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

Article abstract of DOI:10.1016/j.bmc.2011.03.001

Twenty-three resveratrol derivatives possessing chalcone moiety were synthesized and characterized, and their biological activities were also evaluated as potential antiproliferation and tubulin polymerization inhibitors. Compound C19 exhibited the most p

Full text of DOI:10.1016/j.bmc.2011.03.001

Bioorganic & Medicinal Chemistry 19 (2011) 2688–2695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies
of resveratrol derivatives possessing chalcone moiety as potential
antitubulin agents
Ban-Feng Ruan a, Xiang Lu a, Jian-Feng Tang a, Yao Wei a, Xiao-Liang Wang a, Yan-Bin Zhang a,
Li-Sheng Wang b, Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-three resveratrol derivatives possessing chalcone moiety were synthesized and characterized,
and their biological activities were also evaluated as potential antiproliferation and tubulin polymeriza-
tion inhibitors. Compound C19 exhibited the most potent activity in vitro, which inhibited the growth of
Received 2 January 2011
Revised 26 February 2011
Accepted 1 March 2011
Available online 5 March 2011
HepG2, B16-F10, and A549 cell lines with IC50 values of 0.2, 0.1, and 1.4
l
g/mL, respectively. Compound
g/mL). Docking sim-
C19 also exhibited significant tubulin polymerization inhibitory activity (IC50 = 2.6
l
ulation was performed to position compound C19 into the tubulin–colchicine binding site to determine
the probable binding mode.
Keywords:
Resveratrol
Chalcone
Ó 2011 Published by Elsevier Ltd.
Antiproliferative activity
Molecular docking
Antitubulin polymerization
1. Introduction
mitotic arrest in eukaryotic cells by interfering with the normal
microtubule polymerization/depolymerization process. Classic
Resveratrol, a naturally occurring phytoalexin (3,5,40-trans-
trihydr-oxystilbene) present in medicinal plants, grape skin, pea-
nuts, and red wine,1 has been reported to have chemopreventive
activity,2 by exerting antiproliferative and proapoptotic effects in
human cancer cells at all stages of carcinogenesis: initiation,
promotion, and progression.3 These effects are mediated through
several biological receptors, including cyclooxygenase (COX),4 lipo-
oxygenase (LOX),5 nuclear factor kappa-light-chain-enhancer of
antimitotic agents, many of which are tubulin-binding agents, such
as taxanes20 and vinca alkaloids,21 interfere with microtubule
dynamics by targeting tubulin and are widely used to treat human
cancers. However, neurotoxicity, difficult synthesis, and develop-
ment of multidrug resistance, mainly through the expression of
P-glycoprotein (Pgp), have limited their clinical use. Therefore, it
is important to develop new tubulin-binding agents or other anti-
mitotic agents with novel modes of action. Among them, Combre-
tastatin A-4 (CA-4, 1) is a structurally very simple natural product
with potent cytotoxic activity.22 The mechanism of action of CA-4
involves reversible, high affinity binding in the colchicine site of
tubulin. As a result of the relatively simple chemical structure
and unique biological profiles, CA-4 has stimulated a lot of research
in the last two decades23 (Fig. 1).
Herein we report the synthesis and bioactivities of a series of
novel resveratrol derivatives possessing chalcone moiety to mi-
mic the reported antimitotic agents. Their antiproliferative and
inhibition of tubulin polymerization activities were evaluated.
Molecular modeling studies were consequently performed to
understand tubulin–inhibitor interaction. The docking results
confirmed that the combination of static and hydrophobic inter-
actions and hydrogen bonding may contribute to the potent bio-
logical activities.
activated B cells (NF-j
B),6 quinone reductase 2 (QR2),7,8 ornithine
decarboxylase (ODC),9 and aromatase.10
Chalcones (1,3-diaryl-2-propen-1-ones) are flavonoids and
isoflavonoids precursors which are abundant in edible plants and
display a wide variety of anticancer,11–13 anti-inflammatory,14,15
antiinvasive,16 antituberculosis,17 and antifungal activities.18 Their
wide-range biological properties are largely attributed to the
a,b-unsaturated ketone moiety. Introduction of various substitu-
ents into the two aryl rings is also a field of interest to screen
pharmacologically active chalcones.19
The microtubule system of eukaryotic cells is an important tar-
get for development of antitumor agents. Antimitotic agents cause
⇑
Corresponding author. Tel.: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.03.001

B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
2689
OH
H3CO
H3CO
H3CO
OCH3
O2
O2
S
S
N
H3CO
H3CO
N
H
N
H
NH
H3CO
OCH3
HO
H3CO
OH
3
2
1
OCH3
OCH3
O2
OCH3
F
S
F
F
N
H
OCH3
O2
F
F
S
N
H3C
N
H
F
5
4
R
O
R
OCH3
OCH3
O2
S
N
R
N
H
N
OCH3
7
6
Figure 1. Lead compound and antimitotic agents.
HO
HO
HO
HO
CHO
H3CO
H3CO
CHO
i
ii
OH
OH
OCH3
Resveratrol
B
A
R1 R2
O
R3
H3CO
iii
R2 R1
OCH3
O
R3
H3CO
R4
C1-C23
Scheme 1. Synthesis of compounds C1–C23. Reagents and conditions: (i) CH3CN, DMF, POCl3; (ii) CH3COCH3, CH3I, reflux, 24 h; (iii) CH3CH2OH, 40% NaOH, 24 h.
reaction of (A) with CH3I in acetone/K2CO3 under refluxing. The
synthesis of the target compounds C1–C23 were prepared by the
Claisen–Schmidt condensation between (B) and a series of ketones
in the presence of 40% KOH in ethanol26 in 82–90% yields. All the
synthetic compounds were characterized by 1H NMR, elemental
analysis and mass spectrum, which were in full accordance with
their depicted structures.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds C1–C23 is outlined in Scheme 1.
The synthesis of these compounds started from resveratrol. The
key intermediate, (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzal-
dehyde (B), was prepared in two steps as previously reported with
some modification.24,25
2.2. Biological activity
First, (E)-2,4-dihydroxy-6-(4-hydroxystyryl)benzaldehyde (A)
was prepared by the Vilsmeier reaction in CH3CN from resveratrol
in a high yield (98%). The desired intermediate (B) was obtained by
All the synthesized compounds C1–C23 were evaluated for their
antiproliferative activities against human HepG2 hepatoma cells,

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B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
Table 1
R1 R2
Inhibition (IC50) of HepG2, B16-F10 and A549 cells proliferation and inhibition of
tubulin polymerization by compounds C1–C23
O
R3
C
H3CO
H3CO
Compound
IC50 SD (lg/mL)
HepG2a
6.8 0.3
11.6
4.9 0.3
8.0 0.25
B16-F10a
A549a
Tubulinb
34
59 10
43 10
A
B
OCH3
C1
C2
C3
C4
C5
C6
C7
C8
3.7 0.2
4.3 0.7
5.8 0.1
6.4 0.2
5.3 0.13
6.7 0.14
3.9 0.4
2
2
6.1 0.6
4.5 0.3
7.5 0.4
8.7 0.36
9.4 0.6
8.4 0.5
7.9 0.45
6.6 0.3
6.4 0.25
8.3 0.6
5.6 0.23
8.1 0.7
5.9 0.6
7.8 0.42
3.6 0.14
5.9 0.25
0.3 0.11
0.1 0.15
3.8 0.12
5.4 0.28
4.6 0.36
3.8 0.2
26
94
55
5
9
4
C1-C23
12.9
6
4
4
6
2
2
8
11.1
16.2
11.6
12.2
13.6
12.9
R1
H
R2
H
R3
H
10.4
12.7
4
6
131 16
C1
C2
C3
C4
C5
58
64
6
3
H
H
H
H
OCH3
H
H
H
OCH3
OCH2CH3
O(CH2)3CH3
C9
5.3 0.2
4.9 0.12
6.5 0.18
3.3 0.19
5.1 0.24
7.5 0.4
4.3 0.24
5.3 0.3
6.1 0.32
4.0 0.1
1.4 0.08
2.8 0.15
8.1 0.4
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
RES
Colchicine
CA-4
80 13
72
41
76
47
8
2
4
1
H
9.5 0.8
C6
C7
H
H
H
H
OCH(CH3)(CH2)2CH3
13.0
10.8
18.7
3
2
7
O(CH2)5CH3
150 15
C8
C9
H
H
H
H
O(CH2)11CH3
4.8 0.15
5.3
29
42
2
6
F
4
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
Cl
Br
Cl
H
H
H
H
CH3
H
Cl
Br
H
H
Cl
CF3
CH3
H
CH3
0.9 0.15
0.2 0.01
7.5 0.7
6.3 0.5
4.1 0.4
2.6 0.6
36
33
44
38
9
4
5
4
10.5
3
10.6
4
7.3 0.2
3.3 0.25
38.9
6
37.3
3
30.4
0.12 0.01
0.09 0.01
4
158 20
1.3 0.4
0.70 0.2
0.23 0.02
00.19 0.04
0.5 0.04
0.4 0.15
CH3
NH2
a
Inhibition of the growth of tumor cell lines.
Inhibition of tubulin polymerization.
b
H
H
N(CH2CH3)2
H
C23
H
H
N
O
melanoma B16-F10 cells, and human non-small cell lung cancer
A549 cells. The results were summarized in Table 1.
Figure 2. Chemical structures of compounds C1–C23.
For all the studied compounds, we can see that the antiprolifer-
ative activities against HepG2, B16-F10, and A549 cells are, in gen-
eral, much better than resveratrol. Among them, the oxyalkyl
derivatives C2–C8 showed almost the same moderate antiprolifer-
ative activities with 2 to 10-fold higher IC50 values than that of res-
veratrol and it also can be observed that the length of the oxyalkyl
chain affected little on the antiproliferative activities of C2–C8; The
alkylated resveratrol analogues C16–C19, especially C19 displayed
ment of cell division and confirmed compound C19 was a potent
antitubulin agent.
To gain better understanding on the potency of the studied com-
pounds and guide further SAR studies, we proceeded to examine
the interaction of compound C19 with tubulin (PDB code: 1SA0).
The molecular docking was performed by simulation of compound
C19 into the colchicine binding site of tubulin. All docking runs
were applied the Lamarckian genetic algorithm of Auto-Dock 4.0.
The binding modes of compound C19 and tubulin were depicted
in Figure 4. All the amino acid residues which had interactions with
tubulin were exhibited. In the binding mode, compound C19 is ni-
cely bound to the colchicine binding site of tubulin via hydrophobic
interactions and binding is stabilized by two hydrogen bonds. The
the most potent inhibitory activity (IC50 = 0.2
lg/mL for HepG2,
1.4 g/mL for B16-F10, and 0.1 g/mL for A549), compared to the
l
l
positive control. However, structure-relationships in this series of
resveratrol derivatives that possessing chalcone moiety demon-
strated that there was just a slight difference between the antipro-
liferative activities against HepG2, B16-F10, and A549 cells of all
the studied compounds except C18 and C19, indicating that substi-
tuted groups on the C-ring (Fig. 2) may not play a key role in the
antiproliferative activities and the existence of resveratrol skeleton
together with the chalcone moiety might be responsible for the
antiproliferative activities.
To examine whether the compounds interact with tubulin and
inhibit tubulin polymerization in vitro, we performed the tubulin
assembly assay in a cell-free immunoassay system. The inhibition
of tubulin polymerization by resveratrol derivatives were shown in
Table 1. Compound C19 showed the most potent inhibitory effect
oxygen atom of the
a,b-unsaturated carbonyl system formed one
hydrogen bond with the amino hydrogen of Ser 178 (bond length:
Ser178 N–Hꢀ ꢀ ꢀO = 1.971 Å; bond angle: Ser178 N–Hꢀ ꢀ ꢀO = 129.5°)
and the oxygen atom of one of the methoxyl groups on A-ring of
C19 formed another one with the amino hydrogen of Val 355 (bond
length: Val355 N–Hꢀ ꢀ ꢀO = 2.044 Å; bond angle: Val355 N–
Hꢀ ꢀ ꢀO = 135.5°). The 3D model of the interaction between C19 and
the colchicine binding site was depicted in Figure 5, showing well
binding affinity to the target. We can clearly see that the hydropho-
bic pockets of colchicine binding site are occupied by a methoxy
and its 50% tubulin polymerization inhibition was 2.6 lg/mL.
group on A ring and the carboxyl group of the a,b-unsaturated ke-
Furthermore, cell cycle analysis of compound C19 was per-
formed using flow cytometry, see Figure 3. It can be seen that com-
pound C19 strongly induced G2/M arrest in B16-F10 cells, and the
effect was observed in a dose-independent manner after treatment
for 24 h increasing amounts of this compound. About 31.5% of the
cells were arrested in the G2/M phase while 72.1% of the cells were
tone moiety. The two methyl groups on the C-ring may play an aux-
iliary role in stabilizing the interaction between C19 and the
colchicine binding site. The results of the molecular docking study
showed that the resveratrol skeleton and the a,b-unsaturated car-
bonyl system of the chalcone could act synergistically to interact
with the colchicine binding site of tubulin, suggesting that com-
pound C19 is a potential inhibitor of tubulin.
found to be in the G2/M phase in the presence of 1 and 5
lg/mL
C19, respectively. These findings indicated a continuing impair-

B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
2691
Figure 3. Effects of compound C19 on cell cycle progression of B16-F10 cells were determined by flow cytometry analysis. B16-F10 cells were treated with different
concentrations of C19 for 24 h. The percentage of cells in each cycle phase was indicated.
Figure 4. Compound C19 (colored by atom: carbons: gray; oxygens: red) is bond into tubulin (entry 1SA0 in the Protein Data Bank). The dotted lines show the hydrogen
bonds.

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B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
[M+H]+). Anal. Calcd for C15H12O4: C, 51.05; H, 3.86. Found: C,
50.91; H, 3.87.
4.3. Synthesis method for (E)-2,4-dimethoxy-6-(4-methoxysty-
ryl)benzaldehyde (B)
To a solution of A (1.28 g, 0.005 mol) and anhydrous K2CO3
(2.07 g, 0.015 mol) in 10 mL of CH3COCH3, CH3I (2.84 g, 0.02 mol)
was added dropwise. The reaction mixture was refluxed for 24 h.
After cooling to room temperature, the reaction mixture was fil-
trated and the filtrate was dried and evaporated. Purification by sil-
ica gel afforded (B) in 84% yield. Mp: 108–109 °C. 1H NMR (DMSO-
d6): 3.78 (s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 6.63 (s, 1H), 6.91 (s, 1H),
6.97 (d, 2H, J = 7.9 Hz), 7.21 (d, 1H, J = 16.2 Hz), 7.50 (d, 2H,
J = 7.9 Hz), 7.95 (d, 1H, J = 16.2 Hz), 10.41 (s, 1H). MS (ESI): 299.3
(C18H18O4, [M+H]+). Anal. Calcd for C18H18O4: C, 72.47; H, 6.08.
Found: C, 72.69; H, 6.07.
Figure 5. The surface model structure of C19–tubulin complex. The dotted lines
show the hydrogen bonds.
3. Conclusions
4.4. General method of synthesis of compounds C1–C23
In our present work, a series of novel antitubulin polymeriza-
tion inhibitors (C1–C23) containing resveratrol skeleton and
chalcone moiety had been synthesized and evaluated. These com-
pounds exhibited potent tubulin polymerization inhibitory activity
and antiproliferative activities against HepG2, B16-F10, and A549
tumor cell lines. Compound C19 showed the most potent activity
which inhibited the growth of HepG2, B16-F10, and A549 cell lines
The chalcones were synthesized by a base catalyzed Claisen–
Schmidt condensation reaction of substituted acetophenones and
(B) (Scheme 1). An EtOH solution of substituted acetophenones
(1.0 equiv) and (B) (1.0 equiv) was added with 50% KOH
(2.5 equiv). The reaction mixture was stirred overnight at room
temperature; the pH was adjusted to 3–4 with aq 2 M HCl solution;
the precipitate was collected by filtration and purified by recrystal-
lization in EtOH.
with IC50 values of 0.2 0.01, 1.4 0.08, and 0.1 0.15
lg/mL and
inhibited the polymerization of tubulin with IC50 of 2.6 0.6
lg/
mL. Molecular docking was performed to study the inhibitor-tubu-
lin protein interactions. Analysis of the binding conformation of
compound C19 in the colchicine binding site demonstrated that
several interactions with the protein residues led to the antitubulin
polymerization and antiproliferative activity.
4.4.1. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
phenylprop-2-en-1-one (C1)
Yellow solid (0.364 g, 91% yield). Mp 118–120 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H),
6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d, J = 8.0, 2H), 6.97 (d, J = 16.0,
1H), 7.37 (d, J = 16.0, 1H), 7.52 (d, J = 7.6, 1H), 7.43–7.51 (dd, 3H),
7.52 (d, J = 7.6, 1H), 7.60 (d, J = 15.6, 1H), 8.00 (d, J = 7.2, 2H), 8.21
(d, J = 15.6, 1H). MS (ESI): 401.5 (C26H26O4, [M+H]+). Anal. Calcd
for C26H26O4: C, 78.98; H, 6.04. Found: C, 78.23; H, 6.03.
4. Experimental
4.1. Chemistry
All the NMR spectra were recorded on a Bruker DRX 400 or DPX
300 model Spectrometer in CDCl3. Chemical shifts (d) for 1H NMR
spectra were reported in parts per million to residual solvent pro-
tons. 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-Rapid 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).
4.4.2. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(3-methoxyphenyl)prop-2-en-1-one (C2)
Yellow solid (0.353 g, 82% yield). Mp 94–95 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.83 (s, 3H), 3.86 (s, 3H), 3.93 (s, 6H),
6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d, J = 8.4, 2H), 6.97 (d, J = 16.0,
1H), 7.10 (dd, 1H, J1 = 7.6, J2 = 16.4), 7.34 (d, J = 16.0, 1H), 7.37 (d,
J = 16.0, 1H), 7.49 (d, J = 8.4, 2H), 7.54 (d, J = 8.4, 1H), 7.56 (s, 1H),
7.59 (d, J = 7.2, 1H), 8.21 (d, J = 16.0, 1H). MS (ESI): 431.2
(C27H26O5, [M+H]+). Anal. Calcd for C27H26O5: C, 75.33; H, 6.09.
Found: C, 75.57; H, 6.08.
4.2. Synthesis method for (E)-2,4-dihydroxy-6-(4-hydroxystyryl)
benzaldehyde (A)
4.4.3. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-methoxyphenyl)prop-2-en-1-one (C3)
Yellow solid (0.400 g, 92% yield). Mp 136–138 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1H NMR (400 MHz, CDCl3) d (ppm):
3.86 (s, 3H), 3.88 (s, 3H), 3.93 (s, 6H), 6.47 (s, 1H), 6.76 (s, 1H),
6.92 (d, J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.37 (d, J = 16.0, 1H),
7.49 (d, J = 8.0, 2H), 7.58 (d, J = 16.0, 1H), 8.01 (d, J = 8.0, 2H), 8.18
(d, J = 16.0, 1H). MS (ESI): 431.5 (C27H26O5, [M+H]+). Anal. Calcd
for C27H26O5: C, 75.33; H, 6.09. Found: C, 75.13; H, 6.11.
To a solution of resveratrol (2.28 g, 0.01 mol) in 50 mL of CH3CN
and DMF (0.73 g, 0.01 mol), POCl3 (2.30 g, 0.015 mol) was added
dropwise while cooling with an ice/water bath for 0.5 h. The reac-
tion mixture was stirred for another 2 h at room temperature.
Then, the solution was added to cold water (300 mL). The yellow
solution was stirred under 50 °C for 3 h, and extracted with EtOAc
(100 mL ꢁ 3). The combined organic layers were washed with
water, dried over anhydrous sodium sulfate, filtered and evapo-
rated. Purification by silica gel afforded (A) as yellow powder in
96% yield. Mp: 210–212 °C. 1H NMR (DMSO-d6): 6.21 (s, 1H),
6.62 (s, 1H), 6.78 (d, 2H, J = 8.4 Hz), 7.02 (d, 1H, J = 16.0 Hz), 7.49
(d, 2H, J = 8.4 Hz), 7.70 (d, 1H, J = 16.2 Hz), 9.71 (s, 1H), 10.27
(s, 1H), 10.76 (s, 1H), 12.12 (s, 1H). MS (ESI): 257.3 (C15H12O4,
4.4.4. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-ethoxyphenyl)prop-2-en-1-one (C4)
Yellow solid (0.360 g, 81% yield). Mp 131–133 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1.46 (t, J = 6.8, 3H), 3.86 (s, 3H), 3.93
(s, 6H), 4.11 (q, J1 = 14.0, J2 = 7.2, 2H), 6.47 (s, 1H), 6.76 (s, 1H),
6.91 (d, J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.38 (d, J = 16.0, 1H),

B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
2693
7.49 (d, J = 8.0, 2H), 7.58 (d, J = 16.0, 1H), 7.99 (d, J = 8.8, 2H), 8.18
(d, J = 16.0, 1H). MS (ESI): 445.2 (C28H28O5, [M+H]+). Anal. Calcd
for C28H28O5: C, 75.65; H, 6.35. Found: C, 76.19; H, 6.37.
MS (ESI): 435.1 (C26H23ClO4, [M+H]+). Anal. Calcd for C26H23ClO4:
C, 71.80; H, 5.33. Found: C, 72.06; H, 5.35.
4.4.11. (E)-1-(4-Bromophenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C11)
4.4.5. (E)-1-(4-Butoxyphenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C5)
Yellow solid (0.441 g, 92% yield). Mp 159–161 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.87 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H),
6.47 (s, 1H), 6.75 (s, 1H), 6.93 (d, J = 8.4, 2H), 6.96 (d, J = 16.0,
1H), 7.34 (d, J = 16.0, 1H), 7.48 (d, J = 8.0, 2H), 7.54 (d, J = 16.0,
1H), 7.58 (d, J = 8.4, 2H), 7.85 (d, J = 8.0, 2H), 8.21 (d, J = 15.6, 1H).
MS (ESI): 479.1 (C26H23BrO4, [M+H]+). Anal. Calcd for C26H23BrO4:
C, 65.14; H, 4.84. Found: C, 65.37; H, 4.86.
Yellow solid (0.369 g, 78% yield). Mp 109–111 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1.01 (t, 3H, J = 7.2), 1.50–1.55 (m, 2H),
1.77–1.84 (m, 2H), 3.86 (s, 3H), 3.93 (s, 6H), 4.04 (t, 2H, J = 6.8),
6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d, J = 8.0, 4H), 6.97 (d, J = 16.0,
1H), 7.37 (d, J = 16.0, 1H), 7.49 (d, J = 8.0, 2H), 7.58 (d, J = 16.0,
1H), 8.01 (d, J = 8.0, 2H), 8.18 (d, J = 16.0, 1H). MS (ESI): 473.2
(C30H32O5, [M+H]+). Anal. Calcd for C30H32O5: C, 76.25; H, 6.83.
Found: C, 76.51; H, 6.86.
4.4.12. (E)-1-(3-Chlorophenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C12)
4.4.6. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-(pentan-2-yloxy)phenyl)prop-2-en-1-one (C6)
Yellow solid (0.331 g, 76% yield). Mp 121–123 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.82 (s, 3H), 3.94 (s, 6H), 6.24 (s, 1H),
6.47 (s, 1H), 6.91 (d, J = 8.0, 2H), 6.92 (d, J = 16.0, 1H), 7.30 (d,
J = 8.0, 1H), 7.32 (d, J = 16.0, 1H), 7.44 (d, J = 8.0, 2H), 7.52 (d,
J = 16.0, 1H), 7.63 (d, J = 8.0, 1H), 7.82 (dd, 1H, dd, 1H, J1 = 7.6,
J2 = 16.4), 8.10 (s, 1H), 8.21 (d, J = 16.0, 1H). MS (ESI): 435.1
(C26H23ClO4, [M+H]+). Anal. Calcd for C26H23ClO4: C, 71.80; H,
5.33. Found: C, 72.04; H, 5.31.
Yellow solid (0.384 g, 79% yield). Mp 76–77 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 0.98 (t, 3H, J = 7.2), 1.04 (d, 3H,
J = 6.8), 1.54–1.65 (m, 4H), 1.86–1.95 (m, 1H), 3.86 (s, 3H), 3.93
(s, 6H), 4.04 (t, 2H, J = 6.8), 6.47 (s, 1H), 6.76 (s, 1H), 6.92
(d, J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.37 (d, J = 16.0, 1H), 7.49 (d,
J = 8.0, 2H), 7.58 (d, J = 16.0, 1H), 8.01 (d, J = 8.0, 2H), 8.18 (d,
J = 16.0, 1H). MS (ESI): 487.2 (C31H34O5, [M+H]+). Anal. Calcd for
C31H34O5: C, 76.52; H, 7.04. Found: C, 76.33; H, 7.07.
4.4.13. (E)-1-(3-Bromophenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C13)
4.4.7. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-(hexyloxy)phenyl)prop-2-en-1-one (C7)
Yellow solid (0.431 g, 90% yield). Mp 125–127 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.93 (s, 3H), 3.94 (s,
3H), 6.47 (s, 1H), 6.76 (s, 1H), 6.93 (d, J = 8.0, 2H), 6.96 (d,
J = 16.0, 1H), 7.32 (d, J = 8.0, 1H), 7.34 (d, J = 16.0, 1H), 7.49 (d,
J = 8.0, 2H), 7.55 (d, J = 16.0, 1H), 7.67 (d, J = 8.0, 1H), 7.86 (dd,
1H, J1 = 7.6, J2 = 16.4), 8.12 (s, 1H), 8.21 (d, J = 16.0, 1H). MS
(ESI): 450.2 (C26H23BrO4, [M+H]+). Anal. Calcd for C26H23BrO4:
C, 65.14; H, 4.84. Found: C, 64.90; H, 4.86.
Yellow solid (0.416 g, 83% yield). Mp 99–101 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 0.93 (t, 3H, J = 7.2), 1.35–1.38 (m, 4H),
1.47–1.50 (m, 2H), 1.78–1.85 (m, 2H), 3.86 (s, 3H), 3.93 (s, 6H),
4.03 (t, 2H, J = 6.8), 6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d, J = 8.0, 4H),
6.97 (d, J = 16.0, 1H), 7.37 (d, J = 16.0, 1H), 7.49 (d, J = 8.0, 2H),
7.58 (d, J = 16.0, 1H), 8.01 (d, J = 8.0, 2H), 8.18 (d, J = 16.0, 1H).
MS (ESI): 501.3 (C32H36O5 [M+H]+). Anal. Calcd for C32H36O5: C,
76.77; H, 7.25. Found: C, 76.59; H, 7.28.
4.4.14. (E)-1-(3,4-Dichlorophenyl)-3-(2,4-dimethoxy-6-((E)-4-
methoxystyryl)phenyl)prop-2-en-1-one (C14)
4.4.8. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-(dodecyloxy)phenyl)prop-2-en-1-one (C8)
Yellow solid (0.404 g, 86% yield). Mp 140–142 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.87 (s, 3H), 3.93 (s, 3H), 3.95 (s, 3H),
6.47 (s, 1H), 6.75 (s, 1H), 6.93 (d, J = 8.4, 2H), 6.96 (d, J = 16.0,
1H), 7.33 (d, J = 15.6, 1H), 7.49 (d, J = 8.4, 3H), 7.53 (d, J = 16.0,
1H), 7.80 (d, J = 8.0, 1H), 8.07 (s, 1H), 8.23 (d, J = 16.0, 1H). MS
(ESI): 470.4 (C26H22Cl2O4, [M+H]+). Anal. Calcd for C26H22Cl2O4: C,
66.53; H, 4.72. Found: C, 66.32; H, 4.70.
Yellow solid (0.415 g, 71% yield). Mp 82–83 °C. 1H NMR
(400 MHz, CDCl3)
d (ppm): 0.90 (t, 3H, J = 7.2), 1.29–1.34
(m, 16H), 1.44–1.51 (m, 2H), 1.78–1.85 (m, 2H), 3.86 (s, 3H), 3.93
(s, 6H), 4.02 (t, 2H, J = 6.8), 6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d,
J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.37 (d, J = 16.0, 1H), 7.49 (d,
J = 8.0, 2H), 7.58 (d, J = 16.0, 1H), 8.01 (d, J = 8.0, 2H), 8.18
(d, J = 16.0, 1H). MS (ESI): 585.4 (C38H48O5, [M+H]+). Anal. Calcd
for C38H48O5: C, 78.05; H, 8.27. Found: C, 78.32; H, 8.25.
4.4.15. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-(trifluoromethyl)phenyl)prop-2-en-1-one (C15)
Yellow solid (0.436 g, 93% yield). Mp 178–180 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.87 (s, 3H), 3.93 (s, 3H), 3.95 (s,
3H), 6.48 (s, 1H), 6.76 (s, 1H), 6.93 (d, J = 8.8, 2H), 6.96 (d,
J = 16.0, 1H), 7.34 (d, J = 16.0, 1H), 7.48 (d, J = 8.4, 2H), 7.59
(d, J = 16.0, 1H), 7.70 (d, J = 8.0, 2H), 8.07 (d, J = 8.0, 2H), 8.23
(d, J = 16.0, 1H). MS (ESI): 469.5 (C27H23F3O4, [M+H]+). Anal.
Calcd for C27H23F3O4: C, 69.22; H, 4.95. Found: C, 69.46; H,
4.93.
4.4.9. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-fluorophenyl)prop-2-en-1-one (C9)
Yellow solid (0.381 g, 91% yield). Mp 138–140 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.87 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H),
6.47 (s, 1H), 6.76 (s, 1H), 6.93 (d, J = 8.0, 2H), 6.97 (d, J = 16.0,
1H), 7.12 (d, J = 8.4, 2H), 7.36 (d, J = 16.0, 1H), 7.49 (d, J = 8.4, 2H),
7.57 (d, J = 16.0, 1H), 8.02 (d, J = 8.4, 2H), 8.21 (d, J = 16.0, 1H).
MS (ESI): 419.2 (C26H23FO4, [M+H]+). Anal. Calcd for C26H23FO4:
C, 74.63; H, 5.54. Found: C, 74.87; H, 5.53.
4.4.16. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(p-tolyl)prop-2-en-1-one (C16)
4.4.10. (E)-1-(4-Chlorophenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C10)
Yellow solid (0.311 g, 75% yield). Mp 112–114 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.43 (s, 3H), 3.86 (s, 3H), 3.93 (s, 6H),
6.47 (s, 1H), 6.76 (s, 1H), 6.92 (d, J = 8.8, 2H), 6.97 (d, J = 16.0,
1H), 7.25 (d, J = 8.0, 2H), 7.38 (d, J = 16.0, 1H), 7.49 (d, J = 8.4, 2H),
7.58 (d, J = 16.0, 1H), 7.91 (d, J = 8.0, 2H), 8.19 (d, J = 16.0, 1H).
MS (ESI): 415.5 (C27H26O4, [M+H]+). Anal. Calcd for C27H26O4: C,
78.24; H, 6.32. Found: C, 78.51; H, 6.34.
Yellow solid (0.408 g, 94% yield). Mp 147–149 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.87 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H),
6.47 (s, 1H), 6.76 (s, 1H), 6.93 (d, J = 8.0, 2H), 6.96 (d, J = 16.0,
1H), 7.34 (d, J = 16.0, 1H), 7.42 (d, J = 8.4, 2H), 7.48 (d, J = 8.4, 2H),
7.55 (d, J = 16.0, 1H), 7.93 (d, J = 8.4, 2H), 8.21 (d, J = 15.6, 1H).

2694
B.-F. Ruan et al. / Bioorg. Med. Chem. 19 (2011) 2688–2695
4.4.17. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(o-tolyl)prop-2-en-1-one (C17)
(t, J = 4.8, 4H), 3.93 (s, 6H), 6.49 (s, 1H), 6.76 (s, 1H), 6.92
(d, J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.38 (d, J = 16.4, 1H), 7.50 (d,
J = 8.4, 2H), 7.57 (d, J = 15.6, 1H), 7.98 (d, J = 8.4, 2H), 8.17 (d,
J = 15.6, 1H). MS (ESI): 486.22 (C30H31NO5, [M+H]+). Anal. Calcd
for C30H31NO5: C, 74.21; H, 6.43; N, 2.88. Found: C, 73.95; H,
6.45; N, 2.87.
Yellow solid (0.282 g, 68% yield). Mp 106–108 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.46 (s, 3H), 3.87 (s, 3H), 3.91 (s, 6H),
6.44 (s, 1H), 6.72 (s, 1H), 6.90 (d, J = 16.0, 1H), 6.92 (d, J = 8.4,
2H), 7.19 (d, J = 16.4, 1H), 7.25 (d, J = 8.0, 1H), 7.26 (d, J = 16.0,
1H), 7.27 (dd, J = 8.0, J1 = 7.6, J2 = 15.6, 1H), 7.34 (dd, J1 = 7.6,
J2 = 15.6, 1H), 7.39 (d, J = 8.4, 2H), 7.50 (d, J = 7.6, 1H), 7.86 (d,
J = 16.0, 1H). MS (ESI): 415.5 (C27H26O4, [M+H]+). Anal. Calcd for
4.5. Antiproliferation assay
C
27H26O4: C, 78.24; H, 6.32. Found: C, 78.50; H, 6.30.
The antiproliferative activity of the prepared compounds
against HepG2, B16-F10, and A549 cell lines were evaluated as de-
scribed elsewhere with some modifications.27 Target tumor cell
lines were grown to log phase in RPMI 1640 medium supple-
mented with 10% fetal bovine serum. After diluting to
4.4.18. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(2,4-dimethylphenyl)prop-2-en-1-one (C18)
Yellow solid (0.313 g, 73% yield). Mp 112–114 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.36 (s, 3H), 2.46 (s, 3H), 3.86 (s, 3H),
3.92 (s, 3H), 3.95 (s, 3H), 6.41 (s, 1H), 6.78 (s, 1H), 6.92 (d, J = 8.0,
3H), 7.00 (d, J = 16.4, 1H), 7.02 (s, 1H), 7.22 (d, J = 15.6, 1H), 7.41
(d, J = 8.4, 1H), 7.53 (d, J = 8.4, 2H), 7.88 (d, J = 16.0, 1H), 8.08 (d,
J = 16.0, 1H). MS (ESI): 429.20 (C28H28O4, [M+H]+). Anal. Calcd for
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 six wells
with colchicine and CA-4 coassayed as positive reference. After
C28H28O4: C, 78.48; H, 6.59. Found: C, 78.23; H, 6.61.
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
4.4.19. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(3,4-dimethylphenyl)prop-2-en-1-one (C19)
l
Yellow solid (0.330 g, 77% yield). Mp 111–113 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 2.36 (s, 3H), 2.46 (s, 3H), 3.79 (s, 3H),
3.89 (s, 3H), 3.93 (s, 3H), 6.63 (s, 1H), 6.84 (s, 1H), 6.97 (d,
J = 16.0, 1H), 6.99 (d, J = 8.4, 2H), 7.09 (d, J = 16.0, 1H), 7.37 (d,
J = 16.0, 1H), 7.56 (s, 1H), 7.57 (d, J = 8.4, 2H), 7.92 (d, J = 8.8, 2H),
7.98 (d, J = 16.0, 1H). MS (ESI): 429.50 (C28H28O4, [M+H]+). Anal.
Calcd for C28H28O4: C, 78.48; H, 6.59. Found: C, 78.24; H, 6.62.
(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 1.
4.6. Effects on tubulin polymerization
4.4.20. (E)-1-(4-Aminophenyl)-3-(2,4-dimethoxy-6-((E)-4-meth-
oxystyryl)phenyl)prop-2-en-1-one (C20)
Bovine brain tubulin was purified as described previously.28 To
evaluate the effect of the compounds on tubulin assembly
Yellow solid (0.370 g, 89% yield). Mp 198–200 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.92 (s, 6H), 4.11 (s, 2H),
6.27 (s, 1H), 6.64 (d, J = 8.4, 2H), 6.76 (s, 1H), 6.91 (d, J = 8.4, 2H),
6.97 (d, J = 16.0, 1H), 7.38 (d, J = 16.0, 1H), 7.49 (d, J = 8.4, 2H),
7.55 (d, J = 16.0, 1H), 7.89 (d, J = 8.4, 2H), 8.15 (d, J = 16.0, 1H).
MS (ESI): 416.18 (C26H25NO4, [M+H]+). Anal. Calcd for C26H25NO4:
C, 75.16; H, 6.06; N, 3.37. Found: C, 74.99; H, 6.08; N, 3.38.
in vitro,29 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
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.
4.4.21. (E)-1-(4-(Diethylamino)phenyl)-3-(2,4-dimethoxy-6-((E)-
4-methoxystyryl)phenyl)prop-2-en-1-one (C21)
4.7. Docking simulations
Yellow solid (0.434 g, 92% yield). Mp 148–150 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 1.22 (t, J = 7.2, 6H), 3.44 (q, J1 = 7.2,
J2 = 14.4, 4H), 3.86 (s, 3H), 3.92 (s, 6H), 4.11 (s, 2H), 6.46 (s, 1H),
6.65 (d, J = 8.4, 2H), 6.76 (s, 1H), 6.91 (d, J = 8.4, 2H), 7.14 (d,
J = 16.0, 1H), 7.40 (d, J = 16.0, 1H), 7.50 (d, J = 8.4, 2H), 7.57 (d,
J = 16.0, 1H), 7.95 (d, J = 8.4, 2H), 8.13 (d, J = 16.0, 1H). MS (ESI):
472.24 (C30H33NO4 [M+H]+). Anal. Calcd for C30H33NO4: C, 76.41;
H, 7.05; N, 2.97. Found: C, 76.22; H, 7.07; N, 2.87.
Molecular docking of compound C19 into the three-dimen-
sional X-ray structure of tubulin (PDB code: 1SA0) was carried
out using the Auto-Dock software (version 4.0) as implemented
through the graphic user interface Auto-Dock Tool Kit (ADT
1.4.6).30
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.31 For macromole-
cules, generated 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.
4.4.22. (E)-1-([1,10-Biphenyl]-4-yl)-3-(2,4-dimethoxy-6-((E)-4-
methoxystyryl)phenyl)prop-2-en-1-one (C22)
Yellow solid (0.405 g, 85% yield). Mp 173–175 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.86 (s, 3H), 3.93 (s, 6H), 6.47 (s, 1H),
6.76 (s, 1H), 6.92 (d, J = 8.0, 4H), 6.97 (d, J = 16.0, 1H), 7.26
(d, J = 8.0, 2H), 7.43–7.51 (dd, 3H), 7.37 (d, J = 16.0, 1H), 7.49 (d,
J = 8.0, 2H), 7.58 (d, J = 16.0, 1H), 8.01 (d, J = 8.0, 2H), 8.18 (d,
J = 16.0, 1H). MS (ESI): 477.20 (C32H28O4, [M+H]+). Anal. Calcd for
C32H28O4: C, 80.65; H, 5.92. Found: C, 80.35; H, 5.90.
4.4.23. (E)-3-(2,4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-
(4-morpholinophenyl)prop-2-en-1-one (C23)
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, the maps were centered on N1 atom of the
Yellow solid (0.461 g, 95% yield). Mp 167–169 °C. 1H NMR
(400 MHz, CDCl3) d (ppm): 3.33 (t, J = 4.8, 4H), 3.86 (s, 3H), 3.90

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Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4310.
Kcx 219 in the catalytic site of the protein. A grid spacing of 0.375 Å
(approximately one forth 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.
Acknowledgments
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This work was supported by the Jiangsu National Science Foun-
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