Synthesis and bioevaluation and doking study of 1H-pyrrolo[2,3-b]pyridine derivatives bearing aromatic hydrazone moiety as c-Met inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.078

Two series of aromatic hydrazone derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (7a–r, 8a–i, 12a–b, 13a–c, 16a–d and 17a–e) were designed, synthesized and evaluated for the IC₅₀ values against four cancer cell lines (A549, HepG2, MCF-7and PC-3). Two selected compounds (7c and 17e) were further evaluated for the activity against c-Met, Flt-3, VEGFR-2 and EGFR kinases. The data indicated that targets compounds were selective for c-Met kinase. And the most promising compound 7c was further studied in terms of dose-dependent, time-dependent and cell apoptosis. Most of the compounds showed excellent cytotoxicity activity, especially the most promising compound 7c with the IC₅₀ values of 0.82 ± 0.08 μM, 1.00 ± 0.11 μM, 0.93 ± 0.28 μM and 0.92 ± 0.17 μM against A549, HepG2, MCF-7 and PC-3 cell lines and 0.506 μM against c-Met kinase. Structure–activity relationships (SARs) and docking studies indicated that the activities of the phenyl hydrazone derivatives (7a–r and 8a–i) were superior to that of the heterocyclic hydrazone series (12a–b, 13a–c, 16a–d and 17a–e). What's more, the further studies indicated that the target compounds can induce apoptosis of A549 cells and arrest efficiently the cell cycle progression in G2/M phase of A549 cells.

Full text of DOI:10.1016/j.ejmech.2017.12.078

Accepted Manuscript
Synthesis and bioevaluation and doking study of 1H-pyrrolo[2,3-b]pyridine derivatives
bearing aromatic hydrazone moiety as c-et inhibitors
Wenhui Wang, Shan Xu, Yongli Duan, Xiaobo Liu, Xiaojing Li, Caolin Wang, Bingbing
Zhao, Pengwu Zheng, Wufu Zhu
PII:
S0223-5234(17)31109-1
10.1016/j.ejmech.2017.12.078
EJMECH 10056
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2017
Revised Date: 18 December 2017
Accepted Date: 22 December 2017
Please cite this article as: W. Wang, S. Xu, Y. Duan, X. Liu, X. Li, C. Wang, B. Zhao, P. Zheng, W. Zhu,
Synthesis and bioevaluation and doking study of 1H-pyrrolo[2,3-b]pyridine derivatives bearing aromatic
hydrazone moiety as c-et inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.078.
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ACCEPTED MANUSCRIPT
Two series of aromatic hydrazone derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (7a–r, 8a–i, 12a–b, 13a–c, 16a–d
and 17a–e) were designed. What’s more, enzyme-based selectivity, cell cycle, cell apoptosis and molecules docking study
were also carried out in this paper.

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Synthesis and bioevalution and doking study of 1H-pyrrolo[2,3-b]pyridine derivatives bearing aromatic
hydrazone moiety as c-Met inhibitors
Wenhui Wang1,†, Shan Xu1,†, Yongli Duan1,†, Xiaobo Liu1, Xiaojing Li2, Caolin Wang1, Bingbing Zhao1, Pengwu
Zheng1, , Wufu Zhu1,
1 Jiangxi Provincial Key Laboratory of Drug Design and Evaluation,School of Pharmacy, Jiangxi Science &
Technology Normal University, Nanchang 330013, P. R. China.
2 College of Service, Naval Logistics College of PLA ,Tianjin 300450,PR 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).
Two series of aromatic hydrazone derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (7a–r, 8a–i, 12a–b,
13a–c, 16a–d and 17a–e) were designed, synthesized and evaluated for the IC50 values against four cancer cell
lines (A549, HepG2, MCF-7and PC-3). Two selected compounds (7c and 17e) were further evaluated for the
activity against c-Met, Flt-3, VEGFR-2 and EGFR kinases. The data indicated that targets compounds were
selective for c-Met kinase. And the most promising compound 7c was further studied in terms of dose-dependent,
time-dependent and cell apoptosis. Most of the compounds showed excellent cytotoxicity activity, especially the
most promising compound 7c with the IC50 values of 0.82±0.08 µM, 1.00±0.11 µM, 0.93±0.28 µM and 0.92±
0.17 µM against A549, HepG2, MCF-7 and PC-3 cell lines and 0.506 µM against c-Met kinase. Structure–activity
relationships (SARs) and docking studies indicated that the activities of the phenyl hydrazone derivatives (7a–r
and 8a–i) were superior to that of the heterocyclic hydrazone series (12a–b, 13a–c, 16a–d and 17a–e). What’s
more, the further studies indicated that the target compounds can induce apoptosis of A549 cells and arrest
efficiently the cell cycle progression in G2/ M phase of A549 cells.
Key words: Phenyl hydrazone; Heterocyclic hydrazone; 1H-Pyrrolo[2,3-b]pyridine; Synthesis; c-Met inhibitors;
Antitumor activity
† 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(P. Zheng).

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1. Introduction
Cancer is a disease that seriously endangers human life and health[1-2]. According to the World Health
Organization (WHO) statistics, 8.8 million patients died of cancer in 2015[3]. Therefore, it is urgent to find
effective and less toxic side effects of cancer treatment[4]. Because c-Met kinase is located at the intersection of
many tumor signaling pathways and is a key node protein, which can interact with other kinases and receptors,
therefore, the small molecule inhibitors targeting c-Met become the hotspot of research[5].
Foretinib[6], the first small molecule oral inhibitor to enter clinical trials, is in clinical stage II currently,
whose inhibitory effect against c-Met kinase is 0.4 nM. Cabozantinib[7] also belongs to this class of inhibitors and
was approved by FDA for the treatment of patients with progressive metastatic medullary thyroid cancer on
November 29, 2012. And many other kinds of c-Met inhibitor were also reported[8-9]. In our previous study, we
also reported a series of quinoline derivatives and a series of 1H-pyrrolo[2,3-b]pyridine derivativesas potential
c-Met inhibitors[10-12] (the structures are shown in Figure 1). In addition, it was found that this class inhibitor
exhibited the best antitumor activity when the 5 atoms were connected between A and B, which was characterized
by the illustrated “5 atoms regulation”[10]. The design of the compounds in this article is based on this conclusion.
Figure 1 structures of c-Met inhibitors
In this study, further modifications were carried out on 1H-pyrrolo[2,3-b]pyridine derivatives that reported in
our previous study. It has been found that N-acylhydrazones and its analogs containing multiple hydrogen bond
donors and receptors were with different biological activity and strong coordination ability[13]. Therefore, they
were used as a component of anticancer agents, such as PAC-1[14] and compounds I[15] and II[16] (the structures are
shown in Figure 2). Thus, the N-acylhydrazone was introduced into the compounds according to the principle of
flattening and replaced the phenylpicolinamide scaffold and the phenylpyrimidine moiety to obtain the target

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compounds 7a–r and 8a–i. In addition, it was found that a reasonable modification of the phenyl group could
modulate the π-stacking effect with the corresponding receptor and enhance the inhibitory potency of the
compounds. Therefore, we replaced the phenyl group with pyrrole (five-membered heterocycle ) and introduced
the small molecule amine on the N atom to increase the water solubility to design the target compounds 12a–b,
13a–c, 16a–d and 17a–e bearing the heterocyclic hydrazone structure. The design strategy are shown in Figure 3.
Figure 2 Structures of N-acylhydrazone and its mimics.
Figure 3 Design strategy of the target compounds.
Herein, the synthesis and antitumor activity against A549, HepG2, MCF-7 and PC-3cancer cell lines, and
c-Met, Flt-3, VEGFR-2 and EGFR kinases of the target compounds were disclosed. Moreover, docking studies
were presented in this paper as well.
2. Chemistry
The preparation of target compounds 7a–r and 8a–i is described in Scheme 1.
Compounds 3a, 3b, 4a and 4b were synthesized according to the reported procedures[11,12]
.
Compounds 5a and 5b was synthesized by amide 4a or 4b and phenyl chloroformate via substitution in
1,4-dioxane. Then the key intermediates 6a and 6b were obtained via hydrazinolysis of 5a and 5b with 80%
hydrazine hydrate in 1,4-dioxane[17]. The mixture of 6a or 6b, substituted benzaldehyde and catalytic amounts of
acetic acid were refluxed in isopropanol for 3 h to yield the target compounds 7a-r and 8a-i.

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Scheme 1 Synthetic route of target compounds 7a–r and 8a–i.Reagents and conditions: (a) diphenyl ether, 190 ,
.
.
1 h; (b) N2H4 H2O, FeCl3, activated carbon, EtOH, 10 min;(c) K2CO3, 1,4-dioxane, ice bath, rt; (d) N2H4 H2O,
1,4-dioxane; (e) acetic acid, isopropanol.
The preparation of target compounds 12a–b, 13a–c, 16a–d and 17a–e is described in Scheme 2.
The intermediate 1H-pyrrole-2-carbaldehyde 9 was achieved from pyrrole, DMF and POCl3 via
Vilsmeier-Haack reaction. Then the intermediates 9 was reacted with 1-chloro-2-bromoethane or
1-chloro-3-bromopropane in DMSO-D6 under the catalysis of NaH via substitution reaction to yielding the
intermediate 10 or 14, which were then reacted with small molecule amine such as piperidine via substitution
reaction to obtain the compounds 11a–c and 15a–f. 12a–b and 13a–c were obtained by hydrazinolysis of 11a–c
and 6a or 6b. Similarly, 16a–d and 17a–e were achieved from 15a–f and 6a or 6b via hydrazinolysis reaction.
Furthermore, According to our previous work[18], all the target compounds were prepared to the E isomer by the
same reaction condition.
Scheme 2 Synthetic route of target compounds 12a–b, 13a–c, 16a–d and 17a–e. Reagents and conditions: (f)
DMF, POCl3, CH3CHCl2, ice bath, rt, CH3COONa, H2O; (g) DMSO-D6, NaH, rt, 110 ; (h) small molecule amine,

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DMF, 125 ; (i) DMF, microwave, 65 , 100 W.
3. Results and discussion
3.1 Biological evaluation
3.1.1 Antiproliferative assay in vitro
Taking c-Met inhibitor Foretinib as reference compound, the target compounds (12a–b, 13a–c, 16a–d
and17a–e) were evaluated for the cytotoxicity against four cancer cell lines A549 (human lung cancer), PC-3
(human prostatic cancer), MCF-7(human breast cancer) and HepG2 (Human liver cancer) by 3-(4,
5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. In addition, two selected
(7c and 17e) were evaluated for the IC50 values against c-Met kinase in vitro by the Mobility shift assay with ATP
concentration at Km with reference compound Foretinib. The results expressed as IC50 values were summarized in
Tables 1–4 and the values are the average of at least two independent experiments. Furthermore, In order to
examine the effects of time and concentration, the most promising compound 7c was investigated in a
time-dependent and dose-dependent manner. And the results are shown in Figure 4 and Figure 5. In order to
investigate whether the target compounds could induce apoptosis, A549 cell acted with the selected compound 7c
was observed cell morphology by Acridine Orange (AO) staining , which was shown in Figure 6 .
Table 1 Structures and cytotoxicity of compounds 7a–r and 8a–i.
Compound
IC50(µM)a
X
R
A549
HepG2
MCF-7
2.38±0.54
0.049±0.09
0.93±0.28
0.80±0.13
NAc
PC-3
s
F
F
F
F
F
F
F
F
F
H
2.50±0.62
2.22±0.08
0.82±0.08
0.68±0.04
1.36±0.21
3.64±1.01
0.02±0.01
NA
0.64±0.15
0.66±0.14
1.00±0.11
3.88±0.78
0.85±0.19
5.18±0.44
3.03±0.38
NA
7.94±1.47
4.02±1.10
0.92±0.17
2.33±0.21
9.03±1.04
5.14±0.81
0.15±0.04
NA
7a
7b
7c
7d
7e
7f
3-Br
4-Br
4-F
4-CN
3-NO2
4-NO2
2,3-di Cl
3,4-di Cl
NA
4.00±0.85
NA
7g
7h
7i
2.65±0.65
3.70±0.81
0.998±0.22
14.06±1.88

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F
F
F
F
F
F
2-Cl-4-F
4-OH
0.01±0.01
3.76±0.62
3.98±1.07
5.84±1.01
1.45±0.10
34.14±1.03
1.11±0.65
1.95±0.87
1.77±0.20
4.55±1.04
2.99±0.53
NA
7.61±0.41
NA
0.003±0.001
2.78±1.20
6.73±1.09
13.00±0.96
8.97±1.26
NA
7j
7k
7l
4-OCH3
5.75±0.90
47.99±1.23
0.134±0.07
21.87±0.92
2,4-di OH
7m
7n
7o
2,3,4-tri OCH3
3,4,5-tri OCH3
4-OH-3,5-C(CH3)
F
13.64±1.48
7.22±0.50
0.84±0.11
10.39±0.78
7p
3
F
3-Br-4-OH
9.25±1.37
16.24±4.31
17.19±1.03
0.64±0.14
1.43±0.12
2.10±0.52
NA
1.36±0.81
6.52±0.99
0.75±0.12
1.00±0.19
3.43±0.64
4.62±0.82
NA
8.23±1.08
NA
28.61±1.14
NA
7q
F
3,5-di Br-4-OH
H
7r
H
H
H
H
H
H
H
H
H
-
1.51±0.28
NA
10.16±1.18
NA
8a
4-Br
8b
4-F
3.72±1.01
15.59±1.01
NA
7.49±1.07
28.07±0.59
NA
8c
4-NO2
8d
2,3-diCl
4-OH
8e
1.91±0.20
4.30±0.26
3.34±0.59
5.08±1.21
0.64 ± 0.16
32.22±0.42
9.74±0.50
2.96±0.46
NDd
5.80±1.22
NA
4.92±1.14
NA
8f
4-OCH3
2,3,4-tri OCH3
2-Br-4-OH
-
8g
8h
NA
27.60±1.40
NA
NA
8i
Foretinibb
-
9.47 ± 0.22
0.39 ± 0.09
a
c
d
The values are an average of two separate determinations;b Used as a positive controls; No Activity; Not
determined.
Table 2 Structures and cytotoxicity of compounds 12a–b and13a–c.
IC50(µM)a
Compound
X
R
s
A549
NAc
HepG2
MCF-7
NDd
PC-3
F
8.98±1.55
1.60±1.19
12a

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F
H
H
3.41±0.19
11.9±2.35
ND
ND
ND
ND
2.45±1.59
3.93±1.40
1.37±1.41
1.65±0.94
12b
13a
13b
NA
NA
3.57±0.95
5.88±1.12
H
-
3.88±0.94
9.94±0.74
-
13c
Foretinibb
-
0.64 ± 0.16
9.47 ± 0.22 0.39 ± 0.09
a
c
d
The values are an average of two separate determinations;b Used as a positive controls; No Activity; Not
determined.
Table 3 Structures and cytotoxicity of compounds 16a–d and17a–e.
IC50(µM)a
Compound
X
R
s
A549
HepG2
MCF-7
PC-3
F
F
1.21±0.74
2.14±1.33
3.39±1.03
2.95±1.09
16a
2.85±0.85
2.20±0.97
2.01±0.96
2.66±1.10
2.24±0.56
1.56±0.82
1.64±0.96
2.84±1.26
2.74±1.40
3.50±1.10
4.19±1.10
1.69±1.00
3.69±1.30
2.76±1.45
4.84±1.01
3.74±1.08
4.58±1.16
9.14±0.61
NAc
2.84±1.20
2.19±1.32
3.58±1.06
5.15±1.15
14.72±1.10
3.58±1.16
3.74±1.09
4.29±1.15
16b
16c
16d
17a
17b
17c
17d
F
F
H
H
H
H
4.69±1.06
4.28±1.24
4.53±1.16
H
-
1.30±0.85
1.42±1.35
-
17e
Foretinibb
-
0.64 ± 0.16
9.47 ± 0.22 0.39 ± 0.09
a The values are an average of two separate determinations;b Used as a positive controls; c No Activity.
Table 4 Activity against c-Met kinase inhibitory of selected compounds 7c and 17e.

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IC50(µM) a
Compounds
c-Met
0.506
0.907
0.014
7c
17e
Foretinibb
a The values are an average of two separate determinations;b Used as a positive controls.
Table 5 Activity against c-Met, Flt-3, VEGFR-2 and EGFR kinases inhibitory of selected compounds 7c and
17e.
IC50(µM) a
VEGFR-2
Compounds
c-Met
Flt-3
EGFR
0.5
0.9
2.6
3.8
8.9
>100
45.0
7c
14.4
17e
a The values are an average of two separate determinations.
As shown in table 1–3, most of the compounds exhibited moderate to excellent cytotoxic activity against four
cancer cell lines. Among the compounds of the phenyl hydrazone series in Table 1, the inhibitory activity of
compounds 7c, 7g and 7j against the four cells was superior to that of the positive control Foretinib. Compound 7c
showed superior inhibitory activity against four tested cells, with IC50 values of 0.82±0.08 µM, 1.00±0.11 µM,
0.93±0.28 µM and 0.92±0.17 µM against A549, HepG2, MCF-7 and PC-3 cell lines. In addition, the activity
against the four cell lines was different which was caused by the introduction of fluorine atoms on the
aminophenoxy group of the target compounds. Compared to 7a–r and 8a–i, the introduction of fluorine atoms
enhanced the inhibitory activity of the target compounds against MCF-7 and PC-3 cell lines, while played no
significant impact on the activity against A549 and HepG2 cell lines. Furthermore, different substitutions of aryl
group affected the cytotoxicity of target compounds. It’s seemed to be that the presence of electron withdrawing
group at C-4 position increased the activity of the target compounds, while the introduction of electron-donating
group decreased the activity. Moreover, the more the number of electron-donating groups, the lower the activity,
such as compounds 7k and 7m–p. However, the effect of electronegativity on activity was not obvious. What’s
more, compounds 7h, 8e and 8i suggested that the presence of electron withdrawing groups on C-2 position led to
the activity of the compounds was eliminated. The activity of the compounds 7b–c and 7f–g showed that the
activity of the compounds with substituents at the C-4 position was slightly better than that of substituents at the
C-3 position.
The results of the activity of the compounds containing heterocyclic hydrazone structures are shown in table
2-3. It can be seen that most of the compounds exhibited moderate cytotoxic activity against four cancer cell lines
and the selectivity of this series compounds on A549 and HepG2 was stronger than that on MCF-7 and PC-3.

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Compared 12a–b and 13a–c, 16a–d and 17a–e, it was found that the introduction of fluoro atoms to the
aminophenoxy part played no significant impact on the activity. Furthermore, the length of the carbon chain
between the N atom on the pyrrole ring and the small molecule amine affected the activity of the target
compounds. In general, the activity against A549 and HepG2 of the compounds with three C atoms between N
atom and the small molecule amine were superior to that of the compounds with two C atoms, however, the
activity against PC-3 was just opposite. In addition, the type of small molecule amine played no significant impact
on the cytostatic activity of the target compounds.
Activity against c-Met kinase of compounds 7c and 17e was further carried out in this paper to investigate
the target and selectivity of these compounds. According to the results of Table 3, we can easily find that the two
selected compounds showed moderate inhibitory on c-Met kinase, and the activity of compound 7c (IC50 = 0.506
µM) was slightly better than that of compound 17e (IC50 = 0.907 µM), which was similar to the result of cytotoxic
activity.
As shown in Table 5, the selected compounds 7c and 17e were taken into further study by the enzyme-based
experimental. The activity data claimed that the compounds 7c and 17e can inhibit the c-Met selectively compared
with other three kinases (Flt-3, VEGFR-2 and EGFR kinases), with IC50 values of 0.5 µM and 0.9 µM,
respectively.
3.1.2 Concentration-dependentassay
In order to investigate the relationship between activity and concentration, seven concentrations of compound
7c were set and the inhibitory rate of the compounds against four cancer cell lines for 72 h was measured by MTT
assay. The results were shown in Figure 4.
Figure 4 Relationship between activity and concentration of selected compound 7c against four cancer cell lines.

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It can be seen that the inhibition rate of compound 7c against four cancer cell lines increased with increasing
concentration. It can be concluded that the target compounds inhibited the growth of the four tumor cell lines in a
concentration-dependent manner.
3.1.3 Time-dependent assay
In order to examine whether the time can affect the inhibitory effect of the compound against tumor cells,
five concentrations of compound 7c and three time gradients were set and the inhibition rate of A549 cells was
measured by MTT assay. The results were shown in Figure 5.
Figure 5 The inhibitory effect of the selected compound 7c on A549 cell lines at different time points.
At each concentration, the inhibition rate of compound 7c against A549 cell increased with time. In addition,
at the same time, the inhibition rate of compound 7c against A549 cells increased with increasing concentration,
which was same to the results obtained in Table 1. Therefore, for the representative compound 7c, the phenomena
of dose-dependent and time-dependent were observable in Figure 4 and Figure 5 respectively. In other words,
compound 7c can inhibit the proliferation of A549 cells in a time and concentration dependent manner.
3.1.4 Apoptosis-inducing assay
In order to investigate whether the selected compound 7c could induce apoptosis of A549 cells,
morphological changes of A549 cells before and after treated by compound 7c were observed by AO staining. The
cell morphology after staining was shown in Figure 6.

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Figure 6 The cell morphology of A549 cells of the control and test groups. (Fig.6a: A549 cells treated without any
compounds; Fig.6b: A549 cells were treated with 0.80 µM 7c for 12 h; Changes of nuclear morphology were
determined by AO staining.)
3.1.5 Cell cycle analysis
The cell growth inhibitory potency of the tested compounds prompted us to evaluate their effects on the cell
mitosis. The A549 cells treated with the selected compound 7c was tested for cell cycle. The data for each phase
of the cell cycle and the area parameter histogram were shown in Table 6 and Figure 8, respectively. The
percentage of cells was increased from 23.62 to 79.86 in G2/M phase in 5 times IC50 concentration (4 µM), and
the index of Coefficient of Variation (CV) G1 and G2 are below the 7% which indicated that the data was reliable.
Therefore, this result displayed that the compound 7c arrested efficiently the cell cycle progression in G2/ M
phase.
Table 6 Effects of compound 7c on the cell cycle against A549 cell lines.
Freq
Sub-G1
0.06
Compound
RMS Freq G1
Freq S
Freq G2
CV G1 CV G2
s
4.73
27.08
10.67
48.44
7.08
23.62
79.86
3.61%
6.46%
3.36%
6.45%
Control
7c
16.14
0.01

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8a
8b
Figure 7 Cell cycle analysis in A549 cells. (Fig.8a: control; Fig.8b: Cell cycle analysis in compound 7c-treated
A549 cells.)
3.2 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 SURFLEX-DOCK module of SYBYL package version and analysis
by Discover Studio 3.5 viewer. Based on the in vitro inhibition results, we selected compound 7c, 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 [19]).
Figure 8 Binding poses of compound 7c with c-Met. The 3D and 2D graph was depicted in Figure 8, and the
hydrogen was colored with green, the pi-pi stacked bond was colored with blue and the halogen was colored with
pink.
The binding modes of compound 7c and lead compound Foretinib were shown in Figure8. As depicted in

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Figure 8, compound 7c and lead compound can mostly overlap in the binding model and pyrrolo[2,3-b]pyridine
nucleus formed one hydrogen bonds with residues PHE 1223, and the F-atom of oxyphenyl connect with MET
1160 and PRO 1158 via one hydrogen bond and halogen bond. What’s more, the pyrrolo[2,3-b]pyridine moiety
can form two pi-pi bond contact to the PHE 1223, and the phenyl of 7c also can form pi-pi bond intermolecules.
Analysis of compound 7c’s binding mode in the active binding site demonstrated that the docking mode of the 7c
is similar to the lead compound with the same residues MET 1160 and PHE 1223. According these results, it
claimed that the effect of hydrogen bond , pi-pi stacked bond and the halogen bond maybe a rational explanation
that the compound 7c contained excellent antitumour activity. Furthermore, the docking results also give us a new
direction to design new c-Met inhibitors. The above-mentioned results of SARs analysis and molecular docking
study may allow the rational design of more potent c-Met inhibitors.
4. Conclusions
In summary, we designed and synthesized two series of aromatic hydrazone derivatives
bearing1H-pyrrolo[2,3-b]pyridine nucleus and evaluated for the IC50 values against four cancer cell lines and
c-Met, Flt-3, VEGFR-2 and EGFR kinases. Most of them exhibited moderate to excellent cytotoxic activity
against four cancer cell lines. Especially, the most promising compound 7c showed superior activity to Foretinib,
with the IC50 values of 0.82±0.08 µM, 1.00±0.11 µM, 0.93±0.28 µM and 0.92±0.17 µM against A549, HepG2,
MCF-7 and PC-3 cell lines. In addition, the results of the in-depth study of the compound 7c indicated that the
target compounds can induce apoptosis of A549 cells. And the activity on four kinases showed the target
compounds had selectivity for c-Met kinase. Furthermore, the compound 7c arrested efficiently the cell cycle
progression in G2/M phase of A549 cells. Structure–activity relationships (SARs) and docking studies indicated
that the introduction of fluorine atoms on the aminophenoxy groups had an effect on the activity of the
compounds of the phenylhydrazone series while played no significant impact on the activity of the heterocyclic
hydrazone series. Among the compounds of phenylhydrazone series, the position of the substituent on the aryl
group affected the activity of the compounds. For heterocyclic hydrazone series, the distance between the N atom
on the pyrrole and the small molecule amine can affect the activity of the compounds, and the type of small
molecule played no significant impact on the activity. In general, the activity of the compounds of the
phenylhydrazone series was superior to that of the heterocyclic hydrazone series. Further study will be carried out
to identify the exact action mechanism in near future.
5. Experimental
5.1. Chemistry

ACCEPTED MANUSCRIPT
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labor technik, 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). 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 specied. Yields were not optimized.
5.2 General procedure for the preparation of compounds 3a, 3b, 4a and 4b.
Compounds 3a, 3b, 4a and 4b were synthesized according to the reported procedures by our research group.
5.3 Preparation of compounds 5a and 5b.
Phenyl chloroformate (5.48 g, 34.8 mmol) was added drop-wise to a solution of an appropriate aniline 4a or
4b (4.23 g, 17.4 mmol) and potassium carbonate (4.81 g, 34.8 mmol) in 1,4-dioxane (30 mL) in an ice bath. Upon
completion of the addition, the reaction mixture was removed from the ice bath and placed in room temperature
and monitored by thin-layer chromatography (TLC). The insoluble was filtered and washed with water. The
collected solid 5a or 5b was use for the next step without further purification.
5.4 General procedure for the preparation of the key intermediates6a and 6b.
Compounds 5a or 5b (4.18 g, 13.9 mmol) was refluxed with 80% hydrazine hydrate (6.96 g, 13.9 mmol) in
1,4-dioxane (40 mL) for 2 h and monitored by TCL. The solution was evaporated when white solid appeared.
Then the solid was filtered off, washed with water and dried to obtain the key intermediates 6a or 6b.
5.5 General procedure for the preparation of the target compounds 7a-r and 8a-i.
The mixture of 6a or 6b and 1.1 equiv benzaldehyde bearing different substituent was catalyzed with acetic
acid (1 drop) and refluxed in isopropanol for 3 h until TCL showed the completion of the reaction. After cooling
to room temperature, the solid was filtered off and dried to yield the target compounds 7a-r and 8a-i.
5.5.1 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-benzylidenehydrazinecarboxamide (7a)
Yield: 66.6%; ESI-MS [M + H]+ m/z:390.13; m.p. 245.4–246.4 ;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H),
10.89 (s, 1H), 9.18 (s, 1H), 8.06 (d, J = 4.9 Hz, 1H), 7.97 (s, 1H), 7.86 (d, J = 8.8 Hz, 3H), 7.59 (d, J = 8.9 Hz,
1H), 7.41 (t, J = 8.3 Hz, 3H), 7.34 (d, J = 11.0 Hz, 2H), 6.36 (d, J = 5.1 Hz, 1H), 6.24 (d, J = 1.5 Hz, 1H).13C
NMR (101 MHz, DMSO-d6) δ 157.85, 155.22, 153.51, 151.55, 144.70, 141.81, 138.44, 136.01, 134.74, 130.01,
129.06(2C), 127.60(2C), 125.23, 123.91, 116.74, 109.77, 108.64, 101.05, 97.27.
5.5.2 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-nitrobenzylidene)hydrazinecarbox-
amide (7b)

ACCEPTED MANUSCRIPT
Yield: 58.9%; ESI-MS [M + H]+ m/z:435.12;1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 11.23 (s, 1H), 9.34
(s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 4.4 Hz, 2H), 7.84 (d, J = 13.4 Hz, 1H), 7.58
(d, J = 8.8 Hz, 1H), 7.35 (t, J = 6.4 Hz, 2H), 6.37 (d, J = 5.4 Hz, 1H), 6.23 (s, 1H).
5.5.3 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-hydroxybenzylidene)hydrazecarbox-
amide (7c)
Yield: 41.8%; ESI-MS [M + H]+ m/z:406.13; m.p. 239.5–240.9 ;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H),
10.67 (s, 1H), 9.82 (s, 1H), 8.06 (d, J = 5.3 Hz, 1H), 7.85 (d, J = 10.6 Hz, 2H), 7.67 (d, J = 7.9 Hz, 2H), 7.57 (s,
1H), 7.37 – 7.29 (m, 2H), 6.80 (d, J = 7.8 Hz, 2H), 6.36 (d, J = 5.2 Hz, 1H), 6.23 (s, 1H).13C NMR (101 MHz,
DMSO-d6) δ 157.82, 153.44, 151.55, 144.70, 140.57, 138.36, 136.06, 134.07, 132.02(2C), 129.51(2C), 125.24,
123.91, 123.19, 116.86, 109.76, 108.95, 108.72, 101.06, 97.26.
5.5.4 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-bromobenzylidene)hydrazinecarbox-
amide (7d)
Yield: 56.7%; ESI-MS [M + H]+ m/z:468.04;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.96 (s, 1H), 9.22
(s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.94 (s, 1H), 7.86 – 7.81 (m, 3H), 7.63 (s, 1H), 7.61 (s, 1H), 7.57 (d, J = 8.8 Hz,
1H), 7.36 (s, 1H), 7.32 (d, J = 9.2 Hz, 1H), 6.36 (d, J = 5.4 Hz, 1H), 6.23 (s, 1H).
5.5.5 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(2,3-dichlorobenzylidene)hydrazine-
carboxamide (7e)
Yield: 57.4%; ESI-MS [M + H]+ m/z:459.05; m.p. 229.0–230.6 ;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H),
11.21 (s, 1H), 9.29 (s, 1H), 8.39 (s, 1H), 8.36 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.83 (d, J = 13.2 Hz, 1H), 7.67 (d,
J = 7.8 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.37 – 7.31 (m, 2H), 6.36 (d, J = 5.4 Hz, 1H),
6.23 (s, 1H).
5.5.6 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3,4-dichlorobenzylidene)hydrazinecar-
boxamide (7f)
Yield: 61.8%; ESI-MS [M + H]+ m/z:459.05; m.p. 253.0–254.9 ; 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H),
11.07 (s, 1H), 9.29 (s, 1H), 8.24 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.93 (s, 1H), 7.82 (t, J = 11.9 Hz, 2H), 7.67 (d, J
= 8.4 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.38 – 7.32 (m, 2H), 6.36 (d, J = 5.4 Hz, 1H), 6.23 (d, J = 1.7 Hz, 1H).
5.5.7 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(2-chloro-4-fluorobenzylidene)hydrazine
carboxamide (7g)
Yield: 62.1%; ESI-MS [M + H]+ m/z:442.08; m.p. 257.6–258.9 ;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H),
11.10 (s, 1H), 9.27 (s, 1H), 8.48 – 8.43 (m, 1H), 8.32 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.83 (d, J = 13.3 Hz, 1H),
7.56 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.36 (s, 2H), 7.31 (s, 1H), 6.36 (d, J = 5.4 Hz, 1H), 6.23 (s, 1H).

ACCEPTED MANUSCRIPT
5.5.8 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3-bromobenzylidene)hydrazinecarbox-
amide (7h)
Yield: 59.0%; ESI-MS [M + H]+ m/z:468.04;1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 11.00 (s, 1H), 9.28
(s, 1H), 8.18 (s, 1H), 8.07 (d, J = 5.4 Hz, 1H), 7.93 (s, 1H), 7.85 (d, J = 13.3 Hz, 1H), 7.79 (s, 1H), 7.57 (d, J = 4.5
Hz, 2H), 7.40 – 7.36 (m, 2H), 7.33 (d, J = 9.0 Hz, 1H), 6.37 (d, J = 5.3 Hz, 1H), 6.23 (s, 1H).
5.5.9 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-fluorobenzylidene)hydrazine-
carboxamide (7i)
Yield: 55.7%; ESI-MS [M + H]+ m/z:408.12; m.p. 252.4–254.1 ;1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H),
10.89 (s, 1H), 9.19 (s, 1H), 8.07 (d, J = 5.3 Hz, 1H), 7.96 (s, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.84 (d, J = 13.1 Hz,
1H), 7.58 (d, J = 8.8 Hz, 1H), 7.36 (s, 1H), 7.32 (d, J = 9.1 Hz, 1H), 7.26 (t, J = 8.5 Hz, 2H), 6.36 (d, J = 5.4 Hz,
1H), 6.23 (s, 1H).
5.5.10 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-cyanobenzylidene)hydrazine-
carboxamide (7j)
Yield: 56.9%; ESI-MS [M + H]+ m/z:415.13;1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 11.16 (s, 1H), 9.29
(s, 1H), 8.07 (d, J = 7.1 Hz, 3H), 8.00 (s, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 13.3 Hz, 1H), 7.57 (d, J = 8.9
Hz, 1H), 7.36 (s, 1H), 7.33 (d, J = 9.2 Hz, 1H), 6.37 (d, J = 5.2 Hz, 1H), 6.23 (d, J = 1.3 Hz, 1H).
5.5.11 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3-nitrobenzylidene)hydrazinecarbox-
amide (7k)
Yield: 60.8%; ESI-MS [M + H]+ m/z:425.12; 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 11.12 (s, 1H), 9.37
(s, 1H), 8.66 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.10 – 8.06 (m, 2H), 7.84 (d, J = 13.4 Hz,
1H), 7.72 (t, J = 8.0 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 2.7 Hz, 1H), 7.34 (d, J = 9.3 Hz, 1H), 6.37 (d, J
= 5.4 Hz, 1H), 6.24 (s, 1H).
5.5.12(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(2,3,4-trimethoxybenzylidene)hydrazinec
arboxamide (7l)
Yield: 62.0%; ESI-MS [M + H]+ m/z:480.16; 1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.75 (s, 1H), 9.12
(s, 1H), 8.16 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.94 (s, 1H), 7.84 (d, J = 11.9 Hz, 1H), 7.57 (d, J = 9.3 Hz, 1H),
7.36 (s, 1H), 7.31 (d, J = 9.1 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H), 6.36 (d, J = 5.3 Hz, 1H), 6.23 (s, 1H), 3.84 (s, 3H),
3.81 (s, 3H), 3.76 (s, 3H).
5.5.13(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(4-methoxybenzylidene)hydrazinecarbox
amide (7m)

ACCEPTED MANUSCRIPT
Yield: 61.2%; ESI-MS [M + H]+ m/z:420.14;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.74 (s, 1H), 9.13
(s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.91 (s, 1H), 7.85 (d, J = 13.1 Hz, 1H), 7.81 (s, 1H), 7.78 – 7.76 (m, 1H), 7.58 (d,
J = 8.7 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J = 9.1 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H), 6.36 (s, 1H), 6.23 (s, 1H), 3.79 (s,
3H).
5.5.14(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3,4,5-trimethoxybenzylidene)hydrazinec
arboxamide (7n)
Yield: 60.8%; ESI-MS [M + H]+ m/z:480.16; m.p. 237.6–238.1 ;1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H),
10.91 (s, 1H), 9.22 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.86 (d, J = 13.9 Hz, 1H), 7.56 (d, J = 9.2 Hz,
1H), 7.36 (s, 1H), 7.32 (d, J = 9.2 Hz, 1H), 7.15 (s, 2H), 6.35 (d, J = 5.4 Hz, 1H), 6.24 (s, 1H), 3.85 (s, 6H), 3.68
(s, 3H).
5.5.15(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3,5-dibromo-4-hydroxybenzylidene)
hydrazinecarboxamide (7o)
1
Yield: 62.3%; ESI-MS [M + H]+ m/z:564.94; H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H), 10.89 (s, 1H),
10.27 (s, 1H), 9.27 (s, 1H), 8.07 (d, J = 7.0 Hz, 3H), 7.86 (s, 1H), 7.82 (s, 1H), 7.57 (d, J = 9.2 Hz, 1H), 7.36 (s,
1H), 7.32 (d, J = 9.1 Hz, 1H), 6.36 (d, J = 5.3 Hz, 1H), 6.23 (s, 1H).
5.5.16(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3,5-di-tert-butyl-4-hydroxybenzylidene)
hydrazinecarboxamide (7p)
Yield: 60.4%; ESI-MS [M + H]+ m/z:518.25; 1H NMR (400 MHz,DMSO-d6) δ 11.74 (s, 1H), 10.63 (s, 1H), 9.15
(s, 1H), 8.05 (d, J = 5.5 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J = 13.4 Hz, 1H), 7.50 (d, J = 11.7 Hz, 3H), 7.35 (s, 1H),
7.31 (d, J = 8.0 Hz, 2H), 6.35 (d, J = 5.2 Hz, 1H), 6.25 (s, 1H), 1.41 (s, 18H).
5.5.17(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(2,4-dihydroxybenzylidene)hydrazine
carboxamide (7q)
1
Yield: 59.8%; ESI-MS [M + H]+ m/z:422.12; H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 10.53 (s, 1H),
10.00 – 9.95 (m, 1H), 9.70 (s, 1H), 9.08 (s, 1H), 8.15 (s, 1H), 8.06 (d, J = 5.5 Hz, 1H), 7.82 (d, J = 12.8 Hz, 1H),
7.74 (d, J = 7.1 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.35 (s, 2H), 7.31 (d, J = 9.1 Hz, 1H), 6.35 (d, J = 5.4 Hz, 1H),
6.30 (s, 1H), 6.23 (s, 1H).
5.5.18 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-(3-bromo-4-hydroxybenzylidene)hydra
zinecarboxamide (7r)
1
Yield: 58.9%; ESI-MS [M + H]+ m/z:484.04; H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.76 (s, 1H),
10.65 (s, 1H), 9.18 (s, 1H), 8.10 (s, 1H), 8.06 (d, J = 5.6 Hz, 1H), 7.86 (s, 1H), 7.83 (s, 1H), 7.58 (t, J = 7.6 Hz,
2H), 7.35 (s, 1H), 7.31 (d, J = 9.7 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.36 (d, J = 5.2 Hz, 1H), 6.23 (s, 1H).

ACCEPTED MANUSCRIPT
5.5.19 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-benzylidenehydrazinecarboxamide (8a)
Yield: 59.6%; ESI-MS [M + H]+ m/z:372.14; m.p. 239.7–240.8 ;1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H),
10.78 (s, 1H), 9.02 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.95 (s, 1H), 7.85 (d, J = 7.0 Hz, 2H), 7.73 (d, J = 8.2 Hz,
2H), 7.41 (d, J = 7.8 Hz, 3H), 7.33 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.39 (d, J = 5.3 Hz, 1H), 6.19 (s, 1H).
5.5.20 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(4-nitrobenzylidene)hydrazinecarboxamide (8b)
Yield: 60.4%; ESI-MS [M + H]+ m/z:417.13; m.p. 248.7–250.3 ;1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H),
11.13 (s, 1H), 9.20 (s, 1H), 8.35 (d, J = 8.2 Hz, 1H), 8.26 (d, J = 5.2 Hz, 1H), 8.25 (s, 1H), 8.16 (s, 1H), 8.14 (s,
1H), 8.06 (s, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.34 (s, 1H), 7.17 (d, J = 8.5 Hz, 2H), 6.41 (d, J = 5.2 Hz, 1H), 6.19 (s,
1H).13C NMR (101 MHz, DMSO-d6) δ 158.09, 153.59, 151.58, 150.03, 144.67, 140.04, 136.54, 134.21,
132.00(2C), 129.43(2C), 124.97, 123.03, 122.18(2C), 121.09(2C), 110.44, 102.27, 97.63.
5.5.21(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(4-bromobenzylidene)hydrazinecarboxamide (8c)
Yield: 61.5%; ESI-MS [M + H]+ m/z:451.05; m.p. 223.6–225.6 ;1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H),
10.84 (s, 1H), 9.06 (s, 1H), 8.06 (d, J = 5.3 Hz, 1H), 7.92 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.71 (d, J = 8.3 Hz,
2H), 7.60 (s, 2H), 7.33 (s, 1H), 7.14 (d, J = 8.1 Hz, 2H), 6.39 (d, J = 5.4 Hz, 1H), 6.18 (s, 1H).
5.5.22(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(2,3-dichlorobenzylidene)hydrazinecarboxamide
(8d)
Yield: 60.4%; ESI-MS [M + H]+ m/z:441,06; m.p. 260.3–262.0 ;1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H),
11.10 (s, 1H), 9.13 (s, 1H), 8.38 (d, J = 8.0 Hz, 2H), 8.06 (d, J = 5.4 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.66 (d, J =
7.9 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.33 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.39 (d, J = 5.4 Hz, 1H), 6.18 (s, 1H).
5.5.23(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(4-fluorobenzylidene)hydrazinecarboxamide (8e)
Yield: 59.9%; ESI-MS [M + H]+ m/z:390.13;1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H), 10.78 (s, 1H), 9.03
(s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.94 (s, 1H), 7.91 (d, J = 6.2 Hz, 2H), 7.72 (d, J = 8.9 Hz, 2H), 7.33 (s, 1H), 7.24
(d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.39 (d, J = 5.6 Hz, 1H), 6.19 (s, 1H).
5.5.24
(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(4-hydroxybenzylidene)hydrazinecarboxamide
(8f)
Yield: 51.9%; ESI-MS [M + H]+ m/z:388.14; m.p. 216.0–218.7 ;1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H),
10.56 (s, 1H), 9.81 (s, 1H), 8.92 (s, 1H), 8.07 (d, J = 5.0 Hz, 1H), 7.86 (s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.67 (d, J
= 7.9 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 7.7 Hz, 2H), 6.39 (d, J = 5.2 Hz, 1H), 6.20 (s,
1H).
5.5.25
(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(4-methoxybenzylidene)hydrazinecarboxamide
(8g)

ACCEPTED MANUSCRIPT
Yield: 60.4%; ESI-MS [M + H]+ m/z:402.15; m.p. 230.5–231.7 ;1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H),
10.63 (s, 1H), 8.96 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.8 Hz,
2H), 7.33 (s, 1H), 7.13 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 6.39 (d, J = 5.4 Hz, 1H), 6.19 (s, 1H), 3.79 (s,
3H).
5.5.26(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(2,3,4-trimethoxybenzylidene)hydrazinecarbox-
amide (8h)
Yield: 61.3%; ESI-MS [M + H]+ m/z:462.17; m.p. 209.8–211.2 ;1H NMR (400 MHz, DMSO-d6) δ 11.70 (s, 1H),
10.65 (s, 1H), 8.95 (s, 1H), 8.14 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 8.4 Hz,
2H), 7.33 (s, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.9 Hz, 1H), 6.38 (d, J = 5.3 Hz, 1H), 6.19 (s, 1H), 3.84
(s,3H), 3.81 (s, 3H), 3.76 (s, 3H).
5.5.27(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-(2-bromo-4-hydroxybenzylidene)hydrazinecarbox
amide (8i)
Yield: 61.5%; ESI-MS [M + H]+ m/z:467.04; m.p. 250.8–252.6 ;1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H),
10.63 (s, 1H), 10.62 (s, 1H), 9.02 (s, 1H), 8.10 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.82 (s, 1H), 7.72 (d, J = 8.4 Hz,
2H), 7.58 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.14 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H), 6.39 (d, J = 5.4 Hz,
1H), 6.19 (s, 1H).13C NMR (101 MHz, DMSO-d6) δ 158.14, 155.65, 153.72, 151.57, 149.93, 144.66, 140.13,
136.69, 131.42, 128.66, 127.67, 124.95, 122.19(2C), 121.08(2C), 116.62, 110.49, 110.44, 102.23, 97.65.
5.6 General procedure for the preparation of compounds 9.
N,N-dimethylformamide (2.15g, 30.0 mmol) in an ice bath was slowly treated with POCl3(3.38 g, 30.0
mmol). Then, the reaction mixture was removed from the ice bath to room temperature and stirred for 5 min.
Afterwards, anhydrous 1,1-dichloroethane (6.8 mL) was added in the solution in ice bath, followed by a solution
of pyrrole 8 (1.25 g, 18.4 mmol) in 1,1-dichloroethane (6.8 mL) over 30 min. The mixture was refluxed for 15
min and cooled to room temperature. A saturated aqueous NaOAc solution (6.9 g of NaOAc in 15 mL of
deionized water) was added, and the solution was refluxed for 15 min. When it was cooled to room temperature,
the solution was diluted with diethyl ether and washed with saturated aqueous NaHCO3 solution. The organic
layer was dried over anhydrous Na2SO4 and concentrated to yield the title compound 9.
5.7 General procedure for the preparation of compounds 10 and 14.
The compound 9 was dissolved in DMSO-D6 (10mL). 60% NaH (2.53g, 6.0mmol) was slowly added to the
solution and stirred for 30 min in room temperature. Then 1-chloro-2-bromoethane 8 g (42.5mmol) or 1-chloro-
3-bromopropane was added dropwise and refluxed for 1 h at 110 and monitored by TLC. The reaction solution
was added to saturate NaCl and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4

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and concentrated in vacuum to obtain the compound 10 or 14.
5.8 General procedure for the preparation of compounds 11a–c and 15a–f.
A mixture of 10 or 14 (3.53 g, 17.7mmol), small molecule amine such as Piperidine (3.0 g, 35.4mmol) and
DMF (20mL) was heated and stirred for 24 h at 125 , and the reaction was monitored by TLC. The mixture was
added to saturate NaCl, extracted with ethyl acetate. The organic phase was separated, dried, and evaporated to
yield the title compounds 11a–c and 15a–f.
5.9 General procedure for the preparation of compounds 12a–b, 13a–c, 16a–d and 17a–e.
A mixture of intermediate 6a or 6b (0.18 mmol) and substituted 2-pyrrole formaldehyde 11a-c or 15a-f (0.36
mmol) were dissolved in DMF (4 mL), and then reacted in microwave reactor at 65 and 100W. Upon
completion of the reaction, the solution was removed, added water (10 mL) and stirred for 15 min in room
temperature. The target compounds12a–b, 13a–c, 16a–d and 17a–e were obtained by precipitation of solid
crystals.
5.9.1(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(2-(dimethylamino)ethyl)-1H-pyrrol-2-
yl)methylene)hydrazinecarboxamide (12a)
Yield: 48.5%; ESI-MS [M + H]+ m/z:450.20; m.p. 191.6–192.7 ;1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H),
10.65 (s, 1H), 8.96 (s, 1H), 8.07 (s, 1H), 7.90 (s, 1H), 7.76 (s, 1H), 7.43 (d, J = 9.2 Hz, 1H), 7.36 (t, J = 6.6 Hz,
2H), 7.02 (s, 1H), 6.50 (s, 1H), 6.37 (d, J = 5.3 Hz, 1H), 6.23 (s, 1H), 6.09 (s, 1H), 4.41 – 4.33 (m, 2H), 2.16 (s,
4H), 1.23 (s, 4H).13C NMR (101 MHz, DMSO-d6) δ 157.82, 155.27, 153.41, 152.84, 151.55, 144.70, 135.02,
127.97, 126.51, 125.23, 124.07, 116.21, 115.99, 109.75, 108.52, 108.39, 108.16, 101.05, 97.23, 60.11, 47.89,
45.80(2C).
5.9.2 (E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(2-morpholinoethyl)-1H-pyrrol-2-yl)
methylene)hydrazinecarboxamide (12b)
Yield: 47.9%; ESI-MS [M + H]+ m/z:492.21; 1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.58 (s, 1H), 8.07
(d, J = 5.3 Hz, 1H), 7.92 (s, 1H), 7.79 (d, J = 14.2 Hz, 1H), 7.51 – 7.44 (m, 1H), 7.36 (dd, J = 10.5, 6.3 Hz, 2H),
7.02 (s, 1H), 6.54 (s, 1H), 6.42 – 6.37 (m, 1H), 6.35 (d, J = 5.4 Hz, 1H), 6.24 (s, 1H), 6.12 (d, J = 10.6 Hz, 1H),
4.43 (s, 1H), 3.46 (s, 2H), 2.55 (d, J = 9.7 Hz,2H), 2.39 (s, 2H), 1.30 – 1.20 (m, 4H), 0.85 (d, J = 6.5 Hz, 1H).
5.9.3(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(2-(dimethylamino)ethyl)-1H-pyrrol-2-yl)methy
lene)hydrazinecarboxamide (13a)
Yield: 45.3%; ESI-MS [M + H]+ m/z:432.21; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 10.52 (s, 1H), 8.84
(s, 1H), 8.07 (d, J = 5.2 Hz, 1H), 7.89 (s, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.34 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 7.01
(s, 1H), 6.48 (s, 1H), 6.40 (d, J = 5.4 Hz, 1H), 6.19 (s, 1H), 6.08 (s, 1H), 4.40 – 4.34 (m, 2H), 2.15 (s, 5H), 1.23 (s,

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3H).13C NMR (101 MHz, DMSO-d6) δ 158.11, 153.62, 151.58, 149.85, 144.67, 136.74, 134.60, 127.86, 126.58,
124.95, 121.52(2C), 121.22(2C), 115.86, 110.42, 108.46, 102.24, 97.61, 60.11, 47.98, 45.80(2C).
5.9.4(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(2-(diethylamino)ethyl)-1H-pyrrol-2-yl)methyle
ne)hydrazinecarboxamide(13b)
Yield: 46.2%; ESI-MS [M + H]+ m/z:460.24; 1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.59 (s, 1H), 8.80
(s, 1H), 8.07 (d, J = 4.9 Hz, 1H), 7.91 (s, 1H), 7.79 (d, J = 13.1 Hz, 1H), 7.49 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 10.3
Hz, 2H), 7.02 (s, 1H), 6.53 (s, 1H), 6.36 (d, J = 5.1 Hz, 1H), 6.22 (s, 1H), 6.08 (s, 1H), 4.35 (s, 2H), 3.32 – 3.26
(m, 3H), 2.62 (s, 2H), 1.26 (d, J = 19.5 Hz, 2H), 0.87 (t, J = 6.7 Hz, 6H).
5.9.5(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(2-morpholinoethyl)-1H-pyrrol-2-yl)methylene)
hydrazinecarboxamide (13c)
Yield: 43.7%; ESI-MS [M + H]+ m/z:474.22; 1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.61 (d, J = 10.5
Hz, 1H), 8.07 (d, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.80 (d, J = 11.7 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39 – 7.32 (m,
2H), 7.01 (d, J = 11.6 Hz, 1H), 6.53 (s, 1H), 6.35 (d, J = 5.1 Hz, 1H), 6.24 (s, 1H), 6.10 (s, 1H), 4.46 – 4.29 (m,
2H), 3.45 (s, 2H), 2.60 – 2.51 (m, 2H), 2.39 (s, 2H), 2.00 (d, J = 7.3 Hz, 1H), 1.24 (s, 4H), 0.85 (d, J = 6.1 Hz,
1H).
5.9.6(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(3-(diethylamino)propyl)-1H-pyrrol-2-
yl)methylene)hydrazinecarboxamide(16a)
Yield: 44.5%; ESI-MS [M + H]+ m/z:492.71; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 10.43 (s, 1H), 8.58
(s, 1H), 8.06 (d, J = 5.3 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.96
(s, 1H), 6.55 (s, 1H), 6.37 (d, J = 5.4 Hz, 1H), 6.20 (s, 1H), 6.09 (s, 1H), 4.26 (t, J = 7.0 Hz, 2H), 2.42 – 2.36 (m,
4H), 2.32 (d, J = 7.1 Hz, 2H), 1.78 (d, J = 7.0 Hz, 1H), 0.86 (t, J = 7.0 Hz, 6H).
5.9.7(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(3-(piperidin-1-yl)propyl)-1H-pyrrol-2-
yl)methylene)hydrazinecarboxamide(16b)
Yield: 45.9%; ESI-MS [M + H]+ m/z:504.24; 1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.54 (s, 1H), 8.80
(s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.94 (s, 1H), 7.80 (s, 1H), 7.50 (d, J = 8.9 Hz, 1H), 7.37 (s, 1H), 7.32 (d, J = 9.1
Hz, 1H), 6.96 (s, 1H), 6.58 (s, 1H), 6.34 (d, J = 5.4 Hz, 1H), 6.25 (s, 1H), 6.10 (s, 1H), 4.25 (t, J = 6.7 Hz, 2H),
2.22 (s, 4H), 2.13 (d, J = 6.9 Hz, 2H), 1.84 – 1.78 (m, 2H), 1.44 (d, J = 4.8 Hz, 4H), 1.23 (s, 2H).13C NMR (101
MHz, DMSO-d6) δ 157.89, 153.41, 151.54, 144.63, 138.47, 135.72, 135.29, 127.13, 126.89, 125.21, 124.05,
116.11, 114.30, 109.75, 108.60, 108.20, 107.97, 100.90, 97.26, 55.64, 54.45, 46.25, 28.59, 25.98, 24.62.
5.9.8(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(3-(4-methylpiperazin-1-yl)propyl)-1H
-pyrrol-2-yl)methylene)hydrazinecarboxamide(16c)

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Yield: 46.0%; ESI-MS [M + H]+ m/z:519.26; m.p. 192.8–194.9 ;1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H),
10.54 (s, 1H), 8.80 (s, 1H), 8.06 (d, J = 5.2 Hz, 1H), 7.94 (s, 1H), 7.81 (d, J = 13.2 Hz, 1H), 7.49 (d, J = 8.9 Hz,
1H), 7.34 (dd, J = 17.2, 7.9 Hz, 2H), 6.96 (s, 1H), 6.57 (s, 1H), 6.35 (d, J = 5.4 Hz, 1H), 6.26 (s, 1H), 6.09 (s, 1H),
4.26 (t, J = 6.8 Hz, 2H), 2.26 (s, 4H), 2.17 (dd, J = 16.7, 9.9 Hz, 4H), 2.10 (s, 4H), 1.85 – 1.76 (m, 2H), 1.23 (s,
1H).
5.9.9(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-2-((1-(3-(pyrrolidin-1-yl)propyl)-1H-pyrrol-
2-yl)methylene)hydrazinecarboxamide (16d)
Yield: 47.6%; ESI-MS [M + H]+ m/z:490.21;1H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.54 (s, 1H), 8.80
(s, 1H), 8.07 (d, J = 5.1 Hz, 1H), 7.94 (s, 1H), 7.81 (d, J = 12.0 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.39 – 7.30 (m,
2H), 6.97 (s, 1H), 6.57 (s, 1H), 6.34 (d, J = 5.6 Hz, 1H), 6.25 (s, 1H), 6.10 (s, 1H), 4.28 (d, J = 6.8 Hz, 2H), 2.38 –
2.28 (m, 4H), 1.61 (s, 2H), 1.24 (s, 6H).
5.9.10(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(3-(dimethylamino)propyl)-1H-pyrrol-2-yl)met
hylene)hydrazinecarboxamide (17a)
Yield: 45.2%; ESI-MS [M + H]+ m/z:446.32; m.p. 193.1–195.4 ;1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H),
10.44 (s, 1H), 8.58 (s, 1H), 8.06 (s, 1H), 7.92 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.34 (s, 1H), 7.15 (d, J = 8.6 Hz,
2H), 6.96 (s, 1H), 6.54 (s, 1H), 6.38 (d, J = 5.3 Hz, 1H), 6.20 (s, 1H), 6.09 (s, 1H), 4.28 (t, J = 7.0 Hz, 2H), 2.15 (t,
J = 6.8 Hz, 2H), 2.05 (s, 4H), 1.84 – 1.77 (m, 2H), 1.23 (s, 2H).
5.9.11(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(3-morpholinopropyl)-1H-pyrrol-2-yl)methyle
ne)hydrazinecarboxamide (17b)
Yield: 43.9%; ESI-MS [M + H]+ m/z:488.56; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 10.45 (s, 1H), 8.59
(s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.93 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.34 (s, 1H), 7.15 (d, J = 8.8 Hz, 2H), 6.96
(s, 1H), 6.56 (s, 1H), 6.37 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.27 (t, J = 6.4 Hz, 2H), 3.53 (s, 4H),
2.26 (s, 2H), 2.19 (t, J = 6.8 Hz, 2H), 1.84 (d, J = 7.3 Hz, 2H), 1.24 (s, 2H).
5.9.12(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(3-(piperidin-1-yl)propyl)-1H-pyrrol-2-yl)meth
ylene)hydrazinecarboxamide (17c)
Yield: 45.8%; ESI-MS [M + H]+ m/z:486.73; m.p. 191.4–195.0 ;1H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H),
10.43 (s, 1H), 8.57 (s, 1H), 8.06 (d, J = 5.5 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J
= 8.7 Hz, 2H), 6.95 (s, 1H), 6.56 (s, 1H), 6.37 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.25 (t, J = 6.9 Hz,
2H), 2.17 (dd, J = 21.2, 14.4 Hz, 6H), 1.44 (s, 4H), 1.23 (s, 4H).13C NMR (101 MHz, DMSO-d6) δ 158.19, 153.54,
151.56, 149.68, 144.61, 136.73, 134.92, 127.13, 126.93, 124.94, 121.28(2C), 121.25(2C), 114.33, 110.39, 108.56,
102.08, 97.61, 55.63, 54.44(2C), 46.30, 28.56, 25.97(2C), 24.61.

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5.9.13(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrol-
2-yl)methylene)hydrazinecarboxamide (17d)
Yield: 44.3%; ESI-MS [M + H]+ m/z:501.81; m.p. 187.6–188.1 ;1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H),
10.43 (s, 1H), 8.56 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J
= 8.7 Hz, 2H), 6.95 (s, 1H), 6.55 (s, 1H), 6.37 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.26 (t, J = 6.8 Hz,
2H), 2.30 (s, 6H), 2.22 – 2.15 (m, 3H), 2.13 (s, 4H), 1.87 – 1.79 (m, 2H).
5.9.13(E)-N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-2-((1-(3-(pyrrolidin-1-yl)propyl)-1H-pyrrol-2-yl)met
hylene)hydrazinecarboxamide (17e)
Yield: 44.8%; ESI-MS [M + H]+ m/z:472.38; m.p. 193.9–197.6 ;1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H),
10.43 (s, 1H), 8.57 (s, 1H), 8.06 (d, J = 5.4 Hz, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.34 (s, 1H), 7.15 (d, J
= 8.7 Hz, 2H), 6.96 (s, 1H), 6.55 (s, 1H), 6.37 (d, J = 5.4 Hz, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.29 (t, J = 6.8 Hz,
2H), 2.39 – 2.28 (m, 6H), 1.87 – 1.80 (m, 2H), 1.61 (s, 4H).
5. 10 Cytotoxicity assay in vitro
The cytotoxic activities of target compounds (12a–b, 13a–c, 16a–d and17a–e) were evaluated with A549,
PC-3, MCF-7 and HepG2 cell lines by the standard MTT assay in vitro, with compounds 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 5µg/mL and incubated with cells at 37°C for 4
h. The formazan crystals were dissolved in 100 µL DMSO-D6 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. 11 c-Met kinase assay in vitro
The selected compounds (7c and 17e) are tested for their activity against c-Met kinase through the mobility
shift assay[19, 20]. 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-D6, then transfer 10 µL of compound to a new 96-well plate as the intermediate plate, add 90

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µL kinase buffers 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-D6 without compound were used as DMSO-D6
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 °C 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-D6 control;
“min” stands for low control.
5.12 Time-dependent assay
A549 cells were cultured according to the method of 5.10. The test compound 7c at indicated final
concentrations were added to the culture medium and the cell cultures were continued for 24 h, 48 h and 72 h
respectively, and then tested separately by MTT method as described in 5.10.
5.13 Observation of nuclear morphology
The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine
serum (FBS). Approximately 2×104 cells, suspended in MEM medium, were plated onto each well of a 24-well
plate and incubated in 5% CO2 at 37°C for 24 h. Then the medium was removed, and 1mL drug-free medium and
1mL medium with the test compound7c at indicated final concentrations were added to each well in control group
and test group respectively and the cell cultures were continued for 12 h. While the medium was removed,
AO-PBS buffer was added to each well at a terminal concentration of 10µg/mL and stained in dark for 10 min.
Each well was washed with PBS buffer three times and observed under a fluorescence microscope.
5.14 Cell cycle analysis
A549 cells were seeded in 16-well plates at a density of 1 × 106cells/well in DMEM, then treated with 4.0µM
concentration of 7cand treated without compounds for 12 h, respectively. Cultured cells were stained with
propidium iodide (PI) in the dark at 4 for 30 min and analyzed by ACEA NovoCyteTM
5.15 Docking studies
.
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8) were obtained from
RCSB Protein Data Bank[21]. Hydrogen atoms were added to the structure allowing for appropriate ionization at
physiological pH. The protonated state of several important residues, such as CYS919, and ASP1046 were
adjusted by using SYBYL6.9.1 (Tripos, St. Louis, USA) in favor of forming reasonable hydrogen bond with the
ligand. Molecular docking analysis was carried out by the SURFLEX-DOCK module of SYBYL 6.9.1 package to
explore the binding model for the active site of c-Met with its ligand. What’s more, the analysis of bond mode was

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using the Discover Studio viewer 3.5. All atoms located within the range of 5.0 Å from any atom of the cofactor
were selected into the active site, and the corresponding amino acid residue was, therefore, involved into the
active site if only one of its atoms was selected. Other default parameters were adopted in the SURFLEX-DOCK
calculations. All calculations were performed on Silicon Graphics workstation.
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds
(No.21162014),
Natural
Science
Funds
for
Distinguished
Young
Scholar
of
Jiangxi
Province,China(20171BCB23078); Key projects of the youth fund，Natural Science Funds of Jiangxi Province
(20171ACB21052); The Project Supported by Natural Science Foundation of Jiangxi, China (20161BAB215216);
Department Education Science and Technology Research Project of Jiangxi, China(GJJ 160787 ); Jiangxi
Provincial Key Laboratory of Drug Design and Evaluation（20171BCD40015）.

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Research Highlights
► Two series of aromatic hydrazone derivatives bearing 1H-pyrrolo[2,3-b]pyridine
moiety (7a–r, 8a–i, 12a–b, 13a–c, 16a–d and 17a–e) were designed and
characterized.
► Most of the synthesized compounds showed moderate to significant antitumor
activity.
►7c arrest efficiently the cell cycle progression in G2/ M phase of A549 cells.
► Docking study was investigated to explore the binding modes of compounds with
c-Met.