Synthesis and biological evaluation of 2-indolinone derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2011.10.009

Three series of 3-substituted-indolin-2-ones and azaindolin-2-ones have been synthesized and showed potential antiproliferative activity to cancer cell lines. The inhibition activities on VEGF-induced VEGFR phosphorylation were observed for selected 2-indolinones. Among the compounds synthesized, 5-fluoroindolin-2-one derivative 23 with a pyridone unit showed the most significant enzymatic and cellular activities. Flow cytometric analysis indicates that 23 plays a role in suppressing HCT-116 cell proliferation via G1 phase arrest and apoptosis in a dose dependent manner. The binding mode of compound 23 complexed with VEGFR-2 was predicted using FlexX algorithm. Described here are the chemistry and biological testing for these series which will guide the design and optimization of novel 2-indolione antitumor agents.

Full text of DOI:10.1016/j.ejmech.2011.10.009

European Journal of Medicinal Chemistry 46 (2011) 5970e5977
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 2-indolinone derivatives as potential
antitumor agents
a
a
a
b
a,b,
*
Hongbin Zou , Liang Zhang , Jingfeng Ouyang , Marc A. Giulianotti , Yongping Yu
a
College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, PR China
Torrey Pines Institute for Molecular Studies, 11350 Village Parkway, Port St. Lucie, FL 34987, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2011
Received in revised form
Three series of 3-substituted-indolin-2-ones and azaindolin-2-ones have been synthesized and showed
potential antiproliferative activity to cancer cell lines. The inhibition activities on VEGF-induced VEGFR
phosphorylation were observed for selected 2-indolinones. Among the compounds synthesized,
2
October 2011
5-fluoroindolin-2-one derivative 23 with a pyridone unit showed the most significant enzymatic and
Accepted 4 October 2011
Available online 13 October 2011
cellular activities. Flow cytometric analysis indicates that 23 plays a role in suppressing HCT-116 cell
proliferation via G1 phase arrest and apoptosis in a dose dependent manner. The binding mode of
compound 23 complexed with VEGFR-2 was predicted using FlexX algorithm. Described here are the
chemistry and biological testing for these series which will guide the design and optimization of novel 2-
indolione antitumor agents.
Keywords:
2-Indolinones
Antitumor agent
Apoptosis
Ó 2011 Elsevier Masson SAS. All rights reserved.
VEGFR-2
1. Introduction
derivative 4 was found to be orally active, showing better in vivo
antitumor activity in comparison with SU11248 (3) [12]. Herein we
Oxindole and its derivatives are frequently occurring structure
units in natural products and pharmacologically important
compounds that display utilizations in many major therapeutic
reported the synthesis and biological evaluation of 5-
fluorooxindoles and 4-, 5-, 7-azaoxindole derivatives 12e36
(Scheme 1). The oxindole pharmacophore was maintained and
nitrogenwas introduced into the skeleton (9e11, Scheme 1) in order
to improve the water solubility and unique hydrogen bonding
properties which were also seen for the indole scaffold [19,20]. To
obtain an insight into the effect of the 3-substituents of oxindole on
the biological activities of these compounds, the substituted
pyrrole-2-carbaldehydes, pyridone-2-carbaldehydes and indole-3-
aldehydes were chosen for condensation with the various 2-
indolinone units (8e11, Scheme 1). All the synthesized compounds
(12e36, Scheme 1) were used to screen six cultured human cancer
cell lines (PC-3, K562, HL60, HCT-116, A549 and HepG2) and one
human normal cell line QSG-7701. The results showed that the
compounds have cytotoxic potential and compound 23 exhibited
the most significant cytotoxicity against these cancer cell lines,
especially against HCT-116. Further biological analyses and molec-
ularmodelingon selectedcompoundswerecarriedout todetermine
the possible mechanism involved in the cytotoxicities.
areas [1e4]. Among them, SU5416 (1) possessing
a
3-
methyleneindolin-2-one substructure, was found to be a tyrosine
kinase inhibitor targeting vascular endothelial growth factor
receptor (VEGFR) [5]. In the search for novel and potent anticancer
agents, studies involving 3-methyleneindolin-2-ones have been
reported (2e4, Fig. 1) [6e12]. Among them, SU11248 (Sunitinib, 3)
was approved by the FDA for the treatment of renal cell carcinoma
(
[
RCC) and imatinib-resistant gastrointestinal stromal tumors (GIST)
13,14]. Preliminary evidence has also suggested promising activity
of SU11248 in other tumor groups and showed improvement in the
therapeutic efficacy of advanced solid tumors by combination
SU11248 with other chemotherapeutic agents [15e17]. However,
with long time exposure to Sunitinib, adverse effects have been
observed during treatment such as arterial hypertension, fatigue,
oral disorders and diarrhea [18].
Recently Cho found pyrrolo-fused six-, seven-, and eight-
member heterocycle derivatives in which the novel oxindole
2. Chemistry
*
Corresponding author. Institute of Material Medica, College of Pharmaceutical
Sciences, Zhejiang University, 383 Yu Hang Tang Road, Hangzhou 310058, PR China.
Tel./fax: þ86 571 88208452.
The targeted compounds (12e36, Table 1) were obtained by
condensation of substituted aldehydes (5e7, Scheme 1) with
E-mail address: yyu@zju.edu.cn (Y. Yu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.009

H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
5971
Fig. 1. Developed antitumor 3-methyleneindolin-2-ones.
various 2-indolinone derivatives (8e11, Scheme 1) in the presence
of piperidine as shown in Scheme 1 [6]. Among them, the hydro-
chloride derivatives 22e27, which have good water solubility, were
synthesized by additional stirring in the presence of hydrogen
chloride saturated methanol. The substituted pyrrole-2-aldehydes
example, Fig. 2) [26,27]. The results also showed the existence of
the inseparable E/Z mixtures for compounds 28e30 and the E/Z
ratios were determined using the corresponding chemical shifts
and integrals in the H NMR spectra [27]. As shown in Table 1, the
inseparable E/Z mixtures (28e30) contain the 4-aza-2-indolinone
1
(
5) and pyridone-2-carbaldehydes (6) were prepared by amida-
scaffold which suggested that both possible indicated intra-
molecular interactions between H-2 and the scaffold (29, Fig. 2)
might be the main reason for the two isomers.
0
tion of the corresponding 5a and 6a with different aimines
respectively (Scheme 2). The pyrrole-3-carboxylic acid (5a) and the
pyridone-3-carboxylic acid (6a) were synthesized utilizing re-
ported conditions [6,21]. The indole-3-aldehydes (7) were prepared
from substituted indoles as previously described with the yields
over 85% [22]. The 5-fluoroindolin-2-one (8) was commercially
available while the 4-azaindolin-2-one (9) was synthesized from 2-
chloro-3-nitropyridine in the same way that was reported by Finch
3. Results and discussion
All of the synthesized oxindole derivatives were evaluated for
cytotoxic activity in vitro against six human cancer cell lines (PC-3,
K562, HL60, HCT-116, A549 and HepG2) and one human normal cell
line QSG-7701 using SU11248 as a positive control. Results showing
the concentrations required to inhibit cell growth by 50% (IC50
values) are presented in Table 1.
The bioassay results suggest that most of the synthesized
2-indolinones and aza-2-indolinones have cytotoxic potential to
these six human cancer cell lines while only two compounds (30, 31)
show weak cytotoxicities to QSG-7701 cell line (Table 1). Analysis of
the MTT assay results of compounds 12e21 with pyrrole moiety,
[
23]. Bromination of 5-azaindole (10a) and the following PdeC
catalyzed reduction afforded the 5-azaindolin-2-one (10, Scheme
) in which 10a was prepared according to the reported method
24,25]. A similar approach was utilized to obtain 7-azaindolin-2-
one (11, Scheme 2) from the commercially available 7-azaindole
11a).
The configurations of the isolated compounds were confirmed
2
[
(
by mass spectrometry and NMR spectra. Because of the exocyclic
double bond, the oxindole derivatives may exist as the E or Z isomer
or as mixture of both. For the synthesized oxindole derivatives with
either a pyrrole or a pyridone moiety (12e27) a single Z configu-
ration was obtained due to the intramolecular interaction (23 as an
1
suggests that when the R substituent is a piperazine moiety (15e17
and 21, Table 1) the compounds lack cytotoxicity. Comparing
compounds 12,13, 20 with SU11248 (3), the bioassay results indicate
that the aza-2-indolinone scaffold with incorporated nitrogen at
Scheme 1. Synthesis of 2-indolinone derivatives. Reagents and conditions: (a) piperidine, methanol, reflux, 6 h. For 22e27, additional stirring in hydrogen chloride saturated
methanol is needed. Yields 60e92% were obtained.

5972
H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
Table 1
Synthesized 2-indolinones 12e36 and their cytotoxicities on seven human cell lines.
a
Compd
PC-3
K562
HL60
HCT-116
A549
HepG2
QSG-7701
b
3
25.1 ꢀ 2.1
47.0 ꢀ 5.3
21.9 ꢀ 1.3
24.1 ꢀ 1.7
15.7 ꢀ 1.1
14.8 ꢀ 0.8
e
15.5 ꢀ 1.0
45.9 ꢀ 2.3
34.8 ꢀ 1.6
35.0 ꢀ 2.0
e
6.1 ꢀ 0.4
12.4 ꢀ 1.5
9.1 ꢀ 0.4
11.0 ꢀ 1.8
e
18.5 ꢀ 1.1
16.7 ꢀ 2.1
12.5 ꢀ 0.6
13.9 ꢀ 0.3
e
15.4 ꢀ 0.5
20.2 ꢀ 0.6
8.6 ꢀ 0.4
16.4 ꢀ 0.5
e
e
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
2
3
4
e
b
e
e
e
e
5e17
e
e
8
9
0
1
2
3
4
5
6
7
25.6 ꢀ 1.6
18.0 ꢀ 1.2
e
30.2 ꢀ 1.5
27.8 ꢀ 1.3
48.8 ꢀ 2.2
40.0 ꢀ 2.5
32.9 ꢀ 2.8
22.7 ꢀ 1.7
24.7 ꢀ 1.6
38.9 ꢀ 2.3
32.7 ꢀ 1.1
34.3 ꢀ 1.8
e
18.1 ꢀ 1.2
25.5 ꢀ 1.8
9.2 ꢀ 0.5
35.0 ꢀ 0.8
8.2 ꢀ 0.5
2.3 ꢀ 0.1
5.3 ꢀ 0.3
12.8 ꢀ 0.2
9.5 ꢀ 0.4
4.3 ꢀ 0.3
e
25.4 ꢀ 1.3
29.2 ꢀ 1.2
21.0 ꢀ 0.9
48.2 ꢀ 2.7
11.8 ꢀ 0.7
6.2 ꢀ 0.3
12.6 ꢀ 0.2
22.0 ꢀ 0.4
15.2 ꢀ 0.3
13.7 ꢀ 0.6
e
38.6 ꢀ 0.7
e
e
e
e
49.5 ꢀ 3.7
19.8 ꢀ 0.9
e
18.3 ꢀ 0.5
37.3 ꢀ 0.6
12.6 ꢀ 0.2
5.0 ꢀ 0.2
17.1 ꢀ 0.4
35.0 ꢀ 0.7
18.9 ꢀ 0.5
27.3 ꢀ 0.5
e
e
e
e
30.3 ꢀ 1.5
25.3 ꢀ 3.1
13.8 ꢀ 1.1
19.3 ꢀ 1.2
45.1 ꢀ 2.3
26.8 ꢀ 1.4
21.3 ꢀ 0.5
e
e
21.0 ꢀ 1.2
e
25.2 ꢀ 1.1
e
35.6 ꢀ 1.5
e
28.0 ꢀ 2.4
e
26.0 ꢀ 2.2
e
c
d
8 e29
e
e
d
0
e
e
34.2 ꢀ 2.1
37.4 ꢀ 1.9
48.0 ꢀ 2.6
34.1 ꢀ 1.8
e
22.3 ꢀ 1.1
13.6 ꢀ 0.8
17.8 ꢀ 1.0
22.3 ꢀ 1.5
42.1 ꢀ 1.6
28.0 ꢀ 1.5
8.6 ꢀ 0.8
32.5 ꢀ 0.9
17.1 ꢀ 0.7
e
e
44.8 ꢀ 0.5
e
1
e
27.7 ꢀ 1.3
30.0 ꢀ 2.1
e
10.5 ꢀ 0.3
e
42.6 ꢀ 0.7
e
2
e
e
e
e
e
e
e
3
e
31.1 ꢀ 1.2
e
15.4 ꢀ 0.4
e
e
4
e
e
e
5
e
e
43.1 ꢀ 2.1
26.7 ꢀ 1.5
e
e
e
6
26.4 ꢀ 1.8
15.8 ꢀ 0.8
11.0 ꢀ 1.1
28.1 ꢀ 0.3
a
Key to cell lines: PC-3, human prostate cancer cell line; K562, human erythroleukemia cells; HL60, human promyelocytic leukemia cell line; HCT-116, human colon cancer
cell line; A549, human lung adenocarcinoma cell line; HepG2, human liver carcinoma cell line; QSG-7701, human liver cell line.
b
IC50 values greater than 50 mM were considered as inactive and omitted here.
c
d
e
Configuration ration: Z/E 5:2.
Configuration ration: Z/E 1:5.
Z configuration ration.
different positions provides no increase of the cytotoxicity to the
selected cancer cell lines in vitro. Compounds 30e36 with an indole
moiety exhibited selective cytotoxicities to HCT-116 and HL60
cancer cell lines while compound 36 showed potent cytotoxicity to
all of the selected cancer cell lines, similar to that seen for 3.
Analysis of the MTT assay results, also suggests that the HCT-116
cell line is much more sensitive to the synthesized oxindole
derivatives and analogs with a pyridone moiety are generally more
potent than those with pyrrole and indole moiety. Among them,
compound 23 showed the most significant cytotoxicity to these cell
lines, especially to the HCT-116, A549 and HepG2 cell lines, with
most effective of the three compounds against VEGF-induced
VEGFR-2 phosphorylation (Fig. 4). These compounds were further
screened for the in vitro inhibition of VEGFR-2 activity and the
results were shown in Table 2. All of the tested compounds showed
inhibitory potency. Among them, compound 23 exhibited the most
significant activity against VEGFR-2 with an enzymatic IC50 of
0.015
mM which is identical to that seen for Sunitinib (3) with an
enzymatic IC50 of 0.012
mM.
IC50 values of 2.3, 6.2 and 5.0 mM, stronger than that seen for
SU11248 (3).
To gain additional insight into the possible mechanism involved
in the cytotoxicity, HCT-116 cell line treated with compound 23 was
chosen for further biological analysis. The effect of compound 23 on
cell cycle distribution was investigated by flow cytometric anaylsis.
HCT-116 cells were treated with 12.5, 25 and 50
2
3
m
M of compound
3 for 48 h which resulted in the accumulation of G1 phase, from
6.8% to 42.63%, 45.44% and 81.63%, respectively (Fig. 3A). The
apoptotic effect of compound 23 was evaluated using annexin V
and PI staining and a dose dependent relationship was also
observed (Fig. 3B). After treatment with compound 23 for 48 h, the
cell apoptotic population increased from 4.17% of the control group
to 20.48%, 28.63% and 48.67% (Fig. 3B). These results suggest that
compound 23 inhibited HCT-116 cell viability via G1 phase arrest
and apoptosis in a dose dependent manner.
The synthesized targeted compounds 12e36 share the same 2-
indolinone scaffold with compounds 1e4 which are good VEGFR-2
inhibitors [5,6,12]. To determine whether the VEGFR-2 inhibitory
activity plays a rule in the observed cytotoxicity, the representative
compounds 13, 23 and 36 with potent cytotoxicities were selected
for further analysis. HCT-116 cells that naturally express VEGFR-2
were chosen for the cellular inhibition assay of 13, 23 and 36 on
the VEGF-induced VEGFR phosphorylation [28,29]. Interestingly, all
of them showed inhibitory potency and 23 was noted to be the
Scheme 2. Synthesis of several key intermediates. Reagents and conditions: (a) EDC,
HOBt, DMF, amines, rt, 12 h, 66% for 5, 84% for 6; (b) Br , t-BuOH, 20 min, 65% for 10b,
77% for 11b; (d) Pd/C, H , CH CH OH, rt, 12 h, 62% for 10, 76% for 11.
2
2
3
2

H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
5973
observed which was confirmed by a predicted binding mode of 23
complexed with VEGFR-2. The results reported here provide
a foundation for further improvement of the potency of these
compounds to discover more potent novel indolinone antitumor
agents in future studies.
5. Experimental
5.1. Materials
Fig. 2. The intramolecular interaction of the synthesized compounds.
Melting points were measured on a Perkin-Taike X-4 apparatus
and then corrected. H NMR and C NMR spectra were recorded on
a Varian INOVA 400 spectrometer with TMS as an internal standard
1
13
To illustrate the detailed possible interaction mechanism,
a potential binding model was constructed. Compound 23, which
was the compound that showed the most significant enzymatic and
cellularactivities, waschosen anddockedintotheATP-bindingsite of
VEGFR-2 (PDB entry 1Y6A) using FlexX program [30]. The predicted
binding mode of 23 to VEGFR-2 is shown in Fig. 5, in which the
indolinone scaffold forms two hydrogen bonds to the backbone
amine group of residues Lsy866 and Asp1044, while the diethyl-
amine tail group is exposed to solvent without specific contacts with
VEGFR-2. The backbone carbonyl group ofresidue Glu883 potentially
interacts through a hydrogen bond with the amine of pyridone unit.
3
and CDCl as solvent. ESI-MS data were recorded on a Bruker
Esquire 3000þ spectrometer. TLC was performed on silica gel
(
(
GF254). Column chromatography was carried out on silica gel H
10e40 m). All of the silica gel GF254 and silica gel H were
m
purchased from Qingdao Marine Chemical Factory, China. The MTT
assay was measured at 570 nm using a multiskan spectrum
Thermo Electron Co., Vantaa, Finland). The primary antibodies to
VEGFR and p-VEGFR were purchased from Cell Signaling Tech-
nology (Cell Signaling Technology, Beverly, MA). The peroxidase-
coupled secondary antibody was obtained from Southern Biotech
(
(
Birmingham, UK) and ECL-plus kit was from Amersham Biosci-
ences (UK) for detection. The Eu-cryptate labeled anti-
phosphotyrosine antibody (PY20K) was purchased from CAS Bio
International. The modified APC labeled Streptavidin (SV-XL) was
obtained from CIS Bio International.
4
. Conclusions
In summary, three series of novel indolinone-based chemical
entities with potential antiproliferative activities were synthesized
in which the pyridone-type compounds are much more sensitive to
the selected six cancer cell lines. Further bioassay results, especially
involving compound 23, show that the indolinone plays a role in
suppressing HCT-116 cell proliferation via G1 phase arrest and
apoptosis in a dose dependent manner. The inhibition activity of 23
on VEGFR-2 and VEGF-induced VEGFR phosphorylation were also
5.2. Synthesis
5.2.1. General procedure for the preparation of intermediates
5.2.1.1. General procedure for the preparation of aza-oxindolin-2-
ones (10, 11). To a stirred solution of azaindole (10a or 10b,
Fig. 3. Cell cycle progression (A) and apoptosis (B) involved in compound 23 cytotoxicity on HCT-116 cells. Cells were treated with 23 at indicated concentrations for 48 h. (A) Cells
treated with 23 were washed and collected. The cells were stained with propidium iodide and then DNA contents were analyzed by flow cytometry. (B) Flow cytometric analysis of
phosphatidylserine externalization (annexin V-binding) and cell membrane integrity (PI staining). Cell apoptosis rate in each group was marked.

5974
H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
For the targeted compounds 12e21 and 28e36, the mixture was
evaporated to remove the solvent and the residue was purified by
column chromatography (CH Cl /MeOH 100:1e10:1). For the tar-
2
2
geted compounds 22e27, ethyl ether (15 mL) was added to the
residue and the yellow precipitate was collected by filtration. The
obtained precipitate was subjected to the hydrogen chloride satu-
rated methanol to afford it as hydrochloride derivative. All the
targeted compounds 12e36 show moderate to good yields under
this reaction condition with the yields of 60e92%.
Fig. 4. Inhibition of 23 on VEGF-induced VEGFR-2 phosphorylation. HCT-116 cells were
pre-incubated with indicated concentrations of compound for 30 min followed by
5.2.2.1. (Z)-N-(2-(Dimethylamino)ethyl)-2,4-dimethyl-5-((2-oxo-1H-
pyrrolo[3,2-b]pyridin-3(2H)-ylidene)methyl)-1H-pyrrole-3-
50 ng/mL VEGF for 2 h.
ꢁ
1
carboxamide (12). Yield 70%; mp > 250 C; H NMR (500 MHz,
DMSO-d ):
Table 2
6
d
14.56 (1H, br s), 10.62 (1H, br s), 8.24 (1H, dd, J ¼ 5.0,
Enzymatic activities of selected compounds toward VEGFR-2 kinase.
1.5 Hz), 7.56 (1H, t, J ¼ 5.5 Hz), 7.50 (1H, s), 7.28 (1H, dd, J ¼ 8.0,
1.5 Hz), 7.22 (1H, dd, J ¼ 7.5, 5.0 Hz), 3.32e3.30 (2H, m), 2.54 (3H, s),
M)a
Compound
IC50
(
m
Compound
IC50 (mM)
13
2
6
.41e2.38 (5H, m), 2.19 (6H, s); C NMR (125 MHz, DMSO-d ):
3
1
0.012
0.060
23
36
0.015
6.58
d
168.49, 164.96, 143.19, 140.04, 138.51, 136.12, 131.70, 126.53,
3
1
1
25.25, 122.24, 121.71, 116.51, 113.48, 58.72, 45.56, 37.34, 13.94,
a
Determined against isolated VEGFR-2 for three independent experiments (at
0.98. ESI-MS m/z [M þ H]þ 354.
least 8 points per experiment).
5.2.2.2. (Z)-N-(2-(Dimethylamino)ethyl)-2,4-dimethyl-5-((2-oxo-
1
2.7 mmol) in tert-butyl alcohol (100 mL) and H
2
O (100 mL) at
1H-pyrrolo[3,2-c]pyridin-3(2H)-ylidene)methyl)-1H-pyrrole-3-
ꢁ
1
room temperature, Br (2.6 mL, 50.5 mmol) was added dropwise
2
carboxamide (13). Yield 64%; mp > 250 C; H NMR (500 MHz,
DMSO-d ): 13.43 (1H, br s), 11.28 (1H, s), 8.91 (1H, s), 8.22 (1H, d,
over 20 min. The mixture was stirred for 1 h and the yellow
precipitate (10b, or 11b) was collected by filtration. The dried
precipitate (10b, or 11b, 4.8 mmol) was refluxed with 5% PdeC
6
d
J ¼ 5.5 Hz), 7.79 (1H, s), 7.54 (1H, t, J ¼ 5.0 Hz), 6.90 (1H, d, J ¼ 5.5 Hz),
3.30e3.28 (2H, m), 2.43 (3H, s), 2.40 (3H, s), 2.37 (2H, t, J ¼ 6.5 Hz),
13
(
1.0 g) in 60 mL of 8.8% HCOOH/CH
3
OH for 12 h under nitrogen. The
6
2.18 (6H, s); C NMR (125 MHz, DMSO-d ): d 169.72, 164.93, 147.19,
catalyst was removed by filtration through a pad of celite. After
removal of the solvent, the residual was adjusted to pH 8e9 by
addition of saturated K CO solution. The solution was stored in the
2 3
144.46, 139.97, 137.29, 131.12, 126.42, 125.35, 122.40, 121.47, 112.01,
105.51, 58.73, 45.63, 37.32, 13.74, 10.99; ESI-MS m/z [M þ H] 354.
þ
refrigerator overnight and the precipitate was filtered to give 5-
azaindolin-2-one (10) or 7-azaindolin-2-one (11).
5.2.2.3. (Z)-N-(2-(Diethylamino)ethyl)-2,4-dimethyl-5-((2-oxo-1H-
pyrrolo[3,2-c]pyridin-3(2H)-ylidene)methyl)-1H-pyrrole-3-
ꢁ
1
carboxamide (14). Yield 62%; mp > 250 C; H NMR (500 MHz,
DMSO-d ): 13.50 (1H, br s), 11.36 (1H, s), 8.96 (1H, s), 8.28 (1H, d,
5.2.1.2. General procedure for the preparation of substituted alde-
d
6
hydes (5, 6). The substituted acid (5a or 6a, 1.0 mmol), EDC
J ¼ 5.0 Hz), 7.85 (1H, s), 7.68e7.60 (1H, m), 6.96 (1H, d, J ¼ 5.0 Hz),
(
229 mg, 1.2 mmol) and HOBt (160 mg, 1.2 mmol) were dissolved in
3.41e3.39 (2H, m), 2.79e2.64 (6H, m), 2.50 (3H, s), 2.47 (3H, s), 1.07
ꢁ
13
DMF (25 mL). After cooling to 5 C, the substituted amine was
added to the mixture and was stirred at room temperature over-
night followed by evaporation under reduced pressure to remove
DMF. The residue was purified by column chromatography (pet.
ether/EtOAc 7:1 to 2:1) to afford 5 and 6 with the yields of 66% and
(6H, t, J ¼ 7.5 Hz); C NMR (125 MHz, DMSO-d
6
): d 169.51, 164.63,
146.95, 144.17, 139.76, 137.13, 130.77, 126.17, 125.06, 122.15, 121.14,
111.80, 105.23, 51.84, 46.74, 37.19, 13.54, 12.06, 10.81; ESI-MS m/z
[M þ H]þ 382.
8
4% respectively.
5.2.2.4. (Z)-3-((4-(4-(2-Hydroxyethyl)piperazine-1-carbonyl)-3,5-
dimethyl-1H-pyrrol-2-yl)methylene)-1H-pyrrolo[3,2-c]pyridin-
ꢁ
1
5.2.2. General procedure for the preparation of targeted compounds
2(3H)-one (15). Yield 70%; mp 240e243 C; H NMR (500 MHz,
DMSO-d ): 13.38 (1H, br s), 11.23 (1H, s), 8.89 (1H, s), 8.23 (1H, d,
12e36
6
d
Piperidine (0.08 mL) was added to methanol (25 mL) dissolved
J ¼ 5.0 Hz), 7.77 (1H, s), 6.90 (1H, d, J ¼ 5.0 Hz), 4.39 (1H, t,
J ¼ 5.5 Hz), 3.49 (2H, q, J ¼ 6.0 Hz), 3.37e3.70 (4H, m), 2.46e2.33
(4H, m), 2.40 (2H, t, J ¼ 6.0 Hz), 2.26 (3H, s), 2.29 (3H, s); ESI-MS m/z
[M þ H]þ 396.
in substituted aldehydes 5e7 (0.18 mmol) and starting 2-
indolinone derivatives 8e11 (0.18 mmol) which were prepared as
described previously [6,21e25]. The mixture was refluxed for 5 h.
Fig. 5. Binding models of compound 23 bound to VEGFR-2 kinase (PDB entry 1Y6A) and potential interactions between compound 23 and Glu883, Lys866, Asp1044 in VEGFR-2
active site. The hydrogen bonds are labeled as yellow broken lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
5975
5
1
.2.2.5. (Z)-3-((3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-
5.2.2.12. (Z)-N-(2-(Diethylamino)ethyl)-6-((5-fluoro-2-oxoindolin-
3-ylidene)methyl)-2-oxo-1,2-dihydropyridine-3-carboxamide hydro-
H-pyrrol-2-yl)methylene-1H-pyrrolo[3,2-c]pyridin-2(3H)-one (16).
ꢁ
1
ꢁ
1
Yield 60%; mp 170e173 C; H NMR (500 MHz, DMSO-d
6
):
d
13.39
chloride (23). Yield 77%; mp > 250 C; H NMR (500 MHz, DMSO-
): 14.44 (1H, s), 11.49 (1H, s), 9.93e10.00 (1H, br s), 9.92 (1H, t,
(
1H, br s), 11.27 (1H, s), 8.90 (1H, s), 8.23 (1H, d, J ¼ 5.0 Hz), 7.78 (1H,
d
6
d
s), 6.90(1H, d, J ¼ 5.0 Hz), 3.75e3.44 (4H, m), 2.29 (3H, s), 2.26(3H, s),
J ¼ 6 Hz), 8.45 (1H, d, J ¼ 7.5 Hz), 7.82 (1H, s), 7.74 (1H, dd, J ¼ 8.5,
2.5 Hz), 7.20e7.16 (1H, m), 6.95e6.93 (2H, m), 3.71 (2H, q,
J ¼ 6.5 Hz), 3.27e3.23 (2H, m), 3.23e3.16 (4H, m), 1.23 (6H, t,
þ
2
.37e2.20 (4H, m), 2.17 (3H, s); ESI-MS m/z [M þ H] 366.
1
3
5.2.2.6. (Z)-3-((4-(4-(2-Hydroxyethyl)piperazine-1-carbonyl)-3,5-di-
J ¼ 7.5 Hz); C NMR (125 MHz, DMSO-d
6
): d 169.29, 164.05, 161.68,
methyl-1H-pyrrol-2-yl)methylene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-
one (17). Yield 68%; mp > 250 C H NMR (500 MHz, DMSO-d ):
6
d
158.84 (d, J ¼ 235.6 Hz), 144.22, 143.85, 138.44, 131.98, 129.50,
125.29 (d, J ¼ 9.2 Hz), 122.72, 117.94 (d, J ¼ 24.4 Hz), 114.50, 112.12
(d, J ¼ 8.0 Hz), 109.17 (d, J ¼ 25.8 Hz), 49.99, 47.36, 34.34, 8.96; ESI-
MS m/z [M þ H ꢂ HCl]þ 399.
ꢁ
1
13.42 (1H, s), 11.47 (1H, s), 8.09 (1H, d, J ¼ 7.5 Hz), 8.01 (1H, d,
J ¼ 5.0 Hz), 7.69 (1H, s), 7.00 (1H, dd, J ¼ 7.5, 5.0 Hz), 4.44 (1H, t,
J ¼ 5.5 Hz), 3.48 (2H, q, J ¼ 6.0 Hz), 3.70e3.41 (4H, m), 2.38 (2H, t,
J ¼ 6.0 Hz), 2.46e2.31 (4H, m), 2.28 (3H, s), 2.25 (3H, s); ESI-MS m/z
5.2.2.13. (Z)-N-(2-(Dimethylamino)ethyl)-6-((5-fluoro-2-oxoindolin-
þ
[M þ H] 396.
3-ylidene)methyl)-2-oxo-1,2-dihydropyridine-3-carboxamide hydro-
ꢁ
1
chloride (24). Yield 79%; mp > 250 C; H NMR (500 MHz, DMSO-
):
5
.2.2.7. (Z)-2,4-Dimethyl-5-((2-oxo-1H-pyrrolo[2,3-b]pyridin-3(2H)-
d
6
d
14.44 (1H, s), 11.46 (1H, s), 9.91 (1H, t, J ¼ 6.0 Hz), 8.46 (1H, d,
ylidene)methyl)-N-(2-(piperidin-1-yl)ethyl)-1H-pyrrole-3-carboxam-
J ¼ 7.5 Hz), 7.81 (1H, s), 7.74 (1H, dd, J ¼ 8.5, 2.5 Hz), 7.21e7.17 (1H,
m), 6.96e6.93 (2H, m), 3.68 (2H, q, J ¼ 6.0 Hz), 3.22e3.17 (2H, m),
2.77 (6H, s); ESI-MS m/z [M þ H ꢂ HCl]þ 371.
ꢁ
1
ide (18). Yield 60%; mp > 250 C; H NMR (500 MHz, DMSO-d
13.46 (1H, s), 11.46 (1H, s), 8.11 (1H, d, J ¼ 7.5 Hz), 8.01 (1H, d,
J ¼ 5.0 Hz), 7.71 (1H, s), 7.51e7.42 (1H, m), 7.00 (1H, t, J ¼ 6.0 Hz),
.33e3.25 (2H, m), 2.44 (3H, s), 2.41 (3H, s), 2.40e2.24 (6H, m),
6
):
d
3
5.2.2.14. (Z)-6-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-mo-
rpholinoethyl)-2-oxo-1,2-dihydropyridine-3-carboxamide hydrochlo-
13
1
.57e1.42 (4H, m), 1.42e1.30 (2H, m); C NMR (125 MHz, DMSO-
): 169.75, 164.82, 153.10, 145.25, 137.22, 130.81, 126.24, 125.97,
ꢁ
1
d
6
d
ride (25). Yield 80%; mp > 250 C; H NMR (500 MHz, DMSO-d ):
6
1
2
25.63, 121.37, 120.37, 117.79, 113.09, 57.94, 54.38, 36.65, 26.08,
d
14.36 (1H, s), 11.44 (1H, s), 9.86 (1H, t, J ¼ 5.5 Hz), 8.43 (1H, d,
4.49, 13.75, 11.04. ESI-MS m/z [M þ H]þ 394.
J ¼ 7.5 Hz), 7.78 (1H, s), 7.72 (1H, dd, J ¼ 8.5, 2.5 Hz), 7.19e7.15 (1H,
m), 6.92e6.89 (2H, m), 3.57 (4H, t, J ¼ 4.5 Hz), 3.43 (2H, q, J ¼ 6 Hz),
2.45 (2H, t, J ¼ 6.0 Hz), 2.36e2.43 (4H, m); ESI-MS m/z
5.2.2.8. (Z)-2,4-Dimethyl-N-(2-morpholinoethyl)-5-((2-oxo-1H-pyr-
þ
rolo[2,3-b]pyridin-3(2H)-ylidene)methyl)-1H-pyrrole-3-carboxamide
[M þ H ꢂ HCl] 413.
ꢁ
1
(19). Yield 64%; mp > 250 C; H NMR (500 MHz, DMSO-d
6
):
d
13.47 (1H, s), 11.48 (1H, s), 8.12 (1H, d, J ¼ 7.5 Hz), 8.02 (1H, d,
5.2.2.15. (Z)-6-((5-Fluoro-2-oxoindolin-3-ylidene)methyl)-2-oxo-N-
(2-(piperidin-1-yl)ethyl)-1,2-dihydropyridine-3-carboxamide hydro-
J ¼ 5.0 Hz), 7.72 (1H, s), 7.53 (1H, t, J ¼ 5.5 Hz), 7.01 (1H, dd, J ¼ 7.5,
ꢁ
1
5
2
.0 Hz), 3.61e3.52 (4H, m), 3.33e3.29 (2H, m), 2.45 (3H, s),
chloride (26). Yield 78%; mp > 250 C H NMR (500 MHz, DMSO-
):
13
.47e2.43 (2H, m), 2.42 (3H, s), 2.43e2.36 (4H, m); C NMR
): 169.75, 164.88, 153.10, 145.24, 137.16,
d
6
d
14.38 (1H, s), 11.46 (1H, s), 9.87 (1H, t, J ¼ 5.0 Hz), 8.45 (1H, d,
(
125 MHz, DMSO-d
6
d
J ¼ 7.0 Hz), 7.80 (1H, s), 7.76 (1H, dd, J ¼ 9.0, 2.5 Hz), 7.21e7.17 (1H,
1
5
30.85, 126.24, 125.96, 125.62, 121.44, 120.36, 117.79, 113.07, 66.75,
m), 6.95e6.92 (2H, m), 3.52e3.41 (2H, m), 2.46e2.34 (4H, m),
7.72, 53.63, 36.27, 13.74, 11.01; ESI-MS m/z [M þ H]þ 396.
1.60e1.48 (4H, m), 1.48e1.35 (2H, m); ESI-MS m/z [M þ H ꢂ HCl]
þ
411.
5.2.2.9. (Z)-N-(2-(Dimethylamino)ethyl)-2,4-dimethyl-5-((2-oxo-1H-
pyrrolo[2,3-b]pyridin-3(2H)-ylidene)methyl)-1H-pyrrole-3-carboxa-
mide (20). Yield 69%; mp > 250 C; H NMR (500 MHz, DMSO-d ):
6
5.2.2.16. (Z)-N-(2-(Dimethylamino)ethyl)-2-oxo-6-((2-oxo-1H-pyr-
rolo[2,3-b]pyridin-3(2H)-ylidene)methyl)-1,2-dihydropyridine-3-car-
ꢁ
1
ꢁ
1
d
13.47 (1H, br s), 11.47 (1H, s), 8.10 (1H, dd, J ¼ 7.5, 1.0 Hz), 8.01 (1H,
boxamide hydrochloride (27). Yield 70%; mp > 250 C; H NMR
(500 MHz, DMSO-d ): 14.26 (1H, s), 12.02 (1H, br s), 9.91 (1H, t,
dd, J ¼ 5.0, 1.0 Hz), 7.70 (1H, s), 7.56 (1H, t, J ¼ 5.5 Hz), 7.00 (1H, dd,
J ¼ 7.5, 5.0 Hz), 3.35e3.31 (2H, m), 2.48e2.46 (2H, m), 2.43 (3H, s),
6
d
J ¼ 6.0 Hz), 8.47 (1H, d, J ¼ 7.5 Hz), 8.23 (1H, dd, J ¼ 5.0, 1.5 Hz), 8.14
(1H, dd, J ¼ 7.5, 1.5 Hz), 7.83 (1H, s), 7.16 (1H, dd, J ¼ 7.5, 5.0 Hz), 7.00
(1H, dd, J ¼ 7.5, 1.5 Hz), 3.70 (2H, q, J ¼ 6.0 Hz), 3.24 (2H, t,
J ¼ 5.0 Hz), 2.80 (6H, s); ESI-MS m/z [M þ H ꢂ HCl]þ 354.
13
2
6
.39 (3H, s), 2.26 (6H, s); C NMR (125 MHz, DMSO-d ): d 169.77,
165.81, 153.14, 145.37, 137.70, 131.07, 126.26, 126.16, 125.65, 120.30,
1
20.23, 117.84, 113.48, 56.70, 43.02, 34.74, 14.11, 11.27; ESI-MS m/z
þ
[M þ H] 354.
5
.2.2.17. (Z/E)-3-((5-Fluoro-1H-indol-3-yl)methylene)-1H-pyrrolo
ꢁ
1
5
1
.2.2.10. (Z)-3-((3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-
[3,2-b]pyridin-2(3H)-one (28). Yield 86%; mp > 250 C; H NMR
(500 MHz, DMSO-d ): 12.34 (1H, br s, Z), 10.70 (1H, s, Z), 9.59 (1H,
H-pyrrol-2-yl)methylene)-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (21).
6
d
ꢁ
1
Yield 67%; mp > 250 C; H NMR (500 MHz, DMSO-d
6
):
d
13.42 (1H,
s, Z), 8.33 (1H, s, Z), 8.14 (1H, dd, J ¼ 4.5, 1.0 Hz, Z), 7.79 (1H, dd,
J ¼ 10, 2.5 Hz, Z), 7.57 (1H, dd, J ¼ 9.0, 5.0 Hz, Z), 7.19e7.17 (1H, m, Z),
7.14e7.12 (2H, m, Z); 12.33 (0.4 ꢃ1H, br s, E), 10.58 (0.4 ꢃ1H, s, E),
10.09 (0.4 ꢃ1H, s, E), 8.30 (0.4 ꢃ1H, dd, J ¼ 5.0, 1.5 Hz, E), 8.02
(0.4 ꢃ1H, s, E), 7.77 (0.4 ꢃ1H, dd, J ¼ 10, 2.5 Hz, E), 7.57 (0.4 ꢃ1H,
dd, J ¼ 9.0, 5.0 Hz, E), 7.23 (0.4 ꢃ1H, dd, J ¼ 7.5, 1.5 Hz, E), 7.19e7.17
s), 11.47 (1H, s), 8.10 (1H, dd, J ¼ 7.5, 1.0 Hz), 8.01 (1H, dd, J ¼ 5.0,
1
2
.0 Hz), 7.69 (1H, s), 7.00 (1H, dd, J ¼ 7.5, 5.0 Hz), 3.74e3.40 (4H, m),
.34e2.20 (4H, m), 2.28 (3H, s), 2.25 (3H, s), 2.17 (3H, s); ESI-MS m/z
þ
[M þ H] 366.
13
5.2.2.11. (Z)-N-(2-(Diethylamino)ethyl)-2-oxo-6-((2-oxo-1H-pyrrolo
(0.4 ꢃ1H, m, E), 7.12e7.10 (0.4 ꢃ1H, m, E); C NMR (125 MHz,
[
3,2-b]pyridin-3(2H)-ylidene)methyl)-1,2-dihydropyridine-3-carbox-
DMSO-d
6
): Z:
d
168.90, 158.98 (d, J ¼ 233.9 Hz), 144.11, 141.56,
ꢁ
1
amide hydrochloride (22). Yield 75%; mp > 250 C;
500 MHz, DMSO-d ): 15.27 (1H, br s), 11.03 (1H, br s), 9.85 (1H, t,
J ¼ 5.5 Hz), 8.40 (1H, d, J ¼ 7.5 Hz), 8.30 (1H, dd, J ¼ 5.0, 1.5 Hz),
H
NMR
138.18, 137.01, 133.45, 130.50, 129.35 (d, J ¼ 10.0 Hz), 122.12, 118.07,
115.58, 114.30 (d, J ¼ 9.9 Hz), 112.57 (d, J ¼ 4.2 Hz), 111.42 (d,
J ¼ 25.9 Hz), 103.97 (d, J ¼ 24.1 Hz); ESI-MS m/z [M þ H]þ 280.
(
6
d
7
2
[
.46e7.43 (3H, m), 7.21 (1H, d, J ¼ 7.5 Hz), 3.42e3.38 (2H, m),
.59e2.54 (6H, m), 0.99 (6H, t, J ¼ 7.5 Hz); ESI-MS m/z
5.2.2.18. (Z/E)-3-((6-Fluoro-1H-indol-3-yl)methylene)-1H-pyrrolo
þ
ꢁ
1
M þ H ꢂ HCl] 382.
[3,2-b]pyridin-2(3H)-one (29). Yield 88%; mp > 250 C; H NMR

5976
H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
(
500 MHz, DMSO-d
6
):
d
12.19 (1H, br s, E), 10.59 (1H, s, E), 10.02 (1H,
113.93 (d, J ¼ 9.8 Hz), 113.26 (d, J ¼ 23.9 Hz), 112.05 (d, J ¼ 4.4 Hz),
111.10 (d, J ¼ 26.1 Hz), 109.99 (d, J ¼ 8.4 Hz), 106.66 (d, J ¼ 25.2 Hz),
104.50 (d, J ¼ 24.1 Hz); ESI-MS m/z [M þ H]þ 297.
s, E), 8.31 (1H, dd, J ¼ 5.0, 1.5 Hz, E), 8.03 (1H, s, E), 7.96 (1H, dd,
J ¼ 8.5, 5.0 Hz, E), 7.38 (1H, dd, J ¼ 9.5, 2.0 Hz, E), 7.23 (1H, dd,
J ¼ 8.0, 1.5 Hz, E), 7.19 (1H, dd, J ¼ 8.0, 2.5 Hz, E), 7.13e7.09 (1H, m,
E); 12.19 (0.2 ꢃ 1H, br s, Z), 10.71 (0.2 ꢃ 1H, s, Z), 9.53 (0.2 ꢃ1H, s, Z),
5.2.2.24. (Z)-5-Fluoro-3-((5-fluoro-1H-indol-3-yl)methylene)indolin-
ꢁ
1
8
.35 (0.2 ꢃ 1H, s, Z), 8.14 (0.2 ꢃ 1H, dd, J ¼ 5.0, 1.5 Hz, Z), 7.99
2-one (35). Yield 92%; mp > 250 C; H NMR (500 MHz, DMSO-d
6
):
(
7
0.2 ꢃ 1H, dd, J ¼ 8.5, 5.0 Hz, Z), 7.38 (0.2 ꢃ1H, dd, J ¼ 9.5, 2.0 Hz, Z),
d
12.09 (1H, br s, Z), 10.55 (1H, s, Z), 9.45 (1H, s, Z), 8.25 (1H, dd,
13
.19 (0.2 ꢃ1H, dd, J ¼ 8.0, 2.5 Hz, Z), 7.14e7.09 (0.4 ꢃ 2H, m, Z);
C
J ¼ 9.0, 5.5 Hz, Z), 8.21 (1H, s, Z), 7.86 (1H, dd, J ¼ 9.5, 2.5 Hz, Z), 7.34
(1H, dd, J ¼ 10.0, 2.5 Hz, Z), 7.14e7.10 (1H, m, Z), 6.97e6.93 (1H, m, Z),
NMR (125 MHz, DMSO-d
1
1
J ¼ 24.1 Hz), 99.37 (d, J ¼ 25.5 Hz); ESI-MS m/z [M þ H] 280.
6
): E:
d
169.04, 160.00 (d, J ¼ 235.2 Hz),
1
3
43.97, 141.75, 137.18, 136.93, 136.84 (d, J ¼ 12.8 Hz), 130.41, 125.10,
6.81 (H, dd, J ¼ 8.0, 4.5 Hz, Z); C NMR (125 MHz, DMSO-d
6
): Z:
22.25, 119.68 (d, J ¼ 10.1 Hz), 118.40, 115.76, 112.26, 110.30 (d,
d
168.61, 159.96 (d, J ¼ 235. 0 Hz), 158.43 (d, J ¼ 232.5 Hz), 136.31 (d,
þ
J ¼ 12.5 Hz), 135.84, 135.04, 129.10, 127.61 (d, J ¼ 9.2 Hz), 125.42,
1
20.30 (d, J ¼ 10.0 Hz), 119.81 (d, J ¼ 2.8 Hz), 113.37 (d, J ¼ 23.9 Hz),
5
2
.2.2.19. (E/Z)-3-((7-Ethyl-1H-indol-3-yl)methylene)-1H-pyrrolo[3,-
111.75, 110.01 (d, J ¼ 8.2 Hz), 109.65 (d, J ¼ 24.0 Hz), 106.64 (d,
ꢁ
1
J ¼ 25.2 Hz), 99.48 (d, J ¼ 25.8 Hz); ESI-MS m/z [M þ H]þ 296.
-b]pyridin-2(3H)-one (30). Yield 86%; mp 242e244 C; H NMR
): 12.29 (1H, br s, E),10.57 (1H, s, E),10.06 (1H,
(
500 MHz, DMSO-d
6
d
s, E), 8.30 (1H, dd, J ¼ 5.0, 1.5 Hz, E), 8.06 (1H, s, E), 7.76 (1H, d,
J ¼ 7.5 Hz, E), 7.25e7.15 (3H, m, E), 7.11 (1H, d, J ¼ 7.5 Hz, E), 2.92 (2H,
q, J ¼ 7.5 Hz, E), 1.30 (3H, t, J ¼ 7.5 Hz, E) 12.29 (0.2 ꢃ 1H, br s, Z),
5.2.2.25. (Z)-5-Ethyl-3-((5-fluoro-1H-indol-3-yl)methylene)indolin-
ꢁ
1
2-one (36). Yield 86%; mp 246e248 C; H NMR (500 MHz, DMSO-
): 12.09 (1H, br s), 10.51 (1H, s), 9.46 (1H, s), 8.21 (1H, s), 8.06
d
6
d
1
0.67 (0.2 ꢃ 1H, s, Z), 9.54 (0.2 ꢃ 1H, s, Z), 8.37 (0.2 ꢃ1H, s, Z), 8.14
(1H, d, J ¼ 8.0 Hz), 7.85 (1H, dd, J ¼ 9.0, 2.5 Hz), 7.18 (1H, t,
(
7
(
d
0.2 ꢃ 1H, dd, J ¼ 5.0, 1.5 Hz, Z), 7.79 (0.2 ꢃ 1H, d, J ¼ 8.0 Hz, Z),
J ¼ 7.5 Hz), 7.08 (1H, d, J ¼ 7.0 Hz), 6.97e6.90 (1H, m), 6.80 (1H, dd,
1
3
.24e7.11 (0.2 ꢃ 4H, m, Z), 2.92 (0.2 ꢃ 2H, q, J ¼ 7.5 Hz, Z), 1.30
J ¼ 8.0, 5.0 Hz), 2.91 (2H, q, J ¼ 7.5 Hz), 1.29 (3H, t, J ¼ 7.5 Hz);
C
13
0.2 ꢃ 3H, t, J ¼ 7.5 Hz, Z); C NMR (125 MHz, DMSO-d
6
): E:
NMR (125 MHz, DMSO-d
6
):
d
168.65, 158.42 (d, J ¼ 232.1 Hz),
169.15, 144.15, 141.65, 136.76, 136.48, 135.54, 130.95, 128.77,
135.70, 135.04, 134.32, 129.62, 128.76, 128.32, 127.76 (d, J ¼ 9.1 Hz),
121.91, 121.69, 119.02 (d, J ¼ 2.8 Hz), 116.69, 113.11 (d, J ¼ 23.9 Hz),
112.17, 109.92 (d, J ¼ 8.2 Hz), 106.48 (d, J ¼ 25.1 Hz), 24.00, 14.96;
ESI-MS m/z [M þ H]þ 307.
1
1
28.47, 122.42, 122.26, 122.02, 117.59, 115.92, 115.64, 112.71, 23.99,
4.91; ESI-MS m/z [M þ H]þ 290.
5
.2.2.20. (Z)-3-((7-Ethyl-1H-indol-3-yl)methylene)-1H-pyrrolo[2,3-
ꢁ
1
b]pyridin-2(3H)-one (31). Yield 87%; mp > 250 C;
H
NMR
5.3. Cell culture
(
500 MHz, DMSO-d ): 12.13 (1H, br s), 11.10 (1H, s), 9.43 (1H, d,
6
d
J ¼ 3.0 Hz), 8.22 (1H, s), 8.19 (1H, dd, J ¼ 7.5, 1.0 Hz), 8.04e7.99 (2H,
Various human cancer cell lines were cultured in minimum
essential medium (MEM), supplemented with 10% fetal calf serum
(Gibco Laboratories, Grand island, NY), 100 units/mL penicillin and
m), 7.21e7.16 (1H, m), 7.09 (1H, d, J ¼ 7.0 Hz), 7.01 (1H, dd, J ¼ 7.5,
1
3
5
.0 Hz), 2.91 (2H, q, J ¼ 7.5 Hz), 1.30 (3H, t, J ¼ 7.5 Hz); C NMR
(
125 MHz, DMSO-d
6
):
d
168.08, 153.80, 145.54, 135.06, 134.36,
100 mg/mL streptomycin in a humidified atmosphere in 5% CO
2
at
ꢁ
1
31.10, 128.63, 128.41, 125.89, 122.01, 121.82, 120.54, 117.39, 117.12,
37 C. Cell culture media were renewed every three days, up to the
confluence of the monolayer. Cell culture was washed upon
formation of confluent cultures, using trypsineEDTA to detach the
cells from their culture flasks or dishes. Test compounds were
þ
116.56, 112.26, 23.99, 14.95; ESI-MS m/z [M þ H] 290.
5
.2.2.21. (Z)-3-((5-Fluoro-1H-indol-3-yl)methylene)-1H-pyrrolo[2,3-
ꢁ
1
ꢁ
b]pyridin-2(3H)-one (32). Yield 85%; mp > 250 C;
H
NMR
12.20 (1H, s), 11.13 (1H, s), 9.47 (1H, s), 8.21
1H, s), 8.12e8.00 (2H, m), 7.59e7.50 (1H, m), 7.15e7.05 (1H, m),
.05e6.99 (1H, m); 13C NMR (125 MHz, DMSO-d
): 168.07, 158.85
d, J ¼ 232.4 Hz), 153.94, 145.70, 136.11, 132.98, 129.73, 129.61 (d,
J ¼ 10.5 Hz), 126.08, 120.50, 117.50, 117.39, 114.05 (d, J ¼ 9.5 Hz),
stored at ꢂ70 C and solubilized in 100% DMSO.
(
(
6
500 MHz, DMSO-d ): d
5.4. MTT assay for cell viability
7
(
6
d
Exponentially growing cells were seeded into 96-well micro-
4
culture plates at a concentration of 1 ꢃ10 cells per well. After 6 h
1
12.11 (d, J ¼ 3.9 Hz), 111.21 (d, J ¼ 25.6 Hz), 104.43 (d, J ¼ 24.1 Hz);
incubation, cells were exposed to compounds at concentrations
þ
ESI-MS m/z [M þ H] 280.
from 50 to 1.562 mM. After 48 h, 10 mL of MTT solution (5 mg/mL in
phosphate buffered solution) was added to the culture medium and
ꢁ
5
.2.2.22. (Z)-3-((6-Fluoro-1H-indol-3-yl)methylene)-1H-pyrrolo[2, 3-
incubated at 37 C for a further 4 h. After removing unconverted
ꢁ
1
b]pyridin-2(3H)-one (33). Yield 83%; mp > 250 C;
H
NMR
MTT, 200 ml of DMSO was added to each well and the plates shaken
(
(
500 MHz, DMSO-d
1H, s), 8.21e8.16 (2H, m), 8.03 (1H, dd, J ¼ 5.0, 1.0 Hz), 7.33 (1H, dd,
6
):
d
12.12 (1H, s), 11.12 (1H, s), 9.41 (1H, s), 8.22
to dissolve the reduced MTTcrystals (formazan); the optical density
was measured on a microplate reader at a wavelength of 570 nm.
The average 50% inhibitory concentration (IC50) was evaluated by
MTT tetrazolium dye assay. Each experiment was performed three
times.
13
J ¼ 9.5, 2.0 Hz), 7.09e7.13 (1H, m),7.01 (1H, dd, J ¼ 7.0, 5.0 Hz);
C
NMR (125 MHz, DMSO-d
6
):
d
168.00, 159.98 (d, J ¼ 230.4 Hz),
1
1
53.99, 145.80, 136.33 (d, J ¼ 13.0 Hz), 135.07, 129.62, 126.12, 125.33,
20.41, 120.26 (d, J ¼ 9.9 Hz), 117.96, 117.39, 111.82, 109.74 (d,
þ
J ¼ 24.1 Hz), 99.08 (d, J ¼ 25.5 Hz); ESI-MS m/z [M þ H] 280.
5.5. Flow cytometric analysis
5
5
.2.2.23. (Z)-5-Fluoro-3-((5-fluoro-1H-indol-3-yl)methylene)indo-
HCT-116 Cells (1 ꢃ10 /mL) were seeded in a 6-well culture plate,
ꢁ
1
lin-2-one(34). Yield 88%; mp > 250 C; H NMR (500 MHz, DMSO-
): 12.16 (1H, br s),10.54 (1H, s), 9.49 (1H, s), 8.19 (1H, s), 8.11 (1H,
after 24 h incubation, cells were treated with 0, 0.5, 2.0 and 8.0 mM
of compound 23 for 48 h. Cells were collected and washed three
times with D-Hank’s buffer, re-suspended and fixed in 70% ice-cold
d
6
d
dd, J ¼ 10.0, 2.0 Hz), 7.87 (1H, dd, J ¼ 9.5, 2.5 Hz), 7.34 (1H, dd,
ꢁ
J ¼ 9.0, 4.5 Hz), 7.11e7.07 (1H, m), 6.97e6.92 (1H, m), 6.80 (H, dd,
ethanol for 2 h at 4 C. Subsequently they were re-washed three
13
J ¼ 8.5, 4.5 Hz); C NMR (125 MHz, DMSO-d
6
):
d
168.66, 158.86 (d,
times with D-Hank’s buffer and treated with RNase A (100
and propidium iodide (PI) (20 g/mL) for 30 min in the dark.
Finally, cells were analyzed in a FACScan flow cytometer (BD
mg/mL)
J ¼ 232.8 Hz), 158.46 (d, J ¼ 232.4 Hz), 136.08, 135.82, 132.97, 129.73
m
(
d, J ¼ 10.1 Hz), 129.18, 127.74 (d, J ¼ 9.1 Hz), 119.40 (d, J ¼ 2.8 Hz),

H. Zou et al. / European Journal of Medicinal Chemistry 46 (2011) 5970e5977
5977
Biosciences, San Jose, CA, USA). The percentage of cells in G0/G1
phase, S phase, G 2/M and sub-G 1 phase was analyzed. To deter-
mine the cell apoptosis, fluorescent probes Annexin V/propidium
iodide (PI, BioVision, CA, USA) were used following manufacturer’s
instructions.
Major Project “Key New Drug Creation and Manufacturing
Program” of China (No. 2009ZX09501-010) for the financial
support.
Supplementary data
5.6. Immunoblots analyses
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.10.009. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
5
HCT-116 cells (5 ꢃ10 /well) were pre-treated with indicated
concentrations of compound for 30 min followed by 50 ng/mL
VEGF for 2 h. The cells were harvested and re-suspended in lysis
buffer (50 mM TriseHCl, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5%
deoxycholic acid, 0.02% sodium azide, 1% NP-40, 2.0 mg/mL aproti-
nin, 1 mM phenylmethylsulfonylfluoride). The lysates were centri-
fuged at 10,000 ꢃ g for 15 min at 4 C. Equivalent amounts of
References
ꢁ
proteins were analyzed by SDS-PAGE. The following appropriate
primary antibodies to VEGFR and p-VEGFR from Cell Signaling
Technology (Cell Signaling Technology, Beverly, MA) were used.
Proteins were visualized with peroxidase-coupled secondary anti-
body from Southern Biotech (Birmingham, UK), using ECL-plus kit
from Amersham Biosciences (UK) for detection. Actin controls were
used to normalize protein loading.
[
[
[
1] M. Mohammadi, G. McMahon, L. Sun, C. Tang, P. Hirth, B.K. Yeh, S.R. Hubbard,
J. Schlessinger, Science 276 (1997) 955e960.
2] L. Bouérat, J. Fensholdt, X. Liang, S. Havez, S.F. Nielsen, J.R. Hansen, S. Bolvig,
C. Andersson, J. Med. Chem. 48 (2005) 5412e5414.
3] C.V. Galliford, K.A. Scheidt, Angew. Chem., Int. Ed. Engl. 46 (2007) 8748e8758.
[4] V.V. Vintonyak, K. Warburg, H. Kruse, S. Grimme, K. Hübel, D. Rauh,
H. Waldmann, Angew. Chem. Int. Ed. Engl. 49 (2010) 5902e5905.
[
[
5] L. Sun, N. Tran, F. Tang, H. App, P. Hirth, G. McMahon, C. Tang, J. Med. Chem. 41
1998) 2588e2603.
(
6] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J.Y. Fukuda, J.Y. Chu,
A. Nematalla, X. Wang, H. Chen, A. Sistla, T.C. Luu, F. Tang, J. Wei, C. Tang,
J. Med. Chem. 46 (2003) 1116e1119.
5.7. In vitro VEGFR-2 kinase assay
[
7] N. Kammasud, C. Boonyarat, S. Tsunoda, H. Sakurai, I. Saiki, D.S. Griersond,
Into a black 96-well Costar plate was added 2 mL/well of
O. Vajraguptaa, Bioorg. Med. Chem. Lett. 17 (2007) 4812e4818.
compound in DMSO. An amount of 0.5 mmol pGAT-biotin and
e4 ng hVEGFR-2 was added to each well in the presence of 38
of reaction buffer (25 mM Hepes, pH 7.5, 5 mM MgCl , 5 mM MnCl
mM DTT, and 1 mg/mL BSA). The kinase reaction was initiated by
addition of 10 L of 10 M ATP to reaction buffer after 10 min
[8] L. Mologni, R. Rostagno, S. Brussolo, P.P. Knowles, S. Kjaer, J. Murray-Rust,
E. Rosso, A. Zambon, L. Scapozza, N.Q. McDonald, V. Lucchini, C. Gambacorti-
Passerini, Bioorg. Med. Chem. 18 (2010) 1482e1496.
3
mL
2
,
2
[
9] R.R. Khanwelkar, G.S. Chen, H.C. Wang, C.W. Yu, C.H. Huang, O. Lee,
C.H. Chen, C.S. Hwang, C.H. Ko, N.T. Chou, M.W. Lin, L.M. Wang, Y.C. Chen,
T.H. Hseu, C.N. Chang, H.C. Hsu, H.C. Lin, Y.C. Shih, S.H. Chou, H.W. Tseng,
C.P. Liu, C.M. Tu, T.L. Hu, Y.J. Tsai, J.W. Chern, Bioorg. Med. Chem. 18 (2010)
2
m
m
preincubation and the plate is incubated for additional 45 min at
room temperature. The reaction was stopped by the addition of
4674e4686.
[10] A. Andreani, S. Bellini, S. Burnelli, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi,
M. Rambaldi, L. Varoli, N. Calonghi, C. Cappadone, M. Zini, C. Stefanelli,
L. Masotti, R.H. Shoemaker, J. Med. Chem. 53 (2010) 5567e5575.
11] C.C. Chiang, Y.H. Lin, S.F. Lin, C.L. Lai, C. Liu, W.Y. Wei, S.C. Yang, R.W. Wang,
L.W. Teng, S.H. Chuang, J.M. Chang, T.T. Yuan, Y.S. Lee, P. Chen, W.K. Chi,
J.Y. Yang, H.J. Huang, C.B. Liao, J.J. Huang, J. Med. Chem. 53 (2010)
1
00 mM EDTA and 0.36
buffer (50 mM Hepes, pH 7.5, 0.5 M KF, 1 mg/mL BSA). After 30 min,
00 L of 10 nM SV-XL in KF buffer was added, and after an addi-
mg/mL PY20 K in the presence of 50 mL KF
[
1
m
tional 2 h incubation at room temperature, the plate is read on the
BMG Nova star.
5929e5941.
[
12] T.P. Cho, S.Y. Dong, F. Jun, F.J. Hong, Y.J. Liang, X. Lu, P.J. Hua, L.Y. Li, Z. Lei,
H. Bing, Z. Ying, L.F. Qiong, F.B. Bei, L.L. Guang, G.A. Shen, S.G. Hong, S.W. Hong,
M.X. Tai, J. Med. Chem. 53 (2010) 8140e8149.
13] K. Pietras, D. Hanahan, J. Clin. Oncol. 23 (2005) 939e952.
14] M. Atkins, C.A. Jones, P. Kirkpatrick, Nat. Rev. Drug Disc. 5 (2006) 279e280.
15] D.Y. Heng, C. Kollmannsberger, Recent Results Cancer Res. 184 (2010) 71e82.
5.8. Molecular modeling
[
[
[
The crystal structure of VEGFR-2 kinase in complex with a 2-
anilino-5-aryl-oxazole inhibitor was downloaded from the
Protein Data Bank (PDB entry 1Y6A) [30]. All calculations and
manipulations were performed using SYBYL 6.9, running on SGI O2
workstation. Compound 23 was built using the SYBYL Sketcher
model and fully minimized with the Powell method (Tripos force
field and GasteigereHuckel charges) to an energy gradient of
[16] R.J. Motzer, G.R. Hudes, M.S. Ginsberg, M.S. Baum, C.S. Harmon, S.T. Kim,
I. Chen, B.G. Redman, Am. J. Clin. Oncol. 33 (2010) 614e618.
[
17] C.J. Sweeney, E.G. Chiorean, C.F. Verschraegen, F.C. Lee, S. Jones, M. Royce,
L. Tye, K.F. Liau, A. Bello, R. Chao, H.A. Burris, J. Clin. Oncol 28 (2010)
4513e4520.
[
[
18] V. Grünwald, D. Kalanovic, A.S. Merseburger, World J. Urol. 28 (2010)
343e351.
19] J.Y. Merour, B. Joseph, Curr. Org. Chem. 5 (2001) 471e506.
0
.001 kcal/(mol Å). For the FlexX docking calculation, default
[20] H.B. Zou, H.J. Zhu, L. Zhang, L.Q. Yang, Y.P. Yu, J. Stöckigt, Chem. Asian. J. 5
2010) 2400.
(
parameters were used and 30 binding modes were generated and
ranked according to FlexX scoring function. The docking mode was
chosen on the basis of binding affinity rank.
[
[
21] J.P. Sanchez, T.F. Mich, G.G. Huang, J. Heterocyclic. Chem. 31 (1994) 297e303.
22] M. Mor, S. Rivara, C. Silva, F. Bordi, P.V. Plazzi, J. Med. Chem. 41 (1998)
3831e3844.
[
[
[
[
23] N. Finch, M.M. Robison, M.P. Valerio, J. Org. Chem. 37 (1972) 51e54.
24] R.P. Robinson, K.M. Donahue, J. Org. Chem. 56 (1991) 4805e4806.
25] M.T. Cash, P.R. Schreiner, R. Phillips, Org. Biomol. Chem. 3 (2005) 3701e3706.
26] C.Y. Hung, M.H. Hsu, L.J. Huang, C.S. Hwang, O. Lee, C.Y. Wu, C.H. Chen,
S.C. Kuo, Bioorg. Med. Chem. 16 (2008) 4222e4232.
5
.9. Statistical analysis
All data were expressed as mean ꢀ S.D from at least three
[
27] K. Huber, J. Schemies, U. Uciechowska, J.M. Wagner, T. Rumpf,
independent experiments. Differences between groups were
examined for statistical significance using one-way ANOVA analysis
with SPSS 10.0 for WINDOWS. In all cases, P < 0.05 was considered
significant.
F. Lewrick, R. Süss, W. Sippl, M. Jung, F. Bracher, J. Med. Chem. 53 (2010)
1
383e1386.
[28] I.K. Dev, R.E. Dornsife, T.M. Hopper, J.A. Onori, C.G. Miller, L.E. Harrington,
K.M. Dold, R.J. Mullin, J.H. Johnson, R.M. Crosby, A.T. Truesdale, A.H. Epperly,
K.W. Hinkle, M. Cheung, J.A. Stafford, D.K. Luttrell, R. Kumar, Br. J. Cancer 91
(
2004) 1391e1398.
[29] J. Ma, X. Xin, L. Meng, L. Tong, L. Lin, M. Geng, J. Ding, e3774, PLoS One 3
2008).
Acknowledgments
(
[
30] P.A. Harris, M. Cheung, R.N. Hunter 3rd, M.L. Brown, J.M. Veal, R.T. Nolte,
L. Wang, W. Liu, R.M. Crosby, J.H. Johnson, A.H. Epperly, R. Kumar, D.K. Luttrell,
J.A. Stafford, J. Med. Chem. 48 (2005) 1610e1619.
We thank the National Natural Sciences Foundation of China
No. 81072516, 20802066) and National Science & Technology
(