Nicolas Gerst

Inhibitors of sterol synthesis. Effects of fluorine substitution at carbon atom 25 of cholesterol on its spectral and chromatographic properties and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

25-Fluorocholesterol (III) was prepared by treatment of 25-hydroxycholesterol (IV) with hydrogen fluoride-pyridine. Compounds III, IV, and cholesterol (I) were fully characterized by 1H and 13C NMR, and stereochemical assignments were established for the C-22 and C-23 protons. The side-chain proton assignments, which apply to most other sterols with a saturated eight-carbon side chain, were based on conformational analysis and comparisons with NMR data for 25,26,26,26,27,27,27-heptafluorocholesterol (II). The chromatographic behavior of I, II, and III were compared on thin-layer chromatography, high performance liquid chromatography, and gas chromatography. Major fragment ions in electron-impact mass spectra of III were analogous to ions of either cholesterol or desmosterol, and a similar analogy was observed for the trimethylsilyl ethers. The 25-hydroxysterol IV and the 25-fluorosterol III differed markedly in their effects on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. Whereas 25-hydroxycholesterol caused a approximately 66% lowering of reductase activity in cells at 0.1 microM, the 25-fluorosterol III had no effect at this concentration.

Sterol synthesis. Synthesis of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one and its effects on HMG-CoA reductase activity in Chinese hamster ovary cells, on ACAT activity in rat jejunal microsomes, and serum cholesterol levels in rats.

3 beta-Hydroxycholest-5-en-7-one (I; 7-ketocholesterol) is an oxysterol of continuing interest in biology and medicine. In the present study, we have prepared a side-chain fluorinated analog, 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI), with the anticipation that the F7 substitution would block major metabolism of the 7-ketosterol, and thereby enhance its potential in vivo effects on serum cholesterol levels and other parameters. Chromium trioxide/dimethyl pyrazole oxidation of the acetate derivative of the previously described 25,26,26,26,27,27,27-heptafluorocholest-5-en-3 beta-ol (Swaminathan et al., 1993. J. Lipid Res. 34, 1805-1823) followed by mild alkaline hydrolysis gave VI. The effects of VI on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in Chinese hamster ovary (CHO-K1) cells, on acyl coenzyme A-cholesterol acyltransferase (ACAT) activity in rat jejunal microsomes, and on serum cholesterol levels and other parameters in male Sprague-Dawley rats were determined and compared with those obtained with I and with another alpha, beta-unsaturated ketosterol, i.e. 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (II). I and VI showed essentially the same potency, considerably less than that of II, in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells. Whereas addition of II to rat jejunal microsomes inhibited ACAT activity (IC50 approximately 3 microM), I and VI had no effect under the conditions studied (from 1 to 16 microM). Dietary administration of I, at levels of 0.1 and 0.15%, had no effect on food consumption, gain in body weight, or serum cholesterol levels. At 0.2%, I caused a modest decrease in body weight gain and a slight decrease in serum cholesterol levels (relative to ad libitum but not pair-fed control animals). The F7-7-ketosterol VI, at 0.26% in diet (the molar equivalent of 0.2% I), had no effect on food consumption, body weight, or serum cholesterol levels. Administration of I (0.1, 0.15 or 0.2% in diet) caused increases in the weight of small intestine. In contrast, no effect of VI (0.26% in diet) on small intestinal weight was observed.

Inhibitors of sterol synthesis: synthesis and spectral properties of derivatives of 3β-hydroxy-25,26,26,26,27,27,27-heptafluoro-5α-cholest-8(14)-en-15-one fluorinated at carbon 7 or carbon 9 and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

As part of a program to prepare delta 8(14)-15-ketosterols that cannot readily be metabolized to cholesterol or side-chain oxygenated species, we have prepared 3 beta-hydroxy-7 alpha-fluoro-5 alpha-cholest-8(14)-en-15-one (VII) and the 9 alpha-hydroxy (IV), 9 alpha-fluoro (VI) and 7 alpha-fluoro (VIII) derivatives of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (II). Sterol IV was prepared by oxidation of the delta 8,14 dienol ethyl ether of the 3 beta-acetate of II with m-chloroperbenzoic acid, followed by mild alkaline hydrolysis of the 3 beta-acetate derivative of IV. Treatment of IV with hydrogen fluoride-pyridine gave VI. The 7 alpha-fluoro-15-ketosterols VII and VIII were synthesized by treating the 3 beta,15-bis-trimethylsilyl delta 7,14-dienol ether derivative of the appropriate delta 8(14)-15-ketosterol with N-fluoropyridinium triflate, followed by hydrolysis of residual trimethylsilyl ethers and purification by high-performance liquid chromatography. The combined results of 1H and 13C nuclear magnetic resonance (NMR) chemical shifts, 1H-1H coupling constants, 1H-19F long-range coupling constants and molecular modeling indicated that a 7 alpha-fluoro, 9 alpha-fluoro or 9 alpha-hydroxy substituent has negligible effect on the conformation of the 15-ketosterols. 1H and 13C-NMR data are also given for delta 6,8(14)- and delta 8(14),9(11)-15-ketosterols, synthetic byproducts that could not be detected readily in samples of the fluoro-15-ketosterols by chromatographic methods. Mass spectra of VI and of previously reported 9 alpha-fluoro and 9 alpha-hydroxy-delta 8(14)-15-ketosterols showed abundant M-62 or M-60 ions that appear to correspond to loss of ketene and HF or H2O. The 9 alpha-hydroxy-F7-15-ketosterol IV, the 7 alpha-fluoro-15-ketosterol VII and the 7 alpha-fluoro-F7-15-ketosterol VIII were of equivalent potency to the parent 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. The 9 alpha-fluoro-F7-15-ketosterol VI showed high potency but appeared to be slightly less active than I.

Inhibitors of sterol synthesis: effects of a 7α-alkyl analog of 3β-hydroxy-5α-cholest-8(14)-en-15-one on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells and on serum cholesterol levels and other parameters in rats

Abstract The 7α-methyl analog (II) of 3β-hydroxy-5α-cholest-8(14)-en-15-one (I) was prepared by chemical synthesis and evaluated with respect to its effects on HMG-CoA reductase activity in CHO-K1 cells and on serum cholesterol levels in rats. The 7α-methyl substitution had no detectable effect on the potency of I in lowering HMG-CoA reductase activity in the cultured cells. In contrast, the 7α-methyl substitution had a marked effect on the action of I in the suppression of food consumption in rats. Whereas II was less potent than I in lowering serum cholesterol levels in rats, it did so at dosage levels at which only slight or moderate effects on food consumption were observed. Full 1 H and 13 C-NMR assignments for II and intermediates in its synthesis have been presented. Conformational analysis, based on 1 H- 1 H coupling constants, NMR shieldings and force-field calculations, indicated that the 7α-methyl substitution had virtually no effect on the conformation of the 15-ketosterol apart from minor distortions of ring B.

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3β-hydroxy-5α-cholestan-15-one and its 14β-epimer and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

3 beta-Hydroxy-5 alpha-cholestan-15-one (2a) and its 14 beta-epimer 2b were prepared from 3 beta-acetoxy-5 alpha-cholest-8(14)-ene (3). Hydroboration of 3 at 45-50 degrees C gave a mixture of 5 alpha,14 alpha-cholestane-3 beta,15 alpha-diol and 5 alpha,14 beta-cholestane-3 beta,15 beta-diol, which were separated on silica gel as their 3 beta-tert-butyldimethylsilyl ethers 5a and 5b. Oxidation of 5a with pyridinium chlorochromate, followed by desilylation with tetrabutylammonium fluoride gave 2a. Analogous transformations of 5b gave 2b contaminated with 2a. Desilylation of 5b followed by oxidation with pyridinium chlorochromate resulted in a mixture composed mainly of 5 alpha,14 beta-cholestane-3,15-dione and 2b. Successive chromatographic separations on silica gel and reversed phase media gave 2b of high purity. Compound 2a was also prepared by lithium-ammonia reduction of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (96% yield) and by selective reduction of 5 alpha-cholestane-3,15-dione with lithium tri-tert-butoxyaluminum hydride (90% yield). Isomers 2a and 2b were readily epimerized under acidic or basic conditions or under conditions used for gas chromatographic analysis. The purities of 2a and 2b were measured from nuclear magnetic resonance (NMR) spectra; chromatographic methods gave less reliable estimates of purity. NMR data also showed that ring C of the 14 beta sterols is predominantly in a chair conformation. The effects of 2a and 2b on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase have been studied in Chinese hamster ovary cells.