Identification of small molecule inhibitors of the STAT3 signaling pathway: Insights into their structural features and mode of action

Article abstract of DOI:10.1016/j.bmcl.2015.07.063

A series of novel STAT3 inhibitors consisting of Michael acceptor has been identified through assays of the focused in-house library. In addition, their mode of action and structural feature responsible for the STAT3 inhibition were investigated. In parti

Full text of DOI:10.1016/j.bmcl.2015.07.063

Accepted Manuscript
Identification of small molecule inhibitors of the STAT3 signaling pathway:
Insights into their structural features and mode of action
Kyeojin Kim, Su-Jung Kim, Young Taek Han, Sung-Jun Hong, Hongchan An,
Dong-Jo Chang, Taewoo Kim, Jeeyeon Lee, Young-Joon Surh, Young-Ger Suh
PII:
S0960-894X(15)00767-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.063
BMCL 22952
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
27 May 2015
18 July 2015
21 July 2015
Please cite this article as: Kim, K., Kim, S-J., Han, Y.T., Hong, S-J., An, H., Chang, D-J., Kim, T., Lee, J., Surh,
Y-J., Suh, Y-G., Identification of small molecule inhibitors of the STAT3 signaling pathway: Insights into their
structural features and mode of action, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/
1
0.1016/j.bmcl.2015.07.063
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bioorganic & Medicinal Chemistry Letters
Identification of small molecule inhibitors of the STAT3 signaling pathway:
Insights into their structural features and mode of action
a, †
b, †
c
b
a
d
Kyeojin Kim , Su-Jung Kim , Young Taek Han , Sung-Jun Hong , Hongchan An , Dong-Jo Chang ,
a
a
b,
a,
Taewoo Kim , Jeeyeon Lee , Young-Joon Surh , and Young-Ger Suh
a
College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742,
b
Republic of Korea
c
College of Pharmacy, Dankook University, Cheonan 330-714, Republic of Korea
College of Pharmacy, Sunchon National University, Suncheon 540-950, Republic of Korea
d
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel STAT3 inhibitors consisting of Michael acceptor has been identified through
assays of the focused in-house library. In addition, their mode of action and structural feature
responsible for the STAT3 inhibition were investigated. In particular, analog 6 revealed
promising STAT3 inhibitory activity in HeLa cell lines. The analog also exhibited selective
inhibition of STAT3 phosphorylation without affecting STAT1 phosphorylation and cytostatic
effect in human breast epithelial cells (MCF10A-ras), which supports cancer cell-specific
inhibitory properties.
Keywords:
STAT3
Small molecule inhibitors
Apoptosis
2
014 Elsevier Ltd. All rights reserved.
Antitumor
Breast cancer

Corresponding authors. Tel.: +82-2-880-7845 (Y.-J. Surh); tel.: +82-2-880-7875 (Y.-G. Suh); fax: +82-2-888-0649 (Y.-G. Suh).
E-mail address: surh@snu.ac.kr (Y.-J. Surh), ygsuh@snu.ac.kr (Y.-G. Suh).
†
These authors contributed equally to this work.

Signal transducer and activator of transcription 3 (STAT3) is
one of the STAT proteins (STATs 1, 2, 3, 4, 5a, 5b, and 6),
which relay signals from cytokines and growth factor receptors in
the plasma membrane to the nucleus where they regulate gene
1
transcription. Activation of STAT3 signaling is generally
dependent on the phosphorylation of specific tyrosine residue by
upstream cytokine receptor-associated kinase and growth factors
receptor-associated tyrosine kinase. STAT3 undergoes
dimerization via reciprocal interactions between the phosphor-
tyrosine and SH2 domain and then moves to the nucleus resulting
in activation of the target gene transcription, which modulate
critical cellular responses, including cell differentiation,
proliferation, apoptosis, angiogenesis, metastasis, and immune
Figure 1. Chemical structures of the STAT3 inhibitors from in-house library
2
responses. STAT3 is highly implicated in tumorigenesis in a
variety of solid and hematological malignancies through
1b, 3
overexpression and constitutive activation.
Although STAT1
and STAT3 are quite similar in terms of their proteins and target
DNA sequences, STAT1 plays a major role as a pro-
inflammatory, anti-pathogenic, and anti-proliferative factor. The
biological function of STAT1 is mostly antagonistic to that of
4
STAT3. Consequently, STAT3 has been validated as a novel
cancer drug target and development of selective STAT3
inhibitors have been consistently demanded because the
inhibition of abnormally elevated STAT3 activity or expression
5
Scheme 1. Reagents and conditions: (a) K
C, 100%; (b) Br , NaOH, water, DME, 86%; (c) diethylaniline, 215 C,
7%; (d) n-BuLi, aldehdye 13 or 14, -78 C; (e) DMP, DCM, rt, 41% for 1,
3% for 2 for 2 steps.
2 3
CO , propargylbromide, DMF, 60
is an important therapeutic modality.

7
5
2
Recently, we have been working on the identification and mode
of action of novel and selective STAT3 inhibitors from our in-
house chemical library, which consist of a variety of scaffolds
derived through long-term medicinal chemistry works. Thus, we
6
investigated the focused in-house library based on the inhibition
of STAT3 transcriptional activity using HeLa/STAT3-luc cells.
Subsequently, we identified a series of novel STAT3 inhibitors,
which is shown in Figure 1, and investigated their structural
features and mode of action to further develop STAT3-selective
inhibitors with therapeutic potential. Interestingly, all of the
identified inhibitors possess a Michael acceptor as a common
electrophilic moiety, which was in agreement with a recent
Scheme 2. Reagents and conditions: (a) for 17: 3-methyl-2-butenal, pyridine,
acetone, 120 C, 32 %; for 18: i) 3-chloro-3-methyl-1-butyne, K CO , KI,
DMF, 60 C, 100%; ii) diethylaniline, 195 C, 65%; (b) 3,4-
2
3
7
report. It was reported that Michael acceptor of many potent
dimethoxybenzaldehyde for 4 and 5, 2,4,5-trimethoxybenzaldehyde for 8,
STAT3 inhibitors function as a key moiety to react with the
cysteine residues of the active-site. Herein, we describe our
recent work on selective STAT3 inhibitors and present insight
into their structural features responsible for their inhibitory
activities.
KOH, EtOH, reflux, 57 % for 4 and 5, 49% for 8; (c) K
acetone, 60 C, 81% for 3, 88% for 7.
2 3
CO , iodomethane,
The identified enones exhibited different STAT3 inhibitory
potencies, although they all consist of a key enone system as a
Michel acceptor. Analog 6 exhibited the most potent inhibition of
STAT3 transcription as shown in Figure 2.
We initially investigated the structural feature of the eight
identified compounds shown in Figure 1. Enone 6 was prepared
5b
from dimethoxyphenyl acetate by a known procedure. The
syntheses of the other enones are outlined in Schemes 1 and 2.
For the syntheses of enones 1 and 2, dimethoxy phenol 9 was
subjected to propargylation, and the subsequent bromination of
the resulting ether 10 afforded bromo alkyne 11. A regioselective
Claisen rearrangement of 11 followed by a spontaneous
cyclization provided benzopyran 12. Finally, the addition of an
5b
aryl anion, prepared from 12, to aldehyde 13 or 14 followed by
oxidation of the resulting alcohol afforded ketone 1 or 2,
respectively. For the syntheses of enones 3 and 7, benzopyrans
1
7 and 18 were initially prepared by a regioselective
8
electrocyclization of 15 from 3-methyl-2-butenal or by the
procedure described for 12, respectively. Adol condensation of
acetophenone 17 and 18 with 3,4-dimethoxybenzaldehyde or
Figure 2. STAT3 transcriptional activities of the identified enones.
HeLa/STAT3-luc cells were pretreated with DMSO or each compound (10
μM) for 24 h, stimulated with oncostatin M (OSM) 10 ng/mL for 5 h, and
then assayed for the luciferase reporter gene activity.
2
,4,5-trimethoxybenzaldehyde followed by O-methylation of the
resulting enones 4 and 8 produced 3 and 7, respectively.
We assumed that the high inhibitory activity of enone 6 was
likely due to the favorable conformation indicated by the
sterically less-hindered exo-olefin for interaction with the

nucleophilic cysteine residue. This was well supported by our
molecular modeling study of the identified inhibitors as shown in
Figure 3. To investigate the conformational space of the enones,
molecular dynamics simulations were carried out using the
Figure 4. (a) Chemical structures of the analogs without a Michael
acceptor. (b) Effects of structural modifications on STAT3 transcription
activity. HeLa/STAT3-luc cells were pretreated with DMSO or each analog
(10 μM) for 24 h, stimulated with oncostatin M (OSM, 10 ng/mL) for 5 h,
and then assayed for the luciferase reporter gene activity.
9
simulated annealing protocol. The structures with the lowest
energy are shown in Figure 3. Compared to enones 1 and 3,
enone 6, which contains an exo-olefin, appeared to have a
sterically less-hindered electrophilic carbon for Michael type 1,
To investigate the mode of action for 6, STAT3 phosphorylation
was examined by Western blot analysis using H-ras transformed
MCF10A (MCF10A-ras) human breast epithelial cells, which
seemed to serve as an adequate model for studies on mammary
carcinogenesis (Figure 5). Enone 6 exhibited suppression of
STAT3 phosphorylation while 19 and 20, which are devoid of
enone system, showed no suppressive effect on STAT3
phosphorylation.
4
- addition. The nucleophilic attacks on the other enones by the
cysteine residue were less likely to occur due to the steric
hindrance. Figure 3 (b) shows overlaid structures of the final ten
ensembles with enone 6 having the lowest energy among the 500
conformers generated in the simulated annealing process.
Figure 5. Immnunoblot analysis on STAT3 phosphorylation inhibition by
6
1
. MCF10A-ras cells were treated with 5 and 10 μM of 6 and with 10 μM of
9 and 20 for 24 h. (b) Effect of the thiol reducing agents on the suppression
of STAT3 phosphorylation by 6. MCF10A-ras cells were treated with 0.5
mM of DTT for 1 h followed by treatment with 10 μM of 6 for 24 h.
In order to confirm direct interaction between STAT3 and 6, we
prepared biotinylated-6 (30) as shown in Scheme 3. Allylation of
phenol 22 followed by Dess-Martin oxidation of the alcohol
6b
produced aldehyde 23. Anionic addition of benzopyran 24 to
aldehyde 23 followed by oxidation of the resulting alcohol (25)
yielded ketone 26. Cross-metathesis (CM) of 26 with tert-butyl
N-allylcarbamate using Grubbs’ second generation catalyst
afforded the Boc-protected amine 27. Aldol condensation of 27
with paraformaldehyde produced enone 28, which was treated
with hydrochloric acid to yield amine 29. The crude amine was
directly treated with Hünig base and then with biotin, activated
with N-hydroxysuccinimide, to produce 30. Finally, binding of
30 to STAT3 was confirmed by immunoblot analysis as shown in
Figure 6. From these results, the inhibition of the STAT3
Figure 3. (a) The lowest energy conformations of the structures of enones 1,
3
, and 6. (b) Overlaid structures of the final ten ensembles with the lowest
energy conformations of 6 obtained by the SYBYL molecular modeling
program.
To further confirm that the enone system of 6 was a crucial
moiety for interaction with the thiol residue, we prepared analogs
1
9, 20, and 21, which are devoid of a Michael acceptor, by
transcriptional activity by
dimerization and translocation was concluded.
6 through the disruption of
5b
deletion (19) or reduction (20) of the enone-olefin and carbonyl
group (21). As we anticipated, analogs 19, 20, and 21 did not
suppress STAT3 transcription as shown in Figure 4. Obviously,
structural changes in the ,-unsaturated carbonyl moiety
resulted in the complete loss of STAT3 inhibitory activity. This
result supports that the enone system of 6 plays a crucial role for
the STAT inhibitory activity of 6 based on its interaction with the
cysteine residue of STAT3.

9
). Considering that both MCF10A-ras and MDA-MB-231 cell
lines express high levels of pSTAT3 whereas MCF10A and
1
0
MCF7 cell lines express lower levels of pSTAT3, a possible
mechanism for the selective cytotoxicity of 6 in cancer cells
rather than in normal cells is likely due to the enhanced
expression of STAT3 in cancer cells, leading to a greater
reduction in cell proliferation by enone 6.
Figure 8. Evaluation of the cytotoxicities in MCF10A and MCF10A-ras cells
by the identified STAT3 inhibitors. Both cells were treated with each
inhibitor for 24 h.
Scheme 3. Reagents and conditions: (a) allyl bromide, K
reflux, 93%; (b) Dess-Martin periodinane, NaHCO , CH Cl , 0 °C to rt, 23:
9%, 26: 70%; (c) 24, n-BuLi, THF, -78 °C to rt, 55%; (d) tert-butyl N-
allylcarbamate, Grubbs II catalyst, CH Cl , reflux, 40%; (e) (CH O) , K CO
DMF, rt, 72%; (f) 4N HCl in dioxane, CH Cl , rt; (g) (+)-biotin N-
hydroxysuccinimide ester, iPr NEt, DMF, rt, 30% for 2 steps.
2 3
CO , acetone,
3
2
2
6
2
2
2
n
2
3
,
2
2
2
Figure 9. Effect of enone 6 on the viability of MCF7 and MDA-MB-231
cells. Both cells were treated with 6 at the indicated concentrations for 24 h.
Figure 6. Immunoblot analysis of biotin-conjugate 30 binding to STAT3.
MCF10A-ras cells were incubated with 30 for 24 h. The binding of 30 to
STAT3 was detected using HRP-streptavidin.
In summary we report the discovery of novel STAT3-selective
inhibitors through the screening of a focused library. In particular,
enone 6 exhibited a promising inhibition in STAT3-driven
luciferase expression in HeLa cells. We confirmed that the enone
moiety of 6 is essential for the direct interaction with STAT3 via
Michael addition. Most notably, enone 6 selectively inhibited the
activation of STAT3 without affecting STAT1 and showed
selective viability suppression in human breast cell line harboring
constitutively active STAT3. Intensive studies on enone 6
including elucidation of its precise inhibition and development of
more potent STAT3 inhibitors based on the current results are
making good progress.
Next, we investigated the selective inhibition of STAT3 by 6
and subsequent induction of cell death in human breast cancer
cell lines. As anticipated, Western blot analysis shown in Figure
7
supported that enone 6 selectively inhibited phosphorylation of
STAT3 but not STAT1.
Acknowledgments
This work was supported by the National Research Foundation
of Korea (NRF) grant for the Global Core Research Center
Figure 7. Immunoblotting analysis for the selective inhibition of pSTAT3
by enone 6. MCF10A-ras cells were treated with 6 of the indicated
concentrations for 24 h, and the expressions of p-STAT3, STAT3 and p-
STAT1 were analyzed by Western blot analysis.
(
GCRC) funded by the Korea government (MSIP) (No. 2011-
030001) and the National Research Foundation of Korea (NRF)
Grant funded by the Korean Government (MSIP) (No.
013R1A1A2063282).
0
2
Finally, the anti-proliferative effects of the identified STAT3
inhibitors were examined on the untransformed MCF10A and
MCF10A-ras cells. As shown in Figure 8, enone 6 exhibited
selective cytotoxic effects to MCF10A-ras cells at 2 M after 24
h of treatment, whereas other enones were not cytotoxic in both
cells. Moreover, enone 6 showed effective suppression in the
viability of MDA-MB-231 cells rather than MCF7 cells (Figure
References and notes
1. (a) Yu, H.; Jove, R. Nat. Rev. Cancer 2004, 4, 97; (b) Yu, H.;
Pardoll, D.; Jove, R. Nat. Rev. Cancer 2009, 9, 798.
2
6
. Debnath, B.; Xu, S.; Neamati, N. J. Med. Chem. 2012, 55,
645.

3
4
. Siddiquee, K. A. Z.; Turkson, J. Cell Res. 2008, 18, 254.
. (a) Souissi, I.; Ladam, P.; Cognet, J. A. H.; Le Coquil, S.;
Lee, J. H.; Park, S.; Kim, K.; Lim, C.; Han, Y. T.; Yun, H.; Jung,
J.-W.; Park, H.-g.; Kim, H.-D.; Woo, B. Y.; Shim, S. S.; Kim, S.-
Y.; Choi, J. K.; Jeong, Y.-S.; Park, Y.; Park, Y.-H.; Kim, D.-D.;
Choi, S.; Suh, Y.-G. Chem.-Asian J. 2013, 8, 400; (f) Suh, Y.-G.;
Kim, N.-J.; Koo, B.-W.; Lee, K.-O.; Moon, S.-H.; Shin, D.-H.;
Jung, J.-W.; Paek, S.-M.; Chang, D.-J.; Li, F.; Kang, H.-J.; Le, T.
V. T.; Chae, Y. N.; Shin, C. Y.; Kim, M.-K.; Lim, J. I.; Ryu, J.-
S.; Park, H.-J. J. Med. Chem. 2008, 51, 6318.
Varin-Blank, N.; Baran-Marszak, F.; Metelev, V.; Fagard, R.
Mol. Cancer 2012, 11, 12; (b) Stephanou, A.; Latchman, D. S.
Int. J. Exp. Pathol. 2003, 84, 239.
5
. (a) Miklossy, G.; Hilliard, T. S.; Turkson, J. Nat. Rev. Drug
Discov. 2013, 12, 611; (b) Zhang, M.; Zhu, W.; Ding, N.; Zhang,
W.; Li, Y. Bioorg. Med. Chem. Lett. 2013, 23, 2225.
6
. (a) Agrawal, V.; Maharjan, S.; Kim, K.; Kim, N.-J.; Son, J.;
7. (a) Butturini, E.; Cavalieri, E.; de Prati, A. C.; Darra, E.; Rigo,
A.; Shoji, K.; Murayama, N.; Yamazaki, H.; Watanabe, Y.;
Suzuki, H.; Mariotto, S. PLoS One 2011, 6, e20174; (b) Mulliner,
D.; Wondrousch, D.; Schuurmann, G. Org. Biomol. Chem. 2011,
9, 8400; (c) Nakayachi, T.; Yasumoto, E.; Nakano, K.; Morshed,
S. R. M.; Hashimoto, K.; Kikuchi, H.; Nishikawa, H.; Kawase,
M.; Sakagami, H. Anticancer Res. 2004, 24, 737.
8. Adler, M. J.; Baldwin, S. W. Tetrahedron Lett. 2009, 50, 5075.
9. Marques, H. M.; Brown, K. L. Coordin. Chem. Rev. 2002,
225, 123.
Lee, K.; Choi, H. J.; Rho, S.-S.; Ahn, S.; Won, M.-H.; Ha, S.-J.;
Koh, G. Y.; Kim, Y.-M.; Suh, Y.-G.; Kwon, Y.-G. Oncotarget
2014, 5, 2761; (b) Chang, D.-J.; An, H.; Kim, K.-s.; Kim, H. H.;
Jung, J.; Lee, J. M.; Kim, N.-J.; Han, Y. T.; Yun, H.; Lee, S.;
Lee, G.; Lee, S.; Lee, J. S.; Cha, J.-H.; Park, J.-H.; Park, J. W.;
Lee, S.-C.; Kim, S. G.; Kim, J. H.; Lee, H.-Y.; Kim, K.-W.; Suh,
Y.-G. J. Med. Chem. 2012, 55, 10863; (c) Chun, S. K.; Jang, J.;
Chung, S.; Yun, H.; Kim, N.-J.; Jung, J.-W.; Son, G. H.; Suh, Y.-
G.; Kim, K. ACS Chem. Biol. 2014, 9, 703; (d) Han, Y. T.; Choi,
G.-I.; Son, D.; Kim, N.-J.; Yun, H.; Lee, S.; Chang, D. J.; Hong,
H.-S.; Kim, H.; Ha, H.-J.; Kim, Y.-H.; Park, H.-J.; Lee, J.; Suh,
Y.-G. J. Med. Chem. 2012, 55, 9120; (e) Kim, N.-J.; Li, F.-N.;
10. Berishaj, M.; Gao, S. P.; Ahmed, S.; Leslie, K.; Al-Ahmadie,
H.; Gerald, W. L.; Bornmann, W.; Bromberg, J. F. Breast Cancer
Res. 2007, 9, R32.

Graphical Abstract
Identification of small molecule inhibitors of
the STAT3 signaling pathway: Insights into their
structural features and mode of action
Leave this area blank for abstract info.
a, †
b, †
c
b
a
d
Kyeojin Kim , Su-Jung Kim , Young Taek Han , Sung-Jun Hong , Hongchan An , Dong-Jo Chang , Taewoo
a
a
b,
a,
Kim , Jeeyeon Lee , Young-Joon Surh , and Young-Ger Suh
a
College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, 1
b
Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
c
College of Pharmacy, Dankook University, Cheonan 330-714, Republic of Korea
College of Pharmacy, Sunchon National University, Suncheon 540-950, Republic of Korea
d