Discovery of novel 2-aminopyridine derivatives as ROS1 and ALK dual inhibitors to combat drug-resistant mutants including ROS1^G2032R and ALK^G1202R

Figures & data

Figure 1. Structures of representative ROS1/ALK dual inhibitors.

Figure 2. Design strategies of target compounds based on cocrystal structures of Crizotinib with ROS1^WT (PDB 3ZBF) and ALK^WT (PDB 2XP2). (A) Docking structure of Crizotinib binding to apo-G2032R ROS1 receptor. (B) Docking structure of Crizotinib binding to apo-G1202R ALK receptor. (C) 2D structure of Crizotinib. (D) Docking posture of Crizotinib binding to ROS1 receptor. (E) Design strategies for novel 2-aminopyridine analogues as ROS1/ALK dual inhibitors. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article).

Figure 3. (A–C) General synthetic routes of 2-aminopyridine derivatives. Reagents and conditions: (i) (-)-DIP-Cl, THF, −35 °C, 3 h, rt, 18 h; (ii) diethanolamine, TBME, 60 °C, reflux, 1 h, rt, 2 h; (iii) DIAD, PPh₃, 2-nitropyridin-3-ol, dry THF, 0 °C, 8 h; (iv) Fe, AcOH, EtOH, 85 °C, reflux, 2 h; (v) NBS, MeCN, 0 °C, 30 min; (vi) potassium carbonate, Pd(PPh₃)₄, DOX, H₂O, 85 °C, 8 h.

Table 1. The enzymatic and anti-proliferative activities of target compounds A01–A08.

COM	R^1	R^2	Enzymatic activity					Cellular IC50 (μM)^c
			ROS1		ALK		c-MET	HCC-78	NCI-H3122
			Inhibition (1μM)^a	IC50 (nM)^b	Inhibition (1μM)	IC50 (nM)	Inhibition (1μM)
A01	H		101%	173.2	90%	407.1	5%	28.7 ± 5.8	152.1 ± 44.4
A02	H		101%	99.5	65%	457.5	2%	36.4 ± 3.7	148.6 ± 18.5
A03	H		85%	317.2	65%	454.1	14%	50.5 ± 14.0	101.4 ± 22.8
A04	H		64%	963.9	37%	1544.0	5%	ND^d	ND
A05	F		43%	ND	18%	ND	−10%	278.4 ± 53.1	168.2 ± 39.4
A06	F		100%	83.1	73%	231.1	11%	16.0 ± 1.2	10.2 ± 0.1
A07	F		87%	81.4	69%	ND	18%	ND	ND
A08	F		91%	119.6	58%	ND	22%	13.9 ± 6.3	36.8 ± 2.5
Crizotinib	–	–	101%	9.31	100%	17.7	ND	4.5 ± 1.4	4.1 ± 0.5

^aValues indicate the kinase inhibition % at 1 μM, which are the average of two independent experiments.

^bIC₅₀ values are the average of two independent experiments.

^cIC₅₀ values are presented as mean values of at least three independent experiments.

^dNo detection.

Table 2. The enzymatic and anti-proliferative activities of target compounds B01–B10.

COM	R^1	R^2	Enzymatic activity					Cellular IC50 (μM)^c
			ROS1		ALK		c-MET	HCC-78	NCI-H3122
			Inhibition (1μM)^a	IC50 (nM)^b	Inhibition (1μM)	IC50 (nM)	Inhibition (1μM)
B01			81%	103.2	30%	ND^d	0%	33.9 ± 1.8	185.5 ± 8.6
B02			86%	216.2	30%	ND	6%	64.8 ± 4.5	689.4 ± 57.2
B03			74%	374.3	88%	286.0	21%	52.7 ± 15.2	206.8 ± 40.7
B04			ND	80.5	59%	ND	28%	28.0 ± 4.5	127.6 ± 25.5
B05			90%	259.7	42%	1165.0	26%	21.3 ± 0.7	96.2 ± 6.9
B06			90%	192.5	44%	ND	9%	7.1 ± 0.7	24.8 ± 5.0
B07			99%	65.7	99%	216.8	8%	16.9 ± 1.4	14.3 ± 3.6
B08			103%	37.4	100%	67.4	64%	4.6 ± 0.8	5.7 ± 0.6
B09			99%	55. 9	92%	75.9	50%	11.4 ± 0.5	8.5 ± 1.2
B10			98%	147.8	82%	ND	60%	7.7 ± 1.2	13.9 ± 1.2
Crizotinib	–	–	101%	9.31	100%	17.7	ND	4.5 ± 1.4	4.1 ± 0.5

^aValues indicate the kinase inhibition % at 1 μM, which are the average of two independent experiments.

^bIC₅₀ values are the average of two independent experiments.

^cIC₅₀ values are presented as mean values of at least three independent experiments.

^dNo detection.

Table 3. The enzymatic and anti-proliferative activities of target compounds C01–C07.

COM	R^1	Enzymatic activity					Cellular IC50 (μM)^c
		ROS1		ALK		c-MET	HCC-78	NCI-H3122
		Inhibition (1μM)^a	IC50 (nM)^b	Inhibition (1μM)	IC50 (nM)	Inhibition (1μM)
C01		100%	4.7	101%	25.6	70%	3.9 ± 0.5	3.0 ± 0.1
C02		102%	4.6	98%	30.1	69%	4.4 ± 0.5	2.8 ± 0.9
C03		101%	11.6	94%	56.1	63%	4.6 ± 0.23	1.9 ± 0.1
C04		100%	16.5	100%	38.8	43%	ND^d	2.4 ± 0.3
C05		100%	77.3	101%	79.7	59%	4.6 ± 0.1	2.4 ± 0.1
C06		98%	31.2	91%	214.1	58%	3.1 ± 0.3	1.4 ± 0.1
C07		95%	73.6	55%	413.6	18%	4.2 ± 0.2	2.4 ± 0.1
Crizotinib	–	101%	9.31	100%	17.7	ND	4.5 ± 1.4	4.1 ± 0.5

^aValues indicate the kinase inhibition % at 1 μM, which are the average of two independent experiments.

^bIC₅₀ values are the average of two independent experiments.

^cIC₅₀ values are presented as mean values of at least three independent experiments.

^dNo detection.

Figure 4. In vitro activities against Crizotinib-resistant ROS1^G2032R and ALK^G1202R mutants. Dose–response curves for proliferation of Ba/F3 CD74-ROS1^WT cell (A), Ba/F3 CD74-ROS1^L2026M cell (B), Ba/F3 CD74-ROS1^G2032R cell (C), Ba/F3 EML4-ALK^WT cell (D) and Ba/F3 EML4-ALK^L1196M cell (E) after 72 h exposure to B08, C01–C03, and Crizotinib. (F) Enzyme-based activities of B08, C01, C02, and Crizotinib against ALK^G1202R mutant. All data are the average of n ≥ 2 ± standard deviation.

Table 4. Enzymatic and cellular activities to ROS1 and ALK mutants.

Compound	Ba/F3 cellular activities (nM)^a					Enzymatic activities (nM)^b
	CD74-	CD74-	CD74-	EML4-	EML4-	ALK^L1196M	ALK^G1202R
	ROS1^WT	ROS1^L2026M	ROS1^G2032R	ALK^WT	ALK^L1196M
B08	20.9 ± 1.7	168.5 ± 56.3	317.0 ± 92.1	681.3 ± 151.0	1809.7 ± 270.5	>1000	195.2
C01	7.6 ± 0.6	33.1 ± 1.4	42.0 ± 3.1	145.2 ± 36.0	119.7 ± 17.3	404.2	49.1
C02	5.4 ± 1.0	21.3 ± 4.7	107.4 ± 12.6	191.4 ± 51.8	148.2 ± 27.3	643.4	275.5
C03	18.3 ± 1.2	38.3 ± 5.9	94.4 ± 31.9	228.0 ± 47.3	185.1 ± 22.2	ND^c	491.6
Crizotinib	3.8 ± 0.1	31.5 ± 8.2	1266.8 ± 366.0	151.7 ± 26.5	699.9 ± 42.8	206.6	422.0
Lorlatinib	ND	1.1 ± 0.1	55.1 ± 22.8	ND	ND	ND	ND

^aIC₅₀ values are presented as mean values of at least three independent experiments.

^bIC₅₀ values are the average of two independent experiments.

^cNo detection.

The activity of ROS1^G2032R mutant cells is the representative result of compound C01, which is much better than its lead compound Crizotinib are indicated in bold.

Figure 5. Inhibition of cellular phosphorylation of ROS1, ALK, and ERK by C01 in CD74-ROS1^WT (A), CD74-ROS1^L2026M (B), CD74-ROS1^G2032R (C), and EML4-ALK^L1196M (D) cells investigated by western blot analysis, with Crizotinib as the positive control. Cells were treated with Crizotinib or C01 at the indicated concentrations for 4 h and the levels of protein were evaluated by western blot analysis of cell lysates using specific antibodies.

Figure 6. (A) Flow cytometric analysis of apoptosis in HCC-78 cell line treated with C01 in the concentrations of 0 (control), 2.0 μM, 3.9 μM, and 7.8 μM. (B) Graphical representation of total percentage of apoptosis induced by C01 in HCC-78 cell line. (C) Flow cytometric analysis of apoptosis in NCI-H3122 cell line treated with C01 in the concentrations of 0 (control), 1.5 μM, 3.0 μM and 6.0 μM. (D) Graphical representation of total percentage of apoptosis induced by C01 in NCI-H3122 cell line. (*p < 0.05; **p < 0.01; ***p < 0.001 vs control, n = 3).

Figure 7. Representative structures from MD simulation of C01 (A) and Crizotinib (B) in the active site of ROS1^G2032R. (ROS1 in coloured cartoon, ligands, and residues in stick mode. Yellow dots represent hydrogen bonds). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) (C) 2D diagram of the interaction between C01 and the binding site of ROS1^G2032R. (D) 2D diagram of the interaction between Crizotinib and the binding site of ROS1^G2032R.