[Development of aromatase inhibitors and analysis of their inhibitory activities]

Inhibitors of aromatase are of interest in the treatment of advanced estrogen-dependent breast cancers. In addition, the inhibitors are promising to play as conformational and catalytic probes for the active site of this enzyme, aromatase. There fore, we synthesized a number of steroidal aromatase inhibitors, including suicide substrates, and also studied the mechanism for a time-dependent inactivation of aromatase by the suicide substrates. The mechanism for the aromatase inactivation by 6-oxo-androstenedione (AD) (1), one of the first discovered suicide substrates, was explored using the 19-substituted analogs 2-5 as well as stereo- and/or regio-specifically labeled [3H, 14C]-compound 1. The results indicated that the 4 beta,5 beta-epoxy-19-oxo derivative 7 is a reactive electrophile that irreversibly binds to the active site of aromatase. Studies on the aromatase inhibition by regioisomers of AD, 4-en-6-one 17, 5-en-4-one 18 and 5-en-7-one 19, revealed that the C-3 carbonyl function is not essential for the tight binding of an inhibitor to the active site. 3-Deoxy AD (22) and its 6 alpha,7 alpha-cyclopropano steroid 24 as well as some of 6-alkyl-ADs are among the most potent competitive inhibitors reported so for (K(m) for AD/Ki > 6). Structure-activity relationships of the 6-alkyl-ADs and their 3-deoxy-, delta 1-, delta 6-, and delta 1,6-analogs as aromatase inhibitors showed that aromatase has a hydrophobic binding pocket with a limited accessible volume in the active site in the region corresponding to the beta-side rather than the alpha-side of the C-6 position of the substrate. The 6-alkyl-ADs and their delta 1-analogs were converted into the corresponding estrogens with human placental aromatase, whereas the 3-deoxy steroids 22 and 25 were metabolized to the corresponding 19-oxygenated compounds. The relative apparent K(m) values for the androgens are different from the relative Ki values, indicating that there is a difference between the ability to serve as an inhibitor and that to serve as a substrate. Moreover, it seems likely that the alignment of the substrate AD analogs in the active site would be markedly different from that of the 3-deoxy steroids.