Synthesis, biological evaluation and molecular docking of novel 5-phenyl-1H-pyrazol derivatives as potential BRAFV600E inhibitors

Article abstract of DOI:10.1039/c3ob40776d

The RAF-MEK-ERK cascade appears to be intimately involved in the regulation of cell cycle progression and apoptosis. The BRAF^V600E mutant results in constitutive activation of the ERK pathway, which can lead to cellular growth dysregulation. A series of 5-phenyl-1H-pyrazol derivatives (3a-5e) have been designed and synthesized, and their biological activities were evaluated as potential BRAF^V600E inhibitors. All the compounds were reported for the first time except 3e, and compound 1-(4-bromo-2-hydroxybenzyl)- 3-phenyl-1-(5-phenyl-1H-pyrazol-3-yl)urea (5c) displayed the most potent inhibitory activity (BRAF^V600E IC₅₀ = 0.19 μM). Antiproliferative assay results indicated that compound 5c possessed high antiproliferative activity against cell lines WM266.4 and A375 in vitro, with IC₅₀ values of 1.50 and 1.32 μM, respectively, which were comparable with the positive control vemurafenib. Docking simulations showed that compound 5c binds tightly to the BRAF^V600E active site and acts as BRAF^V600E inhibitor. A 3D-QSAR model was also built to provide more pharmacophore understanding towards designing new agents with more potent BRAF^V600E inhibitory activity.

Full text of DOI:10.1039/c3ob40776d

Organic &
Biomolecular Chemistry
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Synthesis, biological evaluation and molecular docking
of novel 5-phenyl-1H-pyrazol derivatives as potential
BRAFV600E inhibitors†
Cite this: Org. Biomol. Chem., 2013, 11,
6328
Jing-Jun Dong,‡a Qing-Shan Li,‡a,c Shu-Fu Wang,a Cui-Yun Li,a Xin Zhao,b
Han-Yue Qiu,a Meng-Yue Zhaoa and Hai-Liang Zhu*a
The RAF–MEK–ERK cascade appears to be intimately involved in the regulation of cell cycle progression
and apoptosis. The BRAFV600E mutant results in constitutive activation of the ERK pathway, which can
lead to cellular growth dysregulation. A series of 5-phenyl-1H-pyrazol derivatives (3a–5e) have been
designed and synthesized, and their biological activities were evaluated as potential BRAFV600E inhibitors.
All the compounds were reported for the first time except 3e, and compound 1-(4-bromo-2-hydroxy-
benzyl)-3-phenyl-1-(5-phenyl-1H-pyrazol-3-yl)urea (5c) displayed the most potent inhibitory activity
(BRAFV600E IC50 = 0.19 μM). Antiproliferative assay results indicated that compound 5c possessed high
antiproliferative activity against cell lines WM266.4 and A375 in vitro, with IC50 values of 1.50 and
1.32 μM, respectively, which were comparable with the positive control vemurafenib. Docking simulations
showed that compound 5c binds tightly to the BRAFV600E active site and acts as BRAFV600E inhibitor.
A 3D-QSAR model was also built to provide more pharmacophore understanding towards designing new
agents with more potent BRAFV600E inhibitory activity.
Received 17th April 2013,
Accepted 5th June 2013
DOI: 10.1039/c3ob40776d
www.rsc.org/obc
A number of activating mutations of the BRAF gene have
been identified, but the most common in human cancer is the
1. Introduction
The Ras/RAF/MEK/ERK mitogen-activated protein kinase substitution of a valine for glutamic acid at position 600
(MAPK) signaling pathway plays an important role in the trans- (V600E; formally identified as V599E).3 This single mutation
duction of signals from cell surface receptors to the nucleus, accounts for a 500-fold activation compared to the wild type in
regulating cell growth, survival, differentiation and prolifer- melanoma, providing cancer cells with proliferation, survival
ation in response to external stimuli (growth factors, cytokines signals, growth and maintenance functions. The large-scale
or hormones).1 Phosphorylation of RAF stimulates its serine/ genomic screens have detected that BRAF was mutated in
threonine activity, triggering and activating the protein kinases approximately 7% of human tumors,4,5 colorectal cancer
MEK and ERK sequentially.2 ERK phosphorylates some tran- (5–22%),4 papillary thyroid cancer (29–83%),4,6,7 cholangiocar-
scription factors such as ELK-1, and controls gene expression, cinoma (22%),8 and ovarian cancer (30%),2 especially in mela-
cytoskeletal rearrangements, and metabolism, thus causing noma (50–70%).9
cell transformation and contributing to tumor initiation and
The RAF family are serine/threonine kinases, which phos-
maintenance. Therefore, targeting this pathway, for example phorylate and activate downstream MEK1/2, including A-RAF,
through inhibition of BRAF, is becoming important in many B-RAF and C-RAF (also known as RAF-1) with different bio-
current human cancer therapeutics approaches.
chemical potencies (B-Raf > C-Raf ≫ A-Raf).1 Several drug
development programs in the field of small molecule kinase
inhibitors have been initiated, with some representatively mar-
keted compounds such as SB-590885,10 AZ628,11 PLX4032
(vemurafenib),12 and RAF26513 (Fig. 1). A detailed account of
the discovery and clinical development of PLX4032, a selective
BRAFV600E inhibitor that binds to the active conformation of
the kinase, has recently been published.14 It uses a common
scaffold that comprises a terminal aromatic group (phenyl or
pyrazole) that fills the allosteric pocket created by the displace-
ment of the DFG loop and an amide linker which couples an
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China. E-mail: zhuhl@nju.edu.cn; Fax: +86 25 83592672;
Tel: +86 25 83592672
bNanjing Institute of Environmental Science, Ministry of Environmental Protection
of China, Nanjing 210042, P. R. China
cSchool of Medical Engineering, Hefei University of Technology, Hefei 230009,
P. R. China
†CCDC 941059 for 3b. For crystallographic data in CIF or other electronic format
see DOI: 10.1039/c3ob40776d
‡Both authors contributed equally to this work.
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BRAFV600E. Docking simulations were also performed using
the X-ray crystallographic structure of BRAFV600E in complex with
an inhibitor to explore the binding modes of these compounds.
A QSAR model was built based on the activity data to study the
structure–activity relationships and guide further studies.
2. Results and discussion
2.1. Chemistry
The synthesis of the 5-phenyl-1H-pyrazol derivatives followed
the general pathway outlined in Scheme 1. First of all, 3-oxo-
3-phenylpropanenitrile (1) was synthesized in THF by treating
methyl benzoate with acetonitrile using NaH as catalyst. Sec-
ondly, treating compound 1 in ethanol with hydrazine hydrate
and refluxing for 1 h gave 5-phenyl-1H-pyrazol-3-amine (2).
Thirdly, compound 2 and substituted salicylaldehyde were dis-
solved in ethanol at room temperature, and the reaction
mixture was stirred until the solids fully formed, then the
Schiff bases were obtained with yields of 80–90%. Next, the
intermediate amines (4a–4g) were obtained from the Schiff
bases after the reduction reactions with sodium borohydride
in 60–80% yield. Finally, the intermediate amines were con-
densed with phenylisocyanate in chloroform to afford the
target urea derivatives (5a–5e) after 2 h, with yields in the
range of 50–70%. All the compounds except 3e are reported for
the first time. All of the synthesised compounds gave satisfac-
tory analytical and spectroscopic data in accordance with their
depicted structures.
Fig. 1 Chemical structures of antitumor agents and lead BRAFV600E inhibitors.
aryl group in the hydrophobic pocket to a hinge-binding het-
erocycle. Linkage via an amide bond would allow for rapid and
efficient screening of several potential hinge-binding groups.15
Pyrazole and its derivatives have attracted constant interest
over the past decades because of their wide range of pharma-
cological activities, such as antitumor, anti-inflammatory, anti-
bacterial, analgesic, fungistatic, and anti-hyperglycemic.16–18
SB-590885 has the most hot spots, containing the pyrazolyl
2.2. Crystal structure of compound 3b
From the perspective of structural chemistry, we can see that a
cis–trans isomerism phenomenon exists for these Schiff base
compounds. Among them, the crystal structure of compound
3b was determined by X-ray diffraction analysis. The crystal
ring, which inhibits BRAFV600E with a Ki of 0.16 nmol L−1
.
Meanwhile, it has been reported that urea derivatives exhibit a
wide range of pharmacological uses such as anticancer
agents,19 anticonvulsants20 and CXCR3 antagonists.21 More-
over, urea derivatives occupy an important position in modern
medicinal chemistry as inhibitors of g-secretase,22 NADH
oxidase23 and receptor tyrosine kinases (RTKs).24 The bi-aryl
urea sorafenib demonstrated a significant activity against BRAF.
This previous research encouraged us to design and syn-
thesize a pyrazole derivative containing the urea structure as
an anticancer/antitumor agent. These combined substructures—
the pyrazole ring along with the urea structure—might
possess synergistic BRAFV600E inhibitory effect. A series of
novel pyrazole derivatives were recently reported as potent anti-
cancer agents targeting BRAF in our group and some of them
demonstrated potent antitumor activity.25,26 In order to extend
our research, we herein describe the synthesis of these target
compounds and the subsequent studies of structure activity
relationships (SAR). Biological evaluation indicated that some
Scheme 1 General synthesis of 5-phenyl-1H-pyrazol derivatives (3a–5e).
Reagents and conditions: (i) CH3CN, NaH, THF, reflux, 4 h; (ii) NH2NH2·H2O,
CH3SO3H, EtOH, reflux, 1 h; (iii) EtOH, rt, 4 h; (iv) NaBH4, EtOH, reflux, 4–6 h;
of the synthesized compounds were potent inhibitors of (v) PhNCO, CH3Cl, rt, 2 h.
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A375, the obtained values for WM1361 were higher by a factor
of several dozens.
Table 1 Crystallographic and experimental data for compound 3b
Compound
3b
2.3.2. BRAF inhibitory activity. The 5-phenyl-1H-pyrazol
derivatives 3a–5e were evaluated for their ability to inhibit the
BRAFV600E kinases by using a solid-phase ELISA assay. The
IC50 values of the compounds are summarized in Table 2. It
was observed that all the synthesized compounds showed inhi-
bition of BRAFV600E with IC50 values between 0.19 and
8.13 μM. Among them, compound 5c displayed the most
potent anti-BRAFV600E kinase activity.
Formula
Formula weight
Crystal system
Space group
Crystal size (mm3)
a (Å)
C
16H12ClN3O
297.74
Monoclinic
P21/n
0.20 × 0.20 × 0.17
15.8817(11)
5.8250(4)
16.5782(12)
117.321(2)
1362.58(17)
4
273
1.451
0.282
616
b (Å)
c (Å)
β (°)
V (Å3)
The subsequent SAR studies were performed by modifi-
cation of the parent compound to determine how the substitu-
ents affect the BRAFV600E inhibitory activities. In general, the
inhibitory activities of urea derivatives were better than the
Schiff base compounds as well as the corresponding inter-
mediate amines. Among Schiff base derivatives (3a–3g), the
compounds (3f, 3g) with two halogen atoms showed more
potent anticancer activity than those with one halogen atom,
and the strength order was 3,5-2Cl > 3,5-Br > 4-Cl > 5-Cl > 4-Br
> 5-Br > H. We also observed that a stronger electron-withdraw-
ing (3b, 3d) substituent slightly improved the BRAFV600E
inhibitory activity compared to a relatively weaker electron-
withdrawing substituent (3c, 3e), whereas a hydrogen atom at
the same position (3a) led to a noteworthy loss of activity.
Moreover, the corresponding intermediate amines (4a–4g)
followed the same trend as the Schiff base, while a significant
loss of activity was observed when the double bond of the
Schiff base was hydrogenated to a single bond. Last but
not least, the inhibitory activity of the pyrazolyl-urea deriva-
tives (5a–5e) with different substituents increased in the
following order: 5-Br < 5-Cl < H < 4-Cl < 4-Br. The results
suggest that stronger electronegativity groups in the 4-position
and weaker groups in the 5-position afford better inhibitory
activity.
Z
Temperature
Dc (g cm−3
)
μ (mm−1
F (000)
)
θ Range (°)
2.41–25.71
12 810
2584
Reflns collected
Reflns unique
Goodness-of-fit on F2
R1, wR2 [I > 2σ(I)]
R1, wR2 [all data]
1.033
0.0391, 0.0959
0.0629, 0.1063
0.239, −0.186
Max, min Δρ (e Å−3
)
Fig. 2 The molecular structure of compound 3b showing 30% probability dis-
placement ellipsoids for non-hydrogen atoms.
data presented in Table 1 and Fig. 2 gave perspective views of
3b in the form of the trans-isomer with an atomic labeling
system.
The above results indicated that the pyrazolyl core, as well
as the phenyl urea motif, play an important role in the
BRAFV600E inhibition. The activity of the compounds was
slightly influenced by the changes of substituents on the
benzene rings, and the differences in the skeleton had greater
2.3. Bioassay
2.3.1. Antiproliferative activity. To test the anticancer effects and this might explain why substituents on the
activity of the synthesized compounds, we evaluated the anti- benzene rings had limited influence on the ability of mole-
proliferative activity of compounds 3a–5e against BRAFV600E cular bonding between the BRAFV600E and the compounds.
cell lines (WM266.4 and A375) and a wild type BRAF cell line Subsequent molecular docking studies of all the synthesized
(WM1361). The results are summarized in Table 2. These compounds demonstrated this point and all of these results
5-phenyl-1H-pyrazol derivatives displayed potent antiproliferative indicated that these compounds were potential BRAFV600E
effects on the BRAFV600E cell lines, and five compounds (5a– inhibitory agents.
5e) containing the urea structure performed better than
Moreover, an acute oral toxicity test was conducted with
others, showing relatively good antiproliferative activities. The mice to determine the toxicity from a single dose via the oral
inhibition constant (IC50) values of WM266.4 and A375 cell route. Based on the results of study (not listed), the single
lines against 5a–5e were between 1.50 and 3.54 μM and 1.32 dose acute oral LD50 (half maximal concentration of lethal
and 3.76 μM, respectively. Among them, compound 5c dis- does) values of the most potent compound 5c is greater than
played the most potent inhibitory activity (IC50 = 1.50 μM for 5000 mg kg−1 of body weight.
WM266.4 and IC50 = 1.32 μM for A375), comparable to the
positive control vemurafenib (IC50 = 0.06 μM for WM266.4 and
2.4. Molecular docking study
IC50 = 0.19 μM for A375, respectively). At the same time, com- To gain a better understanding of the potency of the syn-
pared to the IC50 of the BRAFV600E cell lines WM266.4 and thesized compounds and guide further SAR studies, we
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Table 2 Structural features, inhibition (IC50) of WM266.4, A375 and WM1361 cells proliferation and inhibition (IC50) of BRAFV600E by compounds 3a–5e
IC50 SD (μM)
Compound
R
WM266.4a
A375a
WM1361b
BRAFV600E
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
5a
5b
H
7.08 0.77
5.68 0.72
6.56 0.73
5.88 0.59
6.77 0.69
4.72 0.34
5.02 0.48
10.42 1.29
9.85 1.08
10.53 1.02
9.89 1.21
10.96 1.12
7.09 0.72
8.38 0.98
2.88 0.28
1.68 0.17
1.50 0.12
2.32 0.22
3.54 0.36
0.06 0.008
8.78 1.02
6.24 0.58
8.04 0.79
6.99 0.92
8.23 0.85
4.49 0.35
5.08 0.63
11.09 1.10
9.12 0.93
9.86 1.04
9.37 1.18
9.96 1.03
8.15 0.82
9.08 1.01
3.39 0.33
2.46 0.24
1.32 0.13
3.59 0.35
3.76 0.34
0.19 0.02
16.68 1.58
14.34 1.47
16.62 1.61
14.32 1.43
16.22 1.50
13.16 1.45
14.03 1.45
>20
5.87 0.62
4.13 0.44
5.04 0.67
4.76 0.50
5.30 0.54
3.06 0.33
3.80 0.40
8.13 0.88
6.44 0.68
7.34 0.78
6.67 0.75
7.85 0.82
6.21 0.70
6.22 0.75
0.43 0.04
0.21 0.03
0.19 0.03
0.74 0.04
0.92 0.05
0.03 0.005
4-Cl
4-Br
5-Cl
5-Br
3,5-2Cl
3,5-2Br
H
4-Cl
4-Br
5-Cl
5-Br
3,5-2Cl
3,5-2Br
H
18.32 1.76
>20
>20
>20
17.78 1.72
17.90 1.70
13.14 1.36
12.37 1.38
11.69 1.18
13.78 1.40
13.43 1.38
1.87 0.15
4-Cl
4-Br
5-Cl
5-Br
5c
5d
5e
Vemurafenibc
a BRAFV600E cell lines. b Wild type BRAF cell line. c Used as a positive control.
proceeded to examine the interaction of the 5-phenyl-1H-
pyrazol derivatives with the BRAFV600E crystal structure (PDB
code: 2FB8). The molecular docking was performed by insert-
ing the compound into the SB-590885 binding site of
BRAFV600E. All docking runs were performed by Discovery
Studio 3.1. Residues within a distance of 10 Å around the
BRAFV600E inhibitor SB-590885 were isolated for the construc-
tion of the grid for docking screening. This grid was large
enough to include every residue of the BRAFV600E kinase ATP-
binding pocket. The binding models of compound 5c and
BRAFV600E are depicted in Fig. 3. The amino acid residues
which interact with BRAFV600E are labeled.
In the binding mode, compound 5c binds to BRAFV600E via
two hydrogen bonds, one π–cation interaction and one π–π
interaction. One hydrogen atom on the pyrazolyl ring interact-
ing with the oxygen atom on the main chain of ILE463 (angle
N–H⋯O = 140.5°, distance = 2.24 Å) and another hydrogen
atom of OH interacting with the oxygen atom on the side
chain of ASN581 (angle O–H⋯O = 127.1°, distance = 1.98 Å)
contribute to the hydrogen bonding interactions together; this
will be a probable explanation for the activity. The π–cation
interaction and π–π interaction are formed between the
benzene rings of compound 5c and LYS578 (distance = 5.39 Å)
and HIS539 (angle = 25.0°, distance = 4.48 Å). Meanwhile, the
receptor surface models shown in Fig. 3 revealed that they
both have good shape complementarity with the ATP-binding
pocket of BRAF, and the candidate BRAFV600E inhibitor 5c was
Fig. 3 Molecular docking modeling of compound 5c with BRAFV600E kinase.
(A) 2D model of the interaction between compound 5c and the ATP binding
site. The H-bond (blue arrows and green arrows) is displayed as dotted arrows,
and the π–cation interaction is shown as orange lines. (B) 3D model of the inter-
action between compound 5c and the ATP binding site. For clarity, only the
interacting residues are displayed. H-bonds (green arrows) are displayed as
dotted arrows, and the π–cation interactions are shown as orange lines. (C) The
receptor surface model with compounds 5c (grey), 3c (yellow) and 4c (green).
embedded into the ATP-binding pocket better than its Schiff hydrophobic group from the phenyl isocyanate has enhanced
base derivative 3c and corresponding secondary amine 4c. the affinity of the receptor BRAFV600E for the ligand 5c. This
Extensive hydrophobic interactions are formed between com- molecular docking result, along with the enzyme assay data,
pound 5c and residues VAL471, ALA481, LYS483, LEU514, suggests that compound 5c is
a potential inhibitor of
ILE527, PHE583, suggesting that the introduction of the BRAFV600E
.
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2.5. 3D-QSAR
To evaluate the synthesized compounds as BRAFV600E inhibi-
tors systematically and explore more potent inhibitors, nine-
teen compounds with definite IC50 values against BRAFV600E
were selected as the model dataset by using the Create 3D
QSAR protocol of Discovery Studio 3.1. By convention, the
pIC50 scale (−log IC50), in which higher values indicated expo-
nentially greater potency, was used as a method to measure
inhibitory activity. The training and test set was chosen by the
Diverse Molecules method in Discovery Studio. Because a good
alignment is very important for the analysis of molecular
fields, we applied the CDOCKER protocol to explore each
molecule with the lowest energy before alignment confor-
mation. 5-Phenyl-1H-pyrazol was selected as substructure to
build the alignment conformation before building the QSAR
model.
Fig. 4 Plot of experimental versus predicted BRAFV600E inhibitory activities of
training set and test set.
The correlation coefficient r2 between the observed and pre-
dicted activity of the training set was found to be 0.952, while
that of the test set was found to be 0.758, which proved that
the built QSAR model was acceptable. Predicted pIC50 values
and residual errors for the 19 compounds by this QSAR model
are given in Table 3. The good agreement between predicted
and experimental pIC50 values for both test sets and training
sets is shown in Fig. 4.
The molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on van der Waals grids (Fig. 5a) and electro-
static potential grids (Fig. 5b) were also listed. The electrostatic
map has red contours around the regions where high electron
density (negative charge) is expected to increase activity, and
blue contours around areas where low electron density (partial
positive charge) is expected to increase activity. Similarly, the
Fig. 5 (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue
represents positive coefficients; red represents negative coefficients.
(b) 3D-QSAR model coefficients on van der Waals grids. Green represents posi-
tive coefficients; yellow represents negative coefficients.
steric map has areas where steric bulk is predicted to increase
(green) or decrease (yellow) activity.
According to the maps, a compound with a high negative
charge and small R group would possess higher activity, vali-
dating that halogen disubstitution is a better choice than
mono halogen substituton and Cl is better than Br. The
phenyl urea linker (surrounded by several green grids) would
help obtain better activity, demonstrating that the pyrazole
derivatives which containing the urea motif could be more
effective. As a result, the data summarized above demonstrate
that compounds 5c, the most potent BRAFV600E inhibitor (IC50
= 0.19 μM) with suitable substituents, possesses an outstand-
ing activity. The 3D QSAR models fit the inhibitory activity well
thus providing us with a direction for further modification. It
can be predicted that greater electronegativity substituents (F
and NO2) for the R group and an increasing steric bulk, such
as a naphthalene ring replacing a benzene ring, may help
enhance the BRAFV600E inhibitory activity.
Table 3 Experimental and predicted inhibitory activity of compounds 3a–5e
by 3D-QSAR models based upon active conformation achieved by molecular
docking
BRAFV600E
Compounda
Actual pIC50
Predicted pIC50
Residual error
3a
3b
5.231
5.384
5.298
5.322
5.276
5.514
5.420
5.090
5.191
5.134
5.176
5.105
5.207
5.206
6.367
6.678
6.721
6.131
6.036
5.358
5.466
5.668
5.358
5.329
5.505
5.334
5.172
5.208
5.050
5.114
5.049
5.470
5.157
6.381
5.882
6.476
5.852
6.249
−0.127
−0.082
−0.370
−0.036
−0.053
0.009
3
3d
3e
̲
̲
3̲f̲
3g
4a
4b
4c
4d
4e
0.086
−0.082
−0.017
0.084
0.062
0.056
−0.263
0.049
−0.014
0.796
0.245
4
4g
5a
̲
̲
3. Conclusion
5̲b̲
In this study, we synthesized a series of pyrazole derivatives
including novel Schiff bases, intermediate amines, and their
corresponding new urea derivatives, and then evaluated their
biological activities. Preliminary results showed that most of
the compounds displayed good BRAFV600E inhibitory activity
5c
5
5e
̲
̲
0.279
−0.213
a Underlined compounds were selected as the test set while the rest
were in the training set.
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and antiproliferative activity against WM266.4 and A375 cell 4.3. General procedure for the synthesis of 5-phenyl-1H-
lines. Among these compounds, compound 5c showed the pyrazol-3-amine (2)
most potent BRAFV600E inhibitory activity (IC50 = 0.19 μM) and
antiproliferative activity (IC50 = 1.50 μM for WM266.4 and IC50
= 1.32 μM for A375).
To a stirred mixture of compound 1 (0.8 g, 5 mmol) in EtOH
(25 mL) were added NH2NH2·H2O (0.3 mL, 6 mmol) and
CH3SO3H (0.1 mL, 1 mmol) at room temperature, and the
mixture was refluxed for 1 h. The precipitate was purified by
Molecular docking studies provided further insight into
interactions between the BRAFV600E protein and its ligand.
silica gel column chromatography to give the compound 2 as a
After analyzing the binding model of compound 5c with BRAF,
we found that two hydrogen bonds, one π–cation interaction,
one π–π interaction and the hydrophobic interactions with the
protein residues in the ATP binding site might play an essen-
tial role in its BRAFV600E inhibition and antiproliferative
activity. Therefore, it can be concluded that compound 5c as
well as the other pyrazolyl-urea derivatives are promising leads
for further study as potential anticancer agents. The results of
this study might help with the search for a promising
BRAFV600E inhibitor with potent activity. A QSAR model was
built to provide a reliable tool for the rational design of novel
BRAFV600E inhibitors in the future.
1
brown solid: yield, 32%; m.p. 121–122 °C. H NMR (300 MHz,
DMSO-d6, δ ppm): 4.75 (s, 2H, NH2); 5.75 (s, 1H, C–CH–C);
7.21–7.38 (m, 3H, Ph–H); 7.62–7.65 (m, 2H, Ph–H); 11.72 (s,
1H, C–NH–N). 13C NMR (300 MHz, DMSO-d6) δ: 154.0, 153.2,
130.0, 129.2, 128.7, 127.5, 87.8. MS (ESI): 160.1 ([M + H]+).
Anal. Calcd for C9H9N3: C, 67.90; H, 5.70; N, 26.40%; Found:
C, 67.78; H, 5.66; N, 26.56%.
4.4 General procedure for the synthesis of 5-phenyl-
1H-pyrazol derivatives
A. Synthesis of (E)-N-benzylidene-5-phenyl-1H-pyrazol-3-
amines (3a–3g). Equimolar portions of compound 2 (3 mmol)
and substituted salicylaldehydes (3 mmol) were dissolved in
approximately 30 mL ethanol. The mixture was refluxed for
4 h, and the reaction was monitored by TLC. The precipitate
was filtered and recrystallized in ethanol.
B. Synthesis of N-benzyl-5-phenyl-1H-pyrazol-3-amines
(4a–4g). NaBH4 (4 mmol) was dissolved in ethanol and then
added slowly to a solution of compound (3a–3g) in EtOH. With
the colour of the mixture fading fast, the reaction was finished.
The precipitate was purified by silica gel column chromato-
graphy to give the product.
C. Synthesis of corresponding urea derivatives (5a–5e). To a
chloroform solution of the intermediate amine (4a–4g)
(1 mmol), phenylisocyanate (1 mmol) was slowly added, then
the mixture was stirred at room temperature for 2 h. Isopycnic
hexyl hydride was added to the reaction mixture and the crude
product separated out spontaneously. The precipitate was sub-
sequently filtered and purified by recrystallization to afford
urea derivatives 5a–5e.
4.4.1. (E)-2-((5-Phenyl-1H-pyrazol-3-ylimino)methyl)phenol
(3a). Yellow powder, yield: 85%; m.p. 215–216 °C. 1H NMR
(300 MHz, DMSO-d6, δ ppm): 6.97–7.03 (m, 3H, C–CH–C,
Ph–H); 7.43–7.50 (m, 4H, Ph–H); 7.66 (d, J = 7.86 Hz, 1H,
Ph–H); 7.80 (d, J = 7.32 Hz, 2H, Ph–H); 9.16 (s, 1H, NvCH);
13.07 (s, 1H, OH); 13.35 (s, 1H, NH). 13C NMR (300 MHz,
4. Experimental
4.1. Materials and measurements
All chemicals and reagents used in the current study were of
analytical grade. Thin layer chromatography (TLC) was per-
formed on glass-backed silica gel sheets (Silica Gel 60 GF254)
and visualized under UV light (254 nm). Separation of the
compounds by column chromatography was carried out with
silica gel 60 (200–300 mesh ASTM, E. Merck). The quantity of
silica gel used was 50–100 times the weight charged on the
column. Melting points were determined on a XT4 MP appar-
atus (Taike Corp., Beijing, China). All the proton nuclear mag-
netic resonance (1H NMR) spectra were recorded on a Bruker
PX500 or DPX300 model spectrometer in DMSO-d6 at 25 °C
with TMS and chemical shifts were reported in parts per
million (δ). The solvent signals were allotted as internal stan-
dards. Elemental analyses were performed on a CHN-O-Rapid
instrument. All the compounds gave satisfactory chemical ana-
lyses ( 0.4%).
4.2. General procedure for the synthesis of 3-oxo-
3-phenylpropanenitrile (1)
Methyl benzoate (7 mmol, 0.95 g) and NaH (14 mmol, 0.35 g) DMSO-d6) δ: 161.3, 158.4, 136.2, 134.2, 128.9, 128.2, 124.3,
in boiling tetrahydrofuran (5 mL) were treated with a solution 120.3, 112.8, 91.7. MS (ESI): 264.1 ([M + H]+). Anal. Calcd for
of acetonitrile (7 mmol, 0.29 g, 0.4 mL) in tetrahydrofuran C16H13N3O: C, 72.99; H, 4.98; N, 15.96%; Found: C, 72.78;
(1 mL) dropwise, and the mixture was then refluxed for 4 h. H, 4.88; N, 15.98%.
After cooling to room temperature, the mixture was diluted
4.4.2. (E)-5-Chloro-2-((5-phenyl-1H-pyrazol-3-ylimino)methyl)-
with diethyl ether (15 mL) and left to stand at room tempera- phenol (3b). Yellow powder, yield: 80%; m.p. 260–262 °C.
ture for 48 h. The sodium salt precipitate was filtered and 1H NMR (300 MHz, DMSO-d6, δ ppm): 6.97–7.06 (m, 3H,
washed with diethylether. The dry compound was dissolved in C–CH–C, Ph–H); 7.36–7.51 (m, 3H, Ph–H); 7.68–7.80 (m, 3H,
water (5 mL) and acidified with HCl (1 mol L−1) to pH 2. The Ph–H); 9.12 (d, J = 25.23 Hz, 1H, NvCH); 13.32–13.42 (m, 2H,
collected extracts were crystallized from toluene, filtered and OH, NH). 13C NMR (300 MHz, DMSO-d6) δ: 159.3, 157.4, 136.7,
dried with Na2SO4.
132.9, 128.6, 128.1, 123.6, 117.2, 112.8, 93.2. MS (ESI): 298.1
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([M + H]+). Anal. Calcd for C16H12ClN3O: C, 64.54; H, 4.06; 130.2, 128.6, 127.9, 127.2, 126.4, 124.2, 122.7, 117.2, 89.0, 44.1.
N, 14.11%; Found: C, 64.56; H, 4.14; N, 14.04%.
MS (ESI): 266.1 ([M + H]+). Anal. Calcd for C16H15N3O: C,
4.4.3. (E)-5-Bromo-2-((5-phenyl-1H-pyrazol-3-ylimino)methyl)- 72.43; H, 5.70; N, 15.84%; Found: C, 72.30; H, 5.79; N, 15.77%.
phenol (3c). Yellow powder, yield: 86%; m.p. 256–258 °C. 4.4.9. 5-Chloro-2-((5-phenyl-1H-pyrazol-3-ylamino)methyl)-
1H NMR (300 MHz, DMSO-d6, δ ppm): 6.98 (s, 1H, C–CH–C); phenol (4b). White powder, yield: 67%; m.p. 156–157 °C.
7.20 (d, J = 9.33 Hz, 2H, Ph–H); 7.38–7.49 (m, 3H, Ph–H); 7.63 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.18 (d, J = 6.21 Hz, 2H,
(d, J = 8.04 Hz, 1H, Ph–H); 7.79 (d, J = 7.14 Hz, 2H, Ph–H); 9.15 CH2); 5.70 (s, 1H, C–CH–C); 5.87 (s, 1H, NH–CH2); 6.78–6.82
(s, 1H, NvCH); 13.37 (s, 2H, OH, NH). 13C NMR (300 MHz, (m, 2H, Ph–H); 7.25–7.41 (m, 4H, Ph–H); 7.64 (d, J = 7.68 Hz,
DMSO-d6) δ: 162.5, 157.4, 136.2, 133.4, 129.7, 128.8, 124.6, 2H, Ph–H); 10.42 (s, 1H, OH); 12.07 (s, 1H, C–NH–N). 13C NMR
117.8, 108.4, 91.9. MS (ESI): 342.0 ([M + H]+). Anal. Calcd for (300 MHz, DMSO-d6) δ: 157.4, 152.3, 131.1, 129.6, 129.0, 127.2,
C16H12BrN3O: C, 56.16; H, 3.53; N, 12.28%; Found: C, 56.07; 126.2, 124.5, 122.4, 115.6, 91.2, 43.9. MS (ESI): 300.1
H, 3.50; N, 12.45%.
4.4.4. (E)-4-Chloro-2-((5-phenyl-1H-pyrazol-3-ylimino)methyl)- N, 14.02%; Found: C, 64.23; H, 4.58; N, 14.30%.
([M + H]+). Anal. Calcd for C16H14ClN3O: C, 64.11; H, 4.71;
phenol (3d). Yellow powder, yield: 82%; m.p. 263–265 °C.
4.4.10. 5-Bromo-2-((5-phenyl-1H-pyrazol-3-ylamino)methyl)-
1H NMR (300 MHz, DMSO-d6, δ ppm): 6.95–7.02 (m, 2H, phenol (4c). White powder, yield: 65%; m.p. 162–164 °C.
C–CH–C, Ph–H); 7.39–7.49 (m, 4H, Ph–H); 7.75–7.80 (m, 3H, 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.17 (d, J = 6.24 Hz, 2H,
Ph–H); 9.17 (d, J = 12.06 Hz, 1H, NvCH); 12.96 (s, 1H, OH); CH2); 5.68 (s, 1H, C–CH–C); 5.87 (s, 1H, NH–CH2); 6.91–6.97
13.39 (s, 1H, NH). 13C NMR (300 MHz, DMSO-d6) δ: 162.6, (m, 2H, Ph–H); 7.19–7.41 (m, 4H, Ph–H); 7.64 (d, J = 7.32 Hz,
158.7, 136.1, 134.1, 129.6, 126.3, 124.0, 121.5, 112.4, 95.2. MS 2H, Ph–H); 10.39 (s, 1H, OH); 12.05 (s, 1H, C–NH–N). 13C NMR
(ESI): 298.1 ([M + H]+). Anal. Calcd for C16H12ClN3O: C, 64.54; (300 MHz, DMSO-d6) δ: 160.0, 155.2, 130.1, 128.3, 128.2, 127.0,
H, 4.06; N, 14.11%; Found: C, 64.46; H, 4.17; N, 14.23%.
126.5, 124.0, 123.5, 118.1, 85.7, 44.0. MS (ESI): 344.0
4.4.5. (E)-4-Bromo-2-((5-phenyl-1H-pyrazol-3-ylimino)methyl)- ([M + H]+). Anal. Calcd for C16H14BrN3O: C, 55.83; H, 4.10; N,
phenol (3e). Yellow powder, yield: 89%; m.p. 270–272 °C. 12.21%; Found: C, 55.67; H, 4.25; N, 12.34%.
1H NMR (500 MHz, DMSO-d6, δ ppm): 6.95 (d, J = 12.06 Hz,
4.4.11. 4-Chloro-2-((5-phenyl-1H-pyrazol-3-ylamino)methyl)-
2H, C–CH–C, Ph–H); 7.38–7.87 (m, 7H, Ph–H); 9.13 (s, 1H, phenol (4d). White powder, yield: 73%; m.p. 169–171 °C.
NvCH); 12.96 (s, 1H, OH); 13.37 (s, 1H, NH). 13C NMR 1H NMR (500 MHz, DMSO-d6, δ ppm): 4.18 (d, J = 6.25 Hz, 2H,
(300 MHz, DMSO-d6) δ: 160.6, 159.2, 135.3, 133.5, 129.0, 128.5, CH2); 5.71 (s, 1H, C–CH–C); 5.91 (s, 1H, NH–CH2); 6.78 (d, J =
125.0, 119.0, 110.0, 94.0. MS (ESI): 342.0 ([M + H]+). Anal. 8.30 Hz, 1H, Ph–H); 7.06 (d, J = 8.20 Hz, 1H, Ph–H); 7.25–7.38
Calcd for C16H12BrN3O: C, 56.16; H, 3.53; N, 12.28%; Found: (m, 4H, Ph–H); 7.62 (d, J = 6.40 Hz, 2H, Ph–H); 10.09 (s, 1H,
C, 56.23; H, 3.46; N, 12.32%.
OH); 12.04 (s, 1H, C–NH–N). 13C NMR (300 MHz, DMSO-d6)
4.4.6. (E)-2,4-Dichloro-6-((5-phenyl-1H-pyrazol-3-ylimino)- δ: 157.1, 153.9, 129.6, 128.7, 128.1, 127.6, 127.0, 124.8, 122.3,
methyl)phenol (3f). Yellow powder, yield: 84%; m.p. 276– 116.7, 88.1, 42.3. MS (ESI): 300.1 ([M + H]+). Anal. Calcd for
278 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 6.97 (s, 1H, C16H14ClN3O: C, 64.11; H, 4.71; N, 14.02%; Found: C, 64.02;
C–CH–C); 7.39 (s, 1H, Ph–H); 7.48 (d, J = 7.30 Hz, 2H, Ph–H); H, 4.82; N, 14.18%.
7.72–7.78 (m, 4H, Ph–H); 9.18 (s, 1H, NvCH); 13.48 (s, 1H,
4.4.12. 4-Bromo-2-((5-phenyl-1H-pyrazol-3-ylamino)methyl)-
OH); 14.31 (s, 1H, NH). 13C NMR (300 MHz, DMSO-d6) phenol (4e). White powder, yield: 73%; m.p. 173–175 °C.
δ: 161.5, 159.8, 136.1, 132.7, 128.5, 127.9, 126.3, 122.6, 108.5, 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.20 (d, J = 6.03 Hz, 2H,
96.4. MS (ESI): 332.0 ([M + H]+). Anal. Calcd for C16H11Cl2N3O: CH2); 5.77 (s, 1H, C–CH–C); 5.91 (s, 1H, NH–CH2); 6.76 (d, J =
C, 57.85; H, 3.34; N, 12.65%; Found: C, 57.70; H, 3.45; 8.40 Hz, 1H, Ph–H); 7.19–7.40 (m, 5H, Ph–H); 7.65 (d, J = 7.32
N, 12.72%.
Hz, 2H, Ph–H); 10.15 (s, 1H, OH); 12.08 (s, 1H, C–NH–N).
4.4.7. (E)-2,4-Dibromo-6-((5-phenyl-1H-pyrazol-3-ylimino)- 13C NMR (300 MHz, DMSO-d6) δ: 157.0, 153.2, 126.3, 128.1,
methyl)phenol (3g). Yellow powder, yield: 86%; m.p. 127.9, 127.2, 126.4, 124.3, 122.9, 117.0, 88.3, 42.1. MS (ESI):
271–273 °C. 1H NMR (300 MHz, DMSO-d6, δ ppm): 6.97 (s, 344.0 ([M + H]+). Anal. Calcd for C16H14BrN3O: C, 55.83;
1H, C–CH–C); 7.36–7.51 (m, 3H, Ph–H); 7.75–7.91 (m, 4H, Ph– H, 4.10; N, 12.21%; Found: C, 55.93; H, 4.12; N, 12.03%.
H); 9.14 (s, 1H, NvCH); 13.49 (s, 1H, OH); 14.49 (s, 1H, NH).
4.4.13. 2,4-Dichloro-6-((5-phenyl-1H-pyrazol-3-ylamino)-
13C NMR (300 MHz, DMSO-d6) δ: 163.0, 157.6, 136.2, 134.1, methyl)phenol (4f). White powder, yield: 78%; m.p. 169–
129.1, 127.9, 125.3, 119.7, 111.1, 93.8. MS (ESI): 419.9 171 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 4.22 (s, 2H,
([M + H]+). Anal. Calcd for C16H11Br2N3O: C, 45.64; H, 2.63; CH2); 5.91 (s, 1H, C–CH–C); 6.18 (s, 1H, NH–CH2); 7.26–7.42
N, 9.98%; Found: C, 45.58; H, 2.70; N, 9.87%.
(m, 5H, Ph–H); 7.64 (d, J = 7.35 Hz, 2H, Ph–H); 11.67 (s, 1H,
4.4.8. 2-((5-Phenyl-1H-pyrazol-3-ylamino)methyl)phenol OH); 12.32 (s, 1H, C–NH–N). 13C NMR (300 MHz, DMSO-d6)
(4a). White powder, yield: 70%; m.p. 166–167 °C. 1H NMR δ: 155.3, 153.4, 128.6, 128.0, 127.9, 127.3, 126.7, 124.4, 121.0,
(300 MHz, DMSO-d6, δ ppm): 4.21 (d, J = 6.21 Hz, 2H, CH2); 115.8, 87.6, 43.4. MS (ESI): 334.0 ([M + H]+). Anal. Calcd for
5.65 (s, 1H, C–CH–C); 5.88 (s, 1H, NH–CH2); 6.72–6.81 (m, 2H, C16H13Cl2N3O: C, 57.50; H, 3.92; N, 12.57%; Found: C, 57.34;
Ph–H); 7.03–7.08 (m, 1H, Ph–H); 7.25–7.41 (m, 4H, Ph–H); 7.65 H, 3.88; N, 12.78%.
(d, J = 7.50 Hz, 2H, Ph–H); 9.90 (s, 1H, OH); 12.07 (s, 1H,
4.4.14. 2,4-Dibromo-6-((5-phenyl-1H-pyrazol-3-ylamino)-
C–NH–N). 13C NMR (300 MHz, DMSO-d6) δ: 156.0, 154.8, methyl)phenol (4g). Yellow powder, yield: 66%; m.p. 148–150 °C.
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1H NMR (300 MHz, DMSO-d6,
δ
ppm): 4.21 (d,
J
=
4.4.19. 1-(5-Bromo-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl-
5.13 Hz, 2H, CH2); 5.91 (s, 1H, C–CH–C); 6.27 (s, 1H, 1H-pyrazol-3-yl)urea (5e). White powder, yield: 65%; m.p.
NH–CH2); 7.32–7.42 (m, 4H, Ph–H); 7.58–7.66 (m, 3H, Ph–H); 159–161 °C. 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.33 (d,
12.01 (s, 1H, OH); 12.40 (s, 1H, C–NH–N). 13C NMR (300 MHz, J = 6.39 Hz, 2H, CH2); 6.07 (s, 1H, NH–CvO); 6.79 (d, J = 8.43 Hz,
DMSO-d6) δ: 158.0, 155.2, 129.0, 128.8, 128.6, 128.3, 127.9, 1H, C–CH–C); 7.11–7.26 (m, 2H, Ph–H); 7.34–7.47 (m, 7H,
124.3, 122.1, 116.9, 88.0, 41.4. MS (ESI): 421.9 ([M + H]+). Anal. Ph–H); 7.70 (d, J = 8.43 Hz, 2H, Ph–H); 7.94 (d, J = 7.14 Hz, 2H,
Calcd for C16H13Br2N3O: C, 45.42; H, 3.10; N, 9.93%; Found: Ph–H); 9.93 (s, 1H, OH); 10.09 (s, 1H, C–NH–N). 13C NMR
C, 45.20; H, 3.22; N, 9.67%.
(300 MHz, DMSO-d6) δ: 157.2, 153.6, 152.7, 151.4, 138.4, 133.7,
4.4.15. 1-(2-Hydroxybenzyl)-3-phenyl-1-(5-phenyl-1H-pyrazol- 133.4, 131.5, 129.4, 129.3, 129.2, 128.9, 127.1, 125.3, 125.2,
3-yl)urea (5a). White powder, yield: 60%; m.p. 164–166 °C. 124.4, 120.1, 116.3, 114.5, 113.4, 83.3, 42.9. MS (ESI): 463.1
1H NMR (300 MHz, DMSO-d6, δ ppm): 4.36 (d, J = 6.21 Hz, 2H, ([M + H]+). Anal. Calcd for C23H19BrN4O2: C, 59.62; H, 4.13;
CH2); 6.10 (s, 1H, NH–CvO); 6.75–6.87 (m, 2H, C–CH–C, N, 12.09%; Found: C, 59.45; H, 4.27; N, 12.35%.
Ph–H); 7.08–7.18 (m, 2H, Ph–H); 7.30–7.48 (m, 7H, Ph–H); 7.71
4.5. Crystal structure determination
(d, J = 8.22 Hz, 2H, Ph–H); 7.97 (d, J = 6.57 Hz, 2H, Ph–H); 9.75
(s, 1H, OH); 9.93 (s, 1H, C–NH–N). 13C NMR (300 MHz, DMSO- The crystal structure determination of compound 3b was
d6) δ: 155.8, 152.1, 150.9, 150.5, 137.8, 131.5, 130.9, 128.3, carried out on a Nonius CAD4 diffractometer equipped with
127.9, 127.9, 127.6, 127.3, 126.8, 123.2, 122.9, 122.5, 120.7, graphite-mono chromated MoKα (k = 0.71073 Å) radiation. The
118.6, 115.3, 112.5, 85.4, 42.7. MS (ESI): 385.2 ([M + H]+). Anal. structure was solved by direct methods and refined on F2 by
Calcd for C23H20N4O2: C, 71.86; H, 5.24; N, 14.57%; Found: full-matrix least-squares methods using SHELX-97.27 All non-
C, 71.97; H, 5.36; N, 14.34%.
hydrogen atoms of compound 3b were refined with anisotropic
4.4.16. 1-(4-Chloro-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl- thermal parameters. All hydrogen atoms were placed in geome-
1H-pyrazol-3-yl)urea (5b). White powder, yield: 55%; m.p. trically idealized positions and constrained to ride on their
156–158 °C. 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.34 (d, parent atoms.
J = 6.42 Hz, 2H, CH2); 6.05 (s, 1H, NH–CvO); 6.81–6.87 (m,
2H, C–CH–C, Ph–H); 7.13–7.18 (m, 1H, Ph–H); 7.31–7.47 (m,
4.6. Antiproliferation activity
7H, Ph–H); 7.71 (d, J = 7.68 Hz, 2H, Ph–H); 7.94–7.97 (m, 2H, The antiproliferative activities of the prepared compounds
Ph–H); 9.92 (s, 1H, OH); 10.26 (s, 1H, C–NH–N). 13C NMR against WM1361, WM266.4 and A375 cell lines were evaluated
(300 MHz, DMSO-d6) δ: 156.2, 152.3, 151.9, 150.5, 137.2, 132.0, as described elsewhere with some modifications.28 Target
132.0, 130.2, 128.8, 128.7, 128.6, 128.4, 126.0, 124.7, 124.1, tumor cell lines were grown to log phase in RPMI
124.0, 120.9, 118.6, 114.8, 113.8, 83.8, 43.3. MS (ESI): 419.1 1640 medium supplemented with 10% fetal bovine serum.
([M + H]+). Anal. Calcd for C23H19ClN4O2: C, 65.95; H, 4.57; After diluting to 2 × 104 cells per mL with the complete
N, 13.38%; Found: C, 65.83; H, 4.49; N, 13.55%.
medium, 100 μL of the obtained cell suspension were added to
4.4.17. 1-(4-Bromo-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl- each well of 96-well culture plates. The subsequent incubation
1H-pyrazol-3-yl)urea (5c). White powder, yield: 57%; m.p. was left at 37 °C, 5% CO2 atmosphere for 24 h before the cyto-
169–171 °C. 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.32 (d, toxicity assessments. Tested samples at pre-set concentrations
J = 6.24 Hz, 2H, CH2); 6.04 (s, 1H, NH–CvO); 6.92–7.01 (m, were added to six wells with sorafenib as positive control.
2H, C–CH–C, Ph–H); 7.13–7.28 (m, 2H, Ph–H); 7.36–7.48 (m, After 48 h exposure period, 40 μL of PBS containing 2.5 mg mL−1
6H, Ph–H); 7.69–7.72 (m, 2H, Ph–H); 7.93–7.97 (m, 2H, Ph–H); of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
9.91 (s, 1H, OH); 10.24 (s, 1H, C–NH–N). 13C NMR (300 MHz, bromide) were added to each well. Four hours later, 100 μL
DMSO-d6) δ: 155.7, 151.8, 150.3, 150.2, 139.3, 132.0, 131.9, extraction solution (10% SDS–5% isobutyl alcohol–0.01 M
130.7, 129.3, 129.0, 128.6, 128.2, 126.5, 123.5, 123.1, 122.0, HCl) were added. After overnight incubation at 37 °C, the
118.9, 117.4, 115.0, 112.4, 84.6, 44.9. MS (ESI): 463.1 optical density was measured at a wavelength of 570 nm on an
([M + H]+). Anal. Calcd for C23H19BrN4O2: C, 59.62; H, 4.13; ELISA microplate reader. In all experiments three replicate
N, 12.09%; Found: C, 59.78; H, 4.04; N, 12.00%.
wells were used for each drug concentration. Each assay was
4.4.18. 1-(5-Chloro-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl- carried out for at least three times.
1H-pyrazol-3-yl)urea (5d). White powder, yield: 62%; m.p.
4.7. Kinase assay
167–168 °C. 1H NMR (300 MHz, DMSO-d6, δ ppm): 4.33 (d,
J = 6.39 Hz, 2H, CH2); 6.07 (s, 1H, NH–CvO); 6.84 (d, J = 8.61 The BRAF enzymatic assay was described by J. Dietrich et al.29
Hz, 1H, C–CH–C); 7.10–7.16 (m, 2H, Ph–H); 7.34–7.46 (m, 7H, The assay was performed in triplicate for each tested com-
Ph–H); 7.70 (d, J = 7.50 Hz, 2H, Ph–H); 7.94 (d, J = 6.60 Hz, 2H, pound in this study. Briefly, 7.5 ng Mouse Full-Length GST-
Ph–H); 9.92 (s, 1H, OH); 10.05 (s, 1H, C–NH–N). 13C NMR tagged BRAFV600E (Invitrogen, PV3849) were pre-incubated at
(300 MHz, DMSO-d6) δ: 154.7, 150.5, 149.9, 149.7, 138.7, 134.2, room temperature for 1 h with 1 μL drug and 4 μL assay
134.0, 132.3, 128.8, 128.6, 128.2, 127.9, 125.4, 123.2, 122.3, dilution buffer. The kinase assay was initiated when 5 μL of a
122.7, 121.2, 119.2, 113.8, 113.6, 83.4, 45.2. MS (ESI): 419.1 solution containing 200 ng recombinant human full length,
([M + H]+). Anal. Calcd for C23H19ClN4O2: C, 65.95; H, 4.57; N-terminal His-tagged MEK1 (Invitrogen), 200 μM ATP (0.8 μCi
N, 13.38%; Found: C, 65.78; H, 4.45; N, 13.45%.
hot ATP), and 30 mM MgCl2 in dilution buffer were added.
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Paper
Organic & Biomolecular Chemistry
The kinase reaction was allowed to continue at room tempera- were treated as separate terms. A +1e point charge was used as
ture for 25 min and then quenched with 5 μL 5 × protein dena- the electrostatic potential probe and distance-dependent
turing buffer (LDS) solution. The protein was further dielectric constant was used to mimic the solvation effect. For
denatured by heating for 5 min at 70 °C. 10 μL of each reaction the van der Waals potential, a carbon atom with a 1.73 Å
were loaded into a 15-well, 4–12% precast NuPage gel (Invitro- radius was used as a probe.
gen) and run at 200 V, and upon completion, the front, which
A Partial Least-Squares (PLS) model was built using energy
contained excess hot ATP, was cut from the gel and discarded. grids as descriptors. QSAR models were built by the Create 3D
The gel was then dried and developed onto a phosphor screen. QSAR Model protocol in Discovery Studio 3.1.
The reaction that contained no active enzyme was used as the
negative control, and the reaction without inhibitor was used
as the positive control.
Abbreviations
4.8. Acute oral toxicity
BRAF
V-RAF murine sarcoma viral oncogene homologue
B1
Five thousand milligrams of compound 5c per kilogram of
body weight were administered to ten healthy rats by oral
gavage. The animals were observed for mortality, signs of gross
toxicity and behavioral changes at least once daily for 14 days.
Body weights were recorded prior to administration and again
on Day 7 and 14. All animals survived and appeared active and
healthy throughout the study. With the exception of one male
that exhibited a loss in body weight between Day 7 and 14, all
animals gained body weight over the 14-day observation
period. There were no signs of gross toxicity or abnormal
behavior.
BRAFV600E V600E mutant BRAF
IC50
Half maximal inhibitory concentration
3D-QSAR Quantitative structure–activity relationship
Acknowledgements
Dr. Xin Zhao was dedicated to 2012-NIES Specific Funds for
Basic Scientific Research By Central Nonprofit Research Insti-
tutes. This work was supported by Major Projects on Control
and Rectification of Water Body Pollution (No. 2011ZX07204-
001-004), and was supported by the project (No. BY2012136)
from the Science & Technology Agency of Jiangsu Province and
by the projects (Nos. CXY1213 & CXY1222) from the Science &
Technology Bureau of Lianyuangang City of Jiangsu Province.
4.9. Docking simulations
The three-dimensional X-ray structure of BRAFV600E (PDB code:
2FB8) was chosen as the template for the modeling study of
compound 5c. The pdb file about the crystal structure of the
BRAFV600E kinase domain bound to SB-590885 (2FB8.pdb) was
obtained from the RCSB protein data bank (http://www.pdb.
org). The molecular docking procedure was performed using
the CDOCKER protocol for the receptor–ligand interactions
section of Discovery Studio 3.1 (Accelrys Software Inc, San
Diego, CA).30,31 All bound water and ligands were eliminated
from the protein and the polar hydrogen was added. The
whole BRAFV600E complex was defined as a receptor and the
site sphere was selected based on the ligand binding location
of SB-590885, then the SB-590885 molecule was removed and
replaced with 5c during the molecular docking procedure. The
types of interactions of the docked protein with ligand were
analyzed after the end of molecular docking.
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