Clues to the development of mechanism-based inactivators of 3α-hydroxysteroid dehydrogenase: comparison of steroidal and nonsteroidal Michael acceptors and epoxides

Abstract

A series of steroidal and nonsteroidal Michael acceptors that represent reaction products for 3α-hydroxy steroid dehydrogenase were synthesized and evaluated as potential enzyme-generated inactivators. Introduction of exocyclic olefins either at C-2 or C-6 produced inhibitors with high affinity for the enzyme (0.05 to 5.0 μM). However, despite this affinity, none of these compounds produced time-dependent inactivation of the enzyme. By contrast, analogs based on l-phenyl-2-propen-l-one were stoichiometric inactivators of the enzyme and ease of turnover of the parent latent Michael acceptor depended on the presence of an electron-withdrawing substituent at the para position. A series of steroidal and nonsteroidal epoxides in which the oxiranyl oxygen could be substituted for the 3-ketone (the acceptor carbonyl of a steroid substrate) were also synthesized and evaluated as potential mechanism-based inactivators. Steroidal 2α,3α-, and 3α,4α-epoxides as well as 3α- and 3β-spiroepoxides did not bind to the enzyme and were unable to cause enzyme inactivation in either the presence or absence of pyridine nucleotide. In contrast, nitrostyrene oxides produced time-dependent inactivation, the rate of which was governed by the presence of an electron withdrawing group at the para position. These data indicate that the design of mechanism-based inactivators for 3α-hydroxysteroid dehydrogenase requires the incorporation of electron-withdrawing groups adjacent to the latent enzyme-activated group and, as a result, the turnover and/or reactivity of these compounds is increased. Moreover, these compounds can be modeled on nonsteroids. (Steroids 56:420–427, 1991)