Metabolism of androsta‐1,4,6‐triene‐3,17‐dione and detection by gas chromatography/mass spectrometry in doping control

The urinary metabolism of the irreversible aromatase inhibitor androsta‐1,4,6‐triene‐3,17‐dione was investigated. It is mainly excreted unchanged and as its 17β‐hydroxy analogue. For confirmation, 17β‐hydroxyandrosta‐1,4,6‐trien‐3‐one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post‐administration urines, namely 17β‐hydroxyandrosta‐1,4‐dien‐3‐one (boldenone), 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one (boldenone metabolite), 17β‐hydroxyandrosta‐4,6‐dien‐3‐one, and androsta‐4,6‐diene‐3,17‐dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta‐1,4,6‐triene‐3,17‐dione is classified as a prohibited substance in sports by the World Anti‐Doping Agency (WADA) due to its aromatase‐inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta‐1,4,6‐triene‐3,17‐dione. The samples were initially found suspicious for the boldenone metabolite 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one. Since metabolites of androst‐4‐ene‐3,6,17‐trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like ‘Novedex Xtreme’, which could be easily obtained from the sport supplement market. Copyright © 2008 John Wiley & Sons, Ltd.     

Microbial degradation of deoxycholic acid by pseudomonas sp. ncib 10590. characterisation of products and a postulated pathway

Minor products in the microbial degradation of deoxycholic acid are characterised and a metabolic pathway proposed. In a previous publication1 we reported the transformation of deoxycholic acid 1 by Pseudomonas sp. NCIB 10590, in a mineral salts medium, to a mixture of neutral and acidic steroids. The major neutral product. 12β-hydroxyandrosta - 1,4 - dien - 3.17 - dione 2 and the major acidic product 12α - hydroxypregna - 1,4 - dien - 3 - one - 20 - carboxylic acid 9 were identified. We now report the structures of the minor products and propose a metabolic pathway.     

Degradation of deoxycholic acid by Pseudomonas Sp. NCIB 10590

The microbial degradation of deoxycholic acid 1 by Pseudomonas NCIB 10590 has been studied and two major products have been isolated and identified as 12β-hydroxyandrosta-1,4-dien-3,17-dione 2 and 12α-hydroxypregna-1,4-dien-3-one-20-carboxylic acid 9. Three minor products were isolated and evidence is given for the following structures: 12α-hydroxyandrosta-1,4-dien-3,17-dione 4, 12β-hydroxyandrosta-4-en-3,17-dione 7 and 12?, 17?-dihydroxyandrosta-1,4-dien-3-one 8.     

Addition reactions of nucleophiles to dibenzoylacetylene

Several enamine diones have been prepared through the nucleophilic addition of different amines to dibenzoylacetylene (DBA). Thus, the reaction of aniline, piperidine, o-aminophenol and N-phenylbenzylamine with DBA gave the corresponding 1:1 adducts namely, 1,4 - diphenyl - 2 - (N - phenylamino)but - 2 - ene -1,4 - dione (1), 1,4 - diphenyl - 2 - piperidinobut - 2 - ene - 1,4 - dione (2), 2 - (N - 2 - hydroxyphenylamino)1,4 - diphenylbut - 2 -ene - 1,4 - dione (3) and 1,4 - diphenyl - 2 - (N - phenylbenzylamino)but - 2 - ene - 1,4 - dione (4). UV absorption data reveal that the adducts 1 and 3, formed from aniline and o-aminophenol, respectively, are the E-isomers, arising through a trans-mode of addition whereas the adducts 2 and 4, formed from piperidine and N-phenyl-benzylamine are the Z-isomers, formed through a cis-mode of addition.The reaction of N-phenaeylaniline with DBA gave 2,3 - dibenzoyl - 1,4 - diphenylpyrrole (5), whereas the reaction of 1,8 - diaminonaphthalene with DBA gave a mixture of products consisting of 2 - benzoyl - 2 - phenacyl -2,3 - dihydroperimidine (11) and 2 - benzoylperimidine (12). The reaction of 2-aminopyridine with DBA gave a mixture of two 1:1-adducts, 2 - (2 - imino - 1(2H) - pyridyl) - 1,4 - diphenylbut - 2 - ene - 1,4 - dione (14) and 1,4 -diphenyl - 2 - (N - 2 - pyridylamino)but - 2 - ene - 1,4 - dione (15).     

Indenone chemistry—III: Use of the 2-carboethoxymethyl-6-methoxyindenone in the synthesis of the gibbane skeleton

The 2-carboethoxymethyl-6-methoxyindenone 5 has been synthesized in good yield. The endo head-to-tail dimer 6 and the exo head-to-tail dimer 7 can be synthesized stereoselectively from the indenone 5. Diels Alder addition of 5 to 1,3-butadiene affords the 7-methoxy-9aα-carboethoxymethyl-9a, 1,4aα, 4-tetrahydrofluorenone 8. The corresponding acid 9 can be cyclised to the 2 - methoxy - 4bαH - gibba - A,5 - tetraene - 8,10 - dione 10.     

The metal ion catalysed oxidation of hexahydrocolupulone

The decomposition of 4-hydroperoxyhexahydrocolupulone 4, catalysed by various metal ions, gives mainly 4-hydroxyhexahydrocolupulone 5 and tetrahydrocohulupone 6 in different ratios, depending on the nature of the metal ion. The maximum yield of the bitter-tasting 6 is 45%, if Co(II) acetate in 1,4-dioxan is used. The oxidation of hexahydrocolupulone 3 in 0.5% alcoholic cyclohexane in the presence of Cu(II) acetate affords two new neutral oxidation products, 2 - (2' - methyl)propanoyl - 3,5,5 - tri(3' - methyl)butyl - 2 - cyclopentene -1,4 - dione 7 and 2 -carboalkoxy - 3,5,5 - tri(3' - methyl)butyl - 2 - cyclopentene - 1,4 - dione 8.     

Bufadienolides—19 : 3β-acetoxy-15-oxo-5β,14β-bufa-8,20,22-trienolide

Reaction of 3β - hydroxy - 14α,15α - epoxy - 5β - bufa - 20,22 - dienolide (1a) with 72% perchloric acid was found to yield 14β - artebufogenin (2a), 14α - artebufogenin (2b), 15α - hydroxy-bufalin (3a), and a new substance 3β,15ξ - dihydroxy - 5β,14ξ - bufa - 8,20,22 - trienolide (4a). Similar results were realized by antimony (III) chloride and an iron(III) chloride catalyzed ring opening of epoxide 1a. Selective acetylation of the new bufadienolide (4a) followed by oxidation completed a route to the title substance.     

Soil bacterial hydrolysis leading to genuine aglycone—IX : A steroidal prosapogenol of new type from epigeous part of Metanarthecium luteo-viride maxim. and characterization of 3-epi-metagenin

By virtue of the soil bacterial hydrolysis method, a mixture (ca 12:1) of prosapogenols has been isolated, and on the basis of chemical and physicochemical evidence, the structure of major component (designated as NE-1A) has been established as 2β - acetoxy - 3α - hydroxy - 11α- (tri -O- acetyl - α - L - arabinopyranosyl)oxy - 5β, 25R - spirostane (10), and the minor presumed as its 3-epimer (11). NE-1A (10) is characteristic of possessing a fully acetylated arabinoside linkage at C-11 of a new steroidal aglycone 3-epi-metagenin (9), which is a ninth spirostanol isolated from the titled plant.Finally, NE-1A (10) has been subjected to detailed PMR examinations including homonuclear INDOR, double and triple resonance experiments, and the prominent signals have been assigned.     

A new synthetic method of 1α-hydroxy-7-dehydrocholesterol

Cholesta - 1,4,6 - trien - 3 - one (1) was converted to 3β - hydroxycholesta - 1,5,7 - triene (3) via the deconjugation procedure using t-BuOK in DMSO followed by the subsequent reduction with Ca(BH₄)₂. The compound (3) readily reacted with 4-phenyl-1,2,4-triazoline-3,5-dione to yield the corresponding 1,4-addition product (4). Epoxidation of 4 with m-chloroperbenzoic acid resulted in the formation of the 1α,2α-epoxide (5) and the 1β,2β-epoxide (6) in the ratio 2:3. Reduction of 5 with LAH under reflux in THF afforded the titled compound (7). The same reduction of 6 gave 2β-hydroxy- and 1β - hydroxy - 7 - dehydrocholesterol (8 and 9) in the ratio 8:1.The compound (4) can be obtained in 25% yield from 1 without any purification of the intermediate compounds; cholesta - 1,5,7 - trien - 3 - one (2, a very unstable compound) and 3. Since 1 is obtained readily from cholesterol in high yield, the present study provides a simple and efficient synthetic method of 1α-hydroxycholecalciferol and is reasonably expected to be applicable in the synthesis of 12,25-dihydroxycholecalciferol and the other metabolites of vitamin D₃.     

The anaerobic degradation of deoxycholic acid by PSEUDOMONAS SP. NCIB 10590

The anaerobic metabolism of deoxycholic acid by Pseudomonas sp. NCIB 10590 was studied. The metabolic pathway was similar to that operating under aerobic conditions with 12β-hydroxyandrosta-1,4-dien-3,17-dione as the major neutral product an metabolites which are not produced during aerobic metabolism were isolated and evidence is presented for the following structures: 9α-hydroxyandrost-1-en-3,17-dione, 12α,17)β-dihydroxyandrosta-1,4-dien-3-one; 3β,12β-dihydroxy-5β-androstan-17-one an formation and significance of the phenolic secosteroid is discussed.     

Metabolites of bird's nest fungi. Part 18. new oxygenated cadinane derivatives from cyathus striatus

The bird's nest fungusCyathus striatus Willd. ex Pers. when grown in aerated liquid culture produces three sesquiterpenes which are identified as schizandronol 1, 7α - hydroxy - schizandronol3 and schizandronol - 8,13β - oxide 5. The identification is based on spectral investigation of the compounds and their derivatives. This is the first reported isolation of 1 from a fungal source. Compounds 3 and 5 have not been reported previously.     

Biphenylenes—XXIX: Synthesis of cyclobuta[b]biphenylene-1-carboxylic acid and of cyclopenteno[b]biphenylene-1,2,3-trione

Cyelobuta[b]biphenylene-1-carboxylic acid 3 has been made by photochemical ring-contraction of 2-diazocyclopenteno[b]biphenylene-1-one 10. Evidence for increased strain in the acid 3 compared with 2,3-dimethylbiphenylene is discussed. Cyclopenteno[b]biphenylene-1,2,3-trione 12, an analogue of anhydrous ninhydrin, was obtained by selenium dioxide oxidation of cyclopenteno[b]biphenylene-1-one. The preferred conformation of Z-2-β cyanovinylbiphenylene 19 is such that the cyano group is close to the hydrogen atom at position 1 of the biphenylene ring.     

Two approaches to the enantioselective synthesis of (4R)-(−)-4-hydroxymethyl-4-thiobutyro-1,4-lactone

Enantiomerically pure (4R)-4-hydroxymethyl-4-thiobutyro-1,4-lactone [(5R)-dihydro-5-(hydroxymethyl)-2(3H)-thiophenone (12)] and derivatives were synthesized by two enantiospecific sequences employing d-ribono-1,4-lactone (1) and l-glutamic acid (6) as chiral templates. The key step in the first approach was the SmI₂-promoted 2,3-deoxygenation of a 4-thio-l-lyxono-1,4-lactone derivative, prepared from 1. The other strategy, which starts from 6, involves the (5S)-dihydro-5-(p-tolylsulfonyloxymethyl)-2-(3H)-furanone (8) as chiral precursor. This was converted into a 4,5-thiirane derivative via the corresponding 4,5-epoxide. Regioselective opening of the thiirane ring by acetate followed by O-deacetylation gave 12 (40% overall yield from 8).     

Epoxide configuration in 3β-acetoxy-7α-hydroxy-14,15-epoxy-apotirucallane prepared from tirucalla-7,24-dien-3-one : X-ray analysis

Epoxidation of 3β - Acetoxy - 7α - hydroxy - Δ¹⁴ - apotirucallane, prepared from Δ7-tirucallone, afforded the corresponding 14α,15α epoxide. The α-configuration of the epoxide function has been established by single-crystal X-ray analysis, representing the first such study of a tirucallol derivative with an intact side-chain. Epoxidation of Apotirucalla - 3,7 - dione gave the corresponding 14α,15α - epoxide as well. Eight new tirucallol derivatives were described.     

Steroids from sponges

The sponges Stelleta clarella Tethya aurantia, Lissodendoryx noxiosa, Haliclona permollis and Haliclona sp. were examined for steroids. All sponges contained C₂₇-C₂₉, Δ⁵, mono and diunsaturated sterols. In addition, the sponge Tethya aurantia contained Z - 24 - propylidene - cholest - 5- en -3β-ol (19) and the 5α,8α-peroxides of cholesta - 5,7 - dien - 3β - ol, ergosterol, ergosta - 5,7,24(28) - trien - 3β - ol and 24ξ - ethyl - cholesta - 5,7 - dien - 3β - ol (29, 30, 31 and 32). The sponge Stelleta clarella also contained 24 - nor - cholesta - 4,22 - dien - 3 - one (21), cholesta - 4,22 - dien - 3 - one (22), 24ξ - methyl - cholesta - 4,22 - dien - 3 - one (24), ergosta - 4,24(28) - dien - 3 - one (25), (E) - stigmasta - 4,24(28) - dien - 3 - one (28), 5α - cholestanol (5), 5α - ergostanol (7) and 5α - poriferastanol (9) The possible biosynthetic significance of these hitherto undescribed peroxides and enones from marine sources is discussed. A synthesis of 19 is also described.     

New polyesters from Talaromyces flavus

Six new polyesters, talapolyesters A–F (1–4, 14, and 16), together with 11 known ones 15G256ν (5), 15G256ν-me (6), 15G256π (7), 15G256β-2 (8), 15G256α-2 (9), 15G256α-2-me (10), 15G256ι (11), 15G256β (12), 15G256α (13), 15G256α-1 (15), and 15G256ω (17), were isolated from the wetland soil-derived fungus Talaromyces flavus BYD07-13, and their structures were determined by NMR and MS spectroscopic data. Among them, 1–4 and 16 were assembled in a different manner from that of the known 256 polyesters. All the polyesters are composed of (R)-2,4-dihydroxy-6-(2-hydroxypropyl)benzoic acid and (R)-3-hydroxybutyric acid/(S)-3,4-dihydroxybutyric acid residues. The absolute configurations of the residues were determined by alkaline hydrolysis. The cytotoxicity against five tumor cell lines of these compounds was examined, and a tight structure–activity relationship is proposed.     

Synthesis of pyrrolo[3,2-b]benzofurans and pyrrolo[3,2-b]naphthofurans via addition of a silyloxypyrrole to activated quinones

The uncatalyzed reaction of N-(tert-butoxycarbonyl)-2-tert-butyldimethylsilyloxypyrrole 3 with 1,4-quinones bearing an electron withdrawing group at C-2 has been studied. Use of 1,4-quinones 4, 5 bearing an ester group at C-2 provided an efficient synthesis of the respective pyrrolidinobenzofuran adduct 9 or pyrrolidinonaphthofuran adduct 10 whereas use of 1,4-quinones 6, 7 and 8 bearing an acetyl group at C-2 afforded silyloxypyrroles 11, 12 and 13 resulting from direct electrophilic substitution of the silyloxypyrrole by the electrophilic quinone. Addition of Eu(fod)₃ to the reaction of 2-acetyl-1,4-naphthoquinone 7 and 3-acetyl-5-methoxy-1,4-naphthoquinone 8 with N-(tert-butoxycarbonyl)-2-tert-butyldimethylsilyloxypyrrole 3 provided a method for obtaining the pyrrolidinonaphthofuran adducts 14 and 15 together with silyloxypyrroles 12 and 13. Oxidative rearrangement of pyrrolidinonaphthofuran adduct 15 to pyrrolidino pyranonaphthoquinone 16 using ceric ammonium nitrate in acetonitrile provided a novel approach for the synthesis of an aza-analogue of the pyranonaphthoquinone antibiotic kalafungin.     

A synthesis of 3′-α-fluoro-2′,3′-dideoxyadenosine via a bromine rearrangement during fluorination with MOST reagent

A facile method for the synthesis of 3′-α-fluoro-2′,3′-dideoxyadenosine 6 has been developed. Fluorination of 5′-O-acetyl-3′-β-bromo-3′-deoxyadenosine 3 with MOST gave 2′-β-bromo-3′-α-fluoro-2′,3′-dideoxyadenosine 4 via a rearrangement of the 3′-β-bromine to the 2′-β position during 3′-α fluorination. The 2′-β bromine was reduced by radical reduction and then the 5′-O-acetyl group was removed to afford 3′-α-fluoro-2′,3′-dideoxyadenosine 6 in good yield. A possible mechanism for the rearrangement is discussed.     

First cross-coupling reactions on halogenated 1H-1,2,4-triazole nucleosides

The halogenated 1H-1,2,4-triazole glycosides 6-10 were synthesized by BF₃-activated glycosylation of 3(5)-chloro-1,2,4-triazole (2), 3,5-dichloro-1,2,4-triazole (3), 3,5-dibromo-1,2,4-triazole (4), and 3(5)-bromo-5(3)-chloro-1,2,4-triazole (5) with 1,2,3,4-tetra-O-pivaloyl-β-d-xylopyranose (1). The β-anomeric major products 3-chloro-1-(2,3,4-tri-O-pivaloyl-β-d-xylopyranosyl)-1,2,4-triazole (6β), 3,5-dichloro-1-(2,3,4-tri-O-pivaloyl-β-d-xylopyranosyl)-1,2,4-triazole (7β), and 3,5-dibromo-1-(2,3,4-tri-O-pivaloyl-β-d-xylopyranosyl)-1,2,4-triazole (8β) were used as starting materials for transition metal catalyzed C-C-coupling reactions. Arylations of the triazole ring of 7β, and 8β were successful in 5-position with phenylboronic acid, 4-vinylphenylboronic acid, and 4-methoxyphenylboronic acid, respectively, under Suzuki cross-coupling conditions (products 11-17). Moreover, a Cu-catalyzed perfluoroalkylation of 8β is reported with 1-iodo-perfluorohexane yielding 3-perfluorohexyl-1-(2,3,4-tri-O-pivaloyl-β-d-xylopyranosyl)-1,2,4-triazole (18). Compound 18 was depivaloylated to the trihydroxy derivative 19. The copper-mediated reaction of 8β with Rupert's reagent gave the bis(3-bromo-1-(2,3,4-tri-O-pivaloyl-β-d-xylopyranosyl)-1,2,4-triazol-5-yl) (20).     

Biomimetically inspired total synthesis of (12S)-12-hydroxymonocerin and (12R)-12-hydroxymonocerin

A concise asymmetric total synthesis of (12S)-12-hydroxymonocerin (1) and (12R)-12-hydroxymonocerin (2) were efficiently achieved from the known 4-bromo-2,6-dimethoxyphenol. The synthetic approach was inspired by our biomimetic synthesis of (+)-monocerin (3) and 7-O-demethylmonocerin (4). The cis-fused furobenzopyranones of 1 and 2 was efficiently constructed via an intramolecular nucleophilic trapping of a quinonemethide intermediate, which was obtained by benzylic oxidation of compound 10 using 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).