Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol (excreted as glucuronides) and ETIO, ANDRO and 3β-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.     

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.     

A favorski reaction using iodosobenzene

Iodosobenzene or iodobenzene diacetate and excess base when reacted with 17β-hydroxy-5α-androstane-3-one (1a) unexpectedly gave a good yield of Favorski acid (3a) and some (3b). 17β-hydroxy-5α-19-norandrostan-3-one (1b) gave mainly the expected dimethylketal of the 2α-hydroxy-3-keto steroid (5).     

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.     

Steroids and steroidases—XIX : Comparison of diazomethane and tiffeneau-demjanov homologations of 5α-3-oxosteroids. Evidence for predominant equatorial approach of the C-3 carbonyl group by diazomethane

Quantitative comparisons of the product ratios of the mechanistically similar diazomethane and Tiffeneau-Demjanov homologations of 17β-hydroxy-5α-androstan-3-one and 5α-cholestan-3-one have shown that equatorial approach of diazomethane to the C-3 CO group predominates to the extent of 70–79%. The data for both the C-17β-OH and -C₈H₁₇ substituted steroids are in close agreement thereby confirming that the C-17 substituents do not exert any significant long range effect on the reactions studied.     

5α-androstano [3,2-b]pyridines, 5α-androstano[17,16-b]pyridines and androst-4 and 5-eno[17,16-b]pyridines

A new synthesis of 5α-androstano[3,2-b]pyridin-17β-ol acetate (VIa) and 17-methyl-5α-androstano[3,2-b]pyridin-17β-ol (VIb), first reported by Shimizu, Ohta, Ueno, and Takegoshi, was achieved. The analogous 5α - androstano[17,16-b]pyridin-3β-ol (XII), 5α-androstano[17,16-b]pyridin-3-one (XIVa), and androst-4-eno[17,16-b]pyridin-3-one (XIVb) were also prepared. An illustration of the method follows. Condensation of 3β-hydroxy-5α-androstan-17-one (VIIa) with 3-(2-furyl)acrolein afforded 16-[3-(2-furyl)-2-propenylidene]-3β-hydroxy-5α-androstan-17-one (VIIIa), the oxime (IXa) of which was thermally cyclized to 5α-androstano[17,16-b]-6′-(2-furyl)pyridin-3β-ol (Xa). 3β-Hydroxy-5α-androstano[17,16-b]pyridine-6′-carboxylic acid (XI) was obtained by ozonolysis of Xa. Thermal decarboxylation of XI gave XII. Cinnamaldehyde was used in place of 3-(2-furyl)acrolein to give the corresponding phenylpyridines.     

Steroidal quinoxalines

The treatment of 3β-hydroxy-16α-bromo-5α-androstan-17-one, 3β-acetoxy-16α-bromo-5-androsten-17-one and 21-bromo-5-pregnen-3β-ol-20-one with 4,5-dimethyl-o-phenylenediamine gave substituted quinoxalines. Hydrolysis of 3β-acetoxy-5-androsteno[16,17-b]-6′,7′-dimethylquinoxaline produced the corresponding 3β-hydroxy compound. 3-Oxo-4-androsteno[16,17-b]-6′,7′-dimethylquinoxaline was obtained by Oppenauer oxidation of the corresponding alcohol.     

Synthesis,x-ray structure and molecular mechanics studies of the boar taint steroid (5α -androst-16-en-3-one)

5α-Androst-16-en-3-one has been prepared from 5α-androstan-3β-ol-17-one in an overall yield of 34% by the vinyl iodide route. Accurate molecular dimensions have been determined by X-ray crystal structure analysis and by molecular mechanics calculations. There is significant twisting of the angular methyl groups in the molecule.     

Reaction of a 5α-bromo-6β,19-epoxysteroid with BF3·Et2O/Ac2O: An evidence of a cyclic bromonium cation

Treatment of 3β-acetoxy-5-bromo-6β,19-epoxy-5α-androstan-17-one with Ac₂O and BF₃·OEt₂, produced the cleavage of the epoxy moiety and migration of the bromine atom to afford 3β,19-diacetoxy-6α-bromo-5-hydroxy-5β-androst-17-one in high yield.     

Use of n-lithioethylenediamine in the double bond isomerization and degradation of sterol side chains

Double bond isomerization of stigmasterol ((24S)-24-ethylcholesta-5,22-dien-3β-ol) with N-lithioethy-lenediamine produced stigmasta-5,17(20)-dien-3β-ol, stigmasta-5,20(22)-dien-3β-ol, stigmasta-5,23-dien-3β-ol and stigmasta-5,24-dien-3β-ol. Some starting material was also present in the isomerization mixture along with a small amount of stigmast-5-en-3β-ol. Similar treatment of 3α,5-cyclo-6β-methoxystigmast-22-en (stigmasterol i-methyl ether) followed by ozonization and removal of the ring A-protecting group yielded predominantly 3β-hydroxy-androst-5-en-17-one and 3β-hydroxypregn-5-en-20-one. The isomerization products of fucosterol (24-ethylcholesta- 5, 24(28)E-dien-3β-ol) and 23,24-bisnorchola-5,20-dien-3β-ol are also reported.     

Detection of norbolethone,an anabolic steroid never marketed,in athletes' urine

Norbolethone (13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn-4-en-3-one) is a 19-nor anabolic steroid first synthesized in 1966. During the 1960s it was administered to humans in efficacy studies concerned with short stature and underweight conditions. It has never been reported by doping control laboratories. Norbolethone was identified in two urine samples from one athlete by matching the mass spectra and chromatographic retention times with those of a reference standard. The samples also contained at least one likely metabolite. The samples were also unusual because the concentrations of endogenous steroids were exceptionally low. Since norbolethone is not known to be marketed by any pharmaceutical company, a clandestine source of norbolethone may exist.     

Copper-mediated regio- and stereoselective 12β-hydroxylation of steroids with molecular oxygen and an unexpected 12β-chlorination

It is shown, that copper(I) complexes of 17-(2-iminomethyl)pyridino steroids (17-IMPY steroids) can react with molecular oxygen followed by a regio- and stereoselective γ-hydroxylation in 12β-position. After decomplexation and hydrolysis of the IMPY group 12β-hydroxy-17-ketones are available in practical useful yields. IMPY compounds are simple to prepare by condensation of oxo compounds with (2-aminomethyl)pyridine. In the cases of 17-IMPY steroids the yields in the hydroxylation procedure of an unactivated CH₂ group are higher by starting with copper(II) complexes, reduction with benzoin/triethylamine in acetone and reaction with molecular oxygen in comparison to the direct reaction of copper(I) complexes with molecular oxygen in acetone. Employing the procedure in dichloromethane as solvent starting with copper(II) complexes surprisingly the 12β-chloro compound could be isolated next to the hydroxylation product. This regio- and stereoselective γ-chlorination takes place also in acetone, when triethylammonium chloride is added to the reaction mixture. Oxygen is necessary for this reaction. The mechanistic and stereochemical aspects of these new reactions are discussed. Comparison of the different yields of steroids with different A-ring [3-methoxy-estra-1,3,5(10)-triene and 3β-hydroxy-androst-5-ene] pointed out to a subtle influence of the molecular structure far from the reaction centre on these reactions. The successful hydroxylation of the IMPY derivative of 3β-hydroxy-androst-5-ene-17-one shows the tolerance of a homoallylic system against this oxidation procedure. By Oppenauer oxidation 12β-hydroxy-androst-4-ene-3,17-dione is available. The hydroxylation procedure opens a short way to 12β-hydroxy-17-oxo steroids, which are difficult to obtain by other routes.     

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.     

Dissimilar behaviour of 3β, 16α-dihydroxy-5α-androstan-17-one Diacetate under Basic and Acidic Conditions

The base-catalysed rearrangement of 3β, 16α-dihydroxy-5α-androstan-17-one diacetate ( 1 ) in (D₆)benzene/ CD₃OD to 3β, 17β-dihydroxy-5α-androstan-16-one ( 3 ) is followed by ¹³C-NMR spectroscopy. By the same procedure, it is determined that in (D₆)benzene/CD₃OD, but under acid catalysis, 1 does not rearrange to 3 but yields the intermediate product 3β, 16α-dihydroxy-5α -androstan-17-one 17α -methyl hemiacetal ( 5 ).     

Identification de stérols à chaînes latérales courtes dans l′éponge Damiriana hawaiiana

Identification of short side chain sterols in the sponge Damiriana hawaiiana The steroidal composition of the sponge Damiriana hawaiiana is examined. Twenty-seven components are identified. In addition to the C₂₆-C₂₉, Δ⁵-mono and diunsaturated sterols, the sponge contains sterols without side-chain: androsta-5, 16-dien-3β-ol( 1 ), androst-5-en-3β-ol( 2 ); sterols with a non-functionalized side-chain consisting of two, three, four, five and six carbon atoms: pregna-5, 20-dien-3β-ol( 5 ), pregn-5-en-3β-ol( 6 ), 23, 24-bisnor-chola-5, 20-dien-3β-ol( 7 ), 23, 24-bisnor-chol-5-en-3β-ol( 8 ), 24-nor-chol-5-en-3β-ol( 10 ), chol-5-en-3β-ol( 11 ), 26, 27-bisnor-cholest-5-en-3β-ol( 12 ), and sterols possessing a short oxygenated side-chain such as 3β-hydroxy-androst-5-en-17-one( 3 ), androst-5-en-3β, 17β-diol( 4 ) and 3β-hydroxy-26, 27-bisnor-22-trans-cholesta-5, 22-dien-24-one( 14 ). The probable biological or dietary origin rather than artifact production of these hitherto undescribed components from marine sources is supported by their relatively high concentration and their relative proportions, both very different from those expected for autoxidation.     

Oxidation of Steroidal 5-En-3β-ol with Pyridinium Chlorochromate: Isolation of Key Intermediate,Steroidal 6β-Hydroxy-4-en-3-one

The PCC oxidation of 5-en-3β-ol steroids proceeds to 4-en-3,6-dione through the intermediate 5-en-3-one. The isolation of another key intermediate, steroidal 6β-hydroxy-4-en-3-one guides an understanding of the mechanism involved.     

Synthesis and biological evaluation of 3-beta,20(R)-dihydroxy-protost-24-ene

The possible lanosterol precursor, 3β, 20(R)-dihydroxy-protost-24-ene ( 1 a ) has been prepared, in thirteen steps, from 3α-hydroxy-4α, 8, 14-trimethyl-18-nor-5α, 8α, 9β, 13ξ, 14β-androstan-17-one (mixture of 2 and 3 ). In vitro experiments with rat liver homogenates failed to convert 1 a to lanosterol.     

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.     

Thiosteroids—XXXV : Diastereomers of steroidal thiolsulfinates

17β-Hydroxy-5α-androstan-2α,3α-anti(R)-episulfoxide on treatment with methanol and ethanol in the presence of a trace amount of sulfuric acid gave diastereomers of bis((2β-methoxy- and 2β-ethoxy-17β-hydroxy-5α-androstan-3α-yl) disulfide S-monoxides respectively. The absolute configuration of the compounds was established by their Grignard reactions leading to diastereomeric phenyl sulfoxides stereoaspecifically.     

Steroidal imidazopyridines and lactam imidazopyridines

Condensation of 17β-acetoxy-2α-bromo-5α-androstan-3-one with unsubstituted and substituted amino-pyridines, gives the corresponding 17β-acetoxy-5α-androstanimidazo[1,2-a]pyridines. Treatment of 16α-bromo-3-aza-A-homo-4α-androsten-4,17-dione with 2-aminopyridine or methyl-2-aminopyridine produces the corresponding 3-aza-A-homo-4α-androsten[16,17:2′,3′]imidazo[1,2-a]pyridines. Similarly, from 2α-bromo-17β-acetamido-5α-androstan-3-one and methylaminopyridine the 17β-acetamido-5α-androstan[2,3:2′,3′]imidazo[1,2-a]methylpyridine has been obtained. The structure of the compounds was apparent from their chemical properties and spectral data (ir, uv and nmr).