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Endocrinology 148(1):4 –12 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0843

Pharmacological and X-Ray Structural Characterization of a Novel Selective Androgen Receptor Modulator: Potent Hyperanabolic Stimulation of Skeletal Muscle with Hypostimulation of Prostate in Rats Jacek Ostrowski, Joyce E. Kuhns, John A. Lupisella, Mark C. Manfredi, Blake C. Beehler, Stanley R. Krystek, Jr., Yingzhi Bi, Chongqing Sun, Ramakrishna Seethala, Rajasree Golla, Paul G. Sleph, Aberra Fura, Yongmi An, Kevin F. Kish, John S. Sack, Kasim A. Mookhtiar, Gary J. Grover, and Lawrence G. Hamann Departments of Metabolic Diseases (J.O., J.E.K., J.A.L., B.C.B., R.S., R.G., P.G.S., K.A.M., G.J.G.), Discovery Chemistry (M.C.M., Y.B., C.S., L.G.H.), Macromolecular Structure (S.R.K., Y.A., K.F.K., J.S.S.), and Pharmaceutical Candidate Optimization (A.F.), Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543 A novel, highly potent, orally active, nonsteroidal tissue selective androgen receptor (AR) modulator (BMS-564929) has been identified, and this compound has been advanced to clinical trials for the treatment of age-related functional decline. BMS564929 is a subnanomolar AR agonist in vitro, is highly selective for the AR vs. other steroid hormone receptors, and exhibits no significant interactions with SHBG or aromatase. Dose response studies in castrated male rats show that BMS-564929 is substantially more potent than testosterone (T) in stimulating the growth of the levator ani muscle, and unlike T, highly selective for muscle vs. prostate. Key differences in the binding interactions of BMS-564929 with the AR relative to the native hormones were revealed through x-ray crystallography, includ-

ing several unique contacts located in specific helices of the ligand binding domain important for coregulatory protein recruitment. Results from additional pharmacological studies effectively exclude alternative mechanistic contributions to the observed tissue selectivity of this unique, orally active androgen. Because concerns regarding the potential hyperstimulatory effects on prostate and an inconvenient route of administration are major drawbacks that limit the clinical use of T, the potent oral activity and tissue selectivity exhibited by BMS564929 are expected to yield a clinical profile that provides the demonstrated beneficial effects of T in muscle and other tissues with a more favorable safety window. (Endocrinology 148: 4 –12, 2007)

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ERUM ANDROGEN (TESTOSTERONE, T) levels decline progressively with aging in men, beginning in the third decade of life at the rate of approximately 1% per year (1–3). Furthermore, 98% of T in men is not freely available to most tissues because it is bound with high affinity to SHBG (4), a serum protein the concentration of which increases with age. This decline in available T is associated with alterations in body composition, diminished energy, diminished muscle strength and physical function, reduced sexual function, and depressed mood (5–9). This androgen deficient state in aging males, often called andropause or androgen decline in the aging male, can subsequently lead to age-related functional decline (frailty). Several clinical studies have shown that supplementation of T at physiologic doses in elderly men results in a significant increase in lean body mass, a decrease in adipose tissue, and an increase in muscle strength (10 –15). However, use of T replacement therapy in elderly males by

primary care physicians is limited due to concerns about potential side effects (16 –20). Among the more serious side effects are hyperstimulation of the prostate, which may be a preamble to or exacerbate occult subclinical benign prostatic hypertrophy and/or prostate cancer, and increases in hematocrit. T cannot be administered orally due to its rapid and extensive metabolism, and consequently, is given by injection, transdermal patch, or in a recently introduced gel form (21, 22). Each of these delivery routes has certain drawbacks: the need for needles and physician administration for the former, skin irritation and potential for contact transfer to others with the latter. Several T analogs with either 7␣- or 17␣-alkyl substitution, including 7␣-methyl-19-nortestosterone (23) and oxandrolone (24), have been used clinically in place of T, because these are shown to circumvent metabolism and improve oral bioavailability and half-life. However, 17␣-alkylated androgens have frequently been associated with hepatotoxicity (25), although these effects are hypothesized to be structure related, not mechanism related. There are currently no approved drugs indicated for the prevention of functional decline in the aging male, but the growing desire among the aging population to remain independent has raised awareness within the medical community regarding the potential for therapeutic intervention. Consequently, there exists an unmet medical need for safe and effective oral

First Published Online September 28, 2006 Abbreviations: AR, Androgen receptor; DHT, dihydrotestosterone; E2, estradiol; GR, glucocorticoid receptor; LBD, ligand binding domain; MR, mineralocorticoid receptor; PEC, prostate epithelial; SARM, selective androgen receptor modulator; T, testosterone; TFA, trifluoroacetic acid; TP, T propionate. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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Ostrowski et al. • Muscle Selective AR Agonist

therapies that might offer greater separation between the desired anabolic and undesired androgenic effects. In theory, a selective androgen receptor modulator (SARM) (26) has the potential to maintain or improve muscle strength and function, thereby enabling or facilitating the performance of everyday activities, and to improve body composition, mental health, libido, and sexual function in elderly men without the concomitant deleterious effects on prostate, liver, or erythrocytes associated with steroid treatment regimens. Since the first identification of orally active nonsteroidal androgens (27), several groups have actively pursued the identification and development of SARMs (28 –30), and some of the most advanced compounds to emerge from these efforts have recently reached the clinic (31). However, these compounds exhibit limited efficacy and modest tissue selectivity in preclinical models. As part of our program to discover and develop novel treatments for age-related functional decline, we have discovered a novel, potent, orally bioavailable SARM, BMS-564929 (Fig. 1), which, analogous to the natural hormone agonists T and dihydrotestosterone (DHT), exerts its effects via the regulation of androgen receptor (AR)-mediated gene transcription in tissues that express the AR (32). This highly potent compound exhibits remarkable muscle vs. prostate selectivity in vitro and in chronic in vivo models of androgen action in rodents. The mechanism by which BMS-564929 tissue selectivity is achieved is anticipated to be analogous to that of the more well-characterized selective estrogen receptor modulators (33–36), which involves ligand-based selective activation of target genes through differential recruitment of cofactors present in various tissues expressing the receptor (37, 38). Recent efforts directed at beginning to understand the structural basis for ligand-induced differential gene expression through the AR are making advances toward characterizing these relationships in greater detail at the molecular level (39). X-ray data of the AR ligand binding domain (LBD) bound to BMS-564929 and DHT shows that BMS-564929 has unique binding interactions compared with DHT. Therefore, binding of BMS-564929 to the AR LBD results in a receptor conformation disparate to that induced by DHT, which may thereby allow the BMS-564929-AR complex to interact with a different set of coactivators/corepressors than the DHT-AR complex. Through additional investigations, we have been

FIG. 1. Structures of endogenous AR ligands testosterone (T) and DHT, and selective AR modulator BMS-564929.

Endocrinology, January 2007, 148(1):4 –12

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able to rule out contributions from a number of potential alternative selectivity mechanisms. Based on the present results, we believe that BMS-564929 is the most potent and muscle-selective, orally available AR agonist reported to date. Consequently this compound has advanced into human clinical trials for the treatment of age-related functional decline. Materials and Methods Reagents [3H]DHT, [3H]progesterone, and [3H]estradiol (E2) were obtained from PerkinElmer (Wellesley, MA). SARM compound BMS-564929 [(7R,7aS)-2(3-chloro-4-cyano-2-methylphenyl)-7-hydroxytetrahydro-2H-pyrrolo[1,2e]imidazole-1,3-dione] was synthesized in our laboratories by the following procedures as outlined schematically (Fig. 2). (3S)-N-tert-Butoxycarbonyl-3-hydroxy-l-proline methyl ester. Hydrogen chloride gas was bubbled through a suspension of trans-3-hydroxy-lproline (50 g, 0.38 mol) in MeOH (600 ml) at 0 C for 10 min. The resulting clear solution was stirred at room temperature for 4 h, then concentrated under reduced pressure. The resulting white solid was dried in vacuo overnight to afford 68.3 g of the title compound. The hydroxyproline ester thus obtained was suspended in CH2Cl2 (1.0 liter) cooled to 0 C, and Et3N (105.3 ml, 0.755 mol) was added, followed by portion-wise addition of di-tert-butyl dicarbonate (82.96 g, 0.380 mol). The resulting mixture was stirred at room temperature for 4 h, then partitioned between water and CH2Cl2. The CH2Cl2 layer was washed twice with water, and once each with 20% aqueous citric acid, water, and brine, then dried over Na2SO4 and concentrated under reduced pressure to give an oily residue. The crude product was chromatographed (silica gel) eluting with 15–50% EtOAc/hexane to afford the diprotected compound (73.3 g) as a pale yellow viscous oil. 1H NMR (CDCl3, 400 MHz, 3:2 mixture of rotamers, data for major rotamer) ␦ 1.41 (s, 9H), 1.93 (m, 1H), 2.11 (m, 1H), 2.45 (d, J ⫽ 4.8 Hz, 1H), 3.60 (m, 2H), 3.75 (s, 3H), 4.18 (d, J ⫽ 1.3 Hz, 1H), 4.44 (m, 1H). (3R)-N-tert-Butyloxycarbonyl-3-benzoyloxy-l-proline methyl ester. To a stirred solution of (3S)-N-tert-butoxycarbonyl-3-hydroxy-l-proline methyl ester (69.1 g, 0.282 mol), Ph3P (88.7 g, 0.338 mol), and benzoic acid (41.3 g, 0.338 mol) in anhydrous tetrahydrofuran (1.35 L) cooled to 0 C was added drop-wise over 1 h (through an addition funnel) to a solution of diethylazodicarboxylate (62 ml, 0.33 mol) in anhydrous tetrahydrofuran (50 ml). After addition, the resulting light yellow solution was stirred at room temperature for 8 h. The reaction mixture was then partitioned between EtOAc and aqueous NaHCO3. The organic layer was washed with saturated aqueous NaHCO3, water (2⫻), and brine,

FIG. 2. Synthesis route for the preparation of BMS-564929.

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dried over Na2SO4, and concentrated under reduced pressure to yield a crude product as a semisolid. The crude benzoate product was suspended in 25% EtOAc/hexane and stirred vigorously for 3 h. The resulting suspension was filtered, and the collected white solid (Ph3PO) was rinsed twice with 20% EtOAc/hexane. The combined filtrates were concentrated under reduced pressure to yield an oily residue, which was triturated twice with 20% EtOAc/hexane as described above to yield approximately 150 g of partially purified product as a yellow oil, which was further purified by flash chromatography (silica gel) eluting with 10 –20% EtOAc/hexane to furnish the pure title compound (88.4 g) as a light yellow viscous oil. 1H NMR (400 MHz, CDCl3, 2:1 mixture of rotamers, data for major rotamer) ␦ 1.39 (s, 9H), 2.26 (m, 2H), 3.57–3.75 (m, 2H), 3.62 (s, 3H), 4.65 (d, J ⫽ 4.0 Hz), 5.71 (q, J ⫽ 4.0 Hz), 7.45 (t, J ⫽ 4.0 Hz, 2H), 7.58 (t, J ⫽ 4.0 Hz, 1H), 7.98 (d, J ⫽ 4.0 Hz, 2H). (3R)-3-Hydroxy-l-proline methyl ester. To a solution of (3R)-N-tert-butyloxycarbonyl-3-benzoyloxy-l-proline methyl ester (88.4 g, 0.253 mol) in anhydrous MeOH (700 ml) at 0 C, a freshly prepared 1 n solution of KOH in anhydrous MeOH (367ml, 0.367 mol) was slowly added through an addition funnel over 25 min. After the addition, the resultant light yellow solution was stirred at 0 C for 2 h, then the reaction was quenched by slow addition (over 25 min) of a solution of 1 n HCl in dioxane/ EtOAc (380 ml) through an addition funnel. The resulting white suspension was concentrated under reduced pressure to remove most of the solvent, and the remaining mixture was partitioned between water and EtOAc. The separated organic phase was washed with water (2⫻), saturated aqueous NaHCO3 (2⫻), water, and brine, and dried over Na2SO4. The filtrate was concentrated under reduced pressure to give a light yellow oily residue, which was chromatographed (silica gel) eluting first with 25–30% EtOAc/hexane, then 5% MeOH in 30% EtOAc/ hexane to furnish the N-Boc-protected cis-hydroxy-l-proline methyl ester (44.6 g) as a pale yellow oil. To a solution of the free hydroxy compound thus obtained (44.6 g, 0.182 mol) in CH2Cl2 (450 ml) at 0 C, trifluoroacetic acid (TFA) (275 ml) was slowly added through an addition funnel over 40 min. After the addition, the reaction mixture was stirred at 0 C for 2 h, then concentrated under reduced pressure to give a viscous oily residue, which was evaporated with ether (2⫻), toluene (1⫻), and ether (2⫻), and dried in vacuo overnight to yield the Bocdeprotected TFA salt of cis-hydroxy-l-proline methyl ester (59.5 g) as a light yellow solid. A solution of this TFA salt (12.64 g, 48.8 mmol) in MeOH (150 ml) was free based by treatment with WA21J resin (Diaion WA21J, polyamine resin from Supelco, Bellefonte, PA; 60 g). The resulting suspension was stirred at room temperature for 1 h and then filtered. The collected resin was rinsed with MeOH (2⫻), and combined filtrate was concentrated carefully under reduced pressure to give the desired cis-hydroxy-l-proline methyl ester (7.7 g) as a colorless oil. [␣]D ⫽ ⫹14.9° (c. 1.0, MeOH); 1H NMR (400 MHz, CD3OD) ␦ 1.86 (m, 1H), 2.02 (m, 1H), 2.87 (m, 1H), 3.24 (m, 1H), 3.69 (d, J ⫽ 4.0 Hz, 1H), 3.75 (s, 3H), 4.48 (t, J ⫽ 4.0 Hz, 1H); HPLC: 100% at 0.157 min (retention time) [conditions: Phenominex Luna C18 (4.6 ⫻ 50 mm); eluted with 0 –100% B; 4 min gradient (A ⫽ 90% H2O/10% MeOH/0.1% H3PO4 and B ⫽ 10% H2O/90% MeOH/0.1% H3PO4), flow rate at 4 ml/min, UV detection at 220 nm]; MS (ES): m/z 146 [M⫹H]⫹. N-(4-Bromo-3-chloro-2-methylphenyl)acetamide. To a solution of commercially obtained 3-chloro-2-methylaniline (10.0 g, 70.6 mmol) in EtOH (85 ml) at room temperature, acetic anhydride (8.00 ml, 84.7 mmol) was slowly added, and the reaction mixture was stirred for 15 min. The resulting suspension was concentrated and dried in vacuo to provide the desired acetamide (17.0 g) as a red-brown solid. To a solution of this acetamide (13.0 g, 70.6 mmol) in AcOH (100 ml) at 15 C was added bromine (10.9 ml, 212 mmol) over 20 min. The solution was allowed to warm to room temperature and stir for 1 h. The solution was then poured into ice water while stirring, and the precipitate that formed was filtered, washed with water until the filtrate was at neutral pH, and dried to provide the desired bromide (17.6 g) as a pale orange solid. 1H NMR (400 MHz, DMSO-d6) ␦ 2.05 (s, 3H), 2.28 (s, 3H), 7.29 (d, J ⫽ 8.25 Hz, 1H), 7.56 (d, J ⫽ 8.80 Hz, 1H), 9.60 (s, 1H); 13C NMR (100 MHz, DMSO-d6) ␦ 16.71, 23.14, 118.05, 125.48, 130.36, 132.68, 133.39, 137.14, 168.44; HPLC a) column: Phenominex ODS C18 4.6 ⫻ 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA; 1 min hold, 4 ml/min UV detection at 220 nm, 2.95 min retention time; HPLC b) column: Shimadzu Shim-Pack VP-ODS C18 4.6 ⫻ 50 mm, 4 min


gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/ 0.1% TFA, 1 min hold; 4 ml/min, UV detection at 220 nm, 2.87 min retention time (98%); MS (ES) m/z 263 [M⫹H]⫹. 4-Amino-2-chloro-3-methylbenzonitrile. A suspension of the bromide thus obtained (17.5 g, 66.7 mmol) and copper cyanide (7.16 g, 80.0 mmol) in dimethylformamide (200 ml) was heated to 150 C for 5 h. The solution was then cooled and poured into ice water while stirring. The precipitate that formed was filtered, washed with water, and dried. The solid was triturated in refluxing methanol and filtered, and the filtrate concentrated to provide the nitrile (9.94 g) as a brown solid. A suspension of the nitrile thus obtained (9.90 g, 47.4 mmol) in concentrated HCl (50 ml) and EtOH (50 ml) was heated at reflux for 30 min. The solution was concentrated and dried to provide the hydrochloride salt of the desired aniline (9.41 g) as a brown solid. 1H NMR (400 MHz, DMSO-d6) ␦ 2.12 (s, 3H), 6.30 (s, 2H), 6.61 (d, J ⫽ 8.25 Hz, 1H), 7.36 (d, J ⫽ 8.25 Hz, 1H); 13 C NMR (100 MHz, DMSO-d6) ␦ 13.83, 96.86, 112.11, 118.31, 118.85, 132.16, 135.56, 152.52; HPLC a) column: Phenominex ODS C18 4.6 ⫻ 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/ 10% H2O/0.1% TFA; 1 min hold, 4 ml/min UV detection at 220 nm, 2.42 min retention time; HPLC b) column: Shimadzu Shim-Pack VP-ODS C18 4.6 ⫻ 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA, 1 min hold; 4 ml/min, UV detection at 220 nm, 2.31 min retention time (99%); MS (ES): m/z 167 [M⫹H]⫹. 2-Chloro-4-isocyanato-3-methylbenzonitrile. To a mixture of the hydrochloride salt of 4-amino-2-chloro-3-methylbenzonitrile (5.78 g, 28.4 mmol) in CH2Cl2 (230 ml) at 0 C, NaHCO3 (23.9 g, 284 mmol) was added followed by 20% phosgene in toluene (60.2 ml, 114 mmol) over 30 min. The mixture was allowed to warm to room temperature and stir for 1.5 h. The mixture was filtered and the filtrate was concentrated. The residue was azeotroped with toluene (3 ⫻ 100 ml) and concentrated to provide the isocyanate (5.45 g) as a pale, orange solid, which was used immediately in the next step without further purification. (7R,7aS)-2-Chloro-4-(7-hydroxy-1,3-dioxotetrahydropyrrolo[1,2-c]imidazol2-yl)-3-methylbenzonitrile (BMS-564929). A suspension of 2-chloro-4-isocyanato-3-methylbenzonitrile (5.45 g, 28.3 mmol), diisopropylethylamine (5.95 ml, 34.1 mmol), 4 Å molecular sieves (5 g), and (3R)-3hydroxy-l-proline methyl ester (7.37 g, 28.4 mmol) in CH2Cl2 (300 ml) was stirred at room temperature for 1.5 h. Diazabicycloundecane (5.10 ml) was then added, and after 18 h at room temperature, the suspension was filtered, the solid was triturated with acetone and refiltered, and the combined filtrates were concentrated in vacuo. The residue was chromatographed on silica gel (CH2Cl2/CH3OH; 49:1 to 9:1 gradient) to provide BMS-564929 (4.08 g) as a white solid. 1H NMR (400 MHz, DMSO-d6) ␦ 2.05–2.11 (m, 1H), 2.15–2.22 (m, 1H), 2.20, 2.24 (s, 3H), 3.29 –3.33 (m, 1H), 3.59 –3.68 (m, 1H), 4.42– 4.50 (m, 2H), 5.64, 5.72 (d, J ⫽ 3.85, 3.30 Hz, 1H), 7.22, 7.51 (d, J ⫽ 8.25 Hz, 1H), 7.96 (d, J ⫽ 8.25, 1H); 13 C NMR (100 MHz, DMSO-d6) ␦ 15.44, 15.63, 35.49, 35.62, 43.30, 43.41, 68.76, 69.25, 69.84, 112.86, 113.08, 115.79, 128.11, 128.73, 132.07, 136.27, 136.42, 136.85, 137.12, 158.63, 169.09, 169.60; HPLC a) column: Phenominex ODS C18 4.6 ⫻ 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA; 1 min hold, 4 ml/min UV detection at 220 nm, 2.07, 2.32 min retention time; HPLC b) column: Shimadzu Shim-Pack VP-ODS C18 4.6 ⫻ 50 mm, 4 min gradient, 10% MeOH/90% H2O/0.1% TFA to 90% MeOH/10% H2O/0.1% TFA, 1 min hold; 4 ml/min, UV detection at 220 nm, 1.93, 2.23 min retention time (97%); HPLC c) column: Daicel Chiralcel OD 4.6 ⫻ 250 mm, isocratic 25% isopropanol/hexanes, 30 min, 1 ml/min, UV detection at 220 nm, 10.99 min retention time (98%); MS (ES): m/z 306 [M⫹H]⫹.

Receptor and binding protein binding assays The human cancer epithelial breast cell lines MDA MB-453 and T47D, which endogenously express AR and progesterone receptor (PR), respectively, were used for radioligand competition binding assays. Binding assays were conducted by incubating BMS-564929 at various concentrations with either [3H]DHT or [3H]progesterone with the cells for 2 h at room temperature. For ER␣ and ER␤, fusion proteins expressed in Escherichia coli, consisting of maltose binding protein, a specific biotinylation sequence (BioP), an enterokinase cleavage site, and either the ER␣ or ER␤ LBD was used. Binding reactions were conducted by incubating ER␣ and ER␤ LBD with BMS-564929 and [3H]E2 for 2 h at room


temperature. Specific binding activity to the mineralocorticoid receptor (MR) by BMS-564929 was evaluated by competition binding assay using kidney cytosolic preparations and [3H]aldosterone. The kidneys were obtained from adrenalectomized rats to remove the endogenous source of aldosterone and to increase the MR concentration in the cytosol of kidney cells. Binding reactions were incubated for 2 h on ice in the presence of excess mifepristone (RU486; Sigma, St. Louis, MO) to block nonspecific glucocorticoid receptor (GR) binding. A fluorescence polarization based assay (Glucocorticoid Receptor Competitor Assay Kit Red; PANVERA, Madison, WI) was used for GR binding, as per manufacturer recommendations. Inhibitory constants (Ki, app) defining apparent binding affinity of test compounds to intracellular receptors were calculated from the observed inhibition of natural ligand binding at multiple concentrations of test compound. SHBG (Research Diagnostics) binding was performed using a standard charcoal assay. Reagents: 1 mg lyophilized SHGB powder (Tris), [3H]DHT (NEN Life Science Products, Boston, MA), 3% charcoal, and 0.4% Dextran in PBS; binding buffer: 50 mm Tris, pH 7.6, 100 mm NaCl, 1 mm EDTA, 1 mm DTT, and mock lysate (3.5 ␮g /100 ␮l buffer); stock solutions: stock SHBG protein: 1 mg/ml in water ⫽ 20 ␮m; stock [3H]DHT ligand: 9 ␮m; DHT: 10 mm in DMSO; BMS 564929: 10 mm in DMSO. Compounds diluted in binding buffer were added to 40 nm [3H]DHT and 20 nm SHBG protein in 200 ␮l volume and incubated for 1 h at room temperature. Total binding: 40 nm [3H]DHT and 20 nm SHBG protein in 200 ␮l volume; nonspecific binding: 40 nm [3H]DHT and 20 nm SHBG protein and 1 mm cold DHT in 200 ␮l volume. At the end of the incubation period, 200 ␮l of the charcoal solution (3% containing 0.04% dextran) was added to 200 ␮l of the reactions and shaken for 15 min before centrifugation. Supernatant (200 ␮l) was then transferred to the wells of a 24-well white Optiplate; 200 ␮l of scintillant were added with mixing. Radioactivity counts were read in Topcount.

Stable AR-luciferase reporter transactivation assays Functional transactivation assays were conducted to assess the potency and efficacy of androgen agonists in muscle and prostate cell backgrounds. We stably transfected C2C12 mouse myoblast (ATCC, Manassas, VA) and rat prostate epithelial (PEC) cell lines (obtained from Dr. Douglas Hixson, Rhode Island Hospital, Providence, RI) with both full-length rat AR and the enhancer/luciferase reporter. The enhancer/ reporter construct, containing the 2XDR-1 androgen-specific response element was developed by random mutagenesis of an AR/GR consensus response element sequence and has been used as an AR-specific response element based on its AR selectivity demonstrated in CV-1 cells (40). We plated both cell lines in 96-well format at 6000 cells per well in high glucose DMEM without phenol red (Life Technologies, Inc., Rockville, MD; catalog no. 21063-029) containing 10% charcoal and dextran treated FBS (HyClone, Logan, UT; catalog no. SH30068.02), 50 mm HEPES Buffer (Life Technologies, Inc.; catalog no. 15630-080), 1⫻ MEM Na pyruvate (Life Technologies, Inc.; catalog no. 11360-070), 0.5⫻ antibiotic-antimycotic, 800 ␮g/ml geneticin (Life Technologies, Inc.; catalog no. 10131-035), and 800 ␮g/ml hygromycin ␤ (Life Technologies, Inc.; catalog no. 10687-010). Forty-eight hours later, we added ligands (10 ␮l/well) in fresh media. After an additional 24 h, we used the Steady-Glo Luciferase Assay System to detect activity according to the manufacturer’s instructions (Promega, Madison, WI; catalog no. E2520).

Enzymatic assays A CYP19 aromatase inhibition assay using human CYP19⫹P450 Reductase Supersomes GENTEST P260 was used to measure inhibition of aromatase by BMS-564929. IC50s were determined using assay conditions recommended by the manufacturer (BD Gentest Corp., San Jose, CA).


d 15 after surgery. Animals were dosed (vol/wt) at 1 ml/kg body weight. T propionate (TP) was dosed once daily sc in a 10% ethanol/90% peanut oil vehicle as a reference compound (0.03–10 mg/kg). After 14 d of treatment, the animals were killed by carbon dioxide asphyxiation, the levator ani and the ventral prostate were surgically removed and weighed, and serum was collected for LH measurements. All experiments were conducted in accordance with regulations of the Animal Care and Use Committee of the Bristol-Myers Squibb Co., in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

Rat LH measurements Rat LH was quantitatively determined by RIA using a Biotrak [125I] kit (Amersham Pharmacia Biotech, Piscataway, NJ) under the manufacturer’s protocol.

Statistical analysis Data are presented as means ⫾ sd for each group. Statistical analysis of the in vivo data were performed by a one-way ANOVA. When a significant F ratio was identified, groups were compared using Fisher’s protected least significant difference post hoc test. P ⬍ 0.05 was considered to be significant.

X-ray crystallography The rat AR LBD cDNA, from amino acids 646 –901 (rat numbering, human numbering 664 –919) was cloned as previously described (41) and included an N-terminal polyhistidine tag and a thrombin cleavage site. Because the rat and human AR LBD are identical at the protein level, the human numbering scheme was used for discussion and structural analysis. The rat AR LBD-BMS-564949 complex was crystallized at 20 C by vapor diffusion in the hanging drop mode using a 3 mg/ml concentration of AR LBD-BMS-564929 complex as described previously (41). The crystals have the symmetry of the space group P212121 with unit cell dimensions a ⫽ 55.6 Å, b ⫽ 65.6 Å, c ⫽ 70.8 Å. Data to 3.0 Å resolution were collected on a Rigaku R-AXIS IV2⫹ Imaging Plate system mounted on a Rigaku RU-300 rotating-anode x-ray generator (Rigaku-MSC, Woodlands, TX). The data were processed and scaled using the HKL package (HKL Research, Inc. Charlottesville, VA). The crystals are isostructural to the AR LBD-DHT structures previously determined (41, 42). The initial model was subjected to rigid-body refinement followed by positional and simulated-annealing refinements using CNX (Accelrys, Inc., San Diego, CA). At the end of refinement, the structure has an R factor of 24.9% with a total of 2032 atoms (2003 protein atoms, 21 ligand atoms, and eight solvent atoms). The coordinates of androgen receptor complexed with BMS-564929 have been deposited to the PDB (Protein Data Bank; www.rcsb.org).

Results AR binding affinity of BMS-564929

Competition binding assays with the appropriate radioligands were used to determine the binding affinity of BMS564929 for AR as well as for PR, estrogen receptors ␣ and ␤ (ER␣, ␤), the GR, and the MR. BMS-564929 is a high affinity ligand for AR with a Ki of 2.11 ⫾ 0.16 nm (Table 1). This compound is more than 1000-fold selective for AR vs. ER␣ and ␤, GR, and MR, and approximately 400-fold selective vs. PR (data not shown). TABLE 1. In vitro activities of BMS-564929 (BMS) and T

Rodent muscle and prostate tissue growth assays Matched sets of castrated, sexually mature Harlan Sprague Dawley rats (42–56 d old, 200 –250 g) were dosed once daily by oral gavage with BMS-564929 (0.00001–10 mg/kg) in solution/suspension of 80% PEG 400 and 20% Tween 20 (PEG/TW, both obtained from Sigma Chemical) for 14 d. Two control groups, one sham operated intact and one castrated, were dosed orally with the PEG/TW vehicle only, beginning on

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AR binding Ki (nM) C2C12 EC50 (nM) PEC EC50 (nM) SHBG IC50 (nM) Aromatase IC50 (nM)

BMS

T

2.11 ⫾ 0.16 0.44 ⫾ 0.03 8.66 ⫾ 0.22 ⬎30,000 ⬎30,000

0.25 ⫾ 0.03 2.81 ⫾ 0.48 2.17 ⫾ 0.49 7⫾1 740 ⫾ 2

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Transcriptional activation of AR in rodent skeletal muscle and prostate epithelial stable cell lines by BMS-564929

The potency, efficacy, and in vitro selectivity of BMS-564929 were measured in functional transactivation assays in muscle and prostate cell backgrounds to assess activity in cellular contexts where appropriate levels of endogenous cofactors for each tissue type are expressed. Varying concentrations of T and BMS-564929 were tested using the mouse myoblast (C2C12) and rat PEC cell lines, each stably transfected with rat AR and an androgen-specific response element (ARE)-driven luciferase reporter (Fig. 3) (40). BMS564929 exhibited a potency (EC50, calculated as the concentration at which 50% of the maximum stimulatory effect of DHT is achieved) of 0.44 ⫾ 0.03 nm compared with 2.81 ⫾ 0.48 nm measured for T in the C2C12 myoblast cell line (Table 1). In the PEC cell line, the EC50 for BMS-564929 was 8.66 ⫾ 0.22 nm, compared with 2.17 ⫾ 0.49 nm for T. Both compounds achieved equivalent maximal stimulatory efficacy in each cell line. These data suggest that BMS-564929 is approximately 20-fold more potent at induction of luciferase reporter gene expression in muscle cells vs. prostate cells in vitro. This is in contrast to that for T, which showed similar potency in both cell backgrounds, suggesting that endogenous cofactors complementing the transcriptional machinery in each cell line are differentially recruited by the two ligands. BMS-564929 shows no measurable activity in functional transactivation assays with ER␣/␤, GR, MR, or PR at concentrations up to 30 ␮m (data not shown). BMS-564929 does not bind to SHBG or inhibit CYP19 aromatase

In addition to selective binding to intracellular hormone receptors, androgens and other sex steroid hormones can interact with SHBG to trigger a signal transduction pathway

FIG. 3. In vitro activation of AR-mediated luciferase reporter gene induction in C2C12 and PEC cells with testosterone (A) and selective AR modulator BMS-564929 (B) relative to DHT.


via a membrane receptor for SHBG, using cAMP as a second messenger (43, 44). This mechanism is believed to be partly responsible for non-AR-mediated effects of T on gene activation in target cells. To evaluate BMS-564929 binding to the SHBG we used increasing concentrations of BMS-564929 to compete [3H]DHT from purified SHBG. In addition to its androgenic and anabolic activities, T undergoes metabolic conversion to E2 by CYP19 (aromatase), and this process plays an important role in the regulation of gonadotropin feedback, several brain functions, bone remodeling, and lipid metabolism. We measured the ability of BMS-564929 and T to inhibit aromatase to assess the potential for interference with E2 production. The results (Table 1) show that BMS564929 does not interact appreciably with SHBG, and does not significantly inhibit aromatase activity at concentrations up to 30 ␮m, indicating that BMS-564929 is unlikely to exhibit or interfere with these T-mediated physiological effects in humans. Effects of BMS-564929 on tissue growth and serum LH concentrations after 2 wk of treatment in mature castrated rats

We evaluated the effects of BMS-564929 in sexually mature, castrated male rats, a well-characterized animal model for studying the anabolic or androgenic effects of AR modulators on skeletal muscle and sex accessory tissues (45, 46). Castration of mature male rats results in the rapid involution and atrophy of both levator ani muscle and prostate, reaching a steady state approximately 10 d after castration. Increases in prostate and levator ani wet weights after drug treatment serve as indicators of androgenic and anabolic activities, respectively. We used TP as the positive control due to its superior pharmacokinetic properties and enhanced efficacy both in humans and in preclinical species. In the recovery mode, we dosed rats for 14 d beginning 14 d after surgical castration. Wet weights of prostate and levator ani (normalized by body weight) remained unchanged during the 2-wk treatment period for both sham-operated rats and castrated controls treated with vehicle, relative to pretreatment values. TP treatment (sc) increased both prostate and levator ani weights in a dose-dependent manner beginning with the 0.03 mg/kg dose and reaching normal weight (100% of sham-operated vehicle controls) at 1 mg/kg for the levator ani and 3 mg/kg for the prostate, respectively (Fig. 4A). The potency of TP was calculated as an ED50 value, (defined as the dose at which tissue wet weight reached 50% of the weight of that of sham operated vehicle control animals). TP exhibited an ED50 of 0.21 mg/kg in the levator ani muscle and an ED50 of 0.42 mg/kg in the prostate, exhibiting 2-fold selectivity for the levator ani vs. prostate. In the same model, BMS-564929 (p.o.) showed substantially more potent activity in the levator ani, exhibiting an ED50 of 0.0009 mg/kg in the levator ani and an ED50 of 0.14 mg/kg in the prostate; a net 160-fold selectivity for muscle vs. prostate (Fig. 4B). Approximately 100% muscle stimulation was achieved at 0.1 mg/kg, reaching greater than 125% stimulation at 0.3 and 1 mg/kg. Compared with TP in the same model, BMS-564929 is more than 200 times more potent in stimulation of muscle and 80 times more selective for muscle vs. prostate.



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compound was administered across a wide dose range (1 ␮g to 1 mg/kg) for 8 wk in the same recovery schedule of administration as the 2-wk experiments. A dose-dependent increase in levator ani muscle and prostate wet weight was observed with BMS-564929 treatment, with ED50 values of 0.001 and 0.09 mg/kg for the levator ani and prostate, respectively (Fig. 5). In these same experiments, TP exhibited somewhat less potent and minimally selective activity in each tissue (ED50 of 0.19 and 0.39 mg/kg for the levator ani and prostate, respectively). The results for both compounds are in close agreement with those observed in the 2-wk study, indicating a lack of temporal effect on the relative and absolute potency and selectivity of BMS-564929. In addition to the previously obtained endpoints, whole body adiposity was measured by dual-energy x-ray absorptiometry scan in this experiment. Interestingly, we observed similar dose-dependent decreases in adiposity in both the BMS-564929 and TP treatment groups (4% decrease in fat mass at 1 mg/kg BMS-564929 or 3 mg/kg TP, data not shown). The significant decrease in adiposity suggests the potential for clinical improvements in lean body mass in humans in addition to strictly anabolic effects. Structure of BMS-564929 in complex with the LBD of AR

FIG. 4. Effects of sc testosterone propionate (A) or oral BMS-564929 (B) on wet weight of levator ani muscle and prostate, and on suppression of serum LH (C) in mature castrated male rats after 2 wk of once daily treatment.

The AR LBD structure is analogous to our previously published structures (41, 42), and consists of the nuclear receptor LBD, which has been shown to have three layers of orthogonally packed ␣-helices and four ␤-strands arranged in two ␤-hairpin motifs. For the current structure, AR LBD was initially refined in the absence of ligand. The difference in electron density clearly showed the position of BMS-564929, so the ligand was then fit to the difference in

In man and rodents, LH directly stimulates the production of T in the Leydig cells of the testes. In response to this stimulation, increased levels of T inhibit further production and secretion of LH through feedback inhibition of the hypothalamic-pituitary axis, leading to the suppression of T production in normal healthy individuals. In elderly men treated for age-related sarcopenia, complete inhibition of LH production might possibly lead to a near total depletion of endogenous T, which could subsequently result in E2 deficiency and a consequent hormonal imbalance. To evaluate the potential for BMS-564929 to suppress LH secretion, we measured serum LH levels at the termination of the castrated rat assay. BMS-564929 suppressed secretion of LH with an ED50 of 0.008 mg/kg and showed 9-fold selectivity for levator ani muscle stimulation vs. LH suppression (Fig. 4C). Compared with TP, which has an ED50 of 0.26 mg/kg for LH suppression and essentially no selectivity (1.2-fold) for levator ani stimulation vs. LH suppression, BMS-564929 is approximately 33 times more potent in LH suppression, but 9-fold more selective for levator ani. The selectivity of BMS-564929 on tissue growth is maintained after 8 wk of treatment in mature castrated rats

To determine the effects of longer-term dosing with BMS564929 on androgen-sensitive tissues in castrated rats, the

FIG. 5. Effects of sc testosterone propionate (A) or oral BMS-564929 (B) on wet weight of levator ani muscle and prostate in mature castrated male rats after 8 wk of once daily treatment in recovery mode.

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electron density and the protein-ligand complex was subjected to two additional cycles of CNX refinement. The structure was analyzed after refinement. The His-Tag with the first seven residues of the amino terminus and the carboxylterminal residue of AR LBD were not visible in the electron density and were excluded from the final model. No solvent atoms were found in or near the ligand binding site. The ligand binding site is well defined, and residues L704, N705, M745, M749, R752, F764, M780, and T877 have direct interactions with BMS-564929. There are two possible hydrogen bonds between AR-LDB and BMS-564929 involving residues N705 and R752 (these interactions are described in the Discussion). As seen previously for DHT (41), the majority of the interactions between ligand and protein involve van der Waals interactions. In our previous work, it was seen that, for DHT, hydrophobic residues were shown to interact with the steroid nucleus, and for BMS-564929, the same residues contact the phenyl and bi-cyclic rings while hydrogen bonding interactions anchor each end of the ligand providing affinity and selectivity.


Insight into the molecular basis for tissue selectivity may be gleaned from comparative x-ray crystallographic analysis of various agonists bound to the LBD of the AR (49), as the molecular basis for differential cofactor recruitment is a ligand-mediated process. Comparison of the specific residues in the AR LBD involved in binding to DHT (41) vs. those interacting with BMS-564929 (Fig. 6B) suggest intriguing opportunities for further mechanistic study. Because the x-ray crystal structures of T and DHT with the AR LBD are essentially identical, we chose that with DHT for purposes of discussion, because this is the active androgen in prostate due to local conversion of T by 5␣-reductase. Figure 6A shows the alignment of AR LBD from the DHT (blue) and

Discussion

The results described in this study demonstrate that BMS564929 is a highly potent and selective AR agonist in vitro and in vivo, and it exhibits unprecedented muscle selectivity in an established rodent tissue growth model. In the broadest context, certain components of pharmacological selectivity are inherent to nonsteroidal SARM agents such as BMS-564929, because compounds of this class are not reduced by 5␣reductase or aromatized by aromatase to other hormonally active agents that act to modulate other pathways. The lack of potential nongenomic effects associated with SHBG binding adds a further means of selectivity to BMS-564929 relative to T and DHT. The 20-fold selectivity observed in relevant cell contexts in vitro correlates with the tissue selectivity observed in vivo under varied dosing regimens. This strongly suggests that selectivity has a fundamental molecular basis whereby differential use of endogenous coregulators in each respective cell or tissue type affect the induction of genes responsible for the growth and differentiation of each tissue. In a similar manner, various diverse ER ligands differentially induced unique receptor conformations and subsequently recruited specific coactivators, ultimately leading to tissue selective action (47, 48). Ongoing studies target identification and characterization of the specific AR-regulated genes involved in the growth of each respective tissue, facilitated by the availability of selective tool molecules. We are also conducting further studies to evaluate the potential for additional benefits on bone, body composition, cognition, and sexual function. It is particularly noteworthy that, in both the 2- and 8-wk in vivo experiments in castrated rats, BMS-564929 is able to achieve significant and sustained hyperanabolic activity on skeletal muscle at doses that incompletely restore prostate size to that of normal animals. Should this level of separation of effects extend to the human clinical setting, it would potentially allow more aggressive therapy for abrogation of functional decline than can be presently pursued with T and its analogs.

FIG. 6. A, Protein structural alignment (Maestro; Schrodinger LLC, Portland, OR) of AR LBD from the DHT (blue/cyan) and BMS-564929 (red/yellow) complex. The protein backbone is rendered in ribbon diagram and the active site residues are displayed in stick rendering. B, Superimposition of x-ray cocrystal structures of DHT (green) and BMS-564929 (gray) bound to the AR LBD at 2.0 and 3.0 Å resolution, respectively. Key LBD residues are labeled in yellow. DHT makes binding contacts with R-752, Q-711, and a bifurcated H-bond to N-705 and T-877. BMS-564929 makes contacts with R-752 and N-705, as well as a ␲-edge-face interaction with F-764.


BMS-654929 (red) complexes. Structural alignment shows that the two proteins, including amino acid side-chains, are very similar with a root mean square deviation of 0.58 Å (backbone) and 0.97 Å (all heavy atoms). Both DHT and BMS-564929 engage R752 via the 3-keto group of DHT (2.89 Å) or the CN group of BMS-564929 (3.22 Å). A significant difference for these respective ligands is the interaction of the side chain of F764 with the phenyl ring of BMS-564929 via a ␲-edge to face interaction (5.20 Å, ring centroids). This interaction for BMS-564929 is likely to provide up to 5 kcal/mol binding energy, and may result in steric compression of the LBD in a manner distinct from that induced by DHT. Additionally, the 17␤-hydroxyl group of DHT is within hydrogen bonding distance to both N705 (2.80 Å) and T877 (2.70 Å), whereas the hydroxyl group of BMS-564929 engages only N705 (2.73 Å). Because T877 has been shown to be a critical residue for recognition of agonism/antagonism of AR ligands in the common A877T AR (50) mutant seen in prostate cancer patients, these findings may have significant implications regarding differential activation of AR in prostate. It should also be noted that this particular point mutation allows antagonists to cocrystallize with the AR, which to date has not been possible with the wild-type sequence (42). These key contact differences for the two AR ligands are located in specific helices that cooperatively form a hydrophobic groove critical for recognition of the LxxLL motif of coactivator proteins (51), and ligand binding alters the positioning of these helices, thereby affecting coactivator binding. Because x-ray structural analysis provides only a static picture of ligand-receptor interactions, additional studies are ongoing in these laboratories using dynamic methods to investigate AR helical mobility as it relates to these unique interactions identified through crystallography (52). Several ARregulated genes that are differentially induced in specific tissues by the present SARMs relative to the natural ligands have been identified (Ostrowski, J., J. Lupisella, and W.-P. Yang, unpublished results). We are also investigating the structure-function relationship of additional ligands structurally related to BMS-564929, which induce different phenotypes in rodents to more specifically characterize the importance of selected interactions as it relates to tissue selectivity. Based on the present results, as well as extensive preclinical safety testing, BMS-564929 has advanced to clinical trials. We expect that this compound will be suitable as a once daily oral drug for the treatment of age-related musculoskeletal decline in men with an improved side-effect profile relative to T (e.g. prostate growth, hematopoiesis, liver toxicity). The marked muscle selectivity of BMS-564929 has the potential to provide a greater safety window for the beneficial effects of androgens while reducing the likelihood of deleterious side effects observed with T treatment, most notably the potential for hyperstimulatory effects on the prostate, thereby providing a valuable means for addressing a growing unmet medical need. Acknowledgments We gratefully acknowledge Celia Darienzo and Lifei Wang, and the Department of Pharmaceutical Candidate Optimization at BMS for ex-


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tensive characterization of BMS-564929. We thank Dr. Michael Blanar and Dr. Robert Zahler for their helpful comments. Received June 21, 2006. Accepted September 19, 2006. Address all correspondence and requests for reprints to: Lawrence G. Hamann: Department of Discovery Chemistry, Bristol-Myers Squibb, Pharmaceutical Research Institute, 5 Research Parkway, Wallingford Connecticut 06492. E-mail: [email protected]. Present address for Y.B.: Lexicon Pharmaceuticals, 350 Carter Road, Princeton, New Jersey 08540. E-mail: [email protected]. Present address for K.A.M.: Advinus Therapeutics, Pune, India. Email: [email protected]. Present address for G.J.G.: Product Safety Laboratories, 2394 Route 130, Dayton, New Jersey 08810. E-mail: garygrover@productsafetylabs. com. Disclosure Statement: J.O., J.E.K., J.A.L., M.C.M., B.C.B., S.R.K., C.S., R.S., R.G., P.G.S., A.F., Y.A., K.F.K., J.S.S., and L.G.H. are employed by Bristol-Myers Squibb. Y.B., K.A.M., and G.J.G. were previously employed by Bristol-Myers Squibb. M.C.M., A.F., J.S.S., K.A.M., G.J.G., and L.G.H. have equity interests in Bristol-Myers Squibb.

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