Several 7 alpha-thiosubstituted derivatives of androstenedione have demonstrated effective inhibition of aromatase, the cytochrome P450 enzyme complex responsible for the biosynthesis of estrogens. Introduction of an additional double bond in the A ring resulted in 7 alpha-(4'-amino)phenylthioandrosta-1,4-diene-3,17-dione (7 alpha-APTADD), a potent inhibitor that inactivated aromatase by an enzyme-catalyzed process. Additional 7 alpha-thiosubstituted androsta-1,4-diene-3,17-dione derivatives were designed to further examine enzyme-catalyzed inactivation. Two halogenated and one unsubstituted 7 alpha-phenylthioandrosta-1,4-diene-3,17-diones were synthesized via an acid-catalyzed conjugate Michael addition of substituted thiophenols with androsta-1,4,6-triene-3,17-dione. Two 7 alpha-naphthylthioandrosta-1,4-diene-3,17-diones were synthesized via either acid-catalyzed or based-catalyzed conjugate Michael addition of substituted thionaphthols with androsta-1,4,6-triene-3,17-dione. These agents were evaluated for aromatase inhibitory activity in the human placental microsomal preparation. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent Ki's ranging from 12 to 27 nM. Furthermore, these compounds produced time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no aromatase inactivation was observed in the absence of NADPH. This enzyme-activated irreversible inhibition, also referred to as mechanism-based inhibition, can be prevented by the substrate androstenedione. Thus, the apparent Ki values for these inhibitors are consistent with earlier studies on 7 alpha-substituted competitive inhibitors that indicate bulky substituents can be accommodated at the 7 alpha-position.(ABSTRACT TRUNCATED AT 250 WORDS)