Synthesis and biological evaluation of pyrazole derivatives containing thiourea skeleton as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.05.034

Two series of pyrazole derivatives designing for potential EGFR kinase inhibitors have been discovered. Some of them exhibited significant EGFR inhibitory activity. Compound 3-(3,4-dimethylphenyl)-5-(4-methoxyphenyl)-4,5- dihydro-1H-pyrazole-1-carbothioamide (C5) displayed the most potent EGFR inhibitory activity with IC₅₀ of 0.07 μM, which was comparable to the positive control erlotinib. Docking simulation was performed to position compound C5 into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicating that some of the pyrazole derivatives own high antiproliferative activity against MCF-7. Compound C5 showed significant antiproliferative activity against MCF-7 with IC₅₀ of 0.08 μM. Therefore, compound C5 with potent inhibitory activity in tumor growth inhibition would be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2010.05.034

Bioorganic & Medicinal Chemistry 18 (2010) 4606–4614
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of pyrazole derivatives containing
thiourea skeleton as anticancer agents
*
Peng-Cheng Lv , Huan-Qiu Li , Juan Sun, Yang Zhou, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, 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:
Two series of pyrazole derivatives designing for potential EGFR kinase inhibitors have been discovered.
Some of them exhibited significant EGFR inhibitory activity. Compound 3-(3,4-dimethylphenyl)-5-(4-
methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (C5) displayed the most potent EGFR inhibi-
Received 3 April 2010
Revised 12 May 2010
Accepted 13 May 2010
Available online 20 May 2010
tory activity with IC50 of 0.07 lM, which was comparable to the positive control erlotinib. Docking sim-
ulation was performed to position compound C5 into the EGFR active site to determine the probable
binding model. Antiproliferative assay results indicating that some of the pyrazole derivatives own high
antiproliferative activity against MCF-7. Compound C5 showed significant antiproliferative activity
Keywords:
Pyrazole derivatives
Thiourea
EGFR
Inhibitors
against MCF-7 with IC50 of 0.08
lM. Therefore, compound C5 with potent inhibitory activity in tumor
growth inhibition would be a potential anticancer agent.
Ó 2010 Elsevier Ltd. All rights reserved.
Anticancer
1. Introduction
to the ATP binding site of KDR kinase, the amino thienopyrimidine
core mimics the adenine of ATP in its interaction with the hinge re-
gion of KDR. Hydrogen bonds are formed between the exocyclic
amino group of pyrazolopyrimidine ureas and the backbone car-
bonyl of Glu 917 and the proximal ring nitrogen and the backbone
N–H of Cys 919.14
Cancer chemotherapy has entered a new era of molecularly
targeted therapeutics, which is highly selective and not associated
with the serious toxicities of conventional cytotoxic drugs.1
Receptor protein tyrosine kinases play a key role in signal trans-
duction pathways that regulate cell division and differentiation.
Among the growth factor receptor kinases that have been identi-
fied as being important in cancer is epidermal growth factor
receptor (EGFR) kinase. Activation of EGFR may be because of
overexpression, mutations resulting in constitutive activation, or
autocrine expression of ligand.2,3 The role of EGFR has been most
thoroughly studied in breast cancer, where it is overexpressed in
25–30% of cases and is correlated with a poor prognosis. EGFR
overexpression is also seen in ovarian cancer,4 lung cancer (espe-
cially lung adenocarcinomas)5–7 and in hormone-refractory pros-
tate cancer.8
Compounds that inhibit the kinase activity of EGFR after bind-
ing of its cognate ligand are of potential interest as new therapeu-
tic antitumor agents.9,10
The thiourea and urea derivatives play important role in anti-
cancer agents because of their good inhibitory activity against
receptor tyrosine kinases (RTKs), protein tyrosine kinases (PTKs),
and NADH oxidase, which play critical roles in many aspects of
tumorigenesis.11–13 For example, pyrazolopyrimidine ureas bound
Many pyrazole derivatives are acknowledged to possess a wide
range of bioactivities. The pyrazole motif makes up the core struc-
ture of numerous biologically active compounds. Thus, some repre-
sentatives of this heterocycle exhibit anti-viral/anti-tumor,15–17
antibacterial,18–21 antiinflamatory,22 analgesic,23 fungistatic,24 and
anti-hyperglycemic activity.25,26 Much attention was paid to pyra-
zole as a potential antimicrobial agent after the discovery of the
natural pyrazole C-glycoside, pyrazofurin which demonstrated a
broad spectrum of antimicrobial activity.27 However, to our knowl-
edge, few reports have been dedicated to the synthesis and EGFR
inhibitory activity of pyrazole derivatives containing thiourea skel-
eton. Herein, in continuation to extend our research on anticancer
compounds with EGFR inhibitory activity,28,29 we report in the
present work the synthesis and structure–activity relationships of
a series of pyrazole derivatives containing thiourea skeleton as anti-
cancer agents. Biological evaluation indicated that some of the syn-
thesized compounds are potent inhibitors of EGFR. Compound C5
displayed the most potent EGFR inhibitory activity with IC50 of
0.07
(IC50 = 0.03
l
M, which was comparable to the positive control erlotinib
M). Docking simulations were performed using the
l
X-ray crystallographic structure of the EGFR in complex with an
inhibitor to explore the binding modes of these compounds at the
active site.
* Corresponding author. Tel./fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
Both authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.034

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
4607
of EGFR kinases using a solid-phase ELISA assay. The inhibition
constant (IC50) of the compounds were summarized in Tables 1
and 2. As shown in Table 1, it was observed that pyrazole deriva-
tives containing thiourea skeleton (compounds C1–C30) have been
found to show fairly good inhibitory activity displaying IC50 values
2. Results and discussion
2.1. Chemistry
The synthesis of compounds C1–C30 and D1–D16 followed the
general pathway outlined in Scheme 1. They are prepared in two
steps. Firstly, the chalcones were obtained by direct condensation
between the aromatic aldehydes and the substituted acetophe-
none, using 20% potassium hydroxide as catalyst in ethanol. Sec-
ondly, for compounds C1–C30, cyclization of different chalcones
with thiosemicarbazide under basic condition in refluxing ethanol
leads to the formation of pyrazole derivatives containing thiourea
skeleton. As for compounds D1–D16, a solution of chalcones
(5 mmol) in 30 mL of acetic acid was added dropwise to 0.6 mL
of hydrazine hydrate (12.5 mmol) and kept under stirring at
120 °C for 24 h. The mixture was then poured into ice-water,
obtaining the 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole
derivatives D1–D16. All of the synthetic compounds gave satisfac-
tory analytical and spectroscopic data, which were in full accor-
dance with their depicted structures.
between 0.07 and 13.37 lM. In order to help understand the struc-
ture–activity relationship, a series of 1-acetyl-3,5-diphenyl-4,5-
dihydro-(1H)-pyrazole derivatives (compounds D1–D16) have
been synthesized and also tested for their EGFR kinase inhibitory
activity.
As illustrated in Table 2, compounds D1–D16 displayed moder-
ate EGFR inhibitory activity with IC50 ranging from 15.24 to
28.69 lM. These results indicated that the thiourea skeleton in
the pyrazole derivatives C1–C30 play an important role in the EGFR
inhibitory activity. For example, there is only one difference be-
tween compounds C1–C5 and compounds D9–D13, that is, the for-
mer five compounds have the thiourea skeleton. Compounds C1–
C5 exhibited potent EGFR inhibitory activity with IC50 ranging from
0.07 to 2.17
lM, however, compounds D9–D13 showed moderate
EGFR inhibitory activity with IC50 more that 15.24
l
M. The same
rule also applies to compounds C11–C15 and compounds D1–D5
(see Tables 1 and 2).
2.2. EGFR inhibitory activity and structure–activity relationship
of the pyrazole derivatives C1–C30 and D1–D16
Subsequently SAR studies were performed by modification of
the parent compound to determine how the substituents of the
subunits affected the EGFR inhibitory activities. Inspection of the
The two series of pyrazole derivatives (C1–C30 and D1–D16)
chemical structure of the compounds C1–C30 (Table
1 and
were evaluated for their ability to inhibit the autophosphorylation
i
R1
R2
R1
O
O
A
B
R1
I
ii
II
N
R2
N
S
H2N
C1-C30
R3
III
iii
IV
N
R4
N
O
H3C
D1-D16
Scheme 1. General synthesis of compounds C1–C30 and D1–D16. Reagents and conditions: (i) substituted aromatic aldehyde, 40% NaOH, CH3CH2OH; (ii) thiosemicarbazide,
CH3CH2OH, reflux; (iii) N2H4ꢀH2O, CH3COOH, 120 °C, reflux, 12 h.

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P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
Table 1
Chemical structures and EGFR inhibitory activity of compounds C1–C30
R1
I
II
N
R2
N
S
H2N
M)
Compd
R1
R2
EGFR (IC50
,
l
Compd
R1
R2
EGFR (IC50, lM)
C1
C2
C3
C4
C5
C6
C7
C8
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2Cl
4-F
4-Cl
4-Br
4-CH3
4-OCH3
4-OH
4-NO2
2-F
2-Cl
2-Br
4-F
4-Cl
4-Br
4-CH3
4-OCH3
0.83
1.36
2.17
0.34
0.07
0.13
3.06
5.19
3.87
4.21
6.27
7.32
6.85
7.38
5.74
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
3,4-2Br
4-OH
4-NO2
2-F
2-Cl
2-Br
4-F
4-Cl
4-Br
4-CH3
4-OCH3
4-OH
4-NO2
2-F
5.27
7.78
6.56
7.28
6.43
8.92
8.15
10.06
9.83
8.09
11.27
10.64
13.37
12.26
10.97
C9
C10
C11
C12
C13
C14
C15
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
2-Cl
2-Br
Erlotinib
—
—
0.03
—
—
—
—
Table 2
Chemical structures and EGFR inhibitory activity of compounds D1–D16
R3
III
IV
N
R4
N
O
H3C
Compd
R3
R4
EGFR (IC50
,
lM)
Compd
R3
R4
EGFR (IC50, lM)
D1
D2
D3
D4
D5
D6
D7
D8
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
3,4-2Cl
4-F
4-Cl
4-Br
4-CH3
4-OCH3
2-Cl
4-H
3,5-2OCH3
18.35
16.14
18.45
19.32
17.19
24.15
28.69
25.47
D9
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
3,4-2CH3
4-F
4-Cl
4-Br
4-CH3
4-OCH3
2-Cl
4-H
3,5-2OCH3
15.56
19.73
20.61
17.49
15.24
21.48
20.04
17.31
D10
D11
D12
D13
D14
D15
D16
Erlotinib
—
—
0.03
—
—
—
—
Scheme 1) suggested that it could be divided into two subunits: I-
and II-rings.
As shown in Table 1, structure–activity relationships in com-
pounds C1–C30 demonstrated that compounds bearing two methyl
group at 3-position and 4-position at I-ring (compounds C1–C10,
IC50: 0.07–5.19 M) showed better EGFR inhibitory activity than
those with two halogen substituents (compounds C11–C30, IC50
5.27–13.37 M) at the same position. Among the former, com-
l
:
l

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
4609
pounds C8–C10 (IC50: 3.87–5.19
sition of the II-ring displayed less inhibitory activity than those with
4-position (compounds C1–C7, IC50: 0.07–3.06 M).
lM) with substitution on the 2-po-
l
As for compounds C1–C7, a comparison of the substitution on
II-ring demonstrated that the electron-donating substituents at
4-position derivatives (compounds C4–C6, IC50: 0.07–0.34
have more potent EGFR inhibitory activities than the electron-
withdrawing substituents ones (compounds C1–C3 and C7, IC50
1.06–3.16 M). Most significantly, the stronger electron-donating
lM)
:
l
substituents the compounds contained on II-ring at 4-position,
the more potent it showed, which was illustrated by the potency
order OCH3 > OH > CH3. Among them, compound C5 displayed
the most potent EGFR inhibitory activity with IC50 of 0.07
which was comparable to the positive control erlotinib (IC50
0.03 M).
lM,
=
Figure 2. Binding model of compound C5 and EGFR kinase. The H-bond (yellow
line) is displayed as line, and the dots show the binding sites of EGFR kinase. For
clarity, only interacting residues are displayed.
l
2.3. The antiproliferative activity and molecular docking study
of the top 10 compounds
The binding affinity was evaluated by the hydrogen bonding
and binding free energies (DGb, kcal/mol). Compound C5 which
displayed the most potent EGFR inhibitory activity was selected
for further molecular docking study. Then, molecular docking of
the most potent inhibitor C5 into ATP binding site of EGFR kinase
was performed on the binding model based on the EGFR complex
structure (1M17.pdb). The binding model of compound C5 and
EGFR is depicted in Figures 1–3. In the binding model, compound
C5 is nicely bound to the EGFR kinase with its N–H group project
toward the side chain carbonyl group of D831 (Asp831), forming
a
more optimal H-bond interaction (distance: 1.94 Å, angle:
127.1°). Based on the significant EGFR inhibitory activity of pyra-
zole derivatives containing thiourea skeleton, it can be concluded
that this H-bond plays an important effect in the EGFR inhibitory.
Also, the oxygen atom of the methoxy group of compound C5
forms hydrogen bond with G697 (Gly697) (distance: 2.20 Å, angle:
156.8°). The predicted binding free energy that includes the inter-
molecular energy and torsional free energy was used as the crite-
rion for ranking. The selected pose of compound C5 had an
estimated free energy of binding of ꢁ10.17 kcal/mol. The esti-
mated free energy of other compounds (C1–C4, C6–C10) are rang-
ing from ꢁ9.78 to ꢁ6.57 kcal/mol. Furthermore, the intermolecular
hydrogen bonds of compound C5 (Fig. 1), whose effect has already
been counted in the binding energy, were also investigated in order
to find useful information for drug design. This molecular docking
result, along with the biological assay data, suggesting that com-
pound C5 is a potential inhibitor of EGFR.
Figure 3. 3D model of the interaction between compound C5 and the EGFR kinase
binding site. The EGFR kinase is represented by molecular surface. Compound C5 is
depicted by sticks and balls.
Table 3
Antiproliferative activity and docking parameters of the top 10 compounds
Top compounds
MCF-7 (IC50
,
lM)
Docking parameters
Gb (kcal/mol)
D
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
0.58 0.03
0.71 0.05
0.64 0.05
0.31 0.07
0.08 0.01
0.17 0.02
0.96 0.06
1.54 0.09
1.83 0.12
1.48 0.08
ꢁ7.76
ꢁ8.29
ꢁ7.86
ꢁ8.62
ꢁ10.17
ꢁ9.78
ꢁ8.07
ꢁ7.49
ꢁ6.57
ꢁ7.14
Erlotinib
0.02 0.005
ꢁ10.86
Besides, the in vitro antiproliferative activity of the top 10 com-
pounds (compounds C1–C7 and C9) was studied on a panel of one
human tumor cell line (MCF-7), which overexpresses EGFR. As
shown in Table 3, out of the top 10 compounds, compounds C5
and C6, which have potent inhibitory activity of EGFR showed high
antiproliferative activity against MCF-7 with IC50 of 0.08 and
0.17 lM, indicating that these pyrazole derivatives were potent
inhibitor of EGFR as antitumor agents. In particular, compound
C5 has demonstrated significant inhibitory activity in tumor
growth inhibition and displayed favorable EGFR inhibitory activity.
Figure 1. Molecular docking modeling of compound C5 with EGFR kinase: for
clarity, only interacting residues are displayed. The H-bond (yellow line) is
displayed as line. Compound C5 is nicely bound to the EGFR kinase with its N–H
group project toward the side chain carbonyl group of D831 (Asp831), forming a
more optimal H-bond interaction. Also, the oxygen atom of the methoxy group of
compound C5 forms hydrogen bond with G697 (Gly697).
3. Conclusions
Two series of pyrazole derivatives that may function as inhibi-
tors of EGFR and kinases have been prepared, and some of the

4610
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
synthesized compounds displayed potent EGFR inhibitory. Com-
pound C5 exhibited the most potent EGFR inhibitory activity with
7.14–7.19 (m, 3H); 7.27–7.30 (m, 2H); 7.41–7.44 (m, 1H); 7.50 (s,
1H). MS (ESI): 344.1 (C18H19ClN3S, [M+H]+). Anal. Calcd for
C18H18ClN3S: C, 62.87; H, 5.28; N, 12.22. Found: C, 62.69; H,
5.42; N, 12.41.
IC50 of 0.07
erlotinib.
lM, which was comparable to the positive control
Compound C5 is nicely bound to the EGFR kinase with its N–H
group project toward the side chain carbonyl group of Asp831,
forming a more optimal H-bond interaction. Also, the oxygen atom
of the methoxy group of compound C5 forms hydrogen bond with
Gly697. Antiproliferative assay results indicated that these
pyrazole derivatives own high antiproliferative activity against
MCF-7. In particular, compound C5 has demonstrated significant
EGFR inhibitory activity and inhibitory activity in tumor growth
inhibition as a potential anticancer agent.
4.2.2.3.
5-(4-Bromophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C3). White powder. Mp
231–233 °C. 1H NMR (300 MHz, DMSO-d6): 2.30 (s, 6H); 3.11–
3.18 (dd, J1 = 3.66, J2 = 17.55 Hz, 1H); 3.76–3.86 (dd, J1 = 11.52,
J2 = 17.76 Hz, 1H); 5.94–5.99 (dd, J1 = 3.66, J2 = 11.52 Hz, 1H);
7.08–7.19 (m, 3H); 7.41–7.44 (m, 2H); 7.45–7.47 (m, 1H); 7.51 (s,
1H). MS (ESI): 388.1 (C18H19BrN3S, [M+H]+). Anal. Calcd for
C18H18BrN3S: C, 55.67; H, 4.67; N, 10.82. Found: C, 55.78; H,
4.81; N, 10.64.
4. Experimental section
4.1. General
4.2.2.4. 3-(3,4-Dimethylphenyl)-5-p-tolyl-4,5-dihydro-1H-pyra-
zole-1-carbothioamide (C4). White powder. Mp 198–200 °C. 1H
NMR (300 MHz, DMSO-d6): 2.30 (s, 9H); 3.14–3.21 (dd, J1 = 3.48,
J2 = 17.55 Hz, 1H); 3.74–3.84 (dd, J1 = 11.34, J2 = 17.55 Hz, 1H);
5.96–6.01 (dd, J1 = 3.45, J2 = 11.34 Hz, 1H); 7.08–7.13 (m, 3H);
7.14–7.18 (m, 2H); 7.41–7.44 (m, 1H); 7.51 (s, 1H). MS (ESI):
324.1 (C19H22N3S, [M+H]+). Anal. Calcd for C19H21N3S: C, 70.55;
H, 6.54; N, 12.99. Found: C, 70.74; H, 6.42; N, 12.76.
Separation of the compounds by column chromatography was
carried out with silica gel 60 (200–300 mesh ASTM, E. Merck).
The quantity of silica gel used was 50–100 times the weight
charged on the column. Then, the eluates were monitored using
TLC. Melting points (uncorrected) were determined on a XT4 MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were ob-
tained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spec-
trometer at 25 °C with TMS and solvent signals allotted as internal
standards. Chemical shifts were reported in ppm (d). Elemental
analyses were performed on a CHN-O-Rapid instrument and were
within 0.4% of the theoretical values.
4.2.2.5. 3-(3,4-Dimethylphenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C5). White powder. Mp
207–209 °C. 1H NMR (300 MHz, DMSO-d6): 2.31 (s, 6H); 3.11–
3.19 (dd, J1 = 3.47, J2 = 17.56 Hz, 1H); 3.71 (s, 3H); 3.72–3.81 (dd,
J1 = 11.42,
J2 = 17.55 Hz,
1H);
5.97–6.03
(dd,
J1 = 3.45,
J2 = 11.34 Hz, 1H); 7.06–7.11 (m, 3H); 7.15–7.18 (m, 2H); 7.42–
7.45 (m, 1H); 7.51 (s, 1H). MS (ESI): 340.1 (C19H22N3OS, [M+H]+).
Anal. Calcd for C19H21N3OS: C, 67.23; H, 6.24; N, 12.38. Found: C,
67.48; H, 6.46; N, 12.17.
4.2. Synthesis
4.2.1. General synthetic procedure of chalcones
4.2.2.6. 3-(3,4-Dimethylphenyl)-5-(4-hydroxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C6). White powder. Mp
218–219 °C. 1H NMR (300 MHz, DMSO-d6): 2.30 (s, 6H); 3.12–
3.18 (dd, J1 = 3.67, J2 = 17.55 Hz, 1H); 3.74–3.83 (dd, J1 = 11.53,
J2 = 17.76 Hz, 1H); 5.93–5.99 (dd, J1 = 3.68, J2 = 11.53 Hz, 1H);
7.09–7.19 (m, 3H); 7.41–7.44 (m, 2H); 7.44–7.46 (m, 1H); 7.50 (s,
1H). MS (ESI): 326.1 (C18H20N3OS, [M+H]+). Anal. Calcd for
C18H19N3OS: C, 66.43; H, 5.88; N, 12.91. Found: C, 66.65; H, 5.69;
N, 12.75.
Equimolar portions of the appropriately substituted aromatic
aldehydes (10 mmol, 1 equiv) and ketones (10 mmol, 1 equiv)
were dissolved in approximately 15 mL of ethanol. The mixture
was allowed to stir for several minutes at 5–10 °C. A 10 mL aliquot
of a 40% aqueous potassium hydroxide solution was then slowly
added dropwise to the reaction flask via a self-equalizing addition
funnel. The reaction solution was allowed to stir at room temper-
ature for approximately 4 h. Most commonly, a precipitate formed
and was then collected by suction filtration.
4.2.2.7. 3-(3,4-Dimethylphenyl)-5-(4-nitrophenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C7). White powder. Mp 202–
203 °C. 1H NMR (300 MHz, DMSO-d6): 2.31 (s, 6H); 3.13–3.19
(dd, J1 = 3.66, J2 = 17.55 Hz, 1H); 3.76–3.85 (dd, J1 = 11.51,
J2 = 17.73 Hz, 1H); 5.92–5.97 (dd, J1 = 3.65, J2 = 11.51 Hz, 1H);
7.07–7.16 (m, 3H); 7.40–7.43 (m, 2H); 7.45–7.47 (m, 1H); 7.50 (s,
1H). MS (ESI): 355.1 (C18H19N4O2S, [M+H]+). Anal. Calcd for
C18H18N4O2S: C, 61.00; H, 5.12; N, 15.81. Found: C, 61.18; H,
5.32; N, 15.64.
4.2.2. General synthetic procedure of pyrazole derivatives C1–C30
A mixture of chalcone (0.01 mol), thiosemicarbazide (0.01 mol),
and NaOH (0.025 mol) was refluxed in ethanol (25 mL) for 8 h. The
solution was poured into ice-water. The precipitate was filtered
and crystallized from methanol.
4.2.2.1.
3-(3,4-Dimethylphenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C1). White powder. Mp
223–225 °C. 1H NMR (300 MHz, DMSO-d6): 2.29 (s, 6H); 3.12–
3.18 (dd, J1 = 3.66, J2 = 17.58 Hz, 1H); 3.74–3.83 (dd, J1 = 11.52,
J2 = 17.76 Hz, 1H); 5.92–5.97 (dd, J1 = 3.66, J2 = 11.56 Hz, 1H);
7.09–7.18 (m, 3H); 7.42–7.46 (m, 2H); 7.46–7.48 (m, 1H); 7.50 (s,
1H). MS (ESI): 328.1 (C18H19FN3S, [M+H]+). Anal. Calcd for
C18H18FN3S: C, 66.03; H, 5.54; N, 12.83. Found: C, 66.17; H, 5.72;
N, 12.65.
4.2.2.8.
3-(3,4-Dimethylphenyl)-5-(2-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C8). White powder. Mp
211–213 °C. 1H NMR (300 MHz, DMSO-d6): 2.30 (s, 6H); 3.08–
3.15 (dd, J1 = 4.04, J2 = 17.89 Hz, 1H); 3.88–3.97 (dd, J1 = 11.53,
J2 = 17.96 Hz, 1H); 6.33–6.38 (dd, J1 = 4.09, J2 = 11.43 Hz, 1H);
7.08–7.12 (m, 1H); 7.16–7.26 (m, 3H); 7.39–7.45 (m, 2H); 7.51 (s,
1H). MS (ESI): 328.1 (C18H19FN3S, [M+H]+). Anal. Calcd for
C18H18FN3S: C, 66.03; H, 5.54; N, 12.83. Found: C, 66.19; H, 5.31;
N, 12.74.
4.2.2.2.
5-(4-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C2). White powder. Mp
216–217 °C. 1H NMR (300 MHz, DMSO-d6): 2.30 (s, 6H); 3.11–
3.18 (dd, J1 = 3.66, J2 = 17.73 Hz, 1H); 3.76–3.86 (dd, J1 = 11.52,
J2 = 17.73 Hz, 1H); 5.96–6.01 (dd, J1 = 3.66, J2 = 11.34 Hz, 1H);
4.2.2.9.
5-(2-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C9). White powder. Mp

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
4611
208–210 °C. 1H NMR (300 MHz, DMSO-d6): 2.29 (s, 6H); 3.06–3.14
(dd, J1 = 4.02, J2 = 17.94 Hz, 1H); 3.85–3.95 (dd, J1 = 11.52,
J2 = 17.91 Hz, 1H); 6.30–6.35 (dd, J1 = 4.05, J2 = 11.52 Hz, 1H);
7.04–7.07 (m, 1H); 7.15–7.23 (m, 3H); 7.37–7.44 (m, 2H); 7.50 (s,
1H). MS (ESI): 344.1 (C18H19ClN3S, [M+H]+). Anal. Calcd for
C18H18ClN3S: C, 62.87; H, 5.28; N, 12.22. Found: C, 62.63; H,
5.38; N, 12.46.
J1 = 3.67, J2 = 17.53 Hz, 1H); 3.72–3.83 (dd, J1 = 11.56, J2 = 17.76 Hz,
1H); 5.92–5.98 (dd, J1 = 3.68, J2 = 11.51 Hz, 1H); 7.08–7.19 (m, 3H);
7.42–7.44 (m, 2H); 7.44–7.47 (m, 1H); 7.50 (s, 1H). MS (ESI): 366.0
(C16H14Cl2N3OS, [M+H]+). Anal. Calcd for C16H13Cl2N3OS: C, 52.47;
H, 3.58; N, 11.47. Found: C, 52.62; H, 3.74; N, 11.59.
4.2.2.17. 3-(3,4-Dichlorophenyl)-5-(4-nitrophenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C17). White powder. Mp 203–
205 °C. 1H NMR (300 MHz, DMSO-d6): 3.13–3.18 (dd, J1 = 3.65,
J2 = 17.53 Hz, 1H); 3.74–3.83 (dd, J1 = 11.53, J2 = 17.73 Hz, 1H);
5.94–5.97 (dd, J1 = 3.65, J2 = 11.53 Hz, 1H); 7.07–7.14 (m, 3H);
7.40–7.43 (m, 2H); 7.46–7.48 (m, 1H); 7.50 (s, 1H). MS (ESI):
395.0 (C16H13Cl2N4O2S, [M+H]+). Anal. Calcd for C16H12Cl2N4O2S:
C, 48.62; H, 3.06; N, 14.17. Found: C, 48.76; H, 3.24; N, 14.34.
4.2.2.10. 5-(2-Bromophenyl)-3-(3,4-dimethylphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C10). White powder. Mp
217–219 °C. 1H NMR (300 MHz, DMSO-d6): 2.31 (s, 6H); 3.05–3.12
(dd, J1 = 4.06, J2 = 17.96 Hz, 1H); 3.83–3.94 (dd, J1 = 11.51, J2 =
17.93 Hz, 1H); 6.28–6.32 (dd, J1 = 4.03, J2 = 11.52 Hz, 1H); 7.06–
7.09 (m, 1H); 7.16–7.23 (m, 3H); 7.38–7.45 (m, 2H); 7.50 (s, 1H).
MS (ESI): 388.1 (C18H19BrN3S, [M+H]+). Anal. Calcd for C18H18BrN3S:
C, 55.67; H, 4.67; N, 10.82. Found: C, 55.83; H, 4.54; N, 10.74.
4.2.2.18.
3-(3,4-Dichlorophenyl)-5-(2-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C18). White powder. Mp
235–236 °C. 1H NMR (300 MHz, DMSO-d6): 3.09–3.15 (dd,
J1 = 4.16, J2 = 17.86 Hz, 1H); 3.85–3.96 (dd, J1 = 11.53, J2 = 17.96 Hz,
1H); 6.31–6.38 (dd, J1 = 4.21, J2 = 11.43 Hz, 1H); 7.08–7.14 (m, 1H);
7.18–7.26 (m, 3H); 7.36–7.43 (m, 2H); 7.51 (s, 1H). MS (ESI): 368.0
(C16H13Cl2FN3S, [M+H]+). Anal. Calcd for C16H12Cl2FN3S: C, 52.18;
H, 3.28; N, 11.41. Found: C, 52.32; H, 3.39; N, 11.58.
4.2.2.11.
3-(3,4-Dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C11). White powder. Mp
206–208 °C. 1H NMR (300 MHz, DMSO-d6): 3.11–3.17 (dd, J1 =
3.65, J2 = 17.51 Hz, 1H); 3.72–3.83 (dd, J1 = 11.32, J2 = 17.73 Hz,
1H); 5.91–5.96 (dd, J1 = 3.67, J2 = 11.51 Hz, 1H); 7.07–7.16 (m,
3H); 7.41–7.45 (m, 2H); 7.47–7.48 (m, 1H); 7.50 (s, 1H). MS
(ESI): 368.0 (C16H13Cl2FN3S, [M+H]+). Anal. Calcd for C16H12Cl2-
FN3S: C, 52.18; H, 3.28; N, 11.41. Found: C, 52.35; H, 3.41; N, 11.34.
4.2.2.19. 5-(2-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C19). White powder. Mp
241–243 °C. 1H NMR (300 MHz, DMSO-d6): 3.06–3.12 (dd,
J1 = 4.14, J2 = 17.92 Hz, 1H); 3.87–3.95 (dd, J1 = 11.53, J2 = 17.91 Hz,
1H); 6.31–6.35 (dd, J1 = 4.26, J2 = 11.52 Hz, 1H); 7.03–7.07 (m, 1H);
7.17–7.23 (m, 3H); 7.36–7.44 (m, 2H); 7.50 (s, 1H). MS (ESI): 383.0
(C16H13Cl3N3S, [M+H]+). Anal. Calcd for C16H12Cl3N3S: C, 49.95; H,
3.14; N, 10.92. Found: C, 49.76; H, 3.27; N, 10.81.
4.2.2.12. 5-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C12). White powder. Mp
211–213 °C. 1H NMR (300 MHz, DMSO-d6): 3.11–3.17 (dd,
J1 = 3.68, J2 = 17.75 Hz, 1H); 3.75–3.86 (dd, J1 = 11.51, J2 = 17.73 Hz,
1H); 5.94–6.02 (dd, J1 = 3.65, J2 = 11.37 Hz, 1H); 7.13–7.17 (m, 3H);
7.28–7.32 (m, 2H); 7.40–7.44 (m, 1H); 7.51 (s, 1H). MS (ESI): 383.0
(C16H13Cl3N3S, [M+H]+). Anal. Calcd for C16H12Cl3N3S: C, 49.95; H,
3.14; N, 10.92. Found: C, 49.82; H, 3.26; N, 10.78.
4.2.2.20. 5-(2-Bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C20). White powder. Mp
229–230 °C. 1H NMR (300 MHz, DMSO-d6): 3.07–3.12 (dd,
J1 = 4.16, J2 = 17.93 Hz, 1H); 3.86–3.94 (dd, J1 = 11.53, J2 = 17.91 Hz,
1H); 6.26–6.32 (dd, J1 = 4.13, J2 = 11.51 Hz, 1H); 7.04–7.09 (m, 1H);
7.17–7.23 (m, 3H); 7.36–7.45 (m, 2H); 7.50 (s, 1H). MS (ESI): 427.0
(C16H13BrCl2N3S, [M+H]+). Anal. Calcd for C16H12BrCl2N3S: C,
44.78; H, 2.82; N, 9.79. Found: C, 44.92; H, 2.69; N, 9.93.
4.2.2.13. 5-(4-Bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C13). White powder. Mp
214–216 °C. 1H NMR (300 MHz, DMSO-d6): 3.12–3.18 (dd,
J1 = 3.68, J2 = 17.53 Hz, 1H); 3.74–3.86 (dd, J1 = 11.53, J2 = 17.76 Hz,
1H); 5.92–5.98 (dd, J1 = 3.66, J2 = 11.53 Hz, 1H); 7.07–7.17 (m, 3H);
7.42–7.45 (m, 2H); 7.47–7.48 (m, 1H); 7.51 (s, 1H). MS (ESI): 427.0
(C16H13BrCl2N3S, [M+H]+). Anal. Calcd for C16H12BrCl2N3S: C,
44.78; H, 2.82; N, 9.79. Found: C, 44.96; H, 2.66; N, 9.98.
4.2.2.21. 3-(3,4-Dibromophenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C21). White powder. Mp
246–247 °C. 1H NMR (300 MHz, DMSO-d6): 3.14–3.17 (dd,
J1 = 3.66, J2 = 17.51 Hz, 1H); 3.76–3.83 (dd, J1 = 11.31, J2 = 17.73 Hz,
1H); 5.92–5.96 (dd, J1 = 3.66, J2 = 11.56 Hz, 1H); 7.05–7.16 (m, 3H);
7.42–7.45 (m, 2H); 7.46–7.48 (m, 1H); 7.51 (s, 1H). MS (ESI): 455.0
(C16H13Br2FN3S, [M+H]+). Anal. Calcd for C16H12Br2FN3S: C, 42.04;
H, 2.65; N, 9.19. Found: C, 42.17; H, 2.74; N, 9.35.
4.2.2.14. 3-(3,4-Dichlorophenyl)-5-p-tolyl-4,5-dihydro-1H-pyra-
zole-1-carbothioamide (C14). White powder. Mp 223–225 °C. 1H
NMR (300 MHz, DMSO-d6): 2.30 (s, 3H); 3.12–3.21 (dd, J1 = 3.46,
J2 = 17.55 Hz, 1H); 3.76–3.84 (dd, J1 = 11.37, J2 = 17.53 Hz, 1H);
5.94–6.01 (dd, J1 = 3.46, J2 = 11.34 Hz, 1H); 7.06–7.13 (m, 3H);
7.13–7.17 (m, 2H); 7.41–7.43 (m, 1H); 7.50 (s, 1H). MS (ESI):
364.0 (C17H16Cl2N3S, [M+H]+). Anal. Calcd for C17H15Cl2N3S: C,
56.05; H, 4.15; N, 11.53. Found: C, 56.18; H, 4.26; N, 11.38.
4.2.2.22. 5-(4-Chlorophenyl)-3-(3,4-dibromophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C22). White powder. Mp
216–218 °C. 1H NMR (300 MHz, DMSO-d6): 3.12–3.17 (dd,
J1 = 3.66, J2 = 17.75 Hz, 1H); 3.73–3.86 (dd, J1 = 11.53, J2 = 17.71 Hz,
1H); 5.96–6.04 (dd, J1 = 3.65, J2 = 11.45 Hz, 1H); 7.15–7.17 (m, 3H);
7.26–7.32 (m, 2H); 7.41–7.44 (m, 1H); 7.51 (s, 1H). MS (ESI): 471.0
(C16H13Br2ClN3S, [M+H]+). Anal. Calcd for C16H12Br2ClN3S: C,
40.58; H, 2.55; N, 8.87. Found: C, 40.46; H, 2.68; N, 8.69.
4.2.2.15. 3-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C15). White powder. Mp
209–210 °C. 1H NMR (300 MHz, DMSO-d6): 3.12–3.19 (dd,
J1 = 3.46, J2 = 17.53 Hz, 1H); 3.71 (s, 3H); 3.74–3.81 (dd, J1 = 11.43,
J2 = 17.53 Hz, 1H); 5.94–6.01 (dd, J1 = 3.46, J2 = 11.34 Hz, 1H);
7.07–7.11 (m, 3H); 7.14–7.18 (m, 2H); 7.42–7.45 (m, 1H); 7.51 (s,
1H). MS (ESI): 380.0 (C17H16Cl2N3OS, [M+H]+). Anal. Calcd for
C17H15Cl2N3OS: C, 53.69; H, 3.98; N, 11.05. Found: C, 53.78; H,
3.81; N, 11.24.
4.2.2.23. 5-(4-Bromophenyl)-3-(3,4-dibromophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C23). White powder. Mp
239–241 °C. 1H NMR (300 MHz, DMSO-d6): 3.14–3.18 (dd,
J1 = 3.69, J2 = 17.53 Hz, 1H); 3.77–3.86 (dd, J1 = 11.51, J2 = 17.76 Hz,
1H); 5.91–5.98 (dd, J1 = 3.66, J2 = 11.57 Hz, 1H); 7.09–7.17 (m, 3H);
7.42–7.46 (m, 2H); 7.47–7.48 (m, 1H); 7.50 (s, 1H). MS (ESI): 515.0
4.2.2.16. 3-(3,4-Dichlorophenyl)-5-(4-hydroxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C16). White powder. Mp
235–237 °C. 1H NMR (300 MHz, DMSO-d6): 3.12–3.17 (dd,

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P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
(C16H13Br3N3S, [M+H]+). Anal. Calcd for C16H12Br3N3S: C, 37.09; H,
2.33; N, 8.11. Found: C, 37.18; H, 2.45; N, 8.32.
4.2.3. General synthetic procedure of pyrazole derivatives D1–D16
A solution of chalcone (5 mmol) in 30 mL of acetic acid was
added dropwise to 0.6 mL of hydrazine hydrate (12.5 mmol) and
kept under stirring at 120 °C for 24 h. The mixture was then poured
in ice-water, obtaining the crude pyrazole derivatives D1–D16,
which were crystallized from ethanol.
4.2.2.24. 3-(3,4-Dibromophenyl)-5-p-tolyl-4,5-dihydro-1H-pyra-
zole-1-carbothioamide (C24). White powder. Mp 211–213 °C. 1H
NMR (300 MHz, DMSO-d6): 2.30 (s, 3H); 3.13–3.21 (dd, J1 = 3.46,
J2 = 17.55 Hz, 1H); 3.74–3.84 (dd, J1 = 11.31, J2 = 17.57 Hz, 1H);
5.96–6.01 (dd, J1 = 3.46, J2 = 11.37 Hz, 1H); 7.08–7.13 (m, 3H);
7.13–7.17 (m, 2H); 7.41–7.43 (m, 1H); 7.50 (s, 1H). MS (ESI):
451.0 (C17H16Br2N3S, [M+H]+). Anal. Calcd for C17H15Br2N3S: C,
45.05; H, 3.34; N, 9.27. Found: C, 45.21; H, 3.48; N, 9.36.
4.2.3.1. 1-(3-(3,4-Dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (D1). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.30 (s, 3H); 3.17–3.22 (dd, J1 = 2.91, J2 =
10.98 Hz, 1H); 3.80–3.86 (dd, J1 = 7.32, J2 = 10.98 Hz, 1H); 5.55–
5.58 (dd, J1 = 2.94, J2 = 7.14 Hz, 1H); 7.12–7.16 (t, J = 5.31 Hz, 2H);
7.22–7.25 (m, 2H); 7.72–7.78 (m, 2H); 7.97 (d, J = 11.10 Hz, 1H).
MS (ESI): 351.0 (C17H14Cl2FN2O, [M+H]+). Anal. Calcd for C17H13Cl2-
FN2O: C, 58.14; H, 3.73; N, 7.98. Found: C, 58.16; H, 3.71; N, 7.84.
4.2.2.25. 3-(3,4-Dibromophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C25). White powder. Mp
236–238 °C. 1H NMR (300 MHz, DMSO-d6): 3.11–3.17 (dd, J1 = 3.43,
J2 = 17.53 Hz, 1H); 3.71 (s, 3H); 3.77–3.81 (dd, J1 = 11.46, J2 =
17.53 Hz, 1H); 5.97–6.01 (dd, J1 = 3.43, J2 = 11.34 Hz, 1H); 7.08–
7.11 (m, 3H); 7.16–7.18 (m, 2H); 7.42–7.45 (m, 1H); 7.51 (s, 1H).
MS (ESI): 467.0 (C17H16Br2N3OS, [M+H]+). Anal. Calcd for C17H15Br2-
N3OS: C, 43.52; H, 3.22; N, 8.96. Found: C, 43.68; H, 3.37; N, 8.84.
4.2.3.2. 1-(5-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (D2). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.30 (s, 3H); 3.17–3.22 (dd, J1 = 2.90,
J2 = 10.96 Hz, 1H); 3.81–3.87 (dd, J1 = 7.30, J2 = 10.91 Hz, 1H);
5.54–5.57 (dd, J1 = 2.96, J2 = 7.14 Hz, 1H); 7.21–7.23 (d, J = 5.13 Hz,
2H); 7.37–7.39 (d, J = 4.95 Hz, 2H); 7.72–7.78 (m, 2H); 7.96–7.97
(d, J = 11.10 Hz, 1H). MS (ESI): 367.0 (C17H14Cl3N2O, [M+H]+). Anal.
Calcd for C17H13Cl3N2O: C, 55.54; H, 3.56; N, 7.62. Found: C, 55.52;
H, 3.58; N, 7.53.
4.2.2.26. 3-(3,4-Dibromophenyl)-5-(4-hydroxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C26). White powder. Mp
221–223 °C. 1H NMR (300 MHz, DMSO-d6): 3.11–3.16 (dd,
J1 = 3.63, J2 = 17.51 Hz, 1H); 3.75–3.83 (dd, J1 = 11.53, J2 = 17.76 Hz,
1H); 5.93–5.98 (dd, J1 = 3.68, J2 = 11.53 Hz, 1H); 7.09–7.19 (m, 3H);
7.42–7.45 (m, 2H); 7.46–7.48 (m, 1H); 7.50 (s, 1H). MS (ESI): 453.0
(C16H14Br2N3OS, [M+H]+). Anal. Calcd for C16H13Br2N3OS: C, 42.22;
H, 2.88; N, 9.23. Found: C, 42.45; H, 2.69; N, 9.38.
4.2.3.3. 1-(5-(4-Bromophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (D3). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.30 (s, 3H); 3.17–3.22 (dd, J1 = 2.91,
J2 = 10.96 Hz, 1H); 3.81–3.87 (dd, J1 = 7.34, J2 = 10.98 Hz, 1H);
5.52–5.56 (dd, J1 = 2.96, J2 = 7.17 Hz, 1H); 7.21–7.23 (d, J = 5.13 Hz,
2H); 7.37–7.39 (d, J = 4.95 Hz, 2H); 7.72–7.78 (m, 2H); 7.96–7.97
(d, J = 11.10 Hz, 1H). MS (ESI): 410.9 (C17H14BrCl2N2O, [M+H]+). Anal.
Calcd for C17H13BrCl2N2O: C, 49.55; H, 3.18; N, 6.80. Found: C, 49.57;
H, 3.17; N, 6.68.
4.2.2.27. 3-(3,4-Dibromophenyl)-5-(4-nitrophenyl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C27). White powder. Mp 246–
248 °C. 1H NMR (300 MHz, DMSO-d6): 3.12–3.18 (dd, J1 = 3.69,
J2 = 17.53 Hz, 1H); 3.76–3.83 (dd, J1 = 11.51, J2 = 17.73 Hz, 1H);
5.92–5.97 (dd, J1 = 3.63, J2 = 11.53 Hz, 1H); 7.09–7.14 (m, 3H);
7.41–7.43 (m, 2H); 7.46–7.48 (m, 1H); 7.50 (s, 1H). MS (ESI):
482.0 (C16H13Br2N4O2S, [M+H]+). Anal. Calcd for C16H12Br2N4O2S:
C, 39.69; H, 2.50; N, 11.57. Found: C, 39.78; H, 2.41; N, 11.69.
4.2.3.4. 1-(3-(3,4-Dichlorophenyl)-5-p-tolyl-4,5-dihydro-1H-pyr-
azol-1-yl)ethanone (D4). White powder. 1H NMR (300 MHz,
DMSO-d6): 2.26 (s, 3H); 2.29 (s, 3H); 3.13–3.17 (dd, J1 = 2.76,
J2 = 10.80 Hz, 1H); 3.79–3.85 (dd, J1 = 7.32, J2 = 10.98 Hz, 1H);
5.49–5.52 (dd, J1 = 2.76, J2 = 7.14 Hz, 1H); 7.05–7.13 (dd, J1 = 4.74,
J2 = 17.37 Hz, 4H); 7.71–7.77 (m, 2H); 7.96–7.97 (d, J = 11.1 Hz,
4.2.2.28. 3-(3,4-Dibromophenyl)-5-(2-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C28). White powder. Mp
236–237 °C. 1H NMR (300 MHz, DMSO-d6): 3.07–3.13 (dd,
J1 = 4.18, J2 = 17.86 Hz, 1H); 3.83–3.92 (dd, J1 = 11.51, J2 = 17.96 Hz,
1H); 6.35–6.38 (dd, J1 = 4.36, J2 = 11.43 Hz, 1H); 7.12–7.14 (m, 1H);
7.19–7.26 (m, 3H); 7.36–7.43 (m, 2H); 7.51 (s, 1H). MS (ESI): 455.0
(C16H13Br2FN3S, [M+H]+). Anal. Calcd for C16H12Br2FN3S: C, 42.04;
H, 2.65; N, 9.19. Found: C, 42.16; H, 2.43; N, 9.31.
1H). MS (ESI): 347.1 (C18H17Cl2N2O, [M+H]+). Anal. Calcd for C18H16
Cl2N2O: C, 62.26; H, 4.64; N, 8.07. Found: C, 62.27; H, 4.62; N, 8.21.
-
4.2.3.5.
1-(3-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (D5). White powder. 1H
NMR (300 MHz, DMSO-d6): 2.25 (s, 3H); 3.11–3.16 (dd, J1 = 2.73,
J2 = 10.80 Hz, 1H); 3.68 (s, 3H); 3.74–3.80 (dd, J1 = 7.14,
J2 = 10.80 Hz, 1H); 5.45–5.49 (dd, J1 = 2.73, J2 = 6.93 Hz, 1H); 6.83–
6.85 (d, J = 5.31 Hz, 2H); 7.07–7.08 (d, J = 5.31 Hz, 2H); 7.69–7.70
(d, J = 5.13 Hz, 1H); 7.73–7.75 (dd, J1 = 1.29, J2 = 5.13 Hz, 1H); 7.94
(d, J = 11.1 Hz, 1H). MS (ESI): 363.1 (C18H17Cl2N2O2, [M+H]+). Anal.
Calcd for C18H16Cl2N2O2: C, 59.52; H, 4.44; N, 7.71. Found: C,
59.53; H, 4.42; N, 7.85.
4.2.2.29. 5-(2-Chlorophenyl)-3-(3,4-dibromophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C29). White powder. Mp
219–221 °C. 1H NMR (300 MHz, DMSO-d6): 3.08–3.12 (dd,
J1 = 4.26, J2 = 17.92 Hz, 1H); 3.89–3.95 (dd, J1 = 11.53, J2 = 17.91 Hz,
1H); 6.33–6.35 (dd, J1 = 4.26, J2 = 11.52 Hz, 1H); 7.04–7.07 (m, 1H);
7.17–7.26 (m, 3H); 7.36–7.44 (m, 2H); 7.50 (s, 1H). MS (ESI): 471.0
(C16H13Br2ClN3S, [M+H]+). Anal. Calcd for C16H12Br2ClN3S: C,
40.58; H, 2.55; N, 8.81. Found: C, 40.69; H, 2.38; N, 8.72.
4.2.3.6. 1-(5-(2-Chlorophenyl)-3-(3,4-dichlorophenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (D6). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.30 (s, 3H); 3.15–3.24 (dd, J1 = 2.91,
J2 = 10.96 Hz, 1H); 3.82–3.89 (dd, J1 = 7.34, J2 = 10.98 Hz, 1H);
5.51–5.58 (dd, J1 = 2.96, J2 = 7.17 Hz, 1H); 7.21–7.25 (d,
J = 5.13 Hz, 2H); 7.35–7.39 (d, J = 4.95 Hz, 2H); 7.72–7.78 (m, 2H);
7.94–7.97 (d, J = 11.10 Hz, 1H). MS (ESI): 367.0 (C17H14Cl3N2O,
[M+H]+). Anal. Calcd for C17H13Cl3N2O: C, 55.54; H, 3.56; N, 7.62.
Found: C, 55.56; H, 3.53; N, 7.78.
4.2.2.30. 5-(2-Bromophenyl)-3-(3,4-dibromophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carbothioamide (C30). White powder. Mp
214–216 °C. 1H NMR (300 MHz, DMSO-d6): 3.09–3.15 (dd,
J1 = 4.21, J2 = 17.91 Hz, 1H); 3.84–3.91 (dd, J1 = 11.52, J2 = 17.93 Hz,
1H); 6.29–6.32 (dd, J1 = 4.26, J2 = 11.51 Hz, 1H); 7.06–7.09 (m, 1H);
7.19–7.23 (m, 3H); 7.34–7.45 (m, 2H); 7.50 (s, 1H). MS (ESI): 515.0
(C16H13Br3N3S, [M+H]+). Anal. Calcd for C16H12Br3N3S: C, 37.09; H,
2.33; N, 8.11. Found: C, 37.19; H, 2.42; N, 8.32.

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
4613
4.2.3.7.
1-(3-(3,4-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-
4.2.3.14.
1-(5-(2-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-
pyrazol-1-yl)ethanone (D7). White powder. 1H NMR (300 MHz,
DMSO-d6): 2.31 (s, 3H); 3.14–3.22 (dd, J1 = 4.74, J2 = 18.27 Hz,
1H); 3.79–3.89 (dd, J1 = 11.88, J2 = 18.30 Hz, 1H); 5.53–5.58 (dd,
J1 = 4.77, J2 = 11.91 Hz, 1H); 7.17–7.35 (m, 5H); 7.70–7.79 (m,
2H); 7.70–7.72 (s, 1H). MS (ESI): 333.1 (C17H15Cl2N2O, [M+H]+).
Anal. Calcd for C17H14Cl2N2O: C, 61.28; H, 4.23; N, 8.41. Found: C,
61.26; H, 4.25; N, 8.56.
dihydro-1H-pyrazol-1-yl)ethanone (D14). White powder. 1H
NMR (300 MHz, DMSO-d6): 2.24 (s, 6H); 2.34 (s, 3H); 2.98–3.06
(dd, J1 = 4.74, J2 = 17.91 Hz, 1H); 3.86–3.96 (dd, J1 = 11.88,
J2 = 17.91 Hz, 1H); 5.70–5.76 (dd, J1 = 4.59, J2 = 11.73 Hz, 1H);
7.02–7.05 (m, 1H); 7.19–7.22 (d, J = 7.86 Hz, 1H); 7.27–7.30 (m,
2H); 7.47–7.55 (m, 3H). MS (ESI): 327.1 (C19H20ClN2O, [M+H]+).
Anal. Calcd for C19H19ClN2O: C, 69.83; H, 5.86; N, 8.57. Found: C,
69.85; H, 5.84; N, 8.71.
4.2.3.8.
1-(3-(3,4-Dichlorophenyl)-5-(3,5-dimethoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (D8). White powder. 1H
NMR (300 MHz, DMSO-d6): 2.26 (s, 3H); 3.12–3.16 (dd, J1 = 2.73,
J2 = 10.80 Hz, 1H); 3.68 (s, 6H); 3.73–3.83 (dd, J1 = 7.14,
J2 = 10.80 Hz, 1H); 5.45–5.49 (dd, J1 = 2.73, J2 = 6.93 Hz, 1H); 6.81–
6.85 (d, J = 5.31 Hz, 2H); 7.06–7.08 (d, J = 5.31 Hz, 2H); 7.69–7.70
(d, J = 5.13 Hz, 1H); 7.72–7.75 (dd, J1 = 1.29, J2 = 5.13 Hz, 1H); 7.95
(d, J = 11.1 Hz, 1H). MS (ESI): 393.1 (C19H19Cl2N2O3, [M+H]+). Anal.
Calcd for C19H18Cl2N2O3: C, 58.03; H, 4.61; N, 7.12. Found: C,
58.05; H, 4.59; N, 7.26.
4.2.3.15. 1-(3-(3,4-Dimethylphenyl)-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (D15). White powder. 1H NMR (300 MHz,
DMSO-d6): 2.25 (s, 6H); 2.30 (s, 3H); 3.05–3.12 (dd, J1 = 4.38,
J2 = 18.12 Hz, 1H); 3.77–3.87 (dd, J1 = 11.91, J2 = 18.12 Hz, 1H);
5.49–5.54 (dd, J1 = 4.23, J2 = 11.73 Hz, 1H); 7.15–7.34 (m, 6H);
7.48–7.56 (m, 2H). MS (ESI): 293.2 (C19H21N2O, [M+H]+). Anal. Calcd
for C19H20N2O: C, 78.05; H, 6.89; N, 9.58. Found: C, 78.07; H, 6.87; N,
9.67.
4.2.3.16. 1-(5-(3,5-Dimethoxyphenyl)-3-(3,4-dimethylphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)ethanone (D16). White powder.
1H NMR (300 MHz, DMSO-d6): 2.26 (s, 6H); 2.27 (s, 3H); 3.02–3.10
(dd, J1 = 3.84, J2 = 12.09 Hz, 1H); 3.71 (s, 6H); 3.74–3.81 (dd,
J1 = 11.68, J2 = 18.06 Hz, 1H); 5.42–5.47 (dd, J1 = 4.26, J2 = 10.91 Hz,
1H); 6.84–6.87 (d, J = 7.24, 2H); 7.06–7.10 (d, J = 7.22, 2H); 7.20–
7.22 (d, J = 4.86, 2H); 7.45–7.56 (m, 2H). MS (ESI): 353.2
(C21H25N2O3, [M+H]+). Anal. Calcd for C21H24N2O3: C, 71.57; H,
6.86; N, 7.95. Found: C, 71.55; H, 6.87; N, 7.84.
4.2.3.9. 1-(3-(3,4-Dimethylphenyl)-5-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazol-1-yl)ethanone (D9). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.25 (s, 6H); 2.29 (s, 3H); 3.07–3.14 (dd,
J1 = 2.76, J2 = 10.86 Hz, 1H); 3.76–3.86 (dd, J1 = 7.32, J2 = 10.80 Hz,
1H); 5.50–5.54 (dd, J1 = 2.76, J2 = 6.96 Hz, 1H); 7.09–7.23 (m, 5H);
7.48–7.50 (d, J = 7.86, 1H); 7.56 (s, 1H); MS (ESI): 311.2
(C19H20FN2O, [M+H]+). Anal. Calcd for C19H19FN2O: C, 73.53; H,
6.17; N, 9.03. Found: C, 73.55; H, 6.16; N, 9.16.
4.2.3.10. 1-(5-(4-Chlorophenyl)-3-(3,4-dimethylphenyl)-4,5-di-
hydro-1H-pyrazol-1-yl)ethanone (D10). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.25 (s, 6H); 2.29 (s, 3H); 3.06–3.14 (dd,
J1 = 2.76, J2 = 10.86 Hz, 1H); 3.77–3.87 (dd, J1 = 7.32, J2 = 10.80 Hz,
1H); 5.49–5.55 (dd, J1 = 2.76, J2 = 6.96 Hz, 1H); 7.18–7.23 (m, 3H);
7.36–7.39 (d, J = 8.22, 2H); 7.47–7.50 (d, J = 8.04, 2H); 7.56 (s, 1H);
MS (ESI): 327.1 (C19H20ClN2O, [M+H]+). Anal. Calcd for C19H19ClN2O:
C, 69.83; H, 5.86; N, 8.57. Found: C, 69.84; H, 5.84; N, 8.64.
4.3. EGFR inhibitory assay
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645–1186) were cloned into baculoviral
expression vector pFASTBacHTc. A sequence that encodes (His)6
was located at the 50 upstream to the EGFR sequence. Sf-9 cells
were infected for 3 days for protein expression. Sf-9 cell pellets
were solubilized at 0 °C in a buffer at pH 7.4 containing 50 mM
HEPES, 10 mM NaCl, 1% Triton, 10
100 M sodium vanadate, 10 g/mL aprotinin, 10
10 g/mL pepstatin, and 16 g/mL benzamidine HCl for 20 min fol-
lowed by 20 min centrifugation. Crude extract supernatant was
passed through an equilibrated Ni-NTA superflow packed column
and washed with 10 mM and then 100 mM imidazole to remove
nonspecifically bound material. Histidine tagged proteins were
eluted with 250 and 500 mM imidazole and dialyzed against
l
M ammonium molybdate,
4.2.3.11. 1-(5-(4-Bromophenyl)-3-(3,4-dimethylphenyl)-4,5-di-
hydro-1H-pyrazol-1-yl)ethanone (D11). White powder. 1H NMR
(300 MHz, DMSO-d6): 2.25 (s, 6H); 2.29 (s, 3H); 3.07–3.14 (dd,
J1 = 4.56, J2 = 18.09 Hz, 1H); 3.76–3.86 (dd, J1 = 11.88, J2 = 18.09 Hz,
1H); 5.47–5.53 (dd, J1 = 4.38, J2 = 11.70 Hz, 1H); 7.12–7.15 (d,
J = 8.43, 2H); 7.20–7.23 (d, J = 7.86, 1H); 7.47–7.55 (m, 4H). MS
(ESI): 371.1 (C19H20BrN2O, [M+H]+). Anal. Calcd for C19H19BrN2O:
C, 61.47; H, 5.16; N, 7.55. Found: C, 61.49; H, 5.13; N, 7.69.
l
l
l
lg/mL leupeptin,
l
50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1 lg/mL each of
aprotinin, leupeptin, and pepstatin for 2 h. The entire purification
4.2.3.12. 1-(3-(3,4-Dimethylphenyl)-5-p-tolyl-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (D12). White powder. 1H NMR (300 MHz,
DMSO-d6): 2.22 (s, 6H); 2.25 (s, 6H); 3.01–3.06 (dd, J1 = 2.73,
J2 = 10.80 Hz, 1H); 3.73–3.79 (dd, J1 = 6.96, J2 = 10.80 Hz, 1H);
5.42–5.46 (dd, J1 = 2.55, J2 = 8.14 Hz, 1H); 7.01–7.02 (d, J = 4.77,
2H); 7.07–7.09 (d, J = 4.74, 2H); 7.17–7.19 (d, J = 4.59, 1H);7.45–
7.46 (d, J = 4.74, 1H); 7.53 (s, 1H). MS (ESI): 307.2 (C20H23N2O,
[M+H]+). Anal. Calcd for C20H22N2O: C, 78.40; H, 7.24; N, 9.14. Found:
C, 78.43; H, 7.22; N, 9.25.
procedure was performed at 4 °C or on ice.
The EGFR kinase assay was set up to assess the level of auto-
phosphorylation based on DELFIA/Time-Resolved Fluorometry.
Compounds C1–C30 and D1–D16 were dissolved in 100% DMSO
and diluted to the appropriate concentrations with 25 mM HEPES
at pH 7.4. In each well, 10
10 L (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant enzyme
(1:80 dilution in 100 mM HEPES) for 10 min at room temperature.
Then, 10 L of 5 mM buffer (containing 20 mM HEPES, 2 mM
MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM
lL of compound was incubated with
l
l
l
l
4.2.3.13. 1-(3-(3,4-Dimethylphenyl)-5-(4-methoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (D13). White powder. 1H
NMR (300 MHz, DMSO-d6): 2.25 (s, 6H); 2.27 (s, 3H); 3.04–3.11
(dd, J1 = 3.84, J2 = 12.09 Hz, 1H); 3.71 (s, 3H); 3.76–3.82 (dd,
J1 = 11.68, J2 = 18.06 Hz, 1H); 5.44–5.47 (dd, J1 = 4.26, J2 = 10.91 Hz,
1H); 6.85–6.87 (d, J = 7.24, 2H); 7.07–7.10 (d, J = 7.22, 2H); 7.20–
7.22 (d, J = 4.86, 2H); 7.47–7.56 (m, 2H). MS (ESI): 323.2
(C20H23N2O2, [M+H]+). Anal. Calcd for C20H22N2O2: C, 74.51; H,
6.88; N, 8.69. Found: C, 74.53; H, 6.85; N, 8.76.
ATP-50 mM MgCl2 was added for 1 h. Positive and negative con-
trols were included in each plate by incubation of enzyme with
or without ATP-MgCl2. At the end of incubation, liquid was aspi-
rated, and plates were washed three times with wash buffer. A
75 lL (400 ng) sample of europiumlabeled anti-phosphotyrosine
antibody was added to each well for another 1 h of incubation.
After washing, enhancement solution was added and the signal
was detected by Victor (Wallac Inc.) with excitation at 340 nm
and emission at 615 nm. The percentage of autophosphorylation

4614
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 4606–4614
inhibition by the compounds was calculated using the following
equation: 100% ꢁ [(negative control)/(positive control ꢁ negative
control)]. The IC50 was obtained from curves of percentage inhibi-
tion with eight concentrations of compound. As the contaminants
in the enzyme preparation are fairly low, the majority of the
signal detected by the anti-phosphotyrosine antibody is from
EGFR.
References and notes
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Zoltick, P. W.; Biegel, J. A.; Hayes, R. L.; Wong, A. J. Cancer Res. 1995, 55, 5536.
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determined using a standard (MTT)-based colorimetric assay (Sig-
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concentrations ranging from 0.1 to 100
vival was determined by the addition of an MTT solution (10
5 mg/mL MTT in PBS). After 4 h, 100 L of 10% SDS in 0.01 N HCl
was added, and the plates were incubated at 37 °C for a further
18 h; optical absorbance was measured at 570 nm on an LX300 Ep-
son Diagnostic microplate reader. Survival ratios are expressed in
percentages with respect to untreated cells. IC50 values were deter-
mined from replicates of six wells from at least two independent
experiments.
l
g/mL. After 48 h, cell sur-
l
L of
l
4.5. Molecular docking study
The automated docking studies were carried out using Auto-
Dock version 4.0. First, AutoGrid component of the program pre-
calculates a three-dimensional grid of interaction energies based
on the macromolecular target using the AMBER force field. The cu-
bic grid box of 60 Å size (x, y, z) with a spacing of 0.375 Å and grid
maps were created representing 17 the catalytic active target site
region where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free en-
ergy of the inhibitors within the macromolecules.
21. Bekhit, A. A.; Fahmy, H. T. Y.; Rostom, S. A. F.; Baraka, A. M. Eur. J. Med. Chem.
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D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
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Isakson, P. C. J. Med. Chem. 1997, 40, 1347.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger-Hückel charges of ligands were assigned. The crystal
structures of EGFR (PDB code: 1M17) complex were retrieved from
the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do). All bound waters and ligands were eliminated from
the protein and the polar hydrogens and the Kollman-united
charges were added to the proteins.
23. Menozzi, G.; Mosti, L.; Fossa, P.; Mattioli, F.; Ghia, M. J. Heterocycl. Chem. 1997,
34, 963.
24. Sridhar, R.; Perumal, P. T.; Etti, S.; Shanmugam, G.; Ponnuswamy, M. N.;
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Acknowledgment
The work was financed by a grant (Project 30772627) from
National Natural Science Foundation of China.