Design, synthesis, and biological evaluation of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate derivatives as anti-proliferative agents through ROS-induced cell apoptosis

Article abstract of DOI:10.1016/j.ejmech.2016.09.005

A novel class of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate derivatives were designed and synthesized as potent anti-proliferative agents. Most of these compounds showed potent anti-proliferative activity against some tumor cell lines, including SK-BR-3, MDA-MB-231, HCT-116, SW480, Ovcar-3, HL-60, Saos-2 and HepG2. Compounds 8c and 11h were identified as the most potent ones, while HL-60, HCT116 and MDA-MB-231 were the most sensitive cell lines. Mechanistic study revealed that compound 8c enhanced reactive oxygen species level by inhibiting TrxR and then induced apoptosis by activating apoptosis proteins, bax and cleaved-caspase 3 in HCT116?cells. Preliminary SAR analysis indicated that modifications of the double bond and ester group made great effects on the anti-proliferative activity. Our findings suggested that it was worth further studies on the antitumor potency of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetates.

Full text of DOI:10.1016/j.ejmech.2016.09.005

European Journal of Medicinal Chemistry 124 (2016) 809e819
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of (2E)-(2-oxo-1, 2-
dihydro-3H-indol-3-ylidene)acetate derivatives as anti-proliferative
agents through ROS-induced cell apoptosis
,
b
,
,
b
,
,
, b, **
Zhuang Song a 1, Cai-Ping Chen a 1, Jun Liu c, Xiaoan Wen a b, Hongbin Sun a
,
Haoliang Yuan a
, b, *
a Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tong Jia Xiang, Nan Jing 210009, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nan Jing 210009, PR China
c China Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nan Jing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate derivatives were designed and
synthesized as potent anti-proliferative agents. Most of these compounds showed potent anti-
proliferative activity against some tumor cell lines, including SK-BR-3, MDA-MB-231, HCT-116, SW480,
Ovcar-3, HL-60, Saos-2 and HepG2. Compounds 8c and 11h were identified as the most potent ones,
while HL-60, HCT116 and MDA-MB-231 were the most sensitive cell lines. Mechanistic study revealed
that compound 8c enhanced reactive oxygen species level by inhibiting TrxR and then induced apoptosis
by activating apoptosis proteins, bax and cleaved-caspase 3 in HCT116 cells. Preliminary SAR analysis
indicated that modifications of the double bond and ester group made great effects on the anti-
proliferative activity. Our findings suggested that it was worth further studies on the antitumor po-
tency of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetates.
Received 28 April 2016
Received in revised form
1 September 2016
Accepted 2 September 2016
Available online 3 September 2016
Keywords:
3-Ylideneoxindole
Antitumor agent
Apoptosis
© 2016 Elsevier Masson SAS. All rights reserved.
Reactive oxygen species (ROS)
Thioredoxin reductase (TrxR)
1. Introduction
progression through p53 as the key tumor suppressor [7e11].
Previous studies mainly focused on the 3-spiro-oxindole skel-
Compounds based on indolinone-fragment, which are wide-
spread in natural products and synthetic small molecules, have a
wide range of biological activities (Fig. 1). For example, Indirubin
(1), the major active component of a traditional Chinese herbal
medicine used as an anticancer agent [1,2], is an inhibitor of protein
kinases such as cyclin-dependent kinases (CDKs) and glycogen
eton construction and related biological evaluation. However, the
rarely studied 3-ylideneoxindole skeleton also showed great
bioactivity. For example, Nintedanib (Ofev) (4), a kinases inhibitor
[12e14], has launched for the treatment of idiopathic pulmonary
fibrosis (IPF) and cancer. Oxindole compound 5 and related com-
pounds also showed inhibitory effects on CDKs [15,16]. Recently, 3-
ylideneoxindole acetamides (6) have been reported as antitumor
agents, displaying a similar profile to that of roscovitine [17].
In this study, a new series of (2E)-(2-oxo-1, 2-dihydro-3H-indol-
3-ylidene)acetate derivatives (Chart 1) have been designed, syn-
thesized and evaluated for their inhibitory activity against several
cancer cell lines. Besides, we also attempted to delineate their
mechanism of action. In the presence of compound 8c with the best
in vitro anti-proliferative activity, cell apoptosis and cell cycle dis-
tribution were investigated. The apoptosis proteins were also
analyzed. We preliminarily proved that their anti-proliferative ac-
tivity was attributed to the fact that the enhanced reactive oxygen
species (ROS) level induced cell apoptosis by inhibiting thioredoxin
reductase (TrxR). Moreover, structure-activity relationship (SAR)
was also analyzed for further study of this series of compounds.
synthase kinase-3
b (GSK-3b) [3,4]. Spirotryprostatin B (2), an
indolic alkaloid found in the Aspergillus fumigatus fungus, is a novel
mammalian cell cycle inhibitor in the G2/M phase [5,6]. Spiro-
oxindole compound 3 is an inhibitor of p53-MDM2 interaction
and prevents the ubiquitination and degradation of p53, thus
promoting cancer cell apoptosis and preventing cell cycle
* Corresponding author. Jiangsu Key Laboratory of Drug Discovery for Metabolic
Disease, China Pharmaceutical University, 24 Tong Jia Xiang, Nan Jing 210009, PR
China.
** Corresponding author. Jiangsu Key Laboratory of Drug Discovery for Metabolic
Disease, China Pharmaceutical University, 24 Tong Jia Xiang, Nan Jing 210009, PR
China.
E-mail addresses: hbsun2000@yahoo.com (H. Sun), yhl@cpu.edu.cn (H. Yuan).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.09.005
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

810
Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
Fig. 1. Indolinone-fragment based compounds.
Chart 1. The SAR analysis of (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate derivatives.
2. Results and discussion
reacted with three different substituted 2-bromoacetates to get the
corresponding products 8a-c through a rapid and efficient one-pot
methodology [18], and the trans-type of 8c was confirmed by 1H
NMR, 13C NMR, ESI-MS and NOESY (see the Supplementary mate-
rial). Firstly, compound 8c was respectively alkylated at eNH by
benzyl bromide and iodomethane to give the corresponding N-
2.1. Synthesis of compounds 8a-m, 11a-k and12a-d
Compounds 8a-m, 11a-k and12a-d were prepared according to
the procedure depicted in Schemes 1 and 2 [18,19]. Isatin (7) was
Scheme 1. Synthesis of compounds 8a-m. Reagents and conditions: (a) CH3I, K2CO3, MeCN, 0ꢀC- r.t. overnight or PhCH2Br, NaH, THF, 0ꢀC- r.t.; (b) Ph3P (1.2eq), morpholine (1.2eq),
80 ꢀC; (c) Butadiene sulfone (1.1eq), phenol (0.02eq), EtOH, 150 ꢀC, 12 h; (d) CF3COOH, CH2Cl2, r.t. 12 h; (e) EDC (1.2eq), HoBt (1.3eq), DIEA (3eq), CH2Cl2, r.t. 1 h; then methylamine
hydrochloride (1.2eq), ꢁ2.5ꢀC- r.t. overnight; (f) DCC (1.5eq), DMAP (1.3eq), substituted phenol (1.2eq), CH2Cl2, r.t. 1 he10 h.

Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
811
Scheme 2. Synthesis of compounds 11a-k and 12a-d. Reagents and conditions: (a) NH2OH HCl (3.25 eq), Cl3CCH(OH)2 (1.2eq), Na2SO4 (6eq), 1N HCl (1 mmol/mL), H2O (0.3 mmol/
mL), reflux; (b) con. H2SO4/H2O ¼ 10/1, 80 ꢀC, 10min; (c) Ph3P (1.2eq), morpholine (1.2eq), 80 ꢀC; (d) NaBH4, MeOH, 0 ꢀC, 20 min.
alkylated product 8d, 8e, but it was in low yields (data not shown).
In this situation, we had to adjust the synthesis strategy of 8d and
8e. That is, Isatin (7), firstly, was alkylated with BnBr and MeI
respectively, and then reacted with 2-bromo-tertiary-butylacetate
to get the products 8d and 8e in 76% yields in two steps. In this way,
the yields of products were improved, which might be attributed to
the less reaction sites in the reactions. On the other hand, com-
pound 8c is an active substrate for many reactions because many
unknown site-products could be found by directly alkylation of 8c.
Compound 8f with cis-double bond was obtained through the
reaction shown in Scheme 1. Reaction of compound 8b with
butadiene sulfone in the presence of a catalytic amount of phenol at
150 ꢀC gave 8f in 63% yield, and the structure of 8f was confirmed
by 1H NMR, 13C NMR, NOESY and ESI-MS (see the Supplementary
material). Compound 8f was obtained later than those compounds
listed in Table 1. Therefore, we directly explored its anti-
proliferative activity on HL-60 cell line, one of the most sensitive
cell lines to this series of compounds. The anti-proliferative activity
Compound 8c was reacted with trifluoroacetate in dichloro-
methane to provide 8g in a good yield. In a similar way, we obtained
8h from 8e. These two compounds not only helped us to investigate
the importance of ester group, but also were useful for us to design
and synthesize new derivatives. Therefore, we also synthesized
amide and aryl ester compounds through condensation reactions to
afford compounds 8i-m from 8h (Scheme 1).
The synthetic route to compounds 11a-j is outlined in Scheme 2
[18,19]. In brief, substituted anilines were reacted with hydroxyl-
amine hydrochloride, chloral hydrate,1N HCl (1 mmol/mL) and H2O
(0.3 mmol/mL) in refluex for 1 h, and then treated with con. H2SO4/
H2O ¼ 10/1 at 80 ꢀC for 10 min, pouring into ice-water and stirring
strongly to give the key substituted indoline-2,3-dione in-
termediates 10a-j. It is known that Sandmeyer method has been
described as being no applicable to ortho-hydroxy or ortho-
alkoxyanilines [20]. However, there are also some references
about the synthesis of compound 10f through Sandmeyer method
[21e24]. In these references, we found that compound 10f was in
low yields, as well as in our experiments. The synthetic routes and
analysis data of compound 10f and its intermediate 9f-IM were
shown in Supplementary material.
of this cis-isomer (compound 8f) IC50 > 100
line has been listed in Table 2. Compared with the trans-isomer 8b
M), the cis-isomer 8f was much less active against HL-
mM against HL-60 cell
(IC50 ¼ 4.83
m
60. Consequently, we focused on the studies of the trans-isomers
with better anti-proliferative activity, while only one compound 8f
was prepared and tested.
These intermediates 10a-j and 10k (commercially) reacted with
tert-butyl-2-bromoacetate to give the target compounds 11a-k. On
the other hand, in order to verify the importance of the double

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Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
Table 1
Tumor cell growth inhibition in the presence of compounds 8a-e, 8g, 11a-k and 12b (20 mM).
Compound
Cell line (inhibition rate %)
SK-BR-3
MDA-MB-231
HCT-116
SW480
Ovcar-3
HL-60
Saos-2
HepG2
L-02
8a
8b
8c
8d
8e
8g
11a
11b
11c
11d
11e
11f
11g
11h
11i
86.7
85.5
87.3
88
87.6
17.9
88
88.5
87.8
87.7
87
87.7
88.1
87.6
88.2
87.9
87.9
22.6
1.2
86.8
86.9
87.6
87.1
86.6
1.0
87.8
87.7
87.7
87.6
87.1
86.7
86.9
87.6
87.4
87.3
86.9
3.7
87
78.1
78.3
78.1
78.4
77.8
6.4
54.4
57.3
59.6
58.9
57.1
13.4
59.5
59.7
60.2
59
57.9
57.1
59
59.4
59.3
57.4
55.6
6.4
81.5
77.7
94.5
91.7
94.7
0.9
94.6
94.4
94.7
94.2
91.6
84.2
95.9
94.9
93.7
82.9
86.6
0.5
78.6
81.4
83.3
82.1
81.9
8.9
83.7
82.8
84.2
82
83.2
81.1
80.1
82.3
82.4
82.6
82.8
1.5
88
61.5
63.9
64.1
62
61.4
3.3
63.9
63.8
63.7
63.7
63.6
61.6
61.3
63.3
64
89.3
89.5
87.9
87.5
ꢁ0.7
89.4
89.4
89
89.6
89.5
87.6
87.7
89.7
89.5
89.9
88.1
1.7
90.2
86.1
88
88.4
3.0
90.3
90.5
90.3
90.1
88.3
87.3
88.4
87.1
88.1
69.7
88.2
1.2
79
78.5
78.3
78.7
78.8
77.8
77.8
78.5
78
77.2
77.9
8.8
1.6
76.4
11j
64.3
62
0.1
11k
12b
DMSOa
Taxolb
ꢁ0.6
2.9
88.9
0.1
58.5
ꢁ0.1
1.1
77.4
1
89.4
ꢁ0.2
72.1
85.1
71
61.5
a
Negative control, 0.1%.
Positive control, 10 mg/mL.
b
Table 2
Inhibition of HL-60 and L-02 by compounds 8a-m, 11a-k and 12a-d.
was then chosen for IC50 test of compound 8a-m, 11a-k and 12a-
d (Table 2), as well as cell line L-02. Similarly, the results showed
that all compounds, except 8f, 8g, 8h and 12a-d (IC50 > 80
mM),
Comp. IC50
(
mM)
Comp.
IC50
(mM)
exhibited quite potent activity (IC50 ¼ 2.45e23.83
m
M). Unfortu-
HL-60a
L-02b
HL-60a
L-02b
nately, this series of compounds have not exhibit a good selectivity
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
11a
11b
4.18 0.57
4.83 0.67
2.45 0.19
13.75 2.15 5.03
2.76 0.30
>100
7.05
11c
11d
11e
11f
6.19 0.40 4.4
6.19 0.94 3.82
9.15 1.18 4.34
9.68 1.01 ND
4.48 0.23 4.04
3.16 0.64 4.72 (6.18)
6.12 0.71 4.8
9.86 1.18 3.82
3.64 0.46 6.26
on normal cell line L-02. Three most potent compounds 8c, 8e and
11h got IC50 values of 2.45 mM, 2.76 mM and 3.16 mM against HL-60,
4.23
2.45 (6.21)c
respectively. Based on these, the eight tumor cell lines were further
screened with compound 8c and 11h, and it was found that HL-60,
HCT116 and MDA-MB-231 were the most sensitive cell lines to the
tested compounds (Table 3). We therefore chose the most potent
compound 8c and HCT116 cell line for further mechanistic study.
4.32 (12.21) 11g
NDd
>100
NAe
11h
11i
11k
11j
12a
12b
12c
12d
>100
83.73b
10.71 2.30 19.07
10.20 1.10 22.7
12.16 1.68 ND
10.64 1.25 23.53
23.83 1.08 46.62
114.1
>100
93.1
>100
>100
>100
NA
2.2.2. Apoptosis study by flow cytometry assay
Cell apoptosis analysis was carried out to observe if the growth
inhibitory effects of these compounds on the tumor cells caused by
81.1
5.00 0.28
5.29 0.55
2.68
3.36
Sunitinibf 4.95
4.62
cell apoptosis. HCT116 cells, treated with 0.1
mM, 0.5 mM, 1 mM,
a
4
m
M, 8 M,16 M of compound 8c and DMSO (0.1%) vehicle control
m
m
IC50 values were expressed as the mean standard deviation of three separate
determinations.
for 48 h, were quantified for apoptosis ratios through flow cytom-
etry (BD Accuri C6) by Annexin V-FITC/PI assay [25e27]. As shown
in Fig. 2, exposure to 8 mM and 16 mM of compound 8c resulted in
20.7% and 71.8% of cell apoptosis respectively, compared with 10.5%
of DMSO-treated group, while low doses treatment did not show
any apparent effect. These data indicated that compound 8c could
induce HCT116 apoptosis in a dose-dependent manner.
b
c
d
e
f
IC50 values were determined from a single test.
IC50 data in the parenthesis was determined by another separate test.
Not detected.
No activity.
positive control.
bond, we obtained compound 12a-d through sodium borohydride
(NaBH4) reduction of 8a, 8c, 8e and 11h, respectively (Scheme 2).
2.2.3. Investigation of cell cycle distribution
To further probe the cell state with different drug
2.2. Biological evaluation
Table 3
Inhibition of selected cancer cells by compounds 8c and 11h.
2.2.1. In vitro anti-tumor activity screening
Compounds 8a-e, 8g, 11a-k and 12b were initially evaluated at
single concentration of 20 mM for the growth inhibition activity
Cell line IC50
8c
(
m
M)a
Cell line
IC50
8c
(m
M)a
11 h
11 h
against eight tumor cell lines, including SK-BR-3 (breast), MDA-MB-
231 (breast), HCT-116 (colon), SW480 (colon), Ovcar-3 (ovarian),
HL-60 (leukemic), Saos-2 (osteoblast) and HepG2 (liver), as well as
a normal liver cell line, L-02 (Table 1). The CCK-8 assay showed that
all the compounds, except 8g and 12b, displayed potent inhibition
rates against all cell lines, especially for HL-60. In this regard, HL-60
HL-60
2.45 0.19 3.16 0.64 HCT116
2.64 0.05 5.50 0.45
6.11 0.26 6.51 0.34
SK-BR-3 3.42 0.15 6.48 0.87 Saos-2
SW480 5.68 0.52 5.49 0.43 MDA-MB-231 2.52 0.07 3.11 0.25
HepG2
4.64 0.11 8.99 0.85 Ovcar-3
6.55 0.67 19.03 2.55
a
IC50 values were expressed as the mean standard deviation of three separate
determinations.

Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
813
Fig. 2. Compound design based on the privileged structures.
concentrations, we investigated the effect of compound 8c on cell
cycle in HCT116 cell. HCT116 cell was harvested after treatment
with compound 8c at 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M and
DMSO (0.1%) for 48 h, respectively. The fixed cell was stained with
propidium iodide (PI) and then detected using flow cytometry (BD
Accuri C6) [25,28]. The consequent cell cycle distributions were
shown in Fig. 3. The results indicated that compound 8c got only
marginal effects on cell cycle distribution in HCT116.
2.2.4. Western blotting analysis
Based on the analysis of cell apoptosis and cell cycle distribution
by flow cytometry, it was found that apoptosis pathway was very
likely to be the main reason for the inhibitory activity of compound
8c against HCT116. We detected the expression of apoptosis protein
of HCT116 after 8c treatment for 9 h by western blotting assay. As
shown in Fig. 4, the expression of pro-apoptotic protein Bax was
increased obviously, but not for anti-apoptosis protein Bcl-2. On the
other hand, the cleaved caspase 3 was also activated. This proved
m
m
m
m
m
m
Fig. 3. Apoptosis ratio detection of compound 8c in HCT116 cells by Annexin V-FITC/PI assay.

814
Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
compound 8c with 5 mmol/L N-acetylcysteine (NAC) for 53 h,
respectively. As we expected, the N-acetylcysteine treated group
got higher cell viability (Fig. 5A), which indicated that compound 8c
might enhance the intracellular ROS level and induce the cell
apoptosis. Next, we examined intracellular ROS production using
DCFH-DA, which could be converted into a green fluorescent DCF
by ROS oxidation. As shown in Fig. 5B, compound 8c improved the
level of ROS in a time-dependent manner, while Fig. 5C and D
showed intracellular ROS raised in a does-dependent manner. The
thioredoxin system, composed of thioredoxin reductase (TrxR),
thioredoxin (Trx), and NADPH, is a highly conserved, ubiquitous
network in all cells and plays crucial roles in the redox regulation of
numerous cellular signaling pathways involved in cell survival and
proliferation [29e31]. Fig. 5E demonstrated that compound 8c got
TrxR inhibitory activity in a dose-dependent manner.
2.3. Structure-activity relationship (SAR) and proposed mechanism
of cell death
Fig. 4. Effect of compounds 8c on cell cycle arrest in human colonic cancer cell line
HCT116.
SAR analysis is simplified in Fig. 6. For HL-60 cell line, compar-
ison between compound 12a-d (IC50 > 80
other compounds, such as 8c (IC50 ¼ 2.45
m
M) and most of the
mM) indicates that the
our hypothesis that apoptosis pathway mainly caused the inhibi-
tory activity of compound 8c against HCT116.
double bond in this series of compounds is very important for the
potency. A possible explanation is that the Michael-acceptor
structure contributes to the cytostatic activity, but the specific
mechanism of action is unclear. According to the potency of 8a-c
2.2.5. Compound 8c enhanced intracellular ROS level in time- and
does-dependent manner by inhibition TrxR activity
(IC50 < 10 mM), 8g (IC50 > 100 mM) and 8i-m (IC50 > 10 mM), it seems
ROS has been reported to be involved in early stages of apoptosis
in many cellular systems. To determine whether the apoptosis was
induced by our compounds, we examined intracellular ROS level in
HCT116 cells. Firstly, we examined whether ROS made effect on the
cancer cell viability. HCT116 was incubated with compound 8c and
that alkyl-substituted compounds are more potent than the aryl-
substituted and non-substituted compounds. Moreover, com-
pounds with smaller substituted groups (e.g. 8c: IC50 ¼ 2.45
IC50 ¼ 2.76 M) are more potent than the larger benzyl substituted
compounds (e.g. 8d: IC50 ¼ 13.75 M). No significant effects have
mM, 8e:
m
m
Fig. 5. Expression of the apoptotic proteins in HCT116 after 9 h of compound 8c treatment.

Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
815
recorded at 300/500 and 75/125 MHz respectively on an ACF*300Q
Bruker or ACF*500Q Bruker spectrometer with Me4Si as the inter-
nal reference. Low-resolution and high-resolution mass spectra
(LRMS and HRMS) were given with electron impact mode. The mass
analyzer type used for the HRMS measurements was TOF. Reactions
were monitored by TLC on silica gel60-F254 plates (Qingdao Ocean
Chemical Company, China). Column chromatography was carried
out on silica gel (200e300 mesh, Qingdao Ocean Chemical Com-
pany, China). The purity of the key compounds was determined by
analytical HPLC (Equipment: Agilent 1100 system with a VWD
G1314A UV detector; Column: Chiralpak IC 4.6 mm 250 mm).
Condition a: mobile phase (MeOH/H2O ¼ 9/1), flow rate 0.5 mL/
min, injection volume, 10
mobile phase (MeOH/H2O ¼ 8/2), flow rate 1.0 mL/min, injection
volume, 10 L and detection at 254 nm.
mL and detection at 254 nm; Condition b:
Fig. 6. Effects of compound 8c on intracellular ROS level. A. The proliferation inhibition
(CCK-8 assay) of compound 8c and 8c þ 5 mmol/L N-acetylcysteine treatment for 53 h.
m
B. Compound 8c enhanced intracellular ROS level in
a time-dependent manner;
***P < 0.001 vs DMSO. C and D. Compound 8c enhanced intracellular ROS level in a
dose-dependent manner. E. The effect of compound 8c on TrxR activity of HCT116;
*P < 0.05 vs DMSO.
4.1.2. General procedure
General procedure A for preparing compound 8a-c and 11k.
A mixture of PPh3 (1.1 eq), isatin or 5-nitro-isatin (1.0 eq), and
substituted 2-bromoacetate (1.1 eq) was heated at 80 ꢀC, then
morpholine (1.1 eq) was added. After the reaction completion and
cooling to the room temperature, the mixture was diluted with
water, extracted with CH2Cl2, and the organic phase was dried over
sodium sulfate. The solvent was removed under reduced pressure
and the residue was purified by chromatography using Petroleum
ether/Ethyl acetate to give the target compounds.
been found on the other substituted positions on indoline, no
matter whether electric or steric factors are taken in consideration.
According to all data above, we hypothesized the possible
mechanism of cell apoptosis induced by these compounds (eg. 8c).
They firstly inhibited TrxR activities by Michael addition with the
bioactivity needed thiol groups in TrxR, disrupted cellular redox
balance as well as redox-mediated signal transduction, leading to
the enhanced intracellular ROS level, and finally causing cell
apoptosis by upregulated pro-apoptotic regulators Bax and acti-
vated caspase-cascade pathway.
General procedure B for preparing compound 11a-j.
Substituted aniline (1 mmol), 1N HCl (1 mL), H2O (6 mL), hy-
droxylamine hydrochloride (226 mg, 3.25 mmol), and anhydrous
Na2SO4 (900 mg) was added in 25 ml seal tube, heat to boiling, and
then added chloral hydrate (198 mg, 1.2 mmol), kept boiling for
40 min, cooled, and then extracted with Ethyl acetate. The Ethyl
acetate extracts were dried (Na2SO4) and evaporated. The residue
was added to a stirred solution of concentrated H2SO4 (1 mL) and
H2O (0.1 mL) during 10 min at 65 ꢀC. The mixture was then kept at
80 ꢀC for 10 min and then poured onto cracked ice. Then the
mixture was extracted with Ethyl acetate, and, the organic phase
was dried over sodium sulfate. The solvent was removed under
reduced pressure to afford the substituted isatins as crude product,
and then compound 11a-j was obtained by substituted istins
through the General procedure A.
3. Conclusion
In this study, twenty eight novel (2E)-(2-oxo-1, 2-dihydro-3H-
indol-3-ylidene)acetate derivatives bearing 3-ylideneoxindole-
based moiety have been designed and synthesized. Their in vitro
anti-proliferative activities were evaluated against different cancer
cell lines, including SK-BR-3, MDA-MB-231, HCT-116, SW480,
Ovcar-3, HL-60, Saos-2 and HepG2. The results showed that most of
the compounds got a potent inhibitory activity against these cancer
cell lines. Within this series of compounds, compound 8c
(IC50 ¼ 2.45
m
M), 8e (IC50 ¼ 2.76
m
M) and 11h (IC50 ¼ 3.16
mM) were
General procedure C for preparing compound 8j-m.
the most potent ones against HL-60. Further biological assays
showed that HL-60, HCT116 and MDA-MB-231 were the most
sensitive cell lines for 8c and 11h. Compound 8c could significantly
A
mixture of (E)-2-(1-methyl-2-oxoindolin-3-ylidene)acetic
acid (1.0 eq), DCC (1.5 eq) and DMAP (1.2 eq) was dissolved in 2 mL
of dry CH2Cl2 at 0 ꢀC. Substituted phenol was dissolved in 1 mL of
dry CH2Cl2 and added slowly with stirring at 0 ꢀC for 5 min and then
stirring at r.t until the reaction was completed. The CH2Cl2 was
removed under reduced pressure and then Ethyl acetate was added
to the residue. After filtration to remove the white solids, organic
phase was removed under pressure and the residue was purified by
chromatography using Petroleum ether/Ethyl acetate ¼ 8/1 to get
the target products.
induce cell apoptosis at 16 mM, but got only marginal effects on cell
cycle distribution. Western blotting showed the apoptotic proteins,
bax and cleaved-caspase 3, were activated under compound 8c
treatment for 9 h. This might be attributed to the fact that com-
pound 8c enhanced the intracellular ROS level by inhibiting TrxR
activity. SAR analysis showed that the double bond in this series of
compounds was very important for their cytostatic activity, indi-
cating that the Michael-acceptor structure might contribute greatly
to the anti-proliferative activity. Taken together, (2E)-(2-oxo-1, 2-
dihydro-3H-indol-3-ylidene)acetate derivatives should deserve
further studies to explore their potential utility as antitumor
agents.
4.1.2.1. Methyl (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate
(8a) [32]. Yellow solid, mp: 178e180 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.56 (d, J ¼ 7.4 Hz, 2H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.07 (t, J ¼ 7.6 Hz,
1H), 6.90 (s, 1H), 6.89 (d, J ¼ 7.8 Hz, 1H), 3.90 (s, 3H); ESI-MS: 202.0
(M-Hþ); HPLC: tr 7.24 min (99.8%, condition a).
4. Experimental section
4.1.2.2. Ethyl (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate
4.1. Chemistry
(8b) [33]. Yellow solid, mp: 183e185 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.83 (s, 1H, br), 8.53 (d, J ¼ 7.8 Hz, 1H), 7.32 (t, J ¼ 7.7 Hz, 1H), 7.05
4.1.1. General
(t, J ¼ 7.7 Hz, 1H), 6.88 (d, J ¼ 7.5 Hz, 1H), 6.88 (s, 1H), 4.35 (q,
J ¼ 7.1 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz, 1H); ESI-MS: 240.1 (M þ Naþ);
HPLC: tr 7.68 min (99.7%, condition a).
All commercially available solvents and reagents were used
without further purification. 1H NMR and 13C NMR spectra were

816
Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
4.1.2.3. Tert-butyl (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)ace-
J ¼ 7.7 Hz, 1H), 3.26 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 167.24,
tate (8c) [34]. Yellow solid, mp: 128e131 ꢀC; 1H NMR (300 MHz,
164.24, 146.34, 139.87, 132.98, 131.40, 129.55, 129.28, 128.74, 126.20,
122.96, 121.46, 121.02, 108.21, 26.28; ESI-MS: 302.1 (M þ Naþ); ESI-
HRMS: found: 302.0801 (M þ Naþ); Calc.: 302.0793 C17H13NO3Na;
HPLC: tr 7.64 min (94.6%, condition b).
CDCl3)
1H), 7.04 (t, J ¼ 7.7 Hz, 1H), 6.85 (d, J ¼ 7.8 Hz, 1H), 6.83 (s, 1H), 1.57
(s, 9H); 13C NMR (75 MHz, CDCl3)
169.68, 164.85, 143.21, 137.06,
d
8.51 (d, J ¼ 7.8 Hz, 1H), 8.36 (s, 1H, br), 7.30 (t, J ¼ 7.7 Hz,
d
132.17, 128.82, 124.95, 122.75, 120.52, 110.08, 81.93, 28.09; ESI-MS:
268.1 (M þ Naþ); HPLC: tr 8.70 min (99.6%, condition a).
4.1.2.11. 4-Chlorophenyl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-indol
-3-ylidene)acetate (8k). Orange solid, mp: 131e132 ꢀC; 1H NMR
4.1.2.4. Tert-butyl (2E)-(1-benzyl-2-oxo-1, 2-dihydro-3H-indol -3-
(300 MHz, CDCl3)
(d, J ¼ 8.7 Hz, 2H), 7.09 (s, 1H), 7.06e7.01 (m, 1H), 6.82 (d, J ¼ 7.8 Hz,
1H), 3.25 (s, 3H); 13C NMR (75 MHz, CDCl3)
167.18, 163.95, 148.93,
146.45, 140.28, 133.17, 131.62, 129.60, 129.26, 122.99, 122.84, 120.43,
119.66, 108.28, 26.29; HPLC: tr 15.51min (95.8%, condition b).
d
8.56 (d, J ¼ 7.7 Hz, 1H), 7.41e7.37 (m,3H), 7.18
ylidene)acetate (8d) [35]. Orange solid, mp: 103e104 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
8.53 (d, J ¼ 7.4 Hz, 1H), 7.29e7.21 (m, 6H), 7.02
d
(t, J ¼ 7.6 Hz, 1H), 6.93 (s, 1H), 6.68 (d, J ¼ 7.8 Hz, 1H), 4.94 (s, 2H),
1.57 (s, 9H); ESI-MS: 358.1 (M þ Naþ); HPLC: tr 14.25 min (98.2%,
condition a).
4.1.2.12. 4-Methoxyphenyl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-
4.1.2.5. Tert-butyl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-indol -3-
indol -3-ylidene)acetate (8l). Orange solid, mp: 123e125 ꢀC; 1H
ylidene)acetate (8e) [36]. Orange solid, mp: 90e92 ꢀC; 1H NMR
NMR (300 MHz, CDCl3)
d
8.57 (d, J ¼ 7.7 Hz, 1H), 7.38 (t, J ¼ 7.7 Hz,
(300 MHz, CDCl3)
d
8.52 (d, J ¼ 7.7 Hz, 1H), 7.36 (t, J ¼ 7.7 Hz, 1H),
1H), 7.13 (d, J ¼ 9.2 Hz, 2H), 7.03 (t, J ¼ 7.6 Hz,1H), 6.94 (d, J ¼ 8.9 Hz,
7.06 (t, J ¼ 7.7 Hz, 1H), 6.85 (s, 1H), 6.79 (d, J ¼ 7.7 Hz), 3.23 (s, 3H),
1.57 (s, 9H); ESI-MS: 282.1 (M þ Naþ); HPLC: tr 10.50 min (99.9%,
condition a).
2H), 6.80 (d, J ¼ 7.7 Hz, 1H), 3.82 (s, 3H), 3.25 (s, 3H); 13C NMR
(75 MHz, CDCl3)
d 167.30, 164.62, 157.54, 146.30, 143.93, 139.68,
132.91, 129.25, 122.93, 122.21, 121.12, 119.74, 114.58, 108.19, 55.62,
26.26; ESI-MS: 332.1 (M þ Naþ); ESI-HRMS: found: 332.0909
(M þ Naþ); Calc.: 302.0899 C18H15NO4Na; HPLC: tr 7.79 min (98.3%,
condition b).
4.1.2.6. Ethyl (2Z)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetate
(8f). A mixture of compound 3a-2 (1.0 eq), butadiene sulfone
(1.1eq), and a catalytic amount of phenol (0.02eq) in ethanol, was
heated at 150 ꢀC for 12 h. After the reaction completed, EtOH was
removed under reduced pressure, and the mixture was extracted
with EtOAc, organic phase was dried over sodium sulfate and
removed under pressure, and then the residue was purified by
chromatography using Petroleum ether/Ethyl acetate ¼ 2/1 to get
the target products. White solid, mp: 205e206 ꢀC; 1H NMR
4.1.2.13. Naphthalene-2-yl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-
indol -3-ylidene)acetate (8m). Orange solid, mp: 102e104 ꢀC; 1H
NMR (300 MHz, CDCl3)
d
8.60 (d, J ¼ 7.7 Hz, 1H), 7.93e7.84 (m, 3H),
7.70 (s, 1H), 7.52e7.50 (m, 2H), 7.42e7.33 (m, 1H), 7.19 (s, 1H), 7.04
(t, J ¼ 7.6 Hz, 1H), 6.82 (d, J ¼ 7.8 Hz, 1H), 3.28 (s, 3H); ESI-MS: 352.1
(M þ Naþ); ESI-HRMS: found: 352.0950 (M þ Naþ); Calc.: 352.0974
(300 MHz, DMSO)
d
12.09 (s, 1H), 8.04 (d, J ¼ 8.2 Hz, 1H), 7.55 (t,
C21H15NO3Na; HPLC: tr 13.55 min (99.1%, condition b).
J ¼ 7.6 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 1H), 7.22 (t, J ¼ 7.6 Hz, 1H), 6.88 (s,
1H), 4.38 (q, J ¼ 7.1 Hz, 2H), 1.34 (t, J ¼ 7.1 Hz, 3H); 13C NMR
4.1.2.14. Tert-butyl (2E)-(5-methyl-2-oxo-1, 2-dihydro-3H-indol -3-
(75 MHz, DMSO)
d
165.49, 161.23, 140.64, 139.89, 131.49, 126.25,
ylidene)acetate (11a). Orange solid, mp: 137e138 ꢀC; 1H NMR
124.28, 122.79, 116.30, 115.90, 62.32, 14.36. ESI-MS: 240.2
(300 MHz, CDCl3)
d
8.63 (s, 1H), 8.31 (s, 1H), 7.10 (d, J ¼ 7.9 Hz, 1H),
(M þ Naþ); HPLC: tr 4.15 min (98.2%, condition b).
6.80 (s,1H), 6.75 (d, J ¼ 7.9 Hz,1H), 2.34 (s, 3H),1.58 (s, 9H), 13C NMR
(75 MHz, CDCl3)
d 169.75, 164.90, 140.90, 137.20, 132.65, 132.15,
4.1.2.7. (2E)-(2-oxo-1, 2-dihydro-3H-indol-3-ylidene)acetic acid
129.23, 124.65, 120.51, 109.79, 81.85, 28.11, 21.15; ESI-MS: 282.1
(8g). Orange solid, mp: 190e192 ꢀC; 1H NMR (300 MHz, DMSO)
(M þ Naþ); ESI-HRMS: found: 282.1113 (M þ Naþ); Calc.:282.1106
d
13.19 (br., 1H), 10.72 (s, 1H), 8.30 (d, J ¼ 7.7 Hz, 1H), 7.32 (t,
C15H17NO3Na; HPLC: tr 9.84 min (99.8%, condition a).
J ¼ 7.5 Hz, 1H), 6.98 (t, J ¼ 7.6 Hz, 1H), 6.85 (d, J ¼ 7,7 Hz, 1H), 6.59 (s,
1H); ESI-MS: 188.0(M-Hþ), 144.0(M-2Hþ-CO2).
4.1.2.15. Tert-butyl (2E)-(5-isopropyl-2-oxo-1, 2-dihydro-3H-indol
-3-ylidene)acetate (11b). Orange solid, mp: 123e124 ꢀC; 1H NMR
4.1.2.8. (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-indol-3-ylidene)ace-
(300 MHz, CDCl3)
d
8.37 (s, 1H), 8.25 (s, 1H), 7.16 (d, J ¼ 8.0 Hz, 1H),
tic acid (8h). Orange solid, mp: 165e166 ꢀC; 1H NMR (300 MHz,
6.80 (s, 1H), 6.77 (d, J ¼ 8.0 Hz, 1H), 2.92 (heptet, J ¼ 6.9 Hz, 1H), 1.58
DMSO-d6)
d
13.34 (br, 1H), 8.32 (d, J ¼ 7.7 Hz, 1H), 7.42 (t, J ¼ 7.7 Hz,
(s, 1H), 1.26 (d, J ¼ 6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 169.13,
1H), 7.09e7.02 (m, 2H), 6.67 (s, 1H), 3.15 (s, 3H). ESI-MS: 202.1 (M-
164.42, 143.05, 140.53, 136.48, 129.49, 126.45, 124.14, 120.04,
109.29, 81.34, 33.44, 27.62, 23.65; ESI-MS: 310.1 (M þ Naþ); ESI-
HRMS: found: 310.1413 (M þ Naþ); Calc.: 310.1419 C17H21NO3Na;
HPLC: tr 11.76 min (97.8%, condition a).
Hþ); ESI-HRMS: found: 202.05189 (M-Hþ); Calc.:202.05097
C
11H8NO3; HPLC: tr 2.64 min (99.8%, condition b).
4.1.2.9. Methyl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-indol -3-
ylidene)acetamide (8i). Yellow solid, mp: 202e203 ꢀC; 1H NMR
4.1.2.16. Tert-butyl (2E)-(5-butyl-2-oxo-1, 2-dihydro-3H-indol -3-
(300 MHz, CDCl3)
d
8.67 (d, J ¼ 7.6 Hz, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H),
ylidene)acetate (11c). Orange solid, mp: 105e106 ꢀC; 1H NMR
7.06 (t, J ¼ 7.7 Hz, 1H), 6.93 (s, 1H), 6.78 (d, J ¼ 7.7 Hz, 1H), 6.47 (br,
(300 MHz, CDCl3)
d
8.34 (s, 1H), 8.30 (s, 1H), 7.10 (d, J ¼ 7.9 Hz, 1H),
1H), 3.23 (s, 3H), 2.99 (d, J ¼ 4.9 Hz, 3H); 13C NMR (75 MHz, CDCl3)
6.80 (s, 1H), 6.75 (d, J ¼ 7.9 Hz, 1H), 2.60 (t, J ¼ 7.7 Hz, 2H), 1.64e1.25
d
168.17, 165.22, 145.32, 135.04, 131.66, 128.91, 125.73, 122.93,
(m, 15H), 0.93 (t, J ¼ 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 169.13,
120.19, 107.91, 26.59, 26.17; ESI-MS: 239.1 (M þ Naþ); ESI-HRMS:
found: 239.0801 (M þ Naþ); Calc.: 239.0796 C12H12N2O2Na;
HPLC: tr 3.18 min (99.9%, condition b).
164.41, 140.48, 136.93, 136.53, 131.57, 128.17, 124.15, 120.05, 109.22,
81.35, 34.84, 33.39, 27.62, 21.74, 13.41; ESI-MS: 324.2 (M þ Naþ);
ESI-HRMS: found: 324.1565 (M
þ
Naþ); Calc.: 324.1576
C18H23NO3Na; HPLC: tr 14.27 min (98.5%, condition a).
4.1.2.10. Phenyl (2E)-(1-methyl-2-oxo-1, 2-dihydro-3H-indol -3-
ylidene)acetate (8j). Orange solid, mp: 103e105 ꢀC; 1H NMR
4.1.2.17. Tert-butyl (2E)-(5-methoxy-2-oxo-1, 2-dihydro-3H-indol -3-
(300 MHz, CDCl3)
d
8.57 (d, J ¼ 7.6 Hz, 1H), 7.47e7.36 (m, 3H),
ylidene)acetate (11d). Orange solid, mp: 138e140 ꢀC; 1H NMR
7.31e7.19 (m, 3H), 7.13 (s, 1H), 7.03 (t, J ¼ 7.7 Hz, 1H), 6.81 (d,
(300 MHz, CDCl3)
d
8.48 (s, 1H), 8.20 (d, J ¼ 2.4 Hz, 1H), 6.87 (dd,

Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
817
J ¼ 2.4 Hz, 8.5 Hz, 1H), 6.81 (s, 1H), 6.75 (d, J ¼ 8.5 Hz, 1H), 3.82 (s,
3H), 1.56 (s, 9H); 13C NMR (75 MHz, CDCl3)
169.61, 164.78, 155.64,
137.49, 136.93, 125.19, 121.23, 118.06, 114.62, 110.39, 81.92, 55.89,
28.06; ESI-MS: 298.1 (M þ Naþ); ESI-HRMS: found: 298.1063
(M þ Naþ); Calc.: 298.1055 C15H17NO4Na; HPLC: tr 8.37 min (99.6%,
condition a).
1H), 7.24e7.19 (m, 2H), 7.01 (t, J ¼ 7.6 Hz,1H), 6.91 (d, J ¼ 7.7 Hz,1H),
3.84e3.80 (m, 1H), 3.70 (s, 3H), 3.09 (dAB, J ¼ 4.5 Hz, 16.9 Hz, 1H),
2.84 (dAB, J ¼ 8.0 Hz, 16.9 Hz, 1H); ESI-MS: 228.1 (M þ Naþ); ESI-
d
HRMS: found: 228.06381 (M
þ
Naþ); Calc.: 228.06311
C11H11NO3Na; HPLC: tr 3.27 min (96.8%, condition b).
4.1.2.26. Tert-butyl 2-(2-oxo-1, 2-dihydro-3H-indol -3-yl)acetate
4.1.2.18. Tert-butyl (2E)-(6-methoxy-2-oxo-1, 2-dihydro-3H-indol -3-
(12b). White solid, mp: 105e106 ꢀC; 1H NMR (300 MHz, CDCl3)
ylidene)acetate (11e) [37]. Orange solid, mp: 160e162 ꢀC; 1H NMR
d
8.84 (br, 1H), 7.25e7.19 (m, 2H), 7.01 (t, J ¼ 7.6 Hz, 1H), 6.90 (d,
(300 MHz, CDCl3)
d
8.51 (d, J ¼ 8.7 Hz, 1H), 8.38 (s, 1H), 6.66 (s, 1H),
J ¼ 7.5 Hz, 1H), 3.75 (t, J ¼ 5.7 Hz, 1H), 2.99 (dAB J ¼ 4.7 Hz, 16.4 Hz,
6.52 (dd, J ¼ 2.2 Hz, 8.7 Hz, 1H), 6.40 (d, J ¼ 2.2 Hz, 1H), 3.84 (s, 3H),
1.55 (s, 9H); ESI-MS: 298.3 (M þ Naþ); HPLC: tr 9.57 min (89.2%,
condition a).
1H), 2.81 (dAB J ¼ 7.2 Hz, 16.4 Hz, 1H), 1.34 (s, 9H); 13C NMR
(75 MHz, CDCl3)
d 179.54, 169.98, 141.70, 128.96, 128.18, 124.11,
122.38, 109.74, 109.70, 81.37, 42.63, 35.89, 27.83; ESI-MS: 270.1
(M þ Naþ); ESI-HRMS: found: 270.1112 (M þ Naþ); Calc.: 270.1106
4.1.2.19. Tert-butyl (2E)-(7-methoxy-2-oxo-1, 2-dihydro-3H-indol -3-
C14H17NO3Na; HPLC: tr 3.42 min (99.3%, condition b).
ylidene)acetate (11f). 1H NMR (300 MHz, CDCl3)
d
8.10 (d, J ¼ 7.7 Hz,
1H), 7.53 (s, 1H), 7.00 (t, J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 7.7 Hz, 1H), 6.83
(s, 1H), 3.88 (s, 3H), 1.56 (s, 9H); ESI-MS: 274.1 (M-Hþ); ESI-HRMS:
found: 274.1086 (M-Hþ); Calc.: 274.1079 C15H16NO4; HPLC: tr
8.74 min (99.7%, condition a).
4.1.2.27. Tert-butyl 2-(1-methyl-2-oxo-1, 2-dihydro-3H-indol -3-yl)
acetate (12c). Colourless oil. 1H NMR (300 MHz, CDCl3)
7.29e7.23
d
(m, 2H), 7.01 (t, J ¼ 7.5 Hz,1H), 6.81 (d, J ¼ 7.7 Hz,1H), 3.72e3.68 (m,
1H), 3.20 (s, 3H), 2.96 (dAB, J ¼ 4.8 Hz, 16.4 Hz, 1H), 2.75 (dAB,
J ¼ 7.3 Hz, 16.4 Hz, 1H), 1.31 (s, 9H); 13C NMR (75 MHz, CDCl3)
4.1.2.20. Tert-butyl (2E)-(5-(trifluoromethoxy)-2-oxo-1, 2-dihydro-
3H-indol -3-ylidene)acetate (11g). Orange solid, mp: 122e123 ꢀC;
d 176.98,170.08,144.50,128.30,128.25,123.88,122.48,107.98, 81.16,
42.17, 36.11, 27.93, 26.33; ESI-MS: 284.1 (M þ Naþ); ESI-HRMS:
found: 284.12699 (M þ Naþ); Calc.: 284.12571 C15H19NO3Na;
HPLC: tr 4.64 min (95.9%, condition b).
1H NMR (300 MHz, CDCl3)
d 8.85 (s, 1H), 8.47 (s, 1H), 7.19 (d,
J ¼ 8.3 Hz, 1H), 6.88 (s, 1H), 6.87 (d, J ¼ 8.9 Hz, 1H), 1.58 (s, 9H); 13
C
NMR (75 MHz, CDCl3)
d 168.97, 163.90, 144.10, 141.14, 135.62,
126.42, 124.62, 122.05, 121.78, 120.89, 109.97, 82.06, 27.52; ESI-MS:
352.1 (M þ Naþ); ESI-HRMS: found: 352.0781 (M þ Naþ); Calc.:
352.0773 C15H14NO4NaF3; HPLC: tr 10.31 min (98.7%, condition a).
4.1.2.28. Tert-butyl 2-(5-chloro-2-oxo-1, 2-dihydro-3H-indol -3-yl)
acetate (12d). White solid, mp: 163e164 ꢀC; 1H NMR (300 MHz,
CDCl3)
d
8.97 (s, 1H), 7.27e7.19 (m, 2H), 6.83 (d, J ¼ 8.6 Hz, 1H),
3.77e3.73 (m, 1H), 2.98 (dAB, J ¼ 4.6 Hz, 16.7 Hz, 1H), 2.80 (dAB,
4.1.2.21. Tert-butyl (2E)-(5-chloro-2-oxo-1, 2-dihydro-3H-indol -3-
J ¼ 7.4 Hz, 16.7 Hz, 1H), 1.38 (s, 9H); 13C NMR (75 MHz, CDCl3)
ylidene)acetate (11h). Orange solid, mp: 150e151 ꢀC; 1H NMR
d 179.45, 169.88, 140.45, 130.81, 128.30, 127.90, 124.76, 114.73,
(300 MHz, CDCl3)
J ¼ 1.7 Hz, 8.0 Hz, 1H), 6.84 (s, 1H), 6.80 (d, J ¼ 8.0 Hz, 1H), 1.58 (s,
9H); 13C NMR (75 MHz, CDCl3)
168.93, 163.93, 141.12, 135.67,
d
8.81 (s, 1H), 8.54 (d, J ¼ 1.7 Hz, 1H), 7.28 (dd,
110.89, 81.86, 42.91, 35.80, 28.02; ESI-MS: 304.1 (M þ Naþ); ESI-
HRMS: found: 304.07212 (M
þ
Naþ); Calc.: 304.07109
d
C14H16ClNO3Na; HPLC: tr 5.07 min (94.4%, condition b).
131.36, 128.36, 127.64, 126.05, 121.20, 110.56, 81.97, 27.58; ESI-MS:
278.1 (M-Hþ); ESI-HRMS: found: 278.0575 (M-Hþ); Calc.:
278.0584 C14H13NO3Cl; HPLC: tr 10.61 min (95.5%, condition a).
4.2. Biological studies
4.2.1. Cell lines and cell culture and antibodies
4.1.2.22. Tert-butyl (2E)-(7-chloro-2-oxo-1, 2-dihydro-3H-indol -3-
Cancer cell lines SK-BR-3, MDA-MB-231, HCT-116, SW480,
Ovcar-3, HL-60, Saos-2, HepG2 were purchased from the American
Type Culture Collection. The cells were routinely maintained in
RPMI 1640 (Hyclone) medium supplemented with 10% heat-
inactivated fetal calf serum (Gibco) and 1% streptomycin/peni-
cillin (Hyclone) at 37 ꢀC in a humidified incubator with 5% CO2.
Antibodies were include: anti-Bax (proteintech, 50599-Ig), anti-
Bcl-2 (proteintech, 12789-1-AP), anti-Cleaved Caspase 3 (pro-
teintech, 25546-1-AP) and anti-Caspase 3 (proteintech, 19677-1-
AP).
ylidene)acetate (11i). Orange solid, mp: 186e187 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
8.44 (d, J ¼ 7.7 Hz, 1H), 8.19 (s, 1H), 7.30 (d,
J ¼ 8.2 Hz, 1H), 7.00 (t, J ¼ 8.0 Hz, 1H), 6.88 (s, 1H), 1.57 (s, 8H); 13
C
NMR (75 MHz, CDCl3) d 168.06,164.51,136.62,131.63,127.13,126.67,
123.44, 121.86, 82.27, 28.05; ESI-MS: 278.1 (M-Hþ); ESI-HRMS:
found: 278.0591 (M-Hþ); Calc.: 278.0584 C14H13NO3Cl; HPLC: tr
10.58 min (98.5%, condition a).
4.1.2.23. Tert-butyl (2E)-(6-chloro-7-methyl-2-oxo-1, 2-dihydro-3H-
indol -3-ylidene)acetate (11j). Orange solid, mp: 224e226 ꢀC; 1H
NMR (300 MHz, CDCl3)
d
8.93 (s,1H), 8.33 (d, J ¼ 8.3 Hz, 2H), 7.05 (d,
4.2.2. CCK-8 assay for in vitro cytotoxicity screening
J ¼ 8.3 Hz, 1H), 6.81 (s, 1H), 2.29 (s, 3H), 1.56 (s, 9H); 13C NMR
The cells were seeded into a 96-well plate at a density of 2500
(75 MHz, CDCl3)
d
169.87, 164.76, 143.07, 138.32, 136.97, 127.09,
(100
mL) per well for 24 h, followed by drug treatment for 72 h.
125.20, 123.27, 118.68, 117.78, 82.07, 28.08, 13.78; ESI-MS: 316.1
Next, 10
m
L of CCK-8 solution was added to the medium, and the
(M þ Naþ); ESI-HRMS: found: 316.0721 (M þ Naþ); Calc.: 316.0716
cells were incubated for 4 h at 37 ꢀC. The plates were read using
MicroplateReader (Thermo Scientific Multiskan FC) at 450 nm. The
viability of untreated cells was set as 100%, and the viability in the
other groups was calculated by comparing the optical density
reading with the control. The IC50 values were calculated using
nonlinear regression analysis.
C
15H16ClNO3Na; HPLC: tr 15.17 min (99.0%, condition a).
4.1.2.24. Tert-butyl (2E)-(5-nitro-2-oxo-1, 2-dihydro-3H-indol -3-
ylidene)acetate (11k). Yellow solid, 161e162 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
9.44 (d, J ¼ 1.8 Hz. 1H), 8.88 (s, 1H), 8.29 (dd,
J ¼ 2.2 Hz, 8.6 Hz, 1H), 6.99 (d, J ¼ 8.6 Hz, 1H), 6.96 (s, 1H), 1.61 (s,
9H); ESI-MS: 289.1 (M-Hþ); HPLC: tr 8.33 min (99.6%, condition a).
4.2.3. Cell apoptosis and cell cycle analysis
HCT116 cells were seeded at 2 ꢂ 105 cells/well in 6-well plates
and cultured for 6 h, followed by drug treatment for 48 h. After
harvest, the cells were washed twice with cold PBS. For apoptosis
4.1.2.25. Methyl-2-(2-oxo-1, 2-dihydro-3H-indol -3-yl)acetate (12a)
[38]. White solid, 178e179 ꢀC; 1H NMR (300 MHz, CDCl3)
d 8.87 (s,

818
Z. Song et al. / European Journal of Medicinal Chemistry 124 (2016) 809e819
analysis, the cells were resuspend in 300
added FITC Annexin V (5 mL) and PI (5 m
m
L of 1 ꢂ Binding Buffer,
L) to cells, incubated for
15 min at RT in the dark before adding 400
L of 1 ꢂ Binding Buffer
dx.doi.org/10.1016/j.ejmech.2016.09.005.
m
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D
m
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Acknowledgment
Financial support from National Natural Science Foundation of
China (grants 81373303, 81473080, 81573299 and 31501182) and
the scientific research fund for new teacher of China Pharmaceu-
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