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.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Inhibitors of sterol synthesis. Chemical syntheses and activities of new derivatives of 15-oxygenated sterols

The chemical syntheses of 5α-cholestane-3β,14α,15β-triol, 5α-cholestane-14α-ol-3,15-dione, 5α-cholestane-3β,14α-diol-15-one, 14α,15α-epoxy-5α-cholestan-3β-ol, and 5α-cholest-8(14)-en-3β-ol-15-one oxime are described. All of these compounds were found to be potent inhibitors of sterol synthesis in cultured mouse L cells. However, the former three compounds had little or no effect on the levels of 3-hydroxy-3-methylgutaryl (HMG)-CoA reductase in the same cells. In contrast, in the case of the latter two compounds, the concentrations required to cause a 50% inhibition of the synthesis of digitonin-precipitable sterols were comparable to those required to cause a 50% reduction in the levels of HMG-CoA reductase in the same cells. 5α-Cholest-8(14)-en-3β-ol-15-one oxime had no effect on serum cholesterol levels when administered to male rats at a level of 0.15% in a cholesterol-free diet.     

Inhibitory effect of 15-oxygenated sterols on cholesterol synthesis from 24,25-dihydrolanosterol

Several 15-oxygenated sterols were examined as to their inhibitory activity toward cholesterol synthesis from [24,25-3H]-24,25-dihydrolanosterol in the 10,000 X g supernatant fraction of a rat liver homogenate. At 40 microM, three 15 alpha-hydroxylated compounds, 14 alpha-ethylcholest-7-ene-3 beta,15 alpha-diol, 14 alpha-methylcholest-7-ene-3 beta,15 alpha-diol, and lanost-7-ene-3 beta,15 alpha-diol, were found to be extremely potent inhibitors (more than 90% inhibition) of dihydrolanosterol metabolism. The inhibitory effect of the C-15 substituents appeared to be in the order of: 15 alpha-hydroxyl greater than 15-ketone greater than 15 beta-hydroxyl.     

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

Inhibition of sterol synthesis in L cells by 14alpha-ethyl-5alpha-cholest-7-en-15alpha-ol-3-one at nanomolar concentrations

14α-Ethyl-5α-cholest-7-en-15α-ol-3-one was prepared in 85% yield by selective oxidation of the 3β-hydroxyl function of 14α-ethyl-5α-cholest-7-en-3β,15α-diol by cholesterol oxidase. 14α-Ethyl-5α-cholest-7-en-15α-ol-3-one caused a 50% inhibition of the incorporation of [1-¹⁴C]-acetate into digitonin-precipitable sterols at a concentration of 6 × 10^?9M in L cells and a 50% reduction in level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in the same cells at a concentration of 4 × 10^?8 M.     

Inhibition of sterol biosynthesis by ergosterol and cholesterol in Saccharomyces cerevisiae

When accumulation of squalene was used as a measure of the flow of carbon into the sterol pathway in whole cells of semi-anaerobic Saccharomyces cerevisiae, both ergosterol and cholesterol were found to be inhibitory. However, at equivalent concentrations in the medium ergosterol was substantially the more potent inhibitor. Marked differences found in the absorption and esterification of the two sterols failed to account for the observed difference in their capacities to act as feedback agents. Cholesterol was much more effectively absorbed as well as esterified, but, when the abilities of the two sterols to lower the squalene level were calculated on the basis of free sterol in the cells, ergosterol remained more effective by a factor of four.     

Inhibition of Trypanosoma cruzi growth and sterol biosynthesis by lovastatin

We have studied the effects of lovastatin, an inhibitor of hydroxy-methylglutaryl-CoA-reductase, on cultures of Trypanosoma cruzi epimastigotes. It inhibits growth at 10 and 30 micrograms per ml; these effects are reverted by 100 microM of squalene, but not by 100 microM of cholesterol. Lovastatin at the same amounts inhibits [14C]acetate incorporatin into sterols, isolated either by digitonin precipitation or thin layer chromatography. At 50 micrograms per ml it kills most of the trypanosomes. These concentrations are below reported toxic levels for mammals; this drug and its analogs should, therefore, be tested as chemotherapeutic agents against Chagas' disease.     

Inhibition of growth,lipid, and sterol biosynthesis by monensin in fungi

The effects of monensin on the growth, morphology, and lipid metabolism of four fungi were determined. The growth of Hypomyces chlorinus, Neurospora crassa, Achlya bisexualis, and Taphrina deformans was suppressed by approximately 50% at 1, 2 to 5, 10, and 20 μg/ml monensin, respectively. Total lipid production, and more specifically the sterols, was reduced in each species by monensin. The hyphal tips of H. chlorinus cultured in the presence of monensin became swollen, whereas no morphological responses were observed in A. bisexualis. The results reported here suggest that growth suppression by monensin is possibly due to the interruption of endomembrane function, perhaps Golgi or their equivalents, which may result from the inhibition of sterol biosynthesis.     

Inhibition of sterol biosynthesis in Saccharomyces cerevisiae by N,N-diethylazasqualene and derivatives

The ability of some azasqualene derivatives to inhibit yeast cell growth was compared with their inhibition activity on squalene-2,3-oxide cyclase (EC 5.4.99.7) both in living cells and in microsome preparations. Among the compounds tested, N,N-diethylazasqualene showed the best correlation between the activity on squalene-2,3-oxide cyclase and its inhibition of yeast growth. The N-oxide derivative, N,N-diethylazasqualene N-oxide, which was as active as the amine in microsomes, was much less active in living cells, probably because it could not easily penetrate the cell wall. Kinetic analysis of the inhibitory activity of compounds on squalene-2,3-oxide cyclase revealed a sharp difference between N,N-diethylazasqualene and its N-oxide; the former showed a non-competitive-type inhibition, whereas the latter behaved as a competitive inhibitor.     

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols

The chemical syntheses of a number of C27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterol biosynthesis in cultured mammalian cells. Thus, 5 alpha-cholest-7-ene-3 beta, 11 alpha-diol, 3 alpha-hydroxy-5 alpha-cholest-9(11)-en-12-one, and the previously unreported 11 alpha-hydroxy-5 alpha-cholest-7-en-3-one, 5 alpha-cholest-9(11)-ene-3,12-dione, and 3 beta-hydroxy-5 alpha-cholest-9 (11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5 alpha-Cholest-7-ene-3 beta, 11 alpha-diol was shown to be the most potent inhibitor of sterol synthesis.     

The biosynthesis of the free sterols and sterol esters of Neurospora crassa.

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4,14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.     

Palmitates of isomeric 15-oxygenated delta8(14)-sterols

3 beta-Hexadecanoyloxy-5 alpha-cholest-8(14)-en-15-one, 3 alpha-hexadecanoyloxy-5 alpha-cholest-8(14)-en-15-one, 15 beta- hexadecanoyloxy-5 alpha-cholest-8(14)-en-3 beta-ol, 15 alpha-hexadecanoyloxy-5 alpha-cholest-8(14)-en-3 beta-ol, 15 beta-hexadecanoyloxy-5 alpha-cholest-8(14)-en-3-one, and 15 alpha-hexadecanoyloxy-5 alpha-cholest-8(14)-en-3-one were synthesized and their chromatographic and 1H NMR characteristics were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-(14)C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (alpha-p-chlorophenoxyisobutyrate) and Clofenapate [alpha,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.     

15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. Synthesis, structure, and inhibition of sterol synthesis in animal cells

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene with methyl magnesium iodide gave, as the major product, 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol. The product was characterized as the free sterol and in the form of its 3 beta-acetoxy and 3 beta-p-bromobenzoate derivatives. Unambiguous assignment of structure was based upon X-ray analysis of the latter derivative. 15 beta-Methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol was found to be a potent inhibitor of sterol synthesis in cultured mammalian cells. The 15 beta-methyl-3 beta,15 alpha-dihydroxysterol caused a 50% reduction of the level of HMG-CoA reductase activity and a 50% reduction in the incorporation of labeled acetate into digitonin-precipitable sterols in L cells at a concentration of 3.0 x 10(-6) M.     

Stereospecific 1,4-addition to an alpha,beta-unsaturated steroidal epoxide: syntheses of new 15-oxygenated sterols

3 beta-Benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (1) is a key intermediate in the synthesis of C-7 and C-15 oxygenated sterols. Treatment of 1 with benzoyl chloride resulted in the formation of 3 beta,15 alpha-bis-benzoyloxy-7 alpha-chloro-5 alpha-cholest-8(14)-ene (2). Reaction of 2 with LiAlH4 or LiAlD4 resulted in the formation of 5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3a) or [14 alpha-2H]5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3b). Diol 3b was selectively oxidized by Ag2CO3/celite to [14 alpha-2H]5 alpha-cholest-7-en-15 alpha-ol-3-one (4). Treatment of 1 with MeMgI/CuI gave 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 alpha-diol (5). Selective oxidation of 5 with pyridinium chlorochromate (PCC)/pyridine or oxidation with PCC resulted in the formation of 7 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one (6) and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3,15-dione, respectively. Reduction of 6 with LiAlH4 yielded 5 and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 beta-diol (6). Reaction of 1 with benzoic acid/pyridine gave 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-en-15 alpha-ol (9). Treatment of 9 with LiAlH4 or ethanolic KOH resulted in the formation of 5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol (10). Dibenzoate 9, upon brief treatment with mineral acid, gave 3 beta-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (11). Oxidation of 9 with PCC yielded 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (12). Ketone 12 was also prepared by the selective hydride reduction of 5 alpha-cholest-8(14)-en-7 alpha-ol-3,15-dione (13) to give 5 alpha-cholest-8(14)-ene-3 beta,7 alpha-diol-15-one (14), which was then treated with benzoyl chloride to produce 12.     

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.     

Inhibitors of sterol synthesis. 15-oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one (15 ketosterol) is a potent inhibitor of cholesterol biosynthesis with significant hypocholesterolemic activity. The results of a recent study (Schroepfer, G.J., Jr., Christophe, A., Chu, A.J., Izumi, A., Kisic, A. and Sherrill, B.C. (1988) Chem. Phys. Lipids 48, 29-58) have indicated that, after intragastric administration of the 15-ketosterol in triolein to rats, most of the compound in intestinal lymph occurs in the form of the oleate ester, which is associated with chylomicrons. Moreover, after intravenous administration of chylomicrons containing the oleate ester of 15-[2,4-3H]ketosterol, rapid and selective uptake of 3H by liver was observed, which was associated with the rapid and substantial appearance of labeled free 15-ketosterol in liver. The present study concerns the capabilities of rat liver fractions to catalyze the hydrolysis of 15-ketosteryl oleate. Efficient hydrolysis was observed at acid pH with a digitonin-solubilized extract of rat liver, with a rate similar to that for the hydrolysis of cholesteryl oleate. The distribution of acid 15-ketosteryl oleate hydrolase of whole liver homogenate on a metrizamide isopycnic density gradient was similar to that of acid cholesteryl oleate hydrolase and acid phosphatase, suggesting that the lysosomal acid lipase is the enzyme responsible for the hydrolysis of the 15-ketosteryl oleate at acid pH. At neutral pH, 15-ketosteryl oleate and cholesteryl oleate was hydrolyzed at similar rates by the microsomal fraction of liver homogenate, whereas the 15-ketosteryl oleate was hydrolyzed at a much lower rate than cholesteryl oleate by the cytosolic fraction. The distribution of neutral 15-ketosteryl oleate hydrolase activity of whole liver homogenate on a metrizamide isopycnic density gradient was most correlated to a microsomal esterase, whereas cholesteryl oleate hydrolase activity was most correlated to a cytosolic enzyme. Both 15-ketosteryl oleate and cholesteryl oleate hydrolase activities were correlated to a mitochondrial marker enzyme.     

Concerning the role of 24,25-dihydrolanosterol and lanostanol in sterol biosynthesis by cultured cells

Rat hepatoma cells (H4-II-E-C3) efficiently converted a dietary supplement of [2-3H]24,25-dihydrolanosterol (1) to [3H]cholesterol while [2-3H]lanostanol (4,4,14 alpha-trimethylcholestanol (2) was recovered from the cells without apparent transformation, although it was esterified and induced an accumulation of lanosterol. A comparison of the chromatographic (TLC, GLC and HPLC), spectral (MS and 1H-NMR) and physical properties of 1 and 2 is given for the first time. The inability to detect 2 in nature coupled with our findings that 1 but not 2 is metabolized to cholesterol by H4 cells is interpreted to imply that the biosynthetic inclusion of the delta 8(9)-bond during the cyclization process of squalene-oxide to a tetracyclic product is an evolutionary adaptation selected for because the olefinic linkage is structually important in the subsequent conversion of lanosterol and its stereoisomers, e.g., cycloartenol, to delta 5-sterols.