Synthesis, biological evaluation and molecular docking studies of resveratrol derivatives possessing curcumin moiety as potent antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2011.11.017

A series of resveratrol derivatives possessing curcumin moiety were synthesized and evaluated for their antiproliferative activity against three cancer cell lines including murine melanoma B16-F10, human hepatoma HepG2 and human lung carcinoma A549. Among them, compound C5 displayed the most potent in vitro antiproliferative activity against B16-F10 with IC₅₀ value of 0.71 μg/mL. Compound C5 also exhibited good tubulin polymerization inhibitory activity with IC₅₀ value of 1.45 μg/mL. Furthermore, docking simulation was carried out to position C5 into the tubulin-colchicine binding site to determine the probable binding mode.

Full text of DOI:10.1016/j.bmc.2011.11.017

Bioorganic & Medicinal Chemistry 20 (2012) 1113–1121
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular docking studies of
resveratrol derivatives possessing curcumin moiety as potent antitubulin agents
Ban-Feng Ruan a,b, , Xiang Lu b, , Ting-Ting Li a, Jian-Feng Tang b, Yao Wei b, Xiao-Liang Wang b,
b,
Shi-Li Zheng a, Ri-Sheng Yao a, , Hai-Liang Zhu
⇑
⇑
a School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China
b 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 resveratrol derivatives possessing curcumin moiety were synthesized and evaluated for their
antiproliferative activity against three cancer cell lines including murine melanoma B16-F10, human hep-
atoma HepG2 and human lung carcinoma A549. Among them, compound C5 displayed the most potent
Received 18 October 2011
Revised 8 November 2011
Accepted 9 November 2011
Available online 1 December 2011
in vitro antiproliferative activity against B16-F10 with IC50 value of 0.71
l
g/mL. Compound C5 also exhib-
g/mL. Furthermore, docking
ited good tubulin polymerization inhibitory activity with IC50 value of 1.45
l
simulation was carried out to position C5 into the tubulin-colchicine binding site to determine the prob-
able binding mode.
Keywords:
Resveratrol
Curcumin
Ó 2011 Elsevier Ltd. All rights reserved.
Antiproliferative activity
Antitubulin polymerization activity
Molecular docking
1. Introduction
Curcumin (1, Fig. 1), originally isolated from the rhizomes of
Curcuma longa,14 is the active ingredient in the traditional herbal
a
,b-Tubulin heterodimers are components of both microtubules
remedy and dietary Indian spice turmeric. Besides its wide range
of medicinal properties like antibacterial, antifungal, antiviral,
antioxidative, anti-inflammatory and antiproliferative,15 curcumin
is also known for its strong cancer preventive activity against a
wide range of tumor cells and prevention of tumor initiation, pro-
motion, metastasis, and angiogenesis in experimental animal sys-
tems.16–21 Curcumin is currently in phase II clinical trials in
patients with advanced pancreatic cancer.22
One of the predominant targets of curcumin is the microtubule
system. Recently, Gupta et al. had shown that curcumin inhibited
HeLa and MCF-7 cell proliferation, disrupted microtubule assembly
in vitro, reduced GTPase activity, and partially inhibited colchi-
cine’s binding activity. They also reported that colchicine and pod-
ophyllotoxin partly inhibited the binding of curcumin to tubulin,
while vinblastine had no effect on the curcumin–tubulin interac-
tion, strongly suggesting that curcumin interacts with dimeric
tubulin or microtubules.23 More recently, Banerjee et al. from the
same laboratory reported that curcumin suppressed the dynamic
instability of microtubules in MCF-7 cells.24
and cytoskeleton, which are involved in crucial biological process
such as cell mobility, cell to cell interactions and mitosis.1 There-
fore, microtubule system has been recognized as an established
target of many anticancer drugs. A number of naturally occurring
compounds, such as colchicine, paclitaxel,2 epothilone A,3 vinblas-
tine,4 combretastatin A-4 (CA4),5 and podophyllotoxin,6 all exhibit
their anticancer properties by interfering with the dynamics of
tubulin polymerization or depolymerization, resulting in mitotic
arrest.
However, in most cases, the clinical use of antitubulin drugs is
associated with problems of significant toxicity, drug resistance,
bioavailability and other side effects.7 For this reason, scientists
have been actively exploring new potent antitubulin agents. Inter-
estingly, there is compelling evidence from epidemiological and
other experimental studies that highlight the importance of many
dietary agents such as resveratrol,8 genistein,9 napthoquinone,10
plumbagin,11 and curcumin12 reducing the risk of cancer and inhi-
bition of the development and spread of tumors in experimental
animals. The advantage of using such compounds for cancer
treatment is their relatively nontoxic nature and availability in
an ingestive form.13
Unfortunately, the clinical potential of curcumin remains limited
because of its relatively low potency and poor bioavailability.25 At-
tempts have been made by others to chemically modify curcumin
in order to increase its activity against cancer,26–31 see Figure 1. To
continue our study on resveratrol,32 another famous natural product
that is well-known for its cancer chemopreventive activity,8,33 we
report herein the synthesis and bioactivities of a series of resveratrol
⇑
Corresponding authors.
E-mail addresses: rishengyao@163.com (R.-S. Yao), zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.017

1114
B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
N
O
O
O
O
O
O
O
HO
OH
HO
OH
2
1
3
O
N
O
O
O
O
O
O
O
HO
OH
OCH3
OH
4
O
O
O
O
O
O
O
O
O
O
O
N
H
HO
OH
OH
O
5
6
Figure 1. Curcumin and its analogues.
derivatives possessing curcumin moiety as potent antimitotic
agents. Their antiproliferative and inhibition of tubulin polymeriza-
tion activities were evaluated. Molecular modeling studies were
consequently performed to understand the tubulin-inhibitor
interaction.
activity. Compound C1 without any substituent on C-ring showed
moderate antiproliferative activity with the mean IC50 values of
2.66, 4.79 and 5.25 lg/mL when tested against B16-F10, HepG2
and A549, respectively. Then, we evaluated the effect of introduc-
ing different substituent groups on C-ring in C1. Introducing
methyl or methoxy group(s) on the C-ring in C1 to form a class
of compounds greatly increased the antiproliferative activity
2. Results and discussion
2.1. Synthesis
against B16-F10 (e.g., C5, IC50 = 0.71 lg/mL; C8, IC50 = 1.08 lg/
mL), especially for C5 with two methoxy groups (R2 = R4 = OCH3)
whose antiproliferative activity increased about 50-fold compared
to resveratrol. It can be seen that the number and the position of
the methoxy group may play an important role in affecting the
antiproliferative activity. A comparable increase in antiprolifera-
tive activity was also observed on introducing F and methyl groups
The structures of these resveratrol derivatives possessing curcu-
min moiety were listed in Table 1. General synthesis of them was
outlined in Scheme 1. Compound A was prepared as previously
reported.34 Then, C1–C34 were synthesized by two steps of base-
catalyzed Claisen condensation reaction as previously reported
with some modification.35 The first step consisted of compound
A with excess acetone in the presence of NaOH (2 M) to afford
the intermediate B. Then, the target compounds (C1–C34) were
prepared by sodium ethylate-catalyzed Claisen condensation of B
and suitably substituted aromatic aldehydes or pyridinecarbalde-
hydes in good yield, respectively. All the synthetic compounds
were purified by column chromatography or recrystallization and
characterized by 1H NMR, elemental analysis and mass spectrum,
which were in full accordance with their depicted structures.
on C-ring in C26 (R1 = CH3, R4 = F) with IC50 value of 0.75 lg/mL
against B16-F10. However, a comparable decrease was observed
on introducing ortho- halogen groups on the C-ring. When tested
against A549 cell line, the IC50 values of compounds C10–C12 were
8.01, 15.23 and 9.37 lg/mL, respectively. This decrease was also
observed in C22 (R1 = R6 = Cl). These results demonstrated that
hydrophobic and electron-donating methoxy or methyl group
might increase the antiproliferative activity to some great extent;
however, electron-withdrawing halogen groups had slightly pro-
motion function on the antiproliferative activity of the target
compounds.
To investigate whether this series of compounds were exerting
their antiproliferative activity by interacting with microtubule sys-
tem, the selected compounds (C1–C9, C26) were evaluated for
antitubulin activity in a cell-free in vitro assay. The IC50 values of
the inhibition of tubulin polymerization by these selected com-
pounds were also shown in Table 2. Cohchicine (Col) and combre-
tastatin A4 (CA4), two known tubulin polymerization inhibitors,
were used as references. Consistent with its significant antiprolif-
erative activity, C5 was found to exhibit the most potent tubulin
2.2. Biological evaluation
The antiproliferative activity of C1–C34 against B16-F10, HepG2
and A549 cell lines was evaluated by using the MTT assay. The re-
sults were summarized in Table 2. All the compounds exhibited
fairly good antiproliferative activity with the IC50 values within
0.71–15.23 lg/mL, which were much better than resveratrol.
Among them, the 3,5-dimethoxy derivatives (C5) displayed the
most potent antiproliferative activity against B16-F10 cell line with
polymerization inhibitory effect with IC50 value of 1.45 lg/mL,
the IC50 value of 0.71
control.
lg/mL, which was comparable to the positive
which was almost the similar to colchicine. This suggests that
the two methoxy groups on C-ring in C5 are playing an important
role in the inhibition of tubulin polymerization.
Subsequently SAR studies were performed to determine how
the substituents on the C-ring affected the antiproliferative

B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
1115
Table 1
Chemical structures of compounds C1–C34
R4
R3
R2
O
O
R5
A
C
Y
X
O
R1
B
O
Compd No.
X
Y
R1
R2
R3
R4
R5
Compd No.
X
Y
R1
R2
R3
R4
R5
C1
C2
C3
C4
C5
C6
C7
C8
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
H
OMe
H
H
H
OMe
H
H
H
F
Cl
Br
H
H
H
H
H
OMe
H
OMe
H
Me
H
Me
H
H
H
F
Cl
Br
H
H
H
H
H
OMe
H
H
H
Me
Me
H
H
H
H
H
H
F
Cl
H
H
H
H
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
H
Cl
H
H
Cl
H
H
H
Br
Cl
Cl
H
H
F
F
H
H
H
H
H
H
NO3
(CH3)2
H
H
H
H
Cl
H
H
H
H
F
H
CF3
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
Cl
F
F
H
CF3
H
H
NO3
H
H
H
n
H
Me
Me
H
Cl
NO3
H
C9
C10
C11
C12
C13
C14
C15
C16
C17
H
H
H
H
n
H
H
R4
O
O
R5
R3
R2
O
O
O
O
Y
CHO
X
R1
i
ii
O
O
O
O
O
A
B
C1-34
Scheme 1. Synthesis of compounds C1–C34. Reagents and conditions: (i) NaOH (2 M), acetone, room temperature; (ii) NaOEt, EtOH, substituted benzaldehydes or
pyridinecarbaldehydes, room temperature.
Moreover, cell cycle analysis of C5 was performed using flow
cytometry, see Figure 2. It can be seen that C5 induced G2/M arrest
in B16-F10 cells to some extent. About 52% of the cells were arrested
C-ring so that they selectively inhibit formation of the a,b-dimer.
In the binding mode, C5 is nicely bound to the tubulin-colchicine
binding site via hydrogen bonds and hydrophobic interactions.
The oxygen atom of the monocarbonyl curcumin system formed
one hydrogen bond with the amino hydrogen of GLN11 (bond
length: GLN11N–Hꢀ ꢀ ꢀO = 2.193 Å; bond angle: GLN11N–Hꢀ ꢀ ꢀO =
140.1°). The oxygen atom of one of the methoxyl groups on C-ring
in C5 formed another hydrogen bond with the amino hydrogen of
ASN101 (bond length: ASN101N–Hꢀ ꢀ ꢀO = 1.888 Å; bond angle:
ASN101N–Hꢀ ꢀ ꢀO = 151.3°). The hydrophobic interactions may play
an auxiliary role in stabilizing the interaction between C5 and the
tubulin-colchicine binding site.
The 3D model of the interaction between C5 and the tubulin-
colchicine binding site was depicted in Figure 4, showing well
binding affinity to the target. We can clearly see that the hydro-
phobic pockets of the tubulin-colchicine binding site are occupied
by a methoxy group on C-ring and the monocarboxyl group of the
curcumin moiety. The two methoxyl groups on C-ring may play a
in G2/M phase in the presence of 1.0 lg/mL C5. At the same concen-
tration, 85% of the cells were arrested in G2/M phase induced by col-
chicine. These findings indicated a continuing impairment of cell
division and confirmed C5 was a potent antitubulin agent.
2.3. Binding mode of compound C5 into tubulin-colchicine site
To gain better understanding on the potency of the studied com-
pounds and guide further SAR studies, we proceeded to examine the
interactions of C5 with tubulin-colchicine binding site of b-tubulin
(PDB code: 1SA0). All docking runs were applied the Lamarckian ge-
netic algorithm of Auto-Dock 4.0. The binding mode of C5 and tubu-
lin-colchicine site was depicted in Figure 3. All the amino acid
residues which had interactions with tubulin-colchicine binding
site were exhibited. Our aim was to vary the substitutions on the

1116
B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
Table 2
Antiproliferative activity (IC50) against B16-F10, HepG2 and A549 cell lines of
compounds C1–C34 and antitubulin activity (IC50) of the selected compounds
Compd No.
IC50 SD (lg/mL)
B16-F10a
HepG2a
A549a
Tubulinb
C1
C2
C3
C4
C5
C6
C7
C8
2.66 0.71
2.13 0.25
2.72 0.53
2.40 0.46
0.71 0.11
1.30 0.24
1.67 0.26
1.08 0.13
1.57 0.12
2.51 0.28
5.94 0.62
3.09 0.30
1.67 0.22
2.99 0.45
1.97 0.23
1.81 0.31
3.17 0.42
2.44 0.26
2.11 0.52
2.73 0.47
2.08 0.34
3.07 0.64
1.79 0.28
2.23 0.22
1.80 0.16
0.75 0.12
3.10 0.27
1.85 0.42
3.43 0.67
1.47 0.34
2.35 0.48
2.24 0.37
3.12 0.21
1.48 0.13
2.88 0.36
37.3 3.0
4.79 1.30
5.82 0.53
8.52 2.10
6.99 1.44
1.60 0.12
2.78 0.64
4.58 1.36
2.37 0.42
4.12 1.13
4.85 1.58
12.45 2.43
10.24 4.21
2.65 0.24
6.94 2.11
5.52 0.97
4.35 0.73
8.29 1.62
7.75 0.97
4.67 0.53
9.49 1.29
3.28 0.42
9.31 0.80
9.07 1.82
5.97 1.23
2.74 0.32
1.96 0.24
5.38 0.72
6.79 0.96
6.75 0.81
2.44 0.32
6.95 0.65
4.43 1.17
9.17 2.32
3.50 0.97
8.41 1.78
38.9 6.0
5.25 0.91
6.49 1.22
7.66 1.47
7.65 1.63
2.10 0.53
4.09 0.94
4.08 0.42
2.69 0.44
5.33 0.32
8.01 0.80
15.23 2.52
9.37 1.26
4.47 0.82
8.23 1.84
4.75 1.01
5.83 1.63
11.15 4.65
7.89 2.15
5.05 0.93
6.99 1.68
6.18 1.32
8.93 1.94
4.65 0.85
6.23 1.30
3.98 0.63
2.32 0.21
4.97 0.41
5.96 1.96
11.65 3.43
3.09 0.68
6.69 1.20
6.06 1.12
9.16 2.14
3.48 0.92
6.97 0.76
30.4 4.0
6.28 0.85
5.99 0.62
8.05 1.24
2.89 0.38
1.45 0.22
2.11 0.35
3.08 0.21
2.68 0.42
3.57 0.54
—
—
—
—
—
—
—
—
—
—
—
—
C9
d
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
B
—
—
—
—
1.81 0.43
—
—
—
—
—
—
—
—
—
—
RESc
Colc
CA4c
0.46 0.04
0.38 0.15
0.21 0.02
0.18 0.03
0.11 0.06
0.13 0.02
1.28 0.4
0.69 0.2
a
b
c
Inhibition the growth of tumor cell lines.
Inhibition of tubulin polymerization.
Data from Ref. 32.
Figure 2. Effects of compound C5 on cell cycle progression of B16-F10 cells were
determined by flow cytometry analysis. B16-F10 cells were treated with C5 at the
d
Not measured.
concentration of 1
indicated.
lg/mL for 24 h. The percentage of cells in each cycle phase was
role as anchor to bind with tubulin. The methoxyl group on the B-
ring in the stilbene moiety also exhibited good affinity towards
tubulin via hydrophobic interactions. The results of the molecular
docking study indicated that the resveratrol skeleton and the
monocarbonyl curcumin moiety could act synergistically to inter-
act with the tubulin-colchicine binding site, suggesting that C5 is
a potential inhibitor of tubulin.
3. Conclusions
In summary,
a series of resveratrol derivatives possessing
curcumin moiety were prepared and evaluated for antiproliferative
and tubulin polymerization inhibition activities. (1E,4E)-1-(2,
4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-5-(3,5-dime-
thoxyphenyl) penta-1,4-dien-3-one (C5) showed the most potent
antiproliferative activityagainst B16-F10 cell line with the IC50 value
Figure 3. Binding mode of compound C5 with tubulin-colchicine site. The
molecules are colored by the atoms (carbons, gray; nitrogen, blue; oxygen, red).
The green dotted lines show the hydrogen bonds. Hydrophobic interactions were
shown as red ball.
of 0.71
exhibited the most potent antitubulin activity to the same extent
as colchicine with IC50 value of 1.45 g/mL. Molecular docking anal-
lg/mL. Correlated with its antiproliferative activity, C5 also
l
ysis of the binding conformation of C5 into the tubulin-colchicine
binding site demonstrated that hydrogen bonds and hydrophobic
interactions with the protein residues might lead to the antitubulin
polymerization and antiproliferative activities. The results of this
work might be helpful for discovering novel class of potent antimi-
tomic agents.

B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
1117
4.3.1. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-phenylpenta-1,4-dien-3-one (C1)
Yellow solid (0.346 g, 81.2% yield). Mp 94–95 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H,
J = 2.4 Hz), 6.73 (d, 1H, J = 2.4 Hz), 6.70 (d, 2H, J = 8.8 Hz), 6.94 (d,
1H, J = 16.4), 7.03 (d, 1H, J = 16.0 Hz), 7.10 (d, 1H, J = 16.0 Hz),
7.31 (d, 1H, J = 16.0 Hz), 7.36–7.37 (m, 3H), 7.47–7.52 (m, 4H),
7.65 (d, 1H, J = 16.0 Hz), 8.12 (d, 1H, J = 15.6 Hz). MS (ESI): 427.5
(C28H26O4, [M+H]+). Anal. Calcd for C28H26O4: C, 78.85; H, 6.14.
Found: C, 78.73; H, 6.17.
4.3.2. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(2-methoxyphenyl)penta-1,4-dien-3-one (C2)
Yellow solid (0.368 g, 80.6% yield). Mp 103–105 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.81 (s, 3H), 3.82 (s, 3H), 3.90 (s, 6H),
6.44 (d, 1H, J = 2.0 Hz), 6.73 (d, 1H, J = 2.0 Hz), 6.88–6.97 (m, 5H),
7.10 (d, 1H, J = 7.2 Hz), 7.14 (d, 1H, J = 6.4 Hz), 7.33 (d, 2H,
J = 16.0 Hz), 7.47 (d, 2H, J = 8.4 Hz), 7.54 (dd, 1H, J = 1.2, 7.6 Hz),
8.01 (d, 1H, J = 16.0 Hz), 8.09 (d, 1H, J = 15.6 Hz). MS (ESI): 457.2
(C29H28O5, [M+H]+). Anal. Calcd for C29H28O5: C, 76.30; H, 6.18.
Found: C, 76.13; H, 6.20.
Figure 4. 3D mode of the interaction between compound C5 and the tubulin-
colchicine site. The protein is represented by surface. C5 is depicted by sticks and
balls.
4. Experimental
4.1. General
4.3.3. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(3-methoxyphenyl)penta-1,4-dien-3-one (C3)
All the chemicals used were commercial products employed
without further purification. The 1H NMR spectra were recorded
on a Bruker DRX 400 model spectrometer in CDCl3 solutions at
room temperature with TMS as an internal standard. Chemical
shifts (d) for 1H NMR spectra were reported in parts per million
to residual solvent protons. Melting points were measured on a
Boetius micro melting point apparatus. The ESI-MS spectra were
recorded on a Mariner System 5304 Mass spectrometer. Carbon,
hydrogen and nitrogen assays were carried out with a CHN–O-Ra-
pid instrument and were within 0.4% of the theoretical values.
TLC was run on the silica gel coated aluminum sheets (Silica Gel
60 GF254, E. Merk, Germany) and visualized in UV light (254 nm).
Yellow solid (0.385 g, 84.3% yield). Mp 121–123 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1H NMR (400 MHz, CDCl3) d (ppm):
3.81 (s, 3H), 3.83 (s, 3H), 3.91 (s, 6H), 6.44 (d, 1H, J = 2.1 Hz), 6.72
(d, 1H, J = 2.4 Hz), 6.89–6.94 (m, 4H), 6.97 (d, 1H, J = 2.7 Hz),
7.03–7.12 (m, 3H), 7.25–7.34 (m, 2H), 7.47–7.49 (m, 2H), 7.61 (d,
1H, J = 16.2 Hz), 8.12 (d, 1H, J = 15.9 Hz). MS (ESI): 457.5
(C29H28O5, [M+H]+). Anal. Calcd for C29H28O5: C, 76.30; H, 6.18.
Found: C, 76.49; H, 6.20.
4.3.4. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(4-methoxyphenyl)penta-1,4-dien-3-one (C4)
Yellow solid (0.419 g, 91.7% yield). Mp 150–152 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.84 (s, 3H), 3.90 (s, 6H),
6.44 (d, 1H, J = 2.4 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.87–6.92 (m, 5H),
6.94 (d, 1H, J = 16.6 Hz), 7.07 (d, 1H, J = 16.0 Hz), 7.32 (d, 1H,
J = 16.0 Hz), 7.45–7.49 (m, 4H), 7.62 (d, 1H, J = 15.6 Hz), 8.09 (d,
1H, J = 16.0 Hz).
4.2. Synthesis method for (E)-4-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)but-3-en-2-one (B)
To a solution of (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzal-
dehyde (A) (5.96 g, 20 mmol) in 100 mL of acetone was added 2 mL
of NaOH (2 M), and the reaction mixture was stirred for 12 h at
room temperature. Then, the solvent was concentrated, 100 mL
of water was added, and the yellow solid that formed was collected
by filtration, washed with water and cold hexane, and then dried
under vacuum to afford 4.92 g B. Yield: 72.8%. Mp: 125–127 °C.
1H NMR (CDCl3): 2.35 (s, 3H), 3.83 (s, 3H), 3.87 (s, 3H), 3.88
(s, 3H), 6.41 (s, 1H), 6.69 (s, 1H), 6.72 (d, 1H, J = 12.4 Hz),
6.89–6.93 (dd, 3H, J = 4.0, 12.8 Hz), 7.21 (d, 1H, J = 16.4 Hz), 7.44
(d, 2H, J = 8.8 Hz), 7.87(d, 1H, J = 16.4 Hz). MS (ESI): 339.4
(C21H22O4, [M+H]+). Anal. Calcd for C21H22O4: C, 74.54; H, 6.55.
Found: C, 74.42; H, 6.57.
MS (ESI): 457.3 (C29H28O5, [M+H]+). Anal. Calcd for C29H28O5: C,
76.30; H, 6.18. Found: C, 76.52; H, 6.16.
4.3.5. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(3,5-dimethoxyphenyl)penta-1,4-dien-3-one (C5)
Yellow solid (0.372 g, 76.4% yield). Mp 109–111 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.79 (s, 6H), 3.83 (s, 3H), 3.90 (s, 6H),
6.44 (d, 1H, J = 2.1 Hz), 6.49 (t, 1H, J = 2.1 Hz), 6.66–6.67 (m, 2H),
6.72 (d, 1H, J = 2.1 Hz), 6.90 (m, 2H), 6.93 (d, 1H, J = 10.8), 6.98
(d, 1H, J = 11.1), 7.09 (d, 1H, J = 15.9 Hz), 7.31 (d, 1H, J = 16.2 Hz),
7.46–7.49 (m, 2H), 7.57 (d, 1H, J = 15.9 Hz), 8.11 (d, 1H,
J = 15.9 Hz). MS (ESI): 487.5 (C30H30O6, [M+H]+). Anal. Calcd for
4.3. General procedure: preparation of compounds C1–34
C30H30O6: C, 74.06; H, 6.21. Found: C, 74.26; H, 6.20.
To a solution of B (0.338 g, 1 mmol) and substituted aromatic
aldehydes (1 mmol) in 20 mL of ethanol was added EtONa
(0.136 g, 2 mmol). The reaction mixture was stirred for 12 h at
room temperature. The precipitate was collected by filtration,
washed with water, and recrystallized from methanol or purified
by silica gel chromatography to afford the target products C1–C32.
Compounds C33 and C34 were obtained when the intermediate
B was reacted with 2-pyridinecarbaldehyde or 3-pyridinecarbalde-
hyde by running the same procedure as mentioned above,
respectively.
4.3.6. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(2,5-dimethoxyphenyl)penta-1,4-dien-3-one (C6)
Yellow solid (0.385 g, 79.1% yield). Mp 90–91 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.76 (s, 3H), 3.78 (s, 3H), 3.82 (s, 3H),
3.90 (s, 6H), 6.44 (d, 1H, J = 2.4 Hz), 6.73 (d, 1H, J = 2.4 Hz), 6.81–
6.92 (m, 4H), 6.97–7.04 (m, 1H), 7.10–7.16 (m, 3H), 7.33 (d, 1H,
J = 16.2 Hz), 7.45–7.48 (m, 2H), 7.99 (d, 1H, J = 16.2 Hz), 8.09 (d,
1H, J = 15.9 Hz). MS (ESI): 487.6 (C30H30O6, [M+H]+). Anal. Calcd
for C30H30O6: C, 74.06; H, 6.21. Found: C, 73.94; H, 6.24.

1118
B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
4.3.7. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(m-tolyl)penta-1,4-dien-3-one (C7)
4.3.13. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(3-fluorophenyl)penta-1,4-dien-3-one (C13)
Yellow solid (0.356 g, 80.1% yield). Mp 99–100 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.8 Hz), 6.72 (d, 1H, J = 2.1 Hz), 6.89–7.11 (m, 6H),
7.18–7.22 (m, 1H), 7.28–7.34 (m, 3H), 7.47–7.50 (m, 2H), 7.58 (d,
1H, J = 15.9 Hz), 8.13 (d, 1H, J = 15.9 Hz). MS (ESI): 445.5
(C28H25FO4, [M+H]+). Anal. Calcd for C28H25FO4: C, 75.66; H, 5.67.
Found: C, 75.87; H, 5.68.
Yellow solid (0.326 g, 74.1% yield). Mp 99–101 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.36 (s, 3H), 3.83 (s, 3H), 3.91 (s, 6H),
6.44 (d, 1H, J = 2.8 Hz), 6.72 (d, 1H, J = 2.8 Hz), 6.88–6.98 (m, 4H),
7.06 (d, 1H, J = 15.6 Hz), 7.04–7.23 (m, 2H), 7.28–7.35 (m, 3H),
7.46–7.50 (m, 2H), 7.62 (d, 1H, J = 16.2 Hz), 8.12 (d, 1H,
J = 15.9 Hz). MS (ESI): 441.5 (C29H28O4 [M+H]+). Anal. Calcd for
C29H28O4: C, 79.07; H, 6.41. Found: C, 78.90; H, 6.42.
4.3.14. (1E,4E)-1-(3-Chlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C14)
4.3.8. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(p-tolyl)penta-1,4-dien-3-one (C8)
Yellow solid (0.390 g, 84.5% yield). Mp 98–99 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.91 (s, 6H), 6.44 (d, 1H,
J = 2.4 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.90–7.11 (m, 5H), 7.26–7.39
(m, 4H), 7.47–7.49 (m, 3H), 7.57 (d, 1H, J = 16.0 Hz), 8.13 (d, 1H,
J = 15.6 Hz). MS (ESI): 461.9 (C28H25ClO4, [M+H]+). Anal. Calcd for
Yellow solid (0.332 g, 75.4% yield). Mp 114–115 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.40 (s, 3H), 3.86 (s, 3H), 3.92 (s, 6H),
6.46 (d, 1H, J = 2.0 Hz), 6.74 (d, 1H, J = 2.4 Hz), 6.92–6.95 (m, 2H),
7.01 (d, 2H, J = 15.6 Hz), 7.11 (d, 1H, J = 16.0 Hz), 7.19 (d, 2H,
J = 8.0 Hz), 7.34 (d, 1H, J = 16.4 Hz), 7.43 (d, 2H, J = 8.0 Hz), 7.50
(d, 2H, J = 8.8 Hz), 7.65 (d, 1H, J = 16.0 Hz), 8.13 (d, 1H,
J = 16.0 Hz). MS (ESI): 441.4 (C29H28O4, [M+H]+). Anal. Calcd for
C28H25ClO4: C, 72.96; H, 5.47. Found: C, 72.84; H, 5.48.
4.3.15. (1E,4E)-1-(3-Bromophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C15)
C29H28O4: C, 79.07; H, 6.41. Found: C, 78.27; H, 6.39.
Yellow solid (0.424 g, 83.9% yield). Mp 102–104 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.91 (s, 6H), 6.44 (d, 1H,
J = 2.0 Hz), 6.72 (d, 1H, J = 2.40 Hz), 6.90–7.02 (m, 4H), 7.09 (d,
1H, J = 15.6 Hz), 7.22 (d, 1H, J = 7.6 Hz), 7.30 (d, 1H, J = 16.0 Hz),
7.41–7.49 (m, 4H), 7.55 (d, 1H, J = 16.0 Hz), 7.66 (s, 1H), 8.12 (d,
1H, J = 15.6 Hz). MS (ESI): 506.4 (C28H25BrO4, [M+H]+). Anal. Calcd
for C28H25BrO4: C, 66.54; H, 4.99. Found: C, 66.40; H, 5.00.
4.3.9. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)
-5-(3,4-dimethylphenyl)penta-1,4-dien-3-one (C9)
Yellow solid (0.311 g, 68.5% yield). Mp 119–121 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.26 (s, 3H), 2.28 (s, 3H), 3.83 (s, 3H),
3.90 (s, 6H), 6.44 (d, 1H, J = 2.8 Hz), 6.72 (d, 1H, J = 2.8 Hz), 6.89–
6.95 (m, 3H), 6.98 (d, 1H, J = 8.7 Hz), 7.06–7.14 (m, 2H,), 7.25–
7.27 (m, 2H), 7.32 (d, 1H, J = 15.9 Hz), 7.47–7.50 (m, 2H), 7.61 (d,
1H, J = 15.9 Hz), 8.11 (d, 1H, J = 15.9 Hz). MS (ESI): 455.6
(C30H30O4, [M+H]+). Anal. Calcd for C30H30O4: C, 79.27; H, 6.65.
Found: C, 79.10; H, 6.67.
4.3.16. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(4-fluorophenyl)penta-1,4-dien-3-one (C16)
Yellow solid (0.380 g, 85.6% yield). Mp 106–108 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H,
J = 2.0 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.89–6.96 (m, 4H), 7.03–7.09
(m, 3H), 7.30 (d, 1H, J = 16.4 Hz), 7.46–7.49 (m, 4H), 7.60 (d, 1H,
J = 16.0 Hz), 8.11 (d, 1H, J = 16.0 Hz). MS (ESI): 445.2 (C28H25FO4,
[M+H]+). Anal. Calcd for C28H25FO4: C, 75.66; H, 5.67. Found: C,
75.54; H, 5.68.
4.3.10. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(2-fluorophenyl)penta-1,4-dien-3-one (C10)
Yellow solid (0.325 g, 73.2% yield). Mp 95–96 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.4 Hz), 6.73 (d, 1H, J = 2.4 Hz), 6.90 (d, 2H,
J = 8.7), 6.94 (d, 1H, J = 16.0 Hz), 7.07–7.17 (m, 4H), 7.32 (d, 2H,
J = 16.0 Hz), 7.48 (d, 2H, J = 8.8 Hz), 7.54–7.58 (m, 1H), 7.80 (d,
1H, J = 16.0 Hz), 8.12 (d, 1H, J = 16.0 Hz). MS (ESI): 445.2
(C28H25FO4, [M+H]+). Anal. Calcd for C28H25FO4: C, 75.66; H, 5.67.
Found: C, 75.88; H, 5.65.
4.3.17. (1E,4E)-1-(4-Chlorophenyl)-5-(2,4-dimethoxy-6-((E)-4-
methoxystyryl)phenyl)penta-1,4-dien-3-one (C17)
Yellow solid (0.407 g, 88.3% yield). Mp 137–139 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H,
J = 2.0 Hz), 6.72 (d, 1H, J = 2.4 Hz), 6.89–6.96 (m, 3H), 6.98 (d, 1H,
J = 16.0 Hz), 7.08 (d, 1H, J = 15.6 Hz), 7.28–7.34 (m, 3H), 7.43 (d,
2H, J = 8.4 Hz), 7.48 (d, 2H, J = 8.8 Hz), 7.58 (d, 1H, J = 16.0 Hz),
8.12 (d, 1H, J = 16. 0 Hz). MS (ESI): 461.7 (C28H25ClO4, [M+H]+).
Anal. Calcd for C28H25ClO4: C, 72.96; H, 5.47. Found: C, 72.84; H,
5.48.
4.3.11. (1E,4E)-1-(2-Chlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C11)
Yellow solid (0.352 g, 76.4% yield). Mp 110–112 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.4 Hz), 6.73 (d, 1H, J = 2.4 Hz), 6.89 (d, 2H,
J = 8.8 Hz), 6.94 (d, 1H, J = 16.0 Hz), 6.99 (d, 1H, J = 16.0 Hz), 7.17
(d, 1H, J = 15.6 Hz), 7.27 (d, 1H, J = 2.0 Hz), 7.31 (d, 2H,
J = 16.0 Hz), 7.41 (dd, 1H, J = 1.6, 8.0 Hz), 7.45–7.48 (m, 2H), 7.66
(dd, 1H, J = 2.0, 7.2 Hz), 8.06 (d, 1H, J = 16.0 Hz), 8.12 (d, 1H,
J = 16.0 Hz). MS (ESI): 461.9 (C28H25ClO4, [M+H]+). Anal. Calcd for
4.3.18. (1E,4E)-1-(4-Bromophenyl)-5-(2,4-dimethoxy-6-((E)-4-
methoxystyryl)phenyl)penta-1,4-dien-3-one (C18)
Yellow solid (0.441 g, 87.3% yield). Mp 139–141 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H,
J = 2.0 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.89–6.96 (m, 3H), 6.99 (d, 1H,
J = 16.0 Hz), 7.08 (d, 1H, J = 16.0 Hz), 7.30 (d, 1H, J = 16.0 Hz), 7.36
(d, 2H, J = 8.4 Hz), 7.46–7.50 (m, 4H), 7.56 (d, 1H, J = 16.0 Hz), 8.12
(d, 1H, J = 16.0 Hz). MS (ESI): 505.4 (C28H25BrO4, [M+H]+). Anal. Calcd
for C28H25BrO4: C, 66.54; H, 4.99. Found: C, 66.73; H, 5.00.
C28H25ClO4: C, 72.96; H, 5.47. Found: C, 72.85; H, 5.49.
4.3.12. (1E,4E)-1-(2-Bromophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C12)
Yellow solid (0.395 g, 78.2% yield). Mp 118–120 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.0 Hz), 6.73 (d, 1H, J = 2.0 Hz), 6.89–6.92 (m, 3H),
6.96 (d, 1H, J = 4 Hz), 7.17–7.23 (m, 2H), 7.29–7.33 (m, 2H), 7.46
(d, 2H, J = 8.8 Hz), 7.60 (d, 1H, J = 8.0 Hz), 7.65 (d, 1H, J = 7.6 Hz),
8.01 (d, 1H, J = 16.0 Hz), 8.13 (d, 1H, J = 16.0 Hz). MS (ESI): 506.4
(C28H25BrO4, [M+H]+). Anal. Calcd for C28H25BrO4: C, 66.54; H,
4.99. Found: C, 66.77; H, 4.50.
4.3.19. (1E,4E)-1-(2,4-Dichlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C19)
Yellow solid (0.335 g, 67.6% yield). Mp 117–119 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.92 (s, 3H), 3.93 (s, 3H),
6.44 (d, 1H, J = 2.4 Hz), 6.74 (d, 1H, J = 2.4 Hz), 6.92 (d, 2H,
J = 8.8 Hz), 6.96 (d, 1H, J = 16.0 Hz), 6.99 (d, 1H, J = 16.0 Hz), 7.17

B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
1119
(d, 1H, J = 15.6 Hz), 7.23–7.26 (m, 1H), 7.33 (d, 1H, J = 16.0 Hz), 7.43–
7.48 (m, 3H), 7.61 (d, 1H, J = 8.4 Hz), 8.00 (d, 1H, J = 16 Hz), 8.16 (d,
1H, J = 16.0 Hz). MS (ESI): 496.4 (C28H24Cl2O4, [M+H]+). Anal. Calcd
for C28H24Cl2O4: C, 67.89; H, 4.88. Found: C, 67.78; H, 4.90.
4.3.26. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(5-fluoro-2-methylphenyl)penta-1,4-dien-3-one (C26)
Yellow solid (0.314 g, 68.4% yield). Mp 92–93 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.28 (d, 3H, J = 2.1 Hz), 3.83 (s, 3H),
3.90 (s, 6H), 6.44 (d, 1H, J = 2.1 Hz), 6.72 (d, 1H, J = 2.8 Hz), 6.87–
6.91 (m, 2H), 6.95 (d, 1H, J = 15.6 Hz), 6.98–7.19 (m, 4H), 7.31 (d,
1H, J = 16.2 Hz), 7.38 (d, 1H, J = 7.5 Hz), 7.46 (m, 2H), 7.91 (d, 1H,
J = 15.6 Hz), 8.13 (d, 1H, J = 15.9 Hz). MS (ESI): 459.4 (C29H27FO4,
[M+H]+). Anal. Calcd for C29H27FO4: C, 75.96; H, 5.94. Found: C,
75.75; H, 5.96.
4.3.20. (1E,4E)-1-(3,4-Dichlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C20)
Yellow solid (0.343 g, 69.2% yield). Mp 116–117 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.93 (s, 6H), 6.46 (d, 1H,
J = 2.0 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.92–6.98 (m, 4H), 7.09 (d, 1H,
J = 16.0 Hz), 7.11–7.25 (m, 2H), 7.30–7.34 (m, 2H), 7.50 (d, 2H,
J = 8.8 Hz), 7.55 (d, 1H, J = 16.0 Hz), 8.15 (d, 1H, J = 16.0 Hz). MS
(ESI): 416.18 (C28H24Cl2O4, [M+H]+). Anal. Calcd for C28H24Cl2O4:
C, 67.89; H, 4.88. Found: C, 68.06; H, 4.87.
4.3.27. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl) phenyl)-
5-(3-(trifluoromethyl)phenyl)penta-1,4-dien-3-one (C27)
Yellow solid (0.363 g, 73.5% yield). Mp 147–149 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.85 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H),
6.47 (d, 1H, J = 2.4 Hz), 6.75 (d, 1H, J = 2.4 Hz), 6.93 (d, 2H,
J = 8.8 Hz), 6.97 (d, 1H, J = 16.4 Hz), 7.09 (d, 1H, J = 16.4 Hz), 7.14
(d, 1H, J = 16.0 Hz), 7.34 (d, 1H, J = 16.0 Hz), 7.49–7.57 (m, 3H),
7.63–7.70 (m, 3H), 7.80 (s, 1H), 8.17 (d, 1H, J = 15.6 Hz). MS (ESI):
495.5 (C29H25F3O4, [M+H]+). Anal. Calcd for C29H25F3O4: C, 70.44;
H, 5.10. Found: C, 70.61; H, 5.08.
4.3.21. (1E,4E)-1-(3,5-Dichlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C21)
Yellow solid (0.361 g, 72.8% yield). Mp 161–1630 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.43 (d, 1H, J = 1.8 Hz), 6.71 (d, 1H, J = 2.8 Hz), 6.90–6.96 (m, 3H),
6.99 (d, 1H, J = 15.9 Hz), 7.08 (d, 1H, J = 15.9 Hz), 7.30 (d, 1H,
J = 15.9 Hz), 7.34–7.37 (m, 3H), 7.46–7.51 (m, 3H), 8.13 (d, 1H,
J = 15.9 Hz). MS (ESI): 496.3 (C28H24Cl2O4 [M+H]+). Anal. Calcd for
4.3.28. (1E,4E)-1-(2-Chloro-5-(trifluoromethyl)phenyl)-5-(2,
4-dimethoxy-6-((E)-4-methoxystyryl)phenyl)penta-1,4-dien-
3-one (C28)
C28H24Cl2O4: C, 67.89; H, 4.88. Found: C, 67.74; H, 4.90.
4.3.22. (1E,4E)-1-(2,6-Dichlorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C22)
Yellow solid (0.329 g, 62.2% yield). Mp 120–122 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.91 (s, 3H), 3.92 (s, 3H),
6.44 (d, 1H, J = 2.8 Hz), 6.73 (d, 1H, J = 2.8 Hz), 6.89–6.92 (m, 2H),
6.95 (d, 1H, J = 15.9 Hz), 7.04 (d, 1H, J = 15.9 Hz), 7.17 (d, 1H,
J = 15.9 Hz), 7.32 (d, 1H, J = 15.9 Hz), 7.46–7.50 (m, 2H), 7.53–
7.54 (m, 2H), 7.91 (s, 1H), 8.03 (d, 1H, J = 15.9 Hz), 8.16 (d, 1H,
J = 15.6 Hz). MS (ESI): 529.9 (C29H24ClF3O4, [M+H]+). Anal. Calcd
for C29H24ClF3O4: C, 65.85; H, 4.57. Found: C, 65.75; H, 4.59.
Yellow solid (0.326 g, 65.9% yield). Mp 97–98 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H,
J = 2.4 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.89 (d, 2H, J = 8.4 Hz), 6.93 (d,
1H, J = 16.4 Hz), 7.11 (d, 1H, J = 16.0 Hz), 7.14–7.19 (m, 2H), 7.30
(d, 1H, J = 16.0 Hz), 7.34 (d, 2H, J = 8.0 Hz), 7.46 (d, 2H, J = 8.8 Hz),
7.73 (d, 1H, J = 16.4 Hz), 8.13 (d, 1H, J = 15.6 Hz). MS (ESI): 496.5
(C28H24Cl2O4, [M+H]+). Anal. Calcd for C28H24Cl2O4: C, 67.89; H,
4.88. Found: C,67.80; H, 4.89.
4.3.29. 2-((1E,4E)-5-(2,4-Dimethoxy-6-((E)-4-methoxystyryl )
phenyl)-3-oxopenta-1,4-dien-1-yl)phenyl nitrate (C29)
4.3.23. (1E,4E)-1-(3-chloro-4-fluorophenyl)-5-(2,4-dimethoxy-
6-((E)-4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C23)
Yellow solid (0.360 g, 75.1% yield). Mp 98–100 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.0 Hz), 6.72 (d, 1H, J = 2.0 Hz), 6.90–6.96 (m, 4H),
7.07 (d, 1H, J = 16.0 Hz), 7.09–7.15 (m, 1H), 7.30 (d, 1H,
J = 16.0 Hz), 7.35–7.38 (m, 1H), 7.47–7.56 (m, 4H), 8.12 (d, 1H,
J = 16.0 Hz). MS (ESI): 479.9 (C28H24ClFO4, [M+H]+). Anal. Calcd
for C28H24ClFO4: C, 70.22; H, 5.05. Found: C, 70.08; H, 5.07.
Brown solid (0.426 g, 87.4% yield). Mp 130–132 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.93 (s, 3H),
6.45 (d, 1H, J = 2.4 Hz), 6.73 (d, 1H, J = 2.0 Hz), 6.87–6.91 (m, 3H),
6.93 (d, 1H, J = 16.0 Hz), 7.23 (d, 1H, J = 16.0 Hz), 7.32 (d, 1H,
J = 16.0 Hz), 7.47 (d, 2H, J = 8.8 Hz), 7.51–7.55 (m, 1H), 7.61–7.70
(m, 2H), 8.04 (d, 1H, J = 8.0 Hz), 8.08 (d, 1H, J = 16.0 Hz), 8.13 (d, 1H,
J = 15.6 Hz). MS (ESI): 488.5 (C28H25NO7, [M+H]+). Anal. Calcd for
C28H25NO7: C, 68.98; H, 5.17, N, 2.87. Found: C, 68.90; H, 5.15, N, 2.88.
4.3.30. 3-((1E,4E)-5-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-3-oxopenta-1,4-dien-1-yl)phenyl nitrate (C30)
4.3.24. (1E,4E)-1-(3,4-Difluorophenyl)-5-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)penta-1,4-dien-3-one (C24)
Yellow solid (0.357 g, 77.2% yield). Mp 120–122 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.44 (d, 1H, J = 2.0 Hz), 6.72 (d, 1H, J = 2.4 Hz), 6.90–6.96 (m, 3H),
6.98 (d, 1H, J = 16.0 Hz), 7.08 (d, 1H, J = 15.6 Hz), 7.28–7.33 (m,
2H), 7.42–7.49 (m, 3H), 7.52 (d, 1H, J = 16.0 Hz), 7.58 (d, 1H,
J = 1.6 Hz), 8.13 (d, 1H, J = 16.0 Hz). MS (ESI): 463.5 (C28H24F2O4,
[M+H]+). Anal. Calcd for C28H24F2O4: C, 72.72; H, 5.23. Found: C,
72.56; H, 5.24.
Yellow solid (0.434 g, 89.1% yield). Mp 137–139 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H),
6.44 (d, 1H, J = 2.8 Hz), 6.72 (d, 1H, J = 2.8 Hz), 6.89–6.97 (m, 3H),
7.10 (d, 1H, J = 2.1 Hz), 7.15 (d, 1H, J = 2.1 Hz), 7.31 (d, 1H,
J = 16.2 Hz), 7.46–7.50 (m, 2H), 7.55 (t, 1H, J = 8.1 Hz), 7.66 (d, 1H,
J = 15.9 Hz), 7.79 (d, 1H, J = 7.8 Hz), 8.13–8.23 (m, 2H), 8.38 (s, 1H).
MS (ESI): 488.4 (C28H25NO7, [M+H]+). Anal. Calcd for C28H25NO7: C,
68.98; H, 5.17; N, 2.87. Found: C, 68.88; H, 5.18; N, 2.86.
4.3.31. 4-((1E,4E)-5-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-3-oxopenta-1,4-dien-1-yl)phenyl nitrate (C31)
4.3.25. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(3-fluoro-2-methylphenyl)penta-1,4-dien-3-one (C25)
Yellow solid (0.301 g, 65.6% yield). Mp 101–103 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.34 (s, 3H), 3.83 (s, 3H), 3.90 (s, 6H),
6.44 (d, 1H, J = 2.8 Hz), 6.72 (d, 1H, J = 2.1 Hz), 6.88–6.96 (m, 5H),
7.08 (d, 1H, J = 15.9 Hz), 7.12–7.17 (m, 1H), 7.27–7.34 (m, 2H),
7.45–7.48 (m, 2H), 7.88 (dd, 1H, J = 1.5, 15.6 Hz), 8.13 (d, 1H,
J = 15.9 Hz). MS (ESI): 459.5 (C29H27FO4, [M+H]+). Anal. Calcd for
Brown solid (0.439 g, 90.1% yield). Mp 191–193 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.84 (s, 3H), 3.91 (s, 3H), 3.92 (s, 3H),
6.44 (d, 1H, J = 2.4 Hz), 6.72 (d, 1H, J = 2.4 Hz), 6.90–6.96 (m, 3H),
7.09 (d, 1H, J = 5.2 Hz), 7.13 (d, 1H, J = 5.2 Hz), 7.29 (d, 1H,
J = 16.0 Hz), 7.48 (d, 2H, J = 8.8 Hz), 7.63 (d, 2H, J = 8.4 Hz), 7.66
(s, 1H), 8.16 (d, 1H, J = 16.0 Hz), 8.21 (d, 2H, J = 8.8 Hz). MS (ESI):
488.5 (C28H25NO7, [M+H]+). Anal. Calcd for C28H25NO7: C, 68.98;
H, 5.17; N, 2.87. Found: C, 69.18; H, 5.18; N, 2.86.
C29H27FO4: C, 75.96; H, 5.94. Found: C, 75.78; H, 5.957.

1120
B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
4.3.32. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(4-isopropylphenyl)penta-1,4-dien-3-one (C32)
Yellow solid (0.404 g, 86.2% yield). Mp 74–75 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1.25 (s, 3H), 1.27 (s, 3H), 2.88–2.97
(m, 1H), 3.83 (s, 3H), 3.90 (s, 6H), 6.44 (d, 1H, J = 2.8 Hz), 6.72 (d,
1H, J = 2.8 Hz), 6.89–6.96 (m, 3H), 6.99 (d, 1H, J = 11.7 Hz), 7.08
(d, 1H, J = 15.9 Hz), 7.23 (d, 2H, J = 8.1 Hz), 7.32 (d, 1H,
J = 16.2 Hz), 7.43–7.50 (m, 4H), 7.64 (d, 1H, J = 16.2 Hz), 8.11 (d,
1H, J = 15.9 Hz). MS (ESI): 469.2 (C31H32O4, [M+H]+). Anal. Calcd
for C31H32O4: C, 79.46; H, 6.88. Found: C, 79.66; H, 6.90.
recording spectrophotometer and warmed-up to 30 °C and the
assembly of tubulin was observed turbidimetrically. The IC50 was
defined as the compound concentration that inhibited the extent
of assembly by 50% after 20 min incubation.
4.6. Assessment of cell cycle distribution
The cell cycle distribution of B16-F10 cells was determined by
flow cytometry analysis based on the previously described meth-
od.39 Briefly, the B16-F10 cells were plated at a density of
5 ꢁ 105 cells/mL. After treatments, both the adherent and detached
cells were collected, washed twice with ice-cold PBS buffer (pH
7.4), fixed with 70% alcohol overnight, and then stained with pro-
pidium iodide (1 mg/ml) in the presence of 1% RNAase A for at least
30 min before analysis by flow cytometry (Becton Dickinson,
Franklin Lakes, NJ). Ten thousand cells were counted per sample.
The data were analyzed with CellQuest software (Becton
Dickinson).
4.3.33. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (C33)
Yellow solid (0.223 g, 52.1% yield). Mp 91–92 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H),
6.43 (d, 1H, J = 2.0 Hz), 6.73 (d, 1H, J = 2.4 Hz), 6.90 (d, 2H,
J = 8.4 Hz), 6.94 (d, 1H, J = 16.4 Hz), 7.15 (d, 1H, J = 16.0 Hz), 7.26
(d, 1H, J = 12.0 Hz), 7.32 (d, 1H, J = 16.0 Hz), 7.41 (d, 1H, J = 7.6 Hz),
7.48 (d, 2H, J = 8.8 Hz), 7.55 (d, 1H, J = 15.6 Hz), 7.64–7.73 (m, 2H),
8.16 (d, 1H, J = 16.0 Hz), 8.65 (d, 1H, J = 5.2 Hz). MS (ESI): 428.5
(C27H25NO4, [M+H]+). Anal. Calcd for C27H25NO4: C, 75.86; H, 5.89,
N, 3.28. Found: C, 75.72; H, 5.91; N, 3.29.
4.7. Docking simulations
Molecular docking of compound C5 into the three-dimensional
X-ray structure of tubulin-colchicine site (PDB code: 1SA0) was
carried out using the Auto-Dock software (Version 4.0) as imple-
mented through the graphic user interface Auto-Dock Tool Kit
(ADT 1.4.6).40
The graphical user interface AUTODOCKTOOLS was employed to set-
up the enzymes: all hydrogens were added, Gasteiger charges were
calculated and nonpolar hydrogens were merged to carbon atoms.
The Ni initial parameters are set as r = 1.170 Å, q = +2.0, and van der
Waals well depth of 0.100 kcal/mol.41 For macromolecules, gener-
ated pdbqt files were saved.
The 3D structures of ligand molecules were built, optimized
(PM3) level, and saved in Mol2 format with the aid of the molecu-
lar modeling program SPARTAN (Wavefunction Inc.). These partial
charges of Mol2 files were further modified by using the ADT pack-
age (version 1.4.6) so that the charges of the nonpolar hydrogens
atoms assigned to the atom to which the hydrogen is attached.
The resulting files were saved as pdbqt files.
AUTODOCK 4.0 was employed for all docking calculations. The
AUTODOCKTOOLS program was used to generate the docking input files.
In all docking a grid box size of 60 ꢁ 60 ꢁ 60 points in x, y, and z
directions was built and the coordinate of X, y, and Z is 121.952,
90.314, 5.066. The maps were centered on N1 atom of the Kcx
219 in the catalytic site of the protein. A grid spacing of 0.375 Å
(approximately one fourth of the length of carbon–carbon covalent
bond) and a distances-dependent function of the dielectric con-
stant were used for the calculation of the energetic map. Ten runs
were generated by using Lamarckian genetic algorithm searches.
Default settings were used with an initial population of 50 ran-
domly placed individuals, a maximum number of 2.5 ꢁ 106 energy
evaluations, and a maximum number of 2.7 ꢁ 104 generations. A
mutation rate of 0.02 and a crossover rate of 0.8 were chosen. Re-
sults differing by less than 0.5 Å in positional root-mean-square
deviation (RMSD) were clustered together and the results of the
most favorable free energy of binding were selected as the resul-
tant complex structures.
4.3.34. (1E,4E)-1-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)
phenyl)-5-(pyridin-3-yl)penta-1,4-dien-3-one (C34)
Yellow solid (0.250 g, 58.4% yield). Mp 67–68 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H),
6.43 (d, 1H, J = 2.4 Hz), 6.71 (d, 1H, J = 2.0 Hz), 6.90 (d, 2H,
J = 8.8 Hz), 6.93 (d, 1H, J = 16.4 Hz), 7.06 (d, 1H, J = 5.6 Hz), 7.10
(d, 1H, J = 5.6 Hz), 7.27–7.31 (m, 2H), 7.45–7.48 (m, 2H), 7.62 (d,
1H, J = 16.0 Hz), 7.81–7.83 (m, 1H), 8.14 (d, 1H, J = 16.0 Hz), 8.58
(dd, 1H, J = 1.2, 4.4 Hz), 8.73 (d, 1H, J = 2.0 Hz). MS (ESI): 428.6
(C27H25NO4, [M+H]+). Anal. Calcd for C27H25NO4: C, 75.86; H,
5.89, N, 3.28. Found: C, 76.12; H, 5.88; N, 3.28.
4.4. Antiproliferation assay
The antiproliferative activity of the prepared compounds
against B16-F10, HepG2 and A549 cell lines were evaluated as de-
scribed elsewhere with some modifications.36 Target tumor cell
lines were grown to log phase in RPMI 1640 medium supple-
mented with 10% fetal bovine serum. After diluting to
2 ꢁ 104 cells mLꢂ1 with the complete medium, 100
lL of the ob-
tained cell suspension was added to each well of 96-well culture
plates. The subsequent incubation was permitted at 37 °C, 5%
CO2 atmosphere for 24 h before the cytotoxicity assessments.
Tested samples at pre-set concentrations were added to 6 wells
with colchicine and CA-4 coassayed as positive reference. After
48 h exposure period, 40
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide)) was added to each well. 4 h later, 100 L extraction solution
l
L of PBS containing 2.5 mg mLꢂ1 of
l
(10% SDS–5% isobutyl alcohol–0.01 M HCl) was added. After an
overnight incubation 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 concen-
tration. Each assay was carried out at least three times. The results
were summarized in Table 2.
4.5. Effects on tubulin polymerization
Acknowledgments
Bovine brain tubulin was purified as described previously.37 To
evaluate the effect of the compounds on tubulin assembly
This work was supported by Condition Construction Cost of He-
fei University of Technology (407–037022) and Key Program of
Natural Science Research by Education Department of Anhui Prov-
ince (2011AJZR0919).
in vitro,38 varying concentrations were preincubated with 10
lM
tubulin in glutamate buffer at 30 °C and then cooled to 0 °C. After
addition of GTP, the mixtures were transferred to 0 °C cuvettes in a

B.-F. Ruan et al. / Bioorg. Med. Chem. 20 (2012) 1113–1121
1121
21. Chakraborti, S.; Das, L.; Kapoor, N.; Das, A.; Dwivedi, V.; Poddar, A.;
Chakraborti, G.; Janik, M.; Basu, G.; Panda, D.; Chakrabarti, P.; Surolia, A.;
Bhattacharyya, B. J. Med. Chem. 2011, 54, 6183.
22. Dhillon, N.; Aggarwal, B. B.; Newman, R. A.; Wolff, R. A.; Kunnumakkara, A. B.;
Abbruzzese,J.L.;Ng,C.S.;Badmaev, V.;Kurzrock,R.Clin.CancerRes. 2008,14, 4491.
23. Gupta, K. K.; Bharne, S. S.; Rathinasamy, K.; Naik, N. R.; Panda, D. FEBS J. 2006,
273, 5320.
24. Banerjee, M.; Singh, P.; Panda, D. FEBS J. 2010, 277, 3437.
25. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol. Pharm.
2007, 4, 807.
26. Dimmock, J. R.; Padmanilayam, M. P.; Puthucode, R. N.; Nazarali, A. J.;
Motaganahalli, N. L.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Kraatz, H. B.;
Prisciak, J. S.; Allen, T. M.; Santos, C. L.; Balzarini, J.; De Clercq, E.; Manavathu, E.
K. J. Med. Chem. 2001, 44, 586.
27. Makarov, V. M.; Odinets, I. L.; Lyssenko, K. A.; Rybalkina, Y. E.; Antipin, M. J.;
Timofeeva, T. V. J. Heterocycl. Chem. 2008, 45, 729.
28. Ohtsu, H.; Xiao, Z.; Ishida, J.; Nagai, M.; Wang, H.; Itokawa, H.; Su, C.; Shih, C.;
Chiang, T.; Chang, E. J. Med. Chem. 2002, 45, 5037.
29. Zambre, A.; Kulkarni, V.; Padhye, S.; Sandur, S.; Aggarwal, B. Bioorg. Med. Chem.
2006, 14, 7196.
30. Weber, W.; Hunsaker, L.; Roybal, C.; Bobrovnikova-Marjon, E.; Abcouwer, S.;
Royer, R.; Deck, L.; Vander Jagt, D. Bioorg. Med. Chem. 2006, 14, 2450.
31. Padhye, S.; Chavan, D.; Pandey, S.; Deshpande, J.; Swamy, K. V.; Sarkar, F. H.
Mini-Rev. Med. Chem. 2010, 10, 372.
32. Ruan, B. F.; Lu, X.; Tang, J. F.; Wei, Y.; Wang, X. L.; Zhang, Y. B.; Wang, L. S.; Zhu,
H. L. Bioorg. Med. Chem. 2011, 19, 2688.
33. Signorelli, P.; Ghidoni, R. J. Nutr. Biochem. 2005, 16, 449.
34. Huang, X. F.; Ruan, B. F.; Wang, X. T.; Xu, C.; Ge, H. M.; Zhu, H. L.; Tan, R. X. Eur.
J. Med. Chem. 2007, 42, 263.
35. Cui, M. C.; Ono, M.; Kimura, H.; Liu, B. L.; Saji, H. J. Med. Chem. 2011, 54, 2225.
36. Boumendjel, A.; Boccard, J.; Carrupt, P.-A.; Nicolle, E.; Blanc, M.; Geze, A.;
Choisnard, L.; Wouessidjewe, D.; Matera, E.-L.; Dumontet, C. J. Med. Chem.
2008, 51, 2307.
37. Hamel, E.; Lin, C. M. Biochemistry 1984, 23, 4173.
38. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
39. Guthrie, H. D.; Welch, G. R.; Cooper, B. S.; Zakaria, A. D.; Johnson, L. A. Biol.
Reprod. 1994, 50, 1303.
40. Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 2007, 28, 1145.
41. Musiani, F.; Arnofi, E.; Casadio, R.; Ciurli, S. J. Biol. Inorg. Chem. 2001, 6, 300.
References and notes
1. (a) Hammond, J. W.; Cai, D.; Verhey, K. J. Curr. Opin. Cell Biol. 2008, 20, 71; (b)
Nogales, E.; Wang, H. W. Curr. Opin. Cell Biol. 2006, 18, 179.
2. (a) Cragg, G. M.; Schepartz, S. A.; Suffness, M.; Grever, M. R. J. Nat. Prod. 1993,
56, 1657; (b) Suffness, M.; Wall, M. E. Taxol: Science, Applications; CRC Press,
Inc.: Boca Raton, FL, 1995. pp 3–25.
3. Bode, C. J., Jr.; Gupta, M. L.; Reiff, E. A.; Suprenant, K. A.; Georg, G. I.; Himes, R. H.
Biochemistry 2002, 41, 3870.
4. Pezzuto, J. M. Biochem. Pharmacol. 1997, 53, 121.
5. Duan, J.-X.; Cai, X.-H.; Meng, F.-Y.; Lan, L.; Hart, C.; Matteucci, M. J. Med. Chem.
2007, 50, 1001.
6. Gupta, S.; Das, L.; Datta, A. B.; Poddar, A.; Janik, M. E.; Bhattacharyya, B.
Biochemistry 2006, 45, 6467.
7. Sridhare, M.; Manuel, M.; Sanjay, G.; Dominik, V.-D.; Tito, F.; Mace, R.;
Dimitrios, C. Anticancer Drugs 2004, 15, 553.
8. Jang, M.; Cai, L. N.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W.
W.; Fong, H. H. S.; Farnsworth, N. R. A.; Kinghorn, D.; Mehta, R. G.; Moon, R. C.;
Pezzuto, J. M. Science 1997, 275, 218.
9. Mukherjee, S.; Acharya, B. R.; Bhattacharyya, B.; Chakrabarti, G. Biochemistry
2010, 49, 1702.
10. Thomson, R. H. Naturally Occurring Quinones IV: Recent Advances, 4th ed.;
Blackie: London, 1997.
11. Acharya, B. R.; Bhattacharyya, B.; Chakrabarti, G. Biochemistry 2008, 47, 7838.
12. Agrawal, D. K.; Misbra, P. K. Med. Res. Rev. 2010, 30, 818.
13. Sarkar, F. H.; Li, Y. Cancer Treat. Rev. 2009, 35, 597.
14. Hatcher, H.; Planalp, R.; Cho, J.; Torti, F. M.; Torti, S. V. Cell. Mol. Life Sci. 2008,
65, 1631.
15. Aggarwal, B. B.; Sung, B. Trends Pharmacol. Sci. 2009, 30, 85.
16. Kálai, T.; Kuppusamy, M. L.; Balog, M.; Selvendiran, K.; Rivera, B. K.;
Kuppusamy, P.; Hideg, K. J. Med. Chem. 2011, 54, 5414.
17. Agrawal, D. K.; Misbra, K. Med. Res. Rev. 2010, 30, 818.
18. Simoni, E.; Bergamini, C.; Fato, R.; Tarozzi, A.; Bains, S.; Motterlini, R.; Cavalli,
A.; Bolognesi, M. L.; Minarini, A.; Hrelia, P.; Lenaz, G.; Rosini, M.; Melchiorre, C.
J. Med. Chem. 2010, 53, 7264.
19. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Mol.
Pharmaceutics 2007, 4, 807.
20. Safavy, A.; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R.;
Bonner, J. A. J. Med. Chem. 2007, 50, 6284.