Inhibition of the signal transducer and activator of transcription-3 (STAT3) signaling pathway by 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid esters

Article abstract of DOI:10.1021/jm701271y

The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester 4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the 7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC₅₀ of 170 nM. Compound 8 also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation in K562 cells.

Full text of DOI:10.1021/jm701271y

J. Med. Chem. 2008, 51, 4115–4121
4115
Inhibition of the Signal Transducer and Activator of Transcription-3 (STAT3) Signaling
Pathway by 4-Oxo-1-Phenyl-1,4-Dihydroquinoline-3-Carboxylic Acid Esters
Jun Xu,† Derek C. Cole,‡ Chao-Pei Betty Chang,† Ramzi Ayyad,‡ Magda Asselin,‡ Wenshan Hao,† James Gibbons,†
Scott A. Jelinsky,§ Kathryn A. Saraf,§ and Kaapjoo Park*,‡
Oncology and Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965, Biological
Technologies Department, Wyeth Research, 87 Cambridge Park DriVe, Cambridge, Massachusetts 02140
ReceiVed October 9, 2007
The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising
target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous
Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester
4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the
7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC50 of 170 nM. Compound 8
also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation
in K562 cells.
Introduction
Signal transducers and activators of transcription (STATs)a
are an important family of molecules that mediate signal
transduction in cells.1,2 They are latent in the cytoplasm until
activated by various extracellular signaling proteins such as
cytokines and growth factors. Upon stimulation, STATs are
activated by phosphorylation on specific tyrosine residues by
Janus kinase (JAK), receptor tyrosine kinases such as PDGF,
and nonreceptor tyrosine kinases such as the Src family
members. JAK activation may also be required in the latter two
activation pathways. Upon phosphorylation, STATs dimerize
and translocate into the nucleus, where they function as
transcription factors on their target genes.
Accumulating evidence indicates that STAT family members
play important roles in carcinogenesis and, in particular, STAT3
has emerged as a good target for cancer therapy.3–8 STAT3
Figure 1. Structures of STAT3 pathway inhibitors.
regulates genes that are important in cell proliferation (cyclin
D1, c-myc), cell survival (survivin, Bcl-XL, Mcl-1), angiogenesis
has been linked with hepatocellular carcinoma (SOCS-1 and
SOCS-3)11,12 and anaplastic large cell lymphoma (PIAS3).13
In addition, activating JAK2 (ie. Y617F) and JAK3 mutants
have been recently reported in patients with myeloproliferative
disorders (JAK2)14,15 and acute megakaryoblastic leukemia
(JAK3).16 STAT pathways are important mediators in pathologic
events, thus therapeutics that inhibit the JAK-STAT pathway
may have potential for treating solid tumors and hematological
malignancies. Recently, several inhibitors of STAT3 signaling
including natural products,20–24 peptidomimetics,17 platinum
compounds,18,19 and small molecules (1-3 in Figure 1)25–27
have been reported. The natural product Phaeosphaeride A has
been shown to inhibit STAT3-dependent signaling,20 while
Flavopiridol disrupts STAT3/DNA interactions and attenuates
STAT3-directed transcription21 and Resveratrol blocks constitu-
tive STAT3 protein activation in malignant cells by inhibiting
upstream Src tyrosine kinase activity.22 Cucurbitacin Q selec-
tively inhibits the activation of STAT3 and induces apoptosis
without inhibition of JAK2, Src, Akt, Erk, or JNK activation,23
and Curcumin (diferuloylmethane) inhibits IL-6-induced STAT3
phosphorylation and consequent STAT3 nuclear translocation.24
The synthetic terpenoid CDDO-Im (1) suppresses both constitu-
tive and IL-6-induced STAT3 and STAT5 phosphorylation in
human myeloma and lung cancer cells.25 STA-21 (2) inhibits
(VEGF, HIF-1),6 and modulates p53 transcription.9 STAT3 is
constitutively activated in many forms of hemotopoitic and solid
tumors including, breast, head and neck, prostate, lung, mela-
noma, multiple myeoloma, lymphomas, and leukemia. An
artificially engineered and constitutively active mutant form of
STAT3, STAT3C, induces transformation and cells transformed
by this active STAT3 mutant form tumor in vivo.10 Inhibition
of the STAT3 pathway by the dominant negative form, antisense
oligonucleotides and RNAi of STAT3, and JAK inhibitors has
been shown to inhibit cell growth and induce apoptosis. Loss
of expression of negative regulators of the JAK-STAT3 pathway
* To whom correspondence should be addressed. Phone: 845-602-4331.
Fax: 845-602-3045. E-mail: Parkk3@wyeth.com.
†
Oncology, Wyeth Research.
Chemical and Screening Sciences, Wyeth Research.
Biological Technologies Department, Wyeth Research.
‡
§
a Abbreviation: STAT3, signal transducer and activator of transcription
3; JAK2, Janus kinase 2; BCR, breakpoint cluster region; PDGF, platelet-
derived growth factor; Bcl, B-cell lymphoma; Mcl-1, myeloid cell leukemia-
1; VEGF, vascular endothelial growth factor; HIF-1, hypoxia-inducible
factor 1; SOCS, suppressors of cytokine signaling; PIAS3: protein inhibitor
of activated STAT3; AKT, protein kinase B; mTOR, mammalian target of
rapamycin; IL-6, interleukin-6; INF-R, interferons-R; TYK2, tyrosine kinase
2; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated
protein kinase.
10.1021/jm701271y CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008

4116 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Xu et al.
Scheme 1a
STAT3 dimerization, DNA binding, and luciferase activity in
breast cancer cells that express constitutively active STAT3.26
Also recently, Stattic (3) has been reported to selectively inhibit
activation, dimerization, and nuclear translocation of STAT3
and increases the apoptosis of STAT3-dependent breast cancer
cell lines.27
In the human myeloma cell line U266, STAT3 is constitu-
tively activated through an IL-6 autocrine loop. Inhibition of
the constitutive STAT3 pathway induces the cells into apop-
tosis.28 Using an Apo-ONE Homogenous Caspase 3/7 assay
(Promega) in the U266 cell line, we conducted a high-throughput
screen (HTS) for compounds that inhibit the STAT3 pathway
and cause cell apoptosis. Ethyl 5,6,7,8-tetrafluoro-4-oxo-1-
(pentafluorophenyl)-1,4-dihydroquinoline-3-carboxylate (4) (Fig-
ure 1) induced Apo-ONE signal at low micromolar concentration
in U266 and was also shown to inhibit the STAT3-Y705
phosphorylation by Western blot analysis. Further characteriza-
tions revealed that activation of the upstream activators of
STAT3, the Janus kinases (JAKs), were also inhibited. Previ-
ously, quinolone derivatives, especially C-7 amine derivatives,
have been shown to possess antibacterial activity by inhibiting
DNA gyrase and topoisomerase IV29,30 and have also been
reported as anticancer or antitumor agents.31 Herein we report
the characterization and optimization of the quinolinone class
of JAK-STAT3 pathway inhibitors.
Chemistry
The synthesis of the 1,4-dihydroquinoline derivatives was
carried out using previously reported literature procedures32,33
as shown in Scheme 1. Treatment of 2,3,4,5,6-pentafluoroben-
zoic acid (5a) or 2,3,4,5-tetrafluorobenzoic acid (5b) with thionyl
chloride in the presence of catalytic amount of DMF gave the
corresponding acid chlorides, which were reacted with the
dianion of monoethyl malonate to give the corresponding ꢀ-keto
esters 6a and 6b in high yield (∼90%). These esters were reacted
with triethyl orthoformate in acetic anhydride at 140 °C to
provide the enol ether intermediates 7a and 7b, which were
used in the next step without purification. Reactions of enol
ether 7a with 2,3,4,5,6-pentafluoroaniline and enol ether 7b with
4-amino-2,3,5,6-tetrafluorobenzonitrile in the presence of NaH
afforded dihydroquinoline carboxylates 4 and 8, respectively,
in good yield (∼80%). Ethyl ester 4 was hydrolyzed under acidic
or basic conditions to provide the corresponding dihydroquino-
line carboxylic acid 9. Benzyl ester 10 was also prepared in a
similar manner as described for the synthesis of 8 except using
monobenzyl malonate. Ethyl ester 8 was hydrolyzed to the
corresponding dihydroquinoline carboxylic acid. The carboxylic
acid was activated by ethyl chloroformate, and the resulting
anhydride reacted with 4-fluorobenzylamine to yield benzyl
amide 11 in high yield (85%).
a (a) (i) (SOCl)2, DMF, 10 h; (ii) n-BuLi, -78 °C, CH2CO2Et(COOH),
2 h; (iii) HCl, 90%; (b) triethyl orthoformate, Ac2O, 140 °C, 5 h, 98%; (c)
(i) ArNH2 (2,3,4,5,6-pentafluoroaniline or 4-amino-2,3,5,6-tetrafluoroben-
zonitrile), EtOH, rt, 12 h; (ii) K2CO3, DMF, rt, 12 h, or NaH, THF, 40 °C,
80%; (d) H2SO4, 100 °C, 12 h or LiOH/THF-MeOH, rt, 12 h, 100%; (e) (i)
(SOCl)2, DMF, 10 h; (ii) n-BuLi, -78 °C, CH2CO2Bn(COOH), 2 h; (iii)
HCl, 90%; (f) ethyl chloroformate, N(C2H5)3, THF-CH2Cl2 (v/v′ ) 2:1), 0
°C, 30 min; (iii) 4-fluorobenzylamine, 0 °C, 30 min, 85%.
ONE signal at low micromolar concentration in U266 but did
not induce Apo-ONE signal in the A2780 cell line in which
STAT3-Y705 is not constitutively phosphorylated.34 Western
blot analysis on U266 cell lysates confirmed that compound 4
induced apoptosis through inhibiting the STAT3 pathway. As
shown in Figure 2A, compound 4 inhibited STAT3-Y705
phosphorylation in a dose dependent manner after 1 h treatment
with an EC50 of 4.6 µM (Table 1).
Results and Discussion
Analogue synthesis showed that while removal of the labile
C-5 fluorine only led to a slight reduction in potency, removal
of the C-6, C-7, and C-8 fluorine atoms led to a more significant
loss in activity (data not shown). Also, hydrolysis of the ethyl
ester yielded the corresponding carboxylic acid analogue 9
(Scheme 1), which was 10-fold less active (EC50 ) 46 µM)
than ethyl ester 4 in inhibiting STAT3-Y705 phosphorylation
(Table 1 and Figure 2B). Array synthesis varying the aniline
moiety led to the identification of the 4-cyanophenyl analogue
8, with a 30-fold increase in potency (EC50 ) 170 nM). The
benzyl ester 10 and benzyl amide 11 showed similar potency
with EC50’s of 130 nM and 200 nM, respectively. As shown in
Figure 3, compound 8 inhibited steady state STAT3-Y705
Constitutively active STAT3 pathways have been implicated
in many types of cancer. We surveyed many cultured cell lines
for steady state level of STAT3-Y705 phosphorylation as an
indicator of the constitutively active pathway because phos-
phorylation of this site is required for its transcription activities.
Among the cell lines tested, human multiple myeloma cell line
U266 showed the highest level of STAT3-Y705 phosphorylation
(data not shown) and thus was chosen as our model cell line.
U266 cells have an IL-6 autocrine loop and inhibition of the
IL-6-JAK-STAT3 pathway at various points lead to apoptosis.28
We identified compound 4 via HTS using an Apo-ONE
Homogenous Caspase 3/7 assay. The compound induced Apo-

Inhibition of the STAT3 Signaling Pathway
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4117
Figure 2. STAT3-Y705 phosphorylation affected by compound 4 (A)
and compound 9 (B) in U266 cells.
Table 1. Inhibition of STAT3-Y705 Phosphorylation by Compounds 4,
8-11
Figure 3. Compound 8 potently and specifically inhibits STAT3
phosphorylation in U266 cells (A). Western blot data was quantified
by GraphPad Prism software (B).
compound
R1
R2
R3
STAT3-Pa EC50 (µM)
4
9
8
10
11
F
F
H
H
H
F
F
CN
CN
CN
OEt
OH
OEt
OBn
p-F-BnNH
4.6
46
0.17
0.13
0.20
a Inhibition of STAT3-Y705 phosphorylation as estimated based on
Western blot assay, repeated at least 3 times for each compound.
phosphorylation (P-STAT3-Y705) with an EC50 of 170 nM
while the total level of STAT3 was not affected at this
concentration.35 The inhibitory effects were very fast, being
observed within 30 min, thus excluding indirect secondary
effects by 8 on the pathway. However, 8 did not inhibit the
AKT-mTOR pathway as indicated by the phosphorylation status
of the p70 S6 kinase (P-p70 S6 kinase-T389) and S6 protein
(P-S6-S240/244) nor the MAPK pathway as indicated by the
MAPK phosphorylation (P-p44/42 MAPK-T202/204) status.
Interestingly, STAT3-Ser727 phosphorylation, which has been
reported to be mediated by serine/theonine kinases involving
the MAPK pathway, AKT-mTOR, and PKC,36–41 was not
inhibited. Compound 8 induced apoptosis in U266 cells, but
the caspase activation detected with the Apo-ONE Homogenous
Caspase 3/7 assay did not occur until at least 6 h after compound
treatment. Furthermore, apoptosis could be halted with a general
antioxidant N-acetyl-L-cysteine, while inhibition of STAT3
phosphorylation was not affected (data not shown), suggesting
pathway inhibition is not a result of apoptosis.
Figure 4. Compound 8 inhibits cytokines induced phosphorylation of
JAK1, JAK2, and Tyk2 in U266 cells.
receptors but converge on Janus kinase family members (JAKs)
to activate STAT3. Thus we tested whether the compounds
would inhibit JAK function. The steady state levels of phos-
phorylated JAKs were very difficult to detect using available
antibodies, so we used cytokine treatment to increase the
phospho-JAK levels. Compound 8 inhibited cytokine induced
phosphorylation of JAK1, JAK2, and Tyk2 (P-JAK1-Y1022/
1023, P-JAK2-Y1007/1008, P-Tyk2-Y1054/1055), while the
total protein amounts were not affected (Figure 4). JAK3
Compound 8 also inhibited STAT3-Y705 phosphorylation in
serum starved U266 cells activated by IL-6 (data not shown)
and IFN-R cytokines (Figure 4). These cytokines use different

4118 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Xu et al.
Figure 5. Compound 8 potently and specifically inhibits STAT3 phosphorylation in HeLa (A) and DU145 (B) cells.
phosphorylation still remained undetectable. Furthermore, this
inhibition of JAK activation correlated well with inhibition of
STAT3 activation. However, compound 8 did not inhibit JAK1,
JAK2, and JAK3 enzyme activity in an in vitro kinase assay at
the concentration of 1 and 10 µM.
We further tested the inhibition of STAT3 target gene
transcription by compound 8 using microarray analysis. U266
cells were treated with compound 8 (0.5 µM, ∼EC90) and 100
µM of compound 12 (AG490: ((E)-2-cyano-3-(3,4-dihydrophe-
nyl)-N-(phenylmethyl)-2-propenamide), a well-characterized
inhibitor of the JAK/STAT pathway42 as a control, for 6 h and
RNA samples were extracted for microarray analysis. Using a
1.5 fold as cutoff for significant changes, several STAT3 target
genes, including cyclin D1, myeloid cell leukemia sequence 1
(MCL1), and Bcl2-like 1, were down regulated to a similar
extent by both 8 and 12.
Compound 8 also inhibited steady state (data not shown) and
cytokine induced STAT3-Y705 phosphorylation in HeLa and
DU145 cells, while the phosphorylation status of the AKT-
mTOR and MAPK pathway markers were not affected (Figure
5A,B). Cytokine induced STAT3 nuclear translocation was
inhibited when visualized in a High-content screening assay,43
indicating the inhibitory effects demonstrated by these com-
pounds were not restricted to a particular cell line.
The specificity of compound 8 for inhibition of the JAK-
STAT3 pathway was further tested in K562 and A431 cells. In
K562 cells, STAT5 is constitutively activated by a different
signaling pathway, the oncogene BCR-ABL.44 As expected and
in contrast to a control Src kinase inhibitor, compound 8 did
not inhibit STAT5-Y694/699 phosphorylation (Figure 6A).
When both U266 and K562 cells were treated with compound
8 for 4 days, growth of U266 cells was much more inhibited
than that of K562 cells (Figure 6B). Further, compound 8 did
not inhibit EGF stimulated activation of MAPK in A431 cells,
a cell line widely used for studying the EGFR pathway (data
not shown).
Conclusion
In summary, we used a cell based apoptosis assay to identify
the potential JAK-STAT3 pathway inhibitor 4. Lead optimiza-
tion led to the identification of compound 8 in which cell
apoptosis induction correlates well with inhibition of steady state
and cytokine induced JAK and STAT3 activation. Compound
8 inhibited activation of JAKs but did not inhibit JAK1, JAK2,
and JAK3 enzyme activity in in vitro kinase assay. Biochemical
evidence suggests compound 8 was selective for the JAK-STAT
pathway, and it inhibited many STAT3 target genes. Further
investigation of the specific molecular target is underway.

Inhibition of the STAT3 Signaling Pathway
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4119
antibodies for phosphorylated STAT3 (Tyr705 and Ser727), STAT3,
phosphorylated p70 S6 kinase (Thr389), phosphorylated p44/42
MAPK (Thr202/Tyr204), phosphorylated S6 ribosomal protein
(Ser240/Ser244), phosphorylated JAK2 (Tyr1007/Tyr1008), and
phosphorylated TYK2 (Tyr1054/Tyr1055) were purchased from
Cell Signaling Technology. Antibodies for phosphorylated JAK1
(Tyr1022/Tyr1023) and JAK2 were from Biosource. Antibodies
for JAK1, TYK2, and phosphorylated STAT5 (Tyr694/Tyr699)
were from Upstate. Antibodies for STAT5, JAK3, and phospho-
rylated JAK3 (Tyr980) were from Santa Cruz Biotechnology.
Antibody for actin was from Chemicon. All antibodies were diluted
1:1000 with the exception of the JAK family member antibodies,
which were diluted 1:500, and the actin antibody, which was diluted
1:10000. Secondary antibodies Alexa Fluor 680 conjugated goat
antirabbit IgG and goat antimouse IgG were obtained from
Invitrogen. IRDye 800 conjugated goat antimouse IgG secondary
antibody was from Rockland Immunochemicals. Western analyses
were developed using the Odyssey infrared imaging system (LI-
COR Biosciences) for pictures.
STAT3-Y705 Phosphorylation Analyses. Western blots were
quantified using the Odyssey infrared imaging system (LI-COR
Biosciences). Actin levels were used for normalization. Quantified
data were analyzed using GraphPad Prism software. An arbitrary
10-12 M was used for no compound treatment for plotting purpose.
A representative Western blot analysis was shown from several
repeated experiments.
Figure 6. Compound 8 does not inhibit BCR-ABL activated STAT5
phosphorylation in K562 cells (A) but inhibits growth of U266 cells
selectively over K562 cells (B).
Proliferation Assay. Cells were seeded at a density of 2 × 105
cells per mL, and compounds were dosed at various doses. Four
days later, viable cells were stained using Guava ViaCount reagent
and counted using Guava PCA-96 flow cytometer (Guava Tech-
nologies Inc.). Data were analyzed and plotted using GraphPad
Prism software.
Emerging evidence continues to implicate the JAK-STAT
pathway participation and crosstalk with other oncogenic
pathways for tumor growth and survival, thus JAK-STAT
inhibitors may be promising therapeutics. Compound 8 may be
a potentially useful tool in elucidating the inhibition of JAK-
STAT signaling pathway in cancer or tumor cell lines.
RNA Expression Analysis. U266 cells were seeded at the
density of 1 × 106 cells per mL and treated with 0.5 µM compound
8 or 100 µM compound 12 for 6 h in triplicate. Cells were collected
and RNA purified using an RNeasy kit (QIAGEN). mRNA levels
were determined by microarray analysis. Briefly, 2-5 µg of total
RNA from each individual sample was used to generate biotin-
labeled cRNA using an oligo T7 primer in a reverse transcription
reaction followed by an in vitro transcription reaction with biotin-
labeled UTP and CTP. Then 10 µg of cRNA were fragmented and
hybridized to a human genome array (U133A; Affymetrix).
Hybridized arrays were stained and scanned according to the
manufacturer’s protocols (Fluidics Station 450 and Affymetrix
scanner 3000; Affymetrix). We determined signal values using the
operating system software (GCOS 1.0; Affymetrix). For each array,
all probe sets were normalized to a mean signal intensity value of
100. The default GCOS statistical values were used for all analysis.
Signal values and absolute detection calls were imported into
Expressionist (GeneData) for analysis.
Experimental Section
Cell Culture and Cell Treatment. All cell lines were obtained
from American Type Culture Collection (ATCC). U266 and K562
cells were cultured in RPMI1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS) and Penicillin-Streptomycin
(Pen-Strep). For steady state phosphorylation, exponentially growing
cells (1 × 106 cells at density 2 × 105 cells/mL for U266 and 1 ×
106 cells at density 1 × 106 cells/mL for K562) were treated with
compound for 1 h before being harvested for Western blot analysis.
For cytokine induction, cells were starved in serum free medium
for 2 h before compound treatment. One hour after compound
treatment, human interferon alpha R (IFN-R) was added at 20000
units/mL. Cells were harvested 20 min after cytokine induction for
Western blot analysis. Cells were lysed in 100 µL of PhosphoSafe
extraction reagent (Novagen) with protease inhibitor cocktail after
being washed once with PBS. Then 13 µL for U266 and 16 µL for
K562 of lysate were loaded for each sample for Western blot
analysis. HeLa and DU145 cells were cultured in DMEM supple-
mented with 10% heat-inactivated fetal bovine serum (FBS) and
Penicillin-Streptomycin (Pen-Strep). For cytokine induction, ∼80%
confluent cells were starved in serum free medium overnight before
compound treatment. One hour after compound treatment, IFN-R
was added for cytokine induction at 20000 units/mL. Cells were
harvested 20 min after cytokine induction for Western blot analysis.
Cells from each 12-well plate were collected in 100 µL of 1.1X
NuPAGE LDS sample buffer (Invitrogen) after being washed once
with PBS and were then sonicated. Then 18 microliter of lysates
were loaded for each sample for Western blot analysis. IFN-R was
purchased from PBL Biomedical Laboratories.
General Methods. All reactions were performed under N2
atmosphere using anhydrous solvents. Purification of the products
was carried out by flash chromatography using EM silica gel 60
(230-400 mesh). Semipreparative HPLC was used under the
conditions: A ) 0.02% TFA in water, B ) 0.02% TFA in
acetonitrile, 10-95% B in 8 min, 34 mL/min, 50 °C, 215 nm
detection, Waters Xterra 20 mm × 50 mm column.
Ethyl 3-oxo-3-(2,3,4,5,6-Pentafluoro-phenyl)-propanoate (6a).
Ester 6a was prepared in a similar manner as described for the
synthesis of 6b except using 2,3,4,5,6-pentafluorobenzoic acid. 1H
NMR spectroscopy data was confirmed by reported data. 1H NMR
(400 MHz, CDCl3): δ (two sets of signals) 4.23 (q, J ) 7.6 Hz,
2H), 3.91 (s, 2H), 1.26 (t, J ) 7.6 Hz, 3H); 12.35 (s, OH), 5.42 (s,
1H), 4.39 (q, J ) 7.6 Hz, 2H), 1.38 (t, J ) 7.6 Hz, 3H).
Western Blot Analyses. Cell lysates were separated on NuPAGE
gels (Invitrogen). Proteins were transferred to nitrocellulose mem-
branes (Invitrogen) and then blocked for 1 h in Odyssey blocking
buffer (LI-COR Biosciences). Membranes were incubated with
specific antibodies for 2 h and then secondary antibodies for 1 h at
room temperature in Odyssey blocking buffer with 0.1% Tween-
20, except 5% nonfat milk in PBS was used for blocking and
primary antibody incubation for JAK family antibodies. Specific
Ethyl 3-oxo-3-(2,3,4,5-Tetrafluorophenyl)-propanoate (6b). To
a solution of 2,3,4,5-tetrafluorobenzoic acid (1 g, 5.15 mmol) in
thionyl chloride (10 mL) were added 2 drops of anhydrous DMF
at room temperature. The reaction mixture was stirred for 12 h at
room temperature. The mixture was concentrated, dissolved in
anhydrous THF (10 mL), and saved as an acyl chloride solution.
To a solution of malonic acid half-ethyl ester (1.21 mL, 10.3 mmol)

4120 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Xu et al.
in THF (20 mL) at 0 °C was added a solution of n-BuLi (8.23 mL,
2.5 M in hexanes, 20.6 mmol) and the mixture was stirred for 1 h
at room temperature. The mixture was cooled to -78 °C, and a
solution of acyl chloride, prepared previously, was added to the
mixture slowly. The mixture was warmed to room temperature and
quenched with 1N HCl (30 mL). The solution was extracted with
ethyl ether (3 × 30 mL), and the organic layer was washed with
saturated NaHCO3 (2 × 50 mL) and then water. The organic
solution was dried over Na2SO4 and concentrated. The residue was
purified by short column chromatography with CH2Cl2 to give
1.22 g (90%) of 6b as a yellow solid. 1H NMR spectroscopy data
9.00 (s, 1H), 8.24 (ddd, J ) 10.0, 8.4, 2.0 Hz, 1H). HRMS (ESI)
[M + H]+ calcd for C17H4F7N2O3, 417.0111; found, 417.0117.
To a solution of 1-(4-cyano-2,3,5,6-tetrafluorophenyl)-6,7,8-
trifluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (35 mg, 0.08
mmol) in a mixture of THF (2 mL) and DCM (1 mL) was added
triethylamine (50 µL, 0.4 mmol) and then the mixture was cooled
to 0 °C. To the mixture was added ethyl chloroformate (20 µL,
0.21 mmol). The solution was stirred for 30 min at 0 °C and then
4-fluorobenzylamine (13 µL, 0.11 mmol) was added. The solution
was stirred for 30 min, upon which LC/MS revealed the reaction
was complete. The solvent was removed under vacuum, and the
residue was purified by semipreparative HPLC to give 11 as a white
solid. 1H NMR (400 MHz, DMSO-d6): δ 9.76 (t, J ) 6.0 Hz 1H),
8.91 (s, 1H), 8.20 (m, 1H), 7.40 (m, 2H), 7.16 (m, 2H), 4.56 (d, J
) 6.0 Hz, 2H). HRMS (ESI) [M + H]+ calcd for C24H10F8N3O2,
524.0640; found, 524.0641.
1
was confirmed by reported data. H NMR (400 MHz, CDCl3): δ
(two sets of signals) 7.63 (m, 1H), 4.22 (q, J ) 7.6 Hz, 2H), 3.97
(s, 1H), 3.96 (s, 1H), 1.27 (t, J ) 7.6 Hz, 3H); 12.73 (s, OH), 7.53
(m, 1H), 5.84 (s, 1H), 4.30 (q, J ) 7.6 Hz, 2H), 1.35 (t, J ) 7.6
Hz, 3H).
(Z)-Ethyl 3-Ethoxy-2-(2,3,4,5,6-pentafluoroben-zoyl)acrylate
(7a). (Z)-Ethyl 3-ethoxy-2-(2,3,4,5,6-pentafluorobenzoyl)acrylate
(7a) was prepared in a similar manner as described for the synthesis
of 7b and the product was used for further reaction without
purification.
Supporting Information Available: Table S1 for the inhibition
of STAT3 target genes by compound 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank the members of the Wyeth
Chemical Technologies group for analytical and spectral
determinations and the Wyeth Biological Technologies group,
especially Drs. Steve Haney and Jiang Wu. We also thank
Mairead Young for conducting compound stability experiments
and Drs. John Ellingboe and Clark Eid for their helpful
discussions.
(Z)-Ethyl 3-Ethoxy-2-(2,3,4,5-tetrafluorobenzoyl)-acrylate
(7b). The mixture of 7b (1.20 g, 4.54 mmol), triethylorthoformate
(1.13 mL, 6.81 mmol), and Ac2O (1.85 mL, 19.4 mmol) was heated
at 140 °C for 5 h. The mixture was concentrated to give crude 7b.
Ethyl 1-(4-Cyano-2,3,5,6-tetrafluorophenyl)-6,7,8-trifluoro-
4-oxo-1,4-dihydroquino-line-3-carboxylate (8). To a solution of
7b (1.45 g, 4.54 mmol) in THF (30 mL) were added NaH (174
mg, 60% in oil, 4.54 mmol) and 4-amino-2,3,5,6-tetrafluoroben-
zonitrile (863 mg, 4.54 mmol). The mixture was stirred for 1 h at
room temperature then stirred for 1 h at 40 °C. The reaction was
quenched with water (10 mL), and the mixture was extracted with
ethyl acetate (3 × 30 mL). The organic solution was washed with
brine, dried over Na2SO4, and concentrated. The residue was
purified by column chromatography with 50% ethyl acetate in
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1
hexanes to give 1.31 g (65%) of 8 as an orange solid. H NMR
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1
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MHz, DMSO-d6): δ 8.64 (s, 1H), 4.25 (q, J ) 7.2 Hz, 2H), 1.27
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1
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