SAR Based Design of Nicotinamides as a Novel Class of AndrogenReceptor Antagonists for Prostate CancerSu 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 ofKorea‡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 optimizationof DIMN to provide nicotinamides as a novel series of AR antagonists. Nicotinamides with extended linear scaffold bearingsterically bulky alkoxy groups on isoquinoline end were synthesized for H12 displacement. AR binding affinity and molecularbasis of antiandrogenic effect establish the optimized derivatives, 7au and 7bb, as promising candidates of second generation ARantagonists for advanced prostate cancer.

■ INTRODUCTIONAntiandrogen therapy with first generation androgen receptor(AR) antagonists, such as bicalutamide (BIC), leads to atemporary reduction of prostate cancer with a decrease in thelevel of serum prostate-specific antigen (PSA), a biomarker ofprostate cancer (Figure 1). Unfortunately, cancer cells grow

again in the absence of androgens and progress to castration-resistant prostate cancer (CRPC).1 CRPC is attributed toelevated AR gene expression which can be driven by AR geneamplification,2,3 AR gene mutation,4,5 or ligand-independentAR activation through other factors such as increasedexpression of transcriptional coactivators.6,7 In addition, thefirst generation AR antagonists acquire agonistic property incells engineered to express higher AR amounts. This switchfrom antagonism to partial agonism is demonstrated by theantiandrogen withdrawal syndrome; the serum concentration ofPSA decreases in patients after discontinuation of antiandro-gens.8 Besides, the current combination therapy for CRPCinvolving docetaxel and prednisone increases survival by 2.4months on average, but it is not curative and causes significantadverse effects.9

AR, by itself, is not transcriptionally active; however,androgen signaling is activated by binding of a natural orsynthetic ligand to the active site of AR LBD. It in turnindicates that the pharmacological activity of AR ligands can bedetermined by the impact of ligand binding on AR. Like othernuclear receptors (e.g., estrogen receptor), AR shares a similarthree-dimensional structure containing 11 α-helices and two

short β-turns arranged in three layers to form an antiparallel “α-helical sandwich”.10,11 Upon agonist binding, the carboxyl-terminal helix 12 (H12) is repositioned to serve as a lid tostabilize the ligand, and the second β-turn is then formed tolock the conformation of H12 to allow the formation of anactivation function 2 (AF2) site which is essential forrecruitment of coactivators, thus controlling transcriptionalactivities of the receptor.12 On the other hand, an antagonistsuch as bicalutamide causes a partial unfolding of H12, therebydisrupting the formation of AF2 region in similar pattern asobserved 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 modificationof either agonists or first generation antagonists of AR has beenapplicable because of the lack of structural information of AR inthe antagonistic mode. This limitation has provided themajority of drug candidates share similar chemical scaf-folds.14−16 To address the issue of a possible switch from ARantagonist to agonist induced by structural similarity, weperformed receptor-based virtual screening (VS) to identify atotally new chemical scaffold, the nicotinamide DIMN (Figure1).17 DIMN has been proven to be more potent than thecurrent drug, BIC, and to significantly reduce cell growth ofboth early and late stage prostate cancer. Moreover, to the bestof our knowledge, the nicotinamide DIMN has never beenexposed in the literature with any biological activity, and this isthe first successful attempt to apply VS for a lead identificationby 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 theirbiological evaluation, to suggest that nicotinamides maybecome a new series of AR antagonists effective for thetreatment of advanced prostate cancer.

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

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

Brief Article

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■ RESULTS AND DISCUSSION

To enable a rational design of AR antagonists, a SAR study ofDIMN analogues was performed to identify chemical siteswhich could be derivatized without a significant loss in activity.Variations of the lead DIMN structure were possible in threeparts (Figure 1); the isoquinoline was replaced by heterocyclicamine or substituted isoquinoline, the nicotinamide byterephthalamide, and the pyridylamine by a variety of amines.N-Methylation on the amide bond of nicotinamides was alsoplanned. Such DIMN analogues as nicotinamides or tereph-thalamides (7, 8) were prepared by a similar method which hasbeen recently reported to be favorable for easy isolation of finalproducts with moderate to good chemical yield,17 and N-methylation of nicotinamides (9) was carried out by applying atypical procedure (Scheme 1).18

According to the AR antagonistic effect of 31 syntheticDIMN analogues (4b−e, 7ab−as, 7bl−bn, 8a−b, 9ac, 9aj−al)(see Table 1), compounds which were well tolerated orconferred stronger potency compared to DIMN have isoquino-line or 6,7-dimethoxy isoquinoline (7ab, 7ae, 7ao, 7aq, 7as).Replacement of the isoquinoline (A) by saturated monocyclicamine (7bl−bn) led to lower inhibition (max 65.7%inhibition). The activity was further diminished on removalof the amine (4b−e) (max 43.1% inhibition), suggesting arequirement of a bulky hydrophobic moiety (A). Substitutionof the nicotinamide core (B) by terephthalamide (8a−b) or byN-methyl nicotinamide (9ac, 9aj−9al) led to a considerableloss in activity (max 48.8% inhibition). It indicates that thenicotinamide scaffold is necessary to retain the potency as itpossibly confer the best positioning of amide CO, NH (B) toassociate with the receptor by hydrogen bonding. Next, theinhibitory effect was not relied on the substitution pattern ofthe amine (C). However, in the analysis of the effect ofisoquinolinyl-nicotinamides (DIMN, 7ab−an) and 6,7-dime-

thoxyisoquinolinyl-nicotinamides (7ao−7as), 6′-methyl pyr-idine substitution (C) mostly displayed the strongestantagonistic activity (>89.9% inhibition), probably due totheir involvement in hydrogen bonding (Figure 2; see FigureS1 of Supporting Information (SI)).The active analogues are expected to have similar binding

mode to DIMN.17 They also have a stretched scaffold linkedwith the isoquinoline end which can dislocate H12.Considering the development of AR antagonists with a longchain facing H12,19 we envisioned that the introduction ofsterically bulky groups on the hydrophobic isoquinoline ofnicotinamides would improve AR antagonistic potency.Optimization was further carried out to add large functional

groups on the isoquinoline by varying the size, position, ornumbers of the substituents. Benzaldehydes as startingmaterials were prepared; some aldehydes (10c−d, 10j) werecommercially available and others (10e−g) were simplysynthesized (Scheme 1). Henry reaction on benzaldehydes 10and a subsequent reduction resulted in phenethylamines 12.Intramolecular cyclization followed by the use of concentratedHCl produced isoquinoline salts 13 which were basified toprovide the desired free isoquinolines 6c−g (Scheme 1).20

As another route for intramolecular cyclization, amine 12jwas converted into carbamic acid methyl ester 14j in thepresence of methyl chloroformate. Further cyclization underacidic condition afforded isoquinolone 15j, which was reducedto give the desired isoquinoline 6j.21 Treatment of isoquino-lines 6f−g with sulfuryl chloride produced chlorinatedisoquinolines 6h−i. In addition, some isoquinolines with analkoxy group on C6 were derived from the syntheticisoquinoline salt 13c. Demethylation of 13c followed by N-protection with a Boc group conferred the phenolic OH, whichis susceptible to selective O-alkylation for incorporation ofdifferent alkoxy groups. Application of either Mitsunobu

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

aReagents and conditions: (a) SOCl2, reflux; (a′) SOCl2, cat. DMF, reflux; (b) amine (R2-NH2, 3), Et3N, CH2Cl2, rt; (c) cyclic amine (R1-H, 6),

iPrOH, 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 NaOHsol, 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.

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reaction or an SN2 reaction on the hydroxy isoquinoline 17cand further deprotection of the Boc protecting group producedthe 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 nicotinamidesbearing alkoxy isoquinoline resulted in comparable or strongerantagonistic activity compared to DIMN whereas they did notshow agonistic effect. Particularly, the introduction of an alkoxy

substitution at positions 5 or 6 of the isoquinoline ring (R1)(7au, 7bb−bc, 7bf−bg, 7bk) led to the improved potency withmore than 95% inhibition. However, some derivatives whichcontain 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). Theseobservations suggest that the benzyloxy group or chlorine ofthe isoquinoline moiety would be too bulky to fit into theligand binding pocket of AR. On the other hand, the flexiblealkoxy chain is presumed to ensure proper binding into theactive site and ultimately affect H12 positioning by a strongsteric clash with Met895 in closest proximity (Figure S1,SI).13,22

Prior to verifying the probability of nicotinamides as potentAR antagonists, cytotoxicity on growth of mouse embryonicfibroblast (MEF) cells as normal cells was investigated. Inparticular, among those which inhibited the ligand-induced ARtransactivation by more than 95%, two compounds (7au and7bb) exhibited little or no cytotoxic effect (Figure 3) and were

further tested for AR inhibitory activity and binding affinitytoward AR. 7au and 7bb showed IC50 values of 0.99 and 0.46μM, respectively, which are much lower than that of DIMN(IC50 = 4.45 μM) (Figure 4A); this result is in agreement withtheir 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 equilibriumdissociation constant (Kd) for DHT−AR complex,23 theequilibrium 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 onthe proliferation of human prostate cancer cells was evaluated.In androgen-dependent LNCaP cells, 7au showed a strongerinhibitory effect by suppressing up to 90% while 7bb inhibitedthe proliferation by 80% which is similar to that of DIMN(Figure 4C). The IC50 values of 7au and 7bb are 0.68 and 1.47μM, respectively (Figure S2, SI). More interestingly, inandrogen-independent C4-2 cells which have similar character-istics with CRPC cells,24,25 growth suppression by 7au withIC50 value of 2.50 μM was more apparent whereas 7bb withIC50 of 11.8 μM has comparable effect with respect to DIMN(Figure 4D and Figure S3, SI). Furthermore, the nicotinamides

Table 1. AR Agonist/Antagonist Effect of DIMN Analogues

aData from triplicate experiments. AR transcriptional activity wasdetermined as percentage after treatment with 10 μM compounds inthe absence (AR agonistic effect) or presence of 10 nM DHT (ARantagonistic effect). N.E., no antagonistic effect.

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

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.

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hardly inhibited the growth of androgen-independent but AR-negative PPC-1 cells, implying their selective inhibitory activityon prostate cancer cells which are AR-positive (Figure 4E).The resultant strong antiandrogenic effect of the nicotina-

mides can be explained not only by ligand−receptor bindingaffinity but also by molecular basis for their antagonism. Toexplore molecular basis of the nicotinamides, the dynamic ofAR subcellular distribution was analyzed by green fluorescentprotein (GFP) expression. When a natural agonist DHT bindsto AR, the DHT−AR complex translocates from the cytoplasminto the nucleus, causing the localization of the liganded ARproteins in the nucleus (Figure 5A). However, treatment of 7au

or 7bb in presence of DHT resulted in a distinct distributionpattern in which the liganded AR proteins were insteaddispersed throughout the cytoplasm, indicating that theyeffectively interfere with nuclear translocation of the ARproteins (Figure 5A). Once entering the nucleus, the ligandedAR proteins bind to their target gene promoter as a homodimerwhich can be formed by recruiting AR coactivators. Suchcoactivators as SRC-1 and SRC-2 have been reported to beelevated in the development of more aggressive prostatecancer.26 Next, the inhibition effect of the nicotinamides on ARtransactivation enhanced by overexpression of SRC-1 and SRC-

2 was accessed, and, encouragingly, they thoroughly blockedthe AR transactivation (Figure 5B). Taken together, theseresults suggest that the optimized nicotinamides effectivelyinhibit the AR activation steps, ultimately exhibiting potentantiandrogenic activity.To further elucidate our presumption of antagonism of the

nicotinamides toward AR by dispositioning an amino acidresidue of H12, a molecular modeling study was performed.According to the docking modes of DIMN and 7au, 7au hasthe same hydrogen bonding pattern of DIMN but with anadditional hydrogen bond with Gln711 (Figure 6). While the

linear structure of DIMN (left panel) helps the isoquinolineend to face H12, the sterically bulky methoxy substituents inthe isoquinoline of 7au (right panel) are far more likely todisplace Met895 of H12. Illustrated by the predicted dockingresults, it is obvious that the nicotinamides containing alkoxygroups in the isoquinoline end locate themselves to be closeenough to have a stronger steric clash with Met895 to exhibitpotent antagonism.

■ CONCLUSIONA novel chemical entity, nicotinamides, has been designed anddeveloped as potent candidates of second generation ARantagonists. On the basis of the lead DIMN structure, an initialSAR exploration could be rationalized as well as the importanceof having a nicotinamide core. As expected, the optimizednicotinamides bearing alkoxy substituents at positions 5 or 6 ofthe isoquinoline led to a significant inhibition in growth ofandrogen-dependent or androgen-independent prostate cancercells. This strong antiandrogenic effect of the nicotinamideswould be well explained by their AR binding affinity as well asthe potent inhibition effect on AR activation steps.Furthermore, encouragingly, the assumption of the nicotina-mides retaining strong AR antagonism seems to be clarified asthe alkoxy substituents of the isoquinoline moiety are wellclosely packed on the pocket and located in just a shortdistance, causing a dislocation of Met895. It in turn leads to theimpairment in the reposition of mobile carboxyl-terminal H12and the consequent disruption of AF2 formation. All thesefindings highlight the nicotinamide nucleus containing alkoxygroups on isoquinoline as an excellent scaffold for a novel classof AR antagonists. The provided SAR data and docking modelscan offer a useful guidance for designing of pure AR antagonistsas effective therapeutic agents for advanced prostate cancer.

■ ASSOCIATED CONTENT*S Supporting InformationPreparation and characterization of all synthesized compounds,physical and spectral data, biological assays details and

Figure 4. Effects of the optimized nicotinamides in human prostatecancer cells in vitro. (A) Dose-dependent AR antagonistic activity. (B)Dose−response competitive ligand binding curves of [3H] 5α-DHTequilibrium 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 DHTbinding to AR (B). Error bars indicate the mean ± SEM n.s., notsignificant; ***, p < 0.001.

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

Figure 6. Molecular docking modes of DIMN (A) and 7au (B).

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

■ AUTHOR INFORMATIONCorresponding Author*For W.-J.C.: phone, +82-62-530-2933; fax, +82-62-530-2911;E-mail, wjcho@jnu.ac.kr. For K.L.: phone, +82-62-530-0509;fax, +82-62-530-0500; E-mail, klee@jnu.ac.kr.Author Contributions§These authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science andT e c h n o l o g y (NRF - 2 0 1 1 - 0 0 1 5 5 5 1 a n d NRF -2012R1A2A2A01008388).

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

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