Sorafenib derivatives induce apoptosis through inhibition of STAT3 independent of Raf

Article abstract of DOI:10.1016/j.ejmech.2011.04.007

STAT3 is a transcription factor that modulates survival-directed transcription. It is persistently activated in many human cancers. Literature has shown that sorafenib, Raf kinase inhibitor, reduces Phospho-STAT3 and induces cell death. A series of sorafenib derivatives were synthesized as new inhibitors for STAT3. Urea, sulfonamide, and carboxamide linkers brought out different SARs from the end of sorafenib. Urea and carboxamide linked derivatives showed greater inhibition against STAT3 activity than sulfonamide linked derivatives. In particular, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4- (4-cyanophenoxy)phenyl)urea (1), a urea linker, was as potent as sorafenib in reducing P-STAT3 level and cell death but no inhibition for Raf activity. Such result provides a new lead for the design of STAT3 inhibitors.

Full text of DOI:10.1016/j.ejmech.2011.04.007

European Journal of Medicinal Chemistry 46 (2011) 2845e2851
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Sorafenib derivatives induce apoptosis through inhibition of STAT3
independent of Raf
,
b
,
c
,
d
,
,b,c,d,
Kuen-Feng Chen a
e, Wei-Tien Tai a
e, Jui-Wen Huang f, Cheng-Yi Hsu g, Wei-Lin Chen g,
,
b,
c
,
d
,
,
b,
c
,
d,
,
*
Ann-Lii Cheng a
e, Pei-Jer Chen a
e, Chung-Wai Shiau g
a Department of Medical Research, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei, Taiwan, Republic of China
b Department of Oncology, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei, Taiwan, Republic of China
c Department of Internal Medicine, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei, Taiwan, Republic of China
d Graduate Institute of Molecular Medicine, National Taiwan University College of Medicine, Taiwan
e National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, 7, Chung-Shan South Road, Taipei, Taiwan, Republic of China
f Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, 195, Sec. 4, Chung Hsing Rd., Chutung, Hsinchu, Taiwan
g Institute of Biopharmaceutical Sciences, National Yang-Ming University, 155, Sec. 2, Linong Street, Taipei, Taiwan, Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
STAT3 is a transcription factor that modulates survival-directed transcription. It is persistently activated
in many human cancers. Literature has shown that sorafenib, Raf kinase inhibitor, reduces Phospho-
STAT3 and induces cell death. A series of sorafenib derivatives were synthesized as new inhibitors for
STAT3. Urea, sulfonamide, and carboxamide linkers brought out different SARs from the end of sorafenib.
Urea and carboxamide linked derivatives showed greater inhibition against STAT3 activity than sulfon-
amide linked derivatives. In particular, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophenoxy)
phenyl)urea (1), a urea linker, was as potent as sorafenib in reducing P-STAT3 level and cell death but no
inhibition for Raf activity. Such result provides a new lead for the design of STAT3 inhibitors.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 15 September 2010
Received in revised form
24 March 2011
Accepted 3 April 2011
Available online 14 April 2011
Keywords:
Sorafenib
STAT3
Apoptosis
1. Introduction
suggesting that the SHP-1 gene acts as a tumor suppressor [7]. From
the drug discovery point of view, development of a small molecule
Signal transducer and activator of transcription 3 (STAT3) is
a transcription factor that regulates cell growth and survival by
modulating the expression of target genes [1]. It acts as an oncogene
which is constitutively active in many cancers including liver, lung,
head and neck, prostate, and breast as well as myeloma and
leukemia [2e6]. In a xenograft study, inhibition of STAT3 resulted in
an increase in mouse survival. A key factor that regulates STAT3
activity is Src homology-2 containing protein tyrosine phosphatase-
1(SHP-1). From a mechanistic perspective, SHP-1 exhibits protein
phosphatase activity which reduces the level of Phospho-STAT3 (P-
STAT) and subsequently blocks the dimerization of P-STAT3. There-
fore, expression of target genes, such as cyclin D1 and survivin
transcribed by STAT3, is significantly reduced. In addition, extensive
studies of SHP-1 protein and SHP-1 mRNA showed that expression
level of SHP-1 was low in most cancer cells. Genetic increase in SHP-
1 in cancer cells resulted in the suppression of cell growth,
which can reduce P-STAT3 and increase SHP-1 level is a promising
direction for cancer therapy.
A number of agents, including the natural products 5-hydroxy-
2-methyl-1,4-naphthoquinone (a vitamin K3 analog), betulinic acid
and boswellic acid have been reported to inhibit P-STAT3 level and
reduces cell proliferation [8e13]. Although these agents show the
ability to inhibit STAT3 activity, the dosage needed is higher than
the clinical limitation. In addition, these natural products have the
potential for off-target effects and activation or inactivation of other
enzyme activity at high concentrations.
Sorafenib (BAY43-9006, Nexavar) is used clinically for renal
carcinoma and hepatocellular carcinoma (HCC). It targets the c-Raf
and b-Raf kinases and prolongs the survival of renal and HCC
patients. Studies have shown that sorafenib was able to reduce the
phosphorylation level of STAT3 in medulloblastoma and esopha-
geal carcinoma [14,15]. Further study showed that sorafenib
reduced P-STAT3 through activating SHP-1 phosphatase activity in
HCC cells. To date, no correlation has been found between inhibi-
tion of Raf and activation of SHP-1 by the treatment with sorafenib.
* 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 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.007

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K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
In addition, there are no reports showing that sorafenib derivatives
inhibit STAT3 activity without affecting Raf activity.
In light of the ability of sorafenib to reduce the P-STAT3 level,
here we synthesized novel sorafenib derivatives which retain the
ability to reduce cell survival, but do not affect Raf kinase activity.
We further provide evidence that these novel sorafenib derivates
mediate apoptosis through downregulation of P-STAT3 indepen-
dent of Raf activity.
2. Chemistry
According to the X-ray structure of b-Raf and sorafenib, the
amide group connected to the pyridine ring of sorafenib provides
a hydrogen donor and forms a hydrogen bond with b-Raf in the ATP
binding pocket [16]. The urea moiety of sorafenib also binds to the
allosteric region by hydrogen bonding. To address the relationship
between Raf kinase activity and downregulation of P-STAT3, we
used a chemical approach to reduce the hydrogen bonding inter-
action between the amide group of sorafenib with Raf by replacing
amido group with a phenylcyano group (Fig. 1). The Raf kinase
activity of the resulting sorafenib analog 1 was tested by ELISA. To
test the structure activity relationship of the downregulation of P-
STAT3, we replaced the urea functional group in the sorafenib
backbone with various amide and sulfonamide yielding
compounds 2e11. In addition, we replaced the pyridine ring with
quinoline and used it as a platform to carry out structural modifi-
cation, which generated a series of compounds 12e19 and 20e25.
These sorafenib derivatives were synthesized according to
a general procedure described in Fig. 2. The inhibition of STAT3 and
the Raf-1 activation by these compounds were tested by ELISA and
western blot.
Fig. 2. General synthetic procedure for series AeC compounds: (a), K2CO3, DMF; (b),
pyridine, THF.
12e25. The amide linker showed different conformation from the
sulfonyl linker, exhibiting better activity than sulfonyl linker
compounds. For example, compound 16 showed a better cell toxicity
than compound 12. Compound 25 showed cytotoxicity comparable
to sorafenib and 1. We concluded that the urea and amide linkers
exhibited the most potent cell toxicity in PLC5 cells.
3. Biological evaluations
3.1. Development of sorafenib derivative lacking inhibition of Raf
kinase activation
3.3. Mechanistic validation of the mode of action of sorafenib
derivatives
We synthesized
a sorafenib derivative without providing
hydrogen donor ability by replacing the pyridine ring and amide
functional group with phenyl cyanide and exploring the ability of
sorafenib and compound 1 to inhibit Raf kinase activity in PLC5
cells. Sorafenib was able to inhibit 50% of the Raf-1 kinase activity of
Previously, the sulfonyl linker and amide linker were shown
to exhibit low activity against Raf kinase compared with sor-
afenib [17]. However, some of these compounds showed good
the untreated cells in the PLC5 cells at 5
mM (Fig. 3). In contrast,
compound 1 treated cells showed the same Raf-1 activity as vehicle
control.
3.2. Structure activity relationship of replacement of urea group
and pyridine ring in cell death
The urea functional group linkage of sorafenib was replaced with
various substitutes with sulfonylamide linkage, generating
compounds 2e6 (Fig. 4). None of these derivatives within the elec-
tron donating or electron withdrawing group showed greater cell
toxicity than sorafenib and compound 1. Next, we changed the
pyridine to a quinoline ring and amide linker to generate compounds
Fig. 3. Raf-1 activity of sorafenib and compound 1. Huh-7 cells were exposed to sor-
afenib or compound 1 at 10
mM for 24 h and cell lysates were analyzed for raf-1
Fig. 1. Chemical structure of sorafenib and compound 1.
activity. Columns, mean; bars, SD (n ¼ 3). *P < 0.05.

K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
2847
a
b
c
Fig. 4. Chemical structure of compounds 2e25 and IC50 of growth inhibition in PLC cells.
cytotoxicity in cancer cells. Therefore, we hypothesized that
sorafenib derivatives downregulate P-STAT3 and enhance cell
apoptosis. The sorafenib derivatives were tested for the inhibi-
tion of P-STAT3 by the ELISA. As shown in Fig. 5, sulfonyl linker
compounds showed no appreciable change in P-STAT3.
Compound 1 and some of the amide linker compounds showed
a high degree of dephosphorylation of STAT3. The decreased
level of P-STAT3 induced by these derivatives was correlated
with cell toxicity. In the other words, these derivatives induced
cell death in part through inhibition of STAT3. In addition, we
also tested the downstream signal pathway after the inhibition
of P-STAT3. Expression levels of the cyclin D1 and survivin,
downstream target genes of STAT3, were assessed in compounds
1 and 12. As shown in Fig. 6, compound 1 with STAT3 inhibitory
activity, was able to reduce cyclin D1 and survivin level, but
compound 12 had no effect on either protein. DNA fragmenta-
tion and flow cytometry analysis of PLC5 cells treated with
compound 1 showed that cell death was attributed to the
inhibition of STAT3 and further induced the apoptotic signal
(Fig. 7).
4. Discussion
Recent data suggest that STAT3 serves as a key regulator of
cell survival by activating its downstream survival signal
pathway. This function is protective for chemotherapy cancer
patients and renders STAT3 a good target for drug discovery. For
example, a recent study demonstrated that cisplatin resistance in
head and neck squamous cell carcinoma was associated with
a high expression of STAT3 which played a role in the induction
of reprogrammed survival pathways that circumvent cisplatin
treatment [18]. Thus, this study focuses on the structural modi-
fication of sorafenib to develop a novel class of STAT3 inhibitors.
Our premise that sorafenib inhibition of Raf and STAT3 could be
structurally dissociated was borne out by compound 1, which,
devoid of Raf activity, exhibited the level of downregulation of P-
STAT3 as sorafenib. Presumably, the cyanide group of compound
1 reduces its interaction with Raf. Subsequent modifications of
sorafenib by changing the linker and pyridine ring to amide and
quinoline (compounds 1, 16, and 25, respectively) resulted in
a decrease in STAT3-repressing potency.

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K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
Fig. 5. Elisa analysis of the inhibitory effects of compounds 1e25 versus sorafenib, each at 10
bars, SD (N ¼ 3).
mM, on the IL-6 stimulated P-STAT in PLC5 cells after 24 h of treatment. Columns, mean,
5. Conclusion
We synthesized a series of sorafenib derivatives which showed
the inhibition of STAT3 activity. In addition, these derivatives
showed the cytotoxicity in HCC cells without reducing the Raf
activity. Among these compounds, compound 1 demonstrated the
potency as sorafenib in inhibition of P-STAT3 and cell growth. From
a mechanistic study, compound 1 also showed the reduction
expression of survivin and cyclin D1 after blocking the STAT3
signaling cascade. In addition, it provides a useful pharmacological
tool with which to study therapeutic relevance in the treatment of
HCC with disregulated STAT3 expression and drug resistance. Thus,
these data give the potential use of this anti-STAT3 agent in the
treatment of HCC. Testing of compound 1 in an in vivo HCC model is
currently being pursued.
Fig. 6. Western blot analysis of the effect of compounds 1 and 12, each at 5
10 mM on the phosporylation of P-STAT3, STAT3, cyclin D and survivin in PLC5 cells in
mM and
FBS-containing medium after 24 h of treatment.
A
B
Fig. 7. (A). Elisa analysis of cell death induced by compound 1 and 12, at 5, and 10
compound 1 and 12, at 5, and 10 M, after 24 h of treatment in PLC5 cells.
mM, after 24 h of treatment in PLC5 cells (B). Flow cytometry analysis of cell death induced by
m

K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
2849
6. Experimental section
6.2.2.2. N-methyl-4-(4-(4-nitrophenylsulfonamido)phenoxy)picolin-
amide (3). 1H NMR (400 MHz, CDCl3):
(d, 2H, J ¼ 8.8 Hz), 8.07 (brs, 1H), 7.93 (d, 2H, J ¼ 8.8 Hz), 7.49 (s, 1H),
7.17 (d, 2H, J ¼ 8.8 Hz), 7.01e6.98 (m, 3H), 3.00 (d, 3H, J ¼ 5.2 Hz);
HRMS calculated for C19H16N4O6S (M þ H): 428.0791. Found:
428.0798.
d
8.39 (d, 1H, J ¼ 5.6 Hz), 8.30
6.1. Material
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on Bruker DPX300 (400 MHz) instruments. Chemical
shifts are reported in 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 plate (Merck).
Chromatographic purification was carried on silica gel columns 60
(0.063e0.200 mm or 0.040e0.063 mm, Merck), basic silica gel.
Commercial reagents and solvents were used without additional
purification. Abbreviations are used as follows: CDCl3, deuterated
chloroform; DMSO-d6, dimethyl sulfoxide-d6; EtOAc, ethyl acetate;
DMF, N,N-dimethylformamide; MeOH, methanol; THF, tetrahy-
drofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide; NMP, N-
methylpyrrolidone. High resolution mass spectra were recorded on
a FINNIGAN MAT 95S mass spectrometer.
6.2.2.3. 4-(4-(4-Fluorophenylsulfonamido)phenoxy)-N-methylpicoli-
namide (4). 1H NMR (400 MHz, CDCl3):
d
8.37 (d, 1H, J ¼ 5.6 Hz),
8.00 (brs, 1H), 7.77e7.43 (m, 2H), 7.57 (s, 1H), 7.17e7.09 (m, 4H),
6.99e6.93 (m, 4H), 3.00 (d, 3H, J ¼ 4.8 Hz); NMR (100 MHz, CDCl3):
d
166.5, 165.9, 164.6, 163.9, 152.1, 151.2, 149.7, 135.0, 134.0, 130.0,
129.9, 124.1, 121.7, 116.4, 116.2, 114.5, 109.8, 26.19; HRMS calculated
for C19H16FN3O4S (M þ H): 401.0846. Found: 401.0849.
6.2.2.4. 4-(4-(4-tert-Butylphenylsulfonamido)phenoxy)-N-methylpi-
colinamide (5). 1H NMR (400 MHz, CDCl3):
d
8.33 (d, 1H, J ¼ 6.0 Hz),
8.21 (brs, 1H), 7.79 (brs, 1H), 7.69 (d, 2H, J ¼ 6.8 Hz), 7.62 (s, 1H), 7.44
(d, 2H, J ¼ 6.8 Hz), 7.15 (d, 2H, J ¼ 6.8 Hz), 6.91 (s, 2H, J ¼ 6.8 Hz),
6.88e6.86 (m, 1H), 2.98 (d, 3H, J ¼ 5.2 Hz); 13C NMR (100 MHz,
CDCl3):
d 166.0, 164.6, 156.8, 152.2, 150.8, 149.7, 136.1, 134.4, 127.0,
126.1, 123.6, 121.6, 114.1, 110.1, 35.1, 30.1, 26.1; HRMS calculated for
C23H25N3O4S (M þ H): 439.1566. Found: 439.1564.
6.2. Chemical synthesis
6.2.2.5. N-methyl-4-(4-(naphthalene-2-sulfonamido)phenoxy)picoli-
6.2.1. Procedures for compound 1
namide (6). 1H NMR (400 MHz, CDCl3):
d 8.34 (s, 1H), 8.30 (d, 1H,
To a 50 mL THF solution of triphosgen (0.30 g, 1.0 mmol), 4-
chloro-3-(trifluoromethyl)aniline (0.21g, 1.1mmol) and 2 equiva-
lent of triethyl amine were added. The mixture was heated to 50 ꢀC
for 30 min. After the temperature was back to room temperature, 4-
(4-aminophenoxy)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 1 (0.34 g, 80%).
J ¼ 5.2 Hz), 8.05e8.02 (m,1H), 7.89e7.83 (m, 4H), 7.74 (dd,1H, J ¼ 8.4,
1.6 Hz), 7.60e7.52 (m, 3H), 7.16 (d, 2H, J ¼ 8.8 Hz), 6.88 (d, 2H,
J ¼ 8.8 Hz), 6.84e6.82 (m, 1H); 13C NMR (100 MHz, CDCl3): 165.9,
164.6,152.1,151.0,149.7,135.9,134.9,134.2,132.0,129.4,129.3,128.9,
128.7, 127.9, 127.5, 123.9, 122.2, 121.6, 114.2, 110.1, 26.2; HRMS
calculated for C23H19N3O4S (M þ H): 433.1096. Found: 433.1079.
6.2.2.6. 4-(4-(2-Bromo-4-(trifluoromethyl)phenylsulfonamido)phen-
oxy)-N-methylpicolinamide (7). 1H NMR (400 MHz, CDCl3):
d 8.35
(d, 1H, J ¼ 5.6 Hz), 8.15 (d, 1H, J ¼ 8.0 Hz), 7.79 (brs, 1H), 7.96 (s, 1H),
7.67 (d, 2H, J ¼ 8.0 Hz), 7.57 (s, 1H), 7.18 (d, 2H, J ¼ 9.2 Hz), 6.95 (d,
2H, J ¼ 9.2 Hz), 6.90e6.88 (m, 1H), 2.98 (d, 3H, J ¼ 5.2 Hz); 13C NMR
6.2.1.1. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophe-
noxy)phenyl)urea (1). 1H NMR (400 MHz, CDCl3):
d
9.17 (s,1H), 8.94
(100 MHz, CDCl3): d 165.7, 164.5, 152.2, 151.6, 149.8, 141.5, 136.1,
(s,1H), 8.10 (s,1H), 7.81 (d, 2H, J ¼ 6.8), 7.63e7.59 (m, 2H), 7.54 (d, 2H,
135.8, 135.5, 135.2, 132.7, 132.2 (m), 124.9 (m), 124.1, 123.5, 121.7,
120.8, 120.4114.5, 110.0, 26.1; HRMS calculated for C20H15BrF3N3O4S
(M þ H): 528.9919. Found: 528.9917.
J ¼ 7.2 Hz), 7.10 (d, 2H, J ¼ 6.8 Hz), 7.05 (d, 2H, J ¼ 7.2 Hz); 13C NMR
(100 MHz, methanol-d4):
d 163.7, 163.6, 154.8, 151.4, 151.2, 140.1,
137.7, 137.4, 135.3, 132.9, 129.7, 129.4, 129.1, 128.8, 128.3, 125.6, 125.5,
125.4, 124.2, 122.9, 122.4, 122.3, 122.1, 120.2, 119.7, 118.8, 118.7, 118.6,
118.6, 106.5, 106.4; HRMS calculated for C21H13ClF3N3O2 (M þ H):
431.0648. Found: 431.0656.
6.2.2.7. N-methyl-4-(4-(2-nitrophenylsulfonamido)phenoxy)picolin-
amide (8). 1H NMR (400 MHz, CDCl3):
d
8.37 (d, 1H, J ¼ 6.0 Hz), 7.98
(brs, 1H), 7.86e7.83 (m, 2H), 7.72e7.68 (m, 2H), 7.55 (s, 1H), 7.24 (d,
2H, J ¼ 8.8 Hz), 6.98 (d, 2H, J ¼ 8.8 Hz), 6.94e6.92 (m, 1H), 2.98 (d,
3H, J ¼ 4.8 Hz); HRMS calculated for C19H16N4O6S (M þ H):
428.0791. Found: 428.0796.
6.2.2. General procedures for the synthesis of compounds 2e25
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 70% to 95% yield.
6.2.2.8. 4-(4-(3,5-Bis(trifluoromethyl)benzamido)phenoxy)-N-meth-
ylpicolinamide (9). 1H NMR (400 MHz, CDCl3):
d 9.92 (s, 1H), 8.40 (s,
1H), 8.33 (d, 1H, J ¼ 5.6 Hz), 8.10 (q, 1H, J ¼ 5.2 Hz), 7.90 (s, 1H), 7.71
(d, 2H, J ¼ 8.8 Hz), 7.40 (d,1H, J ¼ 2.8 Hz), 6.99e6.97 (m,1H), 6.93 (d,
2H, J ¼ 8.8 Hz), 2.91 (d, 3H, J ¼ 4.8 Hz); 13C NMR (100 MHz,
methanol-d4):
d 167.8, 166.8, 165.1, 153.4, 151.8151.6, 138.6, 137.6,
133.6, 133.3, 133.0, 132.6, 129.4 (d), 126.2 (m), 126.0, 124.2, 123.2,
122.4, 115.2, 110.7, 26.4; HRMS calculated for C22H15F6N3O3
(M þ H): 483.1018. Found:483.1017.
6.2.2.1. N-methyl-4-(4-(phenylsulfonamido)phenoxy)picolinamide
(2). 1H NMR (400 MHz, CDCl3):
d
8.36 (d, 1H, J ¼ 5.6 Hz), 8.01
(brs, 1H), 7.76 (d, 2H, J ¼ 7.6 Hz), 7.59 (s, 1H), 7.54 (t, 1H,
J ¼ 8.0 Hz), 7.46 (t, 2H, J ¼ 8.0 Hz), 7.12 (d, 2H, J ¼ 8.8 Hz), 6.94 (d,
2H, J ¼ 8.8 Hz), 6.92e6.90 (m, 1H), 3.00 (d, 3H, J ¼ 5.2 Hz); 13C
6.2.2.9. 4-(4-(5-Fluoro-2-(trifluoromethyl)benzamido)phenoxy)-N-me-
thylpicolinamide (10). 1H NMR (400 MHz, CDCl3):
d 8.66 (d, 1H,
NMR (100 MHz, CDCl3):
d
166.0, 164.6, 152.1, 151.0, 149.7, 138.9,
J ¼ 12.4 Hz), 8.31e8.26 (m, 2H), 7.93 (s,1H), 7.70e7.65 (m, 3H), 7.56 (t,
1H, J ¼ 2.4 Hz), 7.24e7.19 (m, 1H), 7.02 (d, 2H, J ¼ 6.4 Hz), 6.89e6.87
(m, 1H), 2.90 (d, 3H, J ¼ 3.2 Hz); 13C NMR (100 MHz, methanol-d4):
134.2, 133.0, 129.0, 127.1, 123.9, 121.6, 114.3, 109.9, 26.1; HRMS
calculated for C19H17N3O4S (M þ H): 383.0940. Found: 383.0941.

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K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
d
166.2, 164.5, 162.9, 160.4, 160.0, 159.9, 152.2, 150.4, 149.7, 135.0, 130.6
7.16 (d, 2H, J ¼ 9.0 Hz), 6.62 (d, 1H, J ¼ 5.0 Hz); HRMS calculated for
C24H13F6N3O4 (M þ H): 521.0810. Found: 521.0814.
(m),129.8 (m),128.3,128.1,127.7,127.4,124.5,122.5,122.4,122.3,121.8,
121.5, 117.2, 117.0, 114.1, 110.1, 26.1; HRMS calculated for C21H15F4N3O3
(M þ H): 433.1050. Found: 433.0152.
6.2.2.17. 2-Fluoro-N-(4-(quinolin-4-yloxy)phenyl)-5-(trifluoromethyl)
benzamide (18). 1H NMR (400 MHz, CDCl3):
d 8.83 (d, 1H,
6.2.2.10. N-methyl-4-(4-(4-(trifluoromethyl)benzamido)phenoxy)pi-
J ¼ 12.8 Hz), 8.68 (d, 1H, J ¼ 5.2 Hz), 8.39e8.30 (m, 2H), 8.11 (d, 1H,
J ¼ 8.4 Hz), 7.79e7.67 (m, 4H), 7.58 (t, 1H, J ¼ 8.0 Hz), 7.27e7.22 (m,
1H), 7.18(d, 2H, J ¼ 9.2 Hz), 6.56 (d, 1H, J ¼ 5.2 Hz); 13C NMR
colinamide (11). 1H NMR (400 MHz, CDCl3):
d 9.45 (s, 1H), 8.31 (d,
1H, J ¼ 5.6 Hz), 8.15 (s, 1H), 8.08 (d, 2H, J ¼ 8.0 Hz), 7.71e7.65 (m,
3H), 7.50 (d, 1H, J ¼ 2.4 Hz), 7.47 (t, 1H, J ¼ 8.0 Hz), 6.96e6.91 (m,
(100 MHz, methanol-d4):
d 164.4, 164.2, 163.8, 161.7, 151.9, 151.8,
3H), 2.92 (d, 3H, J ¼ 5.2 Hz); 13C NMR (100 MHz, CDCl3):
d
166.4,
149.7, 137.5, 132.1 (m), 131.2 (m), 128.9 (m), 128.3, 128.1, 127.9, 126.5,
126.3, 123.7, 123.6, 123.0, 122.7, 122.6, 119.1, 119.8, 118.7, 118.5, 105.2;
HRMS calculated for C23H14F4N2O2 (M þ H): 426.0991. Found:
426.0991.
164.8, 164.7, 151.8, 149.9149.8, 138.8, 135.5, 131.4, 131.1, 130.8, 130.7,
130.5, 129.1, 128.1, 125.0, 124.3 (m), 122.6, 122.3, 121.2, 114.4, 109.5,
26.1; HRMS calculated for C21H16F3N3O3 (M þ H): 415.1144. Found:
415.1146.
6.2.2.18. 2-Fluoro-N-(4-(8-nitroquinolin-4-yloxy)phenyl)-5-(trifluor-
6.2.2.11. 2-Nitro-N-(4-(quinolin-4-yloxy)phenyl)-4-(trifluoromethyl)
omethyl)benzamide (19). 1H NMR (400 MHz, CDCl3):
d 8.81 (d, 1H,
benzenesulfonamide (12). 1H NMR (400 MHz, CDCl3):
d
8.71 (d, 1H,
J ¼ 5.2 Hz), 8.59 (d, 1H, J ¼ 8.8 Hz), 8.53e8.47 (m, 2H), 8.06 (d, 1H,
J ¼ 7.6 Hz), 7.83e7.77 (m, 3H), 7.64 (t, 1H, J ¼ 7.6 Hz), 7.37e7.32 (m,
1H), 7.23e7.20 (m, 2H), 6.68 (d, 1H, J ¼ 5.2 Hz); HRMS calculated for
C23H13F4N3O4 (M þ H): 471.0842. Found: 471.0850.
J ¼ 5.2 Hz), 8.28 (d, 1H, J ¼ 8.4 Hz), 8.12 (d, 1H, J ¼ 8.4 Hz), 8.10 (s,
1H), 8.05 (d, 1H, J ¼ 8.4 Hz), 7.89 (d, 1H, J ¼ 8.4 Hz), 7.80e7.76 (m,
1H), 7.61e7.57 (m, 1H), 7.31 (d, 2H, J ¼ 8.8 Hz), 7.13 (d, 1H,
J ¼ 8.8 Hz), 6.53 (d, 1H, J ¼ 5.2 Hz); HRMS calculated for
C22H14F3N3O5S (M þ H): 489.0606. Found: 489.0610.
6.2.2.19. N-(2-methyl-4-(8-nitroquinolin-4-yloxy)phenyl)-3,5-bis(tri-
fluoromethyl)benzamide (20). 1H NMR (400 MHz, CDCl3):
d 9.86 (s,
6.2.2.12. 2-Nitro-N-(4-(8-nitroquinolin-4-yloxy)phenyl)-4-(trifluoro-
1H), 8.45 (d, 1H, J ¼ 5.2 Hz), 8.38 (s, 2H), 8.31 (d, 1H, J ¼ 8.4 Hz), 7.92
(s, 1H), 7.89 (d, 1H, J ¼ 8.4 Hz), 7.69e7.63 (m, 2H), 7.53 (t, 1H,
J ¼ 7.6 Hz), 7.33 (s,1H), 7.28 (d,1H, J ¼ 8.4 Hz), 6.33 (d,1H, J ¼ 5.2 Hz);
HRMS calculated for C25H15F6N3O4 (M þ H): 535.0967 Found:
535.0956.
methyl)benzenesulfonamide (13). 1H NMR (400 MHz, CDCl3):
d 8.78
(d, 1H, J ¼ 5.2 Hz), 8.51 (d, 1H, J ¼ 8.8 Hz), 8.19 (s, 1H), 8.12e8.02 (m,
3H), 7.89 (t, 1H, J ¼ 9.6 Hz), 7.62 (t, 1H, J ¼ 8.4 Hz), 7.34 (d, 2H,
J ¼ 9.6 Hz), 7.15 (d, 2H, J ¼ 9.6 Hz), 6.91 (d, 1H, J ¼ 6.8 Hz), 6.59 (d,
1H, J ¼ 5.2 Hz), 6.55 (d, 1H, J ¼ 6.8 Hz); HRMS calculated for
C22H13F3N4O7S (M þ H): 534.0457. Found: 534.0423.
6.2.2.20. N-(4-(8-aminoquinolin-4-yloxy)-2-methylphenyl)-3,5-bis(-
trifluoromethyl)benzamide (21). 1H NMR (400 MHz, CDCl3):
d 8.49
6.2.2.13. 2-Bromo-N-(4-(quinolin-4-yloxy)phenyl)-4-(trifluoromethyl)
(d, 1H, J ¼ 2.0 Hz), 8.27 (s, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.65 (d, 1H,
J ¼ 8.0 Hz), 7.48e7.45 (m, 2H), 7.37e7.32 (m, 2H), 6.96 (d, 1H,
J ¼ 7.6 Hz), 4.95 (s, 2H), 2.16 (s, 3H); HRMS calculated for
C25H17F6N3O2 (M þ H): 505.1225. Found: 505.1216.
benzenesulfonamide (14). 1H NMR (400 MHz, CDCl3):
d 8.65 (d, 1H,
J ¼ 5.2 Hz), 8.25 (d, 1H, J ¼ 8.4 Hz), 8.18 (d, 1H, J ¼ 8.4 Hz), 8.07
(d, 1H, J ¼ 8.4 Hz), 7.98 (s, 1H), 7.73 (t, 1H, J ¼ 7.6 Hz), 7.67 (d, 1H,
J ¼ 8.4 Hz), 7.54 (t, 1H, J ¼ 7.6 Hz), 7.24 (d, 2H, J ¼ 8.8 Hz), 7.05 (d,
2H, J ¼ 8.8 Hz), 6.43 (d, 1H, J ¼ 5.2 Hz); 13C NMR (100 MHz,
6.2.2.21. N-(4-(8-acetamidoquinolin-4-yloxy)-2-methylphenyl)-3,5-
CDCl3):
d
161.3, 152.6, 150.9, 149.7, 141.4, 135.9, 135.6, 135.3,
bis(trifluoromethyl)benzamide (22). 1H NMR (400 MHz, CDCl3):
132.7, 132.4, 132.2 (m), 130.3, 129.1, 126.3, 124.9 (m), 124.4, 123.5,
122.1, 122.0, 121.9, 121.6, 121.3, 120.8, 120.4, 116.3, 104.4; HRMS
calculated for C22H14BrF3N2O3S (M þ H): 521.9861. Found:
521.9858.
d
9.77 (s,1H), 9.36 (s,1H), 8.65 (d,1H, J ¼ 7.2 Hz), 8.46 (s, 2H), 8.44 (d,
1H, J ¼ 5.2 Hz), 7.97 (s, 1H), 7.87 (d, 1H, J ¼ 8.4 Hz), 7.72 (d, 1H,
J ¼ 8.4 Hz), 7.50 (d, 1H, J ¼ 2.0 Hz), 7.37 (t,1H, J ¼ 8.0 Hz), 7.28 (d, 1H,
J ¼ 8.4 Hz), 6.41 (d, 1H, J ¼ 5.2 Hz), 2.26 (s, 3H), 2.10 (s, 3H); 13C NMR
(100 MHz, CDCl3):d169.2,163.0,161.2,152.0,148.8,139.5,137.2,136.7,
6.2.2.14. 2-Bromo-N-(4-(8-nitroquinolin-4-yloxy)phenyl)-4-(trifluo-
romethyl)benzenesulfonamide (15). 1H NMR (400 MHz, CDCl3):
133.8, 132.6, 132.3, 132.2, 132.0, 131.6, 127.8, 127.1, 126.3, 125.2 (m),
124.2, 121.5, 120.4, 118.8, 118.3, 116.7, 115.7, 113.8, 25.0, 15.4; HRMS
calculated for C27H19F6N3O3 (M þ H): 547.1331. Found: 547.1325.
d
8.76 (d,1H, J ¼ 5.2 Hz), 8.49 (d,1H, J ¼ 8.4 Hz), 8.18 (d,1H, J ¼ 8.4 Hz),
8.04 (d,1H, J ¼ 7.6 Hz), 7.99 (s,1H), 7.68 (d,1H, J ¼ 8.4 Hz), 7.60 (t,1H,
J ¼ 8.4 Hz), 7.25 (d, 2H, J ¼ 8.4 Hz), 7.07 (d, 2H, J ¼ 8.4 Hz), 6.53 (d,1H,
J ¼ 5.2 Hz); HRMS calculatedfor C22H13BrF3N3O5S (M þ H): 566.9711.
Found: 566.9706.
6.2.2.22. N-(3-(8-nitroquinolin-4-yloxy)phenyl)-3,5-bis(trifluorome-
thyl)benzamide (23). 1H NMR (400 MHz, CDCl3):
d 8.78 (d, 1H,
J ¼ 5.6 Hz), 8.58 (d, 1H, J ¼ 8.4 Hz), 8.52 (s, 1H), 8.31 (s, 2H), 8.22 (s,
1H), 8.08e8.04 (m, 2H), 7.71 (s, 1H), 7.64 (t, 1H, J ¼ 8.0 Hz),
7.53e7.49 (m, 2H), 7.03 (d, 1H, J ¼ 7.2 Hz), 6.71 (d, 1H, J ¼ 4.8 Hz);
HRMS calculated for C24H13F6N3O4 (M þ H): 521.0810. Found:
521.0821.
6.2.2.15. N-(4-(quinolin-4-yloxy)phenyl)-3,5-bis(trifluoromethyl)ben-
zamide (16). 1H NMR (400 MHz, CDCl3):
d 10.02 (s, 1H), 8.59 (d, 1H,
J ¼ 5.6 Hz), 8.37 (s, 2H), 8.34 (d,1H, J ¼ 8.4 Hz), 7.99 (d,1H, J ¼ 8.4 Hz),
7.92 (s, 1H), 7.80 (d, 2H, J ¼ 9.2 Hz), 7.70 (t, 1H, J ¼ 7.6 Hz), 6.56 (t, 1H,
J ¼ 7.6 Hz), 7.14 (d, 2H, J ¼ 9.2 Hz), 6.52 (d, 1H, J ¼ 5.2 Hz); 13C NMR
6.2.2.23. N-(3-(8-aminoquinolin-4-yloxy)phenyl)-3,5-bis(trifluorom-
(100 MHz, DMSO-d6):
d
162.6, 161.0, 151.5, 150.0, 149.2, 137.0, 136.1,
ethyl)benzamide (24). 1H NMR (400 MHz, CDCl3):
d 8.55 (d, 1H,
131.0, 130.7, 130.4, 130.3, 130.0, 128.8, 128.5 (m), 126.4, 125.2 (m),
124.5, 122.5, 121.7, 121.5, 121.3, 120.6, 104.3; HRMS calculated for
C24H14F6N2O2 (M þ H): 476.0959. Found: 476.0958.
J ¼ 4.8 Hz), 8.29 (s, 2H), 8.05 (s, 1H), 7.90 (s, 1H), 7.60 (d, 1H,
J ¼ 8.4 Hz), 7.55 (s, 1H), 7.70e7.43 (m, 2H), 7.34 (t, 1H, J ¼ 8.0 Hz),
7.01 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 1H, J ¼ 7.9 Hz), 6.63 (d, 1H,
J ¼ 4.8 Hz); 13C NMR (100 MHz, CDCl3):
d 163.1, 161.3, 155.3, 147.9,
6.2.2.16. N-(4-(8-nitroquinolin-4-yloxy)phenyl)-3,5-bis(trifluorome-
143.6, 139.9, 138.8, 136.5, 132.7, 132.4, 132.1, 131.7, 130.6, 127.5 (d),
127.0, 126.8, 125.3 (m), 124.1, 122.0, 118.7, 117.4, 117.2, 113.1, 111.1,
110.0, 105.4; HRMS calculated for C24H15F6N3O2 (M þ H): 491.1068.
Found: 491.1068.
thyl)benzamide (17). 1H NMR (400 MHz, CDCl3):
d 9.05 (s, 1H), 8.69
(d, 1H, J ¼ 5.0 Hz), 8.59 (d, 1H, J ¼ 5.0 Hz), 8.35 (s, 2H), 8.06 (d, 1H,
J ¼ 7.8 Hz), 7.97 (s,1H), 7.80 (d, 2H, J ¼ 9.0 Hz), 7.63 (t,1H, J ¼ 8.6 Hz),

K.-F. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 2845e2851
2851
6.2.2.24. N-(3-(8-acetamidoquinolin-4-yloxy)phenyl)-3,5-bis(trifluo-
romethyl)benzamide (25). 1H NMR (400 MHz, CDCl3):
9.77 (s, 1H),
financial support. C.W. S. also thanks the National Research Insti-
tute of Chinese Medicine for NMR support.
d
8.74 (d, 1H, J ¼ 7.6 Hz), 8.54 (d, 1H, J ¼ 5.2 Hz), 8.48 (s, 1H), 8.39 (s,
2H), 8.04 (s, 1H), 7.87 (d, 1H, J ¼ 8.4 Hz), 7.67e7.60 (m, 2H),
7.50e7.43 (m, 2H), 7.37 (t, 1H, J ¼ 8.0 Hz), 7.00 (d, 1H, J ¼ 8.4 Hz),
6.65 (d, 1H, J ¼ 5.2 Hz), 2.30 (s, 3H); 13C NMR (100 MHz, CDCl3):
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We thank the National Science Council, Taiwan (NSC98-2320-B-
010-005-My3) and National Yang Ming University, Taiwan for