally more active than coumarine-derived candidates 109. 3-(arylamino)-1-ferrocenyl-1-propanones 111 ( fig. 21) showed broad spectrum activity against both gram-positive and gram-negative bacteria, the highest degree of growth inhibition being obtained for s. aureus [139] . mic values varied between 0.2 and 12.5 mg/ml, making these compounds approximately 100-fold less active than reference drug tetracycline. because compounds 112 ( fig. 21) with a similar structure but with a phenyl ring instead of ferrocene have been reported to possess moderate to high antibacterial activity [140] , it appears that the replacement of phenyl in 112 with ferrocene could be responsible for the substantial decrease in activity recorded in the case of compounds 111. aminomethylated phenols have also been reported to have antibacterial activity. mannich bases 113 ( fig. 22 ) obtained from 4t-butylcatechol and secondary aliphatic amines have been screened for antibacterial activity against seven types of bacteria, but their potency was rather low to moderate [141] ; their corresponding copper(ii) complexes fared better, as two of them had mics comparable to that of chloramphenicol (12.5 mg/ml). the mannich reaction of a derivative of another dihydroxylic phenol with either secondary aliphatic amines or primary arylamines afforded compounds 114 (fig. 22 ), whose zones of inhibition for three common bacteria, determined at two concentrations (100 and 200 mg/ml) using disc diffusion method, were smaller than those observed for reference drug ofloxacin at 20 mg/ml [142] . because chemical modification of natural antibiotic novobiocin by means of aminomethylation was hypothesized to increase permeability through the outer membrane of gram-negative bacteria, phenolic mannich bases 115 (fig. 22) were designed, synthesized and evaluated against s. aureus (one of novobiocin's usual targets), francisella tularensis and e. coli [143] . candidates 115 were significantly less potent than novobiocin against these three microorganisms, the antibacterial activity of the most potent of these compounds being almost 100 times weaker than that of novobiocin. thirteen novel phenolic mannich bases 116 (fig. 22 ) derived from lawsone, benzaldehydes and primary aliphatic amines, together with their corresponding copper(ii) complexes, were tested for antibacterial activity against seven types of bacteria [144] . with a few exceptions, mannich bases 116 were generally more potent than the corresponding complexes, presumably due to the greater solubility of the pro-ligands. two mannich bases 116 had antibacterial activity comparable or higher to that of chloramphenicol against bacillus subtilis, e. coli and s. aureus, whereas the other compounds inhibited bacterial growth at concentrations greater than 200 mmol/l. antibacterial activity of fused oxazines obtained through the mannich reaction of phenols and naphthols with primary amines has also been examined. 6-acetyl-1,3-benzoxazines 117 ( fig. 22) were screened at 50 mg/ml against three common microorganisms using disc diffusion method, and they showed good growth inhibitory activity towards s. aureus, but only moderate potency against e. coli and b. subtilis [145] . the antibacterial activity was evaluated for 1,3-benzoxazines 118 ( fig. 22 ) substituted with chloro and methyl groups in the aromatic ring, bis(1,3-benzoxazines) 119 ( fig. 22) , naphtho[1,2-e]-1,3-oxazines 120 (fig. 22) , as well as naphtho[2,1-e]-1,3-oxazines 121 (fig. 22 ) using serial dilution method [146] . regardless of the aliphatic or aromatic nature of the moiety at the nitrogen atom, most compounds, and especially naphthoxazines 120 and 121, had mic values greater than 50 mg/ ml, but two benzoxazines 118 derived from 4-chlorophenol and aromatic amines were more potent than ampicillin against e. coli, whereas compound 119 derived from 2,4-dichlorophenol was equipotent to gentamycin against s. aureus. in addition, benzoxazines 118 and naphthoxazines 120 and 121 having benzazole moieties at the nitrogen atom were tested against four common types of bacteria [147] . although the majority of the compounds were inactive, the antibacterial activity of few candidates was 2-folde15-fold greater than that of reference drug tetracycline; the enhancement of activity for these candidates has been associated with the presence of a benzimidazole system at the nitrogen atom and at least one halogen in the phenolic starting material. in contradiction to the previously mentioned results, a number of naphtho[2,1-e]-1,3-oxazines 121 obtained from variously substituted arylamines were found to be quite active as antibacterial agents towards e. coli and b. subtilis [148] . these compounds were generally more active against the latter pathogen, and the candidates derived from 4-fluoroaniline, 4-ethoxyaniline and 2,4,6tribromoaniline were even more potent than reference drug streptomycin. the mannich reaction of phenols fused with heterocycles has also been investigated for the preparation of antibacterials. mannich bases of 8-hydroxyquinoline have been quaternized at the heterocyclic nitrogen atom with bromoacetic acid esters of superior alcohols (r ¼ n-c 12 h 25 , n-c 16 h 33 , n-c 18 h 37 ) to give compounds 122 (x ¼ o, ch 2 ) (fig. 22 ) [149] . the antibacterial activity of candidates 122, determined for three concentrations (1, 2.5 and 5 mg/ml) using disc diffusion method, showed that the compounds were active towards both gram-positive and gram-negative bacteria, but the presence in their structure of long alkyl chains rather than the aminomethyl function is most likely responsible for their activity. phenolic mannich bases 123 (r 1 ¼ h, br) ( fig. 22) were evaluated against e. coli and bacillus cirroflagellosus at 1 mg/ml using disc diffusion method, but their antibacterial activity was generally weak, with the exception of candidates derived from morpholine as amine reagent [150] . aminomethylated derivatives 41 (r ¼ oh, fig. 7 ) also manifested antibacterial activity in various degrees; the most active candidates were the mannich base derived from cyclohexylamine, which was as potent as tetracycline against b. subtilis (mic 3.9 mg/ml), and the mannich base derived from morpholine, which had the same antibacterial activity as ampicillin against s. aureus (mic 2 mg/ml) [58] . phenolic mannich bases derived from 3-hydroxy-4h-pyran-4ones received special attention as potential antibacterials. allomaltol (5-hydroxy-2-methyl-4h-pyran-4-one) was converted into mannich bases 124 (fig. 23) , whose antibacterial activity against four different gram-positive bacteria was only moderate (mic > 32 mg/ml) [151] . on the other hand, the antibacterial activity of mannich bases 125 ( fig. 21 ) derived from chlorokojic acid was superior to that of analogous 124, and the candidates were shown to possess a broad spectrum, while being more active against standard strains (mics between 1 and 32 mg/ml) than towards clinical, drug-resistant isolates [152] . there is no striking difference between the antibacterial activity of aminomethylated derivatives 125 having 4-benzylpiperazines and those having 4-substituted piperidines as amine moiety. the growth inhibition of grampositive bacteria (with the exception of enterococcus faecalis) occurred at mic values that were lower than those required for the growth inhibition of gram-negative bacteria. mannich base 125 with 1-(4-chlorobenzhydryl)piperazine as amine moiety seems to be best compound in this series, but its activity was consistently lower than that of any reference antibacterial drug. subsequently, a novel series of mannich bases 125 was synthesized, this time using various 4-(substituted aryl)piperazines as amine reagent, but the compounds in this collection were weaker antibacterials than analogous 125 derived from 4-benzylpiperazines [153] . screening of another novel set of compounds 125 with either 4benzylpiperazines of 4-arylpiperazines did not identify any compounds with improved or remarkable antibacterial activity, as the best candidates in this study had mic values between 4 and 16 mg/ ml [154] . however, when piperazinyl-substituted fluoroquinolones were employed as amine reagents in the mannich reaction of 3hydroxy-4h-pyran-4-ones as substrates, the resulting hybrids 126 (r ¼ cl) (fig. 23 ) had a broad spectrum and were highly active against a panel comprising both gram-negative and gram-positive bacteria [155] . generally, aminomethylated derivatives of chlorokojic acid (126, r ¼ cl) were more potent antibacterials than aminomethylated derivatives of kojic acid (126, r ¼ oh). mannich base 126 (r ¼ cl) derived from fluoroquinolone ciprofloxacin was the most potent candidate in the series. replacement of the quinolone scaffold in candidates 126 derived from norfloxacin with a naphthyridone moiety (as in mannich bases 126 derived from enoxacin) resulted in decreased antibacterial activity. among the bacteria used in this study, e. coli and klebsiella pneumoniae were the most sensitive microorganisms to hybrids 126. docking of the most active compound into topoisomerase ii dna-gyrase active site (which is the traditional target of quinolones) showed that mannich base 126 derived from ciprofloxacin binds in a manner similar to ciprofloxacin to the enzyme's active site, and that the 3hydroxypyran-4-one moiety is anchored with additional hydrogen bonding within the binding pocket, thus increasing the stability of the inhibitoreenzyme complex. mannich bases of isatin derivatives have also been investigated as antibacterial agents. aminomethylation of isatin semicarbazone with (hetero)arylamines afforded mannich bases 127 (fig. 24 ), whose antibacterial activity was moderate towards e. coli and good towards s. aureus [156] . in addition, a series of mannich bases 128 (r ¼ secondary aliphatic amines) (fig. 24 ) derived from a hydrazone of isatin incorporating the biologically relevant moieties of 1,3,5triazine, sulfa drugs and azacarbazole had moderate to good antibacterial activity against the previously mentioned microorganisms [157] . hydrazidones obtained from isatin and hydrazides of variously substituted acetic acids have also been aminomethylated with a view to synthesize antibacterial mannich bases. for example, mannich bases 129 24 ) derived from substituted isatins and morpholine, piperidine or 1-methylpiperazine generally showed poor antibacterial activity against the two aforementioned types of bacteria, with the exception of compound 129 (r ¼ 4-methylpiperazinyl, r 2 ¼ 5-br), whose inhibition determined by disc diffusion method was approximately half of that for reference drug gentamycin [158] . another collection of mannich bases 129 (r 1 ¼ 4-or 4,5-substituted 2-ethylphenyloxy) generated from substituted isatins as substrates and morpholine or piperidine as amine reagents exhibited weak to moderate antibacterial activity towards e. coli, s. aureus and shigella flemeri (zone of inhibition 12e20 mm) compared to norfloxacin (zone of inhibition 26e28 mm) [159] . schiff bases generated from isatin and various (hetero)aromatic amines represent another type of isatin-derived substrate that was subjected to aminomethylation to obtain potential antibacterial agents. the use of isatin schiff bases in the mannich reaction with 2-((2,6-dichlorophenyl)amino]) phenylacetic acid as the amine reagent yielded compounds 130 (r 1 ¼ substituted phenyl) (fig. 24) , which proved to be 3e12-fold less potent than reference drug ciprofloxacin against an array of bacteria [160] . when 3-chloro-4-fluoroaniline was used to generate schiff bases from isatin and 5-chloroisatin, aminomethylation with secondary aliphatic amines gave mannich bases 131 (fig. 24) ; these compounds had moderate to good activity against e. coli, s. aureus, p. aeruginosa and salmonella typhi in disc diffusion assay, and showed also moderate to good b-lactamase inhibitory activity [161] . schiff base of 5chloroisatin and trimethoprim was aminomethylated to give compounds 131 (r 1 ¼ 4-amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-2-yl), but only mannich bases 131 derived from ciprofloxacin, lomefloxacin or gatifloxacin as amine reagents in the mannich reaction were at least as potent, if not more potent, than the corresponding parent fluoroquinolones against a large panel of bacteria [162] . antibacterial activity of mannich bases 131 (r 1 ¼ 5benzyl-3-thioxo-2h-1,2,4-triazol-4-yl) derived from schiff bases of isatin or 5-halogen-substituted isatins and an aminotriazolethione was generally good against four common microorganisms, as the size of the inhibition zones for some of the candidates (especially those derived from halogenated isatins and having piperidine or morpholine residues in the aminomethyl group) was comparable to the size of the inhibition zone for reference drug ciprofloxacin [163] . recently, mannich bases 132 ( fig. 24 ) obtained using schiff bases derived from 5-fluoroisatin and 4-arylideneaminoanilines as substrates and ciprofloxacin as amine reagent were shown to be generally less potent antibacterials than reference drug ciprofloxacin, although some candidates had mic values comparable to those of ciprofloxacin against the investigated bacteria [164] . a large number of papers published in the last decade report the antibacterial activity of mannich bases of 2,3-dihydro-1,2,4triazole-3-thiones. from a structural point of view, mannich bases from triazolethiones that were synthesized through aminomethylation in these reports fall into two major categories: 5substituted 2-aminomethyl-2h-1,2,4-triazole-3-thiones 133 and 5-substituted 2-aminomethyl-4-arylideneamino-2h-1,2,4triazole-3-thiones 134. the relevant information from these studies (i.e., substituents at position 4 and 5 of the triazole ring, amine reagents used in aminomethylation, microorganisms employed in the antibacterial evaluation) is presented in a condensed manner in table 1 . in the series of compounds 133a, the nature of the amine moiety in the aminomethyl group appears to be critical for the antibacterial activity, as mannich bases from piperazines were consistently more potent than mannich bases from arylamines, and some of them were even more potent than reference drug ampicillin [165] . a comparison between antibacterial activities of 133a and structurally related 134b suggests that the replacement of substituent at position 4 of the triazole ring in 133a with hydroxybenzylideneamino in 134b further enhances the antibacterial activity. although it was shown that substitution with chlorine of the phenyl at position 5 of the triazole ring improves the antibacterial activity in the series of compounds 133b and 133c, and that aminomethylation also increases the bacterial growth of mannich bases 133c compared to the parent triazolethione, no candidates with substantial antibacterial activity could be singled out from these two collections [166e168]. however, use of ciprofloxacin as amine reagent in the synthesis of mannich bases 133c and 133d led to a substantial increased antibacterial activity of these compounds, and some of them were as potent as reference drugs ciprofloxacin and vancomycin against several types of bacteria and methicillin-resistant s. aureus (mrsa), respectively [169] . mannich bases 133e or 134f having a diphenylsulfone moiety at position 5 of the triazole ring had only weak antibacterial activity [170] . a small number of mannich bases 133f, especially those having a halogen as substituent of the phenyl at n-4 of the triazole ring, inhibited the growth of gram-positive bacteria [171] . mannich bases 133g, especially candidates with morpholine, 4benzylpiperazine, n-methylpiperidine and trifluoromethylphenylpiperazine in the aminomethyl group, had very good antibacterial activity (mic 1.56e12 mg/ml) [172] , whereas analogous 133h having 2-furyl instead of 2-thiophenyl were less potent (inhibition zones of 6e13 mm, compared to 10e35 mm for ampicillin) [173] . the three mannich 133i bases reported in a study gave interesting results, as the candidates derived from 1methylpiperazine (inhibition zones of 23e30 mm for all bacteria) and 2-(4-morpholinyl)ethylamine (inhibition zones of 14e22 mm for some of the microorganisms under evaluation) were more potent than reference drug ampicillin, whereas the bis-mannich base 133i derived from piperazine was inactive [174] . however, further elaboration of 133i into compound 133p abolished the antibacterial activity [174] . mannich bases 133j, which feature a 3pyridinyl moiety at position 5 of the triazole ring, were generally poorer antibacterials than reference drug ampicillin, although there were isolated examples amongst these candidates that show more potency than ampicillin against certain bacteria [175] . furthermore, the replacement of 4-pyridinyl with 2-quinolinyl as substituent at position 5 of the triazole ring, and modification of phenyl into benzyl as substituent at position 4 of the triazole ring led to inactive mannich bases 133k [176] . both types of mannich bases 133l and 134l, featuring a 1,2,4-triazole moiety at position 5 of the triazolethione scaffold, showed good antibacterial activity, but compounds 134l were generally more potent than 133l, and two of candidates 134l actually had mic values comparable to those of reference drug ciprofloxacin [177] . mannich bases 133m and 133n (continued on next page) [165] were devoid of effective antibacterial activity [178, 179] , as was structurally related candidate 133o (a singular and notable exception is this compound's activity against bacillus cereus) [179] . mannich bases 133q were also inactive as antibacterials, except for a few candidates that were more potent against p. aeruginosa than reference drug ampicillin [180] . every member of the series of [197] h primary arylamines diphenylamine piperazines s. aureus s. pyogenes [198] mannich bases 134a was active against the tested bacteria, but not as potent as reference drug nitrofurazone [181] . in addition, the nature of substituent at position 5 of the triazole ring in the series of compounds 134a had no noticeable influence on their antibacterial activity, but the data (based on a single example) suggest that presence of chlorine in the aromatic ring of substituent x of the pyrazole moiety could improve the activity. mannich bases 134c were mostly active against s. aureus and b. subtilis, and candidates derived from ethyl piperidine-4-carboxylate were generally more potent than those derived from piperazines [182] . it should be noted that the presence of halogen in the structure of 134c (r 2 ¼ 2,6-difluorophenyl or 2,6-dichlorophenyl) did not enhance the antibacterial activity of these mannich bases. in contrast, substitution with halogen in compounds 134d (e.g., r 2 ¼ 2,4dichlorophenyl) led to the most potent candidates in the series, and their antibacterial activity was comparable to that of reference [203] drug ciprofloxacin [183] . use of 2,4-dichloro-5-fluorophenyl moiety at position 5 of the triazole ring in mannich bases 134e also produced candidates with good to excellent antibacterial activity [184] . compounds 134e having secondary aliphatic amines in the aminomethyl group were generally more potent than those having arylamines, even if the anilines used in aminomethylation were substituted with additional halogen atoms [184] . although mannich base 134g had no antibacterial activity, a close analogue of 134g such as compound 135 (fig. 25 ) was 1.5e3-fold more potent than ampicillin against most bacteria in the panel, with the exception of s. aureus [185] . replacement of 4-methylpiperazin-1yl with 4-morpholinyl in the aminomethyl group of mannich base 134g, and use of substituted phenyl other than 4-fluorophenyl as r 2 afforded structurally related candidates 134h with moderate antibacterial activity [186] . antibacterial activity of mannich bases 134i was moderate to good, but even the most potent compounds in this series (all of them having a 4-methylpiperazin-1-yl moiety in the aminomethyl group) were at least 4-fold less active than reference drug ciprofloxacin [187] . mannich bases 134j also had moderate to good antibacterial activity (inhibition zones of 12e22 mm), but were less potent than tetracycline (inhibition zones of 25 mm) [188] . overall antibacterial activity was reasonable in the series of mannich bases 134k, while a few compounds had mic values against various bacteria close to those determined for reference drugs [189] . a few mannich bases 134m were more potent against p. aeruginosa than reference drugs levofloxacin and amikacin, while most candidates exhibited broad, moderate to good antibacterial activity [190] . evaluation of mannich bases of 2,3-dihydro-1,2,4-triazole-3ones as antibacterial agents received little attention in recent literature. a small series of six mannich bases 136 (fig. 25 ) derived from 4-(substituted (hetero)arylidendeamino)-5-methyl-1,2,4triazole-2-ones was synthesized and tested against seven types of bacteria, and the antibacterial activity of the candidates ranged from moderate to excellent [191] . all compounds were more potent than reference drug ampicillin against e. coli and p. aeruginosa, and candidate 136 (r 1 ¼ 2-hydroxyphenyl, nr 2 ¼ 2-(4-morpholinyl) ethylamino) was at least as active as ampicillin against all types of bacteria used in the study, with the exception of s. aureus. out of mannich bases of type 137 (fig. 25) , only those derived from morpholine or piperidine were excellent, broad spectrum antibacterials, whereas other analogues were either inactive, or weak antibacterials, or moderately active against the tested bacteria [192] . a single mannich base 138 (fig. 25 ) of 1,2,4-triazole-3-ones having a 2-quinolinyl moiety as substituent at position 5 of the triazole ring was synthesized and evaluated for antibacterial activity, and its effect on e. coli and p. aeruginosa was comparable to that of ampicillin [176] . three mannich bases having a 3-pyridinyl moiety at position 5 of 1,2,4-triazole-3-one scaffold, analogous to compounds 133j derived from 1,2,4-triazole-3-thione, had moderate to good activity against most bacteria in the panel, but were not superior to reference drug ampicillin [175] . antibacterial activity of mannich bases 139 of 2,3-dihydro-1,3,4oxadiazole-2-thiones has been investigated extensively. the structural features of the compounds reported in these studies have been summarized in table 2 , along with the microorganisms used for screening. mannich bases 139a had moderate to good antibacterial activity (mic values between 6.25 and 25 mg/ml), but none was as potent as reference drug ciprofloxacin, and no definite sar could be inferred from the biological results [193] . the growth inhibition of mannich bases 139b appears to be modulated by the nature of the amine in the aminomethyl residue; thus, candidates derived from 3-trifluoromethylaniline were the most potent in this series and had antibacterial activity comparable by that of reference drug ciprofloxacin, followed in order by mannich bases 139b derived from morpholine, 2-chloro-5-methylaniline and 4-chloro-2-trifluoroacetylanline [194] . in the case of mannich bases 139c, candidates derived from secondary aromatic amines were inactive, whereas the remaining compounds were less active against gram negative bacteria, and more active against gram positive bacteria (mic values 2e8-fold lesser than those of reference drug ampicillin) [195] . both mannich bases 139d reported in this study [185] had moderate to good antibacterial activity, and both were more potent than ampicillin against e. coli and e. faecalis, but the candidate derived from 1-methylpiperazine was generally more potent than mannich base derived from 2-(4-morpholinyl)ethylamine. in the case of thiazole-containing mannich bases, antibacterial activity of compounds 139e was moderate (mic values between 16 and 62.5 mg/ml) [187] , whereas compounds two of compounds 139f emerged as effective antibacterials, especially against gramnegative bacteria, with mic values 2e4-fold lesser than those for reference drug chloramphenicol [196] . several mannich bases 139g (nr 2 ¼ nhc 6 h 4 f-4, nhc 6 h 4 cl-4, nhc 6 h 4 cf 3 -2, nhc 6 h 3 f 2 -2,5) had antibacterial activity comparable to that of reference drug penicillin, and, for some bacteria, even comparable to reference drug streptomycin [197] . mannich base 139h (nr 2 ¼ 1-piperazinyl) was equipotent to reference drug ciprofloxacin against s. aureus, but the antibacterial activity of the rest of candidates in this series was weaker than that of ciprofloxacin against the tested bacteria [198] . the compounds reported in a study, including mannich bases 139i, are claimed to exhibit moderate antibacterial activity, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [199] . phenylpiperazine-containing mannich base 139j was generally more potent that candidate 139j derived from ethyl isonipecotate, but both had moderate antibacterial activity compared to reference drug ampicillin [176] . three out of eight synthesized mannich bases 139k were screened for antibacterial activity, but their growth inhibition was only weak to moderate (inhibition zones of 12e26 mm) compared to that of reference drug chloramphenicol (inhibition zones of 37e44 mm) [200] . none of the 21 synthesized mannich bases 139l presented any antibacterial activity at the maximum concentration tested (250 mg/ml) [98] . mannich base 139m was the only compound with antibacterial activity in the structural diverse library evaluated in the study, but its effects were moderate at best [189] . screening results for mannich bases 139n were also disappointing, as they only barely inhibited the growth of e. coli at a concentration of 10 mg/ml [174] . out of two synthesized mannich bases 139o, the candidate having a morpholine residue in the aminomethyl group displayed excellent antimicrobial activity against all the tested microorganisms, while the candidate having a methylpiperazine residue had good or moderate activities against most bacteria in the panel, with the exception of e. coli and k. pneumoniae [190] . the growth inhibition exerted by mannich bases 139p was moderate compared to reference drugs, and it was uninfluenced by the nature of the amine moiety in the aminomethyl group [177] . nalidixic acid-based oxadiazolethione mannich bases 139q exhibited very weak antibacterial activity (mic values > 750 mg/ml) compared to reference drug streptomycin (mic ¼ 5 mg/ml), and only towards b. subtilis, but some of the structurally related mannich bases 140 of 2,3-dihydro-1,3,4-thiadiazole-2-thione ( fig. 25 ) were quite active (mic values as high as 6.25 mg/ml) against the microorganisms used in the study [203] . finally, mannich bases 141 having a 3,4dichlorophenyl moiety at position 5 of 2,3-dihydro-1,3,4oxadiazole-2-one ring (fig. 25) were inactive against s. aureus, e. coli, p. aeruginosa and bacillus megaterium [204] . two studies presented the synthesis and antibacterial evaluation of c-mannich bases 142 of thiazolidinones (fig. 26) having a thiazolyl-2-imino substituent at position 2 [205, 206] . while candidates 142 having alkyl or aryl groups as r 1 substituent at c-5 of thiazolidinone ring showed no antibacterial activity against a panel of eight bacteria, some of their analogues unsubstituted at c-5 (r 1 ¼ h) had weak antibacterial activity against s. aureus (mic values of 20e40 mg/ml). two of mannich bases 143 (r 1 ¼ 2-hoc 6 h 4 ) of thiazolidinones featuring a 6,8-dibromo-4oxoquinazolin-3-yl moiety (fig. 26 ) were 2-fold less potent against s. typhimurium (when nr 2 ¼ diethylamino) and 4-fold less potent against b. cereus (when nr 2 ¼ 1-piperidinyl) than reference drug ciprofloxacin [207] . antibacterial activity of mannich bases 144 (fig. 26 ) was found to be more potent (mic values between 6.25 and 100 mg/ml) than that of parent thiazolidinones, and the sar suggests that the nature of substituent r 1 influences significantly the ability of these compounds to inhibit the growth of various bacteria [208] . aminomethylation of pyrimidine-2-thiones and pyrimidine-2ones, substrates that can be easily obtained in a considerable structural variety through a simple biginelli reaction, has also been investigated in relation with the antibacterial activity of the resulting mannich bases. thus, pyrimidine-2-thione mannich base 145 (fig. 26 ) was obtained using natural alkaloid cytosine as amine reagent in aminomethylation, and its evaluation as antibacterial agent showed that it is a potent growth inhibitor of s. aureus and b. subtilis, and it has weak activity against p. aeruginosa and e. coli [209] . double mannich bases 146 (fig. 26 ) of pyrimidine-2-thiones (x ¼ s) were good antibacterial agents (zones of inhibition of 96 to 88% of the zone of inhibition for reference drug streptomycin) against e. coli and b. subtilis [210] , and analogous double mannich bases 146 of pyrimidine-2-ones (x ¼ o) were even more potent, with the exception of the candidate having a 4-methoxyphenyl moiety at position 4 of the pyrimidine ring (r ¼ 4-och 3 , r 1 ¼ h) [211] . taking advantage of the lability of the proton located at the nitrogen atom in the amide function, several n-mannich bases 147 of 5-chloro-2-methoxybenzamide ( fig. 27 ) obtained from either sulfonamides or common secondary amines as amine reagents have been synthesized and evaluated for antibacterial activity against a panel of bacteria [212] . candidates 147 derived from sulfonamides were more potent than the parent compounds, whereas mannich base 147 derived from morpholine was an antibacterial agent with broad spectrum and good activity. n-mannich bases 148 of glutarimides (r 1 ¼ h, c-c 5 h 9 , c-c 6 h 11 , 4-clc 6 h 4 ) (fig. 27 ) have been screened for antibacterial activity, and claimed to be potent very potent, although no comparison to currently-in-use antibacterials was available [213, 214] . compounds 148 derived from sulfonamides showed improved growth inhibition activity of the tested microorganisms over the corresponding parent amines, whereas analogues generated from secondary aliphatic amines were generally equipotent to those obtained from sulfonamides [213] . also, n-mannich bases 149 of succinimide (r 1 ¼ h or c 6 h 5 , nr 2 ¼ nhc 6 h 5 , 2-pyridinyl) (fig. 27 ) and an n-mannich base from phthalimide were weak to moderate antibacterials against e. coli, s. typhi and b. subtilis [215] . well-known antibiotics have also been subjected to aminomethylation with a hope of increasing their antibacterial activity or improving their bioavailability. hybrids 150 of tetracycline and sulfonamides ( fig. 28 ) have been generated using the mannich reaction, and their screening against salmonella enteritidis and pasturella multocida showed that their antibacterial activity is higher than that of the parent sulfonamides; unfortunately, no comparison with the activity of tetracycline or a reference drug is provided in the study [216] . aminomethylation is one of the modifications that have also been explored in the case of vancomycin. after an n-decylaminoethyl moiety was introduced into the structure of vancomycin with a view to restore antibacterial activity against vancomycin-resistant enterococci while retaining potency against mrsa, grafting a hydrophilic group has been considered as a way to reduce the overall lipophilicity of the n-decylaminoethylmodified vancomycin and to restore the initial favorable distribution properties of vancomycin. several types of amine reagents (amino acids, amino sugars, phosphonic acids) have been considered for the synthesis of mannich bases 151 (fig. 28) , and some of these modifications led to candidates active against vancomycinresistant bacteria, and ultimately to the discovery of telavancin (td-6424) as a lead compound with improved absorption, distribution, metabolism and excretion (adme) properties over ndecylaminoethylvancomycin [217] . (fig. 28 ) was subsequently screened against a large number of predominantly gram-positive isolates with developed antibacterial resistance, and showed to be highly active against methicillin-resistant staphylococci (mic 90 0.5e1 mg/ml), streptococci (mic values 0.12 mg/ml), and vanb-type enterococci (mic values 2 mg/ml) [218] . with respect to its mechanism of action, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion, bound to cell wall with high affinity, and perturbed bacterial cell membrane potential and permeability [219] . aminomethylation of vancomycin has also been used as the first step in the construction of glycopeptide/b-lactam heterodimers via linkage of aminomethylated vancomycin and various cephalosporin cores through an adipic acid moiety [220] . these heterodimers were all found to exhibit excellent potency against a range of important gram-positive bacteria, and a subset of these candidates also demonstrated rapid bactericidal activity against mrsa, whereas two of the most attractive compounds have been shown to exhibit in vivo efficacy 40 fold greater than that of vancomycin. finally, a patent claims a series of mannich bases of vancomycin as compounds suitable for oral delivery and having enhanced antibacterial potency [221] . antibacterial activity of mannich bases derived from miscellaneous substrates is covered in the last paragraph of this section. mannich bases 152 (r ¼ ch 3 , oc 2 h 5 ) (fig. 29 ) derived from either ethyl acetoacetate or diethyl malonate as substrates, variously substituted benzaldehydes as aldehyde components and benzyl carbamate as amine reagent had poor to moderate activity against five types of common bacteria [222] . hydrazidones derived from isoniazid and 2-alkoxybenzaldehydes have been aminomethylated at position ortho to the alkoxy group to give mannich bases 153 (fig. 29) . a few members of this collection having an ethoxy group demonstrated at concentration of 100 mg/ml antibacterial activity superior to that of reference drug amoxicillin at 50 mg/ml [223] , whereas candidates having a propoxy group were less potent [224] . mannich bases 154 derived from 7-methyl-2-(substituted aryl) imidazo[1,2-a]pyridines (fig. 29 ) and having two halogens as substituents in the phenyl ring at position 2 of the fused heterocyclic system were the most potent in this series against e. coli, p. aeruginosa, s. aureus and streptococcus pyogenes [225] . mannich bases 155 derived from an imidazo[2,1-b]-1,3,4-thiadiazole ring system (fig. 29) showed weak activity against gram-positive bacteria and moderate activity against vibrio cholera, but their growth inhibition activity of e. coli was comparable to reference drug norfloxacin [226] . evaluation of the antibacterial activity of mannich bases derived from 2-alkyl-3-hydroxy-pyridine-4(1h)-ones showed that only one candidate 156 (fig. 29 ) had moderate activity against s. enteritidis (16 mg/ml) and s. aureus (16 mg/ml) compared to reference drug ciprofloxacin (1 mg/ml and 0.5 mg/ml, respectively, for the mentioned microorganisms) [227] . mono-mannich bases 157 (r 1 ¼ ch 3 ) and double mannich bases 158 generated from sydnones ( fig. 29) were both found to inhibit moderately the growth of four standard microorganisms, regardless of the nature of the amine reagent used in aminomethylation, but no useful conclusions regarding sar in this series could be drawn for further development [228] . also, most mannich bases 157 (r 1 ¼ och 3 ) had weak to moderate activity against both gram-positive and gramnegative bacteria, with the exception of candidate 157 (r 1 ¼ och 3 , nr 2 ¼ 4-nitrobenzothiazole-2-ylamino), which was as potent as reference drugs ciprofloxacin and ampicillin against gram-positive bacteria [229] . in contrast, aminomethylation of another sydnone derivative using various primary and secondary aliphatic amines was reported to occur in the at n-1 in the pyrazoline ring to give mannich bases 159 instead (fig. 29) , which had good antibacterial activity compared to reference drug ampicillin against four common bacteria, and particularly against b. subtilis [230] . the copper(ii) complex of pyrazole c-mannich base 160 (fig. 29 ) inhibited the growth of b. subtilis at concentrations as low as 1.25 mm, most likely by promoting mutagenesis and inducing cell death [231] . screening of a few of pyrazolone n-mannich bases 161 (fig. 29 ) against various bacteria resulted in inhibition zones that were comparable to those of reference drug ciprofloxacin, but the mic values for these candidates were ultimately far greater from those of ciprofloxacin [232] . some of the acetylenic mannich bases 162 (r 1 ¼ c 6 h 5 , 4-clc 6 h 4 ; r 2 ¼ c 6 h 5 , 1-c 10 h 7 ) (fig. 29 ) had antibacterial activity comparable to reference drug ofloxacin against s. aureus, fewer showed good activity against e. coli, and only one candidate was equipotent to ofloxacin against p. aeruginosa [233] . various substrates (e.g., benzimidazole, 3methylpyrazole-5(4h)-one, succinimide, phthalimide or naphthalimide) were aminomethylated using norfloxacin as amine reagent, and some of the corresponding mannich bases were more potent than the parent fluoroquinolone, especially against e. coli and p. aeruginosa [234] . sulfonamides and primary or secondary aliphatic amines were employed as amine reagents in aminomethylation of isoniazid to give mannich bases 163 (fig. 29) , and some of the candidates had antibacterial activity that was superior to that of the parent sulfonamides, whereas mannich base 163 (nr 2 ¼ nhch 3 ) was the most potent compound in the series against s. aureus and b. anthracis [235] . use of hydantoins as substrates in the mannich reaction led to 3-aminomethylated derivatives 164 or to 1-aminomethylated derivatives 165 (fig. 29) , depending on the substituent on n-3 of imidazole ring, and some of these mannich bases had moderate to good antibacterial activity against four common bacteria [236] . aminomethylation and aminoalkylation of saccharin using various types of amine reagents led to mannich bases 166 ( fig. 29 ) with moderate to excellent antibacterial activity against s. aureus (zones of inhibition between 12 and 50 mm, compared to 31 mm for reference drug chloramphenicol) [237] . a note reports in a concise manner the synthesis and antibacterial activity of mannich bases 167 (fig. 29 ) derived from a phenothiazine and primary aromatic amines, and some of the compounds in this study were potent and had a broad spectrum against the tested bacteria [238] . mono-mannich base 168 of quinoxaline-2,3(1h,4h)-dione (fig. 29 ) was screened at concentration of 100 mg/disc, and was found to be more potent than reference drug nalidixic acid at concentration of 50 mg/disc against four common bacteria, with the exception of proteus vulgaris [239] . moderate to good antibacterial activity was determined for mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)one (fig. 29 ), but none of the candidates was as potent as reference drug amikacin against the four common bacteria used in this study [240] . in addition to their anticancer activity evaluation, p-mannich bases 83 (fig. 15 ) were also screened for antibacterial activity, and the mic values were generally approximately 10 mg/ml, regardless of candidate's structure and type of bacteria, but no comparison with reference drugs was provided in the study [106] . a paper claiming the synthesis of s-mannich bases 170 of 4,6-diaryl-2mercaptopyrimidine and secondary aliphatic amines (fig. 29 ) also reports their antibacterial activity weak to moderate, as the most potent candidates are 3-to 7-fold less potent than reference drug ciprofloxacin against four types of bacteria [241] . tuberculosis is a chronic disease whose magnitude and gravity can be gleaned from a 2010 study by world health organization stating that in 2009 alone, 9.4 million new cases of tuberculosis were reported, and 1.7 million deaths were caused by tuberculosis, out of which 0.38 million people were infected with both human immunodeficiency virus and mycobacterium [242] . continuous rise of the number of reported cases of drug-resistant and latent tuberculosis is also alarming and calls for urgent discovery of novel classes of compounds with antimycobacterial activity capable of overcoming the resistance of mycobacterium strains to current drugs. mannich bases are among these novel classes of antimycobacterial compounds that have recently caught the attention of researchers in the field. several examples of papers dealing with antitubercular ketonic mannich bases are available. thus, dimethylamine mannich bases 171 of 2-benzylidenecyclohexanones ( fig. 30 ) were reported to have mic values between 0.39 mg/ml and more than 12.5 mg/ml against mycobacterium tuberculosis h 37 rv (for candidate 171 (n ¼ 0, r 1 ¼ r 2 ¼ h) and candidate 171 (n ¼ 1, r 1 ¼ f, r 2 ¼ h), respectively), which is approximately 20e25% of the efficiency of reference drug rifampin against this microorganism [243] . in addition, the ability of the same candidates to inhibit the growth of mycobacterium avium at concentration of 12.5 mg/ml ranged from 0 to 87%. however, no significant correlations between the structural patterns of mannich bases 171 (e.g., presence or absence of the cinnamoyl double bond, substitution pattern in the phenyl ring, electronic, hydrophobic or steric properties of the substituents in the aromatic ring) and their antimycobacterial activity could be established. mannich base 171 (n ¼ 0, r 1 ¼ och 3 , r 2 ¼ h) emerged from this series as a viable hit compound, having a mic value of 0.78 mg/ml and an ic 50 value for vero cells of 16.4 mg/ml. this compound presents therefore a greater toxicity towards mycobacterium over normal mammalian cells, which has been tentatively explained by its ability to affect respiration in isolated rat liver mitochondria, and had mic values against several drug-resistant strains of mycobacterium that were similar or identical to the values determined for the h 37 rv strain [244] . isocitrate lyase is an enzyme in the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate, and, together with malate synthase, bypasses the two decarboxylation steps of the tricarboxylic acid cycle [245] . glyoxylate cycle is essential for the survival of pathogens, including m. tuberculosis, inside the host [246] , and isocitrate lyase has been identified only in bacteria, fungi, and plants, but no analogous enzyme has been documented in humans. therefore, isocitrate lyase has been pursued as a promising target for the development of novel antimycobacterial agents [247] . a high-throughput screening of a large library of ketonic mannich bases against this enzyme led to the identification of candidate 172 (fig. 30 ) with a potent inhibitory activity (ic 50 ¼ 53.5 mg/ml), but this compound's activity against m. tuberculosis has not been actually evaluated [248] . phenolic mannich bases 173 ( fig. 30 ) derived from the hydrazone generated from isoniazid and 2 0 -hydroxyacetophenone were screened against m. tuberculosis h 37 rv, and found to inhibit its growth with mic values ranging from 0.56 to 4.61 mm [249] . six compounds in this series were more potent than isoniazid (mic ¼ 2.04 mm), and the most potent candidate 173 (nr 2 ¼ 1piperazinyl) was shown to be equipotent to isoniazid in decreasing bacterial load in lungs of infected mice at a dose of 25 mg/kg. anti-mycobacterium activity of novobiocin mannich bases 115 (fig. 22) was evaluated, and two of these candidates (nr 2 ¼ n-acetoxyacetyl-n-methyl and n-acetaminoacetyl-nmethyl) showed an increased potency over novobiocin, although no comparison to established antimycobacterial drugs was provided [143] . phenolic mannich bases 174 derived from allomaltol and piperazines (fig. 30 ) had good activity against mycobacterium smegmatis, with mic values between 4 and 16 mg/ml [250] . although the growth inhibition of mycobacterium by the candidates in the library was moderate to good (mic values between 4 and 128 mg/ml), mannich bases derived from 1-phenylpiperazine and its analogues substituted with halogens in the aromatic ring were among the most potent compounds in the series. departure from substitution of piperazine in candidates 174 with bulky, aromatic rings directly linked at n-4 generally led to decreased potency against mycobacterium, although some substituents (such as tbutoxycarbonyl, ethoxycarbonyl, 2-hydroxyethyl, cyclohexyl, benzoyl, furfuryl or 2-(dimethylamino)ethyl) appear to be tolerated better than others (e.g., isopropyl, allyl, 2-methoxyethyl, 2ethoxyethyl, phenethyl or benzyl). an extension of this study, aimed at evaluating against m. tuberculosis h 37 rv the library of mannich bases of type 124 (fig. 23 ) obtained by replacement of piperazines in compounds 174 with piperidines, led to valuable insight on the sar for this particular type of compounds [251] . inspection of the sar in the subset of mannich bases 124 suggested that the position of the substituents and, to a lesser extent, the nature of these substituents plays an important role in their antimycobacterial activity, as the most potent candidates in this subset are generally those substituted at position 4 of piperidine ring [251] . replacement of allomaltol with chlorokojic acid as substrate afforded piperazine mannich bases 125 (r ¼ 4-substituted piperazin-1-yl) (fig. 23) , analogous to both 124 and 174 and having comparable antimycobacterial activity (mic values between 4 and many papers reporting the antimycobacterial activity of mannich bases of pyrrole derivatives have been published in the last decade. the intensive research in this field stems from the discovery by italian researchers at universit a "la sapienza" in rome of bm212 175 (fig. 31) as a potent agent, not only against several collection strains and clinical isolates of m. tuberculosis, but also against non-tuberculosis mycobacterial strains and drug-resistant mycobacteria, with mic values that are comparable to those of reference drugs isoniazid and streptomycin [252] . although the quest for a more potent antimycobacterial agent through systematic modification of lead compound bm212 was not successful at first, it provided experimental proofs for the importance of the presence and nature of substituents at positions 1 and 5 of pyrrole ring [253] . subsequently, novel pyrrole mannich bases 176 analogous to bm212 (fig. 31) , derived from 1-methylpiperazine but also from thiomorpholine as amine reagents, and having either chlorine [254] or chlorine and fluorine [255] as substituents of the phenyl rings in positions 1 and 5 of pyrrole, have been synthesized. two candidates 176 (r 1 ¼ f, r 2 ¼ h or r 1 ¼ h, r 2 ¼ f) showed an improved activity (mic values of 0.4 and 0.5 mg/ml) against m. tuberculosis over mannich base 175 (mic ¼ 0.7 mg/ml) [255] . investigation of structureeantimycobacterial activity relationship in analogues of 175 and 176 was further pursued with a view to optimize the structure of these candidates. thus, synthesis of novel mannich bases of pyrrole using iterative introduction of more lipophilic 1-naphthyl or 2-fluoro-or 2-chlorophenyl moieties at either one of positions 1 and 5 in pyrrole ring of these lead compounds [256] , or a recombination through shuffling of any of the substituents employed so far on the phenyl rings at these two positions of the pyrrole ring [257] was undertaken. these studies confirmed the superior antimycobacterial activity of mannich bases containing a thiomorpholinyl moiety compared to the activity of the corresponding analogues with a 4-methylpiperazinyl moiety, evidenced the importance of fluorine substitution (especially at position 2 of the phenyl ring) for antimycobacterial activity, and proved the decrease of activity with the introduction of 1-naphthyl moieties in the structure of lead compounds 175 and 176. unfortunately, no novel candidates, more potent than mannich bases 175 and 176, have been identified during these investigations. however, replacement of one halogen in the structure of lead compound 175 with more lipophilic substituents (methyl, ethyl n-propyl, i-propyl) afforded candidates with improved antimycobacterial activity (mic values between 0.125 and 0.5 mg/ml) over 175 and even over reference drug streptomycin [258, 259] . in order to further increase the lipophilicity, replacement of the methyl group at position 2 of the pyrrole ring with ethyl was also examined, but these novel mannich bases 177 (x ¼ s or nch 3 ) (fig. 31) were generally less active against several m. tuberculosis strains than their methylsubstituted analogues, although their cytotoxicity towards normal cells was lower than that of the methyl-substituted counterparts [260] . further exploration of the relationship between the nature of substituents on phenyl rings at positions 1 and 5 of pyrrole ring and the antimycobacterial activity of pyrrole mannich bases led to the identification of a novel lead compound 178 (fig. 31) with a very high activity toward both m. tuberculosis 103471 and h 37 rv strains (mic ¼ 0.125 mg/ml) [261] . antimycobacterial activity of mannich base 178 was comparable to that of reference drugs streptomycin or rifampin, while the candidate demonstrated low cytotoxicity towards normal cells (selectivity index ic 50 /mic > 1000) [261] . subsequently a series of morpholine derivatives were designed and synthesized with a view to lower the clearance rate in mouse microsomal fractions associated with the presence of thiomorpholine [262] . although the replacement of thiomorpholine with morpholine generally resulted in a decrease in potency, candidate 179 (fig. 31 ) was found to be equipotent to any of the lead compounds, showed improved microsomal clearance and lower cytotoxicity to normal cells, and was ultimately selected for in vivo pharmacokinetic and efficacy studies in an acute murine tuberculosis infection model. mannich base 179 had an efficacious dose that results in a 99% colony-forming unit reduction in the lung of 49 mg/kg, which is within the range of commonly employed tuberculosis drugs. a recent study showed that mutations in the mmpl3 gene in mycobacterium strains are responsible for resistance to bm212, and suggested that products of this gene are cellular targets for bm212, which makes mmpl3, a member of the mmpl (mycobacterial membrane protein, large) family, a new potential druggable target for the treatment of tuberculosis [263] . point mutations in mmpl3 gene that confer resistance to bm212 and other analogous pyrrole mannich bases have been later identified in several mycobacterium mutants [262] . a large number of antimycobacterial pyrrole mannich bases were also employed to obtain a final multiprobe 3-d qsar model, which was shown to offer good predictions for antimycobacterial activity of an external, unrelated test set of compounds [264] . accounts at various stages of the endeavors concerning the synthesis of pyrrole mannich bases as antimycobacterial agents, along with the related biological results obtained by the italian researchers, have also made the object of several author's reviews along the years [265e268]. a patent claiming the antimycobacterial activity of pyrrole mannich bases derived from 1-arylpiperazines is also worth mentioning [269] . on the other hand, attempts to make a more drastic departure from these already established structural features of antimycobacterial pyrrole mannich bases (such as the introduction of an oxazolidinone moiety reminding of antibacterial linezolid) led to a series of compounds 180 (x ¼ acetamido of 4-(hetero)aryl-1,2,3-triazol-1yl) (fig. 31 ) that were at best 15e65-fold less potent than mannich base 178 [270] . in addition, pyrrole mannich bases 181 (fig. 31 ) were shown to act as inhibitors (ic 50 values ranging from 1.5 to 15 mm) of mycobacterium protein tyrosine phosphatase b, an enzyme that is essential for the survival of bacteria in the host, but no in vitro evaluation using mycobacterium strains was performed [271] . mannich bases of isatin and its derivatives have been evaluated as antimycobacterial agents as well. several isatin mannich bases 182 (fig. 32) containing a semicarbazide moiety were equipotent (mic ¼ 0.625 mg/ml) to isoniazid against m. tuberculosis, and two of the candidates were more potent in vitro than either isoniazid or rifampicin against a multidrug-resistant mycobacterium strain (mic ¼ 6.25 mg/ml) [272] . schiff bases obtained from 5-substituted isatins (r 1 ¼ f, cl, f) and lamivudine were aminomethylated using fluoroquinolones (r 2 ¼ ethyl, cyclopropyl; r 3 ¼ h, ch 3 ) as amine reagents, and the resulting mannich bases 183 (fig. 32) showed 92e100% growth inhibition of m. tuberculosis h 37 rv at a concentration of 6.25 mg/ml; inhibition for one selected schiff base was 82%, and lamivudine alone did not inhibit the growth of this mycobacterium strain at the given concentration [273] . several morpholine mannich bases 184 of 4-substituted thiosemicarbazones of 5-trifluoromethoxyisatin (r ¼ c-c 6 h 11 , 4-fc 6 h 4 , 4-clc 6 h 4 , 4-brc 6 h 4 ) (fig. 32) were potent antimycobacterial agents (mic values between 3 and 5 mg/ml) [274] . replacement of the trifluoromethoxy group in 184 with nitro led to a slight decrease in potency, which became more drastic when fluorine replaced the trifluoromethoxy group, whereas the substitution of morpholine with piperidine generally decreased the antimycobacterial activity of the candidates [275] . using fluoroquinolone gatifloxacin as amine reagent, sixteen mannich bases derived from isatins, isatin semicarbazones, isatin thiosemicarbazones, isatin hydrazones with isoniazid, and isatin schiff bases with sulfadiazine were prepared and tested for antimycobacterial activity [276] . mic values recorded for 90% growth inhibition of mycobacterium by these compounds ranged from 0.78 mg/ml to 12.5 ng/ml, whereas mic values between 0.78 and 0.1 mg/ml were determined for multidrug-resistant strains. promising results were obtained when the most potent compound in this series was tested in a murine model at a dose of 50 mg/kg, and the analysis of data for inhibition of m. tuberculosis dna gyrase suggested that this enzyme could be the target of these isatinefluoroquinolones hybrids. good results were obtained from the evaluation as antimycobacterial agents of mannich bases obtained from 5-substituted isatins and their semicarbazones and thiosemicarbazones as substrates and ciprofloxacin as amine reagent (mic values between 1.2 and 10.3 nm) [277] , and mannich bases generated from 5-substituted isatins, the corresponding semicarbazones, thiosemicarbazones and oxime ethers using 8methoxyciprofloxacin as amine reagent had comparable potencies [278] . analogous mannich bases derived from moxifloxacin as amine reagent were also potent (mic value of 1 mg/ml for m. tuberculosis, and between 4 and 16 mg/ml for multidrugresistant mycobacterium strains), but all of these isatinemoxifloxacin conjugates were generally less potent antimycobacterials than parent fluoroquinolone [279] . evaluation as antimycobacterial agents of aminomethylated 2,3dihydro-1,3,4-oxadiazole-2-ones, 2,3-dihydro-1,3,4-oxadiazole-2thiones and 2,3-dihydro-1,2,4-triazole-3-thiones has led to interesting results. a direct comparison between mannich bases 185 (x ¼ o) of 2,3-dihydro-1,3,4-oxadiazole-2-one and mannich bases 185 (x ¼ s) of 2,3-dihydro-1,3,4-oxadiazole-2-thione (fig. 33) , having both a 4-pyridinyl substituent at position 5 of the azole ring and derived from cyclic secondary aliphatic amines, showed that the former were significantly more potent against an isoniazidsensitive m. tuberculosis h 37 rv strain than the latter, thus suggesting that the presence of oxygen is crucial for the antimycobacterial activity of this type of compounds [280] . in a continuation of the initial study, the search for better antimycobacterial agents was restricted to mannich bases of 2,3dihydro-1,3,4-oxadiazole-2-ones, while the variety of amine reagents employed in aminomethylation was expanded to include n-(substituted benzyl)methylamines, a structural modification that preserved the good activity of the candidates (mic values of 4e8 mg/ml) [281] . however, replacement of the 4-pyridinyl substituent at position 5 of mannich bases 185 (x ¼ o) with phenyl, 3pyridinyl of pyrazinyl resulted in a significantly decreased antimycobacterial activity, whereas replacement with 2-pyridinyl did not generally affect the activity of the candidates. bis-mannich bases 186 (fig. 33 ) derived from dapsone as amine reagent, benzaldehyde and furan-2-carboxaldehyde as aldehyde reagents and variously 5-substituted 1,3,4-oxadiazole-2-thiones as substrates had excellent antimycobacterial activities against both isoniazidsensitive and isoniazid-resistant strains of m. tuberculosis, as they were 5-fold more potent than isoniazid against the sensitive strain and 10-fold more potent than isoniazid against the resistant strain [282] . a study allowed another direct comparison, this time between mannich bases 139e of 2,3-dihydro-1,3,4-oxadiazole-2thiones ( table 2) and mannich bases 134i (table 1 ) of 2,3dihydro-1,2,4-triazole-3-thiones having the same 4isopropylthiazole-2-yl substituent at position 5 of the aforementioned azoles. some of candidates 134i were 2-to 4-fold more potent than the most potent mannich base 139e against m. tuberculosis h 37 rv strain, but they were still 16-fold less potent than isoniazid [187] . on the other hand, mannich bases 187 (r 1 ¼ h, oh; r 2 ¼ c 2 h 5 , ch 2 ch]ch 2 , c 6 h 5 , 4-brc 6 h 4 ; r 3 ¼ ch 2 c 6 h 5 , c 6 h 5 , substituted phenyl, 2-pyrimidinyl) derived from 1,2,4-triazole-3thiones (fig. 33 ) had low antimycobacterial activity (mic values between 25 and 100 mg/ml) [283] . the majority of mannich bases derived from either 2,3-dihydro-1,2,4-triazole-3-thione or 2,3dihydro-1,2,4-triazole-3-one having a 3-pyridinyl moiety at position 5 of the triazole ring were more potent antimycobacterials (mic between 16 and 32 mg/ml) than reference drug streptomycin against m. smegmatis [175] . in addition, 2,3-dihydro-1,2,4-triazole-3-one mannich base 137 (nr 2 ¼ 1-piperidinyl) (fig. 25 ) was equipotent to reference drug streptomycin (mic ¼ 4 mg/ml) against m. smegmatis [192] . mannich bases 188 of imidazo[2,1-b]-1,3,4-thiadiazole derivatives ( fig. 34 ) have been screened for antimycobacterial activity. evaluation of a library of mannich bases 188 (r 1 ¼ c-c 6 h 11 , 2-furyl, 2-thienyl; r 2 ¼ h, br; nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4morpholinyl) against m. tuberculosis h 37 rv showed that all of the candidates had mic >6.25 mg/ml [284] , but a second collection of aminomethylated imidazo[2,1-b]-1,3,4-thiadiazole derivatives 188 (r 1 ¼ n-c 3 h 7 , c-c 6 h 11 , 2-thienyl; r 2 ¼ cl, ch 3 , och 3 ; nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4-morpholinyl) consisted of mannich bases with good antimycobacterial activity (52e99% growth inhibition) at a concentration of 6.25 mg/ml [285] . amongst mannich bases 188 (r 1 ¼ 4-ch 3 oc 6 h 4 ch 2 ), pyrrolidine-containing derivatives were consistently the best agents against m. tuberculosis h 37 rv [286] . also, compounds 155 (nr 2 ¼ 1-pyrrolidinyl, 1-piperidinyl, 4morpholinyl) (fig. 29 ) had moderate antimycobacterial activity (mic ¼ 25 mg/ml) [226] . in addition to the types of substrates presented so far, miscellaneous other substrates have been subjected to the mannich reaction with a view to synthesizing compounds with antimycobacterial activity. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases 189 (fig. 34) with excellent activity against m. tuberculosis (mic values between 0.2 and 1.56 mg/ml) [287] . pyrazinamide was also aminomethylated at the amide function using piperazines (including four fluoroquinolones) as amine reagents; the resulting mannich bases 190 (fig. 34) were at least as potent as reference drug pyrazinamide against m. tuberculosis, and showed a significantly improved antimycobacterial activity over pyrazinamide against multidrug-resistant mycobacterium strain [288] . anti-hiv drug efavirenz was aminomethylated using either common piperazines or piperazine-containing fluoroquinolones as amine reagents to afford hybrids 191 (fig. 34) , but only the mannich bases derived from fluoroquinolones had good activity against m. tuberculosis (no comparison with standard antimycobaterial drugs was available though) [289] . investigation of antimycobacterial activity of a small number of indole mannich bases 59 obtained from 1-arylpiperazines (fig. 11 ) uncovered a few candidates that inhibited the growth of m. tuberculosis h 37 rv almost completely at a concentration of 6.25 mg/ml [290] . 159 (fig. 29 ) had good antimycobacterial activity (mic < 5 mg/ml) [230] , while mannich base 168 (fig. 29 ) was moderately active against m. tuberculosis h 37 rv [239] . finally, pyrazolone mannich base 161 (nr 2 ¼ nhcoc 6 h 5 ) (fig. 29 ) was more potent than reference drugs ethambutol and ciprofloxacin, but inferior to isoniazid, against m. tuberculosis h 37 rv [232] . the incidence of fungal infections of any variety has been steadily increasing over the last decades, and it has become one of the main causes of morbidity and mortality, especially in patient with compromised immune systems [293] . development of resistance to currently-in-use antifungal drugs also represents a major concern, and the discovery of novel antifungal agents, preferably outside any of the six major classes that are presently available for treatment, is one of the highest priorities for pharmaceutical industry. the latest advances in the evaluation of mannich bases with various structures as potential antifungals are presented in this section. first reports on the antifungal activity of ketonic mannich bases are relatively recent. a few mono-mannich bases of type 8 (fig. 3) and double mannich bases 12 (fig. 4 ) of acetophenone (r 1 ¼ r 2 ¼ h; nr 2 ¼ dimethylamino, 1-piperidinyl, 4-morpholinyl) have been shown to have weak antifungal activity, but the corresponding methiodides were more potent than reference drug amphotericin b against yeasts saccharomyces cerevisiae and candida krusei, and against dermatophytes trichophyton mentagrophytes, trichophyton rubrum and microsporum canis [294] . thus, quaternization of the nitrogen atom in mannich bases of type 8 appears to be a chemical modification leading to efficient antifungals [135] . various substitution patterns in the phenyl ring in ketonic mannich bases 8 also did not improve significantly the antifungal activity of these compounds [295] . bis-mannich bases 13 (r ¼ c 2 h 5 ) derived from various acetophenones (fig. 4) were only active against aspergillus fumigatus, but the antifungal activity of the related piperidinols 14 (r ¼ c 2 h 5 ) (fig. 4) was broader and slightly more potent compared to that of corresponding mono-mannich bases 8 and bis-mannich bases 13, especially against dermatophytes t. rubrum and m. canis [295] . however, a later study showed that a minor modification such as replacement of ethyl with methyl as substituent at nitrogen increased the antifungal activity of bis-mannich bases 13 (r ¼ ch 3 ), while it decreased the potency of piperidinols 14 (r ¼ ch 3 ) against t. rubrum and m. canis [296] . use of phenethylamine as amine reagent in the mannich reaction with various acetophenones afforded secondary ketonic mono-mannich bases 15 (r ¼ ch 2 ch 2 c 6 h 5 ) (fig. 4) along with piperidinols 14 (r ¼ ch 2 ch 2 c 6 h 5 ), but these compounds had relevant activity (most mic values were either 12.5 or 25 mg/ml) only against m. canis, a dermatophyte whose growth was not inhibited by reference drug nystatin in the concentration range used in this study [297] . most members of a library of mannich bases 10 of enones had antifungal effects 2e16-fold more potent than amphotericin b against at least one (if not many) selected plant fungi [298] . ketonic mannich bases derived from arylamines as amine reagents have also been reported to have antifungal effects. thus, mannich bases 108 (r 1 ¼ 2-br, r 2 ¼ h or f) (fig. 21) were almost twofold more potent than reference drug ampicillin against candida albicans [137] , whereas candidates 109 (r ¼ f or cl) derived from 3-acetylcoumarin and 110 (r ¼ cl or n(ch 3 ) 2 ) derived from 2-acetylbenzofuran (fig. 21) had mic values comparable to those of reference drug fluconazole against aspergillus flavus, chrysosporium keratinophilum, and c. albicans [138] . evaluation of mannich bases 194 (fig. 35 ) derived from cyclic ketones (n ¼ 1e4) showed that these compounds had good anti-candida and anti-saccharomyces activity, but they were less potent against aspergillus strains, and none of the compounds were as potent as reference drug amphotericin b [299] . the anti-candida potency of candidates 194 generally diminished with the increasing size of the ring, while the substitution pattern of the aromatic ring or the nature of the amine in the aminomethyl moiety did not influence the antifungal activity considerably. in addition, the a,bunsaturated ketone system is a structural requirement for the antifungal activity of mannich bases 194, as the amino alcohols obtained through the reduction of the carbonyl group in 194 were mostly inactive towards all the fungi used in this study. inhibition of ergosterol synthesis does not appear to be the mechanism by which mannich bases 194 exert their anti-candida activity, although they are able to influence the development of pseudohyphae and induce noticeable changes in the protein composition in c. albicans [299] . inspection of sar disclosed in the previous study also proved valid for mannich bases 195 derived from cyclic ketones fused with an aromatic ring (fig. 35) , except that the mic values for compounds 195 were consistently better than those for mannich bases 194 [300] . transcript profiling of c. albicans cells treated with candidate 195 (n ¼ 0, nr 2 ¼ 4-morpholinyl) showed that the transcriptional response is typical for oxidative stress and similar to that of a c. albicans cap1 transcriptional activator, which suggests that the ability of mannich bases 195 to directly trigger oxidative stress may be at least in part the reason for their antifungal activity [300] . mannich bases 196 of thiocroman-4-ones (fig. 35) obtained either from secondary aliphatic amines or from primary arylamines had good activity against several types of fungi, and two candidates 196 (nr 1 r 2 ¼ n(ch 3 ) 2 , r 3 ¼ 6-cl, r 4 ¼ 5-f or 8-cl) were more potent than reference drug ketoconazole against the fungi investigated in the study [301] . antifungal activity of double mannich bases 105 (fig. 21) against several fungi was also good, although the potency of compounds 105 against c. albicans was only moderate [134] . several studies reporting the antifungal activity of phenolic mannich bases are available. the hydrazone obtained from 2,4dinitrophenylhydrazine and salicylaldehyde was aminomethylated using various secondary amines to give mannich bases 197 (fig. 36 ) [302] the most potent anti-candida candidates 197 (nr 2 ¼ diphenylamino, 4-morpholinyl, 1-piperazinyl) were 5e10fold weaker than reference drug clotrimazole, whereas the growth of aspergillus niger was inhibited by the most potent compounds at mic values in the range of 1.56e3.12 mg/ml [302] . most aminomethylated 4-t-butylcatechols 113 (fig. 22) , along with their copper(ii) complexes, have significant antifungal activity (radial inhibition of mycelial growth ! 70%) against several plant pathogenic fungi, which is comparable, or even higher in a several cases, to that of reference drugs nystatin and terbinafine [141] . even at a concentration 10-fold greater than that of reference drug voriconazole, only three of mannich bases 114 (nr 1 r 2 ¼ 4methoxyphenylamino, 1-piperidinyl, 4-methylpiperazinyl) (fig. 22 ) barely showed anti-candida activity that was comparable to that of voriconazole [142] . aminomethylation of mulundocandin, an antifungal lipopeptide belonging to echinocandin antifungals, was undertaken with a view to improve its solubility in water; the resulting mixture of mannich bases 198 (r 1 ¼ ch 2 nr 2 , r 2 ¼ h) and double mannich bases 198 (r 1 ¼ r 2 ¼ ch 2 nr 2 ) (fig. 36) was separated before the antifungal activity of the compounds was evaluated in vitro, but only one candidate showed good inhibition against c. albicans and a. fumigatus [303] . nonetheless, in vivo testing of the collection was promising, as many compounds showed anti-candida activity comparable to parent mulundocandin, while a significant improvement in anti-candida activity compared to mulundocandin was observed for mono-mannich base 198 (r 1 ¼ 4-phenylpiperazinylmethyl, r 2 ¼ h), which was equipotent to fluconazole. three members of a collection of 6acetyl-1,3-benzoxazines 117 (r 1 ¼ ch 3 , r ¼ 4-br; r 1 ¼ h, r ¼ 2-och 3 or 4-f) (fig. 22 ) had inhibition zones against a. niger comparable to that of reference drug fluconazole [145] . benzoxazines 118 and 119, as well as naphthoxazines 120 and 121 (fig. 22) were either inactive or had weak activity against candida spp., a. fumigatus and cryptococcus neoformans, but a few compounds in this library had moderate activity against sporothrix schenckii and t. mentagrophytes [146] . in contrast, naphtho[2,1-e]-1,3-oxazine 121 (r ¼ 4-c 2 h 5 oc 6 h 4 ) was more potent than reference drug griseofulvin against c. albicans, whereas naphtho[2,1-e]-1,3oxazine 121 (r ¼ 3-ch 3 oc 6 h 4 ) was equipotent to griseofulvin against a. niger [148] . examples of mannich bases from phenols fused with heterocyclic rings that were investigated for antifungal activity include compounds 123 (fig. 22) , which had only moderate to weak activity against a. niger and penicillium spp. compared to reference drug griseofulvin [150] , and compounds 122 (fig. 22) , which were efficient against aspergillus spp. and c. albicans at concentrations as high as 5 mg/ml [149] . mannich bases 125 of chlorokojic acid (fig. 23) had good anti-candida activity (mic values between 4 and 16 mg/ml) [152e154], but the antifungal activity against dermatophytes was slightly poorer [304] . in contrast, mannich bases 174 (fig. 30) derived from allomaltol and 4substituted piperazines had poor antifungal activity against c. albicans and candida parapsilosis compared to that of reference drug fluconazole, but some members of the library were equipotent to fluconazole against c. krusei [305] . antifungal activity of various mannich bases of isatin derivatives has been examined jointly with their antibacterial activity. mannich bases 127 (fig. 24 ) obtained from isatin semicarbazone as substrate and (hetero)arylamines as amine reagents had good anti-candida activity (75e87% of the activity of reference drug fluconazole) [156] , whereas mannich bases 128 (r ¼ secondary aliphatic amines) (fig. 24 ) had moderate antifungal activity against aspergillus spp., and consistently lower than that of reference drug fluconazole [157] . for the most active mannich bases 129 (r 1 ¼ 4-or 4,5-substituted 2-ethylphenyloxy, r ¼ 1-piperidinyl or 4morpholinyl), the antifungal potency against c. albicans and a. niger was 2/3 of the potency of reference drug griseofulvin [159] . a couple of mannich bases 130 (r 1 ¼ substituted phenyl) (fig. 24) were moderately active against a. niger, but the activity against c. albicans and penicillium notatum was generally poor [160] . mannich bases 131 (r 1 ¼ 5-benzyl-3-thioxo-2h-1,2,4-triazol-4-yl) (fig. 24) were moderately potent against c. albicans and a. niger compared to reference drug fluconazole [163] . finally, in their evaluation against a. niger, mannich base 132 (ar ¼ c 6 h 4 oh-4) (fig. 24 ) was equipotent to reference drug ketoconazole against a, fumigatus, whereas mannich base 132 (ar ¼ c 6 h 4 n(ch 3 ) 2 -4) was twofold more potent than ketoconazole, and three other analogues were equipotent to ketoconazole [164] . evaluation of mannich bases of 2,3-dihydro-1,2,4-triazole-3thiones (table 1) as antifungal agents has been reported in conjunction with their antibacterial activity. in the series of mannich bases 133a, with the exception of candidate 133a (r ¼ c 6 h 4 cl-4, nr 2 ¼ nhc 6 h 3 f 2 -2,6) whose anti-candida activity was 2/3 of that of reference drug clotrimazole, all other compounds were inactive, and so were mannich bases 133b investigated in the same study [165] . compared to reference drug fluconazole, most active mannich bases 133h were 3-fold less potent against c. albicans and s. cerevisiae [173] , mannich bases 133j were either inactive or showed low antifungal activity [175] , whereas mannich bases 133k were inactive against c. albicans and s. cerevisiae [176] . mannich bases 133i lacked any anti-candida activity [174] , as did mannich bases 133n and 133o [179] , and most mannich bases 133q [180] . the majority of mannich bases 134a were equipotent to reference drug fluconazole against c. albicans [181] , and a small number of candidates 134c were moderately active against the same fungus, whereas the rest were inactive [182] . while most mannich bases 134d were moderately active against aspergillus spp., c. albicans and penicillium marneffei, two candidates 134d (r 2 ¼ 4-clc 6 h 4 or 2,4-cl 2 c 6 h 3 ) derived from 1-methylpiperazine as amine reagent were equipotent to reference drug ciclopiroxolamine [183] . several mannich bases 134e were also equipotent to reference drug fluconazole against aspergillus spp., t. mentagrophytes and p. marneffei [184] . neither compound 134g, nor candidate 135 (fig. 25 ) exhibited any anti-candida activity [185] , whereas mannich bases 134i had weak to moderate activity against candida tropicalis, s. cerevisiae and a. niger [187] . the antifungal activity of mannich bases 134j against aspergillus spp. and penicillium spp. was moderate to good, and mannich base 134j (x ¼ y ¼ ch 3 , r 2 ¼ 4-no 2 c 6 h 4 ) was more potent than reference drug fluconazole against c. albicans [188] . a few mannich bases 134k also showed good antifungal activity against c. albicans and fusarium solani [189] . the best anti-candida activity in the series of mannich bases 134m was recorded for the candidate with r 2 ¼ c 6 h 4 och 3 -4 (85% of the inhibition zone of reference drug fluconazole) [190] . in addition, 1,2,4-triazolo[5,1-b]-1,3,5-thiadiazines 199 (fig. 36) have been synthesized through a double mannich reaction starting from 5-benzyl-2h-1,2,4-triazole-3(4h)-thione and primary aliphatic and aromatic amines, and these compounds had good antifungal activity against chrysosponium tropicum and t. rubrum, but were completely inactive against a. fumigatus, a. flavus and microsporum nanum [306] . mixed results were obtained for other fungi, as a few candidates 199 were more potent than reference drug clotrimazole against a. niger, but were moderately potent against c. albicans or fusarium oxysporum, whereas the rest of the compounds in the series were totally inactive against these three fungi. as far as the aminomethyl derivatives of 2,3-dihydro-1,2,4-triazole-3-ones are concerned, candidates 136 and 138 (fig. 25 ) had no activity against c. albicans and s. cerevisiae [176, 191] , whereas in the series of mannich bases 137 featuring an imidazole moiety (fig. 25) , candidate 137 (nr 2 ¼ 1-piperidinyl) was twofold more potent against s. cerevisiae and twofold less potent against c. albicans than reference drug fluconazole [192] . antifungal activity of aminomethylated 2,3-dihydro-1,3,4oxadiazole-2-thiones ( table 2 ) has been reported in the same studies dealing with these compounds' antibacterial activity. several mannich bases 139a were equipotent to reference drug ciclopiroxolamine against t. mentagrophytes, a. flavus and a. fumigatus (mic 6.25 mg/ml), but not against p. marneffei [193] . mannich bases 139b derived from either morpholine or 3trifluoromethylaniline showed high activity against p. marneffei, t. mentagrophytes, a. flavus and a. fumigatus compared to reference drug ciclopiroxolamine, whereas those derived from other primary aromatic amines had only weak antifungal activity [194] . only a few of mannich bases 139c derived from piperazines had moderate anti-candida activity, and the rest of the collection was inactive [195] . oxadiazolethione mannich bases 139d [185] and 139j [176] , having at position 5 of the oxadiazole ring either a pyridine or a quinoline moiety, had no noticeable anti-candida activity, and the activity of mannich bases 139e against s. cerevisiae, c. tropicalis and a. niger was moderate at best [187] . most mannich bases 139f had weak antifungal activity against c. albicans, a. niger and aspergillus clavatus, but one candidate 139f (nr 2 ¼ nhc 6 h 4 och 3 -4) was 2-fold more potent than reference drug ketoconazole against the first two fungi, and almost equipotent to ketoconazole against the last fungus [196] . several mannich bases 139g had similar mic values as reference drug amphotericin b against c. albicans, a. fumigatus, t. rubrum and t. mentagrophytes [197] . most imidazole-containing mannich bases 139h exhibited better antifungal activity against c. albicans, t. rubrum and t. mentagrophytes than reference drug fluconazole [198] . the compounds reported in a study, including mannich bases 139i, are claimed to exhibit significant antifungal activity against c. albicans, but lack of access to the original information (which is only provided in the online edition as supplemental material, and is missing) makes data assessment impossible [199] . three mannich bases 139k were moderately active against aspergillus spp. and curvularia lunata [200] , while mannich bases 139l [98] and 139o [202] had no anti-candida activity, and mannich base 139m showed no antifungal activity against c. albicans and s. cerevisiae [201] . in addition, several aminomethylated 2,3dihydro-1,3,4-oxadiazole-2-ones 141 were more potent than reference drug nystatin against fungal plant pathogens aspergillus spp. ( [204] . anti-candida activity of c-mannich bases 142 of thiazolidinone (fig. 26 ) was found to be 16-fold weaker than that of reference drug terbinafine [205] . while three mannich bases 143 were moderately active against c. albicans and a. flavus, one candidate 143 (r 1 ¼ c 6 h 5 , nr 2 ¼ n(c 2 h 5 ) 2 ) (fig. 26) showed against the latter fungus a growth inhibition potency (mic ¼ 1.56 mg/ml) which was half of that determined for reference drug fluconazole [207] . also, aminomethylated thiazolidinones 144 (fig. 26) had moderate activity against aspergillus spp. and c. albicans compared to reference drug ketoconazole [208] . pyrimidine derivatives that have been aminomethylated with a view to evaluate the antifungal activity of the resulting mannich bases include pyrimidin-2-ones, pyrimidin-2-thiones and (thio) barbituric acid. double n-mannich bases 146 (x ¼ o or s) (fig. 26) had 67e77% of the anti-candida activity of the clotrimazolecontaining commercial drug imidil [210, 211] . c-mannich bases 200 of (thio)barbituric acid (fig. 37) , obtained using furan-2carboxaldehyde or indole-3-carboxaldehyde as aldehyde components and 2-aminopyridine or 4-aminoantipyrine as amine reagents in the mannich reaction, showed good activity against aspergillus spp. compared to reference drug salicylic acid [307] . antifungal evaluation of mannich bases from miscellaneous substrates that do not belong to any of the previously mentioned classes is the topic of several studies. p-mannich bases 83 (fig. 15 ) showed good activity against c. albicans and s. cerevisiae (mic ¼ 10 mg/ml) compared to reference drug amphotericin b (mic ¼ 15 mg/ml) [106] . various mannich bases of type 149 (fig. 27) derived from imides presented anti-aspergillus activity that ranged from weak to good, but no correlations between structure and activity for the small number of compounds in this series could be established [215] . anti-candida activity of the most potent mannich bases 152 (fig. 29 ) was only half of the activity of reference drug fluconazole [222] . aminomethylated hydrazidones 153 (r 1 ¼ c 2 h 5 ) derived from isonicotinic acid hydrazide (fig. 29 ) had good anti-candida activity [223] , but their analogues 153 (r 1 ¼ n-c 3 h 7 ) were less active, and their activity against a. niger was even poorer [224] . several dimethylamine mannich bases 154 derived from 7-methyl-2-(substituted aryl)imidazo[1,2-a]pyridines (fig. 29 ) were 4-folde2-fold more active against aspergillus spp. and c. albicans than reference drug griseofulvin [225] . when evaluated against the same fungi, mannich bases 157 (r 1 ¼ ch 3 ) and 158 of sydnones (fig. 29) were consistently less active than both reference drugs griseofulvin and nystatin [228] , and the majority of mannich bases 157 (r 1 ¼ och 3 ) behaved similarly, with the exception of candidate 157 (r 1 ¼ och 3 ; nr 2 ¼ 4-nitrobenzothiazole-2-ylamino), which was more potent than reference drugs fluconazole and nystatin against c. albicans [229] . all of the mannich bases 156 derived from 2-alkyl-3-hydroxy-pyridine-4(1h)-ones (fig. 29) were inactive against aspergillus spp. and c. albicans [227] . a few aminomethylated pyrazolines 159 (fig. 29 ) had anti-candida activity (mics between 1.5 and 4 mg/ml) comparable or even better than that of reference drug clotrimazole (mic ¼ 2 mg/ml) [230] , while several acetylenic mannich bases 162 (fig. 29 ) were equipotent to reference drug ketoconazole against c. albicans [233] . mannich bases 164 and 165 derived from hydantoins ( fig. 29) were generally moderate growth inhibitors for c. albicans and a. niger [236] , and phenothiazine mannich bases 167 (fig. 29) were all active against a. niger (sizes of inhibition zone between 12 and 21 mm) [238] . antifungal activity of mannich base 168 (fig. 29 ) of quinoxaline-2,3(1h,4h)-dione (100 mg/disc) against c. albicans and a. niger was superior to that of reference drug clotrimazole (50 mg/disc) [239] , while antifungal activity of mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)-one (fig. 29) equipotent to reference drug fluconazole against c. albicans (mic 6.25 mg/ml) [240] . as much as 20% of the world population live in areas with high risk of contracting malaria, and millions of people suffer from acute malaria each year, while approximately half million died from the disease in 2012 [308] . malaria has been largely eradicated in many parts of the world, but the total number of recorded cases is still on the rise. one of the causes for this grave situation is the proliferation of malaria parasites that are rapidly becoming resistant to antimalarial drugs, especially those that are most frequently used, such as chloroquine. the development of novel antimalarials has stagnated since the 1970s owing to lack of interest in developed countries for this topic, and limited market potential for these drugs. initiation of numerous programs supported by public funding led to a surge of interest in drugs for neglected diseases, and antimalarials are certainly amongst these drugs. use of the mannich reaction for the synthesis of antimalarials has a long and rich history, the quest for aminomethylated substrates for treatment of malaria starting with amodiaquine 201 (fig. 38) , the first mannich base that was used to treat malaria. many of these early studies focused on derivatives of 4aminoquinolines, and the sar within this class of antimalarials suggested that the chlorine atom at position 7 of the quinoline scaffold and the phenolic mannich bases moiety are structural features that are essential for the antimalarial activity of these compounds. nowadays, the use of amodiaquine has declined owing to its considerable toxicity as a result of its in vivo conversion into a reactive quinone-imine metabolite by cytochrome p450. it was therefore hypothesized that swapping the phenolic hydroxyl and the aminomethyl side chain in amodiaquine should prevent the formation of toxic metabolites, and help circumvent the adverse side effects associated with the use of amodiaquine. this hypothesis led to the design and synthesis of several amodiaquine analogues that retain the antimalarial activity, while lacking the potential to form a quinone-imine derivative [309] . out of these analogues, mannich base 202 (the isomer of amodiaquine henceforth named isoquine, fig. 38 ) expressed in vitro activity against both chloroquine sensitive hb3 strain and highly chloroquine-resistant k1 strain of plasmodium falciparum below 10 nm, and was 20 times more potent that chloroquine. isoquine (as its diphosphate salt) was subsequently tested in vitro against the murine plasmodium yoelii ns strain, and found to be 3-fold more potent than amodiaquine when administered orally. moreover, no toxic metabolites or any excessive accumulation of isoquine have been noticed in animal models, but unacceptably high first pass metabolism of isoquine to n-dealkylated metabolites in four animal species compromised isoquine's activity against parasites, and complicated the development process of isoquine into a marketable drug. a novel candidate, n-tert-butyl isoquine (gsk369796) 203 (fig. 38) , was identified using isoquine as lead, and shown to exhibit low nm activity against chloroquine-resistant and sensitive parasites, as well as in vivo oral activity equivalent to amodiaquine, but with a significantly improved antimalarial exposure profile [310] . in addition, n-tert-butyl isoquine had a better overall preclinical safety profile than chloroquine or amodiaquine, and can be synthesized in a scalable and cost-effective way. the study of disposition of candidates 202 and 203 showed that isoquine 202 undergoes in vivo oxidative n-dealkylation to desethyl-isoquine, a metabolite that had an improved blood clearance over isoquine; unfortunately, desethyl-isoquine also had a more potent inhibitory action of cyp1a2 than isoquine [311] . on the other hand, n-tertbutyl isoquine 203 had human plasma protein binding ability and inhibition of cyp2d6 similar to those of isoquine and desethylisoquine, but a reduced rate of n-dealkylation and a higher oral bioavailability compared to isoquine and desethyl-isoquine, and these properties made n-tert-butyl isoquine 203 a better candidate than isoquine 202 for further development. a study of the in vitro activity of isoquine 202 against p. falciparum clinical isolates collected in kenya in relation to amino acid changes in pfcrt and pfmdr1, the two genes associated with 4-aminoquinoline resistance, showed that isoquine possesses high activity against field isolates (ic 50 was 9 nm, compared with 56 nm for chloroquine, and 8 nm for amodiaquine), and that isoquine's activity could be correlated with polymorphisms in pfcrt, but not in pfmdr1 [312] . starting from n-tert-butyl isoquine 203 as a template, three benzoxazines 204 (r ¼ c 2 h 5 , n-c 3 h 7 , ch 2 c 6 h 5 ) (fig. 38 ) were designed and generated through a double mannich reaction, but despite the good anti-plasmodium activity against both chloroquine-sensitive (ic 50 between 21 and 38 nm) and chloroquine-resistant (ic 50 between 31 and 72 nm) strains, the rapid ring opening of benzoxazine at low ph makes them unsuitable for further development [313] . novel analogues 205 (nr 2 ¼ aliphatic primary and secondary amines, aromatic primary and secondary amines) of amodiaquine have been tested in vitro against chloroquine-sensitive strains of p. falciparum, and while all of the compounds were active, only mannich base 205 (nr 2 ¼ 4morpholinyl) (fig. 38 ) showed good activity (mic ¼ 63 ng/ml) compared to chloroquine (mic ¼ 250 ng/ml) [314] . antimalarial activity of analogues 206 of isoquine (fig. 38) has also been evaluated, but regardless of the nature of the amine in the aminomethyl moiety, their activity was considerably lower than that of reference drug chloroquine [315, 316] . use of primary amines derived from bicyclic scaffolds for the synthesis of novel isoquine analogues led to the discovery of 207 (fig. 38) , which was approximately twofold more potent that either chloroquine or isoquine against chloroquine-sensitive and resistant p. falciparum strains [317] . a series of isotebuquine analogues 208 (r ¼ cl or cf 3 , r 1 ¼ h), the corresponding double mannich bases 208 (r 1 ¼ ch 2 nhc(ch 3 ) 3 ), as well as n-oxides derived from a mono-mannich base and a double mannich base of type 208 (fig. 38) were also evaluated against chloroquine-sensitive and resistant p. falciparum strains [318] . while only a few of candidates 208 were more potent than chloroquine against the chloroquine-sensitive strain, the majority of these mannich bases were more potent than chloroquine against the chloroquine-resistant strain, and mannich bases having one aminomethyl function were generally more potent than double mannich bases. pyronaridine 209 (fig. 39) is another example of antimalarial drug that feature a phenolic mannich bases moiety in its structure, but the aminoquinoline scaffold that was typical for amodiaquine and its congeners has been replaced in pyronaridine by a naphthyridine ring system. pyronaridine was first synthesized in 1970 at the chinese institute of parasitic disease, and has been used solely in china as treatment for malaria with good results for over 30 years [319] . beside the difficultly accessible literature in chinese, there are several recent studies that provide information on its physicochemical properties [320] and adme properties in rats [321] , interaction with other antimalarials [322] , potential use in combination therapy with artesunate [323] , or its mechanism of action based on inhibition of b-hematin formation and subsequent glutathione-dependent hematin degradation process [324] . based on the observation that substitution of the quinoline scaffold in amodiaquine with a napthyridine ring system in pyronaridine preserved the antimalarial action, g€ orlitzer initiated an ambitious synthetic program aiming at exploring the effect on the antimalarial properties of replacement of quinoline in quinoline-based antimalarials with other fused heterocyclic systems containing one pyridine ring. thus, single (r 1 ¼ h) and double mannich bases (r 1 ¼ ch 2 nr 2 ) structurally related to amodiaquine and pyronaridine, respectively, and featuring pyrido[3,2-b]indol-4-yl (compounds 210, x ¼ nh) [325] , benzofuro [3,2[1, 6] naphthyridin-5-yl (compound 215) [332] moieties were synthesized and evaluated against chloroquine-sensitive and resistant p. falciparum strains (fig. 39) . as expected, all of the mannich bases presented in these studies exhibited good antimalarial activity, and double mannich bases were generally more potent than single mannich bases. however, in spite of the variety of fused heterocyclic systems that were investigated, no compound with antimalarial activity at least comparable to that of chloroquine emerged, with the exception of one pyronaridine analogue (210, x ¼ nh, r 1 ¼ ch 2 nr 2 , nr 2 ¼ 1-pyrrolidinyl). this candidate had ic 50 ¼ 50 nm against chloroquine-sensitive strain 3d7, ic 50 ¼ 38 nm against chloroquine-resistant strain dd2, and its evaluation against plasmodium winckei in a murine malaria model (intraperitoneal dosage of 100 mg/kg) showed that no intact parasite could be noticed after three days of treatment [325] . the phenolic mannich base moiety in the previously mentioned antimalarial agents can be also found in candidates that were designed through its incorporation into hybrids with dual mode of action. thus, the combination of a tetraoxane and a phenolic mannich base moiety afforded hybrids 216 called mannoxanes (fig. 40) , which are active at low nanomolar concentrations and surpass the ability of artesunate, tetraoxane rka 182 and a peroxide/amodiaquine combination to cure malaria in mice at 10 mg/kg [333] . hybridization of artemisin with a phenolic mannich base moiety led to candidates 217 (fig. 40) , which are up to 3 times more potent than artemisin against p. yoelii nigeriensis, could be easily converted into water soluble forms, can be administered orally, are presumed to have good bioavailability, and lack the capability to form neurotoxic metabolite dihydroartemisinin [334] . hybrids 218 (fig. 40 ) are structurally very similar to 217, and have been shown to be more active than sodium artesunate against chloroquine-sensitive nf54 and chloroquine-resistant k1 p. falciparum strains [335] . screening of a collection of compounds containing a 2-hydroxyethylamino motif in their structures for inhibition of parasite growth in red blood cells infected with chloroquine-sensitive nf54 p. falciparum strain identified a hit compound, whose further elaboration led to several hybrids featuring several types of phenolic mannich moieties as the second pharmacophore [336] . their evaluation against nf54 strain, k1 strain, and the rodent parasite plasmodium berghei showed that the introduction of the phenolic mannich bases moiety resulted in candidates that were very potent against the parasites in the 72 h assay. for example, compound 219 (fig. 40) the design of hybrids with dual mode of action has also been broadened to include pyrrole mannich bases. thus, hybrids 220 from 4-amino-7-chloroquinoline and pyrrole mannich bases (fig. 40) were evaluated against chloroquine-sensitive d10 and chloroquine-resistant w2 p. falciparum strains, but they were all less potent than isoquine against both strains [317] . however, candidate 220 (r 1 ¼ 4-clc 6 h 4 , nr 2 ¼ n(c 2 h 5 ) 2 ) was equipotent to chloroquine against d-10 strain, and four of the candidates were also more potent than chloroquine against w2 strain [317] . several candidates from a small library of mannich bases 221 (r 1 ¼ h or ch 3 ) of c-10 pyrrole analogues of artemisin (fig. 40 ) had antimalarial activity that was superior to both artemisin and chloroquine against chloroquine-sensitive 3d7 p. falciparum strain, and selected mannich bases from this library were more potent than the aforementioned reference drugs against chloroquine-resistant k1 p. falciparum strain [337] . all of the compounds tested displayed good activity in peter's 4 day suppressive test using 30 mg/kg over days 1e3 post-infection, and candidates 221 ( showed complete elimination of parasites. these three mannich bases were further investigated in vivo, and had effective doses between 4.3 and 5.3 mg/kg that eliminate 90% of p. berghei in mice, whereas candidate 221 (r 1 ¼ ch 3 , nr 2 ¼ 4methyl-1-piperazinyl) reached 100% clearance of parasitemia 24 h after the last treatment and increased mouse survival to 9 days, a biological profile that render it superior to clinically used sodium artesunate [337] . phenolic mannich bases of chalcone analogues (such as 222, fig. 41 ) have been claimed to possess antimalarial activity against chloroquine-sensitive d10 p. falciparum strain at concentrations ranging from 0.3 to 3 mg/ml [44] . taking into account the structural similarity between phenolic mannich bases 222 and ketonic mannich bases of a,b-unsaturated ketones, a possible mechanism action for compounds 222 could be the inhibition of thioredoxin fig. 41 . antimalarial phenolic mannich bases derived from miscellaneous substrates. reductase in p. falciparum [338] . phenolic mannich bases 223 of 2,4dihydroxybenzaldehyde, its thiosemicarbazone and semicarbazone (fig. 41) , as well as related quinoline-containing derivatives 224 (fig. 41) have been screened against chloroquine-resistant w2 p. falciparum strain [339] . only candidates 223 derived from 1-(7chloroquinolin-4-yl)piperazine had antimalarial activity, while the remaining mannich bases 223 were inactive. all of the candidates 224 were potent antimalarial agents, with mannich base 224 (nr 2 ¼ 4-(7-chloroquinolin-4-yl)piperazin-1-yl) featuring two quinoline motifs in its structure being the most potent (ic 50 ¼ 77 nm) [339] . imines of 4(1h)-pyridones 225 (r 1 and r 2 ¼ h or ch 3 ) (fig. 41) having a phenolic mannich base moiety were active against chloroquine-resistant w2 p. falciparum strain (ic 50 values between 9 and 33 mm) and atovaquone-resistant fcr3 p. falciparum strain (ic 50 values as low as 8 mm), but their potency was lower than that of reference drugs chloroquine or atovaquone [340] . in addition, aminoalkylation of lawsone using various aldehydes and n-alkyl-ferrocenylmethylamines afforded mannich bases 226 (r 1 ¼ alkyl) (fig. 41 ) structurally resembling antimalarial drug atovaquone, but evaluation of three candidates 226 (r 1 ¼ n-c 6 h 13 , n-c 7 h 15 , n-c 8 h 17 ) against p. falciparum showed that they were less potent than atovaquone itself [341] . because of their self-limiting nature, most viral diseases (with the exception of those caused by human immunodeficiency virus, hiv) do not require specific therapy, although treatments are used to bring the condition or its symptoms to an end more quickly. currently available drugs are designed to target four main groups of viruses, namely herpes viruses, hepatitis viruses, influenza viruses, and hiv. recent investigations in the antiviral activity of mannich bases deal mostly with aminomethylated phenols and aminomethylated isatins, although examples of mannich bases with antiviral properties derived from other types of substrates are available in literature. phenolic mannich base arbidol 227 (fig. 42) [342] is a broadspectrum antiviral agent commonly used in russia to treat acute respiratory viral infections, which acts by inhibiting virus-mediated fusion with target membrane and subsequent blockage of virus entry into target cells [343, 344] . using arbidol as lead compound, a group at shenyang pharmaceutical university led by gong initiated a program aiming at investigating structureeanti-hepatitis b relationship for a series of phenolic mannich bases derived from various heterocyclic ring systems. in their first report on this topic, the chinese researchers presented a series of analogues of arbidol 228 (x ¼ so, r 1 ¼ ch 3 , c-c 3 h 5 , r 2 ¼ br) (fig. 42) having various substituents r 2 in the aromatic sulfoxide moiety, and presenting amino residues different than the original dimethylamino group in arbidol (e.g., secondary aliphatic amines, nh-azoles belonging to imidazoles and 1,2,4-triazoles) in the aminomethyl function in position 4 [345] . sar investigation was based on the ability of these compounds to inhibit replication of hepatitis b virus (hbv) and the production of surface antigen of the hepatitis b virus (hbsag) and extracellular form of hepatitis b core antigen (hbeag) in hepg2.2.15 cells infected with hbv, and showed that half of these mannich bases exhibited inhibitory effects on hbv that were superior to reference drug lamivudine. replacement of the original methyl group at n-1 in 228 with cyclopropyl led to an increase in antiviral activity, but enhanced the cytotoxicity of these candidates as well, whereas the presence of fluorine or chlorine as substituents in the aromatic sulfoxide moiety resulted in an improvement of the anti-hbv activity. departure from the original phenylmercapto function in arbidol reduced the cytotoxicity of mannich bases 228 (x ¼ so), leaving in the same time the anti-hbv activity practically unchanged. also, the nature of the amino group in the aminomethyl function appears to have little effect on the anti-hbv activity of the mannich bases reported in this study [345] . the relationship between the presence of a linker (containing two or three atoms between the sulfur atom in the side chain and the phenyl moiety) and the anti-hbv activity of mannich bases 228 (x ¼ soch 2 ch 2 y, y ¼ ch 2 , o or null, r 1 ¼ ch 3 , r 2 ¼ br) was also explored, and candidates having a phenethyl residue proved to be inactive, whereas candidates with a 3-phenylpropyl residue presented remarkable anti-hbv activity [346] . a subsequent study [347] of the anti-hbv activity of arbidol analogues 228 (x ¼ so or so 2 , r 1 ¼ ch 3 , c-c 3 h 5 , r 2 ¼ h) unsubstituted at position 5 of the indole scaffold claimed that the nature of the amino function plays an important role, and that pyrrolidine-and imidazole-containing candidates exhibited better anti-hbv activity than candidates having different amino residues. for another set of mannich bases 228 (x ¼ so or so 2 , r 1 ¼ ch 3 , r 2 ¼ h), the authors suggested that the presence of 4-methylpiperazin-1-yl residue as amino moiety, rather than 1-imidazolyl, 2-methyl-1-imidazolyl or 1-piperidinyl, afforded more potent anti-hvb agents [348] . replacement of the sulfinyl group in the side chain with sulfonyl appeared to enhance the antiviral activity while reducing cellular toxicity of these mannich bases [347] , although the results in the later study [348] supported the opposite conclusion. substitution of the indole scaffold with quinoline in mannich bases 229 (x ¼ s or so) (fig. 42 ) resulted in potent inhibition of hbv dna replication (10-to 66-fold compared to reference drug lamivudine) [349] . analysis of sar for compounds 229 revealed that the presence of 4-fluorophenyl moiety linked to sulfur in the side chain at position 2 of the quinoline scaffolds, in conjunction with amine moieties such as 1pyrrolidinyl, 1-piperidinyl, 1-imidazolyl (but not 4fig. 42 . phenolic mannich bases inspired by arbidol and useful as anti-hepatitis b agents. methylpiperazinyl or morpholinyl) in the aminomethyl function are required for good anti-hbv activity. evaluation of mannich bases 230 (fig. 42 ) derived from a tetracyclic ring system comprising the quinoline scaffold fused with a benzothiopyrane ring system that has various substitution patterns in the aromatic ring identified a significant number of candidates with good inhibition of hbv replication [350, 351] . the search for anti-hbv agents with similar structures continued with the evaluation of a series of phenolic mannich bases 231 (r 1 ¼ ch 3 , c 2 h 5 , ch(ch 3 ) 2 , r 2 ¼ h or f, x ¼ s or so) of 5-hydroxybenzimidazoles substituted at position 2 with 4-alkylmercaptophenyl residues (fig. 42 ) [352] . these compounds exhibited inhibitory effect on the secretion of hbsag that was superior to reference drug lamivudine, but lacked the ability to inhibit the replication of hbv dna in hepg2.2.15 cells at concentrations that were inferior to those corresponding to 50% of their cytotoxicity. the presence of fluorine at position 6 of benzimidazole scaffold and the presence of sulfinyl function appears to be critical for the inhibition of hbsag secretion of mannich bases 231. evaluation of another series of mannich bases 232 (x ¼ s, so, so 2 , r 1 ¼ h, 4-f, 4-och 3 , 4-ch 3 ) of 5-hydroxybenzimidazoles (fig. 42) showed that the thioethers in this series did not inhibit the replication of hbv dna, but the candidates with 4-methylpiperazin-1-yl as amino moiety were good inhibitors of secretion of hbsag, and the inhibitory effect significantly decreased upon oxidation of sulfur in thioethers to sulfinyl and sulfonyl [353] . phenolic mannich bases 233 (r 1 ¼ h, 4-f, 4-cl, 4-br, 4-ch 3 , 3-och 3 ) derived from 6bromo-8-hydroxyimidazo[1,2-a]pyridine-3-carboxylates (fig. 42) , especially those having 4-morpholinyl or 4-methylpiperazin-1-yl moieties in the aminomethyl function, inhibited the replication of hbv dna, but had no inhibitory effect on secretion of hbsag or hbeag [354] . the nature of substituents in the arylmercapto residue modulates the anti-hbv activity, as the decrease of bulkiness of the substituents (from bromine to fluorine, for example) and the presence of lipophilic substituents (e.g., a methyl group) enhance the inhibition of replication of hbv dna, whereas the presence of a 3-methoxy group further increases the activity. phenolic mannich bases 125 derived from chlorokojic acid (fig. 23 ) have been evaluated against herpes simplex virus type-1 (hsv-1) and parainfluenza-3 virus (pi-3). amongst the candidates reported in one study, only two mannich bases had inhibitory concentration in the range of 0.1e0.8 mg/ml against hsv-1, whereas all of the compounds were active in various degrees against pi-3 [152] . all of the mannich bases 125 derived from 1arylpiperazines as amine reagents inhibited both hsv-1 and pi-3, and one candidate 125 (r ¼ 4-(4-methoxyphenyl)piperazin-1-yl) was as potent as reference drug acyclovir against hsv-1 [153] . in addition, candidate 125 (r ¼ 4-(3-chlorophenyl)piperazin-1-yl) presented remarkable activity (0.025e0.4 mg/ml) against pi-3. other aminomethyl derivatives of various phenolic substrates have been tested against different viruses. only one phenolic mannich base 57 (r ¼ nhch 3 ) of norvisnagin (fig. 10) was moderately active against hsv-1 [78] . evaluation of aminomethylated 7-hydroxycoumarin derivatives 234 (fig. 43) against flaviviridae and other viruses led to mixed results [355] . phenolic mannich bases 234 (r 1 ¼ h, r 2 ¼ h or ch 3 ) were generally inactive against bovine viral diarrhea virus (bvdv), yellow fever virus (yfv) or respiratory syncytial virus (rsv), and a few other candidates that were active against bvdv or yfw were also cytotoxic. o-alkylated phenolic mannich bases 234 (r 1 ¼ n-c 3 h 7 , r 2 ¼ h or ch 3 ), and especially candidates having a methyl group at position 4 of the coumarin ring system, were active against bvdv, but they were inactive against yfv or rsv. o-acylated phenolic mannich bases 234 (r 1 ¼ coc 6 h 5 , r 2 ¼ h or ch 3 ) were generally inactive against all three viruses, but one candidate 234 (r 1 ¼ coc 6 h 5 , r 2 ¼ h, nr 2 ¼ 1,2,3,4-tetrahydroisoquinolin-2-yl) had a remarkable activity against rsv, comparable to that of reference drug 6-azauridine, and had also very low toxicity. compounds 234 were not active against a panel of viruses comprising hiv-1, coxsackievirus b2, sb-1 strain of marek's disease virus, vesicular stomatitis virus and a reovirus. in addition, screening of an extensive library of compounds identified mannich bases 235 of 5-chloro-8-hydroxyquinoline (fig. 43) as reactivators of latent hiv-1, which could prove helpful in eradicating the latent reservoir of hiv-1 in resting memory cd4þ t cells, either alone or in combination with other treatments [356] . antiviral activity of mannich bases of isatin and its derivatives is the topic of several investigations. screening of twelve mannich bases 236 derived from semithiocarbazones of 5-nitroisatin (fig. 43 ) against a panel of other viruses afforded no compound with antiviral properties, with the exception of candidate 236 (x ¼ o, r 1 ¼ allyl), which had weak activity against yfv (strain 17d) at subtoxic concentrations. this candidate was more potent than reference drug ribavirin, but was also more cytotoxic [357] . based on results of molecular studies aiming at designing inhibitors of hiv reverse transcriptase, a series of mannich bases 237 (fig. 43 ) have been synthesized [358] . candidates 237 having secondary aliphatic amino moieties in the aminomethyl function showed 92 to 69% inhibition of the enzyme, whereas mannich bases 237 (nr 2 ¼ nhc 6 h 5 ) showed no inhibition. aminomethylation of schiff bases obtained from 5-substituted isatins (r 1 ¼ f, cl, f) and lamivudine using fluoroquinolones (r 2 ¼ ethyl, cyclopropyl; r 3 ¼ h, ch 3 ) as amine reagents gave mannich bases 183 (fig. 32) , which were less potent against hiv than the parent schiff bases, and the most potent candidates 183 (r 1 ¼ f) were also 10-fold less potent than lamivudine itself against hiv [273] . starting from schiff bases of trimethoprim, mannich bases 238 (fig. 43) were obtained using fluoroquinolones as amine reagents. their evaluation against hiv and hepatitis c virus (hcv) showed that mannich bases 238 (r 1 ¼ cl) inhibited replication of hiv in mt-4 cells at effective concentrations (ec 50 ) ranging from 9.4 to 56 mg/ml, and most compounds were active against hcv rna replication (80% inhibition at 50 mg/ml) [162] . mannich base 238 (r 1 ¼ ch 3 , nr 2 ¼ 4-(4chlorophenyl)piperazin-1-yl) and three mannich bases 238 (r 1 ¼ ch 3 ) derived from fluoroquinolones as amine reagents showed inhibition against replication of hiv in mt-4 cells at ec 50 ranging from 11.6 to 28.4 mg/ml [359] . in addition, all of the compounds 238 (r 1 ¼ ch 3 ) were active against hcv rna replication (65% inhibition at 50 mg/ml) [359] . furthermore, a study reports candidate 238 (r 1 ¼ h, nr 2 ¼ 4-(4-nitrophenyl)piperazin-1-yl) as inhibitor of japanese encephalitis virus and west nile virus in vitro, and a remarkable inhibitor of japanese encephalitis virus in a murine model [360] . investigations into the antiviral activity of mannich bases derived from substrates other than phenols or isatins are available in literature as well. the majority of mannich bases 80 (r ¼ cl, och 3 ) (fig. 14) , obtained through n-aminomethylation of thiazolidine-2,4-diones with morpholine, piperidine and variously 1substituted piperazines, showed no activity against severe acute respiratory syndrome (sars) coronavirus [103] . also, all of these mannich bases had antiviral activity against types a and b of influenza virus, but virus inhibition occurred almost at cytotoxic concentration. aminomethylation of the amide function in tetracyclines using fluoroquinolones as amine reagents afforded mannich bases 189 (fig. 34) , and four candidates derived from tetracycline and one derived from minocycline were found to inhibit hiv replication with ec 50 values below 20 mm, while their toxicity against mock infected cem cell line was greater than 140 mm [287] . in addition, all mannich bases 189 showed moderate inhibition of both 3 0 -processing and strand transfer steps of hiv-1 integrase. evaluation of efavirenz mannich bases 191 (fig. 34) led to the discovery of three candidates that were at least as potent as the parent compound against hiv [289] . a large number of indole mannich bases 239 (r ¼ aminomethyl, 3-amino-1-propyn-1-yl) (fig. 43 ) derived from a pentacyclic core, along several acetylenic mannich bases with the same core, have been claimed to be active against hcv and members of the flaviviridae family of viruses [361] . none of the mannich bases 240 (fig. 43) derived from indophenazine as substrate and sulfonamides, anthranilic acid or 2aminopyridine as amine reagents either showed any activity against hiv above their cytotoxic concentrations, but some presented weak activity against hsv, vsv, or vaccinia virus [362] . anticonvulsants are drugs used in the treatment of seizures in epilepsy, a common chronic neurological disorder that affects around 50 million people of all ages worldwide. the discovery of novel antiepileptic drugs relies either on rational design based on the use of well-known pharmacophores (e.g., imides), or on random screening of libraries of compounds. ketonic mannich bases 8 (r 1 ¼ 4-substituted aryloxy, 4substituted arylthio, r 2 ¼ h, nr 2 ¼ n(c 2 h 5 ) 2 ; r 1 ¼ 4fluorophenyloxy, r 2 ¼ h, nr 2 ¼ dimethylamino, 1-piperidinyl, 4morpholinyl, 1-pyrrolidinyl) (fig. 3 ) and the related piperidinols 14 (r 1 ¼ 4-substituted aryloxy, r ¼ c 2 h 5 ) (fig. 4) were examined for anticonvulsant activity in the maximal electroshock (mes) and subcutaneous pentylenetetrazole (scptz) screens [20] . none of the compounds provided protection in the scptz screen, but several candidates 8 and 14 demonstrated anticonvulsant activity in the mes screen below their neurotoxic levels (30 mg/kg). four ketonic mannich bases of type 8 derived from acetophenone or 4hydroxyacetophenone as substrates and common secondary aliphatic amines as amine reagents, as well as the corresponding azines 16 (fig. 4) , were assessed as anticonvulsants using the same two tests. the results showed that anticonvulsant activity of candidates 8 was superior to that of azines 16, and compounds derived from 4-hydroxyacetophenone were active at a dose of 300 mg/kg in the mes test, but no candidate showed anticonvulsant activity in the cptz test [363] . bis-mannich bases 13 (r 1 ¼ h, 4-cl, 4-ch 3 , 2thienyl, r ¼ c 2 h 5 ) and the corresponding piperidinols 14 were protective in the mes test at 30 mg/kg and/or above, while candidate 14 (r 1 ¼ 4-cl, r ¼ c 2 h 5 ) was protective in the scmet test at 300 mg/kg after 4 h [364] . the presence of a 4-chlorophenyl moiety appears to be important for the anticonvulsant activity of these compounds, and analogues having the same moiety were identified as good anticonvulsant agents in a previous study [365] . anticonvulsant activity of phenolic mannich bases of 3hydroxy-4-pyranones has been investigated extensively by aytemir's group. screening of derivatives of allomaltol 174 having a 4substituted piperazin-1-ylmethyl group (fig. 30 ) using mes and scptz tests showed that candidate 174 (r ¼ 3-cf 3 c 6 h 4 ) provided excellent protection against pentylenetetrazole-induced seizures, but was neurotoxic at high dose (300 mg/kg), while candidate 174 (r ¼ 4-clc 6 h 4 ) had high anticonvulsant activity in mes test at all doses after 30 min, without being neurotoxic [305] . evaluation of another series of mannich bases of allomaltol revealed that candidate 174 (r ¼ 2,3-(ch 3 ) 2 c 6 h 3 ) was active in scptz test at 300 mg/kg after 4 h, along with candidate 174 (r ¼ 3-clc 6 h 4 ), which was active in mes test at 300 mg/kg after 5 h [366] . although mannich bases of allomaltol obtained using morpholine or 4-(1-piperidinyl)piperidine as amine reagents had no anticonvulsant activity [366] , a subsequent study dealing with novel mannich bases generated from piperidines as amine reagents reported that candidates 124 (r 1 ¼ 3-ch 3 , r 2 ¼ 5-ch 3 ; r 1 ¼ 4-hoch 2 ch 2 , r 2 ¼ h; r 1 ¼ 4-c 6 h 5 ch 2 , r 2 ¼ h) (fig. 23) were protective in scptz test, while only candidate 124 (r 1 ¼ 4-hoch 2 ch 2 , r 2 ¼ h) provided protection in mes test at 300 mg/kg [367] . use of other piperidines as amine reagent in the mannich reaction with allomaltol as substrate led to candidates 124 (r 1 ¼ 4-(un)substituted phenyl, r 2 ¼ oh, cn, coch 3 ), and two of these mannich bases (r 1 ¼ 4-brc 6 h 4 , r 2 ¼ oh; r 1 ¼ 4-clc 6 h 4 , r 2 ¼ oh) were active in scptz test at 300 mg/kg after 4 h, while all of them were active in mes test either after 0.5 h or after 4 h [368] . in contrast, when allomaltol was replaced with kojic acid as substrate in the mannich reaction, but the same piperidines were used as mine reagents, the resulting candidates 241 (nr 2 ¼ 4,4-disubstituted piperidines) (fig. 44 ) were all active in scptz test either after 0.5 h or after 4 h, but only two of them (r 1 ¼ 4-brc 6 h 4 , r 2 ¼ oh; r 1 ¼ 4-clc 6 h 4 , r 2 ¼ oh) were active in the mes test at 300 mg/kg after 0.5 h, and one candidate 241 (r 1 ¼ c 6 h 5 , r 2 ¼ coch 3 ) was active at any dose after 4 h [368] . mannich bases 241 (nr 2 ¼ 4-substituted piperazines) of kojic acid as substrate and piperazines as amine reagents were generally better anticonvulsants than analogous mannich bases 174 of allomaltol, or than mannich bases 241 derived from kojic acid and 4,4disubstituted piperidines [369] . the authors tentatively explain the enhanced anticonvulsant activity of mannich bases of kojic acid compared to the activity of mannich bases of allomaltol through the possible formation of an extra hydrogen bond in the former compounds. however, mannich bases derived from chlorokojic acid were also good anticonvulsants, and they present, like mannich bases of allomaltol, only one hydrogen bond in their structure [154] . hydantoin represents the core structure of the old generation of antiepileptic drugs, such as phenytoin, and substitution with an aminomethyl moiety was shown to improve activity against mes seizures in mice [370] . evaluation of a library of n-mannich bases 242 (fig. 44 ) derived from 5-cyclopropyl-5-arylhydantoins having 4-substituted piperazines in the aminomethyl function showed that the majority of candidates were effective in the mes or/and scptz screens, and quantitative studies in rats after oral administration showed that three mannich bases 242 (r 1 ¼ h, r 2 ¼ c 6 h 5 , c 6 h 5 ch 2 , 3-ch 3 c 6 h 4 ch 2 ) were more potent than phenytoin in mes test [371] . results from another study [372] showed that candidates 242 were generally more active in mes test than in scptz screen, and chlorine-substituted mannich bases (r 1 ¼ cl) were generally less active than those unsubstituted in the aromatic ring (r 1 ¼ h), whereas candidates derived from 1,2,3,4-tetrahydroisoquinoline as amine reagent in the mannich reaction were less potent than those derived from morpholine or piperazines. compared to candidates 242 obtained from arylpiperazines as amine reagents, mannich bases having alkylene, alkenylene, carbonyl or ester linkers between the piperazine moiety and phenyl ring presented enhanced anticonvulsant protection to pentylenetetrazole-induced seizures, which was noticeable not only after 0.5 h, but after 4 h as well [372] . taking into account the established anticonvulsant properties of many spirohydantoins [373e375], mannich bases 243 (x ¼ nh) of spirohydantoins (fig. 44) have been synthesized and evaluated as anticonvulsants, and while some of them were effective in mes or/ and scptz screens, and were more potent than reference drug phenytoin, their high neurotoxicity precluded further testing [376] . besides hydantoin, succinimide presents the structural requirements for the core structure of good anticonvulsant agents (namely, a nitrogen-containing heteroatomic system with a least one carbonyl group), and the well-established drug ethosuximide is an example for this class of antiepileptic drugs. the group of obniska has been developing novel anticonvulsants for a long time, and mannich bases derived from variously substituted succinimides has been one of the classes of compounds that provided some of the most interesting results reported by these researchers. based on the significant number of candidates that have been synthesized, aminomethylated derivatives 244 of 3phenylsuccinimides (fig. 44) [382] , generally showed protection in mes screen, but some of them were also effective in scptz screen. moreover, a few candidates presented activity not only 0.5 h after administration, but also after 4 or 5 h, which is indicative of quick onset and long duration of anticonvulsant activity. mannich bases 244 of 3-arylsuccinimides derived from other secondary aliphatic amines, such as morpholine, 4benzylpiperidine, 4-cyclohexylpiperazine, were generally effective in both screens [381, 382] . despite the large number of compounds evaluated in these studies, no consistent sars could be established. compounds with good anticonvulsant properties emerged from almost all of these studies, but none of these anticonvulsant mannich bases was deemed sufficiently promising to advance to clinical studies. aminomethylated spirosuccinimides have also been investigated as anticonvulsants. candidates 243 (x ¼ ch 2 ) derived from 4-(3-trifluoromethylphenyl)piperazine and 4-(3chlorophenyl)piperazine as amine reagents in the mannich reaction were the most potent anticonvulsants in this series in mes test, and replacement of substituted aryl with 2-hydroxyethyl rendered the candidates active in both tests [376] . the activity of mannich bases 245 of simpler spirosuccinimides (fig. 44 ) appears to depend [377] and candidates 245 (n ¼ 1 or 2) derived from 4-(2methylphenyl)piperazine [383] were devoid of anticonvulsant activity. on the other hand, mannich bases 245 (n ¼ 1 or 2) obtained from 4-(3-trifluoromethylphenyl)piperazine as amine reagent were efficient in mes screen, but not in scptz screen [383] . evaluation as anticonvulsant agents of mannich bases 246 of succinimides 3,3disubstituted with identical ( (fig. 44) has also been the topic of several recent papers [384e387]. generally, mannich bases obtained from 3,3diphenylsuccinimide were more potent than those obtained from 3-alkyl-3-phenylsuccinimides, which, in turn, were more potent than mannich bases derived from 3,3-dialkylsuccinimides, which were actually inactive in most cases. candidates generated from 4arylpiperazines as amine reagents in the mannich reaction were efficient only in mes test, and the nature and position of the substituent in the aromatic ring attached to piperazine modulate the anticonvulsant activity of these compounds. however, mannich bases 246 derived from 4-(2-hydroxyethyl)piperazine or 4benzylpiperidine were efficient in both screens [384] . the effect of mannich bases 246 with potent anticonvulsant activity on na v 1.2 sodium channel currents was also investigated as a potential mechanism of action for these compounds, and the results showed that the anticonvulsant activity of these candidates correlates nicely with their effectiveness as sodium channel blockers [385, 386] . in addition, there was no direct correlation between anticonvulsant properties and 5-ht 1a , 5-ht 2a , 5-ht 1a and/or 5-ht 7 serotonin receptor affinity [383, 387] . besides hydantoins and succinimides, other substrates featuring the ureido motif in their structure have been aminomethylated with a view to obtain anticonvulsant agents. barbituric acid and its thio analogue have been subjected to the mannich reaction with two amine reagents having a quinazolinone moiety, and the resulting candidates 247 and 248 (x ¼ o or s, r ¼ 6-br, 6-i, 6,8-br 2 ) (fig. 45 ) provided good protection (40e80%, and 50e90%, respectively) in both mes and scptz screens at a dose of 50 mg/kg, while lacking neurotoxicity, or sedative and hypnotic effects [388] . mannich bases 169 of 3-aryl-2-thioxo-2,3-dihydroquinazolin-4(1h)-one (fig. 29 ) showed significant anticonvulsant activity in mes test, as some of these candidates afforded results comparable to those obtain for reference drug phenytoin [240] . several studies concerning the anticonvulsant activity of fused seven-membered ring systems containing two heteroatoms are available in literature. aminomethylated 2,3-dihydro-1,5benzoxazepines 249 (fig. 46) provided protection in the mes test in the range of 30e90% at a dose of 30 mg/kg; candidate 249 (r 1 ¼ 3-och 3 -4-oh, r 2 ¼ 2-och 3 ) was equipotent to reference drugs phenytoin and lamotrigine in mes screen, and was also equipotent to reference drug valproate in scptz screen [389] . another study allowed a direct comparison between the anticonvulsant activities of analogous aminomethylated 2,3-dihydro-1,5-benzoxazepines 250 (x ¼ o) and 2,3-dihydro-1,5benzothiazepines 250 (x ¼ s) (fig. 46) [390] . at a dose of 30 mg/ kg, the latter provided more protection (20e90%) in mes test than the former, with mannich base 250 (x ¼ s, r 1 ¼ 2-cl, r 2 ¼ och 3 ) being the most active compound in this series. ketonic mannich bases 251 (r ¼ h, cl, no 2 ; r 1 er 2 ¼ r 3 er 4 ¼ (ch 2 ) 4 or r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r 4 ¼ h) having a 2,3-dihydro-1,5benzodiazepine scaffold as the amino moiety (fig. 46) were evaluated using a model in which the seizures were induced chemically, and some of these compounds provided protection after 0.5 h, whereas only candidates 251 derived from acetophenone (r ¼ h) provided protection up to 2 h [391] . inflammation is part of the complex, nonspecific immune response of vascular tissue that occurs in reaction to any type of injury, such as pathogens, irritants, damaged cells, etc. the initial response of the body to harmful stimuli is initiated by cells already present in all tissues, and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from blood into the injured tissue, where they release a series of cell-derived mediators, triggering afterwards a cascade of biochemical events that propagate the inflammatory response. nonsteroidal antiinflammatory drugs (nsaids) are commonly used for treatment of inflammation, along with its symptoms (fever, pain, swelling), but they present significant side effects after long-term usage, such as gastrointestinal lesions, kidney injury, and cardiovascular risk. along with many other classes of compounds, mannich bases of various structures have been investigated as part of the ongoing search for novel anti-inflammatory drugs. ketonic mannich bases 252 (r 1 ¼ h, ch 3 , so 2 ch 3 ) derived from 2arylidenecyclohexanones and aromatic amines (fig. 47) have been evaluated using xylene-induced ear swelling test and carrageenan-induced paw edema, and candidate 252 (r ¼ h) was superior to reference drug ibuprofen in both tests, whereas several candidates 252 (r ¼ ch 3 ) were less efficient, but still more effective than ibuprofen, in the same tests [392] . administration of ketonic mannich base 253 (fig. 47 ) to rats with a chronic inflammatory process induced by insertion of cotton pellets led to increased phagocytic capacity of peripheric neutrophils, enhanced activity of the serum complement system, and higher catalase activity, while a slightly decrease in the activity of superoxide dismutase was observed [393] . a few double mannich bases 105 of 4,6dimethoxybenzofuran-3(2h)-one (fig. 21) inhibited the production of pro-inflammatory cytokines tnf-a and il-6 by 76e100% at a concentration of 10 mm, but they were more cytotoxic than reference drug dexamethasone, which had comparable effects with candidates 105 on the production of the same cytokines at only 1 mm [134] . investigation of the anti-inflammatory activity of mannich bases 254 (r ¼ h, f, cl, br, or 2 ; r 2 ¼ och 3 and oc 4 h 9 -n) having amino acids isoleucine and methionine as amine moieties (fig. 47 ) was performed using both carrageenan-induced paw edema model and pellet-induced granuloma model, and resulted in the discovery of two anti-inflammatory agents 254 (r ¼ och 3 and oc 4 h 9 -n; r 1 ¼ ch 2 ch 2 sch 3 ) derived from methionine [394] . at a dose of 25 mg/kg, these two mannich bases 254 had 81% and 71%, respectively, of the efficiency of the reference drug diclofenac (dose of 10 mg/kg) on the reduction of paw swelling. at the same dose, these two candidates 254 also showed 88% and 79%, respectively, of the activity of reference drug indomethacin (dose of 3 mg/kg) on chronic inflammation. a continuation of this study was undertaken with a view to broaden the nature of amino acids (cysteine, threonine, methionine, isoleucine, asparagine, and glutamine) as amine moieties in mannich bases 254, and also the nature of the alkoxy substituents in the aromatic ring, but these novel candidates had no anti-inflammatory activity at 5 mg/kg, with the exception of two candidates 254 (r ¼ oc 2 h 5 ; r 1 ¼ ch 2 sh, ch 2 conh 2 ), which displayed mild activity in the acute inflammation model [395] . only one example of mannich bases obtained from simple phenols as potential anti-inflammatory agents is available in recent literature. thus, evaluation of four phenolic mannich bases 255 (fig. 48) using formalin-induced acute inflammation model in mice revealed that one candidate had reliable activity upon parenteral administration (50 mg/kg), whereas reference drug diclofenac sodium exhibited only two-thirds of its activity, albeit at 8 mg/kg [396] . phenolic mannich bases 256 of resveratrol analogues containing a pyridinyl moiety and either one, two or three aminomethyl residues (fig. 48) were tested using xylene-induced ear edema in mice at 200 mg/kg, and two of them had 80% of the efficiency of reference drug ibuprofen, whereas the remaining candidates were less potent [397] . the anti-inflammatory activity of mannich bases of polyhydroxylic phenols was also investigated. at a dose of 10 mg/kg, two candidates 113 (nr 1 r 2 ¼ 1-piperidinyl and 4-morpholinyl) derived from 4,6-diacetylresorcinol (fig. 22 ) had a slightly lower activity than that of reference drug indomethacin in carrageenan-induced paw edema [398] . three candidates 257 (r 1 ¼ 3-cl, nr 2 ¼ 4-methylpiperazin-1-yl; r 1 ¼ 2-br, nr 2 ¼ 1piperidinyl or 1-pyrrolidinyl) (fig. 48) were more efficient at inhibiting the production of tnf-a at a concentration of 10 mm than reference drug dexamethasone at 1 mm [399] ; under the same experimental conditions, most mannich bases in the study were also more efficient at inhibiting the production of il-6 than dexamethasone. the same researchers also investigated the action on enzymes that are involved in inflammation (such as cyclooxygenases (cox), trypsin and b-glucuronidase) of phenolic mannich bases 258 (r 1 ¼ 2-f, 2-cl, 2-br, 3-f, 3-cl, 3-br) of chalcone analogues (fig. 48) having the same substitution pattern in ring a as candidates 257 [400] . with one exception, none of the candidates inhibited the activity of trypsin, whereas most mannich bases in this study inhibited the activity of b-glucuronidase. candidates 258 derived from chalcone analogues having a halogen at position 3 of the b ring were generally more potent than those derived from chalcone analogues having a halogen at position 2 of the b ring. in addition, a few mannich bases 258 were poor inhibitors of cox-1, but excellent inhibitors of cox-2, and the majority of the candidates were more efficient at inhibiting cox-2 than reference drug aspirin. in a different study [401] , two out of four phenolic mannich bases of other chalcone analogues were found to reduce rat paw edema induced by carrageenan, and although these compounds were found to be good inhibitors of trypsin this time, no satisfactory correlation with their antioxidant, free radical scavenging, or lipooxygenase inhibition could be established. because inducible nitric oxide synthase (inos) generates high levels of nitric oxide that modulates inflammations through multiple pathways, the inhibition of this enzyme by phenolic mannich bases of heterocyclic analogues of chalcones with various structures was also investigated. this type of phenolic mannich bases was found to strongly inhibit no production, with ic 50 values ranging between 10.5 and 0.018 mm [402] . benzoxazines 117 (fig. 22) , easily obtained from 4hydroxyacetophenones through a double mannich reaction, inhibited the swelling of rat paw in various degrees when administered orally in doses equimolar to 20 mg/kg of indomethacin [145] ; compared to indomethacin, one candidate 117 (r ¼ 4-f) was more efficient, while another candidate 117 (r ¼ 4-och 3 ) was equipotent. chalcone analogues 259 (r ¼ 4-och 3 or 4-ch 3 ) derived from these benzoxazines (fig. 48) had a more pronounced antiinflammatory activity compared to candidates 117, and all of them displayed 70e90% of the efficiency of reference drug indomethacin [403] . phenolic mannich bases of naturally-occurring benzopyranones have also been tested as anti-inflammatory agents. most mannich bases 260 derived from 7hydroxycoumarin (fig. 48) were superior to reference drug indomethacin at reducing rat paw edema induced by carrageenan, and two candidates 260 (nr 2 ¼ 4-morpholinyl, 1-piperazinyl) were 1.6 times more efficient than indomethacin at 10 mm without significant inhibition of cox-1 [404] . irisolidone is an isoflavone which was isolated from pueraria spp., and was found to exert its antiinflammatory action through suppression of inos gene expression and pro-inflammatory cytokines in activated microglia [405] . chemical modification of irisolidone by means of the mannich reaction using primary aliphatic and aromatic amines as amine reagents led to candidates 261 ( fig. 48) with enhanced ability to inhibit nitric oxide production compared to parent irisolidone [406] . anti-inflammatory activity of mannich bases of 2,3-dihydro-1,2,4-triazole-3-thiones has also been investigated. several candidates 133f (table 1) have been evaluated using carrageenaninduced rat paw edema model, and the activity of one of them (133f, r 2 ¼ 2-ch 3 c 6 h 4 , nr 2 ¼ 4-morpholinyl) was comparable to that of reference drug ibuprofen [171] . mannich bases 133i with secondary aliphatic amino moieties (dimethylamino, diethylamino, 1-pyrrolidinyl) in the aminomethyl function were as efficient as reference drug celecoxib in reducing rat paw edema both after 1 h and after 2 h, and they also had lower ic 50 values (around 1 nm) for the inhibition of cox-2 compared to celecoxib (1.9 nm) [407] . as far as 5-substituted 2-aminomethyl-4-arylideneamino-2h-2,3dihydro-1,2,4-triazole-3-thiones 134 (table 1) are concerned, the majority of mannich bases 134c had, upon administration of either 20 mg/kg or 40 mg/kg, a lower anti-inflammatory activity in carrageenan-induced rat paw edema model than reference drug indomethacin (5 mg/kg), although one candidate (134c, r 2 ¼ 2,6-cl 2 c 6 h 3 , nr 2 ¼ 4-phenylpiperazin-1-yl) inhibited edema formation at the higher dose almost as efficiently as indomethacin [182] . starting from ibuprofen, two separate studies reported the synthesis and anti-inflammatory activity of mannich bases 262 having different arylideneamino moieties at position 4 of the 2,3-dihydro-1,2,4-triazole-3-thione scaffold (fig. 49) . the most potent compounds in this series were those having either a 4-morpholinyl or a 4-methylpiperazin-1-yl residue in the aminomethyl function. when common 4-substituted benzaldehydes (r 2 ¼ 4-clc 6 h 4 , 4-ch 3 c 6 h 4 , 4-brc 6 h 4 , 4-no 2 c 6 h 4 ) were used to generate the azomethine moiety, several candidates 262 had anti-inflammatory activity comparable with that of reference drug diclofenac, and were generally more efficient than ibuprofen at every time interval up to 3 h [408] . also, the most potent compounds in this series were those having either a 4-morpholinyl or a 4-methylpiperazin-1-yl residue in the aminomethyl function. on the other hand, when 3aryl-4-formylsydnones were used to generate the azomethine moiety, the resulting mannich bases 262 were consistently more potent than the analogues in the previously mentioned series of compounds, but all of these candidates were less efficient than reference drug indomethacin in reducing rat paw edema [409] . several mannich bases 134h showed a peak in their antiinflammatory activity at 1 h post-injection (from 50% to 130% edema inhibition compared to indomethacin), whereas the antiinflammatory activity of other candidates 134h peaked at 4 h post-injection (from 84% to 100% edema inhibition compared to indomethacin) [186] . all of the mannich bases 263 (fig. 49 ) had only moderate anti-inflammatory activity, with the most potent compound in the series showing approximately 80% of the efficiency of reference drug indomethacin [410] . a few mannich bases 264 and other structurally related aminomethylated 2,3-dihydro-1,2,4-triazole-3-thiones (fig. 49 ) reduced significantly the inflammatory response (maximum inhibition between 30 and 59%) compared to reference drug diclofenac (inhibition between 42 and 63%), and some of them presented fast onset of their antiinflammatory action, while others had a long-lasting anti-inflammatory effect [411] . the anti-inflammatory activity of mannich bases of 2,3-dihydro-1,3,4-oxadiazole-2-thiones has been scarcely examined. several mannich bases 139a (table 2) , especially those derived from ibuprofen as starting material for the 1,3,4-oxadiazole-2-thione substrate and generated from 4-arylpiperazines as amine reagents in the aminomethylation step, were as efficient as reference drug diclofenac at reducing rat paw edema [193] . also, mannich base 265 (fig. 49 ) had anti-inflammatory activity comparable to that of reference drug indomethacin in carrageenan-induced rat paw edema test [412] . several papers published by g€ okhan's group have reported the anti-inflammatory activity of mannich bases of 2benzoxazolinones. one of their studies showed that candidates 266 (r 1 ¼ ch 3 , r 2 ¼ 2-or 4-clc 6 h 4 co) having an acyl moiety at position 6 of the benzoxazolidinone scaffold were more potent than their analogues 266 (r 1 ¼ 2-or 4-clc 6 h 4 co, r 2 ¼ h) with the same acyl group at position 5 (fig. 49 ); in addition, the antiinflammatory activity of two of these candidates was equipotent to that of reference drug indomethacin [413] . substitution with fluorine in the phenyl ring of the acyl moiety appears to be less favorable for the anti-inflammatory activity of candidates 266 (r 1 ¼ h, r 2 ¼ difluorobenzoyl) [414] . mannich bases 266 (r 1 ¼ ch 3 , r 2 ¼ h) having 4-arylpiperazine residues in the aminomethyl function were all less potent than reference drug indomethacin in carrageenan-induced rat paw edema test, but the results suggest that the nature of the substituent in the aryl group of piperazines plays an important role in the anti-inflammatory activity of these compounds [415] . a few mannich bases 266 (r 1 ¼ no 2 , r 2 ¼ h) had an anti-inflammatory effect comparable to that of reference drug indomethacin 3 h after administration, but their efficiency in reducing the swelling reached a plateau afterwards, whereas that of indomethacin continued to increase in time [416] . two studies have reported the anti-inflammatory activity of mannich bases of isatins. aminomethylation of derivatives of 5methylisatin thiosemicarbazone afforded mannich bases 267 (r 1 ¼ r 2 ¼ h or ch 3 , nr 2 ¼ secondary aliphatic amines) (fig. 50) , which showed moderate anti-inflammatory activity (18e44% inhibition of edema at a dose of 100 mg/kg) compared to reference drug diclofenac (65% inhibition of edema at a dose of 45 mg/kg) [417] . several mannich bases 268 (fig. 50) , which were obtained from a derivative of isatin hydrazone, had anti-inflammatory activity comparable to that of reference drug diclofenac, and the highest level of activity was observed after 2 h [418] . a correlation in this series between anti-inflammatory activity and the nature of the amino moiety in the aminomethyl function was noticed, as the activity decreased with the increasing lipophilicity of the amino group. a series of n-mannich bases 269 of benzimidazole derivatives (fig. 50) were evaluated as anti-inflammatory agents using formalin-induced paw edema method. compared with reference drug diclofenac (50 mg/kg), they all caused significant reduction of paw edema, albeit at different doses (200 mg/kg for mannich base 269 (r 1 ¼ h or ch 3 ) and 40 mg/kg for mannich bases 269 of 2styrylbenzimidazole) [419] . also, several mannich bases 269 (r 1 ¼ c 2 h 5 ), derived from both secondary aliphatic amines and primary arylamines, showed moderate anti-inflammatory activity 4 h after administration (33e57% of the activity of reference drug aspirin at the same dose of 100 mg/kg) [420] . mannich bases obtained from substrates other than those mentioned so far have been examined as anti-inflammatory agents as well. thus, n-mannich bases 270 (nr 2 ¼ 1-pyrrolidinyl, 1piperidinyl, 4-morpholinyl) of pyrimido[1,6-a]azepine derivatives (fig. 51 ) had moderate activity (46e58% reduction of edema compared to that recorded for reference drug diclofenac) [421] , whereas n-mannich bases 271 of a tricyclic system derived from pyrimido[1,6-a]azepine (fig. 51) were less potent (anti-inflammatory activity of approximately 40% of that of diclofenac) [422] . two aminomethylated pyridazinones bearing a thiophene ring, namely compounds 272 and 273 (fig. 51) , showed 65% and 95% reduction of rat paw edema, respectively, compared to reference drug indomethacin [423] . even when administered in a higher doses relative to that of reference drug, none of the mannich bases 274 (r ¼ h, ch 3 , och 3 , cl, x ¼ o or ch 2 ) derived from isoxazolines (fig. 51) was as efficient as indomethacin in reducing rat paw edema [424] . out of the two acetylenic mannich bases 275 (x ¼ o or ch 2 ) with a betulonic acid scaffold (fig. 51 ) that were investigated as antiinflammatory agents, only the piperidine derivative was almost as efficient as reference drug indomethacin in reducing rat paw edema; the morpholine analogue had only half of the activity of the piperidine mannich base [425] . as pain is a symptom of inflammation, the anti-inflammatory and analgesic activities of novel candidates are usually evaluated at the same time. therefore, it is not surprising that many of the studies that report the anti-inflammatory activity of mannich bases also offer information on their analgesic potential. several ketonic mannich bases 252 (r 1 ¼ h, ch 3 , so 2 ch 3 ) derived from 2-arylidenecyclohexanones and aromatic amines (fig. 47) were equipotent to reference drug ibuprofen in both acetic acid-induced writhing test and hot plate test [392] . one of the most potent candidate was compound 252 (r ¼ r 1 ¼ h) derived from ptoluidine, but a few mannich bases derived from 2-(4methylsulfonylbenzylidene)cyclohexanone were also efficient as pain relievers. phenolic mannich bases of 1-and 2-naphthols substituted in either rings of naphthalene system with various functions, which were synthesized using preformed aminomethylation reagents (e.g., imonium salts obtained from aromatic aldehydes and secondary aliphatic amines), were claimed to have analgesic activity [426] . the claim is difficult to assess, because only two candidates have been evaluated, and no comparison with established analgesics was provided. in addition, while candidate 276 (x ¼ ch 2 ) (fig. 52 ) was efficient (92% inhibition of the writhing reaction), the second candidate 276 (x ¼ o) offered only modest protection against pain (30% inhibition of the writhing reaction). mannich bases of 2,3-dihydro-1,2,4-triazole-3-thiones with analgesic activity have been reported in several studies. candidates 133f (r 2 ¼ 2-ch 3 c 6 h 4 , nr 2 ¼ 4-morpholinyl; r 2 ¼ 4-ch 3 oc 6 h 4 , nr 2 ¼ 4-methylpiperazinyl) ( table 1) , which had good antiinflammatory activity, were also tested for analgesic activity; their efficiency, determined using tail flick method in albino rats, was comparable to that of reference drug ibuprofen [171] . several mannich bases 262 (fig. 49) showed analgesic activity (tail flick latency between 6.8 and 7.1 s) that was comparable with that of reference drug pentazocine (tail flick latency of 7.45 s), while the rest of the compounds were moderately active [409] . mannich bases 263 (fig. 49) were less efficient in the tail flick test, as the reaction time for the most potent compound in the series was approximately 80% of the latency provided by reference drug pentazocine [410] . two candidates 139a (table 2) , both derived from ibuprofen as starting material for the 2,3-dihydro-1,3,4oxadiazole-2-thione substrate and generated using either ethyl piperidine-4-carboxylate or 4-(4-fluorophenyl)piperazine as amine reagents in the aminomethylation step, were more efficient analgesics than reference drug diclofenac in hot plate test [193] , while mannich base 265 (fig. 49 ) was equipotent to reference drug diclofenac in acetic acid-induced writhing test [412] . analgesic activity of mannich bases of 2-benzoxazolinones was also determined for the same candidates that were investigated for anti-inflammatory activity. in the library of mannich bases 266 derived from 2-benzoxazolinones (fig. 49 ) carrying a benzoyl moiety in the aromatic ring (r 1 or r 2 ¼ substituted benzoyl), no significant difference in analgesic activity between the 5benzoylated and the 6-benzoylated analogues could be observed [413] . although most candidates provided moderate to low protection in either tests used to determine their analgesic activity (namely acetic acid-induced writhing test and p-benzoquinoneinduced abdominal constriction test), two mannich bases derived from 6-(substituted benzoyl)-2-benzoxazolinones were found to be as efficient as reference drug aspirin. thus, analgesic activity of mannich base 266 (r 1 ¼ h, r 2 ¼ 2,6-f 2 c 6 h 3 co, nr 2 ¼ 4-(4acetylphenyl)piperazin-1-yl) was equipotent to that of aspirin [414] , while analgesic activity of mannich base 266 (r 1 ¼ ch 3 , r 2 ¼ 4-clc 6 h 3 co, nr 2 ¼ 4-(4-fluorophenyl)piperazin-1-yl) was slightly poorer than that of aspirin [413] . in the series of mannich bases 266 derived from 5-methyl-2-benzoxazolinone, several compounds showed better analgesic activity compared to reference drug aspirin, and the analgesic activity for all the compounds was consistently higher than their anti-inflammatory activity, suggesting that these mannich bases might exert their analgesic activity centrally [415] . two mannich bases 266 derived from 5-nitro-2benzoxazolinone were also found to possess analgesic activity comparable to that of aspirin [416] . mannich bases 267 of derivatives of 5-methylisatin thiosemicarbazone (fig. 50) were moderately efficient (16e53% protection) in preventing acetic acid-induced writhing in mice at a dose of 100 mg/kg, while reference drug diclofenac provided 74% protection in the same test at a dose of only 45 mg/kg [417] . mannich bases 268 of isatin hydrazone (fig. 50) carrying a quinazoline moiety and derived from acyclic secondary aliphatic amines (dimethylamine, diethylamine) were the most potent; their analgesic effect was superior or comparable to that of reference drug diclofenac 2 h after administration, but it decreased rapidly afterwards [418] . n-mannich bases 269 (fig. 50 ) obtained from benzimidazole using dimethylamine or diethylamine as amine reagents were found to be moderate analgesics at a dose of 200 mg/kg relative to paracetamol (100 mg/kg), while mannich base 269 derived from 2methylbenzimidazole as substrate and diphenylamine as amine reagent was a poor analgesic candidate [419] . owing to their toxicity, mannich bases derived from 2-styrylbenzimidazole were administered at a lower dose (20 and 40 mg/kg), and their analgesic activity ranged from moderate (nr 2 ¼ 4-morpholinyl) to very good (nr 2 ¼ diethylamino or 1-piperidinyl) when compared to paracetamol [419] . n-mannich bases 269 of 2-ethylbenzimidazole had analgesic activity comparable to that of reference drug pentazocine only when administered in doses that were 25 times greater than that of pentazocine [420] . other mannich bases 269 derived from 2-substituted benzimidazoles (r 1 ¼ ch 2 nhnhc 6 h 5 or 2-hoc 6 h 4 ) also showed moderate to good analgesic activity in acetic-acidinduced writhing test (62e84% of the analgesic activity of diclofenac at the same dose) [427] . evaluation of a first series of n-mannich bases 277 derived from 2h-4,6-dimethyl-3-oxo-2,3-dihydroisothiazolo[5,4-b]pyridine ( fig. 52 ) led to identification of weak to moderate analgesic agents [428] . amongst them, candidates derived from 4-aryl-or 4benzylpiperidine and those having a 4-(2-substituted phenyl) piperazine as the amine moiety were the most efficient analgesics in writhing and hot plate tests. a second series of mannich bases 277 was subsequently synthesized, and some of the candidates displayed significant activity in the writhing test, with analgesic activity 2 to 10 times more potent than that of aspirin and 1.5 to 10 times weaker than that of morphine, used as reference drugs in the study [429] . excess of reactive oxygen species produced in living organisms can cause oxidative stress and damage cells by initiating chain reactions that lead to lipid peroxidation, dna damage or protein oxidation. besides the complex system of antioxidant metabolites and enzymes that naturally prevent cell damage, exogenous antioxidants may sometimes be required to keep reactive oxygen species at an optimum level. use of the mannich reaction to generate novel chemical entities capable of acting as antioxidants is presented in this section. ketonic mannich bases 109 and 110 (fig. 21) , which were obtained from arylamines as amine reagents and 3-acetylcoumarine and 2-acetylbenzofuran as substrates, respectively, were tested for antioxidant activity by evaluating their ability to scavenge 1,1diphenyl-picrylhydrazyl (dpph) radical [138] . the most efficient candidates in each series, namely 109 (r ¼ h) and 110 (r ¼ n(ch 3 ) 2 ), showed moderate potency in scavenging dpph radical (approximately 65%) compared to standard butylated hydroxytoluene (90%). substitution of the aromatic ring with electron-withdrawing groups appears to reduce the antioxidant ability of these mannich bases. a large number of phenolic mannich bases have been reported in the literature as potential antioxidants. antioxidant activity of two phenolic mannich bases 278 derived from thymol (fig. 53) has been assessed by means of xanthine oxidase inhibition test for the cell-free system, and by inhibition of lipid peroxidation using ratliver homogenate [430] . both candidates, and especially mannich base 278 having morpholine as amine moiety, presented enhanced antioxidant activity in both tests compared to parent thymol. three phenolic mannich bases 114 (nr 1 r 2 ¼ 4-ch 3 oc 6 h 4 nh, 1piperidinyl and 4-morpholinyl) derived from 4,6diacetylresorcinol (fig. 22) , designed primarily as antiinflammatory agents, were also evaluated for their ability to inhibit lipid peroxidation; results showed that these candidates were more efficient than indomethacin at preventing lipid peroxidation, and that the antioxidant activity may be correlated with their ulcerogenic activity [398] . only three mannich bases 257 (fig. 48) , all of them having piperidine as the amine moiety (nr 2 ¼ 1-piperidinyl), showed moderate to high ability in scavenging dpph radical (49e74% of dpph scavenging ability of standard gallic acid), while the rest of the candidates were either poor scavengers of dpph radical, or failed to react with dpph radical at all [399] . although all of the phenolic mannich bases 197 showed efficiency as antioxidants in various degrees, candidates 197 (nr 2 ¼ diphenylamino, 4-morpholinyl, 1-piperazinyl) (fig. 36) were the most potent scavengers of hydrogen peroxide, their activity being comparable to that of standard ascorbic acid, but inferior to that of standard butylated hydroxyanisole [302] . selected tacrinee8-hydroxyquinoline hybrids 279 (fig. 53) , that were developed primarily as agents for the treatment of alzheimer's disease, exhibited also potent peroxyl radical absorbance capacities (2.6e4.7 trolox equivalents/mmol of mannich base), as determined by means of orac-fl method (oxygen radical absorbance capacity by fluorescence) [431] . phenolic mannich bases of natural flavanones have been designed and synthesized with the view to improve antioxidant efficiency, bioavailability and water solubility of parent flavanones. only three mannich bases 41 (r ¼ oh, r 1 ¼ h, r 2 ¼ 1-pyrrolidinyl, diethylamino or diisopropylamino) of apigenin (fig. 7) exhibited antioxidant activity greater than that of apigenin, while the rest of candidates 41 presented antioxidant activity comparable to that of apigenin [58] . antioxidant activity of these apigenin derivatives was concentration-dependent, and dpph radical scavenging ability of the most potent mannich bases 41 was almost the same as that of the standard ascorbic acid at a concentration of 1.25 mg/ml. scutellarein was also aminomethylated to give mannich bases 41 (r ¼ r 1 ¼ oh, r 2 ¼ aminomethyl), which had the ability to scavenge 50% of dpph radical in the sample at concentrations ranging from 24 to 33 mm, but no comparison with a well-established antioxidant was provided in the study [432] . mono-and bis-mannich bases 280 fig. 53 ) were synthesized and evaluated as inhibitors of photooxidation of a2e (a pigment of the lipofuscin of retinal pigment cells, thought to play a role in macular degeneration) [433] . these candidates showed sufficient antioxidant ability to inhibit noncellular and intracellular photooxidation of a2e, and were superior as antioxidants to quercetin itself. on the other hand, phenolic mannich bases obtained from other known antioxidants such as sesamol or 1-(2-hydroxy-4-methoxyphenyl)-3phenylprop-2-en-1-one were ineffective inhibitors of a2e photooxidation at 200 mm [433] . phenolic mannich bases of chalcone analogues such as candidates 258 (r 1 ¼ 2-f, 2-cl, 2-br, 3-f, 3-cl, 3-br) (fig. 48) were modest to poor scavengers of dpph radical (3e58% reduction of dpph absorption) compared to standard quercetin (86% reduction of dpph absorption) [400] . results for dpph scavenging ability of phenolic mannich bases derived from other chalcone analogues obtained in a different study are also in line with the antioxidant data recorded for mannich bases 258, as the greatest reduction of dpph absorption was only 13% at concentrations as high as 1 mm of mannich base [401] . however, one candidate in this series scavenged superoxide radical efficiently, while another candidate was a potent inhibitor of heme dependent lipid peroxidation [401] . several mannich bases 133g derived from 5-(2-thienyl)-2,3dihydro-1,2,4-triazol-3-thiones (table 1) were as efficient antioxidants as standard ascorbic acid (over 90% reduction of dpph radical), and showed enhanced antioxidant activity over the parent 2,3-dihydro-1,2,4-triazole-3-thiones [172] . a series of mannich bases 139 [1, 4] dioxin-6-yl, nr 2 ¼ substituted primary arylamines) ( table 2) were designed as antioxidant agents, and were evaluated using scavenging dpph radical assay, scavenging 2,2 0 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) radical cation (abts) assay, and ferric reducing antioxidant power assay [434] . more than half of these mannich bases showed greater dpph radical scavenging ability than butylated hydroxytoluene (ic 50 ¼ 44 mm), and seven of them exhibited greater antioxidant activity than trolox (ic 50 ¼ 30 mm) in the same assay. a few candidates 139 with high dpph scavenging activity, along with other mannich bases 139, were also very good scavengers of abts radical cation (their activity was greater than that of trolox). in ferric reducing antioxidant power assay, three mannich bases 139 showed better activity than trolox, and other seven were more potent than butylated hydroxytoluene. mannich bases 139 nr 2 ¼ 3,4,5-f 3 c 6 h 3 ) were the only compounds to exhibit high potency in all three assays, and they were also more efficient than trolox in inhibiting lipid peroxidation in mice liver microsomes homogenate [434] . n-mannich bases 149 of succinimide (r 1 ¼ h or phenyl, nr 2 ¼ nhc 6 h 5 , 2-pyridinyl) (fig. 27) and an n-mannich base of phthalimide showed moderate dpph scavenging activity compared to standards vitamin c and vitamin e, while their ability to inhibit peroxidation of linoleic acid was moderate to weak [215] . also, several n-mannich bases 166 of saccharin (fig. 29) were antioxidants as potent as standard ascorbic acid in the abts assay, and more potent antioxidants than saccharin itself [237] . n-mannich base 281 derived from a 3,5-disubstituted pyrazoline (fig. 53) had dpph scavenging and nitric oxide scavenging activities comparable to standard antioxidants ascorbic acid and rutin; the presence of the phenolic hydroxyl group could be responsible for the antioxidant activity of this compound [435] . finally, antioxidant activity of two c-mannich bases 282 of uracil (fig. 53) was determined by means of measuring the rate of oxidation of 2-propanol [436] ; addition of compounds 282 to the reaction mixture reduces the rate of oxidation to levels comparable to the rate of oxidation in the presence of butylated hydroxytoluene. a small number of studies deal with the investigation of mannich bases as antihypertensive agents. phenolic mannich bases appear to be particularly remarkable as blood pressure-lowering substances. several single and double mannich bases of various phenols and having thiomorpholine as amine moiety showed a gradual effect on systolic, diastolic and mean arterial pressure, starting at a dose 0.1 mg/kg; in the case of reference drugs captopril, losartan and omapatrilat, the same effect was noticeable at 0.001 mg/kg [437] . in a manner similar to the aforementioned reference drugs, these phenolic mannich bases also induced a gradual (but significant) reduction of heart rate in anesthetized mice. double mannich base 283 (fig. 54 ) emerged as a valuable candidate, with one of the lowest effective dosage in this collection, while exhibiting the highest antihypertensive activity in conscious spontaneous hypertensive rat model [437] . the same researchers examined the antihypertensive effect of double mannich base 284 with a 1,4-dihydropyridine moiety in its structure (fig. 54) , but the results showed that the activity of this candidate was inferior to that of double mannich base 283, presumably due to the change in bulkiness of the substituent para to the phenolic hydroxyl [438] . the presence of two aminomethyl functions in the structure of 284 may have been essential in preserving the antihypertensive activity of this candidate to a reasonable level, while replacement of thiomorpholine in 283 with morpholine in 284 could be also the cause for the poorer antihypertensive properties of candidate 284 relative to those of mannich base 283. in an attempt to synthesize aminomethylated 4-(naphthyloxy) butanoic acids (e.g., compound 285, were obtained when 4-(naphthalen-1-yloxy)butanoic acid was subjected to aminomethylation [439] . at a concentration of 5 mg/ kg, candidate 285 (nr 2 ¼ 1-piperidinyl) lowered blood pressure in anesthetized cats similarly to propranolol, while compound 286 (nr 2 ¼ 4-phenylpiperazin-1-yl) was superior to propanolol at the same dose (fall of blood pressure of 60 mm hg, duration of the effect of 120 min). it is also worth mentioning that the antihypertensive effect of a c-mannich base of 2-naphthol, namely 1fig. 54 . mannich bases as agents for blood pressure regulation. pyrrolidinylmethyl-2-naphthol 287 (fig. 54) , was reported in an earlier publication [440] . a large collection of mannich bases 288 (r 1 ¼ h, ch 3 , c 2 h 5 , ch 2 ch(ch 3 ) 2 , c 6 h 5 , och 3 , cl) (fig. 54) was evaluated for antihypertensive activity by the non-invasive tail cuff method, and several candidates were found to significantly reduce mean arterial blood pressure, albeit at higher doses than reference drug hydralazine [441] . the presence of alkyl groups as substituent r 1 seems to be favorable for the antihypertensive activity, whereas the nature of the amine moiety does not appear to influence the activity. several mannich bases 289 (r 1 ¼ ch 3 , c 2 h 5 , n-c 4 h 9 , ch 2 c 6 h 5 ; r 2 ¼ ch 3 , c 6 h 5 ) of imidazo[1,2-a]benzimidazoles (fig. 54 ) were found to reduce arterial blood pressure in anesthetized rats with 20% at doses that were lower than the dose at which reference drug dibazole produces the same effect (22 mg/kg) [442] . analogous aminomethylated imidazo[1,2-a]benzimidazoles 290 (r 1 ¼ ch 3 , n-c 4 h 9 , ch 2 c 6 h 5 ) (fig. 54 ) were more efficient antihypertensive agents than mannich bases 289, as they reduce arterial blood pressure with 20% at doses lower than those recorded for candidates 289. several octahydroquinazoline derivatives 291 (r ¼ h, 2-f, 4-f, 4-cl, 4-br, 4-ch 3 , 4-oh) (fig. 54 ) were obtained through a double mannich reaction starting from 3-(4-chlorophenylamino)-5,5dimethyl-2-cyclohexenone and arylamines, and were shown to produce insignificant changes in both arterial blood pressure and heart rate at a dose of 5 mg/kg [443] . however, derivatives 291 (r ¼ och 2 conhn]chc 6 h 4 r 1 , r 1 ¼ h, 4-ch 3 , 4-no 2 ) of a candidate in the initial series afforded significant decreases in both systolic and diastolic arterial blood pressure, with rapid onset of action (5 min) and minor decrease of heart rate in anesthetized male adult albino rats [443] . on the other hand, candidate 291 (r ¼ 4-cl) produced a time-dependent significant increase in both systolic and diastolic arterial blood pressure, without causing tachycardia for 30 min, which makes this compound useful for treatment of hypotension. a small number of studies report the activity of mannich bases against parasites other than plasmodium spp. among these parasites, which are the cause of parasitic infections especially in developing countries, different species from the trypanosomatidae family are pathogenic to humans and cause african trypanosomiasis (sleeping sickness, trypanosoma brucei), american trypanosomiasis (chagas' disease, trypanosoma cruzi) or leishmaniasis (leishmania spp.). in addition, one study investigated the activity of mannich bases against entamoeba histolytica, and one study reported the anti-schistosoma activity of mannich bases derived from praziquantel. all parasitic protozoa belonging to trypanosoma spp. have a unique thiol metabolism based on the flavoenzyme trypanothione reductase. this enzyme could be therefore considered a promising target for rational drug design against african sleeping sickness, chagas' disease, and different forms of leishmaniasis, owing to its absence of in the mammalian host, the structural differences to related host enzymes, and its essential role for parasite survival. because unsaturated ketonic mannich bases react readily with thiols, and because several such compounds were shown to be efficient mechanism-based inhibitors of p. falciparum thioredoxin reductase [338] , a study concerning the ability of these compounds to interact with both trypanothione reductase and free trypanothione was undertaken [444] . candidates 292 (nr 2 ¼ dimethylamino, 1-piperidinyl, 4-morpholinyl) (fig. 55 ) inactivated trypanothione reductase, but only in the presence of nadph, suggesting that reduction of this enzyme prior to its interaction with the mannich base is essential. the divinyl ketone arising from the deamination of candidates 292 is the actual inhibitor, and its activity against trypanothione reductase was higher than that of parent mannich bases; unfortunately, this divinyl ketone was also too reactive to be considered a drug candidate. mannich bases 292 displayed only modest activity against all strains of intracellular parasites, which may be explained by reaction with glutathione present in millimolar concentrations in the cytosol of the mammalian host cells. nonetheless, they showed a significant effect against extracellular t. b. rhodesiense, which might (at least partially) be due to their high reactivity toward trypanothione reductase and trypanothione. with trypanothione reductase validated as a drug target in trypanosomiasis, design and synthesis of novel unsaturated mannich bases based on melaminophenyl arsenical drug melarsoprol was subsequently pursued [445] . candidates 293 (r 1 ¼ cl, h, ch 3 ) (fig. 55) were efficient in vitro trypanothione reductase inhibitors, and while they did not display any significant activity in cell-based assays against t. cruzi, they were active towards t. brucei. the presence of the melamine residue para to the enone motif did not result in any improvement in the trypanocidal potency of 293 (r 1 ¼ cl) compared to hit compounds 292. however, the presence of the melamine residue meta to the enone motif significantly lowered the ic 50 against the human cell line used in the study, which is indicative of these compounds' high cytotoxicity. on the other hand, phenolic mannich base 294 (fig. 55) was basically inactive towards all parasites. mannich bases 293 were taken up effectively into cells despite the absence of p2 and hapt1 carriers, which suggests that the main route of entry for these compounds was not through aminopurine transporters. stemming from the observation that heterocyclic pyrazolopyridine ring system can be considered analogous to quinoline, and because aminoquinoline derivatives are efficient antimalarials, several derivatives featuring the pyrazolopyridine scaffold were designed and tested against leishmania amazonensis, in the evolutive form of promastigotes [446] . thus, both mannich bases 295 (r 1 ¼ c 6 h 5 , r 2 ¼ ch 3 , c 6 h 5 ) (fig. 55 ) are active antileishmanial agents with ic 50 values of 390 and 120 nm, whereas aminoquinoline derivative amodiaquine had ic 50 ¼ 890 nm. from the structureeactivity point of view, the results suggest that pyrazolopyridine ring system is a bioisostere of quinoline, and that the presence of the carbethoxy group in the structure of candidates 295 does not influence significantly the biological activity. amebiasis is a contagious disease of the human gastrointestinal tract that is caused by parasitic protozoa e. histolytica. all the candidates in a small library of piperazine mannich bases 139d of 5-(4pyridinyl)-2,3-dihydro-1,3,4-oxadiazole-2-thione ( table 2) were active against hm1:imss strain of e. histolytica, but only three of them were more potent than reference drug metronidazole (ic 50 ¼ 1.81 mm) [447] . the amine residues in the aminomethyl function of these active mannich bases were 4-ethylpiperazin-1-yl (ic 50 ¼ 327 nm), 4-(4-fluorophenyl)piperazin-1-yl (ic 50 ¼ 245 nm), and 4-(2-methoxyphenyl)piperazin-1-yl (ic 50 ¼ 1.06 mm). in addition, cytotoxicity of these antiamoebic candidates was low (in the concentration range of 2.5e250 mm). out of four newly synthesized mannich bases 296 of praziquantel (fig. 55) , two candidates (nr 2 ¼ n(c 3 h 7 -n) 2 and nhch 2 ch 2 oh) exhibited significant in vitro anti-schistosoma activity (100% worm killing at 40 mm and 30 mm, respectively), but they were not as efficient as praziquantel itself (100% worm killing at 10 mm) [448] . platelets are crucial for hemostasis, as they connect to one another through receptor bridges, form aggregates, and finally plug the tear in the interrupted endothelium. however, the aggregation of platelets leading to formation of clots can also be triggered by irregularities on the vessel wall, resulting in abnormal clot formation, which is the primary factor in the development of thrombotic disorders such as unstable angina, myocardial infarction, stroke and peripheral vascular diseases, especially when it occurs in the coronary artery. platelet aggregation is also initiated by endogenous substances, such as collagen, thrombin, prostaglandin endoperoxides, thromboxanes, arachidonic acid, adenosine diphosphate (adp), etc. inhibition of platelet aggregation represents a promising strategy for the treatment of thrombotic diseases, and several studies have reported the activity of mannich bases as inhibitors of platelet aggregation. several types of phenolic mannich bases represented by structures 24e32 (fig. 6) were also evaluated as inhibitors of platelet aggregation induced by adp or collagen at concentrations of 20 mm or 10 mg/ml, respectively [449] . numerous candidates having various structures showed good inhibitory effects and were more potent than reference drug clopidogrel, whereas nine compounds in this collection exhibited inhibitory activity in the range of 90e100% in both models. good platelet aggregation inhibitory activity was associated with the presence of a pyridyl moiety as ring b in the structure of chalcone analogues, and also by the presence in ring a of a hydroxy group meta to the carbonyl function. phenolic mannich bases 41 (r ¼ r 1 ¼ oh, r 2 ¼ aminomethyl) of scutellarein (fig. 7) were investigated as thrombin inhibitors, and their influence on several parameters such as prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen was determined [432] . all of these candidates showed greater thrombin inhibitory activity compared to parent scutellarein. mannich base derived from scutellarein and containing a morpholinyl residue in the aminomethyl function was the most potent of all, and was subsequently selected for molecular docking experiments with thrombin. these experiments revealed that the morpholinylmethyl group occupies deep pocket s3 of the thrombin binding site, whereas the scutellarein part of the inhibitor's molecule is anchored by three hydrogen bonds within the active site. the effect of mannich bases 289 (r 1 ¼ ch 3 , c 2 h 5 , n-c 4 h 9 , ch 2 c 6 h 5 ; r 2 ¼ ch 3 , c 6 h 5 ) and 290 (r 1 ¼ ch 3 , n-c 4 h 9 , ch 2 c 6 h 5 ; (fig. 54 ) on adp-induced aggregation of rabbit thrombocytes was studied in vitro [442] . several candidates 289 and most candidates 290 were found to have lower effective concentrations (ec 50 ) at which they decrease the degree of aggregation by half than reference drug acetylsalicylic acid. cellular effects of thrombin, including platelet aggregation, are mediated through the activation of thrombin receptor par-1 belonging to the family of four g-protein-coupled receptors called protease-activated receptors. par-1 has become an attractive drug discovery target, and peptide-mimetics or small organic molecules with par-1 antagonist properties have already been designed and synthesized. an indole mannich base motif has been incorporated in the structure of a series of novel peptide-mimetics 297 (r ¼ 4-ch 3 oc 6 h 4 ch 2 , 3,4-f 2 c 6 h 3 ch 2 , naphthalen-1-ylmethyl, naphthalen-2-ylmethyl) ( fig. 56 ) capable to bind to par-1 [450] . the ability of these candidates to inhibit par-1-induced platelet aggregation has been tested by measuring the degree of aggregation of human platelets, and also by establishing the degree of inhibition of contraction of aortic rings. compounds 297 (r ¼ 4-ch 3 oc 6 h 4 ch 2 , 3,4-f 2 c 6 h 3 ch 2 ) belonging to the series of 6-aminoindole derivatives were the most potent candidates, with values of inhibitory concentration that were about 5 times lower than reference compound rwj54003 for 75% inhibition of platelet aggregation. in addition, these results were confirmed by the enhanced inhibitory activity of these two candidates on the contraction of aorta rings when compared to the activity of rwj54003, an effect that became more evident with the increase of par-1 dose. candidates in the series of 5-aminoindole derivatives were found inactive or weak inhibitors in both assays. anti-ulcer agents are a class of drugs used to treat ulcers in the stomach and the upper part of the small intestine. acid peptic disease is a chronic pathology that affects millions of people worldwide, and it has been estimated that 10% of the world population will develop this condition in their lifetime [451] . an imbalance between aggressive factors (such as gastric acid, helicobacter pylori infection, excessive intake of anti-inflammatory drugs, immoderate consumption of alcohol, high concentrations of reactive oxygen species) on one hand, and protective factors (e.g., mucus, bicarbonate anion, prostaglandins, good blood flow, efficient cellular repair, endogenous and exogenous antioxidants) on the other hand is considered to be the cause of ulcers. treatment of ulcers and their symptoms relies on pain relievers, antiacids and cytoprotective agents to allow the healing of ulcers, and agents to prevent the recurrence of ulcers, such as proton pump inhibitors, muscarinic antagonist pirenzepine or h2 receptor antagonist cimetidine. the ability of mannich bases to act as anti-ulcer agents has been reported in a small number of studies. anti-ulcer activity of saminomethyl derivatives 170 generated from 4,6-diaryl-2mercaptopyrimidines (fig. 29 ) as substrates and secondary aliphatic amines as amine reagents in the mannich reaction was evaluated in vivo using aspirin-induced ulcer model in albino rats [241] . based on the ulcer score, the mean ulcer index and the degree of protection were calculated and compared to those of reference drug omeprazole. five candidates 170 (r 1 ¼ 4-clc 6 h 4 , r 1 ¼ 3-no 2 c 6 h 4 or 4-ch 3 oc 6 h 4 ) offered more than 50% protection, while other five mannich bases 170 (r 1 ¼ 4-clc 6 h 4 , r 1 ¼ 4-clc 6 h 4 , 3-no 2 c 6 h 4 or 4-(h 3 c) 2 nc 6 h 4 ) had only moderate anti-ulcer activity (approximately 30% protection) compared to omeprazole (99% protection). aminomethylation of 1,4-dihydropyrimidines with sulfanilamide as amine reagent afforded mannich bases 298 (fig. 57) , whose ability to reduce the volume of acid secretion was determined by pyloric ligation method [452] . three candidates 298 (r ¼ 4-ch 3 oc 6 h 4 , 3-ch 3 o-4-hoc 6 h 3 , 2-furyl) presented good antiulcer activity (ulcer indices in the range of 0.18e0.35 at a dose of 10 mg/kg), while reference drug omeprazole had an ulcer index of 0.08 at a dose of 1 mg/kg. a small number of furo [3,2-g] flavones of type 299 (r 1 ¼ c 6 h 5 , 4-clc 6 h 4 , 3-pyridinyl; r 2 ¼ h or ch 3 ) and 300 (r 1 ¼ c 6 h 5 , 3-pyridinyl) (fig. 57) , aminomethylated either at position 6 or 9, have been evaluated as gastroprotective agents using the ethanol-induced gastric ulcer model in rats [453] . the mean values of the protection index for these mannich bases were in the range of 20e44%, and no comparison with an anti-ulcer reference drug was provided in the study. the best three candidates 299 (r 1 ¼ 3-pyridinyl, r 2 ¼ ch 3 , nr 2 ¼ 4-methylpiperazin-1-yl), 300 (r 1 ¼ 3-pyridinyl, r 2 ¼ och 3 , nr 2 ¼ 4-methylpiperazin-1-yl) and 300 (r 1 ¼ c 6 h 5 , r 2 ¼ h, nr 2 ¼ 4-morpholinyl) have a methoxy group at position 4 as a common structural feature; the corresponding mannich bases having a hydroxyl group instead of methoxy were significantly less active. mental disorders comprise a broad range of problems, with different symptoms, generally characterized by some combination of abnormal thoughts, emotions, behavior and relationships with others. common neurological conditions labeled as mental disorders include depression and anxiety, while schizophrenia and bipolar disorder stand out as mental disorders that are severe and disabling. untreated mental, neurological and substance use disorders exact a high toll, accounting for 13% of the total global burden of disease, while unipolar depressive disorder is the third leading cause of disease burden, accounting for 4.3% of the global burden of disease. current predictions indicate that depression will be the leading cause of disease burden globally by 2030 [454] . a range of different types of treatment for mental disorders are available, and the most suitable treatment depends both on the disorder and on the individual. a major option for many mental disorders is psychotherapy, while another option is psychiatric medication. amongst several groups of drugs that are currently employed in psychiatric medication, antidepressants treat clinical depression as well as anxiety and a range of other disorders, anxiolytics (including sedatives) are used for anxiety disorders and related problems such as insomnia, mood stabilizers are used primarily in bipolar disorder, and antipsychotics are used for psychotic disorders, notably for positive symptoms in schizophrenia. selective serotonin reuptake inhibitors (ssris) play an important role in pharmacotherapeutic treatment of depression. in an attempt to modify the general ssri structural motif of g-phenoxypropylamine, two ketonic mannich bases 301 (r ¼ cl, br) (fig. 58 ) derived from 4-chloro-and 4-bromoacetophenone as substrates and 4-benzylpiperidine as amine reagent were synthesized, the carbonyl function in these amino ketones was subsequently reduced, and the resulting secondary hydroxyl was then converted into an ether group through reaction with 1-chloro-4trifluoromethylbenzene [455] . although the designed ssri analogues targeted in this study showed no antidepressant activity, the intermediate ketone mannich bases 301 were as effective as reference drugs fluoxetine, sertraline or imipramine at similar dosage in a validated experimental model of depression in mice such as the forced swimming test. an innovative computer-assisted approach based on the prediction of activity spectra for substances (pass) has been applied for the discovery of new anxiolytics [456] . an initial database comprising 5494 structures was generated by virtual combinatorial design of highly diverse chemical compounds, including different types of heterocycles such as thiazoles, pyrazoles, isatins, fused imidazoles, with the view to increase the probability of finding new chemical entities as anxiolytics. out of the eight hits obtained from this database, four candidates were mannich bases. ketonic mannich base 302, mannich base 303 derived from an imidazo[2,1-b] benzo[d]thiazole and two mannich bases 304 (r 1 ¼ f, r 2 ¼ ch 3 ; r 1 ¼ no 2 , r 2 ¼ c 6 h 5 ) derived from imidazo[1,2-a]pyridines (fig. 58 ) were synthesized and tested as potential anxiolytics using the conflict situation test. all of the candidates showed an anxiolytic effect that was comparable or greater than that of reference drug medazepam. mannich base 304 (r 1 ¼ no 2 , r 2 ¼ c 6 h 5 ) was the most potent anxiolytic in this study, being two times more potent than medazepam. a collection of compounds sharing a benzoxepin scaffold as common structural feature was evaluated for their sedativeehypnotic effect using phenobarbital-induced sleep test [457] . among these compounds, three ketonic mannich bases 305 (nr 2 ¼ 1-piperidinyl, 4-methylpiperazin-1-yl, 4-morpholinyl) (fig. 58 ) decreased the onset of phenobarbital-induced sleep and prolonged the duration of hypnosis when administered in a dose equimolar to that of reference drug phenobarbital. the hypnotic activity of candidates 305 (nr 2 ¼ 1-piperidinyl, 4-methylpiperazin-1-yl) was comparable to that of phenobarbital, while mannich base 305 (nr 2 ¼ dimethylamino) exhibited only moderate activity, and mannich base 305 (nr 2 ¼ 4-(2-chlorophenyl)piperazin-1-yl) was virtually inactive. therefore, the nature of the amino residue in these mannich bases seems to have a significant impact on their hypnotic activity. in addition to biological activities of mannich bases presented so far, isolated studies were found to report various other biological activities for mannich bases. these singular results are covered in this section, without any attempt to arrange them in a systematic order. two phenolic mannich bases derived from 2,4-and 2,6-di-tbutylphenol as substrates and dimethylamine as amine reagent were evaluated as hepatoprotective agents against experimental toxic hepatitis induced by tetrachloromethane. the degree of liver damage was evaluated in terms of alanine aminotransferase activity in blood serum and malonic dialdehyde content in liver homogenates. even at a dose of 10% of the corresponding ld 50 , the ability of these two candidates to diminish the hepatotoxic action of tetrachloromethane was superior to that of reference compound emoxypine [458] . in addition, in an assay using the same tetrachloromethane-induced hepatitis model, two acetylenic mannich bases 275 (x ¼ o or ch 2 ) with a betulonic acid scaffold (fig. 51 ) were shown to decrease alkaline phosphatase activity and lower alanine aminotransferase and aspartate aminotransferase activities in blood serum compared to control [425] . acetylenic mannich base 306 (fig. 59 ) was tested on guinea pig spontaneously beating atria with a view to evaluate its negative chronotropic activity, but the potency of this candidate was approximately four times lower than that of bradycardic agent zatebradine, and was therefore excluded from subsequent testing as blocker of hyperpolarization-activated current [459] . ondansetron analogues 307 (r 1 ¼ ch 3 ) having a piperazine moiety instead of imidazole (fig. 59) were synthesized and evaluated as anti-emetic agents using a retching model [460] . all the candidates were effective to some extent when administered at a dose of 8 mg/kg, but only mannich base 307 (r ¼ 2-pyrimidinyl) had anti-emetic activity comparable to that of reference drug ondansetron at low dosage (2 mg/kg). several 7-aminomethylated b-thujaplicin analogues 308 ( fig. 59) were tested against oxidative stress-induced death of ht22 cells following exposure to glutamate [461] . piperazine-containing mannich bases 308 were more potent than parent b-thujaplicin in protecting glutamate-challenged ht22 cells, with ec 50 values ranging from 0.08 to 1.7 mm. because the most potent candidates were those having a chroman moiety as substituent at n4 in the piperazine residue, the high in vitro neuroprotective activity of these mannich bases may be due to the potent antioxidant effect imparted by the chroman moiety. analogous morpholine mannich base 308 was also active in protecting ht22 cells from oxytosis, but it was less active than the piperazine-containing b-thujaplicin derivatives. the presence of the isopropyl group also seems to be important for the neuroprotective activity of these compounds, since an analogous mannich base derived from tropolone was not active. estrogen deficiency after menopause is one of the most common causes of osteoporosis. hormone replacement therapy is widely used to prevent bone loss, although it presents potential drawbacks such as increased risk of uterine bleeding and/or hyperplasia, increased risk of endometrial, breast or ovarian cancer, higher occurrence of myocardial infarction, cardiovascular disease, etc. conjugate 309 of 17b-estradiol and iminodiacetic acid (fig. 59 ) was designed as an estrogen-containing, bone-seeking agent that could prevent bone loss with lesser side effects, and was subsequently synthesized by means of the mannich reaction [462] . mannich base 309 showed significant affinity for bone, but lower affinity for ovary and uterus than 17b-estradiol, while it maintained 92% of the affinity of 17b-estradiol for osteoblast estrogen receptors. candidate 309 did not induce uterine hypertrophy, and lower levels of biochemical markers of bone turnover (e.g., osteocalcin, alkaline phosphatase, c-terminal telopeptide fragment of type i collagen cterminus) were found in the group of rats treated with 309 than in ovariectomized rats, which suggests decreased bone turnover. administration of candidate 309 improved bone mineral density and trabecular architecture after ovariectomy, but did not suppress body weight increase. these results suggest that compound 309 is effective in preventing ovariectomy-induced bone loss while exhibiting exhibited fewer adverse side effects than 17b-estradiol, making mannich base 309 a better choice for the prevention of postmenopausal osteoporosis. three bis-p-mannich bases 310 (r 1 ¼ r 2 ¼ ch 3 ; r 1 er 2 ¼ (ch 2 ) 3 ; r 1 er 2 ¼ (ch 2 ) 4 ) (fig. 59) , derived from diethyl phosphite as substrate and amino acids as amine reagents in the mannich reaction, have been used to investigate the induction of adipogenic or osteogenic differentiation of mesenchymal stem cells isolated from human adipose tissue (hmads cells) [463] . candidates 310 did not affect the adipocyte differentiation, but induced the inhibition of osteoblast formation without any detectable cytotoxic effect, whereas reference drug sodium alendronate elicited a cytotoxic effect even at the lowest concentration used in the study (10 à7 m). therapeutic efficacy and toxicity profiles of ketonic double mannich base 311 (fig. 59) , a putative immunosuppressant which preferentially inhibited jak3 as opposed to several other kinases, were examined [464] . candidate 311 blocked il-2-induced activation of jak3 and its downstream substrates stat5a/b, while it failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, src family members, and serine/threonine protein kinases. mannich base 311 alone prolonged kidney allograft survival and induced transplantation tolerance, and its combination with cyclosporin a presented therapeutic synergism. candidate 311 showed no nephrotoxicity, did not affect hematopoiesis or lipid metabolism, and was not metabolized by the cytochrome p450 3a4 isoform. therefore, because mannich base 311 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs, it may provide significant clinical benefits for transplant patients. an important number of studies report the activity of mannich bases as enzyme inhibitors. although the studies covered in this section usually validate the importance of the topic through a rational and evidence-supported connection between the enzyme under scrutiny and a certain medical condition, they only examine the action of mannich bases as enzyme inhibitors, and do not provide any experimental evidence that these mannich bases could be actually useful agents for the treatment of specific medical conditions. starting from tacrine, the first drug approved for the treatment of alzheimer's disease and a potent inhibitor of both acetylcholinesterase (ache) and butyrylcholinesterase (buche), multifunctional compounds 279 (fig. 53 ) that combine neuroprotective, antioxidant, metal-binding properties, and dual inhibition of ache and buche in a single small molecule have been designed and synthesized through the mannich reaction [431] . tacrinee8hydroxyquinoline hybrids 279 were potent inhibitors of both ache and buche of bovine origin with ic 50 ranging from submicromolar to nanomolar concentrations. candidates containing an unsubstituted 8-hydroxyquinoline moiety and a methylene tether of 7e10 carbon atoms had the most potent inhibitory activities. selected mannich bases 279 were evaluated as inhibitors of human cholinesterases, and they exhibited ic 50 values in the range of 0.5e5.5 nm against ache, and in the range of 6.5e55 nm against buche. mannich base 279 (r 1 ¼ r 2 ¼ h, n ¼ 7) was the most potent dual inhibitor of human ache and buche in this library [431] . other mannich base hybrids 312 (fig. 60) were designed as dual binding site inhibitors of ache by combining tacrine (a recognized inhibitor of catalytic binding site of ache) with the indanone moiety of donepezil (known to be responsible for the binding of this drug to the peripheral site of ache) [465] . their evaluation as inhibitors of ache (bovine erythrocyte) and buche (human serum) using ellman's method showed that the activity of two of these candidates was modest, while the third candidate 312 (z ¼ h, r ¼ och 3 , x ¼ (ch 2 ) 3 ) exhibited moderate activity against ache (25 nm) and good activity against buche (0.6 nm). apparently, the difference in activity between these candidates is due to the lengthening by one methylene group of the alkyl chain linking the tacrine and indanone moieties. 2-benzoxazolone mono-mannich bases 313 (fig. 60 ) derived from secondary aliphatic amines or primary arylamines, and bis-mannich bases 314 derived either from primary aliphatic amines (x ¼ nr, r ¼ c 2 h 5 , r 1 c 6 h 4 ch 2 ch 2 , r 1 ¼ h, 4-cl, 3,4-(ch 3 o) 2 ) or piperazine have been evaluated for ache inhibitory activity using ellman's method. the degree of inhibition was in the range of 70e82% at a concentration of 1 mm, but decreased to 8e34% at 0.1 mm, whereas the degree of ache inhibition by tacrine was virtually the same at both concentrations (greater than 99%) [466] . the most potent inhibitor in this series at both concentration was bis-mannich base 314 derived from piperazine. inhibitory activity against ache and buche of phenolic mannich bases 58 (r 1 ¼ h, ch 3 , allyl, prenyl; nr 2 ¼ n(ch 3 ) 2 , n(c 2 h 5 ) 2 , 1pyrrolidinyl, 1-piperidinyl, 4-morpholinyl) of xanthone derivatives ( fig. 10 ) was found to be moderate to good compared to that of reference drug galantamine [467] . candidates 58 derived from diethylamine as amine reagent in the mannich reaction were generally the most potent inhibitors of both enzymes. the nature of alkyl moiety r 1 modulates the selectivity of inhibitors 58 towards one of these enzymes; thus, the most potent inhibitors of ache feature a methyl group as r 1 , whereas the increasing bulkiness of this substituent appears to favor the inhibition of buche. mannich base 58 (r 1 ¼ prenyl, nr 2 ¼ n(c 2 h 5 ) 2 ) was the most potent dual inhibitor in this series, and molecular docking studies were performed with the view to garner information on the binding mode of this inhibitor with both enzymes. several phenolic mannich bases 315 of naturally occurring flavanone oroxylin a (fig. 61) were more potent inhibitors of intestinal a-glucosidase than parent oroxylin a, but the same candidates were less potent than oroxylin a against yeast a-glucosidase [468] . nonetheless, the most potent inhibitor of intestinal aglucosidase in this series was still 5.5 less potent than reference drug acarbose. because all of the candidates 315 which failed to inhibit both enzymes were derived from acyclic secondary amines as amine reagents in the mannich reaction, the alicyclic nature of secondary amines may responsible for the good inhibitory activity against a-glucosidases in this series. ketonic mannich bases 316 derived from nabumetone (fig. 61) possess modest inhibitory activity against intestinal a-glucosidase (approximately 20 mm), in addition to modest inhibitory activity against another therapeutic target for type 2 diabetes mellitus, obesity and related states of insulin resistance, namely proteintyrosine phosphatase 1b [469] . however, two mannich bases 316 (r ¼ h or 4-oh) were good agonists of peroxisome proliferatoractivated receptors, which are major regulators of lipid and glucose metabolism. a novel series of inhibitors was designed by retaining the nabumetone moiety, while sulfanilamide and 3amino-5-methylisoxazole served as amine reagents in the synthesis of ketonic mannich bases of type 317 and 318, respectively (fig. 61 ) [470] . candidates 317 were generally more potent inhibitors of a-glucosidase than analogous 318, suggesting that sulfonamide moiety is a more potent pharmacophore than aminoisoxazole. mannich base 317 (r ¼ 4-br) inhibited 80% of a-glucosidase activity at a dose of 10 mg/ml. replacement of the sulfonamide moiety with 4-aminobenzoic acid, with a view to mimic the acidic, hydrophilic part of molecules with known antidiabetic activity, led to a novel series of mannich bases 319 (r 1 ¼ h). a second series of candidates 319 (r 1 ¼ c 2 h 5 ) (fig. 61) was also evaluated as a-glucosidase inhibitors, but their activity was generally poorer than that of mannich bases 319 (r 1 ¼ h) derived from 4-aminobenzoic acid [471] . for example, the most potent a-glucosidase inhibitor (67% inhibition) in the series derived from 4-aminobenzoic acid was candidate 319 purine nucleoside phosphorylase (pnp) catalyzes the phosphorolytic cleavage of inosine, guanosine and their 2 0 -deoxy analogues to (deoxy)ribose 1-phosphate and hypoxanthine or guanine. genetical deficiency in pnp leads to complete t-cell deficiency early in life and eventual death of infants from virus infections, and pnp has been identified as a target for the treatment of t-cell lymphoma, rheumatoid arthritis, psoriasis, multiple sclerosis, and other t-cell mediated disorders. a transition state analogue is a compound that occupies the catalytic site of an enzyme and induces the slow conformational changes that would normally occur to place the catalytic site in the appropriate geometry for the search that would locate the transition state with the normal substrate of the enzyme. schramm et al. have developed a series of mannich bases of 9-deazahypoxanthine as transition state analogues for purine nucleoside phosphorylase inhibition, generally called immucilins. although aminomethylated purines and analogues have been present within the firstand second-generation of pnp inhibitors [472, 473] , they have been obtained through reductive amination, and not through direct mannich reaction. because a more expeditious synthesis of second-generation pnp inhibitors by means of the mannich reaction has been later developed [474] , the third generation of these inhibitors included several examples of mannich bases 320 (fig. 62 ) obtained through direct aminomethylation of substrate 9-deazahypoxanthine with amino alcohols such as ethanolamine, 3-amino-propan-1-ol, 4-amino-butan-1-ol, diethanolamine and 3-(2-hydroxyethylamino)propan-1-ol as amine reagents [475] . unfortunately, all these candidates obtained through direct aminomethylation proved to be modest or poor inhibitors of pnp (dissociation constants k i in the range of 780 to 120,000 pm) compared to other mannich bases previously investigated, such as 321 (dadme-immucilinh, k i ¼ 8.5 pm) or 322 fig. 61 . mannich bases as inhibitors of a-glucosidase. (dadme-immuciling, k i ¼ 7.0 pm) (fig. 62) . nonetheless, this study identified two acyclic, simplified alternatives of immucilin analogues, namely 323 (k i ¼ 9.0 pm) having an open-chain amino alcohol with two asymmetric carbon atoms, and 324 (k i ¼ 5.0 pm) derived from achiral dihydroxyaminoalcohol seramide (fig. 62) , as potent inhibitors of pnp. because the enantiomers of both immu-cilinh and dadme-immucilinh 321 have been shown to have pnp binding properties that differ considerably [476] , and since access to enantiomerically pure starting azasugar employed for the generation of 322 requires a suitable enzyme-based route, the discovery of the two simpler mannich bases 323 and 324 represents a significant step forward in the development of clinically useful pnp inhibitors. sulfur-containing transition state analogues have been developed as well, originally as specific inhibitor of pnp from p. falciparum, but it was later shown that sulfur-containing candidates analogous to dadme-immucilinh 320 or mannich base 323 also bind to human native pnp [477] . although human pnp tolerates substitution of 5 0 -hydroxyl in transition state analogues 321 and 324 with alkylthio and arylthio groups, the slow-onset nature of inhibition is lost, and inhibitor dissociation constants increase by two to three orders of magnitude. fluorine-containing analogue 325 (fig. 62) of dadme-immucilinh 321 has also been evaluated as human pnp inhibitor, and its enantiomers exhibit slow-onset binding constants of 32 pm for [(3s,4s)-325] and 1.82 nm for [(3r,4r)-325] [478] . in addition, direct mannich reaction has been used to generate in a similar fashion potent transition state inhibitors for other enzymes, such as methylthioadenosine phosphorylase methylthioadenosine nucleosidase [479, 480] or purine specific nucleoside hydrolase [481] . a large library of a-, band g-amino ketones was screened with a view to discover inhibitors of transglutaminase, but only b-amino ketones (ketonic mannich bases) strongly inhibited this enzyme [482] . mannich bases derived from aliphatic ketones as substrates, or those derived from alkyl aryl ketones as substrates and primary amines as amine reagents were weak inhibitors (ic 50 > 30 mm) of transglutaminase. potent transglutaminase inhibitory activity resided with ketonic mannich bases derived from alkyl aryl ketones as substrates and secondary aliphatic amines as amine reagents. both the nature of the aryl moiety in the substrate and the nature of the alkyl groups attached to the nitrogen atom influenced transglutaminase inhibitory activity of these candidates. generally, heteroaryl-substituted substrates (2-thienyl, 2-benzothienyl, 2furyl, 3-furyl, pyridinyls, pyrazinyl) fared better than substrates having aryl moieties (phenyl, naphthyls), and t-butyl, isopropyl, benzyl or 2-hydroxyethyl groups (but not phenyl) as alkyl substituent at nitrogen afforded potent transglutaminase inhibitors, such as candidate 326 (ic 50 ¼ 81 nm) (fig. 63) . because disulfides are well-known transglutaminase inhibitors, a novel series of b-amino ketones 327 (r ¼ h, n ¼ 0; r ¼ cooch 3 , n ¼ 0; r ¼ cooch 3 , n ¼ 1) (fig. 63 ) derived from cystamine, dimethyl cystine ester, and dimethyl homocystine ester as amine reagents in the mannich reaction were synthesized and found to be approximately 300 times more active than the starting disulfides [483] . mannich bases derived from 2-acetylthiophenes were once more the most potent inhibitors in this series, while the nature of the disulfide-containing amine moiety did not significantly influence the activity of candidates 327 having the same heterocyclic or carbocyclic moiety r 1 . mannich bases 328 derived from 3,4-dimethylphenol ( fig. 63 ) have been evaluated in vitro as inhibitors of two human carbonic anhydrase (hca, ec 4.2.1.1) isozymes i and ii using the hydratase and esterase assays, respectively [484] . only two candidates exhibit weak hca ii inhibitory effects on esterase activity, whereas other two mannich bases 328 could be used as carbonic anhydrase activators. phenolic mannich bases 280 derived from quercetin (fig. 53 ), 329 derived from baicalein and 330 derived from 1-(2-hydroxy-4methoxyphenyl)-3-phenylprop-2-en-1-one ( fig. 63 ) were screened for inhibition of cyclin-dependent kinases using a biochemical assay method based on fluorescence resonance energy transfer [485] . candidates 280 and 330 are essentially devoid of inhibitory activity of cyclin-dependent kinases; on the other hand, baicalein mannich bases 329 were 6e20 times more potent than the parent flavone, suggesting that the presence of a nitrogen atom at c-8 is crucial for the inhibition of cyclin-dependent kinases, while the presence of a second heteroatom in the amine moiety (e.g., oxygen in morpholine, sulfur in thiomorpholine, nitrogen in n-methylpiperazine) further increases the potency of these candidates. virtual screening of a commercial library containing drugs and drug-like molecules against a 3d model of heparanase led to the identification of several candidates with high scores for binding affinity; they were subsequently tested in nmr competitive inhibition experiments using suramine as a known heparanase inhibitor [486] . one of these candidates is amodiaquine 201 (fig. 38) , and a subset of fourteen representative compounds that retained the characteristic 4-phenylamino-chloroquinoline scaffold of amodiaquine, but presented different functional groups in the phenylamino moiety, was further selected for nmr competitive inhibition experiments. although no candidate with improved heparanase inhibitory activity over that of amodiaquine emerged from this subset, several structural requirements for the binding of an inhibitor to the active site of the enzyme were obtained. four mannich bases 331 of nitroxoline (fig. 63 ) were found to inhibit efficiently the activity of methionine aminopeptidase-1 from burkholderia pseudomallei with ic50 values ranging from low micromolar to 30 nm, and a few candidates in this series also showed in vitro growth inhibition of burkholderia thailandensis [487] . hexacyclic indole mannich bases 332 (r 1 ¼ h or ch 3 , nr 2 ¼ n(c 2 h 5 ) 2 , 1-pyrrolidinyl) and 333 (r 1 ¼ ch 3 , nr 2 ¼ 4morpholinyl) derived from annelated maleimidoindolodiazepines (fig. 63 ) were tested against a panel of 25 human protein kinases, but they were inactive against all of the tested enzymes at micromolar concentrations [488] . out of four synthesized acetylenic mannich bases 334 (nr 2 ¼ n(c 2 h 5 ) 2 , 1-pyrrolidinyl, n-allyl-n-methyl, n-(2dimethylaminoethyl)-n-methyl) derived from 7-(prop-2-ynyloxy) chromen-2-one (fig. 63) , three were inhibitors of squalene-hopene cyclase [489] . candidate 334 (nr 2 ¼ n-allyl-n-methyl) was half as potent as the hoffmanela roche's anticholesteremic drug ro 48-8071, which is an effective inhibitor of both squalene-and oxidosqualene cyclases that features the same secondary amino group in its structure. thiazolidinone c-mannich bases 335 (r 1 ¼ 4-ch 3 c 6 h 4 , 4-no 2 c 6 h 4 , 2-furyl, 3,4,5-(ch 3 ) 3 c 6 h 2 ; nr 2 ¼ n(c 2 h 5 ) 2 , 4morpholinyl, 4-methylpiperazinyl) (fig. 63) were evaluated as inhibitors of schistosoma mansoni cercarial elastase, but they were all inactive [490] . oxadiazolethione n-mannich bases 336 having a naphthalen-1ylmethyl residue at c-5 ( fig. 63 ) were all active against mushroom tyrosinase, and 6 to 10 times more potent than reference compound kojic acid [491] . the presence of a thione group able to chelate with copper, and the ability of the cyclic secondary amine to form extensive hydrophobic contacts within the binding site of tyrosinase appear to be responsible for the activity. another oxadiazolethione n-mannich base, namely 139m (table 2) , presented moderate inhibitory activity against urease within the series of compounds investigated, but no comparison with the activity of a well-established urease inhibitor was provided [201] . 19.1. ligands of 5-hydroxytryptamine receptors 5-hydroxytryptamine receptors (5-ht receptors), also known as serotonin receptors, belong to the group of g protein-coupled receptors (with the exception of 5-ht 3 , which is a ligand-gated ion channel). they are found in the central and peripheral nervous systems, and their function is to mediate both excitatory and inhibitory neurotransmission. 5-ht receptors are activated by their natural ligand, the neurotransmitter serotonin. mannich bases 245 (n ¼ 1 or 2) (fig. 44) were evaluated as ligands for the types of 5-ht receptors that could be involved in the mediation of the anticonvulsant effect of these compounds, but the candidates exhibited only moderate to low affinity for both 5-ht 1a (k i ¼ 81e370 nm) and 5-ht 2a receptors (k i ¼ 126e1370 nm) [383] . the nature of the spiranic cycloalkyl group did not influence serotonin receptor affinity significantly, although cyclohexylsubstituted candidates 245 were slightly more active than cyclopentyl-substituted analogues. the nature of the substituent in the aryl moiety at n-4 of piperazine ring modulates the selectivity towards one of these 5-ht receptors; thus, mannich bases 245 (r ¼ 2-och 3 ) showed the highest affinity for 5ht 1a receptors and the lowest affinity for 5-ht 2a receptors, while the highest 5-ht 2a receptor affinity was shown by mannich bases 245 (r ¼ 3-cf 3 ). in addition, computer-aided design of novel mannich bases of imidazoline-2,4-diones led to the identification of several compounds with potential dual affinity for 5ht 1a receptors and serotonin transporter, whose synthesis and evaluation finally led to candidates 337 (r ¼ h or f) (fig. 64) having the desired pharmacological profile [492] . a series of quinoxaline-2-carboxamides n-substituted with phenolic mannich bases moieties having either one or two aminomethyl functions have been synthesized and tested for 5-ht 3 receptor antagonistic activity in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-ht 3 agonist, 2-methyl-5-ht [493, 494] . candidates 338 (r 1 ¼ cl) (fig. 64 ) exhibited mild to moderate antagonist activity, and double mannich bases were generally less potent than their mono-mannich bases counterparts, but mannich base 338 (r 1 ¼ cl, r 2 ¼ h, nr 2 ¼ 4-methylpiperazin-1-yl) had 5-ht 3 receptor antagonistic activity comparable to that of reference compound ondansetron [493] . replacement of chlorine with methoxy led to less potent candidates 338 (r 1 ¼ och 3 ), whereas candidates with an ethoxy group at position 3 of quinoxaline ring were even less potent [494] . the presence of piperazines in the aminomethyl moiety of candidates 338 seemed to be more favorable for 5-ht 3 receptor antagonistic activity than the presence of other cyclic secondary amines. dopamine receptors are g protein-coupled receptors that are widely distributed in the brain, and whose primary endogenous ligand is the neurotransmitter dopamine. there are at least five subtypes of dopamine receptors, but d 1-2 receptor subtypes usually predominate, as they are 10e100 times more numerous than d [3] [4] [5] subtypes. discovery of l-745,870 339 [495] and fauc 113 340 [496] (fig. 65 ) as potent and selective d 4 receptor ligands has fueled the search for novel chemical entities having the ability to bind to dopamine receptors. based on the observation that these two ligands and many other dopamine receptor ligands feature a methylene bridge between the piperazine ring and the (hetero)aromatic part of the molecule, various analogues have been designed and synthesized. however, the majority of these analogues were obtained either through nucleophilic displacement by an appropriately substituted piperazine of a halogen atom from a halomethyl derivative of a selected (hetero)aromatic substrate, or through reductive amination of a formyl-substituted (hetero)aromatic substrate in the presence of a piperazine. literature search revealed a few examples when the mannich reaction was employed to synthesize the ligands to be evaluated for dopamine receptor binding ability. in addition to pyrrolo [2,3-b] pyridine and pyrazolo [1,5-a]pyridine ring systems used to in the generation of ligands 339 and 340, imidazo[1,2-a]pyridine is another example of an azaindole ring system that was aminomethylated to yield mannich bases 341 (fig. 65) [497] . receptor binding profiles of candidates 341 and several mono-mannich bases 342 derived from pyrrole ( fig. 65) were determined in vitro by measuring their ability to compete with [ 3 h]spiperone for the cloned human dopamine receptor subtypes d 2 long , d 2 short , d 3 and d 4.4 , while d 1 receptor affinities were measured employing an assay that used d 1 selective ligand [ 3 h]sch 23390 and porcine striatal membranes. all of the mannich bases 341 and 342 were selective ligands for the d 4 receptor subtype, and the candidates with an imidazo[1,2-a]pyridine scaffold were more potent than their counterparts having a pyrrole ring. the most potent mannich bases 341 (r ¼ 2-c 2 h 5 o, 2,3-cl 2 , 3-cf 3 ) showed an affinity for the dopamine d 4 receptor in the low nanomolar range, and their selectivity for this receptor subtype exceeded that of reference drug clozapine [497] . several small series of candidates comprising mannich bases 343, 344 and 345 (r 1 ¼ h or cl) having a pyrrolo[2,3-d]pyrimidine scaffold (fig. 65 ) were designed and evaluated as ligands for dopamine receptor subtypes d 1 , d 2 long , d 2 short , d 3 and d 4.4 , but they had no appreciable affinity for any of the dopamine receptors tested [498] . the strongest binding in these series was recorded for candidates 343 (r ¼ 2-ch 3 o) and 345 (r 1 ¼ h, r ¼ 2-ch 3 o) at dopamine d 4.4 receptor, with k i values of 2.4 and 1.9 nm, respectively. novel heteroaromatic scaffolds have been employed in the synthesis of mannich bases 346, 347 and 348 (fig. 65 ) as potential d 4 receptor ligands [499] . candidate 346 derived from pyrrolo[2,3c]pyridine ring system bound selectively at d 4.4 receptor (k i ¼ 6.4 nm). evaluation of mannich bases 347 generated from imidazo [1,2-c] pyrimidines and having various substituents at positions 2, 5 and 7 led to different results, depending on the nature and number of these substituents. thus, mannich base 347 (r 1 ¼ r 2 ¼ r 3 ¼ h) derived from the unsubstituted scaffold and all of the mannich bases 347 (r 1 ¼ ch 3 , r 2 ¼ r 3 ¼ h) obtained from 7methylimidazo[1,2-c]pyrimidine had no effect on dopamine d 4 receptor. the results for the binding assay for mannich bases derived from dimethyl-substituted imidazo [1,2-c] pyrimidines showed that candidates 347 (r 1 ¼ r 3 ¼ ch 3 , r 2 ¼ h) have binding affinities to dopamine d 4 receptor in the range of reference drug clozapine, while candidate 347 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ h) is a weak ligand. further modifications, such as introduction of a carbethoxy group in the structure of candidates 347 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ cooc 2 h 5 ), or two methoxy groups in mannich bases 347 (r 1 ¼ r 2 ¼ och 3 , r 3 ¼ h), led to compounds without affinity for dopamine d 4 receptor. in the series of mannich bases derived from 2,5,7-trimethylimidazo[1,2-c]pyrimidine, only candidate 347 (r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r ¼ 2-och 3 ) had good binding properties for the dopamine d 4 receptor, whereas another candidate 347 (r 1 ¼ r 2 ¼ r 3 ¼ ch 3 , r ¼ 4-och 3 ) exhibited a slight selectivity for dopamine d 3 receptor. use of pyrrolo [3,2-d] pyrimidine as scaffold led to some of the most potent ligands of dopamine d 4 receptor in this study (k i values between 2.4 and 3.8 nm). the nature of the amino group at position 2 of pyrrolo [3,2-d] pyrimidine scaffold modulates the activity; thus, mannich bases 348 (nr 2 ¼ 1pyrrolidinyl) had good affinities for dopamine d 4 receptor, but replacement of pyrrolidine with morpholine resulted in significant loss of dopamine d 4 receptor binding affinity. two novel indole mannich bases 59 (r ¼ 2-c 2 h 5 oc 6 h 4 , 2,3-cl 2 c 6 h 3 ) (fig. 11) were synthesized through direct aminomethylation and evaluated as ligands for several dopamine receptor subtypes, along with other heteroarylmethylpiperazines [500] . both candidates were potent and selective dopamine d 4 receptor ligands, and mannich base 59 (r ¼ 2-c 2 h 5 oc 6 h 4 ) was the most potent compound in this library (k i ¼ 0.03 nm). in addition, mannich base 59 (r ¼ 2,3-cl 2 c 6 h 3 ) was the most potent candidate having a 4-(2,3-dichlorophenyl)piperazine moiety, suggesting the importance of indole as (hetero)aromatic moiety in this type of dopamine d 4 receptor ligands. the effect on the affinity to d 2 , d 3 and d 4 dopamine receptor subtypes of replacement of 4-arylpiperazinyl in mannich bases with other monocyclic or bicyclic amine residues was also investigated [501] . mannich bases 349 (x ¼ ch or n) (fig. 66) derived from 3-(4-chlorophenyl)-8-azabicyclo[3.2.1]octan-3-ol as amine reagent in the mannich reaction had modest affinity and moderate selectivity for dopamine d 3 receptor subtype compared to d 2 and d 4 receptor subtypes. pyrrolidinol analogues 350 (x ¼ ch or n) (fig. 66 ) had poor binding affinities to all subtypes of dopamine receptors, while homopiperazine analogue 351 (fig. 66 ) had moderate binding affinity to dopamine d 4 receptor subtype (k i ¼ 18.6 nm). candidate 352 derived from 3,9-diazabicyclo[4.2.1] nonane ring system as amine reagent in the mannich reaction ( fig. 66 ) bound poorly to all types of dopamine receptor investigated in this study. although candidate 353 (fig. 66 ) derived from 2,5-diazabicyclo[2.2.1]heptane ring system as amine reagent in the mannich reaction had the highest d 3 receptor affinity of all the compounds tested in this paper (k i ¼ 11 nm), it also had poor selectivity towards d 3 (k i d2 ¼ 62 nm, k i d4 ¼ 69 nm). mannich base 354 derived from 2-(2-pyrimidinyl)piperazine (fig. 66 ) had modest binding affinity to dopamine d 4 receptor subtype (k i ¼ 56 nm), but good selectivity over d 2 and d 3 subtypes. thyroid receptors are nuclear receptors belonging to a superfamily whose members function as hormone activated transcription factors. the regulation of transcription for thyroid receptors is controlled by their natural ligand, the thyroid hormone. both unliganded and liganded thyroid receptors can bind to and regulate genes under the control of thyroid response elements, but the liganded thyroid receptor complex can also recruit a particular coactivator protein out of many available, with a view to steer the course of transcriptional regulation. high-throughput screening of a library of compounds identified a number of hits for inhibitors of the interaction between thyroid receptor and its coactivator src2 (steroid receptor coactivator 2), and two of these hit compounds were ketonic mannich bases 355 and 356 (fig. 67) [502] . these candidates represent a new class of thyroid receptor antagonists that have exceptional selectivity towards b isoform and are active in the presence of thyroid hormone, while being able to silence its signaling. mannich bases 355 and 356 are most likely covalently bound to thyroid receptor as a result of an alkylation process, thus inhibiting its hormone-induced gene transcription. subsequent preliminary sar showed that a hydrophobic moiety para to the ketone function in other ketonic mannich bases analogous to 355 and 356 is an important structural feature of potent inhibitors of the thyroid receptorecoactivator interaction [503] . a second series of candidates explored the effect that the nature of the amino group in ketonic mannich bases analogous to 355 and derived from 4-nhexylacetophenone has on the interaction between coregulatory protein src2 and both isoforms of thyroid receptors. all the synthesized candidates inhibited the interaction in the low micromolar range, with a roughly 2-fold selectivity towards isoform b. these ketonic mannich bases most likely function as prodrugs for the true active species, namely the a,b-unsaturated ketones resulted through deamination. therefore, a collection of enones having various substituents at the carbonecarbon double bond was also tested with a view to provide an insight on the mechanism of action of ketonic mannich bases as inhibitors of this interaction. the results showed that the inhibition of the interaction between thyroid receptor and coactivator protein src2 depends strongly on the substitution pattern at the carbonecarbon double bond in these a,b-unsaturated ketones. the most potent was the unsubstituted enone 357 (fig. 67) , which formally arises from 355 through deamination, whereas all other substituted enones were weaker inhibitors than 357. subsequent investigations confirmed that enone 357 is the actual inhibitor, and that compound 357 is generated within the binding site, where it remains bound until it reacts with one of the local cysteine residues [504] . in addition, an attempt to examine a complex between thyroid receptor b and mannich base 355 by x-ray crystallography led to the detection in the activation function-2 cleft of thyroid receptor b of enone 357 instead of mannich base 355. although enone 357 did not react rapidly with thyroid hormone, it positioned close to several cysteine residues. several other experiments showed that, once formed, enone 357 slowly alkylates nearby cys298 residue to occlude activation function-2 pocket of thyroid receptor b. significant improvement of pharmacological properties (especially potency and therapeutic index) of inhibitors of thyroid hormone receptor coactivator binding based on ketonic mannich bases was achieved with a series of novel candidates having in the aromatic ring electron withdrawing substituents associated with high efficacy, and a sulfonyl group to reduce cytotoxicity. also, piperazinone and 4-acetylpiperazine were used as preferred amine moieties with a view to minimize ion channel activity of these inhibitors [505] . the most potent compounds 358 and 359 (r ¼ 2,3-cl 2 or 2,5-cl 2 ) (fig. 67) showed outstanding thyroid hormone receptor coactivator interaction inhibitory potency, good therapeutic index, while no inhibition of kcl-stimulated depolarization of hek293-herg cells was observed for these mannich bases. androgen receptor is a ligand-regulated transcription factor in the nuclear receptor superfamily. the receptor, which represents an important molecular target for the treatment of prostate cancer, is activated through the binding of its two natural endogenous ligands, testosterone or 5a-dihydrotestosterone. subsequent activation of androgen-responsive genes can be blocked by androgen receptor antagonists through competitive inhibition. highthroughput screening of a structurally diverse chemical library comprising 16,000 synthetic and natural compounds led to the identification of 130 hit compounds that exhibited more than 85% competitive inhibition of 5a-dihydrotestosterone binding to androgen receptor [506] . mannich base 360 (r 1 ¼ r 2 ¼ ch 3 , r 3 ¼ c 6 h 5 ) (fig. 68 ) was selected for further development owing to its consistent androgen receptor antagonist activities in a cellbased assay using monkey kidney fibroblast cv-1 cells, and fortynine analogues were designed based on its b-amino ketone core structure. nine of these analogues showed higher binding affinity to androgen receptor than 5a-dihydrotestosterone, and evaluation of the enantiomers of candidate 360 (r 1 ¼ no 2 , r 2 ¼ cl, r 3 ¼ 2furyl) showed that the androgen receptor-modulating activity and binding resided with the dextrorotatory isomer. sar within this library of ketonic mannich bases revealed that the presence of chlorine as substituent r 2 , preferably para to the amino group, is important for androgen receptor antagonistic effects, while the presence of an electron-withdrawing group as substituent r 1 is crucial for high-affinity binding to the receptor. good androgen fig. 67 . mannich bases as inhibitors of the interaction between thyroid receptors and their coregulators. receptor binding activity was found among analogues having phenyl, 2-furyl and 2-thienyl as substituent r 3 , and mannich bases with heteroaryl moiety exhibited less cytotoxicity towards hela cells than mannich bases with r 3 ¼ (substituted)phenyl. candidate 360 (r 1 ¼ no 2 , r 2 ¼ cl, r 3 ¼ 2-furyl) inhibited the growth of sc3 cells (a mouse mammary cell line that responds well to androgens), while its effects on the proliferation of pc3 cells (a human prostate cancer cell line that lacks measurable androgen receptors) were very weak. mannich bases of type 360 were subsequently disclosed as selective non-steroidal antagonists of another member of the nuclear receptor superfamily, namely progesterone receptor, and sar within a collection of mannich bases 360 showed that coordinating effects of substituents r 1 and r 3 can modulate the potency and selectivity for progesterone receptor over androgen receptor [507] . mannich bases that modulate the activity of acetylcholine receptors have been the topic of two recent studies. two quaternary salts 361 (r ¼ ch 3 , allyl) derived from bis-aminomethylated pyrazinodiindole (fig. 68) were synthesized in order to probe their binding to the allosteric binding domain of muscarinic receptor m 2 [508] . these quaternary salts showed only slightly higher binding affinity to muscarinic m 2 receptor subtype than the corresponding parent double mannich bases, which in turn exhibited relatively poor allosteric potency. due to the structural similarity of candidates 361 and neuromuscular-blocking agents, their binding affinity to the muscle-type of the nicotinic acetylcholine receptors was also determined. thus, compound 361 (r ¼ ch 3 ) had a 34-fold higher affinity for the muscle type nicotinic acetylcholine receptor than for the allosteric site of m 2 receptors. double mannich bases 362 (nr 2 ¼ 1-piperidinyl, 2-methyl-1-piperidinyl, 1-azepanyl) derived from phthalamide (fig. 68) had a competitive antagonistic effect to acetylcholine on isolated guinea pig ileum that was poorer than that of reference compound atropine, and behaved as noncompetitive antagonists at higher concentration [509] . in addition, candidates 362 (nr 2 ¼ 1-piperidinyl, 2-methyl-1-piperidinyl, 1-azepanyl) antagonize the motor effect of oxotremorine in mice, while mannich base 362 (nr 2 ¼ 2,6-dimethyl-1-piperidinyl) was inactive in both tests. indole mannich base 59 (r ¼ phenethyl) (fig. 11 ) had moderate binding affinity to both sigma-1 (k i ¼ 14.2 nm) and sigma-2 (k i ¼ 55.8 nm) receptors, and can be considered as a potent, but non-selective ligand for sigma receptors [80] . ketonic mannich bases 363 (fig. 68) derived from cyclic ketones such as a-tetralone (x ¼ ch 2 ch 2 ), chromanone (x ¼ och 2 ), indanone (x ¼ ch 2 ) or benzosuberone (x ¼ (ch 2 ) 3 ) as substrates and either 4benzylpiperidine (z ¼ ch 2 ) or 4-benzylpiperazine (z ¼ n) as amine reagents in the mannich reaction were also evaluated for their affinity towards sigma-1 and sigma-2 receptors [510] . generally, 4-benzylpiperazine-containing mannich bases were higher-affinity sigma receptor ligands than the corresponding 4benzylpiperidine-containing analogues. compared to other alkylpiperidines or alkylpiperazines that were evaluated in this study, ketonic mannich bases 363 had only moderate affinity for sigma receptors. the presence of the carbonyl group in their structure appears to be detrimental, especially for the affinity to sigma-1 receptors, and the binding affinity seems to be influenced by the size of the saturated ring fused to the aromatic ring. thus, sixmembered candidates 363 are more potent than mannich bases 363 derived from indanone or benzosuberone. however, candidate 363 (x ¼ ch 2 ch 2 , z ¼ ch 2 ) was one of the most selective compounds in this study, with a selectivity towards sigma-2 receptors of approximately 0.1 (k i-s1 ¼ 24 nm, k i-s2 ¼ 3.4 nm). in contrast, mannich bases 363 derived from 4-benzylpiperazine were consistently better ligands for sigma-1 than for sigma-2 receptors. mannich bases of indoles substituted in the carbocyclic ring with a carboxamide group were evaluated as human histamine h 3 receptor antagonists [511] . preliminary analysis of the binding affinity of candidates 364 (r ¼ i-c 3 h 7 , c-c 4 h 7 , x ¼ ch 2 , o, nc 3 h 7 -i, nc 4 h 7 -c) (fig. 68) to human h 3 receptor with respect to different positional isomers within this series established a correlation between the 3,6-substitution pattern in 364 and good human h 3 receptor affinity, while the 3,4-substitution pattern was the least favorable for h 3 receptor binding affinity. the presence of piperidine and morpholine in the aminomethyl group led to compounds with excellent histamine h 3 receptor affinity, whereas the presence of 4-substituted piperazines in the aminomethyl group was not well tolerated. several mannich bases 364 of indoles with a 3,7substitution pattern also presented reasonable potency as human h 3 antagonists; in this case, the presence of a cyclobutyl residue in the piperazine ring in the carboxamide group seems to be more favorable for human histamine h 3 binding affinity than substitution with an isopropyl residue. in the search for new ligands able to discriminate among the three a 1 -adrenergic receptor subtypes, a series of mannich bases 365 (r ¼ ch 3 , n-c 3 h 7 , ch 2 c 6 h 5 , ch 2 c 6 h 11 -c, c 6 h 5 ) derived from 1,2,3,4-tetrahydro-4h-carbazol-4-ones (fig. 68) , that are structurally related to the potent a 1 -adrenergic receptor antagonist heat, were synthesized and evaluated [512] . as a general trend, all candidates showed lower affinities than heat for all three a 1 -adrenergic receptor subtypes, and replacement of tyramine with 4-(2substituted phenyl)piperazines did not enhance the affinity towards any subtype in particular. in addition, mannich bases 366 derived from 3-acetylindole and the same amines ( fig. 68 ) were designed as "open chain" analogues of candidates 365, and their evaluation showed a slight improvement in the affinity for all three a 1 -adrenergic receptor subtypes. starting from a hit obtained by screening a large library of compounds, and employing subsequent stepwise refinement of structural features, a series of mannich bases 367 (r ¼ (substituted) phenyl) of pyrazolones (fig. 68 ) was designed as ligands of cc chemokine receptor 3 (ccr3), a potential molecular target for treatment of allergic asthma [513] . the evaluation of binding to human ccr3 receptor of candidates 367 showed that most compounds display ic 50 values around 100 nm, and that only two of these mannich bases (r ¼ 4-fc 6 h 4 and 2-pyridinyl) bound more tightly to ccr3 than the initial hit compound (ic 50 ¼ 32 nm). because the lead compound 367 (r ¼ 4-fc 6 h 4 , ic 50 ¼ 20 nm) suffered from poor water solubility and metabolic stability, less lipophilic analogues were prepared by the introduction of ionizable groups in different parts of the molecule. replacement in the structure of 367 (r ¼ 4-fc 6 h 4 ) of the 4-chlorophenyl moiety with 4-fluorophenyl, 4-nitrophenyl, 4-sulfonamidophenyl, 4aminophenyl or pyridinyl moieties, as well as substitution of the 2,6-diflurophenyl moiety with pyridinyl or pyridinyl-n-oxide led to a second series of pyrazolone mannich bases 368 ( fig. 68) with improved solubility in water and enhanced metabolic stability. although structural modulations in the r 1 moiety generally decreased the binding affinity of candidates 368 to ccr3, modifications in the r 2 part afforded potent compounds (ic 50 ¼ 12 nm). two n-mannich bases 369 (r ¼ h or f) of a pyrazole derivative (fig. 68 ) were reported in a study that examined the interaction of a series of 4-heteroaryl-2-phenylquinolines with neurokinin nk-2 and nk-3 receptors, but they were inactive (k i > 10 mm) [514] . throughout the last decade, a large number of novel mannich bases have been synthesized and evaluated as potential treatments for a multitude of diseases and medical conditions, as prodrugs, or as molecules eliciting a response from biological targets. the vast amount of data concerning the structureeactivity relationship for mannich bases derived from structurally diverse substrates has added to previous knowledge, thus allowing better insight into the design of more effective drug candidates based on the aminomethylation reaction in the future. tremendous progress has been made in the field of anticancer agents and antimicrobials, and the last decade has witnessed the chemical modification of many biologically-active, naturally-occurring substrates or well established drugs by means of the mannich reaction with a view to improve their biological activity. as such, the mannich reaction has earned its rightful place as a powerful tool in medicinal chemistry, both for the synthesis of novel chemical entities endowed with various and interesting biological properties, and for the modification of physico-chemical properties of a candidate, that ultimately influence the candidate's biodisponibility, performance and pharmacological activity as a drug. advances in the chemistry of mannich bases further advances in the chemistry of mannich bases mannich bases: chemistry and uses effect of aminomethyl (n-mannich base) derivatization on the ability of s 6 -acetyloxymethyl-6-mercaptopurine prodrug to deliver 6-mercaptopurine through hairless mouse skin prodrugs e an efficient way to breach delivery and targeting barriers gilmer, b-aminoketones as prodrugs with phcontrolled activation anticancer and cytotoxic properties of mannich bases bioactivities of chalcones cytotoxic thiol alkylators sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds evaluation of some mannich bases of cycloalkanones and related compounds for cytotoxic activity cytotoxic and anticancer activities of some 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides cytotoxic activities of mannich bases of chalcones and related compounds cytotoxic activities of mono and bis mannich bases derived from acetophenone against renca and jurkat cells cytotoxic activities of some mono and bis mannich bases derived from acetophenone in brine shrimp bioassay cytotoxicity of some azines of acetophenone-derived mannich bases against jurkat cells synthesis of some mannich bases with dimethylamine and their hydrazones and evaluation of their cytotoxicity against jurkat cells evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells cytotoxic and anticonvulsant aryloxyaryl mannich bases and related compounds biological evaluation and structureactivity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlorides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic t cells effect of some bis mannich bases and corresponding piperidinols on dna topoisomerase i the design and cytotoxic evaluation of some 1-aryl-3-isopropylamino-1-propanone hydrochlorides towards human huh-7 hepatoma cells cytotoxicity of 1-aryl-3-butylamino-1-propanone hydrochlorides against jurkat and l6 cells synthesis of 1-aryl-3-phenethylamino-1-propanone hydrochlorides as possible potent cytotoxic agents biological activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols on pc-3 cells and dna topoisomerase i enzyme effect of acetophenone derived mannich bases on cellular glutathione level in jurkat cells. a possible mechanism of action syntheses and stability studies of some mannich bases of acetophenones and evaluation of their cytotoxicity against jurkat cells effects of mannich bases on cellular glutathione and related enzymes of jurkat cells in culture conditions the effects of some mannich bases on heat shock proteins hsc70 and grp75, and thioredoxin and glutaredoxin levels in jurkat cells cytotoxic mannich bases of 1-arylidene-2-tetralones cytotoxic and topographical properties of 6-arylidene-2-dimethylaminomethylcyclohexanone hydrochlorides and related compounds potential role of nmyristoyltransferase in cancer inhibition of protein n-myristoylation: a therapeutic protocol in developing anticancer agents synthesis and cytotoxic and mechanistic studies of a-arylidenecyclohex(pent)anone or aarylcyclohexanone a'-mannich bases and their deoxo bisaryl cyclohex(pent) ene analogs synthesis and anticancer activity of 2-alkylaminomethyl-5-diaryl-methylenecyclopentanone hydrochlorides and related compounds dimmock, a-substituted 1-aryl-3-dimethylaminopropanone hydrochlorides: potent cytotoxins towards human widr colon cancer cells 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides and related adducts: a quest for selective cytotoxicity for malignant cells synthesis of 4 0 -hydroxy-3 0 -piperidinomethylchalcone derivatives and their cytotoxicity against pc-3 cell lines synthesis and cytotoxicity of novel 3-aryl-1-(3 0 -dibenzylaminomethyl-4 0 -hydroxyphenyl)-propenones and related compounds design, synthesis and in vitro cytotoxic activity evaluation of new mannich bases design, synthesis, and biological evaluation of mannich bases of heterocyclic chalcone analogs as cytotoxic agents 1-(3-aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones: a novel group of tumour-selective cytotoxins preparation of aminoalkyl substituted chalcones for use in anticancer and antimalarial pharmaceutical compositions preparation of a series of novel bichalcones linked with a 1,4-dimethylenepiperazine moiety and examination of their cytotoxicity new bichalcone analogs as nf-kb inhibitors and as cytotoxic agents inducing fas/cd95-dependent apoptosis azidothymidine is effective against human multiple myeloma: a new use for an old drug hłado n, synthesis and anticancer activity of 5 0 -chloromethylphosphonates of 3 0 -azido-3 0 -deoxythymidine (azt) antileukemic activity and cellular metabolism of the aryl phosphate derivative of bromo-methoxy zidovudine (compound whi-07) synthesis and in vitro cytotoxic activity evaluation of novel mannich bases and modified azt derivatives possessing mannich base moieties via click chemistry synthesis and pharmacological exploitation of clioquinol-derived copper-binding apoptosis inducers triggering reactive oxygen species generation and mapk pathway activation cytotoxicity and antimicrobial activity of some naphthol derivatives synthesis and cytotoxicity evaluation of some 8-hydroxyquinoline derivatives synthesis and structure-activity relationship study of 8-hydroxyquinoline-derived mannich bases as anticancer agents design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators recent advancement in nonsteroidal aromatase inhibitors for treatment of estrogen-dependent breast cancer facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors nitrogen-containing apigenin analogs: preparation and biological activity synthesis and antiproliferative activity of oxazinocarbazole and n,n-bis(carbazolylmethyl)amine derivatives synthesis, antitumour activity and structureeactivity relationships of 5h-benzo[b]carbazoles display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and compare algorithm 10-anthradiquinone as precursor for antitumor compounds the natural anticancer agent plumbagin induces potent cytotoxicity in mcf-7 human breast cancer cells by inhibiting a pi-5 kinase for ros generation anti-tumor effects of novel 5-o-acyl plumbagins based on the inhibition of mammalian dna replicative polymerase activity inhibitory effect of novel 5-o-acyl juglones on mammalian dna polymerase activity, cancer cell growth and inflammatory response cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-o-methyl derivative antiproliferative activity of synthetic naphthoquinones related to lapachol. first synthesis of 5-hydroxylapachol novel platinum(ii) complexes of 3-(aminomethyl)naphthoquinone mannich bases: synthesis, crystal structure and cytotoxic activities exploring the dna binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone mannich bases and their platinum(ii) complexes novel 3-(aminomethyl)naphthoquinone mannich base-platinum(iv) complexes: synthesis, characterization, electrochemical and cytotoxic studies one-pot synthesis and cytotoxicity studies of new mannich base derivatives of polyether antibiotic e lasalocid acid novel hexacyclic camptothecin derivatives. part 1: synthesis and cytotoxicity of camptothecins with an a-ring fused 1,3-oxazine ring optimization of tocotrienols as antiproliferative and antimigratory leads quinone methides tethered to naphthalene diimides as selective g-quadruplex alkylating agents hybrid ligandalkylating agents targeting telomeric g-quadruplex structures synthesis and biological activities of quinazoline derivatives with ortho-phenol-quaternary ammonium salt groups dna binding, antiviral activities and cytotoxicity of new furochromone and benzofuran derivatives synthesis and biological evaluation of 1,3-dihydroxyxanthone mannich base derivatives as potential antitumor agents synthesis and in vitro evaluation of novel indole-based sigma receptors ligands synthesis and cytotoxic activity of novel 3-methyl-1-[(4-substituted-1-piperazinyl)methyl]-1h-indole derivatives design, synthesis, and biological evaluation of indole-based 1,4-disubstituted piperazines as cytotoxic agents amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (icmt) 3-aminomethyl derivatives of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione for circumvention of anticancer drug resistance synthesis and structureeactivity relationship studies of naphtho[2,3-f]indole-5,10-dione aminoalkyl derivatives: a new class of topoisomerase i inhibitors synthesis of 4-substituted 3-[3-(dialkylaminomethyl)indol-1-yl]maleimides and study of their ability to inhibit protein kinase c-a, prevent development of multiple drug resistance of tumor cells and cytotoxicity synthesis and sar of novel 4-morpholinopyrrolopyrimidine derivatives as potent phosphatidylinositol 3-kinase inhibitors cytotoxic and anticancer activities of isatin and its derivatives: a comprehensive review from synthesis and in-vitro cytotoxicity evaluation of gatifloxacin mannich bases hybrid pharmacophore design and synthesis of isatinebenzothiazole analogs for their anti-breast cancer activity synthesis of novel isatin-thiazoline and isatin-benzimidazole conjugates as anti-breast cancer agents design and synthesis of anti-breast cancer agents from 4-piperazinylquinoline: a hybrid pharmacophore approach hydroxyphenyl)-3-substituted-2,3-dihydro-1,3,4-oxadiazole-2-thione derivatives: promising anticancer agents synthesis of some novel 3,5-disubstituted 1,3,4-oxadiazole derivatives and anticancer activity on eac animal model synthesis, quantitative structure-activity relationship and biological evaluation of 1,3,4-oxadiazole derivatives possessing diphenylamine moiety as potential anticancer agents synthesis, antitumor and antimicrobial activity of novel 1-substituted phenyl-3-[3-alkylamino(methyl)-2-thioxo-1,3,4-oxadiazol-5-yl]-b-carboline derivatives synthesis and antitumor activity of fluoroquinolone c3-isostere derivatives: oxadiazole mannich base derivatives synthesis, characterization and in vitro cytotoxic properties of some new schiff and mannich bases in hep g2 cells design, synthesis and antitumor activities of fluoroquinolone c-3 heterocycles (iv): s-triazole schiffemannich bases derived from ofloxacin facile synthesis and quantitative structureeactivity relationship study of antitumor active 2-(4-oxo-thiazolidin-2-ylidene)-3-oxo-propionitriles synthesis and biological activity evaluation of 5-pyrazoline substituted 4-thiazolidinones synthesis, xray crystallographic analysis, and antitumor activity of n-(benzothiazole-2-yl)-1-(fluorophenyl)-o,o-dialkyl-a-aminophosphonates synthesis and biological activities of aaminophosphonates derivatives containing thieno[3,2-c]pyridine (in chinese) synthesis, antimicrobial and anticancer activities of a novel series of diphenyl 1-(pyridin-3-yl) ethylphosphonates design and one-pot synthesis of a-aminophosphonates and bis(aaminophosphonates) by iron(iii) chloride and cytotoxic activity synthesis, antiproliferative activity in cancer cells and theoretical studies of novel 6a,7b-dihydroxyvouacapan-17b-oic acid mannich base derivatives synthesis and anti-tumor activities of novel synthesis and cytotoxicity studies of novel dimethylamino-functionalised and n-heteroaryl-substituted titanocene anticancer drugs: synthesis and cytotoxicity studies synthesis and cytotoxicity studies of new dimethylamino-functionalised and indolylsubstituted titanocene anti-cancer drugs synthesis and cytotoxicity studies of new dimethylamino-functionalized and azolesubstituted titanocene anticancer drugs synthesis and preliminary cytotoxicity studies of achiral indolyl-substituted titanocenes multidrug resistance reverting activity and antitumor profile of new phenothiazine derivatives synthesis of antitumoractive betulinic acid-derived hydroxypropargylamines by copper-catalyzed mannich reactions cytotoxic betulin-derived hydroxypropargylamines trigger apoptosis doxoform and daunoform: anthracyclineformaldehyde conjugates toxic to resistant tumor cells doxsaliform: a novel n-mannich base prodrug of a doxorubicin formaldehyde conjugate design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells antiestrogen binding site and estrogen receptor mediate uptake and distribution of 4-hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells rational design and synthesis of androgen receptortargeted nonsteroidal anti-androgen ligands for the tumor-specific delivery of a doxorubicin-formaldehyde conjugate studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in pc-3 prostate cancer cells bearing androgen receptor-gfp chimera doxorubicin-formaldehyde conjugates targeting a v b 3 integrin poly (ethylene glycol) prodrug for anthracyclines via n-mannich base linker: design, synthesis and biological evaluation synthesis and invitro antitumor activities of some mannich bases of 9-alkyl-1,2,3,4-tetrahydrocarbazole-1-ones synthesis and biological evaluation of novel mannich bases of 2-arylimidazo [2,1-b]benzothiazoles as potential anti-cancer agents cytotoxic mannich bases of 6-(3-aryl-2-propenoyl)-2(3h)-benzoxazolones new synthetic chalcones: cytotoxic mannich bases of 6-(4-chlorocinnamoyl)-2(3h)-benzoxazolone synthesis and cytotoxicity of novel hexahydrothieno-cycloheptapyridazinone derivatives synthesis and antibacterial study of unsaturated mannich ketones synthesis and antibacterial activity of fused mannich ketones novel mannich ketones of oxazolidinones as antibacterial agents synthesis and biological evaluation of a novel series of 2,2-bisaminomethylated aurone analogues as anti-inflammatory and antimicrobial agents evaluation of antimicrobial activities of several mannich bases and their derivatives synthesis and biological evaluation of aminoketones highly efficient one-pot synthesis, antimicrobial and docking studies of newer b-amino carbonyl derivatives catalyzed by silica sulfuric acid synthesis of b-amino carbonyl derivatives of coumarin and benzofuran and evaluation of their biological activity vuki cevi c, antibacterial 3-(arylamino)-1-ferrocenylpropan-1-ones: synthesis, spectral, electrochemical and structural characterization synthesis and characterization of some mannich bases as potential antimicrobial agents synthesis, characterization and antimicrobial evaluation of copper(ii) complexes of 5-tert-butyl-pyrocatechin-derived mannich bases synthesis and microbiological evaluation of mannich bases derived from 4,6-diacetylresorcinol mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities novel aminonaphthoquinone mannich bases derived from lawsone and their copper(ii) complexes: synthesis, characterization and antibacterial activity synthesis, antiinflammatory and antimicrobial activity of some new 1-(3-phenyl-3,4-dihydro-2h-1,3-benzoxazin-6-yl)ethanone derivatives an eco-friendly synthesis and antimicrobial activities of dihydro-2h-benzo-and naphtho-1,3-oxazine derivatives synthesis and antibacterial evaluation of benzazoles tethered dihydro[1,3]oxazines ]oxazine derivatives catalyzed by zirconyl chloride and evaluation of its biological activities biocidal activity of some mannich base cationic derivatives some electrophilic substitution reactions on 1-substituted-3-acetyl/carbethoxy-5-hydroxy-2-methylindole and the antimicrobial activity of these new indole derivatives mannich base derivatives of 3-hydroxy-6-methyl-4h-pyran-4-one with antimicrobial activity a study of cytotoxicity of novel chlorokojic acid derivatives with their antimicrobial and antiviral activities synthesis and biological activities of new mannich bases of chlorokojic acid derivatives design, synthesis and in vivo/ in vitro screening of novel chlorokojic acid derivatives mannich bases of 7-piperazinylquinolones and kojic acid derivatives: synthesis, in vitro antibacterial activity and in silico study synthesis and antimicrobial screening of novel mannich bases of isatin derivative synthesis and antimicrobial evaluation of some novel trisubstituted s-triazine derivatives based on isatinimino, sulphonamido and azacarbazole synthesis and antimicrobial screening of certain novel mannich bases bearing 1,8-naphthyridine moiety synthesis and antimicrobial activities of oxadiazoles, phthalazines and indolinones synthesis and antimicrobial activity of mannich bases of isatin and its derivatives with 2-[(2,6-dichlorophenyl) amino]phenylacetic acid synthesis, antimicrobial screening and beta lactamase inhibitory activity of 3-(3-chloro-4-fluorophenylimino)indolin-2-one and 5-chloroindolin-2-one derivatives, turk synthesis, antiviral and antibacterial activities of isatin mannich bases synthesis and bioevaluation of schiff and mannich bases of isatin derivatives with 4-amino-5-benzyl-2,4-dihydro-3h-1,2,4-triazole-3-thione synthesis, characterization and in vitro antimicrobial activity of some novel 5-substituted schiff and mannich base of isatin derivatives synthesis and antimicrobial activity of novel 5-(1-adamantyl)-2-aminomethyl-4-substituted-1,2,4-triazoline-3-thiones microbiologically active mannich bases derived from 1,2,4-triazoles. the effect of c-5 substituent on antibacterial activity synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their mannich bases bearing 3-chlorophenyl moiety synthesis and antibacterial activity of some novel n2-hydroxymethyl and n2-aminomethyl derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-3h-1,2,4-triazole-3-thione synthesis and in vitro activity of 1,2,4-triazole-ciprofloxacin hybrids against drug-susceptible and drug-resistant bacteria synthesis, characterization and antibacterial activity of some triazole mannich bases carrying diphenylsulfone moieties synthesis of some new s-alkylated 1,2,4-triazoles, their mannich bases and their biological activities synthesis and biological activities of some novel aminomethyl derivatives of 4-substituted-5-(2-thienyl)-2,4-dihydro-3h-1,2,4-triazole-3-thiones design, synthesis and antimicrobial activities of some azole derivatives synthesis of some new 1,2,4-triazoles, their mannich and schiff bases and evaluation of their antimicrobial activities syntheses and biological activities of new hybrid molecules containing different heterocyclic moieties alpay karao glu, preparation and antimicrobial activity evaluation of some quinoline derivatives containing an azole nucleus convenient one pot synthesis and antibacterial evaluation of some new mannich bases carrying 1,2,4-triazolyl moiety synthesis and antimicrobial activities of some new biheterocyclic compounds containing 1,2,4-triazol-3-one and 1,3,4-thiadiazole moieties synthesis of some new 1,3,4-thiadiazol-2-ylmethyl-1,2,4-triazole derivatives and investigation of their antimicrobial activities synthesis and biological activities of methylenebis-4h-1,2,4-triazole derivatives regioselective reaction: synthesis, characterization and pharmacological studies of some new mannich bases derived from 1,2,4-triazoles el-emam, synthesis, antimicrobial and anti-inflammatory activities of novel 5-(1-adamantyl)-4-[(arylidene)amino]-3-mercapto-1,2,4-triazoles and related derivatives convenient one pot synthesis and antimicrobial evaluation of some new mannich bases carrying 4-methylthiobenzyl moiety synthesis and biological activity of schiff and mannich bases bearing 2,4-dichloro-5-fluorophenyl moiety synthesis of some new 1,2,4-triazoles starting from isonicotinic acid hydrazide and evaluation of their antimicrobial activities synthesis, antiinflammatory, analgesic, and antibacterial activities of some triazole, triazolothiadiazole, and triazolothiadiazine derivatives synthesis and pharmacological evaluation of clubbed isopropylthiazole derived triazolothiadiazoles, triazolothiadiazines and mannich bases as potential antimicrobial and antitubercular agents regioselective reaction: synthesis of novel mannich bases derived from 3-(4,6-disubstituted-2-thiomethylpyrimidyl)-4-amino-5-mercapto-1,2,4-triazoles and their antimicrobial properties new class of triazole derivatives and their antimicrobial activity synthesis, characterization, and biological activity of novel schiff and mannich bases of 4-amino-3-(n-phthalimidomethyl)-1,2,4-triazole-5-thione alpay karao glu, reduction, mannich reaction and antimicrobial activity evaluation of some new 1,2,4-triazol-3-one derivatives preparation and antimicrobial activity evaluation of some new bi-and triheterocyclic azoles synthesis and biological evaluation of some 1,3,4-oxadiazole derivatives synthesis, characterization and antimicrobial activity of some disubstituted 1,3,4-oxadiazoles carrying 2-(aryloxymethyl)phenyl moiety synthesis and antimicrobial activity of new 5-(2-thienyl)-1,2,4-triazoles and 5-(2-thienyl)-1,3,4-oxadiazoles and related derivatives synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-oxadiazoles synthesis and antimicrobial activity of linked heterocyclics containing pyrazole and oxadiazoles synthesis and antimicrobial studies of some mannich bases carrying imidazole moiety synthesis and antimicrobial activity of some new 5-(3-chloro-1-benzothiophen-2-yl)-1,3,4-oxadiazole-2-thiol and their derivatives synthesis, antimicrobial and antiinflammatory activities of 1,3,4-oxadiazoles linked to naphtho[2,1-b]furan antimicrobial and antiurease activities of newly synthesized morpholine derivatives containing an azole nucleus synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives synthesis of novel nalidixic acid-based 1,3,4-thiadiazole and 1,3,4-oxadiazole derivatives as potent antibacterial agents synthesis, characterization and antimicrobial activity of novel 3,5-disubstituted-1,3,4-oxadiazole-2-ones synthesis of mannich bases of some 2,5-disubstituted 4-thiazolidinones and evaluation of their antimicrobial activities synthesis, characterization and evaluation of antimicrobial activity of mannich bases of some 2-[(4-carbethoxymethylthiazol-2-yl)imino]-4-thiazolidinones novel 6,8-dibromo-4(3h)quinazolinone derivatives of antibacterial and anti-fungal activities synthesis of 3-{4-[4-dimethylamino-6-(4-methyl-2-oxo-2h-chromen-7-yloxy)-[1,3,5]triazin-2-ylamino]-phenyl}-2-phenyl-5-(4-pyridin-2-yl-piperazin-1-ylmethyl)-thiazolidin-4-one and their biological evaluation synthesis and biological activity of 3,4-dihydropyrimidine-2-thione aminomethylene derivatives based on the alkaloid cytosine synthesis and in vitro study of biological activity of heterocyclic n-mannich bases of 3,4-dihydropyrimidine-2(1h)-thiones synthesis and in vitro study of biological activity of heterocyclic n-mannich bases synthesis and in vitro study of novel mannich bases as antibacterial agents synthetic, spectral, antimicrobial and qsar studies on novel mannich bases of glutarimides evaluation of antimicrobial activity and qsar study of a molecule library of the mannich bases of glutarimides syntheses, spectral, crystallographic, antimicrobial, and antioxidant studies of few mannich bases synthesis and biological study of medicinally important mannich bases derived from 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxynaphthacenecarboxamide hydrophobic vancomycin derivatives with improved adme properties: discovery of telavancin in vitro activity of telavancin against resistant gram-positive bacteria telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillinresistant staphylococcus aureus exploring the positional attachment of glycopeptide/b-lactam heterodimers vancomycin derivatives iodine catalyzed three-component synthesis of b-amino-b-keto-esters and their antimicrobial activity deep, synthesis, characterization and antimicrobial evaluation of novel derivatives of isoniazid synthesis and evaluation of some novel derivatives of 2-propoxybenzylideneisonicotinohydrazide for their potential antimicrobial activity synthesis and antimicrobial screening of novel series of imidazo[1,2-a]pyridine derivatives synthesis and evaluation of antibacterial and antitubercular activities of some novel imidazo synthesis, antimicrobial evaluation and qsar study of some 3-hydroxypyridine-4-one and 3-hydroxypyran-4-one derivatives synthesis of novel biologically active methylene derivatives of sydnones synthesis, characterization, and antimicrobial screening of some mannich base sydnone derivatives facile syntheses of mannich bases of 3-[p-(5-arylpyrazolin-3-yl)phenyl]sydnones, as anti-tubercular and anti-microbial agents, under ionic liquid/tetrabutylammonium bromide catalytic conditions copper(ii) complex with the tridentate ligand n,n-bis(2-ethyl-4-methylimidazol-5-ylmethyl)phenylethylamine (biaq). x-ray crystal structure and biological activity on bacillus subtilis of synthesis, sar study and evaluation of mannich and schiff bases of pyrazol-5(4h)-one moiety containing 3-(hydrazinyl)-2-phenylquinazolin-4(3h)-one design, synthesis, antibacterial activity and physicochemical parameters of novel n-4-piperazinyl derivatives of norfloxacin in vitro study of some medicinally important mannich bases derived from antitubercular agent microwave-assisted synthesis of some novel and potent antibacterial and antifungal compounds with biological screening synthesis and antimicrobial and antioxidant activities of simple saccharin derivatives with nbasic side chains synthesis and antimicrobial activity of 10-arylaminomethyl-3-methoxyphenothiazine-9-carboxylic acid in vitro antitubercular and antimicrobial activities of 1-substituted quinoxaline-2,3(1h,4h)-diones synthesis of some new thioxoquinazolinone derivatives and a study on their anticonvulsant and antimicrobial activities synthesis and anti bacterial and anti-ulcer evaluation of new s-mannich bases of 4,6-diaryl-3,4-dihydropyrimidin-2(1h)-thiones world health organization antimycobacterial arylidenecyclohexanones and related mannich bases methoxyphenylcarbonyloxy)benzylidene]-6-dimethylaminomethyl cyclohexanone hydrochloride: a mannich base which inhibits the growth of some drug-resistant strains of mycobacterium tuberculosis major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence isocitrate lyase: a potential target for anti-tubercular drugs identification of mannich base as a novel inhibitor of mycobacterium tuberculosis isocitrate by high-throughput screening synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones 4h-pyran-4-one derivatives: leading molecule for preparation of compounds with antimycobacterial potential molecular modeling and antimycobacterial studies of mannich bases: 5-hydroxy-2-methyl-4h-pyran-4-ones bactericidal activities of the pyrrole derivative bm212 against multidrug-resistant and intramacrophagic mycobacterium tuberculosis strains synthesis and microbiological activities of pyrrole analogs of bm 212, a potent antitubercular agent new pyrrole derivatives as antimycobacterial agents analogs of bm212 importance of the thiomorpholine introduction in new pyrrole derivatives as antimycobacterial agents analogues of bm 212 antimycobacterial compounds. new pyrrole derivatives of bm212 antimycobacterial compounds. optimization of the bm 212 structure, the lead compound for a new pyrrole derivative class antimycobacterial agents. novel diarylpyrrole derivatives of bm212 endowed with high activity toward mycobacterium tuberculosis and low cytotoxicity botta, 1,5-diphenylpyrrole derivatives as antimycobacterial agents. probing the influence on antimycobacterial activity of lipophilic substituents at the phenyl rings -diaryl-2-ethyl pyrrole derivatives as antimycobacterial agents: design, synthesis, and microbiological evaluation identification of a novel pyrrole derivative endowed with antimycobacterial activity and protection index comparable to that of the current antitubercular drugs streptomycin and rifampin improved bm212 mmpl3 inhibitor analogue shows efficacy in acute murine model of tuberculosis infection mmpl3 is the cellular target of the antitubercular pyrrole derivative bm212 pharmacophore assessment through 3-d qsar: evaluation of the predictive ability on new derivatives by the application on a series of antitubercular agents bm 212 and its derivatives as a new class of antimycobacterial active agents new pyrroles with potential antimycobacterial, antifungal and selective cox-2 inhibiting activities. synthetic methodologies new derivatives of bm212: a class of antimycobacterial compounds based on the pyrrole ring as a scaffold developing pyrrole-derived antimycobacterial agents: a rational lead optimization approach pyrrole derivatives as antimycobacterial compounds, us patent 7 antitubercular agents. part 7: a new class of diarylpyrroleeoxazolidinone conjugates as antimycobacterial agents organocatalytic multicomponent reaction for the acquisition of a selective inhibitor of mptpb, a virulence factor of tuberculosis novel 1-(4-substituted benzylidene)-4-(1-(substituted methyl)-2,3-dioxoindolin-5-yl)semicarbazide derivatives for use against mycobacterium tuberculosis h37rv (atcc 27294) and mdr-tb strain synthesis, anti-hiv and antitubercular activities of lamivudine prodrugs synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-1h-indole-2,3-dione 3-thiosemicarbazone derivatives synthesis and structureeantituberculosis activity relationship of 1h-indole-2,3-dione derivatives gatifloxacin derivatives: synthesis, antimycobacterial activities, and inhibition of mycobacterium tuberculosis dna gyrase synthesis and antimycobacterial evaluation of various 7-substituted ciprofloxacin derivatives synthesis and in vitro antimycobacterial activity of 8-och 3 ciprofloxacin methylene and ethylene isatin derivatives synthesis and in vitro antimycobacterial activity of moxifloxacin methylene and ethylene isatin derivatives antimycobacterial activity of new 3-substituted 5-(pyridin-4-yl)-3h-1,3,4-oxadiazol-2-one and 2-thione derivatives. preliminary molecular modeling investigations antimycobacterial activity of new 3,5-disubstituted 1,3,4-oxadiazol-2(3h)-one derivatives. molecular modeling investigations oxadiazole mannich bases: synthesis and antimycobacterial activity augustynowicz-kope c, synthesis and tuberculostatic activity of some 2-piperazinmethylene derivatives 1,2,4-triazole-3-thiones synthesis and evaluation of antitubercular activity of imidazo mannich bases and novel benzothiazole derivatives of imidazo[2,1-b]-1,3,4-thiadiazoles and their biological evaluation synthesis & evaluation of antitubercular activity of novel mannich bases of imidazo newer tetracycline derivatives: synthesis, anti-hiv, antimycobacterial activities and inhibition of hiv-1 integrase synthesis of pyrazinamide mannich bases and their antitubercular properties efavirenz mannich bases: synthesis, anti-hiv and antitubercular activities indole mannich bases and their antimycobacterial effect synthesis of novel isothiazolopyridines and their in vitro evaluation against mycobacterium and propionibacterium acnes studies on pyrazine derivatives. 39. synthesis, reactions, and tuberculostatic activity of 3 epidemiology of invasive mycoses in north america antifungal activity of some mono, bis and quaternary mannich bases derived from acetophenone antimicrobial evaluation of some mannich bases of acetophenones and representative quaternary derivatives antifungal evaluation of bis mannich base derived from acetophenones and their corresponding piperidinols and stability studies synthesis and antifungal activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols synthesis and antifungal evaluation of 1-aryl-2-[(dimethylamino)methyl]-2-propen-1-one hydrochlorides antifungal unsaturated cyclic mannich ketones and amino alcohols: study of mechanism of action antifungal activity of fused mannich ketones triggers an oxidative stress response and is cap1-dependent in candida albicans synthesis and antifungal activity of 3-substituted thiochromanones deep, synthesis, characterization and evaluation of mannich bases as potent antifungal and hydrogen peroxide scavenging agents mannich reaction: an approach for the synthesis of water soluble mulundocandin analogues evaluation of bioactivities of chlorokojic acid derivatives against dermatophytes couplet with cytotoxicity synthesis and evaluation of anticonvulsant and antimicrobial activities of 3-hydroxy-6-methyl-2-substituted 4h-pyran-4-one derivatives synthesis and studies of triazolothiadiazines. an approach toward new biologically active heterocyclic compounds, phosphorus ecofriendly synthesis of novel antifungal (thio)barbituric acid derivatives world health organization malaria isoquine and related amodiaquine analogues: a new generation of improved 4-aminoquinoline antimalarials candidate selection and preclinical evaluation of ntert-butyl isoquine (gsk369796), an affordable and effective 4-aminoquinoline antimalarial for the 21st century comparative preclinical drug metabolism and pharmacokinetic evaluation of novel 4-aminoquinoline anti-malarials antimalarial activity of isoquine against kenyan plasmodium falciparum clinical isolates and association with polymorphisms in pfcrt and pfmdr1 genes mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents synthesis and in vitro and in vivo antimalarial activity of novel 4-anilinoquinoline mannich base derivatives synthesis and antimalarial activity evaluation of some isoquine analogues synthesis and antimalarial activity study of some new mannich bases of 7-chloro-4-aminoquinoline novel amodiaquine congeners as potent antimalarial agents synthesis and antimalarial activity of new isotebuquine analogues review of pyronaridine anti-malarial properties and product characteristics determination of the physicochemical properties of pyronaridine e a new antimalarial drug absorption, distribution, excretion, and pharmacokinetics of 14 c-pyronaridine tetraphosphate in male and female spra-gueedawley rats in vitro interactions between piperaquine, dihydroartemisinin, and other conventional and novel antimalarial drugs, antimicrob anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo targeting of hematin by the antimalarial pyronaridine pyrido [3,2-b]indol-4-yl-amine e synthese und prüfung auf wirksamkeit gegen malaria benzofuro[3,2-b]pyridin-4-ylamines e synthese und prüfung auf wirksamkeit gegen malaria benzothieno[3,2-b]pyridin-4-yl-amine e synthese und prüfung auf wirksamkeit gegen malaria thieno[3,2-c]chinolin-4-ylamine e synthese und prüfung auf wirksamkeit gegen malaria thieno[3,4-c]chinolin-4-ylamine e synthese und prüfung auf wirksamkeit gegen malaria naphthyridin-5-yl-arylamine e phenol-mannich-basen vom amodiaquin-, cycloquin-und pyronaridin-typ antimalarial mannoxanes: hybrid antimalarial drugs with outstanding oral activity profiles and a potential dual mechanism of action aromatic amino analogues of artemisinin: synthesis and in vivo antimalarial activity artemisinin derivatives bearing mannich base group: synthesis and antimalarial activity novel in vivo active anti-malarials based on a hydroxy-ethyl-amine scaffold modular synthesis and in vitro and in vivo antimalarial assessment of c-10 pyrrole mannich base derivatives of artemisinin mechanism-based inactivation of thioredoxin reductase from plasmodium falciparum by mannich bases. implication for cytotoxicity synthesis and biological evaluation of phenolic mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of plasmodium falciparum design, synthesis and structureeactivity relationships of (1h-pyridin-4-ylidene)amines as potential antimalarials synthesis and in vitro activities of ferrocenic aminohydroxynaphthoquinones against toxoplasma gondii and plasmodium falciparum pershina, indole derivative having antiviral arbidol: a broad-spectrum antiviral compound that blocks viral fusion mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis and in vitro anti-hepatitis b virus activities of some ethyl 6-bromo-5-hydroxy-1h-indole-3-carboxylates synthesis and in vitro anti-hepatitis b virus activity of some ethyl 5-hydroxy-4-substituted aminomethyl-2-sulfinylmethyl-1h-indole-3-carboxylates synthesis and in vitro anti-hepatitis b virus activities of some ethyl 5-hydroxy-1h-indole-3-carboxylates synthesis and in vitro-anti-hepatitis b virus activities of several ethyl 5-hydroxy-1h-indole-3-carboxylates synthesis and anti-hepatitis b virus evaluation of novel ethyl 6-hydroxyquinoline-3-carboxylates in vitro synthesis and in-vitro antihepatitis-b virus activity of 6h synthesis and in vitro anti-hepatitis b virus activity of 6h-[1]benzothiopyrano[4,3-b]quinolin-9-ols synthesis and in vitro anti-hepatitis b virus activity of 1h-benzimidazol-5-ol derivatives synthesis and biological evaluation of 1h-benzimidazol-5-ols as potent hbv inhibitors synthesis and in-vitro anti-hepatitis b virus activity of ethyl 6-bromo-8-hydroxyimidazo[1,2-a] pyridine-3-carboxylates activity of mannich bases of 7-hydroxycoumarin against flaviviridae novel structurally related compounds reactivate latent hiv-1 in a bcl-2-transduced primary cd4þ t cell model without inducing global t cell activation synthesis and primary antiviral activity evaluation of 3-hydrazono-5-nitro-2-indolinone derivatives design of potential reverse transcriptase inhibitor containing isatin nucleus using molecular modeling studies newer aminopyrimidinimino isatin analogues as non-nucleoside hiv-1 reverse transcriptase inhibitors for hiv and other opportunistic infections of aids: design, synthesis and biological evaluation n-methylisatin-beta-thiosemicarbazone derivative (sch 16) is an inhibitor of japanese encephalitis virus infection in vitro and in vivo condensed pentacyclic derivatives for use in the treatment of flaviviridae infections synthesis, antiviral and cytotoxicity studies of novel n-substituted indophenazine derivatives, indian synthesis of some mono-mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity evaluation of anticonvulsant activities of bis(3-aryl-3-oxo-propyl)ethylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinol hydrochlorides synthesis and evaluation of anticonvulsant activities of some bis mannich bases and corresponding piperidinols synthesis of some new hydroxypyranone derivatives and evaluation of their anticonvulsant activities synthesis of some novel mannich bases derived from allomaltol and evaluation of their anticonvulsant activities anticonvulsant and neurotoxicity evaluation of some novel kojic acids and allomaltol derivatives synthesis and anticonvulsant activity of new kojic acid derivatives synthesis of aminomethyl-substituted cyclic imide derivatives for evaluation as anticonvulsants synthesis and anticonvulsant activity of new n-mannich bases derived from 5-cyclopropyl-5-phenylhydantoins synthesis and anticonvulsant activity of new n-mannich bases derived from 5-cyclopropyl-5-phenyl-and 5-cyclopropyl-5-(4-chlorophenyl)-imidazolidine-2,4-diones synthesis and anticonvulsant activity of new n-1 0 synthesis and potential anticonvulsant activity of new n-3-substituted 5,5-cyclopropanespirohydantoins synthesis and pharmacological evaluation of novel 1 0 karolak-wojciechowska, design, synthesis, and anticonvulsant activity of new n-mannich bases derived from spirosuccinimides and spirohydantoins synthesis and anticonvulsant properties of new n-[(4-arylpiperazin-1-yl)-methyl] derivatives of 3-aryl pyrrolidine-2,5-dione and 2-aza-spiro synthesis and anticonvulsant activity of new n-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 3-phenylpyrrolidine-2,5-dione synthesis, physicochemical and anticonvulsant properties of new n-[(4-aryl-1-piperazinyl) alkyl]-1-phenyl-2,5-pyrrolidinedione and n-[(4-aryl-1-piperazinyl)alkyl]-3-(3-methylphenyl)-2,5-pyrrolidinedione derivatives synthesis and anticonvulsant properties of new mannich bases derived from 3-aryl-pyrrolidine-2,5-diones synthesis and anticonvulsant activity of new n-mannich bases derived from 3-(2-fluorophenyl)-and 3-(2-bromophenyl)-pyrrolidine-2,5-diones. part ii design, synthesis and anticonvulsant properties of new n-mannich bases derived from 3-phenylpyrrolidine-2,5-diones duszy nska, synthesis, anticonvulsant activity and 5-ht 1a , 5-ht 2a receptor affinity of new n-[(4-arylpiperazin-1-yl)-alkyl] derivatives of 2-azaspiro synthesis and anticonvulsant properties of new mannich bases derived from 3,3-disubstituted pyrrolidine-2,5-diones. part iv synthesis and anticonvulsant properties of new n-mannich bases derived from 3,3-diphenyl-and 3-ethyl-3-methyl-pyrrolidine-2,5-diones. part iii synthesis and biological properties of new n-mannich bases derived from 3-methyl-3-phenyl-and 3,3-dimethyl-succinimides. part v anticonvulsant activity and 5-ht 1a /5-ht 7 receptors affinity of piperazine derivatives of synthesis of some newer derivatives of substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant agents synthesis and pharmacological evaluation of newer substituted benzoxazepine derivatives as potent anticonvulsant agents synthesis and evaluation of some new substituted benzothiazepine and benzoxazepine derivatives as anticonvulsant agents synthesis and anticonvulsant activity of various mannich and schiff bases of 1,5-benzodiazepines novel 2-(e)-substituted benzylidene-6-(n-substituted aminomethyl)cyclohexanones and cyclohexanols as analgesic and anti-inflammatory agents biochemical effects of some new mannich base from ortho-hydroxyaryl alkyl ketones on rats with chronic inflammation synthesis and pharmacological activity of n-[b-(para-substituted benzoyl)ethyl]isoleucine and -methionine synthesis and anti-inflammatory, analgesic, and antipyretic activities of n-[b-(p-substituted benzoyl)ethyl]amino acids anti-inflammatory activity of isobornylphenol derivatives synthesis and antiinflammatory activity of resveratrol analogs new 4,6-diacetylresorcinol mannich bases: synthesis and biological evaluation synthesis and biological evaluation of nitrogencontaining benzophenone analogues as tnf-a and il-6 inhibitors with antioxidant activity synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents synthesis and antiinflammatory activity of chalcones and related mannich bases inhibitory effects of mannich bases of heterocyclic chalcones on no production by activated raw 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils synthesis of some new 3,4-dihydro-2h-1,3-benzoxazines under microwave irradiation in solvent-free conditions and their biological activity synthesis and antiinflammatory activity of coumarin derivatives anti-inflammatory mechanisms of isoflavone metabolites in lipopolysaccharide-stimulated microglial cells synthesis and no production inhibition of novel mannich base derivatives of irisolidone synthesis and in-silico studies of some diaryltriazole derivatives as potential cyclooxygenase inhibitors regioselective reaction: synthesis and pharmacological study of mannich bases containing ibuprofen moiety regioselective reaction: synthesis, characterization and pharmacological activity of some new mannich and schiff bases containing sydnone synthesis, characterization and pharmacological activity of 4-{[1-substituted aminomethyl-4-arylideneamino-5-sulfanyl-4,5-dihydro-1h-1,2,4-triazol-3-yl] methyl}-2h-1,4-benzothiazin-3(4h)-ones synthesis and anti-inflammatory activity of new polyheterocyclic schiff bases and mannich bases docking, synthesis, and pharmacological investigation of novel substituted thiazole derivatives as non-carboxylic, anti-inflammatory, and analgesic agents synthesis, analgesic and antiinflammatory properties of certain 5-/6-acyl-3-(4-substituted-1-piperazinylmethyl)-2-benzoxazolinones derivatives synthesis and evaluation of analgesic, anti-inflammatory and antimicrobial activities of 6-acyl-3-piperazinomethyl-2-benzoxazolinones some new mannich bases of 5-methyl-2-benzoxazolinones with analgesic and antiinflammatory activities analgesic and antiinflammatory activities of some new mannich bases of 5-nitro-2-benzoxazolinones synthesis and pharmacological evaluation of 1-(1-((substituted)methyl)-5-methyl-2-oxoindolin-3-ylidene)-4-(substituted pyridin-2-yl)thiosemicarbazide synthesis, analgesic, antiinflammatory and ulcerogenic properties of some novel n 0 -((1-(substitutedamino)methyl)-2-oxoindolin-3-ylidene)-4-(2-(methyl/phenyl)-4-oxoquinazolin-3(4h)-yl)benzohydrazide derivatives synthesis, pharmacological screening, quantum chemical and in vitro permeability studies of n-mannich bases of benzimidazoles through bovine cornea synthesis and biological evaluation of mannich bases of benzimidazole derivatives synthesis, antiinflammatory and ulcerogenicity studies of some substituted pyrimido[1,6-a]azepine derivatives potential antiinflammatory activity and ulcerogenicity study of some novel pyrimido synthesis and anti-inflammatory activity of certain piperazinylthienylpyridazine derivatives design and synthesis of 3-[3-(substituted phenyl)-4-piperidin-1-ylmethyl/-4-morpholin-4-ylmethyl-4,5-dihydro-isoxazol-5-yl]-1h-indoles as potent anti-inflammatory agents efficient synthesis of the first betulonic acideacetylene hybrids and their hepatoprotective and anti-inflammatory activity substituted 1 and 2-naphthol mannich bases synthesis and analgesic activity of novel derivatives of 1,2-substituted benzimidazoles synthesis and in vivo pharmacology of new derivatives of isothiazolo[5,4-b] pyridine of mannich base type synthesis, analgesic activity and computational study of new isothiazolopyridines of mannich base type thymol analogues with antioxidant and l-type calcium current inhibitory activity alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties mannich bases of scutellarein as thrombin-inhibitors: design, synthesis, biological activity and solubility synthesis of antioxidants for prevention of age-related macular degeneration synthesis and antioxidant activity of novel mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan design, synthesis, and in vitro antioxidant activity of 1,3,5-trisubstituted-2-pyrazolines derivatives synthesis and antioxidant activity of aminomethylated 6-methyluracil derivatives synthesis and antihypertensive effects of new methylthiomorpholinphenol derivatives antihypertensive and antiarrhythmic properties of a para-hydroxy[bis(ortho-morpholinylmethyl)] phenyl-1,4-dhp compound: comparison with other compounds of the same kind and relationship with logp values 1-and 2-substituted naphthalenes: a new class of potential hypotensive agents studies on the cardiovascular action of tpyb: an antihypertensive agent with antiplatelet activity synthesis, characterization and antihypertensive activity of pyridazinone derivatives synthesis and pharmacological activity of 2-and 3-(aminomethyl)imidazo [1,2-a]benzimidazoles new octahydroquinazoline derivatives: synthesis and hypotensive activity irreversible inactivation of trypanothione reductase by unsaturated mannich bases: a divinyl ketone as key intermediate unsaturated mannich bases active against multidrugresistant trypanosoma brucei brucei strains antileishmanial pyrazolopyridine derivatives: synthesis and structureeactivity relationship analysis mannich base derivatives of 1,3,4-oxadiazole: synthesis and screening against entamoeba histolytica in vitro antischistosomal evaluation of some newly synthesized praziquantel derivatives structureeactivity relationships of chalcone analogs as potential inhibitors of adpand collagen-induced platelet aggregation synthesis and pharmacological evaluation of peptide-mimetic protease-activated receptor-1 antagonists containing novel heterocyclic scaffolds the association of helicobacter pylori infection and nonsteroidal anti-inflammatory drugs in peptic ulcer disease synthesis and antiulcer activity study of 1,4-dihydropyridines and their mannich bases with sulfanilamide synthesis of 6-and 9-alkylaminomethyl furoflavones as gastroprotective agents submitted to the 65 th world health assembly synthesis and antidepressant-like profile of novel 1-aryl-3-[(4-benzyl)piperidine-1-yl]propane derivatives design, synthesis, computational and biological evaluation of new anxiolytics benzoxepin derivatives: design, synthesis, and pharmacological evaluation with sedativeehypnotic effect synthesis and hepatoprotector activity of water-soluble derivatives of aminoalkylphenols design, synthesis and preliminary biological evaluation of zatebradine analogues as potential blockers of the hyperpolarization-activated current synthesis and antiemetic activity of 1,2,3,9-tetrahydro-9-methyl-3-(4-substituted-piperazin-1-ylmethyl)-4h-carbazol-4-one derivatives synthesis of tropolone derivatives and evaluation of their in vitro neuroprotective activity bone selective protective effect of a novel bone-seeking estrogen on trabecular bone in ovariectomized rats a one step/one pot synthesis of n,nbis(phosphonomethyl)amino acids and their effects on adipogenic and osteogenic differentiation of human mesenchymal stem cells the mannich base nc1153 promotes long-term allograft survival and spares the recipient from multiple toxicities donepeziletacrine hybrid related derivatives as new dual binding site inhibitors of ache synthesis and acetylcholinesterase (ache) inhibitory activity of some n-substituted-5-chloro-2(3h)-benzoxazolone derivatives synthesis and biological evaluation of 1,3-dihydroxyxanthone mannich base derivatives as anticholinesterase agents synthesis and biological evaluation of novel 8-aminomethylated oroxylin a analogues as a-glucosidase inhibitors synthesis and evaluation of 5-(6-methoxynaphthalen-2-yl)-1-aryl-1-(4-(trifluoromethyl) phenylamino)pentan-3-ones as potential antidiabetic agents synthesis and antidiabetic performance of b-amino ketone containing nabumetone moiety synthesis of novel b-amino ketones containing a p-aminobenzoic acid moiety and evaluation of their antidiabetic activities exploring structureeactivity relationships of transition state analogues of human purine nucleoside phosphorylase synthesis of second-generation transition state analogues of human purine nucleoside phosphorylase synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the mannich reaction third-generation immucillins: syntheses and bioactivities of acyclic immucillin inhibitors of human purine nucleoside phosphorylase syntheses and bio-activities of the l-enantiomers of two potent transition state analogue inhibitors of purine nucleoside phosphorylases immucillins in custom catalytic-site cavities a bfluoroamine inhibitor of purine nucleoside phosphorylase azetidine based transition state analogue inhibitors of n-ribosyl hydrolases and phosphorylases design and synthesis of potent "sulfur-free" transition state analogue inhibitors of 5 0 -methylthioadenosine nucleosidase and 5 0 -methylthioadenosine phosphorylase augustyns, narylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase potent transglutaminase inhibitors, aryl b-aminoethyl ketones potent transglutaminase inhibitors, dithio b-aminoethyl ketones synthesis and characterization of phenolic mannich bases and effects of these compounds on human carbonic anhydrase isozymes i and ii nitrogen-containing flavonoid analogues as cdk1/cyclin b inhibitors: synthesis, sar analysis, and biological activity hit identification of novel heparanase inhibitors by structure and ligand-based approaches discovery of inhibitors of burkholderia pseudomallei methionine aminopeptidase with antibacterial activity search for inhibitors of bacterial and human protein kinases among derivatives of diazepines[1,4] annelated with maleimide and indole cycles umbelliferone aminoalkyl derivatives, a new class of squalene-hopene cyclase inhibitors synthesis of new 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine derivatives with an incorporated thiazolidinone moiety and testing their possible serine protease and cercarial elastase inhibitory effects with a possible prospective to block penetration of schistosoma mansoni cercariae into the mice skin synthesis and biological activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors novel mannich bases, 5-arylimidazolidine-2,4-dione derivatives with dual 5-ht 1a receptor and serotonin transporter affinity pharmacophore based synthesis of 3-chloroquinoxaline-2-carboxamides as serotonin 3 (5-ht 3 ) receptor antagonist synthesis and biological evaluation of a novel structural type of serotonin 5-ht 3 receptor antagonists ]pyridine: an antagonist with high affinity and selectivity for the human dopamine d 4 receptor azaindole derivatives with high affinity for the dopamine d 4 receptor: synthesis, ligand binding studies and comparison of molecular electrostatic potential maps phenylpiperazinylmethylheterocycle derivatives: synthesis and dopamine receptor binding profiles synthesis of 5-[(4-phenylpiperazin-1-yl)methyl]pyrrolo [2,3-d]pyrimidine derivatives as potential dopamine d 4 receptor ligands design, synthesis and dopamine d4 receptor binding activities of new n-heteroaromatic 5/6-ring mannich bases discovery of highly potent and selective d4 ligands by interactive sar study synthesis and evaluation of ligands for d 2 -like receptors: the role of common pharmacophoric groups discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators inhibitors of the interaction of a thyroid hormone receptor and coactivators: preliminary structureeactivity relationships structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor improvement of pharmacological properties of irreversible thyroid receptor coactivator binding inhibitors discovery and biological characterization of a novel series of androgen receptor modulators aromatic b-amino-ketone derivatives as novel selective non-steroidal progesterone receptor antagonists synthesis and biological evaluation of n 1 ,n 2 -bis-[4-(t-amino)-2-butynyl]phthalamides as oxotremorine and acetylcholine antagonists synthesis and structureactivity relationships of 1-aralkyl-4-benzylpiperidine and 1-aralkyl-4-benzylpiperazine derivatives as potent s ligands indole-and benzothiophene-based histamine h 3 antagonists new 1,2,3,9-tetrahydro-4h-carbazol-4-one derivatives: analogues of heat as ligands for the a 1 -adrenergic receptor subtypes pyrazolone methylamino piperidine derivatives as novel ccr3 antagonists synthesis of new 4-heteroaryl-2-phenylquinolines and their pharmacological activity as nk-2/nk-3 receptor ligands the author declares that there is no conflict of interest.