Studies on the catalytic function of aromatase: aromatization of 6-alkoxy-substituted androgens

Abstract

To gain insight into the catalytic function of aromatase, we studied aromatization of a series of 6α- and 6β-ether-substituted (methoxy, ethoxy, and n-butoxy) androst-4-ene-3,17-dione (AD) steroids ( 1 and 2) and their androsta-1,4-diene-3,17-dione (ADD) derivatives ( 3 and 4) with human placental aromatase by gas chromatography–mass spectrometry (GC–MS). Among the steroids examined, 6β-methoxy and 6β-ethoxyADDs ( 4a and 4b) are suicide substrates of aromatase. All of the steroids were found to be converted into the corresponding 6-alkoxy estrogens. Introduction of the alkoxy groups at C-6 of AD or ADD decreased the ability of these to serve as a substrate of aromatase. In 6α-alkoxy steroid series, compounds 1 and 3, the aromatization rate increased by elongating the 6-methoxy group up to the n-butoxy group whereas, in the 6β-isomers series, 2 and 4, the rate decreased due to this structural modification. 6β-Alkoxy steroids, 2 and 4, including the suicide substrates, were extremely poor substrates for the aromatization reaction. Apparent K m values obtained for 6α-alkoxy compounds 1 and 3 were similar to each other, ranging from 92 to 111 nM, as shown by their previously-obtained K i values. The findings indicate that the stereochemistry as well as the bulkiness of the 6-ether-substituent play an important role in the ability to serve as a substrate. It is also predicted that the aromatization reaction and the mechanism-based inactivation reaction would be related and have a definite partition number which is characteristic to the compound in a series of suicide substrates.