Inhibition of estrogen synthetase (aromatase) by 4-cyclohexylaniline

Abstract

4-Cyclohexylaniline, a structurally simple analog of the drug aminoglutethimide [d, l-3-(4-aminophenyl)3- ethyl-2,6-piperidinedione], was found to be an effective inhibitor of the aromatization of testosterone and androstenedione. With human placental microsomes, 4-cyclohexylaniline was a more potent aromatase inhibitor than d-aminoglutethimide. For androstenedione and testosterone aromatization, competitive inhibition by 4-cyclohexylaniline was observed; a Ki value of 0.14 µm was found with both substrates. A Ki value for d-aminoglutethimide inhibition of androstenedione aromatization of 0.3 µM was obtained. Kinetic analysis of the simultaneous inhibition by 4-cyclohexylaniline and d-aminoglutethimide suggests that both compounds bind to the same site on the enzyme. 4-Cyclohexylaniline and d-aminoglutethimide were also tested for inhibition of androstenedione aromatization in human and rat ovarian microsomes. With the human aromatase, both inhibitors exhibited approximately the same effectiveness; with rat aromatase, however, d-aminoglutethimide was more potent. 4-Cyclohexylaniline and d-aminoglutethimide were also assayed for their inhibition of cytochrome P-450-catalyzed cholesterol side-chain cleavage. When the enzyme from human placenta was used, 4-cyclohexylaniline was 24-fold less effective than d-aminoglutethimide, and when the purified bovine adrenal enzyme was used, it was 16-fold less effective. Thus, 4-cyclohexylaniline exhibits inhibitory specificity toward aromatase. Difference spectral measurements using crude placental microsomes and cholate extracts of these microsomes show that binding of 4-cyclohexylaniline produces a type II spectral change; this is indicative of coordination of the arylamine to the heme-iron of the aromatase cytochrome P-450. Consistent with this spectral finding was the fact that blockade of the amino group of both 4-cyclohexylaniline and d-aminoglutethimide by acetylation resulted in essentially complete loss of inhibitory activity toward aromatase. These findings establish that the arylamine moieties of both 4-cyclohexylaniline and d-aminoglutethimide are essential for their inhibitory actions. The potent aromatase inhibition by 4-cyclohexylaniline indicates that neither the complex glutarimide ring nor the chiral 3-ethyl substituent of d-aminoglutethimide is required for high affinity binding of this class of inhibitor to aromatase P-450. © 1984 by The Endocrine Society.