Abstract
Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC50 = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC50 values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC50 < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC50 = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.
Highlights
Microbial transformation is an effective tool for structural derivatizations that are difficult to achieve by conventional chemical methods
We have reported the biotransformation of several steroids for the synthesis of new anti-inflammatory compounds [31,32,33]
All the compounds were found to be non-cytotoxic against above mentioned cell lines. This is the first report of the fungal transformation of steroidal anti-inflammatory drug medrysone (1) into several new derivatives 2–8 with C. blakesleeana (ATCC 8688a), N. crassa (ATCC 18419), and R. stolonifer (TSY 0471)
Summary
Microbial transformation is an effective tool for structural derivatizations that are difficult to achieve by conventional chemical methods. Microbial systems are extensively employed in the study of drug metabolism and bioremediation [1, 2]. Steroids are among the most widely marketed pharmaceutical products. Several steroids are used as anabolic, contraceptive, anti-androgenic, anti-inflammatory, and anti-cancer agents. Microbial hydroxylation of steroids is an efficient method for the synthesis of new hydroxysteroids with high stereo- and regio-selectivity, and for study of the steroidal metabolism [1, 3,4,5]. The bioconversion of steroids was initiated in 1950. In 1952, progesterone was converted into 11α-hydroxyprogesterone, which was later used as an intermediate in the synthesis of cortisone [6]
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