Studies on anabolic steroids. 9. Tertiary sulfates of anabolic 17 alpha-methyl steroids: synthesis and rearrangement.

A simple and convenient method has been developed to prepare sulfates of anabolic 17 beta-hydroxy-17 alpha-methyl steroids. The sulfates of methandienone, 17 alpha-methyltestosterone, mestanolone, oxandrolone, and stanozolol were prepared. Different A-ring functions were not affected under the sulfation condition. The buffered hydrolyses of these sulfates provided the 17-epimers of the original steroids and 17,17-dimethyl-18-nor-13(14)-ene steroids, presumably via the 17-carbocations.     

Studies on anabolic steroids--12. Epimerization and degradation of anabolic 17 beta-sulfate-17 alpha-methyl steroids in human: qualitative and quantitative GC/MS analysis.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.     

Effects of training and anabolic steroids on collagen synthesis in dog heart.

The effects of endurance training and anabolic steroid (Methandienone 1.5 mg.kg-1 p. o. daily) and their combination on regional collagen biosynthesis and concentration in the hearts of male beagle dogs were studied by measuring prolyl 4-hydroxylase (PH) activity and hydroxyproline (HYP) concentration. The PH (P less than 0.05) and HYP (P less than 0.05) were both greater in the subendocardinal layer than in the subepicardium (EPI) of the left ventricular wall in controls, whereas opposite gradients (P less than 0.05) were observed in the right ventricle. Endurance exercise caused an increase of PH activity in EPI of the left ventricular wall (P less than 0.01). The HYP concentration increased in both layers of the right ventricle in the exercise plus steroid group (P less than 0.05). The results suggest that transmural differences exist in the rate of collagen synthesis and concentration in canine cardiac ventricles and that endurance exercise may accelerate collagen synthesis in EPI of the left ventricle and the combination of exercise and anabolic steroid causes an increase in collagen concentration in the right ventricular wall.     

Mechanistic aspects of rearrangement of 16alpha-hydroxy-17-keto steroids to the 17beta-hydroxy-16-keto isomers

The mechanistic aspects of the alkali-catalyzed rearrangement of 16alpha-hydroxy-17-keto steroid 1 to 17beta-hydroxy-16-keto steroid 2 are elucidated by use of (18)O- and deuterium-labeling experiments. The (18)O-labeling experiments refute the gem-hydration-quasi-diaxial dehydration mechanism for the rearrangement previously proposed and support the conventional enolization mechanism. Moreover, equilibrium by gem-hydration-dehydration occurs at the C-17 carbonyl more efficiently than at the C-16 carbonyl. Enolization rate of a carbonyl group at C-16 of 17beta-ketol 2 toward the C-17 position (k(16,17)) was about 8-10 times higher than those of 16alpha-ketol 1 toward the C-16 position (k(17,16)) and ketol 2 toward the C-15 position (k(16,15)). The marked deuterium-isotope effect on each enolization was observed with k(H)/k(D) ranging between 5.4 and 8.8. The present findings reveal that the initial hydration-dehydration equilibration at the C-17 carbonyl of ketol 1 followed by enolization of the carbonyl gives the ene-diol intermediate that isomerizes quantitatively to the 16-keto isomer of which the 16-carbonyl moiety enolizes preferentially toward the C-17 position rather than the C-15 position, yielding the ene-diol. Computational calculations of ground state energies of ketols 1-M and 2-M, trans-cyclohexane/cyclopentane structures, and their activation energies in the rearrangement support the dynamic aspects of the rearrangement as well as the kinetics data of the enolization.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.     

Synthesis and biological activities of an alpha-methyl and a beta-methyl carbapenem and the corresponding unsubstituted compound

The carbapenem potassium salts 4, 7 and 8 were prepared. Their rates of beta-lactam hydrolysis and their biological activities, particularly their beta-lactamase inhibiting properties, were examined and explained on the basis of their different substitution and pyramidality.     

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.     

Steroids and related products. 39. The functionalization of the 17-methyl group of 17 -methyl steroids. II. The synthesis of 12 ,17 1 -epoxy-17 -methylprogesterone and of 20-oxygenated 17 -hydroxymethyl steroids

The functionalization of the 17-methyl group of 17α-methylated 20-oxygenated steroids is reported. Two methods were employed for that purpose: the Jeger reaction with lead tetraacetate, applied to a 12α-hydroxy 17α-methyletianic ester, leading to a 12,17¹-epoxide and the photolytic Barton reaction, applied to the corresponding 12-nitrite, and leading to a 17¹-nitroso derivative which was transformed into a 17β-methoxycarbonyl lactone of a 12α-hydroxy 17α-etianic acid. The 17β-ester group of this compound can be selectively hydrolyzed and the lactone group can be reduced to a 12α-hydroxy 17α-hydroxymethyl steroid. The synthesis of 12,17¹-epoxy-17α-methylprogesterone is also described.     

Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights

The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse.

This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for “older” steroids with respect to doping control is given.     

Parallel solid-phase synthesis of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-one derivatives for inhibition of type 3 17beta-hydroxysteroid dehydrogenase.

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Delta(4)-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23-58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17beta-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3beta-(N-heptanoyl-L-phenylalanine-L-leucine-aminomethyl)-3alpha-hydroxy-5alpha-androstan-17-one (42) inhibited the enzyme with an IC(50) value of 227nM, which is twice as potent as the natural substrate Delta(4)-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR(+)) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 microM (less than previously reported type 3 17beta-HSD inhibitors) and, interestingly, no proliferation at 0.1 microM.     

Fatty acid esters of steroids: synthesis and metabolism in lipoproteins and adipose tissue

At the end of the last century ideas concerning the physiological role of the steroid fatty acid ester family were emerging. Estrogens, fatty acylated at C-17 hydroxyl group and incorporated in lipoproteins were proposed to provide antioxidative protection to these particles. A large number of studies involving non-estrogenic adrenal steroids, and their fatty acylated forms, demonstrated their lipoprotein-mediated transport into cells and subsequent intracellular activation, suggesting a novel transport mechanism for lipophilic steroid derivatives. After these important advances the main focus of interest has shifted away from C-19 and C-21 steroids to fatty acylated estrogens. However, interest in their lipoprotein-mediated transport has decreased because only minute amounts of these derivatives were detected in circulating lipoproteins, and their antioxidative activity remained unconfirmed under physiological circumstances. It now appears that the overwhelming majority of estradiol in postmenopausal women resides in adipose tissue, most of it in esterified form. This is poorly reflected in plasma levels which are very low. Recent data suggest that estrogen fatty acid esters probably represent a storage form. The future focus of investigation is likely to be on firstly, the enzymatic mechanisms regulating the esterification and de-esterification of estradiol and other steroids residing in adipose tissue and secondly, on the role of insulin and other hormones in the regulation of these enzymatic mechanisms. Thirdly, as a large proportion of fatty acid esterified C-19 and C-21 non-estrogenic steroids is transported in lipoproteins and as they are important precursors of androgens and estrogens, this field should be investigated further.     

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

One- and two-dimensional echocardiography in bodybuilders using anabolic steroids

The object of this study was to investigate the possible concentric increase in the left ventricular (LV) wall thickness by intensive strength training and to differentiate between the specific effect of the strength training itself and the influence of anabolic drugs. In this study 21 top-level bodybuilders [users of anabolic steroids (A): n = 14; non-users (N): n = 7] underwent one-dimensional and two-dimensional echocardiography as well as a cycle ergometer test. In both groups blood pressure at rest and during ergometric exercise was within the normal range. In spite of the same amount of time being spent on training, A showed significantly better power results than N. Total heart volume (A = 11.3 +/- 0.9 ml.kg-1; N = 11.9 +/- 0.9 ml.kg-1) and LV muscle mass were almost identical in A and N and correlated significantly with body weight and lean body mass respectively. The body dimension-related diastolic LV diameter was significantly lower in A (0.567 +/- 0.062 mm.kg-1) than in N (0.639 +/- 0.040 mm.kg-1). An increase in the LV posterior wall (p less than 0.01) and septum thickness (ns) resulted in increased LV wall thickness:diameter (p less than 0.01) and LV muscle mass:volume (p less than 0.05) ratios in A (0.458 +/- 0.590; 1.38 +/- 0.25 g.ml-1) in comparison to N (0.356 +/- 0.077; 1.16 +/- 0.17 g.ml-1). The septal:posterior wall thickness ratio was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggression in male rats receiving anabolic androgenic steroids: effects of social and environmental provocation.

This study examined the effects of anabolic androgenic steroids (AAS) on aggression under different social and environmental conditions. Three AAS were tested in gonadally intact male rats: testosterone propionate (TP), nandrolone (ND), and stanozolol (ST). Doses of 5 mg/kg were given 5 times/week, with gonadally intact controls receiving vehicle only (propylene glycol). Animals received six weekly tests under each condition in a counterbalanced order. Results show that the three AAS differed in their ability to elicit aggression. Males receiving TP were more aggressive than controls, ND males were similar to controls, and ST males were less aggressive than controls. In the social and environmental provocation tests TP-treated males were more aggressive than other groups, but were able to discriminate between intact and castrated opponents and between their home cage and a neutral cage. In the environmental provocation test, TP males were also more aggressive against opponents when tested in the opponent's home cage. It is suggested that chronic exposure to high levels of TP does not eliminate the ability to discriminate between social or environmental cues, as might be expected if it induces a " 'roid rage." However, TP does increase the likelihood that the animal will respond with aggression/dominance in a provoking situation. All three AAS variably affected serum testosterone and LH levels, as well as testes, seminal vesicle, and prostate weights. No effect on body weight was observed.