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.     

Testosterone metabolism revisited: discovery of new metabolites

The metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography–tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors.     

Biotransformation of (+)-androst-4-ene-3,17-dione

Fermentation of (+)-androst-4-ene-3,17-dione (1) with Curvularia lunata for 10 days yielded five oxidative and reductive metabolites, androsta-1,4-diene-3,17-dione (2), 17beta-hydroxyandrosta-1,4-dien-3-one (3), 11alpha-hydroxyandrost-4-ene-3,17-dione (4), 11alpha,17beta-dihydroxyandrost-4-en-3-one (5) and 15alpha-hydroxyandrosta-1,4-dien-17-one (6). The structures of these metabolites were elucidated on the basis of spectroscopic techniques. These microbially transformed products were assayed against the clinically important enzymes, tyrosinase and prolyl endopeptidase.     

Metabolic studies of methenolone acetate in horses

Methenolone acetate (17β-acetoxy-1-methyl-5α-androst-1-en-3-one), a synthetic anabolic steroid, is frequently abused in human sports. It is preferred for its therapeutic efficiency and lower hepatic toxicity compared with its 17α-alkylated analogs. As with other anabolic steroids, methenolone acetate may be used to enhance performance in racehorses. Metabolic studies on methenolone acetate have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the investigation of in vitro and in vivo metabolism of methenolone acetate in racehorses.Studies on the in vitro biotransformation of methenolone acetate with horse liver microsomes were carried out. Methenolone (M1, 1-methyl-5α-androst-1-en-17β-ol-3-one) and seven other metabolites (M2-M8) were detected in vitro. They were 1-methyl-5α-androst-1-ene-3,17-dione (M2), 1-methyl-5α-androst-1-en-6-ol-3,17-dione (M3) and two stereoisomers of 1-methylen-5α-androstan-2-ol-3,17-dione (M4 and M5), 1-methyl-5α-androst-1-en-16-ol-3,17-dione (M6) and monohydroxylated 1-methyl-5α-androst-1-en-17-ol-3-one (M7 and M8). After oral administration of Primobolan^® (80 tablets × 5 mg of methenolone acetate each) to two thoroughbred geldings, the parent steroid ester was not detected in the post-administration urine samples. However, seven metabolites, namely M1, M6-M8, two stereoisomers of M7 (M9 and M10) and 1-methyl-5α-androst-1-en-17α-ol-3-one (M11), could be detected. The metabolic pathway for methenolone acetate is postulated. This study has shown that metabolite M1 could be targeted for controlling the abuse of methenolone acetate in horses.     

Microbial transformation of (+)-androsta-1 ,4-diene-3,17-dione by Cephalosporium aphidicola

Fermentation of (+)-androsta-1,4-diene-3,17-dione ([structure: see text]) with Cephalosporium aphidicola for 8 days yielded oxidative and reductive metabolites, androst-4-ene-3,17-dione ([structure: see text]), 17beta-hydroxyandrosta-1,4-diene-3-one ([structure: see text]), 11alpha-hydroxyandrosta-1,4-diene-3,17-dione ([structure: see text]), 11alpha-hydroxyandrost-4-ene-3,17-dione ([structure: see text]), 11alpha,17beta-dihydroxyandrost-4-ene-3-one ([structure: see text]) and 11alpha,17beta-dihydroxyandrosta-1,4-diene-3-one ([structure: see text]). The fermentation of [structure: see text] with Fusarium lini also yielded metabolites [structure: see text]. The structures of these metabolites were elucidated on the basis of spectroscopic techniques.     

The high resolution mass spectra of α,β-unsaturated and α-chloromercuri-α,β-unsaturated steroidal ketones

The high resolution mass spectra (500 eV) of some α,β-unsaturated steroidal ketones have been studied and compared with the spectra of the corresponding α-chloromercuri ketones. In the latter, the carbon-mercury bond frequently remains intact at the expense of the fission of two carbon-carbon bonds. The abundance of mercury-containing ions allows the use of the mercury atom fingerprint in confirming ring B fragmentation of the steroid nucleus at C(6)–C(7) and C(9)–C(10) for 5α-androst-1-ene-3,17-dione, 1,4-androstadiene-3,17-dione and their 2-chloromercuri derivatives; and at C(7)–C(8) and C(9)–C(10) for 1,4,6-androstatriene-3,17-dione, 1,4,6-androstarien-17 β-ol-3-one and their 2-chloromercuri derivatives. 2-Chloromercuri-1,4,6-androstatriene-3,17-dione and 2-chloromercuri-1,4,6-androstatrien-17 β-ol-3-one also give an abundant ion as the result of ring C fragmentation at C(8)–C(14) and C(11)–C(12), the chloromercuri group being replaced by a hydrogen atom. This ring C cleavage gives the only recognizable distinctive fragmentation ion for 1,4,6-pregnatriene-3,20-dione and 2-chloromercuri-1,4,6-pregnatriene-3,20-dione. For most of the mercurated steroids, the low resolution mass spectra (70 eV) are reported. In these spectra, the fragmentation patterns are similar to those obtained using the higher ionization energy employed for the high resolution spectra.     

Metabolic studies of mesterolone in horses

Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration.In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after acylation or silylation. Five metabolites (M1-M5) were detected. They were 1α-methyl-5α-androstan-3α-ol-17-one (M1), 1α-methyl-5α-androstan-3β-ol-17-one (M2), 1α-methyl-5α-androstane-3α,17β-diol (M3), 1α-methyl-5α-androstane-3β,17β-diol (M4), and 1α-methyl-5α-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1.For the in vivo metabolic studies, Proviron^® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1α-methyl-5α-androstane-3,17α-diol (M6 and M7) and 1α-methyl-5α-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1α-methyl-5α-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.     

Milieux hyperacides—XXIII: Isomerisation dienone-phenol et phenol-phenol en serie steroide. mise en evidence d'un intermediaire reactionnel

In the hyperacid medium HF-SbF₅, 3,17-dione androst-1,4-diene leads first to the expected 1-methyl estrone and 1-hydroxy 4-methyl estra-1,3,5(10)-triene-17-one in a 9:1 ratio. In a second step, 1-methyl estrone isomerises to the more stable 1-methyl(8α, 14β)estrone. Kinetic studies show the influence of anion structure on the rate of the Al step. The mechanism of the phenol-phenol isomerisation is substantiated through trapping the short-lived species involved in the reaction by hydrogen (or deuterium) donor. This reduction gives 1-methyl (5βH or D) estr-1-en-3,17-dione setting up a mechanism involving C-para (C-10) and C-ortho (C-4) diprotonation of the aromatic ring.     

18-甲基腺甾烯酮一类物的酶催化羟化

18-甲基-11α-羟基腺甾-4-烯-3,17-双酮是合成高效口服避孕药的重要中间体。试用黑根霉酶羟化引进11α-羟基于18-甲基-19-失碳雌甾-4-烯-3,17-双酮,得到包括该化合物在内的几种不同位置羟基产物的混合物。改用赭曲霉酶羟化同一底物也得到包括11α-羟基在内的几种不同位置羟基产物的混合物。而用赭曲霉催化羟化18-甲基-17β-羟基腺甾-4-烯-3-酮时,首次得到15α-羟化的主要产物和7β羟化的次要产物。前者可用来合成另一类高效口服避孕药△^15-D-18-甲基炔诺酮。     

Autooxidation of Δ17(20)-20-hydroxy derivatives of steroids. Synthesis of 3β-acetoxy-17α-hydroperoxy-16α-methylpregn-5-en-20-one and its reduction to 17α-hydroxy derivative

An efficient procedure was proposed for the synthesis of 3β-acetoxy-17α-hydroperoxy-16α-methylpregn-5-en-20-one. Optimal conditions were found for the combined process including 1,4-addition of methylmagnesium bromide at the Δ¹⁶-20-oxo fragment of dehydropregnenolone acetate and autooxidation of resulting bromomagnesium 3β-acetoxy-16α-methylpregna-5,17(20)-dien-20-olate. The subsequent reduction of the 17α-hydroperoxy group and hydrolysis of the 3β-acetoxy group afforded 17α-hydroxy-16α-methyl-substituted dehydropregnenolone acetate and its 3-hydroxy analog in high yield.     

Synthesis of oryzalexins a,b and c,the diterpenoidal phytoalexins isolated from rice blast leaves infected with pyricularia oryzae

Naturally occurring enantiomers of three diterpenes isolated as phytoalexins from rice blast leaves were synthesized: (+)-oryzalexin.A [ent-3β-hydroxyisopimara-8(14),15-dien-7-one, 1], (+)-oryzalexin B [ent-7α-hydroxyisopimara-8(14),15-dien-3-one, 2] and (+)-oryzalexin C [ent-isopimara-8(14),15-diene-3,7-dione, 3]. Their antipodes were also synthesized.     

Excretion profile of boldenone and its metabolites after oral administration to veal calves

The residue profiles of boldenone (17β-Bol), its epimer (17α-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17α-Bol decreased rapidly after end of treatment; detectable amounts of 17α-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17β-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.     

Synthesis of all 7αH-guaia-4,11-dien-3-one diastereomers from (+)-dihydrocarvone

All four 7αH-guaia-4,11-dien-3-one diastereomers have been synthesized from the common intermediate 1αH,10α-acetoxy-7αH-guaia-4,11-dien-3-one obtained from (+)-dihydrocarvone. The spectral features of the four diasteromers have been correlated and the structure and absolute configuration of 1βH,10βH,7αH-guaia-4,11-dien-3-one isolated from Pleocarphus revolutus has been confirmed.     

Synthesis and photolysis of doxyl derivatives of 5β-androstan-3-ones

In an unsuccessful attempt to effect remote functionalization at C9, 4,4-dimethyloxazolidine-N-oxyl (doxyl) derivatives at C3 of 5β-androstan-3,17-dione and 5β-androstan-17β-ol-3-one were prepared and photolyzed. The former doxyl yielded its C13 stereoisomer; the latter was converted very slowly to intractable material.     

Steroidal analogues of unnatural configuration—VII : Total synthesis of 17β-hydroxy-9-methyl-9β, 10α-oestr-4-en-3-one,a novel retrotestosterone isomer

Conjugate methylation of 17β-hydroxy-des-a-oestr-9-en-5-one (1) and the derived 4,5-seco-steroid (6b) afforded the respective 9β-methyl compounds. Base-catalysed alkylation of 17β-hydroxy-9-methyl-des-a-9/gb-oestran-5-one (3a) resulted in attack at C(6); this result was used to prepare the anthrasteroid (5). Ring closure of the 9β-methyl-4,5-seco-steroid (8) derived from 6b afforded 17β-hydroxy-9-methyl-9β,10α-oestr-4-en-3-one (9a). Conjugate methylation of 17β-hydroxyoestra-4,9-dien-3-one (11) resulted in 1,4-addition to the dienone system.     

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n?=?67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.     

Epoxidation and reduction of DHEA, 1,4,6-androstatrien-3-one and 4,6-androstadien-3beta,17beta-diol

Dehydroepiandrosterone (DHEA) reacted with m-chloroperoxybenzoic acid(m-CPBA) to form 3beta-hydroxy-5alpha,6alpha-epoxyandrostan-17-one (1), but it did not react with 30% H2O2. 1,4,6-Androstatrien-3,17-dione (2) was obtained from DHEA and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. Compound 2 was reacted with 30%H2O2 and 5% NaOH in methanol to give 1alpha,2alpha-epoxy-4,6-androstadien-3,17-dione (3),which was stereoselectively reduced with NaBH4 to form 1alpha,2alpha-epoxy-4,6-androstadien-3beta,17beta-diol (7) and reacted with Li metal in absolute ethanol-tetrahydrofuran mixture to give 2-ethoxy-1,4,6-androstatrien-3,17-dione (8). Compound 2 was also epoxidized with m-CPBA in dichloromethane to afford 6alpha,7alpha-epoxy-1,4-androstadien-3,17-dione (4),which was reacted with NaBH4 to synthesize 6alpha,7alpha-epoxy-4-androsten-3beta,17beta-diol (9).Compound 4 was reduced with Li metal in absolute ethanol-tetrahydrofuran mixture to form 7beta-ethoxy-6alpha-hydroxy-1,4-androstadien-3,17-dione (10). Compound 2 was reduced with NaBH4 in absolute ethanol to form 4,6-androstadien-3beta,17beta-diol (5), which was reacted with 30% H2O2 to give the original compound, but which reacted with m-CPBAto give 4beta,5beta-epoxy-6-androsten-3beta,17beta-diol (6).     

Urine stability and steroid profile: towards a screening index of urine sample degradation for anti-doping purpose

The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free + conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography–mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL⁻¹ for 5α-androstane-3,17-dione and 20 ng mL⁻¹ for 5β-androstane-3,17-dione. The validity of this approach was confirmed by the analysis of routine samples for more than five months (i.e. on a total of more than 4000 urine samples): samples with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than the threshold values showed a percentage of free testosterone higher than 5 of its total amount; whereas free testosterone in a percentage higher than 5 of its total amount was not detected in urines with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione lower than the threshold values.     

Synthese von 3-Oxacholestanen

Successive treatment of 5α-cholestan-3-one ( 1 ) with O₂ under basic conditions and then NaBH₄ led to 5α-3-oxa-cholestan-2-one ( 5 ). Analogous reactions with 5β-cholestan-3-one ( 6 ) yielded 5α-4-oxa-cholestan-3-one ( 7 ) and 5 ξ-3-oxa-cholestan-4-one ( 8 ). 4-Cholesten-2-one ( 10 ), which was prepared starting from 4-cholesten-3-one, was isomerized by methanolic KOH to give a mixture of 5α-cholest-3-en-2-one ( 11 ) and 5β-cholest-3-en-2-one ( 12 ). 5β-Cholestane-2,3-dione ( 17 ) was synthesized from 4β-bromo-5β-cholestan-3-one ( 13 ). Ozonolysis of the dione 17 and subsequent NaBH₄ reduction of the oxidation product gave both 5β-2-oxa-cholestan-3-one ( 18 ) and 5β-3-oxa-cholestan-2-one ( 19 ).     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.