Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 4-oxoquinoline moiety as potential antitumor inhibitor

Article abstract of DOI:10.1016/j.bmcl.2019.126848

A series of pyrrolo[2,3-b]pyridine derivatives bearing 4-oxoquinoline moiety were designed, synthesized and evaluated for the anti-proliferative on three cancer cell lines (A549, HepG2 and MCF-7) in vitro. Most of the compounds showed moderate to high potency. Some excellent compounds were tested for the inhibitory activity of c-Met kinase. Compound 34 (c-Met IC₅₀ = 17 nM) was investigated the selectivity against Flt-3, c-Kit, VEGFR-2, ALK, PDGFR-β and RON. Structure-activity relationship studies indicated that hydrogen, fluorine atom, and mono-electron-withdrawing groups (mono-EWGs, such as R² = F) on R, R¹ and R², respectively, were beneficial for the anti-proliferative activities of the target compounds. Besides, we have took further study on the combined mode between compound 34 and c-Met kinase through molecular docking.

Full text of DOI:10.1016/j.bmcl.2019.126848

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel pyrrolo[2,3-b]pyridine derivatives bearing 4-
oxoquinoline moiety as potential antitumor inhibitor
Huimin Liua,d, Yongli Duanb,c,d, Hehua Xiongb, Jianqing Zhangb, Shunmin Huangb, Ting Chenb,
⁎
⁎
Pengwu Zhengb, , Qidong Tangb,
a School of Perfume and Aroma Technology, Shanghai Institute of Technology; Engineering Research Center of Perfume & Aroma and Cosmetics of Ministry of Education,
Shanghai 201418, PR China
b Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, PR China
c School of Optoelectronic Science and Engineering, University of Electronic Science and Technology of China (UESTC), Chengdu 610054, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of pyrrolo[2,3-b]pyridine derivatives bearing 4-oxoquinoline moiety were designed, synthesized and
evaluated for the anti-proliferative on three cancer cell lines (A549, HepG2 and MCF-7) in vitro. Most of the
compounds showed moderate to high potency. Some excellent compounds were tested for the inhibitory activity
of c-Met kinase. Compound 34 (c-Met IC50 = 17 nM) was investigated the selectivity against Flt-3, c-Kit, VEGFR-
2, ALK, PDGFR-β and RON. Structure-activity relationship studies indicated that hydrogen, fluorine atom, and
mono-electron-withdrawing groups (mono-EWGs, such as R2 = F) on R, R1 and R2, respectively, were beneficial
for the anti-proliferative activities of the target compounds. Besides, we have took further study on the combined
mode between compound 34 and c-Met kinase through molecular docking.
Synthesis
Pyrrolo[2,3-b]pyridine derivatives
4-Oxoquinoline
Anti-proliferative activity
c-Met
Introduction
preclinical research (Fig. 1). At the same time many small molecule
inhibitors are in the biological activity test phase.10–13 We have also
According to the World Health Organization, the number of new
cancer cases in world in the 2012 was about 14 million. It is estimated
that the quantity of deaths will rise to 222 million in the next 20 years
from 8.2 million to 13 million.1–2 Tyrosine kinase family has received
more attention in cancer research. c-Met is a class of transmembrane
receptors with auto-phosphorylation activity encoded by c-Met gene
and it has been confirmed as a specific HGF ligand.3–5 The HGF/c-Met
signaling pathway plays a significant role in embryogenesis, wound
healing and tissue regeneration. However, c-Met shows abnormalities
activity in many cancers and is associated with gene mutation, ampli-
fication and recombination, which has become an effective target for
antitumor drug development.6–8
carried out relevant research in the previous work, such as compounds
5 and 6.14–15 Foretinib is a multi-targets small molecule tyrosine kinase
inhibitor that has high affinity to c-Met,16–18 which interacted with c-
Met kinase perfectly as shown in Fig. 2. We found that these potential c-
Met inhibitors have common features in structure, including three main
moieties of A, B and C. Moiety B has the characteristic of six chemical
bond distance (5-atom linker), which was characterized by the illu-
strated ‘5 atoms regulation’.14 Another characteristic is the ‘hydrogen-
bond donor or acceptor’ which means that the “5-atom linker” contains
hydrogen, oxygen, and nitrogen atoms to provide hydrogen-bond donor
or acceptor.14
In our previous work, we introduced the pyridazinone to moiety B
based on ‘5 atoms regulation’ and obtained a series of potential c-Met
inhibitors, especially the most promising compounds 5 and 6 with the
IC50 values in the nanomole level (Fig. 1).14–15 In this research, the
structure of pyridazinone was derivatized into the pyridone and in-
corporated with the benzene ring to obtain the ‘connection chain
structure’ of 4-oxoquinoline, the oxygen atom in which had possibility
to form intramolecular hydrogen bond with hydrogen atom (Fig. 3).
Pyrrolo[2,3-b]pyridine was used as the moiety A to study the effect of
c-Met inhibitors can be divided into two broad categories: mono-
clonal antibodies and small molecule kinase inhibitors, the common
mechanism of which is to inhibit the transmission of HGF/c-Met sig-
naling pathway. Cabozantinib (1), a representative small molecule
multi-target c-Met kinase inhibitor, was approved by the U.S. FDA for
the treatment of patients with progressive metastatic medullary thyroid
cancer (MTC) in 2012.9 Apart from cabozantinib, some c-Met inhibitors
such as MGCD-265 (2), BMS-1 (3), and Foretinib (4) are in clinical or
⁎ Corresponding authors.
E-mail addresses: zhengpw@126.com (P. Zheng), tangqidongcn@126.com (Q. Tang).
d These authors contribute equally to this work.
https://doi.org/10.1016/j.bmcl.2019.126848
Received 25 September 2019; Received in revised form 4 November 2019; Accepted 18 November 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:HuiminLiu,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126848

H. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Representative small-molecule c-Met kinase inhibitors and our previous work.
added and refluxed at 100 °C for 3 h to obtain 13a-h. K2CO3 was mixed
in the above reaction system and the temperature was increased to
150 °C to obtain 14a-h, which were reacted with NaOH in water and
1,4-dioxane to give 15a-h at 100 °C. Acid chloride 16a-h was obtained
in SOCl2 at 80 °C. The reaction of anilines 9a-d with acyl chlorides 16a-
h facilitated by DIPEA in CH2Cl2 yielded the target compounds 17–40.
The anti-proliferative activity of the novel pyrrolo[2,3-b]pyridine
derivatives bearing 4-oxoquinoline moiety was determined through
three parallel experiments by the MTT methods against A549 (human
lung cancer), HepG2 (human liver cancer), MCF-7 (human breast
cancer).21 The results expressed as IC50 values are shown in Table 1.
As shown in Table 1, the target compounds 17–40 showed moderate
to excellent inhibitory activities against the three cancer cells. It was
worth mentioning that eight compounds had stronger anti-proliferative
activity than Foretinib against at least one of the three cancer cells in
vitro, such as compounds 18 (IC50
=
0.39
0.01, IC50 = 0.22
0.29 0.02), 34
0.02,
Fig. 2. The docking mode of Foretinib with c-Met protein.
IC50 = 0.45
0.03), 20 (IC50 = 0.45
0.25 0.03, IC50
0.02, IC50 = 0.27
0.02),
27 (IC50
=
=
(IC50 = 0.29
HepG2 cells.
0.03), against A549 cells and
hydrogen bonding formation in this part on antiproliferative potency.
Small substituent R, R1, and R2 were introduced to investigate their
effects on activity of the target compounds. Accordingly, we designed
and synthesized a novel series of pyrrolo[2,3-b]pyridine derivatives
bearing 4-oxoquinoline moiety.
It was meaningful to find that the anti-proliferative activity of the
most promising compound 3422 (IC50
=
0.29
0.02,
IC50 = 0.27
(IC50 = 0.48
0.03, IC50 = 0.52
0.01, IC50 = 0.35
0.02) was superior to Foretinib
0.01, IC50 = 0.73
0.02)
We selected the A549, HepG2 and MCF-7 cells to test the activity of
the anti-proliferative of the target compounds in vitro.19–20 It was found
that 8 of the target compounds are more effective than Foretinib at least
one cancer cell lines. We selected some target compounds (20, 27, 34
and 35) with good anti-proliferative activity for c-Met kinase activity
test and compound 34 was chosen to further examine the selectivity
against six other tyrosine kinases. In addition, compound 34 was se-
lected for docking analysis to further explain the binding mode.
The key intermediate 4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy) ap-
propriate anilines were achieved from the commercially available 4-
chloro-1H-pyrrolo[2,3-b]pyridine or 4-chloro-1-methyl-1H-pyrrolo[2,3-
b]pyridine. Condensation and reduction reactions were used to give
intermediates 9a-d as shown in Scheme 1, which has been explained in
detail in our previous study.14
against all the three cancer cells (A549, HepG2, and MCF-7, respec-
tively).
As listed data in Table 1, the target compounds had a positive anti-
proliferative activity when R was H instead of CH3, such as the activity
of compound 33 (IC50
=
0.42
0.02, R = H) and 25
(IC50 = 1.72
0.13, R = CH3) on A549 cells, which may because it
retains the ability of H to form hydrogen bonds with Met1160. When R1
was F instead of H, the compound had superior inhibition on cancer
cells. For example, the activities of compound 18 (IC50 = 0.39
0.02,
R1 = H) and 19 (IC50 = 0.65
0.03, R1 = H) were weaker than that
of 34 (IC50 = 0.29
0.02, R1 = F) and 35 (IC50 = 0.32
0.01,
R1 = F) against A549 cells, respectively.
It was found that different substituents in the terminal benzene ring
of the target compounds had a great influence on the anti-proliferative
activity. When R2 was mono-electron-withdrawing groups (mono-
EWGs, such as F and Cl), the anti-proliferative activities were stronger
than that of H, such as the activities between compounds 25
The target compounds 17–40 were prepared as illustrated in
Scheme 2. 2-chloroacetophenone and sodium hydride (NaH) were
added to toluene under the condition of ice-cooling. The mixture was
stirred for 0.5 h. Dimethyl carbonate was added and reacted at 120 °C
for 4 h to give intermediate 11, which was reacted with N,N-di-
methylformamide dimethyl acetal (DMF-DMA) to give 12. The key in-
termediate 12 was dissolved in NMP, and appropriate anilines were
(IC50 = 2.17
0.11, R2 = H) and 27 (IC50 = 0.29
0.02, R2 = F)
against HepG2 cells, which indicated that a mono-EWGs of R2 had a
positive anti-proliferative effect on cancer cells. The same trend could
2

H. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 3. Structures and design strategy of the target compounds.
R2 = OCH3) showed the same trend.
To further explore the specific mechanism of action of target com-
pounds on anti-proliferative, we selected compound 34 by Acridine
Orange (AO) single staining with HepG2 cell line. As shown in Fig. 4,
the cells in the control group (Fig. 4a) were stained with AO, and the
cells were complete. The edges were obvious and evenly dispersed with
no abnormalities. However, in the experimental group (Fig. 4b), most of
the cell morphology was abnormally under the conditions of compound
34 at the concentration of 0.24 μM and AO staining. The nucleus was
decomposed and dispersed. Chromatin was concentrated. This proved
that compound 34 had a strong apoptosis-inducing effect on HepG2 cell
line.
Scheme 1. Reagents and conditions: (i) Diphenyl Ether, 190 °C; (ii) FeCl3,
N2H4-H2O, activated carbon, 80 °C.
be observed in compounds 17/18, 25/26, and 33/34. When R2 were
double electron withdrawing groups, the anti-proliferative activities
against cancer cells slightly reduced. For example, the activities of
To effectively investigate whether the compound 34 is concentra-
tion-dependent, the cells were treated by the MTT method and seven
concentration gradients were selected. After 72 h of interaction, the
inhibition rate was tested. As shown in Fig. 5, the inhibitory effect of
compound 34 was associated with concentrations closely, and com-
pound 34 inhibited the three tumor cells in a concentration-dependent
manner.
compound 37 (IC50
=
1.15
0.03, R2
= 2-F-4-Br) and 38
(IC50 = 1.62
(IC50 = 0.42
0.05, R2 = 2-Cl-4-CF3) were weaker than that of 33
0.02, R2 = H) against A549 cells. At the same time,
compound 21 (IC50
=
1.33
0.03, R2
= 2-F-4-Br) and 22
(IC50 = 2.35
0.20, R2 = 2-Cl-4-CF3) also had weaker activities than
that of compound 17 (IC50 = 1.03
0.21, R2 = H). However, the
introduction of mono-electron-donating groups (mono-EDGs) reduced
the anti-proliferative activities. For example, the activity of compound
It was beneficial to comprehensively summarize the SARs of the
target compounds based on the anti-proliferative activity, and com-
pounds 20, 27, 34, 35 were selected to evaluate their c-Met kinase
inhibitory activities through Caliper mobility shift assay. As listed in
Table 2, these four compounds showed good inhibitory effects on c-Met
kinase. Compound 34 showed a strong inhibitory effect on c-Met with
33 (IC50 = 0.42
0.02, R2 = H) was superior to that of 40
(IC50 = 4.15
0.04, R2 = OCH3). At the same time, compounds 17
(IC50 = 1.03
0.21, R2 = H)/24 (IC50 = 4.98
0.25, R2 = OCH3)
and 25 (IC50 = 1.72
0.13, R2 = H)/32 (IC50 = 3.21
0.19,
Scheme 2. Reagents and conditions: (i) Toluene, dimethyl carbonate; NaH, 120 °C; (ii) DMF-DMA, 120 °C; (iii) Suitable aniline, NMP,100 °C; (iv) NMP, K2CO3,
150 °C (v) NaOH, 1,4-dioxane/H2O, 100 °C, (vi) SOCl2, reflux; (vii) DIPEA, CH2Cl2, r.t.
3

H. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
Anti-proliferative activities of the target compounds 17–40 against the A549, HepG2 and MCF-7 cancer cell lines in vitro.
Compd.
R
R1
R2
IC50 (μM)
A549
HepG2
MCF-7
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Foretinib
H
H
H
H
H
H
H
H
H
F
H
1.03
0.39
0.65
0.45
1.33
2.35
4.81
4.98
1.72
1.46
0.25
1.39
1.52
2.05
1.21
3.21
0.42
0.29
0.32
0.95
1.15
1.62
2.15
4.15
0.48
0.21
2.25
0.45
0.25
0.22
0.79
ND
0.31
0.03
0.01
0.02
0.02
3.56
0.72
1.59
0.61
0.26
0.05
0.01
0.04
a
H
4-F
0.02
0.03
0.01
0.03
0.20
0.21
0.25
0.13
0.15
0.03
0.03
0.05
0.02
0.04
0.19
0.02
0.02
0.01
0.04
0.03
0.05
0.03
0.04
0.01
H
4-Cl
H
4-Br
b
H
2-F-4-Br
2-Cl-4-CF3
3-Cl-4-F
4-OCH3
H
ND
H
3.57
1.51
4.65
2.79
ND
0.25
0.17
0.13
0.15
H
1.12
5.95
2.17
2.01
0.29
1.26
2.51
ND
0.02
0.15
0.11
0.23
0.02
0.03
0.03
H
Me
Me
Me
Me
Me
Me
Me
Me
H
F
4-F
F
4-Cl
2.12
1.83
0.42
0.19
ND
0.31
0.13
0.02
0.03
F
4-Br
F
2-F-4-Br
2-Cl-4-CF3
3-Cl-4-F
4-OCH3
H
F
F
ND
F
3.75
0.76
0.27
0.38
0.71
2.20
1.84
3.39
6.53
0.35
0.13
0.01
0.03
0.21
0.06
0.04
0.02
0.02
0.08
0.01
6.72
0.43
0.52
ND
0.33
0.02
0.02
F
H
F
4-F
H
F
4-Cl
H
F
4-Br
0.65
1.39
1.12
ND
0.04
0.02
0.02
H
F
2-F-4-Br
2-Cl-4-CF3
3-Cl-4-F
4-OCH3
H
F
H
F
H
F
6.62
0.73
0.04
0.02
c
a
b
c
Bold values show the IC50 values of the target compounds lower than the values of the positive control.
ND: Not determined.
Used as the positive control.
Fig. 4. Morphologic changes of HepG2 cells under inverted microscopy and fluorescence microscopy. (5a) Control group cells treated with nothing; (5b)
Experimental group cells treated with 0.24 μM concentration of compound 34.
Table 2
c-Met kinase activity of selected compounds 20, 27, 34, 35
and Cabozantinib in vitro.
Compd.
IC50 on c-Met (nM)
20
10
87
17
41
1.4
27
34
35
Cabozantinib
the IC50 value 17 nM. For investigating the selectivity of compound 34
on c-Met, we took enzymatic selective assays including Flt-3, c-Kit,
VEGFR-2, ALK, PDGFR-β, and RON. As shown in Table 3, compound 34
showed good selectivity on c-Met.
To explore the binding mode of the target compound 34, the three-
dimensional structure of c-Met (PDB code: 3LQ8) was obtained from the
Fig. 5. The relationship between compound 34 with different concentration
and cytotoxicity.
4

H. Liu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 3
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the pyrrole ring in compound 34 formed two hydrogen bonds interac-
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In summary, 24 novel compounds were designed, synthesized and
evaluated for their anti-proliferative activity. 8 compounds were more
effective than Forentinib against at least one cancer cell lines. It is
worth mentioning that compound 34 showed good selectivity on c-Met
over other six tyrosine kinases. Analysis by SARs indicated that the anti-
proliferative activity is obviously improved, when R was H and R2 was
single electron withdrawing groups (such as R2 = F). Furthermore, the
most promising compound 34 has a good interaction with c-Met kinase.
21. Cytotoxicity assay: Standard MTT assay was used to measure the cells growth. Cancer
cell lines were cultured in minimum essential medium (MEM) supplemented with
10% fetal bovine serum. Approximately 4 × 103 cells, suspended in MEM medium,
were plated onto each well of a 96-well plate, and incubated in 5% CO2 for 24 h at 37
°C. 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 then incubated with cells at
37 °C for 4 h. The formazan crystals were dissolved in DMSO (100 mL) of each well,
and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for
the reference wavelength) was measured with ELISA reader. The compounds were
tested in triplicate for each cell line. The results, expressed as IC50 (inhibitory con-
centration 50%), were the average of three determinations and calculated by using
the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
Acknowledgments
We gratefully acknowledge the generous support provided by
National Natural Science Foundation of China (NSFC No. 81660572),
Natural Science Foundation of Jiangxi Province (20192ACBL21009,
20171BAB215071), Top-notch talent project of Jiangxi Science &
Technology Normal University (2016QNBJRC002), and Jiangxi
Provincial Key Laboratory of Drug Design and Evaluation
(20171BCD40015).
22. The procedure for preparation of compound 34: O-chloroacetophenone (3.0 g, 0.155
mol) was added to toluene (20 mL) under the condition of ice water and stirred for
30 min. Dimethyl carbonate (5.2 g, 0.210 mol) was added, and then reacted at 120 °C
for 4 h. The mixed system was adjusted to pH = 6 with glacial acetic acid in water
and extracted with CH2Cl2. The organic phase was dried over Na2SO4 and con-
centrated to give methyl 3-(2-chlorophenyl)-3-oxopropanoate as a light yellow oily
liquid. Methyl 3-(2-chlorophenyl)-3-oxopropanoate (2.66 g, 0.175 mol) and DMF-
DMA (1.88 g, 0.361 mol) were added to toluene (25 mL) and stirred at 120 °C for 10
h. The reaction mixture was poured into water and extracted with CH2Cl2 to give
methyl 2-(2-chlorobenzoyl)-3-(dimethylamino)acrylate as brown oily liquid. Methyl
2-(2-chlorobenzoyl)-3-(dimethylamino)acrylate (3.2 g, 0.267 mol), p-fluoroaniline
(1.9 g, 0.111 mol) were added to NMP, and stirred at 100 °C for 3 h. K2CO3 (2.1 g, 0.
361 mol) was added and the temperature was raised to 150 °C to reacte for 12 h. The
reaction solution was filtered, and isopropyl alcohol (5 mL) and petroleum ether (5
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.126848.
5

H. Liu, et al.
mL) was add to filtrate to give 1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
purified by chromatography on silica gel using MeOH/CH2Cl2 to afford compound
34 as white solid. Yield: 59%. 1H NMR (400 MHz, DMSO-d6) δ 12.51 (s, 1H), 11.79
(s, 1H), 8.73 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.05 (dd, J = 11.7, 9.1 Hz, 2H), 7.84 –
7.73 (m, 3H), 7.56 (dd, J = 9.7, 8.1 Hz, 4H), 7.38 (s, 2H), 7.10 (d, J = 8.5 Hz, 1H),
6.40 (d, J = 5.3 Hz, 1H), 6.28 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 176.78, 163.
12, 157.82, 151.64, 148.95, 144.76, 141.33, 137.54, 137.25, 134.15, 130.64, 130.
55, 126.93, 126.67, 126.41, 125.50, 124.47, 119.03(2C), 118.01, 117.78, 116.
96(2C), 111.19, 110.10, 109.25, 109.03, 101.46, 97.43; MS m/z (ESI): 508.13 [M
+H]+; Anal. calcd. for C29H18F2N4O3 (%): C, 68.50; H, 3.57; N, 11.02; Found (%): C,
68.51; H, 3.49; N, 11.19.
carboxylate as a yellow solid, which was dissolved to 1,4-dioxane (15 mL) and H2O
(15 mL) and added NaOH (0.6 g, 0.146 mol). The mixture was reacted at 100 °C for 2
h, and then concentrated. NaCl (aq) was added, and adjusted to pH = 4 with HCl
(37.5%). Yellow solid of 1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid was obtained through filter, which was stirred in SOCl2 at 80 °C for 1 h to give 1-
(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carbonyl chloride as a yellow viscous
oil. 4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluoroaniline were synthesized ac-
cording our previously reported procedures in Ref. 14. Reaction of aromatic amine
with acyl chloride catalyzed by DIPEA in CH2Cl2 to give crude product, which was
6