Metabolism of 19-Methyl-Substituted Steroids by Human Placental Aromatase

The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (lc)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-l-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring 0₂ and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with ¹⁸0₂ established that 3c generated from 2c retained little ¹⁸0 (<3%), while 3c arising from 2e retained a significant amount of ¹⁸0 (≈70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (K₁= 81 nM, 11 μM, 9.9 μM, and 150 nM for lc, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e. The failure of 2c, 2e, and 3c to undergo aromatization was rationalized in the context of a mechanistic proposal for the third oxygenation of aromatase requiring hydrogen abstraction at C₁ of 19,19-dihydroxyandrostenedione, homolytic cleavage of the C₁₀-C_l9bond, and oxygen rebound at C₁₉.