Design, synthesis, and biological evaluation of thiazole/thiadiazole carboxamide scaffold-based derivatives as potential c-Met kinase inhibitors for cancer treatment

Figures & data

Figure 1. Structures of representative small molecule c-Met inhibitors.

Figure 2. Design strategy of target compounds.

Scheme 1. Synthesis of the intermediates 13a–13g; reagents and conditions: (a) conc. HNO₃ (40%), 0 °C, overnight; (b) DMF-DMA, toluene, reflux, 10 h; (c) Fe (powder), AcOH, 80 °C, 2 h; (d) POCl₃, DMF (cat.), reflux, 1 h; (e) (1) 4-nitrophenols, PhCl, 140 °C, 20 h; (2) SnCl₂, EtOH, 70 °C, 6 h.

Scheme 2. Synthesis of the intermediates 17, 22, and 26; reagents and conditions: (a) formamide, 170 °C, 10 h; (b) POCl₃, DMF (cat.), toluene, reflux, 6 h; (c) 4-amino-2-fluorophenol, NaH, DMF, 0 °C, 1.5 h; (d) (1) aq. NaOH, reflux, 30 min; (2) oxalic acid, 1-propanol, 38 °C, 45 min; (e) triethyl orthoformate, 2,2-dimethyl-1,3-dioxane-4,6-dione, 85 °C, overnight; (f) Ph₂O, 240 °C, 30 min; (g) POCl₃, DMF (cat.), 0 °C → reflux, 2 h; (h) (1) 2-fluoro-4-nitrophenol, K₂CO₃, Ph₂O, 160 °C, 6 h; (2) SnCl₂, MeOH, 70 °C, 6 h; (i) HCOOH, Ac₂O, 0 °C → rt, 12 h; (j) HCONH₂, 150 °C, 8 h; (k) oxalyl chloride, DMF (cat.), CH₂Cl₂, 0 °C → reflux, 3 h; (l) 4-amino-2-fluorophenol, NaH, DMF, 80 °C, 2 h.

Scheme 3. Synthesis of the intermediate 30; reagents and conditions: (a) formamide, 150 °C, 8 h; (b) POCl₃, 0 °C → reflux, 6 h; (c) 4-amino-2-fluorophenol, NaH, DMSO, 0 °C → 60 °C, overnight.

Scheme 4. Synthesis of the intermediate 32; reagents and conditions: (a) R = H, 4-amino-2-fluorophenol, NaH, DMF, 0 °C → 80 °C, overnight; R = Cl, 4-amino-2-fluorophenol, NaH, DMF, 0 °C → 100 °C, 3 h.

Scheme 5. Synthesis of the intermediates 37, 42, 46, and 50; reagents and conditions: (a) (1) SOCl₂, 80 °C, 4 h; (2) NH₄OH, THF, 0 °C → rt, 30 min; (b) (chlorothio)formyl chloride, toluene, 100 °C, 3 h; (c) ethyl cyanoformate, n-dodecane, 160 °C, 16 h; (d) aq. LiOH, MeOH, rt, 4 h; (e) ethyl oxalyl monochloride, POCl₃, 70 °C, 6 h; (f) t-BuONO, CuBr₂, CH₃CN, 60 °C, 30 min; (g) Pd(OAc)₂, Xantphos, NMM, toluene, H₂O, rt, 7 h; (h) aq. LiOH, MeOH, 0 °C, 1 h; (i) NBS, p-TsOH, CH₃CN, 50 °C, 24 h; (j) ethyl thiooxamate, EtOH, reflux, 6 h; (k) (1) urotropin, CHCl₃, 50 °C, 2 h; (2) conc. HCl, EtOH, reflux, 2 h; (l) ethyl oxalyl monochloride, TEA, CH₂Cl₂, 0 °C → rt, overnight; (m) P₂S₅, CHCl₃, reflux, 5 h.

Scheme 6. Synthesis of target compounds 51a–51an; reagents and conditions: (a) oxalyl chloride, DMF (cat.), dry CH₂Cl₂, 0 °C → rt, 1.5 h; (b) corresponding amine, TEA, dry CH₂Cl₂, 0 °C → rt, 4–6 h; (c) corresponding amine, HATU, TEA, DMF, rt, overnight.

Table 1. In vitro c-Met inhibitory activities of target compounds 51a–51an.

Compd.	A	R1	Linker	R2	IC50 (nM)^a
51a	A1	H		C6H5	56.64 ± 4.82
51b	A1	H	C1	4-F-C6H5	50.15 ± 5.14
51c	A1	H		C6H5	45.67 ± 3.88
51d	A1	H	C2	4-F-C6H5	41.53 ± 2.76
51e	A1	H		C6H5	34.48 ± 4.65
51f	A1	H	C3	4-F-C6H5	29.05 ± 3.53
51g	A1	H		C6H5	39.36 ± 3.25
51h	A1	H	C4	4-F-C6H5	35.42 ± 5.03
51i	A1	2-F	C3	C6H5	31.25 ± 4.16
51j	A1	3-F	C3	C6H5	29.54 ± 3.42
51k	A1	2-Cl	C3	C6H5	33.05 ± 2.66
51l	A1	3-Cl	C3	C6H5	31.86 ± 4.51
51m	A1	2-Me	C3	C6H5	39.82 ± 5.47
51n	A1	3-Me	C3	C6H5	45.58 ± 6.03
51o	A2	3-F	C3	C6H5	29.64 ± 3.84
51p	A2	3-F	C3	4-F-C6H5	24.67 ± 1.95
51q	A3	3-F	C3	C6H5	38.48 ± 4.23
51r	A3	3-F	C3	4-F-C6H5	35.26 ± 5.19
51s	A4	3-F	C3	C6H5	43.26 ± 6.25
51t	A4	3-F	C3	4-F-C6H5	40.42 ± 3.66
51u	A5	3-F	C3	C6H5	40.36 ± 5.53
51v	A5	3-F	C3	4-F-C6H5	37.56 ± 2.88
51w	A6	3-F	C3	C6H5	24.27 ± 1.67
51x	A6	3-F	C3	4-Me-C6H5	31.54 ± 4.25
51y	A6	3-F	C3	4-OMe-C6H5	36.68 ± 2.97
51z	A6	3-F	C3	3,4-Di-OMe-C6H5	45.51 ± 6.28
51aa	A6	3-F	C3	4-F-C6H5	15.62 ± 2.43
51ab	A6	3-F	C3	3-F-C6H5	23.24 ± 2.16
51ac	A6	3-F	C3	2-F-C6H5	18.49 ± 3.05
51ad	A6	3-F	C3	4-Cl-C6H5	17.78 ± 1.82
51ae	A6	3-F	C3	3-Cl-C6H5	23.42 ± 3.61
51af	A6	3-F	C3	4-Br-C6H5	19.23 ± 1.44
51ag	A6	3-F	C3	4-CF3-C6H5	39.41 ± 5.25
51ah	A6	3-F	C3	3,4-Di-Cl-C6H5	9.26 ± 1.34
51ai	A6	3-F	C3	2-Naphthyl	61.36 ± 4.58
51aj	A6	3-F	C3	2-Thienyl	49.55 ± 5.36
51ak	A6	3-F	C3	3-Cl-4-F-C6H5	3.89 ± 0.52
51al	A6	3-F	C3	3,4-Di-F-C6H5	5.23 ± 0.74
51am	A7	3-F	C3	3-Cl-4-F-C6H5	2.54 ± 0.49
51an	A7	3-F	C3	3,4-Di-F-C6H5	3.73 ± 0.62
Foretinib^b					1.96 ± 0.23

^a IC₅₀ (50% inhibitory concentration) values are expressed as mean ± standard deviation (SD). Each assay was performed in triplicate with duplicated samples.

^b Positive control.

Table 2. In vitro antiproliferative activities of compounds 51a–51an against six different human cell lines.

Compd.	IC50 (µM)^a
	MKN-45^b	A549^c	HT-29^d	MDA-MB-231^e	HUVEC^f	FHC^g
51a	9.25 ± 0.86	26.35 ± 1.24	24.45 ± 1.68	>50	>100	>100
51b	7.89 ± 1.35	22.48 ± 3.15	26.53 ± 1.42	38.65 ± 4.07	>100	>100
51c	6.94 ± 1.06	20.56 ± 1.96	29.26 ± 3.85	34.66 ± 2.58	53.45 ± 4.62	71.34 ± 5.25
51d	6.65 ± 0.94	18.98 ± 2.46	24.63 ± 1.87	33.56 ± 4.35	52.35 ± 3.85	68.63 ± 4.83
51e	5.86 ± 0.93	16.55 ± 2.16	21.75 ± 1.66	>50	56.54 ± 4.81	64.52 ± 6.12
51f	4.95 ± 0.82	14.68 ± 3.27	22.36 ± 1.94	31.84 ± 2.56	45.52 ± 3.83	ND^h
51g	6.21 ± 0.85	17.43 ± 2.23	23.42 ± 1.58	32.76 ± 2.23	48.36 ± 2.54	54.47 ± 3.25
51h	5.97 ± 0.98	16.84 ± 1.58	22.73 ± 3.32	34.25 ± 2.82	42.14 ± 4.35	50.22 ± 2.89
51i	5.35 ± 0.84	15.63 ± 1.86	22.89 ± 2.43	35.53 ± 4.62	44.32 ± 5.64	60.24 ± 5.33
51j	4.58 ± 0.75	13.85 ± 1.76	19.56 ± 2.89	32.34 ± 2.78	45.57 ± 5.12	>100
51k	6.87 ± 0.86	14.52 ± 1.64	23.36 ± 4.32	33.28 ± 4.21	>100	>100
51l	6.23 ± 0.72	13.25 ± 2.41	20.86 ± 3.56	32.11 ± 2.54	40.86 ± 2.32	51.65 ± 3.96
51m	6.43 ± 0.76	14.05 ± 1.87	22.34 ± 1.75	35.25 ± 4.23	38.84 ± 3.55	49.62 ± 5.04
51n	7.21 ± 0.95	17.48 ± 2.25	24.41 ± 2.14	ND	>100	>100
51o	4.23 ± 0.96	12.25 ± 1.63	18.66 ± 3.13	29.58 ± 3.54	36.62 ± 4.23	50.27 ± 3.58
51p	3.17 ± 0.84	9.83 ± 1.22	15.24 ± 2.26	28.13 ± 1.96	33.56 ± 2.68	46.36 ± 6.24
51q	5.95 ± 0.63	12.86 ± 1.82	19.36 ± 2.48	32.53 ± 2.68	69.54 ± 2.68	80.63 ± 4.52
51r	5.02 ± 0.77	10.54 ± 1.76	18.27 ± 3.62	28.86 ± 3.13	40.58 ± 2.25	51.24 ± 4.61
51s	6.05 ± 0.94	18.45 ± 2.33	25.56 ± 1.76	32.78 ± 2.70	43.42 ± 3.65	65.49 ± 5.56
51t	5.45 ± 0.65	14.46 ± 2.87	23.12 ± 3.34	36.62 ± 2.83	41.36 ± 2.39	67.44 ± 4.38
51u	5.53 ± 1.26	16.82 ± 2.28	26.42 ± 3.22	35.89 ± 2.52	38.84 ± 3.53	48.25 ± 5.04
51v	4.23 ± 0.94	12.61 ± 1.83	19.24 ± 2.85	29.11 ± 2.52	54.35 ± 4.16	63.28 ± 3.35
51w	2.53 ± 0.62	7.26 ± 1.37	12.48 ± 2.39	26.29 ± 3.09	38.64 ± 3.53	48.25 ± 5.04
51x	3.46 ± 0.75	8.65 ± 1.72	14.29 ± 2.13	28.57 ± 1.85	30.76 ± 2.43	43.41 ± 3.68
51y	3.22 ± 0.86	9.28 ± 1.85	13.95 ± 2.26	28.49 ± 2.35	32.43 ± 1.98	ND
51z	4.05 ± 1.12	11.26 ± 1.96	15.62 ± 2.51	20.52 ± 3.14	26.52 ± 3.84	>100
51aa	0.95 ± 0.18	4.83 ± 0.72	4.36 ± 0.38	11.68 ± 1.39	15.32 ± 2.16	24.63 ± 4.23
51ab	1.49 ± 0.63	6.02 ± 0.87	6.95 ± 0.82	10.13 ± 1.88	12.74 ± 0.83	20.25 ± 3.62
51ac	1.94 ± 0.26	7.54 ± 0.62	9.53 ± 0.75	18.06 ± 2.14	19.45 ± 0.83	31.34 ± 2.63
51ad	1.74 ± 0.23	6.75 ± 0.55	8.04 ± 0.93	15.20 ± 1.46	18.52 ± 2.04	26.46 ± 1.28
51ae	1.95 ± 0.68	7.08 ± 0.95	9.62 ± 1.56	19.20 ± 2.35	13.05 ± 0.95	24.50 ± 1.84
51af	2.06 ± 0.45	8.23 ± 1.42	11.65 ± 1.63	20.54 ± 3.65	11.45 ± 0.83	21.55 ± 2.82
51ag	5.84 ± 1.21	12.36 ± 2.85	20.37 ± 3.02	30.58 ± 2.64	43.22 ± 3.53	ND
51ah	0.53 ± 0.42	3.17 ± 0.51	2.78 ± 0.49	7.29 ± 1.22	12.36 ± 0.85	20.44 ± 1.86
51ai	6.12 ± 1.38	15.33 ± 2.81	21.49 ± 1.58	30.26 ± 2.16	>100	>100
51aj	4.55 ± 0.91	11.46 ± 2.17	17.53 ± 2.44	25.87 ± 3.41	52.26 ± 3.74	>100
51ak	0.22 ± 0.04	1.81 ± 0.34	1.56 ± 0.32	4.24 ± 0.68	8.52 ± 1.35	13.20 ± 0.84
51al	0.35 ± 0.07	2.26 ± 0.68	1.94 ± 0.65	5.82 ± 0.73	6.14 ± 0.75	10.17 ± 1.65
51am	0.092 ± 0.006	0.83 ± 0.36	0.68 ± 0.09	3.94 ± 1.32	2.54 ± 0.46	8.63 ± 1.52
51an	0.15 ± 0.07	1.32 ± 0.35	0.94 ± 0.27	4.65 ± 0.93	4.83 ± 0.69	9.54 ± 1.26
Foretinib^i	0.058 ± 0.006	0.45 ± 0.05	0.37 ± 0.05	0.89 ± 0.14	0.68 ± 0.09	4.06 ± 0.75

^a Fifty percent inhibitory concentration after 48 h of drug treatment obtained from MTT assay. IC₅₀ values are expressed as mean ± standard deviation (SD). Each assay was performed in triplicate with duplicated samples.

^b Gastric cancer.

^c Lung carcinoma.

^d Colon adenocarcinoma.

^e Triple-negative breast cancer.

^f Human umbilical vein endothelial cells.

^g Human normal colorectal mucosa epithelial cells.

^h Not determined.

ⁱ Positive control.

Figure 3. The effect of 51am and foretinib on MKN-45 cells apoptosis by Annexin V/PI double staining.

Figure 4. The effect of 51am and foretinib on MKN-45 cells cycle arrest by PI staining with RNase.

Figure 5. 51am inhibited c-Met phosphorylation in MKN-45 cells.

Table 3. c-Met mutant and kinase selectivity profile of 51am.

Enzyme	IC50 (nM)	Enzyme	IC50 (nM)
c-Met H1094R	93.6	Ron	3.83
c-Met D1228H	29.4	PDGFRα	425
c-Met Y1230H	45.8	PDGFRβ	513
c-Met Y1235D	54.2	VEGFR-2	527
c-Met M1250T	26.5	EGFR	>10 000
Wild type c-Met	2.54	Flt-3	6.12
c-kit	4.94	Flt-4	276

Table 4. Pharmacokinetic profile of 51am in BALB/c mice^a.

Compd.	51am
Route	i.v. (1.5 mg/kg)	p.o. (10 mg/kg)
T1/2 (h)	3.2	5.6
Cmax (ng/mL)	552	1756
Tmax (h)	–	4.1
AUC0–∞ (µg.h/mL)	2.5	11.5
CL (L/h.kg)^b	0.6	–
F/%^c	–	69

^a Data reported as the average of six animals.

^b CL = dose/AUC_i.v..

^c F = (AUC_p.o./AUC_i.v.) × (Dose_i.v./Dose_p.o.) × 100%.

Figure 6. (a) The proposed binding mode of compound 51a (C1 scaffold as moiety C) with the active site of c-Met. Compound was shown in coloured sticks, green: carbon atom, blue: nitrogen atom, pink: oxygen atom, yellow: sulphur atom; (b) the proposed binding mode of compound 51c (C2 scaffold as moiety C) with the active site of c-Met; (c) the proposed binding mode of compound 51e (C3 scaffold as moiety C) with the active site of c-Met; (d) the proposed binding mode of compound 51g (C4 scaffold as moiety C) with the active site of c-Met; (e) the proposed binding mode of compound 51am with the active site of c-Met; (f) the proposed binding mode of compound 51am with the active site of VEGFR-2.