Sterols in blood of normal and Smith-Lemli-Opitz subjects.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols. The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.

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. Metabolism-based design and construction of a new analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and its effects in cultured mammalian cells and in rats.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent regulator of cholesterol metabolism. In the present study, the 7 alpha-methyl-25,26,26,26,27,27,27-heptafluoro analog (X) of I has been synthesized with the goal of blocking not only the side chain oxidation of I but also its conversion to cholesterol. X was prepared in seven steps from the known 7 alpha-methyl analog (IX) of I. Treatment of the acetate of IX with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid gave 3 beta-acetoxy-7 alpha-methyl-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII) in remarkably high (68%) yield. Dehydration of XII via the orthonitrophenylselenide to the 23-ene, followed by addition of (CF3)2CFI gave (23R)-3 beta-acetoxy-7 alpha-methyl-23-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (XV). Reductive deiodination of XV with tributyltin hydride, followed by hydrolysis of the acetate gave 3 beta-hydroxy-7 alpha-methyl-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (X). The F7-7 alpha-methyl-15-ketosterol X lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells with a potency equivalent to that of I. X showed significant hypocholesterolemic action upon oral administration to rats, with a potency far in excess of the 7 alpha-methyl-15-ketosterol IX lacking the F7 substitution. In marked contrast to I, X showed little or no suppression of food consumption in rats. Upon oral administration of X to rats, low levels of X (relative to cholesterol), characterized by chromatographic and gas chromatography-mass spectrometric methodologies, were observed in serum, liver, and small intestine. No material was observed with the expected properties of F7-7-methylcholesterol (or potential intermediates in its possible formation from X). In contrast to I, X lowered serum cholesterol levels at dosages at which no effect on food consumption was observed.

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholestan-15-one and its 17 beta-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.

Inhibitors of sterol synthesis. Submicromolar 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol causes a major modification of the sterol composition of CHO-K1 cells and a marked change in cell morphology.

Incubation of Chinese hamster ovary cells (CHO-K1) with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol (0.1 microM) in lipid-deficient medium led to a major change in cellular sterol composition, which was characterized by a very marked accumulation of C30 sterols (lanosterol and 24,25-dihydrolanosterol). The accumulation of C30 sterols was associated with a striking change in cell morphology. The change in cell shape (elongation) was similar to that described previously (A. W. Hsie and T. T. Puck, 1971. Proc. Natl. Acad. Sci. USA. 68: 358-361; and confirmed herein) for CHO-K1 cells incubated in the presence of dibutyryl cAMP (1 mM). This change in morphology, induced by dibutyryl cAMP, was not accompanied by a change in cellular sterol composition. The cell elongation and accumulation of C30 sterols, induced by the 14 alpha-ethyl diol, were prevented by the addition of cholesterol (10 microM or 100 microM) and were reversed by removal of the 14 alpha-ethyl diol from the incubation medium. Incubation of the cells with the 14 alpha-ethyl diol had no effect on the levels of cAMP under the conditions studied. Incubation of the cells with miconazole (10 microM) or with lanosterol (10 microM) was also associated with the accumulation of C30 sterols and an elongation of the cells. 24,25-Dihydrolanosterol (10 microM) also induced similar changes in cellular morphology. The results presented herein demonstrate that marked changes in the sterol composition of CHO-K1 cells can be effected by incubation of the cells with 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, miconazole, or lanosterol. In addition, the findings reported herein indicate an important role of sterols in the control of the shape of these cells.

Inhibitors of sterol synthesis. Effects of a new fluorinated analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one in rats.

3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (VII), an analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in which conversion to 26- and 25-oxygenated metabolites is blocked by the F7-substitution, was administered to male Sprague-Dawley rats at levels of from 0.025 to 0.15% by weight in a ground chow diet. Administration of VII resulted in lowering of the levels of serum cholesterol at dosages as low as 0.025% by weight in diet. In marked contrast to I, VII had little or no effect on food consumption. Whereas administration of I at a level of 0.1% by weight in diet resulted in a cessation of growth, VII, at approximately the same molar concentration in diet, had only slight or no effect on changes in total body weight. Significant levels of 25,26,26,26,27,27,27-heptafluorocholesterol (VIII) were observed in serum and liver, indicating the conversion of VII to VIII. Characterization of VIII in liver was based upon the results of gas chromatography, low and high resolution mass spectral studies, infrared spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The levels of VIII in serum appeared to be related to dosage and duration of administration of VII.

Inhibitors of sterol synthesis: synthesis and spectral properties of derivatives of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-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 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.

Inhibitors of sterol synthesis: 3 beta-hydroxy-25,26,26,26,27,27,27- heptafluoro-5 alpha-cholestan-15-one, an analog of a potent hypocholesterolemic agent in which its major metabolism is blocked.

The chemical synthesis of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro- 5 alpha-cholestan-15-one (IV) has been pursued to provide an analog of the potent hypocholesterolemic agent 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in which its major metabolism is blocked. Reduction of 3 beta-acetoxy-5 alpha-chola-8(14),23-dien-15-one with lithium in liquid ammonia gave 3 beta-hydroxy-5 alpha-chol-23-en-15-one (VI). Addition of (CF3)2CFI to VI in the presence of triethylborane gave 3 beta-hydroxy-23R-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholestan- 15-one, which was reduced to IV with tributyltin hydride. IV was found to be highly active in lowering the levels of HMG-CoA reductase activity in CHO-K1 cells, in lowering acyl coenzyme A:cholesterol acyltransferase activity in jejunal microsomes, and in lowering serum cholesterol levels in rats.

Inhibitors of sterol synthesis: effects of a 7 alpha-alkyl analog of 3 beta-hydroxy-5 alpha-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.

The 7 alpha-methyl analog (II) of 3 beta-hydroxy-5 alpha-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 7 alpha-methyl substitution had no detectable effect on the potency of I in lowering HMG-CoA reductase activity in the cultured cells. In contrast, the 7 alpha-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 1H and 13C-NMR assignments for II and intermediates in its synthesis have been presented. Conformational analysis, based on 1H-1H coupling constants, NMR shieldings and force-field calculations, indicated that the 7 alpha-methyl substitution had virtually no effect on the conformation of the 15-ketosterol apart from minor distortions of ring B.

Inhibitors of sterol synthesis. Chemical synthesis and properties of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one and 25,26,26,26,27,27,27-heptafluorocholesterol and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells.

A side-chain fluorinated delta 8(14)-15-ketosterol has been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VII) as part of a program to prepare new analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. 3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (VIII) was prepared in five steps from VII in 38% overall yield. Dehydration of VII via the ortho-nitrophenylselenide to the 23-ene, followed by addition of (CF3)2CFI gave 3 beta-acetoxy-23R-iodo-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one. Reductive deiodination with tributyltin hydride, followed by hydrolysis of the acetate gave VIII. 25,26,26,26,27,27,27-Heptafluorocholest-5-en-3 beta-ol (XXI) was prepared in eight steps in 31% overall yield from 3 alpha,6 alpha-diacetoxy-5 beta-cholanic acid (XIII). Compound XIII was reduced with borane-methyl sulfide to the corresponding 24-hydroxysteroid, which was converted to 3 alpha,6 alpha-diacetoxy-25,26,26,26,27,27,27-heptafluoro-5 beta-cholestane (XVIII) by reactions analogous to those developed for the preparation of VIII from VII. Conversion of XVIII via the 3 alpha,6 alpha-diol to the 3 alpha,6 alpha-ditosylate, followed by heating with potassium acetate in dimethylformamide and subsequent hydrolysis gave XXI. Full 1H and 13C NMR assignments are presented for VIII, XXI, and intermediates involved in their synthesis. 13C NMR assignments for 3 alpha,6 alpha-dihydroxy-5 beta-steroids have been corrected, and stereochemical assignments were established for the side-chain methylene protons of VIII, XXI, and most synthetic intermediates. Compound VIII lowered the levels of HMG-CoA reductase activity in CHO-K1 cells and in HepG2 cells with a potency comparable to that of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I). In contrast, 25,26,26,26,27,27,27-heptafluorocholest-5-en-3 beta-ol had little or no effect on reductase activity in CHO-K1 cells. These combined results indicate that metabolism of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) to 26- and 25-oxygenated species is not required for the suppressive action of I on the levels of HMG-CoA reductase activity in CHO-K1 cells and HepG2 cells.

Differing effects of three oxysterols on low density lipoprotein metabolism in HepG2 cells.

25R)-26-Hydroxycholesterol, 25-hydroxycholesterol, and 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, all extraordinarily potent suppressors of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in mammalian cells, have been studied with respect to their effects on the metabolism of low density lipoproteins (LDL) by human hepatocarcinoma (HepG2) cells. The three oxysterols differed markedly in their effects on LDL metabolism, as measured by the combination of cell-associated plus degraded 125I-LDL. The 26-hydroxysterol, at concentrations from 0.1 microM to 75 microM, lowered LDL metabolism. In contrast, the 25-hydroxysterol and the 15-ketosterol, at concentrations from 0.05 microM to 2.5 microM, caused an increase in LDL metabolism. At higher concentrations of these oxysterols, LDL metabolism was suppressed. However, upon increasing the concentration of the 15-ketosterol further to 75 microM, an extraordinary 9-fold increase in LDL metabolism was observed. In contrast to their effects on LDL metabolism, the 25-hydroxysterol and the 15-ketosterol caused simple concentration-dependent decreases in the levels of HMG-CoA reductase activity under the same conditions.

D-erythro-sphingosine lowers 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.

D-Erythro-sphingosine lowered the levels of HMG-CoA reductase activity in CHO-K1 cells. Significant suppression of reductase activity was observed at 5 microM, 10 microM, and 15 microM concentrations of the sphingolipid base and approximately 50% lowering was found at 10 microM. In contrast, L-threo-sphingosine had no effect on the levels of reductase activity under the conditions studied. Direct addition of D-erythro-sphingosine, at concentrations up to 100 microM, to rat liver microsomes had no effect on the levels of HMG-CoA reductase activity. The concentrations of D-erythro-sphingosine required to lower reductase activity in CHO-K1 cells correspond to reported levels of sphingosine in mammalian cells.

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.

Inhibitors of sterol synthesis. Chemical synthesis and spectral properties of 3 beta-hydroxy-5 alpha-cholesta-8(14),24-dien-15-one, 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one, and 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

Side-chain functionalized delta 8(14)-15-ketosterols have been synthesized from 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (VI) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Oxidation of VI to the 24-aldehyde VII, followed by Wittig olefination with isopropyltriphenylphosphonium iodide gave 3 beta-acetoxy-5 alpha-cholesta-8(14),24-dien-15-one (VIII), which was hydrolyzed to the free sterol IX. Oxymercuration of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,25-dihydroxy-5 alpha-cholest-8(14)-en-15-one (IV). Hydroboration-oxidation of VIII followed by hydrolysis of the 3 beta-acetate gave 3 beta,24-dihydroxy-5 alpha-cholest-8(14)-en-15-one (V) as a 5:4 mixture of the 24R and 24S epimers. 1H and 13C nuclear magnetic resonance (NMR) assignments and mass spectral fragmentation patterns, supported by high-resolution measurements, are presented for IV and its 3 beta-acetate, V, VII, VIII, and IX. Characterization of IV by NMR and of trimethylsilyl ethers of IV and V by gas chromatography-mass spectrometry was compatible with spectral data for samples of IV and V isolated previously after incubation of I with rat liver mitochondria in the presence of NADPH. Sterols IV, V, and IX were very potent in lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells; their potency was comparable to that of I.

Inhibitors of sterol synthesis. Chemical synthesis of 7 alpha-ethyl and 16 alpha-ethyl derivatives of delta 8(14)-15-oxygenated sterols and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase in CHO-K1 cells.

The enolate of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (II), formed upon treatment of II with potassium tert-butoxide in tert-butanol, was alkylated with ethyl iodide. In addition to the major products, 3 beta-hydroxy-14 alpha-ethyl-5 alpha-cholest-7-en-15-one and its 3 beta-ethyl ether, small amounts of 3 beta-hydroxy-7 alpha-ethyl-5 alpha-cholest-8(14)-en-15-one (V), 3 beta-hydroxy-16 alpha-ethyl-5 alpha-cholest-8(14)-en-15-one (VI) and the 3 beta-ethyl ether of VI were isolated. When the enolate of II was formed by treatment with lithium diisopropylamide in tetrahydrofuran, the same alkylation furnished VI as the major product. Reduction of VI with lithium aluminum hydride gave 16 alpha-ethyl-5 alpha-cholest-8(14)-ene-3 beta, 15 alpha-diol (IX) and its 15 beta epimer X, which were separated by column chromatography. Full 1H and 13C nuclear magnetic resonance (NMR) assignments, augmented by nuclear Overhauser effect difference spectra for VI, established the stereochemistry of these diols at C-15 and C-16. The NMR results indicate that the 16 alpha-ethyl group affects the side-chain conformation. The effects of II, V, VI, IX and X on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were studied in CHO-K1 cells. With the exception of IX, each of the compounds reduced the levels of HMG-CoA reductase activity. The order of potency with respect to suppression of the elevated levels of HMG-CoA reductase activity induced by transfer of the cells to lipid-deficient medium, was II greater than V greater than VI greater than X.

Synergistic action of two oxysterols in the lowering of HMG-CoA reductase activity in CHO-K1 cells.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) and (25R)-26-hydroxycholesterol (II), both potent regulators of sterol biosynthesis, have been found to show synergism in the reduction of the levels of HMG-CoA reductase activity in CHO-K1 cells. When equimolar concentrations of I and II were added in combination, synergistic reduction (p less than 0.0001) of enzyme activity was observed at total oxysterol concentrations of 0.1 microM, 0.2 microM, and 0.5 microM. Maximal synergistic effect in the lowering of reductase activity (28% greater than predicted) was observed at 0.1 microM total oxysterol concentration. Five additional experiments conducted with 50 nM oxysterols confirmed the synergistic effect at 0.1 microM total sterol concentration. These results suggest that the in vivo importance of I and II may be greater than that anticipated on the basis of the concentrations of the individual sterols.

Inhibitors of sterol synthesis. Chemical synthesis and spectral properties of (25R)-5 alpha-cholest-8(14)-ene-3 beta,15 beta,26-triol, a potential metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and its effects on 3-hydroxy-3-methylglutaryl-coenzyme A reductase in CHO-K1 cells.

(25R)-5 alpha-Cholest-8(14)-ene-3 beta,15 beta,26-triol (III) was prepared by reduction of (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one with sodium borohydride followed by treatment of the crude product with lithium aluminium hydride. The trihydroxysterol III, a potential metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, was characterized by the results of mass spectral studies and by nuclear magnetic resonance (NMR) spectroscopy. Full 1H and 13C NMR assignments for III and 5 alpha-cholest-8(14)-ene-3 beta,15 beta-diol are given and used to establish the structure of III. The triol was found to be very potent in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.

Inhibitors of sterol synthesis. A highly efficient and specific side-chain oxidation of 3 beta-acetoxy-5 alpha-cholest-8(14)-en-15-one for construction of metabolites and analogs of the 15-ketosterol.

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.