Endocrinology, Vol 114, 2128-2137, Copyright © 1984 by Endocrine Society

ARTICLES

Inhibition of estrogen synthetase (aromatase) by 4-cyclohexylaniline

JT Kellis Jr and LE Vickery

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 microM was found with both substrates. A Ki value for d- aminoglutethimide inhibition of androstenedione aromatization of 0.3 microM 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.

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