Discovery of [1,2,4]triazolo[4,3-a]pyrazine derivatives bearing a 4-oxo-pyridazinone moiety as potential c-Met kinase inhibitors

Article abstract of DOI:10.1039/d0nj00575d

Two series of [1,2,4]triazolo[4,3-a]pyrazine derivatives bearing 4-oxo-pyridazinone moieties (compounds21a-land22a-l) were designed and their IC₅₀values were evaluated against three cancer cell lines (A549, MCF-7 and HeLa) and c-Met kinase. Among them, the compound with most potential,22i,exhibited excellent anti-tumor activity against A549, MCF-7 and HeLa cancer cell lines with IC₅₀values of 0.83 ± 0.07 μM, 0.15 ± 0.08 μM and 2.85 ± 0.74 μM, respectively, and it also possessed superior c-Met kinase inhibition ability at the nanomolar level (IC₅₀= 48 nM). Moreover, dose-dependent experiments, AO fluorescence staining, cell cycle assay, Annexin V-FITC/PI staining and docking studies were carried out in this study. The results demonstrated that compound22icould be a potential c-Met kinase inhibitor.

Full text of DOI:10.1039/d0nj00575d

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Discovery of [1,2,4]triazolo[4,3-a]pyrazine derivatives bearing 4-oxo-pyridazinone moiety asDOpIo: 1t0e.n10t3ia9/lD0cN-MJ0e0t575D
kinase inhibitors
Binliang Zhang a, 1, Xiaobo Liu a, 1, Hehua Xiong , Qian Zhang , Xin Sun , Zunhua Yang , Shan Xu , Pengwu Zheng
a
a
a
b
a
a, *
and Wufu Zhu a, *
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a
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science &
Technology Normal University, 605 Fenglin Road, Nanchang, Jiangxi 330013, China
b
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China
*
Corresponding author.
Tel. & Fax: +86 791 8380-2393;
E-mail: zhengpw@126.com (P. Zheng), zhuwufu-1122@163.com (W. Zhu)
Abstract：
Two series of [1,2,4]triazolo[4,3-a]pyrazine derivatives bearing 4-oxo-pyridazinone moiety (compounds 21a-l and
2
2a-l) were designed and evaluated for the IC50 values against three cancer cell lines (A549, MCF-7 and Hela) and
c-Met kinase. Among them, the most potential compound 22i exhibited excellent anti-tumor activity against A549,
MCF-7 and Hela cancer cell lines with IC50 values of 0.83 ± 0.07 µM, 0.15 ± 0.08 µM and 2.85 ± 0.74 µM, respectively,
and also possessed superior c-Met kinase inhibition at nanomolar level (IC50 = 48 nM). Moreover, dose-dependent
experiments, AO fluorescence staining, cell cycle assay, Annexin V-FITC/PI staining and docking studies were carried
out in this study. The results demonstrated that the compound 22i could be a potential c-Met kinase inhibitor.
Keywords: [1,2,4]Triazolo[4,3-a]pyrazine; c-Met inhibitors; 4-oxo-pyridazinone; Anti-tumor activity

Corresponding author. Tel./fax: +86 791 83802393.
These authors contribute equally to this work.
1
E-mail address: zhengpw@126.com (P. Zheng), zhuwufu-1122@163.com (W. Zhu).

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DOI: 10.1039/D0NJ00575D
1 Introduction
HGF/c-Met signaling plays a prominent role in tumor growth, invasion, proliferation and angiogenesis, which has
been identified as a promising target for the treatment of cancer, providing an effective therapeutic target for
malignancy 1-2. At present, two drugs namely Crizotinib and Cabozantinib had been approved by FDA 3-4. Moreover,
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there are also many c-Met inhibitors undergoing clinical trial or preclinical research, such as Foretinib , AMG337 and
so on (Figures 1 and 2). According to their structures and their binding models with c-Met kinase, the reported c-Met
inhibitors are mainly summarized and classified into Class I (e.g. AMG337), Class II (e.g. Foretinib) and the others
types 7.
Class II c-Met inhibitors are one of the most important type, which can bind to multiple sites of c-Met kinase and
are proved to be multi-target kinase inhibitors. According to the structure characteristics of Class II c-Met inhibitors,
We divided the structures of Class II c-Met inhibitors into four parts A, B, C and D (Figure 1). The main structural
modification of these inhibitors was focused on the moiety A (heterocyclic nucleus) and moiety C. The moiety C is
defined as a 5-atom linker, which has two obvious structural characteristics: on the one hand, the ‘5 atoms regulation’
means that six chemical bonds distance exists between moiety B and moiety D; on the other hand, the linker contains
hydrogen, oxygen and nitrogen atoms which could provide appropriate hydrogen-bond donor or acceptor to form
8
hydrogen bonds with amino acid residues . Based on the above summary, taking Foretinib as lead compound, we had
designed and synthesized several different series of derivatives such as compounds 6a-b 9-10 and 7 11 (Figure 1) in the
past several years, which exhibited excellent anti-tumor activity and kinase activity.
According to principles of scaffold hopping and bioisosterism, various heterocyclic nucleus of c-met inhibitors had
12
13
been developed, such as pyrazolo[1,5-a]pyridine core of compound 2 , imidazo[1,2-a]pyridine core of compound 3 ,
imidazo[1,2-b]pyridazine core of compound 4 14 and imidazo[1,2-b]pyridazine core of compound 5 15 (Figure 1).
Among them, the activity against c-Met kinase of compounds 2, 3 and 5 reached a single digit nanomolar level, which
indicated that the modification of the heterocyclic nucleus had a critical impact on activity. It is worth mentioning that
the triazole heterocyclic structure was found to be a significant group in some Class I c-Met inhibitors which exhibited
excellent biologically activity. Therefore, based on bioisosterism principles, the [1,2,4]triazolo[4,3-a]pyrazine structure
was used as the core of the novel c-Met inhibitor in this research. Furthermore, in our previous study, compound 8 16
showed excellent anti-tumor due to the introduction of 4-oxo-pyridazinone to the 5-atom linker moiety. So the active
structure 4-oxo-pyridazinone was retained in our design (Figure 2). Finally, we introduced electron-withdrawing group
and electron-donating group on the benzene ring to explore the effect on activity, so that the benzene ring was allowed

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to occupy the internal hydrophobic pocket of c-Met and could improve the binding of the compouDnOdI: t1o0.1t0h3e9/pDr0oNtJe0in05.75D
Herein, based on the structure-based drug design (SBDD) strategy and bioisosterism principles, and taking Foretinib as
reference compound, two series of c-Met inhibitors were designed and synthesized, culminating in the discovery of
compound 22i.
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linker
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1 (Foretinib)
2 (WO 2015100117)
3 (WO 2009136663)
Hydrophobic
fragment
Linker 1
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R3
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-atom linker
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linker
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General Structure
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(US 20080045536)
5 (WO 2009096435)
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2b: Z=N, R=CH3
N
N
H
H
the most potential
compound 22i in this research
6
a-b our previous work
7 our previous work
Figure 1. Examples of c-Met kinase inhibitors and the most potential compound 22i in this research.

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DOI: 10.1039/D0NJ00575D
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8
. Our previous research
Molecules, 2018, 23, 1543
N
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R2
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21a-l, 22a-l
C
Linker 1
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fragment
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Nucleus
1
(Foretinib)
Figure 2. Structures and design strategy for target compounds
2
Results and Discussion
2
.1 Chemistry
The synthetic route of intermediate compounds 14a-f was illustrated in Scheme 1. Using different substituted
anilines 9a-f as starting materials, different substituted anilines 9a-f were diazotized and then reacted with ethyl
acetoacetate to obtain compounds 10a-f. Next, compounds 10a-f were dissolved in the DMF-DMA, heated to 100 ºC
and stirred for 6-10 h, and then the mixture was poured to the petroleum ether and filtered to obtain the compounds
11a-f. Then, the cyclization was proceeded to convert 11a-f to compounds 12a-f in the presence of NaOH. Finally,
hydrolysis and acyl chlorination were used to convert compounds 12a-f to the key intermediate 14a-f.
The target compounds 21a-l and 22a-l were prepared as outlined in Scheme 2. The key intermediate 17 was
synthesized from 2,3-dichloropyrazine (compound 15) via substitution reaction with hydrazine hydrate and cyclization
with triethoxy methane, respectively. Then, compounds 18a-b were obtained by substitution reaction with
4
-aminphenol or 2-fluoro-4-aminphenol. Finally, reaction of anilines 18a-b with acyl chloride 14a-f promoted by
9
DIPEA in CH
2
Cl
2
at room temperature yielded the target compounds 21a-l . The synthetic method of compounds 22a-l
was the same as that of the compounds 21a-l.

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DOI: 10.1039/D0NJ00575D
O
N
O
O
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O
NH2
O
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O
O
O
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R2
O
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R2
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a-14a: R = H
2
10a-f
11a-f
b-14b: R = 3-Cl-4-F
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3
12a-f
13a-f
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Scheme 1. Reagents and conditions: (a) Sodium nitrite, EtOH, ethyl acetoacetate, 0 ºC, 0.5 h; (b) DMF-DMA, 100 ºC,
reflux, 6-10 h; (c) EtOH, 10% NaOH, 80 ºC, 1 h; (d) Sodium carbonate, EtOH/H
2
O, 80 ºC, 3-5 h; (e) Oxalyl chloride,
DMF, CH
2
Cl
2
, r.t., 5 min.
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Cl
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H
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1a-l, 22a-l
N
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0a-l: R1 = H
1a-l: R1= CH3
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20a-b
N
Scheme 2. Reagents and conditions: (a) N
2
H
4 2
·H O, EtOH, 85 ºC, reflux; (b) Triethoxy methane, 80 ºC, reflux; (c)
4
2
2 2
-Aminophenol, potassium t-butoxide, KI, THF; (d) Triethyl orthoacetate, 80 ºC, reflux; (n) DIPEA, CH Cl , r.t., 0.5 h.
.2 Biological Evaluation
Three cancer cell lines A549, MCF-7 and Hela were selected for evaluating the anti-tumor activities of the target
compounds by the method of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), and the results were
shown in Table 1. As can be seen, most of the target compounds showed moderate to excellent levels of anti-tumor
activity in vitro against three tested cell lines. In addition, most of the compounds had more potent anti-tumor activities
against both A549 and Hela cell lines than against MCF-7 cell line. Among them, compound 22i exhibited excellent
anti-tumor potency for A549, MCF-7 and Hela cell lines with IC50 values of 0.83 ± 0.07 µM, 0.15 ± 0.08 µM and 2.85
±
0.74 µM, respectively.
Further investigations about the effects of different substituents on the benzene ring were carried out, which
revealed that the electron-withdrawing group on the benzene ring could increase the in vitro anti-tumor activity of the
target compounds, and the opposite was true. For example, the compounds 21e, 21k, 22e and 22k with methoxy group
showed lower in vitro anti-tumor activities than that of the target compounds with electron-withdrawing group. The
target compounds bearing a larger substituent (trifluoromethyl group) on the phenyl ring exhibited better anti-tumor
2 3 2 3
activity such as compound 21i (R =3-CF -4-Cl) and 22i (R =3-CF -4-Cl). Furthermore, in order to investigate the

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cytotoxic activity of the target compounds on normal cells, the in vitro cytotoxic activity against LOD2OnI:o1r0m.1a0l39l/iDve0rNJc0e0l5l75D
line was carried out. The results presenting in Table 1 indicated that the 24 target compounds had little cytotoxic
activity against LO2 normal liver cell line, and their IC50 values were greater than 50 μM. These data indicated that the
three cancer cell lines (A549, MCF-7 and Hela) were more sensitive to the target compounds than LO2 normal liver cell
line.
0
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Table 1
c-Met inhibitory activities and anti-tumor activities of target compounds.
O
H
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N
N
N
R2
O
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R1
IC50 (μM) a
Compounds
21a
X
R
1
R
2
A549
MCF-7
Hela
LO2
c-Met
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
5
.21 ± 0.48
4.78 ± 0.52 10.58 ± 0.85
>50
>50
>50
>50
>50
>50
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>1
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1c
21d
1e
1f
21g
3-Cl-4-F
3-CF -4-Cl
2-F-4-Br
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3.74 ± 1.48 19.01 ± 1.77 25.01 ± 1.93
7.48 ± 1.57 22.17 ± 0.38 28.25 ± 0.85
2
3
>1
>1
2
4-OCH
4-F
3
3.62 ± 1.15 34.73 ± 1.92
.48 ± 0.35 3.18 ± 0.42
>50
>1
2
2
6.68 ± 0.56
0.095
H
1
3.48 ± 0.67 16.95 ± 1.81 23.26 ± 1.53
>50
>50
>1
2
1h
F
3-Cl-4-F
3-CF -4-Cl
2-F-4-Br
4.25 ± 0.62
3.09 ± 0.30
1.64 ± 0.56
8.89 ± 0.96
2.56 ± 0.49
0.120
2
1i
F
F
H
H
3
0.89 ± 0.07
5.15 ± 0.51
>50
>50
>50
0.055
0.280
ND b
2
1j
6.13 ± 0.58 11.87 ± 1.29
2
1k
1l
22a
2b
2c
22d
2e
2f
22g
F
H
4-OCH
4-F
3
14.56 ± 1.04 27.42 ± 2.24
.87 ± 0.19 4.45 ± 0.32
>50
2
F
H
4
8.36 ± 0.54
>50
>50
>50
>50
>50
>50
>50
>50
0.126
0.768
>1
H
H
H
H
H
H
F
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
H
1
3.16 ± 0.77 15.90 ± 1.25 19.27 ± 1.48
12.67 ± 0.92 16.48 ± 1.69 20.74 ± 1.24
.82 ± 0.75 8.77 ± 0.86 13.58 ± 0.92
2
3-Cl-4-F
3-CF -4-Cl
2-F-4-Br
2
3
7
0.440
ND
2
1
1
8.25 ± 1.57 30.48 ± 1.63 35.62 ± 1.78
3.23 ± 1.42 16.09 ± 1.59 29.32 ± 2.17
8.31 ± 1.64 19.85 ± 1.94 33.27 ± 2.31
2
4-OCH
4-F
3
0.880
>1
2
H
2
.04 ± 0.25
2.16 ± 0.39
4.42 ± 0.65
0.089

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2
2h
22i
2j
2k
F
F
F
F
F
-
CH
CH
CH
CH
CH
-
3
3
3
3
3
3-Cl-4-F
3-CF -4-Cl
2-F-4-Br
DOI: 10.1039/D0NJ00575D
6
0
.72 ± 0.85
.83 ± 0.07
6.49 ± 0.79 12.54 ± 0.98
0.15 ± 0.08 2.85 ± 0.74
>50
0.300
0.048
ND
3
>50
>50
>50
>50
ND
2
23.42 ± 0.75 16.48 ± 0.57 24.26 ± 1.87
1.56 ±0.42 16.79 ± 1.27 25.38 ± 1.25
2
4-OCH
3
1
>1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
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4
4
4
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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6
7
8
9
0
1
2
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0
22l
4-F
-
5
.65 ± 0.20
6.42 ± 0.34
0.93 ± 0.09
8.05 ± 0.31
-
ND
Foretinibc
0.71 ± 0.05
0.019
a
b
c
The values are an average of two separate determinations.
ND: Not detected.
Used as a positive control.
®
Through homogeneous time-resolved fluorescence (HTRF ) assays, experiments against c-Met kinase of twenty
selected target compounds as well as the reference compound Foretinib were carried out for further study. The obtained
results were presented in Table 1, which suggested that most of the target compounds exhibited moderate inhibition
activity on c-Met kinase. The results indicated that the target compounds may act through inhibiting the c-Met signal
pathway. Furthermore, the c-Met kinase activity of target compounds 21f (0.095 μM), 21i (0.055 μM), 22g (0.089 μM)
and 22i (0.048 μM) at nanomolar level were slightly lower than that of the reference Foretinib (0.019 μM). The
anti-tumor activity and c-met kinase activity of compound 22i were slightly better than that of compound 21i, which
may be due to the introduction of a methyl group into the nucleus of compound 22i which were advanced for
sufficiently occupied the cavity of c-Met protein.
In addition, the most promising compound 22i was selected as a representative to evaluate the relationship between
the anti-tumor activity and the concentration of the target compounds by the method of MTT. Three cancer cell lines
A549, MCF-7 and Hela were treated with seven different concentrations of compound 22i for 72 h, respectively, and
the results were shown in Figure 3. As can be seen, as the concentration of compound 22i increasing, the inhibition rate
of compound 22i on the three cancer cell lines increased, and the phenomenon of dose-dependent was observed.
Furthermore, we can find that compound 22i had better anti-tumor activity against MCF-7 and A549 than against Hela.
Figure 3. Relationship between activity and concentration of compound 22i against three cancer cell lines.

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DOI: 10.1039/D0NJ00575D
2
.3 Effect on cell cycle progression.
In order to have a deeper understanding of the growth inhibition mechanism of target compounds to cancer cells,
the effect of compound 22i on cell cycle progression was investigated. A549 cells were treated with 1.0 μM of
compound 22i for 72 h, and then the flow cytometric cycle experiment was carried out. Compared with the control
group, the population of G0/G1 phase increased significantly after treatment of A549 with compound 22i, increasing
from 60.54% to 73.86%, while the S phase decreased correspondingly, and there was no significant change in G2/M
phase. It indicated that compound 22i could induce cell cycle arrest of A549 cells at the G0/G1 checkpoint.
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Figure 4. Cell cycle progression analyses of A549 cells treated with compound 22i for 72 h.
2
.4 Apoptosis result analyzing
To further explain the inhibitory effect of compound 22i on cancer cell growth, the apoptosis-inducing effect of
compound 22i on A549 cells was evaluated by flow cytometry. As can be seen from the Figure 5, compound 22i
significantly induced early apoptosis and late apoptosis in A549 cells, especially late apoptosis, reaching 9.16% at the
concentration of 1.00 μM. As the concentration of compound 22i increasing, the total apoptosis rate was also increased,
from 6.43% to 15.87% in a dose-dependent manner. These data demonstrated that compound 22i could induced
apoptosis in A549 cells in a dose-dependent manner.
Figure 5. Apoptosis analyses of A549 cells treated with compound 22i for 72 h.
2.5 Morphologic changes of A549 cells under inverted microscopy and fluorescence microscopy

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To further verify that compound 22i can induce apoptosis in A549 cells, morphological chanDgOesI: 1o0f.10A359/4D90NcJe0l0ls575D
treated with compound 22i for 12 h were observed by AO fluorescence staining. As illustrated in Figure 6A, A549 cells
in the control group grew well, the cells were full and the edges were clear. However, when A549 cells were treated
with 0.83 µM of compound 22i for 12 h, the obtained result was presented in Figure 6B, in which the A549 cells
shrank, the chromatin was condensed and the nucleus was orange. The results further indicated that compound 21i
successfully induced apoptosis in A549 cells.
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Figure 6. Observation of A549 cells morphology by AO fluorescence staining
2
.6 Molecular docking study
According to the results of in vitro anti-tumor activity assays and enzymatic activity assay, we selected compound
2i as the representative to dock with c-Met protein (PDB code 3LQ8) by AutoDock 4.2 software (The Scripps
2
Research Institute, USA), and the docking results were processed and modified in PyMOL 1.8.x software
https://pymol.org). As illustrated in Figure 7a, the whole compound 22i skeleton embedded into hydrophobic pocket
(
of c-Met protein and closely combined with c-Met protein. As can be seen from Figure 7b, the two nitrogen atoms on
the triazolopyrazine core structure can form a bidentate hydrogen bond with the key amino acid residue MET1160 in
the hinge region, demonstrating that the introduction of the triazolopyrazine core ensured the activity of the target
compounds. In the 5-atom linker, the oxygen atom of the pyridazinone group formed a hydrogen bond with LYS1110,
and the two nitrogen atoms on the pyridazinone group formed a bidentate hydrogen bond with residue ASP1222. The
molecular docking studies suggested that compound 22i may be a potential inhibitor of c-Met.

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Figure 7. The docking mode of compound 22i with c-Met (PDB code 3LQ8)
3 Experimental Section
.1 General Information
3
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were uncorrected. Frequently used solvents (Methanol, DCM, EA, DMF, etc.) were absolutely
anhydrous. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China) with
fluorescent indicator 254 nm. Column chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Mass spectrometry (MS) was performed on Waters High Resolution
Quadrupole Time of Flight Tandem Mass Spectrometry (QTOF). The purity of the compound was determined by
Agilent 1260 liquid chromatograph fitted with an Inertex-C18 column. HNMR and 13CNMR spectra were recorded on
Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as
an internal standard.
1
3
3
.2 Chemistry
.2.1 Preparation of intermediate compounds 14a-f
1
7
The preparation of intermediate compounds 14a-f had been described in detail in our previous work .
3
0
.2.2 Preparation of compound 16
2
,3-Dichloropyrazine (20 g, 0.134 mol) was dissolved in ethanol, and a small amount of hydrazine hydrate (21 g,
.42 mol) was added thereto in multiple portions. The mixture was refluxed at 80 ºC and monitored by TLC. After the
reaction mixture was cooled, it was added to ice water, and then the precipitated solid was filtered and dried to give
compound 16 (16.8 g, 84.1%) as yellow powder.
3
.2.3 Preparation of compound 17
Intermediate compound 16 (5.0 g, 0.034 mol) was added to triethyl orthoformate (50 mL) for 2 h at 80 ºC. The

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reaction solution was directly filtered under suction, and the filter cake was washed with petroleumDeOthI:e1r0.a10n3d9/dDr0iNedJ0t0o575D
4
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9
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4
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5
6
yield compound 17 (4.8 g, yield 90.1%) as yellow powder.
3
.2.4 Preparation of compounds 18a-b
-aminophenol and potassium t-butoxide (1.46 g, 13.0 mmol) were added to tetrahydrofuran, and the mixture was
4
0
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8
9
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7
8
9
0
stirred for 1 h at 0 ºC. Intermediate 18 (1.00 g, 6.5 mmol) and potassium iodide (0.12 g, 0.72 mmol) were added to the
flask in which tetrahydrofuran was used as a solvent, then the mixture was heated to 80 ºC, and the above-mentioned
4
-aminophenol was added there under nitrogen atmosphere. After the reaction was completed, the mixture was filtered,
and the filtrate was evaporated to dryness. After evaporation, a small amount of NaOH aqueous solution was added to
the mixture, and a solid was precipitated, filtered, and dried to give compound 18a (0.45 g, 30.6%) as grayish white
solid. The preparation method of compound 18b was the same as that of the compound 18a.
3
3
3
.2.5 Preparation of compound 19
The preparation method of compound 19 was the same as that of the compound 17.
.2.6 Preparation of compounds 20a-b
The preparation method of compounds 20a-b were the same as that of the compound 18a.
.2.7 Preparation of compounds 21a-l
Compounds 14a-f (0.51 mmol) and N,N-diisopropylethylamine (0.49 mmol) were dissolved in dichloromethane
(
10 mL), and then compounds 18a-b were added dropwise under ice bath. Then, the reaction mixture was taken from
ice bath, stirred at room temperature for 0.5 h. The reaction was washed with 10% aqueous K CO (50 mL), and the
organic layer was dried over anhydrous Na SO and concentrated in reduced pressure to obtain the crude product. The
crude product was purified through a column chromatography on silica to yield compounds 21a-l.
2
3
2
4
3
.2.8 Preparation of compounds 22a-l
The preparation method of compounds 22a-l were the same as that of the compounds 21a-l.
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxamide (21a).
1
Light yellow solid in 31.2% yield. m.p: 304.4–306.1 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.80 (s, 1H), 9.34 (s,
1H), 8.89 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 4.4 Hz, 1H), 7.69 (d, J = 8.1 Hz, 4H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (d, J = 7.4
Hz, 1H), 7.24 (t, J = 6.0 Hz, 3H), 6.80 (d, J = 7.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.74, 160.48, 152.01,
147.06, 143.39, 142.31, 139.61, 130.22 (2C), 129.95, 129.14, 127.47, 126.01, 125.13, 121.89 (2C), 120.78, 119.33,
-1
116.78, 116.09, 114.62, 108.88. IR (KBr) cm : 3442, 3038, 1688, 1596, 1565, 1492, 1330, 1205. TOF MS ES+ (m/z):
+
[
M + H] , calcd for C22
H
15
N
7
O
3
: 426.1315, found, 426.1316. HPLC purity: 97.22% (Column: Dikma Luna C18; Mobile

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o
phase: H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/minD)O. I: 10.1039/D0NJ00575D
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-1-(3-chloro-4-fluorophenyl)-4-oxo-1,4-dihydropyridazine-3-.
carboxamide (21b).
1
Brown solid in 51.2% yield. m.p: 248.0–251.3 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.74 (s, 1H), 9.35 (s, 1H),
0
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3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
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8.91–8.82 (m, 1H), 8.20 (d, J = 4.5 Hz, 1H), 8.02 (t, J = 4.5 Hz, 1H), 7.72 (d, J = 8.7 Hz, 3H), 7.57 (s, 1H), 7.29 (d, J =
9
1
1
.1 Hz, 3H), 6.82 (d, J = 7.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.72, 160.47, 151.88, 147.93, 143.36,
42.35, 139.35, 138.61, 137.77, 135.00, 130.22 (2C), 129.14, 126.53 (2C), 125.18, 121.89 (2C), 121.08, 116.73, 115.78,
-
1
+
08.81. IR (KBr) cm : 3441, 3044, 1688, 1601, 1559, 1490, 1326, 1200. TOF MS ES+ (m/z): [M + H] , calcd for
C
22
H
13ClFN
7
O
3
: 478.0831, found, 478.0830. HPLC purity: 97.51% (Column: Dikma Luna C18; Mobile phase:
o
H
2
O:MeCN = 60:40; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-1-(4-chloro-3-(trifluoromethyl)phenyl)-4-oxo-1,4-dihydropy
ridazine-3-carboxamide (21c).
Grayish white solid in 61.2% yield. m.p: 283.5–284.9 ºC. H NMR (400 MHz, DMSO-d
1H), 9.03 (d, J = 7.9 Hz, 1H), 8.23 (d, J = 5.2 Hz, 2H), 8.09 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.74 (d, J =
1
6
) δ 11.71 (s, 1H), 9.39 (s,
8
1
1
.3 Hz, 2H), 7.30 (d, J = 8.6 Hz, 3H), 6.86 (d, J = 7.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d
53.10, 148.42, 142.23, 142.20, 139.14 (2C), 136.30, 133.55 (2C), 130.83, 128.28, 127.96, 127.23, 126.75, 124.17,
6
) δ 169.91, 159.91,
-
1
23.00 (2C), 121.50 (2C), 120.65, 115.10. IR (KBr) cm : 3077, 1687, 1599, 1560, 1488, 1326, 1201. TOF MS ES+
+
(m/z): [M + H] , calcd for C23
H
13ClF
3
N
7
O
3
: 528.0799, found, 528.0799. HPLC purity: 96.91% (Column: Dikma Luna
o
C18; Mobile phase: H
2
O:MeCN = 50:50; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydropyridazine-3-
carboxamide (21d).
1
6
White solid in 31.2% yield. m.p: 286.3–298.5 ºC. H NMR (400 MHz, DMSO-d ) δ 11.60 (s, 1H), 9.38 (s, 1H),
8.65 (s, 1H), 8.23 (d, J = 4.2 Hz, 1H), 7.85 (d, J = 10.2 Hz, 1H), 7.71 (d, J = 8.8 Hz, 3H), 7.62 (s, 1H), 7.28 (s, 3H),
6.81 (d, J = 8.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.45, 159.93, 153.10, 148.36, 144.98, 139.15 (2C),
1
36.29, 130.95, 129.20, 129.14 (2C), 126.75, 123.45, 123.02 (2C), 121.46 (2C), 121.10, 120.87, 120.13, 115.10. IR
-1
+
(
7 3
KBr) cm : 3441, 3074, 1689, 1607, 1569, 1484, 1322, 1202. TOF MS ES+ (m/z): [M + H] , calcd for C22H13BrFN O :
5
2
22.0326, found, 522.0327. HPLC purity: 96.22% (Column: Dikma Luna C18; Mobile phase: H O:MeCN = 60:40;
o
Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydropyridazine-3-carbox
amide (21e).
1
6
Brown solid in 20.2% yield. m.p: 293.5–295.7 ºC. H NMR (400 MHz, DMSO-d ) δ 11.97 (s, 1H), 9.39 (s, 1H),

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.84 (s, 1H), 8.23 (s, 1H), 7.70 (d, J = 37.9 Hz, 3H), 7.44 (s, 1H), 7.30 (s, 2H), 7.08 (s, 2H), 6.84 (s,D2OHI:)1,03.1.07379/(Ds0,N3JH00)5.75D
4
5
13C NMR (101 MHz, DMSO-d
) δ 170.45, 160.82, 153.82, 148.28, 143.12, 142.04, 139.84 (2C), 137.66, 137.10, 127.46,
6
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
2
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-
1
1
1
24.25 (2C), 123.73 (2C), 122.16, 121.80, 120.61, 120.56, 115.90 (2C), 115.78, 56.89. IR (KBr) cm : 3443, 3104, 1688,
+
597, 1567, 1507, 1332, 1202. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
17
N
7
O
4
: 456.1420, found, 456.1417. HPLC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
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2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
purity: 98.93% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 70:30; Column temperature: 26 C;
Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)phenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydropyridazine-3-carboxa
mide (21f).
1
Yellow solid in 41.3% yield. m.p: 309.4–311.7 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.81 (s, 1H), 9.37 (s, 1H),
8
.88 (s, 1H), 8.23 (s, 1H), 7.77 (d, J = 17.4 Hz, 4H), 7.39 (s, 2H), 7.29 (s, 3H), 6.84 (s, 1H). 13C NMR (101 MHz,
6
DMSO-d ) δ 171.82, 160.23, 153.12, 148.04, 145.97, 140.90, 139.13 (2C), 136.35, 130.14, 126.75, 124.47, 124.38,
-1
1
1
23.03 (2C), 121.46 (2C), 120.89, 117.10 (2C), 116.87, 115.09. IR (KBr) cm : 3444, 3107, 1688, 1598, 1564, 1508,
+
328, 1204. TOF MS ES+ (m/z): [M + H] , calcd for C22
H
14FN
7
O
3
: 444.1220, found, 444.1223. HPLC purity: 96.51%
o
(Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm;
Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxami
de (21g).
1
6
White solid in 28.2% yield. m.p: 296.3–298.5 ºC. H NMR (400 MHz, DMSO-d ) δ 11.97 (s, 1H), 9.42 (s, 1H),
8
7
1
1
.94 (d, J = 7.9 Hz, 1H), 8.33–8.25 (m, 1H), 7.89 (d, J = 12.5 Hz, 1H), 7.74 (d, J = 7.8 Hz, 2H), 7.55 (t, J = 7.8 Hz, 2H),
.45 (s, 3H), 7.32 (s, 1H), 6.86 (d, J = 7.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d
) δ 170.55, 160.84, 153.88, 149.11,
6
48.88, 147.48, 142.95 (2C), 140.18, 130.83 (2C), 129.92, 129.80, 126.97, 123.68 (2C), 122.71, 122.60 (2C), 122.17,
-1
+
21.67, 114.63. IR (KBr) cm : 3446, 3108, 1699, 1591, 1563, 1490, 1327, 1205. TOF MS ES+ (m/z): [M + H] , calcd
for C22
H
14FN
7
O
3
: 444.1220, found, 444.1219. HPLC purity: 98.20% (Column: Dikma Luna C18; Mobile phase:
o
H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-1-(3-chloro-4-fluorophenyl)-4-oxo-1,4-dihydropyri
dazine-3-carboxamide (21h).
Light yellow solid in 36.5% yield. m.p: 273.1–275.6 ºC. H NMR (400 MHz, DMSO-d
1
6
) δ 11.86 (s, 1H), 9.42 (s,
1
9
1
H), 8.93 (d, J = 8.2 Hz, 1H), 8.29 (s, 1H), 8.06 (s, 1H), 7.88 (d, J = 12.2 Hz, 1H), 7.78 (d, J = 6.4 Hz, 1H), 7.62 (t, J =
.1 Hz, 1H), 7.45 (s, 2H), 7.31 (s, 1H), 6.86 (d, J = 7.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.68, 160.28,
48.03, 142.46, 140.24, 139.34, 138.64, 128.87, 126.53 (2C), 125.17 (2C), 124.38 (2C), 122.84, 120.84 (2C), 118.45,

New Journal of Chemistry
Page 14 of 22
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2
2
2
2
2
2
2
2
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18.22, 116.80, 115.78, 108.67. IR (KBr) cm : 3445, 3095, 1703, 1604, 1562, 1495, 1333, 1212. TODOFI:M10S.10E3S9/+D0(NmJ/0z0)5:75D
+
[M + H] , calcd for C22
H
12ClF
2
N
7
O
3
: 496.0736, found, 496.0736. HPLC purity: 97.25% (Column: Dikma Luna C18;
o
Mobile phase: H
2
O:MeCN = 50:50; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-1-(4-chloro-3-(trifluoromethyl)phenyl)-4-oxo-1,4-d
ihydropyridazine-3-carboxamide (21i).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Brown solid in 43.2% yield. m.p:239.4–241.7 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.83 (s, 1H), 9.42 (s, 1H),
9
7
1
1
.03 (d, J = 7.8 Hz, 1H), 8.29 (d, J = 4.7 Hz, 1H), 8.22 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.93–7.86 (m, 2H), 7.45 (s, 2H),
.31 (d, J = 4.5 Hz, 1H), 6.86 (d, J = 7.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.01, 159.56, 151.18, 147.65,
41.61, 141.43, 138.63, 137.92, 136.92, 132.83 (2C), 130.14, 126.54, 125.82 (2C), 124.43, 120.74, 120.01 (2C), 116.08,
-1
+
15.08 (2C), 108.18. IR (KBr) cm : 3444, 3036, 1703, 1611, 1558, 1505, 1327, 1189. TOF MS ES+ (m/z): [M + H] ,
calcd for C23
phase: H
H
12ClF
4
N
7
O
3
: 546.0705, found, 546.0706. HPLC purity: 96.48% (Column: Dikma Luna C18; Mobile
o
2
O:MeCN = 50:50; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydropyri
dazine-3-carboxamide (21j).
1
Brown solid in 28.2% yield. m.p: 289.3–292.2 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.76 (s, 1H), 9.40 (d, J =
1
7
1
1
1
6.9 Hz, 1H), 8.67 (s, 1H), 8.25 (d, J = 23.5 Hz, 1H), 7.88 (d, J = 11.3 Hz, 1H), 7.65 (d, J = 21.8 Hz, 1H), 7.44 (s, 1H),
1
3
.29 (d, J = 11.9 Hz, 1H), 6.99 (s, 1H), 6.84 (s, 1H), 6.46–6.33 (m, 1H), 5.33 (s, 1H). C NMR (101 MHz, DMSO-d
6
) δ
69.34, 160.28, 151.89, 145.03, 139.34, 139.21, 129.18, 129.10 (2C), 126.71, 126.53 (2C), 125.16, 124.55, 121.11,
-1
20.88, 120.24, 116.79, 115.78, 115.19, 110.04, 108.65. IR (KBr) cm : 3445, 3100, 1685, 1606, 1560, 1485, 1321,
+
200. TOF MS ES+ (m/z): [M + H] , calcd for C22
H
12BrF
2
N
7
O
3
: 540.0231, found, 540.0232. HPLC purity: 97.06%
o
(
Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 60:40; Column temperature: 26 C; Wavelength: 280 nm;
Flow rate: 1.0 mL/min).
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydropyridazine-
3-carboxamide (21k).
1
Brown solid in 31.2% yield. m.p: 295.6–298.3 ºC. H NMR (400 MHz, DMSO-d
6
) δ 12.09 (s, 1H), 9.41 (d, J =
1
6
1
2.1 Hz, 1H), 8.86 (s, 1H), 8.28 (s, 1H), 7.88 (s, 1H), 7.65 (s, 2H), 7.45 (s, 1H), 7.31 (s, 1H), 7.08 (s, 2H), 6.86 (s, 1H),
.57 (s, 1H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO-d
) δ 169.66, 160.45, 152.31, 147.36, 142.52, 142.43, 139.33,
6
36.93, 128.45, 126.58 (2C), 126.55, 125.16, 123.54 (2C), 121.23, 118.83, 115.78, 115.22 (2C), 110.71, 108.863, 56.16.
-1
+
IR (KBr) cm : 3441, 3029, 1693, 1594, 1569, 14511, 1325, 1203. TOF MS ES+ (m/z): [M + H] , calcd for
23 7 4
C H16FN O
: 474.1326, found, 474.1326. HPLC purity: 98.26% (Column: Dikma Luna C18; Mobile phase:

Page 15 of 22
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DOI: 10.1039/D0NJ00575D
o
H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
4
5
6
N-(4-([1,2,4]triazolo[4,3-a]pyrazin-8-yloxy)-3-fluorophenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydropyridazine-3-
carboxamide (21l).
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
White solid in 42.1% yield. m.p: 300.5–302.4 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.96 (s, 1H), 9.42 (s, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8.90 (d, J = 8.0 Hz, 1H), 8.29 (s, 1H), 7.88 (d, J = 12.5 Hz, 1H), 7.79 (s, 2H), 7.47–7.37 (m, 4H), 7.31 (d, J = 4.7 Hz,
1
1
H), 6.86 (d, J = 8.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.19, 162.84, 159.89, 151.41, 147.32, 142.07,
39.47, 138.84, 137.17, 126.04 (2C), 124.67, 123.96 (2C), 123.87, 120.52, 116.62 (2C), 116.39, 116.27, 115.29, 108.39.
-1
+
IR (KBr) cm : 3441, 3108, 1699, 1595, 1565, 1509, 1328, 1203. TOF MS ES+ (m/z): [M + H] , calcd for
C
22
H
13
F
2
N
7
O
3
: 462.1126, found, 462.1125. HPLC purity: 99.99% (Column: Dikma Luna C18; Mobile phase:
o
H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxa
mide (22a).
Grayish white solid in 49.2% yield. m.p: 289.4–293.9 ºC. H NMR (400 MHz, DMSO-d
J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.77–7.64 (m, 4H), 7.60–7.40 (m, 4H), 7.28 (d, J = 7.0 Hz, 2H), 6.85 (d, J = 8.2 Hz, 1H),
1
6
) δ 11.85 (s, 1H), 8.93 (d,
2
1
3
.66 (s, 3H). 13C NMR (101 MHz, DMSO-d
) δ 169.66, 159.41, 146.05, 142.59, 141.51, 129.49 (2C), 128.57 (2C),
6
-1
28.37, 125.54, 122.24, 121.34 (2C), 121.18, 120.76, 120.23 (2C), 119.42, 115.19, 113.21, 108.53, 9.61. IR (KBr) cm :
+
441, 3089, 1688, 1592, 1563, 1488, 1329, 1216. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
17
N
7
O
3
: 440.1471,
found, 440.1472. HPLC purity: 99.42% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 70:30; Column
o
temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
1
-(3-chloro-4-fluorophenyl)-N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-dihydropy
ridazine-3-carboxamide (22b).
1
Brown solid in 51.2% yield. m.p: 248.0–251.3 ºC. H NMR (400 MHz, DMSO-d
8.16 (s, 1H), 8.09 (s, 1H), 7.72 (d, J = 8.8 Hz, 3H), 7.57 (s, 1H), 7.29 (d, J = 9.0 Hz, 3H), 6.83 (s, 1H), 2.66 (s, 3H). C
NMR (101 MHz, DMSO-d ) δ 169.93, 159.61, 149.59, 143.18, 140.20, 137.24, 135.24 (2C), 126.80, 125.90, 124.48
6
) δ 11.76 (s, 1H), 8.95 (m, 1H),
1
3
6
-1
(
2C), 123.04, 122.97, 120.80, 119.71 (2C), 118.41 (2C), 118.19, 116.07, 108.21, 12.66. IR (KBr) cm : 3441, 3043,
+
1
4
2
687, 1601, 1560, 1490, 1329, 1201. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
15ClFN
7
O
3
: 492.0987, found,
92.0989. HPLC purity: 98.54% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 50:50; Column temperature:
o
6 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
1
-(4-chloro-3-(trifluoromethyl)phenyl)-N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4
-dihydropyridazine-3-carboxamide (22c).
1
6
Brown solid in 33.2% yield. m.p: 257.6–259.8 ºC. H NMR (400 MHz, DMSO-d ) δ 11.72 (s, 1H), 8.70 (d, J = 8.1

New Journal of Chemistry
Page 16 of 22
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1
1
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1
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2
2
2
2
2
2
2
2
2
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3
3
3
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3
3
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Hz, 1H), 8.22 (s, 1H), 8.11 (s, 1H), 8.03 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.7 Hz, 1H), 7D.7O4I: (1d0.,10J3=9/D80.1NJH00z5,75D
1
3
1
1
1
H), 7.28 (d, J = 7.2 Hz, 2H), 6.29 (d, J = 8.1 Hz, 1H), 2.66 (s, 3H). C NMR (101 MHz, DMSO-d
6
) δ 169.85, 159.87,
53.04, 148.36, 142.17, 142.13, 139.08 (2C), 136.24, 133.49 (2C), 130.76, 128.22, 127.86, 127.17, 126.69, 124.11,
-1
22.94 (2C), 121.44 (2C), 120.59, 115.04, 10.35. IR (KBr) cm : 3444, 3088, 1692, 1600, 1561, 1490, 132, 1191. TOF
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
MS ES+ (m/z): [M + H] , calcd for C24
H
15ClF
3
N
7
O
3
: 542.0955, found, 542.0953. HPLC purity: 97.49% (Column:
o
Dikma Luna C18; Mobile phase: H
mL/min).
2
O:MeCN = 50:50; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0
1
-(4-bromo-2-fluorophenyl)-N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-dihydropy
ridazine-3-carboxamide (22d).
1
Light yellow solid in 31.2% yield. m.p: 286.3–298.5 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.71 (s, 1H), 8.78 (s,
1
3
1
H), 7.93 (s, 1H), 7.75 (s, 1H), 7.61 (d, J = 8.6 Hz, 3H), 7.52 (s, 1H), 7.30 (s, 3H), 6.79 (s, 1H), 2.65 (s, 3H). C NMR
(
101 MHz, DMSO-d ) δ 170.11, 160.67, 154.53, 149.11, 145.65, 143.91, 140.15 (2C), 136.98, 129.87 (2C), 129.63,
6
-1
1
1
29.52, 126.95, 123.65 (2C), 122.17 (2C), 121.58, 120.79, 116.71, 114.65, 11.04. IR (KBr) cm : 3439, 3032, 1693,
+
605, 1563, 1502, 1329, 1215. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
15BrFN
7
O
3
: 536.0482, found, 536.0482.
o
HPLC purity: 98.60% (Column: Dikma Luna C18; Mobile phase: H
Wavelength: 280 nm; Flow rate: 1.0 mL/min).
2
O:MeCN = 60:40; Column temperature: 26 C;
1
-(4-methoxyphenyl)-N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridazin
e-3-carboxamide (22e).
1
Light yellow solid in 52.7% yield. m.p: 298.8–301.2 ºC. H NMR (400 MHz, DMSO-d
H), 8.20 (s, 1H), 7.79 (d, J = 27.6 Hz, 4H), 7.56 (d, J = 37.1 Hz, 4H), 7.36 (s, 2H), 6.90 (s, 2H), 2.74 (s, 3H). C NMR
101 MHz, DMSO-d ) δ 169.84, 160.15, 153.18, 148.19, 146.78, 143.42, 142.24 (2C), 136.35, 130.21 (2C), 130.17,
6
) δ 11.93 (s, 1H), 9.03 (s,
1
3
2
(
6
-1
1
1
29.10, 129.00, 126.26, 122.98, 121.90 (2C), 121.46 (2C), 120.95, 113.93, 55.61, 10.33. IR (KBr) cm : 3441, 3041,
+
688, 1592, 1562, 1502, 1329, 1216. TOF MS ES+ (m/z): [M + H] , calcd for C24
H
19
N
7
O
4
: 470.1577, found, 470.1572.
o
HPLC purity: 99.11% (Column: Dikma Luna C18; Mobile phase: H
Wavelength: 280 nm; Flow rate: 1.0 mL/min).
2
O:MeCN = 70:30; Column temperature: 26 C;
1
-(4-fluorophenyl)-N-(4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridazine-
3
-carboxamide (22f).
1
6
Yellow solid in 53.5% yield. m.p: 280.1–283.4 ºC. H NMR (400 MHz, DMSO-d ) δ 11.85 (s, 1H), 8.90 (s, 1H),
8
1
.52 (s, 1H), 8.10 (s, 1H), 7.80–7.72 (m, 3H), 7.61 (s, 1H), 7.40 (s, 1H), 7.29 (s, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.27 (s,
H), 2.66 (s, 3H). 13C NMR (101 MHz, DMSO-d
6
) δ 169.80, 160.05, 153.19, 148.03, 146.77, 142.49, 140.02 (2C),

Page 17 of 22
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39.46, 136.32, 126.26, 124.46 (2C), 124.37, 122.99 (2C), 121.47 (2C), 120.90, 117.10, 116.87, 113.9D2O,I:1100..13023.9I/DR0N(KJ0B0r5)75D
4
5
-1
+
7 3
cm : 3440, 3082, 1688, 1602, 1563, 1487, 1330, 1216. TOF MS ES+ (m/z): [M + H] , calcd for C23H16FN O :
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
2
58.1377, found, 458.1374. HPLC purity: 97.41% (Column: Dikma Luna C18; Mobile phase: H O:MeCN = 70:30;
o
Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3
carboxamide (22g).
-
1
Brown solid in 37.2% yield. m.p: 261.9–262.1 ºC. H NMR (400 MHz, DMSO-d
6
) δ 12.01 (s, 1H), 8.95 (d, J = 7.7
Hz, 1H), 8.16 (s, 1H), 7.88 (d, J = 12.6 Hz, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.44 (s, 3H), 7.31 (s,
1
1
1
H), 6.86 (d, J = 7.7 Hz, 1H), 2.67 (s, 3H). 13C NMR (101 MHz, DMSO-d
42.30, 130.21 (2C), 129.94, 129.12 (2C), 125.99, 125.12 (2C), 121.87 (2C), 121.06, 120.74, 116.76, 114.61, 108.86,
6
) δ 169.73, 160.46, 151.99, 147.01, 143.38,
-1
+
08.63, 10.35. IR (KBr) cm : 3444, 3048, 1689, 1604, 1567, 1489, 1331, 1214. TOF MS ES+ (m/z): [M + H] , calcd
for C23
H
16FN
7
O
3
: 458.1377, found, 458.1371. HPLC purity: 96.30% (Column: Dikma Luna C18; Mobile phase:
o
H
2
O:MeCN = 70:30; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
1
-(3-chloro-4-fluorophenyl)-N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-d
ihydropyridazine-3-carboxamide (22h).
1
6
Brown solid in 47.2% yield. m.p: 298.7–301.4 ºC. H NMR (400 MHz, DMSO-d ) δ 11.88 (s, 1H), 8.13 (s, 1H),
8
.05 (s, 1H), 7.87 (d, J = 12.1 Hz, 1H), 7.78 (s, 1H), 7.60 (s, 2H), 7.45 (s, 2H), 7.30 (s, 1H), 6.85 (s, 1H), 2.67 (s, 3H).
13C NMR (101 MHz, DMSO-d
) δ 169.65, 160.33, 151.99, 147.07, 142.46, 140.25, 138.91 (2C), 126.01, 125.13, 124.40
6
-1
(2C), 122.94, 122.86 (2C), 120.81 (2C), 118.44, 118.22, 116.82, 114.63, 108.90, 10.36. IR (KBr) cm : 3439, 3039,
+
1
5
2
690, 1604, 1565, 1498, 1333, 1219. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
14ClF
2
N
7
O
3
: 510.0893, found,
10.0893. HPLC purity: 96.61% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 50:50; Column temperature:
o
6 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
1
-(4-chloro-3-(trifluoromethyl)phenyl)-N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-
4-oxo-1,4-dihydropyridazine-3-carboxamide (22i).
1
Light yellow solid in 45.2% yield. m.p: 311.2–313.0 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.84 (s, 1H), 9.02 (s,
1
3
H), 8.21 (s, 1H), 8.10 (d, J = 22.5 Hz, 2H), 7.88 (d, J = 19.2 Hz, 2H), 7.44 (s, 2H), 7.30 (s, 1H), 6.88 (s, 1H), 2.67 (s,
1
3
6
H). C NMR (101 MHz, DMSO-d ) δ 169.79, 160.22, 151.99, 148.26, 142.36, 142.16, 138.91, 137.57, 135.20, 133.57
-1
(2C), 131.08, 127.27, 126.00 (2C), 125.16, 121.41, 120.77 (2C), 116.82, 114.65 (2C), 108.91, 10.37. IR (KBr) cm :
+
3
443, 3086, 1707, 1598, 1564, 1489, 1326, 1219. TOF MS ES+ (m/z): [M + H] , calcd for C24
H
14ClF
4
N
7
O
3
: 560.0861,
found, 560.0865. HPLC purity: 97.38% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 50:50; Column

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DOI: 10.1039/D0NJ00575D
o
temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
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-(4-bromo-2-fluorophenyl)-N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-4-oxo-1,4-d
ihydropyridazine-3-carboxamide (22j).
1
Grayish white solid in 33.2% yield. m.p: 290.7–292.5 ºC. H NMR (400 MHz, DMSO-d
6
) δ 11.77 (s, 1H), 8.34 (d,
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
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4
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7
8
9
0
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8
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J = 7.8 Hz, 1H), 7.79 (d, J = 10.5 Hz, 1H), 7.63 (s, 1H), 7.53 (s, 3H), 7.43 (s, 1H), 7.31 (s, 1H), 6.35 (d, J = 7.8 Hz, 1H),
5
.67 (s, 1H), 2.67 (s, 3H). 13C NMR (101 MHz, DMSO-d
37.16, 130.54 (2C), 129.46, 128.77, 126.33, 125.45, 122.21 (2C), 121.41 (2C), 117.09, 114.93, 109.21, 108.97, 10.68.
6
) δ 170.08, 160.76, 147.36, 142.63, 139.24, 138.07 (2C),
1
-1
+
IR (KBr) cm : 3442, 3053, 1686, 1610, 1565, 1486, 1327, 1210. TOF MS ES+ (m/z): [M + H] , calcd for
C
23
H
14BrF
2
N
7
O
3
: 554.0388, found, 554.0388. HPLC purity: 98.65% (Column: Dikma Luna C18; Mobile phase:
o
H
2
O:MeCN = 60:40; Column temperature: 26 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (22k).
White solid in 41.8% yield. m.p: 301.7–307.5 ºC. H NMR (400 MHz, DMSO-d
1
6
) δ 12.16 (s, 1H), 9.14 (s, 1H),
8.35 (s, 1H), 8.07 (d, J = 11.9 Hz, 1H), 7.92 (s, 3H), 7.69 (d, J = 43.0 Hz, 5H), 7.50 (s, 1H), 7.05 (s, 1H), 2.86 (s, 3H).
13C NMR (101 MHz, DMSO-d
6
) δ 168.50, 160.25, 151.99, 147.06, 143.31, 138.91 (2C), 129.11, 128.96, 128.83 (2C),
-1
1
3
4
2
26.00 (2C), 125.12, 120.89 (2C), 120.66, 120.23, 116.77, 116.20, 114.63, 108.65, 55.36, 10.36. IR (KBr) cm : 3442,
+
051, 1689, 1604, 1567, 1489, 1331, 1214. TOF MS ES+ (m/z): [M + H] , calcd for C24
H
18FN
7
O
4
: 488.1483, found,
88.1480. HPLC purity: 99.71% (Column: Dikma Luna C18; Mobile phase: H
2
O:MeCN = 70:30; Column temperature:
o
6 C; Wavelength: 280 nm; Flow rate: 1.0 mL/min).
N-(3-fluoro-4-((3-methyl-[1,2,4]triazolo[4,3-a]pyrazin-8-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydropy
ridazine-3-carboxamide (22l).
1
6
Brown solid in 36.2% yield. m.p: 289.5–295.1 ºC. H NMR (400 MHz, DMSO-d ) δ 11.72 (s, 1H), 8.99 (s, 1H),
8.64 (s, 1H), 8.24 (s, 1H), 7.96 (s, 1H), 7.87 (s, 1H), 7.71 (s, 2H), 7.47 (s, 2H), 7.38 (s, 1H), 6.94 (s, 1H), 2.76 (s, 3H).
13C NMR (101 MHz, DMSO-d
6
) δ 170.37, 161.11, 152.70, 148.58, 147.76, 143.24, 140.67 (2C), 139.61, 126.71, 125.81,
-
1
1
25.14, 125.05 (2C), 121.69, 117.81 (2C), 117.57 (2C), 117.47, 115.31, 109.60, 11.06. IR (KBr) cm : 3443, 3089, 1692,
+
1
604, 1569, 1487, 1331, 1214. TOF MS ES+ (m/z): [M + H] , calcd for C23
H
15
F
2
N
7
O
3
: 476.1283, found, 476.1282.
o
HPLC purity: 98.49% (Column: Dikma Luna C18; Mobile phase: H
Wavelength: 280 nm; Flow rate: 1.0 mL/min).
2
O:MeCN = 70:30; Column temperature: 26 C;
3
.3 Anti-tumor assay
The anti-tumor activities of target compounds (21a-l and 22a-l) were evaluated with A549, MCF-7 and Hela cell

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lines by the standard MTT assay in vitro, with Foretinib as positive control. Specific operations wDeOrIe: 10b.a10s3e9d/Do0nNJo0u0r575D
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previous research .
.4 Cell apoptosis assay by flow cytometry
A549 cells were seeded in a 6-well plate at 1×10 cells per well and incubated for 24 h, and then treated with
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compound 22i for 72 h. Cells were harvested and fixed with ice-cold 70% ethanol at 4 ºC for 12 h, and then ethanol was
removed and the cells were washed with cold PBS. Cells were then incubated in 0.5 mL of PBS containing 1 mg/mL
Rnase for 30 min at 37 ºC. After which, the cells were stained with propidium iodide (PI) in the dark for 30 min. Finally,
the DNA contents were measured by flow cytometer.
3
.5 Kinase selectivity assay
The most compounds were tested for their activity against c-Met through the mobility shift assay, with Foretinib
1
6
being positive controls. Specific operations were based on according to our previous research .
3
.6 Acridine orange single staining
The cancer cell apoptotic of target compound 22i were evaluated with A549 cancer cell line by acridine orange
single staining. Specific operations were based on according to our previous research 18.
3
.7 Molecular docking study
The co-crystal structure of c-Met (PDB code 3LQ8) was chosen as the template to generate the docking modes.
For the preparation of ligands, the 3D structures were generated and their energy minimizations were performed by
AutoDock 4.2 software (The Scripps Research Institute, USA). After the protein preparation process of flexible docking,
the whole c-Met was defined as a receptor and compound 22i was placed during the molecular docking procedure.
Types of interactions of the docked c-Met with ligand was analyzed and then the docking conformations was selected
and saved based on calculated energy. All the docking results were processed and modified in PyMOL 1.8.x software
(https://pymol.org).
4
Conclusions
In summary, based on the structure based drug design (SBDD) strategy and bioisosterism principles, a series of
[1,2,4]triazolo[4,3-a]pyrazine derivatives bearing 4-oxo-pyridazinone moiety as potential c-Met kinase inhibitors were
designed and evaluated for the IC50 values against three cancer cell lines (A549, MCF-7 and Hela). Most compounds
were selected to evaluate for the activity against c-Met kinase. Among them, the most promising compound 22i showed
superior anti-tumor activity for A549, MCF-7 and Hela cell lines with IC50 values of 0.83 ± 0.07 µM, 0.15 ± 0.08 µM
and 2.85 ± 0.74 µM, respectively, which also possessed excellent c-Met kinase inhibition on nanomolar level (IC50 = 48

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nM). According to the result of AO single staining and cell apoptosis assay, it claimed that compoundDO2I2: 1i0c.1o0u3l9d/Di0nNdJu0c0e575D
remarkable apoptosis of A549 cells in a dose-dependent manner. Furthermore, compound 22i could stimulate A549
cells arrest at G0/G1 phase. By far, the data indicated that compound 22i may be a potential c-Met kinase inhibitor and
further research will be carried out to identify more anti-tumor mechanisms.
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Conflicts of interest
There are no conflicts to declare.
6
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No.
1662014, No. 21967009), Natural Science Foundation of Jiangxi, China (20181BBG70003, 20181ACB20025 ，
0192BAB215061), Science and Technology Project Founded by the Education Department of Jiangxi Province, China
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2
(
GJJ190583).
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we disclosed the preparation and biological evaluation of a series of [1,2,4]triazolo[4,3-a]pyrazine derivatives
bearing 4-oxo-pyridazinone moiety, which demonstrated potent inhibition of c-Met kinase, culminating in the
discovery of 22i.