Effect of 3-substituted Delta8(14)-15-ketosterols on cholesterol metabolism in hepatoma Hep G2 cells

The effects of 3-substituted Delta8(14)-15-ketosterols--3beta-(2-hydroxyethoxy)-, 3beta-(2-propenyloxy)-, 3beta-[2(R,S),2,3-oxidopropyloxy]-, 3beta-[2(R,S),2,3-dihydroxypropyloxy]-, 3beta-(2-oxoethoxy)-, 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]-, and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes--on cholesterol metabolism were studied in human hepatoma Hep G2 cells. 3beta-(2-Propenyloxy)-, 3beta-(2-oxoethoxy)-, and 3beta-[2(R,S),2, 3-oxidopropyloxy]-5alpha-cholest-8(14)-en-15-ones inhibited cholesterol biosynthesis without any effect on triglyceride biosynthesis, while 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]- and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes inhibited both cholesterol biosynthesis and triglyceride biosynthesis at concentrations exceeding 10 microM. 3beta-[2(R,S),2, 3-Dihydroxypropyloxy]-5alpha-cholest-8(14)-en-15-one, effectively inhibiting cholesterol biosynthesis, was found also to be toxic in Hep G2 cells at micromolar concentrations. 3beta-[2(R,S),2, 3-Oxidopropyloxy]-5alpha-cholest-8(14)-en-15-one effectively inhibited cholesterol acylation. All the tested compounds decreased the HMG-CoA reductase mRNA level at concentrations exceeding 10 microM; however, they did not affect the LDL receptor mRNA level. Among the compounds tested, only 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one decreased the uptake and internalization of LDL-associated cholesteryl esters, being as effective as 25-hydroxycholesterol.     

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.     

Distribution of exogenous 25-hydroxycholesterol in Hep G2 cells between two different pools

Binding of [26,27-(3)H]25-hydroxycholesterol (25HC) to human hepatoma Hep G2 cells was saturated within 120 min. Two intracellular pools of 25HC were identified in a pulse-chase experiment: (i) an exchangeable pool which was in dynamic equilibrium with 25HC in the medium (t(1/2) of reversible exchange 15 min) and (ii) an unexchangeable pool which remained in cells during incubation in medium containing LPDS. 25HC from the exchangeable pool inhibits cholesterol biosynthesis, decreases the HMG CoA reductase mRNA level and stimulates cholesterol acylation. 25HC from the unexchangeable pool was partially bound to cytosolic proteins and apparently utilized for metabolic transformation. Incubation of Hep G2 cells with [26,27-(3)H]25HC in the presence of a 30-fold molar excess of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one was found to cause (i) 2-fold decrease in the binding of [26,27-(3)H]25HC to cytosolic proteins (sedimentation constant of radioactive complex was 4-5 S) and (ii) the 35% inhibition of 25HC transformation to polar metabolites.     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

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.     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

Secretion of metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one by Hep G2 cells

We demonstrated that two-thirds of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was efficiently taken up and consequently metabolized and secreted by Hep G2 cells when it was pulsed for 16 hrs followed by chasing for another 72 hrs. The metabolism was clearly reflected by the cellular secretion. Approximately 61%, 26% and 10% of uptaken [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one was metabolized to its water-soluble metabolites, polar metabolites in lipid phase and ketosteryl esters, respectively. Ninety-four percent of these metabolites was secreted into media. Interestingly, polar forms of the metabolites of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one accounted for over 95% of the cellular secretes. Limited secretion of ketosteryl esters was also detected. The data strongly suggest that Hep G2 cells have the potential to process 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and could provide a good model for studying its secretion.     

Inhibitors of sterol synthesis. Spectral characterization of derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

Described herein are the chemical syntheses of a number of deuterated derivatives of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one. These include the [2,2,3 alpha,4,4,7,7,9 alpha,16,16-2H10]-, [7 alpha,9 alpha,16,16-2H4]-, [7,7,9 alpha,16,16-2H5]-, and [2,2,3 alpha,4,4-2H5]-analogs of the delta 8(14)-15-ketosterol. Also included are the syntheses of the 3 beta-acetate derivatives of the latter three deuterated analogs and of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, and 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one. Low resolution mass spectral data on these compounds and on 5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, and the trimethylsilyl ethers of the free sterols have been presented. The results of these studies, supplemented with high resolution mass spectral data on five of these compounds, have been used to evaluate the electron impact mass spectral fragmentation of the delta 8(14)-15-ketosterols and their derivatives. Also presented herein are the results of 1H, 2H, and 13C nuclear magnetic resonance studies of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one and its derivatives.     

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.     

The effect of 15-ketosterol analogues with a 5,6-dimethylhept-3-en-2-yl chain at C17 on cholesterol metabolism in Hep G2 hepatoma cells

The effect on cholesterol metabolism in Hep G2 hepatoma cells was studied for new analogues of 15-ketosterol [3beta-hydroxy-5alpha-cholest-8(14)-en-15-one] (I): (24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-diene-15-one (II), (24S)-3alpha-hydroxy-24-methyl-5-alpha-cholesta-8(14),22-diene-15-one (III), and (24S)-24-methyl-5alpha-cholesta-8(14),22-diene-3,15-dione (IV). Analogues (I) and (II) were found to be equally effective inhibitors of cholesterol biosynthesis after a 3-h incubation with Hep G2 cells; however, (II) produced a stronger inhibitory effect after a 24-h incubation or after an incubation of cells preliminarily treated with the inhibitor in a medium containing no ketosterol. The ability of ketosterols to inhibit cholesterol biosynthesis decreased in the order (II) > (IV) > (III). Ketosterol (II) inhibited, whereas ketosterol (III) stimulated the biosynthesis of cholesteryl esters. (IV) stimulated the biosynthesis of cholesteryl esters at a concentration of 1-10 microM and exerted no marked effect at a concentration of 30 microM. These results indicate that delta8(14)-15-ketosterols containing a modified side chain are of interest as regulators of cholesterol metabolism in liver cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.     

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.     

Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent inhibitor of sterol synthesis with significant hypocholesterolemic activity. (25R)-3 beta,26-Dihydroxy-5 alpha-cholest-8(14)-en-15-one (II) has been shown to be a major metabolite of I after incubation with rat liver mitochondria. Described herein is the chemical synthesis of II from diosgenin. As part of this synthesis, improved conditions are described for the conversion of diosgenin to (25R)-26-hydroxycholesterol. Benzoylation of the latter compound gave (25R)-cholest-5-ene-3 beta,26-diol 3 beta,26-dibenzoate which, upon allylic bromination followed by dehydrobromination, gave (25R)-cholesta-5,7-diene-3 beta,26-diol 3 beta,26-dibenzoate. Hydrogenation-isomerization of the delta 5.7-3 beta,26-dibenzoate to (25R)-5 alpha-cholest-8(14)-ene-3 beta,26-diol 3 beta,26-bis(cyclohexanecarboxylate) followed by controlled oxidation with CrO3-dimethylpyrazole gave (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one 3 beta,26-bis(cyclohexanecarboxylate). Acid hydrolysis of the delta 8(14)-15-ketosteryl diester gave II. 13C NMR assignments are given for all synthetic intermediates and several major reaction byproducts. The structure of II was unequivocally established by X-ray crystal analysis. II was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured mammalian cells and to inhibit oleoyl coenzyme A-dependent esterification of cholesterol in jejunal microsomes.     

Metabolism of a novel side chain modified Delta8(14)-15-ketosterol, a potential cholesterol lowering drug: 28-hydroxylation by CYP27A1

The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.     

New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one and its effects on HMG-CoA reductase activity in CHO-K1 cells.

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.     

Further studies on the inhibition of sterol biosynthesis in animal cells by 15-oxygenated sterols

The chemical syntheses of a number of C27 15-oxygenated sterols and their derivatives have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in animal cells in culture. Described herein are chemical syntheses of 3 alpha-benzoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3 alpha-ol-15-one, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-15-one-3 beta-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en-15-one-3 alpha-yl pyridinium sulfate, 5 alpha-cholest-8(14)-en-3 alpha-yl potassium sulfate (monohydrate), 5 alpha-cholest-8(14)-en3,7,15-trione, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha, 14 alpha-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol diacetate, 5 alpha, 14 alpha-cholestan-3 beta, 15 alpha-diol, 5 alpha, 14 alpha-cholestan-15 alpha-ol-3-one, 5 alpha, 14 beta-cholestan-3 beta, 15 beta-diol, 5 alpha, 14 alpha-cholestan-3,15-dione, and 5 alpha, 14 beta-cholestan-3,5-dione. The effects of 8 of the above compounds and of 5 alpha-cholesta-6,8(14)-dien-3 beta-ol-15-one, 3 beta-he misuccinoyloxy-5 alpha-cholest-8(14)-en-15 one, 3 beta-hexadecanoyloxy-5 alpha-cholest-8(14)-en-15-one, 5 alpha-cholest-8(14)-en-3,15-dione, 5 alpha-cholesta-6,8(14)-dien-3,15-dione, 5 alpha-cholest-8-en-3 beta, 15 alpha-diol, 5 alpha-cholest-7-en-3 beta, 15 alpha-diol, 5 alpha-cholest-8(14)-en-15 alpha-ol-3-one, 5 alpha-cholest-8-en-15 alpha-ol-3-one, and 5 alpha-cholest-7-en-15 alpha-ol-3-one on the synthesis of digitonin-precipitable sterols and on levels of HMG-CoA reductase activity have been investigated and compared with previously published data on 7 other C27 15-oxygenated sterols.     

Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs 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

Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.     

Transformation of 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one in a polar metabolite in HepG2 hepatoma cells

Incubation of 3 beta-(2-hydroxy-2[3H]-ethoxy)-5 alpha-cholest-8(14)-en-15-one with Hep G2 cells led to the accumulation of a radioactive polar product in the culture medium, which was identified as 3 beta-(2-hydroxyethoxy)-15-keto-5 alpha-cholest-8(14)-ene-24-oic acid. Its structure was confirmed by a chemical counter synthesis. The labeled ketosterol was rapidly (tau 1/2 = 6 min) and reversibly bound by Hep G2 cells. The intracellular concentration of 15-ketosterol decreased during incubation mainly due to the formation of a polar metabolite, secreted to the medium. The level of cholesterol biosynthesis was 22 +/- 5% of the control value in Hep G2 cells at a 15-ketocholesterol concentration in the medium of 30 microM. However, further incubation for 3 h in the medium without the ketosterol led to restoration of the level of biosynthesis to 85 +/- 11% of the control value. These results suggest that inhibition of the cholesterol biosynthesis by 15-ketocholesterol in Hep G2 cells depends on the intracellular concentration of the inhibitor, which, in turn, is determined by the rate of its conversion into the polar metabolite. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.