General experimental conditions
Silica gel precoated plates (Merck, PF254; 20 × 20, 0.25 mm, Germany) were used for the TLC based separation. Silica gel (70–230 mesh, Merck) was used for column chromatography. Melting points were determined with a Buchi-535, apparatus and are uncorrected. Optical rotations were measured in methanol with a JASCO P-2000 polarimeter. 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. 1H- and 13C-NMR spectra were recorded in C5D5N on a Bruker Avance NMR spectrometer, with residual solvent signal as the internal standard. Standard Bruker pulse sequences were used for 1D- and 2D-NMR experiments. The chemical shifts (δ values) are reported in parts per million (ppm), relative to TMS at 0 ppm. The coupling constants (J values) are reported in Hertz. Electron impact (EI-MS), and high-resolution mass spectra (HREI-MS) were recorded on JEOL JMS-600H mass spectrometer (Japan); in m/z (rel.%). Single-crystal X-ray diffraction data were collected on a Bruker Smart APEX II diffractometer with CCD detector [17]. Data reductions were performed by using SAINT program. The structures were solved by direct methods [18], and refined by full-matrix least squares on F2 by using the SHELXTL-PC package [19]. The figures were plotted with the aid of ORTEP program [20]. The luminometer used was from Luminoskan RS (Labsystem Luminoskan, Helsinki, Finland), and cell harvester and glass fiber filters used were from Inotech (Dottikon, Swetzerland). Liquid scintillation counter used was LS65000 from Beckman Coulter (Fullerton, CA, USA). Microplate reader used was SpectraMax (Molecular Devices, CA, USA).
The chemicals and reagents were purchased from the following sources: Oxymetholone (1) (TCI, Japan), Luminol (Research Organics, OH, USA), Hanks balance salts solution (HBSS), phytohemagglutinin-L (PHA-L), penicillin, and streptomycin (Sigma,St. Louis, USA), lymphocytes separation medium (LSM) (MP Biomedicals, Illkirch, France), zymosan-A (Saccharomyces cerevisiae) (Fluka BioChemika, Buchs, Switzerland), tritiated thymidine (Amersham Pharmacia Biotech, UK) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Amresco, Solon, OH, USA).
Tissue culture plates were obtained from Iwaki (Japan). Mouse fibroblasts (3T3) were obtained from the European American Culture Collection (EACC). Dulbcco’s modified eagle medium (DMEM) and fetal bovine serum were purchased from Gibco-BRL (Grand Island, NY, USA).
Microorganisms and culture medium
The fungi were obtained either from the Northern Regional Research Laboratories (NRRL), or Karachi University Culture Collection (KUCC) or American Type Culture Collection (ATCC).
Macrophomina phaseolina (KUCC 730) and Fusarium lini (NRRL 2204) were grown in a medium composed of the following ingredients and dissolved in distilled H2O (4.0 L): 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). The media (2.0 L) for Aspergillus niger (ATCC 10549), and Rhizopus stolonifer (ATCC 10404) were the same as above, except addition of glycerol (10.0 mL) for A. niger and yeast extract (6.0 g) for R. stolonifer.
General fermentation and extraction conditions
The fungal medium was distributed into 250 mL conical flasks (100 mL each) and autoclaved at 121°C. Mycelia of the fungi were transferred to flasks and incubated at 26 ± 2°C for two-three days on rotary shaking (120 rpm). Compound 1, dissolved in acetone, was evenly distributed among all the flasks which were placed on the rotary shaker (120 rpm) at 26 ± 2°C for fermentation. Parallel control experiments were conducted which included an incubation of the fungus without substrate 1, and an incubation of 1 in the medium without fungus. The degree of transformation was analyzed on TLC after one day. The culture medium and mycelium were separated by filtration. The mycelium was washed with dichloromethane (CH2Cl2, 1.0 L). The aqueous filtrate was extracted with CH2Cl2 (4 L × 3). The CH2Cl2 extract was dried over anhydrous Na2SO4, evaporated under reduced pressures, and the resulting brown gum was analyzed by thin-layer chromatography. The control flasks were also harvested in the same manner, and compared with the test to assure the presence of biotransformed products.
Fermentation of oxymetholone (1) with Macrophomina phaseolina
Oxymetholone (1; 800 mg) was dissolved in 20 mL acetone, and uniformly distributed to 40 flasks containing 2 days old M. phaseolina culture. Fermentation was carried out for twelve days. The gummy material (2.5 g), obtained after filtration, extraction and evaporation, was loaded onto a silica gel column for fractionation. The mobile phase was composed of pet. ether and acetone with a gradient of 10%. Three main fractions (OX-1 − 3) were obtained on the basis of TLC analysis. Fraction OX-1 yielded metabolites 2 (07 mg) and 6 (43 mg) on elution from silica gel column (pet. ether: acetone = 9:1), while OX-2, when subjected to silica gel column chromatography, yielded metabolite 7 (21 mg, pet. ether: acetone = 8:2). Fraction OX-3 yielded metabolite 3 (276 mg, pet. ether: acetone = 7:3) after elution from silica gel column.
17β-Hydroxy-2-(hydroxymethyl)-17α-methyl-5α-androstan-1-en-3-one (2)
Colorless crystalline solid; m.p. 173–174°C; [α]25D -45.4 (c 0.03, MeOH); UV (MeOH): λmax nm (log ε) 236.4 (3.9); IR (KBr): νmax: 3358, 1666, 1629, 1095 cm-1; 1H-NMR: (500 MHz, C5D5N) see Table 2; 13C-NMR: (125 MHz, C5D5N) see Table 2; EI-MS (%): m/z 332 (M+, 30), 274 (25), 216 (20), 176 (39), 174 (52), 161 (34), 147 (24), 123 (32), 108 (31), 91 (56), 71 (100), 55 (95); HREI-MS: m/z 332.2333 (M+ [C21H32O3]+, calcd 332.2351). Crystal data: empirical formula = C21H32O3, Mr = 332.47, orthorhombic, space group P212121, a = 7.715 (4) Å, b = 13.604 (7) Å, c = 36.769 (16) Å, V = 3859 (3) Å3, Z = 8, ρcalc = 1.145 mg m-3, F(000) = 1456, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9948/0.9867, crystal dimensions 0.18 × 0.13 × 0.07 mm, 1.11° < θ < 25.50°, 22,826 reflections were collected, out of which 4,091 reflections were observed (Rint = 0.1228) and 433 parameters were refined. The R-values were; R1 = 0.0610, wR2 = 0.1293 for I > 2σ (I), and R1 = 0.1210, wR2 = 0.1691 for all data, max/min residual electron density; 0.226/-0.229 e Å-3. Crystallographic data for compound 2 can be obtained from the Cambridge Crystallographic Data Center, through the allocated deposition code CCDC 795529 (Additional file 1 and Additional file 2).
2α,17α-Di(hydroxymethyl)-5α-androstan-3β-17β-diol (3)
Amorphous material; [α]25D -14.3 (c = 0.03, MeOH); IR (KBr): νmax 3382, 1381 cm-1. 1H-NMR: (600 MHz, C5D5N) see Table 3; 13C-NMR: (150 MHz, C5D5N) see Table 3; EI-MS (%): m/z 352 (M+, 6), 334 (26), 316 (14), 303 (72), 285 (100), 260 (55), 245 (49), 229 (23), 177 (23), 161 (33), 147 (37), 107 (47), 93 (42), 55 (26); HREI-MS: m/z 352.2623 (M+ [C21H36O4]+, calcd 352.2613) (Additional file 3).
17β-Hydroxy-2α-(hydroxymethyl)-17α-methyl-5α-androstan-3-one (6)
Colorless crystalline solid; m.p. 197–199°C. [lit. 198–200°C] [15]; [α]25D +13.5 (c 0.04, MeOH) [lit. +19.7]. Crystal data: empirical formula = C21H34O3, Mr = 334.48, orthorhombic, space group P212121, a = 7.3859 (3) Å, b = 20.6898 (9) Å, c = 12.4157 (6) Å, V = 1897.27 (15) Å3, Z = 4, ρcalc = 1.171 mg m-3, F(000) = 736, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9962/0.9754, crystal dimensions 0.33 x 0.21 x 0.05 mm, 1.64° < θ < 27.50°, 18840 reflections were collected, out of which 4479 reflections were observed (Rint = 0.0422) and 437 parameters were refined. The R-values were; R1 = 0.0534, wR2 = 0.1354 for I > 2σ (I), and R1 = 0.0707, wR2 = 0.1472 for all data, max/min residual electron density; 0.433/-0.214e Å-3. Crystallographic data for compound 6 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 795530) (Additional file 4 and Additional file 5).
2α-(Hydroxymethyl)-17α-methyl-5α-androstan-3β-17β-diol (7)
Colorless crystalline solid; m.p. 279–281°C. [lit. 280–282°C] [15]; [α]25D -25.4 (c 0.02, MeOH) [lit. – 37.0] (Additional file 6).
Fermentation of oxymetholone (1) with Aspergillus niger and Rhizopus stolonifer
Incubation of 1 (400 mg/10 mL acetone) with 2 days old culture of A. niger in 20 flasks for 6 days produced the previously isolated metabolites 6 (64 mg) and 7 (136 mg), while R. stolonifer (20 flasks) transformed 1 (400 mg/10 mL acetone) into metabolites 3 (86 mg) and 6 (15 mg).
Fermentation of oxymetholone (1) with Fusarium lini
Incubation of 1 (600 mg/15 mL acetone) with 2-day old F. lini culture in 30 flasks for 12 days produced three metabolites which were purified by silica gel column chromatography to obtain metabolites 2 (146 mg), 4 (32 mg, pet. ether: acetone = 7:3) and 5 (15 mg, pet. ether: acetone = 7:3).
17α-Methyl-5α-androstan-2α,3β-17β-triol (4)
Colorless crystalline solid; m.p.: 124–125°C. [α]25D : -29.0 (c = 0.01, MeOH). IR (KBr): νmax 3409, 2927, 1051 cm-1; 1H-NMR: (500 MHz, C5D5N) see Table 3; 13C-NMR: (125 MHz, C5D5N) see Table 3; EI-MS (%): m/z 322 (M+, 98), 307 (100), 304 (43), 264 (39), 249 (95), 229 (30), 215 (53), 181 (54), 171 (58), 169 (51), 123 (55), 109 (33), 95 (41), 81 (40), 57 (30), 43 (42); HREI-MS: m/z 322.2507 (M+ [C20H34O3]+, calcd 322.2508); Crystal data: empirical formula = C20H35O4 [C20H34O3·OH], Mr = 339.48, monoclinic, space group P21, a = 11.5169 (19) Å, b = 6.7843 (12) Å, c = 12.820 (2) Å, V = 945.6 (3) Å3, Z = 2, ρcalc = 1.192 mg m-3, F(000) = 374, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9912/0.9785, crystal dimensions 0.27 × 0.12 × 0.11 mm, 1.68° < θ < 25.50°, 4,363 reflections were collected, out of which 1,585 reflections were observed (Rint = 0.0493) and 221 parameters were refined. The R-values were; R1 = 0.0452, WR2 = 0.0808 for I > 2σ (I), and R1 = 0.0791, WR2 = 0.0914 for all data, max/min residual electron density; 0.147/-0.141 e Å-3. Crystallographic data for compound 4 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 795528) (Additional file 7 and Additional file 8).
17β-Hydroxy-2-(hydroxymethyl)-17α-methylandrost-1,4-dien-3-one (5)
Colorless crystalline solid; m.p. 163–164°C; [α]25D -33.0 (c = 0.01, MeOH); UV (MeOH): λmax nm (log ε): 249.8 (4.0). IR (KBr): νmax 3402, 1664, 1620, 1082, 1033 cm-1. 1H-NMR: (500 MHz, C5D5N) see Table 3. 13C-NMR: (125 MHz, C5D5N) see Table 3. EI-MS (%): m/z 330 (M+, 35), 312 (19), 294 (13), 254 (17), 161 (24), 152 (76), 147 (29), 134 (100), 121 (34), 107 (18), 91 (14). HREI-MS: m/z 330.2183 (M+, [C21H30O3]+; calcd 330.2195). Crystal data: empirical formula = C42H62O8, Mr = 694.92, monoclinic, space group P21, a = 7.7609 (5) Å, b = 13.2141 (8) Å, c = 18.6769 (11) Å, V = 1913.9 (2) Å3, Z = 2, ρcalc = 1.206 mg m-3, F(000) = 756, μ (Mo Kα) = 0.71073 Å, max/min transmission 0.9879/0.9751, crystal dimensions 0.31 × 0.17 × 0.15 mm, 1.09° < θ < 25.00°, 10,983 reflections were collected, out of which 3,534 reflections were observed (Rint = 0.0364) and 458 parameters were refined. The R-values were; R1 = 0.0523, wR2 = 0.1342 for I > 2σ (I), and R1 = 0.0699, wR2 = 0.1544 for all data, max/min residual electron density; 0.320/-0.311 e Å-3. Crystallographic data for compound 5 can be obtained from the Cambridge Crystallographic Data Center (code CCDC 799213) (Additional file 9 and Additional file 10).
T-Cell proliferation inhibition assay
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of healthy adult donors by Ficoll–Hypaque gradient centrifugation [21]. Cells were proliferated as reported earlier [22]. Briefly, cells were cultured at a concentration of 2 × 106/mL in a 96-well round bottom tissue culture plate. Cells were stimulated with 5 μg/mL of phytohemagglutinin. Various concentrations of compounds were added to obtain final concentrations of 0.5, 5, 50 μg/mL, each in triplicate. The plate was incubated for 72 h at 37°C in 5% CO2 environment. After 72 h, cells were pulsed with 0.5 μCi/well, tritiated thymidine, and further incubated for 18 h. Cells were harvested onto a glass fiber filter by using cell harvester. The tritiated thymidine incorporation into the cells, which reflects the proliferation level, was measured by a liquid scintillation counter.
Phagocyte chemiluminescence assay
Luminol-enhanced chemiluminescence assay was performed according to the previous reported method [23]. Briefly 25 μL of whole blood or neutrophils (1 × 106/mL), suspended in Hank’s solution, were incubated with 25 μL compounds (1, 10, 100 μg/mL for whole blood and 0.5, 5, 50 μg/mL for neutrophils) for 30 min. Zymosan 25 μL (20 mg/mL), followed by 25 μL (7 × 10-5 M) of luminol was added to make a final volume of 100 μL. A control without the compound was also run. Peak chemiluminescence was recorded using the luminometer. The luminometer was set with repeated scan mode, 50 scans with 30 s intervals and one second point measuring time.
Cytotoxicity assay
The cytotoxicity of compounds was determined by using the MTT cellular assay [24, 25] against a normal mouse fibroblast (3T3) cell line. Cells were grown in DMEM and MEM (modified Eagle’s medium), containing 10% FBS and 2% antibiotic (penicillin and streptomycin), and maintained at 37°C in 5% CO2 for 24 hours in a flask. Cells were plated (1 × 105 cell/mL) in 96-well flat bottom plates and incubated for 24 hours for cell attachment. Various concentrations of compounds, ranging between 1.25-100 μM, were added into the well and incubated for 48 hours. A 50 μL [2 mg/mL] MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to the well, 4 hours before the end of incubation. Medium and reagents were aspirated and 100 μL DMSO was added and mixed thoroughly for 15 minutes to dissolve the formazan crystals. The absorbance was measured at 570 nm by using a microplate reader. Finally, IC50 (μM) values were calculated, and the experiment was repeated at least three times. Cycloheximide was used as the standard for normal fibroblast cell line.