Article pubs.acs.org/jmc

A New Avenue toward Androgen Receptor Pan-antagonists: C2 Sterically Hindered Substitution of Hydroxy-propanamides Andrea Guerrini,†,# Anna Tesei,‡,# Claudia Ferroni,† Giulia Paganelli,‡ Alice Zamagni,‡ Silvia Carloni,‡ Marzia Di Donato,§ Gabriella Castoria,§ Carlo Leonetti,∥ Manuela Porru,∥ Michelandrea De Cesare,⊥ Nadia Zaffaroni,⊥ Giovanni Luca Beretta,⊥ Alberto Del Rio,† and Greta Varchi*,† †

Institute for the Organic Synthesis and Photoreactivity, Italian National Research Council, Via Gobetti 101, 40129 Bologna, Italy I.R.S.T., Istituto Scientifico Romagnolo per lo Studio e la cura dei Tumori, Via P. Maroncelli, 40, 47014 Meldola, Forlì, Italy § Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Via L. De Crecchio, 7, 80138 Naples, Italy ∥ Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Via delle Messi d’Oro, 156, 00158 Rome, Italy ⊥ Fondazione IRCCS Istituto Nazionale dei Tumori Milano, Via Amadeo, 42, 20133 Milano, Italy ‡

S Supporting Information *

ABSTRACT: The androgen receptor (AR) represents the primary target for prostate cancer (PC) treatment even when the disease progresses toward androgen-independent (AIPC) or castration-resistant (CRPC) forms. Because small chemical changes in the structure of nonsteroidal AR ligands determine the pharmacological responses of AR, we developed a novel stereoselective synthetic strategy that allows sterically hindered C2-substituted bicalutamide analogues to be obtained. Biological and theoretical evaluations demonstrate that C2substitution with benzyl and phenyl moieties is a new, valuable option toward improving pan-antagonist behavior. Among the synthesized compounds, (R)-16m, when compared to casodex, (R)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward five different prostate cancer cell lines, all representative of CPRC and AIPC typical mutations. Despite being less active than (R)-bicalutamide, (R)-16m also displayed marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR panantagonists.

1. INTRODUCTION Prostate cancer (PC) is the second cause of cancer-related death among the male population of Western society, and androgen-deprivation therapy (ADT) represents a first-line treatment option. In spite of androgen receptor (AR) expression throughout the various stages of PC, ADT frequently fails,1,2 and PC progresses toward the androgenindependent (AIPC) or castration-resistant (CRPC) forms.3,4 Because both AIPC and CRPC express AR, therapies decreasing the level of the receptor reduce tumor growth.5−7 Castration resistance is attributed to the reactivation of AR transcriptional activity, most likely due to the receptor gene amplification8,9 or mutation10−13 as well as AR activation by alternative androgens14,15 or signaling effectors.16−18 Therefore, AR represents the primary target for the treatment of PC. Although steroidal and nonsteroidal AR antagonists are used for the treatment of PC, nonsteroidal antagonists, such as bicalutamide and flutamide (Figure 1), are considered to be more efficacious because of their selective blockade of androgen action and because they present fewer side effects.19−21 Clinical evidence, however, suggests that classical AR antagonists are ineffective for the treatment of advanced PC. Therefore, many © 2014 American Chemical Society

Figure 1. Nonsteroidal androgen receptor antagonists: bicalutamide (1), flutamide (2), enzalutamide (MDV3100) (3), and ARN-509 (4).

efforts have been undertaken to develop new AR antagonists that trigger the death of PC cells either at the early stage of androgen dependence or at the later stage of androgen independence.22 Received: March 31, 2014 Published: August 14, 2014 7263

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Scheme 1. Synthesis of (2S,5R)-7a−n Dioxolanone Bromidesa

Reagents: (a)34 LHMDS, THF, HMPA, RX, −78 to −40 °C, 6h; (b) 2-mercapto pyridine-N-oxide, CBrCl3, DCC, reflux, 2 h; (c)35 pivalaldehyde, H2SO4, pentane, reflux; (d) LHMDS, THF, HMPA, CH2I2, −78 to −30 °C, 2 h. a

2. RESULTS AND DISCUSSION 2.1. Chemistry. Exploiting the Seebach’s “self-regeneration of stereocenters” synthetic principle (SRS),33 we synthesized variously substituted (2S,5S) dioxolanone acids (6a−m) starting from commercially available and inexpensive (S)malic acid, resulting in good yields and diastereoselectivities (>96%).34 Subsequent decarboxylative bromination afforded the corresponding bromides (2S,5R)-7a−m (Scheme 1). On the other hand, compound (2R,5R)-7n, bearing a more sterically demanding phenyl group at the C5 position, was synthesized as homochiral material by acetalization of (R)mandelic acid with pivalaldehyde35 followed by addition of diiodomethane to the corresponding enolate (Scheme 1). The (2S,5R) dioxolanone derivatives 7a−n are versatile starting materials for further functionalization. In particular, the reaction of compounds (2S,5R)-7a,b with hydrochloric acid (6 N) quantitatively afforded the corresponding α-hydroxyl acids (R)-8a,b that were then coupled31,36 with 4-cyano-3-trifluoromethylaniline (9) to afford the corresponding amides (R)10a,b in good yields. The subsequent nucleophilic substitution with 4-fluorothiophenol followed by oxidation of the sulfur to sulfone afforded (R)-12a, namely, (R)-bicalutamide and (R)12b (Scheme 2). Alternatively, a one-pot, two-step reaction of bromides (2S,5R)-7c−i with NaOH in the presence of 4-fluorothiophenol afforded the corresponding α-hydroxyl acids (R)-13c−i in very good yields. Afterward, methyl esters (R)-14c−i were obtained by reaction of (R)-13c−i with a (trimethylsilyl)-

A wide range of literature reports and data evidence that PC can spontaneously acquire androgen receptor gain-of-function mutations rather than simply acquiring mutations that preclude inhibitor binding. This behavior underscores the crucial challenge of overcoming AR resistance mechanisms.23 This is also confirmed by the observation that even very small structural modifications in nonsteroidal AR ligands greatly alter the nature of the receptor−ligand interaction and lead to quite divergent pharmacological responses (i.e., antagonist vs agonist activity). Agonists and antagonists, for instance, differently modulate the interaction of AR with transcriptional machinery by positioning the AR-H12 helix in such a way that it seals (agonists) or opens (antagonists) the AR ligand binding pocket.24−27 Structure−activity relationship (SAR) studies on bicalutamide-like molecules have shown that electron-poor A rings, such as p-CN (or NO2), as well as m-CF3 substitutions are required for a strong ligand−AR binding.24 Moreover, SAR studies on novel derivatives with antagonistic activity on both the wild-type and mutant AR (pan-antagonists) have shown that fine tuning the B ring size28 and properly choosing the moiety connecting the B ring with the bicalutamide backbone, e.g., -SO2- vs -O-,29 pushes the AR-H12 helix and induces an antagonistic conformation even on mutated AR. With the aim of obtaining more effective AR pan-antagonists, we synthesized a novel class of C2-substituted bicalutamide-like molecules by inserting molecular fragments with different steric and electronic properties.30 The available procedures for the synthesis of such derivatives are so far unsuitable. In fact, the challenging coupling reaction with the very electron-poor aniline moiety (Aring-NH2, Figure 1) significantly hampers the possibility of achieving C2-substituted derivatives. To our knowledge, only methyl31 and trifluoromethyl32 groups have been successfully introduced at this position. To this end, we developed a novel, straightforward, and diastereoselective methodology able to afford a large variety of C2-substituted compounds, starting from commercially available and inexpensive materials. Among the novel compounds, we have successfully identified a new structure, (R)-16m, which is effective in vitro against various PC-derived cell lines and shows remarkable in vivo antitumor activity with respect to that of casodex. Collectively, our results indicate that (R)-16m is a promising lead derivative for the development of more potent AR pan-antagonists.

Scheme 2. Synthesis of Derivatives (R)-12a and (R)-12ba

Reagents: (a) 6 N HCl, reflux, 4 h; (b) SOCl2, DMA, from −10 °C to rt, 16 h; (c) NaH, THF, p-F-C6H4SH, rt, 3−5 h; (d) mCPBA, CH2Cl2, rt, 12 h. a

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Scheme 3. Synthesis of Novel Non-steroidal Antiandrogens (R)-16c−Ia

a

Reagents: (a) p-F-C6H4SH, iPrOH/NaOH (1 N), rt, 20 h; (b) Me3SiCHN2 (2 M in Et2O), MeOH/toluene, rt, 1 h; (c) 4-amino-2(trifluoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10 °C to rt, 13 h; (d) mCPBA, CH2Cl2, rt, 12 h.

diazomethane ethereal solution in MeOH/toluene mixed solvent in good isolated yields (Scheme 3). The reaction of esters (R)-14c−i with the lithium salt of aniline 9 at low temperature and in the presence of HMPA provided the corresponding amides (R)-15c−i in good isolated yields. It is worth noting that this latter coupling reaction represents the key synthetic step of the procedure. In fact, unlike all previously reported procedures for the synthesis of bicalutamide-like compounds,31 our methodology allows a large variety of C2-subsituted bicalutamide analogues to be accessed in high yields and stereoselectivities starting from inexpensive building blocks (i.e., (S)-malic acid and (R)-mandelic acid). Finally, oxidation of sulfur derivatives (R)-15c−i with mCPBA afforded the corresponding sulfones (R)-16c−i in quantitative yields (Scheme 3). Whenever milder conditions were required, methyl esters (R)-14 were prepared by an alternative protocol (Scheme 4). First, compounds (2S,5R)-7l−n were reacted with 4fluorothiophenol in DMF and in the presence of K2CO3. The obtained cyclic sulfur compounds (2S,5R)-17l−n were then treated with sodium methylate (1 M) at 0 °C to afford the corresponding methyl esters (R)-14l−n. Derivatives (R)-16l−n were then obtained as described previously for compounds (R)16c−i in very good isolated yields (Scheme 4). Besides studying the role of C2 substitutions, we also wanted to evaluate the influence of the SO2 linker on the antagonistic/ agonistic effect by replacing it with oxygen. In fact, Miller and co-workers demonstrated that the nature of the linker plays a pivotal role in controlling the ultimate antagonistic (agonistic) effect of these kinds of molecules.29,37 To this end, bromides (2S,5R)-7c and (2S,5R)-7e were reacted with 4-cyanophenol in DMF and in the presence of K2CO3. The obtained cyclic compounds (2S,5S)-18c and (2S,5S)-18e were then treated with sodium methylate (1 M) at 0 °C to afford the corresponding methyl esters (S)-19c and (S)-19e that, upon reaction with the lithium salt of aniline 9 at low temperature and in the presence of HMPA, provided the corresponding amides (S)-20c and (S)-20e in very good isolated yields (Scheme 5).

Scheme 4. Synthesis of Non-steroidal Antiandrogens (R)16l−na

a

Reagents: (a) p-F-C6H4SH, K2CO3, DMF, rt, 4 h; (b) MeONa (1 M in MeOH), THF, from 0 °C to rt, 2 h; (c) 4-amino-2(trifluoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10 °C to rt, 13 h; (d) mCPBA, CH2Cl2, rt, 12 h.

The reported synthetic strategy is a combination of dioxolanone chemistry with a novel and efficacious coupling between the methyl ester and the electron-poor aniline 9 lithium salt. This methodology allows the synthesis of numerous novel and enantiomerically pure bicalutamide-like derivatives in high yields. 2.2. In Vitro Biological Activity. 2.2.1. AR-Mediated Transcriptional Activity. We first investigated the ability of the synthesized compounds to interfere with AR-mediated transcriptional activity. Therefore, we established an ARE reporter assay in two cell types, LNCaP and Cos-7 cell lines. LNCaP 7265

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cells have been widely used as a model of androgen-responsive growth38 and are the most representative for our studies. ARnegative Cos-7 cells were transfected with pSG5-hAR plasmid, which allows wild-type human AR (WT hAR) to be expressed. Upon transient transfection, Cos-7 cells express high levels of AR, thus mimicking the scenario of CRPC (Figure S1). This cell model system has been widely used to evaluate the effect of androgens, growth factors, and newly emerging antiandrogen compounds.39−41 As reported in Figures 2 and 3, all compounds exhibited an antagonistic effect on ARE-luc activity in both LNCaP and Cos-7 cells. Importantly, the potency of (R)-16m was higher than or similar to that exerted by (R)bicalutamide and by the second generation antagonist MDV3100 (Figure S2 and S3).42 No cytotoxicity was evidenced for (R)-16c−n derivatives toward either LNCaP and Cos-7 cells, thus indicating that the results obtained in the gene transcription assay were not a

Scheme 5. Synthesis of Non-steroidal AR Ligands (S)-20c and (S)-20ea

a Reagents: (a) p-CN-C6H4OH, K2CO3, DMF, 100 °C, 12 h; (b) MeONa (1 M in MeOH), THF, from 0 °C to rt, 2 h; (c) 4-amino-2(trifluoromethyl)benzonitrile (9), THF, LHMDS, HMPA, from −10 °C to rt, 13 h; (d) mCPBA, CH2Cl2, rt, 12 h.

Figure 2. Effect of hydroxy-propanamide derivatives on AR-mediated gene transcription in LNCaP cells. Cells were co-transfected with ARE-luc 3416 reporter gene and β-galactosidase (β-gal) plasmid and stimulated with 10 nM R1881 in the absence or presence of the indicated compounds for 24 h. After treatment, samples were analyzed for luciferase activity. The results are expressed as fold induction with respect to untreated cells and represent the mean ± SD of three independent experiments. 7266

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Figure 3. Effect of hydroxy-propanamide derivatives on gene transcription in hAR-expressing Cos-7 cells. Cells were co-transfected with pSG5 or pSG5-hAR encoding plasmids together with ARE-luc 3416 and β-galactosidase plasmids, and then stimulated with 10 nM R1881 in the absence or presence of the indicated compounds for 24 h. After treatment, samples were analyzed for luciferase activity. The results are expressed as fold induction with respect to the untreated cells and represent the mean ± SD of three independent experiments.

144 h consecutively. (R)-Bicalutamide was more effective in AR-mutated LNCaP cells (IG50 = 1.8 μM) than in WT ARoverexpressing LNCaP-AR cells. Conversely, MDV3100 showed a similar effect in both cell lines, while reaching an IG50 value only in LNCaP-AR cells (IG50 ∼ 18.9 μM). All of the compounds inhibited cell proliferation at a concentration lower than that of the plasma peak level of casodex in both cell lines (Table S1). In addition, (R)-16f, (R)16g, and (R)-16h exhibited a cytocidal effect at the highest concentrations tested (LC50 values ranging from 16.2 to 19.7 μM) in LNCaP cells only. Conversely, the three compounds showed cell killing activity toward LNCaP-AR cells without reaching LC50 values (Figure 4A and Table S1). To gain further insight into the mechanisms of antitumor activity, an apoptosis assay was performed on LNCaP and LNCaP-AR cells. TUNEL assay showed strong cell death induction in both cell lines, ranging from 91.9 to 99.8%, after 144 h exposure to (R)-16f, (R)-16g, and (R)-16h. Conversely, (R)-16m scantly affected the viability and apoptosis of LNCaP and LNCaP-AR cells (Figures 5 and S7). This behavior underpins the selective AR-mediated action of (R)-16m and as well as its lower extent of off-target activity. The cytotoxicity of (R)-bicalutamide and (R)-16c−n derivatives was evaluated on PC3 and DU145 cells, two ARnegative prostate cancer cell lines derived from bone and brain metastasis, respectively. As expected, (R)-bicalutamide did not produce any cytotoxic effect. In contrast, (R)-16c, (R)-16d, (R)-16f, and (R)-16e derivatives affected cell proliferation of both cell lines at concentrations higher than 2 μM (Figure S8). Furthermore, (R)-16g and (R)-16h displayed remarkable cell

result of reduced viability mediated by drug exposure (Figure S4 and data not shown). Among the tested compounds, (S)-20c and (S)-20e exhibited agonistic activity in Cos-7 cells (Figure S5) compared with the activity of (S)-22, the most advanced and promising nonsteroidal molecule acting as an androgen receptor agonist.43 In agreement with the findings of Miller and co-workers,29 these results confirm the essential role of the linker bulk (i.e., -SO2- vs. -O-) in controlling the antagonist/agonist behavior of nonsteroidal AR ligands. Because PSA is a specific marker of proliferation for PC and is currently used to monitor disease recurrence in patients with advanced PC, we further investigated the variations in PSA secretion in culture medium of treated LNCaP and LNCaP-AR cells (Figure S6). The latter are cells derived from LNCaP that are engineered to stably express high levels of AR, a condition considered representative of clinical AR gene amplification reported in 25−30% of patients with CRPC.7−9,44 With the exception of (R)-12b, a significant inhibition of PSA secretion was observed in the culture medium of both cell lines treated with all of the compounds at a concentration of 20 μM. (R)12b did not affect the PSA level in the culture medium of LNCaP cells, whereas it stimulated PSA secretion in the medium of LNCaP-AR cells, thus confirming the agonist behavior of this derivative. 2.2.2. In Vitro Cytotoxicity. The cytotoxic effect of (R)-16c− n derivatives was evaluated on LNCaP and LNCaP-AR cells. Two references, (R)-bicalutamide and MDV3100, were used. As reported in Figure 4A,B, all of the compounds showed cytotoxic activity toward PC cells exposed to the derivatives for 7267

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Figure 4. Cytotoxic activity of novel hydroxy-propanamide derivatives in human PC (A) LNCaP and (B) LNCaP-AR cells. Twenty-four hours after seeding, cells were exposed to the compounds for 144 h. The cytotoxic potency was measured using SRB assay. (R)-Bicalutamide (e.g., (R)-12a) and MDV3100 were used as references. The mean of three independent experiments is reported.

Quiescent LNCaP cells were incubated with 10 nM [3H]R1881 in the absence or presence of excess of radio-inert compounds. (R)-Bicalutamide was used as a reference drug. As reported for (R)-bicalutamide, (R)-16m displaces the [3H]R1881 binding by about 60 and 65% when used at 1 and 2 μM, respectively (Figure 6A). Similar findings were observed using unlabeled R1881 (see Methods), thus confirming that (R)-16m specifically binds AR. Importantly, (R)-16m did not affect the androgen-induced nuclear import of hAR ectopically expressed in Cos-7 cells, as was also observed when cells were treated with (R)bicalutamide (Figure S10). We then evaluated the effect of (R)-16m on two important biological responses challenged by androgens in LNCaP cells: DNA synthesis (Figure 6B) and cell motility (Figure 6C). As demonstrated by Figure 6B, stimulation of quiescent LNCaP cells with 10 nM R1881 increased BrdU incorporation by 4.5fold in comparison with that of unstimulated cells. (R)-16m almost completely inhibited BrdU incorporation of androgentreated LNCaP cells. Importantly, this effect was similar/ stronger than that obtained using the same concentration range (10−20 μM) of (R)-12a and MDV3100 (Figures 7B and S11). A transwell assay was used to evaluate the inhibitory effects on cell migration mediated by (R)-16m. Stimulation of quiescent LNCaP cells with 10 nM R1881 increased the migration by 3.5fold in comparison with that of unstimulated cells. A complete inhibition of cell migration, similar to that produced by (R)bicalutamide and MDV3100, was observed when stimulated LNCaP cells were treated with (R)-16m (Figure 6C and S12). Lastly, a wound-healing assay was used to evaluate the capability of (R)-16m to impair cell migration. To this end,

Figure 5. Apoptosis induction mediated by hydroxy-propanamide derivatives in LNCaP and LNCaP-AR cells. Twenty-four hours after seeding, cells were treated for 144 h with the compounds (20 μM) and analyzed for apoptotic cell death using TUNEL assay. Data represent the mean ± SD of three independent experiments.

killing activity toward both cells lines. These data indicate a general, modest selectivity toward AR for those compounds, whose cytotoxicity appears to be at least partially ARindependent. Importantly, (R)-16m displayed either no or a modest cytotoxic effect on DU145 and PC3 cell lines, and a superimposable dose−effect curve with that of MDV3100 was observed (Figure S9). On the basis of these findings, (R)-16m, the most promising derivative of the series, was selected for further investigations. Accordingly, we evaluated the ability of (R)-16m to displace the ligand binding activity of AR in whole LNCaP cells. 7268

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Figure 6. Biological characterization of antitumor properties of (R)-16m in LNCaP cells. (A) Displacement of the ligand binding assay by (R)-16m in LNCaP cells. Quiescent LNCaP cells were incubated with 10 nM [3H]R1881 in the absence or presence of excess (from 0.25 to 4 μM) radio-inert (R)-bicalutamide ((R)-bic) or (R)-16m. After 4 h of treatment, intracellular radioactivity was measured. The data represent the mean ± SD of the residual binding expressed as the percent of total AR binding sites of three independent experiments; (B) quiescent LNCaP cells were seeded on coverslips and treated for 18 h with 10 nM R1881 in the absence or presence of (R)-bic or (R)-16m (10 or 20 μM). Cells were then pulsed with BrdU, and the amount of BrdU incorporated was evaluated by immunofluorescence and expressed as the percent of total nuclei. (C) Assay on collagen-coated transwell filters. Twenty four hours after seeding, quiescent LNCaP cells were treated with 10 nM R1881, in the absence or presence of 10 μM (R)-bicalutamide or (R)-16m. Cells were stained with Hoechst and counted using a fluorescent microscope. Data were expressed as the relative increase in the number of migrated cells. (D) After seeding, cells were wounded and then left unstimulated, or stimulated for 24 h with 10 nM R1881, in the absence or presence of 10 μM (R)-16m. Phase contrast images are representative of four different experiments.

Figure 7A,B, (R)-16m markedly inhibited cell growth of LNCaP-Rbic and VCaP cell lines without affecting cell survival, as also confirmed by the poor cell death induction observed after 144 h exposure to the drug (Figure 7C). In comparison to MDV3100 and (R)-bicalutamide, (R)-16m was very effective in inhibiting the proliferation of the castration-resistant cell lines tested, thus confirming its pan-antagonistic behavior. MDV3100 showed superior activity only in VCaP cells, the cell model overexpressing WT AR, whereas (R)-bicalutamide was active toward only the LNCaP cell line (Figure 7 and Table 1). We then sought to investigate whether (R)-16m interferes with the transcriptional activity of AR by detecting PSA levels in LNCaP and LNCaP-Rbic cells in the absence or presence of R1881 (Figure 8). As expected, LNCaP and LNCaP-Rbic cells exposed for 24 h to 10 nM R1881 showed significantly (P < 0.05) increased mRNA levels of PSA (1.5- and 1.7-fold, respectively). Unstimulated cells treated with (R)-16m showed a significant reduction of PSA mRNA. However, in the presence of 10 nM R1881, a decreased ability of (R)-16m to reduce intracellular levels of PSA was evidenced. This behavior likely depends on competition between the two molecules for a common binding site on the androgen receptor. Notably, for high dose exposure, (R)-16m also inhibits R1881-stimulated PSA expression in the hormone-refractory LNCaP-Rbic cell line (Figure 8). In order to compare the transcription inhibitory potency of (R)-16m with that of (R)-bicalutamide, LNCaP and LNCaP-

quiescent LNCaP cells were wounded and allowed to migrate in the absence or presence of 10 nM R1881. Phase contrast images show that the wound area is significantly reduced in cells treated with 10 nM R1881 (Figure 6D). The treatment with (R)-16m consistently inhibits the effect induced by 10 nM R1881. As reported in Figure S12, (R)-16m did not affect cell motility of LNCaP cells stimulated by serum. Additionally, as observed for (R)-bicalutamide and MDV3100, no change in BrdU incorporation was evidenced in AR-negative DU145 cells stimulated with serum and treated with (R)-16m (Figures S13). These findings indicated that (R)-16m specifically targets AR without inducing off-target effects. In order to investigate (R)-16m for possible pan-antagonistic behavior, we tested its cytotoxic activity on PC cell lines resistant to bicalutamide, which are thus representative of CRPC. To this end, LNCaP-Rbic cells, a subline derived from LNCaP and selected in our laboratories,45 which are resistant to (R)-bicalutamide, were used.9 In addition, the compound was tested on CWR22Rv1 cells, a model representative of both androgen responsiveness and androgen insensitivity,46 and on VCaP cells, a cell model mimicking PC progression and metastasis.48 Indeed, VCaP cells derive from a vertebral bone metastasis of a patient affected by CRPC and carry an amplified AR gene locus while displaying a chromosomal rearrangement in which the androgen-regulated gene TMPRSS2 is fused to the oncogenic ERG transcription factor, an alteration found in about half of the cases of prostate cancer.47,48 As reported in 7269

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Figure 7. Cytotoxic (A, B) and apoptotic (C) activity of (R)-16m in LNCaP-Rbic cells and VCaP cells. Twenty-four hours after seeding, cells were exposed to the compound for 144 h. Cell proliferation and apoptosis were evaluated using SRB and TUNEL assays, respectively.

Table 1. Growth Inhibition Mediated by (R)-Bicalutamide, MDV3100, and (R)-16m on a Panel of Hormone-Resistant Prostate Cancer Cellsa IG50 144 h (μM) (R)-Bic MDV3100 (R)-16m

IC50 144 h (μM)

LNCaPb

LNCaP-ARb

LNCaP-Rbicb

VCaPb

CW22Rv1c

1.8 ± 0.06 n.r. 1.6 ± 0.03

n.r. 18.9 ± 1.89 4.9 ± 0.08

n.r. n.r. 6.43 ± 0.26

n.r. 0.61 ± 0.039 1.42 ± 0.063

37.4 ± 4.2 9.7 ± 0.81d 5.0 ± 1.2d

a

Twenty-four hours after seeding, cells were exposed to drug for 144 h. bThe reported values are the IG50 expressed in micromolar; n.r., not reached. The reported values are the IC50 expressed in micromolar. dP < 0.05 (ANOVA) (R)-16m vs MDV3100 and (R)-bicalutamide. Data represent mean values ± SD of three independent experiments. c

Figure 8. Evaluation of mRNA levels of PSA after exposure to (R)-16m in LNCaP (A) or LNCaP-Rbic (B) cell lines. Twenty-four hours after seeding, cells were treated for 24 h with (R)-16m in the presence or absence of R1881. Cells were then collected, and the extracted mRNA was analyzed using RT-PCR. The mean ± SD of two independent experiments is reported (*P < 0.05). NT, not treated.

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Figure 9. Evaluation of PSA mRNA levels after exposure of in LNCaP (A) or LNCaP-Rbic (B) cells to (R)-16m or (R)-bicalutamide. Twenty-four hours after seeding, LNCaP (A) or LNCaP-Rbic (B) cells stimulated with 10 nM R1881 were treated for 24 h with 10 and 20 μM (R)-16m or (R)bicalutamide. Cells were then collected, and the extracted mRNA was analyzed using RT-PCR. The mean ± SD of two independent experiments is reported (*P < 0.05).

Figure 10. In vivo antitumor activity of hydroxy-propanamide derivative (R)-16m against VCaP xenografts. Mice were injected subcutaneously with 106 VCaP cells/mouse, and treatment started when a tumor mass (50−150 mg) was evident (day 35 after tumor cell injection). Mice were treated orally by daily gavage at 10 mg/kg for 4 consecutive weeks with (A) vehicle, (B) casodex, (C) (R)-bicalutamide, and (D) (R)-16m. Five mice per group were evaluated: first mouse, white bar; second mouse, light gray bar; third mouse, dark gray bar; fourth mouse, gray striped bar; and fifth mouse, black striped bar.

Rbic cells were exposed for 24 h at 10 and 20 μM (R)-16m or (R)-bicalutamide (Figure 9). Although the compounds significantly reduced the mRNA level of PSA, (R)-16m did so more efficiently than (R)-bicalutamide (P < 0.05) in both LNCaP and LNCaP-Rbic cells. Collectively, these findings indicate that (R)-16m is very effective, more than that of (R)-bicalutamide, in reducing the transcriptional activity of AR in PC cells. Of note, this effect was evident also in (R)-bicalutamide-resistant cell lines. 2.3. In Vivo Biological Activity. VCaP cells were selected to perform in vivo studies on (R)-16m because they better represent the state of androgen signaling most commonly observed in CRPC.47,48 VCaP cells were subcutaneously injected in the flank of 5 to 6-week old CD-1 male nude (nu/nu) mice. When the tumor mass was evident (50−100 mg), mice were randomized in groups of 5 and treated with the

compounds (10 mg/kg for 4 consecutive weeks). As shown in Figure 10, all the mice treated with vehicle showed progressive tumor growth (mean tumor weight of about 2500 mg at day 100). A marked increase of tumor mass in two out of five mice treated with casodex was evidenced, whereas disease stabilization was elicited in the remaining three mice. Three out of five mice treated with (R)-bicalutamide evidenced a complete response, whereas the remaining two mice showed disease stabilization at day 50 after cell injection. The efficacy of (R)bicalutamide further improved, with a complete response registered in all of the treated animals at day 100 after tumor cell implantation. No signs of tumor relapse were observed. Although less effective than (R)-bicalutamide, a marked antitumor activity, superior to that observed in mice treated with casodex, was evidenced in animals treated with (R)-16m. The stabilization of the disease was observed in two out of five 7271

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toward a hydrophobic pocket, called the α7 pocket, formed by helix α7 and β1. Interestingly, induced fit docking (IFD) calculations suggest a binding pose for the T877A and W741L mutants that differs from that of the WT (Figure S15), where the rings are interchanged, so the C ring is replaced by the B ring to bind an adjacent hydrophobic pocket protruding toward α11 (Figures 11, 12, and S15). Remarkably, this α11 pocket has been recently described by Sawyers et al. as a putative region for the binding of new spirocyclic derivatives of MDV3100 that also improve the biological profiles of mutated AR, in particular the F876L mutation.23 To gain additional insight into how (R)-16m can overcome resistance toward AR-mutated cells, mediated by the movement of helix α12, we modeled this compound by performing molecular dynamics (MD) simulations.29 Probing the initial displacement of helix α12 by means of MD simulations has already been described as a useful way to elucidate the mechanism of AR antagonism23,49 (Figure S14A,B). In our simulations, we aimed to understand whether the C2 substitution constitutes a valuable scaffold optimization to induce AR antagonism in WT and in somatic mutations that confer resistance to current antiandrogen treatments. The analysis of the trajectories shows that the ligand and the protein backbones maintain a stable conformation during MD simulations (Figure 13, blue line). On the other hand, the amino acidic residues corresponding to helix α12 undergo sensible movements (Figure 13, red line). These occur in all cases after approximately 20 ns of simulation, indicating that (R)-16m is able to exert its antagonist activity not only toward the WT AR but also in the mutated forms, T877A and W741L. Not surprisingly, the loop connecting helices α11 and α12 (Figure 13, green line) showed a high degree of flexibility as fluctuations occurs during the entire trajectory.

mice treated with the compound (day 60). A complete regression (no palpable tumor) was observed in three mice, two of which were considered definitively cured at the end of the experiment. Finally, the treatment with (R)-16m was welltolerated, as no body weight loss was observed during or after the end of treatment. 2.4. Molecular Modeling. The capacity of AR ligands to act as agonists or antagonists has been ascribed to their capacity to position helix α12, one of the 12 helices contained in the secondary structure of the ligand binding domain (LBD), in either a closed (agonists) or an open (antagonists) conformation in the ligand binding pocket (Figure S14).29,49 Small chemical changes in AR ligands have been reported to underline different the pharmacological responses of WT AR and its mutated forms.49 Thus, in order to better elucidate the mechanism of (R)-16m antagonism at molecular level, we performed molecular modeling calculations and compared them to that of (R)-bicalutamide. Because structural data of AR in its antagonist conformation is not yet available, in silico techniques have been used to derive useful molecular information.23,49,50 In particular, homology modeling was used to derive AR models (Figure S14B) starting from the structure of the glucocorticoid receptor51 and progesterone receptor that were recently exploited to search for novel AR antagonists with structure-based drug design techniques.52 Nevertheless, as pointed out by other groups, difficulties may arise when accounting for subtle structural differences in the antagonist conformation of the LBD when using structures of other nuclear receptors as the template.29 Several somatic mutations confer specific conformational properties to the α12 helix that are believed to ultimately underlie the pharmacological resistance to current antiandrogen treatments. Because modeling the conformational transition from agonist to antagonist forms can be a computationally expensive task and in order to focus on the above-mentioned mutation to gain a clearer picture of the binding mechanism of our C2-substituted ligands, we used structures of the agonist AR and probed their fitness in the LBD and toward the initial transition to the antagonist conformation. In particular, we compared WT AR and two somatic mutations associated with CRPC, T877A and W741L, to carry out modeling simulations. To initially highlight whether the C2 substitution creates a hindrance in the LBD of the agonist AR that would account for the observed biological properties, we performed a rigid docking simulation. Interestingly, the rigid docking failed to provide a binding pose for (R)-16m in all three structures, and the analysis obtained by overlaying the scaffold of this compound with (R)-bicalutamide revealed that the side chain of Met787 hindered the putative location of the 4-cyano-benzyl C ring (Figure 11). The adjustment of the side chain of Met787 allowed binding pose for (R)-16m to be obtained in all structures, as shown in Figure 12A. Rings A and B are accommodated in two binding regions, consistent with that mediated by (R)-bicalutamide, whereas the C ring stretches

3. CONCLUSIONS This study outlines the synthesis and biological in vitro evaluation of a novel class of bicalutamide-like molecules as androgen receptor pan-antagonists. On the basis of the consideration that small chemical changes in the structure of nonsteroidal AR ligands change the molecular mechanisms underling the pharmacological responses of AR and its mutated forms, we developed a novel stereoselective synthetic strategy that allows a wide range of sterically hindered C2-substituted bicalutamide analogues to be obtained starting from inexpensive and commercially available compounds (i.e., (S)-malic acid and (R)-mandelic acid). Taking into account the peculiar behavior of the androgen receptor, which is able to rapidly circumvent the effect of new therapies, the straightforward, diastereoselective,and high yielding synthetic protocol outlined herein might represent an important advance in the field of nonsteroidal pan-antagonists. Collectively, the presented biological data demonstrate that the newly explored substitution of the bicalutamide C2-methyl group with more demanding benzyl and phenyl moieties can be considered a new option toward the modulation and improvement of the agonist/ antagonist behavior of this class of molecules. Moreover, our results confirm the pivotal role of the linker group (i.e., SO2 vs O) in determining AR antagonistic/agonistic activity. Overall, compound (R)-16m proved to be the most promising compound of the series, being active toward five different cell lines, including those bearing CPRC and AIPC typical mutations, while being scarcely active toward androgenindependent cell lines, e.g., PC3 and DU145 cells. Although

Figure 11. (R)-16m structure. 7272

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Figure 12. (A) Binding modes of (R)-bicalutamide and (R)-16m in WT AR and its W741L and T877A mutants. Poses of binding of (R)bicalutamide were taken from the crystallographic structures, whereas poses of (R)-16m were obtained from molecular docking simulations. The binding modes of (R)-16m show a remarkable overlay with the scaffold of (R)-bicalutamide, indicating that the benzyl substitution in C2 suitably fits the LBD of AR WT and its mutated forms. (B) Magnified pose of the (R)-16m in the LBD of the WT androgen receptor. The C ring resulting from the C2 substitution stretches out to a hydrophobic cavity between α7 and β1. This α7 pocket is predicted to be a valuable binding region of AR to conceive further ligands that overcome CRPC.

Figure 13. Molecular dynamics simulation of AR in complex with compound (R)-16m (WT, left; T877A, middle; W741L, right). Starting structures were generated from the agonist conformation and the molecular docking poses of (R)-16m. Blue lines correspond to the root-mean-squared deviation (RMSD) of the backbone, red lines, to the movement of helix α12 (residues 893−907), and green lines, to the movement of the loop bridging the helices α11 and α12 (residues 885−892). After approximately 20 ns of simulation, the ligand induce a fluctuation of helix α12 in all cases, indicating the capability of the compound to act as an antagonist of AR. peak, δ = 77.0 ppm), CD3OD (δ = 49.0 ppm), etc. as the internal standard. Optical rotation was detected on an ADP220 automatic polarimeter from Bellingham & Stanley Limited. Mass spectra were measured in positive mode electrospray ionization (ESI). ESI-HRMS were acquired on an Agilent Dual ESI Q TOF 6520, in positive-ion mode, using methanol. TLC was performed on silica gel 60 F254 plastic sheets. Column chromatography was performed using silica gel (35−75 mesh). Purity of prepared compounds was determined by HPLC-UV analysis (Waters 600 HPLC connected with photodiode array detector 996). All compounds tested in biological assays were >95% pure. Purity of intermediates was >90%, unless otherwise stated. 4.2. Synthetic Procedures. 4.2.1. General Procedure for the Synthesis of (2S,5R)-5-(Bromomethyl)-2-(tert-butyl)-5-substituted1,3-dioxolan-4-one 7a−m. A solution of dicyclohexylcarbodiimide (DCC) in CBrCl3 (2.4 equiv) was added to a suspension of (2S,5R)-6 (1 equiv) and 2-mercapto pyridine-N-oxide (1.4 equiv) in CBrCl3 kept under reflux. The addition was performed over 30 min; afterward, the reaction mixture was stirred under reflux for 2 h. After cooling, the solvent was removed under reduce pressure, and the crude material was purified by silica gel column chromatography. Characterization exemplified by (2S,5R)-7m: eluent: cHex/Et2O, 7:2; yield = 88%; 1 [α]20 D +39 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 7.64−7.62

less effective than (R)-bicalutamide, (R)-16m exhibited a marked antitumor activity, superior to that observed when mice were treated with casodex. Collectively, the results of induced fit docking suggest that the large hydrophobic region comprising the α7 and α11 pockets is highly relevant for the binding of ligands to WT AR and its mutated forms and that C2-substituted molecules may represent a viable strategy toward the optimization of novel antiandrogens able to circumvent resistance to CRPC.

4. EXPERIMENTAL SECTION 4.1. General Chemistry Methods. All solvents and reagents were used as obtained from commercial sources unless otherwise indicated. All reactions were performed under a nitrogen atmosphere unless otherwise stated. The 1H and 13C NMR spectra were recorded on a Varian spectrometer operating at 400 MHz for 1H and 100 MHz for 13 C. Deuterated chloroform was used as the solvent for NMR experiments, unless otherwise stated. 1H chemical shifts values (δ) are referenced to the residual nondeuterated components of the NMR solvents (δ = 7.26 ppm for CHCl3 and δ = 3.31 ppm for CHD2OD, etc.). The 13C chemical shifts (δ) are referenced to CDCl3 (central 7273

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134.0, 128.7, 121.8, 117.3 (q, J = 5 Hz), 116.4 (d, J = 22.1 Hz), 115.7, 104.7, 78.2, 45.1, 32.7, 8.0 HRMS (m/z) calcd for C19H16F4N2O2S [M]+, 412.0869; found, 412.0867. 4.2.6. General Procedure for the Synthesis of (R)-12a,31b. Three equivalents of m-chloro perbenzoic acid (mCPBA) were added to a solution of (R)-11a in CH2Cl2 (0.1 mM). The reaction mixture was stirred at room temperature for 12 h. The solution was then diluted with EtOAc, and the organic layer was washed with aqueous Na2SO3 followed by saturated NaHCO3. The crude compound was purified by silica gel column chromatography or by crystallization. (R)-12b: yield, 1 90%; [α]20 D −57 (c 0.4, EtOH; mp 98−103 °C); H NMR (400 MHz, CDCl3) δ 9.06 (br s, 1H), 7.97 (s, 1H), 7.85−7.91 (m, 2H), 7.90−7.87 (m, 2H), 7.79−7.77 (m, 2H), 7.16 (t, J = 8.0 Hz, 2H), 4.94 (s, 1H), 3.95 (d, J = 14.4 Hz, 1H), 3.45 (d, J = 14.4 Hz, 1H), 1.93−1.83 (m, 2H), 0.95 (t, 3H, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ 171.2, 166.5 (d, J = 257 Hz), 141.1, 136.1, 135.2, 134.8 (q, J = 35 Hz) 131.1(d, J = 37.2 Hz), 122.0, 121.9, 117.3 (q, J = 5 Hz), 117.0 (d, J = 22.1 Hz), 115.5, 104.8, 77.1, 60.8, 33.9, 7.5. HRMS (m/z) calcd for C19H16F4N2O4S [M]+, 444.0767; found, 444.0764. 4.2.7. General Procedure for the Synthesis of (R)-13c−i. A solution of (2S,5R)-7 (1 equiv) in a 1:1 iPrOH (1 N)/NaOH mixed solvent was stirred at room temperature for 3 h; then, 4fluorobenzenethiol (1.6 equiv) was added dropwise. The reaction mixture was additionally stirred at room temperature for 16 h. The solution was then treated with 1 M HCl (until pH = 8) and extracted twice with CH2 Cl 2 . The pure compound was obtained by crystallization (CHCl3/petrol ether). Characterization exemplified by (R)-13e: yield, 98%; 1H NMR (400 MHz, CDCl3) δ 7.45−7.42 (m, 2H), 7.20 (d, J = 5.2 Hz, 1H), 7.0−6.93 (m, 3H), 6.86 (d, J = 3.6 Hz, 1H), 3.40 (d, J = 14.0 Hz, 1H), 3.36 (d, J = 14.8 Hz, 1H), 3.27 (d, J = 14.0 Hz, 1H), 3.23 (d, J = 14.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 177.7, (163.7, 161.3; d), 135.8, (134.0, 133.9; d), (130.5, 130.4; d), 127.9, 127.0, 125.7, 116.4 (d, J = 20 Hz), 78.3, 44.5, 39.2. HRMS (m/z) calcd for C14H13FO3S2 [M]+, 312.0290; found, 312.0294. 4.2.8. General Procedure for the Synthesis of (R)-14c−i. To a solution of (R)-13 (1 equiv) in MeOH/toluene mixed solvent (1:1, 1 mL/mmol) was added Me3SiCHN2 (2 M in Et2O; 1.5 equiv) dropwise at room temperature. The reaction mixture was then stirred for 1 h and then carefully quenched with acetic acid and extracted with EtOAc. The crude material was purified by silica gel column chromatography. Characterization exemplified by (R)-14f: yield, 87%; 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 7.6 Hz, 2H), 7.43−7.40 (m, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.98 (t, J = 8.8 Hz, 2H), 3.51 (s, 3H), 3.40 (d, J = 13.6 Hz, 1H), 3.21 (d, J = 13.6 Hz, 1H), 3.10 (d, J = 13.6 Hz, 1H), 3.04 (d, J = 13.6 Hz, 1H). 13C NMR (100 MHz,CDCl3) δ 173.9, 162.4 (d, J = 20 Hz), 139.4, 134.0 (d, J = 20 Hz), 130.6, 129.6 (d, J = 20 Hz), 125.4 (d, J = 20 Hz), 116.2 (d, J = 20 Hz), 78.3, 52.9, 45.4, 44.5. HRMS (m/z) calcd for C18H16F4O3S [M]+, 388.0756; found, 388.0759. 4.2.9. General Procedure for the Synthesis of (2S,5R)-17l−n. To a solution of (2S,5R)-7 (1 equiv) in dry dimethylformamide (DMF) (6.5 mL/mmol), was added 2.2 equiv of dry K2CO3 followed by 2.0 equiv of 4-fluorobenzenethiol. The reaction mixture was then stirred at room temperature for 3 to 4 h. Reaction completion was followed by TLC using a cerium−molybdenum solution as stain and treated with distilled water when the starting material disappeared. The organic phase was extracted with EtOAc, and the crude material was purified by silica gel column chromatography. Characterization exemplified by (2S,5R)-17m: yield, 79%; 1H NMR (400 MHz, CDCl3) δ 7.61−7.58 (m, 2H), 7.45−7.41 (m, 2H), 7.37−7.34 (m, 2H), 6.99 (t, J = 8.4 Hz, 2H), 3.35 (d, J = 13.6 Hz, 1H), 3.28 (d, J = 14.0 Hz, 1H), 3.19 (d, J = 14.0 Hz, 1H), 3.16 (d, J = 14.0 Hz, 1H), 0.79 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 173.2, 163.8, 161.3, 139.9, 134.1 (d, J = 20 Hz), 132.7, 131.4, 130.9, 118.7, 116.5, 111.9, 109.6, 83.6, 43.0, 40.4, 34.7, 23.5. HRMS (m/z) calcd for C22H22FNO3S [M]+, 399.1304; found, 399.1301. 4.2.10. General Procedure for the Synthesis of Methyl Esters (R)14l−n. To a solution of (2S,5R)-17 (1 equiv) in dry THF (7 mL/ mmol) was added 1.8 equiv of sodium methylate (1 M) dropwise at 0

(m, 2H), 7.40−7.38 (m, 2H), 4.50 (s, 1H), 3.60−3.50 (m, 2H), 3.29− 3.19 (m, 2H), 0.92 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.5, 139.3, 132.7, 131.4, 118.6, 112.1, 109.5, 82.0, 40.2, 34.9, 34.4, 23.7. HRMS (m/z) calcd for C16H18BrNO3 [M]+, 351.0470; found, 351.0467. 4.2.2. Synthesis of (2S,5R)-5-(Iodomethyl)-2-(tert-butyl)-5-phenyl1,3-dioxolan-4-one 7n. To a solution of (2R,5R)-2-tert-butyl-5phenyl-1,3-dioxolan-4-one 553 (1 equiv) in THF (0.6M), cooled at −78 °C, was added LHMDS (1.5 equiv) dropwise. The reaction mixture was stirred at this temperature for 30 min, and then a solution of HMPA/THF (0.4 mL, 1.8:1) was added via syringe followed by a solution of CH2I2 (3.3 equiv) in THF (3 mL/mmol). The temperature was allowed to rise to −30 °C over 2 h, and the mixture was quenched with a saturated solution of NH4Cl and extracted with Et2O. Silica gel column chromatography of the crude material (eluent: cHex/Et2O, 7:2) afforded the pure compound as sticky oil in 70% yield. [α]20 D +33 (c 0.2, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.70−7.68 (m, 2H), 7.44−7.38 (m, 3H), 4.92 (s, 1H), 3.61 (d, J = 11.6 Hz, 1H), 3.48 (d, J = 11.6 Hz, 1H), 0.97 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.2, 161.2, 133.6, 128.5, 128.4, 81.5, 34.9, 33.2, 32.2, 23.8. HRMS (m/z) calcd for C14H17IO3 [M]+, 360.0222; found, 360.0226. 4.2.3. General Procedure for the Synthesis of (R)-8a,54b. Bromide (2S,5R)-7a was dissolved in a large excess of 6 N HCl (6 equiv) and refluxed for 4 h. The reaction mixture was then cooled at room temperature, treated with brine, and extracted with ethyl acetate (EtOAc). The organic layer was then washed with a saturated solution of NaHCO3, and the aqueous solution was acidified with HCl (pH 2) and extracted with EtOAc. The compound was processed to the next step without any further purification. The procedure used for the synthesis of derivatives (R)-8a and (R)-8b can be, in principle, applied to all other derivatives. (R)-8b: yield, 81%; [α]20 D −18.2 (c 0.45, EtOH; mp 107−111 °C); 1H NMR (400 MHz, CDCl3) δ 3.74 (d, J = 10.4 Hz, 1H), 3.52 (d, J = 10.4 Hz, 1H), 1.94−1.90 (m, 1H), 1.84−1.77 (m, 1H), 1,03 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 174.4, 89.2, 40.1, 32.4, 7.1. HRMS (m/z) calcd for C5H9BrO3 [M]+, 195.9735; found, 195.9738. 4.2.4. General Procedure for the Synthesis of (R)-10a,55b. To a solution of (R)-8a in DMA (dimethylacetamide) (0.55 M) cooled at −10 °C was added 1.3 equiv of SOCl2 dropwise under a nitrogen atmosphere. The solution was stirred at this temperature for 3 h, and then a solution of 4-amino-2-(trifluoromethyl)benzonitrile (9) in DMA (1.2 equiv of amine in 1.5 mL of DMA) was added dropwise. The reaction mixture was then allowed to react at room temperature for 16 h. The solvent was then removed under reduced pressure, and the crude material was treated with a saturated solution of NaHCO3 and extracted with EtOAc. The reaction crude was purified by silica gel column chromatography. Yield, 84%. (R)-10b: yield: 65%; [α]20 D −31.7 (c 1.1, EtOH; mp 106−109 °C); 1H NMR (400 MHz, CDCl3) δ 9.03 (br s, 1H), 8.10 (s, 1H), 7.96 (dd, J = 8.4, 1.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 3.99 (d, J = 10.8 Hz, 1H), 3.58 (d, J = 10.8 Hz, 1H), 3.07 (br s, 1H), 2.06−1.99 (m, 1H), 1.86−1.78 (m, 1H), 1.00 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 171.6, 141.4, 136.1, 134.6, 134.1(q, J = 32.5 Hz), 122.2, 117.5 (q, J = 20 Hz), 115.7, 105.0, 78.7, 40.6, 31.3, 8.3. HRMS (m/z) calcd for C13H12BrF3N2O2 [M]+, 364.0034; found, 364.0031. 4.2.5. General Procedure for the Synthesis of (R)-11a,31b. To a suspension of NaH (60% mineral oil; 1.3 equiv) in dry THF cooled at 0−5 °C was added a solution of 4-fluorobenzenethiol (1.0 equiv) in THF dropwise. The reaction mixture was then stirred at room temperature for 30 min; after that, a solution of (R)-10a (1 equiv) in THF was added dropwise at 0−5 °C. The solution was then stirred at room temperature for 3−5 h and then quenched with distilled H2O and saturated NH4Cl. The organic phase was then extracted with EtOAc, and the crude material purified by silica gel column chromatography. (R)-11b: yield, 85%; [α]20 D −45.2 (c 3.0, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 8.97 (br s, 1H), 7.90 (s, 1H), 7.74 (s, 2H), 7.39−7.36 (m, 2H), 6.85 (t, J = 8.4 Hz, 2H), 3.74 (d, J = 14.0 Hz, 1H), 3.45 (br s, 1H), 3.08 (d, J = 14.0 Hz) 1.86−2.00 (m, 1H), 1.63− 1.76 (m, 1H), 0.93 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 172.8, 161.5 (d, J = 256 Hz), 141.3, 135.9, 134.8 (q, J = 35 Hz), 7274

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°C. The reaction mixture was then stirred at room temperature for 2 h and then quenched with a 0.1 N solution of HCl. The organic layer was extracted with EtOAc and dried over anhydrous Na2SO4. After filtration, the crude material was purified by silica gel column chromatography. Characterization exemplified by (R)-14m: yield, 98%; 1H NMR (400 MHz, CDCl3) δ 7.54−7.51 (m, 2H), 7.39−7.43 (m, 2H), 7.29−7.25 (m, 2H), 6.98−6.93 (m, 2H), 3.49 (s, 3H), 3.39 (d, J = 14.0 Hz, 1H), 3.19 (d, J = 13.6 Hz, 1H), 3.09 (d, J = 13.6 Hz, 1H), 3.05 (d, J = 13.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 173.8, 163.7, 161.2, 140.9, 134.1, 134.0, 132.2, 132.1, 131.3, 131.1, 118.9, 116.4, 116.2, 111.3, 78.2, 52.9, 45.5, 44.7. HRMS (m/z) calcd for C18H16FNO3S [M]+, 345.0835; found, 345.0838. 4.2.11. General Procedure for the Synthesis of (R)-15c−n. To a solution of 4-amino-2-(trifluoromethyl)benzonitrile (9) (1.6 equiv) in THF (8.5 mL/mmol), cooled at −10 °C, was added LHMDS (4.5 equiv) dropwise. The reaction mixture was then stirred at this temperature for 40 min, and HMPA (10% of the final amount of THF) was added to the solution. After 5 min stirring, a solution of the ester (R)-14 (1 equiv) in THF (7 mL/mmol) was added to the reaction mixture. After 30 min at −10 °C, the solution was stirred 12 h at room temperature. The solution was then quenched with 0.1 N HCl and extracted with EtOAc. The crude material was purified by silica gel column chromatography. Characterization exemplified by (R)-15m: yield, 77%; 1H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 7.71−7.68 (m, 2H), 7.53.7.48 (m, 3H), 7.39−7.35 (m, 3H), 7.20−7.18 (m, 1H), 6.84−6.82 (m, 2H), 3.90 (d, J = 14.4 Hz, 1H), 3.78 (br s, 1H), 3.26 (d, J = 13.6 Hz, 1H), 3.07 (d, J = 14.4 Hz, 1H), 2.96 (d, J = 13.2 Hz, 1H). 13 C NMR (100 MHz, CDCl3) δ 171.4, 162.5 (d, J = 248.7 Hz), 140.4, 140.1, 135.6, 134.1, 134.0, 131.9, 131.1, 127.41 (d, J = 3.4 Hz), 121.5, 118.5, 117.1, 117.0, 116.5, 116.3, 115.2, 111.3, 77.4, 44.9, 44.7. HRMS (m/z) calcd for C25H17F4N3O2S [M]+, 499.0978; found, 499.0974. 4.2.12. Synthesis of Compounds (R)-16c−n Was Achieved by the Same Procedure Reported for the Preparation of (R)-12a,b. Characterization exemplified by (R)-16m: yield, 87%; [α]20 D +142.5 (c 0.40, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 7.90− 7.88 (m, 2H), 7.79−7.75 (m, 2H), 7.56−7.54 (m, 3H), 7.33−7.30 (m, 2H), 7.17 (d, J = 8.0 Hz, 2H), 5.33 (s, 1H), 4.02 (d, J = 14.4 Hz, 1H), 3.41 (d, J = 14.4 Hz, 1H), 3.22 (d, J = 13.6 Hz, 1H), 3.13 (d, J = 13.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 170.1, 166.6 (d, J = 258.1 Hz), 140.4, 139.0, 136.0, 132.3, 131.6, 131.3, 131.2, 122.1, 118.6, 117.3 (q, J = 4.7 Hz), 115.3, 112.1, 105.8, 77.4, 60.4, 46.1. HRMS (m/z) calcd for C25H17F4N3O4S [M]+, 531.0876; found, 531.0873. 4.2.13. General Procedure for the Synthesis of Derivatives (2S,5S)-18c and (2S,5S)-18e. To a solution of (2S,5R)-7 (1 equiv) in dry dimethylformamide (DMF) (6.5 mL/mmol) was added 2.2 equiv of dry K2CO3 followed by 2.0 equiv of 4-cyanophenol. The reaction mixture was then stirred at 100 °C for 12 h. Reaction completion was followed by TLC using a cerium−molybdenum solution as stain and treated with distilled water when the starting material disappeared. The organic phase was extracted with EtOAc, and the crude material was purified by silica gel column chromatography. Characterization exemplified by (2S,5S)-18c: yield, 30%; 1H NMR (400 MHz, CDCl3) δ 7.58−7.56 (m, 2H), 7.35−7.26 (m, 5H), 6.91−6.89 (m, 2H), 4.41 (s, 1H), 4.25 (d, J = 10.4 Hz, 1H), 4.13 (d, J = 10.4 Hz, 1H), 3.20 (d, J = 14.0 Hz, 1H), 3.05 (d, J = 14.0 Hz, 1H), 0.89 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 172.7, 1661.5, 134.3, 133.6, 130.4, 129.0, 128.1, 115.5, 109.6, 105.1, 86.2, 70.0, 38.7, 34.8, 23.6. HRMS (m/z) calcd for C22H23NO4 [M]+, 365.1627; found, 365.1624. 4.3. Constructs and Cell Lines. cDNA encoding the WT hAR was in pSG5.57 The 3416 construct, containing four copies of the WT slp-HRE2 (59-TGGTCAgccAGTTCT-39), was cloned into the NheI site of pTKTATA-Luc.56 The human LNCaP prostate cancer-derived cell lines (American Tissue Culture Collection; Rockville, MD, USA) were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, insulin (6 ng/mL), L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 U/mL), and hydrocortisone (3.75 ng/mL). The cells were made quiescent by using phenol red-free RPMI and dextran charcoal-treated calf serum.58 Cos7 (American Tissue Culture Collection; Rockville, MD, USA) cells

were grown in DME supplemented with phenol red, 10% fetal calf serum (FCS), insulin (6 ng/mL), L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 U/mL), and hydrocortisone (3.75 ng/ mL). The cells were made quiescent using phenol red-free DME and dextran charcoal-treated calf serum, as described.40 LNCaP-AR cells, derived from LNCaP cells, were kindly provided by Dr. Charles L. Sawyers (Howard Hughes Medical Institute Investigator at Memorial Sloan Kettering Cancer Center, NY, USA), grown in RPMI medium supplemented with 10% fetal bovine serum and glutamine (2 mM), and checked periodically for mycoplasma contamination by MycoAlert mycoplasma detection kit (Lonza). LNCaP-Rbic, the bicalutamideresistant cell line derived from LNCaP and isolated in our laboratory,46 was maintained in the same way in continuous exposure to 20 μM (R)-bicalutamide. 4.4. Transfection, Nuclear Translocation, and Transactivation Assay. For AR nuclear translocation analysis, Cos-7 cells were plated on coverslips at 70% confluence. Cells were then made quiescent and then transfected with 1 μg of purified pSG5-hARexpressing plasmid using the Superfect reagent (Qiagen, Hilden, Germany). Cells were left unstimulated or stimulated with 10 nM R1881 for 60 min in the absence or presence of the study compounds. For the transactivation assay, LNCaP cells and Cos-7 cells were plated at 70% confluence in phenol red-free RPMI (LNCaP) or DME (Cos7) containing 10% charcoal-stripped serum. After 48 h, cells were transfected by Superfect with 0.8 μg of 3416-pTK-TATA-Luc alone or with 0.2 μg of pSG5-hAR-expressing plasmid. After 24 h, transfected cells were stimulated with 10 nM R1881 (dissolved in 0.001% ethanol, final concentration) for 24 h in the absence or presence of the indicated compounds. Control cells were treated with the vehicle alone. Cell lysates were prepared, and luciferase activity was measured using a luciferase assay system (Promega). The results were corrected using CH110-expressed-galactosidase activity. 4.5. AR Ligand Binding Displacement Studies in LNCaP Cells. LNCaP cells were made quiescent using phenol red-free medium and dextran charcoal-stripped serum.58 Cells at 70% confluence were incubated with 10 nM [3H]R1881 (98 Ci/mmole; PerkinElmer) added to the medium in the absence or presence of the indicated excess of radio-inert compounds. After a 4 h incubation at 37 °C, cells were washed three times with ice-cold PBS and collected by gently scraping in a cold room using 600 μL of ice-cold PBS containing 0.05% EDTA (w/v). The number of cells in an aliquot of 100 μL was counted. An aliquot (200 μL) of the cell suspension was submitted in duplicate to the extraction of intracellular radioactivity using 500 μL ice-cold ethanol (100%) for 1 h, as reported.41 After 24 h at 37 °C, radioactivity was counted in a liquid scintillation counter. Nonspecific binding of [3H]R1881 was determined in separate wells by adding the indicated excess of unlabeled R1881 to the incubation medium. The statistical significance of results was also evaluated by paired Student’s t test. P values < 0.005 were considered to be significant. No significance was attributed to the difference in the residual binding between the cells incubated with 10 nM [3H]R1881 in the presence of (R)-16m and that incubated with 10 nM [3H]R1881 in the presence of (R)-bicalutamide. LNCaP cells were also incubated with 10 nM [3H]R1881 in the absence or presence of a 100-fold excess (1 μM) of unlabeled R1881 or casodex (SigmaAldrich). Residual binding was 30 and 35% for unlabeled R1881 or casodex, respectively. 4.6. BrdU Incorporation Analysis, Wound Scratch Analysis, and Transwell Assay. After in vivo labeling with 100 μM BrdU (Sigma), quiescent cells on coverslips were fixed and permeabilized. BrdU incorporation was analyzed by immunofluorescence using diluted (1:50 in PBS) mouse monoclonal anti-BrdU antibody (clone BU-1, from GE Healthcare), as reported.59 Mouse antibody was detected using diluted (1:200 in PBS) Texas red-conjugated goat antimouse antibody (Jackson Laboratories). No significance was attributed to the difference in BrdU incorporation between the control cells and cells stimulated with 10 nM R1881 in the presence of (R)-bic or (R)16m. For wound-healing assay, quiescent LNCaP cell monolayers at confluence were wounded using sterile pipet tips and then washed with PBS. To avoid proliferation, the cells were treated with cytosine 7275

dx.doi.org/10.1021/jm5005122 | J. Med. Chem. 2014, 57, 7263−7279

Journal of Medicinal Chemistry

Article

arabinoside (at 100 μM; Sigma) and then left unstimulated or stimulated for 6 h with 10 nM R1881 in the absence or presence of the indicated compounds. Fields were analyzed with a DMIRB inverted microscope (Leica) using N-Plan 10× objective (Leica). Transwell assay was done as reported.60 4.7. Immunofluorescence Analysis. Cells on coverslips were fixed and permeabilized.40 WT hAR ectopically expressed in Cos-7 cells was visualized60 using the rabbit polyclonal anti-C19 antibody (Santa Cruz). The primary antibody was detected using diluted (1:100 in PBS) Texas red-conjugated goat anti-rabbit antibody (Jackson Laboratories). Coverslips were finally stained with Hoechst 33258, inverted, and mounted in Mowiol (Calbiochem). Fields were analyzed as detailed described59 using a DMBL Leica (Leica) fluorescent microscope equipped with an HCXPL Apo 63× oil objective. Images were captured using a DC480 camera (Leica) and acquired using FW4000 (Leica) software. 4.8. Lysates and Western Blot Analysis. Cell lysates (2 mg/mL protein concentration) were prepared,59 and AR was detected as reported60 using rabbit polyclonal anti-AR antibodies (C-19 or N-20; Santa Cruz). Immune-reactive proteins were revealed using the ECL detection system (from GE Healthcare). 4.9. In Vitro Chemosensitivity Assay. Sulforhodamine B (SRB) assay was used according to the method by Skehan et al.61 Briefly, cells were collected by trypsinization, counted, and plated at a density of 5000 cells/well in 96-well flat-bottomed microtiter plates (100 μL of cell suspension/well). Experiments were run in octuplicate, and each experiment was repeated three times. The optical density of cells was determined at a wavelength of 490 nm by a colorimetric plate reader. Growth inhibition and cytocidal effect of drugs were calculated according to the formula reported by Monks et al.:62 [(ODtreated − ODzero)/(ODcontrol − ODzero)] × 100%, when ODtreated is > ODzero. If ODtreated is below ODzero, then cell killing has occurred. ODzero depicts the cell number at the moment of drug addition, ODcontrol reflects the cell number in untreated wells, and ODtreated reflects the cell number in treated wells on the day of the assay. Cytotoxic potency on CW22Rv1 was assessed by growth inhibition assay. Cells were cultured in RPMI 1640 containing 10% FCS. Twenty-four hours after seeding, cells were exposed to the drugs (concentration range 0.5−100 μM) and counted 144 h later by coulter counter. IC50 is defined as the drug concentration causing 50% cell growth inhibition, determined by dose−response curves. Experiments were performed in triplicate. 4.10. Tunel Assay. At the end of drug exposure, the percentage of apoptotic cells was evaluated by flow cytometric analysis. Briefly, after treatment, cells were trypsinized, fixed in PBS + 1% paraformaldehyde on ice for 15 min, suspended in ice-cold ethanol (70%), and stored overnight at −20 °C. Cells were then washed twice in PBS and incubated with 50 μL of TUNEL reaction mixture (Roche Diagnostic GmbH, Mannheim, Germany) containing TdT and FITC-conjugated dUTP deoxynucleotides in a humidified atmosphere for 60 min at 37 °C in the dark. Samples were then washed in PBS containing Triton X100 (0.1%), counterstained with 2.5 μg/mL propidium iodide (SigmaAldrich) and RNase (10 Kunits/ml, Sigma-Aldrich) for 30 min at 4 °C in the dark, and finally analyzed (FACS Vantage Becton Dickinson). Data acquisition and analysis were performed using CELLQuest software (Becton Dickinson). For each sample, 10 000 events were recorded. 4.11. Quantitative Real-Time PCR. Total cellular RNA was isolated using RNeasy minikit (Qiagen). One microgram of RNA was reverse-transcribed into cDNA using iScript (BioRad, Hercules, CA) according to the manufacturer’s instructions. Real-time PCR was performed using the MyiQ single color real-time PCR detection system (BioRad) and SYBR green I dye chemistry. The stably expressed endogenous β2-microglobulin gene was amplified as a control for quality and quantity of input RNA. Primers for mRNA amplification are as follows: GAPDH forward 5′CGCTACTCTCTCTTTCTGGC-3′, reverse 5′-AGACACATAGCAATTCAGAAT-3′; HPRT forward 5′AGACTTTGCTTTCCTTGGTCAGG-3′, reverse 5′GTCTGGCTTATATCCAACATTCG-3′; PSA forward 5′-GCAGCATTGAACCAGAGGAG-3′, reverse 5′- CCATGACGTGA-

TACCTTGA-3′); primers were designed using Beacon Designer Software (version 7.2, BioRad). Real-time PCR was carried out in triplicate reactions at a final volume of 25 mL containing 50 ng of cDNA template, SYBR Green Mix, and 200 nM of forward and reverse primers. Samples were maintained at 95 °C for 10 min and 30 s, followed by 40 amplification cycles at 95 °C for 15 s, and then at 60 °C for 30 s for GAPDH, HPRT, and PSA. Product specificity was controlled by melting point analysis. Amplification efficiency, which never varied by 5% in the different experiments, was used to determine the relative expression of mRNA obtained using Gene Expression Macro Software (version 1.1) (BioRad). The amount of mRNA was normalized to the endogenous reference genes GAPDH and HPRT and expressed as n-fold levels relative to untreated samples. RT-PCR reactions were performed in triplicate, and the coefficient of variation (CV), calculated from the three Ct values, was always 1.5%. Reproducibility of the relative mRNA expression was calculated from the results of two experiments in which the procedure was carried out on different retrotranscription products derived from the same mRNA sample. CV was always