Gas chromatography-mass spectrometric analysis of oxidative reactions of [19,19-2H2]19-hydroxy-3-deoxy androgens by placental aromatase

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 [19,19-2H2]19-ols 2 and 5 starting from the corresponding non-labeled 19-als 3 and 6 through NaB2H4 reduction of the 19-aldehyde group, followed by oxidation with pyridinium dichromate, and a subsequent NaB2H4 reduction. Approximately 1:1 mixtures of non-labeled (d0) and deuterated (d2) 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 d0 to d2 of the recovered substrate along with that of d0 to d1 of the product were identical to the d0 to d2 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.