Synthesis and cytotoxic studies of novel 5-phenylisatin derivatives and their anti-migration and anti-angiogenic evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.07.032

A number of 5-arylisatin derivatives were synthesized in 5–6 steps from readily available starting materials. Their structures were confirmed by ¹H NMR and ¹³C NMR as well as LC/MS. The cytotoxicity of these novel isatins against human leukemia K562 cells were evaluated by MTT assay in vitro. SAR studies indicated that the N-substituted benzyl and C-5 substituted phenyl groups greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 present in the parent ring is required to maintain such a potency. Particularly, N-(p-methoxybenzyl)-5-(p-methoxyphenyl)isatin (compound 2m) showed the highest antitumor activity against K562 cell lines (IC₅₀ = 0.03 μM). Moreover, treatment with compound 2m significantly inhibited liver cancer HepG2 cells proliferation and migration, which could also reduce the human umbilical vein endothelial cells (HUVEC) tube formation. In conclusion, compound 2m exhibited very good cancer cells proliferation inhibition by angiogenesis responses in vitro, and 2m might be a promising angiogenesis inhibitor for cancer treatment.

Full text of DOI:10.1016/j.ejmech.2018.07.032

European Journal of Medicinal Chemistry 156 (2018) 800e814
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and cytotoxic studies of novel 5-phenylisatin derivatives
and their anti-migration and anti-angiogenic evaluation
Qian Zhang 1, Yuou Teng 1, Yuan Yuan, Tingting Ruan, Qi Wang, Xing Gao, Yao Zhou,
Kailin Han, Peng Yu*, Kui Lu**
China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Tianjin University of Science and
Technology, Tianjin, 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 5-arylisatin derivatives were synthesized in 5e6 steps from readily available starting ma-
terials. Their structures were confirmed by 1H NMR and 13C NMR as well as LC/MS. The cytotoxicity of
these novel isatins against human leukemia K562 cells were evaluated by MTT assay in vitro. SAR studies
indicated that the N-substituted benzyl and C-5 substituted phenyl groups greatly enhance their cyto-
toxic activity, whereas an intact carbonyl functionality on C-3 present in the parent ring is required to
maintain such a potency. Particularly, N-(p-methoxybenzyl)-5-(p-methoxyphenyl)isatin (compound 2m)
Received 22 January 2018
Received in revised form
10 July 2018
Accepted 12 July 2018
showed the highest antitumor activity against K562 cell lines (IC50 ¼ 0.03
mM). Moreover, treatment with
Keywords:
Synthesis
5-phenylisatin
Proliferation
Migration
compound 2m significantly inhibited liver cancer HepG2 cells proliferation and migration, which could
also reduce the human umbilical vein endothelial cells (HUVEC) tube formation. In conclusion, com-
pound 2m exhibited very good cancer cells proliferation inhibition by angiogenesis responses in vitro,
and 2m might be a promising angiogenesis inhibitor for cancer treatment.
Angiogenesis
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
analogues could act as caspase-3,7 inhibitors and SARS-CoV 3C-like
protease inhibitors [11,12]. In addition, Chinnasamy's group found
Isatins are a class of structurally versatile molecules and their
core structure appears in many biologically active molecules and
pharmaceutical agents. Isatin derivatives have been reported to
possess a broad spectrum of activities such as anticancer [1,2],
antidepressant [3], anticonvulsant [4], antifungal [5], anti-HIV [6]
and anti-inflammatory [7], etc. In the last decade, N-substituted
isatins have attracted increasing attention from both industry and
academia. Vine et al. has reported that some N-benzyl isatin de-
rivatives were more cytotoxic than their N-H counterparts toward
some lymphoma cells as well as a series of human cancer cell lines,
including human leukemic (K562, U937 and Jurkat), liver (HepG2),
breast (MDA-MB-231 and MCF-7), prostate (PC-3) and colorectal
(HCT-116) cell lines [8]. Chen's and Chiyanzu's groups reported that
N-substituted isatin derivatives exhibited inhibition activities
against SARS CoV 3CLpro and parasitic cysteine proteases [9,10].
Later on, Limpachayaporn and Liu found that 5-sulfonylisatin
that some N-1 and C-5 disubstituted molecules such as 1-
(substituted benzylidene)-3-(1-(morpholino/piperidinomethyl)-
2,3-dioxoindolin-5-yl)urea derivatives exhibited antiepileptic ac-
tivity and neurotoxicity [13]. Previously, we reported some 5-(2-
carboxyethenyl)isatin derivatives as anticancer agents, and found
that the combination of a 5-[trans-2-(methoxycarbonyl)ethen-1-
yl] group and a 1-(4-methoxybenzyl) group (compound 5e61,
Fig. 1) in isatin significantly enhanced it's cytotoxic activity [14].
However, further research on this molecule indicated that the
presence of a metabolically unstable acrylic ester moiety on the C-5
position is the key to its in vivo efficacy. In order to further improve
the antitumor property of this type of 1,5-disubstituted isatin
molecules, we have synthesized a serious of isatin derivatives and
studied their cytotoxicity. The results showed that although the
substituent of 5-methacrylate could increase the antitumor activity
of the target compound, it is not stable in metabolic processes
(unpublished data). Therefore, we report a series of new com-
pounds in this paper, which can maintain good anti-tumor efficacy
even without the use of 5-methacrylate substituents. Among them,
compound 2m was shown as the most active compound against
human leukemia K562 cells. Moreover, the molecular mechanism
* Corresponding author.
** Corresponding author.
E-mail addresses: yupeng@tust.edu.cn (P. Yu), lukui@tust.edu.cn (K. Lu).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2018.07.032
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
801
The data obtained from 1H NMR, 13C NMR and Mass Spec-
trometry confirmed the proposed structures (Spectral data results
are provided in supporting information).
3. Results and discussion
When contemplating a new class of 1,5-substituted isatin
structure that could avoid the metabolically liabile acrylic acid ester
group found in compound 5e61 while maintaining its biological
potency, we envisaged a general structure compound 5e61 (Fig. 1)
[14]. It can bear a various of aromatic groups at the C-5 position,
that carry some structural similarity in terms of their size and
conjugation property, whereas the in vivo Michael addition liability
of the 5-acrylic acid ester group could be eliminated.
Fig. 1. The structure of 1,5-disubstituted isatin derivative (compound 5e61).
of the cytotoxic activity of compound 2m was explored on anti-
migration and anti-angiogenesis.
2. Chemistry
As illustrated in Fig. 2, with the isatin as the core structure of our
drug design, this strategy would allow us to start with the modi-
fication of the substitution pattern of the benzene ring, especially at
C-5, followed by N-derivatization and then C-3 variation.
The in vitro antitumor activities of the 5-substituted isatins 1a-
1k against two human tumor cells K562 and HepG2 were first
evaluated. As shown in Table 1, the cytotoxic activities of the 5-
phenyl substituted compounds are far better than the parent nu-
cleus. Among them, compound 1i exhibited the highest cytotoxic
Firstly, the target compounds were synthesized according to the
procedures outlined in Scheme 1. Monosubstituted isatins 1a-1g
were obtained in two steps with a improved yield of 55e80% [15].
Moreover, 5-Bromoisatin (1d) was coupled with six different
boronic acids by microwave-assistant Suzuki coupling reaction to
afford compounds 1h-1k in 70e80% isolated yield [16]. Secondly,
N-alkylation of 1i led to the synthesis of compounds 2a-2v in good
to excellent yield (75e90%). Thirdly, compound 3a-3c were pre-
pared from isatin derivatives by suzuki-coupling reaction (Scheme
2). Finally, compound 4a-4s were prepared from 5-bromo-1-(4-
methoxybenzyl)indoline-2,3-dione by Suzuki-coupling (Scheme 3).
Compounds 5a-5k (Scheme 4) were prepared from 2m in good
yield in one or two steps. Compound 2m was reduced with NaBH4
in methanol under room temperature to afford compound 5a.
Compound 5b was prepared by treatment of the compound 2m
with methylmagnesium bromide in tetrahydrofuran at ꢀ78 ꢁC.
Compound 2m was reduced with hydrazine monohydrate in
ethanol under reflux to afford compound 5c. Compound 5d and 5e
were prepared by treatment of the compound 2m with (triphe-
nylphosphoranylidene)acetate in tetrahydrofuran at room tem-
perature. Compound 5f was synthesized by treatment of the
compound 2m with ethylene glycol in toluene at 110 ꢁC. Compound
5g was prepared by treatment of the compound 2m with dieth-
ylaminosulfur trifluoride in DCM at reflux. Compound 5h was
synthesized by treatment of the compound 2m with hydroxyl-
amine hydrochloride in ethanol at reflux. The compounds 5i-5k
were prepared by the treatment of 5c with benzaldehyde or
substituted benzaldehyde and pyridine in ethanol.
activity against HepG2 cell lines (IC50 ¼ 0.96
mM).
After compound 1i was identified as the potential lead, our SAR
study was then focused on the N-derivatization based on the hy-
pothesis that the combination of C-5 substitution with N-alkylation
may further enhance their cytotoxic potency. A series of 5-(4-
methoxyphenyl) N-substituted derivatives (Scheme 1) were
screened for their in vitro cytotoxic activity.
As can be seen from the data in Table 2, introduction of simple
aliphatic groups at N-1 (2a-2c) did not significantly enhance their
potency. Moreover, a p-methoxyphenyl group at N-1 significantly
diminished the potency. However, the introduction of benzyl
groups into the N-1 position (2d-2v) resulted in much better
inhibitory activities, with 10e300 folds improvement compared to
that of 1i, pointing to the tendency that the N-1 position needs a
relatively sterically larger group with certain lipophilic property to
increase the potency. Further examination of the structure differ-
ence and the cytotoxic data of the compounds 2d and 2f-2v against
K562 showed that the substituted benzyl group did increase the
potency. The improvement in potency does not seem to be related
to the electron density of the substituent groups (2f, 2r and 2h, 2l);
however, the substituents' size (2k, 2p) and location (2n and 2m,
2o) plays a more significant role. Moreover, the excellent cytotoxic
activity for the compounds containing N-1 para or ortho mono
methoxy benzyl group (2m, 2o), dimethoxy benzyl group (2q) as
well as a piperonyl group (2v) indicated that hydrogen bond re-
ceptor in this direction of the molecule could significantly enhance
the potency (8e25 folds, compared to that of 2d).
With the identification of the p-methoxybenzyl group as an
optimal substituent at N-1, our attention then turned back to the p-
methoxyphenyl substitution on the benzene ring. Specifically, we
were interested in finding out how the location would influence the
cytotoxic potency. For this purpose, a series of isatins bearing p-
methoxyphenyl group(s) at various position on the benzene ring
were synthesized and their cytotoxic activities were examined. The
compounds 3a-3c were synthesized following the procedure
similar to that of 2m (Scheme 2) and their potency were listed in
Table 3. The 4-, 6- and 7-monosubstituted compounds 3a-3c
exhibited much weaker potency than that of 2m (>220 folds),
which may suggest that the N-1, C-5 disubstituted pattern in 2m
possess a much favorable 2-dimensional orientation toward the
biological targets.
Scheme 1. Synthesis of isatin derivatives (1a-1k, 2a-2v).
The SAR analysis of the compounds in Table 3 revealed that the

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Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
Scheme 2. Synthesis of 3a-3c.
Scheme 3. Synthesis of 4a-4r.
Scheme 4. C-3 modification of isatin derivatives (5a-5k).
N-(p-methoxybenzyl) and 5-(p-substituted phenyl) disubstituted
isatins are the most attractive candidates, with compound 2m
exhibiting the highest potency. To further investigate the activity of
the structural analogues of 2m, a number of 5-aryl N-(p-methox-
yphenyl)satins were prepared (Scheme 3) and their inhibition
against two human tumor cells were tested (Table 4).
4b as well as the 3,5-dimethoxy-phenyl substituted compounds 4c
exihibted a 5e25 folds decrease in potency compared to 2m,
indicating that the para methoxy group is necessary and its linear
orientation maybe a critical factor in maintaining its high potency.
The halogen-containing compounds (4f, 4g, 4h), compounds (4i,
4j, 4l, 4m) possess either electron-donating or electron-
withdrawing groups, all exhibit very similar inhibition, indicating
that variation of the electron density at this position did not
As shown in Table 4, among all the 18 compounds examined, the
cytotoxic activities of the phenyl analogue 4k, region-isomers 4a,

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
803
Fig. 2. Structure modifications of isatin derivatives for SAR study.
Table 1
In vitro antitumor activities of compounds 1a-1k[14].
Table 2
In vitro antitumor activities of compounds 2a-2v.
Compd.
R2
IC50
(
m
М)
Compd.
R; R1
IC50
(
m
M)
K562
HepG2
K562
HepG2
CPT
1i
0.07 0.01
6.92 0.28
4.98 0.92
2.20 0.36
0.39 0.05
0.57 0.11
>10
0.56 0.08
0.43 0.15
0.35 0.13
0.20 0.12
0.38 0.20
0.10 0.15
0.27 0.03
0.03 0.01
0.68 0.27
0.04 0.01
0.14 0.03
0.06 0.01
0.38 0.07
0.27 0.03
0.40 0.06
0.40 0.04
0.03 0.01
0.37 0.06
0.96 0.32
6.67 2.82
4.47 0.23
7.09 1.04
1.36 0.51
>10
1.12 0.33
0.99 0.02
0.25 0.09
0.32 0.12
0.60 0.24
0.70 0.03
0.33 0.03
0.05 0.03
0.70 0.18
0.13 0.02
0.41 0.03
1.35 1.86
1.00 0.29
0.38 0.02
0.70 0.25
0.38 0.05
0.44 0.19
CPT
1a
1b
1c
1d
1e
1f
1g
1h
1i
0.07 0.01
>10
>10
>10
>10
>10
>10
>10
>10
0.37 0.06
>10
>10
>10
>10
>10
>10
>10
6.00 2.16
0.96 0.32
5.33 0.62
5.43 1.45
H
CH3
CH2CH3
H
F
Cl
Br
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
CH2CH¼CH2
CH2C6H5
CH3
OCH3
(CH2)3CH3
CH3
OCH3
H
C6H4-4-OCH3
CH2-4-C6H4F
CH2-3,4-C6H3Cl2
CH2-4-C6H4Cl
CH2-3-C6H4Cl
CH2-2-C6H4Cl
CH2-4-C6H4Br
CH2-4-C6H4CH3
CH2-4-C6H4OCH3
CH2-3-C6H4OCH3
CH2-2-C6H4OCH3
CH2-3,4-C6H3(CH3)2
CH2-3,4C6H3(OCH3)2
CH2-4-C6H4CN
CH2-4-C6H4CF3
CH2CH2OC6H5
CH2-2-naphthyl
CH2-2-piperonyl
6.92 0.28
>10
>10
1j
1k
CF3
significantly change their potency.
The excellent potency 5-(4-pyridyl) substituted compound 4q
and 5-(2-thiophenyl) substituted compound 4r suggest that groups
possessing a Lewis basic site could potentially improve the com-
pound's activity. The hydroxyl group containing compound 4o did
not exhibit any improved activity in comparison to compounds 2m,
4q and 4r.
2s
2t
2u
2v
There was no significant potency change when the 5-phenyl
substitution (4k) was replaced by 5-(4-methyphenyl) group (4i)
or 5-(2-naphthyl) group (4p). However, the cytotoxic activity of the
5-(4-n-butyl)phenyl compound 4e dropped 15 folds from that of
the non-substituted counterpart (4k), which indicated the bulky
aliphatic chain is detrimental.
Derivatization of the C-3 ketone group of 2m led to compounds
5a-5l (Scheme 4) and their cytotoxic activities were evaluated to
establish the importance of this functionality. The testing result
showed that all these derivatives are inferior candidates as in-
hibitors of K562 and HepG2 (Table 5), suggesting that the carbonyl
functionality at C-3 is essential in order to maintain the observed
high antitumor activity.
The IC50 of compounds 2m against K562 cells and HepG2 cells were
30 nM and 50 nM, respectively (Fig. 3A). Meanwhile, a normal hu-
man cell line (umbilical vein endothelial cells, HUVEC) has been
tested for 2m, the IC50 was 920 nM. Then the molecular mechanism
by which compound 2m exerts its cytotoxic activity on cells pro-
liferation, migration and angiogenesis was further investigated.
Firstly, the morphological changes in compound 2m-treated
K562 cells were observed. K562 cells treated by compound 2m
showed the cells morphology change of G2/M phase arrest (such as
elongation of the cells) and typical apoptotic morphology (such as
cell shrinkage and/or blebbing) (Fig. 3B). As shown in Fig. 3B, the
elongated cells increased significantly as early as 6 h after com-
pound 2m treatment at a concentration of 30 nM as compared to
Based on these SAR studies, compound 2m exhibited significant
cancer inhibitory activity in vitro against the tested cancer cell lines.

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Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
Table 3
Table 5
In vitro antitumor activities of compounds 3a-3c.
Antitumor activity of the C-3 derivatized compounds 5a-5k.
Compd.
C-3
IC50
(
m
M)
K562
HepG2
CPT
2m
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
0.07 0.01
0.03 0.01
0.14 0.09
>10
0.37 0.06
0.05 0.03
0.70 0.03
>10
¼ O
-OH$H
-OH$CH3
-H$H
¼ COOCH2CH3
¼ COOCH3
-CH2O(CH2)2OCH2-
-F$F
>10
>10
Compd.
Position
IC50
(
m
M)
1.24 0.54
1.90 0.14
>10
>10
>10
>10
1.90 0.26
>10
3.39 0.27
4.73 1.24
>10
>10
>10
>10
>10
>10
K562
HepG2
CPT
2m
3a
3b
3c
0.07 0.01
0.03 0.01
>10
7.46 1.69
4.43 0.75
0.37 0.06
0.05 0.03
>10
>10
8.56 0.06
5
4
6
7
¼ NOH
¼ C6H5
¼ C6H4-4-CF3
¼ C6H4-4-CH(CH3)2
Table 4
In vitro antitumor activities of compounds 4a-4r.
apoptotic rates of compound 2m-treated K562 cells increased to
43.4% and 95.2% of the total cells whereas only 4.1% and 4.3%
apoptosis respectively at 30 nM and 300 nM. Result also indicated
that the apoptosis was increased obviously in a time-dependent
and dose-dependent manner. We have concluded that compound
2m inhibits the proliferation of K562 cells by inducing apoptosis in
a time-dependent and dose-dependent manner.
To study whether compound 2m could inhibit migration and
repair ability of tumor cells, wound healing assay and transwell
assay were performed by HepG2 cells. After HepG2 cells were
wounded, they were treated with the indicated concentrations of
compound 2m (10 nM, 30 nM, 100 nM) for 0, 6, 12, 24, 36 and 48 h
respectively. As shown in Fig. 6A, after treatment by DMSO for 48 h,
Compd.
R3
IC50
(
m
М)
K562
HepG2
CPT
2m
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
0.07 0.01
0.03 0.01
0.10 0.03
0.37 0.09
0.51 0.21
0.26 0.11
2.25 0.73
0.24 0.11
0.23 0.05
0.38 0.08
0.40 0.12
0.31 0.08
0.15 0.02
0.26 0.04
0.35 0.08
0.06 0.01
0.06 0.02
0.34 0.10
0.04 0.02
0.08 0.04
0.37 0.06
0.05 0.03
1.89 1.48
>10
1.20 0.58
>10
4.58 1.09
3.15 2.31
0.38 0.21
>10
C6H4-4-OCH3
C6H4-3-OCH3
C6H4-2-OCH3
C6H4-3,5-(OCH3)2
C6H4-3,4-(OCH3)2
C6H4-4-CC3H9
C6H4-4-F
the migration distance was 184.89
HepG2 cells treated with compound 2m were 162.89
135.22 m and 96.24 m respectively at 10 nM, 30 nM and 100 nM.
mm. The migration distances of
mm,
m
m
By calculation, we found that the higher concentration of com-
pound 2m, the higher the inhibition ratio. The inhibition ratios of
HepG2 cells treated with compound 2m were 11.9%, 26.9% and
47.9% respectively at 10 nM, 30 nM and 100 nM.
Transwell assay is an experimental technique for studying the
tendency of cells which is applied in cell culture, cytotaxis, cell
migration and cell invasion. As shown in Fig. 6B, basic DMSO did
not affect migration of HepG2 cells. While, the compound 2m could
inhibit the HepG2 cells migration and have a dose-dependent
inhibitory effect.
C6H4-4-Cl
C6H3-3-Cl-4-F
C6H4-4-CHa3
C6H4-4-(O-Pr)
C6H5
>10
>10
1.08 0.60
0.89 0.11
0.82 0.20
0.41 0.32
>10
>10
>10
3.67 1.40
C6H4-4-CNa
C6H4-4-CFa3
C6H4-4-OCFa3
C6H4-4-OHa
2-naphthyl
4-pyridyl
2-thiophenyl
Tube formation plays an important role in the development of
tumors. Angiogenesis is also the path which tumors transition from
benign to malignant. Inhibiting this process can significantly pre-
vent the development and spread of tumor tissue. As shown in
Fig. 7, basic DMSO did not affect tube forming of HUVEC cells.
However, the compound 2m could inhibit the angiogenesis of
HUVEC cells and have a dose-dependent inhibitory effect.
DMSO-treated cells. The apoptotic cells were clearly observed in
K562 cells after the treatment with compound 2m for 24 h. These
results indicated that compound 2m might inhibit the cell prolif-
eration of K562 cells through the cell cycle arrest and apoptosis.
To evaluate the cell cycle distribution of K562 cells with or
without compound 2m treatment, we measured the DNA content
by flow cytometry. As shown in Fig. 4, after treatment with com-
pound 2m (30 and 300 nM) for 48 h, the percentage of K562 cells in
G2/M phase increased. Only 0.02% of cells were in G2/M phase in
basic DMSO cultures. However, the exposure to compound 2m
caused increased cell fraction in G2/M phase in a time-dependent
and dose-dependent manner. These results demonstrated that
compound 2m has cell proliferation inhibitory effect and can
induce the cell cycle arrest of K562 cells in G2/M phase.
4. Conclusions
In conclusion, a series of novel N-1 benzyl and C-5 phenyl
substituted isatin derivatives were synthesized and tested for their
in vitro antitumor activity against two strains of cancer cell lines
(human leukemia K562 cells and liver cancer HepG2 cells). Among
them, compounds 2m exhibited excellent cancer inhibitory activity
in vitro against K562 cell lines (IC50 ¼ 0.03
mM) and HepG2 cell lines
M). SAR analysis showed that the carbonyl group of
The presence of apoptotic K562 cells following compound 2m
treatment was further revealed by flow cytometric analysis of cells
double-labeled with Annexin V-FITC and PI (Fig. 5). After 48 h of
treatment with compound 2m (30 and 300 nM), we observed that
compound 2m showed significant apoptosis against K562 cells. The
(IC50 ¼ 0.05
m
the isatin, N-benzyl and C-5 phenyl substituted pattern, the para-
methoxyl group of the benzyl played a significant role in the anti-
tumor activity.
Both morphological results and flow cytometry analysis showed

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
805
Fig. 3. Morphological changes in compound 2m-treated K562 cells. (A) Structure and in vitro cell proliferation inhibitory activity of compound 2m. (B) Morphological changes
induced by compound 2m treatment (30 nM) were observed.
Fig. 4. Compound 2m induced the cell cycle arrest of K562 cells in G2/M phase. K562 cells were treated with 30 nM and 300 nM compound 2m. At the time points indicated, cells
were labeled with PI and their DNA content was determined using FACS analysis.
that compound 2m induce apoptosis and cause cell cycle arrest in a
time-dependent and dose-dependent manner. Furthermore,
wound healing and transwell experiments showed that compound
2m could inhibit the migration and repair ability of HepG2 cells. In
addition, the results of cell tube formation showed that the com-
pound 2m could inhibit the angiogenesis of HUVEC cells and has a
dose-dependent inhibitory effect.
The effects of indole derivative compound 2m on tumor cell
growth, migration and angiogenesis were studied, which provide a
strong foundation for further study on the antitumor activity and
mechanism of indole compounds.
further purification. 1H NMR and 13C NMR spectra were recorded on
a Bruker AM-400 NMR spectrometer (Billerica, Middlesex, MA,
USA) in CDCl3 or DMSO‑d6. The chemical shifts are reported in
d
(ppm) relative to tetramethylsilane as internal standard. (1H NMR:
TMS at 0.00 ppm, CDCl3 at 7.26 ppm, DMSO‑d6 at 2.50 ppm; 13C
NMR: CDCl3 at 77.23 ppm, DMSO‑d6 at 39.51 ppm). Mass spectra
were obtained on a Q-TOF mass spectrometer (Agilent, Santa Clara,
CA, USA). TLC analyses were carried out on silica gel F254, and the
spots were examined with UV light. All final products had a purity
of ꢂ95%. The purity of the final products was determined by HPLC
(LabTech) on a Diamonsil C18 column (4.6 mm ꢃ 250 mm, 5
mm)
with methanol/H2O (90/10 v/v) at 0.5 mL/min flow rate and 254 nm
detector wavelength.
5. Experimental section
5.1. Chemistry
5.2. Synthesis of compounds 1a-1k and 2a-2v
All reagents and solvents used in this paper were of reagent
grade, were obtained from commercial suppliers and used without
The general procedure for the preparation of isatin derivatives
was as follows. A mixture of 0.05 mol substituted anilines, 0.15 mol

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Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
Fig. 5. Compound 2m induced apoptosis in K562 cells. K562 cells were treated with 30 nM and 300 nM compound 2m for different periods of time. K562 cells were labeled with
Annexin V-FITC and PI and apoptosis was determined using FACS analysis.
Fig. 6. Effect of compound 2m on HepG2 cells migration at 48 h by using a wound healing assay (A) and transwell assay (B).
Hydroxylammonium chloride, 0.35 mol Na2SO4, 2 mol/L Hydro-
chloric acid at 5 mL and Chloral hydrate at 0.06 mol were stirred in
250 mL H2O at 95 ꢁC. The progress of the reaction was monitored by
TLC (petroleum ether/ethyl acetate). After the reaction was
completed, the reaction mixture was cooled to room temperature
and the precipitate was filtered and dried. The crude product was
used directly for the next step without further purification.
and stir at 80 ꢁC for 30 min. The reaction mixture was cooled to
room temperature and then poured onto ice (250 g). The solid
which resulted was filtered out and dried over air to yield the crude
which was purified by dissolving in dilute sodium hydroxide (5%,
100 mL) followed by acidified with 4N hydrochloric acid (20 mL).
The solid which formed was filtered out and dried over air to
provide the purified compounds 1a-1g.
To a flask (100 mL) which contained concentrated sulfuric acid
(20 mL) was added N-2-(hydroxyimino)acetamide derivatives
(7.0 g) in portions at 50 ꢁC with vigorous stirring. The reaction
temperature was maintained at 50 ꢁC-75 ꢁC during the addition.
After the addition was completed, the mixture was heated to 80 ꢁC
To a microwave reactor vial (5 mL) which contained the solution
of 5-bromoindoline-2,3-dione (1.0 g, 4.4 mmol) in 1,4-dioxane
(5 mL) were added PdCl2(dppf) (161 mg, 0.22 mmol), CH3COOK
(0.6 g, 6.2 mmol) and substituted benzeneboronic acid (0.45 g,
5.3 mmol) under the atmosphere of argon. The microwave reactor

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
807
Fig. 7. Suppressive effects of different concentrations compound 2m on tube formation of HUVEC cells. HUVECs cultured on matrigel coated plates were treated with the indicated
concentrations of compound 2m (10 nM, 30 nM, 100 nM) for 12 h. Morphological changes in HUVECs were observed by microscopy and representative images are shown.
vial was caped and placed into the microwave cavity. The reaction
mixture was irradiated at high level for 30 min at 120 ꢁC. The re-
action mixture was cooled to room temperature, poured onto
100 mL ice-water and then extracted with dichloromethane
(3 ꢃ 100 mL). The combined organic layers were washed with wa-
ter (2 ꢃ 100 mL), brine (100 mL) and dried over anhydrous mag-
nesium sulfate. The solvent was removed under vacuum to afford
the crude which was purified by flash column chromatography
(silica gel, petroleum ether/ethyl acetate 3:1) to yield the desired
compound 1h-1k.
To a flask (25 mL) which contained the solution of compound 1i
(0.2 g, 0.79 mmol) in dry N,N-dimethylformamide (2 mL). The re-
action temperature was maintained at 0 ꢁC followed by addition
K2CO3 (0.33 g, 2.37 mmol). Stirring 5 min, was added alkyl or benzyl
(1.0 mmol) and the mixture allowed warm to room temperature.
The reaction mixture was stirred at room temperature for 4 h. The
orange solution was poured water (25 mL) and extracted with
dichloromethane (3 ꢃ 100 mL). The combined extracts were dried
over anhydrous magnesium sulfate, filtered and evaporated.
Chromatography (petroleum ether/ethyl acetate 10:1e3:1) affor-
ded the title compounds 2a-2v.
J ¼ 8.0 Hz), 7.79 (d, 2H, J ¼ 8.4 Hz), 7.87 (s, 1H), 7.90 (d, 2H,
J ¼ 8.0 Hz), 7.99 (d, 1H, J ¼ 8.4 Hz), 11.20 (s, 1H). 13C NMR (100 MHz,
CDCl3) d 102.01,111.59,113.26,119.03,123.40,126.26,126.29,127.48,
127.48, 133.59, 137.24, 143.19, 151.15, 160.01, 184.63. ESI-MS: 292.0
[MþH]þ.
5.2.5. 5-(4-methoxyphenyl)-1-methylindoline-2,3-dione (2a)
Yield 44%; 1H NMR (400 MHz, CDCl3)
d 3.28 (s, 3H), 3.85 (s, 3H),
6.94 (d, 1H, J ¼ 8.0 Hz), 6.98 (d, 2H, J ¼ 8.4 Hz), 7.46 (d, 2H,
J ¼ 8.4 Hz), 7.78 (d, 1H, J ¼ 8.0 Hz), 7.79 (s, 1H). 13C NMR (100 MHz,
CDCl3)
d 26.33, 55.40, 110.23, 114.50, 114.50, 117.87, 123.26, 127.64,
127.64, 131.43, 136.28, 137.09, 149.96, 158.38, 159.59, 183.55. ESI-
MS: 268.1 [MþH]þ, 290.1 [MþNa]þ.
5.2.6. 1-ethyl-5-(4-methoxyphenyl)indoline-2,3-dione (2b)
Yield 35%; 1H NMR (400 MHz, CDCl3)
d 1.32e1.35 (m, 3H),
3.78e3.84 (m, 2H), 3.85 (s, 3H), 6.95 (d, 1H, J ¼ 8.0 Hz), 6.97 (d, 2H,
J ¼ 8.8 Hz), 7.45 (d, 2H, J ¼ 8.8 Hz), 7.74 (s, 1H), 7.76 (d, 1H,
J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3)
d 12.60, 35.08, 55.40, 110.33,
114.50, 114.50, 118.06, 123.47, 127.63, 127.63, 131.47, 136.24, 136.90,
149.16, 158.00, 159.57, 183.91. ESI-MS: 282.1 [MþH]þ, 304.1
[MþNa]þ.
5.2.1. 5-(p-tolyl)indoline-2,3-dione (1h)
Yield 54%; 1H NMR (400 MHz, DMSO‑d6)
d 2.36 (s, 3H), 6.98 (d,
5.2.7. 1-allyl-5-(4-methoxyphenyl)indoline-2,3-dione (2c)
Yield 42%; 1H NMR (400 MHz, CDCl3)
d 3.85 (s, 3H), 4.40 (d, 2H,
1H, J ¼ 8.0 Hz), 7.25 (d, 2H, J ¼ 8.0 Hz), 7.53 (d, 2H, J ¼ 8.0 Hz), 7.72
(s, 1H), 7.87 (d, 1H, J ¼ 8.0 Hz), 11.11 (s, 1H). 13C NMR (100 MHz,
J ¼ 5.6 Hz), 5.30e5.37 (m, 2H), 5.82e5.96 (m, 1H), 6.94 (d, 1H,
DMSO‑d6)
d 21.10, 113.10, 118.84, 122.62, 126.50, 126.50, 130.05,
J ¼ 8.0 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.45 (d, 2H, J ¼ 8.8 Hz), 7.73 (d,
1H, J ¼ 8.0 Hz), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 42.62,
130.05, 135.34, 136.31, 136.64, 137.27, 150.17, 160.00, 184.86. ESI-MS:
238.1 [MþH]þ, 260.1[MþNa]þ.
55.40, 111.17, 114.52, 114.52, 118.02, 118.70, 123.39, 127.67, 127.67,
130.41, 131.49, 136.24, 137.10, 149.34, 158.05, 159.61, 183.44. ESI-MS:
294.1 [MþH]þ, 316.1 [MþNa]þ.
5.2.2. 5-(4-methoxyphenyl)indoline-2,3-dione (1i)
Yield 55%; 1H NMR (400 MHz, DMSO‑d6)
d 3.79 (s, 3H), 6.97 (d,
1H, J ¼ 8.0 Hz), 7.00 (d, 2H, J ¼ 8.0 Hz), 7.59 (d, 2H, J ¼ 8.0 Hz), 7.70
5.2.8. 1-benzyl-5-(4-methoxyphenyl)indoline-2,3-dione (2d)
(s, 1H), 7.84 (d, 1H, J ¼ 8.0 Hz), 11.09 (s, 1H). 13C NMR (100 MHz,
Yield 40%; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H), 4.95 (s, 2H),
CDCl3) d 55.65, 113.08, 114.88, 114.88, 118.84, 122.38, 127.83, 127.83,
131.57, 135.18, 136.40, 149.84, 159.38, 160.01, 184.92. ESI-MS: 254.0
[MþH]þ, 276.0[MþNa]þ.
6.81 (d, 1H, J ¼ 8.4 Hz), 6.95 (d, 2H, J ¼ 8.4 Hz), 7.29e7.36 (m, 5H),
7.40 (d, 2H, J ¼ 8.4 Hz), 7.64 (d, 1H, J ¼ 8.4 Hz), 7.78 (s, 1H). 13C NMR
(100 MHz, CDCl3)
d 44.16, 55.38, 111.30, 114.50, 114.50, 118.12,
123.38, 127.47, 127.47, 127.63, 127.63, 128.20, 129.08, 129.08, 131.42,
134.57, 136.23, 137.14, 149.22, 158.41, 159.60, 183.44. ESI-MS: 344.1
[MþH]þ, 366.1 [MþNa]þ.
5.2.3. 5-phenylindoline-2,3-dione (1j)
Yield 43%; 1H NMR (400 MHz, DMSO‑d6)
d 7.01 (d, 1H,
J ¼ 8.0 Hz), 7.36e7.38 (m, 1H), 7.44e7.47 (m, 2H), 7.64e7.66 (m, 2H),
7.76 (s, 1H), 7.89e7.92 (m, 1H), 11.13 (s, 1H). 13C NMR (100 MHz,
5.2.9. 1,5-bis(4-methoxyphenyl)indoline-2,3-dione (2e)
Yield 45%; 1H NMR (400 MHz, CDCl3)
d 3.85 (s, 3H), 3.87 (s, 3H),
DMSO‑d6)
d 112.65, 118.37, 122.40, 126.18, 126.18, 127.44, 128.97,
128.97, 134.88, 136.44, 138.68, 149.91, 159.51, 184.32. ESI-MS: 224.1
[MþH]þ, 246.1[MþNa]þ.
6.88 (d, 1H, J ¼ 8.0 Hz), 6.98 (d, 2H, J ¼ 8.0 Hz), 7.07 (d, 2H,
J ¼ 8.0 Hz), 7.35 (d, 2H, J ¼ 8.0 Hz), 7.46 (d, 2H, J ¼ 8.0 Hz), 7.70 (d,
1H, J ¼ 8.0 Hz), 7.85 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 55.41,
5.2.4. 5-(4-(trifluoromethyl)phenyl)indoline-2,3-dione (1k)
55.62, 111.50, 114.53, 114.53, 115.25, 115.25, 117.90, 123.40, 125.50,
127.39, 127.39, 127.69, 127.69, 131.45, 136.27, 137.45, 150.59, 157.76,
Yield 44%; 1H NMR (400 MHz, DMSO‑d6)
d 7.04 (d, 1H,

808
Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
159.65, 159.72, 183.34. ESI-MS: 360.1 [MþH]þ, 382.1 [MþNa]þ.
4.91 (s, 2H), 6.82 (d, 1H, J ¼ 8.0 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz), 7.16 (d,
2H, J ¼ 8.0 Hz), 7.25 (d, 2H, J ¼ 8.0 Hz), 7.40 (d, 2H, J ¼ 8.8 Hz), 7.64
5.2.10. 1-(4-fluorobenzyl)-5-(4-methoxyphenyl)indoline-2,3-dione
(d, 1H, J ¼ 8.0 Hz), 7.78 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 21.13,
(2f)
43.94, 55.38, 111.33, 114.49, 114.49, 118.12, 122.44, 123.37, 127.50,
127.50, 127.63, 127.63, 129.73, 129.73, 131.49, 136.22, 137.08, 138.02,
149.29, 158.39, 159.57, 183.57. ESI-MS: 358.2 [MþH]þ.
Yield 42%; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H), 4.92 (s, 2H),
6.81 (d, 1H, J ¼ 8.4 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz), 7.02e7.07 (m, 2H),
7.32e7.36 (m, 2H), 7.40 (d, 2H, J ¼ 8.8 Hz), 7.66 (d, 1H, J ¼ 8.4 Hz),
7.78 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.47, 55.39, 111.12, 114.51,
5.2.17. 1-(4-methoxybenzyl)-5-(4-methoxyphenyl)indoline-2,3-
114.51, 115.96, 116.17, 118.13, 123.49, 127.64, 127.64, 129.26, 129.35,
130.38, 130.42, 131.33, 136.24, 137.29, 148.95, 158.36, 159.63, 183.28.
ESI-MS: 362.2 [MþH]þ, 384.2 [MþNa]þ.
dione (2m)
Yield 50%; 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 3H), 3.83 (s, 3H),
4.88 (s, 2H), 6.84 (d, 1H, J ¼ 8.4 Hz), 6.87 (d, 2H, J ¼ 8.4 Hz), 6.95 (d,
2H, J ¼ 8.8 Hz), 7.28 (d, 2H, J ¼ 8.4 Hz), 7.39 (d, 2H, J ¼ 8.8 Hz), 7.64
5.2.11. 1-(3,4-dichlorobenzyl)-5-(4-methoxyphenyl)indoline-2,3-
(d, 1H, J ¼ 8.4 Hz), 7.76 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.64,
dione (2g)
55.30, 55.38, 111.32, 114.45, 114.45, 114.49, 114.49, 118.12, 123.32,
126.55, 127.61, 127.61, 128.96, 128.96, 131.43, 136.18, 137.04, 149.26,
158.37, 159.50, 159.58, 183.59; IR (KBr) 3435 (CAr-H), 2982, 2887,
1727 (C¼O), 1618 (C¼O), 1513 (CAr¼CAr), 1480 (CAr¼CAr), 1035 (C-O),
821 (CAr-H) cmꢀ1; ESI-MS: 374.2 [MþH]þ, 396.1 [MþNa]þ. HRMS
(ESI, m/z): calcd for C23 H19 N O4Na [MþNa]þ 396.1206, found
396.1195.
Yield 34%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 4.90 (s, 2H),
6.78 (d, 1H, J ¼ 8.0 Hz), 6.97 (d, 2H, J ¼ 8.4 Hz), 7.20 (d, 1H,
J ¼ 8.4 Hz), 7.42 (d, 2H, J ¼ 8.0 Hz), 7.44 (d, 2H, J ¼ 8.0 Hz), 7.69 (d,
1H, J ¼ 8.0 Hz), 7.81 (s,1H). 13C NMR (100 MHz, CDCl3)
d 43.11, 55.39,
110.89, 114.56, 114.56, 118.19, 123.68, 126.74, 127.67, 127.67, 129.40,
131.13, 131.27, 132.58, 133.35, 134.86, 136.32, 137.60, 148.57, 158.33,
159.72, 182.85. ESI-MS: 412.1 [MþH]þ.
5.2.12. 1-(4-chlorobenzyl)-5-(4-methoxyphenyl)indoline-2,3-dione
5.2.18. 1-(3-methoxybenzyl)-5-(4-methoxyphenyl)indoline-2,3-
(2h)
dione (2n)
Yield 43%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 4.92 (s, 2H),
Yield 40%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 3.83 (s, 3H),
6.78 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 2H, J ¼ 8.4 Hz), 7.29 (d, 2H,
4.92 (s, 2H), 6.83 (d, 1H, J ¼ 8.4 Hz), 6.87 (d, 2H, J ¼ 8.4 Hz), 6.96 (d,
2H, J ¼ 8.0 Hz), 6.97 (s, 1H), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.41 (d, 2H,
J ¼ 8.4 Hz), 7.65 (d, 1H, J ¼ 8.0 Hz), 7.79 (s, 1H). 13C NMR (100 MHz,
J ¼ 8.4 Hz), 7.34 (d, 2H, J ¼ 8.4 Hz), 7.41 (d, 2H, J ¼ 8.4 Hz), 7.66 (d,
1H, J ¼ 8.0 Hz), 7.79 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.52,
55.39, 111.11, 114.52, 114.52, 118.13, 123.52, 127.65, 127.65, 128.87,
128.87, 129.30, 129.30, 131.31, 133.10, 134.16, 136.26, 137.35, 148.86,
158.37, 159.64, 183.18. ESI-MS: 378.0 [MþH]þ.
CDCl3)
d 44.12, 55.31, 55.39, 111.34, 113.28, 113.38, 114.50, 114.50,
118.10, 119.68, 123.39, 127.64, 127.64, 130.14, 131.44, 136.12, 136.28,
137.16, 149.24, 158.40, 159.59, 160.16, 183.43. ESI-MS: 374.2
[MþH]þ, 396.2 [MþNa]þ.
5.2.13. 1-(3-chlorobenzyl)-5-(4-methoxyphenyl)indoline-2,3-dione
(2i)
Yield 40%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 4.93 (s, 2H),
5.2.19. 1-(2-methoxybenzyl)-5-(4-methoxyphenyl)indoline-2,3-
dione (2o)
6.80 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.27 (d, 2H,
Yield 42%; 1H NMR (400 MHz, CDCl3)
d 3.83 (s, 3H), 3.90 (s, 3H),
J ¼ 8.4 Hz), 7.32 (d, 2H, J ¼ 8.4 Hz), 7.42 (d, 2H, J ¼ 8.8 Hz), 7.68 (d,
1H, J ¼ 8.0 Hz), 7.81 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.61,
4.97 (s, 2H), 6.90e6.96 (m, 5H), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.41 (d, 2H,
J ¼ 8.4 Hz),7.65 (d, 1H, J ¼ 8.0 Hz), 7.69 (s, 1H). 13C NMR (100 MHz,
55.39, 111.01, 114.52, 114.52, 118.13, 123.58, 125.56, 127.53, 127.67,
127.67, 128.51, 130.42, 131.32, 135.04, 136.34, 136.64, 137.41, 148.83,
158.37, 159.64, 183.11. ESI-MS: 378.1 [MþH]þ.
CDCl3)
d 38.98, 55.38, 55.46, 110.62, 111.54, 114.47, 114.47, 118.07,
120.92, 122.53, 123.14, 127.62, 127.62, 129.00, 129.39, 131.57, 136.27,
136.84, 149.71, 157.15, 158.56, 159.52, 183.82. ESI-MS: 374.2
[MþH]þ, 396.2 [MþNa]þ.
5.2.14. 1-(2-chlorobenzyl)-5-(4-methoxyphenyl)indoline-2,3-dione
(2j)
Yield 44%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 5.08 (s, 2H),
5.2.20. 1-(3,5-dimethylbenzyl)-5-(4-methoxyphenyl)indoline-2,3-
6.81 (d, 1H, J ¼ 8.4 Hz), 6.96 (d, 2H, J ¼ 8.0 Hz), 7.23e7.26 (m, 2H),
dione (2p)
7.41e7.45 (m, 4H), 7.66 (d, 1H, J ¼ 8.0 Hz), 7.81 (s, 1H). 13C NMR
Yield 46%; 1H NMR (400 MHz, CDCl3)
d 2.29 (s, 6H), 3.84 (s, 3H),
(100 MHz, CDCl3)
d
41.53, 55.39, 111.35, 114.51, 118.10,
4.86 (s, 2H), 6.83 (d, 1H, J ¼ 8.0 Hz), 6.93e6.96 (m, 5H), 7.41 (d, 2H,
123.31,127.50,127.61, 128.34, 129.43, 129.98, 131.26, 131.88, 133.03,
136.40, 137.23, 148.95, 158.56, 159.61, 183.16. ESI-MS: 378.2
[MþH]þ.
J ¼ 8.8 Hz), 7.65 (d, 1H, J ¼ 8.0 Hz), 7.78 (s, 1H). 13C NMR (100 MHz,
CDCl3)
d 21.29, 21.29, 44.13, 55.39, 111.37, 114.49, 114.49, 118.11,
123.34, 125.24, 125.24, 127.63, 127.63, 129.85, 131.49, 134.44, 136.28,
137.05, 138.74, 138.74, 149.39, 158.42, 159.57, 183.62. ESI-MS: 372.2
[MþH]þ, 394.2 [MþNa]þ.
5.2.15. 1-(4-bromobenzyl)-5-(4-methoxyphenyl)indoline-2,3-dione
(2k)
Yield 45%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 4.90 (s, 2H),
6.78 (d, 1H, J ¼ 8.4 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.24 (d, 2H,
5.2.21. 1-(3,4-dimethoxybenzyl)-5-(4-methoxyphenyl)indoline-2,3-
J ¼ 8.4 Hz), 7.41 (d, 2H, J ¼ 8.8 Hz), 7.49 (d, 2H, J ¼ 8.4 Hz), 7.66 (d,
dione (2q)
1H, J ¼ 8.0 Hz), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3)
d
43.58,
Yield 40%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 3.86 (s, 3H),
55.39, 111.10, 114.52, 114.52, 118.14, 122.24, 123.55, 127.66, 127.66,
129.17, 129.17, 131.32, 132.26, 132.26, 133.61, 136.27, 137.39, 148.84,
158.36, 159.65, 183.15. ESI-MS: 422.1 [MþH]þ.
3.86 (s, 3H), 4.89 (s, 2H), 6.83 (d, 1H, J ¼ 8.4 Hz), 6.86 (d, 2H,
J ¼ 8.4 Hz), 6.92 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.41 (d,
2H, J ¼ 8.8 Hz), 7.66 (d, 1H, J ¼ 8.4 Hz), 7.79 (s, 1H). 13C NMR
(100 MHz, CDCl3) d 44.10, 55.39, 55.93, 56.03, 110.75, 111.25, 111.32,
5.2.16. 5-(4-methoxyphenyl)-1-(4-methylbenzyl)indoline-2,3-
114.50, 114.50, 118.13, 120.09, 123.42, 127.01, 127.64, 127.64, 131.43,
136.21, 137.15, 149.05, 149.28, 149.60, 158.43, 159.60, 183.56. ESI-
MS: 404.1 [MþH]þ, 426.1 [MþNa]þ.
dione (2l)
Yield 43%; 1H NMR (400 MHz, CDCl3)
d 2.33 (s, 3H), 3.84 (s, 3H),

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
809
5.2.22. 4-((5-(4-methoxyphenyl)-2,3-dioxoindolin-1-yl)methyl)
(100 mL) and dried over anhydrous magnesium sulfate. The solvent
was removed under vacuum to afford the crude which was purified
by flash column chromatography (silica gel, petroleum ether/ethyl
acetate 3:1) to yield the desired phenylisatin derivatives.
To a flask (25 mL) which contained the solution of phenylisatin
derivatives (0.86 mmol) in dry N,N-dimethylformamide (2 mL). The
reaction temperature was maintained at 0 ꢁC followed by addition
K2CO3 (0.35 g, 2.6 mmol). Stirring 5 min, was added 4-
methoxybenzylchloride (0.16 g, 1.0 mmol) and the mixture
allowed warm to room temperature. The reaction mixture was
stirred at room temperature for 6 h. The orange solution was
poured water (25 mL) and extracted with dichloromethane
(3 ꢃ 50 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered and evaporated. Chromatography (pe-
troleum ether/ethyl acetate 8:1e3:1) afforded the title compounds
3a-3c.
benzonitrile (2r)
Yield 29%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 5.01 (s, 2H),
6.74 (d, 1H, J ¼ 8.0 Hz), 6.97 (d, 2H, J ¼ 8.4 Hz), 7.42 (d, 2H,
J ¼ 8.4 Hz), 7.47 (d, 2H, J ¼ 8.4 Hz), 7.67 (d, 2H, J ¼ 8.4 Hz), 7.68 (d,
1H, J ¼ 8.0 Hz), 7.82 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.73,
55.40, 110.84, 112.36, 114.56, 114.56, 118.16, 118.25, 123.73, 127.67,
127.67, 128.06, 128.06, 131.16, 132.92, 132.92, 136.35, 137.68, 139.98,
148.49, 158.37, 159.73, 182.76. ESI-MS: 369.1 [MþH]þ, 391.1
[MþNa]þ.
5.2.23. 5-(4-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)
indoline-2,3-dione (2s)
Yield 41%; 1H NMR (400 MHz, CDCl3)
d 3.84 (s, 3H), 5.01 (s, 2H),
6.77 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 2H, J ¼ 8.4 Hz), 7.42 (d, 2H,
J ¼ 8.4 Hz), 7.48 (d, 2H, J ¼ 8.0 Hz), 7.63 (d, 2H, J ¼ 8.0 Hz), 7.65 (d,
1H, J ¼ 8.0 Hz), 7.82 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.68,
55.40, 110.84, 112.36, 114.56, 114.56, 118.16, 118.25, 123.73, 127.67,
127.67, 128.06, 128.06, 131.16, 132.92, 132.92, 136.35, 137.68, 139.98,
148.49, 158.37, 159.73, 182.76. ESI-MS: 412.2 [MþH]þ.
5.3.1. 1-(4-methoxybenzyl)-4-(4-methoxyphenyl)indoline-2,3-
dione (3a)
Yield 51%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 3.87 (s, 3H),
4.89 (s, 2H), 6.72 (d, 1H, J ¼ 7.6 Hz), 6.88 (d, 2H, J ¼ 8.4 Hz), 6.97 (d,
2H, J ¼ 8.8 Hz), 7.02 (d, 1H, J ¼ 7.6 Hz), 7.29 (d, 2H, J ¼ 8.8 Hz),
7.42e7.46 (m, 1H), 7.49 (d, 2H, J ¼ 8.0 Hz). 13C NMR (100 MHz,
5.2.24. 5-(4-methoxyphenyl)-1-(2-phenoxyethyl)indoline-2,3-
dione (2t)
Yield 41%; 1H NMR (400 MHz, CDCl3)
d
3.85 (s, 3H), 4.16 (t, 2H,
CDCl3) d 43.52, 55.31, 55.34, 108.96, 113.74, 113.83, 113.83, 114.41,
J ¼ 10.4 Hz), 4.27 (t, 2H, J ¼ 10.4 Hz), 6.845 (d, 2H, J ¼ 8.0 Hz),
114.41, 125.61, 126.70, 128.33, 128.90, 128.90, 130.40, 130.40, 137.40,
143.22, 151.50, 158.09, 159.44, 160.53, 182.02. ESI-MS: 374.2
[MþH]þ.
6.95e6.99 (m, 3H), 7.21e7.28 (m, 4H), 7.46 (d, 2H, J ¼ 8.0 Hz), 7.79
(d, 1H, J ¼ 8.0 Hz), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 40.31,
55.40, 65.72, 111.47, 111.50, 114.37, 114.40, 114.52, 118.03, 121.49,
123.21, 127.67, 127.67, 129.60, 129.60, 131.50, 136.15, 137.07, 149.97,
158.02, 158.63, 159.63, 183.28. ESI-MS: 374.2 [MþH]þ, 396.2
[MþNa]þ.
5.3.2. 1-(4-methoxybenzyl)-6-(4-methoxyphenyl)indoline-2,3-
dione (3b)
Yield 46%; 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 3H), 3.86 (s, 3H),
4.91 (s, 2H), 6.88 (d, 2H, J ¼ 8.4 Hz), 6.94 (s, 1H), 6.98 (d, 2H,
J ¼ 8.8 Hz), 7.24 (d, 1H, J ¼ 8.0 Hz), 7.30 (d, 2H, J ¼ 8.8 Hz), 7.45 (d,
2H, J ¼ 8.4 Hz), 7.63 (d, 1H, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3)
5.2.25. 5-(4-methoxyphenyl)-1-(naphthalen-2-ylmethyl)indoline-
2,3-dione (2u)
Yield 48%; 1H NMR (400 MHz, CDCl3)
d
3.83 (s, 3H), 5.12 (s, 2H),
d 43.52, 55.31, 55.45, 108.87, 114.45, 114.45, 114.60, 114.60, 115.96,
6.84 (d, 1H, J ¼ 8.0 Hz), 6.94 (d, 2H, J ¼ 8.4 Hz), 7.39 (d, 2H,
121.92, 125.86, 126.71, 128.50, 128.50, 128.93, 128.93, 131.79, 151.12,
151.47, 158.95, 159.46, 160.77, 182.52. ESI-MS: 374.2 [MþH]þ, 396.2
[MþNa]þ.
J ¼ 8.8 Hz), 7.44 (d, 1H, J ¼ 8.4 Hz), 7.44e7.50 (m, 2H), 7.59e7.62 (m,
1H), 7.80e7.83 (m, 5H). 13C NMR (100 MHz, CDCl3)
d 44.41, 55.37,
113.36, 114.50, 111.50, 118.17, 123.39, 125.04, 126.39, 126.46, 126.63,
127.62, 127.62, 127.77, 127.80, 129.16, 131.41, 131.99, 133.04, 133.32,
136.23, 137.18, 149.22, 158.51, 159.61, 183.42. ESI-MS: 394.2
[MþH]þ.
5.3.3. 1-(4-methoxybenzyl)-7-(4-methoxyphenyl)indoline-2,3-
dione (3c)
Yield 32%; 1H NMR (400 MHz, CDCl3)
d 3.73 (s, 3H), 3.86 (s, 3H),
4.65 (s, 2H), 6.52 (d, 2H, J ¼ 8.4 Hz), 6.63 (d, 2H, J ¼ 8.4 Hz), 6.82 (d,
5.2.26. 1-(benzo[d] [1,3]dioxol-5-ylmethyl)-5-(4-methoxyphenyl)
2H, J ¼ 8.4 Hz), 6.99 (d, 2H, J ¼ 8.4 Hz), 7.07e7.11 (m, 1H), 7.27e7.30
indoline-2,3-dione (2v)
(m, 1H), 7.63 (d, 1H, J ¼ 8.4 Hz). 13C NMR (100 MHz, CDCl3)
d 44.71,
Yield 46%; 1H NMR (400 MHz, CDCl3)
d
3.84 (s, 3H), 4.85 (s, 2H),
55.25, 55.44, 113.49, 113.49, 113.65, 113.65, 119.30, 123.54, 124.42,
126.99, 127.58, 127.58, 127.76, 129.23, 130.72, 130.72, 142.20, 147.24,
158.87, 159.61, 159.99, 183.62. ESI-MS: 374.1 [MþH]þ, 396.1
[MþNa]þ.
5.95 (s, 2H), 6.78 (d, 1H, J ¼ 8.4 Hz), 6.82e6.86 (m, 3H), 6.96 (d, 2H,
J ¼ 8.4 Hz), 7.41 (d, 2H, J ¼ 8.4 Hz), 7.66 (d, 1H, J ¼ 8.0 Hz), 7.78 (s,
1H). 13C NMR (100 MHz, CDCl3)
d 44.00, 55.39, 101.33, 107.98,
108.58, 111.29, 114.50, 114.50, 118.13, 121.11, 123.43, 127.65, 127.65,
128.27, 131.44, 136.23, 137.17, 147.61, 148.35, 149.15, 158.34, 159.60,
183.44. ESI-MS: 388.1 [MþH]þ, 410.1 [MþNa]þ.
5.4. Synthesis of compounds 4a-4r
To
a
flask (25 mL) which contained the solution of 5-
(1 g, 4.4 mmol) in dry N,N-
5.3. Synthesis of compounds 3a-3c
bromoindoline-2,3-dione
dimethylformamide (4 mL). The reaction temperature was main-
tained at 0 ꢁC followed by addition K2CO3 (1.83 g, 13.2 mmol).
Stirring 5 min, was added 4-methoxybenzylchloride (0.82 g,
5.3 mmol) and the mixture allowed warm to room temperature.
The reaction mixture was stirred at room temperature for 8 h. The
orange solution was poured water (25 mL) and extracted with
dichloromethane (3 ꢃ 50 mL). The combined extracts were dried
over anhydrous magnesium sulfate, filtered and evaporated.
Chromatography (petroleum ether/ethyl acetate 10:1) afforded the
To a microwave reactor vial (5 mL) which contained the solution
of 4 or 5 or 6 or 7-bromoindoline-2,3-dione in 1,4-dioxane (5 mL)
were added PdCl2(dppf) (155 mg, 0.22 mmol), CH3COOK (0.6 g,
6.2 mmol) and 4-Methoxyphenylboronic acid (5.3 mmol) under the
atmosphere of argon. The microwave reactor vial was caped and
placed into the microwave cavity. The reaction mixture was irra-
diated at high level for 30 min at 120 ꢁC. The reaction mixture was
cooled to room temperature, pourd onto 100 mL ice-water and then
extracted with dichloromethane (3 ꢃ 100 mL). The combined
organic layers were washed with water (2 ꢃ 100 mL), brine
title
compounds
5-bromo-1-(4-methoxybenzyl)indoline-2,3-
dione.

810
Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
To a microwave reactor vial (2 mL) which contained the solution
5-bromo-1-(4-methoxybenzyl)indoline-2,3-dione (0.2 g,
55.31, 111.32, 114.45, 114.45, 118.12, 123.66, 126.04, 126.04, 126.19,
126.19, 126.52, 128.99, 128.99, 136.02, 136.54, 137.23, 149.58, 151.07,
of
0.6 mmol) in 1,4-dioxane (2 mL) were added PdCl2(dppf) (21 mg,
0.03 mmol), CH3COOK (82 mg, 0.84 mmol) and substituted benze-
neboronic acid (0.72 mmol) under the atmosphere of argon. The
microwave reactor vial was caped and placed into the microwave
cavity. The reaction mixture was irradiated at high level for
30 min at 120 ꢁC. The reaction mixture was cooled to room tem-
perature, poured onto 30 mL ice-water and then extracted with
dichloromethane (3 ꢃ 50 mL). The combined organic layers were
washed with water (2 ꢃ 100 mL), brine (100 mL) and dried over
anhydrous magnesium sulfate. The solvent was removed under
vacuum to afford the crude which was purified by flash column
chromatography (silica gel, petroleum ether/ethyl acetate 3:1) to
yield the desired compound 4a-4r.
158.38, 159.49, 183.58. ESI-MS: 400.1 [MþH]þ.
5.4.6. 5-(4-fluorophenyl)-1-(4-methoxybenzyl)indoline-2,3-dione
(4f)
Yield 41%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.90 (s, 2H),
6.86e6.90 (m, 3H), 7.10e7.15 (m, 2H), 7.29 (d, 2H, J ¼ 8.0 Hz),
7.42e7.46 (m, 2H), 7.64 (d, 1H, J ¼ 8.0 Hz), 7.78 (s, 1H). 13C NMR
(100 MHz, CDCl3)
d 43.69, 55.32, 111.41, 114.46, 114.46, 115.92,
116.13, 118.14, 123.69, 126.40, 128.18, 128.26, 128.97, 128.97, 135.16,
136.39, 136.54, 149.76, 158.27, 159.52, 161.47, 183.44. ESI-MS: 362.2
[MþH]þ, 384.2 [MþNa]þ.
5.4.7. 5-(4-chlorophenyl)-1-(4-methoxybenzyl)indoline-2,3-dione
(4g)
5.4.1. 1-(4-methoxybenzyl)-5-(3-methoxyphenyl)indoline-2,3-
Yield 48%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.90 (s, 2H),
dione (4a)
Yield 47%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 3.85 (s, 3H),
6.87 (d, 1H, J ¼ 8.4 Hz), 6.88 (d, 2H, J ¼ 8.4 Hz), 7.29 (d, 2H,
J ¼ 8.4 Hz), 7.40 (m, 4H), 7.66 (d,1H, J ¼ 8.4 Hz), 7.79 (s,1H). 13C NMR
4.90 (s, 2H), 6.86 (d, 2H, J ¼ 7.6 Hz), 6.91 (d, 2H, J ¼ 8.0 Hz), 6.99 (t,
1H), 7.06 (d, 1H, J ¼ 7.6 Hz), 7.30 (d, 2H, J ¼ 8.0 Hz), 7.35 (d, 1H,
J ¼ 7.6 Hz), 7.70 (d, 1H, J ¼ 8.0 Hz), 7.82 (s, 1H). 13C NMR (100 MHz,
(100 MHz, CDCl3)
d 43.71, 55.32, 111.46, 114.48, 114.48, 118.18,
123.65,126.36,127.79,127.79,128.96,128.96,129.25,129.25,134.09,
136.10, 136.50, 137.43, 149.99, 158.24, 159.54, 183.34. ESI-MS: 378.1
[MþH]þ, 400.1 [MþNa]þ.
CDCl3)
d 43.70, 55.32, 55.36, 111.33, 112.31, 113.29, 114.46, 114.46,
118.09, 118.98, 123.90, 126.45, 128.98, 128.98, 130.11, 136.77, 137.22,
140.42, 149.89, 158.35, 159.51, 160.14, 183.50. ESI-MS: 374.1
[MþH]þ, 396.1 [MþNa]þ.
5.4.8. 5-(3-chloro-4-fluorophenyl)-1-(4-methoxybenzyl)indoline-
2,3-dione (4h)
5.4.2. 1-(4-methoxybenzyl)-5-(2-methoxyphenyl)indoline-2,3-
Yield 40%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.90 (s, 2H),
dione (4b)
6.88 (d, 1H, J ¼ 8.4 Hz), 6.89 (d, 2H, J ¼ 8.4 Hz), 7.21 (d, 1H,
Yield 43%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 3.80 (s, 3H),
J ¼ 8.4 Hz), 7.28e7.34 (m, 3H), 7.51 (d, 1H, J ¼ 8.4 Hz), 7.63 (d, 1H,
4.89 (s, 2H), 6.83 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 2H, J ¼ 8.4 Hz),
J ¼ 8.4 Hz), 7.76 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.73, 55.32,
6.96e7.03 (m, 2H), 7.22 (d, 1H, J ¼ 8.0 Hz), 7.29e7.33 (m, 3H), 7.63
111.54, 114.49, 114.49, 117.09, 117.30, 118.17, 123.62, 126.23, 126.30,
128.78, 128.97, 128.97, 135.06, 136.28, 136.49, 150.15, 156.70, 158.17,
159.65, 183.23. ESI-MS: 396.1 [MþH]þ, 418.1 [MþNa]þ.
(d, 1H, J ¼ 8.0 Hz), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.61,
55.32, 55.51, 110.68, 111.22, 114.42, 114.42, 117.49, 121.03, 126.59,
126.64, 128.23, 129.01, 129.01, 129.37, 130.18, 134.52, 139.26, 149.40,
156.26, 158.49, 159.47, 183.56. ESI-MS: 374.2 [MþH]þ, 396.2
[MþNa]þ.
5.4.9. 1-(4-methoxybenzyl)-5-(p-tolyl)indoline-2,3-dione (4i)
Yield 46%; 1H NMR (400 MHz, CDCl3)
d 2.38 (s, 3H), 3.79 (s, 3H),
4.89 (s, 2H), 6.85 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 2H, J ¼ 8.4 Hz), 7.24 (d,
5.4.3. 5-(3,5-dimethoxyphenyl)-1-(4-methoxybenzyl)indoline-2,3-
2H, J ¼ 8.0 Hz), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.37 (d, 2H, J ¼ 8.0 Hz), 7.68
(d, 1H, J ¼ 8.4 Hz), 7.81 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 21.10,
dione (4c)
Yield 41%; 1H NMR (400 MHz, CDCl3)
d 3.81 (s, 3H), 3.85 (s, 6H),
43.66, 55.31, 111.30, 114.44, 114.44, 118.12, 123.66, 126.38, 126.38,
126.50, 128.96, 128.96, 129.77, 129.77, 136.08, 136.48, 137.35, 137.83,
149.55, 158.37, 159.49, 183.56. ESI-MS: 358.2 [MþH]þ.
4.92 (s, 2H), 6.48 (t, 1H, J ¼ 4.4 Hz), 6.61 (s, 2H), 6.87e6.91 (m, 3H),
7.31 (d, 2H, J ¼ 8.4 Hz), 7.71 (d, 1H, J ¼ 8.4 Hz), 7.84 (s, 1H). 13C NMR
(100 MHz, CDCl3) d 43.68, 55.32, 55.48, 55.48, 99.71, 104.83, 104.83,
111.32, 114.45, 114.45, 118.00, 123.84, 126.44, 128.99, 128.99, 136.78,
137.20, 141.02, 149.97, 158.35, 159.50, 161.28, 161.28, 183.50. ESI-MS:
404.2 [MþH]þ, 426.2 [MþNa]þ.
5.4.10. 5-(4-isopropoxyphenyl)-1-(4-methoxybenzyl)indoline-2,3-
dione (4j)
Yield 43%; 1H NMR (400 MHz, CDCl3)
d 1.34 (s, 3H), 1.36 (s, 3H),
3.79 (s, 3H), 4.55e4.61 (m, 1H), 4.89 (s, 2H), 6.83 (d, 1H, J ¼ 8.4 Hz),
6.88 (d, 2H, J ¼ 8.4 Hz), 6.93 (d, 2H, J ¼ 8.8 Hz), 7.29 (d, 2H,
J ¼ 8.4 Hz), 7.39 (d, 2H, J ¼ 8.8 Hz), 7.64 (d, 1H, J ¼ 8.4 Hz), 7.78 (s,
5.4.4. 5-(3,4-dimethoxyphenyl)-1-(4-methoxybenzyl)indoline-2,3-
dione (4d)
Yield 38%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 3.91 (s, 3H),
1H). 13C NMR (100 MHz, CDCl3)
d 22.04, 22.04, 43.65, 55.31, 70.02,
3.93 (s, 3H), 4.90 (s, 2H), 6.85 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 2H,
J ¼ 8.0 Hz), 6.92 (d, 1H, J ¼ 8.4 Hz), 6.97 (s, 1H), 7.04 (d, 1H,
J ¼ 8.0 Hz), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.67 (d, 1H, J ¼ 8.0 Hz), 7.80 (s,
111.28, 114.44, 114.44, 116.27, 116.27, 118.12, 123.35, 126.54, 127.63,
127.63, 128.96, 128.96, 131.15, 136.15, 137.15, 149.21, 157.92, 158.38,
159.48, 183.62. ESI-MS: 402.2 [MþH]þ.
1H). 13C NMR (100 MHz, CDCl3)
d 43.67, 55.31, 56.01, 56.01, 109.71,
111.33, 111.60, 114.44, 114.44, 118.08, 118.90, 123.48, 126.47, 128.96,
128.96, 131.86, 136.35, 137.22, 149.08, 149.39, 149.39, 158.35, 159.49,
183.65. ESI-MS: 404.2 [MþH]þ.
5.4.11. 1-(4-methoxybenzyl)-5-phenylindoline-2,3-dione (4k)
Yield 45%; 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 3H), 4.89 (s, 2H),
6.87 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 2H, J ¼ 8.0 Hz), 7.30 (d, 2H,
J ¼ 8.8 Hz), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.42 (d, 2H, J ¼ 8.0 Hz), 7.45 (d,
2H, J ¼ 8.8 Hz), 7.46e7.48 (d, 1H, J ¼ 8.0 Hz), 7.71 (s, 1H). 13C NMR
5.4.5. 5-(4-(tert-butyl)phenyl)-1-(4-methoxybenzyl)indoline-2,3-
dione (4e)
Yield 34%; 1H NMR (400 MHz, CDCl3)
d
1.35 (s, 9H), 3.79 (s, 3H),
(100 MHz, CDCl3) d 43.68, 55.32, 111.36, 114.45, 114.45, 118.13,
4.90 (s, 2H), 6.86 (d, 1H, J ¼ 8.4 Hz), 6.89 (d, 2H, J ¼ 8.4 Hz), 7.30 (d,
123.85,126.47,126.55,126.55,127.90,128.98,128.98,129.08,129.08,
136.73, 137.37, 138.96, 149.79, 158.35, 159.50, 183.50. ESI-MS: 344.2
[MþH]þ.
2H, J ¼ 8.4 Hz), 7.41e7.47 (m, 4H), 7.70 (d, 1H, J ¼ 8.4 Hz), 7.83 (s,
1H). 13C NMR (100 MHz, CDCl3)
d 31.31, 31.31, 31.31, 34.61, 43.67,

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
811
5.4.12. 4-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl)benzonitrile
7.37e7.41 (m, 2H), 7.71 (d, 1H, J ¼ 8.0 Hz), 7.82 (s, 1H). 13C NMR
(4l)
(100 MHz, CDCl3) d 43.69, 55.32, 111.38, 114.45, 114.45, 118.07,
Yield 29%; 1H NMR (400 MHz, CDCl3)
d
3.79 (s, 3H), 4.91 (s, 2H),
120.52, 123.09, 125.68, 126.43, 127.01, 128.96, 128.96, 132.15, 135.87,
140.08, 149.43,158.30, 159.50, 183.52. ESI-MS: 350.1 [MþH]þ, 372.0
[MþNa]þ.
6.89 (d, 2H, J ¼ 8.8 Hz), 6.93 (d, 1H, J ¼ 8.4 Hz), 7.29 (d, 2H,
J ¼ 8.8 Hz), 7.59 (d, 2H, J ¼ 8.4 Hz), 7.71 (d, 2H, J ¼ 8.4 Hz), 7.73 (d,
1H, J ¼ 8.4 Hz), 7.84 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.77,
55.32, 111.60, 111.70, 114.50, 114.50, 118.27, 118.54, 123.88, 126.16,
127.13, 127.13, 128.98, 128.98, 132.89, 132.89, 135.10, 136.77, 143.34,
150.76, 158.11, 159.58, 183.06. ESI-MS: 369.2 [MþH]þ.
5.5. Synthesis of compounds 5a-5k
A mixture of 0.4 mmol compound 2m, 0.8 mmol sodium boro-
hydride were stirred in 2 mL dichloromethane and 8 mL ethanol at
room temperature under an argon atmosphere. The progress of the
reaction was monitored by TLC (petroleum ether/ethyl acetate).
After the reaction finished, the reaction mixture was poured water
(25 mL) and extracted with dichloromethane (3 ꢃ 50 mL). The
combined extracts were dried over anhydrous magnesium sulfate,
filtered and evaporated. Chromatography (petroleum ether/ethyl
acetate 3:1) afforded the title compound 5a.
A mixture of 0.4 mmol compound 2m, 0.8 mmol methyl-
magnesiubromide (1.0M solution in THF) were stirred in 2 mL dry
THF at ꢀ78 ꢁC under a argon atmosphere. The progress of the re-
action was monitored by TLC (petroleum ether/ethyl acetate). After
the reaction finished, the reaction mixture was poured ice water
and extracted with dichloromethane (3 ꢃ 50 mL). The combined
extracts were dried over anhydrous magnesium sulfate, filtered and
evaporated. Chromatography (petroleum ether/ethyl acetate 3:1)
afforded the title compound 5b.
A mixture of 0.4 mmol compounds 2m, 1.2 mmol Hydrazine
hydrate were stirred in 5 mL ethanol at reflux under a argon at-
mosphere. The progress of the reaction was monitored by TLC
(petroleum ether/ethyl acetate). After the reaction finished, the
reaction mixture was poured water (25 mL) and extracted with
dichloromethane (3 ꢃ 50 mL). The combined extracts were dried
over anhydrous magnesium sulfate, filtered and evaporated.
Chromatography (petroleum ether/ethyl acetate 6:1) afforded the
title compounds 5c.
To a flask (25 mL) which contained the solution of compound
2m (0.15 g, 0.4 mmol) in dry THF (8 mL). The reaction temperature
was maintained at 0 ꢁC followed by the added triethyl phospho-
noacetate (0.48 mmol) and the mixture allowed warm to room
temperature. The reaction mixture was stirred at room tempera-
ture for 1 h. The orange solution was poured water (25 mL) and
extracted with dichloromethane (3 ꢃ 50 mL). The combined ex-
tracts were dried over anhydrous magnesium sulfate, filtered and
evaporated. Chromatography (petroleum ether/ethyl acetate 8:1)
afforded the title compounds 5d and 5e.
5.4.13. 1-(4-methoxybenzyl)-5-(4-(trifluoromethyl)phenyl)
indoline-2,3-dione (4m)
Yield 31%; 1H NMR (400 MHz, CDCl3)
d 3.81 (s, 3H), 4.94 (s, 2H),
6.91 (d, 2H, J ¼ 8.8 Hz), 6.94 (d, 1H, J ¼ 8.0 Hz), 7.32 (d, 2H,
J ¼ 8.8 Hz), 7.61 (d, 2H, J ¼ 8.0 Hz), 7.72 (d, 2H, J ¼ 8.8 Hz), 7.74 (d,
1H, J ¼ 8.4 Hz), 7.86 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.76,
55.32, 111.57, 114.51, 114.51, 118.24, 123.92, 126.04, 126.08, 126.28,
126.85,126.85,128.98, 128.98, 129.84, 130.16,135.74,136.80,142.46,
150.47, 158.19, 159.58, 183.20. ESI-MS: 412.1 [MþH]þ, 434.1
[MþNa]þ.
5.4.14. 1-(4-methoxybenzyl)-5-(4-(trifluoromethoxy)phenyl)
indoline-2,3-dione (4n)
Yield 39%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.90 (s, 2H),
6.87e6.90 (m, 3H), 7.28e7.30 (m, 4H), 7.49 (d, 2H, J ¼ 8.8 Hz), 7.66
(d, 1H, J ¼ 8.0 Hz), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.72,
55.31, 111.48, 114.48, 114.48, 118.19, 121.15, 121.15, 123.75, 126.35,
127.96, 127.96, 128.97, 128.97, 135.91, 136.62, 136.62, 137.71, 149.03,
150.08, 158.23, 159.55, 183.31. ESI-MS: 428.1 [MþH]þ, 450.0
[MþNa]þ.
5.4.15. 5-(4-hydroxyphenyl)-1-(4-methoxybenzyl)indoline-2,3-
dione (4o)
Yield 28%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.89 (s, 2H),
6.84 (d, 1H, J ¼ 8.4 Hz), 6.87e6.90 (m, 4H), 7.29 (d, 2H, J ¼ 8.4 Hz),
7.36 (d, 2H, J ¼ 8.4 Hz), 7.63 (d, 1H, J ¼ 8.4 Hz), 7.77 (s, 1H). 13C NMR
(100 MHz, DMSO)
d 42.91, 55.53, 112.03, 114.53, 114.53, 116.23,
116.23, 118.68, 121.99, 127.80, 127.92, 127.92, 129.33, 129.33, 129.76,
135.61, 136.16, 149.16, 157.73, 158.84, 159.18, 183.78. ESI-MS: 360.1
[MþH]þ, 382.1 [MþNa]þ.
5.4.16. 1-(4-methoxybenzyl)-5-(naphthalen-2-yl)indoline-2,3-
dione (4p)
Yield 47%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.92 (s, 2H),
6.89 (d, 2H, J ¼ 8.8 Hz), 6.91 (d, 1H, J ¼ 8.4 Hz), 7.31 (d, 2H,
J ¼ 8.8 Hz), 7.49e7.53 (m, 2H), 7.61 (d, 1H, J ¼ 8.4 Hz), 7.82e7.89 (m,
A mixture of 0.4 mmol compounds 2m,1.6 mmol ethylene glycol
and 0.06 mmol toluene-p-sulfonic acid were stirred in 15 mL
toluene at 125 ꢁC under a argon atmosphere. The progress of the
reaction was monitored by TLC (petroleum ether/ethyl acetate).
After the reaction finished, the reaction mixture was poured water
(25 mL) and extracted with ethyl acetate (3 ꢃ 40 mL). The com-
bined extracts were dried over anhydrous magnesium sulfate,
filtered and evaporated. Chromatography (petroleum ether/ethyl
acetate 10:1) afforded the title compounds 5f.
6H). 13C NMR (100 MHz, CDCl3)
d 43.71, 55.32, 111.44, 114.48, 114.48,
118.24, 124.06, 124.57, 125.41, 126.39, 126.47, 126.68, 127.69, 128.16,
128.88,128.98, 128.98,132.76,133.56,136.22,136.90,137.26,149.82,
158.35, 159.54, 183.49. ESI-MS: 394.1 [MþH]þ, 416.1 [MþNa]þ.
5.4.17. 1-(4-methoxybenzyl)-5-(pyridin-4-yl)indoline-2,3-dione
(4q)
Yield 25%; 1H NMR (400 MHz, CDCl3)
d 3.79 (s, 3H), 4.92 (s, 2H),
6.89 (d, 2H, J ¼ 8.4 Hz), 6.94 (d, 1H, J ¼ 8.4 Hz), 7.27 (d, 2H,
To a flask (25 mL) which contained the solution of compound
2m (0.15 g, 0.44 mmol) in dry dichloromethane (8 mL). The reaction
temperature was maintained at 0 ꢁC followed by the added DAST
(77 mg, 0.48 mmol) and the mixture allowed warm to room tem-
perature. The reaction mixture was stirred at room temperature for
2 h. The orange solution was poured water (25 mL) and extracted
with dichloromethane (3 ꢃ 50 mL). The combined extracts were
dried over anhydrous magnesium sulfate, filtered and evaporated.
Chromatography (petroleum ether/ethyl acetate 5:1) afforded the
title compounds 5g.
J ¼ 8.4 Hz), 7.45 (d, 2H, J ¼ 6.0 Hz), 7.78 (d, 1H, J ¼ 8.4 Hz), 7.90 (s,
1H), 8.68 (d, 2H, J ¼ 6.0 Hz). 13C NMR (100 MHz, CDCl3)
d 43.79,
55.33, 111.73, 114.51, 114.51, 118.30, 120.92, 123.73, 126.15, 128.98,
128.98, 133.99, 136.63, 136.63, 146.25, 150.40, 150.40, 151.14, 158.12,
159.59, 183.01. ESI-MS: 345.2 [MþH]þ, 367.2 [MþNa]þ.
5.4.18. 1-(4-methoxybenzyl)-5-(thiophen-3-yl)indoline-2,3-dione
(4r)
Yield 40%; 1H NMR (400 MHz, CDCl3)
d
3.79 (s, 3H), 4.89 (s, 2H),
6.83 (d, 1H, J ¼ 8.0 Hz), 6.88 (d, 2H, J ¼ 8.8 Hz), 7.27e7.30 (m, 3H),
A
mixture of 0.4 mmol compounds 2m, 0.44 mmol

812
Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
hydroxylamine hydrochloride and 0.2 mmol Na2CO3 were stirred in
10 mL ethanol at reflux under an argon atmosphere. The progress of
the reaction was monitored by TLC (petroleum ether/ethyl acetate).
After the reaction finished, the reaction mixture was filtered. The
filtrate was concentrated to dryness and subjected to flash column
chromatography (silica gel), eluting with petroleum ether/ethyl
acetate, afforded compounds 5h.
3.88 (s, 3H), 4.89 (s, 2H), 6.75 (d, 1H, J ¼ 8.0 Hz), 6.85 (d, 2H,
J ¼ 8.8 Hz), 6.96 (d, 2H, J ¼ 8.8 Hz), 7.00 (s, 1H), 7.25 (d, 2H,
J ¼ 8.8 Hz), 7.44 (d, 1H, J ¼ 8.0 Hz), 7.49 (d, 2H, J ¼ 8.8 Hz), 8.82 (s,
1H). 13C NMR (100 MHz, CDCl3)
d 43.74, 52.25, 55.28, 55.37, 109.37,
114.25, 114.25, 114.29, 114.29, 120.45, 122.34, 127.30, 127.47, 127.79,
127.79, 128.71, 128.71, 130.62, 133.13, 135.90, 138.23, 143.94, 159.04,
159.21, 166.07, 167.66. ESI-MS: 430.1 [MþH]þ, 452.2 [MþNa]þ.
To a flask (25 mL) which contained the solution of compound 5c
(0.15 g, 0.42 mmol) in ethanol (8 mL) were added Piperidine (4 mg,
0.04 mmol) and substituted benzaldehyde (0.5 mmol) under the
atmosphere of argon. The reaction mixture was stirred at reflux for
4 h. The orange solution was poured water (25 mL) and extracted
with dichloromethane (3 ꢃ 50 mL). The combined extracts were
dried over anhydrous magnesium sulfate, filtered and evaporated.
Chromatography (petroleum ether/ethyl acetate 10:1e3:1) affor-
ded the title compounds 5i-5k.
5.5.6. 1'-(4-methoxybenzyl)-5'-(4-methoxyphenyl)spiro[ [1,3]
dioxolane-2,30-indolin]-20-one (5f)
Yield 89%; 1H NMR (400 MHz, CDCl3)
d 3.77 (s, 3H), 3.83 (s, 3H),
4.35e4.38 (m, 2H), 4.63e4.66 (m, 2H), 4.78 (s, 2H), 6.72 (d, 1H,
J ¼ 8.0 Hz), 6.85 (d, 2H, J ¼ 8.4 Hz), 6.93 (d, 2H, J ¼ 8.8 Hz), 7.24 (d,
2H, J ¼ 8.4 Hz), 7.41 (d,1H, J ¼ 8.0 Hz), 7.42 (d, 2H, J ¼ 8.8 Hz), 7.55 (s,
1H). 13C NMR (100 MHz, CDCl3)
d 43.04, 55.27, 55.34, 65.94, 65.94,
102.39, 109.96, 114.22, 114.22, 114.27, 114.27, 123.39, 124.53, 127.28,
127.80,127.80,128.64, 128.64,129.75,132.99,136.55,142.59,159.06,
159.18, 173.46. ESI-MS: 418.2 [MþH]þ, 440.2 [MþNa]þ.
5.5.1. 3-hydroxy-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)
indolin-2-one (5a)
Yield 59%; 1H NMR (400 MHz, CDCl3)
d 3.76 (s, 3H), 3.83 (s, 3H),
5.5.7. 3,3-difluoro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)
indolin-2-one (5g)
4.77e4.90 (m, 2H), 5.20 (s, 1H), 6.78 (d, 1H, J ¼ 8.4 Hz), 6.85 (d, 2H,
J ¼ 8.4 Hz), 6.94 (d, 2H, J ¼ 8.8 Hz), 7.26 (d, 2H, J ¼ 8.4 Hz), 7.39 (d,
1H, J ¼ 8.4 Hz), 7.44 (d, 2H, J ¼ 8.8 Hz), 7.65 (s, 1H). 13C NMR
Yield 81%; 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 3H), 3.84 (s, 3H),
4.85 (s, 2H), 6.83 (d, 1H, J ¼ 8.4 Hz), 6.87 (d, 2H, J ¼ 8.4 Hz), 6.96 (d,
(100 MHz, CDCl3) d 43.48, 55.27, 55.35, 70.01, 109.74, 114.26, 114.26,
2H, J ¼ 8.4 Hz), 7.26 (d, 2H, J ¼ 8.4 Hz), 7.42 (d, 2H, J ¼ 8.4 Hz), 7.52
114.28, 114.28, 123.78, 127.36, 127.50, 127.81, 127.95, 127.95, 128.79,
128.79, 133.12, 136.38, 141.79, 159.05, 159.22, 176.97. ESI-MS: 376.2
[MþH]þ.
(d, 1H, J ¼ 8.4 Hz), 7.72 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.61,
55.30, 55.38, 110.83, 114.44, 114.44, 114.44, 114.44, 120.75, 123.10,
123.10, 126.31, 127.83, 127.83, 128.81, 128.81, 131.48, 132.03, 137.27,
141.67, 159.46, 159.48, 165.46. ESI-MS: 396.1 [MþH]þ, 418.1
[MþNa]þ.
5.5.2. 3-hydroxy-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-3-
methylindolin-2-one (5b)
Yield 59%; 1H NMR (400 MHz, CDCl3)
d 1.68 (s, 3H), 3.77 (s, 3H),
3.83 (s, 3H), 4.77e4.93 (m, 2H), 6.78 (d, 1H, J ¼ 8.4 Hz), 6.85 (d, 2H,
J ¼ 8.4 Hz), 6.94 (d, 2H, J ¼ 8.8 Hz), 7.24 (d, 2H, J ¼ 8.4 Hz), 7.37 (d,
1H, J ¼ 8.4 Hz), 7.44 (d, 2H, J ¼ 8.8 Hz), 7.59 (s, 1H). 13C NMR
5.5.8. (Z)-3-(hydroxyimino)-1-(4-methoxybenzyl)-5-(4-
methoxyphenyl)indolin-2-one (5h)
Yield 87%; 1H NMR (400 MHz, DMSO)
d 3.71 (s, 3H), 3.78 (s, 3H),
(100 MHz, CDCl3)
d 25.14, 43.32, 55.26, 55.35, 73.87, 109.83, 114.26,
4.90 (s, 2H), 6.90 (d, 2H, J ¼ 7.8 Hz), 7.00 (d, 2H, J ¼ 7.8 Hz), 7.06 (d,
1H, J ¼ 8.0 Hz), 7.30 (d, 2H, J ¼ 8.0 Hz), 7.50 (d, 2H, J ¼ 8.0 Hz), 7.59
(d, 1H, J ¼ 8.0 Hz), 8.20 (s, 1H), 13.63 (s, 1H). 13C NMR (100 MHz,
114.26, 114.26, 114.26, 122.09, 127.51, 127.78, 127.82, 127.82, 128.64,
128.64, 131.92, 133.19, 136.40, 140.71, 159.02, 159.16, 178.56. ESI-MS:
390.1 [MþH]þ, 412.1 [MþNa]þ.
DMSO)
d 42.56, 55.52, 55.63, 110.51, 114.57, 114.57, 114.88, 114.88,
116.42, 125.15, 127.94, 127.94, 128.57, 129.19, 129.19, 130.26, 132.54,
135.32, 142.01, 143.96, 159.11, 159.19, 163.68. ESI-MS: 389.2
[MþH]þ.
5.5.3. 1-(4-methoxybenzyl)-5-(4-methoxyphenyl)indolin-2-one
(5c)
Yield 54%; 1H NMR (400 MHz, CDCl3)
d 3.65 (s, 2H), 3.77 (s, 3H),
3.83 (s, 3H), 4.87 (s, 2H), 6.78 (d, 1H, J ¼ 8.4 Hz), 6.85 (d, 2H,
J ¼ 8.4 Hz), 6.94 (d, 2H, J ¼ 8.8 Hz), 7.27 (d, 2H, J ¼ 8.4 Hz), 7.33 (d,
1H, J ¼ 8.4 Hz), 7.42 (d, 2H, J ¼ 8.8 Hz), 7.43 (s, 1H). 13C NMR
5.5.9. (Z)-3-benzylidene-1-(4-methoxybenzyl)-5-(4-
methoxyphenyl)indolin-2-one (5i)
Yield 41%; 1H NMR (400 MHz, CDCl3)
d 3.78 (s, 3H), 3.81 (s, 3H),
(100 MHz, CDCl3) d 35.93, 43.33, 55.27, 55.35, 109.22, 114.18, 114.18,
4.96 (s, 2H), 6.79 (d, 1H, J ¼ 8.4 Hz), 6.85e6.91 (m, 4H), 7.28e7.35
114.26, 114.26, 123.04, 125.08, 126.20, 127.80, 127.80, 127.99, 128.80,
128.80, 133.50, 133.55, 143.22, 158.93, 159.11, 175.06. ESI-MS: 360.2
[MþH]þ, 382.2 [MþNa]þ.
(m, 5H), 7.43e7.45 (m, 3H), 7.47 (d, 2H, J ¼ 8.4 Hz), 7.70 (s, 1H), 7.84
(s, 1H). 13C NMR (100 MHz, CDCl3)
d 43.35, 55.28, 55.34, 109.37,
114.19, 114.19, 114.26, 114.26, 121.27, 121.82, 127.28, 127.56, 127.56,
128.09,128.13,128.65,128.65,128.76,128.76,129.34,129.34,129.73,
133.40, 134.82, 134.99, 137.73, 142.25, 158.88, 159.09, 168.60. ESI-
MS: 448.2 [MþH]þ.
5.5.4. (Z)-ethyl 2-(1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-2-
oxoindolin-3-ylidene)acetate (5d)
Yield 76%; 1H NMR (400 MHz, CDCl3)
d 1.35e1.39 (m, 3H), 3.77
(s, 3H), 3.84 (s, 3H), 4.31e4.37 (m, 2H), 4.90 (s, 2H), 6.75 (d, 1H,
J ¼ 8.0 Hz), 6.85 (d, 2H, J ¼ 8.8 Hz), 6.96 (d, 2H, J ¼ 8.4 Hz), 7.01 (s,
1H), 7.25 (d, 2H, J ¼ 8.4 Hz), 7.44 (d, 1H, J ¼ 8.0 Hz), 7.48 (d, 2H,
5.5.10. (Z)-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-3-(4-
(trifluoromethyl)benzylidene) indolin-2-one (5j)
J ¼ 8.8 Hz), 8.81 (s, 1H). 13C NMR (100 MHz, CDCl3)
d
14.24, 43.45,
Yield 39%; 1H NMR (400 MHz, CDCl3)
d 3.77 (s, 3H), 3.85 (s, 3H),
55.28, 55.36, 61.26, 109.34, 114.25, 114.25, 114.28, 114.28, 120.49,
123.00, 127.27, 127.50, 127.81, 127.81, 128.70, 128.70, 130.53, 133.18,
135.88, 137.86, 143.89, 159.02, 159.20, 165.63, 167.73. ESI-MS: 466.3
[MþNa]þ.
4.93 (s, 2H), 6.80 (d, 1H, J ¼ 8.0 Hz), 6.85 (d, 2H, J ¼ 8.4 Hz), 6.98 (d,
2H, J ¼ 8.4 Hz), 7.29 (d, 2H, J ¼ 8.4 Hz), 7.39 (d, 1H, J ¼ 8.0 Hz), 7.48
(d, 2H, J ¼ 8.4 Hz), 7.63 (s, 1H), 7.69e7.71 (m, 3H), 8.36 (d, 2H,
J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3)
d 43.33, 55.28, 55.36, 109.37,
114.21, 114.21, 114.29, 114.29, 118.01, 121.60, 121.88, 127.28, 127.85,
127.85,128.04,128.09,128.81,128.81,128.90,128.90,129.65,129.65,
130.15, 133.50, 134.82, 134.99, 137.75, 142.31, 158.90, 159.06, 168.71.
ESI-MS: 516.2 [MþH]þ.
5.5.5. (Z)-methyl 2-(1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-2-
oxoindolin-3-ylidene)acetate (5e)
Yield 68%; 1H NMR (400 MHz, CDCl3)
d 3.77 (s, 3H), 3.84 (s, 3H),

Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
813
5.5.11. (Z)-3-(4-isopropylbenzylidene)-1-(4-methoxybenzyl)-5-(4-
cytometry. The fraction of cell population in different quadrants
was analyzed using quadrant statistics.
methoxyphenyl)indolin-2-one (5k)
Yield 37%; 1H NMR (400 MHz, CDCl3)
d 1.27 (s, 3H), 1.29 (s, 3H),
2.93e3.00 (m, 1H), 3.76 (s, 3H), 3.85 (s, 3H), 4.95 (s, 2H), 6.78 (d, 1H,
J ¼ 8.0 Hz), 6.84 (d, 2H, J ¼ 8.4 Hz), 6.97 (d, 2H, J ¼ 8.4 Hz), 7.29 (d,
2H, J ¼ 8.4 Hz), 7.31 (d, 2H, J ¼ 8.0 Hz), 7.33 (d, 1H, J ¼ 8.0 Hz), 7.49
(d, 2H, J ¼ 8.4 Hz), 7.63 (s, 1H), 7.68 (s, 1H), 8.29 (d, 2H, J ¼ 8.0 Hz).
5.6.5. Wound healing assay
Wound healing assay was performed to determine migration
rate and repair ability of HepG2 cells. The HepG2 cells were plated
in 6-well plates at a density of 2 ꢃ 105 cells/well and cultured in
Dulbecco's modified Eagle's medium. After 24 h incubation, the
cultured cells grew against the wall of 6-well plates. Then, the
13C NMR (100 MHz, CDCl3)
d 23.87, 23.87, 34.20, 43.34, 55.28, 55.35,
109.30, 114.19, 114.19, 114.22, 114.22, 121.29, 122.03, 126.64, 126.75,
126.75, 127.59, 127.59, 127.81, 128.22, 128.78, 128.78, 129.61, 129.61,
132.49, 133.49, 134.69, 138.08, 142.14, 151.07, 158.87, 159.08, 168.75.
ESI-MS: 490.2 [MþH]þ.
HepG2 cells were scratched with a 10 ml pipette tip to make a
straight line on the HepG2 cells. The medium in the well was
removed and the well of the HepG2 cells was washed for three
times by 1 ꢃ PBS. The cells were treated with DMSO or compound
2m (10 nM, 30 nM, 100 nM) for 0, 6, 12, 24, 36 and 48 h respectively,
and incubated at 37 ꢁC and 5% CO2 atmosphere. Images of the
HepG2 cells were got by a Nikon Eclipse Ti microscope (Nikon,
Tokyo, Japan).
5.6. Biology evaluation
5.6.1. Cell lines and culture conditions
The human leukemia K562 cell line was purchased from the
Shanghai Institutes of Biological Sciences (Shanghai, China). Cells
were maintained in RPMI-1640 supplemented with 10% fetal
bovine serum, 2.05 mM glutamine and 1% penicillin/streptomycin.
The medium was replaced once every third days. The umbilical vein
endothelial cells (HUVECs) were purchased from ATCC (American
type culture collection, USA) and cultured in Ham's F-12 medium
supplemented with 10% fetal bovine serum, 2.05 mM glutamine
and 1% penicillin/streptomycin. The liver cancer cells (HepG2) were
grown in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 2.05 mM glutamine and 1% penicillin/
streptomycin. All cell lines were incubated in a humidified atmo-
sphere of 5% CO2 at 37 ꢁC.
5.6.6. Transwell assay
The transwell assay of HepG2 cells was performed using 24-well
culture plates containing polycarbonate filter inserts (EMD Milli-
pore corporation, Germany). The HepG2 cells were suspended in
Dulbecco's modified Eagle's medium without fetal bovine serum
and 200
m
l (2 ꢃ 105 cells/well) were added to the upper compart-
ment of the chamber. 600 ml Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum was added to the lower
chamber. After 30 min, the cells were treated with DMSO or com-
pound 2m (10 nM, 30 nM,100 nM) for 24 h and incubated in 5% CO2
atmosphere at 37 ꢁC. After 24 h, medium in the chamber was
removed and the well of the HepG2 cells was washed for three
times by 1 ꢃ PBS. Then, use the methanol as fixative and DAPI as
stain. HepG2 cells on the upper side of the filter were removed
using cotton swabs. The photographs of three horizons were taken
in random. The migrated HepG2 cells to the lower side of the filter
were counted.
5.6.2. Cytotoxic activity assay
Inhibition of cell proliferation by compound 2m was measured
by the MTT assay. In brief, 100 ml of K562 cells were plated in 96-
well plates at a density of 5 ꢃ 104 cells/ml for 2 h. Different con-
centrations of compound 2m (10e100,000 nM) were added to each
well to culture for another 48 h. After 48 h of incubation, 10 ml MTT
(5 mg/mL) was added to each well and the plates were further
incubated for 4 h. MTT assay was performed using thermo micro-
plate reader. The DMSO-treated controls were calculated as a cell
viability value of 100%. The inhibitory concentrations (IC50) were
obtained by nonlinear regression using GraphPad Prism 4.0. For
each experiment, IC50 value was calculated from three independent
assays.
5.6.7. Tube forming assay
The capillary-like network formation of HUVEC cells was
investigated with Matrigel-coated 96-well culture plates [17].
Matrigel (13.9 mg/mL; BD Bioscience, San Jose, CA, USA) was
thawed at 4 ꢁC. Fifty microliters of the Matrigel was added to each
well of the 24-well culture plates and incubated for 30 min at 37 ꢁC
for polymerization. The HUVEC cells (1 ꢃ 105 cells/well) were sus-
pended in Ham's F-12 medium containing 10% FBS, and were then
added to the Matrigel-coated wells. After 40 min incubation at
37 ꢁC in 5% CO2 atmosphere, added DMSO or compound 2m
(10 nM, 30 nM, 100 nM) to the cells. Then, the plates were incu-
bated at 37 ꢁC in 5% CO2 atmosphere. After 12 h incubation, the
capillary-like tube formation in each well of the culture plates was
photographed by a Nikon Eclipse Ti microscope (Nikon, Tokyo,
Japan).
5.6.3. Cell cycle analysis
For the DNA content analysis, 5 ꢃ 104 cells were cultured for 2 h
and treated with compound 2m (30 nM, 300 nM) for 0, 6,12, 24 and
48 h respectively. Cells were collected and fixed with 1 mL of 70%
ice-cold ethanol at ꢀ20 ꢁC overnight. Cells then were washed with
PBS, incubated with propidium iodide (PI, 50
(100
temperature and analyzed using a FACS Calibur system (version 2.0,
BD) using the CELLQuest program (Becton Dickinson). Results were
representatives of at least three independent experiments.
mg/mL), RNase A
g/mL) and 0.2% Triton ꢃ 100 for 30 min in dark at room
m
Acknowledgments
The authors sincerely thank the financial support from National
Natural Science Foundation of China (31601203), Undergraduate
Training Program for Innovation and Entrepreneurship of Tianjin
University of Science and Technology (201710057099).
The authors are thankful to the Research Centre of Modern
Analytical Technology, Tianjin University of Science & Technology
for NMR and HRMS analysis.
5.6.4. Flow cytometric analysis of apoptosis
Apoptotic cells were assayed by the Annexin-V-FITC Apoptosis
Detection Kit (Tianjin Sungene Biotech Co., Ltd.) according to the
manufacturer's instructions. K562 cells were seeded in six-well
plates and treated with DMSO or compound 2m (30 nM, 300 nM)
for 0, 6, 12, 24 and 48 h respectively. After the treatment, cells were
harvested, washed twice with ice-cold PBS at 2500 rpm and
resuspended in 1 ꢃ Binding buffer. Then, the cells were stained
Appendix A. Supplementary data
with 5
ml of Annexin-V-FITC for 10 min and 5 ml of PI (50 mg/mL) for
10 min at room temperature in dark and analyzed by flow
Supplementary data associated with this article can be found in

814
Q. Zhang et al. / European Journal of Medicinal Chemistry 156 (2018) 800e814
S. Fu, Discovery of unsymmetrical aromatic disulfides as novel inhibitors of
SARS-CoV main protease: chemical synthesis, biological evaluation, molecular
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