Anti-cancer and immunomodulatory evaluation of new nicotinamide derivatives as potential VEGFR-2 inhibitors and apoptosis inducers: in vitro and in silico studies

Figures & data

Figure 1. (A) Some reported VEGFR inhibitors showing the essential pharmacophoric features that occupy the different regions of the ATP binding site. (B) reported TNFα inhibitor with its pharmacophoric features.

Figure 2. Strategies of molecular design.

Scheme 1. Synthetic route of target compounds 6, 7, 10, and 11.

Table 1. In vitro cytotoxic activities of the target compounds against HCT-116 and HepG-2 cell lines and in vitro inhibitory activities against VEGFR-2.

Comp.	Cytotoxicity IC50 (µM)		VEGFR-2 IC50 (nM)
	HCT-116	HepG-2
6	22.09 ± 0.064	19.50 ± 0.063	291.9
7	15.70 ± 0.058	15.50 ± 0.055	250.2
10	15.40 ± 0.056	9.80 ± 0.05	145.1
11	20.17 ± 0.063	21.60 ± 0.07	86.60
Sorafenib	9.30 ± 0.201	7.40 ± 0.253	53.65

Figure 3. Cell cycle phases after the treatment of HCT-116 Cells with compound 7.

Figure 4. Flow cytometric analysis of cell cycle phases after treatment with compound 7.

Table 2. Effect of compound 7 on cell cycle phases of HCT-116 cells

Sample	Cell cycle distribution (%)
	%G0-G1	% S	%G2-M	%Pre-G1
HCT-116	38.96	33.75	27.29	19.82
Compound 7 /HCT-116	41.92	26.92	31.16	15.74

Figure 5. Apoptosis effect of compound 7 in HCT-116 cells.

Figure 6. Flow cytometric analysis of apoptosis in HCT-116 cells exposed to compound 7.

Table 3. Stages of the cell death process in HCT-116 cells after treatment with compound 7

	Apoptosis			Necrosis
Sample	Total	Early	Late
Control (HCT-116)	3.05	0.7	1.73	0.62
Compound 7 / HCT-116	15.74	2.75	11.26	1.73

Table 4. Effect of compounds 7 and 10 on the levels of TNF-α and IL-6 in HCT-116 cells.

Sample	TNF-α (% inhibition)	IL-6 (% inhibition)
Compound 7	81.64	88.44
Compound 10	84.52	60.98
Dexamethasone	82.47	93.15

Table 5. The calculated ΔG (binding free energies) of the synthesised compounds and reference drug sorafenib against VEGFR-2 (ΔG in Kcal/mole).

Compound	ΔG [Kcal/mole]	Compound	ΔG [Kcal/mole]
6	−16.41	11	−19.30
7	−22.85	Sorafenib	−36.20
10	−19.73

Figure 7. (A) 3D binding mode of compound 6 into VEGFR-2 active site, (B) 2D binding mode of compound 6 into VEGFR-2 active site.

Figure 8. (A) 3D binding mode of compound 7 into VEGFR-2 active site, (B) 2D binding mode of compound 7 into VEGFR-2 active site.

Figure 9. (A) 3D binding mode of compound 7 against TNF-α protein, (B) 2D binding mode of compound 7 against TNF-α protein.

Figure 10. (A) 3D binding mode of compound 10 against TNF-α protein, (B) 2D binding mode of compound 10 against TNF-α protein.

Figure 11. (A) 3D binding mode of compound 7 against IL-6 protein, (B) 2D binding mode of compound 7 against IL-6 protein.

Figure 12. (A) 3D binding mode of compound 10 against IL-6 protein, (B) 2D binding mode of compound 10 against IL-6 protein.

Figure 13. MD simulations experiment; (A) RMSD values of VEGFR-2 -compound 7 before and after binding, (B) RMSF of VEGFR-2 -compound 7 complex, (C) R_g of VEGFR-2 -compound 7 complex (D) SASA of VEGFR-2 -compound 7 complex, (E) H- bonding between VEGFR-2 -compound 7 complex.

Figure 14. MM-PBSA results of VEGFR-2 -compound 7 complex.

Figure 15. MD simulations experiment; (A) RMSD values of IL-6-compound 7 complex before and after binding, (B) R_g of IL-6-compound 7 complex, (C & D) RMSF of IL-6-compound 7 complex, (E) SASA of IL-6-compound 7 complex, (F) H- bonding between IL-6-compound 7 complex.

Figure 16. MD simulations experiment; (A) RMSD values of TNF-α-compound 7 complex before and after binding, (B) R_g of TNF-α-compound 7 complex, (C & D) RMSF of TNF-α-compound 7 complex, (E) SASA of TNF-α-compound 7 complex, (F) H- bonding between TNF-α-compound 7 complex.

Figure 17. The predicted ADMET parameters.

Table 6. In silico toxicity potential of the synthesised compounds

Comp.	Ames mutagenicity prediction	TD50^a	MTD^b	LD50^b	LOAEL^b	Skin irritancy	Ocular irritancy
6	Mutagen	28.360	0.094	2.727	0.077	None irritant	Mild irritant
7	Non-Mutagen	42.483	0.056	2.113	0.091
10		26.437	0.142	3.754	0.639
11		28.065	0.122	6.781	0.315
Sorafenib	Non-Mutagen	19.236	0.089	0.823	0.005	None	Mild

^aUnit: g/kg.

^bUnit: mg/kg body.