Design, synthesis, in silico studies, and biological evaluation of novel pyrimidine-5-carbonitrile derivatives as potential anti-proliferative agents, VEGFR-2 inhibitors and apoptotic inducers

Abstract

A novel series of pyrimidine-5-carbonitrile derivatives bearing benzylidene and hydrazone moieties with different linkers (spacers) were designed and synthesized as possible inhibitors of the vascular endothelial growth factor receptor-2 (VEGFR-2). The newly synthesized compounds were evaluated in vitro for their cytotoxic activities against two human cancer cell lines namely colon cancer (HCT-116) and breast cancer (MCF-7) using sorafenib as a standard anticancer drug. Compounds 9d, 11e, 12b, and 12d showed higher cytotoxic activities than sorafenib with IC₅₀ values ranging from 1.14 to 10.33 μM. In particular, compound 11e exhibited excellent activities against HCT-116 and MCF-7 with IC₅₀ values of 1.14 and 1.54 μM, respectively. Moreover, compound 11e exhibited about 47.32-fold cytotoxic activity against normal human fibroblast (WI-38) cells, lower than the cytotoxicity against the cancer cells. Compounds 11e and 12b were the most potent VEGFR-2 inhibitors with IC₅₀ values of 0.61 and 0.53 μM, respectively, compared to sorafenib. Bedsides, compound 11e arrested the HCT-116 cell growth at S and sub-G1 phases, induced a significant increase in the apoptotic cells, and caused remarkable decrease in the levels of TNF-α, IL-6, and caspase-3. Finally, the binding patterns of the target derivatives were investigated through the docking study against the proposed molecular target (VEGFR-2, PDB ID 1YWN). The results of molecular docking studies showed similar binding modes to sorafenib against VEGFR-2. In addition, molecular dynamic simulations revealed the stability of compound 11e in the active site for 100 ns.

Fig. 1. (A) Some reported VEGFR-2 inhibitors and their necessary four parts. (B) Function of VEGFR-2 and effects of sorafenib.

Fig. 2. Rationale of molecular design for synthesis of novel VEGFR-2 inhibitors.

Scheme 1. General procedure for synthesis of intermediate compounds 4, 5, and 6.

Scheme 2. General procedure for synthesis of target compounds 9a–d.

Scheme 3. General procedure for synthesis of target compounds 11a–e and 12a–g.

Fig. 3. SAR studies of 12a, 12d, and 12b compared to 11a, 11d, and 11b, respectively.

Fig. 4. SAR studies of 9b compared to 9a.

Fig. 5. SAR studies of 9d and 9c compared to 9b and 9a, respectively.

Fig. 6. SAR studies of 11e compared to 11d.

Fig. 7. SAR studies of 11c compared to 11b.

Fig. 8. SAR studies of 12b and 12d compared to 12e and 12g, respectively.

Fig. 9. SAR studies of 12b and 12c compared to 12d.

Fig. 10. The representative histograms show the cell cycle distribution of control (HCT-116), and cells treated with compound 11e.

Fig. 11. Flow cytometric analysis of cell cycle phases post the compound 11e treatment.

Fig. 12. The representative flow cytometric charts for control (HCT-116) and the cells treated with compound 11e.

Fig. 13. Flow cytometric analysis of apoptosis in HCT-116 cells exposed to compound 11e. For each dot plot chart, the quadrant regions represent the cells in each sub-population; lower left quadrant (viable cells), lower right quadrant (late apoptosis), upper right quadrant (early apoptosis), and upper left quadrant (necrosis).

Fig. 14. The representative in vitro immunomodulatory assay charts for dexamethasone as positive control and the tested compound 11e.

Fig. 15. The representative in vitro caspase-3 expression chart for the tested compound 11e.

Fig. 16. Superimposition of the native ligand (turquoise) and the re-docked one (green) in the VEGFR-2 TK active site (RMSD = 0.49 Å).

Fig. 17. The predicted binding mode for the co-crystalized ligand with the active site of VEGFR-2 TK.

Fig. 18. The predicted binding mode for sorafenib with the active site of VEGFR-2 TK.

Fig. 19. The predicted binding mode of compound 9d with the active site of VEGFR-2 TK.

Fig. 20. The predicted binding mode of compound 11e with the active site of VEGFR-2 TK.

Fig. 21. The predicted binding mode of compound 12a with the active site of VEGFR-2 TK.

Fig. 22. MD simulations; (A) RMSD, (B) RMSF (C) R_g (D) SASA, and (E) hydrogen bonding for compound 11e-VEGFR-2 TK complex over the MD run (100 ns).

Fig. 23. MM-PBSA outputs of the compound 11e-VEGFR-2 TK complex.