Synthesis and In Vitro Metabolic Profiling of the Selective Androgen Receptor Modulator (SARM) LY305

2-Chloro-4-[[(1R,2R)-2-hydroxy-2-methyl-cyclopentyl]amino]-3-methyl-benzonitrile (LY305) is a transdermal selective androgen receptor modulator (SARM) that has been under development as a potential therapeutic for conditions involving muscle wasting, osteoporosis, or hypogonadism. Due to its proven anabolic effects, its potential (and illicit) use in sport must be taken into consideration, necessitating information on its biotransformation for the implementation of adequate target analytes into routine doping control analytical procedures. In this study, the synthesis of LY305 and in vitro-derived metabolites of the SARM are described.

LY305 was synthesized by a Buchwald-Hartwig amination using 2-chloro-4-iodo-3-methylbenzonitrile and (1R,2R)-2-amino-1-methyl-cyclopentanol. The in vitro metabolism experiments were conducted using human liver microsomes (HLM) and the S9 fraction to allow for studying phase-I and phase-II biotransformations. For the detection and separation of LY305 and its metabolites, liquid chromatography-high-resolution tandem mass spectrometry was used.

Overall, 18 metabolites were detected, nine of which were identified as phase-I metabolites and an additional nine were attributed to phase-II conjugates. Metabolic reactions were as follows: hydroxylation, dehydrogenation, oxidation, oxidation and hydroxylation, O-glucuronidation, hydroxylation with subsequent glucuronidation, and bis-hydroxylation with subsequent glucuronidation.

The transdermal SARM LY305 was successfully synthesized and subjected to in vitro metabolic studies, yielding chromatographic and mass spectral data that support improving comprehensive anti-doping tests. To the best of our knowledge, no published experimental data exist that report on the in vitro metabolic profile of LY305, a substance that might undergo further (pre)clinical evaluation.