Gas chromatography–mass spectrometric analysis of oxidative reactions of [19,19- 2H 2]19-hydroxy-3-deoxy androgens by placental aromatase : Absence of a deuterium-isotope effect

Abstract

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxidations of the 19-methyl group. 3-DeoxyAD ( 1) and its 5-ene isomer 4 are potent and good competitive aromatase inhibitors, which are converted by aromatase to the aldehyde derivatives 3 and 6, respectively, through 19-hydroxy intermediates 2 and 5, respectively. To study the deuterium isotope effect on the conversions of 19-ols 2 and 5 into the corresponding 19-als 3 and 6, we initially synthesized [19,19- 2H 2]19-ols 2 and 5 starting from the corresponding non-labeled 19-als 3 and 6 through NaB 2H 4 reduction of the 19-aldehyde group, followed by oxidation with pyridinium dichromate, and a subsequent NaB 2H 4 reduction. Approximately 1:1 mixtures of non-labeled ( d 0) and deuterated ( d 2) 19-ols 2 and 5 were separately incubated with human placental microsomes in the presence of NADPH under an air atmosphere, and deuterium contents of the recovered substrates and the 19-aldehyde products were determined by gas chromatography–mass spectrometry. In each experiment, the ratio of d 0 to d 2 of the recovered substrate along with that of d 0 to d 1 of the product were identical to the d 0 to d 2 ratio of the employed substrate irrespective of the incubation time, indicating that the 19-oxygenations of the 3-deoxy steroids 2 and 5 proceeded without a detectable isotope effect, as seen in the aromatization sequence of the natural substrate AD.