Studies directed toward a mechanistic evaluation of aromatase inhibition by androst-5-ene-7,17-dione: Time-dependent inactivation by the 19-nor and 5β,6β-epoxy derivatives

Abstract

To gain further insight into the mechanism for inactivation of aromatase by androst-5-ene-7,17-dione ( 1) and its 19-nor analog 4, 10β-oxygenated steroids 5 and 6, Δ 1(10)-steroid 7, and 19-oxo-5β,6β-epoxy compound 8 were synthesized and tested for their ability to inhibit aromatase in human placental microsomes. All of the steroids studied inhibited the enzyme in a competitive manner with apparent K i values ranging from 1.1 to 35 μM. The Δ 1(10)-compound 7 was the most potent inhibitor among them. All of the inhibitors caused a time-dependent inactivation of aromatase in the presence of NADPH in air with the k inact values ranging from 0.036 to 0.190 min −1. The substrate androstenedione protected the inactivation, but a nucleophile, l-cysteine, did not, in each case. In contrast, each inhibitor did not cause the time-dependent inactivation in the absence of NADPH. These results show that the 5β,6β-epoxide 8 and/or the dienone 7 are not a reactive electrophile involved in the irreversible binding to the active site of aromatase during the mechanism-based inactivation caused by the suicide substrates 1 and/or 4.