New benzoxazole derivatives as potential VEGFR-2 inhibitors and apoptosis inducers: design, synthesis, anti-proliferative evaluation, flowcytometric analysis, and in silico studies

Abstract

A new series of benzoxazole derivatives were designed and synthesised to have the main essential pharmacophoric features of VEGFR-2 inhibitors. Cytotoxic activities were evaluated for all derivatives against two human cancer cell lines, MCF-7 and HepG2. Also, the effect of the most cytotoxic derivatives on VEGFR-2 protein concentration was assessed by ELISA. Compounds 14o, 14l, and 14b showed the highest activities with VEGFR-2 protein concentrations of 586.3, 636.2, and 705.7 pg/ml, respectively. Additionally, the anti-angiogenic property of compound 14b against human umbilical vascular endothelial cell (HUVEC) was performed using a wound healing migration assay. Compound 14b reduced proliferation and migratory potential of HUVEC cells. Furthermore, compound 14b was subjected to further biological investigations including cell cycle and apoptosis analyses. Compound 14b arrested the HepG2 cell growth at the Pre-G1 phase and induced apoptosis by 16.52%, compared to 0.67% in the control (HepG2) cells. The effect of apoptosis was buttressed by a 4.8-fold increase in caspase-3 level compared to the control cells. Besides, different in silico docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

Keywords: Anti-proliferative; VEGFR-2 inhibitors; apoptosis; benzoxazole.

Figure 1.

Some reported VEGFR-2 inhibitors with the essential pharmacophoric features of VEGFR-2 inhibitor agents.

Figure 2.

Summary of the proposed VEGFR-2 inhibitors modifications.

Scheme 1.

General synthetic route of target salts 9a–c.

Scheme 2.

General synthetic route of target intermediates 13a–e.

Scheme 3.

General synthetic route of target final compounds 14a–o.

Figure 3.

In vitro cytotoxic activities of different chemical compounds. *Significant from Sorafenib group at p < 0.001.

Figure 4.

The effect of the most cytotoxic compounds 14b, 14n, 14l, 14i, 14o, and 14a were investigated on VEGFR-2 in HepG2 cells compared to sorafenib as a reference drug. HepG2 cells were treated with sorafenib (3.38 µM), 14b (4.61 µM), 14n (9.93 µM), 14l (6.70 µM), 14i (3.22 µM), 14o (7.01 µM), and 14a (3.95 µM). Data are represented as mean ± SEM of three different experiments. *Significant from the control group at p-value <0.001.

Figure 5.

Effects of compound 14b on endothelial cell migration in HUVEC cells compared to sorafenib. (A) Control/HUVECs, (B) HUVECs were treated with compound 14b for 72 h. (C) HUVECs were treated with sorafenib for 72 h. (D) Represents the graphical illustration for % of wound closure in control, sorafenib and 14b treated cells. Data are represented as mean ± SEM of three different experiments. *Significant from the control group at p < 0.001.

Figure 6.

Flow cytometry analysis for cell cycle distribution of HepG2 cells. (A) Control (HepG2 cells), (B) The representative histogram shows the cell cycle distribution of cells treated with 14b, and (C) Represents the graphical illustration for cell cycle distribution analysis among different treated cells. *Significant from the control group at p < 0.001.

Figure 7.

Compound 14b induced apoptosis in HepG2 cells. (A) Control (HepG2 cells), (B) 14b, and (C) Represent the graphical illustration for % of apoptotic and necrotic cells among cells among control (HepG2) cells and compound 14b treated cells. *Significant from the control group at p < 0.001.

Figure 8.

Effects of compound 14b on Caspase 3 level in HepG2 cells. Values are reported as mean ± SEM of three different experiments. *p < 0.001 indicates statistically significant differences from the control (HepG2) group.

Figure 9.

Superimposition of the co-crystallized molecule (mint green) and the docking pose (red) of the same molecule inside the VEGFR-2 kinase active site.

Figure 10.

3D representation of sorafenib with VEGFR-2 active site.

Figure 11.

The predicted binding pattern of 14b with the active site of VEGFR-2.

Figure 12.

The predicted binding pattern of 14n with the active site of VEGFR-2.

Figure 13.

The predicted binding pattern of 14l with the active site of VEGFR-2.