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.     

Determination of delta4-3-ketosteroids based on oxime formation by difference circular dichroism spectroscopy

delta4-3-Ketosteroids exhibit an intensive negative Cotton effect on the circular dichroism (CD) spectra in the wavelength range for the n-pi* electronic transition (270-350 nm). With hydroxylamine hydrochloride, delta4-3-ketosteroid compounds can be transformed into oxime derivatives. Following oxime formation, positive ellipticity with low intensity can be registered in this wavelength range. The quantitative determination of delta4-3-ketosteroids is based on the considerable difference between the ellipticities before and after oxime formation. The difference ellipticity for the six ketosteroids examined (norethisterone, levonorgestrel, levonorgestrel acetate, methyltestosterone, testosterone phenylpropionate, nortestosterone phenylpropionate) varies linearly with the concentration in the interval 6 x 10(-6)-3 x 10(-3) mol/L. The method can be well applied to determination of delta4-3-ketosteroid contamination of norgestimate [(+)-13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn4-en-20-yn-3-one oxime acetate]; 0.02-10% impurity can be measured.     

Microbial transformation of cortisol and prolyl endopeptidase inhibitory activity of its transformed products

Incubation of cortisol (1) with Gibberella fujikuruoi for 12 days yielded an oxidatively cleaved product, 11beta-hydroxyandrost-4-en-3,17-dione (2), while incubation with Bacillus subtilis and Rhizopus stolonifer yielded the reduced product, 11beta, 17alpha,20,21-tetrahydroxy-(20S)-pregn-4-en-3-one (3). Other reduced products, 11beta, 17alpha, 21-trihydroxy-5alpha-pregnan-3, 20-dione (4) and 3beta, 11beta, 17alpha, 21-tetrahydroxy-5alpha-pregnan-20-one (5) were obtained by incubation of compound 1 with Bacillus cerus. The inhibitory activity of compounds 1-5 against prolyl endopeptidase enzyme (PEP) was also assayed. Compounds 2 (IC50 162.8 microM) and 4 (IC50 157 microM) have shown significant inhibitory activity against PEP.     

Determination of Δ4-3-ketosteroids based on oxime formation by difference circular dichroism spectroscopy

Δ⁴-3-Ketosteroids exhibit an intensive negative Cotton effect on the circular dichroism (CD) spectra in the wavelength range for the n-π* electronic transition (270–¶350 nm). With hydroxylamine hydrochloride, Δ⁴-3-ketosteroid compounds can be transformed into oxime derivatives. Following oxime formation, positive ellipticity with low intensity can be registered in this wavelength range. The quantitative determination of Δ⁴-3-ketosteroids is based on the considerable difference between the ellipticities before and after oxime formation. The difference ellipticity for the six ketosteroids examined (norethisterone, levonorgestrel, levonorgestrel acetate, methyltestosterone, testosterone phenylpropionate, nortestosterone phenylpropionate) varies linearly with the concentration in the interval 6 × 10^–6–3 × 10^–3 mol/L. The method can be well applied to determination of Δ⁴-3-ketosteroid contamination of norgestimate [(+)-13-ethyl-17-hydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one oxime acetate]; 0.02–10% impurity can be measured.     

Two new meliacarpinins from the roots of Melia azedarach

Two new azadirachtin-type limonoids, 1-methacrylyl-3-acetyl-11-methoxymeliacarpinin (1) and 1-(2-methylpropanoyl)-3-acetyl-11-methoxymeliacarpinin (2), together with the known compounds, meliacarpinin D (3), melianin B (4) and 2 beta,3 beta-dihydroxy-5 alpha-pregn-17(20)-(Z)-en-16-one (5), were isolated from the roots of Melia azedarach. The structures of 1 and 2 were elucidated by analysis of spectroscopic data and comparison of their NMR data with those of 3. Compounds 1 and 5 exhibited significant activity in the brine shrimp lethality test (BST).     

Microbial transformation of danazol

Danazol (17beta-hydroxy-17alpha-pregna-2,4-dien-20-yno-[2,3-d] isoxazole) (1) on fermentation with Fusarium lini, Aspergillus niger, and Cephalosporium aphidicola yielded 17beta-hydroxy-2-(hydroxymethyl)-17-alpha-pregn-4-en-20-yn-3-one (2) and 17beta-hydroxy-2-(hydroxymethyl)-17 alpha-pregna-1,4-dien-20-yn-3-one (3), while the fermentation of 1 with Bacillus cerus yielded compound 2 only.     

Microbial hydroxylation of pregnenolone derivatives

Pregnenolone and pregnenolone acetate were incubated with the fungi Cunninghamella elegans, Rhizopus stolonifer and Gibberella fujikuroi. Incubation of with C. elegans yielded metabolites, 3beta,7beta,11alpha-trihydroxypreg-5-en-20-one, 3beta,6alpha,11alpha,12beta,15beta-pentahydroxypreg-4-en-20-one and 3beta,6beta,11alpha-trihydroxypreg-4-en-20-one, while incubation with G. fujikuroi yielded two known metabolites, 3beta,7beta-dihydroxypregn-5-en-20-one and 6beta,15beta-dihydroxypreg-4-ene-3,20-dione. Metabolites and were found to be new. Fermentation of by C. elegans yielded four known oxidative metabolites, androsta-1,4-diene-3,17-dione, 6beta,15beta-dihydroxyandrost-4-ene-3,17-dione and 11alpha,15beta-dihydroxypreg-4-ene-3,20-dione. Fermentation of with R. stolonifer yielded two known metabolites, 11alpha-hydroxypreg-4-ene-3,20-dione and. Compounds were screened for their cholinesterase inhibitory activity in a mechanism-based assay.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry.

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid-liquid and liquid-liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography-mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6 beta-hydroxymethandienone (III), its C-17 epimer (IV) and 16 beta-hydroxymethandienone (V). In addition, three isomers of 6 beta,16-dihydroxymethandienone (VIa-c) were discovered. Apparently, reduction of the delta 4 double bond of 16 beta-hydroxymethandienone (V) in the horse yields 16 beta,17 beta-dihydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (VII). Reduction of the isomers VIa-c results in the corresponding 6 beta,16,17-trihydroxy-17-methyl-5 beta-androst-1-en-3-ones (VIIIa-c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

Two novel steroids from Euphorbia officinarum latex

Two novel steroids, 3beta,7alpha-dihydroxy-4alpha,14alpha-dimethyl-5alpha-cholest-8-en-11-one (2) and 3beta,7beta-dihydroxy-4alpha,14alpha-dimethyl-5alpha-cholest-8-en-11-one (3) were isolated from the latex of Euphorbia officinarum. Their structures were established on the basis of NMR and MS studies.     

Polyhydroxylated steroids and other constituents of the soft coral Nephthea chabroli

Polyhydroxylated steroids and other metabolites were isolated from the soft coral Nephthea chabroli collected in South China Sea. The structures of two new compounds were determined to be 24-methylcholesta-9(11), 24(28)-diene-3beta,12alpha,19-triol and 4alpha-methyl-3beta,14beta-dihydroxy-5alpha-ergost-24(28)-en-23-one on the basis of spectroscopic analysis. Cytotoxic activities of the polyhydroxylated sterols were evaluated on prostate carcinoma LNCaP cell line.     

Synthesis of deuterium-labeled 16 alpha,19-dihydroxy C19 steroids as internal standards for gas chromatography-mass spectrometry.

[2 beta,7,7,16 beta-2H4]16 alpha,19-Dihydroxyandrost-4-ene-3,17-dione (14) and [7,7,16 beta-2H3]3 beta,16 alpha,19-trihydroxyandrost-5-en-17-one (16), with high isotopic purity, respectively, were synthesized from unlabeled 3 beta-(tert-butyldimethylsiloxy)-androst-5-ene-17 beta-yl acetate (1). The deuterium introduction at C-7 was carried out by reductive deoxygenation of the 7-keto compound 3 with dichloroaluminum deuteride and that at C-2 beta and/or C-16 beta by controlled alkaline hydrolysis of 16-bromo-17-ketone 11 or 12 with NaOD in D2O and pyridine. [7,7-2H2]3 beta-Hydroxyandrost-5-en-17-one (6), obtained from compound 1 by a five-step sequence, was converted to compound 14 or 16 by an eight-step or seven-step sequence, respectively. The labeled steroids 14 and 16 are useful as internal standards for gas chromatography-mass spectrometry analysis of the endogenous levels.     

Cytotoxic diterpenoids from Vietnamese medicinal plant Croton tonkinensis GAGNEP

Six new ent-kaurane-type diterpenoids were isolated from the leaves of the endemic Vietnamese medicinal plant Croton tonkinensis GAGNEP. (Euphorbiaceae) together with three known ent-11alpha-acetoxy-7beta,14alpha-dihydroxykaur-16-en-15-one (1), ent-kaur-16-en-15-one 18-oic acid (5) and ent-18-hydroxykaur-16-ene (7). Their structures were determined by spectroscopic analyses to be ent-7beta-acetoxy-11alpha-hydroxykaur-16-en-15-one (2), ent-18-acetoxy-11alpha-hydroxykaur-16-en-15-one (3), ent-11alpha-acetoxykaur-16-en-18-oic acid (4), ent-15alpha,18-dihydroxykaur-16-ene (6), ent-11alpha,18-diacetoxy-7beta-hydroxykaur-16-en-15-one (8), and ent-(16S)-1alpha,14alpha-diacetoxy-7beta-hydroxy-17-methoxykauran-15-one (14). ent-Kaurane-type diterpenoids from Croton tonkinensis 2-4, 6, and 9-13, were tested for toxicity in the brine shrimp lethality assay. Compounds 9, 10, and 12 demonstrated significant activity, compounds 2, 3, 6, and 11 showed weak activity, and compounds 4 and 13 were inactive.     

Improved synthesis and molecular modeling of 4beta,19-dihydroxyandrost-5-en-17-one,an excellent inhibitor of aromatase

4Beta,19-dihydroxyandrost-5-en-17-one (6) is an excellent competitive inhibitor of estrogen synthetase (aromatase). Alternate, improved synthesis of this inhibitor was established. Treatment of 19-(tert-butyldimethylsilyloxy)androst-4-en-17-one (8) with m-chloroperbenzoic acid gave a 1.4:1 mixture of 4alpha,5alpha-epoxide 9 and its 4beta,5beta-isomer 10. The mixture was reacted with diI. HClO4 in dioxane to produce principally 4beta,5alpha-diol 11 (80%) of which acetylation followed by dehydration with SOCl2 yielded 4beta,19-diacetoxy-5-ene compound 14 in good yield. Alkaline hydrolysis of diacetate 14 gave 4beta,19-diol 6. The minimum energy conformation of the powerfull aromatase inhibitor 6 was obtained with the PM3 method and compared with that of the structurally related diol steroid, 4-ene-5beta,19-diol 3, a weak competitive inhibitor.     

Terpenoids from Chloranthus multistachys

Two new diterpenoids, ent-17-hydroxyl-16beta-methoxyl-kauran-3-one (1) and ent-17-acetoxyl-16beta-methoxyl-kauran-3-one (2), along with nine known compounds (3-12), ent-17-hydroxyl-kaur-15-en-3-one (3), ent-3beta-acetoxyl-kaur-15-en-16beta, 17-diol (4), ent-kauran-3beta, 16beta, 17-triol (5), ent-3beta-acetoxyl-kauran-16beta, 17-diol (6), ent-kauran-16beta, 17-diol (7), abbeokutone (8), ent-17alpha-acetyl-16beta-hydroxyl- kauran-3-one (9), shizukaol F (10), cycloshizukaol A (11) and curcolonol (12), were isolated from Chloranthus multistachys (Chloranthaceae). Their structures were elucidated by spectroscopic spectra.     

Brominations of steroidal hormone having alpha,beta-unsaturated ketone, 17-O-acetyltestosterone, in the presence of silver triflate

Bromination of 17-O-acetyltestosterone (17beta-acetoxyandrost-4-en-3-one) (1) was performed with 1, 5, and 10 eq of Br2 in AcOH-Et2O at room temperature. In all cases 2alpha,6beta- (2) and 2alpha,6beta-dibromo-17beta-acetoxyandrost-4-en-3-one (3) were obtained, although the yields were dependent upon the conditions used. Bromination of compound 1 with 10 eq of Br2 in the presence of silver trifluoromethanesulfonate (silver triflate, AgOTf) at room temperature for 12 h gave 2,7alpha-dibromo- (4) and 2,4,7alpha-tribromo-17beta-acetoxy-3-hydroxy-1-methylestra-1,3,5(10)-triene-6-one (5). The formations of the products were inferred on the basis of products obtained under controlled brominations of 1 in the presence of AgOTf, and of those obtained by the brominations of compounds 9-13 also in the presence of AgOTf.     

Microbial transformation of prednisone

The microbial transformation of prednisone (17alpha,21-dihydroxy-pregna-1,4-diene-3,11,20-trione) (1) by Cunninghamella elegans afforded two metabolites, 17alpha,21-dihydroxy-5alpha-pregn-1-ene-3,11,20-trione (2) and 17alpha,20S,21-trihydroxy-5alpha-pregn-1-ene-3,11-dione (3), while the fermentation of 1 with Fusarium lini, Rhizopus stolonifer and Curvularia lunata afforded a metabolite 1,4-pregnadiene-17alpha,20S,21-triol-3,11-dione (4). Compound 3 was found to be a new metabolite. Their structures were elucidated on the basis of spectroscopic techniques. Compound 3 showed inhibitory activity against lipoxygenase enzyme.     

Preparation and Chromatographic Characterization of Tetrahydrogestrinone, a New “Designer” Anabolic Steroid

Tetrahydrogestrinone (THG, (17)-13-ethyl-17-hydroxy-18,19-dinorpregna-4,9,11-trien-3-one, C₂₁H₂₈O₂, 312 m.w.) is a synthetic 19-norsteroid closely related to gestrinone ((17)-13-ethyl-17-hydroxy-18,19-dinorpregna-4,9,11-trien-20-yn-3-one, C₂₁H₂₄O₂, 308 m.w.) which was originally developed as an oral contraceptive for women. Recent press reports detail the probable use of THG by athletes to enhance athletic performance, and the FDA has banned THG, declaring it a designer drug. THG has been difficult to analyze because of its instability, lack of commercially available analytical standards, and the fact that no published synthetic methods for the compound exist. We now report a method for the preparation of THG via the carefully controlled hydrogenation of gestrinone.Published as #350 from the Equine Pharmacology, Therapeutics and Toxicology Laboratory at the Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky.Published as Kentucky Agricultural Experiment Station Article #;04-14-052 with the approval of the Dean and Director, College of Agriculture and the Kentucky Agricultural Experiment Station.     

Isolation of antitrypanosomal compounds from Vitis repens, a medicinal plant of Myanmar

Bioactivity-guided fractionation of an ethanolic extract of Vitis repens led to the isolation of resveratrol (1), 11-O-acetyl bergenin (2), and stigmast-4-en-3-one (3). The compounds were examined for their in vitro antitrypanosomal activities against trypomastigotes of Trypanosoma evansi. Resveratrol showed antitrypanosomal activity with an IC50 value of 0.13 microM, whereas 11-O-acetyl bergenin and stigmast-4-en-3-one exhibited IC50 values of 0.17 and 0.15 microM, respectively.     

Microbial transformation of diosgenin by Syncephalastrum racemosum (Cohn) Schroeter

<正>Microbial transformation of diosgenin(1) by Syncephalastrum racemosum yielded five new polar metabolites,which wereidentified as(25R)-spirost-5-en-3β,7α,9α-triol-12-one(2),(25R)-spirost-5-en-3β,9α,12α-triol-7-one(3),(25R)-spirost-5-en-3β,9α-diol-7,12-dione(4),(25R)-spirost-4-en-9α,12β,14α-triol-3-one(5),and(25S)-spirost-4-en-9α,14α,25β-triol-3-one(6).Compounds 1-6 exhibited moderate cytotoxicity against K562 cells and among them compounds 2,3,and 6 were more potentthan the parent compound 1.     

New cholinesterase inhibiting steroidal alkaloids from the leaves of Sarcococca coriacea of Nepalese origin

From the leaves of Sarcococca coriacea two new steroidal alkaloids, epoxynepapakistamine-A [(20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregna-16alpha,17alpha-epoxy-2beta,4beta-di-O-acetate] (1), and epoxysarcovagenine-D [(20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregna-2-en-16alpha,17alpha-epoxy-4-one] (2), and two known compounds funtumafrine C [(20S)-20-(N,N-dimethylamino)-5alpha-pregna-3-one] (3) and N-methylfuntumine (4) were isolated. Their structures were elucidated on the basis of their spectral properties. The compounds 1, 3 and 4 were found to have cholinesterase inhibitory activity when tested for the inhibition of electric eel acetylcholinesterase and horse serum butyrylcholinesterase. They inhibited both enzymes in a concentration dependent fashion.