Synthesis and biological evaluation of novel oxindole-based RTK inhibitors as anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2014.10.017

Given that receptor tyrosine kinases (RTKs) have emerged as key regulators of all aspects of cancer development, including proliferation, invasion, angiogenesis and metastasis, the RTK family represents an important therapeutic target for anti-cancer drug development. Oxindole structure has been used in RTK inhibitors such as SU4984 and intedanib. In this study, two series of new heterocyclic compounds containing oxindole scaffold have been designed and synthesized, and their inhibitory activity against the proliferation of nine cancer cell lines has been evaluated. Among them, compounds 9a and 9b displayed the strongest anti-proliferative activity with the IC₅₀s below 10 μM. Flow cytometric analysis showed that the compounds 9a and 9b dose-dependently arrested the cell cycle at G0/G1 phase. Although the leading compounds SU4984 and intedanib targets FGFR1, the kinase activity test revealed that these compounds only showed slight inhibitory activity on FGFR1 kinase. Further enzymatic test aided by molecular docking simulation in the ATP-binding site demonstrated that 9a and 9b are potent inhibitors of c-Kit kinase. These compounds are worthy of further evaluation as anticancer agents.

Full text of DOI:10.1016/j.bmc.2014.10.017

Bioorganic & Medicinal Chemistry 22 (2014) 6953–6960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel oxindole-based RTK
inhibitors as anti-cancer agents
⇑
Gaozhi Chen , Qiaoyou Weng , Lili Fu, Zhe Wang, Pengtian Yu, Zhiguo Liu, Xiaokun Li, Huajie Zhang ,
Guang Liang
⇑
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Given that receptor tyrosine kinases (RTKs) have emerged as key regulators of all aspects of cancer devel-
opment, including proliferation, invasion, angiogenesis and metastasis, the RTK family represents an
important therapeutic target for anti-cancer drug development. Oxindole structure has been used in
RTK inhibitors such as SU4984 and intedanib. In this study, two series of new heterocyclic compounds
containing oxindole scaffold have been designed and synthesized, and their inhibitory activity against
the proliferation of nine cancer cell lines has been evaluated. Among them, compounds 9a and 9b dis-
Received 27 July 2014
Revised 13 October 2014
Accepted 14 October 2014
Available online 29 October 2014
Keywords:
played the strongest anti-proliferative activity with the IC50s below 10 lM. Flow cytometric analysis
Anticancer drug
Chemical synthesis
Drug design
Oxindole
Receptor tyrosine kinase inhibitor
showed that the compounds 9a and 9b dose-dependently arrested the cell cycle at G0/G1 phase.
Although the leading compounds SU4984 and intedanib targets FGFR1, the kinase activity test revealed
that these compounds only showed slight inhibitory activity on FGFR1 kinase. Further enzymatic test
aided by molecular docking simulation in the ATP-binding site demonstrated that 9a and 9b are potent
inhibitors of c-Kit kinase. These compounds are worthy of further evaluation as anticancer agents.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
the FDA has approved ten protein kinase inhibitors as drugs for
the treatment of different types of cancer, including both solid
Cancer is continuing to harm human health both in developed
and developing countries.1–2 Surpassing heart disease, it has
become the worldwide number one killer. Several anticancer
agents, such as indomethacin,3 taxol,4 5-fluorouracil,5 vinblastine,6
camptothecin and its derivatives (topotecan and irinotecan),7 eto-
poside8 and mitoxantrone9–10 are used clinically around the world.
However, the usage of these drugs has been linked with various
side effects, such as bone marrow suppression, gastrointestinal
toxicity, low blood pressure, hair loss and constipation. Therefore,
new chemical entities for the effective and safe cure of cancer are
still an active area of research in the scientific community.
Protein kinases are critical components of cellular signal trans-
duction cascades. They form one of the most important target
classes in drug development, as they are directly involved in many
diseases, including cancer.11–12 Therefore, small molecule kinase
inhibitors have been successfully introduced to the drug market
as a selective anticancer agents with few side effects.13–14 To date,
and non-solid tumors.15 Over 300 kinase inhibitors are currently
in clinical development, and many more are at the preclinical study
stage of development. The vast majority of these compounds are
reported to target the kinase ATP binding site, and more than 500
protein kinases identified in the human genome have an ATP
binding site.16 Small molecule kinase inhibitors achieve their inhib-
itory activity against kinases by thoroughly mimicking the binding
mode of the ATP molecule. Therefore, the design of small molecule
kinase inhibitors based on the characteristics of the ATP binding
site is a rational approach to the discovery of anti-cancer agents.
Fibroblast growth-factor receptors (FGFRs) form a sub-family of
the receptor tyrosine kinase (RTK) superfamily, and are encoded by
four genes, FGFR1, FGFR2, FGFR3, and FGFR4. FGFRs are involved in
the regulation of cell proliferation and migration, angiogenesis,
organ development and other processes. Studies have confirmed
that the activation and overexpression of FGFR results in carcino-
genic functions in cells, including excessive proliferation and the
prevention of apoptosis, and are found to be closely related to
the development and progression of tumors in humans.17–18 Con-
sidering the prominent role of FGFRs in cancer development, it
would be desirable to develop potent FGFR inhibitors.
⇑
Corresponding authors. Tel.: +86 13868888091 (H.Z.); tel./fax: +86 577
86699396 (G.L.).
E-mail addresses: HuajieZhang2014@163.com (H. Zhang), wzmcliangguang@
163.com (G. Liang).
Oxindole-based chemical scaffolds have been broadly used
as protein kinase inhibitors because oxindoles generally form
These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.10.017
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

6954
G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
hydrogen bonds with the backbone of the hinge segment of the
kinase ATP binding domain.19 For instance, intedanib20 acts as a
triple kinase inhibitor with inhibitory activity against VEGFR, FGFR
and PDGFR kinases, resulting in suppressed vascularization and
proliferation of tumor tissues causing starvation and death of
tumor cells. SU666821 is a another triple kinase inhibitor of VEGFR,
FGFR and PDGFR kinases and SU4984 was reported to inhibit
stored over 4A molecular sieves. All starting materials and reagents
were commercially available. Unless otherwise noted, chemicals
were obtained from local suppliers and were used without further
purification. All reactions were monitored by thin-layer chroma-
tography (silica gel 60 F254 glass plates). NMR spectra were
recorded on Bruker 600 MHz instruments, and the chemical shifts
were presented in terms of parts per million with TMS as the inter-
nal reference. Electron-spray ionization mass spectra in positive
mode (ESI-MS) data were obtained with a Bruker Esquire 3000+
spectrometer. Column chromatography purifications were carried
out on Silica Gel 60 (E. Merck, 70–230 mesh).
FGFR1 specifically in 199722 with a moderate IC50 of 10–20
lM.
Inspired by the potential inhibitory ability, an oxindole scaffold
has been chosen for further structural modification.
The ATP binding pocket of kinase is located in the cleft between
the N- and C-lobes of the kinase, and is composed of residues from
the N- and C-lobes, the nucleotide-binding loop, and the hinge
region that connects the two lobes.17 Common pharmacophores of
oxindole-based kinase inhibitors usually divided into three main
categories. First, the oxindole of the inhibitors forms a hydrogen
bond with two residues from the hinge region and binds in a similar
fashion to the ATP’s purine ring (this part is denoted by a red rectan-
gle in Fig. 1). Second, the methylpyrrole of SU6668 and the 3-phenyl
ring of SU4984 and Intedanib form van der Waals interactions with
hydrophobic groups present in a hydrophobic pocket located
outside of the ATP binding pocket (the hydrophobic groups of com-
pounds are marked by a blue rectangle in Fig. 1). The third feature is
a nucleotide-binding domain which is located adjacent to the
hydrophobic domain. The long-chain carboxyl group of SU6668
and the piperazinecarboxylate moiety of SU4984 and intedanib
form a hydrogen bond with the amino acid residue in the nucleo-
tide-binding domain (this part is marked by green ovals in Fig. 1).17
Two series of indolin-2-one were designed based on the struc-
ture of SU4984 and the structural features of the ATP-binding
domain, and three pharmacophores were modified according to
the mechanisms of interaction between the group and ATP binding
site in order to determine the most effective kinase inhibitor with
the greatest affinity for the ATP binding site. The main difference
between two series compound is the linker between the oxindole
and the hydrophobic groups.
2.1.2. General procedure for the synthesis of compounds 1a–1g,
2a, 3a
Various aldehydes (1.0 equiv) were added to stirred solutions of
indolin-2-one, 5-chloroindolin-2-one or 7-azaoxindole (1.0 equiv)
in absolute ethanol. After stirring at room temperature for 5 min
NaOEt/EtOH (0.5 mL) was added and the mixture was then stirred
at room temperature overnight. The solvent was then removed
under vacuum. The residue was washed with saturated sodium
chloride solution and then extracted with ethyl acetate. The
organic layer was dried over anhydrous magnesium sulfate and
concentrated under vacuum. The solid part was purified by chro-
matography over silica gel using ethyl acetate/petroleum ether as
the eluent to afford desired compounds 1a–1g, 2a, 3a.
2.1.3. Synthesis of intermediate 4
A reaction mixture of 4-fluorobenzaldehyde (1.0 equiv) and
piperazine (3.0 equiv) in 2-methoxyethanol was stirred at 110 °C
for 3–5 h. The solvent was then removed under vacuum. The resi-
due was washed with saturated sodium chloride solution and
filtrated. The solid part was purified by chromatography over silica
gel using ethyl acetate/methanol as the eluent to generate
intermediate 4.
2.1.4. Synthesis of Intermediates 5 and 6
Intermediate 4 (1.0 equiv) was added to a stirred solution of
indolin-2-one or 7-azaoxindole (1.0 equiv) in absolute ethanol.
After stirring at room temperature for 5 min, NaOEt/EtOH
(0.5 mL) was added, and then the mixture was stirred at room tem-
perature overnight. The solvent was then removed under vacuum.
The residue was washed with saturated sodium chloride solution
and filtrated. The solid part was purified by chromatography over
silica gel using ethyl acetate/methanol as the eluent to generate
intermediates 5 and 6.
2. Material and methods
2.1. Chemical synthesis
2.1.1. General information
Solvents were distilled under the positive pressure of dry argon
before use and dried using standard methods. Acetonitrile (MeCN)
and Tetrahydrofuran (THF) were distilled over calcium hydride and
2.1.5. Synthesis of compounds 7a and 8a
O
A reaction mixture of intermediate 5 or 6 in ethyl formate was
stirred at 50 °C overnight. The solvent was then removed under
vacuum. The residue was purified by chromatography over silica
gel using ethyl acetate/petroleum ether as the eluent to afford
desired compounds 7a and 8a.
N
N
O
N
OH
N
N
O
NH
O
N
H
O
O
N
H
O
N
H
N
2.1.6. General procedure for the synthesis of compounds 9a–9c
and 10a–10c
H
O
SU4984
SU6668
Intedanib(BIBF1120)
Acryloyl chloride, 3-chloropropionyl chloride or Benzoyl chlo-
ride (1.1 equiv) was added to a stirred solution of intermediate 5
or 6 (1.0 equiv) in Tetrahydrofuran (THF). After stirring at room
temperature for 5 min, Et3N (0.5 mL) was added and the mixture
was then stirred at room temperature for 2–4 h. The solvent was
then removed under vacuum. The residue was washed with satu-
rated sodium chloride solution and then extracted with ethyl ace-
tate. The organic layer was dried over anhydrous magnesium
sulfate and concentrated under vacuum. The solid was purified
by chromatography over silica gel using ethyl acetate/petroleum
ether as the eluent to afford desired compounds 9a–9c, 10a–10c.
R'
R'
N
R
O
O
R
N
H
N
H
X
Indole-2-one and 7-aza-2-oxindole derivatives
Figure 1. Structural skeleton and chemical structures of Oxindole-based protein
kinase inhibitors and the designed compound in this paper.

G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
6955
2.1.7. Synthesis of intermediates 11
reaction mixture of 6-chloro-3-nitropyridin-2-amine
over silica gel using ethyl acetate/petroleum ether as the eluent
to afford desired compounds 21a and 21b.
A
(1.0 equiv), morpholine (1.0 equiv) and K2CO3 (3.0 equiv) in MeCN
was stirred at 80 °C for 3–5 h. The mixture was extracted with
CH2Cl2 and the organic layer washed with brine, dried over anhy-
drous magnesium sulfate, and concentrated under vacuum. The
solid part was purified by chromatography over silica gel using
ethyl acetate/petroleum ether to generate intermediate 11.
The spectral data of new or unreported compounds are shown
in Supplementary materials.
2.2. Cell lines
All the cell lines including Human lung adenocarcinoma cell
line H1975, Human glioma cell line U251, Human hepatoma cell
line Bel7402, Human non-small cell lung cancer cell line HCC827,
Human lung tumor cell lines H460, Human breast cancer cell line
SK-BR-3, Human ovarian cancer cell linePM8910, Human gastric
cancer cell line SGC-7901, Human non-small cell lung cancer cell
line A549 and Normal rat kidney cell line NRK-52E were purchased
from Cell Bank (Shanghai Institutes for Biological Sciences, CAS,
Shanghai, China).
2.1.8. Synthesis of intermediates 13
A reaction mixture of 3-chloro-4-nitrobenzoic acid (1.0 equiv)
and H2SO4 in absolute ethanol was stirred at 80 °C for 3–5 h. The
mixture was extracted with ethyl acetate and the organic layer
washed with brine, dried over anhydrous magnesium sulfate, and
concentrated under vacuum. The solid part was purified by chro-
matography over silica gel using ethyl acetate/petroleum ether to
generate intermediate 13.
2.3. Cell proliferation assay
2.1.9. Synthesis of intermediates 14 and 17
A reaction mixture of intermediate 13 (1.0 equiv), morpholine
or 3,5-dimethoxyaniline (1.0 equiv) and K2CO3 (3.0 equiv) in MeCN
was stirred at 80 °C for 3–5 h. The mixture was extracted with
CH2Cl2 and the organic layer washed with brine, dried over anhy-
drous magnesium sulfate, and concentrated under vacuum. The
solid part was purified by chromatography over silica gel using
ethyl acetate/petroleum ether to generate intermediate 14 and 17.
H1975, U251, Bel7402, HCC827, H460, SK-BR-3, PM8910, SGC-
7901 and A549 cell lines (4 Â 104 cells/ml) were cultured and
seeded into 96-well plates. After overnight incubation, the cells
were incubated with compounds at 10 lM dissolved in DMSO at
37 °C in an atmosphere of 5% CO2 for 72 h. The control samples
received the same volume of DMSO. Cell viability was determined
by using an MTS assay (Promega, San Luis Obispo, CA). Briefly, MTS
dye solution was added and incubated in a CO2 incubator for 4 h.
Absorbance was measured by using the spectraMax M2 microplate
reader (MolecularDevices, Sunnyvale, CA) at 490 nm and the
results are expressed as the inhibition of cell proliferation calcu-
lated as the ratio [(1 À (OD490 treated/OD490 control)) Â 100] in
triplicate experiments. For the determination of the IC50 [50% inhi-
bition of cell proliferation], cells were incubated with 9a and 9b at
2.1.10. Synthesis of intermediates 16
A solution of the intermediate 14 (1.0 equiv) in absolute ethanol
was added to a solution of 20% NaOH and stirred at 80 °C for 3–5 h.
The ethanol was then removed under vacuum. The HCl (aq) was
dropped into the residual solution until the pH of the solution
was equal to 7. The solid part was filtered to generate intermediate
16.
concentrations of 30 lM, 10 lM, 3 lM, 1 lM, 0.3 lM, and 0.1 lM
in separate triplicate experiments for 72 h following the protocol
2.1.11. Synthesis of intermediates 12, 15 and 18
mentioned before.
Sodium dithionite (3 equiv) was added to a solution of interme-
diate 11, 14 or 17 (1.0 equiv) in a mixture of ethanol (20 mL) and
water (4 mL). The reaction mixture was refluxed at 90 °C for
2–3 h then cooled to room temperature. The reaction mixture
was diluted with water (40 mL) and then extracted with ethyl ace-
tate. The combined organic layers were washed with brine, dried
over anhydrous magnesium sulfate, and concentrated under vac-
uum. The solid part was purified by chromatography over silica
gel using ethyl acetate/petroleum ether as the eluent to generate
intermediates 12, 15 and 18.
2.4. Kinase inhibition assays
The kinase inhibitory activities were determined using a micro-
fluidic assay (Caliper Mobility Shift Assay) that monitors the sepa-
ration of a phosphorylated product from its substrate. Briefly, the
enzyme, substrate, ATP and compound were mixed in a 384-well
assay plate for the phosphorylation reaction. After 1 h incubation,
EDTA was added to stop the reaction. The plate was read on an
EZ Reader II (Caliper Life Sciences, MA). The percentage conversion
of substrate into the phosphorylated product was generated auto-
matically and the percentage inhibition was calculated relative to
blank wells (containing no enzyme and 2.5% (v/v) DMSO) and total
wells (containing all reagents and 2.5% (v/v) DMSO). The recombi-
nant kinases, including FGFR1, EGFR, c-Kit, PDGFR-b, KDR, AUR-B
and SGK1, were purchased from Carna (Chuo-ku, Kobe, Japan).
The ATP concentration was set at the Km values of FGFR1:
2.1.12. General procedure for the synthesis of intermediate 20
and compounds 19a–19I
A reaction mixture of the various substituted amines, hydrazine
or intermediate 12, 15, 18 (1.0 equiv) and 5-chloroisatin
(1.0 equiv) in methanol was stirred at 60 °C for 3–5 h. The solvent
was then removed under vacuum. The residue was washed with
saturated sodium chloride solution and filtrated. The solid
was purified by chromatography over silica gel using ethyl
acetate/petroleum ether as the eluent to afford intermediate 20
and desired compounds 19a–19I.
262
92
in duplicate at 3 concentrations (10, 1.0, and 0.1
tested from 10 M, 3-fold dilution.
lM, EGFR: 2.3
lM, c-Kit: 20
l
l
M, PDGFR-b: 38
M. Compounds were tested
M). IC50 was
lM, KDR:
l
M, AUR-B: 15 M and SGK1: 38
l
l
l
2.1.13. Synthesis of compounds 21a and 21b
2.5. Cell cycle analysis
A reaction mixture of EDCÁHCl (1.1 equiv) and HOBt (1.3 equiv)
was stirred with intermediate 16 or furan-2-carboxylic acid
(1.0 equiv) at 0–8 °C in CH2Cl2. After 10 min, intermediate 20 was
added, and the mixture was stirred at 0–8 °C for 1 h before being
stirred at room temperature overnight. The residue was washed
with saturated NaHCO3 solution and extracted with CH2Cl2. After
solvent evaporation, the solid was purified by chromatography
Cell cycle distribution was determined by flow cytometric anal-
ysis of PI-stained nuclei. DNA content was determined by FACScan
flow cytometer and CellQuest software (BD Biosciences, San Jose,
CA). Briefly, PM8910, SGC7901 and A549 cells were seeded in
60 mm plates .In the following days, cells were treated with the
desired doses of 9a and 9b for 24 h. Cells were washed twice with

6956
G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
PBS, fixed in ice-cold ethanol (75% vol/vol in water), and stained
with the PI solution (25 mg/ml PI, 180 U/ml RNase, 0.15% Triton
X-100, and 30 mg/ml polyethylene glycol in 4 mmol/l citrate buf-
fer, pH 7.8; all from Sigma–Aldrich, St. Louis, MO) overnight at 4 °C.
to afford compound 13. Compound 13 and 6-chloro-3-nitropyri-
din-2-amine were treated with morpholine or 3,5-dimethoxyani-
line with potassium carbonate in a solution of acetonitrile to
provide intermediates 11, 14 and 17. Reduction of the nitro com-
pounds (11, 14 and 17) to the corresponding amino intermediates
(12, 15 and 18) was achieved using sodium dithionite in a solution
of ethanol–water. Compounds 19g–19h were prepared by the
reaction of commercially available 5-chloroisatin and intermedi-
ates 12, 15 or 18 with a yield of 45–75%.
For the synthesis of compounds 21a and 21b (Scheme 5), inter-
mediate 16 was first produced through the hydrolysis of 14 in
sodium hydroxide solution, and intermediate 20 was obtained
from 5-chloroisatin and hydrazine. One-step amidation was intro-
duced to produce compounds 21a (56%) and 21b (76%) using
furan-2-carboxylic acid and intermediate 16 with compound 20
under the catalytic condition of EDC-HCl and HOBt. The structures
of all compounds were characterized using 1H NMR, 13C NMR and
electrospray ionization mass spectrometry (ESI-MS). Before used in
biological experiments, all synthetic compounds were purified by
recrystallization or silica gel column chromatography, and HPLC
method was used to determine their purity (>95%).
2.6. Molecular docking simulation
All three-dimensional structures of compounds were built using
the SYBYL-X suite. Energy minimizations of small molecules were
performed by the Tripos force field and the Gasteiger–Hückel
charge with a distance-dependent dielectric, and the Powell conju-
gate gradient algorithm with a convergence criterion of 0.05 kcal/
(mol  Å). Molecule docking simulations of compounds with
c-Kit kinase protein (PDB ID: 3G0E) were carried out with the Sur-
flex-Dock program interfaced with SYBYL. The ligand-binding
groove on c-Kit was kept rigid, whereas all torsible bonds of com-
pounds were set free to allow flexible docking to produce more
than 100 structures. Final docked conformations were clustered
within the tolerance of 1 Å root-mean-square deviation.
2.7. Statistical analysis
The results were presented as means SEM. Student’s t-test
was employed to analyze the differences between sets of data. Sta-
tistics were performed using GraphPad Pro (GraphPad, San Diego,
CA). P values less than 0.05 (p <0.05) were considered indicative
of significance. All experiments were repeated at least three times.
3.2. Anti-proliferative activities assay
The in vitro anti-proliferative activities of the synthesized com-
pounds against nine human cancer cell lines (H1975, A549,
SGC7901, U251, Bel7402, HCC827, H460, PM8910 and SK-BR3)
were evaluated using an MTS assay at a compound concentration
of 10 lM (Table S1).
3. Results and discussion
3.1. Chemistry
Compounds 9a and 9b displayed broad spectrum anti-prolifer-
ative activity against various cell lines with a mean growth inhibi-
tions of 51.77% and 50.93%, respectively at a concentration of
10 lM. Other compounds showed low or modest activities. Based
The common synthetic route and structures of compounds
1a–1g, 2a and 3a are outlined in Scheme 1. Compounds were
synthesized by the conventional refluxing ethanol-water solution
of indole-2-ones and substituted benzaldehyde in basic conditions
with a yield ranging from 30% to 80%. The synthesis of compounds
7a, 8a, 9a–9c and 10a–10c began with the preparation of the key
intermediate 4-(piperazin-1-yl) benzaldehyde 4 from 4-fluoro-
benzaldehyde and piperazine in one step (Scheme 2). Indole-2-
one or 7-aza-2-oxindole was refluxed with intermediate 4 in basic
conditions to afford intermediate 5 or 6. Compounds 7a and 8a
were obtained through the general refluxing of intermediate 5 or
6 with ethyl formate, and compounds 9a–9c and 10a–10c were
synthesized by electrophilic substitution reaction of intermediate
5 or 6 with acyl chloride under the catalytic condition of triethyl-
amine (yield 30–50%).
For the synthesis of the amine linked indole-2-one series com-
pounds, 19a–19i and 21a–21b, reasonable synthetic routes were
designed as shown in Schemes 3–5. Mixed reflux of commercially
available 5-chloroisatin and substituted amines in methanol were
applied to produce compounds 19a–19f (yield 67–83%, Scheme 3).
According to Scheme 4, intermediates 12, 15 and 18 were prepared
first, in order to generate compound 19g–19i. 3-chloro-4-nitroben-
zoic acid was esterified by ethanol in the presence of sulfuric acid
on the promising results of compounds 9a and 9b, these com-
pounds were selected for five dose tests against the 7 cell lines.
The half maximal (50%) inhibitory concentration (IC50) of these
compounds was also measured. Compound 9a displayed cellular
activity at concentrations of 6–28
all the cell lines of 10.7 M. This compound inhibited six types of
cancer cell with an IC50 of 8 M, and showed a high IC50 value of
28 M towards the normal kidney cell line, NRK-52E. Similarly,
compound 9b also exhibited potential biological activity at con-
centrations from 2 M to 30 M with a mean IC50 against all the
cell lines at 9.7 M. Compound 9b inhibited six types of cancer cell
with an IC50 of 6.8 M, and more than 30 M towards the normal
lM, with a mean IC50 against
l
l
l
l
l
l
l
l
cell line, NRK-52E. Table 1 shows the IC50 of compounds 9a and 9b
against those the cell lines. These results indicate that compounds
9a and 9b have potential anti-proliferative activities against cancer
cell lines, but low activities towards normal cells. However, the
exact mechanism of action is still unknown.
3.3. Enzyme activity inhibition assay
In order to evaluate the activity of our designed compounds as
kinase inhibitors, kinase inhibition assay was carried out using the
Scheme 1. Synthetic pathway for the new indole-2-ones and 7-aza-2-oxindoles 1a–1g, 2a, 3a.

G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
6957
Scheme 2. Synthetic route for the new indole-2-ones and 7-aza-2-oxindoles 7a, 8a, 9a–9c, 10a–10c.
Scheme 3. Synthetic step for the new amine linked indole-2-ones 19a–19f.
Scheme 4. Synthetic milestone for the new amine linked indole-2-ones 19g–19i.
Caliper Mobility Shift Assay. The percentage inhibition of FGFR1
tyrosine kinase enzymatic activity caused by our compounds was
evaluated against a reference kinase inhibitor, staurosporine, at
an IC50 of 9.7 nM. Unfortunately, the compounds showed weak to
moderate inhibitory activity against the FGFR1 kinase, as shown
in Table 2. It is noteworthy that compound 7a, which exhibited
the highest inhibition of FGFR1 shared the same structure with
the FGFR1inhibitor named SU4984. These data imply that the
cellular activity of active compounds was not due to the inhibition
of FGFR1.
Compounds 9a and 9b showed excellent cellular activity
against the tested cancer cell lines. Since 9a and 9b showed low
inhibition of FGFR1, they were tested against five other kinases
at a dose of 0.1, 1, and 10 lM (Table 3). We selected these kinases
from different kinase families whose inhibitors share some struc-
tural similarities with our lead compound, Intedanib. This assay
was used to determine whether compounds 9a and 9b have an
unanticipated target activity that may be responsible for their high
cellular anti-proliferative activity. Compounds 9a and 9b showed a
superior inhibitory activity against Aurora B kinase at 10 lM but

6958
G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
Scheme 5. Synthetic milestone for the new amine linked indole-2-ones 21a–21b.
Table 1
Table 3
MTS testing results of compound 9a and 9b against representative cell lines
Inhibition rates of compound 9a and 9b against some kinases
Cell name
Compound (IC50
,
lM)
Kinase
% Inhibition of compd 9a (
lM)
% Inhibition of compd 9b (lM)
9a
6.74 0.15
7.00 0.98
9.75 0.49
7.68 0.32
10.13 0.28
11.50 1.65
28.78 0.68
9b
10
1
0.1
10
1
0.1
PM8910
HCC827
SGC7901
Bel7402
A549
2.76 2.83
12.66 1.4
2.85 0.21
3.39 4.13
3.04 0.18
20.05 13.67
>30
c-Kit
88.2
PDGFRb 61.8
55.3
26.2
7.2
41.6
38.3
30.1
19.1
0.4
13.4
4.5
2.2
14.9
57.6
76.0
62.6
88.3
42.4
24.3
56.4
46.7
14.7
53.5
11.8
20.0
31.9
13.6
6.0
14.4
3.56
19.1
KDR
30.6
93.5
90.7
75.4
Aur B
SGK1
EGFR
H460
NRK-52E
Table 2
Enzyme inhibition assay of the synthesized compounds against FGFR1 at 10
Table 4
lM
IC50 of compounds 9a and 9b against Aur B and c-Kit
Compound % Inhibition of FGFR1 at Compound % Inhibition of FGFR1 at
10 10
Kinase
IC50 of 9a (nM)
IC50 of 9b (nM)
lM
lM
c-Kit
128
71
1a
1b
1c
1d
1e
1f
1g
2a
3a
7a
8a
9a
9b
9c
53.2
38.4
6.7
10.9
6.4
10a
10b
10c
19a
19b
19c
19d
19e
19f
19g
19h
19i
21a
21b
22.24
30.3
49.3
Aur B
2205
587
À28.85
indolinone scaffold, and many c-Kit inhibitors are indolinone based
compounds.29 The high inhibitory activity exhibited by compounds
9a and 9b on c-Kit kinase may explain their excellent antiprolifer-
ative profiles against the tested cancer cell lines.
4.8
3.11
9.3
0.58
0.36
21.8
16.6
46.0
8.1
72.5
49.1
48.2
47.4
65.0
16.6
3.4. Cell cycle analysis
À14.8
À1.42
À16.8
11.4
To further research the mechanism responsible for the anti-pro-
liferative effect of compounds 9a and 9b in addition to enzyme
inhibition assay, their apoptotic effects on SGC7901, PM8910 and
A549 was evaluated. Compounds 9a and 9b were incubated in cul-
tured cancer cells for 24 h and then analyzed in order to predict the
phase of the cell cycle at which the growth of cells was inhibited.
showed highest inhibitory effects on c-Kit at lower concentration.
The IC50 of two compounds towards Aur B and c-Kit were further
determined. The result shows that 9a and 9b inhibit Aur B kinase
at the IC50 of 2205 nM and 587 nM, while inhibit c-Kit kinase with
the IC50 of 128 nM and 71 nM, respectively (Table 4). Thus, 9a and
9b showed about 10-fold stronger inhibition against c-Kit than Aur
B and exhibited modest results on the other tested kinases. C-Kit
kinase is one of the receptor tyrosine kinase which can activates
the mitogen-activated protein kinases (MAPKs) and phosphoinosi-
tide-3-kinase(PI3 K) pathway and regulates the gene expression,
cell growth, proliferation and differentiation.23–24 Both the expres-
sion level and the kinase activity of c-Kit kinases were found
to be up-regulated in many human cancers, suggesting a role
for use of pharmacologic inhibitors of c-kit in the treatment of
c-Kit-dependent malignancies.25–26 There are many compounds
that target c-Kit kinase that are now in different stages of clinical
testing for the treatment of many types of tumors.27–28 Fortu-
nately, it was reasonable for 9a and 9b to have inhibitory
activity on the c-Kit enzyme as they were designed based on the
MTS data showed that 9a had an IC50 of 9.75
6.74 M on PM8910, and 10.13 M on A549, and 9b has an IC50
of 2.85 M on SGC7901, 2.76 M on PM8910, and 3.04 M on
lM on SGC7901,
l
l
l
l
l
A549, which are considered as suitable values of compound con-
centration for cell cycle analysis. Figure 2 shows the cell cycle anal-
ysis results obtained after treatment of SGC7901, PM8910 and
A549 cells with 9a or 9b for 24 h (Gefitinib was used as a positive
control). Inspection of the results showed that the cells have halted
in the G1 phase and their transformation into the S phase has
stopped relative to the negative control (Fig. 2). This implies the
likelihood that c-Kit kinase was involved in the anti-proliferative
activity of compounds 9a and 9b.
3.5. Docking study
In order to predict the possible binding mode of compounds 9a
and 9b inside the binding site of c-Kit kinase, compounds were

G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
6959
A
B
C
100
*
100
100
90
80
70
60
50
90
90
80
70
60
50
**
*
**
80
*
*
70
***
**
**
*
***
60
50
M
µ
.5
-3
9a
M
M
M
M
M
7
µM
µ
µ
µ
µ
-
M
M
M
µ
M
M
M
M
0µ
µM
µ
µ
ib
in
-7
9a
-3
9b
-6
9b
µ
µ
µ
10
0
0µ
-1
9a
3
-1
DMSO
Gef
-5
9a
-6
9b
-5
9a
-3
9b
-6
9b
b-
b
i
-1
9a
i
b-
it
DMSO
DMSO
i
n
n
9
i
ti
t
i
f
e
G
Gef
PM8910
SGC7901
A549
Figure 2. Cell cycle analysis of active compounds 9a and 9b. PM8910, SGC7901 and A549 cells were planted in 60 mm plates. In the following days, cells were treated with
desired doses of 9a and 9b for 24 h. Cells were washed twice with PBS, fixed in ice-cold ethanol (75% vol/vol in water), and stained with the PI solution (25 mg/ml PI, 180U/ml
RNase, 0.15%Triton X-100, and 30 mg/ml polyethylene glycol in 4 mmol/l citrate buffer, pH7.8; all from Sigma–Aldrich, St. Louis, MO) overnight at 4 °C. The results were
presented as the percent of G0/G1 phase. Each bar represents mean SEM of three independent experiments. Statistical significance relative to DMSO group was indicated,
⁄p < 0.05, ⁄⁄p < 0.01.
A
B
N
O
N
H
N
H
F
O
Sunitinib
N
H
D
C
Figure 3. Chemical structure of c-Kit kinase inhibitor sunitinib (A) and Molecular docking simulation of sunitinib (B), compound 9a (C) and 9b (D) inside the ATP binding
pocket of c-Kit enzyme.
docked into the ATP binding site of c-Kit kinase (PDB ID:3G0E)
using Sybyl-2.0 software.
Sunitinib is an oxindole c-Kit kinase inhibitor developed by Pfizer
(Fig. 3A). The cocrystallization of sunitinib fitting into the ATP bind-
ing site of c-Kit kinase indicated that the NH and O atoms of the
sunitinib dihydroxyindole ring system make up the donor–acceptor
motif that is frequently part of kinase inhibitors and participates in
hydrogen-bonding interactions with the backbone amides in the
interlobe hinge region of the protein. In detail, the O atoms on the
oxindole ring does form hydrogen bond with Cys673, and the NH

6960
G. Chen et al. / Bioorg. Med. Chem. 22 (2014) 6953–6960
atoms of the oxindole ring makes another hydrogen bond with
Glu671 (Fig. 3B). Comparing the binding mode of 9a and 9b with
the sunitinib illustrated that the oxindole moiety established hydro-
gen bonds with Glu671 and Cys673 of the hinge residue and form
another hydrogen bond with Thr 670. In addition, the carbonyl of
the piperazine form another two hydrogen bonds with Lys593 and
Glu758 which make the chemical 9a and 9b inhibit the c-Kit kinase
strongly (Fig. 3C and D). The docking of c-Kit kinase domain with all
presented compounds was also performed. The docking scores of all
compounds in c-Kit kinase domain were shown in Table S2. Based
on the docking score, 9a and 9b are predicted as the two strongest
c-Kit inhibitors among these compounds, which is consistent with
the experimental data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.017.
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In summary, we synthesized several derivatives of indole-2-one
and screened their anti-proliferative activities against nine human
cancer cell lines. Several compounds showed significant suppres-
sion of the proliferation of the cancer cells. In particular, com-
pounds 9a and 9b exhibited strong activity against many cancer
cell lines and showed a broad spectrum of anticancer properties.
Since 9a and 9b showed low inhibition of FGFR1, they were tested
against five other kinases, and showed significant inhibition of
receptor tyrosine kinase, c-Kit. In addition, evidence of kinase
inhibition was implicated by cell cycle analysis as the 9a and
9b arrested the cell cycle in the G1 phase and the computer
simulation also exhibited a positive result. Further studies should
include detailed examination of the structure–activity relationship
of these compounds, considering cell line inhibitions as well as the
determination of the exact cellular targets of the interesting
anti-proliferative compounds.
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Financial support was provided by the National Natural Science
Funding of China (21202124), High-level Innovative Talent Fund-
ing of Zhejiang Department of Health (G. L.), Project of Wenzhou
Sci&Tech Bureau (Y20120061), Qianjiang Talent Project of Zhejiang
Province (2013R10020), and Zhejiang Key Group Project in Scien-
tific Innovation (2010R50042). We also kindly thank Professor
S.H. Cheon in Chonnam National University for performing the
molecular docking experiments.