Difference in cytotoxicity of (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol is not explained by different patterns of metabolites

Ehrlich ascites tumor cells in suspension culture were incubated with the plant-derived sterol isomers (22R)-cholest-5-ene-3 beta,7 alpha,22-triol and (22R)-cholest-5-ene-3 beta,7 beta,22-triol. Both sterols were 7-dehydroxylated by the neoplastic cells, and the product was identified as (22R)-22-hydroxycholesta-4,6-dien-3-one. At sub-toxic sterol concentrations the conversion of the 7 alpha-hydroxy compound was about 5 times higher than that of the 7 beta-isomer. At higher sterol concentrations the 7 beta-hydroxy compound caused growth inhibition of the Ehrlich ascites cells, whereas the 7 alpha-hydroxylated sterol was ineffective. The rate of 7 alpha-dehydroxylation was, however, too low to be considered a likely pathway for detoxification. No other lipid-extractable products were detected, and no water-soluble products with influence on cell proliferation were present. Thus, the cytotoxicity is probably attributed to a property of the 7 beta-hydroxyl group of the (22R)-cholest-5-ene-3 beta,7 beta,22-triol.     

24Epsilon-methylcholestane-3beta,5alpha,6beta,25-tetrol 25-monoacetate. A novel polyhydroxylated steroid from an alcyonarian

24ξ-Methylcholestane-3β,5a,6β,25-tetrol 25-monoacetate has been isolated from an Alcyonarian and its structure elucidated. A synthesis of the corresponding tetrol, 24ξ-methylcholestane-3β,5a,6β,25-tetrol, confirmed the original structure assignment.     

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.     

Synthesis of (3alpha,7beta,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol, a metabolite of ORG OD14, and its 7-epimer

The syntheses of the 7beta-hydroxy metabolite of ORG OD14 (Livial((R))), (3alpha,7beta,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol (35), and its 7-epimer, (3alpha,7alpha,17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol (11), are described.     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

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.     

Synthesis of (3alpha,7beta, 17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-triol, a metabolite of ORG OD14, and its 7-epimer

The syntheses of the 7beta-hydroxy metabolite of ORG OD14 (Livial), (3alpha,7beta, 17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-t riol (35), and its 7-epimer, (3alpha,7alpha, 17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-t riol (11), are described.     

An efficient one-pot synthesis of 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione from 9-bromide-11-hydroxypregna-1, 4, 16-trien-3, 20-dione by two annulation reactions

An efficient method to prepare 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione in one pot was reported. Treatment of 9-bromide-11-hydroxypregna-1, 4, 16-trien-3, 20-dione with Raney Ni in absolute ethanol afforded 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione by two annulation reactions in reasonable yield. The absolute configuration was also confirmed by X-ray crystal analysis.     

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.     

1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies.

Heating (20R)-3beta,20,26-trihydroxy-27-norcholest-5-en-22-one (1) with hydrazine and KOH at 160 degrees C completely converted the steroid to a diastereoisomeric mixture of the new (20R,22RS)-27-norcholest-5-ene-3beta,20,22-triols (2). Exclusive formation of 2 suggests that the expected Wolff-Kishner reduction to a methylene group at the C-22 ketone in 1 was diverted to the C-26 position by a 1,5-hydride shift. All attempts under acid conditions failed to produce a C-22 phenyl hydrazone from 1. However, reaction of 1 was reacted with phenylhydrazine in hot KOH, gave the C-26 phenylhydrazone 4 as the sole product. Evidently, under alkaline conditions, first a hydride ion undergoes an intramolecular transfer from the C-26 CH(2)OH group to the C-22 ketone in 1, and then the phenylhydrazine traps the newly formed aldehyde. To examine this hypothesis, we constructed computer-simulated transition state models from quantum chemical calculations and then compared data from these models with NMR measurements of the reaction mixtures containing 2. The NMR data showed that the C-22 diastereoisomers of 2 are formed in a nearly 1:1 ratio exactly as predicted from the energy-optimized transition states, which were calculated for intramolecular 1,5-hydride shifts that produced each of the two C-22 diastereoisomers. Accordingly, these results support the hypothesis that an intramolecular 1,5-hydride shift mechanism promotes complete conversion of 1 to 2 under classical Wolff-Kishner reduction conditions.     

Isolation of 3beta,7alpha-dihydroxychol-5-enoic acid, an intermediate of chenodeoxycholic acid biogenesis, and 3alpha,7alpha-dihydroxychol-4-enoic acid from bladder bile of hens

Two Lifschütz-positive C24-bile acids were isolated from bladder bile of hens. One of these was identified by isotope dilution experiments after conversion to a 3H-labeled compound, and also by GLC after methoxylation, as 3beta,7alpha-dihydroxychol-5-enoic acid, a key intermediate of chenodeoxycholic acid biogenesis. The other, to which the structure 3beta,7alpha-dihydroxychol-4-enoic acid had been assigned previously, was proved to be its 3alpha-epimer by several experiments. These findings favor the alternative pathway of chenodeoxycholic acid biogenesis proposed by Yamasaki and his associates.     

Chemical synthesis of (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid and its taurine and glycine conjugates: a major bile acid in the rat

A method for the synthesis of Delta(22)-beta-muricholic acid (Delta(22)-beta-MCA), (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Delta(22)-beta-muricholyltaurine and Delta(22)-beta-muricholylglycine) is described. The key intermediate, 3 alpha,6 beta,7 beta-triformyloxy-23,24-dinor-5 beta-cholan-22-al, was prepared from beta-muricholic acid (beta-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Delta(22)-beta-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Delta(22)-beta-MCA. These synthetic reference compounds are now available for investigation of the metabolism of beta-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-(2)H or (3)H-beta-MCA.     

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.     

Discovery of N-[(3R,5R)-1-azabicyclo[3.2.1]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide as an agonist of the alpha7 nicotinic acetylcholine receptor: in vitro and in vivo activity

A novel alpha7 nAChR agonist, N-[(3R,5R)-1-azabicyclo[3.2.1]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (3a, PHA-709829), has been identified for the potential treatment of cognitive deficits in schizophrenia. The compound shows potent and selective alpha7 in vitro activity, excellent brain penetration, good rat oral bioavailability and robust in vivo efficacy in a rat auditory sensory gating model.     

Biosynthesis of a C21 steroid conjugate in an insect. The conversion of [14C]cholesterol to 5-[14C]pregnen-3 beta,20 beta-diol glucoside in the tobacco hornworm, Manduca sexta

Following injection into Manduca sexta (L.) female pupae (day 16), [14C]cholesterol was converted to a C21 steroid conjugate, 5-[14C]pregnen-3 beta,20 beta-diol glucoside. The conjugate was isolated from ovaries and eggs and contained three glucose units at least one of which is attached to C-20. The distribution of the other two glucose units remains to be determined. Other than the dealkylation of C-24 alkane or alkene substituents, side-chain cleavage of sterols is uncommon to insects. Here we report the first definitive proof of the biosynthesis of a C21 steroid conjugate from cholesterol in an insect species. The capability of M. sexta to so readily convert cholesterol to a C21 steroid suggests a physiological role for 5-pregnen-3 beta,20 beta-diol in this species.