Discovery of new 1H-pyrazolo[3,4-d]pyrimidine derivatives as anticancer agents targeting EGFR^WT and EGFR^T790M

Figures & data

Figure 1. EGFR inhibitors and their pharmacophoric features.

Figure 2. ATP binding site of EGFR-TK cavity composed of five main features⁴¹ and summary of the possible modifications of EGFR-TK inhibitors.

Figure 3. Rationale of molecular design of the new proposed EGFR-TK inhibitors.

Scheme 1. Synthetic protocol of starting compounds. Reagents and conditions: (a) phenyl hydrazine, absolute ethanol, reflux, 2 h; (b) sodium hydroxide, absolute ethanol, reflux, 5 h; (c) urea, fusion; (d) POCl₃, PCl₅, reflux, 28 h; (e) aniline, absolute ethanol, room temperature.

Scheme 2. Synthetic protocol of compounds 7a,b, 8, and 9. Reagents and conditions: (a) aliphatic amines, ethanol, reflux 4 h; (b) aniline, ethanol, reflux 6 h; (c) cyclohexylamine, ethanol, reflux 5 h.

Scheme 3. Synthetic protocol of compounds 10, 11a,b, 12a-c, and 13a,b. Reagents and conditions: (a) hydrazine hydrate 99%, reflux, 6 h; (b) aromatic aldehydes, ethanol, glacial acetic acid, reflux 12 h; (c) acetophenones, ethanol, glacial acetic acid, reflux 14 h.

Table 1. In vitro anti-proliferative activities of the tested compounds against human lung (A549) and colon (HCT-116) cancer cell lines.

Comp.	In vitro cytotoxicity IC50 (µM)^a
	A549	HCT-116
7a	64.42	29.62
7b	67.38	31.49
8	16.75	24.16
9	29.07	39.39
10	15.68	18.78
11a	47.67	46.18
11b	75.71	75.11
12a	13.72	23.33
12b	8.21	19.56
12c	28.79	28.69
13a	43.12	27.03
13b	15.36	36.44
Erlotinib	6.77	19.22

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

Figure 4. SAR study of the target compounds.

Table 2. the inhibitory activities of the tested compounds against EGFR^WT and EGFR^T790M kinases.

Comp.	EGFR^WT	EGFR^T790M
	IC50 (µM)^a	IC50 (µM)^a
8	0.026	NT^b
10	0.023	NT^b
12a	0.021	NT^b
12b	0.016	0.236
Erlotinib	0.006	0.563

^aData were expressed as the mean of three independent experiments.

^bNT: Compounds not tested.

Figure 5. Flow cytometric analysis of cell cycle phases after treatment with compound 12 b.

Table 3. Effect of compound 12 b on cell cycle progression in A549 cells after 48 h treatment.

Sample	Cell cycle distribution (%)^a
	%Sub-G1	%G1	%S	% G2/M
A549	0.87 ± 0.13	53.87 ± 0.58	28.70 ± 2.50	16.56 ± 2.30
Compound 12 b/A549	0.89 ± 0.31	28.04 ± 2.78***	42.39 ± 3.50*	28.55 ± 1.61*

^aValues are given as mean ± SEM of three independent experiments. *p < 0.05 ***p < 0.001 indicate statistically significant differences from the corresponding control (A549) group in unpaired t-tests.

Figure 6. Flow cytometric analysis of apoptosis in A549 cells exposed to compound 12 b.

Table 4. Effect of compound 12 b on stages of the cell death process in A549 cells after 48 h treatment.

Sample	Viable^a (Left Bottom)	Apoptosis^a		Necrosis^a (Left Top)
		Early (Right Bottom)	Late (Right Top)
A549	93.43 ± 0.96	6.03 ± 0.76	0.43 ± 0.19	0.11 ± 0.03
Comp. 12 b/ A549	31.57 ± 4.64	67.69 ± 4.86***	0.64 ± 0.30	0.10 ± 0.02

^aValues are given as mean ± SEM of three independent experiments. ***p < 0.001 indicates a statistically significant difference from the corresponding control (A549) group in unpaired t-tests.

Figure 7. Gene expression analysis for the expression levels of BAX, and Bcl-2 after treatment of A549 with compound 12 b for 48 h. Normalised data are expressed as the fold changes, with the control set to ‘1’. *p < 0.05 **p < 0.01 indicate statistically significant differences from the corresponding control in unpaired t-tests.

Table 5. Effect of Compound 12 b on levels of BAX, and Bcl-2 genes expression in A549 cells treated for48 h.

Sample	Gene expression (Fold Change)^a
	BAX	Bcl-2	BAX/Bcl-2 ratio
A549	1.00 ± 0.22	1.00 ± 0.13	1.00 ± 0.21
12b / A549	3.33 ± 0.37**	0.40 ± 0.08*	8.80 ± 1.41**

^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 6. In vitro cytotoxicity of 12 b and erlotinib against normal cells (WI-38).

Comp.	Cytotoxicity WI-38 IC50 (µM)^a	Selectivity index (SI)
		A549^b	HCT-116^c
12b	39.15	4.77	2.00
Erlotinib	33.75	4.99	1.76

^aThe results were the mean of three replicates.

^bSI = cytotoxicity against WI-38 cells/cytotoxicity against A549 cell line.

^cSI = cytotoxicity against WI-38 cells/cytotoxicity against HCT-116 cell line.

Figure 8. Selectivity indices of compound 12 b.

Table 7. 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	−19.63	−16.09
7b	−20.55	−17.79
9	−21.00	−19.01
8	−21.80	−19.60
10	−17.06	−15.83
11a	−21.58	−20.10
11b	−21.67	−20.19
12a	−23.07	−21.14
12b	−23.09	−20.59
12c	−23.67	−21.66
13a	−19.69	−20.30
13b	−21.74	−20.77
Erlotinib	−22.59	–
TAK-285	–	−21.49

Figure 9. (A) Superimposition of the re-docked conformer of erlotinib (pink) over the co-crystallised conformer (green) with an RMSD value of 1.18 Å. (B) Superimposition of the re-docked conformer of TAK-285 (pink) over the co-crystallised conformer (green) with an RMSD value of 1.66 Å.

Figure 10. (A) 3D interaction of erlotinib docked into the active site of EGFR^WT. The hydrogen bonds were represented in green dashed lines. The pi interactions were represented in orange lines. (B) 2D interaction of erlotinib docked into the active site of EGFR^WT.

Figure 11. (A) 3 D interaction of compound 12 b docked into the active site of EGFR^WT. The hydrogen bonds were represented in green dashed lines. The pi interactions were represented in orange lines. (B) 2 D interaction of compound 12 b docked into the active site of EGFR^WT.

Figure 12. (A) 3 D interaction of TAK-285 docked into the active site of EGFR^T790M. The hydrogen bonds were represented in green dashed lines. The pi interactions were represented in orange lines. (B) 2 D interaction of TAK-285docked into the active site of EGFR^T790M.

Figure 13. (A) 3 D interaction of compound 12 _b docked into the active site of EGFR^T790M. The hydrogen bonds were represented in green dashed lines. The pi interactions were represented in orange lines. (B) 2 D interaction of compound 12 _b docked into the active site of EGFR^T790M.

Table 8. Predicted ADMET for the designed compounds and reference drugs

Comp.	BBB level^a	Solubility level^b	Absorption level^c	CYP2D6 prediction^d	PPB prediction^e
7a	2	2	0	false	false
7b	2	2	0	false	true
9	1	2	0	false	true
8	4	1	1	false	true
10	3	2	0	false	true
11a	4	1	1	false	true
11b	4	1	2	false	true
12a	4	1	1	false	true
12b	4	1	2	false	true
12c	4	1	2	false	true
13a	4	2	0	false	true
13b	4	1	1	false	true
Erlotinib	1	2	0	false	true

^aBBB level, blood brain barrier level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low.

^bSolubility level, 1 = very low, 2 = low, 3 = good, 4 = optimal.

^cAbsorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.

^dCYP2D6, cytochrome P2D6, TRUE = inhibitor, FALSE = non inhibitor.

^ePBB, plasma protein binding, FALSE means less than 90%, TRUE means more than 90%.

Table 9. In silico toxicity studies of the synthesised compounds and erlotinib

Comp.	Ames mutagenicity	Developmental Toxicity Potential	Carcinogenic Potency TD50 (Rat)^a	Rat Maximum Tolerated Dose (Feed)^b	Ocular Irritancy	Skin Irritancy
7a	Non-Mutagen	Non-Toxic	34.965	0.287	Mild	None
7b	Non-Mutagen	Non-Toxic	27.147	0.401	Mild	None
9	Non-Mutagen	Non-Toxic	4.054	0.266	Mild	None
8	Non-Mutagen	Non-Toxic	1.528	0.213	Mild	None
10	Non-Mutagen	Non-Toxic	18.673	0.291	Mild	None
11a	Non-Mutagen	Non-Toxic	1.655	0.152	Mild	None
11b	Non-Mutagen	Non-Toxic	2.675	0.391	Mild	None
12a	Non-Mutagen	Non-Toxic	3.791	0.294	Mild	None
12b	Non-Mutagen	Non-Toxic	2.321	0.358	Mild	None
12c	Non-Mutagen	Non-Toxic	2.98	0.139	Mild	None
13a	Non-Mutagen	Non-Toxic	32.038	0.530	Mild	None
13b	Non-Mutagen	Non-Toxic	24.690	0.735	Mild	None
Erlotinib	Non-Mutagen	Non-Toxic	8.057	0.083	Mild	None

^aUnit: mg/kg body weight/day.

^bUnit: g/kg body weight.

Figure 15. values of different energy components obtained from MM-GBSA analysis. Bars represent the standard deviation values.

Figure 16. The decomposition of the free binding energy of amino acids around 10 Å of compound.12b.

Figure 17. The 3 D interaction between compound 12 b and EGFR in each of the representative frame for each cluster. Amino acids are shown as blue sticks. compound 12 b is shown as brown sticks. Grey dashed lines: hydrophobic interaction. Blue solid lines: hydrogen bonds.

Table 10. The number and types of interactions between compound 12 b and EGFR as obtained from PLIP webserver for the representative frame of each cluster. Amino acids in bold are common in all of the clusters representative.

Cluster number	Number of hydrophobic interactions	Amino acids in receptor	Number of hydrogen bonds	Amino acids in receptor
C1	9	L694 - V702 - K721 (2) - L753 - L764 - R817 - L820 - T830	1	T830
C2	9	L694 - V702 - K721 (2) - L764 - T766 - L768 - R817 - T830	1	T830
C3	11	L694 - V702 - K721 - L764 (2) - T766 - L768 - R817 - L820 - T830 - L834	1	T830

Li R, Tang H, Fu H, et al. Arynes double bond insertion/nucleophilic addition with vinylogous amides and carbodiimides. J Org Chem 2014;79:1344–55.

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