Glomerella fusarioides-catalyzed structural transformation of steroidal drugs mesterolone and methasterone, and anti-inflammatory activity of resulting derivatives.

Transformation of steroidal drug mesterolone (1) with Glomerella fusarioides yielded two new (17α-hydroxy-1α-methyl-5α-androstan-3-one-11α-yl acetate (2) and 15α-hydroxy-1-methyl-5α-androstan-1-en-3,17-dione (3)), and four known derivatives (15α,17ß-dihydroxy-1α-methyl-5α-androstan-3-one (4), 15α-hydroxy-1α-methyl-5α-androstan-3,17-dione (5), 1α-methyl-androsta-4-en-3,17-dione (6) and 15α,17ß-dihydroxy-1-methyl-5α-androstan-1-en-3-one (7). Similarly, G. fusarioides-catalyzed transformation of steroidal drug methasterone (8) afforded four new metabolites, 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (9), 3a,11α,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (10), 1ß,3ß,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (11), and 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (12). Structures of new derivatives were determined by using 1D-, and 2D-NMR, HREI-MS, and IR spectroscopic data. New derivative 3 was identified as a potent inhibitor of NÈ® production with the IC50 value of 29.9 ± 1.8 µM, in comparison to the standard l-NMMA (IC50 = 128.2 ± 0.8 µM) in vitro. In addition, methasterone (8) (IC50 = 83.6 ± 0.22 µM) also showed a significant activity comparable to new derivative 12 (IC50 = 89.8 ± 1.2 µM). New derivatives 2 (IC50 = 102.7 ± 0.5 µM), 9 (IC50 = 99.6 ± 5.7 µM), 10 (IC50 = 123.5 ± 5.7 µM), and 11 (IC50 = 170.5 ± 5.0 µM) showed a moderate activity. NG-MonomethylL-arginine acetate (IC50 = 128.2 ± 0.8 µM) was used as standared NO⋅- free radicals have an important role in the regulation of immune responses and cellular events. Their overproduction is associated with the pathogenesis of numerous ailments, such as Alzheimer's cardiac disorders, cancer, diabetes, and degenerative diseases. Therefore, inhibition of NÈ® production can help in the treatment of chronic inflammation and associated disorders. All derivatives were found to be non-cytotoxic to human fibroblast (BJ) cell line. The results presented here form the basis of further research for the development of new anti-inflammatory agents with improved efficacy through biotransformation approaches.

Liquid chromatography-mass spectrometry behavior of Girard's reagent T derivatives of oxosteroid intact phase II metabolites for doping control purposes.

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17ß-isomers of the sulfate conjugates, but limited differentiation for 17α/17ß-isomers of glucuronide conjugates and for 3α/3ß- and 5α/5ß-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS).

Biotransformation of androgenic steroid mesterolone with Cunninghamella blakesleeana and Macrophomina phaseolina.

Fermentation of mesterolone (1) with Cunninghamella blakesleeana yielded four new metabolites, 1α-methyl-1ß,11ß,17ß-trihydroxy-5α-androstan-3-one (2), 1α-methyl-7α,11ß,17ß-trihydroxy-5α-androstan-3-one (3), 1α-methyl-1ß,6α,17ß-trihydroxy-5α-androstan-3-one (4) and 1α-methyl-1ß,11α,17ß-trihydroxy-5α-androstan-3-one (5), along with three known metabolites, 1α-methyl-11α,17ß-dihydroxy-5α-androstan-3-one (6), 1α-methyl-6α,17ß-dihydroxy-5α-androstan-3-one (7) and 1α-methyl-7α,17ß-dihydroxy-5α-androstan-3-one (8). Biotransformation of 1 with Macrophomina phaseolina also yielded a new metabolite, 1α-methyl, 17ß-hydroxy-5α-androstan-3,6-dione (9). The isolated metabolites were subjected to various in vitro biological assays, such as anti-cancer, inhibition of α-glucosidase, and phosphodiesterase-5 enzymes and oxidative brust. However, no significant results were observed. This is the first report of biotransformation of 1 with C. blakesleeana and M. phaseolina.

Microbial transformation of mesterolone.

The microbial transformation of mesterolone (= (1alpha,5alpha,17beta)-17-hydroxy-1-methylandrostan-3-one; 1), by a number of fungi yielded (1alpha,5alpha)-1-methylandrostane-3,17-dione (2), (1alpha,3beta,5alpha,17beta)-1-methylandrostane-3,17-diol (3), (5alpha)-1-methylandrost-1-ene-3,17-dione (4), (1alpha,5alpha,15alpha)-15-hydroxy-1-methylandrostane-3,17-dione (5), (1alpha,5alpha,6alpha,17beta)-6,17-dihydroxy-1-methylandrostan-3-one (6), (1alpha,5alpha,7alpha,17beta)-7,17-dihydroxy-1-methylandrostan-3-one (7), (1alpha,5alpha,11alpha,17beta)-11,17-dihydroxy-1-methylandrostan-3-one (8), (1alpha,5alpha,15alpha, 17beta)15,17-dihydroxy-1-methylandrostan-3-one (9), and (5alpha,15alpha,17beta)-15,17-dihydroxy-1-methylandrost-1-en-3-one (10). Metabolites 5-10 were found to be new compounds. All metabolites, except 2, 3, 6, and 7, exhibited potent anti-inflammatory activity. The structures of these metabolites were characterized on the basis of spectroscopic studies, and the structure of 5 was also determined by single-crystal X-ray-diffraction analysis.

Studies on anabolic steroids--11. 18-hydroxylated metabolites of mesterolone, methenolone and stenbolone: new steroids isolated from human urine.

New metabolites of mesterolone, methenolone and stenbolone bearing a C18 hydroxyl group were isolated from the steroid glucuronide fraction of urine specimens collected after administration of single 50 mg doses of these steroids to human subjects. Mesterolone gave rise to four metabolites which were identified by gas chromatography/mass spectrometry as 18-hydroxy-1 alpha-methyl-5 alpha-androstan-3,17-dione 1, 3 alpha,18-dihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 2, 3 beta,18-dihydroxy-1-alpha-methyl-5 alpha-androstan-17-one 3 and 3 alpha,6 xi,18-trihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 4. These data suggest that mesterolone itself was not hydroxylated at C18, but rather 1 alpha-methyl-5 alpha-androstan-3,17-dione, an intermediate metabolite which results from oxidation of mesterolone 17-hydroxyl group. In addition to hydroxylation at C18, reduction of the 3-keto group and further hydroxylation at C6 were other reactions that led to the formation of these metabolites. It is of interest to note that in the case of both methenolone and stenbolone, only one 18-hydroxylated urinary metabolite namely 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 5 and 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 6 were both detected in post-administration urine specimens. These data indicate that the presence of a methyl group at the C1 or C2 positions in the steroids studied is a structural feature that seems to favor interaction of hepatic 18-hydroxylases with these steroids. These data provide further evidence that 18-hydroxylation of endogenous steroids can also occur in extra-adrenal sites in man.

The methyl-5 alpha-dihydrotestosterones mesterolone and drostanolone; gas chromatographic/mass spectrometric characterization of the urinary metabolites.

Before including the detection of the methyl-5 alpha-dihydrotestosterones mesterolone (1 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) and drostanolone (2 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) in doping control procedures, their urinary metabolites were characterized by gas chromatography/mass spectrometry. Several metabolites were found after enzymatic hydrolysis and conversion of the respective metabolites to their trimethylsilyl-enol-trimethylsilyl ether derivatives. The major metabolites of mesterolone and drostanolone were identified as 1 alpha-methyl-androsterone and 2 alpha-methyl-androsterone, respectively. The parent compounds and the intermediate 3 alpha,17 beta-dihydroxysteroid metabolites were detected as well. The reduction into the corresponding 3 beta-hydroxysteroids was a minor metabolic pathway. All metabolites were found to be conjugated to glucuronic acid.