Design, synthesis, and anti-cancer evaluation of new pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as potential EGFRWT and EGFRT790M inhibitors and apoptosis inducers

Figures & data

Figure 1. The essential pharmacophoric features of erlotinib as an EGFR inhibitor occupying three pockets in the ATP binding site based on Reference²².

Figure 2. Some reported EGFR-TK inhibitors and their basic pharmacophoric features.

Figure 3. Synthesis of new EGFR inhibitors strategy.

Scheme 1. General procedure for the synthesis of the target compound 7a–e, 8a–d, and 9a–e.

Scheme 2. General procedure for the synthesis of the target compound 10a–d, 11a–e and 12a–d.

Table 1. Percentage of growth inhibition activity of compounds 7a, 8a–d, 9a, 10a–e against A549, PC-3, HCT-116 and MCF-7 at a concentration of 100 μM.

Comp.	Growth inhibition (%)
	A549	PC-3	HCT-116	MCF-7
7a	14	12	18	34
8a	90	98	84	32
8b	94	96	67	9
8c	8	17	9	21
8d	97	95	78	15
9a	42	90	89	68
10a	17	29	21	41
10b	7	15	14	4
10c	9	52	23	8
10d	2	8	5	5
11a	2	30	9	7
11b	28	43	15	29
11c	26	28	3	23
11d	5	20	3	37
11e	30	22	6	23
12a	41	28	35	45
12b	33	36	56	54
12c	22	30	33	55
12d	4	14	5	15
Doxorubicin	100	100	100	100

Figure 4. SAR according to modifiable moieties in the target compounds.

Table 2. IC₅₀ values of compounds 8_a, 8_b, 8_d, 9_a and 12_b against A-549, PC-3, HCT-116 and MCF-7.

Compounds	IC50 (µM)^a
	A-549	PC-3	HCT-116	MCF-7
8a	16.2 ± 2.4	7.98 ± 2.4	25.61 ± 1.3	–
8b	10 ± 2.4	18.01 ±  2.3	26 ± 1.3	–
8d	7.23 ± 2.1	7.12 ± 2.0	70.17 ± 2	–
9a	–	9.26 ± 2.4	–	42 ± 1.2
12b	–	–	86.26 ± 2.2	–
Erlotinib	6.53 ± 0.82	11.05 ± 1.07	5.47 ± 0.3	4.21 ± 0.62

^aAll IC₅₀ values are calculated as the mean of at least three different experiments.

Table 3. In vitro enzymatic inhibitory activities against EGFR^L858R and EGFR^790M.

Comp.	EGFR^WT IC50 (µM)^a	EGFR^T790M IC50 (µM)^a
8a	0.099  ± 0.007	0.123 ± 0.010
8d	0.419  ± 0.029	0.290 ± 0.023
9a	0.594  ± 0.042	0.571 ± 0.046
Erlotinib	0.043  ± 0.003	0.071 ± 0.006

^aThe results were presented as Mean ± Standard error (SE) of three different tests.

Figure 5. PC3distribution upon treatment with compound 8a.

Figure 6. Apoptosis and necrosis percent induced by compound 8a.

Table 4. Effect of compound 8a on active caspase-3 in PC-3 cells after 24 h treatment.

Sample	Caspase-3 (pg/mL)^a
8a/PC-3	452.3 ± 10.5
Staurosporine/PC-3	413.1 ± 11.66
Cont. (PC-3)	84.24 ± 16.5

^aValues are given as mean ± SEM of three independent experiments.

Table 5. The docking binding free energies of the synthesised compounds against EGFR^WT and EGFR^T790M.

Comp.	Binding free energy (kcal/mol)
	EGFR^WT	EGFR^T790M
7a	–16.16	–11.59
7b	–16.61	–11.77
7c	–19.17	–15.03
7d	–21.41	–16.26
7e	–22.42	–19.57
8a	–19.29	–15.63
8b	–19.06	–18.13
8c	–21.92	–19.40
8d	–21.92	–19.30
9a	–15.80	–12.15
9b	–16.62	–15.14
9c	–19.23	–15.18
9d	–21.55	–17.63
9e	–22.46	–17.63
10a	–17.13	–15.27
10b	–19.19	–15.20
10c	–21.28	–16.57
10d	–22.48	–19.86
11a	–17.02	––14.22
11b	–16.92	–15.58
11c	–19.28	–15.89
11d	–21.60	–17.33
11e	–22.32	–19.22
12a	–20.96	–18.50
12b	–21.02	–20.62
12c	–21.67	–21.70
12d	–23.46	–22.39
Erlotinib	–22.12	–
TAK-285	–	–18.70

Figure 7. (A and B) 3D and 2D superimposition of the docked ligand (erlotinib; pink) and the original ligand (green) with RMSD value of 0.88 Å.

Figure 8. (A and B) 3D and 2D superimposition of the docked ligand of mutant EGFR (TAK-285; Pink) and the original ligand (green) with RMSD value of 1.06 Å.

Figure 9. Erlotinib docked into the active site of EGFR^WT.

Figure 10. Compound 8a docked into the active site of EGFR^WT.

Figure 11. Compound 8b docked into the active site of EGFR^WT.

Figure 12. Compound 8d docked into the active site of EGFR^WT.

Figure 13. Compound 9a docked into the active site of EGFR^WT.

Figure 14. Co-crystallised ligand (TAK-285) docked into the active site of EGFR^T790M.

Figure 15. Binding of compound 8c with EGFR^T790M.

Figure 16. Binding of compound 9a with EGFR^T790M.

Figure 17. Binding of compound 10d with EGFR^T790M.

Figure 18. Binding of compound 12d with EGFR^T790M.

Gaber AA, Bayoumi AH, El-Morsy AM, et al. Design, synthesis and anticancer evaluation of 1H-pyrazolo[3,4-d]pyrimidine derivatives as potent EGFRWT and EGFRT790M inhibitors and apoptosis inducers. Bioorg Chem 2018;80:375–95.

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