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Glomerella fusarioides-mediated structural modifications of steroidal anabolic drug, oxandrolone (1), yielded a new product 17β,11α-dihydroxy-17α-methylandrosta-2-oxa-4-ene-3-one (2). Similarly, Cunninghamella elegans-catalyzed transformation of a steroidal anti-epileptic drug ganaxolone (3) afforded a new metabolite 14α-hydroxy-5α-pregnan-1-ene-3,20-dione (4). Structures of new metabolites 2 and 4 were deduced via 1D-, and 2D-NMR, HREI-, HRFAB-MS, and IR spectroscopic techniques. New derivative 2 showed a remarkable anti-aromatase activity with an IC50 = 0.6 ± 0.005 μM, when compared to substrate 1 (IC50 = 0.808 ± 0.07 μM), and standard anti-cancer drug exemestane (IC50 = 0.232 ± 0.031 μM). Ganaxolone (3) (IC50 = 13.76 ± 2.00 μM) also showed a good activity against human aromatase enzyme, while its derivative 4 was found to be inactive. Inhibition of aromatase enzyme is a key approach in the treatment of estrogen positive breast cancers. Compounds 1-4 were non-cytotoxic to 3T3 cell line (mouse fibroblast).

Graphical abstract, Seven new derivatives, 6α-hydroxy-1-methyl-3-oxo-5α-androst-1-en-17-yl acetate (2), 6α,17β-dihydroxy-1-methyl-3-oxo-5α-androst-1-en (3), 7β-hydroxy-1-methyl-3-oxo-5α-androst-1-en-17-yl acetate (4), 15β,20-dihydroxy-1-methyl-3-oxo-5α-androst-1-en-17-yl acetate (5), 15β-hydroxy-1-methyl-3-oxo-5α-androst-1-en-17-yl acetate (6), 12β,17β-dihydroxy-1-methyl-3-oxoandrosta-1,4-dien (11), and 7β,15β,17β-trihydroxy-1-methyl-3-oxo-5α-androst-1-en (14), along with six known metabolites, 17β-hydroxy-1-methyl-3-oxoandrosta-1,4-dien (7), 17β-hydroxy-1-methyl-3-oxo-5α-androst-1-en (8), 17β-hydroxy-1-methyl-3-oxo-5β-androst-1-en (9), 1-methyl-5β-androst-1-en-3,17-dione (10), 1-methyl-3-oxoandrosta-1,4-dien-3,17-dione (12), and 17β-hydroxy-1α-methyl-5α-androstan-3-one (13) of metenolone acetate (1), were synthesized through whole-cell biocatalysis with Rhizopus stolonifer, Aspergillus alliaceous, Fusarium lini, and Cunninghamella elegans. Atamestane (12), an aromatase inhibitor, was synthesized for the first time via F. lini-mediated transformation of 1 as the major product. Hydroxylation, dehydrogenation, and reduction were occurred during biocatalysis. Study indicated that F. lini was able to catalyze dehydrogenation reactions selectively. Structures of compounds 1–14 were determined through NMR, HRFAB-MS, and IR spectroscopic data. Compounds 1–14 were identified as non-cytotoxic against BJ human fibroblast cell line (ATCC CRL-2522). Metabolite 5 (81.0 ± 2.5%) showed a potent activity against TNF-α production, as compared to the substrate 1 (62.5 ± 4.4%). Metabolites 2 (73.4 ± 0.6%), 8 (69.7 ± 1.4%), 10 (73.2 ± 0.3%), 11 (60.1 ± 3.3%), and 12 (71.0 ± 7.2%), also showed a good inhibition of TNF-α production. Compounds 3 (IC50 = 4.4 ± 0.01 µg/mL), and 5 (IC50 = 10.2 ± 0.01 µg/mL) showed a significant activity against T-cell proliferation. Identification of selective inhibitors of TNF-α production, and T-cell proliferation is a step forward towards the development of anti-inflammatory drugs.

A synthetic anabolic-androgenic steroid, methylstenbolone (1), was structurally transformed into a series of nine analogues, 2,17α-dimethyl-7α,17β-dihydroxy-5α-androst-1-en-3-one (2), 2,17α-dimethyl-15β,17β-dihydroxy-5α-androst-1-en-3-one (3), 2,17α-dimethyl-6α,9α,17β-trihydroxy-5α-androst-1-en-3-one (4), 2-methyl-17β-hydroxy-17α-(hydroxymethyl)-5α-androst-1-en-3-one (5), 2-methyl-11β,17β-dihydroxy-17α-(hydroxymethyl)-5α-androst-1-en-3-one (6), 2-methyl-17β-hydroxy-17α-(hydroxymethyl)-5α-androst-1-en-3,6-dione (7), 2-methyl-17β-hydroxy-17α-(hydroxymethyl)-5β-androst-1-en-3,6-dione (8), 2,17α-dimethyl-7β,17β-dihydroxy-5α-androst-1-en-3-one (9), and 2,17α-dimethyl-12β,17β-hydroxy-5α-androst-1-en-3,7-dione (10) by fungal cell suspension cultures, Macrophomina phaseolina and Cunninghamella blakesleeana for the first time. Among those, compounds 2-4 and 6-10 were identified as new. Herein, spectral data of metabolite 5 was reported for the first time. Their structures were elucidated by NMR, MS, UV, and IR spectroscopic methods. Substrate 1 (ICsub50/sub10.1 ± 0.3 µg/mL) was identified as a potent anti-inflammatory agent against nitric oxide (NO) production. Its transformed products 3 (ICsub50/subas 27.8 ± 1.1 µg/mL) and 9 (26.9 ± 0.4 µg/mL) displayed good inhibition. Compounds 2 (ICsub50/sub = 45.9 ± 0.8 µg/mL) and 6 (ICsub50/sub = 36.6 ± 1.2 µg/mL) were also active moderately against NO production, in comparison to standard LNMMA (ICsub50/sub = 24.2 ± 0.8 µg/mL). Cytotoxicity assay showed 1 was active to cancer cell line MCF7 (ICsub50/sub = 12.26 ± 0.35 µg/mL), compared to the standard Doxorubicin having ICsub50/subas 1.25 ± 0.11 µg/mL. However, it is also toxic to human normal cell line (BJ) with ICsub50/subas 8.69 ± 0.02 µg/mL. More importantly, all transformed products are non-cytotoxic on BJ. Therefore, biotransformation can be an efficient approach to reduce the toxicity of methylstenbolone.

Cunninghamella blakesleeana-mediated biotransformation of an oral contraceptive drug, levonorgestrel (1), yielded a new metabolite, 13β-ethyl-17α-ethynyl-10,17β-dihydroxy-4,6-dien-3-one (2), and two known metabolites 3 (13β-ethyl-17α-ethynyl-10β,17β-dihydroxy-4-en-3-one), and 4 (13β-ethyl-17α-ethynyl-6β,17β-dihydroxy-4-en-3-one) at an ambient temperature using aqueous media. Hydroxylation and dehydrogenation of compound 1 was observed during the bio-catalytic transformation. The structure of a new metabolite 2 was determined by 1H, 13C, and 2DNMR and HR-EIMS spectroscopic techniques.

Although the discovery of antibiotics has decreased the spread and severity of infectious diseases, their uncontrolled use has lead to the emergence of bacterial resistance to existing chemotherapeutic agents. Bacterial disease thus remains a challenge for health authorities in worldwide and especially in sub-Saharan Africa. Despite their efficacy, the miss-use of medicinal plants for the treatment of infectious diseases couple to the farming and hunting activities has contribute enormously to the destruction of many medicinal plant species. In search of an alternative for new and effective agents against bacterial infection, norandrostenedion (19-nor-4-androsten-3,17-dione) (1), was biotransformed by Cunninghamella blakesleeana ATCC 8688A and yielded a new metabolite, 6α,10 β -dihydroxy-19-nor-4-androsten-3-one (2), together with three known compounds, 10 β -hydroxy-19-nor-4-androsten-3,17-dione (3), 6 β,10 β,17 β -trihydroxy-19-nor-4-androsten-3-one (4) and 10 β,17 β -dihydroxy-19-nor-4-androsten-3-one (5). Their structures were elucidated on the basis ofspectroscopic techniques: NMR analysis (1D and 2D) and HRIE-MS and by comparison with previously reported data. In addition, the agar diffusion method was used to evaluate the diameter of the inhibition zone and INT colorimetric assay for MIC values. All metabolites obtained showed a potent and varied activity against tested bacteria. These results support the uses of biotransformation to develop new antimicrobial compounds for clinical application.

Biotransformation of an orally active contraceptive drug, desogestrel (1), with Cunninghamella elegans yielded a new metabolite, 13β-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-17β-ol-3,6-dione (2), along with five known metabolites, i.e., 13β-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3β,6β,17β-triol (3), 13β-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,17β-diol-3-one (4), 13β-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-17β-ol-3-one (5), 13β-ethyl-11-epoxy-18,19-dinor-17α-pregn-4-en-20-yn-17β-ol-3-one (6), and 13β-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-10β,17β-diol-3-one (7). The structure of new metabolite 2 was elucidated by using 1H-, 13C-, and 2D-NMR, EI-, and HREI-MS, IR, and UV spectroscopic data. Compounds 1–7 were evaluated for anti-inflammatory activities, i.e., inhibition of T-cell proliferation, and pro-inflammatory cytokine (TNF-α). Compounds 1 (IC50 = 1.12 ± 0.03 µg/mL), 2 (IC50 = 1.15 ± 0.05 µg/mL), 3 (IC50 = 1.15 ± 0.05 µg/mL), 4 (IC50 = 1.40 ± 0.03 µg/mL), 5 (IC50 = 1.78 ± 0.08 µg/mL), and 6 (IC50 = 1.36 ± 0.07 µg/mL) were identified as potent inhibitors of T-cells proliferation, in comparison to the standard drug, prednisolone (IC50 = 3.51 ± 0.03 µg/mL). Compound 7 (IC50 = 6.18 ± 0.04 µg/mL) showed a good activity. In addition, substrate 1 (IC50 ≤ 1 µg/mL), and its metabolites 2 (IC50 = 4.1 ± 0.60 µg/mL), and 6 (IC50 = 6.8 ± 0.8 µg/mL) also showed a potent inhibition of pro-inflammatory cytokine (TNF-α) production, as compared to the standards drug, pentoxifilline (IC50 = 94.8 ± 2.1 µg/mL). Whereas compounds 3 (IC50 = 57.9 ± 7.6 µg/mL), and 5 (IC50 = 27.2 ± 6.8 µg/mL) showed a moderate inhibition of TNF-α production, while compounds 4 and 7 showed no inhibition. Compounds 1–7 were found to be non-cytotoxic to 3T3 normal cell line (mouse fibroblast).

International audience; Staphylococcus aureus is one of the most infectious agents among staphylococcal bacteria. Currently many strains of S. aureus have developed resistance against available antibiotics. Therefore, the treatment of infections caused by them is a major challenge. During current study, desogestrel (1), a contraceptive drug, was found to be a potent growth inhibitor of drug resistant strains of S. aureus. Therefore, in search of new and effective agents against multi-drug resistant S. aureus strains, whole-cell bio-catalytic conversion of desogestrel (1) by Cunninghamella blakesleeana ATCC 8688A at pH 7.0 and 25 °C was carried out, yielding three new metabolites, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,15β,17β-triol (2), 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3β,6β,17β-triol (3), and 13-ethyl-11-methylene-18,19-dinor-17α-pregn-20-yn-3α,5α,6β,17β-tetraol (4), along with a known metabolite, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,17β-dihydroxy-3-one (5). Among them, compounds 1–2 showed a potent activity against S. aureus EMRSA-17, S. aureus NCTC 13277 (MRSA-252), and S. aureus NCTC 13143, and clinically isolated Pakistani strain of S. aureus in an in vitro Microplate Alamar Blue Assay (MABA). Vancomycin was used as the standard drug in this assay. In addition, compound 1 also showed a significant activity against vancomycin-resistant S. aureus (VRSA) ATCC 700699. Compounds 1–5 were also evaluated against 3T3 normal cell line (mouse fibroblast) where they all were identified as non-cytotoxic. The present study thus provides new leads for the development of anti-bacterial drugs against MDR S. aureus.

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.