Oxidation of 3-keto steroids in alkaline medium

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated.Excretion studies were conducted with oral applications of 100 mg of 4-hydroxyandrostenedione or 200 mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography–mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry.Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3β-hydroxy-5α-androstane-4,17-dione (2) and 3α-hydroxy-5β-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3ξ,4ξ-dihydroxy-5ξ-androstan-17-one (4, 6–11) except 3α,4α-dihydroxy-5β-androstan-17-one (5).Out of the 17β-hydroxylated analogs 4-hydroxytestosterone (18), 3β,17β-dihydroxy-5α-androstan-4-one (19), 3α,17β-dihydroxy-5β-androstan-4-one (20), 5α-androstane-3β,4β,17β-triol (21), 5α-androstane-3α,4β,17β-triol (26) and 5α-androstane-3α,4α,17β-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products.Conjugation was diverse and included glucuronidation and sulfatation.