Identification of drostanolone and 17-methyldrostanolone metabolites produced by cryopreserved human hepatocytes

Methyldrostanolone (2alpha,17alpha-dimethyl-17beta-hydroxy-5alpha-androstan-3-one) was synthesized from drostanolone (17beta-hydroxy-2alpha-methyl-5alpha-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3alpha- (major) and 3beta-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3alpha-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d(0) and TMS-d(9)) derivatives. Finally, on the basis of the GC-MS and (1)H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.