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, 2,17 alpha-dimethyl-7 alpha,17fl-dihydroxy-5 alpha-androst-1-en-3-one (2), 2,17 alpha-dimethyl-15fl,17fl-dihydroxy-5 alpha-androst-1-en-3-one (3), 2,17 alpha-dimethyl-6 alpha,9 alpha,17fl-trihydroxy-5 alpha-androst-1-en-3-one (4), 2-methyl-17fl-hydroxy-17 alpha-(hydroxymethyl)-5 alpha-androst-1-en-3-one (5), 2-methyl-11fl,17fl-dihydroxy-17 alpha-(hydroxymethyl)-5 alpha-androst-1-en-3-one (6), 2-methyl-17fl-hydroxy-17 alpha-(hydroxymethyl)-5 alpha-androst-1-en-3,6-dione (7), 2-methyl-17fl-hy-droxy-17 alpha-(hydroxymethyl)-5fl-androst-1-en-3,6-dione (8), 2,17 alpha-dimethyl-7fl,17fl-dihydroxy-5 alpha-androst-1-en-3-one (9), and 2,17 alpha-dimethyl-12fl,17fl-hydroxy-5 alpha-androst-1-en-3,7-dione (10) by fungal cell suspension cul-tures, 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 mu g/mL) was identified as a potent anti-inflammatory agent against nitric oxide (NO) production. Its transformed products 3 (IC50 as 27.8 +/- 1.1 mu g/mL) and 9 (26.9 +/- 0.4 mu g/mL) displayed good inhibition. Compounds 2 (IC50 = 45.9 +/- 0.8 mu g/mL) and 6 (IC50 = 36.6 +/- 1.2 mu g/mL) were also active moderately against NO production, in comparison to standard LNMMA (IC50 = 24.2 +/- 0.8 mu g/mL). Cytotoxicity assay showed 1 was active to cancer cell line MCF7 (IC50 = 12.26 +/- 0.35 mu g/mL), compared to the standard Doxorubicin having IC50 as 1.25 +/- 0.11 mu g/mL. However, it is also toxic to human normal cell line (BJ) with IC50 as 8.69 +/- 0.02 mu g/mL. More importantly, all transformed products are non-cytotoxic on BJ. Therefore, biotransformation can be an efficient approach to reduce the toxicity of methylstenbolone.