Design, synthesis, apoptotic, and antiproliferative effects of 5-chloro-3- (2-methoxyvinyl)-indole-2-carboxamides and pyrido[3,4-b]indol-1-ones as potent EGFR^WT/EGFR^T790M inhibitors

Figures & data

Figure 1. Structures of indole-based EGFR inhibitors I-VI.

Figure 2. Structures of new targets 5a-g, 6a-f, 7a, and 7b.

Scheme 1. Synthesis of compounds 5a-g, 6a-f, 7a, and 7b. Reagents and conditions: (a) NaH, (Boc)₂O, DMF, 0 °C to rt, overnight, 79%; (b) CH₃OCH₂P⁺(C₆H₅)₃Cl^-, t-BuOK, THF, 0 °C to rt, overnight, 76%; (c) 5% NaOH, EtOH, 40 °C, 93%; (d) Amine, BOP, DIPEA, DCM, rt, overnight; (e) PTSA, toluene, reflux, overnight.

Table 1. IC₅₀ of compounds 5a-g, 6a-f, 7a, and 7b against four cancer cell lines.

Comp.	Cell viability %	Antiproliferative activity IC50 ± SEM (nM)
		A-549	MCF-7	Panc-1	HT-29	Average (GI50)
5a	91	75 ± 8	79 ± 8	73 ± 7	75 ± 8	76
5b	89	76 ± 7	79 ± 7	75 ± 7	78 ± 7	77
5c	87	45 ± 5	49 ± 5	45 ± 5	47 ± 5	47
5d	89	34 ± 4	38 ± 3	35 ± 3	38 ± 4	36
5e	90	70 ± 7	73 ± 7	67 ± 7	71 ± 7	70
5f	87	28 ± 3	30 ± 3	27 ± 3	30 ± 3	29
5 g	89	30 ± 3	32 ± 3	30 ± 3	32 ± 3	31
6a	86	89 ± 9	92 ± 9	85 ± 9	89 ± 9	89
6b	87	102 ± 10	105 ± 10	97 ± 10	104 ± 10	102
6c	85	67 ± 6	69 ± 6	62 ± 6	64 ± 6	66
6d	89	85 ± 8	87 ± 8	81 ± 8	85 ± 8	85
6e	91	45 ± 5	47 ± 5	43 ± 4	45 ± 5	45
6f	89	40 ± 4	42 ± 4	38 ± 4	42 ± 4	41
7a	90	53 ± 5	57 ± 5	51 ± 5	55 ± 5	54
7b	89	57 ± 6	59 ± 6	55 ± 5	57 ± 6	57
Erlotinib	ND	30 ± 3	40 ± 3	30 ± 3	30 ± 3	33

ND: Not determined

Table 2. IC₅₀ of compounds 5c, 5d, 5f, 5 g, 6e, and 6f against EGFR^WT and EGFR^T790M.

Compound	EGFR^WT inhibition	EGFR^T790M inhibition
	IC50 ± SEM (nM)	IC50 ± SEM (nM)
5c	102 ± 7	ND
5d	85 ± 5	ND
5f	68 ± 4	9.5 ± 2
5g	74 ± 5	11.9 ± 3
6e	98 ± 7	ND
6f	93 ± 6	ND
Erlotinib	80 ± 5	ND
Osimertinib	–	8 ± 2

ND: Not determined

Table 3. Caspase-3 level for compounds 5f, 5 g, and staurosporine in human pancreatic cancer cell line (Panc-1).

Compd. No.	Caspase-3
	Conc (Pg/ml)	Fold change
5f	560.2 ± 5.0	8.5
5g	542.5 ± 5.0	8.3
Staurosporine	503.2 ± 4.0	7.6
Control	65.6	1

Table 4. Caspase-8, Bax and Bcl-2 levels for compounds 5f, 5 g and staurosporine on human pancreatic cancer cell line (Panc-1).

Compd. No.	Caspase-8		Bax		Bcl-2
	Conc (ng/ml)	Fold change	Conc (Pg/ml)	Fold change	Conc (ng/ml)	Fold reduction
5f	2.10	25	305.50	37	0.80	6
5g	1.97	23	299.40	36	0.84	6
staurosporine	1.75	21	276.20	Fold reduction	0.98	5
Control	0.085	1	8.25	1	5.08	1

Figure 3. Docking representation models for compounds 5f and 5 g; (A) 3D-docked models of compound 5f (green) aligned with 5 g (purple) within the active site of EGFR^WT showing the interaction surface of the protein (electrostatics; green: hydrophobic, blue; positive and red: negative); (B) 2D-docked model of compound 5f within the active site of EGFR^WT; (C) 3D-docked models of compound 5f (green) aligned with 5 g (purple) within the active site of EGFR^T790M showing the interaction surface of the protein (electrostatics; green: hydrophobic, blue; positive and red: negative); (D) 2D-docked model of compound 5f within the active site of EGFR^T790M.

Table 5. Ligand-protein complex interactions of the tested compounds 5c, 5d, 5f, 5 g, 6e and 6f within the active site of EGFR^WT.

Compd.	MOE Score kcal/mol	Hydrogen bond interactions	Hydrophobic interactions	Other interactions
Erlotinib	−9.37	Met769	Leu694, Leu820, Val702,	Lys721
		Gln767	Gly722, Thr766, Thr830
5c	−8.85	Asp831	Leu694, Leu820, Asp776,	----------
			Gly722, Cys773, Asp831.
5d	−9.30	Met769	Leu694, Leu820, Val702,	Glu738 (ionic)
			Gly722, Thr766, Pro770.	Leu694 (Pi-H)
5f	−9.88	Asp831	Leu694, Leu820, Asp776,	Asp776 (ionic)
		Asp776	Gly722, Cys773, Asp831.	Cys773, Gly772 (Pi-H)
5g	−9.47	Asp831	Leu694, Leu820, Asp776,	Cys773, Gly772 (Pi-H)
			Gly722, Cys773, Asp831.
6e	−8.69	Asp831	Leu694, Leu820, Asp776,	Cys773 (Pi-H)
			Gly722, Cys773, Asp831.
6f	−9.01	Met769	Leu694, Leu820, Val702,	Leu820 (Pi-H)
			Gly722, Thr766, Asp776,
			Glu780

Table 6. Ligand-protein complex interactions of the tested compounds 5f and 5 g within the active site of EGFR^T790M.

Compd.	MOE Score kcal/mol	Hydrogen bond interactions	Hydrophobic interactions	Other interactions
Co-crystalized ligand (6HJ)^a	−10.42	Met790	Leu844, Cys797, Leu718, Val726, Met790	Val726 (Pi-H)
		Gln791		Cys797 (covalent)
		Met793
5f	−9.43	Asp855	Asp800, Phe723, Leu844, Cys797, Leu718, Val726, Met790, Lys745.	Asp800 (Ionic)
		Cys797		Leu844 (Pi-H)
		Asp800
5g	−9.31	Asp855	Asp800, Phe723, Leu844, Cys797, Leu718, Val726, Met790	Asp800 (Ionic)
		Asp800		Lys745 (Pi-cation)
				Leu844 (Pi-H)

^a (R)-1–(3-(4-amino-3–(1-methyl-1H-indol-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.

Table 7. Physicochemical and pharmacokinetic properties (Lipinski parameters) of compounds 5f and 5 g.

Compd.	MW	^nROTB	HBA	HBD	Violations	MR	TPSA	Log P
5f	438	8	2	2	0	131.03	57.36	4.68
5g	440	8	3	2	0	127.31	66.59	3.83

Table 8. ADME properties of compounds 5f and 5 g.

Compd.	GI Abs.	BBB	P-gp substrate	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
5f	High	Yes	Yes	No	Yes	Yes	Yes	Yes
5g	High	Yes	Yes	Yes	Yes	Yes	Yes	Yes