Crystal structure of steroidal monoenes obtained by reduction of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol

The crystal structures of 13-ethyl-gona-1(10)-ene-11alpha,17beta-diacetate (3b) and 13-ethyl-10alpha-gona-4-ene-11alpha,17beta-diacetate (5b), two steroidal monoenes obtained as minor products from the reduction, then acetylation, of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol (1), were determined by X-ray diffraction. The conformations of the rings A, B, C, and D and the unusual stereochemistry at C-10 of the 10alpha-gona-4-ene (5b) are discussed.     

The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3beta,17beta-diol was developed. The 19-(3)H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3beta,17beta-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19-(3)H]androst-5-ene-3beta,17beta-diol and the 19-(3)H-labelled triol produced by reduction of 19-oxo[19-(3)H]-androst-5-ene-3beta,17beta-diol with sodium borohydride as 19-hydroxy[(19-S)-19-(3)H]-androst-5-ene-3beta,17beta-diol. 3. In the conversion of the (19-R)-19-(3)H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61.6%) than in water (38.4%). In a parallel experiment with the (19-S)-19-(3)H-labelled compound the order of radioactivity was reversed: formic acid (23.4%), water (76.2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

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.     

Metabolism of metandienone in man: identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites

After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

21beta-Hydroxy-oleanane-type triterpenes from Hippocratea excelsa

Stem bark of Hippocratea excelsa afforded six pentacyclic triterpenes, five oleanane and one ursane types. They were identified as 11beta,21beta-dihydroxy-olean-12-ene-3-one (2), 3alpha,11alpha,21beta-trihydroxy-olean-12-ene (3), 3alpha,21beta-dihydroxy-11alpha-methoxy-olean-12-ene (4), 3alpha,21beta-dihydroxy-olean-9(11),12-diene (5), 3alpha,21beta-dihydroxy-olean-12-ene (6) and 3alpha,21beta-dihydroxy-11alpha-methoxy-urs-12-ene, isolated as its diacetate derivative (7), as well as 3alpha,21beta-dihydroxy-olean-12-ene (1) previously isolated from the root bark. The known alpha- and beta-amyrin, oleanoic and ursolic acids, trans-polyisoprene, and the ubiquitous beta-sitosterol were also isolated. Structures were elucidated on the basis of spectroscopic analyses, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC and HMBC) and comparison with literature data. The antigiardial activity of compounds 2-5 was not significant.     

Synthesis of a precursor for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide

Starting from 11 beta-hydroxytestosterone, we achieved the synthesis of a strategic precursor, C-9 (11) unsaturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (9a), for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide. We optimized the reaction conditions for catalytic reduction employing hydrogen and subsequent base hydrolysis followed by purification on Amberlite XAD-2 resin to obtain the saturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.     

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.     

Synthesis of steroidal diacyl hydrazines and their 1,3,4-oxadiazole derivatives

Homogeneous catalytic hydrazinocarbonylation of some steroid derivatives possessing iodo-alkenyl moiety (17-iodo-androst-16-ene 1, 17-iodo-3-methoxy-estra-1,3,5(10),16-tetraene 2, 17-iodo-4-aza-4-methyl-androst-16-en-3-one 3 and 17-iodo-6beta-hydroxy-3alpha,5alpha-cycloandrost-16-ene 4) were carried out in the presence of a palladium catalyst, a base and acetic or benzoic hydrazide as the nucleophilic reagent. The corresponding N-acetamido-carbamoyl 1a-4a or N-benzamido-carbamoyl derivatives 1b-4b were obtained in high yields. Some of these derivatives served as starting materials for the synthesis of new steroidal 1,3,4-oxadiazole compounds.     

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Isolation of new cytotoxic metabolites from Cleome droserifolia growing in Egypt

The sulforhodamine B (SRB) assay was used to assess the cytotoxicity of the aqueous (AqEx) and ethanolic (AlEx) extracts, respectively, of the aerial parts of Cleome droserifolia (Forssk.) Del. against two human cancer cell lines, breast (MCF7) and colon (HCT116) adenocarcinoma. AqEx exhibited higher cytotoxic activity, thus its four subfractions, namely n-hexane (HxFr), chloroform (ClFr), ethyl acetate (EtFr), and n-butanol (BuFr) fractions, were also tested. Purification of the more active ClFr and EtFr yielded nine compounds. Six terpenoids, guai-7(11),8-diene (C1), 1-hydroxy-guai-3,10(14)-diene (C2), 18-hydroxydollabela-8(17)-ene (C3), (24E)-stigmasta-5,8-dien-3beta-ol (C4), teucladiol [1alpha,5beta-guai-10(14)-ene-4beta,6beta-diol] (C5), and buchariol (4,10-epoxy-6a-hydroxyguaiane) (C6), were isolated from ClFr and three flavonol glycosides, isorhamnetin-3-O-beta-D-glucoside (F1), quercetin-3'-methoxy-3-O-(4"-acetylrhamnoside)-7-O-alpha-rhamnoside (F2), and kaempferol-4'-methoxy-3,7-O-dirhamnoside (F3), were isolated from EtFr. Compounds C3 and F2 are new in nature. The isolated compounds were identified using various spectroscopic methods (UV, IR, 1H NMR, 13C NMR, HMQC, HMBC, and COSY). Compounds C1, C3, F2, and F3 showed significant cytotoxic activities against the two tested cell lines comparable to those of the anticancer drug doxorubicin. The new compound C3 was the most active as it had the lowest IC50 values, (1.9 +/- 0.08) and (1.6 +/- 0.09) microg/ml corresponding to 6.5 and 5.4 microM, against MCF7 and HCT116 cells, respectively.     

Structure and cytotoxicity of new polyhydroxylated sterols from the Caribbean gorgonian Plexaurella grisea.

The gorgonian Plexaurella grisea contains the new steroids 9-hydroxygorgosterol (1), 9,11 alpha,14-trihydroxygorgosterol (2), 5 beta,6 beta-epoxyergost-24(28)-ene-3 beta,7 beta-diol (3), ergost-24(28)-ene-3 beta,5 alpha,6 beta,7 beta-tetrol (4), an unseparable 1:1 mixture of the epimers (25R) and (25S)-26-acetoxy-3 beta,5 alpha-dihydroxyergost-24(28)-en-6-one (5/6), and seven related, known compounds (7-13). The structures of these new compounds were defined by spectroscopic analysis. All the compounds (1-13) isolated from P. grisea were tested against P 388, A 549, and HT 29 tumor cell lines. Compounds 3, 5/6, and 12 exhibited selective activity against the HT 29 cell line (ED(50) = 0.1 microg/ml).     

Influence of a 9-double bond on stereospecific microbial 4,5-reductions

By stereospecific microbial reduction with Rhodosporidium rubrum or Rhodotorula glutinis, 17 alpha-cyano-methyl-4-estren-17 beta-ol-3-one was metabolized to 17 alpha-cyanomethyl-5 alpha-estrane-3 beta,17 beta-diol (50%) and 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha,17 beta-diol (30%). By Clostridium paraputrificum the same substrate was reduced stereospecifically to 17 alpha-cyanomethyl-5 beta-estrane-3 alpha, 17 beta-diol (70%). When the corresponding 9-dehydrogenated compound 17 alpha-cyanomethyl-4,9-estradien-17 beta-ol-3-one (STS 557, a new progestagen) was fermented, yeasts failed in 5 alpha-reducing the 4-double bond. Still Clostridium paraputrificum formed the expected 5 beta-reduced metabolite 17 alpha-cyanomethyl-5 beta-estr-9-ene-3 alpha,17 beta-diol (60%). Structures were elucidated by n.m.r. and mass spectra and partly by circular dichroism. By oxidation of the metabolites, the corresponding 3-oxo compounds 17 alpha-cyanomethyl-5 alpha-estran-17 beta-ol-3-one, 17 alpha-cyanomethyl-5 beta-estran-17 beta-ol-3-one and 17 alpha-cyanomethyl-5 beta-estr-9-en-17 beta-ol-3-one were prepared. The evident influence of the 9-double bond on reduction of the 4-en-3-oxo compound STS 557 preventing 5 alpha-reduction but permitting 5 beta-reduction is discussed in view of the distinctly diminished metabolism of this progestagen in mammals.     

17-Epimerization of 17 alpha-methyl anabolic steroids in humans: metabolism and synthesis of 17 alpha-hydroxy-17 beta-methyl steroids.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.     

Binding, X-ray and NMR studies of the three A-ring isomers of natural estradiol

The effect of the position of the phenolic hydroxyl on the conformations of the three A-ring isomers of estradiol, namely, estra-1,3,5(10)-trien-1,17 beta-diol (10), estra-1,3,5(10)-trien-2,17 beta-diol (3), and estra-1,3,5(10)-trien-4,17 beta-diol (6), has been analyzed by X-ray crystallography. The results of these analyses were correlated with the absorptions of the angular methyl groups in the [1H]NMR spectra of these isomers and natural estradiol (E2). It was observed that the changes in chemical shift of protons at C18 corresponded to skeletal modifications in the steroid structure which changed the anisotropic effect of the hydroxyl group at C17. Examination of the affinity of these A-ring isomers of E2 for the estrogen receptor has shown the 2-hydroxylated isomer 3 to retain 1/5th the affinity of E2 for its binding protein. The 1- and 4-hydroxylated derivatives (10 and 6, respectively) bound to a much lesser extent. The receptor affinities of these estrogen analogues may be related to the angle between the 18-methyl and the 17 beta-hydroxyl groups (or the dihedral angle between the planar A-ring and the angular C18 methyl) as well as the position of the A-ring hydroxyl group.     

Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus

The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform steroid biotransformations under single phase, pulse feed conditions. The steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta hydroxylations. P. chrysosporium was more prone functionalize steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.     

Microbiologic oxidation of estratrienes and estratetraenes by Streptomyces roseochromogenes ATCC 13400

Incubation of estrone (1a) with Streptomyces roseochromogenes ATCC 13400 yielded a mixture of 3,16 alpha-dihydroxyestra-1,3,5(10)-trien-17-one (3a) and 3,17 beta-dihydroxyestra-1,3,5(10)-trien-16-one (4a). Transformation of 3-methoxyestra-1,3,5(10)-trien-17-one (1b), 3-hydroxyestra-1,3,5(10),9(11)-tetraen-17-one (2a), and 3-methoxyestra-1,3,5(10),9(11)-tetraen-17-one (2b) with the same microorganism gave the corresponding mixtures of 16 alpha-hydroxy-17-ketones and 17 beta-hydroxy-16-ketones (3b and 4b, 6a and 7a, 6b and 7b, respectively). In addition, in these three last experiments, the 16 beta-17 beta-dihydroxy derivatives 5b, 8a, and 8b, respectively, were also isolated. The complete assignments of the 13C nuclear magnetic resonance spectra of these compounds are given.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.