Design and biological evaluation of 3-substituted quinazoline-2,4(1H,3H)-dione derivatives as dual c-Met/VEGFR-2-TK inhibitors

Figures & data

Figure 1. Pharmacophoric elements for dual inhibition of c-Met/VEGFR-2 TKs.

Figure 2. Four structural elements of reported dual c-Met/VEGFR-2 inhibitors.

Figure 3. General structures of target compounds.

Scheme 1. Synthesis of target compounds 2a–g.

Scheme 2. Synthesis of target compounds 3 and 4a–j; reagents and conditions: (a) N₂H₄·H₂O, EtOH, reflux. (b) Ar-CHO, piperidine, EtOH, reflux. (c) Cyclohexanone, EtOH, reflux. (d) Isatin, AcOH, reflux. (e) Acetylacetone, EtOH, reflux. (f) CS₂, pyridine, reflux. (g) PhNCS, EtOH, reflux. (h) NaOH.

Figure 4. Suggested mechanism of reaction of acyl azide with primary and secondary amines.

Figure 5. Isomers of N-acylhydrazones.

Table 1. In vitro cytotoxicity of compounds 2a–g, 3, 4a–j, and cabozantinib against HCT116 cell line.

Compound	IC50 (µM)	Compound	IC50 (µM)
2a	15.11 ± 0.80	4b	0.734 ± 0.04
2b	16.02 ± 0.85	4c	31.82 ± 1.68
2c	4.692 ± 0.25	4d	10.83 ± 0.57
2d	47.52 ± 2.51	4e	1.408 ± 0.07
2e	37.99 ± 2.01	4f	44.1 ± 2.33
2f	5.401 ± 0.29	4g	3.697 ± 0.20
2g	12.5 ± 0.66	4h	6.989 ± 0.37
3	24.29 ± 1.28	4i	9.204 ± 0.49
4a	36.94 ± 1.95	4j	18.89 ± 1.00
Cabozantinib	16.350 ± 0.86

Figure 6. Cis/trans amide conformers of hydrazone 4e.

Table 2. In vitro cytotoxicity of compounds 4b and 4e against WI38 normal cell line.

Compound	IC50 (µM)
4b	18.45 ± 1.09
4e	10.55 ± 0.62
Cabozantinib	44.71 ± 2.65

Figure 7. Correlation of the inhibitory activity of c-Met enzyme and VEGFR-2 enzyme.

Table 3. Inhibitory activity of selected compounds against c-Met and VEGFR-2.

Compound	c-Met IC50 (μM)	VEGFR-2 IC50 (μM)
2c	0.084 ± 0.004	0.052 ± 0.002
2f	0.161 ± 0.007	0.210 ± 0.009
4b	0.063 ± 0.003	0.035 ± 0.002
4e	0.069 ± 0.003	0.082 ± 0.004
4g	0.271 ± 0.012	0.103 ± 0.005
4h	0.245 ± 0.011	0.297 ± 0.013
Cabozantinib	0.03 ± 0.002	0.059 ± 0.003

Figure 8. Cell cycle analysis in HCT-116 colon cancer cell line treated with compounds 4b and 4e.

Table 4. Cell cycle analysis in HCT-116 colon cancer cell line treated with compounds 4b and 4e.

Compound	%G0–G1	%S	%G2/M
4b	39.59	51.07	9.34
4e	44.60	46.89	8.51
Cabozantinib	43.12	32.16	24.72
Control	47.41	39.28	13.31

Figure 9. Apoptosis induction analysis using Annexin-V/propidium iodide (PI) staining assay.

Figure 10. Representative cytograms of apoptotic HCT-116 cells induced by 4b and 4e compared to cabozantinib for 24 h.

Figure 11. Representative cytograms of apoptotic HCT-116 cells induced by 4b and 4e compared to cabozantinib for 24 h.

Table 5. Apoptosis induction analysis for compounds 4b, 4e, and cabozantinib.

Compound	Apoptosis			%Necrosis
	%Total	%Early	%Late
4b	41.69	15.28	26.41	4.53
4e	38.78	26.76	12.02	2.28
Cabozantinib	32.13	12.52	19.61	3.63
Control HCT-116	0.52	0.33	0.19	1.33

Figure 12. Docking pose of compound 4b and 4e with c-Met TK (PDB: 3lq8).

Figure 13. Docking pose of compound 4b and 4e with VEGFR-2 TK (PDB: 4asd).

Figure 14. Bioavailability Radar for 4b, 4e, and cabozantinib.

Figure 15. BOILED-Egg model of target compounds and cabozantinib.