The Novel Mnk1/2 Degrader and Apoptosis Inducer VNLG-152 Potently Inhibits TNBC Tumor Growth and Metastasis.

Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC), an aggressive and highly metastatic disease. Activation of eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1/2) play a critical role in the development, progression and metastasis of TNBC. Herein, we undertook a comprehensive study to evaluate the activity of a first-in-class Mnk1/2 protein degraders, racemic VNLG-152R and its two enantiomers (VNLG-152E1 and VNLG-152E2) in in vitro and in vivo models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader, superior to its pure enantiomers. By targeting Mnk1/2 protein degradation (activity), VNLG-152R potently inhibited both Mnk-eIF4E and mTORC1 signaling pathways and strongly regulated downstream factors involved in cell cycle regulation, apoptosis, pro-inflammatory cytokines/chemokines secretion, epithelial-mesenchymal transition (EMT) and metastasis. Most importantly, orally bioavailable VNLG-152R exhibited remarkable antitumor (91 to 100% growth inhibition) and antimetastatic (~80% inhibition) activities against cell line and patient-derived TNBC xenograft models, with no apparent host toxicity. Collectively, these studies demonstrate that targeting Mnk-eIF4E/mTORC1 signaling with a potent Mnk1/2 degrader, VNLG-152R, is a novel therapeutic strategy that can be developed as monotherapy for the effective treatment of patients with primary/metastatic TNBC.

The retinamide VNLG-152 inhibits f-AR/AR-V7 and MNK-eIF4E signaling pathways to suppress EMT and castration-resistant prostate cancer xenograft growth.

VNLG-152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diverse prostate cancer cells via inhibition of androgen receptor signaling and eukaryotic initiation factor 4E (eIF4E) translational machinery. Herein, we report therapeutic effects of VNLG-152 on castration-resistant prostate cancer (CRPC) growth and metastatic phenotype in a CRPC tumor xenograft model. Administration of VNLG-152 significantly and dose-dependently suppressed the growth of aggressive CWR22Rv1 tumors by 63.4% and 76.3% at 10 and 20 mg·kg-1 bw, respectively (P < 0.0001), vs. vehicle with no host toxicity. Strikingly, the expression of full-length androgen receptor (f-AR)/androgen receptor splice variant-7 (AR-V7), mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1/2), phosphorylated eIF4E and their associated target proteins, including prostate-specific antigen, cyclin D1 and Bcl-2, were strongly decreased in VNLG-152-treated tumors signifying inhibition of f-AR/AR-V7 and MNK-eIF4E signaling in VNLG-152-treated CWR22Rv1 tumors as observed in vitro. VNLG-152 also suppressed the epithelial to mesenchymal transition in CWR22Rv1 tumors as evidenced by repression of N-cadherin, ß-catenin, claudin, Slug, Snail, Twist, vimentin and matrix metalloproteinases (MMP-2 and MMP-9) with upsurge in E-cadherin. These results highlight the promising use of VNLG-152 in CRPC therapy and justify its further development towards clinical trials.

Novel galeterone analogs act independently of AR and AR-V7 for the activation of the unfolded protein response and induction of apoptosis in the CWR22Rv1 prostate cancer cell model.

The androgen receptor (AR) has long been the primary target for the treatment of prostate cancer (PC). Despite continuous efforts to block AR activity through ligand depletion, AR antagonism, AR depletion and combinations thereof, advanced PC tumors remain resilient. Herein, we evaluate two galeterone analogs, VNPT-178 and VNLG-74A, in PC cell models of diverse androgen and AR dependence attempting to delineate their mechanisms of action and potential clinical utility. Employing basic biochemical techniques, we determined that both analogs have improved antiproliferative and anti-AR activities compared to FDA-approved abiraterone and enzalutamide. However, induction of apoptosis in these models is independent of the AR and its truncated variant, AR-V7, and instead likely results from sustained endoplasmic reticulum stress and deregulated calcium homeostasis. Using in silico molecular docking, we predict VNPT-178 and VNLG-74A bind the ATPase domain of BiP/Grp78 and Hsp70-1A with greater affinity than the AR. Disruption of 70 kDa heat shock protein function may be the underlying mechanism of action for these galeterone analogs. Therefore, despite simultaneously antagonizing AR activity, AR and/or AR-V7 expression alone may inadequately predict a patient's response to treatment with VNPT-178 or VNLG-74A. Future studies evaluating the context-specific limitations of these compounds may provide clarity for their clinical application.

VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats.

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

Systematic structure modifications of multitarget prostate cancer drug candidate galeterone to produce novel androgen receptor down-regulating agents as an approach to treatment of advanced prostate cancer.

As part of our program to explore the influence of small structural modifications of our drug candidate 3ß-(hydroxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (galeterone, 5) on the modulation of the androgen receptor (AR), we have prepared and evaluated a series of novel C-3, C-16, and C-17 analogues. Using structure activity analysis, we established that the benzimidazole moiety at C-17 is essential and optimal and also that hydrophilic and heteroaromatic groups at C-3 enhance both antiproliferative (AP) and AR degrading (ARD) activities. The most potent antiproliferative compounds were 3ß-(1H-imidazole-1-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (47), 3-((EZ)-hydroximino)-17-(1H-benzimidazol-1-yl)androsta-4,16-diene (36), and 3ß-(pyridine-4-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (43), with GI50 values of 0.87, 1.91, and 2.57 µM, respectively. Compared to 5, compound 47 was 4- and 8-fold more potent with respect to AP and ARD activities, respectively. Importantly, we also discovered that our compounds, including 5, 36, 43, and 47, could degrade both full-length and truncated ARs in CWR22rv1 human prostate cancer cells. With these activities, they have potential for development as new drugs for the treatment of all forms of prostate cancer.

The combination of the histone deacetylase inhibitor vorinostat and synthetic triterpenoids reduces tumorigenesis in mouse models of cancer.

Novel drugs and drug combinations are needed for the chemoprevention and treatment of cancer. We show that the histone deacetylase inhibitor vorinostat [suberoylanilide hydroxamic acid (SAHA)] and the methyl ester or ethyl amide derivatives of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me and CDDO-Ea, respectively) cooperated to inhibit the de novo synthesis of nitric oxide in RAW 264.7 macrophage-like cells and in primary mouse peritoneal macrophages. Additionally, SAHA enhanced the ability of synthetic triterpenoids to delay formation of estrogen receptor-negative mammary tumors in MMTV-polyoma middle T (PyMT) mice. CDDO-Me (50 mg/kg diet) and SAHA (250 mg/kg diet) each significantly delayed the initial development of tumors by 4 (P < 0.001) and 2 (P < 0.05) weeks, respectively, compared with the control group in the time required to reach 50% tumor incidence. CDDO-Ea (400 mg/kg diet), as a single agent, did not delay tumor development. The combination of either triterpenoid with SAHA was significantly more potent than the individual drugs for delaying tumor development, with a 7 week (P < 0.001) delay before 50% tumor incidence was reached. SAHA, alone and in combination with CDDO-Me, also significantly (P < 0.05) inhibited the infiltration of tumor-associated macrophages into the mammary glands of PyMT mice and levels of the chemokine macrophage colony-stimulating factor in primary PyMT tumor cells. In addition, SAHA and the synthetic triterpenoids cooperated to suppress secreted levels of the pro-angiogenic factor matrix metalloproteinase-9. Similar results were observed in mouse models of pancreatic and lung cancer. At concentrations that were anti-inflammatory, SAHA had no effect on histone acetylation. These studies suggest that both SAHA and triterpenoids effectively delay tumorigenesis, thereby demonstrating a promising, novel drug combination for chemoprevention.

Anti-tumor effects of a novel retinoic acid metabolism blocking agent VN/14-1 in the N-methyl-N-nitrosourea-induced rat mammary carcinoma model and its effects on the uterus.

VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed ß-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.

First chemical feature-based pharmacophore modeling of potent retinoidal retinoic acid metabolism blocking agents (RAMBAs): identification of novel RAMBA scaffolds.

The first three-dimensional (3D) pharmacophore model was developed for potent retinoidal retinoic acid metabolism blocking agents (RAMBAs) with IC(50) values ranging from 0.0009 to 5.84nM. The seven common chemical features in these RAMBAs as deduced by the Catalyst/HipHop program include five hydrophobic groups (hydrophobes), and two hydrogen bond acceptors. Using the pharmacophore model as a 3D search query against NCI and Maybridge conformational Catalyst formatted databases; we retrieved several compounds with different structures (scaffolds) as hits. Twenty-one retrieved hits were tested for RAMBA activity at 100nM concentration. The most potent of these compounds, NCI10308597 and HTS01914 showed inhibitory potencies less (54.7% and 53.2%, respectively, at 100nM) than those of our best previously reported RAMBAs VN/12-1 and VN/14-1 (90% and 86%, respectively, at 100nM). Docking studies using a CYP26A1 homology model revealed that our most potent RAMBAs showed similar binding to the one observed for a series of RAMBAs reported previously by others. Our data shows the potential of our pharmacophore model in identifying structurally diverse and potent RAMBAs. Further refinement of the model and searches of other robust databases is currently in progress with a view to identifying and optimizing new leads.

Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model.

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3ß-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.

4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesulfonate) (NSC 88915) and related novel steroid derivatives as tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo when topoisomerase I (Top1) processes DNA. For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes. Tdp1 inhibitors have been regarded as potential therapeutics in combination with Top1 inhibitors, such as the camptothecin derivatives, topotecan, and irinotecan, which are used to treat human cancers. Using a novel high-throughput screening assay, we have identified the C21-substituted progesterone derivative, NSC 88915 (1), as a potential Tdp1 inhibitor. Secondary screening and cross-reactivity studies with related DNA processing enzymes confirmed that compound 1 possesses specific Tdp1 inhibitory activity. Deconstruction of compound 1 into discrete functional groups reveals that both components are required for inhibition of Tdp1 activity. Moreover, the synthesis of analogues of compound 1 has provided insight into the structural requirements for the inhibition of Tdp1. Surface plasmon resonance shows that compound 1 binds to Tdp1, whereas an inactive analogue fails to interact with the enzyme. On the basis of molecular docking and mechanistic studies, we propose that these compounds are competitive inhibitors, which mimics the oligonucleotide-peptide Tdp1 substrate. These steroid derivatives represent a novel chemotype and provide a new scaffold for developing small molecule inhibitors of Tdp1.

Promise and challenges in drug discovery and development of hybrid anticancer drugs.

BACKGROUND: Because cancer is a complex disease, it is unlikely that a single mono functional 'targeted' drug will be effective for treating this most advanced disease. Combined drugs that impact multiple targets simultaneously are better at controlling complex disease systems, are less prone to drug resistance and are the standard of care in cancer treatment. In order to improve the efficiency of using a two-drug cocktail, one approach involves the use of the so-called hybrid drugs, which comprises the incorporation of two drugs in a single molecule with the intention of exerting dual drug action. OBJECTIVE: In the present article, we discuss the design, synthesis and various applications of anticancer hybrid agents and the developments in this field during the last few decades. Additionally, we describe different types of linkers and their role in contributing towards biological effects and the in vivo mechanism of drug release. We also depict some challenges from scientific and regulatory perspectives in the hybrid drug development process. CONCLUSION: In the era of increasing drug resistance in cancer patients, the discovery of hybrid drugs could provide an effective strategy to create chemical entities likely to be more efficacious and less prone to resistance. However, some technical and regulatory challenges will have to be surmounted before hybrid drugs succeed in the clinical settings and justify the considerable promise of this novel concept.

Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer.

We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm(3) in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer.

Design, synthesis, and evaluation of novel mutual prodrugs (hybrid drugs) of all-trans-retinoic acid and histone deacetylase inhibitors with enhanced anticancer activities in breast and prostate cancer cells in vitro.

Novel mutual prodrugs (MPs) of ATRA (all- trans-retinoic acid) and HDIs (histone deacetylase inhibitors) ( 10, 13, 17- 19) connected via glycine acyloxyalkyl carbamate linker (AC linker) or through a benzyl ester linker (1,6-elimination linker) were rationally designed and synthesized. Most of our novel MPs were potent inhibitors of growth of several hormone-insensitive/drug resistant breast cancer cell lines and the hormone-insensitive PC-3 prostate cancer cell line. The novel MPs exhibited differential antiproliferative potencies in both MDA-MB-231 and PC-3 cell lines. Whereas 19 (VNLG/124) [4-(butanoyloxymethyl)phenyl(2 E,4 E,6 E,8 E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI 50 of 10 nM was the most potent MP versus the MDA-MB-231 cells, 13 (VNLG/66) [{ N-[ N-{2-[4-{[3-pyridylmethoxy)carbonyamino]methyl}phenyl) carbonylamino]phenyl} carbamoylcarbamoyloxy}methyl(2 E,4 E,6 E,8 E)-3,7-dimethyl-9-(2,6,6-trimethyl cyclohex-1-enyl)nona-2,4,6,8-tetraenoate] with a GI 50 = 40 nM was the most potent versus the PC-3 cells. MP 19 exhibited the most benefit because its GI 50 of 10 nM versus MDA-MB-231 cells was remarkably 1085-fold lower than that of parent ATRA and over 100000-fold lower than butyric acid (BA).

Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent.

The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.

Improved synthesis of histone deacetylase inhibitors (HDIs) (MS-275 and CI-994) and inhibitory effects of HDIs alone or in combination with RAMBAs or retinoids on growth of human LNCaP prostate cancer cells and tumor xenografts.

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

First pharmacophore-based identification of androgen receptor down-regulating agents: discovery of potent anti-prostate cancer agents.

A qualitative 3D pharmacophore model (a common feature based model or Catalyst HipHop algorithm) was developed for well-known natural product androgen receptor down-regulating agents (ARDAs). The four common chemical features identified included: one hydrophobic group, one ring aromatic group, and two hydrogen bond acceptors. This model served as a template in virtual screening of the Maybridge and NCI databases that resulted in identification of six new ARDAs (EC(50) values 17.5-212 microM). Five of these molecules strongly inhibited the growth of human prostate LNCaP cells. These novel compounds may be used as leads to develop other novel anti-prostate cancer agents.

A new simple and high-yield synthesis of suberoylanilide hydroxamic acid and its inhibitory effect alone or in combination with retinoids on proliferation of human prostate cancer cells.

We have developed a procedure for the synthesis of N-hydroxy-N(1)-phenyloctanediamide (suberoylanilide hydroxamic acid (SAHA)), providing the product in 79.8% yield. SAHA is a potent inhibitor of histone deacetylase, induces differentiation and/or apoptosis in certain transformed cells in culture, and suppressed the growth of human prostate cancer LNCaP and PC-3 cell lines. The combination of SAHA with other compounds inhibited cell proliferation of LNCaP cells in additive fashion and resulted in synergistic growth inhibition.

Novel C-17-heteroaryl steroidal CYP17 inhibitors/antiandrogens: synthesis, in vitro biological activity, pharmacokinetics, and antitumor activity in the LAPC4 human prostate cancer xenograft model.

New chemical entities, steroidal C-17 benzoazoles (5, 6, 9 and 10) and pyrazines (14 and 15) were rationally designed and synthesized. The key reaction for synthesis of the benzoazoles involved the nucleophilic vinylic "addition-elimination" substitution reaction of 3beta-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) and benzoazole nucleophiles, while that for synthesis of pyrazines involved palladium-catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3beta-ol (13) with tributylstannyl diazines. Some of the compounds were shown to be potent inhibitors of human CYP17 enzyme as well as potent antagonist of both wild type and mutant androgen receptors (AR). The most potent CYP17 inhibitors were 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3beta-hydroxy-17-(5(1)-pyrimidyl)androsta-5,16-diene (15) and 17-(1H-benzimidazole-1-yl)androsta-4,16-dien-3-one (6), with IC(50) values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15 were effective at preventing binding of (3)H-R1881 (methyltrienolone, a stable synthetic androgen) to both the mutant LNCaP AR and the wild-type AR, but with a 2.2- to 5-fold higher binding efficiency to the latter. Compounds 5 and 6 were also shown to be potent pure AR antagonists. The cell growth studies showed that 5 and 6 inhibit the growth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC(50) values in the low micromolar range (i.e., <10 microM). Their inhibitory potencies were comparable to that of casodex but remarkably superior to that of flutamide. The pharmacokinetics of compounds 5 and 6 in mice were investigated. Following s.c. administration of 50 mg/kg of 5 and 6, peak plasma levels of 16.82 and 5.15 ng/mL, respectively, occurred after 30 to 60 min, both compounds were cleared rapidly from plasma (terminal half-lives of 44.17 and 39.93 min, respectively), and neither was detectable at 8 h. Remarkably, compound 5 was rapidly converted into a metabolite tentatively identified as 17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 proved to be very effective at inhibiting the growth of androgen-dependent LAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound 5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P = 0.00065) in the mean final tumor volume compared with controls, and it was also significantly more effective than castration. To our knowledge, this is the first example of an antihormonal agent (an inhibitor of androgen synthesis (CYP17 inhibitor)/antiandrogen) that is significantly more effective than castration in suppression of androgen-dependent prostate tumor growth. In view of these impressive anticancer properties, compound 5 is a strong candidate for development for the treatment of human prostate cancer.

Novel retinoic acid metabolism blocking agents endowed with multiple biological activities are efficient growth inhibitors of human breast and prostate cancer cells in vitro and a human breast tumor xenograft in nude mice.

Novel retinoic acid metabolism blocking agents (RAMBAs) have been synthesized and characterized. The synthetic features include introduction of nucleophilic ligands at C-4 of all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid, and modification of terminal carboxylic acid group. Most of our compounds are powerful inhibitors of hamster liver microsomal ATRA metabolism enzyme(s). The most potent compound is methyl (2E,4E,6E,8E)-9-(3-imidazolyl-2,6,6-trimethylcyclohex-1-enyl)-3,7-dimethylnona-2,4,6,8-tetraenoate (5) with an IC(50) value of 0.009 nM, which is 666,667 times more potent than the well-known RAMBA, liarozole (Liazal, IC(50) = 6000 nM). Quite unexpectedly, there was essentially no difference between the enzyme inhibitory activities of the two enantiomers of compound 5. In MCF-7 cell proliferation assays, the RAMBAs also enhance the ATRA-mediated antiproliferative activity in a concentration dependent manner. The novel atypical RAMBAs, in addition to being highly potent inhibitors of ATRA metabolism in microsomal preparations and in intact human cancer cells (MCF-7, T47D, and LNCaP), also exhibit multiple biological activities, including induction of apoptosis and differentiation, retinoic acid receptor binding, and potent antiproliferative activity on a number of human cancer cells. Following subcutaneous administration to mice bearing human breast MCF-7 tumor xenografts, 6 (VN/14-1, the free carboxylic acid of 5) was well-tolerated and caused significant tumor growth suppression ( approximately 85.2% vs control, p = 0.022). Our RAMBAs represent novel anticancer agents with unique multiple mechanisms of action. The most potent compounds are strong candidates for development as therapeutic agents for the treatment of a variety of cancers.