In conclusion, the biotransformation of exemestane (1) with F. lini and M. phaseolina were investigated for the first time which provided an efficient route towards the synthesis of several new metabolites 2–5. Metabolite 2 was found to be moderately active against both cancer cell lines (HeLa and PC3). The work presented here can be helpful for the study of in vivo metabolism of exemestane (1), as well as for the discovery of new anticancer drugs
Substrate and chemicals
Exemestane (1) was purchased from local market as drug (Pfizer Canada Inc., Brand name Aromasin), extracted and further purified by flash chromatography. Thin layer chromatography (TLC) was carried out on silica gel precoated plates (PF254; Merck). Column chromatography (CC) was performed by using silica gel (E. Merck, Germany). Optical rotations were measured in methanol with a JASCO P-2000 polarimeter. 1H- and 13C-NMR spectra were recorded in (CD3)2CO and CD3OD on Bruker Avance spectrometers. The chemical shifts (δ values) are presented in ppm and the coupling constants (J) are in Hz. For 1D- and 2D-NMR experiments, standard Bruker pulse sequences were used. UV Spectra (in nm) were recorded in methanol with a Hitachi U-3200 spectrophotometer. Infrared (IR) spectra (in cm-1) were recorded with an FT-IR-8900 spectrophotometer. JEOL (Japan) JMS-600H mass spectrometer was used for recording of EI-MS and high-resolution mass spectra (HREI-MS) in m/z (rel. %).
The anticancer activity of compounds 2-5 was evaluated in 96-well flat-bottomed micro-titer plates [Iwaki, Japan] by using the standard dye MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) [Sigma-Aldrich Chemicals, St. Louis, USA] through colorimetric analysis. For this purpose, the cells were cultured in Minimum Essential Medium (MEM), supplemented with 10% Fetal Calf Serum (FCS), 1 mmol/l sodium pyruvate, 1% (v/v) antibiotic / antimycotic and passaged weekly, using 0.25% trypsin / EDTA [Sigma-Aldrich Chemicals, St. Louis, USA] in tissue culture flasks T-75 [Iwaki, Japan]. Absorbance was taken at 540 nm wavelength by using microplate reader (Spectra Max plus, Molecular Devices, USA) using software SoftMax Pro 340 [Molecular Devices, CA, USA]. Dimethyl sulfoxide (DMSO) and doxorubicin (standard inhibitor) were purchased from Sigma-Aldrich Chemicals, [St. Louis, USA].
Microorganisms and culture medium
The fungi were purchased from the Northern Regional Research Laboratories (NRRL), or obtained as gift from the Karachi University Culture Collection (KUCC).
Fusarium lini (NRRL 2204), and Macrophomina phaseolina (KUCC 730) were grown in a culture medium prepared by mixing glucose (40.0 g), glycerol (40.0 mL), peptone (20.0 g), yeast extract (20.0 g), KH2PO4 (20.0 g), and NaCl (20.0 g) in distilled H2O (4.0 L).
PC3 (prostate cancer) and HeLa (cervical cancer) cell lines were purchased from the American Type Culture Collection (ATCC) for anticancer activity.
General Fermentation and Extraction Conditions
4 Liters fungal media was prepared and distributed into 40 conical flasks (100 mL in each flask). All flasks were then autoclaved at 121°C. The fungal cultures were then inoculated into each flask containing media and incubated at room temperature on shaker for three days. Compound 1 was dissolved in 40 mL methanol and distributed equally to all 40 flasks. All experimental flasks were then kept for fermentation. Two control experiments, i.e. media + compound 1 and media + fungus were also conducted. The transformation was then checked on TLC. After the detection of transformation on TLC, fungal culture from all 40 flasks was filtered and extracted with CH2Cl2 (12 L) by using liquid-liquid chromatography. The dichloromethane layer was evaporated in vaccue. The obtained gum was analyzed by thin-layer chromatography.
Fermentation and Purification of Exemestane (1) with Fusarium lini
Exemestane (1; 1.0 g) was dissolved in 40 mL methanol, and incubated with culture of F. Lini. The obtained gum (2.3 g) was fractionated (ARC 1-3) by using silica gel column chromatography. The mobile phase was composed of petroleum ether and acetone with a gradient of 10%. Fraction ARC-2 yielded metabolite 2 (4 mg, pet. ether: acetone = 8:2) after elution through silica gel column.
Amorphous material; [α]25D: +81.4 (c = 0.096, MeOH); IR (KBr): νmax 3408, 1657 cm-1; UV (MeOH): λmax nm (log ε) 247 (3.78); 1H- and 13C-NMR: see Tables 1 and 2 (Additional file 1).
Fermentation and Purification of Exemestane (1) with Macrophomina phaseolina
Incubation of 1 (1.0 g / 40 mL methanol) with 3 days old culture of M. phaseolina in 40 flasks for 12 days produced the metabolites 3 (10 mg), 4 (5 mg) and 5 (6 mg).
16β, 17β-Dihydroxy-6-methylene-androsta-1,4-diene-3-one (3)
Amorphous material; [α]25D: +181.6 (c = 0.032, MeOH); IR (KBr): νmax 3388, 1658 cm-1; UV (MeOH): λmax nm (log ε) 249 (4.03); 1H- and 13C-NMR: see Tables 1, and 2 (Additional file 2).
Amorphous material; [α]25D: -56.0 (c = 0.043, MeOH); IR (KBr): νmax 3411, 1749, 1658 cm-1; UV (MeOH): λmax nm (log ε) 247 (4.04); 1H- and 13C-NMR: see Tables 1 and 2 (Additional file 3).
Amorphous material; [α]25D: +174.5 (c = 0.046, MeOH); IR (KBr): νmax 3421, 1657, cm-1; UV (MeOH): λmax nm (log ε) 248 (4.24); 1H- and 13C-NMR: see Tables 1 and 2 (Additional file 4).
Cell Viability Assay
The cytotoxicity of metabolites 1-5 were determined by using MTT-based colorimetric assay in 96-well plate . Both cell lines (PC-3 and HeLa) were cultured in DMEM and MEM media, respectively, in 25 cm3 tissue culture flasks. The media were supplemented with FBS (5%), pencillin (100 IU/mL) and streptomycin (100 mg/mL). The flasks were then incubated at 37°C in an incubator containing 5% CO2. The flask (80% confluence) was processed for MTT-based cytotoxicity assay. The percent viability of the cells was monitored by trypan blue dye. The cells with clear cytoplasm were considered viable. For the assay, the cells (1 × 105) were loaded onto 96-well tissue culture treated plate. The plate was incubated for 24 hours at 37°C. After incubation, the cells were treated with different concentrations (1.56-50 μM dissolved in DMSO) of compounds 1-5 and kept in an incubator for 48 hours at 37°C. At the end of the incubation, the MTT dye (50 μL, 2 mg/mL) was added to each well and the plate was incubated for 4 hours at 37°C in an incubator. Following incubation, the insoluble formazan crystals were dissolved by adding DMSO (100 μL).
The following formula was used to analyze the cytotoxic effects of the compounds.