Discovery of potent anticancer agent HJC0416, an orally bioavailable small molecule inhibitor of signal transducer and activator of transcription 3 (STAT3)

Article abstract of DOI:10.1016/j.ejmech.2014.05.049

In a continuing effort to develop orally bioavailable small-molecule STAT3 inhibitors as potential therapeutic agents for human cancer, a series of novel diversified analogues based on our identified lead compound HJC0149 (1) (5-chloro-N-(1,1-dioxo-1H-1λ⁶-benzo[b]thiophen-6-yl) -2-hydroxybenzamide, Eur. J. Med. Chem. 2013, 62, 498-507) have been rationally designed, synthesized, and pharmacologically evaluated. Molecular docking studies and biological characterization supported our earlier findings that the O-alkylamino-tethered side chain on the hydroxyl group is an effective and essential structural determinant for improving biological activities and druglike properties of these molecules. Compounds with such modifications exhibited potent antiproliferative effects against breast and pancreatic cancer cell lines with IC₅₀ values from low micromolar to nanomolar range. Among them, the newly discovered STAT3 inhibitor 12 (HJC0416) displayed an intriguing anticancer profile both in vitro and in vivo (i.p. & p.o.). More importantly, HJC0416 is an orally bioavailable anticancer agent as a promising candidate for further development.

Full text of DOI:10.1016/j.ejmech.2014.05.049

European Journal of Medicinal Chemistry 82 (2014) 195e203
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of potent anticancer agent HJC0416, an orally bioavailable
small molecule inhibitor of signal transducer and activator of
transcription 3 (STAT3)
,
c
,
,
Haijun Chen a 1, Zhengduo Yang b 1, Chunyong Ding a, Ailian Xiong b, Christopher Wild a,
Lili Wang b, Na Ye a, Guoshuai Cai b, Rudolfo M. Flores a, Ye Ding a, Qiang Shen b
,
,
*
,
*
Jia Zhou a
a Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, United States
b Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Sciences, The University of Texas M.D. Anderson Cancer Center,
Houston, TX 77030, United States
c College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing effort to develop orally bioavailable small-molecule STAT3 inhibitors as potential ther-
Received 10 March 2014
Received in revised form
15 May 2014
Accepted 21 May 2014
Available online 22 May 2014
apeutic agents for human cancer, a series of novel diversified analogues based on our identified lead
compound HJC0149 (1) (5-chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-hydroxybenzamide,
Eur. J. Med. Chem. 2013, 62, 498e507) have been rationally designed, synthesized, and pharmacologically
evaluated. Molecular docking studies and biological characterization supported our earlier findings that
the O-alkylamino-tethered side chain on the hydroxyl group is an effective and essential structural
determinant for improving biological activities and druglike properties of these molecules. Compounds
with such modifications exhibited potent antiproliferative effects against breast and pancreatic cancer
cell lines with IC50 values from low micromolar to nanomolar range. Among them, the newly discovered
STAT3 inhibitor 12 (HJC0416) displayed an intriguing anticancer profile both in vitro and in vivo (i.p. &
p.o.). More importantly, HJC0416 is an orally bioavailable anticancer agent as a promising candidate for
further development.
Keywords:
STAT3 inhibitors
Oral bioavailability
Breast cancer
Pancreatic cancer
Anticancer agents
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
[13e15]. The suitability of the scaffolds and physicochemical
properties are most significant issues in promoting a potent agent
Signal transducer and activator of transcription 3 (STAT3) is the
most studied member of a family of seven proteins (STAT1, 2, 3, 4,
5a, 5b, and 6) that are involved in the regulation of early embryonic
development, angiogenesis, immune response, cell proliferation,
differentiation, and apoptosis [1e5]. From nearly two decades of
research on STAT3, accumulating evidence has demonstrated that
STAT3 is hyperactivated in many human tumors and represents an
attractive therapeutic target for the treatment of various types of
cancer [6e12]. However, despite many years of intensive research
devoted to the discovery of small molecules targeting STAT3, only
several compounds are in early phase clinical trials, and currently
there are no candidates approved for clinical use in oncology
into clinical trials [16]. Thus, there is an urgent need to develop
novel diversified scaffolds, and orally bioavailable drug candidates
capable of targeting STAT3 [17].
Our previous work by utilizing fragment-based drug design
(FBDD) approach has explored several novel scaffolds targeting
STAT3 as potent anticancer agents [18]. The identified drug candi-
dates including HJC0149 (1) with enhanced pharmacological ac-
tivities and especially drug-like properties may act as advanced
chemical leads for further optimization (Fig. 1) [18]. Another pre-
vious effort in our laboratories has demonstrated that it is feasible
to identify new niclosamide derivatives with improved oral
bioavailability through modifications of the hydroxyl group on the
phenol ring tethered by an O-alkylamino side chain, leading to the
identification of HJC0152 with a similar or significantly higher
anticancer potency on proliferation of human breast and pancreatic
cancer cells [19]. Pharmacological evaluation revealed that HJC0152
inhibited STAT3 promoter activity, and increased the expression of
* Corresponding authors.
E-mail addresses: qshen@mdanderson.org (Q. Shen), jizhou@utmb.edu (J. Zhou).
These authors contribute equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.05.049
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

196
H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
Fig. 1. Previous work and drug design strategy for the current work.
active caspase-3. Further in vivo studies in nude mice bearing
triple-negative breast tumor xenografts demonstrated that
HJC0152 significantly suppressed tumor growth (both i.p. & p.o.)
[19]. These results suggest that chemical optimization of the hy-
droxyl group of salicylic amide scaffold in niclosamide may be a
viable strategy to develop novel orally bioavailable agents for hu-
man cancer therapy.
anticancer activity and bioavailability. These studies led to the
discovery of an orally efficacious anticancer agent 12 (HJC0416), a
promising candidate for further drug development.
2. Results and discussion
2.1. Chemistry
In a continuing effort to develop novel diversified analogues
based on the scaffold of lead compound 1, we directed our chemical
optimization involving modification of the hydroxyl group on the
phenol ring by introduction of O-alkylamino side chains as depicted
in Fig. 1. We envisioned that such modifications on the salicylic
amide moiety of 1 by utilizing the same strategy might enhance
both anticancer potency and oral bioavailability. Herein, we report
the lead optimization of compound 1 with a focus on improving
The synthesis of new derivatives based on salicylic amide scaf-
fold with chemical optimization of the hydroxyl group is outlined
in Scheme 1. Analogues 2e6 were conveniently prepared by Mit-
sunobu reaction of the appropriate substituted alcohols with lead
compound 1 in 39e77% yields. Alkylation of the bromide inter-
mediate 2 with piperidine or 1-methylpiperazine in the presence of
K2CO3 and KI in acetone introduced basic functionalities into the
Scheme 1.

H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
197
molecules providing compounds 7 and 8 in yield of 98% and 77%,
respectively. Mitsunobu coupling of lead compound 1 with N-Boc-
protected amino alcohols followed by the Boc-deprotection with
the treatment of TFA afforded analogues 9e13 with diversified O-
alkylamino side chains in 40e52% yields (two steps). Modification
of benzo[b]thiophene 1,1-dioxide motif by introducing an ether
group to the double bond moiety is shown in Scheme 2. Compound
14 was prepared from 1 by the treatment with methanol and 10%
aqueous solution of NaOH in yield of 60% [20].
Among the newly synthesized analogs, compound 12 was
selected for a battery of further in vitro and in vivo characterizations
due to its enhanced antiproliferative effects and druglike properties
including the aqueous solubility. To further study the anticancer
effects of compound 12 on cell growth, cellular morphological
changes were examined in MDA-MB-231 breast cancer cells treated
with compound 12 or stattic for 48 h, under light microscopy. As
shown in Fig. 2, like stattic, 12 significantly inhibited cell growth
and induced apoptosis accompanying cellular morphological
changes at concentration of 1 mM, 5 mM, and 10 mM, respectively.
To determine whether compound 12 acts as a potent small-
molecule inhibitor of STAT3 activation, we further measured the
inhibitory effect on promoter activity using the cell-based transient
transfection and dual luciferase reporter assays. MDA-MB-231 cells
were pre-treated with stattic or 12 at the same concentration
(5
was determined after transient transfecting with pSTAT3-Luc vec-
tor. As shown in Fig. 3, treatment with 5 M of 12 decreased the
STAT3 promoter activity in MDA-MB-231 cells by approximately
51%, while stattic only decreased the STAT3 promoter activity by
39%.
mM) for 24 h. The STAT3 promoter activity in MDA-MB-231 cells
m
Scheme 2.
2.2. Biology
Our previous work and studies from other groups have revealed
that compounds with the 1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl
To explore the structureeactivity relationship (SAR) and
examine how the substitutions of the moiety groups affected bio-
logical activities of newly synthesized analogues, we first evaluated
the in vitro anticancer effects of compounds 2e14 on the prolifer-
ation of breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-
231 (ER-negative and triple-negative), as well as two pancreatic
cancer cell lines AsPC1 and Panc-1 using MTS assays as described in
the Experimental Section. The suitable calculated lipophilicity
(cLogP) and topological polar surface area (tPSA) values shown in
Table 1 suggest that these newly designed analogues are clearly in
good alignment with Lipinski's “Rule of Five” and may have ideal
physicochemical properties. Meanwhile, the introduced basic
functionalities of the target molecules can form HCl salts to facili-
tate the aqueous solubility. The capabilities of these new analogues
to inhibit the growth of cancer cells are summarized in Table 1.
Introduction of an O-bromoalkyl group into the lead compound 1
resulted in a dramatic loss of activity, and the derivative 2 displayed
no significant antiproliferative effects on all tested cell lines.
However, further replacement of the bromo with a hydrophilic
amino group regained the antiproliferative effects to a similar or
significantly enhanced level in comparison with 1. For instance, O-
alkylamino-tethered derivatives 4e13 exhibited promising anti-
proliferative effects against four cancer cell lines with IC50 values
from low micromolar to nanomolar range. Compound 6 with a
morpholine moiety showed potent antiproliferative effect against
fragment might interact with STAT3-SH2 domain [18,22]. In an
attempt to elucidate the structural basis of the inhibition of STAT3
assembly at the SH2 binding site, it was of interest to determine the
possible conformation and binding pose of compound 12 by using
AutoDock Vina docking approach [18,23]. As depicted in Fig. 4, the
1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl moiety interacted with
the pocket of Val637 and Ile659 in a similar fashion as we previ-
ously reported [18]. The salicylic amide of compound 1 and com-
pound 12 formed the H-bond with Trp623, while oxygen of 3-
aminopropoxy side chain of 12 formed an additional H-bond with
Trp623 and terminal amino group formed another H-bond with
Glu638. Furthermore, the alkyl chain might have the hydrophobic
interactions with Val637. These molecular docking studies could
also reasonably explain why the compounds with a hydrophilic
amino group or the certain length of the carbon chain might have
better anticancer activity. It is worth mentioning that the large
pocket formed around Lys591, Glu594, Arg595, Arg609 and Glu612
may be a good starting point for further drug design and structural
optimization.
To further investigate the inhibitory activity of compound 12
against STAT3 pathway, we examined STAT3 phosphorylation and
expression of the known STAT3 target genes in MDA-MB-231 cell
line. The cells were treated with different doses of compound 12 for
12 h, and levels of total STAT3 and phosphorylated STAT3 at Tyr-705
were then examined by Western blot. As shown in Fig. 5, total
STAT3 expression in these cells was reduced after treatment with
compound 12 or stattic. Similarly, phosphorylated STAT3 at Tyr-705
is suppressed by compound 12 or stattic, suggesting that com-
pound 12 has a comparable potency in downregulating STAT3
protein production and phosphorylation at Tyr-705 site. We
observed that compound 12 induced cleaved caspase-3 and
downregulated cyclin D1 levels in MDA-MB-231 cells. These results
suggest that compound 12 inhibits cell cycle progression and pro-
motes apoptosis.
MCF-7 with an IC50 value of 0.49
proliferation of both ER-positive, and ER-negative (triple negative)
breast cancer cells with IC50 values of 1.76 M and 1.97 M,
mM. Compound 12 inhibited the
m
m
respectively. Intriguingly, compound 12 also displayed a marked
antiproliferative effect against pancreatic cancer cell line AsPC1
with an IC50 value of 40 nM. Interestingly, the fluorinated derivative
3 displayed moderate to potent antiproliferative effects against the
tested cancer cells, indicating that this compound might be suitable
for developing as a potential 18F-radiolabeled positron emission
tomography (PET) imaging agent for human cancer [21]. Modifi-
cation of benzo[b]thiophene 1,1-dioxide motif by introducing an
ether group to the double bond moiety has been demonstrated to
generally reduce the antiproliferative effects. For example, com-
pound 14 displayed only moderate activity against all the tested cell
lines, indicating that benzo[b]thiophene 1,1-dioxide is an important
pharmacophore for this class of molecules. Therefore, more
extensive SAR study based upon the scaffold of compound 14 was
not pursued.
Compound 12 was further evaluated for potential antitumor
effects in the MDA-MB-231 triple-negative breast cancer murine
xenograft model, and tumor volume was measured daily in intra-
peritoneal treatment (i.p.) and oral gavage (p.o.) groups. It was
found that mice treated with 10 mg/kg of compound 12 via i.p.
showed a 67% decrease of tumor volume as compared to the control
mice (Fig. 6A). Similarly, we treated the MDA-MB-231 xenograft
mice with oral administration of compound 12 and found that the

198
H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
Table 1
Effects of newly synthesized analogues 2e14 on proliferation of human breast and pancreatic cancer cell lines.
Compound
R
cLog Pa
tPSAb
IC50 (m
M)c
Breast cancer ER-positive
MCF-7
Breast cancer ER-negative
MDA-MB-231
Pancreatic cancer
AsPC1
Panc-1
Stattic
1
1.05
2.88
80.0
83.5
3.60
0.91
2.89
1.64
1.32
1.92
3.77
2.34
H
2
3
3.60
3.14
72.5
72.5
>10d
>10
NDe
ND
4.0
4.42
1.37
9.53
4
3.21
75.7
3.53
2.68
1.04
1.36
5
6
7
8
2.74
2.45
3.65
2.54
75.7
84.9
75.7
79.0
3.5
2.69
5.43
2.51
2.86
1.14
1.37
1.42
1.07
3.38
6.95
1.81
3.01
0.49
3.74
4.08
9
2.16
2.83
87.7
84.5
2.56
4.21
3.29
2.85
1.02
1.6
3.69
4.09
10
11
12
13
1.91
2.24
98.5
98.5
3.47
1.76
3.12
1.97
1.05
0.04
2.25
1.88
2.32
2.43
84.5
92.7
3.15
6.07
3.12
7.05
2.05
8.46
3.09
8.39
14
a
Average cLogP: http://146.107.217.178/lab/alogps/start.html.
tPSA: http://www.molinspiration.com/cgi-bin/properties.
b
c
Breast cancer cell lines: MCF-7 and MDA-MB-231. Pancreatic cancer cell lines: AsPC1 and Panc-1. Software: MasterPlex ReaderFit 2010, MiraiBio, Inc.
If a specific compound is given a value >10, it indicates that a specific IC50 cannot be calculated from the data points collected, meaning ‘no effect’.
ND: not determined.
d
e

H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
199
Fig. 2. Effects of 12 (HJC0416) and stattic on cell growth and cellular morphological changes. Exponentially growing MDA-MB-231 breast cancer cells were incubated with 12 or
stattic for 48 h. Cell morphology was evaluated under light microscopy.
growth of xenograft tumors in mice was also significantly reduced
at a dose of 100 mg/kg by 46% (Fig. 6B). The i.p. route appeared to
have a better reduction of tumor volume, and this observation
might be attributed to the efficiency of bioavailability for com-
pound 12. It is also noteworthy that compound 12 did not show
significant signs of toxicity at a dose of 100 mg/kg. These results
have demonstrated that compound 12 effectively reduced the tu-
mor growth originated from MDA-MB-231 cells in vivo with no
significant body weight loss, indicating its low adverse side effects
as a drug candidate. Further pharmacokinetic studies and preclin-
ical assessment are under way.
compounds is an effective and essential structural determinant for
improving biological activity and druglikeness. Intriguingly, 12
significantly suppressed MDA-MB-231 xenograft tumor growth
in vivo (i.p. & p.o.), indicating its great potential as an orally
bioavailable anticancer agent. This work together with our previous
efforts enabled us to establish a sizable compound library of
druglike STAT3 inhibitors with diversified scaffolds and may open
new venues for further clinical development of promising candi-
dates for human cancer therapeutic regimens.
3. Conclusions
In summary, an appropriate modification of the hydroxyl group
of salicylic amide scaffold enabled us to expand the scope of the
exploration of the series, leading to the identification of several
potent STAT3 inhibitors with enhanced anticancer activities and
druglike properties. Through the optimization of the lead com-
pound 1, a novel O-alkylamino-tethered derivative 12 has been
discovered to exhibit potent antiproliferative effects, inhibit STAT3
promoter activity, and decrease the expression of phosphorylated
STAT3. Molecular docking studies support our findings that the O-
alkylamino side chain on the hydroxyl group of this class of
Fig. 3. Compound 12 (HJC0416) inhibited the STAT3 mediated luciferase reporter ac-
tivity in MDA-MB-231 cells. STAT3 promoter activity was measured using dual lucif-
erase assay with a STAT3 reporter. Promoter activity obtained from DMSO-treated
MDA-MB-231 cells was used as control. Error bars represent standard deviation of
triplicate wells. Representative experiment from at least 3 independent experiments is
shown. RLU: relative luciferase unit.
Fig. 4. Predicted binding modes for HJC0149 (1) and HJC0416 (12). The figures were
generated using Pymol.

200
H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra (HRMS) were obtained from Thermo
Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include
the following: Nano ESI spray voltage was 1.8 kV; Capillary tem-
perature was 275 ꢀC and the resolution was 60,000; Ionization was
achieved by positive mode. Melting points were measured on a
Thermo Scientific Electrothermal Digital Melting Point Apparatus
and uncorrected. Purity of final compounds was determined by
analytical HPLC, which was carried out on a Shimadzu HPLC system
(model: CBM-20A LC-20AD SPD-20A UV/VIS). HPLC analysis con-
ditions: Waters
m
Bondapak C18 (300 ꢁ 3.9 mm); flow rate 0.5 mL/
min; UV detection at 270 and 254 nm; linear gradient from 30%
acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in
20 min followed by 30 min of the last-named solvent. All biologi-
cally evaluated compounds are >95% pure.
4.1.1. 2-(2-Bromoethoxy)-5-chloro-N-(1,1-dioxo-1H-1
l
6-benzo[b]
thiophen-6-yl)benzamide (2)
To a solution of 5-chloro-N-(1,1-dioxo-1H-1
l
6-benzo[b]thio-
phen-6-yl)-2-hydroxybenzamide (1, HJC0149) [18] (200 mg,
0.6 mmol) and PPh3 (314 mg, 1.2 mmol) in THF (5 mL) was added 2-
bromoethanol (150 mg, 1.2 mmol) and DIAD (218 mg, 1.08 mmol).
The mixture was stirred at r.t. for 16 h. The reaction mixture was
diluted with EtOAc (100 mL) and extracted with H2O (40 mL). The
organic layer was washed with brine (10 mL), dried with anhydrous
Na2SO4, and then concentrated to give the crude product. This
residue was purified with silica gel column (Hexane/Acetone ¼ 2/1
to 1/2) to provide 2 (200 mg, 75%) as a yellow solid (mp
Fig. 5. Western blot analysis of biochemical markers for apoptosis induction and in-
hibition of STAT3 activity by stattic and compound 12 in the MDA-MB-231 cell line.
Cells were treated with stattic or 12 for 12 h, levels of pSTAT3, STAT3, cleaved caspase-
3, PARP-1, cleaved PARP-1 and cyclin D1 were probed by specific antibodies. b-actin
was used as the loading control.
235e236 ꢀC). 1H NMR (600 MHz, DMSO-d6)
d 10.59 (s, 1H), 8.26 (s,
4. Experimental section
1H), 7.89 (d, 1H, J ¼ 7.8 Hz), 7.70 (d, 1H, J ¼ 2.4 Hz), 7.58e7.61 (m,
3H), 7.26e7.31 (m, 2H), 4.47 (t, 2H, J ¼ 5.4 Hz), 3.87 (t, 2H,
4.1. Chemistry
J ¼ 5.4 Hz). 13C NMR (150 MHz, DMSO-d6)
d 163.4, 154.1, 141.1, 137.2,
132.8, 132.0, 130.2, 129.3, 126.5, 126.2, 125.9, 125.0, 123.9, 115.5,
112.0, 69.1, 31.1. HRMS (ESI) calcd for C17H14BrClNO4S 441.9510
(M þ H)þ, found 441.9515.
All commercially available starting materials and solvents were
reagent grade, and used without further purification. Reactions
were performed under a nitrogen atmosphere in dry glassware
with magnetic stirring. Preparative column chromatography was
performed using silica gel 60, particle size 0.063e0.200 mm
(70e230 mesh, flash). Analytical TLC was carried out employing
silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the
developed chromatograms was performed with detection by UV
(254 nm). NMR spectra were recorded on a Bruker-600 (1H,
600 MHz; 13C, 150 MHz) spectrometer. 1H and 13C NMR spectra
were recorded with TMS as an internal reference. Chemical shifts
4.1.2. 5-Chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-(2-
fluoroethoxy)benzamide (3)
Compound 3 was prepared in 77% yield by a procedure similar
to that used to prepare compound 2. The title compound was ob-
tained as a pale yellow solid (mp 216e217 ꢀC). 1H NMR (600 MHz,
DMSO-d6)
d 10.63 (s, 1H), 8.21 (s, 1H), 7.85e7.86 (m, 1H), 7.68 (d, 1H,
J ¼ 2.4 Hz), 7.58e7.61 (m, 3H), 7.26e7.31 (m, 2H), 4.74e4.83 (m,
Fig. 6. In vivo efficacy of compound 12 (HJC0416) in inhibiting growth of xenograft tumors (triple-negative breast cancer MDA-MB-231) in mice via A) i.p. or B) oral gavage (p.o.)
routes.

H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
201
2H), 4.37e4.43 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d
163.6,
4.1.7. 5-Chloro-N-(1,1-dioxo-1H-1
(4-methylpiperazin-1-yl)-ethoxy]benzamide (8)
Compound 8 was prepared in 77% yield by a procedure similar
to that used to prepare compound 7. The title compound was ob-
tained as a pale yellow solid (mp 218e219 ꢀC). 1H NMR (600 MHz,
l
6-benzo[b]thiophen-6-yl)-2-[2-
154.3,141.1,137.2, 132.8,131.9, 130.2,129.1, 126.6,126.4,125.9,124.9,
123.7,115.5,111.7, 82.1 (d, J ¼ 166 Hz), 68.5 (d, J ¼ 18 Hz). HRMS (ESI)
calcd for C17H14ClFNO4S 382.0311 (M þ H)þ, found 382.0314.
DMSO-d6) d 10.70 (s, 1H), 8.17 (s, 1H), 7.89e7.90 (m, 1H), 7.73 (d, 1H,
4.1.3. 5-Chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-(1-
methylpiperidin-4-yloxy) benzamide (4)
Compound 4 was prepared in 39% yield by a procedure similar
to that used to prepare compound 2. The title compound was ob-
tained as a pale yellow solid (mp 220e221 ꢀC). 1H NMR (600 MHz,
J ¼ 3.0 Hz), 7.58e7.62 (m, 3H), 7.26e7.31 (m, 2H), 4.25 (t, 2H,
J ¼ 5.4 Hz), 2.73 (t, 2H, J ¼ 4.8 Hz), 2.42e2.50 (m, 4H), 2.12e2.22 (m,
4H), 2.03 (s, 3H). 13C NMR (150 MHz, DMSO-d6)
d 163.3, 154.9, 141.0,
137.2, 132.8, 132.2, 130.2, 129.2, 126.5, 126.0, 125.4, 124.7, 124.2,
115.4, 112.2, 66.9, 56.2, 54.5, 52.7, 45.6. HRMS (ESI) calcd for
22H25ClN3O4S 462.1249 (M þ H)þ, found 462.1253.
DMSO-d6)
d
10.63 (s, 1H), 8.22 (s, 1H), 7.85 (d, 1H, J ¼ 7.2 Hz),
C
7.58e7.61 (m, 3H), 7.51e7.53 (m, 1H), 7.27e7.30 (m, 2H), 4.53e4.55
(m, 1H), 2.42e2.44 (m, 2H), 2.19e2.21 (m, 2H), 2.06 (s, 3H),
1.89e1.91 (m, 2H), 1.69e1.71 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
4.1.8. 5-Chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-(2-
piperazin-1-yl-ethoxy) benzamide (9)
d
164.1, 153.2, 141.2, 137.2, 132.8, 131.5, 130.1, 128.8, 128.0, 126.6,
To a solution of 5-chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thio-
125.8, 124.4, 123.5, 116.8, 111.6, 79.2, 51.6, 45.8, 30.0. HRMS (ESI)
phen-6-yl)-2-hydroxybenzamide (100 mg, 0.3 mmol) and PPh3
(157 mg, 0.6 mmol) in THF (5 mL) was added 4-(2-hydroxyethyl)-
piperazine-1-carboxylic acid tert-butyl ester (138 mg, 0.6 mmol) in
THF (5 mL) and DIAD (109 mg, 0.54 mmol). The mixture was stirred
at r.t. for 2 h, and then was concentrated to give the crude product.
This residue was purified with silica gel column (EtOAc/hexane ¼ 1/
1) to afford 80 mg of the intermediate as a pale yellow solid. To the
solution of the intermediate (80 mg) in CH2Cl2 (5 mL) was added
TFA (1 mL) at 0 ꢀC. The mixture was stirred at r.t. for 3 h, and then
was concentrated. The residue was partitioned between EtOAc
(250 mL) and 1 N NaHCO3 (10 mL). The organic layer was washed
with H2O (10 mL) and dried with Na2SO4. The organic layer was
concentrated. The residue was washed with EtOAc (10 mL) and
then the solid was filtered to give 9 (65 mg, 48%, two steps) as a pale
yellow solid (mp 206e207 ꢀC). 1H NMR (600 MHz, DMSO-d6)
calcd for C21H22ClN2O4S 433.0983 (M þ H)þ, found 433.0989.
4.1.4. 5-Chloro-2-(2-dimethylamino-ethoxy)-N-(1,1-dioxo-1H-1l6
benzo[b]thiophen-6-yl) benzamide (5)
-
Compound 5 was prepared in 62% yield by a procedure similar
to that used to prepare compound 2. The title compound was ob-
tained as a pale yellow solid (mp 189e190 ꢀC). 1H NMR (600 MHz,
CDCl3)
d
10.77 (s, 1H), 8.41 (d, 1H, J ¼ 7.8 Hz), 8.24 (d, 1H, J ¼ 1.8 Hz),
7.99 (s, 1H), 7.43e7.45 (m, 1H), 7.33 (d, 1H, J ¼ 8.4 Hz), 7.20 (d, 1H,
J ¼ 6.6 Hz), 6.97 (d, 1H, J ¼ 9.0 Hz), 6.64 (d, 1H, J ¼ 7.2 Hz), 4.26 (t,
2H, J ¼ 5.4 Hz), 2.84 (t, 2H, J ¼ 4.2 Hz), 2.39 (s, 6H). 13C NMR
(150 MHz, CDCl3)
d 162.4, 155.3, 141.9, 137.6, 133.4, 132.5, 132.5,
129.7, 127.5, 126.0, 124.5, 123.0, 114.3, 66.1, 57.9, 45.2. HRMS (ESI)
calcd for C19H20ClN2O4S 407.0827 (M þ H)þ, found 407.0831.
d
10.71 (s, 1H), 8.52 (s, 1H), 8.21 (s, 1H), 7.87e7.89 (m, 1H), 7.69 (d,
1H, J ¼ 2.4 Hz), 7.58e7.63 (m, 3H), 7.32 (d,1H, J ¼ 7.2 Hz), 7.26 (d,1H,
J ¼ 8.4 Hz), 4.24 (t, 2H, J ¼ 4.8 Hz), 2.96 (t, 4H, J ¼ 4.8 Hz), 2.82 (t, 2H,
J ¼ 4.8 Hz), 2.62e2.64 (m, 4H). 13C NMR (150 MHz, DMSO-d6)
4.1.5. 5-Chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-(2-
morpholin-4-yl-ethoxy) benzamide (6)
Compound 6 was prepared in 67% yield by a procedure similar
to that used to prepare compound 2. The title compound was ob-
tained as a white solid (mp 214e215 ꢀC). 1H NMR (600 MHz, DMSO-
d
163.6, 154.6, 141.1, 137.2, 132.9, 132.0, 130.2, 129.1, 126.6, 126.0,
126.0, 124.6, 123.8, 115.2, 111.9, 66.9, 55.8, 49.4, 42.9. HRMS (ESI)
calcd for C21H23ClN3O4S 448.1092 (M þ H)þ, found 448.1101.
d6)
J ¼ 2.4 Hz), 7.58e7.62 (m, 3H), 7.27e7.32 (m, 2H), 4.26e4.28 (m,
2H), 3.43e3.45 (m, 4H), 2.74e2.76 (m, 2H), 2.42e2.50 (m, 4H). 13
NMR (150 MHz, DMSO-d6) 163.4, 154.8, 141.0, 137.2, 132.8, 132.1,
d
10.72 (s, 1H), 8.18 (s, 1H), 7.90 (d, 1H, J ¼ 8.4 Hz), 7.73 (d, 1H,
4.1.9. 5-Chloro-N-(1,1-dioxo-1H-1
(piperidin-4-yloxy)benzamide (10)
l
6-benzo[b]thiophen-6-yl)-2-
C
d
Compound 10 was prepared in 52% yield by a procedure similar
to that used to prepare compound 9. The title compound was ob-
tained as a pale yellow solid (mp 128e129 ꢀC). 1H NMR (600 MHz,
130.2, 129.2, 126.5, 126.0, 125.5, 124.7, 124.1, 115.4, 112.1, 66.7, 66.0,
56.6, 53.3. HRMS (ESI) calcd for C21H22ClN2O5S 449.0932 (M þ H)þ,
found 449.0942.
DMSO-d6) d 10.73 (s, 1H), 8.26 (s, 1H), 7.81e7.82 (m, 1H), 7.58e7.63
(m, 3H), 7.54e7.61 (m, 1H), 7.29e7.31 (m, 2H), 4.72e4.73 (m, 1H),
3.03e3.07 (m, 2H), 2.87e2.90 (m, 2H), 1.98e2.01 (m, 2H), 1.74e1.77
4.1.6. 5-Chloro-N-(1,1-dioxo-1H-1
piperidin-1-yl-ethoxy)benzamide (7)
l
6-benzo[b]thiophen-6-yl)-2-(2-
(m, 2H). 13C NMR (150 MHz, DMSO-d6)
d 164.2, 152.8, 141.3, 137.2,
132.9, 131.4, 130.2, 128.9, 128.2, 126.6, 125.9, 124.6, 123.6, 116.7,
111.6, 71.7, 41.1, 28.5. HRMS (ESI) calcd for C20H20ClN2O4S 419.0827
(M þ H)þ, found 419.0834.
To a solution of 2 (50 mg, 0.11 mmol), KI (28 mg, 0.17 mmol) and
K2CO3 (32 mg, 0.23 mmol) in acetone (5 mL) was added piperidine
(49 mg, 0.57 mmol) at 0 ꢀC. The mixture was stirred at 75 ꢀC for 18 h.
The solution was diluted with EtOAc (100 mL), washed with 0.1 N
HCl (aq.) (10 mL) and brine (10 mL). The organic layer was dried
over anhydrous Na2SO4, and then concentrated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (hexane/EtOAc ¼ 1/1 to 1/3) to give the desired product 7
(50 mg, 98%) as a pale yellow solid (mp 180e181 ꢀC). 1H NMR
4.1.10. 2-(2-Aminoethoxy)-5-chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]
thiophen-6-yl)benzamide (11)
Compound 11 was prepared in 40% yield by a procedure similar
to that used to prepare compound 9. The title compound was ob-
tained as a pale yellow solid (mp 202e203 ꢀC). 1H NMR (600 MHz,
(600 MHz, DMSO-d6)
d
10.71 (s, 1H), 8.20 (s, 1H), 7.89 (d, 1H,
acetone-d6) d 10.85 (s, 1H), 8.19 (s, 1H), 8.13e8.15 (m, 1H), 8.06 (d,
J ¼ 7.8 Hz), 7.74 (d, 1H, J ¼ 2.4 Hz), 7.59e7.62 (m, 3H), 7.27e7.31 (m,
2H), 4.25 (t, 2H, J ¼ 5.4 Hz), 2.70e2.72 (m, 2H), 2.38e2.40 (m, 4H),
1.35e1.38 (m, 4H),1.22e1.24 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
1H, J ¼ 3.0 Hz), 7.55e7.59 (m, 2H), 7.51e7.52 (m, 1H), 7.33 (d, 1H,
J ¼ 8.4 Hz), 6.99 (d,1H, J ¼ 7.2 Hz), 4.52 (t, 2H, J ¼ 4.8 Hz), 3.80 (t, 2H,
J ¼ 4.8 Hz), 1.92e1.96 (m, 2H). 13C NMR (150 MHz, DMSO-d6)
d
163.3, 155.0, 141.0, 137.2, 132.8, 132.2, 130.2, 129.3, 126.5, 126.0,
d 163.4, 154.8, 141.2, 137.1, 132.6, 132.1, 130.1, 129.5, 126.4, 125.8,
125.3, 124.7, 124.1, 115.4, 112.2, 66.9, 56.9, 54.1, 25.4, 23.8. HRMS
125.8, 124.6, 124.1, 116.0, 112.1, 71.2, 40.4. HRMS (ESI) calcd for
C
(ESI) calcd for C22H24ClN2O4S 447.1140 (M þ H)þ, found 447.1145.
17H16ClN2O4S 379.0514 (M þ H)þ, found 379.0520.

202
H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
4.1.11. 2-(3-Aminopropoxy)-5-chloro-N-(1,1-dioxo-1H-1
l
6-benzo
4.2.2. Transient transfection and dual luciferase reporter assays
[b]thiophen-6-yl)benzamide (12)
MDA-MB-231 cells were pre-treated with stattic or 12 at 5 mM
Compound 12 was prepared in 51% yield by a procedure similar
to that used to prepare compound 9. The title compound was ob-
tained as a pale yellow solid (mp 115e116 ꢀC). 1H NMR (600 MHz,
for 24 h. Then the cells were trypsinized and seeded in 24-well
plate at a density of 5 ꢁ 104 cells/well in RPMI-1640 medium
containing 10% FBS and 1% penicillin-streptomycin. Transient
transfections were performed 4 h after plating, using the previously
described method [18,24]. Total amount of DNA for transfections
DMSO-d6)
d 10.76 (s, 1H), 8.31 (s, 1H), 7.78e7.80 (m, 1H), 7.75 (s,
2H), 7.57e7.64 (m, 4H), 7.31 (d, 1H, J ¼ 6.6 Hz), 7.21 (d, 1H,
J ¼ 8.4 Hz), 4.18 (t, 2H, J ¼ 6.0 Hz), 2.95 (t, 2H, J ¼ 6.6 Hz), 2.00e2.02
was 0.5 mg/well, including pSTAT3-Luc (95%, obtained from Pano-
(m, 2H). 13C NMR (150 MHz, DMSO-d6)
d
164.3, 154.3, 141.3, 137.2,
mics, Cat# LR0077) and internal control vector renilla (5%, from
Promega, Madison, WI, USA). 5 h after transfection, the cells were
treated with stattic or compound 12 for 24 h, and then reporter
activity was evaluated using dual luciferase reporter assay kit
(Promega, Madison, WI, USA) on an Omega™ Microplate Lumin-
ometer (BMG LABTECH Inc., NC, USA). Relative luciferase units were
the ratio of the absolute activity of firefly luciferase to that of renilla
luciferase. Experiments were conducted in triplicates and results
are representatives of at least 3 independent experiments.
132.8, 131.6, 130.2, 128.8, 126.8, 126.6, 125.9, 124.3, 123.8, 114.8,
111.8, 66.0, 36.5, 26.5. HRMS (ESI) calcd for C18H18ClN2O4S 393.0670
(M þ H)þ, found 393.0678.
4.1.12. 5-Chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-
(2-methylaminoethoxy)benzamide (13)
Compound 13 was prepared in 43% yield by a procedure similar
to that used to prepare compound 9. The title compound was ob-
tained as a white solid (mp 175e176 ꢀC). 1H NMR (600 MHz, CDCl3)
4.2.3. Molecular docking studies
d
10.77 (s, 1H), 8.36e8.38 (m, 1H), 8.21 (d, 1H, J ¼ 2.4 Hz), 8.10 (s,
Lead compound 1 and compound 12 were docked with the
STAT3-SH2 domain (PDB code: 1BG1) [25] and AutoDock Vina 1.1.2,
using the method as previously described [18,26,27]. Water mole-
cules within the crystal structure were removed and polar hydro-
gens were added using AutoDockTools. The protein was treated as
rigid. Docking runs were carried out using the standard parameters
of the program for interactive growing and subsequent scoring,
except for the parameters for setting grid box dimensions and
1H), 7.44e7.46 (m, 1H), 7.35 (d, 1H, J ¼ 8.4 Hz), 7.21e7.22 (m, 1H),
6.98 (d, 1H, J ¼ 9.0 Hz), 6.67 (d, 1H, J ¼ 6.6 Hz), 4.31 (t, 2H,
J ¼ 4.8 Hz), 3.18 (t, 2H, J ¼ 5.4 Hz), 2.64 (s, 3H). 13C NMR (150 MHz,
CDCl3)
d 162.6, 155.2, 141.8, 137.6, 133.3, 132.5, 132.4, 129.6, 127.4,
126.0, 124.5, 123.3, 114.3, 114.1, 68.3, 50.8, 36.5. HRMS (ESI) calcd for
C
18H18ClN2O4S 393.0670 (M þ H)þ, found 393.0678.
center. For all of the docking studies,
a grid box size of
4.1.13. 5-Chloro-N-(3-methoxy-1,1-dioxo-2,3-dihydro-1H-1l6
benzo[b]thiophen-6-yl)-2-hydroxybenzamide (14)
-
30 Å ꢁ 30 Å ꢁ 30 Å, centered at coordinates 100.452 (x), 75.972 (y),
and 68.790 (z) of the PDB structure.
To the solution of 5-chloro-N-(1,1-dioxo-1H-1l
6-benzo[b]thio-
phen-6-yl)-2-hydroxybenzamide (335 mg, 1.0 mmol) in MeOH
(8 mL) was added 10% NaOH (2 mL, 5.0 mmol) at 0 ꢀC. The mixture
was stirred at r.t. for 15 min. The mixture was diluted with EtOAc
(100 mL) and washed with 2 N HCl (20 mL) and brine (20 mL). The
organic layer was separated and dried with anhydrous Na2SO4. The
solution was concentrated to afford the crude product, which was
washed with CH2Cl2 (5 mL) to give the desired product (220 mg,
60%) as a pale yellow solid (mp 268e269 ꢀC). 1H NMR (600 MHz,
4.2.4. Western blot analysis
Protein levels were determined by Western blot using the pre-
viously reported methods [18,24]. Total cell lysates were prepared
from MDA-MB-231 cells. Protein concentrations were measured
using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA).
Equal amounts of total cellular protein extract (40
mg) were
resuspended in denaturing sample loading buffer (0.5 M TriseHCl,
pH 6.8, 10% SDS, 0.1% bromophenol blue, and 20% glycerol), sepa-
rated by electrophoresis on a 10% polyacrylamide SDS-PAGE gel and
then electrophoretically transferred to a nitrocellulose membrane
(Thermo Scientific, IL, USA) at 100 V for 1 h at 4 ꢀC. The membrane
was then incubated in a blocking solution containing 5% non-fat
milk and 1% Tween 20 in TBS for 1 h. The membrane was then
incubated with antibodies specific for: phospho-STAT3-PY705
(1:3000, Epitomics, #2236-1), STAT3 (1:2000, Cell Signaling,
#4904), Caspase-3-active (1:2000, Epitomics, #1476-1), Cyclin D1
DMSO-d6)
d 11.52 (s, 1H), 10.73 (s, 1H), 8.24 (s, 1H), 7.95 (d, 1H,
J ¼ 8.4 Hz), 7.88 (s, 1H), 7.70 (d, 1H, J ¼ 9.0 Hz), 7.49 (d, 1H,
J ¼ 9.0 Hz), 7.03 (d, 1H, J ¼ 8.4 Hz), 5.17e5.18 (m, 1H), 3.96e3.99 (m,
1H), 3.65e3.67 (dd, 1H, J ¼ 3.0, 13.8 Hz), 3.40 (s, 3H). 13C NMR
(150 MHz, DMSO-d6)
d 165.2, 156.3, 140.5, 139.8, 133.1, 132.7, 128.6,
128.3, 125.7, 122.8, 120.2, 119.0, 111.1, 74.5, 56.8, 56.0. HRMS (ESI)
calcd for C16H15ClNO5S 368.0354 (M þ H)þ, found 368.0357.
(1:10,000, Epitomics, #2261-1) and b-actin (1:10,000, Sigma, clone
4.2. Biology
AC-15). An anti-rabbit or anti-mouse secondary antibody (Amer-
sham, Piscataway, NJ) was used at 1:4000 dilution. The Western
blotted bands were visualized using ECL procedure according to the
manufacturer's instructions (Amersham).
4.2.1. In vitro determination of effects of synthesized compounds on
cancer cell proliferation
Cancer cells (breast cancer cell lines MCF-7 and MDA-MB-231,
pancreatic cancer cell lines AsPC-1 and Panc-1) were seeded in
96-well plates at a density of 2 ꢁ 103 cells/well and treated with
4.2.5. In vivo antitumor activity assays
All procedures including mice and in vivo experiments were
approved by the Institutional Animal Care and Use Committee
(IACUC) of UT M.D. Anderson Cancer Center (MDACC). Thirty-one
female nude mice were obtained from MDACC and were used for
orthotopic tumor studies at 6 weeks of age. The mice were main-
tained in a barrier unit with 12 h lightedark switch. Freshly har-
vested MDA-MB-231 cells (2.5 ꢁ 106 cells per mouse, resuspended
DMSO, 0.01, 0.1, 1, 5, 10, and 100 mM of individual STAT3 inhibitors
for 72 h. Proliferation was measured by treating cells with the 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-((4-
sulfophenyl)-2H-tetrazolium) (MTS) in a CellTiter 96 Aqueous Non-
Radioactive Cell Proliferation Assay kit (Promega, Madison, WI,
USA). Absorbance of all wells was determined by measuring OD at
550 nm after 1 h incubation at 37 ꢀC on a 96-well iMark™ Micro-
plate Absorbance Reader (BioRad, Hercules, CA). Each individual
compound was tested in quadruplicate wells for each
concentration.
in 100 mL PBS) were injected into the 3rd mammary fat pad of the
mice, and then randomly assigned into 5 groups (5e10 mice per
group). For the intraperitoneal treatment experiment, the mice
were treated daily with 10 mg/kg of compound 12 (HJC0416), or

H. Chen et al. / European Journal of Medicinal Chemistry 82 (2014) 195e203
203
vehicle when the tumor volume reached 150 mm3. Similarly, for the
oral gavage experiment, the mice were given 100 mg/kg of 12, or
vehicle five days per week when the tumor volume reached
110 mm3. All drugs were dissolved in 50% DMSO with 50% poly-
ethylene glycol for in vivo administration. Body weights and tumors
volume were measured daily and tumor volume was calculated
according to the formula V ¼ 0.5 ꢁ L ꢁ W2, where L ¼ length (mm)
and W ¼ width (mm).
[6] H. Yu, D. Pardoll, R. Jove, STATs in cancer inflammation and immunity: a
leading role for STAT3, Nat. Rev. Cancer 9 (2009) 798e809.
[7] P. Yue, J. Turkson, Targeting STAT3 in cancer: how successful are we? Expert
Opin. Investig. Drugs 18 (2009) 45e56.
[8] S. Haftchenary, M. Avadisian, P.T. Gunning, Inhibiting aberrant Stat3 function
with molecular therapeutics: a progress report, Anticancer Drugs 22 (2011)
115e127.
[9] R. Buettner, L.B. Mora, R. Jove, Activated STAT signaling in human tumors
provides novel molecular targets for therapeutic intervention, Clin. Cancer
Res. 8 (2002) 945e954.
[10] J.F. Bromberg, M.H. Wrzeszczynska, G. Devgan, Y. Zhao, R.G. Pestell,
C. Albanese, J.E. Darnell Jr., Stat3 as an oncogene, Cell 98 (1999) 295e303.
[11] H. Yu, R. Jove, The STATs of cancerenew molecular targets come of age, Nat.
Rev. Cancer 4 (2004) 97e105.
[12] J.E. Darnell, Validating Stat3 in cancer therapy, Nat. Med. 11 (2005) 595e596.
[13] B. Debnath, S. Xu, N. Neamati, Small molecule inhibitors of signal transducer
and activator of transcription 3 (Stat3) protein, J. Med. Chem. 55 (2012)
6645e6668.
4.2.6. Statistical analysis
Statistical significance was determined using student t-test in
cell cycle analysis. * represents a p value less than 0.05.
Conflict of interest
[14] J. Deng, F. Grande, N. Neamati, Small molecule inhibitors of Stat3 signaling
pathway, Curr. Cancer Drug Targ. 7 (2007) 91e107.
The authors declare no competing financial interest.
Acknowledgments
[15] K. Al Zaid Siddiquee, J. Turkson, STAT3 as a target for inducing apoptosis in
solid and hematological tumors, Cell Res. 18 (2008) 254e267.
[16] B.D. Page, D.P. Ball, P.T. Gunning, Signal transducer and activator of tran-
scription 3 inhibitors: a patent review, Expert Opin. Ther. Pat. 21 (2011)
65e83.
[17] M.M. Hann, G.M. Keseru, Finding the sweet spot: the role of nature and
nurture in medicinal chemistry, Nat. Rev. Drug. Discov. 11 (2012) 355e365.
[18] H. Chen, Z. Yang, C. Ding, L. Chu, Y. Zhang, K. Terry, H. Liu, Q. Shen, J. Zhou,
Fragment-based drug design and identification of HJC0123, a novel orally
bioavailable STAT3 inhibitor for cancer therapy, Eur. J. Med. Chem. 62 (2013)
498e507.
[19] H. Chen, Z. Yang, C. Ding, L. Chu, Y. Zhang, K. Terry, H. Liu, Q. Shen, J. Zhou,
Discovery of -Alkylamino tethered niclosamide derivatives as potent and
orally bioavailable anticancer agents, ACS Med. Chem. Lett. 4 (2013) 180e185.
[20] A. Innocenti, R. Villar, V. Martinez-Merino, M.J. Gil, A. Scozzafava, D. Vullo,
C.T. Supuran, Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-
associated carbonic anhydrase isozymes I, II, and IX with benzo[b]thiophene
1,1-dioxide sulfonamides, Bioorg. Med. Chem. Lett. 15 (2005) 4872e4876.
[21] D. Tang, E.T. McKinley, M.R. Hight, M.I. Uddin, J.M. Harp, A. Fu, M.L. Nickels,
J.R. Buck, H.C. Manning, Synthesis and structure-activity relationships of 5,6,7-
substituted pyrazolopyrimidines: discovery of a novel TSPO PET ligand for
cancer imaging, J. Med. Chem. 56 (2013) 3429e3433.
This work was supported by grants P50 CA097007, P30
DA028821, R21 MH093844 (JZ) from the National Institutes of
Health, a grant from Duncan Family Institute Seed Funding
Research Program and a startup fund from MD Anderson Cancer
Center (QS), R. A. Welch Foundation Chemistry and Biology
Collaborative Grant from the Gulf Coast Consortia (GCC), a training
fellowship from the Keck Center for Interdisciplinary Bioscience
Training of the GCC (NIGMS grant T32 GM089657-03), Sealy and
Smith Foundation grant (to the Sealy Center for Structural Biology
and Molecular Biophysics), John Sealy Memorial Endowment Fund,
and the Center for Addiction Research (CAR) at UTMB. We thank
Drs. Lawrence C. Sowers, Jacob A. Theruvathu, and Tianzhi Wang for
the NMR spectroscopy assistance, as well as Drs. Huiling Liu and
Carol Nilsson for mass spectrometry assistance.
[22] J. Schust, B. Sperl, A. Hollis, T.U. Mayer, T. Berg, Stattic: a small-molecule in-
hibitor of STAT3 activation and dimerization, Chem. Biol. 13 (2006)
1235e1242.
Appendix A. Supplementary data
[23] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of
docking with
a new scoring function, efficient optimization, and multi-
threading, J. Comput. Chem. 31 (2010) 455e461.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.049.
[24] Q. Shen, I.P. Uray, Y. Li, T.I. Krisko, T.E. Strecker, H.T. Kim, P.H. Brown, The AP-1
transcription factor regulates breast cancer cell growth via cyclins and E2F
factors, Oncogene 27 (2008) 366e377.
[25] S. Becker, B. Groner, C.W. Muller, Three-dimensional structure of the Stat3-
beta homodimer bound to DNA, Nature 394 (1998) 145e151.
References
[26] H. Chen, T. Tsalkova, F.C. Mei, Y. Hu, X. Cheng, J. Zhou, 5-Cyano-6-oxo-1,6-
dihydro-pyrimidines as potent antagonists targeting exchange proteins
directly activated by cAMP, Bioorg. Med. Chem. Lett. 22 (2012) 4038e4043.
[27] H. Chen, C.Z. Wang, C. Ding, C. Wild, B. Copits, G.T. Swanson, K.M. Johnson,
[1] J.E. Darnell Jr., STATs and gene regulation, Science 277 (1997) 1630e1635.
[2] T. Bowman, R. Garcia, J. Turkson, R. Jove, STATs in oncogenesis, Oncogene 19
(2000) 2474e2488.
[3] J. Bromberg, J.E. Darnell Jr., The role of STATs in transcriptional control and
their impact on cellular function, Oncogene 19 (2000) 2468e2473.
[4] J. Turkson, R. Jove, STAT proteins: novel molecular targets for cancer drug
discovery, Oncogene 19 (2000) 6613e6626.
J. Zhou,
A combined bioinformatics and chemoinformatics approach for
developing asymmetric bivalent AMPA receptor positive allosteric modulators
as neuroprotective agents, ChemMedChem 8 (2013) 226e230.
[5] J. Bromberg, Stat proteins and oncogenesis, J. Clin. Invest 109 (2002)
1139e1142.