Using pregnenolone and 7-deoxycholic acid as starting materials, some 17-acetamidoandrostane and against two tumor cell lines: SGC 7901 (human being gastric carcinoma) and Bel 7404 (human being liver carcinoma). for Cell Viability SGC 7901 (human being gastric carcinoma) and Bel 7404 (human being liver carcinoma) cell lines were from Guangxi Traditional Chinese Medical University or college. Cells were cultivated in RPMI-1640 supplemented with 10% cosmic calf serum (Hyclone, Beijing, China) and antibiotics inside a humidified atmosphere of 5% CO2 at 37 C. The viability of these cells was identified using the colorimetric CellTiter 96 aqueous Cell Proliferation Assay (MTT) according to the instructions provided by the manufacturer (Promega, Madison, WI, USA). Briefly, cells (1~3 104 cells per well) were seeded in 96 wells plates. An equal amount of DMSO Rabbit polyclonal to PHC2 was added to the cells used as negative settings. All were treated in triplicate. One day after seeding, the cells were treated with or without different concentration of each compound and reincubated for 72 h. After the cells were washed with sterile phosphate buffer saline (PBS), 190 L of RPMI-1640 and 10 L of the tetrazolium dye (MTT) (5 mg/mL) remedy were added to each well, as well as the cells had been incubated for yet another 4 h. The moderate was discarded; 200 L of DMSO was put into dissolve the crimson formazan crystals produced. The absorbance (A) at 492 nm was assessed utilizing a MULTISCAN MK3 evaluation spectrometer (Thermo Scientific, Shanghai, China). The full total results were summarized as IC50 values in mol/L in Table 1. Desk 1 antiproliferative actions (IC50 in mol/L) from the substances 3C18. (5). K2CO3 alternative (13%, 20 mL) was put into a remedy of substance 4 (438 mg, 3.32 mmol) in CH3OH (200 mL). The response mixture was warmed under reflux for 1 h. After conclusion of the response as indicated by TLC, the solvent was taken out under vacuum. CH2Cl2 (200 mL) was put into dissolve the solid as well as the causing alternative was cleaned with cool water and saturated brine. After drying out over anhydrous sodium sulfate, the 68521-88-0 solvent was taken out under decreased pressure, as well as the causing crude item was purified by chromatography on silica gel using methanol/dichloromethane (30:1) as eluent to provide 709 mg (76%) of 5 being a white solid. mp 196C197 C; IR (KBr) /cm?1: 3354, 2962, 2937, 1646, 1556, 1433, 1372, 1315; 1H-NMR (CDCl3, 300 MHz) = 9.0, C17-H), 5.302 (1H, brd, = 9.9, N-H), 5.367(1H, dd, = 3.6, 1.8, C6-H); 13C-NMR (CDCl3, 75 MHz) (6). Jones reagent (0.7 mL, 0.267 mol/L) was added dropwise to the answer of 5 (709 mg) in acetone (30 mL) in 20 min. The mix was stirred at area heat range for 1 h and neutralized with 10% K2CO3 alternative. The suspension system was poured more than a silica gel column and eluted with ethyl acetate. The solvent was taken out under decreased pressure. The residue was purified by chromatography on silica gel using methanol/dichloromethane (20:1) as elution to provide 590 mg (80%) of 6 as white solid. mp 198C199 C; IR (KBr) /cm?1: 3321, 2941, 2864, 1691, 1654, 1556, 1442, 1384; 1H-NMR (CDCl3, 300 MHz) = 13.8, 5.1, C7-H), 2.662 (1H, dd, = 15.9, 3.9, C2-H), 3.937 (1H, q, = 9.0, C17-H), 5.582 (1H, d, = 9.0, N-H), 6.149 (1H, s, C4-H); 13C-NMR (CDCl3, 300 MHz) (7). NaBH4 (42 mg, 1.1 mmol) was added at area temperature to a remedy of 6 (124 mg, 0.36 mmol) in CH3OH (50 mL) in a single part. After 30 min, the response was stopped. The answer was neutralized with 1 M HCl. After evaporation of 68521-88-0 nearly all MeOH under decreased pressure, the residue was extracted with ethyl acetate (15 mL 3). The organic level was cleaned with cool water 68521-88-0 and saturated brines. After drying over anhydrous 68521-88-0 sodium sulfate, the solvent was eliminated under reduced pressure and a crude product was acquired. After recrystallization from methanol, 95 mg (78%) of compound 7 was acquired like a white solid. mp 198C200 C; IR (KBr) /cm?1: 3331, 2941, 2866, 1654, 1442, 1384, 1217, 1131; 1H-NMR (CDCl3, 300 MHz) = 7.0, 1.5, C17-H), 5.36 (1H, d, = 2.1, C4-H), 5.40 (1H, br s, N-H); 13C-NMR (CDCl3, 75 MHz) (8). NaBH4 (42 mg, 1.1 mmol) was added to a solution of 6 (124 mg, 0.36 mmol) and NiCl26H2O (87 mg, 0.37 mmol) in CH3OH (15 mL) in the interval of 5 min at space temperature. After 5 min, the reaction was stopped. The perfect solution is was neutralized with 1 M HCl. After evaporation of the majority of MeOH under reduced pressure, water of 15 mL was added. Then the residue was extracted.