Synthesis of Deuterium-Labeled Androst-5-ene-17b ,19-diol and Its 4-Ene Isomer as Internal Standards for the Determination of the 19-Oxygenation of Aromatase Inhibitors Using GC-MS

the conversion of androst-4-ene-3,17-dione (androstenedione) to estrone through three sequential oxygenations of the 19-methyl group. Inhibitors of aromatase have recently attracted interest not only in the treatment of advanced estrogen-dependent breast cancer but also in the elucidation of the spatial aspects of the active-site of the enzyme as well as the still unsolved mechanism of the aromatase reaction. We have previously reported that the androst-4-en-17-one (2), 3-deoxy analog of the natural substrate androstenedione, and its 5-ene isomer 1 (Chart 1) are very potent competitive inhibitors of aromatase, although they have no carbonyl group at C-3 which is thought to be essential for tight binding of the substrate to the active site of aromatase. We have recently reported that some competitive inhibitors of aromatase, 6-alkyl and 6-oxo androstenediones, can serve as substrates for the enzyme to afford estrogens when they are incubated with human placental microsomes. Thus, it was of interest to investigate the relationship between the aromatase inhibitory activity of the 3-deoxy compounds, 1 and 2, and their ability to act as substrates. It was postulated that the aromatase products of compounds 1 and 2, obtained by incubation with placental microsomes, would be a mixture of the 19-hydroxy and 19-oxo derivatives as well as their 17b-alcohol analogs produced by the action of 17b-hydroxysteroid dehydrogenase in each incubation system. The aromatase-catalyzed 19-oxygenation activity could be determined by GC-MS analysis of the 17b ,19-dihydroxy steroids obtained after NaBH4 reduction of the mixture of 19-oxygenated products. Thus, we synthesized the deuterium labeled 17b ,19-diols 6 and 15-d4 as internal standards.