SAR Based Design of Nicotinamides as a Novel Class of Androgen

Mar 25, 2013 - SAR Based Design of Nicotinamides as a Novel Class of Androgen Receptor Antagonists for Prostate Cancer. Su Hui Yang† ... Citation da...
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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.

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short β-turns arranged in three layers to form an antiparallel “αhelical sandwich”.10,11 Upon agonist binding, the carboxylterminal helix 12 (H12) is repositioned to serve as a lid to stabilize the ligand, and the second β-turn is then formed to lock the conformation of H12 to allow the formation of an 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 In spite of this probable mechanism, chemical modification of either agonists or first generation antagonists of AR has been applicable because of the lack of structural information of AR in the antagonistic mode. This limitation has provided the majority of drug candidates share similar chemical scaffolds.14−16 To address the issue of a possible switch from AR antagonist to agonist induced by structural similarity, we performed receptor-based virtual screening (VS) to identify a totally new chemical scaffold, the nicotinamide DIMN (Figure 1).17 DIMN has been proven to be more potent than the current drug, BIC, and to significantly reduce cell growth of both early and late stage prostate cancer. Moreover, to the best of our knowledge, the nicotinamide DIMN has never been exposed in the literature with any biological activity, and this is the first successful attempt to apply VS for a lead identification by using AR LBD in the agonistic mode. In this study, we present a SAR study of DIMN analogues on AR, the structural optimization of nicotinamides, and their biological evaluation, to suggest that nicotinamides may become a new series of AR antagonists effective for the treatment of advanced prostate cancer.

INTRODUCTION Antiandrogen therapy with first generation androgen receptor (AR) antagonists, such as bicalutamide (BIC), leads to a temporary reduction of prostate cancer with a decrease in the level of serum prostate-specific antigen (PSA), a biomarker of prostate cancer (Figure 1). Unfortunately, cancer cells grow

Figure 1. Chemical structures of AR antagonists, BIC and DIMN.

again in the absence of androgens and progress to castrationresistant prostate cancer (CRPC).1 CRPC is attributed to elevated AR gene expression which can be driven by AR gene amplification,2,3 AR gene mutation,4,5 or ligand-independent AR activation through other factors such as increased expression of transcriptional coactivators.6,7 In addition, the first generation AR antagonists acquire agonistic property in cells engineered to express higher AR amounts. This switch from antagonism to partial agonism is demonstrated by the antiandrogen withdrawal syndrome; the serum concentration of PSA decreases in patients after discontinuation of antiandrogens.8 Besides, the current combination therapy for CRPC involving docetaxel and prednisone increases survival by 2.4 months on average, but it is not curative and causes significant adverse effects.9 AR, by itself, is not transcriptionally active; however, androgen signaling is activated by binding of a natural or synthetic ligand to the active site of AR LBD. It in turn indicates that the pharmacological activity of AR ligands can be determined by the impact of ligand binding on AR. Like other nuclear receptors (e.g., estrogen receptor), AR shares a similar three-dimensional structure containing 11 α-helices and two © 2013 American Chemical Society

Received: September 28, 2012 Published: March 25, 2013 3414

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

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Reagents and conditions: (a) SOCl2, reflux; (a′) SOCl2, cat. DMF, reflux; (b) amine (R2-NH2, 3), Et3N, CH2Cl2, rt; (c) cyclic amine (R1-H, 6), PrOH, reflux; (d) cyclic amine (R1-H, 6), DMSO, 100 °C; (e) MeI, NaH, THF, 60 °C; (f) alkyl halide, K2CO3, DMSO or THF; (g) CH3NO2, AcONH4, AcOH, reflux; (h) LiAlH4, dry THF, reflux; (i) HCHO, 1N HCl, 30% NaOH, rt; (j) MTBE, conc HCl, iPrOH, rt; (k) NaHCO3 or NaOH sol, rt; (l) ClCOOMe, Et3N, dry THF, rt; (m) PPA, 145 °C; (n) SO2Cl2, acetic acid, rt; (o) HBr (48% in H2O), reflux; (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. i

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RESULTS AND DISCUSSION To enable a rational design of AR antagonists, a SAR study of DIMN analogues was performed to identify chemical sites which could be derivatized without a significant loss in activity. Variations of the lead DIMN structure were possible in three parts (Figure 1); the isoquinoline was replaced by heterocyclic amine or substituted isoquinoline, the nicotinamide by terephthalamide, and the pyridylamine by a variety of amines. N-Methylation on the amide bond of nicotinamides was also planned. Such DIMN analogues as nicotinamides or terephthalamides (7, 8) were prepared by a similar method which has been recently reported to be favorable for easy isolation of final products with moderate to good chemical yield,17 and Nmethylation of nicotinamides (9) was carried out by applying a typical procedure (Scheme 1).18 According to the AR antagonistic effect of 31 synthetic DIMN analogues (4b−e, 7ab−as, 7bl−bn, 8a−b, 9ac, 9aj−al) (see Table 1), compounds which were well tolerated or conferred stronger potency compared to DIMN have isoquinoline or 6,7-dimethoxy isoquinoline (7ab, 7ae, 7ao, 7aq, 7as). Replacement of the isoquinoline (A) by saturated monocyclic amine (7bl−bn) led to lower inhibition (max 65.7% inhibition). The activity was further diminished on removal of the amine (4b−e) (max 43.1% inhibition), suggesting a requirement of a bulky hydrophobic moiety (A). Substitution of the nicotinamide core (B) by terephthalamide (8a−b) or by N-methyl nicotinamide (9ac, 9aj−9al) led to a considerable loss in activity (max 48.8% inhibition). It indicates that the nicotinamide scaffold is necessary to retain the potency as it possibly confer the best positioning of amide CO, NH (B) to associate with the receptor by hydrogen bonding. Next, the inhibitory effect was not relied on the substitution pattern of the amine (C). However, in the analysis of the effect of isoquinolinyl-nicotinamides (DIMN, 7ab−an) and 6,7-dime-

thoxyisoquinolinyl-nicotinamides (7ao−7as), 6′-methyl pyridine substitution (C) mostly displayed the strongest antagonistic activity (>89.9% inhibition), probably due to their involvement in hydrogen bonding (Figure 2; see Figure S1 of Supporting Information (SI)). The active analogues are expected to have similar binding mode to DIMN.17 They also have a stretched scaffold linked with the isoquinoline end which can dislocate H12. Considering the development of AR antagonists with a long chain facing H12,19 we envisioned that the introduction of sterically bulky groups on the hydrophobic isoquinoline of nicotinamides would improve AR antagonistic potency. Optimization was further carried out to add large functional groups on the isoquinoline by varying the size, position, or numbers of the substituents. Benzaldehydes as starting materials were prepared; some aldehydes (10c−d, 10j) were commercially available and others (10e−g) were simply synthesized (Scheme 1). Henry reaction on benzaldehydes 10 and a subsequent reduction resulted in phenethylamines 12. Intramolecular cyclization followed by the use of concentrated HCl produced isoquinoline salts 13 which were basified to provide the desired free isoquinolines 6c−g (Scheme 1).20 As another route for intramolecular cyclization, amine 12j was converted into carbamic acid methyl ester 14j in the presence of methyl chloroformate. Further cyclization under acidic condition afforded isoquinolone 15j, which was reduced to give the desired isoquinoline 6j.21 Treatment of isoquinolines 6f−g with sulfuryl chloride produced chlorinated isoquinolines 6h−i. In addition, some isoquinolines with an alkoxy group on C6 were derived from the synthetic isoquinoline salt 13c. Demethylation of 13c followed by Nprotection with a Boc group conferred the phenolic OH, which is susceptible to selective O-alkylation for incorporation of different alkoxy groups. Application of either Mitsunobu 3415

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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 (IC50 = 4.45 μM) (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] 5αDHT, 7au and 7bb were bound to AR with reduced affinity (Figure 4B). On the basis of the apparent equilibrium dissociation constant (Kd) for DHT−AR complex,23 the equilibrium binding constants (Ki), calculated by Cheng− Prusoff equation, are 25.6 and 1.5 μM for 7au and DIMN, respectively. Next, the inhibitory effect of the nicotinamides on the proliferation of human prostate cancer cells was evaluated. In androgen-dependent LNCaP cells, 7au showed a stronger inhibitory effect by suppressing up to 90% while 7bb inhibited the proliferation by 80% which is similar to that of DIMN (Figure 4C). The IC50 values of 7au and 7bb are 0.68 and 1.47 μM, respectively (Figure S2, SI). More interestingly, in androgen-independent C4-2 cells which have similar characteristics with CRPC cells,24,25 growth suppression by 7au with IC50 value of 2.50 μM was more apparent whereas 7bb with IC50 of 11.8 μM has comparable effect with respect to DIMN (Figure 4D and Figure S3, SI). Furthermore, the nicotinamides

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Data from triplicate experiments. AR transcriptional activity was determined as percentage after treatment with 10 μM compounds in the absence (AR agonistic effect) or presence of 10 nM DHT (AR antagonistic effect). N.E., no antagonistic effect.

reaction or an SN2 reaction on the hydroxy isoquinoline 17c and further deprotection of the Boc protecting group produced the desired O-alkylated isoquinoline 6k−m. A total of 19 nicotinamide derivatives (7at−az, 7ba−bk, 9ax) were prepared and examined for their AR agonistic/ antagonistic effects (Table 1). As expected, most nicotinamides bearing alkoxy isoquinoline resulted in comparable or stronger antagonistic activity compared to DIMN whereas they did not show agonistic effect. Particularly, the introduction of an alkoxy 3416

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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 androgendependent LNCaP cell growth, (D) androgen-independent, but ARpositive, C4-2 cell growth, and (E) androgen-independent and ARnegative 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 ARnegative PPC-1 cells, implying their selective inhibitory activity on prostate cancer cells which are AR-positive (Figure 4E). The resultant strong antiandrogenic effect of the nicotinamides can be explained not only by ligand−receptor binding affinity but also by molecular basis for their antagonism. To explore molecular basis of the nicotinamides, the dynamic of AR subcellular distribution was analyzed by green fluorescent protein (GFP) expression. When a natural agonist DHT binds to AR, the DHT−AR complex translocates from the cytoplasm into the nucleus, causing the localization of the liganded AR proteins in the nucleus (Figure 5A). However, treatment of 7au

linear structure of DIMN (left panel) helps the isoquinoline end to face H12, the sterically bulky methoxy substituents in the isoquinoline of 7au (right panel) are far more likely to displace Met895 of H12. Illustrated by the predicted docking results, it is obvious that the nicotinamides containing alkoxy groups in the isoquinoline end locate themselves to be close enough to have a stronger steric clash with Met895 to exhibit potent antagonism.

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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 nicotinamides retaining strong AR antagonism seems to be clarified as the alkoxy substituents of the isoquinoline moiety are well closely packed on the pocket and located in just a short distance, causing a dislocation of Met895. It in turn leads to the impairment in the reposition of mobile carboxyl-terminal H12 and the consequent disruption of AF2 formation. All these findings highlight the nicotinamide nucleus containing alkoxy groups on isoquinoline as an excellent scaffold for a novel class of AR antagonists. The provided SAR data and docking models can offer a useful guidance for designing of pure AR antagonists as effective therapeutic agents for advanced prostate cancer.

Figure 5. Molecular basis of antiandrogenic effect of the nicotinamides (7au and 7bb). (A) Inhibitory effect on the nuclear translocation of GRP-AR, and (B) SRC-1 and SRC-2 mediated enhancement of AR transactivation.

or 7bb in presence of DHT resulted in a distinct distribution pattern in which the liganded AR proteins were instead dispersed throughout the cytoplasm, indicating that they effectively interfere with nuclear translocation of the AR proteins (Figure 5A). Once entering the nucleus, the liganded AR proteins bind to their target gene promoter as a homodimer which can be formed by recruiting AR coactivators. Such coactivators as SRC-1 and SRC-2 have been reported to be elevated in the development of more aggressive prostate cancer.26 Next, the inhibition effect of the nicotinamides on AR transactivation enhanced by overexpression of SRC-1 and SRC-

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ASSOCIATED CONTENT

S Supporting Information *

Preparation and characterization of all synthesized compounds, physical and spectral data, biological assays details and 3417

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molecular docking methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For W.-J.C.: phone, +82-62-530-2933; fax, +82-62-530-2911; E-mail, [email protected]. For K.L.: phone, +82-62-530-0509; fax, +82-62-530-0500; E-mail, [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2011-0015551 and NRF2012R1A2A2A01008388).

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ABBREVIATIONS USED AR, androgen receptor; BIC, bicalutamide; PSA, prostatespecific antigen; CRPC, castration-resistant prostate cancer; AF2, activation function 2; DHT, dihydrotestosterone; MEF, mouse embryonic fibroblast; SRC, steroid receptor coactivator

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