Synthesis and bioevaluation study of novel N-methylpicolinamide and thienopyrimidine derivatives as selectivity c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.11.039

Four series of N-methylpicolinamide moiety and thienopyrimidine moiety bearing pyridazinone were designed and synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, HepG2 and MCF-7) and some selected compounds were

Full text of DOI:10.1016/j.bmc.2017.11.039

Accepted Manuscript
Synthesis and bioevaluation study of novel N-methylpicolinamide and thieno-
pyrimidine derivatives as selectivity c-Met kinase inhibitors
Linxiao Wang, Shan Xu, Xiuying Chen, Xiaobo Liu, Yongli Duan, Dejia Kong,
Dandan Zhao, Pengwu Zheng, Qidong Tang, Wufu Zhu
PII:
S0968-0896(17)32047-3
https://doi.org/10.1016/j.bmc.2017.11.039
BMC 14091
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
18 October 2017
20 November 2017
24 November 2017
Please cite this article as: Wang, L., Xu, S., Chen, X., Liu, X., Duan, Y., Kong, D., Zhao, D., Zheng, P., Tang, Q.,
Zhu, W., Synthesis and bioevaluation study of novel N-methylpicolinamide and thienopyrimidine derivatives as
selectivity c-Met kinase inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.
2017.11.039
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Synthesis and bioevaluation study of novel N-methylpicolinamide and thienopyrimidine derivatives as
selectivity c-Met kinase inhibitors
Linxiao Wang1,†, Shan Xu1,†, Xiuying Chen2, Xiaobo Liu1, Yongli Duan1, Dejia Kong1, Dandan Zhao1, Pengwu
Zheng1, Qidong Tang1,*, Wufu Zhu1, *
1Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science &
Technology Normal University, Nanchang 330013, PR China
2College of Pharmaceutical Sciences, Zhengzhou University; Key Laboratory of Advanced Drug Preparation
Technologies, Zhengzhou University, Henan, Zhengzhou 450001, China
*Correspondence author.
Tel/Fax: +86 791 8380-2393;
E-mail address: zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu).tangqidongcn@126.com
Abstract: Four series of N-methylpicolinamide moiety and thienopyrimidine moiety bearing pyridazinone were
designed and synthesized and evaluated for the IC50 values against three cancer cell lines (A549, HepG2 and
MCF-7) and some selected compounds were further evaluated for the activity against c-Met, Flt-3, VEGFR-2,
c-Kit and EGFR kinases. Three compounds (35, 39 and 43) showed more active than positive control Foretinib
against A549, HepG2 and MCF-7 cell lines. The most promising compound 43 showed superior activity against
A549, HepG2 and MCF-7, with the IC50 values of 0.58 0.15 µM, 0.47 0.06 µM and 0.74 0.12 µM, which
were 3.73-5.39 fold more activity than Foretinib, respectively. The experiments of enzyme-based showed that 43
restrain the c-Met selectively, with the IC50 values of 16 nM, which showed equal activity to Foretinib (14 nM)
and better than the compound 5 (90 nM). Moreover, AO and Annexin V/PI staining and docking studies were
carried out.
Keywords: N-methylpicolinamide; Thienopyrimidine; Pyridazinone; c-Met; Bioevaluation
1
† These authors contribute equally to this work

1. Introduction
Receptor tyrosine kinases (RTKs) play a significant roles in signal transduction pathways and the treatment of
various types of cancers.[1] c-Met [the receptor for hepatocyte growth factor/scatter factor, (HGF/SF)][2] and
members of the vascular endothelial growth factor receptor (VEGFR)[3] family1 including VEGFR1 (Flt-1),
VEGFR2 (KDR) and VEGFR3 (Flt-4) are among the RTKs under investigation as potential targets for the
development of small molecule cancer therapeutics. Among these kinases, the c-Met kinase is one of hot spot in
the anticancer field.
c-Met inhibitors are multitarget compounds which can inhibit the c-Met kinase selectively and used in the
treatment of various types of cancers[4]. Cabozantinib, the first small-molecule c-Met inhibitor, was approved by
FDA on November 29, 2012. In recent years, many Cabozantinib derivatives were reported, such as Foretinib,
Sorafenib, 4, 5 and 6 (Fig.1 ). [5-8]
Many researches showed that nearly all the Cabozantinib derivatives show excellent activity and contain two
obvious structural characteristics: HD/HR (hydrogen bond donor and hydrogen bond receptor) and HI
(hydrophobic interaction) can be summarized (Fig.1). And the main modification of these different series of
derivatives was focused on HD/HR moiety which could form hydrogen-bond donor or receptor.[9] What's more,
the HI moiety may form hydrophobic bond with c-Met and bound to c-Met nicely.
In our previous research, a series of pyrrolo[2,3-b]pyridine derivatives (compound 7, Fig.1) were designed and
synthesized as potent c-Met inhibitors.[10] Most of these compounds exhibited potent activity, especially the most
promising compound 7 with the IC50 values in the nanomole level. The SARs and docking study exhibited that
∗Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu), tangqidongcn@126.com

6-oxo-pyridazinone may be beneficial to the in vitro activity.
In this work, further modification was concentrated on pyrrolo[2,3-b]pyridine moiety which belonged to the
HD/HR. Firstly, according to our previous work, the 6-oxo-pyridazinone scaffold of compound 7 was retained.
And inspired by Sorafenib, the pyrrolo[2,3-b]pyridine moiety was replaced with N-methylpicolinamide to study
the effects of increasing the molecular flexible to the compounds activity, and the first series compounds 24-29
were yield. Then, inspired by the compound 5 and compound 6, the thieno[3,2-d]pyrimidine was introduced to
investigate the effects of increasing the number of hydrogen donor and receptor to activity and the second series
compounds (30-37 and 38-44) were prepared. Consequently, thieno[3,2-d]pyrimidine was replaced by
thieno[2,3-d]pyrimidine to study the effects on the position of S atom to activity, and the third series compounds
(45-51 and 52-58) were prepared. What's more, according to the SARs of the first three series compounds, we
found that the 4-(2-fluoro-4-phenoxy)thieno[3,2-d]pyrimidine moiety was a privileged structure. Thus, the
privileged structure was retained, and then the 4-oxo-pridazinone was introduced into the HD moiety the fourth
series compounds 59-63 was obtained. As a result, four series of compounds 24-29, 30-37, 38-44, 45-51, 52-58
and 59-63 were synthesized. And the design strategy for all target compounds was described in Fig.2.
Herein we disclosed the synthesis and antitumor activity of N-methylpicolinamide moiety and thienopyrimidine
moiety bearing pyridazinone against A549 (human lung cancer), HepG2 (human liver cancer), MCF-7 (human
breast cancer) cancer cell lines, and c-Met kinase. Moreover, experiments of enzyme-based selectivity, acridine
orange single staining and docking studies were presented within this paper as well.
Fig. 1 Structures characteristic of small-molecule tyrosine kinases inhibitors

Fig. 2 Structures and design strategy for target compounds 24-63.
2. Chemistry
The preparations of target compounds 24-63 were described in scheme 1
Substituted anilines 8a-h were diazotized and then reacted with ethyl-acetoacetate to get compounds 10a-h,
which condensed with DMF-DMA or ethyl (triphenylphosphoranylidene)acetate to yield compounds 11a-h and
12a-e with the absence of different catalyst, respectively. Following by hydrolysis reaction and chlorination
reaction, 15a-h and 16a-e were obtained.
The key intermediates 19b-c were synthesized from methyl 2-amino-2,3-dihydrothiophene-3-carboxylate and
methyl 3-amino-2,3-dihydrothiophene-2-carboxylate 17a-b via cyclization and chlorination with formamidine
acetate and phosphorus oxychloride, respectively. Then, intermediates 21a, 22a-b and 23a-b were obtained by
substitution reaction with 4-aminphenol or 2-fluoro-4-aminphenol 20a-b. Finally, substitution reaction of amides
21a, 22a-b and 23a-b with the carbonyl chloride 15a-h/16a-e. promoted by DIPEA in dichloromethane at room
temperature to obtain target compounds 24-63, respectively.

O
H
N
F
N
N
R
F
NH2
O
O
O
24-29
HN
N
OH
Cl
HN
O
R1
O
N
H
N
O
R1
O
N
HN
21a
N
N
R2
NH2
O
O
19a
15a-h
or 16a-e
20a-b R1=H/F
R1
NH2
X
Y
N
O
g
O
Cl
N
20-37:X=S,Y=C,R1= H
38-44:X=S,Y=C,R1= F
O
N
N
X
X
Y
X
Y
O
NH
N
X
Y
N
Y
f
45-51:X=C,Y=S,R1= H
52-58:X=C,Y=S,R1= F
NH2
e
N
17a-b
19b-c
18a-b
O
H
17a:X=S,Y=C
17b:X=C,Y=S
19b:X=S,Y=C
19c:X=C,Y=S
18a:X=S,Y=C
18b:X=C,Y=S
22a:X=S,Y=C,R1= H
22b:X=S,Y=C,R1= F
F
N
N
N
22a:X=C,Y=S,R1= H
22b:X=C,Y=S,R1= F
R
O
O
N
S
N
59-63
Scheme 1. Synthetic route of target compounds.
Reagents and conditions: (a) sodium nitrite, EtOH, ethyl acetoactate, 0 ℃ , 30 minute; (b) ethyl
(triphenylphosphoranylidene)acetate, Et2NH, 85℃, 6 h; (c) DMF-DMA, 100 ℃, reflux, 6-10 h; (d) sodium
carbonate, ethanol/H2O (5:1), 80℃, 3-5 h; (e) oxalyl chloride, DMF, CH2Cl2, rt, 5 minute; (f) Formamidine
acetate, EtOH, 110℃, 2h, 90％; (g) phosphorus oxychloride, DMF, 110℃, 1h, 95％; (h) sodium carbonate,
sodium hydroxide, tetrahydrofuran, 1,4-dioxane/H2O (5:1), 80℃, 1 h; (i) DIPEA, CH2Cl2, rt, 0.5 h.
3. Results and discussion
3.1. Biological evaluation
Taking Foretinib as reference compounds, the target compounds 24-63 were evaluated for the cytotoxicity
against three cancer cell lines (A549, HepG2 and MCF-7) by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) cell proliferation assay. Compounds 35, 39 and 43 were further evaluated for the activity against

c-Met, Flt-3, VEGFR-2, c-Kit and EGFR kinases. The results expressed as IC50 values were summarized in Tables
1-6 and the values were the average of at least two independent experiments.
Table 1. Structures and activity of target compounds 24-29
Compounds
IC50a (ꢀM)
HepG2
NA
R2
A549
NA
MCF-7
35.74 1.53
9.36 1.28
NA
2-CF3
2-F-4-Br
2-OCF3
3-F
24
7.93 1.73
NA
2.83 0.45
NA
25
26
9.54 1.89 10.21 1.19 6.41 0.92
11.47 1.51 10.64 1.06 8.38 0.93
27
28
4-CH3
2-F
NA
NA
NA
29
Foretinibb
3.13 0.67
2.27 0.25
2.76 0.53
-
aThe values an average of two separate determinations.
bUsed as a positive control.
c NA: Not active (IC50 > 40 ꢀM).
Table 2. Structures and activity of target compounds 30-44
Compounds
IC50a (ꢀM)
R1
R2
A549
HepG2
MCF-7
H
2-CF3
2-F-4-Br
2-OCF3
3-F
8.85 1.38
7.93 1.73
10.14 1.18
9.54 1.89
11.47 1.18
2.36 0.23d
8.18 1.38
9.23 1.25
10.64 1.06
24.99 1.43
10.21 1.19
24.99 1.43
2.11 0.47
9.23 1.25
5.55 0.86
9.36 1.28
23.79 1.49
6.41 0.92
23.79 1.49
4.4 0.63
30
31
32
33
34
35
36
H
H
H
H
H
H
4-CH3
4-F
3,4-diF
5.55 0.86

H
F
F
F
F
F
F
F
2,5-diC
2-CF3
2-F-4-Br
2-OCF3
3-F
16.4 1.16
4.68 0.86
2.98 0.73
5.72 0.62
5.22 0.89
5.89 0.89
0.58 0.15
4.59 1.28
3.13 0.67
17.92 1.62
9.38 1.62
3.35 0.77
15.66 1.45
7.81 1.16
17.25 1.43
0.47 0.06
6.93 0.89
2.27 0.25
11.93 1.06
6.02 0.94
1.55 0.26
11.49 1.13
9.05 1.07
7.25 1.51
0.74 0.12
6.9 1.11
37
38
39
40
41
4-CH3
4-F
42
43
3,4-diF
-
44
Foretinibb
2.76 0.53
aThe values an average of two separate determinations.
bUsed as a positive control.
c NA: Not active (IC50 > 40 ꢀM).
dthe data of bold showed more activity than Foretinib
Table 3. Structures and activity of target compounds 45-58
Compounds
IC50a (ꢀM)
R1
R2
A549
HepG2
NA
MCF-7
NA
H
H
H
H
H
H
H
F
2-CF3
2-F-4-Br
2-OCF3
3-F
19.93 1.52
12.77 1.28
45
46
6.3 0.84
15.2 1.29
15.50 1.36 14.44 1.37 11.04 1.15
16.40 1.76 18.51 1.63 27.18 2.78
47
48
4-CH3
4-F
NAc
NA
NA
49
50
7.05 1.02
6.46 0.85 20.96 0.84
3,4-diF
2-CF3
2-F-4-Br
2-OCF3
3-F
13.50 2.39 16.77 1.92 16.12 2.27
16.28 1.38 14.65 1.20 13.8 1.25
11.47 1.24 13.84 1.18 13.25 1.22
51
52
F
53
54
F
NA
19.51 1.46 24.45 1.42 17.32 1.35
NA NA NA
2.62 0.59 12.47 1.09 12.27 1.32
NA
NA
F
55
F
4-CH3
4-F
56
57
F
58
Foretinibb
F
3,4-diF
-
15.70 2.73
3.13 0.67
NA
20.56 1.96
2.76 0.53
2.27 0.25

aThe values an average of two separate determinations.
bUsed as a positive control.
c NA: Not active (IC50 > 40 ꢀM).
Table 4. Structures and activity of target compounds 59-63
Compounds
IC50a (ꢀM)
HepG2
R2
A549
MCF-7
4-F
3-Cl-4-F
4-Br
8.23 1.34
9.16 1.51
5.72 0.69
59
9.65 1.45 10.64 1.24 6.63 0.43
7.84 1.25 10.73 1.16 9.62 1.72
19.73 1.16 13.52 1.31 12.38 0.73
11.62 1.04 24.25 1.83 23.15 1.73
60
61
62
H
4-OCH3
-
63
Foretinibb
3.13 0.67
2.27 0.25
2.76 0.53
aThe values are were an average of two separate determinations.
bUsed as a positive control.
Table 5. c-Met kinase inhibitory activity of selected compounds 35, 39 and 43.
Compounds
No.
IC50a (nM)
c-Met
R1
R2
35
39
H
F
F
-
4-F
48
32
19
90
14
2-F-4-Br
4-F
43
Compound 5c
Foretinibb
-
-
-
aThe values are an average of two separate determinations.
bUsed as a positive control.
cthe data of c-Met kinase activity is from Gaudette 's work[7]
Table 6.Tyrosine kinases inhibitory activity of 43

Kinase
c-Met
Enzyme IC50 (nM)
19
Flt-3
950
VEGFR-2
c-Kit
450
3700
6900
EGFR
As showed in Tables 1-6, many compounds displayed excellent anticancer activity against three cancer cells
lines (A549, HepG2 and MCF-7) with potency from single-digit µM to nanomole range. Among all the
compounds, three selected compounds (35, 39 and 43) showed better activity against A549, HepG2 and MCF-7
cells lines compared with the positive drug Foretinib. The most promising compound 43 exhibited the best activity
against A549, HepG2and MCF-7 cell lines with the IC50 values of 0.58 0.15 µM, 0.47 0.06 µM and 0.74
0.12 µM, which were 3.73-5.39 fold more activity than the lead drug Foretinib, respectively. The results claimed
that the thieno[3,2-d]pyrimidine derivatives modified with 6-oxo-pyridazinone moiety was a privileged scaffold
for anticancer activity.
What’s more, R1 group of compounds sbstituted with F atom showed more activity than no substituent against
A549 and HepG2 cell lines. The trend also could be observed in compounds 24-29, 30-44 and so on.
Furthermore, different R2 substituent of the aryl group also affected the cytotoxicity of target compounds. In
general, it’s seem to be that target compounds with electron drawing groups(EWGs) have a significant impact on
the in vitro activity. For example, the compounds substituted with F, Cl and Br showed more activity than no
substituent or the electron donating groups (EDGs). Moreover, the F atom on the 4-C position showed the best
activity, such as compounds 35, 43, 57 and so on. The results suggested that the F atom on the 4-C position could
improve the compounds activity against HepG2 significantly, with IC50 values of 0.47 0.06 µM.
Activity against c-Met kinase of compounds 35, 39 and 43 was further carried out in this paper. As shown in
Table 5, we can easily find that the compounds 39 and 43 are more active than compound 35, especially
compound 43 with IC50 value of 19 nM against c-Met kinase. The results prompt us that the target compounds

may be a series of c-Met kinase inhibitors. Furthermore, compound 43 exhibited medium inhibitory effects against
Flt-3 (IC50 = 950 nM), VEGFR-2 (IC50 = 450 nM), c-Kit (IC50 = 3700 nM) and EGFR (IC50 = 6900 nM), which
was 59.4-, 23.7-, 194.8- and 431.3- fold lower than that of c-Met, respectively (Table 6). These data indicated that
compound 43 could inhibit the c-Met kinase selectively.
3.2. Morphologic changes of HepG2 cells under inverted microscopy and fluorescence microscopy
In order to investigate the target compound whether could induce the apoptosis of HepG2, acridine orange (AO)
was carried out. As show in the Fig. 3, the control group cell (Fig. 3a) was treated with nothing and showed
normal HepG2 cell shape. But in the Fig. 3b, the cell shape was abnormal with cell shrinkage, chromatin
condensation after 0.47 ꢀM concentration of compound 43 acted on the HepG2 cells. It claimed that the
compounds 43 could induce apoptosis of HepG2 cells.
Fig. 3 Morphologic changes of HepG2 cells under inverted microscopy and fluorescence microscopy
3.3. Apoptosis result was carried out by flow cytometry
In order to further reveal the mechanism of apoptosis of HepG2 induced by the compound 43,the experiment
of Annexin V/PI staining was carried out ( Fig. 4). Compared to the control group, compound 43 could induce the
late apoptotic significantly, with 21.01% late apoptotic and only 13.89% early apoptotic treated with 0.94 ꢀM.
And the same trend was also observed in other concentration. In addition, the number of apoptotic and dead cells
increased with increasing concentration of the compound. Therefore, it can be concluded that the target compound
43 could induce late apoptosis and in a concentration- dependent manner.

Fig. 4 Annexin V (x-axis) versus PI (y-axis) analyses show in the Fig.4a and 4b was summarized by a drawing
Fig.4b.
3.4. Molecular docking study
To explore the binding modes of target compounds with the active site of c-Met, molecular docking
simulation studies were carried out by using SYBYL 6.9.1 (Tripos, St. Louis, MO, USA), pymol 1.8.6 and
ChemDraw. And basing on the in vitro inhibition results, we selected compound 43, our best c-Met inhibitor in
this study, as the ligand example, and the structure of c-Met was selected as the docking model (PDB ID code:
3LQ8 ). [13] The binding modes of compound 43 and c-Met were depicted in Fig.5. Visual inspection of the pose of
compound 43 into c-Met binding site revealed that compound 43 was tightly embedded into the active binding
pocket. In the binding mode, compound 43 was potently bound to the active binding site of c-Met via three
hydrogen bonds and two pi–pi interactions. The N atoms of thieno[3,2-d]pyrimidine moiety and the O atom
formed one hydrogen bond with the ASP 1222, respectively. And the O atom of 6-oxo-pyridazinone contact with
MET 1160 via one hydrogen bond; contribute to the hydrogen bonding interaction together, being a probable
explanation for its nice activity. On the other hand, the two substituted aryl group formed one pi–pi interaction

with PHE 1223 and TYR 1159 respectively. In general, these results of the molecular docking study showed that
compound 43 may be potential inhibitor of c-Met.
a
Fig. 5 . The 3D interaction map between the 43 and c-Met, the hydrogen bonds was colored with red and the pi-pi
bonds was colored with black, respectively (Fig.5a). 2D interaction map between the 43 and c-Met, the hydrogen
bonds was colored with red and the pi-pi bonds was colored with black (Fig.5b). 43 occupied the activity package
of protein, and the hydrogen bonds was colored with red while the pi-pi interaction was colored with
black(Fig.5c).
4. Conclusions
In summary, we designed and synthesized four series of N-methylpicolinamide moiety and thienopyrimidine
moiety bearing pyridazinone and evaluated for the IC50 values against three cancer cell lines (A549, HepG2 and
MCF-7) and some selected compounds were further evaluated for the activity against c-Met, Flt-3, VEGFR-2,

c-Kit and EGFR kinases. Three compounds (35, 39 and 43) showed more active than positive control Foretinib
against one or more cell lines. The most promising compound 43 showed superior activity against A549, HepG2
and MCF-7, with the IC50 values of 0.58 0.15 µM, 0.47 0.06 µM and 0.74 0.12 µM, which were 3.73-5.39
fold more activity than the lead drug Foretinib, respectively. The experiments of enzyme-based showed that 43
could inhibitor the c-Met selectively, with the IC50 values of 19 nM, which show equal activity to Foretinib and
better than the compound 5. Moreover, SARs and docking studies indicated that thieno[3,2-d]pyrimidine
derivatives bearing 6-oxo-pyridazinone were favorable to the activity. Especially, the 4-C position of aryl group
substituted with F atom show the best activity. What’s more, According to the result of AO single staining and
Annexin V/PI staining, it's claimed that the 43 could induce late apoptosis of HepG2 cells by a concentration-
dependent manner.
5. Experimental
5.1 Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were 4 uncorrected. NMR spectra were performed using Bruker 400 MHz spectrometers (Bruker
Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in ESI mode
on Agilent 1100 LCMS (Agilent, Palo Alto, CA, USA). All the materials were obtained from commercial
suppliers and used without purification, unless otherwise specified. Yields were not optimized. TLC analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). All the materials were obtained from
commercial suppliers and used without purification, unless otherwise specified. Yields were not optimized.
5.2 General procedure for preparation of compounds 10a-h
Appropriate substituted anilines 8a-h (0.101 mol) was added to the mixture solution of hydrogen chloride/water
(1:1, 10 ml), sodium nitrite (0.1mol) and stirred for 5 minute at 0 , respectively. Then the mixture was added to
the ethanol solution which ammonium acetate (1 mol) and ethyl acetoacetate (0.1 mol) were dissolved in. The
reaction was monitored by TLC until completed. Finally the solution was filtered and washed with a plenty of
water to give a yellow solid.
5.3 General procedure for preparation of 11a-h
Intermediate 10a-h (0.105 mol), ethyl (triphenylphosphoranylidene) acetate (0.15 mol) and Et2NH (0.1 mol) were
dissolved in DMSO, heated to 85 from room temperature and stirred for 4 hours. After the solution cooled to the

room temperature, it was poured to water, extracted with dichloromethane, washed with brine (50ml×3), dried
with anhydrous sodium sulfate, then the organic layer was concentrated in vacuum to give the product 11a-h.
5.4 Preparation of compounds 12a-e
The yellow solid 10a-h (0.115 mol) was dissolve in the DMF-DMA (50 ml), heated from 0 to 100 and stirred
for 5-6 hour. After the reaction was completed, poured the mixture to the petroleum ether and filtered to obtain the
compounds 12a-e .
5.5 General procedure for preparation of 13a-h and 14a-e
Appropriate intermediate 11a-h (0.11 mol) or 12a-e (0.104 mol), sodium carbonate (0.2 mol) were dissolved in
Ethanol/H2O (50ml, 5:1), and heated to 80 for 5–6 hour. Subsequently, the mixture solution acidified to pH 2–
3 to yield the compounds 13a-h and 14a-e, respectively.
5.6 General procedure for the key intermediate 15a-h and 16a-e
The compounds 13a-h or 14a-e (0.001 mol) and appropriate DMF (0.0001 mol) were dissolved in
dichloromethane, then appropriate oxalyl chloride was added slowly and monitored by TLC. The solution was
used for next step without further purification.
5.7 General procedure for the key intermediate 18a-b
2-Aminothiophene-3-carboxylate or methyl 3-aminothiophene-2-carboxylate 17a-b (0.1 mol), formamidine
acetate (0.15 mol) were dissolved in ethanol (100 mL), and heated to 100℃ for 2h. The reaction was monitored
by TLC until completed. Finally the solution was cooled and pour into ice water stirred for 0.5 hour to give a gray
solid.
5.8 General procedure for the key intermediate 19a-b
Thieno[2,3-d]pyrimidin-4(3H)-one or thieno[3,2-d]pyrimidin-4(3H)-one 18a-b (0.101 mol) was dissolved in
toluene, and phosphorus oxychloride (3 mL), DMF (0.05 mL) was added slowly. Then the solution was heated to
110℃ and stirring for 1hour. The reaction was monitored by TLC until completed. The solution was cooled to rt
and pour into ice water slowly to obtain a white solid.
5.9 General procedure for the key intermediate 21a, 22a-b and 23a-b

4-Aminophenol or 2-fluoro-4-aminphenol 20a-b (0.1mol) which was dissolved in tetrahydrofuran (50ml), was
added to a 1,4-dioxane/H2O (50ml, 5:1) solution of compounds 19a-c, sodium carbonate and hydrogen sodium at
80 for 2 hours. Then the solution was concentrated in vacuum and washed with water, filtered to give a solid .
5.10. General procedure for the preparation of target compounds 24-29, 30-37, 38-44, 45-51, 52-58 and
59-63.
A solution of phenylpyrdazinone carbonyl chloride 15a-h or 16a-e (0.82 mmol) in dichloromethane (10 mL) was
added drop-wise to a solution of aniline 21a or 22a-b or 23a-b (0.41 mmol) and diisopropylethylamine (0.49
mmol) in dichloromethane (10 mL) in an ice bath. Upon completion of the addition, the reaction mixture was
removed from the ice bath and placed in room temperature for 30 min and monitored by TLC. The mixture was
concentrated in vacuum to yield 24-29, 30-37, 38-44, 45-51, 52-58 and 59-63 which were recrystallized by
isopropanol.
5.10.1 N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethyl)ph
enyl)-1,6-dihydropyridazine-3-carboxamide (24)
This compound was obtained as pale yellow solid in 67% yield; ESI-MS m/z: 541.14[M+H]+. 1HNMR (400 MHz,
DMSO-d6) ꢁ 12.32 (s, 1H), 8.85 (s, 1H), 8.82 (d, J =8.7Hz, 1H), 8.54 (d, J = 5.7 Hz, 1H), 8.08-8.00 (m, 1H),
7.99-7.94 (m, 1H), 7.94-7.90 (m, 1H), 7.87 (d, J = 7.1Hz, 1H), 7.56 (d, J = 8.2Hz, 1H), 7.50-7.43 (m, 1H), 7.41 (s,
1H), 7.21(s, 1H), 2.79 (d, J = 4.6 Hz, 3H), 2.10 (s, 3H).
5.10.2 1-(4-Bromo-2-fluorophenyl)-N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-4-methyl-6-
oxo-1,6-dihydropyridazine-3-carboxamide (25)
This compound was obtained as pale yellow solid in 68% yield;ESI-MS m/z: 569.05[M+H]+. 1HNMR (400 MHz,
DMSO-d6) ꢁ 10.85 (s, 1H), 8.82 (d, J = 4.7 Hz, 1H), 8.54 (d, J =5.6 Hz, 1H), 7.97-7.88 (m, 1H), 7.85 (d, J =9.9
Hz, 1H), 7.68 (m, 2H), 7.60 (d, J = 8.5 Hz, 1H), 7.45 (t, J =9.0 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.21 (s, 1H),
7.11(s, 1H), 2.78 (d, J = 4.6 Hz, 3H),2.42 (s, 3H).
5.10.3 N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethoxy)
phenyl)-1,6-dihydropyridazine-3-carboxamide (26)
This compound was obtained as pale yellow solid in 75% yield; ESI-MS m/z: 557.13[M+H]+. 1HNMR (400 MHz,

DMSO-d6) ꢁ 12.22 (s, 1H), 8.96-8.73 (m, 2H), 8.54 (d, J = 5.5Hz, 1H), 8.08-8.00 (m, 1H), 7.89 (d, J = 7.5Hz, 1H),
7.72 (dd, J = 21.3, 7.1Hz, 3H), 7.56 (d, J = 9.1 Hz, 1H), 7.46 (dt, J = 7.8, 9.1 Hz, 2H), 7.22 (s, 1H), 2.79 (d, J =
4.7 Hz, 3H),2.04 (s, 3H).
5.10.4 N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-1,6-d
ihydropyridazine-3-carboxamide (27)
This compound was obtained as pale yellow solid in 76% yield; ESI-MS m/z: 491.14[M+H]+. 1HNMR (400 MHz,
DMSO-d6) ꢁ 10.87 (s, 1H), 8.82 (d, J = 4.6 Hz, 1H), 8.54 (d, J = 5.5 Hz, 1H), 7.95 (m, 1H), 7.94-7.90 (d, J = 12.9
Hz, 1H), 7.70 (d, J = 7.1Hz, 1H), 7.63 (d, J = 8.1Hz, 2H), 7.59-7.52 (m, 1H), 7.46 (t, J = 9.0 Hz, 1H), 7.39 (s, 1H),
7.33 (t, J = 8.1 Hz, 1H), 7.27-7.19 (m, 1H), 7.09 (s, 1H), 2.78 (d, J = 4.5 Hz, 3H),2.41(s, 3H).
5.10.5
N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-
dihydropyridazine-3-carboxamide (28)
This compound was obtained as pale yellow solid in 73% yield; ESI-MS m/z: 487.17[M+H]+. 1HNMR (400 MHz,
DMSO-d6) ꢁ 10.83 (s, 1H), 8.82 (d, J = 4.6 Hz, 1H), 8.54 (d, J = 5.7 Hz, 1H), 7.94 (d, J = 12.9 Hz, 1H), 7.67-7.50
(m, 3H), 7.46 (t, J = 9.0 Hz, 1H), 7.40-7.26 (m, 3H), 7.22(t, J = 2.7 Hz, 1H), 2.78 (d, J = 4.5 Hz, 3H),2.37 (s, 3H).
5.10.6 N-(3-fluoro-4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl)-1-(2-fluorophenyl)-4-methyl-6-oxo-1,6-d
ihydropyridazine-3-carboxamide (29)
This compound was obtained as pale yellow solid in 75% yield; ESI-MS m/z: 491.14[M+H]+. 1HNMR (400 MHz,
DMSO-d6) ꢁ 10.86 (s, 1H), 8.82 (d, J = 4.7 Hz, 1H), 8.51 (dd, J = 19.4, 5.7 Hz, 1H), 7.93 (d, J = 12.8 Hz, 1H),
7.69 (t, J = 7.4 Hz, 1H), 7.63-7.52 (m, 2H), 7.45 (t, J = 8.4 Hz, 2H), 7.38 (t, J = 12.4 Hz, 2H), 7.26-7.16 (m, 1H),
7.11 (s, 1H), 2.79 (d, J = 4.8 Hz, 3H),2.43 (s, 3H).
5.10.7
4-Methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(2-(trifluoromethyl)phenyl)-1,6-
dihydropyridazine-3-carboxamide (30)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 201.0-203.0 ; ESI-MS m/z: 523.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.31 (s, 1H), 8.98 (s, 1H), 8.81 (s, 1H), 8.58 (d, J = 5.4 Hz, 1H), 8.15 (d, J =
7.7 Hz, 1H), 8.08 (d, J = 7.3 Hz, 1H), 8.05 – 7.95 (m, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 5.3 Hz, 1H), 7.47
(d, J = 8.7 Hz, 2H), 2.21 (s, 3H).

5.10.8
1-(4-Bromo-2-fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1,6-
dihydropyridazine-3-carboxamide (31)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 204.0-205.0 ; ESI-MS m/z: 553.00[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.77 (s, 1H), 8.80 (s, 1H), 8.57 (d, J = 5.2 Hz, 1H), 7.94 (d, J = 9.5 Hz, 1H),
7.89 (d, J = 8.6 Hz, 2H), 7.83 – 7.71 (m, 3H), 7.44 (d, J = 8.3 Hz, 2H), 7.20 (s, 1H), 2.51 (s, 3H).
5.10.9 4-Methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(2-(trifluoromethoxy)phenyl)-1,6-
dihydropyridazine-3-carboxamide (32)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 207.0-208.0 ; ESI-MS m/z: 539.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.20 (s, 1H), 8.97 (s, 1H), 8.81 (s, 1H), 8.58 (d, J = 5.4 Hz, 1H), 7.99 (d, J =
8.0 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.89 – 7.83 (m, 2H), 7.79 (t, J = 6.3 Hz,2H), 7.47 (d, J = 8.8 Hz, 2H), 2.21
(s, 3H).
5.10.10 1-(3-fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (33)
This compound was obtained as pale yellow solid in 65% yield; M.P.: 206.0-207.0 ; ESI-MS m/z: 473.10[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.77 (s, 1H), 8.80 (s, 1H), 8.58 (d, J = 5.4 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H),
7.82 – 7.77 (m, 2H), 7.70 (dt, J = 15.8, 7.3 Hz, 2H), 7.48 – 7.40 (m, 3H), 7.19 (s, 1H), 2.51 (s, 3H).
5.10.11 4-Methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(p-tolyl)-1,6-dihydropyridazine-3-carb
-oxamide (34)
This compound was obtained as pale yellow solid in 59% yield; M.P.: 207.0-209.0 ; ESI-MS m/z: 469.12[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.74 (s, 1H), 8.80 (s, 1H), 8.58 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 8.8 Hz, 2H),
7.78 (d, J = 5.3 Hz, 1H), 7.69 (d, J = 8.2 Hz,2H), 7.44 (t, J = 7.6 Hz,4H), 7.15 (s, 1H), 2.49 (s, 3H), 2.48 (s, 3H).
5.10.12 1-(4-Fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (35)
This compound was obtained as pale yellow solid in 77% yield; M.P.: 209.0-211.0 ; ESI-MS m/z: 473.10[M+H]
1
+. H NMR (400 MHz, DMSO-d6) ꢁ 10.66 (s, 1H), 8.70 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.84 – 7.76 (m, 4H),

7.68 (d, J = 5.3 Hz, 1H), 7.42 – 7.33 (m, 4H), 7.07 (s, 1H), 2.41 (s, 3H); 13C NMR (101 MHz, DMSO-d6) ꢁ 168.99,
163.19, 162.49, 161.62, 160.05, 158.64, 152.85, 148.12, 142.77, 141.73, 137.02, 135.80, 129.60, 127.89, 127.80,
127.29, 122.24(2C), 121.41(2C), 118.40, 115.50, 115.28, 18.11..
5.10.13 1-(3,4-Difluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (36)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 205.0-207.0 ; ESI-MS m/z: 491.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.67 (s, 0H), 8.71 (s, 1H), 8.47 (d, J = 5.3 Hz,1H), 8.02 – 7.93 (m, 1H), 7.83
(d, J = 8.5 Hz, 2H), 7.68 (d, J = 5.4 Hz, 2H), 7.64 – 7.56 (m, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.08 (s, 1H), 2.42 (s,
3H).
5.10.14 1-(2,4-Dimethylphenyl)-4-methyl-6-oxo-N-(4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (37)
This compound was obtained as pale yellow solid in 56% yield; M.P.: 209.0-211.0 ; ESI-MS m/z: 483.14[M+H]
1
+. H NMR (400 MHz, DMSO-d6) ꢁ 10.57 (s, 1H), 8.71 (s, 1H), 8.49 – 8.46 (m, 1H), 7.80 (d, J = 6.7 Hz, 2H),
7.69 – 7.68 (m, 1H), 7.35 (d, J = 6.9 Hz,2H), 7.25 (d, J = 17.2 Hz, 2H), 7.06 (s, 1H), 2.43 (s, 3H), 2.34 (s, 3H),
2.09 (s, 3H).
5.10.15 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethyl)phenyl)-1
,6-dihydropyridazine-3-carboxamide (38)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 210.0-212.0 ; ESI-MS m/z: 541.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.29 (s, 1H), 8.88 (s, 1H), 8.72 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H), 8.04 (d, J =
7.8 Hz, 1H), 8.01 – 7.90 (m, 4H), 7.87 (t, J = 7.8 Hz, 1H), 7.70 (d, J = 5.4 Hz, 1H), 7.58 – 7.48 (m, 2H), 2.09 (s,
3H).
5.10.16 1-(4-Bromo-2-fluorophenyl)-N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1,
6-dihydropyridazine-3-carboxamide (39)
This compound was obtained as pale yellow solid in 54% yield; M.P.: 213.0-215.0 ; ESI-MS m/z: 570.99[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.80 (s, 1H), 8.71 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H), 7.85 (t, J = 11.3 Hz, 2H),
7.70 (d, J = 5.4 Hz, 1H), 7.65 (t, J = 10.0 Hz, 2H), 7.56 (d, J = 8.7 Hz, 1H), 7.53 – 7.47 (m, 1H), 7.10 (s, 1H),

2.41 (s,3H); 13C NMR (101 MHz, DMSO-d6) ꢁ 169.77, 162.80, 162.14, 158.92, 155.09, 153.30, 152.63, 150.57,
148.12, 143.55, 141.81, 137.67, 135.41, 130.34, 128.42, 124.84, 123.26, 118.69, 118.50, 117.84, 117.16, 116.05,
109.19,18.74.
5.10.17 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethoxy)phenyl)
-1,6-dihydropyridazine-3-carboxamide (40)
This compound was obtained as pale yellow solid in 58% yield; M.P.: 214.0-215.0 ; ESI-MS m/z: 557.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.31 (s, 1H), 8.98 (s, 1H), 8.84 (s, 1H), 8.63 (d, J = 5.3 Hz, 1H), 8.06 (d, J =
11.9 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.90 – 7.74 (m, 4H), 7.68 – 7.60 (m, 2H), 2.21 (s, 3H).
5.10.18 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-1,6-dihydro
pyridazine-3-carboxamide (41)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 217.0-218.0 ; ESI-MS m/z: 491.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.92 (s, 1H), 8.83 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 8.00 (d, J = 11.8 Hz, 1H),
7.84 – 7.77 (m, 2H), 7.75 – 7.59 (m, 4H), 7.44 (t, J = 10.3 Hz, 1H), 7.20 (s, 1H), 2.52 (s, 3H).
5.10.19 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-dihydropyridazi
ne-3-carboxamide (42)
This compound was obtained as pale yellow solid in 71% yield; M.P.: 211.0-212.0 ; ESI-MS m/z: 487.11[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.89 (s, 1H), 8.82 (s, 1H), 8.63 (d, J = 5.3 Hz, 1H), 7.99 (d, J = 13.1 Hz, 1H),
7.82 (d, J = 5.3 Hz, 1H), 7.69 (d, J = 8.2 Hz, 3H), 7.62 (t, J = 8.3 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.15 (s, 1H),
2.50 (s, 3H), 2.48 (s, 3H).
5.10.20 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-dihydro
pyridazine-3-carboxamide (43)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 213.0-215.0 ℃;ESI-MS m/z: 491.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.84 (s, 1H), 8.73 (s, 1H), 8.53 (d, J = 5.3 Hz, 1H), 7.91 (d, J = 12.6 Hz, 1H),
7.81 – 7.71 (m, 3H), 7.60 (d, J = 8.8 Hz, 1H), 7.53 (t, J = 8.6 Hz, 1H), 7.38 (t, J = 8.6 Hz, 2H), 7.08 (s,1H), 2.42
(s,3H); 13C NMR (101 MHz, DMSO-d6) ꢁ 168.78, 161.83, 161.35, 159.62, 158.16, 152.31, 142.39, 140.77, 136.72,
136.49, 134.29, 129.21, 127.43 (2C), 127.34 (2C), 123.85, 117.72, 116.14, 115.05, 114.83, 108.18, 107.95, 17.72.
5.10.21 1-(3,4-Difluorophenyl)-N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1,6-dih

ydropyridazine-3-carboxamide (44)
This compound was obtained as pale yellow solid in 78% yield; M.P.: 219.0-220.0 ; ESI-MS m/z: 509.08[M+H]
1
+. H NMR (400 MHz, DMSO-d6) ꢁ 10.83 (s, 1H), 8.73 (s, 1H), 8.53 (d, J = 5.4 Hz, 1H), 8.01 – 7.86 (m, 2H),
7.77 – 7.63 (m, 3H), 7.60 (d, J = 10.2 Hz,1H), 7.54 (t, J = 8.6 Hz, 1H), 7.10 (s, 1H), 2.43 (s, 3H).
5.10.22 4-Methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(2-(trifluoromethyl)phenyl)-1,
6-dihydropyridazine-3-carboxamide (45)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 201.0-203.0 ; ESI-MS m/z: 523.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.19 (s, 1H), 8.86 (s, 1H), 8.61 (s, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.95 (d, J =
5.6 Hz, 2H), 7.93 – 7.84 (m, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 5.8 Hz, 1H), 7.32 (d, J = 8.7 Hz, 3H), 2.09
(s, 3H).
5.10.23 1-(4-Bromo-2-fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1,
6-dihydropyridazine-3-carboxamide (46)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 204.0-205.0 ; ESI-MS m/z: 553.00[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.64 (s, 1H), 8.60 (s, 1H), 7.95 (d, J = 6.0 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H),
7.77 (d, J = 9.4 Hz, 2H), 7.65 (d, J = 4.2 Hz, 3H), 7.30 (d, J = 6.9 Hz, 2H), 7.09 (s, 1H), 2.39 (s, 3H).
5.10.24 4-Methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(2-(trifluoromethoxy)phenyl)-1,
6-dihydropyridazine-3-carboxamide (47)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 207.0-208.0 ; ESI-MS m/z: 539.09 [M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.08 (s, 1H), 8.85 (s, 1H), 8.61 (s, 1H), 7.95 (d, J = 5.9 Hz, 1H), 7.87 (d, J =
7.2 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 7.1 Hz,2H), 7.67 (t, J = 8.1 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H),
2.09 (s, 3H).
5.10.25 1-(3-Fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (48)
This compound was obtained as pale yellow solid in 65% yield; M.P.: 206.0-207.0 ; ESI-MS m/z: 473.10[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.64 (s, 1H), 8.60 (s, 1H), 7.96 (d, J = 3.5 Hz, 1H), 7.79 (d, J = 8.1 Hz, 2H),

7.72 – 7.53 (m, 4H), 7.31 (d, J = 5.1 Hz, 3H), 7.08 (s, 1H), 2.39 (s,3H).
5.10.26 4-Methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(p-tolyl)-1,6-dihydropyridazine-3-
carboxamide (49)
This compound was obtained as pale yellow solid in 59% yield; M.P.: 207.0-209.0 ; ESI-MS m/z: 469.12[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.61 (s, 1H), 8.60 (s, 1H), 7.96 (d, J = 4.8 Hz, 1H), 7.79 (d, J = 7.9 Hz, 2H),
7.65 (d, J = 5.1 Hz, 1H), 7.58 (d, J = 4.1 Hz, 2H), 7.31 (d, J = 1.2 Hz, 4H), 7.03 (s, 1H), 2.38 (s, 3H), 2.36 (s, 3H).
5.10.27 1-(4-Fluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (50)
This compound was obtained as pale yellow solid in 77% yield; M.P.: 209.0-211.0 ; ESI-MS m/z: 473.10[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.57 (s, 1H), 8.52 (s, 1H), 7.88 (d, J = 5.9 Hz, 1H), 7.70 (t, J = 9.7 Hz, 4H),
7.58 (d, J = 5.8 Hz, 1H), 7.29 (t, J = 8.7 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 6.98 (s, 1H), 2.41 (s, 3H).
5.10.28 1-(3,4-Difluorophenyl)-4-methyl-6-oxo-N-(4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1,6-dihydro-
pyridazine-3-carboxamide (51)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 205.0-207.0 ; ESI-MS m/z: 491.09[M+H]
1
+. H NMR (400 MHz, DMSO-d6) ꢁ 10.57 (s, 1H), 8.52 (s, 1H), 7.90 – 7.84 (m, 2H), 7.72 (d, J = 8.9 Hz, 2H),
7.60 – 7.48 (m, 3H), 7.24 (d, J = 8.5 Hz, 2H), 7.00 (s, 1H), 2.32 (s, 3H).
5.10.29 N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethyl)phenyl)-1
,6-dihydropyridazine-3-carboxamide (52)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 210.0-212.0 ; ESI-MS m/z: 541.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.29 (s, 1H), 8.87 (s, 1H), 8.64 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 8.01 (d, J =
6.0 Hz, 1H), 7.98 – 7.91 (m, 3H), 7.87 (t, J = 7.7 Hz, 1H), 7.71 (d, J = 5.9 Hz, 1H), 7.54 – 7.46 (m, 2H), 2.09 (s,
3H).
5.10.30 1-(4-Bromo-2-fluorophenyl)-N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1,
6-dihydropyridazine-3-carboxamide (53)
This compound was obtained as pale yellow solid in 54% yield; M.P.: 213.0-215.0 ; ESI-MS m/z: 570.99[M+H]

+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.79 (s, 1H), 8.62 (s, 1H), 8.01 (d, J = 5.8 Hz, 1H), 7.84 (t, J = 9.9 Hz, 2H),
7.72 – 7.60 (m,3H), 7.55 (d, J = 8.8 Hz, 1H), 7.47 (t, J = 8.8 Hz, 1H), 7.10 (s, 1H), 2.40 (s, 3H).
5.10.31 N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethoxy)phenyl)
-1,6-dihydropyridazine-3-carboxamide (54)
This compound was obtained as pale yellow solid in 58% yield; M.P.: 214.0-215.0 ; ESI-MS m/z: 557.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.19 (s, 1H), 8.86 (s, 1H), 8.64 (s, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.94 (d, J =
11.7 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.70 (mz, 4H), 7.53 – 7.46 (m, 2H), 2.09 (s, 3H).
5.10.32 N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-1,6-dihydro
pyridazine-3-carboxamide (55)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 217.0-218.0 ; ESI-MS m/z: 491.09[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.80 (s, 1H), 8.63 (s, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.88 (d, J = 12.5 Hz, 1H),
7.73 – 7.65 (m, 2H), 7.63 – 7.53 (m, 3H), 7.49 (t, J = 8.7 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H), 7.08 (s, 1H), 2.40 (s,
3H).
5.10.33 N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-dihydropyridazi
ne-3-carboxamide (56)
This compound was obtained as pale yellow solid in 71% yield; M.P.: 211.0-212.0 ; ESI-MS m/z: 487.11[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.76 (s, 1H), 8.62 (s, 1H), 8.00 (d, J = 5.9 Hz, 1H), 7.87 (d, J = 14.4 Hz, 1H),
7.70 (d, J = 5.9 Hz, 1H), 7.57 (d, J = 8.2 Hz, 3H), 7.47 (t, J = 8.8 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 7.03 (s, 1H),
2.38 (s,1H), 2.36 (s,3H).
5.10.34 N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-dihydro
pyridazine-3-carboxamide (57)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 213.0-215.0 ; ESI-MS m/z: 491.09[M+H]
+.1H NMR (400 MHz, DMSO-d6) ꢁ 10.72 (s, 1H), 8.56 (s, 1H), 7.93 (s, 1H), 7.81 (d, J = 12.7 Hz,1H), 7.69 (dt, J
= 8.8, 4.1 Hz, 2H), 7.64 (d, J = 5.7 Hz, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.41 (t, J = 8.6 Hz, 1H), 7.30 (t, J = 9.0 Hz,
2H), 6.99 (s, 1H), 2.42 (s, 3H).
5.10.35 1-(3,4-Difluorophenyl)-N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-4-methyl-6-oxo-1,6-

dihydropyridazine-3-carboxamide (58)
This compound was obtained as pale yellow solid in 78% yield; M.P.: 219.0-220.0 ; ESI-MS m/z: 509.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 10.72 (s, 1H), 8.55 (s, 1H), 7.93 (d, J = 5.9 Hz,1H), 7.87 (dd, J = 11.0, 8.0 Hz,
1H), 7.80 (d, J = 12.6 Hz, 1H), 7.63 (d, J = 5.9 Hz, 1H), 7.59 – 7.46 (m, 1H), 7.41 (t, J = 8.7 Hz, 3H), 7.01 (s, 1H),
2.41 (s, 3H).
5.10.36 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydropyridazine
-3-carboxamide (59)
This compound was obtained as pale yellow solid in 61% yield; M.P.: 223.0-224.0 ; ESI-MS m/z: 477.07[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.04 (s, 1H), 8.98 (d, J = 8.0 Hz, 1H), 8.72 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H),
7.95 (d, J = 12.6 Hz, 1H), 7.89 – 7.81 (m, 2H), 7.71 (d, J = 5.5 Hz, 1H), 7.49 (dt, J = 17.0, 9.6 Hz, 4H), 6.93 (d, J
= 7.8 Hz, 1H).
5.10.37 1-(3-Chloro-4-fluorophenyl)-N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (60)
This compound was obtained as pale yellow solid in 64% yield; M.P.: 220.0-221.0 ; ESI-MS m/z: 511.03[M+H]
1
+. H NMR (400 MHz, DMSO-d6) ꢁ 11.94 (s, 1H), 9.01 (d, J = 7.8 Hz, 1H), 8.73 (s, 1H), 8.52 (d, J = 5.4 Hz,
1H), 8.13 (s, 1H), 7.95 (d, J = 12.5 Hz, 1H), 7.86 (s, 1H), 7.71 (d, J = 6.5 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 6.93
(d, J = 7.8 Hz, 1H).
5.10.38 1-(4-Bromophenyl)-N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-oxo-1,4-dihydro-
pyridazine-3-carboxamide (61)
This compound was obtained as pale yellow solid in 66% yield; M.P.: 225.0-226.0 ; ESI-MS m/z: 538.99[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 11.96 (s, 1H), 9.01 (d, J = 7.8 Hz, 1H), 8.74 (d, J = 12.1 Hz, 1H), 8.52 (d, J =
5.5 Hz, 1H), 7.95 (d, J = 12.3 Hz, 1H), 7.80 (q, J = 8.6 Hz, 4H), 7.71 (d, J = 5.6 Hz, 1H), 7.53 (d, J = 9.5 Hz, 2H),
6.93 (d, J = 7.9 Hz, 1H).
5.10.39 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-
carboxamide (62)

This compound was obtained as pale yellow solid in 67% yield; M.P.: 220.0-221.0 ; ESI-MS m/z: 459.08[M+H]
+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.04 (s, 1H), 9.02 (d, J = 7.8 Hz, 1H), 8.73 (s, 1H), 8.52 (d, J = 5.5 Hz, 1H),
8.00 – 7.92 (m, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 5.4 Hz, 1H), 7.62 (t, J = 7.8 Hz, 2H), 7.53 (d, J = 8.9 Hz,
3H), 6.93 (d, J = 7.8 Hz, 1H).
5.10.40 N-(3-fluoro-4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl)-4-oxo-1-(p-tolyl)-1,4-dihydropyridazine-3-
carboxamide (63)
This compound was obtained as pale yellow solid in 67% yield; M.P.: 222.0-223.0 ; ESI-MS m/z:
473.10[M+H]+. 1H NMR (400 MHz, DMSO-d6) ꢁ 12.15 (s, 1H), 8.94 (d, J = 7.7 Hz, 1H), 8.73 (s, 1H), 8.52 (d, J
= 5.5 Hz, 1H), 7.96 (d, J = 12.4 Hz, 1H), 7.71 (dd, J = 7.2, 4.6 Hz, 3H), 7.53 (q, J = 10.4, 9.5 Hz, 2H), 7.15 (d, J =
8.6 Hz, 2H), 6.92 (d, J = 7.8 Hz, 1H), 3.83 (s, 3H).
5.11. Cytotoxicity assay in vitro
The cytotoxic activities of target compounds (24-29, 30-37, 38-44, 45-51, 52-58 and 59-63) were evaluated with
A549, HepG2, MCF-7 and PC-3 cell lines by the standard MTT assay in vitro, with compounds c-MET inhibitors
Foretinib as positive control.[14] The cancer cell lines were cultured in minimum essential medium (MEM)
supplement with 10% fetal bovine serum (FBS). Approximately 4×103 cells, suspended in MEM medium, were
plated onto each well of a 96-well plate and incubated in 5% CO2 at 37°C for 24 h. The test compounds at
indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h.
Fresh MTT was added to each well at a terminal concentration of 5ꢀg/mL and incubated with cells at 37°C for 4
h. The formazan crystals were dissolved in 100 ꢀL DMSO each well, and the absorbency at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader.
All of the compounds were tested three times in each of the cell lines. The results expressed as inhibition rates or
IC50 (half-maximal inhibitory concentration) were the averages of two determinations and calculated by using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
5.12. Tyrosine kinases assay in vitro
The selected compounds ( 35, 39 and 43) are tested for their activity against one or several Tyrosine kinases
(c-Met, Flt-3, VEGFR-2, c-Kit and EGFR kinases) through the mobility shift assay[8,9]. All kinase assays were
performed in 96-well plates in a 50 µL reaction volume. The kinase buffer contains 50 mM HEPES, pH 7.5, 10

mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer contains 100 mM HEPES, pH 7.5, 0.015% Brij-35,
0.2% Coating Reagent #3 and 50 mM EDTA. Dilute the compounds to 500 µM by 100% DMSO, then transfer 10
µL of compound to a new 96-well plate as the intermediate plate, add 90µL kinase buffer to each well. Transfer 5
µL of each well of the intermediate plate to 384-well plates. The following amounts of enzyme and substrate were
used per well: kinase base buffer, FAM-labeled peptide, ATP and enzyme solution. Wells containing the substrate,
enzyme, DMSO without compound were used as DMSO control. Wells containing just the substrate without
enzyme were used as low control. Incubate at room temperature for 10 min. Add 10 µL peptide solution to each
well. Incubate at 28 for specified period of time and stop reaction by 25 µL stop buffer. At last collect data on
Caliper program and convert conversion values to inhibition values. Percent inhibition = (max - conversion)/(max
- min) × 100. ‘max’ stands for DMSO control; ‘min’ stands for low control.
5.13. Annexin V-FITC staining
After digestion, the cells were centrifuged at 1000 rpm for 5 min. The supernatant was abandoned and the cells
were collected, gently resuspended with PBS and counted. 5-10 million resuspended cells were centrifuged at
1000 rpm for 5 min. Then the supernatant was abandoned and 195 ꢀl of Annexin V-FITC binding solution was
added to gently resuspend the cells. Afterwards, 5 ꢀl of Annexin V-FITC and 10 ꢀl of propidium iodide staining
solution were added and the solution was mixed gently. Finally, the solution was incubated in dark and in room
temperature (20-25℃) for 10-20 min and tested by flow cytometry.[15]
5.14. Docking studies
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8) was obtained from
RCSB Protein Data Bank. Hydrogen atoms were added to the structure allowing for appropriate ionization at
physiological pH and the water was removed. The protonated state of several important residue were adjusted and
docking was carried out by using SYBYL 6.9.1 in favor of forming reasonable hydrogen bond with the ligand.
And using pymol 1.8.6 to analyzing the results. Molecular docking of 43 into the 3D c-Met complex structure
(PDB code: 3LQ8) was carried out using the SYBYL 6.9.1 (Tripos, St. Louis, MO, USA) and analyzing the
results by the pymol 1.8.6. Displaying the hydrogen bond, p-pi interaction bond, surface and cartoon model to
show the relationship between the 43 and c-Met(Fig 5a). According to the 3D figure, the 2D figure was carried out
by chemdraw (Fig 5b). And from the Fig 5C, we can know that the compound 43 occupied the package of the
c-Met.

Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China
(No.21662014), Outstanding Youth Foundation of Jiangxi, Natural Science Foundation of Jiangxi, China
(20171BCB23078), Natural Science Foundation of Jiangxi, China (20171ACB21052&20171BAB215073),
Science and Technology Project Founded by the Education Department of Jiangxi Province (GJJ160787),
Graduate Students' Science and Technology Innovation Project of Jiangxi Science & Technology Normal
University (YC2016-X16).
References
[1] Blume-Jensen P, Hunter T. Oncogenic kinase signalling[J]. Nature, 2001, 411(6835): 355.
[2] Birchmeier C, Birchmeier W, Gherardi E, et al. Met, metastasis, motility and more[J]. Nature reviews.
Molecular cell biology, 2003, 4(12): 915.
[3] Ferrara N, Gerber H P, LeCouter J. The biology of VEGF and its receptors[J]. Nature medicine, 2003, 9(6):
669-676.
[4] Liu X, Newton R C, Scherle P A. Development of c-MET pathway inhibitors[J]. Expert opinion on
investigational drugs, 2011, 20(9): 1225-1241.
[5] Kataoka Y, Mukohara T, Tomioka H, et al. Foretinib (GSK1363089), a multi-kinase inhibitor of MET and
VEGFRs, inhibits growth of gastric cancer cell lines by blocking inter-receptor tyrosine kinase networks[J].
Investigational new drugs, 2012, 30(4): 1352-1360.
[6] Wilhelm S M, Carter C, Tang L Y, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and
targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and
angiogenesis[J]. Cancer research, 2004, 64(19): 7099-7109.
[7] Gaudette F, Raeppel S, Nguyen H, et al. Identification of potent and selective VEGFR receptor tyrosine

kinase inhibitors having new amide isostere headgroups.[J] . Bioorganic & medicinal chemistry letters, 2010,
20(3): 848-52.
[8] Zificsak C A, Theroff J P, Aimone L D, et al. 2,4-Diaminopyrimidine inhibitors of c-Met kinase bearing
benzoxazepine anilines.[J] . Bioorganic & medicinal chemistry letters, 2011, 21(2): 660-663.
[9] Li S, Huang Q, Liu Y, et al. Design, synthesis and antitumour activity of bisquinoline derivatives connected
by 4-oxy-3-fluoroaniline moiety[J]. European journal of medicinal chemistry, 2013, 64: 62-73.
[10] Tang Q, Wang L, Duan Y, et al. Discovery of novel 7-azaindole derivatives bearing dihydropyridazine
moiety as c-Met kinase inhibitors[J]. European Journal of Medicinal Chemistry, 2017, 1 33: 97-106.
[11] Lone S H, Bhat K A, Majeed R, et al. Click chemistry inspired facile synthesis and bioevaluation of novel
triazolyl analogs of Ludartin[J]. Bioorganic & medicinal chemistry letters, 2014, 24(4): 1047-1051.
[12] Ramachary D B, Gujral J, Peraka S, et al. Triazabicyclodecene as an Organocatalyst for the Regiospecific
Synthesis of 1, 4, 5-Trisubstituted N-Vinyl-1, 2, 3-triazoles[J]. European Journal of Organic Chemistry, 2017,
2017(3): 459-464.
− pyridone based inhibitors of Met kinase:
[13] Kim K S, Zhang L, Schmidt R, et al. Discovery of pyrrolopyridine
Synthesis, X-ray crystallographic analysis, and biological activities[J]. Journal of medicinal chemistry, 2008,
51(17): 5330-5341
[14] Zhu W, Wang W, Xu S, et al. Design, synthesis, and docking studies of phenylpicolinamide derivatives
bearing 1H-pyrrolo [2, 3-b] pyridine moiety as c-Met inhibitors[J]. Bioorganic & medicinal chemistry, 2016,
24(4): 812-819.
[15] Casciola-Rosen L, Rosen A, Petri M, et al. Surface blebs on apoptotic cells are sites of enhanced
procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus
erythematosus[J]. Proceedings of the National Academy of Sciences, 1996, 93(4): 1624-1629.

Four series of N-methylpicolinamide moiety and thienopyrimidine moiety bearing pyridazinone were designed
and synthesized Moreover, antitumour activity, enzyme-based selectivity, AO staining, Annexin V/PI staining and
docking study were carried out.