3.2.2. Cytotoxic Activity Using MTT Assay

The cytotoxic activity of the tested compounds (3, 4, 6, 8a, 8b, and 8c) was determined by the MTT assay [81,82]. According to the manufacturer’s protocol, the cells were seeded in 96-well plates at a concentration of 5 × 10⁴ cells per well (200 µL). Different concentrations of the tested compounds were used to test the cytotoxicity. There were three replicates of each concentration, and the standard control was staurosporine, with vehicle DMSO used as the blank. After 48 h of incubation at 37 °C, 10 L of the MTT stock solution was added to each well and incubated again for 4 h. Finally, the optical absorbances at 570 nm were measured, and cell survival was calculated using the following equation: “percentage of cell viability = AsapmpleAcontrol×100”. A non-linear regression of curve fit was used to calculate the IC₅₀.