{Alkylamino and aralkylamino derivatives of avarone and its mimetic as selective agents against non-small cell lung cancer cells, their antibacterial and antifungal potential} J. Serb. Chem. Soc. 83 (11) 1193–1207 (2018) UDC 542.913:547.21.024–304.2+547.53.024– JSCS–5143 304.2:547.567:615.277+615.281 Original scientific paper 1193 Alkylamino and aralkylamino derivatives of avarone and its mimetic as selective agents against non-small cell lung cancer cells, their antibacterial and antifungal potential MARKO JEREMIĆ1#, JELENA DINIĆ2, MILICA PEŠIĆ2, MARIJA STEPANOVIĆ2, IRENA NOVAKOVIĆ3#, DEJAN ŠEGAN4 and DUŠAN SLADIĆ4#* 1Innovation Center of the Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia, 2Institute for Biological Research “Siniša Stanković”, University of Belgrade, Despota Stefana 142, 11060 Belgrade, Serbia, 3Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Center for Chemistry, Njegoševa 12, 11000 Belgrade, Serbia and 4Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia (Received 27 June, accepted 23 July 2018) Abstract: In this paper, the synthesis of fourteen alkylamino and arylamino derivatives of sesquiterpene quinone avarone and its model compound tert-but- ylquinone is described. Branched, cyclic, allylic and benzylic alkylamino/aryl- amino groups were introduced into the quinone moiety. For all the obtained derivatives, their biological activity and redox properties were studied. The cyto- toxic activity of the synthesized derivatives towards multidrug resistant (MDR) human non-small cell lung carcinoma NCI-H460/R cells, their sensitive counter- part NCI-H460 and human normal keratinocytes (HaCaT) was investigated. The antimicrobial activity towards Gram-positive and Gram-negative bacteria, and fungal cultures was determined. Some of the synthesized derivatives showed selectivity for cancer cells, including MDR cells. Regarding their cell death ind- uction potential, the most promising compounds were allylamino derivatives, preferentially triggering apoptosis, with high selectivity for cancer cells, inc- luding MDR cells. Several compounds showed promising antimicrobial activity, comparable to those of commercial antibiotic and antimycotic agents. Keywords: quinones; anticancer activity; multidrug resistant; apoptosis; anti- microbial activity; cyclic voltammetry. INTRODUCTION Cancer is one of the leading causes of death worldwide, with over 8 million deaths in 2015.1 Therefore, a great amount of resources and effort have been * Corresponding author. E-mail: dsladic@chem.bg.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC180627062J ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1194 JEREMIĆ et al. invested in developing novel strategies and drugs for combating this disease. Tackling this serious issue includes various forms of therapies, such as radio- therapy, chemotherapy, immunotherapy, surgery as well as prevention of cancer by reducing the risk of carcinogenesis.2 Chemotherapy involves the application of drugs or cocktails of drugs in order to eliminate developed cancer cells. The limitations of chemotherapy are low efficacy, lack of selectivity, severe toxicity, metastasis and development of drug resistance.3 The phenomenon of multi-drug resistance (MDR) is a particular problem, caused by the fast proliferation and metabolism of cancer cells together with a proneness to mutations, which enables cancer cells to overcome the toxicity of the applied drugs, resulting in resistant forms of cancer.4 Quinones are a class of organic compounds that have a versatile array of act- ivities, some of which are herbicidal,5 antimalarial,6 antiviral,7 antipsoriatic,8 antileishmanial9 and cytotoxic activity.10–12 Biological activity of quinones ori- ginates from dual mode of action. It is a combination of 1,4-Michael addition of cellular nucleophiles to conjugate enone system and the generation of reactive oxygen species (ROS) in redox cycling reactions of quinone and its hydroquin- one pair via a semiquinone anion radical.13 Quinone/hydroquinone core-contain- ing compounds are very abundant in nature, being involved in crucially important processes, such as photosynthesis,14 the mitochondrial electron transport chain15 and protection from oxidizing species.16 Natural products of marine origin are of particular interest for their diversity of structures and a myriad of activities, together with the vast possibilities for their modifications.17–19 As a continuation of previous work on alkylamino, aralkylamino and amino acid derivatives of avarone and its mimetic tert-butylbenzoquinone (TBQ),20,21 the synthesis and investigation of the cytotoxic activity of a series of alkylamino and aralkylamino derivatives of avarone and tert-butylbenzoquinone on three cell lines, i.e., non-small cell lung cancer cells, both the sensitive NCI-H460 and multi-drug resistant NCI-H460/R, and healthy human keratinocytes HaCaT are reported herein. Since in a previous work, benzylamino derivatives of tert-butyl- quinone showed good selectivity for tumour cells, including MDR cells, benzyl- amino derivatives of avarone were synthesized in the present study. Other amines selected for derivatization of both quinones were allylamines due to similar elec- tronic effects of the allyl and benzyl group, and amines leading to sterically more compressed derivatives, i.e., sec-butylamine and pyrrolidine. Additionally, their antibacterial and antifungal activities were determined. Cyclic voltammetry was used to study the redox behaviour of newly synthesized compounds in the system quinone–semiquinone–hydroquinone, in order to gain information on the struc- ture−activity relationship. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1195 RESULTS AND DISCUSSION Chemistry As a continuation of previous work, fourteen aminoquinones 3–10a and 3–10b were synthesized (preparation of compounds 6a and 6b was presented in a previous paper20). Treatment of the parent quinones tert-butylquinone (1) and avarone (2) with various amines produced the corresponding two regioisomers of aminoquinones via 1,4-Michael addition reactions (Scheme 1). Scheme 1. Synthesis of the quinone derivatives. As expected, with tert-butylquinone, the 2,6-disubstituted quinone products were dominant over the 2,5-disubstituted products. On the other hand, with ava- rone, only in the reaction with pyrrolidine was the 2,6-disubstituted product dominant while in the reaction with allylamine, the 2,5-disubstituted product was the main product and with the other two nucleophiles, similar amounts of two products were obtained. Racemic sec-butylamine was used in the reactions. The NMR spectra of the products obtained in its reaction with avarone indicate the presence of only one diastereoisomer. Unfortunately, the configuration of the asymmetric carbon could not be determined, since all attempts to crystallize the products were unsuccessful. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1196 JEREMIĆ et al. Half-wave potentials were recorded at a glassy carbon disk (3 mm diameter) in DMSO towards a silver wire immersed in the electrolyte solution containing 0.01 M silver ions as the reference electrode, and ferrocene as the reference com- pound. The results are given in Table I and shown in Fig. S-1 of the Supple- mentary material to this paper. TABLE I. Voltammetric half-peak potentials and standard redox potentials of the synthesized compounds (V vs. silver/silver chloride electrode) Compound Ec1 Ea1 Ec2 Ea2 E01/Fc 3a –1.210 –1.126 –1.890 –1.741 –1.170 3b –1.218 –1.154 –1.868 –1.744 –1.186 4a –1.180 –1.121 –1.812 –1.712 –1.149 4b –1.210 –1.152 –1.848 –1.733 –1.181 5a –1.276 –1.214 –1.876 –1.694 –1.244 5b –1.276 –1.214 –1.865 –1.749 –1.243 6a20 –1.128 –1.053 –1.800 –1.619 –1.159 6b20 –1.141 –1.071 –1.803 –1.593 –1.174 7a –1.193 –1.127 –1.881 –1.774 –1.158 7b –1.201 –1.133 –1.859 –1.713 –1.168 8a –1.170 –1.113 –1.830 –1.741 –1.142 8b –1.177 –1.117 –1.799 –1.689 –1.150 9a –1.267 –1.163 –1.883 –1.710 –1.217 9b –1.259 –1.177 –1.861 –1.704 –1.218 10a –1.115 –1.043 –1.865 –1.733 –1.147 10b –1.123 –1.054 –1.840 –1.685 –1.157 Typical quinone electrochemical behaviour was observed for all the synthe- sized compounds, i.e., two waves that could be attributed to the reduction of qui- none to the semiquinone radical and, subsequently, to the hydroquinone dianion (Fig. S-1). First reduction wave was fully reversible, indicating a diffusion con- trolled process. However, second reduction process was not reversible, having higher peak separation potentials. The derivatization did not perturb the nature of these two redox processes, but rather shifted the peak potentials. As expected, all the derivatives had more negative reduction potential than the parent quinone. It could be assumed that amino substituents with electron-donating ability destabil- ize the intermediate semiquinone relative to the corresponding quinone. This effect leads to a negative shift in the peak potential. Since the inductive effect of the alkyl part of the alkylamino substituent influences the electron-donating abil- ity of the substituent, the pyrrolidino derivatives have the most negative reduct- ion potentials, while benzylamino and allylamino derivatives have the least nega- tive one. As previously described,20 the position of the substituent plays a parti- cular role in shifting the peak potential. It was observed that the 4′-derivatives generally had a peak potential more negative by about 10 to 20 mV than the cor- responding 3′-derivatives. The probable reason is that the arrangement is more ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1197 favourable when the electron-donating amino and alkyl substituents in semiqui- none anion radical are both in the meta position to the negative oxygen. Anticancer activity Derivatives of TBQ were compared with corresponding avarone derivatives regarding their growth inhibition activity against human cancer and normal cell lines. The cell growth inhibition activity was studied in non-small cell lung car- cinoma cells – NCI-H460/R, their multidrug resistant (MDR) counterpart – NCI- -H460/R and human keratinocytes – HaCaT cells. The differences in response between cancer and normal cells, evaluated by the MTT assay after 72 h treat- ment, are presented in Table II and Fig. S-2 of the Supplementary material. TABLE II. Growth inhibition activity (IC50 in µM) of TBQ, avarone and their derivatives in human non-small cell lung carcinoma cell lines (NCI-H460 – sensitive and NCI-H460/R – MDR variant) and human normal keratinocytes (HaCaT); the IC50 values were calculated from a minimum of three independent experiments (average ± standard deviation) Compound Cell line NCI-H460 NCI-H460/R HaCaT CDDPa 5.2±0.4 1.7±0.1 0.7±0.1 120a >100 72±8 95±4 220a,b 84±9 24±4 37±8 3ab,c 10.5±0.4 16.3±1.3 24.4±1.5 3bb,c 36.6±1.8 96.3±3.9 72.5±1.5 4a 9.6±0.1 12.6±0.4 8.5±0.4 4b 18.3±0.6 21.8±0.2 23.9±0.7 5ac 40.9±0.4 65.9±2.6 36.8±4 5bc 44.4±4.0 85.7±2.6 26.8±2.4 6a20b 14±2 10±2 21±1 6b20b 19±4 15±2 28±2 7a 3.5±0.04 3.8±0.1 4±0.1 7b 37.5±1.1 44.4±0.9 40.6±2 8a 2.8±0.03 3.0±0.1 3.5±0.1 8b 18±1.3 24.2±0.7 19.2±0.4 9a 28.2±3.7 20.4±1.2 19.3±0.8 9bb 8.9±1.5 7.1±0.2 15.1±0.3 10ac 54.4±2.2 26.3±2.6 12±1 10b >100 >100 42.2±3.0 aSelectivity towards MDR cells (higher efficacy in NCI-H460/R compared to NCI-H460 i.e., IC50 of sensitive cancer cells ≥ 1.5 fold than IC50 of corresponding MDR cells); bselectivity towards cancer cells (IC50 of either sensitive or MDR cancer cells ≤ 1.5 fold than IC50 of normal cells. The dose response curves are shown in Fig. S-2); cresistance (lower efficacy in NCI-H460/R compared to NCI-H460 i.e., IC50 of sensitive cancer cells ≤ 1.5 fold than IC50 of the corresponding MDR cells) The growth inhibition abilities of parent compounds TBQ and avarone, as well as CDDP, an FDA-approved drug for non-small cell lung carcinoma treat- ment,22 were investigated in our previous study.20 CDDP showed stronger effect ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1198 JEREMIĆ et al. in MDR cancer cells in comparison with their sensitive counterparts, but it was not selective against cancer cells due to the pronounced activity obtained in nor- mal human keratinocytes. TBQ was largely ineffective in all cell lines while ava- rone showed high selectivity towards MDR cells. The derivatives had generally a higher activity than the parent compounds. Avarone 2,6-disubstituted sec-butyl- amino and allylamino derivatives (7a and 8a, respectively) exhibited the highest activity in all tested cell lines with IC50 values below 5 µM and, importantly, their activity was not affected by the presence of MDR phenotype. Corres- ponding 2,5-disubstituted derivatives 7b and 8b showed the same pattern of acti- vity (without selectivity against cancer cells) but their efficacy was significantly diminished as their IC50 values were around one order of magnitude higher than those obtained by 7a and 8a. Similarly, 2,6-disubstituted allylamino derivative 4a with IC50 values near 10 µM was more efficient than 4b but without selectivity to cancer cells. Benzylamino avarone derivative 10b was the least active com- pound in all cell lines, while the IC50 values for corresponding 10a and two pyr- rolidino tert-butylquinone derivatives 5a and 5b were above 25 µM in cancer cells. These four derivatives were more active in normal cells. Compounds 3a and 9b exhibited both high cytotoxicity and selectivity towards cancer cell lines and most notably MDR phenotype did not reduce their activity. Although direct correlation between redox properties and cytotoxicity of derivatives with differ- ent substituents could not be established, it should be noticed that out of two regioisomers, the isomer with a less negative cathodic potential of the first wave, i.e., the isomer that is more prone to the formation of semiquinone radicals, is more active. In the tert-butylquinone series, the 6-derivatives were always more active to tumour cells than the 5-derivatives. The introduction of the bulky sec-butylamino group into the 6-position, i.e., closer to the tert-butyl group, did not have much influence on the activity to tumour cells, but it decreased the toxicity to normal cells, and hence, contrary to the 6-(butylamino) derivative, the corresponding sec-butylamino substituted quinone showed a pronounced selectivity to tumour cell lines. This did not apply for a more remote 5 substituent. The introduction of allylamino group led to products with higher cytotoxicity than similar derivatives with linear saturated alkylamino substituents, but the selectivity for tumour cells displayed by 6-(ethylamino) derivative was lost.20 5-(Allylamino) and 5-(benzyl- amino) products were the only 5-substituted derivatives that showed a relatively high cytotoxicity, but only the benzylamino derivative showed selectivity for tumour cell lines. The pyrrolidino derivatives were only moderately active, without selectivity for tumour cells. If the whole set of compounds is considered, for a strong cytotoxic activity against tumour cells, an unsaturated or bulky sub- stituent should be present in position 6. For achieving selectivity, the set of sub- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1199 stituents was narrowed to bulky groups, and even more to aralkyl groups (benzyl- amino, phenethylamino) if selectivity to MDR cells is considered. As for avarone derivatives, the 3′-derivatives (2,6-disubstitution) were always much more active than the 4′-derivatives (2,5-disubstitution), with the exception of the pyrrolidino derivatives when the inverse was true. Within this series, the introduction of branching or unsaturation into alkylamino substituent increased the cytotoxicity regardless of the position of the substituent. 3′-(sec- -Butylamino) and 3′-(allylamino) derivatives showed IC50 values in the range 3–4 μM. Unfortunately, the selectivity for MDR cells, shown by 3′-(butylamino)- avarone,20 was lost. The benzylamino derivatives were less active to tumour cells than the phenethylamino ones. The 4′-pyrrolidino derivative (the more active isomer) showed strong activity to the tumour cell lines, with an IC50 value of 7.1 μM against the multi-drug resistant cells, and selectivity for tumour cells. The 3′-derivative showed moderate activity, without selectivity. Summarizing these results, the necessary prerequisite for an avarone derivative to have a strong act- ivity against tumour cells is a substituent with a relatively low number of carbon atoms at the position 3′ or pyrrolidine at position 4′. Of these substituents, only unbranched saturated butylamino derivative and heterocyclic pyrrolidino derivat- ive showed the desired selectivity. Apoptosis and necrosis are the two main forms of cell death. Apoptosis is regarded as a programmed process, with minimal level of ATP required for the assembly of a apoptosome complex and activation of caspases, while necrosis is often referred to as the complete decay of cell metabolism.23 They can be dis- tinguished from one another by various morphological and biochemical charac- teristics, although there is no clear-cut distinction between these two (hence, other forms of cell death, i.e., necroptosis, aponecrosis, etc.).24 The most obvious characteristics of apoptosis are cell shrinkage, DNA fragmentation, condensation of chromatin, formation of apoptotic bodies, disruption of mitochondrial redox processes and generation of ATP. Cells undergoing apoptosis do not release their constituents into the surrounding tissue because the integrity of the cell mem- brane is preserved. In addition, macrophages quickly phagocytose apoptotic bodies and the surrounding cells do not produce anti-inflammatory cytokines.25 Therefore, no inflammatory reaction occurs during the apoptotic process. Nec- rosis, on the other hand, is usually followed by inflammation, because one of the main characteristics of necrosis is the loss of membrane integrity and the release of the cytoplasmic content into the surrounding area, leading to inflammation.26 Inflammation, for its part, can damage cells and even cause cancer.27 Bearing this in mind, apoptosis is clearly the preferred type of cell death, considering the removal of either healthy, aging or tumour cells. Besides inflammation, the other characteristics of necrosis are cell swelling and complete abolition of ATP pro- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1200 JEREMIĆ et al. duction. However, many types of cancer employ anti-apoptotic mechanisms for their survival, so necrosis could be useful in promoting the death of tumour cells. The cell death inducing capacity of 25 µM TBQ and the avarone derivatives was analyzed after 72 h treatment (Table III, Fig. S-3 of the Supplementary material). Both pyrrolidino tert-butylquinone derivatives (5a and 5b) showed sig- nificant activity towards sensitive cancer cells, triggering both apoptosis and nec- rosis, which was not observed in normal cells. The allylamino derivatives 4a and 4b induced apoptotic type of cell death more prominently in both cancer cell lines than in HaCaT cells. The sec-butylamino derivatives 3a and 3b also ind- uced apoptosis, most effectively in NCI-H460 cells. Cells treated with the ava- rone pyrrolidino derivatives 9a and 9b underwent necrosis as the predominant cell death type. Consistent with the cell growth analysis, the inverted efficacy pattern of 9a and 9b as well as the selectivity of 9b towards cancer cells was confirmed by cell death induction. Compounds 8a and 8b (allylamino derivatives of avarone) also predominantly induced necrosis in cancer cells with 8a being the significantly more potent derivative. sec-Butylamino derivative 7a was consider- ably more active against MDR cancer cells in triggering necrotic cell death com- pared to 7b. A similar but reduced effect was also observed in NCI-H460 and HaCaT cells. Importantly, according to the cell death analysis, 7a and 8a were shown to be selective against cancer cells. TABLE III. Cell death induction by 25 μM TBQ, avarone and their derivatives in human non- -small cell lung carcinoma cell lines (NCI-H460 – sensitive and NCI-H460/R – MDR variant) and human normal keratinocytes (HaCaT)). CDDP is included as a positive control. The values represent percentages of viable, apoptotic and necrotic cells Compound Viable cells Early apoptosis Late apoptosis Necrosis AV–PI– AV+PI– AV+PI+ AV–PI+ NCI-H460 Control 95.5 2 1.1 1.3 CDDP 66 1.6 5.2 27.2 120 95.2 0.5 0.5 3.8 220 74 3.4 16.3 6.3 3a 43.8 21.9 28.2 6.1 3b 85.6 3.5 4.9 6 4a 5.1 24.9 61.4 8.6 4b 4.9 23.1 63.9 8.1 5a 67.4 4.7 11.4 16.5 5b 51 5.3 18.1 25.6 7a 53.9 6.6 5.7 33.8 7b 81.4 0.7 3.9 13.9 8a 28.6 12.5 18 40.9 8b 72.1 3.5 7 17.4 9a 83.6 2 3.5 10.9 9b 31 3.4 10 55.6 ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1201 TABLE III. Continued Compound Viable cells Early apoptosis Late apoptosis Necrosis AV˗PI˗ AV+PI˗ AV+PI+ AV˗PI+ NCI-H460/R Control 95.6 1.6 1.9 0.9 CDDP 55.3 3.8 13.3 27.6 120 95.2 0.5 1.2 3.1 220 65.8 2.6 22.7 8.9 3a 68.4 9.2 17.5 4.9 3b 93 2.3 3.2 1.5 4a 7.3 4.1 64.4 24.2 4b 11.8 6.3 63.4 18.5 5a 91.8 2 4.1 2.1 5b 84.8 2 5.5 7.7 7a 38.4 3.8 9.8 48 7b 96.3 1.5 1.4 0.8 8a 21.7 6.6 19.2 52.5 8b 55.1 4.4 7.1 33.4 9a 58.7 5.2 7.7 28.4 9b 26.9 3.9 10.9 58.3 HaCaT Control 93.6 2.9 1 2.4 CDDP 53.5 1.4 9 36.1 120 86.8 1.9 3.8 7.5 220 78.3 2.5 10.1 9.1 3a 64 14.2 9.7 12.1 3b 82 6.8 5.5 5.7 4a 61.5 15.9 8.1 14.5 4b 33.5 18.8 18.9 28.8 5a 85.9 4.8 4.9 4.4 5b 85.3 4.9 4 5.8 7a 72.3 1.1 1 25.6 7b 95.7 0.5 0.6 3.2 8a 73.8 2.4 4.1 19.7 8b 89.3 0.6 0.8 9.3 9a 97.2 0.3 0.3 2.2 9b 83.7 0.9 1.1 14.3 The cell death-inducing activity of compounds 4a and 4b significantly stand out from the others in the series. The majority of the cells treated with 4a or 4b underwent apoptotic cell death, targeting late apoptosis preferentially. Most imp- ortantly, this pair were very selective toward cancer cell lines, both sensitive and resistant ones, particularly the 4a derivative with over 60 % of viable cells in healthy HaCaT cell lines. This distinction could be attributed to the allyl moiety. The probable reason is the additional generation of ROS via allyl radicals, formed by the decomposition of the semiquinone radical generated by the one- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1202 JEREMIĆ et al. electron reduction of allylamino quinone (the other product would be amino qui- none) and/or the formation of cytotoxic acrolein by the oxidative metabolism of allylamino quinones.28 Considering that allylamino TBQ derivatives preferen- tially induce (late) apoptosis, and that cancer cells are usually already in a state of oxidative stress,29 this seems a plausible explanation for the selective action. Antimicrobial activity Compounds 3–10a and 3–10b were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungal strains, and compared to the commercial antibiotics amikacin and antimycotic nystatin. The results are given in Tables IV and V. Most of the derivatives showed weak activity in comparison to amikacin, having at least an order of a magnitude higher MIC. The most active TBQ deri- vatives were allylamino derivatives 4a and 4b, with activity comparable to ami- kacin for E. coli. This pair had almost identical MIC for all strains except M. luteus (ATCC 10240 and 4698) and S. enterica. The pair 3a and 3b showed generally weaker activity than 4a and 4b, with 3a being only slightly more active against B. subtilis, M. luteus (ATCC 10240 and 4698) and E. coli, and 3b for K. rhizophila. The pair 5a and 5b displayed the weakest activity of all the tested TBQ compounds, with some activity only towards K. rhizophila, E. coli and M. luteus, ATCC 4698, (only 5a). The avarone counterparts showed no activity, as expected from previous results.20 The probable reason for inactivity is insufficient hydro- philicity. This conclusion is corroborated by the fact that several amino acid deri- vatives of avarone showed strong antibacterial activity.21 In general, in order to show a relatively broad activity comparable to amikacin, tert-butylquinone deri- vatives should have a non-branched medium length alkylamino group or an aral- kyl group in position 3′. It is interesting that among the avarone amino acid deri- vatives, the most active were those with aromatic amino acids, implying that there is an aromatic binding site in a putative target. All the tested compounds, except 10a and 10b, showed stronger activity toward C. albicans than nystatin, with 3a, 4a, 4b and 5a even having a two orders of a magnitude lower MIC (less than 50 μM). Towards A. brasiliensis, the activities were generally comparable to that of nystatin, with 4a, 4b and 5a displaying stronger activity. Considering S. cerevisiae, 4a, 5a, 7b, 9a and 9b possess stronger activity than amikacin. Of all the tested compounds, 4a showed the strongest antifungal activity, while 10a and 10b showed no activity at all. Based on these results and those from a previous paper,20 it could be concluded that in order to achieve a strong anti-Candida activity, a compound should be a 6-substituted 2-tert-butyl-1,4-benzoquinone (exception are allylamino derivatives, where both isomers have a similar activity), ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1203 ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1204 JEREMIĆ et al. with at least three carbon atoms in the side chain. The avarone derivatives were much less active, but in contrast to antibacterial effects, most of them had some activity, although much lower than the amino acid derivatives.21 The amino acid derivatives of TBQ had weak antimicrobial activity, probably due to their excessive hydrophilicity. TABLE V. Antifungal in vitro activity (MIC/mM) Compound Fungi Candida albicans (ATCC 10231) Aspergillus brasiliensis (ATCC 16404) Saccharomyces cerevisiae (ATCC 9763) 3a 0.042 1.33 1.33 3b 0.166 2.656 2.656 4a 0.047 0.36 0.36 4b 0.047 0.716 1.427 5a 0.042 0.339 0.673 5b 0.673 2.679 2.679 *6a 2.333 2.333 2.333 6b20 – – – 7a 0.41 1.631 0.817 7b 0.41 1.631 1.631 8a 0.852 1.701 3.401 8b 1.701 1.701 3.401 9a 1.638 1.638 0.82 9b 1.638 1.638 0.411 10a – – – 10b – – – Nystatin 2.7 1.35 1.35 EXPERIMENTAL General synthetic procedure The parent quinones were obtained from hydroquinones according to a previously des- cribed procedure.20 Quinones (300mg; 1.83 mmol 1; 0.96 mmol 2) were dissolved in ethanol (50 mL). Amine hydrochloride salts (in large excess, 22×) were prepared as aqueous solut- ions. The pH of the solution was adjusted to 7–8 by the addition of solid sodium bicarbonate, and the solution was added to quinone. Water and ethanol were added to the reaction mixture to a final ratio water:ethanol of 1:1, and total volume of 300 mL. The reaction mixture was stirred at 60–70 °C for 3 h. Ethanol was removed by vacuum evaporation, and the reaction mixture was extracted two times by dichloromethane, with half the volume of the aqueous phase each time. The organic phase was separated, dried with anhydrous calcium chloride, and the solvent was removed by evaporation under vacuum. The crude products were separ- ated by column or low-bar chromatography and purified by preparative thin-layer chromato- graphy, with the indicated solvents. Numbering scheme for assignment of signals in NMR spectra of all compounds is given in Scheme S-1. Cyclic voltammetry Electrochemical behaviour of synthesized aminoquinones was studied as previously des- cribed.20 ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1205 Biological activity Cytotoxic, antibacterial and antifungal activity of synthesized compounds were analyzed according to a previous study.20 CONCLUSIONS Among the 14 newly synthesized compounds, a potential to be antitumor agents was shown for sec-butylamino derivatives of tert-butylquinone (3a and b) because of their selectivity for tumour cells, allylamino derivatives of tert-butyl- quinone (4a and b) because of their selective induction of apoptosis in tumour cells, including MDR cells, 3′-(sec-butylamino)avarone (7a) and 3′-(allylamino)- avarone (8a) because of their higher cytotoxic activity than cisplatin, as well as 4′-pyrrolidinoavarone (9b) because of selectivity to tumour cells, including MDR cells. Some derivatives showed promising antimicrobial properties: sec-butyl- amino (3a) and allylamino (4a and b) derivatives of tert-butylquinone, because of an activity against E. coli similar to that of amikacin, and strong antifungal activity to C. albicans, which also applies to the pyrrolidino derivative of tert- -butylquinone (5a). SUPPLEMENTARY MATERIAL Additional experimental results, as well as spectroscopic and analytical data, are avail- able electronically on the pages of the journal’s website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support (Grant Nos. OI172055 and III41031). ИЗВОД АЛКИЛАМИНО И АРАЛКИЛАМИНО ДЕРИВАТИ АВАРОНА И ЊЕГОВОГ МИМЕТИКА КАО СЕЛЕКТИВНИ АГЕНСИ ПРЕМА ЋЕЛИЈАМА НЕСИТНОЋЕЛИЈСКОГ КАРЦИНОМА ПЛУЋА, ЊИХОВ АНТИБАКТЕРИЈСКИ И АНТИФУНГАЛНИ ПОТЕНЦИЈАЛ MАРКО ЈЕРЕМИЋ1, JЕЛЕНА ДИНИЋ2, MИЛИЦА ПЕШИЋ2, MАРИЈА СТЕПАНОВИЋ2, ИРЕНА НОВАКОВИЋ3, ДЕЈАН ШЕГАН4 и ДУШАН СЛАДИЋ4 1Иновациони центар Хемијског факултета, Универзитет у Београду, Студентски трг 12–16, 11000 Београд, 2Институт за биолошка истраживања „Синиша Станковић“, Универзитет у Београду, Деспота Стефана 142, 11060 Београд, 3Институт за хемију, технологију и металургију, Универзитет у Београду, Центар за хемију, Његошева 12, 11000 Београд и 4Хемијски факултет, Универзитет у Београду, Студентски трг 12–16, 11000 Београд У овом раду, описана је синтеза четрнаест алкиламино и аралкиламино деривата сесквитерпенског хинона аварона и његовог модел-једињења, терц-бутилхинона. Алкил- амино/аралкиламино групе које су уведене у хинонски остатак биле су разграната, цик- лична, алилна и бензилна. За све добијене деривате одређена је биолошка активност и редокс особине. Испитана је цитотоксична активност синтетисаних деривата према резистентним ћелијама неситноћелијског карцинома плућа (NCI-H460/R), њиховом осетљивом пандану (NCI-H460) и нормалним хуманим кератиноцитима (HaCaT). Одре- ђена је и антимикробна активност према Грам-позитивним и Грам-негативним бакте- ријама и културама гљивица. Неки од синтетисаних деривата су показали селективност ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1206 JEREMIĆ et al. према ћелијама канцера, укључујући и резистентне ћелије. Што се тиче потенцијала за индукцију ћелијске смрти, деривати који највише обећавају су алиламино деривати, који преференцијално активирају апоптозу, са великом селективношћу према ћелијама канцера, укључујући и резистентне ћелије. Неколико једињења је показало обећавајућу антимикробну активност, упоредиву са комерцијалним антибиотицима и антимико- тицима. (Примљено, 27. јуна, прихваћено 23. јула 2018) REFERENCES 1. GBD 2015 Mortality and Causes of Death Collaborators, Lancet 388 (2016) 1459 2. L. H. Kushi, C. Doyle, M. McCullough, C. L. Rock, W. Demark-Wahnefried, E. V. Bandera, S. Gapstur, A. V. Patel, K. Andrews, T. Gansler, Ca ̶ Cancer J. Clin. 62 (2012) 30 3. K. Mross, F. Kratz ,in Drug Delivery in Oncology: From Basic Research to Cancer Therapy, Vol. 1, F. Kratz, P. Senter, H. Steinhagen, Eds., Wiley–VCH, Weinheim, Germany, 2011, p. 3 4. K. Nooter, G. Stoter, Pathol., Res. Pract. 192 (1996) 768 5. M. González-Ibarra, N. Farfán, C. Trejo, S. Uribe, B. Lotina-Hennsen, J. Agric. Food Chem. 53 (2005) 3415 6. T.-S. Lin, L.-Y. Zhu, S.-P. Xu, A. A. Divo, A. C. Sartorelli, J. Med. Chem. 34 (1991) 1634 7. P. S. Sarin, D. Sun, A. Thornton, W. E. G. Müller, J. Natl. Cancer Inst. 78 (1987) 663 8. K. Müller, A. Sellmer, W. Wiergrebe, J. Nat. Prod. 62 (1999) 1134 9. A. J. M. da Silva, C. D. Netto, W. Pacienza-Lima, E. C. Torres-Santos, B. Rossi-Berg- man, S. Maurel, A. Valentin, P. R. R. Costa, J. Braz. Chem. Soc. 20 (2009) 176 10. L.-Y. Tao, J.-Y. Zhang, Y.-J. Liang, L.-M. Chen, L.-S. Zheng, F. Wang, Y.-J. Mi, Z.-G. She, K. K. W. To, Y.-C. Lin, L.-W. Fu, Mar. Drugs 8 (2010) 1094 11. V. Kuete, L. K. Omosa, V. R. Sipowo Tala, J. O. Midio, A. T. Mbaveng, S. Swaleh, O. Karaosmanoğlu, H. Sivas, BMC Pharmacol. Toxicol. 17 (2016) 60 12. R. R. Kitagawa, W. Vilegas, I. Z. Carlos, M. S. G. Raddi, Braz. J. Pharmacogn. 21 (2011) 1084 13. X. Wang, B. Thomas, R. Sachdeva, L. Arteburn, L. Frye, P. G. Hatcher, D. G. Cornwell, J. Ma, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 3604 14. C. S. Coates, J. Ziegler, K. Manz, J. Good, B. Kang, S. Milikisiyants, R. Chatterjee, S. Hao, J. H. Golbeck, K. V. Lakshmi, J. Phys. Chem. B 117 (2013) 7210 15. G. Lenaz, M. L. Genova, Biochim. Biophys. Acta, Bioenerg. 1787 (2009) 563 16. M. G. Traber, J. F. Stevens, Free Radical Biol. Med. 51 (2011) 1000 17. A. Kijjoa, P. Sawangwong, Mar. Drugs 2 (2004) 73 18. T. F. Molinski, D. S. Dalisay, S. L. Lievens, J. P. Saludes, Nat. Rev. Drug Discovery 8 (2009) 69 19. M. Gordaliza, Mar. Drugs 8 (2010) 2849 20. M. Jeremić, M. Pešić, J. Dinić, J. Banković, I. Novaković, D. Šegan, D. Sladić, Eur. J. Med. Chem. 118 (2016) 107 21. J. Vilipić, I. Novaković, T. Stanojković, I. Matić, D. Šegan, Z. Kljajić, D. Sladić, Bioorg. Med. Chem. 23 (2015) 6930 22. National Cancer Institute (NCI). Available from: https://www.cancer.gov/about- cancer/treatment/drugs/lung (last accessed 2018/04/10). ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ALKYLAMINO AND ARALKYLAMINO DERIVATIVES OF AVARONE 1207 23. E. White, D. R. Green, in The molecular basis of cancer, 4th ed., J. Mendelsohn, J. W. Gray, P. M. Howley, M. A. Israel, C. B. Thompson, Eds., Elsevier Saunders, Philadelphia, PA, USA, 2015, pp. 209 24. G. Kroemer, L. Galluzzi, P. Vandenabeele, J. Abrams, E. S. Alnemri, E. H. Baehrecke, M. V. Blagosklonny, W. S. El-Deiry, P. Goldstein, D. R. Green. M. Hengartner, R. A. Knight, S. Kumar, S. A. Lipton, W. Malorni, G. Nuñez, M. E. Peter, J. Tschopp, J. Yuan, M. Piacentini, B. Zhivotovsky, G. Melino, Cell Death Differ. 16 (2009) 3 25. S. Elmore, Toxicol. Pathol. 35 (2007) 495 26. K. L. Rock, H. Kono, Annu. Rev. Pathol. 3 (2008) 99 27. L. M. Coussens, Z. Werb, Nature 420 (2002) 860 28. P. J. Boor, R. M. Hysmith, Toxicology 44 (1987) 129 29. S. Toyokuni, K. Okamoto, J. Yodoi, H. Hiai, FEBS Lett. 358 (1995) 1. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice