3.5. Biological Studies

Antitumor activity of the newly prepared imidazole compounds 4a–e and 5 were carried out against MCF-7, HepG2, HCT-116, and MCF-10A using the MTT assay method. Cells at a density of 1 × 10⁴ were seeded in 96-well plates at 37 °C for 48 h under 5% CO₂. After incubation, the cells were treated with different concentrations of the prepared molecules and incubated for 24 h. MTT dye was added at the end of 24 h of drug treatment and incubated for 4 h at 37 °C. Next, 100 µL of dimethyl sulfoxide was added to each well to dissolve the purple formazan formed. The color intensity of the formazan product, which represents the growth condition of the cells, is quantified using an ELISA plate reader at 570 nm. The experimental conditions were carried out with at least three replicates and the experiments were repeated at least three times.

MCF-7 cells (2 × 10⁵/well) were harvested and washed twice in PBS. After that, cells were incubated at 37 °C and 5% CO₂. The medium was replaced with DMSO (1% v/v) containing the tested compounds, then incubated for 48 h, washed twice in PBS, fixed with 70% ethanol, rinsed again with PBS, and then stained with DNA fluorochrome PI for 15 min at 37 °C. DNA content was analyzed by flow cytometry on a FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

Apoptosis in MCF-7 cells was investigated using fluorescent Annexin V-FITC/PI detection kit by flow cytometry assay. Briefly, MCF-7 cells (2 × 10⁵) after incubation for 12 h. Cells were treated with compounds 4d and 5 at their IC₅₀ concentration for 48 h, then the cells were harvested and stained with Annexin V-FITC/PI dye for 15 min in the dark at 37 °C. Flow cytometry analyses were performed using a FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

VEGFR-2 inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using human VEGFR-2 ELISA kits according to the manufacturer’s instructions. A total of 100 µL of standard solution and tested molecules were pipetted into the 96-wells and VEGFR-2 was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman VEGFR-2 antibody was added. After washing away unbound biotinylated antibody, 100 µL of conjugated streptavidin solution was pipetted into the wells. After washing, 100 µL of TMB substrate solution was added to the wells for 30 min at 37 °C. Stop solution (50 µL) was added, and the intensity of the color was measured immediately at 450 nm.

RAF inhibitory activity for compounds 4d, 5, and erlotinib was evaluated using human B-RAF ELISA kits according to the manufacturer’s instructions. Next, 100 µL of standard solution and tested molecules were pipetted into the 96-wells and B-RAF was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman B-Raf antibody was added. After washing away unbound biotinylated antibody, 100 µL of conjugated streptavidin solution was pipetted into the wells. After washing, 100 µL of TMB substrate solution was added to the wells for 30 min at 37 °C. Stop solution (50 µL) was added, and the intensity of the color is measured immediately at 450 nm.