Design, synthesis and neuroprotective activities of novel cinnamide derivatives containing benzylpiperazine moiety

Article abstract of DOI:10.1007/s00044-018-2153-5

A new series of cinnamide derivatives 6a–l were synthesized by the reaction of acyl chlorides with various substituted benzylpiperazines. The structures were characterized by ¹H NMR, ¹³C NMR, and HRMS. The potential neuroprotective activities of cinnamide analogs were evaluated in differentiated rat pheochromocytoma cells (PC12 cells) and in mice subjected to acute cerebral ischemia. Among the series, 6a, 6b, and 6c, featuring a 1,3-benzodioxole moiety, showed potent neuroprotection both in vivo and in vitro. The three compounds were selected and further studied to determine their mechanism of action. MTT assay, Hoechst 33342/PI double staining, and high content screening (HCS) revealed that pretreatment of the cells with 6a, 6b, and 6c has significantly decreased the extent of cell apoptosis in a dose-dependent manner. The results of western blot analysis demonstrated these compounds suppressed apoptosis of glutamate-induced PC12 cells via caspase-3 pathway. These compounds can be lead compounds for further discovery of neuroprotective agents for treating cerebral ischemic stroke.

Full text of DOI:10.1007/s00044-018-2153-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2153-5
ORIGINAL RESEARCH
Design, synthesis and neuroprotective activities of novel cinnamide
derivatives containing benzylpiperazine moiety
Yan Zhong1 Xiaofeng Li Aixia Zhang Yi Xu Ping Li Bin Wu
2 ●
2 ●
2 ●
3 ●
2
●
Received: 17 July 2016 / Accepted: 2 February 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A new series of cinnamide derivatives 6a–l were synthesized by the reaction of acyl chlorides with various substituted
1
13
benzylpiperazines. The structures were characterized by H NMR, C NMR, and HRMS. The potential neuroprotective
activities of cinnamide analogs were evaluated in differentiated rat pheochromocytoma cells (PC12 cells) and in mice
subjected to acute cerebral ischemia. Among the series, 6a, 6b, and 6c, featuring a 1,3-benzodioxole moiety, showed potent
neuroprotection both in vivo and in vitro. The three compounds were selected and further studied to determine their
mechanism of action. MTT assay, Hoechst 33342/PI double staining, and high content screening (HCS) revealed that
pretreatment of the cells with 6a, 6b, and 6c has significantly decreased the extent of cell apoptosis in a dose-dependent
manner. The results of western blot analysis demonstrated these compounds suppressed apoptosis of glutamate-induced
PC12 cells via caspase-3 pathway. These compounds can be lead compounds for further discovery of neuroprotective agents
for treating cerebral ischemic stroke.
●
●
●
●
Keywords Cinnamide Neuroprotection Caspase-3 Cerebral ischemia Apoptosis
Introduction
coagulants, and neuroprotective agents (Yousef et al. 2004).
Neuroprotection is widely recognized to be a potential
strategy for the treatment of ischemic stroke (Beresford
et al. 2003), which may make the brain more resistant to
damage from stroke, and considerable efforts have been
devoted to the development of neuroprotective agents.
However, little of them have reached routine clinical use
because of their limited efficacy or unfavorable risk-benefit
ratio in clinical trials (de Keyser et al. 1999). There is
currently no treatment that has been conclusively proven by
controlled clinical trials to be beneficial, except for throm-
bolytic treatment with recombinant tissue plasminogen
activator for highly selected patients (Alexandrov et al.
2004). Nonetheless, ischemia/reperfusion after thrombolysis
therapy is a big problem, which restrained clinical appli-
cation of thrombolytic drugs (Warach and Latour 2004).
Therefore, it is necessary to search for safer and more
effective neuroprotective agents.
Stroke is a leading cause of death and long-lasting disability
in developed countries (Maki et al. 2013). In China, stroke
is the second cause for mortality in all diseases. Particularly,
ischemic strokes account for 60–80% of these strokes (Jia
et al. 2011; Liu et al. 2007). With the rapid increase of aging
population in China, stroke has become a critical cause for
the health of the old, and stroke is becoming a major burden
facing the government and health providers (Wang et al.
2
011; Liu et al. 2007).
So far, therapeutic options remain limited. The medica-
tions commonly used for stroke can be divided into four
groups: thrombolytic agents, antiplatelet agents, anti-
*
Bin Wu
wubin@njmu.edu.cn
Current status of treating ischemic stroke promoted a
great deal of interest in the area of neuroprotective agents.
Previous work reported by our group suggested that cin-
namide scaffold often affords neuroprotective compounds
(Wu et al. 2008). Especially the compound NY-308, which
has the (E)-p-methoxycinnamoyl moiety, exhibited excel-
lent neuroprotection in vitro PC12 cells and in vivo rat focal
1
2
3
School of Chemistry and Chemical Engineering, Southeast
University, Nanjing 211189, China
School of Pharmacy, Nanjing Medical University,
Nanjing 211166, China
School of Basic Medical Sciences, Nanjing Medical University,
Nanjing 211166, China

Medicinal Chemistry Research
cerebral ischemic animal model (Wu et al. 2012). Structu-
rally, the (E)-p-methoxycinnamoyl moiety in NY-308 was
believed to play a very important role in its activity.
Therefore it became a pattern for target compounds men-
tioned in this paper. New compounds retained α,β-unsatu-
rated carbonyl and basic center piperazine. Furthermore, we
replaced diphenylmethylpiperazine of NY-308 with ben-
zylpiperazine and changed substituent groups of benzene
rings to study of the substituent variability influence on the
biological activity (Fig. 1).
substitutedacrylic acids 2a–c (Tanaka et al. 1983). The key
intermediates benzyl bromides 4a–d, were prepared by free-
radical bromination of commercially available substituted
toluenes 3a–d with N-bromosuccinimide (NBS) (Sahin
et al. 2008; Šterk et al. 2012). Potassium carbonate cata-
lyzed coupling of 4a–d with N-Boc-piperazine afforded the
compounds 5a–d in excellent yield (Long et al. 2010). The
target compounds 6a–l were finally obtained by acylation of
various substituted benzylpiperazines with the correspond-
ing acyl chlorides under mild conditions.
Based on the excellent neuroprotective activity, 6a, 6b,
and 6c were chosen for further study of action mechanism.
The elucidation of the molecular mechanism involved in
this effect might provide novel insight into the process of
stroke and other neurodegeneration and suggest the poten-
tial utility of cinnamide analogs for protection against
neurological insults. Therefore, in vitro neuroprotective
activities in the cell injury induced by glutamate, apoptosis
assays, and the inhibition of caspase-3 were evaluated.
Herein, the synthesis and preliminary biological evaluation
of the novel cinnamide derivatives were reported.
Biological activity
Bilateral common carotid artery occlusion
Kunming mice of both sexes were randomly divided into
groups (10 mice per group). The test agents used in this
study were 6a–l. These compounds above were dissolved in
aqueous 0.5% sodium carboxy methyl cellulose (CMCNa)
solution before use and administered intraperitoneally (i.p.).
Nimodipine was i.p. treated with 80 mg/kg as a positive
control. The control group received normal saline (NS) in
the same volume as other groups. All groups received drugs
twice a day for 3 days. Sixty minutes after the last admin-
istration, all mice were anesthetized by ether. All groups
underwent the operation for common carotid artery and
vagus nerves ligation. Then the survival time of mice were
recorded (Zhang et al. 2006; Yanpallewar et al. 2004).
Materials and methods
Chemistry
The title compounds were synthesized as outlined in the
Scheme 1. Substituted benzaldehydes 1a–c was coupled
with malonic acid via a Knoevenagel reaction to give (E)-
Cell viability assay
PC12 cells obtained from New Drug Research Center of
China Pharmaceutical University (Nanjing, China) were
grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented 10% FBS (heat-inactivated), 5% HS (heat-
inactivated), 100 IU/mL penicillin, and 100 µg/mL strepto-
mycin in a humidified incubator at 37 °C and 5% CO2.
O
R2
N
R1
A
B
N
R3
PC12 cells were seeded in 96-well plates at a density of 1 ×
5
1
0 /mL. Prior to the treatments, PC12 cells were rinsed by
1
2
3
NY-308 (R = 4-OMe; R = 4-F; R = 4-fluorophenyl)
DMEM for three times (5 min each), then covered with
serum-free DMEM in which Glu (10 mmol/L) or Glu
Fig. 1 Structure of novel cinnamide deravatives
CHO
COOH
(
i)
R1
R1
O
(iv)
N
2a-c
5a-d
R1
R2
1
4
a-c
a-d
N
(
ii)
Br
(iii)
N
R2
R2
R2
6
a-l
NH
3
a-d
Scheme 1 Synthesis of cinnamide derivatives (6a–l). Reagents and
conditions: (i) CH2(COOH)2, pyridine, piperidine, reflux, 2 h; (ii)
NBS, AIBN, CCl4, h.v., reflux, 1 h; (iii) N-Boc-piperazine, CH2Cl2,
K2CO3, reflux, 10 h; CH2Cl2, TFA, r.t., 2 h; (iv) SOCl2, CH2Cl2, r.t., 6
h; Et3N, CH2Cl2, CH3COCH3, r.t., 12 h

Medicinal Chemistry Research
(
10 mmol/L) with compounds (0.1, 1.0, and 10 μmol/L) or
Technology, USA) and actin (1:1000) (Beyotime, China).
After washing with TBST, the membranes were incubated
for 2 h at 30 °C with a 1:2000 dilution of horseradish
peroxidase-conjugated antirabbit or antimouse IgG. After
rewashing with TBST, the bands were developed by using a
Immobilon Wester Chemiluminescent HRP Substrate kit
(Millipore, USA) for 5 min, the protein bands were visua-
lized with Tanon 5200 (Tanon Company Ltd., China).
Edaravone (90 μmol/L) were added. After incubation in a
CO incubator for 24 h, PC12 cells were subsequently tes-
ted by MTT assay (Mosmann 1983; Park et al. 2009).
Tetrazolium (10 µL/well; 5 mg/mL of stock in PBS) salt
was added 4 h prior to completion of incubation periods.
Thereafter, the reaction mixture was carefully taken out and
2
1
50 µL of dimethyl sulfoxide (DMSO) solution was added
to each well to achieve solubilization of the formazan
crystals formed in viable cells. The absorbance was read at
4
90 nm on an ELISA plate reader, with DMSO as a blank.
Results and discussion
Chemistry
All assays were performed in triplicate and repeated thrice.
The degree of protection induced by the test compound was
calculated using the following equation: protection (%) =
(
(C–B)/(A–B)) × 100, where A is the viability of control
General
cultures, B is the viability of the cultures treated with Glu,
and C is the viability of the cultures treated with Glu and the
test compound.
All chemicals used for the synthesis were of reagent grade
and procured from Sigma-Aldrich, Shanghai, China. Melt-
ing points were determined on an electrothermal digital
apparatus model WRR-401 (Shanghai, China) without
Assessment with Hoechst 33342 and propidium
iodide (PI) double staining
1
13
correction. H nuclear magnetic resonance (NMR) and
C
NMR were recorded on a Bruker ACF-300 MHz instrument
To further investigate the protective effects, Hoechst 33342
(Bruker) with CDCl as the solvent and tetramethylsilane as
3
and PI double fluorescent staining was assayed (Wang et al.
an internal standard (chemical shifts are expressed as δ
values, J in hertz). High resolution mass spectra (HRMS)
were recorded on a MALDI Micro MX instrument
(Waters). All the reactions were monitored by thin-layer
chromatography (TLC) using precoated silica gel 60 F254
(Merck). The crude product was purified by column
chromatography.
2
010; Ma et al. 2012). The PC12 cells were cultured in 96-
4
well plates at 1 × 10 cells/well for 24 h. After the indicated
treatment, the cells were incubated with 10 μg/mL Hoechst
3
5
3342 for 15 min, washed twice with PBS, incubated with
0 μg/mL PI working solution for an additional 15 min in
the dark, and analyzed by high content screening (HCS)
Thermo Scientific, USA) within 24 h. The apoptotic nuclei
(
were counted in at least 200 cells from nine randomly
chosen fields in each treatment and are expressed as per-
centages of the total numbers of counted nuclei.
General procedures for the synthesis of (E)-
substitutedacrylic acids (2a–c)
A mixture of corresponding substituted benzaldehydes 1
(50 mmol), malonic acid (75 mmol), and piperidine (1 mL)
in 35 mL pyridine was refluxed for 2 h. The solvent was
evaporated under reduced pressure. The residue was added
to a mixture of 35 mL HCl and 70 g ice. The solid product
was filtered and recrystallized in ethanol to obtain corre-
sponding (E)-substitutedacrylic acids 2.
Western blot analysis
At the end of treatments, cells were collected with ice-cold
radioimmunoprecipitation (RIPA) buffer (Beyotime, China).
Then samples were homogenized and centrifuged at
13,000×g for 20 min at 4 °C to remove insoluble material.
Protein concentration was determined using BCA Protein
Assay kit (Beyotime, China) and the absorbance was mea-
sured using a microplate reader with a wavelength of 562 nm.
For western blot analysis (Li et al. 2014), equal amounts
of protein (30 μg) were separated by electrophoresis on 10%
sodium dodecyl sulfate polyacrylamide gels and transferred
onto nitrocellulose membranes. These membranes were
incubated with 5% (w/v) non-fat milk powder in Tris-
buffered saline containing 0.1% (v/v) Tween-20 (TBST) for
General procedures for the synthesis of substituted benzyl
bromides (4a–d)
A mixture of corresponding substituted toluenes 3 (70 mmol),
N-bromosuccinimide (NBS, 70 mmol), and azobisisobutyr-
onitrile (AIBN, 7 mmol) in 108 mL carbon tetrachloride was
refluxed under irradiation conditions for 1 h. 108 mL cyclo-
hexane was added to the mixture and the reaction was stirred
for 10 min. After removal of precipitate by filtration, the fil-
trate was concentrated and recrystallized in ethanol to obtain
corresponding substituted benzyl bromides 4.
1
h to block nonspecific binding sites. The membranes were
then incubated overnight at 4 °C with the primary anti-
bodies against caspase-3 (1:1000) (Cell Signaling

Medicinal Chemistry Research
General procedures for the synthesis of substituted N-
benzylpiperazines (5a–d)
= 15.5 Hz, CO‒CH=), 5.99 (2H, s, O‒CH ‒O), 3.65 (4H,
2
m, piperazine ring-H), 3.50 (2H, s, ArCH ), 2.46 (4H, bs,
2
1
3
piperazine ring-H); C NMR (CDCl , 75 MHz): δ = 165.5
3
To a stirring solution of N-Boc-piperazine (2 mmol) in
(C, C=O), 149.0 (C, Ar–C), 148.2 (C, Ar–C), 142.6 (CH,
Ar‒CH=), 136.2 (C, Ar–C), 133.1 (CH, Ar–C), 130.3 (C,
Ar–C), 129.7 (C, Ar–C), 128.5 (CH, Ar–C), 123.7 (CH,
Ar–C), 115.0 (CH, CO‒CH=), 108.5 (CH, Ar–C), 106.3
(CH, Ar–C), 101.4 (CH , O‒CH ‒O), 62.0 (CH , ArCH ),
CH Cl (20 mL) was sequentially added substituted benzyl
2
2
bromides 4 (1.6 mmol) and K CO (7.2 mmol). The mixture
2
3
was heated to reflux. After 10 h, the mixture was diluted
with EtOAc and poured onto water. The organic layer was
washed once with brine, dried over Na SO , and con-
2
2
2
2
53.2 (CH , piperazine ring-C), 52.8 (CH , piperazine ring-
2
4
2
2
centrated in vacuo. The crude oil was taken up in CH Cl /
C), 45.8 (CH , piperazine ring-C), 42.1 (CH , piperazine
2 2
2
2
TFA (1:1 v/v, 6 mL total). After the mixture was stirred for
h, the crude was concentrated in vacuo to obtain sub-
ring-C); HRESIMS m/z (pos): 385.1322 C H ClN O
21 22 2 3
(calcd. 385.1319).
2
stituted N-benzylpiperazines
purification.
5
and used without
(E)-1-(4-(4-ethoxybenzyl)piperazin-1-yl)-3-(benzo[d][1,3]
dioxol-5-yl)prop-2-en-1-one (6c) Yellow oil; H NMR
1
General procedures for the synthesis of substituted
cinnamoyl benzylpiperazines (6a–l)
(CDCl , 500 MHz): δ = 7.56 (1H, d, J = 15.5 Hz, Ar‒
3
CH=), 6.78 (7H, m, Ar‒H), 6.66 (1H, d, J = 15.5 Hz, CO‒
CH=), 5.98 (2H, s, O‒CH ‒O), 4.01 (2H, q, J = 7.0 Hz,
2
To a stirred solution of corresponding (E)-substitutedacrylic
acids 2 (4 mmol) in 15 mL dichloromethane were added
thionyl chloride (2 mL) and piperadine (1 drop). The reac-
tion mixture was stirred for 6 h at room temperature. After
completion, the solvent was removed under reduced pres-
sure. The residue was dissolved in acetone (15 mL) and
reacted with the solution of corresponding N-(sub-
stitutedbenzyl)piperazines 5 (6 mmol) and triethylamine
CH , OCH CH ), 3.64 (4H, m, piperazine ring-H), 3.49
2
2
3
(2H, s, ArCH ), 2.47 (4H, bs, piperazine ring-H), 1.38 (3H,
2
1
3
t, J = 7.0 Hz, CH , OCH CH ); C NMR (CDCl , 75
3
2
3
3
MHz): δ = 165.5 (C, C=O), 158.3 (C, Ar–C), 148.9 (C,
Ar–C), 148.1 (C, Ar–C), 142.4 (CH, Ar‒CH=), 130.2 (CH,
Ar–C), 129.7 (C, Ar–C), 129.2 (C, Ar–C), 123.6 (CH,
Ar–C), 115.0 (CH, CO‒CH=), 114.2 (CH, Ar–C), 108.4
(CH, Ar–C), 106.3 (CH, Ar–C), 101.3 (CH , O‒CH ‒O),
2
2
(
1
5 mL) in dichloromethane (30 mL). After being stirred for
2 h at room temperature, the solvent was removed under
63.3 (CH , ArCH ), 62.1 (CH , OCH CH ), 53.0 (CH ,
2 2 2 2 3 2
piperazine ring-C), 52.5 (CH , piperazine ring-C), 45.7
2
reduced pressure. The residue was purified by silica gel
(CH , piperazine ring-C), 42.1 (CH , piperazine ring-C),
2
2
column chromatography using ethyl acetate/hexane
14.7 (CH , OCH CH ); HRESIMS m/z (pos): 395.1974
3 2 3
(
2:1 v/v) as eluent to afford target compounds.
C H N O (calcd. 395.1971).
23 27 2 4
(
E)-1-(4-(4-bromobenzyl)piperazin-1-yl)-3-(benzo[d][1,3]
(E)-1-(4-(2-ethoxybenzyl)piperazin-1-yl)-3-(benzo[d][1,3]
dioxol-5-yl)prop-2-en-1-one (6d) Yellow oil; H NMR
1
dioxol-5-yl)prop-2-en-1-one (6a) Yellow solid; mp 124.6‒
1
=
=
1
26.5 °C; H NMR (CDCl , 500 MHz): δ = 7.56 (1H, d, J
(CDCl , 500 MHz): δ = 7.56 (1H, d, J = 15.5 Hz, Ar‒
3
3
15.0 Hz, Ar‒CH=), 6.78 (7H, m, Ar‒H), 6.66 (1H, d, J
CH=), 6.78 (7H, m, Ar‒H), 6.67 (1H, d, J = 15.5 Hz, CO‒
15.0 Hz, CO‒CH=), 5.98 (2H, s, O‒CH ‒O), 3.65 (4H,
CH=), 5.98 (2H, s, O‒CH ‒O), 4.02 (2H, q, J = 7.0 Hz,
2
2
m, piperazine ring-H), 3.49 (2H, s, ArCH ), 2.47 (4H, bs,
CH , OCH CH ), 3.75 (4H, m, piperazine ring-H), 3.65
2
2
2
3
piperazine ring-H); 13C NMR (CDCl , 75 MHz): δ = 165.5
(2H, s, ArCH ), 2.56 (4H, bs, piperazine ring-H), 1.40 (3H,
2
3
1
3
(
C, C=O), 149.0 (C, Ar–C), 148.2 (C, Ar–C), 142.6 (CH,
t, J = 7.0 Hz, CH , OCH CH ); C NMR (CDCl , 75
3 2 3 3
Ar‒CH=), 136.7 (C, Ar–C), 131.5 (CH, Ar–C), 130.7 (C,
Ar–C), 129.7 (C, Ar–C), 123.7 (CH, Ar–C), 121.1 (CH,
Ar–C), 115.0 (CH, CO‒CH=), 108.5 (CH, Ar–C), 106.3
MHz): δ = 165.4 (C, C=O), 157.2 (C, Ar–C), 148.9 (C,
Ar–C), 148.1 (C, Ar–C), 142.3 (CH, Ar‒CH=), 130.6 (CH,
Ar–C), 129.7 (C, Ar–C), 129.1 (C, Ar–C), 128.2 (CH,
Ar–C), 123.6 (CH, Ar–C), 120.2 (CH, Ar–C), 115.1 (CH,
CO‒CH=), 111.5 (CH, Ar–C), 108.4 (CH, Ar–C), 106.3
(
CH, Ar–C), 101.4 (CH , O‒CH ‒O), 62.1 (CH , ArCH ),
2
2
2
2
5
3.2 (CH , piperazine ring-C), 52.8 (CH , piperazine ring-
2 2
C), 45.7 (CH , piperazine ring-C), 42.1 (CH , piperazine
(CH, Ar–C), 101.3 (CH2, O‒CH ‒O), 63.6 (CH2,
2
2
2
ring-C); HRESIMS m/z (pos): 429.0817 C H BrN O
OCH CH ), 55.7 (CH , ArCH ), 53.1 (CH , piperazine
21
22
2
3
2
3
2
2
2
(
calcd. 429.0814).
ring-C), 52.6 (CH , piperazine ring-C), 45.8 (CH , piper-
2 2
azine ring-C), 42.2 (CH , piperazine ring-C), 14.9 (CH ,
2
3
(
E)-1-(4-(4-chlorobenzyl)piperazin-1-yl)-3-(benzo[d][1,3]
OCH CH ); HRESIMS m/z (pos): 395.1975 C H N O
2 3 23 27 2 4
dioxol-5-yl)prop-2-en-1-one (6b) White solid; mp 125.8‒
(calcd. 395.1971).
1
1
28.4 °C; H NMR (CDCl , 500 MHz): δ = 7.57 (1H, d, J
3
=
15.5 Hz, Ar‒CH=), 6.79 (7H, m, Ar‒H), 6.66 (1H, d, J

Medicinal Chemistry Research
(
E)-1-(4-(4-ethoxybenzyl)piperazin-1-yl)-3-(4-bromophenyl)
NMR (CDCl , 500 MHz): δ = 7.58 (1H, d, J = 15.5 Hz,
3
prop-2-en-1-one (6e) Yellow solid; mp 118.9‒119.4 °C;
Ar‒CH=), 7.22 (8H, m, Ar‒H), 6.82 (1H, d, J = 15.5 Hz,
CO‒CH=) 3.64 (4H, m, piperazine ring-H), 3.49 (2H, s,
1
H NMR (CDCl , 500 MHz): δ = 7.57 (1 H, d, J = 15.5 Hz,
3
1
3
Ar‒CH=), 6.85 (8H, m, Ar‒H), 6.82 (1H, d, J = 15.5 Hz,
CO‒CH=), 4.01 (2H, q, J = 7.0 Hz, CH , OCH CH ), 3.63
ArCH ), 2.47 (4H, bs, piperazine ring-H); C NMR
2
(CDCl , 75 MHz): δ = 165.1 (C, C=O), 141.5 (CH, Ar‒
2
2
3
3
(
4H, m, piperazine ring-H), 3.48 (2H, s, ArCH ), 2.47 (4H,
CH=), 134.2 (C, Ar–C), 132.0 (CH, Ar–C), 131.5 (C,
Ar–C), 130.7 (C, Ar–C), 129.6 (CH, Ar–C), 129.3 (CH,
Ar–C), 123.7 (C, Ar–C), 121.2 (CH, Ar–C), 117.7 (CH,
CO‒CH=), 62.0 (CH , ArCH ), 53.2 (CH , piperazine ring-
2
bs, piperazine ring-H), 1.40 (3H, t, J = 7.0 Hz, CH ,
3
1
3
OCH CH ); C NMR (CDCl , 75 MHz): δ = 165.0 (C,
2
3
3
C=O), 158.3 (C, Ar–C), 141.3 (CH, Ar‒CH=), 134.2 (C,
Ar–C), 132.0 (CH, Ar–C), 130.3 (C, Ar–C), 129.1 (CH,
Ar–C), 128.3 (CH, Ar–C), 123.6 (C, Ar-C), 117.8 (CH,
CO‒CH=), 114.3 (CH, Ar–C), 63.4 (CH , OCH CH ), 62.2
2
2
2
C), 52.7 (CH , piperazine ring-C), 45.8 (CH , piperazine
2
2
ring-C), 42.2 (CH , piperazine ring-C); HRESIMS m/z
2
(pos): 463.0025 C H Br N O (calcd. 463.0021).
2
2
3
20 21
2
2
(
CH , ArCH ), 53.1 (CH , piperazine ring-C), 52.6 (CH ,
2 2 2 2
piperazine ring-C), 45.8 (CH , piperazine ring-C), 42.2
(E)-1-(4-(4-ethoxybenzyl)piperazin-1-yl)-3-(4-chlorophenyl)
2
1
(
CH , piperazine ring-C), 14.8 (CH , OCH CH ); HRE-
prop-2-en-1-one (6i) White solid; mp 128.8‒129.3 °C; H
2
3
2
3
SIMS m/z (pos): 429.1182 C H BrN O (calcd.
NMR (CDCl , 500 MHz): δ = 7.59 (1H, d, J = 15.5 Hz,
22
26
2
2
3
4
29.1178).
Ar‒CH=), 6.85 (8H, m, Ar‒H), 6.81 (1H, d, J = 15.5 Hz,
CO‒CH=), 4.01 (2H, q, J = 7.0 Hz, CH , OCH CH ), 3.64
2
2
3
(
E)-1-(4-(2-ethoxybenzyl)piperazin-1-yl)-3-(4-bromophenyl)
(4H, m, piperazine ring-H), 3.49 (2H, s, ArCH ), 2.47 (4H,
2
1
prop-2-en-1-one (6f) White solid; mp 80.8‒82.5 °C; H
NMR (CDCl , 500 MHz): δ = 7.57 (1H, d, J = 15.5 Hz,
bs, piperazine ring-H), 1.40 (3H, t, J = 7.0 Hz, CH ,
3
1
3
OCH CH ); C NMR (CDCl , 75 MHz): δ = 165.0 (C,
3
2 3 3
Ar‒CH=), 6.86 (8H, m, Ar‒H), 6.82 (1H, d, J = 15.5 Hz,
CO‒CH=), 4.02 (2H, q, J = 7.0 Hz, CH , OCH CH ), 3.75
C=O), 158.2 (C, Ar–C), 141.1 (CH, Ar‒CH=), 135.2 (C,
Ar–C), 133.8 (C, Ar–C), 130.2 (CH, Ar–C), 129.3 (CH,
Ar–C), 128.9 (CH, Ar–C), 128.8 (C, Ar–C), 117.7 (CH,
CO‒CH=), 114.3 (CH, Ar–C), 63.4 (CH , OCH CH ), 62.2
2
2
3
(
4H, m, piperazine ring-H), 3.68 (2H, s, ArCH ), 2.57 (4H,
2
bs, piperazine ring-H), 1.40 (3H, t, J = 7.0 Hz, CH ,
3
2
2
3
1
3
OCH CH ); C NMR (CDCl , 75 MHz): δ = 165.0 (C,
(CH , ArCH ), 53.2 (CH , piperazine ring-C), 52.6 (CH ,
2 2 2 2
2
3
3
C=O), 157.3 (C, Ar–C), 141.2 (CH, Ar‒CH=), 134.3 (C,
Ar–C), 132.0 (CH, Ar–C), 131.9 (C, Ar–C), 130.7 (CH,
Ar–C), 129.1 (CH, Ar–C), 128.4 (CH, Ar–C), 123.6 (C,
Ar–C), 120.3 (CH, Ar–C), 117.9 (CH, CO‒CH=), 111.6
piperazine ring-C), 45.9 (CH , piperazine ring-C), 42.2
2
(CH , piperazine ring-C), 14.9 (CH , OCH CH ); HRE-
2
3
2
3
SIMS m/z (pos): 385.1687 C H ClN O (calcd.
2
2
26
2
2
385.1683).
(
CH, Ar–C), 63.7 (CH , OCH CH ), 55.8 (CH , ArCH )
2 2 3 2 2
5
3.2 (CH , piperazine ring-C), 52.6 (CH , piperazine ring-
(E)-1-(4-(2-ethoxybenzyl)piperazin-1-yl)-3-(4-chlorophenyl)
2
2
1
C), 45.9 (CH , piperazine ring-C), 42.3 (CH , piperazine
ring-C), 14.9 (CH , OCH CH ); HRESIMS m/z (pos):
prop-2-en-1-one (6j) Brown solid; mp 70.0‒72.8 °C; H
2
2
NMR (CDCl , 500 MHz): δ = 7.59 (1H, d, J = 15.0 Hz,
3
2
3
3
4
29.1182 C H BrN O (calcd. 429.1178).
Ar‒CH=), 6.86 (8H, m, Ar‒H), 6.81 (1H, d, J = 15.5 Hz,
2
2
26
2
2
CO‒CH=), 4.02 (2H, q, J = 7.0 Hz, CH , OCH CH ), 3.77
2
2
3
(
E)-1-(4-(4-chlorobenzyl)piperazin-1-yl)-3-(4-bromophenyl)
(4H, m, piperazine ring-H), 3.68 (2H, s, ArCH ), 2.59 (4H,
2
1
prop-2-en-1-one (6g) White solid; mp 138.0‒138.7 °C; H
NMR (CDCl , 500 MHz): δ = 7.58 (1H, d, J = 15.5 Hz,
bs, piperazine ring-H), 1.40 (3H, t, J = 7.0 Hz, CH ,
3
1
3
OCH CH ); C NMR (CDCl , 75 MHz): δ = 165.0 (C,
3
2 3 3
Ar‒CH=), 7.28 (8H, m, Ar‒H), 6.82 (1H, d, J = 15.5 Hz,
CO‒CH=), 3.65 (4H, m, piperazine ring-H) 3.51 (2H, s,
C=O), 157.3 (C, Ar–C), 141.3 (CH, Ar‒CH=), 135.4 (C,
Ar–C), 133.8 (C, Ar–C), 130.9 (CH, Ar–C), 129.0 (CH,
Ar–C), 128.9 (C, Ar–C), 128.6 (CH, Ar–C), 120.3 (CH,
Ar–C), 117.7 (CH, CO‒CH=), 111.6 (CH, Ar–C), 63.7
(CH , OCH CH ), 55.6 (CH , ArCH ), 53.0 (CH , piper-
13
ArCH ), 2.47 (4H, bs, piperazine ring-H); C NMR
2
(
CDCl , 75 MHz): δ = 165.0 (C, C=O), 141.4 (CH, Ar‒
3
CH=), 134.2 (C, Ar–C), 133.0 (C, Ar–C), 132.0 (CH,
Ar–C), 131.4 (C, Ar–C), 130.2 (CH, Ar–C), 129.2 (CH,
Ar–C), 128.5 (CH, Ar–C), 123.6 (C, Ar–C), 117.8 (CH,
CO‒CH=), 62.0 (CH , ArCH ), 53.2 (CH , piperazine ring-
2
2
3
2
2
2
azine ring-C), 52.4 (CH , piperazine ring-C), 45.7 (CH ,
2
2
piperazine ring-C), 42.1 (CH , piperazine ring-C), 14.9
2
(CH3, OCH CH ); HRESIMS m/z (pos): 385.1686
2
2
2
2
3
C), 52.6 (CH , piperazine ring-C), 45.7 (CH , piperazine
C H ClN O (calcd. 385.1683).
22 26 2 2
2
2
ring-C), 42.1 (CH , piperazine ring-C); HRESIMS m/z
2
(
pos): 419.0530 C H BrClN O (calcd. 419.0526).
(E)-1-(4-(4-chlorobenzyl)piperazin-1-yl)-3-(4-chlorophenyl)
2
0
21
2
1
prop-2-en-1-one (6k) White solid; mp 139.8‒141.3 °C; H
(
E)-1-(4-(4-bromobenzyl)piperazin-1-yl)-3-(4-bromophenyl)
NMR (CDCl , 500 MHz): δ = 7.59 (1H, d, J = 15.5 Hz,
3
1
prop-2-en-1-one (6h) White solid; mp 136.8‒138.3 °C; H
Ar‒CH=), 7.28 (8H, m, Ar‒H), 6.81 (1H, d, J = 15.0 Hz,

Medicinal Chemistry Research
Table 1 Effects of title compounds on survival time of mice subjected to bilateral common carotid artery ligation
Compd Survival time (min)
00 mg/Kg
2
100 mg/Kg
50 mg/Kg
25 mg/Kg
12.5 mg/Kg
6.25 mg/Kg
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
16.38 ± 1.79*
16.22 ± 2.49*
15.88 ± 2.01*
10.04 ± 2.27*
10.54 ± 2.80*
6.46 ± 1.03*
6.56 ± 1.43*
12.31 ± 1.69*
7.58 ± 1.33*
8.84 ± 2.03*
6.90 ± 2.62*
2.94 ± 1.81
14.92 ± 2.58*
11.85 ± 2.34*
16.47 ± 1.95*
5.14 ± 0.30*
4.28 ± 1.12*
5.14 ± 1.30*
5.06 ± 2.18*
6.18 ± 2.22*
11.03 ± 1.74*
3.19 ± 0.48*
4.14 ± 1.07*
4.37 ± 0.88*
5.97 ± 1.35*
5.96 ± 1.15*
6.92 ± 1.22*
2.51 ± 1.02
2.31 ± 1.28
1.17 ± 0.30
3.00 ± 1.19
8.54 ± 2.45*
6.16 ± 1.81*
1.02 ± 0.44
2.68 ± 1.44
2.26 ± 1.37
5.47 ± 1.26*
6.37 ± 1.32*
5.83 ± 1.54*
NE
3.90 ± 1.29*
2.73 ± 1.66
5.34 ± 1.78*
4.31 ± 1.70*
4.01 ± 0.44*
3.90 ± 1.51*
NE
NE
NE
NE
NE
NE
NE
NE
g
h
i
NE
NE
NE
2.08 ± 0.67
4.27 ± 1.93*
NE
NE
NE
3.06 ± 1.27
NE
1.14 ± 0.30
NE
j
k
l
NE
NE
NE
NE
NE
NE
NS
2.04 ± 0.61
3.52 ± 1.04*,a
Nimodipine
Values are in mean ± SEM; number of animals in each group = 10
NE not evaluated
*
p < 0.05 versus NS
aNimodipine 80 mg/Kg
CO‒CH=), 3.64 (4H, m, piperazine ring-H), 3.50 (2H, s,
As shown in Table 1, most of the target compounds were
active in mice i.p. at different doses, among which com-
pounds 6a, 6b, 6c, and 6i at all doses significantly pro-
longed the survival time of mice subjected to acute cerebral
ischemia and showed potent neuroprotective effects. Com-
pound 6h showed activities at high and median dose.
Compounds 6d, 6e, 6f, 6g, 6j, and 6k only presented pro-
tection at high dose while showed no effect at low or
median dose.
13
ArCH ), 2.47 (4H, bs, piperazine ring-H); C NMR
2
(
CDCl , 75 MHz): δ = 165.1 (C, C=O), 141.4 (CH, Ar‒
3
CH=), 135.4 (C, Ar–C), 133.8 (C, Ar–C), 130.4 (C, Ar–C),
1
29.2 (CH, Ar–C), 129.0 (CH, Ar–C), 128.9 (CH, Ar–C),
1
28.5 (CH, Ar–C) 117.6 (CH, CO‒CH=), 62.0 (CH2,
ArCH ), 53.2 (CH , piperazine ring-C), 52.7 (CH , piper-
2
2
2
azine ring-C), 45.8 (CH , piperazine ring-C), 42.1 (CH ,
2
2
piperazine ring-C); HRESIMS m/z (pos): 375.1034
C H Cl N O (calcd. 375.1031).
In order to study the potential neuroprotective activity of
the title compounds, another screening was performed
investigating neuroprotection on impairment induced by
glutamine (Glu) in PC12 cells, as evaluated by MTT assay.
We damaged the PC12 cells with 10 mM L-glutamate and
assessed the protective actions of twelve cinnamide deri-
vatives. We found that most of the new compounds
exhibited moderate to good protection against Glu-induced
cell damage (Table 2).
As shown in Table 2, 6a, 6b, 6c, 6d, 6e, 6f, and 6i
showed good neuroprotective activity in glutamate-induced
PC12 cells at all doses tested (protection >20%). It was
found that the cumulative addition of the compounds 6a, 6b
and 6c (0.1–10 µM) caused concentration-dependent neu-
roprotective effects with the maximal effect observed at 10
µM. Compounds 6f and 6i showed a pattern of increased
protection with increasing concentrations (1.0–10 and
0.1–1.0 µM, respectively) in terms of cell protection.
Compounds 6d, 6e, and 6l were observed to have the
highest protection at the lowest concentrations of 0.1 µM
(cell protection: 61.02, 71.82, and 34.02%, respectively).
Compounds 6f, 6g, 6h, and 6k were observed to have the
2
0
21
2
2
(
E)-1-(4-(4-bromobenzyl)piperazin-1-yl)-3-(4-chlorophenyl)
1
prop-2-en-1-one (6l) White solid; mp 151.7‒153.1 °C; H
NMR (CDCl , 500 MHz): δ = 7.59 (1H, d, J = 15.0 Hz,
3
Ar‒CH=), 7.21 (8H, m, Ar‒H), 6.81 (1H, d, J = 15.5 Hz,
CO‒CH=), 3.65 (4H, m, piperazine ring-H), 3.49 (2H, s,
13
ArCH ), 2.47 (4H, bs, piperazine ring-H); C NMR
2
(
CDCl , 75 MHz): δ = 165.1 (C, C=O), 141.4 (CH, Ar‒
3
CH=), 136.6 (C, Ar–C), 135.4 (C, Ar–C), 133.8 (C, Ar–C),
1
31.5 (C, Ar–C), 130.7 (CH, Ar–C), 129.0 (CH, Ar–C),
1
28.9 (CH, Ar–C), 121.2 (CH, Ar–C), 117.6 (CH, CO‒
CH=), 62.1 (CH , ArCH ), 53.2 (CH , piperazine ring-C),
2
2
2
5
2.7 (CH , piperazine ring-C), 45.8 (CH , piperazine ring-
2
2
C), 42.2 (CH , piperazine ring-C); HRESIMS m/z (pos):
2
4
19.0530 C H BrClN O (calcd. 419.0526).
20 21 2
Biological evaluation
Firstly, the target compounds were preliminary screened in
mice underwent bilateral common carotid artery occlusion
to study effects of compounds on acute cerebral ischemia.

Medicinal Chemistry Research
Table 2 Neuroprotective effects of all target compounds against
Table 3 Effect of some cinnamide on apoptosis of PC12 cells induced
glutamate-induced neurotoxicity in PC12 cells
by glutamate
Compound R1
R3 R2
Cell viability (%)
.1 μM 1.0 μM 10 μM
Compd
Rate of apoptotic cells (%)
0
0.1 μM
1.0 μM
10 μM
1
6
6
6
6
a
b
c
R -phenyl = 1,3-
H
H
H
H
4-Br 32.06 45.34 63.56
4-Cl 32.84 48.02 72.16
4-OEt 37.64 52.99 85.40
2-OEt 61.02 49.95 35.51
Control
19.27 ± 2.00
43.97 ± 2.69a
benzodioxole
Glutamate
1
R -phenyl = 1,3-
32.11 ± 2.85b
24.42 ± 3.30b
36.44 ± 1.53b
25.42 ± 2.73b
17.34 ± 2.24b
31.95 ± 2.19b
6
6
6
a
b
c
42.82 ± 4.53
30.04 ± 4.11b
43.45 ± 4.00
benzodioxole
1
R -phenyl = 1,3-
benzodioxole
1
aSignificantly different from control group (p < 0.05)
bSignificantly different from glutamate group (p < 0.05)
d
R -phenyl = 1,3-
benzodioxole
6
6
6
6
6
6
6
6
e
f
4-Br
H
H
H
H
H
H
H
H
4-OEt 71.82 62.12 61.17
2-OEt 23.61 25.26 45.01
4-Br
g
h
i
4-Br
4-Cl 8.41
4-Br 7.87
9.46
8.70
52.64
45.61
4-Br
4-Cl
4-OEt 33.68 42.54 35.71
2-OEt 14.62 17.09 19.44
j
4-Cl
k
l
4-Cl
4-Cl 7.92
15.19 22.92
4-Cl
4-Br 34.02 18.54 14.83
71.01 49.66 77.75
33.01a
NY-308
Edaravone
Fig. 2 Caspase-3 activity in PC12 cells pretreated with cinnamide
derivatives
aEdaravone-90 μM
Apoptosis after cerebral ischemia is one of the major
pathways that leads to the process of cell death (Meng et al.
2011). Therefore, we investigated the anti-apoptotic effects
of the compounds 6a, 6b and 6c by Hoechst 33342/PI
double staining assay using high content screening system.
Quantitative analysis of the stained cells indicated that
pretreatment of the cells with 6a, 6b, or 6c has significantly
decreased the extent of cell apoptosis in a dose-dependent
manner. As shown in Table 3, after glutamate insult for
24 h, 43.97% of cultured cells showed typical character-
istics of apoptosis. However, under pretreatments of 6a, 6b,
and 6c at three doses (0.1, 1.0, and 10 µM respectively), the
apoptotic percentages were significantly reduced (Table 3).
The above results suggest that 6a, 6b, and 6c are capable of
rescuing PC12 cells from glutamate-induced apoptotic
death.
Another important finding, which confirmed the protec-
tive effect of these derivatives, was the data obtained from
western blot analysis of procaspase-3 and caspase-3. As
shown in Fig. 2, glutamate induced the appearance of
cleaved active caspase-3, showing the involvement of
caspase-3 in glutamate-induced cell death in PC12 neurons.
In the cells pretreated with 6b at three doses (0.1, 1.0, and
10 µM), bands of cleaved (active) caspase-3 were weaker
compared to glutamate-treated cells, which illustrated that
6b significantly inhibited the expression of caspase-3
induced by glutamate in a dose-dependent manner. The
procaspase-3 level was not depressed significantly by
highest protection at the highest concentrations of 10 µM
cell protection: 45.01, 52.64, 45.61, and 22.92%,
respectively).
(
Derivatives were substituted with different functional
groups to study of the substituent variability influence on
the biological activity and find new compounds with better
neuroprotective activity. The electronic nature of the sub-
stituent group attached to the aromatic ring B led to a sig-
nificant variation in neuroprotective activity. From the data
shown above, it is clear that the presence of electron
donating group at 4th position on aromatic ring B enhanced
the activity when compared to electron withdrawing group
(
cell protection: 6c > 6b, 6c > 6a, 6e > 6g, 6e > 6h). On the
other hand, for the different isoelectronic halogens, the
lipophilic chloro substituent at the 4-position of B ring was
more potent than the corresponding bromo-substituted
derivatives (cell protection: 6g > 6h, 6b > 6a).
It was worthwhile to note that the introduction of 1,3-
benzodioxole exerted more neuroprotective effect, such as
6
a, 6b, 6c, and 6d, which indicated that larger hydrophobic
groups might be favorable for increased neuroprotection.
For further investigation, apoptosis assays and western
blot analysis were performed to determine their mechanism
of action, at three concentration of 0.1, 1.0, and 10 µM,
against glutamate-induced cell death. According to the
in vitro and in vivo biological activities, the compounds 6a,
6
b, and 6c were selected and further studied.

Medicinal Chemistry Research
compounds 6a, 6b, and 6c, while, 6a and 6c significantly
inhibited the expression of caspase-3 at high doses. The
above results demonstrated these compounds inhibited
apoptosis of glutamate-induced PC12 cells via caspase-3
pathway.
inhibitors of endocannabinoid hydrolases.
3:1830–1842
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glutamate-induced cytotoxicity in PC 12 cells via inhibition of
(
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evaluation of pyrrolidine derivatives as novel and potent sodium
channel blockers for the treatment of ischemic stroke. Bioorg
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Meng Y, Guo YB, Ling Y, Zhao YH, Zhang Q, Zhou XY, Ding F,
Yang YM (2011) Synthesis and protective effects of aralkyl
alcoholic 2-acetamido-2-deoxy-β-d-pyranosides on hypoglyce-
mia and serum limitation induced apoptosis in PC12 cell. Bioorg
Med Chem 19:5577–5584
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
Park SJ, Nam KW, Lee HJ, Cho EY, Koo U, Mar W (2009) Neuro-
protective effects of an alkaloid-free ethyl acetate extract from the
root of Sophora flavescens Ait. against focal cerebral ischemia in
rats. Phytomedicine 16:1042–1051
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selective synthesis of quinoline deravatives. Tetrahedron
In summary, twelve novel cinnamide analogs (6a–l) were
designed and synthesized. Most target compounds showed
neuroprotective activity both in vivo and in vitro, among
which 6a, 6b, and 6c, featuring a 1,3-benzodioxole moiety,
exhibited the significant potency. Further studies showed
that analogs 6a, 6b, and 6c efficiently inhibited glutamate
induced cell apoptosis via caspase-3 pathway. These find-
ings may provide insight for our future design of cinnamide
analogs with optimal neuroprotective activities. Cinnamide
compounds might be useful as agents in the treatment of
stroke and other neurological insults.
6
4:10068–10074
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Acknowledgements The authors are grateful to the support from the
National Natural Science Foundation of China (No. 81371451) and the
Natural Science Foundation of Jiangsu Province (No. BK20131390).
Compliance with ethical standards
3
1:2810–2819
Wang CJ, Hu CP, Xu KP, Yuan Q, Li FS, Zou H, Tan GS, Li YJ
2010) Protective effect of selaginellin on glutamate-induced
Conflict of interest The authors declare that they have no conflict of
interest.
(
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