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The present study aimed to identify selective androgen receptor modulators (SARMs) with in vivo pharmacological activity. We examined the in vitro and in vivo pharmacological activity of four chiral, nonsteroidal SARMs synthesized in our laboratories. In the in vitro assays, these compounds demonstrated moderate to high androgen receptor (AR) binding affinity, with K(i) values ranging from 4 to 37 nM, and three of the compounds efficaciously stimulated AR-mediated reporter gene expression. The compounds were then administered subcutaneously to castrated rats to appraise their in vivo pharmacological activity. Androgenic activity was evaluated by the ability of these compounds to maintain the weights of prostate and seminal vesicle, whereas levator ani muscle weight was used as a measure of anabolic activity. The maximal response (E(max)) and dose for half-maximal effect (ED(50)) were determined for each compound and compared with that observed for testosterone propionate (TP). Compounds S-1 and S-4 demonstrated in vivo androgenic and anabolic activity, whereas compounds S-2 and S-3 did not. The activities of S-1 and S-4 were tissue-selective in that both compounds stimulated the anabolic organs more than the androgenic organs. These two compounds were less potent and efficacious than TP in androgenic activity, but their anabolic activity was similar to or greater than that of TP. Neither S-1 nor S-4 caused significant luteinizing hormone or follicle stimulating hormone suppression at doses near the ED(50) value. Thus, compounds S-1 and S-4 were identified as SARMs with potent and tissue-selective in vivo pharmacological activity, and represent the first members of a new class of SARMs with selective anabolic effects.

The present study characterized the in vitro androgen receptor (AR) binding affinity, in vitro and in vivo pharmacological activity, and in vivo pharmacokinetics and metabolism of acetothiolutamide, a nonsteroidal AR ligand. AR binding was determined by a competitive binding assay. In vitro AR agonist activity was examined by a cotransfection assay. Acetothiolutamide displayed high AR binding affinity (K(i) = 4.9 +/- 0.2 nM) and full agonist activity in the in vitro studies. Next, the androgenic, anabolic, and antiandrogenic activity of acetothiolutamide was evaluated in a castrated immature rat model. In this animal model, acetothiolutamide exhibited an overall negligible androgenic effect, but a statistically significant anabolic effect at high subcutaneous doses. Also, acetothiolutamide demonstrated a noticeable antiandrogenic effect in castrated rats supplemented with testosterone propionate. To understand the causes for the observed disparity between in vitro and in vivo activities, pharmacokinetics and metabolism of acetothiolutamide were studied in male Sprague-Dawley rats. Acetothiolutamide was rapidly cleared from rat plasma (clearance of about 45 ml/min/kg) in a concentration-independent manner after i.v. dosing. Acetothiolutamide was completely absorbed after subcutaneous administration, but not bioavailable after oral dose. In the metabolism study, the unchanged molecule and its metabolites in urine and fecal samples were detected by high-performance liquid chromatography-mass spectrometry. The structures of major metabolites were elucidated with liquid chromatography-tandem mass spectrometry. After i.v. administration, acetothiolutamide was excreted in urine and feces as unchanged drug and a variety of metabolites. Oxidation, hydrolysis, and sulfate conjugation of phase I metabolites were the major metabolic pathways of acetothiolutamide in rats. Overall, the high plasma clearance of acetothiolutamide, due to its extensive hepatic metabolism, likely contributed to its lack of androgenic activity in vivo.