The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed using SW620 and HEK293 adherent cell lines. SW620 and HEK293 were cultured in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively, as adherent monolayers in flasks supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified 37°C incubator supplied with 5% CO2. Cells were then harvested with trypsin and dispensed into 96-well microtiter assay plates at 2000 cells per well for the two cell lines and incubated for 18 hours at 37°C with 5% CO2 (to allow cells to attach). Compounds 2 to 5 were dissolved in 5% dimethyl sulfoxide (DMSO) in PBS (v/v), and aliquots (10 μl) were tested over a series of final concentrations ranging from 10 nM to 30 μM. Control wells were treated with 5% aqueous DMSO. After a 68-hour incubation at 37°C with 5% CO2, an aliquot (10 μl) of MTT in PBS (5 mg/ml) was added to each well (final concentration of 0.5 mg/ml), and the microtiter plates were incubated for a further 4 hours at 37°C with 5% CO2. After this final incubation, the medium was aspirated and precipitated formazan crystals were dissolved in DMSO (100 μl per well). The absorbance of each well was then measured. Vinblastine was used as a positive control (20 mg/ml in H2O). All experiments were performed in duplicate.