Probing the binding pocket of the active site of aromatase with 6-ether or 6-ester substituted androst-4-ene-3,17-diones and their diene and triene analogs

Abstract

A series of 6-ester- ( 3 and 4) and 6-ether- ( 7 and 8) substituted androst-4-ene-3,17-diones (androstenediones) and their 1,4-diene analogs ( 5 and 6, and 9 and 10) as well as C6-substituted 4,6-diene and 1,4,6-triene steroids 11 and 12 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between various substituents and inhibitory activity. All of the inhibitors synthesized blocked aromatase in a competitive manner. The inhibitory activities of all of the steroids, except for the 6β-benzoates 4g and 6h and the 6β-acetate 6a, were fairly effective to very powerful ( K i: 7.0–320 nM). The 6α- n-hexanoyloxy- and 6α-benzyloxyandrostenediones ( 3e and 7e) were the most potent inhibitors ( K i: 7.0 nM each). In the series of 4-ene and 1,4-diene steroids, the 6α-substituted steroids had higher affinity for the enzyme than the corresponding 6β-isomers. In the 1,4-diene steroid series, 6β-substituted steroids 6a, e, g, and 10a, b, e caused a time-dependent inactivation of aromatase, whereas their 6α-isomers 5 and 9 essentially did not. The ether-substituted 1,4,6-trienes 12 inactivated the enzyme in a time-dependent manner; in contrast, their 4,6-diene analogs 11 did not. The substrate androstenedione blocked the inactivation, but no significant effect of L-cysteine was observed. Based on molecular modeling with the PM3 method, along with the present inhibition and inactivation results, it is thought that both the steric effects of the 6-substituents as well as the electronic effects of the C-6 oxygen functions play a critical role in the binding of inhibitors to the active site of aromatase.