Man-Made Cytotoxic Steroids: Exemplary Agents for Cancer Therapy

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Man-Made Cytotoxic Steroids: Exemplary Agents for Cancer Therapy Ranju Bansal* and Pratap Chandra Acharya University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India cancer. There are several fundamental techniques available to treat cancer, which include surgery, radiation therapy, immunologic treatment, and chemical-based approaches, and each has its own merits and limits. Generally, a combination of these methods is used, and most of the therapeutic approaches to the treatment of cancer comprise a chemical component.4,5 Although cancer was recognized as a disease as early as 1500 B.C. and herbal management of cancer is recorded in many ancient medical writings, the use of drugs in cancer treatment was not seriously considered prior to the 1930s. Application of androgens and estrogens in the treatment of breast and prostatic cancers as well as other types of neoplastic growth, based on alteration of the hormonal status of the cancer patients, was CONTENTS initiated about a century ago.6,7 Shortly thereafter, the discovery of antileukemic activity of the nitrogen mustards and analogues 1. Introduction 6986 of folic acid triggered the earnest search for cancer chemo1.1. Need of Cytotoxic Agents for Cancer Treattherapeutic agents.8−11 Up to present, several thousand synthetic ment 6986 compounds and numerous natural products have been evaluated 1.2. Cytotoxic Steroids and Cancer: Accounts of as anticancer agents.12 the Mechanistic Insight and Target Specificity 6987 Despite the availability of a large number of chemotherapeutic 1.3. Present Status and Future Perspectives 6988 antineoplastic agents, the medical need remains still largely 2. Medicinal Chemistry of Cytotoxic Steroids 6989 unmet. The main reasons behind the failure of chemotherapy 2.1. Steroid Alkylating Agent Hybrid Molecules 6989 include (i) the lack of selectivity of conventional drugs, (ii) the 2.1.1. Steroid−Nitrogen Mustard Conjugates 6989 metastatic spreading of initial tumors, (iii) the heterogeneity of 2.1.2. Steroidal Nitrosourea Derivatives 6995 the disease, and (iv) multidrug resistance. These drawbacks 2.1.3. Steroidal Cyclophosphamide Derivatives 6996 prompt medicinal chemists to design and develop safer, target2.2. Steroidal Oximes 6996 specific, and effective antineoplastic agents.4 2.3. 16-Arylidene Steroids 6997 The basic approaches to cancer treatment are constantly 2.4. Miscellaneous Synthetic Cytotoxic Steroids 6999 changing. Clinical protocols are now exploring molecular targets, 3. Conclusions 7001 genetic therapies, manipulations of the immune system, Author Information 7001 stimulation of normal hematopoietic elements, induction of Corresponding Author 7001 differentiation in tumor tissue, and inhibition of angiogenesis as Notes 7001 13−18 In the background of appealing areas for cancer therapy. Biographies 7001 new available chemotherapeutic antineoplastic agents, cytotoxic Acknowledgments 7002 drugs remain the mainstay of cancer therapy as they are highly References 7002 specific to their targets.19−22 It is noteworthy that in the late 1980s the idea that cytotoxic drugs had substantially exhausted their potential and their development had reached a plateau was 1. INTRODUCTION gaining ground. However, just in the nineties, important 1.1. Need of Cytotoxic Agents for Cancer Treatment cytotoxic anticancer drugs such as taxanes and camptothecins entered the market and are still milestones in cancer treatment.23 Cancer is one of the most dreaded diseases to haunt mankind in Most of the FDA (Food and Drug Administration)-approved the world today. It is one of the leading causes of death drugs in the last two decades are either reintroduced after the worldwide. Although overall cancer incidence rates in the patent expiry or analogues of the older cytotoxic drugs. Older developing countries are half of those seen in the developed drugs are constantly being reapproved for a wider use, and their world in both sexes, the overall cancer mortality rates are 1−3 analogues are approved for therapy resistant cancers.24 On the generally similar. Breast cancer is the most frequently basis of their targets, new anticancer drugs approved by the FDA diagnosed and the foremost reason for cancer deaths among in the past decade may be classified into three categories. The females, accounting for almost 23% of the total cancer cases and first group comprises cancer-selective or semiselective drugs that 14% of the cancer deaths every year, while lung is the leading cancer site among males, comprising 17% of the total new cancer cases and 23% of the total cancer deaths. The approach used to Received: May 29, 2013 treat depends upon the specific type, location, and stage of the Published: May 28, 2014 © 2014 American Chemical Society

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dx.doi.org/10.1021/cr4002935 | Chem. Rev. 2014, 114, 6986−7005

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Chart 1. Cytotoxic Steroids Obtained from Natural and Synthetic Sources

are effective in rare kinase-addictive cancers. The second type includes analogues of classic cytotoxic agents such as DNA alkylating agents, nucleoside analogues, and antimicrotubule agents, and the third is a diverse group of tissue-selective agents that essentially attack the normal tissues of tumor origin and thus exploit the tissue-specific similarities between normal and cancer cells. In most of the cases, however, two anticancer strategies may be most fruitful, first, combinations of cytotoxic drugs with semiselective agents aimed at matching targets, and, second, tissue-selective therapy aimed at normal and tumor cells of the same tissues.24 For all of these reasons, cytotoxic drugs will continue to form a chief part of the therapy in combination with cancer-selective and tissue-selective innovative agents in the near future. The traditional cytotoxic approaches, which received support in the recent past, will still receive it in the near future due to new gained knowledge as a result of extensive mechanistic exploration in terms of both screening and designing of new analogues. Chart 1 summarizes a variety of cytostatic steroidal derivatives obtained from various resources.

Figure 1. Steroids used as adjuvants in cancer chemotherapy.

Sex hormones are also of interest in the treatment of neoplastic diseases. The growth of malignant neoplasms of the breast, prostate, and endometrium is often dependent on the hormonal balance of the body. It is observed that changing the hormonal balance of the body by administering sex hormones produces useful remission from cancer. 28−30 Androgens such as fluoxymesterone (2) may be used in the treatment of women with advanced and metastatic neoplasm of the breast.30 The use of estrogens in the treatment of advanced metastatic neoplasm of the breast is made only when ovarian function has ceased.31 The antiandrogen cyproterone acetate (3) has been tried in prostatic carcinoma with encouraging results.32 Estrogens such as stilbestrol (4) are generally preferred to control inoperable and metastatic neoplasm of the prostate.33 Aromatase inhibitors like exemestane (5) are used in the treatment of postmenopausal breast cancer and 5α-reductase inhibitors; for example, dutasteride (6) is already in clinical use to alleviate androgen-dependent cancers.34−37

1.2. Cytotoxic Steroids and Cancer: Accounts of the Mechanistic Insight and Target Specificity

Currently, steroidal derivatives occupy the broadest spectrum of clinical utility and are well-known to the global market as the most diversified therapeutic class of compounds. These are being used extensively in modern medicines and have long been used in the treatment of cancer. Figure 1 depicts the structures of various steroidal agents used as adjuvants in cancer chemotherapy. Adrenocorticosteroids possess lympholytic effects and are able to suppress mitosis in lymphocytes, while glucocorticoids are useful in the treatment of malignant lymphoma and acute leukemias in children, for example, prednisone (1). These are usually used in combination with other antineoplastic agents.4,25−27 6987


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Figure 2. Clinically active steroidal alkylating agent conjugates for cancer chemotherapy.

On the basis of the research studies in this area, it is speculated that steroid molecules could be used as biological vectors for the chemotherapeutic agents. This would reduce toxicity and increase antineoplastic activity as the steroidal part could play the role of a “biological platform” on which the alkylator could easily be transported through the biological barrier to the target site. The speculation was further supported by evolution of steroidal alkylating agent conjugates as a strategic approach in the treatment of hormone responsive cancers such as those of breast, prostate, and endometrium. Various successful attempts have been made to obtain antineoplastic compounds by conjugating N,N-bis(2-chloroethyl)amine moiety (nitrogen mustard, nitrosourea, and cyclophospahamide) to cholestane, androstane, estrane, and other steroidal skeletons. Steroidal alkylating agent hybrid molecules have been developed as potential antitumor agents for the past few years with the hope that useful advantage might be made of the lipophilic nature of the steroid molecule in the transport of various nitrogen and sulfur mustards to the target site. It is also anticipated that hormonal steroids such as androgens and estrogens could deliver the alkylating agent to a specific target tissue more easily, reducing systemic toxicity due to reduced dose, increasing the bioavailability, and improving the therapeutic efficacy. Further, the synergistic activity due to these hybrid steroidal structures has also been expected.38 Steroidal alkylating agents like estramustine phosphate sodium (7) and phenesterin (8) were developed as target orientated anticancer agents for the therapy of estrogen receptor-positive breast cancer (Figure 2).39−44 Although estramustine was designed to function as an alkylating drug targeted to estrogen receptor-bearing cells, where the carbamate bridge would be cleaved to release nitrogen mustard, the compound was found to be devoid of alkylating activity due to unanticipated stability of the carbamate bridge. The pronounced cytotoxicity was independent of any hormonal or alkylating activities. Rather, estarmustine binds to tubulin leading to microtubule depolymerization, which produces the antitumor effect.45,46 It has also been shown to increase the acetylation levels of the interphase microtubules and interfere with the microtubule−kinetochore interaction causing activation of the spindle checkpoint causing apoptosis.47 Estramustine was found to cause alterations in the gene expressions associated with the control of iron homeostasis, cytoskeleton, cell cycle, apoptotic process, and cell signaling transduction, suggesting novel molecular mechanisms by which it exerts its inhibitory effects on prostate cancer cells.48 The pharmacological, pharmacokinetic, clinical, and other features of the drug have been reviewed by Hauser et al.41 Extensive studies have also been carried out on phenesterin (8), a nitrogen mustard derivative of cholesterol.42−44 The presence of the ester function is claimed to be important in explaining the action of phenesterin, which was different from that of the parent free acid.41 The clinical studies on phenesterin have also been reported.49,50

Over time, conjugates of nitrogen mustards and various steroidal skeletons such as androstanes, estranes, homoaza steroids, and homoaza-steroidal lactams have been synthesized to generate potential cancer-specific cytotoxic agents especially against leukemia and, in some cases, against colon, lungs, adenocarcinoma, and other malignancies. Most of the time, these conjugates were found to be effective against a certain type of cancer cell or solid tumor model emphasizing the target specific nature of these agents. The role of the steroidal part in the mechanism of action of these promising compounds provided enough evidence to understand that this nucleus is not mere a “biological vector” as speculated for many years, but may have its own specific cellular binding site(s).51 The steroidal module is thought to enhance the formation of covalently bound DNA adducts in the tumor tissues with additional ability to block repairing enzymes by camouflaging them. This eventually leads to enriched DNA damaging effects with reduced toxicity.52−54 Studies regarding the possible mode of action of the steroidal alkylating agents suggest that the DNA target is reached by the whole molecule and not by the nitrogen mustard alone. Hormone receptors present in the cell nucleus might be the main binding site of the steroidal part.55 Moreover, these chemohormonal agents act in a manner different from that of both alkylating agent and the steroid. They are also superior to mixtures of unlinked alkylating agents and hormones.56−60 The increase in sister chromatid exchange rates, cell division delay, and induction of apoptosis are also some of the suggested modes of action in various steroid linked nitrogen mustards. Other than steroid−nitrogen mustard adducts, steroidal derivatives such as oximes, 16-arylidene, and other miscellaneous molecules have also displayed profound antineoplastic activity through some unconfirmed mechanisms. The means by which these steroids act as antitumor agents is still ambiguous; probably they act by a multi mechanistic way, targeting more than one cellular target. 1.3. Present Status and Future Perspectives

A fair number of steroidal agents are being used in the clinical management of malignancies of various origins as detailed in section 1.2. Numerous steroidal derivatives have been discovered with potent antineoplastic activity conferred with high tissue selectivity. Cytotoxic steroids like estramustine are also used in the treatment of prostate cancer as a single chemotherapeutic agent or along with other agents such as 5α-reductase inhibitors. Chemotherapy is the only way to treat inoperable cancer like leukemia, and steroidal derivatives have shown great promise toward leukemia. Modifications in the basic steroid skeleton, derivatization, introduction of heteroatoms, and formation of steroid alkylating agent hybrids have transformed the inactive or already active compounds to strongly active ones. On a similar account, it is also observed that addition of the steroid moiety confers enhanced activity as compared to the alkylating agent alone. In 6988


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some cases, toxicity remained at clinically acceptable levels and was significantly lower than the alkylating agent itself. These agents comprise many aspects of an ideal chemotherapeutic agent including (i) tissue-selective cytotoxicity to achieve reduced peripheral toxicities, (ii) target specificity to produce cytotoxicity in the cancerous cells, and (iii) implication in the treatment of inoperable malignancies. Various research findings advocate the inherent cytotoxic properties of steroids, further strengthening their role as undisputed agents for cancer therapy. Hence, the development of steroidal derivatives as safer and target specific antineoplastic agents could be considered undoubtedly a promising futuristic approach. Although several studies highlighting the significance of steroidal cytotoxic agents have been reported, so far no general review covering synthetic cytotoxic steroids has appeared in the literature. The following section describes medicinal chemistry of cytotoxic steroids along with their discovery and development as prospective anticancer agents. Particular emphasis has been laid on the structural features and modifications required for the antineoplastic activity. A comprehensive literature survey accounting for the reports available from 1951 to 2012 has been carefully carried out to choose the most active moiety from a particular series of compounds. The IUPAC nomenclature along with conventional steroid numbering and ring designations are used throughout the text to describe the steroidal derivatives.

Figure 3. Steroidal nitrogen mustard conjugates with a rigid N−C linkage.

reported the synthesis of a nitrogen mustard derivative of 5cholestene-3β-carboxylic acid; the molecule was made available for assay at Boston University Medical School, but the results obtained were not convincing.79 In his classical treatise on biological alkylating agents, Ross reviewed the few nitrogen mustards prepared up to year 1960 and mentioned their negligible antitumor properties.80 Further, Burstein and Ringold reported the synthesis and biological activity of a series of androstane−nitrogen mustard derivatives.81 Of all, the 2-substituted androstane−nitrogen mustard conjugate 9 was found to be nontoxic to the animals at a daily dose of 500 mg/kg, but the antitumor activity was missing. Rao and Price further incorporated nitrogen mustard moiety in various steroidal skeletons; resulting androstene mustard 10 at 10 mg/kg and the 3-methoxyestratriene mustard 11 at 15 mg/kg produced about 100% extension of survival time in mice infected with Gardner ascites tumor.82 Androstane derivatives 12−14 possessing nitrogen mustard functionality at C17 position of steroidal core were synthesized by Jones et al. and evaluated against DMBA (7,12-dimethylbenz[a]anthracene)-induced mammary tumors in female Sprague−Dawley rats.83 Compounds 12 and 13 were totally ineffective on the rate of growth or proliferation of the tumors, and compound 14 induced only a moderate regression.83 In general, the steroidal nitrogen mustard derivatives with a rigid N−C linkage were found to possess insignificant antitumor activity. These observations led to some major conclusions regarding anticancer activity of steroidal nitrogen mustards, for instance, (a) direct attachment of nitrogen mustard to the steroid nucleus is undesired and aromatic mustards are better than aliphatic ones, (b) choice of appropriate steroidal backbone results in effective transport and may also impart selectivity in action, and (c) anticancer activity increases if the mustard− steroid link is readily cleavable by hydrolysis or other in vivo processes.83 In the same year (1971), another communication by Jones and Leman described the synthesis of some new androstane mustards in which both the ring A Δ4-3-keto and ring D 17β-hydroxyl functions of 17β-hydroxyandrost-4-en-3-one (testosterone) were retained; however, no reference to the biological activity was made.84 2.1.1.2. Steroid−Nitrogen Mustard Conjugates with a Labile Ester Linkage. The above cited reports involved compounds in which the nitrogen mustard moiety was linked to the steroid skeleton by a rigid N−C bond. In most of the cases,

2. MEDICINAL CHEMISTRY OF CYTOTOXIC STEROIDS Many cytotoxic steroids with characteristic structural features have been synthesized or isolated from natural sources over the past few years. Several excellent reviews covering various aspects of anticancer steroids isolated from diverse natural sources such as plants, marine, and bufadienolides of animal origin are available in the literature.61−76 These naturally occurring steroids act through some unprecedented mechanisms. Numerous rationally designed synthetic steroidal derivatives have evolved as clinical contenders for cancer chemotherapy and are discussed under the following subheads. 2.1. Steroid Alkylating Agent Hybrid Molecules

As discussed in section 1.2, steroidal alkylating agent hybrid molecules have shown versatility to be developed as novel antineoplastic agents with unique mode of action and targetspecificity. Besides the two clinically proven steroidal alkylating agents, estramustine phosphate sodium (7) and phenesterin (8), a variety of other cytotoxic steroidal nitrogen mustard derivatives have been designed, synthesized, and studied as antineoplastic agents. 2.1.1. Steroid−Nitrogen Mustard Conjugates. 2.1.1.1. Steroid−Nitrogen Mustard Conjugates with a Rigid N−C Linkage. Steroidal nitrogen mustard conjugates with a rigid N−C linkage have been synthesized in the primeval years of their development, and the structures of some of these compounds have been presented in Figure 3. The synthesis of three nitrogen mustards of the general formula RN(CH2CH2C1)2, where R is a steroidal unit, has been described by Hazen as early as 1952 in his doctoral dissertation. 77 The nitrogen mustard unit N(CH2CH2Cl)2 is linked to the 3-position of cholesterol, ergosterol, and stigmasterol, replacing the hydroxyl group. However, the biological activity data of these steroidal mustards are not available. In the same year, Vavasour et al. also reported the synthesis of nitrogen mustard conjugates of cholesterol without any reference to their biological activity.78 Gensler and Sherman (1958) further 6989


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Figure 4. Structures of steroidal alkylators with nitrogen mustards conjugated to C17 position of steroid.

either the biological data were missing or if available were not significant enough. In contrast to these unpromising results were the reports by a Russian group,85 who described the preparation of 3-{[p-N,N-bis(2-chloroethyl)amino]phenylacetoxy}-cholest5-ene and named it as phenesterin (8). Further detailed studies of its activity against a variety of solid tumors including sarcoma 45, Walker carcinoma, and alveolar liver carcinoma RS-1 and brain tumors have been presented in various literature reports.42,43,85,86 At 100−200 mg/kg sc administration, phenesterin remarkably inhibited the growth of the tumor. However, phenesterin was inactive against Ehrlich and Sarcoma180 mice tumor. The compound was found to be relatively nontoxic with an LD50 (lethal dose, 50%) of 2.0 g/kg for rats. Degteva, Larionov, and coworkers further claimed that the action of the cholesterol ester was different from that of the parent free acid and also stressed the importance of the ester group.41 Keeping all of these results in hand, Wall et al. reinvestigated steroidal nitrogen mustards and prepared a number of steroidal analogues of phenesterin by varying the steroidal core and the location of the p-[N,N-bis(chloroethyl)amino]phenylacetic acid (BCAPAA) ester on steroid nucleus.87 A number of selective steroidal alkylating agents with a rigid N−C bond were also prepared. They were compared under strictly similar conditions in a variety of solid tumors and leukemia such as Walker carcinosarcoma 256 and the L1210 lymphoid leukemia. Some were tested with R3149 acute monocytic leukemia of the Fischer/344 rat or 13762 DMBA-induced and transplantable mammary adenocarcinoma. Compounds with rigid N−C had no activity, while compounds with a labile ester function displayed varying degrees of activity.87 Carroll et al. extended the classical previous work of Wall and co-workers and synthesized ester or amide derivatives of some new steroids with BCAPAA and highly active antineoplastic alkylating agent p-[N,N-bis(2-chloroethyl)amino]phenylbutyric acid (BCAPBA, chlorambucil).87,88 The BCAPAA ester of pregnenolone and epiandrosterone exhibited antitumor activity in the 13762 tumor system and increased survival time of the host for the leukemias, while the BCAPBA esters in cholestane and estrane series exhibited excellent inhibition of growth of tumor in the same system. The BCAPBA ester of cholesterol appeared to be more active than its BCAPAA counterpart phenesterin.88

These results yet again proved the importance of the ester linkage in steroidal nitrogen mustard conjugates. Medicinal chemistry of labile alkylating agents conjugated to different positions of steroidal nucleus with a hydrolyzable ester linkage has been detailed in the succeeding sections. 2.1.1.2.1. Nitrogen Mustard Conjugated to C17 of Steroid. Steroidal derivatives with an alkylating moiety at C17 position have been demonstrated as potent anticancer compounds against many leukemia models (Figure 4). At the same time, conjugating an alkylating agent to the C17 position has also produced newer anticancer compounds with several advantages including reduced toxicity over the existing drug molecules. Prednimustine (15), the C21 prednisolone ester of chlorambucil, has been shown to be active against the L1210 murine leukemia model and has been tried on patients with chronic lymphocytic leukemia and lymphocytic lymphoma.89−91 The usefulness of compound 15 in the treatment of acute leukemia has also been demonstrated.92,93 It has been shown that prednimustine is more effective than chlorambucil against a sensitive line of the Yoshida ascites sarcoma, but ineffective against Ehrlich ascites tumor (EAT), which is intrinsically resistant to alkylating agents.94 Similarly, bestrabucil (16), the benzoate of the estradiol− chlorambucil conjugate, was initially developed as a target orientated anticancer agent for the therapy of estrogen receptorpositive breast cancer.95 Research data supporting the targeting nature of this conjugate depict that the concentration of bestrabucil and some of its derivatives accumulates 5−10 times higher in tumor tissue of the sensitive xenografts than in blood and muscle tissues.96 Two steroidal alkylating agents 17 and 18 were designed to bind and alkylate the glucocorticoid receptors; however the biological study revealed insignificant alkylation of the receptors and other cellular constituents by these compounds. The expected therapeutic advantage of the conjugated steroids over the steroid alone was not observed.97 Similarly, steroidal nitrogen mustard conjugates 19 (ester of estrogenic acid and phenol mustard) and 20 (amide of estrogenic acid and aniline mustard) were synthesized, and their in vitro cytotoxicity was determined against two colon cancer cell lines (HCT116, HT-29) and one lung cancer cell line (A549). The ester conjugate 19 displayed augmented activities against the 6990


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Figure 5. A-Ring modified steroids with nitrogen mustards conjugated to C17 position.

Figure 6. Steroidal alkylating agents with nitrogen mustards conjugated to C3 of androstane nucleus.

synthesis by 81%, and RNA synthesis by 67% of L1210 leukemia cells when investigated by incorporating radioactive precursor.105,106 2.1.1.2.2. Nitrogen Mustard Conjugated to C3 of Steroid. Placement of the nitrogen mustard moiety at the C3 position of steroidal skeleton has generated several potential antineoplastic steroids including estramustine phosphate sodium (7) and phenesterin (8). Various ring modified and nonmodified steroid skeletons have been investigated while positioning a variety of alkylating agents at C3 position. Impact of introducing heteroatoms within B-, C-, and D-rings of steroids conjugated to the alkylating functionality in the A-ring on antineoplastic activity is discussed in the following section. 2.1.1.2.2.1. Androstane Derivatives. Antitumor steroidal compounds with a double bond between positions C5 and C6 of the steroidal nucleus and a C3-alkylating group were found to induce statistically significant enhancement of SCEs and cell division delays. The structures of some of these derivatives are shown in Figure 6. Steroidal amidoester nitrogen mustard hybrid 23, 3β-[p-bis(2-chloroethyl)aminophenyl]acetyloxy-17β-acetamido-androst-5-ene, and its 5α-H analogue were investigated in L1210 and P388 leukemias, Ehrlich ascites tumor, and adenocarcinoma CA-755. Life-span of the tumor-bearing animals was increased and resulted in several long-term survivors especially in the case of adenocarcinoma CA-755.107 An increased genotoxicity toward human lymphocytes and antileukemic activity toward P388 leukemias was observed for the chlorambucil conjugate of 5-androstene-7,17-dione 24. The results suggested that the introduction of the allylic 7-keto group in the steroidal skeleton induces the antitumor activity in the esteric derivatives. To a certain extent, this structural change modifies the inactive or already active compounds to strongly active ones.108 Compound 25, a steroidal ester of p-methyl-m-N,N-bis(2chloroethyl)-aminobenzoic acid, showed a spectrum of activities similar to that of steroid 24 in both the cytogenetic and the antileukemic experiments. This finding indicated that the introduction of ketone at the 7-position of the steroidal ligand

three cell lines in comparison to its precursor, but the amide conjugate 20 exhibited almost no activities.98 2.1.1.2.1.1. A-Ring Modified Steroids. Homoaza-steroids have emerged as important biological carriers for transporting the tethered alkylating moieties to the site of action. The utilization of modified lactam steroids has resulted in the synthesis of highly active antitumor compounds as these could target specific components, biochemical systems, or pathways of tumor cells besides producing high intracellular concentrations of alkylator. Structures of some potential antineoplastic compounds generated by conjugating the nitrogen mustards to C17 of A-ring modified lactam steroids have been depicted in Figure 5. Catasoulacos et al. synthesized homoaza-steroidal ester 21, chemically, 17-[p-N,N-bis(2-chloroethyl)-aminophenoxyacetoxy]-3-aza-A-homo4a(5)-androsten-4-one, which exhibited 125% increase in the mean survival time of treated animals over the untreated (control) ones in the P388 lymphoid leukemia system.99 It has been found effective against L1210 and P388 leukemias, Lewis lung cancer, DNA synthesis of Ehrlich ascites tumors (EAT), L1210, P388, BHK (baby hamster kidney) cell cultures, and on the induction of sister chromatid exchanges (SCE).100 In vivo and in vitro comparable studies with alkylating agents such as p-bis(2-chloroethyl)aminophenoxyacetic acid, cyclophosphamide, melphalan, and chlorambucil also proved the effectiveness of A-homo lactamic steroid 21.101 It is further reported that compound 21, also named as lactestoxate, exhibited very good activity against nine human colon adenocarcinoma cell lines in vitro and against two murine and one human xenograft colon tumor models in vivo.102 In comparison, the modified steroidal alkylating agent, 17-[pN,N-bis(2-chloroethyl)-aminophenylbutoxy]-3-aza-Ahomo4a(5)-androsten-4-one (22), showed activity against P388 leukemia and Lewis lung (LL) carcinoma, but was found inactive in the L1210 leukemia system.103 Remarkable antitumor activity was found against adenocarcinoma CA-755.104 The unlinked Aring modified steroid, 17β-acetamido-3-aza-A-homo4a(5)-androsten-4-one, also proved to be cytostatic against Ehrlich ascites tumor L1210 and P388 leukemia as well as inhibited DNA 6991


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Figure 7. Steroidal alkylating agents with nitrogen mustards conjugated to C3 of estrane nucleus.

mammary adenocarcinoma BN/10232, the estrone urethane 27 showed a 30% inhibition with 15% mortality.111 Estrogen derived C3 conjugated nitrogen mustards 29 and 30 produced the most pronounced antiproliferative effects at 10−5 M on monolayer cultures of transformed L-929 fibroblasts (from murine skin sarcoma) and inhibited cell proliferation up to 3- and 2.6-fold, respectively.112 It is envisaged that attachment of an alkylating moiety to the antiestrogenic steroid through a labile bond capable of in vivo cleavage would be advantageous in the case of hormonedependent tumors such as those of mammary glands and uterus cancers. The nitrogen mustard derived steroidal derivatives 31− 33 of 11α-acyloxyestra-1,3,5(10)-triene exhibited an antitumor activity against murine mammary adenocarcinoma Ca-755 up to 94−99%. These derivatives inhibited the binding of estradiol to the human mammary gland tumor, suggesting that they possess potent antiestrogenic activity. They also demonstrated advantage over estramustine phosphate sodium (cytotoxic estrogen) and tamoxifen (antiestrogen antitumor drug). Most of them were nontoxic in doses above 125 mg/kg, and compound 32 did not exhibit toxicity even with a total dose of 500 mg/kg.113 In contrast to 11α-derivatives, 11β-analogues possessing nitrogen mustard moiety strongly inhibited tumor growth of Ca-755 breast adenocarcinoma but exhibited pronounced estrogenic properties. Compounds 34 and 35 showed high antitumor activity despite the presence of 11β-moiety, which acts as an estrogenic pharmacophore, the cytotoxic fragment probably contributing toward the cytostatic effect.114 Compound 36 with free hydroxyl groups at C3 and C17 positions and an alkylating moiety substituted at position C11 of 11α-hydroxyestra-1,3,5(10)-triene exhibited antitumor activity with total growth inhibition of 92% against mice bearing CA755 breast adenocarcinoma and B-16 melanoma and also possessed antiestrogen properties. Overall the compounds with the alkylating group attached to C3 of steroid are more active,

accounts for its increased activity and is in agreement with several reports stating that the Δ5-7-keto steroids are more toxic toward cancerous cells due to their ability to inhibit cell replication.109 On the basis of the above results and to further explore the effect of the 7-keto group on the antileukemic activity of the nitrogen mustard functionalized steroidal esters, the antileukemic properties of esters of p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE, active metabolite of chlorambucil) with 3β-hydroxy-androst-5-ene-7,17-dione, 3β-hydroxy-17βacetamido-androst-5-en-7-one, as well as with their parental nonoxidized steroids 3β-hydroxy-androst-5-en-17-one and 3βhydroxy-17β-acetamido-androst-5-ene were studied.108 The 7keto derivatives were effective against P-388 and L1210-leukemia bearing mice in comparison to the nonoxidized derivatives. Furthermore, the steroidal phenyl acetate 26 was also found to be the most effective inducer of SCEs with 2-fold higher potency than those of the other oxidized esters. The toxicity was significantly lower than the alkylating agent alone with a clinically acceptable level.60,108 Similarly, incorporation of a 17βacetamido functionality in compound 24 also resulted in potent antileukemic property.58 The reduction of the C5−C6 double bond in compounds 23, 24, and 26 led to decreased antileukemic property in comparison to their parent molecules as determined on leukemia P388-bearing BDF1 mice, although they exert their own significant genotoxic, cytostatic, antineoplastic, and apoptotic effects.55,110 The reduced compounds significantly increased the SCE frequencies both in vitro and in vivo followed by cytostatic activity marked with decreased proliferation rate indices (PRI), antileukemic activity, and apoptosis induction (caspase-2 and 3 activity).110 2.1.1.2.2.2. Estrane Derivatives. Steroidal nitrogen mustard derivatives with estrane backbone such as compounds 27 and 28 (Figure 7) were prepared with the aim to influence estrogendependent tumors more selectively. When screened on 6992


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Figure 8. D-Ring modified steroids with nitrogen mustards conjugated to C3 position.

N-Methyl-derivative of 37 was found to possess antitumor activity against L1210 leukemia, P388 leukemia, Ehrlich ascites tumor (EAT), and Lewis Lung Carcinoma (LLC) and induced marked increase in SCE rates and cell division delays.129,130 Chlorambucil substituted homoaza-steroidal analogue 3-[pbis(2-chloroethyl)-amino]phenylbutoxy-17a-aza-D-homo-5αandrostan-17-one (38) displayed cytostatic action on two tumor systems (B16 melanoma on C57 b1 mice and T8-Guerin on rats) and was active in L1210 and P388 leukemias.118,131 Similarly, the D-ring modified steroidal alkylating agent 3-[pbis(2-chloroethyl)-amino]-phenoxyacetoxy-17a-aza-D-homo5α-androstan-17-one (39) was found to be active in the colon 26 tumor, B16 melanoma, and P388 leukemia system and was effective in causing statistically significant increases in SCE rates and cell division delays.132,133 The lifespan of L1210 and P388 leukemia bearing animals was increased to 100% and 383%, respectively, on a daily treatment schedule. Activity in advanced L1210 (41% increased lifespan) and P388 leukemias (173% increased lifespan) was also observed.134 It was found to induce mutations and somatic recombination in Drosophila melanogaster and dose-dependent increase in base pair substitutions (his+ revertant strains TA100 and TA1535) in the Salmonella/ microsome assay.135,136 The in vitro clastogenic activity in human lymphocyte cultures demonstrated a dose-dependent reduction in the mitotic index and causes chromosome- as well as chromatid-type aberrations.137 The antitumor activity of the esters of lactamic steroids in which the p-N,N-bis(2-chloroethyl)aminophenoxyacetic acid is linked to the C3 (ring D lactam) or C17 (ring A lactam) position has been extensively reviewed by Catsoulacos and Catsoulacos.138 Compound 39 displayed significantly higher cytostatic effects than dacarbazine, carmustine, and semustine, but lower in comparison to cisplatin against malignant melanoma in an in vitro screening.57 In a comparative study, the D-homoaza androstane derivatives were found distinctly superior to the corresponding steroidal 17β-amido-esters of p-N,N-bis(2-chloroethyl)amino-phenylacetic acid and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid when evaluated for acute toxicity in mice, in vivo antileukemic activity against P388 and L1210 murine leukemias, and in vitro

which might be attributed to the less stable ester bond involving the C3 phenolic −OH group, thus favoring an increased accumulation of nitrogen mustard moiety at the tumor site.115 2.1.1.2.2.3. D-Ring Modified Steroids. Several 17a-azasteroidal C3-derived nitrogen mustard esters (Figure 8) have been synthesized and reported by Catsoulacos and co-workers as effective cytotoxic agents.116 D-Ring modified azasteroidal ester 37 derived from 3β-hydroxy-17a-aza-D-homo-5α-androstan-17one and p-bis(2-chloroethyl)-aminophenylacetic acid was found active against B16-melanoma in C57B1 mice and T8-Guerin tumor in rats and showed exceptional results in P388 leukemia and satisfactory results in L1210 leukemia.99,116−119 The compound was found active in colon 26-bearing CD2F1 mice, CD8F1 mammary tumor, and melanoma-bearing B6D2F1 mice as well as in the LX-1 lung and MX-1 breast xenografts.120,121 DHomosteroid 37 was capable of binding covalently to DNA of Ehrlich ascites tumor cells in vitro and inhibited incorporation of 3 H-thymidine to DNA by 71%.122,123 The steroidal nitrogen mustard hybrid 37 was also found to increase SCE rates and cell division delays markedly and act synergistically with caffeine with enhanced cytogenetic damage to Ehrlich ascites tumor cells, when the two components were administered in combination.124,125 It decreases the net protein synthesis in ovaries of Drosophila melanogaster females, and induces increased micronuclei frequencies in human lymphocytes in a linear fashion with dose and duration of treatment.126,127 The pattern of micronuclei induction further suggested that it acts via chromosome breakage mechanism.127 Regardless of the similar designing concept of both 37 (described as lactandrate) and 21 (lactestoxate), A-ring modified lactestoxate yielded better activity than D-ring modified lactandrate.102 However, lactandrate (37) produced significant anticancer activity on breast cancer in vitro (T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines) and in vivo xenograft mammary tumors (human mammary carcinoma MX-1 xenograft).128 Preclinical investigation indicates that lactandrate might be a substantial candidate for the treatment of human breast cancer.102 6993


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Figure 9. B-Ring and D-ring modified steroids with nitrogen mustards conjugated to C3 position.

Figure 10. B-Ring modified steroids with nitrogen mustards conjugated to C3 position.

2.1.1.2.2.4. B-Ring and D-Ring Modified Steroids. To investigate the influence of lactamic modifications in both steroidal B- and D-rings together on the antitumor profile, azasteroid alkylator 3β-[p-N,N-bis(2-chloroethyl)amino]-phenylacetoxy-7a,17a-diaza-B,D-dihomo-androst-5-ene-7,17-dione (43) was synthesized (Figure 9). The compound was found to be effective in the treatment of P388 and L1210 leukemias in vivo and showed high inhibition effect on DNA, RNA, and protein synthesis. The compound also increased the SCEs frequency and reduced the replication index of human lymphocytes.145,146 The meta substituted steroidal ester 3β-{[m-N,N-bis(2chloroethyl)amino]-4-methyl-phenylacetoxy}-7a,17a-diazaB,D-dihomo-androst-5-ene-7,17-dione (44) proved to be a more effective antileukemic agent than para substituted hybrid 43 when tested against leukemias P388 and L1210 in vivo and for the SCE and PRI induction in normal human lymphocytes in vitro.144 2.1.1.2.2.5. B-Ring Modified Steroids. Esters of m-[N,Nbis(2-chloroethyl)amino]benzoic acid, p-[N,N-bis(2chloroethyl)amino]-phenylacetic acid, and chlorambucil with B-ring modified cholestane, amidosteroid, and androstane as exemplified by compounds 45−50 (Figure 10) have also been synthesized and were found to have profound antileukemic activity.59,60,109,144,146 The most potent compound 50 had significantly reduced toxicity level and notable potency against P388 and L1210 leukemia cell lines.144

antileukemic effect on human leukemia cell lines (K562, MOLT4, ML-1).106 D-Homoaza-steroidal esters of N,N-bis(2chloroethyl)aminocinnamic acid have also been synthesized and evaluated for cytotoxic and antileukemic activities along with the study of their structure−anticancer activity relationships.139,140 Similarly, the cytotoxicity and antileukemic activities of several D-homoaza-steroidal esters of N,N-bis(2-chloroethyl)-aminobenzoic acid were also studied.141−144 The ester 40 of ortho substituted benzoic acid was found to be less effective in causing the cytogenetic damage and antineoplastic activity in comparison to that of D-homoaza-steroidal ester 37 of p-bis(2-chloroethyl)aminophenylacetic acid.143 Steroidal ester 41 of p-N,N-bis(2-chloroethyl)aminophenylbutyric acid with a C7 keto group and a double bond between C5 and C6 positions of steroidal nucleus showed efficient genotoxicity toward human lymphocytes and antileukemic activity toward P388 leukemias as compared to the corresponding Δ5-7- keto derivative of 40.108 Similarly, the compound 3β-[p-N,N-bis(2-chloroethyl)aminophenylacetoxy]-17a-aza-D-homo-5-androstene-7,17dione (42) was found to be a more effective inducer of SCEs than its C7 deoxy counterpart 37. It also exhibited antileukemic potency against leukemia P388- and L1210-bearing mice in vivo and inhibited cell proliferation in normal human lymphocytes in vitro.60 6994


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Figure 11. C-Ring modified steroids with nitrogen mustards conjugated to C3 position.

Figure 12. Structures of some steroidal nitrosourea derivatives.

The presence of −NHCO− moiety within steroid ring proved to be an important pharmacophore in contrast to the nonmodified steroids as the antineoplastic properties diminished when the B-ring was transformed to a simple 7-ketone. The enhanced biological activity of esters of the B- or/and D-lactamic steroids and m-[N,N-bis(2-chloroethyl)amino]benzoic acid is probably caused by reduced peripheral hydrolysis by esterases leading to a greater concentration of the alkylating moiety at the binding site. 2.1.1.2.2.6. C-Ring Modified Steroids. C-Ring modified steroidal alkylator, p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid ester of aza-homohecogenin, represented by compound 51, produced a significantly higher antileukemic effect than the nonmodified steroidal counterpart, when evaluated against P388 lymphocytic and L1210 lymphoid murine leukemias.147 Similar esters of o-, m-, and p-N,N-bis(2-chloroethyl)aminocinnamic acid exhibited good cytogenetic effects (SCEs induction and PRIs depression), the ortho and meta substituted acid esters 52 and 53 being the most active ones (Figure 11).148 2.1.2. Steroidal Nitrosourea Derivatives. Gnewuch and Sosnovsky have presented a comprehensive review on the development of various nitrosourea derivatives including the steroidal ones as anticancer drugs.149 Some of the promising steroidal nitrosourea derivatives are presented in this section. The nitrosoureido steroidal derivative 54 bearing the (2chloroethyl)nitrosouriedo moiety at C17 of the steroid nucleus showed moderate antitumor activity. The molecule is thought to be readily cleavable by hydrolysis or some other in vivo process to form an active alkylating agent.88

The 17β- and 20- nitrosourea derivatives in dehydroepiandrosterone, estrone, and pregnenolone series were studied for activity toward calf uterine estradiol and pregnenolone receptors. The pregnolone derivative 55 and estradiol derivative 56 (Figure 12) were found to exhibit high affinity for calf and lamb uterine pregnenolone and estradiol receptors, respectively.150 More steroidal nitrosourea derivatives 57−60 (Figure 12) were studied for antitumor activity against DMBA-induced transplantable rat mammary tumor by another research group.151 Statistically significant growth inhibition was observed for these compounds in comparison to alkylator N-(2-chloroethyl)-N′cyclohexyl-N-nitrosourea (CCNU or lomustine). They also compete with estradiol for binding to cytosolic estrogen receptors in rat uterus. The binding possibly is irreversible.152 The steroidal nitrosourea derivative 61, an alkylating compound consisting of N-[N′-(2-chloroethyl)-N′-nitrosocarbamoyl]-L-alanine as the alkylating moiety and 5α-dihydrotestosterone as a steroid carrier molecule, showed in vivo activity against the androgen, progesterone, and estrogen-receptor containing MNU-induced mammary cancer in rat and transplantable rat leukemia L5222.153,154 The compound showed effective in vitro growth inhibition of malignant cell lines K-562F, HL-60 with IC50 1.1 and 1.8 μmol/L, respectively. Toxicity level was also found negligible as demonstrated in bone marrow of female NMR-mice in vivo.154 The compound was as active as the other clinically used antineoplastic agents at concentrations >10 μmol/L and significantly more active than 5-fluorouracil at 30 μmol/L. It showed activity against a wide spectrum of tumor types; the highest activity was observed against colorectal carcinomas and breast cancer specimens at 30 μmol/L along 6995


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2.2. Steroidal Oximes

with lesser in vitro myelotoxicity in comparison to doxorubicin.154 3-(N-2-Chloroethylcarbamoyl)-3-aza-A-homo-5α-cholestane and N-methyl-(2-chloro-ethyl)-N-nitrosocarbamoyl-3β-amino5α-cholestane were cytotoxic to L-1210 murine lymphoma system,155,156 while 3-oxo-4-estren-17β-yl-N-2-chloro-N-nitrosocarbamate, a nitrosocarbamate of 19-nortestosterone, did not affect the growth of the androgen-dependent R-3327 rat prostate adenocarcinoma.157 2.1.3. Steroidal Cyclophosphamide Derivatives. Another class of steroidal mustards, in which cyclophosphamide moiety has been attached to the steroid nucleus, is exemplified by compounds 62 and 63; however, any reference to the biological activity is missing in the literature (Figure 13).158,159

In 1997, two steroidal oximes, (6E)-hydroximino-cholest-4-en-3one (64) and its 24-ethyl analogue 65 (Figure 14), were isolated from Cinachyrella alloclada and C. apion, respectively.160 The (6E)-hydroximino derivative 65 exhibited a selective cytotoxic activity against several types of cancer cells such as P-388, A-549, HT-29 (IC50 = 1.25 μg/mL for the three cell lines), and MEL-28 tumor cells (IC50 2.5 μg/mL).161 The synthesis and cytotoxic activity of these natural (6E)hydroximino steroids 64 and 65 have been described in the literature.160,162−164 Furthermore, this group of steroids was also reported to show a high affinity for human placental aromatase and to function as a competitive inhibitor of this enzyme.164 The structural features, which appear to be important for the higher bioactivity of 64 and 65 in comparison to other structural motifs, were found to be the presence of a cholesterol-type side chain, the existence of a ketone functionality at C3, and an elevated degree of oxidation in ring A.161 A number of synthetic analogues of natural (6E)-hydroximino steroids have been synthesized to study the structure activity relationship. The influence of the oxygenation in A-ring, additional oxygenation at C7 and C16 positions, and fluorinated C5 position on cytotoxic activity was investigated against A-549, H116, PSN1, and T98G cultured tumor cell lines. The synthetic analogue 66, with an additional oxygenated C2 position with

Figure 13. Structures of some steroidal cyclophosphamide derivatives.

Figure 14. Structures of various steroidal oximes possessing cytotoxic activity. 6996


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Figure 15. Structures of some 16-arylidene steroids as potent cytotoxic agents.

Furthermore, when the 3-hydroxy in 73 or the 6-hydroxy in 74 is substituted by an oxime to obtain a dioxime as in steroid 75, there was no obvious change in the cytotoxic activity. Interestingly, the results indicated that the double bond between positions 4 and 5 on A-ring confers a negative cytotoxic effect on the cancer cells for 3-hydroximino-substituted compounds. The presence of the 4,5-double bond in the analogues with 3-hydroximino-6carbonyl function resulted in a remarkable decrease in the cytotoxicity. Similarly, compounds 74 and 75 with a 3hydroximino group showed a slight increase in cytotoxicity against Sk-Hep-1, H-292, PC-3, and Hey-1B cancer cells in comparison to the analogues possessing a 4,5-double bond.168 The synthesis of some (E)- and (Z)-cholest-4-ene-6-oxime steroids is also described, and compounds have been tested for their possible effects against two human tumor cell lines, cervical carcinoma (HeLa) and chronic myelogenous leukemia (K-562). Despite the presence of a 4,5 double bond, the E-isomer 76 induced high cytotoxicity in the form of apoptosis in targeted tumor sensitive cell line (IC50 = 20.68 ± 3.10 and 11.16 ± 1.24 μM against HeLa and K-562 cell lines, respectively) and the low toxicity without apoptotic events in isolated peripheral blood mononuclear cells from normal human volunteers in vitro.169,170 O-Alkylated derivatives of various oxime precursors, in which oxime functionality is placed at various positions of steroidal skeleton, have been synthesized and evaluated for their cytotoxic activity by Jindal et al.171 Compounds 77 and 78 (Figure 14) were found to be most active ones in the in vitro assay against 60 human tumor cell lines and were further screened for preliminary in vivo hollow fiber assay.171

respect to that of natural compound 65, showed a very promising level of cytotoxicity.165 The configuration of the oxime does not have an influence on cytotoxic activity in view of the fact that the E-isomer 67 and Z-isomer 68 have similar activity patterns. The C4,5 epoxide 69 was found to be the most active derivative with activity similar to that of compound 65.165 The biological activity of 6E-hydroximinosteroid homodimers 70 and 71, where the two 6E-hydroximinosteroid units are linked at C3 of the steroid nucleus via a ruthenium catalyzed crossmetathesis reaction, was found to be less active than the parent natural steroid on evaluation against A549, HCT-116, T98G, and PSN1 cell lines. Compound 71 exhibited a selective cytotoxicity against HCT-116 cell line with IC50 = 11.3 μM.166 The (6E)-hydroximino-24-ethylcholestane-4,22-dien-3-one (72) on evaluation against sk-Hep-1, H-292, PC-3, and Hey1B cell lines exhibited modest cytotoxic activity. The natural 6Ehydroximinosteroids 64 and 65 were also evaluated on these cell lines for comparison. The results indicated that the antineoplastic activity of this class of compounds increases in conjunction with side chain attached in the following order: cholesterol-like side chain 65 > stigmasterol-like side chain 72 > sitosterol-like side chain 64.162 A number of steroidal oximes with hydroximino group located at various positions on rings A and B along with diverse side chains at 17-position of steroidal core have been synthesized, and SAR has been studied.167,168 The presence of an oxime group on ring B, a hydroxy group on ring A or B, and a cholesterol-type side chain at position 17 resulted in higher cytotoxicity than other structural motifs when evaluated against a diversity of cancer cells such as Sk-Hep-1, H-292, PC-3, and Hey-1B cells.167 In general, the analogues with an oxime group at C6 showed a remarkable increase in cytotoxic activity in comparison to the C3 oxime counterparts. However, analogue 73 with a hydroxyl group at C3 and an oxime group at C6 showed a remarkable increase in cytotoxic activity with IC50 values in nanomoles in comparison to compound 64 with a keto group at C3, indicating the importance of the hydroxyl group for the biological activity of the steroidal oxime. Nevertheless, compounds 73 and analogue 74 with an opposite arrangement of the two functional groups at 3- and 6positions did not show a distinct difference in cytotoxic activity.

2.3. 16-Arylidene Steroids

Recently, various 16-arylidene-5-androstene derivatives with a βpyrrolidino functionality at C3 position of the steroid nucleus have emerged as potential antineoplastic agents (Figure 15).172−176 The compounds have been examined for in vitro anticancer activity against various human cancer cell lines at the National Cancer Institute (NCI), U.S. It seems that the introduction of a 16-arylidene moiety and the replacement of the −OH at C3 position with a 3β-pyrrolidino functionality is a promising step toward the development of novel steroidal 6997


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Figure 16. continued

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Figure 16. (A), (B), (C) Structures of miscellaneous synthetic cytotoxic steroids.

anticancer agents.172 Steroidal derivatives possessing 4-nitro and 4-isopropylbenzylidene substituents at C16 (79) displayed potent anticancer activity.172 The anticancer properties of these 16-substituted heterosteroidal derivatives were further extensively explored by synthesizing new analogues with suitable structural modifications by Bansal et al.173−175 The impact of incorporating bis-tertiary amino functionalities on anticancer activity was also studied. The diethylamino derivative 80 with 3β-pyrrolidino moiety reached up to the in vivo xenograft testing stage of Developmental Therapeutic Program of NCI after successfully passing the first two stages, that is, 60 cell line assay and in vivo hollow fiber assay. The test agent suppressed tumor growth but unfortunately failed to meet the established antitumor activity criteria set by NCI. Yet certainly this class of steroids proved its potential to be developed as future drug candidates for cancer therapy.173 On evaluation of the cytotoxic effects of some imidazolyl substituted 16-arylidene derivatives, compound 81 was found to have mean GI50 = 0.32 μM in the 60 cell line assay protocol of NCI and proved to be 5 times more potent than compound 80 without an imidazole moiety.174 Recently, another group reported some new 16E-arylidene3β-amino androstane derivatives in 5α series. The compounds on evaluation of in vitro anticancer activity against four human cancer cell lines (SW480, A549, HepG2, and HeLa) showed moderate to good anticancer potency.176 Among the synthesized derivatives, compounds 82 (IC50 = 6.54 μM against HepG2) and 83 (IC50 = 4.07 μM against A549 cell line) were the most promising ones.

Another series of 16E-arylidene androstene amides with 3chloro substitution exhibited significant growth inhibition of various cancer cell lines in vitro, with particular specificity for leukemia cell lines. The most potent compound of the series, 3chloro-16E-{[4-(4-methylpiperazin-1-yl)-2-oxoethoxy]benzylidene-androst-5-en-17-one (84), showed GI50 of 3.94, 2.61, 6.90, and 1.79 μM against CCRF-CEM, K-562, RPMI8226, and SR leukemia cell lines, respectively.175 2.4. Miscellaneous Synthetic Cytotoxic Steroids

Several miscellaneous anticancer steroidal derivatives have been synthesized by various research groups with potential cytotoxic activity (Figure 16A−C). Estrogen−daunorubicin conjugate 85 was prepared with an aim to bind specifically to estrogen receptor (ER) for the treatment of receptor positive tumors.176 Yet, it showed negligible binding affinity to the receptor probably because of the substitution of C17 by an imino functionality attached to the daunorubicin moiety.177 Because it is established by structure−activity relationship studies that the presence of the 17β-OH group is essential for high binding affinity of a steroidal estrogen to the ER, a novel daunorubicin-estradiol conjugate 86 was synthesized with a long 17α-linker chain to keep away the −OH function of cytotoxic daunorubicin from the binding site. The cytotoxic activity was assessed against the classical estrogen positive breast cancer cell line MCF-7 as target, but showed no preferential inhibition of the proliferation of the estrogen receptor positive MCF-7 cells in comparison to the estrogen receptor negative MDAMB-231.178 Chalcone derivatives 87 and 88 possessing estradiol framework showed potent activity against MCF-7, a hormone6999


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dependent breast cancer cell line with IC50 = 7.3 and 9.88 μM, resepectively.179 Several research groups have been investigating the combination of a platinum complex to an estrogenic moiety to target the estrogen receptor (ER) in hormone-dependent diseases, particularly breast cancer. The overall goal is to improve the selectivity and efficacy of this type of drug and, more importantly, to minimize its toxic side effects. One such effort was made to synthesize a series of 17β-estradiol-platinum(II) hybrid molecules. The hybrids were made of a PEG (poly(ethylene glycol)) linking chain of various length and a 2-(2′-aminoethyl)pyridine ligand, and the cytotoxic activity was evaluated against breast cancer cell lines MCF-7 and MDA-MB-231. It was observed that the derivative 89 (IC50 = 20.61 ± 0.94 μM against MCF-7 and 13.90 ± 1.87 μM against MDA-MB-231 cell lines) with the longest PEG chain showed the best activity, which was comparable to cisplatin with IC50 = 18.97 ± 0.43 μM against MCF-7 and 17.33 ± 2.28 μM against MDA-MB-231 cell lines.180 More steroidal platinum(II) complexes were synthesized by reaction of potassium tetrachloroplatinate with steroidal esters of L-methionine and L-histidine and evaluated for in vitro cytotoxic activity. Unlike the platinum complex 89, none of these complexes demonstrate any significant activity against MCF-7 cell line. Only compound 90 demonstrated significant cytotoxicity against CEM and RPMI 8226 cell lines (IC50 = 14.5 and 20 μmol/L, respectively) comparable to cisplastin (IC50 = 1.6 and 2.4 μmol/L, respectively). In contrast to cisplatin (IC50 = 5 μmol/L), these new platinum complexes did not demonstrate any cytotoxicity against normal human cells as shown on human fibroblast cells BJ, which means they are nontoxic to normal human cells.181 17-Pyrazolinyl derivative 91 of pregnenolone was found to be active, especially against HT-29 (IC50 = 0.24 μM) and HCT-15 (IC50 = 0.25 μM) cancer cell lines, indicating the high degree of selectivity.182 Steroid-anthraquinone hybrids 92 and 93, with a C-3′ hydroxy group, on evaluation of their antiproliferative activity, were found to possess cytotoxic activities comparable to those of doxorubicin.183 In a series of 2-methoxyestradiol analogues, Δ14-derivative 94 was found to be most potent on MDA-MB-435 (breast carcinoma) and SK-OV-3 (ovarian carcinoma) cell lines. The compound was 23 and 12 times more potent in comparison to the parent 2-methoxyestradiol, a promising drug for cancer therapy, with IC 50 = 0.06 and 0.15 μM, respectively. Furthermore, it was also observed that it exhibits an EC50 of 0.3 μM for microtubule depolymerization in A-10 cells.184 Several vitamin D analogues having a spiro ring in the side chain, spirostanols, and furostanols were prepared from diosgenin, and the cytotoxicity was evaluated on HCT 116 and Hep G2 cells; furostanol derivative 95 was observed to be the most potent compound with IC50 1.3 ± 0.2 and 2.8 ± 0.4 μM, respectively, on these cell lines. On investigating the apoptosissignaling pathway activated by the compound in Hep G2 cell lines using the expression of p53, Bax, p21, and Bcl-2 mRNAs, it was found that the furostanol type derivative 95 overexpressed only Bax mRNA but not the other three mRNAs, which suggests that it is capable of mediating cell death in a p53-independent fashion. As p53 gene plays a role in the development of cisplatin resistance in cancer cells; this result implies that it may be a useful chemotherapeutic agent for the treatment of p53-deficient and/ or p53 mutant hepatoma.185 The diosgenyl saponin dioscin is one of the most common steroidal saponins with potent anticancer activities in several

human cancer cells through apoptosis-inducing pathways. Diosgenyl saponin analogues having β-D-2-amino-2-deoxyglucopyranose residue with different substituents at the amino groups were synthesized and tested against neuroblastoma (SKN-SH) cells, breast cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 96 containing α-lipoic acid residue exhibited the highest potency against all three cancer cell lines with IC50 ranging from 4.8 to 7.3 μM, respectively.186 Synthetic analogues of diosgenyl saponin containing either 2amino-2-deoxy-β-D-glucopyranosyl or α-L-rhamnopyranosyl-(1 → 4)-2-amino-2-deoxy-β-D-glucopyranosyl residue with different acyl substituents on the amino group were evaluated for cytotoxic activity in MCF-7 breast cancer cells and HeLa cervical cancer cells. The disaccharide saponin analogues were less active than their corresponding monosaccharide analogues. The incorporation of aromatic nitro functionality into these saponin analogues does not enhance cytotoxic activity. The monosaccharide derivative 97 was found to have potent cytotoxic activity with IC50 6.9 and 7.3 μM, respectively.187 Similarly, more new synthetic diosgenyl glycosides were evaluated against the human myeloid leukemia (HL-60 and U937) and melanoma cell lines (SK-MEL-1). The derivatives showed strong cell growth inhibition associated with alterations in cell cycle progression and induction of apoptosis. Compound 98 was found to be the most potent glycoside with IC50 values of 7 ± 2, 5 ± 1, and 4 ± 2, respectively, cytochrome c release and caspase-9 activation possibly playing the main role in the intrinsic pathway of apoptosis caused by this kind of compound.188 Imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid were screened for in vitro cytotoxicity against HepG2, Jurkat, and HeLa human cancer cell lines. Compound 99 was particularly found to be the most active with IC50 ≈ 1 μM on various cell lines, although a number of compounds had shown IC50 values lower than 2 μM. Oxidation of the hydroxyl group to a ketone at C3 is thought to influence the cytotoxic potential.189 Sulfated polyhydroxysterols in sodium salt forms are known to exert antitumor activity.190−192 The cytotoxicity of disodium 3β,6β-dihydroxy-5α-cholestane disulfate (100) was evaluated against Sk-Hep-1, H-292, PC-3, and Hey-1B cell lines, and an IC50 ≈ 30 nmol/mL was observed. The results also indicated the essentiality of a cholesterol-type side chain at C17 for the biological activity.193 Another series of 3β,7α,11α-trihydroxypregn-21-benzylidene-5-en-20-one derivatives was found to have remarkable activity against EC109 cells.194 Thiazolo, pyrido, pyrano, and lactam derivatives of 17βhydroxy-5α-androstan-3-one (androstanolone) were evaluated for in vitro cytotoxicity against hepatoma cell and in vivo antitumor activity against Ehrlich ascites carcinoma (EAC). The most potent cytotoxic compound 101 with IC50 = 30 μM showed effectiveness against EAC at 25 mg/kg without any toxic symptoms. It completely inhibited growth of tumor, retaining the hemoglobin content, body weight, and WBCs near normal.195 The cytotoxic activity of the cephalostatin/ritterazine analogues was investigated in vitro against MDA-MB-231, A549, and HT-29 tumor cells. The bis-steroid pyrazine derivative 102 on evaluation showed a GI50 ≈ 2.0 μM on these cell lines.196 Titanocene dichloride is the first metallocene-based organometallic anticancer agent possessing significant antitumor properties in cancer cell lines, insensitive to cisplatin, and produced lower toxic effects than cisplatin. Many titanocene complexes have been synthesized to enhance the cytotoxic activity of titanocene dichloride derivatives. Out of many steroid7000


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proven to be one of the most effective strategies to improve the therapeutic profile. Clinically used estramustine and prednimustine are characteristic examples of such agents; both of the molecules are nitrogen mustard functionalized steroidal derivatives possessing a labile ester linkage. These hybrid molecules are particularly useful in the treatment of hormone responsive cancers such as prostate and breast carcinoma. Implications of a different mode of action of the steroid alkylating agent conjugates suggest their beneficial efficacy in the management of drug-resistance tumors. The mechanism of these hybrid-steroid alkylators as antitumor agents is still ambiguous; probably they act by a multi mechanistic way. The role of the steroidal part is no longer considered simply of a biological carrier, but rather it produces its own effects. Besides, various structural modifications in steroid molecules have rendered them with improved cytotoxicity and have proved to be advantageous in many inoperable malignancies. The promise shown by several nonconjugated steroidal derivatives during preclinical evaluation has indicated toward the vast potential of this privileged scaffold for generating potential cytostatic agents in future. Tissue-selective cytotoxicity, target specificity, and implications in the treatment of inoperable malignancies are some of the major criteria, which advocate the development of steroidal derivatives as safer antineoplastic agents. Hopefully, this Review will prove valuable for the scientists and medicinal chemists working in this area for the successful development of steroidal derivatives as novel therapeutic agents for cancer therapy.

functionalized titanocenyls derivatives, the dehydroepiandrosterone complex 103 was found to be the most potent against MCF7 breast cancer (IC50 = 13 ± 2 μM) and HT-29 (IC50 = 22 ± 1 μM) colon cancer cells, respectively.197 Bile acid conjugates of nitrogenous heterocycles such as piperazinyl-bile acid derivatives were synthesized and tested in vitro against human cancer cells (GBM, KMS-11, HCT-116). The N-[4N-cinnamylpiperazin-1-yl)-3α,7α-dihydroxy-5β-cholan-24-amide (104) was found to have an IC50 ranging from 8.5 to 18.5 μM.198 The cytotoxicity was associated with nuclear and DNA fragmentation, demonstrating induction of cell death by an apoptotic process. These studies revealed the possibility of hybrid heterocyclic-steroids as anticancer agents with improved bioactivity.198 On evaluation of the diastereoisomeric pair of androst-4-en17-spiro-1,3,2-oxathiaphospholane-2-sulfide (105) for cytotoxic activity in vitro against three tumor cell lines (human cervix carcinoma HeLa cells and two human breast carcinoma MDAMB-361 and MDA-MB-453 cells), a moderate activity against HeLa and MDA-MB-453 cell lines was observed.199 In a series of hydroquinone and quinone derivatives of free or peracetylated bile acids, when screened for cytotoxic activity, the hydroquinone 106 displayed promising results against the human pancreatic ductal carcinoma cell line PANC1 (IC50 = 12.1 ± 7.5 μM) with cytotoxic activity similar to that of doxorubicin (IC50 = 20.2 ± 3.3 μM).200 Ether hybrids of phenolic steroid [hydroxy-estra-1,3,5(10)trienes] and a N-alkylpyridinium unit with varied chain length were screened for their antiproliferative and cytotoxic activities. The steroid phenol 107 was found to have remarkable antiproliferative effect against L-929 and K-562 cell lines with GI50 4.7 and 0.9 μg/mL, respectively, while the IC50 against HeLa cell lines was found to be 4.1 μg/mL.201 Another research group synthesized and studied a new series of ring A-modified steroidal and azasteroidal analogues for cytotoxic activity. In the NCI’s single dose prescreening, compounds 108 and 109 displayed prominent activity against HL-60 cell line with IC50 values of 0.7 and 0.03 μM, respectively. On further screening against 60 cell lines, the compounds were found to have profound activity with a mean GI50 value of 0.269 and 0.489 μM, respectively.202 Further, several heterocyclyl such as pyridazino, pyrimido, quinazolo, oxirano, and thiazolo fused derivatives of epiandrosterone and testosterone were synthesized and investigated as anti-breast cancer agents against human breast cancer cells (MCF-7). Compound 110 (IC50 = 2.5 μM) exhibited more inhibitory influence on MCF-7 growth than doxorubicin (IC50 = 4.5 μM). Also, the steroid derivative exhibited significant depletion in gene expression of breast cancer related genes (VEGF, CYP19, and hAP-2c) with various intensities.203

AUTHOR INFORMATION Corresponding Author

*Tel.: +91 172 2541142. Fax: +91 172 2543101. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

3. CONCLUSIONS Cancer is the second most dreadful disease leading to death in the world today according to the WHO. Research efforts directed toward anticancer drug discovery program are tremendous, and researchers have extensively explored various areas ranging from natural sources such as plants, marine, and animals to synthetic compounds for fruitful results. Cytotoxic agents remain the mainstay of cancer therapy despite the availability of a large variety of drugs for the treatment of cancer. The two best known groups belonging to this class are the steroid hormones and the alkylators. Because of high toxicity and low specificity of alkylators, their chemical linkage with a steroidal moiety has

Dr. Ranju Bansal (born February 29, 1968 in Haryana, India), a professor of Pharmaceutical Chemistry at Panjab University Chandigarh, India, was awarded B. Pharm. (1988), M. Pharm. (1990), and Ph.D. (1996) degrees from the same institution. Her doctoral work was focused toward medicinally significant heterosteroids and was carried out under the supervision of her research mentor Prof. D. P. Jindal. She joined as a lecturer of Pharmaceutical Chemistry at University Institute Pharmaceutical Sciences, Panjab University, in 1996 and then elevated to the level of professor. She was awarded the prestigious commonwealth academic staff fellowship (2004−2005) under the supervision of Prof. Alan Harvey at University of Strathclyde, UK, where 7001


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(2) Ferlay, J.; Shin, H. R.; Bray, F.; Forman, D.; Mathers, C.; Parkin, D. M. Int. J. Cancer 2010, 127, 2893. (3) Ahmedin, J.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J. Clin. 2011, 61, 69. (4) Fenton, R. G.; Longo, D. L. In Harrison’s Principles of Internal Medicine; Fauci, A. S., Braunwald, E., Isselbachner, K. J., Wilson, J. D., Martin, J. B., Kasper, D. L., Hauser, S. L., Longo, D. L., Eds.; McGraw Hill: New York, 1998; pp 505−511. (5) Slapak, C. A.; Kufe, D. W. In Harrison’s Principles of Internal Medicine; Fauci, A. S., Braunwald, E., Isselbachner, K. J., Wilson, J. D., Martin, J. B., Kasper, D. L., Hauser, S. L., Longo, D. L., Eds.; McGraw Hill: New York, 1998; pp 523−526. (6) Buzdar, A. U. Clin. Breast Cancer 2004, 5, S4. (7) Swinnen, J. V.; Heemers, H.; van de Sande, T.; de Schrijver, E.; Brusselmans, K.; Heyns, W.; Verhoeven, G. J. Steroid Biochem. Mol. Biol. 2004, 92, 273. (8) Zimmer, H.; Sill, A. D. Arzneim.-Forsch. 1964, 14, 150. (9) Schmidt, C. G. Internist (Berlin) 1971, 12, 119. (10) Goldenberg, D. M. Arzneim.-Forsch. 1962, 12, 457. (11) Takimoto, C. H.; Allegra, C. J. Oncology (Williston Park, N.Y.) 1995, 9, 649. (12) Developmental Therapeutics Program. Tools, Products, and Resources; Division of Cancer Treatment and Diagnosis, National Cancer Institute: Bethesda, MD, 2012; http://www.cancer.gov/ cancertopics/factsheet/NCI/drugdiscovery. (13) Budillon, A.; Caponigro, F. Anti-Cancer Drugs 1999, 10, 249. (14) Kawakami, K. Curr. Gene Ther. 2005, 5, 213. (15) Sanz, L.; Cuesta, A. M.; Compte, M.; Alvarez-Vallina, L. Acta Pharmacol. Sin. 2005, 26, 641. (16) Kubota, T.; Koshizuka, K.; Koike, M.; Uskokovic, M.; Miyoshi, I.; Koeffler, H. P. Cancer Res. 1998, 58, 3370. (17) Frohlich, E.; Machicao, F.; Wahl, R. Endocr.-Relat. Cancer 2005, 12, 291. (18) Kountouras, J.; Zavos, C.; Chatzopoulos, D. J. Surg. Oncol. 2005, 90, 249. (19) Gibbs, J. B. Science 2000, 287, 1969. (20) Saijo, N. Cancer Sci. 2004, 95, 772. (21) Sone, S.; Yano, S.; Nishioka, Y. Gan To Kagaku Ryoho 2004, 31, 1034. (22) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002, 295, 2387. (23) Kwong, M. S.; Bleickardt, E.; Murren, J. R. Oncology (Williston Park, N.Y.) 2002, 16, 33. (24) Blagosklonny, M. V. Cell Cycle 2004, 3, 1035. (25) Chabner, B. A.; Ryan, D. P.; Paz-Ares, L.; Garcia-carbonero, R.; Calabresi, P. In The Pharmacological Basis of Therapeutics; Hardman, J. G., Limbird, L. E., Gilman, A., Eds.; McGraw Hill: New York, 2001; pp 1389−1459. (26) Brown, R. E.; Bostrom, B.; Zhang, P. L. Ann. Clin. Lab. Sci. 2004, 34, 203. (27) Toksöz, H.; Ü nal, S.; Kirazli, S.; Gürgey, A. Leuk. Res. 2004, 28, 533. (28) Drudge-Coates, L. Br. J. Nurs. 2005, 14, 368. (29) Biglia, N.; Sgro, L.; Defabiani, E.; De Rosa, G.; Ponzone, R.; Marenco, D.; Sismondi, P. Eur. J. Surg. Oncol. 2005, 31, 467. (30) Kavanagh, J. J.; Wharton, J. T.; Roberts, W. S. Cancer Treat. Rep. 1987, 71, 537. (31) Ingle, J. N.; Twito, D. I.; Schaid, D. J.; Cullinan, S. A.; Krook, J. E.; Mailliard, J. A.; Marschke, R. F.; Long, H. J.; Gerstner, J. G.; Windschitl, H. E. J. Clin. Oncol. 1988, 6, 825. (32) Berrevoets, C. A.; Umar, A.; Brinkmann, A. O. Mol. Cell. Endocrinol. 2002, 198, 97. (33) Oh, W. K. Clin. Prostate Cancer 2002, 1, 81. (34) Mouridsen, H.; Gershanovich, M. Semin. Oncol. 2003, 30, 33. (35) Crucitta, E.; Fornier, M. N.; Locopo, N.; Silvestris, N.; Lorusso, V.; Lena, M. D. Int. J. Oncol. 2002, 20, 1283. (36) Tindall, D. J.; Rittmaster, R. S. J. Urol. 2008, 179, 1235. (37) Wu, C.; Kapoor, A. Expert Opin. Pharmacother. 2013, 14, 1399. (38) Catsoulacos, P.; Catsoulacos, D. Anticancer Res. 1991, 11, 1773.

she worked toward the development of new xanthine-based antiasthmatic agents. Her main areas of research include design, synthesis, and study of new chemical entities of medicinal significance. She has published over 65 research papers and review articles related to these interests in reputed journals, authored one book, and holds three patents. She is collaborating with many national and international laboratories for the design and development of interesting drug molecules. Eight students have been awarded doctoral degrees under her guidance. She has presented her work at various national and international conferences. Recently, her work on steroidal antileukemic agents received the best paper award at the 63rd Indian Pharmaceutical Congress, Bengaluru, India.

Dr. Pratap Chandra Acharya (born December 31, 1982 in Odisha, India) is currently working as Assistant Professor of Pharmaceutical Chemistry at SPP School of Pharmacy & Technology Management, SVKM’s Narsee Monjee Institute of Management Studies (NMIMS), Mumbai. He was awarded B. Pharm. degree from Berhampur University, Berhampur (2006), and M. Pharm. in Pharmaceutical Chemistry from Banaras Hindu University, Varanasi (2008). After working as a lecturer at Apex Institute of Pharmacy, Sitapura, Jaipur for nearly 1 year, he joined Panjab University, Chandigarh for his Ph.D. and completed it in the year 2013 under the supervision of Prof. Ranju Bansal. He is the recipient of a UGC-RFSMS fellowship (University Grants Commission, New Delhi, India, 2009) and a G.A.T.E. Fellowship (Ministry of Human Resource Development, India, 2006). His current research area includes development of heterosteroids as antineoplastic agents and identification of various pharmacophores responsible for the antineoplastic activity of these steroids. The structure activity relationship studies and determination of possible mode of actions of antineoplastic heterosteroids are also an integral part of his research work. He has published his research work in journals of international repute and presented at various conferences. One of his research presentations was adjudged as the best paper in medicinal chemistry for his work on 16arylidene steroids as antileukemic agents at the 63rd Indian Pharmaceutical Congress, Bengaluru. He is actively involved in some collaborative research projects for the synthesis of radiolabeled anticancer drugs for tumor imaging using positron emission tomography and other nuclear medical imaging techniques.

ACKNOWLEDGMENTS We gratefully acknowledge Prof. Akshay Kumar, Department of English, Panjab University, for help with proofreading of this Review. We thank University Grants Commission, New Delhi, India, for financial support. REFERENCES (1) World Health Organization. The Global Burden of Disease: 2004 Update; World Health Organization: Geneva, 2008. 7002


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