Design, synthesis, molecular modeling and biological evaluation of novel Benzoxazole-Benzamide conjugates via a 2-Thioacetamido linker as potential anti-proliferative agents, VEGFR-2 inhibitors and apoptotic inducers

Abstract A novel series of 2-thioacetamide linked benzoxazole-benzamide conjugates 1–15 was designed as potential inhibitors of the vascular endothelial growth factor receptor-2 (VEGFR-2). The prepared compounds were evaluated for their potential antitumor activity and their corresponding selective cytotoxicity was estimated using normal human fibroblast (WI-38) cells. Compounds 1, 9–12 and 15 showed good selectivity and displayed excellent cytotoxic activity against both HCT-116 and MCF-7 cancer cell lines compared to sorafenib, used as a reference compound. Furthermore, compounds 1 and 11 showed potent VEGFR-2 inhibitory activity. The cell cycle progression assay showed that 1 and 11 induced cell cycle arrest at G2/M phase, with a concomitant increase in the pre-G1 cell population. Further pharmacological studies showed that 1 and 11 induced apoptosis and inhibited the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins in both cell lines. Therefore, compounds 1 and 11 might serve as promising candidates for future anticancer therapy development.


Supplementary Materials
Characterization and Spectral analysis of compounds 1-15 2 Elemental Analysis 62 Biological evaluation 63

Anti-proliferative activity against HCT-116 and MCF-7 human cancer cells lines.
The sulforhodamine B (SRB) assays were performed according to Skehan et al. [55]. Briefly, exponentially growing cells were trypsinized, counted and seeded at the appropriate densities (5000 cells/100 μL/ well) into 96-well microtiter plates. Cells were incubated in a humidified atmosphere at 37 °C for 24 h. Then, the cells were exposed to the tested compounds at the desired concentrations (0.01, 0.1, 1, 10, and 100 μM) or to 1% dimethyl sulfoxide (DMSO) for 72 h. At the end of the treatment period, the media were removed, and the cells were fixed with 10% trichloroacetic acid at 4 °C for 1 h. Following, the cells were washed with tap water four times and incubated with SRB 0.4% for 30 min. Excess dye was removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution for (optical density) OD determination at 510 nm using a Spectra Max plus Microplate Reader (Molecular Devices, CA). Cell viability was expressed relative to the untreated control cells [62].

VEGFR-2 Inhibitory activity
IC50s of compounds 1 and 11 were evaluated in vitro using colorimetric assay of human VEGFR-2 ELISA (Enzyme-Linked Immunosorbent Assay) kits (HTScan ® VEGF Receptor-2 Kinase Assay Kit). It includes active VEGFR-2 kinase (a biotinylated peptide substrate and a phospho-tyrosine antibody) for detection of the phosphorylated form of the substrate peptide). On a 96-well plate, a particular VEGFR-2 antibody was seeded and 100 μL of the normal solution or the tested compound tested was applied, incubated at room temperature for 2.5 h and washed. Then, 100 μL of the prepared biotin antibody was added, incubated for an additional 1 h at room temperature and washed. Following, 100 μL of streptavidin solution was added at room temperature, incubated for 45 minutes and then, 100 μL of TMB Substrate solution was applied and incubated at room temperature for 30 minutes. Finally, 50 μL stop solution was added and the absorption was measured at 450 nm instantly. The standard curve, the X-axis concentrations, and the Y-axis absorbance were drawn [14].

Cell cycle analysis.
HCT-116 and MCF-7 cells were seeded at concentrations of 1 x 10 5 cells per well in a 6well plate, and then incubated for 24 h. The cells were treated for 24 h with vehicles (0.1 percent DMSO) or 10 μM of compounds 1 or 11. Using ice-cold, 70 percent ethanol at 4 °C, cells were harvested and fixed for 12 h. Ethanol was removed, and the cells were washed by cold PBS. Then, the cells were incubated in 0.5 mL of PBS containing 1 mg/mL Ranse for 30 min at 37 °C. In the dark, the cells were stained with propidium iodide for 30 min. Flow cytometer was then used to detect contents of DNA [63].

Annexin V-FITC/PI apoptosis assay.
For this study, annexin V-FITC / PI apoptosis detection kit was used; HCT-116 and MCF-7 cells were stained with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) counter-stained.1 x 10 5 HCT-116 cells were 48 h incubated with compound 1 or 11, trypsinized, washed with phosphate-buffered saline (PBS), stained in the dark at 37 °C for 15 minutes. Finally, the cells analyzed with a cytometer of FACS Caliber flow [64].

Optimization of VEGFR active site.
The VEGFR has been prepared for docking experiments by adding hydrogen atoms and their standard geometry. The atom's connections and types were checked with automatic correction for any errors that existed. Selection of the receptor and its potential atoms has been fixed.MOE Alpha Site Finder used all default items to search for the active site in the receptor structure, and then dummy atoms were created from the alpha spheres obtained.

Docking of the target compounds to the VEGFR active sites.
Docking of the tested compounds' conformational database was performed using MOE-Dock software. To ensure a reasonable docking accuracy and to determine the effect of the water molecules, the co-crystallized ligand in the VEGFR (PDB ID: 4ASD) was docked to its corresponding protein (in the absence and in the presence of water) and the RMSD values were determined between the co-crystallized ligand and docked pose. The success rates obtained were highly excellent where the active site of the VEGFR was calculated from the binding of cocrystallized ligand and saved as MOE file. The active site file of the VEGFR was then loaded, and the docking tool was used. The program specifications have been adjusted to the dummy atoms as docking site, triangle matcher as placement methodology, london dG as Scoring methodology have been adjusted to its default values. The MDB file of the ligands to be docked (Sorafenib and target compounds) was loaded, and calculations for docking were run automatically. The poses obtained were studied and the poses which had the best ligand-receptor interactions were selected and stored for calculating energy.