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Jul 15, 2014 - Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, Canada. J. Med. Chem...
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Discovery of 1H‑Indole-2-carboxamides as Novel Inhibitors of the Androgen Receptor Binding Function 3 (BF3) Fuqiang Ban, Eric Leblanc, Huifang Li, Ravi S. N. Munuganti, Kate Frewin, Paul S. Rennie, and Artem Cherkasov* Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, Canada S Supporting Information *

ABSTRACT: To overcome resistance to conventional antiandrogens of human androgen receptor (AR), the allosteric site of the AR binding function 3 (BF3) was investigated as an alternative target for small molecule therapeutics. A library of 1Hindole-2-carboxamides were discovered as BF3 inhibitors and exhibited strong antiproliferative activity against LNCaP and enzalutamide-resistant prostate cancer cell lines. Several of the lead compounds may prove of particular benefit as a novel alternative treatment for castration-resistant prostate cancers.

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this regard, Jehle et al.22 demonstrated that a duplicated GARRPR motif at the N-terminal region of the cochaperone Bag-1L functions through the AR BF-3 pocket and is very important for fine-tuning the activity of the AR. Promising BF3 inhibitors including ours with novel chemical scaffolds have been reported.13,14,18,19 Furthermore, Jehle et al.22 demonstrated that one of our BF3 lead compounds inhibited the binding of Bag-1L peptides to this site on the AR-LBD. On the basis of the binding mode of 2-((2-phenoxyethyl)thio)-1H-benzo[d]imidazole (compound 1 in Figure 1A) revealed by its cocrystal structure in the BF3 pocket in Figure 1B,19 low micromolar AR inhibitor activity has been achieved within this library through structure-based lead optimization.

INTRODUCTION Androgen depletion/withdrawal therapy is the standard of care for metastatic prostate cancers since Huggins and Hodges’s discovery in the early 1940s that androgens promote prostate cancer (PCa) growth.1,2 The growth of PCas is stimulated by androgen receptor (AR) signaling which can be efficiently blocked by anti-androgens such as bicalutamide, a direct competitor of endogenous androgen in the androgen binding site (ABS) of the AR. Initially, anti-androgen treatments are effective in a high proportion of PCa patients, but subsequently a significant number acquire treatment resistance. This state is referred to as castration-resistant prostate cancer (CRPC) and is associated with poor survival rates.3,4 Despite anti-androgen treatments including the newly FDA-approved enzalutamide5,6 for CRPC, it should be noted that the sustained activity of the AR-signaling pathways is conserved in the CRPC and is likely attributable to many aberrant mechanisms such as mutations in the ABS (which can make AR antagonists act as agonists7,8), AR amplifications, and expression of constitutively active AR splice-variants, which lack the ABS and thus are not affected by current anti-androgens.9,10 All current anti-androgens used for PCa treatment, including the first generation anti-androgens flutamide, bicalutamide, nilutamide and the second generation enzalutamide, bind to the same ABS site of the AR. In addition, they share a common molecular scaffold which increases the likelihood of cross resistance when mutations in the ABS occur. Accordingly, alternative sites of the AR have been attempted to overcome resistance to these anti-androgens.11−21 Our recent studies18,19 have focused on targeting the binding function 3 (BF3) module of the ligand binding domain (LBD) of the AR with small molecules, the binding site of which was originally identified by Estebanez-Perpina et al.14 The BF3 binding site is important for the recruitment of AR coregulators critical for androgen-regulated gene expression.13 In © 2014 American Chemical Society

Figure 1. (A) Chemical structures of compounds 1 and 2. (B) BF3 pharmocophore hypothesis (F1:Don in blue is a proton donor, F2:HydA and F3:Hyd in red are hydrophobic features) based on the binding pose of 1 in its AR cocrystal structure (4HLW with 2.5 Å resolution). Green and pink patches represent hydrophobic and hydrophilic potential surfaces, respectively. Received: April 1, 2014 Published: July 15, 2014 6867

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The estimated structure−activity relationship (SAR) demonstrated that a hydrogen-bonding interaction to residue Glu837 and the hydrophobic S atom in the linker fragment of 1 contribute significantly to the AR inhibition activities of the BF3 pocket.19 To avoid possible tautomerization of 1 through protonation and deprotonation on the imidazole N atoms, we synthesized its indole derivative of 2 in Figure 1A by replacing the benzimidazole moiety of 1 (IC50 = 4.2 μM) with indole. Compound 2 demonstrated an IC50 of 5.4 μM that is comparable to its parent molecule.19 This observation prompted us to further develop indoles as a novel series of anti-androgens targeting the BF3 site. In the present study, we expanded the repertoire of BF3 inhibitors by introducing 1,3-nonsubstituted indole-bearing derivatives. Potent BF3-directed inhibitors of 1H-indole-2carboxamide origin were discovered through synergetic use of structure-based pharmacophore modeling, docking, synthesis, and methods for experimental evaluation. Importantly, several lead compounds demonstrated anti-AR activity in the submicromolar range. This new class of AR BF3 inhibitors represents a potentially novel type of drugs for treating the CRPCs.

Table 1. Structures and Measured Activities of 1H-Indole-2carboxamide Analogues

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RESULTS Identification of 1H-Indole-2-carboxamide Hit Series by Pharmacophore Modeling and Molecular Docking. Challenges for docking small molecules into surface-exposed binding pockets lie in correct prediction of binding poses of compounds and in distinction between real and artificial binding.23 Thus, to complement the use of the docking programs, a pharmacophore of three features (Figure 1B) was generated based on the structure of the AR BF3 site (see Supporting Information for details) and employed to select potential BF3 binders. The corresponding pharmacophore features were defined as the following: a proton-donor feature designated by F1:Don and two hydrophobic features designated by F2:HydA and F3:Hyd, respectively (Figure 1B). The working hypothesis behind the choices of the first two features came from the SAR observations that the imidazole NH and the hydrophobic S atom of 1 are important for its BF3 binding. The hydrophobic pharmacophoric feature F3 was established by probing the hydrophobic interaction potential of the BF3 pocket using Grid methodology24,25 (as implemented in MOE software).26 Together, the above three main features describe the current pharmacophore hypothesis for AR BF3 inhibitors. A focused library of 13 049 neutral 1,3-nonsubstituted indole-containing compounds extracted from the eMolecule collection27 were docked into the BF3 binding site by the Glide program.28 Since we observed that the top few binding poses of the same compound could be very different, while they produce similar docking scores, the pharmacophore model was then used to narrow down docked molecular structures. A total of 245 unique compounds were found that justify all criteria of the pharmacophore. These hits were then ranked by a consensus order, representing a sum of the two ranks resulting from the GLIDE-SP docking score and ligand efficiency, respectively. As the next step, 66 compounds (representing the top 30% of the data set) were visually inspected for their fitness of binding into the BF3 pocket and then the eight closest analogues (3−6 and 8−11 in Table 1) of 1H-indole-2-carboxamide nature were selected for further experimental evaluation.

Cell-Based Testing and in Vitro Characterization. The purchased compounds were screened for their ability to inhibit AR transcriptional activity using a nondestructive, cell-based 6868

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enhanced green fluorescent protein (eGFP) AR transcriptional assay.29 All the purchased compounds exhibited >50% inhibition of AR transcription at 3 μM. Further, all compounds were tested in a concentration-dependent titration experiment to establish the respective IC50 values of inhibition of AR transcriptional activity (Table 1). The observed IC50 values were in the range 3.32−15.67 μM. Compound 11 was found to be the most active in this series with an IC50 value of 3.32 μM (Figure 2A).

which may be detrimental to their inhibition activities. To better understand the impact of single F or Me substitution on the benzene ring of the parent 11 and the effect of replacing the methyl group on the furan ring with hydrogen, docking predictions led to the development of 12−20 with various Me or F substitutions and to 7 that contains a larger CF3 substitution at R3 position. Synthesis and Characterization of the Designed Compounds. This new library of 11 analogues was synthesized by Enamine (http://www.enamine.net/) with the following general method (Scheme 1). To the solution of the Scheme 1. Syntheses of Compounds 12−20

corresponding carboxylic acid (1.1 equiv) in dry DMF was added corresponding amine (1 equiv and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.21 equiv). The resulting mixture was stirred at the room temperature for 72 h and diluted with water. If after water addition solids were formed, they were filtered. If after water addition oily products were formed, they were extracted with ethyl acetate. The combined organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Nevertheless the crude product was purified by CombiFlash column chromatography to afford pure compounds (HPLC purity, >95%). The resulting IC50 values estimated with the eGFP AR transcriptional reporter assay were in the range 0.4−5.2 μM. As anticipated from the docking experiments, Me substitution at R4 and R5 in 12 and 13 slightly improved the activity. Compounds 14 and 16 derived by the replacement of the furan methyl with hydrogen enhanced the potency by ∼3-fold when compared to 12 and 13, respectively. The F-substitution at R3, R4, and R5 in 15, 18, and 17 improved the activity by more than 3-fold with reference to their parent molecule 11. Moreover, the replacement of the methyl group with hydrogen on the furan ring of 15 (1.10 μM) and 18 (0.70 μM) with R3-F and R4-F substitution, respectively, led to 19 (0.60 μM) and 20 (0.43 μM), which are ∼2-fold more potent. However, in regard to 7 (eGFP IC50 = 5.20 μM), we have observed that a larger CF3 substitution at R3 is significantly detrimental to the compound’s efficiency. Thus, 18, 19, and 20 with their respective eGFP-IC50 of 0.70, 0.60, and 0.43 μM are the most potent BF3 inhibitors within this synthesized series (Table 1). Reduction of PSA Expression in LNCaP and Enzalutamide-Resistant Cells. To rule out false positive hits in the AR transcriptional eGFP assay, we have also quantified the effect of all compounds on the production of prostate specific antigen (PSA).30 PSA is a serine protease, the expression of which is dependent on AR activity in a prostate epithelial cell. As expected, the developed compounds induced a dosedependent decrease of PSA levels and the estimated PSA IC50 values are well correlated with eGFP IC50 parameters (Table 1). Compound 18 Is a Selective AR-BF3 Inhibitor in LNCaP and Enzalutamide-Resistant Cells. Among the most potent AR BF3 inhibitors, 19 and 20 demonstrated poor

Figure 2. (A) Dose−response curve (0−50 μM) illustrating the inhibiting effect of the 11 on the AR transcriptional activity in cells. (B) BLI dose−response curves (0−50 μM) reflecting the direct binding of the 11. (C) Predicted BF3 binding mode of 11.

These inhibitors were tested with the SRC2 peptide and androgen displacement assays for their ability to displace SRC2 peptide from the activation function 2 (AF2) site and androgen from the ABS, respectively. None of the studied compounds were active in these assays, supporting that they target the intended AR BF3 site. Furthermore, the direct, reversible, dose-dependent interaction between 11 and the AR LBD was confirmed using biolayer interferometry (BLI) (Figure 2B). Finally, by use of a cell proliferation assay to gauge generic toxicity, 11 was also confirmed not to be toxic to non-AR containing cells at 50 μM administered for over 72 h (data not shown). Rational Design of Analogues of Compound 11. On the basis of the above data, 11 was selected as a starting point for medicinal chemistry optimization. The binding mode of 11 was predicted as shown in Figure 2C. Analysis of the predicted binding pose demonstrates that the indole moiety forms an Hbond with Glu837 and the amide carbonyl O forms an H-bond with Arg840. The isopropyl has favorable hydrophobic contact with Ile672. Similarly, the furan ring of 11 is in contact with residues Ile672, Val676, and Pro723, and the indole moiety forms hydrophobic π−π stacking with residues Phe673 and Tyr834. It has also been observed that around R3, R4, and R5 positions of the phenyl moiety of the indole in the BF3 pocket, there are still small hydrophobic volumes to be explored. Therefore, a library of 11 analogues with small substituents of F and Me at R3, R4, and R5 positions were generated and docked to the BF3 pocket. The docking results suggested that these substitutions could be accommodated and may enhance the BF3 inhibition activity. In addition, the predicted binding poses of 11 and its analogues with F or Me substitutions suggest that the methyl group on their furan ring points to the solvent, 6869

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solubility at high concentrations, and hence, 18 was chosen for further evaluation. In the LNCaP system, 18 demonstrated IC50 values of 0.70 and 0.84 μM in eGFP (Figure 3A) and PSA

Figure 4. Dose−response curve of 18 in comparison to enzalutamide illustrating the proliferation inhibiting effect in (A) LNCaP and PC3 cells and (B) enzalutamide-resistant cells.

growth of enzalutamide-resistant cells, with an IC50 of 1.31 μM, while enzalutamide was ineffective even at 10 μM. The above observations make it possible to conclude that the antiproliferative activity of 18 in LNCaP and enzalutamideresistant cells is AR-specific and is not associated with general cell toxicity.

Figure 3. (A) Dose−response curve (0−12.5 μM) of eGFP illustrating the inhibiting effect of the 18 and enzalutamide (Enz) on the AR transcriptional activity in LNCaP cells. Data points represent the mean of two independent experiments performed in triplicate. Error bars represent the standard error of the mean (SEM) for n = 6 values. Data were fitted using log of concentration of the inhibitors vs % activation with GraphPad Prism 6. (B) Dose−response curve (0−6.25 μM) of PSA illustrating the inhibiting effect of the 18 on the AR transcriptional activity in LNCaP cells. (C) Dose−response curve (0−12.5 μM) of PSA illustrating the inhibiting effect of the 18 on the AR transcriptional activity in enzalutamide resistant cell cells. (D) BLI dose−response curves (0−100 μM) reflecting the direct binding of 18 to AD LBD. (E) DHT displacement effect at 1 and 10 μM 18 in comparison with bicalutamide (Bic).

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DISCUSSION All current anti-androgens correspond to the same mode of inhibition of the AR and therefore can suffer from the same limitations. One well-known resistance mechanism is a specific T877A mutation at the ABS which turns conventional antiandrogens into AR agonists.34 Thus, this study addresses a very important question of whether the development of a new type of anti-AR therapeutic, such as drugs that bind to the AR in different regions (and directly disrupt its interaction with the cofactors), can complement or replace the existing antiandrogens when resistance arises. On the basis of the previously reported X-ray crystallographic structure (PDB code 4HLW.pdb),19 we have combined the power of in silico docking and target-based pharmacophore modeling to identify several new potent BF3 binders. The three features of the pharmacophore model have been applied effectively to select the desired binding poses of 1,3nonsubstituted indole-containing compounds. A consensus rank-order of docking score and ligand efficiency has been shown to be effective in hit selection. From the hit series of 1H-indole-2-carboxamides identified from in silico screening, we made further in silico modeling resulting in improved inhibition potency. These findings culminated in the discovery of rather potent compounds 18, 19, and 20 that contain Me and F substitution at R3−R5 positions with the respective improved IC50 values of 0.7, 0.6, and 0.43 μM, compared to an IC50 of 3.32 μM of the parent 11. The activity of these chemicals was further illustrated by their ability to decrease the levels of PSA in LNCaP and enzalutamide-resistant PCa cells. Importantly, 18 was also found to be effective in reducing PSA levels in the enzalutamide-resistant cell line, MR49F, with an estimated IC50 of 2.18 μM. While this number represents a 3-fold increase in these cells compared to native LNCaP cells, the mechanism of resistance in MR49F cells is unclear and 18 is still remarkably effective compared to enzalutamide. Furthermore, 18 not only reduced the AR transcriptional activity but also inhibited cell proliferation in PCa cell lines, including the MR49F cells. Therefore, the reported AR BF3 pharmacophore has successfully guided the hit selection from virtual screening and provided new insight into the BF3 pocket topology, enabling further applications in our ongoing AR BF3 drug discovery efforts.

(Figure 3B) assays, respectively. As can be seen in Figure 3C, 18 decreased the level of PSA in enzalutamide-resistant MR49F prostate cancer cells, 31,32 with the corresponding IC 50 established as 2.18 μM; on the other hand enzalutamide was unable to decrease completely the level of PSA expression in these resistant cells. Thus, enzalutamide resistance does not affect the action of 18. This suggests that enzalutamide and 18 may target different binding sites of the AR and that the newly discovered 1H-indole-2-carboxamides are quite effective in inhibiting AR signaling. To further characterize the mode of action of 18, we performed BLI experiment on the LBD portion of the AR. Figure 3D demonstrates the observed direct reversible interaction between the AR LBD and 18. In addition, 18 was evaluated for its ability to interact with ABS and the AF2 pocket of the AR. Figure 3E illustrates that 18 exhibits no detectable level of DHT displacement; no AF2 peptide displacement activity was detected either. Therefore, the activity of 18 could be regarded as AR specific and facilitated through BF3 interactions. Compound 18 Reduces the Growth of LNCaP and MR49F Cells. To further characterize the anti-AR potency of 18, its effect on cell proliferation was evaluated in the MTS assay using LNCaP,33 MR49F,32 and androgen-independent PC3 cell lines. The cell viability was assessed after 4 days of incubation with the tested molecules administered in serial concentrations. Figure 4A shows that 18 turned out to be very effective (IC50 = 0.55 μM) in inhibiting the growth of LNCaP cells stimulated by androgen but, as anticipated, did not show any effect on ARindependent PC3 cells which confirms its AR specificity. Moreover, Figure 4B indicates that 18 also potently inhibits the 6870

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6-Fluoro-N-isopropyl-N-((5-methylfuran-2-yl)methyl)-1H-indole-2-carboxamide (18). 1H NMR (DMSO + CCl4, 400 MHz): 1.20 (6H, d, J = 6 Hz, iPr), 2.22 (3H, s, CH3, furan), 4.66 (3H, br m, iPr + CH2), 6.00 (1H, s, arom), 6.18 (1H, s, arom), 6.82 (1H, s, arom), 6.90 (1H, m, arom), 7.12 (1H, d, J = 8 Hz, arom), 7.62 (1H, m, arom), 11.66 (1H, s, indole). MS (APCI) [M + H] calcd 315.17, found 315.0. 5-Fluoro-N-(furan-2-ylmethyl)-N-isopropyl-1H-indole-2-carboxamide (19). 1H NMR (DMSO, 400 MHz): 1.20 (6H, d, J = 6 Hz, iPr), 4.62 (1H, m, iPr), 4.72 (2H, br s, CH2), 6.32 (1H, s, arom), 6.42 (1H, s, arom), 7.05 (1H, m, arom), 7.4 (2H, m, arom), 7.6 (1H, s, arom), 11.72 (1H, s, indole). MS (APCI) [M + H] calcd 301.15, found 301.0. 6-Fluoro-N-(furan-2-ylmethyl)-N-isopropyl-1H-indole-2-carboxamide (20). 1H NMR (DMSO + CDCl4, 400 MHz): 1.25 (6H, d, J = 6 Hz, iPr), 4.72 (3H, br s, iPr + CH2), 6.28 (1H, s, arom), 6.37 (1H, s, arom), 6.72 (1H, s, arom), 6.80 (1H, m, arom), 7.12 (1H, d, J = 8 Hz, arom), 7.48 (2H, m, arom) 11.55 (1H, s, indole). MS (APCI) [M + H] calcd 301.15, found 301.2.

CONCLUSIONS We have found that 1H-indole-2-carboxamide derivatives, such as 18, are potent and selective AR-BF3 inhibitors and demonstrate strong activity against wild type and drug resistant prostate cancer cells. Further optimization of pharmacokinetic and pharmacodynamic properties of this class of compounds is currently ongoing to generate improved candidates for future clinical applications.

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EXPERIMENTAL SECTION

Characterization of the Synthsized Compounds. All compounds described had a purity of >95%, from HPLC. LC/MS spectra were recorded using a chromatography/mass spectrometric system that consists of a high-performance liquid chromatograph “Agilent 1100 series” equipped with a diode-matrix and mass-selective detector “Agilent LC/MSD SL”. The parameters of chromatography mass analysis were the following. Column: Zorbax SB-C18, 1.8 mm × 4.6 mm × 15 mm. Solvents: A, acetonitrile/water (95:5), 0.1% TFA; B, water (0.1% of TFA). Eluent flow: 3 mL/s. Volume of injected sample: 1 mL. UV detection at 215, 254, and 265 nm. Ionization method: chemical ionization under atmospheric pressure (APCI). Ionization mode: simultaneous scanning of positive and negative ions in the mass range of m/z 80−1000. N-(Furan-2-ylmethyl)-N-isopropyl-5-(trifluoromethyl)-1H-indole-2-carboxamide (7). 1H NMR (DMSO, 500 MHz): 1.21 (6H, d, J = 6 Hz, iPr), 4.60 (1H, m, iPr), 4.75 (2H, br s, CH2), 6.35 (1H, s, arom), 6.42 (1H, s, arom), 6.95 (1H, s, arom), 7.48 (1H, d, J = 8 Hz, indole), 7.60 (3H, m, indole + arom), 8.05 (1H, s, furan), 12.08 (1H, s, indole). MS (APCI) [M + H] calcd 351.1, found 351.1. N-Isopropyl-6-methyl-N-((5-methylfuran-2-yl)methyl)-1H-indole-2-carboxamide (12). 1H NMR (DMSO, 400 MHz): 1.27 (6H, d, J = 6 Hz, iPr), 2.30 (3H, CH3, furan), 2.42 (3H, s, CH3, indole), 4.65 (2H, br s, CH2), 4.72 (1H, m, iPr), 5.95 (1H, s, arom), 6.12 (1H, s, arom), 6.62 (1H, s, arom), 6.82 (1H, d, J = 8 Hz, arom), 7.20 (1H, s, arom), 7.40 (1H, d, J = 8 Hz, arom), 11.25 (1H, s, indole). MS (APCI) [M + H] calcd 311.2, found 311.2. N-Isopropyl-5-methyl-N-((5-methylfuran-2-yl)methyl)-1H-indole-2-carboxamide (13). 1H NMR (DMSO + CCl4, 400 MHz): 1.25 (6H, d, J = 6 Hz, iPr), 2.25 (3H, s, furan), 2.40 (3H, s, CH3, indole), 4.70 (3H, br m, iPr + CH2), 5.95 (1H, s, arom), 6.12 (1H, s, arom), 6.60 (1H, s, arom), 6.95 (1H, d, J = 8 Hz, arom), 7.3 (2H, m, arom), 11.35 (1H, s, indole). MS (APCI) [M + H] calcd 311.2, found 311.0. N-(Furan-2-ylmethyl)-N-isopropyl-5-methyl-1H-indole-2carboxamide (14). 1H NMR (DMSO, 400 MHz): 1.20 (6H, d, J = 6 Hz, iPr), 2.35 (3H, s, CH3, indole), 4.65 (1H, m, iPr), 4.70 (2H, br s, iPr), 6.30 (1H, s, arom), 6.42 (1H, s, arom), 7.00 (1H, d, J = 8 Hz, arom), 7.30 (1H, d, J = 8 Hz, arom), 7.35 (1H, s, arom), 7.60 (1H, s, arom), 11.45 (1H, s, indole). MS (APCI) [M + H] calcd 297.18, found 297.2. 5-Fluoro-N-isopropyl-N-((5-methylfuran-2-yl)methyl)-1H-indole-2-carboxamide (15). 1H NMR (DMSO, 400 MHz): 1.18 (6H, d, J = 6 Hz, J = 6 Hz, iPr), 2.23 (3H, s, CH3, furan), 4.65 (3H, br m, iPr + CH2), 6.00 (1H, s, arom), 6.17 (1H, s, arom), 6.78 (1H, s, arom), 7.03 (1H, m, arom), 7.40 (2H, m, arom), 11.70 (1H, s, indole). MS (APCI) [M + H] calcd 315.17, found 315.1. N-(Furan-2-ylmethyl)-N-isopropyl-6-methyl-1H-indole-2carboxamide (16). 1H NMR (DMSO, 400 MHz): 1.25 (6H, d, J = 6 Hz, iPr), 2.42 (3H, s, CH3, indole), 4.70 (3H, br m, iPr + CH2), 6.28 (1H, s, arom), 6.35 (1H, s, arom), 6.80 (1H, d, J = 8 Hz, arom), 7.22 (1H, s, arom), 7.38 (1H, d, J = 8 Hz, arom), 7.45 (1H, s, arom) 11.30 (1H, s, indole). MS (APCI) [M + H] calcd 397.18, found 397.1. 7-Fluoro-N-isopropyl-N-((5-methylfuran-2-yl)methyl)-1H-indole-2-carboxamide (17). 1H NMR (CDCl3, 500 MHz): 1.30 (6H, d, J = 6 Hz, iPr), 2.30 (3H, CH3, furan), 4.75 (2H, br s, CH2), 4.88 (1H, m iPr), 5.95 (1H, s, arom), 6.20 (1H, s, arom), 6.95 (1H, s, arom), 7.05 (2H, m, indole + arom), 7.42 (1H, d, J = 8 Hz, arom), 9.7 (1H, s, indole). MS (APCI) [M + H] calcd 315.1, found 315.1.

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ASSOCIATED CONTENT

* Supporting Information S

Pharmacophore development, characterization of synthesized compounds, modeling procedures, and biological assays. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 604-875-4111. Fax: 604-875-5654. E-mail: artc@ interchange.ubc.ca. Author Contributions

P.S.R.’s laboratory and A.C.’s laboratory made equal contributions to this work. F.B. and E.L. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Prostate Cancer Canada with generous support from Canada Safeway Grant SP2013-02, the Department of Defense (Prostate Cancer Research Program) Award W81XWH-12-1-0401, Canadian Institutes of Health Research grant, and Canadian Cancer Society Research Institute Grant F12-03271. We thank Drs. Martin Gleave and Amina Zoubeidi for providing us with enzalutamide-resistant LNCaP cells.

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ABBREVIATIONS USED PCa, prostate cancer; AR, androgen receptor; ABS, androgen binding site; CRPC, castration-resistant prostate cancer; BF3, binding function 3; eGFP, enhanced green fluorescent protein; AF2, activation function 2; BLI, biolayer interferometry; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium); PSA, prostate specific antigen

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REFERENCES

(1) Huggins, C.; Hodges, C. V. Studies on prostatic cancer I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 1941, 1, 293−297. (2) Schmidt, L. J.; Tindall, D. J. Androgen receptor: past, present and future. Curr. Drug Targets 2013, 14, 401−407. (3) Feldman, B. J.; Feldman, D. The development of androgenindependent prostate cancer. Nat. Rev. Cancer 2001, 1, 34−45.

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Journal of Medicinal Chemistry

Brief Article

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dx.doi.org/10.1021/jm500684r | J. Med. Chem. 2014, 57, 6867−6872