Inhibitors of sterol synthesis. Chemical synthesis of 5 beta-cholest-8-ene-3 beta,15 alpha-diol and its effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

5 beta-Cholest-8-ene-3 beta,15 alpha-diol, prepared by hydroboration of 5 beta-cholesta-8,14-dien-3 beta-ol, was determined to have the 14 alpha-H,15 alpha-OH configuration by comparisons of observed and calculated lanthanide-induced shifts for the 3-tertbutyldimethylsilyl derivative. The 3 beta,15 alpha-diol was found to exist partially in a conformation in which ring B is a 5 beta, 6 alpha-half chair and the axial-equatorial orientation of ring A substituents is reversed. This conformation has been observed previously for 3 beta-(p-bromobenzoyloxy)-5 beta-cholesta-8,14-diene and for some cis-decalin derivatives. 5 beta-Cholest-8-ene-3 beta,15 alpha-diol was found to be highly active in the lowering of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells and only slightly less active than the corresponding sterol (5 alpha-cholest-8-ene-3 beta,15 alpha-diol) with the trans A-B ring junction.     

Crystal structure of 3 beta-benzoyloxy-6 alpha-chloro-5 alpha-cholest-7-ene, a key intermediate in the chemical synthesis of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one

The X-ray crystal structure of 3 beta-benzoyloxy-6 alpha-chloro-5 alpha-cholest-7-ene (IV) was determined by the heavy atom method and refined to R = 0.063 (space group P21, a = 11.364, b = 11.089, c = 12.232, beta = 99.43 degrees, Z = 2). IV was previously shown to be an important intermediate in the acid-catalyzed isomerization of 7-dehydrocholesteryl benzoate. The present work unequivocally establishes the location of the double bond and the configuration of the chlorine of IV, information which is essential to the correct formulation of the mechanism of this reaction.     

The role of a cholesta-8,14-dien-3β-ol system in cholesterol biosynthesis

The biosynthesis of cholesterol from squalene and tritiated water is described. Degradation of the cholesterol indicated that C-15 may be involved in cholesterol biosynthesis. In accordance with this view it is shown that in the conversion of [2RS-(3)H(2)]mevalonic acid into cholesterol one of the hydrogen atoms at C-15 is removed. A mechanism for the removal of the 14alpha-methyl group in steroid biosynthesis that involves the labilization of a C-15 hydrogen atom is outlined. In accordance with the requirement of this scheme it is shown that 4,4'-dimethyl-cholesta-8,14-dien-3beta-ol is converted into cholesterol.     

A facile synthesis of 5-thio- -fucose and 5-thio- -arabinose from -arabinose

5-Thio- -fucopyranose tetraacetate was synthesized in 11 steps from or -arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a

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derivative was achieved to give the corresponding 6-deoxy-β- -altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy- -altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio- -fucopyranose. A derivative was also prepared from 6-deoxy-β- -altrofuranose derivatives. 5-Thio- -arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio- -fucose, was also synthesized from in 5 steps. 5-Thio- -arabinose showed weak inhibitory activity against α- -fucosidase from bovine kidney (K_{i = 0.77 mM}).     

Mechanism and stereochemistry of the 5-aminolaevulinate synthetase reaction

1. Two mechanisms for the biosynthesis of 5-aminolaevulinate from glycine and succinyl-CoA (3-carboxypropionyl-CoA) are considered. One of the mechanisms involves the retention of both the C-2 H atoms of glycine during the synthesis of 5-aminolaevulinate, whereas the other predicts the retention of only one of the C-2 H atoms of glycine. 2. Highly purified 5-aminolaevulinate synthetase from Rhodopseudomonas spheroides was used to show that the C-2 H atom of glycine with R configuration is specifically removed during the biosynthesis of 5-aminolaevulinate. 3. The mechanism of the condensation therefore differs from the analogous reaction of the biosynthesis of sphinganine from palmitoyl-CoA and serine, in which the C-2 H of serine is retained (Wiess, 1963).     

Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.     

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.     

Inhibitors of sterol synthesis. Characterization of beta,gamma-unsaturated analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells

Treatment of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (1), a potent regulator of cholesterol metabolism, with perchloric acid in methanol resulted in its partial isomerization to the beta,gamma-unsaturated 15-ketosterols, 3 beta-hydroxy-5 alpha,14 beta-cholest-8-en-15-one (2) and 3 beta-hydroxy-5 alpha,14 beta-cholest-7-en-15-one (3), which were easily separated from 1 by chromatography. Isomers 1, 2, and 3 could be distinguished by their chromatographic retention times as well as by their physical and spectral properties. Reduction of 2 with sodium borohydride gave 5 alpha,14 beta-cholest-8-ene-3 beta,15 beta-diol (4), for which the C-15 configuration was established from the lanthanide-induced shifts of its 3 beta-tert-butyldimethylsilyl ether. 1H and 13C NMR chemical shift differences between 2, 3, and 4 indicated the involvement of variable populations of conformers that differ in the flexible C-D ring system and in the side chain. Compounds 2, 3, and 4 lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.     

Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis

The enzymic conversion of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid (i.e. o-succinylbenzoic acid) to 1,4-dihydroxy-2-naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two-step process, was investigated using chirally labelled samples of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate-lyase (EC 4.1.3.1), 2-oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4-(2'-carboxyphenyl)-4-oxobutyrate synthase system and 4-(2'-carboxyphenyl)-4-oxobutyrate: CoA ligase. Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiomeric samples of 2-oxoglutarate enantiotopically labelled at C-3. These samples were converted enzymically to succinate with retention of configuration at C-2 and C-3, and to 4-(2'-carboxyphenyl)-4-oxobutyric acid with retention of configuration at C-3. Isocitrate glyoxylate-lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2-oxoglutarate enantiotopically tritiated at C-4 or at C-3 and C-4. The four variously labelled samples of 2-oxoglutarate were enzymically converted to the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. The resulting variously labelled coenzyme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRe atom of the oxobutyric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C-3 of 1,4-dihydroxy-2-naphthoic acid. The second step in this ring closure reaction is the rate-limiting step.     

Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts

Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

5-Substituted-2,3-diphenyltetrahydrofurans: a new class of moderately selective COX-2 inhibitors

The nature of C-5 substituent and the configuration at C-5 carbon of 2,3-diphenyltetrahydrofurans, with chiral centres at C-2, C-3 and C-5, show a remarkable influence on their COX-2 inhibition and selectivity. Out of the eight compounds investigated here, 1b with COOH group and R* configuration at C-5, and 2d with CH2SCH2CH3 group and S* configuration at C-5 have been identified as lead molecules for further studies on COX-2 inhibition.     

Stereospecific elimination of hydrogen at C-10 in eicosapentaenoic acid during the conversion to leukotriene C5

(10L)- and (10D)-[1-14C, 10-3H]5,8,11,14,17-eicosapentaenoic acids were synthesized to investigate mechanistic and stereochemical aspects of leukotriene biosynthesis. Experiments with mastocytoma cells showed that a hydrogen is stereospecifically eliminated from C-10 during the conversion of eicosapentaenoic acid to leukotriene C5. The hydrogen lost has the pro-S (D) configuration. 5-Hydroxy-6,8,11,14,17-eicosapentaenoic acid, formed in the same experiments, was enriched in tritium when the (10D), but not when the (10L), isomer of labeled eicosapentaenoic acid was used. This indicates that oxygenation of the acid at C-5 occurred before the elimination of hydrogen and suggests that removal of the pro-S hydrogen at C-10 in 5-hydroperoxy-6,8,11,14,17-eicosapentaenoic acid initiates its transformation to trans-5(S),6(S)-oxido-7,9-trans-11,14,17-cis-eicosapentaenoic acid (leukotriene A5).     

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.     

D-Fructose-L-sorbose interconversions. Access to 5-thio-D-fructose and interaction with the D-fructose transporter, GLUT5.

Epimerisation and subsequent functionalization at C-5 of D-fructopyranose derivatives under Mitsunobu and Garegg's conditions provided efficient access to 5-thio-D-fructose (2) as well as to 5-azido-5-deoxy-1,2-O-isopropylidene-beta-D-fructopyranose (19), a known precursor to 2,5-deoxy-2,5-imino-D-mannitol (3). The interaction of 2 with the D-fructose transporter GLUT5, was found to be weaker than that of D-fructose, a result that suggests involvement of the ring oxygen atom in the recognition of D-fructose by GLUT5.     

Reversible cleavage of the cobalt-carbon bond to coenzyme B12 catalysed by methylmalonyl-CoA mutase from Propionibacterium shermanii. The use of coenzyme B12 stereospecifically deuterated in position 5'

1. (5'R)-(5'-2H1)Adenosine [(5'R):(5'S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6-N-benzoyl-2',3'-O-isopropylidene-5'-oxoadenosine with a reagent obtained from LiAl2H4 and (-)-isoborneol. 2. (5'S)-(5'-2H1)AdoCbl [(5'S):(5'R) = 74:26] (AdoCbl = 5'-deoxyadenosylcobalamin) was synthesized by reacting cobal(I)amin with (5'R)-2'-3'-O-isopropylidene-5'-tosyl-(5'-2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group. 3. (5'R)-(5'-2H1)AdoCbl [(5'R):(5'S) = 77:23] was prepared by reacting cobalt(I)amin with (5'S)-5'-chloro-5'-(5'-2H1)deoxyadenosine [(5'S):(5'R) = 80:20] obtained in turn from (5'R)-(5'-2H1)adenosine. The reaction sequence involved two consecutive inversions at the C-5' atom of adenosine 4. Comparison of the 500-MHz 1H-NMR spectra of unlabelled, (5'S)- and (5'R)-(5'-2H1)AdoCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5'-HRe and 5'-HSi atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C-4'--C-5' bond. 5. Samples of (5'R)- and (5'S)-(5'-2H1)AdoCbl were incubated with methylmalonyl-CoA mutase from Propionibacterium shermanii. Examination by 1H-NMR spectroscopy at 500 MHz revealed partial loss and stereochemical scrambling of the deuterium at the 5' position. This indicates transient conversion of the C-5' atom into a torsiosymmetric group and hence cleavage of the cobalt-carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.     

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β-p-bromobenzoyl ester of diol B was prepared from 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; $\text{a = 9.24}\text{A}\text{?}$, $\text{b = 12.61}\text{A}\text{?}$, $\text{c = 7.03}\text{A}\text{?}$, α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.     

Synthesis of polyhydroxysterols (V): efficient and stereospecific synthesis of 24-methylene-cholest-5-ene-3beta,7alpha-diol and its C-7 epimer

This paper describes the efficient and stereospecific synthesis of cytotoxic dihydroxylated sterols, 24-methylene-cholest-5-ene-3beta,7alpha-diol 1, and its C-7 epimer, 24-methylene-cholest-5-ene-3beta,7beta-diol 2. The crux of the synthesis is that the selective allylic oxidation of 24-methylene-cholesteryl acetate proceeds to 24-methylene-7-keto-cholesteryl acetate without extensive byproduct formation from reaction at the Delta24(28) double bond. This methodology may be useful for the preparation of other oxysterols with non-standard side chains.     

Inhibitors of sterol synthesis. Concerning the structure of 15 beta-methyl-5 alpha,14 beta-cholest-7-ene-3 beta,15 alpha-diol, an inhibitor of cholesterol biosynthesis

The chemical synthesis of 3 beta-p-bromobenzoyloxy-15 beta-methyl-5 alpha,14 beta-cholest-7-en-15 alpha-ol from 15 beta-methyl-5 alpha, 14 beta-cholest-7-ene-3 beta,15 alpha-diol is described. The structure of the former compound was unambiguously determined by X-ray crystallographic analysis. The space group of the crystal was P2 with unit cell parameters a = 12.611 A, b = 9.826 A, c = 13.221 A, b = 91.71 degrees and Z = 2. The structure was solved by the heavy atom method and refined to a final R of 0.041. Asymmetry parameters indicated that ring A is a symmetrical chair, that rings B and C are half chairs, and that ring D is a 15 alpha-envelope.