Structural transformation of methasterone with Cunninghamella blakesleeana and Macrophomina phaseolina

An anabolic-androgenic synthetic steroidal drug, methasterone (1) was transformed by two fungi, Cunninghamella blakesleeana and Macrophimina phaseclina. A total of six transformed products, 6β,7β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (2), 6β,7α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (3), 6α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3,7-dione (4), 3β,6β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-7-one (5), 7α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3-one (6), and 6β,9α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (7) were synthesized. Among those, compounds 2–5, and 7 were identified as new transformed products. MS, NMR, and other spectroscopic techniques were performed for the characterization of all compounds. Substrate 1 (IC50 = 23.9 ± 0.2 μg mL−1) showed a remarkable anti-inflammatory activity against nitric oxide (NO) production, in comparison to standard LNMMA (IC50 = 24.2 ± 0.8 μg mL−1). Whereas, its metabolites 2, and 7 showed moderate inhibition with IC50 values of 38.1 ± 0.5 μg mL−1, and 40.2 ± 3.3 μg mL−1, respectively. Moreover, substrate 1 was found to be cytotoxic for the human normal cell line (BJ) with an IC50 of 8.01 ± 0.52 μg mL−1, while metabolites 2–7 were identified as non-cytotoxic. Compounds 1–7 showed no cytotoxicity against MCF-7 (breast cancer), NCI-H460 (lung cancer), and HeLa (cervical cancer) cell lines.


Introduction
Biotransformation is a cost-effective and robust approach for the production of new and novel biologically active metabolites through living cell cultures of microorganisms, animals, and plants. 1,2 These biological catalysts can trigger multiple reactions, such as hydroxylation, hydrolysis, reduction, 3 aldol and reverse-aldol reactions, 1 esterication, 4 glycosylation, 5 isomerization, epimerization, methylation, 6 Michael addition, epoxidation, and various rearrangement reactions, 7 thus resulting in highly stereo-, regio-, and chemo-selective products. To some extent, the use of toxic chemical catalysts has been reduced by biological catalysts, which are mostly eco-friendly and low-cost enzymes from various organisms. 8 Therefore, biotransformation has become one of the most convincing techniques for the structural modications of existing drugs and chemical intermediates. 9 Methasterone (1) also known as methyldrostanolone due to its synthetic scheme, 10 or trademarked names Methasteron™ and Superdrol™ 11 is an orally active anabolic-androgenic steroid (AAS), used to increase skeletal muscles growth. Consequently, athletes may favour methasterone (1) for its fatburning ability and moderate anabolic properties. [12][13][14] However, its toxicity has been observed in many cases. 11,[15][16][17] Thus methasterone (1) has been banned by World Anti-Doping Agency since 2006, and its use in-and out-of-competition is prohibited. 18 In fact, the potential risk of AAS has become a great challenge due to their toxicity. 19,20 There are several studies on metabolism of methasterone (1) has been reported. 10,18,21,22 In our continuing studies of biotransformation on steroids, 23-31 previously biocatalysis of methasterone (1) was performed by our research group, which resulted into several new anti-inammatory compounds. 32 Based on the previous results of compound 1, biotransformation of 1 was performed again by using Macrophomina phaseolina and Cunninghamella blakesleeana, which have been demonstrated to be effective cell cultures for bioconversion of

Results and discussion
Compound 2 was isolated as a white crystalline solid (Fig. 1   Compound 3 was obtained as a white crystalline solid (Fig. 1). The molecular formula of 3 was deduced to be C 21 H 34 O 4 (M + m/z 350.2439, calcd 350.2457) from HREI-MS. Absorbances in the IR spectrum at 3372 and 1697 cm À1 were due to the presence of hydroxyl and carbonyl ketone groups, respectively. The spectral data of compound 3 was similar to compound 2. The only difference was the orientation of the OH group at C-7. NOESY spectrum displayed a correlation between H-7 (d 3.61) and b-oriented H-8 (d 1.90), but not with a-oriented H-9 (d 1.22) (Fig. 4). Small coupling constant (J) of H-7 (J a/e ¼ 3.0 Hz) in compound 3 also supported a-orientation of OH group, in comparison to coupling constant of H-7 (J a/a ¼ 10.5 Hz; J a/e ¼ 3.5 Hz) in compound 2.
Compound 4 was puried as a white solid (Fig. 1). The molecular composition (C 21 H 32 O 4 ) was evaluated through HREI-MS analysis ([M] + at m/z 348.2305, calcd for 348.2301). The IR absorbances at 3449 and 1700 cm À1 were due to the presence of hydroxyl and carbonyl groups, respectively. 1 H NMR spectrum (Table 1) showed a new downeld signal at d 3.77. In 13 C NMR spectrum (DEPT-135 ), a signal for an oxymethine carbon appeared at d 79.4, which correlated with d 3.77 in the HSQC spectrum. A new signal for a carbonyl group was also observed at d 211.7 in the Broad-brand spectrum ( Table 2) Compound 5 was isolated as a white crystalline solid (Fig. 1). The molecular formula of 5 was deduced to be C 21 H 34 O 4 (M + m/z 350.2454 calcd 350.2457) from HREI-MS analysis. The hydroxyl (3456 cm À1 ) and carbonyl (1701 cm À1 ) groups were identied from the IR spectrum. 1 H NMR spectrum (Table 1) showed two new downeld signals resonating at d 3.10 and 3.67, which correlated with d 77.7 and 81.6 in HSQC, respectively. Beside this, the 13 C NMR spectrum displaced a signal for a new carbonyl carbon at d 215.8 (Table 2) In the IR spectrum, absorbances at 3454 and 1697 cm À1 were due to the presence of hydroxyl and carbonyl groups, respectively. In the 1 H NMR spectrum (Table 1), a new downeld signal was resonated at d 3.64, whereas the 13 C NMR spectrum revealed two new downeld signals at d 71.2 (methine carbon) and 76.6 (quaternary carbon) ( Table 2). An OH group at C-6 (d 71.2) was inferred from HMBC correlations of H-6 (d 3.64) with C-4 (d 43.3), C-7 (d 34.0), C-8 (d 34.4), and C-10 (d 42.5). This was further supported by COSY correlations of H-6 (d 3.64) with H-5  85), indicative of the same transjunction between C-8 and C-9. Therefore, the 9-OH remained the same a-orientation as the substrate 1.
Anti-inammatory activity of all compounds (2-7) was evaluated using a NO production inhibitory assay. Substrate 1 was found to be a potent anti-inammatory agent against nitric oxide (NO) production with an IC 50 of 23.9 AE 0.2 mg mL À1 , when compared to standard LNMMA having an IC 50 ¼ 24.2 AE 0.8 mg mL À1 . While metabolites 2 (38.1 AE 0.5 mg mL À1 ), and 7 (40.2 AE 3.3 mg mL À1 ) exhibited moderate effect on NO production.
Cytotoxicity was determined against MCF-7 (breast cancer), NCI-H460 (lung cancer), HeLa (cervical cancer), and BJ (normal human broblast) cell lines. The results revealed that compound 1 was found to be cytotoxic against human normal cell line (BJ) with an IC 50 of 8.01 AE 0.52 mg mL À1 , but other transformed products (2-7) were found to be non-cytotoxic. Compounds 1-7 were non-cytotoxic against MCF-7, NCI-H460, and HeLa cancer cell lines (having <50% inhibition at 50 mM), compared with the positive control doxorubicin having IC 50 of 1.2 AE 0.11 mg mL À1 against MCF-7, 0.76 AE 0.05 mg mL À1 against NCI-H460, and 0.16 AE 0.01 mg mL À1 against HeLa cell line. It is concluded that the anti-inammatory activity of transformed products was reduced to a certain level, the cytotoxicity was also reduced. Biotransformation was found to be an effective technique for the production of active and safe lead metabolites for the exploration of new drugs.

General protocol
Normal silica gel (100-200 mesh) was used for the initial fractionation using column chromatography (CC). Final purication of compounds was implemented by reverse phase preparative recycling HPLC (Japan Analytical Industry Co., Ltd) on the ODS-H-80 column. Thin-layer chromatography (TLC) was used for the analysis of the compound through pre-coated cards (Silica gel 60 F 254 , Merck, Germany). The NMR spectrum was recorded on a Bruker Advance spectrometer of 400, 500, and 600 MHz in methanol (deuterated). EI-MS and HREI-MS was performed on JEOL JMS-600H (Japan), and JEOL JMS HX-110, respectively. UV (Ultraviolet) spectra were recorded in methanol on Shimadzu UV 240 spectrophotometer (Tokyo, Japan). IR (Infrared) absorbance was recorded on an FTIR-8900 spectrophotometer as a KBr disk. Melting points were determined by Buchi 535 apparatus.

Microorganisms and preparation of medium
Cunninghamella blakesleeana and Macrophomina phaseolina (KUCC 730) were acquired from American Type Culture Collection (ATCC) and Karachi University Culture Collection (KUCC). Sabouraud dextrose agar (Agar slant) was used to grow the fungi at 25 C, and maintained at 4 C.

General fermentation protocol
Previously, Otten & Rosazza reported the general fermentation procedure used during biotransformation studies. 34 In small scale screening, liquid media of 400 mL was prepared for each fungus. Fermentation media was shied among four 250 mL conical asks (100 mL in each), and then autoclaved at 120 C   for sterilization. Out of four asks, two asks were arranged as positive (substrate + fermentation media), one ask as negative (mature fungal growth in media), and one ask used as a seed ask. The fungus spores were added into each ask under sterilized conditions and kept for 3 to 4 days on an incubator shaker (128 rpm) at 24 AE 2 C. Aer the mature growth of fungi, the substrate (20 mg for each positive ask) was dissolved in methanol and transferred to the fungus culture. All asks were again put on a shaker for fermentation. Aer the denite incubation period (7 days and 14 days for two asks, respectively), the process of fermentation was stopped by adding ethyl acetate. The sporangia were separated by ltration and extracted with ethyl acetate 3-4 times. The organic phase was isolated by a separation funnel, and then concentrated on a rotatory evaporator. The extract was analyzed by TLC.

Biotransformation with Cunninghamella blakesleeana
Based on the small scale screening, 10 L media was prepared by mixing the above-mentioned components in water for Cunninghamella blakesleeana growth. It was dispersed among 6 conical asks (3 L size with 1.5 L media in each), and then autoclaved at 120 C for sterilization of media. The media was cooled down until room temperature and then incubated with seeds of C. blakesleeana under pasteurized conditions. The conical asks were kept on a shaker (128 rpm) at 24 AE 2 C for 3-5 days for fungi growth. Aer the mature growth, 1.0 g of methasterone (1) was dissolved in methanol (9 mL), then distributed to 6 asks, and again placed on a shaker (128 rpm) at 24 AE 2 C for 14 days. Then ethyl acetate was added to stop the fermentation, and biomass was ltered. The ltrate was extracted thrice using ethyl acetate, and then the organic phase was concentrated on a high vacuum rotary evaporator. The gummy crude extract (2.0 g) was obtained and then subjected to silica gel CC through gradient elution with hexane/acetone by increasing polarity (5-100% acetone). The fractions were combined based on TLC analysis, and three major fractions  Table 1; 13 C NMR (CD 3 OD, 125 MHz) Table  2.
Cytotoxicity assay MCF-7 (breast cancer), NCI-H460 (lung cancer), HeLa (cervical cancer) cell lines, and BJ (normal human broblast) cell lines were collected from cell culture biobank (PCMD, ICCBS). DMEM medium was used for MCF-7, HeLa, and BJ, and RPMI (ATCC modied medium) was used for NCI-H460. The media was supplemented with 10% FBS, 100 IU mL À1 of penicillin, and 100 mg mL À1 of streptomycin, and kept at 37 C in a 5% CO 2 incubator. The inhibition was investigated by MTT assay. Briey, 100 mL per well of cell solutions (10 Â 10 4 cells per mL MCF-7 cells, 5 Â 10 4 cells per mL NCI-H460 cells, 6 Â 10 4 cells per mL HeLa cells, or 5 Â 10 4 cells per mL BJ cells), were added into 96-well plate and incubated for 24 h at 37 C, before treatment with 6.25-50 mM of compounds or positive control doxorubicin for 48 h. Aer this, 20 mL of MTT (5 mg mL À1 ) was added to each well and then incubated for 4 h at 37 C. Then 100 mL DMSO was added to each well to dissolve the purple formazan crystal. The absorbance was analyzed at 570 nm (MCF-7, NCI-H460, and HeLa) or 550 nm (BJ) using an ELISA plate reader. The IC 50 value of active compounds (inhibition > 50% at 50 mM) was calculated using the EZ-Fit. The unit of mM was nally converted to mg mL À1 to be comparable with anti-inammatory activity.

Conclusion
In conclusion, methasterone (1) was effectively transformed by two fungi, C. blakesleeana and M. phaseolina, which led to the isolation and elucidation of seven transformed products (2-7). Oxidation and hydroxylation were the main routes of transformation. In comparison to standard LNMMA (IC 50 ¼ 24.2 AE 0.8 mg mL À1 ), substrate 1 (IC 50 ¼ 23.9 AE 0.2 mg mL À1 ) was found to be a potent anti-inammatory agent against nitric oxide (NO) production. Metabolites 2 and 7 were moderately active with IC 50 values of 38.1-40.7 mg mL À1 . Substrate 1 displayed cytotoxicity on the human normal cell line (BJ) with an IC 50 of 8.01 AE 0.52 mg mL À1 . While all its transformed products are noncytotoxic. Although the effect of the analogues was not as strong as the parent molecule, the cytotoxicity of the products aer the fungal transformation has been reduced signicantly. Therefore, biotransformation is not only an effective approach to discover potent leads for new drug discovery, but also a reliable method to decrease the toxicity of bioactive molecules.

Conflicts of interest
There are no conicts to declare.