Metabolism of 19-methyl-substituted steroids by human placental aromatase

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Abstract
The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hdyroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substitutent on the enzyme''s regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] 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-hyroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]ester-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 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 18O2 established that 3c generated from 2c retained little 18O (< 3%), while 3c arising from 2e retained a significant amount of 18O (.apprxeq. 70%). After four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 .mu.M, 9.9 .mu.M, and 150 nM for 1c, 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-C19 bond, and oxygen rebound at C19.