Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites

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.


Introduction
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.It is stable to metabolic conversion in the rat ventral prostate.Because of its stability and high affinity binding, it has been used as a ligand for the characterization and quantitation of androgen in prostate, liver, and cultured cells.In addition, mibolerone (1) is more receptor-selective for androgenic receptor than methyltrienolone.The binding interaction of compound 1 with testosterone-estradiol binding globulin of human serum is weaker than 5α-dihydrotestosterone.Mibolerone (1) also acts through the progesterone receptor (PR) as it eliminates progesterone receptor expression at lower doses (1 nM), in contrast to 5α-dehydrotestosterone (10-100 nM), which reduces PR to basal levels.In breast cancer cells, mibolerone (1) has shown a dual action, i.e., androgenic and progestagenic [1][2][3][4].
The regio-, chemo-, and stereo-selective synthesis of organic compounds has been an area of active research since several decades.Many of these conversions are difficult to achieve through conventional synthetic methodologies.However, biocatalysts can carry out these reactions effectively.Biocatalysis has several advantages over chemical synthesis, such as selectivity, mild reaction conditions, and their eco-friendly nature.Various biocatalysts, such as pure enzymes and whole-cell systems, are being used for the transformation of organic compounds.However, whole-cell biocatalysis, especially with fungi, is an efficient choice for regio-and stereo-selective transformations [5][6][7][8][9], as they have P450 cytochrome enzyme systems, which catalyze hydroxylation at various positions of steroids [10][11][12].
β-Glucuronidase is an inducible exoglycosidase enzyme.Its increased level in blood can create problems in the detoxification process of various toxic substances.Toxic carcinogenic substances, along with endogenously produced toxic metabolites such as steroids, are metabolized in the liver.Before their excretion into the small intestine via the bile, these substances undergo conjugation with glucuronic acid.β-Glucuronidase, produced by intestinal bacteria, catalyzes the hydrolysis of these conjugates in the colon.Increased activity of β-glucuronidase is one of the key observations in colon cancer.Hence, β-glucuronidase plays a key role in the etiology of colon cancer [18][19][20][21][22].
Leishmaniasis, a neglected tropical disease (NTD), is a major vector borne disease of protozoal origin.Nearly 350 million people in 88 countries are at risk of the disease.Orally available drugs for the treatment of leishmaniasis are very few and often less effective.Therefore, development of safe and effective new therapeutic agents for leishmaniasis is urgently needed to reduce the burden of the disease [23][24][25].
After breast cancer, cervical cancer is the second most prevalent cancer in women across the world.In this cancer, malignant cells are formed in tissues of the cervix.Currently, radiotherapy, surgery, and most commonly, cisplatin based chemotherapic agents are used for the treatment of cancers.However, the response rate to chemotherapy is often very low due to frequent development of resistance of cancer cells against chemotherapeutic agents.HeLa cell line, obtained from human cervical cancer, provide a useful model to evaluate the cytotoxic potential of chemical compounds against cervical cancer in vitro [26][27][28][29].

Chromatographic protocols
The purity of compound 1 and the degree of its transformations were analyzed by TLC (Thin layer chromatography) (silica gel, 20×20, 0.25 mm thick, PF 254, Merck, Germany), while silica gel (70-230 mesh, Merck, Germany) was used for column chromatography.Compounds were finally purified on a recycling HPLC (JAI LC-908W, Japan), equipped with YMC L-80 (4-5 μm, 20−50 mm i.d.).Ceric sulphate reagent was used for visualizing the compounds on TLCs.All solvents used for chromatography were of analytical grade.Fungal biotransformation of mibolerone, and biological activity evaluation of its metabolites Instrumental analysis 1 H-(400, 500, and 600 MHz), and 13 C-NMR (100, 125, and 150 MHz) and 2D-NMR spectra were recorded on Bruker Avance-NMR spectrometers (France) in CD 3 OD, CD 3 COCD 3 or DMSO-d 6 .Melting points were recorded on Buchi M-560 apparatus (Japan).EI-and HREI-MS were recorded on JEOL JMS-600H (Japan).Optical rotations of all isolated compounds were measured on JASCO P-2000 polarimeter (Japan) in chloroform or methanol.IR analyses were performed on Bruker Vector 22 FT-IR spectrometer (France).Evolution 300 UV-visible spectrophotometer was used to record the UV spectra (Thermo Scientific, England).Single-crystal X-ray diffraction data was collected on Bruker APEXII D8 Venture diffractometer, fitted with PHO-TON 100 detector (CMOS technology), and fine-focus sealed tube having X-ray source [Cu Kα radiation α = 1.54178Å].Reflection intensities were integrated using SAINT software.Absorption correction was done on M-multi-scan.Structures were solved on SHELXTL [30][31].
Crystallographic data for compounds 1, 2, 4, and 8 were deposited with the Cambridge Crystallographic Data Center and can be obtained free of charge from the Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif.

General fermentation protocol
The ingredients used for 1 L culture medium were comprised 10 g glucose, 5 g peptone, 5 g KH 2 PO 4 , 5 g yeast extract, 5 g NaCl, and 10 mL glycerol in 1 L of distilled water.
The aforementioned ingredients were used to prepare the culture medium for the growth of C. blakesleeana, C. echinulata, and M. phaseolina.The experiments were carried out on two scales, i.e., the experimental and the preparative scales.In the experimental scale, 600 mL media was prepared for each fungus, transferred to 6 flasks of 250 mL, and autoclaved.Two flasks served as positive (fungal media + substrate) and negative (fully grown fungus in media) controls, whereas the remaining four flasks were used as test flasks.The fungi were grown in test flasks and negative control by transferring its spores.After mature growth of the fungi, 20 mg of the substrate was dissolved in 0.5 mL of methanol and incubated in each culture-containing test flask.One test flask was harvested every 4 th day, followed by filtration of the mycelia, and extraction with dichloromethane (DCM).Based on the small scale screening, substrate 1 was subjected to preparative scale transformation.
Four liters of liquid media was prepared for each fungus, and distributed equally in 40 flasks of 250 mL each.The media was then autoclaved and inoculated with spores of the appropriate fungus at 22˚C.After 4 days of inoculation, fungus cultures were found to be fully matured.Substrate 1 (600 mg) was dissolved in 20 mL methanol, and dispensed equally (0.5 mL in each) in all flasks.These flasks were then placed on a rotatory shaker for 12 days at 22˚C.

Extraction of metabolites
On the12 th day, the content of all flasks of each fungus were combined and filtered to remove the mycelia.The aqueous layer was extracted thrice with CH 2 Cl 2 (24 L).The crude extract was made moisture free by adding sodium sulphate, filtered, and concentrated on a rotary evaporator, which yielded a thick brown gum like material.

Isolation of metabolites of mibolerone (1) from the incubation of mibolerone (1) with Cunninghamella blakesleeana, and C. echinulata
Incubation of substrate 1 with C. blakesleeana, and C. echinulata yielded about 2 g of crude extracts.The extracts were subjected to silica gel column chromatography.The mobile phase comprised hexanes-acetone mixtures.The polarity of the mobile phase was increased by increasing the concentration of acetone (5-100% gradient of acetone).500 mL of solvent system at each concentration was passed through the column.The fractions obtained were analyzed by TLC analysis, and the fractions of similar composition were pooled together.Three main fractions (1-3) were obtained, which were then purified on a reverse phase recycling HPLC.

Assay protocol for β-glucuronidase inhibition
The inhibition of β-glucuronidase enzyme (E.C. 3.2.1.31,bovine liver) by test compounds was determined on a spectrophotometer by measuring the absorbance of p-nitrophenol at 405 nm, produced from the substrate.The reaction mixture comprised 185 μL of 0.1 M acetate buffer, and 5 μL of test compound solution, and 10 μL of (1U) enzyme solution in a 96-well plate.The mixture was incubated at 37˚C for 30 min.The test compounds were solubilized in DMSO (100%), and 5 μL volume was added in each well (2% of total volume).Similar conditions were used for the standard (D-saccharic acid 1,4-lactone).The plates were read on a multiplate reader (SpectraMax plus 384) at 405 nm and 37˚C, after addition of 50 μL of 0.4 mM p-nitrophenyl-β-D-glucuronide.All assays were performed in triplicate.IC 50 values were calculated through EZ-Fit software (Perrella Scientific Inc., Amherst, MA, USA).

Assay protocol for leishmanicidal activity
Leishmania major (DESTO, Pakistan) was cultured in a mixture of NNN-biphasic medium, and normal physiological saline solution.L. major promastigotes were grown on RPMI 1640 medium, supplemented with fetal bovine serum (FBS) (10% heat inactivated).Parasites were centrifuged (at log phase) for 10 min at 2,000 rpm and washed thrice with saline.The final density of fresh culture (106 cells/mL) was acquired through dilution of parasites.Sample solution of 1 mg of test compounds was prepared in a mixture 50 μL of DMSO and 950 μL of RPMI media.Fully dissolved compounds were transferred to 96-well plate.The first row of 96-well plate received 180 μL of the medium, while the remaining wells received 100 μL medium.Test compounds (20 μL each) were added into the medium containing wells, followed by serial dilution.The parasite culture (100 μL) was then transferred to each well.Negative controls comprised of only the growth medium, while standard leishmanicidal drugs (amphotericin or pentamidine) were used as positive controls.The plate was then incubated for 72 h at 21-22˚C.The inhibitory effect of test compounds on the culture was analyzed microscopically on an improved Neubauer counting chamber.IC 50 values were calculated through Ezfit 5.03 Perella Scientific software (USA).

Assay protocol for cytotoxicity
MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay was employed to study the cytotoxic activity against HeLa (human cervical carcinoma, provided by Prof. Dr. Anwar Ali Siddiqui from Aga Khan University, Karachi, Pakistan), and mouse fibroblast 3T3 (ATCC 1 CRL-1658, purchased from American Type Culture Collection, ATCC, Virginia, USA) cell lines.The cells (1 × 10 5 /well) were plated in 0.2 mL of DMEM high glucose medium/well in 96-well plates.The cells were treated for 24 hours with test compounds in the range of 25, 50, 75, 100, and 200 μM concentrations, respectively.For the MTT assay, the medium from the wells was removed carefully after 24 hours treatment.Each well was washed with 1X PBS for 2-3 times, and 200 μL of MTT (5 mg/mL) was added in to media containing wells (1:10).The plates were incubated for 4 hours at 37˚C, in a 5% CO 2 incubator.After incubation, MTT was removed and 0.1 mL of DMSO was added to each well and mixed by keeping on a stirrer.The presence of viable cells was visualized by the development of purple color due to formation of the formazan crystals.The plates were observed under spectrophotometer (Spectramax) and absorbance was taken at 545 and 570 nm for cancer and normal cell lines, respectively.Measurements were performed and the concentration required for a 50% inhibition of viability (IC 50 ) was determined graphically.
Leishmanicidal activity.Mibolerone (1) and its metabolites 2-4, and 8 were assessed for leishmanicidal activity.Substrate 1 showed a significant leishmanicidal activity against the promastigotes of Leishmania major with IC 50 value of 29.64 ± 0.  showed only weak activity against the promastigotes of Leishmania major, while compounds 4 and 5 were found to be inactive (Table 4).5).

Conclusion
In conclusion, microbial transformations of mibolerone (1)    Substrate 1 was found to be significantly active against β-glucuronidase enzyme, leishmaniasis, and HeLa cancer, and 3T3 normal cell lines in vitro.Metabolites 2, and 8 were found to be potently active against HeLa cancer cell line, while metabolites 3, and 4 were weakly active.Metabolites 2-4 were toxic to 3T3 cell line, whereas metabolite 8 showed no cytotoxicity against 3T3 cell line.In addition, metabolites 3 and 8 also showed weak leishmanicidal activity in vitro against the promastigotes of Leishmania major.The presented study indicated that compound 8 deserves to be further studied for its therapeutic potential.