Design, synthesis and biological evaluation of novel C3-functionalized oxindoles as potential Pim-1 kinase inhibitors

Article abstract of DOI:10.1039/c5ra00177c

A novel series of C3-functionalized oxindoles, 3-(2-oxo-4-phenylbut-3-en-1-ylidene)indolin-2-ones, were designed, synthesized and investigated for inhibition of cell proliferation against different types of human cancer cell lines, including SW620, HeLa and A549. This biological study showed that these compounds containing the scaffolds of indole and aromatic α,β-unsaturated ketone had moderate to significant antitumor activities. Further study suggested that compound 4b, as one of this kind of structure derivative, showed broad-spectrum antitumor activities against MCF-7, PC-3, SKOV-3, U87, SMMC-7721, SY5Y and A875 cancer cell lines. Besides, the results of the inhibition of Pim kinases indicated that compound 4b showed selective and efficient anti-Pim-1 kinase activity (IC₅₀ = 5 μM). Docking simulation, flow cytometry (FCM), and Hoechst 33342 staining assay suggested that the most active compound 4b induced cell death through apoptosis via binding to the active ATP pocket of Pim-1. Moreover, it showed that compound 4b had strong inhibition of tubulin polymerization which may be caused by inhibiting Pim-1. This journal is

Full text of DOI:10.1039/c5ra00177c

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Design, synthesis and biological evaluation of novel
C3-functionalized oxindoles as potential Pim-1
kinase inhibitors
Cite this: RSC Adv., 2015, 5, 29456
a
b
a
c
a
*
Hong-bao Sun,† Xiao-yan Wang,† Guo-bo Li, Li-dan Zhang, Jie Liu
a
*
and Li-feng Zhao
A novel series of C3-functionalized oxindoles, 3-(2-oxo-4-phenylbut-3-en-1-ylidene)indolin-2-ones,
were designed, synthesized and investigated for inhibition of cell proliferation against different types of
human cancer cell lines, including SW620, HeLa and A549. This biological study showed that these
compounds containing the scaffolds of indole and aromatic a,b-unsaturated ketone had moderate to
significant antitumor activities. Further study suggested that compound 4b, as one of this kind of
structure derivative, showed broad-spectrum antitumor activities against MCF-7, PC-3, SKOV-3, U87,
SMMC-7721, SY5Y and A875 cancer cell lines. Besides, the results of the inhibition of Pim kinases
indicated that compound 4b showed selective and efficient anti-Pim-1 kinase activity (IC50 ¼ 5 mM).
Docking simulation, flow cytometry (FCM), and Hoechst 33342 staining assay suggested that the most
active compound 4b induced cell death through apoptosis via binding to the active ATP pocket of Pim-
1. Moreover, it showed that compound 4b had strong inhibition of tubulin polymerization which may be
caused by inhibiting Pim-1.
Received 5th January 2015
Accepted 12th March 2015
DOI: 10.1039/c5ra00177c
www.rsc.org/advances
cancer,9 gastric cancer.10 Thus, inhibition of Pim-1 is considered
as a valuable target for therapy of cancer.11,12 It has attracted
Introduction
great interesting in developing novel inhibitors of Pim-1, and a
large amount structurally different small molecule inhibitors
have been obtained, such as SGI-1776,13 SMI-4a,14 DHPCC-9,15
K00486,16 CX-6258,17 Cpd 14 (ref. 18) (Fig. 1). Despite these
successes, only a few of them have been tested in cell-based
assays or the related mechanisms of Pim kinases function to
demonstrate anti-cancer activity. Only understanding the
processes that can be targeted by Pim inhibition is of great
importance for dening the potential activity of these
compounds. Therefore, designing novel Pim-1 inhibitors that
Pim-1, a member of the Pim kinases family (Pim-1, Pim-2 and
Pim-3), is a serine/threonine protein kinase identied by
cloning the retroviral integration sites in Moloney murine
leukemia virus induced lymphomas. Pim-1 has been implicated
to function in several normal biological process including cell
cycle regulation, cell survival, proliferation and differentiation.
It can phosphorylate Cdc25A and Cdc25C to enhance G1/S
transition and G2/M transition, respectively.1,2 The phosphory-
lation of the pro-apoptotic protein Bad at Ser112 induced by
Pim-1 blocks apoptotic cell death.3 Pim-1 also inactivates ASK1
through phosphorylation at Ser83 leading to less caspase3
activation and reducing cell death.4 Moreover, Pim-1 associates
with tubulin and plays a vital role during mitosis.5 However,
when these processes become disrupted or hyper-activated,
Pim-1 expresses several hallmarks of cancer. Numerous
evidences conrmed Pim-1 kinase as a proto-oncogene. Pim-1
was found over-expression in many of human tumor diseases,
such as hematologic malignancies,6,7 bladder cancer,8 prostate
aState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041,
China. E-mail: liujie2011@scu.edu.cn; lifengzhao@scu.edu.cn; Fax: +86-28-8550-
3817; Tel: +86-28-8550-3817
bAnalytical & Testing Center, Sichuan University, Chengdu 610064, China
cCollege of Chemical Engineering, Sichuan University, Chengdu 610064, China
Fig. 1 Small molecule inhibitors targeting Pim family kinases.
† These authors made equal contributions to this work.
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targeted Pim and inhibited Pim kinases related function will
still be a challenge for current anti-cancer drug discovery.
In the past decades, plentiful efforts have been started for
treating a wide range and types of cancer, and chemotherapy
C3-functionalized oxindole is being continuously discovered had become a common treatment option for cancer patients.36
to be at the core of several natural products, and becomes an One of well-known methods for primary anti-cancer drug
emerging new scaffold for drug discovery with potential anti- discovery is combinatorial chemistry.37 As it's a part of our own
cancer and other biological activities.19–22 Among the represen- interest to develop some C3-functionalized oxindoles,38 we
tative examples are spiro-tetrahydrofuryl oxindole as anti- conceived that combine C3-functionlized oxindoles with a,b-
cancer agent,23 spiro-isoxazolidinyl oxindole with anti-cancer unsaturated ketones to obtain a novel series of compounds as
and anti-tubercular activities,24–26 pyrrolidinooxindole-type potential Pim-1 inhibitors with great biological activities, as
alkaloid CPC-1,27 convolutamydine A with potent activity in shown in Fig. 2. Based on our previous synthesis efforts,39
the differentiation of HL-60 cells,28 celogentin K as potential herein we report the efficient synthesis of 3-(2-oxo-4-phenylbut-
inhibitor of tubulin polymerization,29 CX-6258 as Pim-1, 2, 3 3-en-1-ylidene)indolin-2-ones with efficient and broad-
inhibitor,17 maremycins with anti-cancer activity30 (Fig. 2). spectrum anti-tumor activities in vitro and described the
Another privileged skeletons, known chemically as chalcones, structure–activity relationship (SAR). The most effective
are biogenetic precursors of avonoids in higher plants. These compound 4b was selected for further study. It showed selective
structures contain a ketone functionality and an unsaturated and good inhibition against Pim-1 (IC50 ¼ 5 mM). Docking
group (double bond), which are in conjugated arrangement simulation also suggested that Pim-1 is the potential target for
among the naturally occurring or synthetic anti-cancer agents. compound 4b. Further study, including ow cytometry (FCM),
The much exible structure made it easier to combine with Hoechst 33342 staining assay and immunohistochemistry of
different protein receptors and exhibited good anti-canter tubulin indicated that compound 4b caused cell arrest in G2/M
activity (Fig. 2). Chalcone inhibited the proliferation of phase, induced cell apoptosis and had signicant inhibition on
human breast cancer cell (MCF-7) by blocking cell cycle tubulin polymerization, which may be all associated with the
progression and inducing apoptosis.31 Licochalcone A had inhibition of Pim-1.
excellent activities against MCF-7 and HL-60.32 Isoliquiritigenin
(ISL), isolated from Licorice, suppressed pulmonary metastasis
of mouse renal cell carcinoma,33 induced apoptosis and cell
cycle arrest through p53 dependent pathway in HepG2.34 The
plant polyphenol butein, another nature product of derivative of
chalcone, inhibited testosterone-induced proliferation in breast
cancer cells expressing aromatase.35 These privileged structures
provide us new idea for designing novel drugs.
Result and discussion
The synthetic strategy to prepare the target compounds is
depicted in Scheme 1. The key intermediates 3a–y were
prepared from benzylidene acetones and isatins as previously
reported.39 Then, targeted compounds 4a–y were obtained,
respectively, by the reaction with corresponding 3a–y in the
Fig. 2 Biologically important molecules containing C3-functionalized oxindoles or a,b-unsaturated ketones, and the design strategy of
combinatorial chemistry for new compound.
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Scheme 1 Reagents and conditions: (a) CH3COCH3, H2O, 0.5% NaOH, r. t, overnight; (b) 10% L-Arg, MeOH, r. t, overnight; (c) CCl4, PPh3, THF,
reflux, overnight.
presence of CCl4 and PPh3 reuxing in THF, in moderate to high activities on A549, MCF-7, skov-3 and A875 cell lines. It indi-
1
(up to 96%) yields. All the products were characterized by H cated that these compounds had broad-spectrum anti-
NMR, 13C NMR, 19F NMR and ESI-MS analysis.
proliferative activity against human cancers.
All the newly synthesized compounds 4a–y were investigated
At the beginning of design these compounds, our project was
for their anti-proliferative activity on three human cancer cell to develop new Pim-1 inhibitors as anti-tumor agents. Therefore
lines including SW620, HeLa, and A549 in vitro by MTT assay we performed kinase assay to examine whether the compounds
method. The half maximal inhibitory concentrations (IC50) of
the tested compounds were listed in Table 1. These compounds
exhibited good anti-tumor activities against SW620, and most of
them had moderate to remarkable activities against A549. Their
Table 1 In vitro activities of compounds 4a–y
inhibitory effect on HeLa changed obviously with the different
groups in the positions of R1, R2 and R3. The structure–activity
relationships (SAR) could be observed mainly related to the
inuence of various substituents in R1, R2 and R3. The elec-
tronic properties and steric hindrance of the substituents at the
aromatic ring had great inuence on anti-tumor ability. The
comparison of the activities of the pair of compounds 4b/4j and
4c/4h, the rst of each pair component with a substituent at
a
para-position (4b and 4c) and the second with the correspond-
ing substituent at meta-position (4j) or ortho-position (4h),
showed that the compounds with substituents at para-position
had higher activities (4b/4j IC50 ¼ 1.53 and 6.72 mM, 4c/4h IC50
¼ 2.20 and 2.96 mM, respectively). The activities of 4b, 4c and 4d
were 1.53 mM, 2.20 mM, and >20 mM, suggesting that the size of
substituent at para-position had a huge inuence on the
activity, the bigger the substituent was, the lower the activity
would be, and even lost activity. The IC50 of compound 4g was
4.15 mM and 4d was >20 mM, meaning that the activity changed
little with the size of substituent at meta-position changing.
Compounds 4q–t and 4y lost activities but compound 4p had a
good activity of 3.45 mM, indicating that R1 group must be
aromatic ring and there should be no substituent at the
nitrogen.
To further study the anti-proliferative activity of this series of
compounds, 4b, 4c and 4p were selected for evaluation of their
inhibitory activities against a panel of different types of human
cancer cell lines: A549, HeLa, MCF-7, PC-3, SKOV-3, U87,
SMMC-7721, SY5Y, A875. As shown in Table 2, generally
speaking, all these compounds had much stronger inhibition
activities than the positive control cisplatin. Compound 4b (IC50
¼ 1.53 mM, 0.47 mM, 4.67 mM, 3.29 mM and 1.13 mM for HeLa,
PC-3, U87, SMMC-7721 and SY5Y, respectively) showed the best
anti-proliferative activities against the cancer cell lines of HeLa,
PC-3, U87, SMMC-7721 and SY5Y. Meanwhile, compound 4p
(IC50 ¼ 3.21 mM, 4.02 mM, 2.2 mM and 0.22 mM against A549,
MCF-7, skov-3 and A875, respectively) had most effective
IC50 (mM)
Compound R1
R2 R3
SW620 HeLa A549
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.19
0.13
1.68
1.97
1.56
1.98
1.34
1.21
0.57
0.46
0.49
0.76
0.54
2.31
0.91
0.28
2.03
2.54
3.21
3.87
3.18
1.53
2.20
5.03
2.02
3.86
15.41
3.97
4-F–C6H4
4-Cl–C6H4
4-Br–C6H4
4-CH(CH3)2–C6H4
4-MeO–C6H4
3-Br–C6H4
3-Cl–C6H4
3-CF3–C6H4
2-F–C6H4
3,4-DiCl–C6H4
3,4-DiMe–C6H4
2,4-DiCl–C6H4
2-F–4-Br–C6H4
2,3,4-TriMeO–C6H4
2-Naphthyl
2-Furyl
>20
2.64
8.46 17.32
3.15
2.96
6.13
6.72
7.31
3.01
3.15
5.58
8.86
>20
2.43 10.21
>20 4.34
>20
>20
>20
4.95
2.57
3.85
4.92
10.22
>20
7.81
4.56
3.45
>20
>20
>20
>20
2-Thienyl
Cyclohexyl
CH(CH3)2
Ph
Ph
Ph
4s
4t
4u
4v
4w
4x
4y
4-Cl 0.67
6-Cl 0.35
5-Br 3.91
5-Me 0.03
7.96
3.92
7.52 >20
Ph
Ph
>20
>20
3.14
5.50
H
1.10
a
The anti-proliferation activities of compounds 4a–y in vitro were
determined by the MTT assay on Sw620, HeLa, A549 cell lines, IC50
was average of three determinations and deviation from the average
was <5% of the average value.
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Table 2 The anti-proliferation activities of compounds 4b,c, 4p and cisplatin against various cancer cell lines
IC50a (mM)
Compound
A549
HeLa
MCF-7
PC-3
SKOV-3
U87
SMMC-7721
SY5Y
A875
4b
4c
3.53
4.74
3.21
1.53
2.2
3.45
5.02
4.86
10.93
4.02
0.47
3.28
2.47
10
2.39
3.22
2.2
4.67
9.94
9.08
3.29
6.84
4.32
6.35
1.13
1.74
1.16
1.71
1.03
1.78
0.22
4.15
4p
Cisplatinb
10.05
>20
5.25
11.08
a
b
Values were average of three determinations and deviation from the average is <5% of the average value. Positive control.
(4b, 4c, 4p) interact with Pim kinases and inhibit Pim kinases, in cluster I might be the true binding modes of compound 4b with
vitro. Compound 4b (IC50 ¼ 5 mM) showed more effective than Pim-1. Fig. 3a depicted the binding pose of compound 4b with the
compound 4c (IC50 ¼ 7 mM) and 4p (IC50 ¼ 10 mM) against Pim- highest Goldscore value of 48.231. The amide group of compound
1. In addition, the IC50 of compound 4b against Pim-1, -2, and -3 4b was observed to form strong hydrogen-bonding interactions
were 5 mM, >20 mM and 15 mM, respectively. It suggested that with Lys67 and Glu89. The uorine atom of compound 4b formed
compound 4b had selective and good inhibition against Pim-1. halogen-bonding interaction with Leu44. In addition, compound
Based on these results, compound 4b was selected as a lead 4b formed intensive hydrophobic interactions with residues
compound for further study because of its best potency against Leu93, Leu120, Leu44, Val52, Lys67 and Ile185. The interaction
most of the cancer cells and selective inhibition of Pim-1, in vitro. mode between compound 4b and Pim-1 was corresponded to the
For better understanding of the potency of compound 4b and SAR. Then, we analyzed the binding modes of compound 4b with
guiding further SAR studies, we evaluated the interaction between Pim-2 and Pim-3. The results showed that the compound 4b did
compound 4b and Pim kinases (Pim-1, Pim-2 and Pim-3). The not perfectly match with Pim-2 and Pim-3, comparing with Pim-1
compound 4b bound to the active site of ATP and the binding (see Fig. 3b and c). Goldscore for the compound 4b binding with
modes of compound 4b to Pim kinases were exhibited in Fig. 3. Pim-2 and Pim-3 were 45.241 and 41.398, respectively, which
Firstly, we used molecular docking to analyze the binding mode of slightly lower than that with Pim-1. These results may be used for
compound 4b with Pim-1. A total of 20 docking poses were explaining why the inhibitory activity of compound 4b against
generated by GOLD program. According to the RMSD values, two Pim-1 was better than Pim-2 and Pim-3. Finally, we also further
docking pose clusters (cluster I and cluster II) were obtained. predicted the other possible kinase targets for compound 4b using
Cluster I contained 16 docking poses, while cluster II only had 4 a reverse molecule docking method.40 The top 10 kinases ranked
docking poses. Therefore, we considered that the docking poses in by the reverse molecule docking method were AKT-1, BCR-ABL,
CLK3, KIT, CK2, MAPK14, PEPCK, ALK5, CaMKK2, TGFR1,
respectively. These kinases may be the targets of compound 4b.
Next, compound 4b was used to evaluate other biological
functions associated with Pim-1. Flow cytometric analysis was
used to quantitatively assess and the cell cycle aer PI-stained.
As shown in Fig. 4a signicant accumulation of cells in the S
phase, accompanied by a decrease in G0/G1 phase was observed
aer treatment with compound 4b in HeLa cancer cells at 1 mM.
However a large amount of tetraploid and polyploidies were
appeared in contrast with a sharply decreasing cells in G0/G1
phase and S phase from 2 mM to 3 mM. The results suggested
that compound 4b arrested the cell divided during the DNA
replicating. In addition, uorescence microscopic examination
of Hoechst 33342 stained cell conrmed the inducing apoptosis
effect of compound 4b. The apoptotic cells are oen charac-
terized by its morphological, including cell shrinkage,
membrane blabbing, chromatin condensation and DNA frag-
mentation. Aer treated with compound 4b for 48 hours, HeLa
cells shrank and rounded up. With the increasing of concen-
tration, it became more signicant. In contrast, the untreated
HeLa cells retained their normal size and shape, kept prolifer-
Fig. 3 Molecular docking compound 4b with Pim kinases (a) binding
mode of compound 4b (represented with stick) to the ATP binding
ating with time and became over-crowded by 36 hours (Fig. 5A).
Morphological changes were also characteristic of apoptosis
aer Hoechst 33342 staining. Apoptosis cells, bright blue
pocket of Pim-1 (PDB ID: 1XWS). (b) Binding mode of compound 4b
(represented with stick) to the ATP binding pocket of Pim-2. (c) Binding
mode of compound 4b (represented with stick) to the ATP binding
pocket of Pim-3.
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Fig. 4 Effect of compound 4b on cell cycle distribution: DNA fluorescence histograms of PI-stained HeLa cells. Cells were treated with 1 mM, 2
mM, 3 mM for 48 h.
uorescent and condensed nuclei, were observed in a large cytometry in HeLa cells. As shown in Fig. 6, HeLa cells were
number of treated cells in HeLa, and the change was treated with 4b at the concentrations of 0, 2, 4 and 8 mM for 48 h.
concentration-dependent, whereas there was no signicant We found an obvious concentration-dependent reduction in the
apoptosis in untreated HeLa cells that showed blue, diffusely percentage of surviving cells. As the concentration of compound
stained intact nuclei (Fig. 5B). These results suggested that 4b increased, the early and the late stage apoptotic cells
compound 4b induced apoptosis in HeLa cells.
increased from 9.04% at 2 mM to 62.31% at 8 mM. This result
To further test the apoptosis inducing ability of 4b, an indicated that compound 4b induced apoptosis of HeLa cells in
Annexin-V/PI uorescence staining was analyzed by ow a dose-dependent manner.
Fig. 5 Effect of compound 4b on cell morphology: (A) bright-field microscopy images and (B) fluorescence microscopic appearance of Hoechst
33342 staining nuclei of HeLa cells, after incubation with compound 4b for 48 h at varying concentrations: 2 mM, 4 mM, 8 mM. Apoptosis cells were
observed in treated cells containing condensed and fragmented fluorescent nuclei.
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Fig. 6 Compound 4b induces apoptosis in HeLa cells. Flow cytometric analysis of cells stained with Annexin V-FITC/PI after treatment with
various concentrations (0 mM, 2 mM, 4 mM and 8 mM) of compound 4b for 48 hours.
We also validated the effect of compound 4b on cellular
tubulin immunohistochemistry. Studies were carried out to
examine the in situ effects of compound 4b on cellular microtu-
bules in HeLa cells. HeLa cells seeded on sterile cover slips were
treated with compound 4b at 2 mM for 48 h. As shown in Fig. 7,
untreated HeLa cells displayed the normal distribution of
microtubules. However, cells treated with compound 4b showed
disrupted microtubule organization, thus demonstrated the
inhibition of tubulin polymerization, inuenced the mitosis and
affected the cell division in an orderly and programmed manner.
This process may be all associated with the inhibition of Pim-1.
In dividing cells, Pim-1 is located in the mitotic spindle and
specically the spindle poles, which plays the key role of inu-
encing the function of mitotic spindle.5 When Pim-1 was
inhibited by compound 4b, the function of spindle was disturbed
and then the microtubule constituted spindle bers was disorder
just as observed in Fig. 7. Finally the cells were arrested in G2/M
phase and apoptosis. The result also explained the appearance of
polyploidy in ow cytometric analysis.
Fig. 7 HeLa cells were treated with compounds 4b with 8 mM for 24 h
followed by staining with a-tubulin antibody. Chromosomal DNA
(blue) was stained with DAPI. Microtubule organization was clearly
observed by green tubulin network-like structures in control cells, and
Experimental
Materials and measurements
Melting points were recorded at SGW X-4 Melting point
was found to be disrupted in cells treated with compound 4b.
instrument (Shanghai precision & scientic instrument Co.,
1
Ltd, Shanghai, China). H NMR spectra were recorded at 400
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MHz. The chemical shis were recorded in ppm relative to 6.97 (t, J ¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100
tetramethylsilane and with the solvent resonance as the internal MHz, DMSO) d 190.1, 168.6, 144.8, 144.6, 136.0, 134.1, 133.1,
standard. Data were reported as follows: chemical shi, multi- 131.1, 129.1, 128.8, 127.80, 127.5, 127.2, 121.8, 120.1, 110.3; ESI-
plicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ MS: m/z 274.1 [M ꢁ H]ꢁ.
multiplet), coupling constants (Hz), integration. 13C NMR data
were collected at 100 MHz with complete proton decoupling.
Chemical shis were reported in ppm from the tetramethylsi-
lane with the solvent resonance as internal standard. 19F NMR
data were collected at 376 MHz with complete proton decou-
pling. ESI-HRMS spectra were recorded on a commercial
apparatus and methanol was used to dissolve the sample. All
chemicals were obtained from commercial sources and used
without further purication. Column chromatography was
carried out on silica gel (300–400 mesh, Qingdao Marine
Chemical Ltd, Qingdao, China). Thin layer chromatography
(TLC) was performed on TLC silica gel 60 F254 plates.
(Z)-3-((E)-4-(4-Fluorophenyl)-2-oxobut-3-en-1-ylidene)indolin-
2-one (4b)
Red solid, yield 96%, m. p. 204–205 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.77 (s, 1H), 8.36 (d, J ¼ 8.0 Hz, 1H), 7.90 (dd, J ¼ 8.4,
6.0 Hz, 2H), 7.79 (d, J ¼ 16.0 Hz, 1H), 7.42–7.26 (m, 5H), 6.99 (t, J
¼ 8.0 Hz, 1H), 6.88 (d, J ¼ 8.0 Hz, 1H); 13C-NMR (100 MHz,
DMSO) d 189.9, 168.5, 163.6 (d, J ¼ 248 Hz), 145.0, 143.2, 136.2,
133.1, 131.3 (d, J ¼ 8.6 Hz), 131.0 (d, J ¼ 2.9 Hz), 127.8, 127.7 (d, J
¼ 2.3 Hz), 127.4, 121.7, 120.3, 116.1 (d, J ¼ 21.8 Hz), 110.3; 19F-
NMR (376 MHz, DMSO) d ꢁ108.9; ESI-MS: m/z 292.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-(4-Chlorophenyl)-2-oxobut-3-en-1-ylidene)indolin-
2-one (4c)
General procedure for the synthesis of benzylidene acetones
(2a–t)
Red solid, yield 93%, m. p. 215–216 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.78 (s, 1H), 8.38 (d, J ¼ 7.6 Hz, 1H), 7.86 (d, J ¼ 8.4
Hz, 2H), 7.78 (d, J ¼ 16.0 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 2H), 7.45–
7.35 (m, 3H), 7.00 (t, J ¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-
NMR (100 MHz, DMSO) d 189.8, 168.4, 145.1, 142.9, 136.3,
135.5, 133.3, 133.2, 130.6, 129.1, 128.4, 127.6, 127.4, 121.7,
120.2, 110.3; ESI-MS: m/z 308.2 [M ꢁ H]ꢁ.
To a stirred solution of 1a–t (10 mmol) in 2 mL acetone and 1
mL H2O was added 0.25 mL 10% NaOH aqua and stirred at
room temperature for 12 hours. Then the solvent was removed
under reduced pressure and the residue was extracted with
acetic ether. The combined organic layers were dried (MgSO4),
ltered and the ltrate was concentrated under reduced pres-
sure. The crude product was used for next step.
(Z)-3-((E)-4-(4-Bromophenyl)-2-oxobut-3-en-1-ylidene)indolin-
2-one (4d)
General procedure for the synthesis of 3-hydroxy-3-(2-oxo-4-
phenylbut-3-en-1-yl)indolin-2-ones (3a–y)
Red solid, yield 92%, m. p. 222–223 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.78 (s, 1H), 8.38 (d, J ¼ 7.6 Hz, 1H), 7.78 (dd, J ¼ 12.4,
8.0 Hz, 3H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.48–7.33 (m, 3H), 7.00 (t, J
¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz,
DMSO) d 189.81, 168.4, 145.1, 143.0, 136.3, 133.6, 133.2, 132.0,
130.8, 128.4, 127.6, 127.4, 124.4, 121.7, 120.2, 110.3; ESI-MS: m/z
352.2 [M ꢁ H]ꢁ.
A mixture of isatin (0.1 mmol), (E)-4-phenylbut-3-en-2-one (2a,
0.1 mmol), arginine (0.01 mmol) in MeOH (1.0 mL) was stirred
for 48 hours at 25 ꢀC. Aer completion of the reaction (TLC), the
solvent was removed under vacuum. The crude product was
subjected to column chromatography on silica gel using
petroleum ether/ethyl acetate ¼ 1 : 1 as the eluent to give 3a.
Compounds 3b–y were synthesized by a similar procedure as
described for compound 3a. For the separation of these
compounds, the eluent of silica gel column chromatography
consisted of appropriate mixtures of petroleum ether and ethyl
acetate.
(Z)-3-((E)-4-(4-Isopropylphenyl)-2-oxobut-3-en-1-ylidene)
indolin-2-one (4e)
Red solid, yield 91%, m. p. 184–185 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.77 (s, 1H), 8.37 (d, J ¼ 7.6 Hz, 1H), 7.76 (dd, J ¼ 12.0,
10.4 Hz, 3H), 7.42 (s, 1H), 7.39–7.27 (m, 4H), 6.99 (t, J ¼ 7.6 Hz,
1H), 6.89 (d, J ¼ 7.6 Hz, 1H), 2.92 (m, 1H), 1.22 (d, J ¼ 6.8 Hz,
6H); 13C-NMR (100 MHz, DMSO) d 189.9, 168.5, 151.9, 145.0,
144.5, 136.0, 133.0, 132.0, 129.1, 127.9, 127.3, 127.0, 126.9,
121.7, 120.3, 110.2, 33.4, 23.5; ESI-MS: m/z 316.1 [M ꢁ H]ꢁ.
General procedure for the synthesis of 3-(2-oxo-4-phenylbut-3-
en-1-ylidene)indolin-2-ones (4a–y)
Compound 3a–y (0.2 mmol) was dissolved in anhydrous THF (4
mL). Ph3P (78.7 mg, 0.3 mmol) and CCl4 (0.1 mL) were then
added. The solution was heated to reux for 12 hours. Aer
completion of the reaction (TLC), the solvent was removed
under vacuum. The crude product was subjected to column
chromatography on silica gel using petroleum ether/ethyl
acetate ¼ 2 : 1 as the eluent to give 4a–y.
(Z)-3-((E)-4-(4-Methoxyphenyl)-2-oxobut-3-en-1-ylidene)
indolin-2-one (4f)
Red solid, yield 65%, m. p. 180–181 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.76 (s, 1H), 8.36 (d, J ¼ 7.6 Hz, 1H), 7.84–7.72 (m,
3H), 7.40–7.33 (m, 2H), 7.27 (d, J ¼ 16.0 Hz, 1H), 7.06–6.95 (m,
3H), 6.88 (d, J ¼ 7.6 Hz, 1H), 3.83 (s, 3H); 13C-NMR (100 MHz,
(Z)-3-((E)-2-Oxo-4-phenylbut-3-en-1-ylidene)indolin-2-one (4a)
Red solid, yield 89%, m. p. 176–177 ꢀC; 1H-NMR (400 MHz, DMSO) d 189.7, 168.5, 161.7, 144.9, 144.6, 135.7, 132.9, 130.9,
DMSO) d 10.78 (s, 1H), 8.29 (d, J ¼ 8.0 Hz, 1H), 7.74 (dd, J ¼ 10.0, 128.2, 127.3, 126.8, 125.4, 121.7, 120.3, 114.6, 110.2, 55.4; ESI-
6.0 Hz, 3H), 7.44–7.38 (m, 4H), 7.32 (dd, J ¼ 21.2, 12.0 Hz, 2H), MS: m/z 304.0 [M ꢁ H]ꢁ.
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(Z)-3-((E)-4-(3-Bromophenyl)-2-oxobut-3-en-1-ylidene)indolin-
2-one (4g)
(Z)-3-((E)-4-(3,4-Dimethylphenyl)-2-oxobut-3-en-1-ylidene)
indolin-2-one (4l)
Red solid, yield 84%, m. p. 192–193 ꢀC; 1H-NMR (400 MHz, Red solid, yield 74%, m. p. 198–199 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.78 (s, 1H), 8.39 (d, J ¼ 7.6 Hz, 1H), 8.10 (s, 1H), 7.83 DMSO) d 10.76 (s, 1H), 8.36 (d, J ¼ 7.6 Hz, 1H), 7.72 (d, J ¼ 16.0
(d, J ¼ 7.6 Hz, 1H), 7.76 (d, J ¼ 16.0 Hz, 1H), 7.65 (dd, J ¼ 8.0, 0.8 Hz, 1H), 7.61 (s, 1H), 7.53 (d, J ¼ 7.6 Hz, 1H), 7.44–7.28 (m, 3H),
Hz, 1H), 7.50 (d, J ¼ 16.0 Hz, 1H), 7.46–7.34 (m, 3H), 7.00 (t, J ¼ 7.22 (d, J ¼ 7.6 Hz, 1H), 6.99 (t, J ¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6
7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz, DMSO) Hz, 1H), 2.26 (s, 6H); 13C-NMR (100 MHz, DMSO) d 189.7, 168.4,
d 189.8, 168.4, 145.1, 142.5, 136.9, 136.4, 133.33, 133.2, 131.2, 144.9, 144.7, 140.1, 136.9, 135.9, 132.9, 131.9, 130.1, 129.8,
131.0, 129.1, 127.9, 127.6, 127.4, 122.4, 121.7, 120.2, 110.3. ESI- 127.9, 127.3, 126.7, 126.6, 121.7, 120.3, 110.2, 19.5, 19.2; ESI-MS:
MS: m/z 352.0 [M ꢁ H]ꢁ.
m/z 302.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-(3-Chlorophenyl)-2-oxobut-3-en-1-ylidene)indolin-
(Z)-3-((E)-4-(2,4-Dichlorophenyl)-2-oxobut-3-en-1-ylidene)
2-one (4h)
indolin-2-one (4m)
Red solid, yield 83%, m. p. 192–193 ꢀC; 1H-NMR (400 MHz, Red solid, yield 78%, m. p. 214–215 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.77 (s, 1H), 8.39 (d, J ¼ 7.6 Hz, 1H), 7.95 (s, 1H), 7.78 DMSO) d 10.80 (s, 1H), 8.45 (d, J ¼ 7.6 Hz, 1H), 8.09 (d, J ¼ 8.4 Hz,
(dd, J ¼ 11.6, 8.4 Hz, 2H), 7.58–7.44 (m, 3H), 7.44–7.30 (m, 2H), 1H), 7.93 (d, J ¼ 16.0 Hz, 1H), 7.79 (d, J ¼ 2.0 Hz, 1H), 7.66–7.51
7.00 (t, J ¼ 7.6 Hz, 1H), 6.89 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 (m, 2H), 7.43–7.33 (m, 2H), 7.01 (t, J ¼ 7.6 Hz, 1H), 6.89 (d, J ¼ 7.6
MHz, DMSO) d 189.8, 168.4, 145.1, 142.5, 136.6, 136.4, 133.8, Hz, 1H); 13C-NMR (100 MHz, DMSO) d 189.4, 168.4, 145.2, 136.7,
133.2, 130.8, 130.4, 129.1, 128.3, 127.5, 127.5, 127.4, 121.7, 134.4, 133.4, 130.6, 130.6, 130.2, 130.1, 128.4, 128.3, 127.5, 127.4,
120.2, 110.3; ESI-MS: m/z 308.1 [M ꢁ H]ꢁ.
121.8, 120.2, 119.8, 110.3; ESI-MS: m/z 342.0 [M ꢁ H]ꢁ.
(Z)-3-((E)-2-Oxo-4-(3-(triuoromethyl)phenyl)but-3-en-1-
(Z)-3-((E)-4-(4-Bromo-2-uorophenyl)-2-oxobut-3-en-1-ylidene)
ylidene)indolin-2-one (4i)
indolin-2-one (4n)
Red solid, yield 79%, m. p. 178–179 ꢀC; 1H-NMR (400 MHz, Red solid, yield 75%, m. p. 201–202 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.79 (s, 1H), 8.32 (d, J ¼ 7.6 Hz, 1H), 8.15 (s, 1H), 8.06 DMSO) d 10.79 (s, 1H), 8.41 (d, J ¼ 7.6 Hz, 1H), 7.93 (t, J ¼ 8.0 Hz,
(d, J ¼ 7.6 Hz, 1H), 7.84–7.76 (m, 2H), 7.66 (t, J ¼ 7.6 Hz, 1H), 1H), 7.72 (dd, J ¼ 12.4, 7.6 Hz, 2H), 7.53 (t, J ¼ 12.8 Hz, 2H),
7.48 (d, J ¼ 16.0 Hz, 1H), 7.41–7.29 (m, 2H), 6.97 (dd, J ¼ 11.2, 7.44–7.31 (m, 2H), 7.00 (t, J ¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz,
4.4 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz, DMSO) 1H); 13C-NMR (100 MHz, DMSO) d 189.4, 168.4, 160.6 (d, J ¼
d 190.0, 168.6, 144.9, 142.5, 136.4, 135.3, 133.3, 132.4, 130.1, 254.8 Hz), 145.2, 136.7, 134.4 (d, J ¼ 2.5 Hz), 133.4, 130.6 (d, J ¼
130.0, 129.3, 127.5, 127.3, 127.1 (q, J ¼ 6.3 Hz), 125.3 (q, J ¼ 7.4 2.6 Hz), 130.2 (d, J ¼ 4.7 Hz), 128.4 (d, J ¼ 3.0 Hz), 127.5, 127.4,
Hz), 121.9, 120.1, 110.4, 48.6; 19F-NMR (376 MHz, DMSO) d 124.7 (d, J ¼ 10.0 Hz) 121.8, 121.5 (d, J ¼ 11.4 Hz), 120.2, 119.7
ꢁ61.2; ESI-MS: m/z 342.1 [M ꢁ H]ꢁ.
(d, J ¼ 25.5 Hz), 110.3; 19F-NMR (376 MHz, DMSO) d ꢁ112.4;
ESI-MS: m/z 370.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-(2-Fluorophenyl)-2-oxobut-3-en-1-ylidene)indolin-
2-one (4j)
(Z)-3-((E)-2-Oxo-4-(2,3,4-trimethoxyphenyl)but-3-en-1-ylidene)
indolin-2-one (4o)
Red solid, yield 94%, m. p. 186–187 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.79 (s, 1H), 8.32 (d, J ¼ 7.6 Hz, 1H), 7.88 (t, J ¼ 7.6 Hz, Red solid, yield 75%, m. p. 178–179 ꢀC; 1H-NMR (400 MHz,
1H), 7.76 (d, J ¼ 16.0 Hz, 1H), 7.59–7.17 (m, 6H), 6.97 (t, J ¼ 7.6 DMSO) d 10.78 (s, 1H), 8.31 (d, J ¼ 7.6 Hz, 1H), 7.84 (d, J ¼ 16.0
Hz, 1H), 6.87 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz, DMSO) d Hz, 1H), 7.60 (d, J ¼ 8.8 Hz, 1H), 7.40–7.29 (m, 2H), 7.25 (d, J ¼
189.7, 168.6, 160.9 (d, J ¼ 250.6 Hz), 144.9, 136.4, 135.8 (d, J ¼ 16.0 Hz, 1H), 6.98 (t, J ¼ 7.6 Hz, 1H), 6.93–6.82 (m, 2H), 3.84 (s,
3.3 Hz), 133.3, 133.1 (d, J ¼ 9.0 Hz), 129.4 (d, J ¼ 4.8 Hz), 129.2 3H), 3.82 (s, 3H), 3.80 (s, 3H); 13C-NMR (100 MHz, DMSO) d
(d, J ¼ 1.7 Hz), 127.5, 127.4, 125.1 (d, J ¼ 3.1 Hz), 121.9, 121.8 (d, 189.9, 168.7, 156.2, 153.2, 144.6, 141.7, 139.1, 135.7, 133.0,
J ¼ 11.2 Hz), 120.0, 116.1 (d, J ¼ 21.4 Hz), 110.4; 19F-NMR (376 128.4, 127.3, 125.9, 123.7, 121.9, 120.4, 120.1, 110.3, 108.6, 61.5,
MHz, DMSO) d ꢁ115.4; ESI-MS: m/z 292.1 [M ꢁ H]ꢁ.
60.5, 56.0; ESI-MS: m/z 364.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-(3,4-Dichlorophenyl)-2-oxobut-3-en-1-ylidene)
(Z)-3-((E)-4-(Naphthalen-2-yl)-2-oxobut-3-en-1-ylidene)indolin-
indolin-2-one (4k)
2-one (4p)
Red solid, yield 77%, m. p. 221–222 ꢀC; 1H-NMR (400 MHz, Red solid, yield 93%, m. p. 199–200 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.79 (s, 1H), 8.39 (d, J ¼ 7.6 Hz, 1H), 8.17 (d, J ¼ 2.0 DMSO) d 10.80 (s, 1H), 8.33 (d, J ¼ 7.6 Hz, 1H), 8.27 (s, 1H), 7.97–
Hz, 1H), 7.83 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.75 (t, J ¼ 12.4 Hz, 2H), 7.89 (m, 5H), 7.64–7.51 (m, 2H), 7.45 (d, J ¼ 13.6 Hz, 2H), 7.35 (t,
7.52 (d, J ¼ 16.0 Hz, 1H), 7.43–7.34 (m, 2H), 7.00 (t, J ¼ 7.6 Hz, J ¼ 7.6 Hz, 1H), 6.99 (t, J ¼ 7.6 Hz, 1H), 6.89 (d, J ¼ 7.6 Hz, 1H);
1H), 6.89 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz, DMSO) d 189.7, 13C-NMR (100 MHz, DMSO) d 190.0, 168.6, 144.8, 144.7, 136.0,
168.4, 145.1, 141.4, 136.5, 135.3, 133.3, 133.1, 131.8, 131.1, 134.0, 133.1, 132.8, 131.8, 131.2, 128.7, 128.6, 127.9, 127.8,
130.5, 129.6, 128.8, 127.4, 127.4, 121.7, 120.2, 110.3; ESI-MS: m/z 127.7, 127.4, 127.3, 126.9, 123.9, 121.9, 120.1, 110.4; ESI-MS: m/z
342.1 [M ꢁ H]ꢁ.
324.1 [M ꢁ H]ꢁ.
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3H), 7.51–7.36 (m, 5H), 7.07 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.91 (t, J ¼
5.6 Hz, 1H); 13C-NMR (100 MHz, DMSO) d 189.7, 168.5, 146.4,
144.6, 137.1, 135.1, 134.2, 131.1, 129.0, 128.9, 128.8, 128.2,
127.7, 121.5, 119.1, 110.3; ESI-MS: m/z 308.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-(Furan-2-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one
(4q)
Red solid, yield 88%, m. p. 184–185 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.76 (s, 1H), 8.29 (d, J ¼ 6.0 Hz, 1H), 7.87 (s, 1H), 7.57
(d, J ¼ 15.6 Hz, 1H), 7.32 (d, J ¼ 6.8 Hz, 2H), 7.12–6.82 (m, 4H),
6.66 (s, 1H); 13C-NMR (100 MHz, DMSO) d 189.4, 168.6, 150.5,
146.8, 144.7, 135.9, 133.1, 130.9, 127.7, 127.3, 124.2, 121.8,
120.1, 118.3, 113.4, 110.3; ESI-MS: m/z 264.1[M ꢁ H]ꢁ.
(Z)-5-Bromo-3-((E)-2-oxo-4-phenylbut-3-en-1-ylidene)indolin-2-
one (4w)
Red solid, yield 91%, m. p. 193–194 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.93 (s, 1H), 8.61 (d, J ¼ 1.6 Hz, 1H), 7.94–7.74 (m,
3H), 7.55 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.49–7.43 (m, 5H), 6.86 (d, J ¼
8.4 Hz, 1H); 13C-NMR (100 MHz, DMSO) d 189.7, 168.0, 144.9,
144.2, 135.3, 135.3, 134.3, 131.1, 129.7, 129.2, 129.0, 128.9,
127.6, 122.1, 113.2, 112.2; ESI-MS: m/z 352.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-2-Oxo-4-(thiophen-2-yl)but-3-en-1-ylidene)indolin-2-
one (4r)
Red solid, yield 87%, m. p. 177–178 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.75 (s, 1H), 8.36 (d, J ¼ 7.6 Hz, 1H), 7.98 (d, J ¼ 15.6
Hz, 1H), 7.83 (d, J ¼ 5.2 Hz, 1H), 7.68 (d, J ¼ 3.6 Hz, 1H), 7.41–
7.31 (m, 2H), 7.21 (dd, J ¼ 4.8, 3.6 Hz, 1H), 7.04 (d, J ¼ 15.8 Hz,
1H), 6.99 (t, J ¼ 7.6 Hz, 1H), 6.88 (d, J ¼ 7.6 Hz, 1H); 13C-NMR
(100 MHz, DMSO) d 189.2, 168.5, 144.9, 139.4, 137.2, 135.9,
133.6, 133.0, 131.3, 128.9, 127.8, 127.4, 126.2, 121.7, 120.3,
110.2; ESI-MS: m/z 280.0 [M ꢁ H]ꢁ.
(Z)-5-Methyl-3-((E)-2-oxo-4-phenylbut-3-en-1-ylidene)indolin-
2-one (4x)
Red solid, yield 81%, m. p. 169–170 ꢀC; 1H-NMR (400 MHz,
DMSO) d 10.67 (s, 1H), 8.24 (s, 1H), 7.86–7.77 (m, 3H), 7.50–7.45
(m, 3H), 7.41 (d, J ¼ 14.8 Hz, 2H), 7.18 (dd, J ¼ 8.0, 0.8 Hz, 1H),
6.78 (d, J ¼ 8.0 Hz, 1H), 2.27 (s, 3H); 13C-NMR (100 MHz, DMSO)
d 189.8, 168.5, 144.3, 142.8, 136.5, 134.3, 133.5, 130.9, 130.4,
129.0, 128.9, 127.8, 127.7, 127.6, 120.3, 109.9, 20.7; ESI-MS: m/z
288.1 [M ꢁ H]ꢁ.
(Z)-3-((E)-4-Cyclohexyl-2-oxobut-3-en-1-ylidene)indolin-2-one
(4s)
Red solid, yield 73%, m. p. 196–197 ꢀC; 1H-NMR (400 MHz,
DMSO) d 11.21 (s, 1H), 7.55 (d, J ¼ 7.6 Hz, 1H), 7.40 (s, 1H), 7.20
(t, J ¼ 7.6 Hz, 1H), 7.03 (t, J ¼ 7.6 Hz, 1H), 6.89 (d, J ¼ 7.6 Hz,
1H), 6.27 (d, J ¼ 12.0 Hz, 1H), 6.20 (d, J ¼ 12.0 Hz, 1H), 2.31–2.21
(m, 1H), 1.65–1.47 (m, 4H), 1.36–1.07 (m, 6H); 13C-NMR (100
MHz, DMSO) d 189.9, 170.2, 150.7, 147.6, 140.0, 135.4, 128.3,
124.3, 122.5, 119.4, 118.1, 110.2, 37.4, 29.2, 28.38, 27.6, 26.8,
26.1; ESI-MS: m/z 280.1 [M ꢁ H]ꢁ.
(Z)-1-Methyl-3-((E)-2-oxo-4-phenylbut-3-en-1-ylidene)indolin-
2-one (4y)
Red solid, yield 90%, m. p. 114–115 ꢀC; 1H-NMR (400 MHz,
DMSO) d 8.30 (d, J ¼ 7.6 Hz, 1H), 7.81–7.72 (m, 3H), 7.49–7.40
(m, 5H), 7.32 (d, J ¼ 16.0 Hz, 1H), 7.03 (dd, J ¼ 14.8, 7.6 Hz, 2H),
3.16 (s, 4H); 13C-NMR (100 MHz, DMSO) d 190.1, 167.2, 145.8,
144.9, 135.0, 134.1, 133.0, 131.2, 129.1, 128.9, 128.4, 127.4,
126.8, 122.4, 119.4, 109.1, 26.1; ESI-MS: m/z 290.3 [M + H]+.
(Z)-3-((E)-5-Methyl-2-oxohex-3-en-1-ylidene)indolin-2-one (4t)
Red solid, yield 69%, m. p. 180–181 ꢀC; 1H-NMR (400 MHz,
DMSO) d 11.20 (s, 1H), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.40 (s, 1H), 7.19
(t, J ¼ 7.6 Hz, 1H), 7.03 (t, J ¼ 7.6 Hz, 1H), 6.89 (d, J ¼ 7.6 Hz,
1H), 6.32 (d, J ¼ 10.8 Hz, 1H), 6.14 (d, J ¼ 12.0 Hz, 1H), 2.50 (m,
1H), 1.89 (s, 3H), 1.84 (s, 3H); 13C-NMR (100 MHz, DMSO) d
170.2, 150.3, 140.0, 139.5, 135.4, 128.3, 124.3, 122.63, 121.1,
119.5, 118.8, 110.2, 59.7, 26.6, 18.8; ESI-MS: m/z 240.2 [M ꢁ H]ꢁ.
Anti-tumor assay
Briey, targeted cancer cells lines (3 ꢂ 103 per well) were seeded
in 96-well plates and cultured for 24 h, followed by compounds
treatment for 48 h. A volume of 20 mL of 5 mg mLꢁ1 MTT was
added per well and incubated for another 2 h at 37 ꢀC, then the
supernatant uid was removed and DMSO was added 150 mL
per well for 15–20 min. The absorptions (OD) were measured at
570 nm with Spectra MAX M5 microplate spectrophotometer
(Molecular Devices). The effect of compounds on tumor cells
viability was expressed by IC50 of each cell line. Each assay was
carried out at least three times. The results are summarized in
Tables 1 and 2.
(Z)-4-Chloro-3-((E)-2-oxo-4-phenylbut-3-en-1-ylidene)indolin-
2-one (4u)
1
ꢀ
Yellow solid, yield 95%, m. p. 217–218 C; H-NMR (400 MHz,
DMSO) d 10.90 (s, 1H), 7.80–7.69 (m, 3H), 7.57 (d, J ¼ 16.4 Hz,
1H), 7.43 (dd, J ¼ 5.2, 1.6 Hz, 3H), 7.32 (t, J ¼ 8.0 Hz, 1H), 7.07 (t,
J ¼ 12.4 Hz, 2H), 6.86 (d, J ¼ 7.6 Hz, 1H); 13C-NMR (100 MHz,
DMSO) d 194.9, 165.7, 144.7, 144.2, 136.2, 134.3, 131.7, 130.7,
130.2, 129.2, 128.9, 128.6, 126.8, 122.7, 117.9, 109.0; ESI-MS: m/z
308.2 [M ꢁ H]ꢁ.
Docking simulation
The molecular docking method was used for the analysis of
compound 4b binding with Pim-1, Pim-2 and Pim-3. The GOLD
docking program (version 5.0) was adopted here. The X-ray
crystal structure of Pim-1 (PDB ID: 1XWS) was taken from
PDB database, and the 3D structure of Pim-2 and Pim-3 were
built using the homology modelling module in Discovery Studio
(Z)-6-Chloro-3-((E)-2-oxo-4-phenylbut-3-en-1-ylidene)indolin-
2-one (4v)
Red solid, yield 94%, m. p. 229–230 ꢀC; 1H-NMR (400 MHz, 3.1. The preparation of protein structure, including adding
DMSO) d 11.01 (s, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H), 7.83–7.78 (m, hydrogen atoms, removing water molecules, and assigning
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Charmm force eld, were carried out by using Discovery Studio followed by incubation with a primary anti-tubulin (mouse
3.1 soware package. A sphere containing the residues in ATP monoclonal) antibody and FITC conjugated secondary mouse
binding pocket of protein structure were dened as the binding anti IgG antibody. Photographs were taken using the confocal
site. Goldscore was selected as the score function, and the other microscope, equipped with FITC settings and the pictures were
parameters were set as default. For each docking study, a total analyzed for the integrity of microtubule network.
of 20 docking poses were retained. The root-mean square
deviation (RMSD) between docking poses were calculated.
Conclusions
A reverse docking method was used for predicting the other
possible kinase targets for compound 4b. Detailed description
of the reverse docking method please see ref. 40 (Journal of
Molecular Graphics and Modelling, 2013, 44, 278–285). Here the
In conclusion, we have designed and synthesized a new series
C3-functionalized
oxindoles,
3-(2-oxo-4-phenylbut-3-en-1-
ylidene)indolin-2-ones, and evaluated their anti-cancer activi-
ties against three cancer cell lines. Most of these compounds
exhibited pronounced anti-tumor with IC50 values ranging from
0.03 to 17.32 mM. The most potent compound 4b displayed
remarkable inhibitory activities against a panel of different
types of human cancer cell lines with IC50 values in the low
micromolar range and showed much stronger effect than
Cisplatin, as a positive control, on each cell line. The inhibition
of Pim-1 and the molecular docking to Pim-1, suggested that
compound 4b was a potential Pim-1 inhibitor. Furthermore,
ow cytometry (FCM), Hoechst 33342 staining assay and
immunohistochemistry of tubulin indicated that compound 4b
induces cell death by apoptosis and compound 4b leads to
apoptotic cell death by inhibition of tubulin polymerization and
inuencing cell division, which were accordant to the biological
phenomena resulted from the inhibition of Pim-1. Based on
these results, it's evident that the novel series of 3-(2-oxo-4-
phenylbut-3-en-1-ylidene)indolin-2-ones, particularly compound
4b, have the potential to be developed as a new class of inhibitors
of Pim-1 with efficient anti-cancer activity by further structural
modications. Further study on the anti-tumor activities in vivo
and exact biological mechanism is underway.
target database only includes top 10 kinases.
Hoechst staining
Briey, cells (1 ꢂ 105 per well) were seeded in 6-well plates and
cultured for 24 h, followed by 4b treatment for another 48 h.
Aer rinsing with PBS, the cells were xed using 75% of ethanol.
The morphological change of cell was examined by inverted
microscope, then the cells were stained with Hoechst 33342 (2
lg mLꢁ1, in PBS) and analyzed under uorescence microscope
(Zeiss, Axiovert 200, Germany) to identify the apoptotic cells.
Cell-cycle distribution by ow cytometry
Briey, cells were seeded in 6-well plates at the density of (1 ꢂ
105 per well) and cultured for 24 h, followed by compound 4b
treatment for another 48 h. At the indicated intervals, both
attached cells and oating cells were harvested, sediments were
resuspended in 1 mL hypotonic uorochrome solution con-
taining 50 mg mLꢁ1 PI in 0.1% sodium citrate plus 0.1% Triton
X-100, and then analyzed by ow cytometer (ESP Elite,
Beckman-Coulter, Miami, FL). Cell cycle distribution was esti-
mated with Listmode soware.
Acknowledgements
Flow cytometric analysis of apoptosis
For Annexin V/PI assays, HeLa cells were stained with Annexin
V-FITC and PI and then monitored for apoptosis by ow
cytometry. Briey, 5 ꢂ 103 cells were seeded in 6-well plates for
24 hours and then were treated with compound 4b (0–8 mM) for
48 hours. Then cells were collected and washed twice with PBS
and stained with 5 mL of Annexin V-FITC and 2.5 mL of PI (5 mg
mLꢁ1) in 1 ꢂ binding buffer (10 mM HEPES, pH 7.4, 140 mM
NaOH, 2.5 mM CaCl2) for 30 minutes at room temperature in
the dark. Apoptotic cells were quantied using a FACS can
cytouorometer. Statistical analysis was done using Flowjo 7.6.1
soware. Both early apoptotic (Annexin V-positive, PI-negative)
and late apoptotic (double positive of Annexin V and PI) cells
were detected.
We appreciate the nancial support of National Natural Science
Foundation of China (no. 81202403). We also thank Sichuan
University Analytical & Testing Center for the NMR analysis.
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