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.     

Inhibition of sterol biosynthesis in animal cells by 14 alpha-alkyl-substituted 15-oxygenated sterols

Reported herein are the results of investigations of the effects of a number of 14 alpha-alkyl-substituted 15-oxygenated sterols, prepared by chemical synthesis, on sterol biosynthesis and the levels of 3-hydroxy-3-methylglutaryl CoA reductase activity in L cells and in primary cultures of fetal mouse liver cells grown in serum-free media. Several of the compounds, most notably 14 alpha-ethyl-5 alpha-cholest-7-en-3 beta, 15 alpha-diol and 14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, were found to be extraordinarily potent inhibitors of sterol synthesis in these cells. For example, the latter compound caused a 50% inhibition of the incorporation of labeled acetate into digitonin-precipitable sterols in L cells in culture at a concentration of 6 X 10(-9) M.     

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.     

Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol.

The purified lanosterol 14a-demethylase (P-45014DM) of S. cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol. Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol. The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL. Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.     

Progestins block cholesterol synthesis to produce meiosis-activating sterols.

The resumption of meiosis is regulated by meiosis-preventing and meiosis-activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis-activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF-MAS and T-MAS). These C29 sterols accumulated as a result of inhibition of Delta24-reduction and 4alpha-demethylation. Progesterone, pregnenolone, and 17alpha-OH-pregnenolone were potent inhibitors of Delta24-reduction in an in vitro cell assay and led to the accumulation of desmosterol, a Delta5,24 sterol precursor of cholesterol. A markedly different effect was observed for 17alpha-OH-progesterone, which caused the accumulation of sterols associated with inhibition of 4alpha-demethylation. The flux of 13C-acetate into lathosterol and cholesterol was decreased by progestins as measured by isotopomer spectral analysis, whereas newly synthesized MAS accumulated. The combined evidence that MAS concentrations can be regulated by physiological levels of progestins and their specific combination provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestins in fertility.     

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.     

Chemical synthesis of 4,4'-dimethyl-7-oxygenated sterols. Inhibitors of 3-hydroxy-3-methylglutaryl reductase

The chemical syntheses of 4,4'-dimethylcholest-5-en-3 beta-ol-7-one, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol and 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol are described. All of these compounds were found to be potent inhibitors of 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase activity in cultured mouse L cells. The synthetic scheme developed in this study utilizes commercial cholesterol as the starting material and provides a simplified method for the preparation of 4,4'-dimethyl-7-oxygenated steroids.     

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.     

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.     

Plasma membrane rafts complete cholesterol synthesis by participating in retrograde movement of precursor sterols

Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER.     

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Abstract Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.     

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.     

Inhibitory effect of 1-beta-D-arabinofuranosylthymine on synthesis of Epstein-Barr virus

The effect of 1-beta-D-arabinofuranosylthymine (ara-T) on cell growth and synthesis of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines was determined. The growth of P3HR-1 cells was not inhibited by 1 microgram of the drug per ml; however, infectious virus production was strongly inhibited and was accompanied by decreased expression of early antigen (EA) and viral capsid antigen (VCA). The ability of 12-O-tetradecanoylphorbol-13-acetate or n-butyric acid to induce synthesis of VCA, but not EA, in P3HR-1 cells was inhibited by ara-T. Similarly, VCA synthesis but not EA synthesis was inhibited by ara-T in Jijoye cells superinfected with the P3HR-1 strain of EBV. The results suggest that ara-T has a specific inhibitory action against EBV replication.