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

Abstract

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.

17 -Hydroxysteroid dehydrogenase: inhibitors and inhibitor design

“…1 l(XIV)). This compound did not require oxidation to a ketone to cause enzyme inactivation, indicating that the powerful electron-withdrawing properties of the trifluoromethyl group were suffi¬ cient to activate the compound (Lawate & Covey 1990 (Ricigliano & Penning 1989 …”

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3α-hydroxysteroid dehydrogenase

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The non-steroidal allylic and acetylenic alcohols 1-(4'-nitrophenyl)prop-2-en-l-ol (I) and l-(4'-nitrophenyl)prop-2-yn-lol (II) are oxidized by homogeneous 3a-hydroxysteroid dehydrogenase to the corresponding a/f-unsaturated ketones 1-(4'-nitrophenyl)prop-2-en-1-one (III) and 1-(4'-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J.

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Improved ethynylation of nitrocarbonyl derivatives by cerium trichloride assisted addition of ethynyl magnesium bromide

“…It was found that in the absence of the lanthanide, the ethynylation reaction was a low yielding process. ethynyl magnesium bromide to nitroarylcarbonyls thereby improving the synthesis of tertiary α-acetylenecarbinols, which are useful substrates for biological studies [12]. The results of this investigation are here presented.…”

“…The reaction proceeded smoothly at 0 °C to give the desired ethynyl carbinol 2a in nearly quantitative yield. Compound 2a had been previously prepared via p-nitrobenzaldehyde ethynylation with ethynyl lithium in 86% yield [12] and with ethynylmagnesium bromide (prepared from acetylene and methylmagnesium bromide.) in an unspecified yield [13].…”

“…(1.22 g, 4.95 mmol), ethynylmagnesium bromide (0.64 g, 4.96 mmol), and 4-nitrobenzaldehyde (0.15 g, 0.99 mmol) in THF (5 mL) were combined following the general procedure, to give the title compound as a light yellow oil[12] (0.174 g, 98%).α-Ethynyl-2-nitrophenylmethanol (2b) Cerium chloride (1.22 g, 4.95 mmol), ethynylmagnesium bromide (0.64 g, 4.96 mmol), and 2-nitrobenzaldehyde (0.15 g, 0.99 mmol) in THF (5 mL) were combined following the general procedure, to give the title compound as a coloured oil (0.12 g, 68%). -IR (film): ν/cm -1 = 3 492, 2 116, 1 526, 1 348.…”