Synthesis, biological evaluation and 3D-QSAR studies of novel 4,5-dihydro-1H-pyrazole niacinamide derivatives as BRAF inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.04.047

A series of novel 4,5-dihydropyrazole derivatives containing niacinamide moiety as potential V600E mutant BRAF kinase (BRAF^V600E) inhibitors were designed and synthesized. Results of the bioassays against BRAF ^V600E and WM266.4 human

Full text of DOI:10.1016/j.bmc.2012.04.047

Bioorganic & Medicinal Chemistry 20 (2012) 3746–3755
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and 3D-QSAR studies of novel
4,5-dihydro-1H-pyrazole niacinamide derivatives as BRAF inhibitors
Cui-Yun Li a, , Qing-Shan Li a, , Li Yan a, Xiao-Guang Sun a, Ran Wei a, Hai-Bin Gong b, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR 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 containing niacinamide moiety as potential V600E
mutant BRAF kinase (BRAFV600E) inhibitors were designed and synthesized. Results of the bioassays
against BRAFV600E and WM266.4 human melanoma cell line showed several compounds to be endowed
potent activities with IC50 and GI50 value in low micromolar range, among which compound 27e, (5-(4-
Received 10 March 2012
Revised 21 April 2012
Accepted 23 April 2012
Available online 28 April 2012
Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)6-methylpyridin-3-yl
methanone
(IC50 = 0.20 M, GI50 = 0.89 M) was bearing the best bioactivity comparable with the positive control
l
l
Keywords:
Synthesis
Antitumor activity
4,5-Dihydropyrazole
BRAF inhibitor
Sorafenib. Docking simulation was performed to determine the probable binding model and 3D-QSAR
model was built to provide more pharmacophore understanding that could use to design new agents
with more potent BRAFV600E inhibitory activity.
Ó 2012 Published by Elsevier Ltd.
1. Introduction
growth factor secretion.10 Overall, these data suggest BRAFV600E
as a therapeutic target11 offering opportunities for anticancer drug
The serine threonine kinase BRAF is a member of the RAF kinase
family, which is part of the RAF/MEK/ERK serine threonine kinase
cascade. This kinase cascade, also called the ERK/MAP kinase path-
way (or ‘classical’ MAPK pathway), regulates cell growth, survival
and differentiation,1 and can be hyper-activated in approximately
30% of human cancers by many different membrane-bound recep-
tors,2 such as BRAF, the small G-proteins of the RAS family, and
receptor tyrosine kinases. It has been reported that BRAF is mu-
tated in many types of human tumors, most frequently observed
in 50–70% of cell lines and tumors in melanoma, in 36–69% of pap-
illary thyroid cancer, lower frequency in colorectal cancer (5–12%)
and nonsmall cell lung cancer (1–4%). Approximately 90% of acti-
vating 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, ꢀ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 maintenance functions.9 BRAFV600E also contrib-
utes to neoangiogenesis by stimulating vascular endothelial
development.
Inhibitors of BRAF have been developed, such as SB-590885,12
sorafenib,13 PLX4720,14 and AZ628.15 Among these inhibitors,
SB-590885, one of the researching hot spots, is a novel triaryl-
imidazole derivative, inhibits BRAF kinase activity with a Ki of
0.16 nmol/L, which is greater than 100-fold more potent than
sorafenib. The origin of the selectivity of SB-590885 for BRAF
seems to be due to interactions with several BRAF amino acids,
as well as the presence of the indane-oxime. In particular, a phen-
ylalanine residue (PHE583) in the COOH-terminal lobe forms
favorable p-stacking interactions with the imidazole and pyridine
rings of SB-590885.12 Andrew K. also evaluated the SAR of a series
of imidazole inhibitors based on SB-590885 targeting at BRAF
kinase.16 Lots of BRAF kinase inhibitors have been discovered with
high-throughput screening of a chemical library and some struc-
tural modifications of the existing inhibitor scaffolds. But the inev-
itable high false positive proportion limits the application of virtual
screen.
4,5-Dihydropyrazoles, an important class of heterocyclic small
molecules, are important biological agents with a wide range
of pharmaceutical (antitumor, anti-inflammatory, antifungal,
antibacterial, and antiviral) and agrochemical activities.17–19
Christopher et al. disclosed a 4,5-dihydropyrazole derivative as a
potent, selective inhibitor of KSP (Kinesin spindle protein) to treat
human cancer with good potency, pharmacokinetics and water
solubility.20 Havrylyuk et al. examined the in vitro anticancer
activity of several novel thiazolone-based compounds containing
Abbreviations: BRAF, V-RAF murine sarcoma viral oncogene homolog B1;
BRAFV600E, V600E mutant BRAF; IC50, half maximal inhibitory concentration; GI50
,
the concentration that causes 50% growth inhibition; 3D-QSAR, quantitative
structure–activity relationship.
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail addresses: ghbxzh@yahoo.com.cn (H.-B. Gong), zhuhl@nju.edu.cn (H.-L.
Zhu).
Both authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.04.047

C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
3747
the 5-aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl in the National
Cancer Institute. Most of compounds displayed anticancer activity
on leukemia, melanoma, lung, colon, CNS (central nervous system),
ovarian, renal, prostate and breast cancer cell lines and the most
efficient anticancer compound was found to be active with
selective influence on colon cancer cell lines, especially on HT-29
(logGI50 = À6.37).21 Some dihydropyrazole derivatives used as
potent and selective inhibitor of BRAFV600E with sound IC50 values
have been identified by high-throughput screening.22 Also,
niacinamide (nicotinamide or nicotinic acid amide) is chemically
known as pyridine-3-carboxamide, a form of vitamin B3, playing
a significant role in cancer therapy. Niacinamide acts as a chemo-
and radio-sensitizing agent, inhibits poly (ADP-ribose) polymer-
ases (PARP-1), enzymes involved in the rejoining of DNA strand
breaks induced by radiation or chemotherapy. Niacinamide is also
used by some patients in combination with intravenous vitamin C
therapy for cancer.23 Based on the studies generalized above, a
series of novel 4,5-dihydropyrazole derivatives containing
Scheme 1. Synthesis of compounds 1–35. Reagents and conditions: (a) EtOH, rt; (b) NH2ÁNH2ÁH2O, EtOH, reflux; (c) EDCÁHCl, Cl2H2, reflux.
Table 1
Structural features, BRAFV600E inhibitory activity and antiproliferation activity of 35 compounds
Compound
R1
R2
R3
IC50
(l
M) BRAFV600E
GI50 (lM) WM266.5
26a
26b
26c
26d
26e
27a
27b
27c
27d
27e
28a
28b
28c
28d
28e
29a
29b
29c
29d
29e
30a
30b
30c
30d
30e
31a
31b
31c
31d
31e
32a
32b
32c
32d
32e
Sorafenib
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
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
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4.58 0.31
7.56 1.23
5.14 0.31
6.14 0.37
3.42 0.51
0.38 0.04
0.51 0.07
0.58 0.04
0.47 0.04
0.20 0.03
3.51 0.39
6.89 0.50
6.76 0.46
5.57 0.43
4.23 0.33
4.27 0.58
6.78 0.41
3.01 0.24
6.49 0.47
1.63 0.13
7.61 0.42
16.43 3.12
12.89 1.89
4.42 0.89
7.02 0.61
4.77 0.31
7.16 0.77
7.77 0.42
2.12 0.34
3.58 0.31
7.82 0.39
7.34 0.21
7.35 0.52
8.67 0.39
7.05 0.55
0.06
7.56 0.43
6.45 0.76
5.74 0.51
4.35 0.68
4.93 0.61
1.29 0.04
1.45 0.05
1.44 0.20
0.78 0.06
0.89 0.04
5.63 0.70
7.13 0.97
8.47 00.91
3.05 0.48
4.28 0.68
6.24 0.89
10.64 1.27
10.47 1.45
9.15 1.51
7.95 1.31
8.13 0.80
13.16 2.81
11.36 1.85
7.71 0.99
9.20 1.05
6.09 1.22
9.89 1.31
8.87 1.05
3.58 0.68
3.79 0.88
8.01 1.00
9.87 1.52
10.78 1.36
7.45 1.26
7.65 0.78
8.12
4-Br
4-Br
4-Br
4-Br
4-Br
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Benzyloxy
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Br
4-Br
4-Br
4-Br
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
5-Bromopyridin
6-Chloropyridin
2-Chloro-6-methylpyridin
2-Chloropyridin
6-Methylpyridin
4-Br

3748
C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
Figure 1. 2D diagram of binding model between BRAF and compound 27e.
niacinamide moiety were designed as potential V600E mutant of
BRAF kinase (BRAFV600E) inhibitors and expected to have a sound
cancer therapeutic benefit.
for 8 h to give 3,5-diphenyl-(4,5-dihydropyrazol-1-yl) pyridin-3-
yl methanones. All of the synthetic compounds gave satisfactory
analytical and spectroscopic data, which were in full accordance
with their depicted structures.
In this study, we describe the synthesis and the SAR of
4,5-dihydropyrazoles derivatives. Biological evaluation indicated
that some of the synthesized compounds are potent inhibitors of
BRAFV600E. 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. Based on the activity data, QSAR model was built to study
the structure–activity relationship and guide the further study.
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. The
BRAFV600E inhibitory activities of the synthesized compounds were
evaluated. The results, expressed as concentrations of IC50 (the half
2. Results and discussion
2.1. Chemistry
The synthesis of 3,5-diphenyl-(4,5-dihydropyrazol-1-yl)pyri-
din-3-ylmethanones followed the general pathway outlined in
Scheme 1. First of all, to a stirred solution of acetophenone deriva-
tive and benzaldehyde derivative in ethanol, KOH solution was
added. The reaction mixture was stirred until the solids fully
formed, and then obtained chalcones with yields of 90–95%. Next,
treat chalcone derivatives in refluxing isopropanol with hydrazine
hydrate for 8 h to get 3,5-diphenyl-4,5-dihydro-1H-pyrazoles.
Finally, to a solution of 4,5-dihydropyrazole derivatives in dichlo-
romethane, nicotinic acid derivatives was added, together with
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and HOBt
(hydroxybenzotriazole). The mixture was refluxed under stirring
Figure 2. The receptor surface model with compounds 27e.

C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
3749
maximal inhibitory concentration of BRAFV600E mediated MEK
phosphorylation) and GI50 (the half maximal inhibitory concentra-
tion of WM266.4 human melanoma cell line growth), were sum-
marized in Table 1 and revealed several significant points.
binding model confirms the importance of hydrophobic group in
niacinamide.
2.4. QSAR model
Among the synthesized 4,5-dihydropyrazole derivatives, ana-
logs 26a–32e, bearing the same core, showed a broad range of
potencies, being compound 27e the most potent agent with IC50
In order to give a systematic evaluation on 4,5-dihydropyrazole
derivates as BRAF inhibitors and to explore more potent inhibitors,
3D-QSAR models were built using the creat 3D-QSAR protocol
of Discovery Studio 3.1. In this study, 35 compounds bearing
4,5-dihydropyrazole core with definite IC50 values were selected
as the model dataset. By convention, the pIC50 scale (ÀlogIC50),
in which higher values indicate exponentially greater potency, is
used as a method to measure inhibitory activity. The training and
test set was chosen by the Diverse Molecules method in Discovery
Studio.
Considering a good alignment is very important for the analysis
of molecular fields, the alignment conformation of each molecule
was the one with lowest energy in the docked results of CDOCKER.
And before building the QSAR model, we apply the alignment by
the substructure (4,5-dihydro-1H-pyrazol-1-yl)methanone.
The correlation coefficient r2 between observed and predicted
activity of training set was found to be 0.736, while that of test
set was found to be 0.789, which proves the QSAR model we built
was acceptable. Predicted pIC50 values of 35 compounds by QSAR
model we built have been given in Table 2. The well agreement be-
tween predicted pIC50 value and experimental pIC50 value for both
test sets and training sets are shown in Figure 3.
value of 0.20
lM and GI50 value of 0.89
l
M, in contrast, compound
30b exhibited a weak 16.43
l
M IC50 value and 13.16 M GI50
l
value.
Comprehensive study of the activity data shows that GI50 values
of these compounds share a similar tendency with their relevant
IC50 values. This relationship suggested the anti-proliferative effect
was produced by inhibitory action against BRAF. In the series of
4,5-dihydropyrazole derivatives, the BRAF inhibitory activity of
26a–32e, which were with different substituents on N-1 nitrogen
atom of the 4,5-dihydropyrazole core, increased in the following
order: Cl < Br < Me. Moreover analogs with 4-methoxyl substituent
or 4-chloride substituent on 3-phenyl ring of 4,5-dihydropyrazole
core exhibited thoroughly better activities of the former than the
later. And replacement on 5-phenyl ring of 4,5-dihydropyrazole
core results in a progressive potency, in order of 4-Cl < 4-Br < 4-
benzyloxy < 4-OMe, as electronegativity decreases.
An acute oral toxicity test was conducted with rats to determine
the toxicity from a single dose via the oral route. Based on the re-
sults of study (not listed), the single dose acute oral LD50 of the
most potent compound, 27e is greater than 5000 mg/kg of
bodyweight.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on electrostatic potential grids (Fig. 4a) and van
2.3. Docking study
Molecular docking is an application wherein molecular model-
ing techniques are used to predict how a protein (enzyme)
interacts with small molecules (ligands).24 In the present study,
to understand the interactions between compounds and BRAF
and to explore their binding mode, a docking study was performed
using the CDOCKER protocol in Discovery Studio 3.1 (Discovery
Studio 3.1, Accelrys, Inc., San Diego, CA).
In the current study, we performed docking of thirty-five
4,5-dihydropyrazole 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 27e with
BRAF active site was showed in Figure 1 and Figure 2. In the bind-
ing model, compound 27e is nicely bound to the active site of BRAF
via hydrogen bond with SER536 (angle ClÁ Á ÁH–N = 169.4°,
distance = 2.42 Å), LYS578 (angle OÁ Á ÁH–N = 143.222°, distance =
1.84 Å), which proves compounds 27a–27e substituted with
4-chloro on 5-phenyl ring and 4-methoxy on 3-phenyl ring of
4,5-dihydropyrazole core performs the most potent activity. Also
Table 2
Experimental, predicted inhibitory activity of compounds 26a–32e by 3D-QSAR
models based upon active conformation achieved by molecular docking
Compound
BRAFV600E
Experimental pIC50
Residual error
Predicted pIC50
26a
26b
26c
26d
26e
27a
27b
27c
27d
27e
28a
28b
28c
28d
28e
29a
29b
29c
29d
29e
30a
30b
30c
30d
30e
31a
31b
31c
31d
31e
32a
32b
32c
32d
32e
5.339
5.121
5.289
5.212
5.466
6.420
6.292
6.237
6.328
6.796
5.455
5.162
5.170
5.254
5.373
5.272
5.369
5.521
5.188
5.788
5.119
4.784
4.890
5.355
5.154
5.321
5.145
5.110
5.672
5.446
5.107
5.134
5.134
5.062
5.152
5.400
5.390
5.330
5.285
5.478
6.000
5.999
6.117
6.090
6.267
5.457
5.051
5.676
5.728
5.259
5.045
5.277
5.795
5.056
5.705
4.995
4.779
4.729
5.347
4.804
5.559
5.315
5.397
5.559
5.884
5.118
4.862
5.121
5.147
5.078
À0.061
À0.269
À0.041
À0.074
À0.012
0.421
0.294
0.119
0.238
0.529
À0.002
0.110
À0.506
À0.474
0.114
0.228
0.092
À0.273
0.132
0.083
0.123
0.005
0.160
0.008
0.350
À0.238
À0.170
À0.287
0.113
À0.438
À0.011
0.272
PHE583 plays an important role in the complex involving
interactions (angle = 25.3°, distance = 5.42 Å) with the pyridin ring
at the N1 atom and –sigma interactions (distance = 6.02 Å) with
p–p
p
the 5-phenyl ring of 4,5-dihydropyrazole core, which shares the
same interaction method of SB-590885. The substitutional group
also forms the hydrogen bond with BRAF. The only difference is
that 4,5-dihydropyrazole core plays a structural role in the binding
model.12 The receptor surface model was showed in Figure 2,
which revealed that the molecule occupies the ATP-binding pocket
and binds to an active conformation of BRAF. The hydrophobic
pocket including VAL471, PHE583, ALA481, THR529, LEU514 and
ASN581 is occupied by niacinamide substituted with methyl. This
0.013
À0.085
0.074

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C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
Figure 3. Plot of experimental versus predicted BRAF inhibitory activities of training set and test set.
Figure 4. (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (b) 3D-QSAR model
coefficients on van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.
der Waals grids are listed (Fig. 4b). Electrostatic map indicates red
contours around regions where high electron density (negative
charge) is expected to increase activity, and blue contours repre-
sent areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates areas
where steric bulk is predicted to increase (green) or decrease
(yellow) activity. According to the maps, it suggested the com-
pound with high negative charged and small R3 group, would
show higher activity, validating that 4-Cl substituent being a better
choice than 4-OMe. Whereas, a low negative charged and bulky R2
group would help obtain sound activity, validating the 4-OMe sub-
stituent could be more effective. As a result, data summarized
above demonstrate that compounds 27e, the most potent BRAF
BRAF and WM266.4 human melanoma cell line, with IC50 and
GI50 inhibitory values in low micromolar range. Among them, com-
pound 27e showed the most potent agent with IC50 value of
0.20 lM and GI50 value of 0.89 lM. The docking simulation was
performed to get binding models and poses, and the result shows
compound 27e can bind well with the BRAF active site and act as
BRAF inhibitor. QSAR model was also built by the activity data
and binding conformations to provide a reliable tool for rational
design of novel BRAF inhibitors.
4. Experimental section
4.1. Materials and measurements
inhibitor (IC50 = 0.20 lM, GI50 = 0.89 lM), containing favor substit-
uents has an outstanding activity.
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). 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, Germany) and visualized in ultraviolet (UV) light
(254 nm). Developed plates were visualized by a Spectroline ENF
3. Conclusion
In summary, a series of novel BRAF inhibitors containing
4,5-dihydropyrazole core was designed, prepared and tested for
their inhibitory activity against BRAF. Results showed these
compounds possessed potent antiproliferative activity against

C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
3751
260C/F UV apparatus. Concentration and evaporation of the solvent
after reaction or extraction were carried out on a rotary evaporator
(Büchi Rotavapor) 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°C with TMS and
solvent signals allotted as internal stands. Chemical shifts were re-
ported 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 instrument and were within 0.4% of
the theoretical values.
4.4.3. (3,5-Bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
2-chloro-6-methylpyridin-3-yl methanone (26c)
Yellow powder, yield: 61%. Mp: 161–163 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.59 (s, 3H); 3.18–3.25 (m, 1H); 3.79–3.83 (m, 4H);
5.68 (dd, J1 = 4.65 Hz, J2 = 11.52 Hz, 1H); 6.86–6.90 (m, 4H); 7.14
(d, J = 7.68 Hz, 1H); 7.26–7.31 (m, 2H); 7.56 (d, J = 8.79 Hz, 2H);
7.66 (d, J = 7.68 Hz, 1H). MS (ESI): 436.1 ([M+H]+). Anal. Calcd for
C24H22ClN3O3: C, 66.13; H, 5.09; N, 9.64%; Found: C, 66.49; H,
5.21; N, 9.29%.
4.4.4. (3,5-Bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
2-chloropyridin-3-yl methanone (26d)
4.2. General procedure for synthesis of chalcones
Brown powder, yield: 71%. Mp: 133–135 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.19–3.27 (m, 1H); 3.80 (s, 3H); 3.82–3.85 (m, 4H);
5.67–5.72 (m, 1H); 6.86–6.91 (m, 4H); 7.23–7.32 (m, 3H); 7.55 (d,
J = 8.94 Hz, 2H); 7.75–7.78 (m, 1H); 8.44–8.46 (m, 1H). MS (ESI):
422.1 ([M+H]+). Anal. Calcd for C23H20ClN3O3: C, 65.48; H, 4.78;
N, 9.96%; Found: C, 65.69; H, 4.59; N, 9.74%.
To a stirred solution of acetophenone derivatives (1 mmol) and
a benzaldehyde derivatives (1 mmol) in ethanol (30 mL), 6 M KOH
(4 mL) was added and the reaction mixture was stirred until the
solids fully formed. The products were filtrated and washed care-
fully with ice water and cool ethanol, and purified by crystalliza-
tion from ethanol in refrigerator to give chalcones.
4.4.5. (3,5-Bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
6-methylpyridin-3-yl methanone (26e)
4.3. General method of synthesis of 3,5-diphenyl-4,5-dihydro-
1H-pyrazole
White powder, yield: 68%. Mp: 146–148 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.56–2.67 (m, 3H); 3.19–3.27 (m, 1H); 3.76–3.91
(m, 3H); 3.84 (d, 4H); 5.70–5.76 (m, 1H); 6.87 (d, J = 8.40 Hz,
2H); 6.95–6.98 (m, 2H); 7.24 (s, 1H); 7.26–7.67 (m, 3H); 8.45–
8.75 (m, 2H); 9.20 (s, 1H). MS (ESI): 402.2 ([M+H]+). Anal. Calcd
for C24H23N3O3: C, 71.80; H, 5.77; N, 10.47%; Found: C, 71.47; H,
5.59; N, 10.75%.
To a solution of chalcone derivative (1 mmol) in isopropanol
(5 mL) hydrazine hydrate (0.2 mL, 4 mmol) was added. The
mixture 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
ethanol. The synthesized compound was purified by crystallization
from ethanol in refrigerator and allowed to air dry to give
4,5-dihydropyrazole derivatives.
4.4.6. (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl) 5-bromopyridin-3-yl methanone (27a)
Yellow powder, yield: 79%. Mp: 148–150 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.16–3.24 (m, 1H); 3.75–3.81 (m, 1H); 3.86 (s, 3H);
5.71–5.76(m, 1H); 6.94 (d, J = 8.97 Hz, 2H); 7.25–7.33 (m, 4H); 7.66
(d, J = 8.97 Hz, 2H); 8.46 (s, 1H); 8.76–8.77 (m, 1H); 9.21 (s, 1H). MS
(ESI): 470.0 ([M+H]+). Anal. Calcd for C22H17BrClN3O2:C, 56.13; H,
3.64; N, 8.93%; Found: C, 56.42; H, 3.46; N, 8.87%.
4.4. General method of synthesis of (3,5-diphenyl-4,5-dihydro-
pyrazol-1-yl) pyridine-3-yl methanone
To a solution of pyrazoline derivatives (1 mmol) in dichloro-
methane (5 mL) nicotinic acid derivatives (1 mmol) was added,
together with EDC (1.2–1.5 mmol) and HOBt (1.2–1.5 mmol).
The mixture was refluxed 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 mixture extracted with ethyl acetate (3 Â 5 mL).
The combined ethyl acetate layers were back-extracted with satu-
rated sodium bicarbonate (1 Â 5 mL) and brine (1 Â 5 mL), dried
over MgSO4, filtered, and evaporated in vacuo. The residue was
crystallized from ethanol to give target compounds 26a–32e.
4.4.7. (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-chloropyridin-3-yl methanone (27b)
Grey powder, yield: 65%. Mp: 174–176 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.15–3.23 (m, 1H); 3.72–3.89 (m, 4H); 5.73–5.85
(m, 1H); 6.94 (d, J = 8.97 Hz, 2H); 7.26–7.34 (m, 4H); 7.40–7.42
(m, 1H); 7.64- 7.67 (m, 2H); 8.26–8.29 (m, 1H); 9.14 (s, 1H). MS
(ESI): 426.1 ([M+H]+). Anal. Calcd for C22H17Cl2N3O2: C, 61.98; H,
4.02; N, 9.86%; Found: C, 61.59; H, 4.13; N, 9.59%.
4.4.1. (3,5-Bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
5-bromopyridin-3-yl methanone (26a)
4.4.8. (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl) 2-chloro-6-methylpyridin-3-yl methanone
(27c)
White powder, yield: 65%. Mp: 144–146 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.23 (dd, J1 = 4.77 Hz, J2 = 17.73 Hz, 1H); 3.73–3.83
(m, 4H); 3.86 (s, 3H); 5.73 (dd, J1 = 4.74 Hz, J2 = 11.52 Hz, 1H);
6.86–6.96 (m, 4H); 7.24 (s, 2H); 7.67 (d, J = 8.76 Hz,2H); 8.46–
8.75 (m, 2H); 9.20 (s, 1H). MS (ESI): 466.1 ([M+H]+). Anal. Calcd
for C23H20BrN3O3: C, 59.24; H, 4.32; N, 9.01%; Found: C, 59.46; H,
4.17; N, 9.28%.
Yellow powder, yield: 81%. Mp: 151–153 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.62 (s, 3H); 3.17–3.25 (m, 1H); 3.79–3.89 (m, 4H);
5.63–5.72 (m, 1H); 6.70 (d, J = 8.76 Hz, 2H); 7.14–7.40 (m, 5H);
7.57 (d, J = 8.94 Hz, 2H); 7.68 (d, J = 7.68 Hz, 1H). MS (ESI): 440.1
([M+H]+). Anal. Calcd for C23H19Cl2N3O2: C, 62.74; H, 4.35; N,
9.54%; Found: C, 62.55; H, 4.51; N, 9.36%.
4.4.2. (3,5-Bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-
6-chloropyridin-3-yl methanone (26b)
4.4.9. (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl) 2-chloropyridin-3-yl methanone (27d)
Yellow powder, yield: 68%. Mp: 129–131 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.19–3.27 (m, 1H); 3.73–3.82 (m, 4H); 3.86(s, 3H);
5.73 (dd, J1 = 4.53 Hz, J2 = 11.52 Hz, 1H); 6.86–6.95 (m, 5H); 7.25(s,
1H); 7.41 (d, J = 8.31 Hz, 1H); 7.67 (d, J = 8.70 Hz, 2H); 8.28 (d,
J = 6.78 Hz,1H); 9.14 (s, 1H). MS (ESI): 422.1 ([M+H]+). Anal. Calcd
for C23H20ClN3O3: C, 65.48; H, 4.78; N, 9.96%; Found: C, 65.25; H,
4.99; N, 9.54%.
Red powder, yield: 73%. Mp: 128–130 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.17–3.25 (m, 1H); 3.79–3.89 (m, 4H); 5.70
(dd, J1 = 4.74 Hz, J2 = 11.52 Hz, 1H); 6.88 (d, J = 7.68 Hz, 2H);
7.31–7.43(m, 4H); 7.54 (d, J = 8.43 Hz, 2H); 7.77 (d, J = 8.87 Hz,
1H); 7.91–7.94 (m, 2H). MS (ESI): 426.1 ([M+H]+). Anal. Calcd for
C22H17Cl2N3O2: C, 61.98; H, 4.02; N, 9.86%; Found: C, 61.73; H,
4.21; N, 9.55%.

3752
C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
4.4.10. (5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-methylpyridin-3-yl methanone (27e)
MS (ESI): 542.1 ([M+H]+). Anal. Calcd for C29H24BrN3O3: C, 64.21;
H, 4.46; N, 7.75%; Found: C, 64.40; H, 4.62; N, 7.48%.
Yellow powder, yield: 75%. Mp: 161–163 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.63 (s, 3H); 3.13–3.20 (m, 1H); 3.73–3.85 (m, 4H);
5.72–5.78 (m, 1H); 6.81 (d, J = 8.79 Hz, 1H); 6.93 (d, J = 8.79 Hz, 2H);
7.28–7.33 (m, 3H); 7.61–7.69 (m, 3H); 8.20–8.22 (m, 1H); 9.25 (s,
1H). MS (ESI): 406.1 ([M+H]+). Anal. Calcd for C23H20ClN3O2: C,
68.06; H, 4.97; N, 10.35%; Found: C, 68.32; H, 4.79; N, 10.43%.
4.4.17. (5-(4-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)6-chloropyridin-3-yl methanone (29b)
Yellow powder, yield: 75%. Mp: 144–147 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.23 (dd, J1 = 4.53 Hz, J2 = 17.73 Hz, 1H); 3.72–
3.82(m, 1H; 3.86 (s, 3H); 5.04 (s, 2H); 5.73 (dd, J1 = 4.53 Hz,
J2 = 11.34 Hz, 1H); 6.93–6.96 (m, 4H); 7.26 (d, J = 6.03 Hz, 1H);
7.35–7.42 (m, 7H); 7.67 (d, J = 8.49 Hz, 2H); 8.28 (d, J = 6.42 Hz,
1H); 9.14 (s, 1H). MS (ESI): 498.2 ([M+H]+). Anal. Calcd for
4.4.11. (5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)5-bromopyridin-3-yl methanone (28a)
Yellow powder, yield: 61%. Mp: 170–172 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.17–3.24 (m, 1H); 3.77–3.83 (m, 1H); 3.87 (s, 3H);
5.70–5.76 (m, 1H); 6.95 (d, J = 8.67 Hz, 2H); 7.20–7.22 (m, 2H);
7.49 (d, J = 8.31 Hz, 2H); 7.61–7.68 (m, 2H); 8.47 (s, 1H); 8.77 (s,
1H); 9.22 (s, 1H). MS (ESI): 514.0 ([M+H]+). Anal. Calcd for
C22H17Br2N3O2: C, 51.29; H, 3.33; N, 8.16%; Found: C, 51.43; H,
3.17; N, 8.23%.
C29H24ClN3O3: C, 69.95; H, 4.86; N, 8.44%; Found: C, 69.59; H,
4.69; N, 8.27%.
4.4.18. (5-(4-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)2-chloro-6-methylpyridin-3-yl
methanone (29c)
Yellow powder, yield: 79%. Mp: 207–209 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.60 (s, 3H); 3.20–3.25 (m, 1H); 3.74–3.82 (m, 4H);
5.04 (s, 2H); 5.66–5.71 (m, 1H); 6.86–6.89 (m, 2H); 6.95–6.98 (m,
2H); 7.15 (d, J = 7.68 Hz,1H); 7.28–7.33 (m, 3H); 7.35–7.43 (m,
4H); 7.56 (d, J = 8.58 Hz, 2H); 7.66 (d, J = 7.68 Hz,1H). MS (ESI):
512.2([M+H]+). Anal. Calcd for C30H26ClN3O3: C, 70.38; H, 5.12; N,
8.21%; Found: C, 70.52; H, 5.31; N, 8.46%.
4.4.12. (5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-chloropyridin-3-yl methanone (28b)
Yellow powder, yield: 74%. Mp: 190–192 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.16–3.23 (m, 1H); 3.76–3.82 m, 1H); 3.87 (s, 3H);
5.70–5.75 (m, 1H); 6.94 (d, J = 8.70 Hz, 2H); 7.20–7.22 (m, 2H);
7.41–7.50 (m, 3H); 7.66 (d, J = 6.42 Hz, 2H); 8.28 (s, 1H); 9.15 (s,
1H). MS (ESI): 470.0 ([M+H]+). Anal. Calcd for C22H17BrClN3O2: C,
56.13; H, 3.64; N, 8.93%; Found: C, 56.35; H, 3.48; N, 8.85%.
4.4.19. (5-(4-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)2-chloropyridin-3-yl methanone (29d)
Brown powder, yield: 64%. Mp: 146–147 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.23 (dd, J1 = 4.56 Hz, J2 = 17.55 Hz, 1H); 3.75–3.85
(m, 4H); 5.05 (s, 2H); 5.70 (dd, J1 = 4.38 Hz, J2 = 11.52 Hz, 1H); 6.87
(d, J = 8.58 Hz, 2H); 6.97 (d, J = 8.79 Hz, 2H); 7.29–7.34 (m, 4H);
7.35–7.43 (m, 4H); 7.53–7.60 (m, 2H); 7.77 (dd, J1 = 1.83 Hz,
J2 = 7.5 Hz, 1H); 8.44–8.47 (m, 1H). MS (ESI): 598.2 ([M+H]+). Anal.
Calcd for C29H24ClN3O3: C, 69.95; H, 4.86; N, 8.44%; Found: C,
69.59; H, 4.69; N, 8.63%.
4.4.13. (5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)2-chloro-6-methylpyridin-3-yl methanone
(28c)
Yellow powder, yield: 78%. Mp: 159–161 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.60 (s, 3H); 3.15–3.23 (m, 1H); 3.77–3.87 (m, 4H);
5.65–5.70 (m, 1H); 6.88 (d, J = 8.94 Hz, 2H); 7.16 (d, J = 7.68 Hz,
1H); 7.24 (s, 1H); 7.26 (s, 1H); 7.48–7.51 (m, 2H); 7.55 (d,
J = 8.97 Hz, 2H); 7.66 (d, J = 7.68 Hz, 1H). MS (ESI): 484.0
([M+H]+). Anal. Calcd for C23H19BrClN3O2: C, 56.98; H, 3.95; N,
8.67%; Found: C, 56.64; H, 3.78; N, 8.72%.
4.4.20. (5-(4-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)6-methylpyridin-3-yl methanone
(29e)
4.4.14. (5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)2-chloropyridin-3-yl methanone (28d)
Yellow powder, yield: 75%. Mp: 139–141 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.17–3.24 (m, 1H); 3.79–3.88 (m, 4H); 5.69 (dd,
J1 = 4.77 Hz, J2 = 11.52 Hz, 1H); 6.87 (d, J = 8.43 Hz, 2H); 7.25–7.26
(m, 1H); 7.28 (s, 1H); 7.30–7.34 (m, 1H); 7.49–7.55 (m, 4H); 7.77
(dd, J1 = 2.01 Hz, J2 = 7.50 Hz, 1H); 8.46–8.48 (m, 1H). MS (ESI):
470.0 ([M+H]+). Anal. Calcd for C22H17BrClN3O2: C, 56.13; H, 3.64;
N, 8.93%; Found: C, 56.41; H, 3.48; N, 8.86%.
Grey powder, yield: 69%. Mp: 108–110 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.56 (s, 3H); 3.13–3.27 (m, 1H); 3.85–3.89 (m,
4H); 5.03–5.15 (m, 2H); 5.70–5.79 (m, 1H); 6.96–7.06 (m, 4H);
7.34–7.45 (m, 7H); 7.60 (d, J = 8.79 Hz, 2H); 7.77 (d, J = 15.54 Hz,
1H); 8.02–8.04 (m, 2H). MS (ESI): 478.2 ([M+H]+). Anal. Calcd for
C30H27N3O3: C, 75.45; H, 5.70; N, 8.80%; Found: C, 75.67; H, 5.55;
N, 8.48%.
4.4.21(.5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)5-bromopyridin-3-yl methanone (30a)
White powder, yield: 73%. Mp: 129–131 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.14–3.23 (m, 1H); 3.78–3.83 (m, 1H); 5.06 (s, 2H);
5.71–5.83 (m, 1H); 6.75 (s, 1H); 6.99 (d, J = 8.58 Hz , 3H); 7.49–7.57
(m, 3H); 7.62 (d, J = 8.97 Hz, 2H); 7.7–7.79 (m, 1H); 7.84 (d,
J = 8.83 Hz, 2H); 7.97–8.03 (m, 3H); 9.23–9.25 (m, 1H). MS (ESI):
546.1 ([M+H]+). Anal. Calcd for C28H21BrClN3O2: C, 61.50; H, 3.87;
N, 7.68%; Found: C, 61.73; H, 3.75; N, 7.93%.
4.4.15. (5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-methylpyridin-3-yl methanone (28e)
Grey powder, yield: 71%. Mp: 139–141 °C. 1H NMR (300 MHz,
CDCl3,
d ppm): 2.64 (s, 3H); 3.17–3.24 (m, 1H); 3.85–3.89
(m, 4H); 6.75 (s, 1H); 7.50–7.57 (m, 3H); 7.60–7.63 (m, 2H);
7.70–7.76 (m, 1H); 7.84 (d, J = 8.76 Hz, 2H); 7.96–7.99 (m, 2H);
8.03 (d, J = 8.94 Hz, 1H). MS (ESI): 450.1([M+H]+). Anal. Calcd for
C23H20BrN3O2: C, 61.34; H, 4.48; N, 9.33%; Found: C, 61.61; H,
4.54; N, 9.59%.
4.4.22(.5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-chloropyridin-3-yl methanone (30b)
Yellow powder, yield: 71%. Mp: 154–156 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.19–3.27 (m, 1H); 3.73–3.83 (m, 1H); 5.04 (s, 2H);
5.73–5.79 (m, 1H); 6.91–6.97 (m, 2H); 7.24 (d, J = 9.33 Hz , 2H);
7.31–7.42 (m, 4H); 7.46–7.51 (m, 3H) 7.61–7.66 (m, 3H); 8.24 (d,
J = 6.24 Hz ,1H); 9.10 (s, 1H). MS (ESI): 502.1 ([M+H]+). Anal. Calcd
for C28H21Cl2N3O2: C, 66.94; H, 4.21; N, 8.36%; Found: C, 66.58; H,
4.40; N, 8.52%.
4.4.16. (5-(4-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)5-bromopyridin-3-yl methanone (29a)
Yellow powder, yield: 63%. Mp: 106–108 °C. 1H NMR (300 MHz,
CDCl3,
d ppm): 3.20–3.27 (m, 1H); 3.78 (dd, J1 = 11.52 Hz,
J2 = 17.76 Hz, 1H); 3.86 (s, 3H); 5.04 (s, 2H); 5.71–5.76 (m, 1H);
6.93–6.96 (m, 5H); 7.26 (d, J = 6.03 Hz, 1H); 7.31–7.40 (m, 5H);
7.67 (d, J = 8.67 Hz, 2H); 8.47 (s, 1H); 8.76 (s, 1H); 9.21 (s, 1H).

C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
3753
4.4.23(.5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)2-chloro-6-methylpyridin-3-yl methanone (30c)
Orange powder, yield: 78%. Mp: 161–163 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.61 (s, 3H); 3.23 (d, J = 17.73 Hz ,1H); 3.71–3.85
(m, 1H); 5.04 (d, J = 8.25 Hz ,2H); 5.71 (d, J = 6.75 Hz ,1H); 6.97
(d, J = 8.43 Hz ,2H); 7.15–7.17 (m, 1H); 7.28–7.43 (m, 9H); 7.52
(d, J = 8.40 Hz ,2H); 7.65–7.68 (m, 1H). MS (ESI): 516.1 ([M+H]+).
Anal. Calcd for C29H23Cl2N3O2: C, 67.45; H, 4.49; N, 8.14%; Found:
C, 67.65; H, 4.69; N, 8.34%.
C21H14Cl3N3O: C, 58.56; H, 3.28; N, 9.76%; Found: C, 58.82; H,
3.41; N, 9.39%.
4.4.30. (3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)6-
methylpyridin-3-yl methanone (31e)
White powder, yield: 65%. Mp: 146–148 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.68 (s, 3H); 3.18–3.25 (m, 1H); 3.88–3.92 (m, 1H);
5.56–5.69 (m, 1H); 6.79 (s, 1H); 7.43–7.50 (m, 5H); 7.94 (d,
J = 8.61 Hz, 2H); 7.99–8.05 (m, 2H); 8.56 (s, 1H). MS (ESI):
410.1([M+H]+). Anal. Calcd for C22H17Cl2N3O: C, 64.40; H, 4.18; N,
10.24%; Found: C, 64.71; H, 4.09; N, 10.52%.
4.4.24(.5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)2-chloropyridin-3-yl methanone (30d)
Brown powder, yield: 74%. Mp: 162–164 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.24 (dd, J1 = 4.74 Hz, J2 = 17.73 Hz, 1H); 3.75–3.86
(m, 1H); 5.06 (s, 2H); 5.69–5.75 (m, 1H); 6.98 (d, J = 8.79 Hz , 2H);
7.28–7.32 (m, 4H); 7.35–7.44 (m, 3H); 7.47–7.51 (m, 3H) 7.53 (d,
J = 8.61 Hz , 2H); 7.75–7.78 (m, 1H); 8.46–8.48 (m, 1H). MS (ESI):
502.1 ([M+H]+). Anal. Calcd for C28H21Cl2N3O2: C, 66.94; H, 4.21;
N, 8.36%; Found: C, 66.73; H, 4.40; N, 8.01%.
4.4.31. (5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)5-bromopyridin-3-yl methanone (32a)
Pink powder, yield: 63%. Mp: 156–158 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.18–3.26 (m, 1H); 3.79–3.88 (m, 1H); 5.77 (dd,
J1 = 4.95 Hz, J2 = 11.70 Hz, 1H); 7.21 (d, J = 8.22 Hz, 2H); 7.43 (d,
J = 8.58 Hz, 2H); 7.51–7.66 (m, 4H); 8.44 (s, 1H); 8.79 (s, 1H);
9.20 (s, 1H). MS (ESI): 517.9 ([M+H]+). Anal. Calcd for
C21H14Br2ClN3O: C, 48.54; H, 2.72; N, 8.09%; Found: C, 48.63; H,
2.68; N, 8.41%.
4.4.25(.5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-methylpyridin-3-yl methanone (30e)
White powder, yield: 69%. Mp: 115–117 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.64 (s, 3H); 3.21–3.25 (m, 1H); 3.74–3.81 (m, 1H);
5.16 (s, 2H); 6.76 (m, 1H); 7.02–7.09 (m, 2H); 7.23–7.30 (m, 4H);
7.34–7.47 (m, 4H); 7.62–7.65 (m, 2H); 7.81 (d, J = 8.79 Hz, 1H);
7.91–8.00 (m, 2H); 8.67(s, 1H). MS (ESI): 482.2([M+H]+). Anal.
Calcd for C29H24ClN3O2: C, 72.27; H, 5.02; N, 8.72%; Found: C,
72.52; H, 5.19; N, 8.54%.
4.4.32. (5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)6-chloropyridin-3-yl methanone (32b)
White powder, yield: 68%. Mp: 175–178 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.21 (dd, J1 = 5.13 Hz, J2 = 17.91 Hz, 1H); 3.78–3.88
(m, 1H); 5.74–5.80 (m, 1H); 7.21 (d, J = 8.40 Hz, 2H); 7.41–7.46 (m,
3H); 7.49–7.59 (m, 2H); 7.66 (d, J = 8.40 Hz, 2H); 8.24–8.38 (m,
1H); 9.11–9.13 (m, 1H). MS (ESI): 474.0 ([M+H]+). Anal. Calcd for
C21H14BrCl2N3O: C, 53.08; H, 2.97; N, 8.84%; Found: C, 53.32; H,
2.76; N, 8.71%.
4.4.26. (3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)5-
bromopyridin-3-yl methanone (31a)
White powder, yield: 78%. Mp: 143–145 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.17–3.24 (m, 1H); 3.77–3.87 (m, 1H); 5.74–5.79
(m, 1H); 7.24–7.27 (m, 2H); 7.34 (d, J = 8.61 Hz, 2H); 7.41 (d,
J = 8.40 Hz, 2H); 7.64 (d, J = 8.61 Hz, 2H); 8.42 (s, 1H); 8.77–8.78
(m, 1H); 9.18 (s, 1H). MS (ESI): 474.0 ([M+H]+). Anal. Calcd for
4.4.33. (5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl2-chloro-6-methylpyridin-3-yl methanone
(32c)
Yellow powder, yield: 75%. Mp: 153–155 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.65 (s, 3H); 3.78–2.87 (m, 1H); 3.94–4.09 (m, 1H);
5.73–5.85 (m, 1H); 7.19 (d, J = 7.68 Hz, 2H); 7.39–7.42 (m, 2H);
7.39–7.45 (m, 3H); 7.78–7.89 (m, 2H); 8.14–8.21 (m, 1H). MS
(ESI): 488.0 ([M+H]+). Anal. Calcd for C22H16BrCl2N3O: C, 54.01;
H, 3.30; N, 8.59%; Found: C, 54.22; H, 3.25; N, 8.90%.
C21H14BrCl2N3O: C, 53.08; H, 2.97; N, 8.84%; Found: C, 53.32; H,
2.84; N, 8.79%.
4.4.27. (3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)6-
chloropyridin-3-yl methanone(31b)
4.4.34. (5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl)2-chloropyridin-3-yl methanone (32d)
Grey powder, yield: 73%. Mp: 158–160 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.19–3.27 (m, 1H); 3.73–3.79 (m, 1H); 5.72–5.83
(m, 1H); 7.29–7.33 (m, 2H); 7.53–7.57 (m, 2H); 7.75 (d,
J = 8.45 Hz ,1H); 7.83–8.77 (m, 3H); 7.94 (dd, J1 = 8.58 Hz,
J2 = 11.52 Hz, 2H); 8.23–8.27 (m, 1H). MS (ESI): 474.0 ([M+H]+).
Anal. Calcd for C21H14BrCl2N3O: C, 53.08; H, 2.97; N, 8.84%; Found:
C, 53.21; H, 2.79; N, 8.76%.
White powder, yield: 79%. Mp: 178–180 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 3.18–3.26 (m, 1H); 3.78–3.88 (m, 1H); 5.78 (dd,
J1 = 5.13 Hz, J2 = 11.70 Hz, 1H); 7.26 (s, 1H); 7.28 (d, J = 2.19 Hz,
1H); 7.35 (d, J = 8.40 Hz, 2H); 7.41–7.46 (m, 3H); 7.65 (dd,
J1 = 1.83 Hz, J2 = 6.78 Hz, 2H); 8.24–8.28 (m, 1H); 9.13 (s, 1H). MS
(ESI): 430.0([M+H]+). Anal. Calcd for C21H14Cl3N3O: C, 58.56; H,
3.28; N, 9.76%; Found: C, 58.34; H, 3.32; N, 9.45%.
4.4.28. (3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)2-
chloro-6-methylpyridin-3-yl methanone (31c)
4.4.35. (5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihydro-
1H-pyrazol-1-yl) 6-methylpyridin-3-yl methanone (32e)
White powder, yield: 65%. Mp: 154–156 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.63 (s, 3H); 3.15–3.25 (m, 1H); 3.89–3.93 (m,
1H); 5.58–5.67 (m, 1H); 6.79–6.83 (m, 1H); 7.47–7.56 (m, 4H);
7.67–7.89 (m, 3H); 7.94 (d, J = 8.79 Hz ,2H); 8.12–8.20 (m, 1H).
MS (ESI): 453.0([M+H]+). Anal. Calcd for C22H17BrClN3O: C, 58.11;
H, 3.77; N, 9.24%; Found: C, 58.43; H, 3.69; N, 9.45%.
White powder, yield: 63%. Mp: 187–189 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.65 (m, 3H); 3.78–3.83 (m, 1H); 3.96–4.06 (m, 1H);
5.72–5.81 (m, 1H); 7.16 (d, 2H); 7.29–7.32 (m, 2H); 7.39 (d,
J = 8.40 Hz, 3H); 7.78 (d, J = 8.04 Hz, 3H). MS (ESI): 444.0
([M+H]+). Anal. Calcd for C22H16Cl3N3O: C, 59.41; H, 3.63; N,
9.45%; Found: C, 59.63; H, 3.44; N, 9.78%.
4.4.29. (3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)2-
chloropyridin-3-yl methanone (31d)
4.5. Antiproliferative activity
White powder, yield: 66%. Mp: 178–180 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 2.94 (s, 1H); 3.99–4.03 (m, 1H); 5.67–5.79 (m, 1H);
7.16 (d, J = 8.40 Hz, 2H); 7.29–7.32 (m, 3H); 7.38 (d, J = 7.86 Hz,
3H); 7.68 (s, 3H). MS (ESI): 430.0([M+H]+). Anal. Calcd for
WM266.5 melanoma cells were cultured in DMEM/10% fetal bo-
vine serum, in 5% CO2 water saturated atmosphere at 37 °C. Cell sus-
pensions (10000/mL) were prepared and 100 lL/well dispensed

3754
C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
into 96-well plates (Costar) giving 1000 cells/well. 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.
(1) A series of ligand conformations are generated using high
temperature molecular dynamics with different random
seeds.
Aliquots (200
200
M, and 10 serial dilutions of 3Â prepared. Aliquots (100
of each dilution were added to the wells, giving doses ranging from
100 M–0.005 M. After a further incubated 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-diphenyltet-
razolium 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.
l
L) were diluted into 20 mL culture medium giving
(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.
l
l
L)
l
l
4.6. Kinase Assay
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) 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 per-
formed with a simulated annealing method. The heating
steps were 2000 with 700 of heating target temperature.
The cooling steps were 5000 with 300 cooling target temper-
ature. Ten molecular docking poses 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.
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
lL drug and 4
lL assay
lL of a solu-
l
assay dilution buffer was added. The kinase reaction was allowed
to continue at room temperature for 25 min and was then
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
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.
4.9. QSAR model
A subset of 35 compounds was utilized as a training set for
QSAR modeling. Since it is essential to assess the predictive power
of the resulting QSAR models on an external set of inhibitors, the
remaining 7 molecules (ca. 20% of the dataset) were employed as
an external test subset for validating the QSAR models by the Di-
verse Molecules protocol in Discovery Studio 3.1. The selected test
compounds are: 26a, 28a, 28e, 29b, 30d, 31d, 32b.
The inhibitory abilities of the compounds in these literatures
[IC50 (mol/L)] was changed to the minus logarithmic scale [pIC50
(mol/L)] and then used for subsequent QSAR analyses as the re-
sponse variable.
In Discovery Studio, the CHARMm force field is used and the
electrostatic potential and the van der Waals potential are treated
as separate terms. A +1e point charge is used as the electrostatic
potential probe and distance-dependent dielectric constant is used
to mimic the solvation effect. For the van der Waals potential a
carbon atom with a 1.73 Å radius is used as a probe. The truncation
for both the steric and the electrostatic energies was set to
30 kcal/mol. The standard parameters implemented in Discovery
Studio 3.1 were used.
4.7. Acute oral toxicity
Five thousand milligrams of compound 27e per kilogram of
bodyweight was administered to ten healthy rats by oral gavage.
The animals were observed for mortality, signs of gross toxicity
and behavioral changes at least once daily for 14 days. Bodyweights
were recorded prior to administration and again on Day 7 and 14.
All animals survived and appeared active and healthy throughout
the study. With the exception of one male that exhibited a loss in
body weight between Day 7 and 14, all animals gained bodyweight
over the 14-day observation period. There were no signs of gross
toxicity or abnormal behavior.
4.8. Docking
The pdb file about the crystal structure of the BRAF kinase
domain bound to SB-590885 (3FB8.pdb) was obtained from the
RCSB protein data bank (http://www.pdb.org). The molecular
docking procedure was performed by using CDOCKER protocol
for receptor-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 2008 and optimized with MMFF94 method. For
enzyme preparation, the hydrogen atoms were added with the
pH of the protein in the range of 6.5–8.5. CDOCKER is an imple-
mentation of a CHARMm based molecular docking tool using a
rigid receptor.25 It includes following steps:
A Partial Least-Squares (PLS) model is built using energy grids
as descriptors. QSAR models were built using the created 3D-QSAR
protocol of Discovery Studio 3.1.
Acknowledgment
This work was financed by NSFC (No. J1103512), by Agriculture
Research Project (NY1002), by PCSIRT (IRT1020) and by Natural
Science Foundation of Jiangsu Province (BK2009234).

C.-Y. Li et al. / Bioorg. Med. Chem. 20 (2012) 3746–3755
3755
13. 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.
14. 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.
15. 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.
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
1. Wellbrock, C.; Karasarides, M.; Marais, R. Nat. Rev. Mol. Cell Biol. 2004, 5, 875.
2. Hoshino, R.; Chatani, Y.; Yamori, T.; Tsuruo, T.; Oka, H.; Yoshida, O.; Shimada,
Y.; Ari-i, S.; Wada, H.; Fujimoto, J.; Kohno, M. Oncogene 1999, 18, 813.
3. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.;
Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, N.; Ewing, R.;
Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.;
Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.;
Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.;
Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
Weber, B. L.; Siegler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.;
Wooster, R.; Stratton, M. R.; Futreal, P. A. Nature 2002, 417, 949.
4. Gorden, A.; Osman, I.; Gai, W. M.; He, D.; Huang, W. Q.; Davidson, A.; Houghton,
A. N.; Busam, K.; Polsky, D. Cancer Res. 2003, 63, 3955.
5. Brose, M. S.; Volpe, P.; Feldman, M.; Kumar, M.; Rishi, I.; Gerrero, R.; Einhorn, E.;
Herlyn, M.; Minna, J.; Nicholson, A.; Roth, J. A.; Albelda, S. M.; Davies, H.; Cox,
C.; Brignell, G.; Stephens, P.; Futreal, P. A.; Wooster, R.; Stratton, M. R.; Weber,
B. L. Cancer Res. 2002, 62, 6997.
6. Kimura, E. T.; Nikiforova, M. N.; Zhu, Z. W.; Knauf, J. A.; Nikiforov, Y. E.; Fagin, J.
A. Cancer Res. 2003, 63, 1454.
17. Ridley, R. G.; Hofheinz, W.; Matile, H.; Jaquet, C.; Dorn, A.; Masciadri, R.;
Jolidon, S.; Richter, W. F.; Guenzi, A.; Girometta, M. A.; Urwyler, H.; Huber, W.;
Thaithong, S.; Peters, W. Antimicrob. Agents Chemother. 1846, 1996, 40.
18. De, D. Y. D.; Krogstad, F. M.; Byers, L. D.; Krogstad, D. J. J. Med. Chem. 1998, 41,
4918.
7. Xu, X. L.; Quiros, R. M.; Gattuso, P.; Ain, K. B.; Prinz, R. A. Cancer Res. 2003, 63,
4561.
19. Azarifar, D.; Ghasemnejad, H. Molecules 2003, 8, 642.
20. Cox, C. D.; Torrent, M.; Breslin, M. J.; Mariano, B. J.; Whitman, D. B.; Coleman, P.
J.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao, W.
K.; South, V. J.; Kohl, N. E.; Yan, Y. W.; Kuo, L. C.; Prueksaritanont, T.; Slaughter,
D. E.; Li, C. Z.; Mahan, E.; Lu, B.; Hartman, G. D. Bioorg. Med. Chem. Lett. 2006, 16,
3175.
21. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2009, 44, 1396.
22. Blackburn, C.; Duffey, M. O.; Gould, A. E.; Kulkarni, B.; Liu, J. X.; Menon, S.;
Nagayoshi, M.; Vos, T. J.; Williams, J. Bioorg. Med. Chem. Lett. 2012, 20, 4795.
23. Padayatty, S. J.; Riordan, H. D.; Hewitt, S. M.; Katz, A.; Hoffer, L. J.; Levine, M.
Can. Med. Assoc. J. 2006, 174, 937.
8. Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116, 855.
9. Gray-Schopfer, V. C.; Dias, S. D.; Marais, R. Cancer Metastasis Rev. 2005, 24, 165.
10. Sommers, J. A.; Sharma, S.; Doherty, K. M.; Karmakar, P.; Yang, Q.; Kenny, M. K.;
Harris, C. C.; Brosh, R. M. Cancer Res. 2005, 65, 1223.
11. Karasarides, M.; Chiloeches, A.; Hayward, R.; Niculescu-Duvaz, D.; Scanlon, I.;
Friedlos, F.; Ogilvie, L.; Hedley, D.; Martin, J.; Marshall, C. J.; Springer, C. J.;
Marais, R. Oncogene 2004, 23, 6292.
12. King, A. J.; Patrick, D. R.; Batorsky, R. S.; Ho, M. L.; Do, H. T.; Zhang, S. Y.; Kumar,
R.; Rusnak, D. W.; Takle, A. K.; Wilson, D. M.; Hugger, E.; Wang, L.; Karreth, F.;
Lougheed, J. C.; Lee, J.; Chau, D.; Stout, T. J.; May, E. W.; Rominger, C. M.;
Schaber, M. D.; Luo, L.; Lakdawala, A. S.; Adams, J. L.; Contractor, R. G.; Smalley,
K. S. M.; Herlyn, M.; Morrissey, M. M.; Tuveson, D. A.; Huang, P. S. Cancer Res.
2006, 66, 11100.
24. Kirkpatrick, P. Nat. Rev. Drug Disc. 2004, 3, 299.
25. Wu, G. S.; Robertson, D. H.; Brooks, C. L.; Vieth, M. J. Comput. Chem. 2003, 24,
1549.