Design, synthesis and biological evaluation of 4-(pyridin-4-yloxy)benzamide derivatives bearing a 5-methylpyridazin-3(2H)-one fragment

Article abstract of DOI:10.1016/j.bmcl.2020.127076

A series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a 5-methylpyridazin-3(2H)-one fragment were designed, synthesized, and evaluated for their biological activity. Most compounds showed effective inhibitory activity against cancer cell lines of A

Full text of DOI:10.1016/j.bmcl.2020.127076

Journal Pre-proofs
Design, synthesis and biological evaluation of 4-(pyridin-4-yloxy)benzamide
derivatives bearing a 5-methylpyridazin-3(2H)-one fragment
Hehua Xiong, Jianqing Zhang, Qian Zhang, Yongli Duan, Han Zhang,
Pengwu Zheng, Qidong Tang
PII:
S0960-894X(20)30154-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127076
BMCL 127076
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 January 2020
27 February 2020
1 March 2020
Please cite this article as: Xiong, H., Zhang, J., Zhang, Q., Duan, Y., Zhang, H., Zheng, P., Tang, Q., Design,
synthesis and biological evaluation of 4-(pyridin-4-yloxy)benzamide derivatives bearing 5-
a
methylpyridazin-3(2H)-one fragment, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/
10.1016/j.bmcl.2020.127076
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

O
B
previous
SAR
R
N
N
O
N
R
R
5-atom linker
D
A
C
Structural skeleton of c-Met inhibitor
Combination mode of Golvatinib with c-Met
O
OCF3
H
F
H
H
N
N
N
N
N
O
O
O
O
N
O
N
F
O
O
H
N
N
N
N
H
O
N
Compound 40
N
Lead compound, Golvatinib
2.60 to 6.95 times more active than Golvatinib in vitro
Design, synthesis and biological evaluation of 4-(pyridin-4-yloxy)benzamide
derivatives bearing a 5-methylpyridazin-3(2H)-one fragment
a
a
Hehua Xiong a, †, Jianqing Zhang a, †, Qian Zhang , Yongli Duan a, b, Han Zhang ,
Pengwu Zheng a, * Qidong Tang a, 
a
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of
Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, P.R.
China
b School of Optoelectronic Science and Engineering, University of Electronic Science
and Technology of China (UESTC), Chengdu 610054, P.R. China
Abstract: A series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a
5-methylpyridazin-3(2H)-one fragment were designed, synthesized, and evaluated for
their biological activity. Most compounds showed effective inhibitory activity against
cancer cell lines of A549, HeLa and MCF-7. Among them, the most promising
compound 40 showed excellent activity against A549, HeLa and MCF-7 cell lines
with IC50 values of 1.03, 1.15 and 2.59 μM, respectively, which was 2.60-6.95 times
more active than that of Golvatinib. The structure-activity relationships (SARs)
† These authors contribute equally to this work.
 Corresponding authors. Tel./fax: +86 791 83802393.
E-mail addresses: tangqidongcn@126.com (Q. Tang), zhengpw@126.com (P. Zheng).

showed that the introduction of 5-methylpyridazin-3(2H)-one to "5-atom linker" and
the modification of the amide with morpholine group were beneficial for enhancing
the inhibitory activity of compounds. In addition, the further research on compound
40 mainly include c-Met kinase activity, concentration dependence, apoptosis
(acridine orange staining), and molecular docking.
Key
words:
Synthesis;
4-(pyridin-4-yloxy)benzamide
derivatives;
5-methylpyridazin-3(2H)-one; Inhibitors; c-Met
Cancer has become the primary factor in killing people besides cardiovascular
diseases.1 Receptor tyrosine kinases are closely related to cell growth, reproduction
and metastasis. However, the aberrant activation of receptor tyrosine kinases usually
leads to the occurrence, invasion and metastasis of various cancer diseases.2-3 In
recent years, small molecule inhibitors have become a research hotspot because they
can block the transformation of signal pathways by targeting specific sites (including
receptor tyrosine kinases), and then induce the apoptosis and necrosis of cancer
cells.4-5
With the development of molecular pharmacology and molecular oncology, the
intracellular mechanism of cancer cells has been gradually clarified, which
accelerated the discovery of c-Met inhibitors.6 Notably, Cabozantinib (1, Fig. 1) and
Crizotinib (2, Fig. 1) belong to c-Met inhibitors of type II and type I, respectively,
which were approved by FDA in 2012 and 2011.7-8 The other four representative type
Ⅱ c-Met inhibitors in clinical trials are listed in Fig. 1, including Golvatinib (3),
TAS-115 (4), BMS-777607 (5) and Altratinib (6).9-12

F
H
H
N
NH2
O
N
N
H
H
N
N
O
O
Cl
F
HN
O
N
F
O
O
N
N
O
N
F
O
O
O
Cl
N
N
H
N
N
2, Grizotinib, approved in 2011
3, Golvatinib, Phase II
1, Cabozantinib, approved in 2012
H
H
H
N
H
O
H
F
N
N
F
N
F
N
N
S
O
O
O
O
O
O
O
F
F
O
O
O
F
N
O
H
Cl
N
N
N
H
H2N
N
6, Altratinib, Phase I
4, TAS-115, Phase II
5, BMS-777607, Phase I/II
Fig. 1. The representative small-molecule c-Met kinase inhibitors.
Type II c-Met inhibitors have the characteristics of low toxicity, strong efficacy
and little drug resistance. However, the SAR study on pyridylamide derivatives of
c-Met inhibitors13-15 is not enough to guide the development of drugs. In this study,
Golvatinib was used as a lead compound, and a series of modifications and
optimization were carried out on it to expand the SAR of type II pyridylamide c-Met
inhibitors. First of all, according to our previous research,16 the skeleton of type II
c-Met inhibitors was divided into four moieties of A, B, C, and D, on which our
design was based, as shown in Fig. 2. Subsequently, docking simulation of Golvatinib
with c-Met (PDB code: 3LQ8) was performed to guide our modification. According
to the docking results we found that moieties A and C have the potential to enhance
the binding affinity through forming strong hydrogen boding interactions with
residues Met1160, Asp1222 and Lys1110. Among them, moiety C has two obvious
structural characteristics: 5-atom regulation and hydrogen bond receptor/donor
regulation,16 which means that the structure of moiety C not only needs to maintain
the distance of six chemical bonds between moieties B and D, but also needs to
contain hydrogen bond acceptor/donor atoms to interact with c-Met. In addition, the
space around moieties A and C was large enough for further optimization. On the
contrary, only minor modifications were suitable for implementation in moieties B
and D.

In moiety A, the pyridine ring was retained, it was an important structure to
maintain the biological activity by forming hydrogen bond with residue Met1160. On
this basis, various flexible or hydrophilic amides, such as alkyl chains, morpholine
and thiophene groups, were introduced into moiety A to improve the solubility of the
target compounds. As reported, pyridazinone fragment possessing good biological
activity was widely used in the design of anticancer agents (Fig. 3).17-19 Therefore,
5-methylpyridazin-3(2H)-one fragment was embedded into moiety C to explore its
effect on biological activity, based on the "5-atom regulation" and "hydrogen bond
donor or acceptor" which had been explained clearly in our previous research.19
Considering the steric clash problem involved in binding process, we introduced
various small substituents, such as halogen atoms, CH3, CF3 and OCF3 to moieties B
and D in order to explore their effect on inhibitory activity. Ultimately, a series of
4-(pyridin-4-yloxy)benzamide derivatives containing a 5-methylpyridazin-3(2H)-one
fragment were designed.
B
O
previous
SAR
R
N
N
O
N
5-atom linker
C
R
R
D
A
Structural skeleton of c-Met inhibitor
Combination mode of Golvatinib with c-Met
O
H
R2
O
N
N
N
F
B
H
N
1
H
N
5
R3
4
2
O
3
O
O
O
F
H
N
5-atom linker
C
R1
N
O
D
Target compounds
O
N
N
N
H
N
S
O
A
R1
R2 = H, F
=
N
N
Lead compound, Golvatinib
R3 = H, 2-CH3 , 4-CH3, 3-F, 2-Cl, 3-CF3, 2-OCF3, 2-F-4-Br
Fig. 2. Design strategy for the target compounds using Golvatinib as a lead compound.

F
CN
NH CN
OCH3
OCHF2
N
N
N
O
N
O
O
O
CH3
S
HN
N
HN N
S
O
HN
N
F
O
N
7, A-241611
8, Levosimendan
9, Zardavenine
10, Motapizone
Fig. 3. Some drugs containing pyridazinone fragment.
The key intermediates 17a-17l were prepared according to the sequence outlined
in Scheme 1. The commercially available picolinic acid 11 was converted into the
corresponding acid chloride 12 in thionyl chloride (SOCl2) by an acylation reaction.
Substitution of compound 12 with ethanol yielded 13, which was then reacted with
p-nitrophenol or 2-fluoro-4-nitrophenol to get 14a-14b. Subsequently, 14a-14b were
hydrolyzed under the mixture of 1,4-dioxane/sodium hydroxide (NaOH, 10%) = 10:1
to produce carboxyl analogues 15a-15b, which were acylated and then nucleophilic
substituted with various amines to obtain intermediates 16a-16l. And then 16a-16l
were reduced via catalytic hydrogenation to generate the corresponding amines
17a-17l.
R2
R2
NO2
NO2
Cl
N
Cl
N
i
ii
iii
iv
O
N
O
N
Cl
O
N
COOH
O
O
O
HO
O
O
11
12
13
14a-14b
15a-15b
R2
R2
NH2
NO2
CH3
O
O
R2 = H, F
R 1
=
v
vi
H
H
N
O
S
N
R1
R1
N
N
N
O
O
16a-16l
17a-17l
Scheme 1. Reagents and conditions: (i) SOCl2, N,N-Dimethylformamide (DMF), NaBr, 85 oC, 20
h; (ii) EtOH, trimethylamine (Et3N), dichloromethane (DCM), 25 °C, 0.5 h; (iii) chlorobenzene
o
(PhCl), p-nitrophenol or 2-fluoro-4-nitrophenol, 130 C, 4.5 h; (iv) 1,4-dioxane/NaOH (10%) =
10:1, 25 °C, 0.5 h; (v) SOCl2, DMF, amines, Et3N, DCM, 25 °C, 1 h; (vi) EtOH, FeCl3·6H2O,
activated carbon, hydrazine hydrate (80%), 85 oC, 4 h.
The target compounds 23-45 were prepared according to the route of Scheme 2.
Analog anilines 18a-18h were diazotized with NaNO2 and then nucleophilic

substituted with ethyl 3-oxobutanoate to gain 19a-19h, which were reacted with ethyl
(triphenylphosphoranylidene)acetate (Ph3P=CHCOOC2H5) to give pyridazinones
20a-20h. Then 20a-20h were hydrolyzed with NaOH solution to get carboxyl
analogues 21a-21h. Ultimately, oxalyl chloride was reacted with 21a-21h in the
presence of DMF in DCM to yield the intermediates 22a-22h. Nucleophilic
substitution of the key intermediates 17a-17l and 22a-22h with
o
N,N-Diisopropylethylamine (DIPEA) under DCM at 0 C obtained the target
compounds 23-45.
O
O
O
O
NH2
O
O
HO
O
O
x
vii
viii
ix
N
N
N
HN
N
O
N
O
R3
O
R3
R3
R3
18a-18h
19a-19h
20a-20h
21a-21h
O
O
H
R2
R2 = H, F
N
N
N
CH3
R3
Cl
O
xi
N
O
N
R1
=
N
O
3
H, 2-CH3 ,4-CH3,
3-F, 2-Cl, 3-CF3,
2-OCF3, 2-F-4-Br
O
S
R =
H
R3
N
N
R1
O
22a-22h
Compounds 23-45
Scheme 2. Reagents and conditions: (vii) sodium nitrite (NaNO2), HCl, ethyl 3-oxobutanoate,
EtOH/H2O (2:1), 0 oC, 4 h; (viii) Ph3P=CHCOOC2H5, dioxane, 90 oC, 10 h; (ix)
o
1,4-dioxane/NaOH (10%) = 10:1, 25 °C, 0.5 h; (x) DCM, oxalyl chloride, 0 C, 0.5 h; (xi)
intermediates 17a-17l, DCM, DIPEA, 0 oC, 0.5 h.
The cytotoxic activities of all synthesized compounds against A549, HeLa and
MCF-7 cell lines were determined through MTT method.20 As shown in Table 1,
most of the compounds showed moderate to potent inhibitory activities on these three
cell lines, especially A549 and HeLa. It is worth noting that compounds 30, 36, 38, 39
and 40 showed stronger cytotoxic activities than Golvatinib. Among them, the most
promising compound 4021 showed excellent activity against A549, HeLa and MCF-7
cell lines with IC50 values of 1.03, 1.15 and 2.59 μM, respectively, which was
2.60-6.95 times more active than that of Golvatinib (IC50 values were 6.89, 4.14 and
20.61 μM, respectively).
According to the results of cytotoxic activity (Table 1), the SARs were
summarized as follows. Firstly, the longer alkyl amide chains have stronger cytotoxic

activity (propyl > ethyl > methyl), such as compounds 35 > 29 > 25. Secondly,
embedding of morpholine group with strong hydrophilicity in the terminal of
alkylamide chain was beneficial to the improvement of inhibition activity. For
example, the IC50 values of compounds 38-40 were lower than that of Golvatinib. The
introduction of F/OCF3/CF3 (electron absorption substituent) to the benzene ring of
moiety D had a great contribution to the inhibition activity, as represented compounds
28-30 and 34-36. Conversely, the introduction of H/CH3 or two substituents to the
same region reduced the inhibitory activity, such as compounds 23, 26-27, 31-33 and
etc. Overall, the introduction of propylmorpholine and OCF3 to moieties A and D,
respectively, led to the best conformation (compound 40), which possessed the best
cytotoxic activity.
Table 1: Cytotoxic activities of the target compounds 23-45 and Golvatinib against the A549,
HeLa and MCF-7 cell lines.
IC50 (μM) ± SD
Compd.
R1
R2
R3
A549
HeLa
MCF-7
23
24
25
26
27
28
29
30
31
32
33
34
Me
Me
Me
Et
F
H
40.80 ± 2.11
11.80 ± 1.32
10.24 ± 0.51
34.84 ± 0.27
11.55 ± 0.38
7.41 ± 0.30
10.04 ± 0.31
5.28 ± 0.16
34.49 ± 1.55
25.69 ± 0.35
17.97 ± 2.14
7.02 ± 0.21
NA b
NA
F
2-Cl
9.87 ± 0.36
11.77 ± 0.44
27.34 ± 5.56
10.74 ± 0.11
6.87 ± 0.14
10.73 ± 0.22
3.99 ± 0.10
NA
135.74 ± 3.27
45.78 ± 3.36
44.27 ± 4.56
51.25 ± 7.44
18.88 ± 2.65 a
40.57 ± 2.20
15.45 ± 0.45
35.14 ± 12.80
39.59 ± 2.82
34.55 ± 3.53
13.34 ± 1.92
F
3-CF3
2-CH3
4-CH3
3-F
H
H
H
H
H
H
F
Et
Et
Et
3-CF3
2-OCF3
2-F-4-Br
2-CH3
4-CH 3
3-F
Et
Et
n-Pr
n-Pr
n-Pr
24.55 ± 0.53
14.24 ± 2.27
6.66 ± 0.65
F
F
n-Pr
n-Pr
n-Pr
35
36
37
F
F
F
3-CF3
9.24 ± 0.51
8.00 ± 0.44
3.69 ± 0.10
53.54 ± 3.43
30.02 ± 2.25
7.42 ± 0.18
NA
2-OCF3
2-F-4-Br
4.22 ± 0.09
234.92 ± 12.82
N
N
N
38
39
40
H
H
H
4-CH3
3-CF3
2.94 ± 0.26
2.50 ± 0.22
1.03 ± 0.14
1.92 ± 0.14
1.65 ± 0.19
1.15 ± 0.06
3.57 ± 0.11
5.21 ± 0.22
2.59 ± 0.09
O
O
O
2-OCF3

N
S
41
42
43
44
H
H
H
H
H
2-F-4-Br
4-CH3
6.83 ± 0.17
4.25 ± 0.36
8.23 ± 1.10
NA
O
965.49 ± 22.37
722.36 ± 23.45
585.46 ± 11.99
660.36 ± 10.99
885.20 ± 14.90
387.21 ± 15.76
S
S
S
3-CF3
NA
2-F-4-Br
2-OCF3
969.43 ± 12.86
45
876.23 ± 15.67
6.89 ± 0.45
433.89 ± 14.38
4.14 ± 0.17
NA
Golvatinib c
20.61 ± 0.30
a Bold values show the IC50 values of the target compounds lower than the values of the positive
control.
b NA: Low inhibitory activity.
c Used as the positive control.
Inspired by the results of cytotoxic activity, compounds 38-41 were selected to
evaluate their c-Met kinase activity based on Mobility Shift Assay.22 Staurosporine
was used as positive control to insure the reliability of experimental data. Among
them, compound 40 showed potent c-Met kinase activity with IC50 value of 0.807 μM,
as shown in Table 2.
Table 2: c-Met kinase activity of selected compounds 38, 39, 40, 41 and Staurosporine.
Compd.
IC50 on c-Met (μM)
38
>10
39
>10
40
0.807
>10
41
Staurosporine
0.057
Next, HeLa cells were treated with seven different concentrations of compound
40 for 72 hours by MTT method,20 and the relationship between concentration and
inhibition rate was discussed. As shown in Fig. 4, compound 40 was observed in a
concentration-dependent manner. The inhibition rate of cells was more than 50% after
the treatment with compound 40 at the concentration of 1.23 μmol/L, while it was
around 20% when treated the cells with Golvatinib at same concentration.

Fig. 4. Concentration-dependent test of compound 40 against HeLa cells.
To further investigate the ability of compound 40 to induce cells apoptosis, we
performed AO (acridine orange) staining experiment23 with HeLa cells. In the control
group, the cells were arranged densely and orderly, with uniform and regular
morphology, as shown in Fig. 5. After treated with compound 40 at the concentrations
of 1 and 5 μg/L for 12 hours, a series of apoptotic phenomena appeared, such as
cytoplasmic contraction, membrane swelling and apoptotic bodies increasing.
Generally, compound 40 induced apoptosis of HeLa cells in
a
concentration-dependent manner.
Fig. 5. Morphological study of HeLa cells after treated with compound 40.
In order to explore the interaction mode of compound 40 with c-Met, we
performed the docking study24 of compound 40 with apoenzyme (c-Met without
endogenous ligand, PDB code: 3LQ8) and holoenzyme (c-Met with endogenous
ligand, PDB code: 3LQ8) by Autodock 4.0 software, and the binding energy were
calculated as -10.80 and -11.23 kcal/mol, respectively. In the binding mode,

compound 40 was completely bound into the internal cavity of c-Met in an extended
conformation (Fig. 6A). Interestingly, pyridylamide structure formed a bidentate
hydrogen bond with the residue Met1160. Meanwhile, the amide and
5-methylpyridazin-3(2H)-one segments of moiety C formed two hydrogen bonds with
residues Lys1110 and Asp1222 (Fig. 6A). On the other hand, the binding of
endogenous ligand to c-Met formed three hydrogen bonds (Fig. 6B). Generally, the
docking configuration of compound 40 was basically consistent with that of
endogenous ligand in Fig. 6C, and compound 40 has strong interaction with c-Met.
Fig. 6. (A) The binding mode of compound 40 with apoenzyme (c-Met, PDB code: 3LQ8). (B)
The binding mode of endogenous ligand belonged with c-Met. (C) The binding mode of
compound 40 with holoenzyme (c-Met with endogenous ligand). The protein and compound 40
were showed as cartoon and sticks, respectively, and hydrogen bonding interactions were
indicated with dashed lines in red.
In conclusion, a series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a
5-methylpyridazin-3(2H)-one fragment were designed, synthesized, and their
biological activity was evaluated. By analyzing the cytotoxic activity and structural
characteristics of the compounds, we have obtained some new SARs of pyridinamide
derivatives. The SARs revealed that the introduction of 5-methylpyridazin-3(2H)-one
structure and morpholine group to the "5-atom linker" and hydrophilic region,
respectively, played an important role in the enhancement of inhibitory activity.
Among them, the inhibitory activity of the promising compound 40 was 2.60-6.95
times higher than that of Golvatinib, and the IC50 values were 1.03, 1.15 and 2.59 μM

against A549, HeLa and MCF-7 cell lines, respectively. Moreover, compound 40
shown excellent biological activities in pharmacological experiments such as c-Met
kinase activity, concentration dependence and apoptosis. Compound 40 will be further
studied in our laboratory in the near future.
Acknowledgments
We acknowledge the generous support provided by National Natural Science
Foundation of China (NSFC No. 81660572), Natural Science Foundation of Jiangxi
Province (20192ACBL21009, 20171BAB215071) and Top-notch talent project of
Jiangxi Science & Technology Normal University (2016QNBJRC002).
References and notes
1. Balakumar P, Maung-U K, Jagadeesh G, Pharmacologist SE. Prevalence and prevention of
cardiovascular disease and diabetes mellitus. Pharmacol Res. 2016; 113: 600.
2. Zhang YZ, Xia MF, Jin K, Wang SF, Wei H, Fan CM, Wu YF, Li XL, Li XY, Li GY, Zeng
ZY, Xiong W. Function of the c-Met receptor tyrosine kinase in carcinogenesis and associated
therapeutic opportunities. Mol Cancer. 2018; 17: 45.
3. Akalu YT, Rothlin CV, Ghosh S. TAM receptor tyrosine kinases as emerging targets of innate
immune checkpoint blockade for cancer therapy. Immunol Rev. 2017; 276: 165.
4. Wu P, Nielsen TE, Clausen MH. FDA-approved small-molecule kinase inhibitors. Trends
Pharmacol Sci. 2015; 36: 422.
5. Fabbro D. 25 years of small molecular weight kinase inhibitors: potentials and limitations.
Mol Pharmacol. 2015; 87: 766.
6. Sarvagalla S, Cheung CHA, Tsai JY, Hsieh HP, Coumar MS. Disruption of protein-protein
interactions: hot spot detection, structure-based virtual screening and in vitro testing for the
anti-cancer drug target-survivin. Rsc Adv. 2016; 6: 31947.
7. Xie JS, Pan HM, Yao JL, Zhou YB, Han WD. SOCE and cancer: Recent progress and new
perspectives. Int J Cancer. 2016; 138: 2067.
8. Pan ST, Li ZL, He ZX, Qiu JX, Zhou SF. Molecular mechanisms for tumour resistance to
chemotherapy. Clin Exp Pharmacol P. 2016; 43: 723.
9. Tannir NM, Gisela S, Grünwald V. Cabozantinib: an active novel multikinase inhibitor in
renal cell carcinoma. Curr Oncol Rep. 2017; 19: 14.
10. Cuneo KC, Mehta RK, Kurapati H, Thomas DG, Lawrence TS, Nyati MK. Enhancing the
radiation response in KRAS mutant colorectal cancers using the c-Met inhibitor crizotinib.
Transl Oncol. 2019; 12: 209.
11. Parikh PK, Ghate MD. Recent advances in the discovery of small molecule c-Met Kinase
inhibitors. Eur J Med Chem. 2018; 143: 1103.
12. Yuan HL, Liu QF, Zhang L, Hu S, Chen TT, Li HF, Chen YD, Xu YC, Lu T. Discovery,
optimization and biological evaluation for novel c-Met kinase inhibitors. Eur J Med Chem.
2018; 143: 491.

13. Adam SJ, Jacob PH, J. Blade H, Taylor HH, Carter JP, Collin AR, Steven RH, Colette Q,
Jeffrey TS, Marc DHH. Pyridine-pyrimidine amides that prevent HGF-induced epithelial
scattering by two distinct mechanisms. Bioorg Med Chem Lett. 2017; 27: 3992.
14. Adam SJ, Jacob PH, David WC, Marc DH. Pharmacology and in vivo efficacy of
pyridine-pyrimidine amides that inhibit microtubule polymerization. Bioorg Med Chem Lett.
2018; 28: 934.
15. Zhang DY, Ai J, Liang ZJ, Li CP, Peng X, Ji YC, iang HL, Geng MY, Luo C, Liu H.
Discovery of novel 2-aminopyridine-3-carboxamides as c-Met kinase inhibitors. Bioorg Med
Chem. 2012; 20: 5169.
16. Wang LX, Liu XB, Duan YL, Zhao BB, Wang CL, Xiao Z, Zheng PW, Tang QD, Zhu WF.
Discovery of novel pyrrolopyrimidine/pyrazolopyrimidine derivatives bearing 1,2,3-triazole
moiety as c-Met kinase inhibitors. Chem Biol Drug Des. 2018; 92: 1301.
17. Ahmed EM, Kassab AE, Ei-Malah AA, Hassan MSA. Synthesis and biological evaluation of
5-methylpyridazin-3(2H)-one derivatives as selective COX-2 inhibitors and potential
anti-inflammatory agents. Eur J Med Chem. 2019; 171: 25.
18. Yaseen R, Ekinci D, Senturk M, Hameed A, Ovais S, Rathore P, Samim M, Javed K, Supuran
CT. 5-methylpyridazin-3(2H)-one substituted benzenesulfonamides as potent carbonic
anhydrase inhibitors. Bioorg Med Chem Lett. 2016; 26: 1337.
19. Taniguchi T, Inagaki H, Baba D, Yasumatsu L, Toyota A, Kaneta Y, Kiga M, Shin L, Odagiri
T, Shibata Y, Ueda K, Seo M, Shimizu H, Imaoka T, Nakayama K. Discovery of Novel
Pyrido-5-methylpyridazin-3(2H)-one Derivatives as FER Tyrosine Kinase Inhibitors with
Antitumor Activity. Acs Med Chem Lett. 2019; 10: 737.
20. Cytotoxic activity assay: The cancer cells lines were cultured in DMEM/1640 medium
containing 10% fetal bovine serum (FBS). Approximate 4×103 suspended cells were plated
into 96-well plates and then incubated in 5% CO2 at 37 °C for 24 h. Compounds being diluted
to the appropriate concentrations with medium or DMSO were added to 96-well plates and the
cells were incubated continually for 72 h. Fresh MTT (5 μg/mL) was added to each well and
incubated continually for 3.5 hours at 37 °C. The formazan crystals reduced by MTT were
dissolved in DMSO (150 μL), and the absorbance at 492 nm (for absorbance of MTT
formazan) and 630 nm (for the reference wavelength) was measured with an ELISA reader.
All of the target compounds were tested in triplicate in each cell line. The results,
demonstrating as IC50 values, were tested evenly by three times and calculated by using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
21. Analytic data of potent inhibitor 40: The commercial pyridinic acid (0.041 mol), DMF (0.32
mL), NaBr (0.004 mol) were dissolved in SOCl2 (25 mL) and stirred at 85 °C for 20 h. The
mixture was then concentrated in vacuum, dissolved in DCM, added dropwise to the mixture
of EtOH (2 mL), Et3N (1.61 mL), DCM (25 mL), and stirred at room temperature for 0.5 h.
After that, the reaction solution was adjusted with hydrochloric acid (aq) to pH 6-7, extracted
with DCM, concentrated to obtain ethyl 4-chloroacetate (0.022 mol), which was reacted with
p-nitrophenol (0.033 mol) at 130 °C in PhCl for 4.5 h. The mixture was concentrated,
extracted with DCM/NaOH mixture, and the organic layer was concentrated to obtain solid
(0.026 mol), then it was dissolved in the mixture of 1,4-dioxane (50 mL), NaOH (0.033 mol),
H2O (1 mL), and stirred at 25 °C for 0.5 h. The reaction solution was concentrated, dissolved
in saturated salt water, adjusted pH to 2-3 to obtain light yellow solid

4-(4-nitrophenoxy)picolinic acid (0.014 mol), which was reacted with SOCl2 (30 mL) at 85
°C for 0.5 h. The mixture was concentrated, dissolved in DCM (5 mL), added dropwise to a
mixture of DCM (30 mL), Et3N (2.91 mL) and 4-(3-aminopropyl)morpholine (0.021 mol) to
react at 25 °C for 0.5 h. Subsequently, the reaction solution was concentrated and
recrystallized to obtain a brown solid, which was reduced by hydrazine hydrate to get the
intermediate 4-(4-aminophenoxy)-N-(3-morpholinopropyl)picolinamide. The commercially
available 2-(trifluoromethoxy)aniline (0.028 mol), 37.5% HCl (0.5 mL), NaNO2 (0.056 mol),
3-oxobutanoate (0.036 mol) were dissolved in EtOH (30 mL), and stirred at 0 °C for 4 h. The
solution was then concentrated and extracted with solution of H2O/DCM to obtain ethyl
(E)-3-oxo-2-(2-(2-(trifluoromethoxy)phenyl)hydrazono)butanoate (0.020 mol), which was
dissolved in the mixture of dioxane (50 mL) and Ph3P=CHCOOC2H5 (0.022 mol), and stirred
at 90 °C for 10 h. The mixture was then concentrated and recrystallized to a yellow solid,
which was performed the same hydrolysis method as above to get the corresponding acid
analogue (0.0016 mol). Then it was dissolved in a mixture of oxalyl chloride (0.0016 mol)
and DCM (10 mL), and reacted at 0 °C for 0.5 h. The mixture was then added dropwise to a
solution
of
DIPEA
(one
drop),
DCM
(30
mL),
4-(4-aminophenoxy)-N-(3-morpholinopropyl)picolinamide (0.0016 mol), and stirred at 0 °C
for 0.5 h. The solution was coagulated under vacuum to obtain a yellow solid, which was
further purified by a chromatographic column with PE/EA to get compound 40. Purity:
1
97.99%; H NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 9.08 (s, 1H), 8.51 (d, J = 4.9 Hz,
1H), 7.86 (d, J = 7.3 Hz, 3H), 7.65 (d, J = 7.4 Hz, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.39 (s, 1H),
7.23 (d, J = 8.0 Hz, 2H), 7.15 (s, 1H), 7.09 (s, 1H), 3.61 (s, 4H), 3.33 (s, 2H), 2.41 (s, 9H),
1.68 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 166.25, 163.56, 161.95, 158.67, 152.95,
150.86, 149.73, 143.84, 142.74, 136.47, 133.36, 131.49 (2C), 130.40, 130.52, 128.45 (2C),
122.58 (2C), 121.83 (2C), 121.15, 114.63, 109.39, 66.40 (2C), 56.95, 53.67 (2C), 38.48,
25.70, 18.77; TOF MS ES+ (m/z): [M + H]+, calcd for C30H29F4N7O5: 653.2345, found,
653.2344.
22. Zhao F, Zhang LD, Hao Y, Chen N, Bai R, Wang YJ, Zhang CC, Li GS, Hao LJ, Shi C,
Zhang J, Mao Y, Fan Y, Xia GX, Yu JX, Liu YJ. Identification of
3-substituted-6-(1-(1H-[1,2,3]triazolo[4,5-b]pyrazin- 1-yl)ethyl)quinoline derivatives as
highly potent and selective mesenchymal-epithelial transition factor (c-Met) inhibitors via
metabolite profiling-based structural optimization. Eur J Med Chem. 2017; 134: 147.
23. Yang TM, Ng WH, Chen H, Chomchopbun K, Huynh TH, Go ML, Kon OL.
Mitochondrial-Targeting MET Kinase Inhibitor Kills Erlotinib-Resistant Lung Cancer Cells.
J Med Chem. 2019; 7: 807.
24. Xing WQ, Ai J, Jin SY, Shi ZX, Peng X, Wang L, Ji YC, Lu D, Liu Y, Geng MY, Hu YH.
Enhancing the cellular anti-proliferation activity of pyridazinones as c-Met inhibitors using
docking analysis. Eur J Med Chem. 2015; 95: 302.