Discovery of novel conjugates of quinoline and thiazolidinone urea as potential anti-colorectal cancer agent

Abstract

Based on the obtained SARs, further structural optimisation of compound BC2021-104511-15i was conducted in this investigation, and totally ten novel quinoline derivates were designed, synthesised and optimised for biological activity. Among them, compound 10a displayed significant in vitro anticancer activity against COLO 205 cells with an IC₅₀ value of 0.11 μM which was over 90-fold more potent than that of Regorafenib (IC₅₀>10.0 μM) and Fruquintinib (IC₅₀>10.0 μM). Furthermore, compound 10a exhibited over 90-fold selectivity towards COLO 205 relative to human normal colorectal mucosa epithelial cell FHC cells. Flow cytometry study demonstrated that compound 10a could induce apoptosis in COLO 205 cells, however, it could not induce cell cycle arrest in COLO 205 cells. The results of preliminary kinase profile study showed that compound 10a was a potential HGFR and MST1R dual inhibitor, with IC₅₀ values of 0.11 μM and 0.045 μM, respectively.

Keywords:

• Anticancer
• HGFR
• MST1R
• quinoline
• structure modification

1. Introduction

Colorectal cancer (CRC) is one of the most predominant malignancies with a high mortality rate globally¹. It is estimated that the number of CRC patients will reach 2.5 million in 2035². Approximately 25% of CRC patients presented metastatic disease at diagnosis, while almost 50% of them will develop metastases. According to the statistics, the 5-year survival rate ranged from 90% to 14% if CRC is diagnosed at a localised or metastatic stage³.

Nowadays, chemotherapy is the most extensively applied approach for the treatment of primary CRC and/or metastatic CRC (mCRC)⁴. The drugs used in chemotherapy were divided into cytotoxic drugs, tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and programmed cell death protein-1/programmed cell death 1 ligand 1 (PD1/PD-L1) inhibitors, etc^5,6. Among them, TKIs could significantly improve major efficacy parameters which included response rate (RR), progression free survival (PFS) and overall survival (OS). Unfortunately, only Regorafenib and Fruquintinib are successfully utilised in clinic as TKIs for the treatment of patients with mCRC (Figure 1). Regorafenib is an orally bioavailable multitarget TKI which mainly inhibits the activity of vascular endothelial growth factor receptor1-3 (VEGFR1-3), tunica intima endothelial kinase 2 (TIE2), rearranged during transfection (RET), mast/stem cell growth factor receptor (Kit), and platelet-derived growth factor receptor (PDGFR), etc⁷. It has been approved by US Food and Drug Administration (US FDA) and European Medicines Agency (EMA) for the treatment of mCRC patients who had already been treated with fluoropyrimidine, oxaliplatin, anti-VEGF therapy and/or irinotecan-based chemotherapy⁸. Fruquintinib also is a small molecule multitarget TKI with high affinity for VEGFR1-3⁹. In 2018, it was approved by National Medical Products Administration (NMPA) for the treatment of mCRC patients who had suffered at least two unsuccessful standard therapies¹⁰. Despite the recent advances in the chemotherapy of primary CRC and mCRC, the survival benefit is still limited due to the high heterogeneity, resistance and severe side effects. Accordingly, there is still urgent need to develop alternative and potential therapeutic strategies with high efficacy and acceptable side effects for the treatment of CRC.

Figure 1. The structures of Regorafenib, Fruquintinib, Foretinib, BMS-777607, MK-8033, Crizotinib, Cabozantinib and BC2021-104511-15i.

Hepatocyte growth factor receptor (HGFR, also known as mesenchymal-epithelial transition factor [c-Met]), and macrophage-stimulating protein receptor (MST1R, also known as recepteur d’Origine nantais [Ron]), belong to a unique subfamily of receptor tyrosine kinases (RTKs)¹¹. It was reported that they shared 34% overall homology and the tyrosine kinase region shared 80% homology¹². The phosphorylated HGFR and MST1R could activate several transduction proteins and trigger their downstream signalling cascades which mainly included phosphatidylin-ositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, mitogen-activated protein kinase (MAPK) pathway, signal transducer and activator of transcription (STAT) pathway, cell sarcoma (c-Src), and extracellular regulated protein kinase1/2 (ERK1/2), etc^13–17. The dysregulation of HGFR and/or MST1R were extensively implicated in multiple cancer oncogenic processes, such as proliferation, migration, invasion, angiogenesis, epithelial-to-mesenchymal transition (EMT) and drug resistance, etc^18–22. It is noteworthy that HGFR and MST1R played an important role in the CRC’s progression, malignancy, and stemness. Therefore, HGFR/MST1R dual inhibitors might be optimal agents for the treatment of CRC. Pharmaceutically, numerous HGFR and/or MST1R kinase inhibitors have been evaluated for the treatment of different types of cancers, such as Foretinib, BMS-777607, MK-8033, Crizotinib, Cabozantinib (Figure 1)^23–25. However, no small molecular HGFR and MST1R dual inhibitors have been discovered as agents for the treatment of CRC.

Based on the above survey, a study on developing novel HGFR/MST1R dual inhibitors as anti-CRC agents was carried out by our group. As shown in Figure 1, HGFR/MST1R dual inhibitor BC2021-104511-15i was discovered by our group^26,27. It exhibited potential in vitro anticancer activity against several cancer cell lines, especially human colorectal carcinoma cell line HT-29 cells. In order to obtain a more potent HGFR/MST1R dual inhibitor as an agent for the treatment of CRC, further modification on the fragments I, II, and III of BC2021-104511-15i was performed (Figure 2). The details of design, synthesis, biological evaluation, docking study and anticancer mechanism were all discussed in the following sections.

Figure 2. The binding mode of BC2021-104511-15i with HGFR (PBD ID: 3LQ8), the SARs of lead compound BC2021-104511-15i and the modification of fragments I, II and III. Lead compound was shown by blue sticks, and the H-bonds were represented by green dotted lines, and the H-arene interaction was shown by red dotted lines.

2. The modification of lead compound

As shown in Figure 2, preliminary SARs were summarised based on the biological evaluation in our previous research. The SARs indicated that the groups attached to the piperidine ring (I) could significantly influence the HGFR and MST1R kinases inhibitory activity, in vitro anticancer activity and water solubility. Thus, compounds 10a–h bearing variant substituents on piperidine rings were designed and synthesised in the beginning of this work. Docking study showed that two significant H-bonds were formed by quinoline ring and urea moiety with Met1160 and Lys1110, respectively (Figure 2). The terminal difluoro-substituted phenyl ring reached into a hydrophobic pocket, and H-π interaction was formed. We assumed that the spatial position change of the H-bond donors and acceptors in the moiety of thiazolidine-4-one urea might strengthen the H-bond and/or lead to additional H-bonds. Thus, the oxygen atom linked quinoline ring and 2-fluorophenyl ring (III) was replaced to deflect the dihedral angel formed by the aromatic rings, and compound 10i was designed and synthesised. Additionally, a quinazoline derivate 19 was designed and prepared to investigate the influence on the kinase inhibitory activity when the electron density distribution changed in the quinoline ring.

Based on the above assumption, totally ten novel compounds were designed, synthesised and evaluated for their biological activity in the present work. Moreover, the anticancer mechanism was also investigated preliminarily.

3. Results and discussion

3.1. Chemistry

Target compounds 10a–i and 19 were successfully prepared by the synthetic routes outlined in Scheme 1 and Scheme 2²⁶. Commercially available 7-(benzyloxy)-4-chloro-6-methoxyquinoline was reacted with 2-fluoro-4-nitrophenol in refluxing chlorobenzene to afford 4-aryloxyquinoline 1a. Subsequently, intermediate 1 was cleanly debenzylated by 33% HBr in acetic acid to obtain phenol 2a, which was alkylated with 1-Boc-4-methanesulfonyloxypiperidine in the presence of Cs₂CO₃ to provide 3a. The amine 4a was achieved by reduction of nitro group in 3a. Then, the semicarbazide 6a was acquired using a two-step procedure involving acylation reaction with phenyl chloroformate in the presence of pyridine and subsequent hydrazinolysis reaction with 50% hydrazine hydrate in xylene with vigorous agitation. Condensation of 6a with 2,6-difluorobenzaldehyde in favour of catalytic HOAc was carried out to provide 7a. The N-Boc group in the semicarbazone 7a was deprotected by CF₃COOH to afford piperidine derivate 8a, which was then acylated or alkylated with corresponding acyl chlorides or chlorides to give intermediates 9a–h. Finally, the target compounds 10a–g were prepared by cyclisation reaction with mercaptoacetic acid in the presence of SiCl₄. Target compound 10h was obtained by the hydrolysis of compound 10g under basic condition in MeOH.

Scheme 1. Synthesis of target compounds 10a–i. Reagents and conditions: (i) 1a: 2-fluoro-4-nitrophenol, PhCl, reflux, 14 h; 1b: 2-fluoro-4-nitroaniline, i-PrOH, conc. HCl, reflux, 3 h; (ii) 33% HBr in HOAc, rt, 3 h; (iii) 1-Boc-4-methanesulfonyloxypiperidine, Cs₂CO₃, DMF, 110 °C, 6 h; (iv) Fe, 90% EtOH-H₂O, conc. HCl (cat.), reflux, 4–6 h; (v) phenyl chloroformate, pyridine, CH₂Cl₂, rt, 2 h; (vi) 50% hydrazine hydrate, xylene, 70 °C, 2 h; (vii) 2, 6-difluorobenzaldehyde, i-PrOH, HOAc (cat.), reflux, 2–3 h; (viii) CF₃COOH, CH₂Cl₂, rt, 2 h; (ix) RCOCl, Et₃N, CH₂Cl₂, rt, 4–5 h; R-Cl, Cs₂CO₃, DMF, 90 °C, 6–8 h; (x) mercaptoacetic acid, SiCl₄, CH₂Cl₂, reflux, 6–8 h; (xi) MeOH, NaOH, 50 °C, 1 h.

Scheme 2. Synthesis of target compound 19. Reagents and conditions: (i) 2-fluoro-4-nitrophenol, PhCl, reflux, 14 h; (ii) 33% HBr in HOAc, rt, 3 h; (iii) 1-Boc-4-methanesulfonyloxypiperidine, Cs₂CO₃, DMF, 110 °C, 6 h; (iv) Fe, 90% EtOH-H₂O, conc. HCl (cat.), reflux, 4 h; (v) (1) phenyl chloroformate, pyridine, CH₂Cl₂, rt, 2 h; (2) 50% hydrazine hydrate, xylene, 70 °C, 2 h; (vi) 2, 6-difluorobenzaldehyde, i-PrOH, HOAc (cat.), reflux, 3 h; (vii) CF₃COOH, CH₂Cl₂, rt, 2 h; (viii) 2-chloroacetamide, Cs₂CO₃, DMF, 90 °C, 8 h; (ix) mercaptoacetic acid, SiCl₄, CH₂Cl₂, reflux, 6 h.

Reaction of commercially available 7-(benzyloxy)-4-chloro-6-methoxyquinoline with 2-fluoro-4-nitroaniline in favour of catalytic concentrated HCl gave 7-(benzyloxy)-N-(2-fluoro-4-nitrophenyl)-6-methoxyquinolin-4-amine 1b²⁸. In the following procedures, target compound 10i was prepared by similar methods of the synthesis of compounds 10a–g.

Taking commercially available 7-(benzyloxy)-4-chloro-6-methoxyquinazoline and 2-fluoro-4-nitrophenol as starting materials, quinazoline derivate 11 was acquired by nucleophilic substitution in refluxing chlorobenzene. Subsequently, target compound 19 was synthesised by similar routes outlined in Scheme 2.

3.2. Structure-Activity relationship

Lead compound BC2021-104511-15i, Fruquintinib, Regorafenib, Cabozantinb and Foretinib were chosen as positive controls in the study of biological evaluation. In our previous research, it was revealed that the substituent on the piperidine ring could significantly influence the inhibitory activity against both kinases and cancer cells. Thus, eight novel compounds bearing diversified R groups (10a–h) were designed and synthesised. Biological activity study indicated that no obvious differences on kinase inhibitory activity could be found between the heterocyclic groups (10e–f) and catenoid groups (10a–d and 10g–h). As a general trend, the introduction of N-CH₂-CO fragment was beneficial for the HGFR and MST1R kinase inhibitory activity, such as compounds 10a, 10c and 10f. Docking study showed that additional H-bond formed by the carbonyl group and the residue His1094 might lead to the increased activity (Figure 3). Among the eight compounds, compound 10a was identified as the most potent HGFR and MST1R inhibitor, with IC₅₀ values of 0.11 μM and 0.045 μM, respectively (Table 1).

Figure 3. Mimetic binding mode of compound 10a (yellow sticks) within HGFR (PDB ID: 3LQ8). The H-bonds were represented by green dotted line and the H-π was represented by red dotted lines.

Table 1. The structures of target compounds 10a–i and 19 and their inhibitory activity against HGFR, MST1R, HT-29, HCT-116 and COLO 205 cells.

Compd.	R	X	Y	IC50 (μM)
				HGFR^a	MST1R^a	HT-29^b	HCT-116^b	COLO 205^b
10a		O	CH	0.11	0.045	1.3 ± 0.11	0.87 ± 0.11	0.11 ± 0.024
10b		O	CH	0.18	0.085	3.0 ± 0.24	3.7 ± 0.34	0.31 ± 0.021
10c		O	CH	0.14	0.072	4.1 ± 0.27	5.9 ± 0.53	0.44 ± 0.038
10d		O	CH	0.90	0.44	2.9 ± 0.19	2.4 ± 0.26	4.9 ± 0.38
10e		O	CH	0.61	0.15	7.7 ± 0.56	2.4 ± 0.26	0.48 ± 0.042
10f		O	CH	0.14	0.078	1.9 ± 0.11	2.6 ± 0.27	0.42 ± 0.034
10g		O	CH	0.18	0.11	6.9 ± 0.47	4.8 ± 0.44	0.59 ± 0.045
10h		O	CH	0.22	0.16	6.5 ± 0.43	–	0.85 ± 0.073
10i		NH	CH	–	–	>10.0	>10.0	–
19		O	N	–	–	8.6 ± 0.67	–	5.3 ± 0.47
BC2021-104511-15i		O	CH	0.27^c	0.11^c	0.79 ± 0.058^c	4.8 ± 0.44	0.72 ± 0.065
Fruquintinib	–	–	–	–	–	1.57	2.2 ± 0.17	>10.0
Regorafenib	–	–	–	–	–	8.49	8.2 ± 0.75	>10.0
Cabozantinib	–	–	–	0.0019		–	–	–
Foretinib	–	–	–		0.0024	–	–	–

^aThe IC₅₀ values are expressed as the mean of two independent experiments. ^bThe IC₅₀ values were an average of three separate determinations and standard deviations were shown. ^cReported IC₅₀ values were 0.20 μM (HGFR), 0.092 μM (MST1R) and 0.19 μM (HT-29 cells), respectively.

Compared with compound 10a, replacement of the oxygen atom linked the quinoline ring and 2-fluorophenyl ring by NH (10i) led to a significant decrease in anticancer activity (HT-29 IC₅₀>10.0 μM and HCT-116 IC₅₀>10.0 μM). Additionally, the quinazoline derivate 19 also showed weaker anticancer activity (HT-29 IC₅₀=8.6 μM and COLO 205 IC₅₀=5.3 μM, Table 1). The decrease of the biological activity might result from the conversion of electron density distribution in the quinoline ring which might weaken the H-bond between the nitrogen atom in quinoline and the residue Met1160.

3.3. The cytotoxicity against FHC cells

In order to investigate the cell selectivity index, the cytotoxicity of potent compounds against human normal colorectal mucosa epithelial cell FHC cells was determined. As could be seen in Table 2, all the anticancer agents 10a–b, 10d, 10f and 10h displayed no obvious cytotoxicity against FHC cells (IC₅₀>10.0 μM). Notably, the cell selectivity index of the most potent compound 10a was over 90 (FHC IC₅₀ value vs COLO 205 IC₅₀ value).

Table 2. The cytotoxicity of selected compounds against FHC cells.

Cells	IC50 (μM)^a
	10a	10b	10d	10f	10h
FHC	>10.0	>10.0	>10.0	>10.0	>10.0

^aThe IC₅₀ values were an average of three separate determinations.

3.4. Molecular docking study

Docking of the most potent compound 10a into HGFR was performed by Molecular Operating Environment (MOE). As shown in Figure 3, compound 10a adopted an extended conformation as type II kinase inhibitor exemplified by Cabozantinib and Foretinib. Totally three key hydrogen bonds were formed: nitrogen atom in quinoline with residue Met1160, oxygen atom in urea moiety with residue Lys1110, and oxygen atom in the terminal amido group with residue His1094. The hydrophobic pocket was occupied by the terminal 2,6-difluorophenyl ring, and weak H-π interaction was formed between 2,6-difluorophenyl fragment with residue Phe1134. Additionally, weak H-arene interaction was also formed between the quinoline ring and residue Val1092.

3.5. In vitro kinase profile

To further investigate the kinase selectivity of novel target compounds, the inhibitory activity of compounds 10a and 10b against another seven kinases was evaluated, including ABL, PDGFRβ, AXL, FLT3, RET, c-Src and VEGFR-2. As indicated in Table 3, compounds 10a and 10b showed much weaker inhibitory activity against the above kinases. The above results suggested that compounds 10a and 10b were potential HGFR and MST1R dual inhibitors. Certainly, only preliminary kinase profile was studied in this work, and further study will be conducted in the following structural modification.

Table 3. The kinase inhibitory activity of compounds 10a and 10b against ABL, PDGFRβ, AXL, FLT3, RET, c-Src and VEGFR-2.

Compd.	IC50 (μM)^a
	ABL	RET	AXL	FLT3	PDGFRβ	VEGFR-2	c-Src
10a	1.68 ± 0.13	1.27 ± 0.10	0.95 ± 0.086	6.93 ± 0.73	1.13 ± 0.12	1.16 ± 0.11	0.42 ± 0.035
10b	3.28 ± 0.26	1.52 ± 0.14	1.50 ± 0.15	>10.0	1.20 ± 0.095	2.36 ± 0.18	0.69 ± 0.057

^aValues are expressed as the mean of two independent experiments and standard deviations were shown.

3.6. Cell apoptosis assay by flow cytometry

Cell apoptosis assay was conducted to investigate whether the cytotoxic activity of the most potent compound 10a was caused by the activation of cellular apoptosis in COLO 205 cells. Quantitative analysis of early-apoptotic cells, advanced-apoptotic cells and necrotic cells was determined. COLO 205 cells were stimulated with different concentrations of compound 10a for 72 h. As depicted in Figure 4, compound 10a could effectively induce COLO 205 cells apoptosis in a dose-dependent manner. The total apoptosis including early apoptosis and advanced apoptosis accounted for 58.8%, 29.3% and 21.0% (the mean value of three independent determinations) when COLO 205 cells were treated with compound 10a at the concentration of 1.0 μM, 0.33 μM and 0.11 μM, respectively.

Figure 4. Cell apoptosis analyses of COLO 205 cells treated with compound 10a for 72 h.

3.7. Cell cycle arrest assay by flow cytometry

The antiproliferative activity of compound 10a was evaluated by flowcytometry analysis of COLO 205 cells. COLO 205 cells were treated with different concentrations (1.0 μM, 0.33 μM and 0.11 μM) of compound 10a for 24 h. As is shown in Figure 5, compound 10a could not arrest the cell-cycle progression at the concentration of 0.33 μM and 0.11 μM. At the concentration of 1.0 μM, cell cycle arrest could not be determined due to the massive dead cells (data not shown). The above results indicated that the anticancer mechanism of compound 10a might be cytotoxicity rather than antiproliferation.

Figure 5. Cell cycle arrest analyses of COLO 205 cells treated with compound 10a for 24 h.

4. Experimental

4.1. Chemistry

Unless otherwise noted, all chemicals were obtained from commercial vendors and used directly without further purification. Analytical reagent (AR) grade solvents were used for all reactions. Reaction progress was monitored by TLC on pre-coated silica plates (Huanghai HSGF254, 0.20 mm, pH 6.2–6.8) and spots were visualised by UV (254 nm). Flash column chromatography was done using silica gel (Qingdao Ocean Chemical Company, 200–300 mesh). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AVANCE neo 600. High resolution ESI-MS were recorded on Orbitrap Exploris 240 (Thermo Fisher Science, MA, USA).

4.1.1. 7-(Benzyloxy)-4–(2-fluoro-4-nitrophenoxy)-6-methoxyquinoline (1a)²⁶

The mixture of 7-(benzyloxy)-4-chloro-6-methoxyquinoline (21.0 g, 0.07 mol) and 2-fluoro-4-nitrophenol (14.2 g, 0.09 mol) in 120 ml chlorobenzene was refluxed for 14 h. The reaction mixture was cooled to room temperature, and the solvent was concentrated under reduced pressure. The brown residue was dissolved in 300 ml dichloromethane, washed with 10% NaOH aqueous solution (3 × 30 ml) and 50 ml water. The dichloromethane were dried over anhydrous MgSO₄ and concentrated under reduced pressure to give 21.4 g (yield 72.8%) of the title intermediate as light brown solid. HRMS (m/z), [M + H]⁺ calculated for C₂₃H₁₈FN₂O₅, 421.1200, found, 421.1189.

4.1.2. 7-(Benzyloxy)-N-(2-fluoro-4-nitrophenyl)-6-methoxyquinolin-4-amine (1b)

2-Fluoro-4-nitroaniline (5.7 g, 0.036 mol) and concentrated HCl (cat.) was added to a solution of 7-(benzyloxy)-4-chloro-6-methoxyquinoline (9.0 g, 0.03 mol) in isopropanol, and the reaction mixture was refluxed for 3 h. After completion of the reaction, the mixture was cooled to 0 °C, and the precipitate was filtered off, washed with cold isopropanol, and dried to yield the title intermediate (8.6 g, 68.3%) as a yellow solid. HRMS (m/z), [M + H]⁺ calculated for C₂₃H₁₉FN₃O₄, 420.1360; found, 420.1349.

4.1.3. General procedure for the synthesis of intermediates (2a–b)

Intermediates 1a–b was dissolved in 33% HBr in acetic acid and the mixtures were stirred for 3 h at room temperature. The precipitate was filtered off, and washed with isopropyl ether to afford target products.

4.1.3.1. 4–(2-Fluoro-4-nitrophenoxy)-6-methoxyquinolin-7-ol (2a)²⁶

Beige solid, yield: 61.3%. HRMS (m/z), [M + H]⁺ calculated for C₁₆H₁₂FN₂O₅, 331.0730, found, 331.0708.

4.1.3.2. 4-((2-Fluoro-4-nitrophenyl)amino)-6-methoxyquinolin-7-ol (2b)

Yellow solid, yield: 59.5%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.54 (s, 1H), 9.16 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.74 (m, 1H),7.62–7.65 (m, 1H), 7.48–7.51 (m, 1H), 7.45 (s, 1H), 7.10 (s, 1H), 6.42 (d, J = 5.4 Hz, 1H), 3.94 (s, 3H). HRMS (m/z), [M + H]⁺ calculated for C₁₆H₁₃FN₃O₄, 330.0890, found, 330.0861.

4.1.4. General procedure for the synthesis of intermediates (3a–b)

The suspension of 2a–b (0.05 mol) and Cs₂CO₃ (0.12 mol) in DMF (70 ml) was stirred at room temperature for 15 min, and 1-Boc-4-methanesulfonyloxypiperidine (0.075 mol) was added. After stirred at 110 °C for 6 h, the reaction mixture was cooled to room temperature and poured into cold water, filtered, and washed with cold water to give crude products, which were purified by flash chromatography (eluent with 10–20% MeOH in DCM) to afford the title intermediates.

4.1.4.1. Tert-butyl 4-((4–(2-fluoro-4-nitrophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (3a)

Yellow solid, yield: 57.1%. ¹H NMR (600 MHz, DMSO-d₆) δ 8.45 (d, J = 5.4 Hz, 1H), 7.74 (m, 1H),7.62–7.65 (m, 1H), 7.49 (m, 1H), 7.44 (s, 1H), 7.08 (s, 1H), 6.41 (d, J = 5.4 Hz, 1H), 3.93 (s, 3H), 3.82–3.87 (m, 4H), 3.67 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.07 (m, 2H), 1.44 (s, 9H). HRMS (m/z), [M + H]⁺ calculated for C₂₆H₂₉FN₃O₇, 514.1990, found, 514.1968.

4.1.4.2. Tert-butyl 4-((4-((2-fluoro-4-nitrophenyl)amino)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (3b)

Yellow solid, yield: 53.6%. HRMS (m/z), [M + H]⁺ calculated for C₂₆H₃₀FN₄O₆, 513.2149, found, 513.2129.

4.1.5. General procedure for the synthesis of intermediates (4a–b)

The suspension of intermediates 3a–b (0.02 mol), powered iron (0.06 mol) and concentrated HCl (2 drops) in 90% EtOH (100 ml) was refluxed for 4–6 h with vigorously stirred. After the reaction was completed, the hot mixture was filtered through celites, and the filtrate was evaporated under reduced pressure to afford the title intermediates.

4.1.5.1. Tert-butyl 4-((4–(4-amino-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (4a)

Yellow solid, yield: 74.6%. ¹H NMR (600 MHz, DMSO-d₆) δ 8.44 (d, J = 5.4 Hz, 1H), 7.44 (s, 1H), 7.08 (s, 1H), 6.86 (m, 1H), 6.74 (m, 1H), 6.65 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 5.74 (br, 2H), 3.93 (s, 3H), 3.83–3.87 (m, 4H), 3.67 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.08 (m, 2H), 1.44 (s, 9H). HRMS (m/z), [M + H]⁺ calculated for C₂₆H₃₁FN₃O₅, 484.2248, found, 484.2227.

4.1.5.2. Tert-butyl 4-((4-((4-amino-2-fluorophenyl)amino)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (4b)

Yellow solid, yield: 77.8%. HRMS (m/z), [M + H]⁺ calculated for C₂₆H₃₂FN₄O₄, 483.2408, found, 483.2385.

4.1.6. General procedure for the synthesis of intermediates (6a–b)

Phenyl chloroformate (20.0 mmol) was added to a solution of amines 4a–b (10.0 mmol) and dry pyridine (30.0 mmol) in dry CH₂Cl₂ (40 ml) at 0 °C. After the addition was completed, the mixture was allowed to warm to room temperature for another 2 h, and then saturated NaHCO₃ aqueous solution was added to the solution. The CH₂Cl₂ phase was separated, washed with water, dried over anhydrous MgSO₄, and concentrated under reduced pressure to afford intermediates 5a–b, which were immediately used in the following step without further purification.

To a mixture of esters 5a–b in 20 ml xylene was added hydrazine monohydrate (50%, 20 ml). The reaction mixture was stirred vigorously at 70 °C for 2 h. The solvent and excessive hydrazine monohydrate were evaporated under reduced pressure, and the residue was purified by flash chromatography (eluent with 1–10% MeOH in DCM, 1% Et₃N) to afford semicarbazides 6a–b.

4.1.6.1. Tert-butyl 4-((4–(2-fluoro-4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (6a)

Yellow solid, yield: 35.4%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.83 (s, 1H), 8.44 (d, J = 5.4 Hz, 1H), 7.64 (m, 1H), 7.44 (s, 1H), 7.15 (m, 1H), 7.08 (s, 1H), 6.86 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 5.74 (br, 2H), 4.62 (s, 2H), 3.95 (s, 3H), 3.83–3.87 (m, 4H), 3.65 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.08 (m, 2H), 1.42 (s, 9H). HRMS (m/z), [M + H]⁺ calculated for C₂₇H₃₃FN₅O₆, 542.2415, found, 542.2387.

4.1.6.2. Tert-butyl 4-((4-((2-fluoro-4-(hydrazinecarboxamido)phenyl)amino)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (6b)

Yellow solid, yield: 39.1%. HRMS (m/z), [M + H]⁺ calculated for C₂₇H₃₄FN₆O₅, 541.2575, found, 541.2554.

4.1.7. General procedure for the synthesis of intermediates (7a–b)

The mixture of 6a–h (10.0 mmol), 2,6-difluorobenzaldehyde (12.0 mmol) and acetic acid (cat.) in dry i-PrOH (50 ml) was refluxed for 2–3 h. The mixture was cooled to 0 °C, and the resultant precipitate was filtered, washed with cold i-PrOH and dried in vacuo to give semicarbazones 7a–b.

4.1.7.1. Tert-butyl 4-((4–(4-(2–(2,6-difluorobenzylidene)hydrazine-1-carboxamido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (7a)

White solid, yield: 80.1%. HRMS (m/z), [M + H]⁺ calculated for C₃₄H₃₅F₃N₅O₆, 666.2539, found, 666.2518.

4.1.7.2. Tert-butyl 4-((4-((4–(2–(2,6-difluorobenzylidene)hydrazine-1-carboxamido)-2-fluorophenyl)amino)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxylate (7b)

Light yellow solid, yield: 75.4%. HRMS (m/z), [M + H]⁺ calculated for C₃₄H₃₆F₃N₆O₅, 665.2699, found, 665.2676.

4.1.8. General procedure for the synthesis of intermediates (8a–b)

The solution of 7a–b (0.015 mol) and CF₃COOH (0.15 mol) in CH₂Cl₂ (50 ml) was stirred for 2 h at room temperature Evaporation of the solvent and excessive CF₃COOH provided yellow oil. The residue was diluted with 100 ml CH₂Cl₂, and 20% NaOH aqueous solution was added until the pH reached to 9. The organic phase was washed with water, dried over anhydrous MgSO₄, concentrated in vacuo, and the residue was used for the next step without further purification.

4.1.8.1. 2-(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-(piperidin-4-yloxy)quinolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (8a)

Light yellow solid, yield: 83.7%. ¹H NMR (600 MHz, DMSO-d₆) δ 8.92 (s, 1H), 8.83 (s, 1H), 8.44 (d, J = 5.4 Hz, 1H), 8.31 (s, 1H), 7.62 (m, 1H), 7.44 (s, 1H), 7.31 (m, 1H), 7.12–7.16 (m, 3H), 7.08 (s, 1H), 6.86 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 3.95 (s, 3H), 3.83–3.87 (m, 4H), 3.65 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.08 (m, 2H), 1.96 (br, 1H). HRMS (m/z), [M + H]⁺ calculated for C₂₉H₂₇F₃N₅O₄, 566.2015; found, 566.1987.

4.1.8.2. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-(piperidin-4-yloxy)quinolin-4-yl)amino)phenyl)hydrazine-1-carboxamide (8b)

Light yellow solid, yield: 81.4%. HRMS (m/z), [M + H]⁺ calculated for C₂₉H₂₈F₃N₆O₃, 565.2175, found, 565.2149.

4.1.9. General procedure for the synthesis of intermediates (9a–h)

Method A for the preparation of 9b, 9d, 9e and 9g. To a cold solution of 8a (0.01 mol) and Et₃N (0.015 mmol) in dry 30 ml CH₂Cl₂ was added acyl chloride (0.013 mol) dropwise. Then, the reaction mixture was stirred at room temperature for 4–5 h, saturated NaHCO₃ aqueous solution was added, and the CH₂Cl₂ phase was separated, dried over anhydrous MgSO₄, and concentrated in vacuum to afford the title intermediates.

Method B for the preparation of 9a, 9c, 9f and 9h. To a suspension of 8a–b (0.01 mol) and Cs₂CO₃ (0.015 mol) in 30 ml DMF was added 2-chloroamides (0.015 mol). The resulting mixture was stirred for 6–8 h at 90 °C, and then poured into cold water. The precipitate was filtered off, washed with water, and dried to afford the title intermediate.

4.1.9.1. N-(4-((7-((1–(2-amino-2-oxoethyl)piperidin-4-yl)oxy)-6-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)-2–(2,6-difluorobenzylidene)hydrazine-1-carboxamide (9a)

Yellow solid, yield: 68.8%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.14 (s, 1H), 8.91 (s, 1H), 8.43 (d, J = 5.4 Hz, 1H), 8.26 (s, 1H), 7.62–7.65 (m, 1H), 7.53 (s, 1H), 7.48–7.51 (m, 1H), 7.45 (s, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.21 (m, 1H), 7.16–7.19 (m, 1H), 7.10 (m, 1H), 6.45 (d, J = 5.4 Hz, 1H), 4.62–4.65 (m, 1H), 3.93 (s, 3H), 2.89 (s, 2H), 2.75–2.76 (m, 2H), 2.37–2.41 (m, 2H), 2.04–2.07 (m, 2H), 1.76–1.81 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₁H₃₀F₃N₆O₅, 623.2230, found, 623.2205.

4.1.9.2. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-((1–(2-methoxyacetyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (9b)

Yellow solid, yield: 79.7%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.22 (s, 1H), 8.87 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.25 (s, 1H), 7.62–7.65 (m, 1H), 7.55 (s, 1H), 7.52 (s, 1H), 7.48–7.53 (m, 1H), 7.34–7.37 (m, 1H), 7.27 (m, 1H), 7.16–7.19 (m, 1H), 6.44 (d, J = 5.4 Hz, 1H), 4.88–4.91 (m, 1H), 4.52 (br, 1H), 4.13 (s, 2H), 3.94 (s, 3H), 3.62–3.64 (m, 1H), 3.32 (s, 3H), 3.05–3.10 (m, 2H), 2.00–2.05 (m, 2H), 1.64–1.72 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₂H₃₁F₃N₅O₆, 638.2226, found, 638.2203.

4.1.9.3. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-((1–(2-(methylamino)-2-oxoethyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (9c)

Yellow solid, yield: 59.7%. HRMS (m/z), [M + H]⁺ calculated for C₃₂H₃₂F₃N₆O₅, 637.2386, found, 637.2357.

4.1.9.4. 2–(2,6-Difluorobenzylidene)-N-(4-((7-((1-(dimethylglycyl)piperidin-4-yl)oxy)-6-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)hydrazine-1-carboxamide (9d)

Yellow solid, yield: 69.4%. HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₄F₃N₆O₅, 651.2543, found, 651.2522.

4.1.9.5. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-((1-(morpholine-4-carbonyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (9e)

Yellow solid, yield: 66.3%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.10 (s, 1H), 8.91 (s, 1H), 8.44 (d, J = 5.4 Hz, 1H), 8.27 (s, 1H), 7.62–7.65 (m, 1H), 7.55 (s, 1H), 7.52 (s, 1H), 7.48–7.50 (m, 2H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.16–7.19 (m, 2H), 6.42 (d, J = 5.4 Hz, 1H), 4.81–4.85 (m, 1H), 3.95 (s, 3H), 3.57 (m, 4H), 3.49–3.52 (m, 2H), 3.13–3.16 (m, 4H), 3.09–3.12 (m, 2H), 2.01–2.05 (m, 2H), 1.65–1.70 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₄H₃₄F₃N₆O₆, 679.2492, found, 679.2471.

4.1.9.6. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-((1–(2-morpholino-2-oxoethyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (9f)

Yellow solid, yield: 55.9%. HRMS (m/z), [M + H]⁺ calculated for C₃₅H₃₆F₃N₆O₆, 693.2648, found, 693.2627.

4.1.9.7. 2–(4-((4–(4-(2–(2,6-Difluorobenzylidene)hydrazine-1-carboxamido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidin-1-yl)-2-oxoethyl acetate (9g)

Yellow solid, yield: 65.5%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.22 (s, 1H), 8.76 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 8.26 (s, 1H), 7.63–7.65 (m, 1H), 7.55 (s, 1H), 7.53 (s, 1H), 7.48–7.53 (m, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.16–7.19 (m, 2H), 6.45 (d, J = 5.4 Hz, 1H), 4.88–4.90 (m, 1H), 4.52 (s, 1H), 4.13 (s, 2H), 3.95 (m, 4H), 3.62 (m, 1H), 3.08 (m, 1H), 2.26 (s, 3H), 2.05 (m, 2H), 1.62–1.71 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₁F₃N₅O₇, 666.2176, found, 666.2148.

4.1.9.8. N-(4-((7-((1–(2-amino-2-oxoethyl)piperidin-4-yl)oxy)-6-methoxyquinolin-4-yl)amino)-3-fluorophenyl)-2–(2,6-difluorobenzylidene)hydrazine-1-carboxamide (9h)

Yellow solid, yield: 52.4%. HRMS (m/z), [M + H]⁺ calculated for C₃₁H₃₁F₃N₇O₄, 622.2390, found, 622.2369.

4.1.10. General procedure for the synthesis of target compounds 10a–g and 10i

To a suspension of intermediates 9a–h (0.3 mmol) in dry CH₂Cl₂ (5 ml), mercaptoacetic acid (0.3 ml) and SiCl₄ (15 drops) were added subsequently at 0 °C. The reaction mixture was allowed to cooled to room temperature, and refluxed for 6–8 h. The mixture was cooled to room temperature, and quenched by 2 ml cold water. After stirred for 5 min, 10% NaOH aqueous solution was added until pH reached to 10. The CH₂Cl₂ phase was separated and washed with water (2 × 5 ml), concentrated under reduced pressure to yield crude products which were purified by flash chromatography (eluent with 5–10% MeOH in DCM, 1% Et₃N) to give target compounds.

4.1.10.1. 2–(4-((4–(4-(3–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidin-1-yl)acetamide (10a)

White solid, yield: 32.7%. HPLC purity: 99.75%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.93 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.62–7.65 (m, 1H), 7.53 (s, 1H), 7.48–7.51 (m, 1H), 7.45 (s, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.21 (m, 1H), 7.16–7.19 (m, 1H), 7.10 (m, 1H), 6.42 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.62–4.65 (m, 1H), 3.94 (s, 3H), 3.81–3.87 (m, 2H), 2.89 (s, 2H), 2.75–2.77 (m, 2H), 2.37–2.41 (m, 2H), 2.05–2.07 (m, 2H), 1.76–1.82 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.8, 168.4, 161.6, 159.9, 159.3, 154.3, 153.6, 152.7, 150.1, 150.1, 148.7, 146.2, 138.2, 138.1, 134.7, 134.6, 131.5, 124.0, 115.1, 114.5, 112.3, 110.2, 101.8, 99.3, 73.0, 61.1, 55.7, 51.8, 50.8 (2 C), 30.2 (2 C), 29.2. HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₂F₃N₆O₆S, 697.2056, found, 697.2023.

4.1.10.2. 1–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)-3–(3-fluoro-4-((6-methoxy-7-((1–(2-methoxyacetyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)urea (10b)

White solid, yield: 29.4%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.29 (s, 1H), 8.93 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 7.62–7.65 (m, 1H), 7.55 (s, 1H), 7.54 (s, 1H), 7.48–7.53 (m, 1H), 7.34–7.37 (m, 1H), 7.27 (m, 1H), 7.16–7.19 (m, 1H), 6.44 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.88–4.91 (m, 1H), 4.52 (br, 1H), 4.13 (s, 2H), 3.95 (s, 3H), 3.81–3.87 (m, 2H), 3.62–3.64 (m, 1H), 3.33 (s, 3H), 3.05–3.10 (m, 2H), 2.00–2.05 (m, 2H), 1.62–1.72 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.3, 167.9, 161.0, 159.4, 158.9, 153.8, 153.2, 152.1, 149.6, 149.4, 148.3, 145.5, 137.7, 137.7, 134.1, 134.0, 131.0, 123.5, 114.5, 114.1, 111.8, 109.9, 101.4, 98.9, 72.2, 59.4, 55.3, 51.2, 44.9 (2 C), 40.2, 30.0, 29.4, 28.7. HRMS (m/z), [M + H]⁺ calculated for C₃₄H₃₃F₃N₅O₇S, 712.2053; found, 712.2039.

4.1.10.3. 2–(4-((4–(4-(3–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidin-1-yl)-N-methylacetamide (10c)

White solid, yield: 30.8%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.15–8.17 (m, 1H), 7.85–7.83 (m, 1H), 7.72 (m, 1H), 7.55 (s, 1H), 7.50–7.53 (m, 1H), 7.46 (s, 1H), 7.42–7.45 (m, 2H), 7.22–7.25 (m, 2H), 6.45 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.63–4.65 (m, 1H), 3.95 (s, 3H), 3.81–3.84 (m, 2H), 2.93 (s, 2H), 2.75–2.77 (m, 2H), 2.64 (s, 3H), 2.37–2.41 (m, 2H), 2.05–2.07 (m, 2H), 1.79–1.84 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₄H₃₄F₃N₆O₆S, 711.2213; found, 711.2184.

4.1.10.4. 1–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)-3–(4-((7-((1-(dimethylglycyl)piperidin-4-yl)oxy)-6-methoxyquinolin-4-yl)oxy)-3-fluorophenyl)urea (10d)

White solid, yield: 31.4%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.17 (s, 1H), 8.91 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.61–7.65 (m, 1H), 7.54 (s, 1H), 7.53 (s, 1H), 7.48–7.52 (m, 1H), 7.34–7.37 (m, 1H), 7.26–7.28 (m, 1H), 7.16–7.19 (m, 1H), 6.42 (d, J = 5.4 Hz, 1H), 6.16 (1H), 4.87–4.90 (m, 1H), 3.94 (s, 3H), 3.81–3.89 (m, 3H), 3.41–3.44 (m, 1H), 3.24–3.26 (m, 2H), 3.08–3.16 (m, 2H), 2.21 (s, 6H), 1.98–2.08 (m, 2H), 1.70 (m, 1H), 1.58(m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.5, 167.6, 161.6, 160.0, 159.4, 153.7, 152.7, 152.2, 150.1, 148.8, 146.2, 138.2, 138.2, 134.7, 134.7, 131.5, 124.0, 115.1, 114.8, 114.7, 112.4, 110.5, 101.9, 99.5, 72.9, 61.9, 55.8, 51.7, 45.1 (2 C), 42.3, 38.5, 31.0, 30.2, 29.2. HRMS (m/z), [M + H]⁺ calculated for C₃₅H₃₆F₃N₆O₆S, 725.2369; found, 725.2345.

4.1.10.5. 1–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)-3–(3-fluoro-4-((6-methoxy-7-((1-(piperidine-1-carbonyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)urea (10e)

White solid, yield: 33.6%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.16 (s, 1H), 8.90 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.62–7.65 (m, 1H), 7.54 (s, 1H), 7.52 (s, 1H), 7.48–7.50 (m, 2H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.16–7.19 (m, 2H), 6.42 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.81–4.85 (m, 1H), 3.94 (s, 3H), 3.81–3.87 (m, 2H), 3.57 (m, 4H), 3.49–3.52 (m, 2H), 3.13–3.16 (m, 4H), 3.10–3.12 (m, 2H), 2.01–2.05 (m, 2H), 1.65–1.71 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.9, 162.5, 161.0, 159.4, 158.8, 153.8, 153.1, 152.1, 149.6, 148.3, 145.6, 137.7, 137.6, 134.2, 134.1, 131.0, 123.5, 114.6, 114.2, 114.1, 111.7, 110.0, 101.4, 98.9, 72.6, 65.3, 55.3 (2 C), 51.1, 46.5 (2 C), 43.0 (2 C), 29.6 (2 C), 28.7. HRMS (m/z), [M + H]⁺ calculated for C₃₇H₃₈F₃N₆O₆S, 753.2318; found, 753.2291.

4.1.10.6. 1–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)-3–(3-fluoro-4-((6-methoxy-7-((1–(2-morpholino-2-oxoethyl)piperidin-4-yl)oxy)quinolin-4-yl)oxy)phenyl)urea (10f)

White solid, yield: 30.8%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.21(s, 1H), 8.93 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.62–7.65 (m, 1H), 7.53 (s, 1H), 7.48–7.51 (m, 1H), 7.45 (s, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.16–7.19 (m, 2H), 6.41 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.60–4.63 (m, 1H), 3.94 (s, 3H), 3.81–3.87 (m, 2H), 3.59 (m, 4H), 3.54 (m, 2H), 3.44 (m, 2H), 3.19 (s, 2H), 2.75 (m, 2H), 2.35–2.38 (m, 2H), 2.04–2.06 (m, 2H), 1.69–1.74 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.9, 167.2, 161.0, 159.4, 158.8, 153.8, 153.1, 152.1, 149.6, 148.2, 145.7, 137.7, 137.6, 134.2, 134.1, 131.0, 123.5, 114.5, 114.2, 114.0, 111.8, 109.7, 101.3, 98.8, 72.5, 65.9, 65.7, 59.8, 55.2 (2 C), 51.2, 49.6 (2 C), 45.3 (2 C), 41.1, 29.9 (2 C), 28.7. HRMS (m/z), [M + H]⁺ calculated for C₃₇H₃₈F₃N₆O₇S, 767.2475; found, 767.2445.

4.1.10.7. 2–(4-((4–(4-(3–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)piperidin-1-yl)-2-oxoethyl acetate (10g)

White solid, yield: 31.6%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.28 (s, 1H), 8.92 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 7.63–7.65 (m, 1H), 7.55 (s, 1H), 7.54 (s, 1H), 7.48–7.53 (m, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.16–7.19 (m, 2H), 6.43 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.88–4.90 (m, 1H), 4.52 (s, 1H), 4.13 (s, 2H), 3.95 (m, 4H), 3.81–3.87 (m, 2H), 3.62 (m, 1H), 3.08 (m, 1H), 2.27 (s, 3H), 2.05 (m, 2H), 1.62–1.72 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.4, 169.0, 162.1, 160.5, 159.9, 154.8, 154.2, 153.2, 150.7, 150.5, 149.3, 146.6, 138.8, 138.7, 135.2, 135.1, 132.0, 124.6, 115.6, 115.2, 112.9, 111.0, 102.5, 100.0, 72.3, 60.5, 56.3, 52.2, 46.0 (2 C), 40.4, 31.0, 30.5, 29.7. HRMS (m/z), [M + H]⁺ calculated for C₃₅H₃₃F₃N₅O₈S, 740.2002; found, 740.1979.

4.1.10.8. 2–(4-((4-((4–(3-(2–(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenyl)amino)-6-methoxyquinolin-7-yl)oxy)piperidin-1-yl)acetamide (10i)

White solid, yield: 31.9%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.21 (s, 1H), 9.12 (s, 1H), 8.93 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H), 7.62–7.65 (m, 1H), 7.54 (s, 1H), 7.48–7.51 (m, 1H), 7.46 (s, 1H), 7.34–7.37 (m, 1H), 7.26 (m, 1H), 7.21 (m, 1H), 7.16–7.19 (m, 1H), 7.10 (m, 1H), 6.43 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.62–4.65 (m, 1H), 3.93 (s, 3H), 3.81–3.87 (m, 2H), 2.88 (s, 2H), 2.75–2.77 (m, 2H), 2.37–2.40 (m, 2H), 2.04–2.07 (m, 2H), 1.76–1.82 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₃F₃N₇O₅S, 696.2216; found, 696.2187.

4.1.11. 1–(2-(2,6-Difluorophenyl)-4-oxothiazolidin-3-yl)-3–(3-fluoro-4-((7-((1–(2-hydroxyacetyl)piperidin-4-yl)oxy)-6-methoxyquinolin-4-yl)oxy)phenyl)urea (10h)

To a suspension of compound 10g (30 mg, 0.04 mmol) in 1 ml MeOH, NaOH solution (4.8 mg in 0.5 ml H₂O) was added. After stirred for 1 h at 50 °C, the solution was concentrated under reduced pressure, and then 1 ml water was added to the residue. Concentrated HCl was added cautiously until pH reached to 4–5, and the resultant precipitate was filtered, washed with water and dried in vacuo to give the title compound as a white solid, yield: 75.9%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.24 (s, 1H), 8.93 (s, 1H), 8.45 (d, J = 5.4 Hz, 1H), 7.63–7.65 (m, 1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.48–7.53 (m, 1H), 7.34–7.37 (m, 1H), 7.25 (m, 1H), 7.16–7.19 (m, 2H), 6.43 (d, J = 5.4 Hz, 1H), 6.16 (s, 1H), 4.88–4.90 (m, 1H), 4.75 (br, 1H), 4.52 (s, 1H), 4.12 (s, 2H), 3.95 (m, 4H), 3.81–3.87 (m, 2H), 3.62 (m, 1H), 3.07 (m, 1H), 2.05 (m, 2H), 1.62–1.72 (m, 2H). HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₁F₃N₅O₇S, 698.1896, found, 698.1871.

4.1.12. 7-(Benzyloxy)-4–(2-fluoro-4-nitrophenoxy)-6-methoxyquinazoline (11)

The mixture of 7-(benzyloxy)-4-chloro-6-methoxyquinazoline (9.0 g, 0.03 mol) and 2-fluoro-4-nitrophenol (6.3 g, 0.04 mol) in 60 ml chlorobenzene was refluxed for 13 h. The reaction mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was dissolved in 300 ml CH₂Cl₂, washed with 10% NaOH aqueous solution (3 × 30 ml) and 50 ml water. The CH₂Cl₂ phase was dried over anhydrous MgSO₄ and concentrated under reduced pressure to give 8.2 g (yield 64.9%) of the title intermediate as dark yellow solid. HRMS (m/z), [M + H]⁺ calculated for C₂₂H₁₇FN₃O₅, 422.1152; found, 422.1128.

4.1.13. 4–(2-Fluoro-4-nitrophenoxy)-6-methoxyquinazolin-7-ol (12)

Intermediate 11 (8.2 g, 0.019 mol) was dissolved in 33% HBr in acetic acid (45 ml) and the mixtures were stirred for 3 h at room temperature. The precipitate was filtered off, and washed with isopropyl ether to afford target products as beige solid (4.1 g, 64.1%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.59 (s 1H), 7.74 (m, 1H),7.62–7.65 (m, 1H), 7.45 (s, 1H), 7.32 (s, 1H), 7.10 (m, 1H), 3.92 (s, 3H). HRMS (m/z), [M + H]⁺ calculated for C₁₅H₁₁FN₃O₅, 332.0683; found, 332.0662.

4.1.14. Tert-butyl 4-((4–(2-fluoro-4-nitrophenoxy)-6-methoxyquinazolin-7-yl)oxy)piperidine-1-carboxylate (13)

The suspension of 12 (4.1 g, 0.012 mol) and Cs₂CO₃ (8.2 g, 0.025 mol) in DMF (25 ml) was stirred at room temperature for 15 min, and 1-Boc-4-methanesulfonyloxypiperidine (5.0 g, 0.018 mol) was added. After stirred at 110 °C for 6 h, the reaction mixture was cooled to room temperature and poured into ice water, filtered, and washed with cold water to give crude products, which were purified by flash chromatography (eluent with 10–20% MeOH in DCM) to afford the title intermediate 13 as a yellow solid (3.0 g, 49.6%). HRMS (m/z), [M + H]⁺ calculated for C₂₅H₂₈FN₄O₇, 515.1942; found, 515.1921.

4.1.15. Tert-butyl 4-((4–(4-amino-2-fluorophenoxy)-6-methoxyquinazolin-7-yl)oxy)piperidine-1-carboxylate (14)

The suspension of intermediate 13 (0.02 mol), powered iron (0.06 mol) and concentrated HCl (2 drops) in 90% EtOH (100 ml) was refluxed for 4–6 h with vigorously stirred. After the reaction was completed, the hot mixture was filtered through celites, and the filtrate was evaporated under reduced pressure to afford the title intermediate as dark yellow solid (6.9 g, 71.2%). ¹H NMR (600 MHz, DMSO-d₆) δ 8.57 (s, 1H), 7.44 (s, 1H), 7.08 (s, 1H), 6.86 (m, 1H), 6.74 (m, 1H), 6.65 (m, 1H), 5.64 (s, 2H), 3.95 (s, 3H), 3.83–3.87 (m, 4H), 3.65 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.08 (m, 2H), 1.43 (s, 9H). HRMS (m/z), [M + H]⁺ calculated for C₂₀H₂₀FN₄O₅, 485.2200; found, 485.2178.

4.1.16. Tert-butyl 4-((4–(2-fluoro-4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinazolin-7-yl)oxy)piperidine-1-carboxylate (15)

Phenyl chloroformate (28.2 mmol) was added to a solution of amine 14 (6.9 g, 14.2 mmol) and dry pyridine (42.6 mmol) in dry CH₂Cl₂ (350 ml) at 0 °C. After the addition was completed, the mixture was allowed to warm to room temperature for another 2 h, and then saturated NaHCO₃ aqueous solution was added to the solution. The CH₂Cl₂ phase was separated, washed with water, dried over anhydrous MgSO₄, and concentrated under reduced pressure to afford brown oil, which were immediately used in the following step without further purification.

The above oil was dissolved in 20 ml xylene was added hydrazine monohydrate (50%, 20 ml). The reaction mixture was stirred vigorously at 70 °C for 2 h. The solvent and excessive hydrazine monohydrate were evaporated under reduced pressure, and the residue was purified by flash chromatography (eluent with 1–10% MeOH in DCM, 1% Et₃N) to afford semicarbazide 15 as a light yellow solid (2.7 g, 35.5%). HRMS (m/z), [M + H]⁺ calculated for C₂₆H₃₂FN₆O₆, 543.2367; found, 543.2346.

4.1.17. Tert-butyl 4-((4–(4-(2–(2,6-difluorobenzylidene)hydrazine-1-carboxamido)-2-fluorophenoxy)-6-methoxyquinazolin-7-yl)oxy)piperidine-1-carboxylate (16)

The mixture of 15 (2.0 g, 3.7 mmol), 2,6-difluorobenzaldehyde (4.4 mmol) and acetic acid (cat.) in dry i-PrOH (12 ml) was refluxed for 3 h. The mixture was cooled to 0 °C, and the resultant precipitate was filtered, washed with cold i-PrOH and dried in vacuum to give semicarbazone 16 as a beige solid (1.8 g, 75.6%). HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₄F₃N₆O₆, 667.2492; found, 667.2471.

4.1.18. 2–(2,6-Difluorobenzylidene)-N-(3-fluoro-4-((6-methoxy-7-(piperidin-4-yloxy)quinazolin-4-yl)oxy)phenyl)hydrazine-1-carboxamide (17)

The solution of 16 (1.5 g, 2.2 mmol) and CF₃COOH (22.0 mmol) in CH₂Cl₂ (10 ml) was stirred for 2 h at room temperature Evaporation of the solvent and excessive CF₃COOH provided yellow oil. The residue was diluted with 20 ml CH₂Cl₂, and 20% NaOH aqueous solution was added until the pH reached to 9. The organic phase was washed with water, dried over anhydrous MgSO₄, and concentrated under reduced pressure to yield yellow residue (1.0 g, 83.1%). ¹H NMR (600 MHz, DMSO-d₆) δ 8.92 (s, 1H), 8.83 (s, 1H), 8.58 (s, 1H), 8.32 (s, 1H), 7.62 (m, 1H), 7.44 (s, 1H), 7.31 (m, 1H), 7.14 (m, 3H), 7.08 (s, 1H), 6.85 (m, 1H), 3.93 (s, 3H), 3.83–3.87 (m, 4H), 3.65 (m, 1H), 2.37–2.41 (m, 2H), 2.05–2.08 (m, 2H), 1.92 (br, 1H). HRMS (m/z), [M + H]⁺ calculated for C₂₈H₂₆F₃N₆O₄, 567.1968; found, 567.1948.

4.1.19. N-(4-((7-((1–(2-amino-2-oxoethyl)piperidin-4-yl)oxy)-6-methoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-2–(2,6-difluorobenzylidene)hydrazine-1-carboxamide (18)

To a suspension of 17 (1.0 g, 1.8 mmol) and Cs₂CO₃ (1.8 g, 5.4 mmol) in 10 ml DMF was added 2-chloroacetamide (0.33 g, 3.6 mmol). The resulting mixture was stirred for 8 h at 90 °C, and then poured into cold water. The precipitate was filtered off, washed with water, and dried to afford the title intermediate 18 as a dark yellow solid (0.68 g, 61.9%). HRMS (m/z), [M + H]⁺ calculated for C₃₀H₂₉F₃N₇O₅, 624.2182; found, 624.2160.

4.1.20. 2–(4-((4–(4-(3–(2-(2,6-Difluorophenyl)-4-oxotetrahydrothiophen-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinazolin-7-yl)oxy)piperidin-1-yl)acetamide (19)

To a suspension of 18 (0.31 g, 0.5 mmol) in dry CH₂Cl₂ (5 ml), mercaptoacetic acid (0.3 ml) and SiCl₄ (15 drops) were added subsequently at 0 °C. The reaction mixture was allowed to cooled to room temperature, and refluxed for 6 h. The mixture was cooled to room temperature, and quenched by 2 ml cold water. After stirred for 5 min, 10% NaOH aqueous solution was added until pH reached to 10. The CH₂Cl₂ phase was separated and washed with water (2 × 5 ml), concentrated under reduced pressure to yield crude products which were purified by flash chromatography (eluent with 5–10% MeOH in DCM, 1% Et₃N) to give target compound as a white solid (96.0 mg, 27.6%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.90 (s, 1H), 8.53 (s, 1H), 7.57 (s, 1H), 7.55 (m, 1H), 7.48–7.53 (m, 2H), 7.35 (m, 1H), 7.21 (m, 1H), 7.17 (m, 2H), 6.16 (s, 1H), 4.77 (m, 1H), 3.98 (s, 3H), 3.81–3.85 (m, 2H), 3.07 (s, 2H), 2.73–2.94 (m, 4H), 2.10 (m, 2H), 1.84 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.9, 163.5, 161.0, 159.3, 153.8, 153.2, 153.0, 150.3, 148.2, 137.6, 137.5, 133.2, 133.1, 131.0, 123.7, 114.1, 114.0, 111.8, 108.5, 108.0, 100.4, 73.5, 61.6, 56.2, 52.1, 51.1 (2 C), 30.7 (2 C), 29.7. HRMS (m/z), [M + H]⁺ calculated for C₃₃H₃₂F₃N₆O₆S, 698.2008; found, 698.1991.

4.2. MTT assay

Taking lead compound BC2021-104511-15i, Fruquintinib and Regorafenib as positive controls, the cytotoxic activity against HT-29, HCT-116, COLO-205 and FHC cell lines by MTT assay. Detailed operation could be found in our previous study²⁹.

4.3. Mobility shift assay

Kinase inhibitory activity against HGFR, MST1R, ABL, PDGFRβ, AXL, FLT3, RET, c-Src, and VEGFR-2 was evaluated by the mobility shift assay. Detailed operation could be found in our previous research²⁹.

4.4. Molecular docking study

Docking study were conducted by Molecular Operating Environment 2018.01 (MOE, Chemical Computing Group ULC, Montreal, QC, Canada) using default settings. The structure of HGFR kinase was prepared (protonation, modelling of missing elements) from the original PDB files using Quickprepare. The binding site was defined within 5.0 Å of the cocrystallized ligands coordinates. The docking forcefield was Amber10: EHT. Ligand conformations were placed in the site with the Triangle Matcher method and ranked using the London dG scoring function.

4.5. Cell-Cycle analysis

COLO 205 cells (2.5 × 10⁵) were seeded in two 12-well plates and treated with DMSO and compound 10a (0.11 μM, 0.33 μM and 1.0 μM) for 24 h (37 °C, 5% CO₂). Cells were collected, centrifuged at 1000 rpm for 5 min, washed with cold PBS for twice, and then fixed with 500 μL 75% cold ethanol at 4 °C. The cells were washed with cold PBS and stained with propidium iodide for 30 min in the dark. Cell-cycle analyses were conducted with Cytoflex S (Beckman Coulter).

4.6. Annexin V-FITC/PI apoptosis assay

COLO 205 cells (2.5 × 10⁵) were seeded in two 12-well plates and treated with DMSO and compound 10a (0.11 μM, 0.33 μM and 1.0 μM) for 72 h (37 °C, 5% CO₂). The cells were collected, centrifuged at 1000 rpm for 5 min, and then washed with cold PBS for twice. Apoptosis assays were conducted with CytoFLEX S (Beckman Coulter).

5. Conclusions

Starting from the obtained HGFR and MST1R dual inhibitor BC2021-104511-15i, ten novel quinoline derivates were designed, synthesised and evaluated for their biological activity. More detailed SARs were summarised based on the kinase inhibitory activity and in vitro anticancer activity. Among these compounds, 10a was identified as the most potent HGFR/MST1R dual inhibitor (HGFR IC₅₀=0.11 μM and MST1R IC₅₀=0.045 μM) with excellent anti-colorectal cancer activity (COLO 205 IC₅₀=0.11 μM). Furthermore, it exhibited over 90-fold selectivity towards COLO 205 cells relative to human normal colorectal mucosa epithelial cell FHC cells. Docking study indicated that compound 10a adopted an extended conformation as type II kinase inhibitor. H-bond, hydrophobic interaction and H-π interaction were the key contributors led to the strong binding affinity to kinase. Flow cytometry study demonstrated that compound 10a could induce apoptosis in COLO 205 cells; however, it could not induce cell cycle arrest in COLO 205 cells. The results indicated that the anti-colorectal cancer activity against COLO 205 cells mainly depended on its cytotoxicity rather than antiproliferation. Preliminary kinase profile study showed that compound 10a was a potential HGFR and MST1R dual inhibitor, its inhibitory activity against HGFR and MST1R was more potent than that of ABL, PDGFRβ, AXL, FLT3, RET, c-Src, and VEGFR-2 kinases.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Science and Technology Projects of Guizhou Province (Qian Ke He Foundation-ZK[2021]key project 015).