Discovery of novel bromophenol hybrids as potential anticancer agents through the ros-mediated apoptotic pathway: Design, synthesis and biological evaluation

Article abstract of DOI:10.3390/md15110343

A series of bromophenol hybrids with N-containing heterocyclic moieties were designed, and their anticancer activities against a panel of five human cancer cell lines (A549, Bel7402, HepG2, HCT116 and Caco2) using MTT assay in vitro were explored. Among t

Full text of DOI:10.3390/md15110343

marine drugs
Article
Discovery of Novel Bromophenol Hybrids as
Potential Anticancer Agents through the
Ros-Mediated Apoptotic Pathway: Design,
Synthesis and Biological Evaluation
Li-Jun Wang 1,2,†, Chuan-Long Guo 1,2,3,†, Xiang-Qian Li 1,2, Shuai-Yu Wang 1,2, Bo Jiang 1,2
Yue Zhao 1,2, Jiao Luo 1,2,3, Kuo Xu 1,2, Hua Liu 1,2,3, Shu-Ju Guo 1,2, Ning Wu 1,2 and
,
Da-Yong Shi 1,2,3,
*
1
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, Shandong, China; wanglijun@qdio.ac.cn (L.-J.W.); gcl_cpu@126.com (C.-L.G.);
lnu101@163.com (X.-Q.L.); 12-12sy@163.com (S.-Y.W.); jiangbo@qdio.ac.cn (B.J.);
zhaoyue19931104@163.com (Y.Z.); luojiao2012@163.com (J.L.); xukuoworld@126.com (K.X.);
baihualin55100@sina.cn (H.L.); guoshuju@qdio.ac.cn (S.-J.G.); wuning@qdio.ac.cn (N.W.)
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology,
Qingdao 266071, Shandong, China
2
3
University of Chinese Academy of Sciences, Beijing 10049, China
*
†
Correspondence: shidayong@qdio.ac.cn; Tel.: +86-532-8289-8719; Fax: +86-532-8289-8741
These authors contributed equally to this work.
Received: 23 September 2017; Accepted: 30 October 2017; Published: 1 November 2017
Abstract: A series of bromophenol hybrids with N-containing heterocyclic moieties were designed,
and their anticancer activities against a panel of five human cancer cell lines (A549, Bel7402, HepG2,
HCT116 and Caco2) using MTT assay in vitro were explored. Among them, thirteen compounds
(
17a, 17b, 18a, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, and 23b) exhibited significant inhibitory
activity against the tested cancer cell lines. The structure-activity relationships (SARs) of bromophenol
derivatives were discussed. The promising candidate compound 17a could induce cell cycle arrest
at G0/G1 phase and induce apoptosis in A549 cells, as well as caused DNA fragmentations,
morphological changes and ROS generation by the mechanism studies. Furthermore, compound
17a suppression of Bcl-2 levels (decrease in the expression of the anti-apoptotic proteins Bcl-2 and
down-regulation in the expression levels of Bcl-2) in A549 cells were observed, along with activation
caspase-3 and PARP, which indicated that compound 17a induced A549 cells apoptosis in vitro
through the ROS-mediated apoptotic pathway. These results might be useful for bromophenol
derivatives to be explored and developed as novel anticancer drugs.
Keywords: bromophenol hybrids; anticancer; structure-activity relationships; ROS; apoptotic pathway
1. Introduction
The development of anticancer agents is the hot topic in medicinal chemistry based on the high
incidence and lethality of cancer today. Although many effective drugs have been approved to treat
cancer, therapies for it are less satisfactory due to side effects, thus there is need for novel therapeutic
agents. Certain new targeted agents can block the growth of cancer cells by interfering with specific
targeted molecules and have less toxicity than chemotherapy drugs, which offer a promise of treating
cancer. Reactive oxygen species (ROS) play vital roles in cell growth, which associate with multiple
changes in cellular functions (cell proliferation, migration, differentiation, apoptosis, etc.) in cancer
cells [1]. Agents targeting ROS-mediated apoptotic pathway have proven to be attractive for cancer
Mar. Drugs 2017, 15, 343; doi:10.3390/md15110343
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 343
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therapy [2]. Some natural and synthetic effective anticancer agents targeting ROS-mediated apoptotic
pathway were reported [3–5].
Bromophenols, a class of natural marine products, and their derivatives possessed various potent
activities, including antioxidative, anticancer, antithrombotic, antimicrobial, and anti-inflammatory
activities, which have attracted much attention [6]. A series of bromophenol hybrids with indolin-2-one
moiety were designed and synthesized as potential anticancer agents in our previous work [
7].
Among of them, compound WLJ18 (Figure 1) displayed excellent antitumor activities against various
human cancer cell lines and could inactivate invasion and metastasis. These encouraging results
prompted us to further design and synthesize a new class of bromophenol derivatives as potential
anticancer agents.
Figure 1. Structures of N-containing heterocyclic anticancer drugs, WLJ18 and compounds 17–25.
N-containing heterocyclic compounds are important compounds with excellent biological
activities, such as antiviral, anti-inflammatory and antitumor activities [8]. Especially in new
drug discovery, the N-containing heterocyclic moieties were widely introduced into pharmaceutical
molecules. For example, the rings of piperidine, morpholine and piperazine were introduced into
anticancer drugs (Gefitinib, Vandetanib, SU11274, Sunitinib, etc. (Figure 1)) and had positive effects
on activities of drugs along with several properties of molecules, including drug-target interactions,
metabolic stability and toxicity [9].

Mar. Drugs 2017, 15, 343
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Usually, the combination of different biological units leads to synergistic activity, which is an
efficient strategy for the design of novel antitumor agents [10 13]. Based on the above, we designed and
–
synthesized a series novel of bromophenol hybrids with heterocyclic molecules containing nitrogen in
our continuing research for novel anticancer agents. The antitumor activities of bromophenol hybrids
were screened against a series of cancer cell lines in vitro using MTT assay, and the structure-activity
relationships (SARs) of these analogs are also discussed. The mechanism studies of the promising
candidate compound 17a were further investigated.
2. Results and Discussion
2.1. Chemistry
The general synthetic methods of compounds are shown in Scheme 1. Firstly, oxindole (1)
was reacted with ClSO3H to yield compound
heated for 3 h in THF at 80 C to afford N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (
2
. Then, compound
2
and 4-bromoaniline were
) [ ].
◦
3
7
The aldehyde
yield intermediates
Et3N in DMF to give compounds
and compound wereperformed under the condition of Knoevenagel condensation in ethanol with
a catalytic amount of piperidine to give the desired derivatives 17 25 in good yields. The structures
4
was reacted with alkyl dibromide under K2CO3 in dimethylformamide (DMF) to
7 [14]. Then compounds were treated with the appropriate amine under
16 15]. Finally, the reactions between intermediates (8–16)
5
–
5–7
8
–
[
3
–
of compounds 8–25 are shown in Table 1. All of the synthesized derivatives were purified and their
structures were characterized by spectroscopic means (1H, 13C-nuclear magnetic resonance (NMR),
and high-resolution mass spectrometer (HRMS)). The configuration of the double bond in compounds
7– ,17]. Physicochemical properties (including
25 was assigned to E based on the spectra of 1H NMR [16
calculated logarithm of partition coefficient between n-octanol and H2O (cLogP), H2O solubility
in mol/L (cLogS), polar surface area (TPSA), hydrogen bond acceptor (Ha), and hydrogen bond
donor (Hd)), toxicity profiles (including mutagenic effect, tumorigenic effect, irritating effect and
reproductive effect) and drug-likeness scores of these compounds were calculated and predicted
using OSIRIS Property Explorer software at URL http://www.organic-chemistry.org/prog/peo/ [18].
The calculation of physicochemical properties and prediction of toxicity risks are shown in Table 2.
H
N
H
N
a
Br
b
O
O
O
O
O
N
H
S
O
S
Cl
R
O
N
O
H
n
1
2
3
OCH3
e
Br
CHO
H
N
CHO
CHO
O
S
c
d
O
O
Br
N
Br
OCH3
Br
OCH3
Br
OCH3
H
O
R
O
OH
17-25
n
n=2,3,4
n
n=2,3,4
Br
4
5-7
8-16
◦
Scheme 1. Reagents and conditions: (
a
) ClSO3H, 65 C, 1 h; (
b
) 4-bromoaniline, THF, reflux; (
c) K2CO3,
◦
◦
DMF, 60–80 C; (d) Et3N, DMF, 40–60 C; and (e) EtOH, piperidine, reflux.

Mar. Drugs 2017, 15, 343
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Table 1. Structures of synthesized compounds 8–25.
Compd.
n
R
8a, 17a
8b, 17b
2
3
9a, 18a
9b, 18b
9c, 18c
2
3
4
10a, 19a
10b, 19b
2
3
11a, 20a
11b, 20b
2
3
12a, 21a
2
12b, 21b
12c, 21c
3
4
13a, 22a
13b, 22b
2
3
14a, 23a
14b, 23b
2
3
15, 24
16, 25
2
2
Table 2. Calculated physicochemical properties and predicted toxicity of compounds 17–25.
Toxicity Risks f
CLogS b
Hd e
TPSA (Å2) c
Ha d
Compd.
CLogP a
Drug-Likeness g
M/T/I/R
17a
17b
18a
18b
18c
19a
19b
20a
20b
4.71
5.16
3.54
4.00
4.45
5.21
5.66
4.89
5.34
−6.79
−7.06
−5.90
−6.17
−6.44
−6.97
−7.24
−6.62
−6.89
105.4
105.4
114.6
114.6
114.6
108.6
108.6
108.6
108.6
8
8
9
9
9
9
9
9
9
2
2
2
2
2
2
2
2
2
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
4.44
3.63
4.80
4.51
1.00
5.95
5.96
8.88
8.91

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Table 2. Cont.
Toxicity Risks f
M/T/I/R
CLogS b
Hd e
TPSA (Å2) c
Ha d
Compd.
CLogP a
Drug-Likeness g
21a
21b
21c
22a
22b
23a
23b
24
3.65
4.11
4.56
3.95
4.41
3.50
3.95
2.53
3.35
−5.40
−5.67
−5.94
−6.51
−6.78
−6.18
−6.44
−5.42
−4.82
108.6
108.6
108.6
134.4
134.4
134.4
134.4
145.81
111.83
9
9
9
11
11
11
11
10
10
2
2
2
2
2
2
2
4
2
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
N/N/N/H
H/H/L/H
N/N/N/H
9.42
9.84
6.56
8.98
9.85
7.58
8.23
3.32
9.50
25
cLogP, calculated logarithm of partition coefficient between n-octanol and water; b cLogS, S: water solubility in
a
mol/L, pH = 7.5, 25 ◦C; c TPSA, topological polar surface area; d Ha, hydrogen bond acceptor; e Hd, hydrogen bond
donor; f Toxicity risks: M, mutagenic effect; T, tumorigenic effect; I, irritating effect; R, reproductive effect; N, none;
L, low; M, medium; H, high; g The approach is based on a list of about 5300 distinct substructure fragments with
associated dårug-likeness scores.
2.2. Biological Activity
2.2.1. Cytotoxicity
All of the target compounds (17–25) were investigated for their in vitro anti-cancer activity
against five human cancer cell lines, A549 (human lung cancer cell line), HepG2 (human hepatocellular
carcinoma cell line), Bel7402 (human hepatocellular carcinoma cell line), HCT116 (human colorectal
cancer cell line), and Caco2 (Human colonic epithelial cell line), using MTT method with sunitinib as
a positive control. The IC50 values of these compounds are listed in Table 3.
Table 3. IC50 values of bromophenol derivatives against five human cancer cell lines.
IC50 (µg/mL) a
Compd.
A549
Bel7402
HepG2
HCT116
Caco2
NA c
44.8 ± 1.45
9.68 ± 0.76
6.10 ± 0.78
9.98 ± 1.57
11.8 ± 1.23
48.6 ± 3.23
NA
10.9 ± 0.77
6.78 ± 0.76
9.40 ± 0.88
8.67 ± 0.37
15.1 ± 1.87
3.25 ± 0.32
NA
17.3 ± 1.98
44.8 ± 2.44
33.7 ± 2.22
NA
32.7 ± 0.24
12.9 ± 1.38
4.50 ± 0.58
NA
31.2 ± 1.56
9.78 ± 0.29
5.74 ± 0.26
3.78 ± 0.91
6.22 ± 0.23
16.8 ± 0.65
NA
4.89 ± 0.35
3.82 ± 0.43
5.07 ± 0.69
5.80 ± 0.21
5.37 ± 0.79
4.43 ± 0.53
43.3 ± 0.23
3.59 ± 0.25
34.2 ± 2.23
8.90 ± 0.43
22.3 ± 2.18
15.0 ± 1.36
10.1 ± 0.81
3.70 ± 0.27
25.6 ± 0.78
9.11 ± 1.23
4.23 ± 0.32
4.70 ± 0.35
5.61 ± 0.89
10.2 ± 1.28
NA
5.76 ± 0.82
1.70 ± 0.24
5.94 ± 1.32
8.24 ± 1.42
6.89 ± 0.55
7.52 ± 0.99
41.9 ± 3.89
4.09 ± 0.76
5.60 ± 0.46
8.50 ± 1.81
8.97 ± 0.29
22.7 ± 2.11
9.3 ± 1.43
2.7 ± 0.35
TTEDM b
wlj-18
17a
7.10 ± 0.53
3.15 ± 0.43
4.78 ± 0.56
10.8 ± 1.21
7.62 ± 0.76
4.49 ± 0.73
12.6 ± 0.98
3.60 ± 0.38
8.10 ± 1.11
6.99 ± 1.42
11.5 ± 0.27
5.20 ± 0.76
16.7 ± 3.39
7.40 ± 0.46
7.10 ± 1.02
14.9 ± 3.26
5.96 ± 1.02
29.9 ± 2.72
11.5 ± 2.41
11.6 ± 1.24
14.1 ± 1.35
4.42 ± 0.72
9.60 ± 0.34
6.63 ± 0.75
25.0 ± 1.66
42.4 ± 1.86
8.79 ± 0.65
5.42 ± 0.92
8.42 ± 0.76
7.83 ± 1.56
8.67 ± 0.53
5.83 ± 1.11
40.7 ± 2.13
6.80 ± 1.11
41.1 ± 3.12
21.6 ± 1.73
NA
17b
18a
18b
18c
19a
19b
20a
20b
21a
21b
21c
22a
22b
23a
23b
24
NA
25
14.9 ± 0.87
Sunitinib d
5.80 ± 1.08
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 2,20,3-tribromo-30,4,40,5-tetrahydroxy-60-ethyloxymethyldiphenylmethane
c
(TTEDM) is a marine bromophenol compound derived from marine algae. P.; NA: Compound showing IC50
value >50 µg/mL; d Sunitinib as the positive control.
As shown in Table 3, the rings of piperidine were introduced to bromophenol derivatives 17a and
17b, which exhibited excellent anticancer activities against the test cancer cell lines. Compounds 18a

Mar. Drugs 2017, 15, 343
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and 18b with ring of morpholine showed better activities against HCT116, Caco2 and A549 cell lines
than that of compound WLJ18. On the contrary, the activity of compound 18c displayed weak activities
against Bel7402, HepG2, HCT116 and Caco2, except A549 with the IC50 value of 4.49 ± 0.73 µg/mL
When 1,40-bipiperidine unit was introduced to bromophenol, the activities of compound 19a and 19b
were increased. The introduction of N,N-diethylpiperidine moiety with two and three carbon chains
.
(20a and 20b) could increase the activities against the test cancer cell lines with excellent IC50 values.
The anticancer activity of compound 21a containing ring of 4-methylpiperazine with two-carbon
chain slightly increased comparing to compound WLJ18. When the number of the carbon chain was
two atoms, compound 21b exhibited excellent anticancer activities, inhibiting the five cancer cell lines
with IC50 values of 5.20
±
0.76
µ
g/mL, 3.25
±
0.32
µ
g/mL, 5.83
±
1.11
µ
g/mL, 4.43
±
0.53 µg/mL
and 7.52 ± 0.99 µg/mL, respectively. Compound 22a with 4-(pyrimidin-2-yl)piperazin-1-yl ring and
two carbon chain showed potent activities against cancer cell lines of HCT116 and Caco2 with the IC50
values of 3.59
±
0.25
µg/mL and4.09
±
0.76 µg/mL. However, its analogs (22b) with three carbon
chains showed weak anticancer activities. The chain length could affect the anticancer activities of
these hybrids based on the above results. In an effort to gain more potent bromophenol hybrids
and their information of the SARs, we probed additional structural changes. The 4-(pyrazin-2-yl)
piperazin-1-yl, 4-(2-(dimethylamino)ethyl) piperazin-1-yl and bis (2-hydroxyethyl) amino groups were
incorporated with WLJ18. Disappointingly, the anticancer activities of compounds 24 and 25 obviously
decreased, which indicated that the moieties of incorporation could affect the steric clash, electron
density, or hydrogen-bonding capacity, resulting different anticancer activities of bromophenol hybrids.
2.2.2. Compound 17a Induce Morphological Changes in A549 Cells
Morphological changes of cancer cells are always associated with the growth inhibition induced
by cytotoxic agents. We also took photos for the cells after treating compound 17a for 48 h. As shown
in Figure 2A, compound 17a treated A549 cells showed morphological changes such as cell shrinkage,
deformation and reduced number of viable cells.
Figure 2. Effects of compound 17a on cell morphology and colony formation in A549 cell. (
cells were treated with compound 17a for 48 h; the representative fields were photographed at 100
magnification. ( ) A549 cells were treated with compound 17a (0, 5, 10, 20 g/mL) for 10 days and
A) A549
×
B,
C
µ
colony formation was determined by staining with crystal violet. The data represent mean values
(±SD) obtained from three separate experiments. ** p < 0.01 vs. control group.

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2.2.3. Compound 17a Inhibits Colony Formation Ability of A549 Cells
The colony formation experiment was performed to determine the long-term impact of compound
17a on A549 cells growth. A 10-day colony formation assay was performed in this study. A549 cells
were seeded in six-well plates (500 cells/well). Cells were treated with various concentrations of
compound 17a (0, 5, 10, 20
revealed that the colony-forming ability of A549 cells was significantly and dose-dependently
suppressed after compound 17a treatment (Figure 2B,C). As shown in Figure 2C, 464.5 10 of
colonies were present in the control panel, whereas after treatment with 5 g/mL compound 17a the
number decreased to 263 37. Further decrease to 133 5 and 55 17 occurred after treatment for
10 and 20 g/mL, respectively. The results indicated that compound 17a had a significant inhibitory
µg/mL), and incubated for 10 days to allow colony formation. The results
±
µ
±
±
±
µ
effect on the colony formation of A549 cells.
2.2.4. Compound 17a Induce Apoptosis in A549 Cells
To determine whether the compound 17a-induced reduction in cell viability was responsible
for the induction of apoptosis, A549 cells were co-stained with PI and Annexin-V FITC, and the
number of apoptotic cells was estimated by flow cytometry. The flow cytometric detection of
phosphatidylserine (PS) expression in early apoptosis was employed (using fluorescence-conjugated
annexin-V). This combination allows the differentiation among viable cells (AV
apoptotic cells (AV+/PI ), late-phase apoptotic cells (AV+/PI+), and necrotic cells (AV
A dose-dependent increase in the percentage of apoptotic cells was noted after the cells were treated
for 48 h with compound 17a (0, 5, 10, 20 g/mL). As shown in Figure 3A,B, 11.45 1.20% of
apoptotic cells were present in the control panel, whereas, after treatment with 5 g/mL compound
17a, the population rose to 14.65 1.06%. Further increase to 24.3 6.36% and 63.8 7.21% occurred
after treatments of 10 and 20 µg/mL, respectively.
−
/PI
−
), early-phase
−
−
/PI+).
µ
±
µ
±
±
±
Figure 3. Compound 17a induces intrinsic apoptosis in A549 cells. (
of cells stained with Annexin V-FITC and PI. A549 cells were treated with various concentrations of
compound 17a (0, 5, 10, and 20 g/mL) for 48 h. Cells were harvested and processed by annexin V-FITC
and PI staining followed by flow cytometry analysis. (C) A549 cells were treated with compound 17a
(0, 5, 10, and 20 g/mL) for 48 h. Hoechst 33258 staining was used to detected the apoptosis and
photographed using an fluorescence microscopy (bar = 50 µm). ** p < 0.01 vs. control group.
A,B) Flow cytometric analysis
µ
µ

Mar. Drugs 2017, 15, 343
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2.2.5. Compound 17a Causes DNA Fragmentations and Morphological Changes
An essential hallmark of apoptosis is DNA fragmentation and morphological changes.
Morphological changes of apoptotic cells such as nuclear apoptotic bodies were analyzed by
fluorescence microscopy with Hoechst 33258 staining. A549 cells were treated with 5, 10 and 20 µg/mL
compound 17a for 48 h. As shown in Figure 3C, the treatment of the A549 cells with compound 17a
resulted in the induction of chromatin condensation, fragmentation and clear apoptotic bodies that
were visualized in fluorescence microscopy.
2.2.6. Compound 17a Induce G0/G1 Cell Cycle Arrest in A549 Cells
To elucidate whether the cytotoxicity induced by the derivatives was due to cell cycle arrest,
A549 cells were treated with compound 17a (0, 5, 10, 20
µg/mL) for 48 h. Flow cytometry
analysis showed A549 cells, which were treated with compound 17a, arrested in G0/G1 phase in
a dose-dependent manner (Figure 4). When compared with control, compound 17a increased the
population in the G1 phase from 57.85% to 80.63% at concentration of 20
µg/mL, while the G2/M phase
was decreased. These findings denote that compound 17a can induce cell cycle arrest in G0/G1 phase.
Figure 4. Compound 17a induces G0/G1 cell cycle arrest in A549 cells. (
with compound 17a (0, 5, 10, and 20 g/mL) for 48 h. Cells were harvested and fixed in 70% ethanol
overnight, and then cells were stained with PI and analysis by FACS. ( ) Western blot analysis of cell
cycle-related proteins, including cyclin D1 and CDK4. -actin was used to normalize protein content.
A,B) A549 cells were treated
µ
C
β
The data represent mean values (±SD) obtained from three separate experiments.
Cell cycle is regulated by a family of protein kinase complexes, including CDKs and cyclins,
in eukaryotic cells [19]. The previous study has reported that the reduced activities of CDK 4 and
cyclin D1 are the hallmarks of cell cycle arrest at the G1/S phase [20
analysis showed that treatment of A549 cells with compound 17a at 20
,
21]. In this study, Western blot
g/mL significantly decreased
µ
the level of cyclin D1 and CDK4.
2.2.7. Compound 17a Triggers ROS Generation
ROS are highly harmful elements to cells as they initiate oxidative stress and ultimately cause
cellular damage. Excessive ROS generation renders cells vulnerable to apoptosis. To determine whether

Mar. Drugs 2017, 15, 343
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compound 17a triggers ROS generation in A549 cells to induce apoptosis, the ROS level was measured
using 20,70-dichlorodihydrofluoresce in diacetate (DCFH-DA) as fluorescent probe. DCFH-DA is
cleaved by intracellular esterases into its non-fluorescent form (DCFH), which is converted to a green
fluorescent product, carboxy-DCF, via oxidation. A rapid production of ROS occurred after the
exposure of A549 cells to compound 17a (Figure 5A,B). When compared with control (100%), the mean
DCF fluorescence increased by 150.10
with 17a for 48 h. Treatment of compound 17a also increased the green fluorescence intensity in
±
22.60%, 177.32
±
15.60% and 214.60
±
18.90%, when treated
A549 cells (Figure 5C).
Figure 5. Compound 17a induces ROS generation in A549 cells. A549 cells were treated with various
concentrations of compound 17a (0, 5, 10, and 20
were incubated at 37 C in the dark for 20 min with culture medium containing DCFH-DA. (
µ
g/mL) for 48 h; the medium was discarded and cells
) Cells
) Cells were washed twice with PBS and analyzed using
m). All data were representative of three independent experiments.
◦
A
,B
were harvested and analyzed using FACS. (
C
fluorescence microscopy (bar = 50
µ
* p < 0.05; ** p < 0.01 vs. control group.
2.2.8. Effect of Compound 17a on the Expression of Apoptosis-Related Proteins
The Bcl-2 family members are important regulators of mitochondrial function during apoptosis.
It has been recognized that accumulation of ROS does not kill cells directly, it triggers an apoptotic
signing pathway that leads to cell death, such as increase the Bax/Bcl-2 ratio and activate caspase-3
and PARP [22]. In our study, the treatment of A549 cells induces decrease in the expression of
the anti-apoptotic proteins Bcl-2 and down-regulation in the expression levels of Bcl-2 (Figure 6).
Treatment with compound 17a also activated caspase-3 and PARP in A549 cells (Figure 6). All these
data demonstrated that compound 17a induced A549 cells apoptosis in vitro, probably through the
ROS-mediated apoptotic pathway.

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Figure 6. Western blot analysis of the effect of compound 17a on apoptosis-related proteins. A549
cells were treated with various concentrations of compound 17a (0, 5, 10, and 20 g/mL) for 48 h.
Western blot analyses were performed, and -actin was used to normalize protein content. All data
µ
β
were representative of three independent experiments.
3. Materials and Methods
3.1. Chemistry
Reaction reagents were purchased from J&K Scientific Ltd. (Beijing, China). Organic
solvents were analytical reagent grade and purchased from Tianjin Chemical Reagent Co., Ltd.
(Tianjin, China) 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. 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).
Melting points were determined on a SGW X-4 Melting Point Apparatus (Shanghai Precision Science
Instrument Co., Ltd., Shanghai, China). The synthesized compounds were named using ChemBioDraw
Ultra software (v 12.0, PerkinElmer, Waltham, MA, USA).
3.1.1. General Procedures for the Preparation of Compounds 5–7
The mixture of anhydrous K2CO3 (1.00 eq.) and the 3-bromo-4-hydroxy-5-methoxybenzaldehyde
4 (1.00 eq.) were suspended in dry DMF. Then, an alkyl dibromide (1.10 eq.) was added in several
portions (neat) during 0.5 h. This reaction mixture was stirred at 60–80 ◦C overnight. After complete
turnover, H2O was added and the aqueous solution was extracted three times with chloroform.

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The combined organic layers washed with H2O, HCl and brine. Then, the organic layer was dried
with Na2SO4 and removed in vacuo. The resulting residue was purified by column chromatography
on silica gel to provide compounds
Supplementary Data.
5–7. Details of synthesis of compounds 5–7 are given in the
3.1.2. General Procedure for the Preparation of Compounds 8–16
To a solution of compounds (0.1 mmol) in DMF, the appropriate amine (0.3 mmol) and Et3N
L, 1.17 mmol) was added. After stirring at room temperature (unless otherwise indicated)
overnight, the mixture was poured into H2O. Then, the organic phase was extracted three times with
EtOAc (4 30 mL) and washed with H2O (2 30 mL) and with brine (2 30 mL). The solvent was
5–7
(160
µ
×
×
×
evaporated, and the residue was purified by column chromatography on silica gel to provide the
compounds 8–16. Details of synthesis of compounds 8–16 are given in the Supplementary Data.
3.1.3. General Procedures for the Preparation of Compounds 17–25
The synthesis of compounds
was added to a mixture of compound
2
and
3
3
were performed as our previous report [
(0.5 mmol) and appropriate aldehydes
7
8
]. Piperidine (50 µL
)
–
16 (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 compounds 17–25 as yellow solid.
(E)-3-(3-Bromo-5-methoxy-4-(2-(piperidin-1-yl)ethoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
(
17a). Intermediate 8a was treated with compound 3 following the general procedure to give the
desired product 17a. Yield: 56.4%; mp.: 210–215 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
11.05 s,
1H), 7.58–7.63 overlap, 2H), 7.49 (s, 1H), 7.42 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H, J = 8.5 Hz),
7.05 (d, 1H, J = 9.0 Hz), 6.94 (d, 2H, J = 8.5 Hz), 4.13 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 2.68 (t, 2H, J =
6.0 Hz), 2.41 (m, 4H), 1.43 (m, 4H) 1.32 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 152.6,
147.7, 146.9, 138.4, 138.2, 137.3, 132.4(2C), 131.1, 129.6, 126.6, 125.6, 122.3(2C), 121.4, 117.8, 116.8, 113.3,
110.7, 110.2, 71.0, 58.5, 56.6, 54.7(2C), 25.9(2C), 24.4; ESIMS: m/z 690 [M + H]+ HRESIMS: calc. for
C29H29Br2N3O5S [M + H]+ 690.0289, found 690.0267.
(E)-3-(3-Bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
(
17b). Intermediate 8b was treated with compound 3 following the general procedure to give the
desired product 17b. Yield: 61.6%; mp.: 198–202 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
11.08
(s, 1H), 7.59–7.63 (overlap, 2H), 7.49 (s, 1H), 7.42 (s, 1H), 7.38 (d, 1H, J = 9.0 Hz), 7.30 (d, 2H, J = 8.5 Hz),
7.05 (d, 1H, J = 9.0 ), 6.94 (d, 2H, J = 8.5 Hz), 4.07 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 2.43 (t, 2H, J = 6.0 Hz),
2.34 (m, 4H), 1.88 (m, 2H), 1.45 (m, 4H), 1.34 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9,
152.9, 147.6, 146.8, 138.4, 138.2, 137.2, 132.4(2C), 131.1, 129.6, 126.5, 125.4, 122.3(2C), 121.3, 117.8, 116.8,
113.4, 110.8, 110.2, 72.1, 56.6, 55.5, 54.4(2C), 27.6, 26.0(2C), 24.5; ESIMS: m/z 704 [M + H]+ HRESIMS:
calc. for C30H31N3O5SBr2 [M + H]+ 704.0424, found 704.0466.
(E)-3-(3-Bromo-5-methoxy-4-(2-morpholinoethoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
(
18a). Intermediate 9a was treated with compound
desired product 18a. Yield: 65.2%; mp.: 238–242 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
s, 1H), 7.58–7.64 (overlap, 2H), 7.50 (s, 1H), 7.43 (s, 1H), 7.40 (d, 1H, J = 9.0 Hz), 7.32 (d, 2H, J = 8.5 Hz),
7.05 (d, 1H, J = 9.0 Hz), 6.95 (d, 2H, J = 8.5 Hz), 4.12 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 3.51 (m, 4H), 2.67
(t, 2H, J = 6.0 Hz), 2.46 (m, 4H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
168.9, 153.6, 147.6, 146.8, 144.5,
3
following the general procedure to give the
δ
11.08
(
δ
137.8, 132.4(2C), 132.1, 131.1, 129.6, 126.6, 125.6, 122.3(2C), 121.3, 119.1, 117.8, 116.8, 113.2, 110.9, 70.6,
66.6(2C), 58.2, 56.6, 53.9(2C); ESIMS: m/z 692 [M + H]+ HRESIMS: calc. for C28H27N3O6SBr2 [M + H]+
692.0060, found 692.0072.

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(E)-3-(3-Bromo-5-methoxy-4-(3-morpholinopropoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
18b). Intermediate 9b was treated with compound following the general procedure to give the
desired product 18b. Yield: 55.3%; mp.: 125–130 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
11.08
(
3
δ
(s, 1H), 7.58–7.64 (overlap, 2H), 7.49 (s, 1H), 7.39–7.42 (overlap, 2H), 7.31 (d, 2H, J = 8.5 Hz), 7.05 (d, 1H,
J = 9.0 Hz), 6.94 (d, 2H, J = 8.5 Hz), 4.09 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 3.55 (m, 4H), 2.48 (m, 2H),
2.36 (m, 4H)), 1.89 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.7, 153.7, 147.6, 146.8, 144.5,
137.8, 132.4(2C), 132.0, 131.1, 129.6, 126.6, 125.5, 122.3(2C), 121.3, 119.1, 117.8, 116.9, 113.4, 110.8, 71.9,
66.7(2C), 56.6, 55.2, 53.8(2C), 27.3; ESIMS: m/z 706 [M + H]+ HRESIMS: calc. for C29H29N3O6SBr2
[M + H]+ 706.0217, found 706.0248.
(E)-3-(3-Bromo-5-methoxy-4-(4-morpholinobutoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide
(
18c). Intermediate 9c was treated with compound
3
following the general procedure to give the
11.10
desired product 18c. Yield: 67.7%; mp.: 170–175 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
(s, 1H), 7.59–7.64 (overlap, 2H), 7.49 (s, 1H), 7.38–7.42 (overlap, 2H), 7.31 (d, 2H, J = 8.5 Hz), 7.06 (d, 1H,
J = 9.0 Hz), 6.94 (d, 2H, J = 8.5 Hz), 4.06 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 3.53 (m, 4H), 2.26–2.32 (overlap,
6H), 1.74 (m, 2H), 1.60 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 153.7, 147.6, 146.8, 144.4,
138.0, 132.4(2C), 132.4, 131.2, 129.6, 126.6, 125.5, 122.3(2C), 121.3, 119.1, 117.8, 116.9, 113.4, 110.8, 73.4,
66.7(2C), 58.3, 56.6, 53.8(2C), 28.1, 22.9; ESIMS: m/z 720 [M + H]+ HRESIMS: calc. for C30H31N3O6SBr2
[M + H]+ 720.0373, found 720.0396.
(E)-3-(4-(2-([1,40-Bipiperidin]-10-yl)ethoxy)-3-bromo-5-methoxybenzylidene)-N-(4-bromophenyl)-2-oxoindoline-
5-sulfonamide (19a). Intermediate 10a was treated with compound
3 following the general procedure to
give the desired product 19a. Yield:59.1%; mp.: 202–207 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ 11.08 (s, 1H), 7.65–7.67 (overlap, 3H), 7.39 (d, 1H, J = 9.0 Hz), 7.38 (s, 1H), 7.27 (d, 2H, J = 8.5 Hz), 6.99
(d, 1H, J = 9.0 Hz), 6.93 (d, 2H, J = 8.5 Hz), 4.22 (t, 2H, J = 6.0 Hz), 3.92 (s, 3H), 2.90 (t, 2H, J = 6.0 Hz),
2.44–2.48 (overlap, 9H), 1.54 (m, 8H), 1.30 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 169.7,
153.5, 146.9, 146.2, 137.4, 136.8, 132.3, 131.8(2C), 130.9, 129.0, 126.4, 126.2, 122.6(2C), 121.4, 117.7, 117.3,
112.2, 110.9, 110.2, 69.9, 63.4, 56.9, 55.7, 51.9(2C), 49.9(2C), 29.6(2C), 26.0(2C), 23.5; ESIMS: m/z 773
[M + H]+ HRESIMS: calc. for C34H38N4O5SBr2 [M + H]+ 773.1002, found 773.1114.
(E)-3-(4-(3-([1,40-Bipiperidin]-10-yl)propoxy)-3-bromo-5-methoxybenzylidene)-N-(4-bromophenyl)-2-oxoindoline-
5-sulfonamide (19b). Intermediate 10b was treated with compound
3 following the general procedure to
give the desired product 19b. Yield: 67.5%; mp.: 160–165 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ 11.11 (s, 1H), 7.65–7.67 (overlap, 3H), 7.43 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.30 (d, 2H, J = 8.5 Hz), 7.07
(d, 1H, J = 9.0 Hz), 7.00 (d, 2H, J = 8.5 Hz), 4.20 (t, 2H, J = 6.0 Hz), 3.88 (s, 3H), 2.99 (t, 2H, J = 6.0 Hz),
2.48–2.53 (overlap, 6H), 2.25 (m, 1H), 1.89–1.97 (overlap, 4H), 1.75 (m, 2H), 1.55 (m, 6H), 1.38 (m, 2H);
13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 153.7, 146.9, 146.7, 137.7, 137.2, 132.4, 132.3(2C), 131.2,
129.6, 126.4, 125.4, 122.3(2C), 121.3, 117.7, 116.9, 113.4, 110.8, 110.2, 71.9, 62.5, 56.6, 54.4, 52.5(2C),
49.7(2C), 27.6, 26.7(2C), 24.6(2C), 23.4; ESIMS: m/z 787 [M + H]+ HRESIMS: calc. for C35H40N4O5SBr2
[M + H]+ 787.1159, found 787.1232.
(E)-3-(3-Bromo-4-(2-(4-(diethylamino)piperidin-1-yl)ethoxy)-5-methoxybenzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (20a). Intermediate 11a was treated with compound 3 following the general
◦
1
procedure to give the desired product 20a. Yield: 64.2%; mp.: 148–152 C; H-NMR (DMSO-d6,
500 MHz, ppm): 11.10 (s, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.58 (s, 1H), 7.28 (d, 1H, J = 9.0 Hz), 7.24
δ
(s, 1H), 7.20 (d, 2H, J = 8.5 Hz), 7.03 (d, 1H, J = 9.0 Hz), 6.90 (d, 2H, J = 8.5 Hz), 4.21 (t, 2H, J = 6.0 Hz),
3.89 (s, 3H), 3.16 (d, 2H, J = 11.5 Hz), 2.82 (t, 2H, J = 6.0 Hz), 2.62–2.69 (overlap, 5H), 2.10 (t, 2H, J = 6.0
Hz), 1.80 (d, 2H, J = 11.5 Hz), 1.57 (m, 2H), 1.06 (t, 6H, J = 7.0 Hz); 13C-NMR (DMSO-d6, 125 MHz,
ppm):
δ 169.7, 153.5, 146.8, 146.1, 138.0, 137.4, 133.0, 131.7(2C), 130.9, 129.1, 126.3, 125.5, 122.6(2C),
121.4, 117.7, 116.5, 112.2, 110.9, 110.1, 70.1, 57.8, 57.4, 55.6, 53.0(2C), 43.3(2C), 27.0(2C), 11.2(2C); ESIMS:
m/z 761 [M + H]+ HRESIMS: calc. for C33H38N4O5SBr2 [M + H]+ 761.1002, found 761.1140.

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(E)-3-(3-Bromo-4-(3-(4-(diethylamino)piperidin-1-yl)propoxy)-5-methoxybenzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (20b). Intermediate 11b was treated with compound
procedure to give the desired product 20b. Yield: 61.3%; mp.: 135–142 C; H-NMR (DMSO-d6,
3
following the general
◦
1
500 MHz, ppm):
δ 11.12 (s, 1H), 7.63–7.60 (overlap, 3H), 7.42 (s, 1H), 7.38 (d, 1H, J = 9.0 Hz), 7.29 (d,
2H, J = 8.5 Hz), 7.05 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H, J = 8.5 Hz), 4.07 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 2.94
(d, 2H, J = 11.0 Hz), 2.48–2.60 (overlap, 7H), 1.82–1.98 (m, 4H), 1.73 (d, 2H, J = 11.0 Hz), 1.50 (m, 2H),
1.00 (t, 6H, J = 7.0 Hz); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 153.5, 147.0, 146.8, 144.4, 138.0,
137.2, 132.2(2C), 131.1, 129.6, 126.6, 125.5, 122.3(2C), 121.3, 117.7, 116.7, 113.4, 110.8, 110.2, 71.9, 58.4,
56.6, 54.6, 52.8(2C), 43.6(2C), 27.7(2C), 26.8, 12.6(2C); ESIMS: m/z 773 [M + H]+ HRESIMS: calc. for
C34H40N4O5SBr2 [M + H]+ 773.1013, found 773.0970.
(E)-3-(3-Bromo-5-methoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-
5-sulfonamide (21a). Intermediate 12a was treated with compound
3 following the general procedure to
give the desired product 21a. Yield: 58.6%; mp.: 123–127 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
11.08 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.30 (d, 2H,
J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.95 (d, 2H, J = 8.5 Hz), 4.12 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 2.71
(t, 2H, J = 6.0 Hz), 2.48 (m, 4H), 2.33 (m, 4H), 2.11 (s, 3H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ
168.8, 153.6, 147.7, 146.8, 144.4, 138.0, 132.3(2C), 132.0, 131.2, 129.6, 126.6, 125.6, 122.3(2C), 121.3, 119.1,
117.8, 116.8, 113.3, 110.8, 70.0, 57.8(2C), 56.5, 55.1(2C), 53.2, 46.1; ESIMS: m/z 705 [M + H]+ HRESIMS:
calc. for C29H30N4O5SBr2 [M + H]+ 705.0376, found 705.0399.
(E)-3-(3-Bromo-5-methoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-
5-sulfonamide (21b). Intermediate 12b was treated with compound
3 following the general procedure to
give the desired product 21b. Yield: 64.2%; mp.: 120–125 ◦C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
11.08 (s, 1H), 7.63 (s, 1H), 7.60 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.30 (d, 2H,
J = 8.5 Hz), 7.05 (d, 1H, J = 9.0 Hz), 6.95 (d, 2H, J = 8.5 Hz), 4.09 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 2.23–2.50
(overlap, 10H), 2.12 (s, 3H), 1.83 (m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 153.6, 147.6,
146.8, 144.5, 137.9, 132.4(2C), 132.2, 131.2, 129.6, 126.4, 125.5, 122.2(2C), 121.3, 119.1, 117.8, 116.8, 113.4,
110.8, 71.9, 56.6, 55.2(2C), 54.7, 53.1(2C), 46.1, 27.6; ESIMS: m/z 719 [M + H]+ HRESIMS: calc. for
C30H32N4O5SBr2 [M + H]+ 719.0533, found 719.0579.
(E)-3-(3-Bromo-5-methoxy-4-(4-(4-methylpiperazin-1-yl)butoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline
-5-sulfonamide (21c). Intermediate 12c was treated with compound
3
following the general procedure
◦
to give the desired product 21c. Yield: 58.7%; mp.: 135–140 C; 1H-NMR (DMSO-d6, 500 MHz, ppm):
δ
11.09 (s, 1H), 7.82 (s, 1H), 7.60 (s, 1H), 7.48 (s, 1H), 7.42 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H,
J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H, J = 8.5 Hz), 4.05 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 2.33–2.48
(overlap, 10H), 2.21 (s, 3H), 1.59–1.79 (m, 4H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 169.2, 153.6,
148.4, 146.4, 143.5, 139.6, 137.8, 132.4(2C), 131.9, 128.2, 126.4, 125.5, 122.2(2C), 120.8, 118.0, 116.5, 116.2,
115.6, 110.5, 73.5, 57.5, 56.3, 54.7(2C), 52.5(2C), 45.6, 28.0, 26.7; ESIMS: m/z 733 [M + H]+ HRESIMS: calc.
for C31H34N4O5SBr2 [M + H]+ 733.0689, found 733.0711.
(E)-3-(3-Bromo-5-methoxy-4-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethoxy)benzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (22a). Intermediate 13a was treated with compound
procedure to give the desired product 22a. Yield: 61.2%; mp.: 184–190 C; H-NMR (DMSO-d6,
3
following the general
◦
1
500 MHz, ppm):
δ 11.08 (s, 1H), 8.31 (d, 2H, J = 4.5 Hz), 7.64 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 7.43 (s,
1H), 7.39 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H, J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H, J = 8.5 Hz), 6.58
(t, 1H, J = 4.5 Hz), 4.18 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 3.66 (t, 4H, J = 5.5 Hz), 2.73 (t, 2H, J = 5.5 Hz), 2.50
(t, 4H, J = 4.5 Hz); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 161.6, 158.3(2C), 153.6, 147.6, 146.7,
144.5, 137.6, 132.4(2C), 132.1, 131.2, 129.6, 126.6, 125.6, 122.3(2C), 121.4, 119.1, 117.8, 116.8, 113.4, 110.5,
110.2, 70.9, 57.8, 56.6, 53.1(2C), 43.8(2C); ESIMS: m/z 769 [M + H]+ HRESIMS: calc. for C32H30N6O5SBr2
[M + H]+ 769.0438, found 769.0447.

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(E)-3-(3-Bromo-5-methoxy-4-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (22b). Intermediate 13b was treated with compound
procedure to give the desired product 22b. Yield: 66.5%; mp.: 123–127 C; H-NMR (DMSO-d6,
3
following the general
◦
1
500 MHz, ppm):
δ 11.06 (s, 1H), 8.32 (d, 2H, J = 4.5 Hz), 7.64 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 7.43 (s,
1H), 7.39 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H, J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H, J = 8.5 Hz), 6.58
(t, 1H, J = 4.5 Hz), 4.13 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 3.69 (t, 4H, J = 5.5 Hz), 2.55 (t, 2H, J = 5.5 Hz),
2.42 (t, 4H, J = 4.5 Hz), 1.93 (2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 161.6, 158.3(2C), 153.6,
147.6, 146.7, 144.4, 137.7, 132.4(2C), 132.0, 131.2, 129.6, 126.6, 125.5, 122.3(2C), 121.3, 119.1, 117.8, 116.8,
113.4, 110.5, 110.3, 71.9, 56.6, 54.9, 53.0(2C), 43.8(2C), 27.6; ESIMS: m/z 783 [M + H]+ HRESIMS: calc. for
C33H32N6O5SBr2 [M + H]+ 783.0594 found 783.0601.
(E)-3-(3-Bromo-5-methoxy-4-(2-(4-(pyrazin-2-yl)piperazin-1-yl)ethoxy)benzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (23a). Intermediate 14a was treated with compound
procedure to give the desired product 23a. Yield: 56.2%; mp.: 143–151 C; H-NMR (DMSO-d6,
3
following the general
◦
1
500 MHz, ppm):
δ 11.07 (s, 1H), 8.27 (s, 1H), 8.03 (s, 1H), 7.79 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.50
(s, 1H), 7.43 (s, 1H), 7.38 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H, J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96 (d,
2H, J = 8.5 Hz), 4.18 (t, 2H, J = 6.0 Hz), 3.88 (s, 3H), 3.52 (t, 4H, J = 5.5 Hz), 2.78 (t, 2H, J = 5.5 Hz), 2.60
(t, 4H, J = 4.5 Hz);13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 155.0, 153.6, 147.5, 146.8, 144.5, 141.9,
137.6, 132.8, 132.4(2C), 132.1, 131.7, 131.2, 129.6, 126.6, 125.6, 122.3(2C), 121.4, 119.1, 117.8, 116.8, 113.4,
110.2, 70.9, 57.8, 56.6, 52.9(2C), 44.5(2C); ESIMS: m/z 769 [M + H]+ HRESIMS: calc. for C32H30N6O5SBr2
[M + H]+ 769.0438, found 769.0457.
(E)-3-(3-Bromo-5-methoxy-4-(3-(4-(pyrazin-2-yl)piperazin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (23b). Intermediate 14b was treated with compound
procedure to give the desired product 23b. Yield: 64.1%; mp.: 124–130 C; H-NMR (DMSO-d6,
3
following the general
◦
1
500 MHz, ppm):
δ 11.08 (s, 1H), 8.27 (s, 1H), 8.04 (s, 1H), 7.80 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H), 7.50 (s,
1H), 7.43 (s, 1H), 7.39 (d, 1H, J = 9.0 Hz), 7.31 (d, 2H, J = 8.5 Hz), 7.06 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H,
J = 8.5 Hz), 4.11 (t, 2H, J = 6.0 Hz), 3.87 (s, 3H), 3.52 (t, 4H, J = 5.5 Hz), 2.45-2.56 (overlap, 6H), 1.93
(m, 2H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
δ 168.9, 155.0, 153.7, 147.5, 146.8, 144.5, 141.9, 137.6,
132.8, 132.4(2C), 132.3, 131.7, 131.2, 129.6, 126.6, 125.3, 122.3(2C), 121.3, 119.1, 117.8, 116.5, 113.4, 110.8,
71.9, 56.6, 54.8, 52.8(2C), 44.5(2C), 27.6; ESIMS: m/z 783 [M + H]+ HRESIMS: calc. for C33H32N6O5SBr2
[M + H]+ 783.0594 found 783.0634.
(E)-3-(4-(2-(Bis(2-hydroxyethyl)amino)ethoxy)-3-bromo-5-methoxybenzylidene)-N-(4-bromophenyl)-
2-oxoindoline-5-sulfonamide (24). Intermediate 15 was treated with compoun◦d
3
following the general
1
procedure to give the desired product 24. Yield: 53.4%; mp.: 135–140 C; H-NMR (DMSO-d6,
500 MHz, ppm):
7.28 (d, 2H, J = 8.5 Hz), 7.03 (d, 1H, J = 8.5 Hz), 6.92 (d, 2H, J = 8.5 Hz), 4.08 (t, 2H, J = 5.0 Hz), 3.88 (s,
3H), 3.44 (m, 4H), 2.67 (t, 2H, J = 5.0 Hz), 2.48 (m, 4H); 13C-NMR (DMSO-d6, 125 MHz, ppm):
168.9,
δ 11.08 (s, 1H), 7.58–7.63 (overlap, 2H), 7.50 (s, 1H), 7.44 (s, 1H), 7.37 (d, 1H, J = 8.5 Hz),
δ
153.5, 147.6, 146.8, 144.3, 138.1, 132.3(2C), 132.2, 131.2, 129.6, 126.7, 125.5, 122.4(2C), 121.3, 119.1, 117.7,
116.8, 113.4, 110.7, 71.9, 59.8(2C), 57.5(2C), 56.6, 54.8; ESIMS: m/z 710 [M + H]+ HRESIMS: calc. for
C28H29N3O7SBr2 [M + H]+ 710.0166, found 710.0222.
(E)-3-(3-Bromo-4-(2-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)ethoxy)-5-methoxybenzylidene)-N-
(4-bromophenyl)-2-oxoindoline-5-sulfonamide (25). Intermediate 16 was treated with compou◦nd
3
following the general procedure to give the desired product 25. Yield: 50.5%; mp.: 125–130 C;
1H-NMR (DMSO-d6, 500 MHz, ppm):
δ 11.08 (s, 1H), 7.63 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 7.42 (s,
1H), 7.39 (d, 1H, J = 9.0 Hz), 7.30 (d, 2H, J = 8.5 Hz), 7.05 (d, 1H, J = 9.0 Hz), 6.95 (d, 2H, J = 8.5 Hz),
4.14 (t, 2H, J = 6.0 Hz), 3.86 (s, 3H), 2.70 (t, 2H, J = 6.0 Hz), 2.48 (m, 4H), 2.34 (m, 8H), 2.14 (s, 6H);
13C-NMR (DMSO-d6, 125 MHz, ppm):
δ
168.9, 153.6, 147.6, 146.6, 144.4, 138.0, 132.4(2C), 132.0, 131.2,
129.7, 126.5, 125.2, 122.3(2C), 121.3, 119.1, 117.8, 116.8, 113.3, 110.8, 70.9, 57.9(2C), 56.6, 56.5, 56.0, 53.5,

Mar. Drugs 2017, 15, 343
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53.4(2C), 45.7(2C); ESIMS: m/z 762 [M + H]+ HRESIMS: calc. for C32H37N5O5SBr2 [M + H]+ 762.0955,
found 762.1093.
3.2. Biological Activity
3.2.1. Cell Culture
A549 (human lung cancer cell line), HepG2 (human hepatocellular carcinoma cell line), Bel7402
(human hepatocellular carcinoma cell line), HCT116 (human colorectal cancer cell line), Caco2
(Human colonic epithelial cell line) were obtained from Shanghai Institute of Cell Biology and were
cultured in RPMI 1640 or DMEM medium (Gibco, Big Cabin, OK, USA), supplemented with 10% fetal
◦
bovine serum (Hyclone, Logan, UT, USA), in 25 cm2 culture flasks at 37 C in a humidified atmosphere
with 5% CO2. Sunitinib was used as a reference compound.
3.2.2. MTT Assay
The cell viability was assessed by MTT assay as described previously [
7
◦ ]. Briefly, the cells were
plated at a density of 3
×
103 cells/well in 96-well plates and incubated at 37 C overnight before drug
exposure. Cells were incubated with the tested compounds to achieve final concentrations (0, 2.5, 5, 10,
20, 40, 80 g/mL) for 48 h. Twenty microliters of MTT (5 mg/mL) was added to each well and allowed
to react for another 4 h. After removing the supernatant, 150 L of DMSO was added to dissolve
µ
µ
the formazan and the plates were read at 490 nm. All experiments were carried out in triplicate and
the viability of the control cells was taken as 100% cell survival. The IC50 values were analyzed by
GraphPad Prime 5.0.
3.2.3. Colony Forming Assay
A549 cells (500 cells/well) were seeded in six-well plates. After 24 h, cells were treated with
various concentrations of compound 17a (0, 5, 10, and 20 µg/mL) and incubated for 15 days to allow
colony formation. The cells were then washed with PBS, fixed with 4% paraformalclehyde for 10 min.
Next, colonies were stained with 0.1% crystal violet for 15 min at room temperature. The excel crystal
violet solution was washed away with distilled H2O, colonies containing more than 50 cells were
counted and evaluated [23].
3.2.4. Cell Cycle Analysis
The cell cycle was determined by flow cytometry with DNA staining to reveal the total amount
of DNA. Briefly, A549 cells were seeded into six-well plates at a density of 5
×
105 cells per well.
The cells were treated with 5, 10 and 20
washed twice with cold PBS, and fixed in cold 75% ethanol at
were washed twice with cold PBS, re-suspended with cold PBS and stained with 20
(Sigma, St. Louis, MO, USA) for 30 min at 37 ◦C. The cells were washed and re-suspended with cold
PBS containing 50 g/mL propidium iodide (PI, Sigma, St. Louis, MO, USA) for 30 min at room
µ
g/mL compound 17a for 48 h. Then the cells were harvested,
◦
−
20 C overnight. The next day, the cells
µg/mL RnaseA
µ
temperature in the dark. The cell cycle phase distribution was analyzed in three different experiments
using flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
3.2.5. Cell Apoptosis Analysis
A549 cells (2
×
105/well) were plated in six-well plates and incubated for 24 h, then treated
with test compounds with various concentration compound 17a (0, 5, 10, 20 µg/mL) for 48 h. Then
1~5 × 105 cells were harvested, washed twice with cold PBS, re-suspended in 500
µL Annexin V
Binding Buffer, and 5 µL Annexin V-FITC and 5 µL PI were added. After being stained in the dark
for 10min at room temperature, the cells were analyzed in three different experiments using flow
cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Mar. Drugs 2017, 15, 343
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3.2.6. Apoptosis Assessment by Hoechst 33258 Staining
Cell morphology was evaluated by fluorescence microscopy following Hoechst 33258 staining [
A549 cells were cultured in six-well plates, containing 10% fetal bovine serum in the absence or presence
of compound 17a (0, 5, 10, 20 g/mL). After 48 h incubation, the plates were washed twice with PBS
9].
µ
and stained with Hoechst 33258 (Sigma, St. Louis, MO, USA) for 15 min in the dark. After being
washed twice with PBS, the cells were visualized under a fluorescence microscope.
3.2.7. Reactive Oxygen Species (ROS) Levels Assay
The production of ROS was monitored by flow cytometry using 20,70-dichlorohydrofluorescein
(DCFH-DA) (Beyotime Biotechnolog, Shanghai, China). Briefly, A549 cells were plated in six-well
plates and incubated for 24 h; then, cells were treated with compound 17a at various concentrations
(0, 5, 10, and 20 µg/mL) for 48 h. Ten micromolar DCFH-DA was added to the plates and incubated
for 30 min. After incubation, cells were washed twice with ice cold PBS and harvested. Cells were
analyzed by flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and fluorescence microscope
(Olympus, Tokyo, Japan).
3.2.8. Western Blot Analysis
A549 cells were treated with compound 17a for various times. Then, cells were harvested in
lysis buffer RIPA for whole-cell lysates, and then incubated on ice for 15 min. The homogenates
were centrifuged at 12,000 rpm for 15 min, and the supernatant was used for Western blot analysis.
The protein concentration of the supernatant was determined by BCA (Beyotime Biotechnolog,
Nantong, China) assay. Supernatants were mixed with SDS–PAGE sample buffer and boiled for 5 min.
Samples were subsequently loaded into each lane of a 10% SDS–PAGE and transferred onto PVDF
membranes (Bio-Rad, Philadelphia, PA, USA). Membranes were blocked for 1 h in 5% (w/v) non-fat milk
and then incubated with a primary antibody at room temperature for 1 h. The following antibodies
were used: Bcl-2 (Cell Signaling), Bax (Cell Signaling), PARP (Cell Signaling), caspase-3 (Cell Signaling),
cyclin D1 (Cell Signaling), CDK4 (Cell Signaling), β-actin (Sangon Biotech). Having been washed
3 times in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbit
IgG (diluted 1:10,000) for 1 h at room temperature, and then washed 3 times. Finally, proteins were
detected using an enhanced chemiluminescence system BeyoECL Plus (Beyotime). The load protein
was normalized to β-actin. All experiments were performed in triplicate.
4. Conclusions
In summary, our design and synthesis have led to a series of novel anticancer agents by attaching
of N-containing heterocyclic moieties to bromophenol. Among them, thirteen compounds (17a
, 17b,
18a 19a 19b 20a 20b 21a 21b 22a 22b 23a, and 23b) exhibited significant inhibitory activity against
,
,
,
,
,
,
,
,
,
the tested cancer cell lines. The SARs of bromophenol derivatives were discussed: the chain length and
types of the moieties of incorporation could affect the anticancer activities. The promising candidate
compound 17a could induce cell cycle arrest at G0/G1 phase and induce apoptosis in A549 cells,
as well as caused DNA fragmentations, morphological changes and ROS generation. Furthermore,
compound 17a suppression of Bcl-2 levels in A549 cells induced cells apoptosis in vitro through the
ROS-mediated apoptotic pathway. These results might be useful for bromophenol derivatives to be
explored and developed as novel anticancer drugs. In the future, we will further investigate that
compound 17a was developed as anticancer agents by more experiments.
Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/15/11/343/s1,
Details of synthesis of intemediates 5–7, HPLC Characterization of purity for the promising candidate
compound 17a.
Acknowledgments: This work was supported by the National Natural Science Foundation of China
(Nos. 81773586 and 81703354); Key research and development project of Shandong province (2016GSF201193,

Mar. Drugs 2017, 15, 343
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2016ZDJS07A13, 2016GSF115002, and 2016GSF115009); Key Research Program of Frontier Sciences, CAS
(QYZDB-SSW-DQC014); the Project of Discovery, Evaluation and Transformation of Active Natural Compounds,
Strategic Biological Resources Service Network Programme of Chinese Academy of Sciences (ZSTH-026),
and NSFC-Shandong Joint Fund for Marine Science Rearch Centers (U1606403); the Scientific and Technological
Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology
(No. 2015ASKJ02); Aoshan Talents Program Supported by Qingdao National Laboratory for Marine Science and
Technology (NO. 2015ASTP); and National Program for Support of Top-notch Young Professionals, and Taishan
scholar Youth Project of Shandong province.
Author Contributions: Li-Jun Wang, Chuan-Long Guo, and Da-Yong Shi contributed to the study concept and
design, and the manuscript preparation. Li-Jun Wang and Chuan-Long Guo performed the experimental studies
and analyzed the data. Xiang-Qian Li, Shuai-Yu Wang, Bo Jiang, Yue Zhao, Jiao Luo, Kuo Xu, Shu-Ju Guo,
and Ning Wu 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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