73 © 2020 Adama Science & Technology University. All rights reserved Ethiopian Journal of Science and Sustainable Development e-ISSN 2663-3205 Volume 8 (1), 2021 Journal Home Page: www.ejssd.astu.edu.et ASTU Review Paper Recent Trend in Synthesis of Indenoisoquinoline Analoges as Topoisomerase I Inhibitor and Cytotoxic Property: Review Digafie Zeleke, Yadessa Melaku Department of Applied Chemistry, School of Applied Natural Science, Adama Science and Technology University, P. O. Box 1888, Adama, Ethiopia Article Info Abstract Article History: Received 20 November 2020 Received in revised form 18 December 2020 Accepted 02 January 2021 Indenoisoquinolinediones are a class of non-camptothecin topoisomerase I inhibitors that display marked cytotoxic and potent antitumor properties. Currently phase 2 clinical trial of three indenoisoquinoline derivatives LMP400 (indotecan, NSC724998), LMP776 (indimitecan, NSC725776) and LMP744 (MJ-III-65, NSC706744) is on progress. In the last two decades, various synthetic methodologies have been developed for syntethesis of indenoisoquinolines and its analogs. These compounds are of great research significance owing to their novel structures and broad biological activities including antitumor, cytotoxic and topomerase I inhibitory properties. This review address the recent trend in synthesis experimental protocols of indenoisoquinolines and their cytotoxicity, antitumor and topomerase I inhibitory efficacy during the period 2015-2020. The synthetic methodologies and bioactivities reviewed herein might serve as a reference for researchers who are interested to work in the area. Keywords: Indenoisoquinolinediones Cytotoxic Antitumor 1. Introduction Cancer is a major health problem projected to affect about 22 million people by 2030 and has become the second leading cause of morbidity and mortality after cardiac disease.(Bray et al., 2012) It is believed that one of the causes of caner is a genetic disease caused due to mutations in genes associated with cell proliferation and cell death that results in DNA damage.(Baikar and Malpathak, 2010) Among the variety of molecular targets for cancer therapy, DNA topoisomerases (topos) are well-characterized targets owing to their essential roles in triggering, controlling, and modifying a wealth of topological DNA problems during cell proliferation, differentiation and survival.(Chen et al., 2013; Hu et al. 2018) The human genome encodes six topoisomerases whereas E. coli encodes four.(Pommier et al., 2010)On  Corresponding author, e-mail: yadessamelaku2010@gmail.com https://doi.org/10.20372/ejssdastu:v8.i1.2021.314 the basis of their mechanisms, eukaryotic topoisomerases can be classified into two major classes, type I and type II, but there are subtypes under each class.(Delgado et al., 2018; Hevener et al., 2018; Liu et al., 1980). 1.1. DNA Topoisomerase I Inhibitors Camptothecin (CPT) is a potent antitumor drug, an alkaloid isolated from the Chinese tree, Camptotheca acuminate, also known as the “tree of joy” by Monroe Wall and coworkers (Figure 1) (Baikar and Malpathak, 2010). Camptothecin carboxylate was tested clinically in the mid-1970s and showed anticancer activity, but was discontinued because of its side effects(Pommier, 2006; Wall et al., 1993; Yu et al., 2012).Two water- soluble camptothecin derivatives (irinotecan and topotecan) are presently approved by the Food and drug http://www.ejssd.astu.edu/ https://doi.org/10.20372/ejssdastu:v8.i1.2021.............. Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 74 administration (FDA) for intravenously administration (Figure 1). Topotecan is used to treat ovarian cancers and small-cell lung cancers (SCLC), but yet with some side effects (Pommier, 2009). Irinotecan is approved by the FDA for colorectal tumors. It is a prodrug and needs to be converted to its active metabolite SN-38 by carboxylesterase (Figure 1) and it has also some undesired effects (Pommier, 2009). Two other newer camptothecin derivatives, gimatecan and belotecan are in clinical trials (Figure 1) (Beecham and Corp, 1995; Pommier, 2009; Zhang et al., 2005). 1.2. Indenoisoquinolines as topoisomerase I inhibitors Though camptothecin derivatives are effective against previously resistant tumors and are the only class of topoisomerase I (Top1) inhibitors approved for cancer treatment in 1970s, yet, they have pharmacologic and clinical limitations that restrict the dose of active drug that can reach the tumor while sparing normal tissues (Pommier, 2012; Staker et al., 2005). A major limitation to the clinical efficacy of camptothecin-containing therapies is represented by drug resistance (Beretta et al., 2006). The “classical” mechanisms of resistance to CPTs have been extensively studied and include: i) pre- target events, which result in reduced accumulation or inadequate subcellular localization of drug in the cell (i.e. drug efflux, metabolism and intracellular drug distribution); ii) target related events, which result in reduced drug-target interaction (e.g., TopoI down- regulation or gene mutation); iii) post-target events, which result inalterations in the cellular response to DNA damage generated by the formation of the ternary complex (e.g., tyrosyl DNA phosphodiesterase 1, TDP- 1) were typical (Beretta et al., 2013; Sharma et al., 2015). In 1978, Pommier and Cushman synthersized the first indenoisoquinolines (later named as NSC 314622) unexpectedly while treating a cis substituted isoquinolone with SOCl2 afforded the aforementioned indenoisoquinolineinstead of its acid chloride (Figure 2). The topoisomerase I inhibitory activity of indenoisoquinolines was identified in 1998 when a COMPARE algorithm analysis was performed on NSC 314622, which indicated that it may act in a manner similar Figure 1: Camptothecin and its clinically approved derivatives Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 75 Figure 2: The first synthesized indenoisoquinoline to that of camptothecin and derivatives. After being confirmed in vitro test, more potent indenoisoquinoline derivative was developed with an the maximal concentration of drug to cause 50% inhibition of biological activity of cancer cells (IC50) of 20 μmol/L (Burke and Mi 1994; Cushman and Cheng 1978; Marzi et al. 2018; Morrell et al. 2006; Strumberg et al. 1999). 1.3. Biological activities of indenoispquinolines Irinotecan and topotecan are the only current approved Top1 inhibitors approved by FDA for the treatment of cancer. However, these campotothecin derivatives are not ideal drug molecules owing to reversablity of the ToP-DNA cleavage complex and structurally they suffered from lactone ring opening to form a hydroxy acid that has a high affinity for human serum albumin. As a result of this pharmacokenetic problem, there is a great intrest in the development of non-campotothecin Top1 inhibitors among which indenoisoquinolinediones are one of them. Structurally, indenoisoquinolinediones are highly fused compounds which contain a planar tetracyclic heteroring system equipped with multifarious functionalities as exemplified by the lead compound (NCS 314622) (Figure 2) (Morrell et al., 2006). They have been demonstrated to inhibit topoisomerase I enzymes by intercalating between the DNA base pairs and to stabilize a ternary complex consisting of the drug molecule, DNA and topoisomerase I (Morrell et al., 2006). The indenoisoquinolines, like the camptothecins, stabilize DNA–Top1 cleavage complexes by intercalating at the DNA cleavage site, resulting in inhibition of the re-ligation reaction (Marzi et al., 2018). Motivated by this discovery, around 400 Indenoisoquinoline derivatives have been synthesized and evaluated for Top1 inhibition using recombinant enzyme and purified DNA substrates and cellular assays in the NCI-60 cell line panel by various scientists across the globe (Pommier and Cushman, 2009). Most of the synthesized indenoisoquinolines were assessed for antiproliferative activity against 55 different human cancer cell lines of diverse tumor origins at the USA National Cancer Institute screen. The results of topoisomerase I DNA cleavage experiments were reported semiquantitatively and provide a means of comparison with the biological activity of camptothecin (++++) and with the the lead compound (NSC 314622) (++) as follows: +: weak activity; ++: similar activity as the parent lead compound (NSC 314622); +++ and ++++: greater activity than the lead compound (NSC 314622); ++++: similar activity as 1 μM camptothecin (Morrell et al., 2006; Strumberg et al., 1999). 2. Synthesis and biological evaluation of Indenoisoquinolines Because of the multiple disease nature of cancer, one chemotherapeutic agent does not work on all cancer types. Thus, specific biomolecule targeted therapies have become more popular which requires various effective anticancer agents (Foto et al., 2020). Since the discovery of indenoisoquinolines as a novel class of potential anticancer drug candidates, extensive structural modifications have been introduced by altering the substituent of the tetracyclic pharmacophore. Out of nearly 400 synthesized indenoisoquinoline derivatives, currently three of them namely, indotecan (LMP400), indimitecan (LMP744) and LMP776 (MJ-III-65, NSC706744)) were promoted to a Phase I clinical trial in 2010 (Figure 3) and their Phase 2 clinical trial is on progress (Marzi et al., 2019, 2020). Indenoisoquinolines offer a number of potential advantages over the camptothecins, including greater chemical stability, formation of more persistent cleavage complexes, and induction of a unique pattern of DNA cleavage sites (Nagarajan et al., 2003). A varieties of reactions and synthetic methodologies that Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 76 Figure 3: Indenoisoquinoline derivatives in the firstphase clinical trial have been developed to construct indenoisoquinolines including condensation (Conda-Sheridan et al., 2013) of indenoisochromenone with a primary amine, Suzuki- Miyaura cross-coupling (Lebrun et al., 2011) reaction followed by ring-closing metathesis, (Liu et al., 2015) oxidative cyclization of cis acid produced by the condensation of a homophthalic anhydride and a Schiff base. Several other reactions such nucleophilic substitution, sandmeyer reaction, reduction and oxidation techniques have been employed to introduce and optimize varied functional groups on the scaffold (Kiselev et al., 2010; Lebrun et al., 2011; Nagarajan et al., 2003, 2004). Among the latest synthetic efforts, Nguyen et al. (2015) synthesized eighteen nitrated 7-, 8-, 9-, and 10- hydroxyindenoisoquinolines bearing a 3-nitro substituent (Figure 4), all of which were potent dual Top1−TDP1 inhibitors, (Conda-Sheridan et al., 2013; Eun-jung et al., 2012; Morrell et al., 2007) using oxidative cyclization of cis acid produced by the condensation of a homophthalic anhydride and a schiff base (Scheme 1) (Nguyen et al., 2015). 2.1. Synthesis of 8-Hydroxy-9-methoxy-3- nitroindenoisoquinolines and 9-Hydroxy-8- methoxy-3-nitroindenoisoquinolines The synthesis of nitrated 9-hydroxy-8- methoxyindenoisoquinolines 10, 12, and 14, (Scheme1 and 2) and the synthesis of 8-hydroxy-9- methoxyindenoisoquinolines 21, 23, and 25 (Scheme 3 and 4) were outlined. These synthesis began with commercially available homophthalic acid 1 which was nitrated with fuming HNO3 to provide the diacid 2, which underwent dehydration in AcCl to provide anhydride 3 (Scheme 1). The reactive hydroxyl groups in vanillin (4) and isovanillin (15) were protected with a benzylgroup. Benzylvanillin (5) and benzylisovanillin (16) reacted with 3-bromopropylamine hydrobromide to give Schiff bases 6 and 17, which uponcondensation with anhydride 3 in CHCl3 furnished cis acids 7 and 18 N O2N O O OH OMe R N O2N O O OMe OH R N O2N O O R3 9 8 3 8 9 OH 3 7 8 910 10 R = Morpholine 12 R = imidazole 14 R = NH2 22 R = Morpholine 23 R = imidazole 25 R = NH2 31 R = Morpholine 32 R = imidazole 33 R = NH2 7-hydroxy 57 R = Morpholine 58 R = imidazole 59 R = NH2 9-hydroxy 43 R = Morpholine 47 R = imidazole 51 R = NH2 8-hydroxy 44 R = Morpholine 48 R = imidazole 52 R = NH2 10-hydroxy Figure 4: Proposed Top1−TDP1 inhibitors Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 77 in good yields with excellent diastereoselectivities. Treatment of 7 or 18 with SOCl2 (neat) provided a mixture of indenoisoquinoline 8 or 19 and their regioisomers, which was confirmed by 1H NMR spectroscopy. Isomers 8 and 19 appeared as the major products after column chromatography separation. The yields were low due tothe nitro group activating epimerization to the transdiastereomers, which exist in pseudodiaxial conformations and do not oxidize and cyclize in SOCl2 (Nguyen et al., 2015). The displacement of the terminal bromide in 8 or 19 with morpholine or imidazole in 1,4-dioxane, or azide in DMSO, yielded the benzyl-protected compounds 9, 11, and 13 (from 8) (Scheme 2), or 20, 22, and 24 (from 19), (Scheme 4) (Morrell et al., 2007). The treatment of the benzyl- protected starting intermediates with aqueous HBr at 70 °C for 4−5 h, followed by dilution with acetone and then concentration (iterated three times), afforded a mixture that was suitable for vacuum filtration to provide the desired phenols 10, 12, 14, 21, 23, and 25 in high yields (80−100%) and excellent purity (Nguyen et al., 2015). Reagents and conditions: (a) fuming HNO3, 0−23°C; (b) AcCl; reflux; (c) BnCl, DMF, K2CO3, 23°C; (d) 3-bromopropylamine hydrobromide, Et3N, Na2SO4, CHCl3, 23°C; (e) CHCl3, 0−23°C; (f) SOCl2, 0−23°C; Scheme 1. Synthesis of intermediate 8 Reagents and conditions: (a) morpholine, 1,4-dioxane, 23°C; (b) aqueous HBr, 70°C; (c) imidazole, 1,4-dioxane, 70°C; (d) NaN3, DMSO, 23°C; (e)(i)P(OEt)3, benzene, reflux, (ii) aqueous HBr, 70°C. Scheme 2. Synthesis of Nitrated 9-Hydroxy-8-methoxyindenoisoquinolines Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 78 Reagents and conditions: (a) BnCl, DMF, K2CO3, 23°C; (b) 3-bromopropylamine hydrobromide, Et3N, Na2SO4, CHCl3, 23°C; (c) anhydride 23c, CHCl3, 0−23°C; (d) SOCl2, 0 to 23°C Scheme 3. Synthesis of intermediate 19 Reagents and conditions: (a) morpholine, 1,4-dioxane, 23°C; (b) aqueous HBr, 70°C; (c) imidazole,1,4-dioxane, 70°C; (d) NaN3, DMSO, 23°C; (e) (i) P(OEt)3, benzene, reflux, (ii) aqueous HBr, 70°C. Scheme 4. Synthesis of Nitrated 8-Hydroxy-9-methoxyindenoisoquinolines 2.1.1. Synthesis of 7-Hydroxy-3-nitroindenoisoquinolines Commercially available salicylaldehyde 26 was O- benzyl protected to give 27, which reacted with 3- bromopropylamine to afford Schiff base 28 (Scheme 5). Condensation of 28 with anhydride 3 in CHCl3 yielded cis acid 29, which upon treatment with SOCl2, followed by AlCl3 in 1,2-dichloroethane, provided indenoisoquinoline bromide 30 in good yield (Kang et al., 2014; Mancuso at al., 1978). The displacement of the bromide in 23 with morpholine, imidazole, or NaN3, followed by a Staudinger reduction of the azide intermediate and acidic hydrolysis with methanolic HCl, provided the desired amines 31, 32, and 23, respectively. The pure products were isolated without chromatographic purification (Nguyen et al. 2015). 2.1.2. Synthesis of 8- and 10-Hydroxy-3-nitroindenoisoquinolines A similar approach was implemented to prepare 8- hydroxy-3-nitroindenoisoquinolines as shown in scheme 6 (Conda-Sheridan et al., 2013) The condensation of 36 with anhydride 3 provided a mixture of the desired cis acid 37 and its trans diastereomer. Boiling the mixture in CHCl3, followed by filtration, helped to remove the unwanted trans acid and provide the pure cis acid 37 as a sole product. Unfortunately, the treatment of 37 with SOCl2 0 to 23°C, Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 79 Reagents and conditions: (a) BnBr, DMF, K2CO3, 23°C; (b) 3-bromopropylaminehydrobromide, Et3N, Na2SO4, CHCl3, 23°C; (c) anhydride 3, CHCl3, 0 to 23°C; (d) (i) SOCl2, 0−23°C, (ii) AlCl3, 1,2-dichloroethane, reflux; (e) SOCl2, reflux. Scheme 6. Synthesis of 8- and 10-Hydroxy-3-nitroindenoisoquinoline intermediates. followed by AlCl3 in refluxing 1,2-dichloroethane, did not yield the desired bromide 39. However, transformation was observed when cis acid 37 was heated with SOCl2 (neat) at reflux for 4 h, during which the solution turned to clear orange (Nguyen et al., 2015). The treatment of the mixture of bromides 39 and 40 with morpholine and imidazole, followed by chromatographic purification, allowed the isolation of each pure morpholinyl and imidazolyl 8-and 10-benzyloxy compounds 41, 42, 45, and 46 (Scheme 7). After chromatographic separation, all of the benzylprotected materials were subjected to a 3 h debenzylation with aqueous HBr (48 wt %) to provide the desired 8- and 10-hydroxyindenoisoquinolines 44, 44, 47and 48 in good yields and purities (Nguyen et al., 2015). 2.1.3. Synthesis of 9-Hydroxy-3-nitroindenoisoquinolines The treatment of 53 with 3-bromopropylamine hydrobromide and Et3N provided Schiff base 54, which upon condensation with anhydride 3 produced cis acid 55 in good yield and excellent purity (Scheme 8) (Nguyen et al., 2015). The treatment of 55 with morpholine Reagents and conditions: (a) morpholine, 1,4-dioxane, 70°C; (b) HBr, H2O, 70°C; (c) imidazole, 1,4-dioxane, 70°C; (d) NaN3, DMSO, 23°C; (e) (i) P(OEt)3, benzene, reflux, (ii) HBr, H2O, 70°C. Scheme 7. Synthesis of 8- and 10-Hydroxy-3-nitroindenoisoquinoline Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 80 or imidazole in THF provided the corresponding displacement products, which were then stirred in freshly made methanolic HBr or HCl to afford the HBr and HCl salts 57 and 58, respectively. The synthesis of amine 59 by the previous methodology involving reduction of the azide intermediate with P(OEt)3 in benzene (Scheme 4) was not successful due to complications in purification and isolation of the compound in solid form. The Staudinger reaction was therefore reattempted by treating the azide intermediate, obtained from 56, with PPh3 in THF (instead of P(OEt)3 in benzene), followed by 4 h acidic hydrolysis with methanolic HBr. This modification provided the desired amine 59 in 32% yield with excellent purity. 2.1.4. Top I inhibitory and Antiproferative properties of indenoisoquinolines All of the synthesized compounds (collectively shown in Figure 4) were evaluated for Top1inhibitory, TDP1 inhibitory potencies and antiproliferative activities (Nguyen et al., 2015). The results revealed that the 3-nitro group seems to facilitate intercalation into free DNA so that 43, 44, 47, and 48 with large substituents on the side chain, all act as Top1 suppressors at 0.1, 1.0, 10, and 100 μM, respectively (Nguyen et al., 2015). The nitrated compounds also displayed a significant improvement in terms of cytotoxicity when compared to their corresponding dimethoxy analogues, with the 9-hydroxy- 8-methoxy series 10, 12 and 14 possessing low nanomolar antiproliferative potencies (MGM values of 16−21 nM). Indeed, the order of Top1 inhibition and cytotoxicity went from the 9-hydroxyl series 56−59 as the most active and cytotoxic (Top1 inhibition ++++ or more, MGM 14−117 nM) to the 8-hydroxyl series 43, 47, and 51 (+++ to ++++, 56−407 nM), and in the 7-hydroxyl series 31−33 and the 10-hydroxyl series 44, 48, and 52 (++ to +++ for both series, 234 to 3550 nM for the 10- hydroxyl) as the least active and least cytotoxic (Morrell et al., 2007). 2.2. Discovery of Potent Indenoisoquinoline Topoisomerase I Poisons Lacking the 3- NitroToxicophore The NCI-60 screening service recently proposed a policy wherein submission of molecules containing “problematic” functionalities, (Morrell et al., 2006) including Reagents and conditions: (a) 3-bromopropylamine hydrobromide, Et3N, Na2SO4, CHCl3, 23°C; (b) anhydride 3, CHCl3, 0−23°C; (c) (i) SOCl2, 23°C, (ii) AlCl3, 1,2-dichloroethane, 0−23°C; (d) (i) morpholine, THF, 70°C, (ii) HBr, MeOH, 23°C; (e) (i) imidazole, THF, 70°C, ( HCl, MeOH, 23°C; (f) (i) NaN3, DMSO, 23°C, (ii) PPh3, THF, 70°C, (iii) HBr, MeOH, 70°C Scheme 8. Synthesis of 9-Hydroxy-3- nitroindenoisoquinolines e f Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 81 nitro groups, are discouraged. Accordingly, Beck et al. (2015) adopted a research to discover a suitable bioisosteric replacement for the 3-nitro group on the indenoisoquinoline system that would maintain or improve Top1 poisoning activity and growth inhibitory potency (Beck et al., 2015). The synthesis was commenced with benzaldehyde 60, which was reduced with NaBH4 to provide the bromobenzylic alcohol 61 (Scheme 9). Rosenmund−von Braun reaction with CuCN and in situ hydrolysis and lactonization of the intermediate yielded compound 62. Phthalide 62 was subjected to radical bromination, and the obtained 3- bromophthalide intermediate 63 was hydrolyzed to produce 3-hydroxyphthalide 65 which was condensed with phthalide (64) in refluxing methanol−EtOAc with NaOMe and then dehydratively cyclized in refluxing Ac2O to afford 66 (Beck et al., 2015). Indenobenzopyrans 72a-d were synthesized by nearly identical synthetic routes, where the only point of difference was in step c (Scheme 10) (Conda-Sheridan et al., 2013; Robert et al., 1988). Phthalic acid 73 was converted to its anhydride 74 in refluxing AcCl. Anhydride 74 was reduced with NaBH4 in THF to give a 1:1 mixture of 5- and 6-(trifluoromethyl) phthalides 75a and 75b which were readily separated by silica gel column chromatography (Marzi et al., 2002). Phthalides 75a and 75b were separately subjected to radical bromination and subsequent hydrolysis to provide 3-hydroxyphthalides 76a and 76b. Indenobenzopyrans 78a and 78b were obtained by the condensation of 3-hydroxyphthalide 77a and 77b with phthalide (4), and dehydrative cyclization of the unisolated intermediate was carried out as before in refluxing Ac2O (Scheme 11) (Beck et al., 2015). Reagents and conditions: (a) NaBH4, MeOH, 0°C to room temp; (b) (i) CuCN, DMF, reflux, (ii) H2O, reflux; (c) NBS, AIBN, CCl4, reflux; (d) KOH, H2O, reflux; (e) (i) NaOMe, MeOH, EtOAc, reflux, (ii) Ac2O, reflux. Scheme 9. Synthesis of 3-Fluoroindenobenzopyran (66) Reagents and conditions: (a) KNO3, H2SO4, 0°C to room temp; (b) H2, Pd/C, EtOAc; (c) (i) NaNO2, 37% HCl, 0°C, (ii) CuCl, 37% HCl, 0°C to reflux (69a), or (i) NaNO2, 48% HBr, 0°C, (ii) CuBr, 48% HBr, 0−80°C (69b), or (i) NaNO2, 37% HCl, 0°C, (ii) KI, 0°C to room temp (69c), or (i) NaNO2, 37% HCl, 0°C, (ii) NaCN, CuCN, 0°C to room temp (69d); (d) NBS, AIBN, CCl4, reflux; (e) KOH, H2O, reflux; (f) (i) NaOMe, MeOH, EtOAc, reflux, (ii) Ac2O, reflux. Scheme 10. Synthesis 3-substituted indenobenzopyran 72a-d Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 82 3-Hydroxyphthalide 65 was used to generate 81b through nucleophilic substitution and condensation reaction (Scheme 12). (S)-3-Amino-1, 2-propanediol (82) was condensed with indenobenzopyrans 66, 72a-d, 78a-b, 80 and 81, to produce indenoisoquinolines 83-86 (Scheme 13). 1-(3- Aminopropyl) imidazole (87) was condensed with the same indenobenzopyrans, to yield indenoisoquinolines 88-91. In the same way, 3-Morpholinopropylamine (92) was condensed with indenobenzopyrans 66, 72a and 72c to produce indenoisoquinolines 93a-c (Scheme 14). Anhydride 94 was reduced with NaBH4 in PhMe- DMF and the reduction product was cyclized to provide phthalide 95 which was converted 98 in three steps (Scheme 15). In similar way to its carbocyclic analogues, lactone 98 was condensed with primary amines 87, 92 and 10 to yield indenoisoquinolines 99, 100, and 102 (Scheme 16). Reagents and conditions: (a) AcCl, reflux; (b) (i) NaBH4, THF, 0°C, (ii) HCl; (c) NBS, AIBN, CCl4, reflux; (d) KOH, H2O, reflux; (e) (i) NaOMe, MeOH, EtOAc, reflux, (ii) Ac2O, reflux. Scheme 11. Synthesis of indenobenzopyran 78a, b Reagents and conditions: (a) NaSMe, DMF, 120°C; (b) (i) NaOMe, MeOH, EtOAc, reflux, (ii) Ac2O, reflux; (c) m-CPBA, CHCl3, room temp Scheme 12. Synthesis of indenobenzopyran 80 and 81. Reagents and conditions: (a) MeOH−CHCl3, reflux; (b) CHCl3, reflux. Scheme 13. Synthesis of indenoisoquinolines 83-91 a b Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 83 Scheme 14. Synthesis of indenoisoquinolines 93a-c Reagents and conditions: (a) (i) NaBH4, PhMe, DMF, −20 to 35°C, (ii) 5 M HCl, reflux; (b) NBS, AIBN, CCl4, reflux; (c) H2O, reflux; (d) (i) NaOMe, MeOH, EtOAc, reflux, (ii) Ac2O, reflux. Scheme 15. Synthesis of indenobenzopyran 98 Scheme 16. Synthesis of indenoisoquinolines 99, 100and 102 2.2.1. Top I inhibitory and Antiproferative activities of indenoisoquinolines 88-102 Among the bioisosteric compounds, 88, 89a, and 90a-c display the best Top1 poisoning activities. The Top1-mediated DNA cleavage induced by these four compounds is between 75 and 95% that of 1 μM CPT (i.e. +++). MGM concentration of drug to cause 50% reduction in proliferation of cancer cells i.e. growth (GI50) values were calculated to be 0.692 μM for 88, 0.229 μM for 89, and 2.75 μM for 93a. Compounds 88, 89a and 93a are substituted with the halogens F and Cl. The other potential bioisosteres (i.e. I, CN, CF3, SMe and SO2Me) did not display Top1-mediated DNA cleavage assay scores above ++. So the researchers had discosed that only fluorine and chlorine were identified as bioisosteres of nitro group on the basis of Top1 poisoning activities and growth inhibitory potencies (Antony et al., 2007; Beck et al., 2015; Pommier et al., 2014; Sirivolu et al., 2012). 2.3. Synthesis and Cytotoxic Evaluation of Novel Indenoisoquinoline-Propan-2-Ol Hybrids Functionalized propanes are often part of biologically active agents. Particularly, the class of the β-amino propanols consists of multiple representatives with antimalarial, anticancer, Src kinase inhibiting, antimicrobial, and antifungal properties (Chennakesava et al., 2014; Pham et al., 2016; Robert et al., 1988). Inspired by above literature reports, Thi et al. (2016) developed a variety of novel indenoisoquinolines by combining the indenoisoquinoline scaffold with 2- hydroxypropane unit (Pham et al., 2016). In the process, the key starting material, benz[d]indeno[1,2-b]pyran- 5,11-dione 105, was synthesized using a two-step methodology. Condensation of 2-carboxybenzaldehyde 93 with phthalide 64 in the presence of sodium methoxide in methanol/ethyl acetate (2:1) under reflux furnished intermediate 104, which could, after dehydrative acid-catalyzed lactonization in toluene, efficiently be Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 84 Scheme 17. Synthesis of indenoisoquinolines 106a–i. converted to indenobenzopyran 105 in 58% yield after recrystallization from ethyl acetate. Derivatives 106a-i generated in high yields (81–96%) upon treatment of 105 with the appropriate primary amines, as shown in Scheme 17 (Morrell et al., 2006; Pham et al., 2016). Multiple indenoisoquinoline derivatives SAR studies have demonstrated that the presence of hydrogen bonding groups (e.g., hydroxyl) on the lactam side chains correlates well with an increase in biological activity. To prove this principle emperically,the researchers derivatized N-allyl-substituted indenoisoquinoline 109g via hydroxybromination (Pham et al., 2016). Then 108 was converted into 109a-k in moderate to good yields (45–78%) after base-catalyzed nucleophilic substitution of the primary bromide by a series of primary or secondary amines or primary thiols in acetone or DMF at 65℃ (Scheme 18). Furthermore, intermediate 108 was converted to 110 via K2CO3- catalyzed nucleophilic substitution by water. Compound 110 was then further acylated using acetic and isobutyric anhydride in the presence of 3 equiv of triethylamine to provide esters 111a, b in 62–65% yield. On the other hand, treatment of 2,3-propanediol 110 with tosyl or mesyl chloride resulted in the formation of the monosulfonylated diols 113 (54–58%) (Pham Thi et al. 2016). A final option involved the reaction of 108 with 3 equiv of sodium azide in order to furnish the corresponding azide 112 in good yield (81%) (Pham et al., 2016). 2.3.1. Top I inhibitory and antiproferative activities of indenoisoquinolines 106a-I, and 109-113 The synthesized indenoisoquinolines were evaluated in terms of their cytotoxicity profile against two human cancer cell lines, KB and Hep-G2 (Alley et al., 1988; Kiselev et al., 2011; Monks et al., 1991; Vann et al., 2016). Ellipticine was used as a reference compound. The results of this biological assessment, revealed the majority of the compounds exhibit at least moderate cytotoxicity against both cancer cell lines. 105i and 109a,c,e,k exhibited equal cytotoxic activity with the reference, 109a and 109e were the most promising (IC50 values of 0.82 and 0.47 μM and 0.82 and 0.69 μM, respectively, against KB and Hep-G2) (Pham et al., 2016). In the same year, Thi et al. (2016) adopting another project wherein twenty three new indenoisoquinoline substituted triazole hybrids were prepared.(Pham et al., 2016). 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 (Solomon et al., 2010). They hypothesized that the introduction of triazole group into N-functionalized three-carbon side chain of indenoisoquinoline, especially indenoisoquinoline-propan-2-ols, could give potent biological compounds (Monks et al., 1991). To confirm their hypothesis, they synthesized and evaluated novel triazole-indenoisoquinolines hybrids (Monks et al., 1991). In their synthesis strategy, novel triazole- indenoisoquinoline hybrids were developedbased on a CuI-catalyzed 1,3-cycloaddition between propargyl- substituted derivatives and the azidecontaining indenoisoquinoline 112 which was prepared by previous four-step methodology (Haldón et al., 2015; Pham et al., 2016). Azidoindenoisoquinoline112 was transformed to triazole–indenoisoquinolinehybrids 113a–n in high Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 85 yields (60–80%) upon treatment with the appropriate 1- propargyl derivative (Scheme 19) (Pham et al., 2016). Scheme 18. Synthesis of indenoisoquinoline 109-113 and Ellipticine. Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 86 Reagents and conditions: (a) 1.1 equiv ethynyl derivatives, 0.2 equiv DIPEA, 0.1 equiv CuI, THF, reflux, 24 h; (b) 3 equiv Ac2O, 2 equiv Et3N, DMF, rt, 24 h. Scheme 19. Synthesis of triazole–indenoisoquinoline hybrids 113a–n and 114a, b. 2.3.2. Top I inhibitory and Antiproferative activities of indenoisoquinolines 113a-n and 114a, b The in vitro biological assessment of compounds (Scheme 18 and 19) against two human cancer cell lines (KB-CCL-17, HepG2-HB-8065) (Pham et al., 2016) revealed that most of the derivatives possess at least moderate cytotoxic activity. It is important to note that these separate pharmacophores display considerably less potent cytotoxic activities as compared to the most promising conjugates 113a-n. 2.4. Synthesis and Biological Evaluation of New Fluorinated and Chlorinated Indenoisoquinoline Topoisomerase I Poisons In 2016, Beck D. et al. contined their effort to develop highly active analogs of the indenoisoquinolines in Phase 1 clinical trial drugs indotecan (LMP400) (Beck et al., 2014; Maris et al., 2012) and indimitecan (LMP776) (Maris at al., 2012; Pommier and Cushman, 2009), as well as the clinical trial candidate MJ-III-65 (LMP744) (Beck et al., 2016). In the process, structural modifications of fluorinated and chlorinated indenoisoquinolines (Pommier et al., 2014) were incoporatedto enhance their Top1 poisoning activities. They synthesized a series of pentacyclic lactone intermediates that could be used to probe the effects of having different A-ring substitution patterns and different side chains on the lactam nitrogen. In these Synthesis, 3-Hydroxyphthalides 65 (Pommier et al., 2014) and 69a (Pommier et al., 2014) were each condensed with phthalide 64 under basic conditions (Scheme 20) (Shapiro et al., 1961). The 1,3-indanedione intermediates 116a and 116b were each cyclized in situ in refluxing Ac2O to yield lactones 117a and 117b (Shapiro et al., 1961) which were condensed with various primary amines (Antony et al., 2007; Beck et al., 2016; Strumberg et al., 1999) provided indenoisoquinolines 118a-i (Beck et al., 2016). Besides monofloro indenoisoquinoline derivatves, this team synthesized 2,3-diflouro and 2,3-diocloroindenoisoquinoline derives (119a-b and 120a-b) by application related procedure discussed here (Figure 5 ). 2.4.1. Top I inhibitory and Antiproferative activities of indenoisoquinolines 118a-120b The result of Top1 inhibitory assement revealed that out of new compounds, 118d achieved a ++++ score, whereas 118h, and 118i achieved a +++ score (Beck et al., 2016). Among 3-fluoro- or 3-chloro-substituted analogs, two Top1 inhibitors (118h and 118i) were also found to be active in both recombinant TDP1 and TDP2 inhibition assays (Beck et al., 2016). The most cytotoxic compound of the present series was the 3-fluoro derivative 118d having an imidazole-containing side chain, which displayed a GI50 MGM of 11 nM (Beck et al., 2016). Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 87 Reagents and conditions: (a) (i) NaOMe, MeOH, EtOAc, reflux; (ii) Ac2O, reflux; (b) CHCl3, MeOH, reflux. Scheme 20. Synthesis of indenoisoquinolines 118a-i Figure 5: Diflouro and dicloroindenoisoquinolines by Beck et al. (2016). 2.5. Design and Synthesis of Chlorinated and Fluorinated 7-Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I Motivated better bioactivity of 3-flouro and 3-chloro substituted analogs, Elsayed et al. (2017), synthesized eighteen chlorinated and fluorinated 7‑Azaindenoisoquinolines by avoiding indenoisoquinolines with nitro groups on aromatic systems to mitigate the toxic effect of nitro groups (Elsayed et al., 2017). In their work, two strategies were involved to incorporate ring nitrogen and for the replacement of the 3-nitro group with halogens (Elsayed et al., 2017). The anhydrides 121a and 121b (Scheme 21) were prepared by published literature procedures (Gwong-Jen, 2010; Kang et al., 2014). Bromination of 5-methoxy-3-methylpicolinonitrile in the presence of the radical initiator AIBN produced intermediate bromide 122 (Kiselev et al., 2011) which was used directly in the next step without additional purification. The condensation of 122 with 121a and 121b in acetonitrile promoted by Et3N afforded compounds 123a and 123b. Oxidation of 123a and 123b with selenium dioxide provided azaindenoisoquinoline intermediates 124a and 124b. Treating compounds 124a and 124b with NaH in DMF at 0 °C, followed by reaction with 1-chloro-3-bromopropane, yielded the common intermediates 125a and 125b. The common intermediates 125a and 125b were used for the synthesis of the final compounds 126a−j and 127a−i by alkylation of the corresponding amines in DMF as shown in Scheme22 (Elsayed et al., 2017). 2.6. Synthesis and Cytotoxic Evaluation of Carboxylic Acid-Functionalized Indenoisoquinolines Functionalized carboxylic acids are often part of biologically active agents. They might provide a point of attachment for the synthesis of prodrugs so that the pharmacokinetics could be modulated and optimized. The importance of the carboxylic acid functional group Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 88 in drug design is illustrated by the fact that > 450 marketed drugs are containing carboxylic acid functional group (Ballatore et al., 2013; Dung et al., 2019). Inspired by this fact, Dung et al. (2019) synthesized a library of indenoisoquinoline acids in order to find out the influence of carboxylic acid functionalities in the N-lactam side chains of indenoisoquinolines on cytotoxic activities (Dung et al., 2019). Dung et al. (2019) first synthesized indenoisoquinoline acetic acid (Scheme 23). Piperonal 128 reacted with glycine methyl ester hydrochloride to give Schiff base 130, which upon condensation with homophthalic acid anhydride furnished cis-acid 132 in good yield with excellent diastereoselectivity. The treatment of cis-acid 132 with thionyl chloride resulted in conversion to the acid chloride, dihydrogenation, and intramolecular Friedel-Crafts cyclization to provide the indenoisoquinoline ester 133 (Nguyen et al., 2015). The ester 133 was then subjected to hydrolysis by sodium hydroxide in MeOH/H2O (1:1) at 60°C to afford indenoisoquinoline acetic acid 134 in good yield. Similary, the indenoisoquinoline acetic acids containing methoxy group in D ring were synthesized by a procedure as illustrated in Scheme 24 (Kohlhagen et al. 1998; Thi et al. 2016). The cis-acid 136 reacted with thionyl chloride to provide a mixture of indenoisoquinoline esters 137 and 138, which were separated by column chromatography. The latter were then hydrolyzed by sodium hydroxide to afford acids 139 and 140, respectively (Dung et al. 2019). Scheme 23. Synthesis of indenoisoquinoline acetic acid Scheme 24. Preparation of indenoisoquinoline acids 139 and 140. Digafie Zeleke and Yadessa Melaku Ethiop.J.Sci.Sustain.Dev., Vol. 8 (1), 2021 89 Scheme 25. Synthetic route for the preparation of indenoisoquinolines 140a-e. A group of indenoisoquinolines containing carboxylic acid functionalized groups were also synthesized starting from indenobenzopyran 105 using the same protocol (Scheme 25) (Dung et al., 2019). 2.6.1. Cytotoxicity of 134, 139, 140 and 142a-e All the synthesized compounds were subjected to in vitro biological assessment against two human cancer cell lines (KB, epidermoid carcinoma; HepG2, hepatoma carcinoma) and Ellipticine was as positive control. The results revealed that compounds constituting carboxylic acid groups on ring D (compounds 142a-e) displayed moderate cytotoxic activity against KB cell line and high activity against HepG2 cell line with IC50 values ranging from 10 to 32 μM with compound 142c being the most active to KB and HepG2 cell lines (IC50 value of 4.55 and 10.46 μM, respectively). However, the presence of the methylenedioxy or methoxy group on D- ring of indenoisoquinoline scaffold decreased the observed cytotoxicity activity. 4. Conclusion A new class of Topisomerase I inhibitors, indenoisoquinolines, emerged with the accidental isolation of the first lead indenoisoquinoline (NSC 314622) as a byproduct of nitidine chloride synthesis in 1978. Since 1999 indenoisoquinolines were investigated extensively as a noncamptothecin topoisomerase I (Top1) inhibitors which later on succeeded to find the first lead indenoisoquinoline (NSC 314622) with prominent activity back in 1998. Over the years several synthesis reports have been developed leading to synthesis of indenoisoquinolines scaffold and it was pointed out that incorporating electron-donating alkoxy substituents on the A- and D-rings and including nitrogen hetrocycles on the nitrogen side chain of the lactam moeity of the core indenoisoquinolines improved cytotoxic properties. Significant cytotoxicity was observed for 3-nitro indenoisoquinolines compared to their corresponding dimethoxy analogues, in the synthesis reports of 9-hydroxy-8-methoxy series, worth mentioning among the recent achivements in the synthesis reports of the scaffold. The 7- azaindenoisoquinoline derivatives bearing a 3-nitro group and 9-methoxy group were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. On comparative basis, 3-flourinated or 3- chlorinated indenoisoquinolines were found to be safer and better topoisomerase I inhibitors than their respectiive 3-nitro analogs with the most active being the one having immidazole ring on the nitogen side chain of the lactam moeity. Incorporating carboxylic acid moeity in the N-lactam side chain of 3-flourinated indenoisoquinolines afforded compound 142c with significant activity towards KB and HepG2 cell lines (IC50 value of 4.55 and 10.46 μM, respectively). In this review efforts have been made to recap the synthetic methods, reactions and the biological results of various indenoisoquinoline derivatives during 2015-2020. 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