Design, synthesis, anticancer evaluation, and molecular modelling studies of novel tolmetin derivatives as potential VEGFR-2 inhibitors and apoptosis inducers

Abstract

Novel tolmetin derivatives 5a-f to 8a-c were designed, synthesised, and evaluated for antiproliferative activity by NCI (USA) against a panel of 60 tumour cell lines. The cytotoxic activity of the most active tolmetin derivatives 5b and 5c was examined against HL-60, HCT-15, and UO-31 tumour cell lines. Compound 5b was found to be the most potent derivative against HL-60, HCT-15, and UO-31 cell lines with IC₅₀ values of 10.32 ± 0.55, 6.62 ± 0.35, and 7.69 ± 0.41 µM, respectively. Molecular modelling studies of derivative 5b towards the VEGFR-2 active site were performed. Compound 5b displayed high inhibitory activity against VEGFR-2 (IC₅₀ = 0.20 µM). It extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. It induced apoptosis in HCT-15 cells (52.72-fold). This evidence was supported by an increase in the level of apoptotic caspases-3, -8, and -9 by 7.808-, 1.867-, and 7.622-fold, respectively. Compound 5b arrested the cell cycle in the G0/G1 phase. Furthermore, the ADME studies showed that compound 5b possessed promising pharmacokinetic properties.

Keywords: Tolmetin; VEGFR-2; anticancer activity; apoptosis; synthesis.

Figure 1.

The biological active apoptosis inducers and VEGFR-2 inhibitors.

Figure 2.

Structural similarities and pharmacophoric features of VEGFR-2 inhibitor (sunitinib) and designed compounds.

Scheme 1.

Synthetic pathway of tolmetin hydrazide 4, tolmetin hydrazones 5a–f, 6a–c, tolmetin derivatives7a–c and tolmetin semicarbazide derivatives 8a–c.

Figure 3.

Tautomerism in tolmetin hydrazone derivatives 5a–f and 6a–c.

Figure 4.

Effects of compound 5b on endothelial cell migration in HUVEC cells compared to sunitinib. (A) HUVECs were treated with 2.4 μM compound 5b and 3.2 μM sunitinib for 72 h. (B) Represents the graphical illustration for % of wound closure in control, sunitinib and 5b treated cells. Data are represented as mean ± SD, *significant from control group at p-values <0.001.

Figure 5.

5b induces cell cycle arrest in HCT-15 cells. HCT-15 cells were incubated with 5b or vehicle for 24 h and subjected to cell cycle analysis by flow cytometry. (A) Control and (B) 5b treated cells. (C) Represents the graphical illustration for cell cycle distribution analysis in control and 5b treated cells.

Figure 6.

Apoptosis in HCT-15 cells by the treatment with 5b. (A) Control and (B) 5b treated cells. (C) Represents the graphical illustration for % of apoptotic and necrotic cells in control and 5b treated cells. Different cellular distributions are shown in the quadrants, (Q1: Left top) Necrotic cells (Annexin^−, PI⁺), (Q2: Right top) Late apoptotic and secondary necrotic cells (Annexin⁺, PI⁺), (Q3: Left bottom) Normal cells (Annexin⁻,PI⁻), and (Q4: Right bottom) Early apoptotic cells (Annexin⁺, PI⁻).

Figure 7.

Effects of compound 5b on the caspase-3, caspase-8 and caspase-9 activity (A, B and C, respectively) in HCT-15 cells compared to sunitinib. Data are represented as mean ± SD, *significant from control group at p-values <0.0001.

Figure 8.

2D interaction diagram showing sunitinib docking pose interactions with the key amino acids in the VEGFR-2 binding site.

Figure 9.

2D diagram (A) and 3D representation (B) of the superimposition of the co-crystallised (red) and the docking pose (blue) of sunitinib in the VEGFR-2 binding site with RMSD of 1.48 Å.

Figure 10.

2D diagram (A) and 3D representation (B) of compound 5b in the VEGFR-2 binding site.

Figure 11.

Predicted Boiled-Egg plot from SwissADME online web tool for compound 5b.