Discovery of Novel Topoisomerase II Inhibitors by ... - ACS Publications

Perspective Cite This: J. Med. Chem. 2018, 61, 8947−8980

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Discovery of Novel Topoisomerase II Inhibitors by Medicinal Chemistry Approaches Wei Hu,#,‡ Xu-Sheng Huang,#,δ Ji-Feng Wu,§ Liang Yang,† Yong-Tang Zheng,*,δ Yue-Mao Shen,† Zhi-Yu Li,ξ and Xun Li*,†

J. Med. Chem. 2018.61:8947-8980. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 10/25/18. For personal use only.

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Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012, Ji’nan, Shandong, P. R. China ‡ State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 27 South Shanda Road, 250100 Ji’nan, Shandong, P. R. China § Institute of Criminal Science and Technology, Ji’nan Public Security Bureau, 21 South QiliShan Road, 250000 Ji’nan, Shandong, P. R. China δ Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Science and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China ξ Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, Philadelphia, Pennsylvania 19104, United States ABSTRACT: DNA topoisomerase II (topo II) is an important enzyme involved in DNA replication, recombination, and repair. Despite the popular applications of topo II inhibitors in cancer therapy, there is still an urgent need to upgrade topo II inhibitors to cope with drug resistance and severe adverse effects. Accordingly, novel topo II catalytic or multitarget topo II inhibitors are gaining more attention and make it possible to ease the toxic limitations of topo II poisons. In this review, medicinal chemistry approaches are mainly discussed toward the development of potent topo II inhibitors with low toxicities.

1. INTRODUCTION Globally, cancer is a major health problem that will affect approximately 22 million people by 2030 and has become the second leading cause of morbidity and mortality after cardiac disease.1 Accordingly, effective chemotherapeutic agents to treat cancers with fewer adverse effects that counteract multidrug resistance (MDR) are still urgently desirable. 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.2 1.1. Topoisomerases. Topoisomerases are ubiquitous enzymes responsible for resolving sophisticated DNA topological intermediates generated during DNA recombination, replication, transcription, and repair processes, such as relaxed, supercoiled, knotted, and catenated DNA.3 All topoisomerases exert their biochemical functions by catalyzing DNA cleavage and religation.4 On the basis of their mechanisms, topoisomerases can be classified into two major classes: type I and type II DNA topoisomerases. Type I DNA topoisomerases cleave one single-stranded DNA during each catalytic cycle. Type II topoisomerases break one double-stranded DNA strand, allowing another segment of duplex DNA to pass through the transient breakage before © 2018 American Chemical Society

resealing the broken strand to resolve DNA knots and tangles. Type II topoisomerases induce topological changes via multiple steps that require Mg2+ and ATP hydrolysis.5 The human genome possesses two type II topoisomerases: topoisomerase IIα and topoisomerase IIβ (topo IIα and topo IIβ). Apparently, they are encoded by two different genes and fulfill different cellular functions. In contrast, lower eukaryotes, e.g., singlecelled yeast, insects, vertebrates, and Xenopus laevis, only possess one type II topoisomerase (topo II). Topo IIα is overexpressed in many rapidly proliferating cancer cells. Inhibiting the function of topo IIα by either inducing double-stranded DNA breaks or blocking ATP hydrolysis is a common approach for cancer therapy.6 1.2. Catalytic Mechanism and Functions of Topo II. Topo II finishes one round of a catalytic reaction via multiple steps at the compensation of ATP hydrolysis.7 A catalytic cycle of topo II is roughly composed of seven steps:8 (i) topo II recognizes and binds to the first double-stranded DNA (G-segment) to form a transient DNA-enzyme complex; (ii) traps the second double-stranded DNA (T-segment) and then binds two ATP molecules, which results in the dimerization of the ATPase Received: August 16, 2017 Published: June 5, 2018 8947

DOI: 10.1021/acs.jmedchem.7b01202 J. Med. Chem. 2018, 61, 8947−8980

Journal of Medicinal Chemistry

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Figure 1. ATP binding domain of topo IIα (PDB ID: 1ZXM) complexed with an ATP analogue AMPPNP (left) and the major H-bonding interactions between topo IIα and AMPPNP (right).

domain and closure of the N-gate; (ii) recruits Mg2+ and cleaves the G-segment resulting in a transient DNA-enzyme covalent complex; (iv) passes the T-segment through the transient break in the G-segment by utilizing energy released from the hydrolysis of one ATP molecule; (v) reconnects the G-segment break facilitated by the hydrolysis of another ATP molecule; (vi) discharges two ADP molecules and induces the opening of the C-gate to release the T-segment; and (vii) disassociates the G-segment and opens the N-gate for another round of reactions. Despite sharing a similar primary sequence and tertiary structure, human topo IIα and β distribute in different tissues, cells, and subcellular locations. They also take different cellular functions. Topo IIα is mainly located in the nuclear plasma and is preferentially overexpressed in proliferating cells.8 Moreover, as a specific and sensitive biomarker for cell proliferation, topo IIα is essential for tumor cell growth and division and abundantly functions in rapidly growing solid tumors. Topo IIα is also essential for chromosomal segregation in mitosis to disentangle two sister chromosomes during the late G2 and M phases, a function that cannot be replaced by topo IIβ. In addition, topo IIα is a cell cycle-dependent enzyme that has the lowest expression levels during the G0/G1 phase. The expression of topo IIα starts to increase in the S phase, reaches its peak in the G2/M phase, and then sharply decreases at the end of mitosis.9 In contrast, topo IIβ is mainly distributed within nucleoli in both normal and malignant tumor cells; however, its expression appears to be consistent in proliferating and normal cells. Topo IIβ is expressed at relatively low and steady levels during all stages of the cell cycle. Although topo IIβ is not essential for cell survival and proliferation, it plays apparent roles in cell development, transcriptional regulation, and differentiation and is implicated in neuronal differentiation and longevity.10 Moreover, human topo IIα relaxes positively supercoiled plasmids faster than negatively supercoiled plasmids; however, this was not observed for topo IIβ.11 For these reasons, topo IIα seems to be the more attractive biotarget for exploiting potent antineoplastic drugs.12 1.3. Structure of Topo II. Human topo IIα is a homodimer. Its monomer is composed of 1531 amino acids including four sections, N-gate, central-gate (DNA-gate), C-gate, and CTD.13 The N-terminal portion possesses an ATPase domain, which belongs to the GHKL (Gyrase, HSP90, histidine Kinase, mutL) superfamily of proteins. Upon ATP binding, two ATPase domains from each monomer move to close the N-gate via dimerization. The central core DNA gate, also known as the DNA binding/cleavage gate, contains a Mg2+-binding TOPRIM (Topoisomerase/Primase)

fold, a winged helix domain (WHD), which includes the tyrosine active site, and a “TOWER” domain, which interacts with another monomer of the dimer. The C-gate holds and controls the passage of the T-segment. The CTD is essential for nuclear localization and possesses sites subject to post-translational modifications. The ATPase domain is situated at the N-terminus of topo II, and it is highly conserved between human topo IIα and topo IIβ. Although the DNA gate is the principal target of topo II inhibitors, because it is highly conserved between the topo II isoforms, it is a challenge to design topo II inhibitors with high specificity against topo IIα. The differences between these two isoforms are mainly reflected in the C-terminal portion. The C-terminus of topo IIβ contains 443 amino acid residues and is slightly longer than that of topo IIα (360 amino acids).14 Detailed structural studies of topo II provide a valuable guide to discover topo II inhibitors to inhibit different catalytic steps. Among the many drug discovery strategies, rational drug design based on the tertiary structure is an efficient and economical practice. Targeting the ATPase domain is one of the most widely accepted approaches to obtain potent topo II catalytic inhibitors that interfere with the ATP-dependent catalytic steps instead of inducing double-stranded DNA breaks. For clarity, a crystallographic structure of the ATP binding domain of topo IIα complexed with an ATP analogue AMPPNP (PDB ID: 1ZXM) and the major H-bonding contacts between topo IIα and ligand was provided, which is shown in Figure 1. The ATP binding domain of topo IIα is illustrated as a T-shaped cavity that is composed of three binding pockets. The relatively broad and shallow pocket I is surrounded by Ser149, Asn150, Arg98, Ser148, Lys157, and Tyr34. Among these residues, Ser149 and Tyr34 are the key residues. Tyr34 binds with the 3-N of the adenine ring of ATP through water bridges, whereas the ribose ring of AMPPNP is stabilized by H-bonding interactions between the 2′- and 3′-hydroxyl groups and side chains of Ser149. Pocket II, which is composed of Asn91, Gln97, Asn120, Pro126, Ile125, and Thr215, appears to form a long and narrow channel, and Asn91 and Asn120 take on important roles. Asn 91 not only generates a H-bonding interaction with the phosphate part of the ligand but also contributes to a coordinative bond with the catalytic Mg2+. As for the Asn120, it interacts with the adenine ring through both direct H-bonding and water-mediated contacts. Solvent-exposed pocket III is a binding region for polar groups and is encircled by Ala167, Gly166, Lys168, Tyr165, and Thr147, which are all favorable contributors to the binding affinity by producing effective interactions with the phosphate 8948



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moieties of the ligand. Additionally, a Mg2+ ion is also situated in pocket III.15 The “enhancing protein backbone binding” concept, by identifying binding factors (e.g., hydrogen bonds, electrostatic interactions, steric effects, etc.) and their essential influences on bioactivity and specificity, has been considered as an effective means of identifying potent drug candidates with the capability to cope with drug resistance.16 Many novel topo II inhibitors have been developed following this strategy.

base pairs and maintains the scissile phosphate groups away from the DC-8 portion at a distance of 7.9 Å, with the polycyclic core (rings A−D) lying between base pairs, and the glycosidic moiety and the E-ring individually extending into the DNA major and minor grooves. As a result, the formation of this stable but reversible ternary complex upgrades the binding affinity between drug and the active cleavage site of topo II by forming H-bonding, hydrophobic and π−π stacking interactions, leading to the blockade of DNA repair.20 As far as the DNA intercalating agent MTZ-involved complex is concerned (Figure 2B and D), the incorporation of MTZ also affects the stacking interaction of DNA base pairs and maintains the phosphate groups away from the DC-8 part at a distance of 7−8 Å with the two polyamine side chains on the C-ring projecting into the DNA major and minor grooves, respectively. Interestingly, the polycyclic anthraquinone nucleus (rings A−C) plays a dual role. It not only prevents DNA repair by physically blocking the stacking effect of base pairs but also stabilizes the structure by forming effective H-bonding contacts with Gln778 and Gly504 in the DNA major and minor grooves. Additionally, the side chain on the C-ring that stretches into the DNA minor groove makes a major contribution to the stability of the enzyme-DNA adduct by adjusting its flexibility with a preponderant conformation while also forming more favorable H-bonding interactions with several key residues, including Glu522, Asn520, and Arg503. In contrast, the side chain on the C-ring that inserts into the DNA major groove fails to take full advantage of the extra space and only generates several water-mediated interactions.21 Unlike topo II poisons, the other class, which is described as topo II “catalytic inhibitors” or “suppressors”, is thought to kill tumor cells by inhibiting the essential enzymatic activity of topo II. These inhibitors work by impeding the conjunction of topo II with DNA without generating increased levels of topo II covalent complexes, blocking the enzyme ATP-binding site (e.g., purine analogues), preventing the cleavage of DNA (e.g., merbarone), or inhibiting the hydrolysis of ATP (e.g., bisdioxipiperazine analogues), thus showing decreased cytotoxicity relative to that of topo II poison and confronting MDR. Between the two topo II isoforms, the inhibition of topo IIα, rather than topo IIβ, has been recognized as a well-defined approach to develop novel antineoplastic agents. The reason, to a certain degree, is that topo IIα is necessary for the hypercompaction of mitotic chromosomes in human cells, whereas inhibitors that target topo IIβ are frequently found to be associated with undesirable side effects, such as drug resistance, cardiotoxicity, or secondary malignancies.22,23 In recent years, efforts aimed at interfering with topo II-mediated processes have exploited numerous small molecule topo II inhibitors. Figure 3 presents the chemical structures of some representative compounds that have been approved for the treatment of varied malignancies or which have entered into clinical trials. Among them, the imidazoacridinone derivative C-1311 (13, Symadex) was recommended for phase II a few years ago.24,25 The quinolone derivative vosaroxin (12, SNS-595), which is considered as a first-in-class antineoplastic drug, has entered phase III trials for the treatment of relapsed and refractory acute myeloid leukemia (AML).26−28 The quinoxaline derivative XK469 (9, NSC697887), as a selective topo IIβ inhibitor, has entered phase I evaluation.29 The third generation synthetic topo II inhibitor 9-aminoanthracycline derivative amrubicin (10, SM-5887) has been shown to have comparable efficacy to doxorubicin in adult soft tissue sarcoma (STS) without obvious cardiac toxicity in a phase II study.30 Acting as DNA intercalators, two naphthalimide

2. TOPO II POISONS AND CATALYTIC INHIBITORS/SUPPRESSORS Evidence has shown that topo I and topo II are expressed at different levels in different cancer types. For example, topo I is mainly expressed in colorectal cancer, whereas topo II is mostly expressed in ovarian and breast carcinomas.17 Accordingly, both topo I and topo II inhibitors have been clinically used in cancer therapy. Topo I inhibitors stabilize the covalent cleavage complexes of topo I and DNA and generate single-stranded DNA cleavages. Although Topo I can be removed from these topo I and DNA covalent adducts by tyrosyl-DNA phosphodiesterase 1 (TDP1) followed by DNA repairing to heal single-stranded DNA nicks, this DNA repair pathway is inefficient for removing all topo I inhibitor-induced DNA adducts. Eventually, DNA replication forks collapse after encountering these DNA adducts and then generate double-stranded DNA breaks. This is why topo I inhibitors in clinical studies, such as camptothecin (CPT) and its derivatives topotecan, 10-hydroxy camptothecin (OPT), irinotecan (CPT-11), and rubitecan (9-NC), are normally toxic for rapidly growing tissues. Moreover, the cytotoxic essence of these drugs easily induces significant therapy-related secondary effects, including cardiotoxicity, myelosuppression, gastrointestinal toxicity, and even secondary leukemia.18 Hence, recent studies have concentrated on the exploitation of drugs or agents targeting topo II. On the basis of different modes of action, topo II inhibitors are classified into two broad categories: topo II poisons and topo II catalytic inhibitors. The first type, which covers the majority of clinical antitumor agents (e.g., etoposide, doxorubicin, mitoxantrone, salvicine, and teniposide) and represents frontline therapies for a wide spectrum of solid and hematological malignancies, can kill cancer cells by increasing the level of covalent “topo II-DNA complexes” and prevents the cleaved DNA strand(s) from religation, allowing the accumulation of undesired double strand breaks. Because these agents can convert functional topos into lethal “lesions”, which induces DNA strand breaks that eventually lead to apoptosis of tumor cells, they are termed topo poisons. On the basis of the differing DNA binding patterns, topo II poisons can be further divided into two types: DNA intercalating and nonintercalating agents. DNA nonintercalating agents (e.g., etoposide (1), teniposide (2), and quinolone) have relatively weak interactions with DNA and exert their functions by trapping “topo II-DNA complexes”. In contrast, DNA intercalating agents, which usually contain coplanar aromatic frameworks such as adriamycin, amsacrine, and mitoxantrone (MTZ), can reversibly insert into the base pairs of DNA and disrupt enzymes involved in DNA transcription and replication.19 For explaining the binding patterns of two types of topo II poisons better, the 3D crystallographic structures of ternary “drug-enzyme-DNA” complexes (A: etoposide, PDB iD: 3QX3; B: MTZ, PDB ID: 4G0V) are exemplified, respectively, in Figure 2. For the DNA nonintercalating agent etoposide (Figure 2A and C), the insertion of etoposide affects the stacking interaction of DNA 8949



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Figure 2. Cocrystal structures of ternary “drug-enzyme-DNA” complexes (A: etoposide, PDB ID: 3QX3; B: MTZ, PDB: 4G0V), and the binding contacts are also schematized in a simplified form (C: DNA nonintercalating agent etoposide; D: DNA intercalating agent MTZ).

the function of topo II. In this Perspective, we elucidate the design strategies, binding modes, as well as the SAR exploration of recently reported topo II inhibitors (2013−2017), including both topo II poisons and catalytic inhibitors, on the basis of their different mechanisms of action and structural scaffolds with the expectation of providing useful insight into the identification of more diversified topo II inhibitors as potent antitumor leads. 3.1. Strategies for the Identification of Novel Topo II Inhibitors/Poisons. 3.1.1. Using Available SAR Results to Guide the Exploration of Novel Topo II Inhibitors. Using available SAR outcomes has always been a quick, simple but effective approach to detect pharmacophores and/or structural segments that can be directly utilized for exploiting new topo II inhibitors. In the process of identifying novel chemotypes, clear pharmacophoric elements, whether working as scaffolding cores or peripheral substitutions, are useful structural determinants to improve drug properties and avoid the shortfalls of prototypes. For this reason, topo II inhibitors identified recently have largely benefited from available SAR studies. 3.1.1.1. α-Terpyridine Analogues. 2,4,6-Triarylpyridines (α-terpyridines) containing a phenolic moiety at the 2- and 4-positions have been established as a promising skeleton for developing novel topo II inhibitors. Lee’s investigations on the

derivatives, amonafide (11, AS1413) and mitonafide (14, NSC 300288), have been studied in phase III and phase II clinical trials, respectively.31,32 Resistance to chemotherapeutic drugs has been a major obstacle in cancer chemotherapy. Most topo II poisons have frequently suffered from MDR, which is related to the overexpression of MDR-associated proteins (i.e., P-gp, MDR1, MRP1, and BCRP), resulting in enhanced efflux of chemotherapeutic drugs out of the cells.33 Moreover, topo II poisons may also trigger chromosomal translocations that lead to specific secondary leukemias.34 Thus, the development of topo II catalytic inhibitors that will not only kill cancer cells but also generate much less DNA damage due to topo II-mediated DNA cleavage, lower cytotoxicity to normal cells, and lower risk of secondary tumors, has become a key focus of recent research.

3. STRATEGIES TO IDENTIFY AND DEVELOP CHEMICAL AGENTS TARGETING TOPO II WITH HIGH EFFICACY AND SPECIFICITY Because of the pivotal role of topo II in correlation with various malignant carcinomas, intensive efforts attempting to explore effective tumor therapeutics have led to a panel of chemical agents with diversified structures that are capable of modulating 8950



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Figure 3. Illustrative examples of representative topo II inhibitors that have been used in clinical trials or are at various stages of development.

Figure 4. 2,4,6-Triarylpyridine (α-terpyridine) is a promising backbone for exploring dual topo inhibitors.

α-terpyridine chemotype have presented several series of derivatives (compounds 15−17). Taking compound 17 as an example (Figure 4), it can be observed that the position of the hydroxyl group in the 2-phenyl ring affects inhibitory potency. Specifically, a meta- or para-phenolic moiety at the 2-position in conjunction with a 6-chlorophenyl group is more favorable for topo I/II

inhibition and cytotoxicity. The monophenolic moiety, which is indispensable for bioactivity, was retained at the 2-position of the pyridine backbone, and the addition of hydroxyl groups at different positions was also explored. In addition, because chlorinated compounds are frequently included in drug design and might contribute to increased protein−ligand binding affinity 8951



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Figure 5. SAR investigation of the α-terpyridine motif led to a series of diphenyl-6-thiophen-2-yl-pyridine derivatives (18) with high selectivity against topo II over topo I and the X-ray crystallographic structure of 18a complexed with human topo IIα (PDB ID: 1ZXM).

bisnaphthalimides (e.g., 19 and 20), and fused naphthalimides (e.g., R16, B1 and 21)38,39 have been reported to display remarkable antineoplastic properties, and some of them have entered clinical trials,40 as illustrated in Figure 6. Xu et al. have presented a pool of naphthalimide-based derivatives (22)41 by combining the merits of a cytotoxic topo inhibitor and a cytostatic antiangiogenic agent directed toward producing advantageous complementarity. This is based on the fact that antiangiogenic agents can normalize tumor vessels and promote the delivery of cytotoxic agents to tumor sites.42,43 However, because tumors are involved in complicated physiological and pathological networks, it is not easy to produce long-term curative effects only using antiangiogenic agents. In the target compounds shown in Figure 7, long alkyl chains and polyamines are contributors to antiproliferative activity and the naphthalimide backbone contributes to the cytotoxic efficacy by inhibiting topo II. As expected, the target compounds, especially 22a, have been shown to be effective chemotherapeutics by targeting both topo II and angiogenesis-related receptor tyrosine kinases (KDR and EGFR1). 3.1.1.3. 2-Phenylnaphthalene Analogues. 2-Phenylnaphthalene was found to be another attractive pharmacophoric skeleton for the development of potent topo II inhibitors, ideally topo IIα-targeting inhibitors. Shen et al. have reported a spectrum of 2-aryl-substituted naphthalenoids (23) to expand the molecular diversity and enrich the SAR exploration on 2-phenylnaphthalene-based pharmaceuticals.44 The SAR results of these derivatives on the topo II-sensitive breast cancer cell line MDA-MB-231 delineated that the hydroxyl substitution at the 6-position of the phenylnaphthalene moiety was required for cytotoxicity. For example, compound 23b with a 6-hydroxyl substituent gave increased potency compared to the 6-methoxyl counterpart 23c. As far as the substituents on the 2-phenyl ring are concerned, polar substituents (e.g., methoxycarbonyl, hydroxymethyl, and hydroxyl) are generally favorable for antiproliferative activity, whereas the strong hydrophilic acidic groups (e.g., carboxyl and carboxymethyl), in contrast, seriously impaired the bioactivity. As to the impact of the 4′-substitution on the 2-phenyl ring, polar substituents are still applicable with an

and binding site specificity, chlorophenyl moieties were introduced at the 6-position of the pyridine core. Moreover, a small set of 4-aryl substituents were fixed at the 6-position to determine its influence on activity from which the 2-furyl moiety demonstrated the best binding affinity for topo II and had higher selectivity.35 Kwon and co-workers also exploited a spectrum of diphenyl6-thiophen-2-yl-pyridine derivatives (18), which manifested excellent inhibition and superior inhibitory specificity toward topo II over topo I.36 SAR exploration demonstrated that, when the phenyl ring at the 6-position of the central pyridine is replaced by a thienyl or furyl unit or when the phenyl groups at the 2- and 4-positions are substituted with a hydroxyl group, potent and specific topo inhibition could be achieved. The orientation of the hydroxyl group has a marked influence on the potency and cytotoxicity. Specifically, compound 18a manifested the most potent and elevated efficacy compared with the controls (1 and 86) and can be regarded as a potential lead for further optimization. On the whole, systematic QSAR analysis demonstrated that both the hydrogen-bond donor and acceptor elements of the hydroxyl group contribute to the potency and cytotoxicity; it also supported the pivotal roles of the meta- and/or parahydroxyl groups in enhancing the integral bioactivity. However, extended SAR investigation of these pyridine-based inhibitors by making subtle modifications to the disposition of the hydroxyl substituents and altering the aryl groups changed the mechanism of action of the compounds.37 For example, compound 18a acts as a topo II catalytic inhibitor with high selectivity toward topo IIα (more than 98% inhibition) over the β-isoform. In contrast, compound 18b has been shown to act as a topo II poison. Figure 5 displays the design and SAR patterns associated with topo II inhibition by 18 as well as the binding mode of 18a complexed with human topo IIα (PDB ID: 1ZXM), indicating that the p-phenolic hydroxyl at the 2-position of pyridine core forms two important H-bonding interactions with Lys123 and Asn120. 3.1.1.2. Naphthalimide Analogues. Naphthalimides have been characterized as a class of DNA intercalators and topo II inhibitors; many mononaphthalimides (e.g., 11 and 14, see Figure 3), 8952



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Figure 6. Chemical structures of naphthalimide-bearing topo II inhibitors.

Figure 7. Naphthalimide-based derivatives 22 were designed as multifunctional molecules by targeting both topo II and receptor tyrosine kinases (RTKs).

Figure 8. SAR exploration of 2-phenylnaphthalene-based derivatives (23).

by the insertion of a “-O-CH2-” linker between the 2′,4′-dichloro phenyl fragment and thiadiazole core (24e and 24f), leading to a loss in both activity and DNA biosynthesis in normal fibroblast cells. As to the substituents on the 5-phenyl motif, both the substitution pattern and electronic environment were explored. This survey showed that the electron-donating meta-hydroxyl substituent provided higher affinity than the electron-withdrawing meta-chlorine counterpart (24a vs 24b). Furthermore, the hydroxyl group has the ability to enable the potential formation of a hydrogen bond, resulting in increased bioactivity. Compounds with 2′,4′-dichloro substituents (24c and 24d) displayed better potencies than their monosubstituted counterpart 24b, which confirmed that the second chloro has a conducive effect on

activity order of hydroxyl (23a, 23d, 23e, 23f) > amino (23g) > 1-hydroxymethyl (23h), as evinced in Figure 8. 3.1.1.4. 1,3,4-Thiadiazole Analogues. The 1,3,4-thiadiazole skeleton with a 2,5-disubstituted pattern has been validated as an attractive structural motif for identifying potent topo II inhibitors with weaker cytotoxicity toward normal cells. Inspired by this, Plech and co-workers have developed a limited spectrum of 1,3,4-thiadiazole derivatives (24) and evaluated their antiproliferative activities on two kinds of breast cancer cell lines, MDA-MB231 and MCF-7, which are sensitive to topo II inhibitors.45 The SAR exploration, as shown in Figure 9, demonstrated that the incorporation of the 1,3,4-thiadiazole core with a 5-phenyl motif is essential for acquiring potent antitumor potency, as evidenced 8953



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Figure 9. Modification of the 1,3,4-thiadiazole core with 2,5-disubstituted groups led to a series of derivatives (24) with different topo II-interacting patterns.

subjected to further biochemical and antiproliferative assessments.48 The results showed that both compounds are topo IIαtargeted poisons without any topo I inhibition and displayed significant antiproliferative activities toward melanoma and prostate tumor cell lines. With respect to the AMTAC series, the introduction of para-methoxy on the phenyl ring (26b) provided enhanced potency and selectivity in contrast with its unsubstituted counterpart (26a). This may be due to the methoxy group, which contributes to the stabilization of the ternary complex, resulting in elevated topo II inhibition. Further molecular binding studies were conducted by using 26b as a tool compound. The result was that the methoxyl O and the N atom of the C=N bond participate in the formation of H-bonding interactions with the key Ser149 residue while the carbonyl O atom chelates with the catalytic Mg2+ in the active binding domain. In addition, several hydrophobic contacts between 26b and the residues Tyr34, Asn91, Asp94, Arg98, Ile141, Phe142, and Thr215 were also favorable contributors to the enhanced binding affinity. 3.1.1.6. Fused Aryl Phenazine Analogues. Compounds containing fused aryl phenazine skeletons, including but not limited to benzo[a]phenazine, pyrido[a]phenazine, benzo[a]phenazine diones, and tetrahydropyrido[a]phenazine, are capable of interfering with DNA metabolism by intercalating between DNA base pairs; therefore, these skeletons are typically used in the design of topo inhibitors. For example, the benzophenazine derivatives XR11576 (27), NC-190 (28), and NC-182 (29) have entered clinical studies based on their broad antitumor activity that is not affected by MDR. Among these, the NC series exhibited selective and superior topo II inhibition upon induction of topo II-dependent DNA fragmentation, whereas XR11576 works as a topo I/II poison.49 Enlightened by these findings, Huang et al. have developed an array of benzophenazine derivatives (30) by initially modifying rings B and D in the benzo[a]phenazine core.50 By introducing various terminal amino groups (e.g., dimethylamino, diethylamino, piperidinyl, N-methylpiperazinyl, and pyrrolidinyl) onto ring B and decorating ring D with substituents with different electronic properties (e.g., NO2, F,

binding affinity. As far as the impact of the substituents on the phenylamine group are concerned, there is a general trend with an order of decreasing activity Br > Cl. Most notably, compound 24c, with a bulkier bromine substituent that has lower electronegativity, is equipotent to control 1 against the two tested breast cancer cell lines. Further investigation of the mechanism of action revealed that compounds with electron-withdrawing substituents (e.g. 2′,4′-dichloro, 24c and 24d) on the 5-phenyl group of the 1,3,4-thiadiazole core were identified to be topo II poisons, whereas the electron-donating group-bearing compounds (e.g. metahydroxyl, 24a) were shown to be topo II catalytic inhibitors. Nevertheless, limited by the number of available 1,3,4-thiadiazole derivatives, a definitive correlation between the structural elements and target recognition requires further validation. 3.1.1.5. Acridine Analogues. The acridine moiety has emerged as a promising chemotype for exploring new bioactive molecules based on the fact that amsacrine is the first and only approved topo II poison for treating AML so far. However, some amsacrine deficiencies, such as its ineffectivity for solid tumors, poor bioavailability, and drug resistance, have stimulated further chemical optimization of this skeleton. In addition, the thiosemicarbazide (TSC) motif is known as an important scaffold due to its various biological properties. However, the precise mechanism of action of TSC derivatives against tumors is not fully understood. The current majority viewpoints are that TSC derivatives exert antitumor effects by inhibiting the increased activity of iron-associated proteins, such as ribonucleotide reductase (RR), which is a crucial enzyme in DNA synthesis and has a high correlation with tumor cell proliferation by catalyzing the rate-limiting step of dNTP synthesis46 or as an iron chelator.47 With these observations in mind, Almeida et al. explored a spectrum of acridine-thiosemicarbazone derivatives by incorporating a variety of TSC units into an acridine backbone. A SAR investigation revealed that compound 25 with an unsubstituted phenyl group gave the best potency in antiproliferative assays. They further developed two acridine-based compounds 26a and 26b from the lead 25 via linker transformation and subsequent spontaneous spirocyclization (Figure 10), which were then 8954



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Figure 10. Discovery of the novel spiro-acridines 26a and 26b as topo IIα-mediated poisons derived from the acridine and TSC chemotypes.

Figure 11. Benzo[a]phenazine heterocycle has been shown as a promising backbone for exploring dual topo I and II inhibitors (e.g., 30 and 31).

Cl, and OCH3), the resulting SAR profile revealed that the basic terminal alkylamino groups on ring B have more significant effects on activity than the substituents on ring D, as the removal of these basic side chains resulted in a complete loss of activity. Moreover, it appears that the disposition and electronic effects of the substituents on the D ring as well as the length of the side chain did not have an impact on the cytotoxic effects. For gaining further insight into the SAR profile, subsequent chemical modifications, which occurred on rings A, C, and D of the benzo[a]phenazine backbone by transferring the alkylamino groups from the 5- to 7-position of ring C, with or without installing substituents on rings A and D, further clarified the indispensable effects of the alkyl amino groups for cellular activity.51 It is also notable that all benzo[a]phenazine derivatives (30 and 31, Figure 11) have been shown to be dual topo I and II inhibitors. Therefore, exploration of these benzo[a]phenazine derivatives has the potential to identify potent antineoplastic agents with improved antitumoral regimens. 3.1.2. Conformational Restriction Approach Constitutes a Core Aspect of Drug Discovery. In connection with a recent work on the exploitation of novel topo inhibitors, researchers from Kwon’s lab exploited several 2,4,6-trisubstituted pyridines via a conformational restriction (or rigidification) strategy using a cyclization approach at the 5,6-position of the pyridine core (32−36). The resultant compounds showed enhanced topo inhibition as well as cytotoxicity when compared to their flexible terpyridine prototypes (Figure 12). It is very likely that the

reconstructed rigid structure tends to have less conformational entropy than the flexible prototypes. Therefore, a molecule with a rigid architecture usually has more chance to realize a desired selectivity for certain isoforms and also has a reduced possibility of drug metabolism compared with its flexible counterparts. It goes without saying that this rule also applies to the identification of topo II inhibitors with improved metabolic stability. In other words, more rigid topo II inhibitors with less conformational entropy are thus more easily able to insert into the DNA-enzyme complex as possible DNA intercalators. The systematic SAR exploration manifested that substituents at both the 2- and 4-positions of the pyridine core have a marked influence on the potency and selectivity of topo inhibition. When the 4-substitution was fixed as a heterocyclic furyl or thienyl motif, compound 32a with a hydroxyphenyl group at the 2-position of the 5H-indeno[1,2-b]pyridine moiety was identified as a dual topo I and IIα inhibitor, whereas the replacement of the 2-hydroxyphenyl with a 2-furyl or thienyl unit (32b) exhibited topo II-selective inhibition. In compounds 32c−32f, the 4-substituent was anchored as a m-hydroxyphenyl group, and chemical diversity was achieved by installing a thienyl or furyl motif at the 2-position. The result was that they generally showed preferential and strong inhibition toward topo IIα. Compared with compounds 32g−32i, the position of the hydroxyl groups in the 4-phenyl ring as well as the disposition of the 2-fury substituent had an impact on binding affinity. Compounds 32g and 32h displayed dual topo I and topo IIα inhibition, whereas 32i exhibited lower 8955



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Figure 12. Discovery of terpyridine-based libraries 32−36 through a conformational restriction approach and methylation modifications.

3.1.3. Scaffold-Hopping Strategy through Optimization of Parent Molecules. Scaffold-hopping, also called lead hopping, has been widely applied in the discovery of novel chemotypes with equipotent or improved potencies.55 For example, the therapeutic effectiveness of the topo I inhibitor CPT has been severely compromised to some extent by rapid inactivation through hydrolysis of the E-ring lactone under physiological conditions. To overcome this deficiency, You et al. developed a wealth of lomefloxacin analogues via a step-by-step structural optimization,56 as displayed in Figure 13. They initially simplified the CPT framework and separated it into two bioactive fragments,

enzyme inhibitory activity, implying that the 2′-furyl motif is essential for cytotoxicity. Following up these discoveries, they continuously put forth two series of terpyridine-based analogues: benzo[4,5]furo[3,2-b]pyridine (35) and chromeno[4,3-b]pyridine (36).52−54 In these series, compound 35a was validated as a nonintercalating topo IIαspecific inhibitor, and 36a was determined to be a nonintercalating dual topo I and IIα inhibitor. It is interesting that, although the methoxy-substituted compounds 35b and 36b lost topo inhibitory potency, they generally gave significant improvements in metabolic stability and PK properties, as shown in Table 1. 8956



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Table 1. Comparison of Stability, Metabolic, and PK Parameters between the Methylated Compounds (35b and 36b) and Their Hydroxyl Precursors (35a and 36a) 35a human dog rat mouse

0.30 ± 0.08 2.28 ± 0.27 1.50 ± 0.31 4.55 ± 5.61

human dog rat mouse

31.1 min 37.6 min 36.3 min 16.7 min

AUC0−24 (μg h−1 mL−1) Cmax (μg/mL) Tmax (h) t1/2 (h) MRT (h) Ft (%)

0.15 ± 0.00 0.02 ± 0.01 1.00 ± 0.00 12.4 ± 1.32 15.3 ± 1.2 2.70

35b Stabilitya 43 59 22 16 Half-Lives (t1/2)/h 1.81 h 2.26 h 1.09 h 0.82 h PK Parametersb 0.50 ± 0.01 0.04 ± 0.00 1.33 ± 0.59 20.10 ± 0.29 31.50 ± 0.31 33.20

36a

36b

1.34 ± 0.01 4.29 ± 0.70 1.50 ± 0.31 4.58 ± 5.58

50 59 19 18

31.4 min 38.9 min 37.7 min 14.0 min

1.79 h 2.25 h 0.92 h 0.72 h

0.10 ± 0.01 0.02 ± 0.01 4.00 ± 0.00 5.49 ± 0.14 6.11 ± 0.24 0.85

0.42 ± 0.00 0.03 ± 0.01 1.33 ± 0.58 17.40 ± 0.58 28.82 ± 0.67 41.30

a For 35a and 36a, stability means % remaining after 60 min, whereas for 35b and 36b, stability means % remaining after 120 min. bCompounds 35a and 36a were given by oral administration at 5 mg/kg to male rats, and 35b and 36b were given by oral administration at 3 mg/kg to rats (n = 5).

Figure 13. Systematic optimization of CPT led to a range of lomefloxacin derivatives (38) with potent topo II inhibitory potencies.

the benzimidazole (fragment A) and quinolone (fragment B). The subsequent incorporation of two fragments led to the formation of a new molecular hybrid (37) as a topo I inhibitor, and the most active compound 37a was eventually established as

a potent lead. For furthering the potential of this lead and identifying more chemical entities with improved efficacy, the 1-ethylquinolin-4(1H)-one backbone remained unchanged while fluoro atoms and 3-methylpiperazine substituent were 8957



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Figure 14. An illustrative example of 2-phenylbenzofuranoid 40 designed from 2-phenylnaphthalenoids 39 using a scaffold-hopping strategy.

Figure 15. Application of a molecular hybridization strategy led to new chemotypes with equipotent or more potent topo II inhibitory efficacies owing to the synergistic effects.

effects on potency while also offering high affinity for topo IIα.60 The resulting pyrazoline derivatives 41 turned out to be selective ATP-competitive catalytic topo II inhibitors. In addition to the aforementioned five-membered skeletal structures, the nitrogen-containing fused heterocycles benzimidazole and pyrrolo[2,3-b]pyrazine are also pharmacophoric functionalities that are frequently used in designing potential catalytic topo II inhibitors. In the benzimidazole structure, the phenyl ring is especially essential and indispensable for the bioactivity, which may exert topo II inhibitory effects by blocking the enzyme ATP-binding domain. Similar to the pyrrolo[2,3b]pyrazine motif, it probably plays a role in topo and kinase inhibition by competitively interacting with the ATP-binding site of the enzymes. Inspired by their structural characteristics as well as topo II inhibitory strengths, the two skeletons (benzimidazole and pyrrolo[2,3-b]pyrazine), which share similar mechanisms of action and binding modes, were combined into a new structural hybrid according to the combination principle.61 As expected, the resulting 1,3-benzoazolyl-substituted pyrrolo[2,3-b]pyrazine derivatives 42 functioned as nonintercalative topo II catalytic inhibitors and were determined to be more potent than the single framework, as depicted in Figure 15. Amsacrine (5) and ametantrone (7) are two identified topo II poisons that have a broad spectrum of antitumor activities. Acting as excellent DNA intercalators, both planar-fused ring systems can intercalate into DNA, leading to alterations in the groove proportions and prevention binding of topo II. Zagotto et al. have reported ametantrone-amsacrine-related hybrids 43 by incorporating the methanesulfonamidoaniline substituent of 5 into the anthracenedione core of 7. The resulting hybrids were

introduced. As a functional surrogate of the nitrile, carbinol, and carbonyl groups, a fluoro atom can endow a molecule with improved metabolic stability.57 As for the piperazine moiety, it is frequently found in pharmacological agents owing to its ability to increase water solubility.58 Subsequently, a ring-opening modification on the benzo[d]oxazole unit was implemented to expose the amide functionality, which was beneficial to the amelioration of issues associated with metabolic stability and water solubility. This resulted in the lomefloxacin derivatives 38 as potent topo II inhibitors with electron-withdrawing substitutions (R1 to R4) on the heteroaryl ring conducive to both selectivity and efficacy. Another example is from the 2-phenylbenzofuranoid derivatives 39, which also confirmed the effectiveness of this approach. The newly discovered 2-phenylbenzofuranoids 40 were shown to produce dramatically increased topo IIα inhibition as well as improved antiproliferative efficacy (Figure 14).59 3.1.4. The Molecular Hybridization Strategy Appears to be a Simple but Powerful Way To Identify Bioactive Molecules. In the pursuit of new molecular hybrids with desirable bioactivities toward specific biotargets, the search and selection of two or more complementary chemical entities is critical to generate a synthetic benefit and fewer side effects. For example, the fivemembered heterocycle pyrazole and its bioisosteres, including pyrazoline, pyrazol-3-one, and pyrazoline-3,5-dione, have been frequently utilized in drug development programs. The chalcone unit has been shown to play an important role in producing a catalytic inhibitory activity toward topo IIα. To address the need for more effective topo inhibitors, Rahman et al. hypothesized that the incorporation of a chalcone element (or its analogues) into the pyrazoline core might afford a new hybrid with synergistic 8958



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Figure 16. Structures of the ametantrone-amsacrine-related hybrid (43) as topo II-induced poisons. The binding mode of compound 43b in complex with topo II (PDB ID: 1ZXM) was also shown.

promising pharmacophore among bioorganometallic compounds. Research on ferrocifen chemotypes, which contain a tamoxifen diphenylmethylene pharmacophoric fragment, e.g., compound 45, has been rapidly increasing in recent years due to their remarkable antiproliferative activity in cancer cells.66−69 Ferrocene and tertiary amino units were incorporated into the indenoisoquinoline-5,11-dione core with the expectation of achieving a synergistic and/or complementary benefit.70 The results demonstrated that hybrids 45−49 were topo II inhibitors, and the monoferrocenyl unit is indeed a favorable contributor in controlling topo II inhibition when it is located at the end of the lactam side chain (47). However, when the ferrocenyl group acts as a linker (entries 46 and 48), the length of lactam side chain, disposition, and size of the ferrocenyl unit’s neighboring substituents were all important determinants of the bioactivity with the best one (47a) possessing a four-methylene spacer and a terminal monoferrocenyl group. It is noteworthy that, although the ferrocenyl motif is very important, it is not “the more, the better” in controlling the bioactivity. Taking 47a and 49a as examples, 49a, which has a terminal diferrocenyl group, showed impaired potency compared with its monoferrocenyl counterpart 47a, indicating certain space requirements of the biotarget. The same situation also occurred in the cases of 46a−c, where the replacement of the terminal dimethylamino group in 46a by a piperidine (46b) or N-methylpiperazine (46c) with a larger volume led to decreased activity. As far as the topo II inhibition associated with 47a−c was concerned, 47a with a free amine (R’=H) displayed similar potency to its alkylated analogues 47b (R’=CH3) and 47c (R’=C2H5), suggesting that the monoferrocenyl group might play a leading part in affecting bioactivity. Collectively, the indenoisoquinoline-5,11-dione skeleton, four carbon-based spacer, and terminal monoferrocenyl motif are all beneficial elements to promote topo II inhibition (Figure 17). β-Carboline (9H-pyrido[3,4-b]indole), also known as norharmane, has been recognized as an important pharmacophoric structure for obtaining potent cytotoxicity, as its planar N-containing

identified to be topo IIβ-preferring poisons and DNAintercalating agents, and the properties of the lateral groups linked to the anthracenedione backbone play a major role in modulating DNA binding as well as cellular cytotoxicity.62 Although all hybrids showed decreased potencies when compared with compound 7, three compounds (43a−c) bearing tertiary noncyclic amine groups, particularly 43b, demonstrated comparable cytotoxic effects to compound 5, suggesting the opportunity for development into potent isoform-selective topo II inhibitors. The binding pattern of compound 43b with topo IIα, as evinced in Figure 16, revealed that the effective H-bonding contacts formed between the inhibitor and several key residues (Tyr34, Asn150, Asp94, and Tyr186) contribute to the bioactivity. The acridine skeleton decorated with a 9-anilino motif, e.g., compound 5, displayed DNA binding abilities and topo II-mediated cytotoxicity through interplaying with the DNA-enzyme ternary complex. In terms of the linkers between the acridine and phenyl ring, methyleneamine (NHCH2) can enhance topo II affinity in comparison with a simple amine linker. Additionally, the incorporation of 6-chloro and 2-methoxyl substituents into the acridine scaffold produced significant antiproliferative efficacy. Thus, a suite of 9-benzylamino acridine derivatives 44 was developed as effective DNA intercalators based on the hypothesis that the combination of an acridine core with 9-benzylamino, 6-chloro and 2-methoxyl substitution patterns might afford improved efficacy. As expected, all of the derivatives exhibited strong topo II inhibition. In particular, compound 44a displayed the most potent DNA-intercalating affinity and potent cytotoxicity and could induce DNA damage and A549 cell apoptosis by blocking tumor cells in the G2/M phase.63 Indenoisoquinoline-5,11-dione has also been shown to be a useful framework in topo inhibitor design.64,65 Notably, the most potent DNA binding affinity can be acquired by introducing a basic tertiary amino pattern to the indenoisoquinoline core via an ethoxy linker. Meanwhile, the sandwich-type ferrocenyl functional group, which possesses a good reversible redox profile, suitable lipophilicity, and kinetic stability, has been regarded as a 8959



Perspective

Figure 17. Design of acridine 44 and indeno[1,2-c]isoquinoline-5,11(6H)-dione derivatives 46−49 involves the combination of several topo II-oriented fragments.

the R1-substituted phenyl ring at the 1-position and an R3 group (H or methyl) at the 9-position. The SAR investigation revealed that most of the derivatives showed significant synergistic cytotoxicity in the DU-145 prostate cancer cell line over other cell types, such as A549, MCF-7, and HeLa. Moreover, the incorporation of a DTC group at the R2 position facilitated the cytotoxicity (50d vs 50i; 50g vs 50h), which can be comparable with the control Doxo, implying the importance of the DTC portion. The lipophilic as well as electronic profiles are basic influencing factors that affect bioactivity. The results, to a certain extent, also validated the effectiveness of the molecular hybridization protocol for the development of potential topo II inhibitors. Additionally, the nature of the substituents at the 1-, 3-, and 9-positions also impacts the cytotoxicity to a large extent. Generally, for R1 substituents, the lipophilic groups, 4-fluoro (50a), 4-trifluoromethyl (50b), and 4-methoxyl (50d and 50e) favorably contribute to potency, whereas for R2 and R3 substituents, methyl substitution (50b and 50e) seems to play a crucial role for cellular bioactivity, suggesting that a methyl group is suitable for this sort of derivative. Further bioactivity evaluation of 50a and 50b indicated that, although both presented similar topo II inhibition and cytotoxicity, they have different mechanisms of action. Specifically, 50a acts as a catalytic inhibitor, whereas 50b functions as a poison.

heterocycle can serve as an effective DNA intercalator and stack between DNA base pairs. Additionally, introducing substituents at the 1-, 3- and 9-positions of the β-carboline backbone is conducive to cytotoxic activity. Many β-carboline derivatives with appropriate substitutions at these positions have been shown to exhibit impressive topo II inhibition by damaging DNA.71 Meanwhile, the biologically active dithiocarbamate (DTC) motif is characterized as another frequently utilized pharmacophore for the design of novel molecules with diverse biological activities. For example, both brassinin, an indole-derived dithiocarbamate that is isolated from cruciferous vegetables, and sulforamate, a phase II enzyme inducer, have been shown to possess chemopreventive activities.72 Moreover, because of the presence of the zinc-binding CS2Me group, the DTC unit can also be exploited in the design of potent inhibitors of zinc-dependent metalloenzymes, e.g., carbonic anhydrase (CA) and tyrosinase, which have been verified to be associated with various pathological conditions, such as epilepsy, osteoporosis, glaucoma, neoplasia, and carcinoma progression.73 For expanding the chemical diversity and SAR profiles of β-carboline derivatives, a spectrum of molecular hybrids 50 was explored as topo II inhibitors via conjugation of β-carboline and the DTC segments (Figure 18).74 Further modifications were mainly implemented on the β-carboline nucleus by incorporating 8960



Perspective

Figure 18. Optimization of the pharmacophoric β-carboline ring by incorporating the biologically active DTC and trifluoromethylphenyl moieties, which are spatially adaptive to the active binding domain of topo II, led to the hybrid β-carboline derivatives (50a, 50b, 50d, 50e) with selective cytotoxicity toward DU-145 prostate cancer cell.

profiles as well as attenuated toxicity.75,76 Specifically, following esterification and amidation modifications at the cyclohexane (C ring) of the DMEP motif, the 9-hydroxyl group was transformed into ester 51 and bivalent bioisostere amides 52, respectively, as displayed in Figure 19. Generally, most of the new DMEP analogues, with the exception of 52d, had improved antiproliferative efficacies, enhanced water solubility, and attenuated toxicity compared with the parent DMEP and the control 1 at submicromolar concentrations in cellular assays. According to the IC50 values against various tumor cells and octanol−water partition coefficients (logP) of each compound, the introduction of an ester group gave better potency than the amide counterparts (compounds 51a−c vs 52a−c). In addition, electronwithdrawing substituents, e.g., F and Cl (51d), are beneficial contributors to the antitumor potency and selective index (SI) toward normal cells. It is noteworthy that compound 52d displayed the worst antitumor activity among these DMEP analogues, but offered the best water solubility, which might be attributable to the presence of the three phenolic hydroxyl groups. The most potent compound, 51a, showed strong topo II inhibitory activity and blocked HeLa cells in the G2/M phase by inducing apoptosis, providing a new promising template for further lead optimization. For learning about the strong topo II inhibition of these analogues, a molecular modeling analysis of the topo II active domain (PDB ID: 3QX3) with 51a was examined, as shown in Figure 19. The result revealed that 51a adopted a very similar binding pattern as 1 by forming three H-bonds with Asp479 and one H-bond with Arg503. Hydrophobic interactions between the trisubstituted phenyl moiety (E ring) and a relatively empty hydrophobic cavity surrounded by six residues, Arg503, Lys456, Asp479, Leu502, Gly478, and Gly504, completed the binding

The ligand−protein interactions were rationalized in detail through docking simulations utilizing 50b as a tool to acquire the pharmacophore for further structural optimization. The AutoDock 4.2 program was used, and the results, which were visualized through the PyMOL software (http://www.pymol.org/), evinced that each part of the molecule fits well into the topo II ATP binding cavity (PDB ID: 1ZXN) and generates favorable interactions with topo II. Specifically, the β-carboline backbone not only forms a π−π stacking contact with Phe142 but also forms hydrophobic interactions with Tyr34, Asn91, and so forth, whereas the terminal methyldithiocarbamate chain contributes to the hydrophobic contact and forms two hydrogen bonds with neighboring amino acids. Additionally, the trifluoromethylphenyl moiety participates in the hydrophobic contacts with Asp94, Ser149, and Arg98. 3.1.5. Natural Products Offer Great Promise for Unearthing Potent Topo II Inhibitors. 3.1.5.1. DMEP Analogues. Natural product-based drug exploration is an attractive but challenging way to discover novel drug-like molecules because, in most cases, a naturally occurring extract needs step-by-step chemical optimization to make it a lead or drug candidate. In the past few years, a large fraction of topo II inhibitors, either originated directly from natural extracts or more often chemically modified from natural prototypes, have been explored. For example, many podophyllotoxin derivatives (e.g., 1 and 2) are derived from the 4′-demethylepipodophyllotoxin (DMEP) framework, which is a natural aryltetralin lignan with antitumor activity. Recently, Tang et al. have applied a prodrug strategy followed by a bioisosteric exchange of the prototype DMEP, aiming to procure new DMEP derivatives with improved pharmacological and therapeutic potency, bioavailability, and pharmacokinetics 8961



Perspective

Figure 19. Chemical modification of the 9-hydroxyl group of the DMEP prototype led to the DMEP esters 51 and amides 52 by means of prodrug principles combined with bioisosteric manipulation.

interactions. Furthermore, a π−π stacking interaction between the coplanar DMEP backbone (C ring) and the adjacent pyrazine moiety was also observed. All of these driving forces were believed to account for remarkable topo II affinity by forming a stable “DNA-topo II-inhibitor” ternary complex. The incorporation into DMEP of three nitrogen mustard alkylating agents chlorambucil (53), melphalan (54), and bendamustine (55) at the C-4 hydroxyl group of the cyclohexane also led to the three epipodophyllotoxin-N-mustard hybrids 57−59 and the five piperazine-bearing derivatives 60−64 (Figure 20).77 The N-mustard modulators were expected to exert antineoplastic effects through attachment of the alkyl portion to uncomplexed or complexed DNA (“DNA-topoII inhibitor” covalent ternary adduct), resulting in irreversible inactivation of topo II.78 These hybrids 56−59 and 62 that were submitted for NCI-60 cellular screening displayed markedly improved potencies compared with the controls (1, 12 μM; 53, 60 μM; 54, 29 μM; and 55, 70 μM) with GI50 values in a low submicromolar range. All of the hybrids exhibited strong topo II inhibitory activities and some of them, compound 62 in particular, providing enhanced cellular growth inhibitory effects toward K562 and etoposide-resistant K/VP.5 cells. As one of the most effective topo II poisons in the clinic that targets both topo IIα and β, DMEP analogue 1 frequently suffers from a risk of therapy-related secondary malignancies following long-term treatment. The nonspecific-isoform targeting of topo II, to a great extent, might account for this limitation.79

To overcome these shortcomings and determine the precise binding pattern of 1 with topo II, Chan and co-workers determined a high-resolution X-ray crystal structure of topo IIβ in complex with DNA and 1.20 According to this cocrystal structure, although most drug-interacting residues are conserved between the α and β isoforms, the glycosidic part of 1 anchors next to the polar Q778 residue in topo IIβ, which is a key druginteracting position for this enzyme, as shown in Figure 21. However, for the α isozyme, this position is taken up by M762 residue, a structural variation that might offer the opportunity to explore isoform-selective topo II inhibitors. For example, because of the fact that the sulfur atom of methionine readily reacts with Pt(II), Pt(II)-coordinative compounds with suitable stereochemistry might be introduced into the DMEP core to deliver the reactive Pt(II) center toward M762 by forming a stable Pt(II)-thioether coordinative bond, making it possible to accomplish selectivity for topo IIα over the β isoform.80 Another example comes from the discovery of the DMEP analogues 65 and 66 as potent and selective topo IIβ inhibiting agents by replacing the sugar unit with triazole-bearing side chains.81 As displayed in Figure 21, the design concept is summarized as follows: (1) the 1,2,3-triazole entity, which can readily be prepared by a CuAAc click reaction of azides and terminal alkynes, has been verified as a useful pharmacophore and building block in drug design owing to its notable biocompatibility and target-specificity profile. More importantly, this motif might generate binding interactions with the key Q778 residue 8962



Perspective

Figure 20. Combination of naturally available topo II inhibitor DMEP backbone with nitrogen mustard alkylating agents led to several DMEP analogues (56−64) with strong topo II inhibitory activities.

through any of the three N atoms similar to the sugar motif; (2) introducing various substituents (R2 and R3) with different physicochemical properties (polarity, steric hindrance, and linker length) to the 1,2,3-triazole unit to simulate the interaction between the glycosidic part of 1 with the polar Q778; and (3) the R1 group is comprised of the 4′-demethyl and -methylated analogues to further examine the topo II-targeting effect and achieve chemical diversity. The biological assays showed that the 4′-methylated counterparts totally lost their bioactivity, implying an indispensable function for the 4’-OH group via the formation of effective interactions with the protein. For example, when the R2 or R3 group was fixed as polar substituents that were negatively charged at physiological pH (e.g., carboxylic acid), the resulting compound (e.g., 66b) displayed impaired affinity presumably due to electrostatic repulsion with the DNA phosphate motif. Compounds with positively charged substituents (e.g., 65a and 66a), by contrast, contributed to topo IIβ-related selectivity and toxicity.

Similarly, according to the determined ternary complex structure (PDB ID: 3QX3), in which two molecules of 1 complexed with a cleaved DNA-topo IIβ, two intercalated molecules of 1 were separated by four DNA base pairs. Meanwhile, both glycosidic motifs from the two molecules of 1 fill the spacious domains in the complex, suggesting that the glycosidic part can be replaced by other appropriate substituents to provide strong interactions with the active binding region. This binding mode hinted that an analogue of 1 decorated with two DMEP segments separated by linkers of varying sizes may promote effective bisintercalation into the two binding sites and subsequently promote stronger topo II inhibition with more a selective and cytotoxic effect toward tumor cells (Figure 22). Regarding the linkers, two piperazines were selected and connected by a methylene of varying lengths (C3, C6, C8, C10, and C12) with the aim of procuring increased solubility while allowing the polyamine transport system (PTS) to selectively deliver the molecule to tumor sites similar to the polyamine-containing analogue 8963



Perspective

Figure 21. Design and SAR for topo IIβ-specific DMEP analogues 65 and 66 that involved replacing the prototype sugar motif with 1,2,3-triazolebridging segments, which highlights the importance of effective interaction with the polar topo IIβ Q778 residue, a position that is different between topo IIα and β subtypes.

Figure 22. Design of mono- and bis-epipodophyllotoxin derivatives 67a−c and 68a−e involved replacement of the glycosidic part of archetype 1 with methylene-linked piperazines to enhance activity by allowing more effective interactions with the topo II active binding area.

of 1, F14512.82 Moreover, for validating the two-site intercalation hypothesis and determining the optimum linker length, monoepipodophyllotoxin derivatives were also prepared and compared with bis- epipodophyllotoxin counterparts.

SAR explorations on the obtained products 67a−c and 68a−e revealed that the diepipodophyllotoxins, as predicted, were generally more active than their monosubstituted counterparts in inhibiting both leukemia K562 cell growth and 1-resistant K/VP.5 cells 8964



Perspective

Figure 23. Semisynthetic processes for the desired ether intermediates (71 and 73) and silyl ethers (70, 72, and 74) from naturally available 69.

identified by the National Cancer Institute (NCI) that have functions in counteracting or preventing the formation of malignant tumors. Mansonone E (ME, 75) and F (MF, 76) are two naturally occurring sesquiterpene o-quinones that contain a fused pyran ring. These fused pyran or furan units are especially important owing to their irreplaceable roles in retaining the cytotoxic effects as well as topo inhibitory activities. A systematic SAR exploration of the mansonone skeleton confirmed that the 3- and 9-substituted mansonone derivatives displayed heightened cytotoxic and topo inhibitory potencies. A halogenation modification at the C-9 position generated a strengthened efficacy compared with the 9-alkyl or 9-aryl counterparts. Inspired by these findings, Huang et al. equipped the ME backbone with a C-3 triazole unit as well as C-9 halogenated substituents (Cl or Br). The resulting ME derivatives 77 produced comparable or even better antitumor efficacies toward the tumor lines A549, HL-60, K562, and Hela, as summarized in Figure 24.85 3.1.5.4. p-Terphenyl Analogues. To unravel the basic pharmacophore as well as the detailed mechanism of action of p-terphenyl, which mainly exists in mycomycetes from A. candidus, Shen et al. discovered a subset of p-terphenyl derivatives (78), as evinced in Figure 25.86 Cytotoxicity studies elucidated that compound 78a, which has phenolic hydroxyl groups at the distal end, displayed the most potent antineoplastic potency toward tumor cells (MDA-MB-435, IC50 < 1 μM) by strongly inhibiting both topo I and topo IIα. In contrast, compounds 78b and 78c with hydroxyl groups at both the proximal and distal ends only furnished selective inhibition against topo IIα, though they were less efficient than 78a, suggesting that they might be used as potential hits for further chemical optimization. 3.1.5.5. Pentacyclic triterpenoid analogues. Pentacyclic triterpenoids, e.g., betulinic acid (79), boswellic acid (80), ursolic acid (81), and oleanolic acid (82), are oleanane-type antineoplastic agents that target both topo I and topo IIα. An SAR exploration revealed that the common pentacyclic ring is a strong determinant for achieving topo inhibition, whereas carboxylation of this pentacyclic structure has been suggested to be another determining factor. For identifying more potent topo inhibitors from the oleanolic skeleton, a computer-aided molecular modeling

at lower micromolar concentrations. For the bisubstituted derivatives 68a−e, the length of the methylene linkers indeed influenced cellular activity. The minimum chain length was six (68b) and the optimum was eight methylenes (68c), which was ∼10-fold more active than 1. This SAR result was in agreement with that for the epipodophyllotoxin-acridine hybrids identified in the preceding work, which confirmed the structure-based design hypothesis to a certain extent. In terms of the monosubstituted derivatives 67a−c, the SAR tendencies differ slightly from the disubstituted ones.83 Although the longer linker enhanced cellular potency, the ten methylene-bearing derivative 67c showed the best activity, which was 6-fold more potent than the control. The activities of the monosubstituted derivatives might be due to the presence of dipiperazine moieties that can be forwardly transported by the PTS similar to polyamines. Three compounds, 68a, 68b, and 68d, were selected for examination with the NCI-60 cell line screening to determine which tumor types were sensitive to these compounds and identify possible mechanisms by which these compounds exerted their activity. As a consequence, the ten methylene-containing bisepipodophyllotoxin derivative 68d afforded the best average activity, which was 25-fold more active than prototype 1. Finally, a preliminary mechanism study indicated that all of the monoand bis-epipodophyllotoxin derivatives were topo IIα poisons that could inhibit topo IIα decatenation and cause DNA DSBs in tumor cells. 3.1.5.2. Andrographolide Analogues. Andrographolide (69), a diterpenoid lactone from Andrographis paniculata, is an herbal medicine that can inhibit tumor growth and proliferation as well as induce caspase-independent cell death via inhibition of topo IIα activity. Piyachaturawat et al. have semisynthesized several andrographolide analogues 70−74 via a step-by-step etherification process (Figure 23).84 Further studies of these andrographolide analogues revealed that five of them (70b, 72c, 74a−c) presented potent topo IIα inhibition and led to cytotoxicity toward several tumor cell lines (BCA1, CHO, HeLa, and HepG2) in vitro. 3.1.5.3. Naphthoquinone Analogues. 1,4-Naphthoquinones and 1,2-naphthoquinones are pharmacophoric fragments initially 8965



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Figure 24. Combination of a sesquiterpene ME ring and a 1,2,3-triazole motif led to novel ME derivatives 77 with decent antitumoral efficacies.

bioavailability, pharmacokinetic/pharmacodynamic (PK/PD) properties owing to the tautomerimic nature to a large extent have greatly hindered the clinical use of this compound.88,89 Jha et al. have explored a variety of curcumin derivatives and concluded that derivatives with the keto-form (or oxygenation pattern) were more potent than those with the enol-form with respect to the antioxidant and antineoplastic efficacy. Additionally, because topo IIα is a thiol-rich enzyme and is crucial for sustained cell proliferation, Michael acceptors (e.g., an α,β-unsaturated carbonyl group) and the enone unit are suitable for incorporation into the curcumin motif with the expectation of furnishing selective affinity for topo IIα upon forming interactions with auxiliary binding sites. On the basis of these data, a spectrum of keto-type curcumin derivatives with a maleic diamide tether (85, Figure 27) were prepared and assessed.90 These derivatives manifested heightened top IIα-related cytotoxicity by approximately 93- to 280-fold compared with the controls (curcumin and melphalan), suggesting the possibility, although questionable, of developing more potent curcumin-derived compounds. Moreover, acting as a structural and functional analogue of curcumin, diarylheptanoid hirsutanone, which is isolated from Alnus glutinosa leaves and contains a Michael acceptor (α,β-unsaturated ketone structure), was shown to be a topo II-mediated poison.91 3.1.5.7. Ellipticine Analogues. Ellipticine (5,11-dimethyl6H-pyrido[4,3-b]carbazole, 86), an alkaloid isolated from Ochrosia elliptica, is a polyaromatic DNA intercalating inhibitor of topo II that forms covalent adducts with DNA. Although its cytotoxicity has a marked influence on the antineoplastic potency, 86 has difficulty in clinical applications due to its poor bioavailability, lack of target specificity, and drug resistance in

Figure 25. p-Terphenyl derivatives 78 with phenolic hydroxyl groups at different positions gave differing selectivity of inhibition toward topo IIα and β isoforms.

method was applied to analyze the active binding site characteristics of topo I and topo IIα and dock the proposed analogues into this domain. By this approach, ten desired inhibitors 83 and 84a−i with optimal parameters (Figure 26), e.g., electrostatic and H-bonding interactions and spatial topology, were selected and subjected to semi- or total synthesis. The subsequent bioevaluation showed that compounds 84a, 84b, 80d, 80f, and 80h showed higher topo I inhibitory activities than CPT, whereas compounds 84a, 84b, 84d, and 80e gave increased topo II inhibition compared with 1. Collectively, 84a, 84b, and 84d stood out among these oleanolic analogues and might be used for exploring more dual topo I and topo IIα inhibitors with heightened antitumor efficacies and MDR reversal potency.87 3.1.5.6. Curcumin Analogues. Curcumin is an active component of phytochemical turmeric (Curcuma longa) that has been used for centuries as a food additive or dietary supplement. Although curcumin and its derivatives have been used extensively in pharmaceutical applications with negligible toxicity issues, its shortcoming, such as poor metabolic stability (t1/2 < 5 min), 8966



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Figure 26. Chemical structures of several oleanane-type triterpenoids (79−82) and oleanolic acid-based analogues 83 and 84a−i.

Figure 27. Structural modification of naturally occurring keto-type curcumin led to a myriad of curcumin-inspired analogues 85 with significantly elevated cytotoxicity compared with curcumin and melphalan. 8967



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Figure 28. Chemical structures of ellipticine-derived analogues 89−92 and the binding mode of 92a with topo IIα (PDB ID: 1ZXM). Some of these compounds, exemplified by 90b and 92, presented significant antineoplastic improvements compared with the parent ellipticine for targeting topo IIα.

DNA with their intercalating potency in the order of 90b > 90a ≥ 86. Guided by these discoveries, the four carbazole derivatives 91a, 91b, 92a, and 92b, inspired by the structure of 86, were prepared. Screening revealed that the urea-linked analogues 92a and 92b exhibited notable thermal stability, strong and selective topo II inhibitory potencies, as well as low cytotoxicity toward normal cells, emphasizing the fundamental role of the urea functionality in promoting topo II inhibition.95 The binding mode of 92a with topo IIα, as shown in Figure 28, clearly illustrates that, apart from efficacious H-bonds between the compound and residues Asp94, Asn150, Ser149, and Lys157, the coordinative force between the methoxyl O and catalytic Mg2+, together with a π-cation contact between the aromatic backbone and Lys157, are all beneficial contributors to the bioactivity. In addition, several residues (Tyr34, Ile33, Thr159, and Arg98) participated in the formation of hydrophobic interactions, which were also beneficial contributors to the improved binding affinity. 3.1.5.8. Evodiamine Analogues. Evodiamine (EVO) is a natural quinazolinocarboline alkaloid extracted from the fruit of Evodia rutaecarpa Benth. (Rutaceae)96 has been shown to have multitarget and broad-spectrum antineoplastic profiles via multiple mechanisms of action, including apoptosis,97 β-cateninmediated angiogenesis,98 cell cycle progression, and the induction of oxidative stress.99 It is therefore a potentially useful lead for modifying and exploring additional chemotypes. For example, the Sheng group has developed the spectrum of EVOinspired diverse scaffolds 93−105 (Figure 29).100 Some important SAR information on these EVO-derived backbones is summarized as follows: (1) Generally, among the 11 novel EVOinspired scaffolds, the oxo- and thio-bearing scaffolds presented predominant efficacy. Installation of a 10-hydroxyl group could induce elevated antitumor activity compared with the 10-methoxyl

prolonged treatment. Even though derivatizing 86 will compensate for these deficiencies to a certain degree, the derivatives of 86 still suffer from a cluster of undesirable adverse effects, such as weight loss, renal toxicity, and hemolysis.92 It is plausible that salification may be a good alternative to avoid these pitfalls as exemplified by celiptium (87), the most clinically successful acetate salt of elliptinium, that displays both increased aqueous solubility and enhanced antineoplastic efficacy. More endeavors to address these limitations led to the identification of several ellipticine-derived bromonium salts (89, Figure 28) derived from 86 by quaternization with alkyl bromides at N-1 of the pyridine ring. Among the obtained ellipticinium salts, both 89a and 89b showed strong topo II inhibition and comparable antiproliferative effect to irinotecan and 1. However, both modified ellipticinium salts and two controls showed attenuated antiproliferative activities compared with their N-1-unsubstituted counterpart 6-methylellipticine (88), which displayed submicromolar cellular potency. This result suggested that the balance between cellular efficacy and water solubility should be considered by further modifying 86.93 To acquire further insights into the SAR profile of 86, Vann et al. attempted to prepare the two analogues 90a and 90b, where the methyl group at the C5 position was removed with the intention of generating improved antineoplastic properties compared to 86 by targeting topo IIα.94 Compound 90b is a quaternary ammonium salt of 90a that aimed to improve its water solubility and enhance its bioavailability. The subsequent topo II-mediated kinetoplast DNA (kNDA) decatenation evaluation of 90a and its salt 90b revealed that they are approximately 5- to 25-fold more potent than prototype 86, respectively, and both act as catalytic inhibitors of topo IIα. It appears that such inhibition toward topo IIα is associated with their ascending ability to intercalate into the double helix of 8968



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Figure 29. Natural product evodiamine (EVO) inspired scaffold diversity and led to a large variety of derivatives 93−105 via multiple design protocols. Among them, the 3-chloro-10-hydroxyl derivatives 98d−h showed particularly excellent potencies, highlighting their promise as leads for further exploitation.

counterparts. (2) With respect to structural changes on the B/C ring, surprisingly, when the indole motif (93) was transformed into a benzofuran core (94), the same R substituent gave an opposite result, presumably due to a different antitumor mechanism. (3) When the D ring remained constant as a 2H-1,3-oxazin4(3H)-one core (95), only moderate potency was acquired. The only exceptions are the 3-chloro substituted analogues 95a and 95b, which expressed nanomolar inhibitory activity toward A549 cells. In contrast, the 3-fluoro (95c) and 3-bromo (95d) substituted analogues were less potent, whereas the 3-nitro counterpart 95e lost activity. When the nitro group was transformed into an amino group at the same position, the resulting compound 95f demonstrated elevated potency. However, further acylation or substitution of the amino functionality led to an overall decrease in bioactivity (95g and 95h). Moreover, both the thiocarbonyl group (96) and five-membered pyrrolidin-2-one 97 were unfavorable moieties for bioactivity. Intriguingly, among the modifications to the D ring, the 2H-1,3-thiazin-4(3H)-one scaffold 98 demonstrated the most potent antitumor profile. Unfortunately, when the sulfur was oxidized to a sulfone (99) or a phenyl moiety was replaced by pyridinyl (100), the corresponding derivatives were almost inactive. Collectively, 10-hydroxyl was found to be the favorite unit for determining cellular activity, closely followed by the 3-chloro and 3-amino substituents on the

phenyl (E) ring. (4) When the E ring was modified, both the pyrrole and thiophene series 101−103 and 105 exhibited broadspectrum antitumor potencies and had a similar SAR profile to the EVO archetype. However, replacement of the pyrrole and thiophene rings with a furan scaffold (104) attenuated activity. Moreover, for the thiophene series 101−103, movement of the S atom in the thiophene ring resulted in a significant antitumor improvement. 3.1.6. Combination of High Throughput and in Silico Screening. High-throughput screening (HTS) by means of an in silico approach is a growing and efficient method in drug discovery that involves performing systematic screening to predict the bioactivity between molecules and drug targets, aiming to discover potent hits and/or leads for further exploitation. Using a virtual screening protocol, Perdih et al. explored a category of 4-amino-6-(phenylamino)-1,3,5-triazines (106) that function as monocyclic mimetics of the purine motif and act as potent topo IIα inhibitors by targeting the ATP binding site.101 To do this, as shown in Figure 30A, the crystal structure of human topo IIα (PDB ID: 1ZXM) was initially selected from the PDB database, and a subsequent reconstruction and calibration was performed to successfully redock the ligand (purine-based compound A) into the ATPase pocket of the target protein. The validated binding mode was then utilized to generate a structure- and 8969



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Figure 30. (A) Schematic flowchart for the discovery of potent topo IIα inhibitors possessing the skeletons of triazines (106), triazoles (107), pyrimidines (108), 9H-purines (109), and 1H-pyrazolo[3,4]pyrimidines (110) via an in silico protocol. (B) Binding modes of the two representative examples 109a and 110a with human topo IIα.

ligand-based pharmacophore model, which was finally employed for further virtual screening procedures. As a consequence, three types of hits (monocyclic scaffold triazines (106), triazoles (107), and pyrimidines (108)) were eventually chosen and subjected to biological assessments based on the orientations obtained from the docking study, the “Lipinski rule of five”, and ligand efficiency selection. When a fluorophenyl or methoxyphenyl was appended onto the 1,3,5-triazine motif, significant topo IIα inhibitory enhancement was observed. Additionally, the bicyclic heterosystems, e.g., 9H-purines (109) and 1H-pyrazolo[3,4]pyrimidines (110), created by fusing 1,3,5-triazines with other

chemotypes that behave as isosteres of the purine ring as exemplified by compounds 109a and 110a, were also shown to be topo IIα inhibitors comparable with positive control 1.102 The binding patterns of these two compounds with human topo IIα, as presented in Figure 30B, suggested that, in addition to the hydrophobic interactions, several H-bonding contacts mainly contribute to the topo IIα binding affinity. Xanthones are naturally oxygenated flavonoids with a variety of pharmaceutical applications as antitumor, antidepressant, hepatoprotective, and antioxidant agents. Even though their antitumor potency was once thought to be relevant to the inhibition of 8970



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Figure 31. (A) A hierarchical computational screening used for the discovery of xanthone derivatives 111a−d with potent topo IIα inhibitory potencies from a 50-member xanthone combinatorial library; the X-ray structures of 111a and 111b complexed with topo IIα (PBD ID: 1ZXM) are also presented. (B) Putative binding modes of xanthone derivatives 111a−d with topo IIα.

tetracyclic indeno[1,2-c]quinoline derivatives 113 via structural replacement of the tetracyclic backbone of 112 and subsequent oximation to the corresponding O-substituted oximes. Among them, 113a−c had the most potent antiproliferative activities toward A549 or HeLa cells, acting as DNA intercalating agents. Further exploration on the SAR led to the discovery of a new series of indeno[1,2-b]quinoxaline analogues 114 via bioisosteric modification and subsequent local manipulation of substituents (Figure 32). It is noteworthy that these changes significantly improved the antiproliferative potencies, although only having moderate topo II inhibition at 10 μM by the incorporation of the aminoalkyl fragments. Compound 114a, with the N,N-dimethylpropan-1-amine side chain, had the best in vitro cellular and in vivo antitumor activity as well as topo II affinity and could be a potential lead for further structural optimization.105 3.1.8. Mining Novel Candidates from Inorganic Complexes. Inorganic coordination compounds or metal complexes, which typically consist of a central metal ion (called the coordination center) and a surrounding pool of bound molecules (called ligands

topo IIα, the exact biotarget(s) of antineoplastic xanthones have not been clearly elucidated. To better understand the SAR and find more effective inhibitors of this type, Khan et al. explored a number of xanthone-derived hits (111a−d) with higher topo IIα docking scores (120.382−138.108) than the control 3 (71.47) from a 50-member xanthone combinatorial library via a hierarchical computational screening (Figure 31A).103 A QSAR model with good predictable outcomes was built, and this model was utilized as a guide for the further development of structurally diverse molecules from the xanthone family. The putative binding modes of these compounds, as shown in Figure 31B, manifested that the H-bonding interactions are beneficial factors to the enhancement of binding affinity. 3.1.7. Isoelectronic or Isostructural Replacement Is a Typical but Effective Approach to Identify Potent Topo II Inhibitors. Guided by the tetracyclic indenoquinoline-derived ellipticine (86) and batracylin (BAT, NSC320846, 112),104 which have emerged as notable DNA intercalators with significant topo II inhibitory activities, Chen and co-workers developed a panel of 8971



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Figure 32. Design of indeno[1,2-b]quinoxaline analogues 113 and 114 via bioisosteric modification protocol.

Figure 33. Formation of complexes provided more potent DNA binding affinities with an affinity order of heterobimetallic complexes > monometallic counterparts > free ligands.

heterobimetallic complexes are more prominent DNA binders than their monometallic counterparts with an affinity order of 117 > 118 > 115 > 116 followed by the free ligand. This result evinced that bioelements as hard Lewis acids (particularly transition metals) are well-suited for covalent coordination with DNA double helix nucleobases. Similarly, evidence has shown that, when the bioactive thiosemicarbazones (e.g., APY and APZ), which can block the cell cycle in the G2/M phase or inhibit ATPase hydrolysis by targeting topo IIα, were utilized as ligands, the resulting copper(II) complexes 119 and 120 were more potent antiproliferative agents than the parent ligands.107,108 Recently, there has emerged evidence that topo IIα is very sensitive to soft metal-containing thiol-reactive agents based on the fact that topo IIα possesses at least two disulfide-bonded Cys pairs and five free Cys residues. Additionally, an electrophilic reaction between the sulfhydryl groups and the metal center is believed to be responsible for the inhibitory potency. Rocha et al. investigated the topo II inhibitory potencies of the four palladium(II)-based, cationic thiol-reactive complexes 121a−d (Figure 34)109 because Pd(II) appears to be an appropriate candidate owing to its high affinity for sulfur-bearing ligands. Moreover, the four Pd(II) complexes were also able to inhibit cathepsin B, another Cys-containing cysteine protease that is also

or complexing agents), are extensively found in nature, including animals, plants, and minerals. Recently, the chemistry of biologically important ligands and their metal complexes is gaining increasing attention. Given that the biological activities of newly formed complexes rely heavily on the nature of organic ligands, bioligands with excellent bioactivity and biocompatibility should be selected to coordinate to central metal ion(s). In addition, for building a structurally stable chelation complex, the ligands should have well-defined stereochemistry, and the coordination sites should have suitable allocation and disposition. For instance, Tabassum and colleagues set out to develop a variety of biocompatible carbohydrate-conjugate heterobimetallic Cu(II)−Sn(IV) and Ni(II)−Sn(IV) complexes 115−118 (Figure 33).106 To accomplish this, they utilized nontoxic saccharides, which are readily recognized by cell-surface receptors as ligands (L1) and coordinate with biologic elements such as Cu(II), Ni(II), and Sn(IV). These elements, often referred to as bioelements, are validated to demonstrate remarkable antiproliferative profiles and preferential selective affinity toward tumor cells. In the newly obtained complexes, an ethylenediamine unit was introduced to function as the coordination site, and a bridging linkage was inserted to connect the two carbohydrate functionalities. A DNA binding affinity assay showed that 8972



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and 1,4- benzoquinonediimine-bearing 127, via the structural integration of two prototype drugs 110 and 111.112 The result was that 126 displayed superior efficacy at inhibiting tumor growth with EC50 values of 50−160 μM without affecting normal cells, implying that the free amine groups might play an indispensable role in maintaining the antitumor efficacy compared with the oxidation product, diimine-containing 127. As far as other ruthenium-based complexes are concerned, Ru(II)-arene complexes with polypyridyl ligands (i.e., bidentate 2,2′-bipyridine and 1,10-phenanthroline) that can furnish two N atoms as coordination sites to create chiral ring structures are especially heightened. These complexes typically display notable antitumor effects owing to their strong DNA-intercalating abilities, leading to effective inhibition of topo II. For example, the four chiral Ru(II) enantiomers bearing 2-furanyl-imidazo[4,5-f ][1,10]phenanthroline and 2,2′-bipyridine ligands 128− 131 were developed as topo IIα poisons. The five-membered heterocyclic entities (e.g., furan and 1H-imidazole), which are verified to have DNA binding affinities, were introduced into the ligand with the expectation of acquiring enhanced topo II inhibitory efficacy. Among them, the best iodofuran 131 gave decent topo II inhibition, antiproliferation, as well as cytotoxic effects comparable with VP-16 and cisplatin.116 For gaining insight into a detailed SAR of Ru(II)-polypyridyl complexes, which would provide clues for the exploration of optimal ligands, six novel Ru(II)-based DNA-intercalative complexes, 132a, 132b, and 132d−g, were prepared and subjected to activity assessment as well as quantum-chemistry calculations. Generally, compound 132a with the cyclohexane unit had the weakest DNA binding affinity and enzymatic inhibition compared with its aromatic-bearing counterparts 132b and 132d−g, suggesting that a coplanar aromatic conjugation with fewer steric effects is favorable for arene-based ligand design. Compounds with electronwithdrawing F and NO2 groups (132e and 132f) were found to be more potent than electron-donating CH3 groups (132d), implying that the electronic effect greatly affects DNA binding interactions. Among them, compound 132b stands out from all the bioevaluations, suggesting that DNA intercalative ligands with less steric hindrance and better planarity are beneficial to DNA binding affinity as well as DNA-dependent enzyme inhibition.117 Enlightened by this SAR information, He et al. have reported a Ru(II)-polypyridyl complex with an unsubstituted phenyl ring (133a) and its ester-containing counterpart 133b and evaluated their topo inhibitory and cytotoxic activities.118 The results showed that both of the complexes possess moderate cytotoxicity toward tumor cells (HeLa and HepG2) and can induce apoptosis in HepG2 cells by acting as dual inhibitors against topo I and II via intercalation into DNA base pairs. However, 133b showed weaker DNA affinity and topo inhibition due to a possible steric effect from an ester functional group. The DNA binding order of the Ru(II)-centered complexes 134−138 containing asymmetric tridentate N-based ligands (134 < 135 < 136 < 137 < 138) further confirmed the important role of coplanar aromatic conjugation in ligand design through enhancing DNA binding affinity and cytotoxicity. Typically, larger π frameworks afford higher DNA affinity. Complex 134 gave the lowest affinity because the two phenyl groups on the 1,2,4-triazine core are noncoplanar. In contrast, other attachments on the 1,2,4-triazine motif complex demonstrated better planarity and higher hydrophobicity, leading to elevated DNA affinities. As far as topo inhibition was concerned, among the test complexes with notable DNA

Figure 34. Pd(II)-based thiol-reactive complex has great potential to be developed as potent topo IIα inhibitor 121.

closely connected to tumor invasion.110 The four complexes were initially subjected to cytotoxic assessment using three tumor cell lines, two murine-derived cell lines (mammary adenocarcinoma LM3 and lung adenocarcinoma LP07), and one human MCF-7 (breast adenocarcinoma) cell line. The results showed that the Pd(II) complexes displayed remarkable and elevated cytotoxicities compared with the positive controls (cisplatin and 1) in the LM3 and MCF-7 cells, and they exhibited comparable cytotoxicity to cisplatin in LP07 cells at submicromolar concentrations. A preliminary mechanistic investigation showed that the Pd(II) complexes 121a−d did not inhibit topo I and cathepsin B. Instead, these molecules were more inclined to be ATP competitive catalytic inhibitors of topo IIα. The SAR results indicate that the polarity, strength of the coordinative Pd-X bond, spatial volume and kinetic differences among different ligands, and steric hindrance derived from thiosemicarbazide ligand might play a key role in achieving topo II inhibition. As a category of novel organometallic alternatives for platinumderived chemotherapeutics, ruthenium-based compounds also function as topo-targeting catalytic inhibitors or poisons, which is supported by the evidence that some limitations of Pt-derived complexes might be overcome by the use of a ruthenium (Ru) substitute.111 Although organoruthenium-based complexes are usually designed to simulate the spatial configuration of platinum drugs, they have more advantages than platinum-derived complexes. On one hand, ruthenium has three oxidation states (II, III, and IV), which are all available in physiological environments. The nontoxic but less active Ru(III) complex can be easily transformed into its toxic and active Ru(II) counterpart. Despite this transformation, both Ru(II) and Ru(III) complexes exhibit relatively slower ligand exchange rates than most of the transition metal complexes, endowing Ru complexes with higher kinetic stability and minimized side effects. On the other hand, Ru complexes typically adopt octahedral geometries, which are more stable than the square planar geometry of platinum complexes. More importantly, this geometry allows the central Ru atom to coordinate more ligands, thereby expanding the chemical diversity and also regulating the complexes’ steric and/ or electronic properties.112 To date, two ruthenium-based candidates, NAMI-A (122) and KP1019 (123), have entered clinical trials, and another two compounds, RAPTA-C (124) and RM175 (125), are currently in preclinical evaluation (Figure 35). As a prodrug, 122 is activated by the reduction of Ru(III) to Ru(II) and exerts its anticancer effects upon entering tumor cells, and 123 is particularly effective for metastatic and cisplatin-resistant tumors.113 As a pH-dependent DNA damage agent, 124 only functions under hypoxic conditions with antimetastatic hypotoxicity.114 Compound 125 displays comparable or greater cytotoxicity than carboplatin and induces tumor growth arrest and apoptosis without any cross-resistance to other platinum drugs.115 Inspired by the favorable antitumor efficacies of 124 and 125, Lida et al. prepared two Ru(II)coordinated analogues, 1,4-phenylenediammine-containing 126 8973



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Figure 35. For the Ru-based complexes, coplanar aromatic conjugation and hydrophobicity are fundamental elements for achieving both notable DNA binding affinities and topo IIα inhibitory profiles.

inhibit the growth of tested tumor cells in vitro as well as the growth of a solid Ehrlich tumor in vivo. Although the efficacy of compound 139 is weaker than that of control cisplatin, it is still a promising candidate for further evaluation.120 Metal-centered coordinated compounds serve as important complements to organic molecules and have played a key role that cannot be ignored in the development of anticancer drugs. Enlightened by the successfully developed platinum-based metallodrugs, concerns about the exploration of novel complexes has aroused many researchers’ interests.121 Chao’s lab has explored

binding activities (137 and 138), compound 137 exhibited the best topo IIα inhibitory activity with an IC50 value of 10 μM.119 To date, the majority of Ru(II)-based arene complexes are mononuclear-type and demonstrate relatively higher cytotoxicities. Muthná et al. exploited a dinuclear, thiolato-bridged, arene Ru-complex 139 by incorporating p-cymene and phenol functional groups as coordinative ligands with the expectation of achieving improved water solubility and bioavailability as well as decreased cytotoxicity compared with mononuclear-type Ru complexes. A systematic evaluation showed that 139 can significantly 8974



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Figure 36. Ru(II)-chelated compounds 140 and 141 are topo I/IIα poisons, note that the mode of action of Au(III) complex 142 changes with the concentration.

fact that topo II poisons can transform the enzyme into a DNAdamaging toxin by inducing the formation of a stable “DrugTopo-DNA” complex and promote the accumulation of irreversible “double-stranded breaks” that are highly toxic to cells, resulting in permanent DNA damage and triggering apoptotic cell death in both normal and cancer cells. Given that long-term treatment with topo II poisons might cause therapy-related side effects, e.g., drug resistance and secondary malignancies, one of the strategies to overcome this drawback is to develop topo II catalytic agents that do not serve as topo II-mediated DNA “toxins” but still effectively inhibit enzyme activity by disturbing the catalytic cycle, which may expand the antitumor spectrum and offer an optimal therapeutic outcome. However, because cancer is an exceedingly complex, multigenetic, and multifactorial disease involving numerous processes that cause a variety of physiological and pathological changes, topo II-targeted chemotherapeutics, either poisons or catalytic inhibitors, might not provide the optimal curative effects for cancer management when used alone as a monotherapy. Combination therapy with different mechanisms (e.g., topo II/I, topo II/microtubule inhibitors, etc.) is in many cases an effective regimen to achieve synergistic effects while avoiding possible drug resistance. On the other hand, the risk of complicated drug metabolism owing to the inherent PK profiles of individual drugs makes it difficult to determine the best remedy in terms of dosing and scheduling. In contrast to combination therapy using two or more therapeutics, a hybrid molecule that simultaneously modulates two or more biotargets (e.g., topo II/I or topo II/ microtubule) through proper hybridization of two or more bioactive fragments may exert complementary or improved efficacy and reduced drug resistance when compared with single-target agents. At present, this approach still remains largely empirical. Although the efficacy of topo II poisons is limited, topo II poisons are still popular in cancer treatment owing to their strong ability to induce DNA damage. Hence, the exploration of novel chemotypes serving as topo II poisons is still an active area of research. The quinolone-based 12, for example, is a topo II poison and first-in-class chemotherapeutic agent that has been studied in phase III clinical trials for both relapsed or refractory and previously untreated AML and platinum-resistant ovarian carcinoma.125 Even more intriguing, although 12 exhibits comparable antitumor efficacy to anthracyclines, 12 has a stronger capacity to counteract drug resistance, a lower potential to conflict with other treatments, and less cardiotoxicity than most anthracyclines. It is thereby a promising new agent for the treatment of AML in both monotherapy and combination regimens. Supporting the quest for new topo II inhibitors, the emphasis of this review focused on the elucidation of possible strategies, such as scaffold hopping, molecular hybridization, bioisosteric

the preliminary SAR of a limited series of Ru(II) complexes with polypyridyl phenolic hydroxyl ligands (140 and 141) by varying the number and disposition of the hydroxyl groups (Figure 36).122 Biological evaluation demonstrated that the complexes act as dual topo I and topo IIα poisons with prominent and elevated topo-mediated DNA binding properties relative to those of the controls (CPT, 1, and novobiocin); more potent topo inhibition was achieved with increased numbers of hydroxyl groups. Isoelectronic gold(III) complexes that have a similar squareplanar configuration to platinum(II) counterparts are expected to exert a similar mode of action as platinum(II) analogues and can overcome cisplatin resistance. Munro et al. have reported four Au(III) complexes in which the AuIII ion was chelated to a bidentate ligand via an anionic amido and pyridyl or isoquinolylamido N-donor atom, leaving a pair of cis-chlorine ions to complete the square-planar coordination geometry. Au(III) complexes with this chelation mode have been found to have promising cytotoxic effects via DNA binding.123 Notably, the isoquinolylamido-containing complex 142, which gave the best cytotoxicity in the NCI-60 screening with an average IC50 value of 23 μM, demonstrated a rare mode of action toward topo based on the drug concentration. It can act as either a topo IIα poison below 1 μM or a dual catalytic inhibitor of topo I/II at relatively high concentrations (>5 μM).

4. CONCLUSIONS AND FUTURE RESEARCH PROSPECTS Compared with traditional topo I inhibitors (e.g., CPT) that have significant dose-limiting toxicities and drug resistance, topo II inhibitors with lower nonspecific toxicities are more desirable. Two subclasses of human topo II, α and β isoforms, which share ∼70% sequence identity, have distinct functions in the cell. The α isoform is a cell cycle-dependent enzyme that is highly expressed in proliferating cells, whereas β isoform expression does not change during the cell cycle. More recently, there is evidence that inhibition of topo IIβ might lead to some unwanted side effects, including secondary leukemias, myelodysplastic syndrome (MDS), and cardiac toxicity, which have been the major limitations associated with clinical applications of topo II inhibitors. For these reasons, topo IIα has been validated as an important cellular biotarget for exploiting potential antiproliferation agents. At present, most topo II-related antineoplastic agents in clinical use are topo II poisons that target both the α- and β-isoforms, including anthracycline-derived 3, 4, 6, and 10 as well as epipodophyllotoxin-based 1 and 2. However, these drugs are frequently associated with unwanted drawbacks, including the induction of cumulative cardiotoxic side effects (e.g., cardiomyopathy), the potential development of therapy-related drug resistance and secondary malignancies.124 This is based on the 8975



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replacement, conformational restriction, and so forth for the design of topo II inhibitors. Meanwhile, the SAR profiles and binding patterns of representative compounds were also described to explore detailed covalent and/or noncovalent interactions (e.g. H-bonds, hydrophobic forces, electrostatic attractions, π−π stacking contacts), which may offer inspiration for discovering more potent topo II inhibitors. For more potent topo II inhibitors to be discovered, selection of proper hits or leads is particularly important. To this end, a library of pharmacophoric entities that have been shown to possess topo II inhibition and/or antiproliferative profiles, including but not limited to α-terpyridine, naphthalimide, 2-phenylnaphthalene, 1,3,4-thiadiazole, benzimidazole, pyrrolo[2,3-b]pyrazine motifs as well as the naturally available DMEP, 1,4- or 1,2-naphthoquinones, pentacyclic triterpenoids, ellipticine, xanthones, and so forth in conjunction with various medicinal chemistry approaches can be reasonably utilized to generate new chemotypes with more beneficial efficacies and fewer adverse effects. There is no doubt that metal-based inorganic complexes are also of primary relevance, identifying potent topo II-directed antineoplastic metallodrugs. Novel topo II-targeting compounds, whether catalytic inhibitors or poisons, are still urgently needed for optimal outcomes in cancer therapy. It should also be noted that topo IIα inhibitors with lower toxicity and a broader anticancer spectrum are actually more desirable compared with topo II-mediated DNA poisons, which might be correlated with undesirable side effects. Nevertheless, topo IIα inhibitors are also toxic to normal cells. In this respect, the research of topo II inhibitor-tethered drug delivery vehicles through installation of tumor-targeting conjugates or attachments has been the subject of extensive research.

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Liang Yang obtained his B.Sc. degree in pharmaceutical science from Ji’nan University, China, in 2015. He is now an M.S. candidate at the College of Pharmacy, Shandong University. His research interests involve the design and synthesis of small molecules with antitumor and antiviral properties. Yong-Tang Zheng obtained his M.D. degree at Jiangxi Medical College in 1983 and Ph.D. at Kunming Institute of Zoology, Chinese Academy of Sciences, in 1997. He is now a professor at the Kunming Institute of Zoology, Chinese Academy of Sciences. His research interests include antiviral agents, molecular immunopharmacology, and primate models. Yue-Mao Shen obtained his Ph.D. degree at Kunming Institute of Botany, Chinese Academy of Sciences, China, in 1999. He has awarded the Distinguished Young Scholars of China and is now a professor at the College of Pharmacy, Shandong University. His research interests involve the discovery and biosynthesis of novel microbial natural products. Zhi-Yu Li obtained his Ph.D. degree at the College of Pharmacy, University of Maryland, USA, in 1999. He is now an associate professor at the University of the Sciences in Philadelphia. His research interests involve the evaluation of novel molecules with antitumor properties. Xun Li obtained her M.Sc. degree at Shandong University in 2001 and Ph.D. at Tianjing University, China, in 2004. She then worked for the College of Pharmacy, Shandong University as a senior associate professor to date. From 2007 to 2008, she worked as a visiting scholar at Tokyo University, Japan, funded by the Japan Medical Association. From 2012 to 2013, she further worked as a visiting scholar in the Department of Chemistry, Texas A&M University, USA. Her research interests include rational design, synthesis, and evaluation of bioactive molecules with antitumor, antiviral, and antibacterial agents.

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ACKNOWLEDGMENTS Authors are thankful for the financial support from the Natural Science Foundation of Shandong Province (No. ZR2018MH042 to X.L.), the Key Research and Development Programs of Shandong Province (No. 2016GSF201175 to X.L.; No. 2017CXGC1401 to X.Y.L.; No. 2015ZDJS04001 to F.S.W.), the Fundamental Research Funds for the Central Universities− “CACHSR for China−Australia” (No. 2018GJ07 to X.L.), the National Natural Science Foundation of China (No. 31370110 to W.H.), and the Rolling program of “ChangJiang Scholars and Innovative Research Team in University” (No. IRT_17R68 to Y.M.S.).

AUTHOR INFORMATION

Corresponding Authors

*For Y.-T.Z. E-mail: [email protected]. *For X.L. Tel: 86-531-88382005; fax: 86-531-88382548; e-mail: [email protected]. ORCID

Yue-Mao Shen: 0000-0002-3881-0135 Xun Li: 0000-0002-5582-8628 Author Contributions

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# W.H. and X.-S.H. contribute equally to this manuscript. All figures illustrating binding patterns were generated by Sylbyl and displayed with PyMol (www.pymol.org).

ABBREVIATIONS USED ADR, adriamycin; AML, acute myeloid leukemia; CA, carbonic anhydrase; CPT, camptothecin; CPT-11, irinotecan; CuAAC, copper(I)-catalyzed azide−alkyne cycloaddition; DMEP, 4′- demethylepipodophyllotoxin; DSBs, double-strand breaks; DTC, dithiocarbamate; EPT, ellipticine; EVO, Evodiamine; HAP, hypoxia-activated prodrug; MDR, multidrug resistance; MDS, myelodysplastic syndrome; MTZ, mitoxantrone; 9-NC, rubitecan; NCI, National Cancer Institute; OPT, 10-hydroxy camptothecin; PTS, polyamine transport system; RR, ribonucleotide reductase; RTKs, receptor tyrosine kinases; SI, selective index; STS, soft tissue sarcoma; TDP1, tyrosyl-DNA phosphodiesterase 1; Topo, topoisomerase; TSC, thiosemicarbazide; WHD, winged helix domain.

Notes

The authors declare no competing financial interest. Biographies Wei Hu obtained his Ph.D. degree in microbial sciences from Shandong University, China, in 2004. He is now a professor at the School of Life Science, Shandong University. His research interests involve the discovery of novel molecules with antitumor and antibacterial properties. Xu-Sheng Huang obtained his bachelor’s degree of Pharmacy at Gannan Medical University in 2015. He is now a Ph.D. student at the University of Chinese Academy of Sciences. His research interests involve antiviral agents and molecular immunopharmacology.

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Ji-Feng Wu obtained his M.Sc. degree in Analytical Chemistry from Shandong University, China, in 2006. His research interests involve the determination of bioactive components from naturally available products.

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