Design, synthesis and biological evaluation of pyrazolyl-thiazolinone derivatives as potential EGFR and HER-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.01.051

A series of pyrazolyl-thiazolinone derivatives (E1-E36) have been designed and synthesized and their biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Thirty-four of the 36 compounds were reported for the first time. Among them, compound 2-(5-(4-bromophenyl)-3-p-tolyl-4,5- dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (E28) displayed the most potent inhibitory activity (IC₅₀ = 0.24 μM for EGFR and IC₅₀ = 1.07 μM for HER-2). Antiproliferative assay results indicated that compound E28 owned high antiproliferative activity against MCF-7, B16-F10 and HCT-116 in vitro, with IC₅₀ value of 0.30, 0.54, and 0.70 μM, respectively. Docking simulation was further performed to position compound E28 into the EGFR active site to determine the probable binding model. Based on the preliminary results, compound E28 with potent inhibitory activity in tumor growth would be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2012.01.051

Bioorganic & Medicinal Chemistry 20 (2012) 2010–2018
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of pyrazolyl-thiazolinone
derivatives as potential EGFR and HER-2 kinase inhibitors
⇑
⇑
Ke-Ming Qiu , Hai-Hong Wang , Li-Ming Wang, Yin Luo, Xian-Hui Yang, Xiao-Ming Wang , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazolyl-thiazolinone derivatives (E1–E36) have been designed and synthesized and their
biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Thirty-four of
the 36 compounds were reported for the first time. Among them, compound 2-(5-(4-bromophenyl)-3-
p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (E28) displayed the most potent inhibitory activ-
Received 31 December 2011
Revised 26 January 2012
Accepted 27 January 2012
Available online 4 February 2012
ity (IC50 = 0.24
compound E28 owned high antiproliferative activity against MCF-7, B16-F10 and HCT-116 in vitro, with
IC50 value of 0.30, 0.54, and 0.70 M, respectively. Docking simulation was further performed to position
lM for EGFR and IC50 = 1.07 lM for HER-2). Antiproliferative assay results indicated that
Keywords:
Pyrazolyl-thiazolidinone
EGFR
HER-2
l
compound E28 into the EGFR active site to determine the probable binding model. Based on the prelimin-
ary results, compound E28 with potent inhibitory activity in tumor growth would be a potential antican-
cer agent.
Antitumor
Ó 2012 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
FDA for treatment of patients with non small-cell-lung cancer
(NSCLC).11,12
Receptor protein tyrosine kinases (RPTKs), also called phospho-
tyrosine kinase, play a key role in signal transduction pathways
that regulate cell division and differentiation. EGFR, a transmem-
brane protein tyrosine kinase (PTK) that is activated by ligand-
induced dimerization, plays a critical role in regulating cell
proliferation, differentiation, and migration.1 EGFR family com-
prise four members, including: EGFR (HER-1/ErbB-1), HER-2
(ErbB-2/neu), HER-3 (ErbB-3), and HER-4 (ErbB-4).2 EGFR tyrosine
kinase-mediated cell growth signaling pathway plays an important
role in the formation and development of many types of solid tu-
mors, including head and neck, lung, breast, bladder, prostate,
and kidney cancers.3–6 Therefore, EGFR tyrosine kinase represents
an attractive target for the development of novel anticancer agents.
EGFR and HER-2 are the hottest targets in current cancer research
and their overexpression or abnormal activation often cause cell
malignant transformation.7 Besides, they have relationship with
postoperative adverse, radiotherapy and chemotherapy resistance
and tumor angiogenesis.8 Compounds that inhibit the kinase activ-
ity of EGFR and/or HER-2 after binding of its ATP binding site are of
potential interest as new therapeutic antitumor agents.9,10
Gefitinib (Iressa) and erlotinib (Tarceva) (Fig. 1) are the representa-
tive drugs for this kind of inhibitors and have been approved by US
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 antiviral/antitumor,13–15
antibacterial,16,17 antiinflamatory,18 analgesic,19 fungistatic,20 and
anti-hyperglycemic activity.21 Ducray et al. reported that 3-alk-
oxy-1H-pyrazolo[3,4-d]pyrimidines analogues (3A-1-PP) (Fig. 1)
showed potent EGFR and HER-2 receptor tyrosine kinase inhibitory
activity, with IC50 values reach to single digit nanomolar.22 Besides,
a series of novel pyrazole derivatives containing thiourea skeleton
(PD, Fig. 1) were recently reported as potent anticancer agents tar-
geting EGFR TK in our group and some of them had demonstrated
potent antitumor activity.23
Thiazolinone and their derivatives have attracted continuing
interest over the years because of their varied biological activities,
such as anti-inflammatory, antimicrobial, antiproliferative, antivi-
ral, anticonvulsant, antifungal, and antibacterial.24–26 Recent years,
thiazolinone derivatives with their antitumor activity have become
a new hot spot. Havrylyuk et al. reported that thiazolidinones con-
taining benzothiazole moiety has anticancer activity on leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate and breast
cancers cell lines.27 Moreover, our group’s previous work had
showed that thiazolidinone derivatives (TD, Fig. 1) were potent
inhibitors of EGFR and HER-2.28
In addition, many pyrazole or thiazolinone derivatives
displayed potent biological activities and low toxicities in previous
reports.23,25,28–30 But to our knowledge, few reports have been
⇑
Corresponding authors. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors contributed equally to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.051

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
2011
Figure 1. Chemical structures of some reported compounds.
dedicated to design and synthesize an anticancer/antitumor com-
pound which contains pyrazole and thiazolinone simultaneously.
The pyrazole ring along with the thiazolinone ring, the two com-
bined substructures, might exhibit synergistic anticancer effect.
All of these encouraged us to integrate these two heterocycles
and screen new pyrazolyl-thiazolinone derivatives as potential
EGFR and HER-2 inhibitory agents. Herein, we report in the present
work the design, synthesis and biological evaluation of pyrazolyl-
thiazolinone derivatives as potential EGFR and HER-2 kinase inhib-
itors. Biological evaluation indicated that some of the synthesized
compounds were potent inhibitors of EGFR and HER-2.
obtained by direct condensation by the aromatic aldehydes and
the substituted acetophenone, using 40% potassium hydroxide as
catalyst in ethanol. Secondly, cyclization of different chalcones
with thiosemicarbazide under basic condition leads to the forma-
tion of pyrazole derivatives containing thiourea skeleton. Thirdly,
pyrazolyl-thiazolinone derivatives E1–E42 were obtained by react-
ing compound D (1 equiv) with bromoacetic acid (1.2 equiv) in
acetic acid and keeping under stirring at 80 °C for 6–8 h. Moreover,
acetic anhydride (2 equiv) was added as dehydrating agent and so-
dium acetate (2 equiv) was used as acid-binding agent. Among
these compounds, E1–E5, E7–E20 and E22–E36 are reported for
the first time. All of the synthetic compounds gave satisfactory
analytical and spectroscopic data, which were in full accordance
with their depicted structures.
2. Results and discussion
2.1. Chemistry
2.2. In vitro enzyme inhibition activity
The synthesis of compounds E1–E36 are followed the general
pathway outlined in Scheme 1. Firstly, the chalcones were
The synthesized derivatives were evaluated for their ability to
inhibit the autophosphorylation of EGFR and HER-2 kinases using
Scheme 1. General synthesis of pyrazolyl-thiazolinone derivatives (E1–E36). Reagents and conditions: (i) 40% aqueous potassium hydroxide solution, ethanol, rt; (ii)
thiosemicarbazide, KOH, ethanol, reflux; (iii) bromoacetic acid, acetic anhydride, sodium acetate, acetic acid, 80 °C, 6–8 h.

2012
K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
a solid-phase ELISA assay. The results were summarized in Table 2.
It was observed that pyrazolyl-thiazolinone derivatives containing
thiazolinone and pyrazoline rings showed fairly good inhibiting
EGFR activities displaying IC50 values between 0.24 and
the bromine atom was replaced by a chlorine/fluorine atom
(IC50 = 1.66–10.92 M/2.01–16.92 M; 3.11–13.16 M/3.53–18.
12 M for EGFR and HER-2, respectively), which was a relatively
strong electron-withdrawing substituent. Moreover, with
Me/OMe group at para-position showed relatively poor activity
(IC50 = 1.16–8.36 M/4.24–10.69 for EGFR, IC50 = 2.52–10.
26 M/6.23–12.43 M for HER-2, respectively), and this due to
its electron-donating effect.
Herein, at the same position for the given compounds, we ob-
served that the IC50 value for inhibition of HER-2 kinase was, in
general, higher than that observed for EGFR kinase but had the
same trends. This was possibly due, in part, to the fact that in
the enzyme assays we used higher concentration of the purified
HER-2 kinase than EGFR kinase. It was evident that there was also
a reasonable correlation between the EGFR and HER-2 inhibitory
activities. Thus, this was not surprising in view of the high
sequence homology of the catalytic domains of these two kinases.
l
l
l
l
a
16.92
lM. Among them, compound E28 displayed the most potent
inhibitory activity (IC50 = 0.24
l
M for EGFR and IC50 = 1.07 M for
l
l
lM
HER-2). Subsequently SAR studies were performed by modification
of the parent compound to determine how the substituents of the
subunits affect the EGFR inhibitory activities. Inspection of the
chemical structures of the compounds E1–E36 suggested that they
could be divided into two subunits: A- and B-rings (Scheme 1 and
Table 1).
As showed in Table 2, structure–activity relationships in com-
pounds E1–E36 demonstrated that compounds bearing one methyl
group at para-position at A-ring showed better EGFR and HER-2
l
l
inhibitory activity (E25–E30, IC50 = 0.24–4.24
lM for EGFR and
IC50 = 1.07–6.23 M for HER-2) than those with hydrogen (E1–
l
E6), fluorin (E7–E12), chlorine (E13–E18), bromine (E19–E24)
and methoxyl (E31–E36) substituents, basically in the order of
Me > H > Br > Cl > F and Me > OMe. This meant that compound
with a faintish electron-donating substituents at para-position of
A-ring (para-Me) had better EGFR activity than that with a rela-
tively stronger electron-donating substituents (para-OMe). We
could also get the result that a methyl group at the para-position
(compound E25–E30) of A ring might have slightly improved EGFR
inhibitory activity compared to a hydrogen atom at the same
position (compound E1–E6), whereas a halogen atom at the same
position led to a noteworthy loss of activity, which was an elec-
tron-withdrawing substituent.
Among all the compounds E1–E36, compound E28 (IC50 = 0.24
lM
for EGFR and IC50 = 1.07 M for HER-2) showed the best activity
l
while the substituents in the A ring and B ring are respectively
methyl and bromine substituents at para-position.
The above results indicated that thiazolinone and pyrazoline
rings in the pyrazolyl-thiazolinone derivatives might play an
important role in the EGFR and HER-2 inhibitory activity. However,
the changes of substituents on the benzene rings had effects on the
activity of the compound, but it was not so obviously as possible.
This might be explained that substituents on the benzene rings
had limited influence on the molecular bonding between the pro-
tein and the compounds. Molecular docking of all the compounds
of this series had demonstrated this point and this was conformed
to our estimate. Nevertheless, the thiazolinone and pyrazoline dif-
ferent from classical EGFR inhibitors (e.g., gefitinib and erlotinib)
with their potential EGFR inhibit effect could be the parent nucleus
of novel efficient EGFR inhibitors.
In the case of constant A ring substituents, changes of substitu-
ents on B ring can also affect the activities of these compounds.
Contrary to A ring, bromine at para-position derivatives had the
best activity (IC50 = 0.24–4.79
lM for EGFR, and IC50 = 1.07–
6.24 M for HER-2), which was a faintish electron-withdrawing
l
substituent. While a significant loss of activity was observed when
2.3. Antiproliferative activity and molecular docking study
Table 1
The in vitro antiproliferative activities of nine compounds (E4,
E16, E22, E25–E29, E34) with better inhibition activities against
EGFR were tested using MCF-7, B16-F10, and HCT-116 cancer cell
lines. As was shown in Table 3, out of the top nine compounds,
compounds E28 and E25, which had potent inhibitory activity of
EGFR showed high antiproliferative activity (IC50 = 0.30 and
Chemical structures of pyrazolyl-thiazolinone derivatives
0.73
lM
for MCF-7, IC50 = 0.54 and 0.86
lM for B16-F10,
IC50 = 0.70 and 1.16
l
M for HCT-116), indicating that these pyraz-
olyl-thiazolinone derivatives were potent antitumor agents. In par-
ticular, compound E28 had demonstrated significant inhibitory
activity in tumor growth inhibition and displayed potential EGFR
inhibitory activity.
To gain better understanding on the potency of the compound
E28 and guide further SAR studies, we proceeded to examine the
interaction of it with EGFR (PDB code: 1M17) by molecular dock-
ing, which was performed by simulation of the compound into
the ATP binding site in EGFR. The binding model of compound
E28 and EGFR was depicted in Figure 2A. The amino acid residues
which had interaction with EGFR were labeled. In the binding
mode, compound E28 was nicely bound to the ATP binding site
of EGFR through hydrophobic interaction and the binding was sta-
Compounds
R1
R2
Compounds
R1
R2
E1
E2
E3
E4
E5
E6
E7
E8
H
H
H
H
H
H
F
F
F
F
F
H
F
Cl
Br
Me
OMe
H
F
Cl
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
E31
E32
E33
E34
E35
E36
Br
Br
Br
Br
Br
Br
Me
Me
Me
Me
Me
Me
OMe
OMe
OMe
OMe
OMe
OMe
H
F
Cl
Br
Me
OMe
H
F
Cl
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
Br
Br
bilized by a hydrogen bond and a p–cation interaction.
Me
OMe
H
F
Cl
Br
Me
OMe
Me
OMe
H
F
Cl
Br
Me
OMe
In the binding model, compound E28 was nicely bound to the
EGFR kinase with its carbonyl group project toward the trunk chain
amino-group of A721 (LYS 721), forming a more optimal H-bond
interaction (distance: N–HÁ Á ÁO = 1.93 Å, angle: 118.7°). Based on
the favorable EGFR inhibitory activity of pyrazolyl-thiazolinone
derivatives, it could be concluded that this H-bond played an
important effect in the EGFR inhibitory. The end amino cation of
F
Cl
Cl
Cl
Cl
Cl
Cl

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
2013
Table 2
Inhibition activities of compounds E1–E42 against EGFR and HER-2 (IC50
, lM)
Compounds
EGFR
HER-2
Compounds
EGFR
HER-2
E1
E2
E3
E4
E5
E6
E7
E8
3.38 0.34
4.86 0.50
3.49 0.32
1.35 0.12
3.03 0.35
4.27 0.45
8.14 0.86
16.92 1.58
10.92 1.11
4.79 0.51
8.36 0.85
10.69 1.12
5.34 0.55
14.21 1.41
8.16 0.86
2.28 0.24
6.67 0.72
8.58 0.84
0.03 0.002
5.12 0.58
6.35 0.65
4.83 0.45
3.05 0.33
4.64 0.42
6.21 0.70
10.53 0.98
18.12 1.69
13.16 1.46
6.24 0.58
10.26 1.12
12.43 1.36
7.05 0.73
16.42 1.73
9.96 1.02
3.84 0.42
8.13 0.85
10.34 1.12
0.14 0.02
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
E31
E32
E33
E34
E35
E36
3.20 0.35
6.48 0.70
4.12 0.45
2.03 0.21
5.58 0.52
7.96 0.82
1.08 0.13
2.01 0.22
1.66 0.18
0.24 0.04
1.16 0.12
4.24 0.45
2.37 0.23
5.95 0.64
5.35 0.55
1.26 0.15
5.46 0.58
8.89 0.91
4.87 0.52
8.14 0.83
5.87 0.60
3.65 0.35
7.04 0.72
9.27 1.01
2.24 0.25
3.53 0.33
3.11 0.33
1.07 0.12
2.52 0.23
6.23 0.68
4.12 0.43
8.04 0.86
7.59 0.77
2.75 0.24
7.35 0.75
10.48 1.13
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
Erlotinib
Table 3
Antiproliferative activities of compounds E4, E16, E22, E25–E29, E34 (IC50
, lM)
Compounds
MCF-7
B16-F10
HCT-116
E3
0.93 0.08
2.12 0.19
2.13 0.23
0.73 0.08
1.83 0.19
1.53 0.15
0.30 0.41
1.17 0.13
1.51 0.17
0.07 0.008
1.40 0.13
2.10 0.18
1.49 0.15
0.86 0.06
1.50 0.12
1.22 0.12
0.54 0.04
1.01 0.08
2.68 0.22
0.10 0.012
1.70 0.13
2.34 0.18
2.19 0.24
1.16 0.10
1.93 0.21
1.79 0.18
0.70 0.08
1.26 0.13
3.92 0.41
0.14 0.012
E16
E22
E25
E26
E27
E28
E29
E34
Erlotinib
LYS721 was also formed a
p–cation interaction with the benzene
ring of compound E28 (distance: 4.53 Å), which enhanced the
binding action between receptor EGFR and ligand compound E28.
The electron-donating substituent amino-NH2 strengthened the
p–cation binding and the results indicated that it was primarily
due to direct through-space interaction between the substituent
and the cation.
Meanwhile, we chose erlotinib as the positive control in the
docking procedure. In Figure 2B, it was found that there was only
one sigma–
otinib formed one sigma–
p
bond in the binding pocket. The benzene ring of erl-
bond with VAL702 (distance: 2.80 Å).
p
Figure 2A. Molecular docking modeling of compound E28 with EGFR kinase: for
clarity, only interacting residues are displayed. Above: 3D model of the interaction
between compound E28 and the ATP binding site. The H-bond (green lines) is
The model between compound E28 and the ATP binding site was
similar to that with erlotinib. Comparing these models, it was
found that the hydrophobic pockets of ATP binding site were all ni-
cely occupied by these compounds, and the difference was the
combination mode. This molecular docking result, along with the
biological assay data, suggesting that compound E28 was a poten-
tial inhibitor of EGFR.
displayed as dotted lines, and the
p–cation interaction is shown as yellow lines.
Below: 2D model of the interaction between compound E28 and the ATP binding
site. The H-bond (blue arrows) is displayed as dotted arrows, and the
interaction is shown as orange lines.
p–cation
anticancer activities (IC50 = 0.30
lM for MCF-7, IC50 = 0.54 lM for
3. Conclusions
B16-F10 and IC50 = 0.70 M for HCT-116).
l
Molecular docking was further performed to study the inhibi-
tor-EGFR protein interactions. After analysis of the binding model
of compound E28 with EGFR, it was found that a hydrogen bond
In this paper, a series of pyrazolyl-thiazolinone derivatives that
may function as inhibitors of EGFR and HER-2 kinases have been
synthesized and their biological activities were evaluated. And
some of them displayed potent EGFR and HER-2 inhibitory activi-
ties and antiproliferative activities against MCF-7 cell lines, B16-
F10 cell lines and HCT-116 cell lines. Among them, compound
E28 showed the most potent EGFR inhibition activities
and a
p–cation interaction with the protein residues in the ATP
binding site might play a crucial role in its EGFR inhibition and
antiproliferative activities. Among these compounds, it could be
concluded that compound E28 had been demonstrated to show
significant EGFR and tumor growth inhibitory activity as a poten-
tial anticancer agent. The result of this work might be helpful for
(IC50 = 0.24 lM for EGFR and IC50 = 1.07 lM for HER-2) and

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K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
cooling, the solution was poured into mass of ice-water and stirred
for a few minutes. The precipitate was filtered and crystallized
from ethanol.
4.2.3. General synthetic procedure of pyrazolyl-thiazolinone
derivatives (E1–E36)
A
mixture of compound D (1 mmol), bromoacetic acid
(1.2 mmol), acetic anhydride (2 mmol), and sodium acetate
(2 mmol) was dissolved in acetic acid (20 mL) and kept stirring
in 80 °C for 6–8 h. After cooling, the solution was poured into
mass of ice-water and stirred for a few minutes. The precipitate
was filtered and crystallized from methylene dichloride/ethanol =
1:1.
4.2.3.1.
4(5H)-one (E1).
2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-
Yield 65%; mp 208–210 °C. 1H NMR (CDCl3,
300 MHz): 3.38 (dd, J1 = 4.02 Hz, J2 = 18.04 Hz, 1H), 3.87 (s, 2H),
3.97 (dd, J1 = 7.04 Hz, J2 = 15.56 Hz, 1H), 5.86 (s, 1H), 7.25–7.30
(m, 3H), 7.45–7.52 (m, 3H), 7.46 (d, J = 8.02 Hz, 2H),7.62(d,
J = 5.34 Hz, 2H). ESI-MS: 322.40 (C18H16N3OS, [M+H]+). Anal. Calcd
for C18H15N3OS: C, 67.27; H, 4.70; N, 13.07. Found: C, 67.38; H,
4.72; N, 13.13.
4.2.3.2. 2-(5-(4-Fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyra-
zol-1-yl)thiazol-4(5H)-one (E2).
Yield 70%; mp 213–215 °C.
1H NMR (CDCl3, 300 MHz): 3.38 (d, J = 16.83 Hz, 1H), 3.85 (s, 2H),
3.95 (dd, J1 = 7.14 Hz, J2 = 14.28 Hz, 1H), 5.83 (d, J = 8.79 Hz, 1H),
7.14–7.17 (m, 4H), 7.44–7.51 (m, 3H), 7.80 (d, J = 6.6 Hz, 2H). ESI-
MS: 340.39 (C18H15FN3OS, [M+H]+). Anal. Calcd for C18H14FN3OS:
C, 63.70; H, 4.16; N, 12.38. Found: C, 63.57; H, 4.14; N, 12.33.
Figure 2B. Molecular docking modeling of erlotinib with EGFR kinase: or clarity,
only interacting residues are displayed. Above: 3D model of the interaction
between erlotinib and the ATP binding site. The
orange lines. Below: 2D model of the interaction between erlotinib and the ATP
binding site. The –sigma bond is displayed as orange lines.
p–sigma bond is displayed as
p
4.2.3.3. 2-(5-(4-Chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyra-
the design and synthesis of EGFR inhibitors with stronger
activities.
zol-1-yl)thiazol-4(5H)-one (E3).
Yield 70%; mp 218–220 °C.
1H NMR (CDCl3, 300 MHz): 3.71 (dd, J1 = 6.96 Hz, J2 = 14.10 Hz,
1H), 3.86 (s, 2H), 4.11 (dd, J1 = 7.14 Hz, J2 = 14.28 Hz, 1H), 5.79 (s,
1H), 7.19 (d, J = 8.04, 2H), 7.30 (d, J = 7.68, 2H), 7.45–7.52 (m,
3H), 7.79 (d, J = 7.68 Hz, 2H). ESI-MS: 356.84 (C18H15ClN3OS,
[M+H]+). Anal. Calcd for C18H14ClN3OS: C, 60.76; H, 3.97; N,
11.81. Found: C, 60.64; H, 3.96; N, 11.76.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker
DPX300 model Spectrometer in CDCl3 or DMSO-d6 and chemical
shifts were reported in ppm (d). ESI-MS spectra were performed
on a CHN-O-Rapid instrument. TLC was performed on the glass-
backed silica gel sheets (Silica Gel 60 GF254) and visualized in
UV light (254 nm). Column chromatography was performed using
silica gel (200–300 mesh) eluting with ethyl acetate and petroleum
ether.
4.2.3.4. 2-(5-(4-Bromophenyl)-3-phenyl-4,5-dihydro-1H-pyra-
zol-1-yl)thiazol-4(5H)-one (E4).
Yield 72%; mp 244–246 °C.
1H NMR (CDCl3, 300 MHz): 3.37 (d, J = 16.83 Hz, 1H), 3.87 (s, 2H),
4.11(dd, J1 = 7.14 Hz, J2 = 14.25 Hz, 1H), 5.81 (s, 1H), 7.15 (d,
J = 7.86 Hz, 2H), 7.45–7.52 (m, 5H), 7.80 (d, J = 7.32 Hz, 2H). ESI-
MS: 401.29 (C18H15BrN3OS, [M+H]+). Anal. Calcd for C18H14BrN3OS:
C, 54.01; H, 3.53; N, 10.50. Found: C, 54.16; H, 3.55; N, 10.56.
4.2.3.5.
yl)thiazol-4(5H)-one (E5).
2-(3-Phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-
Yield 75%; mp 213–215 °C. 1H
4.2. Synthesis
NMR (CDCl3, 300 MHz): 2.04 (s, 3H), 3.37 (dd, J1 = 3.66 Hz,
J2 = 17.91 Hz, 1H), 3.87 (s, 2H), 3.97 (dd, J1 = 6.39 Hz,
J2 = 17.55 Hz, 1H), 5.88 (d, J = 10.08 Hz, 1H), 7.02 (t, J = 8.58 Hz,
2H), 7.24–7.28 (m, 1H), 7.45–7.52 (m, 3H), 7.79–7.83 (m, 2H).
ESI-MS: 336.42 (C19H18N3OS, [M+H]+). Anal. Calcd for C19H17N3OS:
C, 68.03; H, 5.11; N, 12.53. Found: C, 68.18; H, 5.13; N, 12.61.
4.2.1. General synthetic procedure of chalcones (C1–C36)
Equimolar portions of the appropriately aromatic aldehydes
(3 mmol, 1 equiv) and substituted acetophenone (3 mmol, 1 equiv)
were dissolved in approximately 20 mL of ethanol. The mixture was
allowed to stir for several minutes at 0 °C to let dissolve. Than a
1 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 temperature for approximately 4–6 h. Most commonly,
a precipitate formed and was then collected by suction filtration.
4.2.3.6. 2-(5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyra-
zol-1-yl)thiazol-4(5H)-one (E6).
Yield 76%; mp 214–216 °C.
1H NMR (CDCl3, 300 MHz): 3.41(d, J = 10.62 Hz, 1H), 3.87 (s, 3H),
3.92 (s, 2H), 3.96 (dd, J1 = 4.24 Hz, J2 = 14.23 Hz, 1H), 5.99 (s, 1H),
6.85 (d, J = 5.04 Hz, 2H), 7.25 (d, J = 3.93 Hz, 2H), 7.47–7.50 (m,
2H), 7.52 (d, J = 4.41 Hz, 1H), 7.82 (d, J = 4.47 Hz, 2H). ESI-MS:
352.42 (C19H18N3O2S, [M+H]+). Anal. Calcd for C19H17N3O2S: C,
64.94; H, 4.88; N, 11.96. Found: C, 64.99; H, 4.87; N, 12.01.
4.2.2. General synthetic procedure of pyrazole derivatives
(D1–D36)
A mixture of chalcone (2 mmol), thiosemicarbazide (3 mmol),
and KOH (2 mmol) was refluxed in ethanol (30 mL) for 12 h. After

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
2015
4.2.3.7. 2-(3-(4-Fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyra-
zol-1-yl)thiazol-4(5H)-one (E7).
2H), 7.44 (d, J = 7.86 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H). ESI-MS:
374.83 (C18H14ClFN3S, [M+H]+). Anal. Calcd for C18H13ClFN3S: C,
57.83; H, 3.51; N, 11.24. Found: C, 57.71; H, 3.49; N, 11.27.
Yield 68%; mp 232–235 °C.
1H NMR (CDCl3, 300 MHz): 3.37 (dd, J1 = 8.28 Hz, J2 = 14.24 Hz,
1H), 3.87 (s, 2H), 3.95 (dd, J1 = 5.94 Hz, J2 = 17.24 Hz, 1H), 5.84 (d,
J = 10.04 Hz, 1H), 7.24 (s, 1H), 7.31 (d, J = 4.28 Hz, 2H), 7.28–7.36
(m, 4H), 7.83 (d, J = 8.24 Hz, 2H). ESI-MS: 340.39 (C18H15FN3OS,
[M+H]+). Anal. Calcd for C18H14FN3OS: C, 63.70; H, 4.16; N, 12.38.
Found: C, 63.85; H, 4.18; N, 12.45.
4.2.3.15. 2-(3,5-Bis(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)thiazol-4(5H )-one (E15).
Yield 82%; mp 253–255 °C. 1H
NMR (CDCl3, 300 MHz): 3.32 (d, J = 17.37 Hz, 1H), 3.87 (s, 2H),
4.12 (dd, J1 = 7.14 Hz, J2 = 14.28 Hz, 1H), 5.79 (s, 1H), 7.19 (d,
J = 8.22 Hz, 2H), 7.31 (d, J = 8.07 Hz, 2H), 7.45 (d, J = 8.22 Hz, 2H),
7.73 (d, J = 8.61 Hz, 2H). ESI-MS: 391.29 (C18H14Cl2N3OS, [M+H]+).
Anal. Calcd for C18H13Cl2N3OS: C, 55.39; H, 3.36; N, 10.77. Found:
C, 55.23; H, 3.34; N, 10.71.
4.2.3.8.
yl)thiazol-4(5H)-one (E8).
2-(3,5-Bis(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-
Yield 80%; mp 247–249 °C. 1H
NMR (CDCl3, 300 MHz): 3.35 (d, J = 15.35 Hz, 1H), 3.87 (s, 2H),
3.96 (d, J = 17.19 Hz, 1H), 5.82 (d, J = 9.33 Hz, 1H), 6.91–7.05 (m,
2H), 7.14–7.17 (m, 2H), 7.20–7.24 (m, 2H), 7.60–7.83 (m, 2H). ESI-
MS: 358.38 (C18H14F2N3OS, [M+H]+). Anal. Calcd for C18H13F2N3OS:
C, 60.49; H, 3.67; N, 11.76. Found: C, 60.37; H, 3.66; N, 11.70.
4.2.3.16.
2-(5-(4-Bromophenyl)-3-(4-chlorophenyl)-4,5-dihy-
Yield 80%;
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E16).
mp 257–259 °C. 1H NMR (CDCl3, 300 MHz): 3.32 (d, J = 17.37 Hz,
1H), 3.87 (s, 2H), 4.12 (dd, J1 = 7.14 Hz, J2 = 14.28 Hz, 1H), 5.78 (s,
1H), 7.13 (d, J = 8.22 Hz, 2H), 7.44–7.48 (m, 4H), 7.43 (d,
J = 8.43 Hz, 2H). ESI-MS: 435.74 (C18H14BrClN3OS, [M+H]+). Anal.
Calcd for C18H13BrClN3OS: C, 49.73; H, 3.01; N, 9.67. Found: C,
49.61; H, 3.02; N, 9.74.
4.2.3.9. 2-(5-(4-Chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-
1H -pyrazol-1-yl)thiazol-4(5H)-one (E9).
Yield 82%; mp
256–258 °C. 1H NMR (CDCl3, 300 MHz): 3.33 (d, J = 14.07 Hz, 1H),
3.87 (s, 2H), 3.96 (d, J = 17.37 Hz, 1H), 5.79 (d, J = 7.5 Hz, 1H),
6.91–7.13 (m, 4H), 7.31 (d, J = 8.25 Hz, 2H), 7.60–7.82 (m, 2H).
ESI-MS: 374.83 (C18H14ClFN3OS, [M+H]+). Anal. Calcd for
4.2.3.17. 2-(3-(4-Chlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyr-
C
18H13ClFN3OS: C, 57.83; H, 3.51; N, 11.24. Found: C, 57.74; H,
azol-1-yl)thiazol-4(5H)-one (E17).
Yield 85%; mp 242–
3.52; N, 11.19.
244 °C. 1H NMR (CDCl3, 300 MHz): 2.31(s, 3H), 3.38 (d,
J = 12.74 Hz, 1H), 3.86–3.95 (m, 3H), 5.81 (s, 1H), 7.13–7.15 (m,
4H), 7.44 (d, J = 8.04 Hz, 2H), 7.73 (d, J = 8.43 Hz, 2H). ESI-MS:
370.87 (C19H17ClN3OS, [M+H]+). Anal. Calcd for C19H16ClN3OS: C,
61.70; H, 4.36; N, 11.36. Found: C, 61.92; H, 4.39; N, 11.44.
4.2.3.10. 2-(5-(4-Bromophenyl)-3-(4-fluorophenyl)-4,5-dihydro-
1H -pyrazol-1-yl)thiazol-4(5H)-one (E10).
Yield 83%; mp
262–264 °C. 1H NMR (CDCl3, 300 MHz): 3.33 (d, J = 17.16 Hz, 1H),
3.87–3.99 (m, 3H), 5.84 (s, 1H), 7.02 (d, J = 8.61 Hz, 1H), 7.13–
7.20 (m, 2H), 7.22–7.26 (m, 1H), 7.47 (d, J = 8.22 Hz, 1H), 7.60–
7.68 (m, 2H), 7. 80 (dd, J1 = 5.31 Hz, J2 = 8.79 Hz, 1H). ESI-MS:
419.28 (C18H14BrFN3OS, [M+H]+). Anal. Calcd for C18H13BrFN3OS:
C, 51.69; H, 3.13; N, 10.05. Found: C, 51.58; H, 3.13; N, 10.01.
4.2.3.18. 2-(3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E18).
Yield 85%;
mp 245–247 °C. 1H NMR (CDCl3, 300 MHz): 3.36 (d, J = 14.24 Hz,
1H), 3.87 (s, 3H), 3.91 (s, 2H), 4.01 (dd, J1 = 4.34 Hz, J2 = 14.18 Hz,
1H), 5.87 (d, J = 9.88 Hz, 1H), 6.98 (d, J = 6.34 Hz, 2H), 7.22 (d,
J = 4.54 Hz, 2H), 7.54 (d, J = 6.34 Hz, 2H), 7.96 (d, J = 8.46 Hz, 2H).
ESI-MS: 386.87 (C19H17ClN3O2S, [M+H]+). Anal. Calcd for
4.2.3.11. 2-(3-(4-Fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H -pyr-
azol-1-yl)thiazol-4(5H)-one (E11).
Yield 80%; mp 229–
231 °C. 1H NMR (CDCl3, 300 MHz): 2.31 (s, 3H), 3.35 (d,
J = 14.64 Hz, 1H), 3.85 (s, 2H), 3.94 (d, J = 17.01 Hz, 1H), 5.83 (d,
J = 8.22 Hz, 1H), 7.14–7.19 (m, 6H), 7. 81 (dd, J1 = 5.28 Hz,
J2 = 8.76 Hz, 2H). ESI-MS: 354.41 (C19H17FN3OS, [M+H]+). Anal.
Calcd for C19H16FN3OS: C, 64.57; H, 4.56; N, 11.89. Found: C,
64.65; H, 4.59; N, 11.94.
C19H16ClN3O2S: C, 59.14; H, 4.18; N, 10.89. Found: C, 59.28; H,
4.20; N, 10.97.
4.2.3.19. 2-(3-(4-Bromophenyl)-5-phenyl-4,5-dihydro-1H-pyra-
zol-1-yl)thiazol-4(5H)-one (E20).
Yield 72%; mp 242–244 °C.
1H NMR (CDCl3, 300 MHz): 3.37 (d, J = 14.08 Hz, 1H), 3.87 (s, 2H),
3.98 (dd, J1 = 4.48 Hz, J2 = 10.34 Hz, 1H), 5.85 (d, J = 8.56 Hz, 1H),
7.25–7.35 (m, 3H), 7.42 (d, J = 5.85 Hz, 2H), 7.60 (d, J = 6.34 Hz,
2H), 7.72 (d, J = 14.18 Hz, 2H). ESI-MS: 401.29 (C18H15BrN3OS,
[M+H]+). Anal. Calcd for C18H14BrN3OS: C, 54.01; H, 3.53; N,
10.50. Found: C, 54.18; H, 3.55; N, 10.57.
4.2.3.12. 2-(3-(4-Fluorophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E12).
Yield 85%;
mp 240–242 °C. 1H NMR (CDCl3, 300 MHz): 3.37 (d, J = 17.01 Hz,
1H), 3.67 (s, 2H), 3.76 (s, 3H), 4.10 (dd, J1 = 6.93 Hz, J2 = 15.62 Hz,
1H), 5.83 (d, J = 10.24, 1H), 6.85 (d, J = 8.40 Hz, 2H), 7.14–7.22 (m,
4H), 7.81 (dd, J1 = 5.31 Hz, J2 = 8.61 Hz, 2H). ESI-MS: 370.41
(C19H17FN3O2S, [M+H]+). Anal. Calcd for C19H16FN3O2S: C, 61.77;
H, 4.37; N, 11.37. Found: C, 61.62; H, 4.36; N, 11.32.
4.2.3.20. 2-(3-(4-Bromophenyl)-5-(4-fluorophenyl)-4,5-dihydro-
1H -pyrazol-1-yl)thiazol-4(5H)-one (E20).
Yield 84%; mp
250–252 °C. 1H NMR (CDCl3, 300 MHz): 3.33 (d, J = 15.18 Hz, 1H),
3.87–3.93 (m, 3H), 5.83 (d, J = 8.58 Hz, 1H), 6.99–7.05 (m, 2H),
7.21–7.25 (m, 2H), 7.64 (dd, J1 = 8.61 Hz, J2 = 17.58 Hz, 4H). ESI-
MS: 419.28 (C18H14BrFN3OS, [M+H]+). Anal. Calcd for
4.2.3.13. 2-(3-(4-Chlorophenyl)-5-phenyl-4,5-dihydro-1H -pyra-
zol-1-yl)thiazol-4(5H)-one (E13).
Yield 70%; mp 236–238 °C.
1H NMR (CDCl3, 300 MHz): 3.35 (dd, J1 = 3.66 Hz, J2 = 17.73 Hz, 1H),
3.92 (s, 2H), 4.12 (dd, J1 = 7.14 Hz, J2 = 14.28 Hz, 1H), 5.82(d,
J = 7.50 Hz, 1H), 7.22–7.26 (m, 2H), 7.31–7.39 (m, 3H), 7.44 (d,
J = 8.58 Hz, 2H), 7.73 (d, J = 11.70 Hz, 2H). ESI-MS: 356.84
(C18H15ClN3OS, [M+H]+). Anal. Calcd for C18H14ClN3OS: C, 60.76;
H, 3.97; N, 11.81. Found: C, 60.58; H, 3.95; N, 11.70.
C18H13BrFN3OS: C, 51.69; H, 3.13; N, 10.05. Found: C, 51.86; H,
3.14; N, 10.13.
4.2.3.21.
2-(3-(4-Bromophenyl)-5-(4-chlorophenyl)-4,5-dihy-
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E21).
Yield 86%;
mp 256–258 °C. 1H NMR (CDCl3, 300 MHz): 3.32 (d, J = 17.37 Hz,
1H), 3.71 (s, 2H), 3.87 (dd, J1 = 6.78 Hz, J2 = 13.56 Hz, 1H), 5.83 (s,
1H), 7.19 (d, J = 8.43 Hz, 2H), 7.31(d, J = 8.07 Hz, 2H), 7.59–7.66
(m, 4H). ESI-MS: 435.74 (C18H14BrClN3OS, [M+H]+). Anal. Calcd
for C18H13BrClN3OS: C, 49.73; H, 3.01; N, 9.67. Found: C, 49.86;
H, 3.02; N, 9.72.
4.2.3.14. 2-(3-(4-Chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-
1H -pyrazol-1-yl)thiazol-4(5H)-one (E14).
Yield 81%; mp
245–247 °C. 1H NMR (CDCl3, 300 MHz): 3.37 (s, 1H), 3.73–3.87
(m, 3H), 5.79 (d, J = 8.24 Hz, 1H), 7.03 (d, J = 7.32 Hz, 2H), 7.26 (s,

2016
K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
4.2.3.22. 2-(3,5-Bis(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-
yl)thiazol-4(5H )-one (E22).
Yield 85%; mp 266–268 °C. 1H
NMR (CDCl3, 300 MHz): 3.31 (d, J = 17.91 Hz, 1H), 3.87 (s, 2H),
3.95 (dd, J1 = 6.42 Hz, J2 = 17.37 Hz, 1H), 5.75 (d, J = 8.04 Hz, 1H),
7.12 (d, J = 8.25 Hz, 2H), 7.46 (d, J = 8.22 Hz, 2H), 7.59–7.67 (m,
4H). ESI-MS: 480.19 (C18H14Br2N3OS, [M+H]+). Anal.Calcd for
300 MHz): 2.43 (s, 6H), 3.36 (dd, J1 = 4.08 Hz, J2 = 16.98 Hz, 1H),
3.86 (s, 2H), 4.08 (dd, J1 = 6.94 Hz, J2 = 15.08 Hz, 1H), 5.86 (d,
J = 10.08 Hz, 1H), 7.16–7.20 (m, 4H), 7.27 (d, J = 5.88 Hz, 2H), 7.72
(d, J = 9.02 Hz, 2H). ESI-MS: 350.45 (C20H20N3OS, [M+H]+). Anal.
Calcd for C20H19N3OS: C, 68.74; H, 5.48; N, 12.02. Found: C,
68.86; H, 5.51; N, 12.11.
C18H13Br2N3OS: C, 45.12; H, 2.73; N, 8.77. Found: C, 45.21; H,
2.72; N, 8.83.
4.2.3.30. 2-(5-(4-Methoxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H -
pyrazol-1-yl)thiazol-4(5H)-one (E30).
Yield 84%; mp 224–
4.2.3.23.
2-(3-(4-Bromophenyl)-5-(4-chlorophenyl)-4,5-dihy-
226 °C. 1H NMR (CDCl3, 300 MHz): 2.42 (s, 3H), 3.36 (dd,
J1 = 3.66 Hz, J2 = 17.94 Hz, 1H), 3.77 (s, 3H), 3.86 (s, 2H), 4.12 (dd,
J1 = 7.28 Hz, J2 = 14.14 Hz, 1H), 5.79 (s, 1H), 6.84 (d, J = 8.58 Hz,
2H), 7.20 (d, J = 8.62 Hz, 2H), 7.27 (d, J = 6.96 Hz, 2H), 7.70 (d,
J = 8.25 Hz, 2H). ESI-MS: 366.45 (C20H20N3O2S, [M+H]+). Anal. Calcd
for C20H19N3O2S: C, 65.73; H, 5.24; N, 11.50. Found: C, 65.55; H,
5.21; N, 11.41.
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E23).
Yield 84%;
mp 238–240 °C. 1H NMR (CDCl3, 300 MHz): 2.31 (s, 3H), 3.34 (dd,
J1 = 3.84 Hz, J2 = 17.91 Hz, 1H), 3.85 (s, 2H), 3.91 (dd, J1 = 5.94 Hz,
J2 = 10.78 Hz, 1H), 5.82 (dd, J1 = 3.84, J2 = 11.16 Hz, 1H), 7.13–7.19
(m, 4H), 7.59–7.68 (m, 4H). ESI-MS: 415.32 (C19H17BrN3OS,
[M+H]+). Anal. Calcd for C19H16BrN3OS: C, 55.08; H, 3.89; N,
10.14. Found: C, 55.16; H, 3.88; N, 10.19.
4.2.3.31. 2-(3-(4-Methoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyr-
4.2.3.24. 2-(3-(4-Bromophenyl)-5-(4-methoxyphenyl)-4,5-dihy-
azol-1-yl)thiazol-4(5H)-one (E31).
Yield 68%; mp 205–
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E24).
Yield 86%;
207 °C. 1H NMR (CDCl3, 300 MHz): 3.72 (d, J = 14.02 Hz, 1H),
3.85–3.97 (m, 5H), 4.02 (dd, J1 = 8.02 Hz, J2 = 15.48 Hz, 1H), 5.86
(s, 1H), 7.11 (d, J = 6.18 Hz, 2H), 7.25–7.30 (m, 3H), 7.42 (d,
J = 5.44 Hz, 2H), 7.93 (d, J = 8.24 Hz, 2H). ESI-MS: 352.42
(C19H18N3O2S, [M+H]+). Anal. Calcd for C19H17N3O2S: C, 64.94; H,
4.88; N, 11.96. Found: C, 65.12; H, 4.89; N, 12.03.
mp 240–242 °C. 1H NMR (CDCl3, 300 MHz): 3.35 (dd, J1 = 4.24 Hz,
J2 = 17.82 Hz, 1H), 3.78 (s, 3H), 3.85–3.97 (m, 3H), 5.82 (d,
J = 7.32 Hz, 1H), 6.85 (d, J = 8.61 Hz, 2H), 7.20 (d, J = 8.61 Hz, 2H),
7.55–7.62 (m, 4H). ESI-MS: 431.32 (C19H17BrN3O2S, [M+H]+). Anal.
Calcd for C19H16BrN3O2S: C, 53.03; H, 3.75; N, 9.76. Found: C,
53.17; H, 3.77; N, 9.83.
4.2.3.32. 2-(5-(4-Fluorophenyl)-3-(4-methoxyphenyl)-4,5-dihy-
4.2.3.25.
yl)thiazol-4(5H)-one (E25).
2-(5-Phenyl-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-
Yield 63%; mp 210–212 °C. 1H
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E32).
Yield 83%;
mp 210–212 °C. 1H NMR (CDCl3, 300 MHz): 3.77 (s, 1H), 3.85–
3.97 (m, 6H), 5.84 (s, 1H), 6.84 (d, J = 8.04 Hz, 1H), 6.96–7.04 (m,
4H), 7.21–7.32 (m, 1H), 7.75 (d, J = 8.76 Hz, 2H). ESI-MS: 370.41
(C19H17FN3O2S, [M+H]+). Anal. Calcd for C19H16FN3O2S: C, 61.77;
H, 4.37; N, 11.37. Found: C, 61.86; H,4.36; N, 11.43.
NMR (CDCl3, 300 MHz): 2.43 (s, 3H), 3.36 (dd, J1 = 4.08 Hz,
J2 = 16.84 Hz, 1H), 3.87 (s, 2H), 3.97 (dd, J1 = 5.35 Hz, J2 = 14.24 Hz,
1H), 5.89 (d, J = 6.48 Hz, 1H), 7.25–7.35 (m, 3H), 7.27–7.30 (m,
4H), 7.45 (d, J = 5.34 Hz, 2H), 7.72 (d, J = 6.94 Hz, 2H). ESI-MS:
336.42 (C19H18N3OS, [M+H]+). Anal. Calcd for C19H17N3OS: C,
68.03; H, 5.11; N, 12.53. Found: C, 68.16; H, 5.14; N, 12.63.
4.2.3.33. 2-(5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihy-
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E33).
Yield 84%;
4.2.3.26. 2-(5-(4-Fluorophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyr-
mp 224–226 °C. 1H NMR (CDCl3, 300 MHz): 3.71 (dd, J1 = 6.96 Hz,
J2 = 13.89 Hz, 1H), 3.85–3.90 (m, 5H), 4.11(dd, J1 = 7.14 Hz,
J2 = 14.25 Hz, 1H), 5.79 (s, 1H), 6.97 (d, J = 8.61 Hz, 2H), 7.21 (d,
J = 8.22 Hz, 2H), 7.30 (d, J = 8.22 Hz, 2H), 7.74 (d, J = 8.79 Hz, 2H).
ESI-MS: 386.87 (C19H17ClN3O2S, [M+H]+). Anal. Calcd for
azol-1-yl)thiazol-4 (5H)-one (E26).
Yield 78%; mp 228–
230 °C. 1H NMR (CDCl3, 300 MHz): 2.43 (s, 3H), 3.34 (dd,
J1 = 3.63 Hz, J2 = 17.53 Hz, 1H), 3.87(s, 2H), 3.94 (dd, J1 = 6.25 Hz,
J2 = 12.84 Hz, 1H), 5.85(d, J = 7.32 Hz, 1H), 6.98–7.04 (m, 2H),
7.23 (s, 2H), 7.28 (d, J = 4.16 Hz, 2H), 7.69 (d, J = 8.22 Hz, 2H). ESI-
MS: 354.41 (C19H17FN3OS, [M+H]+). Anal.Calcd for C19H16FN3OS:
C, 64.57; H, 4.56; N, 11.89. Found: C, 64.68; H, 4.58; N, 11.95.
C
19H16ClN3O2S: C, 59.14; H, 4.18; N, 10.89. Found: C,59.29;
H,4.20; N, 10.96.
4.2.3.34. 2-(5-(4-Bromophenyl)-3-(4-methoxyphenyl)-4,5-dihy-
4.2.3.27. 2-(5-(4-Chlorophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyr-
dro-1H -pyrazol-1-yl)thiazol-4(5H)-one (E34).
Yield 88%;
azol-1-yl)thiazol-4 (5H)-one (E27).
Yield 81%; mp 234–
mp 230–232 °C. 1H NMR (CDCl3, 300 MHz): 3.36(d, J = 10.04 Hz,
1H), 3.85–3.96 (m, 5H), 4.08 (dd, J1 = 7.14 Hz, J2 = 14.25 Hz, 1H),
5.79 (s, 1H), 6.91–6.98 (m, 2H), 7.15 (s, 2H), 7.46 (s, 2H), 7.74 (d,
J = 6.39 Hz, 2H). ESI-MS: 431.32 (C19H17BrN3O2S, [M+H]+). Anal.
Calcd for C19H16BrN3O2S: C, 53.03; H, 3.75; N, 9.76. Found: C,
53.16; H, 3.77; N, 9.82.
236 °C. 1H NMR (CDCl3, 300 MHz): 2.40 (s, 3H), 3.15 (dd,
J1 = 3.84 Hz, J2 = 17.73 Hz, 1H), 3.76 (s, 2H), 3.83 (dd, J1 = 11.52,
J2 = 17.73 Hz, 1H), 5.99 (dd, J1 = 3.66 Hz, J2 = 11.52 Hz, 1H), 7.16
(d, J = 8.4 Hz, 2H), 7.23–7.26 (m, 2H), 7.28–7.31 (m, 2H), 7.61 (d,
J = 6.42 Hz, 2H). ESI-MS: 370.87 (C19H17ClN3OS, [M+H]+). Anal.
Calcd for C19H16ClN3OS: C, 61.70; H, 4.36; N, 11.36. Found: C,
61.82; H, 4.34; N, 11.43.
4.2.3.35. 2-(3-(4-Methoxyphenyl)-5-(p-tolyl)-4,5-dihydro-1H -
pyrazol-1-yl)thiazol-4(5H)-one (E35).
Yield 80%; mp 217–
4.2.3.28. 2-(5-(4-Bromophenyl)-3-p-tolyl-4,5-dihydro-1H -pyra-
219 °C. 1H NMR (CDCl3, 300 MHz): 2.31 (s, 3H), 3.35 (d,
J = 16.83 Hz, 1H), 3.84–3.95 (m, 6H), 5.82 (d, J = 8.61 Hz, 1H), 6.97
(d, J = 8. 10 Hz, 2H), 7.11–7.18 (m, 4H), 7.74 (d, J = 8.43 Hz, 2H).
ESI-MS: 366.45 (C20H20N3O2S, [M+H]+). Anal. Calcd for
C20H19N3O2S: C, 65.73; H, 5.24; N, 11.50. Found: C, 65.56; H,
5.22; N, 11.42.
zol-1-yl)thiazol-4(5H)-one (E28).
Yield 83%; mp 245–247 °C.
1H NMR (CDCl3, 300 MHz): 2.42 (s, 3H), 3.33 (d, J = 8.79 Hz, 1H),
3.90 (s, 2H), 3.95 (dd, J1 = 6.60 Hz, J2 = 10.62 Hz, 1H), 5.86 (d,
J = 4.68 Hz, 1H), 7.16 (d, J = 5.04 Hz, 2H), 7.26–7.28 (m, 2H),
7.46(d, J = 5.04 Hz, 2H), 7.69 (d, J = 4.86 Hz, 1H). ESI-MS: 415.32
(C19H17BrN3OS, [M+H]+). Anal. Calcd for C19H16BrN3OS: C, 55.08;
H, 3.89; N, 10.14. Found: C, 55.21; H, 3.92; N, 10.20.
4.2.3.36. 2-(3-(4-Chlorophenyl)-5-phenyl-4,5-dihydro-1H -pyra-
zol-1-yl)thiazol-4(5H)-one (E36).
Yield 85%; mp 220–222 °C.
4.2.3.29. 2-(3,5-Dip-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-
1H NMR (CDCl3, 300 MHz): 3.36 (dd, J1 = 3.48 Hz, J2 = 17.73 Hz, 1H),
3.92 (s, 2H), 4.13 (dd, J1 = 4.38 Hz, J2 = 15.78 Hz, 1H), 5.88 (d,
4(5H)-one (E29).
Yield 75%; mp 220–222 °C. 1H NMR (CDCl3,

K.-M. Qiu et al. / Bioorg. Med. Chem. 20 (2012) 2010–2018
2017
J = 10.95 Hz, 1H), 6.84 (d, J = 8.79 Hz, 2H), 6.97 (d, J = 8.79 Hz, 2H),
7.21–7.26 (m, 2H), 7.76 (d, J = 8.97 Hz, 2H). ESI-MS: 382.45
(C20H20N3O3S, [M+H]+). Anal. Calcd for C20H19N3O3S: C, 62.97; H,
5.02; N, 11.02. Found: C, 63.05; H, 5.05; N, 11.07.
with erlotinib as positive reference. After 48 h exposure period,
40
L of PBS containing 2.5 mg mLÀ1 of MTT (3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to each
well. Four hours later, 100 L extraction solution (10% SDS–5% iso-
l
l
butyl alcohol–0.01 M HCl) was added. After an overnight incuba-
tion at 37 °C, the optical density was measured at a wavelength
of 570 nm on an ELISA microplate reader. In all experiments three
replicate wells were used for each drug concentration. Each assay
was carried out for at least three times. The results were summa-
rized in Table 3.
4.3. Preparation, purification of HER-2 and EGFR and inhibitory
assay
A 1.7 kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) and 1.6 kb cDNA encoded for
the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186)
were cloned into baculoviral expression vectors pBlueBacHis2B
and pFASTBacHTc (Huakang Company, China), separately. A se-
quence that encodes (His)6 was located at the 50 upstream to the
HER-2 and EGFR sequences. 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,
4.5. Molecular docking study
Molecular docking of compounds into the 3D EGFR complex
structure (PDB code: 1M17) was carried out using the Discovery
Stutio (version 3.1) as implemented through the graphical user
interface DS-LigandFit protocal.
10
l
M ammonium molybdate, 100
l
M sodium vanadate, 10
l
l
g/mL
g/mL
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 crystal structures of EGFR complex were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.-
do). All bound water and ligands were eliminated from the protein
and the polar hydrogen was added. The whole EGFR complex was
defined as a receptor and the site sphere was selected based on the
ligand binding location of ATP, then the ATP molecule was re-
moved and E28 was placed during the molecular docking proce-
dure. Types of interactions of the docked protein with ligand
were analyzed after the end of molecular docking.
aprotinin, 10
lg/mL leupeptin, 10
lg/mL pepstatin, and 16
benzamidine HCl for 20 min followed 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 imid-
azole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glyc-
erol, and 1 lg/mL each of aprotinin, leupeptin, and pepstatin for
2 h. The entire purification procedure was performed at 4 °C or
on ice.
Both EGFR and HER-2 kinase assays were set up to assess the le-
vel of autophosphorylation based on DELFIA/Time-Resolved Fluo-
rometry. Compounds E1–E36 were dissolved in 100% DMSO and
diluted to the appropriate concentrations with 25 mM HEPES at
pH 7.4. In each well, 10
lL compound was incubated with 10
lL
Acknowledgment
(12.5 ng for HER-2 or 5 ng for EGFR) recombinant enzyme (1:80
dilution in 100 mM HEPES) for 10 min at room temperature. Then,
This work was supported by The National Natural Science Foun-
dation of China (NSFC J1103512).
10
lL of 5 mM buffer (containing 20 mM HEPES, 2 mM MnCl2,
100
l
M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM ATP–
l
50 mM MgCl2 were added for 1 h. Positive and negative controls
were included in each plate by incubation of enzyme with or with-
out ATP-MgCl2. At the end of incubation, liquid was aspirated, and
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