Synthesis of 3α,7α-dihydroxy-5β-androstan-17-one

A short and efficient method for the stereospecific synthesis of 3α,7α-dihydroxy-5β-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.     

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.     

Isolated Sertoli cells from immature rats produce 20α-hydroxy-pregn-4-en-3-one from progesterone and 3β,20α-dihydroxy-5α-pregnane from pregnenolone

Sertoli cells from 17 day old rats convert progesterone to 20α-hydroxy-pregn-4-en-3-one and pregnenolone to 3β,20α-dihydroxy-5α-pregnane after 72 hours $\text{in vitro}$. The metabolites were identified by several systems of thin layer and gas chromatography, derivative formation and crystallization with authentic steroids. The production of 20α-hydroxy-pregn-4-en-3-one and 3β,20α-dihydroxy-5α-pregnane amounted to 1380 and 740 pmoles/h/mg protein which can account for the total amounts of these steroids reported in the testis. It is the first direct evidence that Sertoli cells can metabolize progesterone and pregnenolone and suggests that Sertoli cells contain the major, if not the only, amounts of 20α-hydroxysteroid dehydrogenase in the immature rat 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.     

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.     

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).     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex     

Chemical syntheses of 5α-cholesta-6,8(14)-dien-3β-ol-15-one and related 15-oxygenated sterols

Hydroboration of 5α-cholesta-8,14-dien-3β-ol (I) gave 5α-cholest-8-en-3β,15α-diol (IV) in 89% yield. 5α-Cholest-7-en-3β,15α-diol (V) was prepared in 91% yield by hydroboration of 5α-cholesta-7,14-dien-3β-ol (II). Hydroboration of 27:63 mixture of I and II gave IV and V in 18% and 70% yields, respectively. 5α-Cholest-8-en-15α-ol-3-one and 5α-cholest-7-en-15α-ol-3-one were prepared in high yields from IV and V, respectively, by either selective oxidation with silver carbonate-celite or by enzymatic oxidation using cholesterol oxidase. 7α,8α-Epoxy-5α-cholestan-3β,15α-diol (VIII) was prepared in 93% yield by treatment of V with m-chloroperbenzoic acid. 5α-Cholest-8(14)-en-7α-ol-3,15-dione (IX) was prepared in 56% yield by oxidation of VIII with pyridinium chlorochromate followed by treatment of the crude product with acid. Compound IX was also obtained in 72% yield by selective chemical oxidation of 5α-cholest-8(14)-en-3β,7α,15α-triol. 5α-Cholesta-6,8(14)-dien-3,15-dione (X) was prepared in 89% yield by treatment of IX with p-toluenesulfonic acid under controlled conditions. Reduction of X with lithium tri-tert-butoxyaluminum hydride under controlled conditions gave 5α-cholesta-6,8(14)-dien-3β-ol-15-one in 84% yield.     

Metabolism of (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid in guinea pigs

[7β-³H]-(24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and [7β-³H]-27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid (C₂₇ and C₂₆ bile acids having the same nuclear configuration as cheno-deoxycholic acid and its precursor, 3α,7α-dihydroxy-5β-cholestan-26-oic-acid) were synthesized and administered intraperitoneally to bile fistula guinea pigs. The biliary bile acids formed were hydrolyzed and analyzed by thin layer chromatography, and the metabolites were identified by the inverse isotope dilution method. The results showed that both (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids were not metabolized by the liver and were excreted unchanged as their taurine and glycine conjugates whereas 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid was converted to chenodeoxycholic acid.     

Synthesis of 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their cytotoxic activities

A series of novel 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives were synthesized and characterized by NMR, HRMS. The pregnenolone (1) was first biotransformed by Mucor circinelloides var lusitanicus to 3β, 7α, 11α-trihydroxy-pregn-5-en-20-one (3), then 3 was treated with various benzaldehydes to produce 3β, 7α, 11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives. These derivatives showed remarkable activity against EC109 cells. The absolute configuration of 3 was also confirmed by signal-crystal X-ray analysis.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

Summary A fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15-hydroxylation of lithocholic acid to a new bile acid (3,15-dihydroxy-5-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3, 15-dihydroxy-5-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.Abbreviations LCA lithocholic acid (3-hydroxy-5-cholanic acid) - 3, 15-DHC (3, 15-dihydroxy-5-cholanic acid) - DMSO dimethyl sulfoxide - CHES 2-[N-cyclohexylamino]ethanesulfonic acid     

6β-Hydroxy-4-stigmasten-3-one and 6β-hydroxy-4-campesten-3-one

The first naturally occurring 6-hydroxylated Δ⁴-3-oxo steroids with intact sterol side chains have been isolated as a molecular complex from the bark extracts of Melia azedarach L. The complex has been characterized by UV, IR, NMR and MS analyses to consist of 6β-hydroxy-4-stigmasten-3-one and 6β-hydroxy-4-campesten-3-one, and these structures confirmed by partial synthesis.     

Synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxyandrost-5-en-17-one via microbial 7α-hydroxylation

The synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7α-hydroxylation has been accomplished. At the first stage, 3β,7α-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7α-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC_,¹H NMR, MS, UV- and IR-spectrometry.The combination of microbial and chemical steps ensured 54.6% yield of the target 3β-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.     

Enhanced biotransformation of dehydroepiandrosterone to 3β,7α,15α-trihydroxy-5-androsten-17-one with Gibberella intermedia CA3-1 by natural oils addition

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.     

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.     

Stereospecific Reduction of Keto Group in 3β-Triphenylmethoxy-5α-cholest-8(14)-en-15-one

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

Hepatic microsomal conversion of pregnenolone to 3β,5,6β-trihydroxy-5α-pregnan-20-one via pregnenolone α- and β-epoxides

In the presence of ferrous ion, ADP, and an NADPH-generating system, [4-¹⁴C]pregnenolone was oxidized by bovine liver microsomes to its α-epoxide (5,6α-epoxy-3β-hydroxy-5α-pregnan-20-one), β-epoxide (5,6β-epoxy-3β-hydroxy-5β-pregnan-20-one), trihydroxypregnanone (3β,5,6β-trihydroxy-5β-pregnan-20-one) which were separated, isolated on an octadecylsilicone column in 70% aq. methanol by high performance liquid chromatography, identified with respective synthetic specimens by gas-liquid chromatography-mass spectrometry. The microsomal Δ⁵-oxidation products of pregnenolone were detected in trace yield either when EDTA was added to the incubation mixture or when ferrous ion was omitted from the mixture. The microsomal oxidation system generated malondialdehyde significantly. It, however, was retarded to a negligible extent either by the addition of EDTA or by the omission of ferrous ion. Therefore, the microsomal formation of the significant yields of Δ⁵-oxygenated pregnenolones was reasonably attributed to a reaction linked to microsomal lipid peroxidation. The ratio of pregnenolone α- to β-epoxides formed was 1:3. A comparable study carried out under the same conditions by using [4-¹⁴C]cholesterol as the substrate resulted in the similar Δ⁵-epoxidation with concomitant formation of cholestane-3β,5α,6β-triol; cholesterol α- and β-epoxides formed were in the ratio 1:4.Both pregnenolone α- and β-epoxides were hydrolyzed by the microsomes to trihydroxypregnanone as the sole metabolite at a relative rate of 0.6:1. A similar relative value was also obtained in the microsomal hydrolysis of cholesterol α- and β-epoxides to the cholestanetriol.