Design, synthesis and biological evaluation of novel potent STAT3 inhibitors based on BBI608 for cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2020.112428

Persistently activated signal transducer and activator of transcription 3 (STAT3) plays an important role in the development of multiple cancers, and therefore is a potential therapeutic target for cancer prevention. Herein, we report the rational design, synthesis, and biological evaluation of novel potent STAT3 inhibitors based on BBI608. Among them, compound A11 exhibited the most potent in vitro tumor cell growth inhibitory activities toward MDA-MB-231, MDA-MB-468 and HepG2 cells with IC₅₀ values as low as 0.67 ± 0.02 μM, 0.77 ± 0.01 μM and 1.24 ± 0.16 μM, respectively. Fluorescence polarization (FP) assay validated the binding of compound A11 in STAT3 SH2 domain with the IC₅₀ value of 5.18 μM. Further mechanistic studies indicated that A11 inhibited the activation of STAT3 (Y705), and thus reduced the expression of STAT3 downstream genes CyclinD1 and C-Myc. Simultaneously, it induced cancer cell S phase arrest and apoptosis in a concentration-dependent manner. An additional in vivo study revealed that A11 suppressed the MDA-MB-231 xenograft tumor growth in mice at the dose of 10 mg/kg (i.p.) without obvious body-weight loss. Finally, molecular docking study further elucidated the binding mode of A11 in STAT3 SH2 domain.

Full text of DOI:10.1016/j.ejmech.2020.112428

Journal Pre-proof
Design, synthesis and biological evaluation of novel potent STAT3 inhibitors based on
BBI608 for cancer therapy
Kai-Rui Feng, Feng Wang, Xin-Wei Shi, Yun-Xuan Tan, Jia-Ying Zhao, Jian-Wei
Zhang, Qing-Hua Li, Guo-Qiang Lin, Dingding Gao, Ping Tian
PII:
S0223-5234(20)30399-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112428
EJMECH 112428
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 January 2020
Revised Date: 24 April 2020
Accepted Date: 5 May 2020
Please cite this article as: K.-R. Feng, F. Wang, X.-W. Shi, Y.-X. Tan, J.-Y. Zhao, J.-W. Zhang, Q.-H. Li,
G.-Q. Lin, D. Gao, P. Tian, Design, synthesis and biological evaluation of novel potent STAT3 inhibitors
based on BBI608 for cancer therapy, European Journal of Medicinal Chemistry (2020), doi: https://
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Design, synthesis and biological evaluation of novel potent
STAT3 inhibitors based on BBI608 for cancer therapy
#
#
Kai-Rui Feng , Feng Wang , Xin-Wei Shi, Yun-Xuan Tan, Jia-Ying Zhao, Jian-Wei Zhang,
Qing-Hua Li *, Guo-Qiang Lin, Dingding Gao *, and Ping Tian *

Design, synthesis and biological evaluation of novel potent
STAT3 inhibitors based on BBI608 for cancer therapy
a
a
Kai-Rui Feng a, #, Feng Wang a, #, Xin-Wei Shi , Yun-Xuan Tan, b Jia-Ying Zhao , Jian-Wei
Zhang a, Qing-Hua Li a, *, Guo-Qiang Lin a, b, Dingding Gao a , *, and Ping Tian a, b,
*
a
The Research Center of Chiral Drugs, Innovation Research Institute of Traditional Chinese
Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
b
CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of
Sciences, Chinese Academy of Sciences, Shanghai 200032, China
# These authors contributed equally to this work.
* Corresponding authors:
E-mail addresses: gaodingding@shutcm.edu.cn (Dingding Gao), qinghuali@shutcm.edu.cn
(Qing-Hua Li), tianping@shutcm.edu.cn (Ping Tian)
Abstract: Persistently activated signal transducer and activator of transcription 3
(STAT3) plays an important role in the development of multiple cancers, and therefore
is a potential therapeutic target for cancer prevention. Herein, we report the rational
design, synthesis, and biological evaluation of novel potent STAT3 inhibitors based
on BBI608. Among them, compound A11 exhibited the most potent in vitro tumor cell
growth inhibitory activities toward MDA-MB-231, MDA-MB-468 and HepG2 cells
with IC50 values as low as 0.67±0.02 µM, 0.77±0.01 µM and 1.24±0.16 µM,
respectively. Fluorescence polarization (FP) assay validated the binding of compound
A11 in STAT3 SH2 domain with the IC50 value of 5.18 µM. Further mechanistic
studies indicated that A11 inhibited the activation of STAT3 (Y705), and thus reduced
the expression of STAT3 downstream genes CyclinD1 and C-Myc. Simultaneously, it
induced cancer cell S phase arrest and apoptosis in a concentration-dependent manner.
An additional in vivo study revealed that A11 suppressed the MDA-MB-231
xenograft tumor growth in mice at the dose of 10 mg/kg (i.p.) without obvious
body-weight loss. Finally, molecular docking study further elucidated the binding
mode of A11 in STAT3 SH2 domain.
Keywords: STAT3 inhibitors; BBI608; anti-tumor activity; molecular docking.

1. Introduction
Signal transducer and activator of transcription 3 (STAT3) is a member of the
STAT family and play a pivotal role in tumor initiation, progression and maintenance
[1-4]. Once upon activated by upstream kinases (growth factor receptors, tyrosine
kinases, Janus kinases, Src family kinases, etc.), STAT3 monomers are
phosphorylated at specific tyrosine residues (Y705) in the C-terminal domain and
form transcriptionally active homodimers in a symmetric reciprocal manner. Then
these homodimers translocate into the nucleus and bind to a specific DNA sequence,
regulating target genes transcription [5-7,]. Abnormally activated STAT3 signaling is
found in a variety of solid tumors and hematological malignancies, and can induce
tumor angiogenesis and suppresses anti-tumor immune responses [8-11]. In addition,
[12,13]
aberrant STAT3 correlates with resistance to chemotherapy and poor prognosis
.
Abundant evidence suggests that STAT3 is an ideal target for cancer therapy, and
various small inhibitors were identified using multiple approaches [14], such as
[20]
STA-21 [15], LLL-12 [16], LY-5 [17], Stattic [18], Niclosamide [19], S3I-201
,
.
S3I-201-1066 [21], 8 [22], BBI608 [23], and other representative STAT3 inhibitors [24-31]
It is worth noting that Wang and co-workers have recently reported some potent,
selective, and efficacious small-molecule STAT3 degraders based upon the proteolysis
targeting chimera (PROTAC) concept [32,33]. However, there has been no
STAT3-targeting drug approved by the FDA to date. This may be partly due to the
lack of understanding of signaling crosstalk and adverse events related to
STAT3-specific activity in normal tissues. Also, the scarcity of membrane
permeability and stability, and weak binding affinity were found to be the main
roadblocks of these inhibitors [34-36]. Consequently, development of novel potent
STAT3 inhibitors will bring enormous challenges and opportunities in the cancer
prevention field.

Fig. 1. Representative of known STAT3 inhibitors
Among all the reported STAT3 inhibitors, only BBI608 (Napabucasin) has been
shown to block cancer stem cell pathway activity and is currently in Phase II/III
clinical trials for the treatment of a variety of cancers [37]. To our knowledge, the
previous structural modifications of BBI608 mainly focused on the acetyl group at
[38-42]
2-position of furan ring
(Fig. 2), and lack of structural diversity. These finding
prompted us to develop new BBI608 derivatives with novel scaffolds and potent
antitumor activities using structure-based drug design in this work. Firstly, BBI608
was docked into STAT3 SH2 domain (PDB code: 1BG1) and the binding mode was
shown in Fig. 3A. The 2-acetylfuran group was located at the pY705 (also named
pY+0) site, which is related to the disruption of STAT3 phosphorylation and
dimerization of inhibitors, and formed a hydrogen bond with Arg609. However, the
other important pocket (pY+X site) stayed vacant and was not occupied by any other
group. Accordingly, to improve the binding affinity and find new STAT3 inhibitors,
the benzene ring in BBI608 was selected to be cleaved and diverse functional groups
were directly introduced into the quinone ring via suitable linkers for reaching the
pY+X site (Fig. 2). The binding modes of designed compounds harboring different
linkers (a1−a5) were predicted using docking studies and shown in Fig. 3B−3F.
Considering that the free amine, ie -NH- (linker a1), in the compound A1 could form
hydrogen bond with ILE634, a1 was determined as a privileged linker. As a
consequence, various benzene ring derivatives and fragments were then carefully
investigated.

Fig. 2. Previous and our structure-based drug design of STAT3 inhibitors based on BBI608
Fig. 3. Docking modes of BBI608 and designed compounds A1−A5.
2. Results and discussion
2.1 Chemistry

The synthetic route for designed compounds was described in Scheme 1. Upon
treatment of commercially available, starting material 10 with bromine and
aluminium trichloride, the electrophilic bromination of phenol occurred smoothly to
deliver the dibromide 11 [43]. The p-bromophenol 11 was then easily oxidized to
quinone 12 as a key intermediate by treatment of chromium oxide and acetic acid [43]
.
Finally, the target compounds were readily prepared through Pd-catalyzed C−N
cross-coupling reaction with the use of various aromatic or aliphatic amines [44,45]. As
a representative example, the exact structure of A24 was unambiguously established
by X-ray crystallography (Fig. 4) [46]
.
Regents and conditions: (a) Br2, AlCl3, CH2Cl2, 40 ; (b) CrO3, CH3COOH, 60 ; (c) Pd(OAc)2,
BINAP, Cs2CO3, Toluene, 120
.
Scheme 1. Synthesis of Compounds A1, A6-A30
Fig. 4. X-ray crystallography of a representative compound A24.
2.2 Biological assay
2.2.1 In vitro cell growth inhibitory activity
We firstly evaluated the inhibitory activities of our designed compounds against
three STAT3 over-expressed human cancer cell lines (breast cancer cell lines:
MDA-MB-231 and MDA-MB-468; human liver carcinoma cell line: HepG2) via
established CCK8 assay. The IC50 values were listed in Table 1 with BBI608 and
Stattic as positive controls. In line with our expectations, all compounds displayed
potent antiproliferative activities against tested cancer cells and their IC50 values

ranged from sub-micromole to micromole. Apparently, compounds A6 and A11
exhibited considerable or even better inhibitory activities against two breast cancer
cell lines compared to BBI608. The preliminary SAR that the additional functional
groups occupied the side pocket was summarized as follows: Compound A6
possessing a 2-CH3 and compound A11 containing a 4-OCH3 showed better
efficiency than other compounds with a single substituent group installed on the
benzene ring (A7-A10, A12-A20). Incorporation of two or more substituents on the
benzene ring (A21-A25) caused a slight decrease in activities. Changing phenyl
substituent to different size of cycloalkyl substituents (A26-A29) led to the poor
outcome that their corresponding derivatives except for cyclopropyl compound A26
(0.94-1.97 µM) had relatively low potency. In addition, replacement of cyclohexyl
group with tetrahydropyranyl substituent had no obvious effect on activities
(compounds A29 vs A30). Taken together, compound A11 showed comprehensive
potency in vitro anti-tumor activities and was selected for further biological study.
Table 1. Antiproliferative activity of the designed compounds
O
R
O
O
N
H
O
IC50 ± SD (µM) a
Compd.
R
MDA-MB-231
MDA-MB-468
HepG2
-Ph
1.14±0.16
0.56±0.10
2.62±0.10
0.90±0.08
0.92±0.08
1.48±0.06
0.67±0.02
1.03±0.06
0.73±0.04
4.54±0.17
3.12±0.02
1.05±0.11
1.55±0.05
0.77±0.01
1.54±0.24
A1
2-CH3-Ph
3-CH3-Ph
4-CH3-Ph
2-OCH3-Ph
3-OCH3-Ph
4-OCH3-Ph
1.21±0.01
5.50±0.20
1.85±0.07
2.41±0.33
2.36±0.33
1.24±0.16
A6
A7
A8
A9
A10
A11

2-F-Ph
2.34±0.19
3.50±0.18
3.21±0.10
3.12±0.23
1.51±0.17
3.70±0.30
2.00±0.25
1.53±0.03
1.11±0.19
2.00±0.25
2.50±0.43
7.77±0.32
1.75±0.12
1.16±0.17
2.27±0.50
2.46±0.02
1.83±0.24
1.18±0.25
2.55±0.10
5.34±0.45
8.01±0.11
4.04±0.21
1.99±0.06
6.17±0.13
3.38±0.52
1.51±0.12
1.04±0.18
A12
A13
A14
A15
A16
A17
A18
A19
A20
3-F-Ph
4-F-Ph
2-CF3-Ph
3-CF3-Ph
4-CF3-Ph
2-C2H5-Ph
4-N(CH3)2-Ph
4-isopropoxy-Ph
2-CH3,4-OCH3-Ph
2-CH3,6-CH3-Ph
2.46±0.28
1.88±0.05
2.92±0.05
3.3±0.14
5.90±0.26
8.03±0.41
A21
A22
2-CH3,4-OCH3,6-
0.67±0.02
1.53±1.22
2.01±0.02
A23
CH3-Ph
3-CH3,4-CH3-Ph
3-CH3,5-F-Ph
2.54±0.28
2.62±0.37
0.94±0.09
1.85±0.02
2.11±0.26
1.97±0.16
8.12±0.40
5.66±0.46
1.83±0.03
A24
A25
A26
6.75±0.06
7.17±0.37
6.55±0.18
6.80±0.17
A27
A28
A29
A30
4.87±0.20
1.23±0.03
8.84±0.64
2.47±0.11
2.88±0.07
2.07±0.28
4.13±0.44
3.23±0.06
BBI608
Stattic
-
-
0.70±0.06
2.64±0.03
1.14±0.04
1.92±0.08
0.84±0.03
ND
a The inhibitory effects of these compounds on the proliferation of cancer cell lines were determined by the CCK8
assay. The data are the mean ± SD from at least three independent experiments.
2.2.2 Fluorescence Polarization (FP) Assay

It has been reported that STAT3 SH2 domain can bind to
5-FAM-GpYLPQTV-NH2 derived peptide with a high affinity and then disrupted
STAT3-STAT3 interaction and DNA-binding activity [47]. To verify the directly
binding of the representative compounds A6 and A11 to the STAT3 SH2 domain,
FP-based competition binding assay was conducted according to previously reported
method (Fig. 5). Firstly, the 5-FAM-GpYLPQTV-NH2derived peptide was applied as
the fluorescent probe and showed the Kd value of 174 nM in our experiment, which
was almost equivalent to the reported value (Kd=150 nM) by Berg and coworker [47]
.
Then, compound binding assays were performed in a 100 µL volume at a
concentration of 10 nM 5-FAM-GpYLPQTV-NH2 probe and 160 nM STAT3 protein.
As demonstrated in Fig. 5, compounds A6 and A11 bound to STAT3 protein in a
concentration-dependent manner with IC50 values of 5.55 µM and 5.18 µM,
respectively, which indicating the interaction of compounds A6 and A11 with the
STAT3 SH2 domain.
Fig. 5. (A) Binding of the fluorescent probes 5-FAM-GpYLPQTV-NH2 to STAT3 SH2 domain.
The probe was incubated at 10 nM with increasing amounts of STAT3 protein. (B) Dose−response
competitive binding curve of compounds A6 and A11 to STAT3 using the FP-based binding assay.
2.2.3 Compounds A11 and A6 inhibited the phosphorylation of STAT3 and its
downstream target proteins
As STAT3 inhibitors can suppress the STAT3 phosphorylation on Tyr705 residue
and thus restrain the expression of its downstream target proteins, then we turned our
effort to the effects of compound A11 and A6 in MDA-MB-231 cells adopting
western blot analysis. As shown in Fig. 6, compound A11 and A6 decreased the
STAT3-Y705 phosphorylation in a dose-dependent manner without affecting the total

amount of STAT3 protein after 24 hrs incubation. These results clearly manifested that
the decrease of Tyr705 phosphorylated STAT3 was not owing to the constitutional
drop of total STAT3 protein, Also, these two representative compounds could decrease
the expression of STAT3 target genes, including C-Myc and Cyclin D1, in a
dose-dependent manner. It is noteworthy that compound A11 and A6 had little impact
on the level of STAT1 and its phosphorylation on Tyr701, which suggested that they
had a good selectivity against the tumor suppressor STAT1.
Fig. 6. Western blot analysis of the inhibition of STAT3-Y705 phosphorylation, the selective
inhibition against STAT1 and the downstream target proteins (C-Myc and Cyclin D1) by
compound A11 in the MDA-MB-231 cell line. Cells were treated with A11 or A6 for 24 hrs, and
levels of STAT3, pSTAT3, STAT1, p-STAT1, C-Myc and Cyclin D1 were probed by specific
antibodies. GAPDH was used as the loading control.
2.2.4 Immunofluorescent assay
To further explore the effect of compound A11 on STAT3 phosphorylation in
MDA-MB-231 cells, immunofluorescent assay was conducted. As expected, after
incubation with MDA-MB-231 cells in 3.0 µM for 24 hrs, compound A11 markedly
reduced the expression of p-STAT3 (p-Tyr705) both in nucleus and cytoplasm
compared to the control group (Fig. 7). The results provided another evidence that
A11 had an intense inhibition on the phosphorylation of STAT3 in MDA-MB-231
cells.

Fig. 7. MDA-MB-231 cells were incubated with 3.0 µM A11 for 24 hrs and stained with
anti-phospho-STAT3 (p-STAT3) and Hoechst before subjected to analysis. Red: p-STAT3; blue:
nucleus (Scale bar: 10 μm).
2.2.5 Compound A11 induced apoptosis in cancer cells
As we can see in Table 1, compound A11 displayed potent anti-proliferative
potency toward three cell lines, we then inquired the effect of A11 in the induction of
MDA-MB-231 cells apoptosis through Annexin-V-FITC/PI staining assay by flow
cytometry. As depicted in Fig. 8, compound A11 induced the apoptosis of
MDA-MB-231 cell in a dose-dependent manner. The apoptosis rates at 0, 1, 2, 4 µM
were 6.4%, 11.72%, 17.38%, and 32.28%, respectively, which was consistent with its
antitumor efficacy in MDA-MB-231 cells.

Fig. 8. Flow cytometric analysis of the apoptotic effect of compound A11 in MDA-MB-231 cells
through Annexin-V-FITC/PI staining assay. (A) MDA-MB-231 cells were treated with compound
A11 at tested concentrations for 24 h. (B) The histograms for apoptosis rate (early and late stages
of apoptosis). Data are the mean ± SD of three independent experiments. ***, p < 0.001, **, p <
0.01.
2.2.6 Analysis of cell cycle effect
The effect of compound A11 on cell cycle progression of MDA-MB-231 cells
was assessed using flow cytometry and the results were shown in Fig. 9. After treating
A11 with MDA-MB-231 cells at the concentration of 0 µM, 1 µM, 2 µM and 4 µM
for 24 hrs, the proportion of cells in S phase increased from 24.65 % to 53.40%
accompanying with a decrease of cells in G0/G1 phase and G2/M phase. These results
suggested that A11 could dose-dependently cause a significant S phase arrest in
MDA-MB-231 cells.

Fig. 9. Cell cycle analysis of compound A11 by flow cytometry. MDA-MB-231 cells were treated
with increasing concentrations of compound A11 for 24 hrs.
2.2.7 In Vivo Study of Compound A11
We then estimated the in vivo anti-tumor activity of compound A11 using a
mouse xenograft model bearing inoculation of human breast cancer cells
MDA-MB-231. After the solid tumors were established, compound A11 was
intraperitoneally (i.p.) administered once daily at two doses (5 and 10 mg/kg) for 21
days. As demonstrated in Fig. 10A-B, the treatment with compound A11 brought in an
obvious reduction of the tumor volume at the dose of 10 mg/kg compared to control
group on the twenty-first day (TGITV=54.62%). Besides, the tumor weights of mice
were notably abated by 45.19% with a dosage of A11 at 10 mg/kg (Fig. 10C). What's
more, there was no apparent body-weight loss for those mice treated with compound
A11 at both dosage (Fig. 9D). Furthermore, the immunofluorescent assay of the tumor
tissue revealed that the level of p-STAT3 (Y705) was obviously inhibited by
compound A11 at a dose of 10 mg/kg (Fig. 11). To sum up, compound A11 exhibited
good in vivo anti-tumor capacity and deserved further pharmaceutical studies.

Fig. 10. Compound A11 inhibited the growth of human xenograft tumor in vivo. MDA-MB-231
xenograft mouse models were treated with A11 or vehicle control daily at indicated dosages for 21
consecutive days. (A) Anatomical nude mice’s tumor tissues untreated or treated with A11. (B)
The tumor volume and the tumor growth inhibition values (TGITV) were measured on the final day
of the study. (C) The tumor weight and the tumor growth inhibition values (TGITW) were
measured on the final day of the study. (D) The growing curves of mice’s body weight. Data are
shown as mean ± SEM, n=6; *, P < 0.05.
Fig. 11. Immunofluorescent assay revealed that A11 inhibited the levels of p-STAT3 in tumor
tissues. Data are shown as mean ± SEM, n=4; **, p < 0.01 (Scale bar: 50 μm).

2.2.8 Molecular modeling simulations
In an attempt to elucidate the binding mode of compound A11 with STAT3 SH2
domain, docking study was carried out on the basis of the crystal structure of STAT3
homo dimer (PDB code: 1BG1). For a comparison, the binding mode of BBI608 was
also generated and superimposed on that of A11 (Fig. 12A). As represented, the
p-methoxyphenyl in compound A11 was extended to the pY+X site, which was not
occupied by BBI608 as expected. Such a transformation subsequently gained a
hydrogen bond interaction between the methoxy group of A11 and the guanidyl of
ARG595. Moreover, the NH formed a strong hydrogen bond with ILE634 and the
distance was 2.0 Å. The other one hydrogen bond (2.5 Å) was engendered between
the oxygen atom of acetyl and the amino group of LYS591. (Fig. 12B) In general, all
these interactions ensured the activities of compound A11 toward STAT3 protein.
Fig. 12. Molecular modeling study of A11 in STAT3 SH2 domain (PDB code: 1BG1). (A)
Superimposed pose of A11 (green) and BBI606 (pink) bound in the surface of binding site. (B)
Predicted interaction of A11 (green) within STAT3 SH2 domain. The figures were generated using
Pymol.
3. Conclusions
In summary, structure-based drug design strategy was applied to generate new
scaffold STAT3 inhibitors based on BBI608. Among them, compound A11 was
evinced to be remarkable anti-tumor activities against MDA-MB-231, MDA-MB-468
and HepG2 cells in vitro with an IC50 range of 0.67−1.24 µM. The direct interaction
between compound A11 and STAT3 SH2 domain was validated using fluorescence
polarization assay with the IC50 value of 5.18 µM. Cellular mechanistic studies

showed that A11 can suppress the expression level of phosphorylated STAT3
(p-STAT3) and then downregulated its downstream gene C-Myc and Cyclin D1
without influence the total STAT3 protein. Furthermore, compound A11 manifested
good selectivity against STAT1 which was a member of STAT family and a tumor
suppressor. Moreover, A11 displayed preferable anti-tumor efficacy in vivo toward
MDA-MB-231 xenograft mouse model at a low dose of 10 mg/kg. Finally, molecular
docking study clarified the binding mode of compound A11 in STAT3 SH2 domain.
Collectively, these studies provided more structural reference for the development of
STAT3 inhibitors and suggested that compound A11 might be a highly potent and
minimally toxic anti-tumor lead compound worthy of further investigation.
4. Experimental section
4.1. Chemistry
All solvents and reagents were purchased from commercial suppliers such as
Bide pharmatech, Adamas-beta®, etc., and directly used without further purification
unless specified. Flash chromatography was performed on silica gel (200-300 mesh)
and visualized under UV light monitor (λ = 254 nm and 365 nm). The nuclear
magnetic resonance (NMR) spectroscopy were recorded in CDCl3 or DMSO-d6 with
Bruker 600 MHz spectrometer (TMS as internal standard) at ambient temperature.
The chemical shifts (δ) were expressed in parts per million (ppm) downfield and
coupling constants (J) values were described as hertz. MS was measured on Shimadzu
8040 quadrupole LC/MS system. High-resolution mass spectra (HRMS) data were
given by Agilent 6545 Accurate-Mass Q-TOF LC/MS system. The X-Ray crystal
structures of compound A24 was measured on X-ray Single Crystal Diffractometer
(Bruker D8 Venture).
4.1.1. Synthesis of 1-(4,6-dibromo-7-hydroxybenzofuran-2-yl)ethan-1-one (11)
To a solution of 1-(7-hydroxybenzofuran-2-yl)ethan-1-one (10, 5.0 g, 28.29 mmol) in
CH2Cl2 (200 mL) was added AlCl3 (15.1 g, 113.24 mmol) and the mixture was
keeping stirred for 1 h at 40 . Then, Br2 (10.0 g, 62.57 mmol) was added dropwise

to the reaction mixture over 90 min period at 26 . The resulting mixture was stirred
for 18 hrs at 26 , and poured into ice water, and followed by treatment of sodium
thiosulfate. The resulting precipitate was collected by filtration and washed with water,
then recrystallized from ethyl acetate to give the compound 11. Colorless solid; yield:
1
93.1%. H NMR (600 MHz, DMSO-d6) δ 11.40 (s, 1H)，7.86 (s, 1H), 7.72 (s, 1H),
2.61 (s, 3H). ESI-LCMS [M+H]+ calcd for C10H7Br2O3 332.88, found: 332.75.
4.1.2. Synthesis of 2-acetyl-6-bromobenzofuran-4,7-dione (12)
To a solution of compound 11 (8.8 g, 26.28 mmol) in 100 mL of 80 % acetic acid in
water was added CrO3 (5.8 g, 52.56 mmol, a solution in 20 mL of water). The
reaction mixture was stirred at room temperature for 3 hrs. After cooling to room
temperature, the reaction solution was concentrated in vacuo and the residue was
dissolved in chloroform. The chloroform phase was washed with water, dried over
sodium sulfate, and concentrated in vacuo. The residue was purified by column
chromatography (PE/EAꢀ=ꢀ4:1) to give the compound 12. Yellow solid; yield: 34.5%.
1H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H)，7.34 (s, 1H)，2.64 (s, 3H). ESI-LCMS
[M+H]+ calcd for C10H6BrO4 268.94, found: 268.85.
4.1.3. Synthesis of compounds A1, A6-A30
To a Schlenk flask was added the corresponding amine (0.20 mmol),
2-acetyl-6-bromobenzofuran-4,7-dione (12, 60.0 mg, 0.22 mmol), Pd(OAc)2 (5.5 mg,
0.02 mmol), BINAP (14.3 mg, 0.02 mmol), Cs2CO3 (293.2 mg, 0.9 mmol) and
anhydrous toluene (6.0 mL) under nitrogen atmosphere, respectively. The reaction
mixture was refluxed at 120 °C for 8 hrs. After cooling to room temperature, the
mixture was filtered through a pad of Celite and washed with ethyl acetate. The
organic solvent was removed under vacuum and the residue was purified by silica-gel
column chromatography to give the compounds A1, A6-A30.
4.1.3.1. 2-Acetyl-6-(phenylamino)benzofuran-4,7-dione (A1)
1
Purple solid; yield: 32.3%. H NMR (600 MHz, CDCl3) δ 7.46 (d, J = 4.2 Hz, 2H),
7.43 (d, J = 7.8 Hz, 2H), 7.27 (s, 1H), 7.26 (d, J = 3.8 Hz, 2H), 6.17 (s, 1H), 2.64 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 187.30, 180.63, 171.62, 155.97, 149.09, 144.46,

137.02, 131.53, 129.82, 126.22, 122.85, 112.53, 100.66, 26.71. ESI-HRMS [M+H]+
calcd for C16H12NO4 282.0761, found: 282.0768.
4.1.3.2. 2-Acetyl-6-(o-tolylamino)benzofuran-4,7-dione (A6)
1
Purple solid; yield: 23.2%, H NMR (600 MHz, CDCl3) δ 7.45 (s, 1H), 7.31 (d, J =
7.5 Hz, 1H), 7.28 (s, 1H), 7.25-7.22 (m, 3H), 5.70 (s, 1H), 2.63 (s, 3H), 2.28 (s, 3H).
13C NMR (150 MHz, DMSO-d6) δ 187.54, 180.08, 171.97, 155.10, 150.36, 148.56,
136.65, 135.10, 131.56, 130.66, 127.89, 127.40, 127.30, 114.64, 99.06, 27.12, 17.86.
ESI-HRMS [M+Na]+ calcd for C17H13NNaO4 318.0737, found: 318.0754.
4.1.3.3. 2-Acetyl-6-(m-tolylamino)benzofuran-4,7-dione (A7)
1
Purple solid; yield: 36.6%, H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H), 7.42 (s, 1H),
7.31 (t, J = 7.4 Hz, 1H), 7.06 (d, J = 10.9 Hz, 3H), 6.17 (s, 1H), 2.63 (s, 3H), 2.39 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 187.31, 180.66, 171.67, 155.97, 149.10, 144.48,
139.97, 136.94, 131.57, 129.62, 127.04, 123.33, 119.85, 112.54, 100.62, 26.70, 21.44.
ESI-HRMS [M+Na]+ calcd for C17H13NNaO4 318.0737, found: 318.0745.
4.1.3.4. 2-Acetyl-6-(p-tolylamino)benzofuran-4,7-dione (A8)
1
Purple solid; yield: 35.4%, H NMR (600 MHz, CDCl3) δ 7.45 (s, 1H), 7.41 (s, 1H),
7.23 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 6.10 (s, 1H), 2.63 (s, 3H), 2.37 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 187.31, 180.54, 171.69, 155.94, 149.10, 144.76,
136.31, 134.34, 131.62, 130.36, 122.90, 112.56, 100.29, 26.69, 21.03. ESI-HRMS
[M+Na]+ calcd for C17H13NNaO4 318.0737, found: 318.0744.
4.1.3.5. 2-Acetyl-6-((2-methoxyphenyl)amino)benzofuran-4,7-dione (A9)
Purple solid ; yield: 30.8%, 1H NMR (600 MHz, CDCl3) δ 7.87 (s, 1H), 7.46 (s, 1H),
7.40-7.38 (m, 1H), 7.20-7.16 (m, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.2 Hz,
13
1H), 6.24 (s, 1H), 3.93 (s, 3H), 2.63 (s, 3H). C NMR (150 MHz, CDCl3) δ 187.35,
180.68, 171.70, 155.84, 151.27, 149.24, 143.62, 131.45, 126.44, 126.13, 121.34,
120.92, 112.52, 111.23, 100.79, 55.79, 26.71. ESI-HRMS [M+H]+ calcd for
C17H14NO5 312.0866, found: 312.0859.
4.1.3.6. 2-Acetyl-6-((3-methoxyphenyl)amino)benzofuran-4,7-dione (A10)
1
Purple solid; yield: 32.2%, H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H), 7.42 (s, 1H),
7.35-7.32 (m, 1H), 6.85 (d, J = 7.4 Hz, 1H), 6.79-6.78(m, 2H), 6.21 (s, 1H), 3.83 (s,

3H), 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.28, 180.63, 171.61, 160.69,
155.98, 149.09, 144.30, 138.19, 131.51, 130.60, 114.97, 112.52, 111.51, 108.76,
101.09, 55.48, 26.69. ESI-HRMS [M+H]+ calcd for C17H14NO5 312.0866, found:
312.0866.
4.1.3.7. 2-Acetyl-6-((4-methoxyphenyl)amino)benzofuran-4,7-dione (A11)
1
Purple solid; yield: 15.1%, H NMR (600 MHz, CDCl3) δ 7.45 (s, 1H), 7.35 (s, 1H),
7.19 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 5.98 (s, 1H), 3.84 (s, 3H), 2.63 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 187.31, 180.42, 171.68, 158.03, 155.94, 149.11,
145.41, 131.72, 129.59, 124.97, 115.01, 112.58, 99.83, 55.58, 26.69. ESI-HRMS
[M+H]+ calcd for C17H14NO5 312.0866, found: 312.0867.
4.1.3.8. 2-Acetyl-6-((2-fluorophenyl)amino)benzofuran-4,7-dione (A12)
Purple solid; yield: 27.5%, 1H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H), 7.41-7.39 (m,
2H), 7.24-7.21 (m, 3H), 6.05 (s, 1H), 2.64 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
187.31, 180.62, 171.26, 156.03, 154.65, 149.15, 144.18, 131.35, 127.32, 125.29,
124.92, 124.07, 116.74, 112.44, 101.85, 26.71. ESI-HRMS [M+H]+ calcd for
C16H11FNO4 300.0667, found: 300.0679.
4.1.3.9. 2-Acetyl-6-((3-fluorophenyl)amino)benzofuran-4,7-dione (A13)
1
Purple solid; yield: 15.7%, H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H), 7.43 (s, 1H),
7.40 (d, J = 6.8 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 6.95 (t, J =
7.7 Hz, 1H), 6.21 (s, 1H), 2.64 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.27, 180.65,
171.38, 164.11, 162.46, 156.07, 149.06, 143.86, 138.67, 131.13, 118.23, 112.94,
112.50, 109.83, 101.60, 26.72. ESI-HRMS [M-H]- calcd for C16H9FNO4 298.0516,
found: 298.0517.
4.1.3.10. 2-Acetyl-6-((4-fluorophenyl)amino)benzofuran-4,7-dione (A14)
1
Purple solid; yield: 15.3%, H NMR (600 MHz, CDCl3) δ 7.49 (s, 1H), 7.45 (s, 1H),
7.25-7.23 (m, 2H), 7.15 (t, J = 8.4 Hz, 2H), 6.01 (s, 1H), 2.62 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 187.26, 175.92, 168.99, 161.90, 160.26, 155.02, 152.69, 143.62,
132.83, 127.12, 124.78, 115.68, 115.53, 111.55, 102.64, 26.73. ESI-HRMS [M+H]+
calcd for C16H11FNO4 300.0667, found: 300.0675.
4.1.3.11. 2-Acetyl-6-((2-(trifluoromethyl)phenyl)amino)benzofuran-4,7-dione (A15)

1
Orange solid; yield: 35.8%, H NMR (600 MHz, CDCl3) δ 7.76 (d, J = 7.9 Hz, 1H),
7.65 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H), 7.46 (s, 1H), 7.41 (t, J
= 7.5 Hz, 1H), 5.94 (s, 1H), 2.64 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.34,
180.65, 171.02, 156.06, 149.06, 145.13, 135.18, 133.15, 131.32, 127.45, 127.42,
126.72, 126.25, 112.36, 101.67, 26.71. ESI-HRMS [M+H]+ calcd for C17H11F3NO4
350.0635,found: 350.0632.
4.1.3.12. 2-Acetyl-6-((3-(trifluoromethyl)phenyl)amino)benzofuran-4,7-dione (A16)
Purple solid; yield: 29.9%, 1H NMR (600 MHz, CDCl3) δ 7.60-7.56 (m, 1H), 7.51 (s,
2H), 7.48 (d, J = 7.3 Hz, 3H), 6.16 (s, 1H), 2.64 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 187.25, 180.60, 171.25, 156.13, 149.01, 143.93, 137.82, 131.43, 130.54, 125.78,
122.73, 122.70, 119.57, 119.55, 112.48, 101.55, 26.73. ESI-HRMS [M+H]+ calcd for
C17H11F3NO4 350.0635, found: 350.0630.
4.1.3.13. 2-Acetyl-6-((4-(trifluoromethyl)phenyl)amino)benzofuran-4,7-dione (A17)
Red solid; yield: 39.1%, 1H NMR (600 MHz, CDCl3) δ 7.76 (d, J = 7.8 Hz, 1H), 7.65
(t, J = 7.7 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H), 7.45 (s, 1H), 7.41 (t, J = 7.7
13
Hz, 1H), 5.94 (s, 1H), 2.64 (s, 3H). C NMR (150 MHz, CDCl3) δ 187.35, 180.66,
171.03, 156.08, 149.08, 145.14, 135.19, 133.17, 131.34, 127.44, 126.74, 126.27,
112.38, 101.69, 26.73. ESI-HRMS [M+H]+ calcd for C17H11F3NO4 350.0635, found:
350.0638.
4.1.3.14. 2-Acetyl-6-((2-ethylphenyl)amino)benzofuran-4,7-dione (A18)
1
Purple solid; yield: 24.4%, H NMR (600 MHz, CDCl3) δ 7.50 (s, 1H), 7.37 (s, 1H),
7.35-7.33 (m, 1H), 7.29-7.28 (m, 2H), 7.25-7.23 (m, 1H), 5.75 (s, 1H), 2.63-2.60 (m,
5H), 1.22 (t, J = 7.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 187.38, 175.97, 169.02,
154.90, 153.03, 144.12, 139.73, 135.09, 128.80, 127.82, 127.12, 125.92, 124.59,
111.60, 101.26, 26.77, 24.82, 13.87. ESI-HRMS [M-H]- calcd for C18H14NO4
308.0923, found: 308.0933.
4.1.3.15. 2-Acetyl-6-((4-(dimethylamino)phenyl)amino)benzofuran-4,7-dione (A19)
1
Purple solid; yield: 65.5%, H NMR (600 MHz, CDCl3) δ 7.56 (s, 1H), 7.48 (s, 1H),
7.14 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H), 6.05 (s, 1H), 3.00 (s, 6H), 2.62 (s,
3H).13C NMR (150 MHz, CDCl3) δ 187.63, 178.41, 174.93, 155.01, 154.34, 149.08,

146.00, 125.33, 124.44, 124.30, 112.83, 111.49, 98.88, 40.52, 26.72. ESI-HRMS
[M+H]+ calcd for C18H17N2O4 325.1183, found: 325.1169.
4.1.3.16. 2-Acetyl-6-((4-isopropoxyphenyl)amino)benzofuran-4,7-dione (A20)
1
Purple solid; yield: 21.7%, H NMR (600 MHz, CDCl3) δ 7.48 (s, 1H), 7.46 (s, 1H),
7.16 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.02 (s, 1H), 4.58-4.54 (m, 1H),
2.62 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.42, 178.26,
175.16, 156.57, 154.60, 154.49, 146.23, 129.11, 124.98, 124.47, 116.89, 111.40,
99.43, 70.43, 26.69, 21.97. ESI-HRMS [M+H]+ calcd for C19H18NO5 340.1179, found:
340.1165.
4.1.3.17. 2-Acetyl-6-((4-methoxy-2-methylphenyl)amino)benzofuran-4,7-dione (A21)
1
Purple solid; yield: 22.2%, H NMR (600 MHz, CDCl3) δ 7.42 (s, 1H), 7.18 (s, 1H),
7.06 (d, J = 8.4 Hz, 1H), 6.76 (s, 1H), 6.73 (d, J = 8.2 Hz, 1H), 5.51 (s, 1H), 3.75 (s,
3H), 2.55 (s, 3H), 2.16 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.38, 175.96, 169.02,
159.02, 154.83, 153.22, 144.26, 136.43, 128.45, 128.39, 124.40, 115.55, 111.57,
111.24, 100.05, 55.44, 26.77, 18.66. ESI-HRMS [M+H]+ calcd for C18H16NO5
326.1023, found: 326.1021.
4.1.3.18. 2-Acetyl-6-((2,6-dimethylphenyl)amino)benzofuran-4,7-dione (A22)
1
Red solid; yield: 11.5%, H NMR (600 MHz, CDCl3) δ 7.49 (s, 1H), 7.25 (s, 1H),
7.13 (d, J = 8.6 Hz, 1H), 6.83 (s, 1H), 6.80 (dd, J = 8.6, 2.3 Hz, 1H), 5.58 (s, 1H),
3.82 (s, 3H), 2.62 (s, 3H), 2.23 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.61, 178.26,
175.09, 158.88, 154.70, 154.48, 147.42, 135.45, 127.50, 126.82, 124.55, 116.63,
112.45, 111.40, 99.55, 55.52, 26.72, 18.02. ESI-HRMS [M+Na]+ calcd for
C18H15NNaO4 332.0893, found: 332.0892.
4.1.3.19.
2-Acetyl-6-((4-methoxy-2,6-dimethylphenyl)amino)benzofuran-4,7-dione
(A23)
1
Purple solid; yield: 25.6%, H NMR (600 MHz, CDCl3) δ 7.43 (s, 1H), 6.93 (s, 1H),
6.67 (s, 2H), 5.14 (s, 1H), 3.80 (s, 3H), 2.63 (s, 3H), 2.17 (s, 6H). 13C NMR (150
MHz, CDCl3) δ 187.34, 180.21, 171.61, 159.09, 155.91, 149.28, 146.99, 136.97,
131.90, 126.02, 113.99, 113.97, 112.64, 99.96, 55.39, 26.68, 18.28. ESI-HRMS
[M+H]+ calcd for C19H18NO5 340.1179, found: 340.1179.

4.1.3.20. 2-acetyl-6-((3,4-dimethylphenyl)amino)benzofuran-4,7-dione (A24)
1
Purple solid; yield: 26.1%, H NMR (600 MHz, CDCl3) δ 7.45 (s, 1H), 7.40 (s, 1H),
7.17 (d, J = 8.0 Hz, 1H), 7.03 (s, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.12 (s, 1H), 2.63 (s,
3H), 2.28 (s, 3H), 2.27 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 187.32, 180.57,
171.73, 155.93, 149.10, 144.74, 138.35, 135.02, 134.58, 131.65, 130.76, 123.99,
120.27, 112.56, 100.22, 26.69, 19.93, 19.36. ESI-HRMS [M+H]+ calcd for C18H16NO4
310.1074, found: 310.1083.
4.1.3.21. 2-Acetyl-6-((3-fluoro-5-methylphenyl)amino)benzofuran-4,7-dione (A25)
1
Purple solid; yield: 22.2%, H NMR (600 MHz, CDCl3) δ 7.61 (s, 1H), 7.48 (s, 1H),
6.79 (d, J = 9.2 Hz, 1H), 6.69 (s, 1H), 6.62 (d, J = 9.1 Hz, 1H), 2.64 (s, 3H), 2.36 (s,
3H).13C NMR (150 MHz, CDCl3) δ 187.26, 175.92, 169.00, 163.22, 155.06, 152.50,
143.30, 140.48, 137.79, 124.98, 121.10, 113.94, 111.58, 109.14, 104.38, 26.75, 21.46.
ESI-HRMS [M+H]+ calcd for C17H13FNO4 314.0823, found: 314.0834.
4.1.3.22. 2-Acetyl-6-(cyclopropylamino)benzofuran-4,7-dione (A26)
1
Red solid; yield: 39.1%, H NMR (600 MHz, CDCl3) δ 7.42 (s, 1H), 6.08 (s, 1H),
13
5.88 (s, 1H), 2.61 (s, 3H), 2.52 (m, 1H), 0.93 (d, J = 6.7 Hz, 2H), 0.69 (s, 2H). C
NMR (150 MHz, CDCl3) δ 187.57, 177.91, 174.71, 154.75, 154.34, 149.74, 124.59,
111.35, 99.82, 26.69, 24.55, 7.26. ESI-HRMS [M+H]+ calcd for C13H12NO4 246.0761,
found: 246.0766.
4.1.3.23. 2-Acetyl-6-(cyclobutylamino)benzofuran-4,7-dione (A27)
1
Red solid; yield: 26.3%, H NMR (600 MHz, CDCl3) δ 7.43 (s, 1H), 6.12 (s, 1H),
5.40 (s, 1H), 3.92 (dd, J = 14.2, 7.0 Hz, 1H), 2.60 (s, 3H), 2.49 (d, J = 7.4 Hz, 2H),
2.05-2.02 (m, 2H), 1.94-1.88 (m, 2H). 13C NMR (150 MHz, CDCl3) δ 187.58, 177.91,
174.45, 154.96, 154.29, 147.25, 124.36, 111.38, 98.31, 47.96, 29.92, 26.69, 15.63.
ESI-HRMS [M+H]+ calcd for C14H14NO4 260.0917, found: 260.0906.
4.1.3.24. 2-Acetyl-6-(cyclopentylamino)benzofuran-4,7-dione (A28)
Purple solid; yield: 6.2%, 1H NMR (600 MHz, CDCl3) δ 7.43 (s, 1H), 5.98 (d, J = 4.6
Hz, 1H), 5.53 (s, 1H), 3.80-3.74 (m, 1H), 2.60 (s, 3H), 2.10-2.05 (m, 2H), 1.77-1.64
(m, 6H). 13C NMR (150 MHz, CDCl3) δ 187.59, 177.97, 174.34, 155.09, 154.28,

148.08, 124.28, 111.41, 98.29, 54.43, 32.71, 26.69, 24.10. ESI-HRMS [M+H]+ calcd
for C15H16NO4 274.1074, found: 274.1076.
4.1.3.25. 2-Acetyl-6-(cyclohexylamino)benzofuran-4,7-dione (A29)
1
Purple solid; yield: 25.7%, H NMR (600 MHz, CDCl3) δ 7.43 (s, 1H), 5.96 (d, J =
5.2 Hz, 1H), 5.53 (s, 1H), 3.30-3.25 (m, 1H), 2.60 (s, 3H), 2.03 (d, J = 11.4 Hz, 2H),
1.82-1.80 (m, 2H), 1.70-1.68 (m, 2H), 1.39-1.31 (m, 4H).13C NMR (150 MHz, CDCl3)
δ 187.62, 178.06, 174.48, 155.11, 154.28, 147.45, 124.32, 111.42, 97.69, 51.94, 31.73,
26.70, 25.34, 24.49. ESI-HRMS [M+H]+ calcd for C16H18NO4 287.1230, found:
288.1220.
4.1.3.26. 2-Acetyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4,7-dione (A30)
1
Red solid; yield: 35.3%, H NMR (600 MHz, CDCl3) δ 7.37 (s, 1H), 5.84 (s, 1H),
5.48 (s, 1H), 3.96 (d, J = 10.8 Hz, 2H), 3.44 (t, J = 10.6 Hz, 3H), 2.54 (s, 3H), 1.96 (d,
J = 12.3 Hz, 2H), 1.57 (d, J = 10.6 Hz, 2H).13C NMR (150 MHz, CDCl3) δ 187.54,
177.84, 174.56, 154.70, 154.38, 147.09, 124.48, 111.41, 98.13, 66.23, 49.25, 31.75,
26.69. ESI-HRMS [M+H]+ calcd for C15H16NO5 290.1023, found: 290.1028.
4.2. Biological evaluation
4.2.1. Cell lines culture
All cancer cell lines (MDA-MB-231, MDA-MB-468 and HepG2) were
purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese
Academy of Sciences (Shanghai, China). The MDA-MB-231 and MDA-MB-468 cell
lines were cultured in Dulbecco Modified Eagle Medium/ Nutrient Mixture
F-12(DMEM/F-12, Biological Industries, Beit Haemek, Israel), supplemented with 10%
(v/v) fetal bovine serum (FBS, Gibco), 50 mg/mL penicillin and 50 mg/ mL
streptomycin (Biological Industries, Beit Haemek, Israel). While the HepG2 cell line
was maintained in high glucose Dulbecco's Modified Eagle Medium (Biological
Industries, Beit Haemek, Israel) replenished with 10% fetal bovine serum, and
penicillin/streptomycin. All the cell lines were incubated at 37
atmosphere containing 5% CO2.
in a humidified

4.2.2. In vitro cell growth inhibitory activity assays
All of these cancer cells were seeded in 96-well culture plates at a density of
5000-7000 cells per well (100 μL/well) and incubated at 37
in a humidified
atmosphere containing 5% CO2 for 8h. In succession, different concentrations of test
compounds were added in triplicate to the plates in 100 μL fresh mediums (the total
volume was 200 μL, DMSO <0.1%) and incubated at 37 for 48 h. After removing
the cell culture medium, 10% Cell Counting Kit-8 (CCK-8, APExBIO, Houston, USA)
solution (100 μL) was administered in the 96-well plates and re-incubated for 4 h
(MDA-MB-468, MDA-MB-231) and 1h (HepG2). Finally, the absorbance was
measured at the wavelength of 450 nm by a microplate spectrophotometer (MK3,
Thermo, Germany). Each treatment was performed in triplicate. The half inhibitory
concentration IC50 values were calculated by Prism 7.0 (GraphPad Software).
4.2.3. Fluorescence polarization assay
The STAT3 protein (His-tag 127-722 amino acid, DetaiBio, China) was diluted
to a concentration of 1600 nM and the fluorescently labelled peptide probe
(5-FAM-GpYLPQTV-NH2, ChinaPeptides, China) was deliquated to a concentration
of 100 nM. Different concentrations of tested compound (10 µL) and prepared STAT3
protein (10 µL) were added to 70 µL assay buffer (Hepes 10 mM PH=7.5, EDTA 1
mM, NaCl 50 mM, 0.1% Nonidet P40) in 96-well, black round-bottom plates at 37
for 30 minutes. Then 10 µL of fluorescently labelled peptide probe was added and
incubated at 37
for another 1 h. The polarization values were measured at an
excitation wavelength of 485 nm and an emission wavelength of 530 nm, which was
detected by Tecan Spark instrument. The half inhibitory concentration IC50 values
were by Prism 7.0 (GraphPad Software).
4.2.4. Western blot analysis
MDA-MB-231 cells were incubated with various concentrations of compound
A11 for 24 h and rinsed twice with PBS and then lysed with RIPA buffer (Beyotime
Biotechnology) containing a protease inhibitor (PMSF) mixture at 1:100 dilution on

ice for 10 min. The lysates were cleared by centrifugation (4 , 12,000 rmp,10 min),
and BCA protein assay kit was applied to measure the protein concentration. Equal
amount of proteins from the total cell lysates (40 μg per lane) was separated by
sodium dodecyl sulfate (10 %) polyacrylamide gel electrophoresis (SDS-PAGE,
BioRad Laboratories, Hercules, CA), and then electrically transferred to
polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories, Hercules, CA).
The membranes were blocked with 5% non-fat powdered milk in TBST buffer for 2 h
at room temperature, and blotted with primary antibodies specific for STAT3,
p-STAT3(Y705), STAT1, p-STAT1(Y701), C-Myc, Cyclin D1 and GAPDH at 4 for
overnight. After washing out three times (5 min each) with TBST, the corresponding
HRP-conjugated secondary antibodies were incubated for 1h at room temperature.
Enhanced chemiluminescence (ECL) and bioanalytical imaging system (Azure
biosystems, Inc, C600) were applied for assay of target proteins.
4.2.5. Cell immunofluorescent assay
After MDA-MB-231 cells were cultured and incubated in confocal dishes at a
density of 1×104 cells per dish overnight, compound A11 was added and incubated for
24 h. Then, cells were fixed with 4 % paraformaldehyde and permeabilized with 0.5 %
Triton X-100 for 15 min respectively. Subsequently, they were sealed with 5% BSA
for 1 h and fostered with the specific primary antibody against STAT3 overnight at
4°C. Next, the Alexa-conjugated secondary antibody (Alexa Fluor 546 goat
anti-rabbit IgG, Invitrogen, A-11035) was added and incubated at room temperature
for 1 h. Cell nucleus were stained with Hoechst for 10 min. Finally, the cells images
were tested and analyzed by a fluorescence microscope (Nikon Eclipse Ti2).
4.2.6. Flow cytometry analysis of apoptotic cells
MDA-MB-231 cells at a density of 2×105 per well were cultured in regular
growth medium in 6-well plates for 24 h and disposed in duplicate with various
concentrations of compound A11 for 24 h. Then the cells were trypsinized, was rinsed
twice with PBS (centrifugation at 2000 rpm, 5 min) and collected for the next step.

After resuspending the cells in 500 µL of binding buffer, 5 µL Annexin V-FITC and 5
µL propidium iodide were added and mixed gently. Finally, the mixture was incubated
for 15 min at room temperature in dark and detected by a flow cytometer (Beckman
coulter, Inc, A00-1-1102).
4.2.7 cell cycle effect
MDA-MB-231 cells were seeded in 6-well plates at density of 2×105 cells/well.
After overnight adherence, they were incubated with various concentrations of A11
for 24h. The treated cells were collected by centrifugation, washed with PBS and
fixed in ice-cold 70% ethanol. Then incubated for 30 min at 37°C with PI containing
RNase (MA0334, Dalian Meilun Biotdchnology Co, LTD). The samples were then
analyzed by flow cytometry (Beckman coulter, Inc, A00-1-1102).
4.2.8. In vivo studies
All procedures were performed according to the National Institutes of Health
Guidelines for the Care and Use of Laboratory Animals and were approved by the
local animal ethics committees. Human breast tumor MDA-MB-231cells (5×106)
were injected into the subcutaneous tissue of five weeks old female BALB/c nude
mice (16–18g). When the tumor volume reached around 50 mm3, the mice were
randomly classified into three groups (six mice per group) and intraperitoneally
treated with compound A11 at a dosage of 5 mg/kg or 10 mg/kg daily for 21 days.
Simultaneity, the tumor volume was measured once every three days and numerated
by the formula: length × width2/2, and the body weight of mice was meted and
registered. At terminal phase, all mice were sacrificed and the tumor was segregated,
and weighed. The levels of p-STAT3 in tumor tissues were analyzed by the
immunofluorescent assay.
4.2.9. Tissue immunofluorescent assay
The pre-treated and fixed tumor sections were incubated with antibody against
p-STAT3 at 4
overnight. After scouring with PBST three times, the

Alexa-conjugated secondary antibody (Cy3-AffiniPure Goat Anti-Rabbit IgG(H+L),
Jackson, 111-165-003) was added and reared at 37 for 40 minutes. Cell nucleus was
dyed with DAPI (4’, 6-Diamidino-2-Phenylindole) for 10 min. Finally, the cells
images were detected and resolved by a fluorescence microscope (Nikon Eclipse Ti2).
4.3. Molecular docking
Molecular docking study was executed applying Schrodinger software package.
Firstly, the X-ray crystal structure of STAT3 was retrieved from Protein Data Bank
(PDB code: 1BG1) and prepared with the Protein Preparation Wizard model including
the removement of one monomer, the addition of missing hydrogen atoms, the
assignment of bond order, assessment of the correct protonation states, and a
restrained minimization using the OPLS-2005 force field. Then, the receptor grid was
generated at the centroid of selected residues (LYS591, ARG595) and the grid box
size was set to 20 Å. After preparing the ligands, molecular docking was carried out
using the standard precision (SP) with the default settings. Finally, the pictures were
generated using pymol software.
4.4. X-ray crystal structure determination of compound A24
The D8 venture diffractometer view of compound A24 is shown in Fig. 4. The
crystal data and structure refinement were emerged in supporting information. The
compound was crystallized in the monoclinic space group P2(1)/c with 4 molecules in
the unit cell and the structure was solved by direct method with the SHELXTL
program package. All the crystallographic parameters (excluding structure factors) of
this structure have been deposited in the Cambridge Crystallographic Data Center as
supplementary publication number CCDC 1973547. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK [fax: +44 (0) 223 336033 or e-mail: deposit@ccdc.cam.ac.uk.
Declaration of competing interest

The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Acknowledgement
Financial support was generously provided by the National Natural Science
Foundation of China (Nos. 81903423 and 21871184), the China Postdoctoral Science
Foundation (2018M642064), the Shanghai Sailing Program (19YF1449300), the
Shanghai Municipal Education Commission (2019-01-07-00-10-E00072), and the
Science and Technology Commission of Shanghai Municipality (18401933500).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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