New anti-inflammatory and non-cytotoxic metabolites of methylstenbolone obtained by microbial transformation

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 (IC₅₀ 10.1 ± 0.3 µg/mL) was identified as a potent anti-inflammatory agent against nitric oxide (NO) production. Its transformed products 3 (IC₅₀ as 27.8 ± 1.1 µg/mL) and 9 (26.9 ± 0.4 µg/mL) displayed good inhibition. Compounds 2 (IC₅₀ = 45.9 ± 0.8 µg/mL) and 6 (IC₅₀ = 36.6 ± 1.2 µg/mL) were also active moderately against NO production, in comparison to standard LNMMA (IC₅₀ = 24.2 ± 0.8 µg/mL). Cytotoxicity assay showed 1 was active to cancer cell line MCF7 (IC₅₀ = 12.26 ± 0.35 µg/mL), compared to the standard Doxorubicin having IC₅₀ as 1.25 ± 0.11 µg/mL. However, it is also toxic to human normal cell line (BJ) with IC₅₀ as 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.