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

Abstract

As part of our continuous efforts to discover novel c-Met inhibitors as antitumor agents, four series of thiazole/thiadiazole carboxamide-derived analogues were designed, synthesised, and evaluated for the in vitro activity against c-Met and four human cancer cell lines. After five cycles of optimisation on structure–activity relationship, compound 51am was found to be the most promising inhibitor in both biochemical and cellular assays. Moreover, 51am exhibited potency against several c-Met mutants. Mechanistically, 51am not only induced cell cycle arrest and apoptosis in MKN-45 cells but also inhibited c-Met phosphorylation in the cell and cell-free systems. It also exhibited a good pharmacokinetic profile in BALB/c mice. Furthermore, the binding mode of 51am with both c-Met and VEGFR-2 provided novel insights for the discovery of selective c-Met inhibitors. Taken together, these results indicate that 51am could be an antitumor candidate meriting further development.

Graphical Abstract

Keywords:

• Thiazole
• thiadiazoles
• c-Met inhibitors
• biological evaluation
• docking study

Introduction

Cancer, a leading cause of death second only to cardiovascular diseases, is a serious public health issue to human worldwide according to WHO.¹ The uncontrolled proliferation of abnormal cells is a predominant feature of cancer initiation and progression. Protein kinases, which transfer the terminal phosphate group of ATP to tyrosine, threonine, or serine residues of proteins, play critical roles in plenty of processes such as cell growth, differentiation, metastasis, homeostasis, and death.^2–5 Protein kinases have been intensively explored for drug discovery, resulting ∼80+ small molecule kinase inhibitors (SMKIs) that have been commercialised for targeted therapy of cancer and other diseases.⁶ However, SMKIs are still emerged with two critical challenges: (i) lack of exquisite selectivity for target kinase versus other kinases and (ii) acquired resistance after different periods of clinical usage.

Cellular-mesenchymal epithelial transition factor (c-Met) is a structurally unique transmembrane member of the receptor tyrosine kinases.^7,8 Upon activation by hepatocyte growth factor (HGF) and/or cross-talk with other signalling pathways, the intracellular C-terminal docking domain recruits and then activates a broad range of downstream signalling effectors and adaptors.^9–11 Deregulated HGF/c-Met signalling hijacks a series of cellular processes and promotes the tumours initiation and progression. Moreover, the overexpression of HGF and/or c-Met has been detected in various human solid tumours and is closely associated with poor prognosis and resistance to other kinase inhibitors.¹² Small-molecule c-Met inhibitors have attracted considerable attention because it can inhibit c-Met irrespective of the activation mechanism.^13–16 Based on the structural characteristics and bonding modes to c-Met, the reported c-Met inhibitors can be categorised into two types as shown in Figure 1. Type I c-Met inhibitors bind to the ATP binding pocket of c-Met with a U-shaped conformation. On the contrary, type II c-Met inhibitors not only bind to the ATP active site but also extend the inactive DFG out conformation engaging in hydrophobic interactions. Similar to other kinase inhibitors, the efficacy of c-Met inhibitors is also compromised due to the spot mutations at the ATP-binding site, such as Y1230C and D1228V/N/H for type I inhibitors,^17–19 and due to low selectivity for type II inhibitors.^11,12

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

Accumulating evidencesuggests that the mutations near the active site of c-Met may be overcome by type II inhibitors since their binding interactions with c-Met extend beyond the entrance of c-Met’s active site resulting in additional hydrophobic interactions.^20–24 The structure of type II c-Met inhibitors can be disconnected into four moieties (A–D) based on their interactions with c-Met (Figure 1), among which moiety C serves as a linker to form H-bonds and plays a vital role against c-Met.^25–27 Moreover, the low kinase selectivity associated with type II c-Met inhibitors could be improved significantly by modifying the moiety C.^28–30 By analysing the conformation of type II inhibitors with c-Met, we found that the linear moiety C commonly assumed a pseudocyclic via intramolecular H-bonding; therefore, incorporation of various rings into the moiety C could possess the inherent advantage to generate novel c-Met inhibitors, exemplified by BMS-777607 (6) ³¹ and AMG-458 (7) .^28,32 Pharmacophore merging, an efficient strategy to boost efficacy and overcome drug resistance, has been applied widely in drug design and discovery.³³ The amide functional group is found in many biologically active molecules, including peptides, proteins and clinically approved drugs, and displays unique ability to form H-bonding interaction.³⁴ Thiazole and thiadiazole are two classes of five-membered heterocycles and their derivatives exhibit important pharmaceutical activities such as antitumor, antibacterial, antifungal, antitubercular, and analgesic.^35–37 Thiadiazole is considered as the bioisostere of pyrimidine and oxadiazole; its mesoionic nature allows analogues to cross cellular membranes and interact with biological targets and the sulphur atom imparts improved liposolubility.^38–41 Recent studies have demonstrated the utilisation of thiadiazole as a promising scaffold for antitumor drug discovery by inhibiting diverse targets, such as histone deacetylase (HDAC), c-Src/Abl tyrosine kinase, focal adhesion kinase (FAK) and tubulin polymerisation for 1,3,4-thiadiazoles,^38,39 as well as cathepsin K and glycogen synthase kinase-3β for 1,2,4-thiadiazoles.⁴² Thiazole is another privileged scaffold in lead identification given its ability to interact with crucial biological targets involved in diseases ^43,44 and is also found in several approved drugs and many drug candidates.^45,46 Moreover, introducing thiadiazole/thiazole moiety can be used to tune whole physicochemical and pharmacokinetic properties of candidates. In addition, thiazole and thiadiazole are assumed to preferentially form H-bonding with c-Met given its electron-rich characteristics.⁴⁷

Given the above considerations and insights, in this work, we postulated that incorporation of the intrinsic antitumor functional fragments, thiazole/thiadiazole carboxamide, into the moiety C could facilitate the discovery of novel type II c-Met inhibitors with improved efficacy and selectivity, and reduced toxicity. Meanwhile, modifications of the moieties A, B, and D were performed simultaneously to investigate the effect on activity and improve the potential drug-likeness. Therefore, based on the general chemical structure of type II c-Met inhibitors and the rational drug design, a small-molecular library of 40 novel derivatives was prepared and evaluated (Figure 2).

Figure 2. Design strategy of target compounds.

Results and discussion

Chemistry

The anilines 13a–13g were synthesised starting from 3,4-dimethoxyacetophenone 8 as shown in Scheme 1.^22,29,30,48 Compound 8 was subjected to regioselective nitration to afford o-nitroacetophenone 9, which underwent aminomethylenation with DMF-DMA to give α,β-unsaturated ketone 10. Cyclisation of 10 in the presence of Fe powder and AcOH followed by chlorination with POCl₃ furnished chloride 12. Intermediate 12 was coupled with various substituted p-nitrophenols and the resulting aryl ethers were reduced with SnCl₂ to give the desired 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.

The synthesis of intermediates 17, 22 and 26 is depicted in Scheme 2 using 3-amino-2-thiophene carboxylic acid methyl ester 14 as starting material.^30,49,50 Cyclisation of 14 with formamide yielded ketone 15, which was converted into 17 by employing a two-step strategy through chlorination and nucleophilic substitution under basic conditions. Material 14 was converted into 18 via hydrolysation followed by decarboxylation. Condensation of 18 with 2,2-dimethyl-1,3-dioxane-4,6-dione in the presence of triethyl orthoformate produced imine 19, which was used directly. Heating of 19 in Ph₂O generated ketone 20, which was chlorinated with POCl₃ to afford chloride 21. Treatment of 21 through nucleophilic substitution and reduction yielded compound 22. On the other hand, material 14 underwent an efficient sequence of formylation, cyclisation, chlorination, and condensation to provide 26 in good overall yield.

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.

The intermediate 30 was prepared according to the synthetic route described in Scheme 3.^30,51 The starting material 2-amino-4,5-dimethoxybenzoic acid 27 underwent a sequence of cyclisation and chlorination under basic conditions to afford 4-chloroquinazoline 29 in good overall yield. Treatment of 29 with 4-amino-2-fluorophenol in the presence of NaH gave the desired compound.

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.

The synthesis of 32a–32b was similar to those described for 17, 26, and 30. For access to 4-phenoxy substituted pyridine 32, 4-chloropyridines 31a–31b were subjected to SnAr conditions in the presence of 4-amino-2-fluorophenol, which displaced the halogen at the 4-position of pyridine (Scheme 4).^31,52

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.

The intermediates 37, 42, 46, and 50 were synthesised according to the synthetic sequence outlined in Scheme 5. Acid 33 underwent acylation and animation to afford amide 34. Condensation of 34 with (chlorothio)formyl chloride in toluene at 100 °C yielded 1,3,4-oxathiazol-2-one 35,⁵³ which was treated with ethyl cyanoformate (ECF) in n-dodecane to generate 1,2,4-thiadiazole-5-carboxylic acid ethyl ester 36.⁵⁴ 1,2,4-Thiadiazole-5-carboxylic acid 37 was obtained smoothly via hydrolysation of 36 in the mixture of aq. LiOH and MeOH at room temperature.⁵⁵ Cyclisation of thiosemicarbazide 38 with ethyl oxalyl monochloride on exposure of POCl₃ produced 5-amino-1,3,4-thiadiazol-2-carboxylic acid ethyl ester 39, which was converted into 5-bromo-1,3,4-thiadiazole-2-carboxylic acid ethyl ester 40 via Sandmeyer bromination in the presence of t-BuONO and CuBr₂.⁵⁶ Suzuki–Miyaura coupling of 40 with corresponding boronic acid was performed using a highly active catalyst system Pd(OAc)₂/Xantphos in combination with NMM to give 41 in high yield and excellent quality,⁵⁶ which was then hydrolysed in the mixture of aq. LiOH/MeOH at 0 °C for 1 h to afford lithium salt 42. Treatment of ketone 43 with NBS afforded the α-bromoketone 44, which was subjected to ethyl thiooxamate to generate thiazole-2-carboxylic acid ethyl ester 45.⁵⁷ α-Bromoketones 44a–44b were converted to the corresponding α-aminoketones 47a–47b via Delépine reaction in which the α-bromoketone was reacted with urotropin followed by cleavage of the resulting salt with conc. HCl,⁵⁸ treatment of which with ethyl oxalyl monochloride produced 48. Cyclisation of 48 with phosphorus pentasulfide in CHCl₃ furnished thiazole-2-carboxylic acid ethyl ester 49.⁵⁹ The intermediates 46 and 50 were prepared from 45 and 49 respectively using a similar procedure for the preparation of 37.

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.

The synthesis of target compounds 51a–51an is summarised in Scheme 6. Compounds 51a–51b and 51e–51an were prepared by acylation of various amines (13, 17, 22, 26, 30, and 32) with corresponding carbonyl chloride, which was generated by the reaction of prepared acid (37, 46, and 50) with oxalyl chloride catalysed by DMF. On the contrary, given the fact that the 1,3,4-thiadiazole-2-carboxylic acid is unstable in solution because it undergoes a spontaneous decarboxylation process, therefore, 1,3,4-thiadiazole carboxamide scaffold-based derivatives 51c–51d were synthesised by direct condensation of lithium salt 42 with corresponding aniline.⁶⁰ All target compounds were purified by flash column chromatography on silica gel using hexane–ethyl acetate as eluent, and their structures were characterised by NMR, MS, IR and elemental analysis.

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.

Biology

In vitro c-Met enzymatic assay and SAR

Inhibition of target compounds on c-Met kinase activity was measured by using the homogeneous time-resolved fluorescence (HTRF) assay. Foretinib was used as the reference compound, and the screening results (IC₅₀ ± SD) as mean values of experiments performed in triplicate are shown in Table 1.

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.

The results in Table 1 suggested that most of the target compounds displayed varying degrees of potency against c-Met, with IC₅₀ values ranging from 2.54 to 61.36 nM. It is worth to note that five of them (51ah, IC₅₀ = 9.26 nM; 51ak, IC₅₀ = 3.89 nM; 51al, IC₅₀ = 5.23 nM; 51am, IC₅₀ = 2.54 nM; 51an, IC₅₀ = 3.73 nM) exhibited comparable potency against c-Met relative to foretinib, indicating that introduction of thiazole-2-carboxamide (C3) as the 5-atom linker could obtain potent c-Met inhibitory activity. Using the type II kinase inhibitor binding element hybrid design approach, we started to explore the structure–activity relationships (SARs) by varying the moieties A, B, C and D. Considering the critical role of 5-atom linker on activity, the SAR study of moiety C was initially performed. Meanwhile, 6,7-dimethoxyquinoline core from the launched c-Met inhibitor cabozantinib was introduced into the moiety A to synergistically generate better inhibitory activity. Compounds 51a–51b (51a, IC₅₀ = 56.64 nM, R₁ = H, R₂ = C₆H₅, A1, C1; 51b, IC₅₀ = 50.15 nM, R₁ = H, R₂ = 4-F-C₆H₅, A1, C1) and 51c–51d (51c, IC₅₀ = 45.67 nM, R₁ = H, R₂ = C₆H₅, A1, C2; 51d, IC₅₀ = 41.53 nM, R₁ = H, R₂ = 4-F-C₆H₅, A1, C2) only displayed moderate inhibitory activity against c-Met compared to foretinib. Based on these results, replacement of the thiadiazole carboxamides (C1 and C2) with thiazole carboxamides (C3 and C4) was performed to extend our investigation for the discovery of c-Met inhibitors. To our delight, compounds 51e–51h (51e, IC₅₀ = 34.48 nM, R₁ = H, R₂ = C₆H₅, A1, C3; 51f, IC₅₀ = 29.05 nM; R₁ = H, R₂ = 4-F-C₆H₅, A1, C3; 51g, IC₅₀ = 39.36 nM, R₁ = H, R₂ = C₆H₅, A1, C4; 51h, IC₅₀ = 35.42 nM; R₁ = H, R₂ = 4-F-C₆H₅, A1, C4) exhibited greater potency than compounds 51a–51d, suggesting that the thiazole carboxamide moiety was more suitable for H-binding interactions with c-Met than that of thiadiazole carboxamide. Notably, C3-based derivatives exhibited higher potency than that of C4 derived analogues (51e, IC₅₀ = 34.48 nM vs. 51g, IC₅₀ = 39.36 nM; 51f, IC₅₀ = 29.05 nM vs. 51h, IC₅₀ = 35.42 nM) when other moieties of compounds were fixed, revealing that the relative position of heteroatom in thiazole ring had a non-negligible influence on c-Met. Therefore, C3-based derivatives were further studied. Investigation of the effect of the substituents and its positions (R₁) on the middle benzene ring was performed. Addition of one halogen atom (such as F and Cl) on middle benzene ring (51i, IC₅₀ = 31.25 nM, R₁ = 2-F, R₂ = C₆H₅, A1, C3; 51j, IC₅₀ = 29.54 nM, R₁ = 3-F, R₂ = C₆H₅, A1, C3; 51k, IC₅₀ = 33.05 nM, R₁ = 2-Cl, R₂ = C₆H₅, A1, C3; 51l, IC₅₀ = 31.86 nM, R₁ = 3-Cl, R₂ = C₆H₅, A1, C3) increased activity relative to 51e (51e, IC₅₀ = 34.48 nM), especially for compound 51j in which one F atom was located at the 3-position of benzene ring. In contrast, changing the halogen atom to a methyl group (51m, IC₅₀ = 39.82 nM, R₁ = 2-Me, R₂ = C₆H₅, A1, C3; 51n, IC₅₀ = 45.58 nM, R₁ = 3-F, R₂ = C₆H₅, A1, C3) led to a decrease of activity.

With the promising moiety C (C3) and suitable moiety B (3-F-Phenyl) in hand, the screening of moiety A was subsequently conducted. Replacing the 6,7-dimethoxyquinoline A1 to 6,7-dimethoxyquinazoline A2 displayed comparable inhibitory activity against c-Met (51o, IC₅₀ = 29.64 nM, R₁ = 3-F, R₂ = C₆H₅, A2, C3 vs. 51j, IC₅₀ = 29.54 nM, R₁ = 3-F, R₂ = C₆H₅, A1, C3). However, replacement of the 6,7-dimethoxyquinoline A2 with thieno[3,2-b]pyridine A3 (51q, IC₅₀ = 38.48 nM, R₁ = 3-F, R₂ = C₆H₅, C3; 51r, IC₅₀ = 35.26 nM, R₁ = 3-F, R₂ = 4-F-C₆H₅, C3), thieno[3,2-d]pyrimidine A4 (51s, IC₅₀ = 43.26 nM, R₁ = 3-F, R₂ = C₆H₅, C3; 51t, IC₅₀ = 40.42 nM, R₁ = 3-F, R₂ = 4-F-C₆H₅, C3) and thieno[2,3-d]pyrimidine A5 (51u, IC₅₀ = 40.36 nM, R₁ = 3-F, R₂ = C₆H₅, C3; 51v, IC₅₀ = 37.56 nM, R₁ = 3-F, R₂ = 4-F-C₆H₅, C3) resulted in a slight loss of potency in comparison with 51o–51p, suggesting that sulphur-containing fused rings were not suitable for moiety A when C3 was adopted as the 5-atom linker. To our delight, switching the 6,7-dimethoxyquinoline A2 to 2-chloropyridine A6 (51w, IC₅₀ = 24.27 nM, R₁ = 3-F, R₂ = C₆H₅, C3; 51aa, IC₅₀ = 15.62 nM, R₁ = 3-F, R₂ = 4-F-C₆H₅, C3) resulted in an increase of the activity against c-Met compared to 51o–51p. Further studies on the inhibitory potency of moiety A are currently in progress in our laboratory.

After three optimisation cycles, the effect of moiety D (R₂) on c-Met was further extensively studied. Using 51w (IC₅₀ = 24.27 nM, R₁ = 3-F, R₂ = C₆H₅, C3) as a reference compound, incorporation of mono-electron-donating groups (mono-EDGs) showed a decreased potency, such as 51x (IC₅₀ = 31.54 nM, R₁ = 3-F, R₂ = 4-Me-C₆H₅, C3, A6), 51y (IC₅₀ = 36.68 nM, R₁ = 3-F, R₂ = 4-OMe-C₆H₅, C3, A6) and 51z (IC₅₀ = 45.51 nM, R₁ = 3-F, R₂ = 3,4-di-OMe-C₆H₅, C3, A6). Therefore, investigation of EDGs was not conducted. In contrast, introducing mono-electron-withdrawing groups (such as F, Cl, and Br) led to improved activity. It should be noted that the activity was correlated with the position of substituents on benzene ring, such as the mono-EWG at the 4-position of benzene ring (51aa, IC₅₀ = 15.62 nM, R₁ = 3-F, R₂ = 4-F-C₆H₅, C3, A6) displayed higher inhibitory activity than that of mono-EWG at the 3- (51ab, IC₅₀ = 23.24 nM, R₁ = 3-F, R₂ = 3-F-C₆H₅, C3, A6) and 2-position of benzene ring (51ac, IC₅₀ = 18.49 nM, R₁ = 3-F, R₂ = 2-F-C₆H₅, C3, A6), the similar trend was also observed in chlorine substituted compounds 51ad (IC₅₀ = 17.78 nM, R₁ = 3-F, R₂ = 4-Cl-C₆H₅, C3, A6) and 51ae (IC₅₀ = 23.42 nM, R₁ = 3-F, R₂ = 3-Cl-C₆H₅, C3, A6). Addition of a strong EWG (51ag, IC₅₀ = 39.41 nM, R₁ = 3-F, R₂ = 4-CF₃-C₆H₅, C3, A6) led to a significant loss in activity in comparison to 51w. 2-Thiophene analogue 51aj (IC₅₀ = 49.55 nM, R₁ = 3-F, R₂ = 2-thienyl, C3, A6) exhibited 2.1-fold loss of activity compared to 51w, revealing that the electron-rich five-membered ring was not favourable for c-Met inhibitory activity. The bulky substituent (51ai, IC₅₀ = 46.87 nM, R₁ = 3-F, R₂ = 2-naphthyl, C3, A6, decreased 2.6-fold) also weakened the potency, indicating the hydrophobic pocket of c-Met was fairly sensitive to the size of terminal tail. Based on the above results, modification of moiety D was further explored by introducing double electron-withdrawing groups, which showed an additional increase on c-Met inhibitory efficacy than that of mono-electron-withdrawing-based derivatives (51ak, IC₅₀ = 3.89 nM, R₁ = 3-F, R₂ = 3-Cl-4-F-C₆H₅, C3, A6; 51al, IC₅₀ = 5.23 nM, R₁ = 3-F, R₂ = 3,4-di-F-C₆H₅, C3, A6), which showed that the terminal benzene ring substituted with double-electron withdrawing groups was preferable for c-Met inhibitory potency. Inspired by the promising core of BMS-777607 (6), two other derivatives (51am, IC₅₀ = 2.54 nM, R₁ = 3-F, R₂ = 3-Cl-4-F-C₆H₅, C3, A7; 51an, IC₅₀ = 3.73 nM, R₁ = 3-F, R₂ = 3,4-di-F-C₆H₅, C3, A7) designed by addition of another chlorine atom at the 3-position of pyridine exhibited positive influence on inhibitory potency. In summary, of these 40 newly synthesised compounds, 51am was the most active compound with an IC₅₀ value of 2.54 nM against c-Met, demonstrating that the favourable moiety A, a suitable linker (moiety C) as well as an appropriate terminal hydrophobic tail were all crucial for the discovery of novel type II c-Met inhibitors.

In vitro antiproliferative activity

MET amplification has been reported in gastric cancer, one of the solid tumours with the high incidence rate, and is associated with poor prognosis. Lung cancer is also closely related to the overexpression of HGF and/or MET and poor prognosis. Recent studies have showed that the c-Met activation in colon cancer occurs in a ligand-dependent paracrine manner. Moreover, c-Met inhibitor SGX523 effectively inhibited the phosphorylation of c-Met and its downstream protein Akt in MDA-MB-231 cells.⁶¹ Based on these findings, four cancer cell lines (MKN-45, A549, HT-29, and MDA-MB-231) with confirmed high expression of c-Met ^62,63 were selected for in vitro cytotoxicity assays to determine the antiproliferative effect of the target compounds and further examine whether it was a specific consequence of the dysregulation of c-Met. Additionally, the selectivity of target compounds towards cancer cells versus human normal cell lines (HUVEC and FHC) was determined concurrently. As shown in Table 2, all target compounds were evaluated for in vitro cytotoxicity and selectivity relative to normal cell lines, using foretinib as the positive control. The results showed that all target compounds displayed moderate to significant cytotoxicity against four types of tumour cell lines and demonstrated a certain degree of selectivity for the normal cell lines, HUVEC and FHC. Among them, the most promising compound 51am showed remarkable antiproliferative activity against A549, HT-29, and MDA-MB-231 cell lines with IC₅₀ values of 0.83, 0.68, and 3.94 μM, respectively, and possessed a higher selectivity index (SI, IC₅₀ of normal cells/IC₅₀ of tumour cells) than foretinib. These positive results indicated that utilising thiazole/thiadiazole carboxamide as the moiety C was favourable for cytotoxicity. In addition, some of the target compounds (51ak, 51am–51an) exhibited acceptable cytotoxicity against MDA-MB-231 cells compared with foretinib, indicating that these compounds have potential to overcome drug resistance in some specific tumour types. Moreover, most of the target compounds showed greater cytotoxicity against MKN-45 cells than other three cell lines, indicating that the MKN-45 cells were more sensitive to these compounds.

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.

Preliminary SAR correlations were formulated to facilitate further identification of more efficient c-Met inhibitors. It was observed that compounds 51e–51f containing thiazole-2-carboxamide scaffold (C3) conferred a better effect on cytotoxicity than compounds 51a–51d and 51g–51h. Regarding the effect of substituents on the benzene ring (moiety B), the cytotoxicity of compounds 51i–51n followed the rank order of F > Cl > Me in all tested cancer cell lines. Except for methyl derivatives 51m–51n, the results suggested that incorporating a halogen atom at 3-position of benzene ring resulted in better cytotoxicity than that its 2-position (51j > 51i, 51l > 51k). Moreover, the pyridine core was favoured because of the promising cytotoxicity results for 51w–51an. The SAR of moiety D was then examined, and the results demonstrated that weak electron withdrawing groups (51aa and 51ac–51ad) on the benzene ring were favourable for cytotoxic activity. However, electron donating groups (51x–51z), strong electron withdrawing group (51ag), and bulky group (51ai) were detrimental to cytotoxic potency. The substitution positions on the benzene ring also resulted in significant changes in cytotoxicity, with the cytotoxic results of compounds 51aa–51ae showing a clear preference (4-F > 3-F > 2-F, 4-Cl > 3-Cl). Furthermore, the dual substituted electron withdrawing groups on the phenyl ring (51ah, 51ak–51an) generally afforded greater inhibitory activity than mono electron withdrawing groups (51aa–51af). When exploring the heterocycle (2-thienyl, 51aj) and fused ring (2-naphthyl, 51ai) as moiety D, we found that both of them were unfavourable for toxicity. On the basis of the in vitro results, compound 51am was selected for further biological evaluation.

Induction of apoptosis and cycle arrest

As shown in Table 2, compound 51am displayed higher sensitivity to MKN-45 cells than other three tested cancer cell lines in the preliminary cytotoxicity profile, therefore, MKN-45 cells were used in the subsequent mechanistic study. Apoptosis is one of the main modes of cell death after drug treatment, which is controlled by the expression of many regulatory factors. To determine the antiproliferative potency of 51am on human cancer cells, its effect on MKN-45 cells apoptosis and cycle arrest was determined using Annexin V/PI double staining and analysed by flow cytometer. As shown in Figure 3, after MKN-45 cells were treated with different concentrations of compound 51am (0.4, 0.8, and 1.2 μM) for 24 h, the percentages of apoptotic cells were determined to be 14.74%, 19.83%, and 38.54%, respectively, which were higher than that of the control group (6.02%), suggesting that 51am induced MKN-45 cell apoptosis in a dose-dependent manner. More importantly, the percentage of apoptotic cells following treatment with 51am was comparable to that of foretinib (51am vs. foretinib, 38.54% vs. 41.21%) at the same concentration (1.2 μM). We then investigated the effect of 51am on cell cycle distribution of MKN-45 cells. As depicted in Figure 4, the percentage of cells in G2/M phase significantly increased in a dose-dependent manner compared with the control group after treatment with 51am (0.4, 0.8, and 1.2 μM) for 24 h. Notably, cell cycle distribution of MKN-45 cells after treatment with 1.2 μM of 51am exhibited a similar trend to that observed for foretinib. Taken together, these results suggested that 51am could effectively induce cell cycle arrest and apoptosis in a dose-dependent manner, consequently inhibiting the proliferation of cancer cells.

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.

Western blotting analysis of c-Met phosphorylation

To obtain further insight into the mechanism of 51am induced apoptosis, western blot assay was carried out to investigate the effect of 51am on the phosphorylation of c-Met in living cells. MKN-45 cells were treated with 51am for 24 h in a series of concentrations (0, 2.5, 5.0, and 10.0 μM) using DMSO as the negative control, and the level of GAPDH served as the loading control. As depicted in Figure 5, compound 51am inhibited c-Met phosphorylation in a dose-dependent manner, which was consistent with the observed in vitro activity. This suggested that the antiproliferative activity of 51am may be partially due to the inhibition of c-Met kinase.

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

In vitro kinase profile of compound 51am

The emerging oncogenic mutations of c-Met that confer resistance to small molecule c-Met inhibitors have become critical issues to be solved urgently. Consequently, 51am was tested for anti-drug resistance against a spectrum of mutations. As shown in Table 3, 51am maintained potency against mutations H1094R, D1228H, Y1230H, Y1235D, and M1250T, with IC₅₀ values of 93.6, 29.4, 45.8, 54.2, and 26.5 nM, respectively. On the other hand, comprehensive understanding the off-target kinase inhibition is critical for any kinase inhibitors, particularly to help explain the correlations between efficacy and potential side effects. Given the remarkable potency of 51am against wild type c-Met and its mutants, we moved forward to investigate the kinase selectivity profile of 51am against other protein kinases. In addition to high potency against c-Met, it displayed good inhibitory effects against c-Kit (IC₅₀ = 4.94 nM), Flt-3 (IC₅₀ = 6.12 nM), and Ron (IC₅₀ = 3.83 nM). In contrast to its high potency against c-Met, 51am afforded 108–207-fold less potent against PDGFRα, PDGFRβ, VEGFR-2, and Flt-4. It also showed negligible inhibitory activity against EGFR with IC₅₀ > 10 μM. Notably, in terms of selectivity between c-Met and VEGFR-2 for candidate compounds, 51am (c-Met, IC₅₀: 2.54 nM; VEGFR-2, IC₅₀: 527 nM) showed greater selectivity than foretinib (c-Met, IC₅₀: 1.96 nM; VEGFR-2, IC₅₀: 4.58 nM), suggesting that the C3 scaffold-based type II c-Met inhibitors could confer selectivity against c-Met, overcome acquired drug resistances, and possibly reduce VEGFR-2 related side effects.

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

Pharmacokinetic profile of compound 51am

Identification of drugs with an acceptable balance of potency, physical properties, and pharmacokinetics (PK) has always been a challenge that often hinders evaluation of the compounds in xenograft models and therapeutic use in clinical practice. Therefore, the PK profile of compound 51am was evaluated in BALB/c mice after intravenous (i.v.) injection and oral administration (p.o.) because of its excellent in vitro activity. As shown in Table 4, after p.o. (10 mg/kg), 51am showed a promising overall PK profile, including rapid absorption (T_max = 4.1 h), high maximum concentration (C_max = 1756 ng/mL), good plasma exposure (AUC_0–∞ = 11.5 µg.h/mL), acceptable elimination half-life (T_1/2 = 5.6 h), and favourable clearance (CL = 0.87 L/h.kg). When administered via the i.v. route (1.5 mg/kg), 51am was characterised by slightly better maximum concentration (C_max = 552 ng/mL) and plasma exposure (AUC_0–∞ = 2.5 µg.h/mL), and a good half-life (T_1/2 = 3.2 h). Moreover, 51am exhibited good oral bioavailability (F = 69%) in BALB/c mice. Based on the good overall PK profile, 51am could be a potential candidate for cancer therapy.

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%.

Binding mode analysis

To further explore the binding modes of target compounds to the specific kinases, molecular docking simulation studies were performed concurrently. Amino acid sequences for c-Met and VEGFR-2 were retrieved from the Uniprot database with accessible numbers P08581 and P35968, respectively. 3D crystal structures for c-Met (PDB ID: 3zcl) and VEGFR-2 (PDB ID: 1ywn) with the lowest resolutions at 1.4 Å and 1.7 Å, respectively, were chosen for docking and molecular dynamics simulations. Given the fact that C3-based derivatives generally exhibited favourable potency in both biochemical and cellular assays in vitro than other three types of moiety C, molecular docking study was further performed to explore the binding details of block C. Compounds 51a, 51c, 51e, and 51g were selected as template molecules, in which the moieties A, B, and D were fixed only the moiety C was varied. As shown in Figure 6(a), compound 51a bearing the C1 moiety only formed one hydrogen bond between the oxygen atom of carbonyl (moiety C) and residue Lys1110 of c-Met (estimated binding energy: –9.46 kcal/mol), which could be attributed to the intramolecular repulsion by the conformational change induced by 1,2,4-thiadiazole moiety. For compound 51c, the delicate structural difference between 51a and 51c gave rise to the movement of 51c towards the left in the active site of c-Met (Figure 6(b)), resulting in formation of another hydrogen bond between the nitrogen atom of quinoline core and residue Met1160 in addition to the hydrogen bond formed by the H atom of NH (moiety C) and Asp1222 (estimated binding energy: –10.25 kcal/mol). As shown in Figure 6(c), we clearly observed that there were three hydrogen bonds between 51e and c-Met kinase, the nitrogen atom of quinoline core and residue Met1160, the oxygen atom of carbonyl (moiety C) and residue Lys1110, and the nitrogen atom of thiazole and residue Asp1222, most likely due to the elimination of intramolecular repulsion when changing the thiadiazole ring to a thiazole ring and the newly formed favourable hydrophobic interactions (estimated binding energy: –11.85 kcal/mol). Conversely, when exchanging the positions of the S and N atoms, the nitrogen atom of thiazole of compound 51g formed one hydrogen bond with Lys1110 instead of Asp1222 (Figure 6(d)) (estimated binding energy: –13.06 kcal/mol). Subsequently, the binding modes of 51am with c-Met or VEGFR-2 were analysed as shown in Figure 6(e,f), since VEGFR-2 associated side-effects have been a vital concern in designing and discovering novel antitumor candidates. As shown in Figure 6(e), 51am generated three hydrogen bonds with c-Met and also formed excellent hydrophobic interactions with hydrophobic amino acid residues; whereas, only two hydrogen bonds were produced between 51am and VEGFR-2. The hydrophobic interactions of 51am/VEGFR-2 complex decreased significantly because the hydrophobic amino acids in the hydrophobic cavity of c-Met were replaced with amino acids with shorter side chains for VEGFR-2 (Figure 6(f)). In summary, these docking results could provide a basis for the rational design of novel c-Met inhibitors with high selectivity.

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.

Conclusions

By combining the type II c-Met inhibitors binding element hybrid design approach with the pharmacophore merging strategy, thiazole/thiadiazole carboxamide scaffold-based derivatives were designed, synthesised, and evaluated for their antitumor activity. After five cycles of optimisation, 51am was found to be as the most potent c-Met inhibitor among the synthesised target compounds in both enzyme- and cell-based assays. Further biological evaluation demonstrated that 51am not only induced cell cycle arrest and cell apoptosis but also inhibited c-Met activity in cell and cell-free systems. 51am simultaneously possessed acceptable inhibitory efficacy against a spectrum of c-Met mutations and moderate selectivity for wild type c-Met compared with other test kinases. More importantly, 51am achieved a better balance between cell-based activity and the favourable pharmacokinetic profile. In addition, the binding modes of 51am to both c-Met and VEGFR-2 offered insights for designing novel c-Met inhibitors with high selectivity. Taken together, these positive results suggested that 51am could be a potential c-Met inhibitor deserving further development.

Experimental

Chemistry

Common chemicals (reagents and solvents) used in this paper were purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. (Shanghai, China). All reactions were monitored by thin layer chromatography (TLC) on silica gel 60 GF254 plates (Shanxi Nuotai Biotechnology Co., Ltd, Yuncheng, China) and the spots were visualised using UV light (254 nm). Flash column chromatography was performed using silica gel (200–300 mesh, Qingdao, China) with a linear solvent gradient. Mass spectra were recorded on a Bruker Daltonics APEXII49e spectrometer (Billerica, MA). ¹H and ¹³C NMR spectra were measured on a Bruker Avance III spectrometer (Billerica, MA) and referenced to TMS. Chemical shifts are recorded as δ in units of parts per million (ppm) and coupling constants are given in Hz. Peak multiplicities are defined as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). Melting points were measured by using a Gongyi X-5 microscopy digital melting point apparatus and are uncorrected. IR spectra were obtained on an NIC-5DX spectrophotometer. The purity of all tested compounds was determined by HPLC (Agilent Technologies 1100 series, Santa Clara, CA) equipped with a C-18 bound-phase column (Eclipse Plus C18, 5 μM particle size, 4.6 mm × 250 mm). A gradient elution was performed with MeOH and water as a mobile phase and was monitored at 254 nm. All tested target compounds were >95% pure.

General procedure for the preparation of 4-((6,7-dimethoxyquinolin-4-yl)oxy)anilines (13a–13g)

The key intermediates 13a–13g were prepared according to the methods of our previous works (Supporting Information),^22,29,30 therefore, only characterisation data for the title compounds are presented here.

4-((6,7-Dimethoxyquinolin-4-yl)oxy)aniline (13a).^22,29

Light yellow solid; m.p. 212–214 °C. IR (KBr) ν_max/cm^–1 3375, 3152, 1582, 1509, 1478, 1431, 1344, 1248, 1216, 994, 898, 849, 818. ¹H NMR (400 MHz, DMSO-d₆) δ 8.42 (d, J = 4.0 Hz, 1H), 7.50 (s, 1H), 7.36 (s, 1H), 6.92 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 8.0 Hz, 2H), 6.36 (d, J = 4.0 Hz, 1H), 5.17 (br s, 2H), 3.93 (s, 6H).

4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-methylaniline (13b)

Greyish yellow solid; m.p. 206–208 °C. IR (KBr) ν_max/cm^–1 3402, 3211, 1650, 1579, 1505, 1479, 1461, 1431, 1344, 1299, 1247, 1228, 1208, 1167, 1001, 948, 892, 852, 817. ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J = 5.2 Hz, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 6.85 (s, 1H), 6.81 (dd, J = 2.4, 8.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.36 (d, J = 5.2 Hz, 1H), 4.90 (br s, 2H), 3.92 (s, 6H), 2.08 (s, 3H).

4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-methylaniline (13c)

Yellow solid; m.p. 209–211 °C. IR (KBr) ν_max/cm^–1 3331, 3189, 1623, 1578, 1507, 1481, 1432, 1349, 1300, 1272, 1254, 1211, 1167, 993, 892, 850, 824. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J = 5.2 Hz, 1H), 7.54 (s, 1H), 7.36 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.55 (s, 1H), 6.50 (dd, J = 2.4, 8.4 Hz, 1H), 6.25 (d, J = 5.2 Hz, 1H), 5.07 (br s, 2H), 3.93 (s, 6H), 1.94 (s, 3H).

2-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)aniline (13d)

Orange solid; m.p. 234–236 °C. IR (KBr) ν_max/cm^–1 3405, 3191, 1579, 1505, 1478, 1459, 1432, 1344, 1300, 1247, 1215, 1164, 994, 913, 849, 816. ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 5.2 Hz, 1H), 7.48 (s, 1H), 7.36 (s, 1H), 7.19 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 2.8, 8.8 Hz, 1H), 6.92–6.90 (m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 5.42 (br s, 2H), 3.93 (s, 3H), 3.92 (s, 3H).

3-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)aniline (13e)

Pale yellow solid; m.p. 213–215 °C. IR (KBr) ν_max/cm^–1 3419, 3343, 1625, 1592, 1580, 1508, 1477, 1427, 1347, 1302, 1255, 1219, 1158, 989, 843, 826. ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.38 (s, 1H), 7.09 (d, J = 8.8 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 2.4, 8.4 Hz, 1H), 6.28 (d, J = 5.2 Hz, 1H), 5.48 (s, 2H), 3.93 (s, 6H).

4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-fluoroaniline (13f)

Brown solid; m.p. 204–206 °C. IR (KBr) ν_max/cm^–1 3405, 3168, 1596, 1582, 1505, 1479, 1460, 1431, 1344, 1302, 1247, 1209, 1185, 1168, 996, 955, 849, 817. ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 5.2 Hz, 1H), 7.48 (s, 1H), 7.36 (s, 1H), 7.05 (dd, J = 2.8, 12.0 Hz, 1H), 6.90–6.82 (m, 2H), 6.42 (d, J = 5.2 Hz, 1H), 5.21 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H).

4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluoroaniline (13g).^22,30

Pale yellow solid; m.p. 193–195 °C. IR (KBr) ν_max/cm^–1 3336, 3149, 1625, 1579, 1510, 1486, 1462, 1431, 1351, 1304, 1257, 1213, 1167, 990, 895, 838, 824. ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d, J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.38 (s, 1H), 7.09–7.04 (m, 1H), 6.56 (dd, J = 2.4, 13.2 Hz, 1H), 6.47 (dd, J = 2.0, 8.8 Hz, 1H), 6.39 (d, J = 5.2 Hz, 1H), 5.50 (s, 2H), 3.93 (s, 6H).

Synthesis of thieno[2,3-d]pyrimidin-4(3H)-one (15)

A mixture of 14 (3.14 g, 20 mmol) and formamide (20 mL) was heated at 170 °C for 10 h. After cooling to room temperature, cooled water (30 mL) was added to the reaction mixture. The solid was removed by filtration, washed with water, and dried under vacuum for 12 h. The crude residue was suspended in ethyl ether, stirred for 30 min and filtered. The resultant grey solid (1.88 g, 62% yield) was used for the next step without further purification. M.p. 261–263 °C. IR (KBr) ν_max/cm^–1 3071, 1666, 1592, 1578, 1466, 1368, 1287, 1167, 981, 800, 703, 636, 565. ¹H NMR (400 MHz, DMSO-d₆) δ 12.64 (br s, 1H), 8.14 (s, 1H), 7.56 (d, J = 5.2 Hz, 1H), 7.35 (d, J = 5.2 Hz, 1H).

Synthesis of 4-chlorothieno[2,3-d]pyrimidine (16)

A mixture of 15 (1.52 g, 10 mmol) and POCl₃ (6 mL) with 2–3 drops of DMF was refluxed for 6 h. After the mixture was cooled to room temperature, POCl₃ was removed under vacuum, the obtained residue was poured into a saturated solution of NaHCO₃, and the suspension was neutralised with aq. NaOH (6 M). The mixture was extracted with dichloromethane and the organic phase was washed with water and brine, dried over Na₂SO₄ and concentrated in vacuum. The crude product was purified by silica gel column chromatography using hexane–ethyl acetate (8:1 to 3:1, v/v) as eluent to yield the title compound (1.21 g, 71% yield) as a beige solid. M.p. 110–112 °C. IR (KBr) ν_max/cm^–1 3434, 3113, 1552, 1509, 1477, 1417, 1352, 1276, 1246, 1162, 1131, 877, 844, 715. ¹H NMR (400 MHz, CDCl₃) δ 8.89 (s, 1H), 7.65 (d, J = 6.4 Hz, 1H), 7.44 (d, J = 6.4 Hz, 1H).

Synthesis of 3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)aniline (17)

To a solution of 4-amino-2-fluorophenol (0.95 g, 7.5 mmol) in dry DMF (15 mL) was added NaH (216 mg, 9 mmol). The resulting mixture was stirred at 0 °C for 10 min and 16 (1.02 g, 6 mmol) was then added in portions. The reaction mixture was stirred at 0 °C for 1.5 h. Cold water (50 mL) was added to quench the reaction and the mixture was extracted by ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated under vacuum and purified by silica gel column chromatograph using hexane–ethyl acetate (6:1 to 3:1, v/v) as eluent to give compound 17 (1.33 g, 85% yield) as a brown solid. M.p. 158–160 °C. IR (KBr) ν_max/cm^–1 3401, 3337, 1638, 1574, 1530, 1508, 1421, 1365, 1337, 1323, 1197, 1166, 968, 953, 845, 822, 695, 612. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.94 (d, J = 5.6 Hz, 1H), 7.64 (d, J = 6.0 Hz, 1H), 7.07–7.03 (m, 1H), 6.51 (dd, J = 2.8, 13.2 Hz, 1H), 6.43 (dd, J = 2.4, 8.8 Hz, 1H), 5.43 (br s, 2H).

General procedure for the preparation of 3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline (22), 3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline (26), and 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (30)

The intermediates 22, 26, and 30 were synthesised according to our previous procedures and some other literature (Supporting Information),^30,49,50 as such only characterisation data for the title compounds are listed here.

3-Fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)aniline (22).³⁰

Pale yellow solid; m.p. 137–139 °C. IR (KBr) ν_max/cm^–1 3451, 3329, 3202, 1651, 1621, 1586, 1552, 1505, 1450, 1381, 1293, 1270, 1211, 1161, 1026, 846, 820, 800, 774, 700. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J = 5.2 Hz, 1H), 8.13 (d, J = 5.6 Hz, 1H), 7.57 (d, J = 5.6 Hz, 1H), 7.12–7.07 (m, 1H), 6.57 (d, J = 5.2 Hz, 1H), 6.53 (dd, J = 2.4, 13.2 Hz, 1H), 6.45 (dd, J = 2.0, 8.4 Hz, 1H), 5.55 (br s, 2H).

3-Fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)aniline (26).^30,49

Brown solid; m.p. 165–167 °C. IR (KBr) ν_max/cm^–1 3319, 1633, 1575, 1508, 1466, 1441, 1388, 1337, 1326, 1292, 1227, 1211, 1072, 1037, 847, 799, 543. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.66 (d, J = 5.6 Hz, 1H), 7.10–7.06 (m, 1H), 6.50 (dd, J = 2.0, 12.8 Hz, 1H), 6.42 (dd, J = 1.6, 8.4 Hz, 1H), 5.47 (br s, 2H).

4-((6,7-Dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (30).^30,50

Brown solid; m.p. 182–184 °C. IR (KBr) ν_max/cm^–1 3353, 3179, 1624, 1573, 1511, 1465, 1448, 1419, 1376, 1256, 1242, 1210, 1163, 1138, 989, 907, 855, 843, 829. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.52 (s, 1H), 7.40 (s, 1H), 7.05–7.00 (m, 1H), 6.52 (dd, J = 2.4 Hz, J = 12.8 Hz, 1H), 6.42 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 5.39 (br s, 2H), 3.96 (s, 3H), 3.94 (s, 3H).

Synthesis of 4-((2-chloropyridin-4-yl)oxy)-3-fluoroanilines (32a–32b)

To a solution of 4-amino-2-fluorophenol (1.39 g, 11 mmol) in DMF (15 mL), NaH (0.29 g, 12.1 mmol) was added and the resulting mixture was stirred at 0 °C for 0.5 h. Then, 2,4-dichloropyridine 31a (1.48 g, 10 mmol) was added and the reaction mixture was stirred at 80 °C overnight. After cooling to room temperature, the mixture was poured into H₂O (100 mL) and extracted with ethyl acetate (3 × 80 mL). The organic layer was washed with brine (3 × 100 mL), dried over Na₂SO₄, evaporated under reduced pressure and purified by flash column chromatography on silica gel using hexane–ethyl acetate (4:1, v/v) as eluent to give compound 32a (1.47 g, 62% yield) as a yellow solid. M.p. 74–76 °C. IR (KBr) ν_max/cm^–1 3409, 3224, 1617, 1591, 1559, 1509, 1459, 1388, 1300, 1260, 1237, 1204, 1162, 1117, 1064, 920, 844, 820. ¹H NMR (400 MHz, DMSO-d₆) δ 8.25 (d, J = 5.6 Hz, 1H), 7.02–6.98 (m, 1H), 6.94–6.88 (m, 2H), 6.52 (dd, J = 2.8, 13.2 Hz, 1H), 6.43 (dd, J = 2.4, 8.8 Hz, 1H), 5.52 (br s, 2H). Compound 32b was prepared following the similar synthetic procedure of 32a, only the reaction conditions 80 °C overnight were replaced with 100 °C for 3 h. Yellow solid; yield 57%; m.p. 134–136 °C. IR (KBr) ν_max/cm^–1 3459, 3350, 3220, 1637, 1593, 1573, 1509, 1454, 1381, 1293, 1216, 1205, 1164, 1122, 1054, 956, 937, 841, 825. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (d, J = 5.6 Hz, 1H), 7.07–7.03 (m, 1H), 6.73 (d, J = 5.6 Hz, 1H), 6.54 (dd, J = 2.4, 13.2 Hz, 1H), 6.44 (dd, J = 2.0, 8.4 Hz, 1H), 5.56 (br s, 2H).

Synthesis of 1,3,4-oxathiazol-2-ones (35a–35b)

A mixture of acid 33 (5 mmol) in excess SOCl₂ (2.18 mL, 30 mmol) was stirred at 80 °C for 4 h. After cooling to room temperature, the mixture was concentrated in vacuum to remove excess SOCl₂ and then suspended in THF (5 mL). The THF solution was added to an ice cooled NH₄OH (37%, 10 mL). After stirring for 30 min at room temperature, the mixture was extracted with CHCl₃ (3 × 50 mL). The combined organic extracts were dried and evaporated. The resultant solid was soaked with hexane and filtered to obtain the desired amide 34. Then, (chlorothio)formyl chloride (4.5 mmol, 0.38 mL) was added to a solution of amide 34 (3 mmol) in toluene (5 mL) under N₂ atmosphere. The reaction mixture was heated to 100 °C for 3 h. After cooling to room temperature, the mixture was quenched with H₂O (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄ and concentrated under reduced pressure. The residue was further purified by flash column chromatography on silica gel using hexane–ethyl acetate (12:1 to 6:1, v/v) as eluent to afford the title compounds.

5-Phenyl-1,3,4-oxathiazol-2-one (35a)

White solid; yield 57% (three steps); m.p. 66–68 °C. IR (KBr) ν_max/cm^–1 3472, 1741, 1593, 1560, 1493, 1447, 1322, 1291, 1091, 1070, 983, 886, 791, 770, 723, 689, 681, 663, 573, 473. ¹H NMR (400 MHz, DMSO-d₆) δ 7.92 (d, J = 7.2 Hz, 2H), 7.67–7.63 (m, 1H), 7.59–7.55 (m, 2H).

5-(4-Fluorophenyl)-1,3,4-oxathiazol-2-one (35b)

White solid; yield 53% (three steps); m.p. 101–103 °C. IR (KBr) ν_max/cm^–1 3472, 1784, 1755, 1601, 1574, 1505, 1413, 1293, 1240, 1223, 1160, 1093, 984, 891, 844, 781, 722, 676, 592, 508. ¹H NMR (400 MHz, DMSO-d₆) δ 7.99–7.94 (m, 2H), 7.44–7.38 (m, 2H).

Synthesis of 1,2,4-thiadiazole-5-carboxylates (36a–36b)

A solution of 35 (2 mmol) and ECF (1.97 mL, 20 mmol) in n-dodecane (4 mL) was heated at 160 °C for 16 h. After removal of the excess ECF and n-dodecane under reduced pressure, the residue was purified by flash column chromatography on silica gel using hexane–ethyl acetate (9:1 to 4:1, v/v) as eluent to afford the desired compounds.

3-Phenyl-1,2,4-thiadiazole-5-carboxylic acid ethyl ester (36a)

White solid; yield 75%; m.p. 68–70 °C. IR (KBr) ν_max/cm^–1 3002, 2984, 2907, 1712, 1498, 1470, 1414, 1366, 1325, 1307, 1287, 1258, 1119, 1095, 1008, 843, 764, 711, 685. ¹H NMR (400 MHz, DMSO-d₆) δ 8.24–8.22 (m, 2H), 7.58–7.55 (m, 3H), 4.46 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H).

3-(4-Fluorophenyl)-1,2,4-thiadiazole-5-carboxylic acid ethyl ester (36b)

Pale yellow solid; yield 68%; m.p. 70–72 °C. IR (KBr) ν_max/cm^–1 3471, 3057, 2987, 1742, 1599, 1518, 1488, 1414, 1294, 1237, 1156, 1121, 1095, 1008, 850, 823, 744, 695. ¹H NMR (400 MHz, DMSO-d₆) δ 8.28–8.23 (m, 2H), 7.40–7.35 (m, 2H), 4.46 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H).

Synthesis of 1,2,4-thiadiazole-5-carboxylic acids (37a–37b)

Aqueous solution (1 mL, 2 N LiOH) was added to a solution of carboxylic ester 36 (1 mmol) in methanol (2 mL) at room temperature. The reaction mixture was stirred for 4 h and methanol was then removed by evaporation in vacuum. The resulting residue was adjusted to pH = 2–3 with 1 N HCl. The precipitated solid was collected by filtration and dried to give carboxylic acid 37, which was used directly in the next step without further purification.

3-Phenyl-1,2,4-thiadiazole-5-carboxylic acid (37a)

White solid; yield 71%; m.p. 90–92 °C. IR (KBr) ν_max/cm^–1 3650, 3444, 1718, 1693, 1513, 1429, 1411, 1314, 1258, 1123, 1105, 776, 711, 688. ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 8.27–8.23 (m, 2H), 7.56–7.52 (m, 3H).

3-(4-Fluorophenyl)-1,2,4-thiadiazole-5-carboxylic acid (37b)

Off-white solid; yield 64%; m.p. 106–108 °C. IR (KBr) ν_max/cm^–1 3650, 3446, 1737, 1664, 1600, 1518, 1489, 1422, 1409, 1332, 1233, 1161, 837, 746. ¹H NMR (400 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.30–8.24 (m, 2H), 7.38–7.33 (m, 2H).

Synthesis of 5-amino-1,3,4-thiadiazole-2-carboxylic acid ethyl ester (39)

To a stirred solution of thiosemicarbazide 38 (1.64 g, 18 mmol) in POCl₃ (8.4 mL, 90 mmol), ethyl oxalyl monochloride was added (2.1 mL, 18.9 mmol). The reaction mixture was heated to 70 °C for 6 h. Excess POCl₃ was completely removed under reduced pressure. The residue was poured into cold water (50 mL), basified to pH = 8 with saturated NaHCO₃ solution and extracted with ethyl acetate (60 mL). The organic layer was separated, dried over Na₂SO₄ and concentrated in vacuum. The residue was purified by flash chromatography on silica gel using hexane–ethyl acetate (4:1, v/v) as eluent to afford 39 (1.31 g, 42% yield) as a yellow solid. M.p. 195–197 °C. IR (KBr) ν_max/cm^–1 3423, 3321, 3198, 2986, 2899, 1644, 1619, 1539, 1458, 1398, 1283, 1219, 1125, 1098, 1014, 897, 780, 749, 488. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (br s, 2H), 4.32 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H).

Synthesis of 5-bromo-1,3,4-thiadiazole-2-carboxylic acid ethyl ester (40)

To a solution of 39 (1.21 g, 7 mmol) in CH₃CN (10 mL), CuBr₂ was added (3.12 g, 14 mmol) and the mixture was stirred at room temperature for 20 min. tert-Butyl nitrite (1.68 mL, 14.1 mmol) was then added for 10 min, and the resulting mixture was heated to 60 °C for 30 min. The reaction mixture was concentrated, diluted with water (80 mL) and extracted with ethyl acetate (2 × 60 mL). The combined organic layer was separated, dried over Na₂SO₄ and evaporated to give the title compound 40 (1.16 g, 70% yield) as a dark yellow solid. M.p. 83–85 °C. IR (KBr) ν_max/cm^–1 3463, 2987, 2699, 1748, 1474, 1451, 1360, 1265, 1140, 1107, 1068, 1036, 1012, 831, 772, 757. ¹H NMR (400 MHz, DMSO-d₆) δ 4.42 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H).

Synthesis of 5-substituted 1,3,4-thiadiazole-2-carboxylic acid ethyl esters (41a–41b)

To a mixture of 40 (543 mg, 2.3 mmol), corresponding boronic acid (2.76 mmol), Pd(OAc)₂ (11.3 mg, 0.05 mmol) and Xantphos (29 mg, 0.05 mmol) was added N-methyl morpholine (0.56 mL, 5.1 mmol), toluene (4 mL), and water (2 mL) at room temperature. The reaction mixture was strongly stirred at room temperature for 7 h and then extracted with ethyl acetate (3 × 10 mL). The organic layer was washed with water (20 mL) and brine (20 mL), dried over MgSO₄, filtered and concentrated in vacuum. The residue was purified by flash chromatography on silica gel using hexane–ethyl acetate (12:1 to 6:1, v/v) as eluent to give product 41.

5-Phenyl-1,3,4-thiadiazole-2-carboxylic acid ethyl ester (41a)

Pale yellow solid; yield 83%; m.p. 74–76 °C. IR (KBr) ν_max/cm^–1 3428, 2988, 1725, 1455, 1441, 1405, 1306, 1276, 1233, 1116, 1090, 1001, 762, 689, 637, 566. ¹H NMR (400 MHz, DMSO-d₆) δ 8.07 (d, J = 6.8 Hz, 2H), 7.65–7.55 (m, 3H), 4.44 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H).

5-(4-Fluorophenyl)-1,3,4-thiadiazole-2-carboxylic acid ethyl ester (41b)

Golden yellow solid; yield 76%; m.p. 93–95 °C. IR (KBr) ν_max/cm^–1 3421, 2986, 1716, 1597, 1509, 1479, 1445, 1422, 1396, 1369, 1309, 1219, 1157, 1111, 1086, 1016, 843, 603, 564. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18–8.14 (m, 2H), 7.46–7.41 (m, 2H), 4.45 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H).

Synthesis of thiazole-2-carboxylic acids (46a–46p)

To a solution of ketone 43 (8 mmol) in CH₃CN (4 mL), NBS (1.5 g, 8.4 mmol) and p-toluenesulfonic acid (1.38 g, 8 mmol) were added. The reaction mixture was heated to 50 °C and stirred for 24 h. The solvent was evaporated under reduced pressure and saturated NaHCO₃ (30 mL) was added to the residue. The mixture was extracted with CH₂Cl₂ (3 × 20 mL); the combined organic layers were dried over Na₂SO₄ and evaporated in vacuum. The crude product was purified by flash column chromatography on silica gel using hexane–ethyl acetate (15:1 to 6:1, v/v) as eluent to afford α-bromoketone 44. Afterwards, 44 (5 mmol) was added to a solution of ethyl thiooxamate (692 mg, 5.2 mmol) in ethanol (20 mL), the resulting mixture was heated to reflux for 6 h. The reaction mixture was then concentrated in vacuum, diluted with ethyl acetate (30 mL), and washed with 1 N NaHCO₃ (3 × 20 mL) and brine (2 × 20 mL). The organic layer was dried over Na₂SO₄, filtered and evaporated under vacuum to obtain compound 45, which underwent a similar procedure as described for 37 to afford the title compounds.

4-Phenylthiazole-2-carboxylic acid (46a)

White solid; yield 38% (three steps); m.p. 76–78 °C. IR (KBr) ν_max/cm^–1 3423, 3117, 1894, 1720, 1662, 1487, 1439, 1317, 1262, 1205, 1110, 1073, 1025, 764, 689, 672, 634. ¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (s, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.49–7.42 (m, 3H).

4-(4-Fluorophenyl)thiazole-2-carboxylic acid (46b)

White solid; yield 45% (three steps); m.p. 110–112 °C. IR (KBr) ν_max/cm^–1 3424, 3112, 1870, 1727, 1670, 1602, 1523, 1445, 1325, 1218, 1202, 1164, 1120, 843, 805, 771. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43, 8.03 (dd, J = 5.6, 8.8 Hz, 2H), 7.32–7.27 (m, 2H).

4-(p-Tolyl)thiazole-2-carboxylic acid (46c)

Pale yellow solid; yield 31% (three steps); m.p. 117–119 °C. IR (KBr) ν_max/cm^–1 3467, 3104, 1963, 1697, 1613, 1490, 1447, 1282, 1254, 1126, 1066, 822, 783, 772, 758. ¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 2.32 (s, 3H).

4-(4-Methoxyphenyl)thiazole-2-carboxylic acid (46d)

Off-white solid; yield 42% (three steps); m.p. 116–118 °C. IR (KBr) ν_max/cm^–1 3467, 3110, 2837, 2558, 1973, 1682, 1609, 1530, 1490, 1450, 1435, 1304, 1280, 1252, 1178, 1125, 1100, 1067, 1026, 770. ¹H NMR (400 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H).

4-(3,4-Dimethoxyphenyl)thiazole-2-carboxylic acid (46e)

Pale yellow solid; yield 27% (three steps); m.p. 85–87 °C. IR (KBr) ν_max/cm^–1 3748, 3503, 2939, 1716, 1527, 1496, 1463, 1436, 1405, 1289, 1267, 1244, 1166, 1147, 1112, 1021, 766. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 7.56–7.54 (m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 3.83 (s, 3H), 3.79 (s, 3H).

4-(3-Fluorophenyl)thiazole-2-carboxylic acid (46f)

White solid; yield 48% (three steps); m.p. 84–86 °C. IR (KBr) ν_max/cm^–1 3448, 1703, 1646, 1616, 1591, 1490, 1455, 1376, 1305, 1264, 1235, 1175, 1131, 1079, 944, 816, 776, 756. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.85–7.78 (m, 2H), 7.48 (d, J = 7.2 Hz, 1H), 7.18 (s, 1H).

4-(2-Fluorophenyl)thiazole-2-carboxylic acid (46g)

Yellow solid; yield 35% (three steps); m.p. 78–80 °C. IR (KBr) ν_max/cm^–1 3443, 1704, 1646, 1495, 1456, 1435, 1378, 1299, 1255, 1216, 1127, 1061, 868, 811, 785, 749. ¹H NMR (400 MHz, DMSO-d₆) δ 8.15–8.12 (m, 2H), 7.44–7.39 (m, 1H), 7.35–7.29 (m, 2H).

4-(4-Chlorophenyl)thiazole-2-carboxylic acid (46h)

Pale yellow solid; yield 48% (three steps); m.p. 118–120 °C. IR (KBr) ν_max/cm^–1 3478, 3114, 1946, 1688, 1597, 1486, 1448, 1294, 1278, 1257, 1123, 1092, 1064, 1013, 832, 782, 772. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (s, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H).

4-(3-Chlorophenyl)thiazole-2-carboxylic acid (46i)

Yellow solid; yield 39% (three steps); m.p. 103–105 °C. IR (KBr) ν_max/cm^–1 3421, 1885, 1722, 1659, 1595, 1573, 1510, 1481, 1442, 1424, 1318, 1207, 1119, 1075, 874, 771. ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (s, 1H), 8.04 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.51–7.42 (m, 2H).

4-(4-Bromophenyl)thiazole-2-carboxylic acid (46j)

Pale yellow solid; yield 55% (three steps); m.p. 117–119 °C. IR (KBr) ν_max/cm^–1 3496, 3111, 1684, 1508, 1495, 1450, 1442, 1398, 1280, 1261, 1097, 1069, 1009, 845, 831, 773. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H).

4-(4-(Trifluoromethyl)phenyl)thiazole-2-carboxylic acid (46k)

Yellow solid; yield 21% (three steps); m.p. 116–118 °C. IR (KBr) ν_max/cm^–1 3449, 1953, 1685, 1618, 1492, 1454, 1322, 1260, 1170, 1125, 1072, 1060, 1018, 851, 781. ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (s, 1H), 8.20 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H).

4-(3,4-Dichlorophenyl)thiazole-2-carboxylic acid (46l)

Pale yellow solid; yield 52% (three steps); m.p. 126–128 °C. IR (KBr) ν_max/cm^–1 3435, 3112, 1729, 1638, 1507, 1482, 1434, 1294, 1223, 1139, 1114, 1028, 871, 827, 775. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.18 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H).

4-(Naphthalen-2-yl)thiazole-2-carboxylic acid (46m)

Pale red solid; yield 54% (three steps); m.p. 128–130 °C. IR (KBr) ν_max/cm^–1 3435, 3110, 1708, 1676, 1477, 1434, 1319, 1287, 1256, 1212, 1127, 1100, 892, 863, 836, 779, 761, 751. ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (s, 1H), 8.56 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.02–7.99 (m, 2H), 7.94–7.92 (m, 1H), 7.55–7.50 (m, 2H).

4-(3-Chloro-4-fluorophenyl)thiazole-2-carboxylic acid (46n)

White solid; yield 40% (three steps); m.p. 119–121 °C. IR (KBr) ν_max/cm^–1 3435, 1733, 1706, 1677, 1519, 1489, 1443, 1297, 1246, 1110, 1054, 927, 872, 833, 774. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.15 (d, J = 7.2 Hz, 1H), 7.97 (s, 1H), 7.50–7.46 (m, 1H).

4-(Thiophen-2-yl)thiazole-2-carboxylic acid (46o)

Yellow solid; yield 47% (three steps); m.p. 118–120 °C. IR (KBr) ν_max/cm^–1 3452, 3083, 2872, 1680, 1547, 1489, 1460, 1412, 1293, 1280, 1219, 1097, 1056, 899, 817, 769, 730, 696. ¹H NMR (400 MHz, DMSO-d₆) δ 8.29 (s, 1H), 7.65 (d, J = 3.6 Hz, 1H), 7.58 (d, J = 4.8 Hz, 1H), 7.14–7.12 (m, 1H).

4-(3,4-Difluorophenyl)thiazole-2-carboxylic acid (46p)

White solid; yield 29% (three steps); m.p. 125–127 °C. IR (KBr) ν_max/cm^–1 3436, 3107, 1720, 1707, 1608, 1533, 1502, 1460, 1416, 1379, 1313, 1275, 1208, 1170, 1121, 1061, 885, 819, 775. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (s, 1H), 8.02–7.96 (m, 1H), 7.83 (br s, 1H), 7.54–7.48 (m, 1H).

Synthesis of 2-oxo-1-ethylamine hydrochlorides (47a–47b)

α-Bromoketone (44a–44b, 10 mmol) and urotropine (1.4 g, 10 mmol) were dissolved in dry CHCl₃ (50 mL). The mixture was stirred at 50 °C for 2 h. The white precipitate was isolated by filtration and washed with CHCl₃ and ethanol to yield the urotropin salt, which was then dissolved in a mixture of absolute ethanol (50 mL) and concentrated HCl (5 mL). The resulting mixture was heated to reflux for 2 h. The residual solid was removed by filtration and the filtrate was evaporated under reduced pressure to yield the title compounds.

2-Amino-1-(phenyl)ethanone hydrochloride (47a)

Brown solid; yield 82%; m.p. 209–211 °C. IR (KBr) ν_max/cm^–1 3375, 2924, 2609, 1697, 1596, 1548, 1504, 1448, 1431, 1373, 1242, 1120, 1076, 964, 900, 761, 690. ¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (br s, 2H), 8.01 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.2 Hz, 1H), 7.59–7.55 (m, 2H), 4.60 (s, 2H).

2-Amino-1-(4′-fluorophenyl)ethanone hydrochloride (47b)

White solid; yield 67%; m.p. 238–240 °C. IR (KBr) ν_max/cm^–1 3435, 2982, 2584, 1682, 1600, 1509, 1470, 1430, 1417, 1302, 1255, 1237, 1167, 1124, 1105, 974, 841. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (br s, 2H), 8.11 (dd, J = 5.6, 8.8 Hz, 2H), 7.44–7.39 (m, 2H), 4.56 (s, 2H).

Synthesis of amides (48a–48b)

To a stirred solution of 47 (6 mmol) in CH₂Cl₂ (18 mL) under 0 °C, ethyl oxalyl monochloride (0.73 mL, 6.5 mmol) and Et₃N (1.66 mL, 12 mmol) were added. The reaction mixture was warmed to room temperature and stirred overnight. The mixture was partitioned between CH₂Cl₂ and aq. HCl (1 N). After extraction with CH₂Cl₂ (2 × 30 mL), the combined organic layers were washed with water and saturated brine, dried over Na₂SO₄ and concentrated in vacuum to obtain the crude product 48, which was used directly in the next step without further purification.

Ethyl 2-oxo-2-((2-oxo-2-phenylethyl)amino)acetate (48a)

Off-white solid; yield 78%; m.p. 95–97 °C. IR (KBr) ν_max/cm^–1 3390, 3066, 2983, 2914, 1733, 1724, 1683, 1589, 1519, 1448, 1432, 1357, 1293, 1238, 1199, 1019, 994, 858, 754, 688, 510. ¹H NMR (400 MHz, DMSO-d₆) δ 9.11 (s, 1H), 8.01 (d, J = 6.8 Hz, 2H), 7.70–7.66 (m, 1H), 7.57–7.53 (m, 2H), 4.71 (d, J = 6.0 Hz, 2H), 4.28 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H).

Ethyl 2-((2-(4-fluorophenyl)-2-oxoethyl)amino)-2-oxoacetate (48b)

Off-white solid; yield 83%; m.p. 121–123 °C. IR (KBr) ν_max/cm^–1 3391, 3076, 2985, 2919, 1734, 1723, 1682, 1596, 1511, 1415, 1357, 1293, 1237, 1198, 1161, 1103, 1018, 991, 857, 836, 601, 568. ¹H NMR (400 MHz, DMSO-d₆) δ 9.11 (s, 1H), 8.10 (dd, J = 5.6, 8.8 Hz, 2H), 7.40–7.35 (m, 2H), 4.69 (d, J = 5.6 Hz, 2H), 4.27 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H).

Synthesis of 4-phenylthiazole-2-carboxylic acid ethyl esters (49a–49b)

Phosphorus pentasulfide (1.78 g, 8 mmol) was added to a solution of 48 (4 mmol) in dry CHCl₃ (10 mL) and the mixture was heated to reflux for 5 h. After cooling to room temperature, the mixture was quenched with water and extracted with CHCl₃. The organic phase was washed with water and brine, dried over Na₂SO₄ and concentrated in vacuum. The residue was purified by flash column chromatography on silica gel using hexane–ethyl acetate (7:1 to 3:1, v/v) as eluent to afford the desired compounds.

5-Phenylthiazole-2-carboxylic acid ethyl ester (49a)

Golden-yellow solid; yield 64%, m.p. 70–72 °C. IR (KBr) ν_max/cm^–1 3434, 2974, 1702, 1518, 1476, 1446, 1419, 1392, 1364, 1296, 1164, 1118, 1086, 1016, 861, 760, 690, 610, 555. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (s, 1H), 7.79–7.76 (m, 2H), 7.50–7.41 (m, 3H), 4.38 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H).

5-(4-Fluorophenyl)thiazole-2-carboxylic acid ethyl ester (49b)

Yellow solid; yield 57%, m.p. 110–112 °C. IR (KBr) ν_max/cm^–1 3434, 3087, 1702, 1599, 1527, 1478, 1426, 1411, 1394, 1323, 1298, 1230, 1182, 1166, 1086, 1019, 838, 757, 595. ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (s, 1H), 7.82 (dd, J = 5.6, 8.8 Hz, 2H), 7.33–7.28 (m, 2H), 4.37 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H).

Synthesis of 5-phenylthiazole-2-carboxylic acids (50a–50b)

Compound 50 was prepared from intermediate 49 following the similar procedure as described for 37.

5-Phenylthiazole-2-carboxylic acid (50a)

Off-white solid; yield 71%; m.p. 113–115 °C. IR (KBr) ν_max/cm^–1 3427, 2459, 1852, 1736, 1705, 1448, 1426, 1270, 1153, 1118, 870, 790, 770, 759, 685. ¹H NMR (400 MHz, DMSO-d₆) δ 9.08 (s, 1H), 8.31 (s, 1H), 7.69–7.67 (m, 2H), 7.49–7.42 (m, 3H).

5-(4-Fluorophenyl)thiazole-2-carboxylic acid (50b)

Pale yellow solid; yield 76%; m.p. 128–130 °C. IR (KBr) ν_max/cm^–1 3106, 2535, 1705, 1600, 1534, 1465, 1419, 1404, 1326, 1303, 1244, 1189, 1161, 1111, 881, 826, 785. ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (s, 1H), 8.26 (s, 1H), 7.74–7.69 (m, 2H), 7.33–7.26 (m, 2H).

Synthesis of target compounds 51a–51an

1. The prepared acid (37, 46, and 50, 1 mmol) was suspended in dry CH₂Cl₂ (5 mL); oxalyl chloride (0.17 mL, 2 mmol) and DMF (one drop) were added at 0 °C. The reaction mixture was stirred at room temperature for 1.5 h and concentrated in vacuum to afford the corresponding acyl chloride. The acyl chloride without further purification was dissolved in dry CH₂Cl₂ (2 mL) and cooled to 0  °C. The corresponding amine (13, 17, 22, 26, 30, and 32, 1 mmol) was added thereinto, the mixture was warmed to room temperature and stirred for 4–6 h. The solvent was concentrated in vacuum and the residue was purified by flash chromatography on silica gel using hexane–ethyl acetate (8:1 to 2:1, v/v) as eluent to give the target compounds 51a–51b and 51e–51an.

2. Aqueous solution (1 mL, 2 N LiOH) was added to a solution of carboxylic ester 41 (1 mmol) in methanol (2 mL) at 0  °C and the resulting mixture was continuously stirred for 1 h. Then, the solvent was removed by evaporation in vacuum to afford the lithium salt 42 which was used directly. Then, HATU (570 mg, 1.5 mmol) and Et₃N (0.21 mL, 1.5 mmol) were added to a stirred solution of 42 (1 mmol) in DMF (4 mL) at room temperature. The mixture was stirred for 10 min followed by addition of 13a (296 mg, 1 mmol). The resulting mixture was stirred at room temperature overnight. Water (10 mL) was added to the reaction solution and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated in vacuum and purified by flash chromatography on silica gel using hexane–ethyl acetate (5:1 to 2:1, v/v) as eluent to yield the target compounds 51c–51d.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-3-phenyl-1,2,4-thiadiazole-5-carboxamide (51a)

Pale yellow solid; yield 63% (two steps); m.p. 145–147 °C. HPLC purity 95.2%. IR (KBr) ν_max/cm^–1 3381, 1691, 1626, 1579, 1528, 1506, 1482, 1431, 1414, 1350, 1306, 1253, 1214, 1202, 1174, 1119, 1080, 996, 849, 713. ¹H NMR (600 MHz, DMSO-d₆) δ 11.14 (s, 1H), 8.50 (d, J = 4.8 Hz, 1H), 8.38 (dd, J = 2.4, 7.2 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H), 7.63–7.59 (m, 3H), 7.51 (s, 1H), 7.40 (s, 1H), 7.35 (d, J = 9.0 Hz, 2H), 6.53 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 184.9, 173.0, 159.6, 156.2, 152.6, 150.7, 149.3, 148.8, 146.4, 134.6, 131.7, 131.1, 129.0, 128.1, 122.9, 121.3, 115.2, 107.8, 103.4, 99.0, 55.7, 55.6; ESI-MS: m/z 485.2 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-3-(4-fluorophenyl)-1,2,4-thiadiazole-5-carboxamide (51b)

Yellow solid; yield 74% (two steps); m.p. 113–115 °C. HPLC purity 96.5%. IR (KBr) ν_max/cm^–1 3435, 1667, 1605, 1559, 1546, 1538, 1505, 1479, 1430, 1409, 1349, 1305, 1253, 1214, 1154, 1116, 1080, 995, 849, 746. ¹H NMR (600 MHz, DMSO-d₆) δ 11.13 (s, 1H), 8.50 (d, J = 5.4 Hz, 1H), 8.42 (dd, J = 5.4, 8.4 Hz, 2H), 8.01 (d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.47–7.44 (m, 2H), 7.40 (s, 1H), 7.35 (d, J = 9.0 Hz, 2H), 6.52 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 185.0, 172.0, 163.8 (d, J = 247.6 Hz), 159.6, 156.1, 152.6, 150.8, 149.3, 148.8, 146.4, 134.6, 130.5 (d, J = 8.7 Hz), 128.4 (d, J = 1.9 Hz), 122.9, 121.3, 116.1 (d, J = 21.9 Hz), 115.2, 107.8, 103.4, 99.0, 55.7, 55.6; ESI-MS: m/z 503.1 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-5-phenyl-1,3,4-thiadiazole-2-carboxamide (51c)

Yellow solid; yield 72% (two steps); m.p. 141–143 °C. HPLC purity 96.1%. IR (KBr) ν_max/cm^–1 3423, 1725, 1658, 1630, 1560, 1513, 1476, 1435, 1402, 1364, 1325, 1255, 1210, 1185, 1132, 1074, 985, 832, 694. ¹H NMR (600 MHz, DMSO-d₆) δ 10.99 (s, 1H), 8.54 (s, 1H), 8.49 (d, J = 5.4 Hz, 1H), 8.04 (d, J = 7.8 Hz, 2H), 7.84 (d, J = 7.0 Hz, 2H), 7.53–7.44 (m, 5H), 7.31 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.6, 160.3, 158.2, 153.0, 150.6, 149.8, 149.3, 145.0, 140.5, 136.0, 130.6, 129.9, 127.4, 123.6, 122.9, 121.8, 115.6, 108.3, 103.7, 99.6, 56.2, 56.1; ESI-MS: m/z 485.1 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)-1,3,4-thiadiazole-2-carboxamide (51d)

Pale yellow solid; yield 63% (two steps); m.p. 136–138 °C. HPLC purity 97.0%. IR (KBr) ν_max/cm^–1 3450, 1699, 1646, 1605, 1504, 1488, 1465, 1432, 1389, 1357, 1316, 1254, 1210, 1172, 1135, 1074, 982, 841, 725. ¹H NMR (600 MHz, DMSO-d₆) δ 11.01 (s, 1H), 8.51 (s, 1H), 8.49 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.91 (dd, J = 5.4, 8.3 Hz, 2H), 7.52 (s, 1H), 7.38–7.35 (m, 2H), 7.31 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 4.8 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.0 (d, J = 246.1 Hz), 162.6, 160.3, 158.1, 153.0, 150.5, 149.8, 149.3, 146.9, 140.6, 135.9, 129.7 (d, J = 7.9 Hz), 127.2 (d, J = 2.4 Hz), 122.8, 121.8, 116.9 (d, J = 21.7 Hz), 115.6, 108.3, 103.6, 99.5, 56.2, 56.1; ESI-MS: m/z 503.3 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-4-phenylthiazole-2-carboxamide (51e)

Yellow solid; yield 56% (two steps); m.p. 63–65 °C. HPLC purity 97.4%. IR (KBr) ν_max/cm^–1 3434, 2922, 1672, 1580, 1559, 1531, 1505, 1479, 1431, 1349, 1305, 1252, 1215, 1168, 1080, 994, 853, 755. ¹H NMR (600 MHz, DMSO-d₆) δ 10.79 (s, 1H), 8.52 (s, 1H), 8.49 (d, J = 5.4 Hz, 1H), 8.18 (d, J = 7.2 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.53–7.50 (m, 3H), 7.43 (d, J = 7.2 Hz, 1H), 7.40 (s, 1H), 7.33 (d, J = 9.0 Hz, 2H), 6.52 (d, J = 4.8 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 163.2, 159.7, 157.7, 155.5, 152.5, 150.2, 149.3, 148.8, 146.5, 135.2, 133.4, 128.8, 128.6, 126.4, 122.6, 121.3, 120.3, 115.2, 107.8, 103.3, 99.0, 55.7, 55.6; ESI-MS: m/z 484.2 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51f)

Yellow solid; yield 65% (two steps); m.p. 84–86 °C. HPLC purity 95.9%. IR (KBr) ν_max/cm^–1 3434, 2924, 1667, 1604, 1584, 1559, 1531, 1506, 1481, 1431, 1350, 1253, 1216, 1157, 1080, 995, 849. ¹H NMR (600 MHz, DMSO-d₆) δ 10.79 (s, 1H), 8.50–8.49 (m, 2H), 8.23 (dd, J = 6.0, 7.8 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.52 (s, 1H), 7.40 (s, 1H), 7.38–7.35 (m, 2H), 7.33 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 163.3, 162.3 (d, J = 244.2 Hz), 159.7, 157.7, 154.4, 152.5, 150.3, 149.3, 148.8, 146.5, 135.2, 130.0 (d, J = 2.7 Hz), 128.6 (d, J = 8.1 Hz), 122.6, 121.3, 120.1, 115.7 (d, J = 21.7 Hz), 115.2, 107.8, 103.3, 99.0, 55.7, 55.6; ESI-MS: m/z 502.3 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-5-phenylthiazole-2-carboxamide (51g)

Pale yellow solid; yield 75% (two steps); m.p. 260–262 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3338, 2923, 1661, 1590, 1527, 1506, 1478, 1449, 1431, 1420, 1343, 1303, 1240, 1219, 1198, 1178, 1158, 1078, 991, 853, 825. ¹H NMR (600 MHz, DMSO-d₆) δ 10.98 (s, 1H), 8.53 (s, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 7.2 Hz, 2H), 7.51–7.50 (m, 3H), 7.46–7.44 (m, 1H), 7.40 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 4.8 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 162.1, 159.8, 157.6, 152.5, 150.1, 149.3, 148.8, 146.4, 144.5, 140.0, 135.4, 130.1, 129.4, 126.9, 123.1, 122.4, 121.3, 115.1, 107.8, 103.2, 99.0, 55.7, 55.6; ESI-MS: m/z 484.3 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-5-(4-fluorophenyl)thiazole-2-carboxamide (51h)

Pale yellow solid; yield 59% (two steps); m.p. 252–254 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3336, 2938, 1678, 1622, 1601, 1532, 1508, 1494, 1478, 1428, 1348, 1305, 1273, 1246, 1233, 1214, 1168, 1078, 992, 836, 816. ¹H NMR (600 MHz, DMSO-d₆) δ 10.99 (s, 1H), 8.50 (s, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.89 (dd, J = 5.4, 8.4 Hz, 2H), 7.51 (s, 1H), 7.39 (s, 1H), 7.37–7.34 (m, 2H), 7.30 (d, J = 9.0 Hz, 2H), 6.48 (d, J = 4.8 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 162.6 (d, J = 246.3 Hz), 162.2, 159.8, 157.6, 152.5, 150.1, 149.3, 148.8, 146.4, 143.4, 140.1, 135.5, 129.2 (d, J = 8.2 Hz), 126.7 (d, J = 2.5 Hz), 122.4, 121.3, 116.4 (d, J = 21.9 Hz), 115.1, 107.8, 103.2, 99.0, 55.7, 55.6; ESI-MS: m/z 502.2 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-fluorophenyl)-4-phenylthiazole-2-carboxamide (51i)

Pale yellow solid; yield 73% (two steps); m.p. 112–114 °C. HPLC purity 95.5%. IR (KBr) ν_max/cm^–1 3378, 2926, 1683, 1600, 1531, 1505, 1478, 1430, 1350, 1303, 1253, 1211, 1167, 1144, 1076, 995, 958, 853, 823, 752. ¹H NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.52 (s, 1H), 8.14 (d, J = 7.6 Hz, 2H), 7.85–7.81 (m, 1H), 7.53–7.48 (m, 3H), 7.45–7.40 (m, 3H), 7.19 (dd, J = 2.8, 8.8 Hz, 1H), 6.65 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.4, 159.1, 157.9, 156.1 (d, J = 248.4 Hz), 155.5, 152.7, 152.6 (d, J = 10.5 Hz), 149.5, 148.9, 146.5, 133.3, 128.9, 128.7, 127.9, 126.4, 121.9 (d, J = 12.3 Hz), 120.5, 116.7 (d, J = 3.3 Hz), 115.3, 109.2 (d, J = 22.7 Hz), 107.8, 104.2, 99.0, 55.8, 55.7; ESI-MS: m/z 502.1 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-4-phenylthiazole-2-carboxamide (51j)

Pale yellow solid; yield 65% (two steps); m.p. 90–92 °C. HPLC purity 97.1%. IR (KBr) ν_max/cm^–1 3434, 2928, 1668, 1623, 1598, 1559, 1538, 1509, 1480, 1431, 1349, 1305, 1251, 1212, 1165, 1076, 994, 853, 833, 754. ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (s, 1H), 8.53 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.17 (d, J = 7.6 Hz, 2H), 8.09 (d, J = 12.8 Hz, 1H), 7.87 (d, J = 9.6 Hz, 1H), 7.54–7.50 (m, 4H), 7.44–7.40 (m, 2H), 6.51 (d, J = 5.2 Hz, 1H), 3.95 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.8, 162.3, 159.2, 158.0, 155.6, 153.3 (d, J = 244.3 Hz), 152.7, 149.5, 148.9, 146.4, 136.8 (d, J = 9.6 Hz), 136.6 (d, J = 12.0 Hz), 133.3, 128.8, 126.5, 124.0, 120.7, 117.7, 114.6, 109.7 (d, J = 23.2 Hz), 107.8, 102.2, 98.9, 55.7; ESI-MS: m/z 502.3 [M + H]⁺.

N-(2-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-phenylthiazole-2-carboxamide (51k)

Yellow solid; yield 53% (two steps); m.p. 201–203 °C. HPLC purity 96.8%. IR (KBr) ν_max/cm^–1 3435, 2924, 1690, 1586, 1531, 1505, 1479, 1430, 1349, 1304, 1248, 1210, 1194, 1165, 1090, 995, 915, 874, 819, 749. ¹H NMR (600 MHz, DMSO-d₆) δ 10.41 (s, 1H), 8.56 (s, 1H), 8.55 (d, J = 4.8 Hz, 1H), 8.12 (d, J = 7.2 Hz, 2H), 8.02 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 3.0 Hz, 1H), 7.53–7.51 (m, 2H), 7.49 (s, 1H), 7.44–7.41 (m, 2H), 7.36 (dd, J = 3.0, 9.0 Hz, 1H), 6.64 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 162.3, 158.9, 157.5, 155.4, 152.6, 152.1, 149.5, 148.9, 146.6, 133.1, 131.2, 128.9, 128.7, 128.5, 127.1, 126.3, 121.9, 120.8, 120.1, 115.3, 107.9, 104.1, 99.0, 55.7; ESI-MS: m/z 518.1 [M + H]⁺.

N-(3-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-4-phenylthiazole-2-carboxamide (51l)

Pale yellow solid; yield 58% (two steps); m.p. 146–148 °C. HPLC purity 97.0%. IR (KBr) ν_max/cm^–1 3375, 2926, 1668, 1596, 1530, 1505, 1477, 1431, 1385, 1348, 1305, 1251, 1211, 1165, 1084, 1060, 994, 852, 753. ¹H NMR (600 MHz, DMSO-d₆) δ 10.93 (s, 1H), 8.54 (s, 1H), 8.49 (d, J = 4.8 Hz, 1H), 8.29 (d, J = 2.4 Hz, 1H), 8.18 (d, J = 7.2 Hz, 2H), 8.03 (dd, J = 3.0, 9.0 Hz, 1H), 7.55 (s, 1H), 7.53–7.51 (m, 3H), 7.43 (d, J = 7.2 Hz, 1H), 7.42 (s, 1H), 6.41 (d, J = 4.8 Hz, 1H), 3.95 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ 162.8, 158.8, 158.0, 155.5, 152.6, 149.4, 148.8, 146.4, 145.4, 136.7, 133.3, 128.8, 128.7, 126.4, 125.7, 123.8, 122.4, 121.2, 120.6, 114.6, 107.8, 102.5, 98.9, 55.7; ESI-MS: m/z 518.1 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-methylphenyl)-4-phenylthiazole-2-carboxamide (51m)

Yellow solid; yield 46% (two steps); m.p. 209–211 °C. HPLC purity 95.2%. IR (KBr) ν_max/cm^–1 3435, 2924, 1668, 1574, 1505, 1480, 1428, 1349, 1303, 1253, 1207, 1168, 1075, 996, 947, 886, 851, 818, 762. ¹H NMR (600 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.52–8.51 (m, 2H), 8.15 (d, J = 7.2 Hz, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.52–7.50 (m, 3H), 7.42 (d, J = 7.8 Hz, 1H), 7.41 (s, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.17 (dd, J = 2.4, 8.4 Hz, 1H), 6.54 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H), 2.34 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 163.0, 159.5, 157.7, 155.4, 152.6, 151.9, 149.3, 148.8, 146.5, 135.7, 133.3, 132.5, 128.8, 128.6, 127.5, 126.3, 122.4, 120.1, 118.4, 115.3, 107.8, 103.6, 99.0, 55.7, 55.6, 17.8; ESI-MS: m/z 498.2 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-methylphenyl)-4-phenylthiazole-2-carboxamide (51n)

Yellow solid; yield 58% (two steps); m.p. 136–138 °C. HPLC purity 98.3%. IR (KBr) ν_max/cm^–1 3435, 2924, 1667, 1620, 1580, 1559, 1538, 1505, 1477, 1430, 1348, 1303, 1250, 1212, 1165, 1078, 993, 850, 826, 753. ¹H NMR (600 MHz, DMSO-d₆) δ 10.71 (s, 1H), 8.52 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.18 (d, J = 7.8 Hz, 2H), 7.94 (s, 1H), 7.88 (dd, J = 2.4, 9.0 Hz, 1H), 7.58 (s, 1H), 7.53–7.50 (m, 2H), 7.43 (d, J = 7.2 Hz, 1H), 7.41 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.34 (d, J = 5.4 Hz, 1H), 3.95 (s, 6H), 2.15 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 163.2, 159.4, 157.7, 155.4, 152.5, 149.3, 148.9, 148.1, 146.3, 135.5, 133.3, 130.4, 128.8, 128.6, 126.4, 123.9, 121.9, 120.3, 120.2, 114.8, 107.8, 102.2, 99.1, 55.7, 15.7; ESI-MS: m/z 498.0 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-4-phenylthiazole-2-carboxamide (51o)

Pale yellow solid; yield 74% (two steps); m.p. 115–117 °C. HPLC purity 97.6%. IR (KBr) ν_max/cm^–1 3373, 2925, 1672, 1619, 1586, 1537, 1509, 1466, 1446, 1418, 1376, 1307, 1237, 1209, 1133, 1077, 994, 850, 753. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.58 (s, 1H), 8.53 (s, 1H), 8.17 (d, J = 7.2 Hz, 2H), 8.03 (dt, J = 2.4, 12.8 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.55–7.50 (m, 3H), 7.44–7.40 (m, 2H), 3.99 (s, 3H), 3.98 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9, 162.9, 157.9, 155.9, 155.6, 153.4 (d, J = 243.5 Hz), 152.1, 150.3, 149.0, 136.7 (d, J = 9.9 Hz), 135.4 (d, J = 12.7 Hz), 133.3, 128.8, 128.7, 126.5, 124.4, 120.6, 117.2 (d, J = 2.7 Hz), 109.3, 109.1, 106.7, 100.5, 56.2, 56.0; ESI-MS: m/z 503.2 [M + H]⁺.

N-(4-((6,7-Dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51p)

White solid; yield 69% (two steps); m.p. 188–190 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3434, 3091, 1683, 1621, 1579, 1559, 1539, 1506, 1447, 1419, 1381, 1239, 1215, 1079, 995, 913, 855. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.58 (s, 1H), 8.51 (s, 1H), 8.21 (dd, J = 5.6, 8.8 Hz, 2H), 8.03–7.99 (m, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.54–7.50 (m, 1H), 7.40 (s, 1H), 7.38–7.34 (m, 2H), 3.99 (s, 3H), 3.98 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.0, 163.0, 162.4 (d, J = 244.2 Hz), 157.9, 155.9, 154.5, 153.4 (d, J = 243.6 Hz), 152.1, 150.3, 149.0, 136.7 (d, J = 10.1 Hz), 135.5 (d, J = 12.8 Hz), 129.9 (d, J = 2.7 Hz), 128.6 (d, J = 8.3 Hz), 124.4, 120.4, 117.2 (d, J = 3.0 Hz), 115.7 (d, J = 21.7 Hz), 109.3, 109.1, 106.8, 100.5, 56.2, 56.0; ESI-MS: m/z 521.1 [M + H]⁺.

N-(3-Fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)phenyl)-4-phenylthiazole-2-carboxamide (51q)

Pale yellow solid; yield 63% (two steps); m.p. 251–253 °C. HPLC purity 96.8%. IR (KBr) ν_max/cm^–1 3453, 2922, 1668, 1606, 1585, 1547, 1503, 1484, 1452, 1440, 1407, 1381, 1296, 1267, 1202, 1074, 1025, 816, 754. ¹H NMR (400 MHz, DMSO-d₆) δ 10.95 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.54 (s, 1H), 8.19–8.16 (m, 3H), 8.10 (dt, J = 2.4, 12.8 Hz, 1H), 7.89–7.85 (m, 1H), 7.62 (d, J = 5.2 Hz, 1H), 7.58–7.50 (m, 3H), 7.44–7.42 (m, 1H), 6.70 (d, J = 5.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.8, 159.1, 158.9, 158.0, 155.6, 153.3 (d, J = 244.1 Hz), 149.6, 137.1 (d, J = 10.1 Hz), 135.9 (d, J = 12.2 Hz), 133.3, 132.2, 128.8, 128.7, 126.4, 125.0, 123.9, 120.8, 120.7, 117.6 (d, J = 3.3 Hz), 109.5 (d, J = 23.0 Hz), 103.3; ESI-MS: m/z 448.0 [M + H]⁺.

N-(3-Fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)phenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51r)

Pale yellow solid; yield 58% (two steps); m.p. 223–225 °C. HPLC purity 96.4%. IR (KBr) ν_max/cm^–1 3469, 2923, 1668, 1604, 1548, 1527, 1504, 1484, 1449, 1406, 1382, 1298, 1269, 1233, 1202, 1156, 1130, 1027, 844, 812, 767. ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (s, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.50 (s, 1H), 8.21 (dd, J = 5.6, 8.8 Hz, 2H), 8.17 (d, J = 5.6 Hz, 1H), 8.11–8.07 (m, 1H), 7.88–7.84 (m, 1H), 7.61 (d, J = 5.2 Hz, 1H), 7.57–7.53 (m, 1H), 7.37–7.32 (m, 2H), 6.69 (d, J = 5.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.8, 162.3 (d, J = 244.2 Hz), 159.1, 158.9, 157.9, 154.5, 153.3 (d, J = 244.7 Hz), 149.6, 137.1 (d, J = 10.1 Hz), 135.9 (d, J = 12.2 Hz), 132.2, 129.9 (d, J = 3.0 Hz), 128.6 (d, J = 8.1 Hz), 124.9, 123.9, 120.8, 120.4, 117.5 (d, J = 3.0 Hz), 115.7 (d, J = 21.6 Hz), 109.5 (d, J = 22.6 Hz), 103.4; ESI-MS: m/z 466.3 [M + H]⁺.

N-(3-Fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-phenylthiazole-2-carboxamide (51s)

Pale yellow solid; yield 67% (two steps); m.p. 95–97 °C. HPLC purity 95.2%. IR (KBr) ν_max/cm^–1 3434, 2920, 1683, 1608, 1577, 1559, 1529, 1506, 1466, 1439, 1338, 1332, 1294, 1195, 1078, 1033, 870, 799, 752. ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 8.75 (s, 1H), 8.53–8.51 (m, 2H), 8.17 (d, J = 7.6 Hz, 2H), 8.04 (d, J = 12.0 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 5.6 Hz, 1H), 7.59–7.50 (m, 3H), 7.44–7.40 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 162.9, 162.8, 158.0, 155.6, 154.1, 153.3 (d, J = 243.9 Hz), 137.7, 137.1 (d, J = 10.0 Hz), 134.7 (d, J = 12.8 Hz), 133.3, 128.8, 128.7, 126.5, 124.3, 120.6, 117.3 (d, J = 2.5 Hz), 117.2 (d, J = 3.0 Hz), 116.2, 109.2 (d, J = 23.2 Hz); ESI-MS: m/z 449.1 [M + H]⁺.

N-(3-Fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51t)

Pale yellow solid; yield 75% (two steps); m.p. 146–148 °C. HPLC purity 97.6%. IR (KBr) ν_max/cm^–1 3373, 3098, 1678, 1607, 1578, 1530, 1509, 1484, 1467, 1441, 1392, 1334, 1294, 1224, 1196, 1079, 837, 795. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.74 (s, 1H), 8.52–8.51 (m, 2H), 8.23–8.19 (m, 2H), 8.02 (d, J = 12.4 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 5.6 Hz, 1H), 7.58–7.54 (m, 1H), 7.38–7.33 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.3, 162.9, 162.8, 162.3 (d, J = 244.2 Hz), 157.9, 154.5, 154.1, 153.3 (d, J = 243.8 Hz), 137.7, 137.1 (d, J = 10.0 Hz), 134.7 (d, J = 12.6 Hz), 129.9 (d, J = 2.5 Hz), 128.6 (d, J = 8.5 Hz), 124.4, 124.3, 120.4, 117.3 (d, J = 3.3 Hz), 116.2, 115.7 (d, J = 21.4 Hz), 109.2 (d, J = 22.9 Hz); ESI-MS: m/z 467.0 [M + H]⁺.

N-(3-Fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-phenylthiazole-2-carboxamide (51u)

Yellow solid; yield 49% (two steps); m.p. 78–80 °C. HPLC purity 95.8%. IR (KBr) ν_max/cm^–1 3453, 1719, 1683, 1626, 1559, 1531, 1505, 1477, 1430, 1363, 1335, 1192, 1075, 966, 749. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.67 (s, 1H), 8.53 (s, 1H), 8.17 (dd, J = 1.6, 8.4 Hz, 2H), 8.05–8.01 (m, 2H), 7.84–7.80 (m, 1H), 7.72 (d, J = 6.0 Hz, 1H), 7.56–7.49 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.3, 162.9, 162.3, 157.9, 155.6, 153.3 (d, J = 244.0 Hz), 152.9, 136.9 (d, J = 10.2 Hz), 135.0 (d, J = 12.4 Hz), 133.3, 128.8, 128.7, 128.0, 126.5, 124.3, 120.6, 118.2, 118.0, 117.3 (d, J = 3.1 Hz), 109.3 (d, J = 23.1 Hz); ESI-MS: m/z 449.1 [M + H]⁺.

N-(3-Fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51v)

Yellow solid; yield 62% (two steps); m.p. 154–156 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3452, 3100, 1677, 1607, 1584, 1530, 1506, 1484, 1430, 1364, 1343, 1294, 1223, 1192, 1159, 1070, 968, 839, 763, 707. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.66 (s, 1H), 8.50 (s, 1H), 8.21 (dd, J = 5.6, 8.4 Hz, 2H), 8.04–8.00 (m, 2H), 7.83–7.79 (m, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.55–7.33 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.2, 162.9, 162.4 (d, J = 244.2 Hz), 162.3, 157.9, 154.5, 153.3 (d, J = 243.5 Hz), 152.8, 136.9 (d, J = 10.1 Hz), 135.0 (d, J = 12.5 Hz), 129.9 (d, J = 2.9 Hz), 128.6 (d, J = 8.3 Hz), 127.9, 124.3, 120.4, 118.2, 118.0, 117.3 (d, J = 3.4 Hz), 115.7 (d, J = 21.6 Hz), 109.3 (d, J = 23.2 Hz); ESI-MS: m/z 467.4 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-phenylthiazole-2-carboxamide (51w)

Yellow solid; yield 64% (two steps); m.p. 205–207 °C. HPLC purity 97.0%. IR (KBr) ν_max/cm^–1 3436, 3105, 2917, 2850, 1672, 1606, 1582, 1560, 1533, 1509, 1484, 1459, 1440, 1393, 1303, 1266, 1235, 1194, 1071, 920, 758. ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 8.52 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.16 (d, J = 6.8 Hz, 2H), 8.10–8.06 (m, 1H), 7.87–7.83 (m, 1H), 7.53–7.49 (m, 2H), 7.47–7.39 (m, 2H), 7.10 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4, 6.0 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 166.1, 163.2, 158.4, 156.0, 153.5 (d, J = 244.2 Hz), 152.1, 151.9, 137.6 (d, J = 9.8 Hz), 136.1 (d, J = 12.4 Hz), 133.8, 129.3, 129.2, 126.9, 124.1, 121.1, 118.1 (d, J = 3.0 Hz), 111.5, 111.2, 110.1 (d, J = 22.9 Hz); ESI-MS: m/z 426.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-methylphenyl)thiazole-2-carboxamide (51x)

Pale yellow solid; yield 48% (two steps); m.p. 157–159 °C. HPLC purity 98.1%. IR (KBr) ν_max/cm^–1 3435, 3103, 2923, 2854, 1676, 1604, 1591, 1566, 1531, 1508, 1476, 1442, 1387, 1308, 1267, 1229, 1199, 1065, 922, 822. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.45 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 8.10–8.06 (m, 3H), 7.86 (d, J = 9.2 Hz, 1H), 7.50–7.46 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.12–7.11 (m, 1H), 7.02 (d, J = 4.4 Hz, 1H), 2.37 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.6, 158.0, 155.7, 153.0 (d, J = 244.6 Hz), 151.6, 151.4, 138.1, 137.1 (d, J = 10.2 Hz), 135.6 (d, J = 12.3 Hz), 130.6, 129.3, 126.4, 123.6, 119.8, 117.7 (d, J = 3.0 Hz), 111.0, 110.7, 109.6 (d, J = 23.0 Hz), 20.8; ESI-MS: m/z 440.2 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-methoxyphenyl)thiazole-2-carboxamide (51y)

Pale yellow solid; yield 53% (two steps); m.p. 191–193 °C. HPLC purity 96.4%. IR (KBr) ν_max/cm^–1 3435, 3105, 2924, 1676, 1612, 1591, 1566, 1531, 1508, 1476, 1438, 1387, 1308, 1267, 1248, 1199, 1177, 1068, 1028, 923, 827. ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 8.35 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.09–8.05 (m, 3H), 7.84 (d, J = 8.8 Hz, 1H), 7.48–7.44 (m, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.00 (dd, J = 2.4, 6.0 Hz, 1H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.5, 159.6, 158.0, 155.6, 153.0 (d, J = 244.2 Hz), 151.6, 151.4, 137.1 (d, J = 9.8 Hz), 135.6 (d, J = 12.2 Hz), 127.9, 126.1, 123.6, 118.7, 117.6 (d, J = 3.5 Hz), 114.1, 111.0, 110.7, 109.6 (d, J = 23.0 Hz), 55.2; ESI-MS: m/z 456.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(3,4-dimethoxyphenyl)thiazole-2-carboxamide (51z)

Yellow solid; yield 65% (two steps); m.p. 120–122 °C. HPLC purity 95.9%. IR (KBr) ν_max/cm^–1 3377, 2928, 1678, 1589, 1559, 1537, 1506, 1491, 1460, 1431, 1299, 1264, 1235, 1195, 1161, 1146, 1120, 1073, 1027, 922, 758. ¹H NMR (400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 8.40 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.08–8.05 (m, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.73–7.70 (m, 2H), 7.48–7.44 (m, 1H), 7.09–7.05 (m, 2H), 7.00 (d, J = 4.8 Hz, 1H), 3.89 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.4, 158.0, 155.7, 153.0 (d, J = 244.3 Hz), 151.7, 151.4, 149.4, 148.9, 137.1 (d, J = 9.9 Hz), 135.6 (d, J = 12.1 Hz), 126.2, 123.6, 119.3, 118.9, 117.7 (d, J = 3.3 Hz), 111.8, 111.0, 110.7, 110.4, 109.7 (d, J = 22.4 Hz), 55.7, 55.5; ESI-MS: m/z 486.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-fluorophenyl)thiazole-2-carboxamide (51aa)

White solid; yield 70% (two steps); m.p. 150–152 °C. HPLC purity 96.5%. IR (KBr) ν_max/cm^–1 3434, 3090, 2923, 1674, 1605, 1585, 1560, 1536, 1508, 1485, 1445, 1302, 1266, 1233, 1200, 1162, 1121, 1072, 920, 840. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.50 (s, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.20 (dd, J = 5.6, 8.8 Hz, 2H), 8.06 (dd, J = 2.4, 13.2 Hz, 1H), 7.84–7.81 (m, 1H), 7.49–7.32 (m, 3H), 7.09 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.8, 162.3 (d, J = 244.2 Hz), 157.9, 154.5, 153.0 (d, J = 244.6 Hz), 151.6, 151.4, 137.1 (d, J = 9.8 Hz), 135.6 (d, J = 12.3 Hz), 129.9 (d, J = 2.9 Hz), 128.6 (d, J = 8.1 Hz), 123.6, 120.4, 117.6 (d, J = 3.4 Hz), 115.7 (d, J = 21.7 Hz), 111.0, 110.7, 109.6 (d, J = 23.0 Hz); ESI-MS: m/z 444.3 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(3-fluorophenyl)thiazole-2-carboxamide (51ab)

Yellow solid; yield 46% (two steps); m.p. 189–191 °C. HPLC purity 97.3%. IR (KBr) ν_max/cm^–1 3327, 2917, 1668, 1607, 1582, 1561, 1537, 1509, 1480, 1456, 1435, 1407, 1393, 1303, 1268, 1235, 1197, 1176, 1117, 1071, 919, 771. ¹H NMR (400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 8.62 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.08–8.04 (m, 2H), 7.99 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.57–7.51 (m, 1H), 7.49–7.44 (m, 1H), 7.26–7.21 (m, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.4, 6.0 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.8, 162.6 (d, J = 241.4 Hz), 157.8, 154.1 (d, J = 2.9 Hz), 153.0 (d, J = 244.5 Hz), 151.6, 151.4, 137.1 (d, J = 10.0 Hz), 135.7 (d, J = 10.8 Hz), 135.6 (d, J = 6.7 Hz), 130.8 (d, J = 8.5 Hz), 123.6, 122.4 (d, J = 2.1 Hz), 121.8, 117.7 (d, J = 3.2 Hz), 115.3 (d, J = 21.0 Hz), 113.2 (d, J = 23.0 Hz), 111.0, 110.7, 109.6 (d, J = 22.6 Hz); ESI-MS: m/z 444.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(2-fluorophenyl)thiazole-2-carboxamide (51ac)

Yellow solid; yield 52% (two steps); m.p. 168–170 °C. HPLC purity 98.6%. IR (KBr) ν_max/cm^–1 3351, 2924, 2854, 1674, 1606, 1582, 1560, 1531, 1511, 1488, 1460, 1429, 1394, 1303, 1268, 1235, 1199, 1120, 1071, 920, 763. ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (s, 1H), 8.41 (td, J = 2.0, 8.0 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.06 (dd, J = 2.0, 12.8 Hz, 1H), 7.85–7.81 (m, 1H), 7.51–7.44 (m, 2H), 7.40–7.34 (m, 2H), 7.09 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.0, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.3, 159.6 (d, J = 247.6 Hz), 157.8, 153.0 (d, J = 244.2 Hz), 151.6, 151.4, 149.1 (d, J = 1.8 Hz), 137.0 (d, J = 9.9 Hz), 135.7 (d, J = 12.2 Hz), 130.5 (d, J = 8.6 Hz), 130.3 (d, J = 2.8 Hz), 124.8 (d, J = 3.4 Hz), 124.7 (d, J = 13.5 Hz), 123.6, 121.0 (d, J = 11.4 Hz), 117.7 (d, J = 3.4 Hz), 116.2 (d, J = 21.7 Hz), 111.0, 110.7, 109.7 (d, J = 22.9 Hz); ESI-MS: m/z 444.0 [M + H]⁺.

4-(4-Chlorophenyl)-N-(4-((2-chloropyridin-4-yl)oxy)-3-fluorophenyl)thiazole-2-carboxamide (51ad)

Pale yellow solid; yield 71% (two steps); m.p. 167–169 °C. HPLC purity 98.0%. IR (KBr) ν_max/cm^–1 3435, 3105, 2924, 1674, 1591, 1566, 1538, 1508, 1476, 1441, 1388, 1308, 1268, 1230, 1200, 1096, 1073, 923, 829. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 8.56 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.06 (dd, J = 2.4, 12.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.48–7.44 (m, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.0, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.9, 157.8, 154.2, 153.0 (d, J = 244.9 Hz), 151.6, 151.4, 137.1 (d, J = 9.7 Hz), 135.6 (d, J = 12.3 Hz), 133.3, 132.1, 128.8, 128.1, 123.6, 121.2, 117.7 (d, J = 3.3 Hz), 111.0, 110.7, 109.6 (d, J = 22.7 Hz); ESI-MS: m/z 460.1 [M + H]⁺.

4-(3-Chlorophenyl)-N-(4-((2-chloropyridin-4-yl)oxy)-3-fluorophenyl)thiazole-2-carboxamide (51ae)

Pale yellow solid; yield 65% (two steps); m.p. 171–173 °C. HPLC purity 94.8%. IR (KBr) ν_max/cm^–1 3431, 2924, 1673, 1593, 1560, 1530, 1508, 1476, 1389, 1309, 1267, 1229, 1200, 1163, 1073, 923, 772. ¹H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 8.65 (s, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.29–8.27 (m, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.06 (dd, J = 2.4, 12.8 Hz, 1H), 7.85–7.81 (m, 1H), 7.55–7.51 (m, 1H), 7.49–7.45 (m, 2H), 7.10 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.0, 157.8, 153.8, 153.0 (d, J = 244.1 Hz), 151.6, 151.4, 137.0 (d, J = 9.7 Hz), 135.7 (d, J = 12.4 Hz), 135.2, 133.8, 130.6, 128.4, 126.1, 124.9, 123.6, 121.9, 117.8 (d, J = 3.4 Hz), 111.0, 110.8, 109.7 (d, J = 22.7 Hz); ESI-MS: m/z 460.0 [M + H]⁺.

4-(4-Bromophenyl)-N-(4-((2-chloropyridin-4-yl)oxy)-3-fluorophenyl)thiazole-2-carboxamide (51af)

Pale yellow solid; yield: 78% (two steps); m.p. 192–194 °C. HPLC purity 95.9%. IR (KBr) ν_max/cm^–1 3434, 3102, 2924, 1672, 1592, 1566, 1538, 1508, 1476, 1460, 1441, 1389, 1308, 1268, 1230, 1200, 1166, 1117, 1073, 923, 827. ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 8.58 (s, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 8.06 (dd, J = 2.4, 12.8 Hz, 1H), 7.85–7.81 (m, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.49–7.44 (m, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.0, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.0, 157.8, 154.3, 153.0 (d, J = 244.6 Hz), 151.6, 151.4, 137.1 (d, J = 10.1 Hz), 135.7 (d, J = 12.0 Hz), 132.5, 131.7, 128.4, 123.6, 122.0, 121.3, 117.7 (d, J = 3.3 Hz), 111.0, 110.7, 109.6 (d, J = 23.0 Hz); ESI-MS: m/z 504.2 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(4(trifluoromethyl)phenyl)thiazole-2-carboxamide (51ag)

Yellow solid; yield 41% (two steps); m.p. 212–214 °C. HPLC purity 97.4%. IR (KBr) ν_max/cm^–1 3413, 2924, 1672, 1606, 1591, 1560, 1538, 1509, 1476, 1450, 1409, 1388, 1325, 1307, 1270, 1231, 1201, 1166, 1119, 1071, 923, 833. ¹H NMR (400 MHz, DMSO-d₆) δ 10.96 (s, 1H), 8.73 (s, 1H), 8.37 (d, J = 8.4 Hz, 2H), 8.32 (d, J = 6.0 Hz, 1H), 8.06 (dd, J = 2.4, 13.2 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.84–7.81 (m, 1H), 7.49–7.44 (m, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.4, 6.0 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.3, 157.8, 153.8, 153.0 (d, J = 244.4 Hz), 151.6, 151.4, 137.1, 137.0, 135.8 (d, J = 12.3 Hz), 128.7 (q, J = 31.6 Hz), 127.0, 125.7 (q, J = 3.6 Hz), 124.2 (q, J = 270.4 Hz), 123.6, 123.0, 117.7 (d, J = 3.4 Hz), 111.0, 110.8, 109.7 (d, J = 23.0 Hz); ESI-MS: m/z 494.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(3,4-dichlorophenyl)thiazole-2-carboxamide (51ah)

Pale yellow solid; yield 68% (two steps); m.p. 195–197 °C. HPLC purity 96.5%. IR (KBr) ν_max/cm^–1 3452, 3100, 2924, 2854, 1667, 1593, 1566, 1532, 1508, 1474, 1435, 1391, 1310, 1271, 1230, 1201, 1167, 1071, 923, 825. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.67 (s, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.11 (dd, J = 2.0, 8.4 Hz, 1H), 8.04 (dd, J = 2.4, 13.2 Hz, 1H), 7.83–7.79 (m, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.48–7.44 (m, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.0, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.1, 157.7, 153.0 (d, J = 244.2 Hz), 152.8, 151.6, 151.4, 137.0 (d, J = 10.0 Hz), 135.7 (d, J = 12.3 Hz), 133.8, 131.8, 131.1, 131.0, 128.1, 126.3, 123.6, 122.4, 117.7 (d, J = 3.4 Hz), 111.0, 110.7, 109.7 (d, J = 23.0 Hz); ESI-MS: m/z 493.9 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(naphthalen-2-yl)thiazole-2-carboxamide (51ai)

Pale yellow solid; yield 86% (two steps); m.p. 182–184 °C. HPLC purity 95.9%. IR (KBr) ν_max/cm^–1 3435, 2923, 1673, 1589, 1559, 1549, 1531, 1506, 1476, 1440, 1387, 1308, 1265, 1230, 1200, 1167, 1071, 924, 817. ¹H NMR (400 MHz, DMSO-d₆) δ 11.00 (s, 1H), 8.73 (s, 1H), 8.66 (s, 1H), 8.33 (d, J = 5.6 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.13–7.96 (m, 4H), 7.88 (d, J = 8.4 Hz, 1H), 7.60–7.47 (m, 3H), 7.12 (d, J = 1.6 Hz, 1H), 7.02 (dd, J = 2.4, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.9, 158.0, 155.5, 153.0 (d, J = 244.3 Hz), 151.6, 151.4, 137.1 (d, J = 10.2 Hz), 135.7 (d, J = 12.3 Hz), 133.0, 132.9, 130.7, 128.4, 128.1, 127.7, 126.7, 126.5, 125.3, 124.4, 123.6, 121.1, 117.7 (d, J = 2.6 Hz), 111.0, 110.8, 109.7 (d, J = 22.9 Hz); ESI-MS: m/z 476.2 [M + H]⁺.

N-(4-((2-chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(thiophen-2-yl)thiazole-2-carboxamide (51aj)

Yellow solid; yield 56% (two steps); m.p. 184–186 °C. HPLC purity 95.7%. IR (KBr) ν_max/cm^–1 3435, 3102, 2924, 2853, 1672, 1606, 1584, 1559, 1538, 1509, 1458, 1406, 1394, 1303, 1264, 1234, 1196, 1120, 1070, 920, 712. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.34 (s, 1H), 8.32 (d, J = 6.0 Hz, 1H), 8.05 (dd, J = 2.4, 12.8 Hz, 1H), 7.84–7.80 (m, 1H), 7.74 (dd, J = 1.2, 3.6 Hz, 1H), 7.63 (dd, J = 1.2, 5.2 Hz, 1H), 7.49–7.44 (m, 1H), 7.18 (dd, J = 3.6, 5.2 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.4, 6.0 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.0, 157.8, 153.0 (d, J = 244.1 Hz), 151.6, 151.4, 150.3, 137.0 (d, J = 10.0 Hz), 136.6, 135.7 (d, J = 12.2 Hz), 128.0, 126.9, 125.8, 123.6, 119.3, 117.8 (d, J = 3.1 Hz), 111.0, 110.8, 109.8 (d, J = 22.8 Hz); ESI-MS: m/z 431.9 [M + H]⁺.

4-(3-Chloro-4-fluorophenyl)-N-(4-((2-chloropyridin-4-yl)oxy)-3-fluorophenyl)thiazole-2-carboxamide (51ak)

White solid; yield 76% (two steps); m.p. 168–170 °C. HPLC purity 96.6%. IR (KBr) ν_max/cm^–1 3435, 3096, 2924, 1683, 1585, 1559, 1538, 1506, 1482, 1442, 1328, 1299, 1266, 1232, 1200, 1166, 1123, 1073, 919, 820. ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 8.62 (s, 1H), 8.41 (dd, J = 2.4, 7.2 Hz, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.17–8.13 (m, 1H), 8.05 (dd, J = 2.4, 12.8 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.58–7.45 (m, 2H), 7.09 (d, J = 2.0 Hz, 1H), 7.00 (dd, J = 2.0, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 163.0, 157.7, 157.3 (d, J = 246.9 Hz), 153.1 (d, J = 244.8 Hz), 153.0, 151.6, 151.4, 137.0 (d, J = 10.1 Hz), 135.7 (d, J = 12.0 Hz), 131.1 (d, J = 3.6 Hz), 128.4, 127.0 (d, J = 7.5 Hz), 123.6, 121.6, 120.2 (d, J = 18.0 Hz), 117.7 (d, J = 3.4 Hz), 117.3 (d, J = 21.0 Hz), 111.0, 110.8, 109.7 (d, J = 22.9 Hz); ESI-MS: m/z 478.1 [M + H]⁺.

N-(4-((2-Chloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(3,4-difluorophenyl)thiazole-2-carboxamide (51al)

Pale yellow solid; yield 67% (two steps); m.p. 170–172 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3434, 3086, 2923, 1667, 1607, 1584, 1559, 1537, 1509, 1459, 1302, 1268, 1234, 1199, 1120, 1073, 920, 774. ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 8.59 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 8.29–8.23 (m, 1H), 8.06–7.99 (m, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.62–7.55 (m, 1H), 7.50–7.45 (m, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4, 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 165.6, 162.9, 157.7, 153.2, 153.0 (d, J = 244.2 Hz), 151.6, 151.4, 150.8 (dd, J = 10.4, 243.7 Hz), 148.3 (dd, J = 10.7, 243.9 Hz), 137.0 (d, J = 10.1 Hz), 135.7 (d, J = 12.2 Hz), 130.9 (dd, J = 3.5, 6.5 Hz), 123.6, 123.2 (dd, J = 3.5, 6.5 Hz), 121.5, 118.0 (d, J = 17.3 Hz), 117.7 (d, J = 3.3 Hz), 115.6 (d, J = 18.4 Hz), 111.0, 110.8, 109.6 (d, J = 22.6 Hz); ESI-MS: m/z 462.2 [M + H]⁺.

4-(3-Chloro-4-fluorophenyl)-N-(4-((2,3-dichloropyridin-4-yl)oxy)-3-fluorophenyl)thiazole-2-carboxamide (51am)

Yellow solid; yield 78% (two steps); m.p. 123–125 °C. HPLC purity 97.3%. IR (KBr) ν_max/cm^–1 3434, 2928, 2850, 1684, 1610, 1568, 1559, 1549, 1538, 1506, 1485, 1460, 1310, 1263, 1199, 1130, 1075, 935, 820, 728. ¹H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 8.62 (s, 1H), 8.41 (dd, J = 2.4, 7.6 Hz, 1H), 8.24 (d, J = 5.6 Hz, 1H), 8.17–8.13 (m, 1H), 8.07 (dd, J = 2.4, 13.2 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.58–7.49 (m, 2H), 6.89 (d, J = 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9, 161.2, 157.8, 157.3 (d, J = 246.9 Hz), 153.0, 152.7 (d, J = 244.7 Hz), 149.4, 148.7, 137.2 (d, J = 10.0 Hz), 135.8 (d, J = 12.2 Hz), 131.1 (d, J = 3.8 Hz), 128.4, 127.0 (d, J = 7.6 Hz), 123.4, 121.6, 120.2 (d, J = 18.0 Hz), 117.8, 117.7 (d, J = 3.3 Hz), 117.3 (d, J = 21.2 Hz), 110.4, 109.7 (d, J = 22.5 Hz); ESI-MS: m/z 512.1 [M + H]⁺.

N-(4-((2,3-Dichloropyridin-4-yl)oxy)-3-fluorophenyl)-4-(3,4-difluorophenyl)thiazole-2-carboxamide (51an)

Yellow solid; yield 69% (two steps); m.p. 156–158 °C. HPLC purity 96.2%. IR (KBr) ν_max/cm^–1 3364, 2924, 2855, 1678, 1608, 1559, 1538, 1506, 1482, 1453, 1379, 1328, 1300, 1199, 1116, 1077, 936, 821, 773. ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 8.61 (s, 1H), 8.30–8.27 (m, 1H), 8.24 (d, J = 5.2 Hz, 1H), 8.07 (dd, J = 2.4, 12.8 Hz, 1H), 8.04–8.00 (m, 1H), 7.85–7.81 (m, 1H), 7.63–7.51 (m, 2H), 6.90 (d, J = 5.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.8, 161.2, 157.8, 153.2, 152.7 (d, J = 244.7 Hz), 150.8 (dd, J = 9.7, 243.8 Hz), 149.4, 148.7, 148.4 (dd, J = 10.2, 244.3 Hz), 137.2 (d, J = 10.2 Hz), 135.9 (d, J = 12.3 Hz), 130.9 (dd, J = 3.6, 6.4 Hz), 123.4, 123.3 (dd, J = 2.9, 6.5 Hz), 121.6, 118.0 (d, J = 17.4 Hz), 117.8, 117.7 (d, J = 2.8 Hz), 115.6 (d, J = 18.6 Hz), 110.4, 109.6 (d, J = 22.8 Hz); ESI-MS: m/z 496.2 [M + H]⁺.

Biology

Cell proliferation assay

MTT assay was used to determine the cytotoxic activity of target compounds against different cell lines. The cells (∼4 × 10³ cells/well) were seeded in 96-well plates filled with minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS) and incubated in 5% CO₂ at 37 °C for 24 h. Then, the cells were treated with five doses of test compounds (pre-dissolved in DMSO), and 0.1% DMSO was used as a negative control. After incubation at 37 °C for 48 h, MTT was added to each well and cells were cultured for an additional 4 h. Next, the formazan crystal in the well was dissolved with 100 µL of DMSO for optical density reading at 492 nm (for the absorbance of formazan) and 630 nm (for the reference wavelength). The results expressed as IC₅₀ were the average of three determinations calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

Tyrosine kinases assay

The tyrosine kinases inhibitory activities of target compounds were evaluated in 384-well microtiter plates using purified kinases purchased from Invitrogen (Waltham, MA) by homogeneous time-resolved fluorescence (HTRF) assay. The HTRF KinEASE TK kit was performed according to the instructions. After the kinases and test compounds were incubated at 25–30 °C for 5 min, the kinase reactions were initiated by addition of 39 μL of kinase reaction buffer solution and incubated at 25–30 °C for 30 min. The reactions were quenched by addition 10 µL mixed detection solution. Then, the plates were read using an Envision plate reader at 615 nm and 620 nm, respectively. The inhibition rate (%) was calculated using the following equation: inhibition rate = [(activity of enzyme with tested compound – min)/(max – min)] × 100 (max: the enzyme activity in the presence of enzyme, substrates and cofactors; min: the enzyme activity in the presence of substrates, cofactors and in the absence of enzyme). IC₅₀ value was calculated from the inhibition curve using GraphPad Prism 5.0 software (La Jolla, CA).

Analysis of cellular apoptosis

Apoptosis was detected by an Annexin V/propidium iodide (PI) double staining kit (Beyotime Biotechnology, Shanghai, China). Briefly, MKN-45 cells were treated with different doses of test compounds for 24 h. Cells were harvested by trypsinisation and washed with ice-cold PBS solution. Cells were labelled with annexin V and PI and incubated at room temperature for 30 min in the dark. The labelled cells were analysed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Analysis of cell cycle distribution

After MKN-45 cells were incubated in the presence of test compounds at the indicated concentrations for 24 h, cells were fixed with ice-cold 70% EtOH at 4 °C overnight, incubated with RNase at 37 °C for 30 min and stained with PI working solution for 30 min in the dark. The cells were analysed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ) and the distribution and percentages of cells in the G1, S, and G2/M phases of the cell cycle were analysed using the ModFit LT software.

Western blot analysis

MKN-45 cells were incubated with the test compound at various concentrations for 24 h. After incubation, cells were harvested in PBS solution containing proteinase inhibitors and phosphatase inhibitors, and then sonicated. Whole cell lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Beyotime Biotechnology, Shanghai, China). The membrane was incubated with primary antibody followed by labelling with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Biotechnology, Shanghai, China). The blots were developed with enhanced chemiluminescence and visualised by an LAS4000 imager (Fuji Photo Film, Minato City, Japan).

Pharmacokinetic study

The experimental procedures and the animal use and care protocols were approved by the Committee on Ethical Use of Animals of Shenzhen Second People’s Hospital. Each male BALB/c mice received a single compound 51am as a solution in PEG 400/water (70:30) by either oral route (10 mg/kg) or i.v. route (1.5 mg/kg). Blood samples were collected into the microcentrifuge tube containing heparin at time points 0 (prior to dosing) 5, 10, 20, 30, 60, 180, 360, 480, 600, 720, 1080, and 1440 min after dosing. The blood samples were centrifuged; the obtained plasma was separated and stored at –20 °C. All samples for 51am were measured by liquid chromatography–tandem mass spectrometry (LC/MS/MS).

Docking study

The three-dimensional structures of c-Met and VEGFR-2 were retrieved from the RCSB Protein Data Bank (http://www.pdb.org). Under physiological pH, the binding water and ligand were deleted and the polar hydrogen was added, the protonated state of the important residues was also adjusted by using Sybyl 6.9.1. Molecular docking analysis was carried out by Autodock 4.2 package to explore the binding mode for the active site of c-Met/VEGFR-2 with ligands.

Author contributions

The manuscript was written through the contributions of all authors. All authors approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Shenzhen High-level Hospital Construction Fund [1801025] and the National Key Research and Development Program for 2019 Special Fund [2019YFF0302403]

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