19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxyge nations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent com petitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated w ere analyzed by gas chromatography-mass spectrometry. Inhibitors 1 and 4 we re found to be oxygenated with aromatase to produce the corresponding 19-hy droxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17 beta-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that t he 5-ene steroid 4 was surprisingly a good substrate for the aromatase-cata lyzing 19-oxygenation with the V-max value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K-m val ue for steroids 1 and 4 obtained in this study is opposite from the relativ e K-i value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an in hibitor of aromatase and a binding suitable for serving as a substrate of t he enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromat ase. (C) 2000 Elsevier Science Ltd. All rights reserved.