New diarylureas and diarylamides containing 1,3,4-triarylpyrazole scaffold: Synthesis, antiproliferative evaluation against melanoma cell lines, ERK kinase inhibition, and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2011.08.013

Synthesis of a new series of diarylureas and diarylamides possessing 1,3,4-triarylpyrazole scaffold is described. Their in vitro antiproliferative activities against 9 human melanoma cell lines were tested. Compounds 12, 13, 15, and 21-23 showed the highe

Full text of DOI:10.1016/j.ejmech.2011.08.013

European Journal of Medicinal Chemistry 46 (2011) 5754e5762
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New diarylureas and diarylamides containing 1,3,4-triarylpyrazole scaffold:
Synthesis, antiproliferative evaluation against melanoma cell lines, ERK kinase
inhibition, and molecular docking studies
,
,b,
Won-Kyoung Choi a, Mohammed I. El-Gamal a b, Hong Seok Choi c, Daejin Baek d, Chang-Hyun Oh a
*
a Center for Biomaterials, Korea Institute of Science and Technology, P.O. Box 131,Cheongryang, Seoul 130-650, Republic of Korea
b Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
c College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea
d Department of Chemistry, Hanseo University, Seosan 356-706, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a new series of diarylureas and diarylamides possessing 1,3,4-triarylpyrazole scaffold is
described. Their in vitro antiproliferative activities against 9 human melanoma cell lines were tested.
Compounds 12, 13, 15, and 21e23 showed the highest potency against A375P melanoma cell line. In
addition, compounds 10e15 and 19e24 showed high potency over the NCI 8 tested melanoma cell-lines
Received 31 March 2011
Received in revised form
3 August 2011
Accepted 7 August 2011
Available online 12 August 2011
panel. The IC50 values for compound 23 were 0.36 mM and 0.84 mM over LOX IMVI and M14 cell lines,
respectively. Compounds 21 and 23 showed high, dose-dependent inhibition of ERK kinase. Virtual
screening was carried out through docking of compound 21 into the domain of V600E-B-RAF and the
binding mode was studied.
Keywords:
1,3,4-Triarylpyrazole
Melanoma
Ó 2011 Elsevier Masson SAS. All rights reserved.
Diarylurea
Diarylamide
B-RAF kinase
ERK kinase
1. Introduction
a median survival time of less than one year, and the estimated 5-year
survival rate is less than 15% [7,8]. With the incidence of melanoma
The RAS-RAF-MEK-ERK signalling pathway (ERK pathway) plays
an important role in tumorigenesis and cancer progression [1].
Sorafenib (Nexavar), a diarylurea derivative, targets ERK pathway. It
inhibits basal phosphorylation of ERK (pERK) in numerous cancer
cell lines in vitro, including melanoma cell lines, independent of
their K-RAS and B-RAF mutational status [2]. In addition, sorafenib
is a well known inhibitor of B-RAF. Dysregulated signaling through
RAF kinase isoforms has been detected in w30% of human cancers
[3]. Constitutive B-RAF activity can be caused by activating onco-
genic mutations, such as B-RAF V600E mutation, which is prevalent
in melanomas (63%) [4].
rapidly rising in the United States and other developed countries,
there is an urgent need to develop more effective drugs [9e11].
A number of reports have recently highlighted diarylureas and
diarylamides as potential antiproliferative agents against mela-
noma cell line [12e19]. Sorafenib (Nexavar) is a diarylurea deriva-
tive that has been extensively used in clinical trials [20].
Encouraged by the interesting antiproliferative activity of diary-
lurea and diarylamide derivatives, a new series of diarylureas and
diarylamides containing 1,3,4-triarylpyrazole scaffold was synthe-
sized (Fig. 1). Their in vitro antiproliferative activities against nine
human melanoma cell lines are reported. ERK kinase inhibitory
activity of compounds 21 and 23, in addition to molecular docking
of compound 21 into the domain of V600E-B-RAF are also reported.
Melanoma is the most serious type of skin cancer as a malignant
tumor of melanocytes [5]. Early stage melanoma can be cured
surgically. However, melanoma metastasizing to major organs (stage
IV) is virtually incurable [6]. Patients with advanced melanoma have
2. Results and discussion
2.1. Chemistry
* Corresponding author. Center for Biomaterials, Korea Institute of Science and
Technology, P.O. Box 131,Cheongryang, Seoul 130-650, Republic of Korea. Tel.: þ82 2
958 5160; fax: þ82 2 958 5189.
The target triarylpyrazole compounds 9e24 were synthesized
according to the sequence of reactions illustrated in Scheme 1.
E-mail address: choh@kist.re.kr (C.-H. Oh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.013

W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
5755
OR1
with the appropriate benzoic acid derivatives in the presence of
HOBt, EDCI, and triethylamine. Demethylation of the methoxy group
of 9e16 using BBr3 afforded the corresponding hydroxyl derivatives
17e24.
O
CF3
Cl
H3C
O
Cl
O
N
H
O
R2
N
N
N
NH
N
H
N
H
N
Sorafenib
9-24
2.2. Biological evaluation
R
1 = H, CH3
R2 = Ar, ArNH
2.2.1. Antiproliferative activity against A375P human melanoma
cell line
Fig. 1. Structures of Sorafenib and the target compounds 9e24.
The antiproliferative activity of the target compounds against
A375P human melanoma cell line was tested. Their ability to inhibit
the growth of A375P cell line is summarized in Table 1. The results
are expressed as IC50 values. Sorafenib was selected as a reference
standard.
Methylation of the phenolic hydroxyl group of 2-chloro-5-
methylphenol (1) using dimethyl sulfate gave 1-chloro-2-methoxy-
4-methylbenzene (2) [21]. Oxidation of the methyl group of 2 using
KMnO4 produced 4-chloro-3-methoxybenzoic acid (3) [22], which
upon esterification with methanol in the presence of acetyl chloride
afforded the corresponding methyl ester 4. The pyridyl derivative 5
was obtained by treatment of 4 with 4-picoline in the presence of
lithium bis(trimethylsilyl)amide (LHMDS). Cyclization to the pyr-
azole compound 6 was carried out by treatment of 5 with dime-
thylformamide dimethyl acetal (DMF-DMA), and subsequent
treatment with hydrazine monohydrate. 1,3,4-Triarylpyrazole
derivative 8 with amino group was prepared through N-arylation
of 6 using 1-iodo-4-nitrobenzene in the presence of anhydrous
As listed in Table 1, most of the compounds showed moderate
activity, while compounds 15 and 21e23 having IC50 values ranging
from 6.7 to 9.8
m
M were more potent than Sorafenib (IC50 ¼ 11.5
mM).
In addition, compounds 12 and 13 exhibited similar potency to that
of Sorafenib. Compounds 12, 13 and 15 possess a methoxy group
while compounds 21e23 possess a hydroxyl group. In addition,
compound 12 possesses a urea linker while compounds 13, 15, and
21e23 have an amide group as a linker.
The hydroxyl compounds 17, 18, and 21e23 were more potent
than the corresponding methoxy derivatives 9, 10, and 13e15.
These results suggest that the hydroxyl group on position 3 of the p-
chlorophenyl ring at pyrazole ring is optimal for the activity. This
may be attributed to hydrogen bond formation at the receptor site.
Docking of 21 structure into the domain of V600E-B-Raf kinase
crystal structure revealed the formation of one hydrogen bond by
the hydroxyl group at the binding site (molecular docking part). Or
K2CO3, CuI, and L-proline, and subsequent reduction of the nitro
group of 7 using palladium over carbon in hydrogen atmosphere.
Reaction of the amino group of 8 with the appropriate aryl isocya-
nate derivatives afforded the corresponding urea derivatives 9e12.
Synthesis of the methoxy compounds 13e16 with amide moiety as
a linker was carried out by condensation of the amino compound 8
OH
OCH3
OCH3
OCH3
OCH3
Cl
Cl
Cl
Cl
Cl
a
c
b
d
OH
O
CH3
CH3
CH3
O
O
5
O
N
4
1
2
3
OCH3
OCH3
OCH3
Cl
Cl
Cl
N
N
N
e
g
f
N
NH2
N
NO2
NH
N
N
N
6
8
7
OCH3
OH
Cl
Cl
N
N
j
N
NH Ar
NH
O
N
NH Ar
NH
O
h
N
N
9-12
17-20
i
OH
OCH3
Cl
Cl
N
N
N
N
j
NH
Ar
O
NH
Ar
O
N
N
21-24
13-16
Scheme 1. Reagents and conditions: (a) (CH3)2SO4, K2CO3, acetone, reflux, 1 h; (b) KMnO4, C5H5N, H2O, 50 ꢀC, 24 h, then rt, 13 h; (c) acetyl chloride, CH3OH, rt, 15 h; (d) 4-picoline,
LHMDS, THF, rt, overnight; (e) (i) DMF-DMA, rt, 18 h, (ii) hydrazine monohydrate, C2H5OH, rt, overnight; f) 1-iodo-4-nitrobenzene, K2CO3, CuI, L-proline, DMSO, 90 ꢀC, 8 h; g) H2, Pd/
C, THF, rt, 2 h; h) appropriate aryl isocyanate, THF, rt, 12 h; i) appropriate benzoic acid derivative, HOBt, EDCI, TEA, DMF, 80 ꢀC, 12 h; j) BBr3, CH2Cl2, -78 ꢀC, 30 min; then rt, 1 h.

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W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
Table 1 (continued)
Table 1
Antiproliferative activity of the target compounds 9e24 against A375P cell line.
Compound No.
R1
R2
IC50 (mM)
R1
O
Cl
Cl
21
H
6.7
6.8
O
R2
N
Cl
N
N
H
Cl
22
23
H
N
Cl
Cl
Compound No.
R1
R2
IC50 (mM)
H
H
6.8
N
9
CH3
CH3
CH3
>20
H
F3C
F3C
Cl
Cl
Cl
24
>20
N
H
10
11
>20
F3C
Cl
Sorafenib
11.5
Cl
N
H
14.0
11.8
H
F3C
F3C
the o-chloro-phenolic moiety may induce additional DNA damage
effect in the presence of copper and oxygen.
The effects of the amide and urea moieties as linkers on the
activity were also investigated. The amide derivatives 13, 15, 21, and
23 showed higher potency than the corresponding urea derivatives
10, 11, 18, and 19. In addition, the most potent compounds 15 and
21e23 possess an amide linker. They are more potent than the
diarylurea reference standard, Sorafenib.
As observed from the data, the best results were obtained with
compounds 21e23 which possess hydroxyl group on the p-chlor-
ophenyl ring and amide linker. So we can conclude that these two
moieties are optimum for antiproliferative activity of this series of
compounds against A375P human melanoma cell line.
N
12
CH3
F3C
Cl
13
14
CH3
CH3
13.0
Cl
Cl
>20
Cl
Cl
2.2.2. In vitro anticancer screening over 8 melanoma cell lines at
the NCI
After initial single-dose screening of the 16 target compounds at
the National Cancer Institute (NCI) [23], Bethesda, Maryland, USA,
12 compounds, 10e15 and 19e24, with interesting inhibitory
activity in single-dose testing were further tested in a five-dose
testing mode, in order to determine their potency over 8 mela-
noma cell lines. For each of these compounds, the IC50 (the
concentration producing 50% inhibition) values were recorded. The
antiproliferative activity of these twelve compounds over 8 mela-
noma cell lines are summarized in Table 2.
15
16
CH3
CH3
9.8
F3C
F3C
>20
F3C
N
As shown in Table 2, the twelve tested compounds have shown
good potency over the 8 tested cell lines. The IC50 values of the
17
18
19
H
H
H
16.8
13.5
14.9
H
Cl
Cl
Cl
tested compounds were less than 10
mM over all the cell lines,
except for compounds 13 and 14 which exhibited lower potency
against few cell lines. Of special interest, the IC50 of compound 23
N
H
H
was in sub-micromolar scale against 2 cell lines. It was 0.36
mM and
Cl
0.84 M in case of LOX IMVI and M14 cell lines, respectively. This
m
high potency of compound 23 together with its result against
A375P cell line suggest that hydroxyl group, amide linker, and 4-
chloro-3-(trifluoromethyl)phenyl terminal moiety are the most
optimum for antiproliferative activity of this series of compounds
against melanoma cell lines.
Cl
F3C
N
F3C
N
20
H
>20
H
2.2.3. ERK kinase inhibition
F3C
In order to study the mechanism of action of this series of
compounds, the most potent compound against A375P human
melanoma cell line 21 and the most potent derivative against the

W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
5757
Table 2
Antiproliferative activity of compounds 10e15 and 19e24 over 8 melanoma cell lines.
Compound No.
Melanoma cell line (IC50, mM)
LOX IMVI
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
10
11
12
13
14
15
19
20
21
22
23
24
1.98
1.43
1.63
2.76
3.09
1.56
1.44
1.63
1.45
1.19
0.36
2.54
2.56
1.74
1.99
22.40
7.39
1.92
1.43
1.76
2.35
1.51
0.84
6.34
2.87
1.60
1.88
3.44
1.63
1.70
3.94
1.88
1.93
2.84
1.57
1.53
1.65
12.00
1.59
1.53
1.67
1.15
1.40
1.07
1.55
3.17
1.64
2.30
3.35
1.77
1.55
6.34
4.58
2.18
1.70
2.23
3.22
2.73
2.03
7.13
5.74
40.30
>100
2.80
>100
>100
5.83
>100
2.52
>100
3.15
1.32
1.77
2.73
3.78
1.20
4.11
1.70
1.80
4.05
6.91
1.58
6.60
1.63
2.01
2.94
2.75
1.08
4.88
1.51
2.01
4.74
5.70
2.55
8.94
14.4
NCI 8-melanoma cell line panel 23 were screened for ERK kinase
inhibitory activity. The ERK-containing A375P cell lysate was
treated with three different concentrations of the test compounds
formed between the phenolic hydroxyl hydrogen atom and Val-
B590 (1.75 Ǻ); and between the pyrazole N2 atom and Lys-B591
(1.61 Ǻ). The areneecation interaction occurred between the
phenolic benzene ring of 21 and Lys-B591 protonated amino group.
Figs. 3 and 4 demonstrate the binding model of compound 21 with
the binding site of V600E-B-RAF. The results of this molecular
docking study can support the postulation that our active
compounds may inhibit the growth of melanoma cell lines through
inhibition of B-RAF kinase, similar to sorafenib.
(1, 3, and 10 mM) and their inhibitory activities were compared with
that of sorafenib. The results showed that 21, 23, and sorafenib
significantly suppressed phosphorylation of ERK1/2 in a dose-
dependent manner. The dichlorophenyl derivative 21 demonstrated
higher ERK inhibition than 23 with 4-chloro-3-(trifluoromethyl)
phenyl terminal moiety (Fig. 2). These compounds may inhibit
melanoma cells proliferation through ERK kinase inhibition.
3. Conclusions
2.2.4. Molecular docking
V600E-B-RAF kinase is over-expressed in most of melanoma
cases [4]. We assumed that the active target compounds might
demonstrate antiproliferative activity against melanoma cell lines
through inhibition of V600E-B-RAF. In this study, we performed
molecular docking of compound 21, as a representative example of
this series of compounds, into the domain of V600E-B-RAF kinase.
All calculations were performed using MOE 2008.10 software [24]
installed on 2.0G Core 2 Duo. The crystal structure of V600E-B-
RAF kinase in complex with PLX4032 (PDB code: 3OG7) was
obtained from protein data bank (PDB) [25]. The automated dock-
ing program of MOE 2008.10 was used for docking of 21 into the
domain of V600E-B-RAF kinase. The complex was energy-
minimized with a MMFF94x force-field [26] till the gradient
convergence 0.01 kcal/mol was reached. The docking study has
revealed that the ligand 21 has bound in the active site of one of the
protomers in the protein dimer through the formation of two
strong hydrogen bonds and one areneecation interaction between
the binding site and the ligand. The hydrogen bonds have been
We have designed and synthesized a new series of diarylureas and
diarylamides containing 1,3,4-triarylpyrazole scaffold. A structure-
activity relationship (SAR) study has been made to correlate
between the compounds structures and antiproliferative activities.
Compounds12,13,15, and 21e23showed the highestpotencyagainst
A375P human melanoma cell line. In addition, all the twelve tested
compounds 10e15 and 19e24 showed good potency over the NCI 8
tested melanoma cell lines panel. Compounds 21 and 23 showed
significant and dose-dependent ERK kinase inhibitory activity. In
addition, we have studied the binding mode of compound 21 with
V600E-B-RAF crystal structure (molecular docking). Based on ERK
inhibition data together with the docking results, we may assume
that the target compounds may inhibit the growth of melanoma cell
lines through inhibition of ERK and/or V600E-B-RAF kinases. Among
all the target compounds, compound 23 with hydroxyl group, amide
linker, and 4-chloro-3-(trifluoromethyl)phenyl terminal moiety
showed the best potencyagainst almost all the tested cell lines. It may
be considered as a promising lead for future design of anti-
proliferative agents for melanoma.
4. Experimental
4.1. General
All melting points were obtained on a Walden Precision Appa-
ratus Electrothermal 9300 apparatus and are uncorrected. Mass
spectra (MS) were taken in ESI mode on a Waters 3100 Mass
Detector (Waters, Milford, MA, USA). Nuclear magnetic resonance
(NMR) spectroscopy was performed using a Bruker ARX-300,
300 MHz and a Bruker ARX-400, 400 MHz spectrometers (Bruker
Bioscience, Billerica, MA, USA) with TMS as an internal standard.
Purities of the target compounds 9e24 (>95%) were determined by
LC-MS analysis using the following system: Waters 2998 photodiode
array detector, Waters 3100 mass detector, Waters SFO system
fluidics organizer, Waters 2545 binary gradient module, Waters
reagent manager, Waters 2767 sample manager, SunfireÔ C18
Fig. 2. Inhibition of ERK kinase activity by compounds 21, 23, and sorafenib.

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W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
375 mmol) in acetone (250 mL) was heated under reflux for 1 h. The
mixture was filtered, and the filtrate was evaporated under reduced
pressure. Water (150 mL) was added to the residue, and the
resulting mixture was carefully extracted with Et2O. The organic
layer was separated and the aqueous layer was extracted with Et2O
(3 ꢁ 50 mL). The combined Et2O extracts were washed with brine,
dried over anhydrous Na2SO4, and filtered. The organic solvent was
evaporated under reduced pressure, and the residue was used in
the next step without further purification. MS m/z: 157.5 [M þ H]þ.
4.3. 4-Chloro-3-methoxybenzoic acid (3)
A stirred mixture of compound 2 (11.17 g, 71 mmol), potassium
permanganate (35.0 g, 221 mmol), pyridine (36 mL), and water
(107 mL) was heated at 50 ꢀC for 24 h. The mixture was then stirred
at room temperature for 13 h. The mixture was filtered and MnO2
was suspended in hot water and again filtered off. The combined
aqueous filtrates were washed with ethyl acetate (3 x 75 mL), and
then acidified with 2 N H2SO4. The precipitate was filtered off,
washed with water, and dried to give the title compound (11.98 g,
90%). mp: 215e216 ꢀC (lit. mp: 217e219 ꢀC [22]).
4.4. Methyl 4-chloro-3-methoxybenzoate (4)
Acetyl chloride (1.9 mL, 28.1 mmol) was added dropwise to
a solution of 3 (1.0 g, 5.4 mmol) in MeOH (40 mL) at 0 ꢀC and the
reaction mixture was then stirred at room temperature for 15 h.
After evaporation of the organic solvent, the residue was purified
by flash column chromatography (silica gel, hexane-ethyl acetate
5:1 v/v) to give 4 (0.91 g, 85%) as a crystalline solid. mp: 49e50 ꢀC;
Fig. 3. Interaction between compound 21 (green) and V600E-B-RAF, hydrogen bonds
are shown as purple lines. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article).
1H NMR (400 MHz, DMSO-d6)
d 7.62e7.55 (m, 3H), 3.94 (s, 3H), 3.88
column (4.6 ꢁ 50 mm, 5 m particle size); Solvent gradient ¼ 95% A
m
(s, 3H).
at 0 min,1% A at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA) in
water; solvent B: 0.035% TFA in CH3OH; flow rate ¼ 3.0 mL/min; the
AUC was calculated using Waters MassLynx 4.1 software. Unless
otherwise noted, all solvents and reagents were commercially
available and used without further purification.
4.5. 1-(4-Chloro-3-methoxyphenyl)-2-(pyridin-4-yl)ethanone (5)
To a solution of compound 4 (1.0 g, 5.0 mmol) and 4-picoline
(0.5 mL, 5.6 mmol) in THF (5 mL) in a cooled bath at ꢂ25 ꢀC,
LHMDS (3.7 mL,1.0 M solution in THF,19.9 mmol) was slowly added
to maintain the temperature at ꢂ25 ꢀC. The resulting mixture was
stirred overnight at room temperature. The mixture was quenched
with saturated aqueous NH4Cl. Ethyl acetate was added and the
organic layer was separated. The aqueous layer was extracted with
4.2. 1-Chloro-2-methoxy-4-methylbenzene (2)
A mixture of 2-chloro-5-methylphenol (1, 21.5 g, 150 mmol),
dimethyl sulfate (20.8 g, 165 mmol), and anhydrous K2CO3 (51.8 g,
Fig. 4. 2D-presentation for the binding interactions of compound 21 with V600E-B-RAF kinase domain.

W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
5759
ethyl acetate (3 ꢁ 10 mL). The combined organic layer extracts were
washed with brine and dried over anhydrous Na2SO4. The organic
solvent was evaporated under reduced pressure and the residue
was purified by flash column chromatography (silica gel, hexane-
ethyl acetate 1:1 v/v then switching to hexane-ethyl acetate 1:5
v/v) to yield compound 5 (0.58 g, 45%). mp: 85e88 ꢀC; 1H NMR
under N2. The reaction mixture was stirred at room temperature for
12 h. The mixture was evaporated under reduced pressure, and the
residue was purified by column chromatography (silica gel, hexane-
ethyl acetate 1:5 v/v) to yield compound 9 (40 mg, 53.3%). mp:
132e135 ꢀC (dec.); 1H NMR (400 MHz, CDCl3)
d 8.57 (d, 2H,
J ¼ 6.0 Hz), 8.13e8.10 (m, 2H), 7.76 (d, 2H, J ¼ 9.0 Hz), 7.72 (d, 1H,
J ¼ 7.5 Hz), 7.64 (d, 2H, J ¼ 9.0 Hz), 7.51 (d, 1H, J ¼ 10.8 Hz), 7.52 (s,
1H), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.15 (s, 1H), 7.07 (d, 1H, J ¼ 8.0 Hz), 3.82
(s, 3H); ESI-MS: 566.0 [M þ 1]þ.
(400 MHz, DMSO-d6)
7.30 (d, 2H, J ¼ 4.1 Hz), 4.53 (s, 2H), 3.96 (s, 3H).
d
8.53 (d, 2H, J ¼ 4.5 Hz), 7.69e7.64 (m, 3H),
4.6. 4-(3-(4-Chloro-3-methoxyphenyl)-1H-pyrazol-4-yl)pyridine (6)
The synthesis of compounds 10e12 was carried out by the same
procedure as described for preparation of 9.
Compound 5 (1.0 g, 3.8 mmol) was added to DMF-DMA (5.14 mL,
38.2 mmol) and the mixture was stirred at room temperature for
18 h. The resulting solution was concentrated to dryness to furnish
an oil which was used in the next step without purification. To
a portion of the oil from the previous step (0.137 g, 0.457 mmol) in
EtOH (3 mL) was added hydrazine monohydrate (0.04 mL,
0.76 mmol) and the reaction mixture was stirred overnight at room
temperature. Water (5 mL) was added to the reaction mixture and
the organics were extracted with ethyl acetate (3 ꢁ 5 mL). The
combined organic layer extracts were washed with brine and dried
over anhydrous Na2SO4. After evaporation of the organic solvent,
the residue was purified by column chromatography (silica gel,
hexane-ethyl acetate 1:1 v/v then switching to hexane-ethyl
4.9.1. 1-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(3,4-dichlorophenyl)urea (10)
Yield 43%; mp: 225e227 ꢀC (dec.); 1H NMR (400 MHz, CDCl3)
d 8.58
(d, 2H, J ¼ 5.2 Hz), 8.10 (s,1H), 7.78 (d, 2H, J ¼ 8.7 Hz), 7.63 (s,1H), 7.53
(d, 2H, J ¼ 8.0 Hz), 7.40e7.35 (m, 3H), 7.15 (s,1H), 7.07 (d,1H, J ¼ 8.5 Hz),
6.53 (s, 1H), 6.48 (s, 1H), 3.82 (s, 3H); ESI-MS: 566.0 [M þ 1]þ.
4.9.2. 1-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(4-chloro-3-(trifluoromethyl)phenyl)urea
(11)
Yield 29%; mp: 139e140 ꢀC (dec.); 1H NMR (300 MHz, CDCl3)
d
8.58 (d, 2H, J ¼ 6.1 Hz), 8.09 (s, 1H), 7.76 (d, 2H, J ¼ 8.9 Hz), 7.70 (d,
acetate 1:5 v/v) to yield compound
6
(0.11 g, 81%). mp:
13.39 (brs, 1H), 8.48
1H, J ¼ 2.4 Hz), 7.61 (d, 1H, J ¼ 8.8 Hz), 7.51 (d, 2H, J ¼ 8.9 Hz), 7.44
(d, 1H, J ¼ 8.6 Hz), 7.36 (d, 1H, J ¼ 8.1 Hz), 7.14 (s, 1H), 7.06 (d, 1H,
J ¼ 8.1 Hz), 6.91 (s, 1H), 6.82 (s, 1H), 3.81 (s, 3H); 13C NMR (75 MHz,
248e251 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
(d, 2H, J ¼ 6.0 Hz), 8.14 (brs, 1H), 7.45 (d, 1H, J ¼ 8.0 Hz), 7.28 (d, 2H,
J ¼ 5.9 Hz), 7.18 (s, 1H), 6.97 (dd, 1H, J ¼ 1.5 Hz, J ¼ 1.6 Hz), 3.77 (s,
DMSO-d6)
d 162.3, 155.0, 152.8, 150.3, 148.5, 140.5, 139.7, 138.5,
3H); 13C NMR (75 MHz, DMSO-d6)
d
154.9, 150.2, 141.3, 130.5, 122.8,
134.3, 132.4, 129.8, 129.2, 128.7, 126.3, 123.6, 122.9, 121.6, 119.8,
121.6, 117.0, 112.9, 56.4; MS m/z: 287.0 [Mþ þ 1].
119.7, 119.4, 117.3, 113.3, 56.6; ESI-MS: 599.1 [M þ 1]þ.
4.7. 4-[3-(4-chloro-3-methoxyphenyl)-1-(4-nitrophenyl)-1H-pyraz-
ol-4-yl]pyridine (7)
4.9.3. 1-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(3,5-bis(trifluoromethyl)phenyl)urea (12)
Yield 36%; mp: 140e143 ꢀC (dec.); 1H NMR (400 MHz, CDCl3)
A
mixture of compound
nitrobenzene (0.9 g, 3.5 mmol), K2CO3 (0.7 g, 5.2 mmol), CuI
(0.033 g, 0.2 mmol), and -proline (0.04 g, 0.2 mmol) in DMSO
6
(0.5 g, 1.7 mmol), 1-iodo-4-
d
8.59 (d, 2H, J ¼ 6.1 Hz), 8.11 (s, 1H), 7.92 (s, 2H), 7.78 (d, 2H,
J ¼ 8.9 Hz), 7.55 (t, 3H, J ¼ 10.2 Hz), 7.36 (d, 2H, J ¼ 12.6 Hz), 7.15 (s,
L
1H), 7.08 (s, 1H), 6.84 (s, 1H), 3.82 (s, 3H); ESI-MS: 633.0 [M þ 1]þ.
(7 mL) was heated at 90 ꢀC under nitrogen atmosphere for 8 h. The
cooled reaction mixture was partitioned between water and ethyl
acetate. The organic phase was washed with brine (3 times) and
dried over anhydrous Na2SO4. After evaporation of the organic
solvent, the residue was purified by column chromatography (silica
gel, hexane-ethyl acetate 1:5 v/v) to yield compound 7 (0.8 g, 86%).
4.10. 3,4-Dichloro-N-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-
4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (13)
A
mixture of compound 8 (50 mg, 0.1 mmol), 3,4-
dichlorobenzoic acid (38 mg, 0.2 mmol), HOBt (36 mg, 0.3 mmol),
and EDCI (38 mg, 0.2 mmol) in DMF (1.0 mL) was cooled to 0 ꢀC
under nitrogen atmosphere. Triethylamine (0.03 mL, 0.2 mmol)
was added thereto at the same temperature. The mixture was then
stirred at 80 ꢀC for 12 h. The reaction mixture was cooled and then
partitioned between H2O and ethyl acetate. The organic layer was
separated and the aqueous layer was extracted with ethyl acetate (3
x 5 mL). The combined organic layer extracts were washed with
brine and dried over anhydrous Na2SO4. After evaporation of the
organic solvent, the residue was purified by column chromatog-
raphy (silica gel, hexane-ethyl acetate 1:1 v/v) to yield compound
13 (26 mg, 36.6%). mp: 201e202 ꢀC (dec.); 1H NMR (300 MHz,
1H NMR (300 MHz, DMSO-d6):
d
9.26 (s, 1H), 8.58 (d, 2H, J ¼ 3.3 Hz),
8.43 (d, 2H, J ¼ 6.9 Hz), 8.24 (d, 2H, J ¼ 6.9 Hz), 7.50 (d, 1H,
J ¼ 6.1 Hz), 7.37 (d, 2H, J ¼ 3.7 Hz), 7.26 (s, 1H), 7.06 (d, 1H,
J ¼ 6.1 Hz), 3.78 (s, 3H).
4.8. 4-[3-(4-chloro-3-methoxyphenyl)-1-(4-aminophenyl)-1H-pyra-
zol-4-yl]pyridine (8)
A mixture of compound 7 (0.5 g,1.2 mmol) and Pd/C (0.5 g) inTHF
(5 mL) was stirred at room temperature under hydrogen atmo-
sphere for 2 h. The mixture was filtered through celite and the
filtrate was evaporated under reduced pressure to give compound 8
CDCl3)
d
8.58 (d, 2H, J ¼ 4.5 Hz), 8.13 (s, 1H), 8.00 (d, 1H, J ¼ 2.1 Hz),
(0.4 g, 86.4%). 1H NMR (400 MHz, CDCl3):
d
8.55 (d, 2H, J ¼ 4.6 Hz),
7.91 (brs, 1H), 7.81 (brs, 4H), 7.73 (d, 1H, J ¼ 8.3 Hz), 7.60 (d, 1H,
J ¼ 8.3 Hz), 7.36 (d, 1H, J ¼ 8.1 Hz), 7.28 (brs, 1H), 7.15 (s, 1H), 7.08 (d,
1H, J ¼ 8.1 Hz), 3.82 (s, 3H); ESI-MS: 551.0 [M þ 1]þ.
7.99 (s,1H), 7.54 (d, 2H, J ¼ 8.7 Hz), 7.34 (d,1H, J ¼ 8.1 Hz), 7.24 (s,1H),
7.15 (s,1H), 7.06 (d,1H, J ¼ 8.1 Hz), 6.78 (d, 2H, J ¼ 8.7 Hz), 3.81 (s, 3H).
The synthesis of compounds 14e16 was carried out by the same
procedure as described for preparation of 13.
4.9. 1-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(2,3-dichlorophenyl)urea (9)
4.10.1. 3,5-Dichloro-N-{4-[3-(4-chloro-3-methoxyphenyl)-4-(pyridin-
4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (14)
To a solution of compound 8 (50 mg, 0.1 mmol) in anhydrous
THF (1 mL), a solution of 2,3-dichlorophenyl isocyanate (25 mg,
0.1 mmol) in THF (1 mL) was added dropwise at room temperature
Yield 36%; mp: 216e218 ꢀC (dec.); 1NMR (300 MHz, CDCl3)
d
8.58 (d, 2H, J ¼ 6.0 Hz), 8.13 (s, 1H), 7.96 (brs, 1H), 7.81 (s, 4H), 7.77

5760
W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
(d, 2H, J ¼ 1.7 Hz), 7.56 (brs, 1H), 7.36 (d, 1H, J ¼ 8.1 Hz), 7.28 (brs,
1H), 7.16 (s, 1H), 7.07 (d, 1H, J ¼ 8.1 Hz), 3.82 (s, 3H); ESI-MS: 551.1
[M þ 1]þ.
133.8, 132.4, 132.0, 130.0, 128.8, 126.9, 123.2, 122.5, 119.9, 119.8,
119.3, 119.2, 119.1, 116.9, 116.8, 116.2; ESI-MS: 585.2 [M þ 1]þ.
4.11.3. 1-{4-[3-(4-chloro-3-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(3,5-bis(trifluoromethyl)phenyl)urea (20)
Yield 42%; mp: 216e217 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
4.10.2. 4-Chloro-3-trifluoromethyl-N-{4-[3-(4-chloro-3-
methoxyphenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}
benzamide (15)
d
10.34 (s, 1H), 9.49 (s, 1H), 9.23 (s, 1H), 8.92 (s, 1H), 8.55 (d, 2H,
Yield 29%; mp: 190e193 ꢀC (dec.); 1H NMR (300 MHz, CDCl3)
J ¼ 6.1 Hz), 8.17 (s, 2 H), 7.87 (d, 2H, J ¼ 9.1 Hz), 7.67 (d, 2H,
d
8.58 (d, 2H, J ¼ 4.5 Hz), 8.22 (brs, 1H), 8.13 (s, 1H), 8.02 (d, 1H,
J ¼ 8.9 Hz), 7.66 (s, 1H), 7.40 (s, 1H), 7.35 (d, 2H, J ¼ 6.1 Hz), 7.15 (s,
J ¼ 8.3 Hz), 7.93 (brs, 1H), 7.82 (brs, 4H), 7.68 (d, 1H, J ¼ 8.2 Hz), 7.36
(d, 1H, J ¼ 8.1 Hz), 7.28 (brs, 1H), 7.16 (s, 1H), 7.08 (d, 1H, J ¼ 8.1 Hz),
3.82 (s, 3H); ESI-MS: 584.1 [M þ 1]þ.
1H), 6.92 (d, 1H, J ¼ 8.2 Hz); 13C NMR (75 MHz, DMSO-d6)
d 153.6,
152.9, 150.4, 149.2, 142.3, 140.4, 138.4, 134.5, 132.9, 131.4, 131.0,
130.5, 129.4, 123.0, 122.0, 120.4, 120.3, 120.1, 119.7, 119.6, 118.6,
116.7; ESI-MS: 619.0 [M þ 1]þ.
4.10.3. 3,5-Bis(trifluoromethyl)-N-{4-[3-(4-chloro-3-methoxyp-
henyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (16)
Yield 42%; mp: 206e208 ꢀC (dec.); 1H NMR (300 MHz, CDCl3)
4.11.4. 3,4-Dichloro-N-{4-[3-(4-chloro-3-hydroxyphenyl)-4-
(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (21)
d
8.58 (d, 2H, J ¼ 4.5 Hz), 8.36 (brs, 2H), 8.14 (s, 1H), 8.09 (brs, 1H),
Yield 47%; mp: 231e233 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
8.00 (brs, 1H), 7.84 (s, 4H), 7.37 (d, 1H, J ¼ 8.1 Hz), 7.28 (d, 1H,
J ¼ 1.6 Hz), 7.16 (d, 1H, J ¼ 1.8 Hz), 7.08 (d, 1H, J ¼ 8.1 Hz), 3.82 (s,
d
10.57 (s, 1H),10.32 (s, 1H), 8.95 (s, 1H), 8.55 (d, 2H, J ¼ 4.6 Hz), 8.25
(d, 1H, J ¼ 2.0 Hz), 7.94 (brs, 5H), 7.85 (d, 1H, J ¼ 8.4 Hz), 7.35e7.40
3H); 13C NMR (100 MHz, DMSO-d6)
d 162.9, 160.6, 150.4, 148.5,
(m, 3H), 7.15 (s, 1H), 6.93 (d, 1H, J ¼ 8.2 Hz); ESI-MS: 537.2 [M þ 1]þ.
140.2, 137.8, 137.3,135.6, 134.5, 131.1,130.8, 129.5, 129.0,125.6, 123.1,
122.2, 121.9, 120.5, 120.0, 119.5, 114.3, 113.4, 56.1; ESI-MS: 618.0
[M þ 1]þ.
4.11.5. 3,5-Dichloro-N-{4-[3-(4-chloro-3-hydroxyphenyl)-4-
(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (22)
Yield 87%; mp: 250e252 ꢀC (dec.); 1H NMR (300 MHz, DMSO-
d6)
d
10.59 (s, 1H), 10.31 (s, 1H), 8.95 (s, 1H), 8.55 (d, 2H, J ¼ 4.5 Hz),
4.11. 1-{4-[3-(4-chloro-3-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(2,3-dichlorophenyl)urea (17)
8.01 (d, 2H, J ¼ 1.9 Hz), 7.94 (s, 4H), 7.89 (t, 1H, J ¼ 1.9 Hz), 7.40e7.35
(m, 3H), 7.15 (s, 1H), 6.93 (d, 1H, J ¼ 8.2 Hz); 13C NMR (75 MHz,
DMSO-d6)
d 163.2, 153.5, 150.4, 149.3, 140.4, 138.4, 137.8, 135.6,
To a solution of compound 9 (50 mg, 0.1 mmol) in methylene
chloride (1 mL), BBr3 (0.08 mL of a 1M solution in methylene
chloride, 1.2 mmol) was added dropwise at ꢂ78 ꢀC under N2 and
the reaction mixture was stirred at the same temperature for
30 min. The mixture was allowed to warm to room temperature
and stirred for 1 h. The mixture was quenched with saturated
aqueous NaHCO3. Ethyl acetate was added and the organic layer
was separated. The aqueous layer was extracted with ethyl acetate.
The combined organic layer extracts were washed with brine, dried
over anhydrous Na2SO4. After evaporation of the organic solvent,
the residue was purified by short column chromatography (silica
gel, hexane-ethyl acetate 1:5 v/v) to yield compound 17 (36 mg,
73.6%). mp: 143e146 ꢀC (dec.). 1H NMR (300 MHz, DMSO-d6)
134.8, 132.8, 131.5, 130.5, 129.5, 127.0, 123.0, 121.7, 120.4, 120.3,
119.8, 119.4, 116.7; ESI-MS: 536.8 [M þ 1]þ.
4.11.6. 4-Chloro-3-trifluoromethyl-N-{4-[3-(4-chloro-3-hydroxy-
phenyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (23)
Yield 73%; mp: 169e171 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
d
10.70 (s, 1H),10.34 (s, 1H), 8.97 (s, 1H), 8.55 (d, 2H, J ¼ 6.0 Hz), 8.42
(brs, 1H), 8.29 (d, 1H, J ¼ 9.3 Hz), 7.95 (brs, 5H), 7.41e7.35 (m, 3H),
7.15 (s, 1H), 6.93 (d, 1H, J ¼ 8.2 Hz); 13C NMR (75 MHz, DMSO-d6)
d
163.6, 153.6, 150.4, 149.3, 140.4, 137.9, 135.6, 134.4, 133.9, 132.8,
132.5, 130.5, 129.5, 127.6, 123.0, 121.8, 120.4, 120.3, 119.8, 119.4,
116.7; ESI-MS: 570.5 [M þ 1]þ.
d
10.37 (s, 1H), 9.49 (s, 1H), 8.97 (s, 1H), 8.55 (s, 2H), 7.96 (d, 1H,
4.11.7. 3,5-Bis(trifluoromethyl)-N-{4-[3-(4-chloro-3-hydroxyp-
henyl)-4-(pyridin-4-yl)-1H-pyrazol-1-yl]phenyl}benzamide (24)
Yield 43%; mp: 289e291 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
J ¼ 8.0 Hz), 7.91 (d,1H, J ¼ 8.6 Hz), 7.83 (d,1H, J ¼ 8.0 Hz), 7.74 (d,1H,
J ¼ 7.9 Hz), 7.55 (d, 2H, J ¼ 7.4 Hz), 7.45 (d,1H, J ¼ 7.9 Hz), 7.39 (d, 2H,
J ¼ 7.4 Hz), 7.37 (s, 2H), 7.15 (s, 1H), 6.92 (d, 1H, J ¼ 8.3 Hz); ESI-MS:
552.2 [M þ 1]þ.
d
10.82 (s, 1H), 10.34 (s, 1H), 8.97 (s, 1H), 8.64 (s, 2H), 8.55 (d, 2H,
J ¼ 4.6 Hz), 8.34 (s, 1H), 7.97 (s, 4H), 7.40e7.35 (m, 3H), 7.15 (s, 1H),
The synthesis of compounds 18e24 was carried out by the same
procedure as described for preparation of 17.
6.93 (d, 1H, J ¼ 8.2 Hz); 13C NMR (100 MHz, DMSO-d6)
d 163.1, 153.5,
150.4, 150.0, 149.7, 140.4, 137.8, 137.7, 137.4, 137.1, 135.8, 132.8, 131.1,
130.8, 130.5, 129.5, 129.1, 123.0, 122.0, 120.4, 119.4, 116.7; ESI-MS:
604.16 [M þ 1]þ.
4.11.1. 1-{4-[3-(4-chloro-3-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(3,4-dichlorophenyl)urea (18)
Yield 77%; mp: 191e194 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
9.10 (s, 1H), 9.05 (s, 1H), 8.91 (s, 1H), 8.55 (d, 2H, J ¼ 6.0 Hz), 7.92
4.12. Evaluation of the antiproliferative activity against A375P
human melanoma cell line
d
(d, 1H, J ¼ 2.4 Hz), 7.85 (d, 2H, J ¼ 9.0 Hz), 7.63 (d, 2H, J ¼ 9.0 Hz),
7.53 (d, 1H, J ¼ 12.7 Hz), 7.40 (s, 1H), 7.34e7.37 (m, 4H), 7.15 (d, 1H,
J ¼ 1.9 Hz), 6.92 (d, 1H, J ¼ 8.2 Hz); ESI-MS: 552.0 [M þ 1]þ.
A375P cells were purchased from American Type Culture
Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s
modified eagle medium (DMEM, Welgene, Daegu, Korea) supple-
mented with 10% foetal bovine serum (FBS, Welgene, Daegu, Korea)
and 1% penicillin/streptomycin (Welgene, Daegu, Korea) in
a humidified 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 ꢁ 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 with various concentrations (3-fold serial dilu-
tion, 12 points) of test compounds. The cells were incubated for
4.11.2. 1-{4-[3-(4-chloro-3-hydroxyphenyl)-4-(pyridin-4-yl)-1H-
pyrazol-1-yl]phenyl}-3-(4-chloro-3-(trifluoromethyl)phenyl)urea
(19)
Yield 69%; mp: 127e128 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6)
10.36 (s, 1H), 9.24 (s, 1H), 9.07 (s, 1H), 8.91(s, 1H), 8.54 (d, 2H,
d
J ¼ 5.6 Hz), 8.14 (s, 1H), 7.85 (d, 2H, J ¼ 8.9 Hz), 7.63 (brs, 4H),
7.34e7.39 (m, 3H), 7.14 (s, 1H), 6.91 (d, 1H, J ¼ 8.2 Hz); 13C NMR
(75 MHz, DMSO-d6)
d 153.0, 152.4, 149.8, 148.6, 140.0, 139.2, 138.0,

W.-K. Choi et al. / European Journal of Medicinal Chemistry 46 (2011) 5754e5762
5761
48 h after treatment with the test compounds. The A375P 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 manufacturer’s instructions. The absorbance at 590 nm was
recorded using EnVision 2103 (Perkin Elmer; Boston, MA, USA). The
IC50 was calculated using GraphPad Prism 4.0 software.
sonication and centrifuged at 12,000 rpm for 10 min. The quantity
of protein was determined with DC protein assay kit (Bio-Rad Lab.,
Hercules, CA). Protein samples were subjected to SDS-PAGE and
immunoblotting.
4.14.2. Suppression of RAF-1/MEK/ERK signaling pathway in A375P
cells
To assess the effect of compounds 21 and 23 on the RAF-1/MEK/
ERK signaling pathway, A375P cells were treated with 21, 23, and
4.13. NCI 8-melanoma cell line screening
sorafenib (1, 3, and 10
mM) for 24 h and immunoblotted with
8-Melanoma cell line screening was applied at the National
Cancer Institute (NCI), Bethesda, Maryland, USA [23], applying the
following procedure. The human cell lines are grown in RPMI 1640
medium containing 5% fetal bovine serum and 2 mM L-glutamine. For
a typical screening experiment, cells are inoculated into 96-well
antibodies against phospho-ERK1/2 and b-actin, respectively.
4.15. Molecular docking methodology
Docking studies were performed using MOE 2008.10. With this
purpose, the crystal structure of V600E-B-RAF Kinase oncogenic
mutant was obtained from Protein Data Bank [25] (PDB ID: 3OG7)
in order to prepare the protein for docking study. Docking proce-
dure was followed using the standard protocol implemented in
MOE 2008.10 and the geometry of resulting complex was studied
using the MOE’s pose viewer utility.
microtiter plates in 100 mL at plating densities ranging from 5000 to
40,000 cells/well depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates are incubated at
37 ꢀC, 5% CO2, 95% air and 100% relative humidity for 24 h prior to
addition of experimental drugs. After 24 h, two plates of each cell line
are fixed in situ with TCA, to represent a measurement of the cell
population for each cell line at the time of drug addition (Tz).
Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold
the desired final maximum test concentration and stored frozenprior
to use. At the time of drug addition, an aliquot of frozen concentrate is
thawed and diluted to twice the desired final maximum test
Acknowledgements
We would like to thank the National Cancer Institute (NCI),
Bethesda, Maryland, USA, for performing the anticancer testing of
the target compounds over eight melanoma cell lines. We thank
the Chemical Computing Group Inc, 1010 Sherbrooke Street West,
Suite 910, Montreal, H3A 2R7, Canada, for its valuable agreement
to use the package of MOE 2008.10 software. We are also grateful
to Korea Institute of Science and Technology (KIST) for financial
support.
concentration with complete medium containing 50 mg/mL genta-
micin. Additional four, 10-fold or 1/2 log serial dilutions are made to
provide a total of five drug concentrations plus control. Aliquots of
100
mL of these different drug dilutions are added to the appropriate
microtiter wellsalreadycontaining 100
mL ofmedium, resulting inthe
required final drug concentrations. Following drug addition, the
plates are incubated for an additional 48 h at 37 ꢀC, 5% CO2, 95% air,
and 100% relative humidity. For adherent cells, the assay is termi-
nated by the addition of cold TCA. Cells are fixed in situ by the gentle
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