Design, synthesis, docking, MD simulations, and anti-proliferative evaluation of thieno[2,3-d]pyrimidine derivatives as new EGFR inhibitors

Figures & data

Figure 1. FDA-approved and reported EGFR inhibitors with their essential pharmacophoric features.

Figure 2. Compound 5f as a representative example of the synthesised compounds occupying the ATP active cavity of EGFR (schematic presentation).

Figure 3. Design strategies of the new compounds.

Scheme 1. Synthetic pathway for the preparation of the target compounds 5a–f, 6, 7a,b, and 8.

Table 1. In vitro anti-proliferative and EGFR inhibitory activities of the tested compounds.

Samples	Cell lines IC50 (µM)^a			Enzymes IC50 (nM)^a
	A549	MCF-7	WI-38	EGFR^WT	EGFR^T790M
5a	20.61	29.32	NT^b	NT^b	NT^b
5b	17.79	22.66	70.13	37.19	204.10
5c	27.14	30.04	NT^b	NT^b	NT^b
5d	34.77	35.48	NT^b	NT^b	NT^b
5e	19.90	29.23	NT^b	NT^b	NT^b
5f	17.46	21.40	NT^b	35.64	NT^b
6	21.15	26.55	NT^b	NT^b	NT^b
7a	28.69	35.68	NT^b	NT^b	NT^b
7b	18.39	23.70	NT^b	NT^b	NT^b
8	43.06	56.19	NT^b	NT^b	NT^b
Erlotinib	4.18	14.27	28.48	5.90	212.20

^aData are presented as the mean of the IC₅₀ values from three different experiments.

^b NT = Not tested.

Figure 4. Flow cytometry analysis of A549 cell cycle phases post treatment with 5b.

Table 2. Effect of 5b on cell cycle progression in A549 cells after 72 h treatment.

Sample	Cell cycle distribution (%)^a
	%Sub-G1	%G1	%S	% G2/M
A549	1.14 ± 0.12	9.12 ± 1.34	71.62 ± 1.64	18.12 ± 2.86
5b/A549	0.54 ± 0.09	23.19 ± 2.18*	37.66 ± 4.19*	38.61 ± 2.11*

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

*p < 0.05 indicates statistically significant differences from the corresponding control (A549) group in unpaired t-tests.

Figure 5. Flow cytometry analysis of apoptosis in A549 cells exposed to compound 5b.

Table 3. Effect of compound 5b on stages of the cell death process in A549 cells after 72 h treatment.

Sample	Viable^a (left bottom)	Apoptosis^a		Necrosis^a (left top)
		Early (right bottom)	Late (right top)
A549	98.93 ± 0.04	0.07 ± 0.03	0.98 ± 0.03	0.02 ± 0.01
5b/ A549	34.33 ± 2.20	17.84 ± 3.68	45.71 ± 5.77**	2.12 ± 0.11

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

**p < 0.01 indicates a statistically significant difference from the corresponding control (A549) group in unpaired t-tests.

Figure 6. Gene expression analysis of BAX and Bcl-2 the expression levels after treatment of A549 with compound 5b for 72h.

Table 4. Effect of 5b on levels of BAX and Bcl-2 genes expression in A549 cells treated for 72 h.

Sample	Gene expression (fold change)^a
	BAX	Bcl-2	BAX/Bcl-2 ratio
A549	1.00 ± 0.19	1.00 ± 0.17	1.00 ± 0.03
5b/A549	2.97 ± 0.14**	0.37 ± 0.06*	8.35 ± 1.09**

^aValues are given as changes from the corresponding control (A549) group, which is set to “1”.

*p < 0.05 **p < 0.01 indicate statistically significant differences from the corresponding control in unpaired t-tests.

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
5a	−17.54	−16.01
5b	−17.22	−16.08
5c	−18.64	−17.27
5d	−17.41	−16.27
6	−18.81	−17.61
7a	−15.02	−12.95
5e	−19.84	−16.11
5f	−17.49	−17.37
7b	−12.87	−12.29
8	−26.27	−21.08
Erlotinib	−23.94	–
TAK-285	–	−21.55

Figure 7. Erlotinib docked into the active site of EGFR^WT forming two HBs with Met769 and Cys773 and seven HIs with Lys721, Val702, Ala719, Leu820, and Leu694.

Figure 8. Compound 5b docked into the active site of EGFR^WT forming 1 HB with Met769 and 12 HIs with Lys721, Val702, Ala719, Leu820, Cys773, and Leu694.

Figure 9. Compound 5f docked into the active site of EGFR^WT, forming 1 HB with Met769, and 10 HIs with Val702, Ala719, Leu820, Cys773, and Leu694.

Figure 10. Co-crystallised ligand (TAK-285) docked into the active site of EGFR^T790M forming formed 3 HBs with Met793, Ser720, and Lys745 and 14 HIs with Lys745, Glu762, Leu788, Ile759, Leu844, Ala743, Val726, Met790, and Ala743.

Figure 11. Binding of compound 5f with EGFRT^790M, forming two HBs with Met793 and Lys745, nine HIs with Met766, Leu788, Ile759, Val726, Met790, Ala763, and Ala743, and three electrostatic attractions with Met790, Lys745, and Glu762.

Figure 13. MM-GBSA analysis of the EGFR-5b complex.

Figure 14. Binding free energy decomposition of the EGFR-5b complex.

Figure 15. PLIP analysis of the EGFR-5b complex for each cluster representative. HB: blue solid line, HI: dashed grey line, amino acids: blue sticks, and 5b: orange sticks.

Table 6. The number and types of interactions detected from the PLIP webserver.

Cluster number	No. of HBS	Amino acids in EGFR	No. of HIS	Amino acids in EGFR
C1	1	Met769	5	Leu694–Val702–Lys721–Thr766–Leu820
C2	1	Met769	4	Leu694–Ala719–Lys721–Thr766
C3	1	Met769	6	Leu694–Val702–Ala719–Lys721–Leu764–Thr766
C4	1	Met769	5	Leu694–Val702–Lys721–Thr766–Leu820

Note: Bold amino acids are the amino acids with the largest number of interactions in all cluster representatives.

Figure 16. The expected ADMET study.

Table 7. Calculated ADMET descriptors.

Comp.	BBB	Sol. L	Abs. L	CYP2D6	PPB. L
5a	Very high	Very low	Good	Maybe an inhibitor	>90%
5b
5c	High			Non-inhibitor
5d
6	Very high			Maybe an inhibitor
7a	Medium	Low		Non-inhibitor	<90%
5e	Very low	Very low			>90%
5f
7b	Medium	Low			< 90%
8	Very low	Very low	Very Poor		>90%
Erlotinib	High	Low	Good

Table 8. Toxicity properties of the synthesised compounds.

Comp.	MC-TD50^a	MTDRF^b	DTP	RLD50^b	CLOAEL^b	OI	SI
5a	3.7461	0.129531	Non-toxic	0.483059	0.0375019	Irritant	Non-irritant
5b	10.4772	0.138151		0.211542	0.0746137
5c	1.41938	0.0606517		0.450393	0.048643
5d	2.41863	0.137552		0.610853	0.0630163
6	29.8656	0.100448		0.359547	0.0709221		Irritant
7a	10.0095	0.221518		0.169431	0.12173		Non-irritant
5e	29.2894	0.108657		2.94856	0.323061
5f	10.6751	0.108657		2.61225	0.177435
7b	9.14814	0.133206		0.466603	0.129244
8	0.347134	0.0912244		0.705064	0.0718845
Erlotinib	8.05746	0.0827884		0.662169	0.0359487

^aUnit: mg/kg/day.

^bUnit: g/kg.