Design, synthesis, and biological evaluation of pyridineamide derivatives containing a 1,2,3-triazole fragment as type II c-Met Inhibitors

Article abstract of DOI:10.3390/molecules25010010

A series of 4-(pyridin-4-yloxy)benzamide derivatives containing a 1,2,3-triazole fragment were designed, synthesized, and their inhibitory activity against A549, HeLa, and MCF-7 cancer cell lines was evaluated. Most compounds exhibited moderate to potent antitumor activity against the three cell lines. Among them, the promising compound B26 showed stronger inhibitory activity than Golvatinib, with IC₅₀ values of 3.22, 4.33, and 5.82 μM against A549, HeLa, and MCF-7 cell lines, respectively. The structure-activity relationships (SARs) demonstrated that the modification of the terminal benzene ring with a single electron-withdrawing substituent (fluorine atom) and the introduction of a pyridine amide chain with a strong hydrophilic group (morpholine) to the hinge region greatly improved the antitumor activity. Meanwhile, the optimal compound B26 showed potent biological activity in some pharmacological experiments in vitro, such as cell morphology study, dose-dependent test, kinase activity assay, and cell cycle experiment. Finally, the molecular docking simulation was performed to further explore the binding mode of compound B26 with c-Met.

Full text of DOI:10.3390/molecules25010010

molecules
Article
Design, Synthesis, and Biological Evaluation of
Pyridineamide Derivatives Containing a 1,2,3-Triazole
Fragment as Type II c-Met Inhibitors
Hehua Xiong †, Jianxin Cheng †, Jianqing Zhang, Qian Zhang, Zhen Xiao, Han Zhang,
Qidong Tang * and Pengwu Zheng *
School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China;
18296154955@163.com (H.X.); cjx3159@163.com (J.C.); zhang1405474771@163.com (J.Z.);
15797966937@163.com (Q.Z.); xz950420@163.com (Z.X.); zbl1045762244@163.com (H.Z.)
*
Correspondence: tangqidongcn@126.com (Q.T.); zhengpw@jxstnu.edu.cn (P.Z.); Tel.: +86-791-8380-2393 (P.Z.)
† These authors contribute equally to this work.
Academic Editor: Qiao-Hong Chen
Received: 21 November 2019; Accepted: 10 December 2019; Published: 18 December 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of 4-(pyridin-4-yloxy)benzamide derivatives containing a 1,2,3-triazole fragment
were designed, synthesized, and their inhibitory activity against A549, HeLa, and MCF-7 cancer cell
lines was evaluated. Most compounds exhibited moderate to potent antitumor activity against the
three cell lines. Among them, the promising compound B26 showed stronger inhibitory activity
than Golvatinib, with IC50 values of 3.22, 4.33, and 5.82 µM against A549, HeLa, and MCF-7 cell
lines, respectively. The structure–activity relationships (SARs) demonstrated that the modification of
the terminal benzene ring with a single electron-withdrawing substituent (fluorine atom) and the
introduction of a pyridine amide chain with a strong hydrophilic group (morpholine) to the hinge
region greatly improved the antitumor activity. Meanwhile, the optimal compound B26 showed
potent biological activity in some pharmacological experiments in vitro, such as cell morphology
study, dose-dependent test, kinase activity assay, and cell cycle experiment. Finally, the molecular
docking simulation was performed to further explore the binding mode of compound B26 with c-Met.
Keywords: 4-(pyridin-4-yloxy)benzamide; 1,2,3-triazole; c-Met; inhibitor
1. Introduction
Cancer has become a serious threat to human life and health [
of type III tyrosine kinase, which is closely related to cellular activities [
metastasis, etc. However, abnormal expression of c-Met kinases in cells usually triggers the occurrence,
invasion, and metastasis of various cancer diseases [ ]. c-Met kinase has been found overexpressed in
various cancer cells and has become an attractive target for cancer treatment [ ]. Nowadays, developing
1,
2]. c-Met tyrosine kinase is a kind
3
] such as growth, reproduction,
4
5,6
small molecule c-Met kinase inhibitors has become a hotspot in the treatment of human cancer [7].
c-Met inhibitors are classified as type I and type II inhibitors according to the binding mode of
inhibitors with c-Met. Usually, c-Met inhibitors of type I are single-target inhibitors that bind to the
hinge region of the ATP pocket, and c-Met inhibitors of type II are multitarget inhibitors that bind
to the hinge region and an extra hydrophobic pocket [8,9]. With the development of biology and
pharmacology, the intracellular mechanisms have been elucidated, and research in small molecule
inhibitors has made significant progress. Type II c-Met inhibitors possess more potent inhibitory activity
and better tolerance to drug resistance through binding to kinases of multiple types. Cabozantinib,
a c-Met inhibitor of type II, was approved by the FDA for treatment of prostate cancer in 2012. Some
representative c-Met inhibitors (type II) in the clinical trial phase are listed in Figure 1, including
Molecules 2020, 25, 10; doi:10.3390/molecules25010010
www.mdpi.com/journal/molecules

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Golvatinib, BMS-777607, Altriatinib, and TAS-115 [10
–
12]. Furthermore, according to the structure
characteristics of inhibitors, the skeleton of c-Met inhibitor (type II) was summarized into blocks A, B,
C, and D by our research group, as shown in Figure 1 [13].
Figure 1. Some representative c-Met inhibitors of type II and the summarized skeleton.
We explored the SARs of c-Met inhibitors (type II) bearing pyridinamide structure using Golvatinib
as a lead compound in this study. In order to guide our modification of the lead compound, a molecular
docking simulation of Golvatinib and c-Met protein was performed, as shown in Figure 2. The docking
results indicated that blocks A and C, which possessed hydrogen bonding interactions with residues
Met1160, Asp1222, and Lys1110, played a key role in maintaining the inhibitory activity against c-Met.
Therefore, the modification was mainly concentrated on blocks A and C. Firstly, a pyridylamide
structure in block A, including hydrogen bond donor and hydrogen bond acceptor atoms, was
introduced into the meta position of pyridine in order to enhance the interaction with the hinge region
residues, and then some alkyl chains bearing morpholine or thiophene groups were connected to the
terminal of the pyridine amide structure for better water solubility of compounds. Then, a 1,2,3-triazole
fragment with good biological activity and two hydrogen bond acceptor atoms was introduced to
block C for more tight combination between the compounds and c-Met. In addition, considering
the steric hindrance problem [14], H/F atoms and different substituents were introduced in blocks
B and D, respectively, to explore their effects on biological activity of target compounds. Finally,
a series of small molecule inhibitors containing the 1,2,3-triazole fragment were designed, synthesized,
and biologically evaluated.
Figure 2. Design strategy of target compounds based on the molecular simulation and structure–activity
relationships (SARs).

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2. Results and Discussion
2.1. Chemistry
The synthesis route of target compounds B1–B27 is shown in Scheme 1. Firstly, the commercially
available pyridinecarboxylic acid was reacted with SOCl2 using NaBr as catalyst to yield
4-chloropicolinoyl chloride , which was then converted into ethyl 4-chloropicolinate with ethanol. Next,
4-chloropicolinate was subjected to nucleophilic substitute with p-nitrophenol or 2-fluoro-4-nitrophenol
using chlorobenzene as solvent to give picolinate analogues 9a–9b, which were then dissolved in
6
7
8
8
1,4-dioxane and hydrolyzed with NaOH aqueous solution to obtain picolinic acid analogues 10a–10b
Intermediates 10a–10b were reacted with SOCl2 to get the corresponding acyl chlorides and then
connected with amines (including propan-1-amine, 2-(thiophen-2-yl)ethan-1-amine, pyrrolidine and
3-morpholinopropan-1-amine) to obtain picolinamide analogues 11a–11e. Finally, the key intermediates
12a–12e were produced by the reduction of 11a–11e with hydrazine hydrate as reducing agent. Other
intermediates 13a–13i, which have been reported in our previous research [15], were combined with
intermediates 12a–12e by a nucleophilic substitute reaction to get the target compounds B1–B27.
.
1
The structural information of the target compounds was confirmed by H-NMR, 13C-NMR,
and TOF MS (ES+), which were consistent with the structures depicted.
Scheme 1. The synthetic route for target compounds B1–B27. Reagents and conditions: (i) NaBr,
SOCl2, 85 ◦C, 18 h; (ii) triethylamine, dichloromethane (DCM), EtOH, 0 ◦C, 1 h; (iii) p-nitrophenol
or 2-fluoro-4-nitrophenol, chlorobenzene, 135 ◦C, 4 h; (iv) 1,4-dioxane, sodium hydroxide (10%), r.t.,
0.5 h; (v) SOCl2, amines, Et3N, DCM, 0–85 ◦C, 1 h; (vi) EtOH, activated carbon, FeCl3
·
6H2O, hydrazine
hydrate (80%), 90 ◦C, 4 h. (vii) EtOH, activated carbon, FeCl3
4 h; (vii) DCM, NaHCO3, 0 ◦C, 1 h.
·
6H2O, hydrazine hydrate (80%), 90 ◦C,
2.2. Antitumor Activity of Compounds B1–B27 Against A549, HeLa, and MCF-7 Cell Lines and SAR Analysis
The antitumor activity of target compounds against A549, HeLa, and MCF-7 cell lines was
evaluated to investigate their inhibitory activity against cancer cells [16]. The antitumor activities of
compounds were displayed as the IC50 (half-inhibitory concentration) values, as shown in Tables 1 and 2.
Most compounds exhibited moderate to potent antitumor activity against three cell lines. Compounds
B25–B27 possessed more potent inhibitory activity than Golvatinib, as shown in Table 2, with IC50
values in the range of 3.22–13.60
against A549, HeLa, and MCF-7 cell lines with IC50 values of 3.22
respectively, which were more efficient than Golvatinib (8.14 0.45, 15.17
µ
M. The optimal compound B26 showed excellent inhibitory activity
0.12, 4.33 0.09, and 5.82 0.09 M,
0.17, and 16.91 0.29 M).
±
±
±
µ
±
±
±
µ

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Table 1. Antitumor activities of the target compounds B1–B18 against A549, HeLa, and MCF-7 cell lines.
IC50 (µM) ± SD
Compd.
R2
R3
Z
A549
HeLa
MCF-7
B1
B2
B3
B4
B5
B6
B7
B8
B9
4-H
4-F
2-OCF3
4-Cl
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
46.55 ± 1.90
21.30 ± 0.66
32.67 ± 1.16
56.26 ± 2.26
35.57 ± 1.03
256.30 ± 10.37
221.98 ± 21.46
238.07 ± 17.84
357.24 ± 14.50
52.31 ± 5.42
42.06 ± 6.03
53.94 ± 4.23
48.23 ± 1.59
59.55 ± 2.94
170.38 ± 13.69
295.65 ± 15.37
NA
63.05 ± 2.44
32.13 ± 0.86
48.71 ± 3.26
120.76 ± 1.25
270.40 ± 12.35
NA a
H
2-CF3
3-F-4-F
3-Cl-4-F
2-Cl-5-CF3
4-Cl-3-CF3
NA
NA
NA
NA
B10
B11
B12
B13
B14
B15
B16
B17
B18
4-H
4-F
2-OCF3
4-Cl
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
33.31 ± 1.25
19.70 ± 0.89
24.24 ± 0.71
46.13 ± 3.59
32.94 ± 2.33
123.07 ± 10.23
150.58 ± 12.21
223.34 ± 10.89
246.70 ± 21.23
41.55 ± 2.26
46.57 ± 1.59
53.04 ± 1.11
55.43 ± 2.30
40.68 ± 1.09
240.60 ± 22.49
270.24 ± 13.56
NA
60.50 ± 3.99
29.11 ± 1.30
42.26 ± 1.09
82.48 ± 2.34
84.45 ± 12.34
332.01 ± 20.08
NA
F
2-CF3
3-F-4-F
3-Cl-4-F
2-Cl-5-CF3
4-Cl-3-CF3
NA
NA
NA
Golvatinib b
8.14 ± 0.45
15.17 ± 0.17
16.91 ± 0.29
a
NA: Low inhibitory activity, b Used as the positive control.
Table 2. Antitumor activities of the target compounds B10–B12 and B19–B27 against A549, HeLa,
and MCF-7 cell lines.
IC50 (µM) ± SD
Compd.
R1
R3
A549
HeLa
MCF-7
B10
B11
B12
4-H
4-F
2-OCF3
33.31 ± 1.25
19.70 ± 0.89
24.24 ± 0.71
41.55 ± 2.26
46.57 ± 1.59
53.04 ± 1.11
60.50 ± 3.99
29.11 ± 1.30
42.26 ± 1.09
B19
B20
B21
4-H
4-F
2-OCF3
27.25 ± 0.79
12.10 ± 0.31
17.72 ± 0.42
32.31 ± 2.11
21.84 ± 1.93
29.55 ± 2.04
39.08 ± 0.97
19.12 ± 0.21
41.10 ± 1.21
B22
B23
B24
4-H
4-F
2-OCF3
19.88 ± 0.75
12.52 ± 0.37
13.34 ± 0.58
45.18 ± 3.89
15.19 ± 0.96 a
19.73 ± 0.83
33.80 ± 1.11
35.11 ± 3.05
44.82 ± 2.96
B25
B26
B27
4-H
4-F
2-OCF3
6.43 ± 0.21
3.22 ± 0.12
4.91 ± 0.09
8.14 ± 0.45
10.51 ± 0.68
4.33 ± 0.09
5.72 ± 0.17
15.17 ± 0.17
13.60 ± 0.86
5.82 ± 0.09
9.34 ± 0.52
16.91 ± 0.29
Golvatoinib b
a
Bold values show the IC50 values of the target compounds lower than the values of the positive control; b Used as
the positive control.

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According to the antitumor activity of compounds B1–B27 listed in Tables 1 and 2, the SARs
were summarized as follows. Overall, the F atom on the meta-central benzene ring provided stronger
inhibitory activity against cancer cells than H atom, for example, compounds B1–B8 displayed less
inhibitory activity than compounds B9–B18. A single substitution (such as H/F/OCF3/Cl/CF3) linked
to the terminal benzene ring was favorable for maintaining the biological activity, such as compounds
B1–B5 and B10–B15 that possessed moderate antitumor activity with IC50 values less than 60 µM
against A549 and HeLa cell lines. However, the double substituents connected to the terminal benzene
ring obviously impaired the inhibitory activity of compounds B6–B9 and B15–B18, whose IC50 values
were more than 120 µM against three cell lines. A possible explanation would be that the introduction
of two substituents to the terminal benzene ring may create steric hindrance, which made it difficult
for compounds to extend into the hydrophobic pocket and to bind tightly to c-Met kinase.
Inspired by the inhibitory activity of compounds B1–B3 and B10–B12, as shown in Table 1, the F
atom of the central benzene ring and the single group (H, F, or OCF3) of the terminal benzene ring
were reserved to perform a SAR study of the pyridine amide moiety. Hydrophilic groups, including
morpholine or thiophene groups attached to the terminal of pyridine amide, have a great contribution
to inhibitory activity of the compounds B22–B27 (with IC50 values in the range of 3.22–45.18 µM).
In summary, introducing morpholino groups and F atoms to the pyridylpropyl amide and the
terminal benzene ring, respectively, made significant advances in the inhibitory activity.
2.3. Dose-Dependent Test of Compound B26 Against A549 Cells
To explore the relationship between concentrations and antitumor activity of compound B26, we
carried out a dose-dependent test based on the MTT method [16], in which A549 cells were treated with
seven different concentrations of compound B26 (0.137, 0.411, 1.234, 3.704, 11.11, 33.33, and 100 µM),
using Golvatinib as a positive control, as seen in Figure 3. The results revealed that compound B26
could effectively inhibit cancer cells in a dose-dependent manner. Notably, compound B26 inhibited
A549 cells more than 50% at the concentration of 3.704
less than 40% at the same concentration.
µM, while the inhibitory rate of Golvatinib was
Figure 3. Relationship between concentration and inhibition rate of compound B26 against A549 cells.
2.4. Cell Cycle Study of A549 Cells Treated with Compound B26
To further investigate the inhibitory pattern of compound B26 on the growth of cancer cells, cell
cycle distribution analysis [17] was carried out on A549 cells, as shown in Figure 4. Compound B26 at
concentrations of 5, 10, and 15 µM and Golvatinib at a concentration of 5 µM were delivered to A549
cells for 24 h, as shown in Figure 4. With the increased concentration of compound B26, the percentage
of cells blocked in G0/G1 phase was increased from 65.44% to 79.18%, while the percentage in the S
phase decreased from 21.91% to 16.56%. There was no significant change in G2/M phase. The result

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showed that compound B26 (74.41%) and Golvatinib (74.77%) had the same inhibitory effect on A549
cells at the concentration of 5 µM, both of which could block cells in the G0/G1 phase.
Figure 4. Cell cycle study of A549 cells treated with compound B26 and Golvatinib.
2.5. c-Met Enzyme Assay of Compounds B25–B27 and Staurosporine
Compounds B25, B26, and B27, possessing potent antitumor activity against A549, HeLa,
and MCF-7 cell lines, were selected to investigate their c-Met kinase assay by mobility shift assay
method [18] with staurosporine as a positive control, which was used to insure the reliability of the
experimental data. Compound B26 exhibited potent inhibitory activity against c-Met with an inhibition
rate of 36% at the concentration of 0.625 µM, as shown in Table 3.
Table 3. c-Met kinase activity of selected compounds B25, B26, B27, and staurosporine.
Compound (0.625 µM)
Rates (%)
B25
B26
B27
4.3
36.0
23.8
87.3
Staurosporine
2.6. Molecular Docking Simulation of Compounds (B26 and Golvatinib) and c-Met
Next, we further explored the binding mode of compound B26 and Golvatinib with c-Met
kinase (PDB: 3LQ8, extracted from the PDB database) through molecule docking simulation using
AutoDock 4.2 software [19], as shown in Figure 5. The docking results indicated that compound
B26 and Golvatinib in an extended conformation formed strong hydrogen bonding interactions with
residues Met1160, Asp1222, and Lys1110, as shown in Figure 5a–d. Interestingly, the pyridylamide
structure of compound B26 formed a bidentate hydrogen bond with residue Met1160, as shown in
Figure 5a,b, which may offer a stronger ability of compound B26 to bind with c-Met. Compound B26
formed more hydrogen bonding interactions with Met1160 than Golvatinib, which may explain the
better antitumor activity of compound B26.

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Figure 5. Figure 5a, 5b, and 5c are partial views, two-dimensional partial views, and integral cavity
views of the docking results of c-Met (PDB code: 3LQ8) and compound B26, respectively. Figure 5d
shows partial views of c-Met and Golvatinib. The c-Met protein and compounds are displayed by
cartoons and sticks, respectively. Hydrogen bonding interactions between compounds and c-Met are
indicated with dashed lines or arrows in red.
3. Experimental Section
3.1. Chemistry
Unless specifically required, all reagents used in the experiments were purchased as commercial
analytical grade and used without further purification. Frequently used solvents (dichloromethane,
ethyl acetate, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, etc.) were absolutely anhydrous. Flash
column chromatography was performed on silica gel (300 mesh) using a mixture of petroleum ether
(PE) and ethyl acetate (EA). The reaction process was monitored by thin-layer chromatography (TLC,
1
SH-GH254) and spots were visualized with ultraviolet analyzer (light in 254 or 365 nm). H-NMR
and 13C-NMR spectra analysis of compounds was implemented in Bruker 400 MHz spectrometer
(Bruker Bioscience, Billerica, MA, USA) using tetramethylsilane (TMS) as an internal standard at room
temperature. Mass spectrometry (MS) of target compounds was carried out Waters High Resolution
Quadrupole Time of Flight Tandem Mass Spectrometry (Waters, Xevo G2-XS Tof). The melting point of
the compounds was measured by SGW X-4 micro melting point instrument. The purity of compounds
was determined by an Agilent 1260 liquid chromatograph equipped with an Inertex-C18 column,
and all were more than 95%.
3.1.1. Preparation of 4-Chloropicolinoyl Chloride (7)
Pyridinecarboxylic acid
6 (10.00 g, 0.081 mol), NaBr (0.10 g, 0.001 mol), and a drop of
N,N-dimethylformamide (DMF) were added in thionyl chloride (SOCl2, 50 mL) and stirred at
85 ◦C for 18 h. The reaction was monitored by TLC. After completion of the reaction, the solvent was
removed under vacuum to obtain a yellow liquid, which was dissolved in DCM for further use.
3.1.2. Preparation of Ethyl 4-Chloropicolinate (8)
Dichloromethane (30 mL), ethanol (16.43 g, 0.170 mol), and triethylamine (17.17 g, 0.170 mol)
◦
were successively added to a beaker and stirred at 0 C for 0.5 h. Subsequently, a mixture of

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4-chloropicolinoyl chloride
7
(16.43 g, 0.110 mol) and DCM (5 mL) was added dropwise, stirred at 0 ◦C
for 0.5 h, and monitored by TLC. After completion of the reaction, NaOH solution was added to the
mixture to adjust the pH value 9–10. Ultimately, the organic layers were combined and dried to obtain
a brown liquid.
3.1.3. Preparation of Ethyl 4-(4-Nitrophenoxy)picolinate (9a) and Ethyl4-(2-Fluoro-4-Nitrophenoxy)
picolinate (9b)
Intermediate 8 (13.22 g, 0.071 mol) and p-nitrophenol or 2-fluoro-4-nitrophenol (0.026 mol) were
dissolved in chlorobenzene (40 mL) and then stirred at 130 ◦C for 4 h. The completion of the reaction
was monitored by TLC. After completion of the reaction, the cold petroleum ether (100 mL) was poured
into the reaction solvent and stirred at room temperature for 0.5 h. Next, petroleum ether (100 mL) was
poured off to give a viscous liquid, which was then dissolved in DCM (300 mL) and extracted with
aqueous solution of NaOH. The organic layer was combined, dried, and concentrated under vacuum
to get a pale yellow solid.
3.1.4. Preparation of 4-(4-Nitrophenoxy)picolinic Acid (10a) and 4-(2-Fluoro-4-Nitrophenoxy)picolinic
Acid (10b)
Intermediates 9a–9b (0.035 mol) were dissolved in 1,4-dioxane (50 mL) and stirred at room
temperature for 0.5 h. A solution of 10% NaOH (2.28 mL, 0.057 mol) was then added dropwise to the
above mixture, stirred at room temperature for 0.5 h, and monitored by TLC. After completion of the
reaction, the solvent was evaporated under vacuum to give a white solid. The white solid was then
dissolved in saturated NaCl solution (500 mL) and stirred at room temperature for 10 h. Subsequently,
after the pH of the solution was adjusted to 2–3 by 75% hydrochloric acid (HCl), a yellow solid was
precipitated, filtered, and dried.
3.1.5. Preparation of 4-(4-Nitrophenoxy)picolinamide or 4-(2-Fluoro-4-Nitrophenoxy)picolinamide
Analogues (11a–11e)
Intermediates 10a–10b (0.020 mol) and one drop of DMF were successively dissolved in SOCl2
(50 mL) and stirred at 85 ◦C for 0.5 h. Then reaction solvent was concentrated in vacuum, dissolved
in DCM (8 mL), and added dropwise to a mixture of DCM (50 mL), ammonia analogs (0.020 mol),
and triethylamine (0.020 mol). The reaction solution was stirred at 0 ◦C for 0.5 h, and monitored by
TLC. Finally, a gray slid was obtained by the same post-treatment method as that used for preparation
of ethyl 4-chloropicolinate 8.
3.1.6. Preparation of 4-(4-Aminophenoxy)picolinamide or 4-(2-Fluoro-4-aminophenoxy)picolinamide
Analogues (12a–12e)
Intermediates 11a–11e (0.016 mol), activated carbon (1.92 g, ◦0.160 mol), and FeCl3
·
6H2O (0.43 g,
0.016 mol) were dissolved in ethanol (30 mL) and stirred at 90 C for 0.5 h. Then, 80% hydrazine
hydrate (8.00 g, 0.128 mol) was added dropwise and stirred for 3.5 h. The reaction was monitored by
TLC. After completion of the reaction, the solution was filtered, concentrated, and recrystallized to
give a green solid.
3.1.7. Preparation of 5-Methyl-1-Phenyl-1H-1,2,3-Triazole-4-Carbonyl Chloride or 1-Phenyl-5-
(trifluoromethyl)-1H-1,2,3-Triazole-4-Carbonyl Chloride Analogues (13i–13e)
The preparation of intermediates 13a–13i was carried out according to our previous research [15].
3.1.8. Preparation of Target Compounds B1–B27
Intermediates 12a–12e (0.002 mol) and sodium bicarbonate (4.20 g, 0.005 mol) were dissolved
in DCM and stirred at 0 ◦C for 0.5 h. The solution of intermediates 13a–13i (0.002 mol) and DCM
were added dropwise to the above mixture, and stirred at 0 ◦C for 0.5 h. The reaction was monitored

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by TLC. After completion of the reaction, the solution was extracted with a mixture of DCM/NaOH
(500 mL) three times and then the organic layers were combined, dried over sodium sulfate, evaporated,
and purified by chromatographic column to possess a light yellow or white solid.
4-(4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide (B1) Light yellow
solid; Yield: 78.5%; m.p.: 116.8–117.1 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.75 (s, 1H), 8.82 (s,
1H), 8.52 (d, J = 5.5 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.67 (s, 2H), 7.64 (d, J = 6.4 Hz, 2H), 7.43 (s, 1H),
7.23 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 4.5 Hz, 1H), 3.22 (dd, J = 12.8, 6.3 Hz, 2H), 2.59 (s, 3H), 1.50 (dt,
J = 14.1, 7.0 Hz, 2H), 0.84 (t, J = 7.2 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C25H24N6O3:
457.1988, found, 457.1986.
4-(4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide (B2) Light
yellow solid; Yield: 59.3%; m.p.: 110.4–110.8 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.64 (s, 1H),
8.71 (s, 1H), 8.40 (d, J = 5.6 Hz, 1H), 7.91 (s, 1H), 7.89 (s, 1H), 7.65 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 4.6 Hz,
1H), 7.39 (t, J = 8.7 Hz, 2H), 7.31 (d, J = 2.1 Hz, 1H), 7.11 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 3.1 Hz, 1H), 3.10
(dd, J = 13.3, 6.6 Hz, 2H), 2.46 (s, 3H), 1.38 (dt, J = 14.2, 7.1 Hz, 2H), 0.72 (t, J = 7.3 Hz, 3H). TOF MS ES+
(m/z): [M + H]+, calcd for C25H23FN6O3: 475.1894, found, 475.1896.
4-(4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B3) White solid; Yield: 78.2%; m.p.: 97.7–98.0 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.81 (s,
1H), 8.81 (s, 1H), 8.52 (d, J = 5.3 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.84 (d,
J = 7.6 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 7.2 Hz, 1H), 7.42 (s, 1H), 7.23 (d, J = 8.6 Hz, 2H), 7.18
(d, J = 0.8 Hz, 1H), 3.22 (d, J = 6.2 Hz, 2H), 2.46 (s, 3H), 1.51 (dd, J = 14.0, 7.0 Hz, 2H), 0.84 (t, J = 7.2 Hz,
3H). TOF MS ES+ (m/z): [M + H]+, calcd for C26H23F3N6O4: 541.1811, found, 541.1813.
4-(4-(1-(4-chlorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B4) White solid; Yield: 66.4%; m.p.: 159.0–159.2 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 11.19
(s, 1H), 8.80 (s, 1H), 8.52 (d, J = 5.5 Hz, 1H), 7.99 (s, 1H), 7.97 (d, J = 1.2 Hz, 1H), 7.79 (d, J = 4.4 Hz,
3H), 7.75 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.27 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 5.5 Hz, 1H), 3.23 (dd,
J = 13.2, 6.7 Hz, 2H), 1.57–1.47 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ
165.73, 163.13, 156.58, 152.54, 150.36(2,C), 149.46, 142.31, 136.19, 135.84, 134.09, 129.70(2,C), 128.22(2,C),
122.40(2,C), 121.33(2,C), 114.18(2,C), 108.95, 40.58, 22.34, 11.27. TOF MS ES+ (m/z): [M + H]+, calcd for
C25H20ClF3N6O3: 545.1316, found, 545.1317.
N-propyl-4-(4-(5-(trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)
picolinamide (B5) White yellow solid; Yield: 69.4%; m.p.: 163.4–163.7 ◦C; 1H-NMR (400 MHz, DMSO-d6,
ppm)
(d, J = 7.7 Hz, 3H), 7.43 (s, 1H), 7.27 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 2.7 Hz, 1H), 3.23 (dd, J = 12.4,
6.0 Hz, 2H), 1.57–1.47 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
165.72,
δ 11.26 (s, 1H), 8.80 (s, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.15–8.09 (m, 2H), 8.05 (d, J = 7.2 Hz, 1H), 8.00
δ
163.14, 156.20, 152.53, 150.36(2,C), 149.53, 141.89, 135.72, 134.28(2,C), 132.86, 132.02, 130.02(2,C), 127.71,
122.60(2,C), 121.29(2,C), 114.17(2,C), 108.96, 40.57, 22.33, 11.24. TOF MS ES+ (m/z): [M + H]+, calcd for
C26H20F6N6O3: 579.1580, found, 579.1573.
4-(4-(1-(3,4-difluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B6) Light yellow solid; Yield: 85.0%; m.p.: 97.6–97.8 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 11.21
(s, 1H), 8.80 (s, 1H), 8.52 (d, J = 4.9 Hz, 1H), 8.12 (s, 1H), 7.98 (d, J = 6.9 Hz, 2H), 7.81 (d, J = 8.9 Hz,
1H), 7.74 (s, 1H), 7.41 (s, 1H), 7.27 (d, J = 6.9 Hz, 2H), 7.20 (d, J = 2.3 Hz, 1H), 3.26–3.19 (m, 2H), 1.52
(dd, J = 13.7, 6.8 Hz, 2H), 0.84 (t, J = 7.1 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ 165.73,
163.13, 156.48, 152.54, 150.36(2,C), 149.48, 142.09, 135.81, 131.59, 124.44, 122.46(2,C), 121.32(2,C), 118.63,
118.44, 117.06, 116.86, 114.19(2,C), 108.93, 40.58, 22.34, 11.26. TOF MS ES+ (m/z): [M + H]+, calcd for
C25H19F5N6O3: 547.1517, found, 547.1517.
4-(4-(1-(3-chloro-4-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B7) White solid; Yield: 81.6%; m.p.: 153.6–153.9 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 11.23 (s,

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1H), 8.80 (t, J = 5.9 Hz, 1H), 8.52 (d, J = 5.6 Hz, 1H), 7.99 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 4.0 Hz, 3H),
7.42 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 3.8 Hz, 1H), 3.23 (dd, J = 13.3, 6.5 Hz, 2H), 1.57–1.47 (m,
2H), 0.84 (t, J = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ 165.73, 163.15, 156.61, 152.52,
150.33(2,C), 149.45, 142.38, 136.20, 135.87, 134.06, 129.69(2,C), 128.18(2,C), 122.39(2,C), 121.29(2,C),
114.13(2,C), 108.99, 40.58, 22.34, 11.25. TOF MS ES+ (m/z): [M + H]+, calcd for C25H19ClF4N6O3:
563.1222, found, 563.1216.
4-(4-(1-(2-chloro-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-
propylpicolinamide (B8) White solid; Yield: 56.7%; m.p.: 143.3–143.7 ◦C; 1H-NMR (400 MHz, DMSO-d6,
ppm)
δ 11.30 (s, 1H), 8.81 (s, 1H), 8.65 (s, 1H), 8.53 (d, J = 4.9 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.15
(d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.42 (s, 1H), 7.27 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 2.1 Hz,
1H), 3.22 (dd, J = 12.2, 6.0 Hz, 2H), 1.52 (dq, J = 13.9, 7.1 Hz, 2H), 0.84 (t, J = 7.1 Hz, 3H). 13C-NMR
(100 MHz, DMSO-d6, ppm) δ 165.72, 163.14, 156.13, 152.54, 150.37(2,C), 149.56, 141.97, 135.71, 135.05,
133.58, 131.78(2,C), 131.49, 130.44, 127.03, 122.63(2,C), 121.28(2,C), 114.18(2,C), 108.96, 40.58, 22.33,
11.25. TOF MS ES+ (m/z): [M + H]+, calcd for C26H19ClF6N6O3: 613.1190, found, 613.1189.
4-(4-(1-(4-chloro-3-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-
propylpicolinamide (B9) Light yellow solid; Yield: 66.3%; m.p.: 155.5–155.8 ◦C; 1H-NMR (400 MHz,
DMSO-d6, ppm) δ 11.21 (s, 1H), 8.81 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 8.44 (s, 1H), 8.17 (d, J = 8.1 Hz, 1H),
8.10 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 8.7 Hz, 2H), 7.41 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 2.9 Hz,
1H), 3.22 (dd, J = 12.8, 6.2 Hz, 2H), 1.52 (dq, J = 14.8, 7.4 Hz, 2H), 0.84 (t, J = 7.3 Hz, 3H). TOF MS ES+
(m/z): [M + H]+, calcd for C26H19ClF6N6O3: 613.1190, found, 613.1188.
4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide (B10) White
solid; Yield: 76.8%; m.p.: 143.3–143.7 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.96 (s, 1H), 8.83 (s,
1H), 8.54 (d, J = 5.4 Hz, 1H), 8.11 (d, J = 13.2 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 2.6 Hz, 5H),
7.48–7.39 (m, 2H), 7.23 (d, J = 4.7 Hz, 1H), 3.23 (dd, J = 12.9, 6.3 Hz, 2H), 2.59 (s, 3H), 1.57–1.47 (m, 2H),
0.84 (t, J = 7.3 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ 165.66, 163.43, 160.15, 153.09, 150.87(2,C),
138.46, 138.29, 135.61, 130.48, 130.09(3,C), 125.84(3,C), 124.07, 117.64, 113.82, 109.37, 108.46, 41.02, 22.73,
11.67, 9.86. TOF MS ES+ (m/z): [M + H]+, calcd for C25H23FN6O3: 475.1894, found, 475.1894.
4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B11) White solid; Yield: 65.1%; m.p.: 143.5–143.8 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.95 (s,
1H), 8.82 (s, 1H), 8.54 (d, J = 5.1 Hz, 1H), 8.10 (d, J = 12.7 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.76 (s, 2H),
7.51 (t, J = 8.4 Hz, 2H), 7.44 (d, J = 12.9 Hz, 2H), 7.22 (d, J = 2.5 Hz, 1H), 3.22 (d, J = 6.0 Hz, 2H), 2.58 (s,
3H), 1.51 (dd, J = 13.9, 6.9 Hz, 2H), 0.84 (t, J = 7.2 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6, ppm)
δ
165.65, 164.20, 163.42, 161.74, 160.11, 153.08, 150.88, 138.75, 138.22, 131.99, 128.44, 128.35, 124.08, 117.65,
117.18(2,C), 116.95, 113.83, 109.60, 109.38, 108.44, 41.01, 22.73, 11.67, 9.78. TOF MS ES+ (m/z): [M + H]+,
calcd for C25H22F2N6O3: 493.1800, found, 493.1797.
4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-propylpicolinamide
(
B12) Light yellow solid; Yield: 63.2%; m.p.: 132.6–132.9 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ
10.99 (s, 1H), 8.82 (s, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.10 (d, J = 13.2 Hz, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.85
(d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.41 (d,
J = 2.5 Hz, 1H), 7.23 (dd, J = 5.5, 2.6 Hz, 1H), 3.26–3.20 (m, 2H), 2.46 (s, 3H), 1.58–1.47 (m, 2H), 0.85 (t,
J = 7.4 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C26H22F4N6O4: 559.1717, found, 559.1717.
4-(4-(1-(4-chlorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-N-propylpicolinamide
(
B13) Light yellow solid; Yield: 76.3%; m.p.: 176.5–176.8 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ
11.37 (s, 1H), 8.81 (s, 1H), 8.55 (d, J = 5.3 Hz, 1H), 8.06 (d, J = 12.8 Hz, 1H), 7.79 (d, J = 7.4 Hz, 5H),
7.48 (d, J = 8.9 Hz, 1H), 7.44 (s, 1H), 7.27–7.20 (m, 1H), 3.23 (d, J = 6.1 Hz, 2H), 1.52 (dd, J = 14.1,
7.1 Hz, 2H), 0.84 (t, J = 7.3 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C25H19ClF4N6O3: 563.1222,
found, 563.1216.

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4-(2-fluoro-4-(5-(trifluoromethyl)-1-(2-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-
propylpicolinamide (B14) Light yellow solid; Yield: 79.6%; m.p.: 172.8–173.1 ◦C; 1H-NMR (400 MHz,
DMSO-d6, ppm) δ 11.43 (s, 1H), 8.82 (s, 1H), 8.55 (d, J = 5.3 Hz, 1H), 8.12 (s, 1H), 8.09 (d, J = 4.7 Hz, 1H),
8.05 (s, 1H), 8.00 (d, J = 9.1 Hz, 2H), 7.84 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H), 7.44 (s, 1H), 7.24 (d,
J = 2.5 Hz, 1H), 3.23 (d, J = 5.6 Hz, 2H), 1.55–1.49 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). 13C-NMR (100 MHz,
DMSO-d6, ppm)
δ 165.15, 163.01, 156.39, 152.70, 150.48(2,C), 141.52, 134.25(2,C), 134.18, 132.86, 132.00,
129.99(2,C), 127.65, 123.86(2,C), 117.59, 113.43(2,C), 109.61, 109.39, 108.12, 40.60, 22.31, 11.21. TOF MS
ES+ (m/z): [M + H]+, calcd for C26H22F4N6O3: 543.1614, found, 543.1616.
4-(4-(1-(3,4-difluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-N-propylpicolinamide
(
B15) White solid; Yield: 55.4%; m.p.: 134.4–134.8 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 11.40
(s, 1H), 8.83 (s, 1H), 8.55 (d, J = 5.4 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 13.0 Hz, 1H), 7.82
(d, J = 7.0 Hz, 2H), 7.74 (s, 1H), 7.48 (t, J = 8.8 Hz, 1H), 7.42 (s, 1H), 7.25 (d, J = 2.0 Hz, 1H), 3.22 (d,
J = 6.2 Hz, 2H), 1.52 (d, J = 7.1 Hz, 2H), 0.84 (t, J = 7.1 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for
C26H19F7N6O3: 597.1485, found, 597.1486.
4-(4-(1-(3-chloro-4-fluorophenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-fluorophenoxy)-N-
propylpicolinamide (B16) White solid; Yield: 59.7%; m.p.: 163.3–163.5 ◦C; 1H-NMR (400 MHz, DMSO-d6,
ppm)
δ 11.39 (s, 1H), 8.82 (t, J = 5.8 Hz, 1H), 8.56 (d, J = 5.6 Hz, 1H), 8.23 (d, J = 4.8 Hz, 1H), 8.06
(d, J = 12.9 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.77 (t, J = 8.9 Hz, 1H), 7.48 (t,
J = 9.0 Hz, 1H), 7.42 (s, 1H), 7.25 (d, J = 5.4 Hz, 1H), 3.23 (dd, J = 13.3, 6.5 Hz, 2H), 1.53 (dt, J = 14.5,
7.4 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C25H18ClF5N6O3: 581.1127,
found, 581.1128.
4-(4-(1-(2-chloro-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-
fluorophenoxy)-N-propylpicolinamide (B17) White solid; Yield: 64.5%; m.p.: 154.1–154.4 ◦C; 1H-NMR
(400 MHz, DMSO-d6, ppm) δ 11.48 (s, 1H), 8.83 (s, 1H), 8.65 (s, 1H), 8.55 (d, J = 5.0 Hz, 1H), 8.21 (d,
J = 7.8 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.07 (d, J = 12.9 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.49 (t,
J = 8.8 Hz, 1H), 7.43 (s, 1H), 7.25 (d, J = 1.4 Hz, 1H), 3.23 (d, J = 6.0 Hz, 2H), 1.52 (dd, J = 13.9, 6.9 Hz,
2H), 0.84 (t, J = 7.3 Hz, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C26H18ClF7N6O3: 631.1089,
found, 631.1089.
4-(4-(1-(4-chloro-3-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamido)-2-
fluorophenoxy)-N-propylpicolinamide (B18) White solid; Yield: 55.9%; m.p.: 153.4–153.7 ◦C; 1H-NMR
(400 MHz, DMSO-d6, ppm) δ 11.41 (s, 1H), 8.83 (s, 1H), 8.56 (d, J = 5.3 Hz, 1H), 8.45 (s, 1H), 8.18 (d,
J = 8.4 Hz, 1H), 8.12–8.01 (m, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 8.9 Hz, 1H), 7.42 (s, 1H), 7.25
(s, 1H), 3.26–3.19 (m, 2H), 1.52 (dq, J = 14.4, 7.0 Hz, 2H), 0.84 (t, J = 7.2 Hz, 3H). TOF MS ES+ (m/z):
[M + H]+, calcd for C26H18ClF7N6O3: 631.1089, found, 631.1096.
N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy)phenyl)-5-methyl-1-phenyl-1H-1,2,3-triazole-4-
carboxamide (B19) Light yellow solid; Yield: 63.2%; m.p.: 169.7–170.0 ◦C; 1H-NMR (400 MHz, DMSO-d6,
ppm) δ 10.94 (s, 1H), 8.50 (d, J = 4.4 Hz, 1H), 8.09 (d, J = 12.9 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.65 (d,
J = 14.4 Hz, 5H), 7.42 (t, J = 8.6 Hz, 1H), 7.13 (d, J = 14.2 Hz, 2H), 3.59 (s, 2H), 3.45 (s, 2H), 2.59 (s, 3H),
1.82 (s, 4H). TOF MS ES+ (m/z): [M + H]+, calcd for C26H23FN6O3: 487.1897, found, 487.1902.
N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-5-methyl-1H-1,2,3-
triazole-4-carboxamide (B20) Light yellow solid; Yield: 66.4%; m.p.: 164.3–164.7 ◦C; 1H-NMR (400 MHz,
DMSO-d6, ppm) δ 10.94 (s, 1H), 8.51 (d, J = 5.3 Hz, 1H), 8.09 (d, J = 13.2 Hz, 1H), 7.84 (d, J = 8.4 Hz,
1H), 7.75 (d, J = 3.2 Hz, 2H), 7.51 (t, J = 8.4 Hz, 2H), 7.42 (t, J = 8.9 Hz, 1H), 7.18 (s, 1H), 7.12 (d,
J = 4.6 Hz, 1H), 3.59 (s, 2H), 3.45 (s, 2H), 2.57 (s, 3H), 1.81 (s, 4H). TOF MS ES+ (m/z): [M + H]+, calcd
for C26H22F2N6O3: 505.1800, found, 505.1807.
N-(3-fluoro-4-((2-(pyrrolidine-1-carbonyl)pyridin-4-yl)oxy) phenyl)-5-methyl-1-(2-(trifluoromethoxy)phenyl)-
1H-1,2,3-triazole-4-carboxamide (B21) White solid; Yield: 66.5%; m.p.: 176.3–176.6 ◦C; 1H-NMR (400 MHz,

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DMSO-d6, ppm)
δ
11.00 (s, 1H), 8.51 (d, J = 4.9 Hz, 1H), 8.09 (d, J = 12.8 Hz, 1H), 7.89 (d, J = 7.3 Hz,
1H), 7.85 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.75–7.70 (m, 1H), 7.43 (t, J = 8.6 Hz, 1H), 7.16
(s, 1H), 7.11 (d, J = 1.9 Hz, 1H), 3.60 (s, 2H), 3.46 (s, 2H), 2.46 (s, 3H), 1.82 (s, 4H). TOF MS ES+ (m/z):
[M + H]+, calcd for C27H22F4N6O4: 571.1717, found, 571.1722.
4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-2-yl)ethyl)picolinamide
(
B22) Light yellow solid; Yield: 61.9%; m.p.: 124.6–125.0 ◦C; 1H NMR (400 MHz, DMSO-d6, ppm)
δ
10.97 (s, 1H), 8.98 (t, J = 5.9 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.11 (d, J = 13.2 Hz, 1H), 7.86 (d, J = 8.8 Hz,
1H), 7.67 (d, J = 2.3 Hz, 5H), 7.48–7.41 (m, 2H), 7.32 (d, J = 4.9 Hz, 1H), 7.24 (d, J = 3.0 Hz, 1H), 6.96-6.91
(m, 1H), 6.89 (d, J = 2.6 Hz, 1H), 3.54 (dd, J = 13.4, 6.8 Hz, 2H), 3.06 (t, J = 7.1 Hz, 2H), 2.59 (s, 3H). TOF
MS ES+ (m/z): [M + H]+, calcd for C28H23FN6O3S: 543.1614, found, 543.1616.
4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-2-yl)
ethyl)picolinamide (B23) White solid; Yield: 51.7%; m.p.: 110.4–110.8 ◦C; 1H-NMR (400 MHz, DMSO-d6,
ppm)
δ 10.99 (s, 1H), 9.00 (t, J = 5.7 Hz, 1H), 8.57 (d, J = 5.6 Hz, 1H), 8.13 (d, J = 13.2 Hz, 1H), 7.88
(d, J = 8.8 Hz, 1H), 7.79 (d, J = 4.8 Hz, 1H), 7.77 (d, J = 4.8 Hz, 1H), 7.54 (t, J = 8.7 Hz, 2H), 7.47 (d,
J = 9.1 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 4.6 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 6.98–6.94 (m,
1H), 6.91 (s, 1H), 3.55 (dd, J = 13.3, 6.8 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.60 (s, 3H). TOF MS ES+ (m/z):
[M + H]+, calcd for C28H22F2N6O3S: 561.1520, found, 561.1529.
4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(2-(thiophen-
◦
1
2-yl)ethyl)picolinamide (B24) White solid; Yield: 56.6%; m.p.: 103.4–103.7 C; H-NMR (400 MHz,
DMSO-d6, ppm) 10.98 (s, 1H), 8.94 (s, 1H), 8.55 (d, J = 5.3 Hz, 1H), 8.10 (d, J = 13.2 Hz, 1H), 7.88 (d,
δ
J = 6.4 Hz, 2H), 7.84 (d, J = 7.0 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 8.8 Hz,
2H), 7.31 (d, J = 4.1 Hz, 1H), 7.23 (d, J = 2.3 Hz, 1H), 6.96–6.92 (m, 1H), 6.90 (s, 1H), 3.55 (d, J = 6.2 Hz,
2H), 3.07 (t, J = 6.9 Hz, 2H), 2.47 (s, 3H). TOF MS ES+ (m/z): [M + H]+, calcd for C29H22F4N6O4S:
627.1437, found, 627.1439.
4-(2-fluoro-4-(5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3-morpholinopropyl)picolinamide
(
B25) White solid; Yield: 42.6%; m.p.: 108.3–108.6 ◦C; 1H-NMR (400 MHz, DMSO-d6, ppm)
δ 10.67
(s, 1H), 9.05 (s, 1H), 8.53 (s, 1H), 8.03 (d, J = 7.5 Hz, 2H), 7.67 (s, 5H), 7.45 (s, 1H), 7.23 (d, J = 7.6 Hz,
1H), 7.17 (s, 1H), 3.64 (s, 4H), 3.36 (s, 2H), 2.60 (s, 3H), 2.44 (s, 6H), 1.72 (s, 2H). 13C-NMR (100 MHz,
DMSO-d6, ppm) δ 166.25, 163.53, 159.92, 152.85, 150.72, 149.29, 138.56, 138.11, 136.82, 135.67, 130.44,
130.10(2,C), 125.83(2,C), 122.70(2,C), 121.51(2,C), 114.53, 109.30, 66.25(2,C), 56.81, 53.53(2,C), 38.36,
25.59, 9.85. TOF MS ES+ (m/z): [M + H]+, calcd for C29H30FN7O4: 560.2421, found, 560.2416.
4-(2-fluoro-4-(1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3-morpholinopropyl)
◦
1
picolinamide (B26) White solid; Yield: 39.3%; m.p.: 108.5–108.9 C; H-NMR (400 MHz, DMSO-d6,
ppm) 10.73 (s, 1H), 9.10 (s, 1H), 8.52 (d, J = 3.7 Hz, 1H), 8.03 (d, J = 7.9 Hz, 2H), 7.76 (s, 2H), 7.52
(d, J = 6.9 Hz, 2H), 7.43 (s, 1H), 7.23 (d, J = 7.9 Hz, 2H), 3.61 (s, 4H), 3.35 (s, 2H), 2.59 (s, 3H), 2.34 (s,
6H), 1.68 (s, 2H). 13C-NMR (100 MHz, DMSO-d6, ppm)
166.25, 163.48, 159.87, 152.90, 150.69, 149.33,
δ
δ
138.38, 136.78, 132.06, 128.39, 128.30, 122.71(2,C), 121.46(2,C), 117.16(2,C), 116.93(2,C), 114.50, 109.32,
66.54(2,C), 57.08, 53.77(2,C), 38.55, 25.80, 9.74. TOF MS ES+ (m/z): [M + H]+, calcd for C29H29F2N7O4:
578.2327, found, 578.2347.
4-(2-fluoro-4-(5-methyl-1-(2-(trifluoromethoxy)phenyl)-1H-1,2,3-triazole-4-carboxamido)phenoxy)-N-(3-
morpholinopropyl)picolinamide (B27) White solid; Yield: 41.0%; m.p.: 97.6–97.9 ◦C; 1H-NMR (400 MHz,
DMSO-d6, ppm) δ 10.78 (s, 1H), 9.05 (s, 1H), 8.54 (s, 1H), 7.99 (d, J = 5.2 Hz, 2H), 7.86 (d, J = 3.8 Hz,
2H), 7.79 (s, 1H), 7.73 (s, 1H), 7.41 (s, 1H), 7.23 (d, J = 6.1 Hz, 2H), 3.91 (d, J = 8.3 Hz, 2H), 3.85 (d,
J = 11.1 Hz, 2H), 3.36 (d, J = 5.0 Hz, 2H), 3.06 (s, 4H), 2.45 (s, 2H), 1.98 (s, 2H). TOF MS ES+ (m/z):
[M + H]+, calcd for C30H29F4N7O5: 643.1059, found, 643.1094.
1H-NMR spectra of representative target compounds (B1
13C-NMR spectra of representative target compounds (B6
B7
,
B6
,
B12
,
,
B13
,
B20
,
B25, and B26),
B25, and B26),
,
,
B10
B11
,
B14
,

Molecules 2020, 25, 10
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and TOF-MS spectra of representative target compounds (B13, B17, B19, B21, B22, B25, and B26) can
be seen in the Supplementary Materials.
3.2. Antitumority Assay
The antitumor activities of target compounds were determined by the MTT method using
Golvatinib as a positive control. All cancer cell lines (A549, HeLa, and MCF-7) were cultured with
Dulbecco Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (1640) containing
10% fetal bovine serum and 0.1% penicillin–streptomycin, under ambient conditions of 5% CO2 and
37 ◦C. Cells were digested with an appropriate amount of Trypsin–EDTA solution to obtain the cell
suspension, which was diluted with medium and inoculated into 96-well plates at 5*104 cells per well.
After cells were incubated for 24 h, the target compounds diluted by medium to suitable concentrations
were added into 96-well plates, and the cells were cultured continue for 72 h. Next, the medium
was removed, and then thiazolyl blue tetrazolium bromide (MTT) was added to each well to treat
cells for 3.5 h. Ultimately, dimethyl sulfoxide (DMSO) was added to each well after removal of MTT
and the absorbance values were measured with the ELISA (enzyme-linked immunosorbent assay)
reader. All antitumor activities were tested for three times. The IC50 values were the average of
three measurements and were calculated using the Bacus Laboratories Incorporated Slide Scanner
(Bliss) software.
3.3. Dose-Dependent Test
The dose-dependent effect of compound B26 on A549 cells was tested by the MTT method using
Golvatinib as a positive control. The experimental procedure was identical to that of the cytotoxic
activity, wherein the concentration of the test compound was configured to be seven and the cytotoxic
activity was five. Seven different concentrations of compound B26 treated on A549 cells to obtain
the corresponding inhibition rates. Experimental data was obtained by Orange (2018 64 bit) software
based on the inhibition rate.
3.4. Cell Morphology Studies
Cell morphology studies aimed to explore the morphological changes of A549 cells with and
without treatment with compound B26, and the cell morphology was visualized by acridine orange
(AO) staining. The culture environment of A549 cells was consistent with that in the antitumor activity
experiments. Cells were digested with Trypsin–EDTA solution to get the cell suspension, which was
diluted with 1640 medium and inoculated into a 24-well plate at 2*104 cells per well. After incubating
for 12 h, compound B26 diluted to suitable concentrations by 1640 medium was added into a 24-well
plate, and cell culture was continued for 12 h. Then, the 1640 medium in the well plate was removed
and every well were washed three times with phosphate buffer saline (PBS). After washing with PBS
three times, A549 cells were treated with AO for 15 min. A549 cells were washed three times with
PBS again, and the cell morphology distribution was observed directly by a fluorescence microscope.
Ultimately, the picture is exported via computer.
3.5. Cell Cycle Study
A549 cells were seeded into six-well plates at 1*105 cells per well and incubated for 24 h.
Then, the medium containing DMEM (control, without compound) or compound B26 with different
concentrations was added to the culture plate, and A549 cells were further cultured for 24 h. Then,
A549 cells were collected into a centrifuge tube and fixed with a small amount of ice-cold 70% ethanol
at 4 ◦C for 6–12 h. Cells washed three times with PBS were incubated with Rnase (1 mg/mL, diluted
with PBS) for 30 min at room temperature. Finally, propidium iodide (PI) was added to staining
without light for 30 min at room temperature, and the DNA content was measured by flow cytometry
within 1 h. Experimental data was obtained by Modify software.

Molecules 2020, 25, 10
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3.6. c-Met Kinase Assay
The kinase assay was implemented through Mobility shift assay. The Mixture the configured
available Brij-35 (0.0015%) and 50 mM HEPES (pH = 7.5) to get a kinase buffer base. Then, compounds
B25, B26, and B27 were configured to five concentrations using dimethyl sulfoxide. The kinase buffer
containing the compounds or dimethyl sulfoxide was added to 96-well plates and mixed on the shaker
for fifteen minutes. Next, the solution was transferred in duplicate from a 96-well plate to a 384-well
plate, and then an enzyme solution (c-Met kinase mixed with kinase buffer) was added to each well.
After incubation of the 384-well plate for 10 min at room temperature, a 2.5*peptide solution (formed
by the addition of FAM-labeled peptide and ATP to the kinase base buffer) was added to each well.
After incubation at 28 ◦C for a certain period of time, a stop buffer was added to each well for stop the
reaction. The inhibition value is obtained by converting the conversion data on the caliper. The formula
is that percent inhibition = (max
DMSO control, “min” stands for low control. Finally, the IC50 values were obtained by fitting the
inhibition rate data using the XLFit excel add-in version software.
−
conversion) / (max
−
min) * 100. Among them, “max” stands for
3.7. Molecular Docking Study
All the molecular docking simulations were performed by the AutoDock 4.2 software. The crystal
structure of c-Met (PDB code: 3LQ8) used in the docking was downloaded from http://www.rcsb.org/.
The preparation process of the protein for docking mainly involves the addition of hydrogen atoms
and charges, elimination of unrelated water molecules, immobilization of exact residues, and removal
of endogenous ligands (Foretinib), etc. The preparation process of the molecules for docking mainly
includes the addition of hydrogen atoms and charges. Then the prepared molecules (target compounds)
were docked to the certified binding site of c-Met protein. Subsequently, the genetic algorithm was
used for energy optimization. All the docking results were modified and processed by PyMOL 1.8.x
software (https://pymol.org).
4. Conclusions
In conclusion, a series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a triazole fragment
were designed and synthesized. In addition, we evaluated them for antitumor activity against three
cancer cell lines and c-Met kinase activity (only for compounds B25–B27) in vitro. The pharmacological
results indicated that most compounds showed moderate antitumor activity against the three cancer
cell lines. In particular, compound B26 showed excellent inhibitory activity with IC50 values of
3.22, 4.33, and 5.82 µM against A549, HeLa, and MCF-7 cell lines, which were more potent than
Golvatinib, respectively. The SARs study indicated that the introduction of a morpholino group in the
hydrophilic region was more favorable than an alkane chain in terms of antitumor activity, and a single
electron-withdrawing substituent (such as a fluorine atom) on the terminal phenyl ring improved the
inhibitory activity of the target compounds. Further studies will be carried out in the near future.
Supplementary Materials: The Supplementary Materials are available online at http://www.mdpi.com/1420-3049/
25/1/10/s1.
Author Contributions: P.Z., Q.T. and J.C. conceived and designed the experiments; H.X., J.Z., Q.Z., Z.X. and H.Z.
performed the experiments and statistics analysis; J.C. and H.X. completed the molecular docking simulation
and H.X. started the project and wrote the paper. All authors have read and agreed to the published version of
the manuscript.
Funding: We gratefully acknowledge the generous support provided by Natural Science Foundation of National
Natural Science Foundation of China (NSFC No. 81660572), Jiangxi Province (20171BAB215071, 20192ACBL21009)
and Top-notch talent project of Jiangxi Science & Technology Normal University (2016QNBJRC002).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 10
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Sample Availability: Samples of all target compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).