Discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as mitochondria-targeting antitumor STAT3 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.04.024

STAT3 has been extensively studied as a potential antitumor target. Though studies on regulating STAT3 mainly focus on the inhibition of STAT3 phosphorylation at Tyr705 residue, the phosphorylation at Ser727 residue of STAT3 protein is also closely associated with the mitochondrial import of STAT3 protein. N, N-diethyl-7-aminocoumarin is a fluorescent mitochondria-targeting probe. In this study, a series of STAT3 inhibitors were developed by connecting N, N-diethyl-7-aminocoumarin fluorophore with benzo [b]thiophene 1, 1-dioxide moiety. All designed compounds displayed potent anti-proliferative activity against cancer cells. The representative compound 7a was mainly accumulated in mitochondria visualized by its fluorescence. STAT3 phosphorylation was inhibited by compound 7a at both Tyr705 and Ser727 residues. Compound 7a inhibited STAT3 phosphorylation whereas had no influence on the phosphorylation levels of STAT1, JAK2, Src and Erk1/2, indicating good selectivity of compound 7a. Moreover, compound 7a down-regulated the expression of STAT3 target genes Bcl-2 and Cyclin D1, increased ROS production and remarkably reduced the mitochondrial membrane potential to induce mitochondrial apoptotic pathway. Furthermore, compound 7a in vivo suppressed breast cancer 4T1 implanted tumor growth. Taken together, these results highlighted that compound 7a might be a promising mitochondria-targeting STAT3 inhibitor for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.04.024

Accepted Manuscript
Discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as
mitochondria-targeting antitumor STAT3 inhibitors
Guiping Cai, Wenying Yu, Dongmei Song, Wenda Zhang, Jianpeng Guo, Jiawen Zhu,
Yuhao Ren, Lingyi Kong
PII:
S0223-5234(19)30330-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.024
EJMECH 11259
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2019
Revised Date: 18 March 2019
Accepted Date: 10 April 2019
Please cite this article as: G. Cai, W. Yu, D. Song, W. Zhang, J. Guo, J. Zhu, Y. Ren, L. Kong, Discovery
of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as mitochondria-targeting antitumor
STAT3 inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.024.
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ACCEPTED MANUSCRIPT
Compound 7a acted as a mitochondria-targeting STAT3 inhibitor to exert antitumor
activities in vitro and in vivo.

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Discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide
conjugates as mitochondria-targeting antitumor STAT3 inhibitors
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Guiping Cai1, Wenying Yu1**, Dongmei Song, Wenda Zhang, Jianpeng Guo, Jiawen
Zhu, Yuhao Ren and Lingyi Kong*
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Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’ s Republic of China.
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*Corresponding Author.
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**Corresponding Author.
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Tel/Fax: +86-25-83271405; E-mail: cpu_lykong@126.com (Lingyi Kong);
ywy@cpu.edu.cn (Wenying Yu).
1 Both authors contributed equally to this paper.
Abstract
STAT3 has been extensively studied as a potential antitumor target. Though
studies on regulating STAT3 mainly focus on the inhibition of STAT3 phosphorylation
at Tyr705 residue, the phosphorylation at Ser727 residue of STAT3 protein is also
closely associated with the mitochondrial import of STAT3 protein. N,
N-diethyl-7-aminocoumarin is a fluorescent mitochondria-targeting probe. In this
study, a series of STAT3 inhibitors were developed by connecting N,
N-diethyl-7-aminocoumarin fluorophore with benzo[b]thiophene 1, 1-dioxide moiety.
All designed compounds displayed potent anti-proliferative activity against cancer
cells. The representative compound 7a was mainly accumulated in mitochondria
visualized by its fluorescence. STAT3 phosphorylation was inhibited by compound
7a at both Tyr705 and Ser727 residues. Compound 7a inhibited STAT3
phosphorylation whereas had no influence on the phosphorylation levels of STAT1,
JAK2, Src and Erk1/2, indicating good selectivity of compound 7a. Moreover,
compound 7a down-regulated the expression of STAT3 target genes Bcl-2 and Cyclin
D1, increased ROS production and remarkably reduced the mitochondrial membrane
potential to induce mitochondrial apoptotic pathway. Furthermore, compound 7a in

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vivo suppressed breast cancer 4T1 implanted tumor growth. Taken together, these
results highlighted that compound 7a might be a promising mitochondria-targeting
STAT3 inhibitor for cancer therapy.
Keywords: STAT3, Benzo[b]thiophene 1, 1-dioxide, N, N-diethyl-7-aminocoumarin
fluorophore, Ser727 phosphorylation, Mitochondrial apoptosis pathway, Antitumor
activity.

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1. Introduction
Signal transducer and activator of transcription 3 (STAT3), a latent cytoplasmic
transcription factor, has been documented to play vital roles in a series of biological
processes, such as cell proliferation, differentiation, development, survival, apoptosis
and immunization [1-5]. Persistent activation of STAT3 by cytokines or growth
factors exists in many solid and hematopoietic malignancies [5-8], making it a
promising target for cancer therapy.
As the powerhouse of cells and important intracellular organelles, mitochondria
exert both crucial and lethal functions in physiological and pathological processes that
generate metabolic energy via oxidative phosphorylation, maintain calcium
homeostasis, produce cellular signaling by reactive oxygen species (ROS) and
regulate the intrinsic pathway of apoptosis [9-14]. High level of ROS irreversibly
results in DNA damage and ultimately causes apoptosis and necrosis of cancer cell
[15]. And changes in mitochondrial membrane potential (∆ψm) play an important role
in mitochondrial apoptosis [16]. Emerging evidence indicates that a portion of STAT3
is located in the mitochondria and exerts an influence on the levels of ROS and ∆ψm
to modulate mitochondrial apoptotic pathways [17-24].
STAT3 activation is regulated by phosphorylation on tyrosine 705 (Tyr705)
through receptor tyrosine kinases, which leads to its homodimerization, translocation
into nucleus, DNA-binding and downstream transcriptional activities [17]. Whereas,
except for Tyr705 phosphorylation, phosphorylation at serine 727 (Ser727) residue is
also associated with the transcription, mitochondrial import and other biological
functions of STAT3 protein [20, 21, 25-27]. In consequence, we speculated that
simultaneous suppression of phosphorylation at Tyr705 and Ser727 residues might
suppress STAT3 activation to a greater extent and thereby treat cancer.
The benzo[b]thiophene 1, 1-dioxide (BTP) moiety has the potential to inhibit
Tyr705 phosphorylation of STAT3 as well as prominently suppress proliferation and
enhance apoptosis of tumor cell by means of promoting ROS production. This makes
BTP moiety an important pharmacophore of multiple STAT3 inhibitors, such as
Stattic, HJC0123 and HJC0146 [28-34].

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Fluorescent probes targeting on subcellular organelles could function as real-time
monitors of the therapeutic outcomes [35-37]. Coumarins were selected as fluorescent
probes owing to their good physical properties, such as good water-solubility,
photostability, small size and nontoxicity [38, 39]. Fluorescent N,
N-diethyl-7-aminocoumarin with delocalized lipophilic cation can permeate the living
cell membranes and accumulate preferentially in the mitochondria of cancer cells to
be fluorescent mitochondria-targeted theranostic probe [40-42]. In 2015, Xu Zhang et
al. reported that conjugating N, N-diethyl-7-aminocoumarin fluorophore with
antimalarial drug artemisinin could enhance anticancer activity, owing to the
mitochondrial accumulation of artemisinin by the probe [42]. Similarly, Hong Yao et
al. reported the possible anticancer mechanism of 23-hydroxybetulinic acid
derivatives by combining fluorescent N, N-diethyl-7-aminocoumarin with
23-hydroxybetulinic acid derivatives in 2018 [43].
Herein, a series of coumarin-benzo[b]thiophene 1, 1-dioxide hybrids were
designed, synthesized and biologically evaluated as STAT3 mitochondria-targeting
inhibitors (Figure 1), triggering dual suppression of STAT3 phosphorylation on
Tyr705 and Ser727. To the best of our knowledge, no fluorescent dual STAT3
phosphorylation inhibitors have been reported [29, 30, 34, 44-51]. These hybrids were
discovered to accumulate in mitochondria, cause ∆ψm depolarization, increase
intracellular ROS and further induce apoptosis and necrosis. These new inhibitors
might be promising candidates for cancer therapy.

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Figure 1. Design strategy of the coumarin-benzo[b]thiophene 1, 1-dioxide conjugates for targeting
mitochondria and tumor therapy.
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2. Results and Discussion
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2.1. Chemistry
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Schemes 1 outlined the synthetic routes for compounds 7a-7o. The methyl
benzo[b]thiophene-2-carboxylates 2a-2n were prepared from the commercially
available starting materials 1a-1n and methyl thioglycolate, and were conveniently
hydrolyzed to provide the corresponding acids 3a-3n as described previously [31, 34].
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Using
2-(7-Azabenzotriazol-1-yl)-N,N,Nʹ,Nʹ-tetra-methyluronium
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hexafluorophosphate (HATU) as a coupling reagent, compounds 3a-3o were reacted
with tert-butyl-1-piperazinecarboxylate to afford intermediates 4a-4o, which were
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then
oxidized
by
3-chloroperoxybenzoic
acid
to
produce
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2-carbonylbenzo[b]thiophene 1, 1-dioxides 5a-5o [31, 34]. After deprotecting N-Boc
protection of 5a-5o with trifluoroacetic acid (TFA), the desired compounds 7a-7o
were prepared by coupling with 7-(Diethylamino)coumarin-3-carboxylic acid 6 in the
1
presence of HATU. All hybrids were confirmed by H NMR, 13C NMR, IR, and
HRMS (ESI) spectrum.

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Scheme 1. Synthesis of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates compound
7a-7o. Reagents and conditions: (a) HSCH2COOCH3, DMF, 60 °C, 15 h; (b) 10% KOH, reflux, 3 h, 1
M HCl; (c) tert-butyl-1-piperazine-carboxylate, HATU, DIEA, DCM, rt., 12 h; (d) m-CPBA, DCM,
45 °C, 5 h; (e) (i) TFA, DCM, rt., 2 h; (ii) HATU, DIEA, DCM, rt., 12 h.
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2.2. In vitro cell growth inhibition activity
To evaluate in vitro tumor cell growth inhibitory activity of all compounds and
explore the structure-activity relationships, four STAT3 over-activated human cancer
cell lines, including human breast carcinoma MDA-MB-231 cells and MCF-7 cells,
human colonic carcinoma HCT-116 cells and human hepatocellular carcinoma
HepG2 cells, were treated with designed compounds and the positive control Stattic,
by MTT assay. As shown in Table 1, all designed compounds displayed potent
anti-proliferative activity against four cancer cells. In contrast to BTP (IC50 = 27.65
µM for MDA-MB-231 cells), the intercalation of fluorescent coumarin (7o)
significantly enhanced cell growth inhibitory activity. In general, compounds with
electron-withdrawing substitutes such as nitro substituent (7j and 7l) or halogen
substituents (7c, 7e, 7g, 7h and 7i) were less active than those with electron-donating
substitutes such as methoxy substituent (7m and 7n) except compound 7a, which
bears a bromo substituent at 4 position of the BTP ring. Compared with compound 7o,

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compounds with the methoxy substituent (such as 7m and 7n) exhibited enhanced
anticancer activity, while that with nitro substituent (7j，7k and 7l) led to a decreased
activity. The compound with a 4-bromo substitute on the BTP ring (7a) had a slightly
stronger activity than compounds with a bromine substitute at 5, 6 or 7 position on the
BTP ring (7b, 7c and 7d). The most potent compound 7a inhibited the growth of
MDA-MB-231, HCT-116, HepG2, and MCF-7 cells with IC50 values of 1.43, 1.89,
2.88, and 3.33 µM respectively, which showed 4.6- to 19-fold improvement in
activity than compound 8 (BTP). The anti-proliferative effects of compound 7a on
normal cells were investigated by MTT assays subsequently. As shown in Table 2,
Stattic has growth inhibition activity in both tumor and normal cells, while compound
7a exhibited selective inhibitory activity against cancer cells (IC50 = 14.62 µM for
MCF-10A cells and IC50 = 35.60 µM for L02 cells) and selectivity index was 10.2 and
12.4 respectively, indicating in vitro comparatively better safety of compound 7a than
Stattic.
Table 1
Anti-proliferative activity of the designed compounds 7a-7o, compound 8 and the reference compound
Stattic.
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Compd.
R
IC50 ± SD (µM)a
MDA-MB-231
1.43 ± 0.30
2.23 ± 0.12
2.74 ± 0.37
2.32 ± 0.19
2.24 ± 0.08
2.35 ± 0.16
2.03 ± 0.09
HCT-116
HepG2
MCF-7
4-Br
5-Br
6-Br
7-Br
4-Cl
5-Cl
6-Cl
1.89 ± 0.42
3.83 ± 0.79
2.79 ± 0.41
2.97 ± 0.62
1.57 ± 0.05
4.12 ± 0.60
4.44 ± 0.66
2.88 ± 0.69
5.15 ± 1.00
5.52 ± 0.89
4.74 ± 0.77
4.49 ± 0.46
5.55 ± 0.89
5.29 ± 0.56
3.33 ± 0.23
3.90 ± 0.31
5.04 ± 0.76
3.20 ± 0.32
4.44 ± 0.31
4.39 ± 0.91
5.23 ± 0.41
7a
7b
7c
7d
7e
7f
7g

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4-F
6-F
2.13 ± 0.28
3.20 ± 0.31
3.46 ± 0.28
4.92 ± 0.41
5.22 ± 0.30
1.84 ± 0.16
1.92 ± 0.22
2.32 ± 0.21
27.65 ± 8.93
1.81 ± 0.21
2.33 ± 0.42
4.26 ± 0.73
3.24 ± 0.30
6.74 ± 1.10
4.95 ± 0.39
2.02 ± 0.15
2.30 ± 0.13
3.78 ± 0.37
9.33 ± 1.64
2.16 ± 0.06
3.30 ± 0.11
4.90 ± 1.25
4.91 ± 0.10
7.47 ± 0.92
7.09 ± 0.12
2.94 ± 0.12
4.21 ± 0.95
4.31 ± 0.76
22.87 ± 4.90
3.52 ± 0.79
4.04 ± 0.60
6.35 ± 0.64
4.41 ± 0.60
6.33 ± 0.68
6.76 ± 0.57
3.95 ± 0.80
2.28 ± 0.13
3.00 ± 0.23
15.19 ± 2.18
3.71 ± 0.19
7h
7i
4-NO2
5-NO2
6-NO2
4-OCH3
6-OCH3
H
7j
7k
7l
7m
7n
7o
8
Stattic
a
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The inhibitory effects of individual compounds on the proliferation of the four cell lines were
determined by the MTT assay. SD: standard deviation, all experiments were independently performed
at least three times.
Table 2
Anti-proliferative activity and selectivity index of compound 7a and Stattic on MCF-10A and L02
cells.
Compd.
IC50 ± SD (µM)
MCF-10A
Selectivity Index
IC50 (MCF-10A)/IC50
(MDA-MB-231)
10.2
Selectivity Index
IC50 (L02)/IC50
(HepG2)
12.4
L02
14.62 ± 1.14
6.11 ± 0.18
35.60 ± 0.81
5.20 ± 0.32
7a
3.4
1.5
Stattic
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2.3 Compound 7a reduced colony formation in vitro
STAT3 has been demonstrated to drive colony formation, which was mediated by
its phosphorylation of Tyr705 and Ser727 [20]. Consequently, we performed colony
survival assay to further explore the anti-proliferative activity of compound 7a. As
shown in Figure 2, treatment with 1 µM of compound 7a markedly inhibited the
proliferation of MDA-MB-231 cells and there was almost a cessation of colony
formation at 2 µM compound 7a. Therefore, similar to data from the MTT assays,
compound 7a could suppress the proliferation of STAT3 dependent MDA-MB-231

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Figure 2. Compound 7a inhibited MDA-MB-231 cells proliferation in vitro. MDA-MB-231 cells
were treated with compound 7a at tested concentrations for 24 h. Cells were subcultured to fresh
medium and allowed to grow until the colonies were visible.
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2.4 Compound 7a induced apoptosis in cancer cells
To explore whether the anti-proliferative potency of compound 7a was consistent
with the induction of apoptosis, Annexin V-FITC/PI staining assay was performed by
flow cytometry. The results showed that compound 7a could induce the apoptosis of
MDA-MB-231 cells dose-dependently and the apoptosis rates (early and late
apoptosis) at 0, 1, 2, 4 µM were 4.2%, 6.6%, 9.2% and 20.4% (Figure 3A). To
confirm that apoptotic effects in MDA-MB-231 cells by compound 7a were exerted
via the mitochondrial pathway, we evaluated the protein levels of Cleaved Caspases-9
and Cleaved Caspases-3 and Cleaved PARP in MDA-MB-231 cells after treatment
with compound 7a (1-4 µM) or Stattic (4 µM) for 24 h by western blotting. As shown
in Figure 3B, compound 7a could induce the cleavage of Caspase-9, Caspase-3 and
PARP, which were similar to the positive STAT3 inhibitor Stattic (Figure S1A in the
Supporting Information). Thus, we supposed that cytotoxicity of compound 7a in
cancer cells is mainly through the mitochondrial Caspase-dependent apoptotic
pathway, which was potentially regulated by the deactivation of STAT3 protein.

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Figure 3. Apoptotic effects of compound 7a on MDA-MB-231 cells. MDA-MB-231 cells were
treated with compound 7a at tested concentrations for 24 h. (A) Apoptotic effects were detected using
Annexin V-FITC/PI staining by flow cytometry. (B) The histograms for % of apoptosis (early and late
stages of apoptosis). ***, p < 0.001, **, p < 0.01. (C) The effects of compound 7a on protein levels of
Caspase-9, Cleaved Caspase-9, Caspase-3, Cleaved Caspase-3, PARP and Cleaved PARP. Cell lysates
were harvested and analyzed using the corresponding antibodies by western blotting. (D) Quantitative
analysis of western blotting by Image Lab program with GAPDH as the internal control. ***, p < 0.001.
**, p < 0.01.
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2.5. Compound 7a accumulated in mitochondria
It
was
reported
that
compounds
conjugated
with
the
N,
N-diethyl-7-aminocoumarin fluorophore might accumulate in mitochondria [41, 42].
To determine the role of compound 7a as a mitochondrial probe, we investigated the

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subcellular localization of compound 7a. MitoTracker (mitochondria-specific dye;
Figure 4b), and LysoTracker (Lysosome-specific dye; Figure S2b in the Supporting
Information) were employed for colocalization experiments in human breast
carcinoma MDA-MB-231 cells and MCF-7 cells. The preferential concentration of
compound 7a for study was 2 µM and 5 µM respectively. MDA-MB-231 cells and
MCF-7 cells were stained by compound 7a after 60 min of incubation and strong blue
fluorescence was detected as shown in Figure 4a and Figure S2a (Supporting
Information). The colocalization study revealed that the fluorescence of compound 7a
overlapped exactly with that of MitoTracker Red, with an extensive magenta color in
the merged image (Figure 4c) but that of Lysosome, with only partial colocalization
(Figure S2c in the Supporting Information). The results confirmed that compound 7a
was accumulated to mitochondria by the aid of the fluorescent N,
N-diethyl-7-aminocoumarin moiety.
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Figure 4. Subcellular localization studies of compound 7a in MDA-MB-231 cells and MCF-7 cells.
Images of cells pretreated, respectively, with compound 7a for 60 min, and subsequently, 1 µM
MitoTracker Red (Figure 4b) or 1 µM LysoTracker Red (Figure S2b in the Supporting Information) for
30 min. (a) Probe fluorescence, blue channel; (b) MitoTracker, red channel; (c) The overlap.
2.6. Compound 7a affected the Mitochondrial Membrane Potential (∆ψm)
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The level of mitochondrial membrane potential (∆ψm) was more elevated in cancer
cells than that in normal cells, which could be exploited therapeutically to selectively
target compounds to the mitochondria of cancer cells [52]. And changes in ∆ψm have
been shown to play an important role in mitochondrial apoptosis [16]. Taking into
consideration that the hybrids were designed to target mitochondria and to induce
mitochondrial apoptosis, we continued to investigate the effect of compound 7a on
∆ψm of MDA-MB-231 cells using JC-1 assay kit. Changes in ∆ψm were reflected by
the increase of J-monomers (green fluorescence) and concurrent decrease of
J-aggregates (red fluorescence). As shown in Figure 5, treatment with compound 7a
led to increased intensity of green fluorescence in a dose-dependent manner. These
data suggested that compound 7a induced the depolarization of mitochondrial
membrane potential and subsequently induced mitochondrial damage and apoptosis of
cancer cells.
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Figure 5. Compound 7a affected mitochondrial membrane potential in MDA-MB-231 cells. (A)
MDA-MB-231 cells were treated with compound 7a at tested concentrations for 24 h analyzed using
flow cytometer after JC-1 staining. (B) The ratios of red fluorescence to green fluorescence. **, p <
0.01, ***, p < 0.001.
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2.7. Compound 7a induced the ROS generation in cancer cells
Mitochondria are the main organelle that produce ROS and previous studies have
suggested that constitutively activated STAT3 phosphorylated on Ser727 in the

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mitochondria could decrease the amount of ROS, causing decreased apoptosis and
enhanced tumor cell growth [5, 21, 53]. We next evaluated ROS production after
treating with compound 7a at different concentrations in MDA-MB-231 cells for 24 h
by flow cytometry analysis. As shown in Figure 6, compound 7a induced ROS
accumulation in a dose-dependent manner, indicating that compound 7a could
increase ROS production which is also responsible for mitochondrial apoptosis and
necrosis of cancer cell.
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Figure 6. Compound 7a induced ROS generation. MDA-MB-231 cells were treated with compound
7a at tested concentrations for 24 h. (A) The ROS accumulation in MDA-MB-231 cells measured by
flow cytometer using DCFH-DA staining. (B) Relative ROS levels of MDA-MB-231 cells after
treatment with compound 7a for 24 h. *, p < 0.05, **, p < 0.01.
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2.8. Compound 7a inhibited STAT3 tyrosine phosphorylation and serine
phosphorylation
BTP moiety could suppress the phosphorylation of STAT3 on Tyr705 but have
minimal impact on STAT3 phosphorylation on Ser727 [33]. To examine whether the
aforementioned improved antitumor activities in vitro were derived from addition of
the mitochondria-targeting N, N-diethyl-7-aminocoumarin fluorophore to inhibit
STAT3 phosphorylation at Ser727 residue, the p-STAT3 (Tyr705 and Ser727) protein
levels were investigated on MDA-MB-231 cells by western blotting assays.
MDA-MB-231 cells were treated with compound 7a (1-4 µM) or Stattic (4 µM) for
24 h and the phosphorylated and total STAT3 protein levels were examined. As

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shown in Figure 7A, compound 7a markedly inhibited STAT3 phosphorylation at
Tyr705 residue as well as STAT3 phosphorylation at Ser727 residue in a
dose-dependent manner but total STAT3 proteins remain unaffected, while Stattic
only inhibited STAT3 phosphorylation at Tyr705 residue (Figure S1B in the
Supporting Information). These results indicated that compound 7a was a dual
STAT3 inhibitor that including BTP moiety to suppress the phosphorylation of
STAT3 on Tyr705 and the mitochondria-targeting N, N-diethyl-7-aminocoumarin
fluorophore to make compound accumulate to mitochondria, inhibit STAT3
phosphorylation on Ser727 and thereby induce mitochondrial damage and apoptosis.
2.9. Compound 7a inhibited STAT3 DNA-binding activity
Maximal transcriptional activity of STAT3 requires both phosphorylation on
tyrosine 705 and serine 727 [27]. In consideration of the inhibitory effect of
compound 7a on p-STAT3 in tumor cells, we performed electrophoretic mobility shift
assay (EMSA) to evaluate the inhibition of STAT3:STAT3 DNA-binding activity.
After treated with compound 7a (1-4 µM) or Stattic (4 µM) for 24 h, nuclear extracts
of MDA-MB-231 cells were incubated with hSIE probe and EMSA assay was
performed. The capability of STAT3:STAT3 binding DNA was decreased by
compound 7a at a start concentration at 1 µM and an obvious diminish at 4 µM as
compared with Stattic at 4 µM (Figure 7B), which indicated that compound 7a
effectively suppressed STAT3 activation.
2.10. Compound 7a did not affect STAT1 phosphorylation and typical upstream or
other kinases of STAT3
STAT1, another member of the STATs family, can inhibit cancer cell
proliferation and induce apoptosis. As shown in Figure 7A and Figure S1B by western
blotting assays, compound 7a and Stattic had little effect on STAT1 phosphorylation.
Upstream kinases (such as JAK2, Src) can phosphorylate tyrosine residues of STAT3
and the Erk family of mitogen-activated protein kinases can stimulates STAT3
phosphorylation on Ser727 [54]. We evaluated the effects of compound 7a on the
phosphorylation of JAK2, Src and Erk1/2 by western blotting assays to investigate the
selectivity of compound 7a targeting the phosphorylation of STAT3. As shown in

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Figure 7C, compound 7a had no effect on p-JAK2, p-Src and p-Erk1/2 as same as
Stattic (Figure S1C). These results showed that the phosphorylation inhibition of
STAT3 by compound 7a was selective and independent of these related kinases.
2.11. Molecular docking study
Given that compound 7a could inhibit the phosphorylation of STAT3 without
influencing other related kinases, to determine whether compound 7a is a direct
STAT3 SH2 domain inhibitor, we performed docking assay for predicting potential
binding modes of compound 7a with STAT3 SH2 domain (PDB: 1BG1). As shown in
Figure 8, the benzo[b]thiophene 1, 1-dioxide and coumarin fragments of compound
7a perfectly overlapped with the native hot spot pTyr705 site and side pocket of
STAT3 SH2 domain via forming four hydrogen bonding interactions with the Arg609
residues (distance = 1.593 Å and 2.144 Å), Lys591 residues (distance = 2.146 Å) and
Arg595 residues (distance = 2.185 Å). The predicted binding energy was -7.8
kcal/mol. Moreover, suitable length and angle of piperazine fragment contributed to
interact with the two binding pocket of STAT3. Therefore, compound 7a was
speculated to be a direct STAT3 SH2 domain inhibitor.
2.12. Compound 7a affected the expression of STAT3 targeted genes
Several proteins such as anti-apoptotic Bcl-2 protein and Cyclin D1 involved in
cell cycle progression have been found to associate directly with STAT3. Hence,
looking for more insights in the mechanism of compound 7a on the STAT3 pathway,
we estimated the effects of compound 7a on the expression levels of Bcl-2 and Cyclin
D1 using western blotting and compared with Stattic (Figure S1A). After treatment
with compound 7a for 24 h, Bcl-2 and Cyclin D1 protein levels were decreased in a
concentration-dependent manner (Figure 7D). These results demonstrated that
compound 7a suppressed the protein levels of p-STAT3 (Y705) and p-STAT3 (S727),
inhibited STAT3:STAT3 DNA-binding activity and consequently down-regulated the
expression of STAT3 target genes.

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Figure 7. Compound 7a inhibited STAT3 activation and the effects of compound 7a on protein
levels of STAT3 related proteins. MDA-MB-231 cells were treated with compound 7a at tested
concentrations for 24 h and then the cell lysates were harvested and analyzed using the corresponding
antibodies by western blotting or EMSA analysis. (A) The effects of compound 7a on STAT3, p-STAT3
(Y705), p-STAT3 (S727), STAT1 and p-STAT1 (Y701) protein levels in MDA-MB-231 cells by
western blotting analysis using GAPDH as a control. (B) EMSA analysis of nuclear extracts of equal
total protein containing activated STAT3 from MDA-MB-231 cells treated with compound 7a or
Stattic for 24 h after incubation with hSIE probe that binds STAT3. (C) The effects of compound 7a on
protein levels of upstream tyrosine kinases p-JAK2, Src, p-Src and bypass tyrosine kinase Erk1/2 and
p-Erk1/2. (D) The effects of compound 7a on expression levels of STAT3 related downstream gene
Bcl-2 and Cyclin D1 and the quantitative analysis of western blotting by Image Lab program with
GAPDH as the internal control. **, p < 0.01, ***, p < 0.001.
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Figure 8. Molecular docking model of compound 7a binding to SH2 domain of STAT3 generated
by Autodock4.2. (A) Carbon atoms of compound 7a were colored magenta. STAT3 protein was
represented as white ribbon and the key residues were shown. Hydrogen bonds were represented by
green lines. (B) Compound 7a bound to hot spot pTyr705 site and side pocket of STAT3 SH2 domain
(For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article.)
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2.13. Compound 7a exhibited antitumor activity in vivo
To evaluate in vivo antitumor effect of compound 7a, we established a
mouse-xenograft model bearing inoculation of STAT3 over-expressed and
over-activated mouse breast cancer 4T1 cells as reported previously [34, 50, 55]. The
mice were randomly assigned and treated with doses (10 mg/kg, 20 mg/kg) of
compound 7a or the vehicle daily by intraperitoneal injection for 14 consecutive days.
As shown in Figure 9, compound 7a significantly inhibited tumor volume (52.29%
decrease at 10 mg/kg, 61.20% decrease at 20 mg/kg) and weight (39.67% decrease at
10 mg/kg, 56.86% decrease at 20 mg/kg). Western blotting assays showed that
compound 7a inhibited the p-STAT3 (Y705) level in tumor tissue. All these results
indicated that compound 7a possessed potent antitumor activity against implanted
4T1 breast tumors growth.

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Figure 9. Compound 7a inhibited the growth of mouse sarcoma xenograft tumors in vivo. (A) The
weight of tumor tissues of control and treated mice. (B) The tumor growth inhibitory rates of
compound 7a (10 mg/kg, 20 mg/kg). (C) The growing curves of mice’s tumor volume. (D) Anatomical
mice’s tumor tissue untreated or treated with compound 7a (10 mg/kg, 20 mg/kg). (E) Western blotting
analysis of p-STAT3 (Y705) and STAT3 levels in tumor tissues prepared from control or mice treated
with compound 7a (10 mg/kg, 20 mg/kg). **, p < 0.01, ***, p < 0.001.
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3. Conclusions
In the present study, we have designed and synthesized a series of
mitochondria-targeting STAT3 inhibitors by conjugating BTP moiety with N,
N-diethyl-7-aminocoumarin fluorophore, and evaluated their anticancer activities in
vitro and in vivo. In contrast to the compound BTP, new STAT3 inhibitors revealed
remarkable higher potency, represented by the most potent compound 7a. Compound

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7a showed more anticancer activity and comparatively better safety than the positive
STAT3 inhibitor Stattic. Subcellular localization studies visualized that compound 7a
was accumulated to mitochondria by the aid of the fluorescent N,
N-diethyl-7-aminocoumarin moiety. The promoted ROS and reduced ∆ψm by
compound 7a induced mitochondrial apoptosis pathway and the cessation of tumor
cells colony formation. Further biological evaluation showed that compound 7a
inhibited Tyr705 and Ser727 STAT3 phosphorylation, STAT3:STAT3 DNA-binding
activity and regulated the expression of the STAT3 downstream genes Bcl-2 and
Cyclin D1, with little effect on the phosphorylation of STAT1 and related kinases
(JAK2, Src and Erk1/2). Docking studies revealed that compound 7a might be a
STAT3-SH2 domain inhibitor. Furthermore, compound 7a significantly inhibited
breast cancer 4T1 implanted tumor growth in vivo. In summary, all evidences
highlight that compound 7a is a promising mitochondria-targeting STAT3 inhibitor
for cancer therapy.
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4. Materials and Methods
4.1. Chemistry
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All chemicals were obtained from commercial suppliers and used without further
purification. Reaction progress was monitored using analytical thin layer
chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang
Chemical Plant, Qingdao, China) and the spots were detected under UV light (254
nm). Melting point was measured on an XT-4 point instrument and uncorrected. IR
1
(KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer. H NMR and
13C NMR spectra were measured on a Bruker ACF-500 spectrometer at 25 °C and
referenced to TMS. Chemical shifts are reported in ppm (δ) using the residual solvent
line as internal standard. Splitting patterns were designed as s, singlet; br.s, broad
single; 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 1100 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.

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4.1.1. General procedures for the preparation of 2a-2n
To a stirred solution of compound 1a-1n (9.90 mmol) in DMF (20 mL) were
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-2n [31, 34].
4.1.1.1. Methyl 4-bromobenzo[b]thiophene-2-carboxylate (2a)
Yield 89%, white solid; m.p. 77-78 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.12 (d,
J = 8.2 Hz, 1H), 8.01 (d, J = 0.6 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.46 (t, J = 7.9 Hz,
1H), 3.91 (s, 3H).
4.1.1.2. Methyl 5-bromobenzo[b]thiophene-2-carboxylate (2b)
Yield 92%, white solid; m.p. 82-84 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.28 (d,
J = 1.9 Hz, 1H), 8.17 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 8.7, 2.0 Hz, 1H),
3.89 (s, 3H).
4.1.1.3. Methyl 6-bromobenzo[b]thiophene-2-carboxylate (2c)
Yield 94%, white solid; m.p. 79-81 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.40 (s,
1H), 8.21 (s, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.64 (dd, J = 8.6, 1.8 Hz, 1H), 3.89 (s,
3H).
4.1.1.4. Methyl 7-bromobenzo[b]thiophene-2-carboxylate (2d)
1
Yield 94%, white solid; m.p. 87-88 °C; H NMR (600 MHz, CDCl3) δ 8.17 (s,
1H), 7.86 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.31 (dd, J = 16.2, 8.4 Hz, 1H),
3.99 (s, 1H).
4.1.1.5. Methyl 4-chlorobenzo[b]thiophene-2-carboxylate (2e)
1
Yield 89%, white solid; m.p. 80-82 °C; H NMR (600 MHz, CDCl3) δ 8.78 (s,
1H), 8.32 (s, 1H), 7.89 (d, J = 7.4 Hz, 1H), 7.44 (dd, J = 8.9, 6.9 Hz, 2H), 3.89 (s,
3H).
4.1.1.6. Methyl 5-chlorobenzo[b]thiophene-2-carboxylate (2f)

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Yield 91%, gray solid; m.p. 72-74 °C; H NMR (600 MHz, DMSO-d6) δ 8.15 (s,
1H), 8.11 (s, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.55 (dd, J = 8.7, 2.2 Hz, 1H), 3.89 (s,
3H).
4.1.1.7. Methyl 6-chlorobenzo[b]thiophene-2-carboxylate (2g)
Yield 90%, white solid; m.p. 67-69 °C; 1H NMR (600 MHz, CDCl3) δ 8.02 (d, J =
6.4 Hz, 1H), 7.88-7.84 (m, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.39 (dd, J = 8.6, 1.8 Hz,
1H), 3.97 (s, 3H).
4.1.1.8. Methyl 4-fluorobenzo[b]thiophene-2-carboxylate (2h)
Yield 85%, white solid; m.p. 75-77 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.11 (s,
1H), 7.93 (t, J = 14.4 Hz, 1H), 7.64-7.49 (m, 1H), 7.29 (dd, J = 10.1, 8.1 Hz, 1H),
3.90 (s, 3H).
4.1.1.9. Methyl 6-fluorobenzo[b]thiophene-2-carboxylate (2i)
1
Yield 93%, orange solid; m.p. 89-90 °C; H NMR (600 MHz, DMSO-d6) δ 8.22
(d, J = 7.2 Hz, 1H), 8.11-8.05 (m, 1H), 8.04-7.99 (m, 1H), 7.42-7.34 (m, 1H), 3.89 (s,
3H).
4.1.1.10. Methyl 4-nitrobenzo[b]thiophene-2-carboxylate (2j)
Yield 70%, brown solid; m.p. 111-114 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.63
(d, J = 8.8 Hz, 1H), 8.12 (d, J = 28.1 Hz, 1H), 7.98-7.88 (m, 1H), 7.80 (t, J = 8.0 Hz,
1H), 3.95 (s, 3H).
4.1.1.11. Methyl 5-nitrobenzo[b]thiophene-2-carboxylate (2k)
1
Yield 76%, pale yellow solid; m.p. 87-88 °C; H NMR (600 MHz, DMSO-d6) δ
9.00 (s, 1H), 8.44 (s, 1H), 8.37 (d, J = 8.9 Hz, 1H), 8.32 (d, J = 8.6 Hz, 1H), 3.92 (s,
3H).
4.1.1.12 Methyl 6-nitrobenzo[b]thiophene-2-carboxylate (2l)
Yield 70%, brown solid; m.p. 67-68 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.38 (s,
1H), 8.28-8.25 (m, 1H), 7.76-7.71 (m, 1H), 7.26 (dd, J = 25.2, 10.0 Hz, 1H), 3.93 (s,
3H).
4.1.1.13. Methyl 4-methoxybenzo[b]thiophene-2-carboxylate (2m)

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Yield 91%, white solid; m.p. 69-71 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.06 (s,
1H), 7.62-7.58 (m, 1H), 7.51-7.47 (m, 1H), 6.97 (d, J = 7.9 Hz, 1H), 3.94 (s, 3H),
3.87 (s, 3H).
4.1.1.14. Methyl 6-methoxybenzo[b]thiophene-2-carboxylate (2n)
1
Yield 84%, white solid; m.p. 77-79 °C; H NMR (600 MHz, CDCl3) δ 7.97 (s,
1H), 7.74 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 7.05-6.99 (m, 1H), 3.92 (s,
3H), 3.89 (s, 3H).
4.1.2. General procedures for the preparation of 3a-3n
The solution of compound 2a-2n (1.1 mmol) in water (10 mL) was stirred and
then potassium hydroxide pellets (5.4 mmol) were added, which was refluxed for 3 h.
The aqueous layer was then acidified to pH 1 with 1 M 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-3n [31, 34].
4.1.2.1 4-bromobenzo[b]thiophene-2-carboxylic acid (3a)
Yield 97%, white solid; m.p. 140-143 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.78
(s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.96 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.44 (dt, J =
13.1, 2.7 Hz, 1H).
4.1.2.2. 5-bromobenzo[b]thiophene-2-carboxylic acid (3b)
Yield 95%, white solid; m.p. 133-135 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.18
(d, J = 1.8 Hz, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.85 (s, 1H), 7.57 (dd, J = 8.6, 1.9 Hz,
1H).
4.1.2.3. 6-bromobenzo[b]thiophene-2-carboxylic acid (3c)
Yield 96%, white solid; m.p. 122-124 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.27
(s, 1H), 7.91-7.85 (m, 1H), 7.84 (s, 1H), 7.55 (dd, J = 8.5, 1.7 Hz, 1H).
4.1.2.4. 7-bromobenzo[b]thiophene-2-carboxylic acid (3d)
Yield 93%, white solid; m.p. 143-146 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.27
(s, 1H), 8.09-8.03 (m, 1H), 7.78 (dd, J = 7.6, 0.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H).
4.1.2.5. 4-chlorobenzo[b]thiophene-2-carboxylic acid (3e)
Yield 90%, white solid; m.p. 213-216 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.97

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(s, 1H), 7.96 (d, J = 3.9 Hz, 1H), 7.50 (dd, J = 7.6, 1.0 Hz, 1H), 7.48 (dd, J = 9.3, 4.4
Hz, 1H).
4.1.2.6. 5-chlorobenzo[b]thiophene-2-carboxylic acid (3f)
1
Yield 95%, pink solid; m.p. 233-236 °C; H NMR (600 MHz, DMSO-d6) δ
8.08-8.06 (m, 1H), 8.08-8.03 (m, 3H), 8.05 (d, J = 3.4 Hz, 1H), 7.50 (dd, J = 8.6, 2.1
Hz, 1H).
4.1.2.7. 6-chlorobenzo[b]thiophene-2-carboxylic acid (3g)
Yield 94%, white solid; m.p. 113-116 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.12
(d, J = 1.8 Hz, 1H), 7.93 (t, J = 9.4 Hz, 1H), 7.89 (s, 1H), 7.43 (dd, J = 8.5, 2.0 Hz,
1H).
4.1.2.8. 4-fluorobenzo[b]thiophene-2-carboxylic acid (3h)
Yield 95%, white solid; m.p. 238-240 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.72
(s, 1H), 8.05 (s, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.55 (td, J = 8.1, 5.2 Hz, 1H), 7.28 (dd,
J = 10.3, 8.0 Hz, 1H).
4.1.2.9 6-fluorobenzo[b]thiophene-2-carboxylic acid (3i)
1
Yield 94%, orange solid; m.p. 193-196 °C; H NMR (600 MHz, DMSO-d6) δ
13.30 (s, 1H), 8.11 (s, 1H), 8.05 (dd, J = 9.0, 5.3 Hz, 1H), 7.97 (dd, J = 9.4, 2.2 Hz,
1H), 7.35 (td, J = 9.0, 2.4 Hz, 1H).
4.1.2.10. 4-nitrobenzo[b]thiophene-2-carboxylic acid (3j)
Yield 89%, white solid; m.p. 243-246 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.85
(s, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 7.76 (t, J = 8.0
Hz, 1H).
4.1.2.11. 5-nitrobenzo[b]thiophene-2-carboxylic acid (3k)
Yield 75%, white solid; m.p. 145-146 °C; 1H NMR (600 MHz, CDCl3) δ 13.70 (s,
1H), 8.85 (d, J = 2.2 Hz, 1H), 8.22 (d, J = 9.0 Hz, 1H), 8.18 (t, J = 2.7 Hz, 1H), 8.17
(d, J = 2.1 Hz, 1H).
4.1.2.12. 6-nitrobenzo[b]thiophene-2-carboxylic acid (3l)
1
Yield 78%, brown solid; m.p. 161-164 °C; H NMR (600 MHz, DMSO-d6) δ
13.93 (s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.24 (dd, J = 12.7, 5.5 Hz, 1H), 7.56 (d, J =
8.2 Hz, 1H), 7.21-7.12 (m, 1H).

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4.1.2.13. 4-methoxybenzo[b]thiophene-2-carboxylic acid (3m)
Yield 91%, white solid; m.p. 199-201 °C;1H NMR (600 MHz, DMSO-d6) δ 13.46
(s, 1H), 8.01 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 6.98 (d, J = 7.9
Hz, 1H), 3.96 (s, 3H).
4.1.2.14. 6-methoxybenzo[b]thiophene-2-carboxylic acid (3n)
Yield 93%, white solid; m.p. 143-146 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.25
(s, 1H), 8.01 (s, 1H), 7.89-7.84 (m, 1H), 7.60 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.8,
2.4 Hz, 1H), 3.84 (s, 3H).
4.1.3. General procedures for the preparation of 4a-4o
To a solution of 3a-3o (1.94 mmol) in anhydrous dichloromethane (20.0 mL) and
HATU (2.33 mmol) in the presence of DIEA (3.89 mmol),
tert-butylpiperazine-1-carboxylate (2.14 mmol) was added and stirred for 12 h. After
completion, dilute hydrochloric acid solution was added, extracted with
dichloromethane and washed with saturated sodium carbonate solution three times.
The combined organic layers were washed with brine and dried with sodium sulfate,
filtered, concentrated, and purified by silica gel chromatography with petroleum
ether/ethyl acetate as eluent to give pure 4a-4o [34].
4.1.3.1.
tert-butyl
4-(4-bromobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4a)
Yield 93%, white solid; m.p. 113-116 °C; 1H NMR (600 MHz, CDCl3) δ 7.81 (d,
J = 8.1 Hz, 1H), 7.63 (s, 1H), 7.59 (d, J = 7.6 Hz, 1H), 3.82-3.74 (m, 4H), 3.55 (s, 4H),
1.50 (s, 9H).
4.1.3.2.
tert-butyl
4-(5-bromobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4b)
Yield 95%, white solid; m.p. 148-151 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.15
(d, J = 1.8 Hz, 1H), 8.01 (t, J = 9.9 Hz, 1H), 7.72 (s, 1H), 7.59 (dt, J = 11.9, 5.9 Hz,
1H), 3.63 (d, J = 22.6 Hz, 4H), 3.43 (s, 4H), 1.42 (s, 9H).
4.1.3.3.
tert-butyl
4-(6-bromobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4c)

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Yield 85%, white solid; m.p. 120-124 °C; 1H NMR (600 MHz, CDCl3) δ 7.86 (t, J
= 3.2 Hz, 1H), 7.74 (dd, J = 8.0, 1.6 Hz, 1H), 7.29 (dd, J = 18.6, 8.2 Hz, 2H), 3.61 (s,
4H), 3.51 (s, 4H), 1.47(s, 9H).
4.1.3.4.
tert-butyl
4-(7-bromobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4d)
Yield 84%, white solid; m.p. 156-158 °C; 1H NMR (600 MHz, CDCl3) δ 7.77 (d,
J = 8.0 Hz, 1H), 7.58 (d, J = 7.1 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.29 (t, J = 7.8 Hz,
1H), 3.74 (t, J = 4.6 Hz, 4H), 3.52 (s, 4H), 1.48 (s, 9H).
4.1.3.5.
tert-butyl
4-(4-chlorobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4e)
Yield 89%, white solid; m.p. 107-109 °C; 1H NMR (600 MHz, CDCl3) δ 7.75 (t, J
= 10.6 Hz, 1H), 7.63 (s, 1H), 7.40 (dd, J = 7.7, 0.6 Hz, 1H), 7.36-7.31 (m, 1H),
3.80-3.72 (m, 4H), 3.50 (d, J = 25.6 Hz, 4H), 1.48 (s, 9H).
4.1.3.6.
tert-butyl
4-(5-chlorobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4f)
Yield 88%, white solid; m.p. 124-126 °C; 1H NMR (600 MHz, CDCl3) δ 7.79 (d,
J = 1.8 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.37 (dd, J = 8.6, 2.0 Hz, 1H),
3.73 (s, 4H), 3.47 (s, 4H), 1.47 (d, J = 8.2 Hz, 9H).
4.1.3.7.
tert-butyl
4-(6-chlorobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (4g)
Yield 65%, white solid; m.p. 119-121 °C; 1H NMR (600 MHz, CDCl3) δ 7.83 (d,
J = 0.6 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.37 (dd, J = 8.5, 1.2 Hz, 1H),
3.78-3.69 (m, 4H), 3.50 (d, J = 13.7 Hz, 4H), 1.48 (s, 9H).
4.1.3.8. tert-butyl 4-(4-fluorobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate
(4h)
Yield 92%, yellow solid; m.p. 126-130 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.87
(dd, J = 15.5, 6.6 Hz, 1H), 7.76 (d, J = 0.5 Hz, 1H), 7.48 (t, J = 13.3, 6.7 Hz, 1H),
7.26 (dt, J = 13.9, 7.0 Hz, 1H), 3.67 (s, 4H), 3.43 (s, 4H),1.45 (s, 9H).
4.1.3.9. tert-butyl 4-(6-fluorobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate
(4i)

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Yield 94%, white solid; m.p. 155-157 °C; H NMR (600 MHz, DMSO-d6) δ 7.96
(t, J = 3.2 Hz, 1H), 7.95 (s, 1H), 7.77 (s, 1H), 7.33 (td, J = 9.1, 2.2 Hz, 1H), 3.67 (s,
4H), 3.43 (s, 4H), 1.42 (s, 9H).
4.1.3.10. tert-butyl 4-(4-nitrobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate
(4j)
Yield 88%, yellow solid; m.p. 120-122 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.56
(d, J = 8.1 Hz, 1H), 8.38 (t, J = 11.0 Hz, 1H), 8.22 (s, 1H), 7.75-7.67 (m, 1H),
3.71-3.63 (m, 4H), 3.44 (s, 4H), 1.41 (d, J = 8.1 Hz, 9H).
4.1.3.11. tert-butyl 4-(5-nitrobenzo[b]thiophene-2-carbonyl) piperazine-1-carboxylate
(4k)
Yield 84%, white solid; m.p. 123-126 °C; 1H NMR (600 MHz, CDCl3) δ 8.70 (d,
J = 2.0 Hz, 1H), 8.24 (dd, J = 8.9, 2.2 Hz, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.61 (s, 1H),
3.74 (s, 4H), 3.52 (s, 4H), 1.46 (d, J = 7.3 Hz, 9H).
4.1.3.12. tert-butyl 4-(6-nitrobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate
(4l)
Yield 89%, yellow solid; m.p. 144-146 °C; 1H NMR (600 MHz, DMSO-d6) δ 9.13
(d, J = 1.7 Hz, 1H), 8.26 (dd, J = 8.8, 2.1 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 7.93 (s,
1H), 3.66 (s, 4H), 3.44 (s, 4H), 1.42 (s, 9H).
4.1.3.13.
tert-butyl
4-(4-methoxybenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate (4m)
Yield 92%, orange solid; m.p. 92-96 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.66 (s,
1H), 7.56 (d, J = 8.1 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 7.9 Hz, 1H), 3.93
(s, 3H), 3.66 (s, 4H), 3.42 (s, 4H), 1.42 (s, 9H).
4.1.3.14.
tert-butyl
4-(6-methoxybenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate (4n)
Yield 71%, white solid; m.p. 103-106 °C; 1H NMR (600 MHz, CDCl3) δ 7.64 (m,
1H), 7.40 (s, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.00 (dd, J = 8.8, 2.3 Hz, 1H), 3.86 (s, 3H),
3.78-3.70 (m, 4H), 3.53-3.46 (m, 4H), 1.47 (s, 9H).
4.1.3.15. tert-butyl 4-(benzo[b]thiophene-2-carbonyl) piperazine-1-carboxylate (4o)
1
Yield 92%, white solid; m.p. 136-138 °C; H NMR (600 MHz, CDCl3) δ

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7.89-7.78 (m, 2H), 7.48 (s, 1H), 7.43-7.37 (m, 2H), 3.78-3.70 (m, 4H), 3.51 (s, 4H),
1.47 (d, J = 8.9 Hz, 9H).
4.1.4. General procedures for the preparation of 5a-5o and 8
3-Chloroperbenzoic acid (2.5 mmol) was added to the stirred solution of
compound 4a-4o and benzo[b]thiophene (1.0 mmol) in dichloromethane (20 mL) in
portions over a 20-30 min 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 min
and extracted with dichloromethane. The organic phase was washed with aqueous
sodium bicarbonate (2 × 10 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-5o and 8 [31, 34].
4.1.4.1.
tert-butyl
4-(4-bromo-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5a)
Yield 84%, white solid; m.p. 178-180 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.98
(dd, J = 7.8, 5.6 Hz, 2H), 7.79 (d, J = 0.7 Hz, 1H), 7.65-7.61 (m, 1H), 3.57 (d, J =
13.1 Hz, 4H), 3.45-3.35 (m, 4H), 1.41 (s, 9H).
4.1.4.2.
tert-butyl
4-(5-bromo-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5b)
Yield 79%, white solid; m.p. 138-141 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.93
(d, J = 8.3 Hz, 1H), 7.91 (t, J = 4.0 Hz, 2H), 7.74 (s, 1H), 3.57 (s, 4H), 3.39 (s, 4H),
1.41 (s, 9H).
4.1.4.3.
tert-butyl
4-(6-bromo-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5c)
Yield 85%, white solid; m.p. 156-158 °C; 1H NMR (600 MHz, CDCl3) δ 7.86 (t, J
= 3.2 Hz, 1H), 7.74 (dd, J = 8.0, 1.6 Hz, 1H), 7.29 (dd, J = 18.6, 8.2 Hz, 2H), 3.61 (d,
J = 56.7 Hz, 4H), 3.51 (dd, J = 15.2, 10.5 Hz, 4H), 1.49-1.45 (m, 9H).
4.1.4.4.
piperazine-1-carboxylate (5d)
Yield 71%, white solid; m.p. 127-129 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.86
tert-butyl
4-(7-bromo-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)

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(dd, J = 7.4, 1.5 Hz, 1H), 7.77 (s, 1H), 7.66 (qd, J = 7.5, 2.3 Hz, 1H), 3.57 (s, 4H),
3.38 (s, 4H), 1.41 (s, 9H).
4.1.4.5.
tert-butyl
4-(4-chloro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5e)
Yield 47%, white solid; m.p. 117-119 °C; 1H NMR (600 MHz, CDCl3) δ 7.64 (d,
J = 7.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.53 (dd, J = 8.7, 6.8 Hz, 2H), 3.68 (s, 4H),
3.56-3.50 (m, 4H), 1.47 (s, 9H).
4.1.4.6.
tert-butyl
4-(5-chloro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5f)
Yield 47%, white solid; m.p. 183-186 °C; 1H NMR (600 MHz, CDCl3) δ 7.67 (d,
J = 8.1 Hz, 1H), 7.58-7.51 (m, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.23 (d, J = 12.8 Hz, 1H),
3.60 (s, 4H), 3.51 (s, 4H), 1.47 (s, 9H).
4.1.4.7.
tert-butyl
4-(6-chloro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)
piperazine-1-carboxylate (5g)
1
Yield 53%, white solid; m.p. 132-134 °C; H NMR (600 MHz, CDCl3) δ
7.71-7.69 (m, 1H), 7.56 (dt, J = 9.4, 4.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.28 (d, J =
11.7 Hz, 1H), 3.66 (s, 4H), 3.54-3.48 (m, 4H), 1.46 (s, 9H).
4.1.4.8.
tert-butyl
4-(4-fluoro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate (5h)
1
Yield 85%, white soild; m.p. 203-206 °C; H NMR (600 MHz, DMSO-d6) δ7.91
(d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.76 (td, J = 7.9, 4.7 Hz, 1H), 7.65 (t, J =
8.7 Hz, 1H), 3.58 (s, 4H), 3.39 (s, 4H), 1.41 (s, 9H).
4.1.4.9.
4-(6-fluoro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate (5i)
1
Yield 91%, white solid; m.p. 163-166 °C; H NMR (600 MHz, DMSO-d6) δ 8.07
(dd, J = 7.1, 2.1 Hz, 1H), 7.84 (s, 1H), 7.77 (dd, J = 8.4, 4.7 Hz, 1H), 7.64 (td, J = 8.8,
2.4 Hz, 1H), 3.60 (s, 4H), 3.42 (s, 4H), 1.44 (s, 9H).
4.1.4.10.
4-(4-nitro-1,
1-dioxidobenzo[b]thiophene-2-carbonyl)piperazine-1-carboxylate (5j)