Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase

Two non-steroidal mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver have been synthesized: 1-(4'-nitrophenyl)-2-propen-1-ol (I), and 1-(4'-nitrophenyl)-2-propyn-1-ol (II). Both of these compounds inactivate homogeneous 3 alpha-HSD in a time- and concentration-dependent manner only in the presence of NAD+. Analysis of the pseudo-first-order inactivation data gave a Kd of 1.2 mM for the allylic alcohol and a t1/2 (time required to promote a 50% loss of enzyme activity) for the enzyme of less than 10 s at saturation. Similar inactivation studies with the acetylenic alcohol gave a Kd of 1.5 mM and a t1/2 for the enzyme of 9.9 min at saturation. The allylic alcohol and acetylenic alcohol are oxidized stereoselectively by the enzyme, yielding a Km of 2.0 mM and a Vmax. of 0.58 mumol/min per mg for the allylic alcohol and a Km of 0.75 mM and a Vmax. of 0.29 mumol/min per mg for the acetylenic alcohol. Effective partition ratios (kcat./kinact.) are low for both alcohols: for the allylic alcohol, 5.3; and for the acetylenic alcohol, 141. H.p.l.c. indicates that the Michael acceptors 1-(4'-nitrophenyl)-2-propen-1-one (III) and 1-(4'-nitrophenyl-2-propyn-1-one (IV) are the products of the enzymic oxidation of the corresponding alcohols. The latter compound (IV) was trapped as its monothioether adducts before h.p.l.c. analysis. The Michael acceptors III and IV inactivate the 3 alpha-HSD in the absence of NAD+ at a rate too high to accurately measure and titrate the enzyme in a stoichiometric manner. Enzyme inactivated by I and NAD+, II and NAD+, III or IV is not re-activated by gel filtration or dialysis, implying a stable covalent bond has been formed between the enzyme and the inactivators. A screen of five other HSDs, and two aliphatic alcohol dehydrogenases, indicates that alcohol I is a selective inactivator of rat liver 3 alpha-HSD. It is concluded that 3 alpha-HSD generates non-steroidal alkylating agents (III and IV) that potently inactivate the enzyme with low effective partition coefficients. This report of non-steroidal mechanism-based inactivators of 3 alpha-HSD may provide a precedent for the development of related compounds to act as suicide substrates of other HSDs.