Enzyme-activated inhibitors of steroidal hydroxylases.

Cytochrome P450 monooxygenases (CYP450) of the steroid biosynthetic pathways are highly substrate specific in comparison to the variable specificities of hepatic CYP450 enzymes. Both groups of enzymes catalyze the reductive cleavage of molecular oxygen with transfer of oxygen to the substrate to form hydroxylated derivatives. Those steroids formed in endocrine tissues represent highly specific endocrine/autocrine hormones with enhanced biological potency, while hepatic hydroxylation of steroids reduces their endocrine bioactivities and enhances urinary elimination. Changes of the hormonal milieu of endocrine and peripheral tissues are associated with the development of hyperplastic and/or malignant conditions. Hormone deprivation induces regression of endocrine dependent growth via apoptosis and may also alter growth of hormone insensitive cells by the induction of negative growth factors. Biosynthetic CYP450 enzymes of those steroids that mediate specific disease processes are potential therapeutic targets for selective intervention. This objective can be accomplished by the design of specific pseudo-substrate analogs that will be activated during enzyme-directed catalysis to produce a reactive functional group in the enzyme's active site that will either tightly or irreversibly bind and inactivate the host enzyme. The CYP450 enzymes that hydroxylate the C19 carbon of androgens (aromatase) and the C18 carbon of corticosterone (aldosterone synthase) were selected as target enzymes because they are terminal enzymes of biosynthetic pathways which hydroxylate specific angular methyl groups. Hypersecretion of their respective hormonal products, estrogens and aldosterone, are associated with specific disease conditions. Substrate analogs containing ethynyl, vinyl, or nitrile groups attached to the C19 or C18 methyl groups were enzyme-activated inhibitors. The ethynyl analogs, 19-acetylenic androstenedione (Plomestane) and 18-acetylenic deoxycorticosterone, had nanomolar inhibitory constants (Ki values) and were irreversible inactivators of their target enzymes in animal models.

Enzyme inactivation by potential metabolites of an aromatase-activated inhibitor (MDL 18,962).

MDL 18,962, 19-acetylenic androstenedione, is an enzyme-activated inhibitor of estrogen biosynthesis which is in Phase I clinical evaluations as a potential therapeutic agent for estrogen-dependent cancers. 19-Acetylenic analogs corresponding to the major metabolites of androstenedione were synthesized as potential metabolites of MDL 18,962. These compounds were 19-acetylenic testosterone, the product of 17 beta-hydroxy steroid oxidoreductase, 6 beta-hydroxy- and 6-oxo-19-acetylenic androstenedione, products of P450 steroid 6 beta-hydroxylase and alcohol dehydrogenase, respectively. All of these analogs showed time-dependent inactivation of human placental aromatase activity. The time-dependent Ki and t1/2 at infinite inhibitor concentration (tau 50) were 4.3 nM, 12.0 min for MDL 18,962; 28 nM, 7.8 min for 17-hydroxy analog; 13 nM, 37 min for 6 beta-hydroxy analog; and 167 nM, 6.1 min for the 6-oxo analog. The 19-acetylenic testosterone, a confirmed metabolite from primate studies, was 25% as efficient as MDL 18,962 for aromatase inactivation, while 6 beta-hydroxy- and 6-oxo analogs were 11% and 5%, respectively as efficient as their parent compound. These data indicate that first-pass metabolism of MDL 18,962 does not cause an obligatory loss of time-dependent inhibition of human aromatase activity.