Potent Inhibitors Of Sterol Synthesis Research Articles

Effects of ketoconazole on ovulatory changes in the rat: implications on the role of a meiosis-activating sterol.

In-vitro studies on mouse oocytes have shown that human follicular fluid and bull testes contain an activity which partially overrides the inhibitory action of hypoxanthine on meiosis. This activity was ascribed to two closely related sterols, subsequently named meiosis-activating sterols (MAS). We have used a potent inhibitor of sterol synthesis, ketoconazole, in order to test in vivo and in vitro whether MAS play a necessary physiological role in the resumption of meiosis in the rat. When administered systemically, ketoconazole (8.3-16.6 mg/rat) suppressed ovulation by 40%. Local unilateral administration of the drug into the ovarian bursa (1.25 mg/bursa) resulted in 75% inhibition of ovulation in comparison with the contralateral ovary. All the ovulated ova in the oviduct were mature. Histological examination of the ketoconazole-treated ovaries revealed mature oocytes trapped in follicles which failed to ovulate. Furthermore, extraction of oocytes from the large follicles of such ovaries revealed that 79% of them were mature. Addition of ketoconazole (0.0001-0.01 mM) to the culture medium did not affect significantly the spontaneous maturation of rat oocytes. However, ketoconazole at a higher concentration (0.1 mM) caused the degeneration of oocytes. Ketoconazole (0.01 mM) did not affect luteinizing hormone (LH)-stimulated oocyte maturation in explanted preovulatory follicles, even though it inhibited follicular progesterone production to levels below the hormone-free control follicles. At higher levels, ketoconazole caused the degeneration of follicles and the enclosed oocytes. In conclusion, using a potent inhibitor of MAS we have failed to confirm the suggested obligatory role of MAS in the resumption of meiosis in the rat both in vivo and in vitro.

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC 50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC 50 value of 110 ± 38 nM). Through inhibition of mevalonate production, these compounds have a distinct inhibiting effect on cell proliferation. Because proliferation of myoblasts is important in the repair of damaged skeletal muscle, experiments were performed to investigate the effect of lovastatin, simvastatin, and pravastatin on cell proliferation and cell viability. The more potent inhibitors of sterol synthesis, lovastatin, and simvastatin, were able to inhibit the proliferation of these cells during 3 days of incubation with drug concentrations of 1 μM for lovastatin and 0.1 μM or 1 μM for simvastatin. DNA synthesis was decreased by more than 80% in the presence of 1 μM of lovastatin or simvastatin. In contrast, under these conditions, pravastatin had no influence on cell proliferation or DNA synthesis, which is probably related to the lack of inhibition of sterol synthesis by pravastatin on extended incubation. The three 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors did not disturb cell viability because mitochoridrial dehydrogenase activity and ATP content remained proportional to the number of cells in the culture at any concentration used.

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.

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

The chemical syntheses of a number of C 27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterols biosynthesis in cultured mammalian cells. Thus, 5α-cholest-7-ene-3β,11α-diol, 3α-hydroxy-5α-cholest-9(11)-en-12-one and the previously unreported 11α-hydroxy-5α-cholest-7-en-3-one, 5α-cholest-9(11)-ene-3,12-dione, and 3β-hydroxy-5α-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. 5α-Cholest-7-ene-3β,11α-diol was shown to be the most potent inhibitor of sterol synthesis.

Inhibitors of sterol synthesis. Reverse phase high performance liquid chromatography for the separation of cholesterol, 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, and their fatty acid esters.

A relatively simple and rapid method was required for the separation of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of sterol synthesis, from its major metabolites. Conditions have been determined which permit the resolution of the 15-ketosterol and cholesterol and fatty acid esters of the two sterols by reverse phase high performance chromatography. This methodology also permits the resolution of the major esters of the 15-ketosterol and of cholesterol.

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.

Effect of oxygenated sterol compounds on human bone marrow granulocytic progenitor cells

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C). 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10(-7) M. This compound, as well as 6-ketocholestanol, 7- ketocholesterol, and 20 alpha-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.

Effect of Oxygenated Sterol Compounds on Human Bone Marrow Granulocytic Progenitor Cells

Oxygenated sterol compounds are potent inhibitors of sterol and DNA synthesis in mammalian cells. We studied the effects of oxygenated sterols on human marrow granulocytic progenitor cells in vitro (CFU-C)*. 25-Hydroxycholesterol was found to be a potent inhibitor of sterol synthesis in marrow mononuclear cells, with 50% inhibition occurring at approximately 10–7M. This compound, as well as 6-ketocholestanol, 7-ketocholesterol, and 20α-hydroxycholesterol, also demonstrated marked inhibition of CFU-C proliferation. The latter effect, which was not a result of direct cytoxicity of the compounds, was reversible by cholesterol, but not by mevalonic acid. We conclude that inhibition of sterol synthesis by oxygenated sterol compounds may be insufficient to explain their suppression of CFU-C proliferation.

Inhibition of sterol biosynthesis in animal cells by 14 alpha-hydroxymethyl sterols.

The chemical syntheses of a number of 14 alpha-hydroxymethyl sterols and 14 alpha -hydroxymethyl-15 alpha-hydroxysterols 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 7 alpha,8 slpha-epoxy-14 alpha-methyl-5 alpha-cholestan-3 beta,15 alpha-diol, 14 alpha-methyl-5 alpha-cholestan-3 beta,7 alpha,15 alpha-triol, 3 beta,15 alpha-diacetoxy-14 alpha-methyl-5 alpha-cholestan-7 alpha-ol, 3 beta,15 alpha-diacetoxy-7 alpha,32-epoxy-14 alpha-methyl-5 alpha-cholestane, 14 alpha-hydroxymethyl-5 alpha-cholest-6-en-3 beta,15 alpha-diol, 14 alpha-hydroxymethyl-5 alpha-cholest-7-en-3 beta,15 alpha-diol, 7 alpha,32-epoxy-14 alpha-methyl-5 alpha-cholestan-3 beta,15 alpha-diol, 14 alpha-hydroxymethyl-5 alpha-cholest-6-en-3-one, 14 alpha-hydroxymethyl-5 alpha-cholest-7-en-3-one, and 14 alpha-hydroxymethyl-5 alpha-cholets-7-en-15 alpha-ol-3-one. The effects of eight of the above compounds and of 14 alpha-hydroxymethyl-5 alpha-cholest-8-en-3 beta-ol, 14 alpha-hydroxymethyl-5 alpha-cholest-7-en-3 beta-ol, 14 alpha-hydroxymethyl-5 alpha-cholest-6-en-3 beta-ol, and 7 alpha,32-epoxy-14 alpha-methyl-5 alpha-cholestan-3 beta-ol on the synthesis of digitonin-precipitalbe sterols and on levels of HMG-CoA reductase activity in L cells and in primary cultures of fetal mouse liver cells have been investigated. All of the 14 alpha-hydroxymethyl sterols and 14 alpha-hydroxymethyl-15 alpha-hydroxysterols were found to be potent inhibitors of sterol synthesis and to reduce the levels of HMG-CoA reductase activity in these cells. Since hydroxylation of the 14 alpha-methyl group of 14 alpha-methyl sterol precursors of cholesterol can be considered as an obligatory step in the biosynthesis of cholesterol, the finding that 14 alpha-hydroxymethyl sterols are potent inhibitors of cholesterol biosynthesis and cause a reduction in the levels of HMG-CoA reductase activity raises the possibility that oxygenated sterol precursors of cholesterol, such as 14 alpha-hydroxymethyl sterols, may play an important role in the regulation of cholesterol synthesis and in the regulation of processes dependent upon mevalonate and sterol formation.

15-Oxygenated sterols with the unnatural cis-C-D ring junction. Studies of the metabolism of 5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol and 5 alpha,14 beta-cholest-7-ene-3 beta,15 beta-diol.

Four 15-oxygenated sterols with the "unnatural" cis-C-D ring juncture have recently been shown to be potent inhibitors of sterol synthesis in animal cells in culture (Schroepfer, G.J., Jr., Parish, E.J., Chen, H.W., and Kandutsch, A.A. (1977) J. Biol. Chem. 252, 8975-8980; Schroepfer, G.J., Jr., Pascal, R.A., Jr., and Kandutsch, A.A. (1979) Biochem. Pharmacol. 28, 249-252). In the present study we have prepared two of these sterols, [2,4-3H]5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol and [2,4-3H]5 alpha,14 beta-cholest-7-ene-3 beta,15 beta-diol, in labeled form, 5 alpha,14 beta-Cholest-7-ene-3 beta, 15 alpha-diol, but not its 15 beta-hydroxy epimer, was shown to be efficiently converted to cholesterol in 10,000 x g supernatant fractions of liver homogenate preparations from both male and female rats. After incubation of [2,4-3H]5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol with these enzyme preparations a number of labeled products, in addition to cholesterol, were isolated and characterized. These included 5 alpha-cholesta-8,14-dien-3 beta-ol, cholesta-5,7-dien-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, and 5 alpha-cholest-7-en-3 beta-ol. A scheme to account for the enzymatic formation of cholesterol and the other sterol precursors of cholesterol is presented.

Inhibition of human lymphocyte transformation by oxygenated sterol compounds

Oxygenated sterol compounds (OSC), potent inhibitors of sterol synthesis in both resting and mitogen-stimulated human lymphocytes, are capable of suppressing the DNA-synthetic response of human peripheral blood lymphocytes to mitogenic lectins, anti-human thymocyte antiserum, and the mixed lymphocyte culture. The most potent OSC are 20α-hydroxycholesterol and 25-hydroxycholesterol, which inhibit DNA synthesis in mitogenstimulated lymphocytes at 7.4 and 3.9 × 10 −6 M, respectively. Lymphocytes which have been exposed to OSC for 18 hr and washed free of inhibitor are fully capable of DNA synthesis when subsequently challenged with mitogen. Suppression of DNA synthesis by OSC is not apparent during the first 24–40 hr of culture. The inhibition of lymphocyte DNA synthesis by OSC can be partially reversed by the addition of 10 −2 M mevalonate to the culture. Sterol synthesis by mitogen-stimulated lymphocytes is enhanced by culturing them in medium supplemented with lipoprotein (and cholesterol-)-depleted serum. In such medium, the 50% inhibitory doses of 25-hydroxycholesterol for suppression of both mitogen-stimulated lymphocyte DNA and sterol synthesis are approximately equal (−3 × 10 −7 M ). Sterol synthesis is a necessary, but not sufficient, part of the program of lymphocyte proliferative response to mitogens; the evidence presented suggests that lymphocytes can utilize exogenous sterol at least partially for the purpose of cell replication.

Effects of various oxygenated sterols on cellular sterol biosynthesis in chinese hamster lung cells resistant to 25-hydroxycholesterol

The effects of a wide variety of oxygenated sterols upon sterol biosynthesis and hydroxymethylglutaryl-CoA reductase (mevalonate: NADP + oxidoreductase (CoA-acylating), EC 1.1.1.34) activity in a wild-type clone and in a 25-hydroxycholesterol-resistant clone of Chinese hamster lung (Dede) cells are described. Derivatives of cholesterol which were oxygenated in the 6, 7 or 15 positions of the sterol nucleus or in the 20, 22, 24 or 25 positions of the sterol side chain were shown to be potent inhibitors of sterol synthesis and reductase activity in the wild-type cells but none of these substitutions had any effect on the 25-hydroxy cholesterol-resistant A2 clone. A 32-hydroxylated derivative of lanosterol also suppressed sterol synthesis and reductase activity in wild-type cells but had no significant effect upon the A2 line. It was also apparent that a complete sterol side chain was necessary for inhibitory activity. Studies of a wide range of inhibitory sterols indicated that there was a close correlation between their effects upon sterol synthesis and reductase activity and that their inhibitory action was specific for sterol biosynthesis since little effect was observed upon fatty acid or CO 2 synthesis. Previous studies had shown that the uptake of 25-hydroxycholesterol by the resistant A2 line was unimpaired and the present results indicate that metabolism of this oxygenated sterol is also unaltered. These results, in conjunction with previous studies, suggest that the resistant A2 line is defective in feedback regulation of cholesterol synthesis and that all of the oxygenated sterols tested suppress the biosynthetic pathway through at least one common step.

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.

Inhibition of sterol biosynthesis by 9α-fluoro and 9α-hydroxy derivatives of 5α-cholest-8(14)-en-3β-ol-15-one

5α-Cholest-8(14)-en-3β,9α-diol-15-one has been prepared by chemical synthesis in two steps from 5α-cholest-8(14)-en-3β-ol-15-one. 9α-Fluoro-5α-cholest-8(14)-en-3β-ol-15-one was prepared in 91% yield from the corresponding 9α-hydroxysterol. 9α-Fluoro-5α-cholest-8(14)-en-3,15-dione was prepared, in 84% yield, from the corresponding 3β-hydroxy compound. These compounds have been found to be potent inhibitors of sterol synthesis in animal cells in culture. The concentrations of the steroids required to cause a 50% inhibition of the synthesis of digitonin-precipitable sterols from labeled acetate were comparable to those required to cause a 50% reduction in the levels of HMG-CoA reductase activity in the same cells.

7α,15β-Dichloro-5α-cholest-8(14)-en-3β-ol, a novel inhibitor of sterol biosynthesis in animal cells

7α,15β-Dichloro-5α-cholest-8(14)-en-3β-ol has been prepared by controlled lithium aluminum hydride reduction of 3β-benzoyloxy-7α,15β-dichloro-5α-cholest-8(14)-ene. The latter compound was prepared by treatment of 3β-benzoyloxy-14α,15α-epoxy-5α-cholest-7-ene with gaseous HCl in chloroform at −40°. The structure of the dichloro compound was unequivocally established by X-ray crystallographic analysis of the 3β- p-bromobenzoate derivative. 7α,15β-Dichloro-5α-cholest-8(14)-en-3β-ol was found to be a potent inhibitor of sterol synthesis in animal cells in culture. The dichlorosterol caused a 50% reduction of the level of HMG-CoA reductase in L cells in culture at a concentration of 6 × 10 −7 M and a 50% inhibition of the synthesis of digitonin-precipitable sterols from labeled acetate in the same cells at a concentration of 2 × 10 −6 M.

Inhibition of cholesterol synthesis by oxygenated sterols.

Sterols derived from cholesterol by introducing a ketone or hydroxyl function in the 6, 7, 15, 20, 22, 24, or 25 positions are known to be potent inhibitors of sterol synthesis in cell cultures. To gain more information regarding structural requirements for inhibitory activity, inhibitory potencies were determined for a series of 18 C27-steroids with various combinations of ketone and hydroxyl functions substituted in positions 3, 4, 5, 6, and 7, or with a single ketone or hydroxyl function in one of these positions. The effects of nuclear double bonds upon inhibitory activity were also examined. A ketone or hydroxyl function in position 3 and a second ketone or hydroxyl function in position 6 or 7 was required for inhibitory activity with two kinds of cell culture. A 3beta5alpha6beta-triol was not more inhibitory than a comparable 3beta,6beta-diol. Cholestane-3beta 5alpha-diol inhibited sterol synthesis in L cells but not in liver cell cultures. The inhibitory activities of 7-oxygenated sterols were not markedly affected by the presence of a double bond in position 4 or 5. Current knowledge of the mechanism through which the oxygenated sterols suppress cholesterol synthesis is reviewed.

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 Δ 6-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.

Inhibition of sterol biosynthesis in L cells and mouse liver cells by 15-oxygenated sterols.

Described herein are the chemical syntheses of 5alpha-cholest-8(14-en-3beta-ol-15-one, 14alpha-methyl-5alpha-cholest-7-en-3beta-ol-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15beta-ol, 3beta-methoxy-14alpha-methyl-15alpha-ol, and 5alpha-cholest-8(14)-en-3beta, 7xi,15xi-triol. The effects of these compounds and of 5alpha-cholest-8(14)-en-3beta,15beta-diol, 5alpha-cholest-8(14)-en-3beta,15alpha-diol, 5alpha,14beta-cholest-7-en-3beta,15beta-diol, 5alpha,14beta-cholest-7-en-3beta, 15alpha-diol, 14alpha-methyl-5alpha-cholest-7-en-3beta,15beta-diol, and 14alpha-methyl-5alpha-cholest-7-en-3beta,15alpha-diol on sterol synthesis in L cells and primary cultures of fetal mouse liver cells grown in serum-free media have been studied. With the exception of 3beta-methoxy-5alpha-cholest-7-en-15-one, all of the compounds were found to be potent inhibitors of sterol synthesis. With a few exceptions, the concentrations required to cause a 50% reduction in sterol synthesis were similar to those required to cause a 50% reduction in the level of HMG-CoA reductase activity.