Discovery and Pharmacophore Studies of Novel Pyrazole-Based Anti-Melanoma Agents

Article abstract of DOI:10.1002/cbdv.201400143

Due to the rising incidence and lack of effective treatments, malignant melanoma is the most dangerous form of skin cancer, so that new treatment strategies are urgently needed. Several recent developments indicate that the V600E mutant BRAF (BRAF^V60

Full text of DOI:10.1002/cbdv.201400143

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Discovery and Pharmacophore Studies of Novel Pyrazole-Based Anti-
Melanoma Agents
by Qing-Shan Lia)b), Xian-Hai Lꢀc)d), Yang Yanga), Ban-Feng Ruana)b), Ri-Sheng Yao*a), and
Chen-Zhong Liao*a)
a) School of Medical Engineering, Hefei University of Technology, Hefei 230009, P. R. China
(phone/fax: þ86-551-62904675, e-mail: liqs@hfut.edu.cn)
b) Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, P. R. China
c) College of Science, Anhui Agricultural University, Hefei 230036, P. R. China
d) State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
P. R. China
Due to the rising incidence and lack of effective treatments, malignant melanoma is the most
dangerous form of skin cancer, so that new treatment strategies are urgently needed. Several recent
developments indicate that the V600E mutant BRAF (BRAFV600E
) is a validated target for
antimelanoma-drug development. Based on in silico screening results, a series of novel pyrazole
derivatives has been designed, synthesized, and evaluated in vitro for their inhibitory activities against
BRAFV600E melanoma cells. Compound 3d exhibited the most potent inhibitory activity with an IC50
value of 0.63 mm for BRAFV600E and a GI50 value of 0.61 mm for mutant BRAF-dependent cells.
Furthermore, the QSAR modeling and the docking simulation of inhibitor analogs provide important
pharmacophore clues for further structural optimization.
1. Introduction1). – Cancers arise owing to the accumulation of mutations in critical
genes that alter normal programs of cell proliferation, differentiation, and death [1].
Frequent findings of the BRAF mutations in melanoma rendered BRAF as the most
studied drug target in melanoma therapy [2][3]. The importance of BRAF activation
was highlighted by a more recent study showing that it is mutated in ca. 7% of human
cancers, most notably in melanoma (50–70%) [4][5]. The most common BRAF
mutation in malignant melanoma is the replacement of valine by glutamic acid in
position 600 (termed BRAFV600E), resulting in a protein that has 500-fold elevated
protein kinase activity compared to that of the wild-type. BRAFV600E stimulates
sustained and constitutive activation of the ERK pathway, inducing uncontrolled cell
proliferation, increased cell survival, and tumor progression [6][7]. Inhibition of
mutant BRAF signaling, through either direct inhibition of the enzyme or inhibition of
MEK (mitogen/extracellular signal-regulated kinase), has displayed a preclinical effect
by inhibiting tumor growth [8][9]. Recently, some active BRAFV600E inhibitors (Fig. 1)
1
)
Abbreviations: BRAF, V-RAF murine sarcoma viral oncogene homolog B1; pERK, phosphorylated
extracellular signal-regulated kinase; BRAFV600E, V600E mutant BRAF; BRAFWT, BRAF wild-
type; IC50, half maximal inhibitory concentration; GI50, the concentration that causes 50% growth
inhibition; QSAR, quantitative structureꢀactivity relationship.
ꢀ 2015 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
117
Fig. 1. Recently disclosed BRAFV600E inhibitors
were obtained, and treatment of BRAF mutant melanoma patients with selective
BRAF inhibitors has resulted in antitumor activity [10][11]. These findings strongly
implicate the BRAF protein kinase as an important target for the development of
small-molecule inhibitors to treat human cancers, particularly melanoma.
Many pyrazole derivatives have been shown to possess a wide range of bioactivities
[12–14], and such a pyrazole core has increasingly attracted the attention of synthetic
chemists. Some small chemical molecules containing a pyrazole skeleton have been
identified as selective inhibitors of BRAFV600E and exhibited potent anticancer
activities [15][16]. Encouraged by the promising results of previous research, we
sought to design diverse novel potential BRAFV600E inhibitors as antitumor agents
based on the pyrazole skeleton. For this purpose, we have designed more than 300
compounds with a pyrazole moiety, including amide, (thio)urea, Schiff bases, chalcone,
salicylamide, amine, thiazole, and other derivatives of pyrazole. Virtual screening
program and evaluation of the in vitro protein-kinase inhibitory activities were
introduced to initially screen these candidates. Then, the candidate compounds Hit 1
and Hit 2 (cf. Table 1, below) were obtained and selected for further optimization and
pharmacophore studies. Herein we report the synthesis and bioactivities of a series of
3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives to extend our research on anti-melano-
ma agents with BRAFV600E inhibitory activity [17][18]. Furthermore, molecular
simulation and QSAR modeling were performed to attain a structural framework for
understanding the structureꢀactivity relationships (SARs) of these compounds. These
results provided crucial clues to design new pyrazol derivatives with higher melanoma
inhibitory activities.
2. Results and Discussion. – 2.1. Virtual Screening. In this procedure, the docking
program CDOCKER (Discovery Studio 3.1, Accelrys, Inc., San Diego, CA) [19][20]

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 1. Initially Positive Results of Virtual and Protein-Kinase Inhibitory Activity Screening
Compound
Structure
Binding energy [kcal molꢀ1
]
IC50(BRAFV600E ) [mm]
3.08ꢁ0.35
Hit 1
ꢀ26.2084
Hit 2
6.9ꢁ0.95
ꢀ19.4668
was used as the in silico screening tool. The BRAFV600E/SB-590885 crystal structure
with the inhibitor being removed from the coordinates (Protein Data Bank (PDB) ID:
2FB8) [21] was used as a receptor for compound binding. Residues within a distance of
10 ꢂ 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 kinase ATP-binding pocket [15]. After screening our designed pyrazole
derivatives, five initial candidate compounds, Hit 1–Hit 5, with the lowest binding
energies were selected for protein-kinase inhibitory activity 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, and BRAFV600E
inhibitory activities are compiled in Table 1.
2.2. Chemistry. To obtain a better understanding of SARs of 3,5-diaryl-4,5-dihydro-
1H-pyrazoline derivatives as BRAFV600E inhibitors and for further rational structural
optimization, several analogs of candidate compounds were synthesized. The synthetic
route to target compounds is outlined in the Scheme. The ClaisenꢀSchmidt condensa-
tion of a ketone with an aldehyde in the presence of NaOH give a,b-unsaturated
ketones 1a–1h in generally good yields. 4,5-Dihydro-1H-pyrazole derivatives 2a–2h
were synthesized by cyclization of 1a–1h, respectively, with N2H4 ·H2O in EtOH.
Unfortunately, when the cyclization of 1e with N2H4 ·H2O was attempted under neutral
condition, the target pyrazole derivative 2e was not obtained. Next, urea and thiourea
derivatives, 3a–3h and 4a–4h, respectively, were prepared as described in [22].
Compounds 2a–2h were reacted with isocyanatobenzene or isothiocyanatobenzene in
CHCl3 to affording the target compounds 3a–3h and 4a–4h, respectively. On the other
hand, nicotinamide and isonicotinamide derivatives, 5a–5h and 6a–6h, respectively,

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Scheme. Synthesis of 3,5-Diaryl-4,5-dihydro-1H-pyrazole Derivatives
a) 40% KOH, EtOH, 5–108, 4 h; R1 ¼4-BnO, 4-MeO, 4-Cl, 4-Br; R2 ¼4-MeO, 4-Cl. b) N2H4 · H2O,
EtOH, reflux, 2 h. c) Cyanatobenzene or isothiocyanatobenzene, CHCl3, reflux, overnight; R3 ¼O or S.
d) EDC·HCl and HOBt, CH2Cl2, reflux, overnight; R4 ¼pyridine-3-carboxylic acid or pyridine-4-
carboxylic acid.
were synthesized by coupling appropriate (iso)nicotinic acid with equimolar quantities
of 2a–2h, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDC·HCl) and anhydrous 1-hydroxybenzotriazole (HOBt) as condensing agents.
Except for 3a, 3b, 4a, 4b, 6b, and 6f, the other target compounds are reported for the
1
first time. The structures of all novel compounds were determined by H-NMR, ESI-
MS, and elemental analysis. Moreover, 6g was characterized by X-ray diffraction
analysis; crystallographic data (excluding structure factors) of this structure have
been deposited with the Cambridge Crystallographic Data Centre (CCDC-996024).
The crystallographic data are collected in Table 2, and the ORTEP plot is presented in
Fig. 2.
2.3. Bioassays and SAR Studies. As compiled in Tables 3 and 4, a number of
synthesized 3,5-diaryl-4,5-dihydro-1H-pyrazole analogs displayed potent BRAFV600E
kinase inhibitory activities in the low mm range. We observed that the inhibitory
activities of (thio)urea derivatives were, in general, more potent than those of
(iso)nicotinamide derivatives, but had the same trends. Thiourea derivatives 4a–4h
displayed higher IC50 values than urea derivatives 3a–3h, indicating that the C¼O
group of urea was important for the interaction with the active cave of mutant BRAF
kinase. A comparison of substitutents on ring A of the diphenyl substituted pyrazole
skeleton revealed that a 4-MeO group may lead to a slightly improved inhibitory

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 2. Crystallographic Data of 6g
Empirical formula
Mr
C21H15BrClN3O
440.0
Crystal size [mm]
Crystal system
Space group
0.20ꢂ0.26ꢂ0.31
Triclinic
¯
P1
Z
2
Unit cell parameters:
a [ꢂ]
b [ꢂ]
9.449(6)
9.977(7)
c [ꢂ]
a [8]
b [8]
10.693(7)
77.379(6)
84.935(6)
69.517(5)
921.4(10)
2.569
g [8]
V [ꢂ3]
Dx (calc.) [gcmꢀ3
Absorption coefficient [mmꢀ1
]
]
10.493
F(000)
444.0
q Range for data collection [8]
Reflections collected
Unique reflections
2.30–28.30
7923
4273
Parameters
244
1.025
Goodness-of-fit on F2
Final R indices [I>2s(I)]
R Indices (all data)
R1 ¼0.0448, wR2 ¼0.0939
R1 ¼0.0878, wR2 ¼0.1075
0.436; ꢀ0.635
Largest diff. peak and hole [eꢂꢀ3
]
Fig. 2. ORTEP Plot of 6g

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Table 3. BRAFV600E Kinase and Cellular Activities of 3,5-Diaryl-4,5-dihydro-1H-pyrazole Derivatives 3a–
3h and 4a–4h
Compound
R1
R2
R3
IC50 ꢁSD (BRAFV600E ) [mm]
GI50 ꢁSD (WM266.4) [mm]
3a
4a
4-MeO
4-MeO
4-Cl
4-Cl
4-Br
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-Cl
O
S
O
S
O
S
O
S
0.92ꢁ0.1
1.53ꢁ0.24
3.08ꢁ0.35
3.4ꢁ0.58
1.92ꢁ0.15
–b)
0.63ꢁ0.07
0.77ꢁ0.05
3.39ꢁ0.69
–b)
1.18ꢁ0.25
2.06ꢁ0.19
2.47ꢁ0.1
3.21ꢁ0.27
2.43ꢁ0.12
–b)
0.61ꢁ0.1
1.1ꢁ0.19
4.8ꢁ0.8
–b)
3b (Hit 1)
4b
3c
4c
3d
4d
3f
4f
4-Br
4-BnO
4-BnO
4-Cl
O
S
4-Cl
4-Cl
3g
4g
3h
4h
4-Br
4-Br
4-BnO
4-BnO
4-Cl
4-Cl
4-Cl
4-Cl
O
S
O
S
3.07ꢁ0.51
2.91ꢁ0.33
2.13ꢁ0.19
1.35ꢁ0.2
0.06
2.68ꢁ0.34
3.39ꢁ0.72
2.6ꢁ0.4
1.93ꢁ0.41
8.1
Sorafeniba)
a) Positive control. b) –, Not determined.
activity than a Cl group, suggesting that weakly electronic-withdrawing substituents on
phenyl ring Awere beneficial for the activity. On the other side, compounds bearing the
same substituents on ring A exhibited distinct kinase inhibitory activities depending
upon different substituents introduced into ring B. The inhibitory activities of
compounds with different para-substituents on ring B increased in the following order:
4-Cl<4-Br<4-MeO<4-BnO. The results indicated that the introduction of groups
with higher electronegativity results in less active analogs. It is worth noting that these
trends were observed in all compounds, whether they were urea or nicotinamide
analogs. Most significantly, urea derivative 3d exhibited the most potent inhibitory
activity in the whole series, with an IC50 value of 0.63 mm.
Furthermore, all synthesized 3,5-diaryl-4,5-dihydro-1H-pyrazole analogs were
evaluated for their antiproliferative activities against WM266.4 human melanoma cell
line expressing V600E mutant BRAF. As illustrated in Tables 3 and 4, most of the
compounds displayed GI50 values in the low mm range. We observed that these
compounds, which have potent BRAFV600E kinase inhibitory activities, displayed
corresponding optimal cytotoxic activities against mutant BRAF-dependent WM266.4

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Table 4. BRAFV600E Kinase and Cellular Activity of 3,5-Diaryl-4,5-dihydro-1H-pyrazole Derivatives 5a–
5h and 6a–6h
Compound
R1
R2
R4
IC50 ꢁSD (BRAFV600E
[mm]
)
GI50 ꢁSD (WM266.4)
[mm]
5a
6a
4-MeO
4-MeO
4-Cl
4-Cl
4-Br
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-3-yl
Pyridin-4-yl
2.96ꢁ0.71
3.58ꢁ0.63
6.9ꢁ0.95
6.56ꢁ1.1
4.81ꢁ0.35
4.77ꢁ0.73
5.71ꢁ0.55
4.16ꢁ0.19
7.31ꢁ0.5
5.71ꢁ0.71
7.17ꢁ0.58
6.53ꢁ0.9
4.23ꢁ0.4
3.59ꢁ0.2
0.06
3.1ꢁ0.39
2.41ꢁ0.2
4.32ꢁ0.43
4.75ꢁ0.68
4.3ꢁ0.5
5b (Hit 2)
6b
5c
6c
5d
6d
5f
6f
4-Br
3.36ꢁ0.67
3.35ꢁ0.42
2.02ꢁ0.94
8.26ꢁ1.45
7.04ꢁ1.01
7.96ꢁ0.86
5.67ꢁ0.33
5.95ꢁ0.37
3.38ꢁ0.44
8.1
4-BnO
4-BnO
4-Cl
4-Cl
4-Br
4-Br
4-BnO
4-BnO
5g
6g
5h
6h
4-Cl
Sorafeniba)
a) Positive control.
cells. These results indicated that these compounds were potential anti-melanoma
agents for inhibiting mutant BRAF.
To provide evidence for the specificity of the compounds for mutant BRAF-driven
cell proliferation, the antiproliferative activities for the BRAF wild-type WM1361
melanoma cell line (GI50 WM1361) were determined for selected compounds, i.e., 3d
and 4d, and the results were compiled in Table 5. Compared to the GI50 values on
mutant BRAF melanoma cell line WM266.4, the obtained GI50 (WM1361) values of
these inhibitors were all up to dozens of times. Moreover, in order to assess the
inhibition of the target signaling pathway (RAF-ERK (rapidly accelerated fibrosarco-
ma-extracellular signal-regulated kinase)) by selected compounds, the phosphorylation
level of ERK was determined in a cell-based assay (IC50 pERK). As shown in Table 5,
Table 5. BRAFWT Cellular Activity and BRAFV600E Cell-Based pERK Activities of Selected Compounds
Compound
GI50 (WM1361) [mm]
IC50 (pERK) [mm]
3d
4d
6.63ꢁ0.57
7.16ꢁ0.64
2.15ꢁ0.2
2.9ꢁ0.38

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
123
combined with their GI50 (WM266.4) values, 3d and 4d exhibited a close correlation
between the inhibition of both ERK phosphorylation and cell proliferation in a BRAF
mutant cell line. These results strongly support the assumption that the synthesized 3,5-
diaryl-4,5-dihydro-1H-pyrazole derivatives possess antiproliferative activities against
BRAF melanoma cell line through the selective inhibition of oncogenic BRAF.
For a better understanding of the observed inhibitory-activity differences among
four series of 3,5-diaryl-4,5-dihydro-1H-pyrazole analogs and to guide further SAR
studies, molecular docking was performed on the binding model based on the
BRAFV600E complex structure (cf. Sect. 2.1). Each ligand was docked as described
previously, and the pose with the highest ꢀECD was considered as the optimum pose for
it. The binding models of 3d, 4d, and 5d with the BRAF structure are shown in Fig. 3.
Visual inspection of the pose of selected compounds into the BRAF-binding site
revealed that 3d and 4d were tightly embedded into the ATP binding pocket. In
contrast, as shown in Fig. 3, nicotinamide analogs adopted different orientations in the
BRAF-binding pocket, and occupied significantly less space within the BRAF active
site than the corresponding (thio)urea analogs. Accordingly, we propose that the
introduction of the (thio)urea motif may mediate additional protein interaction and
possess more shape complementarity with the activation loop, then this structural
modification would likely increase BRAF inhibitor potency and specificity. This
conclusion was verified by different inhibitory activities obtained for (thio)urea and
(iso)nicotinamide analogs of pyrazole and further confirmed by their binding models.
According to the docking results, a comparison of 3d with 4d in complex with
BRAFV600E shows significant similarities in their BRAF binding models (Fig. 4,a and
b). Their models suggest that extensive hydrophobic interactions are formed between
ligands and the ATP-binding pocket of BRAF kinase. In particular, residues Ile463,
Val471, Ala481, Lys483, Leu514, Val528, Thr529, Trp531, Cys532, Gly534, Ser535,
Fig. 3. Overlap of 3d (red) and 4d (orange) with 5d (green) in BRAFV600E (BRAFV600E/SB-590885 crystal
structure with the inhibitor removed from the coordinates; Protein Data Bank (PDB) code: 2FB8)
surface model

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Fig. 4. a) Projection of the binding model between BRAF and 4d. b) Projection of the binding model
between BRAF and 3d.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Phe583, and Gly593 are predicted to make Van der Waals contacts with 3d and 4d.
In their binding models, the dihydropyrazole nuclei of both 3d and 4d entered two
p ···cation interactions with NH2 of Lys473 and Lys483. Furthermore, p ···p
interactions with the aromatic portion of Phe583 were also detected in the simulation
results of 3d and 4d. Particularly, a stronger p ···s interaction was established between
3d and Trp531-NH, indicating that C¼O group in the urea skeleton could facilitate the
cohesive docking of dihydropyrazole urea analogs into the ATP binding pocket of
inactive BRAFV600E. These results could possibly explain of the urea analogs 3a–3h, the
higher BRAFV600E inhibitory potencies compared to those of the corresponding
thiourea analogs 4a–4h.
To perform a systematic evaluation on 3,5-diaryl-4,5-dihydro-1H-pyrazole analogs
as BRAFV600E inhibitors, and to explore the more potent and selective BRAFV600E
inhibitors, QSAR models were built using the corresponding minus logarithmic scale
pIC50 (ꢀ logIC50, mollꢀ1, transformed from obtained BRAFV600E IC50 values) and
performed by the software of DS 3.1 (Discovery Studio 3.1). The training sets were
composed of 21 inhibitors, and the test sets comprised five compounds as listed in
Table 6. Experimental and Predicted Inhibitory Activities by QSAR Models
Compounda)
BRAFV600E
Residual error
Exper. pIC50
Predicted pIC50
3a
4a
5a
6a
3b
4b
5b
6b
3c
5c
6c
3d
4d
5d
6d
3f
6.036
5.815
5.529
5.446
5.511
5.468
5.161
5.183
5.717
5.318
5.321
6.201
6.114
5.243
5.381
5.470
5.136
5.243
5.513
5.536
5.144
5.185
5.672
5.870
5.374
5.445
6.009
5.649
5.467
5.480
5.548
5.486
5.160
5.144
5.697
5.370
5.281
6.177
6.121
5.240
5.337
5.441
5.180
5.202
5.520
5.532
5.107
5.238
5.713
5.933
5.383
5.414
ꢀ0.027
ꢀ0.167
ꢀ0.062
0.035
0.036
0.018
ꢀ0.001
ꢀ0.039
ꢀ0.019
0.052
ꢀ0.040
ꢀ0.024
0.008
ꢀ0.004
ꢀ0.044
ꢀ0.028
0.044
5f
6f
ꢀ0.041
3g
4g
5g
6g
3h
4h
5h
6h
0.007
ꢀ0.004
ꢀ0.037
0.053
0.041
0.063
0.010
ꢀ0.031
a) Underlined compounds were selected as test sets, while the rest ones were in the training sets.

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Table 6. For the development of the QSAR models, the statistical techniques used were
genetic function algorithm (GFA) and partial least squares (PLS). The underlined
compounds in Table 6 were selected as test sets, and the rest was in training sets. We
compared two different QSAR methods: PLS algorithm and GFA. The model with the
highest R2 value was acquired using GFA as follows (Eqn. 1):
GFATempModel_1¼11.892þ1.4059·CICþ0.037126·Dipole_Xþ0.037082
·Dipole_magþ0.0073239·Jurs_PPSA_1ꢀ0.2362·Num_H_Acceptorsꢀ0.082225
·SC_3_Pꢀ8.1871·Shadow_XYfracꢀ0.28939·Shadow_Zlength for this model,
r2 ¼0.9892, r2(adj)¼0.9820, r2(pred)¼0.9701, Friedman LOF¼0.006.
(1)
This model is a 2D and 3D hybrid QSAR model containing eight descriptors,
including 2D topological descriptors and other 3D descriptors. In our study, r2, r2 (adj),
and r2 (pred) were used to evaluate the regression model. Eqn. 1 can explain 98.2% of
the variance (r2(adj)), while it could predict 97% of the variance (r2(pred) is the
predicted r2, equivalent to q2 from a leave-1-out cross-validation). The difference
between r2 and r2(pred) values is not very high (less than 0.2) [23]. It can be seen from
Eqn. 1 that CIC, Dipole_X, Dipole_mag, and Jurs_PPSA_1 have a positive
contribution to the bioactivity of the ligands; however, Num_H_Acceptors, SC_3_P,
Shadow_XZfrac and Shadow_Zlength have a negative effect on the bioactivity of the
ligands. The observed and predicted results, and the pIC50 values of physiochemical
properties of the 26 ligands are collected in Table 6. The plot of the observed pIC50
values vs. the predicted data is shown in Fig. 5, and it reveals that this model owns
Fig. 5. Plot of the experimental pIC50 values vs. the predicted data generated from QSAR model

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
127
reliable predictive ability which can be used in prediction of the activity for new 3,5-
diaryl-4,5-dihydro-1H-pyrazole derivatives as BRAFV600E inhibitors.
3. Conclusions. – In this study, a series of 3,5-diaryl-4,5-dihydro-1H-pyrazole
(thio)urea and (iso)niacinamide analogs have been discovered based on virtual
screening results. Among these compounds, urea analogs 3a–3h displayed more potent
BRAFV600E inhibitory activities. Particularly, 3d displayed the most potent inhibitory
activity, with an IC50 value of 0.63 mm for BRAFV600E and a GI50 value of 0.61 mm for
mutant BRAF-dependent WM266.4 cells. The BRAFWT cellular activities and
BRAFV600E cell-based pERK activities for selected compounds, which exhibited good
inhibitory activities, were evaluated; the results indicated that those compounds could
selectively inhibit proliferation of mutant BRAF-dependent melanoma cell line
through inhibition of oncogenic BRAF. Molecular dockings of 3d, 4d, and 5d into
BRAFV600E were performed and the generated binding modes could explain their
different BRAFV600E inhibitory potencies. Furthermore, the QSAR analysis was
performed to provide crucial pharmacophore clues that could be used to design new
3,5-diaryl-4,5-dihydro-1H-pyrazole derivatives with higher BRAFV600E inhibitory
activities. Above all, the results obtained from this study suggest that urea analog 3d
may serve as a lead compound for further development of more potent and selective
BRAFV600E inhibitors which could be used as mutant BRAF-dependent melanoma
therapeutic agents.
This work was funded by the Fundamental Research Funds for the Central Universities
(2013 HGCH0015 and JZ2014 HGBZ0030), the China Postdoctoral Science Foundation (2014M552387
and 2013M530409), and the National Natural Science Foundation of China (21302036 and 21302002).
Experimental Part
General. All chemicals (reagent grade) used were purchased from Aldrich (USA). M.p.: XT4 MP
apparatus (Taike Corp., Beijing, China); uncorrected. TLC: Silica gel-coated aluminum sheets (SiO2; 60
GF254; Merck, Germany); visualized by UV light at 254 nm. Column chromatography (CC): SiO2 60
(200–300 mesh ASTM; Merck, Germany). The quantity of SiO2 used was 50–100 times the weight
charged on the column. 1H-NMR Spectra: 1H-Varian-Mercury-300 spectrometer (300 MHz, in CDCl3, at
258); d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Mariner System 5304 mass
spectrometer; in m/z. Elemental analyses: CHN-O-Rapid instrument; within ꢁ0.4% of the theoretical
values; in %.
Synthesis of Chalcones 1a–1h. Equimolar portions of the appropriately substituted aromatic
aldehydes (40 mmol, 1 equiv.) and acetophenones (40 mmol, 1 equiv.) were dissolved in ca. 15 ml of
EtOH. The mixture was stirred for several min at 5–108. A 40-ml aliquot of 40% aq. KOH soln. was then
slowly added to the reaction flask via a self-equalizing addition funnel. The soln. was stirred at r.t. for ca.
4 h. Most commonly, a precipitate formed, which was then collected by suction filtration.
Preparation of Pyrazoline Derivatives 2a–2h. The chalcones 1a–1h (20 mmol, 1 equiv.) and N2H4 ·
H2O (40 mmol, 2 equiv.) in 50 ml of EtOH were heated under reflux for 2 h. Excess EtOH was distilled
off, and the resulting residue was collected. The corresponding pyrazole derivatives 2a–2h were obtained
through recrystallization from EtOH.
General Procedure for the Synthesis of 3,5-Diaryl-4,5-dihydro-1H-pyrazole Derivatives 3a–3h and
4a–4h. To a CHCl3 soln. of 2a–2h (1 mmol), isocyanatobenzene or isothiocyanatobenzene (1 mmol) was
slowly added under stirring. The mixture was heated under reflux overnight. Then, the product was
filtered and purified by recrystallization from EtOH. If the solid was not formed, the mixture was

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evaporated under reduced pressure to give a residue. The residue was purified by CC (SiO2; petroleum
ether (PE)/AcOEt 10 :1) to afford pure products.
4,5-Dihydro-3,5-bis(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carboxamide (3a). Yield: 61%.
White powder. M.p. 131–1338 ([24]: 1368).
4,5-Dihydro-3,5-bis(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carbothioamide (4a). Yield: 65%.
White powder. M.p. 148–1508 ([24]: 143–1458).
5-(4-Chlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carboxamide (3b).
Yield: 71%. Brown powder. M.p. 192–1958 ([25]: 1968).
5-(4-Chlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carbothioamide
(4b). Yield: 68%. White powder. M.p. 183–1858 ([25]: 1788).
5-(4-Bromophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carboxamide (3c).
1
Yield: 78%. Pink powder. M.p. 200–2028. H-NMR: 3.12–3.20 (m, 1 H); 3.78–3.84 (m, 1 H); 3.87 (s,
3 H); 5.49–5.55 (m, 1 H); 6.91–7.05 (m, 3 H); 7.20 (d, J¼7.95, 3 H); 7.45–7.52 (m, 5 H); 7.69 (d, J¼8.67,
2 H); 8.10 (s, 1 H). ESI-MS: 450.1 ([MþH]þ ). Anal. calc. for C23H20BrN3O2: C 61.34, H 4.48, N 9.33;
found: C 61.52, H 4.64, N 9.46.
5-[4-(Benzyloxy)phenyl]-4,5-dihydro-3-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carboxamide
(3d). Yield: 74%. White powder. M.p. 153–1548. 1H-NMR: 3.16–3.23 (m, 1 H); 3.79 (dd, J¼11.91, 17.94,
1 H); 3.87 (s, 3 H); 5.03 (s, 2 H); 5.50–5.55 (m, 1 H); 6.02–7.03 (m, 6 H); 7.22–7.40 (m, 7 H); 7.52 (d, J¼
7.74, 3 H); 7.70 (d, J¼8.49, 2 H); 8.10 (s, 1 H). ESI-MS: 478.2 ([MþH]þ ). Anal. calc. for C30H27N3O3: C
75.45, H 5.70, N 8.80; found: C 75.18, H 5.52, N 8.95.
5-[4-(Benzyloxy)phenyl]-4,5-dihydro-3-(4-methoxyphenyl)-N-phenyl-1H-pyrazole-1-carbothio-
amide (4d). Yield: 63%. Yellow powder. M.p. 176–1788. 1H-NMR: 3.21 (dd, J¼3.66, 17.73, 1 H); 3.84–
3.93 (m, 4 H); 5.15 (s, 2 H); 6.13–6.24 (m, 1 H); 6.92–6.99 (m, 3 H); 7.21–7.36 (m, 4 H); 7.31–7.46 (m,
7 H); 7.62–7.78 (m, 4 H); 9.26 (s, 1 H). ESI-MS: 494.2 ([MþH]þ ). Anal. calc. for C30H27N3O2S: C 73.00,
H 5.51, N 8.51; found: C 73.32, H 5.70, N 8.82.
3,5-Bis(4-chlorophenyl)-4,5-dihydro-N-phenyl-1H-pyrazole-1-carboxamide (3f). Yield: 76%. White
powder. M.p. 187–1898. 1H-NMR: 2.95 (d, J¼6.93, 1 H); 3.96–4.06 (m, 1 H); 5.73–5.79 (m, 1 H); 7.16
(m, 3 H); 7.30 (d, J¼8.40, 2 H); 7.38 (d, J¼8.04, 4 H); 7.68 (s, 4 H); 8.67–8.79 (m, 1 H). ESI-MS: 410.1
([MþH]þ ). Anal. calc. for C22H17Cl2N3O: C 64.40, H 4.18, N 10.24; found: C 64.73, H 4.32, N 10.46.
5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-N-phenyl-1H-pyrazole-1-carboxamide (3g).
Yield: 72%. White powder. M.p. 209–2118. 1H-NMR: 3.12–3.20 (m, 1 H); 3.71–3.87 (m, 1 H); 5.55
(dd, J¼5.85, 12.09, 1 H); 7.01–7.06 (m, 1 H); 7.18 (d, J¼8.40, 2 H); 7.29–7.33 (m, 2 H); 7.41–7.59 (m,
6 H); 7.67 (d, J¼8.58, 2 H); 8.05 (s, 1 H). ESI-MS: 454.0 ([MþH]þ ). Anal. calc. for C22H17BrClN3O: C
58.11, H 3.77, N 9.24; found: C 58.43, H 3.59, N 9.13.
5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-N-phenyl-1H-pyrazole-1-carbothioamide (4g).
Yield: 61%. White powder. M.p. 195–1978. 1H-NMR: 3.18 (dd, J¼3.84, 17.73, 1 H); 3.87 (dd, J¼
11.70, 17.76, 1 H); 6.14–6.19 (m, 1 H); 7.15–7.24 (m, 3 H); 7.35–7.40 (m, 2 H); 7.46–7.50 (m, 4 H); 7.64
(d, J¼8.22, 2 H); 7.71 (d, J¼8.61, 2 H); 9.23 (s, 1 H). ESI-MS: 470.0 ([MþH]þ ). Anal. calc. for
C22H17BrClN3S: C 56.12, H 3.64, N 8.93; found: C 56.47, H 3.54, N 8.75.
5-[4-(Benzyloxy)phenyl]-3-(4-chlorophenyl)-4,5-dihydro-N-phenyl-1H-pyrazole-1-carboxamide
(3h). Yield: 83%. White powder. M.p. 186–1888. 1H-NMR: 3.17–3.24 (m, 1 H); 3.76–3.86 (m, 1 H); 5.05
(s, 2 H); 5.57 (dd, J¼5.67, 12.06, 1 H); 6.95 (dd, J¼2.01, 6.75, 2 H); 7.01–7.06 (m, 1 H); 7.23–7.28 (m,
3 H); 7.30–7.38 (m, 4 H); 7.38–7.45 (m, 4 H); 7.51–7.54 (m, 2 H); 7.68–7.71 (m, 2 H); 8.07 (s, 1 H). ESI-
MS: 482.1 ([MþH]þ ). Anal. calc. for C29H24ClN3O2: C 72.27, H 5.02, N 8.72; found: C 72.43, H 5.13, N
8.69.
5-[4-(Benzyloxy)phenyl]-3-(4-chlorophenyl)-4,5-dihydro-N-phenyl-1H-pyrazole-1-carbothioamide
(4h). Yield: 69%. Yellow powder. M.p. 196–1998. 1H-NMR: 3.21 (dd, J¼3.66, 17.73, 1 H); 3.84 (dd, J¼
11.52, 17.73, 1 H); 5.05 (s, 2 H); 6.14–6.19 (m, 1 H); 6.94–6.98 (m, 2 H); 7.18–7.26 (m, 3 H); 7.31–7.46
(m, 9 H); 7.64–7.74 (m, 4 H); 9.23 (s, 1 H). ESI-MS: 498.1 ([MþH]þ ). Anal. calc. for C29H24ClN3OS: C
69.94, H 4.86, N 8.44; found: C 69.76, H 4.68, N 8.71.
General Procedure for the Synthesis of 3,5-Diaryl-4,5-dihydro-1H-pyrazole Derivatives 5a–5h and
6a–6h. A stirred soln. of 2a–2h (1.0 mmol, 1 equiv.) in CH2Cl2 (10 ml) was treated with equimolar
quantities of nicotinic acid and isonicotinic acid, and EDC·HCl (1.2 mmol, 1.2 equiv.) and HOBt

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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(1.2 mmol, 1.2 equiv.) were added. The mixture was heated under reflux overnight. The product was
collected by filtration, and the crude residue was purified by recrystallization from EtOH. After
completion of the reaction, if the solid was not formed, the mixture was evaporated under reduced
pressure to give a residue. The residue was purified by CC (SiO2; PE/AcOEt 4 :1) to afford pure
products.
[4,5-Dihydro-3,5-bis(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-3-yl)methanone (5a). Yield: 64%.
1
Yellow powder. M.p. 141–1438. H-NMR: 3.15–3.25 (m, 1 H); 3.78–3.82 (m, 4 H); 3.86 (s, 3 H); 5.76
(dd, J¼4.54, 11.54, 1 H); 6.76–6.90 (m, 4 H); 7.29 (d, J¼8.22, 2 H); 7.46 (d, J¼7.86, 3 H); 7.67–7.79 (d,
J¼6.93, 2 H); 8.78 (d, J¼8.76, 1 H). ESI-MS: 388.2 ([MþH]þ ). Anal. calc. for C23H21N3O3: C 71.30, H
5.46, N 10.85; found: C 71.48, H 5.58, N 10.76.
[4,5-Dihydro-3,5-bis(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-4-yl)methanone (6a). Yield: 65%.
White powder. M.p. 183–1858. 1H-NMR: 3.20–3.28 (m, 1 H); 3.74–3.84 (m, 4 H); 3.89 (s, 3 H); 5.78 (dd,
J¼4.77, 11.70, 1 H); 6.97 (d, J¼8.79, 2 H); 7.25 (s, 2 H); 7.33–7.44 (m, 4 H); 7.81 (d, J¼8.04, 2 H); 8.76
(d, J¼7.50, 2 H). ESI-MS: 388.2 ([MþH]þ ). Anal. calc. for C23H21N3O3: C 71.30, H 5.46, N 10.85; found:
C 71.61, H 5.32, N 10.57.
[5-(4-Chlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-3-yl)methanone
1
(5b). Yield: 78%. Brown powder. M.p. 199–2018. H-NMR: 3.15–3.22 (m, 1 H); 3.74–3.80 (m, 1 H);
3.85 (s, 3 H); 5.71–5.77 (m, 1 H); 6.94 (d, J¼8.79, 2 H); 7.25–7.28 (m, 2 H); 7.33 (d, J¼8.43, 2 H); 7.64
(d, J¼8.79, 2 H); 7.61 (d, J¼5.85, 2 H); 8.74 (d, J¼5.85, 2 H). ESI-MS: 392.1 ([MþH]þ ). Anal. calc. for
C22H18ClN3O2: C 67.43, H 4.63, N 10.72; found: C 67.61, H 4.48, N 10.44.
[5-(4-Chlorophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-4-yl)methanone
(6b). Yield: 65%. White powder. M.p. 186–1878 ([26]: 189–1918).
[5-(4-Bromophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-3-yl)methanone
(5c). Yield: 38%. Red powder. M.p. 173–1758. 1H-NMR: 3.28–3.36 (m, 1 H); 3.81–3.93 (m, 4 H); 5.72–
5.77 (m, 1 H); 6.88–7.00 (m, 5 H); 7.70 (d, J¼8.70, 2 H); 7.98 (s, 1 H); 8.82 (d, J¼4.53, 1 H); 8.98 (d, J¼
7.17, 2 H); 9.67 (s, 1 H). ESI-MS: 436.1 ([MþH]þ ). Anal. calc. for C22H18BrN3O2: C 60.56, H 4.16, N
9.63; found: C 60.33, H 4.44, N 9.51.
[5-(4-Bromophenyl)-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl](pyridin-4-yl)methanone
(6c). Yield: 65%. Grey powder. M.p. 211–2148. 1H-NMR: 3.15–3.22 (m, 1 H); 3.76–3.81 (m, 1 H); 3.86
(s, 3 H); 5.74 (dd, J¼4.53, 11.31, 1 H); 6.94 (d, J¼8.49, 2 H); 7.21 (d, J¼8.32, 2 H); 7.49 (d, J¼7.95, 2 H);
7.64 (d, J¼8.49, 2 H); 7.84 (d, J¼4.71, 2 H); 8.66 (d, J¼4.71, 2 H). ESI-MS: 436.1 ([MþH]þ ). Anal.
calc. for C22H18BrN3O2: C 60.56, H 4.16, N 9.63; found: C 60.79, H 4.34, N 9.47.
{5-[4-(Benzyloxy)phenyl]-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl}(pyridin-3-yl)methan-
one (5d). Yield: 72%. Yellow powder. M.p. 147–1518. 1H-NMR: 3.14–3.20 (m, 1 H); 3.74–3.88 (m,
4 H); 5.24 (s, 2 H); 5.85–5.89 (m, 1 H); 7.90 (d, J¼8.97, 3 H); 7.29–7.32 (m, 2 H); 7.37–7.49 (m, 6 H); 7.65
(m, 3 H); 8.29 (d, J¼8.22, 1 H); 8.78 (d, J¼8.61, 2 H). ESI-MS: 464.2 ([MþH]þ ). Anal. calc. for
C29H25N3O3: C 75.14, H 5.44, N 9.07; found: C 75.37, H 5.54, N 9.38.
{5-[4-(Benzyloxy)phenyl]-4,5-dihydro-3-(4-methoxyphenyl)-1H-pyrazol-1-yl}(pyridin-4-yl)methan-
one (6d). Yield: 76%. Yellow powder. M.p. 159–1628. 1H-NMR: 3.18–3.25 (m, 1 H); 3.71–3.81 (m,
1 H); 3.85 (s, 3 H); 5.03 (s, 2 H); 5.74–5.76 (m, 1 H); 6.92–6.96 (m, 4 H); 7.24–7.27 (m, 2 H); 7.31–7.39
(m, 5 H); 7.64 (d, J¼8.79, 2 H); 7.83 (d, J¼5.85, 2 H); 8.73 (d, J¼5.67, 2 H). ESI-MS: 464.5 ([MþH]þ ).
Anal. calc. for C29H25N3O3: C 75.14, H 5.44, N 9.07; found: C 75.51, H 5.23, N 9.35.
[3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl](pyridin-3-yl)methanone (5f). Yield: 73%.
1
White powder. M.p. 168–1708. H-NMR: 3.14–3.21 (m, 1 H); 3.75–3.86 (m, 1 H); 5.78 (dd, J¼4.53,
11.73, 1 H); 7.26 (s, 1 H); 7.28–7.35 (m, 3 H); 7.40–7.48 (m, 3 H); 7.64–7.73 (m, 2 H); 8.24–8.28 (m, 3 H).
ESI-MS: 396.1 ([MþH]þ ). Anal. calc. for C21H15Cl2N3O: C 63.65, H 3.82, N 10.60; found: C 63.36, H
3.78, N 10.46.
[3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl](pyridin-4-yl)methanone (6f). Brown pow-
der. Yield: 68%. M.p. 114–1178 ([26]: 110–1128).
[5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl](pyridin-3-yl)methanone
(5g). Yield: 68%. Grey powder. M.p. 152–1548. 1H-NMR: 3.21 (dd, J¼5.31, 17.73, 1 H); 3.78–3.88 (m,
1 H); 5.76–5.82 (m, 1 H); 7.23 (d, J¼8.61, 2 H); 7.41 (d, J¼8.58, 2 H); 7.51 (d, J¼8.40, 2 H); 7.66 (d, J¼

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8.61, 2 H); 8.28 (d, J¼7.86, 1 H); 8.73 (d, J¼3.48, 2 H); 9.29 (s, 1 H). ESI-MS: 440.0 ([MþH]þ ). Anal.
calc. for C21H15BrClN3O: C 57.23, H 3.43, N 9.53; found: C 57.41, H 3.52, N 9.36.
[5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl](pyridin-4-yl)methanone
1
(6g). Yield: 61%. Brown powder. M.p. 223–2258. H-NMR: 3.17–3.24 (m, 1 H); 3.77–3.87 (m, 1 H);
5.77 (dd, J¼4.92, 11.70, 1 H); 7.22 (d, J¼8.40, 2 H); 7.40–7.44 (m, 2 H); 7.51 (dd, J¼1.83, 6.57, 2 H); 7.63
(d, J¼8.58, 2 H); 7.81 (d, J¼5.49, 2 H); 8.77 (d, J¼5.31, 2 H). ESI-MS: 440.0 ([MþH]þ ). Anal. calc. for
C21H15BrClN3O: C 57.23, H 3.43, N 9.53; found: C 57.42, H 3.31, N 9.19.
{5-[4-(Benzyloxy)phenyl]-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl}(pyridin-3-yl)methan-
one (5h). Yield: 78%. Yellow powder. M.p. 156–1588. 1H-NMR: 3.18–3.26 (m, 1 H); 3.78 (dd, J¼11.70,
17.73, 1 H); 5.04 (s, 2 H); 5.75–5.81 (m, 1 H); 6.95 (d, J¼8.79, 2 H); 7.23–7.27 (m, 2 H); 7.31–7.40 (m,
8 H); 7.65 (d, J¼8.97, 2 H); 8.26 (d, J¼5.67, 1 H); 8.70 (s, 1 H); 9.27 (s, 1 H). ESI-MS: 468.1 ([MþH]þ ).
Anal. calc. for C28H22ClN3O2: C 71.87, H 4.74, N 8.98; found: C 71.77, H 4.65, N 8.81.
{5-[4-(Benzyloxy)phenyl]-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl}(pyridin-4-yl)methan-
one (6h). Yield: 79%. Brown powder. M.p. 178–1818. 1H-NMR: 3.18–3.26 (m, 1 H); 3.78 (dd, J¼11.52,
17.76, 1 H); 5.04 (s, 2 H); 5.73–5.79 (m, 1 H); 6.96 (d, J¼8.61, 2 H); 7.23 (d, J¼7.68, 2 H); 7.31–7.41 (m,
7 H); 7.62 (d, J¼8.43, 2 H); 7.80 (s, 2 H); 8.76 (s, 2 H). ESI-MS: 468.2 ([MþH]þ ). Anal. calc. for
C28H22ClN3O2: C 71.87, H 4.74, N 8.98; found: C 71.69, H 4.57, N 8.66.
Molecular Docking. The X-ray crystal structure of the BRAF kinase domain in an active
configuration (BRAFV600E/SB-590885; Protein Data Bank (PDB) ID 2FB8), was used as the target
structure in this approach. The molecular docking procedure was performed by using CDOCKER
protocol for receptorꢀligand interactions, section of DS 3.1 [19][20].
Initially both the ligands and the receptor were pretreated. For ligand preparation, the 3D structures
of all compounds were generated with ChemBioOffice. For receptor preparation, the H-atoms were
added with the pH of the protein in the range of 6.5–8.5. CDOCKER is an implementation of a
CHARMm-based molecular-docking tool using a rigid receptor. It includes the following steps.
i) A series of ligand conformations are generated using high-temperature molecular dynamics (MD)
with different random seeds.
ii) Random orientations of the conformations are generated by translating the center of the ligand to
a specified position within the receptor active site, and conducting a series of random rotations. A
softened energy is calculated, and when it is less than a specified limit, the orientation is kept. This
process is repeated until either the desired number of low-energy orientations is obtained, or the test
times of ꢃbadꢄ orientations reached the maximum number.
iii) Each orientation is subjected to simulated annealing MD. The temp. is increased then reduced to
the target value. A final energy minimization of the ligand in the rigid receptor using non-softened
potential is performed.
iv) For each of the final pose, the CHARMm energy (interaction energy plus ligand strain) and the
interaction energy alone are figured out. The poses are sorted according to CHARMm energy, and the
top-scoring (most negative, thus favorable to binding) poses are retained. Residues around SB-590885 at
a radius of 10 ꢂ were isolated for the construction of the grid for docking screening. This radius was large
enough to include all of the residues involved in inhibitor binding. CHARMm was selected as the force
field. The molecular docking was performed with a simulated annealing method. The heating steps were
2000 with 700 of heating target temp. The cooling steps were 5000 with 300 cooling target temp. No
attempt was made to minimize the ligandꢀenzyme complex (rigid docking). After the molecular
docking, the types of interactions of the docked enzyme with ligand were analyzed. Ten molecular
docking poses saved for each ligand were ranked according to their dock score function. The pose with
the highest ꢀCDOCKER interaction energy (ꢀ ECD) was chosen as the most suitable pose.
Biological Assays. Antiproliferative Activity. These assays were conducted by using a modified
procedure as described in [27]. WM266.4 and WM1361 melanoma cells (ATCC) were cultured in
Dulbeccoꢄs modified Eagleꢄs medium (DMEM)/10% fetal bovine serum (FBS), in 5% CO2, H2O-sat.
atmosphere at 378. Cell suspensions (10,000 mlꢀ1) were prepared and, at 100 ml/well, dispensed into 96-
well plates (Costar) to give 1,000 cells/well. The plates were returned to the incubator for 24 h to allow the
cells to reattach. The compounds were initially prepared at 20 mm in DMSO. Aliquots (200 ml) were
diluted into 20 ml of culture medium to afford a 200 mm soln., and ten serial dilutions were prepared in

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
131
triplicate. Aliquots (100 ml) of each dilution were added to the wells, giving doses ranging from 100 to
0.005 mm. After further incubation at 378 for 24 h in a humidified atmosphere with 5% CO2, the cell
viability was assessed by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) reduction assay according to the manufacturerꢄs instructions (Sigma). The absorbance
at 590 nm was recorded with a LX300 Epson Diagnostic microplate reader. Then, GI50 value was
calculated using SPSS 13.0 software.
Kinase Assay. The BRAF enzyme assay was conducted by using a modified procedure as described
by Dietrich et al. [28]. This V600E mutant BRAF kinase assay was performed in triplicate for each tested
compound. Briefly, 7.5 ng of Mouse Full-Length GST-tagged BRAFV600E (Invitrogen, PV3849) was
preincubated at r.t. for 1 h with 1 ml of drug and 4 ml of assay dilution buffer. The kinase assay was
initiated, when a soln. (5 ml) containing 200 ng of recombinant human full length, N-terminal His-tagged
MEK1 (Invitrogen), 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 r.t. for 25 min and was then quenched with 5 ml of
fivefold protein denaturing buffer (LDS) soln. Protein was further denatured by heating for 5 min at 708.
Ten ml of each soln. was loaded into a 15-well, 4–12% precast NuPage gel (Invitrogen) and run at 200 V,
and, upon completion, the front, which contained excess hot ATP, was cut from the gel and discarded.
The gel was then dried and developed onto a phosphor screen. A soln. that contained no active enzyme
was used as negative control, and a soln. without inhibitor was used as positive control.
Detection of the effect of compounds on cell-based pERK1/2 activity in WM266.4 cells was
performed by using ELISA kits (Invitrogen) according to the manufacturerꢄs instructions.
QSAR.The QSAR study was performed with the software of DS 3.1 [19]. The training sets were
composed of 21 inhibitors with the corresponding pIC50 (ꢀ logIC50) values, which were transformed from
the obtained IC50 values (in mollꢀ1), and test sets comprised five compounds with data sets as listed in
Table 6. All the definitions of the descriptors can be seen in the Help of DS 3.1 software, and they were
calculated according to the QSAR protocol of DS 3.1. For the development of the QSAR models, the
statistical techniques used were GFA and PLS.
In this study, we collected more than 60 parameters of the ligand physiochemical properties. QSAR
Software of DS 3.1 provides various scoring functions such as r2, r2(adj), r2(pred), and Friedman LOF
values to evaluate the quality of the obtained equations. An ideal QSAR model should be robust enough
to be capable of enable accurate and reliable predictions of the biological activities of new ligands. Thus,
QSAR models constructed from the training set should be validated with some statistical indexes in order
to check the predictive ability of the developed models. The statistical qualities of the obtained models
were evaluated by the parameters such as squared correlation coefficient (r2), adjusted squared
correlation coefficient (r2(adj)), and Friedman LOF [23].
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Received April 9, 2014