Design, synthesis and molecular docking of α,β-unsaturated cyclohexanone analogous of curcumin as potent EGFR inhibitors with antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2012.11.031

A type of novel α,β-unsaturated cyclohexanone analogous, which designed based on the curcumin core structure, have been discovered as potential EGFR inhibitors. These compounds exhibit potent antiproliferative activity in two human tumor cell lines (Hep G2 and B16-F10). Among them, compounds I ₃ and I₁₂ displayed the most potent EGFR inhibitory activity (IC₅₀ = 0.43 μM and 1.54 μM, respectively). Molecular docking of I₁₂ into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.

Full text of DOI:10.1016/j.bmc.2012.11.031

Bioorganic & Medicinal Chemistry 21 (2013) 388–394
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and molecular docking of
a,b-unsaturated cyclohexanone
analogous of curcumin as potent EGFR inhibitors with antiproliferative activity
Yun-Yun Xu a, , Yi Cao b, , Hailkuo Ma a, Huan-Qiu Li a, , Gui-Zhen Ao a,
⇑
⇑
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR China
b School of Public Health, Soochow University, Suzhou 215123, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A type of novel a,b-unsaturated cyclohexanone analogous, which designed based on the curcumin core
structure, have been discovered as potential EGFR inhibitors. These compounds exhibit potent antiprolif-
erative activity in two human tumor cell lines (Hep G2 and B16-F10). Among them, compounds I3 and I12
displayed the most potent EGFR inhibitory activity (IC50 = 0.43 lM and 1.54 lM, respectively). Molecular
Received 9 October 2012
Revised 21 November 2012
Accepted 22 November 2012
Available online 2 December 2012
docking of I12 into EGFR TK active site was also performed. This inhibitor nicely fitting the active site
might well explain its excellent inhibitory activity.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
a,b-Unsaturated cyclohexanone analogous
Curcumin
EGFR
Antiproliferative
Molecular docking
1. Introduction
358774), Erlotinib (ZD 1839)13 and Lapatinib (GW572016)14 are
reported to be in clinical trial.
Protein kinase plays a fundamental role in the aberrant signal-
ing that is a hallmark of the uncontrolled proliferation of a variety
of human tumors. So far they are considered as the second largest
class of therapeutic targets. Among the growth factor receptor
kinases that have been identified as being important in cancer is
epidermal growth factor receptor (EGFR) kinase. Activation of EGFR
may be because of overexpression, mutations resulting in constitu-
tive activation, or autocrine expression of ligand.1,2 Expression of a
dominant negative Ras mutant in EGFR overexpressing cells also
results in a significant potentiation of EGFR induced apoptosis sug-
gesting that Ras activation is a key survival signal generated by the
EGFR. The role of EGFR has been most thoroughly studied in breast
cancer,3 lung cancer (especially lung adenocarcinomas)4–6 and in
hormone-refractory prostate cancer.7 Therefore, the EGFR kinase
represents an attractive target for the development of novel thera-
pies for the treatment of cancers. Several classes of inhibitors have
been reported with the most potent and selective being the
4-(phenylamino)quinazolines.8–10 Kinetic studies11 show that
these compounds selectively inhibit EGF-stimulated signal
transduction by reversibly binding at the ATP site of EGFR. As we
all know, three 4-(phenylamino)quinazolines, Gefitinb12 (CP
In the search for effective EGFR inhibitors, several natural prod-
uct lead compounds had been identified. Curcumin (diferuloylme-
thane), the primary bioactive compound isolated from the rhizome
of turmeric (Curcuma longa Linn.), is of particular interest.15 Sev-
eral clinical trials of curcumin are currently conducted in patients
with pancreatic cancer, multiple myeloma, rheumatoid arthritis,
cystic fibrosis, inflammatory bowel disease, psoriasis, and other
disorders.16,17 Curcumin has also been shown to downregulate
the activity of EGFR and HER2 and to deplete cells of EGFR pro-
tein.18–20 EGFR is expressed at high levels in colorectal cancer
and prostate cancer. Curcumin inhibits the growth of human colon
cancer-derived Moser cells by suppressing expression of the
cyclinD1 and EGFR genes.21 Curcumin also downregulates EGFR
signaling in prostate cancer cells by suppressing levels of EGFR
protein, inhibiting the intrinsic EGFR tyrosine kinase activity, and
attenuating ligand-induced activation of EGFR.22
Although curcumin is remarkably non-toxic and has promising
anti-cancer activities, preclinical and clinical studies indicated that
its poor bioavailability and pharmacokinetic profiles due to its
instability under physiological conditions had limited its applica-
tion in anti-cancer therapies.23–25 For example, in a phase I trial,
the concentrations of curcumin in plasma and target tissues were
as low as 11.1 nM and 1.3 lM, respectively, even with an oral
administration of 3.6 g/day.17 Thus, our study focused on the
improvement of its both pharmacokinetic profiles and bioavailabil-
ity. It is suggested that the stability and metabolic profiles of
curcumin could be enhanced by deleting the b-diketone moiety.
⇑
Corresponding authors. Tel./fax: +86 512 65882092.
E-mail addresses: huanqiuli@suda.edu.cn (H.-Q. Li), aoguizhen@suda.edu.cn
(G.-Z. Ao).
Equally contributed to the work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.031

Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
389
Table 1
Structure of
curcumin
a, b-unsaturated cyclohexanone analogous and the biological effects
O
O
Compd
R
Antiproliferative EGFR inhibition IC50
assay IC50 M) M)
O
O
(l
(l
Hep
G2
A16-
F10
HO
OH
I1
I2
I3
I4
I5
I6
I7
I8
Ph
3-ClPh
2-ClPh
2-FPh
2-BrPh
3-BrPh
4-BrPh
4-FPh
4.74
6.11
5.67
7.53
2.45
5.17
3.23
5.64
12.42
10.78
12.27
7.63
18.41
12.21
9.72
0.71
>100
3.28
5.84
7.04
32.65
4.71
3.58
7.69
1.6 0.3
2.2 0.5
1.5 0.2
2.1 0.4
8.0 1.6
6.8 1.8
7.1 0.9
5.6 1.8
15.3 3.1
7.5 1.1
6.1 1.2
0.4 0.1
20.8 4.9
4.6 0.5
3.9 0.2
5.3 0.7
18.6 2.2
2.0 0.4
1.9 0.2
4.8 0.6
OH
32.20
16.67
29.16
12.09
25.48
17.17
17.24
1.01
>100
10.47
11.76
11.05
58.07
8.56
HO
O
Ar
OH
I9
4-ClPh
c-C6H11
I10
I11
I12
I13
I14
I15
I16
I17
I18
I19
I20
3-CH3OPh
3,4-diHOPh
2-CH3OPh
4-HOPh
4-(CH3)2NPh
3-HO-4-CH3OPh
4-CH3OPh
3,5-Di CH3OPh
4-CH3SO2Ph
3,5-(CH3)3C-4-
HOPh
O
O
O
O
R
O
O
6.68
11.11
I
Ia
Figure 1. Design of the novel
a,b-unsaturated cyclohexanone analogous.
Curcumin
Erlotinib
26.99
0.12
18.65
0.2
8.6 2.1
0.03
Altering the b-diketone of curcumin with bioactive
a,
b-unsaturated cyclohexanone may increase the stability and anti-
dehydes and Ia were dissolved in 10% NaOH ethanol solution at
room temperature to give the target compounds I1–I20. All com-
pounds were purified by silicagel column and identified by ele-
mental H and C NMR and HRMS.
The hydrolytic stabilities of the curcumin analogues were inves-
tigated in buffered 0.3% CMCNa solution at pH 7.4 and 37 °C in
25
proliferative activity.
Resveratrol, a phytoalexin is present in
medicinal plants, grape skin, peanuts, and red wine, has the same
stilbene structure and bioactivities with curcumin. Thus, a series
of curcumin analogues had been designed through altering its
b-diketones and aromatic ring, their structures contain both
b-unsaturated cyclic ketone and phenolic hydroxyl of resveratrol
(Fig. 1). Herein, we report the synthesis of ,b-unsaturated cyclo-
a,
sodium dihydrogen phosphate buffer. The
a,b-unsaturated cyclo-
a
hexanone analogous lacking the b-diketone moiety were much
more stable in the pH 7.4 situation. As a result, the stability of cur-
cumin could be enhanced through deleting the b-diketone moiety.
hexanone analogous of curcumin, these compounds are potent in
inhibition of cell growth in two cancer cell lines (Hep G2 and
B16-F10) and with high levels of EGFR proteins and effectively in-
duces apoptosis in a dose-dependent manner in the Hep G2 cell
line, molecular docking was built to discuss the structure–activity
relationship.
2.2. Antiproliferative activity in vitro
The synthesizeda, b-unsaturated cyclohexanone analogous of
curcumin were evaluated for their antiproliferative activities
against Hep G2 and B16-F10 cells by applying the MTT colorimetric
assay. The results were summarized in Table 1. As expected, most
2. Results and discussion
2.1. Chemsitry
a,b-unsaturated cyclohexanone analogous exhibited remarked ef-
fects on antiproliferative activities, and generally the results
showed which those applied to B16-F10 cells would performed
better than that in Hep G2 cells. Especially, (2E,6E)-2-(3,5-dihydr-
oxybenzylidene)-6-(3,5-dimethoxybenzylidene)cyclohexanone
Twenty a,b-unsaturated cyclohexanone analogous of curcumin
were designed and synthesized to be screened for the antiprolifer-
ative activity. All of them were synthesized for the first time. As
show in Scheme 1, the intermediates Ia was prepared by the Stork
reaction. Enamine Ia0 was synthesized by cyclohexanone and mor-
pholine in benzene.
(I12) showed themost potent inhibitory activity (IC50 = 1.01
Hep G2 and IC50 = 0.71 M for B16-F10), much better than curcu-
min (IC50 = 26.99 M for Hep G2 and IC50 = 18.65 M for B16-
F10) and comparable to the positive control erlotinib.
lM for
l
l
l
Ia was subsequently obtained by hydrolysis of 3,5-dimethoxy-
benzaldehyde and Ia0.
Preliminary SAR (Structure activity relationship) studies were
performed to deduce how the structure variation and modification
could affect the antiproliferative activity. Firstly, a main trend was
Then the
a,b-unsaturated cyclohexanone analogous were syn-
thesized by Claisen-Schimidt reaction, various substitute benzal-
O
O
O
R
CHO
CH3O
CH3O
O
1. 3,5-OCH3PhCHO,
R
Morpholine
N
reflux,8h
reflux,5h
2.HCl, rt, 2h
NaOH or HCl,
rt, 30min
OCH3
OCH3
I1~I20
Ia
Scheme 1. Synthetic routes of a, b-unsaturated cyclohexanone analogous I1–I20.

390
Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
that compounds with hydroxyl substitution on R-phenyl ring (I12
and I14) displayed more potent antiproliferative activity compared
to other compounds, while compounds with methoxyl substitution
on R-phenyl ring (I11 and I13) did not input substantial effects on
the antiproliferative capability, so the increased lipophilicity of
methoxy derivatives at R1 position may be detrimental to the
inhibitory activity.
also suggested that there is
benzene ring of compound I12 and the side chain of LYS721,
-cation interaction energies are of the same order of magnitude
a p-cation interaction between
p
as hydrogen bonds or salt bridges and play an important role in
stabilizing the three dimensional structure of a protein.26
2.4. Analysis of apoptosis induced by compounds I12
Secondly, as for compounds with halogen substitution on R-
phenyl ring, we could perceive the tendency that Cl P F > Br in
the series. Compounds with R1 substitution at the meta position
(I2, I6, I11 and I16) showed better activities than those with substi-
tution at the para position (I7, I8, I9 and I14) based both on cytotox-
icity and enzyme inhibitory activity, respectively. Taken together,
We evaluated compound I12 for their ability to induce apoptosis
in the Hep G2 cell line using Annexin-V and propium iodide (PI)
double staining by flow cytometry.
The results are shown in Figure 3. As can be seen, compound I12
is effective in induction of apoptosis in a dose-dependent manner.
the designed and synthesized curcumin
a
,b-unsaturated cyclohex-
Treatment of the Hep G2 cells by 1 and 2 lM of I12 for 3 days re-
anone analogous did demonstrate fairly potent antiproliferative
activity against two tested cancer cell lines. Therefore, more struc-
turally diversified derivatives would be synthesized and evaluated
to provide clues on the antagonist–protein interaction.
sults in 32.8% and 39.8% of apoptotic cells (early + late), as com-
pared to 0.98% of apoptotic cells in an untreated control. This is
consistent with its nice binding affinity to EGFR TK and its potent
activity in inhibition of cell growth.
2.3. EGFR inhibitory activity and molecular docking
3. Conclusion
To evaluate the EGFR inhibitory potency of new compounds,
their ability to block EGFR was tested in an EGFR TK assay, their
EGFR inhibitory activities were displayed at Table 1. This result
basically correlates well with that from the cancer cell based assay.
As shown in Table 1, compounds I3 and I12 displayed the most po-
In this study, a series of a,b-unsaturated cyclohexanone analo-
gous of curcumin were synthesized and evaluated for inhibitory
activity against EGFR TK kinases as well as antiproliferative activity
of two cancer cell lines. As expected, these compounds exhibited
remarkable effects on antiproliferative activity and EGFR TK inhib-
itory activity. Especially, compound I12 showed the most potent
tent inhibitory activity (IC50 = 0.43
comparable to the positive control erlotinib (IC50 = 0.03
EGFR). This biological assay indicated that , b-unsaturated cyclo-
lM and 1.54
lM for EGFR), less
l
M for
inhibitory activity (IC50 = 1.01
for B16-F10), much better than that of curcumin (IC50 = 26.99
for Hep G2 and IC50 = 18.65
l
M for Hep G2 and IC50 = 0.71
l
l
M
M
a
hexanone analogous I12 and I3 are potential small-molecule EGFR
inhibitors as anticancer agents.
l
M for B16-F10) and comparable to
the positive control erlotinib. The EGFR molecular docking model
suggested that compound I12 is nicely bound to the region of EGFR.
Thus, compound I12 is potent EGFR TK inhibitor and effectively in-
duces apoptosis in a dose-dependent manner in the Hep G2 cell
line as a potential antiproliferative agent.
In order to explore probable interaction model of inhibitors and
enzyme active site, molecular docking of the most potent inhibitor
I12 into ATP binding site of EGFR kinase was performed on the
binding model based on the EGFR complex structure (1M17.pdb).
All docking runs were applied CDOCKER protocol of Discovery Stu-
dio 3.1. The 2D and 3D binding model of compound I12 and EGFR
was depicted in Figure 2. In the binding model, compound I12 is ni-
cely bound to the region of EGFR with binding interaction energy of
ꢀ51.38 KJ molꢀ1. Visual inspection of the pose of I12 into the active
site revealed that an optimal intermolecular hydrogen bond were
observed (ASP776: N–Hꢁ ꢁ ꢁO: 2.3 Å, angle: 137.6°). The modeling
4. Experimental section
4.1. General chemistry
All chemicals (reagent grade) used were purchased from
Sigma–Aldrich (USA) and Sinopharm Chemical Reagent Co., Ltd.
Figure 2. The 3D model structure of compound I12 binding model with EGFR TK complex. (PDB ID: 1M17) using Discovery Studio program with the essential amino acid
residues at the binding site are tagged in circles. The purple circles show the amino acids which participate in hydrogen bonding, electrostatic or polar interactions and the
green circles show the amino acids which participate in the Van der Waals interactions.

Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
391
Figure 3. Analysis of apoptosis induced by compounds I12 in the Hep G2 liver cancer cell line. Data represent the percentage of apoptotic cells. (a) control, (b) 0. 5
lM, (c)
1.0 lM, (d) 2.0 lM.
(China). 1H NMR spectra were measured on Varian Unity Inova
300/400 MHz NMR Spectrometer at 25 °C and referenced to TMS.
Chemical shifts are reported in ppm (d) using the residual solvent
line as internal standard. Splitting patterns are designed as s, sin-
glet; d, doublet; t, triplet; m, multiplet. HRMS spectra were ac-
quired on Bruker Esquire Liquid Chromatography-Ion Trap Mass
Spectrometer. Analytical thin-layer chromatography (TLC) was
performed on the glass-backed silica gel sheets (silica gel 60 Å
GF254). All compounds were detected using UV light (254 or
365 nm). Analytical HPLC was conducted on SHIMADZU LC-20AD.
Prior to biological evaluation, all compounds were determined to
be >95% pure using appropriate analytical methods (MeOH/H2O
80% v/v, MeOH/H2O 75% v/v, MeOH/H2O 60% v/v) based on the
peak area percentage.
137.123, 136.898, 136.406, 136.196, 130.653, 128.891, 128.657,
108.582, 101.032, 55.673, 28.802, 28.721, 23.229. HR-MS: Calcd
For C21H20O3[M+H]+: 321.1485. Found: 321.1475.
4.2.2. (2E,6E)-2-(3-Chlorobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I2)
Mp: 107.5–108.4 °C. Yield: 84%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.72 (s, 1H, @CH), 7.70 (s, 1H, @CH), 7.43 (s, 1H, ArH),
7.33 (m, 3H, ArH), 6.61 (d, 2H, J = 2.1 Hz, ArH), 6.47 (t, 1H,
J = 2.1 Hz, ArH), 3.82 (s, 6H, OCH3), 2.92 (m, 4H, CH2), 1.81 (quint,
2H, J = 6.5 Hz, CH2). 13C NMR(400 MHz, CDCl3), d (ppm): 190.257,
160.841, 137.985, 137.876, 137.577, 137.541, 136.631, 135.558,
134.564, 130.172, 129.936, 128.795, 108.625, 101.106, 55.707,
28.762, 28.684, 23.117. HR-MS: Calcd For C22H21ClO3 [M+H]+:
369.1252, Found: 369.1257.
4.2. General procedure for the preparation of compounds
4.2.3. (2E,6E)-2-(2-Chlorobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I3)
Various substitute benzaldehydes (1.22 mmol) and Ia (300 mg,
1.22 mmol) were dissolved in 10% NaOH ethanol solution
(10 ml), and stirring at rt for 30 min. Then water (50 ml) was
added, the product extraction was carried out with EtOAc
(3 ꢂ 20 ml). The organic layer was dried over Na2SO4, and after
solvent evaporation the residue was purified by flash column chro-
matography (silica gel 60, 35–70 mesh) using CHCl3 as the eluent.
Mp: 104.0–104.7 °C. Yield: 82%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.88 (s, 1H, @CH), 7.74 (s, 1H, @CH), 7.44 (m, 1H, ArH),
7.33 (m, 1H, ArH), 7.28 (m, 2H, ArH), 6.61 (d, 2H, J = 2.0 Hz, ArH),
6.47 (t, 1H, J = 2.1 Hz, ArH), 3.82 (s, 6H, OCH3), 2.94 (t, 2H,
J = 5.6 Hz, CH2), 2.76 (t, 2H, J = 5.5 Hz, CH2), 1.77 (quint, 2H,
J = 6.2 Hz, CH2). 13C NMR (400 MHz, CDCl3), d (ppm): 190.280,
160.837, 138.169, 137.948, 137.752, 136.739, 135.260, 134.694,
133.913, 130.818, 130.009, 129.827, 126.555, 108.627, 101.090,
4.2.1. (2E,6E)-2-Benzylidene-6-(3,5-dimethoxybenzylidene)-
cyclohexanone (I1)
55.706, 28.983, 28.483, 23.363.HR-MS: Calcd For
C22H21ClO3
Mp: 90.2–91.0 °C. Yield: 87%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.80 (s, 1H, @CH), 7.72 (s, 1H, @CH), 7.47 (m, 2H, ArH),
7.41 (t, 2H, J = 7.2 Hz, ArH), 7.35 (t, 1H, J = 7.1 Hz, ArH), 6.60 (d,
2H, J = 1.8 Hz, ArH), 6.46 (s, 1H, ArH), 3.82 (s, 6H, OCH3), 2.92 (t,
4H, J = 5.7 Hz, CH2), 1.79 (quint, 2H, J = 6.1 Hz, CH2). 13C NMR
(400 MHz, CDCl3), d (ppm): 190.529, 160.834, 138.020, 137.278,
[M+H]+: 369.1252, Found: 369.1251.
4.2.4. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(2-fluorobenzy-
lidene)cyclohexanone (I4)
Mp: 83.0–83.6 °C. Yield: 85%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.82 (s, 1H, @CH), 7.72 (s, 1H, @CH), 7.30–7.39 (m, 2H,

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Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
ArH), 7.16 (t, 1H, J = 7.5 Hz, ArH), 7.11 (t, 1H, J = 9.3 Hz, ArH), 6.60
(d, 2H, J = 2.1 Hz, ArH), 6.47 (t, 1H, J = 2.0 Hz, ArH), 3.82 (s, 6H,
OCH3), 2.94 (t, 2H, J = 5.4 Hz, CH2), 2.80 (t, 2H, J = 5.8 Hz, CH2),
1.78 (quint, 2H, J = 6.5 Hz, CH2). 13C NMR (400 MHz, CDCl3), d
(ppm): 190.173, 160.836, 138.570, 137.967, 137.597, 136.772,
131.009, 130.670, 130.589, 129.727, 124.009, 116.154, 115.937,
108.617, 101.088, 55.715, 28.953, 28.749, 23.246. HR-MS: Calcd.
For C22H21FO3 [M+H]+: 353.1547, Found: 353.1549.
CH2), 1.79 (quint, 2H, J = 6.5 Hz, CH2). 13C NMR (400 MHz, CDCl3),
d (ppm): 190.341, 160.852, 137.923, 137.417, 136.847, 136.702,
135.900, 134.816, 134.636, 131.895, 128.949, 108.613, 101.074,
55.713, 28.752, 23.144. HR-MS: Calcd For C22H21ClO3 [M+H]+:
369.1252, Found: 369.1269.
4.2.10. (2E,6E)-2-(Cyclohexylmethylene)-6-(3,5-dimethoxyben-
zylidene)cyclohexanone (I10)
Mp: 94.0–95.1 °C. Yield: 86%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.64 (s, 1H, @CH), 6.73 (d, 1H, J = 9.8 Hz, ArH), 6.60 (d,
2H, J = 1.9 Hz, ArH), 6.45 (s, 1H, ArH), 3.81 (s, 6H, OCH3), 2.86 (t,
2H, J = 5.6 Hz, CH2), 2.60 (t, 2H, J = 5.5 Hz, CH2), 2.29 (m, 1H, CH),
1.74–1.77 (m, 4H, CH2), 1.67 (m, 2H, CH2), 1.14–1.35 (m, 6H,
CH2). 13C NMR (400 MHz, CDCl3), d (ppm): 190.633, 160.785,
146.497, 138.165, 137.117, 136.557, 134.262, 108.505, 100.872,
55.694, 37.558, 32.046, 28.853, 26.466, 26.180, 25.941, 23.040.
HR-MS: Calcd For C22H28O3 [M+H]+: 341.2111, Found: 341.2111.
4.2.5. (2E,6E)-2-(2-Bromobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I5)
Mp: 111.7–112.5 °C. Yield: 81%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.82 (s, 1H, @CH), 7.74(s, 1H, @CH), 7.63 (d, 1H, J = 7.9 Hz,
ArH), 7.30 (m, 2H, ArH), 7.20 (t, 1H, J = 8.0 Hz, ArH), 6.60 (d, 2H,
J = 1.9 Hz, ArH), 6.47 (t, 1H, J = 1.9 Hz, ArH), 3.82(s, 6H, –OCH3),
2.93 (t, 2H, J = 5.6 Hz, –CH2), 2.74 (t, 2H, J = 5.3 Hz, –CH2), 1.77
(quint, 2H, J = 6.5 Hz, –CH2). 13C NMR (400 MHz, CDCl3), d (ppm):
190.273, 160.844, 137.958, 137.894, 137.796, 136.730, 136.559,
136.196, 133.208, 130.848, 129.961, 127.178, 125.408, 108.638,
101.102, 55.720, 28.996, 28.381, 23.362. HR-MS: Calcd. For
4.2.11. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(3-methoxy-
benzylidene)cyclohexanone (I11
)
C
22H21BrO3 [M+H]+: 413.0747, Found: 413.0765.
Mp: 92.0–92.6 °C. Yield: 83%; 1H NMR d (ppm): 7.76 (s, 1H,
@CH), 7.71 (s, 1H, @CH), 7.32 (t, 1H, J = 7.9 Hz, ArH), 7.06 (d, 1H,
J = 7.7 Hz, ArH), 7.00 (s, 1H, ArH), 6.90 (d, 1H, J = 8.2 Hz, ArH),
6.60 (d, 2H, J = 1.6 Hz, ArH), 6.50 (s, 1H, ArH), 3.84 (s, 3H, CH3),
3.82 (s, 6H, OCH3), 2.90 (t, 4H, J = 5.2 Hz, CH2), 1.80 (quint, 2H,
J = 6.4 Hz, CH2). 13C NMR d (ppm): 190.429, 160.792, 159.652,
137.955, 137.471, 137.107, 136.832, 136.619, 129.600, 123.085,
116.005, 114.455, 108.548, 100.994, 55.630, 55.507, 28.755,
28.728, 23.156. HR-MS: Calcd For C23H24O4 [M+H]+: 365.1747,
Found: 365.1747.
4.2.6. (2E,6E)-2-(3-Bromobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I6)
Mp: 111.9–112.7 °C. Yield: 87%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.72 (s, 1H, @CH), 7.69 (s, 1H, @CH), 7.59 (s, 1H, ArH),
7.46 (d, 1H, J = 7.7 Hz, ArH), 7.36 (d, 1H, J = 7.6 Hz, ArH), 7.28 (t,
1H, J = 7.9 Hz, ArH), 6.60 (s, 2H, ArH), 6.47 (s, 1H, ArH), 3.82 (s,
6H, OCH3), 2.91 (m, 4H, CH2), 1.79 (quint, 2H, J = 6.3 Hz, CH2). 13C
NMR (400 MHz, CDCl3), d (ppm): 190.151, 160.825, 138.269,
137.846, 137.558, 136.593, 135.402, 133.047, 131.667, 130.167,
129.157, 122.721, 108.617, 101.105, 55.676, 28.728, 28.622,
23.097. HR-MS: Calcd. For C22H21BrO3 [M+H]+: 413.0747, Found:
413.0747.
4.2.12. (2E,6E)-2-(3,4-Dihydroxybenzylidene)-6-(3,5-dimeth-
oxybenzylidene)cyclohexanone (I12
)
Mp: 157.6–158.7 °C. Yield: 91%; 1H NMR d (ppm): 7.79 (s, 1H,
@CH), 7.71 (s, 1H, @CH), 7.19 (s, 1H, ArH), 7.04 (d, 1H, J = 8.3 Hz,
ArH), 6.93 (d, 1H, J = 8.2 Hz, ArH), 6.60 (s, 2H, ArH), 6.47 (d, 1H,
J = 1.6 Hz, ArH), 3.82 (d, 6H, J = 1.6 Hz, OCH3), 2.90 (m, 4H, CH2),
1.79 (quint, 2H, J = 5.7 Hz, CH2). 13C NMR d (ppm): 189.808,
160.872, 147.521, 145.617, 137.848, 137.753, 137.630, 135.768,
133.608, 127.474, 124.263, 118.335, 116.360, 108.601, 101.240,
4.2.7. (2E,6E)-2-(4-Bromobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I7)
Mp: 126.4–128.6 °C. Yield: 83%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.71 (s, 1H, @CH), 7.70 (s, 1H, @CH), 7.53 (d, 2H,
J = 8.3 Hz, ArH), 7.32 (d, 2H, J = 8.3 Hz, ArH), 6.60 (d, 2H,
J = 1.8 Hz, ArH), 6.47 (s, 1H, ArH), 3.82 (s, 6H, OCH3), 2.93 (t,
2H, J = 5.4 Hz, CH2), 2.88 (t, 2H, J = 5.4 Hz, CH2), 1.79 (quint, 2H,
J = 6.4 Hz, CH2). 13C NMR (400 MHz, CDCl3), d (ppm): 190.323,
160.845, 137.912, 137.454, 136.967, 136.685, 135.922, 135.069,
132.115, 131.905, 123.159, 108.617, 101.084, 55.718, 28.755,
23.137. HR-MS: Calcd For C22H21BrO3 [M+H]+: 413.0747, Found:
413.0761.
55.840, 28.557, 28.367, 22.934. HR-MS: Calcd For
C22H22O5
[M+H]+: 367.1540, Found: 367.1541.
4.2.13. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(2-methoxy-
benzylidene)cyclohexanone (I13
)
Mp: 90.8–91.2 °C. Yield: 85%; 1H NMR d (ppm): 7.99 (s, 1H,
@CH), 7.71 (s, 1H, @CH), 7.33 (m, 2H, ArH), 6.97 (t, 1H, J = 7.4 Hz,
ArH), 6.92 (d, 1H, J = 8.2 Hz, ArH), 6.60 (d, 2H, J = 2.0 Hz, ArH),
6.46 (t, 1H, J = 2.0 Hz, ArH), 3.87 (s, 3H, OCH3), 3.82 (s, 6H, OCH3),
2.92 (t, 2H, J = 5.7 Hz, CH2), 2.84 (t, 2H, J = 5.5 Hz, CH2), 1.77 (quint,
2H, J = 6.2 Hz, CH2). 13C NMR d (ppm): 190.502, 160.769, 158.596,
138.117, 137.105, 136.911, 136.417, 132.909, 130.533, 130.388,
125.110, 120.152, 110.817, 108.510, 100.880, 55.686, 55.611,
4.2.8. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-fluorobenzy-
lidene)cyclohexanone (I8)
Mp: 107.2–108.3 °C. Yield: 77%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.73 (s, 1H, @CH), 7.70 (s, 1H, @CH), 7.44 (d, 2H,
J = 8.6 Hz, ArH), 7.08 (d, 2H, J = 8.5 Hz, ArH), 6.59 (d, 2H,
J = 1.7 Hz, ArH), 6.45 (s, 1H, ArH), 3.80 (s, 6H, OCH3), 2.86–2.93
(m, 4H, CH2), 1.78 (quint, 2H, J = 6.1 Hz, CH2). 13C NMR (400 MHz,
CDCl3), d (ppm): 190.421, 160.876, 137.996, 137.260, 136.801,
136.156, 132.622, 132.540, 132.375, 115.923, 115.709, 108.616,
101.072, 55.708, 28.765, 28.702, 23.196. HR-MS: Calcd For
28.956, 28.735, 23.421. HR-MS: Calcd For
365.1747, Found: 365.1750.
C
23H24O4 [M+H]+:
4.2.14. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-
hydroxybenzylidene)cyclohexanone (I14
22H21FO3 [M+H]+: 353.1547, Found: 353.1575.
)
C
Mp: 155.0–155.6 °C. Yield: 84%; 1H NMR d (ppm): 7.76 (s, 1H,
@CH), 7.72 (s, 1H, @CH), 7.39 (d, 2H, J = 8.3 Hz, ArH), 6.90 (d, 2H,
J = 8.4 Hz, ArH), 6.59 (s, 2H, ArH), 6.46 (s, 1H, ArH), 6.27 (s, 1H,
OH), 3.81 (s, 6H, OCH3), 2.90 (m, 4H, CH2), 1.79 (m, 2H, CH2). 13C
NMR d (ppm): 191.373, 160.838, 157.381, 138.314, 138.045,
137.259, 137.102, 134.065, 133.039, 128.507, 115.982, 108.648,
4.2.9. (2E,6E)-2-(4-Chlorobenzylidene)-6-(3,5-dimethoxybenzy-
lidene)cyclohexanone (I9)
Mp: 124.4–124.8 °C. Yield: 84%; 1H NMR (400 MHz, CDCl3), d
(ppm): 7.72 (s, 1H, @CH), 7.71 (s, 1H, @CH), 7.38 (m, 4H, ArH),
6.60 (d, 2H, J = 2.1 Hz, ArH), 6.47 (t, 1H, J = 2.1 Hz, ArH), 3.82 (s,
6H, OCH3), 2.93 (t, 2H, J = 5.6 Hz, CH2), 2.87 (t, 2H, J = 5.5 Hz,

Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
393
101.149, 55.748, 28.860, 28.718, 23.219. HR-MS: Calcd For
22H22O4 [M+H]+: 351.1591, Found: 351.1590.
(s, 1H, ArH), 5.48 (s, 1H, OH), 3.82 (s, 6H, OCH3), 2.903 (m, 4H,
CH2), 1.80 (quint, 2H, J = 5.8 Hz, CH2), 1.46 (s, 18H, CH3). 13C NMR
d (ppm): 190.493, 160.829, 155.078, 139.015, 138.272, 137.232,
136.481, 136.151, 133.471, 128.514, 127.638, 108.539, 100.878,
55.704, 34.704, 30.539, 28.925, 28.785, 23.435. HR-MS: Calcd For
C
4.2.15. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(dimethyl-
amino)benzylidene)cyclohexanone (I15
)
Mp: 109.6–110.3 °C. Yield: 80%; 1H NMR d (ppm): 7.78 (s, 1H,
@CH), 7.70 (s, 1H, @CH), 7.46 (d, 2H, J = 8.8 Hz, ArH), 6.72 (d, 2H,
J = 8.8 Hz, ArH), 6.60 (d, 2H, J = 2.0 Hz, ArH), 6.44 (s, 1H, ArH),
3.81 (s, 6H, OCH3), 3.03 (s, 6H, NCH3), 2.95 (t, 2H, J = 6.3 Hz, CH2),
2.90 (t, 2H, J = 5.6 Hz), 1.80 (quint, 2H, J = 6.2 Hz, CH2). 13C NMR d
(ppm): 190.267, 160.794, 150.851, 138.751, 138.411, 137.439,
135.885, 133.004, 131.900, 124.155, 111.895, 108.468, 100.776,
55.675, 40.389, 29.099, 28.733, 23.333. HR-MS: Calcd For
C
30H38O4 [M+H]+: 463.2843, Found: 463.2829.
4.3. Cell proliferation assay
The antiproliferative activities of chalcone thiosemicarbazide
derivatives were determined using a standard (MTT)-based colori-
metric assay (Sigma). Briefly, cell lines were seeded at a density of
7 ꢂ 103 cells/well in 96-well microtiter plates (Costar). After 24 h,
exponentially growing cells were exposed to the indicated com-
pounds at final concentrations ranging from 0.1 to 40 mg/mL. After
48 h, cell survival was determined by the addition of an MTT solu-
C
24H27NO3 [M+H]+: 378.2064, Found: 378.2060.
4.2.16. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(3-hydroxy-4-
methoxybenzylidene)cyclohexanone (I16
)
tion (20 lL of 5 mg/mL MTT in PBS). After 6 h, 100 mL of 10% SDS in
Mp: 131.2–131.6 °C. Yield: 79%; 1H NMR d (ppm): 7.72 (s, 1H,
@CH), 7.69 (s, 1H, @CH), 7.01 (d, 1H, J = 8.2 Hz, ArH), 6.97 (s, 1H,
ArH), 6.93 (d, 1H, J = 8.2 Hz, ArH), 6.58 (d, 2H, J = 1.5 Hz, ArH),
6.43 (s, 1H, ArH), 5.86 (s, 1H, OH), 3.90 (s, 3H, OCH3), 3.79 (s, 6H,
OCH3), 2.87 (m, 4H, CH2), 1.77 (quint, 2H, J = 6.2 Hz, CH2). 13C
NMR d (ppm): 190.406, 160.764, 146.897, 146.642, 138.046,
137.833, 136.960, 136.735, 134.218, 128.600, 124.835, 114.805,
113.616, 108.524, 100.915, 56.175, 55.635, 28.827, 28.664,
23.182. HR-MS: Calcd For C23H24O5 [M+H]+: 381.1697, Found:
381.1692.
0.01 N HCl was added, and the plates were incubated at 37 °C for a
further 4 h; optical absorbance was measured at 570 nm on an
LX300 Epson Diagnostic microplate reader. Survival ratios are ex-
pressed in percentages with respect to untreated cells. IC50 values
were determined from replicates of 6 wells from at least two inde-
pendent experiments.
4.4. General procedure for preparation, purification of EGFR
inhibitory assay
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, separately.
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
4.12.17. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-methoxy-
benzylidene)cyclohexanone (I17
)
Mp: 97.7–98.1 °C. Yield: 82%; 1H NMR d (ppm): 7.77(s, 1H,
@CH), 7.71 (s, 1H, @CH), 7.46 (d, 2H, J = 8.7 Hz, ArH), 6.94 (d, 2H,
J = 8.7 Hz, ArH), 6.60(d, 2H, J = 1.9 Hz, ArH), 6.46 (s, 1H, ArH),
3.85 (s, 3H, CH3), 3.82 (s, 6H, OCH3), 2.92 (t, 4H, J = 5.9 Hz, CH2),
1.80 (quint, 2H, J = 6.4 Hz, CH2). 13C NMR d (ppm): 190.280,
160.745, 160.225, 138.045, 137.273, 136.970, 136.560, 134.257,
132.562, 128.771, 114.127, 108.460, 100.849, 55.573, 28.774,
28.657, 23.150. HR-MS: Calcd. For C23H24O4 [M+H]+: 365.1747,
Found: 365.1747.
pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10
ammonium molybdate, 100 M sodium vanadate, 10 g/mL apro-
tinin, 10 g/mL leupeptin, 10 g/mL pepstatin, and 16 g/mL ben-
lM
l
l
l
l
l
zamidine 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. His-
tidinetagged proteins were eluted with 250 and 500 mM imidazole
and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glycerol, and
4.2.18. (2E,6E)-2,6-Bis(3,5-dimethoxybenzylidene)-
cyclohexanone (I18
)
Mp: 135.7–136.5 °C. Yield: 78%; 1H NMR d (ppm): 7.71 (s, 2H,
@CH), 6.60 (d, 4H, J = 2.1 Hz, ArH), 6.46 (t, 2H, J = 2.1 Hz, ArH),
3.82 (s, 12H, OCH3), 2.92 (t, 4H, J = 5.5 Hz, CH2), 1.78 (quint, 2H,
J = 6.6 Hz, CH2). 13C NMR d (ppm): 190.525, 160.833, 137.995,
137.243, 136.866, 108.592, 101.041, 55.702, 28.813, 23.181. HR-
MS: Calcd For C24H26O5 [M+H]+: 395.1853, Found: 395.1869.
1 lg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The en-
tire purification procedure was performed at 4 °C or on ice.27
EGFR kinase assay was set up to assess the level of autophos-
phorylation based on DELFIA/Time-Resolved Fluorometry. Com-
pounds were dissolved in 100% DMSO and diluted to the
appropriate concentrations with 25 mM HEPES at pH 7.4. In each
well, 10 íL of compound was incubated with 10
of recombinant enzyme (1:80 dilution in 100 mM HEPES) for
10 min at room temperature. Then, 10
L of 5ꢂ buffer (containing
20 mM HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and
20 L of 0.1 mM ATP-50 mM MgCl2 was added for 1 h. Positive and
negative 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
lL (5 ng for EGFR)
4.2.19. (2E,6E)-2-(3,5-Dimethoxybenzylidene)-6-(4-(methylsul-
fonyl)benzylidene)cyclohexanone (I19
)
l
Mp: 162.0–163.5 °C. Yield: 86%; 1H NMR d (ppm): 7.79 (d, 2H,
J = 8.2 Hz, ArH), 7.76 (s, 1H, @CH), 7.73 (s, 1H, @CH), 7.61 (d, 2H,
J = 8.2 Hz, ArH), 6.61 (s, 2H, ArH), 6.48 (s, 1H, ArH), 3.82 (s, 6H,
OCH3), 3.09 (s, 3H, SO2CH3), 2.95 (t, 2H, J = 5.1 Hz, CH2), 2.89 (t,
2H, J = 5.3 Hz, CH2), 1.80 (quint, 2H, J = 5.6 Hz,CH2). 13C NMR d
(ppm): 189.744, 160.713, 141.522, 139.929, 139.108, 137.843,
137.523, 136.255, 134.311, 130.899, 127.501, 108.540, 101.071,
55.543, 44.536, 28.541, 28.499, 22.860. HR-MS: Calcd For
l
l
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 autophos-
phorylation inhibition by the compounds was calculated using
the following equation: 100%ꢀ[(negative control)/(positive con-
trolꢀnegative control)]. The IC50 was obtained from curves of per-
centage inhibition with eight concentrations of compound. As the
C
23H24O5S [M+H]+: 413.1417, Found: 413.1396.
4.2.20. (2E,6E)-2-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-6-
(3,5-dimethoxybenzylidene)cyclohexanone (I20
)
Mp: 148.2–149.9 °C. Yield: 82%; 1H NMR d (ppm): 7.79 (s, 1H,
@CH), 7.70 (s, 1H, @CH), 7.37 (s, 2H, ArH), 6.60 (s, 2H, ArH), 6.45

394
Y.-Y. Xu et al. / Bioorg. Med. Chem. 21 (2013) 388–394
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ity of the signal detected by the anti-phosphotyrosine antibody is
from EGFR.
4.5. Docking study
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The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2010)], then they were energetically minimized by using
MMFF94 with 5000 iterations and minimum RMS gradient of 0.10.
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were eliminated from the protein and the polar hydrogen was
added to the proteins. Molecular docking of all twenty compounds
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The work was supported by National Natural Science Founda-
tion of China (Grant No.81102316); the Natural Science Foundation
of the Jiangsu Higher Education Institutions of China (Grant
No.11KJD350002) and PAPD (A Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education
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