Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine derivatives as novel EGFR^L858R/T790M inhibitors

Figures & data

Figure 1. Structures of EGFR kinase inhibitors.

Figure 2. The binding models of Omutinib with EGFR^T790M (PDB code: 3IKA). (A) (B) Docking of compound Omutinib with 3IKA; (C) 2D diagram of the interaction between Omutinib and 3IKA.

Figure 3. The design strategy of target compounds.

Scheme 1. Reagents and conditions: (a) urea, 160 °C, 4–6 h; (b) POCl₃, 160 °C, 6 h; (c) 4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, CH₃OH/H₂O, K₂CO₃, rt, 4 h; (d) 4-fluoro-2-methoxy-5-nitroaniline, TsOH, EtOH, 90 °C, 3–5 h; (e) various aliphatic amines, K₂CO₃, DMF, 50 °C, 2–4 h; (f) Fe, NH₄Cl, EtOH/H₂O, 70 °C, 6 h; (g) various acyl chlorides, TEA, DMF, rt, 6 h.

Scheme 2. Reagents and conditions: (a) urea, 190 °C, 4–6 h; (b) POCl₃, 110 °C, 6 h; (c) different heterocyclic structures, EtOH/H₂O, K₂CO₃, rt, 4 h; (d) 4-fluoro-2-methoxy-5-nitroaniline, TsOH, EtOH, 150 °C, 3–5 h; (e) various aliphatic amines, K₂CO₃, DMF, 50 °C, 2–4 h; (f) Fe, HCl, EtOH/H₂O, 70 °C, 6 h; (g) various acyl chlorides, DIPEA, DMF, rt, 6 h.

Table 1. Cytotoxic activity studies^a.

				IC50^a (μM) ± SD			Inhibitory activity [%]
Compd	R^1	R^2	R^3	H1975	A549	H460	0.1 µM
A1				0.596 ± 0.075	21.901 ± 1.819	5.676 ± 0.239	19.2
A2				0.810 ± 0.058	1.251 ± 0.288	2.461 ± 0.146	22.5
A3				0.150 ± 0.045	0.393 ± 0.106	0.558 ± 0.099	36.0
A4				3.838 ± 0.856	>50	4.949 ± 0.475	6.0
A5				>50	>50	>50	–
A6				0.198 ± 0.055	21.924 ± 1.56	1.156 ± 0.065	19.2
A7				1.363 ± 0.143	1.423 ± 0.351	0.652 ± 0.019	23.4
A8				1.605 ± 0.201	7.013 ± 0.625	>50	10.0
A9				0.694 ± 0.106	>50	>50	19.4
A10				0.439 ± 0.089	7.905 ± 0.762	14.301 ± 1.378	28.8
A11				0.312 ± 0.057	23.880 ± 1.765	0.229 ± 0.013	23.9
A12				1.898 ± 0.409	>50	>50	7.6
A13				0.214 ± 0.050	>50	>50	23.5
A14				0.471 ± 0.067	>50	>50	30.3
A15				0.906 ± 0.236	5.314 ± 0.065	8.031 ± 1.087	2.0
B1				0.087 ± 0.016	1.508 ± 0.199	2.335 ± 0.268	90.3
B2				15.629 ± 1.03	>50	>50	–
B3				9.931 ± 0.866	27.853 ± 1.694	0.533 ± 0.060	19.5
B4				6.649 ± 0.587	1.960 ± 0.380	0.913 ± 0.083	25.6
B5				>50	7.061 ± 0.551	>50	–
B6				4.545 ± 0.374	>50	>50	6.7
B7				0.023 ± 0.003	0.441 ± 0.027	3.218 ± 0.173	96.2
B8				0.297 ± 0.024	0.440 ± 0.039	2.250 ± 0.196	34.6
B9				0.4185 ± 0.042	1.562 ± 0.243	1.317 ± 0.030	25.1
B10				3.072 ± 0.133	25.917 ± 2.13	>50	18.3
B11				0.106 ± 0.012	0.655 ± 0.056	0.239 ± 0.035	54.1
B12				>50	>50	>50	–
B13				37.509 ± 2.160	>50	>50	–
B14				0.120 ± 0.024	0.107 ± 0.031	1.844 ± 0.176	57.8
B15				0.165 ± 0.944	0.098 ± 0.019	1.315 ± 0.065	50.2
B16				0.126 ± 0.032	4.538 ± 0.378	6.480 ± 0.744	12.7
AZD9291				0.067 ± 0.035	0.379 ± 0.045	1.966 ± 0.071	98.7
Olmutinib				0.458 ± 0.045	4.219 ± 0.315	0.642 ± 0.048	98.4

^aThese values were the average obtained from three isolated experiments.

Table 2. EGFR tyrosine kinase inhibitory activities.

Compd	EGFR IC50 (nM)^a
	L858R/T790M	WT	L858R/T790M/C797S	TL^b/WT selectivity
A3	349	>1000	ND	>2.9
A10	>1000	>1000	ND	>1.0
B1	13	996	>1000	76.6
B7	5.9	100	>1000	16.9
B11	173	>1000	>1000	>5.9
B14	63	>1000	>1000	>15.9
AZD9291	0.48	4.56	309	9.5
Olmutinib	12.5	>1000	>1000	>80

ND: Not determined; IC₅₀: half maximal inhibitory concentration; WT: wild-type.

^aThe values were obtained from the mean of three replicated assays.

^bTL represents EGFR^L858R/T790M.

Table 3. Cytotoxic activity studies^a.

Compound No.	IC50(μM)^a ±  SD
	B1	B7	AZD9291	Olmutinib
LO2	>40	>40	28.33 ± 0.42	21.93 ± 0.36

^aThese values were the average obtained from three isolated experiments.

Figure 4. The cell morphology of B1 and B7 was observed using an orthotropic fluorescence microscope.

Figure 5. In vitro wound healing assays were performed on A549 cells with different concentrations of B1 and B7.

Figure 6. Effect of compound B1 on apoptosis.

Figure 7. Graph the apoptosis results of B1 by GraphPad Prism: mechanically damaged cell(Q1); necrotic cells(Q2); viable apoptotic cells(Q3); total apoptotic cells(Q2 + Q3).

Figure 8. Effect of compound B1 on the cell cycle distribution of H1975 cell line for 24 h.

Figure 9. The binding models of B1 with EGFR^T790M (PDB code: 3IKA). (A,B) Docking of compound B1 with 3IKA; (C) 2D diagram of the interaction between B1 and 3IKA; (D) B1 (Red) overlapping with olmutinib (yellow).