Discovery of novel pyrrolo-pyridine/pyrimidine derivatives bearing pyridazinone moiety as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.10.027

In continue to our previous research, eight series of pyrrolo[2,3-b]pyridine and pyrrolo[2,3-d]pyrimidine derivatives bearing pyridazinone moiety were designed, synthesized, and the in vitro antitumor activity was evaluated against four cancer cell lines (A549, HepG2, MCF-7 and PC-3). Some selected compounds (22f, 22g, 26c and 26e) were evaluated for the activity against c-Met kinase, and according to the results of kinase inhibitory activity, the compound 22g was further evaluated for other four tyrosine kinases (Flt-3, VEGFR-2, c-Kit and EGFR) to test the enzyme-based selectivity. The most promising compound 22g showed excellent activity than lead compound Foretinib against A549, HepG2, MCF-7 and PC-3 cell lines, with the IC₅₀ values of 2.19 ± 0.45 μM, 1.32 ± 0.26 μM, 6.27 ± 1.04 μM and 4.63 ± 0.83 μM. The structure–activity relationships (SARs) and docking studies indicated that the pyrrolo[2,3-b]pyridine derivatives bearing 4-oxo-pyridazinone moiety was superior to the pyrrolo[2,3-d]pyrimidine derivatives bearing 6-oxo-pyridazinone moiety. What's more, the target compounds modified with X and Y (X = H, Y = H) were favorable to the activity. And electron drawing groups (EWGs) of 4-Cl-3CF₃ on the aryl group show the best activity.

Full text of DOI:10.1016/j.ejmech.2017.10.027

Accepted Manuscript
Discovery of novel pyrrolo-pyridine/pyrimidine derivatives bearing pyridazinone moiety
as c-Met kinase inhibitors
Lin Xiao Wang, Xiaobo Liu, Shan Xu, Qidong Tang, Yongli Duan, Zhen Xiao, Jia Zhi,
Liwen Jiang, Pengwu Zheng, Wufu Zhu
PII:
S0223-5234(17)30820-6
10.1016/j.ejmech.2017.10.027
EJMECH 9819
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 May 2017
Revised Date: 7 October 2017
Accepted Date: 9 October 2017
Please cite this article as: L.X. Wang, X. Liu, S. Xu, Q. Tang, Y. Duan, Z. Xiao, J. Zhi, L. Jiang, P.
Zheng, W. Zhu, Discovery of novel pyrrolo-pyridine/pyrimidine derivatives bearing pyridazinone
moiety as c-Met kinase inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.027.
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ACCEPTED MANUSCRIPT
6
Eight series of pyrrolo[2,3-b]pyridine and pyrrolo[2, 3-d]pyrimidine derivatives bearing pyridazinone moiety
were designed, synthesized. Moreover, antitumour activity, enzyme-based selectivity, AO staining, Annexin V/PI
staining and docking study were carried out.

ACCEPTED MANUSCRIPT
Discovery of novel pyrrolo-pyridine/pyrimidine derivatives bearing pyridazinone moiety as c-Met kinase
inhibitors
Lin Xiao Wang1,†, Xiaobo Liu1,†, Shan Xu, Qidong Tang, Yongli Duan, Zhen Xiao, Jia Zhi, Liwen Jiang, Pengwu Zheng
* ,Wufu Zhu*
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology
Normal University, Nanchang 330013,P.R. China;.
*Correspondence author.
Tel/Fax: +86 791 8380-2393;
E-mail address: zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu).zhengpw@126.com(P. Zheng)
Abstract: In continue to our previous research, eight series of pyrrolo[2,3-b]pyridine and pyrrolo[2,3-d]pyrimidine
derivatives bearing pyridazinone moiety were designed, synthesized, and the in vitro antitumor activity was evaluated
against four cancer cell lines (A549, HepG2 , MCF-7 and PC-3). Some selected compounds ( 22f, 22g, 26c and 26e )
were evaluated for the activity against c-Met kinase, and according to the results of kinase inhibitory activity, the
compound 22g was further evaluated for other four tyrosine kinases ( Flt-3, VEGFR-2, c-Kit and EGFR ) to test the
enzyme-based selectivity. The most promising compound 22g showed excellent activity than lead compound Foretinib
against A549, HepG2 , MCF-7 and PC-3 cell lines, with the IC50 values of 2.19 ± 0.45 µM, 1.32 ± 0.26 µM, 6.27 ± 1.04
µM and 4.63 ± 0.83 µM. The structure–activity relationships (SARs) and docking studies indicated that the
pyrrolo[2,3-b]pyridine derivatives bearing 4-oxo-pyridazinone moiety was superior to the pyrrolo[2,3-d]pyrimidine
compounds derivatives bearing 6-oxo-pyridazinone moiety. What’s more, the target compounds modified with X and Y
(X = H, Y = H) were favorable to the activity. And electron drawing groups (EWGs) of 4-Cl-3CF3 on the aryl group
show the best activity.
Keywords: Pyrrolo[2,3-b]pyridines; Pyrrolo[2,3-d]pyrimidines; Pyridazinone; c-Met; Antitumor activity; Docking
Study1
† These authors contribute equally to this work
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address:zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com

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1. Introduction
c-Met inhibitors, a class of small molecules which can inhibit the enzymatic activity of the c-Met kinase and have
therapeutic application in the treatment of various types of cancers[1]. 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, compounds 3 and 4 (The structures are shown in Fig.1[2-4] ).
Many researches showed that nearly all of the Cabozantinib derivatives show excellent activity and contain an
5-atom linker between the aminophenoxy at C-4 position of quinoline and aryl group, which we called ‘5-atom
regulation’. The ‘5-atom regulation’ have two obvious structural characteristics. The first one is that there is six
chemical bonds distance between aminophenoxy at C-4 position of quinoline moiety and aryl group moiety, and the
other one is that this “six chemical bonds linker” contains hydrogen, oxygen, and nitrogen atoms which could form
hydrogen-bond donor or acceptor. (Fig. 1)[5].
In our previous research , several series of pyrrolo[2,3-b]pyridine derivatives (compounds 5 and 6, Fig.1) were
designed and synthesized as potent c-Met inhibitors according to the ‘5 atom regulation’[6-7]. Most of these compounds
exhibited potent activity, especially the most promising compound 6 with the IC50 values in the nanomole level. The
SARs and docking study exhibited that pyrrolo[2,3-b]pyridine moiety and 6-oxo-pyridazinone moiety may be benefit to
the in vitro activity.
From the 3D model of Foretinib (Fig.2), we can know that Foretinib contain a intramolecular hydrogen bond
(distance = 1.64 Ao) in the 5 atom linker. And inspired by compound 6, further modification was concentrated on
pyrrolo[2,3-b]pyridine and the 5-atom linker moiety. According to the inspiration from the Foretinib and compound 6,
we retained the feature of intramolecular hydrogen bond and the pyrrolo[2,3-b]pyridine moiety. Then
4-oxo-pyridazinone was introduced into 5 atom linker moiety to limit the conformation deeply, and then modified with
X (X = H\F), Y (Y = H\F) and R to get the first four series compounds (22a-g, 23a-g, 24a-g and 25a-g). Furthermore, in
order to investigate the effect of hydrogen acceptor and electron-deficient to the target compounds, the
pyrrolo[2,3-b]pyridine moiety and 4-oxo-pyridazinone moiety were replaced by the pyrrolo[2,3-d]pyrimidine and
6-oxo-pyridazinone, respectively. As a result, the compounds 26a-f, 27a-f, 28a-f and 29a-f were designed.
Herein we disclosed the synthesis and antitumor activity of pyrrolo-pyridine/pyrimidine derivatives bearing
pyridazinone moiety against A549 (human lung cancer), HepG2 (human liver cancer), MCF-7 (human breast cancer),
PC-3 (human prostate cancer) cancer cell lines, and c-Met kinase. Moreover, AO single staining, AnnexinV/PI staining,
enzyme-based selectivity and docking studies were presented in this paper as well.

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Fig. 1. Structures of small-molecule c-Met inhibitors
Fig. 2. Structures and design strategy for target compounds 22a-g, 23a-g, 24a-g, 25a-g, 26a-f, 27a-f, 28a-f and 29a-f.
2. Chemistry
The preparation of target compounds 22ag, 23a—g, 24a-g, 25a-g, 26a-f, 27a-f, 28a-f and 29a-f was described in Scheme
1.
The key intermediates 19a-d and 21a-d were synthesized from 4-cholor-1-substituted-pyrrolo[2,3-b]pyridine
17a-b or 4-cholor-1-substituted-pyrrolo[2,3-d]pyrimidine 20a-b via substitution reaction with p-aminphenol or
2-fluoro-4-aminphenol 18a-b as shown in scheme 1.

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Substituted anilines 6a-g and 12a-j were diazotized and then reacted with ethyl aceto-acetate to get compounds
8a-g and 13a-j, which condensed with DMF-DMA or ethyl-triphenylphosphoranylidene-acetate to yield compounds
9a-g and 14a-j with the present of different catalyst, respectively. Following by hydrolysis reaction and chlorination
reaction, 11a-g and 16a-j were obtained. Finally, substitution reaction of amides 19a-d and 21a-d with
4-oxo-dihydropyrizadine-3-carbonyl-chloride 11a-g or 6-oxo-dihydropyrizadine-carbonyl-chloride 16a-j promoted by
DIPEA in dichloromethane at room temperature to obtain target compounds 22a-g, 23a-g, 24a-g, 25a-g, 26a-f, 27a-f,
28a-f and 29a-f, respectively.
Reagents and conditions: (a) sodium nitrite, EtOH, ethyl acetoactate, 0 ，30 minute ; (b) DMF-DMA, 100 ℃, reflux,
6-10 h; (c) ethyl (triphenylphosphoranylidene) acetate, Et2NH, 85℃, 6 h; (d) sodium carbonate , EtOH/H2O(5:1), 80℃，
3-5 h; (e) oxalyl chloride , DMF, CH2Cl2 , rt, 5 minute; (f) sodium carbonate, sodium hydroxide, tetrahydrofuran ,
1,4-dioxane/H2O (5:1), 80℃, 1 h; (g) DIPEA, CH2Cl2 , rt,0.5 h.
Scheme 1 Synthetic route of target compounds.
3. Results and discussion
3.1. Biological evaluation
Taking Foretinib as reference compound, the target compounds (22a-g, 23a-g, 24a-g, 25a-g, 26a-f, 27a-f, 28a-f and
29a-f) were evaluated for the cytotoxicity against four cancer cell lines A549, HepG2, MCF-7 and PC-3 by
3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Some selected

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compounds ( 22f, 22g, 26c and 26e ) were evaluated for the activity against c-Met kinase, and according to the results
of kinase inhibitory activity, the compound 22g was further evaluated for other four tyrosine kinases ( Flt-3, VEGFR-2,
c-Kit and EGFR ) to test the enzyme-based selectivity . The results expressed as IC50 values were summarized in Tables
1-3, and the values are the average of at least two independent experiments. Moreover, discussion of the
structure–activity relationships of target compounds was depicted in Fig.3. In addition, AO single staining was carried
out in this study, and in order to further verify the AO single staining's inference, cell apoptosis of the selected
compound 22g was analyzed by Annexin V/PI staining.
Table 1. Structures and activity of target compounds 22a-g, 23a-g, 24a-g, 25a-g, 26a-f, 27a-f, 28a-f and 29a-f.
Compounds
No.
R
IC50a (µM)
MCF-7
A549
HepG2
PC-3
4-Cl
25.12±1.10
53.09±0.11
6.24±1.19
10.45±0.92
26.09±0.91
2.58±1.10
2.19±0.45
NAc
19.48±1.36
50.84±2.26
4.28±1.18
17.78±1.24
46.38±0.98
5.24±1.13
1.32±0.26
NA
10.58±0.98
21.648±3.29
7.89±1.11
15.64±0.86
17.86±0.95
3.11±1.03
6.27±1.28
NA
32.39±1.57
NA
22a
4-OCH3
4-Br
22b
8.04±0.93
14.97±1.20
12.79±1.08
7.78±1.02
4.63±0.83
42.45±0.97
46.42±0.87
40.80±0.91
38.63±1.04
8.80±1.04
13.57±0.87
22c
H
22d
2-F-4-Br
3-F-4-Cl
4-Cl-3-CF3
4-Cl
22e
22f
22g
23a
23b
23c
4-OCH3
4-Br
NA
NA
55.36±2.76
11.58±1.15
34.86±1.26
23.48±0.94
11.55±0.87
NA
67.96±1.46
24.91±0.99
56.54±1.17
9.61±1.48
H
56.77±2.25
55.68±0.97
10.27±1.57
23d
23e
2-F-4-Br
3-F-4-Cl
23f

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4-Cl-3-CF3
4-Cl
13.43±1.05
11.87±1.26
11.62±1.07
NA
5.50±0.95
NA
23g
24a
24b
24c
24d
24e
24f
NA
NA
4-OCH3
4-Br
NA
NA
NA
NA
59.36±1.42
51.72±2.076
35.70±1.34
NA
39.23±1.08
56.18±1.13
31.17±0.92
NA
16.25±1.24
45.15±1.68
19.76±1.38
16.84±0.99
15.16±0.96
NA
23.04±1.47
58.20±3.36
20.49+1.59
32.68+0.91
10.27±0.91
NA
H
2-F-4-Br
3-F-4-Cl
4-Cl-3-CF3
4-Cl
11.36±0.90
NA
23.37±1.29
NA
24g
25a
25b
25c
25d
25e
25f
4-OCH3
4-Br
NA
NA
NA
NA
NA
NA
33.638±2.158
NA
NA
H
NA
NA
NA
2-F-4-Br
3-F-4-Cl
4-Cl-3-CF3
4-F
26.80±1.04
NA
23.95±1.00
22.14±0.95
13.50±1.29
NA
14.13±1.25
16.58±0.94
18.38±0.99
4.80±0.92
9.67±1.06
3.62±1.24
32.01±0.99
3.21±1.01
4.56±1.28
24.49±1.02
32.03±1.01
18.87±0.96
11.70±0.91
26.61±0.91
11.78±0.96
26.97±0.97
27.71±0.86
30.82±0.92
10.78±0.912
16.34±1.16
17.87±1.17
20.87±0.85
NA
17.66±1.01
NA
25g
26a
26b
26c
26d
26e
26f
3-F
34.81±0.83
6.40±1.41
16.17±0.99
3.77±1.53
7.57±1.26
42.43±1.70
31.45±2.20
18.50±2.25
16.20±0.87
6.12±0.93
35.06±0.93
25.15±0.95
47.26±1.21
23.38±0.86
NA
3.18±0.83
7.46±1.58
22.75±0.96
3.66±1.59
9.47±1.73
12.85±1.59
23.05±1.69
18.82±1.58
NA
14.61±1.05
9.61±0.95
22.47±0.91
2.73±0.98
3.46±1.23
17.31±0.99
6.68±0.97
20.35±1.35
NA
4-CH3
2-OCF3
2-F-4-Br
3-CF3
4-F
27a
27b
27c
27d
27e
27f
3-F
4-CH3
2-OCF3
2-F-4-Br
3-CF3
32.01±1.12
26.29±1.02
NA
13.30±0.98
15.60±0.92
5.16±0.84
18.75±0.93
NA
2-OCF3
2-F-4-Br
3-F
28a
28b
28c
28d
33.58±0.96
NA
4-CH3
33.76±1.01
NA

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2-CF3
2-Cl
5.86±0.86
5.12±0.92
NA
27.23±1.17
11.19±0.99
NA
7.84±0.90
4.13±0.87
NA
19.08±0.87
16.42±0.87
NA
28e
28f
2-OCF3
2-F-4-Br
3-F
29a
14.42±0.84
NA
NA
NA
6.67±0.88
8.99±0.97
50.46±0.88
25.91±0.88
NA
29b
NA
NA
29c
4-CH3
2-CF3
2-Cl
NA
NA
NA
29d
35.37±1.55
NA
NA
NA
29e
NA
NA
29f
Foretinibb
-
1.74 ± 0.38
2.88 ± 0.67
3.92 ± 0.93
1.48 ± 0.47
aThe values are an average of two separate determinations.
bUsed as a positive control.
c NA: Not active (IC50 > 60 µM).
Fig.3. The structure–activity relationships of target compounds

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Table 2. c-Met kinase inhibitory activity of selected compounds 22f, 22g, 26c and 26e.
IC50a (µM)
Compounds No.
R
c-Met
0.17
3-F-4-Cl
4-Cl-3-CF3
4-CH3
22f
0.073
27.03
20.69
0.019
22g
26c
2-F-4-Br
-
26e
Foretinibb
aThe values are an average of two separate determinations.
bUsed as a positive control.
Table 3. Inhibition of tyrosine kinases by compound 22g
Kinase
Enzyme IC50 a (µM)
Flt-3
0.77
2.4
VEGFR-2
c-Kit
EGFR
2.2
>10
aThe values are an average of two separate determinations.
As showed in Tables 1-3 and Fig.3, most of compounds displayed excellent anticancer activity against four cancer
cells lines (A549, HepG2 , MCF-7 and PC-3) with potency from single-digit µM to nanomole range. And the first four
series of compounds (22a-g, 23a-g, 24a-g and 25a-g) show more activity than the other four series of compounds
(26a-f, 27a-f, 28a-f and 29a-f). Among these compounds, four selected compounds (22f, 22g, 26c and 26e) show better
activity against one or more cells lines compared with the positive drug Foretinib. The most promising compound 22g
exhibits the best activity against A549, HepG2, MCF-7 and PC-3 cell lines with the IC50 values of 2.19 ± 0.45 µM, 1.32
± 0.26 µM, 6.27 ± 1.04 µM and 4.63 ± 0.83 µM, which were equal to the lead drug Foretinib, respectively. The results
suggested that the pyrrolo[2,3-b]pyridine moiety and 4-oxo-pyridazinone moiety were a privileged scafford for
anticancer activity.
What’s more, those compounds containing no substituent (X = H) at N-7 position of pyrrolo-pyridine/pyrimidine
moiety (A ring) show more activity than that substituting with -CH3 group (X = CH3). That’s why the first and second
series compounds (22a-g and 23a-g) are more activity than that of the third and fourth series (24a-g and 25a-g), and the
similar regulation can be found among the other four series of compounds (26a-f, 27a-f, 28a-f and 29a-f).
Then, the investigation of activity effect of introducing Y group (Y = H/F) to C-2 position of aminophenoxy moiety

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(B ring) of the target compounds were also taken into consideration. The result revealed that the aminophenoxy moiety
substituting without any substituent (X = H) is benefit to the activity of the target compounds (22a-22g) compared with
F-atom substituent (23a-23g, X = F), and the same trend also was observed in compounds 24a-g/25a-g and
26a-f/27a-f.
Furthermore, different substituents of aryl group (D ring) also affected the cytotoxicity of target compounds. In
general, it’s seem to be that target compounds with no less than one electron drawing groups (EWGs) show better in
vitro activity, such as 22f-g, 23e-g, 24c, 24e-g and so on. Among these compounds, the aryl group (D ring) substituted
with two EWGs is more preferred, such as compounds 22-25g, 22-23f, 26-27e, 29b and so on. Especially, the EWGs
were substituted at the C-3 and C-4 position, such as compounds 22g, 23g, 22f, and 23f. And the compounds 22g (X =
H, Y = H, R = 4-Cl-3-CF3) show the best activity. In addition, electron donating groups (EDGs) are unfavorable for the
anticancer activity, such as compounds 22b, 23b, 24b, 26d, 27c, 28a and so on.
Activity against c-Met kinase of compounds 22f, 22g, 26c and 26e was further carried out in this paper. As shown in
Table 2, we can easily find that the four selected compounds showed moderate to excellent inhibitory on c-Met kinase.
Compounds 22f and 22g are more active than compounds 26c and 26e, especially compound 22g with IC50 value of
0.073 µM. The results prompt us that the target compounds may be a series of c-Met kinase inhibitors.
In order to examine whether compound 22g is a selective c-Met inhibitor, it was screened against other 4 tyrosine
kinases. As shown in Table 3, compound 22g exhibited medium inhibitory effects against Flt-3 (IC50, 0.77 µM),
VEGFR-2 (IC50 , 2.4 µM), c-Kit (IC50 , 2.2 µM) and EGFR (IC50 > 10 µM), which was 10.5-, 32.9-, and 136.9 -fold
lower than that of c-Met. These data indicated that compound 22g is a multitarget inhibitor and could inhibit the c-Met
kinase selectively.
3.2.Relationship between activity and concentration of selected compounds 22g against four cancer cell lines
In order to examine the relationship between the antitumor activity and the concentrations. We used the method of
MTT and selected seven different concentrations. The inhibition rate was measured after 72 h, and the phenomenon of
dose-dependent was observed of representative compound 22g. What’s more, we can find that the compound 22g
inhibits HepG2 cancer cell line the best.

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Fig.4. Relationship between activity and concentration of selected compounds 22g against our
cancer cell lines
3.3. Morphologic changes of HepG2 cells under inverted microscopy and fluorescence microscopy
According to the results of Fig. 4, the apoptotic experiment would be carried to exam the effect of compound 22g
on HepG2 cell by acridine orange (AO) single staining.
As shown in Fig.5, the control group cell (Fig.5a) was stained with acridine orange (AO) and shape of the cell was
full and the edge was clear, which was added with nothing. But in the right picture(Fig.5b), the cell shape was abnormal
with cell shrinkage, chromatin condensation and the DNA fragments was stained with Orange after 1.3 µM
concentration of compound 22g acted on the HepG2 cells. It claimed that the compounds 22g can induce apoptosis of
HepG2 cells.
Fig. 5 Morphologic changes of HepG2 cells under inverted microscopy and fluorescence microscopy. (5a) The
control group cell treated with nothing; (5b) Experimental group treated with 1.3 μM concentration of 22g.

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3.4. Apoptosis result analyzing
Compared to the control group, the number of early apoptotic cells of Foretinib and 22g was increased, and
increased the number of late apoptotic and death cells are more significant with the number of mechanical damage cells
was within the error range. It claimed that our data is credible. In addition , the number of early apoptotic cells of 22g is
equal to the Foretinib, and the number of late apoptotic and death cells (23.01%) was higher than that of Foretinib
(16.09%). It can be concluded that the target compound 22g can induce apoptosis of HepG2 and more efficient than
Foretinib.
Figure.6 Representative dot plot of Annexin V (x-axis) versus PI (y-axis) analyses. Q2-1: Mechanical damage; Q2-2:
Early apoptotic; Q2-3: Living cells; Q2-4: Late apoptotic and death.
3.5. 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 AutoDock vina 1.1.2, Open Babel and Discover Studio 3.5 visualizer. The four selected
compounds (22f, 22g, 26c and 26e) occupy the ATP site of c-Met kinase (Fig.7a). And basing on the in vitro inhibition
results, we selected compound 22g, 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 ).The binding modes of compound 22g and c-Met were
depicted in Fig.7b, Fig.7c and pharmacophore model Fig.7d. Visual inspection of the pose of compound 22g into c-Met
binding site revealed that compound 22g was tightly embedded into the active binding pocket. In the binding mode,
compound 22g is potently bound to the active binding site of c-Met via two hydrogen bonds and one pi–pi interactions.
The oxygen atoms of the 5 atom linker moiety (C ring) formed one hydrogen bond with the amino hydrogen of MET A:
1160; contribute to the hydrogen bonding interaction together, being a probable explanation for its nice activity. On the
other hand, the substituted aryl group (D ring) formed one hydrogen bond and one pi–pi interaction with Met A: 1211
and PHE A: 1223 respectively. In general, these results of the molecular docking study showed that 4-oxo-pyridazinone

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derivatives bearing pyrrolo[2,3-b]pyridine moiety could act synergistically to interact with the active binding site of
c-Met , suggested that compound 22g may be a potential inhibitor of c-Met.
Fig. 7 The protonated state of several important residue were adjusted by using AutoDock vina 1.1.2 in favor of
forming reasonable hydrogen bond with the ligand and Molecular docking analysis was carried out by the Discover
Studio 3.5 visualizer to explore the binding model for the active site of c-Met with its ligand. Docking simulations
show that the four selected compounds (22f, 22g, 26c, and 26e) occupy the ATP site of c-Met kinase (Fig.7a). Then We
employed 3D interaction map (Fig.7b) , 2D diagram (Fig.7c) and pharmacophore model (Fig.7d) to display the
interaction between 22g and the targeted protein (3LQ8). In the binding model, compound 22g is nicely bound to c-Met
via two hydrogen bonds and one pi–pi interaction.
4. Conclusions
In summary, we designed and synthesized eight series of pyrrolo-pyridine/pyrimidine derivatives bearing
pyridazinone moiety and evaluated for the IC50 values against four cancer cell lines (A549, HepG2 , MCF-7 and PC-3).
Some selected compounds ( 22f, 22g, 26c and 26e ) were evaluated for the activity against c-Met kinase, and according
to the results of kinase inhibitory activity, the compound 22g was further evaluated for other four tyrosine kinases

ACCEPTED MANUSCRIPT
( Flt-3, VEGFR-2, c-Kit and EGFR ) to test the enzyme-based selectivity. SARs and docking studies indicated that
pyrrolo[2,3-b]pyridine derivatives bearing 4-oxo-pyridazinone moiety show better antitumour activity than
pyrrolo[2,3-d]pyrimidine derivatives bearing 4-oxo-pyridazinone moiety. What’s more, the target compounds modified
with X and Y (X = H, Y = H) were favorable to the activity. And EWGs on aryl group (D ring) can increase the activity
compounds. In addition, the experiments of enzyme-based selectivity confirmed that 22g inhibit the c-Met selectively.
According to the result of AO single staining and Annexin V/PI staining, it's claimed that the 22g could induce
remarkable apoptosis of HepG2 cells. And further study will be carried out to identify the exact action mechanism in
near future.
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 8a-g and 13a-j
Appropriate substituted anilines 6a-g or 12a-j (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 Preparation of compounds 9a-g
The yellow solid 8a-g (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
9a-g .
5.4 General procedure for preparation of 14a-g
Intermediate13a-j (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

ACCEPTED MANUSCRIPT
anhydrous sodium sulfate, then the organic layer was concentrated in vacuum to give the product 14a-g.
5.5 General procedure for preparation of 10a-g and 15a-j
Appropriate intermediate 9a-g (0.11 mol) or 14a-j (0.104 mol) , sodium carbonate (0.2 mol) were dissolved in
Ethanol/H2O (50 ml, 5:1) ,and heated to 80 for 5-6 hour. Subsequently, the mixture solution acidified to pH 2-3 to
yield the compounds 10a-g or 15a-j, respectively.
5.6 General procedure for the key intermediate 11a-g and 16a-j
The compounds 10a-g or 15a-j (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 19a-d and 21a-d
P-aminophenol or 2-fluoro-4-aminphenol 18a-b (0.1 mol) which was dissolved in tetrahydrofuran (50 ml), was added to
a 1,4-dioxane/H2O(50 ml, 5:1) solution of compounds 17a-b or 20a-b, 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 .
5.7.1. 4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (19a)
Light yellow solid; Yield: 55%; M.P.: 182-185 ;1 H NMR (400 MHz, DMSO-d6) δ11.61 (s, 1H), 8.01 (d, J = 5.3 Hz,
1H), 7.30 (d, J = 2.3 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.63(d, J = 8.5 Hz, 2H), 6.28 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H),
5.08 (s, 2H); MS (ESI) m/z(%):226.5 [M+H] +.
5.7.2. 3-Fluoro-4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (19b)
Light yellow solid; Yield: 49%; M.P.: 184-188 ;1 H NMR (400 MHz, DMSO-d6) δ11.67 (s, 1H), 8.03 (d, J = 5.3 Hz,
1H), 7.33 (s, 1H), 7.02 (t, J = 9.0 Hz, 1H), 6.52 (d, J = 13.3Hz, 1H), 6.44 (d, J = 8.6 Hz, 1H), 6.29 (d, J = 5.5 Hz, 1H),
6.24 (s, 1H), 5.42 (s, 2H); MS(ESI) m/z(%): 244.2 [M+H] +.
5.7.3. 4-((1-Methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (19c)
Light yellow solid; Yield: 53%; M.P.: 188-191 ;1 H NMR (400 MHz, DMSO-d6) δ8.01 (d, J = 5.3 Hz, 1H), 7.31 (d, J
= 2.5 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 8.6 Hz,2H), 6.26 (d, J = 5.3 Hz, 1H), 6.23 (m, 1H), 5.10 (s, 2H),
3.96 (s, 3H); MS (ESI) m/z(%):240.8 [M+H] +.
5.7.4. 3-Fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)aniline (19d)
Light yellow solid; Yield: 56%; M.P.: 192–195 ; 1H NMR (400 MHz,DMSO-d6) 8.02 (m, 1H),7.32 (s, 1H), 7.01 (t, J
= 9.0, 1H), 6.51 (dd, J = 13.2, 1H), 6.43 (dd, J = 8.6, 1H), 6.28 (d, J =5.4 Hz, 1H), 6.24 (m, 1H), 5.42 (s, 2H), 4.09 (s,
3H); MS (ESI) m/z(%): 258.7 [M+H] +.
5.7.5. 4-((1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (21a)
Light yellow solid; Yield: 61%; M.P.: 186-188℃;1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.26 (s, 1H), 7.39 –

ACCEPTED MANUSCRIPT
7.33 (m, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H), 6.23 (dd, J = 3.3, 1.7 Hz, 1H), 5.04 (s, 2H) ; MS (ESI)
m/z(%):227.09 [M+H] +.
5.7.6. 3-Fluoro-4-((1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (21b)
Light yellow solid; Yield: 56%; M.P.: 189-191 ;1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 8.26 (s, 1H), 7.43 (s,
1H), 6.99 (t, J = 8.9 Hz, 1H), 6.44 (m, 3H), 5.34 (s, 2H) ; MS (ESI) m/z(%):245.08[M+H] +.
5.7.7. 4-((1-Methyl-1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (21c)
Light yellow solid; Yield: 58%; M.P.: 193-195 ;1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.53 (d, J = 3.5 Hz,
1H), 6.97 (d, J = 8.7 Hz, 2H), 6.69 (d, J = 8.7 Hz,2H), 6.34 (d, J = 3.5 Hz, 1H), 5.17 (s, 2H), 3.89 (s,3H) ; MS (ESI)
m/z(%):241.10 [M+H] +.
5.7.8. 3-Fluoro-4-((1-methyl-1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (21d)
Light yellow solid; Yield: 59%; M.P.: 194-197℃;1H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J = 3.2 Hz, 1H), 8.47 (s,
1H), 8.32 (d, J = 9.0 Hz, 1H), 7.89 (t, J = 8.4 Hz, 1H), 7.73 (d, J = 3.5 Hz,1H), 6.82 (d, J = 3.5 Hz,1H), 5.27 (s, 2H),
3.96 (s,3H) ; MS (ESI) m/z(%):259.10 [M+H] +.
5.8. General procedure for the preparation of target compounds 22a-g,23a-g, 24a-g, 25a-g,26a-f, 27a-f, 28a-f and
29a-f.
A solution of phenylpyrdazinone carbonyl chloride 11a-g or 16a-j (0.82 mmol) in dichloromethane (10 mL) was added
drop-wise to a solution of aniline 19a-d or 21a-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 22a-g,23a-g, 24a-g, 25a-g,26a-f, 27a-f, 28a-f and 29a-f which were recrystallized by isopropanol.
5.8.1.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-chlorophenyl)-4-oxo-1,4-dihydropyridazine-3
-carboxamide (22a)
This compound was obtained as pale yellow solid in 79% yield; M.P.: 215.3-216.0℃; ESI-MS m/z:459.08[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 11.75 (s, 1H), 9.00 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 5.4 Hz, 1H), 7.84 (d, J
= 8.9 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.9 Hz, 2H), 7.34 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 7.9
Hz, 1H), 6.44 (d, J = 5.4 Hz, 1H), 6.19 (s, 1H).
5.8.2.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydropyridazine-3-carb
oxamide (22b)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 211.0-212.0 ℃; ESI-MS m/z:454.15[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 11.73 (s, 1H), 8.91 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 5.4 Hz, 1H), 7.79 (d, J
= 8.8 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 7.34 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 7.8

ACCEPTED MANUSCRIPT
Hz, 1H), 6.43 (d, J = 5.4 Hz, 1H), 6.18 (s, 1H), 3.82 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.73, 159.51, 157.66,
155.34, 153.32, 152.62, 150.47, 143.22, 141.63, 137.54, 133.65, 130.36, 129.86(2C), 125.68(2C), 124.82, 121.85（2C）,
121.62(2C), 120.32, 110.84, 102.72, 97.56, 31.78, 21.26.
5.8.3.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-bromophenyl)-4-oxo-1,4-dihydropyridazine-3
-carboxamide (22c)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 217.0-218.0 ℃; ESI-MS m/z:503.04[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.79 (s, 1H), 11.73 (s, 1H), 9.02 (d, J = 7.9 Hz, 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.83 –
7.76 (m, 6H), 7.36 (s, 1H), 7.24 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 7.8 Hz, 1H), 6.46 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H).
5.8.4.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxamide
(22d)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 212.0-213.0 ℃; ESI-MS m/z:424.14[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 11.71 (s, 1H), 9.00 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 5.4 Hz, 1H), 7.85 –
7.75 (m, 4H), 7.61 (t, J = 7.9 Hz, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 2.5 Hz, 1H), 7.21 (d, J = 8.9 Hz, 2H), 6.90
(d, J = 7.8 Hz, 1H), 6.44 (d, J = 5.4 Hz, 1H), 6.19 (d, J = 1.8 Hz, 1H).
5.8.5.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydropyrid-azine
-3-carboxamide (22e)
+
1
This compound was obtained as pale yellow solid in 79% yield; M.P.: 210.0-211.0℃; ESI-MS m/z:521.03[M+H] . H
NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 11.66 (s, 1H), 8.75 (s, 1H), 8.09 (d, J = 5.3 Hz, 1H), 7.98 (d, J = 10.4 Hz,
1H), 7.76 (m, 4H), 7.36 (s, 1H), 7.23 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 7.9 Hz, 1H), 6.46 (d, J = 5.5 Hz, 1H), 6.20 (s,
1H).
5.8.6.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-chloro-3-fluorophenyl)-4-oxo-1,4-dihydropyridazine-
3-carboxamide (22f)
This compound was obtained as pale yellow solid in 70% yield; M.P.: 214.0-215.0℃; ESI-MS m/z:477.08[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H), 11.79 (s, 1H), 9.02 (d, J = 7.9 Hz, 1H), 8.14 (d, J = 3.8 Hz, 1H), 8.09 (d, J
= 5.4 Hz, 1H), 7.98 (d, J = 12.7 Hz, 1H), 7.86 (s, 1H), 7.71 (t, J = 9.0 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.42 (dd, J =
17.3, 8.3 Hz, 2H), 6.94 (d, J = 7.9 Hz, 1H), 6.43 (d, J = 5.3 Hz, 1H), 6.26 (s, 1H);13C NMR (101 MHz, DMSO) δ
169.75, 159.86, 157.75, 151.29, 150.53, 148.30, 144.56, 142.28, 140.19, 135.56, 129.13, 124.28, 122.81, 122.73,
121.82(2C), 121.42(2C), 120.67, 118.40, 118.17, 110.80, 102.83, 96.65.
5.8.7.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-chloro-3-(trifluoromethyl)phenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (22g)
This compound was obtained as pale yellow solid in 77% yield; M.P.: 212.0-214.0 ℃; 1H NMR (400 MHz, DMSO-d6)

ACCEPTED MANUSCRIPT
11.73 (s, 1H), 11.71 (s, 1H), 9.10 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 2.2 Hz, 1H), 8.15 (d, J = 11.2 Hz, 1H), 8.07 (d, J =
5.4 Hz, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.36 – 7.33 (m, 1H), 7.22 (d, J = 8.8 Hz, 2H), 6.93 (d, J
= 7.9 Hz, 1H), 6.43 (d, J = 5.4 Hz, 1H), 6.18 (s, 1H);13C NMR (101 MHz, DMSO) δ 169.35, 159.30, 157.14, 151.15,
150.92, 148.09, 144.16, 143.02, 141.65, 134.97, 133.02, 130.31, 130.12, 127.77, 126.64, 124.64, 123.64, 121.34(2C),
120.90(2C), 120.08, 110.14, 102.24, 97.09. HRMS (ESI): m/z [M+H]+ calcd for C25H15ClF3N5O3, 525.0816; found
525.0823.
5.8.8.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-chlorophenyl)-4-oxo-1,4-dihydropyridazine-
3-carboxamide (23a)
+
1
This compound was obtained as pale yellow solid in 72% yield; M.P.: 219.0-220.0;ESI-MS m/z:477.08[M+H] . H
NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 11.80 (s, 1H), 9.02 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 5.3 Hz, 1H), 7.98 (d, J
= 12.7 Hz, 1H), 7.85 (t, J = 9.7 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 9.2 Hz, 1H), 7.47 – 7.35 (m, 2H), 6.93 (d,
J = 7.8 Hz, 1H), 6.43 (d, J = 5.3 Hz, 1H), 6.26 (s, 1H).
5.8.9.
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-methoxyphenyl)-4-oxo-1,4-dihydropyridazi
ne-3-carboxamide (23b)
This compound was obtained as pale yellow solid in 68% yield; M.P.: 221.0-223.0 ℃; ESI-MS m/z:472.14[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 12.08 (s, 1H), 11.78 (s, 1H), 8.93 (d, J = 7.4 Hz, 1H), 8.07 (d, J = 5.2 Hz, 1H), 7.97 (d, J
= 13.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.48 (s, 1H), 7.45 – 7.33 (m, 2H), 7.14 (d, J = 9.1 Hz, 2H), 6.91 (d, J = 7.3 Hz,
1H), 6.40 (d, J = 5.2 Hz, 1H), 6.24 (s, 1H), 3.82 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.43, 159.65, 157.63,
157.35, 154.32, 152.68, 149.43, 143.15, 141.68, 137.34, 133.52, 130.15, 129.28(2C), 125.63(2C), 124.72, 121.87,
121.38, 120.19, 118.48, 110.84, 102.67, 97.68, 21.37.
5.8.10. N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-bromophenyl)-4-oxo-1,4-dihydropyridazine-
3-carboxamide (23c)
This compound was obtained as pale yellow solid in 74% yield; M.P.: 218.0-220.0 ℃; ESI-MSm/z:521.03[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 11.78 (s, 1H), 9.00 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 5.4 Hz, 1H), 7.96 (d, J
= 12.7 Hz, 1H), 7.79 (q, J = 8.9 Hz, 5H), 7.49 (d, J = 9.0 Hz, 1H), 7.44 – 7.36 (m, 2H), 6.91 (d, J = 7.8 Hz, 1H), 6.40 (d,
J = 5.4 Hz, 1H), 6.24 (s, 1H).
5.8.11. N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carboxa
mide (23d)
This compound was obtained as pale yellow solid in 72% yield; M.P.:216.0-217.0 ℃; ESI-MS m/z:442.13[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 11.83 (s, 1H), 9.22 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 5.6 Hz, 1H), 7.83 (d, J

ACCEPTED MANUSCRIPT
= 7.9 Hz, 2H), 7.64 (dt, J = 17.4, 7.7 Hz, 5H), 7.52 (t, J = 9.5 Hz, 2H), 7.41 (s, 1H), 7.33 (s, 1H), 6.51 (d, J = 5.4 Hz,
1H), 6.31 (s, 1H).
5.8.12. N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-bromo-2-fluorophenyl)-4-oxo-1,4-dihydrop
yridazine-3-carboxamide (23e)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 221.0-222.0 ℃; ESI-MS m/z:539.02[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 11.77 (s, 1H), 8.74 (d, J = 7.9 Hz, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.94 (dd,
J = 17.1, 11.5 Hz, 2H), 7.75 (t, J = 8.3 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 8.9 Hz,
1H), 7.36 (s, 2H), 6.90 (d, J = 7.8 Hz, 1H), 6.39 (d, J = 5.4 Hz, 1H), 6.23 (s, 1H).
5.8.13. N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-chloro-3-fluorophenyl)-4-oxo-1,4-dihydropy
ridazine-3-carboxamide (23f)
This compound was obtained as pale yellow solid in 72% yield; M.P.: 216.0-218.0 ℃; 1H NMR (400 MHz, DMSO-d6)
δ 11.90 (s, 1H), 11.80 (s, 1H), 9.03 (s, 1H), 8.12 (d, J = 14.7 Hz, 2H), 7.98 (d, J = 12.9 Hz, 1H), 7.87 (s, 1H), 7.72 (d, J
= 8.5 Hz, 1H), 7.52 (s, 1H), 7.46 – 7.37 (m, 2H), 6.94 (d, J = 6.7 Hz, 1H), 6.43 (s, 1H), 6.26 (s, 1H); HRMS (ESI): m/z
[M+H]+ calcd for C24H14ClF2N5O3, 493.0753; found 493.0741.
5.8.14. N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-chloro-3-(trifluoromethyl)phenyl)-4-oxo-1,4
-dihydropyridazine-3-carboxamide (23g)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 217.0-219.0 ℃; ESI-MS m/z:541.09[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.79 (s, 1H), 11.72 (s, 1H), 9.05 (d, J = 7.9 Hz, 1H), 8.23 (s, 1H), 8.10 (d, J = 8.4 Hz,
1H), 8.01 (d, J = 5.4 Hz, 1H), 7.92 (t, J = 10.6 Hz, 2H), 7.45 (d, J = 8.3 Hz, 1H), 7.35 (dd, J = 17.3, 8.4 Hz, 2H), 6.88 (d,
J = 7.9 Hz, 1H), 6.35 (d, J = 5.3 Hz, 1H), 6.18 (s, 1H).
5.8.15. 1-(4-Chlorophenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridazin
e-3-carboxamide (24a)
This compound was obtained as pale yellow solid in 68% yield; M.P.: 232.0-234.0 ℃; 1H NMR (400 MHz, DMSO-d6)
δ 11.78 (s, 1H), 8.99 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 5.4 Hz, 1H), 7.84 (d, J = 9.0 Hz, 2H), 7.79 (d, J = 8.9 Hz, 2H),
7.68 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 3.4 Hz, 1H), 7.21 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 7.8 Hz, 1H), 6.47 (d, J = 5.4 Hz,
1H), 6.19 (d, J = 3.4 Hz, 1H), 3.80 (s, 3H); HRMS (ESI): m/z [M+H]+ calcd for C24H16ClN5O3, 457.0942; found
457.0939.
5.8.16. 1-(4-Methoxyphenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridaz
ine-3-carboxamide (24b)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 229.0-232.0 ℃; ESI-MS m/z:468.16[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 5.4 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H),

ACCEPTED MANUSCRIPT
7.58 (d, J = 8.9 Hz, 2H), 7.27 (d, J = 3.3 Hz, 2H), 7.08 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.77 (d, J = 7.8 Hz,
2H), 6.35 (d, J = 5.4 Hz, 1H), 6.06 (d, J = 3.3 Hz, 1H), 3.70 (s, 3H), 3.67 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ
169.75, 159.53, 157.62, 155.32, 153.28, 152.72, 150.43, 143.25, 141.59, 137.48, 133.58, 130.29, 129.73(2C),
125.65(2C), 124.86, 121.83（2C）, 121.56(2C), 120.36, 110.78, 102.68, 97.52, 31.76, 21.34.
5.8.17. 1-(4-Bromophenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydropyridazin
e-3-carboxamide (24c)
This compound was obtained as pale yellow solid in 78% yield; M.P.: 231.0-233.0 ℃; ESI-MS m/z:517.06[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.86 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 5.4 Hz, 1H), 7.69 – 7.63 (m, 5H),
7.27 (d, J = 3.5 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 7.9 Hz, 1H), 6.35 (d, J = 5.4 Hz, 1H), 6.06 (d, J = 3.5 Hz,
1H), 3.67 (s, 3H).
5.8.18. N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-3-carbox
amide (24d)
This compound was obtained as pale yellow solid in 76% yield; M.P.: 234.0-236.0 ℃; ESI-MS m/z:438.15[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 8.87 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 5.2 Hz, 1H), 7.67 (d, J = 6.3 Hz, 4H),
7.49 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.1 Hz, 1H), 7.27 (d, J = 3.2 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 7.8 Hz,
1H), 6.35 (d, J = 5.3 Hz, 1H), 6.06 (d, J = 3.3 Hz, 1H), 3.68 (s, 3H).
5.8.19. 1-(4-Bromo-2-fluorophenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (24e)
This compound was obtained as pale yellow solid in 75% yield; M.P.: 233.0-235.0 ℃; ESI-MS m/z:535.05[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 5.4 Hz, 1H), 7.83 (d, J = 10.4 Hz,
1H), 7.62 (dd, J = 11.3, 8.7 Hz, 3H), 7.55 (d, J = 10.5 Hz, 1H), 7.27 (d, J = 3.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.76
(d, J = 7.8 Hz, 1H), 6.34 (d, J = 5.4 Hz, 1H), 6.05 (d, J = 3.4 Hz, 1H), 3.67 (s, 3H).
5.8.20. 1-(4-Chloro-3-fluorophenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (24f)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 231.0-232.0 ℃; ESI-MS m/z:491.10[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 9.01 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 5.1 Hz, 2H), 7.86 (s, 1H), 7.81 (d, J
= 8.5 Hz, 2H), 7.70 (t, J = 8.8 Hz, 1H), 7.42 (s, 1H), 7.23 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 7.7 Hz, 1H), 6.50 (d, J = 5.3
Hz, 1H), 6.21 (d, J = 3.2 Hz, 1H), 3.82 (s, 1H);13C NMR (101 MHz, DMSO-d6) δ 169.75, 159.86, 157.75, 156.05,
151.29, 150.53, 148.30, 144.56, 142.28, 140.21, 135.56, 129.13, 124.28, 122.81, 122.73, 121.82(2C), 121.42(2C),
120.77, 118.29, 110.80, 102.83, 96.65, 31.63.
5.8.21. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,

ACCEPTED MANUSCRIPT
4-dihydropyridazine-3-carboxamide (24g)
+
1
This compound was obtained as pale yellow solid in 70% yield; M.P.: 230.0-231.0 .ESI-MS m/z:541.09[M+H] . H
NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 9.05 (d, J = 7.5 Hz, 1H), 8.23 (s, 1H), 8.13 – 8.06 (m, 2H), 7.94 (d, J = 8.5
Hz, 1H), 7.74 (d, J = 8.6 Hz,2H), 7.34 (s, 1H), 7.16 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 5.1 Hz,
1H), 6.14 (d, J = 3.2 Hz, 1H), 3.75 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.83, 159.83, 157.75, 151.32, 150.53,
148.61, 144.57, 142.16, 135.54, 133.53, 130.81, 129.14, 128.26, 127.94, 127.15, 124.14, 121.82(2C), 121.42(2C),
121.31, 120.58, 110.80, 102.85, 96.64, 31.63.
5.8.22. 1-(4-Chlorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydro
pyridazine-3-carboxamide (25a)
This compound was obtained as pale yellow solid in 68% yield; M.P.: 230.0-231.0 ; ESI-MS m/z:491.10[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1H), 9.00 (d, J = 7.9 Hz, 1H), 8.12 (d, J = 5.4 Hz, 1H), 7.96 (d, J = 12.0 Hz,
1H), 7.84 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 9.0 Hz, 1H), 7.43 – 7.39 (m, 2H), 6.91 (d, J = 7.9 Hz,
1H), 6.44 (d, J = 5.4 Hz, 1H), 6.25 (d, J = 3.3 Hz, 1H), 3.80 (s, 1H).
5.8.23. 1-(4-Methoxyphenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4-dihydr
opyridazine-3-carboxamide (25b)
This compound was obtained as pale yellow solid in 69% yield; M.P.: 235.0-236.0 ; ESI-MS m/z:486.15[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.92 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 5.4 Hz, 1H), 7.95 (s, 1H), 7.71 (d, J
= 9.0 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.14 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 7.7 Hz, 1H), 6.44 (d,
J = 5.4 Hz, 1H), 6.24 (d, J = 3.4 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.56, 159.67,
157.59, 157.32, 154.28, 152.76, 149.41, 143.11, 141.64, 137.36, 133.46, 130.11, 129.47(2C), 125.88(2C), 124.90,
121.81, 121.36, 120.03, 118.36, 110.80, 102.62, 97.66, 31.74, 21.25.
5.8.24. N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-1-(4-bromophenyl)-4-oxo-1,4-dihydrop
yridazine-3-carboxamide (25c)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 234.0-235.0 ; ESI-MS m/z:535.05[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 9.00 (d, J = 7.9 Hz, 1H), 8.12 (d, J = 5.4 Hz, 1H), 7.96 (d, J = 12.7 Hz,
1H), 7.83 – 7.76 (m, 5H), 7.50 (d, J = 9.5 Hz, 1H), 7.44 – 7.40 (m, 2H), 6.91 (d, J = 7.9 Hz, 1H), 6.44 (d, J = 5.4 Hz,
1H), 6.24 (d, J = 3.5 Hz, 1H), 3.80 (s, 3H).
5.8.25. N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1-phenyl-1,4-dihydropyridazine-
3-carboxamide (25d)
+
This compound was obtained as pale yellow solid in 65% yield; M.P.: 229.0-232.0 ; ESI-MS m/z:456.14[M+H]
.
1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H), 9.22 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 5.6 Hz, 1H), 7.83 (d, J = 7.9 Hz,

ACCEPTED MANUSCRIPT
2H), 7.64 (dt, J = 17.4, 7.7 Hz, 5H), 7.52 (t, J = 9.5 Hz, 2H), 7.41 (s, 1H), 7.33 (s, 1H), 6.51 (d, J = 5.4 Hz, 1H), 6.31 (s,
1H), 3.80 (s, 3H).
5.8.26. 1-(4-Bromo-2-fluorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4
-dihydropyridazine-3-carboxamide (25e)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 233.0-234.0 ; ESI-MS m/z:553.04[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 8.74 (d, J = 6.1 Hz, 1H), 8.11 (d, J = 5.4 Hz, 1H), 7.94 (dd, J = 17.6, 11.6
Hz, 2H), 7.75 (t, J = 8.4 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.44 – 7.39 (m, 2H), 6.90 (d, J =
7.8 Hz, 1H), 6.43 (d, J = 5.4 Hz, 1H), 6.24 (d, J = 3.5 Hz, 1H), 3.80 (s,3H).
5.8.27. 1-(4-Chloro-3-fluorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-oxo-1,4
-dihydropyridazine-3-carboxamide (25f)
1
This compound was obtained as pale yellow solid in 72% yield; M.P.: 232.0-233.0 ;ESI-MS m/z:509.09[M+H] +. H
NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 9.02 (d, J = 7.3 Hz, 1H), 8.14 (d, J = 5.3 Hz, 2H), 7.98 (d, J = 12.8 Hz,
1H), 7.87 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 7.5 Hz, 1H),
6.47 (d, J = 4.9 Hz, 1H), 6.27 (s, 1H), 3.83 (s, 3H).
5.8.28. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)
-4-oxo-1,4-dihydropyridazine-3-carboxamide (25g)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 285.3-286.0 ; ESI-MS m/z:559.08[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 9.05 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 2.1 Hz, 1H), 8.08 (t, J = 9.4 Hz, 2H),
7.91 (dd, J = 20.3, 5.5 Hz, 2H), 7.45 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 11.5, 6.2 Hz, 2H), 6.88 (d, J = 7.9 Hz, 1H), 6.39
(d, J = 5.4 Hz, 1H), 6.19 (d, J = 3.4 Hz, 1H), 3.75 (s, 3H).
5.8.29. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-dihydropyridazi
ne-3-carboxamide (26a)
+
This compound was obtained as pale yellow solid in 74% yield; M.P.: 235.0-236.0 ; ESI-MS m/z: 457.14[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 10.73 (s, 1H), 8.89 (s, 1H), 8.57 (s, 1H), 8.23 (d, J = 3.5 Hz, 1H), 7.93
– 7.87 (m, 2H), 7.75 (s, 1H), 7.46 (dd, J = 18.1, 8.9 Hz, 3H), 7.17 (s, 1H), 6.97 (d, J = 3.7 Hz, 1H), 6.89 (s, 1H), 2.51 (s,
3H).
5.8.30. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-1,6-dihydropyridazi
ne-3-carboxamide (26b)
+
This compound was obtained as pale yellow solid in 77% yield; M.P.: 236.0-237.0 ; ESI-MS m/z: 457.14 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 10.75 (s, 1H), 8.88 (s, 1H), 8.57 (s, 1H), 8.22 (d, J = 3.5 Hz, 1H), 7.90
(d, J = 8.6 Hz, 2H), 7.76 (d, J = 5.0 Hz, 1H), 7.59 – 7.47 (m, 2H), 7.43 (d, J = 8.9 Hz, 2H), 7.20 (s, 1H), 6.97 (d, J = 3.6

ACCEPTED MANUSCRIPT
Hz, 1H), 2.52 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.69, 161.53, 160.45, 158.48, 152.49, 150.07, 148.82,
142.86, 141.97, 135.43, 130.21, 129.78, 129.25, 122.13(2C), 121.64, 121.45(2C), 115.10, 112.98, 112.10, 104.71, 97.39,
18.15.
5.8.31. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-dihydropyridazine-3-car
boxamide (26c)
This compound was obtained as pale yellow solid in 74% yield; M.P.: 231.0–232.0 ; ESI-MS m/z: 453.16[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 10.61 (s, 1H), 8.79 (s, 1H), 8.13 (d, J = 3.6 Hz, 1H), 7.82 (d, J = 8.8 Hz,
2H), 7.66 (s, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 8.5 Hz, 3H), 7.05 (s, 1H), 6.88 (d, J = 3.7 Hz, 1H), 2.41 (s, 3H),
2.39 (s, 3H);13C NMR (101 MHz, DMSO-d6) δ 162.15, 159.13, 152.94, 150.54, 149.20, 143.02, 142.07, 138.84, 138.20,
136.03, 130.03, 129.68, 129.43(2C), 125.83(2C), 125.64, 122.64(2C), 121.74(2C), 105.18, 97.86, 21.15, 18.63.
5.8.32. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethoxy)phenyl)-1,6-dih
ydropyridazine-3-carboxamide (26d)
+
This compound was obtained as pale yellow solid in 73% yield; M.P.: 237.0-238.0 ; ESI-MS m/z: 523.13[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 12.09 (s, 1H), 8.79 (s, 1H), 8.14 (d, J = 3.7 Hz, 1H), 7.90 (d, J = 7.8
Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.78 – 7.65 (m, 3H), 7.37 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 4.0 Hz, 1H), 6.81 (s, 1H),
2.12 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.73, 161.52, 159.36, 152.49, 150.08, 149.97, 148.76, 145.64, 144.62,
142.45, 141.67, 135.88, 135.42, 131.80, 130.50, 129.25, 128.73, 128.16, 122.29(2C), 120.87(2C), 104.71, 97.62, 18.58.
5.8.33. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-bromo-2-fluorophenyl)-4-methyl-6-oxo-1,6-dihydr
opyridazine-3-carboxamide (26e)
This compound was obtained as pale yellow solid in 72% yield; M.P.: 238.0-239.0 ; 1H NMR (400 MHz, DMSO-d6)
δ 12.45 (s, 1H), 10.65 (s, 1H), 8.79 (s, 1H), 8.13 (d, J = 4.0 Hz, 1H), 7.83 (dd, J = 17.2, 9.6 Hz, 2H), 7.69 (dd, J = 15.0,
7.5 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.11 (s, 1H), 6.88 (d, J = 3.6 Hz, 1H), 6.80 (s, 1H), 2.43 (s, 3H); 13C NMR (101
MHz, DMSO) δ 162.13, 161.74, 158.46, 158.06, 155.51, 152.95, 150.54, 149.27, 144.03, 143.07, 135.87, 131.14,
129.70, 129.54, 128.84, 122.65(2C), 121.81(2C), 120.31, 120.09, 105.18, 97.86, 18.83; HRMS (ESI): m/z [M+H]+
calcd for C25H17BrFN5O3, 533.0499; found 533.0506.
5.8.34. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(3-(trifluoromethyl)phenyl)-1,6-dihy
dropyridazine-3-carboxamide (26f)
This compound was obtained as pale yellow solid in 78% yield; M.P.: 232.0–233.0 ; ESI-MS m/z: 507.13[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 10.57 (s, 1H), 8.26 (s, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 10.2 Hz,
1H), 7.58 – 7.46 (m, 2H), 7.43 (d, J = 3.1 Hz, 1H), 7.24 (t, J = 8.5 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.01 (s, 1H), 6.39
(d, J = 3.3 Hz, 1H), 2.32 (s, 3H).

ACCEPTED MANUSCRIPT
5.8.35. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-4-methyl-6-oxo-1,6-dihydropyridazi
ne-3-carboxamide (27a)
+
This compound was obtained as pale yellow solid in 69% yield; M.P.: 235.0-236.0 ; ESI-MS m/z: 457.14 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.74 (s, 1H), 8.27 (s, 1H), 7.83 (d, J = 12.4 Hz, 1H), 7.77 – 7.74 (m,
2H), 7.55 – 7.49 (m, 2H), 7.43 (d, J = 8.8 Hz, 1H), 7.36 (t, J = 8.7 Hz, 2H), 7.06 (s, 1H), 6.58 (s, 1H), 2.39 (s, 3H).
5.8.36. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-1,6-dihydropyridazi
ne-3-carboxamide (27b)
This compound was obtained as pale yellow solid in 71% yield; M.P.: 230.0-231.0 ; ESI-MS m/z: 457.14[M+H] +. 1H
NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.75 (s, 1H), 8.27 (s, 1H), 7.84 (d, J = 12.1 Hz, 1H), 7.67 (d, J = 10.4 Hz,
1H), 7.64 – 7.48 (m, 3H), 7.47 – 7.39 (m, 1H), 7.39 – 7.28 (m, 1H), 7.08 (s, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.58 (s, 1H),
2.40 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.89, 161.66, 160.73, 160.48, 158.42, 154.83, 152.46, 149.95, 142.94,
141.46, 136.79, 130.27, 130.18, 129.87, 129.59, 124.45, 121.65, 116.54, 115.15, 112.87, 108.53, 104.18, 97.23, 18.27.
5.8.37. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-dihydropyridazine-3-car
boxamide (27c)
+
This compound was obtained as pale yellow solid in 78% yield; M.P.: 236.0-237.0 ; ESI-MS m/z: 453.16[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.72 (s, 1H), 8.27 (s, 1H), 7.83 (d, J = 12.6 Hz, 1H), 7.57 (d, J = 8.1
Hz, 2H), 7.51 (d, J = 10.0 Hz, 2H), 7.41 (t, J = 8.8 Hz, 1H), 7.32 (d, J = 8.1 Hz, 2H), 7.03 (s, 1H), 6.58 (s, 1H), 2.39 (s,
3H), 2.36 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.43, 161.36, 159.15, 155.26, 152.94, 150.45, 143.18, 141.69,
138.83, 138.34, 137.38, 135.97, 130.09, 129.52(2C), 125.93(2C), 124.94, 116.87, 109.13, 108.83, 104.66, 97.68, 21.25,
18.73.
5.8.38. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethoxy)phenyl)-1,6-dih
ydropyridazine-3-carboxamide (27d)
+
This compound was obtained as pale yellow solid in 70% yield; M.P.: 233.0-234.0 ; ESI-MS m/z: 523.13[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 12.15 (s, 1H), 8.85 (s, 1H), 8.28 (s, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.87
(d, J = 7.8 Hz, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 9.5 Hz, 2H), 7.43 (t, J = 8.4 Hz, 1H),
6.59 (s, 1H), 2.09 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.52, 160.75, 159.75,154.89, 152.45, 149.98, 144.52,
142.47, 141.65, 136.59, 135.85, 131.86, 130.72, 129.65, 128.74, 128.14, 124.63, 122.36, 116.19, 108.52, 104.12, 97.26,
18.83.
5.8.39. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-bromo-2-fluorophenyl)-4-methyl-6-oxo-1,6-dihydr
opyridazine-3-carboxamide (27e)
+
This compound was obtained as pale yellow solid in 74% yield; M.P.: 235.0-236.0 ; ESI-MS m/z: 535.05[M+H] .

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 10.80 (s, 1H), 8.27 (s, 1H), 7.82 (d, J = 10.8 Hz, 2H), 7.72 – 7.60 (m,
2H), 7.58 – 7.48 (m, 2H), 7.41 (t, J = 8.9 Hz, 1H), 7.09 (s, 1H), 6.57 (s, 1H), 2.40 (s, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 161.83, 161.25, 158.47, 158.13, 155.25, 152.96, 150.43, 144.15, 142.64, 139.92, 137.27, 135.98, 131.15,
130.13, 129.63, 128.95, 124.92, 123.19, 120.42, 116.96, 109.16, 104.57, 97.68, 18.86.
5.8.40. N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(3-(trifluoromethyl)phenyl)-1,6-dihy
dropyridazine-3-carboxamide (27f)
+
This compound was obtained as pale yellow solid in 78% yield; M.P.: 231.0-232.0 ; ESI-MS m/z: 507.13 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.79 (s, 1H), 8.28 (s, 1H), 8.16 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.88
– 7.82 (m, 2H), 7.78 (t, J = 7.7 Hz, 1H), 7.55 – 7.49 (m, 2H), 7.43 (t, J = 8.8 Hz, 1H), 7.11 (s, 1H), 6.59 (s, 1H), 2.42 (s,
3H); 13C NMR (101 MHz, DMSO-d6) δ 162.67, 160.85, 159.47, 154.95, 152.52, 149.95, 143.43, 142.71, 140.68, 136.74,
135.55, 134.29, 131.22, 130.68, 129.65, 128.89, 127.66, 124.67, 124.23, 121.42, 116.24, 108.35, 104.09, 97.24, 18.65.
5.8.41. 4-Methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-1-(2-(trifluoromethoxy)phen
yl)-1,6-dihydropyridazine-3-carboxamide (28a)
+
This compound was obtained as pale yellow solid in 72% yield; M.P.: 244.0-245.0 ; ESI-MS m/z: 537.15[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.86 (s, 1H), 8.34 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.9 Hz,
1H), 7.72 (t, J = 8.4 Hz, 2H), 7.69 – 7.66 (m, 1H), 7.50 (d, J = 3.4 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 3.4 Hz,
1H), 3.81 (s, 3H), 2.09 (s, 3H);13C NMR (101 MHz, DMSO-d6) δ 162.67, 161.63, 159.36, 152.49, 150.08, 149.97,
148.76, 145.64, 144.62, 142.45, 141.67, 135.88, 135.42, 131.80, 130.50, 129.25, 128.73, 128.16, 122.37(2C),
120.91(2C), 104.71, 97.38, 31.17, 18.67.
5.8.42. 1-(4-Bromo-2-fluorophenyl)-4-methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-
1,6-dihydropyridazine-3-carboxamide (28b)
This compound was obtained as pale yellow solid in 73% yield; M.P.: 244.0-245.0 ; 1H NMR (400 MHz, DMSO-d6)
δ 10.63 (s, 1H), 8.33 (s, 1H), 7.83 (d, J = 9.8 Hz, 1H), 7.75 (d, J = 8.9 Hz, 2H), 7.71 – 7.65 (m, 1H), 7.63 (d, J = 8.5 Hz,
1H), 7.50 (d, J = 3.4 Hz, 1H), 7.24 (d, J = 8.9 Hz, 2H), 7.09 (s, 1H), 6.45 (d, J = 3.4 Hz, 1H), 3.81 (s, 3H), 2.39 (s, 3H);
13C NMR (101 MHz, DMSO-d6) δ 162.13, 161.74, 158.46, 158.06, 155.51, 152.95, 150.54, 149.30, 144.03, 143.07,
135.88, 131.14, 129.62, 128.84, 128.49, 123.11, 122.66(2C), 121.81(2C), 120.20, 105.19, 97.87, 31.66, 18.84; HRMS
(ESI): m/z [M+H]+ calcd for C26H19BrFN5O3, 547.0655; found 547.0664.
5.8.43. 1-(3-Fluorophenyl)-4-methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-1,6-dihyd
ropyridazine-3-carboxamide (28c)
+
This compound was obtained as pale yellow solid in 70% yield; M.P.: 248.0-249.0 ; ESI-MS m/z: 471.15[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.26 (s, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 10.2 Hz, 1H), 7.58 –

ACCEPTED MANUSCRIPT
7.46 (m, 2H), 7.43 (d, J = 3.1 Hz, 1H), 7.24 (t, J = 8.5 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.01 (s, 1H), 6.39 (d, J = 3.3
Hz, 1H), 3.74 (s,3H), 2.32 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.83, 161.57, 160.41, 158.47, 152.49, 150.07,
148.82, 142.86, 141.97, 135.43, 130.21, 129.78, 129.25, 122.16(2C), 121.67, 121.38(2C), 115.10, 112.98, 112.10,
104.71, 97.39, 31.18, 18.15.
5.8.44. 4-Methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-1-(p-tolyl)-1,6-dihydropyrida
zine-3-carboxamide (28d)
+
This compound was obtained as pale yellow solid in 71% yield; M.P.: 242.0-243.0 ; ESI-MS m/z: 467.18 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.26 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.42 (d,
J = 3.4 Hz, 1H), 7.24 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 6.95 (s, 1H), 6.38 (d, J = 3.3 Hz, 1H), 3.74 (s, 3H),
2.31 (s, 3H), 2.29 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.15, 159.13, 157.10, 152.94, 150.54, 149.20, 143.02,
142.07, 138.84, 138.20, 136.03, 130.03, 129.68, 129.43(2C), 125.83(2C), 122.64(2C), 121.74(2C), 105.18, 97.86, 31.65,
21.15, 18.63.
5.8.45. 4-Methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-1-(2-(trifluoromethyl)phenyl
)-1,6-dihydropyridazine-3-carboxamide (28e)
+
This compound was obtained as pale yellow solid in 59% yield; M.P.: 246.0-247.0 ; ESI-MS m/z: 521.15[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.79 (s, 1H), 8.27 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.93 – 7.87 (m, 1H),
7.86 – 7.76 (m, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 3.2 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 6.40 (d, J = 3.2 Hz, 1H),
3.74 (s, 3H), 2.02 (s,3H).
5.8.46. 1-(2-Chlorophenyl)-4-methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-6-oxo-1,6-dihy
dropyridazine-3-carboxamide (28f)
+
This compound was obtained as pale yellow solid in 67% yield; M.P.: 245.0-246.0 ; ESI-MS m/z: 488.12 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.77 (s, 1H), 8.27 (s, 1H), 7.72 (dd, J = 15.5, 8.8 Hz, 4H), 7.62 – 7.51
(m, 2H), 7.44 (d, J = 3.3 Hz, 1H), 7.19 (d, J = 8.7 Hz, 2H), 6.41 (d, J = 3.1 Hz, 1H), 3.74 (s, 3H), 2.02 (s, 3H).
5.8.47. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluorometho
xy)phenyl)-1,6-dihydropyridazine-3-carboxamide (29a)
+
This compound was obtained as pale yellow solid in 82% yield; M.P.: 246.0-247.0 ; ESI-MS m/z: 555.14 [M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.80 (s, 1H), 8.27 (s, 1H), 7.86 – 7.78 (m,2H), 7.70 – 7.65 (m, 2H),
7.63 – 7.58 (m, 1H), 7.49 (d, J = 3.5 Hz, 1H), 7.42 (dd, J = 8.9, 1.9 Hz, 1H), 7.37 (t, J = 8.6 Hz, 1H), 6.55 (d, J = 3.5 Hz,
1H), 3.76 (s, 3H), 2.02 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.56, 160.82, 159.70,154.92, 152.51, 149.93,
144.46, 142.51, 141.66, 136.65, 135.83, 131.84, 130.64, 129.67, 128.74, 128.14, 124.63, 122.32, 116.16, 108.44,
104.15, 97.17, 31.25, 18.66.

ACCEPTED MANUSCRIPT
5.8.48. 1-(4-Bromo-2-fluorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-meth
yl-6-oxo-1,6-dihydropyridazine-3-carboxamide (29b)
+
This compound was obtained as pale yellow solid in 74% yield; M.P.: 244.0-245.0 ; ESI-MS m/z: 568.05[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.25 (s, 1H), 7.79 – 7.72 (m,2H), 7.64 – 7.54 (m, 2H), 7.47 (dd, J =
10.5, 6.2 Hz, 2H), 7.34 (t, J = 8.8 Hz, 1H), 7.03 (s, 1H), 6.53 (d, J = 3.4 Hz, 1H), 3.75 (s, 3H), 2.33 (s, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 161.93, 161.27, 158.43, 158.04, 155.49, 155.25, 152.96, 150.39, 144.11, 142.59, 137.28,
131.15, 130.14, 129.61, 128.87, 124.88, 120.33, 120.10, 116.96, 109.08, 108.86, 104.61, 97.64,31.74, 18.90.
5.8.49. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3-fluorophenyl)-4-methyl-6-oxo-
1,6-dihydropyridazine-3-carboxamide (29c)
+
This compound was obtained as pale yellow solid in 75% yield; M.P.: 248-249.0 ; ESI-MS m/z: 489.14[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.26 (s, 1H), 7.77 (dd, J = 12.7, 2.1 Hz, 1H), 7.61 (d, J = 10.3 Hz, 1H),
7.54 (t, J = 6.0 Hz, 1H), 7.49 (dd, J = 9.1, 6.1 Hz,2H), 7.36 (t, J = 8.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.01 (s, 1H),
6.54 (d, J = 3.4 Hz, 1H), 3.76 (s, 3H), 2.33 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.84, 161.67, 160.82, 160.42,
158.45, 154.79, 152.51, 149.91, 142.91, 141.43, 136.85, 130.25, 130.16, 129.85, 129.66, 124.41, 121.68, 116.55,
115.11, 112.98, 108.54, 104.15, 97.15, 31.25, 18.20.
5.8.50. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(p-tolyl)-1,6-dihy
dropyridazine-3-carboxamide (29d)
+
This compound was obtained as pale yellow solid in 72% yield; M.P.: 242.0-243.0 ;ESI-MS m/z: 485.17[M+H] .
1H NMR (400 MHz, DMSO) δ 10.67 (s, 1H), 8.25 (s, 1H), 7.77 (dd, J = 12.6, 2.1 Hz, 1H), 7.49 (dd, J = 13.3, 8.4 Hz,
4H), 7.35 (t, J = 8.8 Hz, 1H), 7.25 (d, J = 8.2 Hz, 2H), 6.97 (s, 1H), 6.54 (d, J = 3.4 Hz, 1H), 3.75 (s, 3H), 2.32 (s, 3H),
2.29 (s,3H); 13C NMR (101 MHz, DMSO-d6) δ 162.37, 161.30, 159.12, 155.28, 152.96, 152.85, 150.41, 143.11, 141.64,
138.79, 137.36, 130.16, 130.11, 129.47(2C), 125.88(2C), 124.90, 116.94, 109.03, 108.80, 104.62, 97.66, 31.74, 21.16,
18.69.
5.8.51. N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-1-(2-(trifluoromethy
l)phenyl)-1,6-dihydropyridazine-3-carboxamide (29e)
+
This compound was obtained as pale yellow solid in 79% yield; M.P.: 246.0-247.0 ; ESI-MS m/z: 539.14[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 8.81 (s, 1H), 8.27 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.93 – 7.88 (m, 1H),
7.87 – 7.77 (m, 3H), 7.49 (d, J = 3.5 Hz, 1H), 7.42 (d, J = 9.0 Hz, 1H), 7.36 (t, J = 8.6 Hz, 1H), 6.55 (d, J = 3.5 Hz, 1H),
3.76 (s, 3H), 2.02 (s, 3H);13C NMR (101 MHz, DMSO-d6) δ 162.72, 160.81, 159.57, 154.92, 152.50, 149.93, 143.42,
142.68, 140.71, 136.70, 135.53, 134.32, 131.20, 130.64, 129.68, 128.94, 127.68, 124.64, 124.18, 121.46, 116.20,
108.28, 104.14, 97.17, 31.25, 18.68.

ACCEPTED MANUSCRIPT
5.8.52. 1-(2-Chlorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-4-methyl-6-oxo-
1,6-dihydropyridazine-3-carboxamide (29f)
+
This compound was obtained as pale yellow solid in 71% yield; M.P.: 245.0-246.0 ; ESI-MS m/z: 506.11[M+H] .
1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.78 (s, 1H), 8.27 (s, 1H), 7.88 – 7.80 (m, 1H), 7.74 (dd, J = 12.4, 4.7
Hz, 2H), 7.57 (tt, J = 13.1, 6.6 Hz, 2H), 7.49 (d, J = 3.4 Hz, 1H), 7.42 (d, J = 9.0 Hz, 1H), 7.37 (t, J = 8.6 Hz, 1H), 6.55
(d, J = 3.5 Hz, 1H), 3.76 (s, 3H), 2.02 (s, 3H).
5.9 Cytotoxicity assay in vitro
The cytotoxic activities of target compounds (22a-g, 23a-g, 24a-g, 25a-g, 26a-f, 27a-f, 28a-f and 29a-f) were
evaluated with A549, HepG2, MCF-7 and PC-3 cell lines by the standard MTT assay in vitro, with c-MET inhibitors
Foretinib as positive control. 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 5 µg/mL and incubated with cells at 37°C for 4 h. The formazan crystals were dissolved in
100 µL 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.
5.10 Tyrosine kinases assay in vitro
The selected compounds ( 22f, 22g, 26c and 26e ) 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 90 µL 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’

ACCEPTED MANUSCRIPT
stands for low control.
5.11 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 µL of Annexin V-FITC binding solution was added to gently
resuspend the cells. Afterwards, 5 µL of Annexin V-FITC and 10 µL 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.
5.12 Docking studies
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8) was obtained from RCSB
Protein Data Bank[10]. Autodock Vina 1.12, Discover Studio 3.5 visualizer and Open Babel were used for the docking
study. Firstly, using the Discover Studio 3.5 visualizer to prepare ligands (22f, 22g, 26c, and 26e) and acceptor protein
(PDB code: 3LQ8), then saved as pdb format after energy minimization for ligand and clean protein, defined the
activity site for acceptor protein. Secondly, the pdb file of ligand and acceptor protein were converted to the pdbqt
format by the Open Babel tool. Thirdly, docking was carried out by uploading the pdbqt file to the Autodock vina 1.1.2,
and the result was named out.pdbq. Moreover, out.pdbpt file was uploaded to the Discover Studio 3.5 visualizer for
analyzing the result. In the first place, hide the residues and displayed the activity pocket to get the Fig.7a. Next, using
the receptor-ligand interaction part to defined the 3LQ8 as receptor to show the interaction between the 22g and c-Met,
then display the residues that contact with 22g and hide others to produce the Fig.7b after adding the activity pocket.
following by using the "show 2D diagram" to obtain the Fig.7c. Finally, the "Receptor-Ligand Pharmacophore
Generation" moiety was used to get the Fig. 7d. All calculations were performed on Silicon Graphics workstation.
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No.
21662014,81460527), Outstanding Youth Foundation of Jiangxi, Natural Science Foundation of Jiangxi, China
(20171BCB23078), Natural Science Foundation of Jiangxi, China (20171ACB21052 & 20171BAB215073 &
20161BAB215216)，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