Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.02.059

A series of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety were designed, synthesized, and evaluated for their c-Met kinase inhibitory activities and antiproliferative activities against 4 cancer cell lines (HT-29, A549, MCF-7, and PC-3) in vitro. Most compounds showed moderate to excellent potency, with the most promising analog 34 showing a c-Met IC₅₀value of 1.68?nM. Structure–activity relationship studies indicated that electron-withdrawing groups (X?=?CF₃, R¹?=?F, R²?=?4-F) were required to decrease the higher electron density on the 5-atom linker to a proper degree to improve the inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2016.02.059

Accepted Manuscript
Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole
moiety as c-Met kinase inhibitors
Qidong Tang, Linxiao Wang, Yayi Tu, Wufu Zhu, Rong Luo, Qidong Tu, Ping
Wang, Chunjiang Wu, Ping Gong, Pengwu Zheng
PII:
S0960-894X(16)30172-X
http://dx.doi.org/10.1016/j.bmcl.2016.02.059
BMCL 23613
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
6 January 2016
30 January 2016
19 February 2016
Please cite this article as: Tang, Q., Wang, L., Tu, Y., Zhu, W., Luo, R., Tu, Q., Wang, P., Wu, C., Gong, P., Zheng,
P., Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety as c-Met kinase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.059
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Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety as
c-Met kinase inhibitors
a
a
a
Qidong Tang a,c,,†, Linxiao Wang a,†, Yayi Tu a, Wufu Zhu , Rong Luo b, Qidong Tu , Ping Wang ,
Chunjiang Wu a, Ping Gong c, Pengwu Zheng a,*
a School of Pharmacy, Jiangxi Science and Technology Normal University, Nanchang 330013, PR China
b Jiangxi Province Institute of Materia Medica, Nanchang 330000, PR China
c
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
Abstract
A series of novel pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety were
designed, synthesized, and evaluated for their c-Met kinase inhibitory activities and
antiproliferative activities against 4 cancer cell lines (HT-29, A549, MCF-7, and PC-3) in
vitro. Most compounds showed moderate to excellent potency, with the most promising
analogue 34 showing a c-Met IC50 value of 1.68 nM. Structure-activity relationship studies
indicated that electron-withdrawing groups (X = CF3, R1 = F, R2 = 4-F) were required to
decrease the higher electron density on the 5-atom linker to a proper degree to improve the
inhibitory activity.
Keywords:
Synthesis; pyrrolo[2,3-b]pyridine
derivatives; 1,2,3-triazole; c-Met;
antiproliferative activity
Receptor tyrosine kinases (RTKs) play crucial roles in signal transduction pathways and
cellular processes, and many are implicated in cancer.1 c-Met [the receptor for hepatocyte
growth factor/scatter factor, (HGF/SF)]2 belongs to a subfamily of RTKs which composed of
an extracellular α chain and a membrane-spanning β chain connected through a disulfide
bond.3–4 c-Met has been shown to be frequently amplified or overexpressed in various cancers,
including brain, colorectal, gastric, lung, head, neck, and stomach cancers. Both
amplifications/overexpression are associated with poor clinical outcomes, underscoring the
importance of increased c-Met signaling in these cancer types.5–8 Therefore, c-Met shows
 Corresponding authors. Tel./fax: +86 791 83802393.
E-mail addresses: tangqidongcn@126.com (Q. Tang), zhengpw@126.com (P. Zheng).
† These authors contribute equally to this work.

high potential as a therapeutic target for human cancer.
Recently, significant progress has been made in the development of small-molecule
c-Met inhibitors, resulting in the marketing of cabozantinib9 (1, a small molecule that inhibits
the activity of multiple tyrosine kinases, including c-Met, RET, and VEGFR-2/KDR,
marketed in 2012),10 and several candidates currently under clinical trials，such as foretinib (2,
GSK1363089), compounds 3 and 4.11–13 According to the structure characteristics of these
inhibitors, general structure which contained four parts: moiety A, B, C, and D can be
summarized (Fig. 1).
Figure 1. The representative c-Met kinase inhibitors and the corresponding General Structure.
Many structure types of these derivatives were included judging from moiety A, such as
substituted quinoline, substituted pyridine, thieno[2,3-b]pyridine, and pyrrolo[2,3-b]pyridine
series (1-2, 3, 4, and 5, respectively). However, the main modification of these different series
of derivatives was focused on moiety C (a 5-atom linker) which has two obvious structural
characteristics. One is the ‘5 atoms regulation’, which means six chemical bonds distance
existing between moiety B and moiety D; the other is the linker containing hydrogen, oxygen,
and nitrogen atoms which could form hydrogen-bond donor or acceptor.14-15
1,2,3-triazole fragment was widely used as a building block in the design of anticancer
agents. For example, compounds 6, 7, 8 (Fig. 2) displayed a multitude of biological
activities.16–18 In this work, 1,2,3-triazole was introduced to moiety C to form the 5-atom
linker, because the three nitrogen atoms in 1,2,3-triazole as the hydrogen-bond acceptor have
high ability to form hydrogen-bonding interactions with c-Met. Pyrrolo[2,3-b]pyridine was
used as the moiety A. Substituted phenyl ring was reserved as the moiety B and D. Small

substituent X, R1, and R2 were introduced to investigate their effects on activity of the target
compounds. Accordingly, we designed a novel series of pyrrolo[2,3-b]pyridine derivatives
bearing 1,2,3-triazole moiety (Fig. 3).
Figure 2. Anticancer agents bearing 1,2,3-triazole fragments.
Figure 3. Design strategy for the pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety.
The target compounds synthesized were evaluated for their c-Met kinase activities and
antiproliferative activities against 4 cancer cell lines included the HT-29, A549, MCF-7, and
PC-3. Apparent growth inhibition on c-Met and the 4 cancer cell lines was observed for most
of the compounds. The most promising compound 34 were chosen to screen against 5 other
tyrosine kinases (Flt-3, PDGFR-β, KDR, c-Kit, and EGFR) to examine its selectivity on
c-Met. Docking analysis of compounds with c-Met was performed using compound 34.
The key intermediates 4-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)anilines 12a-b was prepared
by
coupling
4-chloro-1H-pyrrolo[2,3-b]pyridine
9
with
4-nitrophenol
or
2-fluoro-4-nitrophenol 10a-b, followed by reduction of the nitro group of intermediate 11a-b
using zinc metal as shown in Scheme 1.19 The intermediates 1-aryl-5-methyl (or
trifluoromethyl)-1,4,5-trisubstituted-1,2,3-triazoles 15a-p were synthesized in high yield by a
one-pot three-component reaction of arylboronic acids 13a-g, sodium azide, and active
methylene ketones, such as ethyl acetoacetate (or ethyl 4,4,4-trifluoroacetoacetate) 14a-b in

the presence of Cu(OAc)2 and piperidine using a DMSO/H2O (10/1) mixture as solvent.20
Simple procedures such as hydrolysis and acyl chlorination were used to convert ethyl
5-methyl(or trifluoromethyl)-1-substitutedphenyl-1H-1,2,3-triazole-4-carboxylate 15a-p to
the corresponding acyl chloride 17a-p; the reactions proceeded with 20% H2SO4 and thionyl
chloride, respectively. Reaction of anilines 12a-b with acyl chloride 17a-p promoted by
DIPEA in dichloromethane at room temperature yielded the target compounds 18-45.21
Scheme 1. Reagents and conditions: (ⅰ) i-Pr2NEt, microwave, 200 °C, NMP, 42–44 ; (ⅱ) Zn, NH4Cl,
MeOH/THF, rt., overnight, 77–80 ; (ⅲ) Cu(OAc)2, DMSO/H2O (10:1), Piperidine, rt. to 80 °C, 24h,
88–92 ；(ⅳ) 20%H2SO4, 100 °C, 5–10 h, 72–76 ; (ⅴ) SOCl2, reflux, 6 h, 92–94 ; (ⅵ) appropriate
aniline, carbonyl chloride, DIPEA, 0 °C, 1 h, rt., 7–10 h, 56–61 .
The c-Met enzymatic assays of all synthesized compounds were evaluated using
homogeneous time-resolved fluorescence (HTRF) assay.22 The results expressed as the
half-maximal inhibitory concentration (IC50) values presented in Table 1; the mean values of
experiments performed in triplicate are shown.
As illustrated in Table 1, these novel pyrrolo[2,3-b]pyridine derivatives bearing
1,2,3-triazole moiety were found to be active against c-Met kinase with IC50 values ranging
from 1.6842.56 nM; 4 of them (3423, IC50 = 1.68 nM; 33, IC50 = 5.24 nM; 41, IC50 = 6.39
nM; 32, IC50 = 7.25 nM) showed high potency with IC50 values in single-digit nanomole range,
which indicated that it’s a good design strategy to use pyrrolo[2,3-b]pyridine as moiety A and
introduce 1,2,3-triazole fragment to moiety C to form the 5-atom linker.
According to the data shown in Table 1, enzymatic assays data showed a preference for
activity when the X group on 1,2,3-triazole fragment was trifluoromethyl instead of methyl,
indicating that the introduction of electron-withdrawing groups (EWGs, such as CF3) on the
5-atom linker had a positive effect. For example, the activity of compound 39 (IC50 = 11.37
nM; X = CF3, R1 = H, R2 = H) was higher than compound 25 (IC50 = 19.36 nM; X = CH3, R1

= H, R2 = H).
Moreover, compounds substituted with fluoro atom (R1 group) on moiety B are more
active than that of substituted with hydrogen atom, which indicated that the EWGs (such as F)
on moiety B benefited to the potency. For example, the activity of compound 32 (IC50 = 7.25
nM; X = CF3, R1 = F, R2 = H) was higher than compound 39 (IC50 = 11.37 nM; X = CF3, R1 =
H, R2 = H), and the same trend was observed in compounds 18/25, 19/26, 20/27 and so on.
Further studies were performed to examine the effect of different substituents R2 on the
phenyl ring (moiety D) on potency. The mono-electron-withdrawing groups (mono-EWGs)
introduced to the phenyl ring increased the c-Met inhibitory efficacy, while incorporation of
the mono-EWGs at 4-position of the phenyl showed a higher preference. For example,
compound 32, with no substituent on the phenyl ring, showed a c-Met IC50 value 7.25 nM.
Introduction of mono-EDGs at 4-position of the phenyl (34, R2 = 4-F, IC50 = 1.68 nM,
increased 4.3-fold) increased the inhibitory efficacy to a greater extent than that of
mono-EDGs at 2-position of the phenyl (36, R2 = 2-F, IC50 = 5.24 nM, increased 1.4-fold).
This was further confirmed by introduction of these 2 types of groups in compounds 19/20,
26/27, and 40/41. However, incorporation of mono-donating groups (mono-EDGs) and
double electron-withdrawing groups (double-EWGs) showed opposite trend in potency of the
compounds, such as 21 (R2 = 4-OCH3, IC50 = 24.14 nM), 22 (R2 = 3,4-(F)2, IC50 = 21.63 nM),
23 (R2 = 3-Cl-4-F, IC50 = 29.27 nM) and so on. Similarly to our previous study, regulating
electron density on the 5-atom linker to a proper degree was a key factor in improving the
potency.21 The 1,2,3-triazole ring, which is part of the 5-atom linker, has higher electron
density because of the three nitrogen atoms. Therefore, EWGs (X = CF3, R1 = F, R2 = 4-F)
were required to decrease the higher electron density on the 5-atom linker to a proper degree
to improve the inhibitory activity.
Table 1
c-Met kinase activities of the target compounds 18-45
X
R1
R2
c-Met IC50 (nM)
16.82 ± 1.16
14.34 ± 1.22
12.78 ± 1.42
24.14 ± 1.42
21.63 ± 1.27
Compd.
18
CH3
CH3
CH3
CH3
CH3
F
H
F
2-F
19
F
4-F
20
F
4-OCH3
3,4-(F)2
21
F
22

CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
F
F
3-Cl-4-F
3-CF3-4-Cl
H
29.27 ± 1.34
37.71 ± 1.84
19.36 ± 1.51
16.25 ± 1.03
14.74 ± 0.42
25.42 ± 1.25
21.49 ± 0.32
22.33 ± 1.27
42.56 ± 1.91
7.25 ± 0.59 a
5.24 ± 0.51
1.68 ± 0.36
12.56 ± 0.35
11.26 ± 1.23
15.69 ± 1.46
18.15 ± 1.38
11.37 ± 1.31
10.73 ± 1.62
6.39 ± 0.43
16.38 ± 1.85
21.27 ± 1.72
19.39 ± 1.64
21.26 ± 1.78
1.92 ± 0.21
23
24
H
H
H
H
H
H
H
F
25
2-F
26
4-F
27
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
H
28
29
30
31
32
F
2-F
33
F
4-F
34
F
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
H
35
F
36
F
37
F
38
H
H
H
H
H
H
H
39
2-F
40
4-F
41
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
42
43
44
45
Foretinib
a Bold values show the IC50 values of the target compounds lower than 10 nM
The cytotoxic activities of all target compounds 18–45 have been evaluated against the
HT-29 and A549 cell lines using the MTT assay.24 Some potent compounds were further
evaluated against the MCF-7 and PC-3 cell lines. The results expressed as IC50 values are
shown in Table 2 as the mean values of triplicate experiments.
As illustrated in Table 2, all target compounds showed moderate to excellent cytotoxic
activity against different cancer cells with potencies in the single-digit μM range. The IC50
values of the most promising compound 34 were 0.029, 0.082, 0.11, and 1.52 μM against the
HT29, A549, MCF-7, and PC-3 cell lines, respectively. Examination of the structure-activity
relationships (SARs) indicated that these analogs had similar SARs as summarized in the
c-Met kinase level: (a) The target compounds showed excellent selectivity toward HT29 cell
line; (b) the 1,2,3-triazole ring, which was a part of the 5-atom linker, required EWGs (such as
F and CF3) to decrease the electron density; (c) the number and electronic property of
substituents (R2 group) on the phenyl ring (moiety D) were key factors in improving the

potency.
Table 2
Cytotoxic activities of compounds 18–45 against HT-29, A549, MCF-7 and PC-3 cancer cell lines in vitro.
IC50 (μM) ± SD
X
R1
R2
Compd.
HT29
A549
MCF-7
0.35 ± 0.02
0.42 ± 0.02
0.17 ± 0.03
PC-3
2.34 ± 0.04
3.24 ± 0.09
4.35 ± 0.12
ND
0.29 ± 0.03
0.36 ± 0.02
0.39 ± 0.02
0.15 ± 0.01
0.71 ± 0.04
0.57 ± 0.03
0.73 ± 0.03
2.37 ± 0.06
0.42 ± 0.02
0.32 ± 0.02
0.22 ± 0.03
2.42 ± 0.18
2.26 ± 0.12
0.98 ± 0.07
2.83 ± 0.15
0.22 ± 0.04
0.19 ± 0.02
0.082 ± 0.003
0.36 ± 0.04
0.35 ± 0.02
0.82 ± 0.03
1.29 ± 0.10
0.29 ± 0.01
0.21 ± 0.03
0.18 ± 0.02
0.46 ± 0.05
0.79 ± 0.03
1.12 ± 0.09
2.34 ± 0.13
0.49 ± 0.08
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
F
F
H
18
a
2-F
0.18 ± 0.02
0.12 ± 0.03
0.59 ± 0.04
0.61 ± 0.03
1.06 ± 0.04
1.12 ± 0.05
0.35 ± 0.03
0.36 ± 0.05
0.18 ± 0.03
1.16 ± 0.10
1.07 ± 0.06
2.18 ± 0.12
3.29 ± 0.11
0.091 ± 0.003
0.073 ± 0.004
0.029 ± 0.002
0.29 ± 0.04
0.32 ± 0.003
0.63 ± 0.03
1.05 ± 0.12
0.19 ± 0.02
0.12 ± 0.03
0.093 ± 0.006
0.29 ± 0.04
0.52 ± 0.03
1.02 ± 0.04
1.53 ± 0.11
0.26 ± 0.03
19
F
4-F
20
b
F
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
H
ND
21
0.82 ± 0.02
ND
6.54 ± 0.11
ND
F
22
F
23
ND
ND
F
24
0.32 ± 0.04
0.28 ± 0.02
0.39 ± 0.05
ND
7.26 ± 0.15
3.45 ± 0.28
3.45 ± 0.32
ND
H
H
H
H
H
H
H
F
25
2-F
26
4-F
27
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
H
28
ND
ND
29
6.31 ± 0.15
8.41 ± 0.12
1.15 ± 0.11
0.28 ± 004
0.11 ± 0.02
0.51 ± 0.05
0.59 ± 0.03
ND
12.59 ± 0.13
10.92 ± 0.15
2.32 ± 0.11
2.13 ± 0.12
1.52 ± 0.14
1.85 ± 0.16
2.48 ± 0.12
ND
30
31
32
F
2-F
33
F
4-F
34
F
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
H
35
F
36
F
37
ND
ND
F
38
0.26 ± 0.04
0.29 ± 0.03
0.21 ± 0.04
0.58 ± 0.03
0.69 ± 0.05
ND
3.57 ± 0.12
3.69 ± 0.14
2.83 ± 0.16
4.85 ± 0.11
7.31 ± 0.14
ND
H
H
H
H
H
H
H
39
2-F
40
4-F
41
4-OCH3
3,4-(F)2
3-Cl-4-F
3-CF3-4-Cl
42
43
44
ND
ND
45
6.25 ± 0.36
0.89 ± 0.12
Foretinib
a Bold values show the IC50 values of the target compounds lower than 0.2 μM.
b ND: Not determined.
To examine the selectivity of compound 34 on c-Met over other kinases, this compound
was screened against 5 other tyrosine kinases (Table 3). Compared with its high potency
against c-Met (IC50 = 1.68 nM), 34 also exhibited high inhibitory effects against Flt-3 (IC50 =
2.06 nM) and PDGFR-β (IC50 = 4.57 nM). Moreover, compound 34 showed inhibitory effects
against KDR, c-Kit and EGFR although the potency was 51.3-, 193.2-, and 346.5-fold lower

than that of c-Met. These data suggested that compound 34 is a promising multi-target kinase
inhibitor.
Table 3
Inhibition of tyrosine kinases by compound 34.
Kinase
Flt-3
Enzyme IC50 (nM)
2.06
PDGFR-β
KDR
4.57
86.25
c-Kit
324.58
EGFR
582.17
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8)
were obtained from RCSB Protein Data Bank. The docking simulation was conducted using
SURFLEX-DOCK module of SYBYL 8.1 package version. The binding model was
exemplified by the interaction of compound 34 with c-Met. As shown in Fig. 4, the
pyrrolo[2,3-b]pyridine fragment formed two hydrogen bonds with MET1160. In the 5-atom
linker, two nitrogen atoms of 1,2,3-triazole and hydrogen atom on amide linkage formed three
hydrogen bonds with ASP1222. In addition, the trifluoromethyl group formed two hydrogen
bonds with LYS1110. Therefore, compound 34 formed seven hydrogen-bonding interactions
with c-Met.
Figure 4. Binding poses of compound 34 with c-Met. The proteins were displayed by grid
ribbon. Compound 34 were displayed by multicolor sticks. H-bonding interactions between
the 34 and c-Met were indicated with dashed lines in yellow.
In summary, we designed and synthesized 28 pyrrolo[2,3-b]pyridine derivatives bearing

1,2,3-triazole moiety. c-Met kinase and 4 human cancer cell lines (HT29, A549, MCF-7, and
PC-3) were used to evaluate the potency of the synthesized compounds. 4 of the derivatives
showed high potency with IC50 values in single-digit nanomole range. Compound 34 (c-Met
IC50 = 1.68 nM, a multi-target tyrosine kinase inhibitor) showed the strongest cytotoxic
activities against HT29, A549, and MCF-7 cell lines (IC50 values: 0.029, 0.082, and 0.11 μM,
respectively). Analysis of SARs indicated that EWGs (X = CF3, R1 = F, R2 = 4-F) were
required to decrease the higher electron density on the 5-atom linker to a proper degree to
improve the inhibitory activity.
Acknowledgments
This work was supported by Research Fund for the Doctoral Program of Jiangxi Science
and Technology Normal University (No. 3000990116).
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22. In vitro kinase assay: The kinase assays were performed by homogeneous time-resolved fluorescence
(HTRF) assay as previously reported protocol. Briefly, 20 μg/mL poly (Glu, Tyr) 4:1 (Sigma) was
preloaded as a substrate in 384-well plates. Then 50 μL of 10 mM ATP (Invitrogen) solution diluted in
kinase reaction buffer (50 mM HEPES, pH 7.0, 1 M DTT, 1 M MgCl2, 1 M MnCl2, and 0.1% NaN3)
was added to each well. Various concentrations of compounds diluted in 10 μL of 1% DMSO (v/v)
were used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine
kinase proteins diluted in 39 μL of kinase reaction buffer solution. The incubation time for the
reactions was 30 min at 25 °C, and the reactions were stopped by the addition of 5 μL of
Streptavidin-XL665 and 5 μL Tk Antibody Cryptate working solution to all of wells. The plates were
read using Envision (PerkinElmer) at 320 nm and 615 nm. The inhibition rate (%) was calculated
using the following equation: % inhibition = 100 - [(Activity of enzyme with tested compounds -
Min)/ (Max - Min)] × 100 (Max: the observed enzyme activity measured in the presence of enzyme,
substrates, and cofactors; Min: the observed enzyme activity in the presence of substrates, cofactors
and in the absence of enzyme). IC50 values were calculated from the inhibition curves.
23. Analytic data of potent inhibitor 34:1H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 8.42 (d, J = 5.6 Hz,
1H), 8.35 (d, J = 4.1 Hz, 1H), 7.99 (d, J = 4.9 Hz, 1H), 7.96 (s, 1H), 7.93 (d, J = 4.9 Hz, 1H), 7.87 (d, J =
4.9 Hz, 1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.67 (d, J = 2.2 Hz, 2H), 6.95 (d, J = 4.1 Hz, 1H), 6.83 (d, J = 5.6
Hz, 1H); 13C NMR (100 MHz, DMSO) δ 163.86, 162.58, 161.40, 159.71, 159.16, 157.46, 140.45, 146.32,
141.03, 139.07, 138.32, 137.83, 136.64, 131.35, 128.04 (2C), 123.54, 116.72 (2C), 112.20, 108.88, 104.39,
101.79; MS m/z (ESI): 501.3 [M+H]+; Anal. calcd. for C23H13F5N6O2 (%): C, 55.25; H, 2.63; N, 18.79.
Found (%): C, 55.19; H, 2.58; N, 18.76.
24. Cytotoxicity assay: A standard MTT assay was used to measure cell growth. The cancer cell lines were
cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS).
Approximate 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 compounds tested at the 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 mL DMSO of each well, and the absorbency at 492 nm
(for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with an
ELISA reader. All of the compounds were tested in triplicate in each cell line. The results expressed as
IC50 (inhibitory concentration 50%) were the averages of three determinations and calculated by using
the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

Graphical abstract
A series of pyrrolo[2,3-b]pyridine derivatives bearing 1,2,3-triazole moiety (18–45) were designed,
synthesized and evaluated for their activity against c-Met kinase and cancer cell lines. The most promising
compound 34 showed excellent in vitro activity.