Design and synthesis of thiazolidine-2,4-diones hybrids with 1,2-dihydroquinolones and 2-oxindoles as potential VEGFR-2 inhibitors: in-vitro anticancer evaluation and in-silico studies

Figures & data

Figure 1. Design of target compounds based on FDA-approved VEGFR-2 inhibitors and molecular hybridisation strategy.

Scheme 1. Synthesis of compounds 8a–c.

Scheme 2. Chemical synthesis of compounds 12a and b.

Table 1. In vitro anti-proliferative activities of 8a–c and 12a,b against Caco-2, HepG2, and MDA-MB-231 cell lines.

Compounds	Anti-proliferative activity (IC50 µM)^a
	Caco-2	HepG2	MDA-MB-231
8a	9 ± 0.001	86 ± 0.001	150 ± 0.01
8b	150 ± 0.013	60 ± 0.008	60 ± 0.003
8c	90 ± 0.004	156 ± 0.004	70 ± 0.009
12a	2 ± 0.005	10 ± 0.001	40 ± 0.002
12b	120 ± 0.001	49 ± 0.008	37 ± 0.002
Doxorubicin	3.46 ± 0.003	1.15 ± 0.02	0.98 ± 0.01

^aThe results were the mean of three replicates.

Table 2. IC₅₀ values of the tested compounds against VEGFR-2 and Vero cell line and their selectivity index (SI) against different cancer cell lines.

Compounds	VEGFR-2 IC50 (nM)	Cytotoxicity against Vero (IC50 µM)	Selectivity index (SI)
			(Caco-2)^a	(HepG2)^b	(MDA-MB-231)^c
8a	96.64	1590 ± 0.068	176.67	18.49	10.60
8b	87.37	890 ± 0.015	5.93	14.83	14.83
8c	317.7	390 ± 0.020	4.33	2.50	5.57
12a	116.3	730 ± 0.015	365	73.00	18.25
12b	84.05	480 ± 040	4	9.80	12.97
Sorafenib	53.65	–	–

^aSI = Cytotoxicity against Vero cells / Cytotoxicity against Caco-2 cell line.

^bSI = Cytotoxicity against Vero cells / Cytotoxicity against HepG2 cell line.

^cSI = Cytotoxicity against Vero cells / Cytotoxicity against MDA-MB-231 cell line.

Figure 2. Selectivity indices of the synthesised compounds.

Figure 3. Effect of compound 12a on cells migration and healingefficacy of Caco-2 cells.

Figure 4. Relative gene expression levels of 4 different genes (BCL2, BCLXL, Survivin, and TGF) in Caco-2 cell line treated with 12a using RT-qPCR.

Table 3. Docking binding free energies (ΔG) of the synthesised candidates with VEGFR-2 enzyme.

Compounds	ΔG (kcal/mol)
8a	–26.60
8b	–23.97
8c	–23.96
12a	–27.44
12b	–26.44
Sorafenib	–26.30

Figure 5. 3D and 2D binding mode of sorafenib into VEGFR-2 active site.

Figure 6. 3D and 2D binding mode of 8a with the active site of VEGFR-2.

Figure 7. 3D and 2D binding mode of 12a with the active site of VEGFR-2.

Figure 8. M D simulations experiment: (A) RMSD values of compound VEGFR-2-compound 12a complex before and after binding, (B) RMSF of VEGFR-2-compound 12a complex, (C) R_g of VEGFR-2-compound 12a complex, D) SASA of VEGFR-2-compound 12a complex, E) H- bonding between VEGFR-2-compound 12a complex.

Figure 9. MM-PBSA analysis.

Figure 10. In silico predicted ADMET parameters for the synthesised compounds and references.

Table 4. ADMET parameters for the synthesised compounds and reference molecules.

Compound	BBB^a	Sol.^b	Ab.^c	CYP2D6^d	PPB^e
8a	3	2	0	NI	M
8b	3	2	0	NI	M
8c	4	2	2	NI	M
12a	3	2	0	NI	M
12b	4	2	2	NI	M
Sorafenib	4	1	0	NI	M
Sunitinib	2	2	0	NI	L

^aBBB, blood brain barrier penetration level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low.

^bSol, Solubility level, 1 = very low, 2 = low, 3 = good, 4 = optimal.

^cAbs., Absorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.

^dCYP2D6, cytochrome P2D6 inhibition, I = inhibitor, NI = non inhibitor.

^ePBB, plasma protein binding, L = less than 90%, M = more than 90%.

Table 5. In silico toxicity of the synthesised compounds and reference molecules.

Compound	FDA Rodent Carcinogenicity (Rat- Female)	TD50 (Rat)^a	MTD (Feed)^b	Rat Oral LD50^b	Rat Chronic LOAEL^b	Ocular Irritancy	Skin Irritancy
8a	Non-Carcinogen	122.38	0.04	2.01	0.020	Mild	None
8b	Non-Carcinogen	812.63	0.04	0.95	0.030	Moderate	None
8c	Non-Carcinogen	75.48	0.03	0.77	0.014	Mild	None
12a	Non-Carcinogen	18.26	0.04	4.07	0.046	Mild	None
12b	Non-Carcinogen	11.31	0.04	1.38	0.039	Mild	None
Sorafenib	Non-Carcinogen	14.24	0.09	0.82	0.005	Mild	None
Sunitinib	Non-Carcinogen	4.13	0.18	2.88	0.040	Severe	None

^aUnit: mg/kg body weight/day.

^bUnit: g/kg body weight.