Inhibition of estrogen synthetase (aromatase) by 4-cyclohexylaniline.
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.
D000616:Aminoglutethimide / Q000494:pharmacology; D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D047072:Aromatase Inhibitors; D002458:Cell Fractionation; D004958:Estradiol / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010053:Ovary / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D051381:Rats; D013739:Testosterone / Q000378:metabolism
D000814:Aniline Compounds; D047072:Aromatase Inhibitors; D000616:Aminoglutethimide; D013739:Testosterone; D004958:Estradiol; C028146:4-cyclohexylaniline; D010088:Oxidoreductases