Design, synthesis, and cytotoxic evaluation of quinazoline-based derivatives as VEGER-2 inhibitors: comparative study against EGFR kinase activity, induction of apoptosis, and molecular docking study

Abstract

A novel series of quinazoline-based compounds were designed and synthesized as modified analogues to certain known VEGFR-2 inhibitors, as an extension of our work on the design and synthesis of new VEGFR-2 inhibitors. The anti-proliferative properties of the synthesized compounds were assessed in vitro against three tumor cell lines (MCF-7, HepG-2, and K-562). Compounds 8a (IC₅₀ = 0.6955, 0.1871, 0.1884 μM), 8b (IC₅₀ = 0.1908, 0.2242, 0.4642 μM), 8c (IC₅₀ = 0.1875, 0.7344, 0.5444 μM), 8e (IC₅₀ = 0.5523, 1.4357, 0.2664 μM), 9a (IC₅₀ = 0.2824, 0.1871, 0.3858 μM), 9b (IC₅₀ = 0.2090, 0.1944, 0.1902 μM), and 9d (IC₅₀ = 0.2042, 0.3227, 2.2742 μM) showed the highest levels of the cytotoxic activity against the cell lines under investigation respectively, with IC₅₀ values that were comparable to those of sorafenib (IC₅₀ = 0.1283, 0.0844, 0.0606 μM). Next, the inhibitory action against VEGFR-2 kinase activity was also estimated for the synthesized compounds to confirm their mechanism of action to induce antiproliferative actions. The cytotoxicity and VEGFR-2 inhibition results were highly consistent, identifying compounds 8a (IC₅₀ = 67.623, 74.864, 62.505 nM), 8b (IC₅₀ = 80.740, 88.327, 78.668 nM), 9a (IC₅₀ = 80.036, 85.240, 64.017 nM), 9b (IC₅₀ = 19.320, 66.436, 43.052 nM), and 9d (IC₅₀ = 47.042, 58.752, 80.182 nM) as top VEGFR-2 inhibitors comparing to sorafenib (IC₅₀ = 87.993, 92.775, 95.735 nM). In addition, the implemented comparative study against EGFR kinase activity specifies the inhibition of VEGFR-2 kinase activity as the major mechanism correlated to the cytotoxic activity of the synthesized compounds. Furthermore, extra mechanistic studies were conducted for the synthesized compounds, including cell cycle analysis that revealed the ability of compounds 8a and 9b to arrest the HepG-2 cells at the sub-G1 phase while increasing the population of the cells to 96.3% for 8a and 94.68% for 9b in comparison to the control 68.12%. Additionally, the titled compounds caused a significant decrease in Bcl-2 expression levels, a significant increase in caspase-3, caspase-9, and BAX gene expression levels, and a suppression of TNF-α and IL-6R protein levels, indicating their significant apoptotic impact. Ultimately, the synthesized compounds' high affinity and proper binding manner inside the VEGFR-2 active site were demonstrated by molecular docking modeling.

Fig. 1. Sorafenib, as an FDA-approved anti-VEGFR-2, has the pharmacophoric features of VEGFR-2 inhibitors.

Fig. 2. The proposed design of the target compounds.

Scheme 1. Synthetic pathways for the target compounds.

Fig. 3. Analysis of cell cycle phases in HepG-2 cells after compounds 8a (left) and 9b (right) treatment.

Fig. 4. The impact of the synthesized compounds on the levels of caspase-3, caspase-9, TNF-alpha, and IL-6R gene expression in HepG-2 cells.

Fig. 5. The impact of compounds 8a and 9a on HepG-2 cell expression levels of the BAX and Bcl-2 genes.

Fig. 6. (A) 3D binding mode of compound 8a into VEGFR-2. (B) 2D binding mode of compound 8a into VEGFR-2.

Fig. 7. (A) 3D binding mode of compound 8b into VEGFR-2. (B) 2D binding mode of compound 8b into VEGFR-2.

Fig. 8. (A) 3D binding mode of compound 9a into VEGFR-2. (B) 2D binding mode of compound 9a into VEGFR-2.

Fig. 9. (A) 3D binding mode of compound 9b into VEGFR-2. (B) 2D binding mode of compound 9b into VEGFR-2.