Synthesis, molecular docking and evaluation of thiazolyl-pyrazoline derivatives containing benzodioxole as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2012.11.020

A series of novel thiazolyl-pyrazoline derivatives containing benzodioxole (C1-C20) have been designed and synthesized. Among of the synthesized compounds, 2-(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)- 4-(4-bromophenyl)thiazole (C6) displayed the most potent inhibitory activity for HER-2 (IC₅₀ = 0.18 μM for HER-2). Antiproliferative assay results indicated that compound C6 owned high antiproliferative activity against MCF-7 and B16-F10 in vitro, with IC₅₀ value of 0.09 and 0.12 μM, respectively, being comparable with the positive control Erlotinib. Docking simulation was further performed to determine the probable binding model. Based on the preliminary results, compound C6 with potent inhibitory activity in tumor growth would be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2012.11.020

Bioorganic & Medicinal Chemistry 21 (2013) 448–455
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular docking and evaluation of thiazolyl-pyrazoline
derivatives containing benzodioxole as potential anticancer agents
Hai-Hong Wang a, , Ke-Ming Qiu a, , Hong-En Cui a, Yu-Shun Yang a, Yin-Luo a, Man Xing a, Xiao-Yang Qiu b,
,
⇑
a,
Li-Fei Bai c, , Hai-Liang Zhu
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Department of chemistry, Shangqiu Normal University, Shangqiu 476000, PR China
c School of Life Sciences and Chemistry, Jiangsu Institute of Education, Nanjing 210013, 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 thiazolyl-pyrazoline derivatives containing benzodioxole (C1–C20) have been designed
and synthesized. Among of the synthesized compounds, 2-(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-bromophe-
nyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole (C6) displayed the most potent inhibitory
Received 6 September 2012
Revised 11 November 2012
Accepted 12 November 2012
Available online 24 November 2012
activity for HER-2 (IC50 = 0.18
owned high antiproliferative activity against MCF-7 and B16-F10 in vitro, with IC50 value of 0.09 and
0.12 M, respectively, being comparable with the positive control Erlotinib. Docking simulation was
lM for HER-2). Antiproliferative assay results indicated that compound C6
l
Keywords:
Benzodioxole
Thiazole
Pyrazoline
HER-2
further performed to determine the probable binding model. Based on the preliminary results, compound
C6 with potent inhibitory activity in tumor growth would be a potential anticancer agent.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
attention in medicinal and pharmaceutical research as this struc-
tural scaffold is found in a variety of drugs, and we could suppose
Cancer is a multifactor disease with superfluous and robust
biological networks.1 It may require treatment with compounds
that could target multiple intracellular components.2 Recently,
these novel anticancer drugs are those targeting mutant or aber-
rantly expressed oncogenic growth factor receptor and non-recep-
tor tyrosine kinases involved in mitogenic or proliferative signal
transduction pathways in cancer cells.3
Receptor tyrosine kinases (RTKs) and epidermal growth factor
receptor (EGFR) play very important roles in regulating tumor cell
proliferation, differentiation, survival and apoptosis.4–6 EGFR
family comprises four members, including: EGFR (HER-1/ErbB-1),
ErbB-2 (HER-2/neu), ErbB-3 (HER-3) and ErbB-4 (HER-4).7 Com-
pounds that inhibit the kinase activity of EGFR and/or HER-2 after
binding to its ATP binding site are of potential interest as new
therapeutic antitumor agents.8 For example, Erlotinib ( Fig. 1)
inhibits EGFR/HER-2 that is overexpressed in tumors and is ap-
proved antitumor agent.
the benzo[d][1,3]dioxole-5-carbaldehyde and the 2,3-dihydro-
benzo[b][1,4]dioxine-6-carbaldehyde as 3,4-dimethoxy derivative
and 3,4-diethoxy derivative, respectively, which was reported in
our previous research.11
On the other hand, thiazoles and their derivatives have been
frequently discovered as a vital component of novel that recently
found application in drug development for treatment of allergies,12
schizophrenia13 and more recently for the treatment of cancer.14
For example, Micheal, D.G. et al. reported that 6-thiazolylquinazo-
line derivatives (Fig. 1) showed modest to HER-2/EGFR TK inhibi-
tory activity.15 Meanwhile, pyrazole derivatives have attracted
continuing attention over the years because of their broad
spectrum biological activities and strong efficacy. Thus, some rep-
resentatives of this heterocycle exhibit antitumor,16 anti-hypergly-
cemic activity17 and sedative-hypnotic activity.18 For example,
Claudio, N.C. et al. have discovered N1 (Fig. 1) showed modest
EGFR/HER-2 inhibitory activity.19 And as previous reported in our
laboratory, N2 (Fig. 1) displayed the most potent EGFR TK inhibi-
Small and simple hetero-aromatics often have surprisingly
complex biological properties and belong to one of the most
important classes of compounds in medicinal chemistry.9 Benzodi-
oxole, thiazoles and pyrazoles are widely found in both natural
products and drugs.10 Benzodioxole has received significant
tory activity (IC50 = 0.06 l
M for EGFR).20
In addition, benzodioxole is found in a variety of anticancer
drugs with excellent bioavailability and low cytotoxicity,21,22 which
will enhance the anticancer activity of thiazole and pyrazole motif,
thus might exhibit synergistic anticancer effect and the modifica-
tion in this paper also creates new possibility to reinforce the
combination between our compounds and the receptor.23 In an ef-
fort to extend our research on anticancer compounds with HER-2
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.020

H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
449
R
NH
N
O
O
O
Ar
N
S
N
N
O
NH
NH
N
6-thiazolylquinazoline
Erlotinib
derivatives
F
N
N
S
NH
O
N
S
N
NH
Cl
N-[3-(4-Methylphenyl)-1H-pyrazol-5-yl]
-2-thiophenecarboxamide (N1)
4-(4-chlorophenyl)-2-(3-(3,4-dimethylphenyl)-5-p-
tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (N2)
Figure 1. Chemical structures of some reported compounds.
inhibitory activity, we synthesized a series of compounds building
of benzodioxole combined with thiazolyl-pyrazoline derivatives
as potential anticancer agents. But as we know, as a member of
the EGFR family, although in recent years it has evolved to become
an important biomarker and target of therapy for the disease, there
is a little literature reported about HER-224–26 and we want to
investigate the effect of HER-2, so we choose HER-2 to test the
inhibitory activity and do docking simulation. And the HER-2 is
the homologous protein of human EGFR, it can heterodimerise with
any of the other three receptors and is considered to be the pre-
ferred dimerisation partner of the other ErbB receptors, and the
activity of HER-2 is often stronger than the other heterodimer.
And the drugs we study eventually will be acting on the human
body, so it will has more practical significance of the study on
HER-2. According to the activity data and biological evaluation re-
sult, the compounds have potent HER-2 inhibitory activity, which
eventually would be proved to be potential anticancer agents.
2. Results and discussion
2.1. Chemistry
Synthesis of compounds C1–C20 is followed the general path-
way outlined in Scheme 1. Firstly, the benzodioxole compounds
were obtained by protocatechuic aldehydes reacting with dibro-
momethane or 1,2-dibromoethane in acetonitrile. Secondly, the
chalcone derivatives were obtained by direct condensation by the
benzodioxole compounds and the substituted acetophenone, using
40% potassium hydroxide as catalyst in ethanol. Thirdly, cycliza-
tion of different chalcone derivatives with thiosemicarbazide un-
der basic condition leads to the formation of pyrazole derivatives
containing thiourea skeleton. Finally, thiazolyl-pyrazoline deriva-
tives C1–C20 were obtained by reacting compound B with two dif-
ferent kinds of substituted 2-bromoacetophenone. All of these
compounds are reported for the first time. All of the synthesized
OHC
O
O
+
CH2
n
R1
R1
CH2
n
O
O
O
O
A1-A10
R1
R1
O
O
N
CH2
N
n
N
CH2
N
S
n
O
O
S
N
H2N
B1-B10
R2
C1-C20
Scheme 1. General synthesis of thiazolyl-pyrazoline derivatives containing benzodioxole (C1–C20). Reagents and conditions: (i) 40% aqueous potassium hydroxide solution,
ethanol, 4 h rt; (ii) thiosemicarbazide, KOH, ethanol, reflux, 12 h; (iii) substituted 2-bromoacetophenone, ethanol, reflux, 8 h.

450
H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
Table 2
compounds gave satisfactory analytical and spectroscopic data,
which were in full consistent with their depicted structures.
Antiproliferative activities of compounds C1–C20 against MCF-7 and B16-F10
(IC50 M)
,
l
Compounds MCF-7
B16-F10
Compounds MCF-7
B16-F10
2.2. Anticancer activity and structure–activity relationship of
the thiazolyl-pyrazoline derivatives containing benzodioxole
C1–C20
C1
3.65 0.36 3.98 0.41 C11
3.43 0.38 3.81 0.35 C12
2.41 0.25 2.32 0.25 C13
1.72 0.18 1.51 0.16 C14
0.27 0.02 0.32 0.04 C15
0.09 0.008 0.12 0.01 C16
1.43 0.14 1.52 0.14 C17
1.41 0.12 1.39 0.12 C18
2.73 0.27 2.67 0.28 C19
2.47 0.25 2.54 0.24 C20
0.02 0.004 0.05 0.008 Erlotinib
4.53 0.46 4.43 0.48
4.20 0.41 4.11 0.43
3.03 0.32 3.12 0.33
2.23 0.20 2.15 0.22
0.87 0.07 1.12 0.14
0.32 0.05 0.43 0.06
2.68 0.24 2.87 0.26
1.59 0.14 1.68 0.18
2.73 0.30 3.01 0.28
2.54 0.24 2.52 0.26
0.02 0.004 0.05 0.008
C2
C3
C4
C5
C6
C7
C8
C9
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds C1–C20
against MCF-7 (human breast cancer cell lines) and B16-F10 cells
(mouse melanoma cell lines). The results were summarized in
Table 2. It was observed that thiazolyl-pyrazoline derivatives
which containing benzodioxole showed fairly good inhibiting
MCF-7 and B16-F10 activities displaying IC50 values between
C10
Erlotinib
0.09 and 4.53
compound C6 displayed the most potent inhibitory activity
(IC50 = 0.09 M for MCF-7, IC50 = 0.12 M for B16-F10). Subse-
quently SAR studies were performed by modification of the parent
compound to determine how the substituents of the subunits
affect the HER-2 inhibitory activities. Inspection of the chemical
structures of the compounds C1–C20 suggested that they could
be divided into three subunits: A-, B- and C-rings (Scheme 1 and
Table 1).
lM, 0.12 and 4.43 lM, respectively. Among them,
In the case of constant A ring substituents, changes of substitu-
ents on B ring can also affect the activities of these compounds.
Compared to the six-membered ring compounds (the value of
IC50 range from 0.32 to 4.53 lM, 0.43 to 4.43 lM for MCF-7 and
B16-F10, respectively), the five-membered ring compounds
showed more inhibitory activity (the value of IC50 range from
0.09 to 3.65 lM, 0.12 to 3.98 lM for MCF-7 and B16-F10, respec-
l
l
tively). We can arrive at the conclusion that five-membered ring
structure is more similar to the receptor (the five-membered ring
is smaller in volume and is more hydrophobic, which would be
easier to insert into receptor’s hydrophobic pocket), so that they
showed better affinity properties than the six-membered ring
compounds.
At the same time, changes of substituents on C ring can also af-
fect the activities of these compounds. According to the results of
activities data, we know that electron-withdrawing substituents
at 4-position derivatives had more potent HER-2 inhibitory activi-
ties than the electron-donating substituents ones.
The above results indicated that benzodioxole, thiazole and pyr-
azole rings in the thiazolyl-pyrazoline derivatives containing ben-
zodioxole might play an important role in the process of apoptosis.
And the changes of substituents on the A-, B-, C-rings had effects
on the activity of the compound, which might be explained that
substituents on the three different rings had certain influence on
the molecular bonding between the receptor and the compounds.
Molecular docking of all the compounds of this series had demon-
strated the point and this was conformed to our estimate.
As shown in Table 1, structure–activity relationships in com-
pounds C1–C20 demonstrated that compounds bearing one bro-
mine atom at para- at A-ring (compounds C5, C6, C15, C16 and
the value of IC50, respectively: 0.85, 0.18, 1.32 and 0.46 lM)
showed better HER-2 inhibitory activity than those with fluorin (
C1, C2, C11, C12), chlorine ( C3, C4, C13, C14), methyl ( C7, C8,
C17, C18) and methoxyl ( C9, C10, C19, C20) substituents at the
same position, basically in the order of Br > Cl > F, Br > Me > OMe.
This meant that a weaker electron-withdrawing substituent at
para-position of A-ring (Br) had better HER-2 inhibitory activity
than that with a relatively stronger electron-withdrawing substit-
uents (Cl, F), and that compound with a faintish electron-donating
substituents at para-position of A-ring (Me) had better HER-2
inhibitory activity than that with a relatively stronger electron-
donating substituents (OMe).
Table 1
Chemical structures of thiazolyl-pyrazoline derivatives containing benzodioxole
2.3. HER-2 inhibitory activity and molecular docking study
R1
The series of thiazolyl-pyrazoline derivatives containing benzo-
dioxole (C1–C20) were evaluated for their ability to inhibit the
autophosphorylation of HER-2 kinases using a solid-phase ELISA
assay. The inhibition constant (IC50) of the compounds were sum-
marized in Table 3. As was shown in Table 3, out of the top six
compounds, compounds C6 and C16, showed potent inhibitory
O
CH2
N
N
S
n
O
N
activity of HER-2 (IC50 = 0.18 and 0.46 lM, respectively), indicating
R2
Table 3
Compounds
R1
n
R2
Compounds
R1
n
R2
Inhibition activities of compounds C4, C5, C6, C8, C15,
C16 (IC50 M)
,
l
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
F
F
Cl
Cl
Br
Br
Me
Me
OMe
OMe
1
1
1
1
1
1
1
1
1
1
OMe
Br
OMe
Br
OMe
Br
OMe
Br
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
F
F
Cl
Cl
Br
Br
Me
Me
OMe
OMe
2
2
2
2
2
2
2
2
2
2
OMe
Br
OMe
Br
OMe
Br
OMe
Br
Compounds
HER-2
C4
C5
C6
C8
C15
C16
Erlotinib
2.38 0.22
0.85 0.09
0.18 0.02
2.23 0.25
1.32 0.16
0.46 0.06
0.16 0.007
OMe
Br
OMe
Br

H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
451
that these thiazolyl-pyrazoline derivatives containing benzodiox-
ole were potent antitumor agents. In particular, compound C6
had demonstrated significant inhibitory activity in tumor growth
inhibition and displayed potential HER-2 inhibitory activity.
To determine whether target compounds suppress activation of
HER-2, selected compound C6 were tested for its suppression on
HER-2 and Cleaved Caspase 3 (an important enzyme in apoptosis).
As shown in Figure 2, HER-2 was dose-dependent lower regulated
upon C6 treated for 24 h. The activity of Cleaved Caspase 3 was
dose-dependent up-regulated upon C6 treated. It can be conferred
from the result that the apoptosis induced by C6 may be mediated
by the inhibition of PI3K/AKT pathway.27,28
Figure 2. Western blotting was performed to detect the effect of compound C6 on
HER-2.
In order to gain better understanding on the potency of the com-
pound C6 and guide further SAR studies, we proceeded to examine
the interaction of it with HER-2 (PDB code: 1UOM) by molecular
docking, which was performed by simulation of the compound into
the ATP binding site in HER-2. The binding model of compound C6
and HER-2 was depicted in Figures 3A and 3B. The amino acid
residues which had interaction with HER-2 were labeled. In the
binding mode, compound C6 was nicely bound to the ATP binding
site of HER-2 through hydrophobic interaction, and the two
H-bonds played an important effect in strengthening the interac-
tion: Nꢀ ꢀ ꢀH–O/Thr347 (distance: 2.17612 Å, angle: 162.968°) and
Brꢀ ꢀ ꢀH–N/Arg394 (distance: 2.15122 Å, angle: 145.903°). Based on
the favorable HER-2 inhibitory activity of thiazolyl-pyrazoline
derivatives, it could be concluded that the hydrophobic pockets of
ATP binding site were all nicely occupied by these compounds. This
molecular docking result, along with the enzyme assay data, sug-
gesting that compound C6 is a potential inhibitor of HER-2.
3. Conclusions
In this paper, a series of thiazolyl-pyrazoline derivatives con-
taining benzodioxole that may function as inhibitors of HER-2 ki-
nases have been synthesized and their biological activities were
evaluated. And some of them displayed potent HER-2 inhibitory
activities and antiproliferative activities against MCF-7 cell lines
and B16-F10 cell lines. Among them, compound C6 showed the
Figure 3A. Molecular docking 3D modeling of compound C6 with HER-2: for
clarity, only interacting residues are displayed. The H-bond (green lines) is
displayed as dotted lines.
most potent HER-2 inhibition activities (IC50 = 0.18
l
M) and anti-
M for
cancer activities (IC50 = 0.09 M for MCF-7 and IC50 = 0.12
l
l
B16-F10). Molecular docking was further performed to study the
inhibitor-HER-2 protein interactions. After analysis of the binding
model of compound C6 with HER-2, it was found that two hydro-
gen bonds interaction with the protein residues in the ATP binding
site might play a crucial role in its HER-2 inhibition and antiprolif-
erative activities. Among these compounds, it could be concluded
that compound C6 had been demonstrated to show significant
HER-2 and tumor growth inhibitory activity as a potential antican-
cer agent. The result of this work might be helpful for the design
and synthesis of HER-2 inhibitors with stronger activities.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were of analyt-
ical grade. Separation of the compounds by column chromatogra-
phy 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 obtained on a Mariner System 5304 mass spectrometer,
and 1H NMR spectra were recorded on a Bruker PX500 or
DPX300 spectrometer at 25 °C with TMS and solvent signals
Figure 3B. Molecular docking 2D modeling of C6 with HER-2: for clarity, only
interacting residues are displayed. The H-bond (blue lines) is displayed as arrow
dots.

452
H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
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.
J2 = 18.04 Hz, 1H), 5.92 (d, J = 5.35 Hz, 1H), 5.98 (s, 2H), 6.78–6.84
(m, 3H), 7.28 (s, 1H), 7.32 (d, J = 9.34 Hz, 2H), 7.69 (d, J = 6.74 Hz,
2H), 7.68 (d, J = 6.32 Hz, 2H), 7.83 (d, J = 7.81 Hz, 2H). ESI-MS:
523.39 (C25H18BrFN3O2S, [M+H]+). Anal. Calcd for C25H17BrFN3O2S:
C, 57.48; H, 3.28; N, 8.04. Found: C, 57.56; H, 3.24; N, 8.01.
4.2. Synthesis
4.2.4.3. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-chlorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C3).
4.2.1. General synthetic procedure of benzodioxole compounds
(benzo[d][1,3]dioxole-5-carbaldehyde and 2,3-
dihydrobenzo[b][1,4]dioxine-6-carbaldehyde)
Yield: 75%. Mp 198–200 °C. 1H NMR (CDCl3, 500 MHz):
3.29 (dd, J1 = 5.95 Hz, J2 = 17.55 Hz, 1H), 3.86 (s, 3H), 3.88 (d,
J = 12.05 Hz, 1H), 5.82 (s, 1H), 5.93 (d, J = 4.25 Hz, 2H), 6.68 (s,
1H), 6.79 (d, J = 7.90 Hz, 1H), 6.87–6.89 (m, 3H), 6.97 (d,
J = 7.90 Hz, 1H), 7.41 (d, J = 8.40 Hz, 2H), 7.65–7.71 (m, 4H). ESI-
MS: 490.97 (C26H21ClN3O3S, [M+H]+). Anal. Calcd for
Equimolar portions of the appropriately protocatechuic alde-
hydes (50 mmol, 1 equiv) and dibromomethane or 1, 2-dibromo-
ethane (50 mmol, 1 equiv) were dissolved in acetonitrile. The
mixture was allowed to stir for several minutes at 90 °C to let dis-
solve. Then potassium carbonate (20.7 g, 3 equiv) was slowly
added to the reaction as acid binding agent. The reaction solution
was refluxed approximately 24 h. Most commonly, filtered and re-
moved the precipitation, spin dry the solvent, extraction and
recrystallization, then you will get benzodioxole compounds as
reaction raw materials.
C26H20ClN3O3S: C, 63.73; H, 4.11; N, 8.58. Found: C, 63.85; H,
4.03; N, 8.62.
4.2.4.4. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-chlorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole
(C4).
Yield: 78%. Mp 220–222 °C. 1H NMR (CDCl3, 500 MHz):
3.34 (d, J = 17.55 Hz, 1H), 3.90 (d, J = 16.83 Hz, 1H), 5.94 (s, 2H),
6.14 (s, 1H), 6.80 (s, 2H), 6.86 (s, 1H), 7.04 (d, J = 7.50 Hz, 1H),
7.43 (d, J = 8.35 Hz, 2H), 7.49 (d, J = 8.40 Hz, 2H), 7.62 (d,
J = 8.25 Hz, 2H), 7.71 (d, J = 8.40 Hz, 2H). ESI-MS: 538.84
(C25H18BrClN3O2S, [M+H]+). Anal. Calcd for C25H17BrClN3O2S: C,
55.72; H, 3.18; N, 7.80. Found: C, 55.84; H, 3.11; N, 7.72.
4.2.2. General synthetic procedure of chalcones derivatives (A1–
A10)
Equimolar portions of the appropriately benzodioxole com-
pounds (5 mmol, 1 equiv) and substituted acetophenone (5 mmol,
1 equiv) were dissolved in approximately 20 mL of ethanol. The
mixture was allowed to stir for several minutes at 10 °C to let dis-
solve. Than a 1 mL aliquot of a 40% aqueous potassium hydroxide
solution was then slowly added dropwise to the reaction flask
via a selfequalizing addition funnel. The reaction solution was al-
lowed to stir at room temperature for approximately 4–6 h. Most
commonly, a precipitate formed and was then collected by suction
filtration.
4.2.4.5. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C5).
Yield: 81%. Mp 226–228 °C. 1H NMR (CDCl3, 500 MHz):
3.28 (dd, J1 = 6.25 Hz, J2 = 17.40 Hz, 1H), 3.82 (s, 3H), 3.87 (d,
J = 11.90 Hz, 1H), 5.77 (s, 1H), 5.93 (d, J = 4.10 Hz, 2H), 6.68 (s,
1H), 6.79 (d, J = 7.95 Hz, 1H), 6.86–6.89 (m, 3H), 6.96 (d,
J = 7.45 Hz, 1H), 7.56 (d, J = 8.55 Hz, 2H), 7.62–7.66 (m, 4H). ESI-
4.2.3. General synthetic procedure of pyrazole derivatives
(B1–B10)
MS:
535.42(C26H21BrN3O3S,
[M+H]+).
Anal.
Calcd
for
C26H20BrN3O3S: C, 58.43; H, 3.77; N, 7.86. Found: C, 58.59; H,
A mixture of chalcone derivatives (3 mmol), thiosemicarbazide
(4.5 mmol), and KOH (3 mmol) was refluxed in ethanol (30 mL) for
12 h. After cooling, the solution was poured into mass of ice-water
and stirred for a few minutes. The precipitate was filtered and crys-
tallized from ethanol.
3.70; N, 7.81.
4.2.4.6. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole
(C6).
Yield: 84%. Mp 241–243 °C. 1H NMR (CDCl3, 500 MHz):
3.29 (dd, J1 = 6.25 Hz, J2 = 17.40 Hz, 1H), 3.88 (d, J = 12.05 Hz, 1H),
5.77 (s, 1H), 5.93 (d, J = 4.90 Hz, 2H), 6.79 (d, J = 7.95 Hz, 1H),
6.83 (d, J = 14.50 Hz, 2H), 6.95 (d, J = 7.95 Hz, 1H),
7.46 (d, J = 8.05 Hz, 2H), 7.56–7.68 (m, 6H). ESI-MS: 584.29
(C25H18Br2N3O2S, [M+H]+). Anal. Calcd for C25H17Br2N3O2S: C,
51.48; H, 2.94; N, 7.20. Found: C, 51.32; H, 2.98; N, 7.14.
4.2.4. General synthetic procedure of benzodioxole thiazolyl-
pyrazoline derivatives (C1–C20)
A mixture of compound B (1 mmol) and substituted 2-bromo-
acetophenone (1 mmol) was dissolved in ethanol (20 mL) and kept
stirring in 80 °C for 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.
4.2.4.7. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)-4,5-dihydro-
1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole (C7).
Yield:
74%. Mp 207–209 °C. 1H NMR (CDCl3, 500 MHz): 2.34 (s, 3H),
3.32 (dd, J1 = 9.75 Hz, J2 = 18.95 Hz, 1H), 3.40 (s, 3H), 3.90 (d,
J = 19.85 Hz, 1H), 5.91 (d, J = 7.55 Hz, 1H), 6.07 (s, 2H), 6.66 (s,
1H), 6.80 (d, J = 12.65 Hz, 2H), 6.91 (s, 1H), 6.98 (d, J = 13.70 Hz,
2H),7.14 (d, J = 14.60 Hz, 2H), 7.75 (d, J = 8.85 Hz, 2H), 7.78 (d,
J = 8.80 Hz, 2H). ESI-MS: 470.55 (C27H24N3O3S, [M+H]+). Anal. Calcd
for C27H23N3O3S: C, 69.06; H, 4.94; N, 8.95. Found: C, 68.95; H,
4.90; N, 9.01.
4.2.4.1. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C1).
3.32 (dd, J1 = 5.35 Hz, J2 = 17.40 Hz, 1H), 3.83 (s, 3H), 3.90 (dd,
J1 = 11.90 Hz, J2 = 17.35 Hz, 1H), 5.93 (d, J = 2.15 Hz, 3H), 6.65 (s,
1H), 6.79 (d, J = 8.10 Hz, 2H), 6.87–6.91 (m, 2H), 7.06 (s, 1H),
7.12–7.16 (m, 2H), 7.69 (d, J = 8.65 Hz, 2H), 7.63–7.69 (m, 2H).
ESI-MS: 474.52 (C26H21FN3O3S, [M+H]+). Anal. Calcd for
Yield: 78%. Mp 189–191 °C. 1H NMR (CDCl3, 500 MHz):
C
26H20FN3O3S: C, 65.95; H, 4.26; N, 8.87. Found: C, 66.07; H,
4.2.4.8. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)-4,5-dihydro-
4.28; N, 8.81.
1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole
(C8).
Yield:
82%. Mp 219–221 °C. 1H NMR (CDCl3, 500 MHz): 2.42 (s, 3H),
3.33 (dd, J1 = 5.80 Hz, J2 = 17.55 Hz, 1H), 3.90 (dd, J1 = 11.70 Hz,
J2 = 17.50 Hz, 1H), 5.93 (d, J = 4.45 Hz, 1H), 5.98 (s, 2H), 6.78–6.81
(m, 2H), 6.87 (s, 1H), 7.00 (d, J = 7.95 Hz, 1H), 7.24–7.27 (m, 2H),
7.47 (d, J = 8.40 Hz, 2H), 7.61 (d, J = 8.55 Hz, 2H), 7.67 (d,
4.2.4.2. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole
(C2).
3.32 (dd, J1 = 5.43 Hz, J2 = 17.69 Hz, 1H), 3.92 (dd, J1 = 8.78 Hz,
Yield: 82%. Mp 218–220 °C. 1H NMR (CDCl3, 500 MHz):

H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
453
J = 8.10 Hz, 2H). ESI-MS: 519.42 (C26H21BrN3O2S, [M+H]+). Anal.
Calcd for C26H20BrN3O2S: C, 60.24; H, 3.89; N, 8.11. Found: C,
60.39; H, 3.82; N, 8.03.
ESI-MS: 553.87 (C26H20BrClN3O2S, [M+H]+). Anal. Calcd for
26H19BrClN3O2S: C, 56.48; H, 3.46; N, 7.60. Found: C, 56.32; H,
3.51; N, 7.69.
C
4.2.4.9. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C9).
4.2.4.15. 2-(3-(4-Bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C15).
Yield: 74%. Mp 190–192 °C. 1H NMR (CDCl3, 500 MHz):
Yield: 75%. Mp 247–249 °C. 1H NMR (CDCl3, 500 MHz):
3.33 (d, J = 17.55 Hz, 1H), 3.83 (s, 6H), 3.88 (d, J = 14.25 Hz, 1H),
5.91 (s, 1H), 5.94 (s, 2H), 6.63 (s, 1H), 6.79 (d, J = 7.95 Hz, 1H),
6.89 (d, J = 10.70 Hz, 3H), 6.96 (d, J = 8.85 Hz, 2H), 7.06 (s, 1H),
7.69–7.78 (m, 4H). ESI-MS: 486.55 (C27H24N3O4S, [M+H]+). Anal.
Calcd for C27H23N3O4S: C, 66.79; H, 4.77; N, 8.65. Found: C,
66.64; H, 4.70; N, 8.71.
3.32 (d, J = 15.85 Hz, 1H), 3.83 (s, 3H), 3.91 (dd, J1 = 7.10 Hz,
J2 = 17.45 Hz, 1H), 4.26 (s, 4H), 5.91 (s, 1H), 6.72 (s, 1H), 6.75 (d,
J = 8.25 Hz, 2H), 6.97 (d, J = 8.10 Hz, 1H), 6.91–6.94 (m, 2H), 7.03
(d, J = 8.35 Hz, 2H), 7.38 (d, J = 6.25 Hz, 2H), 7.71 (d, J = 8.75 Hz,
2H). ESI-MS: 549.45 (C27H23BrN3O3S, [M+H]+). Anal. Calcd for
C27H22BrN3O3S: C, 59.13; H, 4.04; N, 7.66. Found: C, 59.01; H,
4.09; N, 7.72.
4.2.4.10. 2-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-1-yl)-4-(4-bromophenyl)thiazole
(C10).
3.34 (d, J = 17.55 Hz, 1H), 3.83 (s, 3H), 3.94 (d, J = 10.25 Hz, 1H),
5.93 (d, J = 9.15 Hz, 1H), 5.98 (s, 2H), 6.79–6.99 (m, 3H), 7.21 (d,
J = 7.65 Hz, 1H), 7.47 (d, J = 5.60 Hz, 2H), 7.53 (d, J = 8.10 Hz, 2H),
7.68 (d, J = 7.30 Hz, 2H), 7.75 (d, J = 8.40 Hz, 2H). ESI-MS: 535.42
(C26H21BrN3O3S, [M+H]+). Anal. Calcd for C26H20BrN3O3S: C,
58.43; H, 3.77; N, 7.86. Found: C, 58.31; H, 3.71; N, 7.92.
4.2.4.16. 4-(4-Bromophenyl)-2-(3-(4-bromophenyl)-5-(2,3-dihy-
drobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-yl)thia-
zole (C16).
500 MHz): 3.31 (dd, J1 = 5.95 Hz, J2 = 16.05 Hz, 1H), 3.92 (d,
J1 = 6.35 Hz, J2 = 14.35 Hz, 1H), 4.28 (s, 4H), 5.94 (s, 1H), 6.79–
6.82 (m, 3H), 6.95 (d, J = 7.25 Hz, 1H), 7.06 (d, J = 7.45 Hz, 2H),
7.24 (d, J = 8.05 Hz, 2H), 7.56 (d, J = 7.35 Hz, 2H), 7.71 (d,
J = 7.95 Hz, 2H). ESI-MS: 598.32 (C26H20Br2N3O2S, [M+H]+). Anal.
Calcd for C26H19Br2N3O2S: C, 52.28; H, 3.21; N, 7.03. Found: C,
52.14; H, 3.28; N, 7.09.
Yield: 76%. Mp 207–209 °C. 1H NMR (CDCl3, 500 MHz):
Yield: 74%. Mp 261–263 °C. 1H NMR (CDCl3,
4.2.4.11. 2-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-fluo-
rophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxy-
phenyl)thiazole (C11).
Yield: 70%. Mp 218–220 °C. 1H NMR
4.2.4.17. 2-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(p-tolyl)-
4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(CDCl3, 500 MHz): 3.28 (dd, J1 = 6.15 Hz, J2 = 14.25 Hz, 1H), 3.83
(s, 3H), 3.94(dd, J1 = 7.55 Hz, J2 = 15.20 Hz, 1H), 4.12 (s, 2H), 4.15
(s, 2H), 5.95 (s, 1H), 6.70 (s, 1H), 6.79 (d, J = 7.05 Hz, 2H), 6.88 (s,
1H), 6.93 (d, J = 5.80 Hz, 2H), 7.03–7.08 (m, 2H), 7.22 (d,
J = 9.35 Hz, 2H), 7.68 (d, J = 7.95 Hz, 2H). ESI-MS: 488.55
(C27H23FN3O3S, [M+H]+). Anal. Calcd for C27H22FN3O3S: C, 66.51;
H, 4.55; N, 8.62. Found: C, 66.69; H, 4.50; N, 8.55.
(C17).
Yield: 68%. Mp 217–219 °C. 1H NMR (CDCl3, 500 MHz):
2.33 (s, 3H), 3.29 (dd, J1 = 7.05 Hz, J2 = 17.45 Hz, 1H), 3.83 (s, 3H),
3.90 (d, J1 = 7.34 Hz, J2 = 16.85 Hz, 1H), 4.25 (s, 4H), 5.85 (s, 1H),
6.63 (s, 1H), 6.77 (d, J = 7.75 Hz, 2H), 6.82 (d, J = 8.15 Hz, 1H),
6.98 (d, J = 5.70 Hz, 2H), 7.14 (d, J = 6.60 Hz, 2H), 7.35 (d,
J = 7.85 Hz, 2H), 7.67 (d, J = 7.80 Hz, 2H). ESI-MS: 484.58
(C28H26N3O3S, [M+H]+). Anal. Calcd for C28H25N3O3S: C, 69.54; H,
5.21; N, 8.69. Found: C, 69.42; H, 5.27; N, 8.61.
4.2.4.12. 4-(4-Bromophenyl)-2-(5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole
4.2.4.18.
dioxin-6-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole
(C18).
Yield: 77%. Mp 231–233 °C. 1H NMR (CDCl3, 500 MHz):
4-(4-Bromophenyl)-2-(5-(2,3-dihydrobenzo[b][1,4]
(C12).
Yield: 72%. Mp 230–232 °C. 1H NMR (CDCl3, 500 MHz):
3.29 (dd, J1 = 6.75 Hz, J2 = 17.25 Hz, 1H), 3.87 (d, J = 12.55 Hz, 1H),
4.16 (s, 2H), 4.22 (s, 2H), 5.88 (s, 1H), 6.76–6.80 (m, 2H), 6.86 (s,
1H), 6.98 (d, J = 8.05 Hz, 1H), 7.18 (d, J=7.35 Hz, 2H), 7.40 (d,
J = 7.30 Hz, 2H), 7.64 (d, J = 6.95 Hz, 2H), 7.70 (d, J = 8.60 Hz, 2H).
ESI-MS: 537.42 (C26H20BrFN3O2S, [M+H]+). Anal. Calcd for
2.33 (s, 3H), 3.32 (dd, J1 = 7.15 Hz, J2 = 16.75 Hz, 1H), 3.93 (dd,
J1 = 5.75 Hz, J2 = 17.25 Hz, 1H), 4.24 (s, 4H), 5.81 (s, 1H), 6.67 (s,
1H), 6.74 (d, J = 7.25 Hz, 2H), 6.85 (d, J = 6.75 Hz, 1H), 7.13 (d,
J = 7.05 Hz, 2H), 7.31 (d, J = 7.85 Hz, 2H), 7.53 (d, J = 7.55 Hz, 2H),
7.70 (d, J = 8.25 Hz, 2H). ESI-MS: 533.45 (C27H23BrN3O2S, [M+H]+).
Anal. Calcd for C27H22BrN3O2S: C, 60.90; H, 4.16; N, 7.89. Found:
C, 60.84; H, 4.10; N, 7.95.
C26H19BrFN3O2S: C, 58.22; H, 3.57; N, 7.83. Found: C, 58.38; H,
3.51; N, 7.74.
4.2.4.13. 2-(3-(4-Chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)thiazole
(C13).
3.32(dd, J1 = 8.75 Hz, J2 = 15.95 Hz, 1H), 3.83 (s, 3H), 3.92 (d,
J = 5.20 Hz, 1H), 4.22 (s, 4H), 5.85 (s, 1H), 6.71–6.73 (m, 1H), 6.77
(d, J = 8.15 Hz, 1H), 6.88 (d, J = 8.25 Hz, 2H), 6.99 (d, J = 8.05 Hz,
2H), 7.17 (d, J = 5.85 Hz, 2H), 7.23 (d, J = 6.75 Hz, 2H), 7.68 (d,
J = 9.15 Hz, 2H). ESI-MS: 505.00 (C27H23ClN3O3S, [M+H]+). Anal.
Calcd for C27H22ClN3O3S: C, 64.34; H, 4.40; N, 8.34. Found: C,
64.47; H, 4.46; N, 8.27.
4.2.4.19. 2-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-methoxy-
phenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-methoxyphenyl)
thiazole (C19).
500 MHz): 3.29 (dd, J1 = 7.45 Hz, J2 = 18.75 Hz, 1H), 3.82 (s, 6H),
3.90 (d, J = 15.35 Hz, 1H), 4.24 (s, 4H), 5.91 (d, J = 4.45 Hz, 1H),
6.67 (s, 1H), 6.76 (d, J = 8.05 Hz, 2H), 6.84 (d, J = 7.30 Hz, 1H),
7.01 (d, J = 5.35 Hz, 2H), 7.04 (d, J = 7.15 Hz, 2H), 7.41 (d,
J = 6.75 Hz, 2H), 7.73 (d, J = 7.25 Hz, 2H). ESI-MS: 500.58
(C28H26N3O4S, [M+H]+). Anal. Calcd for C28H25N3O4S: C, 67.32; H,
5.04; N, 8.41. Found: C, 67.20; H, 5.09; N, 8.48.
Yield: 69%. Mp 228–230 °C. 1H NMR (CDCl3, 500 MHz):
Yield: 72%. Mp 220–222 °C. 1H NMR (CDCl3,
4.2.4.14. 4-(4-Bromophenyl)-2-(3-(4-chlorophenyl)-5-(2,3-dihy-
drobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-yl)thia-
zole (C14).
500 MHz): 3.33 (d, J = 15.25 Hz, 1H), 3.90 (d, J = 11.75 Hz, 1H),
4.25 (s, 4H), 5.94 (s, 1H), 6.75 (d, J = 8.25 Hz, 2H), 6.87 (d,
J = 6.75 Hz, 1H), 7.02 (d, J = 8.35 Hz, 1H), 7.39–7.41 (m, 2H), 7.51
(d, J = 7.30 Hz, 2H), 7.67 (d, J = 8.65 Hz, 2H), 7.78–7.81 (m, 2H).
4.2.4.20. 4-(4-Bromophenyl)-2-(5-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole
(C20).
3.31 (d, J = 17.15 Hz, 1H), 3.83 (s, 3H), 3.92 (d, J = 16.35 Hz, 1H),
4.24 (s, 4H), 5.92 (s, 1H), 6.66 (s, 1H), 6.76 (d, J = 7.05 Hz, 1H),
6.86–6.90 (m, 2H), 7.02 (d, J = 7.15 Hz, 2H), 7.32 (d, J = 7.30 Hz,
Yield: 75%. Mp 245–247 °C. 1H NMR (CDCl3,
Yield: 78%. Mp 235–237 °C. 1H NMR (CDCl3, 500 MHz):

454
H.-H. Wang et al. / Bioorg. Med. Chem. 21 (2013) 448–455
2H), 7.58 (d, J = 7.35 Hz, 2H), 7.75 (d, J = 6.65 Hz, 2H). ESI-MS:
549.45 (C27H23BrN3O3S, [M+H]+). Anal. Calcd for C27H22BrN3O3S:
C, 59.13; H, 4.04; N, 7.66. Found: C, 59.28; H, 3.98; N, 7.60.
4.5. Western blotting
After incubation, cells were harvested and washed with PBS,
then lysed in lysis buffer (30 mm Tris, pH 7.5, 150 mm NaCl,
1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1% Nonidet
P-40, 10% glycerol, and phosphatase and protease inhibitors). After
centrifugation at 12,000 g for 5 min, the supernatant was collected
as total protein. The concentration of the protein was determined
by a BCA™ protein assay kit (Pierce, Rockford, IL, USA). The protein
samples were separated by 10% SDS–PAGE and subsequently elec-
trotransferred onto a polyvinylidene difluoride membrane (Milli-
pore, Bedford, MA, USA). The membrane was blocked with 5%
nonfat milk for 2 h at room temperature. The blocked membrane
was probed with the indicated primary antibodies overnight at
4 °C, and then incubated with a horse radish peroxidase (HRP)-
coupled secondary antibody. Cleaved Caspase 3 and HER-2 were
measured as shown in Figure 2.
4.3. Preparation, purification of HER-2 and inhibitory assay
A 1.7 Kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) was cloned into baculoviral
expression vector pBlueBacHis2B. A sequence that encodes (His)6
was located at the 50 upstream to the HER-2 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
l
M ammonium molybdate,
l
l
lg/mL leupeptin,
l
l
followed by 20 min centrifugation. Crude extract supernatant
was passed through an equilibrated Ni-NTA superflow packed col-
umn and washed with 10 mM and then 100 mM imidazole to re-
move nonspecifically bound material. Histidinetagged proteins
were eluted with 250 and 500 mM imidazole and dialyzed against
4.6. Molecular docking study
50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1
aprotinin, leupeptin, and pepstatin for 2 h. The entire purification
procedure was performed at 4 °C or on ice.
The HER-2 kinase assay was set up to assess the level of auto-
phosphorylation based on DELFIA/Time-Resolved Fluorometry.
Compounds ( C4, C5, C6, C8, C15, C16) were dissolved in 100%
DMSO and diluted to the appropriate concentrations with
25 mM HEPES at pH 7.4. The final percentage of DMSO in the buf-
l
g/mL each of
Molecular docking of compounds into the 3D HER-2 complex
structure (PDB code: 1UOM) was carried out using the Discovery
Studio (version 3.1) as implemented through the graphical user
interface CDocker protocal. The three-dimensional structures of
the aforementioned compounds were constructed using Chem 3D
ultra 11.0 software [Chemical Structure Drawing Standard; Cam-
bridge Soft corporation, USA (2009)], then they were energetically
minimized by using MOPAC with 5000 iterations and minimum
RMS gradient of 0.10. The crystal structures of HER-2 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 HER-2 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 removed and C6 was placed
during the molecular docking procedure. Types of interactions of
the docked protein with ligand were analyzed after the end of
molecular docking.
fer solution is less than 0.1%. In each well, 10
was incubated with 10 L (12.5 ng for HER-2) of recombinant en-
zyme (1:80 dilution in 100 mM HEPES) for 10 min at room tem-
lL of compound
l
perature. Then, 10
2 mM MnCl2, 100
l
l
L of 5 mM buffer (containing 20 mM HEPES,
M Na3VO4, and 1 mM DTT) and 20 L of
l
0.1 mM ATP-50 mM MgCl2 was added for 1 h. Positive and nega-
tive controls were included in each plate by incubation of enzyme
with or without ATP-MgCl2. At the end of incubation, liquid was
aspirated, and plates were washed three times with wash buffer.
A 75 lL (400 ng) sample of europium labeled anti-phosphotyro-
sine antibody was added to each well for another 1 h of incuba-
tion. 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 autophospho-
rylation inhibition by the compounds was calculated using the
following equation: 100% ꢁ [(negative control)/(positive con-
trol ꢁ negative control)]. The IC50 was obtained from curves of
percentage inhibition 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 HER-2.
Acknowledgment
This work was supported by NSFC J1103512, PCSIRT IRT1020
and FRFCU 1082020803. Dr. Xiao-Yang Qiu thanks Henan Research
Program of Foundation and Advanced Technology (112300410324,
122300410386).
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4. Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med. Chem.
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