Synthesis of 18-hydroxy-19-norcorticosterone and 18-deoxy-19-noraldosterone. Structure determination of related 19-nor steroids by means of 2-D 1H nmr

The compounds named in the title have been synthesized from the di-(ethylene ketal) of 21-hydroxy-3,20-dioxo-19-norpregn-5-ene-18, 11 beta-lactone and its 5(10)-ene isomer. Reduction of this mixture 1 with sodium aluminum bis-(methoxyethoxy)hydride furnished the 11 beta, 18, 21-triol 2a. Conversion to the 18,21-diacetate 2b, followed by deketalization to the free dione 3 and hydrolysis, afforded 18-hydroxy-19-norcorticosterone 4a which, in the solid state and probably in solution, has the 18,20-hemiacetal structure. Periodate oxidation of 4a gave 11 beta-hydroxy-3-oxo-19-norandrost-4-ene-17 beta, 18-carbolactone 5a, and acid treatment of 4a or its precursor 2a yielded 18-deoxy-19-noraldosterone 6a. The structure of 5a was confirmed by mass spectrometry and 1H nmr, and compared with that of its C-19 methyl homolog 5b and 19-noraldosterone-gamma-etiolactone 8. In particular, 2-D nmr COSY 45 experiments, affording full 1H line assignments, have rigorously established the "natural" beta (axial) configuration of the C-10 hydrogen in the 19-nor lactones 5a and 8, and therefore also in the related 4a, 6a and 19-noraldosterone 7.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

Structure elucidation of new compounds from acidic treatment of the progestins gestodene and drospirenone

Gestodene acidic treatment afforded a single rearrangement product, namely 13-beta-ethyl-18,19-dinorpregna-4,14,16-trien-3,20-dione 3, which was originated through HCl-catalyzed Rupe rearrangement. Drospirenone acidic treatment yielded two epimeric lactones by addition of HCl to the 6beta,7beta-cyclopropane ring, namely 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17beta-pregn-4-ene-21,17-carbolactone 4 and 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17alpha-pregn-4-ene-21,17-carbolactone 5. The structure of the compounds was assessed by spectroscopic and crystallographic methods.     

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%     

18,19-Dihydroxydeoxycorticosterone; A novel product of cytochrome P-45011β-catalyzed reaction

After incubating 18-hydroxydeoxycorticosterone (18-OH-DOC) with cytochrome P-450_11β in the reconstituted system, the products were analyzed with HPLC. There appeared two product-peaks on the chromatogram, one of which was identified as a peak of 18-hydroxycorticosterone (18-OH-B), an expected product of the 11β-hydroxylation. Another peak did not coincide with those of any known corticoids. This unidentified product was further purified, and the purified material was analyzed by gas chromatography-mass spectrometry ($\text{GC}\text{MS}$). The mass spectrum showed that the unidentified product is one of the structural isomers of 18-OH-B. A further analysis with ¹H-NMR spectrometry indicated that a proton resonance peak of 19-CH₃ in 18-OH-DOC disappeared in the product and the methyl group of the substrate seemed to be converted to -CH₂OH. These results suggested that the unidentified product generated from 18-OH-DOC by P-450_11β-linked hydroxylase system may be 18,19-dihydroxydeoxycorticosterone (18,19,21-trihydroxypregn-4-ene-3,20-dione; 18,19-diOH-DOC), a hitherto unreported corticoid.     

Effect of 5 alpha-dihydrocorticoids on enzymes of gluconeogenesis in rat liver

5 alpha-Dihydrocortisol (11 beta, 17, 21-trihydroxy-5 alpha-pregnane-3,20-dione), 5 alpha-dihydrocorticosterone (11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione) as well as cortisol (11 beta, 17, 21-trihydroxy-4-pregnene-3,20-dione) and corticosterone (11 beta, 21-dihydroxy-4-pregnene-3,20-dione) were administered for seven days to male rats. Blood glucose increased in cortisol- and corticosterone-treated rats and blood insulin decreased after 5 alpha-dihydrocorticosteroid treatment. In the liver, total protein was elevated after cortisol, corticosterone and 5 alpha-dihydrocorticosterone application. Phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in liver were significantly lowered after treatment with 5 alpha-dihydrocortisol and 5 alpha-dihydrocorticosterone.     

Synthesis and structure elucidation of potential 6-oxygenated metabolites of (22R)-6alpha,9alpha-difluoro-11beta,21-dihydroxy-1 6alpha,17alpha-propyl methylenedioxypregn-4-ene-3,20-dione, and related glucocorticosteroids

(22R)-6alpha,9alpha-Difluoro-11beta,21-dihydroxy-16 alpha,17alpha-propylmethylenedioxypregn-4-ene-3,20-dione (rofleponide) is a synthetic glucocorticosteroid with high affinity for the rat thymus glucocorticoid receptor and a very high biotransformation rate demonstrated through incubation with a human liver S9 subcellular fraction. Because oxidation in the 6-position is an important metabolic pathway of glucocorticosteroids, the potential 6beta-hydroxy and 6-oxo metabolites of rofleponide were synthesized to be used as reference compounds. Three alternative routes were used to reach the 6-hydroxy compound: (a) a one-step procedure involving allylic oxidation of rofleponide by selenium dioxide, (b) selenium dioxide oxidation of the corresponding 1,4-diene followed by selective 1,2-hydrogenation using Wilkinson's catalyst, and (c) autoxidation of a 3-methoxypregna-3,5-diene derivative. All three routes proceeded stereospecifically. Routes (a) and (c) gave approximately the same overall yield of the 6beta-hydroxy epimer, whereas the overall yield from route (b) was much lower, primarily because of incomplete 1,2-hydrogenation. The 6-oxo compound was prepared through Pfitzner/Moffat oxidation of the 6-hydroxy compound. The stereochemistry of the 6-hydroxy substituent is discussed on the basis of 1H-NMR spectroscopy and supplementary 2D NOESY experiments.     

Stereospecific 1,4-addition to an alpha,beta-unsaturated steroidal epoxide: syntheses of new 15-oxygenated sterols

3 beta-Benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (1) is a key intermediate in the synthesis of C-7 and C-15 oxygenated sterols. Treatment of 1 with benzoyl chloride resulted in the formation of 3 beta,15 alpha-bis-benzoyloxy-7 alpha-chloro-5 alpha-cholest-8(14)-ene (2). Reaction of 2 with LiAlH4 or LiAlD4 resulted in the formation of 5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3a) or [14 alpha-2H]5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3b). Diol 3b was selectively oxidized by Ag2CO3/celite to [14 alpha-2H]5 alpha-cholest-7-en-15 alpha-ol-3-one (4). Treatment of 1 with MeMgI/CuI gave 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 alpha-diol (5). Selective oxidation of 5 with pyridinium chlorochromate (PCC)/pyridine or oxidation with PCC resulted in the formation of 7 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one (6) and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3,15-dione, respectively. Reduction of 6 with LiAlH4 yielded 5 and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 beta-diol (6). Reaction of 1 with benzoic acid/pyridine gave 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-en-15 alpha-ol (9). Treatment of 9 with LiAlH4 or ethanolic KOH resulted in the formation of 5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol (10). Dibenzoate 9, upon brief treatment with mineral acid, gave 3 beta-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (11). Oxidation of 9 with PCC yielded 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (12). Ketone 12 was also prepared by the selective hydride reduction of 5 alpha-cholest-8(14)-en-7 alpha-ol-3,15-dione (13) to give 5 alpha-cholest-8(14)-ene-3 beta,7 alpha-diol-15-one (14), which was then treated with benzoyl chloride to produce 12.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

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.     

New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC₅₀ = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC₅₀ values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC₅₀ < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC₅₀ = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.     

Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent inhibitor of sterol synthesis with significant hypocholesterolemic activity. (25R)-3 beta,26-Dihydroxy-5 alpha-cholest-8(14)-en-15-one (II) has been shown to be a major metabolite of I after incubation with rat liver mitochondria. Described herein is the chemical synthesis of II from diosgenin. As part of this synthesis, improved conditions are described for the conversion of diosgenin to (25R)-26-hydroxycholesterol. Benzoylation of the latter compound gave (25R)-cholest-5-ene-3 beta,26-diol 3 beta,26-dibenzoate which, upon allylic bromination followed by dehydrobromination, gave (25R)-cholesta-5,7-diene-3 beta,26-diol 3 beta,26-dibenzoate. Hydrogenation-isomerization of the delta 5.7-3 beta,26-dibenzoate to (25R)-5 alpha-cholest-8(14)-ene-3 beta,26-diol 3 beta,26-bis(cyclohexanecarboxylate) followed by controlled oxidation with CrO3-dimethylpyrazole gave (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one 3 beta,26-bis(cyclohexanecarboxylate). Acid hydrolysis of the delta 8(14)-15-ketosteryl diester gave II. 13C NMR assignments are given for all synthetic intermediates and several major reaction byproducts. The structure of II was unequivocally established by X-ray crystal analysis. II was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured mammalian cells and to inhibit oleoyl coenzyme A-dependent esterification of cholesterol in jejunal microsomes.     

Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors

The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5α-reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor.The 3,20-dioxopregna-4-ene-17α-yl acetate 4 containing an acetoxy group in C-17 and steroid 17α-hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes. On the other hand, 17α-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited only the type 1 enzyme. Steroid 4-chloro-17α-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at C-4 respectively, inhibited both types of 5α-reductase. These results indicate that an increase in the electronegativity of ring A produces a major inhibitory activity for 5α-reductase type 1; however this increase was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds 3,20-dioxo-4-chloropregn-4-ene-17α yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17α yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5α-reductase type 2 enzyme.Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however the IC₅₀ values of these compounds were higher as compared to that of finasteride.These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas compounds 6, 8a and 8b failed to do so.The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.     

中国南海侧扁软柳珊瑚中孕甾烷类化学成分的研究(英文)

从南海侧扁软柳珊瑚Subergorgia suberosa的二氯甲烷-甲醇提取物中首次分离鉴定了8个孕甾烷类化合物,经波谱鉴定为3β-O-palmitoyl-pregn-5-ene-20-one-3-ol (1),3β-O-palmitoyl-5α-pregn-20-one-3-ol (2),5α-pregn-1-ene-3,20-dione (3),3β,5α-pregn-20-one-3-ol (4),3β-pregn-5-ene-20-one-3-ol (5),3β,5β-pregn-20-one-3-ol (6),5β-pregn-3,20-dione (7),pregn-4-ene-3,20-dione (8).其中化合物1,2为新化合物.     

Isolation and partial synthesis of a new metabolite of medroxyrogesterone acetate.

3 alpha-Hydroxy-17-acetoxy-6 alpha-methyl-5 beta-pregnan-20-one (IIIa) has been isolated from urine of patients receiving medroxyprogesterone acetate (MPA). It was characterized by partial synthesis from MPA by catalytic reduction with palladium-charcoal to 17-acetoxy-6 alpha-methyl-5 beta-pregnan-3,20-dione (IV) and reduction of the latter with sodium borohydride. The isolation of 6 beta, 17,21-trihydroxy-6 alpha-methyl-pregn-4-ene-3,20-dione (IIc) is reported for the first time. The 17- and 21-monoacetates of this compound have been isolated and characterized earlier by other investigators. 7 alpha-3H-Medroxyprogesterone acetate was administered to 4 subjects by intravenous and intramuscular injections and by mouth. The ring A saturated metabolite IIIa was excreted in 0.1% to 4.0% of the administered dose; the highest excretion was after the intravenous dose and lowest after oral ingestion. 6 beta, 17,21-Trihydroxy-6 alpha-methylpregn-4-ene-3,20-dione (IIc) and its 17- and 21-monoacetates were excreted in about 5% of the doses in all subjects. No increase in 6 beta-hydroxylation was observed in the patient treated with o,p'-DDD,2,2-bis(2-chlorophenyl, 4'-chlorophenyl)-l,1-dichloroethane.     

Cholinesterase-inhibiting withanolides from Ajuga bracteosa

Three new withanolides, bracteosin A (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 1), bracteosin B (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxy-1,26-dioxoergost-24-en-19-oic acid; 2), and bracteosin C (= (22R)-22,26-epoxy-4beta,6beta,27-trihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 3), have been isolated from the whole plants of Ajuga bracteosa. Their structures were deduced by spectral analysis, including 1D- and 2D-NMR techniques. In addition, dihydroclerodin-1, clerodinin A, lupulin A, and dihydroajugapitin have also been isolated for the first time from this species. Compounds 1-3 exhibited evident inhibitory potential against cholinesterase enzymes in a concentration-dependent fashion.     

Various 18-substituted progesterones

In order to test the potential biological activity of 18-substituted progesterones, 3,20-dioxo-4-pregene-18-carbonitrile (ld approximately) was converted to 3,20-dioxo-4-pregnene-18-carboxylic acid (lb approximately) and methyl 3,20-dioxo-4-pregnene-18-carboxylate (ld approximately) via a sequence of reactions involving an intramolecular hydrolysis of the 18--arbonitrile. Lithium aluminum hydride reduction of the bisethylene ketal derived from la approximately furnished 18-aminomethyl-5-pregnene-3,20-dione 3,20-bisethylene ketal (8 approximately). Acetylation and hydrolysis furnished 18-acetamidomethyl-4-pregnene-3,20-dione (lf approximately) and simple hydrolysis of 8 approximately furnished 3'alpha H-5' 6'-dihydro-2',19 beta-dimethyl-3-oxo-4-goneno [13,17-c]pyridine (9 approximately). None of the compounds exhibited any activity in Clauberg or anti-Clauberg tests.     

An improved synthesis of (20R,22R)-cholest-5-ene-3beta,20,22-triol, an intermediate in steroid hormone formation and an activator of nuclear orphan receptor LXR alpha.

Asymmetric dihydroxylation of (20(22)E)-cholesta-5,20(22)-dien-3beta-ol acetate (2a), prepared from pregnenolone, gave a 1:1 mixture (67% yield) of (20R,22R)-cholest-5-ene-3beta,20,22-triol 3-acetate (3a) and its 20S,22S isomer 3b. Highly purified 3a and 3b were obtained by semipreparative silver ion high performance liquid chromatography. Saponification of 3a and 3b gave (20R,22R)-cholest-5-ene-3beta,20,22-triol (4a) and its 20S,22S isomer 4b. This simple approach provided the natural isomer 4a more efficiently than previously described chemical or enzymatic syntheses. Full 1H and 13C nuclear magnetic resonance data were presented for triols 4a and 4b and their synthetic precursors. Side-chain conformations of 2a, its 20(22)Z isomer, 4a, and 4b were studied by molecular mechanics and nuclear Overhauser effect difference spectroscopy.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Simultaneous determination of 6β- and 6α-hydroxycortisols and 6β-hydroxycortisone in human urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-²H₅]OHF (6β-OHF-d₅), 6α-[1,1,19,19,19-²H₅]OHF (6α-OHF-d₅) and 6β-[1,1,19,19,19-²H₅]OHE (6β-OHE-d₅) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]⁺) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.