Blockade of STAT3 activation by sorafenib derivatives through enhancing SHP-1 phosphatase activity

Article abstract of DOI:10.1016/j.ejmech.2012.07.023

Previously, we demonstrated that the multiple kinase inhibitor sorafenib mediates the repression of phospho-STAT3 in hepatocellular carcinoma cells. In this study, we used this kinase-independent mechanism as a molecular basis to use sorafenib as scaffold to develop a novel class of SHP-1-activating agents. The proof of principle of this premise was provided by SC-1, which on replacement of N-methylpicolinamide by a phenylcyano group showed abolished kinase activity while retaining phospho-STAT3 repressive activity. Structural optimization of SC-1 led to compound 6, which repressed phospho-STAT3 through SHP-1 activation and inhibited PLC5 cell proliferation at sub-micromolar potency. In light of the pivotal role of phospho-STAT3 in promoting tumorigenesis and drug resistance, this novel SHP-1-activating agent may have therapeutic relevance in cancer therapy.

Full text of DOI:10.1016/j.ejmech.2012.07.023

European Journal of Medicinal Chemistry 55 (2012) 220e227
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Blockade of STAT3 activation by sorafenib derivatives through enhancing SHP-1
phosphatase activity
,
,
,
,h
Kuen-Feng Chen b c, Wei-Tien Tai c d, Cheng-Yi Hsu a, Jui-Wen Huang f, Chun-Yu Liu a e, Pei-Jer Chen b
,
InKi Kim g, Chung-Wai Shiau a
,
*
a Institute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei, Taiwan
b Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
c National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan
d Graduate Institute of Molecular Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
e Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
f Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan
g ASAN Institute for Life Science, ASAN Medical Center, Seoul, Republic of Korea
h Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously, we demonstrated that the multiple kinase inhibitor sorafenib mediates the repression of
phospho-STAT3 in hepatocellular carcinoma cells. In this study, we used this kinase-independent
mechanism as a molecular basis to use sorafenib as scaffold to develop a novel class of SHP-1-
activating agents. The proof of principle of this premise was provided by SC-1, which on replacement
of N-methylpicolinamide by a phenylcyano group showed abolished kinase activity while retaining
phospho-STAT3 repressive activity. Structural optimization of SC-1 led to compound 6, which repressed
phospho-STAT3 through SHP-1 activation and inhibited PLC5 cell proliferation at sub-micromolar
potency. In light of the pivotal role of phospho-STAT3 in promoting tumorigenesis and drug resistance,
this novel SHP-1-activating agent may have therapeutic relevance in cancer therapy.
Received 2 May 2012
Received in revised form
2 July 2012
Accepted 15 July 2012
Available online 23 July 2012
Keywords:
Sorafenib
STAT3
Ó 2012 Elsevier Masson SAS. All rights reserved.
SHP-1
HCC
1. Introduction
The Src-homology protein tyrosine phosphatase (PTP) SHP-1 is
a non-receptor phosphatase that negatively regulates cytokine
Overexpression of signal transducers and activators of tran-
scription 3 (STAT3) has been linked to tumorigenesis in a variety of
human cancers [1e7]. Evidence indicates that STAT3 is directly
recruited to ligand-induces cytokine receptors. Phospho-STAT3
then dimerizes in the cytoplasm and translocates to the nucleus,
where the dimer binds to specific DNA-response elements and
regulates STAT3-targeted gene expression [8,9]. For example, the
anti-apoptotic proteins, Bcl-2, cyclin D1, and Mcl-1 are transcribed
by the activated STAT3 and preclude cell apoptosis upon the treat-
ment with chemotherapeutic agents. The action of interleukin-6-
STAT3 signaling underscores the oncogenic potential of STAT3
induction in many forms of cancer, and inhibition of STAT3 activa-
tion by small molecules has been shown to block tumorigenesis and
reverse the transformed phenotype of human cancer cells [10e13].
signaling such as that of IL-3R, the PDGF- and EGF receptors, and
other tyrosine kinase receptors [14e16]. It has been shown that
SHP-1 dephosphorylates STAT3 by IL-6 signaling the preventing
STAT3 activation. Moreover, genetic transfection of SHP-1 in breast
cancer cells has been shown to reduce cell proliferation [17].
Conversely, siRNA knockdown of SHP-1 expression in prostate
cancer cells resulted in increased cellular proliferation [18]. In light
of the function of SHP-1 as a tumor suppressor gene, increasing its
activity represents a promising strategy for cancer therapy. Previ-
ously, we reported that the multiple kinase inhibitor, sorafenib,
activated SHP-1 activity independently of kinase inhibition [12,13].
The dissociation of these two pharmacological activities, i.e., SHP-1
activation versus kinase inhibition, provides a molecular basis for
using sorafenib as a scaffold to develop novel agents that can
effectively activate SHP-1 expression. Here, structural optimization
of compounds led to a novel class of SHP-1-activating agents with
sub-micromolar potency.
* Corresponding author. Tel.: þ886 2 28267930; fax: þ886 2 28250883.
E-mail address: cwshiau@ym.edu.tw (C.-W. Shiau).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.023

K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
221
2. Chemistry
example, the concentration required for significant enhancement of
SHP-1 activation was approximately the same for SC-1 as for sor-
afenib. In a previous report, we demonstrated that sorafenib-
mediated p-STAT3 downregulation was caused by an increase in
SHP-1 activity. Together, these findings suggest that the effect of
this agent on SHP-1 activation was independent of kinase activa-
tion. Therefore, SC-1 was used as a starting point from which to
generate SHP-1-activating agents with low-micromolar potency.
In our previous report, we demonstrated that the replacement
of N-methylpicolinamide by a phenylcyano group in the sorafenib
scaffold abolished kinase inhibition activity without compromising
SHP-1-activating activity. In this study the resulting kinase-inactive
analog SC-1 (Fig. 1A) was used as a lead compound. First, to
understand the role of the urea group of SC-1 in SHP-1 activation,
different substituents were added to the terminal urea of SC-1
yielding compounds 1e12. Among them, addition of a methylene
next to the urea group resulted in compounds 1e5. Further, we
replaced the urea in SC-1 with sulfonylamide, carbonyl amide and
amino groups to obtain compounds 7e11, and a sorafenib moiety
was synthesized by the replacement of the urea group with sulfo-
nylamide, carbonyl amide and amino groups to generate
compounds 13e17. We then used the nitro-, amino-, benzoimida-
zol-2-one substitutions of SC-1 as platforms to carry out structural
optimization, which generated compounds 18e24. These SC-1
derivatives were synthesized according to the general procedure
described in Fig. 1B. All derivatives synthesized were racemic
compounds except compound 5.
3.2. Lead optimization
SC-1 was first conjugated with different substituents through
various linkers at the terminal amino group, generating compounds
1e12. These derivatives were analyzed by MTT to determine cell
growth inhibition within the dose range of 0.1e15
mM in PLC5 cell
(Table 1). Among these derivatives, urea derivatives showed robust
cell growth inhibition, while the sulfonylamide counterparts
showed moderate potency even at 7.5 mM, and amide and amino
linkers showed little effect. This differential increase in potency
suggests a unique mode of ligand recognition that prefers planar
geometry. The 4- to 5-fold improvement in the cell growth inhi-
bition of compound 2 over SC-1 indicates the importance of this
planar geometry interaction in ligand binding. Our data also
showed that the meta substituent of benzene was better than the
para substituent at inhibiting cell growth. For example, the only
difference between compound SC-1 and compound 6 is the meta
and para position for the conjunction in the benzene, however, the
cell growth inhibition of compound 6 was at least one order of
magnitude greater than that of SC-1.
3. Results
3.1. Pharmacological exploitation of kinase-independent effect of
sorafenib on SHP-1 activation to generate a novel class of SHP-1-
enhancer agents
The dose-dependent effect of SC-1 on SHP-1 activation in PLC5
cells was examined using an IP-SHP-1 phosphatase activity kit after
drug treatment for 24 h (Fig. 2). SC-1, though devoid of kinase
inhibition activity, exhibited potent activation of SHP-1. For
With the pyridinyl ring moiety of sorafenib, we further exam-
ined the effect of modifying the urea moiety on cell growth inhi-
bition. Among the four different analogs of sorafenib examined, the
Fig. 1. (A) Representative structures of sorafenib and derivatives and (B) the general synthetic procedure for sorafenib derivatives.

222
K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
Table 1
Chemical structure of compounds 1e12 and IC50 of growth inhibition in PLC5 cells.
Cells were treated with drugs for 24 h and IC50 were measured in MTT assay.
Cpd
SC-1
1
R1
R2
R3
IC50 (mM)
in PLC5 cells
7.5
H
H
H
H
12.3
2.1
Fig. 2. Effects of sorafenib and SC-1 on SHP-1 activity. Dose effects of sorafenib and SC-
1 on SHP-1 activation in PLC5 cells. Cells were treated with drugs at the indicated
concentrations for 24 h. Columns, mean; bars, SD (N ¼ 3).
2
sulfonylamide with phenyl trifluoromethane derivative (compound
14) exhibited the highest potency, while the amide, amino meth-
ylene and phenyl sulfonyl derivative counterparts (15e17,
respectively) showed no appreciable reduction in cell growth
(Table 2). Next, we replaced the phenylcyano group of SC-1 with
di-amino, nitro-amino and benzoimidazol-2-one to generate
compounds 18e24. As compared to these derivatives, compounds
18e20 bearing the nitro substituents showed more potent growth
3
4
H
H
H
H
10.5
>15
inhibition against PLC5 cells with IC50 values of 8.3, 3.9, and 1.2 mM,
respectively, whereas electron-donating substituted compounds
such as 21, and 22 revealed no appreciable cell growth inhibition
(Table 3). These results indicate that a certain degree of electron
withdrawing in the aromatic system is favorable for growth-
inhibitory activity.
5
6
H
H
H
>15
3.3. Effect of derivatives on downregulation of p-STAT3 through
SHP-1 activation
H
0.5
Previously, we demonstrated that sorafenib and SC-1 repressed
STAT3 phosphorylation through increasing SHP-1 activity. Here, we
carried out three experiments to validate the mechanism by which
the newly synthesized SC-1 derivatives mediate dephosphorylation
of STAT3. First, to demonstrate the dephosphorylation ability of
these derivatives, we assessed p-STAT3 inhibition by ELISA. As
shown in Fig. 3, the derivatives which inhibited cell growth most
effectively significantly reduced the level of p-STAT3, whereas the
derivatives that did not inhibit cell growth did not inhibit p-STAT3.
For example, compound 6 and compound 2 showed close correla-
tion between reduction of p-STAT3 and cell growth.
7
8
H
H
H
H
2.2
>15
9
H
H
3.3
Second, to understand the effect of the new derivatives on SHP-
1, we further examined their induction of SHP-1 phosphatase
activity. PLC5 cells were exposed to compounds 2, 6, and 16 at
10
11
H
H
H
H
>15
>15
10 mM for 24 h and then cell lysates were collected for detection of
SHP-1 activity. Compounds 2, and 6 showed significantly increased
SHP-1 activity in PLC5 cells, whereas compound 16 showed no
appreciable change (Fig. 4A). Also, compound 6 enhanced SHP-1
activation in a dose dependent manner (Fig. 4B). These data
imply that these compounds enhanced SHP-1 phosphatase activity
and further reduced the p-STAT3 level.
Third, we validated the downstream signals of compounds 6 and
12
H
Me
4.5
16 in PLC5 cells. As shown, at concentrations of 5 and 10
mM
compound 6 was effective in inhibiting p-STAT3 and subsequently
repressing the expression of its target genes, such as Mcl-1, cyclin

K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
223
Table 2
100
Chemical structure of compounds 13e17 and IC50 of growth inhibition in PLC5 cells.
Cells were treated with drugs for 24 h and IC50 were measured in MTT assay.
80
60
40
20
0
Cpd
R1
H
R2
IC50 (mM) in PLC5 cells
13
>15
Cpd
Fig. 3. ELISA analysis of the inhibitory effects of compounds 1e24 versus DMSO, each
at 10 M, on the IL-6 stimulated p-STAT3 in PLC5 cells after 24 h of treatment.
Columns, mean; bars, SD (N ¼ 3).
14
15
H
H
8.4
m
>15
D1 and survivin. On the other hand, compound 16 had no effect on
the reduction of p-STAT3 and further signaling cascades (Fig. 5).
Moreover, PLC5 treated with compound 6 showed significant
cleavage of PARP-1 after a 24 h exposure suggesting apoptotic cell
death. We also examined SOCS proteins, the negative regulator of
Jak2/STAT pathway, upon drug treatment. Our data showed that
compounds 6 and 16 did not affect the protein levels of SOCS-1 or
SOCS-3.
16
17
H
H
>15
>15
Taken together, these data confirmed that the SC-1 derivatives
mediated the repression of p-STAT3 through the same mechanism
of action as SC-1, i.e., through SHP-1 activation. However, the exact
mechanism of SC-1 derivative-induced SHP-1 activation remains
unclear and will be further investigated in our lab.
Table 3
Chemical structure of compounds 18e24 and IC50 of growth inhibition in PLC5 cells.
Cells were treated with drugs for 24 h and IC50 were measured in MTT assay.
3.4. Effects of compound 2 in a PLC5 xenograft nude mouse model
To assay whether the biological effect of compound 2, is
potentially clinically relevant, we tested the in vivo effect of
compound 2 on tumor growth in PLC5 bearing xenograft mice.
PLC5-bearing mice were treated with vehicle (Cremophor EL/95%
ethanol (50:50; Sigma)) or compound 2 perorally (p.o.) at 10 mg/
kg/day for three weeks. As shown in Fig. 6A, compound 2 strongly
inhibited the growth of PLC5 xenograft tumors. To further confirm
the molecular mechanism of compound 2 and its anti-cancer effect
in xenograft nude mice model, tumor extracts from vehicle and
compound 2-treated mice were assayed for p-STAT3 by western
blot. p-STAT3 was repressed in compound 2-treated tumor (Fig. 6B,
right panel). We further examined SHP-1 activity in compound 2-
treated PLC5 xenograft. Compound 2-treated tumors showed
significant upregulation of SHP-1 activity in vivo.
Cpd
R1
R2
R3
IC50
8.3
(mM) in PLC5 cells
18
NO2
NH2
19
20
NO2
NO2
NH2
NH2
3.9
1.2
4. Discussion
21
22
NH2
NH2
NH2
NH2
>15
>15
On the basis of previous mechanistic data showing that the
effect of sorafenib on SHP-1 activation was independent of kinase
inhibition, in this study we structurally modified sorafenib to
develop a novel class of SHP-1-activating agents. Our hypothesis
that the SHP-1 activation and p-STAT3 repression seen in sorafenib
could be structurally dissociated was borne out by the compound
SC-1, which, devoid of kinase activity, exhibited the same SHP-1-
activating activity as sorafenib. Presumably, the phenylcyano
component of SC-1, which has no hydrogen donor ability, reduces
its interaction with kinase in the ATP binding domain. Subsequent
modification of SC-1 by adding various linkers, including urea,
23
24
10.2
11.5

224
K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
A
B
6
16
4
3
2
1
0
4
3
2
1
0
2
6
1
5
16
10
15
DMSO
DMSO
Cpd
CONC(µM)
Fig. 4. Effects of new derivatives on SHP-1. (A) SHP-1 activity assay with compounds 2, 6, and 16 at 10
or 16 on SHP-1 activation in PLC5 cells. Cells were treated with drugs at the indicated concentrations for 24 h. Columns, mean; bars, SD (N ¼ 3).
m
M. Columns, mean; bars, SD (n ¼ 3)/P < 0.05. (B) Dose effects of compound 6
methylene urea, sulfonylamide, amide, and amino groups resulted
in different geometries and hydrogen bond proton donor ability.
Among them, urea, methylene urea showed a multifold increase in
SHP-1 activity in comparison with sulfonylamide, and amide
groups. This increase suggests that planar geometry favors SHP-1
activation. Moreover, a comparison of the substitution position of
benzene in SC-1 and compound 6 showed that meta substitution
greatly enhanced SHP-1 activation. Further modifications of the N-
methylpicolinamide moiety revealed that electron withdrawing
groups activate SHP-1 more strongly than electron-donating
groups. For example, nitro and benzoimidazol-2-one resulted in
significantly stronger SHP-1 activation than amino groups. It is also
noteworthy that the SHP-1 activation by compound 6 was linked to
biological and pharmacological activity in vivo and in vitro. i.e.,
compound 6 activates SHP-1 phosphatase and removes the phos-
phor group of STAT3 in PLC5 cells. This action impedes STAT3
transcription activation and subsequently represses proliferation
and survival genes such as Mcl-1, cyclin D1 and survivin in PLC5
cells. Equally important, compound 2 was shown to mediate tumor
growth inhibition in a nude mouse xenograft model. Thus, from
a mechanistic perspective, compound 2 provides a useful phar-
macological tool to study SHP-1 activation.
5. Conclusion
SC-1 derivatives represent the first series of small-molecule
agents that exhibit potent enhancement of SHP-1 activation.
These agents may have clinical relevance in light of the pivotal role
of STAT3 in promoting tumorigenesis and drug resistance. In HCC,
overexpression of STAT3 confers resistance to chemotherapy drugs
and represents an oncoprotein drug target. The findings presented
here point to the potential use of SHP-1-activating agents in the
treatment of hepatocellular carcinoma.
6. Experimental section
6.1. Materials
All commercially available starting materials were purchased
from SigmaeAldrich, or Acros and were used without further
purification. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on Bruker DPX400 (400 MHz) instruments. Chem-
ical shifts are reported as ppm. 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. High resolution mass spectra were recorded on a Finnigan
MAT 95S mass spectrometer. Sorafenib (Nexavar) was kindly
provided by Bayer Pharmaceuticals (West Haven, CT). Sodium
vanadate and SHP-1 inhibitor were purchased from Cayman
Chemical (Ann Arbor, MI). Antibodies for immunoblotting such as
Cyclin D1, and PARP were purchased from Santa Cruz Biotech-
nology (San Diego, CA). Other antibodies such as anti-SOCS-3, Mcl-
1, survivin, phospho-STAT3 (Tyr705), and STAT3 were from Cell
Signaling (Danvers, MA).
6.2. Chemical synthesis
6.2.1. General procedures for the synthesis of compounds 1e13
To a 50 mL THF solution of triphosgen (0.30 g, 1.0 mmol), aniline
(1.1 mmol) and 2 equivalent of triethyl amine were added. The
mixture was heated to 50 ꢀC for 30 min. After the temperature was
back to room temperature, aminophenoxyl-benzonitrile in the
10 mL THF solution was added to the mixture and heated to 50 ꢀC
for another 30 min. The mixture was evaporated, diluted with
water and extracted with EtOAc. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure to give desires compound 1e13.
Fig. 5. Western blot analysis of the effect of compounds 6 and 16 each at 5
10 M on the phosporylation of STAT3, STAT3, Mcl-1, cyclin D1, survivin, SOCS-1, SOCS-
3, and PARP-1 in PLC5 cells with serum-free medium after treatment for 24 h.
mM and
m

K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
225
Fig. 6. In vivo effect of compound 2 on PLC5 xeonograft nude mice. (A) Compound 2 shows a significant antitumor effect on PLC5 tumors. Top panel, points, mean (n ¼ 6); bars, SE.
Lower right panel, (B) Western blot analysis of p-STAT3, STAT3, and SHP-1 in PLC5 tumors. (C) SHP-1 activity in PLC5 tumors.
6.2.1.1. 1-(4-(4-Cyanophenoxy)phenyl)-3-(3,4-dimethoxybenzyl)urea
(1). 1H NMR (400 MHz, CDCl3):
J ¼ 8.8 Hz, 2H), 6.98e6.94 (m, 4H), 6.88e6.75 (m, 4H), 6.56 (brs,1H),
4.36 (s, 2H), 3.84 (s, 6H); HRMS calculated for C23H20N3O4
[M ꢁ H]ꢁ: 402.1454. Found: 402.1462.
2H), 7.58 (d, J ¼ 7.2 Hz, 1H), 7.53 (d, J ¼ 7.2 Hz, 1H), 7.50e7.45 (m,
2H), 7.41 (d, J ¼ 8.8 Hz, 2H), 6.99 (t, J ¼ 9.2 Hz, 4H), 5.74 (d,
J ¼ 6.8 Hz, 1H), 1.63 (d, J ¼ 6.8 Hz, 3H); HRMS calculated for
C26H20N3O2 [M ꢁ H]ꢁ: 406.1556. Found: 406.1563.
d
7.56 (d, J ¼ 6.8 Hz, 2H), 7.34 (d,
6.2.1.6. 1-(3-(4-Cyanophenoxy)phenyl)-3-(4-chloro-3-(trifluorom-
6.2.1.2. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophe-
ethyl)phenyl)urea (6). 1H NMR (400 MHz, DMSO):
d 9.17 (s, 1H),
noxy)benzyl)urea (2). 1H NMR (400 MHz, CDCl3):
d
7.76 (s, 1H),
9.03 (s, 1H), 8.04 (d, J ¼ 2.4 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz, 2H),
7.64e7.55 (m, 2H), 7.41e7.32 (m, 2H), 7.23 (d, J ¼ 7.2 Hz, 1H), 7.11
(d, J ¼ 8.0 Hz, 2H), 6.75 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H); 13C NMR
7.51e7.39 (m, 3H), 7.29 (dd, J ¼ 8.8 Hz, 2.4 Hz, 1H), 7.13 (d,
J ¼ 8.4 Hz, 3H), 6.83 (dd, J ¼ 8.8 Hz, 4.8 Hz, 4H), 5.93 (t, J ¼ 6.0 Hz,
1H), 4.24 (d, J ¼ 6.0 Hz, 2H); 13C NMR (100 MHz, MeOD):
d
163.2,
(100 MHz, DMSO-d6): d 161.0, 154.8, 152.3, 141.2, 139.1, 134.6,
157.5, 155.3, 140.6, 137.9, 135.4, 132.9, 130.3, 129.1 (m), 125.6,
124.9, 123.8, 122.9, 122.7, 1215, 119.6, 119.0, 118.4 (m), 106.7, 43.9;
HRMS calculated for C22H14N3O2F3Cl [M ꢁ H]ꢁ: 444.0727. Found:
444.0732.
132.0, 130.5, 127.3, 126.8, 126.5, 126.2, 124.1, 123.2, 122.5, 121.4,
118.7, 118.1, 116.9 (m), 115.1, 113.7, 110.0, 105.1; HRMS calculated
for C21H12N3O2F3Cl [M ꢁ H]ꢁ: 430.0570. Found: 430.0576.
6.2.1.7. 4-(3-(3-(Trifluoromethyl)benzen-sulfonylamino)phenoxy)
6.2.1.3. 1-(4-(4-Cyanophenoxy)benzyl)-3-(3,4-dimethoxybenzyl)urea
benzonitrile (7). 1H NMR (400 MHz, CDCl3):
d 8.00 (s, 1H), 7.96 (d,
(3). 1H NMR (400 MHz, DMSO):
d
7.81 (d, J ¼ 9.2 Hz, 2H), 7.33 (d,
J ¼ 8.0 Hz, 1H), 7.81 (d, J ¼ 8.0 Hz, 1H), 7.65e7.54 (m, 3H), 7.26 (t,
J ¼ 8.0 Hz, 1H), 7.08 (s, 1H), 7.05e6.97 (m, 1H), 6.94e6.86 (m, 3H),
6.84 (t, J ¼ 2.0 Hz, 1H), 6.81 (dd, J ¼ 8.4 Hz, 2.0 Hz, 1H); HRMS
J ¼ 8.4 Hz, 2H), 7.06 (dd, J ¼ 15.6 Hz, 9.2 Hz, 4H), 6.86 (d, J ¼ 9.2 Hz,
2H), 6.76 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.45 (t, J ¼ 6.0 Hz, 1H), 6.38 (t,
J ¼ 6.0 Hz, 1H), 4.23 (d, J ¼ 5.2 Hz, 2H), 4.14 (d, J ¼ 5.2 Hz, 2H), 3.69
calculated for C20H12N2O3F3S [M
417.0518.
ꢁ
H]ꢁ: 417.0521. Found:
(s, 6H); 13C NMR (100 MHz, DMSO-d6):
d 161.4, 158.0, 152.9, 148.6,
147.6, 138.1, 134.6, 133.2, 128.9, 120.2, 119.0, 118.7, 117.7, 111.7, 111.1,
104.8, 55.5, 55.3, 42.8, 42.3; HRMS calculated for C24H24N3O4
[M þ H]þ: 418.1767. Found: 418.1773.
6.2.1.8. 4-(3-(3-(Trifluoromethoxy)benzylamino)phenoxy)benzoni-
trile (8). 1H NMR (400 MHz, CDCl3):
d
7.61 (d, J ¼ 8.8 Hz, 2H), 7.43 (t,
J ¼ 8.0 Hz, 1H), 7.33 (d, J ¼ 8.4 Hz, 1H), 7.29e7.16 (m, 3H), 7.04 (d,
J ¼ 8.8 Hz, 2H), 6.55 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 6.46 (dd, J ¼ 8.0 Hz,
2.0 Hz, 1H), 6.34 (t, J ¼ 2.4 Hz, 1H), 4.41 (s, 2H); 13C NMR (100 MHz,
6.2.1.4. 1-(4-(4-Cyanophenoxy)benzyl)-3-(3-(trifluoromethoxy)ben-
zyl)urea (4). 1H NMR (400 MHz, CDCl3):
d
7.49 (d, J ¼ 9.2 Hz, 2H),
7.17 (t, J ¼ 8.0 Hz, 1H), 7.10 (d, J ¼ 8.4 Hz, 2H), 7.00e6.92 (m, 3H),
6.88 (d, J ¼ 9.2 Hz, 2H), 6.84 (d, J ¼ 8.4 Hz, 2H), 6.17e6.05 (m, 2H),
CDCl3): d 161.5, 155.9, 149.5 (m), 141.3, 133.9, 130.7, 130.0, 125.4,
124.2, 121.6, 119.7, 119.6, 119.1, 118.9, 117.9, 116.5, 109.8, 109.2, 105.5,
104.4, 47.4; HRMS calculated for C21H16N2O2F3 [M þ H]þ: 385.1164.
Found: 385.1157.
4.10 (m, 4H); 13C NMR (100 MHz, DMSO-d6):
d 163.3, 160.8, 155.2,
150.7, 150.7, 144.4, 138.5, 135.4, 131.0, 130.1, 126.8, 125.7, 123.1,
121.4, 120.6, 10.5, 120.3, 119.6, 118.9, 118.1, 106.7, 44.1, 44.1; HRMS
calculated for C23H19N3O3F3 [M þ H]þ: 442.1379. Found: 442.1381.
6.2.1.9. 1-(3-(4-Cyanophenoxy)phenyl)-3-(3-fluorophenyl)urea (9).
1H NMR (400 MHz, MeOD):
d
7.66 (d, J ¼ 9.2 Hz, 2H), 7.60 (s, 1H),
6.2.1.5. (R)-1-(4-(4-Cyanophenoxy)phenyl)-3-(1-(naphthalen-1-yl)
7.41e7.34 (m, 2H), 7.22 (q, J ¼ 8.0 Hz, 1H), 7.18 (dd, J ¼ 8.0 Hz,
ethyl)urea (5). 1H NMR (400 MHz, MeOD):
7.87 (d, J ¼ 8.0 Hz, 1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 7.65 (d, J ¼ 8.8 Hz,
d
8.17 (d, J ¼ 8.0 Hz, 1H),
2.0 Hz, 1H), 7.10e7.02 (m, 3H), 6.71 (dd, J ¼ 8.8 Hz, 2.4 Hz, 2H); 13C
NMR (100 MHz, MeOD):
d 165.6, 163.2, 162.9, 156.7, 154.6, 142.3,

226
K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
142.2, 142.1, 135.3, 131.4, 131.1, 131.0, 119.6, 119.1, 116.5, 115.5, 115.3,
112.1, 110.1, 109.9, 107.2, 106.9, 106.7; HRMS calculated for
C20H13N3O2 [M ꢁ H]ꢁ: 346.0992. Found: 346.0999.
7.25 (t, J ¼ 8.4 Hz, 1H), 6.98e6.94 (m, 2H), 6.91 (dd, J ¼ 5.6 Hz,
2.4 Hz, 1H), 6.82 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 3.00 (d, J ¼ 5.2 Hz, 3H);
HRMS calculated for C20H17N3O4F3S [M þ H]þ: 452.0892. Found:
452.0891.
6.2.1.10. N-(3-(4-Cyanophenoxy)phenyl)benzamide (10). 1H NMR
(400 MHz, CDCl3):
d
8.82 (s, 1H), 7.75 (d, J ¼ 7.6 Hz, 2H), 7.53 (s, 1H),
6.2.2.3. 4-(3-(Benzene-carbonylamino)phenoxy)-N-methylpyridine-
7.46e7.35 (m, 4H), 7.28 (t, J ¼ 8.0 Hz, 2H), 7.22 (t, J ¼ 8.0 Hz, 1H),
6.90 (d, J ¼ 8.8 Hz, 2H), 6.72 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H); HRMS
calculated for C20H13N2O2 [M ꢁ H]ꢁ: 313.0977. Found: 313.0971.
2-carboxamide (16). 1H NMR (400 MHz, CDCl3):
d
8.37 (d, J ¼ 5.2 Hz,
1H), 8.05 (s, 1H), 8.00 (brs, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.83 (s, 1H),
7.68 (d, J ¼ 2.4 Hz, 1H), 7.62 (t, J ¼ 2.0 Hz, 1H), 7.34e7.60 (m, 5H),
6.99 (dd, J ¼ 5.2 Hz, 2.4 Hz, 1H), 6.86 (dt, J ¼ 7.2 Hz, 2.0 Hz, 1H), 2.98
6.2.1.11. N-(3-(4-Cyanophenoxy)phenyl)benzenesulfonamide (11).
(d, J ¼ 4.8 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 166.0, 165.7, 164.6,
1H NMR (400 MHz, CDCl3):
d
7.79 (d, J ¼ 8.4 Hz, 2H), 7.53 (t,
154.2, 152.1, 149.7, 139.8, 134.5, 132.0, 130.6, 128.8, 127.0, 117.0, 116.6,
114.4, 112.7, 110.3, 26.1; HRMS calculated for C20H18N3O3 [M þ H]þ:
348.1348. Found: 348.1345.
J ¼ 4.4 Hz, 3H), 7.42 (t, J ¼ 8.0 Hz, 2H), 7.20 (t, J ¼ 8.0 Hz, 1H), 6.93
(dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.86e6.83 (m, 3H), 6.73 (dd, J ¼ 8.0 Hz,
2.0 Hz, 1H); HRMS calculated for C19H13N2O3S [M ꢁ H]ꢁ: 349.0647.
Found: 349.0643.
6.2.2.4. 4-(3-(Benzene-sulfonylamino)phenoxy)-N-methylpyridine-
2-carboxamide (17). 1H NMR (400 MHz, MeOD):
d 8.43 (d,
6.2.1.12. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-(4-cyanophe-
J ¼ 5.6 Hz, 1H), 7.76 (d, J ¼ 8.0 Hz, 2H), 7.60e7.54 (m, 1H), 7.50 (t,
J ¼ 8.0 Hz, 2H), 7.46 (d, J ¼ 2.4 Hz, 1H), 7.31 (t, J ¼ 8.0 Hz, 1H),
6.99 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.91 (dd, J ¼ 5.6 Hz, 2.4 Hz, 1H),
6.88 (t, J ¼ 2.0 Hz, 1H), 6.83 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 2.94 (s,
3H); HRMS calculated for C19H18N3O4S [M þ H]þ: 384.1018.
Found: 384.1013.
noxy)-4-methylphenyl)urea (12). 1H NMR (400 MHz, MeOD):
d 7.87
(d, J ¼ 2.8 Hz, 1H), 7.60 (d, J ¼ 9.2 Hz, 2H), 7.54 (dd, J ¼ 8.4 Hz,
2.4 Hz, 1H), 7.39 (d, J ¼ 8.8 Hz, 1H), 7.25 (d, J ¼ 2.4 Hz, 1H), 7.18 (d,
J ¼ 8.0 Hz, 1H), 7.12 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 6.93 (d, J ¼ 9.6 Hz,
2H), 2.02 (s, 3H); 13C NMR (100 MHz, MeOD):
d 163.0, 154.5, 153.8,
139.9, 139.7, 135.4, 132.9, 129.7, 129.4, 129.1, 128.8, 126.2, 125.7,
125.5 (m), 124.1, 122.8, 120.1, 119.7, 118.6 (m), 118.0, 117.6, 117.5,
113.2, 113.1, 106.2, 15.5; HRMS calculated for C22H14N3O2F3Cl
[M ꢁ H]: 444.0727. Found: 444.0725.
6.2.2.5. N-(3-(Trifluoromethoxy)benzyl)-3-(3-amino-4-nitrophen-
oxy)benzenamine (18). 1H NMR (400 MHz, CDCl3):
d 8.05 (d,
J ¼ 9.2 Hz, 1H), 7.34 (t, J ¼ 8.0 Hz, 1H), 7.30e7.20 (m, 2H), 7.19e7.13
(m, 2H), 7.11 (d, J ¼ 8.0 Hz, 1H), 6.47 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H),
6.40 (dd, J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.32e6.26 (m, 2H), 6.12 (d,
J ¼ 2.8 Hz, 1H), 6.06 (brs, 2H), 4.33 (d, J ¼ 5.2 Hz, 2H), 4.27 (d,
J ¼ 5.2 Hz, 1H); HRMS calculated for C20H17N3O4F3 [M þ H]þ:
420.1171. Found: 420.1165.
6.2.1.13. 1-(3-(2-(Methylcarbamoyl)pyridin-4-yloxy)phenyl)-3-(3-
fluorophenyl)urea (13). 1H NMR (400 MHz, DMSO-d6):
d 8.97 (d,
J ¼ 9.6 Hz, 2H), 8.77 (d, J ¼ 4.8 Hz, 1H), 8.51 (d, J ¼ 5.6 Hz, 1H),
7.50e7.35 (m, 4H), 7.28 (t, J ¼ 8.4 Hz, 2H), 7.18 (d, J ¼ 5.6 Hz, 1H),
7.09 (d, J ¼ 8.4 Hz,1H), 6.82 (d, J ¼ 8.0 Hz,1H), 6.77 (t, J ¼ 8.4 Hz,1H),
2.77 (d, J ¼ 4.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
d
165.5,
6.2.2.6. N-(3-(Trifluoromethyl)benzene-sulfonyl)-3-(3-amino-4-nitro-
163.9, 163.5, 161.2,153.7, 152.5, 152.3, 150.6, 141.6, 141.4,141.3, 130.8,
130.5, 130.4, 115.7, 114.51, 114.2, 110.4, 109.1, 108.6, 108.4, 105.3,
105.0, 26.1; HRMS calculated for C20H18N4O3F [M þ H]þ: 381.1363.
Found: 381.1358.
phenoxy)benzenamine (19). 1H NMR (400 MHz, CDCl3):
d 8.06 (d,
J ¼ 9.6 Hz, 1H), 8.00 (s, 1H), 7.96 (d, J ¼ 8.0 Hz, 1H), 7.81 (d, J ¼ 8.0 Hz,
1H), 7.61 (t, J ¼ 8.0 Hz, 1H), 7.27 (t, J ¼ 8.0 Hz, 1H), 6.91e6.80 (m, 3H),
6.19 (dd, J ¼ 9.6 Hz, 2.4 Hz, 1H), 6.14 (d, J ¼ 2.4 Hz,1H), 6.10 (brs, 2H);
13C NMR (100 MHz, CDCl3):
d 163.1, 155.5, 146.7, 140.0, 137.6, 132.0,
6.2.2. General procedures for the synthesis of compounds 14e24
In a 25 mL two-necked round flask, aniline derivatives (1 mmol)
and catalytic amount of pyridine were placed in anhydrous THF
(10 mL) at room temperature. Acyl chloride or sulfonyl chloride
compounds were added to the mixture and stirred for 2 h at room
temperature. The solvent was removed under vacuum and the crude
residue purified by chromatography on a silica gel column using
EtOAc/hexane as eluent (1/10 to 1/2). This procedure afforded the
expected coupling product as a white solid from 80% to 95% yield.
131.6, 130.9, 130.3, 130.0, 129.9 (m), 128.8, 128.0, 124.4, 124.3, 124.2,
124.2, 124.0, 121.6, 117.8, 117.6, 113.6, 107.6, 104.3; HRMS calculated
for C19H13N3O5F3S [M ꢁ H]ꢁ: 452.0528. Found: 452.0529.
6.2.2.7. 1-(3-(3-Amino-4-nitrophenoxy)phenyl)-3-(4-chloro-3-(tri-
fluoromethyl)phenyl) urea (20). 1H NMR (400 MHz, DMSO):
d 9.19
(s, 1H), 9.05 (s, 1H), 8.06 (d, J ¼ 1.6 Hz, 1H), 8.00 (d, J ¼ 9.6 Hz, 1H),
7.67e7.55 (m, 2H), 7.47 (s, 1H), 7.42e7.32 (m, 2H), 7.27 (d, J ¼ 8.4 Hz,
1H), 6.78 (q, J ¼ 8.0 Hz, 1H), 6.40 (d, J ¼ 2.8 Hz, 1H), 6.29 (dd,
J ¼ 9.6 Hz, 2.4 Hz, 1H); HRMS calculated for C20H13N4OClF3
[M ꢁ H]ꢁ: 465.0577. Found: 465.0584.
6.2.2.1. 4-(3-(3-(Trifluoromethoxy)benzylamino)phenoxy)-N-meth-
ylpyridine-2-carbo-xamide (14). 1H NMR (400 MHz, CDCl3):
d 8.31
(d, J ¼ 5.6 Hz, 1H), 7.98 (brs, 1H), 7.70 (d, J ¼ 2.4 Hz, 1H), 7.34 (t,
J ¼ 8.0 Hz, 1H), 7.26 (d, J ¼ 7.6 Hz, 1H), 7.18e7.14 (m, 2H), 7.10 (d,
J ¼ 8.0 Hz, 1H), 6.90 (dd, J ¼ 5.6 Hz, 2.4 Hz, 1H), 6.47 (dd, J ¼ 8.0 Hz,
2.0 Hz, 1H), 6.39 (dd, J ¼ 7.6 Hz, 2.0 Hz, 1H), 6.28 (t, J ¼ 2.0 Hz, 1H),
4.31 (s, 2H), 4.27 (s, 1H), 2.98 (d, J ¼ 4.8 Hz, 3H); 13C NMR (100 MHz,
6.2.2.8. 4-(3-(3-(Trifluoromethoxy)benzylamino)phenoxy)benzene-
1,2-diamine (21). 1H NMR (400 MHz, CDCl3):
d
7.33 (t, J ¼ 8.0 Hz,
1H), 7.27e7.22 (m, 1H), 7.18 (s, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 7.04 (t,
J ¼ 8.0 Hz, 1H), 6.62 (d, J ¼ 8.4 Hz, 1H), 6.39 (d, J ¼ 2.4 Hz, 1H), 6.36
(dd, J ¼ 8.4 Hz, 2.4 Hz,1H), 6.31e6.24 (m, 2H), 6.19 (t, J ¼ 2.4 Hz,1H),
4.28 (d, J ¼ 5.6 Hz, 2H), 4.08 (t, J ¼ 5.6 Hz, 1H), 3.44 (brs, 2H), 3.17
CDCl3):
d 166.2, 164.6, 154.9, 152.1, 149.7, 149.6, 149.5, 149.5, 141.3,
130.8, 130.0, 125.5, 124.2, 121.6, 119.7, 119.7, 119.1, 116.5, 114.1, 110.4,
110.3, 109.6, 104.8, 47.6, 26.0; HRMS calculated for C21H19N3O3F3
[M þ H]þ: 418.1379. Found: 418.1385.
(brs, 2H); 13C NMR (100 MHz, MeOD):
d 161.2, 151.4, 151.1, 150.7,
150.7, 144.5, 137.8, 131.3, 130.9, 130.7, 126.9, 125.7, 123.1, 120.5, 120.1,
118.4, 111.2, 109.2, 108.1, 107.1, 103.1, 47.9; HRMS calculated for
C20H19N3O2F3 [M þ H]þ: 390.1429. Found: 390.1421.
6.2.2.2. 4-(3-(3-(Trifluoromethyl)benzene-sulfonylamino)phenoxy)-
N-methylpyridine-2-carboxamide (15). 1H NMR (400 MHz, CDCl3):
6.2.2.9. 4-(3-(3-(Trifluoromethoxy)benzene-sulfonylamino)-phe-
noxy)benzene-1,2-diamine (22). 1H NMR (400 MHz, CDCl3):
d
8.36 (d, J ¼ 5.6 Hz, 1H), 8.20e8.08 (m, 2H), 7.93 (d, J ¼ 8 Hz, 1H),
7.77 (d, J ¼ 8 Hz, 1H), 7.60 (t, J ¼ 8 Hz, 1H), 7.54 (d, J ¼ 2.4 Hz, 1H),
d
7.96 (s, 1H), 7.88 (d, J ¼ 8.0 Hz, 1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 7.56

K.-F. Chen et al. / European Journal of Medicinal Chemistry 55 (2012) 220e227
227
(t, J ¼ 8.0 Hz, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H), 6.70 (td, J ¼ 8.0 Hz,
2.0 Hz, 2H), 6.61 (d, J ¼ 8.0 Hz, 1H), 6.55 (t, J ¼ 2.0 Hz, 1H), 6.32 (d,
J ¼ 2.8 Hz, 1H), 6.25 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 3.84 (brs, 2H), 3.28
6.3.5. Phosphatase and kinase activity
The RediPlate 96 EnzChek_Tyrosine Phosphatase Assay Kit (R-
22067) was used for SHP-1 activity assay (Molecular Probes,
Carlsbad, CA).
(brs, 2H); HRMS calculated for C19H17N3O3F3S [M
424.0943. Found: 424.0936.
þ
H]þ:
6.3.6. Xenograft tumor growth
6.2.2.10. 5-(3-((3-(Trifluoromethoxy)benzyl)amino)phenoxy)-1H-
benzo[d]imidazol-2 (3H)-one (23). 1H NMR (400 MHz, MeOD):
Male NCr athymic nude mice (5e7 weeks of age) were obtained
from the National Laboratory Animal Center (Taipei, Taiwan). All
experimental procedures using these mice were performed in
accordance with protocols approved by National Taiwan University.
When PLC5 tumors reached 100 mm3, mice received compound 2
(10 mg/kg) p.o. once daily. Controls received vehicle.
d
7.33 (t, J ¼ 8.0 Hz, 1H), 7.26 (d, J ¼ 7.6 Hz, 1H), 7.16 (s, 1H), 7.08 (d,
J ¼ 8.0 Hz, 1H), 7.01e6.97 (m, 1H), 6.93 (d, J ¼ 8.4 Hz, 1H), 6.66e6.60
(m, 2H), 6.31 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 6.18 (dd, J ¼ 8.0 Hz, 2.4 Hz,
1H), 6.09 (d, J ¼ 2.0 Hz, 1H), 4.27 (s, 2H); HRMS calculated for
C21H17N3O3F3 [M þ H]þ: 416.1222. Found: 416.1221.
Acknowledgments
6.2.2.11. N-(3-((2-Oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)
phenyl)-3-(trifluoromethyl)benzenesulfonamide
(400 MHz, CDCl3):
1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.53 (t, J ¼ 8.0 Hz, 1H), 7.10 (t, J ¼ 8.0 Hz,
1H), 6.85 (d, J ¼ 8.0 Hz, 1H), 6.79 (d, J ¼ 8.0 Hz, 1H), 6.66 (dd,
J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.62e6.53 (m, 2H), 6.49 (s, 1H); HRMS
(24). 1H
NMR
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.
d
9.65 (brs, 2H), 7.99 (s, 1H), 7.91 (d, J ¼ 8.0 Hz,
calculated for C20H13N3O4F3S [M
448.0588.
ꢁ
H]ꢁ: 448.0579. Found:
Appendix A. Supplementary material
6.3. Biological assays
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.07.023.
6.3.1. Cell culture
PLC5 cells were maintained in DMEM supplemented with 10%
FBS, 100 units/mL penicillin G, 100 g/mL streptomycin sulfate and
25
g/mL amphotericin B in a 37 ꢀC humidified incubator in an
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