Structure based design and syntheses of amino-1H-pyrazole amide derivatives as selective Raf kinase inhibitors in melanoma cells

Article abstract of DOI:10.1016/j.bmc.2011.01.067

The synthesis of a novel series of N-(5-amino-1-(4-methoxybenzyl)-1H- pyrazol-4-yl amide derivatives 6a-o, 7a-s and their antiproliferative activities against A375P melanoma cell line were described. Most compounds showed competitive antiproliferative act

Full text of DOI:10.1016/j.bmc.2011.01.067

Bioorganic & Medicinal Chemistry 19 (2011) 1915–1923
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure based design and syntheses of amino-1H-pyrazole amide
derivatives as selective Raf kinase inhibitors in melanoma cells
Mi-hyun Kim a, Minjung Kim a, Hana Yu a, Hwan Kim b, Kyung Ho Yoo b, Taebo Sim b, Jung-Mi Hah a,
⇑
a Department of Pharmacy, College of Pharmacy, Hanyang University, 1271 Sa 3-Dong, Sangnok-gu, Ansan-si, Gyunggi-do 426-791, South Korea
b Life Sciences Research Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a novel series of N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl amide derivatives
6a–o, 7a–s and their antiproliferative activities against A375P melanoma cell line were described. Most
compounds showed competitive antiproliferative activities to sorafenib, the reference standard. Among
Received 3 January 2011
Revised 28 January 2011
Accepted 29 January 2011
Available online 3 February 2011
them,
N-(5-amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-(3-(4-chloro-3-(trifluoromethyl)phenyl)
ureido)-2-methylbenzamide 7c exhibited potent activities (GI50 = 0.27
be a potent and selective B-Raf V600E and C-Raf inhibitor (IC50 = 0.26
showing a possibility as melanoma therapeutics.
l
M). Especially, 7c was found to
l
M, IC50 = 0.11 M, respectively),
l
Keywords:
Aminopyrazole amide
Antiproliferative activity
Melanoma cell line
Kinase inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Kinase selectivity
1. Introduction
activation segment in an inactive conformation5, namely type II
inhibition.
The ‘Ras/Raf/Mek/Erk pathway’ is a well-known cell signaling
network for cell survival, growth, and proliferation.1 The Ras pro-
teins are membrane-bound small G-protein, whereas Raf, Mek,
and Erk are cytosolic protein kinases that compose a sequential
signaling cascade. Out of these complicated cascade, Raf kinase
has been the most studied drug target since mutations of the Raf
protein were found in approximately 7% of human cancers2,3 with
particularly high frequency in melanoma (50–70%), ovarian (35%),
thyroid (30%), and colorectal (10%) cancers.
The discovery of the most frequent V600E (>85%) B-Raf muta-
tions in 50% of melanoma have raised the expectation for targeted
therapy.2 The V600E B-Raf mutations show a 500-fold increase in
catalytic activity, providing cancer cells with both proliferation
and survival signals.4 Therefore, B-Raf V600E is a high-interest
therapeutic target for the treatment of human cancers.
In our melanoma program, we were intrigued by the well char-
acterized Raf inhibitor sorafenib, the bi-aryl urea compound from
Bayer. Sorafenib is a potent inhibitor of preactivated C-Raf, B-Raf
and oncogenically activated B-Raf kinases (V600E B-Raf:
IC50 = 43 nM), as well as it holds a unique binding mode for Raf
protein. Crystal structure of V600E B-Raf kinase domains in
complex with sorafenib showed that the inhibitor held the
The primary part of pharmacophore was still considered to be
hinge binder in designing a new type II inhibitor for B-Raf V600E,
and we recently reported the imidazolopyrazole bicyclic ring6 as
a novel scaffold for Raf inhibitor. Although the hinge hydrogen
bonding in B-Raf active site is known less critical compared with
that in other kinase inhibitors, we concluded that the imidazolopy-
razole bicyclic ring was not the best scaffold based on SAR study.
Furthermore, the docking analysis showed that the length of the
imidazolopyrazole bicycle was short for an effective hydrogen
bonding with Glu500 and Asp593 and hydrophobic interaction with
secondary pocket.
In an attempt to find a better hinge binding scaffold with
optimal length for effective hydrogen bonding and hydrophobic
interactions (Fig. 1), we tried to crack the imidazole ring in pyraz-
oleimidazole scaffold6, and relieve the strain; therefore, we came
up with novel amino-1H-pyrazole amide derivatives. In docking
experiment (Fig. 2), this new scaffold showed not only proper hinge
binding, but also plausible hydrogen bonding and effective hydro-
phobic interactions.
The structure of this series comprises N-(5-amino-1-alkyl-1H-
pyrazol-4-yl amide part, the middle phenyl ring moiety and
aromatic tail part connected by amide or urea linkage. Specifi-
cally, we modified the structures by introducing two different
direction of connectivity (4-, 5-nitro) in middle phenyl group,
changing the spacer (amide or urea) and various aromatic tails
to understand the relative interaction of hydrophobic tail groups.
⇑
Corresponding author. Tel.: +82 31 400 5803; fax: +82 31 400 5958.
E-mail address: jhah@hanyang.ac.kr (J.-M. Hah).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.067

1916
M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
and 3-methoxyacrylonitrile in one pot process as reported in refer-
O
O
Cl
ence.7 Then, the nitroso group was reduced to give diamine (3)
using SnCl2.8 The pyrazolediamine 3 was then reacted with
2-methyl 5- (or 4-) nitro benzoic acid to provide single amide
product 4. The selective amidation at 4-amino group was consid-
ered to be due to the steric effect of PMB and stronger nucleophi-
licity by resonance. Then the nitro group in 4 was reduced to amino
group and linked with various aromatic acids under EDCI/HOBt
conditions to give amide (6a–6n) or directly aromatic isocynate
to give urea (7a–7p) analogues.
N
O
H
Sorafenib
N
N
H
N
H
CF3
R2
N
O
H
N
Imidazolopyrazole
R3
N
N
H
H
N
N
R1
3. Results and discussion
R2
O
H2N
R1
O
H
N
The synthesized amino-1H-pyrazole amide derivatives 6a–6o
were evaluated for antiproliferative activities against human mel-
anoma cell line, and Table 1 shows the antiproliferative activities9
of amino-1H-pyrazole amide derivatives linked with various aro-
matic tail groups by amide bond. The activities are compared with
sorafenib as a reference (GI50 values) against A375P10 human
melanoma cell line and also normal fibroblast cell line HS27. In
general, the amide derivatives 6 showed poor antiproliferative
activities against both cell lines, while 5-amino substituted 6a,
6b, 6g obtained medieval potency against A375P. With the same
hydrophobic tail group, the substitution on 5-position seems better
in hydrophobic interaction (6g > 6h).
Aminopyrazole amide
N
H
N
R3
N
H
N
Figure 1. Anatomy of sorafenib and imidazolopyrazole derivatives, amino-
1H-pyrazole amide derivatives.
Continuously, we determined antiproliferative activity of
amino-1H-pyrazole ureido amide derivatives and 7a–7s with vari-
ous aromatic tail groups by urea linkage (Table 2). Interestingly,
when we replaced the PMB-group of 1-N in pyrazole moiety with
benzyl group, the antiproliferative potency was dropped dramati-
cally (8c vs 7c), and it was surprising since we found very different
results in our old SAR data of pyrazoloimidazole scaffold.6 There-
fore, we kept the PMB group in our scaffold and continued varia-
tion in substitution of middle phenyl ring and hydrophobic tails.
Urea derivatives were more potent than amide derivatives in gen-
eral, having preference of 2-methyl 5-aminopheurea linked middle
phenyl ring (7c > 7b; 7i > 7h; 7k > 7j) like in amide series 6. Also,
the mono-aromatic tails ( 7b, 7c, 7d, 7h, 7i) were preferred as a
hydrophobic tail rather than bulky bi-aryl tails.
Figure 2. Docking structures of designed amino-1H-pyrazole amide scaffold (bold,
green) overlayed with sorafenib (thin, cyan) and imidazolopyrazole derivatives
(thin, orange) in B-Raf V600E.
We further investigated enzymatic activities (B-Raf V600E,
C-Raf) of several selected compounds. Indeed, we found amide
derivatives 6a, 6b were only C-Raf inhibitors, but urea 7b is inhib-
itors of both Raf kinases (Table 3).
2. Chemistry
Considering that C-Raf is also associated significantly with
disease progression and cell proliferation in
a subset of
melanoma,11–13 the finding of dual Raf-kinase inhibitor 7c was
valuable, and it was selected as a lead compound for further study.
Shown in Table 4, the representative compound 7c were screened
The general synthesis of amino-1H-pyrazole amide derivatives
is shown in Scheme 1. The 1-(4-methoxybenzyl)-4-nitroso-1H-
pyrazol-5-amine (2) was made with p-methoxybenzyl hydrazine
NH2
NH2
NH2
NH2
OMe
PMB
PMB
PMB
i
ii
iii
O
O
N
N
N
N
N
N
NO
N
CN
NO2
H
1
2
3
4
NH2
NH2
NH2
PMB
PMB
O
O
PMB
O
N
N
O
N
N
iv
N
N
v
R
N
N
N
R
N
H
N
H
N
H
H
H
NH2
H
5
6
7
Scheme 1. Reagents and reaction conditions: (i) PMBHNNH2, NaNO2, EtOH, 50 °C; (ii) SnCl2 2H2O, EtOH, 80 °C; (iii) 2-CH3-5-NO2-C6H3-CO2H, HOBt, EDCI, TEA, DMF, 80 °C,
4 h; (iv) HCl, SnCl2ꢀ2H2O, EtOH, 80 °C, 1.5 h; (v) RCO2H, EDCI, TEA, HOBt, DMF or RNCO, THF.

M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
1917
Table 1
Antiproliferative activity of amino-1H-pyrazole amide derivatives amide derivatives 6a–6n
NH2
O
O
N
N
MeO
N
H
R
N
H
6
Compd
Substitution
5-
R
A375P (GI50
,
l
M)
HS27 (GI50, lM)
N
N
6a
4.51
>30
CF3
CF3
6b
6c
5-
5-
5.53
11.3
>30
>30
N
N
CF3
CF3
6d
6e
5-
4-
27.9
NA
>30
>30
NO2
CF3
N
N
Cl
O
6f
5-
NA
>30
CF3
6g
6h
5-
4-
1.39
24.9
>30
>30
Cl
N
O
CF3
N
6i
5-
>30
>30
Cl
N
N
O
6j
4-
4-
NA
NA
>30
>30
N
6k
N
Cl
6l
5-
5-
4-
NA
NA
>30
>30
>30
>30
N
S
6m
6n
N
N
S
O
6o
4-
>30
>30
Sorafenib
5.58
7.85
on selected 30 different kinases panel at a single dose concentra-
tion of 10 M and it was revealed that the compound has an excel-
4. Conclusions
l
lent selectivity profile. While this compound has completely
inhibited C-Raf and more than 97% at this concentration, the inhi-
bition exerted in most other kinases tested activity was below 20%.
A series of amino-1H-pyrazole amide derivatives 6a–6o and
7a–7s, 8c based on the structural features of sorafenib has been
synthesized and evaluated their antiproliferative activities against

1918
M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
Table 2
Antiproliferative activity of amino-1H-pyrazole amide derivatives with urea linkage 7a–7s
NH2
NH2
PMB
O
Bn
O
O
N
N
O
N
N
R
N
H
R
N
N
H
N
H
N
H
N
H
H
7
8
Compd
Substitution
R
A375P (GI50
,
l
M)
HS27 (GI50, lM)
Cl
Cl
7a
5-
11.4
>30
Cl
7b
7c
4-
5-
33.2
0.27
>30
>30
CF3
Cl
CF3
8c
5-
4-
15.9
4.12
>30
>30
Cl
Cl
7d
Cl
Cl
7e
5-
NA
>30
Cl
7f
5-
4-
>30
>30
>30
>30
CF3
CF3
7g
7h
7i
4-
5-
36.5
0.21
>30
>30
Cl
7j
7k
4-
5-
NA
8.73
>30
>30
Cl
7l
5-
5-
NA
NA
>30
>30
7m
F3C
CF3
N
N
7n
7o
4-
4-
98.3
>30
>30
CF3
CF3
N
>100
O
O
N
N
7p
4-
51.5
>30
CF3
N
7q
7r
4-
5-
5.71
NA
>30
>30
CF3
O
N

M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
1919
M)
Table 2 (continued)
Compd
Substitution
4-
R
A375P (GI50
NA
,
l
M)
HS27 (GI50, l
N
CF3
7s
>30
N
Sorafenib
5.58
7.85
Germany). The quantity of silica gel used was 50–100 times the
weight charged on the column. Thin layer chromatography (TLC)
was run on the silica gel coated aluminum sheets (silica gel 60
GF254, E. Merck, Germany) and visualized in ultraviolet (UV) light
(254 nm). 1H NMR and 13C NMR spectra were recorded on Varian
unity Plus 300 MHz and Brucker Avance 400 MHz spectrometer
at 25 °C, using tetramethylsilane (TMS) as the internal standard.
High-resolution MS (HR/MS) experiments were conducted with a
Finnigan LTQ Orbitrap mass spectrometry (Thermo Fisher
Scientific Inc., MA, USA) operated in positive-ion electrospray
mode.
Table 3
Enzymatic activities of selected compounds
IC50 (nM) B-Raf V600E
IC50 (nM) C-Raf
6a
6b
7b
7c
NA
NA
NA
264
3.86
240.3
22.41
8319
107.1
2.87
GW5074
Table 4
Percentages of enzymatic inhibitions by compound 7c (10 lM) on selected protein
kinases
5.1.1. Synthesis of 1-(4-methoxybenzyl)-4-nitroso-1H-pyrazol-
5-amine (2)
Kinase
% Inhibition
Staurosporine IC50 (nM)
AKT1 (dPH, S473D)
ALK
Aurora A
b-RAF (V599E)
c-MET
3.7
7.7
0
97
13
16
0
0
16
0
1.5
0.1
13
1.4
0.7
0
7.1
13
0
4.72
2.71
In round bottom flask, 35% HCl (6 mL, 71.6 mmol) was stirred at
ꢁ15 °C, and
a mixture of 3-methoxyacrylonitrile 1 (1.3 g,
<1.0
15.9 mmol) and 30% NaNO2 solution (3.6 g, 15.9 mmol) in MeOH
(6 mL) was added dropwise at ꢁ15 to ꢁ5 °C. Then the mixture
was stirred at same temperature for 1 h. To remove excess amount
of nitrosyl chloride, the reaction mixture was flushed with N2 for
5 min. Then a mixture of 4-methoxybenzylhydrazine hydrochlo-
ride (3 g, 15.9 mmol), water (4 mL) and MeOH (4 mL) was added,
stirred at 50 °C for 2 h. After cooling the reaction mixture to
10 °C, more water (4 mL) was added, and neutralized with NH4OH.
The reaction mixture was cooled to 5 °C, stirred for 30 min, filtered
off and washed with cold water and MeOH to give desired com-
pound 2 (3 g, 81%) as red solid. 1H NMR (300 MHz, CDCl3) 8.58
(1H, s), 7.13 (2H, d, J = 16.75 Hz), 6.93 (2H, d, J = 13.36 Hz), 6.23
(2H, br s), 5.06 (2H, s), 3.80 (3H, s).
3.32a
193.5
4.79
c-Src
CDK1/cyclin B
CDK2/cyclinE
EGFR/ERBB1
ERK2/MAPK1/P42MAPK
FAK/PTK2
FGFR3
FLT3
GSK3b
IGF-1R
JAK3
3.20
2.13
101.40
11380.0
4.13
21.50
<1.0
2.73
51.62
<1.0
1592
>20000
9.42
18.19
3018b
<1.0
JNK1a1
JNK3/MAPK10
KDR/VEGFR2
MEK1
mTOR/FRAP1
p70S6K
0
1.2
0
5.1.2. Synthesis of 4,5-diamino-1-(4-methoxy-benzyl)pyrazole
(3)
PKA
PLK1
RAF1
RON/MST1R
ROS/ROS1
SYK
0
<1.0
A suspension of 2 (100 mg, 0.43 mmol) in EtOH (0.86 mL) was
acidified with 35% HCl (0.22 mL, 2.15 mmol), then it was stirred
at 80 °C. A solution of SnCl2ꢀ2H2O (213.4 mg, 0.95 mmol) in EtOH
(0.25 mL) was added to dissolved adduct over 15 min. Stirring
was continued for 30 min and the clear solution was cooled to
room temperature, then was poured into ice. The pH was made
slightly basic (pH 7–8) by addition of saturated aqueous sodium
bicarbonate before being extracted with organic solvent. Because
compound 2 is very polar, IPA/CHCl3 = 4:1 solution was used for
extraction. The organic phase is thoroughly washed with brine,
dried over sodium sulfate. Evaporation of the solvent leaves crude
100 mg of 4,5-diamino-1-(4-methoxy-benzyl)pyrazole 3, which
gives one spot on TLC. Purification was not necessary at this step.
(1H NMR (400 MHz, CDCl3) 7.15 (1H, s), 7.11 (2H, d, J = 8.55 Hz),
6.85 (2H, d, J = 8.64 Hz), 5.11 (2H, s), 3.76 (3H, s).
6.3
100
0
0
0
167.40
2.87a
277.10
<1.0
<1.0
<1.0
TRKB/NTRK2
6.6
Data of GW5074.14
Data of LY294002.15
a
b
A375P human cell line, together with normal cell lines in control.
Several compounds in this scaffold showed potent antiproliferative
activities, and furthermore, one of the best compound 7c has been
confirmed as
a potent and selective Raf kinases inhibitor
(IC50 = 264 nM on V600E B-Raf, 107 nM on C-Raf), suggesting that
the amino-1H-pyrazole amide derivatives could serve as a promis-
ing scaffold for new therapeutics of melanoma, having absolutely
impressive kinase profiling.
5.1.3. Synthesis of N-(5-amino-1-(4-methoxybenzyl)-1H-pyr-
azol-4-yl)-2-methyl-5-nitrobenzamide (4)
A solution of 3 compound (348 mg, 1.59 mmol), 2-methyl-5-
nitrobenzoic acid (289 mg, 1.59 mmol), HOBt (323 mg, 2.39 mmol),
EDCI (397 mg, 2.07 mmol) and TEA (0.26 mL, 1.91 mmol) in DMF
(5 mL) was heated at 80 °C for overnight. The reaction mixture
diluted with ethyl acetate and washed with saturated aqueous so-
dium bicarbonate. The organic layer dried over Na2SO4. Purification
of column chromatography with hexane/ethyl acetate = 1:1. Giving
5. Materials and methods
5.1. Chemistry general
All chemicals (reagent grade) used were purchased from Aldrich
(USA). Separation of the compounds by column chromatography
was carried out with silica gel 60 (200–300 mesh ASTM, E. Merck,

1920
M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
12 compound (520 mg, 85.7%), white solid. 1H NMR (400 MHz,
DMSO-d6) 9.56 (1H, s), 8.16 (1H, s), 7.45 (1H, d, J = 7.43 Hz),
7.346 (1H, s), 7.33 (1H, d, J = 9.748 Hz), 7.31 (2H, d, J = 7.11 Hz),
6.88 (2H, d, J = 17.96 Hz), 4.78 (2H, s), 3.66 (3H, s), 2.26 (3H, s).
5.1.5.5. N-(3-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-4-methylphenyl)-1-phenyl-5-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (6e). 1H NMR (400 MHz, DMSO-d6)
10.54 (1H, s), 9.55 (1H, s), 8.23 (1H, s), 7.76 (1H, d), 7.43 (2H, d,
J = 6.3 Hz), 7.29 (1H, d, J = 8.11 Hz), 7.14–7.05 (4H, m), 6.86 (2H,
d, J = 8.57 Hz), 5.14 (2H, s), 5.05 (2H, s), 3.83 (3H, S), 3.72 (3H, d,
J = 1.62 Hz), 2.40 (3H, s); HRMS calcd for C30H26O3N7F3 590.2128.
Found 590.2123.
5.1.4. Synthesis of 5-amino-N-(5-amino-1-(4-methoxybenzyl)-
1H-pyrazol-4-yl)-2-methylbenzamide (5)
To a suspension of 12 (1.36 mmol) in EtOH (5 mL) a solution of
SnCl2ꢀ2H2O (0.95 mmol) in EtOH (0.25 mL) was added over 15 min.
Stirring continued for 30 min then the clear solution was cooled
room temperature. The pH is made slightly basic (pH 7–8) by addi-
tion of saturated aqueous sodium bicarbonate before being
extracted with ethyl acetate. The organic phase is thoroughly
washes with brine, dried over sodium sulfate. Evaporation of the
solvent leaves crude of 4,5-diamino-1-(4-methoxy-benzyl)pyra-
zole 13, which gives one spot on TLC. 1H NMR (400 MHz,
DMSO-d6) 9.75 (1H, s), 7.14 (1H, d, J = 2.5 Hz), 7.45 (1H, d,
J = 7.432 Hz), 7.346 (1H, s), 7.33 (1H, d, J = 9.748 Hz), 7.31 (2H, d,
J = 7.11 Hz), 6.88 (2H, d, J = 17.96 Hz), 4.78 (2H, s), 3.66 (3H, s),
2.26 (3H, s).
5.1.5.6. N-(3-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-4-methylphenyl)-5-(2-chloro-5-(trifluoro-
methyl)phenyl)furan-2-carboxamide (6f). 1H NMR (400 MHz,
DMSO-d6) 10.46 (1H, s), 9.48 (1H, s), 8.47 (1H, s), 7.88 (1H, d,
J = 8.5 Hz), 7.80 (1H, d, J = 8.5 Hz), 7.70 (1H, d, J = 8.5 Hz), 7.65
(1H, s), 7.54–7.49 (2H, m), 7.42 (1H, s), 7.15 (2H, d, J = 8.5 Hz),
6.88 (2H, d, J = 8.5 Hz), 5.15 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.41
(3H, s); HRMS calcd for
624.1624.
C31H25O4 N5ClF3 624.1626. Found
5.1.5.7. N-(3-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-4-methylphenyl)-5-(4-chlorophenyl)isoxazole-3-
carboxamide (6g). 1H NMR (400 MHz, DMSO-d6) 10.80 (1H, s),
9.58 (1H, s), 8.09 (2H, d, J = 7.98 Hz), 7.93 (1H, d, J = 9.93 Hz) 7.71
(2H, s) 7.66 (2H, d, J = 8.58 Hz), 7.48 (2H, d, J = 8.11 Hz), 7.29 (1H,
d, J = 8.11 Hz), 7.15 (2H, d, J = 8.57 Hz), 6.88 (2H, d, J = 8.57 Hz),
5.14 (2H, s), 5.05 (2H, s), 3.72 (3H, d, J = 1.62 Hz), 2.40 (3H, s);
HRMS calcd for C29H25O4N6Cl 557.1705. Found 557.1696.
5.1.5. General synthses of N-(5-amino-1-(4-methoxybenzyl)-1H-
pyrazol-4-yl)-2-methyl-benzamido benzamide (6a–6o)
A solution of 5 compound (10 mg, 0.028 mmol), substituted
benzoic acid (0.028 mmol), HOBt (6.8 mg, 0.05 mmol), EDCI
(8.05 mg, 0.04 mmol) and TEA (10 lL, 0.07 mmol) in DMF
(0.5 mL) was stirred at room temperature for overnight. The reac-
tion mixture was dilluted with ethyl acetate and washed with sat-
urated aqueous sodium bicarbonate. The organic layer dried over
Na2SO4. Purification of column chromatography with Hexane/ethyl
acetate = 1:1 to afford compound 6 as a white solid.
5.1.5.8. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-4-methylphenyl)-5-(4-chlorophenyl)isoxazole-3-
carboxamide (6h). 1H NMR (400 MHz, DMSO-d6) 10.86 (1H, s),
9.50 (1H, s), 8.01 (2H, d, J = 8.28 Hz), 7.71 (2H, s), 7.66 (2H, d,
J = 8.58 Hz), 7.56 (1H, s), 7.49 (1H, d, J = 8.07 Hz), 7.42 (1H, s),
7.15 (2H, d, J = 8.57 Hz), 6.88 (2H, d, J = 8.57 Hz), 5.14 (2H, s),
5.05 (2H, s), 3.72 (3H, d, J = 1.62 Hz), 2.40 (3H, s); HRMS calcd for
C29 H25O4N6Cl 557.1705. Found 557.1696.
5.1.5.1. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-5-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benz-
amido)benzamide (6a). 1H NMR (400 MHz, DMSO-d6) 10.30 (1H,
s), 9.57 (1H, s), 8.22 (1H, s), 7.92 (2H, m), 7.82 (1H, s), 7.79 (1H, s),
7.74 (1H, s), 7.62 (1H, s), 7.44 (1H, s) 7.37 (1H, s), 7.27 (1H, d,
J = 8.51 Hz), 7.15 (2H, d, J = 8.83 Hz), 6.88 (2H, d, J = 8.46 Hz), 5.13
(2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.34 (3H, s); HRMS calcd for
5.1.5.9. N-(3-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-4-methylphenyl)-1-phenyl-5-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (6i). 1H NMR (400 MHz, DMSO-d6)
10.86 (1H, s), 9.50 (1H, s), 8.01 (2H, d, J = 8.28 Hz), 7.71 (2H, s),
7.66 (2H, d, J = 8.58 Hz), 7.56 (1H, s), 7.49 (1H, d, J = 8.07 Hz),
7.42 (1H, s), 7.15 (2H, d, J = 8.57 Hz), 6.88 (2H, d, J = 8.57 Hz),
5.14 (2H, s), 5.05 (2H, s), 3.72 (3H, d, J = 1.62 Hz), 2.40 (3H, s);
HRMS calcd for C31H28O4N7F3 620.2234. Found 620.2233.
C32H34O3N7F3 622.2754. Found 621.2759.
5.1.5.2. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-5-(3-(trifluoromethyl)benzamido) benzamide (6b). 1H
NMR (400 MHz, DMSO-d6) 9.54 (1H, s), 9.14 (1H, s), 8.92 (1H, s),
8.01 (1H, s), 7.55 (2H, s), 7.52 (1H, s), 7.44 (1H, s) 7.30 (1H, d,
J = 8.05 Hz), 7.18 (1H, d, J = 8.06 Hz), 7.15 (2H, d, J = 8.22Hz), 6.88
(2H, d, J = 8.588 Hz), 5.13 (2H, s), 5.05 (2H, s), 3.72 (3H, s),
2.34 (3H, s); HRMS calcd for C27H24O3N5F3 524.1910. Found
524.1902.
5.1.5.10. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)-5-methylisoxazole-3-carboxamide
(6j). 1H NMR1H NMR (400 MHz, DMSO-d6) 10.71 (1H, s), 7.68
(1H, s), 7.67 (1H, d, J = 7.83 Hz), 7.46 (1H, d, J = 8.07 Hz), 7.41
(1H, s), 7.15 (2H, d, J = 8.5 Hz), 6.88 (2H, d, J = 8.5 Hz), 6.66 (1H,
s), 5.14 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.37 (3H, s); HRMS calcd
for C24H24O4N6 461.1938. Found 461.1931.
5.1.5.3. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-5-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)-
benzamido)benzamide (6c). 1H NMR (400 MHz, DMSO-d6)
10.33 (1H, s), 9.52 (1H, s), 8.1 (1H, s), 7.90 (1H, s), 7.81 (1H, s),
7.79 (1H, s), 7.77 (1H, s), 7.64 (1H, s), 7.44 (1H, s), 7.27 (1H, d,
J = 8.12 Hz), 7.15 (2H, d, J = 8.83 Hz), 6.88 (2H, d, J = 8.50Hz), 5.13
(2H, s), 5.05 (2H, s), 3.72 (3H, s),, 2.34 (3H, s), 2.27 (3H, s) HRMS
calcd for C31H28O3N7F3 604.2285. Found 604.2288.
5.1.5.11. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)benzo[b]thiophene-2-carboxamide
(6k). 1H NMR (400 MHz, DMSO-d6) 10.41 (1H, s), 9.47 (1H, s),
8.87 (1H, s), 8.77 (1H, s), 8.66 (1H, s), 7.87–7.80 (2H, m), 7.51
(1H, d, J = 8.12 Hz), 7.48 (1H, s), 7.15 (2H, d, J = 8.54 Hz), 6.88
(2H, d, J = 8.59 Hz), 5.14 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.37
(3H, s); HRMS calcd for C24H23O3N7 458.1941. Found 458.1937.
5.1.5.4. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-5-(4-nitro-3-(trifluoromethyl) benzamido) benzamide
(6d). 1H NMR (400 MHz, DMSO-d6) 10.47 (1H, s), 9.54 (1H, s),
8.32 (2H, m), 8.16 (1H, d, J = 9.01 Hz), 8.01 (1H, s), 7.55 (2H, s),
7.52 (1H, s), 7.44 (1H, s), 7.15 (2H, d, J = 8.22 Hz), 6.88 (2H, d,
J = 8.588 Hz), 5.13 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.34 (3H, s);
HRMS calcd for C27 H23 O5 N6F3 569.1819. Found 569.1758.
5.1.5.12. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)-2-chloroisonicotinamide (6l). 1H
NMR (400 MHz, DMSO-d6) 10.63 (1H, s), 9.47 (1H, s), 8.63 (1H, d,
J = 5.08 Hz), 8.01 (1H, s), 7.87 (1H, dd), 7.68–7.65 (2H, m), 7.50

M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
1921
(1H, d, J = 8.32 Hz), 7.41 (1H, s), 7.15 (2H, d, J = 8.57 Hz), 6.88 (2H,
d, J = 8.57 Hz), 5.14 (2H, s), 5.05 (2H, s), 3.72 (3H, d, J = 1.62 Hz),
2.40 (3H, s); HRMS calcd for C25H23O3N6Cl 491.1599. Found
491.1593.
5.1.6.4. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-4-
(3-(3,4-dichlorophenyl)ureido)-2-methylbenzamide (7d). 1H
NMR (400 MHz, DMSO-d6) 9.39 (1H, s), 9.05 (1H, s), 8.93 (1H, s),
7.89 (1H, d, J = 2.51 Hz), 7.50 (2H, d, J = 5.7 Hz), 7.40 (2H, s),
7.35–7.31 (2H, m), 7.14 (2H, d, J = 8.62 Hz), 6.87 (2H, d,
J = 8.62 Hz), 5.12 (2H, s), 5.04 (2H, s), 3.72 (3H, s), 2.36 (3H, s);
HRMS calcd for C26H24O3N6Cl2 539.1366. Found 539.1361.
5.1.5.13. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)-2-(pyridin-4-yl)thiazole-4-carbox-
amide (6m). 1H NMR (400 MHz, DMSO-d6) 10.35 (1H, s), 9.47
(1H, s), 8.80–8.78 (2H, m), 8.66 (1H, s), 8.13 (2H, m), 7.84–7.72
(2H, m), 7.51 (1H, d, J = 8.41 Hz), 7.42 (1H, s), 7.15 (2H, d,
J = 8.5 Hz), 6.88 (2H, d, J = 8.5 Hz), 6.66 (1H, s), 5.14 (2H, s), 5.05
(2H, s), 3.72 (3H, s), 2.37 (3H, s); HRMS calcd for C28H25O3N7S
540.1819. Found 540.1817.
5.1.6.5. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(2,6-dichlorophenyl)ureido)-2-methyl benzamide (7e). 1H
NMR (400 MHz, DMSO-d6) 9.58 (1H, s), 9.55 (1H, s), 8.53 (1H, s),
7.77 (1H, s), 7.53 (2H, d, J = 5.69Hz), 7.43 (1H, s), 7.35 (1H, d,
J = 5.89 Hz), 7.22 (1H, s), 7.15 (2H, d, J = 8.42 Hz), 7.11 (1H, s),
6.88 (2H, d, J = 8.7 Hz), 5.13 (2H, s), 5.05 (2H, s), 3.72 (3H, s),
2.44 (3H, s); HRMS calcd for C26H24O3N6Cl2 539.1366 Found
539.1359
5.1.5.14. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)pyrazine-2-carboxamide (6n). 1H
NMR (400 MHz, DMSO-d6) 10.59 (1H, s), 9.47 (1H, s), 8.39 (1H,
s), 8.07 (1H, d, J = 8.09 Hz), 8.02 (1H, d, J = 8.09 Hz), 7.7 (1H, m),
7.67 (2H, m), 7.50 (2H, d, J = 7.23 Hz), 7.42 (1H, s), 7.15 (2H, d,
J = 8.57 Hz), 6.88 (2H, d, J = 8.57 Hz), 5.14 (2H, s), 5.05 (2H, s),
3.72 (3H, d, J = 1.62 Hz), 2.41 (3H, s); HRMS calcd for C28H25O3N5S
512.1757. Found 512.1748.
5.1.6.6. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-4-(3-(3-(trifluoromethyl)phenyl)ureido)
benzamide
(7f). 1H NMR (400 MHz, DMSO-d6) 9.54 (1H, s), 9.14 (1H, s),
8.92 (1H, s), 8.01 (1H, s), 7.55 (2H, s), 7.52 (1H, s), 7.44 (1H, s)
7.30 (1H, d, J = 8.05 Hz), 7.18 (1H, d, J = 8.06 Hz), 7.15 (2H, d,
J = 8.22 Hz), 6.88 (2H, d, J = 8.588 Hz), 5.13 (2H, s), 5.05 (2H, s),
3.72 (3H, s), 2.34 (3H, s); HRMS calcd for C27H25O3N6F3 539.2019.
Found 539.2012.
5.1.5.15. N-(4-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-ylc-
arbamoyl)-3-methylphenyl)benzofuran-2-carboxamide
(6o). 1H NMR (400 MHz, DMSO-d6) 10.60 (1H, s), 9.46 (1H, s),
7.84 (1H, d, J = 7.66 Hz), 7.79 (1H, d, J = 0.65 Hz) 7.74 (1H, s), 7.72
(2H, s), 7.54–7.48 (2H, m), 7.42 (1H, s), 7.39–7.36 (1H, m) 7.15
(2H, d, J = 8.57 Hz), 6.88 (2H, d, J = 8.57 Hz), 5.14 (2H, s), 5.05
(2H, s), 3.72 (3H, d, J = 1.62 Hz), 2.40 (3H, s); HRMS calcd for
5.1.6.7. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-5-(3-(4-(trifluoromethyl)phenyl)ureido)
benzamide
(7g). 1H NMR (400 MHz, DMSO-d6) 9.14 (1H, s), 7.55 (2H, m),
7.38 (1H, s), 7.21 (1H, d, J = 8.55Hz), 7.14 (2H, d, J = 8.44 Hz), 6.89
(2H, s), 6.39 (2H, s), 5.37 (2H, s), 5.15 (2H, s), 5.05 (2H, s), 3.72
(3H, s), 2.32 (3H, s); HRMS calcd for C27H25O3N6F3 539.2019. Found
539.2011.
C28H25O4N5 496.1986. Found 496.1978.
5.1.6. General synthses of N-(5-amino-1-(4-methoxybenzyl)-1H-
pyrazol-4-yl)-2-methyl-ureido benzamide (7a–7s)
A mixture of 5-amino-N-(5-amino-1-(4-methoxybenzyl)-1H-
pyrazol-4-yl)-2-methylbenzamide 5 and substituted isocyanate in
THF was stirred at room temperature overnight. The reaction mix-
ture was cooled, extracted with CH2Cl2. The organic layer was
washed with water and brine. After drying over anhydrous MgSO4,
and the mixture was evaporated under reduced pressure to give a
residue. The residue was purified by silica gel chromatography.
5.1.6.8. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-4-
(3-(3-chlorophenyl)ureido)-2-methylbenzamide (7h). 1H NMR
(400 MHz, DMSO-d6) 9.92 (1H, s), 9.45 (1H, s), 8.55 (1H, s), 7.77
(1H, d, J = 3.34 Hz), 7.48 (2H, d, J = 4.88 Hz), 7.31 (2H, m), 7.14
(2H, d, J = 8.62 Hz), 6.87 (2H, d, J = 8.62 Hz), 5.12 (2H, s), 5.04
(2H, s), 3.72 (3H, s), 2.36 (3H, s); HRMS calcd for C26H25O3N6Cl3
505.1756. Found. 505.1748.
5.1.6.1. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
5.1.6.9. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(3-chlorophenyl)ureido)-2-methylbenzamide (7i). 1H NMR
(400 MHz, DMSO-d6) 9.68 (1H, s), 9.54 (1H, s), 8.45 (1H, s), 7.65–
7.55 (2H, m), 7.39 (1H, m), 7.35 (1H, d, J = 2.6 Hz), 7.34 (1H, s),
7.31 (1H, s), 7.29–7.27 (1H, m), 7.22 (1H, s), 7.14 (2H, d,
J = 8.52 Hz), 6.87 (2H, d, J = 8.50 Hz), 5.10 (2H, s), 5.05 (2H, s),
methyl-5-(3-(2,4,5-trichlorophenyl)ureido)
benzamide
(7a). 1H NMR (400 MHz, DMSO-d6) 9.61 (1H, s), 9.55 (1H, s),
8.53 (1H, s), 7.90 (1H, s), 7.77 (1H,s), 7.68–7.62 (3H, m), 7.54
(1H, s), 7.43 (1H, s), 7.35 (1H, d, J = 5.89 Hz), 7.21 (2H, d,
J = 8.86 Hz), 7.15 (2H, d, J = 8.42 Hz), 7.10 (1H, s), 6.88 (2H, d,
J = 8.7 Hz), 5.13 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.44 (3H, s); HRMS
calcd for C26H23O3N6Cl3 573.0976. Found 573.0970.
3.72 (3H, s), 2.32 (3H, s); HRMS calcd for
505.1756. Found 505.1747.
C26H25O3N6Cl3
5.1.6.2. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-4-
(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-2-methylben-
zamide (7b). 1H NMR (400 MHz, DMSO-d6) 9.10 (1H, s), 7.40 (1H,
m), 7.21 (1H, d, J = 8.688 Hz), 7.14 (2H, d, J = 8.44 Hz), 6.89 (2H, s),
6.39 (2H, s), 5.37 (2H, s), 5.15 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.32
(3H, s); HRMS calcd for C27H24O3N6ClF3 573.1629. Found 573.1623.
5.1.6.10. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-4-
(3-(4-chlorophenyl)ureido)-2-methylbenzamide (7j). 1H NMR
(400 MHz, DMSO-d6) 10.21 (1H, s), 10.04 (1H, s), 8.89 (1H, s),
8.10 (1H, d, J = 2.38 Hz), 7.54 (1H, d, J = 2.26 Hz), 7.44 (1H, s),
7.41 (1H, d, J = 2.132 Hz), 7.17 (2H, d, J = 9.21 Hz), 7.15 (2H, d,
J = 8.67 Hz), 6.87 (2H, d, J = 8.67 Hz), 6.44 (2H, d, J = 9.24 Hz), 5.12
(2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.32 (3H, s); HRMS calcd for
5.1.6.3. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-2-methylben-
zamide (7c). 1H NMR (400 MHz, DMSO-d6) 9.53 (1H, s), 9.19 (1H,
s), 8.89 (1H, s), 8.10 (1H, d, J = 2.38 Hz), 7.65–7.59 (3H, m), 7.54
(1H, d, J = 2.26 Hz), 7.44 (1H, s), 7.41 (1H, d, J = 2.132 Hz), 7.19
(2H, d, J = 8.492 Hz), 7.15 (2H, d, J = 8.67 Hz), 6.87 (2H, d,
J = 8.67 Hz), 5.12 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.32 (3H, s);
HRMS calcd for C27H24O3N6ClF3 573.1629. Found 573.1622.
C26H25O3N6Cl3 505.1756. Found 505.1749.
5.1.6.11. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(4-chlorophenyl)ureido)-2-methylbenzamide (7k). 1H NMR
(400 MHz, DMSO-d6) 9.68 (1H, s), 9.54 (1H, s), 8.45 (1H, s), 7.65–
7.55 (2H, m), 7.39 (1H, m), 7.35 (1H, d, J = 2.6 Hz), 7.34 (1H, s),
7.31 (1H, s), 7.29–7.27 (1H, m), 7.22 (1H, s), 7.14 (2H, d,
J = 8.52 Hz), 6.87 (2H, d, J = 8.50 Hz), 5.10 (2H, s), 5.05 (2H, s),

1922
M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
3.72 (3H, s), 2.32 (3H, s); HRMS calcd for C26H25O3N6Cl3 505.1756.
Found 505.1748.
5.07 (2H, s), 3.72 (3H, s), 2.35 (3H, d, J = 5.58Hz), 2.34 (3H, s); HRMS
calcd for C24H25O4N7 476.2047. Found 476.2041.
5.1.6.12. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
5.1.6.19. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-4-(3-(4-(trifluoromethyl)pyrimidin-2-yl) ureido)benz-
amide (7s). 1H NMR (400 MHz, DMSO-d6) 9.84 (1H, s), 9.11
(1H, s), 8.12 (1H, d, J = 4.56 Hz), 7.21 (1H, d, J = 8.12 Hz), 7.14
(2H, d, J = 8.44 Hz), 6.89 (2H, d, J = 8.50 Hz), 6.39 (1H, d), 5.37
(2H, s), 5.15 (2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.32 (3H, s); HRMS
calcd for C25H23O3N8F3 541.1924. Found 541.1919.
(3-cyclohexylureido)-2-methylbenzamide
(7l). 1H
NMR
(400 MHz, DMSO-d6) 9.51 (1H, s), 8.47 (1H, s), 7.39 (1H, s), 7.35
(1H, d, J = 2.6 Hz), 7.34 (1H, s), 7.29–7.27 (1H, m), 7.22 (1H, s),
7.14 (2H, d, J = 8.57 Hz), 6.87 (2H, d, J = 8.56 Hz), 5.10 (2H, s),
5.05 (2H, s), 3.72 (3H, s), 2.32 (3H, s), 1.81–1.64 (4H, m), 1.28–
1.14 (6 H, m); HRMS calcd for C26H32O3N6 477.2615. Found
477.2607.
5.1.6.20. N-(5-Amino-1-benzyl-1H-pyrazol-4-yl)-5-(3-(4-chloro-
3-(trifluoromethyl)phenyl)ureido)-2-methylbenzamide
5.1.6.13. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(2,4-bis(trifluoromethyl)phenyl)ureido)-2-methylbenzamide
(7m). 1H NMR (400 MHz, DMSO-d6) 9.50 (1H, s), 9.17 (1H, s), 8.89
(1H, s), 8.10 (1H, d, J = 2.38 Hz), 7.69–7.61 (4H, m), 7.54 (1H, d,
J = 2.26 Hz), 7.47 (1H, s), 7.43 (1H, d, J = 2.56 Hz), 7.31 (1H, d,
J = 9.23 Hz), 7.15 (2H, d, J = 8.67 Hz), 6.87 (2H, d, J = 8.67 Hz), 5.12
(2H, s), 5.05 (2H, s), 3.72 (3H, s), 2.32 (3H, s); HRMS calcd for
(8c). 1H NMR (400 MHz, DMSO-d6 9.58 (1H, s), 9.22 (1H, s), 8.93
(1H, s), 8.10 (1H, d, J = 2.31 Hz), 7.63–7.61 (2H, m), 7.55 (1H, d,
J = 2.26 Hz), 7.47 (1H, s), 7.43 (1H, dd, J = 2.22 Hz, 9.8 Hz), 7.31
(2H, d, J = 7.74 Hz), 7.26 (1H, d, J = 6.88 Hz), 7.20 (1H, s), 7.17
(2H, d, J = 6.8 Hz), 5.16 (2H, s), 5.12 (2H, s), 2.32 (3H, s); HRMS
calcd for C26H22O2N6ClF3 543.1524. Found 543.1517.
C28H24O3N6F6 607.1893. Found 607.1885.
5.2. Antiproliferative activity
5.1.6.14. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-5-
(3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen-
yl)ureido)-2-methylbenzamide (7n). 1H NMR (400 MHz, DMSO-
d6) 9.58 (1H, s), 9.26 (1H, s), 9.02 (1H, s), 7.65–7.56 (4H, m), 7.40
(1H, dd, J = 2.33 Hz, 8.32 Hz) 7.22 (1H, d, J = 6.6 Hz), 7.16 (2H, d,
J = 8.75 Hz), 6.89 (2H, d, J = 8.67 Hz), 5.13 (2H, s), 5.08 (2H, s),
3.72 (3H, s), 3.16 (2H, s), 3.14 (3H, m), 2.93 (4H, t, J = 12.4 Hz),
2.40 (4H, t, J = 12.3 Hz), 2.34 (3H, s), 2.29 (3H, s), 1.18 (3H, t,
J = 7.24 Hz); HRMS calcd for C34 H39O3N8F3 665.3176. Found
665.3187.
A375P cells were purchased from American Type Culture
Collection (ATCC, Rockville, MD, US) and maintained in DMEM
medium (Welgene, Daegu, Korea) supplemented with 10% FBS
(Welgene) and 1% penicillin/streptomycin (Welgene) in a humidi-
fied atmosphere with 5% CO2 at 37 °C. A375P cells were taken from
culture substrate with 0.05% trypsin–0.02% EDTA and plated at a
density of 5 ꢂ 103 cells/well in 96 well plates and then incubated
at 37 °C for 24 hours in a humidified atmosphere with 5% CO2 prior
to treatment of various concentration (3-fold serial dilution, 12
points) of test compounds. The A357P cell viability was assessed
by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) reduction assay. MTT assays were carried
out with CellTiter 96Ò (Promega) according to the manufacturer’s
instructions. The absorbance at 590 nm was recorded using
EnVision 2103 (Perkin Elmer; Boston, MA, US). The IC50 was calcu-
lated using GRAPHPAD PRISM 4.0 software.
5.1.6.15. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-4-(3-(2-morpholino-5-(trifluoromethyl)phenyl)ureido)-
benzamide (7o). 1H NMR (400 MHz, DMSO-d6) 9.91 (1H, s), 9.43
(1H, s), 8.47 (1H, s), 8.35 (1H, s), 7.49–7.30 (6 H, m), 7.15 (2H, d,
J = 8.5 Hz), 6.88 (2H, d, J = 8.5 Hz), 5.15 (2H, s), 5.05 (2H, s), 3.72
(3H, s), 3.88 (4H, s), 2.86 (4H, s), 2.41 (3H, s); HRMS calcd for
C31H32O4N7F3 624.2547. Found 624.2540.
5.3. Docking simulations
5.1.6.16. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-4-(3-(3-morpholino-5-(trifluoromethyl)phenyl)ureido)-
benzamide (7p). 1H NMR (400 MHz, DMSO-d6) 9.39 (1H, s), 9.11
(1H, d, J = 9.24 Hz), 8.95 (1H, s), 8.87 (1H, s), 7.40 (1H, s), 7.35 (2H,
s), 7.32 (1H, s), 7.20 (1H, m), 7.16–7.13 (2H, m), 6.88–6.84 (2H, m),
6.39 (1H, s), 5.42 (1H, s), 5.12 (1H, s), 5.05 (2H, t, J = 7.88 Hz), 3.74
(4H, t, J = 4.78 Hz) 3.72 (3H, d, J = 1.62 Hz), 3.16 (4H, t, J = 4.78 Hz),
2.36 (3H, s); HRMS calcd for C31H32O4N7F3 624.2547. Found
624.2545.
Molecular docking of compound 7c into 3D X-ray structure of
V600E B-Raf (PDB code: 1uwj) was carried out using the Glide
SCHRODINGER software package Version 9.1).
(
5.4. Selected kinase profiling
We used Reaction Biology Corp. Kinase HotSpotSM service
(http://www.reactionbiology.com) for screening of 7b, and IC50
Profiler Express for IC50 measurement. Assay protocol: In a final
reaction volume of 25
25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic pro-
tein, 10 mM Mg Acetate and [
33P-ATP] (specific activity approx.
500 cpm/pmol, concentration as required). The reaction is initiated
by the addition of the Mg-ATP mix. After incubation for 40 min at
lL, C-RAF (h) (5–10 mU) is incubated with
5.1.6.17. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
methyl-4-(3-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluorometh-
yl)phenyl)ureido)benzamide (7q). 1H NMR (400 MHz, DMSO-
d6) 9.40 (1H, s), 9.26 (1H, s), 9.12 (1H, s), 8.95 (1H, s), 8.87 (1H,
s), 7.40 (1H, s), 7.35 (2H, s), 7.32 (1H, s), 7.20 (1H, m), 7.16–7.13
(2H, m), 6.88–6.84 (2H, m), 6.39 (1H, s), 5.42 (1H, s), 5.12 (1H, s),
5.05 (2H, t, J = 7.88 Hz), 3.74 (4H, t, J = 4.78 Hz) 3.72 (3H, d,
J = 1.62 Hz), 3.16 (4H, t, J = 4.78 Hz), 2.36 (3H, s); HRMS calcd for
c
-
room temperature, the reaction is stopped by the addition of 5
lL
of a 3% phosphoric acid solution. 10 L of the reaction is then spot-
l
ted onto a P30 filtermat and washed three times for 5 min in
75 mM phosphoric acid and once in methanol prior to drying and
scintillation counting.
C31H29O3N8F3 619.2394. Found 619.2398.
5.1.6.18. N-(5-Amino-1-(4-methoxybenzyl)-1H-pyrazol-4-yl)-2-
Acknowledgments
methyl-5-(3-(3-methylisoxazol-5-yl)ureido)benzamide
(7r).
1H NMR (400 MHz, DMSO-d6) 9.58 (1H, s), 9.26 (1H, s), 7.65–7.56
(3H, m), 7.40 (1H, s) 7.22 (1H, d, J = 6.6 Hz), 7.16 (2H, d,
J = 8.75 Hz), 6.89 (2H, d, J = 8.64 Hz), 6.61 (1H, s), 5.13 (2H, s),
This work was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2009-

M. Kim et al. / Bioorg. Med. Chem. 19 (2011) 1915–1923
1923
were taken from culture substrate with 0.05% trypsin–0.02% EDTA and plated
at a density of 5 ꢂ 103 cells/well in 96 well plates and then incubated at 37 °C
for 24 h in a humidified atmosphere with 5% CO2 prior to treatment of various
concentration (three-fold serial dilution, 12 points) of test compounds. The
A357P cell viability was assessed by the conventional 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assays
were carried out with CellTiter 96Ò (Promega) according to the manufacturer0s
instructions. The absorbance at 590 nm was recorded using EnVision 2103
(Perkin Elmer; Boston, MA, US). The IC50 was calculated using GRAPHPAD PRISM 4.0
software.
0087992; J.M.H.). We also thank Professor Hye Hyun Yoo in
Hanyang University for HRMS data acquisition and interpretation.
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