Design, synthesis and biological evaluation of (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives as potential BRAFV600E inhibitors

Article abstract of DOI:10.1039/c4ra08708a

A series of (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives (6a-10d) were designed, synthesized and evaluated as BRAF^V600E inhibitors. Biological evaluation assays indicated that compound 10a showed the most potent inhibitory activity against A375, WM266.4 and BRAF^V600Ein vitro with IC₅₀ values of 1.36 μM, 0.94 μM and 0.11 μM respectively compared with the positive compound vemurafenib. Furthermore, compound 10a showed highly selective BRAF^V600E inhibitory activity in vitro. A docking simulation displayed that compound 10a could tightly bind the crystal structure of BRAF^V600E at the active site. 3D-QSAR would provide a guideline to design and optimize more potent and positive BRAF^V600E inhibitors based on the (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives skeleton.

Full text of DOI:10.1039/c4ra08708a

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Design, synthesis and biological evaluation of
(1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate
Cite this: RSC Adv., 2014, 4, 52702
derivatives as potential BRAFV600E inhibitors
a
a
a
a
b
*
Ya-Juan Qin,† Man Xing,† Ya-Liang Zhang, Jigar A. Makawana, Ai-Qin Jiang
a
*
and Hai-Liang Zhu
A series of (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives (6a–10d) were designed, synthesized
and evaluated as BRAFV600E inhibitors. Biological evaluation assays indicated that compound 10a showed
the most potent inhibitory activity against A375, WM266.4 and BRAFV600E in vitro with IC50 values of
1.36 mM, 0.94 mM and 0.11 mM respectively compared with the positive compound vemurafenib.
Furthermore, compound 10a showed highly selective BRAFV600E inhibitory activity in vitro. A docking
simulation displayed that compound 10a could tightly bind the crystal structure of BRAFV600E at the active
site. 3D-QSAR would provide a guideline to design and optimize more potent and positive BRAFV600E
inhibitors based on the (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives skeleton.
Received 15th August 2014
Accepted 15th October 2014
DOI: 10.1039/c4ra08708a
www.rsc.org/advances
In melanoma, BRAFV600E stimulates proliferation and
survival; inhibition of BRAF signaling with siRNA inhibits
1. Introduction
proliferation and induces apoptosis. These data validate BRAF
as an important and exciting therapeutic target in human
melanoma.11,12 Lots of potent biochemical inhibitors of RAF
were in clinical trials. Among them, the most studied BRAF
inhibitor, Sorafenib (Bay 43-9006, Nexavar), was approved by the
FDA in 2005 for the treatment of renal cell carcinoma and in
2007 for the treatment of hepatocellular carcinoma, and is still
undergoing multiple clinical trials in other types of cancer.6,13,14
It is probable because it also inhibits a number of other kinases
(such as VEGFR, PDGFR).15 So more potent and more selective
inhibitors of BRAFV600E, able to produce clinical results in
melanoma, are needed.16–20
The general structure of BRAF kinase inhibitors (such as
PLX4032 (ref. 21), RAF265, XL281 (ref. 22), L-779450, SB-590885
(ref. 23 and 24) and sorafenib) (Fig. 1) contains a H-bond donor,
usually pyrazole, pyridine or imidazole heterocycle and a
substituted aromatic group interacting with the hydrophobic
pocket. In an attempt to nd a better scaffold with optimal
length for effective hydrogen bonding and hydrophobic inter-
actions, we came up with (1,3-diphenyl-1H-pyrazol-4-yl) methyl
benzoate derivatives. This series compounds were envisaged as
having two purposes: the length of pyrazol bicycle was short for
an effective hydrogen bonding with Glu500 and Asp593 (the sor-
afenib binding site).18 In addition, the substituted benzoic acid
skeleton can afford a hydrophobic complementarity interaction
with secondary pocket of BRAF protein.
The MAPK signaling pathway consists of the Ras/Raf/MEK/ERK
signal transduction cascade which is a vital mediator of a
number of cellular fates including growth, proliferation, survival
and differentiation in response to many different external
stimuli such as growth factors, cytokines, and hormones. The
MAPK pathway is also a common pathway that is activated at
abnormally high levels in various human cancers. RAF kinases
are vital players in the MAPK signal transduction pathway and
are also important for regulating cell proliferation, survival and
angiogenesis in a variety of cancer models. If this pathway is
mutated and constantly switched on, the cell proliferation can
have damaging effects, resulting in cancer.1–3 This pathway is
becoming an important point of current human cancer thera-
peutics and our group has been doing research on the pathway
for many years, especially the RAF.
In mammalian cells there are three members of the RAF
family, ARAF, BRAF and CRAF. The discovery of the most
frequent V600E (>85%) BRAF mutations in 66% of melanoma,
45% of sporadic papillary thyroid cancers, 33% of KRAS
mutated pancreatic cancers, and 15% of sporadic colorectal
cancer have raised the expectation for targeted therapy. The
V600E BRAF mutations show a 500-fold increase in catalytic
activity, providing cancer cells with both proliferation and
survival signals.4–10
aState Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing
University, Nanjing 210093, P. R. China. E-mail: zhuhl@nju.edu.cn; Fax: +86-25-
8359-2672; Tel: +86-25-8359-2572
Many pyrazole derivatives were recently reported as
potent BRAFV600E inhibitors in our group.25,26 In order to extend
our research, herein we report the synthesis of a series of
(1H-pyrazol-4-yl) methyl benzoate derivatives as BRAFV600E
bSchool of Medicine, Nanjing University, Nanjing, 210093, P. R. China
† These two authors equally contributed to this paper.
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2.2. Crystal structure of compound 9c
Among all compounds, the crystal structure of compound 9c
was determined by X-ray diffraction analysis. The crystal data
presented in Table 1 and Fig. 2 gives perspective views of
compound 9c with the atomic labeling system.
2.3. Pharmacology
2.3.1. Antiproliferative activities. Antiproliferative activity
of synthesized compounds 6a–10d was carried out against two
cancer cell lines (A375 and WM266.4). The results were
summarized in Table 2. (1,3-Diphenyl-1H-pyrazol-4-yl) methyl
benzoate derivatives (6a–10d) showed remarkable anti-
proliferative effects. Among them, compound 10a displayed the
most potent inhibitory activity against A375 and WM266.4 (IC50
¼ 1.36 mM, 0.94 mM respectively) as compared to the positive
Fig. 1 Examples of chemical structures of BRAFV600E inhibitors.
inhibitors. Biological evaluation indicated that compound 10a
control vemurafenib (IC50 ¼ 0.06 mM for WM266.4 and IC50
¼
0.20 mM for A375 respectively).
2.3.2. BRAF inhibitory activity. The (1,3-diphenyl-1H-
pyrazol-4-yl) methyl benzoate 6a–10d were evaluated for their
inhibitory for BRAFV600E using a solid-phase ELISA assay. 10c,
10b and 10a showed strong inhibitory effect (IC50 ¼ 1.15 mM,
0.63 mM and 0.11 mM, respectively). Compound 10a displayed
the most potent anti-BRAF activity (Table 2). This result indi-
cated that the anti-proliferative effect was produced by direct
connection of BRAF protein and the compounds.
was found to be a potent and selective inhibitor of BRAFV600E
.
Docking simulations were performed using the X-ray crystallo-
graphic structure of BRAFV600E in complex with an inhibitor to
explore the binding modes of these compounds at the active
site. In addition, based on the biological activity data, QSAR
model was built to study the structure–activity relationship and
guide the further study.
Structure–activity relationships of synthesized derivatives
indicate that compounds with para-electron-donating substit-
uents (10a, 9a) showed more potent activities than those
with electron-withdrawing substituents (7a, 6a) in A-ring. A
comparison of the para-substituents on A-ring demonstrated
that an electron-donating group (10a, 9a) has slightly improved
BRAFV600E inhibitory activity and the potency order is –OMe >
–Me, whereas a Cl (7a) and F (6a) group substituent has minimal
2. Results and discussion
2.1. Chemistry
The synthetic route of the (1H-pyrazol-4-yl) methyl benzoate
derivatives (6a–10d) is outlined in Scheme 1. Synthesis of
(1,3-diphenyl-1H-pyrazol-4-yl) methanol (6–10) was obtained
from 1,3-diphenyl-1H-pyrazole-4-carbaldehyde using NaBH4
as a reducing agent. (1,3-Diphenyl-1H-pyrazol-4-yl) methanol
(6–10), benzoyl chloride and triethylamine were dissolved
in dichloromethane and reuxed to give the desired
compounds 6a–10d. All synthesized compounds give satis-
factory elementary analytical and spectroscopic data. 1H
NMR, 13C NMR and ESI-MS spectra were consistent with the
assigned structures.
Table 1 Crystallographical and experimental data for compound 9c
Compounds
9c
Empirical formula
Formula weight
Crystal system
Space group
C
24H20N2O2
368.42
Triclinic
P1ꢀ
˚
a (A)
8.2744(9)
9.9739(12)
12.4229(13)
81.884(4)
78.048(3)
77.788(4)
975.22(19)
2
˚
b (A)
˚
c (A)
a (ꢀ)
b (ꢀ)
c (ꢀ)
˚
V (A)
Z
Dcalcd/g cmꢁ3
1.255
1.68–25.20
388
H range (ꢀ)
F(000)
Reections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢁ1
R1; wR2 [I > 2s(I)]
R1; wR2 (all data)
GOF
9365/3473
3473/0/254
0.081
0.0497/0.1222
0.0942/0.1433
1.024
)
Scheme
1 General synthesis of (1,3-diphenyl-1H-pyrazol-4-yl)
methyl benzoate derivatives (6a–10d). Reagents and conditions: (i)
ethanol, 50–60 ꢀC, 3 h; (ii) DMF, POCl3, 50–60 ꢀC, 5 h; (iii) NaBH4,-
ethanol, 0 ꢀC; (iv) EDC, HOBt, CH2Cl2, refluxed, 8–10 h.
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10a with para-OMe and F group in the A and B ring respectively,
leads to a noteworthy best activity. The following molecular
docking of all the synthesized compounds also showed this
result and indicated that these compounds were potential
BRAFV600E inhibitory agents.
2.3.3. Kinase selectivity. To evaluate potential inhibition of
other kinases beyond BRAFV600E, we tested the compound 10a
Table 3 Kinase selectivity of compound 10a
Kinase
IC50 (mM)
Kinase
IC50 (mM)
Fig. 2 Crystal structure diagram of compound 9c. H atoms are shown
as small spheres of arbitrary radii.
p38a
N.i.a
0.11
13
15
N.i.
CDK1/CycB
CDK2/CycA
VEGFR2
PDGFRa
cSrc
11
18
N.i.
N.i
N.i.
BRAFV600E
Aurora-A
Aurora-B
MEK1
effects compared with 8a. In the case of constant A ring
substituents, change of substituents on B ring could also affect
the activities of compounds. Among compounds 10a–10d,
compounds with para electron-withdrawing substituted (10a,
10b) showed stronger anticancer activities and the strength
order was different from A ring: –F > –Cl > –H > –OMe, followed
that 10d showed the lowest activity. Among all the compounds,
a
N.i.: no inhibition, IC50 > 20 mM.
Table
2 Inhibition (IC50)
of A375, WM266.4 and BRAFV600E by
compounds (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate deriva-
tives (6a–10d)
IC50 ꢂ SD (mM)
Compounds R1
R2
A375
WM266.4
BRAFV600E
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
F
F
F
F
Cl
Cl
Cl
Cl
H
H
H
H
Me
Me
Me
Me
OMe
OMe
F
Cl
H
7.72 ꢂ 0.59 5.12 ꢂ 0.45 3.82 ꢂ 0.91
8.63 ꢂ 0.85 6.01 ꢂ 0.67 4.01 ꢂ 0.71
8.81 ꢂ 1.01 7.23 ꢂ 0.77 12.0 ꢂ 1.12
OMe 11.71 ꢂ 2.25 13.44 ꢂ 1.12 10.9 ꢂ 1.13
F
Cl
H
7.08 ꢂ 0.91 5.23 ꢂ 0.23 3.86 ꢂ 0.38
8.64 ꢂ 1.15 6.33 ꢂ 1.23 3.56 ꢂ 0.32
9.04 ꢂ 1.37 8.34 ꢂ 1.04 6.06 ꢂ 0.65
OMe 16.91 ꢂ 1.57 14.33 ꢂ 1.22 8.02 ꢂ 0.88
F
Cl
H
5.83 ꢂ 0.89 5.33 ꢂ 0.25 3.79 ꢂ 0.07
7.92 ꢂ 0.91 5.23 ꢂ 0.73 4.12 ꢂ 0.38
7.05 ꢂ 1.35 6.33 ꢂ 0.45 5.01 ꢂ 0.59
OMe 9.13 ꢂ 1.63 8.83 ꢂ 0.98 5.91 ꢂ 0.71
F
Cl
H
5.78 ꢂ 0.51 3.44 ꢂ 0.17 1.41 ꢂ 0.04
8.88 ꢂ 0.66 8.33 ꢂ 1.21 6.56 ꢂ 0.05
8.09 ꢂ 0.73 6.44 ꢂ 0.77 5.68 ꢂ 0.06
Fig.
3 The binding mode between the active conformation of
compound 10a and the target protein BRAFV600E (PDB code: 2FB8)
provided by the CDOCKER protocol (Discovery Studio 3.5, Accelrys,
Co. Ltd). We employed 2D diagram (A), 3D interaction map (B) to
display the interaction between 10a and the targeted protein. In the
binding model, compound 10a is nicely bound to 2FB8 via one
hydrogen bond, three p–p interaction and one p–sigma interaction.
OMe 9.65 ꢂ 0.91 8.33 ꢂ 1.02 5.78 ꢂ 0.07
10a
10b
10c
10d
F
H
1.36 ꢂ 0.07 0.94 ꢂ 0.05 0.11 ꢂ 0.01
2.09 ꢂ 0.43 1.33 ꢂ 0.78 0.63 ꢂ 0.22
2.78 ꢂ 0.37 2.45 ꢂ 0.32 1.15 ꢂ 0.01
OMe Cl
OMe OMe 8.76 ꢂ 0.49 7.34 ꢂ 0.89 4.38 ꢂ 0.41
Vemurafenib
0.20 ꢂ 0.02 0.06 ꢂ 0.007 0.03 ꢂ 0.006
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in a selectivity study towards a panel of 10 different kinases, and active binding pocket (Fig. 4). In the binding mode, compound
the results were reported in Table 3. Four kinases (p38a, MEK1, 10a is potently bound to the active binding site of BRAFV600E via
VEGFR2, PDGFRa, and cSrc) were inhibited by compound 10a one hydrogen bond, three p–p interactions and one p–sigma
with IC50 over 20 mM. Selectivity for BRAFV600E was also more interaction. The oxygen atoms of the A ring formed one
than 1000-fold as well as other protein kinases.
hydrogen bond with the amino hydrogen of Lys A: 483 (bond
˚
length: Lys A: 483 N–H/O ¼ 1.6 A); contribute to the hydrogen
bonding interaction together, being a probable explanation for
its nice activity. On the other hand, the A ring formed one p–p
interaction and one p–sigma interaction with ARG A: 462 (bond
2.4. Molecular docking study
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded to
examine the interaction of compounds 6a–10d with BRAFV600E
(PDB code: 2FB8). All docking runs were applied LigandFit Dock
protocol of Discovery Studio 3.5. The binding modes of
compound 10a and BRAF were depicted in Fig. 3 and 4. Visual
inspection of the pose of compound 10a into BRAFV600E binding
site revealed that compound 10a was tightly embedded into the
˚
˚
length: 6.0 A) and ARG A: 464 (bond length: 2.0 A) respectively.
The binding model also showed that there were three p–p
interactions between phenyl pyrazole ring of compound 10a
and PHE A: 583. Overall, these results of the molecular
docking study showed that (1,3-diphenyl-1H-pyrazol-4-yl)
methyl benzoate derivatives could act synergistically to
interact with the active binding site of BRAFV600E, suggested
that compound 10a is a potential inhibitor of BRAFV600E
.
2.5. 3D-QSAR
In order to obtain a systematic SAR prole on (1,3-diphenyl-1H-
pyrazol-4-yl) methyl benzoate derivatives as BRAFV600E inhibi-
tors as well as to explore the more potent and selective
BRAFV600E inhibitors, 3D-QSAR model was built using the cor-
responding pIC50 values which were converted from the
obtained IC50 (mM) values of BRAFV600E kinase inhibition and
performed by built-in QSAR soware of DS 3.5 (Discovery Studio
3.5, Accelrys, Co. Ltd). The way of this transformation was
derived from an online calculator developed from an indian
medicinal chemistry lab (http://www.sanjeevslab.org/tools-
IC50.html). The training and test set was divided by the
random diverse molecules method of DS 3.5, in which the
training set accounts for 80% and the test set was set to 20%.
The training set was composed of 16 agents and the relative test
set comprised 4 agents. In default situation, the alignment
Fig. 4 The surface model structure to display the interaction between
10a and the BRAFV600E
.
Fig. 5 The predicted versus experimental pIC50 values for the inhibition of BRAFV600E (PDB: 2FB8).
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role in its BRAFV600E inhibition and antiproliferative activity.
This study might be helpful for the design and synthesis of
BRAFV600E and tumor growth inhibitors with stronger activities.
QSAR model was built to provide a reliable tool for reasonable
design of novel BRAFV600E inhibitors in future.
4. Experimental protocols
Fig. 6 (A) Isosurface of the 3D-QSAR model coefficients on electro- 4.1. Materials and measurements
static potential grids. The blue triangle mesh represents positive elec-
trostatic potential and the red area represents negative electrostatic
potential. (B) Isosurface of the 3D-QSAR model coefficients on van der
All chemicals and reagents used in current study were of
analytical grade. All the 1H NMR and 13C NMR spectra were
Waals grids. The green triangle mesh representation indicates positive recorded on a Bruker DPX300 model Spectrometer in DMSO-d6
at 25 ꢀC with TMS and chemical shis were reported in ppm (d).
coefficients; the yellow triangle mesh indicates negative coefficients.
ESI-MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN–O-
conformation of each molecule was the one that possessed the
lowest CDOCKER_INTERACTION_ENENGY among the twenty
docked poses. The 3D-QSAR model generated from DS 3.5,
dened the critical regions (steric or electrostatic) affecting the
binding affinity. It was a PLS model built on 400 independent
variables (conventional r2 ¼ 0.978). Moreover, their graphical
relationship had been illustrated in Fig. 5, in which the plot of
the observed IC50 versus the predicted values showed that this
model could be used in prediction of activity for (1,3-diphenyl-
1H-pyrazol-4-yl) methyl benzoate derivatives as BRAFV600E
inhibitors.
A contour plot of the electrostatic eld region favorable (in
blue) and unfavorable (red) for anticancer activity based on
BRAFV600E protein target were shown in Fig. 6A while the energy
grids corresponding to the favorable (in green) or unfavorable
(yellow) steric effects for the BRAFV600E affinity were shown in
Fig. 6B. It was widely acceptable that a better inhibitor based on
the 3D-QSAR model should have strong van der Waals attrac-
tion in the green areas and a polar group in the blue electro-
static potential areas (which were dominant close to the
skeleton). Thus, this promising model would provide a guide-
line to design and optimize more effective BRAFV600E inhibitors
based on the (1,3-diphenyl-1H-pyrazol-4-yl) methyl benzoate
derivatives skeleton and provide us the direction of further
modication.
Rapid instrument. Elemental analyses were performed on a
CHN–O-Rapid instrument. All the compounds gave satisfactory
chemical analyses (ꢂ0.4%). TLC was performed on the glass-
backed silica gel sheets (Silica Gel 60 GF254) and visualized in
UV light (254 nm). Column chromatography was performed
using silica gel (200–300 mesh) eluting with ethyl acetate and
petroleum ether.
4.2. Synthesis
4.2.1. General synthetic procedure of (1,3-diphenyl-1H-
pyrazole-4-carbaldehyde) (6–10). The starting material 1,3-
diphenyl-1H-pyrazole-4-carbaldehyde (6–10) was synthesized
based on a literature method.27–29 1-Phenyl-2-(1-phenylethylidene)
hydrazine (1–5) (3.6 g, 0.015 mol) was added to a cold solution of
DMF (25 ml), then POCl3 (5 ml) was added and the resulting
mixture was stirred at 50–60 ꢀC for 6 h. The mixture was poured
into ice-cold water. A saturated solution of sodium hydroxide was
added to neutralize the mixture, and then the solid precipitate
was ltered, washed with water, dried and recrystallized from
ethanol.
4.2.2. General synthetic procedure of (1,3-diphenyl-1H-
pyrazol-4-yl) methanol. A solution of 0.76 g (0.02 mol) of sodium
tetrahydridoborate in ethanol (100 ml) was added with stirring
to a solution of 0.02 mol of 1,3-diphenyl-1H-pyrazole-4-
carbaldehyde (6–10) in ethanol (60 ml). The mixture was stir-
red for 1 h, 200 ml of water was added, and the mixture was le
to stand for 12 h at 0 ꢀC. The precipitate was ltered off, washed
3. Conclusion
In our present work, a series of anticancer inhibitors (1,3- with water, dried, and recrystallized from ethanol.
diphenyl-1H-pyrazol-4-yl) methyl benzoate derivatives (6a–10d) 4.2.3. General synthetic procedure of (1,3-diphenyl-1H-
has been synthesized and biologically evaluated. These pyrazol-4-yl) methyl benzoate (6a–10d). Compounds 6a–10d
compounds exhibited BRAFV600E inhibitory activities and anti- were synthesized by coupling (1,3-diphenyl-1H-pyrazol-4-yl)
proliferative activities against A375 and WM266.4 cell lines. methanol with substituted benzoic acid, using 1-ethyl-3-(3-
Among them, compound 10a demonstrated the most potent dimethylaminopropyl) carbodiimidehydrochloride and N-
activity which inhibited the growth of A375 and WM266.4 hydroxybenzotriazole (HOBt) as condensing agent. The mixture
cancer cell lines with IC50 values of 1.36 and 0.94 mM respec- was reuxed in anhydrous CH2Cl2 for 8–10 h. The products were
tively, and inhibited BRAFV600E inhibitory activity with an IC50 of extracted with ethyl acetate. The extract was washed succes-
0.11 mM. Furthermore, compound 10a showed highly selective sively with 10% HCl, saturated NaHCO3 and water, dried over
potent BRAFV600E inhibitory activity in vitro. Molecular docking anhydrous Na2SO4, ltered and evaporated. The residue was
showed that compound 10a bound to the binding site by one puried by column chromatography using petroleum ether and
hydrogen bond, three p–p bonds which might play a crucial ethyl acetate (3 : 1).
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[M + H]+). Anal. calcd for C23H17Cl2N2O2: C, 65.26; H, 3.81; Cl,
16.75; N, 6.62; O, 7.56%. Found: C, 65.25; H, 3.80; Cl, 16.74; N,
6.62; O, 7.59%.
4.3.7. (3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
benzoate (7c). White powder, yield 76%, mp: 147–148 ꢀC; 1H
NMR (300 MHz, CDCl3) d ppm: 7.31–7.33 (m, 1H); 7.41–7.49 (m,
6H); 7.54–7.60 (m, 1H); 7.72–7.77 (m, 2H); 7.78–7.83 (m, 2H);
8.03–8.06 (m, 2H); 8.16 (s, 1H). ESI-MS: 389.85 (C23H18ClN2O2
[M + H]+). Anal. calcd for C23H18ClN2O2: C, 71.04; H, 4.41; Cl,
9.12; N, 7.20; O, 8.23%. Found: C, 71.04; H, 4.42; Cl, 9.11; N,
7.21; O, 8.22%.
4.3. Spectral properties of (1,3-diphenyl-1H-pyrazol-4-yl)
methyl benzoate derivatives
4.3.1. (3-(4-Fluorophenyl)-1-phenyl-1-pyrazol-4-yl) methyl
4-uorobenzoate (6a). Light yellow powder, yield 79%, mp: 151–
154 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 5.39 (s, 2H); 7.10 (t, J
¼ 6.03 Hz, 2H); 7.16 (t, J ¼ 4.98 Hz, 2H); 7.31 (d, J ¼ 4.20 Hz, 1H);
7.46 (t, J ¼ 4.57 HZ, 2H); 7.74 (d, J ¼ 4.86 HZ, 2H); 7.81(t, J ¼ 3.93
HZ, 2H); 8.05 (t, J ¼ 4.13 HZ, 2H); 8.14 (s, 1H). ESI-MS: 391.38
(C23H17F2N2O2 [M + H]+). Anal. calcd for C23H17F2N2O2: C,
70.76; H, 4.13; F, 9.73; N, 7.18; O, 8.20%. Found: C, 70.75; H,
4.14; F, 9.72; N, 7.19; O, 8.20%.
4.3.8. (3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
4-methoxybenzoate (7d). Light yellow powder, yield 81%, mp:
150–152 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 3.90 (s, 3H); 5.37
(s, 2H); 6.91 (d, J ¼ 5.22 Hz, 2H); 7.30 (t, J ¼ 4.44 Hz, 1H); 7.43–
7.47 (m, 4H); 7.74 (d, J ¼ 4.77 Hz, 2H); 7.80 (d, J ¼ 5.04 Hz, 2H);
7.99 (d, J ¼ 5.22 Hz, 2H); 8.14 (s, 1H). ESI-MS: 419.87
(C24H20ClN2O3 [M + H]+). Anal. calcd for C24H20ClN2O3: C,
68.82; H, 4.57; Cl, 8.46; N, 6.69; O, 11.46%. Found: C, 68.81; H,
4.58; Cl, 8.45; N, 6.68; O, 11.48%.
4.3.9. (1,3-Diphenyl-1H-pyrazol-4-yl) methyl 4-uorobenzoate
(8a). Light yellow powder, yield 83%, mp: 94–95 ꢀC; 1H NMR
(300 MHz, CDCl3) d ppm: 5.42 (s, 2H); 7.10 (t, J ¼ 5.04 Hz, 2H); 7.30
(t, J ¼ 4.39 Hz, 1H); 7.40 (t, J ¼ 4.39 Hz, 1H); 7.44–7.48 (m, 4H); 7.76
(d, J ¼ 4.86 Hz, 2H); 7.83 (d, J ¼ 4.68 Hz, 2H); 8.06 (t, J ¼ 3.93 Hz,
2H); 8.15 (s, 1H). 13C NMR (100 MHz, CDCl3) d ppm: 167.14,
165.62, 164.62, 152.61, 139.81, 132.63, 132.30, 132.20, 129.73,
129.50, 128.79, 128.38, 127.98, 126.77, 126.40, 126.37,
119.21, 115.77, 115.61, 115.56, 57.92. ESI-MS: 373.39 (C23H18FN2O2
[M + H]+). Anal. calcd for C23H18FN2O2: C, 74.18; H, 4.60; F, 5.10; N,
7.52; O, 8.59%. Found: C, 74.17; H, 4.61; F, 5.11; N, 7.53; O, 8.58%.
4.3.10. (1,3-Diphenyl-1H-pyrazol-4-yl) methyl 4-chloroben-
zoate (8b). Light yellow powder, yield 79%, mp: 125–126 ꢀC; 1H
NMR (300 MHz, CDCl3) d ppm: 5.43 (s, 2H); 7.30 (t, J ¼ 8.68 Hz,
1H); 7.39–7.50 (m, 7H); 7.76 (d, J ¼ 7.50 Hz, 2H); 7.83 (t, J ¼ 4.20
Hz, 2H); 7.96–7.99 (m, 2H); 8.15 (s, 1H). ESI-MS: 389.85
(C23H18ClN2O2 [M + H]+). Anal. calcd for C23H18ClN2O2: C,
71.04; H, 4.41; Cl, 9.12; N, 7.20; O, 8.23%. Found: C, 71.03; H,
4.42; Cl, 9.11; N, 7.21; O, 8.23%.
4.3.11. (1,3-Diphenyl-1H-pyrazol-4-yl) methyl benzoate (8c).
White powder, yield 80%, mp: 75–77 ꢀC; 1H NMR (300 MHz,
CDCl3) d ppm: 5.44 (s, 2H); 7.30 (t, J ¼ 7.41 Hz, 1H); 7.40–7.50 (m,
7H); 7.56 (t, J ¼ 7.41 Hz, 1H); 7.77 (d, J ¼ 7.50 Hz, 2H); 7.86 (d, J ¼
7.14 Hz, 2H); 8.06 (t, J ¼ 4.38 Hz, 2H); 8.17 (s, 1H). ESI-MS: 355.40
(C23H19N2O2 [M + H]+). Anal. calcd for C23H19N2O2: C, 77.95; H,
5.12; N, 7.90; O, 9.03%. Found: C, 77.94; H, 5.11; N, 7.91; O,
9.04%.
4.3.12. (1,3-Diphenyl-1H-pyrazol-4-yl) methyl 4-methoxy-
benzoate (8d). Light yellow powder, yield 76%, mp: 91–93 ꢀC; 1H
NMR (300 MHz, CDCl3) d ppm: 3.87 (s, 3H); 5.37 (s, 2H); 6.91 (d,
J ¼ 5.21 Hz, 2H); 7.15 (t, J ¼ 5.19 Hz, 2H); 7.30 (t, J ¼ 7.41 Hz, 1H)
7.40–7.50 (m, 2H); 7.46 (t, J ¼ 6.41 Hz, 1H); 7.76 (d, J ¼ 4.50 Hz,
2H); 7.84 (d, J ¼ 7.02 Hz, 2H); 8.05 (t, J ¼ 4.02 Hz, 2H); 8.17 (s,
1H). ESI-MS: 385.15 (C24H20N2O3 [M + H]+). Anal. calcd for
4.3.2. (3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
4-chlorobenzoate (6b). White powder, yield 81%, mp: 180–182 ꢀC;
1H NMR (300 MHz, CDCl3) d ppm: 5.40 (s, 2H) 7.16 (t, J ¼ 8.70 Hz,
2H); 7.31 (t, J ¼ 7.41 Hz, 1H); 7.40–7.49 (m, 4H); 7.73–7.83 (m, 4H);
7.97 (t, J ¼ 4.38 Hz, 2H); 8.16 (s, 1H). ESI-MS: 407.84
(C23H17ClFN2O2 [M + H]+). Anal. calcd for C23H17ClFN2O2: C, 67.90;
H, 3.96; Cl, 8.71; F, 4.67; N, 6.89; O, 7.87%. Found: C, 67.91; H,
3.95; Cl, 8.70; F, 4.68; N, 6.88; O, 7.88%.
4.3.3. (3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
benzoate (6c). White powder, yield 80%, mp: 131–133 ꢀC; 1H
NMR (300 MHz, CDCl3) d ppm: 5.40 (s, 2H); 7.17 (t, J ¼ 8.68 Hz,
2H); 7.30 (t, J ¼ 7.32 Hz, 1H); 7.42–7.49 (m, 4H); 7.57 (t, J ¼ 7.41
Hz, 1H); 7.75 (d, J ¼ 8.07 Hz, 2H); 7.81–7.85 (m, 2H); 8.04 (t, J ¼
4.38 Hz, 2H); 8.16 (s, 1H). 13C NMR (100 MHz, CDCl3) d ppm:
166.52, 164.18, 161.72, 151.74, 139.75, 133.24, 130.06, 129.82,
129.78, 129.70, 129.67, 129.52, 128.85, 128.81, 128.53, 126.81,
119.19, 115.88, 115.66, 115.57, 57.68. ESI-MS: 373.39
(C23H18FN2O2 [M + H]+). Anal. calcd for C23H18FN2O2: C, 74.18;
H, 4.60; F, 5.10; N, 7.52; O, 8.59%. Found: C, 74.17; H, 4.61; F,
5.11; N, 7.52; O, 8.59%.
4.3.4. (3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
4-methoxybenzoate (6d). White powder, yield 81%, mp:
1
ꢀ
152–153 C; H NMR (300 MHz, CDCl3) d ppm: 3.85 (s, 3H); 5.37
(s, 2H); 6.91 (d, J ¼ 5.31 Hz, 2H); 7.15 (t, J ¼ 5.17 Hz, 2H); 7.30
(t, J ¼ 4.33 Hz, 1H); 7.46 (t, J ¼ 4.66 Hz, 2H); 7.75 (d, J ¼ 4.83 Hz,
2H); 7.82 (t, J ¼ 4.22 Hz, 2H); 7.99 (d, J ¼ 5.31 Hz, 2H); 8.14 (s, 1H).
13C NMR (100 MHz, CDCl3) d ppm: 166.25, 164.15, 163.56, 161.70,
151.68, 139.77, 131.72, 129.77, 129.69, 129.50, 128.90, 128.87,
126.76, 122.42, 119.16, 115.85, 115.79, 115.63, 113.77, 57.38, 55.50.
ESI-MS: 403.42 (C24H20FN2O3 [M
+
H]+). Anal. calcd for
C24H20FN2O3: C, 71.63; H, 4.76; F, 4.72; N, 6.96; O, 11.93%. Found:
C, 71.63; H, 4.77; F, 4.71; N, 6.97; O, 11.92%.
4.3.5. (3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
4-uorobenzoate (7a). Light yellow powder, yield 85%, mp: 136–
138 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 5.40 (s, 2H); 7.11 (t, J
¼ 5.16 Hz, 2H); 7.31 (t, J ¼ 4.49 Hz, 1H); 7.44–7.48 (m, 4H); 7.74–
7.79 (m, 4H); 8.03–8.06 (m, 2H); 8.15 (s, 1H). ESI-MS: 408.84
(C23H17ClFN2O2 [M + H]+). Anal. calcd for C23H17ClFN2O2: C,
67.90; H, 3.96; Cl, 8.71; F, 4.67; N, 6.89; O, 7.87%. Found: C,
67.91; H, 3.95; Cl, 8.70; F, 4.68; N, 6.88; O, 7.88%.
4.3.6. (3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methyl
4-chlorobenzoate (7b). White powder, yield 77%, mp:
175–177 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 5.40 (s, 2H); 7.31
(t, J ¼ 7.41 Hz, 1H); 7.40–7.49 (m, 6H); 7.73–7.79 (m, 4H);
7.95–7.98 (m, 2H); 8.15 (s, 1H). ESI-MS: 424.29 (C23H17Cl2N2O2
C
24H20N2O3: C, 74.98; H, 5.24; N, 7.29; O, 12.49%. Found: C,
74.96; H, 5.22; N, 7.31; O, 12.51%.
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7.42–7.46 (m, 4H); 7.56 (t, J ¼ 4.62 Hz, 1H); 7.72–7.79 (m, 4H);
8.06 (d, J ¼ 4.95 Hz, 2H); 8.14 (s, 1H). 13C NMR (100 MHz,
CDCl3) d ppm: 166.61, 159.80, 152.48, 139.89, 133.17, 130.19,
129.70, 129.65, 129.47, 129.25, 128.51, 126.59, 125.28, 119.14,
115.39, 114.22, 57.91, 55.39. ESI-MS: 385.43 (C24H21N2O3 [M +
H]+). Anal. calcd for C24H21N2O3: C, 74.98; H, 5.24; N, 7.29; O,
12.49%. Found: C, 74.97; H, 5.23; N, 7.30; O, 12.50%.
4.3.13. (1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl) methyl 4-uo-
robenzoate (9a). Light yellow powder, yield 80%, mp: 117–
119 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 2.41 (s, 3H); 5.41 (s,
2H); 7.10 (t, J ¼ 5.08 Hz, 2H); 7.29 (t, J ¼ 4.71 Hz, 3H); 7.45 (t, J ¼
4.68 Hz, 2H); 7.71–7.76 (m, 4H); 8.04–8.07 (m, 2H); 8.14 (s, 1H).
ESI-MS: 387.42 (C24H20FN2O2 [M + H]+). Anal. calcd for
C
24H20FN2O2: C, 74.60; H, 4.96; F, 4.92; N, 7.25; O, 8.28%.
4.3.20. (3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl 4-methoxybenzoate (10d). White powder, yield 72%, mp:
100–103 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 3.83 (s, 6H); 5.43
(s, 2H); 6.85 (d, J ¼ 4.22 Hz, 2H); 7.02–7.10 (m, 4H); 7.45 (t, J ¼
4.22 Hz, 1H); 7.62 (t, J ¼ 4.62 Hz, 4H); 7.91 (d, J ¼ 5.14 Hz, 2H);
8.10 (s, 1H). ESI-MS: 415.45 (C25H23N2O4 [M + H]+). Anal. calcd
for C25H23N2O4: C, 72.42; H, 5.38; N, 6.71; O, 15.49%. Found: C,
72.44; H, 5.37; N, 6.71; O, 15.50%.
Found: C, 74.61; H, 4.95; F, 4.93; N, 7.24; O, 8.28%.
4.3.14. (1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl) methyl 4-chlor-
1
ꢀ
obenzoate (9b). White powder, yield 74%, mp: 139–140 C; H
NMR (300 MHz, CDCl3) d ppm: 2.41 (s, 3H); 5.42 (s, 2H); 7.22–
7.32 (m, 3H); 7.39–7.48 (m, 4H); 7.71–7.77 (m, 4H); 7.98 (d, J ¼
8.58 Hz, 2H); 8.14 (s, 1H). ESI-MS: 403.87 (C24H20ClN2O2 [M +
H]+). Anal. calcd for C24H20ClN2O2: C, 71.55; H, 4.75; Cl, 8.80; N,
6.95; O, 7.94%. Found: C, 71.56; H, 4.74; Cl, 8.81; N, 6.94; O,
7.95%.
4.4. Crystal structure determination
4.3.15. (1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl) methyl benzoate
1
ꢀ
(9c). Light yellow powder, yield 77%, mp: 133–136 C; H NMR
(300 MHz, CDCl3) d ppm: 2.35 (s, 3H); 5.42 (s, 2H); 7.30–7.36 (m,
3H); 7.50–7.55 (m, 4H); 7.64–7.72 (m, 3H); 7.90–7.97 (m, 5H). ESI-
MS: 370.43 (C24H20N2O2 [M + H]+). Anal. calcd for C24H20N2O2: C,
78.24; H, 5.47; N, 7.60; O, 8.69%. Found: C, 78.23; H, 5.48; N,7.59;
O, 8.70%.
Crystal structure determination of compound 9c was carried out
on a Nonius CAD4 diffractometer equipped with graphite-mono
˚
chromated MoKa (k ¼ 0.71073 A) radiation. The structure was
solved by direct methods and rened on F2 by full matrix least-
squares methods using SHELX-97.30 All non-hydrogen atoms of
compound 9c were rened with anisotropic thermal parame-
ters. All hydrogen atoms were placed in geometrically idealized
positions and constrained to ride on the parent atoms.
4.3.16. (1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)
methyl
4-
methoxybenzoate (9d). Light yellow powder, yield 82%, mp: 99–
100 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 2.41 (s, 3H); 3.86 (s,
3H); 5.40 (s, 2H); 6.91 (d, J ¼ 5.13 Hz, 2H); 7.29 (t, J ¼ 4.21 Hz,
4H); 7.45 (t, J ¼ 4.53 Hz, 2H); 7.75 (t, J ¼ 4.62 Hz, 4H); 8.01 (d, J ¼
5.04 Hz, 2H); 8.14 (s, 1H). 13C NMR (100 MHz, CDCl3) d ppm:
166.32, 163.49, 152.63, 139.90, 138.13, 131.74, 129.83, 129.57,
129.45, 127.86, 126.58, 122.58, 119.16, 115.83, 113.71, 57.57,
55.49, 21.38. ESI-MS: 399.45 (C25H23N2O3 [M + H]+). Anal. calcd
for C25H23N2O3: C, 75.36; H, 5.57; N, 7.03; O, 12.05%. Found: C,
75.35; H, 5.56; N, 7.04; O, 12.05%.
4.3.17. (3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl 4-uorobenzoate (10a). White powder, yield 75%, mp:
104–107 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 3.80 (s, 3H); 5.40
(s, 2H); 7.03 (d, J ¼ 5.12 Hz, 2H); 7.35 (t, J ¼ 4.24 Hz, 1H); 7.45 (d,
J ¼ 4.13 Hz, 2H); 7.65 (t, J ¼ 4.75 Hz, 2H); 7.74–7.77 (m, 4H); 8.03
(d, J ¼ 4.31 Hz, 2H); 8.13 (s, 1H). ESI-MS: 403.14 (C24H19FN2O3
[M + H]+). Anal. calcd for C24H19FN2O3: C, 71.63; H, 4.76; F, 4.72;
N, 6.96; O, 11.93%. Found: C, 71.60; H, 4.79; F, 4.70; N, 6.97; O,
11.94%.
4.3.18. (3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl 4-chlorobenzoate (10b). White powder, yield 85%, mp:
94–95 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 3.80 (s, 3H); 5.40 (s,
2H); 7.00 (d, J ¼ 5.22 Hz, 2H); 7.28 (t, J ¼ 4.44 Hz, 1H); 7.41 (d, J
¼ 5.13 Hz, 2H); 7.45 (t, J ¼ 4.75 Hz, 2H); 7.74–7.77 (m, 4H); 7.98
(d, J ¼ 5.22 Hz, 2H); 8.12 (s, 1H). ESI-MS: 419.87 (C24H20ClN2O3
[M + H]+). Anal. calcd for C24H20ClN2O3: C, 68.82; H, 4.57; Cl,
8.46; N, 6.69; O, 11.46%. Found: C, 68.81; H, 4.58; Cl, 8.47; N,
6.68; O, 11.46%.
4.3.19. (3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl benzoate (10c). White powder, yield 78%, mp:
107–109 ꢀC; 1H NMR (300 MHz, CDCl3) d ppm: 3.81 (s, 3H); 5.41
(s, 2H); 7.01 (d, J ¼ 4.77 Hz, 2H); 7.28 (t, J ¼ 4.44 Hz, 1H);
4.5. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against A375 and WM266.4 cells were evaluated as described
elsewhere with some modications.31 Target tumor cell lines
were grown to log phase in DMEM medium supplemented with
10% fetal bovine serum. Aer diluting to 2 ꢃ 104 cells per ml with
the complete medium, 100 mL of the obtained cell suspension
was added to each well of 96-well culture plates. The subsequent
incubation was permitted at 37 ꢀC, 5% CO2 atmosphere for 24 h
before the cytotoxicity assessments. Tested samples at pre-set
concentrations were added to six wells with vemurafenib as
positive references. Aer 48 h exposure period, 40 mL of PBS
containing 0.5 mg mlꢁ1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was added to each well. Aer 4 h
incubation, the optical absorbance was measured at 570 nm on
an ELISA microplate reader. In all experiments three replicate
wells were used for each drug concentration. Each assay was
carried out for at least three times. The results were summarized
in Table 1.
4.6. BRAFV600E enzymatic assay
7.5 ng Mouse Full-Length GST-tagged BRAFV600E (Invitrogen,
PV3849) was preincubated at room temperature for 1 h with 1
mL drug and 4 mL assay dilution buffer. The kinase assay was
initiated when 5 mL of a solution containing 200 ng recombi-
nant human full length, N-terminal His-tagged MEK1 (Invi-
trogen, PV3093), 200 mM ATP (0.8 mCi hot ATP), and 30 mM
MgCl2 in assay dilution buffer was added. The kinase reaction
was allowed to continue at room temperature for 25 min and
was then quenched with 5 mL 5 ꢃ protein denaturing buffer
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(LDS) solution. Protein was further denatured by heating for 3% phosphoric acid. The plates were dried, followed by the
5 min at 70 ꢀC. 10 mL of each reaction was loaded into a 15 well, addition of 40 ml of MicroScint0 (PerkinElmer). The radioac-
4–12% precast NuPage gel (Invitrogen) and run at 200 V, and tivity was counted by a Top Count scintillation counter (Perki-
upon completion, the front, which contained excess hot ATP, nElmer). The results were summarized in Table 3.
was cut from the gel and discarded. The gel was then dried and
4.7.2. Tyrosine kinase proling by IC50 measurement. The
developed onto a phosphor screen, which was scanned on a cytoplasmic domain of vascular endothelial growth factor
Storm 820 scanner and quantitated from optical densitometry receptor 2 (VEGFR2), platelet-derived growth factor receptor a
using Image Quant v5.0. A reaction that contained no active (PDGFRa) and cSRC were purchased from Millipore Corp.
enzyme was used as a negative control, and a reaction without Assays for 3 tyrosine kinases were performed in 384 well plates
inhibitor was used as the positive control. Final compound using the Alphascreen® system (PerkinElmer) at room
concentrations were 100 mM, 31.6 mM, 10 mM, 3.16 mM, 1 mM, temperature. Enzyme reactions were performed in 50 mM Tris–
316 nM, 100 nM, 31.6 nM, 10 nM, 3.16 nM, 1 nM, 316 pM, and HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20, 2 mM
100 pM.32,33 The results were summarized in Table 2.
dithiothreitol and 0.1 mg mlꢁ1 biotinylated poly-GluTyr (4 : 1)
containing optimized concentration of enzyme and ATP as
described below.
4.7. Kinase selectivity studies34–36
Prior to the kinase reaction, compound and enzyme were
4.7.1. Serine/threonine kinase proling by IC50 measure- incubated for 5 min at room temperature. The reactions were
ment. Assays for seven serine/threonine kinases using radio initiated by adding ATP. Aer the reaction period as described
labeled [g-32P] ATP (GE Healthcare, Piscataway, NJ) were below at room temperature, the reactions were stopped by the
performed in 96 well plates. Mitogen-activated protein kinase addition of 25 ml of 100 mM EDTA, 10 mg mlꢁ1 Alphascreen
p38a (p38a), was expressed as N-terminal FLAG-tagged protein streptavidin donor beads and 10 mg mlꢁ1 acceptor beads
using a baculovirus expression system. Aurora-A and -B were described below in 62.5 mM HEPES, pH 7.4, 250 mM NaCl, and
expressed as N-terminal 6xHis tagged protein using a baculo- 0.1% BSA. Plates were incubated in the dark for more than 12 h
virus expression system. MEK1 was expressed as N-terminal and then read by EnVision 2102 Multilabel Reader (Perki-
GST fusion protein using a freestyle 293 expression system. nElmer). The well containing substrate and enzyme without
Cyclin-dependent kinase 1 (CDK1)/CycB and CDK2/CycA were compound was used as total reaction control. The reaction
expressed as C-terminal 6His-tagged CDK2, and N-terminal conditions for these 10 kinases were optimized for each kinase:
GST-tagged Cyclin A protein using a baculovirus expression VEGFR2 (19 ng mlꢁ1 of enzyme, 10 mM ATP, 10 min reaction, PY-
system.
100 conjugated acceptor beads (PY-100)); PDGFRa (50 ng mlꢁ1 of
The reaction conditions were optimized for each kinase: enzyme, 10 mM ATP, 30 min reaction, PT66 conjugated acceptor
p38a (100 ng per well of enzyme, 1 mg per well of MBP (Wako beads (PT66)); cSrc (0.33 ng mlꢁ1 of enzyme, 2 mM ATP, 10 min
Pure Chemical Ind., Osaka, Japan), 0.1 mCi per well of [g-32P] reaction, PY-100). The results were summarized in Table 3.
ATP, 60 min reaction at 30 ꢀC); BRFV600E (25 ng per well of
enzyme, 1 mg per well of GST-MEK1(K96R), 0.1 mCi per well of
[g-33P] ATP, 20 min reaction at room temperature); Aurora-A
and -B (50 ng per well of enzyme, 30 mM of Aurora substrate
peptide, 0.2 mCi per well of [g-32P] ATP, 60 min reaction at room
temperature); MEK1 (100 ng per well of enzyme, 0.3 mg per well
of GST-ERK1 (K71A) 0.2 mCi per well of [g-33P] ATP, 20 min
reaction at room temperature); CDK1/CycB (4.2 ng per well of
enzyme, 1 mg per well of Histone H1 (Calbiochem), 0.2 mCi/well
of [g-33P] ATP, 20 min reaction at room temperature); CDK2/
CycA (1.8 mUnits per well of enzyme, 1 mg per well of Histone
H1 (Calbiochem), 0.2 mCi per well of [g-32P] ATP, 20 min reac-
tion at room temperature).
All the seven enzyme reactions were performed in 25 mM
HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol
and 0.5 mM ATP containing optimized concentration of enzyme,
substrate and radio labeled ATP as described above in a total
volume of 50 ml. Prior to the kinase reaction, compound and
enzyme were incubated for 5 min at reaction temperature as
described above. The kinase reactions were initiated by adding
4.8. Molecular docking study
Molecular docking of compounds into the 3D BRAFV600E
complex structure (PDB code: 2FB8) was carried out using the
Discovery Studio (version 3.5) as implemented through the
graphical user interface CDOCKER protocol. The three-
dimensional structures of the aforementioned compounds
were constructed using Chem 3D ultra 11.0 soware [Chemical
Structure Drawing Standard; Cambridge So corporation, USA
(2010)], then they were energetically minimized by using
MOPAC with 5000 iterations and minimum RMS gradient of
0.10. The crystal structures of BRAF complex were retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/
home/home.-do). All bound water and ligands were elimi-
nated from the protein and the polar hydrogen was added. The
whole BRAFV600E complex was dened as a receptor and the site
sphere was selected based on the ligand binding location.
4.9. 3D-QSAR
ATP. Aer the reaction period as described above, the reactions Ligand-based 3D-QSAR approach was performed by QSAR
were terminated by the addition of 10% (nal concentration) soware of the DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd).
trichloroacetic acid. The [g-32P]-phosphorylated proteins were The training sets were composed of 16 inhibitors with the cor-
ltrated in Harvest Plate (Millipore Corp.) with a Cell Harvester responding pIC50 values which were converted from the
(PerkinElmer) and then free of [g-32P] ATP was washed out with obtained IC50 (mM), and test sets comprised 4 compounds. All
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the denition of the descriptors can be seen in the help of DS
3.5 soware and they were calculated by QSAR protocol of DS
R. Marais, C. J. Marshall, R. Wooster, M. R. Stratton and
P. A. Futreal, Nature, 2002, 417, 949–954.
3.5. The alignment conformation of each molecule was the one 11 M. Karasarides, A. Chiloeches, R. Hayward, D. Niculescu-
with lowest interaction energy in the docked results of
CDOCKER (generated in molecular CDOCKER). The predictive
ability of 3D-QSAR modeling can be evaluated based on the
Duvaz, I. Scanlon, F. Friedlos, L. Ogilvie, D. Hedley,
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respectively. The models were also validated by test sets, in 581–586.
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Usually, one can believe that the modeling is reliable, when the
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Acknowledgements
This work was supported by the open foundation from State Key 16 I. Niculescu-Duvaz, E. Roman, S. R. Whittaker, F. Friedlos,
Laboratory of Pollution Control and Resource Reuse, Nanjing
University, Nanjing 210093, PR China.
R. Kirk, I. J. Scanlon, L. C. Davies, D. Niculescu-Duvaz,
R. Marais and C. J. Springer, J. Med. Chem., 2006, 49, 407–
416.
17 T. Ahmad and T. Eisen, Clin. Cancer Res., 2004, 10, 6388S–
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