V., Rush J. inoculum comprising the potential inhibitors at 5 M or 0.5 M for 4 h. Concentrations were chosen according to the highest non-cytotoxic concentration of the given compound. Cells were washed, complete medium was added and cells were cultured for 48 h. Later on cells were lysed for luciferase assay as previously explained . Chemistry The carboxylic acid was stirred with thionyl chloride (10 mL) at space heat for 1h. After eliminating the excess of thionyl chloride under reduced pressure, dry dichloromethane (15 mL) and the benzene derivative (1 comparative, referring to the carboxylic acid) were added. The perfect solution is was cooled to 0 C and AlCl3 (1.2 equivalents) were added. After stirring at that heat for 45 min, the combination was hydrolyzed, the layers separated and the water phase was extracted with dichloromethane two times. The combined organic layers were dried and the solvent eliminated. Adobe flash chromatography using 1/15 ethyl acetate/n-hexane as eluent led to the desired compounds. Azacyclonol (1 Exatecan mesylate comparative), ketone (1 comparative), potassium carbonate (5 equivalents), a catalytic amount of potassium iodide and 18-crown-6 were stirred in dry acetonitrile (4 mL) in the microwave (CEM Discover) at 150 Watt, 6.5 bar, 175 C for 45 min or (MultiSYNTH) at 100 Watt, 140 C, 3 h (2g, 2h, 2t, 2u, 2v, 2w) or at 50 Watt, 100 C, 5 min (8a). The microwave aided reactions were performed in a continuous air flow stream cooled closed vessel. Reaction heat was monitored via an IR sensor in both microwave ovens and an additional dietary fiber optic component in case of the MultiSYNTH. After filtering off the remaining precipitate the Rabbit polyclonal to AMACR solvent was eliminated. The impurities could not be eliminated completely by adobe flash chromatography (6/1 ethyl acetate/n-hexane + 3 % NEt3), so preparative HPLC (isocratic 70/30 MeOH/H2O) was performed and the real product isolated. Sodium borohydride (5 equivalents) and ketone (1 comparative) were added to 2 mL methanol at 0 C. After stirring for 30 min a spatula tip of sodium borohydride was added to the combination which was stirred for another 30 min at 0 C. The combination was hydrolyzed using 2 mL NH4Cl-solution. After eliminating the solvents under reduced pressure, the remaining solid was washed two times with methanol and ethyl acetate (2 mL each). The combined organic layers Exatecan mesylate were freed from solvent and the natural product was purified by preparative HPLC (isocratic MeOH/H2O: 70/30) to give the racemates in satisfying yields. The carboxylic acid (1 comparative) was stirred with an excess of thionyl chloride for 1 hour at space temperature. The producing clear answer was freed from remaining thionyl chloride under reduced pressure. The acid chloride was dissolved in dry dichloromethane and added dropwise at 0 C to a solution of the related alcohol or amine (1.2 equivalents) and an equimolar amount of triethylamine in dry dichloromethane. After stirring for 30 minutes at 0 C the combination was warmed to space heat and stirred for 1 hour. The precipitated solid was filtered off. The solvent was eliminated and the natural product purified by column adobe flash chromatography using an ethyl acetate/n-hexane combination. (1a). Yield: 26 %; 1H-NMR: 7.88C7.85 Exatecan mesylate (2 H, m), 7.49C7.45 (1 H, m), 7.39C7.35 (2 H, m), 3.35C3.32 (2 H, m), 2.92C2.88 (2 H, m), 1.86C1.79 (2 H, m), 1.72C1.64 (2 H, m), 1.48C1.40 (2 H, m) [Lit.  400 MHz, CDCl3: 7.94 (2 H, m), 7.55 (1 H, m), 7.45 (2 H, m), 3.41 (2 H, t, = 6.80), 2.98 (2 H, t, = 7.00), 1.90 (2 H, m), 1.76 (2 H, m), 1.53 (2 H, m)]; 13C-NMR: 199.96, 136.95, 133.02, 128.61, 128.02, 38.29, 33.69,.