Design, synthesis, and biological evaluation of benzo[b]thiophene 1,1-dioxide derivatives as potent STAT3 inhibitors

Article abstract of DOI:10.1111/cbdd.13939

As a member of the signal transducer and activator of transcription (STAT) family, STAT3 plays a critical role in several biological pathways such as cell proliferation, migration, survival, and differentiation. Due to abnormal continuous activation in tumors, inhibition of STAT3 has emerged as an attractive approach for the treatment of various cancer cells. Herein, we report a series of novel STAT3 inhibitors based on benzo[b]thiophene 1,1-dioxide scaffold and evaluated their anticancer potency. Among them, compound 8b exhibited the best activity against cancer cells. Compound 8b induced apoptosis and blocked the cell cycle. Meanwhile, 8b reduced intracellular ROS content and caused the loss of mitochondrial membrane potential. Further research revealed that 8b significantly blocked STAT3 phosphorylation and STAT3-dependent dual-luciferase reporter gene experiments showed that compound 8b has a marked inhibition of STAT3-mediated Firefly luciferase activity. Molecular modeling studies revealed compound 8b occupied the pocket well with the SH2 domain in a favorable conformation.

Full text of DOI:10.1111/cbdd.13939

Received: 22 February 2021
DOI: 10.1111/cbdd.13939
Revised: 6 July 2021
Accepted: 23 July 2021
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R E S E A R C H A R T I C L E
Design, synthesis, and biological evaluation of benzo[b]thiophene
1,1-dioxide derivatives as potent STAT3 inhibitors
Wen-Zhen Li1*
Hui-Zhi Xi1*
Yi-Jie Wang1*
Hong-Bo Ma2
Zhi-Qiang Cheng3
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Yu Yang1
Meng-Ling Wu1
Ting-Mei Liu1
Yi-Wen Zhang1
Wen Yang1
Qin Wang1
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Meng-Ya Liao4
Yong Xia1
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1Cancer Center, West China Hospital,
Sichuan University, Chengdu, China
Abstract
2West China School of Pharmacy, Sichuan
University, Chengdu, China
As a member of the signal transducer and activator of transcription (STAT) family,
STAT3 plays a critical role in several biological pathways such as cell proliferation,
migration, survival, and differentiation. Due to abnormal continuous activation in
tumors, inhibition of STAT3 has emerged as an attractive approach for the treat-
ment of various cancer cells. Herein, we report a series of novel STAT3 inhibitors
based on benzo[b]thiophene 1,1-dioxide scaffold and evaluated their anticancer po-
tency. Among them, compound 8b exhibited the best activity against cancer cells.
Compound 8b induced apoptosis and blocked the cell cycle. Meanwhile, 8b reduced
intracellular ROS content and caused the loss of mitochondrial membrane potential.
Further research revealed that 8b significantly blocked STAT3 phosphorylation and
STAT3-dependent dual-luciferase reporter gene experiments showed that compound
8b has a marked inhibition of STAT3-mediated Firefly luciferase activity. Molecular
modeling studies revealed compound 8b occupied the pocket well with the SH2 do-
main in a favorable conformation.
3Department of Pharmacology and
Molecular Sciences, Johns Hopkins
University School of Medicine, Baltimore,
MD, USA
4Center of Gerontology and Geriatrics,
West China Hospital, Sichuan University,
Chengdu, China
Correspondence
Yi-Wen Zhang and Yong Xia, Cancer
Center, West China Hospital, Sichuan
University, Chengdu, China.
Email: yiwenzhang@scu.edu.cn;
scuxiayong@163.com
Meng-Ya Liao, Center of Gerontology and
Geriatrics, West China Hospital, Sichuan
University, Chengdu, China.
K E Y W O R D S
Email: 507162040@qq.com
apoptosis, cell cycle, inhibitors, mitochondrial membrane potential, ROS, STAT3, tumors
Funding information
Support Program of Science & Technology
Department of Sichuan Provincial, Grant/
Award Number: 2021YFH0173
1
INTRODUCTION
transmits them through phosphorylation of the tyrosine
kinase site (Tyr705) in the SH2 domain. Once in the nu-
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Signal transducer and activator of transcription (STAT)
family consist of seven members, STAT1, 2, 3, 4, 5α, 5β, 6
(Darnell, 1997). Among them, STAT1, STAT3, and STAT5
are highly associated with human tumorigenesis and de-
velopment due to their frequent hyperactivation (Benekli
et al., 2009). In normal cells, STAT3 responds to the stim-
ulation of various extracellular signaling factors and then
cleus, STAT3 binds to DNA and subsequently activates
transcription and translation, thus performing various phys-
iological functions (Furtek et al., 2016; Ren et al., 2015;
Yamamoto et al., 2002). However, according to previous
research, STAT3 is involved in the process of growth,
proliferation, angiogenesis, and invasion of tumor cells
(Bromberg & Chen, 2001). Abnormally high expression
*These authors contribute equally.
Chem Biol Drug Des. 2021;00:1–15.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons Ltd.
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LI et aL.
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and persistent activation of STAT3 have been observed in
many human solid and hematological tumors, such as ovar-
ian cancer (Huang et al., 2000), pancreatic cancer (Sun
et al., 2014), prostate cancer (Liao & Nevalainen, 2011),
head and neck cancer (Cohen-Kaplan et al., 2012), breast
cancer (Barash, 2012), leukemia (Casetti et al., 2013), and
non-small cell lung carcinoma (Sanchez-Ceja et al., 2006).
In general, hyperactivation of STAT3 upregulates the ex-
pression of many downstream transcriptional genes, in-
cluding the anti-apoptotic proteins Bcl-xL and the Bcl-2,
proliferation-related proteins cyclin D1 and c-Myc, MMP-
2, and MMP-9, which are associated with migration and
invasion-related proteins, and the pro-angiogenesis factor
VEGF (Germain & Frank, 2007; Yu et al., 2009). More im-
portantly, the expression of above oncogenes is not the only
way leading to cancer. Activation of STAT3 also promotes
the occurrence of cancer through immunosuppression in
immune cells (Wang et al., 2018).
During the past decades, the development of STAT3 in-
hibitors has been a popular strategy for the treatment of
various tumors. Although plenty of STAT3 inhibitors has
been reported, there are still no FDA-approved drugs on the
market. STAT3 contains 6 domains, including N-terminated
conserved domain, coiled-coil domain, DNA-binding site
domain, linker domain, SH2 domain, and C-terminated ac-
tivated domain (Furtek et al., 2016). Among them, the key
SH2 domain is responsible for STAT3 phosphorylation and
homodimerization. The process of phosphorylation and
homodimerization could be disrupted by small molecules
therefore providing a feasible way for STAT3 inhibitor drug
discovery. (Furtek et al., 2016).
Stattic is the first small molecule inhibitor targeting the
SH2 domain (Schust et al., 2006). Based on Stattic, several
derivatives were synthesized, such as HJC0416, HJC0123,
and E28 (Chen et al., 2014; Chen et al., 2013; Ji et al.,
2015). Besides, STA-21, LLL-3, LLL-12, and BBI608
which share common moiety were also representative in-
hibitors binding to SH2 domain(Bhasin et al., 2008; Chen
et al., 2007; Lin et al., 2010; Song et al., 2005; Zhang
et al., 2016)
Previous works have indicated that pY and pY-X cav-
ities in the SH2 domain are critical for inhibitor activity
and selectivity (Maritano et al., 2004; Park & Li, 2011).
We found that Stattic only occupied the pY-X cavity, leav-
ing the pY cavity empty (Figure S1). We hired a virtual
screening program to find the appropriate fragment tar-
geting the pY cavity. In accordance with the results, ligu-
strazine, reported to treat tumors through ROS mediation
(Han et al., 2015), was induced into pY cavity to obtain
superior activity and selectivity. Finally, 28 compounds
were synthesized, among them 8b was proved the best
compound possessing exceptional property as a potential
candidate.
2
MATERIALS AND METHOD
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All chemical reagents and solvents were bought from
Energy Chemical or Chron Chemicals. Thin-layer chroma-
tography (TLC) was inducted to monitor reaction process,
and column chromatography was carried out on 200–300
mesh silica gel. UV light was used to visualize products.
All compounds were characterized by 1H-NMR (401 MHz,
Bruker Avance400) and 13C-NMR (101 MHz, Bruker
Avance400). TMS was used as internal standard. Q-TOF
Premier mass spectrometer (Micromass) was applied to
measure mass spectrum.
2.1
Chemicals
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We synthesized these compounds according to the synthetic
route described in Scheme 1. In short, 2-fluorobenzaldehyde
and its derivatives were used as the starting materials and
reacted with methyl thioglycolate in DMF to obtain 2a–
2m. Subsequently, 2a–2m removed the methyl ester in
NH4Cl/MeOH solvent leading to 3a–3m. Various amino
derivatives reacted with 3a–3m to obtain 4a–4o. Then,
4a–4o were oxidized by 3-chloroperoxybenzoic acid in
DCM, giving 5a–5o. The boc protecting group was sub-
sequently removed by trifluoroacetic acid to form 6a–6o.
6a–6o were coupled with 3,5,6-trimethylpyrazine-2-carbo
xylic acid to get 7a–7o. 8a–8m were obtained through the
alkylation of 6a–6m. 3,5,6-Trimethylpyrazine-2-carboxyl
ic acid and 2-(bromomethyl)-3,5,6-trimethylpyrazine were
prepared from tetramethylpyrazine (Ligustrazine). Details
can be found in the reported method (Chen et al., 2015;
Wang et al., 2019).
2.2
Cell culture and materials
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All cell lines including human colorectal cancer cell line
HCT116, human breast adenocarcinoma cell lines MCF-7
and MDA-MB-231, human liver hepatocellular carci-
noma HepG2 were obtained from American Type Culture
Collection (ATCC). The cells were cultured in DMEM con-
taining 10% fetal bovine serum (v/v) and maintained at 37℃
under 5% CO2.
2.3
Cell antiproliferation assays
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The antiproliferation activities of synthesized compounds
were determined in the HCT116 cells, MCF-7 cells,
MDA-MB-231 cells, and HepG2 cells. Cells (5 × 107/L)
were seeded in 96-well plates and allowed to incubate
for 24 hr. Then, cells were treated with compounds

LI et aL.
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SCHEME 1 (a) HSCH2COOCH3, DMF, 60℃, 15 hr, 72%-94%. (b) THF, H2O, LiOH, HCl, 98%–100%. (c) HATU, DIEA, DMF, rt,
overnight, 95%–100%. (d) m-CPBA, DCM, 3 hr, 45℃, 45%–60%. (e) DCM, TFA (98%), rt, 3hr. (f) HATU, DIEA, 3,5,6-trimethylpyrazine-2-
carboxylic acid, DCM, rt, 40%–60%. (g) KI, CH3CN, 2-(bromomethyl)-3,5,6-trimethylpyrazine, reflux, 1.5 hr, 35%–47%
(0–20 μM) for 72 hr. After that, 10 μl of MTT reagent
(5 mg/ml) was added per well followed by extra 2- to
4-hr incubation. Formazan precipitates were dissolved in
150 μl of 100% DMSO, and absorbance was determined at
570 nm by a microplate reader 3550-UV (Thermo Fisher
Scientific).
the samples were analyzed by flow cytometer (ACEA
Biosciences Inc).
2.5
ROS detections
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MCF-7 cells and HepG2 cells were exploited to detect intra-
molecular ROS level. Cells (5 × 107/L) were cultured in six-
well plates and treated with compound 8b or vehicle. After
treatment, the growth media was replaced with serum-free
medium containing DCF-DA (10 μM). The cells were har-
vested and washed with PBS. The ROS level was measured
by flow cytometer (ACEA Biosciences Inc).
2.4
Flow cytometry analysis of apoptotic
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cells and cell cycle
Apoptosis assays were determined in HCT116, MCF-7 cells,
MDA-MB-231 cells, and HepG2 cells. Cells (5 × 107/L)
were seeded in six-well plates and treated with compound
8b (0, 2.5, 5, 10 μM) for 48 hr. Then, the cells were har-
vested and washed with PBS. The apoptosis-induced effect
was checked by Annexin V/FITC Apoptosis Detection Kit
I (Keygen Tec).
2.6
Mitochondrial membrane
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potential detections
Cell cycle assays were performed in HCT116, MCF-7
cells, and HepG2 cells. Cells (5 × 107/L) were seeded in
six-well plates and treated with compound 8b (0, 2.5, 5,
10 μM) for 48 hr. After collection and PBS washing, the
cells were fixed by 70% ethanol at −20℃ for 8 hr. The cells
were harvested and washed with PBS and resuspended in
propidium iodide (PI)/RNase-free staining solution. After,
MCF-7 cells and HepG2 cells were used to detect mitochon-
drial membrane potential. Cells (5 × 107/L) were cultured
in six-well plates and treated with compound 8b or DMSO
vehicle. After treatment, cells were incubated with DMEM
containing Rh123 (5 μg/ml) for 30 min. The cells were har-
vested and washed with PBS. The ΔΨm was measured by
flow cytometer (ACEA Biosciences Inc).

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LI et aL.
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2.7
Western Blotting assays
brine (10 ml × 3), the organic layer was collected, dried by
anhydrous sodium sulfate, purified by silica gel column, and
concentrated under reduced pressure. Yield 72%–94%. White
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Western Blotting assay was performed in HepG2 cells.
The cells (5 × 107/L) were cultured in six-well plates and
treated with compound 8b (1.5, 2.5, 5 μM) or vehicle for
24 hr. Then, the cells were collected and washed by ice-cold
PBS and lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM
NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA,
and protease inhibitors). Lysates were collected through cen-
trifugation (13, 400 g, 10 min, 4℃). An aliquot of samples
(30 μg) was subjected to 10% SDS-PAGE and subsequently
transferred onto PVDF membranes, blocked by non-fat milk
for 1 hr, and blotted with primary antibodies specific for
STAT3, p-STAT3, cleaved caspase 3, caspase 3, Bax, Bcl-2,
E-cadherin, vimentin cylin B, p-CDC2, γH2AX, p53, and
GAPDH at 4℃ for 8 hr. After incubating with correspond-
ing secondary antibodies, bound immuno-complexes were
visualized using ELC.
1
solid. 2a: H NMR (401 MHz, CDCl3) δ 8.04 (s, 1H), 7.85
(dd, J = 7.3, 5.8 Hz, 2H), 7.41 (m, 2H), 3.93 (s, 3H).
3.1.2
General procedures for preparation of
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3a-3m
MeOH (10 ml) and water (10 ml) were added to the round-
bottom flask which contained 3(a-m) (1 mmol, 1.0 eq);
then, LiOH (3 mmol, 3.0 eq) solid was added; mixture
solution was stirred overnight at room temperature. Upon
completion, adjusting the pH of the solution to 3 with di-
lute hydrochloric acid (1 N). The solution was extracted
by ethyl acetate (15 ml × 2), collecting the organic phase
and concentrating under reduced pressure. Yield 100%.
White solid. 3a: 1H NMR (401 MHz, DMSO-d6):13.43 (s,
3H), δ 8.54 (s, 1H), 7.82 (dd, J = 7.3, 5.8 Hz, 2H), 7.46
(m, 2H).
2.8
STAT3 dual-luciferase reporter assays
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HepG2 cells were cultured in 24-well plates (2 × 105 cells/
well). After 24 hr of culture, the cells were transiently
transfected with pLuc-TK/STAT3 (0.2 mg) and pRL-TK
(0.04 mg). 24 hr later, the cells were treated compound 8b
for 48 hr. Then, the cells were washed and lysed according to
the protocol. Luciferase activity was measured using a multi-
functional enzyme marker (Bio-Rad).
3.1.3
General procedures for preparation of
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4a-4o
A mixture of 3(a-m) (1.0 mmol, 1.0 eq), HATU (1.50 mmol,
1.5 eq), DIEA (4.0 mmol, 4.0 eq) were stirred in anhy-
drous DMF (20 ml) for 15 min at room temperature; amino
(1.0 mmol, 1.1 eq) was added. Reaction was stirred over-
night at room temperature. Upon completion, DMF was
removed by rotary evaporation under reduced pressure;
the residues were dissolved in ethyl acetate (30 ml) and
washed with saturated brine (10 ml × 3), collecting the
organic layer and drying with anhydrous sodium sulfate;
organic phase was evaporated in vacuo. The crude product
was purified using silica gel chromatography to obtain 4(a-
2.9
Molecular docking study
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The crystal structure of STAT3 in complex with DNA was
obtained from Protein Data Bank (PDB 1BG1); Schrödinger
10.1 was used to predict the binding modes of 8b. All dock-
ing programs were run with default settings.
1
o). Yield 75%–88%. White solid. 4a: H NMR (401 MHz,
DMSO-d6) δ 8.02 (m, 1H), 7.93 (dt, J = 6.4, 2.6 Hz, 1H),
7.76 (s, 1H), 7.45 (m, 2H), 3.68 (d, J = 4.7 Hz, 4H), 3.45
(m, 4H), 1.43 (s, 9H).
3
RESULT AND DISCUSSION
Chemistry
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3.1
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3.1.1
General procedures for preparation of
3.1.4
General procedures for preparation of
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2a-2m
5a-5o
To a solution of 1(a-m) (1 mmol, 1.0 eq) in 10 ml DMF,
methyl thioglycolate (1.1 mmol, 2.0 eq) and anhydrous po-
tassium carbonate 1 (2 mmol, 2.0 eq) were added; the re-
action were carried out at 60℃ for 8 hr. The reaction was
monitored by TLC. After completion, DMF was removed
by rotary evaporation under reduced pressure, residues were
dissolved in ethyl acetate (30 ml) and washed with saturated
To a solution of 4(a-o) (1.0 mmol, 1.0 eq) in DCM (5 ml),
m-CPBA (3.0 mmol, 3.0 eq) was added slowly in 30 min.
After addition, the reaction was heated to 45℃ for 5 hr. Upon
completion, solution was allowed to cool to room tempera-
ture. Saturated aqueous sodium bisulfite solution (50 ml) was
added to the stirring solution for 15 min, subsequently adding
saturated aqueous sodium bicarbonate solution (50 ml). The

LI et aL.
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mixture was quenched with water and extracted by DCM.
Anhydrous sodium sulfate was used to remove water; or-
ganic phase was evaporated in vacuo. The crude product was
purified using silica gel chromatography to obtain 5(a-o).
White solid.
H, m), 3.22 (2 H, d, J = 6.7 Hz), 2.51 (1 H, s), 1.76 (2 H, dd,
J = 13.9, 3.8 Hz), 1.46 (9 H, s).
1
5o: Yield, 85%. H NMR (401 MHz, CDCl3) δ 7.87–
7.78 (2 H, m), 7.46 (1 H, s), 7.42–7.36 (2 H, m), 4.51 (1 H,
s), 4.40 (2 H, s), 3.75 (1 H, s), 3.13 (2 H, s), 2.04 (2 H, d,
J = 10.6 Hz), 1.62 (1 H, s), 1.45 (9 H, s).
5a: Yield, 77%. 1H NMR (401 MHz, DMSO-d6) δ 7.92 (d,
J = 7.4 Hz, 1H), 7.81 (s, 1H), 7.75 (dd, J = 11.1, 3.7 Hz, 1H),
7.69 (t, J = 6.7 Hz, 2H), 3.58 (s, 4H), 3.40 (m, 4H), 1.42 (s, 9H).
5b: Yield, 81%. 1H NMR (401 MHz, CDCl3) δ 7.76 (d, J
8.1 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.24 (s, 1H), 3.58 (s,
4H), 3.97–3.38 (m, 4H), 1.42 (s, 9H).
3.1.5
General procedures for preparation of
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6a-6o
5c: Yield, 79%. 1H NMR (401 MHz, CDCl3) δ 7.73 (dd,
J 8.1 Hz, 1H), 7.60 (dd, J = 8.3 Hz, 2H), 7.52–7.41 (m, 4H),
3.58 (s, 4H), 3.97–3.38 (m, 4H), 1.42 (s, 9H).
To a solution of 5(a-o) (1.0 mmol, 1.0 eq) in DCM (5 ml),
TFA (0.5 ml) was added. Solution was stirred for 3 hr at room
temperature. Upon completion, TFA and DCM were evapo-
rated in vacuo. The residue was washed with ether; yellow
oily solid was obtained without further purification.
5d: Yield, 77%. 1H NMR (401 MHz, CDCl3) δ 8.00 (1 H,
s), 7.66 (1 H, s), 7.51 (1 H, dd, J = 8.5, 1.6 Hz), 7.44 (1 H, s),
3.78–3.70 (4 H, m), 3.57–3.49 (4 H, m), 1.48 (9 H, s).
5e: Yield, 80%. 1H NMR (401 MHz, CDCl3) δ 7.54 (1 H,
dd, J = 8.4, 7.5 Hz), 7.21 (1 H, s), 7.07 (1 H, d, J = 8.5 Hz),
6.98 (1 H, d, J = 7.4 Hz), 3.67 (4 H, s), 3.54–3.50 (4 H, m),
1.47 (9 H, s).
3.1.6
General procedures for preparation of
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7a-7o
1
5f: Yield, 64%. H NMR (401 MHz, CDCl3) δ 7.65 (1
A mixture of 3,5,6-trimethylpyrazine-2-carboxylic acid
(1.0 mmol, 1.0 eq), HATU (1.50 mmol 1.5 eq), DIEA
(4.0 mmol, 4.0 eq) was stirred in anhydrous DMF (20 ml)
for 15 min at room temperature; then, 6(a-o) (1.0 mmol,
1.1 eq) was added. Reaction was stirred overnight at room
temperature. Upon completion, DMF was removed by rotary
evaporation under reduced pressure, the residue was dis-
solved in ethyl acetate (30 ml) and washed with saturated
brine (10 ml × 3), collecting the organic layer and drying
with anhydrous sodium sulfate, organic phase was evapo-
rated in vacuo. The crude product was purified using silica
gel chromatography. Pale yellow solid.
H, d, J = 8.0 Hz), 7.45 (1 H, t, J = 7.7 Hz), 7.37 (1 H, d,
J = 7.5 Hz), 7.22 (1 H, s), 3.70 (4 H, d, J = 22.9 Hz), 3.56–
3.50 (4 H, m), 1.47 (11 H, s).
5g: Yield, 67%. 1H NMR (401 MHz, CDCl3) δ 7.68 (1 H,
d, J = 8.1 Hz), 7.56 (1 H, dd, J = 8.1, 1.7 Hz), 7.42 (1 H, d,
J = 1.6 Hz), 7.25 (1 H, s), 3.66 (4 H, s), 3.56–3.48 (4 H, m),
1.47 (9 H, s).
5h: Yield, 74%. 1H NMR (401 MHz, CDCl3) δ 7.72 (1 H,
s), 7.58 (1 H, dd, J = 8.0, 1.8 Hz), 7.38 (1 H, d, J = 8.0 Hz),
7.29 (1 H, s), 3.67 (4 H, s), 3.56–3.49 (4 H, m), 1.47 (9 H, s).
5i: Yield, 65%. 1H NMR (401 MHz, CDCl3) δ 7.51 (2 H,
dt, J = 8.1, 7.7 Hz), 7.32 (1 H, dd, J = 7.1, 0.9 Hz), 7.26 (1 H,
s), 3.67 (4 H, s), 3.57–3.49 (4 H, m), 1.47 (9 H, s).
1
7a: Yield, 50%. H NMR (401 MHz, CDCl3) δ 7.74 (d,
J = 5.6 Hz, 1H), 7.57 (s, 2H), 7.52 (m, 1H), 7.38 (s, 1H), 3.92
(d, J = 5.7 Hz, 2H), 3.84 (s, 2H), 3.74 (s, 2H), 3.41 (s, 2H),
2.59 (d, J = 8.6 Hz, 6H), 2.54 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ: 166.77, 158.67, 152.59, 148.70, 147.67, 144.11,
136.26, 134.41, 134.14, 131.72, 130.25, 129.26, 126.21,
121.81, 46.78, 42.64, 21.79, 21.10, 20.53. ESI-MS for
C21H22N4O4S m/z: 449.1197[M + Na]+.
1
5j: Yield, 57%. H NMR (401 MHz, CDCl3) δ 7.54 (2
H, t, J = 7.9 Hz), 7.31 (1 H, d, J = 7.5 Hz), 7.10 (1 H, d,
J = 8.4 Hz), 3.94 (3 H, s), 3.68 (4 H, s), 3.55–3.49 (4 H, m),
1.47 (9 H, s).
5k: Yield, 54%. 1H NMR (401 MHz, CDCl3) δ 7.36–7.29
(2 H, m), 7.27 (1 H, s), 7.06 (1 H, dd, J = 8.4, 2.3 Hz), 3.90 (3
H, s), 3.72–3.65 (4 H, m), 3.55–3.50 (4 H, m), 1.47 (9 H, s).
5l: Yield, 68%. 1H NMR (401 MHz, CDCl3) δ 7.54 (1 H,
dd, J = 8.4, 7.5 Hz), 7.21 (1 H, s), 7.07 (1 H, d, J = 8.5 Hz),
6.98 (1 H, d, J = 7.4 Hz), 4.00 (3 H, s), 3.67 (4 H, s), 3.55–
3.50 (4 H, m), 1.47 (9 H, s).
1
7b: Yield, 49%. H NMR (401 MHz, CDCl3) δ 7.77 (d,
J = 8.1 Hz, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.52 (s, 1H), 7.47
(t, J = 7.8 Hz, 1H), 3.94 (dd, J = 6.2, 3.6 Hz, 2H), 3.85 (s,
2H), 3.75 (s, 2H), 3.41 (s, 2H), 2.58 (d, J = 8.5 Hz, 6H),
2.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 166.84, 158.22,
152.55, 148.66, 147.80, 144.03, 140.52, 139.81, 134.20,
131.43, 131.23, 130.52, 126.37, 122.82, 46.53, 42.88, 21.79,
21.32, 20.32. ESI-MS for C21H21ClN4O4S m/z: 483.0864
[M + Na]+.
1
5m: Yield, 38%. H NMR (401 MHz, CDCl3) δ 7.74 (1
H, dd, J = 8.3, 4.6 Hz), 7.27 (2 H, q, J = 2.5 Hz), 7.24 (1 H,
d, J = 2.8 Hz), 7.14 (1 H, dd, J = 7.7, 2.2 Hz), 3.66 (4 H, d,
J = 11.6 Hz), 3.56–3.49 (4 H, m), 1.49 (9 H, s).
5n: Yield, 52%. 1H NMR (401 MHz, CDCl3) δ 7.74 (1 H,
dd, J = 6.8, 1.1 Hz), 7.59 (2 H, td, J = 6.4, 1.2 Hz), 7.42 (1 H,
dd, J = 6.4, 1.7 Hz), 7.26 (1 H, d, J = 0.6 Hz), 4.46–3.91 (2
7c: Yield, 37%. 1H NMR (401 MHz, CDCl3) δ 7.73 (d,
J = 8.0 Hz, 1H), 7.61 (d, J = 7.3 Hz, 2H), 7.30 (s, 1H), 3.92 (d,
J = 5.1 Hz, 2H), 3.83 (s, 2H), 3.73 (s, 2H), 3.40 (s, 2H), 2.53

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LI et aL.
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(t, J = 8.6 Hz, 9H). 13C NMR (101 MHz, CDCl3) δ: 166.79,
158.37, 152.83, 148.73, 147.75, 143.93, 139.52, 138.26,
134.25, 132.03, 131.33, 130.50, 126.59, 122.43, 46.65,
43.10, 21.85, 21.36, 20.31. ESI-MS for C21H21ClN4O4S m/z:
483.0865 [M + Na]+.
42.10, 21.81, 21.09, 20.78. ESI-MS for C21H21BrN4O4S m/z:
527.0355 [M + Na]+.
7j: Yield, 53%. 1H NMR (401 MHz, CDCl3) δ(ppm): 7.53
(2 H, t, J = 7.9 Hz), 7.31 (1 H, d, J = 7.5 Hz), 7.09 (1 H, d,
J = 8.4 Hz), 3.67 (4 H, s), 3.54–3.48 (4 H, m), 2.55 (9 H, t,
J = 7.9 Hz). 13C NMR (101 MHz, CDCl3) δ: 166.81, 164.16,
158.63, 152.47, 148.58, 147.57, 144.20, 139.23, 131.67,
131.50, 127.49, 123.26, 115.39, 112.51, 55.98, 46.92, 46.37,
42.80, 41.91, 21.92, 21.38, 20.47. ESI-MS for C22H24N4O5S
m/z: 479.1358 [M + Na]+.
7d: Yield, 47%. 1H NMR (401 MHz, DMSO-d6) 8.35 (1
H, s), 7.86 (1 H, d, J = 8.5 Hz), 7.78 (1 H, s), 7.60 (1 H, dd,
J = 8.6, 1.5 Hz), 3.79 (4 H, s), 3.66 (2 H, s), 3.30 (2 H, d,
J = 5.0 Hz), 2.48 (3 H, d, J = 4.5 Hz), 2.45 (3 H, d, J = 5.3 Hz),
2.40 (3 H, s). 13C NMR (101 MHz, CDCl3) δ: 166.87, 158.29,
152.56, 148.55, 147.68, 143.84, 138.44, 138.09, 137.53,
134.41, 132.10, 127.62, 126.99, 122.89, 46.83, 42.42, 21.89,
21.36, 20.39. ESI-MS for C21H21ClN4O4S m/z: 483.0862
[M + Na]+.
7k: Yield, 44%. 1H NMR (401 MHz, CDCl3) δ 7.66 (1 H,
d, J = 8.5 Hz), 7.26–7.24 (1 H, m), 7.01 (1 H, dd, J = 8.4,
1.8 Hz), 6.92 (1 H, s), 3.93 (2 H, dd, J = 6.3, 3.5 Hz), 3.90
(3 H, s), 3.84 (2 H, s), 3.73 (2 H, t, J = 6.9 Hz), 3.42–3.36
(2 H, m), 2.52 (9 H, t, J = 7.6 Hz). 13C NMR (101 MHz,
CDCl3) δ: 166.73, 163.08, 158.42, 152.49, 148.69, 147.57,
144.14, 138.19, 135.99, 133.12, 127.45, 121.14, 119.24,
107.90, 56.23, 46.81, 41.94, 21.91, 21.30, 20.36. ESI-MS for
C22H24N4O5S m/z: 479.1355 [M + Na]+.
1
7e: Yield, 56%. H NMR (401 MHz, CDCl3) 7.67 (1 H,
d, J = 3.4 Hz), 7.49–7.42 (1 H, m), 7.37 (1 H, d, J = 7.5 Hz),
7.26 (1 H, s), 3.99–3.90 (2 H, m), 3.84 (2 H, s), 3.74 (2 H, s),
3.44–3.37 (2 H, m), 2.60–2.45 (9 H, m). 13C NMR (101 MHz,
CDCl3) δ: 166.89, 158.31, 152.59, 148.69, 147.71, 143.97,
138.57, 138.13, 137.56, 134.23, 131.41, 127.73, 127.09,
122.42, 46.52, 42.12, 22.02, 21.33, 20.54. ESI-MS for
C21H21ClN4O4S m/z: 483.0865 [M + Na]+.
7l: Yield, 68%. 1H NMR (401 MHz, CDCl3) δ 7.57–7.51
(1 H, m), 7.24 (1 H, s), 7.07 (1 H, d, J = 8.5 Hz), 6.98 (1 H, d,
J = 7.3 Hz), 4.00 (3 H, s), 3.93 (2 H, dd, J = 6.4, 3.6 Hz), 3.83
(2 H, s), 3.73 (2 H, t, J = 7.0 Hz), 3.41–3.37 (2 H, m), 2.52
(9 H, t, J = 7.9 Hz). 13C NMR (101 MHz, CDCl3) δ: 166.69,
158.71, 155.58, 152.51, 148.68, 147.54, 144.12, 138.61,
136.16, 131.41, 131.07, 121.23, 118.27, 115.14, 56.43,
46.69, 42.05, 21.77, 21.27, 20.29. ESI-MS for C22H24N4O5S
m/z: 479.1357 [M + Na]+.
1
7f: Yield, 48%. H NMR (401 MHz, CDCl3) δ 7.70 (d,
J = 8.2 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.55(m, 2H),
3.93 (s, 2H), 3.82 (s, 2H), 3.72 (s, 2H), 3.40 (s, 2H), 2.54
(t, J = 7.5 Hz, 9H). 13C NMR (101 MHz, CDCl3) δ: 166.94,
162.93, 158.68, 154.26, 150.31, 149.88, 136.21, 134.69,
133.85, 130.45, 128.61, 128.59, 128.43, 123.94, 46.57,
42.74, 21.69, 21.44, 20.43. ESI-MS for C21H21BrN4O4S m/z:
527.0361 [M + Na]+.
1
7m: Yield, 38%. H NMR (401 MHz, CDCl3) δ 7.75 (1
H, dd, J = 8.0, 4.7 Hz), 7.29 (2 H, q, J = 2.5 Hz), 7.15 (1
H, d, J = 7.7 Hz), 3.93 (2 H, dd, J = 6.4, 3.6 Hz), 3.83 (2
H, s), 3.76–3.71 (2 H, m), 3.42–3.37 (2 H, m), 2.53 (9 H, t,
J = 7.8 Hz). 13C NMR (101 MHz, CDCl3) δ: 166.96, 160.12,
155.73, 148.56, 147.65, 143.51, 139.46, 139.26, 135.36,
133.06, 130.98, 130.40, 125.98, 122.43, 46.29, 42.93, 21.85,
21.39, 20.58. ESI-MS for C21H21FN4O4S m/z: 467.1260
[M + Na]+.
7g: Yield, 64%. 1H NMR (401 MHz, CDCl3) δ 7.68 (2 H,
d, J = 7.8 Hz), 7.57 (1 H, d, J = 9.1 Hz), 7.44 (1 H, s), 7.29
(1 H, s), 3.97–3.90 (2 H, m), 3.83 (2 H, s), 3.72 (5 H, dd,
J = 14.0, 7.0 Hz), 2.60–2.46 (9 H, t). 13C NMR (101 MHz,
CDCl3) δ: 166.95, 160.90, 160.68, 157.26, 152.71, 149.04,
134.95, 134.24, 133.99, 130.11, 129.35, 128.88, 128.66,
123.07, 46.53, 43.80, 21.79, 21.56, 20.30. ESI-MS for
C21H21BrN4O4S m/z: 527.0354 [M + Na]+.
7n: Yield, 55%. 1H NMR (401 MHz, CDCl3) δ 8.02 (1 H,
d, J = 8.3 Hz), 7.75 (1 H, dd, J = 6.9, 1.0 Hz), 7.60 (2 H, td,
J = 6.4, 1.2 Hz), 7.43 (1 H, dd, J = 6.4, 1.7 Hz), 4.62 (1 H,
s), 4.22 (2 H, dd, J = 7.7, 3.6 Hz), 2.90 (3 H, s), 2.55 (6H, d,
J = 10.4 Hz), 2.14 (2 H, d, J = 10.5), 1.82 (2 H, s), 1.68 (2
H, d, J = 9.5 Hz). 13C NMR (101 MHz, CDCl3) δ: 164.38,
158.14, 154.39, 151.37, 147.71, 138.84, 138.49, 136.33,
133.94, 131.42, 131.06, 129.56, 125.92, 121.61, 46.55,
46.26, 41.74, 32.88, 31.67, 22.87, 22.02, 21.35. ESI-MS for
C22H24N4O4S m/z: 463.1423 [M + Na]+.
7h: Yield, 65%. 1H NMR (401 MHz, CDCl3) δ 7.72 (1 H,
s), 7.59 (1 H, dd, J = 8.0, 1.7 Hz), 7.39 (1 H, d, J = 8.0 Hz),
7.33 (1 H, s), 3.96–3.88 (2 H, m), 3.81 (2 H, d, J 14.6),
3.73 (2 H, t, J = 6.9 Hz), 3.45–3.36 (2 H, m), 2.55 (9 H, t,
J = 7.7 Hz). 13C NMR (101 MHz, CDCl3) δ: 166.89, 163.71,
152.55, 148.67, 147.80, 144.14, 141.67, 137.25, 136.38,
128.62, 125.80, 125.21, 124.94, 120.18, 46.67, 41.86, 21.99,
21.41, 20.57. ESI-MS for C21H21BrN4O4S m/z: 527.0364
[M + Na]+.
1
7i: Yield, 42%. H NMR (401 MHz, CDCl3) δ 7.64 (t,
7o: Yield, 52%. 1H NMR (401 MHz, CDCl3) δ 7.73 (1 H,
dd, J = 6.8, 1.1 Hz), 7.56 (2 H, td, J = 6.4, 1.2 Hz), 7.41 (1
H, dd, J = 6.4, 1.7 Hz), 7.25 (1 H, d, J = 0.6 Hz), 4.45–3.93
(2 H, m), 3.21 (2 H, d, J = 6.7 Hz), 2.51 (1 H, s), 1.77 (2 H,
dd, J = 13.9, 3.8 Hz), 2.55 (9 H, t, J = 7.8 Hz). 13C NMR
(101 MHz, CDCl3) δ: 166.47, 157.24, 155.39, 150.27, 144.55,
J = 9.2 Hz, 2H), 7.40 (d, J = 8.3 Hz 1H), 7.35 (s, 1H), 3.95
(s, 2H), 3.84 (s, 2H), 3.76 (s, 2H), 3.40 (s, 2H), 2.55 (t,
J = 8.0 Hz, 9H). 13C NMR (101 MHz, CDCl3) δ: 166.82,
158.16, 152.57, 148.70, 147.71, 144.03, 139.73, 132.48,
131.80, 130.33, 123.85, 118.40, 118.08, 113.82, 46.52,

LI et aL.
7
|
141.24, 139.29, 139.12, 131.24, 130.22, 131.89, 127.64,
125.32, 122.21, 49.69, 45.24, 40.38, 32.11, 23.01, 22.17,
21.89. ESI-MS for C21H22N4O4S m/z: 449.1261 [M + Na]+.
(1 H, s), 3.99–3.91 (2 H, m), 3.84 (2 H, s), 3.74 (2 H, s), 3.44–
3.37 (2 H, m), 2.60–2.45 (9 H, m). 13C NMR (101 MHz,
CDCl3) δ: 158.36, 149.64, 149.37, 148.96, 146.67, 138.32,
136.14, 134.43, 131.54, 131.36, 129.40, 125.64, 121.34,
60.31, 47.64, 42.21, 21.43, 21.41, 20.45. ESI-MS for
C21H24N4O3S m/z: 469.2372 [M + Na]+.
3.1.7
General procedures for preparation of
|
1
8a-8m
8f: Yield, 48%. H NMR (401 MHz, CDCl3) δ 7.64 (d,
J = 7.3 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.7 Hz,
1H), 7.50 (s, 1H), 3.70 (s, 6H), 2.63 (s, 4H), 2.57 (s, 3H), 2.49
(d, J = 1.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 157.26,
150.13, 149.99, 149.74, 147.80, 146.57, 140.11, 139.82,
138.11, 136.85, 132.52, 129.36, 119.96, 61.26, 53.04, 46.53,
22.72, 21.54, 20.78. ESI-MS for C21H23BrN4O3S m/z:
513.1667 [M + Na]+.
A mixture of 6(a-o) (1.0 mmol, 1.0 eq), KI (0.01 mmol, 0.01
eq), TEA (3.0 mmol, 3.0 eq) were stirred in CH3CN (20 ml)
for 15 min at room temperature; then, 2-(bromomethyl)-3,5,
6-trimethylpyrazine (1.1 mmol, 1.1 eq) was added. Reaction
was heated to 80℃. 1 hr later, CH3CN was removed by ro-
tary evaporation under reduced pressure, the residue was
dissolved in DCM (30 ml) and washed with saturated brine
(10 ml × 3), collecting the organic layer and drying with an-
hydrous sodium sulfate, and organic phase was evaporated in
vacuo. The crude product was purified using silica gel chro-
matography. White solid.
8g: Yield, 64%. 1H NMR (401 MHz, CDCl3) δ 7.68 (2 H,
d, J = 7.9 Hz), 7.56 (1 H, d, J = 9.0 Hz), 7.44 (1 H, s), 7.29 (1
H, s), 3.95–3.91 (2 H, m), 3.83–3.76 (4 H, m), 3.72 (4 H, dd,
J = 14.9, 7.1 Hz), 2.61–2.45 (9 H, t). 13C NMR (101 MHz,
CDCl3) δ: 157.54, 149.89, 149.81, 149.74, 147.68, 146.14,
140.36, 139.72, 138.36, 136.65, 132.31, 129.28, 120.14,
61.12, 52.95, 46.78, 22.81, 21.24, 20.96. ESI-MS for
C21H23BrN4O3S m/z: 513.1666 [M + Na]+.
1
8a: Yield, 46%. H NMR (401 MHz, CDCl3) δ 7.73 (d,
J = 6.8 Hz, 1H), 7.58 (m, 2H), 7.42 (m, 1H), 7.28 (s, 1H),
3.70 (d, J = 6.2 Hz, 6H), 2.60 (s, 4H), 2.55 (s, 3H), 2.49 (d,
J = 2.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 158.17,
149.93, 149.73, 148.29, 146.98, 138.59, 136.32, 134.08,
131.42, 131.19, 129.40, 125.94, 121.65, 59.91, 47.44, 42.45,
21.53, 21.43, 20.88. ESI-MS for C21H24N4O3S m/z: 435.1461
[M + Na]+.
8h: Yield, 65%. 1H NMR (401 MHz, CDCl3) δ 7.72 (1 H,
s), 7.59 (1 H, dd, J = 8.0, 1.7 Hz), 7.38 (1 H, d, J = 8.0 Hz),
7.33 (1 H, s), 3.93–3.85 (2 H, m), 3.80 (2 H, d, J = 14.6 Hz),
3.73 (2 H, t, J = 6.9 Hz), 3.72 (2 H, s),3.45–3.37 (2 H, m),
2.54 (9 H, t, J = 7.7 Hz). 13C NMR (101 MHz, CDCl3) δ:
157.78, 149.97, 149.87, 149.64, 147.24, 146.31, 140.25,
139.73, 138.31, 136.63, 132.44, 129.66, 119.89, 62.20, 52.68,
45.29, 23.12, 22.03, 21.05. ESI-MS for C21H23BrN4O3S m/z:
513.1667 [M + Na]+.
1
8b: Yield, 48%. H NMR (401 MHz, CDCl3) δ 7.75 (d,
J = 8.1 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.45 (dd, J = 9.1,
6.5 Hz, 2H), 3.72 (s, 6H), 2.65 (m, 4H), 2.59 (s, 3H), 2.50
(s, 6H). 13C NMR (101 MHz, CDCl3) δ: 158.26, 149.73,
149.71, 148.38, 146.65, 138.87, 136.55, 134.21, 131.12,
131.05, 129.65, 126.21, 121.76, 60.59, 47.49, 43.20, 21.96,
21.53, 20.23. ESI-MS for C21H23ClN4O3S m/z: 469.2372
[M + Na]+.
1
8i: Yield, 47%. H NMR (401 MHz, CDCl3) δ 7.50 (dt,
J = 15.7, 7.9 Hz, 2H), 7.31 (d, J = 7.1 Hz, 3H), 7.23 (s, 1H),
7.23 (s, 1H), 3.76 (s, 6H), 2.71 (s, 4H), 2.58 (s, 3H), 2.49 (d,
J = 1.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ: 158.21,
149.37, 149.31, 148.64, 147.54, 146.15, 140.31, 139.76,
138.35, 136.64, 132.27, 129.38, 120.16, 61.14, 52.69, 46.78,
22.34, 21.34, 20.99. ESI-MS for C21H23BrN4O3S m/z:
513.1667 [M + Na]+.
1
8c: Yield, 39%. H NMR (401 MHz, CDCl3) δ 7.70 (d,
J = 8.1 Hz, 1H), 7.62 (d, J = 7.5 Hz, 2H), 7.28 (s, 1H), 3.74
(s, 6H), 2.69 (m, 4H), 2.56 (s, 3H), 2.49 (d, J = 2.1 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ: 158.19, 150.03, 149.93,
148.81, 147.64, 139.24, 136.66, 134.28, 132.0, 131.79,
128.41, 125.84, 122.31, 60.96, 47.39, 42.41, 21.87, 21.65,
20.87. ESI-MS for C21H24N4O3S m/z: 469.2372 [M + Na]+.
1
8j: Yield, 53%. H NMR (401 MHz, CDCl3) δ 7.51 (2
H, t, J = 7.9 Hz), 7.31 (1 H, d, J = 7.5 Hz), 7.11 (1 H, d,
J = 8.4 Hz), 3.94 (3 H, s), 3.73 (6 H, s), 3.47 (2 H, s), 2.66 (2
H, s), 2.54 (9 H, t, J = 7.9 Hz). 13C NMR (101 MHz, CDCl3)
δ: 166.48, 158.95, 154.86, 152.37, 148.51, 147.10, 144.16,
137.41, 135.70, 133.72, 128.88, 117.49, 116.32, 113.14,
60.60, 56.21, 46.99, 41.87, 21.93, 21.41, 20.48. ESI-MS for
C22H26N4O4S m/z: 481.1302 [M + K]+.
1
8d: Yield, 47%. H NMR (401 MHz, CDCl3) 8.35 (1 H,
s), 7.87 (1 H, d, J = 8.4 Hz), 7.77 (1 H, s), 7.60 (1 H, dd,
J = 8.8, 1.9 Hz), 3.79 (4 H, s), 3.78 (2 H, s), 3.65 (2 H, s),
3.30 (2 H, d, J = 5.5 Hz), 2.50 (3 H, d, J = 4.5 Hz), 2.46 (3 H,
d, 5.3), 2.41 (3 H, s). 13C NMR (101 MHz, CDCl3) δ: 158.67,
150.26, 149.65, 148.31, 146.33, 138.67, 136.68, 134.54,
131.29, 131.11, 128.20, 125.91, 121.11, 61.13, 48.01, 43.55,
21.56, 21.54, 20.56. ESI-MS for C21H24N4O3S m/z: 469.2372
[M + Na]+.
1
8k: Yield, 44%. H NMR (401 MHz, CDCl3) δ 7.35 (2
H, t, J = 4.1 Hz), 7.27 (1 H, d, J = 1.8 Hz), 7.07 (1 H, dd,
J = 8.4, 2.3 Hz), 3.92 (2 H, dd, J = 6.4, 3.4 Hz), 3.88–3.82
(4 H, m), 3.74 (2 H, dd, J = 8.7, 3.7 Hz), 3.41–3.36 (2 H,
m), 2.54(9 H, t, J = 7.9 Hz). 13C NMR (101 MHz, CDCl3)
δ: 162.71, 158.53, 150.42, 149.84, 149.58, 148.23, 146.87,
8e: Yield, 56%. 1H NMR (401 MHz, CDCl3) 7.65 (1 H, d,
J = 3.8), 7.47–7.40 (1 H, m), 7.35 (1 H, d, J = 7.7 Hz), 7.25

8
LI et aL.
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TABLE 1 Antiproliferation activity of
compounds 7a-7o and 8a-8m
N
O
O
N
N
N
N
N
O
N
N
HN
N
N
O
S
NH
O
S
O
7
O
7
O
S
N
O
O
S
O
N
O
O
6
5
6
5
N
O
N
O
R
R
4
4
8a-8m
7a-7m
7n
7o
Antiproliferative activity (IC50 SD, μM)
Compounds
R
HCT116
MCF-7
MDA-MB-231
HepG2
7a
7b
7c
7d
7e
H
>10
5.08 0.15
>10
>10
>10
6.44 0.61
5.81 0.04
>10
>10
4.31 0.26
>10
3.24 0.17
9.54 0.98
3.29 0.08
3.35 0.11
>10
>10
4.69 0.13
>10
>10
>10
4.86 0.27
6.41 0.21
>10
>10
4.90 0.17
>10
>10
>10
>10
>10
>10
>10
4-Cl
5-Cl
6-Cl
7-Cl
4-Br
5-Br
6-Br
7-Br
7f
7g
7h
7i
>10
>10
>10
7j
7k
7l
7m
7n
7o
8a
8b
8c
8d
8e
8f
8g
8h
8i
4-OCH3 >10
6-OCH3 >10
7-OCH3 >10
3.58 0.20
>10
>10
3.85 0.05
>10
>10
3.45 0.09
1.50 0.02
3.20 0.25
3.57 0.06
2.21 0.07
4.26 0.09
3.52 0.06
>10
2.42 0.02
4.56 0.41
>10
5.12 0.31
5.22 0.21
2.08 0.07
3.58 0.03
>10
>10
>10
>10
5.81 0.09
>10
4.81 0.05
6.12 0.20
>10
>10
>10
5.94 0.38
>10
>10
5-F
H
6.65 0.12
>10
H
>10
>10
H
4.93 0.04
2.67 0.05
7.72 0.91
>10
3.78 0.03
3.91 0.39
3.06 0.51
>10
4.82 0.13
2.20 0.16
7.86 0.59
8.02 0.24
2.86 0.36
4.44 0.41
5.39 0.16
>10
3.42 0.07
7.41 0.88
>10
5.40 0.07
5.96 0.23
3.90 0.04
4-Cl
5-Cl
6-Cl
7-Cl
4-Br
5-Br
6-Br
7-Br
>10
>10
4.26 0.36
>10
4.67 0.28
4.80 0.67
>10
4.26 0.05
>10
6.75 0.15
3.36 0.33
8j
8k
8l
8m
Stattic
4-OCH3 4.93 0.05
6-OCH3 >10
7-OCH3 3.05 0.26
5-F
\
6.69 0.62
1.71 0.06
Note: IC50 values represent the mean SD for each compound based on three independent experiments.
138.27, 132.13, 126.86, 121.49, 119.15, 107.32, 61.26, 56.21,
46.53, 43.10, 21.50, 21.42, 20.85. ESI-MS for C22H26N4O4S
m/z: 465.1567 [M + Na]+.
(3 H, s), 3.83 (2 H, s),3.76 (2 H,s), 3.73 (2 H, t, J = 7.0 Hz),
3.41–3.37 (2 H, m), 2.52 (9 H, t, J = 7.9 Hz). 13C NMR
(101 MHz, CDCl3) δ: 162.87, 158.63, 150.29, 149.91,
149.90, 148.33, 146.67, 138.81, 132.22, 126.62, 121.45,
119.54, 107.37, 61.25, 57.31, 46.29, 42.59, 21.90, 21.68,
20.75. ESI-MS for C22H26N4O4S m/z: 465.1565 [M + Na]+.
8l: Yield, 68%. 1H NMR (401 MHz, CDCl3) δ 7.52 (1 H,
dd, J = 8.4, 7.5 Hz), 7.18 (1 H, s), 7.05 (1 H, d, J = 8.5 Hz),
6.98 (1 H, d, J = 7.3 Hz), 3.93 (2 H, dd, J = 6.4, 3.6 Hz),3.90

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1
8m: Yield, 38%. H NMR (401 MHz, CDCl3) δ 7.76 (1
H, dd, J = 8.1, 46 Hz), 7.29 (2 H, q, J = 2.3 Hz), 7.15 (1
H, d, J = 7.5 Hz), 3.94 (2 H, dd, J = 6.4, 3.5 Hz), 3.83
(2 H, s), 3.76–3.71 (2 H, m), 3.40–3.36 (4 H, m), 2.54 (9
H, t, J = 7.8 Hz). 13C NMR (101 MHz, CDCl3) δ: 160.12,
153.21, 149.70, 148.23, 144.44, 139.21, 139.55, 135.66,
133.71, 130.68, 125.98, 122.24, 61.54, 46.21, 42.64, 21.55,
21.10, 20.18. ESI-MS for C21H21FN4O4S m/z: 453.1278
[M + Na]+.
3.3
Compound 8b induced apoptosis in
|
cancer cells
To explore the ability of compound 8b to induce apoptosis,
Annexin V-FITC/PI staining assay was performed. The re-
sult of flow cytometry assay showed that compound 8b can
induce apoptosis in a dose-dependent manner in cancer cells
(Figure 2a). At the concentration of 2.5 μM, the apoptosis
rates of MDA-MB-231 cells, HepG2 cells, MCF-7 cells,
and HCT116 cells reached to 30.90%, 37.33%, 6.43%, and
21.90%, respectively (Figure 1b).
3.2
In vitro cell growth inhibition activities
|
The antiproliferation activity of all synthetic compounds
was evaluated using four cell lines (HCT-116, MCF-7,
MDA-MB-231, and HepG2). Stattic was used as positive
control. As shown in Table 1, the anticancer activity of
each compound was presented as IC50. No inhibition was
observed in 7n and 7o groups, indicating that piperazine
linker is more appropriate for promoting activity. 8a-8m
showed significantly better antiproliferation activity than
7a-7o, reveling the flexibility of the methylene group be-
tween ligustrazine and piperazine. The substituents in the
benzene ring have a significant effect on the activity, with
the substituents for C6 and C7 positions in benzene lead-
ing directly to the loss of activity. However, the species of
benzene substituents have little impacts on activity. Among
these compounds, 8b exhibited excellent activities against
HepG2, MCF-7, MDA-MB-231, and HCT116 cancer cell
lines with IC50 ranging from 1.50 to 5.94 μM. Considering
the potent anticancer activity, we performed further mecha-
nistic studies based on 8b.
3.4
Compound 8b induced G2-phase arrest
|
in cancer cells
We investigated the cell cycle effects of compound 8b on
HepG2, MCF-7 and HCT116 cells by flow cytometry. As
shown in Figure 3, the percentage of cells in G2 phase in-
creased significantly after treatment. These data revealed that
compound 8b could cause G2 arrest in HepG2, MCF-7 and
HCT116 cells in a dose-dependent manner.
3.5
Compound 8b induced the
|
generation of ROS in cancer cells
The induction of apoptosis through elevated ROS levels
has been highlighted as the core of targeted cancer therapy.
Considering the ROS generation function of ligustrazine
(Yu et al., 2018), we detected the ROS level in HepG2 cells
and MCF-7 cells. As shown in Figure 4, the ROS level was
FIGURE 1 Chemical structures of SH2 domain targeting STAT3 inhibitors

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FIGURE 2 Compound 8b induced apoptosis in cancer cells. (a) HepG2 cell line, MDA-MB-231 cell line, MCF-7 cell line, and HCT116 cell
line were treated with compound 8b at tested concentration for 24 hr and analyzed by flow cytometer. (b) The ratio of apoptosis cells in HepG2,
MDA-MB-231, MCF-7, and HCT116, respectively. (*p < .05, **p < .01, compared with the vehicle group)
significantly up-regulated in MCF-7 cells and HepG2 cells
when compared to vehicle, indicating that the introduction
of ligustrazine empowered 8b to generate active oxygen,
thereby resulting in significant cancer cells apoptosis.
apoptosis, we measured the ∆ψm in HepG2 cells and
MCF-7 cells. As shown in Figure 5, compound 8b in-
duced the loss of mitochondrial membrane potential in a
dose-dependent manner, indicating the apoptosis of above
cancer cells may be caused by the change of mitochon-
drial membrane potential.
3.6
Compound 8b caused the loss of
|
mitochondrial membrane potential (∆ψ m)
3.7
Compound 8b inhibits the activation of
|
It has been found that many cancer-derived cells (lung
cancer, breast cancer, melanomas) possess a higher mito-
chondrial membrane potential than normal cells (Bonnet
et al., 2007; Chen, 1988; Hockenbery, 2002; Kroemer
& Pouyssegur, 2008). In contrast, loss of mitochondrial
membrane potential is a sign of the early apoptosis me-
diated by the mitochondrial pathway (Ye et al., 2017).
To investigate the mechanism of compound 8b-induced
STAT3 and regulates the expression of proteins
related to apoptosis, cell cycle, and DNA
damage in HepG2
Western blot assay was conducted to exam whether com-
pound 8b functioned as an STAT3 inhibitor. As shown in
Figure 6a, the phosphorylation of STAT3(Tyr705) was sig-
nificantly blocked at 2.5 μM. Meanwhile, E-cadherin and

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FIGURE 3 Compound 8b induced G2-phase arrest in cancer cells. (a) HCT116 cell line, HepG2 cell line, and MCF-7 cell line were treated
with compound 8b at tested concentration for 24 hr and analyzed by flow cytometer. (b) The ratio of cell cycle phase in HCT116, HepG2, and
MCF-7 respectively. (*p < .05, **p < .01, compared with the vehicle group)
vimentin proteins associated with migration and invasion rep-
resented uptrend and downtrend, respectively, validating the
antiproliferation abilities of 8b in cancer cells. p53 protein is
the product of a critical tumor-suppressor gene; the activation
of p53 can result in DNA damage, apoptosis, and cell cycle
arrest in cancer cells. This demonstrated that 8b could effec-
tively upregulate the expression level of p53 in HepG2 cells.
To further investigate the anti-tumor mechanism, we deter-
mined the expression of proteins related to cell cycle, DNA
damage, and apoptosis. As shown in Figure 6b, 8b reduced
the expression of p-CDC2 and increased the expression of
cylin B, which were consistent with G2/M arrest results in
3.4. Besides, 8b promoted the expression of γ2HAX, demon-
strating 8b increased the DNA double-strand breaks. Cleaved
caspase 3, Bcl-2, and Bax are pivotal proteins of intrinsic ap-
optosis. In Figure 6c, the ratio of Bax/Bcl-2 and the expres-
sion of cleaved caspase 3 were significantly increased dose
dependently, indicating the apoptotic pathway might a vital
role in 8b-induced cancer cell death. Western blot assays have
proved that compound 8b regulates the expression level of p-
STAT3; thus, we carried out dual-luciferase reporter assays to
verify whether compound 8b was worked in a targeted man-
ner. The ratio of Firefly luciferase activity and Renilla lucif-
erase activity can reflect that constitutive STAT3 transcription
activity was blocked by 8b-treated HepG2 cells; compound
8b had stronger inhibitory ability then Stattic (Figure 6c). In
general, all these results proved that compound 8b is an effec-
tive inhibitor targeting STAT3.
3.8
Molecular docking study
|
To figure out the binding model of 8b to STAT3, we docked
compound 8b to the SH2 domain (PDB:1BG1). As shown
in Figure 7, the BTP part of compound 8b binds to pY-X
tightly. On this basis, the piperazine ring elongates the mo-
lecular length; methylene turns and provides ligustrazine
fragment excellent flexibility and angle to bind into pY cav-
ity. Generally, compound 8b forms hydrogen bonds with
Lys591 and Ser636, and Pi-Pi conjugations with other amino
acid residues can also be found.
4
CONCLUSION
|
In this study, we successfully designed and synthesized
a series of novel inhibitors based on benzo[b]thiophene
1,1-dioxide scaffold. Compound 8b was found to be the most

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FIGURE 4 Compound 8b induced
the generation of ROS in cancer cells.
(a) MCF-7 cell line was treated with
compound 8b at tested concentration for
24 hr, ROS accumulation was analyzed
by flow cytometer; relative ROS level in
MCF-7 was represented by a histogram.
(b) HepG2 cell lines were treated with
compound 8b at tested concentration for
24 hr, ROS accumulation was analyzed
by flow cytometer, and relative ROS level
in HepG2 was represented by a histogram
(*p < .05, **p < .01, compared to the
vehicle group)
FIGURE 5 Compound 8b caused the
loss of mitochondrial membrane potential
(∆ψm). (a) MCF-7 cell was treated with
compound 8b at tested concentration
for 24 hr, ∆ψm was analyzed by flow
cytometer. The loss of mitochondrial
membrane potential (%) was counted with
histogram. (b) HepG2 cell were treated
with compound 8b at tested concentration
for 24 hr; ∆ψm was analyzed by flow
cytometer. The loss of mitochondrial
membrane potential (%) was counted with
histogram. (*p < .05, **p < .01, compared
to the vehicle group)
potent compound in cell antiproliferation assay in four can-
cer cell lines. Further mechanism research proved that com-
pound 8b significantly induced the apoptosis in HCT116
cells, MCF-7 cells, MDA-MB-231 cells, and HepG2 cells.
Meanwhile, G2/M-phase blocking was observed after treat-
ment. Subsequently, we detected the ROS levels and ∆ψm
of mitochondrial membrane potential and observed the up-
trend and downtrend, respectively. Western blot assays and

LI et aL.
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FIGURE 6 Western blot results of
compound 8b. (a) The protein level of
STAT3, p-STAT3, E-cadherin, vimentin,
p53 in HepG2 cells. (b) The protein level of
cylin B, p-CDC2, γ2HAX in HepG2 cells.
(c) The protein level of cleaved caspase
3, caspase 3, Bax, Bcl-2 in HepG2 cells.
(d) HepG2 cells were transfected with
pLuc-TK/STAT3 and pRL-TK before the
treatment with 8b for additional 48 hr; the
results were expressed as relative luciferase
activity (*p < .05, **p < .01, ***p < .001
compared to the vehicle group)
FIGURE 7 Molecular docking results. Predicted binding model of 8b in STAT3 SH2 domain (PDB:1BG1), the figure was generated by using
pymol
dual-luciferase reporter assays demonstrated that 8b inhibited
the phosphorylation of STAT3 and regulated the expression
of proteins associated with apoptosis, cell cycle, and DNA
damage. Docking study revealed that compound 8b bound to
pY and pY-X cavities of the STAT3 SH2 domain. In sum-
mary, all these evidences suggested compound 8b a novel
potent STAT3 inhibitor and a potential candidate for tumor
treatment.
ACKNOWLEDGMENTS
We appreciate the Support Program of Science & Technology
Department of Sichuan Provincial (no. 2021YFH0173).

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CONFLICT OF INTEREST
The authors declare no conflict of interests.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Li, W.-Z., Xi, H.-Z.,
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Liao, M.-Y., Xia, Y., & Zhang, Y.-W. (2021).
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