Studies directed towards a mechanistic evaluation of inactivation of aromatase by the suicide substrates androsta-1,4-diene-3,17-diones and its 6-ene derivatives aromatase inactivation by the 19-substituted derivatives and their enzymic aromatization.

To gain insight into the mechanistic features for aromatase inactivation by the typical suicide substrates, androsta-1,4-diene-3,17-dione (ADD, 1) and its 6-ene derivative 2, we synthesized 19-substituted (methyl and halogeno) ADD and 1,4,6-triene derivatives 8 and 10 along with 4,6-diene derivatives 9 and tested for their ability to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. 19-Methyl-substituted steroids were the most powerful competitive inhibitors of aromatase (K(i): 8.2-40 nM) in each series. Among the 19-substituted inhibitors examined, 19-chloro-ADD and its 6-ene derivatives (7b and 9b) inactivated aromatase in a time-dependent manner in the presence of NADPH in air while the other ones did not. The time-dependent inactivation was blocked by the substrate AD and required NADPH. Only the time-dependent inactivators 7b and 9b in series of 1,4-diene and 1,4,6-triene steroids as well as all of 4,6-diene steroids 9, except for the methyl compound 9a, served as a substrate for aromatase to yield estradiol and/or its 6-ene estradiol with lower conversion rates compared to the corresponding parent steroids 1,4-diene, 1,4,6-triene and 4,6-diene derivatives. The present findings strongly suggest that the aromatase reaction, 19-oxygenation, at least in part, would be involved in the time-dependent inactivation of aromatase by the suicide substrates 1 and 2, where the 19-substitutent would play a critical role in the aromatase reaction probably though steric and electronic reasons.