Abstract
Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2–4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7 μM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1–4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.
Highlights
Mibolerone (7α,17α-dimethyl-19-nortestosterone) (1) is a potent synthetic anabolic and androgenic steroid, marketed by the Upjohn Company under the brand name of Check Drops, for the treatment of estrous in female dogs
The analysis revealed that metabolite 4 was crystalized out as a water solvate, and comprised three six-membered rings A (C1-C5/C10), and B (C5-C10), C (C8-C9/ C11-C14), existing in half chair and chair conformations, respectively, while a five-membered ring D (C13-C17) exists in an envelope conformation (Fig 7)
Substrate 1 showed a significant leishmanicidal activity against the promastigotes of Leishmania major with IC50 value of 29.64 ± 0.88 μM as compared to the standard drugs, pentamidine (IC50 = 5.09 ± 0.09 μM), and amphotericin B (IC50 = 0.29 ± 0.05 μM) (Fig 10)
Summary
Mibolerone (7α,17α-dimethyl-19-nortestosterone) (1) is a potent synthetic anabolic and androgenic steroid, marketed by the Upjohn Company under the brand name of Check Drops, for the treatment of estrous (heat) in female dogs. Substrate 1 was found to be active against β-glucuronidase enzyme, Leishmania major promastigotes, and HeLa (cervical cancer) and 3T3 (normal) cell lines in preliminary assays. Biological activity evaluation of its metabolites solution of 1 mg of test compounds was prepared in a mixture 50 μL of DMSO and 950 μL of RPMI media.
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