Synthesis and biological activity of obatoclax derivatives as novel and potent SHP-1 agonists

Article abstract of DOI:10.1016/j.ejmech.2012.08.024

Obatoclax is a linear oligopyrrole compound which antagonizes the antiapoptotic effects of the Bcl-2 family. Herein we describe the synthesis of obatoclax derivatives by replacement of the pyrrole and indole ring of obatoclax with thiophene, furan and thiazolidinedione. The in vitro cytotoxicity of the newly synthesized compounds is evaluated against hepatocellular carcinoma cells. Pyrrole and indole substituents of obatoclax analogues exhibited potent inhibition of cell growth. Among the tested compounds, 5d and 5e were active at 6.3 and 13.2 μM against PLC5 cells. Further assays confirmed a correlation between cell death, and p-STAT3 inhibition and SHP-1 activation by these analogues.

Full text of DOI:10.1016/j.ejmech.2012.08.024

European Journal of Medicinal Chemistry 56 (2012) 127e133
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of obatoclax derivatives as novel and potent
SHP-1 agonists
Jung-Chen Su , Kuen-Feng Chen b , Wei-Lin Chen , Chun-Yu Liu , Jui-Wen Huang , Wei-Tien Tai c,e,
a
,
1
,
c
,
1
a
a
,
d
g
b
,
f
h
a,
*
Pei-Jer Chen , InKi Kim , Chung-Wai Shiau
a
Institute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei, Taiwan
Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan
Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
Graduate Institute of Molecular Medicine, National Taiwan University College of, Medicine, Taipei, Taiwan
Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan
ASAN Institute for Life Science, ASAN Medical Center, Seoul, Republic of Korea
b
c
d
e
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 December 2011
Received in revised form
Obatoclax is a linear oligopyrrole compound which antagonizes the antiapoptotic effects of the Bcl-2
family. Herein we describe the synthesis of obatoclax derivatives by replacement of the pyrrole and
indole ring of obatoclax with thiophene, furan and thiazolidinedione. The in vitro cytotoxicity of the
newly synthesized compounds is evaluated against hepatocellular carcinoma cells. Pyrrole and indole
substituents of obatoclax analogues exhibited potent inhibition of cell growth. Among the tested
15 August 2012
Accepted 17 August 2012
Available online 27 August 2012
compounds, 5d and 5e were active at 6.3 and 13.2
mM against PLC5 cells. Further assays confirmed
a correlation between cell death, and p-STAT3 inhibition and SHP-1 activation by these analogues.
Keywords:
Obatoclax
STAT3
Ó 2012 Elsevier Masson SAS. All rights reserved.
SHP-1
Cytotoxicity
1. Introduction
drugs such as lapatinib (dual inhibitors of EGFR and HER-2/neu),
gifitinib (inhibitor of EGFR), bortezomib (inhibitor of proteasome),
Hepatocellular carcinoma is a major global health problem.
Advanced or recurrent HCC is resistant to chemotherapy and has
a poor prognosis. Despite increased attention being placed on
target therapy for HCC, sorafenib, a multi-kinase inhibitor, is
currently the only by FDA-approved anti-HCC targeted drug [1e3].
The discovery of new agents with tolerable toxicity may provide
new target therapies for the treatment of advanced HCC.
Obatoclax is an oligopyrrole compound that antagonizes the anti-
apoptotic function of Bcl-2, Bcl-xL and Mcl-1 [4]. Obatoclax binds to
the BH3 domain of Bcl-2 and disrupts its interaction with proapo-
ptotic proteins, such as Bax and Bak. In addition to Bcl-2 antagonism,
obatoclax has been reported to synergize with clinical drugs by
regulating several other biological and pharmacological effects [5e8].
For example, the combination of obatoclax and molecular targeted
and entinostat (inhibitor of histone deacetylase) showed synergistic
repression of cell growth in MCF-7 human breast cancer cells and the
tamoxifen-resistant variant (MTR-3). In addition, obatoclax also
synergizes chemotherapy agents such as cisplatin, Ara C and
topotecan in cancer cell lines [9,10].
For the current project, we started with typical pyrrole-based
Mcl-1 inhibitors containing an indole moiety. Aiming to expand
the structural diversity of the pyrrole substituents, we added
various pyrrole with indole ring. We found that Mcl-1 expression
level decrease and the p-STAT3, the upstream regulator of Mcl-1,
show the same reduction in HCC. Therefore, our hypothesis is
that indoleepyrrole ring can be a scaffold for STAT3 inhibitor. We
designed and synthesized novel oligopyrrole derivatives with the
aim of examining their ability to induce growth inhibition in HCC
cells. We further investigated the structure-activity relationship
(SAR) and found that these agents mediate apoptosis in association
with p-STAT3 downregulation in HCC cells. Western blot analyses
of downstream signaling in the human HCC cell-line PLC-5 are also
presented.
*
Corresponding author. Tel.: þ886 2 28267930; fax: þ886 2 28250883.
E-mail address: cwshiau@ym.edu.tw (C.-W. Shiau).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.024

128
J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
2
. Chemistry
Table 1
Chemical structure of compounds 5 and 8 and IC50 of growth inhibition in PLC-5
cells.
The synthesis of conjugated heterocyclic obatoclax derivatives
(Fig. 1) was initiated by converting the readily accessible 2-
carbaldehyde heterocycle (1) to the corresponding condensation
product (3) with 4-methoxy-1H-pyrrol-2(5H)-one (2). The
carbonyl groups of the corresponding products were then smoothly
converted to trifluoromethanesulfonate (4) by using triflate anhy-
dride. Use of the Suzuki coupling reaction permitted the position of
CPD
Ar
1
R
Cell death MTT assay (mM)
3 4
trifluoromethanesulfonate with the boron acid and Pd(PPh ) to
obtain compound 5 [11e13]. Using methodologies outlined in the
literature, the key intermediates 6 for the aryl-4-methoxy-5-
carboxaldehyde (7) were generated from the coupling reaction of
compound 2, N,N-diethylformamide and phosphoryl tribromide
Obatoclax
12.1 ꢀ 2.5
[14]. The condensation of aldehyde (7) and appropriate substrates
under the acidic conditions resulted in the final products (8).
8
8
a
>40
>40
3
. Biological activity
All the newly synthesized obatoclax analogues were assessed by
b
MTT assay for in vitro antitumor activity against HCC cancer cells
PCL5). The compound concentrations causing 50% cell growth
(
inhibition (IC50 values) are summarized in Tables 1e3. IC50 values
were determined by interpolation from doseeresponse curves.
A set of analogues 8ae8c (Table 1) were generated in which
a Boc-indole, thiophen and furan replaced the indole ring on the
left-hand side of the core moiety. In vitro analysis of the activity of
these compounds against PLC5 cells demonstrated significantly
8
c
28.4 ꢀ 1.2
diminished activity (IC50 > 40
mM) relative to that of obatoclax
5a
>40
>40
>40
(
IC50 ¼ 13.2 M). In addition, the removal of the dimethyl group of
m
pyrrole in obatoclax did not increase the potency of the resulting
compounds against PLC5 cells (compounds 5ae5c). The presence of
a bromide substituent in the indole ring, however, to led to an
increase in activity (5d). These results suggest that the left-hand
side indole ring of obatoclax is important for antitumor activity.
We next tested the hypothesis that the pyrrole on the right-hand
side of obatoclax might induce potent antitumor activity with the
heteroaryl ring. Subsequent analogues were thus synthesized to
obtain a SAR with the replacement of the right-hand side pyrrole of
obatoclax with thiophene, furan, and indole groups. These
compounds were screened for cell viability in PLC5 cells and the
5b
5c
5d
6.3 ꢀ 2.1
Fig. 1. General synthetic procedure for obatoclax derivatives.

J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
129
Table 2
Chemical structure of compounds 5 and 8 and IC50 of growth inhibition in PLC-5
cells.
CPD
Ar
1
R
Cell death MTT assay (mM)
5e
13.2 ꢀ 4.7
Fig. 2. ELISA analysis of the inhibitory effects of obatoclax derivatives versus obatoclax,
each at 10 mM, on IL-6 stimulated p-STAT3 in PLC5 cells.
5f
>40
5
g
d
25.9 ꢀ 3.4
15.9 ꢀ 3.6
on either the right-hand or the left-hand side. Analogues 8ee8i,
which contained indolin and thiazolidinedione on the right-hand
side of obatoclax were also tested for cell viability. Among these
analogues, compounds 8f and 8g showed a moderate effect but
with lower potency than obatoclax.
8
4
. Mechanistic study of obatoclax analogues in PLC5 cells
results are demonstrated at Table 2. The pyrrole ring of compound
e exhibited equal potency to the dimethyl pyrrole of obatoclax.
5
Originally, obatoclax was shown to inhibit the proteineprotein
Compound 5f, with thiophene introduced to replace dimethyl
pyrrole, led to a reduction in the antitumor effect. Interestingly, the
introduction of thiophene rings on both sides of the obatoclax
moiety resulted in better growth inhibition than a single thiophene
interactions of the Bcl-2 family. The analogues synthesized here
showed more potent cytotoxicity than obatoclax. In addition, the
expression level of Mcl-1 was repressed by the new compounds.
We, therefore, analyzed the upstream regulator STAT3 activity with
a p-STAT3 ELISA kit. The result showed that potency of p-STAT3
inhibition by the new analogues correlated with cell death (Fig. 2).
Therefore, we applied four agents, 5a, 5d, 5e and 8d, to PLC5 cells
and studied the effect of the upstream and downstream signals of
STAT3. As shown in Fig. 3, 5d, 5e and 8d resulted in a high degree
inhibition of p-STAT3 and subsequent repression of downstream
targets such as Mcl-1, survivin and cyclin D1. On the other hand, 5a,
Table 3
Chemical structure of compounds 8 and IC50 of growth inhibition in PLC-5 cells.
5
e
5a
10 (μM)
CPD
Ar
1
R
Cell death MTT assay (
m
M)
5
10
5
5d 8d
p-STAT3
Y705)
p-STAT3
(Y705)
(
8e
>40
STAT-3
SHP-1
STAT-3
SHP-1
8
f
35.4 ꢀ 4.6
27.5 ꢀ 4.2
Mcl-1
Survivin
Cyclin D1
β-actin
Mcl-1
Survivin
Cyclin D1
β-actin
8g
8h
>40
>40
8
i
Fig. 3. Obatoclax derivatives downregulate p-STAT3 and its downstream signaling
pathway. Cells were treated with 5a, 5d, 5e and 8d at 10 M for 12 h. p-STAT3, STAT3,
SHP-1, survivin, cyclin D1 and -actin were examined by western blot.
m
b

130
J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
which had no effect on cell toxicity, demonstrated no appreciable
effects on regulating p-STAT3 and its downstream targets. We then
further explored the expression level of SHP-1, a protein tyrosine
phosphatase that acts as negative regulator of STAT3. Compound
obatoclax analogues enhance SHP-1 activity may lead to new
targets for cancer therapy. These agents may provide structure
activity relationships for compound modification of SHP-1 agonists.
5
d, 5e and 8d significantly increased the level of SHP-1 in PLC5
6. Experimental section
cells, but 5a had no effect on SHP-1. We further confirmed that
down-regulated p-STAT3 in PLC5 cells resulted from phosphatase
activation (Fig. 4).
6.1. Materials
1
Proton nuclear magnetic resonance ( H NMR) spectra were
5
. Discussion
recorded on Bruker DPX400 (400 MHz) instruments. Chemical
shifts are reported as ppm. Peak multiplicities are expressed as
follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublet; ddd, doublet of doublet of doublets; dt, doublet of triplet;
brs, broad singlet; m, multiplet. Coupling constants (J values) are
given in hertz (Hz). Reaction progress was determined by thin layer
chromatography (TLC) analysis on silica gel 60 F254 plates (Merck).
Chromatographic purification was carried out on silica gel 60
(0.063e0.200 mm or 0.040e0.063 mm, Merck) basic silica gel.
Commercial reagents and solvents were used without additional
Highly phosphorylated STAT3 has been linked to cancer inci-
dence. Several factors have been reported to activate STAT3. For
example, IL-6 induces Jak2 phosphorylation through its receptor and
activates STAT3. In addition, growth factors, such as EGF and VEGF
are able to activate STAT3. STAT3 activity is also regulated through
negative feedback by SHP-1. The literature shows that loss-of-
function of SHP-1 leads to STAT3 activation contributing to tumor
formation [15e17]. In this study, we generated a series of obatoclax
analogues that exhibit anticancer activity against PLC5 cells. Our data
have several implications: (1) The mechanism of apoptosis induction
driven by obatoclax analogues occurred, at least in part, through
downregulation of p-STAT3. Obatoclax is known to induce apoptosis
through targeting the Bcl-2 family; however, in addition to new
analogues, 5d, 5e and 8d inhibited phosphorylation of STAT3; (2)
Compounds 5d, 5e and 8d acted as SHP-1 agonists which enhanced
SHP-1 phosphatase activity and further dephosphorylated p-STAT3.
At present, kinase and growth factor inhibitors are common strate-
gies for anticancer therapy whereas upregulation of negative regu-
lators of STAT3 have seldom been considered. The enhancement of
SHP-1 expression antagonizing the function of STAT3 may provide
a new direction for HCC treatment; (3) We prepared a set of oba-
toclax analogues via two synthetic pathways. These standardized
procedures used commercially available starting materials and
reagents to generate large amounts of obatoclax analogues that can
be used in future in vivo studies. Our current findings suggest that
the indole ring on the left-hand side of the obatoclax analogue is
important for biological activity. Replacement with thiophene and
furan resulted in loss of anticancer activity and SHP-1 expression. On
the other hand, dimethyl pyrrole and pyrrole on the right-hand side
of the obatoclax moiety exhibited greater activity than thiophene,
indolin and furan.
3
purification. Abbreviations are used as follows: CDCl , deuterated
chloroform; DMSO-d6, dimethyl sulfoxide-d6; EtOAc, ethyl acetate;
DMF, N,N-dimethylformamide; MeOH, methanol; THF, tetrahy-
drofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide; DCM,
dichloromethane. High resolution mass spectra were recorded on
a Finnigan MAT 95S mass spectrometer.
6.2. Chemical synthesis
6.2.1. General procedure for the synthesis of compound 5
2 3
PdCl (0.1 equiv, 59%) and PPh (0.45 equiv) were added to
a nitrogen passed solution of toluene (1 mL). The mixture became
ꢁ
bright yellow and was stirred at 70 C for 20 min under nitrogen.
3 4
The Pd(PPh ) in toluene suspension was transferred into a solution
of 1.0 equiv triflate (compound 4) and 1.2 equiv aryl boronic acid
(compound 5 or commercially available) in 10% water/dioxane
(5 mL) purged with nitrogen. One equiv solid sodium carbonate
ꢁ
was added. The reaction mixture was stirred at 100 C for 90 min,
then poured into 10 mL water and extracted with ethyl acetate
(20 mL) three times. The organic layer was collected, washed with
brine, dried over MgSO and concentrated. The residue was then
4
chromatographed by silica gel with the eluent ethyl acetate:hexane
(1:20 to 1:5). This procedure afforded the expected coupling
product in 81%e94% yield.
In conclusion, the obatoclax analogues synthesized in this study
exhibited anti-HCC through a novel mechanism that directly
enhanced SHP-1 expression level and consequently suppressed p-
STAT3. This indicates that targeting STAT3 is a promising strategy
for HCC and could be applied to other unregulated STAT3-driven
cancers. Further studies of the detailed mechanism by which
6.2.1.1. 2-((5-(furan-2-yl)-3-methoxy-2H-pyrrol-2-ylidene)methyl)-
1
1H-pyrrole (5a). H NMR (400 MHz, CDCl
3
):d
7.56 (d, J ¼ 2.0 Hz,
1H), 7.13 (d, J ¼ 2.4 Hz, 1H), 7.00 (d, J ¼ 3.2 Hz, 1H), 6.92 (s, 1H), 6.61
(d, J ¼ 3.6 Hz, 1H), 6.54 (dd, J ¼ 3.6 Hz, J ¼ 2.0 Hz, 1H), 6.26 (dd,
J ¼ 3.6 Hz, J ¼ 2.4 Hz, 1H), 5.96 (s, 1H), 3.88 (s, 3H); LCMS(ESI): m/z
þ
þ
2
2
41.2 (100, M þ H ); HRMS calculated for C14
H
12
N
2
O
2
(M þ H ):
5
4
3
2
1
00
00
00
00
00
0
þ
41.0972. Found 241.0964 (M þ H ).
6
.2.1.2. 2-((3-methoxy-5(-thiophen-2-yl)-2H-pyrrol-2-ylidene)
1
methyl)-1H-pyrrole (5b). H NMR (400 MHz, CDCl
J ¼ 3.6 Hz, 1H), 7.41 (d, J ¼ 4.8 Hz, 1H), 7.14 (d, J ¼ 2.0 Hz, 1H), 7.10
dd, J ¼ 5.2 Hz, J ¼ 4.0 Hz, 1H), 6.90 (s, 1H), 6.61 (d, J ¼ 3.6 Hz, 1H),
3
):d 7.50 (d,
(
6
13
.27 (t, J ¼ 3.6 Hz, J ¼ 2.4 Hz, 1H), 5.95 (s, 1H), 3.89 (s, 3H); C NMR
(100 MHz, CDCl 58.4, 95.2, 110.9, 117.4, 118.8, 126.1 127.7, 127.9,
3
) d
1
28.1, 128.4, 130.7, 140.3, 161.5, 168.6; LCMS(ESI): m/z 257.2 (100,
þ
þ
M þ H ); HRMS calculated for C14
H
12
N
2
OS (M þ H ): 257.0743.
Found 257.0731.
DMSO Ob
5a
5e
5d
8d
6
.2.1.3. tert-butyl-2-(2-((1H-pyrrol-2-yl)methylene)-3-methoxy-2H-
Fig. 4. SHP-1 activity in obatoclax, 5a, 5d, 5e and 8d-treated PLC5 cells. Cells were
treated with obatoclax, 5a, 5d, 5e and 8d at 10 mM for 6 h and then cell lysates were
1
pyrrol-5-yl)-1H-indol-1-carboxylate (5c).
H
NMR (400 MHz,
analyzed by phosphatase activity assay.
3
CDCl ):d
8.12 (d, J ¼ 8.0 Hz, 1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.36 (t,

J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
131
J ¼ 8.0 Hz, 1H), 7.25 (t, J ¼ 7.6 Hz, 1H), 7.08 (d, J ¼ 2.4 Hz, 1H), 7.01 (s,
H), 6.98 (s, 1H), 6.64 (d, J ¼ 3.6 Hz, 1H), 6.26 (dd, J ¼ 3.6 Hz,
6.2.2.2. 3-Methoxy-5-(thiophen-2-yl)-1H-pyrrole-2-carbaldehyde
1
1
(7b). H NMR (400 MHz, CDCl
3
):
d
9.46 (s, 1H), 7.32 (d, J ¼ 5.2 Hz,
13
J ¼ 2.4 Hz, 1H), 5.82 (s, 1H), 3.88 (s, 3H), 1.51 (s, 9H); C NMR
100 MHz, CDCl 27.5 (3C), 58.1, 83.7, 98.0, 110.7, 112.0, 114.4,
18.4, 118.7 121.0, 122.8, 125.2, 126.0, 128.6, 130.3, 136.0, 138.11,
1H), 7.30 (d, J ¼ 3.6 Hz, 1H), 7.07 (dd, J ¼ 5.2 Hz, J ¼ 3.6 Hz, 1H), 6.06
(
3
)
d
(s, 1H), 3.89 (s, 3H).
1
þ
141.5, 150.0, 161.1, 167.1; LCMS(ESI): m/z 388.4 (100, M ꢂ H ). HRMS
6.2.2.3. 5-(1H-Indol-2-yl)-3-methoxy-1H-pyrrole-2-carbaldehyde
þ
1
calculated for C23
23
H N
3
O
3
(M þ H ): 389.1739. Found: 389.1744.
(7c). H NMR (400 MHz, DMSO-d
6
):
d
11.84 (bs, 1H), 11.43 (bs, 1H),
9
.42 (s, 1H), 7.53 (d, J ¼ 8.0 Hz, 1H), 7.38 (d, J ¼ 8.0 Hz, 1H), 7.12 (t,
6
.2.1.4. 2-(2-((1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-
J ¼ 7.6 Hz, 1H), 7.10 (s, 1H), 7.00 (t, J ¼ 7.6 Hz, 1H), 6.55 (d, J ¼ 2.8 Hz,
1
yl)-5-bromo-1H-indol (5d). H NMR (400 MHz, CDCl
3
):
d
7.65 (d,
1H), 3.88 (s, 3H).
J ¼ 1.6 Hz, 1H), 7.15 (dd, J ¼ 8.8 Hz, J ¼ 2.0 Hz, 1H), 7.07 (s, 1H), 6.88
(
s, 1H), 6.83 (d, J ¼ 8.4 Hz, 1H), 6.73 (s, 1H), 6.60 (d, J ¼ 3.6 Hz, 1H),
6.2.2.4. tert-Butyl-2-(5-formyl-4-methoxy-1H-pyrrol-2-yl)-1H-
1
3
1
6
CDCl
1
.22 (s, 1H), 6.12 (t, J ¼ 3.2 Hz, 1H), 4.04 (s, 3H); C NMR (100 MHz,
59.0, 96.2, 105.6, 111.6, 113.0, 113.4, 119.2, 121.4 123.8, 127.1
27.7, 129.6, 130.3 134.4, 136.5, 140.2, 160.8, 169.5; LCMS(ESI): m/z
indole-1-carboxylate (7d). H NMR (400 MHz, CDCl
3
):
d
9.98 (bs,
3
)
d
1H), 9.57 (s, 1H), 8.08 (d, J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 8.0 Hz, 1H), 7.33
(t, J ¼ 7.6 Hz, 1H), 7.25 (t, J ¼ 7.6 Hz, 1H), 6.89 (s, 1H), 6.13 (d,
J ¼ 2.8 Hz, 1H), 3.90 (s, 3H), 1.59 (s, 9H).
þ
þ
3
68.2 (100, M þ H ); HRMS calculated for C18
H14BrN
3
O (M þ H ):
3
68.0393. Found 368.0345.
6.2.3. General procedure for the synthesis of compound 8ae8b
6
.2.1.5. 2-(2-((1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-
1.2 equiv 2,4-dimethyl pyrrole was added to a solution of 1.0
1
yl)-1H-indol (5e). H NMR (400 MHz, CDCl
H), 7.10 (t, J ¼ 7.2 Hz, 1H), 7.04e6.97 (m, 4H), 6.78 (s, 1H), 6.58 (d,
J ¼ 3.6 Hz, 1H), 6.23 (s, 1H), 6.12 (dd, J ¼ 3.6 Hz, J ¼ 2.4 Hz, 1H), 4.03
s, 3H); 13C NMR (100 MHz, CDCl
58.6, 95.8, 106.4, 111.1, 111.4,
18.0, 120.0, 120.4, 121.3, 124.1, 127.0, 128.4, 129.5, 133.0, 137.8, 140.1,
3
):
d
7.54 (d, J ¼ 8.0 Hz,
equiv aldehydes (compound 7) in 4 mL methanol. The mixture was
stirred and 1N methanolic HCl was added. The reaction was stirred
continuously overnight at room temperature under nitrogen.
Methanol was removed under rotary evaporation. The residue was
then purified by a flash column. This procedure afforded the ex-
pected coupling product in 81% to quantitative yield.
1
(
1
3
) d
þ
1
60.7, 168.8; LCMS(ESI): m/z 290.3 (100, M þ H ); HRMS calculated
þ
for C18
15
H N
3
O (M þ H ): 290.1288. Found 290.1281.
6
.2.3.1. 2-((3,5-Dimethyl-2H-pyrrol-2-ylidene)methyl)-5-(furan-2-
1
6
1
.2.1.6. 2-(3-methoxy-2-(thiophen-2-ylmethylene)-2H-pyrrol-5-yl)-
H-indol (5f). 1H NMR (400 MHz, CDCl ): 9.32 (bs, 1H), 7.64 (d,
4
yl)-3-methoxy-1H-pyrrole (8a). H NMR (400 MHz, MeOD-d ):
3
d
d
7.67 (d, J ¼ 1.6 Hz,1H), 7.03 (d, J ¼ 3.2 Hz,1H), 6.92 (s,1H), 6.60 (dd,
J ¼ 8.0 Hz,1H), 7.61 (d, J ¼ 5.2 Hz,1H), 7.47 (d, J ¼ 4.0 Hz,1H), 7.43 (d,
J ¼ 3.2 Hz, J ¼ 1.6 Hz, 1H), 6.10 (s, 1H), 5.89 (s, 1H), 3.90 (s, 3H), 2.35
þ
J ¼ 8.0 Hz, 1H), 7.26 (t, J ¼ 7.2 Hz, 1H), 7.23 (s, 1H), 7.12e7.07 (m, 2H),
(s, 3H), 2.19 (s, 3H); LCMS(ESI): m/z 269.6 (100, M þ H ); HRMS
þ
7.00 (s, 1H), 6.05 (s, 1H), 3.94 (s, 3H); LCMS(ESI): m/z 307.3 (100,
calculated for C16
(M þ H ).
H
16
N
2
O
2
(M þ H ): 269.1285. Found 269.1284
þ
þ
þ
M þ H ); HRMS calculated for C18
14
H N
2
OS (M þ H ): 307.0900.
Found: 307.0895.
6
.2.3.2. 2-((3,5-Dimethyl-2H-pyrrol-2-ylidene)methyl)-3-methoxy-
1
6
.2.1.7. 3-ethoxy-5-(thiophen-2-yl)-2-(thiophen-2-ylmethylene)-2H-
3
5-(thiophen-2-yl)-1H-pyrrole (8b). H NMR (400 MHz, CDCl ):d
1
pyrrole (5g). H NMR (400 MHz, CDCl
.55 (d, J ¼ 4.0 Hz,1H), 7.47 (d, J ¼ 5.2 Hz,1H), 7.45 (d, J ¼ 3.6 Hz,1H),
.20 (s, 1H), 7.11 (dd, J ¼ 5.2 Hz, J ¼ 4.0 Hz, 1H), 7.05 (dd, J ¼ 5.2 Hz,
3
):
d
7.59 (d, J ¼ 5.2 Hz, 1H),
7.44 (d, J ¼ 3.6 Hz, 1H), 7.35 (d, J ¼ 5.2 Hz, 1H), 7.08 (dd, J ¼ 5.2 Hz,
7
7
J ¼ 3.6 Hz, 1H), 6.88 (s, 1H), 5.96 (s, 1H), 5.83 (s, 1H), 3.89 (s, 3H),
þ
2.35 (s, 3H), 2.20 (s, 3H); LCMS(ESI): m/z 285.3 (100, M þ H );
13
þ
J ¼ 3.6 Hz, 1H), 5.96 (s, 1H), 3.89 (s, 3H); C NMR (100 MHz, CDCl
3
)
HRMS calculated for C16
285.1063.
H
16
N
2
OS (M þ H ): 285.1056. Found:
d
58.5, 96.5, 120.8, 127.2, 128.0, 128.6, 129.7, 133.8, 134.3, 139.2,
þ
1
40.6, 145.6, 164.4, 169.1; LCMS(ESI): m/z 274.2(100, M þ H ).
þ
HRMS calculated for C14
H11NOS
2
(M þ H ): 273.0282. Found:
6.2.4. General procedure for the synthesis of compound obatoclax,
8c and 8d
2
73.0281.
2,4-dimethyl pyrrole or indolyl-pyrrole (1.2 equiv) was added to
6
.2.2. Preparation of compound 7
a solution of 1.0 equiv aldehydes (7d) in 4 mL methanol. The
mixture was stirred and 1N methanolic TFA was added. The reac-
tion was stirred continuously overnight at room temperature under
nitrogen. Methanol was removed under rotary evaporation. The
residue was then purified by a flash column. This procedure affor-
ded the expected coupling product in 70% to quantitative yield.
PdCl (0.1 equiv, 59%) and PPh (0.45 equiv) was added to
2
3
a degassed solution of toluene (1 mL). The mixture became bright
yellow and was stirred at 70 C for 20 min under nitrogen. The
freshly prepared Pd(PPh ) in toluene suspension was transferred
3 4
into a solution of 1.0 equiv bromo pyrrole enamine (compound 6)
and 1.5 equiv aryl boronic acid in 10% water/dioxane (5 mL) purged
with nitrogen. Three-equiv sodium carbonate was added. The
ꢁ
6.2.4.1. 2-(5-((3,5-Dimethyl-2H-pyrrol-2-ylidene)methyl)-4-
ꢁ
1
reaction mixture was stirred at 100 C for 90 min and then poured
methoxy-1H-pyrrol-2-yl)-1H-indole
(400 MHz, MeOD-d ):
(obatoclax).
H
NMR
onto 50 mL ice-water. The pH of the mixture was adjusted to 7.0
using 2N HCl (5 mL) and stirred for 20 min. The slurry was extracted
with ethyl acetate (20 mL) twice. The precipitate was recovered by
filtration, washed with water and collected from acetone. The solid
was recrystallized by 1:2 chloroform:ether (5 mL). This procedure
afforded the expected coupling product in 71% to quantitative yield.
4
d
7.56 (d, J ¼ 8.0 Hz, 1H), 7.38 (d, J ¼ 8.0 Hz,
1H), 7.15 (t, J ¼ 7.6 Hz, 1H), 7.00 (t, J ¼ 7.6 Hz, 1H), 6.96 (s, 1H), 6.90
(s, 1H), 6.25 (s, 1H), 5.87 (s, 1H), 3.95 (s, 3H), 2.38 (s, 3H), 2.20 (s,
þ
3H); LCMS(ESI): m/z 318.2 (100, M þ H ).
6.2.4.2. tert-Butyl-2-(5-((3,5-Dimethyl-2H-pyrrol-2-ylidene)methyl)
1
4
-methoxy-1H-pyrrol-2-yl)-1H-indole-1-carboxylate (8c). H NMR
6
.2.2.1. 5-(Furan-2-yl)-3-methoxy-1H-pyrrole-2-carbaldehyde (7a).
(400 MHz, CDCl
3
):
d
8.10 (d, J ¼ 8.0 Hz, 1H), 7.56 (d, J ¼ 8.0 Hz, 1H),
1
H NMR (400 MHz, CDCl
J ¼ 1.6 Hz,1H), 6.82 (d, J ¼ 3.6 Hz,1H), 6.47 (dd, J ¼ 3.6 Hz, J ¼ 1.6 Hz,
H), 6.10 (d, J ¼ 2.4 Hz, 1H), 3.89 (s, 3H).
3
):
d
10.03 (bs, 1H), 9.50 (s, 1H), 7.43 (d,
7.32 (t, J ¼ 7.6 Hz, 1H), 7.22 (t, J ¼ 7.6 Hz, 1H), 6.95 (s, 1H), 6.93 (s,
1H), 5.83 (s, 1H), 5.82 (s, 1H), 3.88 (s, 3H), 2.30 (s, 3H), 2.21 (s, 3H),
1.47 (s, 9H); DIP(EI-70ev): m/z 417.3 (53, M), 360.1 (89), 317.2 (100),
1

132
J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
2
86.2 (84); HRMS calculated for C25
H
27
N
3
O
3
417.2052. Found:
dichloromethane was stirred under nitrogen, followed by addition
of 0.1 equiv para-toluenesulfonic acid. The solution was stirred for
417.2048 (M).
2
h at room temperature under nitrogen. The mixture was diluted
with ethyl acetate, washed with three 20 mL portions each of
saturated Na CO , and brined. Purification by flash chromatography
6
.2.4.3. 2-(5-((5-(1H-Pyrrol-2-yl)-2H-pyrrol-2-ylidene)methyl)-4-
1
methoxy-1H-pyrrol-2-yl)-1H-indole (8d).
H
NMR (400 MHz,
2
3
MeOD-d ):
4
d
8.81 (bs, 1H), 7.63 (s, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.44
using an eluent ethyl acetate:hexane 1:20 to 1:5 yielded dithiane.
(
1
3
d, J ¼ 8.4 Hz, 1H), 7.40e7.38 (m, 1H), 7.25 (m, 2H), 7.18 (t, J ¼ 7.6 Hz,
The yield was 31%.
1
H), 7.13 (s, 1H), 7.05 (m, 2H), 6.63 (d, J ¼ 3.6 Hz, 1H), 6.35 (m, 2H),
3
H NMR (400 MHz, CDCl ):d 8.33 (bs, 1H), 8.20 (bs, 1H), 7.55 (d,
þ
.99 (s, 3H); LCMS(ESI): m/z 405.3 (100, M þ H ); HRMS calculated
J ¼ 7.6 Hz, 1H), 7.32 (d, J ¼ 8.0 Hz, 1H), 7.14 (t, J ¼ 8.0 Hz, 1H), 7.08 (t,
þ
for C26
20
H N
4
O (M þ H ): 405.1710. Found: 405.1723.
J ¼ 7.6 Hz,1H), 6.52 (d, J ¼ 2.0 Hz,1H), 6.11 (d, J ¼ 2.8 Hz,1H), 5.55 (s,
1
H), 3.81 (s, 3H), 3.12e3.05 (t, J ¼ 13.2 Hz, 2H), 2.92e2.87 (m, 2H),
þ
6.2.5. General procedure for the synthesis of compound 8ee8h
0.91e0.82 (m, 2H); LCMS(ESI): m/z 329.5 (100, M ꢂ H ). HRMS
þ
Piperidine (0.15 equiv) and benzoic acid (0.13 equiv) was added to
calculated for C25
H25NOS
2
(M þ H ): 273.0282. Found: 273.0281.
a mixture of 1.0 equiv aldehydes (compound 7c or 7d) and 1.0 equiv
oxindole or thiazolidinedione in 3 mL toluene. The mixture was
reacted in a closed system in a microwave reactor. The reaction
6.3. Biological assays
ꢁ
condition was set at 50 W, 150 C holding for 25 min, and stirring at
6.3.1. Cell culture
high speed. After cooling to room temperature, the reaction mixture
was poured into 10 mL water to quench the reaction. The organic
layer was extracted with 20 mL ethyl acetate, brined, dried over
PLC5 cells were purchased from ATCC and maintained in DMEM
supplemented with 10% FBS, 100 units/mL penicillin G, 100
streptomycin sulfate and 25
m
g/mL
ꢁ
m
g/mL amphotericin B in a 37
C
4
anhydrous MgSO , filtered and concentrated under rotary evapora-
2
humidified incubator in an atmosphere of 5% CO in air.
tion. The orange solid was recrystallized with 1:10 ethyl acetate:-
hexane (2 mL). The residue was then purified by a flash column. This
procedure afforded the expected coupling product in 70%e85% yield.
6.3.2. Western blot
PLC5 cells were treated with compounds 5a, 5e and 8d at 10
mM
for 12 h. Cell lysates were analyzed by western blot. p-STAT3,
STAT3, SHP-1, Mcl-1, Survivin, Cyclin D1, PARP and actin antibodies
were purchased from Cell Signaling.
6
.2.5.1. tert-Butyl-2-(4-methoxy-5-((2-oxoindolin-3-ylidene)
1
methyl)-1H-pyrrol-2-yl)-1H-indole-1-carboxylate (8e).
H
NMR
13.40 (bs 1H), 9.01 (bs, 1H), 8.22 (d, J ¼ 8.0 Hz,
H), 7.57 (s, 1H), 7.56 (d, J ¼ 6.8 Hz, 1H), 7.49 (d, J ¼ 6.8 Hz, 1H), 7.36
(
3
400 MHz, CDCl ):d
1
6.3.3. SHP-1 phosphatase activity
Ò
(
1
(
1
t, J ¼ 8.0 Hz, 1H), 7.26 (t, J ¼ 8.0 Hz, 1H), 7.07e7.00 (m, 2H), 6.87 (s,
H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.14 (d, J ¼ 2.8 Hz, 1H), 3.95 (s, 3H), 1.47
s, 9H); 13C NMR (100 MHz, CDCl
27.8, 58.1, 84.2, 96.1, 109.3,
11.5, 111.9, 115.6, 117.6, 118.1, 120.8, 121.2, 121.5, 123.2, 125.1, 125.5,
26.3, 128.9, 129.9 131.7, 137.1, 137.8, 149.1, 155.2, 169.8; LCMS(ESI):
A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R-
22067) was used for SHP-1 activity assay (Molecular Probes,
Carlsbad, CA). The method was as described previously [18].
3
) d
1
6.3.4. STAT3 activity assay
PLC5 cells were pretreated with the indicated compounds in
10 mM for 24 h and then stimulated with IL-6 (10 ng/mL) for 30 min.
The PathScan Phospho-Stat3 (Tyr705) Sandwich ELISA Kit was
purchased from Cell Signaling, Danvers, MA.
þ
m/z 478.0 (100, M þ Na ); HRMS calculated for C27
H
25
N
3
O
4
þ
(
M ꢂ H ): 454.1772. Found: 454.1779.
6
.2.5.2. 3-((5-(1H-Indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)methy-
1
lene)indolin-2-one (8f). H NMR (400 MHz, DMSO-d
H), 11.72 (bs, 1H), 10.84 (bs, 1H), 7.59 (d, J ¼ 7.6 Hz, 2H), 7.49 (s, 1H),
.41 (d, J ¼ 8.0 Hz, 1H), 7.14 (t, J ¼ 8.0 Hz, 1H), 7.09 (t, J ¼ 7.6 Hz, 1H),
6
):d 13.94 (bs
1
7
Acknowledgments
7
.03 (t, J ¼ 7.6 Hz, 1H), 6.97 (t, J ¼ 7.6 Hz, 1H), 6.91 (d, J ¼ 7.6 Hz, 1H),
We thank the National Science Council, Taiwan (NSC98-2320-B-
010-005-My3 and NSC-100-2325-B-010-007) and National Yang
Ming University, Taiwan for financial support. C.W.S. also thanks
the National Research Institute of Chinese Medicine for NMR
support.
13
6
.79 (d, J ¼ 1.6 Hz, 1H), 6.65 (d, J ¼ 2.8 Hz, 1H), 3.95 (s, 3H); C NMR
57.5, 92.9, 97.9, 108.8, 110.7, 111.5, 116.9, 117.1,
19.0, 119.2, 119.7, 120.4, 121.8, 125.0, 125.0, 127.7, 128.8, 129.5, 136.7,
(
100 MHz, DMSO) d
1
þ
1
37.5, 154.9, 168.8; LCMS(ESI): m/z 354.4 (100, M ꢂ H ); HRMS
calculated for C22
17 3 2
H N O
(M þ H): 356.1394. Found: 356.1410.
References
6
.2.5.3. 5-((5-(1H-Indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)methy-
1
[1] L.R. Roberts, Sorafenib in liver cancerejust the beginning, N. Engl. J. Med. 359
lene)thiazolidine-2,4-dione (8g). H NMR (400 MHz, DMSO-d
6
):d
(2008) 420e422.
7
.56 (d, J ¼ 7.2 Hz, 1H), 7.39 (d, J ¼ 8.0 Hz, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H),
[
2] J.M. Llovet, S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.F. Blanc, A.C. de Oli-
veira, A. Santoro, J.L. Raoul, A. Forner, M. Schwartz, C. Porta, S. Zeuzem,
L. Bolondi, T.F. Greten, P.R. Galle, J.F. Seitz, I. Borbath, D. Haussinger,
T. Giannaris, M. Shan, M. Moscovici, D. Voliotis, J. Bruix, Sorafenib in advanced
hepatocellular carcinoma, N. Engl. J. Med. 359 (2008) 378e390.
7.01 (t, J ¼ 7.2 Hz, 2H), 6.72 (s, 1H), 6.59 (d, J ¼ 2.4 Hz, 1H), 3.87 (s,
þ
3
H); LCMS(ESI): m/z 338.5 (100, M ꢂ H ).
6
.2.5.4. tert-Butyl-2-(5-((2,4-dioxothiazolidin-5-ylidene)methyl)-4-
[3] S. Wilhelm, C. Carter, M. Lynch, T. Lowinger, J. Dumas, R.A. Smith, B. Schwartz,
R. Simantov, S. Kelley, Discovery and development of sorafenib: a multikinase
inhibitor for treating cancer, Nat. Rev. Drug Discov. 5 (2006) 835e844.
1
methoxy-1H-pyrrol-2-yl)-1H-indole-1-carboxylate (8h).
400 MHz, CDCl ):
H
NMR
(
3
d
12.15 (bs, 1H), 8.12 (d, J ¼ 8.8 Hz, 1H), 7.55 (d,
[4] M.H. Kang, C.P. Reynolds, Bcl-2 inhibitors: targeting mitochondrial apoptotic
J ¼ 7.6 Hz, 1H), 7.32 (t, J ¼ 8.4 Hz, 1H), 7.22 (t, J ¼ 7.6 Hz, 1H), 6.92 (s,
pathways in cancer therapy, Clin. Cancer Res. 15 (2009) 1126e1132.
1
H), 6.83 (s, 1H), 6.11 (d, J ¼ 2.4 Hz, 1H), 3.88 (s, 3H), 1.53 (s, 9H);
[5] L.M. Witters, A. Witkoski, M.D. Planas-Silva, M. Berger, J. Viallet, A. Lipton,
Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-
HER-2/neu and a Bcl-2 inhibitor, Oncol. Rep. 17 (2007) 465e469.
þ
LCMS(ESI): m/z 438.0 (100, M ꢂ H ); HRMS calculated for
C
H
22 21
N
3
O
5
S (M þ H): 438.1129. Found: 438.1137.
[
6] A.P. Martin, C. Mitchell, M. Rahmani, K.P. Nephew, S. Grant, P. Dent, Inhibition
of MCL-1 enhances lapatinib toxicity and overcomes lapatinib resistance via
BAK-dependent autophagy, Cancer Biol. Ther. 8 (2009) 2084e2096.
6
.2.5.5. 2-(5-(1,3-dithian-2-yl)-4-methoxy-1H-pyrrol-2-yl)-1H-
[
7] P. Perez-Galan, G. Roue, M. Lopez-Guerra, M. Nguyen, N. Villamor,
E. Montserrat, G.C. Shore, E. Campo, D. Colomer, BCL-2 phosphorylation
modulates sensitivity to the BH3 mimetic GX15-070 (obatoclax) and reduces
indole (8i). A mixture of 1.0 equiv indolyl methoxypyrrole aldehyde
compound 7c) and 1.0 equiv propanedithiol in mL
(
2

J.-C. Su et al. / European Journal of Medicinal Chemistry 56 (2012) 127e133
133
its synergistic interaction with bortezomib in chronic lymphocytic leukemia
cells, Leukemia 22 (2008) 1712e1720.
[13] E. Rioux, X. Billot, K. Dairi, G. Gonzalez, J.-F. Lavallee, S. Tripathy, A. Babineau,
S. Bailly, H. Chan, G. Chen, G. Gagnon, A. Jang, A. Khadir, R. Marcellus,
D. Paquette, A. Roulston, B. St-Denis, N. Steenaart, M. Watson, Z. Zhang,
D. Goulet, P. Beauparlant, G. Shore, G. Attardo, SAR study on aryl and het-
eroaryl bipyrrole inhibitors of Bcl antiapoptotic proteins with potent anti-
tumor activity in vivo (IUPAC-ICOS-16 special issue), J. Mex. Chem. Soc. 50
(2006) 209.
[
8] A. Jona, N. Khaskhely, D. Buglio, J.A. Shafer, E. Derenzini, C.M. Bollard,
L.J. Medeiros, A. Illes, Y. Ji, A. Younes, The histone deacetylase inhibitor enti-
nostat (SNDX-275) induces apoptosis in Hodgkin lymphoma cells and syn-
ergizes with Bcl-2 family inhibitors, Exp. Hematol. 39 (2011) 1007e1017.
9] J. Li, J. Viallet, E.B. Haura, A small molecule pan-Bcl-2 family inhibitor, GX15-
[
0
70, induces apoptosis and enhances cisplatin-induced apoptosis in non-small
[14] K. Daïri, Y. Yao, M. Faley, S. Tripathy, E. Rioux, X. Billot, D. Rabouin,
G. Gonzalez, J.-F. Lavallee, G. Attardo, A scalable process for the synthesis of
the Bcl inhibitor obatoclax, Org. Process. Res. Dev. 11 (2007) 1051e1054.
[15] H. Tassidis, Z. Culig, A.G. Wingren, P. Harkonen, Role of the protein tyrosine
phosphatase SHP-1 in Interleukin-6 regulation of prostate cancer cells, Pros-
tate 70 (2010) 1491e1500.
cell lung cancer cells, Cancer Chemother. Pharmacol. 61 (2008) 525e534.
[
10] P.K. Paik, C.M. Rudin, A. Brown, N.A. Rizvi, N. Takebe, W. Travis, L. James,
M.S. Ginsberg, R. Juergens, S. Markus, L. Tyson, S. Subzwari, M.G. Kris,
L.M. Krug, A phase I study of obatoclax mesylate, a Bcl-2 antagonist, plus
topotecan in solid tumor malignancies, Cancer Chemother. Pharmacol. 66
(
2010) 1079e1085.
[16] Q. Zhang, H.Y. Wang, M. Marzec, P.N. Raghunath, T. Nagasawa, M.A. Wasik,
STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1
tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes,
Proc. Natl. Acad. Sci. U S A 102 (2005) 6948e6953.
[17] H. Tassidis, L.J. Brokken, K. Jirstrom, R. Ehrnstrom, F. Ponten, D. Ulmert,
A. Bjartell, P. Harkonen, A.G. Wingren, Immunohistochemical detection of
tyrosine phosphatase SHP-1 predicts outcome after radical prostatectomy for
localized prostate cancer, Int. J. Cancer 126 (2010) 2296e2307.
[
[
11] G. Tang, C.Y. Yang, Z. Nikolovska-Coleska, J. Guo, S. Qiu, R. Wang, W. Gao,
G. Wang, J. Stuckey, K. Krajewski, S. Jiang, P.P. Roller, S. Wang, Pyrogallol-
based molecules as potent inhibitors of the antiapoptotic Bcl-2 proteins,
J. Med. Chem. 50 (2007) 1723e1726.
12] M. Nguyen, R.C. Marcellus, A. Roulston, M. Watson, L. Serfass, S.R. Murthy
Madiraju, D. Goulet, J. Viallet, L. Belec, X. Billot, S. Acoca, E. Purisima,
A. Wiegmans, L. Cluse, R.W. Johnstone, P. Beauparlant, G.C. Shore, Small
molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-
mediated resistance to apoptosis, Proc. Natl. Acad. Sci. U S A 104 (2007)
[18] K.F. Chen, W.T. Tai, T.H. Liu, H.P. Huang, Y.C. Lin, C.W. Shiau, P.K. Li, P.J. Chen,
A.L. Cheng, Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma
cells through the inhibition of STAT3, Clin. Cancer Res. 16 (2010) 5189e5199.
1
9512e19517.