Antagonizing STAT3 activation with benzo[b]thiophene 1, 1-dioxide based small molecules

Article abstract of DOI:10.1016/j.ejmech.2016.09.068

STAT3 is an attractive therapeutic target for cancer therapy. However, due to low potency or poor druggability, none of its inhibitors are clinically available. Herein, a series of aminobenzo[b]thiophene 1, 1-dioxides with good drug-likeness properties were designed, synthesized and evaluated as STAT3 inhibitors. Most of them exhibited higher antitumor activity than the small-molecule STAT3 inhibitor, Stattic. Compound 15 was the most potent and had an IC₅₀range in 0.33–0.75?μM in various cancer cell lines. The overexpressed and IL-6 induced phosphorylation levels of STAT3 were both inhibited by 15 without influencing the phosphorylation levels of the upstream kinases Src and Jak2. 15 also suppressed the expressions of STAT3 downstream gene, Bcl-2. 15 effectively increased the ROS levels of cancer cells, induced cancer cell apoptosis and abolished the colony formation ability of cancer cells without affecting bypass kinase p-Erk. Furthermore, 15 in?vivo induced significant antitumor responses, and exhibited less toxicity than Doxorubicin. Together, this study described a class of new STAT3 inhibitors as antitumor agents.

Full text of DOI:10.1016/j.ejmech.2016.09.068

Accepted Manuscript
Antagonizing STAT3 activation with benzo[b]thiophene 1, 1-dioxide based small
molecules
Wenda Zhang, Ting Ma, Shanshan Li, Yanwei Yang, Jianpeng Guo, Wenying Yu,
Lingyi Kong
PII:
S0223-5234(16)30804-2
10.1016/j.ejmech.2016.09.068
EJMECH 8934
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2016
Revised Date: 20 September 2016
Accepted Date: 21 September 2016
Please cite this article as: W. Zhang, T. Ma, S. Li, Y. Yang, J. Guo, W. Yu, L. Kong, Antagonizing STAT3
activation with benzo[b]thiophene 1, 1-dioxide based small molecules, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.068.
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ACCEPTED MANUSCRIPT
Compound 15 selectively inhibited STAT3 activation to exert antitumor activity.

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Antagonizing STAT3 activation with benzo[b]thiophene 1, 1-dioxide
based small molecules
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Wenda Zhang, Ting Ma, Shanshan Li, Yanwei Yang, Jianpeng Guo, Wenying Yu*
and Lingyi Kong*
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State Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’ s Republic of China.
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*Corresponding Authors. Tel/Fax: +86-25-83271405; E-mail: cpu_lykong@126.com
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(Lingyi Kong); ywy@cpu.edu.cn (Wenying Yu).
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Abstract
STAT3 is an attractive therapeutic target for cancer therapy. However, due to low
potency or poor druggability, none of its inhibitors are clinically available. Herein, a
series of aminobenzo[b]thiophene 1, 1-dioxides with good drug-likeness properties
were designed, synthesized and evaluated as STAT3 inhibitors. Most of them
exhibited higher antitumor activity than the small-molecule STAT3 inhibitor, Stattic.
Compound 15 was the most potent and had an IC50 range in 0.33-0.75 ꢀM in various
cancer cell lines. The overexpressed and IL-6 induced phosphorylation levels of
STAT3 were both inhibited by 15 without influencing the phosphorylation levels of
the upstream kinases Src and Jak2. 15 also suppressed the expressions of STAT3
downstream gene, Bcl-2. 15 effectively increased the ROS levels of cancer cells,
induced cancer cell apoptosis and abolished the colony formation ability of cancer
cells without affecting bypass kinase p-Erk. Furthermore, 15 in vivo induced
significant antitumor responses, and exhibited less toxicity than Doxorubicin.
Together, this study described a class of new STAT3 inhibitors as antitumor agents.
Keywords: Antitumor agents, Aminobenzo[b]thiophene 1, 1-dioxides, Drug-likeness
properties, STAT3 phosphorylation, Apoptosis.

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1. Introduction
Signal transducer and activator of transcription 3 (STAT3) is a member of STAT
protein family, which includes STAT1, 2, 3, 4, 5a, 5b and 6. STAT3 is closely
associated with the occurrence of cancers [1]. Especially, aberrantly activated STAT3
has been found in numerous cancers such as breast, prostate, lung, ovarian, leukemia
and lymphoma cancers [2-6]. STAT3 is widely regarded as a master regulator of the
cellular events which is correlated with cell proliferation, differentiation, apoptosis,
angiogenesis and immune response [7-12].
STAT3 can be phosphorylated at Tyr705 and then be activated which is mediated
by the cytoplasmic kinases such as Janus kinases (Jaks) and Src family kinases [11,
13]. Two phosphorylated STAT3 proteins form homo-dimeric activated transcription
factor complex via reciprocal binding of pTyr-SH2 domains. The p-STAT3 dimers
translocate to the nucleus, where they bind to DNA and induce target gene expression
[5, 11-14]. More evidences have shown that the constitutive activation of STAT3
plays an essential role in cancer cell growth and survival and inhibiting the activation
of STAT3 may be a novel and powerful weapon in the cancer therapies [15-19].
Over the past decade, several strategies have been explored to directly or indirectly
target and inhibit STAT3, which are: (a) the direct inhibitors targeting the SH2 domain
or dimerization of STAT3 (such as LY5 [5], STA-21 [20], LLL12 [21-23],
Cryptotanshinone [24], Stattic [25], Niclosamide [26-27], HJC0146 [28], BP-1-102
[29]), the inhibitors targeting the DNA binding domain (such as S3-54A18) [30], and
the inhibitors targeting the N-terminal domain (a category of short peptides) [31, 32];
and (b) the indirect inhibitors targeting the upstream kinases or factors on the STAT3
pathway (such as resveratrol [33], curcumin [34, 35]). However, there’s still no
STAT3 inhibitor available in the market. The challenges include the lack of membrane
permeability and stability, low water-solubility, weak binding affinity, or low
specificity. Therefore, discovery of novel potent and druggable STAT3 inhibitors is
still highly valuable.
The benzo[b]thiophene 1, 1-dioxide （BTP） moiety is a crucial pharmacophore of
many STAT3 inhibitors (such as Stattic, HJC0123 and HJC0146), which not only

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significantly inhibited STAT3 phosphorylation but also markedly exhibited
anti-proliferative activity [20, 22, 28, 36]. Besides, compounds with BTP moiety
could increase ROS levels in cancer cells, which further induced cancer cell apoptosis.
What’s more, its low molecular weight is more feasible for further optimization. In
view of this, we were promoted to use this fragment as a starting point for the design
of novel antitumor STAT3 inhibitors.
Using structure-based drug design methods, we discovered 6 or 7-substituted
aminobenzo[b]thiophene 1, 1-dioxides as a new series of STAT3 inhibitors with
potent antitumor activity. These new inhibitors are promising candidates for further
drug development, due to their drug-like properties and potent bioactivity in vitro and
in vivo.
2. Results and discussion
2.1. Design
Initially, BTP was docked into STAT3 protein (PDB: 1BG1) and the binding mode
was carefully analyzed by AutoDock4.2 software. As shown in Figure 1, this planner
scaffold only occupied in the essential pTyr705 binding site by forming three
hydrogen bonds with residues Arg609, Ser611 and Ser613. In order to increase the
binding affinity of the designed compounds, we were promoted to introduce
fragments at the C6 or C7 position of BTP and to develop BTP derivatives binding to
two “hot spots” of STAT3 SH2 domain (pTyr705 binding site and side pocket) (Figure
2) [5]. Aiming to target the side pocket of STAT3, according to docking the poses of
known inhibitors, substituted aromatic benzene rings were introduced to fit the side
pocket. To search for suitable linkers, benzene ring was initially introduced to the
BTP fragment. Linkers were chosen from the known inhibitors. Then, the merged
potential candidates were quickly screened via computational docking for the final
decision of the most suitable linker (Figure 3). The criteria are as follows: (1) the
linker should maintain the binding of BTP to the pTyr705 site and ensure the new
introduced fragments fitting in the side pocket as well; (2) the linker should be as
simple and small-size as possible according to the theory of ligand efficiency, the
smaller the linker is, the higher the LE is; (3) the linker should be feasible for

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synthesis. Finally, a secondary amine was chosen as the linker to evolve new STAT3
inhibitors.
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2.2. Chemistry
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The synthetic routes for compounds 6-29 were shown in Scheme 1. 2a or 2b was
obtained by the reaction of commercially available 3-bromo-2-fluorobenzaldehyde
(1a) or 4-bromo-2-fluorobenzaldehyde (1b) and methyl thioglycolate catalyzed by
potassium carbonate. Compound 2a or 2b was hydrolyzed in 10% potassium
hydroxide solution and acidified by concentrated hydrochloric acid to produce 3a or
3b. Compound 4a or 4b was obtained by the decarboxylation of 3a or 3b, which was
then reacted with 3-chlorine peroxide benzoic acid for the synthesis of 5a or 5b.
Compounds 6-29 were obtained by Buchwald--Hartwig C−N coupling reaction of 5a
or 5b using different anilines. All of the BTP derivatives were confirmed by 1H NMR,
IR, HRMS (ESI) spectra, and 13C NMR spectrum.
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2.3 Biological Evaluation
2.3.1 In vitro cell growth inhibitory activity.
To summarize the structure-activity relationships of the synthetic compounds, four
STAT3 over-activated human cancer cell lines, including lung carcinoma A549, breast
carcinoma MDA-MB-231 and MCF-7 and colon carcinoma HCT116, were treated
with designed compounds and the positive control, Stattic, by MTT assay. The IC50
values were summarized in Table 1. Notably, most of the compounds exhibited better
activity than Stattic. The IC50 values were within 0.26 to 4.40 ꢀM. Among them, the
most potent compound 15 inhibited the growth of MDA-MB-231, A549, MCF-7, and
HCT116 cells with IC50 values of 0.68, 0.36, 0.75, and 0.33 ꢀM, which had 1.5- to
12-fold improvement in activity than Stattic, respectively. Compound 19 possessing a
4’-Cl was 1.4- to 7.2-fold more potent than the reference Stattic in four tested cell
lines. Comparison of the two series of compounds, generally, 7-substituted
aminobenzo[b]thiophene 1, 1-dioxides were more potent than 6-substituted
compounds. The substitution of aniline groups significantly contributed to the
antitumor activity. In general, the compounds with electron-withdrawing group such
as chlorine (11, 19 and 27), fluorine (12 and 26), or trifluoromethyl group (6, 7, 15

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and 18) usually showed better inhibitory activity than those with electron-donating
group such as cyano (8) or methyl groups (20 and 23). Replacement of a hydrogen
atom with methoxy or trifluoromethoxy group at 3’ or 4’ position (9, 14, 16 and 24)
was tolerated but 2’-OCH3 in compound 25 led to a slight decrease in activity. And
replacement with three methoxy groups at 3’, 4’ and 5’ position in compound 13 and
17 increased the activity. Adding a chlorine at C3’, as in compound 27, led to a slight
decrease of anti-proliferative activity as compared to 19 indicating that C3’-Cl was
not favored. Compounds (6, 7, 15, and 18) with trifluoromethyl group showed an
improvement in the anti-proliferative activity, however, the activity of 28 or 29 led to
a slight decrease.
2.3.2 Molecular docking.
To further understand the structure-activity relationship, the potential binding
modes of all compounds with the STAT3 SH2 domain were predicted by docking
experiments. Most compounds could bind to the pTyr705 site and the side pocket well,
except of the 2’ or 3’, 5’-substituted compound 25, 28 and 29 due to the steric
hindrance of the substituents to the side pocket (Figure S1), which reasonably
explained their decreasing bioactivity. As shown in Figure 4, the representative
compound 15 could bind tightly to the STAT3 SH2 domain by forming four hydrogen
bonding interactions with the Arg609, Lys591 and Ser636 residues. In addition, its
3’-trifluoromethylaniline could fit deeply into the side pocket. Furthermore, it also
formed two cation-π interactions with Lys591 and Arg609 (one was formed by the
charged amine of Lys591 residue to the phenyl ring of BTP and the other was formed
by the charged amine of Arg609 residue to C7 position of the other phenyl group)
providing a rationale explaining the best activity of compound 15 among all.
2.3.3 Compound 15 inhibited STAT3 activity in tumor cells.
To evaluate whether these compounds were STAT3 inhibitors, the most potent
compound 15 was selected to study its antitumor mechanism. Firstly, the effect of 15
on the p-STAT3 levels was investigated by western blot analysis. MDA-MB-231 cells
were incubated with 15 (1-5 ꢀM) or Stattic (5 ꢀM) for 24 h and the levels of p-STAT3
and total STAT3 were then examined. It was found that compound 15 markedly

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suppressed STAT3 phosphorylation at Tyr705 residue in a concentration-dependent
manner, but had no effect on the total STAT3 (Figure 5A).
2.3.4 Compound 15 inhibited STAT3 phosphorylation induced by interleukin-6 (IL-6).
The effect of 15 on IL-6 induced STAT3 phosphorylation was also assessed as
reported [37]. MDA-MB-231 cells were starved overnight, followed by treatment
with 15 for 12 h before IL-6 stimulation. As shown in Figure 5B, IL-6 significantly
increased the phosphorylation levels of STAT3, which was decreased by 15 in a
dose-dependent manner. Compound 15 at 5 ꢀM almost completely abolished STAT3
phosphorylation induced by IL-6.
2.3.5 Compound 15 inhibited STAT3 DNA-binding activity.
Given the inhibitory effect of 15 on p-STAT3 in tumor cells, we evaluated the
inhibitory activity against STAT3 DNA-binding activity by electrophoretic mobility
shift assay (EMSA). Nuclear extracts from MDA-MB-231 cells incubated with 15
prior to incubating with hSIE probe and preforming EMSA analysis.
STAT3:STAT3/DNA-binding activity was diminished by 15 at a start concentration at
1 ꢀM and an obvious decrease at 5ꢀM as compared with Stattic at 5ꢀM (Figure 5C).
2.3.6 Typical upstream or other kinases of STAT3 were not inhibited by 15.
The activation of STAT3 phosphorylated at Tyr705 may be mediated by upstream
kinases (such as Jak2, Src) or other kinases (such as Erk1/2). To investigate the
selectivity of 15 targeting p-STAT3, we evaluated the effect of 15 on p-Jak2, p-Src,
p-Erk1/2 by western blot analysis. Data showed that compound 15 had no effect on
p-Jak2, p-Src, and p-Erk1/2 in MDA-MB-231 cells (Figure 6). These results indicated
that the inhibition of p-STAT3 by 15 was selective and independent of the above
kinases.
2.3.7 Compound 15 inhibited the expression of the downstream gene of STAT3, Bcl-2,
and induced the apoptosis of cancer cells.
We had elucidated that compound 15 could effectively inhibit the activation of
STAT3. To further gain more insights into the mechanism of 15 on the STAT3
pathway, we examined its effect on the expressions of STAT3-targeted gene, Bcl-2,
and the levels of Cleaved PARP and Cleaved Caspase-3 which are related to apoptosis.

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MDA-MB-231 cells were treated with compound 15 and processed for western
blotting. As shown in Figure 7, compound 15 inhibited the expression of Bcl-2 and
induced the cleavage of PARP and Caspase-3 in MDA-MB-231 cells.
The effect of compound 15 on inducing tumor cell apoptosis was analyzed.
MDA-MB-231 cells were incubated with 15 at different concentrations for 24 h.
Annexin V-FITC/PI staining was carried out and the percentages of apoptotic cells
were further determined using flow cytometry. The results showed that 15
dose-dependently induced the apoptosis of MDA-MB-231 cells. The induced
apoptosis rates at 0, 1, 3, 5 and 7 ꢀM were 5.6%, 9.7%, 10.9%, 17.5% and 20.9%,
respectively (Figure 8). Together, these data showed that compound 15 induced the
apoptosis of cancer cells through activating the caspase-dependent pathway relating to
Bcl-2, which was regulated by the deactivation of STAT3 protein.
2.3.8 Compound 15 induced the ROS generation in tumor cells.
It was reported that benzo[b]thiophene 1, 1-dioxide derivatives could induce the
generation of reactive oxygen species, which would partially contribute to the cell
apoptosis [38]. Subsequently, we evaluated the effect of 15 on the ROS levels in
tumor cells. MDA-MB-231 cells were incubated with 15 at different concentrations
for 24 h, and then cells were collected and stained by DCFH-DA and subjected to
flow cytometry. Expectedly, the levels of ROS showed a 1.37, 1.74 or 1.94 fold
increase at 1, 3 and 5 ꢀM of 15 (Figure 9).
2.3.9 Compound 15 inhibited tumor cells colony formation.
To further investigate the anti-proliferative activity of compound 15 on cancer cells,
colony survival assay was performed. MDA-MB-231 cells were treated with
compound 15 at 0.5-6 ꢀM for 24 h and cultured for 14 d until the colonies were
visible. Crystal violet solution (Sigma, St. Louis, MO, USA) was used to stain the
colonies for 4 h and imaged. As shown in Figure 10, a significant reduction in
clonogenic ability at 0.5 and 1.0 ꢀM of compound 15 and an almost cessation of
colony formation at 6 ꢀM.
2.3.10 Compound 15 exhibited antitumor activity in vivo.
We tested the in vivo antitumor efficacy of 15 against mouse breast tumor

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xenograft. After the solid tumors were established, the mice were randomized and
treated intraperitoneally with 15 10 mg/kg per day, 1 mg/kg of Doxorubicin every
four days or the same volume of vehicle consisting of PBS/DMSO (8:2) daily for 11
consecutive days. The tumor weights of mice were both significantly reduced 61%
treated with 15 and 67% decrease treated with Doxorubicin (Figure 11A), and the
p-STAT3 levels in the tumor tissues from mice treated with 15 was also inhibited
(Figure 11B). However, 15 exhibited much less side effect than Doxorubicin, since
dramatic loss of mice body weight was observed from the treated by Doxorubicin,
whereas for 15 without any such toxicity (Figure 11C and D).
3. Conclusions
In summary, a series of 6 or 7-substituted aminobenzo[b]thiophene 1, 1-dioxides
were designed, synthesized, and biologically evaluated as STAT3 inhibitors.
According to our screening results, most of the designed compounds exerted higher
antitumor activity than Stattic. SAR analysis indicated that 6 or 7-substituted anilines
of aminobenzo[b]thiophene 1, 1-dioxides were important to the bioactivities. Docking
studies revealed that most compounds could tightly bind to the SH2 domain of STAT3
and further provided a rationale for SAR. Especially, 15 was well fitted in the SH2
domain by four hydrogen bonding interactions with the Arg609, Lys591 and Ser636
residues and two cation-π interactions with Lys591 and Arg609 residues, and its
additional 7-substituted aniline was deeply buried into the side binding pocket. The
most potent compound 15 exhibited remarkable anti-proliferation activity against four
cancer lines with an IC50 range in 0.33-0.75 ꢀM and was selected to investigate the
mechanism of its antitumor activity. It inhibited STAT3 phosphorylation, IL-6 induced
STAT3 phosphorylation, STAT3-DNA binding activity and the expression of STAT3
downstream gene, Bcl-2, to induce tumor apoptosis. The increased levels of ROS by
15 further induced the tumor apoptosis and the cessation of the tumor colony
formation. Furthermore, 15 in vivo induced significant antitumor responses, and
exhibited less toxicity than Doxorubicin. Compared with Stattic, 15 exhibited a
significant increase of bioactivity and better binding affinity, which is worth of further
investigations toward the discovery of novel STAT3 inhibitors as antitumor drugs.

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4. Materials and Methods
4.1 Chemistry
All chemicals (reagent grade) used were purchased from Sinopharm Chemical
Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin
layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Plant, Qingdao, China) plates and the spots were detected under UV light
(254 nm). Melting point was measured on an XT-4 micromelting point instrument and
uncorrected. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer.
1H NMR and 13C NMR spectra were measured on a Bruker ACF-500 spectrometer at
25°C and referenced to TMS. Chemical shifts were reported in ppm (δ) using the
residual solvent line as internal standard. Splitting patterns were designed as s, singlet;
d, doublet; t, triplet; m, multiplet. The purity of all compounds used for biological
evaluation was confirmed to be higher than 95% through analytical HPLC performed
with Agilent 1200 HPLC System. Mass spectra were obtained on a MS Agilent 1100
Series LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner ESI-TOF
spectrometer (HRESI-MS), respectively. Column chromatography was performed on
silica gel (90-150 ꢀm; Qingdao Marine Chemical Inc).
4.1.1. General procedures for the preparation of 2a and 2b
To a stirred solution of compound 1a or 1b（9.90 mmol）in DMF (20 mL) was
added methyl thioglycolate (10.9 mmol) and potassium carbonate (39.6 mmol). The
resulting mixture was stirred at 60 °C for 15 h. The DMF was removed under reduced
pressure, water (50 mL) was added and the mixture was extracted with ethyl acetate
(2 × 40 mL). The combined organic layers were dried with sodium sulfate, filtered,
and the solvents were removed under reduced pressure to afford the title compound
2a or 2b.
methyl 6-bromobenzo[b]thiophene-2-carboxylate (2a)
1
Yield 92%, white solid; H NMR (500 MHz, DMSO) δ 8.39 (s, 1H, Ar-H), 8.20 (s,
1H, Ar-H), 7.96 (d, J = 8.6 Hz, 1H, Ar-H), 7.63 (dd, J = 8.6, 1.7 Hz, 1H, Ar-H), 3.89
(s, 3H, OCH3).
methyl 7-bromobenzo[b]thiophene-2-carboxylate (2b)

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Yield 95%, white solid; 1H NMR (500 MHz, CDCl3) δ 8.14 (s, 1H, Ar-H), 7.83 (d, J =
8.0 Hz, 1H, Ar-H), 7.60 (d, J = 7.6 Hz, 1H, Ar-H), 7.29 (t, J = 7.8 Hz, 1H, Ar-H), 3.96
(s, 3H, OCH3).
4.1.2. General procedures for the preparation of 3a and 3b
The solution of compound 2a or 2b (1.1 mmol) in water (10 mL) was stirred and
then potassium hydroxide pellets (5.4 mmol) was added, which was refluxed for 3
hours. The aqueous layer was then acidified to pH 1 with 1M hydrochloric acid
solution. The aqueous layer was extracted with dichloromethane (3 × 15 mL). The
combined organic layers were dried with sodium sulfate, filtered, and the solvents
were removed under reduced pressure to afford the title compound 3a or 3b.
6-bromobenzo[b]thiophene-2-carboxylic acid (3a)
Yield 96%, white solid; 1H NMR (500 MHz, DMSO) δ 13.57 (s, 1H, COOH), 8.36 (s,
1H, Ar-H), 8.10 (s, 1H, Ar-H), 7.95 (d, J = 8.6 Hz, 1H, Ar-H), 7.61 (dd, J = 8.6, 1.6
Hz, 1H, Ar-H).
7-bromobenzo[b]thiophene-2-carboxylic acid (3b)
Yield 96%, white solid; 1H NMR (500 MHz, DMSO) δ 13.68 (s, 1H, COOH), 8.26 (s,
1H, Ar-H), 8.05 (d, J = 8.0 Hz, 1H, Ar-H), 7.76 (d, J = 7.6 Hz, 1H, Ar-H), 7.43 (t, J =
7.8 Hz, 1H, Ar-H)
4.1.3. General procedures for the preparation of 4a and 4b
A mixture 3a or 4b (0.5 mmol), Ag2CO3 (0.05 mmol), AcOH (0.025 mmol) in
DMSO (1.0 mL) was stirred at 120 ºC. After 16 hours, the reaction was cooled down
to room temperature and quenched with 1 M hydrochloric acid solution (2 mL) and
the aqueous phase extracted with ethyl acetate. The combined organic layers were
washed with brine and dried sodium sulfate, filtered, and the solvents were removed
under reduced pressure to give analytically pure 4a or 4b.
6-bromobenzo[b] thiophene (4a)
Yield 78%, white solid; 1H NMR (500 MHz, DMSO) δ 8.10 (s, 1H, Ar-H), 7.95 (d, J
= 8.6 Hz, 1H, Ar-H), 7.61 (dd, J = 8.6, 1.6 Hz, 1H, Ar-H), 7.56 (d, J = 6.8 Hz, 1H,
Ar-H), 7.30 (d, J = 6.8 Hz, 1H, Ar-H).
7-bromobenzo[b] thiophene (4b)

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Yield 76%, white solid; 1H NMR (500 MHz, CDCl3) δ 8.06 (s, 1H, Ar-H), 7.96 (d, J =
8.1 Hz, 1H, Ar-H), 7.65 (dd, J = 8.1, 1.0 Hz, 1H, Ar-H), 7.50 – 7.46 (m, 2H, Ar-H).
4.1.4. General procedures for the preparation of 5a and 5b
3-Chloroperbenzoic acid (2.5 mmol) was added to the stirred solution of compound
4a or 4b (1.0 mmol) in dichloromethane (50 mL) in portions over a 20-30 minutes
period. The mixture was heated to reflux for about 5 h and monitored by TLC. After
completion, the mixture was cooled to room temperature and sodium hydrogen sulfite
(20 mL) was added. The solution was stirred for 15 minutes and extracted with
dichloromethane. The organic phase was washed with aqueous sodium bicarbonate (2
× 30 mL). The organic phase was separated, washed with brine and dried with sodium
sulfate, filtered, and the solvents were removed under reduced pressure to give the
sulfone compound 5a or 5b.
6-bromobenzo[b]thiophene 1, 1-dioxide (5a)
Yield 81%, light yellow solid; 1H NMR (500 MHz, DMSO) δ 8.18 (s, 1H, Ar-H), 7.90
(dd, J = 8.0, 1.6 Hz, 1H, Ar-H), 7.63 (d, J = 6.9 Hz, 1H, Ar-H), 7.54 (d, J = 8.0 Hz,
1H, Ar-H), 7.40 (d, J = 6.9 Hz, 1H, Ar-H).
7-bromobenzo[b]thiophene 1, 1-dioxide (5b)
1
Yield 81%, light yellow solid; H NMR (500 MHz, DMSO) δ 7.78 (m, 1H, Ar-H),
7.59 (m, 3H, Ar-H), 7.44 (d, J = 6.9 Hz, 1H, Ar-H)
4.1.5. General procedures for the preparation of 6-29
To a solution of 5a or 5b (0.05 mmol) in toluene (10 mL) was added aniline (0.06
mmol), Cs2CO3 (0.07 mmol), BINAP (0.004 mmol), and Pd(OAc)2 (0.004 mmol) at
room temperature. The reaction mixture was allowed to stir at 120 °C for 4-48 h.
Once the reaction appeared to be complete by consumption of the bromide by TLC,
the mixture was allowed to cool to room temperature, diluted with ethyl acetate,
washed with 1M hydrochloric acid solution, brine, and dried over sodium sulfate. The
solution was concentrated, loaded on silica gel, and purified by silica gel
chromatography with petroleum/ethyl acetate as eluent to give 6-29.
6-((3-(trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (6)
Yield 45%, yellow solid; m.p. 118-120 °C; IR (KBr) ν 3375, 1601, 1529, 1496, 1412,

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1337, 1283, 1206, 1124, 1070, 868, 827, 792, 743, 699, 623 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.45 (t, J = 8.1 Hz, 1H, Ar-H), 7.38 – 7.30 (m, 4H, Ar-H), 7.22 (d, J = 8.2
Hz, 1H, Ar-H), 7.15 (d, J = 6.9 Hz, 1H, Ar-H), 7.12 (dd, J = 8.2, 1.9 Hz, 1H, Ar-H),
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6.57 (d, J = 6.9 Hz, 1H, Ar-H), 6.24 (s, 1H, NH). C NMR (126 MHz, DMSO) δ
145.54 (s), 142.36 (s), 138.29 (s), 132.99 (s), 130.62 (s), 128.08 (s), 127.04 (s),
122.06 (s), 121.53 (s), 119.12 (s), 117.67 (s), 114.41 (JCF = 3.9 Hz), 108.82 (s). MS
(ESI) m/z 326.0 [M+H]+; HRMS (ESI) m/z 326.0460 [M+H]+ (calcd for 326.0457
C14H11F3NO2S).
6-((4-(trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (7)
Yield 73%, yellow solid; m.p. 112-114 °C; IR (KBr) ν 3375, 1601, 1509, 1496, 1412,
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1321, 1283, 1206, 1124, 1070, 868, 792, 743, 699, 559 cm-1; H NMR (500 MHz,
DMSO) δ 9.25 (s, 1H, NH), 7.63 (d, J = 8.5 Hz, 2H, Ar-H), 7.55 (d, J = 6.8 Hz, 1H,
Ar-H), 7.47 (d, J = 8.2 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.38 (dd, J = 8.2, 1.9 Hz,
1H, Ar-H), 7.28 (d, J = 8.5 Hz, 2H, Ar-H), 7.15 (d, J = 6.8 Hz, 1H, Ar-H). 13C NMR
(126 MHz, DMSO) δ 145.33, 144.88, 138.20, 132.93, 128.39, 126.98, 126.67 (JCF
=3.7 Hz), 124.56 (JCF =270.0 Hz), 122.61, 120.83, 120.221, 117.09, 109.16. MS (ESI)
m/z 326.0 [M+H]+; HRMS (ESI) m/z 326.0460 [M+H]+ (calcd for 326.0457
C14H11F3NO2S).
3-((1, 1-dioxidobenzo[b]thiophen-6-yl) amino) benzonitrile (8)
Yield 43%, yellow solid; m.p. 122-124 °C; IR (KBr) ν 3449, 2923, 2852, 1609, 1529,
1262, 1491, 1262, 1188, 1081, 967, 791, 546 cm-1; 1H NMR (500 MHz, DMSO) δ
9.13 (s, 1H, NH), 7.54 (d, J = 6.8 Hz, 1H, Ar-H), 7.52 – 7.49 (m, 2H, Ar-H), 7.45 (d, J
= 8.4 Hz, 2H, Ar-H), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 7.32 (dd, J = 8.3, 1.9 Hz, 1H,
Ar-H), 7.13 (d, J = 6.8 Hz, 1H, Ar-H). 13C NMR (126 MHz, DMSO) δ 145.27, 142.45,
138.26, 132.95, 130.73, 128.17, 127.00, 124.82, 122.82, 122.23, 120.62, 119.32,
118.66, 112.28, 109.08. MS (ESI) m/z 326.0 [M+H]+; HRMS (ESI) m/z 326.0460
[M+H]+ (calcd for 326.0457 C14H10Cl2NO2S).
6-((4-methoxyphenyl) amino) benzo[b]thiophene 1, 1-dioxide (9)
Yield 32%, yellow solid; m.p. 170-172 °C; IR (KBr) ν 3345, 1607, 1509, 1496, 1412,

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1337, 1276, 1206, 1135, 1032, 821, 742, 684，597 cm-1; 1H NMR (500 MHz, DMSO)
δ 8.61 (s, 1H, NH), 7.46 (d, J = 6.8 Hz, 1H, Ar-H), 7.31 (d, J = 8.3 Hz, 1H, Ar-H),
7.13 (d, J = 8.8 Hz, 2H, Ar-H), 7.07 (s, 1H, Ar-H), 7.02 – 6.93 (m, 4H, Ar-H), 3.75 (s,
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3H, OCH3). C NMR (126 MHz, DMSO) δ 155.55, 148.64, 138.44, 133.49, 133.31,
126.89, 126.60, 123.17, 119.39, 116.20, 114.80, 105.97, 55.22. MS (ESI) m/z 288.1
[M+H]+; HRMS (ESI) m/z 288.0688 [M+H]+ (calcd for 288.0689 C15H14NO3S).
6-((4-chlorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (10)
Yield 73%, yellow solid; m.p. 118-120 °C; IR (KBr) ν 3421, 2853, 1589, 1521, 1471,
1289, 1201, 1137, 1113, 816, 628, 520 cm-1; 1H NMR (500 MHz, DMSO) δ 8.94 (s,
1H, NH), 7.51 (d, J = 6.8 Hz, 1H, Ar-H), 7.38 (m, 3H, Ar-H), 7.27 (s, 1H, Ar-H), 7.22
(dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.17 (d, J = 8.7 Hz, 2H, Ar-H), 7.07 (d, J = 6.8 Hz,
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1H, Ar-H). C NMR (126 MHz, DMSO) δ 146.27, 140.18, 138.30, 133.06, 129.25,
127.61, 126.93, 125.37, 121.18, 120.53, 118.22, 107.91. MS (ESI) m/z 292.0 [M+H]+;
HRMS (ESI) m/z 292.0192 [M+H]+ (calcd for 292.0194 C14H11ClNO2S).
6-((3, 4-dichlorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (11)
Yield 45%, yellow solid; m.p. 165-167 °C; IR (KBr) ν 3421, 2924，2853，1589, 1521,
1471, 1276, 1201, 1137, 1023, 816，628，531 cm-1; 1H NMR (500 MHz, DMSO) δ
9.07 (s, 1H, NH), 7.53 (d, J = 7.4 Hz, 2H, Ar-H), 7.44 (d, J = 7.9 Hz, 1H, Ar-H), 7.34
– 7.31 (m, 3H, Ar-H), 7.23 – 7.08 (m, 2H, Ar-H). 13C NMR (126 MHz, DMSO) δ
145.28, 141.83, 138.24, 132.93, 131.59, 131.12, 128.16, 127.00, 122.59, 122.20,
119.40, 118.25, 109.04. MS (ESI) m/z 281.0 [M+H] +; HRMS (ESI) m/z 281.0390
[M+H]+ (calcd for 281.0390 C14H9Cl2NO2S).
6-((3, 4-difluorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (12)
Yield 44%, yellow solid; m.p. 132-134 °C; IR (KBr) ν 3370, 2923, 2852, 1601, 1571,
1510, 1407, 1366, 1292, 1265, 1162, 1137, 1061, 1029，962, 824, 746, 622, 600, 561
cm-1; 1H NMR (500 MHz, DMSO) δ 8.93 (s, 1H, NH), 7.51 (d, J = 6.8 Hz, 1H, Ar-H),
7.38 (dd, J = 18.0, 8.7 Hz, 2H, Ar-H), 7.27 (d, J = 1.6 Hz, 1H, Ar-H), 7.24 – 7.14 (m,
2H, Ar-H), 7.08 (d, J = 6.8 Hz, 1H, Ar-H), 7.01 – 6.93 (m, 1H, Ar-H). 13C NMR (126
MHz, DMSO) δ 149.68 (JCF = 245.0 Hz), 146.33, 144.39 (JCF = 245.0 Hz), 138.45
(JCF = 8.5 Hz), 138.32, 133.06, 127.74, 127.00, 121.39, 118.20, 118.03 (JCF = 18.0

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Hz), 115.68 (JCF = 5.9 Hz), 108.28 (JCF = 19.4 Hz), 108.02. MS (ESI) m/z 294.0
[M+H]+; HRMS (ESI) m/z 294.0397 [M+H]+ (calcd for 294.0395 C14H10F2NO2S).
7-((3, 4, 5-trimethoxyphenyl) amino) benzo[b]thiophene 1, 1-dioxide (13)
Yield 56%, yellow solid; m.p. 166-167 °C; IR (KBr) ν 3348, 2923, 1585, 1505, 1472,
1427, 1393, 1314, 1279, 1127, 1000, 964, 836, 790, 728, 683, 649, 600, 549 cm-1; 1H
NMR (500 MHz, DMSO) δ 8.74 (s, 1H, NH), 7.49 (d, J = 6.8 Hz, 1H, Ar-H), 7.35 (d,
J = 8.1 Hz, 1H, Ar-H), 7.20 (d, J = 10.3 Hz, 2H, Ar-H), 7.03 (d, J = 6.8 Hz, 1H, Ar-H),
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6.45 (s, 2H, Ar-H), 3.76 (s, 6H, OCH3), 3.66 (s, 3H, OCH3). C NMR (126 MHz,
DMSO) δ 153.40, 153.23, 147.42, 138.34, 136.90, 133.24 (JCF = 19.8 Hz), 127.10,
126.91, 120.21, 117.31, 107.53, 98.16, 91.82, 60.07, 55.80, 55.36. MS (ESI) m/z
348.1 [M+H]+; HRMS (ESI) m/z 348.0902 [M+H]+ (calcd for 348.0900,
C17H18NO5S).
6-((3-(trifluoromethoxy ) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (14)
Yield 34%, yellow solid; m.p. 132-134 °C; IR (KBr) ν 3372, 3098, 2924, 1599, 1526,
1494, 1408, 1327, 1250, 1210, 850, 829, 797, 744, 726, 682, 627, 527 cm-1; 1H NMR
(500 MHz, DMSO) δ 9.10 (s, 1H, NH), 7.53 (d, J = 6.8 Hz, 1H, Ar-H), 7.44 (dt, J =
8.1, 4.0 Hz, 2H, Ar-H), 7.33 (s, 1H, Ar-H), 7.29 (dd, J = 8.2, 1.9 Hz, 1H, Ar-H), 7.18
(d, J = 8.1 Hz, 1H, Ar-H), 7.12 (d, J = 6.8 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.93 (d,
J = 8.2 Hz, 1H, Ar-H). 13C NMR (126 MHz, DMSO) δ 149.22, 145.52, 143.32,
138.26, 132.97, 131.09, 128.06, 127.00, 121.99, 119.18, 116.78, 113.31, 110.31,
108.75. MS (ESI) m/z 342.0 [M+H]+; HRMS (ESI) m/z 342.0407 [M+H]+ (calcd for
342.0406 C15H11F3NO3S).
7-((3-(trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (15)
Yield 55%, yellow solid; m.p. 120-122 °C; IR (KBr) ν 3335, 2920, 2851, 1611, 1587,
1562, 1524, 1494, 1394, 1332, 1281, 1223, 1169, 1136, 1113, 1076, 895, 795, 717,
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698, 682, 660, 642, 600, 551 cm-1; H NMR (500 MHz, DMSO) δ 8.39 (s, 1H, NH),
7.55 (t, J = 5.0 Hz, 1H, Ar-H), 7.54 – 7.45 (m, 3H, Ar-H), 7.41 (d, J = 8.3 Hz, 1H,
Ar-H), 7.31 (dd, J = 12.2, 7.6 Hz, 2H, Ar-H), 7.26 (d, J = 7.5 Hz, 1H, Ar-H), 7.12 (m,
1H, Ar-H). MS (ESI) m/z 326.0 [M+H]+; HRMS (ESI) m/z 326.0455 [M+H]+ (calcd
for 326.0457, C15H11F3NO2S). 13C NMR (126 MHz, DMSO) δ 143.29, 138.55,

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135.09, 133.33, 132.26, 131.61, 130.10, 129.83, 125.15, 124.20 (JCF =245.0 Hz),
122.99, 121.99, 119.18, 117.59, 115.22 (JCF = 3.8 Hz).
7-((4-(trifluoromethoxy) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (16)
Yield 55%, yellow solid; m.p. 108-110 °C; IR (KBr) ν 3343, 1590, 1516, 1470, 1270,
851, 786, 677, 613, 557, 521 cm-1; 1H NMR (500 MHz, DMSO) δ 9.25 (s, 1H, NH),
7.63 (d, J = 8.5 Hz, 2H, Ar-H), 7.55 (d, J = 6.8 Hz, 1H, Ar-H), 7.47 (d, J = 8.2 Hz, 1H,
Ar-H), 7.42 (s, 1H, Ar-H), 7.38 (dd, J = 8.2, 1.9 Hz, 1H, Ar-H), 7.28 (d, J = 8.4 Hz,
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2H, Ar-H), 7.15 (d, J = 6.8 Hz, 1H, Ar-H). C NMR (126 MHz, DMSO) δ 142.84,
141.15, 139.39, 134.97, 133.22, 132.33, 131.52, 122.71, 121.96, 121.05, 120.86,
118.22. MS (ESI) m/z 342.0 [M+H]+; HRMS (ESI) m/z 342.0405 [M+H]+ (calcd for
342.0406, C15H11F3NO3S).
7-((3, 4, 5-trimethoxyphenyl) amino) benzo[b]thiophene 1, 1-dioxide (17)
Yield 60%, yellow solid; m.p. 168-170 °C; IR (KBr) ν 3348, 2923, 1585, 1505, 1472,
1427, 1393, 1314, 1279, 1233, 1180, 1127, 1000, 964, 836, 790, 728, 683, 649, 600,
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549 cm-1; H NMR (500 MHz, DMSO) δ 7.76 (s, 1H, NH), 7.50 (d, J = 6.8 Hz, 1H,
Ar-H), 7.45 – 7.37 (m, 1H, Ar-H), 7.29 – 7.22 (m, 2H, Ar-H), 6.93 (d, J = 7.1 Hz, 1H,
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Ar-H), 6.57 (s, 2H, Ar-H), 3.73 (s, 6H, OCH3), 3.64 (s, 3H, OCH3). C NMR (126
MHz, DMSO) δ 153.16, 140.31, 136.66, 134.85, 133.45, 133.06, 132.46, 131.35,
120.31, 119.23, 116.73, 99.05, 60.05, 55.77, 39.52, 39.35, 39.19, 39.02. MS (ESI) m/z
348.1 [M+H]+; HRMS (ESI) m/z 348.0901 [M+H]+ (calcd for 348.0900,
C17H18NO5S).
7-((4-(trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (18)
Yield 73%, yellow solid; m.p. 166-168 °C; IR (KBr) ν 3356, 2923, 1608, 1532, 1471,
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1323, 1292, 1294, 1162, 1144, 1107, 1065, 795, 678, 619, 555, 520 cm-1; H NMR
(500 MHz, DMSO) δ 8.49 (s, 1H, NH), 7.57 (d, J = 7.0 Hz, 4H, Ar-H), 7.41 (d, J =
8.0 Hz, 1H, Ar-H), 7.34 – 7.29 (m, 1H Ar-H), 7.24 (d, J = 7.9 Hz, 2H Ar-H), 7.20 (d, J
= 7.1 Hz, 1H Ar-H). 13C NMR (126 MHz, DMSO) δ 146.79, 137.88, 135.16, 133.35,
132.29, 131.69, 126.33 (d, JCF = 3.7 Hz), 125.80, 123.89, 120.19, 117.32. MS (ESI)
m/z 326.0 [M+H]+; HRMS (ESI) m/z 326.0455 [M+H]+ (calcd for 326.0457,
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7-((4-chlorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (19)
Yield 56%, yellow solid; m.p. 120-122 °C; IR (KBr) ν 3347, 3096, 1598, 1598, 1517,
1491, 1468, 1352, 1321, 1295, 1215, 1170, 1135, 1092，1009, 812, 791, 728, 698, 621,
599, 555, 520 cm-1; 1H NMR (500 MHz, DMSO) δ 8.11 (s, 1H, NH), 7.52 (d, J = 6.8
Hz, 1H, Ar-H), 7.48 – 7.42 (m, 1H, Ar-H), 7.33 (d, J = 8.7 Hz, 2H, Ar-H), 7.28 (d, J
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= 6.8 Hz, 1H, Ar-H), 7.23-7.19 (m, 3H, Ar-H), 7.03 (d, J = 6.8 Hz, 1H, Ar-H). C
NMR (126 MHz, DMSO) δ 140.75, 139.48, 134.96, 133.24, 132.38, 131.52, 128.94,
125.77, 122.38, 121.68, 120.61, 118.03. MS (ESI) m/z 292.0 [M+H]+; HRMS (ESI)
m/z 292.0192 [M+H]+ (calcd for 292.0194, C14H11ClNO2S).
7-((3, 5-dimethylphenyl) amino) benzo[b]thiophene 1, 1-dioxide (20)
Yield 45%, yellow solid; m.p. >250 °C; IR (KBr) ν 3346, 2919, 1587, 1519, 1469,
1333, 1283, 1216, 1135, 1032, 857, 838, 786, 680, 649, 621, 601, 551 cm-1; 1H NMR
(500 MHz, DMSO) δ 7.67 (s, 1H, NH), 7.50 (d, J = 6.8 Hz, 1H, Ar-H), 7.44 – 7.39 (m,
1H, Ar-H), 7.26 (d, J = 6.8 Hz, 1H, Ar-H), 7.18 (d, J = 8.4 Hz, 1H, Ar-H), 6.95 (d, J =
13
6.8 Hz, 1H, Ar-H), 6.83 (s, 2H, Ar-H), 6.68 (s, 1H, Ar-H), 2.23 (s, 6H, CH3). C
NMR (126 MHz, DMSO) δ 140.87, 138.27, 134.81, 132.47, 131.39, 124.43, 119.75,
118.49, 116.99, 40.02, 39.52, 39.35, 39.19, 39.02, 20.93. MS (ESI) m/z 286.1 [M+H]+;
HRMS (ESI) m/z 286.0895 [M+H]+ (calcd for 286.0896, C16H16NO2S).
7-((perfluorophenyl ) amino)benzo[b]thiophene 1, 1-dioxide (21)
Yield 56%, yellow solid; m.p. 189-191 °C; IR (KBr) ν 3397, 3081, 2922, 1612, 1568,
1512, 1368, 1288, 1182, 1133, 1088, 1034，986, 843, 790, 738, 701, 652, 589, 534
cm-1; 1H NMR (500 MHz, DMSO) δ 8.37 (s, 1H, NH), 7.49 (d, J = 6.8 Hz, 1H, Ar-H),
7.49-7.41 (m, 1H, Ar-H), 7.21 (d, J = 6.8 Hz, 1H, Ar-H), 6.96 (d, J = 7.2 Hz, 1H,
Ar-H), 6.90 (d, J = 7.2 Hz, 1H, Ar-H). 13C NMR (126 MHz, DMSO) δ 140.94, 135.02,
133.19, 132.54, 131.10, 119.09, 118.88, 117.09. MS (ESI) m/z 346.0 [M+H]+; HRMS
(ESI) m/z 345.9968 [M+H]+ (calcd for 345.9967, C14H6NF5O2S).
7-((3-fluorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (22)
Yield 56%, yellow solid; m.p. 143-145 °C; IR (KBr) ν 3396, 2923，2852, 1605, 1467,
1261, 1098, 801, 681, 633, 548, 520 cm-1; 1H NMR (500 MHz, DMSO) δ 8.18 (s, 1H,
NH), 7.54 (d, J = 6.8 Hz, 1H, Ar-H), 7.52 – 7.46 (m, 1H, Ar-H), 7.36 – 7.31 (m, 1H,

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Ar-H), 7.31 – 7.28 (m, 2H), 7.09 (d, J = 7.2 Hz, 1H, Ar-H), 7.02 – 6.94 (m, 2H, Ar-H),
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6.76 (td, J = 8.5, 2.3 Hz, 1H, Ar-H). C NMR (126 MHz, DMSO) δ 144.17 (JCF
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10.8 Hz), 138.86, 134.97, 133.21, 132.31, 131.54, 130.54 (JCF = 9.8 Hz), 121.78,
118.76, 114.76, 108.12, 107.95, 105.99, 105.79. MS (ESI) m/z 276.0 [M+H]+; HRMS
(ESI) m/z 276.0490 [M+H]+ (calcd for 276.0489, C14H11FNO2S).
7-(p-tolylamino) benzo[b]thiophene 1, 1-dioxide (23)
Yield 45%, yellow solid; m.p. >250 °C; IR (KBr) ν 3376, 3350, 2921, 2851, 1596,
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1519, 1473, 1331, 1288, 1262, 1156, 1128, 1097, 816, 777, 627, 548, 491 cm-1; H
NMR (500 MHz, DMSO) δ 7.79 (s, 1H, NH), 7.49 (d, J = 6.8 Hz, 1H, Ar-H), 7.40 –
7.34 (m, 1H, Ar-H), 7.25 (d, J = 6.8 Hz, 1H, Ar-H), 7.15-7.11 (m,4H, Ar-H), 7.08 (d,
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J = 8.5 Hz, 1H, Ar-H), 6.90 (d, J = 7.1 Hz, 1H, Ar-H), 2.28 (s, 3H, CH3). C NMR
(126 MHz, DMSO) δ 140.74, 138.07, 134.74, 133.05, 132.46, 132.29, 131.34, 129.61,
121.83, 118.55, 119.91, 116.39, 20.35. MS (ESI) m/z 272.1 [M+H]+; HRMS (ESI)
m/z 272.0741 [M+H]+ (calcd for 272.0740 C15H14NO2S).
7-((4-methoxyphenyl) amino) benzo[b]thiophene 1, 1-dioxide (24)
Yield 68%, yellow solid; m.p. 142-144 °C; IR (KBr) ν 3444, 3335, 3085, 2929, 2852,
1612, 1512, 1468, 1395, 1354, 1321, 1284, 1258, 1138, 1084, 1036, 846, 823, 788,
1
721, 683, 640, 607, 555, 505 cm-1; H NMR (500 MHz, DMSO) δ 7.74 (s, 1H, NH),
7.47 (d, J = 6.8 Hz, 1H, Ar-H), 7.35 – 7.30 (m, 1H, Ar-H), 7.23 (d, J = 6.8 Hz, 1H,
Ar-H), 7.17 (d, J = 8.5 Hz, 2H, Ar-H), 6.94 (t, J = 6.1 Hz, 2H, Ar-H), 6.91 (d, J = 8.5
13
Hz, 1H, Ar-H), 6.83 (d, J = 7.1 Hz, 1H, Ar-H), 3.75 (s, 3H, OCH3). C NMR (126
MHz, DMSO) δ 156.05, 141.66, 134.71, 133.02, 132.48, 131.29, 124.88, 118.50,
117.41, 115.50, 114.91, 114.49, 55.21. MS (ESI) m/z 288.1 [M+H]+; HRMS (ESI)
m/z 288.0688 [M+H]+ (calcd for 288.0689 C15H14NO3S).
7-((2-methoxyphenyl) amino) benzo[b]thiophene 1, 1-dioxide (25)
Yield 53%, yellow solid; m.p. 116-118 °C; 3393, 2923, 2852, 1589, 1518, 1463, 1286,
1136, 1024, 797, 747, 631, 526 cm-1; 1H NMR (500 MHz, DMSO) δ 7.54 (d, J = 6.8
Hz, 1H, Ar-H), 7.46 – 7.41 (m, 1H, Ar-H), 7.31 (t, J = 7.2 Hz, 2H, Ar-H), 7.15 – 7.06
13
(m, 4H, Ar-H), 6.96 (d, J =6.8 Hz, 1H, Ar-H), 3.83 (s, 3H, OCH3). C NMR (126
MHz, DMSO) δ 150.42, 139.72, 134.98, 132.91, 132.69, 131.16, 128.55, 123.98,

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120.68, 120.12, 119.72, 117.75, 116.74, 111.85, 55.78. MS (ESI) m/z 288.1 [M+H]+;
HRMS (ESI) m/z 288.0687 [M+H]+ (calcd for 288.0689 C15H14NO3S).
7-((3, 4-difluorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (26)
Yield 53%, yellow solid; m.p. 176-177 °C; 3444, 3350, 1608, 1560, 1514, 1464, 1386,
1355, 1277, 1202, 1172, 1138, 1103, 1078, 946, 855, 840, 791, 709, 687, 687, 633,
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598, 572, 546, 515, 484, 445 cm-1; H NMR (500 MHz, DMSO) δ 8.15 (s, 1H, NH),
7.53 (d, J = 6.8 Hz, 1H, Ar-H), 7.46 (t, J = 7.8 Hz, 1H, Ar-H), 7.36-7.32 (m, 1H,
Ar-H), 7.28 (d, J = 6.8 Hz, 1H, Ar-H), 7.26 – 7.19 (m, 2H, Ar-H), 7.04 (d, J = 7.2 Hz,
13
1H, Ar-H), 7.00 (d, J = 8.6 Hz, 1H, Ar-H). C NMR (126 MHz, DMSO) δ 149.48
(JCF = 244.1 Hz), 144.95 (JCF = 239.9 Hz), 139.42, 138.99 (JCF = 8.7 Hz), 135.02,
133.22, 132.35, 131.51, 122.45, 120.62, 118.18, 117.58 (JCF = 17.7 Hz), 116.40 (JCF
= 5.9 Hz), 109.36 (JCF = 19.7 Hz). MS (ESI) m/z 294.0 [M+H]+; HRMS (ESI) m/z
294.0393 [M+H]+ (calcd for 294.0395 C14H10F2NO2S).
7-((3, 4-dichlorophenyl) amino) benzo[b]thiophene 1, 1-dioxide (27)
Yield 66%, yellow solid; m.p. 165-167 °C; 3354, 1590, 1504, 1466, 1375, 1318, 1286,
1138, 862, 798, 686, 625, 556, 520 cm-1; 1H NMR (500 MHz, DMSO) δ 8.34 (s, 1H,
NH), 7.55 (d, J = 6.8 Hz, 1H, Ar-H), 7.54 – 7.49 (m, 1H, Ar-H), 7.47 (d, J = 8.8 Hz,
1H, Ar-H), 7.35 (d, J = 2.6 Hz, 1H, Ar-H), 7.33 (d, J = 8.3 Hz, 1H, Ar-H), 7.30 (d, J =
6.8 Hz, 1H, Ar-H), 7.14 (d, J = 7.2 Hz, 1H, Ar-H), 7.11 (dd, J = 8.8, 2.6 Hz, 1H,
Ar-H). 13C NMR (126 MHz, DMSO) δ 147.28, 140.80, 138.24, 132.93, 131.59,
131.12, 128.16, 127.00, 122.59, 122.20, 119.40, 118.25, 109.04. MS (ESI) m/z 326.0
[M+H]+; HRMS (ESI) m/z 325.9803 [M+H]+ (calcd for 325.9804 C14H10Cl2NO2S).
7-((3, 5-bis (trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (28)
Yield 70%, yellow solid; m.p. 202-204 °C; 3366, 2921, 2850, 1601, 1539, 1476, 1384,
1278, 1218, 1182, 1166, 1144, 1119, 931, 892, 878, 836, 801, 719, 705, 682, 642, 552,
1
522, 484 cm-1; H NMR (500 MHz, DMSO) δ 8.87 (s, 1H, NH), 7.66 – 7.58 (m, 4H,
Ar-H), 7.49 (s, 1H, Ar-H), 7.46 (d, J = 8.2 Hz, 1H, Ar-H), 7.34 (d, J = 6.8 Hz, 1H,
Ar-H), 7.28 (d, J = 6.8 Hz, 1H, Ar-H). 13C NMR (126 MHz, DMSO) δ 145.53, 136.83,
135.54, 133.49, 132.24, 131.72, 131.08 (JCF = 32.6 Hz), 124.27 (JCF = 13.2 Hz),

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122.15, 121.16, 116.67, 112.74. MS (ESI) m/z 394.0 [M+H]+; HRMS (ESI) m/z
394.0332 [M+H]+ (calcd for 390.0331 C16H9F6NO2S).
7-((2-(trifluoromethyl) phenyl) amino) benzo[b]thiophene 1, 1-dioxide (29)
Yield 76%, yellow solid; m.p. 125-127 °C; 3412, 3087, 2922, 2851, 1599, 1556, 1522,
1468, 1326, 1281, 1219, 1143, 1113, 1036, 855, 793, 761, 687, 651, 631, 596, 551,
1
526 cm-1; H NMR (500 MHz, DMSO) δ 7.77 (d, J = 7.8 Hz, 1H, Ar-H), 7.68 (t, J =
7.7 Hz, 1H, Ar-H), 7.56 (d, J = 6.8 Hz, 1H, Ar-H), 7.47 (d, J = 8.8 Hz, 1H, Ar-H),
7.43 (d, J = 7.7 Hz, 1H, Ar-H), 7.37 (t, J = 7.7 Hz, 1H, Ar-H), 7.32 (m, 2H, NH,
Ar-H), 7.02 (d, J = 7.2 Hz, 1H, Ar-H), 6.93 (d, J = 8.8 Hz, 1H, Ar-H). 13C NMR (126
MHz, DMSO) δ 139.76, 138.33, 135.11, 133.79, 132.86, 132.80, 131.32, 127.01 (JCF
= 5.2 Hz), 125.02, 124.69, 120.75, 118.96, 117.63. MS (ESI) m/z 326.0 [M+H]+;
HRMS (ESI) m/z 326.0458 [M+H]+ (calcd for 326.0457 C14H10Cl2NO2S).
4.2. Docking
Computational docking program AutoDock4.2 was used to dock our designed
small molecules to predict their binding modes and approximate binding free energies
to the STAT3 SH2 dimerization sites [5]. Briefly, compounds were docked using the
Lamarckian Genetic Algorithm. The ligand and macromolecule were prepared using
the Schrödinger software. Gasteiger charges were assigned to the ligands by Auto
Dock Tools. Then, AutoGrid maps were precomputed for all atom types in the ligand
set. After 10 million energy evaluations were completed, the root-mean-square
deviation threshold was set as 1.5Å and all the resulting conformations of the ligands
in the binding pocket of the macromolecule were clustered. Low energy clusters were
identified and binding energies were evaluated.
4.3. Maintenance of cell lines culture and cell viability assays
All cell lines were purchased from Cell Bank of Shanghai Institute of Biochemistry
and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human
non-small cell lung carcinoma cell (A549), human breast carcinoma cell (MCF-7) and
(MDA-MB-231), human colon carcinoma cell (HCT116) were maintained in
RPMI-1640 medium. All cells were supplemented with 10% fetal bovine serum
containing 50ꢀg/ml penicillin and 50ꢀg/ml streptomycin. Cells were grown to 80%

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confluency in a tissue culture flask at 37 °C in a humidified atmosphere containing
5% CO2, and then were trypsinized with 1×Trypsin-Versene and splitted.
Cells (MDA-MB-231, A549, MCF-7, HCT116) were seeded in 96-well plates at a
density of 3000-5000 cells per well. The cells were incubated at 37 °C overnight (16 h)
in a humidified 5% CO2 incubator. After medium removal, different concentrations of
test compounds were added in triplicate to the plates in 200ꢀL fresh mediums, the
plates were incubated at 37 °C for 72 h. The percentage of DMSO in the medium not
exceeded 0.1%. 3-(4, 5-Dimethylthiazolyl)-2, 5-diphenyltetrazoliumbromide (MTT)
was added to evaluate cell viability. The absorbance was read by an ELISA reader
(SpectraMax Plus384, Molecular Devices, Sunnyvale, CA) at a test wavelength of
570 nm and a reference wavelength of 630 nm. Cell viability was calculated by the
following formula:
% Cell viability = (At/As) × 100%
At and As denoted the absorbance of the test substances and solvent control,
respectively.
4.4. Western blot analysis
MDA-MB-231 cells were incubated with various concentrations of 15 for 24 h.
Harvesting after trypsinisation, cells were treated with 1× RIPA lysis buffer (50mM
Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA
and protease inhibitors) (Amresco, Solon, USA) to extract the total proteins. An
aliquot of proteins from the total cell lysates (30 to 60ꢀg/lane) was separated by
sodium dodecyl sulfate (10%) polyacryl amide gel electrophoresis (SDS–PAGE,
BioRad Laboratories, Hercules, CA), wet-transferred to NC membrane (BioRad
Laboratories, Hercules, CA) and blotted with primary antibodies specific for STAT3,
p-STAT3, p-Jak2, Jak2, p-Src, Src, Erk, P-Erk, Cleaved Caspase-3, Caspase-3, PARP,
Cleaved PARP, Bcl-2 and GAPDH. Bound immuno-complexes were detected using
ChemiDOC™ XRS+system (BioRad Laboratories, Hercules, CA).
4.5. IL-6 induction of STAT3 Phosphorylation
MDA-MB-231 cells were seeded in 6-well plates and allowed to adhere overnight.
The following day, the cells were serum-starved. The cells were then left untreated or

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were treated with 15 (1-5 ꢀM). After 12 h, the untreated and 15 treated cells were
stimulated by IL-6 (25 ng/mL). The cells were harvested after 30 min and analyzed by
western blot.
4.6. Electrophoretic Mobility Shift Assay (EMSA) of DNA-binding activity
MDA-MB-231 cells were cultured in culture dish overnight and then treated with
compound 15 in fresh regular growth medium for 24 h. The cells were harvested and
nuclear extract preparations and EMSA analysis were carried out as previously
described [10]. The 32Plabeled oligonucleotide hSIE probe was used to bind STAT3.
4.7. Flow cytometry analysis of apoptotic cells
MDA-MB-231 cells at a density of 2.5 × 105 per well were cultured in regular
growth medium in 6-well plates for 24 h and treated in duplicate with different
concentrations of 15 for 24 h. The cells were harvested, washed and stained with 5ꢀL
Annexin V-FITC and 5 ꢀL propidium iodide at room temperature for 15 min. Cells
were then analyzed by flow cytometry (488 nm excitation and 600 nm emission filters)
using BD FACSCalibur flowcytometer (Becton & Dickinson Company, Franklin
Lakes, NJ).
4.8. ROS detection by flow cytometry
MDA-MB-231 cells were seeded at a density of 3×105 per well of 6-well plates.
The cells were treated with various concentrations of 15 (0, 1, 3, 5 ꢀM) for 24h.
DCFH-DA was dissolved in Serum-free medium and diluted to a final concentration
of 10 ꢀM. After treatment with 15, the growth media was replaced with Serum-free
medium containing the probe. After incubation for 20 min at 37 oC, cells were washed
with Serum-free medium twice, digested by trypsin and resuspended in the
pre-warmed PBS buffer. The samples were then subjected to a flow cytometry assay
using BD FACSCalibur flowcytometer (Becton & Dickinson Company, Franklin
Lakes, NJ).
4.9. Colony survival assay
MDA-MB-231 cells were cultured 800-1000 per well in 6-well plate with regular
growth medium. Cells were treated by vehicle and 15 at 0.5-6 ꢀM on the following
day for 24 h. Cells were allowed to grow for 10-14 d until the colonies were visible.

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Crystal violet solution (Sigma, St. Louis, MO, USA) was used to stain the colonies
for 4 hours and colonies were scored manually as described previously [39].
4.10. In vivo studies
Mouse breast tumor 4T1 inoculation (2 × 106 cells at right armpit) was performed
on 5-week old ICR male mice. Twenty-four hours after inoculation, the mice were
divided randomly into three groups (6 mice/group) and intraperitoneally administered
with 15 10 mg/kg daily, Doxorubicin 1 mg/kg every four days, respectively. Body
weight was measured daily. On the 12th day, all mice were killed, and the tumor was
segregated, weighed, and stored in −80 °C for later use. Tumor inhibitory ratio TI (%)
was calculated as TI (%) = (1 −WT/WC) × 100, where WT and WC were the average
tumor weights of the treated and control group. Body weight inhibitory ratio BI (%)
was calculated as BI (%) = (1 −MT/MC) × 100, where MT and MC were the average
body weights of the treated and control group, respectively. The levels of p-STAT3
and STAT3 in tumor tissues prepared from control or mice treated with DOX or
compound 15 were analyzed by western blot assay.
The experimental mice were cared and handled strictly according to obligations of
the Animal Ethics Committee of China Pharmaceutical University and the National
Institutes of Health (NIH) standard guidelines for the Care and Use of Laboratory
Animal.
Acknowledgments
This research work was supported by National Natural Science Foundation of
China (Grant No. 81402791 and No. 81673298), Natural Science Foundation of the
Jiangsu Higher Education Institutions of China (Grant No. BK20140670), Program
for Changjiang Scholars and Innovative Research Team in University (IRT_15R63),
Project Funded by the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
Abbreviations Used
DMSO, dimethyl sulfoxide; AKT (protein kinase B), a serine/threonine protein
inase; Erk, extracellular regulated kinase; rt, room temperature; MTT,
3-4,5-dehyl-al-2-y]-2,5-dphnetrazolium bromide; SH2, Src hmology 2; STAT, signal

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transducer and activator of transcription; PI, propidium iodide; GAPDH,
glyceraldehyde-3-phosphatedehydrogenase.
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Legends
Table 1. Anti-proliferative activity of the designed compounds and reference Stattic.
Scheme 1. Synthesis of 6 or 7-substituted aminobenzo[b]thiophene 1, 1-dioxides.
Reagents and conditions: (a) SHCH2COOCH3, DMF, 60 °C, 16 h; (b) 10% KOH,
reflux, 3 h, 1 M HCl; (c) Ag2CO3, DMSO, reflux, 16 h; (d) m-CPBA, DCM, reflux, 8
h; (e) Pd(OAc)2, Cs2CO3, BINAP, toluene, reflux, 4-48 h.
Figure 1.The binding mode of BTP and STAT3 SH2 domain by AutoDock4.2.
Figure 2.Design strategy of the new STAT3 inhibitors.
Figure 3.The selection of the most suitable linker by docking experiments. Two
fragments, BTP and benzene, were merged by different linkers derived from known
STAT3 inhibitors.
Figure 4.Docking model of 15 binding to the STAT3 SH2 domain (PDB 1BG1),
generated by AutoDock4.2, carbon atoms of 15 were colored magenta (A) and (B)
viewed by Discovery studio, carbon atoms of 15 were colored gray. The key residues
were shown. Hydrogen bonds were shown as dashed lines and cation-π interactions
were shown as green lines.
Figure 5. Compound 15 inhibited STAT3 activation. (A and B) Western blot analysis
of p-STAT3, STAT3 and IL-6 induced p-STAT3 levels in whole-cell lysates of equal
total protein prepared from MDA-MB-231 cells treated with compound 15 for 24 h.
(C) EMSA analysis of nuclear extracts of equal total protein containing activated
STAT3 from MDA-MB-231 cells treated with compound 15 for 24 h after incubation
with hSIE probe that binds STAT3.
Figure 6.Effects of compound 15 on direct upstream tyrosine kinases p-Jak2 and
p-Src and bypass tyrosine kinase p-Erk. Western blot analysis of p-Jak2, Jak2, p-Src,
Src and p-Erk levels in whole-cell lysates of equal total protein prepared from
MDA-MB-231 cells treated with compound 15 for 24 h.
Figure 7.Effects of compound 15 on the expression of downstream gene, Bcl-2 and
the levels of Cleaved-PARP and Cleaved-Caspase 3. Western blot analysis of Bcl-2,
Cleaved-PARP and Cleaved-Caspase 3 levels in whole-cell lysates of equal total
protein prepared from MDA-MB-231 cells treated with compound 15 for 24 h.
Figure 8.Compound 15 induced human carcinoma MDA-MB-231 cells apoptosis in
Vitro. MDA-MB-231 cells were incubated with 15 at different concentrations for 24 h.
Annexin V-FITC/PI staining was carried out and the percentages of apoptotic cells
were further determined using flow cytometry. ***p < 0.001.
Figure 9.Compound 15 induced ROS generation. MDA-MB-231 cells were incubated

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with 15 at different concentrations for 24 h, and then cells were collected and stained
by DCFH-DA and subjected to flow cytometry. ***p < 0.001, **p < 0.01.
Figure 10.Compound 15 inhibited the colony formation of MDA-MB-231 carcinoma
cells. MDA-MB-231 cells were treated with compound 15 at 0.5-6 ꢀM for 24 h and
cultured for 14 d until the colonies were visible. Crystal violet solution was used to
stain the colonies for 4 h and imaged. ***p < 0.001.
Figure 11.Compound 15 inhibited the growth of mouse sarcoma xenograft tumor in
vivo. (A) The weight of tumor tissues of treated and control mice. (B) Western blot
analysis of p-STAT3 and STAT3 levels in tumor tissues prepared from control (C1,
C2) or mice treated with DOX (D1, D2) or compound 15 (15-1, 15-2). (C) The
growing curves of mice’s body weight. (D) The mice’s body weight before sacrificed.
***p < 0.001.