Design, synthesis and biological evaluation of (E)-3-(3,4-dihydroxyphenyl) acrylylpiperazine derivatives as a new class of tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.05.029

A series of novel (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives had been synthesized and evaluated their biological activities as potential tubulin polymerization inhibitors. Among these compounds, compound 3q exhibited potent antiproliferative activities against three cancer cell lines in vitro, and antitubulin polymerization activity with IC₅₀ of 0.92 μM, which was superior to that of colchicine (IC₅₀ = 1.34 μM). Docking simulation was performed to insert compound 3q into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. These results suggested that compound 3q may be a promising antitubulin agent for the potential treatment of cancer.

Full text of DOI:10.1016/j.bmc.2014.05.029

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation
of (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine
derivatives as a new class of tubulin polymerization inhibitors
⇑
⇑
Yong Yin , Fang Qiao , Lu-Yi Jiang, She-Feng Wang, Shao Sha, Xun Wu, Peng-Cheng Lv , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel (E)-3-(3,4-dihydroxyphenyl)acrylylpiperazine derivatives had been synthesized and
evaluated their biological activities as potential tubulin polymerization inhibitors. Among these com-
pounds, compound 3q exhibited potent antiproliferative activities against three cancer cell lines
Received 3 April 2014
Revised 13 May 2014
Accepted 13 May 2014
Available online xxxx
in vitro, and antitubulin polymerization activity with IC50 of 0.92
lM, which was superior to that of col-
chicine (IC50 = 1.34 M). Docking simulation was performed to insert compound 3q into the crystal struc-
l
ture of tubulin at colchicine binding site to determine the probable binding model. These results
suggested that compound 3q may be a promising antitubulin agent for the potential treatment of cancer.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Dihydroxyphenylacrylylpiperazine
Antiproliferative activity
Antitubulin agent
Molecular docking
1. Introduction
destabilizers (e.g., vinblastine and vincristine). They are widely
used clinically as important chemotherapeutic drugs for the treat-
In recent decades, cancer has become one of the most serious
diseases which has done great threaten to human health in the
world.1 Although there are a large number of anticancer drugs cur-
rently used, more and more people die of cancer.2 Developing some
new potent anticancer drugs is necessary. Strategies to block cell
division by affecting the mitotic spindle have historically been a
successful area of research for the advancement of cancer drugs.3,4
ment of kinds of malignancies.16–18 Colchicine (Fig. 1) is the first
drug that is well known to bind tubulin, which played an impor-
tant role in the elucidation of the properties and functions of tubu-
lin and microtubules, and its binding site on tubulin has been
characterized recently.19 In the past few years, small molecular
colchicines site binders such as CA-4, HMN-214, MKC-1 were
actively pursued as antimitotic agents which exerted potent
cytotoxic activities.20,21 In particularly these compounds such as
Microtubules, cylindrical protein polymers composed of
a- and
b-tubulin heterodimers, are a dynamic cellular compartment in both
neoplastic and normal cells.5,6 And it plays an important role in the
formation and function of the spindle apparatus, which realizes the
controlled segregation of the chromosomes in dividing cells.7,8
Microtubules are also key components of cytoskeleton involved
in a variety of other cellular functions, such as cell movement,
maintenance of the cell shape, as well as the transport of organelles
inside the cell.9–11 Inhibition of microtubule formation, spindle
poisons arrest dividing cells in G2/M phases of the cell cycle, caus-
ing mitotic catastrophy and finally apoptotic cell death.3,12–14 Con-
sequently, microtubules or tubulins are the most important
molecular targets of attack for many anticancer drugs.15
Millepachine, compound 7, compound 8, compound 9 and
compound 10 (Fig. 1), chalcone is a promising skeleton that has
shown the good antitubulin activity.22,23 Chalcones are considered
as precursors in the biosynthesis of flavonoids and isoflavonoids,
and it exhibits a variety of biological activities, such as anti-cancer,
antimitotic, anti-inflammatory, anti-bacterial and anti-fungal
activities.24–28 Over the past few years, the development of
chalcones as tubulin-binding agents has been attracted widely
attention. Recent studies have shown that some compounds
containing the chalcone skeleton and imides fragment act as
microtubule destabilizing agents, targeting the colchicine binding
site.23,29–31
There are two major groups of these antitumor agents, microtu-
bule stabilizers (e.g., paclitaxel and docetaxel), and microtubule
Chalcone and caffeic acid amide own similar part of the active
structure, and it was found in a variety of biologically active
substances.32,33 Caffeic acid (Fig. 2) is one of cinnamic acid, and
antitumor activities of various cinnamic acid derivatives were also
explored by our research group.34,35 Piperazine moiety was found
in kinds of biologically substance and it showed widely biological
⇑
Corresponding authors. Tel.: +86 25 83592572; fax: +86 25 83592672 (H.-L. Zhu).
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.05.029
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yin, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.029

2
Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
MeO
MeO
OMe O
OMe
MeO
MeO
NHAc
O
OMe
OMe
OH
N
OMe
OMe
Colchicine
1
2 Combretastain A-4 (CA-4)
3 MDL-27048
N
O2N
HN
O
N
O
O
O
O
N
N
NH
O
Cl
MeO
OMe
5 Indibulin
6
4
Millepachine
MKC-1
OMe
CONH2
I
OMe
OMe
O
O
N
H
N
H
OMe
7
8
O
O
OMe
OMe
H
N
N
O
F
OMe
10
9
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
O
These compounds were synthesized from caffeic acid (compound
1) and kinds of N-substituted piperazine 2a-2s. Caffeic acid,
N-substituted piperazine, N-hydroxybenzotriazole (HOBt) and
1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide hydrochloride
(EDCI) were dissolved in dichloromethane and refluxed to give
the desired compounds 3a–3s (Table 1).
HO
HO
OH
Caffeic acid
Figure 2. The chemical structures of caffeic acid.
2.2. Antiproliferative effects against cancer cells
activities such as anticancer, antibacterial, anti-inflammatory and
antidepressant activities.36 It encouraged us to combine the two
substructures, caffeic acid and N-substituted piperazine, and
screen out the compounds which have potent anticancer activities.
In this article, a new series of caffeic acid amide derivatives
containing piperazine moiety were synthesized and evaluated for
their anticancer and antitubulin activities toward three different
human cancer cell lines. Docking simulation was also performed
using X-ray crystallographic structure of the tubulin to explore
the binding modes of the synthetic compounds at the active site
of the enzyme.
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 3a–3s
against MCF-7, A549 and B16-F10 cancer cells. The results were
summarized in Table 2.
A number of (E)-3-(3,4-dihydroxy-
phenyl)acrylylpiperazine compounds showed remarkable antipro-
liferative effects. The results revealed several interesting points
including the broad range of potencies in this series.
Among compounds 3a–3p, compounds with substituted on
phenyl ring exhibited antiproliferative activities in order of elec-
tron-donating groups (OMe, Me) > electron-withdrawing groups
(NO2, CF3) > halogen groups (F, Cl). Compounds with chain hydro-
carbon or heterocyclic ring such as Pyridine, it showed unfavorable
inhibitory activity. Compounds (3a, 3f, 3g, 3h, 3k) with electron-
donating groups on phenyl ring showed antiproliferative activities
in order of para > ortho > meta and OMe > Me > H. Compounds
(3c–3d, 3i, 3l, 3m) with disubstituted halogen groups on phenyl
ring revealed antiproliferative activities which were superior to
that of mono-substituted halogen groups, and the strength order
was Cl > F.
Moreover, compounds 3q–3s which have benzhydryl group
exhibited stronger anticancer activities than any other compounds.
To our delight, compound 3q showed the most potent antiprolifer-
ative activity, which was superior to that of colchicine. Based on
the data obtained, it can be found that compounds with no substi-
tuted group showed slightly more potent activities than those of
2. Results and discussion
2.1. Chemistry
The synthetic route for the novel (E)-3-(3,4-dihydroxy-
phenyl)acrylylpiperazine derivatives 3a–3s is outlined in Scheme 1.
O
O
HO
HO
HO
HO
a
OH
N
HN
N R
N
R
2a-2s
3a-3s
1
Scheme 1. Reagents and conditions: (a) CH2Cl2, EDCI, HOBt, ref.
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Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
Table 2
Structure of compounds 3a–3s
Inhibition of cancer cells proliferation and tubulin polymerization by compounds 3a–
3s
O
Compound
IC50 SD (lg/mL)
IC50 SD (lM)
HO
HO
N
MCF-7a
A549b
B16-F10c
Tubulind
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
1.03 0.15
1.38 0.21
1.44 0.12
1.36 0.11
1.34 0.18
0.54 0.08
0.92 0.11
0.52 0.09
1.24 0.16
1.14 0.14
0.78 0.13
1.12 0.13
1.28 0.13
1.82 0.17
2.06 0.19
1.48 0.22
0.32 0.04
0.48 0.09
0.43 0.06
0.39 0.05
0.21 0.06
1.32 0.11
1.43 0.19
1.25 0.20
1.44 0.18
1.29 0.14
0.98 0.12
1.27 0.07
0.93 0.08
1.74 0.24
1.56 0.24
1.02 0.09
1.68 0.09
1.74 0.18
2.32 0.15
6.54 0.23
2.98 0.18
0.54 0.09
0.8 0.04
1.78 0.23
2.43 0.19
1.68 0.21
1.82 0.17
1.71 0.24
1.23 0.21
1.46 0.14
1.21 0.11
2.01 0.38
1.79 0.19
1.43 0.15
2.01 0.24
1.66 0.16
3.01 0.26
6.44 0.48
3.12 0.32
0.44 0.07
0.68 0.10
0.52 0.09
0.48 0.07
0.42 0.06
3.14 0.25
7.78 0.44
7.39 0.48
5.46 0.38
5.28 0.39
1.72 0.19
3.08 0.21
1.86 0.31
5.89 0.42
4.86 0.28
2.98 0.22
4.42 0.33
4.93 0.32
11.61 0.72
12.78 0.81
9.28 0.47
0.92 0.12
1.56 0.18
1.44 0.21
1.34 0.23
0.68 0.16
R
Compound
R
Compound
R
3a
3k
H3C
CH3
3b
3c
3l
F
Cl
Cl
Cl
3m
F
Cl
3d
3e
3f
3n
3o
3p
N
Cl
3r
3s
Cl
0.73 0.07
0.58 0.08
0.46 0.04
CH3CH2À
Colchicine
CA-4
a
b
c
Inhibition of the growth of MCF-7 cell lines.
Inhibition of the growth of A549 cell lines.
Inhibition of the growth of B16-F10 cell lines.
Inhibition of tubulin polymerization.
OCH3
H3CO
3g
3h
3q
3r
d
H3CO
F3C
treatment with 3q of 1 and 2 lg/mL for 24 h, respectively. These
findings indicated a continuing impairment of cell division and
Cl
confirmed compound 3q was a potent antitubulin agent.
3i
3j
3s
F
F
2.5. Binding model of compound 3q with tubulin
O2N
To help understanding the SARs observed at the tubulin and
guide further SAR studies, we proceeded to examine the interac-
tion of compound 3q with tubulin (PDB code: 1SA0). The molecular
docking was performed by simulation of synthetic compounds into
the colchicine binding site in tubulin, and the result was showed in
Figure 4. All docking runs were applied LigandFit Dock protocol of
Discovery Studio 3.5. The binding model of compound 3q and
tubulin was depicted in Figure 5. The amino acid residue which
had interaction with tubulin was labeled. In the binding mode,
compound 3q was nicely bound to the colchicine binding site of
tubulin via hydrophobic interaction and the binding was stabilized
by three hydrogen bonds. The two hydrogen atom of the two
hydroxy formed two hydrogen bonds with the carboxyl oxygen
atoms of Glu A: 183(bond length: C@OÁ Á ÁH = 1.96 Å, bond angle:
C@OÁ Á ÁH = 130.68°; bond length: CAOÁ Á ÁH = 2.03 Å, bond angle:
CAOÁ Á ÁH = 143.31°). Another one hydrogen bond was formed by
the oxygen atom of the one hydroxyl and the amino hydrogen of
ASN A: 101 (bond length: NAHÁ Á ÁO = 2.16 Å, bond angle:
NAHÁ Á ÁO = 168.72°). The 3D model of the interaction between
compound 3q and colchicine bonding site was showed in Figure 6,
it was found that the hydrophobic pockets of colchicine binding
site were occupied by compounds. The enzyme surface model
was showed in Figure 6B, which revealed that compound 3q was
well inserted into the active pocket of tubulin. Overall, the binding
model suggested that compound 3q is a potential inhibitor of
tubulin.
halogen substituted group, and the order was Cl > F. It could be a
promising lead for the further development of novel anti-tubulin
polymerization agents.
2.3. Tubulin polymerization inhibition
In order to examine whether the compounds interact with
tubulin and inhibit tubulin polymerization in vitro, we performed
the abilities of the synthesized compounds in inhibiting the
polymerization of tubulin. As shown in Table 2, compounds 3q,
3r and 3s showed strong inhibitory effect and their 50% tubulin
polymerization inhibition concentration of 0.92, 1.56 and
1.44
anti-tubulin polymerization activity, which was superior to that
of colchicine (IC50 = 1.34 M). This result indicated that the anti-
lM, respectively. Compound 3q displayed the most potent
l
proliferative effect was produced by direct connection of tubulin
and the compound.
2.4. Effects on cell cycle against cancer cells
In order to gain further insight into the action mode of these
compounds, the most potent one was further assayed for their
effects on cell cycle using flow cytometry (Fig. 3). As shown in
Figure 3, compound 3q strongly induced G2/M arrest in MCF-7
cells, and the effect was observed in a dose-independent manner
after treatment with increasing dose of the compound. About
63.39% of the cells were arrested in the G2/M phase while
84.36% of the cells were found to be in the G2/M phase after
3. Conclusion
In summary, a series of (E)-3-(3,4-dihydroxyphenyl)acrylylpip-
erazine derivatives had been synthesized and evaluated their
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Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Effects of compound 3q on cell cycle progression of MCF-7 cells were determined by flow cytometry analysis. MCF-7 cells were treated with different
concentrations of 3q for 24 h. The percentage of cells in each cycle phase was indicated.
biological activities. Among these compounds, compound 3q
exhibited potent antiproliferative activities against three cancer
cells and antitubulin polymerization activity with IC50 of
0.92 lM, which was superior to that of colchicine (IC50 = 1.34 lM).
Molecular docking of the most potent inhibitor 3q into the colchi-
cine binding site of tubulin was further performed to study the
inhibitor–tubulin protein interactions, and it was found that sev-
eral interactions with the protein residues in the colchicine binding
site might play an important role in its antitubulin polymerization
and antiproliferative activities. The information of this work might
be helpful for discovering novel new class of tubulin polymeriza-
tion inhibitors with stronger activities.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer
Figure 4. The docking calculation of the synthesized compounds (3a–3s).
Figure 5. 2D molecular docking model of compound 3q with tubulin (entry 1SA0 in the Protein Data Bank).
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Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 6. (A) Binding mode of compound 3q with tubulin (entry 1SA0 in the Protein Data Bank). The dotted lines show the hydrogen bonds. (B) 3D model of the interaction
between compound 3q and tubulin. The protein is represented by molecular surface. Compound 3q is depicted by sticks and balls.
chromatography (TLC) on Merck pre-coated silica GF254 plates.
Melting points (uncorrected) were determined on XT4MP
and was washed successively with 5% HCl, saturated NaHCO3
and brine, respectively, then dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was subjected to silica
gel chromatography or crystallization if necessary to afford the
compounds 3a–3s.
a
apparatus (Taike Corp., Beijing, China). ESI mass spectra were
obtained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were collected on a Bruker DPX400 spectrometer at
room temperature with TMS and solvent signals allotted as inter-
nal standards. Chemical shifts are reported in ppm (d). Elemental
analyses were performed on a CHN-O-Rapid instrument, and were
within 0.4% of the theoretical values.
4.2.1. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-phenylpiperazi-
ne (3a)
Yield: 67%; mp 171–174 °C. 1H NMR (400 MHz, DMSO): 9.12
(s, br, 2H, 2OH), 7.36 (d, J = 15.24 Hz, 1H, ArH), 7.23 (m, 2H,
2ArH), 7.12–7.05 (m, 1H, ArH), 7.01–6.90 (m, 4H, 4ArH),
6.82–6.74 (m, 2H, 2ArH), 3.79 (s, 2H, CH2), 3.72 (s, 2H, CH2),
3.14 (m, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.31,
150.28, 147.82, 145.92, 142.81, 130.62(2), 127.16, 122.93,
121.17, 116.02, 115.34, 115.08(2), 114.57, 53.24(2), 49.61(2).
MS (ESI): 325.34 (C19H21N2O3, [M+H]+). Anal. Calcd for
4.2. General procedure for synthesis of compounds 3a–3s
To a solution of caffeic acid (compound 1) (2 mmol) and
N-substituted piperazine (2 mmol) in dichloromethane (30 mL),
N-hydroxybenzotriazole (HOBt) (1 mmol) and 1-[3-(dimethyami-
no)-propyl]-3-ethylcarbodiimide hydrochloride (EDCI) (3 mmol)
were added. The mixture was refluxed under stirring for 8–12 h.
After completion of the reaction, the solution was cooled,
C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found: C, 70.51; H,
6.23; N, 8.62.
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Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.2.2. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(2-fluoropheny-
l)piperazine (3b)
2ArH), 7.01–6.95 (m, 2H, 2ArH), 6.75 (d, J = 8.12 Hz, 1H, ArH), 6.55
(dd, J1 = 8.32 Hz, J2 = 1.48 Hz, 1H, ArH), 6.48 (s, 1H, ArH), 6.40 (dd,
J1 = 8.08 Hz, J2 = 1.80 Hz, 1H, ArH), 3.78 (s, 2H, CH2), 3.72 (s, 5H,
CH2, OCH3), 3.14 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO):
165.38, 161.54, 151.67, 147.91, 145.90, 142.93, 131.62, 127.16,
121.19, 116.02, 115.36, 114.63, 111.28, 107.61, 98.43, 55.86,
53.38(2), 49.51(2). MS (ESI): 355.29 (C20H23N2O4, [M+H]+). Anal.
Calcd for C20H22N2O4: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.81;
H, 6.33; N, 7.78.
Yield: 52%; mp 180–181 °C. 1H NMR (400 MHz, DMSO): 9.42 (s,
1H, OH), 8.95 (s, 1H, OH), 7.35 (d, J = 15.24 Hz, 1H, ArH), 7.18–7.06
(m, 4H, 4ArH), 7.03–6.95 (m, 3H, 3ArH), 6.75 (d, J = 8.16 Hz, 1H,
ArH), 3.81 (s, 2H, CH2), 3.73 (s, 2H, CH2), 3.01 (s, 4H, 2CH2). 13C
NMR (100 MHz, DMSO): 165.48, 156.62, 147.79, 145.94, 142.96,
138.21, 127.22, 126.18, 123.02, 121.24, 117.33, 116.08, 115.98,
115.38, 114.62, 53.32(2), 49.58(2). MS (ESI): 343.34 (C19H20FN2O3,
[M+H]+). Anal. Calcd for C19H19FN2O3: C, 66.66; H, 5.59; N, 8.18.
Found: C, 66.51; H, 5.63; N, 8.22.
4.2.8. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(4-methoxyphe-
nyl)piperazine (3h)
Yield: 71%; mp 177–179 °C. 1H NMR (400 MHz, DMSO): 9.18 (s,
br, 2H, 2OH), 7.35 (d, J = 15.24 Hz, 1H, ArH), 7.10 (d, J = 1.88 Hz, 1H,
ArH), 7.01–6.91 (m, 4H, 4ArH), 6.84 (m, 2H, 2ArH), 6.75 (d,
J = 8.16 Hz, 1H, ArH), 3.78 (s, 2H, CH2), 3.68 (s, 5H, CH2, OCH3),
3.01 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.44, 153.81,
147.92, 146.28, 145.89, 142.86, 127.18, 121.18, 116.48(2), 116.07,
115.89(2), 115.28, 114.62, 55.84, 53.31(2), 49.52(2). MS (ESI):
355.29 (C20H23N2O4, [M+H]+). Anal. Calcd for C20H22N2O4: C,
67.78; H, 6.26; N, 7.90. Found: C, 67.81; H, 6.33; N, 7.78.
4.2.3. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(4-fluoropheny-
l)piperazine (3c)
Yield: 48%; mp 181–183 °C. 1H NMR (400 MHz, DMSO): 9.41 (s,
1H, OH), 8.96 (s, 1H, OH), 7.35 (d, J = 15.24 Hz, 1H, ArH), 7.10–7.04
(m, 3H, 3ArH), 7.01–6.95 (m, 4H, 4ArH), 6.75 (d, J = 8.16 Hz, 1H,
ArH), 3.79 (s, 2H, CH2), 3.72 (s, 2H, CH2), 3.01 (s, 4H, 2CH2). 13C
NMR (100 MHz, DMSO): 165.52, 157.84, 147.89, 146.24, 145.94,
142.81, 127.18, 121.11, 117.46(2), 116.38(2), 116.05, 115.27,
114.46, 53.41(2), 49.67(2). MS (ESI): 343.34 (C19H20FN2O3,
[M+H]+). Anal. Calcd for C19H19FN2O3: C, 66.66; H, 5.59; N, 8.18.
Found: C, 66.51; H, 5.63; N, 8.22.
4.2.9. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(3-(trifluorome-
thyl)phenyl)piperazine (3i)
4.2.4. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(2-chloropheny-
l)piperazine (3d)
Yield: 54%; mp 134–136 °C. 1H NMR (400 MHz, DMSO): 9.46
(s,1H, OH), 8.98 (s, 1H, OH), 7.44 (t, J = 7.98 Hz, 1H, ArH), 7.35 (d,
J = 15.24 Hz, 1H, ArH), 7.25 (d, J = 8.44 Hz, 1H, ArH), 7.21 (s, 1H,
ArH), 7.10 (t, J = 3.94 Hz, 2H, 2ArH), 7.01–6.96 (m, 2H, 2ArH),
6.75 (d, J = 8.12 Hz, 1H, ArH), 3.82 (s, 2H, CH2), 3.71 (s, 2H, CH2),
3.26 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.31, 152.18,
147.89, 145.89, 142.91, 132.91, 129.72, 127.19, 125.21, 121.17,
118.94, 116.07, 115.69, 115.38, 114.61, 113.28, 53.39(2),
49.57(2). MS (ESI): 393.42 (C20H20F3N2O3, [M+H]+). Anal. Calcd
for C20H19F3N2O3: C, 61.22; H, 4.88; N, 7.14. Found: C, 61.31; H,
4.74; N, 7.21.
Yield: 53%; mp 172–173 °C. 1H NMR (400 MHz, DMSO): 9.42 (s,
1H, OH), 8.96 (s, 1H, OH), 7.43 (dd, J1 = 7.88 Hz, J2 = 1.28 Hz, 1H,
ArH), 7.38–7.29 (m, 2H, 2ArH), 7.17(m, H, ArH), 7.10–7.05 (m,
2H, 2ArH), 7.01–6.95 (m, 2H, 2ArH), 6.75 (d, J = 8.12 Hz, 1H, ArH),
3.82 (s, 2H, CH2), 3.74 (s, 2H, CH2), 2.98 (s, 4H, 2CH2). 13C NMR
(100 MHz, DMSO): 165.33, 151.81, 147.86, 145.96, 142.87,
131.86, 130.12, 127.74, 127.14, 124.91, 123.76, 121.18, 116.04,
115.42, 114.72, 52.84(2), 49.54(2). MS (ESI): 359.79 (C19H20ClN2O3,
[M+H]+). Anal. Calcd for C19H19ClN2O3: C, 63.60; H, 5.34; N, 7.81.
Found: C, 63.51; H, 5.63; N, 7.72.
4.2.10. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(4-nitropheny-
l)piperazine (3j)
4.2.5. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(3-chloropheny-
l)piperazine (3e)
Yield: 69%; mp 184–185 °C. 1H NMR (400 MHz, DMSO): 9.47 (s,
1H, OH), 8.98 (s, 1H, OH), 8.08 (d, J = 9.44 Hz, 2H, 2ArH), 7.36 (d,
J = 15.24 Hz, 1H, ArH), 7.10 (d, J = 1.96 Hz, 1H, ArH), 7.11–6.94
(m, 4H, 4ArH), 6.75 (d, J = 8.12 Hz, H, ArH), 3.85 (s, 2H, CH2), 3.71
(s, 2H, CH2), 3.54 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO):
165.50, 154.89, 147.95, 145.91, 142.95, 137.41, 127.12, 126.22(2),
121.26, 116.04, 115.38, 114.50, 113.00(2), 54.12(2), 49.41(2). MS
(ESI): 370.33 (C19H20N3O5, [M+H]+). Anal. Calcd for C19H19N3O5:
C, 61.78; H, 5.18; N, 11.38. Found: C, 61.82; H, 5.23; N, 11.44.
Yield: 66%; mp 182–185 °C. 1H NMR (400 MHz, DMSO): 9.21 (s,
br, 2H, 2OH), 7.35 (d, J = 15.2 Hz, 1H, ArH), 7.23 (t, J = 8.2 Hz, 1H,
ArH), 7.10 (d, J = 1.94 Hz, 1H, ArH), 7.01–6.91 (m, 4H, 4ArH), 6.81
(dd, J1 = 7.88 Hz, J2 = 1.12 Hz, 1H, ArH), 6.75 (d, J = 8.16 Hz, 1H,
ArH), 3.80 (s, 2H, CH2), 3.70 (s, 2H, CH2), 3.20 (s, 4H, 2CH2). 13C
NMR (100 MHz, DMSO): 165.46, 150.08, 147.82, 145.91, 142.92,
136.28, 132.04, 127.12, 122.81, 121.22, 116.07, 115.32, 114.61,
113.42, 111.39, 53.29(2), 49.56(2). MS (ESI): 359.79 (C19H20ClN2O3,
[M+H]+). Anal. Calcd for C19H19ClN2O3: C, 63.60; H, 5.34; N, 7.81.
Found: C, 63.51; H, 5.63; N, 7.72.
4.2.11. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(2,4-dimethyl-
phenyl)piperazine (3k)
4.2.6. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(2-methoxyphe-
nyl)piperazine (3f)
Yield: 57%; mp 187–189 °C. 1H NMR (400 MHz, DMSO): 9.38(s,
1H, OH), 8.99 (s, 1H, OH), 7.35 (d, J = 15.24 Hz, 1H,ArH), 7.09 (d,
J = 1.80 Hz, H, ArH), 7.01–6.90 (m, 5H, 5ArH), 6.75 (d, J = 8.12 Hz,
H, ArH), 3.78 (s, 2H, CH2), 3.70 (s, 2H, CH2), 2.78 (s, 4H, 2CH2),
2.25 (s, 3H, CH3), 2.21 (s, 3H, CH3). 13C NMR (100 MHz, DMSO):
165.62, 149.04, 147.89, 145.94, 142.86, 138.31, 132.81, 127.72,
127.18, 126.84, 121.37, 121.10, 116.08, 115.43, 114.61, 53.74(2),
49.59(2), 22.63, 19.21. MS (ESI): 353.49 (C21H25N2O3, [M+H]+).
Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86; N, 7.95. Found: C,
71.63; H, 6.92; N, 8.01.
Yield: 57%; mp 143–144 °C. 1H NMR(400 MHz, DMSO): 9.45
(s,1H, OH), 8.97 (s, 1H, OH), 7.34 (d, J = 15.2 Hz, 1H, ArH), 7.09 (d,
J = 1.92 Hz, 1H, ArH), 7.01–6.95 (m, 2H, 2ArH), 6.91–6.85(m, 2H,
2ArH), 6.74 (d, J = 8.12 Hz, 1H, ArH), 3.80 (s, 5H, CH2, OCH3), 3.69
(s, 2H, CH2), 2.95 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO):
165.27, 152.51, 147.85, 145.89, 142.77, 141.26, 127.20, 123.36,
121.11, 118.81, 116.04, 115.82, 115.39, 114.68, 112.36, 55.83,
53.62(2), 49.57(2). MS (ESI): 355.29 (C20H23N2O4, [M+H]+). Anal.
Calcd for C20H22N2O4: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.81;
H, 6.33; N, 7.78.
4.2.12. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(2,3-dichlorop-
henyl)piperazine (3l)
4.2.7. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(3-methoxyphe-
nyl)piperazine (3g)
Yield: 59%; mp 154–156 °C. 1H NMR (400 MHz, DMSO): 9.46
(s,1H, OH), 8.97 (s, 1H, OH), 7.38–7.32 (m, 3H, 3ArH), 7.16 (m,
1H, ArH), 7.10 (d, J = 1.92 Hz, 1H, ArH), 7.01–6.96 (m, 2H, 2ArH),
Yield: 62%; mp 183–185 °C. 1H NMR (400 MHz, DMSO): 9.11 (s,
br, 2H, 2OH), 7.36 (d, J = 15.24 Hz, 1H, ArH), 7.12 (t, J = 8.12 Hz, 2H,
Please cite this article in press as: Yin, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.029

Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
6.74 (d, J = 8.16 Hz, 1H, ArH), 3.83 (s, 2H, CH2), 3.73 (s, 2H, CH2),
2.98 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.57, 151.04,
147.88, 145.89, 142.79, 134.28, 130.11, 128.33, 127.24, 124.94,
121.18, 118.62, 116.08, 115.32, 114.68, 52.81(2), 49.68(2). MS
(ESI): 394.32 (C19H19Cl2N2O3, [M+H]+). Anal. Calcd for C19H18Cl2N2
O3: C, 58.03; H, 4.61; N, 7.12. Found: C, 58.08; H, 4.72; N, 7.18.
1H, CH), 3.66 (s, 2H, CH2), 3.57 (s, 2H, CH2), 2.30 (s, 4H, 2CH2).
13C NMR (100 MHz, DMSO): 165.45, 147.92, 145.90, 143.74(2),
142.96, 130.18(4), 128.42(4), 127.14, 125.44(2), 121.16, 116.08,
115.42, 114.53, 85.61, 52.43(2), 51.82(2). MS (ESI): 415.42
(C26H27N2O3, [M+H]+). Anal. Calcd for C26H26N2O3: C, 75.34; H,
6.32; N, 6.76. Found: C, 75.31; H, 6.41; N, 6.47.
4.2.13. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(3,4-dichlorop-
henyl)piperazine (3m)
4.2.18. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-((4-chlorophe-
nyl)(phenyl)methyl)piperazine (3r)
Yield: 64%; mp 187–189 °C. 1H NMR (400 MHz, DMSO): 9.46 (s,
1H, OH), 8.98 (s, 1H, OH), 7.42–7.33 (m, 2H, 2ArH), 7.17 (d,
J = 2.8 Hz, 1H, ArH), 7.10 (d, J = 1.6 Hz, 1H, ArH), 7.01–6.95 (m,
3H, 3ArH), 6.74 (d, J = 8.16 Hz, 1H, ArH), 3.79 (s, 2H, CH2), 3.68 (s,
2H, CH2), 3.22 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.37,
150.96, 147.91, 145.91, 142.93, 132.00, 130.96, 127.15, 121.21,
120.37, 117.01, 116.38, 116.02, 115.40, 114.55, 53.38(2),
49.51(2). MS (ESI): 394.32 (C19H19Cl2N2O3, [M+H]+). Anal. Calcd
for C19H18Cl2N2O3: C, 58.03; H, 4.61; N, 7.12. Found: C, 58.08; H,
4.72; N, 7.18.
Yield: 49%; mp 208–210 °C. 1H NMR (400 MHz, DMSO): 9.41 (s,
1H, OH), 8.94 (s, 1H, OH), 7.47–7.35(m, 7H, ArH), 7.33–7.28 (m, 3H,
ArH), 7.04 (d, J = 1.80 Hz, 1H, ArH), 6.94 (dd, J1 = 8.20 Hz,
J2 = 1.80 Hz, 1H, ArH), 6.86 (d, J = 15.28 Hz, 1H, ArH), 6.73 (d,
J = 8.16 Hz, 1H, ArH), 4.39 (s, 1H, CH), 3.66 (s, 2H, CH2), 3.58 (s,
2H, CH2), 2.29 (s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.38,
147.86, 145.93, 143.79, 142.93, 141.28, 132.78, 130.61(2),
129.37(2), 129.06(2), 128.34(2), 127.14, 126.36, 121.11, 116.04,
115.38, 114.54, 84.58, 51.94(2), 50.08(2). MS (ESI): 449.75
(C26H26ClN2O3, [M+H]+). Anal. Calcd for C26H25ClN2O3: C, 69.56;
H, 5.61; N, 6.24. Found: C, 69.61; H, 5.72; N, 6.41.
4.2.14. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(pyridin-2-yl)-
piperazine (3n)
4.2.19. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-(bis(4-fluorop-
henyl)methyl)piperazine (3s)
Yield: 46%; mp 161–164 °C. 1H NMR (400 MHz, DMSO): 9.44
(s,1H, OH), 9.01 (s, 1H, OH), 8.13 (dd, J1 = 4.8 Hz, J2 = 1.36 Hz, 1H,
ArH), 7.55 (m, 1H, ArH), 7.35 (d, J = 15.24 Hz, 1H, ArH), 7.10 (d,
J = 1.96 Hz, 1H, ArH), 7.01–6.95 (m, 2H, 2ArH), 6.85 (d,
J = 8.64 Hz, 1H, ArH), 6.74 (d, J = 8.12 Hz, 1H, ArH), 6.66 (m, 1H,
ArH), 3.77 (s, 2H, CH2), 3.66 (s, 2H, CH2), 3.52 (s, 4H, 2CH2). 13C
NMR (100 MHz, DMSO): 165.43, 159.21, 148.05, 147.89, 145.91,
142.82, 138.10, 127.18, 121.19, 116.04, 115.37, 114.69, 113.71,
107.70, 49.32(2), 46.48(2). MS (ESI): 326.48 (C18H20N3O3,
[M+H]+). Anal. Calcd for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91.
Found: C, 66.54; H, 5.92; N, 12.99.
Yield: 58%; mp 213–215 °C. 1H NMR (400 MHz, DMSO): 9.40 (s,
1H, OH), 8.93 (s, 1H, OH), 7.47–7.42 (m, 4H, 4ArH), 7.29 (m, 1H,
ArH), 7.16–7.11 (m, 4H, 4ArH), 7.03 (m, 1H, ArH), 6.94 (m, 1H,
ArH), 6.86 (m, 1H, ArH), 6.72 (d, J = 8.12 Hz, 1H, ArH), 4.40 (s, 1H,
CH), 3.66 (s, 2H, CH2), 3.57 (s, 2H, CH2), 2.27 (s, 4H, 2CH2). 13C
NMR (100 MHz, DMSO): 165.42, 161.38(2), 147.85, 145.89,
142.86, 139.42(2), 130.89(4), 127.12, 121.25, 117.12(4), 116.04,
115.38, 114.58, 85.52, 51.98(2), 50.12(2). MS (ESI): 451.42
(C26H25F2N2O3, [M+H]+). Anal. Calcd for C26H24F2N2O3: C, 69.32;
H, 5.37; N, 6.22. Found: C, 69.42; H, 5.29; N, 6.29.
4.2.15. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-
4.3. Antiproliferation assay
ethylpiperazine (3o)
Yield: 51%; mp 143–144 °C. 1H NMR (400 MHz, DMSO): 9.14 (s,
br, 2H, 2OH), 7.31 (d, J = 15.24 Hz, 1H, ArH), 7.07 (d, J = 1.60 Hz, 1H,
ArH), 6.97 (dd, J1 = 8.20 Hz, J2 = 1.68 Hz, 1H, ArH), 6.91 (d,
J = 15.24 Hz, 1H, ArH), 6.74 (d, J = 8.12 Hz, 1H, ArH), 3.63 (s, 2H,
CH2), 3.54 (s, 2H, CH2), 2.35 (m, 6H, 3CH2), 1.01 (t, J = 7.18 Hz,
3H, CH3). 13C NMR (100 MHz, DMSO): 165.32, 147.87, 145.89,
142.86, 127.18, 121.20, 116.07, 115.28, 114.61, 56.58(2),
50.28(2), 48.42, 14.24. MS (ESI): 277.51 (C15H21N2O3, [M+H]+).
Anal. Calcd for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14. Found: C,
65.31; H, 7.41; N, 10.33.
The antiproliferative activity of the prepared compounds
against A549, MCF-7 and B16-F10 cells was evaluated as described
elsewhere with some modifications.37 Target tumor cell line was
grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 2 Â 104 cells/mL with
the complete medium, 100 lL of the obtained 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
co-assayed as positive reference. After 48 h exposure period,
4.2.16. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-benzylpiperaz-
ine (3p)
40
zol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to each
well. Four hours later, 100 L extraction solution (10% SDS-5%
isobutyl alcohol-0.01 M HCl) was added. After an overnight incuba-
tion at 37 °C, the optical density was measured at a wavelength of
570 nm on an ELISA microplate reader. In all experiments, three
replicate wells were used for each drug concentration. Each assay
was carried out at least three times. The results were summarized
in Table 2.
lL of PBS containing 2.5 mg/mL of MTT (3-(4,5-dimethylthia-
Yield: 56%; mp 154–157 °C. 1H NMR (400 MHz, DMSO): 9.40 (s,
1H, OH), 8.93 (s, 1H, OH), 7.32 (m, 5H, 5ArH), 7.27 (m, 1H, ArH),
7.06 (d, J = 1.76 Hz, 1H, ArH), 6.97 (dd, J1 = 8.20 Hz, J2 = 1.80 Hz,
1H, ArH), 6.90 (d, J = 15.24 Hz, 1H, ArH), 6.73 (d, J = 8.12 Hz, 1H,
ArH), 3.64 (s, 2H, CH2), 3.55 (s, 2H, CH2), 3.50 (s, 2H, CH2), 2.36
(s, 4H, 2CH2). 13C NMR (100 MHz, DMSO): 165.48, 147.89,
145.91, 142.79, 139.61, 129.42(2), 128.38(2), 127.12, 126.19,
121.19, 116.09, 115.38, 114.61, 65.31, 54.72(2), 49.76(2). MS
(ESI): 339.54 (C20H23N2O3, [M+H]+). Anal. Calcd for C20H22N2O3:
C, 70.99; H, 6.55; N, 8.28. Found: C, 71.06; H, 6.49; N, 8.32.
l
4.4. Effects on tubulin polymerization
Bovine brain tubulin was purified as described previously.38
To evaluate the effect of the compounds on tubulin assembly
in vitro, varying concentrations were preincubated with 10 lM
bovine brain tubulin in glutamate buffer at 30 °C and then cooled
to 0 °C. After addition of 0.4 mM 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
4.2.17. 1-((E)-3-(3,4-Dihydroxyphenyl)acrylyl)-4-benzhydrylpi-
perazine (3q)
Yield: 63%; mp 201–204 °C. 1H NMR (400 MHz, DMSO): 9.40 (s,
1H, OH), 8.93 (s, 1H, OH), 7.44 (d, J = 7.32 Hz, 4H, 4ArH), 7.32–7.28
(m, 5H, 5ArH), 7.20 (t, J = 14.60 Hz, 2H, 2ArH), 7.03 (d, J = 1.56 Hz,
1H, ArH), 6.94 (m, 1H, ArH), 6.72 (d, J = 8.16 Hz, 1H, ArH), 4,34 (s,
Please cite this article in press as: Yin, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.029

8
Y. Yin et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
turbidimetrically at 350 nm.39 The IC50 was defined as the com-
pound concentration that inhibited the extent of assembly by
50% after 20 min incubation.
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Acknowledgment
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This work was supported by Natural Science Foundation of
Jiangsu Province (No. BK20130554) and supported by Major
Projects on Control and Rectification of Water Body Pollution
(No. 2011ZX07204-001-2214 004).
31. Raffa, D.; Maggio, B.; Plescia, F.; Cascioferro, S.; Plescia, S.; Raimondi, M. V.;
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Please cite this article in press as: Yin, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.029