SAR Based Design of Nicotinamides As a Novel Class of Androgen

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SAR Based Design of Nicotinamides as a Novel Class of Androgen Receptor Antagonists for Prostate Cancer † § ‡ § † ‡ † Su Hui Yang, , Chin-Hee Song, , Hue Thi My Van, Eunsook Park, Daulat Bikram Khadka, ‡ ‡ † Eun-Yeung Gong, Keesook Lee,*, and Won-Jea Cho*, † College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwang-ju 500-757, Republic of Korea ‡ Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwang-ju 500-757, Republic of Korea

*S Supporting Information

ABSTRACT: Molecular knowledge of pure antagonism and systematic SAR study offered a direction for structural optimization of DIMN to provide nicotinamides as a novel series of AR antagonists. Nicotinamides with extended linear scaffold bearing sterically bulky alkoxy groups on isoquinoline end were synthesized for H12 displacement. AR binding affinity and molecular basis of antiandrogenic effect establish the optimized derivatives, 7au and 7bb, as promising candidates of second generation AR antagonists for advanced prostate cancer.

■ INTRODUCTION short β-turns arranged in three layers to form an antiparallel “α- ” 10,11 Antiandrogen therapy with first generation androgen receptor helical sandwich . Upon agonist binding, the carboxyl- (AR) antagonists, such as bicalutamide (BIC), leads to a terminal helix 12 (H12) is repositioned to serve as a lid to β temporary reduction of prostate cancer with a decrease in the stabilize the ligand, and the second -turn is then formed to level of serum prostate-specific antigen (PSA), a biomarker of lock the conformation of H12 to allow the formation of an prostate cancer (Figure 1). Unfortunately, cancer cells grow activation function 2 (AF2) site which is essential for recruitment of coactivators, thus controlling transcriptional activities of the receptor.12 On the other hand, an antagonist such as bicalutamide causes a partial unfolding of H12, thereby disrupting the formation of AF2 region in similar pattern as observed in the crystal structure of estrogen receptor (ER) LBD in complex with the selective antagonist raloxifene.10,13 Figure 1. Chemical structures of AR antagonists, BIC and DIMN. In spite of this probable mechanism, chemical modification of either agonists or first generation antagonists of AR has been again in the absence of androgens and progress to castration- applicable because of the lack of structural information of AR in resistant prostate cancer (CRPC).1 CRPC is attributed to the antagonistic mode. This limitation has provided the majority of drug candidates share similar chemical scaf- elevated AR gene expression which can be driven by AR gene 14−16 amplification,2,3 AR gene mutation,4,5 or ligand-independent folds. To address the issue of a possible switch from AR AR activation through other factors such as increased antagonist to agonist induced by structural similarity, we 6,7 performed receptor-based virtual screening (VS) to identify a expression of transcriptional coactivators. In addition, the ff first generation AR antagonists acquire agonistic property in totally new chemical sca old, the nicotinamide DIMN (Figure 1).17 DIMN has been proven to be more potent than the cells engineered to express higher AR amounts. This switch fi from antagonism to partial agonism is demonstrated by the current drug, BIC, and to signi cantly reduce cell growth of antiandrogen withdrawal syndrome; the serum concentration of both early and late stage prostate cancer. Moreover, to the best PSA decreases in patients after discontinuation of antiandro- of our knowledge, the nicotinamide DIMN has never been 8 exposed in the literature with any biological activity, and this is gens. Besides, the current combination therapy for CRPC fi fi involving docetaxel and prednisone increases survival by 2.4 the rst successful attempt to apply VS for a lead identi cation months on average, but it is not curative and causes significant by using AR LBD in the agonistic mode. adverse effects.9 In this study, we present a SAR study of DIMN analogues on AR, by itself, is not transcriptionally active; however, AR, the structural optimization of nicotinamides, and their biological evaluation, to suggest that nicotinamides may androgen signaling is activated by binding of a natural or ff synthetic ligand to the active site of AR LBD. It in turn become a new series of AR antagonists e ective for the indicates that the pharmacological activity of AR ligands can be treatment of advanced prostate cancer. determined by the impact of ligand binding on AR. Like other nuclear receptors (e.g., estrogen receptor), AR shares a similar Received: September 28, 2012 three-dimensional structure containing 11 α-helices and two Published: March 25, 2013

a Scheme 1. Synthesis of DIMN Analogues (7−9) and Synthesis of Substituted Isoquinolines (6c−m)

a fl ′ fl 2 1 Reagents and conditions: (a) SOCl2,re ux; (a ) SOCl2, cat. DMF, re ux; (b) amine (R -NH2, 3), Et3N, CH2Cl2, rt; (c) cyclic amine (R -H, 6), i fl 1 ° ° PrOH, re ux; (d) cyclic amine (R -H, 6), DMSO, 100 C; (e) MeI, NaH, THF, 60 C; (f) alkyl halide, K2CO3, DMSO or THF; (g) CH3NO2, fl fl i AcONH4, AcOH, re ux; (h) LiAlH4, dry THF, re ux; (i) HCHO, 1N HCl, 30% NaOH, rt; (j) MTBE, conc HCl, PrOH, rt; (k) NaHCO3 or NaOH ° fl sol, rt; (l) ClCOOMe, Et3N, dry THF, rt; (m) PPA, 145 C; (n) SO2Cl2, acetic acid, rt; (o) HBr (48% in H2O), re ux; (p) Boc anhydride, Et3N, THF, rt; (q) alcohol, PPh3, DIAD, dry THF, rt; (r) alkyl halide, NaH, DMF, rt; (s) 3 M HCl, NaOH, EtOH, rt. ■ RESULTS AND DISCUSSION thoxyisoquinolinyl-nicotinamides (7ao−7as), 6′-methyl pyr- To enable a rational design of AR antagonists, a SAR study of idine substitution (C) mostly displayed the strongest DIMN analogues was performed to identify chemical sites antagonistic activity (>89.9% inhibition), probably due to which could be derivatized without a significant loss in activity. their involvement in hydrogen bonding (Figure 2; see Figure Variations of the lead DIMN structure were possible in three S1 of Supporting Information (SI)). The active analogues are expected to have similar binding parts (Figure 1); the isoquinoline was replaced by heterocyclic 17 ff amine or substituted isoquinoline, the nicotinamide by mode to DIMN. They also have a stretched sca old linked terephthalamide, and the pyridylamine by a variety of amines. with the isoquinoline end which can dislocate H12. Considering the development of AR antagonists with a long N-Methylation on the amide bond of nicotinamides was also 19 planned. Such DIMN analogues as nicotinamides or tereph- chain facing H12, we envisioned that the introduction of thalamides (7, 8) were prepared by a similar method which has sterically bulky groups on the hydrophobic isoquinoline of been recently reported to be favorable for easy isolation of final nicotinamides would improve AR antagonistic potency. products with moderate to good chemical yield,17 and N- Optimization was further carried out to add large functional methylation of nicotinamides (9) was carried out by applying a groups on the isoquinoline by varying the size, position, or typical procedure (Scheme 1).18 numbers of the substituents. Benzaldehydes as starting − According to the AR antagonistic effect of 31 synthetic materials were prepared; some aldehydes (10c d, 10j) were − DIMN analogues (4b−e, 7ab−as, 7bl−bn, 8a−b, 9ac, 9aj−al) commercially available and others (10e g)weresimply (see Table 1), compounds which were well tolerated or synthesized (Scheme 1). Henry reaction on benzaldehydes 10 conferred stronger potency compared to DIMN have isoquino- and a subsequent reduction resulted in phenethylamines 12. line or 6,7-dimethoxy isoquinoline (7ab, 7ae, 7ao, 7aq, 7as). Intramolecular cyclization followed by the use of concentrated Replacement of the isoquinoline (A) by saturated monocyclic HCl produced isoquinoline salts 13 which were basified to 20 amine (7bl−bn) led to lower inhibition (max 65.7% provide the desired free isoquinolines 6c−g (Scheme 1). inhibition). The activity was further diminished on removal As another route for intramolecular cyclization, amine 12j of the amine (4b−e) (max 43.1% inhibition), suggesting a was converted into carbamic acid methyl ester 14j in the requirement of a bulky hydrophobic moiety (A). Substitution presence of methyl chloroformate. Further cyclization under of the nicotinamide core (B) by terephthalamide (8a−b)orby acidic condition afforded isoquinolone 15j, which was reduced N-methyl nicotinamide (9ac, 9aj−9al) led to a considerable to give the desired isoquinoline 6j.21 Treatment of isoquino- loss in activity (max 48.8% inhibition). It indicates that the lines 6f−g with sulfuryl chloride produced chlorinated nicotinamide scaffold is necessary to retain the potency as it isoquinolines 6h−i. In addition, some isoquinolines with an possibly confer the best positioning of amide CO, NH (B) to alkoxy group on C6 were derived from the synthetic associate with the receptor by hydrogen bonding. Next, the isoquinoline salt 13c. Demethylation of 13c followed by N- inhibitory effect was not relied on the substitution pattern of protection with a Boc group conferred the phenolic OH, which the amine (C). However, in the analysis of the effect of is susceptible to selective O-alkylation for incorporation of isoquinolinyl-nicotinamides (DIMN, 7ab−an) and 6,7-dime- different alkoxy groups. Application of either Mitsunobu

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Table 1. AR Agonist/Antagonist Eﬀect of DIMN Analogues

Figure 2. SAR of DIMN analogues for AR antagonistic eﬀects.

substitution at positions 5 or 6 of the isoquinoline ring (R1) (7au, 7bb−bc, 7bf−bg, 7bk) led to the improved potency with more than 95% inhibition. However, some derivatives which contain a benzyloxy group at position 5 or 6 of the isoquinoline (7bd, 7bj, 9ax) and chlorinated isoquinoline (7ay, 7az) afforded the reduced activity (30.5−86.4% inhibition). These observations suggest that the benzyloxy group or chlorine of the isoquinoline moiety would be too bulky to fit into the ligand binding pocket of AR. On the other hand, the flexible alkoxy chain is presumed to ensure proper binding into the active site and ultimately affect H12 positioning by a strong steric clash with Met895 in closest proximity (Figure S1, SI).13,22 Prior to verifying the probability of nicotinamides as potent AR antagonists, cytotoxicity on growth of mouse embryonic fibroblast (MEF) cells as normal cells was investigated. In particular, among those which inhibited the ligand-induced AR transactivation by more than 95%, two compounds (7au and 7bb) exhibited little or no cytotoxic effect (Figure 3) and were

Figure 3. Cytotoxicity of nicotinamides on MEF cell proliferation. Error bars indicate the mean ± SEM n.s., not signiﬁcant; **, p < 0.01; ***, p < 0.001.

further tested for AR inhibitory activity and binding affinity toward AR. 7au and 7bb showed IC50 values of 0.99 and 0.46 μM, respectively, which are much lower than that of DIMN μ (IC50 = 4.45 M) (Figure 4A); this result is in agreement with their potent inhibitory effect on AR transactivation (Table 1). In a competitive ligand binding assay performed with [3H] 5α- DHT, 7au and 7bb were bound to AR with reduced affinity (Figure 4B). On the basis of the apparent equilibrium − 23 dissociation constant (Kd) for DHT AR complex, the a − Data from triplicate experiments. AR transcriptional activity was equilibrium binding constants (Ki), calculated by Cheng determined as percentage after treatment with 10 μM compounds in Prusoff equation, are 25.6 and 1.5 μM for 7au and DIMN, the absence (AR agonistic effect) or presence of 10 nM DHT (AR respectively. Next, the inhibitory effect of the nicotinamides on antagonistic effect). N.E., no antagonistic effect. the proliferation of human prostate cancer cells was evaluated. In androgen-dependent LNCaP cells, 7au showed a stronger ff reaction or an SN2 reaction on the hydroxy isoquinoline 17c inhibitory e ect by suppressing up to 90% while 7bb inhibited and further deprotection of the Boc protecting group produced the proliferation by 80% which is similar to that of DIMN − the desired O-alkylated isoquinoline 6k m. (Figure 4C). The IC50 values of 7au and 7bb are 0.68 and 1.47 A total of 19 nicotinamide derivatives (7at−az, 7ba−bk, 9ax) μM, respectively (Figure S2, SI). More interestingly, in were prepared and examined for their AR agonistic/ androgen-independent C4-2 cells which have similar character- antagonistic effects (Table 1). As expected, most nicotinamides istics with CRPC cells,24,25 growth suppression by 7au with μ bearing alkoxy isoquinoline resulted in comparable or stronger IC50 value of 2.50 M was more apparent whereas 7bb with μ ff antagonistic activity compared to DIMN whereas they did not IC50 of 11.8 M has comparable e ect with respect to DIMN show agonistic effect. Particularly, the introduction of an alkoxy (Figure 4D and Figure S3, SI). Furthermore, the nicotinamides

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2 was accessed, and, encouragingly, they thoroughly blocked the AR transactivation (Figure 5B). Taken together, these results suggest that the optimized nicotinamides eﬀectively inhibit the AR activation steps, ultimately exhibiting potent antiandrogenic activity. To further elucidate our presumption of antagonism of the nicotinamides toward AR by dispositioning an amino acid residue of H12, a molecular modeling study was performed. According to the docking modes of DIMN and 7au, 7au has the same hydrogen bonding pattern of DIMN but with an additional hydrogen bond with Gln711 (Figure 6). While the

Figure 4. Effects of the optimized nicotinamides in human prostate cancer cells in vitro. (A) Dose-dependent AR antagonistic activity. (B) Dose−response competitive ligand binding curves of [3H] 5α-DHT equilibrium binding to AR. (C) Inhibitory effects on androgen- dependent LNCaP cell growth, (D) androgen-independent, but AR- positive, C4-2 cell growth, and (E) androgen-independent and AR- negative PPC-1 cell growth. IC50 is the concentration of compounds, which inhibits 50% of AR transactivation (A) and radioligand DHT binding to AR (B). Error bars indicate the mean ± SEM n.s., not significant; ***, p < 0.001. Figure 6. Molecular docking modes of DIMN (A) and 7au (B). hardly inhibited the growth of androgen-independent but AR- negative PPC-1 cells, implying their selective inhibitory activity on prostate cancer cells which are AR-positive (Figure 4E). linear structure of DIMN (left panel) helps the isoquinoline The resultant strong antiandrogenic effect of the nicotina- end to face H12, the sterically bulky methoxy substituents in mides can be explained not only by ligand−receptor binding the isoquinoline of 7au (right panel) are far more likely to affinity but also by molecular basis for their antagonism. To displace Met895 of H12. Illustrated by the predicted docking explore molecular basis of the nicotinamides, the dynamic of results, it is obvious that the nicotinamides containing alkoxy AR subcellular distribution was analyzed by green fluorescent groups in the isoquinoline end locate themselves to be close protein (GFP) expression. When a natural agonist DHT binds enough to have a stronger steric clash with Met895 to exhibit to AR, the DHT−AR complex translocates from the cytoplasm potent antagonism. into the nucleus, causing the localization of the liganded AR proteins in the nucleus (Figure 5A). However, treatment of 7au ■ CONCLUSION A novel chemical entity, nicotinamides, has been designed and developed as potent candidates of second generation AR antagonists. On the basis of the lead DIMN structure, an initial SAR exploration could be rationalized as well as the importance of having a nicotinamide core. As expected, the optimized nicotinamides bearing alkoxy substituents at positions 5 or 6 of the isoquinoline led to a significant inhibition in growth of androgen-dependent or androgen-independent prostate cancer cells. This strong antiandrogenic effect of the nicotinamides would be well explained by their AR binding affinity as well as the potent inhibition effect on AR activation steps. Furthermore, encouragingly, the assumption of the nicotina- Figure 5. Molecular basis of antiandrogenic effect of the nicotinamides mides retaining strong AR antagonism seems to be clarified as ff (7au and 7bb). (A) Inhibitory e ect on the nuclear translocation of the alkoxy substituents of the isoquinoline moiety are well GRP-AR, and (B) SRC-1 and SRC-2 mediated enhancement of AR closely packed on the pocket and located in just a short transactivation. distance, causing a dislocation of Met895. It in turn leads to the impairment in the reposition of mobile carboxyl-terminal H12 or 7bb in presence of DHT resulted in a distinct distribution and the consequent disruption of AF2 formation. All these pattern in which the liganded AR proteins were instead findings highlight the nicotinamide nucleus containing alkoxy dispersed throughout the cytoplasm, indicating that they groups on isoquinoline as an excellent scaffold for a novel class effectively interfere with nuclear translocation of the AR of AR antagonists. The provided SAR data and docking models proteins (Figure 5A). Once entering the nucleus, the liganded can offer a useful guidance for designing of pure AR antagonists AR proteins bind to their target gene promoter as a homodimer as effective therapeutic agents for advanced prostate cancer. which can be formed by recruiting AR coactivators. Such coactivators as SRC-1 and SRC-2 have been reported to be ■ ASSOCIATED CONTENT elevated in the development of more aggressive prostate *S Supporting Information cancer.26 Next, the inhibition effect of the nicotinamides on AR Preparation and characterization of all synthesized compounds, transactivation enhanced by overexpression of SRC-1 and SRC- physical and spectral data, biological assays details and

3417 dx.doi.org/10.1021/jm3014103 | J. Med. Chem. 2013, 56, 3414−3418 Journal of Medicinal Chemistry Brief Article molecular docking methods. This material is available free of Schafer, M.; Egner, U.; Carrondo, M. A. Structural evidence for ligand charge via the Internet at http://pubs.acs.org. specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations. J. Biol. Chem. 2000, 275, ■ AUTHOR INFORMATION 26164−26171. (12) Moras, D.; Gronemeyer, H. The nuclear receptor ligand-binding Corresponding Author domain: structure and function. Curr. Opin. Cell Biol. 1998, 10, 384− *For W.-J.C.: phone, +82-62-530-2933; fax, +82-62-530-2911; 391. E-mail, [email protected]. For K.L.: phone, +82-62-530-0509; (13) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T. fax, +82-62-530-0500; E-mail, [email protected]. Structural basis for antagonism and resistance of bicalutamide in − Author Contributions prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 6201 6206. § (14) Kinoyama, I.; Taniguchi, N.; Toyoshima, A.; Nozawa, E.; These authors contributed equally to this work. Kamikubo, T.; Imamura, M.; Matsuhisa, A.; Samizu, K.; Kawanimani, Notes E.; Niimi, T.; Hamada, N.; Koutoku, H.; Furutani, T.; Kudoh, M.; The authors declare no competing financial interest. Okada, M.; Ohta, M.; Tsukamoto, S. (+)-(2R,5S)-4-[4-Cyano-3- (trifluoromethyl)phenyl]-2,5-dimethyl-N-[6-(trifluoromethyl)pyridin- ■ ACKNOWLEDGMENTS 3- yl]piperazine-1-carboxamide (YM580) as an orally potent and peripherally selective nonsteroidal androgen receptor antagonist. J. This research was supported by Basic Science Research Med. Chem. 2006, 49, 716−726. Program through the National Research Foundation of Korea (15) Goto, T.; Ohta, K.; Fujii, S.; Ohta, S.; Endo, Y. Design and (NRF) funded by the Ministry of Education, Science and synthesis of androgen receptor full antagonists bearing a p-carborane Technology (NRF-2011-0015551 and NRF- cage: promising ligands for anti-androgen withdrawal syndrome. J. 2012R1A2A2A01008388). Med. Chem. 2010, 53, 4917−4926. (16) Jung, M. E.; Ouk, S.; Yoo, D.; Sawyers, C. L.; Chen, C.; Tran, ■ ABBREVIATIONS USED C.; Wongvipat, J. Structure−activity relationship for thiohydantoin androgen receptor antagonists for castration-resistant prostate cancer AR, androgen receptor; BIC, bicalutamide; PSA, prostate- (CRPC). J. Med. Chem. 2010, 53, 2779−2796. specific antigen; CRPC, castration-resistant prostate cancer; (17) Song, C. H.; Yang, S. H.; Park, E.; Cho, S. H.; Gong, E. Y.; AF2, activation function 2; DHT, dihydrotestosterone; MEF, Khadka, D. B.; Cho, W. J.; Lee, K. Structure-based virtual screening mouse embryonic fibroblast; SRC, steroid receptor coactivator and identification of a novel androgen receptor antagonist. 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