Synthesis, crystal structure, Hirshfeld surfaces analysis and anti-ischemic activity of cinnamide derivatives

Article abstract of DOI:10.1016/j.molstruc.2017.09.110

Two cinnamide derivatives, namely, (E)-1-(4-(bis(4-methylphenyl)- methyl)piperazin-1-yl)-3-(3,4-diethoxyphenyl)prop-2-en-1-one (5) and (E)-1-(4-(bis- (4-fluorophenyl)methyl)piperazin-1-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (6), have been synthesized and characterized by IR spectra, High resolution mass spectra, ¹H NMR spectra, ¹³C NMR spectra. The compound 5 is a novel compound and has never been reported in the literature. Their crystal structures were studied by single-crystal X-ray diffraction. They all crystallize in the monoclinic system. The single-crystal X-ray revealed that compound 5 has infinite X-shaped 1-D polymeric chains structure and compound 6 has a layered 3-D structure by intermolecular interactions. Hirshfeld surface analysis demonstrated the presence of H?H, O?H, C?H, F?H, C[sbnd]H?π and π?π intermolecular interactions. In addition, the MTT assay results indicated that the compounds 5 and 6 display effective activities against neurotoxicity which is induced by glutamine in PC12 cells. The in vivo experiment indicated that the compound 6 has a good protective effect on cerebral infarction.

Full text of DOI:10.1016/j.molstruc.2017.09.110

Accepted Manuscript
Synthesis, crystal structure, Hirshfeld surfaces analysis and anti-ischemic activity of
cinnamide derivatives
Jian-gang Zhong, Jia-pei Han, Xiao-feng Li, Yi Xu, Yan Zhong, Bin Wu
PII:
S0022-2860(17)31321-2
10.1016/j.molstruc.2017.09.110
MOLSTR 24359
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 June 2017
Revised Date: 27 September 2017
Accepted Date: 27 September 2017
Please cite this article as: J.-g. Zhong, J.-p. Han, X.-f. Li, Y. Xu, Y. Zhong, B. Wu, Synthesis, crystal
structure, Hirshfeld surfaces analysis and anti-ischemic activity of cinnamide derivatives, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.09.110.
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Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis, crystal structure, Hirshfeld surfaces analysis and
anti-ischemic activity of cinnamide derivatives
1
1
2
2
1,
2,
Jian-gang Zhong , Jia-pei Han , Xiao-feng Li , Yi Xu , Yan Zhong *, Bin Wu *
1
School of Chemistry and Chemical Engineering, Southeast University, Nanjing
2
11189, China
School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
Corresponding author. Tel/fax: +86 025 5209 0619.
E-mail: 101009944@seu.edu.cn (Y. Zhong); wubin@njmu.edu.cn (B. Wu).
Abstract
Two
methyl)piperazin-1-yl)-3-(3,4-diethoxyphenyl)prop-2-en-1-one (5) and (E)-1-(4-(bis-
4-fluorophenyl)methyl)piperazin-1-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (6),
have been synthesized and characterized by IR spectra, High resolution mass spectra,
2
*
cinnamide
derivatives,
namely,
(E)-1-(4-(bis(4-methylphenyl)-
(
1
13
H NMR spectra, C NMR spectra. The compound 5 is a novel compound and has
never been reported in the literature. Their crystal structures were studied by
single-crystal X-ray diffraction. They all crystallize in the monoclinic system. The
single-crystal X-ray revealed that compound 5 has infinite X-shaped 1-D polymeric
chains structure and compound 6 has a layered 3-D structure by intermolecular
interactions. Hirshfeld surface analysis demonstrated the presence of HꢀH, OꢀH,
CꢀH, FꢀH, C-Hꢀπ and πꢀπ intermolecular interactions. In addition, the MTT
assay results indicated that the compounds 5 and 6 display effective activities against
neurotoxicity which is induced by glutamine in PC12 cells. The in vivo experiment

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indicated that the compound 6 has a good protective effect on cerebral infarction.
Keywords: Cinnamide derivative; Crystal structure; Hirshfeld surface; MTT assay;
Anti-ischemic activity
1
. Introduction
Stroke, commonly known as cerebrovascular accident, is caused by
cerebrovascular obstruction or rupture which will lead to brain blood circulation
disorders and brain tissue function and structure damages. Stroke is the second
primary reason of mortality and becomes the most common reason of long-term
disability in the world [1-5]. There are two main types of stroke: ischemic, because of
being short of blood flowing into the cerebrum, and hemorrhagic, because of bleeding
into the cerebrum or into the ventricular system [6]. Especially, in China, ischemic
stroke accounts for about 70% of total strokes [7-9]. Ischemic stroke mainly includes
thrombotic cerebral infarction (cerebral thrombosis), embolic cerebral infarction,
lacunar infarction, multiple cerebral infarction and transient ischemic attack [10,11].
The pathogenesis of ischemic stroke involves complex time and spatial cascade
reactions that can initiate many links in the disease. Although the pathogenesis is not
clear, scientists generally believe that activation of glutamate receptors which leads to
increasing intracellular calcium concentration is the main cause of death in the infarct
cells [8,12].
So far, the current methods of treating ischemic stroke are six major categories:
antiplatelet therapy, thrombolytic therapy, anticoagulant therapy, neuroprotective
therapy, traditional Chinese medicine treatment and other drug therapy. Among them,

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traditional Chinese medicine treatment and other drug treatment are not obvious.
Thrombolytic therapy can make the blood vessels recanalization, but it also produces
reperfusion injury [13]. Hundreds of neuroprotective agents have been developed for
stroke. Almost all of the neuroprotective agents are effective in animal experiments,
however, the clinical effect is poor even ineffective or the serious side effects limit its
clinical application. Therefore, the search for new neuroprotective agents has become
the focus of current research.
Natural products are biologically interesting molecules that play an important
role in modern drug discovery [14]. S.R. Kim et al. obtained some natural products by
extracting dried roots of Scrophularia buergeriana [15]. Studies have shown that these
compounds exhibit strong neuroprotective activity. Then the structure-activity
relationship of these compounds indicated that the (E)-p-methoxycinnamoyl structure
has neuroprotective activity [16]. Some literature reported that substituted
benzhydrylpiperazine structures also have antioxidant neuroprotective activity [17].
Based on the above information, we used the splicing principle to incorporate
cinnamoyl moiety and benzhydrylpiperazine structure into the same molecule, and
found that cinnamide scaffold often affords neuroprotective compounds. Especially
(E)-1-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-3-(4-methoxyphenyl)prop-2-en-
1
-one (compound 6 in this paper), which has the (E)-p-methoxycinnamoyl moiety,
exhibited good neuroprotection in vitro [18]. In subsequent work, we selected 6 as
lead compound, and designed and synthesized several novel series of cinnamide
derivatives substituted with different functional groups to study of the substituent

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variability influence on the biological activity. Compound 6 showed good
neuroprotective activity at three test concentrations (0.1, 1.0, 10 µM) (protection >
4
0%) and exhibited better neuroprotection than positive control Edaravone.
In this study, compound 5 and 6 were synthesized and characterized by using
different spectroscopic methods. The in vitro bioassay activity of compounds 5 and 6
was studied by MTT assay. Moreover, compound 6 was conducted on an animal
experiment for further research.
2
. Experimental section
2
.1. General procedures
All chemicals were of analytical grade and used without further purification.
1
Melting point was determined on a WRR-401 apparatus and was uncorrected. The H
1
3
and C NMR spectra were recorded on a Bruker AV-500 instrument (CDCl solution;
3
TMS internal standard). High resolution mass spectra (HRMS) were obtained on a
MALDI Micro MX instrument. IR was obtained on Bruker Tensor 27 FT-IR with KBr
pellets. Fetal bovine serum (FBS) was got from HyClone (Logan, Utah, USA).
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was got from
Sigma Chemical Company (Saint Louis, Missouri, USA).
2
2
.2. Synthesis
.2.1. Synthesis of (E)-1-(4-(bis(4-methylphenyl)methyl)piperazin-1-yl)-3-
(3,4-diethoxyphenyl)prop-2-en-1-one 5
The synthesis step of the target compound is shown in Scheme 1. The substituted
1
-benzhydrylpiperazine 2 was prepared from substituted benzophenone 1 according to

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the reference [19]. The substituted cinnamic acid 4 was synthesized from substituted
benzaldehydes 3 by the Knoevenagel reactions [20]. The detailed synthesis step of the
target compound is shown below.
A stirred solution of compound (4, 5 mmol) in dichloromethane (15 mL) was
treated dropwise with oxalyl dichloride (2.5 mL) and N,N-dimethylformamide (2
drop), continuing to stir at room temperature for 1 hour. After completion of the
reaction, the solvent was evaporated under reduced pressure. The compound (2, 6
mmol) and triethylamine (3 mL) were placed in tetrahydrofuran (30mL) and the
residue that was dissolved in tetrahydrofuran (15 mL) of the previous step was added
dropwise at 0 � . Then the reaction solution was stirred overnight at ambient
temperature. The solution was removed under reduced pressure for a small volume
after completion of the reaction and the residue was purified by column
chromatography (66% EtOAc in petroleum ether) to yield the white solid compound 5
1
(
1.58 g, 63.2%). m.p.: 131~133 � . H NMR (CDCl ) δ: 1.42~1.48 (m, 6H,
3
2
4
1
CH CH O), 2.26~2.29 (s, 6H, 2CH -Ph), 2.40~2.43 (m, 4H, N(CH ) ), 3.65~3.72 (m,
3 2 3 2 2
H, CON(CH ) ), 4.05~4.13 (m, 4H, 2CH CH O), 4.19 (s, 1H, CH(Ph) ), 6.64 (d, J =
2
2
3
2
2
5.3 Hz, 1H, COCH=), 6.81~7.30 (m, 11H, Ar-H), 7.55 (d, J = 15.3 Hz, 1H, PhCH=).
1
3
C NMR (CDCl ) δ: 14.72, 21.16, 42.96, 45.99, 64.67, 75.25, 112.36, 112.95, 114.70,
3
1
21.69, 127.63, 128.24, 129.16, 136.54, 139.31, 142.49, 148.69, 150.31, 165.47. IR
-1
(
KBr, cm ): 3060.2, 3018.7, 2980.7, 2814.2, 1637.9, 1588.5, 1512.7, 1458.7, 1429.5,
1
266.5, 1234.0, 1138.4, 1043.2, 799.0. HRMS (ESI, m/z): Calcd. for C H N O Na
32
38
2
3
+
[
M + Na ] 521.2780. Found: 521.2768.

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2
.2.2. Synthesis of (E)-1-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-3-
(4-methoxyphenyl)prop-2-en-1-one 6
In similar procedure, the target compound 6 was obtained in a 56% yield as a
1
white solid. m.p.: 170ꢁ172 ℃. H NMR (CDCl ) δ: 2.38~2.41 (m, 4H, N(CH ) ),
3
2 2
3
1
.68 (br, 4H, CON(CH ) ), 3.82 (s, 3H, OCH ), 4.25 (s, 1H, CH(Ph) ), 6.67 (d, J =
2 2 3 2
5.3 Hz, 1H, COCH=), 6.86~7.45 (m, 12H, Ar-H), 7.60 (d, J = 15.3 Hz, 1H, PhCH=).
1
3
C NMR (CDCl ) δ: 42.20, 45.81, 45.90ꢂ51.53ꢂ51.59ꢂ51.90ꢂ51.94ꢂ55.25, 74.19,
3
1
1
1
14.15, 114.42, 115.34ꢂ115.62ꢂ127.94, 129.13, 129.19ꢂ129.23, 137.59, 142.38,
-1
60.23, 160.79ꢂ163.49, 165.56. IR (KBr, cm ): 3075.5, 3028.0, 2969.7, 2821.3,
647.4, 1600.8, 1573.1, 1510.4, 1449.5, 1279.6, 1221.2, 1173.8 1003.4, 823.7. HRMS
+
(
ESI, m/z): Calcd. for C H F N O Na [M + Na ] 471.1860. Found: 471.1816.
2
7
26
2
2
2
H
N
O
O
1
(
(
(
1) NaBH
2) SOCl /CH Cl
2
3) piperazine/cyclohexane
4
/EtOH
R
1
R
3
N
N
2
2
N
R
2
R3
R3
R3
R3
2
(
1) (COCl)
2 2 2
/CH Cl
O
(2) Et N/THF
3
R1
CHO
R1
R3
COOH (1) pyridine/piperidine
COOH
OH
+
5 R =R =CH CH O,R =CH
1
2
3
2
3
=F
3
R
2
R2
3
4
6 R
1
=H,R
2
=CH
3
O,R
3
Scheme 1. Synthesis of the title cinnamide compounds 5 and 6.
2
.3. Crystal structure determination
Suitable crystals were obtained by slow evaporation in ethanol (compound 5) or
a mixture of chloroform and cyclohexane (compound 6) solution at room temperature.
The single-crystal X-ray diffraction data of title compounds 5 and 6 were collected at
2
93(2)K with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) on an
Enraf-Nonius CAD4/PC four-circle diffractometer, using the ω-scan mode [21]. The
2
structure was decided by using SHELXS-97 and refined against F by full-matrix

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least-squares method [22]. Hydrogen atoms were generated geometrically. The
Diamond [23] and Mercury programs [24] were used to describe the molecular
structures. The crystal data and parameters of the crystal structure refinement statistics
were summarized in Table 1.
Table 1 The crystal data and structure refinement for compounds 5 and 6.
Compound
Formula
Formula weight
Crystal system
Space group
a/Å
5
6
C H N O
C H F N O
27 26 2
3
2
38
2
3
2
2
498.64
448.50
Monoclinic
Monoclinic
P2 /c
P2 /n
1
1
13.592(3)
21.263(4)
9.939(2)
90.63(3)
2872.3(10)
4
10.171(2)
16.280(3)
14.053(3)
97.12(3)
2309.0(8)
4
b/Å
c/Å
β/°
3
V/Å
Z
-3
Dcalc (mg m )
1.153
1.290
T/K
293(2)
293(2)
-
1
ꢀ
(mm )
0.074
0.092
Cryst dimensions
No. of reflections
collected
0.3×0.2×0.1
5268
0.3×0.2×0.2
4247
No. of unique
reflections
2485
2946
No. of parameters
335
298
2
Goodness-of-fit (GOF) on F
1.003
1.006
R ,wR (I > 2σ(I))
0.0683, 0.1569
0.1610, 0.1996
867525
0.0542, 0.1460
0.0827, 0.1661
806575
1
2
R , wR (all data)
1
2
CCDC NO.
2
.4. Pharmacology
2
.4.1. In vitro experiment
In order to investigate the in vitro anti-ischemic activity, the title compounds 5
and 6 were screened in Glu-induced PC12 cells. The cellular viability was assessed
by MTT assay [25-28].
PC12 cells were vaccinated in 96-well plates (104 cells/pore). Dulbecco’s

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modified Eagle’s Medium (DMEM) containing 10% FBS was put into the cell
incubator under the condition of 5% CO . When the degree of cell fusion reached
2
about 60% under the microscope after 24 hours of cultivating, the culture medium
was removed. Glutamine at a concentration of 10 mmol/mL was incubated in
serum-free DMEM at 37 ꢃ for 1 hour. Then glutamine (10 mmol/L) with
compounds 5 and 6 (0.1, 1, 10 ꢀmol/L) or Edaravone (90 ꢀmol/L) were cultivated in
serum-free DMEM for 24 hours separately. The culture medium was removed. The
MTT (100 ꢀL, 0.5 mg/mL) was added to each culture well at 37 ꢃ. Finally, 100 ꢀL
of DMSO solution was added to each well after 4 hours. The cells were dissolved in
Scroll instrument to read with a microplate reader (the measurement wavelength was
570 nm and the reference wavelength was 630 nm) [29, 30]. Each sample is made of
five holes.
2
.4.2. In vivo experiment
The in vivo anti-ischemic activity of the title compound 6 was tested using
middle cerebral artery occlusion (MCAO) model. Infarction area was assessed by
TTC staining assay [31, 32].
Kunming mice of both sexes were randomly divided into 6 groups (10 mice per
group) which are sham-operated group, MCAO model group, C6/4 group (4 mg/Kg),
C6/12 group (12 mg/Kg), C6/36 group (36 mg/Kg) and Edaravone group (6 mg/Kg)
respectively. Focal cerebral ischemia was induced according to the MCAO method.
Reperfusion was directed by using suture 2 hours after MCAO. After 24 h of
reperfusion, the brain of the rats was isolated to estimate the infarct size.

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Sham-operated groups were performed in the same situation to avoid the influence of
the suture. The brain tissues were stained by TTC, which were photographed
immediately with an HD digital camera, and infarction area was analyzed with
Image-pro Plus Version 5.1.
3
. Result and discussion
3
.1. Chemistry
Substituted benzaldehydes 1 were used as starting materials, which were treated
with malonic acid to afford substituted cinnamic acid 4. Cinnamic acid chlorides,
which were obtained by oxalyl dichloride in dichloromethane at room temperature,
were allowed to react with 2 in the presence of triethylamine to afford the target
compounds 5 and 6. The chemical structures of these compounds were confirmed by
spectroscopic methods.
3
.2. Crystal structures of compounds 5 and 6
The single-crystal X-ray diffraction study reveals that compound 5 crystallizes as
colorless block crystals in the monoclinic system in space group P2 /c with Z = 4, the
1
asym-metric unit (ASU) consisting of an entire compound 5 molecular (Fig. 1a). The
title molecule exists in an E configuration with respect to the C21=C22 ethene bond
(distance of 1.317(4) Å). The piperazine ring adopts a chair conformation. The
geometrical parameters for the hydrogen bonds in the two moleculars are summarized
in Table 2.
The crystal can be described as every two molecules of compound 5 are crossed
together to form a “X” structure (Fig. 1b). The X structures are further stacked by

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C-HꢀO intermolecular interactions, with average distances of 3.358 Å between the
carbonyl oxygen of one molecule and the carbon (C17, C21 and C14) of another in
the adjacent X units, into an interlaced chain structure (Fig. 1c). The plane separation
between the stacked compound 5 molecules in the adjacent X units is 9.939 Å.
Additionly, these molecules are connected in an ꢀX X X X ꢀ fashion by means of
1
-1
1
-1
C-HꢀO (distance of 3.486 Å for the carbonyl group and 3.531 Å for the ethoxy
group) and C-Hꢀπ (distance of 3.571 Å) interactions to form infinite X-shaped 1-D
polymeric chains (Fig. 1d). The two X units in the chains form a head-to-tail type
interaction and every two molecules in the “X” units parallel with each other are
reverse.
Fig. 1 (a) Molecular structure of 5 with atom labeling of the asymmetric unit; (b) two compound 5 molecules are
crossed together to form a “X” structure; (c) The X structures are further stacked by C-HꢀO intermolecular
interactions; (d) infinite X-shaped 1-D polymeric chains.
The single-crystal X-ray diffraction study reveals that compound 6 crystallizes as
colorless block crystals in the monoclinic system in space group P2 /n with Z = 4, the
1

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asym-metric unit (ASU) consisting of an entire compound 6 molecular (Fig. 2a). The
title molecule also has an E configuration and a chair conformation.
The structure can be described as follows: the molecules of compound 6 are
connected by means of C14-H14AꢀF2 (distance of 3.207 Å) interactions to form
infinite 1-D chains structure (Fig. 2b). As shown in Fig. 2c, The 1-D chain structure
formed a 2-D structure through two hydrogen bonds C8-H8ꢀO2 (distance of 3.453 Å)
where the phenyl ring acts as an H donor and the corresponding carbonyl group of the
adjacent molecule acts as an H acceptor. The 2-D structure are further stacked in a
parallel fashion to form a layered 3-D structure by C18-H18ꢀF2 (distance of 3.472
Å) and πꢀπ (distance of 3.672 Å) interactions (Fig. 2d).
Fig. 2 (a) Molecular structure of 6 with atom labeling of the asymmetric unit; (b) 1-D chain
structure of compound 6; (c) perspective view of 2-D structure of compound 6; (d) 3-D pack
structure of compound 6.
Table 2 Geometrical parameters for the hydrogen bonds in the compounds 5 and 6.
D-HꢀA
D-H(Å)
DꢀA(Å)
HꢀA(Å)
ꢄD-HꢀA(°)
compound 5
C4‒H4ꢀO1
0.930
3.486
2.696
143.28

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C17‒H17BꢀO1
C21‒H21ꢀO1
C24‒H24ꢀO1
C14‒H14BꢀO2
compound 6
0.969
0.930
0.930
0.961
3.288
3.281
3.506
3.531
2.365
2.357
2.698
2.647
158.90
171.95
145.81
153.07
C8‒H8ꢀO2
0.930
0.969
0.930
3.453
3.207
3.472
2.587
2.452
2.597
155.12
134.59
156.95
C14‒H14AꢀF2
C18‒H18ꢀF2
3
.3. Hirshfeld surfaces analysis
The intermolecular interactions of compounds 5 and 6 can be visualized by
Hirshfeld surface analysis which is often used to explore molecular crystals [33-35].～
As is shown in Fig. 3, the Hirshfeld surfaces of the two moleculars have been mapped
over dnorm(-0.304 to 1.552, -0.131 to 1.416 Å for moleculars 5 and 6, separately) and
the fingerprint plots of moleculars 5 and 6 are illustrated in Fig. 4. For the compounds
5
and 6, the red regions mainly indicate strong OꢀH and FꢀH interactions and the
green color regions in the 2-D fingerprint plots mainly represent HꢀH, OꢀH, and
FꢀH interactions.
The OꢀH intermolecular interactions look like a “bat”, which contribute 10.1%
and 10.2% to the total Hirshfeld surfaces for compounds 5 and 6. The FꢀH
interactions comprise 16.2% of the total Hirshfeld surfaces, which is the red area on
the dnorm surface for compound 6. The HꢀH interactions have a more significant
contribution to the total Hirshfeld surfaces of compounds 5 and 6, comprising 67.8%
and 44.9% and reflect in the middle of the 2-D fingerprint plots. The “butterfly” are
identified as a consequence of CꢀH interactions, contributing 21.4%, 24.6% to the
total Hirshfeld surfaces for compounds 5 and 6 at the upper right corner of the
fingerprint plots. The CꢀC interactions is belong to πꢀπ interactions and have a

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lower contribution to the two moleculars, of 0.2%, and 2.2% in the total Hirshfeld
surfaces, respectively. The percentage of CꢀO contacts of 5 and 6 is 0.2%, and 0.1%,
respectively. Besides the contacts mentioned above, the presence of FꢀC, FꢀF,
NꢀH and other interactions is summarized in Table 3.
Fig. 3 Hirshfeld surface mapped with dnorm for the compounds 5 and 6.
Fig. 4 The 2D fingerprint plots of the compounds 5 and 6.
Table 3 The percentages of the various interactions contributions to the total Hirshfeld surface
area of the compounds 5 and 6.
Percentage (%)
Interactions
Compound 5
67.8
Compound 6
44.9
Hꢀ
Oꢀ
Cꢀ
Nꢀ
Cꢀ
H
H
H
H
C
10.1
10.2
21.4
24.6
0.2
0.4
0.2
2.2

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C
ꢀ
O
C
H
0.2
—
0.1
0.7
Fꢀ
Fꢀ
—
16.2
0.1
F
ꢀ
F
—
other
0.1
0.6
3
.4. Pharmacological evaluation
3
.4.1. MTT assay
The inhibitory effect of the compounds 5 and 6 on glutamate-induced PC12 cell
injury was performed by MTT assay. As shown in Fig. 5, we can clearly find the
compound 5 has a weak inhibitory effect on glutamate-induced PC12 cells injury in a
dose-dependent manner. Although the dose-effect relationship of the compound 6 is
not obvious, it could significantly inhibit the damage of PC12 cells induced by
glutamate, is stronger than the positive control drug Edaravone. No matter in the
previous work or the new work, the inhibitory effect of the compounds on
glutamate-induced PC12 cell injury was performed by MTT assay and compared to
Edaravone. Cell protection of compound 6 at four test concentrations (0.1, 1.0, 10,
1
00 µM) was 91.2%, 70.4%, 97.4% and 4.8%, respectively (Chinese Chemical
Letters 2008) [18]. Cell protection of compound 6 at three test concentrations (0.1, 1.0,
0 µM) was 71.01%, 49.66%, and 77.75%, respectively. No matter in the previous
1
work or now, this compound showed good neuroprotective activity at three test
concentrations (0.1, 1.0, 10 µM) (protection > 40%) and exhibited better
neuroprotection than Edaravone. Next, we conducted an animal experiment on
compound 6 for further research.

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Fig. 5 The inhibitory effect of the compounds 5 and 6 on glutamate-induced PC12 cell injury, the
cell viability (%) of the compounds 5 and 6 in three dose groups is 10.18% (0.1 ꢀmol/L,5),
1
9.62% (1.0 ꢀmol/L,5), 23.17% (10 ꢀmol/L,5), 71.01% (0.1 ꢀmol/L,6), 49.66% (1.0 ꢀmol/L,6)
and 77.75% (10 ꢀmol/L,6) respectively; the cell viability (%) of the Edaravone is 33.04% (90
mol/L).
ꢀ
3
.4.2. Anti-ischemic activity
The model of cerebral ischemia-reperfusion was established by middle cerebral
artery occlusion (MCAO), and the pharmacological evaluation of compound 6 was
performed. After the experiment, the color of the brain tissue infarction area became
white without staining, but the normal brain tissue was stained with dark-red (Fig. 6a).
As shown in Fig. 6b, the area of cerebral infarction in each dose group was
significantly lower than that in MCAO model group (p < 0.01). When compound 6
was administered at a dose of 36 mg/kg, the infarction area reached to the maximal
reduction with an infarction area of 11.12
±
3.3%. This indicated that compound 6 has
a good protective effect on MCAO cerebral infarction.

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Fig. 6 (a) The infarction area on each section of rat brain was determined on digital images
using a computerized image analyzer, and infarction area was assessed calculated; (b) the
—
infarction area (%, X ± SD) is as follows: 0.97 ± 0.42% (sham-operated), 15.64 ± 3.83%*
#
#
#
(
MACO), 11.87 ± 2.07% (C6/4), 11.96 ± 2.92% (C6/12), 11.12 ± 2.76% (C6/36) and 9.56 ±
#
#
3
.78% (Edaravone). *P < 0.01, compared with sham-operated; P < 0.01, compared with MACO.
4
. Conclusion
In this work, we have designed and synthesized two cinnamide derivatives. The
compound 5 is a novel compound, while the compound 6 has been reported in our
previous study. These compounds were characterized by IR spectra, High resolution
1
13
mass spectra, H NMR spectra, C NMR spectra and single-crystal X-ray diffraction.
Crystal structures analysis revealed that the stacking motif of the compound 5 is
constructed by C-HꢀO and C-Hꢀπ interactions and that of the compound 6 is
constructed by C-HꢀO, C-HꢀF and πꢀπ stacking interactions. Hirshfeld surface
analysis demonstrated the presence of HꢀH, OꢀH, CꢀH, FꢀH, C-Hꢀπ and πꢀπ
intermolecular interactions. Furthermore, the MTT assay results indicated that the
compound 5 has a weak inhibitory effect on glutamate-induced PC12 cells injury in a
dose-dependent manner, while the compound 6 significantly inhibits the damage of
PC12 cells. Moreover, the compound 6 was conducted on an animal experiment for

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further research. The results revealed that compound 6 has a good protective effect on
MCAO cerebral infarction. Finally, the further research will be in the following paper
on the basis of the present study.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(No. 81371451) and the Natural Science Foundation of Jiangsu Province (No.
BK20131390).
Supporting information
CCDC 867525 and 806575 contains the supplementary crystallographic data for 5
and 6. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/
conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
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Highlights
ꢀ
ꢀ
ꢀ
Two cinnamide derivatives have been synthesized and characterized.
Their crystal structures were studied by single-crystal X-ray diffraction.
Intermolecular interactions of compounds have been quantificationally
visualized.
ꢀ
The bioassay activity of the compounds in vitro and in vivo was studied.