Design, synthesis and biological evaluation of novel (E)-α- benzylsulfonyl chalcone derivatives as potential BRAF inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.02.007

Activating mutations in the BRAF serine/threonine kinase are found in more than 70% of human melanomas, >90% of which are BRAF^V600E. It provides new therapeutic opportunities in malignant melanoma. In silico and in vitro screening of our compound collection has identified Hit 2 as BRAF ^V600E inhibitor. Based on its structure, a series of novel (E)-α-benzylsulfonyl chalcone derivatives (13-40) were designed and synthesized. Compound 38 exhibited the most potent inhibitory activity with an IC₅₀ value of 0.17 μM for BRAF^V600E and GI₅₀ value of 0.52 μM for mutant BRAF-dependent cells. The results of cell based pERK activity and cellular selectivity suggested that those compounds could selectively inhibit proliferation of mutant BRAF-dependent melanoma cell line through inhibition of oncogenic BRAF.

Full text of DOI:10.1016/j.ejmech.2012.02.007

European Journal of Medicinal Chemistry 50 (2012) 288e295
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel (E)-a-benzylsulfonyl chalcone
derivatives as potential BRAF inhibitors
*
Qing-Shan Li, Cui-Yun Li, Xiang Lu, Hui Zhang, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Activating mutations in the BRAF serine/threonine kinase are found in more than 70% of human mela-
nomas, >90% of which are BRAFV600E. It provides new therapeutic opportunities in malignant melanoma.
In silico and in vitro screening of our compound collection has identified Hit 2 as BRAFV600E inhibitor.
Received 17 September 2011
Received in revised form
2 February 2012
Accepted 3 February 2012
Available online 11 February 2012
Based on its structure, a series of novel (E)-
and synthesized. Compound 38 exhibited the most potent inhibitory activity with an IC50 value of
0.17 M for mutant BRAF-dependent cells. The results of cell
M for BRAFV600E and GI50 value of 0.52
a-benzylsulfonyl chalcone derivatives (13e40) were designed
m
m
based pERK activity and cellular selectivity suggested that those compounds could selectively inhibit
proliferation of mutant BRAF-dependent melanoma cell line through inhibition of oncogenic BRAF.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
BRAF
Mutant
Inhibitors
Melanoma
Virtual screening
1. Introduction
thyroid, colorectal, and ovarian [10e12]. Melanomas in particular
show a high incidence of BRAF mutations, the most common being
The mitogen-activated protein kinase (MAPK) signal trans-
duction pathway regulates 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]. V-RAF murine sarcoma viral oncogene homolog B1 (termed as
BRAF) [5] is a serine/threonine specific protein kinase that has long
been viewed as key players in the MAPK pathway [6,7]. Under
normal circumstances, BRAF is activated downstream of receptors
in the cell membrane in a RAS small G-protein dependent manner.
It then phosphorylates and activates the protein kinase MEK and
ERK orderly, regulating gene expression and controlling how cells
respond to extracellular signals [6,8].
a substitution of glutamic acid for valine at position 600 (V600E).
This substitution, which lies within the kinase activation loop,
essentially precludes the need for phosphorylation and results in
constitutively active BRAF that displays approximately a 500-fold
increase in kinase activity over the wild-type isoform [13e15].
Oncogenic BRAFV600E bypasses the requirement for upstream
regulation by phosphorylation that results in a MAPK signaling
pathway that is constitutively activated in the absence of extra-
cellular growth factor signals, inducing uncontrolled cell prolifer-
ation, increased cell survival, and tumor progression. Inhibition of
mutant BRAF signaling, through either direct inhibition of the
enzyme or inhibition of MEK, has been demonstrated preclinical to
inhibit tumor growth [16e18]. Very recently, treatment of BRAF
mutant melanoma patients with a selective BRAF inhibitor has
resulted in antitumor activity [19,20], validating mutant BRAF as
a therapeutic target and offering opportunities for anti- melanoma
drug development.
The interest in developing new drugs targeting the MAP kinase
pathway has increased considerably, and inhibitors are currently
being tested in preclinical and clinical trials [17]. The most intensely
studied, sorafenib, was used in the treatment of advanced renal
cancer, proved to be active against BRAF in cellular assays [21].
Several BRAF inhibitors that were identified through solution
screening have been described in the literature [22,23]. The avail-
ability of the crystal structure of the BRAF protein kinase [23] now
Cancers arise owing to the accumulation of mutations in critical
genes that alter normal programmes of cell proliferation, differ-
entiation and death [9]. Clinically, BRAF has a high rate of activating
mutation in a number of cancers, such as melanoma, papillary
Abbreviations: BRAFhomolog B1, V-RAF murine sarcoma viral oncogene; 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.
* Corresponding author. Tel./ fax: þ86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.007

Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
289
provides an opportunity for utilizing the virtual screening strategy to
identify oncogenic BRAF inhibitors from our compound collection.
Based on calculated binding abilities with BRAF structure
which generated from the in silico screening, five hits (Table 1)
were selected for initial protein kinase inhibitory activities scree-
ning. Among them (E)-2-(Benzylsulfonyl)-3-(4-fluorophenyl)-1-
phenylprop-2-en-1-one (Hit 2) exhibited the best BRAFV600E inhib-
crystal 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 6 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 kinase ATP-binding pocket [26]. The binding affinity of
candidate compounds was evaluated by the binding free energies
itoryability, with IC50 valueof 1.04
BRAFV600E inhibitors, various substitutions were introduced to the
aromatic systems of (E)- -benzylsulfonyl chalcone skeleton. Then
twenty-eight (E)- -benzylsulfonyl chalcone derivatives were
m
M. Inorder to obtain more potent
(
D
Gb, kcal/mol, Binding Energy ¼ Intermolecular Energy þ Internal
Energy þ Torsional Energy - Unbound Extended Energy). The
compounds which revealed the highest binding affinities were
owned lowest predicted binding free energies. After screening from
our compound collection, on the basis of their binding affinities
with the ATP-binding pocket of BRAF, five initially hits were
selected for protein kinase inhibitory activity screening. Their
structure, predicted binding free energies and IC50 BRAFV600E were
a
a
synthesized and evaluated as oncogenic BRAFV600E inhibitors, their
antiproliferative activitiesagainst BRAF mutant melanoma cells were
also measured. Some compounds exhibited favorable activities
deserve further research as therapeutic of BRAF mutant melanoma.
summarized in Table 1; the results indicated that (E)-
fonyl chalcone derivative Hit 2 exhibited optimal activity with IC50
value of 1.04 M. However, the N-nitrosoureas derivative Hit 1,
a-benzylsul-
2. Results and discussion
m
thiazolidinyl derivatives Hit 3 and Hit 4, and luteolin derivative Hit
5, those compounds owned in silico calculated binding free ener-
gies comparable to Hit 2, did not shown favorable protein kinase
inhibitory activities. To obtain more direct insight into the binding
2.1. Virtual screening
In this procedure, the docking program AutoDock 4.0 was used
as the in silico screening tool [24,25]. The BRAFV600E/SB-590885
Table 1
Initially virtual screening and protein kinase inhibitory activities screening results.
Compound
Structure
Binding free energies
DGb, kcal/mol
BRAFV600E IC50
, mM
Hit 1
ꢀ21.87
8.61, 10.92b
Hit 2
ꢀ21.75
ꢀ21.36
1.04a
Hit 3
>25b
Hit 4
ꢀ21.02
>25b
Hit 5
ꢀ19.49
10.63, 14.77b
a
Result values are shown as mean values of three independent determinations.
Result values are shown as mean values of two independent determinations.
b

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Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
Table 2
mode of Hit 2 to mutant BRAF, the binding model of Hit 2 docked
into the active site of BRAF is shown in Fig. 1, which was stimulated
by using AutoDock 4.0. Visual inspection of the pose of Hit 2 into
BRAF binding site revealed that this candidate BRAF inhibitor was
tightly embedded into the ATP-binding pocket. As shown in Fig. 1b,
the model suggests that extensive hydrophobic interactions are
formed between Hit 2 and residues Val 471, Ala 481, Lys 483, Glu
501, Leu 514, Ile 527, Thr 529, Trp 531, Cys 532, Phe 583 and Asp 594
of the ATP-binding pocket of BRAF kinase. Furthermore, in addition
BRAFV600E kinase and cellular activity of (E)-
a
-Benzylsulfonyl chalcone analogs
13e40.
Compound
R1
R2
IC50 GI50
BRAFV600E
(
m
M)a
WM266.4 (m
M)a
13 (Hit 2)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
H
H
H
H
H
4-F
4-NO2
4-Br
4-Cl
4-CF3
2-Cl
1.04
0.73
1.93
1.47
2.44
7.3
1.07
0.54
1.41
1.58
3.44
6.54
6.7
4.37
0.71
1.26
4.27
0.6
0.51
3.44
3.58
2.03
3.89
3.36
4.70
2.06
5.84
1.95
0.79
1.84
1.32
0.52
0.67
4.75
8.3
to
p
e
p
stacking interactions with Trp 531 and Phe 583, which were
-benzylsulfonyl chal-
H
2-NO2
2-NO2
4-Cl
4-Br
4-CF3
4-F
4-NO2
2-Cl
4-Br
4-NO2
4-Cl
4-CF3
2-NO2
4-F
2-Cl
4-CF3
4-Cl
2-NO2
4-Br
4-NO2
4-F
3.6
established by aromatic ring A and C of (E)-
a
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3.58
1.06
2.01
3.67
0.79
0.57
4.18
2.42
1.57
1.92
2.94
5.95
1.86
6.6
3.01
0.63
1.44
1.7
0.17
0.29
3.79
0.06
cone skeleton, catione
p
interaction was also found between the
face of an electron-rich
p system of aromatic ring B and Lys 483.
2.2. Chemistry
On the basis of the positive results obtained with (E)-
a-ben-
zylsulfonyl chalcone derivative Hit 2, the (E)- -benzylsulfonyl
a
chalcone skeleton could serve as a promising scaffold for devel-
oping new kinase inhibitors of V600E mutant BRAF. A serial of
novel (E)-a-benzylsulfonyl chalcones prepared as described else-
where with some modifications [27], the general pathway outlined
in Scheme 1. Several substitution were introduced into the A ring
and B ring of sulfonyl chalcone skeleton and then 28 (E)-a-ben-
zylsulfonyl chalcones were synthesized in four steps. For the
synthesis of compounds 13e40, phenacyl bromides 1e4 were
synthesized from acetophenones and then reacted with benzyl
mercaptan to produce phenacyl benzylsulfides 5e8. Oxidation of
phenacyl benzylsulfides 5e8 with 30% hydrogen peroxide in the
presence of glacial acetic acid gave phenacyl benzylsulfonyl
compounds 9e12. Knoevenagel condensation of 9e12 with benz-
aldehyde in glacial acetic acid in the presence of ammonium
38
39
40
2-Cl
Sorafenibb
a
Result values are presented from at least three separated experiments.
Used as a positive control.
b
acetate yielded (E)-a-benzylsulfonyl chalcones 13e40. All the
(E)-a-benzylsulfonyl chalcone derivatives were synthesized for the
first time and all the compounds were fully characterized by 1H
NMR, ESI-MS and elemental analysis.
2.3. Bioassays
The biological activities of the 28 compounds were determined
using two assays: (a) the BRAFV600E inhibitory activities of these
synthesized compounds were determined as their ability to inhibit
BRAF mediated MEK phosphorylation (IC50 BRAFV600E); (b) the
determination of the growth inhibition in WM266.4 human
melanoma cells expressing V600E mutant BRAF with MTT assay
(GI50 WM266.4). The data are summarized in Table 2.
A number of synthesized analogs displayed potent BRAFV600E
kinase inhibitory activities comparable to positive control Sor-
afenib. In general, six compounds (14, 24, 25, 35, 38 and 39)
demonstrated IC50 values being less than Hit 2. Compound 38
exhibited the most potential inhibitory activity in tumor growth
inhibition (IC50 ¼ 0.17
mM). Compounds those contain electronic-
withdrawing halogen substituents (Cl or Br) on phenyl ring A
showed slightly more potent inhibitory activities but did not
improved significantly. Compounds bearing the same substituents
Table 3
BRAFWT cellular activity and BRAFV600E cell based pERK activity of selected
compounds.
Compound
14a
38
39
GI50 WM1361 (
m
M)
4.24, 5.16
1.37, 1.63
3.56
1.23
3.1
1.78
IC50 pERK (
mM)
a
Fig. 1. Hit 2 bound into ATP pocket of BRAF. Both side chains of important active site
Result values are presented from two separated experiments. Others were
presented from three separated experiments.
amino acids and interaction between ligand and receptor are shown.

Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
291
Scheme 1. Synthesis route of (E)-
a
-benzylsulfonyl chalcone derivatives 13e40. R1 ¼ 4-Br, 4-Cl, 4-Br and 4-OMe; R2 ¼ 4-F, 4-NO2, 4-Br, 4-Cl, 4-CF3, 2-NO2 and 2-Cl. Reagents and
conditions: (i) bromine, methanol, 0 ꢁC, 5e6 h; (ii) benzyl mercaptan, sodium hydroxide, methanol, rt, 2 h; (iii) 30% hydrogen peroxide, acetic acid, 70 ꢁC, 2 h; (iiii) benzaldehyde,
ammonium acetate, acetic acid, reflux, overnight.
on phenyl ring A exhibited distinct kinase inhibitory activity due to
the difference of the substituents on the ring B. The inhibitory
activity of compounds with different substituents on ring B
increased in the following order: 2-Cl < 2-NO2 < 4-CF3 < 4-Br < 4-
Cl < 4-F < 4-NO2. This result indicated that the strongly para
electronic-withdrawing substituents on phenyl ring B were bene-
ficial for the activity. It is worth mentioning that, compounds 14, 25,
28 and 38 which with para-nitro substituents on phenyl ring B of
benzylsulfonyl chalcone skeleton exhibited better potent BRAFV600E
inhibitory activity, which suggested that para-nitro substituents on
ring B was beneficial for kinase inhibitory activities to BRAF. These
results clearly showed that the modification of the phenyl ring B
can greatly enhance the activity of the compounds.
In order to evaluate the toxic nature of compound 38, acute oral
toxicity test was introduced. The tested animals appeared normal
immediately after administration and did not exhibit any indication
of acute toxicity for 14 days afterward. The body weights in treated
mice were similar to that of the control mice dosed with an equal
volume of vehicle. The result indicated that compound 38 was
nontoxic.
3. Conclusion
Virtual screening of our compound collection resulted in the
identification of (E)-a-benzylsulfonyl chalcone derivative Hit 2,
which served as starting point for the design of new V600E mutant
BRAF inhibitors. Then a series of novel analogs of hits have been
designed and prepared based on the structure of hit compound.
Some of the synthesized compounds displayed potent BRAFV600E
inhibitory activities. Compound 38 displayed the most potent
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 of human cancers, particularly
melanoma. The synthesized (E)-a-benzylsulfonyl chalcone deriva-
tives 13e40 were evaluated for antiproliferative activities against
WM266.4 human melanoma cell line expressing V600E mutant
BRAF. As illustrated in Table 2, all the compounds displayed IC50
inhibitory activity, with IC50 value of 0.17
value of 0.52 M for mutant BRAF-dependent WM266.4 cells,
m
M for BRAFV600E and GI50
m
comparable to the positive control Sorafenib. Selected compounds
which exhibited good inhibitory activities were evaluated their
BRAFWT cellular activity and BRAFV600E cell based pERK activity, the
results suggested those compounds could selectively inhibit
proliferation of mutant BRAF-dependent melanoma cell line
through inhibition of oncogenic BRAF. Above all, the results ob-
tained from this study suggest that benzylsulfonyl chalcone skel-
eton may serve as a novel scaffold for the further development of
more potent and selective BRAFV600E inhibitors which use as
mutant BRAF-dependent melanoma therapeutic agents.
ranging from 0.51 to 6.7 mM. We observed that these compounds,
which have potent BRAFV600E kinase inhibitory activities, displayed
corresponding optimal cytotoxic activities against mutant BRAF-
dependent WM266.4 cells. These results indicated that these
compounds were potential anti-melanoma agents by inhibiting
BRAF enzyme, and further validated that V600E mutant BRAF was
an effective target of melanoma therapy.
In order to provide evidence for the specificity of the
compounds for mutant BRAF-driven cell proliferation, the anti-
proliferative activities for the BRAF wild-type WM1361 melanoma
cell line (GI50 WM1361) that did not express mutant BRAF was
determined for selected compounds (14, 38 and 39) and these
results 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 were all up to dozens of times.
Furthermore, for the sake of assess the inhibition of the target
signaling pathway (RAF-ERK) by selected compounds, the phos-
phorylation level of extracellular signal-regulated kinase (ERK) was
measured in a cell based assay (IC50 pERK). As shown in Table 3,
combined with their GI50 WM266.4, selected compounds exhibited
close correlation between the inhibition of both ERK phosphory-
lation and cell proliferation in a BRAF mutant cell line. These
positive results strongly support those designed and synthesized
benzylsulfonyl chalcone derivatives possess antiproliferative
activities against BRAF melanoma cell line through the selective
inhibition of oncogenic BRAF.
4. Materials and methods
4.1. Chemistry
4.1.1. Chemistry general
All chemicals (reagent grade) used were purchased from Aldrich
(USA). Separation of the compounds by column chromatography
was carried out with silica gel 60 (200e300 mesh ASTM, E. Merck,
Germany). The quantity of silica gel used was 50e100 times the
weight charged on the column. Thin layer chromatography (TLC)
was run on the silica gel coated aluminum sheets (silica gel 60
GF254, E. Merck, Germany) and visualized in ultraviolet (UV) light
(254 nm). Melting points (uncorrected) were determined with an
XT4 MP apparatus (Taike Corp. Beijing, China). 1H and 13C NMR
spectra (300 MHz) were recorded on a 1H-Varian-Mercury-300
spectrometer at 25 ꢁC, using tetramethylsilane (TMS) as the

292
Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
internal standard. ESI-MS were recorded with a Mariner System
5304 mass spectrometer. Elementary analyses were performed on
a CHN-O-Rapid instrument within ꢂ0.4% of the theoretical values.
(m, 3H); 7.89e7.91 (m, 2H); 8.03 (d, J ¼ 8.79 Hz, 2H). MS (ESI): 381.1
([M þ H]þ). Anal. Calcd for C22H17FO3S: C, 69.46; H, 4.50. Found: C,
69.23; H, 4.16.
4.1.2. Synthesis of 2-benzylthio-1-phenylethanone (5e8)
4.1.4.5. (E)-2-(Benzylsulfonyl)-1-phenyl-3-(4-(trifluoromethyl)
Bromine (3 mL, 60 mmol) was slowly added to a stirred solution
of acetophenone (60 mmol) in methanol (40 mL) and temperature
of the reaction mixture maintained below 20 ꢁC. After addition of
bromine, the contents were stirred at ice bath for further 1e2 h.
Then the contents were poured on to ice-water and the solid ob-
tained was collected by filtration. The crude product was recrys-
phenyl)prop-2-en- 1-one (17). Yellow solid, yield: 77%. m.p.
92e95 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 4.62 (s, 2H); 7.21e7.57
(m, 11H); 7.75 (d, J ¼ 8.04 Hz, 2H); 7.90e7.93 (m, 2H); 8.21 (d,
J ¼ 8.04 Hz, 2H). MS (ESI): 431.1 ([M þ H]þ). Anal. Calcd for
C23H17F3O3S: C, 64.18; H, 3.98. Found: C, 63.94; H, 4.13.
tallized from ethanol to afford pure
(1e4).
a
-bromo phenylethanone
4.1.4.6. (E)-2-(Benzylsulfonyl)-3-(2-chlorophenyl)-1-phenylprop-2-
en-1-one (18). Crystal, yield: 82%. m.p. 130 ꢁC 1H NMR (300 MHz,
To a solution of sodium hydroxide (30 mmol) in methanol
(60 mL) benzyl mercaptan (30 mmol) was added and the contents
were stirred at room temperature for 10 min. To this reaction
CDCl3, d ppm): 4.69 (s, 2H); 6.95e6.99 (m, 2H); 7.09e7.15 (m, 1H);
7.23e7.35 (m, 6H); 7.43e7.48 (m, 1H); 7.56e7.59 (m, 2H); 7.73 (s,
1H); 7.80e7.83 (m, 2H). MS (ESI): 397.0 ([M þ H]þ). Anal. Calcd for
C22H17ClO3S: C, 66.58; H, 4.32. Found: C, 66.81; H, 4.58.
mixture 20 mmol a-Bromo phenylethanone (1e4) was added and
stirred for further 1e2 h. After completion of the reaction, the
contents were cooled, poured on to ice-water and the solid obtained
was collected by filtration. The crude product was recrystallized
from isopropanol to get pure 2-benzylthio -1-phenylethanone
(5e8).
4.1.4.7. (E)-2-(Benzylsulfonyl)-3-(2-nitrophenyl)-1-phenylprop-2-
en-1-one (19). White solid, yield: 73%. m.p. 173e176 ꢁC 1H NMR
(300 MHz, CDCl3, d ppm): 4.69 (s, 2H); 6.96e7.21 (m, 5H); 7.23e7.59
(m, 7H); 7.71e7.96 (m, 3H). MS (ESI): 408.1 ([M þ H]þ). Anal. Calcd
for C22H17NO5S: C, 64.85; H, 4.21; N, 3.44. Found: C, 65.07; H, 4.03;
N, 3.58.
4.1.3. Synthesis of 2- benzylsulfonyl -1-phenylethanone (9e12)
To a mixture of 5e8 (30 mmol) in glacial acetic acid (60 mL), 30%
hydrogen peroxide (36 mL) was added and the contents were
refluxed for 1 h. After completion of the reaction, the cooled
reaction contents were poured on to ice-water and the separated
solid was filtered and dried. The crude product was recrystallized
from methanol to obtain pure sample of 9e12.
4.1.4.8. (E)-2-(4-Bromobenzylsulfonyl)-3-(2-nitrophenyl)-1-phenyl-
prop-2-en-1- one (20). White solid, yield: 90%. m.p. 181e184 ꢁC 1H
NMR (300 MHz, CDCl3,
7.30e7.45 (m, 7H); 7.59e7.67 (m, 4H); 7.83 (s, 1H); 8.01e8.04 (m,
1H). 13C NMR (300 MHz, CDCl3,
ppm): 61.3, 125.2, 128.1, 128.2,
128.7, 129.1, 130.3, 130.7, 131.1, 131.2, 132.1, 133.9, 134.6, 137.3, 143.4,
146.6, 191.2.MS (ESI): 485.9 ([M
H]þ). Anal. Calcd for
d ppm): 4.69 (s, 2H); 6.96e6.99 (m, 1H);
d
4.1.4. General procedure for the preparation of (E)-a-benzylsulfonyl
chalcones (13e40)
þ
C22H16BrNO5S: C, 54.33; H, 3.32; N, 2.88. Found: C, 54.57; H, 3.14;
N, 2.99.
A
mixture of 2-Benzylsulfonyl-1-phenylethanone (9e12)
(1 mmol), benzaldehyde (1 mmol) and ammonium acetate
(2.5 mmol) in glacial acetic acid (5 mL) was refluxed overnight.
After completion of the reaction, the mixture was evaporated under
reduced pressure to give a residue. The residue was purified by
silica gel chromatography (PE/EtOAc ¼ 10:1) and recrystallized
from isopropanol to afford pure compounds 13e40.
4.1.4.9. (E)-2-(4-Bromobenzylsulfonyl)-3-(4-chlorophenyl)-1-phe-
nylprop-2-en-1-one (21). White solid, yield: 72%. m.p. 113e115 ꢁC
1H NMR (300 MHz, CDCl3,
d
ppm): 4.63 (s, 2H); 7.03e7.06 (m, 2H);
7.17e7.19 (m, 2H); 7.30e7.33 (m, 4H); 7.50e7.55 (m, 4H);
7.76e7.79 (m, 2H). 13C NMR (300 MHz, CDCl3,
ppm): 61.1,
d
128.1, 128.6, 128.9, 129.0, 129.6, 129.8, 131.1, 131.2, 134.8,
135.2, 135.6, 137.3, 143.1, 193.2. MS (ESI): 475.0 ([M þ H]þ).
Anal. Calcd for C22H16BrClO3S: C, 55.54; H, 3.39. Found: C, 55.26;
H, 3.33.
4.1.4.1. (E)-2-(Benzylsulfonyl)-3-(4-fluorophenyl)-1-phenylprop-2-
en-1-one (13, Hit 2). Crystal, yield: 76%. m.p. 129e131 ꢁC 1H NMR
(300 MHz, CDCl3, d ppm): 4.61 (s, 2H); 6.83e6.89 (m, 2H); 7.10e7.15
(m, 2H); 7.29e7.41 (m, 6H); 7.51e7.57 (m, 3H); 7.91e7.94 (m, 2H).
MS (ESI): 381.1 ([M þ H]þ). Anal. Calcd for C22H17FO3S: C, 69.46; H,
4.50. Found: C, 69.23; H, 4.16.
4.1.4.10. (E)-2-(4-Bromobenzylsulfonyl)-3-(4-bromophenyl)-1-phe-
nylprop-2- en-1-one (22). White solid, yield: 75%. m.p. 107e110 ꢁC
1H NMR (300 MHz, CDCl3,
d
ppm): 4.63 (s, 2H); 6.97 (d, J ¼ 8.61 Hz,
4.1.4.2. (E)-2-(Benzylsulfonyl)-3-(4-nitrophenyl)-1-phenylprop-2-
2H); 7.24e7.36 (m, 8H); 7.50e7.55 (m, 4H); 7.77 (d, J ¼ 8.58 Hz, 2H).
MS (ESI): 518.9 ([M þ H]þ). Anal. Calcd for C22H16Br2O3S: C, 50.79;
H, 3.10. Found: C, 50.98; H, 2.92.
en-1-one (14). Yellow powder, yield: 81%. m.p. 154e157 ꢁC 1H NMR
(300 MHz, CDCl3, d ppm): 4.63 (s, 2H); 7.29e7.42 (m, 8H); 7.54e7.56
(m, 3H); 7.89e7.92 (m, 2H); 8.01e8.03 (m, 2H). MS (ESI): 408.1
([M þ H]þ). Anal. Calcd for C22H17NO5S: C, 64.85; H, 4.21; N, 3.44.
Found: C, 64.59; H, 4.00; N, 3.68.
4.1.4.11. (E)-2-(4-Bromobenzylsulfonyl)-1-phenyl-3-(4-(trifluoro-
methyl)phen-yl)prop-2-en-1-one (23). Yellow solid, yield: 53%.
m.p. 113e115 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 4.62 (s, 2H);
4.1.4.3. (E)-2-(Benzylsulfonyl)-3-(4-bromophenyl)-1-phenylprop-2-
7.02e7.31 (m, 8H); 7.51e7.78 (m, 4H); 7.97e8.03 (m, 2H). MS (ESI):
509.0 ([M þ H]þ). Anal. Calcd for C23H16BrF3O3S: C, 54.24; H, 3.17.
Found: C, 54.51; H, 3.36.
en-1-one (15). Yellow powder, yield: 70%. m.p. 138e140 ꢁC 1H NMR
(300 MHz, CDCl3,
d ppm): 4.60 (s, 2H); 6.97e7.00 (m, 2H);
7.24e7.42 (m, 8H); 7.51e7.58 (m, 3H); 7.91e7.93 (m, 2H). MS (ESI):
441.0 ([M þ H]þ). Anal. Calcd for C22H17BrO3S: C, 59.87; H, 3.88.
Found: C, 59.63; H, 3.98.
4.1.4.12. (E)-2-(4-Bromobenzylsulfonyl)-3-(4-fluorophenyl)-1-phe-
nylprop-2- en-1-one (24). White solid, yield: 69%. m.p.129e131 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 4.61 (s, 2H); 6.89e7.13 (m, 4H);
4.1.4.4. (E)-2-(Benzylsulfonyl)-3-(4-chlorophenyl)-1-phenylprop-2-
7.25e7.53 (m, 6H); 7.81e8.04 (m, 4H). MS (ESI): 459.9 ([M þ H]þ).
Anal. Calcd for C22H16BrFO3S: C, 57.53; H, 3.51. Found: C, 57.41;
H, 3.60.
en-1-one (16). Yellow solid, yield: 70%. m.p. 126e128 ꢁC 1H NMR
(300 MHz, CDCl3, d ppm): 4.63 (s, 2H); 7.29e7.42 (m, 8H); 7.54e7.56

Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
293
4.1.4.13. (E)-2-(4-Bromobenzylsulfonyl)-3-(4-nitrophenyl)-1-phe-
6.89e6.97 (m, 2H); 7.09e7.20 (m, 2H); 7.43e7.79 (m, 7H); 7.81e7.85
(m, 1H); 7.91e7.94 (m, 2H). MS (ESI): 465.1 ([M þ H]þ). Anal. Calcd
for C23H16ClF3O3S: C, 59.42; H, 3.47. Found: C, 59.66; H, 3.29.
nylprop-2-en-1-one (25). Yellow solid, yield: 66%. m.p. 145e147 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 4.63 (s, 2H); 7.05e7.21 (m, 4H);
7.29e7.59 (m, 7H); 7.62e7.91 (m, 3H). MS (ESI): 485.1 ([M þ H]þ).
Anal. Calcd for C22H16BrNO5S: C, 54.33; H, 3.32; N, 2.88. Found: C,
54.09; H, 3.27; N, 3.03.
4.1.4.23. (E)-2-(4-Chlorobenzylsulfonyl)-3-(4-chlorophenyl)-1-phen-
ylprop-2- en-1-one (35). White solid, yield: 79%. m.p. 137e140 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 4.58 (s, 2H); 6.96e6.99 (d,
4.1.4.14. (E)-2-(4-Bromobenzylsulfonyl)-3-(2-chlorophenyl)-1-phe-
J ¼ 8.58 Hz, 2H); 7.24e7.38 (m, 8H); 7.50e7.53 (m, 2H); 7.84e7.87
(d, J ¼ 8.58 Hz, 2H). MS (ESI): 431.1 ([M þ H]þ). Anal. Calcd for
C22H16Cl2O3S: C, 61.26; H, 3.74. Found: C, 61.13; H, 3.80.
nylprop-2- en-1-one (26). White solid, yield: 58%. m.p. 131e133 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 4.60 (s, 2H); 6.85e6.91 (m, 2H);
7.03e7.11 (m, 2H); 7.21e7.73 (m, 10H). MS (ESI): 474.8 ([M þ H]þ).
Anal. Calcd for C22H16BrClO3S: C, 55.54; H, 3.39. Found: C, 55.43;
H, 3.21.
4.1.4.24. (E)-2-(4-Chlorobenzylsulfonyl)-3-(2-nitrophenyl)-1-phenyl-
prop-2- en-1-one (36). Yellow solid, yield: 65%. m.p. 159e161 ꢁC 1H
NMR (300 MHz, CDCl3,
d
ppm): 4.61 (s, 2H); 6.97 (d, J ¼ 8.6 Hz, 2H);
4.1.4.15. (E)-3-(4-Bromophenyl)-2-(4-methoxybenzylsulfonyl)-1-
7.23e7.41 (m, 9H); 7.81e7.86 (m, 3H). MS (ESI): 442.1 ([M þ H]þ).
Anal. Calcd for C22H16ClNO5S: C, 59.80; H, 3.65; N, 3.17. Found: C,
59.64; H, 3.05; N, 3.43.
phenylprop- 2-en-1-one (27). White solid, yield: 65%. m.p.
127e129 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 3.85 (s, 3H); 4.63 (s,
2H); 6.97e7.02 (m, 2H); 7.12e7.21 (m, 5H); 7.23e7.45 (m, 3H);
7.59e7.71 (m, 4H). MS (ESI): 471.1 ([M þ H]þ). Anal. Calcd for
C23H19BrO4S: C, 58.61; H, 4.06. Found: C, 58.46; H, 4.21.
4.1.4.25. (E)-3-(4-Bromophenyl)-2-(4-chlorobenzylsulfonyl)-1-phenyl-
prop-2- en-1-one (37). White solid, yield: 56%. m.p. 131e133 ꢁC 1H
NMR (300 MHz, CDCl3,
d ppm): 4.63 (s, 2H); 6.83e6.91 (m, 2H);
4.1.4.16. (E)-2-(4-Methoxybenzylsulfonyl)-3-(4-nitrophenyl)-1-phe-
7.21e7.49 (m, 10H); 7.61e7.65 (m, 2H). MS (ESI): 474.9 ([M þ H]þ).
Anal. Calcd for C22H16BrClO3S: C, 55.54; H, 3.39. Found: C, 55.71;
H, 3.45.
nylprop-2- en-1-one (28). Yellow solid, yield: 78%. m.p. 163e165 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 3.85 (s, 3H); 4.61 (s, 2H); 7.11e7.15
(m, 2H); 7.27e7.36 (m, 6H); 7.57e7.59 (m, 4H); 7.91e8.03 (m, 2H). MS
(ESI): 438.1 ([M þ H]þ). Anal. Calcd for C23H19NO6S: C, 63.15; H, 4.38;
N, 3.20. Found: C, 62.97; H, 4.50; N, 3.12.
4.1.4.26. (E)-2-(4-Chlorobenzylsulfonyl)-3-(4-nitrophenyl)-1-phenyl-
prop-2- en-1-one (38). Yellow solid, yield: 84%. m.p. 104e107 ꢁC 1H
NMR (300 MHz, CDCl3,
d ppm): 4.61 (s, 2H); 6.83e6.89 (m, 2H);
4.1.4.17. (E)-3-(4-Chlorophenyl)-2-(4-methoxybenzylsulfonyl)-1-
7.10e7.15 (m, 2H); 7.29e7.41 (m, 6H); 7.51e7.60 (m, 2H); 7.91e7.94
(m, 2H). MS (ESI): 442.1 ([M þ H]þ). Anal. Calcd for C22H16ClNO5S:
C, 59.80; H, 3.65. Found: C, 59.67; H, 3.72.
phenylprop-2-en-1-one (29). White solid, yield: 54%. m.p.
112e113 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 3.83 (s, 3H); 4.60 (s,
2H); 7.15e7.77 (m, 12H); 7.91e8.01 (m, 2H). MS (ESI): 427.1
([M þ H]þ). Anal. Calcd for C23H19ClO4S: C, 64.71; H, 4.49. Found: C,
64.61; H, 4.26.
4.1.4.27. (E)-2-(4-Chlorobenzylsulfonyl)-3-(4-fluorophenyl)-1-phen-
ylprop-2- en-1-one (39). Yellow solid, yield: 42%. m.p. 123e125 ꢁC
1H NMR (300 MHz, CDCl3,
d
ppm): 4.59 (s, 2H); 6.99 (d, J ¼ 8.6 Hz,
4.1.4.18. (E)-2-(4-Methoxybenzylsulfonyl)-1-phenyl-3-(4-(trifluorom-
ethyl)ph-enyl)prop-2-en-1-one (30). Yellow solid, yield: 60%. m.p.
2H); 7.21e7.89 (m, 12H). MS (ESI): 415.1 ([M þ H]þ). Anal. Calcd for
C22H16ClFO3S: C, 63.69; H, 3.89. Found: C, 63.45; H, 4.03.
107e110 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 3.85 (s, 3H); 4.61 (s,
2H); 6.97e7.13 (m, 3H); 7.21e7.61 (m, 7H); 7.79e7.92 (m, 4H). MS
(ESI): 461.1 ([M þ H]þ). Anal. Calcd for C24H19F3O4S: C, 62.60; H,
4.16. Found: C, 62.79; H, 4.21.
4.1.4.28. (E)-2-(4-Chlorobenzylsulfonyl)-3-(2-chlorophenyl)-1-phen-
ylprop-2- en-1-one (40). Yellow solid, yield: 66%. m.p. 139e141 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 4.65 (s, 2H); 6.83e6.99 (m, 2H);
6.97e7.21 (m, 4H); 7.32e7.59 (m, 5H); 7.69e7.87 (m, 3H). MS (ESI):
431.1 ([M þ H]þ). Anal. Calcd for C22H16Cl2O3S: C, 61.26; H, 3.74.
Found: C, 61.47; H, 3.90.
4.1.4.19. (E)-2-(4-Methoxybenzylsulfonyl)-3-(2-nitrophenyl)-1-phe-
nylprop-2- en-1-one (31). Yellow solid, yield: 72%. m.p. 147e149 ꢁC
1H NMR (300 MHz, CDCl3,
d ppm): 3.83 (s, 3H); 4.63 (s, 2H); 7.07e7.21
(m, 7H); 7.35e7.42 (m, 4H); 7.71e7.80 (m, 1H); 8.05 (d, J ¼ 8.5 Hz, 2H).
MS (ESI): 438.1 ([M þ H]þ). Anal. Calcd for C23H19NO6S: C, 63.15; H,
4.38; N, 3.20. Found: C, 62.94; H, 4.24; N, 3.42.
4.2. Biological assays
4.2.1. Antiproliferative activity
WM266.4 and WM1361 melanoma cells (ATCC) were cultured
in DMEM/10% fetal bovine serum, in 5% CO2 water saturated
atmosphere at 37 ꢁC. Cell suspensions (10,000/mL) were prepared
4.1.4.20. (E)-3-(4-Fluorophenyl)-2-(4-methoxybenzylsulfonyl)-1-ph-
enylprop- 2-en-1-one (32). White solid, yield: 80%. m.p. 119e120 ꢁC
1H NMR (300 MHz, CDCl3,
d
ppm): 3.85 (s, 3H); 4.63 (s, 2H); 6.95 (d,
and 100 mL/well dispensed into 96-well plates (Costar) giving 1000
J ¼ 8.6 Hz, 2H); 7.11e7.59 (m, 10H); 7.97e8.11 (m, 2H). MS (ESI):
411.1 ([M þ H]þ). Anal. Calcd for C23H19FO4S: C, 67.30; H, 4.67.
Found: C, 67.22; H, 4.35.
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
culture medium giving 200 M, and 10 serial dilutions of
3 ꢃ prepared. Aliquots (100 L) of each dilution were added to the
mL) were diluted into 20 mL
m
4.1.4.21. (E)-3-(2-Chlorophenyl)-2-(4-methoxybenzylsulfonyl)-1-phe-
m
nylprop- 2-en-1-one (33). White solid, yield: 67%. m.p.130e131 ꢁC 1H
wells, giving doses ranging from 100 mM to 0.005 mM. After
NMR (300 MHz, CDCl3,
d
ppm): 3.85 (s, 3H); 4.63 (s, 2H); 7.12e7.18 (m,
a further incubated at 37 ꢁC for 24 h in a humidified atmosphere
2H); 7.25e7.91 (m, 12H). MS (ESI): 427.1 ([M þ H]þ). Anal. Calcd for
with 5% CO2, the cell viability was assessed by the conventional
C23H19ClO4S: C, 64.71; H, 4.49. Found: C, 64.96; H, 4.38.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) reduction assay and carried out strictly according to the
manufacturer instructions (Sigma). The absorbance at 590 nm was
recorded using LX300 Epson Diagnostic microplate reader. Then
GI50 was calculated using SPSS 13.0 software.
4.1.4.22. (E)-2-(4-Chlorobenzylsulfonyl)-1-phenyl-3-(4-(trifluorome-
thyl)phen- yl)prop-2-en-1-one (34). Yellow solid, yield: 52%. m.p.
127e129 ꢁC 1H NMR (300 MHz, CDCl3,
d ppm): 4.60 (s, 2H);

294
Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
4.2.2. Kinase assay
water only. Compound 38, suspended with carboxymethyl cellu-
lose, (CMC, 0.5% w/v) was administered orally at doses of 50, 250, or
1000 mg kgꢀ1. Appearance and mortality of test animals were
observed for 14 days.
This V600E mutant kinase assay was performed in triplicate for
each tested compound in this study. Briefly, 7.5 ng Mouse Full-
Length GST-tagged BRAFV600E (Invitrogen, PV3849) was pre-
incubated at room temperature for 1 h with 1
dilution buffer. The kinase assay was initiated when 5
containing 200 ng recombinant human full-length, N-terminal His-
tagged MEK1 (Invitrogen), 200 M ATP (0.8 Ci hot ATP), and 30 mM
MgCl2 in assay dilution buffer was added. The kinase reaction was
allowed to continue at room temperature for 25 min and was then
m
L drug and 4
mL assay
mL of a solution
Acknowledgments
m
m
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for the
Central Universities (No. 1092020804 & 1106020824).
quenched with 5
m
L 5 ꢃ protein denaturing buffer (LDS) solution.
Protein was further denatured by heating for 5 min at 70 ꢁC. 10
mL of
Appendix. Supplementary data
each reaction was loaded into a 15-well, 4e12% 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 reaction
that contained no active enzyme was used as a negative control, and
a reaction without inhibitor was used as the positive control.
Detection of the effect of compounds on cell based pERK1/2
activity in WM266.4 cells was performed using ELISA kits (Invi-
trogen) and strictly according to the manufacturer instructions.
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.02.007.
References
[1] M.J. Robinson, M.H. Cobb, Mitogen-activated protein kinase pathways, Current
Opinion in Cell Biology 9 (1997) 180e186.
[2] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal
transduction pathways activated by stress and inflammation, Physiological
Reviews 81 (2001) 807e869.
4.3. Molecular docking
[3] G.L. Johnson, R. Lapadat, Mitogen-activated protein kinase pathways mediated
by ERK, JNK, and p38 protein kinases, Science 298 (2002) 1911e1912.
[4] G. Pearson, F. Robinson, T.B. Gibson, B.E. Xu, M. Karandikar, K. Berman,
M.H. Cobb, Mitogen-activated protein (MAP) kinase pathways: regulation and
physiological functions, Endocrine Reviews 22 (2001) 153e183.
[5] P. Cohen, Protein kinases - the major drug targets of the twenty-first century?
Nature Reviews Drug Discovery 1 (2002) 309e315.
[6] C. Wellbrock, M. Karasarides, R. Marais, The RAF proteins take centre stage,
Nature Reviews Molecular Cell Biology 5 (2004) 875e885.
[7] S.S. Sridhar, D. Hedley, L.L. Siu, Raf kinase as a target for anticancer thera-
peutics, Molecular Cancer Therapeutics 4 (2005) 677e685.
The compound library we used for virtual screening of novel
BRAF inhibitors in this study was established by our laboratory. This
library was a collection of more than 1130 compounds which we
designed or synthesized.
The X-ray crystal structure of the BRAF kinase domain in an
active configuration (BRAFV600E/SB-590885, PDB code: 2FB8,
Protein Data Bank) was used as the target structure in this
approach. We used the active site of the crystal structure with the
inhibitor excluded from the coordinates as a receptor for compound
binding. Residues around SB-590885 at a radius of 6 Å were iso-
lated for the construction of the grid for docking screening. This
radius was large enough to include all of the residues that are
involved in inhibitor binding.
Studies were carried out on only one subunit of the enzymes. The
graphical user interface Autodocktools(ADT) was employed to setup
the enzymes: all hydrogens were added, Gasteiger charges were
calculated and nonpolar hydrogens were merged to carbon atoms.
For macromolecules, generated pdbqt files were saved. Studies were
carried out on only one subunit of the enzymes. The graphical user
interface ADT was employed to setup the enzymes: all hydrogens
were added, Gasteiger charges were calculated and nonpolar
hydrogens were merged to carbon atoms. For macromolecules,
generated pdbqt files were saved. AutoDock 4.0 software was
introduced as the primary screening tool for docking calculations.
Default settings were used with an initial population of 50 randomly
placed individuals, a maximum number of 2.5 ꢃ 106 energy evalu-
ations, and a maximum number of 2.7 ꢃ 104 generations. A mutation
rate of 0.02 and a crossover rate of 0.8 were chosen. Results differing
by less than 0.5 Å in positional root-meansquare deviation (RMSD)
were clustered together and the results of the most favorable free
energy of binding were selected as the resultant complex structures.
[8] J. Avruch, A. Khokhlatchev, J.M. Kyriakis, Z.J. Luo, G. Tzivion, D. Vavvas,
X.F. Zhang, Ras activation of the Raf kinase: tyrosine kinase recruitment of the
MAP kinase cascade, Recent Progress in Hormone Research 56 (2001)
127e155.
[9] H. Davies, G.R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague,
H. Woffendin, M.J. Garnett, W. Bottomley, N. Davis, N. Dicks, R. Ewing,
Y. Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes, V. Kosmidou, A. Menzies,
C. Mould, A. Parker, C. Stevens, S. Watt, S. Hooper, R. Wilson, H. Jayatilake,
B.A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K. Pritchard-Jones,
N. Maitland, G. Chenevix-Trench, G.J. Riggins, D.D. Bigner, G. Palmieri,
A. Cossu, A. Flanagan, A. Nicholson, J.W.C. Ho, S.Y. Leung, S.T. Yuen, B.L. Weber,
H.F. Siegler, T.L. Darrow, H. Paterson, R. Marais, C.J. Marshall, R. Wooster,
M.R. Stratton, P.A. Futreal, Mutations of the BRAF gene in human cancer,
Nature 417 (2002) 949e954.
[10] J.H. Lee, E.S. Lee, Y.S. Kim, Clinicopathologic significance of BRAF V600E
mutation in papillary carcinomas of the thyroid - a meta-analysis, Cancer 110
(2007) 38e46.
[11] M.J. Garnett, R. Marais, Guilty as charged: B-RAF is a human oncogene, Cancer
Cell 6 (2004) 313e319.
[12] P.T.C. Wan, M.J. Garnett, S.M. Roe, S. Lee, D. Niculescu-Duvaz, V.M. Good,
C.M. Jones, C.J. Marshall, C.J. Springer, D. Barford, R. Marais, P. Cancer Genome,
Mechanism of activation of the RAF-ERK signaling pathway by oncogenic
mutations of B-RAF, Cell 116 (2004) 855e867.
[13] C. Wellbrock, L. Ogilvie, D. Hedley, M. Karasarides, J. Martin, D. Niculescu-
Duvaz, C.J. Springer, R. Marais, B-V599E-RAF is an oncogene in melanocytes,
Cancer Research 64 (2004) 2338e2342.
[14] J.S. Sebolt-Leopold, R. Herrera, Targeting the mitogen-activated protein kinase
cascade to treat cancer, Nature Reviews Cancer 4 (2004) 937e947.
[15] V. Gray-Schopfer, C. Wellbrock, R. Marais, Melanoma biology and new tar-
geted therapy, Nature 445 (2007) 851e857.
[16] M. Karasarides, A. Chiloeches, R. Hayward, D. Niculescu-Duvaz, I. Scanlon,
F. Friedlos, L. Ogilvie, D. Hedley, J. Martin, C.J. Marshall, C.J. Springer, R. Marais,
B-RAF is a therapeutic target in melanoma, Oncogene 23 (2004) 6292e6298.
[17] B.B. Friday, A.A. Adjei, Advances in targeting the Ras/Raf/MEK/Erk mitogen-
activated protein kinase cascade with MEK inhibitors for cancer therapy,
Clinical Cancer Research 14 (2008) 342e346.
The binding affinity was evaluated by the binding free energies (DGb,
kcal/mol). The compounds which exhibited the highest binding
affinities, which means lowest binding free energies, were chosen.
[18] A.E. Gould, R. Adams, S. Adhikari, K. Aertgeerts, R. Afroze, C. Blackburn,
E.F. Calderwood, R. Chau, J. Chouitar, M.O. Duffey, D.B. England, C. Farrer,
N. Forsyth, K. Garcia, J. Gaulin, P.D. Greenspan, R.B. Guo, S.J. Harrison,
S.C. Huan, N. Iartchouk, D. Janowick, M.S. Kim, B. Kulkarni, S.P. Langston,
J.X. Liu, L.T. Ma, S. Menon, H. Mizutani, E. Paske, C.C. Renou, M. Rezaei,
R.S. Rowland, M.D. Sintchak, M.D. Smith, S.G. Stroud, M. Tregay, Y.A. Tian,
O.P. Veiby, T.J. Vos, S. Vyskocil, J. Williams, T.L. Xu, J.J. Yang, J. Yano, H.B. Zeng,
D.M. Zhang, Q. Zhang, K.M. Galvin, Design and optimization of potent and
4.4. Acute oral toxicity of compound 38
Acute oral toxicity studies were performed according to the
method of Vasudevan et al. [28] Briefly, mice of either sex, selected
by a random sampling technique, were employed in this study.
Mice (n ¼ 3) in each group were fasted for 4 h with free access to

Q.-S. Li et al. / European Journal of Medicinal Chemistry 50 (2012) 288e295
295
orally bioavailable Tetrahydronaphthalene Raf inhibitors, Journal of Medicinal
Chemistry 54 (2011) 1836e1846.
J.C. Lougheed, J. Lee, D. Chau, T.J. Stout, E.W. May, C.M. Rominger,
M.D. Schaber, L.S. Luo, A.S. Lakdawala, J.L. Adams, R.G. Contractor,
K.S.M. Smalley, M. Herlyn, M.M. Morrissey, D.A. Tuveson, P.S. Huang,
Demonstration of a genetic therapeutic index for tumors expressing onco-
genic BRAF by the kinase inhibitor SB-590885, Cancer Research 66 (2006)
11100e11105.
[19] G. Bollag, P. Hirth, J. Tsai, J.Z. Zhang, P.N. Ibrahim, H.N. Cho, W. Spevak,
C. Zhang, Y. Zhang, G. Habets, E. Burton, B. Wong, G. Tsang, B.L. West,
B. Powell, R. Shellooe, A. Marimuthu, H. Nguyen, K.Y.J. Zhang, D.R. Artis,
J. Schlessinger, F. Su, B. Higgins, R. Iyer, K. D’Andrea, A. Koehler, M. Stumm,
P.S. Lin, R.J. Lee, J. Grippo, I. Puzanov, K.B. Kim, A. Ribas, G.A. McArthur,
J.A. Sosman, P.B. Chapman, K.T. Flaherty, X.W. Xu, K.L. Nathanson, K. Nolop,
Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-
mutant melanoma, Nature 467 (2010) 596e599.
[20] C. Wellbrock, A. Hurlstone, BRAF as therapeutic target in melanoma,
Biochemical Pharmacology 80 (2010) 561e567.
[21] K.S.M. Smalley, M. Xiao, J. Villanueva, T.K. Nguyen, K.T. Flaherty, R. Letrero,
P. Van Belle, D.E. Elder, Y. Wang, K.L. Nathanson, M. Herlyn, CRAF inhibition
induces apoptosis in melanoma cells with non-V600E BRAF mutations,
Oncogene 28 (2008) 85e94.
[22] I. Niculescu-Duvaz, E. Roman, S.R. Whittaker, F. Friedlos, R. Kirk, I.J. Scanlon,
L.C. Davies, D. Niculescu-Duvaz, R. Marais, C.J. Springer, Novel inhibitors of
B-RAF based on a disubstituted pyrazine scaffold. Generation of a nanomolar
lead, Journal of Medicinal Chemistry 49 (2006) 407e416.
[23] A.J. King, D.R. Patrick, R.S. Batorsky, M.L. Ho, H.T. Do, S.Y. Zhang, R. Kumar,
D.W. Rusnak, A.K. Takle, D.M. Wilson, E. Hugger, L.F. Wang, F. Karreth,
[24] R. Huey, G.M. Morris, A.J. Olson, D.S. Goodsell, A semiempirical free energy
force field with charge-based desolvation, Journal of Computational Chem-
istry 28 (2007) 1145e1152.
[25] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, Journal of Computational Chemistry 30 (2009)
2785e2791.
[26] C. Luo, P. Xie, R. Marmorstein, Identification of BRAF inhibitors through in
silico screening, Journal of Medicinal Chemistry 51 (2008) 6121e6127.
[27] M.V.R. Reddy, V.R. Pallela, S.C. Cosenza, M.R. Mallireddigari, R. Patti,
M. Bonagura, M. Truongcao, B. Akula, S.S. Jatiani, E.P. Reddy, Design, synthesis
and evaluation of (E)-alpha-benzylthio chalcones as novel inhibitors of BCR-
ABL kinase, Bioorganic & Medicinal Chemistry 18 (2010) 2317e2326.
[28] M. Vasudevan, K.K. Gunnam, M. Parle, Antinociceptive and anti-inflammatory
effects of Thespesia populnea bark extract, Journal of Ethnopharmacology 109
(2007) 264e270.