Design, synthesis and biological evaluation of novel N-[4-(2-fluorophenoxy)pyridin-2-yl]cyclopropanecarboxamide derivatives as potential c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112244

Three series of novel 4-phenoxypyridine derivatives containing 4-methyl-6-oxo-1,6-dihydropyridazine- 3-carboxamide, 5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide and 4-methyl-3,5-dioxo-2,3,4,5- tetrahydro-1,2,4-triazine-6-carboxamide moieties were synthesized and evaluated for their in vitro inhibitory activitives against c-Met kinase and cytotoxic activitives against A549, H460, HT-29 cancer cell lines. The results indicated that most of the compounds showed moderate to good antitumor activitives. The most promising compound 26a (with c-Met IC₅₀ value of 0.016 μM) showed remarkable cytotoxicity against A549, H460, and HT-29 cell lines with IC₅₀ values of 1.59 μM, 0.72 μM and 0.56 μM, respectively. Their preliminary structure-activity relationships (SARs) studies indicate that 4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide was more preferred as linker part, and electron-withdrawing groups on the terminal phenyl rings are beneficial for improving the antitumor activitives. Furthermore, the colony formation, acridine orange/ethidium bromide (AO/EB) staining, apoptosis, and wound-healing assay of 26a were performed on HT-29 and/or A549 cell lines.

Full text of DOI:10.1016/j.ejmech.2020.112244

Journal Pre-proof
Design, synthesis and biological evaluation of novel N-[4-(2-fluorophenoxy)pyridin-2-
yl]cyclopropanecarboxamide derivatives as potential c-Met kinase inhibitors
Ju Liu, Yilin Gong, Jiantao Shi, Xuechen Hao, Yang Wang, Yunpeng Zhou, Yunlei
Hou, Yajing Liu, Shi Ding, Ye Chen
PII:
S0223-5234(20)30211-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112244
EJMECH 112244
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2019
Revised Date: 8 March 2020
Accepted Date: 16 March 2020
Please cite this article as: J. Liu, Y. Gong, J. Shi, X. Hao, Y. Wang, Y. Zhou, Y. Hou, Y. Liu, S. Ding,
Y. Chen, Design, synthesis and biological evaluation of novel N-[4-(2-fluorophenoxy)pyridin-2-
yl]cyclopropanecarboxamide derivatives as potential c-Met kinase inhibitors, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112244.
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Graphical abstract
Three series of novel 4-phenoxypyridine derivatives were synthesized and evaluated for their c-Met kinase
activities and cytotoxic activities against A549, H460, HT-29 cancer cell lines. Furthermore, colony formation,
acridine orange/ethidium bromide staining, apoptosis, and wound-healing assay of 26a were performed.

Design, synthesis and biological evaluation of novel
N-[4-(2-fluorophenoxy)pyridin-2-yl]cyclopropanecarboxamide derivatives as potential c-Met
kinase inhibitors
Ju Liua, Yilin Gonga, Jiantao Shia, Xuechen Haoa, Yang Wanga, Yunpeng Zhoua,
Yunlei Houb, Yajing Liub, Shi Dinga,*, Ye Chena,**
a College of Pharmacy of Liaoning University, API Engineering Technology Research Center of Liaoning Province, 66 Chongshan Road,
Huanggu District, Shenyang 10036, P. R. China.
b Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103
Wenhua Road, Shenhe District, Shenyang 110016, PR China
∗Corresponding author: Shi Ding. Tel./Fax: 86-24-6220 2469. E-mail: dingshi_destiny@163.com.
*Corresponding author: Ye Chen. Tel./Fax: 86-24-6220 2469. E-mail: sy-chenye@163.com.
∗
Abstract
Three series of novel 4-phenoxypyridine derivatives containing 4-methyl-6-oxo-1,6-dihydropyridazine-
3-carboxamide,
5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide
and
4-methyl-3,5-dioxo-2,3,4,5-
tetrahydro-1,2,4-triazine-6-carboxamide moieties were synthesized and evaluated for their in vitro inhibitory
activitives against c-Met kinase and cytotoxic activitives against A549, H460, HT-29 cancer cell lines. The results
indicated that most of the compounds showed moderate to good antitumor activitives. The most promising
compound 26a (with c-Met IC50 value of 0.016 μM) showed remarkable cytotoxicity against A549, H460, and HT-29
cell lines with IC50 values of 1.59 μM, 0.72 μM and 0.56 μM, respectively. Their preliminary structure-activity
relationships (SARs) studies indicate that 4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide was
more preferred as linker part, and electron-withdrawing groups on the terminal phenyl rings are beneficial for
improving the antitumor activitives. Furthermore, the colony formation, acridine orange/ethidium bromide (AO/EB)
staining, apoptosis, and wound-healing assay of 26a were performed on HT-29 and/or A549 cell lines.
Keywords: Synthesis, 4-Phenoxypyridine derivatives, c-Met inhibitors, Antitumor activity, Docking study.
1. Introduction
c-Mesenchymal epithelial transition factor (c-Met) is a receptor tyrosine kinase that is normally activated by its
natural ligand hepatocyte growth factor/scatter factor (HGF/SF) [1]. The HGF/c-Met axis plays an important role in
normal embryonic development and organ regeneration. However, aberrant activation of c-Met can increase the
tumorigenicity and metastatic potential of tumor cells [2]. Like other oncogenic pathway, overexpression or mutation
of protein members of this pathway is a driving factor for numerous cancers [3]. Consequently, the HGF/c-Met
signaling pathway has been emerging as an attractive target for cancer therapies [4].
To date, numerous examples of successful therapeutic interventions with small molecule c-Met kinase
inhibitors have been reported [5]. These small molecule c-Met kinase inhibitors were mainly divided into Type I and
Type II, according to the structural features and binding mode to the c-Met kinase. As disclosed recently, certain
mutations near the active site of c-Met may cause resistance of type I inhibitors. In contrast, type II inhibitors are
postulated to be more effective against these mutations because their binding interactions extend beyond the entrance
to c-Met’s active site [6-8]. During the past ten years, numerous type II c-Met inhibitors have been discovered and
some of them are launched or in clinical trials, such as Cabozantinib [9], BMS-777607 [10], BMS-794833 [11],
Foretinib [12] and NPS-1034 [13] (Fig. 1). Among them, 4-phenoxypyridines play a key role and have been
extensively studied [14-18]. A good example of 4-phenoxypyridine-based type II c-Met inhibitors is BMS-777607,

which inhibits the kinase activity of c-Met (IC50 = 3.9 nM), as well as that of Axl (IC50 =1.1 nM), Ron (IC50 = 1.8
nM), and Tyro3 (IC50 = 4.3 nM), is currently undergoing phase II clinical trials for various cancers [10].
As described in Fig. 1, structurally, most of type II c-Met inhibitors may be disconnected into three moieties: a
4-phenoxypyridine core (moiety A, usually substituted 4-phenoxypyridine, 4-phenoxyquinoline,
4-phenoxypyrrolopyridine, and 4-phenoxythienopyridine), a phenyl or substituted phenyl group (moiety B) and a
linker bridge (moiety C). Importantly, there should be two structural characteristics on the linker bridge (moiety C)
between moiety A and B. One was ‘5-atom regulation’, which means six chemical bonds distance existing between
moiety A and B; the other was that the linker should contain both hydrogen-bond donor or acceptor and at least one
amide group. Various linear chains and heterocyclic rings can be introduced to the main chain of 5-atom linker
[19-21].
Fig. 1. The representative c-Met kinase inhibitors and the corresponding general structure.
With
the
goal
of finding
more
4-phenoxypyridine-based
was used
type
as
II c-Met
the
inhibitors,
A.
N-[4-(2-fluorophenoxy)pyridin-2-yl]cyclopropanecarboxamide
moiety
4-Methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide, 5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide and
4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide fragments were introduced into the moiety C
via cyclization strategy based on the ‘5 atoms regulation’. Furthermore, various substituents were introduced at the
phenyl ring (moiety B) to investigate their effects on activitives. Accordingly, three series of 4-phenoxypyridine
derivatives were designed and synthesized to study the structure-activity relationships (SARs) and find promising
antitumor agents (Fig. 2).
In the current study, all target compounds were synthesized and evaluated for their in vitro inhibitory activitives
against c-Met kinase and cytotoxic activitives against human lung adenocarcinoma cell line (A549), human lung
cancer cell lines (H460) and human colon cancer cell line (HT-29) cancer cell lines. Their structure-activity
relationships (SARs) were further explored. Furthermore, the colony formation, acridine orange/ethidium bromide
(AO/EB) staining, apoptosis, and wound-healing assays of 26a were performed on HT-29 and/or A549 cell lines.
Additionally, a docking analysis was also performed to elucidate the binding mode of the target compound 26a with
c-Met kinase.

Fig. 2. Design strategy and structures of the target compounds.
2. Results and discussion
2.1 Chemistry
The key intermediate 10 was synthesized according to the route outlined in Scheme 1. Commercially available
4-chloropyridin-2-amine was condensed with cyclopropanecarbonyl chloride in the presence of Et3N to provide 8,
which underwent a nucleophilic substitution with 2-fluoro-4-nitrophenol to give the desired intermediate 9.
Reduction of the nitro group of 9 with iron powder and acetic acid in ethyl acetate /water (10:1 v/v) provided aniline
compound 10.
Scheme 1. Reagents and conditions: (i) cyclopropanecarbonyl chloride, Et3N, CH2Cl2, rt, 12 h; (ii) 2-fluoro-4-nitrophenol,
chlorobenzene, reflux, 40 h; (iii) Fe (powder), HOAc, ethyl acetate /water (5:1 v/v), reflux, 2 h.
The synthesis of target compounds 15a-f were summarized in Scheme 2. The condensation of commercially
available ethyl acetoacetate with different substituted diazotized anilines in the presence of sodium acetate in
ethanol/water mixture afforded intermediates 12a-f as yellow solids. The cyclization of 12a-f with
(ethoxycarbonylmethylene)triphenylphosphorane in refluxing toluene gave compounds 13a-f, which were converted
to acids 14a-f using sodium hydroxide solution at 50 °C for 5 h [22, 23]. Acids 14a-f were then condensed with
intermediate 10 in the presence of HATU and Et3N in DMF at room temperature overnight to afford the target
compounds 15a-f.

Scheme 2. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) Ethyl acetoacetate, AcONa, EtOH, 0-25 °C, 2 h; (iii)
Ph3P=CHCOOEt, PhMe, reflux, 12 h; (iv) NaOH, THF / H2O, 50 °C, 5 h; (v) 10, HATU, Et3N, DMF, rt,12 h.
The target compounds 20a-h were synthesized according to the method summarized in Scheme 3. According to
the similar synthesis procedure of 12a-h, diazotization of the substituted anilines with sodium nitrite in aqueous
hydrochloric acid, followed by condensation with ethyl 3-oxopentanoate in basic medium afforded the corresponding
intermediates 17a-h in good to excellent yields. The cyclization of 17a-h with DMF-DMA afforded
1-aryl-4-oxo-1,4-dihydropyridazine-3-carboxylic acid esters 18a-h, which were converted to acid analogues 19a-h
by hydrolysis in aqueous NaOH [24]. Finally, intermediates 19a-h were coupled with intermediate 10 using HATU
as coupling reactant to afford the target compounds 20a-h.
Scheme 3. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) ethyl 3-oxopentanoate, AcONa, EtOH, 0-25 °C, 2 h; (iii)
DMF-DMA, PhMe, reflux, 12 h; (iv) 10% NaOH, 50 °C, 4 h; (v) 10, HATU, Et3N, DMF, rt, 12 h.
The target compounds 26a-i were obtained using a five-step synthetic route outlined in Scheme 4. Ethyl
(2-cyanoacetyl)carbamate was added to a freshly synthesized different substituted phenyl diazonium chloride,
obtained by action of sodium nitrite on substituted aniline hydrochloride, in the presence of sodium acetate in
ethanol to give the ethyl [2-cyano-2-(2-arylhydrazono)acetyl]carbamate derivative 22a-i, which were converted into
analogues 23a-i by intramolecular cyclization in the presence of sodium acetate in refluxed acetic acid.
N-methylation of 23a-i with iodomethane in anhydrous DMF, in the presence of sodium hydride, gave the desired
intermediates 24a-i, which were hydrolyzed with acetic acid and hydrochloric acid gave rise to carboxylic acid

derivatives 25a-i [15, 25]. Coupling reaction of 25a-i with intermediate 10 produced the corresponding target
compounds 26a-i in the presence of HATU and Et3N in DMF at room temperature.
Scheme 4. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) ethyl (2-cyanoacetyl)carbamate, AcONa, EtOH, 0 °C, 2 h;
(iii) HOAc, AcONa, reflux, 2 h; (iv) NaH, CH3I, DMF, 0 °C, then rt. 5 h; (v) HOAc, HCl, H2O, reflux,4 h; (vi) 10, HATU, Et3N, DMF,
rt, 12 h.
2.2 Biological evaluation
2.2.1 In vitro c-Met kinase assays and analysis of the structure-activity relationships
All the newly synthesized 4-phenoxypyridine derivatives (15a-f, 20a-h, and 26a-i) were evaluated for their in
vitro inhibitory activity toward c-Met kinase using mobility shift assay. Foretinib was used as a positive control, with
the results expressed as half-maximal inhibitory concentration (IC50) values presented in Table 1. The IC50 values are
the average of at least three independent experiments.
As illustrated in Table 1, all the tested compounds showed moderate to excellent c-Met kinase activity with IC50
values ranging from 0.016 to 3.54 μM, which indicated that introduction of the 4-methyl-3,5-dioxo-2,3,4,5-
tetrahydro-1,2,4-triazine-6-carboxamide, 5-methyl-4-oxo-1,4-dihydropyrid azine-3-carboxamide or 4-methyl-6-oxo-
1,6-dihydropyridazine-3-carboxamide framework to “5-atom linker” moiety of pyridine-based c-Met kinase
inhibitor maintained the c-Met inhibitory efficacy. Notably, Nine of them (20a-d, 26a-d, 26f, 26g) exhibited
promising activity against c-Met kinase with IC50 values less than 0.10 μM, and compound 26a demonstrated the
best activity with an IC50 value of 0.016 μM.
The structure-activity relationship (SAR) was commenced by the introduction of “5-atom linker” that contains
different kinds of nitrogen aromatic heterocyclic between moiety A and moiety B. Based on the pharmacological
data in Table 1, we could deduce that different “5-atom linker” had a marked influence on c-Met kinase potency
with the following rank order generally: 4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide >
5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide > 4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide. For
example, the activity of compound 26b (IC50 = 0.031 μM, R = 4-F) was higher than compound 20b (IC50 = 0.044
μM, R = 4-F) and compound 15b (IC50 = 0.15 μM, R = 4-F), which suggested 4-methyl-3,5-dioxo-2,3,4,5-
tetrahydro-1,2,4-triazine-6-carboxamide was more preferred.
Further analysis revealed that compounds with the same “5-atom linker” but different substituents on the
terminal phenyl ring (moiety B) showed different c-Met inhibitory efficacy. Introduction of
mono-electron-withdrawing groups (mono-EWGs), especially halogen groups, on the phenyl ring led to a positive
effect in activity, such as compounds 20c (R = 4-Cl, IC50 = 0.057 μM) and 26b (R = 4-F, IC50 = 0.031 μM). However,

strong-withdrawing groups and double-electron-withdrawing groups (double-EWGs) and mono-electron-donating
groups (mono-EDGs) exhibited a negative effect compared to no substituent on the phenyl ring, such as compound
20f (R = 4-CF3, IC50 = 0.24 μM), compound 26h (R = 3-Cl-4-F, IC50 = 0.37 μM) and 20e (R = 4-OCH3, IC50 = 0.28
μM). Incorporation of double-electron-donating groups (double-EDGs) such as 3, 4-dimethoxy analogue (20h: IC50
= 3.54 μM; 26i: IC50 = 2.08 μM) lowered the efficiency even further. Moreover, it was worth noting that the position
of R group was closely related to c-Met inhibitory efficiency as well. Compounds with 4-substituted phenyl group
(15b, R = 4-F, IC50 = 0.15 μM; 20b, R = 4-F, IC50 = 0.044 μM; 26c, R = 4-Cl, IC50 = 0.046 μM) displayed higher
potency than those with 2-substituted phenyl group (15d, R = 2-F, IC50 = 0.88 μM; 20d, R = 2-F, IC50 = 0.062 μM)
or 3-substituted phenyl group (26g, R = 3-Cl, IC50 = 0.098 μM).
Table 1
Cytotoxicity of target compounds against A549, H460 and HT-29 cells and c-Met kinase inhibition in vitro.
IC50 (μM) ± SD a
Compd.
c-Met
A549
H460
HT-29
15a
15b
15c
H
0.35 ± 0.04
0.15 ± 0.02
0.23 ± 0.05
0.88 ± 0.10
1.05 ± 0.09
2.72 ± 0.13
0.076 ± 0.004
0.044 ± 0.007
0.057 ± 0.005
0.062 ± 0.008
0.28 ± 0.04
0.24 ± 0.06
0.47 ± 0.09
3.54 ± 0.37
0.016 ± 0.002
0.031 ± 0.004
0.046 ± 0.006
0.058 ± 0.008
0.94 ± 0.07
0.073 ± 0.003
0.098 ± 0.005
0.37 ± 0.09
2.08 ± 0.19
0.004 ± 0.0003
4.85 ± 0.33
3.07 ± 0.24
6.38 ± 0.72
4.64 ± 0.41
5.47 ± 0.38
10.56 ± 0.73
4.67 ± 0.16
1.82 ± 0.12
6.74 ± 0.44
2.79 ± 0.11
7.91 ± 0.64
6.84 ± 0.50
4.64 ± 0.37
8.75 ± 0.46
1.59 ± 0.09
3.42 ± 0.16
3.02 ± 0.25
2.56 ± 0.13
5.86 ± 0.59
2.13 ± 0.18
4.18 ± 0.37
4.67 ± 0.29
6.18 ± 0.45
1.05 ± 0.08
4.30 ± 0.27
2.61 ± 0.14
3.49 ± 0.31
5.84 ± 0.49
2.62 ± 0.09
13.09 ± 0.96
2.85 ± 0.15
0.79 ± 0.09
2.23 ± 0.12
1.83 ± 0.14
17.16 ± 1.08
7.45 ± 0.52
3.89 ± 0.25
5.26 ± 0.31
0.72 ± 0.05
0.64 ± 0.06
1.19 ± 0.11
1.37 ± 0.13
6.56 ± 0.46
3.01 ± 0.18
1.96 ± 0.14
3.36 ± 0.27
7.94 ± 0.66
0.81 ± 0.05
2.93 ± 0.21
3.94 ± 0.26
2.07 ± 0.17
4.11 ± 0.30
4.47 ± 0.22
8.36 ± 0.61
1.56 ± 0.08
1.43 ± 0.10
0.63 ± 0.04
2.31 ± 0.18
5.35 ± 0.13
2.92 ± 0.27
7.83 ± 0.56
9.87 ± 0.72
0.56± 0.04
1.03 ± 0.08
0.83 ± 0.07
2.46 ± 0.16
2.69 ± 0.13
1.63 ± 0.11
2.43 ± 0.20
4.61 ± 0.38
3.78 ± 0.23
0.98 ± 0.07
4-F
4-Cl
15d
15e
2-F
2-Cl
15f
4-OCH3
H
20a
20b
20c
4-F
4-Cl
20d
20e
2-F
4-OCH3
4-CF3
3-Cl-4-F
3,4-diOCH3
H
20f
20g
20h
26a
26b
26c
4-F
4-Cl
26d
26e
2-F
4-OCH3
3-F
26f
26g
26h
26i
3-Cl
3-Cl-4-F
3,4-diOCH3
--
Foretinibb
--
a Data presented is the mean ± SD value of three independent determinations.
b Used as a positive control.

2.2.2 In vitro antiproliferative activity
All the target compounds were further evaluated for their antitumor activitives against three c-Met
overexpressed human cancer cell lines, namely A549 (human lung adenocarcinoma cell line), H460 (human lung
cancer cell line) and HT-29 (human colon cancer cell line) together with foretinib as the positive control by a MTT
assay. The data listed in Table 1 revealed that all the target compounds (15a-f, 20a-h, and 26a-i) possessed moderate
to strong cytotoxicity against the three tested cell lines, and high selectivity for of inhibition H460 and HT29 cells,
and five of them were more potent than foretinib against one or more cell lines. Among them, compound 26a
exhibited remarkable inhibitory activity against H460 and HT-29 cell lines with IC50 value of 0.72 and 0.56 μM,
respectively, which were more potent than that of the positive control foretinib (IC50 = 0.81 μM and 0.98 μM). The
cytotoxic activities of these compounds showed similar structure–activity relationships (SARs) with summarized in
the c-Met kinase level: (a) compounds with 4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide as
“5-atom linker” exhibited strong cytotoxicity than the other two linkers (b) compounds bearing mono-EWGs on the
phenyl ring(F or Cl) were generally more active than those with double-EWGs or EDGs; (d) the cytotoxicity of
compounds with substituent at 4-position of phenyl ring were higher than those with substituents at other positions.
2.2.3. Colony formation inhibition ability of 26a
Colony formation assay is commonly used to determine longterm effects of cytotoxic agents on cancer cell
growth in vitro. Colony formation assay can be used not only to measure direct immediate impact of compounds on
cancer cells but also to estimate anti-proliferative potency or long-term recurrence prevention efficacy of compounds
[26]. According to the results of MTT, in vitro clonogenic assay was performed to measure the cell proliferation
capability i.e. the ability of a single cell to grow into a colony. This assay tests each and every cell in the population
for its ability to undergo unlimited divisions [27]. Based on the most excellent activity against HT-29 cells, cell
proliferation inhibiting ability of compound 26a was measured using colony formation assay. The results from the
Fig.3 showed that the colony formation efficiency from 25.3±1.21 % in control decreased to 12.2 ± 1.27 % at 1.0
μM and 4.8 ± 0.45 % at 10.0 μM, respectively. These experiments indicated that compound 26a suppressed the
colony formation and inhibited the unrestricted growth of HT-29 cancer cells in a concentration-dependent manner.
Fig. 3. Various concentrations of compound 26a inhibited colony formation of HT-29 cells for 7 days.
2.2.4. Apoptosis induction ability of 26a
Compound 26a was further confirmed by an acridine orange (AO)/ethidium bromide (EB) assay to examine the
occurrence of apoptosis in HT-29 cells. EB was able to penetrate through intact membranes of live cells and colors
DNA as green fluorescence, while AO was only taken up by apoptotic cells with damaged membranes coloring
DNA as orange fluorescence. Therefore, normal live cells appeared uniformly stained green in color. Early apoptotic
cells contained bright green condensed bodies in their nuclei representing nuclear DNA fragmentation and later
apoptotic cells presented colored orange nuclei indicating that their membranes are broken [28, 29]. It can be

inferred from Fig.4 that the control cells showed normal morphology and appeared green in color. Fluorescence
microscopic images of cells treated with 1.0 μM and 10.0 μM of compound 26a clearly showed the morphological
changes such as cell shrinkage, membrane blebbing, chromatin condensation and apoptotic body formation,
suggesting that compound 26a induced dose-dependent cell death in HT-29 cancer cells via apoptosis.
Fig. 4. AO/EB stained apoptosis of HT-29 cell lines with different concentrations of compound 26a for 48 h.
Subsequently, apoptosis was measured by Annexin V/PI double staining by flow cytometry to quantify the
percentage of cells undergoing apoptosis by compound 26a according to known procedures [30]. This assay
facilitate the detection of live cells, early apoptotic cells, late apoptotic cells and necrotic cells. HT-29 and A549
cells were seeded per well in six-well plates and were treated with compound 26a of different concentrations (0, 1.0
and 10.0 μM) for 48 h, respectively. The result was shown in Fig.5. The upper right section represents the cells
which were in the late apoptotic cells, and the lower right section represents the early or middle apoptosis. Results
from the Fig.5 indicated that the percentage of total apoptotic cells (early and late apoptotic cells) from 9.7%
(control) increased to 15.8 % at 1.0 μM, 29.2 % at 10.0 μM, in HT-29 cells, and the same trend was observed in
A549 cells. The increase of total apoptotic cells indicated that compound 26a induced apoptosis in HT-29 and A549
cells in a dose dependent manner.
Fig. 5. Compound 26a induces cell apoptosis of HT-29 and A549 cell line in vitro.

2.2.5. Cell migration inhibition ability of 26a
Cell migration is a highly coordinated and multi-step process that plays a critical role in progression of cancer.
Cancer cells often have a highly metastatic nature. Wound-healing assay is a simple and inexpensive method to
study the directional migration of cells in vitro [31]. Thus, we investigated the antimigratory effect of compound 26a
on A549 cells in vitro using wound-healing assay. As shown in Fig. 6, after 0, 12, 36 and 72 h at 0 and 1.0 μM study
revealed that compound 26a was sufficient to block the movement of most A549 cells at a dose of 1.0 μM compared
to the control. These results indicated that compound 26a strongly inhibited A549 cell motility.
Fig. 6. Effect of compound 26a in vitro migration potential of A549 cells.
2.2.6. Binding mode analysis
To further elucidate the binding mode of compounds, a detail docking analysis was performed. In our study, the
co-crystal structure of foretinib (GSK1363089) with c-Met was selected as the docking model (PDB ID code: 3LQ8).
The docking simulation was conducted using Glide XP (Schrödinger 2014), since Glide uses a hierarchical series of
filters to search for possible locations of the ligand in the active-site region of the receptor. The shape and properties
of the receptor are represented on a grid by several different sets of fields that provide progressively more accurate
scoring of the ligand poses. The image files were generated using Accelrys DS visualizer 4.0 system. The binding
mode was exemplified by the interaction of compound 26a with c-Met. As shown in Fig. 7, the N atom of pyridine
and the NH of cyclopropanecarboxamide interacted with Met1160 via two hydrogen bonds, and the
4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide moiety formed three hydrogen bonds with
Asp1222 and Lys1110, respectively. Meanwhile, pyridyl ring, the phenyl ring at 4-position of pyridine and the
phenyl ring at 1-position of triazine formed three π–π interactions with Tyr1159, Phe1223 and Phe1134, respectively.
All these interactions contribute to the tight binding and greatly enhance the inhibitory potency of 26a.
Fig. 7. Binding poses of compound 26a with c-Met (A); 2D interactions of the docking model of 26a to c-Met (B)

3. Conclusion
In summary, twenty-three novel 4-phenoxypyridine derivatives containing 4-methyl-6-oxo-1,6-
dihydropyridazine-3-carboxamide, 5-methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide and 4-methyl-3,5-
dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide moieties as c-Met inhibitors were designed, synthesized and
evaluated for their biological activitives. The screening of c-Met kinase activities and cytotoxicity led to the
identification of a most promising compound 26a (c-Met IC50 = 0.016 μM) with IC50 values of 1.59 μM, 0.72 μM
and 0.56 μM against A549, H460, and HT-29 cells, representing a promising lead for further optimization. The initial
SARs analysis disclosed that the 4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4- triazine-6-carboxamide scaffold was
more preferred as linker part, and electron-withdrawing groups on the terminal phenyl rings are beneficial for
improving the antitumor activitives. Meanwhile, colony formation assays, AO/EB assays, cell apoptosis assays by
flow cytometry, and wound-healing assays on HT-29 and/or A549 cells results indicated that compound 26a could
suppress the colony formation on HT-29 cells, induce HT-29 and A549 cells apoptosis, and inhibit A549 cells
motility. Further studies on structural optimization and biological activities about these derivatives are still underway
in our laboratory and will be reported in the future.
4. Experimental
4.1. Chemistry
Unless otherwise specified, all melting points were obtained on a Beijing Taike X-4 microscopy melting point
1
apparatus and were uncorrected. H NMR spectra were recorded on a Bruker Biospin 600 MHz or Bruker Biospin
400 MHz instrument using TMS as the internal standard. All chemical shifts were reported in ppm. IR spectra were
recorded as KBr pellets on a Perkin-Elmer Spcetrum one FT-IR spectrometer. MS spectra were obtained on an
Agilent 6460 QQQ mass spectrometer (Agilent, USA) analysis system. All materials were obtained from commercial
suppliers and were used without further purification. Reactions’ time and purity of the products were monitored by
TLC on FLUKA silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm. Column
chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China).
The key intermediates 11a-f, 12a-f, 13a-f and 14a-f were synthesized based on the previous literatures methods [22,
23]. The key intermediates 16a-c, 17a-h, 18a-h and 19a-h were synthesized according to our previous reported
procedures [24]. The key intermediates 21a-b, 22a-i, 23a-i, 24a-i and 25a-i were synthesized according to previous
reported procedures [15, 25].
4.1.1. N-(4-Chloropyridin-2-yl)cyclopropanecarboxamide (8)
Cyclopropanecarbonyl chloride (9.30 g, 89.00 mmol) was dissolved in dried CH2Cl2 (30 mL) and drop-wise
added to a mixture of 4-chloropyridin-2-amine (8.80 g, 68.45 mmol), Et3N (20.78 g, 205.35 mmol) and CH2Cl2 (80
mL) in an ice bath, which was then removed to raise the temperature to room temperature and stirred for 12 h. The
resulting mixture was sequentially washed with 20% K2CO3 (50 mL × 3) and brine (50 mL × 3), and the organic
phase was separated, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated under reduced pressure.
The crude product obtained was purified by silica gel chromatography to give 9.78 g (72.66%) of 13 as a white solid.
1
IR (KBr) cm-1: 3241.8 (NH), 1706.5 (C=O), 1670.1, 1588.4 (C=N), 1573.5, 1537.4, 1403.5; H-NMR (400 MHz,
DMSO-d6) δ 11.04 (s, 1H), 8.30 (d, J = 5.2 Hz, 1H), 8.15 (s, 1H), 7.21 (d, J = 5.2 Hz, 1H), 2.00 (m, 1H), 0.85 (m,
4H); MS (ESI) m/z(%): 197.1 [M+H]+.
4.1.2. N-(4-(2-Fluoro-4-nitrophenoxy)pyridin-2-yl)cyclopropanecarboxamide (9)
A stirring mixture of compound 8 (8.00 g, 40.68 mmol) and 2-fluoro-4-nitrophenol (15.98 g, 101.71 mmol) in
chlorobenzene (100 mL) was refluxed for about 40 h. After cooling to room temperature, the reaction mixture was

concentrated under reduced pressure to yield a pale solid. The solid was dissolved in CH2Cl2 (150 mL), and washed
with saturated K2CO3 aqueous solution (80 mL × 4), then brine (60 mL × 4), and dried over anhydrous Na2SO4,
concentrated under reduced pressure to afford a brown solid, which was was purified by silica gel chromatography to
give 7.13 g (55.24%) of 9 as a light yellow solid. IR (KBr) cm-1: 3417.7 (NH), 1687.3 (C=O), 1616.7 (C=N), 1528.7
(C=Carom), 1492.5 (C=Carom); 1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.43 (dd, J = 10.3, 2.2 Hz, 1H), 8.30
(d, J = 5.7 Hz, 1H), 8.19 (dd, J = 9.0, 1.9 Hz, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.61 (t, J = 8.5 Hz, 1H), 6.86 (dd, J = 5.6,
2.2 Hz, 1H), 2.04 – 1.95 (m, 1H), 0.78 (t, J = 6.3 Hz, 4H); MS (ESI) m/z(%): 318.1 [M+H]+.
4.1.3. N-(4-(4-Amino-2-fluorophenoxy)pyridin-2-yl)cyclopropanecarboxamide(10)
A mixture of compound 9 (6.00 g, 18.91 mmol), iron powder (5.28 g, 94.56 mmol), acetic acid (11.36 g, 189.10
mmol), water (20 mL) and ethyl acetate (100 mL) was heated to reflux for 2 h. After completion of the reaction as
indicated by TLC, the mixture was filtered immediately. The organic layer of the filtrate was separated, washed with
water, dried over anhydrous Na SO filtered, and the filtrate was evaporated under reduced pressure when white
，
2
4
solid appeared, which was filtered to obtain 3.51 g (64.61%) of 10 as light yellow solid. IR (KBr) cm-1: 3411.2 (NH),
1
3025.9 (CHarom), 1736.6 (C=O), 1617.8 (C=N), 1510.4 (C=Carom); H NMR (600 MHz, DMSO-d6) δ 10.79 (s, 1H),
8.15 (d, J = 5.7 Hz, 1H), 7.59 (s, 1H), 6.95 (t, J = 9.0 Hz, 1H), 6.67 – 6.61 (m, 1H), 6.49 (dd, J = 13.1, 2.2 Hz, 1H),
6.40 (d, J = 8.7 Hz, 1H), 5.44 (s, 2H), 2.03 – 1.88 (m, 1H), 0.76 (br, 4H); MS (ESI) m/z(%): 288.1 [M+H]+, 310.1
[M+Na]+.
4.1.4. General procedure for Preparation of the target compounds (15a-f, 20a-h and 26a-i)
A mixture of the corresponding acids (14a-f, 19a-h or 25a-h, 1.30mmol), compound 10 (1.00 mmol), HATU
(1.50 mmol), Et3N (3.00 mmol), and DMF (8 mL) was stirred at room temperature for 12 h. The residue was
dissolved in CH2Cl2 (50 mL) and the resulting mixture was sequentially washed with 20% K2CO3 (30 mL × 3) and
brine (30 mL × 3), and the organic phase was separated, dried, and evaporated. The crude product obtained was
purified by silica gel chromatography to afford 15a-f, 20a-h or 26a-i as white solids.
4.1.4.1.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-6-oxo-1-phenyl-1,6-
dihydropyridazine-3-carboxamide (15a)
Yield: 74.6%; M.p.: 212–215 °C; IR (KBr, cm-1): 3388.9 (NH), 1676.1 (C=O), 1575.8 (C=N), 1500.6
1
(C=Carom); H NMR (600 MHz, CDCl3) δ 8.97 (s, 1H), 8.62 (s, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.84 – 7.67 (m, 2H),
7.61 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 7.8 Hz, 2H), 7.47 (t, J = 7.4 Hz, 1H), 7.25 (s, 1H), 7.14 (t, J = 8.6 Hz, 1H), 6.91
(d, J = 1.0 Hz, 1H), 6.62 (dd, J = 5.8, 2.3 Hz, 1H), 2.66 (d, J = 0.7 Hz, 3H), 1.51 (m, 1H), 1.03 (dt, J = 8.0, 4.0 Hz,
2H), 0.88 – 0.80 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 172.30, 166.08, 160.32, 159.31, 154.23 (d, J = 249.7 Hz),
153.21, 148.87, 144.42, 140.51, 137.78, 137.08 (d, J = 12.5 Hz), 135.69 (d, J = 9.6 Hz), 130.88, 129.03, 128.88,
125.35, 123.51, 116.11, 109.40 (d, J = 23.1 Hz), 107.88, 100.54, 20.36, 15.78, 8.29; MS (ESI) m/z (%): 500.2
[M+H]+.
4.1.4.2.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide (15b).
Yield: 78.3%; M.p.: 200–202 °C; IR (KBr, cm-1): 3392.8 (NH), 3255.8 (NH), 1674.2 (C=O), 1585.5 (C=N),
1504.5 (C=Carom); 1H NMR (600 MHz, CDCl3) δ 8.92 (s, 1H), 8.64 (s, 1H), 8.12 (d, J = 5.7 Hz, 1H), 7.82 – 7.68 (m,
2H), 7.60 (dd, J = 8.8, 4.7 Hz, 2H), 7.26 – 7.18 (m, 3H), 7.14 (t, J = 8.6 Hz, 1H), 6.91 (s, 1H), 6.64 (dd, J = 5.7, 2.2
Hz, 1H), 2.66 (s, 3H), 1.57 – 1.45 (m, 1H), 1.11 – 0.97 (m, 2H), 0.93 – 0.79 (m, 2H); 13C NMR (150 MHz, CDCl3) δ
172.33, 166.06, 162.30 (d, J = 249.6 Hz), 160.20, 159.24, 154.23 (d, J = 249.7 Hz), 153.20, 148.87, 144.57, 137.99,
137.12 (d, J = 12.3 Hz), 135.62 (d, J = 9.5 Hz), 130.85, 129.63, 128.52, 127.30 (d, J = 8.8 Hz), 123.53, 115.97 (d, J
= 23.1 Hz), 109.43 (d, J = 23.2 Hz), 107.97, 100.43, 20.35, 15.78, 8.30; MS (ESI) m/z (%): 518.1[M+H]+,
540.1[M+Na]+, 556.1[M+K].

4.1.4.3.
1-(4-Chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide (15c)
Yield: 81.9%; M.p.: 191–193 °C; IR（KBr, cm-1）：3207.6 (NH), 1664.6 (C=O), 1589.3 (C=N), 1494.8
1
(C=Carom); H NMR (600 MHz, CDCl3) δ 8.89 (s, 1H), 8.44 (s, 1H), 8.12 (d, J = 5.8 Hz, 1H), 7.82 – 7.66 (m, 2H),
7.59 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.24 (s, 1H), 7.15 (t, J = 8.6 Hz, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.63
(m, 1H), 2.66 (s, 3H), 1.55 – 1.43 (m, 1H), 1.07 – 1.00 (m, 2H), 0.89 – 0.81 (m, 2H); 13C NMR (150 MHz, CDCl3) δ
172.37, 166.05, 160.17, 159.09, 154.21 (d, J = 249.6 Hz), 153.26, 148.85, 144.57, 138.87, 138.21, 137.10 (d, J =
12.3 Hz), 135.65 (d, J = 9.4 Hz), 134.69, 130.85, 129.14, 126.62, 123.50, 116.15, 109.41 (d, J = 23.2 Hz), 107.99,
100.44, 20.33, 15.73, 8.29; MS (ESI) m/z (%): 534.1 [M+H]+, 556.1 [M+Na]+.
4.1.4.4.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(2-fluorophenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide(15d).
Yield: 74.6%; M.p.: 171–174 °C; IR (KBr, cm ): 3408.3 (NH), 1670.4 (C=O), 1589.3 (C=N), 1502.6 (C=Carom);
1H NMR (600 MHz, CDCl3) δ 8.94 (s, 1H), 8.78 (s, 1H), 8.11 (d, J = 5.7 Hz, 1H), 7.74 (d, J = 12.1 Hz, 2H), 7.48 (t,
J = 6.8 Hz, 2H), 7.39 – 7.26 (m, 3H), 7.12 (t, J = 8.6 Hz, 1H), 6.92 (s, 1H), 6.67 – 6.57 (m, 1H), 2.66 (s, 3H), 1.56 –
1.45 (m, 1H), 1.07 – 0.97 (m, 2H), 0.91 – 0.78 (m, 2H); MS (ESI) m/z (%): 518.1 [M+H]+, 540.1 [M+Na]+;
4.1.4.5.
1-(2-Chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide (15e).
Yield: 83.7%; M.p.: 173–176 °C; IR (KBr, cm-1): 3402.4 (NH), 3298.9 (NH), 1672.3 (C=O), 1575.8 (C=N),
1
1517.9 (C=Carom); H NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 8.78 (s, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.74 (dd, J =
12.0, 2.1 Hz, 2H), 7.63 – 7.54 (m, 1H), 7.52 – 7.40 (m, 3H), 7.25 (s, 1H), 7.12 (t, J = 8.7 Hz, 1H), 6.94 (d, J = 0.9 Hz,
1H), 6.61 (dd, J = 5.8, 2.3 Hz, 1H), 2.68 (s, 3H), 1.56 – 1.43 (m, 1H), 1.06 – 0.98 (m, 2H), 0.87 – 0.80 (m, 2H); MS
(ESI) m/z (%): 534.1 [M+H]+, 556.1 [M+Na]+.
4.1.4.6.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-methoxyphenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide (15f).
Yield: 82.1%; M.p.: 245–247 °C; IR (KBr, cm-1): 3392.8 (NH), 3290.6 (NH), 1656.9 (C=O), 1595.1 (C=N),
1
1527.6 (C=Carom), 1502.6 (C=Carom); H NMR (600 MHz, CDCl3) δ 8.98 (s, 1H), 8.55 (s, 1H), 8.11 (d, J = 5.8 Hz,
1H), 7.82 – 7.68 (m, 2H), 7.52 (d, J = 8.9 Hz, 2H), 7.25 (s, 1H), 7.14 (t, J = 8.6 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H),
6.90 (s, 1H), 6.62 (dd, J = 5.7, 2.2 Hz, 1H), 3.87 (s, 3H), 2.65 (s, 3H), 1.55 – 1.44 (m, 1H), 1.08 – 1.00 (m, 2H), 0.89
– 0.83 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 172.2, 166.08, 160.38, 159.70, 159.47, 154.24 (d, J = 249.7 Hz),
153.19, 148.89, 144.27, 137.52, 137.05 (d, J = 12.6 Hz), 135.73 (d, J = 9.4 Hz), 133.48, 130.70, 126.58, 123.50,
116.08, 114.16, 109.37 (d, J = 22.9 Hz), 107.88, 100.53, 55.54, 20.34, 15.79, 8.28; MS (ESI) m/z (%): 530.2
[M+H]+.
4.1.4.7.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-5-methyl-4-oxo-1-phenyl-1,4-
dihydropyridazine-3-carboxamide (20a).
Yield: 74.7%; M.p.: 262-265 °C; IR (KBr, cm-1): 3248.1 (NH), 3047.5 (CHarom), 1685.8 (C=O), 1543.1 (C=N);
1H NMR (600 MHz, DMSO-d6) δ 12.30 (s, 1H), 10.88 (s, 1H), 9.11 (d, J = 0.6 Hz, 1H), 8.21 (d, J = 5.7 Hz, 1H),
7.99 (dd, J = 12.7, 2.3 Hz, 1H), 7.84 (d, J = 7.7 Hz, 2H), 7.71 – 7.60 (m, 3H), 7.53 (t, J = 7.4 Hz, 2H), 7.40 (t, J =
8.9 Hz, 1H), 6.74 (dd, J = 5.7, 2.4 Hz, 1H), 2.14 (s, 3H), 2.01 – 1.93 (m, 1H), 0.81 – 0.73 (m, 4H); 13C NMR (150
MHz, CDCl3) δ 172.14, 171.18, 166.22, 159.47, 154.20 (d, J = 248.3 Hz), 153.08, 148.90, 143.34, 142.24, 138.21,
136.97 (d, J = 12.7 Hz), 136.76 (d, J = 9.6 Hz), 133.17, 129.91, 129.63, 129.19, 128.52, 123.38, 121.62, 116.56,
109.83 (d, J = 23.6 Hz), 107.58, 100.95, 15.84, 13.61, 8.27; MS (ESI) m/z (%): 500.1 [M+H]+, 522.1 [M+Na]+.
4.1.4.8.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-5-methyl-
4-oxo-1,4-dihydropyridazine-3-carboxamide (20b).
Yield: 68.3%; M.p.: 179-181 °C; IR (KBr, cm-1): 3423.7 (NH), 1683.9 (C=O), 1545.0 (C=N), 1496.8 (C=Carom);

1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.81 (s, 1H), 9.00 (s, 1H), 8.14 (d, J = 5.7 Hz, 1H), 7.92 (dd, J =
12.7, 2.3 Hz, 1H), 7.86 – 7.77 (m, 2H), 7.59 (d, J = 2.1 Hz, 1H), 7.44 (dd, J = 14.4, 5.0 Hz, 3H), 7.33 (t, J = 8.9 Hz,
1H), 6.67 (dd, J = 5.7, 2.4 Hz, 1H), 2.48 – 2.40 (m, 3H), 1.95 – 1.84 (m, 1H), 0.74 – 0.65 (m, 4H); 13C NMR (150
MHz, DMSO-d6) δ 173.27, 170.02, 165.70, 162.01 (d, J = 246.0 Hz), 160.51, 154.32, 153.89 (d, J = 246.2 Hz),
149.98, 144.21, 140.23, 139.97 (d, J = 2.7 Hz), 137.42 (d, J = 9.9 Hz), 136.26 (d, J = 12.3 Hz), 131.78, 124.62,
124.28 (d, J = 9.0 Hz), 116.97, 116.82, 108.93 (d, J = 22.9 Hz), 107.57, 99.65, 14.59, 13.31, 8.09; MS (ESI) m/z (%):
518.2 [M+H]+, 540.1 [M+Na]+.
4.1.4.9.
1-(4-Chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-5-methyl-4-
oxo-1,4-dihydropyridazine-3-carboxamide. (20c).
Yield: 84.0%; M.p.: 204-207 °C; IR (KBr, cm-1): 3417.9 (NH), 1672.3 (C=O), 1550.8 (C=N), 1492.9 (C=Carom);
1H NMR (600 MHz, DMSO-d6) δ 12.21 (s, 1H), 10.88 (s, 1H), 9.11 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 7.98 (dd, J =
12.7, 2.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 2.1 Hz, 1H), 7.56 – 7.49 (m, 1H),
7.40 (t, J = 8.9 Hz, 1H), 6.74 (dd, J = 5.7, 2.4 Hz, 1H), 2.13 (s, 3H), 2.01 – 1.94 (m, 1H), 0.81 – 0.73 (m, 4H); 13
C
NMR (150 MHz, CDCl3) δ 172.28, 171.16, 166.19, 159.26, 154.18 (d, J = 248.9 Hz), 153.24, 148.86, 142.20,
141.66, 137.76, 137.00 (d, J = 12.3 Hz), 136.65 (d, J = 9.4 Hz), 135.18, 133.30, 130.02, 123.40, 122.69, 116.53,
109.79 (d, J = 22.9 Hz), 107.64, 100.92, 15.76, 13.58, 8.27; MS (ESI) m/z (%): 534.1 [M+H]+, 556.1 [M+Na]+.
4.1.4.10.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(2-fluorophenyl)-5-methyl-4-
oxo-1,4-dihydropyridazine-3-carboxamide . (20d).
Yield: 75.4%; M.p.: 178-180 °C; IR (KBr, cm-1): 3477.7 (NH), 3261.6 (NH), 3030.2 (CHarom), 1687.7 (C=O),
1496.8 (C=Carom), 1417.7; 1H NMR (600 MHz, CDCl3) δ 12.66 (s, 1H), 8.57 (s, 1H), 8.22 (d, J = 1.0 Hz, 1H), 8.11
(d, J = 5.8 Hz, 1H), 7.99 (dd, J = 12.2, 2.3 Hz, 1H), 7.81 (s, 1H), 7.74 (td, J = 7.9, 1.3 Hz, 1H), 7.48 (dd, J = 21.2,
14.4 Hz, 2H), 7.39 – 7.28 (m, 2H), 7.16 (t, J = 8.7 Hz, 1H), 6.59 (dd, J = 5.8, 2.3 Hz, 1H), 2.26 (s, 3H), 1.57 – 1.47
(m, 1H), 1.10 – 1.03 (m, 2H), 0.89 – 0.82 (m, 2H); MS(ESI) m/z (%): 518.1 [M+H]+, 540.1 [M+Na]+.
4.1.4.11.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-methoxyphenyl)-5-methyl-
4-oxo-1,4-dihydropyridazine-3-carboxamide (20e).
Yield: 76.6%; M.p.: 173-176 °C; IR (KBr, cm-1): 3420.6 (NH), 3010.8 (CHarom), 1687.7 (C=O), 1504.5
(C=Carom); 1H NMR (600 MHz, DMSO-d6) δ 12.44 (s, 1H), 10.88 (s, 1H), 9.03 (s, 1H), 8.22 (d, J = 5.6 Hz, 1H), 8.00
(d, J = 12.6 Hz, 1H), 7.76 (d, J = 8.9 Hz, 2H), 7.67 (s, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 8.8 Hz, 1H), 7.17 (d,
J = 8.9 Hz, 2H), 6.75 (d, J = 3.5 Hz, 1H), 3.85 (s, 3H), 2.14 (s, 3H), 2.03 – 1.92 (m, 1H), 0.84 – 0.68 (m, 4H); 13
C
NMR (150 MHz, DMSO-d6) δ 173.27, 169.96, 165.71, 160.55, 159.68, 154.32, 153.90 (d, J = 246.2 Hz), 149.97,
143.62, 140.18, 137.46 (d, J = 9.7 Hz), 136.92, 136.23 (d, J = 12.3 Hz), 132.06, 124.60, 123.40, 116.95, 115.06,
108.93 (d, J = 22.9 Hz), 107.56, 99.67, 56.04, 14.59, 13.32, 8.09; MS (ESI) m/z (%): 530.2 [M+H]+, 552.1 [M+Na]+.
4.1.4.12.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-5-methyl-4-oxo-1-(4-(trifluoro
methyl)phenyl)-1,4-dihydropyridazine-3-carboxamide (20f).
Yield: 69.2%; M.p.: 172-174 °C; IR (KBr, cm-1): 3238.5 (NH), 1687.7 (C=O), 1560.4 (C=N), 1510.3 (C=Carom),
1321.2 (CF3); 1H NMR (600 MHz, DMSO-d6) δ 12.07 (s, 1H), 10.88 (s, 1H), 9.20 (d, J = 0.7 Hz, 1H), 8.21 (d, J =
5.7 Hz, 1H), 8.10 (d, J = 8.6 Hz, 2H), 8.05 – 7.95 (m, 3H), 7.65 (d, J = 2.3 Hz, 1H), 7.54 (dd, J = 8.9, 1.4 Hz, 1H),
7.41 (t, J = 8.9 Hz, 1H), 6.75 (dd, J = 5.7, 2.4 Hz, 1H), 2.14 (s, 3H), 2.01 – 1.93 (m, 1H), 0.77 (t, J = 5.6 Hz, 4H);
MS (ESI) m/z (%): 568.2 [M+H]+, 590.1 [M+Na]+.
4.1.4.13.
1-(3-Chloro-4-fluorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-5-
methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide (20g).
Yield: 81.5%; M.p.: 194-197 °C; IR (KBr, cm-1): 3427.5 (NH), 1695.4 (C=O), 1552.7 (C=N), 1500.6 (C=Carom);
1H NMR (600 MHz, DMSO-d6) δ 12.17 (s, 1H), 10.88 (s, 1H), 9.11 (s, 1H), 8.26 – 8.11 (m, 2H), 8.05 – 7.94 (m, 1H),
7.92 – 7.84 (m, 1H), 7.68 (dd, J = 28.8, 5.5 Hz, 2H), 7.54 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 8.9 Hz, 1H), 6.74 (dd, J =

5.7, 2.3 Hz, 1H), 2.12 (s, 3H), 2.02 – 1.92 (m, 1H), 0.82 – 0.73 (m, 4H); MS (ESI) m/z (%): 552.1 [M+H]+, 574.1
[M+Na]+.
4.1.4.14.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-1-(3,4-dimethoxyphenyl)-5-
methyl-4-oxo-1,4-dihydropyridazine-3-carboxamide (20h).
Yield: 86.1%; M.p.: 288-290 °C; IR (KBr, cm-1): 3248.1 (NH), 1687.7 (C=O), 1563.2 (C=N), 1502.6 (C=Carom);
1H NMR (600 MHz, DMSO-d6) δ 12.42 (s, 1H), 10.88 (s, 1H), 9.03 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 8.00 (dd, J =
12.7, 2.3 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 8.8, 1.3 Hz, 1H), 7.43 – 7.31 (m, 3H), 7.16 (d, J = 8.8 Hz,
1H), 6.74 (dd, J = 5.7, 2.4 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 2.14 (s, 3H), 2.02 – 1.93 (m, 1H), 0.81 – 0.73 (m, 4H);
13C NMR (150 MHz, DMSO-d6) δ 173.27, 169.96, 165.71, 160.54, 154.32, 153.90 (d, J = 246.2 Hz), 149.97, 149.47,
149.38, 143.59, 140.28, 137.46 (d, J = 9.7 Hz), 136.90, 136.23 (d, J = 12.3 Hz), 131.97, 124.59, 116.95, 114.21,
112.14, 108.93 (d, J = 22.9 Hz), 107.57, 106.26, 99.66, 56.34, 56.25, 14.59, 13.32, 8.09; MS (ESI) m/z (%): 560.2
[M+H]+, 582.2 [M+Na]+.
4.1.4.15.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-3,5-dioxo-2-phenyl-
2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26a).
Yield: 76.4%; M.p.: 247-249 °C; IR (KBr, cm-1): 3416.7 (NH), 3246.5 (NH), 3060.4 (CHarom), 2851.6 (CHaryl),
1
1738.7 (C=O), 1698.5 (C=O), 1660.6(C=O), 1578.5 (C=N), 1507.5 (C=Carom); H NMR (600 MHz, DMSO-d6) δ
10.96 (s, 1H), 10.88 (s, 1H), 8.22 (d, J = 5.6 Hz, 1H), 7.90 (d, J = 12.5 Hz, 1H), 7.66 (s, 1H), 7.63 – 7.52 (m, 5H),
7.48 (t, J = 7.1 Hz, 1H), 7.41 (t, J = 8.9 Hz, 1H), 6.74 (d, J = 5.5 Hz, 1H), 3.31 (s, 3H), 2.06 – 1.92 (m, 1H), 0.89 –
0.67 (m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 173.27, 165.65, 158.91, 155.06, 154.33, 153.80 (d, J = 246.1 Hz),
149.98, 148.47, 140.46, 137.09 (d, J = 9.7 Hz), 136.51, 136.45, 129.25, 128.93, 126.20, 124.57, 117.14, 109.08 (d, J
= 22.9 Hz), 107.57, 99.66, 27.76, 14.60, 8.09; MS (ESI) m/z (%): 517.2 [M+H]+.
4.1.4.16.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-fluorophenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26b).
Yield: 82.4%; M.p.: 265-268 °C; IR (KBr, cm-1): 3416.9 (NH), 3236.3 (NH), 2934.0 (CHaryl), 1725.7 (C=O),
1
1695.2 (C=O), 1610.3 (C=N), 1552.8 (C=N), 1508.0 (C=Carom); H NMR (600 MHz, DMSO-d6) δ 10.93 (s, 1H),
10.88 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 7.89 (dd, J = 12.6, 2.3 Hz, 1H), 7.70 – 7.60 (m, 3H), 7.55 (dd, J = 8.8, 1.3 Hz,
1H), 7.45 – 7.34 (m, 3H), 6.74 (dd, J = 5.7, 2.4 Hz, 1H), 3.30 (s, 3H), 2.03 – 1.90 (m, 1H), 0.77 (t, J = 5.9 Hz, 4H);
13C NMR (150 MHz, DMSO-d6) δ 173.28, 165.65, 161.84 (d, J = 246.0 Hz), 158.85, 155.01, 154.32, 153.79 (d, J =
246.5 Hz), 149.98, 148.53, 137.07 (d, J = 9.9 Hz), 136.76 (d, J = 2.9 Hz), 136.52, 128.58, 128.52, 124.57, 117.14,
116.22, 116.07, 109.10 (d, J = 23.1 Hz), 107.59, 99.63, 27.76, 14.59, 8.09; MS (ESI) m/z (%): 535.2 [M+H]+, 557.1
[M+Na]+.
4.1.4.17.
2-(4-Chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26c).
Yield: 85.3%; M.p.: 284-286 °C; IR (KBr, cm-1): 3414.1 (NH), 3250.2 (NH), 3076.9 (CHarom), 2923.1(CHaryl),
1734.6 (C=O), 1691.1 (C=O), 1665.3(C=O), 1579.4 (C=N), 1504.9 (C=Carom); 1H NMR (600 MHz, CDCl3) δ 10.92
(s, 1H), 8.45 (s, 1H), 8.12 (d, J = 5.7 Hz, 1H), 7.88 (dd, J = 11.9, 2.2 Hz, 1H), 7.77 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H),
7.48 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.19 (t, J = 8.6 Hz, 1H), 6.63 (dd, J = 5.7, 2.2 Hz, 1H), 3.54 (s,
3H), 1.55 – 1.45 (m, 1H), 1.12 – 0.97 (m, 2H), 0.91 – 0.76 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 172.37, 166.05,
156.43, 156.16, 154.19 (d, J = 249.8 Hz), 153.26, 148.83, 147.35, 138.02, 137.53 (d, J = 12.4 Hz), 135.71 (d, J = 9.4
Hz), 134.97 132.40, 129.25, 126.35, 123.56, 116.55, 109.90 (d, J = 23.3 Hz), 107.83, 100.75, 28.21, 15.75, 8.30; MS
(ESI) m/z (%): 551.1[M+H]+, 573.2[M+Na]+.
4.1.4.18.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-2-(2-fluorophenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26d).
Yield: 71.9%; M.p.: 249-252 °C; IR (KBr, cm-1): 3336.9 (NH), 1737.9 (C=O), 1697.4 (C=O), 1595.1 (C=N),

1498.7 (C=Carom); 1H NMR (600 MHz, DMSO-d6) δ 10.98 (s, 1H), 10.87 (s, 1H), 8.20 (d, J = 5.7 Hz, 1H), 7.87 (dd,
J = 12.6, 2.2 Hz, 1H), 7.68 – 7.34 (m, 7H), 6.73 (dd, J = 5.7, 2.4 Hz, 1H), 3.30 (s, 3H), 1.96 (m , 1H), 0.77 (t, J = 6.3
Hz, 4H); MS (ESI) m/z (%): 535.1 [M+H]+, 557.1 [M+Na]+.
4.1.4.19.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-methoxyphenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26e).
Yield: 83.7%; M.p.: 210-212 °C; IR (KBr, cm-1): 3415.5 (NH), 3238.4 (NH), 3065.9 (CHarom), 2917.6 (CHaryl),
1736.4 (C=O), 1697.7 (C=O), 1663.0 (C=O), 1608.6 (C=N), 1578.3 (C=N), 1541.8 (C=Carom), 1511.8 (C=Carom); 1H
NMR (600 MHz, DMSO-d6) δ 10.93 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 7.90 (dd, J = 12.6, 2.2 Hz, 1H),
7.65 (d, J = 2.2 Hz, 1H), 7.59 – 7.52 (m, 1H), 7.49 (d, J = 8.9 Hz, 2H), 7.40 (t, J = 8.9 Hz, 1H), 7.08 (d, J = 9.0 Hz,
2H), 6.73 (dd, J = 5.7, 2.4 Hz, 1H), 3.82 (s, 3H), 3.30 (s, 3H), 2.03 – 1.91 (m, 1H), 0.77 (t, J = 6.7 Hz, 4H); 13C
NMR (150 MHz, DMSO-d6) δ 173.27, 165.65, 159.46, 158.93, 155.16, 154.33, 153.80 (d, J = 246.1 Hz), 149.98,
148.60, 137.10 (d, J = 9.9 Hz), 136.47 (d, J = 12.2 Hz), 136.08, 133.42, 127.62, 124.56, 117.12, 114.35, 109.06 (d, J
= 23.0 Hz), 107.57, 99.66, 55.88, 27.77, 14.59, 8.09; MS (ESI) m/z (%): 547.2 [M+H]+.
4.1.4.20.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3-fluorophenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26f).
Yield: 74.1%; M.p.: 255-257 °C; IR (KBr, cm-1): 3416.9 (NH), 3243.6 (NH), 3065.9 (CHarom), 2934.1 (CHaryl),
1
1740.7 (C=O), 1700.8 (C=O), 1662.9 (C=O), 1601.0 (C=N), 1547.6 (C=Carom); H NMR (600 MHz, DMSO-d6) δ
10.92 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.6 Hz, 1H), 7.90 (d, J = 12.5 Hz, 1H), 7.72 – 7.29 (m, 7H), 6.81 – 6.64 (m,
1H), 3.31 (s, 3H), 1.97 (d, J = 4.9 Hz, 1H), 0.86 – 0.70 (m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 173.28, 165.65,
161.97 (d, J = 244.3 Hz), 158.80, 154.78, 154.33, 153.79 (d, J = 246.1 Hz), 149.98, 148.33, 141.64 (d, J = 10.5 Hz),
137.06 (d, J = 9.6 Hz), 136.84, 136.52 (d, J = 12.2 Hz), 130.95 (d, J = 9.0 Hz), 124.56, 122.28, 117.20, 115.82 (d, J
= 20.8 Hz), 113.50 (d, J = 25.0 Hz), 109.14 (d, J = 23.1 Hz), 107.58, 99.65, 27.77, 14.59, 8.09; MS (ESI) m/z (%):
535.2 [M+H]+.
4.1.4.21.
2-(3-Chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-methyl-
3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26g).
Yield: 81.4%; M.p.: 175-178 °C; IR (KBr, cm-1): 3415.8 (NH), 3263.7 (NH), 3071.4 (CHarom), 2851.6 (CHaryl),
1
1735.1 (C=O), 1688.6 (C=O), 1661.0 (C=O), 1578.9 (C=N), 1548.1 (C=Carom), 1506.1 (C=Carom); H NMR (600
MHz, CDCl3) δ 10.90 (s, 1H), 8.76 (s, 1H), 8.12 (d, J = 5.7 Hz, 1H), 7.88 (d, J = 11.8 Hz, 1H), 7.78 (s, 1H), 7.64 (s,
1H), 7.52 (d, J = 3.5 Hz, 1H), 7.47 – 7.34 (m, 3H), 7.18 (t, J = 8.6 Hz, 1H), 6.71 – 6.55 (m, 1H), 3.53 (s, 3H), 1.59 –
1.47 (m, 1H), 1.10 – 1.00 (m, 2H), 0.90 – 0.79 (m, 2H); MS (ESI) m/z (%): 551.1 [M+H]+, 573.1 [M+Na]+.
4.1.4.22.
2-(3-Chloro-4-fluorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-
methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26h).
Yield: 79.4%; M.p.: 174-176 °C; IR (KBr, cm-1): 3420.5 (NH), 3257.8 (NH), 3070.9 (CHarom), 2923.1 (CHaryl),
1
1741.8 (C=O), 1660.3 (C=O), 1600.9 (C=N), 1578.4 (C=N), 1547.2 (C=Carom), 1499.2 (C=Carom); H NMR (600
MHz, DMSO-d6) δ 10.91 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 7.97 – 7.80 (m, 2H), 7.63 (dt, J = 17.8, 6.6
Hz, 3H), 7.55 (d, J = 8.7 Hz, 1H), 7.40 (t, J = 8.9 Hz, 1H), 6.74 (dd, J = 5.7, 2.3 Hz, 1H), 3.30 (s, 3H), 2.02 – 1.91
(m, 1H), 0.81 – 0.71 (m, 4H); MS (ESI) m/z (%): 569.1 [M+H]+, 591.1 [M+Na]+.
4.1.4.23.
N-(4-((2-(Cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3,4-dimethoxyphenyl)-4-
methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (26i).
Yield: 82.2%; M.p.: 208-211 °C; IR (KBr, cm-1): 3415.0 (NH), 3252.7 (NH), 2923.1 (CHaryl), 2846.2 (CHaryl),
1
1722.9 (C=O), 1702.2 (C=O), 1604.0 (C=N), 1550.1 (C=Carom), 1508.5 (C=Carom); H NMR (600 MHz, CDCl3) δ
10.96 (s, 1H), 8.57 (s, 1H), 8.12 (d, J = 5.8 Hz, 1H), 7.89 (dd, J = 11.9, 2.2 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.39 (d,
J = 8.8 Hz, 1H), 7.21 – 7.11 (m, 2H), 7.07 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 8.7 Hz, 1H), 6.63 (dd, J = 5.8, 2.3 Hz,
1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.54 (s, 3H), 1.57 – 1.50 (m, 1H), 1.09 – 1.03 (m, 2H), 0.89 – 0.83 (m, 2H); 13C

NMR (150 MHz, CDCl3) δ 172.34, 166.12, 156.57, 156.43, 154.19 (d, J = 249.7 Hz), 153.20, 149.64, 149.08,
148.73, 147.70, 137.42 (d, J = 12.6 Hz), 135.84 (d, J = 9.2 Hz), 132.59, 131.79, 123.53, 117.96, 116.50, 110.75,
109.87 (d, J = 23.2 Hz), 108.93, 107.81, 100.75, 56.09, 28.18, 15.76, 8.32; MS (ESI) m/z (%): 577.2[M+H]+,
599.2[M+Na]+.
4.2. Pharmacology
4.2.1. c-Met kinase assay
The in vitro kinase assays were performed by mobility shift assay. The solution of peptide substrates, ATP,
appropriate kinase (Carna), and various concentrations of tested compounds were mixed with the kinase reaction
buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT), with blank DMSO as the negative
control. The kinase reaction was initiated by the addition of tyrosine kinase proteins diluted in 39 µL of kinase
reaction buffer solution and incubated at 28 °C for 1 h. Then, 25 µL of stop buffer (100 mM HEPES, pH = 7.5,
0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) was added to stop reaction. The data were cellected on
Caliper at 320 nm and 615 nm and converted to inhibition values. IC50 was presented in MS Excel and the curves
fitted by XLfit excel add-in version
4.2.2. MTT assay in vitro
The anti-proliferative activities of compounds 15a-f, 20a-h or 26a-h were evaluated against A549, H460 and
HT-29 cell lines using the standard MTT assay in vitro, with Foretinib as the positive control. The cancer cell lines
were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximate
4 × 103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at
37 °C for 24 h. The tested compounds at the indicated final concentrations were added to the culture medium and the
cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 μg/mL, and
incubated with cells at 37 °C for 4 h. The formazan crystals were dissolved in 100 mL DMSO each well, and the
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured
with an ELISA reader. All compounds were tested three times in each of the cell lines. The results expressed as IC50
(inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus
Laboratories Incorporated Slide Scanner (Bliss) software.
4.2.3. Soft agar colony-formation assay
The colony formation assay has been the gold standard for determining the effects of cytotoxic agents on cancer
cell growth in vitro [32]. Briefly, HT-29 cells (1×105) in 300 μL RPMI1640 15% FBS medium were diluted in 300
μL of 0.4% agar containing various concentrations of 26a to give a final agar concentration of 0.2%. The cells-agar
mixture was poured over a hardened agar base in wells of 24-well plates and allowed to solidify. Once the top layer
solidified, 500 μL of RPMI 1640 medium containing 15% FBS was placed on top to keep the agar moist. Cells were
grown at 37 °C in a 5% CO2 humidified atmosphere for 7 days. Then 100 μL of MTT solution was added to each
well, and after 4 hours of incubation at 37 °C, photographs were captured
4.2.4. AO/ EB assay
HT-29 cells were added to a final concentration of 1×106/mL in a 6-well plate, and the plate was incubated for
24 h. Cells were treated with various concentrations of compound 26a. After being cultured for 48 h, control cells
and treated cells were washed with PBS which stored at 4 , and then dual fluorescent staining solution (20 μL)
containing 100 μg/mL AO and 100 μg/mL EB was added to each well for 10 min, and then covered with a coverslip.
The morphology of apoptotic cells was examined using fluorescent microscope.
4.2.5. Apoptosis assay

Apoptosis was measured by flow cytometry using Annexin V/propidium iodide (PI) double staining. HT-29 or
A549 cells were added to a final concentration of 1×106/mL in a 6-well plate, and the plate was incubated for 24 h.
Cells were treated with various concentrations of compound 26a. After being cultured for 48 h, control cells and
treated cells were harvested and washed with PBS, and then resuspended in 100μL 1×binding buffer incubated in the
mixture of 5μL Annexin V-FTIC and 5μL PI for 10 min at room temperature in dark place. The cells were
resuspended in 400μL 1×binding buffer just before flow cytometric analysis.
4.2.6. Wound-healing assay
A549 cells were added to a final concentration of 1×106/mL in a 6-well plate, and the plate was incubated for 24
h. Twenty-four hours later, when the cells reached confluency, scratches were created with sterile 1.0 mL pipette tips
and images were captured using phase contrast microscopy at 0 h, 12 h, 36 h and 72 h after treatment with 1.0 μM of
compound 26a.
Acknowledgments
The authors thank the financial support of Shenyang Science & Technology project (project no. 18-013-0-03) ,
National Natural Science Foundation of China (project no. 21807055), Natural Science Foundation of Liaoning
Provincial Department of science and technology (project no. 2019-ZD-0191), and Pre-declaration Foundation of
Liaoning University (project no. LDGY2019002).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version,
References and notes
[1] D.P. Bottaro, J.S. Rubin, D.L. Faletto, A.M. Chan, T.E. Kmiecik, G.F. Vande Woude, S.A. Aaronson, Identification of the
hepatocyte growth factor receptor as the c-met proto-oncogene product, Science. 251 (1991) 802-804.
[2] M. Jeffers, S. Rong, G.F. Vande Woude, Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter
factor-met signalling in human cells concomitant with induction of the urokinase proteolysis network, Mol. Cell. Biol. 16 (1996)
1115-1125.
[3] K. Matsumoto, K. Date, H. Shimura, T. Nakamura, Acquisition of invasive phenotype in gallbladder cancer cells via mutual
interaction of stromal fibroblasts and cancer cells as mediated by hepatocyte growth factor, Jpn. J. Cancer Res. 87 (1996) 702–710.
[4] P.M. Comoglio, S. Giordano, L. Trusolino, Drug development of MET inhibitors: targeting oncogene addiction and expedience, Nat
Rev. Drug. Discov. 7 (2008) 504-516.
[5] P.K. Parikh, M.D. Ghate, Recent advances in the discovery of small molecule c-Met Kinase inhibitors. Eur. J. Med Chem. 143(2018)
1103-1138.
[6] S. Berthou, D.M. Aebersold, L.S. Schmidt, D. Stroka, C. Heigl, B. Streit, D. Stalder, G. Gruber, C. Liang, A.R. Howlett, D. Candinas,
R.H. Greiner, K.E. Lipson, Y. Zimmer, The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different
receptor mutated variants, Oncogene. 23 (2004) 5387-5393.
[7] S.G. Buchanan, J. Hendle, P.S. Lee, C.R. Smith, P.Y. Bounaud, K.A. Jessen, C.M. Tang, N.H. Huser, J.D. Felce, K.J. Froning, M.C.
Peterman, B.E. Aubol, S.F. Gessert, J.M. Sauder, K.D. Schwinn, M. Russell, I.A. Rooney, J. Adams, B.C. Leon, T.H. Do, J.M.
Blaney , P.A. Sprengeler, D.A. Thompson, L. Smyth, L.A. Pelletier, S. Atwell, K. Holme, S.R. Wasserman, S. Emtage, S.K. Burley,
S.H. Reich, SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor
activity in vivo, Mol. Cancer Ther. 8 (2009) 3181-3190.
[8] M.H. Norman, L. Liu, M. Lee, N. Xi, I. Fellows, N.D. D'Angelo, C. Dominguez, K. Rex, S.F. Bellon, T. S. Kim, I. Dussault,
Structure-based design of novel class II c-Met inhibitors: 1. Identification of pyrazolone-based derivatives, J. Med. Chem. 55(2012)

1858-1867.
[9] F.M. Yakes, J. Chen, J. Tan, K. Yamaguchi, Y. Shi, P. Yu, F. Qian, F. Chu, F. Bentzien, B. Cancilla, J. Orf, A. You, A.D. Laird, S.
Engst, L. Lee, J. Lesch, Y.C. Chou, A.H. Joly, Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously
suppresses metastasis, angiogenesis, and tumor growth, Mol. Cancer. Ther. 10(2011) 2298-2308.
[10] Y. Dai, D.W. Siemann, BMS-777607, a small-molecule met kinase inhibitor, suppresses hepatocyte growth factor-stimulated
prostate cancer metastatic phenotype in vitro, Mol. Cancer Ther. 9 (2010) 1554-1561.
[11] Z. W. Cai, R. M. Borzilleri, 4-pyridinone compounds and their use for cancer, WO2009094417 [P]. 2009-07-30.
[12] F. Qian, S. Engst, K. Yamaguchi, P. Yu, K.A. Won, L. Mock, T. Lou, J. Tan, C. Li, D. Tam, J. Lougheed, F.M. Yakes, F. Bentzien, W.
Xu, T. Zaks, R. Wooster, J. Greshock, A.H. Joly, Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880,
GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases, Cancer Res. 69 (2009) 8009-8016.
[13] J.K. Rho, Y.J. Choi, S.Y. Kim, T.W. Kim, E.K. Choi, S.J. Yoon, B.M. Park, E. Park, J.H. Bae, C.M. Choi, J.C. Lee, MET and AXL
Inhibitor NPS-1034 Exerts Efficacy against Lung Cancer Cells Resistant to EGFR Kinase Inhibitors Because of MET or AXL
Activation, Cancer Res. 74(2014) 253-262.
[14] Z. Zhan, J. Ai, Q. Liu, Y. Ji, T. Chen, Y. Xu, M. Geng, W. Duan, Discovery of anilinopyrimidines as dual inhibitors of c-Met and
VEGFR-2: synthesis, SAR, and cellular activity, ACS Med. Chem. Lett. 5 (2014) 673-678.
[15] S. Zhao, Y. Zhang, H. Zhou, S. Xi, B. Zou, G. Bao, L. Wang, J. Wang, T. Zeng, P. Gong, X. Zhai, Synthesis and biological
evaluation of 4-(2-fluorophenoxy)-3,30-bipyridine derivatives as potential c-Met inhibitors, Eur. J. Med. Chem. 120 (2016) 37-50,
[16] W. Gu, Y. Dai, H. Qiang, W. Shi, C. Liao, F. Zhao, W. Huang, H. Qian, Discovery of novel 2-substituted-4-(2-fluorophenoxy)
pyridine derivatives possessing pyrazolone and triazole moieties as dual c-Met/VEGFR-2 receptor tyrosine kinase inhibitors,
Bioorg. Chem. 72 (2017) 116-122.
[17] Y. Zhao, J. Zhang, R. Zhuang, R. He, J. Xi, X. Pan, Y. Shao, J. Pan, J. Sun, Z. Cai, S. Liu, W. Huang, X. Lv, Synthesis and
evaluation of a series of pyridine and pyrimidine derivatives as type II c-Met inhibitors, Bioorg. Med. Chem. 25 (2017) 3195-3205,
[18] N. She, L. Zhuo, W. Jiang, X. Zhu, J. Wang, Z. Ming, X. Zhao, X. Cong, W. Huang, Design, synthesis and evaluation of highly
selective pyridone-based class II MET inhibitors, Bioorg. Med. Chem. Lett. 24 (2014) 3351-3355.
[19] J. Liu, M.H. Nie, Y.J. Wang, J.X. Hu, F. Zhang , Y.L. Gao, Y.J. Liu, P. Gong, Design, synthesis and structure-activity relationships
of novel 4-phenoxyquinoline derivatives containing 1,2,4-triazolone moiety as c-Met kinase inhibitors, Euro. J. Med. Chem. 123
(2016) 431-446.
[20] S. Li, Q. Huang, Y.J. Liu, X. Zhang, S. Liu, C. He, P. Gong, Design, synthesis and antitumour activity of bisquinoline derivatives
connected by 4-oxy-3-fluoroaniline moiety, Euro. J. Med. Chem. 64 (2013) 62-73.
[21] J. Liu, D. Yang, Yang X.X. , M.H. Nie, G. Wu, Z. Wang, W. Li, Y.J. Liu, P. Gong, Design, synthesis and biological evaluation of
novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety as c-Met kinase inhibitors, Bioorg.
Med.Chem. 25 (2017) 4475-4486.
[22] S.G. Zhou, H. Liao, C. He, Y. Dou, M. Jiang, L.L. Ren, Y. F. Zhao, P. Gong, Design, synthesis and structureeactivity relationships of
novel 4-phenoxyquinoline derivatives containing pyridazinone moiety as potential antitumor agents, Euro. J. Med. Chem. 83 (2014)
581-593
[23] Q.D. Tang, L.X. Wang, Y.L. Duan, W.H. Wang, S.M. Huang, J. Zhi, S. Jia, W.F. Zhu, P. Wang, R. Rong, P.W. Zheng, Discovery
of novel 7-azaindole derivatives bearing dihydropyridazine moiety as c-Met kinase inhibitors, Eur. J. Med. Chem. 133 (2017)
97-106.
[24] J. Liu, Y.T. Liu, X.C. Hao, Y. Wang, J.C. Ji, Y.J. Liu, S. Ding, Y. Chen, Design, synthesis and biological evaluation of novel
4-phenoxypyridine derivatives as potential antitumor agents, Arch. Pharm (Weinheim). 352 (2019) e1800338.
[25] H. Xu, Preparation of quinoline compounds containing 1,2,4-triazine-dione and use as c-Met kinase inhibitors for treating
proliferative diseases, US2013/0252958 A1
[26] D. Katz, E. Ito, K.S. Lau, J.D. Mocanu, C. Bastianutto, A.D. Schimmer, F.F. Liu, Increased efficiency for performing colony

formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening, Biotechniques
(2008) 44 (ix-xiv).
[27] R.L. Vinall, C.G. Tepper, X.B. Shi, L.A. Xue, R. Gandour-Edwards, R.W. deVere White, The R273H p53 mutation can facilitate
the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7, Oncogene 25
(2006) 2082-2093.
[28] S. Shrivastava, M.K. Jeengar, V.S. Reddy, G.B. Reddy, V.G.M. Naidu, Anticancer effect of celastrol on human triple negative
breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways, Exp. Mol.
Pathol. 98 (2015) 313-327.
[29] S. Shrivastava, P. Kulkarni, D. Thummuri, M.K. Jeengar, V.G.M. Naidu, M. Alvala, G.B. Redddy, S. Ramakrishna,
Piperlongumine, an alkaloid causes inhibition of PI3K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human
triple-negative breast cancer cells, Apoptosis.19 (2014) 1148-1164.
[30] A. Kamal, V. Srinivasulu, V.L. Nayak, M. Sathish, N. Shankaraiah, C. Bagul, N.V.S. Reddy, N. Rangaraj, N. Nagesh, Design and
synthesis of C3-pyrazole/chalcone linked b-carboline hybrids: antitopoisomerase I, DNA interactive and apoptosis inducing
anticancer agents, ChemMedChem 9 (2014) 2084-2098.
[31] L.G. Rodriguez, X. Wu, J.L. Guan, Wound-healing assay, Meth. Mol. Biol. 294 (2005) 23-29.
[32] D. Katz, E. Ito, K.S. Lau, J.D. Mocanu, C. Bastianutto, A.D. Schimmer, F.F. Liu, Increased efficiency for performing colony
formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening, Biotechniques. 44
(2008) (ix-xiv).
Legends
Fig. 1. The representative c-Met kinase inhibitors and the corresponding general structure.
Fig. 2. Design strategy and structures of the target compounds.
Scheme 1. Reagents and conditions: (i) cyclopropanecarbonyl chloride, Et3N, CH2Cl2, rt, 12 h; (ii) 2-fluoro-4-nitrophenol,
chlorobenzene, reflux, 40 h; (iii) Fe (powder), HOAc, ethyl acetate /water (5:1 v/v), reflux, 2 h.
Scheme 2. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) Ethyl acetoacetate, AcONa, EtOH, 0-25 °C, 2 h; (iii)
Ph3P=CHCOOEt, PhMe, reflux, 12 h; (iv) NaOH, THF / H2O, 50 °C, 5 h; (v) 10, HATU, Et3N, DMF, rt,12 h.
Scheme 3. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) ethyl 3-oxopentanoate, AcONa, EtOH, 0-25 °C, 2 h; (iii)
DMF-DMA, PhMe, reflux, 12 h; (iv) 10% NaOH, 50 °C, 4 h; (v) 10, HATU, Et3N, DMF, rt, 12 h.
Scheme 4. Reagents and conditions: (i) NaNO2, HCl, H2O, 0 °C, 30 min; (ii) ethyl (2-cyanoacetyl)carbamate, AcONa, EtOH, 0 °C, 2 h;
(iii) HOAc, AcONa, reflux, 2 h; (iv) NaH, CH3I, DMF, 0 °C, then rt. 5 h; (v) HOAc, HCl, H2O, reflux,4 h; (vi) 10, HATU, Et3N, DMF,
rt, 12 h.
Table 1 Cytotoxicity of target compounds against A549, H460 and HT-29 cells and c-Met kinase inhibition in vitro.

Fig. 3. Various concentrations of compound 26a inhibited colony formation of HT-29 cells for 7 days.
Fig. 4. AO/EB stained apoptosis of HT-29 cell lines with different concentrations of compound 26a for 48 h.
Fig. 5. Compound 26a induces cell apoptosis of HT-29 and A549 cell line in vitro.
Fig. 6. Effect of compound 26a in vitro migration potential of A549 cells.
Fig. 7. Binding poses of compound 26a with c-Met (A); 2D interactions of the docking model of 26a to c-Met (B)

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Three series of novel 4-phenoxypyridine derivatives were designed and synthesized.
The target compounds showed c-Met kinase activities and cytotoxic activities
Compound 26a showed an IC50 value of 0.016 μM against c-Met kinase.
The cytotoxic activities of 26a were more potent than foretinib against H460 and HT-29 cells.
Compound 26a could suppress the colony formation, induce cells apoptosis and inhibit cells motility.

Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.