Biosynthesis of 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol in Datura

Datura meteloides; plants were fed with tiglic acid-[-¹⁴C] via the roots and after 2 days the percentage incorporation into the alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, meteloidine and 3α,6β-ditigloyloxytropan-7β-ol were 15·2, 1·82, 2·2 and 1·8 respectively. 3α,6β-Ditigloyloxytropane was partially hydrolysed to 6β-hydroxy-3α-tigloyloxytropane which contained 58·1% of the radioactivity of the original base, whereas 3α,6β-ditigloyloxytropan-7β-ol gave meteloidine containing only 9·2% of the original activity. The results suggest that the di- and tri-hydroxytropanes may be formed by different routes.     

Urinary excretion of 5β-pregnane-3α,6α,20α-triol in human gestation

The 5β-pregnane-3α,6α,20α-iriol and 5β-pregnane-3α,6α,20β-triol obtained by reduction of 3α,6α-dihydroxy-5β-pregnan-20-one were converted to trimethylsilyl ether derivatives and analysed by gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS).After extraction, solvolysis and enzymatic hydrolysis of urinary steroid conjugates (from 19 normal pregnant women), the liberated steroids were separated by liquid-gel chromatography and were analysed by GLC and GLC-MS. The 5β-pregnane-3α,6α,20α-triol isolated and identified in the Sephadex LH-20 fractions 8 and 9, was present in urine from 15 pregnant women after the 16th week of gestation. After this time, this metabolite was found in a quantity between 0.20 to 2.90 mg/24 h, with a significant increase between the 26th to 30th week of the gestation.With the present in vivo data, it is not possible to establish the exact enzymatic pathway involved in the biosynthesis of the 5β-pregnane-3α,6α,20α-triol. However, it is probable that the immediate precursor of this compound was the 3α,6α-dihydroxy-5β-pregnan-20-one, and that urinary excretion of 5β-pregnane-3α,6α,20α-triol reflected one part of hepatomaternal metabolism of 6-hydroxyprogesterone formed in the foeto-placental unit.     

5α,6α- AND 5β,6β-dichloromethylene adducts of 3β-acetoxy-5-androsten-17-one

Both the 5α, 6α- and 5β, 6β-dichloromethylene adducts ($\text{2a}$ and $\text{2b}$) of 3β-acetoxy-5-androsten-17-one ($\text{1}$) are produced when the latter is exposed to dichlorocarbene generated from chloroform and base by Phase Transfer Catalysis using ultrasound as a means of agitation. The ¹H NMR substituent effects of 5α, 6α- and 5β, 6β-dichloromethylene on the angular methyl groups (Zürcher values) are given. The ¹³C NMR spectra for both compounds are presented and discussed.     

Metabolism of 3α, 7α-dihydrexy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyi-5β-cholanoic acid in hamsters

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.     

Effects of bile salts on cholestan-3β,5α,6β-triol-induced apoptosis in dog gallbladder epithelial cells

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3β,5α,6β-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14±11% (control) to 48±12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63±16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31±12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).     

Absolute configuration and stereochemical analysis of 3α,6β-dibenzoyloxytropane

Both enantiomers of 3α,6β-dibenzoyloxytropane (1) have been prepared from optical active 6β-hydroxyhyoscyamines establishing their absolute configurations as (?)-(3R,6R) and (+)-(3S,6S)-dibenzoyloxytropane. Independent stereochemical confirmation was obtained by vibrational circular dichroism measurements, since bands characteristic of (3R,6R) and (3S,6S) configurations of tropanediols derivatives were observed. In addition, a chiral HPLC method was developed for determining absolute configurations of tropane-related natural substances at the microgram (μg) level. The complete ¹H NMR characterization of the scaffold of 1 is also reported.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

5α-Estrane-3β,17β-diol and 5β-estrane-3α,17β-diol: definitive screening biomarkers to sign nandrolone abuse in cattle?

17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.     

C-19-steroid 7α-hydroxylation by rat testes. isolation and identification of: 7α, 17β-dihydroxy-5α-androstan-3-one, 5α-androstan-3α,7α,17β-triol and 5α-androstane-3β,7α, 17β-triol

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7α-hydroxylated metabolites could be isolated and identified: 7α,17β-dihydroxy-4-androsten-3-one (7α-hydroxy-testosterone), 7α-17β-dihydroxy-5α-androstan-3-one (7α-hydroxy-Dht), 5α-androstan-3α,7α,17β-triol (7α-hydroxy-3α-A'DIOL) and 5α-androstane-3β,7α,l7β-triol (7α-hydroxy-3β-A'DIOL). To our knowledge this is the first demonstration of the formation of 5α-reduced-7α-hydroxylated metabolites of testosterone in the male gonad. These 5α-reduced-7α-hydroxylated metabolites could also be isolated after incubations of 5α-androstane-3α,17β-diol (3α-A'D10L) with testicular homogenates in the presence of a NADPH-generating system.Measured as the sum of 7α-hydroxy-testosterone, 7α-hydroxy-Dht. 7α-hydroxy-3α-A'DIOL and 7α-hydroxy-3β-A'DIOL formed using testosterone as substrate, total 7α-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3α-A'DIOL as substrate total 7α-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation,Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27^Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.     

Synthesis and acetylcholinesterase inhibitory activity of 2β,3α-disulfoxy-5α-cholestan-6-one

Disodium 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (8) has been synthesized using cholesterol (1) as starting material. Sulfation was performed using trimethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. The acetylcholinesterase inhibitory activity of compound 8 was evaluated and compared to that of disodium 2β,3α-dihydroxy-5α-cholestane disulfate (10) and diols 7 and 9. Compounds 8 and 10 were active with IC(50) values of 14.59 and 59.65 μM, respectively. Diols 7 and 9 showed no inhibitory activity (IC(50)>500 μM).     

(+)-Eudesm-3-ene-6β,7α-diol from the liverwort Lepidozia reptans

The structure of a new sesquiterpene diol from the liverwort Lepidozia reptans has been established as eudesm-3-ene-6β,7α-diol on the basis of its ¹H and ¹³CNMR spectroscopic properties.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine