Novel thienopyrimidine derivatives as dual EGFR and VEGFR-2 inhibitors: design, synthesis, anticancer activity and effect on cell cycle profile

Figures & data

Figure 1. Structures of potent dual EGFR and VEGFR-2 inhibitors.

Figure 2. Design strategy of the targeted thieno[2,3-d]pyrimidines derivatives as dual EGFR and VEGFR-2 inhibitors.

Figure 3. The synthetic pathway and reagents for the preparation of the target compounds 4a–c and 5a–k.

Figure 4. Graphical representation of IC₅₀ expressed in µM of test compounds against MCF-7 cell line compared to doxorubicin and erlotinib.

Table 1. Results of in vitro anticancer activity of the newly synthesized compounds 4a–c and 5a–k against MCF-7 cell line.

Compound No.	R	R^1	IC50^a, µM*±SD	IC50^b, µM*±SD	Selectivity index
4a	H	–	39.75 ± 4.11	–	–
4b		–	7.12 ± 0.55	–	–
4c		–	10.98 ± 1.24	–	–
5a	–	3-NH2	1.98 ± 0.21	–	–
5b	–	4-NH2	1.47 ± 0.11	72.74 ± 3.98	49.5
5c	–	4-Br	6.65 ± 0.41	–	–
5d	–	2-Cl	1.34 ± 0.07	–	–
5e	–	4-Cl	2.76 ± 0.33	–	–
5f	–	4-Cl-3-CH3	0.66 ± 0.05	153.16 ± 7.57	232.1
5g	–	4-F	1.23 ± 0.04	–	–
5h	–	2-CH3	87.43 ± 5.4	–	–
5i	–	4-CH3	21.07 ± 2.7	–	–
5j	–	4-OCH3	5.01 ± 0.31	–	–
5k	–	4-NO2	2.76 ± 0.19	–	–
Doxorubicin	–	–	3.06 ± 0.19	27.58 ± 1.55	–
Erlotinib	–	–	1.14 ± 0.05	69.41 ± 4.24	–

^a IC₅₀ on MCF-7 cell line.

^b IC₅₀ on normal mammary epithelial cell line (MCF-10A).

* The results given are means of three experiments.

Figure 5. Graphical representation for IC₅₀ of EGFR assay in µM of compounds 5a, 5b, 5d–g and 5k.

Table 2. Results of in vitro EGFR kinase activity of compounds 5a, 5b, 5d–g and 5k.

Compound No.	IC50, µM* ± SD
5a	0.07 ± 0.003
5b	0.042 ± 0.002
5d	0.07 ± 0.004
5e	0.069 ± 0.003
5f	0.028 ± 0.003
5g	0.20 ± 0.011
5k	0.46 ± 0.018
Erlotinib	0.03 ± 0.002
Lapatinib	0.05 ± 0.003
Gefitinib	0.03 ± 0.002

* The results given are means of three experiments.

Figure 6. Graphical representation for IC₅₀ of VEGFR-2 assay in µM of compounds 5a, 5b and 5d–f.

Table 3. Results of in vitro VEGFR-2 kinase activity of compounds 5a, 5b and 5d–f.

Compound No.	IC50, µM*±SD
5a	1.71 ± 0.062
5b	0.51 ± 0.016
5d	0.74 ± 0.025
5e	0.34 ± 0.013
5f	1.23 ± 0.028
Vandetanib	0.43 ± 0.012

* The results given are means of three experiments.

Figure 7. (A) Superposition of 5b (green) with erlotinib (magenta) inside the EGFR tyrosine kinase ATP-binding site. (B) Superposition of 5f (pink) with erlotinib (magenta) inside the EGFR tyrosine kinase ATP-binding site. (C) Binding interactions of 5b (green) with EGFR showing good binding energy score value (score: −10.13). (D) Binding interactions of 5f (pink) with EGFR showing the best binding energy score value (score: −10.51).

Figure 8. (A) Superposition of vandetanib (orange) with tivozanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding site. (B) Superposition of 5b (green) with tivozanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding site. (C) Superposition of 5f (pink) with tivozanib (blue) inside the VEGFR-2 tyrosine kinase ATP-binding site. (D) 2D binding interactions of vandetanib (orange) with EGFR showing the best binding energy score value (score: −18.48). (E) 2D binding interactions of 5b (green) with VEGFR-2 showing good binding energy score value (score: −12.02). (F) 2D binding interactions of 5f (pink) with EGFR showing the best binding energy score value (score: −12.90).

Figure 9. Effect of compound 5f (0.66 μM) on DNA-ploidy flow cytometric analysis of MCF-7 cells after 24 h.

Table 4. Results of cell cycle inhibition activity of compound 5f.

Compound No.	%G0–G1	%S	%G2/M	%Pre-G1
5f/MCF-7	34.19	33.16	32.65	17.58
Control/MCF-7	55.93	37.2	6.87	1.49

Figure 10. Representative dot plots of MCF-7 cells treated with 5f (0.66 μM) for 24 h and analyzed by flow cytometry after double staining of the cells with annexin-V FITC and PI.

Table 5. Results of apoptotic activity of compound 5f.

Compound No.	Apoptosis
	%Total	%Early	%Late	Necrosis
5f/MCF-7	17.58	5.34	9.37	2.87
Control/MCF-7	1.49	0.88	0.38	0.23