Fragment-based drug design and identification of HJC0123, a novel orally bioavailable STAT3 inhibitor for cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2013.01.023

Fragment-based drug design (FBDD) is a promising approach for the generation of lead molecules with enhanced activity and especially drug-like properties against therapeutic targets. Herein, we report the fragment-based drug design, systematic chemical synthesis and pharmacological evaluation of novel scaffolds as potent anticancer agents by utilizing six privileged fragments from known STAT3 inhibitors. Several new molecules such as compounds 5, 12, and 19 that may act as advanced chemical leads have been identified. The most potent compound 5 (HJC0123) has demonstrated to inhibit STAT3 promoter activity, downregulate phosphorylation of STAT3, increase the expression of cleaved caspase-3, inhibit cell cycle progression and promote apoptosis in breast and pancreatic cancer cells with low micromolar to nanomolar IC ₅₀ values. Furthermore, compound 5 significantly suppressed estrogen receptor (ER)-negative breast cancer MDA-MB-231 xenograft tumor growth in vivo (p.o.), indicating its great potential as an efficacious and orally bioavailable drug candidate for human cancer therapy.

Full text of DOI:10.1016/j.ejmech.2013.01.023

European Journal of Medicinal Chemistry 62 (2013) 498e507
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fragment-based drug design and identification of HJC0123,
a novel orally bioavailable STAT3 inhibitor for cancer therapy
,
,
Haijun Chen a 1, Zhengduo Yang b 1, Chunyong Ding a, Lili Chu b, Yusong Zhang b,
**
Kristin Terry b, Huiling Liu 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Fragment-based drug design (FBDD) is a promising approach for the generation of lead molecules with
enhanced activity and especially drug-like properties against therapeutic targets. Herein, we report the
fragment-based drug design, systematic chemical synthesis and pharmacological evaluation of novel
scaffolds as potent anticancer agents by utilizing six privileged fragments from known STAT3 inhibitors.
Several new molecules such as compounds 5, 12, and 19 that may act as advanced chemical leads have
been identified. The most potent compound 5 (HJC0123) has demonstrated to inhibit STAT3 promoter
activity, downregulate phosphorylation of STAT3, increase the expression of cleaved caspase-3, inhibit
cell cycle progression and promote apoptosis in breast and pancreatic cancer cells with low micromolar
to nanomolar IC50 values. Furthermore, compound 5 significantly suppressed estrogen receptor (ER)-
negative breast cancer MDA-MB-231 xenograft tumor growth in vivo (p.o.), indicating its great potential
as an efficacious and orally bioavailable drug candidate for human cancer therapy.
Received 12 November 2012
Received in revised form
14 January 2013
Accepted 17 January 2013
Available online 26 January 2013
Keywords:
Fragment-based drug design
Anticancer agents
Drug discovery
STAT3
Breast cancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
biological processes such as the immune response, angiogenesis,
cell proliferation, differentiation, and apoptosis [1e7]. It contains
STAT3 (signal transducers and activators of transcription 3) is
a member of a family of seven transcription factors (STATs 1, 2, 3, 4,
5a, 5b, and 6) that transmit signals from cell surface receptors to the
nucleus, and are crucial for the signaling of many cytokines and
growth factors that are mediators in the fundamental cellular and
four functional domains that contribute to its oligomerization, DNA
binding, Src homology 2 (SH2) dimerization, and transactivation,
respectively. Once activated by extracellular signaling proteins,
STAT3 is phosphorylated at a tyrosine residue 705 (Tyr-705) and
phosphorylated STAT3 forms dimers via a reciprocal phosphotyr-
osines (pTyr)-SH2 domain interaction. Then the dimers translocate
to the nucleus where they bind to specific DNA response elements
and induce transcription [8]. In contrast to the transient nature of
STAT3 activation in normal cells, constitutive STAT3 activity has
been observed and reported in many human solid and hemato-
logical tumors [9,10]. Aberrant STAT3 activation is found to be
correlated with worse prognosis by promoting tumorigenesis,
inducing tumor invasion and metastasis, and driving the malignant
progression in many carcinomas. Thus, STAT3 is considered to be
a promising target for prevention and treatment of cancer, thereby
providing the rationale to develop novel anticancer agents target-
ing the STAT3 signaling pathway [11e18].
Abbreviations: FBDD, fragment-based drug design; STATs, signal transducers
and activators of transcription; ER, estrogen receptor; HBTU, 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIPEA, N,N-diisopropyleth-
ylamine; THF, tetrahydrofuran; SAR, structureeactivity relationships; RLU, relative
luciferase unit; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium); IC50
, half maximal inhibitory concentration; PI,
propidium iodide; HRMS, high-resolution mass spectrometry; HPLC, high perfor-
mance liquid chromatography; TFA, trifluoroacetic acid; DMSO, dimethyl sulfoxide;
TLC, thin layer chromatography; NMR, nuclear magnetic resonance; TMS, tetra-
methylsilane; EtOAc, ethyl acetate; DMF, dimethylformamide; PBS, phosphate-
buffered saline; BCA, bicinchoninic acid; SDS-PAGE, sodium dodecyl sulfate poly-
acrylamide gel electrophoresis.
* Corresponding author. Tel.: þ1 409 772 9748; fax: þ1 409 772 9818.
** Corresponding author. Tel.: þ1 713 834 6357; fax: þ1 713 834 6350.
E-mail addresses: Qshen@mdanderson.org (Q. Shen), jizhou@utmb.edu
(J. Zhou).
Over the past decade, several peptidic and non-peptidic STAT3
inhibitors that directly inhibit the different structural domains of
STAT3 protein or indirectly inhibit the upstream components of the
STAT3 activation [19] such as PY*LKTK [20], STX-0119 [21], and S31-
1
These authors contribute equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.023

H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
499
201 [22] to inhibit STAT3 dimerization, Stattic [23] to inhibit
H
N
O
phosphorylation, CAP-1 [24] and IS3 295 [25] to inhibit DNA-
binding, niclosamide [26] to inhibit transcriptional function of
STAT3, WP1066 [27] and AG490 [28] to inhibit the upstream Janus
kinases activity have been explored (Fig. 1). Despite these signifi-
cant advances, no STAT3 inhibitor drugs have reached the market.
The challenges include the lack of membrane permeability and
stability, low water-solubility, weak binding affinity, or low spe-
cificity of effects [29e38]. Therefore, it remains highly valuable to
identify novel potent and efficacious STAT3 inhibitors with new
scaffolds.
OH
Cl
O
O
Br
N
N
H
N
H
CN
N
O
S
O
H
O
N
O
N
H
N
N
Fragment-based drug design (FBDD) is a promising approach for
the generation of lead molecules against therapeutic targets in the
past decade. New molecules with enhanced drug-likeness could be
identified by chemically merging privileged-fragments from small
molecule libraries that bind cooperatively in a given binding pocket
[39e42]. These favorable fragments may maintain their original
orientation in the protein binding pocket, and thus contribute
jointly to the final binding energy. Till now, FBDD has proven to be
a successful approach to identify potent inhibitors for specific tar-
gets [43,44]. Herein, we describe our efforts for the fragment-based
design, synthesis and characterization of new molecules as STAT3
inhibitors for cancer therapy. Compound 5 (HJC0123) was dis-
covered as a potent and orally bioavailable anticancer agent in this
endeavor.
Fig. 2. Privileged fragments selected from known STAT3 inhibitors including niclosa-
mide, STX-0119, WP1066, and stattic.
selection of the privileged fragment library was governed by data
from literature with respect to the most active inhibitors of STAT3
and the drug-like properties such as molecular weight of these
fragments, which ranges from 120 to 251 Da. Taking inspiration
from the FBDD approach, we designed a number of novel molecules
with diversified scaffolds for structureeactivity relationship (SAR)
studies in an attempt to identify new anticancer compounds with
enhanced potency and drug-like properties.
2.2. Chemistry
2. Results and discussion
As shown in Schemes 1 and 2, coupling of 1,1-dioxo-1H-1l6
-
benzo[b]thiophen-6-ylamine (3) with cyanoacetic acid (1) or 2-
phenyl-quinoline-4-carboxylic acid (2) in the presence of HBTU
and DIPEA in CH2Cl2 generated the corresponding amides 4 and 5 in
2.1. Design
We initiated our studies with a small privileged fragment library
containing six fragments selected from representative non-peptidic
STAT3 inhibitors including stattic, WP1066, STX-0119, and niclo-
samide as potential binding pharmacophores to STAT3 (Fig. 2). The
moderate to high yields. Condensation of
L
(ꢀ)-
a-methylbenzyl-
amine (7) with 2-phenylquinoline-4-carboxylic acid (2) or 5-
chloro-2-hydroxybenzoic acid (6) in the same fashion provided
new compounds 8 and 9, respectively. The synthesis of compound
H
NO2
O
O
N
O
OH
Cl
O
O
O
S
N
O
N
N
H
Br
N
O2N
N
H
Cl
CN
WP1066
N
Stattic
Niclosamide
STX-0119
O
O
O
O
O
O O
S
H
SO2NH2
O
HO
HO
N
O
O
HO
O
LLL12
OH
STA-21 (Phase I/II)
O
O
Cryptotanshinone
S31-201
O
R1
O
R3
H
N
R2
O
Cl
H
NH2
N
N
N
NC
O
O
N
O
O
N
H
H2N
N
Pyrimethamine
Bardoxolone methyl, RTA 402
OPB analogues (OPB-31121 on Phase I)
(Phase I/II)
(Phase I/II)
Fig. 1. Chemical structures of representative non-peptidic STAT3 inhibitors.

500
H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
O
O
S
H
N
1, 4: R1 = NC
O
O
O
H2N
O
a
S
+
R1
OH
OH
R1
2, 5: R1 =
1,2
3
4,5
N
Me
Me
O
O
2, 8: R1 =
a
R1
N
H
R1
+
H2N
N
OH
2,6
7
8,9
6, 9: R1 =
Cl
a Reagents and conditions: (a) HBTU, DIPEA, CH2Cl2, rt, 39-94%.
Scheme 1.
10 was achieved in moderate yield via Knoevenagel condensation
of 6-bromopyridine-2-carbaldehyde with the intermediate 4 in the
presence of piperidine as the catalyst. The above mentioned cou-
pling methods failed to give the desired compounds 12 and 13,
which were successfully obtained by an alternative protocol in
three steps. First, the carboxylic acid 6 was converted to the acid
chloride by the treatment with SOCl2 in toluene at 120 ꢁC for 16 h.
After concentration, the acid chloride, which contained some ester
formed from the hydroxyl group of phenol, was used directly for
next step without further purification because the hydrolysis
of the ester appendage afterward could lead to the same
in THF to generate the desired compounds 12 and 13 in yield of 39%
and 50% (three steps), respectively.
2.3. Biology
To explore the SAR, we first evaluated the in vitro anticancer
effects of the compounds 4, 5, 8e10 and 12, 13 on the proliferation
of human breast cancer cell lines MCF-7 (ER-positive) and MDA-
MB-231 (ER-negative and triple-negative), as well as pancreatic
cancer cell lines AsPC1 and Panc-1 using MTS assays as described in
the Experimental section. The ability of these new scaffolds to
inhibit the growth of cancer cells is summarized in Table 1. It is
noteworthy that most of the newly synthesized compounds
described herein exhibited promising antiproliferative activity
with low micromolar to nanomolar IC50 values. Among them,
desired product. 1,1-Dioxo-1H-1l
6-benzo[b]thiophen-6-ylamine or
5-furan-2-yl-[1,3,4]oxadiazol-2-ylamine was then treated with the
above acid chloride in the presence of pyridine as the base. The
crude products were subsequently saponified using 1 N LiOH (aq.)
O
S
H
N
O
O
a
NC
Br
N
S
O
N
H
O
O
CN
10
4
O
OH
O
OH
O
OH
Cl
O
O
S
12: R2 =
13: R2 =
NHR2
OH
b
c, d
Cl
O
N
O
Cl
12, 13
Cl
6
N
11
a Reagents and conditions: (a) 6-bromo-pyridine-2-carbaldehyde, piperidine (cat.), EtOH, 90 oC, 72%; (b)
o
o
SOCl2, toluene, reflux; (c) R2NH2, pyridine, DMF, 0 C to rt; (d) 1 N LiOH (aq.), THF, H2O, 0 C to rt,
39-50% (three steps).
Scheme 2.

H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
501
Table 1
Effects of newly synthesized compounds 4, 5, 8e10 and 12, 13 on proliferation of human breast and pancreatic cancer cell lines.
OH
Br
N
A =
NC
A2
N
CN
O
Cl
A4
B
A1
A3
A
N
H
O
S
O
O
O
B =
N
N
B2
B1
B3
Compound
A
B
IC50 (m
M)a
Breast cancer ER-Positive
Breast cancer ER-Negative
MDA-MB-231
Pancreatic cancer
AsPC1
MCF-7
Panc-1
4
5
8
9
10
12
13
A2
A1
A1
A4
A3
A4
A4
B1
B1
B2
B2
B1
B1
B3
>10b
0.1
2.24
0.9
3.31
0.91
>10
1.06
>10
0.29
86.0
8.88
1.53
1.64
>10
0.79
NDc
1.25
>10
7.54
1.54
1.92
>10
1.47
ND
0.26
>10
8.44
1.64
2.34
>10
1.73
Niclosamide
a
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, indicates that a specific IC50 cannot be calculated from the data points collected, meaning ‘no effect’.
ND: not determined.
b
c
compounds 5, 10, and 12 possessing the 1,1-dioxo-1H-1
l
6-benzo[b]
The obtained SAR results suggest that the hydrophobic sub-
stituent at C2 of the quinoline or pyridine framework is crucial for
targeting STAT3. For example, compounds 20, 21 and 23 without
the 2-Ph moiety displayed moderate to low antiproliferative ac-
tivity against the tested cancer cells. Instead, compound 22 with 2-
Ph group in contrast with compound 21 regained antiproliferative
activity with low micromolar IC50 values. Compound 19 with an
additional phenyl substituent at the 6-position of pyridine ring
exhibited further enhanced anticancer activity when compared
with compound 22, indicating the important role of hydrophobic
substituents on the quinoline and pyridine fragments.
Through the SAR studies, compound 5 has been identified with
most desirable antiproliferative activity and physicochemical pa-
rameters (see Table S1 in the Supporting information), and was
subjected to further biological characterization. Cellular morpho-
logical change in MDA-MB-231 breast cancer cells treating with
compound 5 for 48 h was examined under light microscopy. As
shown in Fig. S1, compound 5 significantly inhibited cell prolifer-
ation and induced apoptosis accompanying cellular morphological
changes in a dose-dependent manner.
thiophen-6-yl fragment (B1) exhibited a similar or significantly
higher potency than the reference compound niclosamide. Inter-
estingly, compound 4 containing the fragment B1 and the moiety
A2 instead of the 6-bromopyridin-2-yl fragment (A3) was found
inactive with a dramatic loss of antiproliferative activity in com-
parison with compound 10. Compounds 8 and 9 with (S)-(1-
phenylethyl) amide moiety (B2) showed moderate to potent anti-
proliferative effects against the tested cancer cells. For example,
compound 9 displayed an IC50 value of 0.9 mM against ER-positive
breast cancer cell line MCF-7. The salicylic amide compound 13
with the 5-furan-2-yl-[1,3,4]oxadiazol-2-yl moiety (B3) was found
inactive, while compound 12 with the fragment B1 instead
exhibited significant antiproliferative activity, indicating that frag-
ment B1 rather than B3 is more favorable for the anticancer activity
of molecules with the salicylic amide scaffold.
Among the active new scaffolds discussed above, compound 5
notably exhibited remarkable potency against all the tested cancer
cells. Since STX-0119 with the 2-phenylquinoline-4-carboxylic acid
amide fragment (A1) [21,45] and stattic with the 1,1-dioxo-1H-1l6
-
benzo[b]thiophen-6-yl fragment (B1) [23] have been reported as
the STAT3-SH2 domain inhibitors, the molecular docking studies of
compound 5 with both A1 and B1 fragments were performed to
investigate the possible conformations and the required spatial
relationship between the scaffold and STAT3-SH2 domain Ref. [46]
using AutoDock Vina [47] docking approach. Examination of the
predicted binding model for 5 complexed with STAT3 revealed that
the 2-phenyl group on the quinoline ring could fit effectively into
the hydrophobic cleft around Ile634 (Fig. 3). To further verify our
findings, chemical optimization of 5 was carried out to gain insights
on this potential series. Additional five compounds (19e23) with
different hydrophobic groups were prepared (Scheme 3), analo-
gously to the synthesis of 4 and 5 described above, and their anti-
cancer activities were evaluated in the same fashion (Table 2).
To determine whether compound 5 acts as a potent small-
molecule inhibitor of STAT3 activation, we measured the effect of
5 on promoter activity using the cell-based transient transfection
and dual luciferase reporter assays. The STAT3 promoter activity in
MDA-MB-231 cells was determined after transient transfecting
with pSTAT3-Luc vector. As shown in Fig. 4, treatment with 5 mM of
compound 5 inhibited the STAT3 promoter activity in MDA-MB-231
cells by approximately 65% compared with control.
To further investigate the inhibitory activity of compound 5
against the 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
5 for 24 h and 48 h, and levels of total STAT3 and phosphorylated
STAT3 at Tyr-705 were then examined by Western blot. It was

502
H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
found that the total STAT3 expression in these cells was reduced
after the treatment with compound 5 (Fig. 5). Similarly, phos-
phorylated STAT3 at Tyr-705 was suppressed by compound 5.
Blocking STAT3 signaling in many different tumor cells leads to
growth arrest and apoptosis [7,22,48e50]. To investigate whether
compound 5 induces apoptosis, we first determined its ability in
inducing cleaved caspase 3 in MDA-MB-231 breast cancer cells. As
shown in Fig. 5, compound 5 induced a higher cleaved caspase-3
level in MDA-MB-231 cells, suggesting that compound 5 pro-
moted the apoptosis of cancer cells. We further confirmed these
results using annexin V-based measurement using flow cytometry.
As depicted in Fig. S2, compound 5 activated apoptosis in MDA-MB-
231 breast cancer cells in a dose-dependent manner. Further flow
cytometry analysis revealed that compound 5 arrested MDA-MB-
231 cells at S phase in a dose-dependent manner (Fig. S3). These
results suggested that compound 5 was conferred the ability to
arrest cells. The more extensive mechanism studies on compound 5
including STAT3 upstream targets and related signaling pathways
are undergoing, and the results will be reported somewhere else in
due course.
Compound 5 was next evaluated for its antitumor activity in
inhibition of tumor growth in the MDA-MB-231 xenograft model.
MDA-MB-231 xenograft tumors were developed in immunodefi-
cient nude mice and tumor volume was measured daily in oral
gavage (p.o.) group. We treated the MDA-MB-231 xenograft mice
through oral administration of compound 5 (50 mg/kg) and found
that the growth of xenograft tumors in mice was significantly
suppressed by compound 5 (Fig. 6). Notably, compound 5 did not
show significant signs of toxicity even at the dose of 150 mg/kg
(data not shown). These results have demonstrated that compound
5 is a potent, efficacious and orally bioavailable anti-cancer drug
candidate that is promising for further clinical development.
3. Conclusions
Fig. 3. Predicted binding mode for compound 5. (A) Surface of the electrostatic map.
(B) Residues of STAT3. Compound 5 is shown in small sticks and in pink color.
Hydrogen bonds are indicated by dashed lines. The figures were generated using
Pymol. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Taken together,
a fragment-based drug design, systematic
chemical synthesis and pharmacological evaluation of novel scaf-
folds as potent anticancer agents have been carried out by utilizing
O
O
S
H
N
O
O
O
H2N
S
O
a
+
R1
OH
R1
14-18
3
19-23
14, 19: R1 =
15, 20: R1 =
16, 21: R1 =
N
N
N
17, 22: R1 =
18, 23: R1 =
N
N
a Reagents and conditions: (a) HBTU, DIPEA, CH2Cl2, rt, 39-56%.
Scheme 3.

H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
503
Table 2
Effects of newly synthesized compounds 19e23 on proliferation of human breast and pancreatic cancer cell lines.
O
S
H
N
O
R1
O
19-23
Compound
R1
IC50 (m
M)a
Breast cancer ER-Positive
MCF-7
Breast cancer ER-Negative
MDA-MB-231
Pancreatic cancer
AsPC1
Panc-1
0.26
5
0.1
0.29
1.25
N
19
0.65
0.45
0.12
0.31
N
20
21
>10b
>10
>10
>10
>10
N
N
>10
NDc
ND
22
3.78
2.97
1.85
6.21
1.3
3.35
7.92
N
23
6.97
N
a
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, indicates that a specific IC50 cannot be calculated from the data points collected, meaning ‘no effect’.
ND: not determined.
b
c
six privileged fragments from known STAT3 inhibitors. Several new
bioavailable drug candidate for human cancer therapy. This prom-
ising compound has been selected for further preclinical assessment
and the results will be reported somewhere else in due course.
molecules such as compounds 5,12, and 19 that may act as advanced
chemical leads have been identified. The most potent compound 5
has demonstrated to inhibit STAT3 promoter activity, down-regulate
phospho-STAT3, increase the expression of cleaved caspase-3,
inhibit cell cycle progression and promote apoptosis in breast and
pancreatic cancer cells with low micromolar to nanomolar IC50
values. Furthermore, compound 5 significantly suppressed ER-
negative breast cancer MDA-MB-231 xenograft tumor growth
in vivo (p.o.), indicating its great potential as an efficacious and orally
4. Experimental
4.1. Chemistry
All commercially available starting materials and solvents were
reagent grade, and used without further purification. Reactions

504
H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
Fig. 4. Compound 5 (HJC0123) inhibited the STAT3 mediated luciferase reporter ac-
tivity in MDA-MB-231 cells. STAT3 promoter activity was measured using dual luci-
ferase 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. 6. In vivo efficacy of compound 5 (HJC0123) in inhibiting growth of xenograft
tumors (Breast cancer MDA-MB-231) in mice (p.o.).
temperature 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-
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 (70e
230 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 Brucker-600 (1H,
600 MHz; 13C, 150 MHz) spectrometer. 1H and 13C NMR spectra
were recorded with TMS as an internal reference. Chemical shifts
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
ditions: Waters
m
Bondapak C18 (300 ꢂ 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 bio-
logically evaluated compounds are >95% pure.
4.1.1. 2-Cyano-N-(1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-yl)
acetamide (4)
To a solution of cyanoacetic acid (340 mg, 4.0 mmol) and 1,1-
dioxo-1H-1
l
6-benzo[b]thiophen-6-ylamine (362 mg, 2.0 mmol)
in 10 mL of CH2Cl2 was added DIPEA (774 mg, 6.0 mmol). HBTU
(1.14 g, 3.0 mmol) was added at 0 ꢁC. The resulting mixture was
stirred at r.t. for 28 h. The reaction mixture was diluted with CH2Cl2
(80 mL) and washed with water (20 mL). The organic layer was
separated and dried with anhydrous Na2SO4. The solution was
concentrated to give a crude product, which was purified with silica
gel column (EtOAc/hexane ¼ 1/1) to obtain the desired product
(400 mg, 81%) as a yellow solid (mp 207e208 ꢁC). HPLC purity
98.6% (tR ¼ 10.93 min). 1H NMR (600 MHz, DMSO-d6)
d 10.79 (s,1H),
8.04 (s, 1H), 7.67e7.69 (m, 1H), 7.60 (d, 1H, J ¼ 7.2 Hz), 7.57 (d, 1H,
J ¼ 7.8 Hz), 7.30 (d, 1H, J ¼ 7.2 Hz), 3.98 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) d 162.0, 140.7,137.3, 132.8, 130.3, 126.7, 126.1, 123.4, 115.6,
111.4, 27.0. HRMS (ESI) calcd for C11H19N2O3S 249.0328 (M þ H)þ,
found 249.0330.
4.1.2. 2-Phenylquinoline-4-carboxylic acid (1,1-dioxo-1H-1l6
benzo[b]thiophen-6-yl)amide (5)
-
To a solution of 2-phenylquinoline-4-carboxylic acid (249 mg,
1.0 mmol) and 1,1-dioxo-1H-1
l
6-benzo[b]thiophen-6-ylamine
(181 mg, 1.0 mmol) in 10 mL of CH2Cl2 was added DIPEA
(387 mg, 3.0 mmol). HBTU (759 mg, 2.0 mmol) was added at 0 ꢁC.
The resulting mixture was stirred at r.t. for 48 h. The reaction
mixture was diluted with EtOAc (300 mL) and washed with water
Fig. 5. Western blot analysis of biochemical markers for apoptosis induction and in-
hibition of STAT3 activity by compound 5 (HJC0123) in the MDA-MB-231 cell line. Cells
were treated with compound 5 for 24 h and 48 h, and levels of STAT3, pSTAT3, cleaved
caspase-3 were probed by specific antibodies. b-Actin was used as the loading control.

H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
505
(50 mL). The organic layer was separated and dried with anhydrous
Na2SO4. The solution was concentrated to give a crude product,
which was further purified with short silica gel column (EtOAc/
hexane ¼ 1/1 to 4/1) to obtain the desired product (160 mg, 39%)
as a pale yellow solid (mp 277e278 ꢁC). HPLC purity 99.2%
overnight. The mixture was concentrated to give a crude product
as a pale yellow oil. To the solution of pyridine (869 mg, 11 mmol)
and 1,1-dioxo-1H-1l
6-benzo[b]thiophen-6-ylamine (200 mg,
1.1 mmol) was added the solution of the acid chloride (500 mg,
2.6 mmol) in DMF (15 mL) dropwise at 0 ꢁC. The mixture was
stirred at r.t. for 24 h. The mixture was added to the water so-
lution dropwise. The yellow solid was formed and filtrated. To the
mixture of the crude product in THF (8 mL) was added 1 N LiOH
(2.2 mL, 2.2 mmol) at 0 ꢁC. The mixture was stirred at 0 ꢁC for
30 min. The mixture was diluted with EtOAc (50 mL) and washed
with 2 N HCl (10 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 (20 mL) to
give the desired product (150 mg, 39%) as a yellow solid (mp
268e269 ꢁC). HPLC purity 98.3% (tR ¼ 17.24 min). 1H NMR
(tR ¼ 32.39 min). 1H NMR (600 MHz, DMSO-d6)
d 11.29 (s, 1H), 8.44
(s, 1H), 8.38 (d, 2H, J ¼ 7.2 Hz), 8.34 (s, 1H), 8.20 (dd, 2H, J ¼ 5.4 Hz,
13.8 Hz), 7.98 (d,1H, J ¼ 7.8 Hz), 7.88 (t,1H, J ¼ 7.2 Hz), 7.54e7.70 (m,
6H), 7.34 (d, 1H, J ¼ 6.6 Hz). 13C NMR (150 MHz, DMSO-d6)
d 165.8,
155.8, 147.9, 142.2, 141.2, 138.0, 137.2, 132.9, 130.5, 130.4, 130.0,
129.7, 129.0, 127.6, 127.3, 126.6, 126.3, 125.1, 124.2, 123.0, 117.1, 117.1,
112.3, 112.3. HRMS (ESI) calcd for C24H17N2O3S 413.0954 (M þ H)þ,
found 413.0959.
4.1.3. (S)-2-Phenylquinoline-4-carboxylic acid (1-phenylethyl)
amide (8)
(600 MHz, DMSO-d6)
d 11.50 (s, 1H), 10.74 (s, 1H), 8.25 (s, 1H),
To a solution of 2-phenyl-quinoline-4-carboxylic acid (249 mg,
7.92 (d, 1H, J ¼ 8.4 Hz), 7.86 (d, 1H, J ¼ 2.4 Hz), 7.59e7.62 (m, 2 H),
1.0 mmol) and
L(ꢀ)-
a-methylbenzylamine (127 mg, 1.05 mmol) in
7.48 (d, 1H, J ¼ 9.0 Hz), 7.31 (d, 1H, J ¼ 6.6 Hz), 7.04 (d, 1H,
10 mL of CH2Cl2 was added DIPEA (388 mg, 3.0 mmol). HBTU
(569 mg, 1.5 mmol) was added at 0 ꢁC. The resulting mixture was
stirred at r.t. for 3 h. The reaction mixture was diluted with CH2Cl2
(80 mL) and washed with water (20 mL). The organic layer was
separated and dried with anhydrous Na2SO4. The solution was
concentrated to give a crude product, which was purified with silica
gel column (EtOAc/hexane ¼ 1/3) to obtain 8 (330 mg, 94%) as
a white solid (mp 157e158 ꢁC). HPLC purity 99.8% (tR ¼ 17.22 min).
J ¼ 8.4 Hz). 13C NMR (150 MHz, DMSO-d6)
d 165.1, 156.2, 140.7,
137.1, 133.1, 132.8, 130.3, 128.6, 126.5, 126.2, 124.6, 122.8, 120.4,
119.0, 112.7. HRMS (ESI) calcd for C15H11ClNO4S 336.0092
(M þ H)þ, found 336.0098.
4.1.7. 5-Chloro-N-(5-furan-2-yl-[1,3,4]oxadiazol-2-yl)-2-
hydroxybenzamide (13)
Compound 13 was prepared in 50% yield by a procedure similar
to that used to prepare compound 12. The title compound was
obtained as a white solid (mp 196e197 ꢁC). HPLC purity 96.0%
1H NMR (600 MHz, CDCl3)
d 8.03e8.07 (m, 3H), 7.97 (d, 1H,
J ¼ 8.4 Hz), 7.71 (s, 1H), 7.67 (t, 1H, J ¼ 7.2 Hz), 7.28e7.48 (m, 9H),
6.78 (d,1H, J ¼ 7.2 Hz), 5.38e5.43 (m,1H),1.66 (d, 3H, J ¼ 6.6 Hz). 13C
(tR ¼ 18.99 min). 1H NMR (600 MHz, CDCl3)
d 10.11 (s, 1H), 8.13 (d,
NMR (150 MHz, CDCl3)
d
166.9, 156.9, 148.7, 143.0, 142.6, 138.9,
1H, J ¼ 2.4 Hz), 8.02 (d, 1H, J ¼ 2.4 Hz), 7.64 (dd, 1H, J ¼ 2.4 Hz and
8.4 Hz), 7.49 (dd, 1H, J ¼ 2.4 Hz and 8.4 Hz), 7.21 (d, 1H, J ¼ 9.0 Hz),
130.3, 130.2, 129.8, 129.0, 127.8, 127.6, 127.4, 126.4, 125.0, 123.4,
116.4, 49.8, 21.9. HRMS (ESI) calcd for C24H21N2O 353.1648
(M þ H)þ, found 353.1653.
6.99 (d, 1H, J ¼ 9.0 Hz). 13C NMR (150 MHz, DMSO-d6)
d 164.4, 158.4,
148.3, 135.2, 133.6, 130.8, 130.6, 130.2, 126.2, 125.9, 122.6, 119.7,
115.4. HRMS (ESI) calcd for C13H9ClN3O4 306.0276 (M þ H)þ, found
306.0274.
4.1.4. (S)-5-Chloro-2-hydroxy-N-(1-phenylethyl)benzamide (9)
Compound 9 was prepared in 39% yield by a procedure similar
to that used to prepare compound 8. The title compound was
obtained as a white solid (mp 124e125 ꢁC). HPLC purity 97.1%
4.1.8. N-(1,1-Dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2,6-diphenyl-
isonicotinamide (19)
(tR ¼ 19.75 min). 1H NMR (600 MHz, CDCl3)
d
12.22 (s, 1H), 7.28e
Compound 19 was prepared in 50% yield by a procedure similar
to that used to prepare compound 4. The title compound was
obtained as a pale yellow solid (mp 235e236 ꢁC). HPLC purity 97.2%
7.48 (m, 7H), 6.93 (d, 1H, J ¼ 9.0 Hz), 6.55 (d, 1H, J ¼ 6.6 Hz),
5.29e5.34 (m, 1H), 1.64 (d, 3H, J ¼ 7.2 Hz). 13C NMR (150 MHz,
CDCl3)
d
168.2, 160.4, 142.3, 134.3, 129.1, 128.0, 126.3, 125.1, 123.4,
(tR ¼ 22.05 min). 1H NMR (600 MHz, CDCl3)
d 8.76 (s, 1H), 8.34 (d,
1H, J ¼ 6.6 Hz), 8.23 (d, 4H, J ¼ 7.2 Hz), 8.14 (s, 2H) 7.93 (s, 1H), 7.41e
120.3, 115.3, 49.5, 21.7. HRMS (ESI) calcd for C15H15ClNO2 276.0786
(M þ H)þ, found 276.0790.
7.55 (m, 6H), 7.40 (d, 1H, J ¼ 8.4 Hz), 7.13e7.15 (m, 1H), 6.26e6.28
(m, 1H). 13C NMR (150 MHz, DMSO-d6)
d 164.7, 156.6, 143.9, 141.1,
4.1.5. 3-(6-Bromopyridin-2-yl)-2-cyano-N-(1,1-dioxo-1H-1l6
benzo[b]thiophen-6-yl)acrylamide (10)
-
138.1,137.1, 132.8, 130.4, 129.7,129.0, 126.9,126.6,126.3,124.6,116.7,
112.7. HRMS (ESI) calcd for C26H19N2O3S 439.1111 (M þ H)þ, found
439.1116.
To a solution of 4 (100 mg, 0.40 mmol) and 6-bromo-pyridine-2-
carbaldehyde (112 mg, 0.60 mmol) in EtOH (5 mL) was added
piperidine (3 mg, 0.04 mmol) at 0 ꢁC. The mixture was stirred at
90 ꢁC for 0.5 h. A yellow suspension formed during the reaction. The
solid was filtered and washed with H2O. The desired product
(120 mg, 72%) was obtained as a yellow solid (mp 219e220 ꢁC).
HPLC purity 96.0% (tR ¼ 16.57 min). 1H NMR (600 MHz, DMSO-d6)
4.1.9. Acridine-9-carboxylic acid (1,1-dioxo-1H-1l
6-benzo[b]
thiophen-6-yl)amide (20)
Compound 20 was prepared in 39% yield by a procedure similar
to that used to prepare compound 4. The title compound was
obtained as a yellow solid (mp 237e238 ꢁC). HPLC purity 99.4%
d
10.94 (s, 1H), 8.24 (s, 1H), 8.14 (s, 1H), 7.98 (t, 1H, J ¼ 7.8 Hz), 7.91e
(tR ¼ 14.48 min). 1H NMR (600 MHz, DMSO-d6)
d 8.71 (d, 1H,
7.93 (m, 2H), 7.84 (d, 1H, J ¼ 7.8 Hz), 7.62 (t, 2H, J ¼ 7.2 Hz), 7.34 (d,
J ¼ 8.4 Hz), 8.32 (d, 2H, J ¼ 8.4 Hz), 8.08e8.13 (m, 5 H), 7.97 (t, 2H,
1H, J ¼ 7.2 Hz). 13C NMR (150 MHz, DMSO-d6)
d 160.7, 150.8, 147.2,
J ¼ 7.8 Hz), 7.82 (t, 1H, J ¼ 8.4 Hz), 7.68 (t, 2H, J ¼ 7.8 Hz). 13C NMR
141.3, 140.8, 140.6, 137.1, 132.7, 130.9, 130.5, 126.7, 126.6, 126.6,
124.8, 114.6, 112.8, 110.8. HRMS (ESI) calcd for C17H11BrN3O3S
415.9699 (M þ H)þ, found 415.9703.
(150 MHz, DMSO-d6) d 163.4, 147.9, 136.1, 133.9, 132.9, 131.1, 129.5,
128.0, 128.0, 125.2, 121.7, 116.5, 115.5. HRMS (ESI) calcd for
C22H15N2O3S 387.0798 (M þ H)þ, found 387.0802.
4.1.6. 5-Chloro-N-(1,1-dioxo-1H-1
l
6-benzo[b]thiophen-6-yl)-2-
4.1.10. N-(1,1-Dioxo-1H-1l
6-benzo[b]thiophen-6-yl)
hydroxybenzamide (12)
isonicotinamide (21)
A solution of 5-chloro-2-hydroxybenzoic acid (2.0 g, 11 mmol)
Compound 21 was prepared in 56% yield by a procedure similar
to that used to prepare compound 4. The title compound was
and 4 mL of SOCl2 in 4 mL of toluene was stirred at 110 ꢁC

506
H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
obtained as a pale yellow solid (mp 251e252 ꢁC). HPLC purity 97.0%
4.2.3. Transient transfection and dual luciferase reporter assays
MDA-MB-231 cells were seeded in 24-well plate at a density of
5 ꢂ 104 cells/well in RPMI-1640 medium containing 10% FBS and 1%
penicillinestreptomycin. Transient transfections were performed
4 h after plating, using the method described previously [40,51].
(tR ¼ 8.81 min). 1H NMR (600 MHz, DMSO-d6)
d 10.92 (s, 1H), 8.81e
8.82 (m, 2H), 8.27 (s, 1H), 8.00 (d, 1H, J ¼ 7.8 Hz), 7.88 (t, 2H,
J ¼ 2.4 Hz), 7.62 (t, 2H, J ¼ 6.6 Hz), 7.33 (d, 1H, J ¼ 6.6 Hz). 13C NMR
(150 MHz, DMSO-d6)
d 164.6, 150.4, 141.3, 141.1, 137.1, 132.8, 130.4,
126.5, 126.3, 124.5, 121.6, 112.6. HRMS (ESI) calcd for C14H11N2O3S
Total amount of DNA for transfections was 0.5 mg/well, including
287.0485 (M þ H)þ, found 287.0488.
pSTAT3-Luc (95%, obtained from Panomics, Cat# LR0077) and in-
ternal control vector renilla (5%, from Promega, Madison, WI, USA).
5 h after transfection, the cells were treated with compound 5 for
24 h, then reporter activity was evaluated using dual luciferase
reporter assay kit (Promega, Madison, WI, USA) on an OmegaÔ
Microplate Luminometer (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
with triplicates and results are representatives of at least 3 inde-
pendent experiments.
4.1.11. N-(1,1-Dioxo-1H-1l
6-benzo[b]thiophen-6-yl)-2-phenyl-
isonicotinamide (22)
Compound 22 was prepared in 46% yield by a procedure similar
to that used to prepare compound 4. The title compound was
obtained as a pale yellow solid (mp 285e286 ꢁC). HPLC purity 98.0%
(tR ¼ 14.72 min). 1H NMR (600 MHz, DMSO-d6)
d 10.97 (s, 1H), 8.89
(d, 1H, J ¼ 4.8 Hz), 8.42 (s, 1H), 8.28 (s, 1H), 8.19 (d, 2H, J ¼ 7.8 Hz),
8.03e8.05 (m, 1H), 7.82 (d, 1H, J ¼ 4.8 Hz), 7.63 (d, 2H, J ¼ 7.8 Hz),
7.56 (t, 1H, J ¼ 7.2 Hz), 7.51 (t, 2H, J ¼ 7.2 Hz), 7.34 (d, 1H, J ¼ 6.6 Hz).
13C NMR (150 MHz, DMSO-d6)
d
164.6, 156.9, 150.4, 142.6, 141.1,
4.2.4. Western blot analysis
138.1, 137.1, 132.8, 130.4, 129.6, 128.9, 126.8, 126.5, 126.3, 124.6,
120.5, 118.0, 112.7. HRMS (ESI) calcd for C20H15N2O3S 363.0798
(M þ H)þ, found 363.0791.
Protein levels were determined by Western blot using the pre-
viously described methods [51]. Total cell lysates were prepared
from MDA-MB-231 cells. Protein concentrations were measured
using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA).
4.1.12. Quinoline-3-carboxylic acid (1,1-dioxo-1H-1
l
6-benzo[b]
Equal amounts of total cellular protein extract (40 mg) were
thiophen-6-yl)amide (23)
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-cleaved (1:2000, Epitomics, #1476-1), Cyclin D1
Compound 23 was prepared in 48% yield by a procedure similar
to that used to prepare compound 4. The title compound was
obtained as a pale yellow solid (mp 209e210 ꢁC). HPLC purity 98.8%
(tR ¼ 11.01 min). 1H NMR (600 MHz, acetone-d6)
d 10.29 (s, 1H), 9.03
(d, 1H, J ¼ 4.2 Hz), 8.42 (s, 1H), 8.34 (d, 1H, J ¼ 8.4 Hz), 8.14 (d, 1H,
J ¼ 8.4 Hz), 8.03e8.05 (m, 1H), 7.83e7.86 (m, 1H), 7.79 (d, 1H,
J ¼ 4.8 Hz), 7.68e7.70 (m, 1H), 7.63 (d, 1H, J ¼ 7.8 Hz), 7.57 (d, 1H,
J ¼ 7.2 Hz), 7.04 (d, 1H, J ¼ 7.2 Hz). 13C NMR (150 MHz, acetone-d6)
d
166.7, 151.0, 149.7, 142.4, 139.1, 133.1, 131.6, 130.7, 128.5, 127.7,
127.2, 126.3, 125.1, 124.8, 124.7, 120.0, 113.3, 113.2. HRMS (ESI) calcd
(1:10,000, Epitomics, #2261-1) and b-actin (1:10,000, Sigma, clone
for C18H13N2O3S 337.0641 (M þ H)þ, found 337.0646.
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. Biology
4.2.1. In vitro determination of effects of synthesized compounds on
cancer cell proliferation
4.2.5. In vivo antitumor activity assays
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 ꢂ 103 cells/well and treated with
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). Female
nude mice were obtained from MDACC and were used for
orthotopic tumor studies at 4e6 weeks of age. The mice were
maintained in a barrier unit with 12 h lightedark switch.
Freshly harvested MDA-MB-231 cells (2.5 ꢂ 106 cells per mouse,
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-sul-
fophenyl)-2H-tetrazolium) (MTS) in a CellTiter 96t 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 iMarkÔ
Microplate Absorbance Reader (BioRad, Hercules, CA). Each indi-
vidual compound was tested in quadruplicate wells for each
concentration.
resuspended in 100 mL PBS) were injected into the 3rd mammary
fat pad of the mice, and then randomly assigned into 2 groups (6e
7 mice per group). The mice were given 50 mg/kg compound 5 or
vehicle five days per week when the tumor volume reached
200 mm3. All drugs were dissolved in 50% DMSO with 50%
polyethylene 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 ꢂ L ꢂ W2, where
L ¼ length (mm) and W ¼ width (mm).
4.2.2. Molecular docking studies
Compound 5 was docked with the STAT3eSH2 domain using the
X-ray structure (PDB code: 1BG1) and AutoDock Vina 1.1.2. Water
molecules within the crystal structure were removed and polar
hydrogens 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 center. For all of the docking studies, a grid box size of
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
ꢀ
30 Å ꢂ 30 Å ꢂ 30 A, centered at coordinates 100.452 (x), 75.972 (y),
and 68.790 (z) of the PDB structure.
None.

H. Chen et al. / European Journal of Medicinal Chemistry 62 (2013) 498e507
507
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This work was supported by grants P50 CA097007,
P30DA028821, R21MH093844 (J.Z.) from the National Institute of
Health, R.A. Welch Foundation Chemistry and Biology Collaborative
Grant from Gulf Coast Consortia (GCC) for Chemical Genomics, John
Sealy Memorial Endowment Fund (JZ), the Duncan Family Institute
(DFI) Seed Funding Program, and startup fund from MD Anderson
Cancer Center (QS). We thank Dr. Tianzhi Wang at the NMR core
facility of UTMB for the NMR spectroscopy assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.023.
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