Design, synthesis and biological evaluation of novel bromophenol derivatives incorporating indolin-2-one moiety as potential anticancer agents

Article abstract of DOI:10.3390/md13020806

A series of bromophenol derivatives containing indolin-2-one moiety were designed and evaluated that for their anticancer activities against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines using MTT assay in vitro. Among them, seven compounds (4g-4i, 5h, 6d, 7a, 7b) showed potent activity against the tested five human cancer cell lines. Wound-healing assay demonstrated that compound 4g can be used as a potent compound for inactivating invasion and metastasis by inhibiting the migration of cancer cells. The structure-activity relationships (SARs) of bromophenol derivatives had been discussed, which were useful for exploring and developing bromophenol derivatives as novel anticancer drugs.

Full text of DOI:10.3390/md13020806

Mar. Drugs 2015, 13, 806-823; doi:10.3390/md13020806
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Design, Synthesis and Biological Evaluation of Novel
Bromophenol Derivatives Incorporating Indolin-2-One Moiety
as Potential Anticancer Agents
†
†
Li-Jun Wang , Shuai-Yu Wang , Bo Jiang, Ning Wu, Xiang-Qian Li, Bao-Cheng Wang,
Jiao Luo, Meng Yang, Shui-Hua Jin and Da-Yong Shi *
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
E-Mails: wanglijun@qdio.ac.cn (L.-J.W.); 12-12sy@163.com (S.-Y.W.);
jiangbo@qdio.ac.cn (B.J.); wuning@qdio.ac.cn (N.W.); lnu101@163.com (X.-Q.L.);
1
3365456626@163.com (B.-C.W.); luojiao2012@163.com (J.L.); qwyangmeng@163.com (M.Y.);
shisheng14226@126.com (S.-H.J.)
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: shidayong@qdio.ac.cn;
Tel.: +86-0532-8289-8719; Fax: +86-0532-8289-8741.
Academic Editor: Yoshihide Usami
Received: 31 December 2014 / Accepted: 27 January 2015 / Published: 2 February 2015
Abstract: A series of bromophenol derivatives containing indolin-2-one moiety were
designed and evaluated that for their anticancer activities against A549, Bel7402, HepG2,
HeLa and HCT116 cancer cell lines using MTT assay in vitro. Among them, seven
compounds (4g–4i, 5h, 6d, 7a, 7b) showed potent activity against the tested five human
cancer cell lines. Wound-healing assay demonstrated that compound 4g can be used as a
potent compound for inactivating invasion and metastasis by inhibiting the migration of
cancer cells. The structure–activity relationships (SARs) of bromophenol derivatives had
been discussed, which were useful for exploring and developing bromophenol derivatives as
novel anticancer drugs.
Keywords:
bromophenol;
indolin-2-one;
anticancer;
molecular
hybrid;
structure–activity relationship

Mar. Drugs 2015, 13
. Introduction
Cancer is a significant health problem throughout the world, which is a leading cause of death. It is
807
1
estimated that 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012
according to the WHO report. The incidence of cancer has been increasing in most regions of the
world, which will further increase to 19.3 million new cancer cases per year by 2025 [1]. Although many
effective anticancer agents have been developed, therapies for cancer are less than satisfactory due to
side effects. Therefore, novel effective agents for treating cancer disease are urgently needed today.
Bromophenols, natural marine products isolated from various marine biology, are known to possess
various potent activities including antioxidative, protein tyrosine kinase (PTK) inhibitory, anticancer,
protein tyrosine phosphatase 1B inhibitory, antithrombotic, antimicrobial, anti-inflammatory [2],
which have attracted much attention, also due to their unique structures. For example,
3
-bromo-4,5-dihydroxybenzyl alcohol (Figure 1) showed significant cytotoxicity to KB cells
(
IC50 = 8.9 μg/mL) [3]. 2,5-dibromo-3,4-dihydroxybenzyl n-propyl ether (Figure 1) synthesized from
natural bromophenol demonstrated potent activity against DLD-1 and HCT-116 Cells lines with
IC50 values 1.72 and 0.80 µM, respectively [4]. Xu and co-workers reported a series of natural
bromophenols (3,4-dibromo-5(methoxymethyl)-1,2-benzenediol, Figure 1) which showed vigorous
activities against KB, Bel7402, HELF and A549 cancer cells with IC50 values under 10 μg/mL [5].
In our previous study, a variety of bromophenols (Figure 1) isolated from various marine algae, which
exhibited excellent anticancer activity against A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780,
Bel7402 and HCT-8 human cancer cell lines, could be used as potent antitumor agents for PTK
over-expression of c-kit and is considered as part of a new therapeutic strategy for treatment
of cancer [6].
Figure 1. Structures of bromophenols possessing potent activity against cancer.
The structural scaffold of oxindole is proven as a core structure in various active compounds that
exhibit a wide variety of biological activities including anti-inflammatory [7,8], tuberculosis [9],
antiglycation [10], antitumor [11–14], neurological disorders [15], antioxidant [16], antimicrobial [17],
and anticonvulsant activity [18]. Among them, indolin-2-one derivatives have aroused great attention
for their anticancer activities and were developed as antitcancer drugs, such as SU5416, SU5402,
SU6668and SU14813 (Figure 2), which have been reported as potent and selective inhibitors of

Mar. Drugs 2015, 13
808
different protein kinases and showed a significant cytotoxic activity [19,20]. SU5416 is the first
selective kinase insert domain receptor (KDR) inhibitor used in clinical trials. Its derivative SU11248
(
sunitinib, Figure 2), an inhibitor of RTK approved by the US Food and Drug Administration (FDA),
has been approved and marketed for the treatment of gastrointestinal stromal cancers and renal cell
carcinoma [21]. SU11274 and PHA665752 (Figure 2) containing 3-methylene indolin-2-one scaffold
with sulfonyl group located on the 5-position were proved as an effective inhibitor of Met tyrosine
kinase and have entered preclinical and multi-center clinical studies for anticancer activity [22,23].
Therefore, the indolin-2-one moiety is the pharmacophore in developing anticancer agents.
Figure 2. Selected indolinone derivatives as anticancer agents in preclinical or clinical trials.
Molecular hybrids with two pharmacophores often lead to synergistic activity. It has been observed
that the incorporation of indolin-2-one moiety into the active anticancer moiety (such as pyrrole,
oxadiazole, thiazolidinone, etc.) demonstrated profound growth inhibitory activity against different
cancer cells, which was an efficient strategy for thedesign of novel antitumor agents [24–26].
In view of these observations, a novel series of bromophenols derivatives containing indolin-2-one
moiety was designed and evaluated for in vitro anticancer activity against cancer cell lines (Human
lung cancer cell line, A549; Human hepatoma cell lines, Bel-7402 and HepG2; human cervical cancer
cell line, HeLa; human colon cancer cell line, HCT116) using MTT assay. The most potent activity
compound 4g on cancer cell migration was investigated by using the wound-healing assay.
Structure–activity relationships (SARs) of these bromophenols analogs are also discussed in this paper.

Mar. Drugs 2015, 13
809
2
. Results and Discussion
2
.1. Chemistry
The synthetic procedures for the preparation of aldehydes (Scheme 1) have been publised in our
previous reports [27–30]. As shown in Scheme 2, a series of bromophenol derivatives were synthesized
by Knoevenagel condensation between different substituted indolin-2-one and the corresponding
aldehydes in order to explore the SARs of these derivatives and obtain the potencial lead compounds.
Firstly, oxindole (1) was reacted with ClSO H to yield compound 2. Then, compound 2 and amines
3
were heated for 3 h in tetrahydrofuran (THF) at 80 °C to afford 5-substituted-indolin-2-one (3). At last,
the reaction between 5-substituted-indolin-2-one (3) and the synthesized aldehydes was performed
under the condition of Knoevenagel condensation in ethanol with a catalytic amount of piperidine
to give the desired bromophenol derivatives 4–7 in good yields. All of the synthesized derivatives
were purified and their structures were characterized by spectroscopic means ( H, 13C NMR, MS
and HRMS).
1
Scheme 1. Synthesis of aldehydes. Reagents and conditions: (a) Br
2
, CH
O, KOH, diglycol, 120 °C;
, AcOH, Fe, reflux, 12 h; (g) AlCl
, DCM, −78 °C; (i) NBS, AIBN, CCl , hv; (j) K CO , 1,4-dioxane,
O, 90 °C; (k) PCC, DCM, rt; (l) Br , AcOH, rt.
3
OH, 0 °C;
(
b) CH
d) Br , AcOH, 60 °C; (e) NaBH
Cl , rt; (h) BBr
3
I, K
2
CO
3
, DMF, room temperature (rt); (c) 80% N
2
H
4
·H
2
(
2
4
, CH OH, 0 °C; (f) Br
3
2
3
,
CH
2
2
3
4
2
3
H
2
2

Mar. Drugs 2015, 13
810
Scheme 2. Synthesis of bromophenol derivatives 4–7. Reagents and conditions:
a) ClSO H, 65 °C , 1 h; (b) R NH, THF, reflux; (c) EtOH, aldehyde, piperidine, reflux.
(
3
1
R
2
2
.2. Anti-Cancer Activity
2
.2.1. Antiproliferative Activity
All the synthesized compounds were investigated for their anticancer activity in vitro on five human
cancer cell lines, namely against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines using
MTT assay and sunitinib as a positive control. The results of inhibited ration of comounds 4–7 were
listed in Figure 3, and the IC50 values of selective compounds (4g–4i, 5h, 6d, 6e, 7a, 7b) were listed
in Table 1.
As shown in Figure 3, compounds 4g–4i, 5h, 6d, 6e, 7a and 7b exhibited potent anticancer activity
against A549, Bel7402, HepG2, HeLa and HCT116 cancer cell lines at 10 μg/mL, respectively.
Of them, compound 4g showed significant activity against A549, Bel7402, HepG2, HeLa and HCT116
cancer cell lines with the inhibition ratio of 88.2%, 71.9%, 85.2% and 93.0% at 10 μg/mL,
respectively, which were comparable to those of sunitinib. Compounds 4a–4c, 5a–5c, and 6a with two
hydroxy groups on phenol ring showed weaker activity against of five cancer cells at 10 μg/mL.
After methylation of 3-hydroxyl group on phenol ring of compounds 4d–4f, 5d–5f, 6b, and 6c, the activity
have not obviously changed (4d vs. 4a, 4e vs. 4b, 4f vs. 4c, 5d vs. 5a, 5e vs. 5b, 5f vs. 5c, 6b, 6c vs. 6a).

Mar. Drugs 2015, 13
811
）
）
（
100
1
（
00
A
8
0
A549
B
8
0
A549
6
0
BEL7402
HepG2
HCT116
Hela
6
0
BEL7402
HepG2
HCT116
Hela
4
0
4
0
2
0
0
20
0
4
a
4
b
4
c
4
d
5a
4
e
5b
4
f
5c
4
g
5d
4
h
5e
5
f
4
i
5g
5
h
）
5
i
100
（
C
8
0
A549
6
0
BEL7402
HepG2
HCT116
Hela
4
0
2
0
0
6
a
6
b
6
c
6
d
6
e
6
f
7
a
7
b
7
c
SN
Figure 3. Inhibitory activity of bromophenol derivatives (A: compounds 4a–4i;
B: compounds 5a–5i; C: compounds 6a–6f, 7a–7c and Sunitinib) against five human
cancer cell lines; the percent inhibition rate of tumor cell at 10 µg/mL inhibitor; the cells
were seeded in a 96-well plate and incubated overnight. Then, the cells were treated with
various amounts of compounds and incubated for 48 h. Cell proliferation was evaluated
with the MTT Assay. Sunitinib (SN) as the positive control.
Table 1. IC50 values of selected bromophenol derivatives against five human cancer cell lines.
IC50 (μg/mL) a
Compd
A549
Bel7402
HepG2
Hct116
9.1 ± 0.13
9.8 ± 0.55
NA
Hela
7.4 ± 0.22
8.3 ± 0.67
NA
4
g
6.6 ± 0.82
14.4 ± 1.86
9.9 ± 0.11
10.1 ± 0.72
25.4 ± 0.82
NA
9.2 ± 0.84
13.2 ± 2.42
4
h
12.3 ± 0.23 14.3 ± 0.86
b
4
i
22.3 ± 1.11
9.7 ± 2.35
18.6 ± 0.91
NA
NA
11.2 ± 1.26
NA
5
6
h
d
8.6 ± 0.26 18.0 ± 0.13
5.6 ± 0.42 17.6 ± 0.89
6
7
e
NA
9.8 ± 0.20
6.1 ± 0.23
8.2 ± 0.54
NA
a
12.5 ± 0.19
12.5 ± 0.45
NA
7.9 ± 0.26
25.0 ± 0.18
8.6 ± 0.14
9.3 ± 0.47
7
b
12.5 ± 0.39 14.2 ± 0.77
c
TTEDM
Sunitinib
44.1 ± 1.19
3.2 ± 0.15
NA
32.3 ± 2.03 23.5 ± 1.41
2.8 ± 0.27 1.6 ± 0.08
d
10.4± 0.54
4.5 ± 0.23
a
IC50: Concentration of the compound producing 50% cell growth inhibition after 48 h of drug exposure,
as determined by the MTT assay. Each experiment was run at least three times, and the results
are presented as average values ± standard deviation; b NA: Compound showing IC50 value > 50 μg/mL;
c
2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (TTEDM) is a marine bromophenol
d
compound derived from marine algae. P.; Sunitinib as the positive control.

Mar. Drugs 2015, 13
812
It was inspired that methylation of 4-hydroxyl group on phenol ring (4g, 4h, 5g, 5h, 6d, 6e, 7a, 7b)
could significantly enhance their anti-cancer activity, which indicated that the hydrophobic parameter
may affect their anticancer activity. However, compounds 4i, 5i, 6f, 7c with four methoxy groups in
diphenyl methane moiety showed low anticancer activity, suggesting that many of the hydrophobic
groups have a deleterious effect. The steric hindrance may affect their activity also. The derivatives of
mono-brominated on phenol ring exhibited better activity than those of di-brominated compounds
(
4b vs. 4c, 4e vs. 4f, 4g vs. 4h, 5b vs. 5c, 5e vs. 5f, 6d vs. 6e, 7a vs. 7b). On the contrary, compound 5h
with di-brominated on phenol moiety showed better inhibitory activity against cancer cells than those
of compound 5g with mono-brominated on phenol moiety. These results indicated that the number of
bromine atom on phenol moiety could affect the anticancer activities of these hybrid derivatives.
Furthermore, it is notable that most compounds introducing secondary amino groups in 5-position of
indolin-2-one (4a–4i and 7a–7c) displayed significant activity compared to those of compounds (5a–6f)
with tertiary sulfonamide groups, indicating that types of amino groups at 5-position of indolin-2-one
could influence the activities of these derivatives. From Table 1, it is also seenthat these synthesized
bromophenol derivatives containing indolin-2-one moiety (4g–4i, 5h, 6d, 6e, 7a, 7b) showed better
activities than 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (TTEDM)
which was a marine bromophenol compound isolated from marine algae Polysiphonia urcedata.
2
.2.2. Inhibitory Effect of Compound 4g on Cancer Cell Migration
Cancer cell invasion, a hallmark of cancer, plays a critical role during the carcinomas arising from
epithelial tissues progressed to higher pathological grades of malignancy [31]. To further investigate
the anticancer effect of 4g, we studied the inhibitory effect on cancer cell migration; an invasive
process often observed in cancer by using the wound-healing assay [32]. We found that the migration
of HepG2 cells was significantly inhibited by 48 h treatment with 4g at 3 μg/mL concentration
compared to the untreated control (Figure 4A).
Control
4g 3μg/mL
160
40
120
00
A
B
1
0
h
*
1
80
60
40
20
0
*
2
4
4 h
8 h
Control 4g 3μg/mL
Control 4g 3μg/mL
2
4 h
48 h
Figure 4. (A) The HepG2 cells were scraped by a pipette tip to generate wounds.
After treatment with or without 4g 3 μg/mL in the presence of 1 μg/mL Mitomycin C,
cells migration was recorded by microscopy at 0, 24, 48 h; (B) Results were obtained from
three separate experiments. The bar represents the mean ± SE, * (p < 0.05) indicates
a significant difference between the control and 4g treated samples.

Mar. Drugs 2015, 13
813
The migration width (the distances of migrated cells) of cancer cells treated with/without 4g was
summarized in Figure 4B. It was demonstrated that ~20% of cell migration was inhibited by treatment
with 4g at 3 μg/mL concentration for 48 h. These results suggested that 4g might be used as a potent
compound for inactivating invasion and metastasis by inhibiting the migration of cancer cells.
3
. Experimental Section
3
.1. Chemistry
1H and 13C NMR spectra were recorded on Bruker DRX 500 MHz spectrometers with
tetramethylsilane (TMS) as the internal standard (Bruker, Bremerhaven, Germany). MS and HRMS
spectra were determined on a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Column
chromatography (CC): Silica gel (200–300 mesh; Qingdao Makall Group CO., Ltd; Qingdao; China).
All reactions were monitored using thin-layer chromatography (TLC) on silica gel plates. Reaction
reagents were purchased from J&K Scientific Ltd. (Chaoyang District, Beijing, China) Organic solvents
were analytical reagent grade and purchased from Tianjin Chemical Reagent Co., Ltd. (Jinnan District,
Tianjin, China). The synthesized compounds were named using ChemBioDraw Ultra software
(version 12.0, Waltham, MA, USA).
General Procedure for the Preparation of Derivatives (4–7)
To a 100 mL flask charged with chlorosulfonic acid (25 mL), 2-oxindole (50 mmoL) at 0 °C was
added slowly. After the addition, the reaction mixture was stirred at room temperature for 1.5 h. Then,
the reaction mixture was heated to 68 °C for 1 h, cooled, and poured into ice water (200 mL). The
precipitate was washed with water and dried in a vacuum oven to give 5-chlorosulfonyl-2-oxindole (2)
which was used without further purification.
A mixture of 5-chlorosulfonyl-2-oxindole (5 mmoL) and appropriate amine (10 mmoL) in
tetrahydrofuran (THF, 50 mL) was heated to refluxing and stirred for 3 h. Then, this mixture was
concentrated under reduced pressure, and HCl (pH = 3, 25 mL) was added and stirred for 15 min.
The crude product was filtered, washed with ice water (100 mL) and dried in a vacuum oven to give
5-sulfamoyl-2-oxindole (3) which was used without further purification.
Piperidine (50 μL) was added to a mixture of compound 3 (0.5 mmoL) and appropriate aldehyde
(0.55 mmoL) in ethanol (5 mL). The reaction mixture was heated to refluxing and stirred for 2 h,
and TLC analysis indicated when the reaction was complete. The crude product was filtered, washed
with ethanol and dried in a vacuum (if no solid precipitated, the crude product was chromatographed
using a silica gel column) to afford the title compound 4–7 as a yellow solid.
(
E)-N-(4-Bromophenyl)-3-(3,4-dihydroxybenzylidene)-2-oxoindoline-5-sulfonamide (4a). Yield:
1
6
6.1%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 10.96 (s, 1H), 10.21 (s, 1H), 8.10 (s, 1H), 7.75 (s, 1H),
7
.58 (dd, 1H, J = 1.5, 8.5 Hz), 7.56 (s, 1H), 7.38–7.40 (overlap, 3H), 7.06 (d, 1H, J = 9.0 Hz), 7.00
13
(d, 1H, J = 9.0 Hz), 6.97 (d, 2H, J = 8.5 Hz); C NMR (DMSO-d , 125 MHz, ppm): δ 169.4, 150.3,
6
1
1
4
48.9, 146.1, 145.2, 137.7, 140.2, 132.4 (2C), 128.8, 125.4, 123.2, 122.5, 122.3 (2C), 121.9, 120.8,
−
O
S [M − H]−
18.0, 116.5, 116.3, 110.5; ESIMS: m/z 484 [M − H] HRESIMS: Calc for C21
H
15BrN
2
5
84.9805, found 484.9812.

Mar. Drugs 2015, 13
814
(
E)-3-(3-Bromo-4,5-dihydroxybenzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (4b).
1
Yield: 67.6%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.00 (s, 1H), 10.24(s, 1H), 8.11 (s, 1H),
7
.79(s, 1H), 7.56(dd, 1H, J = 1.5, 8.5 Hz), 7.54 (s, 1H), 7.39 (d, 2H, J = 9.0 Hz), 7.32 (s, 1H), 7.06 (d, 2H,
1
3
J = 9.0 Hz), 6.99 (d, 1H, J = 8.5 Hz); C NMR (DMSO-d , 125 MHz, ppm): δ 169.1, 147.4, 146.6,
6
1
1
5
45.8, 139.7, 137.9, 132.4 (2C), 132.1, 131.7,126.6, 126.0, 124.8, 122.7, 122.0 (2C), 121.6, 116.3,
−
O
S [M − H]−
16.1, 110.7, 110.4; ESIMS: m/z 562 [M − H] HRESIMS: Calc for C21
H13BrN
2
5
62.8915, found 562.8917.
(
E)-N-(4-Bromophenyl)-3-(2,3-dibromo-4,5-dihydroxybenzylidene)-2-oxoindoline-5-sulfonamide (4c).
1
Yield: 77.4%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.08 (s, 1H), 10.30 (s, 1H), 7.74 (s, 1H), 7.55
(
(
d, 1H, J = 8.5 Hz), 7.54 (s, 1H), 7.33 (d, 2H, J = 8.5 Hz), 7.15(s, 1H), 6.96 (d, 1H, J = 8.5 Hz), 6.93
1
3
d, 2H, J = 8.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 168.7, 147.0, 146.9, 146.0, 138.0, 137.6,
1
32.4 (2C), 132.1, 129.6, 126.7, 126.0, 121.8 (2C), 121.5, 121.2, 116.4, 115.8, 115.3, 114.4, 110.8;
−
−
ESIMS: m/z 640 [M − H] HRESIMS: Calc for C21
H12Br
3
N
2
5
O S [M − H] 640.8014, found 640.8022.
(
E)-N-(4-Bromophenyl)-3-(4-hydroxy-3-methoxybenzylidene)-2-oxoindoline-5-sulfonamide (4d).
1
Yield: 68.0%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.12 (s, 1H), 10.30 (s, 1H), 8.18 (s, 1H), 7.60
(
s, 1H), 7.56 (dd, 1H, J = 1.5, 8.5 Hz), 7.32 (s, 1H), 7.31 (d, 1H, J = 8.5 Hz), 7.24 (d, 2H, J = 8.5 Hz),
1
3
6
.99 (d, 1H, J = 8.5 Hz), 6.92 (d, 1H, J = 8.5 Hz), 6.87 (d, 2H, J = 8.5 Hz), 3.83 (s, 3H); C NMR
, 125 MHz, ppm): δ 169.6, 150.8, 148.2, 147.8, 145.5, 140.8, 139.6, 132.0 (2C), 128.5,
25.4, 124.8, 122.9, 122.5 (2C), 122.2, 120.7, 116.4, 116.1, 113.4, 110.1, 55.9; ESIMS: m/z 498
(DMSO-d
6
1
−
−
[M − H] HRESIMS: Calc for C22
H16BrN
2
O S [M − H] 498.9970, found 498.9969.
5
(
E)-3-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
1
(
4e). Yield: 73.5%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.08 (s, 1H), 10.30 (s, 1H), 7.92 (s, 1H),
7
.55(s, 1H), 7.48 (s, 1H), 7.47 (s, 1H), 7.37 (d, 2H, J = 8.5 Hz), 7.32 (d, 1H, J = 8.5 Hz), 7.05 (d, 2H,
13
J = 8.5 Hz), 6.97 (d, 1H, J = 8.5 Hz); C NMR (DMSO-d , 125 MHz, ppm): δ 168.0, 150.3, 144.8,
6
1
1
5
41.7, 140.8, 138.3, 138.2, 132.3 (2C), 131.2, 128.0, 124.2, 123.1, 121.9 (2C), 119.7, 116.0, 115.9,
−
O
S [M − H]−
13.7, 111.0, 108.7, 55.8; ESIMS: m/z 576 [M − H] HRESIMS: Calc for C22
H15Br
2
N
2
5
76.9062, found 576.9074.
(
E)-N-(4-Bromophenyl)-3-(2,3-dibromo-4-hydroxy-5-methoxybenzylidene)-2-oxoindoline-5-sulfonamide
1
(
(
(
4f). Yield: 68.9%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.08 (s, 1H), 10.24 (s, 1H), 7.68 (s, 1H), 7.58
d, 1H, J = 8.5 Hz), 7.55 (s, 1H), 7.53 (s, 1H),7.32 (d, 2H, J = 8.5 Hz), 6.97 (d, 1H, J = 8.5 Hz), 6.92
1
3
d, 2H, J = 8.5 Hz), 3.87 (s, 3H); C NMR (DMSO-d , 125 MHz, ppm): δ 168.5, 152.6, 147.1, 146.0,
6
1
1
6
37.7, 137.6, 132.4 (2C), 132.2, 131.4, 129.8, 126.8, 122.1 (2C), 121.6, 121.2, 116.9, 116.4, 115.6,
−
O
S [M − H]−
14.2, 110.9, 65.4; ESIMS: m/z 654 [M − H] HRESIMS: Calc for C22
H14Br
3
N
2
5
54.8168, found 654.8179.
(
E)-3-(3-Bromo-4,5-dimethoxybenzylidene-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (4g).
1
Yield: 67.2%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.08 (s, 1H), 10.24 (s, 1H), 7.63 (s, 1H),
7
6
1
.58–7.61 (overlap, 2H), 7.50 (s, 1H), 7.40 (s, 1H), 7.31 (d, 2H, J = 8.5 Hz), 7.05 (d, 1H, J = 9.0 Hz),
1
3
.94 (d, 2H, J = 8.5 Hz), 3.87 (s, 3H), 3.84 (s, 3H); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 168.9,
53.8, 148.3, 147.5, 137.3, 132.3 (2C), 132.1, 131.5, 131.4, 129.6, 126.7, 126.3, 122.4 (2C), 121.4,

Mar. Drugs 2015, 13
815
−
1
21.3, 117.6, 116.5, 113.5, 110.7, 60.8, 56.6; ESIMS: m/z 590 [M − H] HRESIMS: Calc for
−
C
23
H17Br
2
N
2
O S [M − H] 590.9219, found 590.9230.
5
(
E)-N-(4-Bromophenyl)-3-(2,3-dibromo-4,5-dimethoxybenzylidene)-2-oxoindoline-5-sulfonamide (4h).
1
Yield: 82.5%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.16 (s, 1H), 10.24 (s, 1H), 7.66 (s, 1H),
7
6
1
1
.60 (d, 1H, J = 8.5 Hz), 7.57 (s, 1H), 7.52 (s, 1H), 7.31 (d, 2H, J = 8.5 Hz), 6.99 (d, 1H, J = 8.5 Hz),
1
3
.90 (d, 2H, J = 8.5 Hz), 3.86 (s, 3H), 3.82 (s, 3H); C NMR (DMSO-d , 125 MHz, ppm): δ 168.5,
6
52.8, 148.6, 147.2, 137.5, 136.8, 132.4 (2C), 132.2, 131.7, 130.0, 128.0, 122.2 (2C), 122.1, 121.7,
−
21.2, 117.0, 116.6, 113.9, 110.9, 60.9, 60.7; ESIMS: m/z 668 [M − H] HRESIMS: Calc for
−
C
23
H16Br
3
N O
2 5
S [M − H] 668.8334, found 668.8335.
(
E)-3-(3-Bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl-4,5-dimethoxybenzylidene)-N-(4-bromophenyl)-
1
2
-oxoindoline-5-sulfonamide (4i). Yield: 88.2%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 10.99
(
s, 1H), 10.35 (s, 1H), 7.63 (s, 2H), 7.52 (s, 1H), 7.32-7.34 (overlap, 3H), 6.92-6.95 (overlap, 3H), 6.33
13
(s, 1H), 4.22 (s, 2H), 3.85(s, 3H), 3.80(s, 3H), 3.58(s, 3H), 3.31(s, 3H); C NMR (DMSO-d , 125
6
MHz, ppm): δ 168.3, 152.4, 152.3, 147.5, 146.9, 145.8, 137.6, 136.9, 136.2, 132.3 (3C), 131.2, 130.5,
1
29.8, 127.8, 122.5, 121.6 (3C), 121.5, 121.3, 117.0, 116.2, 113.2, 112.9, 110.7, 60.8, 60.2, 56.7, 56.1,
−
−
41.3; ESIMS: m/z 896 [M − H] HRESIMS: Calc for C32
4 2 7
H25Br N O S [M − H] 896.8107,
found 896.8121.
1
(
E)-3-(3,4-Dihydroxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5a). Yield: 52.3%; H NMR
6
(DMSO-d , 500 MHz, ppm): δ 11.04 (s, 1H), 8.04 (s, 1H), 7.63 (s, 1H), 7.56 (dd, 1H, J = 1.5, 8.5 Hz),
7
.14 (s, 1H), 7.06–7.11 (overlap, 2H), 6.87 (d, 1H, J = 8.5 Hz), 3.62 (t, 4H, J = 4.5 Hz), 2.84 (t, 4H,
1
3
J = 4.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 169.4, 148.9, 145.9, 145.1, 140.2, 129.8, 126.8,
1
25.4, 123.2, 122.3, 121.5, 121.1, 117.5, 116.2, 110.6, 65.7 (2C), 46.5 (2C); ESIMS: m/z 401 [M − H]−
−
HRESIMS: Calc for C19
H
17
N
2
6
O S [M − H] 401.0813, found 401.0813.
(E)-3-(3-Bromo-4,5-dihydroxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5b). Yield: 66.3%;
1H NMR (DMSO-d
, 500 MHz, ppm): δ 11.10 (s, 1H), 8.08 (s, 1H), 7.60 (s, 1H), 7.54 (dd, 1H, J = 1.5,
6
8
.0 Hz), 7.42 (s, 1H), 7.07 (s, 1H), 7.01 (d, 1H, J = 8.0 Hz), 3.62 (t, 4H, J = 4.5 Hz), 2.84(t, 4H,
1
3
J = 4.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 169.1, 147.0, 145.8, 144.3, 140.0, 130.0, 129.4,
1
26.8, 126.3, 124.7, 122.7, 119.4, 116.5, 110.7, 110.3, 65.7 (2C), 46.5 (2C); ESIMS: m/z 478 [M − H]−
−
HRESIMS: Calc for C19
H16BrN
2
O
6
S [M − H] 478.9901, found 478.9918.
(
E)-3-(2,3-Dibromo-4,5-dihydroxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5c). Yield: 68.3%;
1H NMR (DMSO-d
, 500 MHz, ppm): δ 10.95 (s, 1H), 7.79 (s, 1H), 7.67 (s, 1H), 7.55 (dd, 1H, J = 1.5,
.5 Hz), 7.10 (s, 1H), 6.95 (d, 1H, J = 8.5 Hz), 3.66 (t, 4H, J = 4.5 Hz), 2.85 (t, 4H, J = 4.5 Hz);
6
8
13C NMR (DMSO-d
, 125 MHz, ppm): δ 169.7, 146.2, 146.0, 144.4, 139.7, 129.9 128.7, 127.3, 126.6,
6
−
1
25.8, 122.2, 121.9, 117.3, 114.8, 110.2, 65.9 (2C), 46.0 (2C); ESIMS: m/z 556 [M − H] HRESIMS:
−
Calc for C19H15Br
2
N
2
O S [M − H] 556.9015, found 556.9023.
6
(
E)-3-(4-Hydroxy-3-methoxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5d). Yield: 77.5%;
1H NMR (DMSO-d
, 500 MHz, ppm): δ 11.03 (s, 1H), 8.00(s, 1H), 7.69 (s, 1H), 7.52(dd, 1H, J = 1.5,
.5 Hz), 7.38 (s, 1H), 7.26 (dd, 1H, J = 1.5, 8.0 Hz), 7.09 (d, 1H, J = 8.0 Hz), 6.90 (d, 1H, J = 8.0 Hz),
6
8
3
1
3
.84 (s, 3H), 3.62 (t, 4H, J = 4.5 Hz), 2.86(t, 4H, J = 4.5 Hz); C NMR (DMSO-d , 125 MHz, ppm):
6

Mar. Drugs 2015, 13
816
δ 169.8, 150.4, 147.8, 144.3, 140.4, 129.9, 127.2, 125.7, 125.6, 123.6, 122.5, 121.6, 116.7, 113.9,
−
1
10.0, 66.0 (2C), 56.4, 46.7 (2C); ESIMS: m/z 415 [M − H] HRESIMS: Calc for C20
H
19
N
2
6
O S
−
[M − H] 415.0955, found 415.0969.
(
E)-3-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5e). Yield:
1
6
7.2%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.10 (s, 1H), 8.07 (s, 1H), 7.67 (s, 1H), 7.59 (s, 1H), 7.55
(
d, 1H, J = 8.0 Hz), 7.40 (s, 1H), 7.04 (d, 1H, J = 8.0 Hz), 3.90 (s, 3H), 3.62 (t, 4H, J = 4.5 Hz),
1
3
2
1
.86 (t, 4H, J = 4.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 169.1, 148.7, 148.0, 144.2, 139.8,
31.0, 128.8, 127.0, 126.4, 124.9, 123.0, 119.3, 115.6, 109.8, 109.3, 65.7 (2C), 56.6, 46.4 (2C);
−
−
ESIMS: m/z 493 [M − H] HRESIMS: Calc for C20
H18BrN
2
O S [M − H] 493.0073, found 493.0074.
6
(
E)-3-(2,3-Dibromo-4-hydroxy-5-methoxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5f).
1
Yield: 78.3%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 10.95 (s, 1H), 8.02 (s, 1H), 7.67 (s, 1H), 7.56
(
dd, 1H, J = 1.5, 8.0 Hz), 7.39 (s, 1H), 7.04 (d, 1H, J = 8.0 Hz), 3.90 (s, 3H), 3.62 (t, 4H, J = 4.5 Hz),
1
3
2
1
.86 (t, 4H, J = 4.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 169.2, 148.8, 147.5, 144.3, 139.8,
30.9 127.1, 126.4, 124.9, 123.0, 121.9, 121.7, 119.3, 113.1, 109.9, 65.7 (2C), 56.6, 46.4 (2C);
−
−
ESIMS: m/z 570 [M − H] HRESIMS: Calc for C20
H17Br
2
N
2
O
6
S [M − H] 570.9172, found 570.9180.
(5g). Yield:
, 500 MHz, ppm): δ 11.00 (s, 1H), 8.07 (s, 1H), 7.70 (s, 1H), 7.62 (dd, 1H,
J = 1.5, 8.5 Hz), 7.60 (s, 1H), 7.46 (s, 1H), 7.05 (d, 1H, J = 8.5 Hz), 3.89 (s, 3H), 3.82 (s, 3H), 3.62
(
E)-3-(3-Bromo-4,5-dimethoxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one
1
8
2.6%; H NMR (DMSO-d
6
13
(t, 4H, J = 4.5 Hz), 2.87 (t, 4H, J = 4.5 Hz); C NMR (DMSO-d , 125 MHz, ppm): δ 168.9, 153.9,
6
1
6
5
48.3, 144.8, 138.7, 131.7, 130.7, 126.9, 126.0, 125.5, 122.1, 121.7, 116.6, 114.4, 110.1, 65.7 (2C),
−
O
S [M − H]−
0.8, 56.8, 46.4 (2C); ESIMS: m/z 507 [M − H] HRESIMS: Calc for C21
H20BrN
2
6
07.0227, found 507.0231.
(
E)-3-(2,3-Dibromo-4,5-dimethoxybenzylidene)-5-(morpholinosulfonyl)indolin-2-one (5h). Yield: 88.2%;
1H NMR (CDCl
, 500 MHz, ppm): δ 10.96 (s, 1H), 7.89 (s, 1H), 7.67 (dd, 1H, J = 1.5, 8.5 Hz), 7.63
s, 1H), 7.29 (s, 1H), 7.11 (d, 1H, J = 8.5 Hz) , 3.97 (s, 3H) , 3.93 (s, 3H), 3.75 (t, 4H, J = 4.5 Hz), 2.93
3
(
(
1
3
t, 4H, J = 4.5 Hz); C NMR (CDCl
3
, 125 MHz, ppm): δ 169.7, 153.0, 149.4, 147.1, 138.6, 131.9,
1
30.8, 128.3, 128.0, 123.0, 122.9, 122.0, 117.9, 112.9, 111.0, 66.3 (2C), 61.1, 56.8, 46.3 (2C); ESIMS:
−
−
m/z 584 [M − H] HRESIMS: Calc for C21
H19Br
2
N
2
O
6
S [M − H] 584.9325, found 584.9336.
(
E)-3-(3-Bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dimethoxybenzylidene)-5-(morpholinosulfonyl)
1
indolin-2-one (5i). Yield: 84.3%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 10.96 (s, 1H), 7.62 (s, 1H), 7.53
(dd, 1H, J = 1.5, 8.5 Hz), 7.31 (s, 1H), 7.21 (s, 1H), 6.99 (d, 1H, J = 8.5 Hz), 6.56 (s, 1H), 4.26 (s, 2H),
3
.80 (s, 3H), 3.79 (s, 3H), 3.60 (t, 4H, J = 4.5 Hz), 3.54 (s, 3H), 3.52 (s, 3H), 2.75 (t, 4H, J = 4.5 Hz);
, 125 MHz, ppm): δ 168.2, 152.3 (2C), 147.4, 147.1, 145.8, 137.5, 136.8, 131.5,
30.7, 130.6, 127.9, 126.9, 122.6, 122.3, 121.6, 121.3, 116.7, 113.7, 113.4, 110.6, 65.7 (2C), 60.7,
13C NMR (DMSO-d
6
1
6
−
0.1, 56.9, 56.2, 41.4, 46.2 (2C); ESIMS: m/z 812 [M − H] HRESIMS: Calc for C30
H28Br
3
N
2
8
O S
−
[M − H] 812.9113, found 812.9122.
(
E)-3-(3,4-Dihydroxybenzylidene)-5-((4-(pyrimidin-2-yl)piperazin-1-yl) sulfonyl) indolin-2-one (6a).
1
Yield: 55.1%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.11 (s, 1H), 8.30 (d, 2H, J = 5.0 Hz), 7.77
(s, 1H), 7.57 (s, 1H), 7.52 (d, 1H, J = 8.0 Hz), 7.14 (s, 1H), 7.02 (d, 1H, J = 8.0 Hz), 6.94 (d, 1H,

Mar. Drugs 2015, 13
817
J = 8.0 Hz), 6.73 (d, 1H, J = 8.0 Hz),6.60 (t, 1H, J = 5.0 Hz), 2.95 (t, 4H, J = 4.5 Hz), 2.92 (t, 4H,
13
J = 4.5 Hz); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 167.9, 161.2, 158.4 (2C), 147.1, 145.9, 143.0,
1
41.1, 130.5, 127.7, 126.5, 125.4, 122.9, 117.6, 116.4, 115.8, 111.1, 110.2, 109.0, 46.2 (2C), 43.0 (2C);
−
−
ESIMS: m/z 478 [M − H] HRESIMS: Calc for C23
20 5 5
H N O S [M − H] 478.1184, found 478.1191.
(
E)-3-(4-Hydroxy-3-methoxybenzylidene)-5-((4-(pyrimidin-2-yl) piperazin-1-yl) sulfonyl) indolin-2-one
1
(
6b). Yield: 61.5%; H NMR (DMSO-d
.00 (s, 1H), 7.68 (s, 1H), 7.58 (d, 1H, J = 8.5 Hz), 7.39 (s, 1H), 7.05 (d, 1H, J = 8.5 Hz), 6.99 (d, 1H,
J = 8.0 Hz), 6.87 (d, 1H, J = 8.0 Hz), 6.60 (t, 1H, J = 5.0 Hz), 3.84 (s, 3H), 2.94 (t, 4H,
6
, 500 MHz, ppm): δ 11.00 (s, 1H), 8.30 (d, 2H, J = 5.0 Hz),
8
13
J = 4.5 Hz), 2.89 (t, 4H, J = 4.5 Hz); C NMR (DMSO-d , 125 MHz, ppm): δ 167.9, 161.2, 158.4 (2C),
6
1
5
4
50.1, 147.5, 144.0, 141.4, 129.8, 127.2, 126.3, 125.3, 123.4, 121.5, 121.3, 116.7, 115.7, 111.1, 110.7,
−
O
S [M − H]−
6.1, 46.2 (2C), 43.0 (2C); ESIMS: m/z 492 [M − H] HRESIMS: Calc for C24
H N
22 5
5
92.1341, found 492.1347.
(
E)-3-(3-Bromo-4-hydroxy-5-methoxybenzylidene)-5-((4-(pyrimidin-2-yl)piperazin-1-yl) sulfonyl)
1
indolin-2-one (6c). Yield: 70.2%; H NMR (DMSO-d , 500 MHz, ppm): δ 10.70 (s, 1H), 8.29 (d, 2H,
6
J = 5.0 Hz), 7.88 (s, 1H), 7.70 (s, 1H), 7.50 (s, 1H),7.36 (d, 1H, J = 8.0 Hz), 7.14 (s, 1H), 6.91 (d, 1H,
J = 8.0 Hz), 6.58 (t, 1H, J = 5.0 Hz), 3.81 (s, 3H), 2.97 (t, 4H, J = 4.5 Hz), 2.91 (t, 4H, J = 4.5 Hz);
13C NMR (DMSO-d
, 125 MHz, ppm): δ 168.0, 161.2, 158.4 (2C), 150.4, 145.1, 142.0, 141.4, 128.4,
6
1
26.2, 125.1, 123.5, 120.0, 117.8, 116.7, 113.7, 111.0, 1109.8, 108.7, 55.7, 46.2 (2C), 43.0 (2C);
−
−
ESIMS: m/z 570 [M − H] HRESIMS: Calc for C24
H21BrN
5
5
O S [M − H] 570.0433, found 570.0452.
(
E)-3-(3-Bromo-4,5-dimethoxybenzylidene)-5-((4-(pyrimidin-2-yl)piperazin-1-yl)
sulfonyl)
1
indolin-2-one (6d). Yield: 76.2%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.11 (s, 1H), 8.31 (d, 2H,
J = 5.0 Hz), 8.05 (s, 1H), 7.68(s, 1H), 7.58-7.60 (overlap, 2H), 7.45 (s, 1H), 7.01 (d, 1H, J = 8.5 Hz),
.60 (t, 1H, J = 5.0 Hz), 3.89(s, 3H), 3.82(s, 3H), 2.95 (t, 4H, J = 4.5 Hz), 2.91 (t, 4H, J = 4.5 Hz);
13C NMR (DMSO-d
, 125 MHz, ppm): δ 168.9, 161.2, 158.4 (2C), 154.0, 148.3, 144.8, 137.3, 131.7,
30.7, 127.6, 127.3, 125.5, 121.7, 122.0, 116.6, 114.4, 111.1, 110.1, 60.8, 56.8, 46.1 (2C), 42.9 (2C);
6
6
1
−
−
ESIMS: m/z 584 [M − H] HRESIMS: Calc for C25
H23BrN
5
O S [M − H] 584.0606, found 584.0609.
5
(
E)-3-(2,3-Dibromo-4,5-dimethoxybenzylidene)-5-((4-(pyrimidin-2-yl)piperazin-1-yl)
sulfonyl)
1
indolin-2-one (6e). Yield: 78.3%; H NMR (Pyridine-d
5
, 500 MHz, ppm): δ 10.16 (s, 1H), 8.14 (d, 2H,
J = 5.0 Hz), 7.99 (s, 1H), 7.67 (d, 1H, J = 8.0 Hz), 7.42 (s, 1H), 7.03 (s, 1H), 6.97 (d, 1H, J = 8.0 Hz),
.25 (t, 1H, J = 5.0 Hz), 3.84 (s, 3H), 3.78 (s, 3H), 3.04 (t, 4H, J = 4.5 Hz), 2.90 (t, 4H, J = 4.5 Hz);
13C NMR (Pyridine-d
, 125 MHz, ppm): δ 169.6, 161.6, 158.2 (2C), 153.4, 150.3, 148.3, 137.3, 132.4,
31.0, 128.9, 128.4, 123.2, 123.1, 122.3, 118.2, 113.8, 110.93, 110.86, 60.9, 57.0, 46.5 (2C), 43.2 (2C);
6
5
1
−
−
ESIMS: m/z 661 [M − H] HRESIMS: Calc for C25
H
22Br
2
N
5
O
5
S [M − H] 661.9690, found 661.9714.
(
E)-3-(3-Bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dimethoxybenzylidene)-5-((4-(pyrimidin-2-yl)
1
piperazin-1-yl) sulfonyl) indolin-2-one (6f). Yield: 85.2%; H NMR (DMSO-d
6
, 500 MHz, ppm):
δ 10.90 (s, 1H), 8.31 (d, 2H, J = 5.0 Hz), 7.59 (s, 1H), 7.53 (dd, 1H, J = 1.5, 8.5 Hz), 7.28 (s, 1H), 7.21
(
s, 1H), 6.96 (d, 1H, J = 8.5 Hz), 6.60–6.62 (overlap, 2H), 4.26 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H), 3.54
1
3
(s, 3H), 3.51 (s, 3H), 2.92 (t, 4H, J = 4.5 Hz), 2.81 (t, 4H, J = 4.5 Hz); C NMR (DMSO-d , 125 MHz,
6
ppm): δ 168.2, 161.1, 158.4 (2C), 152.3, 152.2, 147.3, 147.0, 145.7, 137.6, 137.0, 131.5, 130.6, 127.7,
27.2, 122.7, 122.2, 121.6, 121.2, 116.6, 114.0, 113.4, 111.1, 110.5, 60.7, 60.0, 56.9, 56.2, 46.1 (2C),
1

Mar. Drugs 2015, 13
818
S [M − H] 889.9522,
−
−
4
2.8 (2C), 41.5; ESIMS: m/z 889 [M − H] HRESIMS: Calc for C34
H31Br
3
N
5
O
7
found 889.9500.
(
E)-N-(Adamantan-1-yl)-3-(3-bromo-4,5-dimethoxybenzylidene)-2-oxoindoline-5-sulfonamide (7a).
1
Yield: 42.6%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.09 (s, 1H), 8.07 (s, 1H), 7.78–7.71 (overlap,
2
H), 7.66 (s, 1H), 7.46 (s, 1H), 7.02 (d, 1H, J = 8.0 Hz), 3.94 (s, 3H), 3.93 (s, 3H), 1.99 (m, 6H), 1.57
1
3
(m, 3H), 1.25 (m, 6H); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 168.1, 153.8, 148.3, 144.7, 138.2,
1
37.6, 130.9, 130.1, 126.7, 126.0, 121.7, 121.6, 118.0, 115.3, 110.3, 60.8, 56.3, 55.3, 43.1 (3C), 35.8
−
−
(3C), 29.5 (3C); ESIMS: m/z 571 [M − H] HRESIMS: Calc for C27
H28BrN
2
5
O S [M − H] 571.0907,
found 571.0908.
(
E)-N-(Adamantan-1-yl)-3-(2,3-dibromo-4,5-dimethoxybenzylidene)-2-oxoindoline-5-sulfonamide (7b).
1
Yield: 52.5%; H NMR (DMSO-d
6
, 500 MHz, ppm): δ 11.08 (s, 1H), 7.85 (s, 1H), 7.70 (d, 1H, 8.0 Hz),
7
.64 (s, 1H), 7.28 (s, 1H), 7.01(d, 1H, J = 8.0 Hz), 3.80 (s, 3H) , 3.83 (s, 3H), 1.85 (m, 6H), 1.70
1
3
(m, 3H), 1.40 (m, 6H); C NMR (DMSO-d
6
, 125 MHz, ppm): δ 168.6, 152.9, 148.4, 146.1, 136.3,
1
2
32.1, 129.6, 128.8, 128.6, 121.9, 121.4, 120.8, 116.6, 113.9, 111.7, 60.8, 56.9, 54.1, 42.9 (3C), 35.9 (3C),
−
−
9.3 (3C); ESIMS: m/z 649 [M − H] HRESIMS: Calc for C27
H27Br
2
N
2
5
O S [M − H] 648.9996,
found 649.0013.
(
E)-N-(Adamantan-1-yl)-3-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dimethoxybenzylidene)-
1
2
9
-oxoindoline-5-sulfonamide (7c). Yield: 57.6%; H NMR (CDCl
3
, 500 MHz, ppm): δ 10.00 (s, 1H),
.43 (s, 1H), 7.87 (s, 1H), 7.69 (s, 1H), 7.61 (d, 1H, J = 8.0 Hz), 7.26 (s, 1H), 7.21 (s, 1H), 7.16 (d, 1H,
J = 8.0 Hz), 4.29 (s, 2H), 3.90 (s, 3H), 3.75 (s, 3H), 3.54 (s, 3H), 3.52 (s, 3H), 2.03 (m, 6H), 1.75
1
3
(
m, 3H), 1.25 (m, 6H); C NMR (CDCl
3
, 125 MHz, ppm): δ 169.4, 152.5, 152.3, 148.4, 146.1, 144.7,
1
6
37.1, 135.4, 131.4, 129.2 130.6, 127.7, 127.6, 123.0, 122.3, 121.6, 121.3, 117.6, 111.8, 111.6, 110.1,
−
0.9, 60.4, 56.5, 56.1, 55.3, 46.9, 43.1 (3C), 35.8 (3C), 29.5 (3C); ESIMS: m/z 876 [M − H] HRESIMS:
−
Calc for C36
H36Br
3
N
2
O
7
S [M − H] 876.9793, found 876.9793.
3
.2. Antiproliferative Activities Assays
3
.2.1. Cell Culture and Proliferation Assay
The cells including Human lung cancer cell line, A549; Human hepatoma cell lines, Bel-7402
and HepG2; human cervical cancer cell line, HeLa; human colon cancer cell line, HCT116 were
purchased from BOSTER, Ltd (Wuhan, China). The cells were maintained in DMEM supplemented
with 10% FBS and 1% antibiotics at 37 °C and in an atmosphere containing 5% CO
split 1:3 when they reached 80%–90% confluence. Cell proliferation was measured using the
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The IC50 was
2
. The cells were
3
calculated by using the SPSS 17.0 (SPSS, Inc., Chicago, IL, USA).
3
The cells (4 × 10 cells/well) with 10% FBS culture medium were seeded in a 96-well plate and
incubated overnight. Next, the cells were treated with various amounts of compounds and incubated for
48 h. Subsequently, 20 μL of 5 mg/mL MTT was transferred into each well, and the cells were incubated
for 4 h. The medium in each well was carefully removed, and 150 μL DMSO was then added to each

Mar. Drugs 2015, 13
819
well. The samples were thoroughly agitated for 10 min on a shaker. Finally, the absorbance of the samples
at 490 and 690 nm was measured against a background control (blank) using a microplate reader.
3
.2.2. Wound-Healing Assay
5
The HepG2 cells (1 × 10 cells/well) were cultured in 6-well plates rinsed with PBS and then
starved overnight in 2% FBS medium until they reached 95% confluence. A single wound was then
scratched in the center of the cell monolayers with a 200 μL sterile plastic pipette tip. The wounded
monolayers were washed twice to remove the non-adherent cells and were incubated with various
concentrations of compound 4g in the presence of 1 μg/mL of mitomycin C. To measure the length of
the endothelial cells that had migrated from the edge of the injured monolayer, images were obtained
immediately after wounding and after a 24, 48 h incubation period, using a phase-contrast microscope
(Olympus, Tokyo, Japan). The length was measured by the Image-Pro Plus v 6.0 (Media Cybernetics, Inc.,
Bethesda, MD, USA). Each experiment was repeated at least three times.
3
.2.3. Statistical Analysis
All the experiments were performed at least three times, and the data are presented as mean ± SD
values. Differences between the mean values were assessed using one-way analysis of variance. For all
the analyses, p < 0.05 was considered significant. Statistical analyses were performed using SPSS 17.0
(SPSS, Inc., Chicago, IL, USA).
4
. Conclusions
In summary, a series of bromophenols derivatives containing indolin-2-one moiety were designed
and evaluated for their anticancer activities against A549, Bel7402, HepG2, HeLa and HCT116 cancer
cell lines using MTT assay in vitro. The preliminary SAR analysis (summarized in Figure 5) reveals
that: (i) the hydrophobic parameter may affect their anticancer activity; (ii) the steric hindrance may
also affect their activity; (iii) the number of bromine atoms on phenol moiety could affect the
anticancer activities of these hybrid derivatives; (iv) the types of amino groups at 5-position of
indolin-2-one could influence the activities; (v) the bromophenol moiety played a crucial role in
maintaining anticancer activities of the conjugated derivatives. Among them, seven compounds (4g–4i,
5
h, 6d, 7a, 7b) showed potent activity. Wound-healing assay demonstrated that compound 4g can be
used as a potent compound for inactivating invasion and metastasis by inhibiting the migration of cancer
cells. These active derivatives targeting properties needs to be further investigated. Potentially,
this finding may aid in the design of novel agents for the intervention of cancer.

Mar. Drugs 2015, 13
820
Figure 5. Structure–Activity relationships of bromophenol derivatives anticancer activity.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (No. 41276167,
1206066 and 41306157), China Postdoctoral Science Foundation (No. 2014M551971 and
014M551972), Innovation Project Special Fund for Post Doctors of Shandong Province (No. 201302017
4
2
and 201401019), the National science and technology support project (2013BAB01B02-2) and the Natural
Science Foundation of Jiangsu Province (Grant No. BK 2012223).
Author Contributions
Li-Jun Wang, Shuai-Yu Wang and Da-Yong Shi contributed to the study concept and design,
and the manuscript preparation. Li-Jun Wang and Shuai-Yu Wang performed the experimental studies
and analyzed the data. Bo Jiang, Ning Wu, Xiang-Qian Li, Bao-Cheng Wang, Jiao Luo, Meng Yang
and Shui-Hua Jin contributed in critical reading and discussion on the manuscript. All the authors
approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.
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2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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(