Design, synthesis and antitumor activities of novel bis-aryl ureas derivatives as Raf kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.051

A series of novel bis-aryl ureas containing trifluoromethyl imidazolyl group targeting Raf kinase were designed and synthesized based on the lead compound of Sorafenib. All the prepared compounds were evaluated for their in vitro antiproliferative activities against three human cancer cell lines including MDA-MB-231 (breast), BGC-823 (gastric), and SMMC-7721 (liver). Several compounds from the series exhibited excellent antitumor activities against all three tested cancer lines. Further their inhibitory activities against Raf kinase were investigated, and three compounds (11c, 11d, and 11p) demonstrated better activities than contrast drug Sorafenib. Especially compound 11c was found to be a potent and selective Raf kinase inhibitor and could be considered as a candidate compound for further development.

Full text of DOI:10.1016/j.bmc.2012.05.051

Bioorganic & Medicinal Chemistry 20 (2012) 4323–4329
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antitumor activities of novel bis-aryl ureas derivatives
as Raf kinase inhibitors
Wenhu Zhan a,b, Yanyang Li a, Weiping Huang a,b, Yanjin Zhao a, Zhenglin Yao a,b, Shanyou Yu a,b
,
Shoujun Yuan a, Falong Jiang a,b, Shan Yao a,b, Shuxin Li a,b,
⇑
a Institute of Radiation and Irradiation Medicine, Academy of Military Medical Science, Beijing 100850, China
b Department of Medicinal Chemistry, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel bis-aryl ureas containing trifluoromethyl imidazolyl group targeting Raf kinase were
designed and synthesized based on the lead compound of Sorafenib. All the prepared compounds were
evaluated for their in vitro antiproliferative activities against three human cancer cell lines including
MDA-MB-231 (breast), BGC-823 (gastric), and SMMC-7721 (liver). Several compounds from the series
exhibited excellent antitumor activities against all three tested cancer lines. Further their inhibitory
activities against Raf kinase were investigated, and three compounds (11c, 11d, and 11p) demonstrated
better activities than contrast drug Sorafenib. Especially compound 11c was found to be a potent and
selective Raf kinase inhibitor and could be considered as a candidate compound for further development.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 18 November 2011
Revised 22 May 2012
Accepted 22 May 2012
Available online 30 May 2012
Keywords:
Bis-aryl ureas
Raf kinase
Synthesis
Antitumor activity
1. Introduction
extensively investigated following the success of Sorafenb.11
Sorafenib (BAY 43-9006) is the first and only Raf kinase inhibitor
Although cancer remains a devastating diagnosis, successfully
developed target-based therapies have significantly changed can-
cer treatment.1 The Ras/Raf/MEK/ERK mitogen-activated protein
kinase (MAPK) signaling cascade transmits mitogenic stimuli to
the nucleus through a series of phosphorylation events, which is
critical to the survival, growth, proliferation and apoptosis of cells
and has been implicated in up to 30% of human cancers.2–5 Raf
serine/threonine protein kinase, existing in three isoforms (A-Raf,
B-Raf, C-Raf or Raf-1), is a crucial component of this signal trans-
duction pathway. All three Raf isoforms are able to interact with
Ras and activate the MAPK signaling pathway.6 Mutations of Raf
lead to an aberrant activation of the signaling pathway, which ulti-
mately results in increased proliferation and survival of cancer
cells. Activating mutations in Raf have been observed in malignant
melanomas (66%), papillary thyroid cancers (40–70%), and ovarian
cancers (35%).7 Therefore Raf kinase has been recognized as an
attractive target for drug discovery in the treatment of cancer.8
A number of small molecule Raf kinase inhibitors containing
diverse scaffolds have emerged in the recent past, which can be di-
vided into several structural classes such as ureas, urea bioisoster-
es, imidazoles, benzamides, oxindoles, and aza-stilbenes.9,10
Among them, ureas, especially bis-aryl ureas, have been most
that has received clinical approval by the Food and Drug Adminis-
tration (FDA) and European Medicine Agency (EMEA) for the treat-
ment of advanced renal cell carcinoma (RCC) and unresectable
hepatocellular carcinoma (HCC) thus far.12,13 However, it has been
pointed out that its activity against certain tumor types may be
through inhibition of other kinases, for example, VEGFR, rather
than Raf for its lack of efficacy in patients expressing the V600E
(formerly termed V599E) B-Raf mutation.14,15 This prompted our
efforts for developing more potent and selective Raf kinase
inhibitors.
According to the crystal structure of V600E B-Raf kinase do-
mains in complex with Sorafenib, the distal pyridyl ring of Sorafe-
nib occupies the ATP adenine binding pocket and interacts with
three amino acids residues (Trp530, Phe582, and Phe594), the ring
nitrogen atom accepts a hydrogen bond from main chain nitrogen
of Cys531 and the methyl amide side group contracts the main car-
bonyl of Cys531. The lipophilic trifluoromethyl phenyl ring at end
side of the molecule inserts into a hydrophobic pocket. Impor-
tantly, the urea moiety of Sorafenib forms two hydrogen bonds
with B-Raf, one with the backbone aspartate, and the other with
the glutamate side chain.16 We explored structural modification
of Sorafenib on the basis of retaining the pyridyl ring as well as
the urea functional moiety with the goal to optimize the activity
on Raf kinase even further. Principal changes focused on hinge
binding moiety, the linear methyl amide side group of pyridyl moi-
ety was replaced with rigid trifluoromethyl imidazolyl group for
⇑
Corresponding author. Tel./fax: +86 10 68214653.
E-mail address: lisx28@163.com (S. Li).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.051

4324
W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
Table 1
enhancing interaction with Raf. Subsequently,
a variety of
The preparation of target bis-aryl ureas compounds 11a–11r
hydrophobic aromatic rings were introduced to the opposite end
of the inhibitors for the study of structure–activity relationships
(SAR). Moreover, we investigated chloro group on central phenyl
ring with a view to adding potency of inhibitors (Fig. 1). Accord-
ingly, a series of novel bis-aryl ureas derivatives were designed
and synthesized. All the prepared compounds (Table 1) were eval-
uated for their antiproliferative activities against three human can-
cer cell lines including MDA-MB-231 (breast), BGC-823 (gastric)
and SMMC-7721 (liver) in vitro. Several compounds from the series
exhibited excellent antitumor activities against all three tested
cancer lines. Further their inhibitory activities against Raf kinase
were investigated, and the selective profile of the best compound
11c was assessed against a panel of 20 protein kinases.
N
F3C
O
N
H
O
N
Ar
N
N
H
N
H
X
Compound
X
Ar
Compound
X
Ar
O
11a
H
11j
H
CH3
OCH3
CF3
CF3
11b
11c
11d
11e
H
H
H
H
11k
11l
H
2. Chemistry
Cl
The synthetic route of the target compounds 11a–11r is illus-
trated in Scheme 1. The commercially available starting material
4-chloropicolinic acid 1 was treated with SOCl2 in the presence
of a catalytic amount of DMF and then ammonolyzed in CH2Cl2
to give intermediate 2. Next, compound 2 was treated with triflu-
oroacetic anhydride in dry EtOAc to afford compound 3 as a white
solid. Compound 3 was reacted with corresponding acetylamino
phenols of general formula by SNAr reaction to produce com-
pounds 4 and 5. The trifluoromethyl imidazolyl rings on compound
6 and 7 were prepared according to the literature.17 which were
one-pot reaction of the corresponding nitrile 3 with NH4Ac and
3-bromo-1,1,1-trifluoroacetone in the presence of CH3ONa as cata-
lyst. Further, the obtained compound 6 and 7 were hydrolyzed in
the dilute sulfuric acid to give the key intermediates 8 and 9. A ser-
ies of substituted aromatic isocyanates 10 were synthesized by
treating corresponding commercial aromatic amines with tricar-
bonyl chloride in toluene. Finally, the target compounds 11a–11r
were successfully obtained via the reaction of key intermediates
8 and 9 with corresponding substituted aromatic isocyanates 10
in the presence of Et3N in DMF. The products obtained were puri-
fied by column chromatography on silica gel. The chemical struc-
tures of these novel compounds were all confirmed by 1H NMR,
MS spectra and elementary analysis and the results are presented
in Section 5.
Cl
F
Cl
Cl
Cl
CF3
Cl
CF3
CF3
Cl
11m
11n
Cl
Cl
F
Cl
CF3
CH3
F
11f
H
H
11o
11p
Cl
Cl
Br
F
CF3
Cl
11g
Cl
Cl
11h
11i
H
H
11q
11r
Cl
Cl
CF3
H3C
OCH3
O
O
OCH3
OCH3
O
CF3
O
Cl
N
O
H
N
N
N
3. Results and discussion
H
H
Sorafenib
The in vitro antitumor activities of all the prepared compounds
11a–11r were evaluated against three cancer cell lines consisting
of MDA-MB-231 (breast), BGC-823 (gastric) and SMMC-7721 (li-
ver) by the MTT assay and Sorafenib was used as the positive con-
trol drug. The IC50 (lM) values (concentration required to achieve
50% inhibition of the tumor growth) of the tested compounds on
each cell line were presented in Table 2.
N
F3C
As shown in Table 2, most of the prepared compounds demon-
strated moderate to excellent antiproliferative activities against
three different cancer cell lines, and seven compounds (11b–11d,
11l–11n and 11p) exhibited more or similar potent activities
against certain cancer lines in comparison with Sorafenib. Appar-
ently, almost all of the tested compounds displayed better activi-
ties against MDA-MB-231 and SMMC-7721 than against BGC-823
cell line, which reflects selective behavior of the target compounds
O
N
O
H
N
Ar
N
N
H
H
X
Target compounds 11a-11r
Figure 1. The structure of Sorafenib and the target compounds 11a–11r.

W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
4325
Cl
N
HO
Cl
O
Cl
N
H
OH
X
NH2
a
N
c
b
N
CN
O
O
1
2
3
N
NC
O
F3C
O
O
N
O
H
N
N
N
d
e
N
H
X
H
6 X=H
7 X=Cl
X
4 X=H
5 X=Cl
N
N
F3C
O
F3C
Ar NCO
g
O
N
H
O
N
H
N
Ar
N
N
N
NH2
NH2
H
H
X
X
8 X=H
9 X=Cl
11a-11r
Ar N C O
Ar
f
10
Scheme 1. Synthetic route for the preparation of the target compounds. Reagents and conditions: (a) (1) SOCl2, DMF, 80 °C, 3 h; (2) NH3ÁH2O, CH2Cl2, 1 h; (b) Et3N,
trifluoroacetic anhydride, EtOAc, 0 °C; (c) K2CO3, DMF, 85 °C, 6 h; (d) NH4Ac, CH3ONa, 3-bromo-1,1,1-trifluoroacetone, n-PrOH, 85 °C, 21 h; (e) 20% H2SO4, 85 °C, 5 h; (f)
tricarbonyl chloride, toluene, 80 °C, 8 h; (g) Et3N, rt, 24 h.
Table 2
A diverse electron withdrawing groups like fluoro, chloro, bro-
Antiproliferative activities of the target compounds 11a–11r against MDA-MB-231,
mo, trifluoromethyl and electron donating groups like methyl,
methoxy were introduced to the phenyl ring that connects with
the urea group in the end of the inhibitors for investigation of
structure–activity relationships (SAR). Compound 11a bearing a
trifluoromethyl group at the meta position of phenyl ring showed
reasonable activities against both MDA-MB-231 and SMMC-7721
BGC-823 and SMMC-7721 cells in vitro
a
Compound
IC50 (lM)
MDA-MB-231
BGC-823
SMMC-7721
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
10.65 0.27
7.83 0.31
1.21 0.14
2.03 0.40
11.26 0.48
25.43 1.13
38.67 1.87
76.57 2.39
63.24 2.13
44.80 1.63
95.36 2.61
8.21 0.46
7.03 0.59
3.77 0.27
10.11 0.40
1.77 0.19
14.24 1.07
>100b
26.19 1.55
18.37 1.81
13.15 1.34
10.70 1.67
15.66 1.08
66.80 2.08
23.74 1.89
83.98 2.60
>100b
11.73 0.69
6.84 0.54
1.38 0.11
3.06 0.33
9.69 1.20
44.62 2.03
36.24 1.83
57.87 1.92
92.36 2.27
87.66 2.73
>100b
5.72 0.36
5.29 0.53
6.52 0.18
8.38 0.67
2.49 0.15
9.72 0.88
>100b
cell lines (with IC50 values = 10.65, 11.73 lM). Introduction of
additional substituent like fluoro, chloro, methyl in other positions
along with the trifluoromethyl group in the meta position were
well tolerated (11b, 11c, 11l, 11o). Introduction of a chloro group
at para position (11c) was more effective than a fluoro group
(11l) or a methyl group (11o), suggesting the importance of the
substituent size at this position. Replacement of the trifluoro-
methyl group with chloro group at the meta position (11d) re-
tained high potency. However, introduction of electron donating
groups to phenyl ring or replacing phenyl ring with isoxazolyl ring
led to remarkable decrease in antitumor activities (11i, 11k, 11r,
11j). Surprisingly, introduction of a chloro group on the central
phenyl ring did show moderate improvements in antitumor activ-
ities of the most molecules (compare 11a and 11q, 11b and 11m,
and 11d and 11p), but led to a slight reduced potency of compound
11n (compare 11c).
97.71 2.03
>100b
17.67 1.57
14.21 1.32
11.10 1.03
33.35 1.20
12.69 1.96
23.18 2.57
>100b
Sorafenib
7.18 0.17
10.91 1.25
6.23 0.21
a
Results are expressed as means SD (standard deviation) of three independent
experiments.
b
Compounds with IC50 values >100 lM are considered to be inactive.
In order to investigate whether these prepared compounds
11a–11r inhibit Raf kinase or not, their inhibitory activities against
C-Raf kinase further evaluated by the FRET assay.18 Sorafenib was
also used as the positive control drug. The IC50 (nM) values were
summarized in Table 3. As clearly shown in Table 3, most of the
tested compounds exhibited evident inhibitory potency against
C-Raf. Moreover, compounds 11c, 11d, 11p (with IC50 val-
ues = 10.6, 13.1, 16.5 nM) demonstrated better inhibitory activities
than contrast drug Sorafenib (with IC50 values = 23.3 nM).
The kinase selectivity profile of these prepared compounds was
assessed over 20 different protein kinases at a single dose concen-
to some extent. To our delight, compound 11c (with IC50 val-
ues = 1.21, 1.38 M) displayed superior activities to Sorafenib
(with IC50 values = 7.18, 6.23 M) in both MDA-MB-231 and
SMMC-7721 cell lines, which indicated that replacement of linear
methylamide side group of pyridyl moiety with rigid trifluoro-
methyl imidazolyl ring was beneficial for improvement of antitu-
mor activities. We hypothesized that the enhanced potency could
arise from a series of contributing factors, including differences
in size, electronic property, hydrogen-bond forming capability,
and aqueous solubility.
l
l
tration of 10
lM, using the best compound 11c as a representative.
The results listed in Table 4 revealed that compound 11c has a very

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W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
Table 3
cal shifts (d) are reported in parts per million (ppm) relative to
tetramethylsilane (TMS), used as an internal standard. Mass spec-
tra (MS) were obtained from Agilent 1100 LC/MS Spectrometry
Services. Elemental analyses were carried out on a CHN-O-rapid
elemental analyzer (GmbH-Germany) for C, H and N, and the re-
sults are within 0.4% of the theoretical values.
C-Raf kinase inhibitory activities of the target compounds 11a–11r with Sorafenib as
positive control
a
b
Compound
IC50 (nM)
Compound
IC50 (nM)
11a
11b
11c
11d
11e
11f
11g
11h
11i
87.8
75.9
10.6
13.1
69.2
81.5
94.9
120.3
NTb
11k
11l
11m
11n
11o
11p
11q
11r
Sorafenib
NTb
77.7
35.9
22.1
46.7
16.5
64.8
NTb
5.1.1. 4-Chloropicolinamide (2)
A mixture of 4-chloropicolinic acid (10.0 g, 63.4 mmol), thionyl
chloride (40 ml) and a catalytic amount of DMF were heated at
80 °C for 3 h. Then the reaction mixture was cooled to room tem-
perature and evaporated under reduced pressure. The residue
was dissolved in 30 ml of CH2Cl2 in an ice-bath, then added to a
25% aqueous solution of ammonia (60 ml) at a rate which kept
the internal temperature below 7 °C with constant stirring. After
1 h, the precipitate was filtrated, washed with water (3 Â 50 ml)
and dried in vacuo to afford compound 2 (9.2 g, 93% yield) as a yel-
low solid, mp 161–162 °C; 1H NMR (DMSO-d6, 400 MHz): d 7.74–
7.76 (dd, J = 1.8 and 5.3 Hz, 1H), 7.80 (br s, 1H), 8.03 (d,
J = 1.8 Hz, 1H), 8.20 (br s, 1H), 8.62 (d, J = 5.3 Hz, 1H); ESI-MS m/
z: 157[M+H]+; Anal. Calcd for C6H5ClN2O (%): C 46.03, H 3.22, N
17.89; Found: C 45.98, H 3.27, N 17.94.
24.3
11j
147.9
a
Values are means of three experiments.
NT: not tested.
b
good selectivity profile. While the compound displayed an inhibi-
tion percentage of 95.3% on C-Raf, the inhibitions exhibited on
most other kinases were below 40%.
4. Conclusions
5.1.2. 4-Chloropicolinonitrile (3)
In conclusion, we have designed and synthesized a series of no-
vel bis-aryl ureas derivatives, evaluated their antiproliferative
activities against three human cancer cell lines (MDA-MB-231,
BGC-823 and SMMC-7721) and inhibitory activities against C-Raf
kinase in vitro. The test results showed that most of the prepared
compounds demonstrated moderate to excellent antiproliferative
activities and C-Raf inhibitory activities. Compounds 11c, 11d,
11p exhibited more potent activities in comparison with contrast
drug Sorafenib, especially compound 11c showed a very good
The compound 2 (7.0 g, 44.7 mmol) and triethylamine (13 ml,
93.7mmol) were dissolved in dry EtOAc (50 ml) and the stirred
solution was cooled to À5 °C in an ice-salt bath. Trifluoroacetic
anhydride (13 ml, 92.2 mmol) was added dropwise to the chilled
mixture over 45 min. The ice-salt bath was then removed and
the reaction was warmed to room temperature and then stirred
for another 30 min. The completion of reaction was detected by
TLC, 10% aqueous K2CO3 (100 ml) was added, and the reaction mix-
ture was allowed to stir for further 20 min. The mixture was ex-
tracted with EtOAc (3 Â 50 ml), the combined organic layers
were washed with brine (2 Â 50 ml), dried over anhydrous Na2SO4,
and concentrated under reduced pressure to give compound 3 (5.6
g, 90% yield) as a white solid, mp 84–85 °C; 1H NMR (DMSO-d6,
400 MHz): d 7.54–7.56 (dd, J = 5.0 and 2.0 Hz, 1H), 7.74 (m, 1H),
8.65 (d, J = 5.0 Hz, 1H); ESI-MS m/z: 139[M+H]+; Anal. Calcd for
C6H3ClN2 (%): C 52.01, H 2.18, N 20.22; Found: C 51.94, H 2.14, N
20.28.
selectivity profile and could be considered as
a candidate
compound for further development. Moreover, from the struc-
ture–activity relationships (SAR) we may conclude that replace-
ment of linear methylamide side group of Sorafenib with rigid
trifluoromethyl imidazolyl ring was beneficial for improvement
of antitumor activities, and introduction of a chloro group on the
central phenyl ring further optimized inhibitory potency to some
extent. The in vivo anticancer activities and detailed SAR of the
compounds are currently under investigation in our laboratory.
5.1.3. Procedure for preparation of compound (4) and (5)
The compound 3 (2.8 g, 20.2 mmol) was added to a suspension
of N-(4-hydroxyphenyl) acetamide (4.6 g, 30.3 mmol) and K2CO3
(5.6 g, 40.4 mmol) in dry DMF (30 ml), and the reaction was stirred
at 85 °C for 5 h. The resulting mixture was cooled to room temper-
ature, and then poured into ice water. The precipitate was collected
by filtration and washed with water (3 Â 50 ml) to give compound
4 (4.3 g, 85% yield) as a white solid, mp 173–175 °C; 1H NMR
(DMSO-d6, 400 MHz): d 2.08 (s, 3H), 7.14(d, J = 8.6 Hz, 2H), 7.53
(d, J = 8.6 Hz, 2H), 7.55–7.57 (dd, J = 2.1 and 5.3 Hz, 1H), 7.77 (d,
J = 2.1 Hz, 1H), 8.59 (d, J = 5.3 Hz, 1H), 9.23 (s, 1H); ESI-MS m/z:
254[M+H]+; Anal. Calcd for C14H11N3O2 (%): C 66.40, H 4.38, N
16.59; Found: C 66.33, H 4.35, N 16.65.
5. Experimental section
5.1. Chemistry: General procedures
All reagents and solvents were purchased from commercial
sources and used without further purification. Melting points were
measured in open capillaries and are uncorrected. 1H-NMR was re-
corded in DMSO-d6 on a Bruker Avance 400 spectrometer; chemi-
Table 4
Inhibition percentages of compound 11c at a single dose concentration of 10 lM over
20 protein kinases
Compound 5. Yield: 83%, off-white solid, mp 161–163 °C; 1H
NMR (DMSO-d6, 400 MHz): d 2.11 (s, 3H), 7.15 (m, 1H), 7.37–
7.44 (m, 2H), 7.54–7.56 (dd, J = 1.8 and 4.9 Hz, 1H), 7.75 (d,
J = 1.8 Hz, 1H), 8.57 (d, J = 4.9 Hz, 1H), 9.25 (s, 1H); ESI-MS m/z:
289[M+H]+ ; Anal. Calcd for C14H10ClN3O2 (%): C 58.45, H 3.50, N
14.61; Found: C 58.39, H 3.54, N 14.68.
Kinase
Inhibition (%)
Kinase
Inhibition (%)
ABL1
AKT1
c-Kit
c-MET
C-Raf
CDK2
FLT3
44.3
11.7
23.9
2.2
95.3
7.8
17.7
50.8
0.4
IKKb
JAK2
PDGFR
PTK2
PKA
PLK1
KDR
SYK
5.6
0
0
4.9
1.3
8.7
3.5
20.4
27.5
36.8
a
5.1.4. Procedure for preparation of compound (6) and (7)
A mixture of compound 4 (4.0 g, 15.8 mmol) and NH4Ac (3.6 g,
47.4 mmol) in n-PrOH was stirred under nitrogen atmosphere at
room temperature during addition of CH3ONa (1.0 g, 19.0 mmol).
FMS
FYN
TIE2
VEGFR2
FGFR1
20.2

W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
4327
The reaction mixture was stirred at 70 °C for 1 h, then treated with
dropwise addition of 3-bromo-1,1,1-trifluoroacetone (4 ml
5.1.7.1. 1-(4-(2-(5-(Trifluoromethyl)-1H-imidazol-2-yl)pyridin-
,
4-yloxy)phenyl)-3-(3-(trifluoromethyl)phenyl)urea
28.4 mmol). The mixture was heated to 85 °C and the completion
of reaction was proved by TLC after stirring for another 20 h. The
mixture was poured into water (50 ml), extracted with EtOAc
(3 Â 50 ml). The combined organic layers were dried over anhy-
drous Na2SO4, and evaporated under reduced pressure, the crude
residue was purified by chromatography on a silica gel column
using EtOAc/Hexane (1/2) as eluent to give 6 (3.3 g, 58% yield) as
(11a).
White solid, yield: 55%, mp 115–118 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.07–7.09 (dd, J = 1.8 and 5.0 Hz, 1H),
7.22 (d, J = 7.7 Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 1.7 Hz,
1H), 7.51–7.53 (m, 1H), 7.59–7.63 (m, 3H), 7.88 (s, 1H), 8.05 (s,
1H), 8.55 (d, J = 5.0 Hz, 1H), 8.98 (s, 1H), 9.13 (s, 1H), 13.58 (s,
1H); ESI-MS m/z: 508[M+H]+; Anal. Calcd for C23H15F6N5O2 (%): C
54.44, H 2.98, N 13.80; Found: C 54.49, H 2.94, N 13.78.
a
light yellow solid, mp 182–184 °C; 1H NMR (DMSO-d6,
400 MHz): d 2.09 (s, 3H), 7.05–7.07 (dd, J = 2.0 and 5.3 Hz, 1H),
7.22 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.7 Hz,
2H), 7.87 (s, 1H), 8.55 (d, J = 5.3Hz, 1H), 9.32 (s, 1H), 13.45 (s,
1H); ESI-MS m/z: 363[M+H]+; Anal. Calcd for C17H13F3N4O2 (%): C
56.36, H 3.62, N 15.46; Found: C 56.29, H 3.67, N 15.49.
5.1.7.2. 1-(2-Chloro-5-(trifluoromethyl)phenyl)-3-(4-(2-(5-(tri-
fluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11b).
White solid, yield: 57%, mp 219–220 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.07–7.09 (dd, J = 1.7 and 5.0 Hz, 1H),
7.24 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 1.7 Hz, 2H), 7.62–7.75 (m, 3H),
7.88 (s, 1H), 8.54–8.56 (m, 2H), 8.68 (s, 1H), 9.75 (s, 1H), 13.58
(s, 1H); ESI-MS m/z: 543[M+H]+; Anal. Calcd for C23H14ClF6N5O2
(%): C 50.98, H 2.60, N 12.93; Found: C 51.04, H 2.57, N 12.88.
Compound 7. Yield: 63%, yellow solid, mp 197–199 °C, 1H NMR
(DMSO-d6, 400 MHz): d 2.06 (s, 3H), 7.04–7.06 (dd, J = 2.0 and
5.1 Hz, 1H), 7.19–7.22 (m, 1H), 7.34–7.46 (m, 3H), 7.84 (s, 1H),
8.53 (d, J = 5.3 Hz, 1H), 9.37 (s, 1H), 13.45 (s, 1H); ESI-MS m/z:
398[M+H]+; Anal. Calcd for C17H12ClF3N4O2 (%): C 51.46, H 3.05,
N 14.12; Found: C 51.42, H 3.01, N 14.18.
5.1.7.3. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(5-(tri-
fluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11c).
White solid, yield: 61%, mp 122–124 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.07–7.09 (dd, J = 1.7 and 5.1 Hz, 1H),
7.23 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 1.7 Hz, 1H), 7.60–7.66 (m, 4H),
7.87 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 8.55 (d, J = 5.1 Hz ,1H), 9.03
(s, 1H), 9.25 (s, 1H), 13.57 (s, 1H); ESI-MS m/z: 543[M+H]+; Anal.
Calcd for C23H14ClF6N5O2 (%): C 50.98, H 2.60, N 12.93; Found: C
51.02, H 2.65, N 12.67.
5.1.5. Procedure for preparation of compound (8)and (9)
A mixture of compound 6 (3.3 g, 9.1 mmol) in 20% of dilute sul-
furic acid (20 ml) was stirred at 85 °C under nitrogen atmosphere
for 5 h. The resulting mixture was cooled to room temperature
and alkalized with a 10% NaOH aqueous solution to pH 8–9. The
precipitate was filtrated, washed with water (3 Â 20 ml), and dried
in vacuo to give 8 (2.6 g, 90% yield)as a red brown solid, mp 165–
167 °C, 1H NMR (DMSO-d6, 400 MHz): d 5.19 (br s, 2H), 6.82 (d,
J = 9.5 Hz, 2H), 7.05–7.07 (dd, J = 1.8 and 5.2 Hz, 1H), 7.20 (d,
J = 9.5 Hz, 2H), 7.41 (d, J = 1.8 Hz, 1H), 7.85 (s, 1H), 8.56 (d,
J = 5.2 Hz, 1H), 13.43 (s, 1H); ESI-MS m/z: 321[M+H]+; Anal. Calcd
for C15H11F3N4O (%): C 56.25, H 3.46, N 17.49; Found: C 56.22, H
3.50, N 17.46.
5.1.7.4. 1-(3,4-Dichlorophenyl)-3-(4-(2-(5-(trifluoromethyl)-1H-
imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11d).
White
solid, yield: 70%, mp 116–118 °C; 1H NMR (DMSO-d6, 400 MHz):
d 7.07–7.40 (m, 5H), 7.53–7.62 (m, 3H), 7.88–7.91 (m, 2H), 8.55
(d, J = 5.2 Hz, 1H), 8.99 (s, 1H), 9.09 (s, 1H), 13.58 (s, 1H); ESI-MS
m/z: 509[M+H]+; Anal. Calcd for C22H14Cl2F3N5O2 (%): C 51.99, H
2.78, N 13.78; Found: C 52.02, H 2.74, N 13.73.
Compound 9. Yield: 92%, red solid, mp 173–175 °C, 1H NMR
(DMSO-d6, 400 MHz): d 5.43 (br s, 2H), 6.82 (m, 2H) 7.04–7.06
(dd, J = 2.0 and 5.0 Hz, 1H), 7.20 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H),
7.89 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 13.51 (s, 1H); ESI-MS m/z:
356[M+H]+; Anal. Calcd for C15H10ClF3N4O (%): C 50.79, H 2.84, N
15.79; Found: C 50.84, H 2.80, N 15.77.
5.1.7.5.
methyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11e).
White solid, yield: 75%, mp 210–211 °C; 1H NMR
1-(3-Chloro-4-fluorophenyl)-3-(4-(2-(5-(trifluoro-
(DMSO-d6, 400 MHz): d 7.06–7.07 (dd, J = 1.9 and 5.3 Hz, 1H),
7.20 (d, J = 7.7 Hz, 2H), 7.33–7.41 (m, 3H), 7.60 (d, J = 7.7 Hz, 2H),
7.81–7.85 (m, 2H), 8.55 (d, J = 5.3 Hz, 1H), 8.92 (s, 1H), 8.94 (s,
1H), 13.53 (s, 1H); ESI-MS m/z: 493[M+H]+; Anal. Calcd for
5.1.6. General procedure for preparation of substituted
aromatic isocyanate (10)
C22H14ClF4N5O2 (%): C 53.73, H 2.87, N 14.27; Found: C 53.77, H
To a stirred and cooled to 0 °C solution of tricarbonyl chloride
(1 equiv) in toluene (20 ml), a solution of different aromatic
amines (3 equiv) in 10 ml of CH2Cl2 was added dropwise under
nitrogen atmosphere in an ice-bath. Upon completion of addition,
the reaction mixture was stirred for 0.5 h at room temperature, and
for another 6–8 h at 80–85 °C. The completion of reaction was de-
tected by TLC, the solvent was removed under vacuum to obtain
(10) as an oil which solidified and was stable at 0–7 °C.
2.85, N 14.23.
5.1.7.6.
1-(4-Bromo-2-fluorophenyl)-3-(4-(2-(5-(trifluoro-
methyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11f).
White solid, yield: 69%, mp 232–234 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.05–7.07 (dd, J = 1.8 and 5.2 Hz, 1H),
7.21 (d, J = 8.0 Hz, 2H), 7.36–7.40 (m, 2H), 7.57–7.60 (m, 3H),
7.85 (s, 1H), 8.12–8.17 (m, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.70 (s,
1H), 9.23 (s, 1H), 13.53 (s, 1H); ESI-MS m/z: 537[M+H]+; Anal. Calcd
for C22H14BrF4N5O2 (%): C 49.27, H 2.63, N 13.06; Found: C 49.23, H
2.59, N 13.09.
5.1.7. General procedure for the synthesis of compounds
(11a–11r)
To a solution of compound 8 or 9 (1 equiv) in DMF (20 ml),
triethylamine (2 equiv) and corresponding substituted aromatic
isocyanate 10 (1.1 equiv) were added under nitrogen atmosphere,
and the mixture was stirred overnight at room temperature. The
reaction mixture was quenched by addition of water and diluted
with EtOAc. The organic layer was washed with brine and dried
over anhydrous Na2SO4. After filtration and concentration, the
crude product was purified by column chromatography (EtOAc/
hexane) to obtain the desired bis-aryl ureas compounds 11a–11r
in 50–80% yield.
5.1.7.7. 1-(4-Fluorophenyl)-3-(4-(2-(5-(trifluoromethyl)-1H-imi-
dazol-2-yl)pyridin-4-yloxy)phenyl)urea (11g).
White solid,
yield: 56%, mp 236–238 °C; 1H NMR (DMSO-d6, 400 MHz): d
7.07–7.21 (m, 5H), 7.39 (d, J = 1.8 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H),
7.67–7.73 (m, 1H), 7.88 (s, 1H), 8.55 (d, J = 5.0 Hz, 1H), 8.78 (s,
1H), 9.25 (s, 1H), 13.57 (s, 1H); ESI-MS m/z: 458[M+H]+; Anal. Calcd
for C22H15F4N5O2 (%): C 57.77, H 3.31, N 15.31; Found: C 57.74, H
3.36, N 15.33.

4328
W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
5.1.7.8.
1-(2-Chloro-6-methylphenyl)-3-(4-(2-(5-(trifluoro-
C
23H13Cl2F6N5O2 (%): C 47.94, H 2.27, N 12.15; Found: C 47.97,
methyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11h).
H 2.24, N 12.19.
White solid, yield: 77%, mp 243–245 °C; 1H NMR
(DMSO-d6, 400 MHz): d 2.28 (s, 3H), 7.03–7.41 (m, 7H), 7.60 (d,
J = 7.9 Hz, 2H), 7.85 (s, 1H), 8.01 (s, 1H), 8.53 (d, J = 4.9 Hz, 1H),
9.06 (s, 1H), 13.52 (s, 1H); ESI-MS m/z: 489[M+H]+; Anal. Calcd
for C23H17ClF3N5O2 (%): C 56.62, H 3.51, N 14.36; Found: C 56.65,
H 3.48, N 14.39.
5.1.7.15. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(4-methyl-3-(trifluoromethyl)
phenyl)urea (11o).
White solid, yield: 64%, mp 263–264 °C;
1H NMR (DMSO-d6, 400 MHz): d 2.50 (s, 3H), 7.09–7.11 (dd,
J = 2.1 and 5.2 Hz, 1H), 7.25–7.28 (m, 1H), 7.39 (d, J = 2.1 Hz, 1H),
7.43–7.44 (m, 1H), 7.49–7.54 (m, 2H), 7.88 (s, 1H), 7.97 (d,
J = 8.9 Hz, 1H), 8.25–8.27 (m, 1H), 8.44 (s, 1H), 8.57 (d, J = 5.2 Hz,
1H), 9.67 (s, 1H), 13.59 (s, 1H); ESI-MS m/z: 557[M+H]+; Anal. Calcd
for C24H16ClF6N5O2 (%): C 51.86, H 2.90, N 12.60; Found: C 51.81, H
2.88, N 12.63.
5.1.7.9.
methyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11i).
White solid, yield: 59%, mp 212–213 °C; 1H NMR
1-(Benzo[d][1,3]dioxol-5-yl)-3-(4-(2-(5-(trifluoro-
(DMSO-d6, 400 MHz): d 5.97 (s, 2H), 7.06–7.07 (dd, J = 1.9 and
5.3 Hz, 1H), 7.17–7.23 (m, 3H), 7.32–7.38 (m, 2H), 7.74 (d,
J = 7.2 Hz, 2H), 7.87 (s, 1H), 8.31 (s, 1H), 8.50 (s, 1H), 8.53 (d,
J = 5.3 Hz, 1H), 10.38 (s, 1H), 13.49 (s, 1H); ESI-MS m/z:
484[M+H]+; Anal. Calcd for C23H16F3N5O4 (%): C 57.15, H 3.34, N
14.49; Found: C 57.13, H 3.38, N 14.37.
5.1.7.16. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(3,4-dichlorophenyl)urea
(11p).
White solid, yield: 58%, mp 269–270 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.08–7.10 (dd, J = 2.0 and 5.0 Hz, 1H),
7.22–7.56 (m, 5H), 7.88 (s, 1H), 7.92 (d, J = 9.5 Hz, 1H), 8.22 (d,
J = 9.5 Hz, 1H), 8.50 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 9.75 (s, 1H),
13.56 (s, 1H); ESI-MS m/z: 544[M+H]+; Anal. Calcd for
5.1.7.10. 1-(5-Methylisoxazol-3-yl)-3-(4-(2-(5-(trifluoromethyl)-
1H-imidazol-2-yl)pyridin-4-yloxy)phenyl)urea (11j).
White
solid, yield: 64%, mp 238–240 °C; 1H NMR (DMSO-d6, 400 MHz):
d 2.33–2.37 (s, 3H), 6.55 (s, 1H), 7.05–7.07 (dd, J = 1.9 and 5.3 Hz,
1H), 7.21 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 1.9 Hz, 1H), 7.60 (d,
J = 7.8 Hz, 2H), 7.85 (s, 1H), 8.54 (d, J = 5.3 Hz, 1H), 8.97 (s, 1H),
9.50 (s, 1H), 13.53 (s, 1H); ESI-MS m/z: 445[M+H]+; Anal. Calcd
for C20H15F3N6O3 (%): C 54.06, H 3.40, N 18.91; Found: C 54.03, H
3.38, N 18.96.
C22H13Cl3F3N5O2 (%): C 48.69, H 2.41, N 12.90; Found: C 48.63, H
2.48, N 12.83.
5.1.7.17. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(3-(trifluoromethyl)phenyl)urea
(11q).
White solid, yield: 67%, mp 213–215 °C; 1H NMR
(DMSO-d6, 400 MHz): d 7.07–7.09 (dd, J = 2.0 and 5.1 Hz, 1H),
7.23 (d, J = 9.1 Hz, 2H), 7.32–7.34 (m, 1H), 7.40 (d, J = 2.0 Hz, 1H),
7.51–7.55 (m, 1H), 7.59–7.63 (m, 3H) , 7.88 (s, 1H), 8.04 (s, 1H),
8.55 (d, J = 5.1 Hz, 1H), 9.13 (s, 1H), 13.58 (s, 1H); ESI-MS m/z:
543[M+H]+; Anal. Calcd for C23H14ClF6N5O2 (%): C 50.98, H 2.60,
N 12.93; Found: C 51.02, H 2.64, N 12.88.
5.1.7.11. 1-(4-Methoxyphenyl)-3-(4-(2-(5-(trifluoromethyl)-1H-
imidazol-2-yl)pyridin-4-yloxy)phenyl)urea
(11k).
White
solid, yield: 72%, mp 240–241 °C; 1H NMR (DMSO-d6, 400 MHz):
d 3.91 (s, 3H), 7.03–7.43 (m, 8H), 7.60 (d, J = 8.0 Hz, 2H), 7.86 (s,
1H), 8.03 (s, 1H), 8.53 (d, J = 5.2 Hz, 1H), 9.06 (s, 1H), 13.54 (s,
1H); ESI-MS m/z: 470[M+H]+; Anal. Calcd for C23H18F3N5O3 (%): C
58.85, H 3.86, N 14.92; Found: C 58.90, H 3.88, N 14.88.
5.1.7.18. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(3,4,5-trimethoxyphenyl)urea
(11r).
White solid, yield: 59%, mp 255–258 °C; 1H NMR
5.1.7.12. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(4-fluoro-3-(trifluoromethyl)
(DMSO-d6, 400 MHz): d 3.77 (s, 9H), 6.66 (s, 2H), 7.06–7.08 (dd,
J = 1.9 and 5.2 Hz, 1H), 7.19–7.22 (m, 1H), 7.44–7.45 (m, 3H),
7.87 (s, 1H), 8.22 (s, 1H), 8.27 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H),
13.60 (s, 1H); ESI-MS m/z: 565[M+H]+; Anal. Calcd for
phenyl)urea (11l).
White solid, yield: 67%, mp 132–135 °C;
1H NMR (DMSO-d6, 400 MHz): d 7.08–7.10 (dd, J = 1.9 and 5.3 Hz,
1H), 7.25–7.28 (dd, J = 8.0 and 7.3 Hz, 1H), 7.44–7.65 (m, 4H),
7.87 (s, 1H), 8.02–8.04 (dd, J = 8.0 and 7.5 Hz, 1H), 8.24 (d,
J = 7.5 Hz, 1H), 8.48 (s, 1H), 8.56 (d, J = 5.3 Hz, 1H), 9.79 (s, 1H),
13.56 (s, 1H); ESI-MS m/z: 561[M+H]+; Anal. Calcd for
C25H21ClF3N5O5 (%): C 53.25, H 3.75, N 12.42; Found: C 53.22, H
3.74, N 12.49.
5.2. Biological assay
C23H13ClF7N5O2 (%): C 49.35, H 2.34, N 12.51; Found: C 49.38, H
2.29, N 12.56.
5.2.1. The assay of antiproliferative activities
The antiproliferative activities of the target compounds 11a–
11r were evaluated with MDA-MB-231, BGC-823 and SMMC-
7721 cell lines by the MTT assay in vitro, with Sorafenib as the
positive control drug. The cancer cell lines were cultured in
RPMI-1640 medium, supplemented with 10% fetal calf serum
(FCS), and maintained in a 5% CO2, 95% humidity atmosphere
at 37 °C. In 96-well plates were seeded 5.0 Â 103 cells/well and
incubated for 24 h. The cells were then incubated for another
72 h with various concentrations of compounds 11a–11r. Subse-
5.1.7.13. 1-(2-Chloro-4-(2-(5-(trifluoromethyl)-1H-imidazol-2-
yl)pyridin-4-yloxy)phenyl)-3-(2-chloro-5-(trifluoromethyl)
phenyl)urea (11m).
White solid, yield: 68%, mp 249–250 °C;
1H NMR (DMSO-d6, 400 MHz): d 7.11–7.12 (dd, J = 1.9 and 5.3 Hz,
1H), 7.26–7.55 (m, 4H), 7.75 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 8.19
(m, 1H), 8.57–8.58 (m, 2H), 9.35 (s, 1H), 9.37 (s, 1H), 13.61 (s,
1H); ESI-MS m/z: 577[M+H]+; Anal. Calcd for C23H13Cl2F6N5O2
(%): C 47.94, H 2.27, N 12.15; Found: C 47.91, H 2.24, N 12.19.
quently, 20 lL of fresh MTT solution (5 mg/ ml) was added to
5.1.7.14. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-chloro-4-
each well and incubated with cells at 37 °C for additional 4 h.
(2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)phe-
nyl)urea (11n).
The supernatant was carefully removed from each well and
100 lL of DMSO was added to each well to dissolve the formazan
White solid, yield: 75%, mp 200–203 °C; 1H
NMR (DMSO-d6, 400 MHz): d 7.10–7.12 (dd, J = 1.8 and 5.3 Hz,
1H), 7.26–7.28 (dd, J = 6.5 and 7.9 Hz, 1H), 7.53 (d, J = 8.0 Hz,
2H), 7.63–7.66 (m, 2H), 7.92 (s, 1H), 8.15 (s, 1H), 8.22 (d,
J = 6.9 Hz, 1H), 8.57 (d, J = 5.2 Hz, 1H), 8.70 (s, 1H), 10.34 (s, 1H),
13.64 (s, 1H); ESI-MS m/z: 577[M+H]+; Anal. Calcd for
crystals which were formed by the cellular reduction of MTT.
After mixing with a mechanical plate mixer, the absorbance of
each well was measured by a plate reader at a test wavelength
of 570 nM. All of the compounds were tested three times in each
of the cell lines. The IC50 values were calculated by linear regres-
sion analysis, expressed in mean SD.

W. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 4323–4329
4329
4. McCubrey, J. A.; Steelman, L. S.; Chappell, W. H.; Abrams, S. L.; Wong, E. W. T.;
Chang, F.; Lehmann, B.; Terrian, D. M.; Milella, M.; Tafuri, A.; Stivala, F.; Libra,
M.; Basecke, J.; Evangelisti, C.; Martelli, A. M.; Franklin, R. A. Biochim. Biophys.
Acta 2007, 1773, 1263.
5.2.2. The assay of inhibitory activities against C-Raf kinase
The inhibitory activities against C-Raf kinase of the target com-
pounds 11a–11r were investigated by the FRET assay, Sorafenib
was also used as positive control drug. Active C-Raf and inactive
Mek were diluted together with kinase dilution buffer (25 mM Tris,
PH 7.5, 0.02 mM EGTA, 0.66 mg/ml myelin basic protein, 1 mM
5. Roberts, P. J.; Der, C. J. Oncogene 2007, 26, 3291.
6. Chong, H.; Vikis, H. G.; Guan, K. L. Cell. Signalling 2003, 15, 463.
7. Smith, A. L.; Demorin, F. F.; Paras, N. A.; Huang, Q.; Petkus, J. K.; Doherty, E. M.;
Nixey, T.; Kim, J. L.; Whittington, D. A.; Epstein, L. F.; Lee, M. R.; Rose, M. J.;
Babij, C.; Fernando, M.; Hess, K.; Le, Q.; Beltran, P.; Carnahan, J. J. Med. Chem.
2009, 52, 6189.
8. Sridhar, S. S.; Hedley, D.; Siu, L. L. Mol. Cancer Ther. 2005, 4, 677.
9. Li, H. F.; Lu, T.; Zhu, T.; Jiang, Y. J.; Rao, S. S.; Hu, L. Y.; Xin, B. T.; Chen, Y. D. Eur. J.
Med. Chem. 2009, 44, 1240.
10. Wong, K. K. Recent Patents Anticancer Drug Discov. 2009, 4, 28.
11. Smith, R. A.; Dumas, J.; Adnane, L.; Wilhelm, S. M. Curr. Top. Med. Chem. 2006,
1071, 6.
12. Montagut, C.; Settleman, J. Cancer Lett. 2009, 283, 125.
13. Ramnath, N.; Adjei, A. Update Cancer Ther. 2007, 2, 111.
14. Eisen, T.; Ahmad, T.; Flaherty, K. T.; Gore, M.; Kaye, S.; Marais, R.; Gibbens, I.;
Hackett, S.; James, M.; Schuchter, L. M.; Nathanson, K. L.; Xia, C.; Simantov, R.;
Schwartz, B.; Poulin-Costello, M.; O’Dwyer, P. J.; Ratain, M. J. Br. J. Cancer 2006,
95, 581.
15. Ramurthy, S.; Subramanian, S.; Aikawa, M.; Amiri, P.; Costales, A.; Dove, J.;
Fong, S.; Jansen, J. M.; Levine, B.; Ma, S.; Mcbride, C. M.; Michaelian, J.; Pick, T.;
Poon, D. J.; Girish, S.; Shafer, C. M.; Stuart, D.; Sung, L.; Renhowe, P. A. J. Med.
Chem. 2008, 51, 7049.
16. Wan, P. T.; Garnett, M. J.; Ros, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116,
856.
DTT, 0.1 mg/ml BSA) to 4 and 20
of this enzyme-substrate mixture was added to each well of a
96-well plate. Test compound (5 L) of desired concentration
was added to the mixture and the kinase reaction was initiated
by adding 25 L of 10
-[33P] ATP (specific activity approx.
lg/ml, respectively, and 20 lL
l
l
lM c
500 cpm/pmol) for incubation at 30 °C for 20 min. The reaction
was spotted onto a phosphocellulose mat, washed with 1% phos-
phoric acid solution, and counted the radioactivity in the presence
of scintillation fluid in a scintillation counter.
Acknowledgment
The generous financial support of this work by the National
Natural Science Foundation of China (30973016) is gratefully
acknowledged.
17. Dimitroff, M.; Miller, B. R.; Stillwell, B. S.; Siesel, D. A.; Swiftney, T.; Diaz, B.; Gu,
D.; Ryckman, D.; Poon, D. J.; Pick, T. E. US20070049622A1.
18. Khire, U. R.; Bankston, D.; Barbosa, J.; Brittelli, D. R.; Caringal, Y.; Carlson, R.;
Dumas, J.; Gane, T.; Heald, S. L.; Hibner, B.; Johnson, J. S.; Katz, M. E.; Kennure,
N.; Kingery-Wood, J.; Lee, W.; Liu, X. G.; Lowinger, T. B.; Mcalexander, I.;
Monahan, M. K.; Natero, R.; Renick, J.; Riedl, B.; Rong, H.; Sibley, R. N.; Smith, R.
A.; Wolanin, D. Bioorg. Med. Chem. Lett. 2004, 14, 783.
References and notes
1. Ma, W. W.; Adjei, A. A. CA Cancer J. Clin. 2009, 59, 111.
2. Weber, C. K.; Slupsky, J. R.; Herrmann, C.; Schuler, M.; Rapp, U. R.; Block, C.
Oncogene 2000, 19, 169.
3. McCubrey, J. A.; Steelman, L. S.; Abrams, S. L.; Chappell, W. H.; Russo, S.; Ove, R.;
Michele, M.; Tafuri, A.; Lunghi, P.; Bonati, A.; Stivala, F.; Nicoletti, F.; Libra, M.;
Martelli, A. M.; Montalto, G.; Cervello, M. Expert Opin. Emerg. Drugs 2009, 14,
633.