Identification of novel 3,5-diarylpyrazoline derivatives containing salicylamide moiety as potential anti-melanoma agents

Article abstract of DOI:10.1016/j.bmcl.2012.09.004

There is an accumulating body of experimental evidences validating oncogenic BRAF^V600E as a therapeutic target and offering opportunities for anti-melanoma drug development. Encouraged by the positive results of pyrazole derivatives as BRAF

Full text of DOI:10.1016/j.bmcl.2012.09.004

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6596–6601
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel 3,5-diarylpyrazoline derivatives containing
salicylamide moiety as potential anti-melanoma agents
Qing-Shan Li a, , Xian-Hai Lv b, , Yan-Bin Zhang a, Jing-Jun Dong a, Wen-Ping Zhou a, Yang Yang a,
Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b College of Science, Anhui Agricultural University, Hefei 230036, PR China
a r t i c l e i n f o
a b s t r a c t
There is an accumulating body of experimental evidences validating oncogenic BRAFV600E as a therapeutic
target and offering opportunities for anti-melanoma drug development. Encouraged by the positive
results of pyrazole derivatives as BRAFV600E inhibitors, we sought to design diverse novel potential
BRAFV600E inhibitors as antitumor agents based on pyrazole skeleton. In silico and in vitro screening of
our designed pyrazole derivatives has identified Hit 1 as BRAFV600E inhibitor. Based on its structure
and through further structure modification, compound 25, which exhibited the most potent inhibitory
Article history:
Received 18 June 2012
Revised 17 August 2012
Accepted 1 September 2012
Available online 10 September 2012
Keywords:
BRAF
Inhibitors
Melanoma
Mutant
activity with an IC50 value of 0.16 l lM for mutant BRAF-
M for BRAFV600E and GI50 value of 0.24
dependent melanoma cells, was obtained. The 3D-QSAR models and the molecular docking simulation
were introduced to analyze the structure–activity relationship.
Ó 2012 Elsevier Ltd. All rights reserved.
Virtual screening
3D-QSAR
The RAS-RAF-MEK-ERK (MAP kinases) cascade plays a key role
in cellular growth, proliferation, and differentiation in response
to many different external stimuli.1,2 MAP kinases are regulated
by phosphorylation cascades whereby activation of an upstream
kinase leads to phosphorylation of a downstream substrate which
itself has protein-kinase activity.3,4 The RAS proteins are mem-
brane-bound small G-protein, whereas RAF, MEK, and ERK are
cytosolic protein-kinases that compose a sequential signaling cas-
cade. Under normal circumstances, RAF is activated in a RAS small
G-protein dependent manner. Then activated RAF activates (phos-
phorylates) MEK, which in turn activates a third protein-kinase
called ERK. ERK phosphorylates transcription factors such as
ELK-1, regulating gene expression and controlling how cells
respond to extracellular signals.5,6
forms named ARAF, c-RAF-1, and BRAF. The importance of BRAF
activation was highlighted by more recent studies that showed
that it is mutated approximately in 7% of human cancer, most
notably in ovarian (ꢀ35%), thyroid (ꢀ30%), and colorectal (ꢀ10%)
cancers, particularly in melanoma (50–70%).8,9 The most common
BRAF mutation in melanoma is the substitution of a valine for glu-
tamic acid at position 600 (termed BRAFV600E), resulting in a pro-
tein that has 500-fold elevated protein-kinase activity compared
to the wild-type. BRAFV600E stimulates sustained and constitutive
activation of the ERK pathway, inducing uncontrolled cell prolifer-
ation, increased cell survival, and tumor progression.10,11 Inhibition
of mutant BRAF signaling, through either direct inhibition of the
enzyme or inhibition of MEK, has been demonstrated preclinical
applications for inhibit melanoma development.12,13 Recently,
treatment of BRAF mutant melanoma patients with a selective
BRAF inhibitor has resulted in antitumor activity,14,15 These find-
ings strongly implicate the BRAF protein-kinase as an important
target for the development of small molecule inhibitors in the
treatment of human melanoma.
Cancers arise owing to the accumulation of mutations in critical
genes that alter normal programs of cell proliferation, differentia-
tion and death.7 Mutations of RAF kinase were frequently found
in cancer cells, results in it being the most studied drug target in
this cascade. The RAF protein-kinase family consists of three iso-
Many pyrazole derivatives are acknowledged to possess a wide
range of bioactivities,16–18 and such pyrazole core has increasingly
attracted the attention of synthetic chemists. Some small chemical
molecules containing pyrazole skeleton have been identified as
selective inhibitors of BRAFV600E and exhibited potent anti-cancer
activities.19,20 Encouraged by the positive results of previous re-
search, we sought to design diverse novel potential BRAFV600E
inhibitors for anti-melanoma agents based on pyrazole skeleton.
Abbreviations: BRAF, V-RAF murine sarcoma viral oncogene homologue B1;
pERK, phosphorylated extracellular regulated kinase; BRAFV600E, V600E mutant
BRAF; BRAFWT, BRAF wild-type; IC50, half maximal inhibitory concentration; GI50
,
the concentration that causes 50% growth inhibition; 3D-QSAR, Quantitative
Structure–Activity Relationship.
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Both authors contributed equally to the work
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.004

Q.-S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6596–6601
6597
Table 1
Initially positive results of virtual and protein-kinase inhibitory activities screening
Compound
Structure
Binding energies kcal/mol
BRAFV600E IC50
, lM
Br
N
HO
OMe
N
O
Hit 1
À20.97
7.22 0.59
O
OMe
HO
N
OMe
N
O
Hit 2
À17.66
8.38 0.44
MeO
Result values are shown as mean values of three independent determinations.
For this purpose, we have designed more than 300 compounds
based on pyrazole skeleton, including amide, (thio)urea, Schiff
bases, chalcone, salicylamide, amine, thiazole and other derivatives
of pyrazole. Virtual screening program and in vitro protein-kinase
inhibitory activities evaluation were introduced to initial screening
these candidate compounds.
aromatic side chain were deeply embedded into the pocket. How-
ever, different interactions with amino acid residues in binding site
were contributed to their combination with BRAF. For SB-590885
(Fig. 1c), in addition to H-bonds formed with Glu 501 and Cys
532,
p–p
interaction was found between its aromatic imidazole
interaction of SB-590885
ring and Phe 583. This additional
p
In the virtual screening procedure, the docking program CDOC-
KER (Discovery Studio 3.1, Accelrys, Inc., San Diego, CA)21,22 was
used as the in silico screening tool. The BRAFV600E/SB-590885 crys-
tal structure with the inhibitor removed from the coordinates (PDB
ID: 2FB8)23 was used as a receptor for compound binding. Residues
within a distance of 10 Å around the BRAF inhibitor SB-590885
were isolated for the construction of a grid for docking simulation.
This grid was large enough to include every residue of the BRAF ki-
nase ATP-binding pocket.19 After screening our designed pyrazole
derivatives, six initially candidate compounds with the lowest
binding energies were selected for protein-kinase inhibitory activ-
ity screening. Two candidate compounds (Hit 1 and Hit 2) which
displayed the lowest IC50 BRAFV600E values were chosen for further
study, and their structures, binding energies, BRAFV600E inhibitory
activities were summarized in Table 1. Both Hit 1 and Hit 2 own
pyrazoline salicylamide skeleton, so we focus on the structure
and activity optimization of pyrazoline salicylamide derivatives
in this study.
To take more directly insight into the binding mode of pyrazo-
line salicylamide derivatives and mutant BRAF, the binding model
of (5-(4-(benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)(5-bromo-2-hydroxyphenyl)meth-anone (Hit 1)
with BRAF structure are shown in Figure 1a and b. Visual inspec-
tion of the pose of Hit 1 into BRAF binding site revealed that this
candidate BRAF inhibitor was tightly embedded into the ATP bind-
ing pocket. The model suggests that extensive hydrophobic inter-
actions are formed between Hit 1 and residues Val 471, Ala 481,
Lys 483, Leu 514, Ile 527, Thr 529, Phe 583 and Asp 594 of the
ATP-binding pocket of BRAF kinase. Furthermore, a more optimal
H-bond interaction was formed between its methoxyl group and
the carbonyl group of Ser 535. Also, the phenolic hydroxyl group
of Hit 1 forms hydrogen bond with Ser 465. Compared with the
binding mode of SB-590885 (Fig. 1a), they both have good shape
complementarity with ATP-binding pocket of BRAF, and their
possibly results in its orientation and conformation was different
from that of Hit 1 in BRAF.
On the basis of the positive results obtained with Hit 1 and Hit
2, the 3,5-diarylpyrazoline skeleton could serve as a promising
scaffold for developing new kinase inhibitors of V600E mutant
BRAF. In order to obtain more pharmacophore understandings
and carry on further rational structure optimization, various sub-
stituents were introduced to the aromatic systems of 3,5-diarylpy-
razoline salicylamide. From the binding model, both the methoxyl
and phenolic hydroxyl groups of candidate compound were bene-
ficial for its binding affinity with mutant BRAF. So these two sub-
stituents were retained in the structure, and then several
substituents were introduced into the ring A and ring B of pyrazo-
line salicylamide skeleton. The synthesis of twenty pyrazoline
derivatives were synthesized in three steps, the synthesis followed
the general pathway outlined in Scheme 1. The pyrazoline core
(5–8) was synthesized via a two-step process in good yields: 1) al-
dol condensation between an appropriately substituted benzalde-
hyde and 4’-Methoxyacetophenone; 2) followed by pyrazoline
formation by addition of hydrazine hydrate. Target pyrazole sali-
cylamide derivatives 9–28 were synthesized by coupling appropri-
ate substituted salicylic acid with equimolar quantities of 5–8,
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDCÁHCl) and 1-hydroxybenzotriazole anhydrous (HOBt)
as condensing agent. Analytical and spectroscopic data of all com-
pounds were reported in part 1 of Supplementary data, and were
in full accordance with their depicted structures.
The biological activities (the data are summarized in Table 2) of
these novel 3,5-diarylpyrazoline derivatives were determined
using two assays: (1) the BRAFV600E inhibitory activities of these
synthesized compounds were determined as their ability to inhibit
BRAF mediated MEK phosphorylation (IC50 BRAFV600E); (2) the
determination of the growth inhibition in WM266.4 human mela-
noma cells expressing V600E mutant BRAF with MTT assay (GI50

6598
Q.-S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6596–6601
Figure 1. (a). Binding model of Hit 1 (purple) and SB-590885 (blue) in the active site of the BRAFV600E protein-kinase. The H-bond is displayed as dashed line. (b). and (c). 2D
projection drawing of Hit 1 and SB-590885 docked into BRAFV600E active site, respectively.
O
O
O
H
i
+
R1
OMe
MeO
R1
1-4
R2
N
B
HN
ii
iii
HO
1-4
OMe
N
OMe
O
N
R1
5-8
9-28
A
R1
Scheme 1. Synthesis route of 4, 5-dihydro-1H-pyrazole salicylamide derivatives 9–28. Reagents and conditions: (i) 40% KOH, ethanol, 5–10 °C, 4 h; R1 = 4-benzyloxy, 4-OMe,
4-Cl, 4-Br; (ii) N2H4ÁH2O, ethanol, reflux, 2 h; (iii) EDCÁHCl and HOBt, CH2Cl2, reflux, overnight; R2 = 5-Cl, 4-Cl, 5-Br, 5-I, 5-OMe.

Q.-S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6596–6601
6599
Table 2
BRAFV600E kinase and cellular activity of 4, 5-dihydro-pyrazole salicylamide analogs 9–28
Compound
R1
R2
BRAFV600E IC50
,
lM
WM266.4 GI50, lM
9
10
11 (Hit 1)
12
13
14
15
16
17
18 (Hit 2)
19
20
21
22
23
24
25
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-Br
4-Br
4-Br
4-Br
4-Br
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
4-Cl
5-Br
5-I
5-OMe
5-Cl
4-Cl
5-Br
5-I
5-OMe
5-Cl
4-Cl
5-Br
5-I
5-OMe
5-Cl
4-Cl
5-Br
5-I
5.69 0.92
5.01 0.75
7.22 0.59
5.9 0.31
10.34 1.2
9.3 1.5
7.04 0.44
4.92 0.61
13.3 0.9
8.38 0.44
2.51 0.27
1.38 0.1
3.69 0.18
4.1 0.72
6.94 1.21
0.57 0.13
0.16 0.03
1.21 0.09
1.30 0.53
3.72 0.64
12.9 0.52
7.89 0.31
12.86 0.97
13.03 0.29
13.92 1.1
12.63 0.64
13.3 0.5
18.4 1.6
15.6 0.78
>20
3.48 0.17
2.89 0.56
7.4 0.33
7.35 0.3
12.9 0.82
1.52 0.5
0.24 0.07
2.39 0.82
3.27 0.13
5.04 0.6
26
27
28
4-Cl
5-OMe
Sorafeniba
0.06
8.1
a
Used as a positive control.
inhibition (IC50 = 0.16 lM), being exactly comparable to the posi-
tive control (Sorafenib).
Table 3
BRAFWT cellular activity, BRAFV600E cell based
pERK activity and kinase inhibitory activities
against selected kinases of compound 25
As described above, the V600E mutant BRAF protein-kinase was
considered as an important target for the development of small
molecule inhibitors in the treatment human melanoma. Then 9–
28 were evaluated for antiproliferative activities against
WM266.4 human melanoma cell line expressing V600E mutant
BRAF. As illustrated in Table 2, most of the compounds displayed
GI50 in the low micromolar range. We observed that these com-
pounds, which have potent BRAFV600E kinase inhibitory activities,
displayed corresponding optimal cytotoxic activities against mu-
tant BRAF-dependent WM266.4 cells. These results indicated that
these compounds were potential anti-melanoma agents for
WM1361 (
pERK
EGFR
HER-2
FAK
l
M)
12.1 0.98
0.81 0.19
10.49 0.5
>25
>25
>25
>25
Aurora-A
IC50 VEGFR-2
The inhibitory activities are displayed as IC50
M).
(l
WM266.4). The protocols of biological assay were described in part
3 of Supplementary data.
Table 4
As shown in Table 2, a number of synthesized 3,5-diarylpyraz-
oline salicylamide analogues displayed potent BRAFV600E kinase
inhibitory activities in the low micromolar range. In general, sev-
eral analogues exhibited good activity with IC50 values of less than
that of Hit 1, two compounds (24, 25) demonstrated IC50 values of
Experimental, predicted inhibitory activity of compounds 9–28 by 3D-QSAR models
based upon active conformation achieved by molecular docking
Compounda
BRAFV600E
Predicted pIC50
Residual error
Actual pIC50
9
10
11
5.24489
5.30016
5.14146
5.22915
4.93531
5.21236
4.90011
5.05946
0.30958
0.0878
0.24135
0.16969
<1 lM. A comparison of the para substitution on A ring demon-
strated that a para halogen group (19–28) may have more slightly
improved inhibitory activity than a benzyloxy or a methoxy group,
and it showed the most potent inhibitory activity when the para
position substituted by chlorine (24–28). The inhibitory activity
of compounds with different para substituents on B ring increased
in the following order: 4-methoxy < 4-benzyloxy < 4-Br < 4-Cl. The
result suggested that strongly electronic-withdrawing substituents
on phenyl B ring were beneficial for the activity. SAR also indicated
that compounds bearing the same substituent on phenyl A ring
exhibited distinct kinase inhibitory activity due to different sub-
stituents which introduced into salicylic B ring. Introduction of
groups with greater electronegativity results in more active ana-
logs. The fact that the stronger electron-withdrawing substituents
at 5-position on B ring, the more potent it showed, was illustrated
by the potency order methoxy < I < Br < Cl. Most significantly,
move of Cl substituent to 4-position of salicylic B ring results in
the most active analog (25) in the whole series. Compound 25
exhibited the most potent inhibitory activity in BRAFV600E kinase
12
13
14
15
4.98548
5.03152
5.15243
5.3078
4.92129
5.06312
5.49426
5.43737
0.06419
À0.0316
À0.34183
À0.12957
16
17
4.87615
5.07654
4.90588
5.22072
À0.02973
À0.14418
18
19
20
21
22
23
24
25
26
5.60033
5.86012
5.43297
5.38722
5.15864
6.24413
6.79588
5.91721
5.88606
5.75179
5.82435
5.58784
5.62821
5.38962
5.99389
6.31049
5.77
À0.15146
0.03577
À0.15487
À0.24099
À0.23098
0.25024
0.48539
0.14721
0.14425
5.74181
27
28
5.42946
5.86954
À0.44008
a
Underlined compounds were selected as the test sets while the rest ones were
in the training sets.

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Q.-S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6596–6601
Figure 2. (a) The predicted versus experimental pIC50 value for the inhibition of BRAFV600E; (b) Isosurface of the 3D-QSAR model coefficients on electrostatic potential grids
with positive electrostatic potential in blue triangle mesh representation and the negative in red area for the aligned molecular structures. (c) Isosurface of the 3D-QSAR
model coefficients on Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates negative coefficients.
inhibiting mutant BRAF, and further validated that V600E mutant
BRAF was an effective target for melanoma therapy.
with an IC50 value of more than 10 lM. Taken together, our data
establish compound 25 as a potent and selective BRAFV600E
inhibitor.
For the sake of assess the inhibition of the target signaling path-
way (RAF-ERK) by compound 25, the phosphorylation level of
extracellular signal-regulated kinase (ERK) was measured in a
cell-based assay (IC50 pERK). As shown in Table 3, compound 25
exhibited obviously inhibitory activity of ERK phosphorylation in
BRAF mutant cell line. Furthermore, in order to provide evidence
to the specificity of the compounds in inhibition of mutant BRAF-
driven cell proliferation, the antiproliferative activities for the
BRAF wild-type WM1361 melanoma cell line (GI50 WM1361) that
did not express mutant BRAF was determined for compound 25
which had the most potent BRAFV600E inhibitory activity and the
result were exhibited in Table 3. Compared to the GI50 values on
mutant BRAF melanoma cell line WM266.4, the obtained GI50
WM1361 of these inhibitors was up to dozens of times. These po-
sitive results strongly support compound 25 possess antiprolifera-
tive activities against BRAF melanoma cell line through the
selective inhibition of oncogenic BRAF. To probe the kinase selec-
tivity of compound 25 against BRAF over other kinases, we selected
five kinases, EGFR, HER-2, FAK, Aurora-A, and VEGFR-2 which val-
idated as key regulators in cell cycle. The results revealed that the
compound has an excellent selectivity profile. As shown in Table 3,
compound 25 did not show significant inhibitory activities against
HER-2, FAK, Aurora-A, and VEGFR-2, and it showed EGFR inhibition
Based on the binding models, the 3D-QSAR (Quantitative Struc-
ture–Activity Relationship) modeling was performed to give fur-
ther validation of the binding mode and provided a structural
framework for understanding the structure–activity relationship
of these compounds. The 3D-QSAR models were built using the
corresponding pIC50 (ÀlogIC50) values which were transformed
from obtained BRAFV600E IC50 (in Mol/L) and performed by QSAR
software of DS 3.1 (Discovery Studio 3.1, Accelrys, Inc., San Diego,
CA). The training and test set was chosen by the Diverse Molecules
method in DS 3.1. The training sets were composed of 16 inhibitors
and test sets comprised 4 compounds of data sets as list in Table 4.
The underlined compounds in Table 4 were selected into test sets
and the rest were in training sets. One of the important steps in
developing 3D-QSAR modeling is the determination of active con-
formation and alignment of molecules. An efficient solution in this
study is to depend on docking programs and the reliability of this
method has been documented in previous studies.22,24 The align-
ment conformation of each molecule was the one with lowest en-
ergy in the docked results of CDOCKER.
The 3D-QSAR model generated from DS 3.1, defines the critical
regions (steric or electrostatic) affecting the binding affinity. It was
a PLS model built on 400 independent variables (conventional

Q.-S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6596–6601
6601
r2 = 0.754). The observed and predicted values and their residual
References and notes
values for the training set and test set molecules in 3D-QSAR model
are given in Table 4 and their graphical relationship is illustrated in
Figure 2A, respectively. The plot of the observed IC50 vs. the pre-
dicted results shows that this model has a good predictive power
which can be used in prediction of activity for new pyrazole sali-
cylamide derivatives as BRAFV600E inhibitors.
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contour plot of the electrostatic field region favorable
(in blue) or unfavorable (red) for the BRAFV600E affinity is shown
in Figure 2B. The energy grids corresponding to the favorable (in
green) or unfavorable (yellow) steric effects for the BRAFV600E affin-
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blue electrostatic potential areas (which are dominant close to
the skeleton). Several key features of the 3D-QSAR contour map
are predicted to increase BRAFV600E affinity: (1) More bulk near
the phenolic hydroxyl group and less bulk 5-substituent group of
ring B (steric study); (2) More bulk group substituted in the ortho
and meta position of ring A (steric study); (3) A more positive envi-
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and ring B (electronic study); (4) A more negative environment
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In conclusion, virtual screening of our designed pyrazole deriv-
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icylamide derivatives as BRAFV600E inhibitors and rational struc-
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inhibitory activity, with IC50 value of 0.16
GI50 value of 0.24 M for mutant BRAF-dependent WM266.4 cells.
l
M for BRAFV600E and
l
The BRAFWT cellular activity and BRAFV600E cell based pERK activity
of compound 25 suggested it could selectively inhibit proliferation
of mutant BRAF-dependent melanoma cell line through inhibition
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mization. Above all, the results obtained from this study suggest
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vel scaffold for the further development of more potent and selec-
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Acknowledgment
This work was financed by National Natural Science Foundation
of China (No. J1103512).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
004.