Synthesis, properties and reactions of 3 beta-benzoyloxy-7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-ene.

Treatment of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (I) with gaseous HCl in chloroform at -40 degrees C gave, in 87% yield, 3 beta-benzoyloxy-7 alpha,15 beta-dichloro-5 alpha cholest-8(14)-ene (III). Reduction of the latter compound with lithium aluminum hydride in ether at room temperature for 20 min gave, in 86% yield, 7 alpha-15 beta-dichloro-5 alpha-cholest-8(14)-en-3 beta-ol (IV). The latter compound was fully characterized and assignments of the individual carbon peaks in the 13C nuclear magnetic resonance spectra of this sterol have been completed. Reduction of III with excess lithium aluminum hydride in refluxing ether for 4 days gave, in 74% yield, 5 alpha-cholesta-7,14-dien-3 beta-ol (VI). Reduction of the dichloro-steryl benzoate III with lithium triethylborohydride in tetrahydrofuran gave, in 88% yield, 5 alpha-cholest-8(14)-en-3 beta-ol (VII). A similar reduction using lithium triethylborodeuteride led to the formation of [7 beta, 15 xi-2 H2]-VIIa. Treatment of III with concentrated HCl in a mixture of chloroform and methanol gave, in 79% yield, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-15-one (II) which was characterized as such and as the corresponding free sterol.     

Synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one: evaluation as potential hypocholesterolemic agents

An efficient procedure for the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one is described. These ketosterols have been shown to have possible significant hypocholesterolemic effects when fed to normal rats at a level of 0.15% in a laboratory chow diet. The diets containing the steroids caused significant decreases in food consumption which were associated with decreases in the rate of gain in body weight.     

Concerning the structure of 3 beta-benzoyloxy-5 beta-cholesta-8,14-diene, a major byproduct in the chemical synthesis of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

The X-ray crystal structure of 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene (space group P21, a = 10.698 A, b = 9.487 A, c = 15.024 A, beta = 96.05 degrees, Z = 2) was determined by the heavy atom method and refined to R = 0.075. This heavy atom derivative was synthesized from 5 beta-cholesta-8,14-diene-3 beta-ol, the benzoate ester of which was previously shown to be the major byproduct in the low-temperature isomerization of 7-dehydrocholesteryl benzoate in HCl/chloroform. The work presented here establishes unequivocally that the configuration of this isomerization byproduct at C-5 is 5 beta-H and that the configuration at C-17 was unchanged.     

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.     

Transformation of 5 alpha-cholest-7-en-3 beta-ol to cholesterol and cholestanol in cerebrotendinous xanthomatosis

The metabolism of Delta(7)-cholestenol, cholesterol, and cholestanol was examined in a patient with cerebrotendinous xanthomatosis after intravenous pulse-labeling with a mixture of dl-[2-(14)C]mevalonate and stereospecific 3S,4S,3R,4R-[4-(3)H]mevalonate. Silver nitrate and reversed-phase thin-layer chromatography were used to purify the sterols isolated from the feces, and their identities were confirmed by gas-liquid chromatography-mass spectrometry. The specific activities were determined and plotted as a function of time. Isotope ratio measurements and specific activity decay curves showed that sterol synthesis proceeded in the following sequence: mevalonate, squalene, lanosterol, Delta(7)-cholestenol, cholesterol, cholestanol. Labeled cholesterol precursors might be advantageously used to measure changes in cholesterol synthesis because they appear to equilibrate rapidly and have very short turnover times.     

Sterol synthesis: studies of the metabolism of 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol

[3 alpha-3H]14 alpha-Methyl-5 alpha-cholest-7-en-3 beta-ol has been prepared by chemical synthesis. The metabolism of this compound has been studied in the 10,000 g supernatant fraction of liver homogenates of female rats. Efficient conversion to cholesterol was observed. Other labeled compounds recovered after incubation of [3 alpha-3H]14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol with the enzyme preparations include the unreacted substrate, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, cholesta-5,7-dien-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, and 5 alpha-cholest-7-en-3 beta-ol. In addition, significant amounts of incubated radioactivity were recovered in steryl esters. The steroidal components of these esters were found to contain labeled 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, 5 alpha-cholest-7-en-3 beta-ol, and cholesterol.     

A new chemical synthesis of 5alpha-cholest-8(14)-en-3beta,5alpha-diol

Reduction of 3beta-benzoyloxy-14alpha,15alpha-epoxy-5alpha-cholest-7-ene with lithium in ethylenediamine gave 5alpha-cholest-8(14)-en-3beta, 5alpha-diol in high yield. This procedure offers an alternate synthesis through the reductive rearrangement of an alpha,beta-unsaturated steroidal epoxide.     

Synthesis of the marine epoxy sterol 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol

The synthesis of 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol (1), a highly oxygenated marine sterol containing a 9,11-epoxide moiety in the nucleus, is described. Epoxy sterol 1 was synthesized from cholesta-5,7-dien-3 beta-ol. Oxidation of this sterol with m-chloroperbenzoic acid followed by hydrolysis and acetylation furnished 5 alpha-cholest-7-ene-3 beta,5,6 alpha-triol 3,6-diacetate (2). Mercuric acetate dehydrogenation of diacetate 2, followed by oxidation with manganese dioxide and epoxidation with m-chloroper-benzoic acid, afforded 9 alpha,11 alpha-epoxy-3 beta,5-dihydroxy-5 alpha-cholest-7-en-6-one (5). Reduction of 5 with lithium aluminum hydride gave the desired compound 1. The structures of all synthetic intermediates were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A reassignment of resonances for carbons 1, 8, and 15 in the 13C NMR spectrum of 1, based on 2D-NMR correlation spectroscopy, has been accomplished.     

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.     

Changes in adenosine 5'-triphosphate, adenylate energy charge and adenosine 3'5'-cyclic monophosphate during the freezing of buffalo semen

In freshly ejaculated buffalo semen (N = 4) there were 24.61 +/- 5.28 nmol ATP and 40.39 +/- 5.94 nmol total adenylate/10(8) spermatozoa, and 97.75 +/- 7.06 pmol cAMP/10(9) spermatozoa. The semen was frozen in 4 steps (I, dilution; II, cooling; III, glycerolization and equilibration; IV, freezing and thawing). Motility, ATP, total adenylate and cAMP were significantly lower after Step IV than after Step I. Motility and ATP concentration were significantly correlated in egg-yolk--Tris (r = 0.530, P less than 0.05), skim milk--egg yolk (r = 0.754, P less than 0.01), egg yolk--citrate--glucose (r = 0.784, P less than 0.01) and citric acid--whey (r = 0.551, P less than 0.05). Cyclic AMP and motility in egg yolk--Tris were also correlated (r = 0.714, P less than 0.01). The adenylate energy charge was stable in all 4 freezing steps.     

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.     

Interaction of mammalian deoxyribonuclease V, a double strand 3' to 5' and 5' to 3' exonuclease, with deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma

Novikoff hepatoma stimulatory factor IV has been resolved from the DNA polymerase-beta on a single-stranded DNA-cellulose column and then purified to > 95% homogeneity on hydroxylapatite. A single band of Mr = 12,000 is found on sodium dodecyl sulfate-polyacrylamide gels. Addition of factor IV to a DNA synthesis reaction causes (i) an increase in initial velocity, (ii) a prolongation of linear synthesis, and (iii) an increase in extent of incorporation. In the absence of factor IV, the reaction reaches a plateau in approximately 1 h. Factor IV, added at this point, causes resumption of synthesis with kinetics similar to when factor IV was present from the start. When factor IV is present, synthesis is followed by DNA degradation, indicating nuclease activity. Factor IV is shown to be an exonuclease which hydrolyzes double-stranded substrates in both the 3' to 5' and 5' to 3' directions at similar rates. Factor IV interacts with the 3.3 S beta-polymerase forming an aggregate sedimenting at 4.1 S and containing both polymerase and exonuclease activities. Analysis of fractions containing a beta-polymerase . exonuclease complex on polyacrylamide gels suggests a stoichiometry of 1:1. The exonuclease shows a strong preference for double-stranded substrates and is most active on poly(dA-dT). It can hydrolyze chains containing either a 3'- or 5'-phosphoryl or a 5'- or 3'-hydroxyl terminus. The product of digestion is predominantly 5'-nucleoside monophosphates. The enzyme cannot hydrolyze di- or trinucleotides, lacks RNase-H activity, and will not liberate thymine dimers from UV-irradiated DNA. The exonuclease has an alkaline pH optimum and requires a divalent cation. Since the properties of this exonuclease are unlike those of previously described mammalian DNases, we have named this enzyme mammalian DNase V.     

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. A highly efficient and specific side-chain oxidation of 3 beta-acetoxy-5 alpha-cholest-8(14)-en-15-one for construction of metabolites and analogs of the 15-ketosterol.

As part of a program directed towards the chemical syntheses of potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism, several routes have been explored for the preparation of 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (IV). These investigations led to a remarkably specific and efficient side-chain oxidation of I. For example, treatment of the acetate of I with a mixture of trifluoroacetic anhydride, hydrogen peroxide, and sulfuric acid for 3.5 h at -2 degrees C gave a crude product consisting of 3 beta-acetoxy-24-trifluoroacetoxy-5 alpha-chol-8(14)-en-15-one (XI), 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (XII), and 3 beta, 24-diacetoxy-5 alpha-chol-8(14)-en-15-one (XIII) in yields of 58%, 8%, and 3%, respectively, by HPLC analysis. XI was readily hydrolyzed to XII upon treatment with triethylamine in methanol at room temperature. Oxidation of XII with Jones reagent gave 3 beta-acetoxy-15-keto-5 alpha-chol-8(14)-en-24-oic acid (XVIII) from which its methyl ester (IX) was prepared by treatment with diazomethane. Mild alkaline hydrolysis of XVIII gave the 3 beta-hydroxy-delta 8(14)-15-keto C24 acid (IV). Hydrolysis of the crude product of the side-chain oxidation with K2CO3 in methanol gave 3 beta,24-dihydroxy-5 alpha-chol-8(14)-en-15-one (XIV) which was oxidized with Jones reagent to yield 3,15-diketo-5 alpha-chol-8(14)-en-24-oic acid (XV). Treatment of XV with diazomethane gave its methyl ester (XVI) which, upon controlled reduction with NaBH4, yielded methyl 3 beta-hydroxy-15-keto-5 alpha-chol-8(14)-en-24-oate (XVII). Compound IX was also prepared by an independent route. Full 1H and 13C NMR assignments are presented for 12 new compounds. IV caused a approximately 56% reduction of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells at a concentration of 2.5 microM. In contrast, the corresponding 3,15-diketo acid XV had no detectable effect on reductase activity under the same conditions.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

5''-3'' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.     

Synthesis and cytotoxicity of platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin and 3-aminocycloheptanespiro-5-hydantoin

Four new platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin (acpsh) and 3-aminocycloheptanespiro-5-hydantoin (achpsh) were synthesized and characterized by elemental analysis, IR and 1NMR spectra. The spectral analyses indicated a cis-square planar structure of the complexes with ligands coordinated via the NH2 group. The complexes were evaluated for in vitro cytotoxicity in murine erythroleukemia (MEL) cells, clone F4N, using cell-growth and macromolecular synthesis assay. The compounds, with exception of [Pt(NH3)(achpsh)Cl2] (IV), exhibited much lower cytotoxicity than that of cisplatin (DDP). Compound IV was nearly as cytotoxic as DDP. The new complexes exerted low antibacterial activity as assessed by seven bacterial strains.     

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.     

Stereospecific reduction of keto group in 3beta-triphenylmethoxy-5alpha-cholest-8(14)-en-15-one

The reduction of 3 beta-triphenylmethoxy-5 alpha-cholest-8(14)-en-15-one with lithium aluminum hydride resulted in a quantitative yield of 3 beta-triphenylmethoxy-5 alpha-cholest-8(14)-en-15 beta-ol.