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.     

Side-chain cleavage of C27-3-oxo-4-ene sterols

In this study various C27 sterols with a 3-oxo-4-ene structure were incubated with adrenal cortex mitochondrial preparations. (22R)-22-Hydroxy-4-cholesten-3-one and (20R,22R)-20,22-dihydroxy-4-cholesten-3-one were found to be converted into progesterone. This suggests the existence of a pathway for adrenal progesterone formation analogous to the normal 3 beta-hydroxy-5-ene pathways. (20S)-20-Hydroxy-4-cholesten-3-one was hydroxylated at C25. 4-Cholesten-3-one, 25-hydroxy-4-cholesten-3-one and (22S)-22-hydroxy-4-cholesten-3-one were not converted to a measurable extent. With 3-oxo-4-ene C27 sterols as substrates, the cholesterol side-chain cleaving enzyme system seems to require the presence of a 22R-hydroxyl group in the substrate. The clinical relevance of these observations is discussed.     

Minor and trace sterols in marine invertebrates XVI. 3 epsilon-hydroxymethyl-A-nor-5 alpha-gorgostane, a novel sponge sterol

The free sterol mixture of the sponge Stylotella agminata contained a series of 3-hydroxymethyl-A-nor sterols of 5 alpha-cholestane, 5 alpha-cholest-22-ene, 24-methyl-5 alpha-cholestane, 24-ethyl-5 alpha-cholestane, and 24-ethyl-5 alpha-cholest-22-ene. A new cyclopropane-containing sterol was isolated and shown to be 3 epsilon-hydroxymethyl-A-nor-5 alpha-gorgostane. Sterols with conventional nuclei were also present as minor constituents;     

Concerning the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, a novel regulator of cholesterol metabolism

A four-step synthesis of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) from 7-dehydrocholesterol is described. This synthesis, which is efficient and suitable for kilogram scale work, was carried out in a 33% overall average yield (39% overall best yield). A major byproduct of the hydrolysis of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene to I was found to be the ring C aromatic sterol 12-methyl-18-nor-5 alpha-cholesta-8,11,13-trien-3 beta-ol. Several other intermediates and byproducts of these reactions were also identified. All new sterols were characterized by 1H- and 13C-NMR.     

3β-Hydroxysteroid dehydrogenase activity in bovine adrenal cortex mitochondria: conversion of oxygenated 3β-hydroxy-sterols

Adrenal cortex 3β-hydroxysteroid dehydrogenase (3β-HSD) is able to convert many C₁₉ and C₂₁ 3β-OH-5-ene steroids into products with a 3-keto-4-ene structure. In the present investigation we describe the conversion of a number of C₂₇ and C₂₄ 3β-5-ene sterols by adrenal milochondrial 3β-HSD. Among these substrates were (20S)-5-cholestcne-3β,20-diol. (22R)-5-cholestene-3β,22-diol and (20R,22R)-5-cholestene-3β,20.22-triol, compounds occurring as intermediates in the cholesterol side-chain cleavage reaction. Cholesterol itself was not converted to a measurable extent.     

Profiling sterols in cerebrotendinous xanthomatosis: utility of Girard derivatization and high resolution exact mass LC-ESI-MS(n) analysis

In this study we profile free 3-oxo sterols present in plasma from patients affected with the neurodegenerative disorder of sterol and bile acid metabolism cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girard's P (GP) reagent 1-(carboxymethyl) pyridinium chloride hydrazide, an approach uniquely suited to studying the 3-oxo sterols that accumulate in CTX, as Girard's reagent reacts with the sterol oxo moiety to form charged hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MS(n) analysis of these sterols in the presence of a large excess (3 orders of magnitude) of cholesterol. Often cholesterol detected in biological samples makes it challenging to quantify minor sterols, with cholesterol frequently removed prior to analysis. We derivatized plasma (10 μl) without SPE removal of cholesterol to ensure detection of all sterols present in plasma. We were able to measure 4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of bile acid precursors were identified in plasma using this technique. GP-tagged sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS(3) ([M](+)→[M-79](+)→) spectra that provided additional structurally informative fragment ions.     

Production of phytosterols by mature Digitalis purpurea L. plants

Summary The relative rates of production by mature Digitalis purpurea plants of cholesterol, campesterol, stigmasterol and sitosterol isolated from the glycoside and lipid fractions of the plant extract, were estimated. Plants were exposed to an atmosphere of ¹⁴CO₂ in a growth chamber and the radioactivity of the individual sterols assessed at intervals over 25 days on a gas-liquid radio chemical chromatography (GLRC). Incorporation of ¹⁴CO₂ occurred within 12 hours into both fractions of the extract. The 5-ene sterols were produced at a similar rate over a period of 25 days but the lipid fraction was about 100% more radioactive than the glycoside fraction.     

A simple synthesis of 3beta-acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene

3beta-Acetoxy-20-oxomethylpregn-5-ene and 3beta-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R,23R)-sitost-5-ene-3beta,22,23-triol in 66% overall yields.     

The Chemical Constituents of Amoora yunnanensis

Two new sterols, 3β,7α,16β-trihydroxy-stigmast-5,22-diene (1), 3β,7α,16β-trihydroxy-stigmast-5-ene (2), were isolated together with six known compounds, ergosta-5,24(28)-dien-3β,7α-diol (3), ergosta-5,24(28)-dien-3β,7β,16β-triol (4), β-amyrone (5), β-amyrin (6), 11α,12α-epoxy-14-taraxeren-3-one (7), and 6-guaiene-4α,10α-diol (8) from the EtOH extract of the bark of Amoora yunnanensis (H. L. Li) C. Y. Wu. Their structures were deduced on the basis of spectral data.     

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.     

A simple synthesis of 3β-acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene

3β-Acetoxy-20-oxomethylpregn-5-ene and 3β-acetoxy-20-hydroxymethylpregn-5-ene were synthesized from (22R, 23R)-sitost-5-ene-3β,22,23-triol in 66% overall yields.     

Further evidence that there is more than one adrenal 21-hydroxylase system

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.     

Evidence for the Presence of "Metabolic Sterols" in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii , respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β -sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C₂₈ and C₂₉ sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii , probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own "metabolic sterols"     

Triterpenes from Maytenus horrida

The new triterpene 1β,3β,11α-trihydroxyolean-12-ene and the already known compounds, lupeol, germanicol, 3β-hydroxy-glutin-5-ene, β-amyrin, 3β-hydroxyolean-9(11),12-diene, 3-oxo-olean-9(11),12-diene, 3β,11α-dihydroxyolean-18-ene,3β,11α-dihydroxyolean-12-ene, 3β,29-dihydroxy-glutin-5-ene and dulcitol, were isolated from the root bark of Maytenus horrida.     

Correlation between oxysterol binding to a cytosolic binding protein and potency in the repression of hydroxymethylglutaryl coenzyme A reductase

Support for the role of a cytosolic oxysterol-binding protein in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was obtained by correlating the relative binding affinities of a wide range of oxysterols to their potency in suppressing HMG-CoA reductase activity in mouse fibroblast cell cultures. Forty-seven oxysterols encompassing a 100-fold range of activity in both assays were tested and the two parameters were closely correlated for 35 of the sterols. Twelve sterols showed poor binding when compared to their ability to suppress HMG-CoA reductase activity in cell cultures. Among these were seven sterols with a ketone function at C-3. For this group, the discrepancy could be explained by their rapid conversion within cells to the 3 beta-hydroxy derivatives which have a much higher affinity for the binding protein. One sterol with 3-keto-4-ene grouping was not reduced to its 3 beta-hydroxy derivative in cells and thereby showed no discrepancy in the two assays. The remaining five sterols exhibiting discordant activities in the two tests contained 4,4-dimethyl moieties and were relatively weak suppressors of HMG-CoA reductase activity. Cellular metabolism of these sterols was not detected. Possible reasons for their apparent inactivity in the binding assay are discussed.     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

Sesqui- and diterpenes from the liverwort Gackstroemia decipiens

Six rosanes, 5 beta,11 beta-dihydroxy-ros-15-ene, 5 beta,12 beta-dihydroxy-ros-15-ene, 11 beta-hydroxy-7-oxo-rosa-5,15-diene, 1 alpha,5 beta,11 beta-trihydroxy-7-oxo-ros-15-ene, 5 beta,20-epoxy-20-hydroxy-ros-15-ene and 5 beta,20-epoxy-20-methoxy-ros-15-ene along with the enantiomer of the already reported 11 beta-hydroxy-rosa-5,15-diene and the known 5 beta-hydroxy-ros-15-ene have been isolated from the liverwort Gackstroemia decipiens. Furthermore, the sesquiterpenes 3-acetoxy-7,11-dihydroxy-farnesa-1,5,9-triene and 1 beta,10 beta-epoxy-nardosin-7,11-diene were identified. Their structures were elucidated by NMR spectroscopy.     

Sterol and Squalene Content of a Docosahexaenoic-Acid-Producing Thraustochytrid: Influence of Culture Age, Temperature, and Dissolved Oxygen

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T₋₄, T₋₂, and T_p, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly. Received January 8, 2001; accepted March 6, 2001.     

The sterols of the echinoderm Asterias rubens

1. Twenty-two sterols were identified in the starfish Asterias rubens (Phylum, Echinodermata; Class, Asteroidea). 2. The major 4-demethyl sterols had a Delta(7) bond and the C(27) compound 5alpha-cholest-7-en-3beta-ol predominated over other mono- and di-unsaturated sterols belonging to the C(26), C(27), C(28) and C(29) series. 3. Small amounts of cholest-5-en-3beta-ol and 5alpha-cholestan-3beta-ol were also present. 4. The minor sterols identified all contained either one or two methyl groups at C-4 and are considered to be potential biosynthetic precursors of 5alpha-cholest-7-en-3beta-ol. 5. Three sterols possessing a 9beta,19-cyclopropane ring were also isolated and were probably derived by the starfish from a dietary source.     

Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.