Synthesis of 6β-hydroxy androgens from a 3,5-diene steroid precursor to test for cytochrome P450 3A4-catalyzed hydroxylation of androstenedione

6 beta-Hydroxytestosterone is a biomarker for the activity of human cytochrome P450 3A4 (P450 3A4), the major drug metabolizing cytochrome P450 enzyme. Despite its significance, efficient routes for the chemical synthesis of 6 beta-hydroxytestosterone are rare. In this study, 6 beta-hydroxytestosterone was synthesized through the oxidation of a 3,5-diene precursor under the Uemura-Doyle reaction conditions using a dirhodium catalyst in the presence of tert-butylhydroperoxide. Mechanistic studies showed that some oxygen is incorporated from molecular oxygen and C-H abstraction is partially rate-limiting. This reaction was used to synthesize 6 beta-hydroxyandrostenedione, which was used as a standard to test the hypothesis of whether P450 3A4 catalyzes the hydroxylation of androstenedione. Upon incubation of P450 3A4 with androstenedione, a hydroxylated product was formed, which matched the retention time of synthetic 6 beta-hydroxyandrostenedione. This reaction can be exploited to study other biochemical processes involving compounds with a 6 beta -hydroxy-3-keto-A4 steroid backbone.

Automated summary

In this study, 6 beta-hydroxytestosterone was synthesized through oxidation reaction and used to synthesize 6 beta-hydroxyandrostenedione as a standard substance. The synthesis route and catalytic activity of P450 3A4 were successfully investigated.