Novel imidazopyridine suppresses STAT3 activation by targeting SHP-1

Article abstract of DOI:10.1080/14756366.2018.1497019

The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthesized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16 on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.

Full text of DOI:10.1080/14756366.2018.1497019

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
018, VOL. 33, NO. 1, 1248–1255
2
https://doi.org/10.1080/14756366.2018.1497019
Novel imidazopyridine suppresses STAT3 activation by targeting SHP-1
a,bꢀ
c
a
a,d
ꢀ
Jung-Chen Su , Chuan-Hsun Chang , Szu-Hsien Wu and Chung-Wai Shiau
a
b
Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan; Faculty of Pharmacy, National Yang-Ming University,
Taipei, Taiwan; Chairman of the Surgical Department, Cheng Hsin General Hospital, Taipei, Taiwan; Department of Chemistry, Chung-Yuan
Christian University, Chungli, Taiwan
c
d
ABSTRACT
ARTICLE HISTORY
The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances
tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers
this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthe-
sized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of
phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of
hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16
on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.
Received 18 January 2018
Revised 1 June 2018
Accepted 26 June 2018
KEYWORDS
STAT3; SHP-1; HCC;
imidazopyridine
1
6
17
Introduction
bortezomib and rituximab in various cancers, and also com-
1
8
19
bined with Olaparib and Lapatinib in preclinical studies.
Previously, we designed and synthesized obatoclax derivatives by
the replacement of the indole ring with thiophene and furan, result-
ing in a new chemical entity possibly suitable for the development of
anticancer agents . Moreover, these derivatives induced cell apop-
tosis through a novel mechanism by targeting the SHP-1/STAT3 path-
way, a different mechanism from obatoclax to Mcl-1, Bcl-2, and Bcl-xL
Recent cancer drug discovery has focused on molecular target
therapy to reduce nonspecific cytotoxicity in patients. Targeting
oncogenes or activating tumour suppressor genes with small mole-
cules is a new approach for cancer treatment. Signal transducer and
activator of transcription 3 (STAT3) is a transcription factor that
plays a central role in tumour cell proliferation, survival, invasion,
20
1
and immunosuppression . The STAT3 pathway is activated by sev-
21
inhibition . Therefore, our hypothesis is that pyrrole-benzimidazole
eral receptors, including those for the interleukin-6 (IL-6) family
could be a new pharmacophore for SHP-1 enhancement which
represses STAT3 activation and induces cell apoptosis (Figure 1). In
this study, we designed a short synthetic scheme for pyrrole-benzimi-
dazole and synthesized various pyrrole-benzimidazole-based com-
pounds aiming to find compounds that enhance SHP-1 activity
2
3,4
cytokines , G-protein-coupled receptors (GPCRs) , and Toll-like
5
receptors (TLRs) . Once the signal is activated, STAT3 is phosphory-
lated at the tyrosine 705 residue, resulting in automatic dimerization
in the cytosol. The dimeric STAT3 translocates from the cytosol to
the nucleus for the transcription of its target genes, such as Mcl-1,
(
Scheme 1). The structure–activity relationship of these compounds
6–8
Bcl-xl, and VEGF-A and further regulates cell proliferation, apop-
tosis, and migration. This phosphorylation activation of STAT3 is
negatively regulated by protein tyrosine phosphatases, such as
SHP-1, SHP-2, and PTP1B by targeting the phosphor group at tyro-
was investigated using hepatocellular carcinoma (HCC) cancer cells.
We further studied the SHP-1 activity and p-STAT-3 inhibition by
treating HCC cells with the effective compounds.
9
,10
sine 705 . Therefore, the enhancement of protein tyrosine phos-
phatases is another approach for target therapy.
Materials and methods
Potentiating the effect of traditional chemotherapy agents and
targeting chemotherapy-induced signalling cascades is a practical
Chemistry
1
13
approach for combating resistant cancer cells. In gastric cancer Proton nuclear magnetic resonance ( H-NMR and C-NMR) spec-
cells, inhibition of STAT3 phosphorylation re-sensitized the chemo- tra were recorded on Bruker Avance III (400 MHz, Bruker, Billerica,
11
therapeutic agent treatment .
MA) instruments. Reaction progress was determined by thin layer
Recently, not only prodigiosin, a natural product extracted from chromatography (TLC) analysis on a silica gel 60 F254 plate
Serratia marcescens, but also obatoclax, a synthetic oligopyrrole (Merck, Darmstadt, Germany). Chromatographic purification was
scaffold derived from prodigiosin, have been shown to regulate bio- carried out on silica gel columns 60 (0.063–0.200 mm or
12–14
logical and pharmacological effects (Figure 1)
. The combination 0.040–0.063 mm; Merck), basic silica gel. Commercial reagents and
of DNA damaging doxorubicin and prodigiosin showed significant solvents were purchased from Aldrich (Darmstadt, Germany) and
growth inhibition in human small-cell lung doxorubicin-resistant Acros Chemical Corporation (Bridgewater, NJ) without additional
1
5
cancer cells . Obatoclax, currently in the clinical trial stage, has also purification. High-resolution mass spectra were recorded on a
been applied to synergize with targeted agents such as FINNIGAN MAT 95S mass spectrometer.
CONTACT Chung-Wai Shiau
cwshiau@ym.edu.tw
jjjaannee@hotmail.com
Institute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Li-Nong Street,
Faculty of Pharmacy, National Yang-Ming University, No. 155, Sec. 2, Li-Nong Street, Taipei, Taiwan.
Taipei, Taiwan; Jung-Chen Su
ꢀ
These authors contributed equally to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1249
Figure 1. The structure of obatoclax, prodigiosin, SC-2001, and the core structure of pyrrole-benzimidazole.
Tert-butyl-2-(5-(1H-benzo[d]imidazol-2-yl)-4-methoxy-1H-pyrrol-
-yl)-1H-indole-1-carboxylate (6)
2
Tert-butyl-2–(5-(1H-benzo[d]imidazol-2-yl)-4-methoxy-1H-pyrrol-2-
1
yl)-1H-indol. H NMR (400 MHz, CDCl
J ¼ 8.4 Hz, 1H), 7.52 (d, J ¼ 7.2 Hz, 1H), 7.29 (t, J ¼ 7.2 Hz, 1H), 7.04
d, J ¼ 7.6 Hz, 1H), 6.96 (t, J ¼ 7.2 Hz, 1H), 6.83 (s, 1H), 6.73 (d,
J ¼ 7.2 Hz, 2H), 6.19 (s, 1H), 3.88 (s, 3H), 1.59 (s, 10H).
3
): d ¼ 8.42 (s, 1H), 8.08 (d,
(
2
–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
benzo[d] imidazole (7)
1
H NMR (400 MHz, MeOH-d ): d ¼ 7.63 (d, J ¼ 1.6 Hz, 1H), 7.52 (dd,
4
J ¼ 6.0, 3.2 Hz, 2H), 7.25 (d, J ¼ 8.4 Hz, 1H), 7.1 5–7.18 (m, 3H), 6.67
1
3
4
(s, 1H), 6.49 (s, 1H), 4.02 (s, 3H) ppm. C NMR (100 MHz, MeOH-d ):
d ¼ 151.4, 146.5, 139.5, 136.8, 133.3, 131.9, 127.1, 125.1, 123.0, 122.8,
1
15.3, 115.1, 114.8, 114.1, 113.5, 113.1, 108.2, 97.6, 94.3, 58.5 ppm.
2
–(5-(1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
benzo[d]imidazole (8)
1
H NMR (400 MHz, MeOH-d
4
): d ¼ 7.5 1–7.54 (m, 3H), 7.36 (d,
J ¼ 8.2 Hz, 1H), 7.1 7–7.19 (m, 2H), 7.10 (t, J ¼ 8.2 Hz, 1H), 7.01 (t,
13
J ¼ 7.6 Hz, 1H), 6.73 (s, 1H), 6.51 (s, 1H), 4.05 (s, 3H) ppm. C NMR
100 MHz, MeOH-d ): d ¼ 151.9, 146.8, 138.4, 132.0, 130.3, 128.2,
(
1
4
23.1, 122.8, 121.0, 120.7, 114.9, 111.8, 107.8, 98.4, 94.1, 58.7 ppm.
16 4
HRMS calculated for C20H N O (M þ H): 329.1402, Found: 329.1403.
Scheme 1. General synthetic procedure for pyrrole-benzimidazole.
General procedure for the synthesis of compound 5
2
–(4-methoxy-1H, 1'H-[2, 2'-bipyrrol]-5-yl)-1H-benzo[d]imidazole (9)
1
H NMR (400 MHz, MeOH-d ): d ¼ 7.4 7 ꢂ 7.49 (m, 2H), 7.1 3 ꢂ 7.15
4
3
Compound 3 (1.0 equiv.) and NaHSO (2.0 equiv.) were mixed in
(
6
m, 2H), 6.78 (dd, J ¼ 3.2, 1.6 Hz, 1H), 6.42 (dd, J ¼ 3.2, 1.6 Hz, 1H),
ethanol solution. After the mixtures were heated with a 50 Watt
.23 (s, 1H), 6.16 (t, J ¼ 3.2 Hz, 1H), 4.01 (s, 3H) ppm. HRMS calcu-
ꢁ
microwave at 90 C for 10 min, the solution was evaporated with
lated for C16
H
14
N
4
O (M þ H): 279.1246, Found: 279.1248.
rotavapor. The crude mixtures were added with a solution of
dimethylformamide and 1.2 equiv benzene-diamine. The reaction
ꢁ
was stirred in 50 Watt microwave reactor at 125 C for 30 min,
6
3
1
-bromo-2-(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-
H-imidazo[4,5-b]pyridine (10)
poured into 20 ml water and extracted with ethyl acetate (30 ml) 3
times. The organic layer was collected, washed with brine, dried
with MgSO
4
. The concentrated crude product was purified by sil-
H NMR (400 MHz, MeOH-d
4
): d ¼ 8.30 (s, 1H), 7.98 (s, 1H), 7.67 (d,
ica gel with elute (ratio: ethyl acetate/hexane ¼1/6 to 1/2). The J ¼ 2.0 Hz, 1H), 7.30 (d, J ¼ 8.8 Hz, 1H), 7.20 (dd, J ¼ 8.8, 2.0 Hz, 1H),
1
3
yield of coupling product in this procedure was 4–24%.
6.77 (s, 1H), 6.54 (s, 1H), 4.07 (s, 3H) ppm. C NMR (100 MHz,

1
250
J.-C. SU ET AL.
MeOH-d
4
): d ¼ 136.8, 132.8, 131.8, 129.0, 125.4, 123.1, 113.5, 113.2, 2–(5-(1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-3H-imidazo[4,5-
9
8.4, 96.7, 94.2, 58.5 ppm.
b]pyridine (17)
1
H NMR (400 MHz, MeOH-d ): d ¼ 8.23 (d, J ¼ 4.4 Hz, 1H), 7.84 (d,
4
J ¼ 8.0 Hz, 1H), 7.53 (d, J ¼ 7.6 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.17
(dd, J ¼ 8.0, 4.8 Hz, 1H), 7.11 (t, J ¼ 7.6 Hz, 1H), 7.01 (t, J ¼ 7.6 Hz,
2
–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-3H-
imidazo[4,5-b]pyridine (11)
1
3
1
H), 6.77 (s, 1H), 6.50 (s, 1H), 4.06 (s, 3H) ppm. C NMR (100 MHz,
1
H NMR (400 MHz, MeOH-d ): d ¼ 8.24 (d, J ¼ 3.6 Hz, 1H), 7.85 (d,
4
MeOH-d
4
): d ¼ 153.2, 143.5, 138.5, 131.7, 130.3, 129.5, 122.9, 121.1,
J ¼ 8.0 Hz, 1H), 7.65 (s, 1H), 7.26 (d, J ¼ 8.8 Hz, 1H), 7.1 6–7.19 (m,
1
20.7, 118.2, 111.8, 107.4, 98.9, 93.9, 58.7 ppm. HRMS calculated
13
2
H), 6.72 (s, 1H), 6.50 (s, 1H), 4.07 (s, 1H) ppm. C NMR (100 MHz,
for C19H
15
5
N O (M þ H): 330.1355, Found: 330.1344.
MeOH-d ): d ¼ 153.1, 143.7, 137.1, 133.2, 132.1, 128.7, 125.6, 123.4,
4
1
20.1, 118.3, 113.8, 113.4, 107.9, 98.4, 94.4, 58.8 ppm. HRMS calcu-
lated for C19 14BrN
H
5
O (M þ H): 408.0460, Found: 408.0459.
2
–(5-(1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-5-fluoro-1H-
benzo[d] imidazole (18)
1
2
1
–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-5-fluoro-
H-benzo[d]imidazole (12)
H NMR (400 MHz, MeOH-d ): d ¼ 7.50 (d, J¼ 8.0 Hz, 1H), 7.43 (dd,
4
J¼ 8.4, 4.8 Hz, 1H), 7.34 (d, J¼ 8.0 Hz, 1H), 7.20 (dd, J ¼ 9.2, 2.4 Hz, 1H),
7.08 (td, J¼ 7.6, 1.2 Hz, 1H), 6.99 (td, J¼ 7.6, 1.2 Hz, 1H), 6.91 (td, J¼ 9.2,
1
H NMR (400 MHz, MeOH-d
4
): d ¼ 7.60 (d, J ¼ 1.6 Hz, 1H), 7.43 (dd,
13
2
.4 Hz, 1H), 6.70 (s, 1H), 6.45 (s, 1H), 4.00 (s, 3H) ppm. C NMR
J ¼ 8.8, 4.8 Hz, 1H), 7.22 (d, J ¼ 8.8 Hz, 1H), 7.19 (dd, J ¼ 9.2, 2.4 Hz,
(
1
100 MHz, MeOD-d ): d ¼ 161.9, 159.5, 152.1, 138.4, 131.9, 130.3, 128.4,
1
H), 7.14 (dd, J ¼ 8.8, 1.6 Hz, 1H), 6.91 (td, J ¼ 9.2, 2.4 Hz, 1H), 6.63
4
13
22.8, 121.0, 120.7, 111.8, 110.7, 110.5, 98.6, 94.0, 58.7 ppm. HRMS cal-
4
(s, 1H), 6.43 (s, 1H), 3.98 (s, 3H) ppm. C NMR (100 MHz, MeOH-d ):
d ¼ 161.9, 159.5, 151.8, 137.0, 133.5, 133.4, 132.1, 132.0, 127.5, 125.4, culated for C20
23.2, 113.7, 113.3, 110.7, 110.5, 108.3, 97.9, 94.5, 58.7 ppm. HRMS
calculated for C20 14BrFN O (M–H): 423.0257, Found: 423.0243.
4
H15FN O (M–H): 345.1152, Found: 345.1143.
1
H
4
3
–((2-(5-(1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
benzo[d]imidazol-6-yl)oxy)aniline (19)
2
–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
1
H NMR (400 MHz, MeOH-d ): d ¼ 7.52 (d, J ¼ 7.6 Hz, 1H), 7.41 (d,
4
benzo[d] imidazole-5-carbonitrile (13)
J ¼ 8.4 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.15 (d, J ¼ 2.4 Hz, 1H), 7.10
1
H NMR (400 MHz, MeOH-d ): d ¼ 7.86 (s, 1H), 7.66 (d, J ¼ 2.0 Hz, (t, J ¼ 7.2 Hz, 1H), 6.9 9–7.07 (m, 2H), 6.90 (dd, J ¼ 8.4, 2.4 Hz, 1H),
4
1
H), 7.64 (d, J ¼ 8.0 Hz, 1H), 7.49 (d, J ¼ 8.0 Hz, 1H), 7.28 (d, 6.73 (s, 1H), 6.51 (s, 1H), 6.43 (dd, J ¼ 8.0, 2.0 Hz, 1H), 6.36 (t,
J ¼ 8.4 Hz, 1H), 7.19 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.75 (s, 1H), 6.54 (s, J ¼ 2.4 Hz, 1H), 6.30 (dd, J ¼ 8.0, 2.4 Hz, 1H), 4.05 (s, 3H) ppm.
1
4
H), 4.07 (s, 3H) ppm. HRMS calculated for C21
30.0303, Found: 430.0326
5
H14BrN O (M-H):
6
–bromo-2-(4-methoxy-1H, 1'H-[2, 2'-bipyrrol]-5-yl)-3H-
imidazo[4,5-b] pyridine (20)
3
–((2–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
1
H NMR (400 MHz, MeOH-d
4
): d ¼ 8.24 (s, 1H), 7.92 (d, J ¼ 2.0 Hz,
benzo[d] imidazol-6-yl)oxy)aniline (14)
1
H), 6.82 (dd, J ¼ 2.8, 1.2 Hz, 1H), 6.50 (dd, J ¼ 3.6, 1.2 Hz, 1H), 6.26
1
H NMR (400 MHz, MeOH-d
4
): d ¼ 7.65 (d, J ¼ 1.6 Hz, 1H), 7.48 (d,
13
(
(
s, 1H), 6.18 (dd, J ¼ 3.6, 2.8 Hz, 1H), 4.03 (s, 3H) ppm. C NMR
J¼ 8.8 Hz, 1H), 7.27 (d, J¼ 8.4 Hz, 1H), 7.18 (dd, J¼ 8.4, 1.6 Hz, 1H),
100 MHz, MeOH-d ): d ¼ 154.2, 146.3, 131.6, 129.1, 125.7, 120.1,
4
7
1
.15 (d, J¼ 2.0 Hz, 1H), 7.03 (t, J¼ 8.0 Hz, 1H), 6.90 (dd, J¼ 8.8, 2.0 Hz,
H), 6.69 (s, 1H), 6.52 (s, 1H), 6.43 (dd, J¼ 8.0, 2.0 Hz, 1H), 6.36 (t,
1
12.9, 110.1, 106.8, 106.5, 105.4, 91.8, 89.8, 81.5, 58.7 ppm. HRMS
calculated for C H BrN O (M–H): 356.0147, Found: 356.0152.
1
5
12
5
J¼ 2.4 Hz, 1H), 6.30 (dd, J¼ 8.0, 2.4 Hz, 1H), 4.04 (s, 1H) ppm.
2
5
1
–(4-methoxy-1H, 1'H-[2, 2'-bipyrrol]-5-yl)-1H-benzo[d]imidazole-
-carbonitrile (21)
3
–((2–(5-(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-1H-
benzo[d] imidazol-6-yl)oxy)benzonitrile (15)
H NMR (400 MHz, MeOH-d ): d ¼ 7.80 (s, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H),
1
4
H NMR (400 MHz, MeOH-d
4
): d ¼ 7.65 (d, J ¼ 2 Hz, 1H), 7.57 (d,
7
.45 (dd, J¼ 8.4, 1.6 Hz, 1H), 6.81 (dd, J¼ 3.2, 1.6 Hz, 1H), 6.47 (dd,
J ¼ 8.4 Hz, 1H), 7.49 (t, J ¼ 7.6 Hz, 1H), 7.40 (d, J ¼ 7.6 Hz, 1H), 7.2
J¼ 3.2, 1.6 Hz, 1H), 6.25 (s, 1H), 6.17 (t, J¼ 3.2 Hz, 1H), 4.03 (s, 3H) ppm.
6
6
ꢂ 7.28 (m, 3H), 7.24 (d, J ¼ 2.4 Hz, 1H), 7.18 (dd, J ¼ 8.8, 2 Hz, 1H),
.94 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.71 (s, 1H), 6.53 (s, 1H), 4.05 (s, 3H)
1
3
ppm. C NMR (100 MHz, MeOH-d ): d ¼ 152.2, 151.9, 137.0, 133.5,
4
4
(E)-N -((5–(5-bromo-1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-
1
1
32.1, 127.0, 125.4, 123.3, 123.1, 121.3, 120.5, 119.2, 116.0, 114.5,
13.7, 113.4, 98.0, 94.6, 58.8 ppm.
yl)methylene) pyrimidine-4,5-diamine (22)
Compound 3 (1.0 equiv.), pyrimidine-4,5-diamine (1.2 equiv) and
NaHSO (2.0 equiv.) were mixed in the solution of ethanol. After
the mixtures were heated with a 50 Watt microwave at 100 C for
3
2
–(5-(1H-indol-2-yl)-3-methoxy-1H-pyrrol-2-yl)-6-bromo-3H-
ꢁ
imidazo[4,5-b]pyridine (16)
3
0 min, the solution was evaporated with rotavapor. The crude
1
mixtures were poured into 20 ml water and extracted with ethyl
acetate (30 ml) 3 times. The organic layer was collected, washed
with brine, dried with MgSO . The concentrated crude product
4
H NMR (400 MHz, MeOH-d ): d ¼ 8.25 (s, 1H), 7.91 (s, 1H), 7.51 (d,
4
J ¼ 8.0 Hz, 1H), 7.34 (d, J ¼ 8.0 Hz, 1H), 7.10 (t, J ¼ 8.0 Hz, 1H), 7.02
1
3
(t, J ¼ 8.0 Hz, 1H), 6.76 (s, 1H), 6.47 (s, 1H), 4.04 (s, 3H) ppm.
C
NMR (100 MHz, MeOH-d ): d ¼ 153.7, 144.2, 138.5, 131.6, 130.3, was purified by silica gel with elute (ratio:ethyl acetate/hexane
4
1
1
30.1, 123.1, 121.2, 120.8, 111.9, 107.2, 99.3, 94.0, 58.6 ppm. HRMS ¼1/1). Yield: 8.2%. H NMR (400 MHz, MeOH-d
4
): d ¼ 8.44 (d,
calculated for C19
H14BrN
5
O (M-H): 406.0303, Found: 406.0302.
J ¼ 1.6 Hz, 1H), 8.17 (d, J ¼ 1.6 Hz, 1H), 7.95 (d, J ¼ 1.6 Hz, 1H), 7.67

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1251
Table 1. Chemical structure of compounds 6–9 and IC50 of cell death in
PLC5 cells.
Table 2. Chemical structure of compounds 7, and 10–15 and IC50 of cell death
in PLC5 cells.
(
6
s, 1H), 7.29 (d, J ¼ 8.4 Hz, 1H), 7.21 (d, J ¼ 8.8 Hz, 1H), 6.86 (s, 1H),
.45 (d, J ¼ 1.6 Hz, 1H), 3.94 (s, 3H) ppm.
(
Z)-3-(2-((1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-
yl)pyridine (23)
Pd(PP ) (0.1 equiv) was added to a solution of 1.0 equiv (Z)-2-((1H-pyr-
3
4
rol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl trifluoromethanesulfonate
0.086 g, 0.267 mmole), Na CO (0.028 g, 0.264 mmole), and 1.2 equiv
(
2
3
pyridin-3-ylboronic acid (0.039 g, 0.317 mmole) in 10% water/dioxane
ꢁ
(
5 ml) purged with nitrogen. After the mixtures were heated at 100 C bromide (MTT) assay in five replicates. Cells were seeded and incu-
for 90 min, the reaction was quenched with water. The mixture was bated in 96-well, flat-bottomed plates for 24 h and were exposed to
extracted with ethyl acetate (30 ml) 3 times. The organic layer was col- various concentrations (2.5, 5, 10, 20, 40 lM) of test agents dissolved
lected, washed with brine, and dried with MgSO . The concentrated in DMSO (final concentration, 0.1%) in media. Controls received
4
crude product was purified by silica gel with elute (ratio: ethyl acetate/ DMSO vehicle at a concentration equal to that in drug-treated cells.
1
hexane ¼1/1). Yield: 35.6%. H NMR (400 MHz, CDCl
3
): d ¼ 9.18 (d, For the MTT assay, 10 lL of MTT dye was directly added to the wells.
J¼ 1.6 Hz, 1H), 8.61 (dd, J¼ 4.8, 1.6 Hz, 1H), 8.26 (d, J¼ 8.0 Hz, 1H), 7.36 After 2 h, media were removed and then the cells were lysed with
(
dd, J¼ 8.0, 4.8 Hz, 1H), 7.24 (s, 1H), 7.18 (s, 1H), 7.01 (s, 1H), 6.68 (d, 100 lL of DMSO. The absorbance at 570 nm and 630 nm (back-
J¼ 3.6 Hz, 1H), 6.29 (dd, J¼ 3.6, 2.8 Hz, 1H), 6.05 (s, 1H), 3.91 (s, 3H) ppm. ground) were read with a microplate reader.
Biological assays
Western blot
Cell culture
Lysates of PLC5 treated with compounds at the indicated concen-
Hepatoma cells PLC/PRF/5 (PLC5) were purchased from ATCC trations for 24 h were analysed by western blot. p-STAT3 (#9145,
(Manassas, VA) (CRL-8024) and maintained in DMEM supple- 1:1000), STAT3 (#4904, 1:1000), Mcl-1 (#4572, 1:1000), survivin
mented with 10% FBS, 100 units/mL penicillin G, 100 mg/mL (#2803, 1:1000), JAK2 (#3230, 1:1000), GAPDH (#8884, 1:5000), and
ꢁ
streptomycin sulphate and 25 mg/mL amphotericin B in a 37 C actin (#4967, 1:5000) antibodies were purchased from Cell
humidified incubator in an atmosphere of 5% CO
2
in air.
Signaling (Danvers, MA). SHP-1 (ab32559, 1:1000) and cyclin D1
(
(
ab24249, 1:1000) antibodies were purchased from Abcam
Cambridge, MA). PTP1B (2066–1, 1:2000) antibody was purchased
Cell viability assay
from Epitomics (Cambridge, MA). p-JAK2(Tyr1007/1008) (44–42 G,
The effect of individual test agents on cell viability was assessed by 1:1000) antibody was purchased from ThermoFisher Scientific
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Waltham, MA).

1
252
J.-C. SU ET AL.
Table 3. Chemical structure of compounds 8, and 16–19 and IC50 of cell death Table 5. Chemical structure of compounds 9, 11, 16, 17, 22, and 23 and IC50 of
in PLC5 cells.
cell death in PLC5 cells.
Table 4. Chemical structure of compounds 9, 20, and 21 and IC50 of cell death
in PLC5 cells.
STAT3 activity assay
PLC5 cells were treated with the indicated compounds in 10 lM
for 24 h and then analysed by the PathScan Phospho-Stat3
(Tyr705) Sandwich ELISA Kit (Cell Signaling)
Gene knockdown using siRNA
Smart-pool small interfering RNAs (siRNAs), including the con-
trol (D-001810–10), SHP-1, were purchased from Dharmacon
(
Chicago, IL). The knockdown procedure was as described
2
3
previously
.
SHP-1 phosphatase activity
Statistical analysis
A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R-22067) Data are expressed as mean ± SD. All statistical analyses were per-
was used for SHP-1 activity assay (Molecular Probes, Carlsbad, CA). formed using SPSS for Windows version 12.0 software (SPSS Inc,
22
The method was as described previously .
Chicago, IL).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1253
Figure 2. Effects of compounds on p-STAT3 inhibition. (A) PLC5 cells were exposed to the indicated compounds at a dose of 10 lM for 24 h and cell lysates were
assayed by p-STAT3 ELISA kit. (B) PLC5 cells were exposed to the indicated compounds (cpd 16, 17, 22, 9, 23, 11) at 10 lM for 24 h and cell lysates were assayed by
western blot.
Figure 3. Effects of compounds on SHP-1 activity. PLC5 cells were exposed to
cpd 11, 16, 17, and SC-2001 at 10 lM for 24h and cell lysates were assayed by
SHP-1 phosphatase activity kit.
Results and discussion
Chemistry
The synthesis of the pyrrole-benzimidazole-based compounds was
initiated with 4-methoxy-1H-pyrrol-2-one and dimethyl amide in relative downstream target in PLC5 cells. (A) Cells were exposed to cpd 16 and
the presence of phosphoryl tribromide to generate two functional SC-2001 at the indicated doses for 24 h. Cell lysates were assayed by western
groups, enamine, and bromine in the pyrrole ring 2. The position
of the brominated pyrrole ring was soon conjugated with aro-
Figure 4. Effects of cpd 16 on protein levels of SHP-1, p-STAT3, and p-STAT3
blot. (B) PLC5 cells were transfected, respectively, with control siRNA or SHP-1
siRNA for 48h. After transfection, the cells were treated w/wo cpd 16 (10 lM) for
2
4 h. The protein levels were analysed by western blot assay.
matic boron acid in the presence of Pd(PPh ) by using the Suzuki
3
4
coupling reaction and enamine was hydrolysed to the carbonyl
group in acid conditions, resulting in the key intermediate 3. The
condensation reaction of the carbonyl group of 3 and various aro-
matic diamines under the different conditions resulted in the final
imine 4 or pyrrole-imidazopyridine 5 products.
vitro test of cell death activity with these compounds against
PLC5 cells showed differentiated activity with various substituents.
Among these substituents, the indole ring 8 exhibited slightly bet-
ter activity than bromo-indole 7 and pyrrole 9 (Table 1). In add-
ition, the Boc-indole substituent 6 showed no activity against cell
growth compared with the indole ring, suggesting the nitrogen
atom plays an important role in the potency of cell growth.
Biological evaluation
All the new pyrrole-benzimidazole derivatives were analysed by We next tested our hypothesis that the presence of various func-
MTT assay for growth inhibition against HCC cancer cells. The vari- tional groups on benzimidazole might exhibit anti-cancer cell
ous substituents attached to the scaffold are summarized in growth effects. A series of substituents, such as bromo, fluoro,
Tables 1–5. The growth inhibition of each of the compounds is cyano, and phenyl-cyano groups were thus introduced to the ben-
shown with an IC50 value which was calculated by interpolation zene and pyridine ring of benzimidazole to obtain a structure–ac-
from the dose–response curve in MTT assay.
tivity relationship. These agents were tested in an MTT assay with
A set of pyrrole-benzimidazole derivatives was generated in PLC5 cells and the inhibition results are shown in Table 2. The
which an indole, bromoindole, pyrrole, and Boc-indole connected pyridine ring of compound 11 demonstrated better activity than
to the methoxyl-pyrrole ring in the centre of the core moiety. In the benzene ring. In addition, Compound 13 with a cyano group

1
254
J.-C. SU ET AL.
26–28
connected to the benzene ring led to a reduction in inhibition of eliminating the phosphor group from active p-STAT3
. These
cell growth. Interestingly, the introduction of aniline 14 or phenyl- phosphatase agonists provide a new direction for future clinical
cyano 15 to benzimidazole resulted in no activity against PLC5 approaches instead of only kinase inhibition. Our discovery also
cells, implying that the linear electron-withdrawing group and provides evidence that imidazopyridine derivatives mediate both
phenyl substituent impede the anticancer activity. Analogues SHP-1 activation and also increase SHP-1 expression. Importantly,
17–19 that contained indole and imidazopyridine on both sides the reduction of cell survival genes, such as cyclin D1, survivin,
of pyrrole were also assayed for the inhibition of cell growth. As and Mcl-1 regulated by SHP-1 were consistent with cell survival in
shown in Table 3, compounds 16 and 17 showed equal potency the treatment of imidazopyridine derivatives.
to compound 11. Compound 20, with imidazopyridine as a sub-
In summary, this study showed that the structure of indole–-
stituent to replace benzimidazole, led to a significant increase in pyrrole-imidazopyridine is a novel anticancer agent. We also dem-
anticancer activity. (Tables 4 and 5).
onstrated that these compounds can enhance SHP-1 expression
and activation and consequently reduced the level of p-STAT3 in
HCC cells. This indicates that the strategy of SHP-1 activation is a
possible drug target in cancer patients who have no or low SHP-1
activity. In addition, these agents might be applied along with
therapeutic cancer drugs to increase efficacy and reduce toxicity.
Mechanistic study of pyrrole-imidazopyridine in PLC 5 cells
Previously, we have shown that indole-pyrrole reduces STAT3
phosphorylation in western blot assay and further represses can-
2
0
cer cell growth . To further study whether the newly synthesized
compounds had STAT3 inhibition activity, we screened these com-
pounds using a p-STAT3 ELISA kit. Compounds 11, 16, and 17 sig- Disclosure statement
nificantly repressed STAT3 activity on PLC 5 cells (Figure 2(A)).
No potential conflict of interest was reported by the authors.
These results correlated with the potency of cell growth inhibition.
We further analysed 6 compounds to PLC5 cells with western blot
assay and studied the relationship of cell growth and the status of
STAT3. As shown in Figure 2(B), compounds 11, 16, and 17
resulted in a significant reduction of p-STAT3. In contrast, com-
pounds 9, 22, and 23, which were showed no cell growth inhib-
ition, showed no appreciable change in inhibition of STAT3 as
compared with the control panel. We further examined SHP-1
activity, the negative regulator of STAT3, by analysing the dephos-
phorylating effect in PLC5 cells. Compounds 11, 16, and 17
increased SHP-1 activity two-fold compared with the control panel
Funding
This work was supported by grants from the Ministry of Science
and Technology, Taiwan [MOST 104-3113-B-076–001, MOST
1
0
04–2321-B-010-017, MOST105-2321-B-010-008, MOST105-2325-B-
10-007, MOST 106-2321-B-010–005, MOST 106-3011-B-010-001,
MOST 106-2320-B-010-018] and the Ministry of Education, Aiming
for the Top University Plan [106AC-P645, 106AC-P632, 105AC-
P645, 104AC-P693] and Cheng Hsin General Hospital Foundation
[CY10625, CY10727] and Yen Tjing Ling Medical Foundation [CI-
(Figure 3). Meanwhile, we also explored the downstream signal
1
07-9].
cascade induced by compound 16 and SC-2001 in PLC5 cells. The
downstream targets of STAT3, such as cyclin D1, survivin, and Mcl-
1
2
, were decreased in the treatment of compound 16 and SC-
001, indicating that SHP-1 phosphatase activation and further p-
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