Biomedical subjects
Publications and source records attributed to J E Herz.
The 17O NMR spectra of cholesterol and 31 other steroid alcohols, esters, ketones, and acids enriched by synthesis with 17O from H2(17)O have been observed under ordinary operating conditions, and correlations between 17O chemical shift and structure have been adduced. Spectra-structure correlations for these steroids are in conformity with those previously adduced with simpler compounds by others.
As part of a program directed towards the chemical syntheses of potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism, several routes have been explored for the preparation of 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (IV). These investigations led to a remarkably specific and efficient side-chain oxidation of I. For example, treatment of the acetate of I with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid for 3.5 h at -2 degrees C gave a crude product consisting of 3 beta-acetoxy-24-trifluoroacetoxy-5 alpha-chol-8(14)-en-15-one (XI), 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII), and 3 beta, 24-diacetoxy-5 alpha-chol-8(14)-en-15-one (XIII) in yields of 58%, 8%, and 3%, respectively, by HPLC analysis. XI was readily hydrolyzed to XII upon treatment with triethylamine in methanol at room temperature. Oxidation of XII with Jones reagent gave 3 beta-acetoxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (XVIII) from which its methyl ester (IX) was prepared by treatment with diazomethane. Mild alkaline hydrolysis of XVIII gave the 3 beta-hydroxy-delta 8(14)-15-keto C24 acid (IV). Hydrolysis of the crude product of the side-chain oxidation with K2CO3 in methanol gave 3 beta,24-dihydroxy-5 alpha-chol-8(14)-en-15-one (XIV) which was oxidized with Jones reagent to yield 3,15-diketo-5 alpha-chol-8(14)-en-24-oic acid (XV). Treatment of XV with diazomethane gave its methyl ester (XVI) which, upon controlled reduction with NaBH4, yielded methyl 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oate (XVII). Compound IX was also prepared by an independent route. Full 1H and 13C NMR assignments are presented for 12 new compounds. IV caused a approximately 56% reduction of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells at a concentration of 2.5 microM. In contrast, the corresponding 3,15-diketo acid XV had no detectable effect on reductase activity under the same conditions.
A simple, three-step synthesis of the 25-aza analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is described. Treatment of 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (III) with 1.75 equivalents of tosyl chloride in pyridine for 24 h at 5 degrees C gave 3 beta-acetoxy-24-tosyloxy-5 alpha-chol-8(14)-en-15-one (IV). In contrast, treatment of III with 3.95 equivalents of tosyl chloride in pyridine for 12 h at 48 degrees C gave 3 beta-acetoxy-24-chloro-5 alpha-chol-8(14)-en-15-one (V). Treatment of IV with dimethylamine in dioxane yielded 3 beta-acetoxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VI). Hydrolysis of VI with LiOH.H2O in methanol gave 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VII). 1H- and 13C-NMR assignments are presented for compounds IV-VII. The 25-aza analog (VII) of the 15-ketosterol (I), at a concentration of 1.0 microM, caused a 47% lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.
The synthesis of ten esters and two ethers of testosterone (17 beta-hydroxyandrost-4-en-3-one) is described. All these possess some form of alpha - and/or beta - substitution in the ester/ether side-chain. The work was undertaken in order to evaluate the long-acting antifertility effect of such compounds in males.
The synthesis of eighteen esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) is described. These all possess some form of alpha- and/or beta-substitution in the ester side-chain. The work was undertaken in order to evaluate any long-acting fertility control effect intrinsic in such compounds. A pentamethyl disiloxy ether was also included in the group of substances prepared for testing because of its similar substitution pattern.
The synthesis of the esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) with three bile acids and of the cholesteryl carbonate of norethisterone are described.
The preparation of three carbonates and two carbamates of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) are described. Due to instability of the carbonates and the very low solubility of the carbamates these compounds could not be submitted to biological testing.
Methyl 1 alpha,3 alpha-dihydroxy 5 beta-cholan-24-oate was synthesized to provide a model compound for the mass spectrometric identification of 1 alpha-hydroxylated bile acids.
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The scope and limitation of some recent acylation methods were investigated as applied to the preparation of steroidal esters of highly hindered acids. As representative steroids, 19-nor-17 alpha-ethinyl testosterone (19-NET) and testosterone, and as representative acid, pivalic (trimethylacetic) acid were used.
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The metabolism of 3beta-hydroxy-5alpha-pregnan-20-one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography--mass spectrometry, in combination with specific deuterium-labelling, was employed to follow the metabolic transformations. Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver. The major metabolic pathway in male rats involved the steps: hydrolysis, 2alpha-hydroxylation, oxidoreduction at C-3 and glucuronide conjugation, yielding 2alpha, 3alpha-dihydroxy-5alpha-pregnan-20-one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3beta,15beta-dihydroxy-5alpha-pregnan-20-one sulphate. A minor metabolite, 3beta,16alpha-dihydroxy-5alpha-pregnan-20-one, was found as a monosulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15beta-and 16alpha-hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate. The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver.
A synthesis is reported of 3beta-hydroxy-5alpha-pregnan-20-one sulphate and the disulphate and 3-monosulphate of 5alpha-pregnane-3beta,20alpha-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3beta-hydroxy-5alpha-25R-spirostan-12-one (hemcogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 3beta-hydroxy-5alpha-[11,11-2H2]pregn-16-en-20-one (VII). Further deuterium atoms were introduced at the 3alpha and 20beta positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3beta-hydroxy-5alpha-[3alpha,11,11-2H3]pregnan-20-one (X; isotopic purity 87.2%) and 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly form the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol 20-acetate (XVII).
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