*2.7. Clonogenic Assay*

To measure the long-term growth inhibitory effect of compound **5** against cancer cells, the clonogenic survival assay was performed as reported previously with a minor modification [42]. Briefly, HCT116 human colon cancer cells were plated at a density of 4 × 10<sup>3</sup> cells per well in 24-well culture plates (BD FalconTM; Becton Dickson Immunocytometry System). After attachment, the cells were incubated in the presence or absence of compound **5** at different concentrations (0, 1, 5, and 10 µM) for 7 days, and fixed with 6% glutaraldehyde, followed by staining with 0.1% crystal violet. Its half-maximal clonogenic growth inhibitory concentration (GI50) was determined using the SigmaPlot software (SYSTAT, Chicago, IL, USA) [43].
