Study on aromatase reaction mechanism using the inhibitors as probes

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In order to gain insight into the aromatase reaction mechanisms, we explored aromatase reactions of the powerful aromatase inhibitors 3-deoxyandrogens, 3-deoxyandrostenedione (1)and 3-deoxydehydroepiandrosterone (2), in relation to the stereomechanisms of the 19-oxygenation and the C(10)-C(19)bond cleavage reaction.1.Treatment of the 19-oxo derivatives of compounds 1 and 2, steroids 5 and 6, with NaB^2H_4 or NaB^3H_4 produced the corresponding [19,19^<-2>H_2] 19-ols 3 and 4 or [19R^<-3>H]-ols 3 and 4 as well as their [19S^<-3>H] isomers.2.Incubation of [19, 19^<-2>H_4] 3 and 4 with human placental microsomes in the presence of NADPH produced the 19-oxo metabolites 5 and 6, respectively, where the deuterium isotope effect was not observed in each case.3.[19R^<-3>H]- and [19S^<-3>H] 3 and 4 were also incubated with the microsomes described above, and ^3H_2O and ^3HCOOH released into the incubation mixtures were analyzed. The results indicate that a 19-pro-R hydrogen in stereospecifically eliminated in the conversion of Steroid 4 into the 19-oxo steroid 6 whereas a selectivity of 75 : 25 for pro-R : pro-S is observed in the case of steroid 3. In both cases, the C(10)-C(19) bond cleavage reaction, yielding ^3HCOOH, was established.4.Structure-activity relationships of 4-substituted derivatives of inhibitor 1,2α-substituted ADs as well as 2, 4, or 6-substituted estrogens as aromatase inhibitors were also studied in relation to elucidate the spatial nature of the active site of aromatase.