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. 2020 Jun 29;5(27):16482–16490. doi: 10.1021/acsomega.0c00838

Discovery of Triazolo-pyridazine/-pyrimidine Derivatives Bearing Aromatic (Heterocycle)-Coupled Azole Units as Class II c-Met Inhibitors

Qian Zhang †, Xiaobo Liu †, Wenhui Gan †, Jinjin Wu †, Hualan Zhou †, Zunhua Yang ‡, Yiling Zhang ‡, Min Liao †, Ping Yuan †, Shan Xu †, Pengwu Zheng †,*, Wufu Zhu †,*
PMCID: PMC7364574  PMID: 32685812

Abstract

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Two series of novel triazolo-pyridazine/-pyrimidine derivatives were designed, synthesized, and evaluated for their inhibitory activity against c-Met kinase, as well as three c-Met overexpressed cancer cell lines (A549, MCF-7, and HeLa) and one normal human hepatocytes cell line LO2 in vitro. The pharmacological data indicated that most of the tested compounds showed moderate cytotoxicity, and the most promising compound 12e exhibited significant cytotoxicity against A549, MCF-7, and HeLa cell lines with IC50 values of 1.06 ± 0.16, 1.23 ± 0.18, and 2.73 ± 0.33 μM, respectively. Moreover, the inhibitory activity of compound 12e against c-Met kinase (IC50 = 0.090 μM) was equal to that of Foretinib (IC50 = 0.019 μM). The result of the acridine orange (AO) single staining test demonstrated that compound 12e could remarkably induce apoptosis of A549 cells. The results of apoptosis and cycle distribution of cells showed that compound 12e could induce late apoptosis of A549 cells and stimulate A549 cells arresting in the G0/G1 phase. Structure–activity relationships (SARs), pharmacological results, and docking studies indicated that the introduction of 5-methylthiazole fragment to the five-atom moiety was beneficial for the activity. So far, the existing data indicated that compound 12e may become a potential class II c-Met inhibitor.

1. Introduction

c-Met, a receptor tyrosine kinase, is normally activated by binding to its natural ligand hepatocyte growth factor (HGF).1 The binding of HGF to c-Met leads to the activation of signaling pathways.2 However, the dysregulated c-Met/HGF pathway usually caused tumorigenesis and metastasis. Among different cancers, multiple kinds of mechanisms may lead to abnormal activity of c-Met, including activating mutations in the kinase domain, overexpression of c-Met and/or its ligand, hepatocyte growth factor (HGF), MET gene amplification, and cross-talk with other receptor tyrosine kinases.3,4 Prevention of c-Met overexpression is a potentially effective method for treating cancers related to c-Met abnormal activation.5−7

There are a variety of therapies aimed at the c-Met signaling pathway, including antibodies, antagonists, and small-molecule kinase inhibitors. Among them, small-molecule c-Met kinase inhibitors were one of the most effective therapeutics for cancer. They are mainly classified into class I and class II according to their structures and their binding modes. Class II inhibitors such as Foretinib (1),8 Cabozantinib (2),9 and 3(10,11) (Figure 1) are completely embedded in c-Met kinases, and their conformations extend from the hinge region to the deep hydrophobic pockets near the C-helix region. Foretinib (1) is the first oral c-Met inhibitor that entered clinical trials.8,12 Cabozantinib (2) was approved as a novel oral multikinase inhibitor for the treatment of progressive metastatic medullary thyroid cancer.9,13 According to the structural characteristics of class II c-Met inhibitors, they are divided into moieties A, B, and C.13,14 Class I c-Met inhibitors showed well c-Met selectivity by binding to the ATP-binding site at the entrance of the kinase pocket in a U-shaped conformation and wrapping MET-1211, such as compounds 4,155,166,17 and 7.18 (Figure 1). In contrast, the binding area between class II inhibitors and c-Met protein is more extensive, which will help to overcome the drug resistance of inhibitors to achieve a better therapeutic effect.19−21

Figure 1.

Figure 1

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Representative c-Met small-molecule kinase inhibitors.

Previous studies showed that 1H-pyrrolo[2,3-b]pyridine derivatives11 (class II) exhibited good activity. Inspired by this, the structure–activity relationship of class II c-Met inhibitors has been further studied based on moieties A, B, and C. As reported, the class I c-Met inhibitors (compounds 4 and 5) with an active pharmacophore fragment (triazolo-pyrazine) had excellent biological activity. Therefore, the above structure was introduced into the A-region as the core to enhance the binding ability with the c-Met hinge region based on the principle of heterozygosity. In addition, different five-membered heterocycles that were be found possessing excellent biological activity, such as thiazolyl and pyrazolyl, were introduced into the five-atom linker (moiety C) as azole units to replace the linear chains of class II c-Met inhibitors. Ultimately, a series of triazolo-pyridazine/-pyrimidine derivatives with a novel structure were designed. As a result, a series of triazolo-pyridazine compounds 12a–12g and 13a–13h were designed by substituting pyridine or substituted phenyl for the benzene ring (moiety B) extending into the hydrophobic cavity. Then, according to bioisosterism, the triazolo-pyrimidine compounds 18a–18g and 19a–19e containing different azoles units were designed.

The cytotoxicities of these novel compounds against A549, MCF-7, and HeLa cancer cell lines were evaluated, and most of them exhibited moderate cytotoxicity. Most of them exhibited low cytotoxicity against LO2 cells. Moreover, selected compounds 12a–12g and 13a–13h were evaluated for c-Met enzyme in vitro. Acridine orange (AO) staining, apoptosis and cell cycle, and molecular docking of compound 12e were also studied (Figure 2).

Figure 2.

Figure 2

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Design strategies and structures of target compounds.

2. Results and Discussion

2.1. Chemistry

The synthetic routes of the key intermediates 11a–11b and 17a–17b and the target compounds 12a–12g, 13a–13h, 18a–18g, and 19a–19e are outlined in Scheme 1. Intermediate 9 was synthesized by cyclization of 4-amino-1,2,4-triazole (8) and ethyl acetoacetate. 8-Chloro-6-methyl-[1,2,4]triazolo[4,3-b]pyridazine (10) was prepared by the chlorination reaction of intermediate 7 with phosphorus oxychloride (POCl3). Intermediate 11a could be achieved by the substitution reaction of intermediate 8 and 4-aminophenol. Intermediates 11b and 17a–17b were prepared in the same manner as intermediate 11a. Finally, the target compounds 12a–12g, 13a–13h, 18a–18g, and 19a–19e were prepared by intermediates 11a–11b and 17a–17b with different acyl chlorides a–h promoted by N,N-diisopropylethylamine (DIPEA) in dichloromethane (DCM) at room temperature.

Scheme 1. Synthetic Routes of the Target Compounds.

Scheme 1

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Reagents and conditions: (a) ethyl acetoacetate, 110 °C, 1 h; (b) POCl3, 100 °C, reflux, 6 h; (c) 4-aminophenol, potassium t-butoxide (KTB), potassium iodide (KI), tetrahydrofuran (THF), 80 °C, 1.1 h; and (d) DIPEA, Z-COOH, oxalyl chloride, dimethylformamide (DMF), DCM, r.t., 0.5 h.

2.2. Biological Evaluation

The cytotoxicities of compounds depicted in Tables 1 and 2 were evaluated in vitro using Foretinib as a positive control. The cytotoxicities of all target compounds against A549, HeLa, MCF-7, and LO2 cells were determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, the enzymatic activities of compounds 12a–12g and 13a–13h against c-Met were determined using Foretinib as a positive control.

Table 1. In Vitro Cytotoxicity and c-Met Kinase Inhibitory Activities of Compounds 12a–12g and 13a–13ha,b,c,d.

2.2.

2.2.

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a

The values are an average of two separate determinations.

b

ND: not detected.

c

Bold shows IC50 ≤ 5 μM.

d

Used as a positive control.

Table 2. In Vitro Cytotoxicity of Compounds 18a–18g and 19a–19ea,b,c.

2.2.

2.2.

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a

The values are an average of two separate determinations.

b

ND: not detected.

c

Used as a positive control.

As illustrated in Tables 1 and 2, most target compounds showed moderate cytotoxicity against A549, HeLa, and MCF-7 cancer cell lines with potencies in the micromole range. Fifteen compounds with better cytotoxicity were selected to evaluate their antiproliferative effect against LO2 cells in vitro. These compounds did not show significant cytotoxicity (IC50 > 50.00 μM) to normal human cells. Compounds 12b and 18b exhibited moderate cytotoxicity against A549, MCF-7, and HeLa cancer cell lines. Interestingly, compounds 13b and 19b with F substituted on the phenyl group (X = F) had improved cytotoxicity against A549, MCF-7, and HeLa cancer cell lines. This indicates that fluorine substitution at this position is favorable for cytotoxicity. Compound 12e exhibited significant cytotoxicity against A549, MCF-7, and HeLa cancer cell lines with IC50 values of 1.06 ± 0.16, 1.23 ± 0.18, and 2.73 ± 0.33 μM, respectively. Compound 19e, of which the triazolo-pyridazine structure was replaced by the triazolo-pyrimidine structure, exhibited significant improvement in cytotoxicity. Compounds 12c–12d and 12f–12g displayed poor cytotoxicity than compound 12e. These results showed that the compounds substituted with 2-pyridyl group on the 5-methyl-thiazole group have stronger cytotoxicity than those substituted with benzene, 3-pyridine group, and 4-pyridyl group. Moreover, compounds 12e, 13e, and 19e showed the most promising cytotoxicity with IC50 values lower than 10 μM. Further studies on structure–activity relationships (SARs) showed that 2-pyridyl group played an important role in cytotoxicity. Moderate cytotoxicity was achieved for compounds 12c–12d, 13–13d, and 19c–19d against all tested cell lines. This indicates that halogen (F) substitution at the benzene ring had a little effect on cytotoxicity.

Compounds 12a, 12c–12e, 13a–13c, 13e, and 13g were selected for further testing of c-Met kinase activity. Compounds 12e and 13b exhibited significant c-Met enzymatic potency with IC50 values ranging from 0.09 to 0.21 μM. Moreover, the data of the inhibitory activity of the compounds 12e and 13b to c-Met kinase were consistent with their cell cytotoxicity. These data suggested that these derivatives were effective in inhibiting c-Met kinase.

2.3. Acridine Orange (AO) Staining on A549 Cells of Compound 12e

To explore the ability of target compounds to induce apoptosis of A549 cells, the most promising compound 12e was selected for the AO fluorescence assay. In the control group (Figure 3A), all cells were growing in a regular round shape with clear edges. After the treatment of A549 cells with compound 12e at a concentration of 0.50 μM for 8 h, a series of apoptosis phenomena such as cell atrophy, chromatin agglutination, and sharp edge appeared in cells. This result indicated that compound 12e had a significant effect on inducing apoptosis, as shown in Figure 3B.

Figure 3.

Figure 3

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Cell morphology of A549 cells. (A) A549 cells treated without any compounds. (B) A549 cells treated with compound 12e for 8 h. Changes in the nuclear morphology were determined by AO staining.

2.4. Effect on Apoptosis and Cell Cycle Progression of Compound 12e

To further verify the ability of compound 12e to induce apoptosis of A549 cells, the flow cytometry assay was performed. The results show that compound 12e could induce late apoptosis of cells in a dose-dependent manner, as shown in Figure 4. The total apoptosis rates of cells treated with compound 12e were 13.73, 24.87, and 35.87% at concentrations of 3.75, 7.50, and 15.00 μM, respectively, compared with the control group (5.16%).

Figure 4.

Figure 4

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Apoptosis analyses of A549 cells treated with compound 12e for 24 h.

Furthermore, cell cycle distribution analysis was carried out on A549 cells to explore the mechanism of cell proliferation inhibition by compound 12e. The G0/G1 phase population of the A549 cancer cells in the control group was 56.06%, as shown in Figure 5. After treatment with compound 12e at the concentrations of 3.75, 7.50, and 15.00 μM, the G0/G1 phase population of cells increased to 60.25, 66.32, and 76.84%, respectively, while the S phase of cell population decreased. Meanwhile, no obvious population changes were seen in the G2/M phase of cells. It is found that compound 12e could stimulate the A549 cells in the G0/G1 phase in a dose-dependent manner.

Figure 5.

Figure 5

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Cell cycle progression analyses of A549 cells treated with compound 12e for 24 h.

2.5. Molecular Docking Study

To explore the binding modes of target compounds with c-Met, molecular docking simulation studies of the most promising compound 12e was carried out by AutoDock 4.2 software and the docking results were processed and modified by PyMOL 1.8.x software. The results are shown in Figure 6.

Figure 6.

Figure 6

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Docking mode of Foretinib and compound 12e. (A, B) Binding model of Foretinib bound to c-Met (PDB code: 3LQ8). (C, D) Binding model of compound 12e bound to c-Met (PDB code: 3LQ8).

The binding mode indicated that compound 12e bound to c-Met in a similar way to Foretinib (Figure 6A and B). As shown in Figure 6C and D, the two nitrogen atoms of the triazolo-pyridazine core structure made a bidentate hydrogen bond with the key amino acid residues of MET-1160 in the hinge, while the nitrogen atom on the thiazole ring of the five-atom moiety and the nitrogen atom on 2-pyridine made a bidentate hydrogen bond with ASP-1222. This indicated that the triazolo-pyridazine core structure can improve the activity of the target compounds and the amino-heterocycles can improve the ATP-binding ability with the amino acid residues. In addition, the carbonyl oxygens group of the five-atom moiety formed a hydrogen bond with the LYS-1110 residue, which was critical for c-Met inhibition. These results of the molecular docking study showed that compound 12e may be a potential class II c-Met inhibitor.

3. Conclusions

Two series of novel triazolo-pyridazine/-pyrimidine derivatives were designed and synthesized, and the cytotoxicities of all of the compounds were evaluated by the MTT method. Most compounds showed moderate cytotoxicity, and compounds 12e and 19e exhibited significant cytotoxicity. Specifically, compound 12e showed optimal cytotoxicity against A549, MCF-7, and HeLa cancer cell lines, with the IC50 values of 1.06 ± 0.16, 1.23 ± 0.18, and 2.73 ± 0.33 μM, respectively. The results of apoptosis and cycle distribution of cells showed that compound 12e could induce late apoptosis of A549 cells and stimulate A549 cell arrest in the G0/G1 phase. In conclusion, our research confirmed the potential of the triazolo-pyridazine/-pyrimidine derivatives for the discovery of class II c-Met inhibitors, and further studies on the structural optimization and biological activities of these derivatives will be carried out.

4. Experimental Section

4.1. General Information

Common reagents and materials were purchased commercially. Melting points were measured using a Büchi melting point B-540 instrument. 1H NMR spectra were obtained using a Bruker 400 MHz spectrometers with tetramethylsilane (TMS) as an internal standard. Mass spectra (MS) were recorded on electrospray ionization (ESI) mode using an Agilent 1100 Liquid chromatography–mass spectrometer (LC–MS). Thin-layer chromatography (TLC) plates were visualized by exposure to ultraviolet light (UV). Thin-layer chromatography (TLC) analysis was carried out on silica gel plates GF254 (Qingdao Haiyang Chemical, Qingdao, China). All materials were obtained from commercial suppliers and used without purification unless otherwise specified. Yields were optimized.

4.2. Chemistry

4.2.1. Preparation of Compounds 9 and 15

A mixture of 4-amino-1,2,4-triazole (10 g, 0.12 mol) and ethyl acetoacetate (100 mL) was added into a 250 mL flask and stirred for 1 h at 110 °C. The reaction was monitored by TLC (DCM/MeOH = 15:1), and the mixture was cooled down, filtered, and dried to give a white solid compound 9, yield 98%. Also, using the same method, we acquired a gray solid compound 15 from 14 with a yield of 97%.

4.2.2. Preparation of Compounds 10 and 16

To a stirred solution of compound 7 (1 g, 4.5 mmol) in phosphorus oxychloride (30 mL), a few drops of DMF were added. The reaction mixture was heated to 100 °C for 6 h and monitored by TLC. After cooling to room temperature, most of the phosphorus oxychloride was concentrated. The concentrate was poured into water (20 mL) and extracted with EtOAc (3 × 100 mL). The extracting solution was concentrated and dried to obtain compound 10. Using the same method as above, compound 15 was obtained from compound 16 with a yield of 87%.

4.2.3. Preparation of Compounds 11a–11b and 17a–17b

t-BuOK (1.46 g, 13.0 mmol) and KI (0.12 g, 0.72 mmol) were added to the mixture of THF (20 mL) with 4-aminophenol or with 4-amino-2-fluorophenol (0.78 g, 7.2 mmol) at 0 °C and stirred for 1 h. Then, 10 (1 g, 6.5 mmol) was added and the reaction was continued for 0.08 h at 80 °C. The TLC (DCM/MeOH =15:1) method was used to test whether the reaction was completed; then, t-BuOK was filtered out. Compound 11a was extracted from the filtrate with DCM under alkaline conditions. The extracting solution was concentrated to get a gray solid compound 11a in 88% yield. The synthesis method for compounds 11b and 17a–17b was the same as that for compound 11a. Compounds 11b and 17a–17b were obtained as yellow solids in 69–80% yield.

4.2.4. Preparation of Compounds 12a–12g, 13a–13h, 18a–18g and 19a–19e

Compounds a–h (0.041 g, 0.62 mmol) and a few drops of DMF were dissolved in dichloromethane; then, an appropriate amount of oxalyl chloride was added slowly and monitored by TLC. The aniline compound 11a (0.05 g, 0.51 mmol) and DIPEA (0.2 mL) were added to DCM (10 mL) in an ice bath. After completion, the solution was concentrated to give yellow solid compounds 12a–12g. The synthesis method for compounds 13a–13h, 18a–18g, and 19a–19e was the same as that for compounds 12a–12g.

4.2.5. N-(4-((6-Methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-3-(thiophen-2-yl)isoxazole-5-carboxamide (12a)

A light yellow solid in 33% yield, mp 269.5–271.3 °C, 1H NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) δ = 11.02 (s, 1H), 9.59 (s, 1H), 7.99 (d, J = 8.9 Hz, 2H), 7.95–7.85 (m, 2H), 7.45–7.37 (m, 3H), 7.31 (d, J = 3.8 Hz, 1H), 6.31 (s, 1H), 2.41 (s, 3H). Time-of-flight (TOF) MS ES+ (m/z): [M + H]+, calcd for C20H14N6O3S: 419.0926; found, 419.0848.

4.2.6. 1-(4-Chlorophenyl)-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (12b)

A light yellow solid in 42% yield; mp 248.0–249.2 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.76 (s, 1H), 9.59 (s, 1H), 8.36 (s, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 6.31 (s, 1H), 2.41 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H15ClF3N7O2: 514.1006; found, 514.0928.

4.2.7. 4-Methyl-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-2-phenylthiazole-5-carboxamide (12c)

A light yellow solid in 80% yield; mp 95.6–98.2 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.52 (s, 1H), 9.60 (s, 1H), 8.00 (d, J = 5.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 3.3 Hz, 3H), 7.39 (d, J = 8.7 Hz, 2H), 6.30 (s, 1H), 2.68 (s, 3H), 2.41 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H18N6O2S: 443.1290; found, 443.1312.

4.2.8. 2-(4-Fluorophenyl)-4-methyl-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)thiazole-5-carboxamide (12d)

A light yellow solid in 38% yield; mp 265.7–267.9 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.58 (s, 1H), 8.13 (d, J = 5.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 3.3 Hz, 3H), 7.42 (d, J = 8.7 Hz, 2H), 6.54 (s, 1H), 2.68 (s, 3H), 2.41 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H17FN6O2S: 461.1196; found, 461.1245.

4.2.9. 4-Methyl-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-2-(pyridin-2-yl)thiazole-5-carboxamide (12e)

A light yellow solid in 21% yield; mp 205.3–207.6 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.53 (s, 1H), 9.59 (d, J = 1.6 Hz, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.01 (t, J = 7.6 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.57 (t, J = 6.3 Hz, 1H), 7.40 (d, J = 8.6 Hz, 2H), 6.30 (s, 1H), 2.69 (s, 3H), 2.41 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H17N7O2S: 444.1243; found, 444.1357.

4.2.10. 4-Methyl-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-2-(pyridin-3-yl)thiazole-5-carboxamide (12f)

A light yellow solid in 79% yield; mp 238.1–239.7 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.22 (s, 1H), 9.60 (s, 2H), 8.00 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.94–7.59 (m, 3H), 7.56 (d, J = 8.9 Hz, 2H), 6.45 (s, 1H), 4.34 (t, J = 7.1 Hz, 3H), 2.42 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H17N7O2S: 444.1243; found, 444.1241.

4.2.11. 4-Methyl-N-(4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-2-(pyridin-4-yl)thiazole-5-carboxamide (12g)

A light yellow solid in 53% yield; mp 196.3–197.2 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.19 (s, 1H), 9.63 (s, 1H), 8.16–8.12 (m, 2H), 8.02–7.87 (m, 2H), 7.71–7.59 (m, 2H), 7.52–7.45 (m, 2H), 6.37 (s, 1H), 2.55 (d, J = 1.9 Hz, 3H), 2.46 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H17N7O2S: 444.1243; found, 444.1247.

4.2.12. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-3-(thiophen-2-yl)isoxazole-5-carboxamide (13a)

A light yellow solid in 28% yield; mp 253.8–255.6 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.22 (s, 1H), 9.62 (s, 1H), 8.07 (d, J = 12.7 Hz, 1H), 7.95–7.85 (m, 2H), 7.80 (d, J = 9.1 Hz, 1H), 7.59 (t, J = 8.9 Hz, 1H), 7.40 (s, 1H), 7.31 (d, J = 4.9 Hz, 1H), 6.47 (s, 1H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C20H13FN6O3S: 437.0832; found, 437.0754.

4.2.13. 1-(4-Chlorophenyl)-N-(3-fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (13b)

A light yellow solid in 29% yield; mp 232.2–234.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.94 (s, 1H), 9.62 (s, 1H), 8.37 (s, 1H), 7.98 (d, J = 13.3 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 9.2 Hz, 4H), 6.47 (s, 1H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H14ClF4N7O2: 532.0912; found, 532.0919.

4.2.14. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide (13c)

A light yellow solid in 41% yield; mp 165.7–167.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.67 (s, 1H), 9.62 (s, 1H), 8.02–7.94 (m, 3H), 7.63 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 6.5 Hz, 4H), 6.46 (s, 1H), 2.68 (s, 3H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H17FN6O2S: 461.1196; found, 461.1197.

4.2.15. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-2-(4-fluorophenyl)-4-methylthiazole-5-carboxamide (13d)

A light yellow solid in 30% yield; mp 251.3–253.7 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.63 (s, 1H), 8.57 (d, J = 4.0 Hz, 1H), 8.36 (q, J = 7.6 Hz, 2H), 7.89 (d, J = 12.7 Hz, 1H), 7.57 (d, J = 21.2, 8.5 Hz, 2H), 7.45 (p, J = 9.4, 8.9 Hz, 3H), 2.75 (s, 3H), 2.67 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H16F2N6O2S: 479.1102; found, 479.1036.

4.2.16. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-4-methyl-2-(pyridin-2-yl)thiazole-5-carboxamide (13e)

A light yellow solid in 50% yield; mp 274.3–278.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.75 (s, 1H), 9.61 (s, 1H), 8.66 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 13.1 Hz, 2H), 7.49 (d, J = 37.8 Hz, 3H), 6.40 (s, 1H), 2.70 (s, 3H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H16FN7O2S: 462.1148; found, 462.1169.

4.2.17. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-4-methyl-2-(pyridin-3-yl)thiazole-5-carboxamide (13f)

A light yellow solid in 51% yield; mp 223.3–224.2 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.37 (s, 1H), 9.41 (s, 1H), 8.72 (s, 1H), 8.45 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 6.41 (s, 1H), 2.79 (s, 3H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H16FN7O2S: 462.1148; found, 462.1150.

4.2.18. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-4-methyl-2-(pyridin-4-yl)thiazole-5-carboxamide (13g)

A light yellow solid in 31% yield; mp 110.8–111.2 °C, 1H NMR (400 MHz, DMSO-d6) δ = 9.63 (s, 1H), 8.72 (d, J = 5.2 Hz, 2H), 7.96–7.87 (m, 3H), 7.78–7.71 (m, 2H), 7.67 (d, J = 8.8 Hz, 1H), 6.55 (s, 1H), 2.68 (s, 3H), 2.27 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H16FN7O2S: 462.1148; found, 462.1147.

4.2.19. N-(3-Fluoro-4-((6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)oxy)phenyl)-3-(thiophen-2-yl)-1H-pyrazole-5-carboxamide (13h)

A light yellow solid in 30% yield; mp 253.8–255.6 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.19 (s, 1H), 9.63 (s, 1H), 8.16–8.12 (m, 2H), 8.02–7.87 (m, 2H), 7.71–7.59 (m, 2H), 7.52–7.45 (m, 2H), 6.37 (s, 1H), 2.55 (d, J = 1.9 Hz, 3H), 2.46 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C20H14FN7O2S: 436.0992; found, 436.0989.

4.2.20. N-(4-((5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-3-(thiophen-2-yl)isoxazole-5-carboxamide (18a)

A light yellow solid in 23% yield; mp 239.6–242.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.14 (s, 1H), 8.74 (s, 1H), 8.05 (d, J = 12.7 Hz, 1H), 7.91 (d, J = 5.1 Hz, 1H), 7.75 (s, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.35 (s, 1H), 7.29–7.26 (m, 2H), 6.45 (s, 1H), 2.51 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C20H14N6O3S: 419.0926; found, 419.0847.

4.2.21. 1-(4-Chlorophenyl)-N-(4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (18b)

A light yellow solid in 33% yield; mp 203.1–205.7 °C, 1H NMR (400 MHz, DMSO-d6) δ = 13.26 (s, 1H), 8.25 (s, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.66 (dd, J = 8.8, 2.1 Hz, 4H), 7.62–7.60 (m, 2H), 7.58 (d, J = 2.0 Hz, 2H), 6.87–6.71 (m, 2H), 5.82 (s, 1H), 2.36–2.30 (m, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H15ClF3N7O2: 514.1006; found, 514.1012.

4.2.22. 4-Methyl-N-(4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-2-(pyridin-3-yl)thiazole-5-carboxamide (18f)

A light yellow solid in 37% yield; mp 213.7–215.8 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.29 (s, 1H), 9.24 (s, 1H), 8.75 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.41 (s, 1H), 2.79 (s, 3H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H17FN7O2S: 444.1243; found, 444.1164.

4.2.23. 4-Methyl-N-(4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-2-(pyridin-4-yl)thiazole-5-carboxamide (18g)

A light yellow solid in 21% yield; mp 203.1–205.7 °C, 1H NMR (400 MHz, DMSO-d6) δ = 9.88 (s, 1H), 8.63 (s, 1H), 8.54 (s, 1H), 7.81 (d, J = 4.8 Hz, 1H), 7.29–7.18 (m, 4H), 6.70 (d, J = 9.0 Hz, 2H), 4.67 (s, 1H), 2.60 (s, 3H), 2.38 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H17N7O2S: 444.1243; found, 444.1164.

4.2.24. N-(3-Fluoro-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-3-(thiophen-2-yl)isoxazole-5-carboxamide (19a)

A light yellow solid in 58% yield; mp 255.9–258.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.24 (s, 1H), 8.62 (s, 1H), 8.08 (d, J = 12.7 Hz, 1H), 7.90 (d, J = 5.1 Hz, 1H), 7.85 (s, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.38 (s, 1H), 7.30–7.27 (m, 1H), 6.54 (s, 1H), 2.51 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C20H13FN6O3S: 437.0832; found, 437.0754.

4.2.25. 1-(4-Chlorophenyl)-N-(3-fluoro-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide (19b)

A light yellow solid in 26% yield; mp 252.3–254.6 °C, 1H NMR (400 MHz, DMSO-d6) δ = 11.43 (s, 1H), 8.64 (s, 1H), 8.39 (s, 1H), 8.17 (d, J = 12.5 Hz, 1H), 8.04 (s, 1H), 7.93 (s, 1H), 7.69 (s, 4H), 6.56 (s, 1H), 2.53 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H14ClF4N7O2: 532.0912; found, 532.0834.

4.2.26. N-(3-Fluoro-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-4-methyl-2-phenylthiazole-5-carboxamide (19c)

A light yellow solid in 56% yield; mp 169.2–171.3 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.69 (s, 1H), 9.53 (s, 1H), 8.01–7.92 (m, 3H), 7.66 (d, J = 7.4 Hz, 1H), 7.54 (d, J = 6.5 Hz, 4H), 6.48 (s, 1H), 2.68 (s, 3H), 2.43 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H17FN6O2S: 461.1196; found, 461.1118.

4.2.27. N-(3-Fluoro-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-2-(4-fluorophenyl)-4-methylthiazole-5-carboxamide (19d)

A light yellow solid in 44% yield; mp 209.9–213.1 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.54 (s, 1H), 8.12 (d, J = 4.0 Hz, 1H), 7.98 (q, J = 7.6 Hz, 2H), 7.65 (d, J = 12.7 Hz, 1H), 7.43 (d, J = 21.2, 8.5 Hz, 2H), 7.26 (p, J = 9.4, 8.9 Hz, 3H), 2.75 (s, 3H), 2.67 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C23H16F2N6O2S: 479.1107; found, 479.1196.

4.2.28. N-(3-Fluoro-4-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)-4-methyl-2-(pyridin-2-yl)thiazole-5-carboxamide (19e)

A light yellow solid in 31% yield; mp 304.1–306.0 °C, 1H NMR (400 MHz, DMSO-d6) δ = 10.73 (s, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.64 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.06–7.97 (m, 2H), 7.66 (d, J = 5.6 Hz, 2H), 7.57 (t, J = 6.3 Hz, 1H), 6.55 (s, 1H), 2.69 (s, 3H), 2.53 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C22H16FN7O2S: 462.1148; found, 461.1070.

4.3. Tyrosine Kinases Assay In Vitro

The target compounds 12a–12g and 13a–13h were tested for their activity against c-Met tyrosine kinases through the mobility shift assay, with Foretinib as a positive control. Specific operations were performed based on our previous research.22

4.4. Cytotoxicity Assay In Vitro

The in vitro cytotoxic activities of all compounds 12a–12g, 13a–13h, 18a–18g, and 19a–19e were evaluated with A549, MCF-7, and HeLa cancer cell lines by the standard MTT assay, with Foretinib as a positive control. Specific operations were performed according to our previous research.23

4.5. Acridine Orange (AO) Single Staining

The effect of target compound 12e on the apoptosis of tumor cells of A549 cells was examined by single staining with acridine orange. Specific operations were performed according to our previous research.24

4.6. Effect on Apoptosis and Cell Cycle Progression

A flow cytometer (BD Accuri C6, BD Biosciences) was used to detect the apoptosis of A549 cells by compound 12e and analyze the progress of cell cycle inhibition.25

4.7. Docking Studies

The three-dimensional structure of c-Met (PDB code: 3LQ8) was obtained from the RCSB Protein Data Bank. The protein preparation process of flexible docking mainly includes fixing the exact residues, adding hydrogen atoms, removing irrelevant water molecules, adding charges, etc. Molecular docking simulation studies were carried out using AutoDock 4.2 software (The Scripps Research Institute).

Acknowledgments

We gratefully acknowledge the generous support provided by the National Natural Science Foundation of China (21662014), the Natural Science Foundation of Jiangxi, China (20181ACB20025, 20181BBG70003), the Youth Top Talent Support Program of Jiangxi Science & Technology Normal University (2019QNBJRC008), and Nanchang Key Laboratory of Molecular Targeted Anticancer Drug Design and Evaluation (2019-NCZDSY-007).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c00838.

  • 1H NMR spectra and TOF MS analytical data (PDF)

Author Contributions

§ Q.Z. and X.L. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ao0c00838_si_001.pdf (543.4KB, pdf)

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Supplementary Materials

ao0c00838_si_001.pdf (543.4KB, pdf)

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