Discovery of a potential anti-ischemic stroke agent: 3-pentylbenzo[c] thiophen-1(3H)-one

Article abstract of DOI:10.1021/jm300681r

The development of novel antithrombotic agents with strong free radical scavenging activity is of great significance for the treatment of ischemic stroke. In the present study, 3-alkyl/arylalkyl-substituted benzo[c]thiophen- 1(3H)-ones (5a-h) were designed and synthesized. The most active compound 5d significantly inhibited the adenosine diphos-phate (ADP) induced and arachidonic acid (AA) induced in vitro platelet aggregation, superior to clinically used anti-platelet drug aspirin (ASP) and anti-ischemic stroke drugs 3-n-butylphthalide (NBP) and edaravone (Eda). More importantly, in comparison with both NBP and Eda, 5d exhibited stronger antithrombotic and free radical scavenging activities and better or comparable neuroprotective effects against ischemia/reperfusion (I/R) in rats by ameliorating neurobehavioral function, reducing infarct size and brain-water content, attenuating cerebral damage, and normalizing the levels of oxidative enzymes. Overall, our findings may provide an alternative strategy for the design of novel anti-ischemic stroke agents more potent than drugs like NBP and Eda.

Full text of DOI:10.1021/jm300681r

Article
pubs.acs.org/jmc
Discovery of a Potential Anti-Ischemic Stroke Agent:
3‑Pentylbenzo[c]thiophen-1(3H)‑one
Jing Wu,†,‡,# Jingjing Ling,†,§,# Xuliang Wang,∥ Tingting Li,†,§ Jingchao Liu,†,‡ Yisheng Lai,†,‡ Hui Ji,*,†,§
Sixun Peng,†,‡ Jide Tian,⊥ and Yihua Zhang*,†,‡
‡
§
†State Key Laboratory of Natural Medicines, Center of Drug Discovery, and Department of Pharmacology, China Pharmaceutical
University, Nanjing 210009, P. R. China
∥CSPC Central Institute of Pharmaceutical Research, Shijiazhuang 050035, P. R. China
⊥Department of Molecular and Medical Pharmacology, University of CaliforniaLos Angeles, Los Angeles, California 90095, United
States
S
* Supporting Information
ABSTRACT: The development of novel antithrombotic
agents with strong free radical scavenging activity is of great
significance for the treatment of ischemic stroke. In the present
study, 3-alkyl/arylalkyl-substituted benzo[c]thiophen-1(3H)-
ones (5a−h) were designed and synthesized. The most active
compound 5d significantly inhibited the adenosine diphos-
phate (ADP) induced and arachidonic acid (AA) induced in
vitro platelet aggregation, superior to clinically used anti-
platelet drug aspirin (ASP) and anti-ischemic stroke drugs 3-n-
butylphthalide (NBP) and edaravone (Eda). More impor-
tantly, in comparison with both NBP and Eda, 5d exhibited stronger antithrombotic and free radical scavenging activities and
better or comparable neuroprotective effects against ischemia/reperfusion (I/R) in rats by ameliorating neurobehavioral function,
reducing infarct size and brain-water content, attenuating cerebral damage, and normalizing the levels of oxidative enzymes.
Overall, our findings may provide an alternative strategy for the design of novel anti-ischemic stroke agents more potent than
drugs like NBP and Eda.
induced lipid peroxidation, and proteins and nucleic acid
oxidation.8 The oxidative stress promotes reactive oxygen
species (ROS), which are extremely detrimental to the
surrounding tissues. Although ROS can be neutralized by a
defense system in normal tissues, increased production of ROS
can lead to neuronal damage/death under an ischemic
condition because the antioxidant defense system in ischemic
tissues is interrupted. Along these lines, the efficacy of available
antiplatelet aggregation and thrombolytic therapies for ischemic
stroke in the clinic is limited. Thus, the development of novel
antiplatelet aggregation and antithrombotic agents with free
radical scavenging activity will be of importance in this field.
The racemic 3-n-butylphthalide (NBP, Figure 1) has been
approved by the SFDA of China as a new drug mainly for the
treatment of ischemic stroke since 2002. It is well-known that
NBP can inhibit platelet aggregation and thrombosis, improve
microcirculation, and reduce brain infarct volume, thus
benefiting patients with ischemic stroke.9−13 Despite these
advantages, NBP is often administered with antioxidant and/or
antiplatelet drug(s) to improve its therapeutic efficacy.14
Therefore, new anti-ischemic stroke drugs with pronouncedly
INTRODUCTION
■
Ischemic stroke, the most common cerebrovascular disorder,
accounts for more than 80% of all strokes and is one of the
leading causes of morbidity and mortality worldwide.1,2
Previous studies have demonstrated that there are three
progressive stages involved in ischemic stroke:3,4 stage 1, the
formation of a thrombus usually initiated by blood vessel injury,
which triggers platelet aggregation and adhesion to the vessel
wall; stage 2, continued growth of the arterial thrombus,
resulting in the blood vessel occlusion; stage 3, narrowing and
obstruction of the microvasculature due to edema. Hence, the
development of new drugs with antiplatelet aggregation and
antithrombotic activities will be of great significance for the
treatment of ischemic stroke. Despite the remarkable progress
achieved in the past 2 decades in understanding the
pathogenesis of ischemic stroke, there is only one FDA-
approved medicine for the treatment of ischemic stroke, i.e., the
serine protease tissue-type plasminogen activator (t-PA, a
thrombolytic agent). Unfortunately, treatment of ischemic
stroke with t-PA for thrombolysis may cause hemorrhage and t-
PA has adverse effects on neuronal cell survival.5−7
Among multiple mechanisms implicated in the pathogenesis
of ischemia/reperfusion (I/R) injury, increased oxidative stress
has been considered as the primary cause because of free radical
Received: May 15, 2012
Published: July 24, 2012
© 2012 American Chemical Society
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5a−h with a purity of >95% were used for subsequent
biological experiments.
Pharmacology. In Vitro Antiplatelet Effect. The
individual compounds were evaluated for inhibition of platelet
aggregation in rabbit platelet rich plasma (PRP) in response to
adenosine diphosphate (ADP, 10 μM) and arachidonic acid
(AA, 1 mM) using Born’s turbidimetric method.25 For the
ADP-induced platelet aggregation, three out of eight com-
pounds displayed significant inhibitory effects, which were
superior to one classical antiplatelet drug, aspirin (ASP), and
two clinically used anti-ischemic stroke drugs, NBP and
edaravone (Eda) at the same dosage. Eda, a free radical
scavenger, has been demonstrated to have therapeutic effects
on ischemic stroke in the clinic and has been marketed in Japan
since June 2001.26−28 As shown in Table 1, the inhibitory
Figure 1. Structure of NBP.
inhibitory activities on both platelet aggregation and oxidation
are clinically needed.
Previous studies have demonstrated that the brain vessel I/R-
related oxidative stress is a key factor to secondary brain
injury.15,16 Many approaches have been documented to reduce
ROS generation and to block the ROS-mediated neuronal cell
death induced by ischemic insults, including utilization of
antioxidants or inhibitors of cell death signaling molecules.17−19
Given that a number of drug molecules containing sulfur
exhibit a better efficacy in several aspects, especially for
antioxidation, antifree radicals, and neuroprotection,20−23 we
therefore designed and synthesized a novel class of compounds
by bioisosteric replacement of the oxygen atom at the 2-
position of phthalide, the scaffold of NBP, with sulfur and,
meanwhile, introduction of various lengths of alkyl, including
straight/branch chain alkyl or arylalkyl groups, into 3-position
of phthalide. It is anticipated that these novel compounds could
synergistically inhibit thrombosis and oxidation and be more
potent than NBP.
Table 1. Effects of 5a−h on the Inhibition of Platelet
a
Aggregation Induced by ADP or AA in Vitro
IC50 (mM)
compd
ADP (10 μM)
AA (1 mM)
control
ASP
NBP
Eda
5a
0.88
>1.0
0.85
>1.0
>1.0
>1.0
0.42
>1.0
0.80
0.72
>1.0
0.15
0.48
0.25
>1.0
0.40
0.35
0.21
>1.0
0.83
>1.0
>1.0
5b
5c
5d
5e
5f
RESULTS AND DISCUSSION
■
5g
Chemistry. The synthesis of target compounds 5a−h is
outlined in Scheme 1. Starting from isobenzofuran-1,3-dione 1,
benzo[c]thiophene-1,3-dione 2 was prepared in 92% yield by
treatment with Na2S·9H2O as described previously,24 followed
by reaction with the Grignard reagents 3a−h, which were
prepared in a two-step sequence from the corresponding
bromoalkanes, to generate hydroxyl compounds 4a−h in 33−
73% yield. These compounds were converted to 5a−h in 45−
69% yield via one-pot reaction of dehydration and reduction by
reaction with hydriodic acid. The target products 5a−h were
purified by column chromatography, and their structures were
identified by MS, 1H NMR, 13C NMR, HRMS, and HPLC. The
5h
a
Data are expressed as IC50 values (the dose achieving 50% inhibition
of platelet aggregation).
effects of 5d, 5f, and 5g (IC50 of 0.42, 0.80, and 0.72 mM,
respectively) on the ADP-induced platelet aggregation were
stronger than those of Eda (IC50 = 0.85 mM), ASP (IC50 = 0.88
mM), and NBP (IC50 > 1 mM). For the AA-induced platelet
activation, three compounds exerted much greater inhibition
than NBP at the same concentration. Furthermore, 5b−d (IC50
of 0.40, 0.35, and 0.21 mM, respectively) inhibited AA-induced
a
Scheme 1. Synthesis of Compounds 5a−h
a
Reagents and conditions: (a) Na2S·9H2O, room temp, 5 h; (b) anhydrous ether, reflux, 2 h, then to room temp, 6 h; (c) AcOH, aqueous HI (57%),
125 °C, 1 h.
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Figure 2. Effect of 5d on the thrombus wet weight (A) and dry weight (B) in rats. Data are expressed as the mean SD of individual group of rats
(n = 12) from two separate experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey test: (∗∗) P <
0.01, (∗∗∗) P < 0.001 vs the control group.
platelet aggregation more effectively than NBP (IC50 = 0.48
mM). Notably, the inhibitory effect of 0.1 mM 5d (15.64%) on
the ADP-induced platelet aggregation was 1.65-fold, 1.45-fold,
and 1.36-fold stronger than that of NBP (9.50%), ASP
(10.82%), and Eda (11.50%), whereas its inhibition (35.23%)
on the AA-induced platelet activation was 2.02-fold more
potent than that of NBP (17.43%) and comparable with that of
ASP (37.20%) and Eda (36.12%). Interestingly, we found that
these compounds did not affect the lengths of activated partial
thromboplastin time (data not shown). These data suggest that
these compounds inhibited platelet aggregation without
affecting blood coagulation. These data also indicated that 5d
was a potent inhibitor of both the ADP- and AA-induced
platelet aggregation. Therefore, 5d was selected as the
candidate compound for the following investigations.
(i.e., copper ion) in biological systems via the Fenton reaction
to produce hydroxyl radical (·OH), and H2O2 and ·OH can
break DNA and have strong cytotoxicity.31 We hypothesized
that 5d with sulfur-containing molecular structure might have
free radical scavenging activity against ROS. To test this
hypothesis, PC12 cells were treated with H2O2 or ·OH in the
presence or absence of 5d for the evaluation of its H2O2 and
·OH scavenging activities, and the cytotoxicity of H2O2 or ·OH
against PC12 cells was determined by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay.32,33 Treat-
ment with 10 μM 5d remarkably increased the percentages of
cell viability in the H2O2-treated or ·OH-treated PC12 cells to
77.23% or 86.08%, compared with 52.45% and 61.95% of
H2O2-treated or ·OH-treated alone cells, respectively (Figure
3). The protective effect of 5d was superior to that of NBP
Antithrombotic Activity in Rats. Next, we investigated
the antithrombotic activity of compound 5d in a rat
arteriovenous (A-V) shunt model.29,30 Male SD rats were
subjected to the surgical induction of A-V shunt, randomized,
and treated orally with normal saline (negative control), ASP
(80 mg/kg), NBP (80 mg/kg), Eda (3.0 mg/kg), and 5d (30
and 90 mg/kg, respectively; the higher dose of 5d was
equimolar to that of NBP) for 5 consecutive days. Both of the
wet and dry thrombus weights in individual rats were measured.
As shown in Figure 2, treatment with 90 mg/kg 5d reduced the
weight of wet thrombus by 39.70% (from 48.31 9.36 to 29.13
3.92 mg) and the antithrombosis activity of 5d was much
greater than that of ASP (from 48.31 9.36 to 32.15 6.32
mg, 33.45%) and NBP (from 48.31 9.36 to 36.92 7.28 mg,
23.58%), and slightly higher than that of Eda (from 48.31
9.36 to 30.85 6.37 mg, 36.14%). In addition, treatment with
5d at 30 mg/kg also lowered the thrombus wet weight by
27.35% (from 48.31 9.36 to 35.01 6.80 mg), which was
more potent than NBP. Similarly, 5d at 90 mg/kg decreased
the thrombus dry weight by 43.33% (from 9.74 1.36 to 5.52
1.01 mg), which was much more effective than that of NBP
(from 9.74 1.36 to 7.12 1.06 mg, 26.90%), Eda (from 9.74
1.36 to 6.81 1.52 mg, 30.08%), and ASP (from 9.74 1.36
to 6.41 1.16 mg, 34.19%). Moreover, treatment with 30 mg/
kg 5d also more effectively reduced the thrombus dry weight by
32.85% (from 9.74 1.36 to 6.54 1.03 mg), compared with
that by NBP and Eda but not by ASP. These data clearly
indicated that 5d possessed a strong antithrombotic activity in
vivo.
Figure 3. Effect of 5d on H2O2-induced or ·OH-induced cytotoxicity
in PC12 cells. Data are expressed as the mean SD of each group of
cells from four separate experiments. Data were analyzed by one-way
analysis of variance (ANOVA) followed by post hoc Tukey test: (∗∗)
P < 0.01 vs the H2O2-treated group; (##) P < 0.01 vs the ·OH-treated
group.
(68.05%, 73.05%, respectively) and comparable with that of
Eda (78.05%, 83.30%, respectively) at the same dosage. These
findings suggested that 5d might act as a scavenger of H2O2 and
·OH and be protected from H2O2 or ·OH-mediated
cytotoxicity against PC12 cells in vitro.
Anticerebral Ischemic Activity in Rats. We further
investigated the anticerebral ischemic activity of 5d in a rat
model of transient focal cerebral ischemia by intraluminal
occlusion of the middle cerebral artery (MCAO) for 2 h
followed by recirculation, which has been widely used to
evaluate the protective effects of antistroke agents.34 SD rats
were treated intragastrically (ig) with normal saline (negative
control), NBP (80 mg/kg), Eda (3.0 mg/kg), or 5d (30 and 90
Free Radical Scavenging Activity in PC12 Cells. Various
ROS can induce cellular and tissue damage following the I/R
induction. Hydrogen peroxide (H2O2) can react with metal
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mg/kg; the higher dose of 5d was equimolar to that of NBP)
for 7 consecutive days, and the rats were subjected to sham
surgery or the MCA occlusion and reperfusion. At 24 h after
reperfusion, the neurological deficits of individual rats were
assessed using Longa’s method.35 As shown in Table 2, no
Table 2. Effect of 5d on Neurological Deficit Score in the
a
Rats Subjected to I/R
group
dose (mg/kg)
neurological deficit scores
sham
model
NBP
Eda
0
2.67 0.65***
1.83 0.39###
1.75 0.45###
1.67 0.49###
2.00 0.43###
Figure 5. Effect of 5d on brain edema after cerebral I/R in rats. Data
are expressed as the mean SD of individual groups of rats (n = 6)
and were analyzed by one-way analysis of variance (ANOVA) followed
by post hoc Tukey test: (∗∗) P < 0.01 vs the sham-operated group;
(#) P < 0.05 vs the model group.
80
3.0
90
30
5d
5d
a
Data are expressed as the mean SD of each group of rats (n = 12):
(∗∗∗) P < 0.001 vs the sham group; (###) P < 0.001 vs the model
attenuated the cerebral damage of rats, as compared with that
in the vehicle-treated model group. As shown in Figure 6,
hematoxylin−eosin stain of the ipsilateral hemisphere of the
ischemic brains showed the loss of hippocampal neurons,
neuronal perikarya shrinkage, and macrophage infiltration,
whereas an obviously smaller loss of hippocampal neurons
occurred in the drug-treated groups, especially in the animals
treated with 90 mg/kg 5d.
Antioxidative Effect. To further explore the mechanism
underlying the anticerebral ischemic effect of 5d, we evaluated
the levels of glutathione (GSH), superoxide dismutase (SOD),
and malondialdehyde (MDA) in the brain tissues. In
comparison with that in the sham group of rats, the levels of
GSH in the model group were decreased by 52.70% (from
60.38 to 28.56 nmol/mg). However, pretreatment with 5d (90
mg/kg) significantly enhanced GSH levels by about 82.67% to
52.17 nmol/mg (Figure 7A), which were slightly higher than
that in the Eda-treated rats (58.33%, 45.22 nmol/mg) and
significantly greater than that in the NBP-treated rats (40.83%,
40.22 nmol/mg).
Furthermore, the SOD activity in the ischemic brains from
the model group of rats was significantly decreased by 26.77%
(from 156.75 to 114.79 U/mg, Figure 7B) relative to that in the
sham-operated rats. In contrast, treatment with 30 or 90 mg/kg
5d significantly increased the SOD activities by 25.87% (from
114.79 to 144.49 U/mg) and 39.77% (160.44 U/mg),
respectively, as compared with that in the model group.
Notably, the SOD activity of the 90 mg/kg 5d-treated rats was
group.
obvious neurological deficit was observed in sham-operated and
saline-treated animals. Compared with that in the sham-
operated group, the I/R model and saline-treated group of rats
showed prominent neurological deficits 24 h after reperfusion
(P < 0.001). In contrast, pretreatment with 5d (30 or 90 mg/
kg) significantly improved the neurobehavioral function in the
rats, as evidenced by 25.09% and 37.45% reduction in
neurological deficit scores (P < 0.001). The neuroprotective
effect of higher dose of 5d was more potent than that of NBP
(31.47%) and Eda (34.46%).
Reduction of Infarct Size and Brain-Water Content in
Ischemic Brains. The infarct size of individual rats were
evaluated by the 2,3,5-triphenyltetrazolium chloride (TTC)
assay. There was no infarct damage in the sham group of rats.
In comparison with that in the model group, the infarct size in
the 5d-treated rats was reduced by 35.50% (30 mg/kg) and
59.62% (90 mg/kg, Figure 4), respectively. Furthermore, the
effect of 5d at a higher dose (59.62%) was more pronounced
than that of NBP (40.04%) and Eda (37.31%). No infarction
damage was observed in the sham-operated animals. Addition-
ally, treatment with 5d (90 mg/kg) and Eda, but not 5d (30
mg/kg) and NBP, also markedly reduced the water content in
the brains from ischemic rats (Figure 5).
Attenuation of Cerebral Damage. Histopathological
analysis revealed that treatment with 5d considerably
Figure 4. Effect of 5d on infarction after cerebral I/R in rats. (A) TTC staining analysis of the infarcted brain regions. Data shown are representative
examples from each treatment group of rats. (B) Quantitative analysis of the infracted brain regions. The ratios of infarct area to whole brain areas in
individual rats were calculated. Data are expressed as the mean SD of individual groups of rats (n = 6) and were analyzed by one-way analysis of
variance (ANOVA) followed by post hoc Tukey test: (∗∗∗) P < 0.001 vs the model group; (#) P < 0.05 vs the NBP group; (&) P < 0.05 vs the Eda
group.
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Figure 6. Effect of 5d on neuronal injury in the ischemic cerebral cortex of rats. The rat brain tissue sections were stained by hematoxylin and eosin
(HE) staining, and data shown are representative images (magnification, ×200) from individual groups of rats (n = 6).
Figure 7. Effect of 5d on the levels of brain GSH (A), SOD activity (B), and MDA (C) in rats after I/R in ischemic cerebral cortex. Data are
expressed as the mean SD of individual groups of rats (n = 12) and were analyzed by one-way analysis of variance (ANOVA) followed by post hoc
Tukey test: (∗∗∗) P < 0.001 vs the sham-operated group; (#) P < 0.05, (##) P < 0.01, (###) P < 0.001 vs the model group.
slightly greater than those in the NBP-treated (33.61%, 153.37
U/mg) or Eda-treated (34.20%, 154.06 U/mg) group.
Further analysis revealed that the content of brain MDA, an
index of lipid peroxidation, was significantly elevated by 47.80%
(from 10.44 to 15.43 nmol/mg) in the model group, as
compared with that in the sham-operated animals. However,
pretreatment with 5d (30 or 90 mg/kg) remarkably decreased
levels of brain MDA by 23.66% (from 15.43 to 11.78 nmol/
mg) and 40.90% (9.12 nmol/mg), compared with that in the
model group (Figure 7C). More importantly, the effect of 5d at
a higher dose on the brain lipid peroxidation was much stronger
than that of NBP (25.73%, 11.46 nmol/mg) or Eda (28.52%,
11.03 nmol/mg).
For the inhibition of AA-induced platelet aggregation,
compounds 5b−d bearing three- to five-carbon side chains
showed stronger activity (19.43%, 25.24%, 35.23%) than NBP
(17.43%), among which 5d had antiplatelet aggregation activity,
similar to that of Eda (36.12%), and interestingly, the inhibitory
activity of those compounds was correlated with the lengths of
their side chains. When compounds have the lengths of alkyl
side chains with less than three or greater than five carbons,
their antiplatelet aggregation activities were dramatically
diminished (5a, 5e−h). Besides, no obvious difference in the
antiplatelet aggregation activity was observed between 5h
(1.83%) with benzyl at the 3-posion and 5a (1.82%) with ethyl
group at the same position; notably, the introduction of the
methyl moiety into the second end carbon in the alkyl side
chain of 5d (35.23%) greatly impaired its activity (5e, 2.54%).
These results suggest that different activation mechanisms may
be involved in platelet aggregation induced by these two
aggregators, leading to various inhibitory activities of the target
compounds. We are interested in preparing more derivatives of
5d by introducing electron withdrawing and/or electron
donating group(s) into the benzene ring and in chiral
synthesizing the enantiomers of 5d to intensively reveal the
SAR in future.
CONCLUSIONS
■
Analysis of structure and antiplatelet aggregation activity
relationship (SAR) revealed that the target compounds 5a−h
bearing various alkyl groups displayed variable antiplatelet
aggregation activity. Significantly, the various lengths of side
chain at the 3-posion of those compounds appeared to affect
their antiplatelet aggregation activity: (1) For the inhibition of
ADP-induced platelet aggregation, generally, the longer the
alkyl side chains of those compounds are, the stronger is the
activity, with the exception of 5d (15.64%), which was superior
to both NBP (9.50%) and Eda (11.50%). An increase in steric
hindrance at the terminus of side chains, however, greatly
reduced the antiplatelet aggregation activity, for example, 5a
(6.94%) vs 5h (3.14%), and 5d (15.64%) vs 5e (6.52%). (2)
Our previous investigations have showed that several NBP
ring-opening compounds inhibit platelet aggregation in vitro
and thrombosis in vivo.36,37 In this study, we designed and
synthesized a series of novel 3-alkyl/arylalkyl-substituted
benzo[c]thiophen-1(3H)-ones (5a−h) and found that 5d not
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mmol) in anhydrous Et2O (30 mL) at −5 °C. The reaction mixture
was stirred at room temperature for 5 h. Subsequently, the reaction
was quenched by addition of a saturated aqueous solution of NH4Cl
(10 mL), acidified with 2 M HCl (15 mL) to pH 2, and stirred for 1 h
at room temperature. The solution was extracted with Et2O (50 mL ×
3), dried over anhydrous Na2SO4, and evaporated in vacuo. The
resulting residue was purified by column chromatography (petroleum
ether−EtOAc, 15: 1 v/v) to give the title compounds.
( )-3-Ethyl-3-hydroxybenzo[c]thiophen-1(3H)-one (4a). The
title compound was obtained as a light yellow solid, 46% yield. Mp
92−94 °C. ESI-MS: m/z 193 [M − H]−. 1H NMR (300 MHz,
CDCl3): δ 1.01 (t, 3H, CH3, J = 7.1 Hz), 2.11−2.48 (m, 2H, CH2),
3.49 (s, 1H, OH), 7.48 (m, 1H, ArH), 7.59−7.68 (m, 3H, ArH). 13C
NMR (300 MHz, CDCl3): δ 195.36, 152.63, 134.97, 134.20, 129.85,
123.64, 123.29, 96.38, 37.09, 9.89. ESI-HRMS (m/z): [M − H]− calcd
for C10H9O2S, 193.0323; obsd 193.0325.
only significantly inhibited ADP- and AA-induced platelet
aggregation in vitro and reduced both the wet and dry
thrombus weights in vivo but also exhibited a potent free
radical scavenging activity against the ROS-mediated cytotox-
icity in PC12 cells. More importantly, the antiplatelet
aggregation, antithrombosis, and free radical scavenging
activities of 5d were superior to those of NBP or Eda or
even both of them. Furthermore, treatment with 5d improved
neurobehavioral function, reduced the infarct brain size and
brain-water content, and cerebral damage in a rat model of
MCAO, and the effect of 5d on inhibiting the brain I/R injury
was similar to that of Eda but greater than that of NBP. In
addition, treatment with 5d significantly mitigated the I/R-
related oxidative stress in rat brains by increasing the levels of
brain antioxidant SOD and GSH but reducing the levels of
brain MDA, an index of reduced lipid peroxidation in the
brains. Apparently, 5d inhibited oxidative stress, which at least
partially contributed to its anti-I/R brain injury in rats. Our data
suggest that 5d may be a potential agent for the intervention of
ischemic stroke and such 3-substituted benzo[c]thiophen-
1(3H)-ones may represent a novel class of compounds for
the treatment of ischemic stroke and other vascular diseases.
We recognized that while many compounds have therapeutic
effects on ischemic stroke in animal models, only few have been
demonstrated to benefit patients with ischemic stroke in the
clinic. We also understand that our studies had limitations,
including the lack of systemic pharmacokinetic and pharmaco-
logical studies of 5d, particularly for the mechanisms underlying
the therapeutic action of 5d. Therefore, further studies are
necessary to reveal other pharmacological activities of 5d and
elucidate the molecular mechanism(s) underlying the anti-
ischemic stroke activity of 5d.
(
)-3-Hydroxy-3-propylbenzo[c]thiophen-1(3H)-one (4b).
The title compound was obtained as a light yellow solid, 46% yield.
1
Mp 78−80 °C. ESI-MS: m/z 207 [M − H]−. H NMR (300 MHz,
CDCl3): δ 0.98 (t, 3H, CH3, J = 7.1 Hz), 1.23−1.34 (m, 2H, CH2),
2.03−2.43 (m, 2H, CH2), 3.25 (s, 1H, OH), 7.52 (m, 1H, ArH), 7.62−
7.73 (m, 3H, ArH). 13C NMR (300 MHz, CDCl3): δ 197.15, 152.70,
134.79, 134.17, 129.88, 123.69, 123.34, 95.56, 44.80, 19.14, 13.92. ESI-
HRMS (m/z): [M − H]− calcd for C11H11O2S, 207.0480; obsd
207.0482.
( )-3-Butyl-3-hydroxybenzo[c]thiophen-1(3H)-one (4c). The
title compound was obtained as a light yellow solid, 47% yield. Mp
65−67 °C. ESI-MS: m/z 221 [M − H]−. 1H NMR (300 MHz,
CDCl3): δ 0.90 (t, 3H, CH3, J = 7.1 Hz), 1.11−1.72 (m, 4H, 2 ×
CH2), 2.03−2.41 (m, 2H, CH2), 3.94 (s, 1H, OH), 7.45 (m, 1H,
ArH), 7.58−7.65 (m, 3H, ArH). 13C NMR (300 MHz, CDCl3): δ
195.57, 152.96, 134.74, 134.19, 129.74, 123.70, 123.31, 95.82, 42.66,
29.68, 22.67, 13.84. ESI-HRMS (m/z): [M − H]− calcd for
C12H13O2S, 221.0636; obsd 221.0638.
(
)-3-Hydroxy-3-pentylbenzo[c]thiophen-1(3H)-one (4d).
The title compound was obtained as a light yellow solid, 43% yield.
1
Mp 61−63 °C. ESI-MS: m/z 235 [M − H]−. H NMR (300 MHz,
EXPERIMENTAL SECTION
■
CDCl3): δ 0.86 (t, 3H, CH3, J = 6.9 Hz), 1.07−1.70 (m, 6H, 3 ×
CH2), 2.00−2.42 (m, 2H, CH2), 3.39 (s, 1H, OH), 7.46 (m, 1H,
ArH), 7.60−7.68 (m, 3H, ArH). 13C NMR (300 MHz, CDCl3): δ
195.32, 152.95, 134.78, 134.26, 129.90, 123.72, 123.36, 95.78, 42.61,
31.62, 25.41, 22.41, 13.96. ESI-HRMS (m/z): [M − H]− calcd for
C13H15O2S, 235.0793; obsd 235.0795.
1
Chemistry. H and 13C NMR spectral data were obtained from a
Bruker Avance 300 MHz spectrometer at 300 K using TMS as an
internal standard. MS spectra were recorded on a Mariner mass
spectrometer. Analytical and preparative TLC was performed on silica
gel GF/UV 254, and the chromatograms were conducted on silica gel
(200−300 mesh) and visualized under UV light at 254 and 365 nm.
The purities of the compounds were characterized by HPLC analysis
(LC-10A HPLC system consisting of LC-10ATvp pumps and SPD-
10Avp UV detector) and HRMS (Agilent Technologies LC/MSD
TOF). 2 was synthesized as previously described.24 The target
compounds 5a−h with a purity of >95% were used for subsequent
experiments (see the ESI results).
All animal experiments were performed in accordance with the
Animal (Scientific Procedures) Act 2009 (P. R. China), and all animal
experimental protocols were approved by the Animal Research
Protection Committee of China Pharmaceutical University, Nanjing,
China.
( )-3-Hydroxy-3-isopentylbenzo[c]thiophen-1(3H)-one (4e).
The title compound was obtained as a light yellow oil, 45% yield. ESI-
MS: m/z 235 [M − H]−. H NMR (300 MHz, CDCl3): δ 0.90 (m,
1
6H, 2 × CH3), 1.23−1.52 (m, 3H, CH2CH), 2.07−2.44 (m, 2H,
CH2), 3.55 (br s, 1H, OH), 7.47 (m, 1H, ArH), 7.53−7.63 (m, 2H,
ArH), 7.78 (d, 1H, ArH, J = 7.8 Hz). 13C NMR (300 MHz, CDCl3): δ
195.52, 152.93, 134.72, 134.11, 129.66, 123.60, 123.15, 95.89, 40.54,
34.41, 27.97, 22.41, 22.28. ESI-HRMS (m/z): [M − H]− calcd for
C13H15O2S, 235.0793; obsd 235.0795.
( )-3-Hexyl-3-hydroxybenzo[c]thiophen-1(3H)-one (4f). The
title compound was obtained as a light yellow solid, 43% yield. Mp
56−58 °C. ESI-MS: m/z 249 [M − H]−. 1H NMR (300 MHz,
CDCl3): δ 0.89 (t, 3H, CH3, J = 7.1 Hz), 1.07−1.72 (m, 8H, 4 ×
CH2), 2.05−2.43 (m, 2H, CH2), 3.36 (s, 1H, OH), 7.48 (m, 1H,
ArH), 7.60−7.69 (m, 3H, ArH). 13C NMR (300 MHz, CDCl3): δ
195.36, 152.97, 134.76, 134.18, 129.79, 123.75, 123.32, 95.79, 42.67,
31.45, 29.11, 25.70, 22.47, 13.97. ESI-HRMS (m/z): [M − H]− calcd
for C14H17O2S, 249.0949; obsd 249.0952.
Benzo[c]thiophene-1,3-dione (2).24 Isobenzofuran-1,3-dione
(15.0 g, 0.1 mol) was mixed with Na2S·9H2O (30.0 g, 0.125 mol)
by mechanical stirring at room temperature for 5 h. The reaction was
quenched by addition of H2O (200 mL), and then the mixture was
acidified with 2 M HCl (100 mL). The solid crude product was
collected by filtration, as described in the workup procedure, and 14.9
g (92%) of 2 was obtained as a white powder solid. Mp 112−114 °C.
ESI-MS: m/z 187 [M + Na]+. 1H NMR (300 MHz, CDCl3): δ 7.74−
7.79 (m, 2H, ArH), 7.92−7.95 (m, 2H, ArH).
General Procedure for the Preparation of 4a−h. The
corresponding bromoalkanes (20.0 mmol) were added to a solution
of magnesium (0.48 g, 20.0 mmol) in anhydrous Et2O (5 mL) under
nitrogen atmosphere until the Grignard reaction started. The reaction
mixture was stirred at room temperature for 2 h, and then the
Grignard solution was added dropwise to a solution of 2 (1.65 g, 10.0
(
)-3-Heptyl-3-hydroxybenzo[c]thiophen-1(3H)-one (4g).
The title compound was obtained as a light yellow solid, 33% yield.
1
Mp 56−58 °C. ESI-MS: m/z 263 [M − H]−. H NMR (300 MHz,
CDCl3): δ 0.86 (t, 3H, CH3, J = 7.2 Hz), 1.31−1.60 (m, 10H, 5 ×
CH2), 2.02−2.44 (m, 2H, CH2), 3.56 (s, 1H, OH), 7.45 (m, 1H,
ArH), 7.52−7.62 (m, 2H, ArH), 7.77 (d, 1H, ArH, J = 7.5 Hz). 13C
NMR (300 MHz, CDCl3): δ 197.87, 152.70, 135.14, 133.70, 130.12,
124.38, 122.59, 96.07, 42.67, 29.71, 29.43, 29.02, 25.60, 22.58, 14.03.
7178
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Journal of Medicinal Chemistry
Article
ESI-HRMS (m/z): [M − H]− calcd for C15H19O2S, 263.1106; obsd
7.5 Hz), 7.62 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C NMR
(300 MHz, CDCl3): δ 199.96, 156.37, 140.22, 133.18, 127.98, 126.10,
123.65, 49.81, 37.78, 31.62, 29.85, 29.04, 22.59, 14.07. ESI-HRMS (m/
z): [M + H]+ calcd for C14H19OS, 235.1155; obsd 235.1157.
263.1109.
(
)-3-Benzyl-3-hydroxybenzo[c]thiophen-1(3H)-one (4h).
The title compound was obtained as a light yellow solid, 73% yield.
Mp 126−128 °C. ESI-MS: m/z 255 [M − H]−. 1H NMR (300 MHz,
CDCl3): δ 3.27−3.61 (m, 2H, CH2Ar), 3.79 (s, 1H, OH), 7.25−7.37
(m, 5H, ArH), 7.48 (m, 1H, ArH), 7.60−7.71 (m, 3H, ArH). 13C
NMR (300 MHz, CDCl3): δ 194.71, 152.35, 135.45, 134.99, 133.97,
130.82, 129.98, 128.55, 128.20, 127.99, 127.00, 124.34, 123.38, 94.96,
49.08. ESI-HRMS (m/z): [M − H]− calcd for C15H11O2S, 255.0480;
obsd 255.0482.
General Procedure for the Preparation of 5a−h. To a solution
of 4a−h (1.0 mol) in AcOH (10 mL) was added aqueous 57% HI (5
mL). After complete addition, the reaction mixture was refluxed for 0.5
h and allowed to cool to room temperature. Then a solution of 5%
NaHSO3 (50 mL) was added to the mixture. The solution was
extracted with Et2O (50 mL × 3), dried over anhydrous Na2SO4, and
evaporated in vacuo. The resulting residue was purified by column
chromatography (petroleum ether−EtOAc, 50:1 v/v) to give the title
compounds.
( )-3-Heptylbenzo[c]thiophen-1(3H)-one (5g). The title com-
pound was obtained as a yellow oil, 45% yield. ESI-MS: m/z 249 [M +
1
H]+, 271 [M + Na]+. H NMR (300 MHz, CDCl3): δ 0.85 (t, 3H,
CH3, J = 7.2 Hz), 1.27−1.58 (m, 10H, 5 × CH2), 1.74−2.34 (m, 2H,
CH2), 4.83 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.52 (d, 1H, ArH, J =
7.5 Hz), 7.62 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C NMR
(300 MHz, CDCl3): δ 197.12, 151.43, 135.92, 132.86, 128.06, 125.14,
123.55, 51.57, 36.49, 31.70, 29.29, 29.02, 28.02, 22.58, 14.04. ESI-
HRMS (m/z): [M + H]+ calcd for C15H21OS, 249.1313; obsd
249.1315.
( )-3-Benzylbenzo[c]thiophen-1(3H)-one (5h). The title com-
pound was obtained as a yellow solid, 62% yield. Mp 118−120 °C.
1
ESI-MS: m/z 241 [M + H]+, 263 [M + Na]+. H NMR (300 MHz,
CDCl3): δ 3.02−3.59 (m, 2H, CH2), 5.07 (m, 1H, CH), 7.24−7.36
(m, 5H, ArH), 7.47 (m, 2H, ArH), 7.62 (m, 1H, ArH), 7.80 (d, 1H,
ArH, J = 8.1 Hz). 13C NMR (300 MHz, CDCl3): δ 196.71, 150.46,
137.99, 136.28, 133.14, 129.14, 129.14, 128.67, 128.67, 128.42, 127.23,
125.49, 123.82, 52.65, 42.95. ESI-HRMS (m/z): [M + H]+ calcd for
C15H13OS, 241.0687; obsd 241.0689.
( )-3-Ethylbenzo[c]thiophen-1(3H)-one (5a). The title com-
pound was obtained as a yellow oil, 46% yield. ESI−MS: m/z 179 [M
1
+ H]+, 201 [M + Na]+. H NMR (300 MHz, CDCl3): δ 1.05 (t, 3H,
CH3, J = 7.2 Hz), 1.85−2.38 (m, 2H, CH2), 4.83 (m, 1H, CH), 7.45
(m, 1H, ArH), 7.52 (d, 1H, ArH, J = 7.5 Hz), 7.64 (m, 1H, ArH), 7.80
(d, 1H, ArH, J = 7.5 Hz). 13C NMR (300 MHz, CDCl3): δ 197.26,
151.08, 136.17, 133.25, 128.13, 125.06, 123.61, 52.85, 29.19, 11.62.
ESI-HRMS (m/z): [M + H]+ calcd for C10H11OS, 179.0531; obsd
179.0532.
Antiplatelet Aggregation in Vitro. Blood was drawn from rabbit
carotid artery and mixed with 3.8% sodium citrate (9:1, v/v). After
centrifugation at 500g for 10 min at room temperature to obtain
platelet-rich plasma (PRP), the remaining blood was further
centrifuged at 3000g for another 10 min to obtain platelet-poor
plasma (PPP). Platelet aggregation was measured by Born’s
turbidimetric method in a platelet aggregometer (LG-PABER-I platelet
aggregometer, Beijing) at 37 °C within 3 h after blood collection.
Briefly, PRP (240 μL) was preincubated in duplicate for 5 min at 37
°C with vehicle, the individual compounds, or reference drugs (ASP,
NBP, and Eda) at the same concentrations (0.1, 0.2, 0.4, 0.8, and 1.6
mM) and the platelet aggregation in individual PPR samples was
induced by ADP (final concentration of 10 μM) or AA (1 mM),
followed by recording of light transmission at maximal aggregation
within 5 min. The inhibition rate of the tested individual compounds
on the platelet aggregation was calculated as the following formula:
inhibition rate (%) = 100 × [(1 − (the platelet aggregation in samples
with the tested compound)/(the platelet aggregation in control
samples)]. In addition, the concentrations of individual compounds
that inhibited the platelet aggregation to 50% (IC50) were calculated.
In Vitro Free Radical Scavenging Assay. The free radical
scavenging capacities of 5d, NBP, and Eda were tested against H2O2
and ·OH-mediated cytotoxicity against PC12 cells. PC12 cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% heat inactivated horse serum (Hyclone), 5% fetal
bovine serum (GIBCO), 1.0 mM sodium pyruvate, 100 U/mL
penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2
atmosphere (Thermo Scientific, 3110, OH, U.S.). During the
exponential phase of growth, PC12 cells (20 000 cells/well) were
cultured in 96-well plates that had been coated with poly-L-lysine for
24 h. The cells were treated in triplicate with different concentrations
(0.1−10 μM) of the tested compounds for 2 h and exposed to 800 μM
H2O2 or 1 mM H2O2/20 μM Fe2+ (pH 7.4) for 1 h. After replacement
with fresh medium, the cells were incubated for 14 h and the cell
viability was determined by MTT, as described previously.38
( )-3-Propylbenzo[c]thiophen-1(3H)-one (5b). The title com-
pound was obtained as a yellow oil, 55% yield. ESI−MS: m/z 193 [M
1
+ H]+, 215 [M + Na]+. H NMR (300 MHz, CDCl3): δ 0.99 (t, 3H,
CH3, J = 7.5 Hz), 1.44−1.61 (m, 2H, CH2), 1.73−2.30 (m, 2H, CH2),
4.84 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.52 (d, 1H, ArH, J = 7.5 Hz),
7.65 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C NMR (300
MHz, CDCl3): δ 196.57, 151.44, 135.96, 133.20, 128.10, 125.09,
123.67, 51.32, 38.62, 21.35, 13.80. ESI-HRMS (m/z): [M + H]+ calcd
for C11H13OS, 193.0687; obsd 193.0689.
( )-3-Butylbenzo[c]thiophen-1(3H)-one (5c). The title com-
pound was obtained as a yellow oil, 67% yield. ESI−MS: m/z 207 [M
1
+ H]+, 229 [M + Na]+. H NMR (300 MHz, CDCl3): δ 0.92 (t, 3H,
CH3, J = 7.2 Hz), 1.35−1.56 (m, 4H, 2 × CH2), 1.74−2.35 (m, 2H,
CH2), 4.83 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.53 (d, 1H, ArH, J =
7.5 Hz), 7.65 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C NMR
(300 MHz, CDCl3): δ 197.30, 151.47, 136.05, 133.22, 128.13, 125.10,
123.71, 51.55, 36.22, 30.12, 22.48, 13.86. ESI-HRMS (m/z): [M + H]+
calcd for C12H15OS, 207.0844; obsd 207.0846.
( )-3-Pentylbenzo[c]thiophen-1(3H)-one (5d). The title com-
pound was obtained as a yellow oil, 65% yield. ESI-MS: m/z 221 [M +
H]+, 243 [M+Na]+. 1H NMR (300 MHz, CDCl3): δ 0.89 (t, 3H, CH3,
J = 6.9 Hz), 1.32−1.57 (m, 6H, 3 × CH2), 1.75−2.33 (m, 2H, CH2),
4.83 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.52 (d, 1H, ArH, J = 7.5 Hz),
7.62 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C NMR (300
MHz, CDCl3): δ 197.30, 151.44, 135.90, 133.26, 128.10, 125.14,
123.57, 51.60, 36.44, 31.49, 27.70, 22.41, 13.96. ESI-HRMS (m/z): [M
+ H]+ calcd for C13H17OS, 221.1000; obsd 221.1002.
( )-3-Isopentylbenzo[c]thiophen-1(3H)-one (5e). The title
compound was obtained as a yellow oil, 69% yield. ESI-MS: m/z
1
221 [M + H]+. H NMR (300 MHz, CDCl3): δ 0.90 (m, 6H, 2 ×
Antithrombotic Activity Assay in Rats. Male Sprague−Dawley
(SD) rats (250−280 g) were purchased from B&K Universal Group,
Shanghai, China. All procedures were performed following institu-
tional approval in accordance with the NIH Guide for the Care and
Use of Laboratory Animals. Rats were randomized and divided into six
groups (n = 12 per group): (1) control group, (2) model group (A-V),
(3) A-V + 5d (30 mg/kg) group, (4) A-V + 5d (90 mg/kg) group, (5)
A-V + NBP (80 mg/kg) group, (6) A-V + Eda (3.0 mg/kg) group.
Groups 3−6 were treated by gavage with the indicated dose of the
compound in saline daily for 5 consecutive days. Subsequently, the rats
were anesthetized by intraperitoneal (ip) injection of pentobarbital (50
mg/kg). The control group of rats received a sham surgery, while the
CH3), 1.22−1.30 (m, 2H, CH2), 1.48 (m, 1H, CH), 1.74−2.35 (m,
2H, CH2), 4.83 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.53 (d, 1H, ArH, J
= 7.5 Hz), 7.63 (m, 1H, ArH), 7.79 (d, 1H, ArH, J = 7.5 Hz). 13C
NMR (300 MHz, CDCl3): δ 197.10, 151.30, 135.89, 133.13, 128.00,
125.02, 123.50, 51.64, 36.82, 34.23, 27.86, 22.53, 22.13. ESI-HRMS
(m/z): [M + H]+ calcd for C13H17OS, 221.1000; obsd 221.1002.
( )-3-Hexylbenzo[c]thiophen-1(3H)-one (5f). The title com-
pound was obtained as a yellow oil, 59% yield. ESI-MS: m/z 235 [M +
1
H]+; 257 [M + Na]+. H NMR (300 MHz, CDCl3): δ 0.90 (t, 3H,
CH3, J = 7.2 Hz), 1.30−1.58 (m, 8H, 4 × CH2), 1.74−2.34 (m, 2H,
CH2), 4.83 (m, 1H, CH), 7.45 (m, 1H, ArH), 7.52 (d, 1H, ArH, J =
7179
dx.doi.org/10.1021/jm300681r | J. Med. Chem. 2012, 55, 7173−7181

Journal of Medicinal Chemistry
Article
remaining rats were subjected to the A-V shunt procedure. Briefly,
individual rats were inserted with an arteriovenous (A-V) shunt tube
that directly connected the right carotid artery with the left jugular
vein of rats. The polyethylene tube (14 cm, containing 6 cm long of
four braided silk threads) was filled with saline before installation.
After 15 min of circulation of blood through the shunt tube, both ends
of the tubing were pinched and the cotton thread was removed from
the shunt tube. The wet weight of the cotton thread was measured
immediately, and the cotton thread was dried at the room temperature
for 6 h, followed by measurement of the dry weight. The wet and dry
weights of thrombus formed on the cotton threads were determined
by subtracting the pre-experiment weight of the dry 6 cm long threads.
Focal Cerebral Ischemia. Rats were anesthetized ip with 350 mg/
kg chloral hydrate (Sinopharm Chemical Reagent, Beijing, China) and
subjected to a procedure of the middle cerebral artery occlusion
(MCAO), as described previously with minor modification. Briefly, the
right common carotid artery, internal carotid artery (ICA), and
external carotid artery (ECA) of individual rats were surgically
exposed. The MCAO was achieved by inserting a 4−0 monofilament
nylon suture (Beijing Sunbio Biotech, Beijing, China) with a round tip
into the ICA through the ECA stump and gently advancing to the
MCA. After 2 h of MCAO, the suture was withdrawn to restore blood
flow (reperfusion) in the MCA. The distal 5 mm of the suture is
coated with silicone to a diameter of 0.26 mm. During the experiment,
the cerebral blood flow (CBF) was monitored by laser Doppler
flowmetry (LDF 100C, BIOPAC Systems) to ensure that occlusion of
the MCA with the specific suture resulted in >80% decline in CBF.
The control rats received a sham surgery. The core body temperature
of individual rats was monitored with a rectal probe and maintained at
37 0.5 °C during the whole procedure.
In Vivo Antioxidant Activity Assay. At 24 h after reperfusion,
the rats were sacrificed and their brains were rapidly removed, rinsed
with 0.9% cold saline, and blotted with filter paper before being kept at
−70 °C. After quantification of protein concentrations, the levels of
GSH and SOD activities and the levels of MDA content in brain
tissues were determined using commercial analysis kits (Jiancheng
Institute of Biotechnology, Nanjing, China) according to the
manufacturer’s protocol.
ASSOCIATED CONTENT
■
S
* Supporting Information
Inhibition of platelet aggregation, table of compound purity by
HPLC. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For H.J.: phone, +86-25-86021369; fax, +86-25-86635503; e-
mail, huijicpu@163.com. For Y.Z.: phone, +86-25-83271015;
fax, +86-25-83271015; e-mail, zyhtgd@ hotmail.com.
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the grants from the Fundamental
Research Funds for the Central Universities (No.
JKY2011010), the Research and Innovation Project for College
Graduates of Jiangsu Province (No. CXLX12_0319), and
Project Program of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. ZJ11176).
Rats were randomly divided into six groups (n = 12 per group): (1)
sham group, (2) model group (I/R), (3) I/R + 5d (30 mg/kg, ig)
group, (4) I/R + 5d (90 mg/kg, ig) group, (5) I/R + NBP (80 mg/kg,
ig) group, (6) I/R + Eda (3.0 mg/kg, ig) group. The tested compound
5d (30 and 90 mg/kg), reference drugs NBP (80 mg/kg), and Eda
(3.0 mg/kg) were dissolved in normal saline and administrated orally
to rats for 7 successive days before ischemia.
Evaluation of the Neurological Deficit. The neurological
deficits of individual rats were evaluated by Longa’s method with
minor modification in a blinded manner at 24 h after reperfusion. The
neurologic deficits were scored on a five-point scale: 0, normal
function; 1, flexion of the torso and contralateral forelimb on lifting the
animal by the tail; 2, circling to the contralateral side but normal
posture at rest; 3, reclination to the contralateral side at rest; 4,
absence of spontaneous motor activity.
Measurement of Infarct Size. At 24 h after reperfusion, the rats
were sacrificed and their brains were rapidly dissected on ice and sliced
into 2-mm-thick coronal sections. The brain sections were stained with
2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) for 30 min at 37
°C, followed by overnight immersion in 10% formalin in PBS. The
infarcted tissues remained unstained (white), whereas normal tissues
were stained red. The infarct zone was demarcated and analyzed by an
image processing software. The infarct areas were calculated as a
percentage of the total area of whole brain.
ABBREVIATIONS USED
■
ADP, adenosine diphosphate; AA, arachidonic acid; NBP, 3-n-
butylphthalide; Eda, edaravone; ASP, aspirin; I/R, ischemia/
reperfusion; ROS, reactive oxygen species; PRP, platelet rich
plasma; PPP, platelet-poor plasma; MTT, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide; MCAO, middle
cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium
chloride; GSH, glutathione; SOD, superoxide dismutase;
MDA, malondialdehyde; ICA, internal carotid artery; ECA,
external carotid artery
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