Synthesis, biological evaluation of novel 4,5-dihydro-2H-pyrazole 2-hydroxyphenyl derivatives as BRAF inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.08.020

A series of novel 4,5-dihydropyrazole derivatives (3a-3t) containing hydroxyphenyl moiety as potential V600E mutant BRAF kinase (BRAF ^V600E) inhibitors were designed and synthesized. Docking simulation was performed to insert compounds 3d (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p- tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) and 3m (1-(3-(4-chlorophenyl)-5-(3, 5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) into the crystal structure of BRAF^V600E to determine the probable binding model, respectively. Based on the preliminary results, compound 3d and 3m with potent inhibitory activity in tumor growth may be a potential anticancer agent. Results of the bioassays against BRAF^V600E, MCF-7 human breast cancer cell line and WM266.4 human melanoma cell line all showed several compounds had potent activities IC₅₀ value in low micromolar range, among them, compound 3d and compound 3m showed strong potent anticancer activity, which were proved by that 3d: IC₅₀ = 1.31 μM for MCF-7 and IC₅₀ = 0.45 μM for WM266.5, IC₅₀ = 0.22 μM for BRAF^V600E, 3m: IC₅₀ = 0.97 μM for MCF-7 and IC₅₀ = 0.72 μM for WM266.5, IC₅₀ = 0.46 μM for BRAF^V600E, which were comparable with the positive control Erlotinib.

Full text of DOI:10.1016/j.bmc.2012.08.020

Bioorganic & Medicinal Chemistry 20 (2012) 6089–6096
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation of novel 4,5-dihydro-2H-pyrazole
2-hydroxyphenyl derivatives as BRAF inhibitors
Jia-Jia Liu a, Hui Zhang a, Juan Sun a, Zhong-Chang Wang a, Yu-Shun Yang a, Dong-Dong Li a,
a,
Fei Zhang a, Hai-Bin Gong b, , Hai-Liang Zhu
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b Xuzhou Central Hospital, Xuzhou 221009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4,5-dihydropyrazole derivatives (3a–3t) containing hydroxyphenyl moiety as potential
V600E mutant BRAF kinase (BRAFV600E) inhibitors were designed and synthesized. Docking simulation
was performed to insert compounds 3d (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyr-
azol-1-yl)ethanone) and 3m (1-(3-(4-chlorophenyl)-5-(3,5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyr-
azol-1-yl)ethanone) into the crystal structure of BRAFV600E to determine the probable binding model,
respectively. Based on the preliminary results, compound 3d and 3m with potent inhibitory activity in
tumor growth may be a potential anticancer agent. Results of the bioassays against BRAFV600E, MCF-7
human breast cancer cell line and WM266.4 human melanoma cell line all showed several compounds
had potent activities IC50 value in low micromolar range, among them, compound 3d and compound
Received 24 July 2012
Revised 14 August 2012
Accepted 16 August 2012
Available online 25 August 2012
Keywords:
Synthesis
Antitumor activity
4,5-Dihydropyrazole
BRAF inhibitor
3m showed strong potent anticancer activity, which were proved by that 3d: IC50 = 1.31 lM for MCF-7
and IC50 = 0.45
IC50 = 0.72 M for WM266.5, IC50 = 0.46
trol Erlotinib.
lM for WM266.5, IC50 = 0.22 l lM for MCF-7 and
M for BRAFV600E, 3m: IC50 = 0.97
l
l
M for BRAFV600E, which were comparable with the positive con-
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
stimulating vascular endothelial growth factor secretion.10 Overall,
these data suggest BRAFV600E as a therapeutic target11 has offered
many valuable and important opportunities for anticancer drug
development.
Cancers arise owing to the accumulation of mutations in critical
genes that alter normal programs of cell proliferation, differentia-
tion and death.1 The RAS/RAF/MEK/ERK/MAP kinase pathway
mediates cellular responses to growth signals. RAS is mutated to
form about 15% of human cancer. BRAF is a serine/threonine kinase
that belongs to this kinase pathway, which is involved in the trans-
duction of mitogenic signals from the cell membrane to the nu-
cleus. It has been reported that this kinase cascade can regulate
cell growth, survival and differentiation in a wonderful way.2
Approximately 90% of activating BRAF mutations in cancer lines
are a glutamic acid to valine substitution at position 600 (V600E;
formally identified as V599E).3–7 In cancer cells, BRAFV600E, which
is ꢀ500-fold more active than the wild-type protein,8 stimulates
constitutive ERK activity and drives proliferation and survival,
thereby providing essential tumor growth and remain good func-
tions.9 BRAFV600E can make contribution to neoangiogenesis by
Inhibitors of BRAF have been developed, such as SB-590885,12
AZ628,13 sorafenib14 and PLX4720.15 Among these inhibitors, SB-
590885, one of the hot spots be researched nowadays, is a novel
triarylimidazole derivative, and Andrew K. has also evaluated the
SAR of a series of imidazole inhibitors based on SB-590885 target-
ing at BRAF kinase.16
Dihydropyrazole, a small bioactive molecule, is a prominent
structural motif found in numerous pharmaceutically active com-
pounds.17 Of late, the dihydropyrazole derivatives used as potent
and selective inhibitors have great effect on causing cancer cell
death have also been reported.18–22 Among them, it has been re-
ported many times and have successfully caught many researchers’
attention that 4,5-dihydropyrazoles as an important class of het-
erocyclic small molecules, are important biological agents with a
wide range of pharmaceutical (antifungal, antibacteria, antitumor,
anti-inflammatory, and antiviral) and agrochemical activities.23–25
Cox et al. has disclosed a series of 4,5-dihydropyrazole derivative
as a potent, selective inhibitor of KSP (Kinesin spindle protein)
which is aim to treat human cancer with good potency, pharmaco-
kinetics and water solubility.26 Some small chemical molecules
Abbreviations: BRAF, V-RAF murine sarcoma viral oncogene homologue B1;
BRAFV600E, V600E mutant BRAF; IC50, half maximal inhibitory concentration; GI50
the concentration that causes 50% growth inhibition.
,
⇑
Corresponding authors. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.020

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J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
O
O
R3
OHC
R3
a
+
R1
HO
R1
HO
1a-1t
R2
R2
b
O
R3
R3
H
N
N
N
N
c
R2
R2
HO
HO
R1
R1
3a-3t
2a-2t
Scheme 1. The synthetic routes of compounds 3a–3t. Reagents and conditions: (a), ethanol, NaOH, rt, 4–6 h; (b) NH2ÁNH2ÁH2O, EtOH, 80 °C; (c) EDCÁHCl, CH2Cl2, rt.
Table 1
Table 2
Chemical structures of 3a–3t
Inhibition (IC50) of MCF-7 and WM266.5 cells proliferation and inhibition of
BRAFV600E by compounds 3a–3t
O
Compound
IC50 SD (
MCF-7a
lM)
R3
WM266.5a
BRAFV600E,b
N
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3.44 0.06
2.70 0.01
2.35 0.02
1.31 0.01
3.58 0.05
3.69 0.04
3.32 0.11
3.46 0.03
23.83 0.06
28.07 0.34
19.02 0.25
19.04 0.22
0.97 0.02
4.73 0.17
4.57 0.17
3.38 0.15
7.74 0.13
8.21 0.19
5.87 0.13
6.60 0.09
8.97
2.88 0.12
2.55 0.15
2.45 0.22
0.45 0.03
3.16 0.28
2.55 0.36
2.35 0.18
2.46 0.13
23.52 0.23
20.22 1.1
19.35 1.2
16.34 0.22
0.72 0.07
4.43 0.5
4.23 0.07
3.53 0.08
7.50 0.15
11.69 0.38
6.82 0.22
6.50 0.37
8.09
2.68 0.12
2.87 0.18
2.41 0.11
0.22 0.06
3.02 0.22
2.56 0.17
2.08 0.13
2.65 0.15
20.83 0.15
18.31 0.51
18.62 0.39
17.31 0.32
0.46 0.35
2.62 0.22
3.34 0.24
3.22 0.13
8.14 0.13
8.68 0.12
8.01 0.62
7.86 0.39
0.08
B
C
A
HO
R2
R1
Compound
R1
R2
R3
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
F
Br
Cl
H
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
H
Br
Cl
H
H
Br
Cl
H
3s
3t
3j
F
3k
3l
F
F
Erlotinib
H
3m
3n
3o
3p
3q
3r
3s
3t
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Cl
H
a
Inhibition of the growth of tumor cell lines.
Inhibition of BRAFV600E
b
.
H
Br
Cl
H
V600E mutant of BRAF kinase (BRAFV600E) inhibitors and expected
to have a sound cancer therapeutic benefit.
H
In organic chemistry, phenols, sometimes called phenolics, are a
class of chemical compounds consisting of a hydroxyl group (–OH)
bonded directly to an aromatic hydrocarbon group. The phenolic
hydroxyl group is very important in many chemical reactions
and has very good bioactivities. It has been reported that the phe-
nolic hydroxyl group of carvacrol is essential for action against the
food-borne pathogen Bacillus cereus.29 Polyphenols, such as
containing pyrazole skeleton have been identified as selective
inhibitors of BRAFV600E and displayed potent anticancer activi-
ties.27,28 Based on the studies generalized above, a series of novel
4,5-dihydropyrazole derivatives were designed as potential

J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
6091
flavones and isoflavones, constitute one of the most represented
classes of compounds in higher plants, including medicinal and
edible plants. Not only extensive studies but also many in vitro
experiments with polyphenols have indicated their broad variety
of biological activities of them, including anticancer,30 anti-inflam-
matory,31 antibacterial,32 cardioprotective,33 anti-osteoporotic34
and enzyme-inhibitory35 activities.
In this study, we want to share our experience about the
synthesis, bioassay and the SAR of 4,5-dihydropyrazoles deriva-
tives containing hydroxyphenyl moiety. We have successfully
synthesized 20 compounds, and 17 of them are new. Biological
evaluation indicated that some of the synthesized compounds
are potent inhibitors of BRAFV600E. To gain better understanding
about the potency of the studied compounds and guide further
SAR studies, we proceeded to examine the interaction of
compounds
with
SB-590885
(2FB8.pdb),
and
docking
simulations were performed using the X-ray crystallographic
structure of the BRAF in complex with an inhibitor to
explore the binding modes of these compounds at the active
site.
Figure 1. (a) Compound 3d (colored by atom: carbons: gray; nitrogen: blue; oxygen: red; sulfur: yellow) is bond into BRAFV600E. The dotted lines show the hydrogen bond
and the yellow line show the
–cation interactions. (b) 2D Ligand interaction diagram of compound 3d with BRAFV600E using Discovery Studio program with the essential
p
amino acid residues at the binding site are tagged in circles. The purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions
and the green circles show the amino acids which participate in the Van der Waals interactions.

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J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
Secondly, treat chalcone derivatives in refluxing isopropanol with
hydrazine hydrate for 8 h to obtain the desired compounds
(2a–2t). Finally, to a solution of compounds (2a–2t) in dichloro-
methane, Acetic acid derivatives was added, together with EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
and
HOBt
(hydroxybenzotriazole). The mixture was refluxed under stirring
for 8 h to obtain the desired compounds (3a–3t). All the synthetic
compounds were characterized by 1H NMR, elemental analysis and
mass spectrum, which were in full accordance with their depicted
structures.
2.2. Bioassay
As described above, the BRAFV600E was considered as an impor-
tant target for the development of small molecule inhibitors in the
treatment of human cancers, particularly melanoma. To test the
anticancer activities of the synthesized compounds, we evaluated
antiproliferative activities of compounds 3a–3t against MCF-7
and WM266.5 cells. The results were summarized in Table 2. These
compounds showed remarkable antiproliferative effects. Among
them, compound 3d and compound 3m displayed the strong po-
tent inhibitory activity (3d: IC50 = 1.31
lM
for MCF-7 and
IC50 = 0.45
IC50 = 0.72
l
l
M for WM266.5, 3m: IC50 = 0.97 lM for MCF-7 and
M for WM266.5), respectively, comparing with the po-
sitive control Erlotinib (IC50 = 8.97
for WM266.5, respectively).
lM for MCF-7, IC50 = 8.09 lM
Structure–activity relationships in these dihydropyrazole
derivatives displayed that compounds with para electron-donat-
ing substituents (3a–3h) showed more potent activities than
those with electron-withdrawing substituents (3i–3t) in the A-
ring. A comparison of the para substituents on the A-ring dem-
onstrated that an electron-donating group (3a–3h) have slightly
improved antiproliferative activity and the potency order is
CH3 > OCH3. Comparing with CH3 (3a–3d) and OCH3 (3e–3h), F
(3i–3l), Cl (3m–3p) and Br (3q–3t) substituents mostly had min-
imal effects, which was clearly viewed through Table 2. In the
case of constant A ring substituents, change of substituents on
B ring could also affect the activities of these compounds. It
can be found that compounds with a halogen atom on the 5-po-
sition of salicylaldehyde mostly displayed higher anticancer
activity than compounds with two halogen atoms on the
3-position and 5-position of salicylaldehyde, however, 1-(5-
(3,5-dibromo-2-hydroxyphenyl)-3-(4-fluorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (3m) showed better anticancer activity
than the compounds with a single halogen atom on the 5-posi-
tion of salicylaldehyde. Among compounds with a halogen atom
on the 5-position of salicylaldehyde and a halogen atom on the
5-position of salicylaldehyde, the strength order is Cl > Br. Thus,
the compound 3d with para-Me group in the A ring and Cl in
the B ring showed the best activity and compound 3m also dis-
played a good anticancer activity. To examine whether the com-
pounds interact with BRAFV600E and inhibit BRAFV600E in vitro,
we did the anti-BRAFV600E research and the data had also shown
in Table 2, compound 3d and compound 3m showed strong po-
tent anti-BRAFV600E activity and 50% BRAFV600E inhibition about
Figure 2. (a) Compound 3m (colored by atom: carbons: gray; nitrogen: blue;
oxygen: red; sulfur: yellow) is bond into BRAFV600E. The dotted lines show the
hydrogen bond and the yellow line show the
p–cation interactions. (b) 2D Ligand
interaction diagram of compound 3m with BRAFV600E using Discovery Studio
program with the essential amino acid residues at the binding site are tagged in
circles. The purple circles show the amino acids which participate in hydrogen
bonding, electrostatic or polar interactions and the green circles show the amino
acids which participate in the Van der Waals interactions.
3d: 0.22
Erlotinib with an IC50 of 0.08
l
M, 3m: 0.46
l
M, respectively (the positive control
2. Results and discussion
2.1. Chemistry
l
M for BRAFV600E). These results
indicated the anti-proliferative effect was produced by the direct
connection of BRAFV600E and the compounds.
The synthesis of compounds (3a–3t) is followed the general
pathway outlined in Scheme 1 and the structure of these com-
pounds have been shown in Table 1. Compounds (3a–3t) are pre-
pared in three steps. Firstly, the chalcones (1a–1t) were obtained
by the substituted salicylaldehydes and the substituted acetophe-
none, using 40% potassium hydroxide as catalyst in ethanol.36
2.3. Docking study
Molecular docking is an application wherein molecular model-
ing techniques are used to predict how a protein (enzyme) inter-
acts with small molecules (ligands).37 In the present study, to
understand the interactions between compounds and BRAF and

J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
6093
to explore their binding mode, a docking study was performed
4. Experimental section
using the CDOCKER protocol in Discovery Studio 3.1 (Discovery
Studio 3.1, Accelrys, Inc., San Diego, CA).
4.1. Materials and measurements
In the current study, we performed docking of 20 4,5-dihydro-
pyrazole derivatives into the active site of the receptor BRAF using
CDOCKER in the receptor–ligand Interactions protocol section of
Discovery Studio 3.1. The crystal structure of BRAF (PDB Code:
2FB8.pdb)12 was obtained from the RCSB protein data bank
(http://www.pdb.org). After preparing the receptor and ligands,
the site sphere was selected based on the ligand binding location
of SB-590885.
Binding model between the most potent compound 3d with
BRAF active site was showed in Figure 1. All the amino acid resi-
dues which had interactions with BRAFV600E were exhibited in
Figure 1b. In the binding model, compound 3d is nicely bound to
the active site of BRAF via hydrogen bond with SER465 (angle
OÁ Á ÁH–N = 126.66°, distance = 2.1 Å). The receptor surface model
was showed in Figure 1a, which revealed that the molecule occu-
pies the ATP-binding pocket and binds to an active conformation
of BRAF. The hydrophobic pocket including VAL471, PHE583,
ALA481, THR529, LEU514, VAL482 and GLY464 is occupied by acet-
amide moiety. This binding model confirms the importance of
hydrophobic group.
Binding model between the most potent compound 3m with
BRAF active site was showed in Figure 2. All the amino acid resi-
dues which had interactions with BRAFV600E were exhibited in
Figure 2b. In the binding model, compound 3m is nicely bound
to the active site of BRAF via hydrogen bond with SER465 (angle
OÁ Á ÁH–N = 104.7°, distance = 2.20 Å) and ASP594(distance = 3.9 Å).
The receptor surface model was showed in Figure 2a, which reveals
that the molecule occupies the ATP-binding pocket and binds to an
active conformation of BRAF. The hydrophobic pocket including
LYS483, ASN580, SER467, ASN581 and GLY466 is occupied by acet-
amide moiety. This binding model also confirms the importance of
hydrophobic group as well.
All chemicals (reagent grade) used were commercially avail-
able. Separation of the compounds by column chromatography
was carried out with silica gel 60 (200–300 mesh ASTM, E.
Merck, Germany). Developed plates were visualized by
a
Spectroline ENF 260C/F UV apparatus. The quantity of silica
gel used was 50–100 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, Ger-
many) and visualized in ultraviolet (UV) light (254 nm). Con-
centration and evaporation of the solvent after reaction or
extraction were carried out on a rotary evaporator (Büchi Rota-
vapor) operating at reduced pressure. Melting points were
measured on
a Boetius micro melting point apparatus. All
the Proton nuclear magnetic resonance (1H NMR) and spectra
were recorded on a DPX300 model Spectrometer at 25 °Cwith
TMS and solvent signals allotted as internal stands. Chemical
shifts were reported in parts per million(d). ESI-MS spectra
were recorded on a Mariner System 5304 Mass spectrometer.
Elemental analyses were performed on a CHN–O-Rapid instru-
ment and were within 0.4% of the theoretical values.
4.2. General procedure for synthesis of chalcones (1a–1t)
Equimolar portions of the appropriately substituted salicylal-
dehydes (2 mmol, 1 equiv) and substituted acetophenone
(2 mmol, 1 equiv) were dissolved in approximately 10 mL of eth-
anol. The mixture was allowed to stir for several minutes at 0 °C
to let it dissolve. Then, 0.5 mL aliquot of a 40% aqueous sodium
hydroxide solution was slowly added dropwise to the reaction
flask via a self-equalizing addition funnel. The reaction solution
was allowed to stir at room temperature for approximately 4–
6 h. The mixture was adjusted to pH 4.0 with dilute hydrochloric
acid until the reaction was complete. The reaction was moni-
tored by TLC. Mostly, a precipitate formed and was then col-
lected by suction filtration.
Overall, these results of the molecular docking study showed
that the compound 3d and 3m could act synergistically to interact
with the binding site of BRAFV600E, suggested that compound 3d
and 3m are potential inhibitors of BRAFV600E
.
4.3. General method of synthesis of 3,5-diphenyl-4,5-dihydro-
3. Conclusion
1H-pyrazole (2a–2t)
In summary, a series of novel compounds containing 4,5-dihyd-
ropyrazole core with hydroxyphenyl moiety have been
synthesized and evaluated for their B-Raf inhibitory and anti-pro-
liferation activities. Results showed these compounds possessed
potent antiproliferative activity against BRAF, WM266.4 human
melanoma cell line, MCF-7 human breast cancer cell line with
IC50 inhibitory values in low micromolar range. Among them, com-
pound 3d and 3m showed strong potent agent with IC50 value (3d:
To a solution of chalcone derivative (1 mmol) in isopropanol
(5 mL) hydrazine hydrate (0.2 mL, 4 mmol) was added. The mix-
ture was refluxed under stirring for 8 h, stored at 4–5 °C for 24 h,
and the precipitate formed was filtered off, washed with cool eth-
anol. The synthesized compound was purified by crystallization
from ethanol in refrigerator and allowed to air dry to obtain 4,5-
dihydropyrazole derivatives.
IC50 = 1.31
IC50 = 0.97
l
l
M for MCF-7 and IC50 = 0.45
M for MCF-7 and IC50 = 0.72
l
M for WM266.5, 3m:
4.4. General method of synthesis of compounds (3a–3t)
l
M for WM266.5, respec-
tively), comparing with the positive control Erlotinib
(IC50 = 8.97 M for MCF-7 and IC50 = 8.09 M for WM266.5). And
To a solution of pyrazoline derivatives (1 mmol) in dichloro-
methane (5 mL) acetic acid (5 ml) was added, together with EDC
(1.2–1.5 mmol) and HOBt (1.2–1.5 mmol). The mixture was re-
fluxed under stirring for 8 h. After completion of the reaction, the
contents were cooled, and then evaporated to dryness in vacuo.
Aqueous hydrochloric acid (0.1 M, 30 mL) was added and the mix-
ture extracted with ethyl acetate (3 Â 5 mL). The combined ethyl
acetate layers were back-extracted with saturated sodium bicar-
bonate (3 Â 5 mL) and brine (3 Â 5 mL), dried over MgSO4, filtered,
and evaporated in vacuo. The residue was crystallized from ethanol
to obtain target compounds.
l
l
compound 3d and compound 3m also showed strong potent
anti-BRAFV600E activity and 50% BRAFV600E inhibition about 3d:
0.22
l
M, 3m: 0.46
l
l
M, respectively (the positive control Erlotinib
M for BRAFV600E), this result could well show
with an IC50 of 0.08
the anti-proliferative effect was produced by direct connection of
BRAFV600E and the compounds.
The docking simulation was performed to get binding models
and poses, and the result showed compound 3d and compound
3m could bind well with the BRAF active site and acted as BRAF
inhibitor.

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J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
4.4.1. 1-(5-(3,5-Dibromo-2-hydroxyphenyl)-3-(p-tolyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone(3a)
4.4.8. 1-(5-(5-Chloro-2-hydroxyphenyl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3h)
White powder, yield: 63%. Mp: 246–248 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.33 (d, J = 13.3 Hz, 6H), 3.77 (s, 1H), 4.31 (d,
J = 4.58 Hz, 2H), 5.65–5.68 (m, 1H), 6.98 (d, J = 2.15 Hz, 1H), 7.27
(d, J = 8.05 Hz, 2H), 7.63–7.67 (m, 3H), MS (ESI): 451
(C18H17Br2N2O2, [M+H]+). Anal. C18H16Br2N2O2. Calcd for: C,
47.82; H, 3.57; N, 6.20. Found: C, 47.83; H, 3.58; N, 6.15.
White powder, yield: 65%. Mp: 262–264 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.31 (s, 3H), 3.76 (t, J = 13.88 Hz, 3H), 4.32 (t,
J = 5.03 Hz, 3H), 5.57 (d, J = 7.3 Hz, 1H), 6.78 (s, 1H), 6.84 (d,
J = 8.55 Hz, 1H), 6.85 (s, 2H), 6.99–7.01 (m, 1H), 7.71 (d,
J = 8.7 Hz, 2H), MS (ESI): 345 (C18H18ClN2O3, [M+H]+). Anal.
C18H17ClN2O3. Calcd for: C, 62.70; H, 4.97; N, 8.12. Found: C,
62.73; H, 4.98; N, 8.19.
4.4.2. 1-(5-(3,5-Dichloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone(3b)
4.4.9. 1-(5-(3,5-Dibromo-2-hydroxyphenyl)-3-(4-fluorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3i)
White powder, yield: 61%. Mp: 237–239 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.33 (d, J = 13.3 Hz, 6H), 3.77 (s, 1H), 4.32 (d,
J = 4.58 Hz, 2H), 5.66 (s, 1H), 6.95 (d, J = 2.15 Hz, 1H), 7.26 (d,
J = 8.05 Hz, 2H), 7.63–7.67 (m, 3H), MS (ESI): 363 (C18H17Cl2N2O2,
[M+H]+). Anal. C18H16Cl2N2O2. Calcd for: C, 59.52; H, 4.44; N,
7.71. Found: C, 59.53; H, 4.48; N, 7.79
White powder, yield: 68%. Mp: 215–216 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.33 (s, 3H), 3.61–3.68 (m, 3H), 5.81–5.83 (m, 1H),
6.94 (d, J = 2.1 Hz, 1H), 7.08 (t, J = 8.55 Hz, 2H), 7.51 (d, J = 2.1 Hz,
1H), 7.69–7.74 (m, 2H), MS (ESI): 455 (C17H14Br2FN2O2, [M+H]+).
Anal. C17H13Br2FN2O2. Calcd for: C, 44.77; H, 2.87; N, 6.14. Found:
C, 44.73; H, 2.88; N, 6.19.
4.4.10. 1-(5-(3,5-Dichloro-2-hydroxyphenyl)-3-(4-
4.4.3. 1-(5-(5-Bromo-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (3c)
fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3j)
White powder, yield: 61%. Mp: 209–211 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.33 (s, 3H), 3.61–3.66 (m, 3H), 5.81–5.82 (m, 1H),
6.88 (d, J = 2.1 Hz, 1H), 6.99 (t, J = 8.55 Hz, 2H), 7.47 (d, J = 2.1 Hz,
1H), 7.65–7.70 (m, 2H), MS (ESI): 367 (C17H14Cl2FN2O2, [M+H]+).A-
nalC17H13Cl2FN2O2. Calcd for: C, 55.60; H, 3.57; N, 7.63. Found: C,
55.53; H, 3.58; N, 7.69.
White powder, yield: 69%. Mp: 215–216 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.31 (s, 6H), 4.32 (t, J = 5.03 Hz, 3H), 5.57 (d, J = 7.3 Hz,
1H), 6.78 (s, 1H), 6.84 (d, J = 8.55 Hz, 1H), 6.85 (s, 2H), 6.99–7.01
(m, 1H), 7.71 (d, J = 8.7 Hz, 2H), MS (ESI): 345 (C18H18BrN2O2,
[M+H]+). Anal. C18H17BrN2O2. Calcd for: C, 57.92; H, 4.59; N, 7.51.
Found: C,57.93; H, 4.58; N,7.49.
4.4.11. 1-(5-(5-Bromo-2-hydroxyphenyl)-3-(4-fluorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3k)
4.4.4. 1-(5-(5-Chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (3d)
White powder, yield: 71%. Mp: 233–235 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.33 (s, 3H), 3.61–3.65 (m, 3H), 5.79–5.81 (m, 1H),
6.77 (d, J = 1.8 Hz, 2H), 7.07–7.18 (m, 3H), 7.68–7.76 (m, 2H), MS
(ESI): 377 (C17H15BrFN2O2, [M+H]+). Anal. C17H14BrFN2O2. Calcd
for: C, 54.13; H, 3.74; N, 7.43. Found: C, 54.15; H, 3.78; N, 7.39.
White powder, yield: 78%. Mp: 209–211 °C. 1H NMR (500 MHz,
DMSO-d6)d: 2.32 (s, 6H), 4.32 (t, J = 5.03 Hz, 3H), 5.57 (d, J = 7.3 Hz,
1H), 6.78 (s, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.85 (s, 2H), 6.99–7.02 (m,
1H), 7.71 (d, J = 8.55 Hz, 2H), MS (ESI): 329 (C18H18ClN2O2, [M+H]+).
Anal. C18H17ClN2O2. Calcd for: C, 65.75; H, 5.21; N, 8.52. Found: C,
65.73; H, 5.18; N, 8.49.
4.4.12. 1-(5-(5-Chloro-2-hydroxyphenyl)-3-(4-fluorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3l)
White powder, yield: 68%. Mp: 212–214 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.61–3.68 (m, 3H), 5.79–5.81 (m, 1H),
6.80 (d, J = 1.8 Hz, 2H), 7.07–7.18 (m, 3H), 7.72–7.76 (m, 2H), MS
(ESI): 333 (C17H15ClFN2O2, [M+H]+). Anal. C17H14ClFN2O2. Calcd
for: C, 61.37; H, 4.24; N, 8.42. Found: C, 61.33; H, 4.28; N, 8.39.
4.4.5. 1-(5-(3,5-Dibromo-2-hydroxyphenyl)-3-(4-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3e)
White powder, yield: 65%. Mp: 214–215 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.41 (d, J = 13.5 Hz, 3H), 4.33 (s, 6H), 5.68 (d, J = 7 Hz,
1H), 7.01 (s, 1H), 7.30 (t, J = 8.85 Hz, 2H), 7.64 (d, J = 2.15 Hz, 1H),
7.83 (t, J = 7.18 Hz, 2H), MS (ESI): 467 (C18H17Br2N2O3, [M+H]+).
Anal. C18H16Br2N2O3. Calcd for: C, 46.18; H, 3.44; N, 5.98. Found:
C, 46.13; H, 3.48; N, 5.99.
4.4.13. 1-(3-(4-Chlorophenyl)-5-(3,5-dibromo-2-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3m)
White powder, yield: 78%. Mp: 208–210 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.79–3.81 (m, 1H), 4.31 (t, J = 5.1 Hz, 2H),
5.65–5.68 (m, 1H), 6.88 (d, J = 8.55 Hz, 1H), 7.41 (d, J = 2.4 Hz, 1H),
7.65(s, 2H), 7.69(t, J = 12.88 Hz, 2H), MS (ESI): 471
(C17H14Br2ClN2O2, [M+H]+). Anal. C17H13Br2ClN2O2. Calcd for: C,
43.21; H, 2.77; N, 5.93. Found: C, 43.23; H, 2.78; N, 5.99.
4.4.6. 1-(5-(3,5-Dichloro-2-hydroxyphenyl)-3-(4-
methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3f)
White powder, yield: 64%. Mp: 206–208 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.41 (d, J = 13.5 Hz, 3H), 3.79–4.32 (m, 6H), 5.66–5.69
(m, 1H), 7.01 (s, 1H), 7.29 (t, J = 5.22 Hz, 2H), 7.63 (s, 1H), 7.83 (t,
J = 4.17 Hz, 2H), MS (ESI): 379 (C18H17Cl2N2O3, [M+H]+). Anal.
4.4.14. 1-(3-(4-Chlorophenyl)-5-(3,5-dichloro-2-
C18H16Cl2N2O3. Calcd for: C, 57.01; H, 4.25; N, 7.39. Found: C,
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3n)
White powder, yield: 75%. Mp: 209–210 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.33 (s, 3H), 3.79–3.81 (m, 1H), 4.31 (t, J = 8.55 Hz,
2H), 5.65–5.67 (m, 1H), 6.86 (d, J = 8.55 Hz, 1H), 7.39 (d,
J = 2.4 Hz, 1H), 7.64 (s, 2H), 7.67 (t, J = 5.1 Hz, 2H), MS (ESI): 385
(C17H14Cl3N2O2, [M+H]+). Anal. C17H13Cl3N2O2. Calcd for: C,
53.22; H, 3.42; N, 7.30. Found: C, 53.23; H, 3.38; N, 7.29.
56.93; H, 4.28; N, 7.43.
4.4.7. 1-(5-(5-Bromo-2-hydroxyphenyl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3g)
White powder, yield: 67%. Mp: 261–263 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.31 (s, 3H), 3.56 (t, J = 8.55 Hz, 3H), 4.22 (t,
J = 5.03 Hz, 3H), 5.55 (d, J = 8.7 Hz, 1H), 6.77 (s, 1H), 6.82 (d,
J = 8.55 Hz, 1H), 6.85 (s, 2H), 6.99–7.10 (m, 1H), 7.68 (d,
J = 8.7 Hz, 2H), MS (ESI): 389 (C18H18BrN2O3, [M+H]+). Anal.
4.4.15. 1-(5-(5-Bromo-2-hydroxyphenyl)-3-(4-chlorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3o)
White powder, yield: 74%. Mp: 270–272 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.79–3.81 (m, 1H), 4.31 (t, J = 5.1 Hz, 2H),
C18H17BrN2O3. Calcd for: C, 55.54; H, 4.40; N, 7.20. Found: C,
55.58; H, 4.48; N, 7.19.

J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
6095
5.65–5.68 (m, 1H), 6.88 (d, J = 8.55 Hz, 1H), 7.41 (d, J = 2.4 Hz, 1H),
4.6. Kinase assay
7.65 (s, 2H), 7.69 (t, J = 12.88 Hz, 2H), MS (ESI): 393 (C17
H15BrClN2O2, [M+H]+). Anal. C17H14BrClN2O2. Calcd for: C, 51.87;
H, 3.58; N,7.12. Found: C,51.93; H, 3.62; N, 7.07.
This V600E mutant BRAF kinase assay was performed in tripli-
cate for each tested compound in this study. Briefly, 7.5 ng Mouse
Full-Length GST-tagged BRAFV600E (Invitrogen, PV3849) was prein-
4.4.16. 1-(5-(5-Chloro-2-hydroxyphenyl)-3-(4-chlorophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3p)
cubated at room temperature for 1 h with 1
dilution buffer. The kinase assay was initiated when 5
tion containing 200 ng recombinant human full length, N-terminal
His-tagged MEK1 (Invitrogen), 200 M 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
l
L drug and 4
lL assay
l
L of a solu-
White powder, yield: 71%. Mp: 263–265 °C. 1H NMR (500 MHz,
DMSO-d6) d: 2.33 (s, 3H), 4.29–4.34 (m, 3H), 5.60 (s, 1H), 6.84 (d,
J = 8.55 Hz, 2H), 7.11 (d, J = 8.55 Hz, 1H), 7.52 (d, J = 8.55 Hz, 2H),
7.79 (d, J = 8.55 Hz, 2H), MS (ESI): 349 (C17H15ClN2O2, [M+H]+).
Anal. C17H14ClN2O2. Calcd for: C, 58.47; H, 4.04; N, 8.02. Found:
C, 58.43; H, 3.98; N, 7.97.
l
quenched with 5
Protein was further denatured by heating for 5 min at 70 °C.
10 L of each reaction was loaded into a 15-well, 4–12% precast
l
L 5Â protein denaturing buffer (LDS) solution.
l
4.4.17. 1-(3-(4-Bromophenyl)-5-(3,5-dibromo-2-
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 phos-
phor 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 (Invitro-
gen) and strictly according to the manufacturer instructions.
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3q)
White powder, yield: 72%. Mp: 223–225 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.35 (s, 2H), 3.76–3.80 (m, 1H), 5.64–5.66
(m, 1H), 7.01 (d, J = 2.19 Hz, 1H), 7.64–7.78 (m, 5H), MS (ESI): 515
(C17H14Br3N2O2, [M+H]+). Anal. C17H13Br3N2O2. Calcd for: C, 39.49;
H, 2.53; N, 5.42. Found: C, 39.48; H, 2.48; N, 5.43.
4.4.18. 1-(3-(4-Bromophenyl)-5-(3,5-dichloro-2-
hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) ethanone (3r)
White powder, yield: 76%. Mp: 237–239 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.32–2.50 (m, 3H), 3.76–3.86 (m, 1H), 4.33 (s, 2H),
5.64–5.67 (m, 1H), 7.01 (d, J = 2.19 Hz, 1H), 7.64–7.73 (m, 5H),
MS (ESI): 427 (C17H14BrCl2N2O2, [M+H]+). Anal. C17H14BrCl2N2O2.
Calcd for: C, 47.69; H, 3.06; N, 6.54. Found: C, 47.68; H, 3.05; N,
6.53.
4.7. Docking
The pdb file about the crystal structure of the BRAF kinase do-
main bound to SB-590885(2FB8.pdb) was obtained from the RCSB
protein data bank (http://www.pdb.org). The molecular docking
procedure was performed by using CDOCKER protocol for recep-
tor–ligand interactions section of Discovery Studio 3.1 (Accelrys
Software Inc, San Diego, CA). Initially both the ligands and the
receptor were pretreated. For ligand preparation, the 3D structures
of all the steroidal compounds were generated with ChemBioOffice
2010 and optimized with MMFF94 method. For enzyme prepara-
tion, the hydrogen 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.38
It includes the following steps:
4.4.19. 1-(5-(5-Bromo-2-hydroxyphenyl)-3-(4-bromophenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3s)
White powder, yield: 76%. Mp: 297–299 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.75–3.85 (m, 3H), 5.56–5.61 (m, 1H),
6.83 (t, J = 4.38 Hz, 2H), 7.10–7.13 (m, 1H), 7.64–7.73 (m, 4H), MS
(ESI): 437 (C17H15Br2N2O2, [M+H]+). Anal. C17 H14Br2N2O2. Calcd
for: C, 46.60; H, 3.22; N, 6.39. Found: C, 46.68; H, 3.28; N, 6.33.
4.4.20. 1-(3-(4-Bromophenyl)-5-(5-chloro-2-hydroxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (3t)
(1) A series of ligand conformations are generated using high
temperature molecular dynamics with different random
seeds.
White powder, yield: 65%. Mp: 270–272 °C. 1H NMR (300 MHz,
DMSO-d6) d: 2.32 (s, 3H), 3.75–3.85 (m, 3H), 5.56–5.61 (m, 1H),
6.63 (t, J = 4.47 Hz, 2H), 7.10–7.13 (m, 1H), 7.64–7.63 (m, 4H), MS
(ESI): 393 (C17H15BrClN2O2, [M+H]+). Anal. C17H14BrClN2O2. Calcd
for: C, 51.87; H, 3.58; N, 7.12. Found: C, 51.78; H, 3.58; N, 7.13.
(2) Random orientations of the conformations are generated by
translating the center of the ligand to a specified position
within the receptor active site, and making a series of ran-
dom rotations. A softened energy is calculated and the orien-
tation is kept when it is less than a specified limit. This
process repeats until either the desired number of low-
energy orientations is obtained, or the test times of bad ori-
entations reached the maximum number.
(3) Each orientation is subjected to simulated annealing molec-
ular dynamics. The temperature is heated up to a high tem-
perature then cooled to the target temperature. A final
energy minimization of the ligand in the rigid receptor using
non-softened potential is performed.
(4) 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. The whole BRAFkinase domain
defined as a receptor and the site sphere was selected based
on the ligand binding location of SB-590885, then the SB-
590885 removed and the ligands prepared by us was placed
during the molecular docking procedure. CHARMm was
selected as the force field. The molecular docking was
4.5. Antiproliferative activity
Target tumor cells were cultured in DMEM/10% fetal bovine ser-
um, with 5% CO2 water saturated atmosphere at 37 °C. After dilut-
ing to 2 Â 104cells mLÀ1with the complete medium, 100
l
L of the
obtained cell suspension was added to each well of 96-well culture
plates. The plates were returned to the incubator for 24 h to allow
the cells to reattach. These compounds were initially prepared at
20 mM in DMSO. Aliquots (200
medium giving 200
M, and 10 serial dilutions of 3Â prepared. Ali-
quots (100 L) of each dilution were added to the wells, giving
doses ranging from 0.005 M to 100 M. After further incubated
lL) were diluted into 20 mL culture
l
l
l
l
at 37 °C 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-diphenyltetrazolium bromide (MTT) reduction assay and
carried out strictly according to the manufacturer instructions (Sig-
ma). The absorbance at 590 nm was recorded using LX300 Epson
Diagnostic microplate reader. Then IC50 was calculated using SPSS
13.0 software.

6096
J.-J. Liu et al. / Bioorg. Med. Chem. 20 (2012) 6089–6096
K. S. M.; Herlyn, M.; Morrissey, M. M.; Tuveson, D. A.; Huang, P. S. Cancer Res.
performed with a simulated annealing method. The heating
2006, 66, 11100.
steps were 2000 with 700 of heating target temperature.
The cooling steps were 5000 with 300 cooling target temper-
ature. Ten moleculardockingposes saved for each ligand were
ranked according to their dock score function. The pose with
the highest-CDOCKER energy was chosen as the most suitable
pose.
13. McDermott, U.; Sharma, S. V.; Dowell, L.; Greninger, P.; Montagut, C.; Lamb, J.;
Archibald, H.; Raudales, R.; Tam, A.; Lee, D.; Rothenberg, S. M.; Supko, J. G.;
Sordella, R.; Ulkus, L. E.; Iafrate, A. J.; Maheswaran, S.; Njauw, C. N.; Tsao, H.;
Drew, L.; Hanke, J. H.; Ma, X.-J.; Erlander, M. G.; Gray, N. S.; Haber, D. A.;
Settleman, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19936.
14. Wilhelm, S. M.; Carter, C.; Tang, L. Y.; Wilkie, D.; McNabola, A.; Rong, H.; Chen,
C.; Zhang, X. M.; Vincent, P.; McHugh, M.; Cao, Y. C.; Shujath, J.; Gawlak, S.;
Eveleigh, D.; Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.;
Gedrich, R.; Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Cancer
Res. 2004, 64, 7099.
Acknowledgment
15. Ibrahim, P. N.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.; Kumar, A.;
Fong, D.; Wang, W.; Zhu, Y.-L.; Marimuthu, A.; Suzuki, Y.; Lam, B.; Rice, J.;
Tsai, J.; Settachatgul, C.; Shelloe, R.; Cantwell, J.; West, B.; Powell, B.; Habets,
G.; Zhang, C.; Artis, D. R.; Hirth, P.; Bollag, G. Abstr. Paper Am. Chem. Soc. 2006,
232.
The work was financed by a grant (No. J1103512) from National
Natural Science Foundation of China.
16. Takle, A. K.; Brown, M. J. B.; Davies, S.; Dean, D. K.; Francis, G.; Gaiba, A.; Hird, A.
W.; King, F. D.; Lovell, P. J.; Naylor, A.; Reith, A. D.; Steadman, J. G.; Wilson, D.
M. Bioorg. Med. Chem. Lett. 2006, 16, 378.
References and notes
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