Studies on the enantiomers of ZJM-289: Synthesis and biological evaluation of antiplatelet, antithrombotic and neuroprotective activities

Article abstract of DOI:10.1039/c2ob26511g

ZJM-289 is a potent racemic agent which inhibits both platelet aggregation and thrombosis superior to a known anti-ischemic stroke drug 3-n-butylphthalide (NBP). Herein, the enantiomers of ZJM-289, (S)-ZJM-289 and (R)-ZJM-289, were synthesized and evaluat

Full text of DOI:10.1039/c2ob26511g

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PAPER
Studies on the enantiomers of ZJM-289: synthesis and biological evaluation
of antiplatelet, antithrombotic and neuroprotective activities†
Xiaoli Wang,‡a,b Qian Zhao,‡a,c,d Xuliang Wang,e Tingting Li,a,c Yisheng Lai,a,b Sixun Peng,a,b Hui Ji,*a,c
Jinyi Xu*a,b and Yihua Zhang*a,b
Received 1st August 2012, Accepted 14th September 2012
DOI: 10.1039/c2ob26511g
ZJM-289 is a potent racemic agent which inhibits both platelet aggregation and thrombosis superior to a
known anti-ischemic stroke drug 3-n-butylphthalide (NBP). Herein, the enantiomers of ZJM-289,
(S)-ZJM-289 and (R)-ZJM-289, were synthesized and evaluated for their biological activities. It was
observed that the two enantiomers appeared to be almost as effective as ZJM-289 in inhibiting platelet
aggregation in vitro and thrombus formation in vivo. Moreover, like ZJM-289, its enantiomers could
regulate the ratio of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α, and enhanced levels of nitric
oxide (NO), cAMP and cGMP, suggesting that the anti-platelet and antithrombotic activities of the
enantiomers and ZJM-289 are associated with both the arachidonic acid cascade and cGMP–NO signal
pathway. Furthermore, it was found that oral administration of the enantiomers and ZJM-289 for three
days significantly reduced the infarct size, brain water content and neurological deficit in rats after cerebral
ischemia reperfusion. Importantly, the two enantiomers equally improved blood flow in the ischemic
stroke model and modulated endothelial function through releasing moderate levels of NO, which might,
at least partially, contribute to their neuroprotection. Collectively, the present study demonstrates that the
two enantiomers are as potent as ZJM-289 in inhibition of platelet aggregation and thrombosis and in
neuroprotection, and (S)-ZJM-289 shows somewhat better effects than (R)-ZJM-289 and ZJM-289 in a
few cases. These findings may provide new insights into the development of therapeutic agents like
ZJM-289 for the intervention of thrombosis-related ischemic stroke.
restricts or blocks the flow of blood and can result in acute
1. Introduction
ischemic stroke, which accounts for 80% of all forms of stroke
patients.2 It has been generally accepted that the activation of
Thromboembolic events are the major cause of morbidity and
mortality worldwide.1 Cerebral thrombosis involves the for-
platelets and the resultant aggregation play a major role in the
pathogenesis of intravascular thrombosis.3 Furthermore, platelet
mation of a blood clot or thrombus inside a cerebral artery that
activation contributes significantly to ischemic microvascular
occlusion and causes secondary injury after the initial ischemic
insult.4 Thus, platelet adhesion and aggregation have been ident-
ified as important targets for the development of antithrombotic
drugs in the treatment of ischemic stroke.
aState Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing 210009, P.R. China. E-mail: zyhtgd@163.com;
Fax: +86-025-83271015; Tel: +86-25-83271015 (Y.Z.);
Racemic 3-n-butylphthalide (NBP) is an anti-cerebral
ischemic drug which was received by the State Food and Drug
Administration (SFDA) of China in 2002.5 A large variety of
studies have demonstrated that NBP possesses multiple actions
beneficial for ischemic stroke patients, such as improvement of
brain microcirculation, inhibition of platelet aggregation and
thrombosis, regulation of energy metabolism, and reduction in
the brain infarct volume.6 However, the clinical application of
NBP is limited due to its moderate potency, and it has been
found that the therapeutic effects of NBP could be improved by
co-administration with other antiplatelet drugs.7 Therefore, new
derivatives of NBP with strong potency may be of great signifi-
cance for the treatment of ischemic stroke.
E-mail: huijicpu@163.com; Fax: +86-25-86635503; Tel: +86-25-
86021369 (H.J.); E-mail: jinyixu@china.com; Fax: +86–25-83302827;
Tel: +86-25-83271445 (J.X.)
bCenter of Drug Discovery, China Pharmaceutical University, Nanjing
210009, P.R. China
cDepartment of Pharmacology, China Pharmaceutical University,
Nanjing 210009, P.R. China
dSchool of Life Sciences, Shandong University of Technology, Zibo
255049, P.R. China
eCSPC Central Institute of Pharmaceutical Research, Shijiazhuang
050035, P.R. China
†Electronic supplementary information (ESI) available: HPLC con-
ditions chromatograms of ZJM-289, (S)- and (R)-ZJM-289. Chiral
HPLC conditions and chromatograms of (S)- and (R)-NBP, (S)- and (R)-
ZJM-289. See DOI: 10.1039/c2ob26511g
‡These authors contributed equally to this work.
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Scheme 1 Resolution of NBP. Reagents and conditions: (a) (i) 2 M NaOH, CH3OH–H2O, 50 °C, 0.5 h; (ii) 5% HCl, −10 to 0 °C; (b) (i) (S)-FEA,
Et2O, −20 to −10 °C; (ii) acetone, recrystallization repeated 2 times; (c) (i) (R)-FEA, Et2O, −20 to −10 °C; (ii) acetone, recrystallization repeated 2
times; (d) (i) 2 M NaOH, r.t., 0.5 h; (ii) 3 M HCl, r.t., 0.5 h.
Recently, we have synthesized a series of nitric oxide (NO)
releasing ring-opening derivatives of NBP that possessed
marked activities of antiplatelet aggregation and antithrombosis
both in vitro and in vivo. Among all the tested compounds,
(E)-2-{[1-(2-diethylamino)acetoxy]pentyl}benzoic acid-{2-methoxy-
4-[2-(4-nitrooxybutoxycarbonyl)vinyl]}phenyl ester hydrochlo-
ride (ZJM-289) showed the strongest inhibitory activity on the
ADP- and TH-induced platelet aggregation in vitro, superior to
NBP. In addition, ZJM-289 could reduce infarct volume and
brain-water content in ischemic brains and promote functional
recovery after ischemia-reperfusion through inhibition of iNOS
and stimulation of the NO–sGC–cGMP pathway.8 Furthermore,
preliminary pharmacokinetic study demonstrated the ester bonds
of ZJM-289 could be cleaved in vivo by esterases to generate
NBP, substituted ferulic acid and NO, all of which function
synergetically to improve the efficacy of NBP.9
It is estimated that about half of all therapeutic agents are
chiral, but most of these drugs are administered in the form of
the racemic mixture, i.e. a 50 : 50 mixture of its enantiomers.10
However, chirality is one of the main features of biology, and
many of the processes essential for life are stereoselective,
implying that two enantiomers may work differently from
each other in a physiological environment. Several reviews of
the importance and value of developing a single enantiomer
rather than a racemate have been documented.11,12 Hence,
we would like to investigate the two enantiomers of ZJM-289,
(S)-ZJM-289 and (R)-ZJM-289, for their biological and pharma-
cological behavior. In this paper, we report the synthesis of
the two enantiomers of ZJM-289 and their antiplatelet and
antithrombotic activities as well as neuroprotective effects.
lactonization under acid conditions to give (S)-NBP ([α]2D7 −66.6
(c 1.0, CH3Cl), ee > 99%). (R)-NBP ([α]2D7 +68.4 (c 1.0,
CH3Cl), ee > 98%) was similarly obtained with (R)-α-methyl-
benzylamine ((R)-FEA, ee > 98%).
The (S)- or (R)-NBP was subjected to alkali hydrolysis and
subsequent acidification to form the acid (S)- or (R)-2. Treatment
of (S)- or (R)-2 with chloroacetyl chloride yielded the acylated
(S)- or (R)-5, which was esterified with phenol compound 6
in the presence of 1,3-dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) to offer ester (S)- or (R)-7.
The ester (S)- or (R)-7 was condensed with diethylamine, fol-
lowed by treatment with anhydrous ethereal HCl to provide the
corresponding hydrochloride (S)- or (R)-ZJM-289, as depicted in
Scheme 2. The racemic ZJM-289 was synthesized starting from
2-formylbenzoic acid by a reported six-step-reaction sequence.14
All of the new compounds were purified by column chromato-
1
graphy and characterized by IR, ESI-MS, H NMR, 13C NMR,
and HRMS, and individual compounds with chemical purity of
>99% and optical purity (ee) of >97% (determined by chiral
HPLC analysis) were used for subsequent experiments.
2.2. In vitro antiplatelet aggregation effects of (S)- and
(R)-ZJM-289
The in vitro inhibitory activities of (S)- and (R)-ZJM-289 at
concentrations ranging from 10 μM to 1.0 mM against adenosine
diphosphate (ADP)-, thrombin (TH)- and arachidonic acid (AA)-
induced platelet aggregation were evaluated using the Born’s
turbidimetric assay, and the IC50 values are listed in Table 1.
As shown in Table 1, the IC50 values of (S)- and (R)-ZJM-289
against ADP-, TH- and AA-induced platelet aggregation are
0.21, 0.44; 0.43, 0.42; and 0.09, 0.34 mM; respectively. The two
enantiomers and ZJM-289 (0.24, 0.41, 0.16 mM) displayed
almost comparable inhibitory effects on ADP- and TH-induced
platelet aggregation while the IC50 value of (S)-ZJM-289 against
AA-induced platelet aggregation was slightly lower than that of
(R)-ZJM-289 or ZJM-289.
2. Results
2.1. Chemistry
Synthesis of the enantiomers of ZJM-289 started from the
chemical resolution of racemic NBP.13 As depicted in Scheme 1,
NBP was subjected to saponification and sequential acidification
to pH 3–4 using dilute HCl solution at −10 to 0 °C to form acid
2. Treatment of 2 with the optically active base (S)-α-methyl-
benzylamine ((S)-FEA, ee > 98%) at −20 to −10 °C yielded the
diastereoisomeric salt 3. Scission of 3 with NaOH and finally
2.3. Antithrombotic activities of (S)- and (R)-ZJM-289 in vivo
We next investigated the effects of (S)- and (R)-ZJM-289 on the
formation of thrombus in a rat extra-corporeal circulation of
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Scheme 2 Synthesis of (S)- and (R)-ZJM-289. Reagents and conditions: (a) (i) 2 M NaOH, CH3OH–H2O, 50 °C, 0.5 h; (ii) 5% HCl, −10 to 0 °C;
(b) ClCH2COCl, Et3N, DMAP, CH2Cl2, −10 to 0 °C, 5 h; (c) DCC, DMAP, CH2Cl2, r.t., 5 h; (d) (i) diethylamine, Et3N, DMF, r.t., 8 h; (ii) HNO3,
Ac2O, EtOAc, 0 °C, 12 h.
Table 1 The IC50 values of (S)- and (R)-ZJM-289 against rabbit
platelet aggregation in vitro
IC50 (mM)
Compd.
ADP (10 μM)
TH (10 μM)
AA (1.0 mM)
NBP
>1.0a
0.24
0.21
0.44
>1.0b
0.41
0.43
0.42
0.10
0.16
0.09
0.34
ZJM-289
(S)-ZJM-289
(R)-ZJM-289
a A 51.3% inhibition of platelet aggregation was observed at maximal
concentration of 1.0 mM. b A 31.3% inhibition of platelet aggregation
was observed at maximal concentration of 1.0 mM.
arteriovenous (A-V) cannula model. Male SD rats were random-
ized and treated orally with 0.1% CMC-Na (negative control),
NBP, ZJM-289, (S)- and (R)-ZJM-289 at equimolar doses (2.63
× 10−4 mol kg−1) for five days. The formation of thrombus in
these rats was induced by a surgical procedure and the thrombus
weights in individual rats were measured. As shown in Fig. 1,
the thrombus wet weights in the rats receiving (S)-ZJM-289
(21.0 8.8 mg), (R)-ZJM-289 (24.5 7.8 mg) and ZJM-289
(22.6 6.6 mg) were almost equal and less than that of the nega-
tive control group (32.2 8.8 mg, P < 0.05), indicating that the
two enantiomers and ZJM-289 have comparable antithrombotic
activity in a rat model.
Fig. 1 Effects of (S)- and (R)-ZJM-289 on thrombus formation in rats.
Male SD rats were randomized and treated orally with 0.1% CMC-Na,
NBP, ZJM-289, (S)- and (R)-ZJM-289 for consecutive 5 days and the
formation of thrombus in individual rats was induced. Subsequently, the
thrombus weights were measured. Data are expressed as mean SD of
individual groups of rats (n = 10) from two separate experiments.
Control and experimental groups of rats were tested simultaneously.
#P < 0.05 vs. control group, determined with one-way ANOVA followed
by Tukey’s post hoc test.
hemiplegia and death in the rats were monitored within
fifteen min. Apparently, treatment with the two enantiomers and
ZJM-289 significantly reduced the onset of hemiplegia and
death in mice. In comparison with (S)-ZJM-289 and ZJM-289,
(R)-ZJM-289 displayed a relatively weak protective effect in this
model (Table 2), but there was no significant difference between
the three treatment groups.
We further evaluated the antithrombotic effects of (S)- and
(R)-ZJM-289 on a mouse model of thromboembolism. Male
ICR mice were randomized and administered orally with vehicle
(0.1% CMC-Na) alone, equimolar doses of NBP, ZJM-289,
(S)- and (R)-ZJM-289, respectively. Two hours after the treat-
ment, the mice were injected intravenously with collagen and
adrenaline to induce thromboembolism, and the development of
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Table 2 Effects of (S)- and (R)-ZJM-289 on death and hemiplegia
induced by i.v. collagen–adrenaline in mice
Number of death
in 5 min and
hemiplegia
in 15 min
Dose
Protection
rate (%)
Group
(mg kg−1
)
n
Control
NBP
ZJM-289
(S)-ZJM-289
(R)-ZJM-289
—
17
17
18
20
19
11
5
5
5
7
35.3
100
342
342
342
70.6a
72.2a
75.0a
68.4a
a P < 0.05 vs. control group.
Fig. 2 Changes of cerebral blood flow in the ischemic cortex before,
during and after MCAO measured by laser Doppler flowmetry. Male SD
rats were randomized and treated orally with 0.1% CMC-Na, NBP,
ZJM-289, (S)- and (R)-ZJM-289 2 h before MCAO. The animals were
subjected to 2 h MCAO and 2 h reperfusion. Data are expressed as mean
SD (n = 6). *P < 0.05 vs. vehicle group, △P < 0.05 vs. NBP group,
determined with one-way ANOVA followed by Tukey’s post hoc test.
2.4. Neuroprotection of (S)- and (R)-ZJM-289 against focal
cerebral ischemia/reperfusion (I/R)
The neuroprotection of (S)- and (R)-ZJM-289 against focal cere-
bral I/R was studied using a model of middle cerebral artery
occlusion (MCAO). Male SD rats were randomized and treated
orally with 0.1% CMC-Na (sham group and vehicle group),
NBP, ZJM-289, (S)- and (R)-ZJM-289 at equimolar doses
(2.63 × 10−4 mol kg−1).
accompanied with decreased density. However, treatment with
(S)- and (R)-ZJM-289, as well as ZJM-289 obviously increased
the amount of normal neurons, and significantly diminished the
extent of damage, respectively.
Since cerebral blood flow (CBF) has a close relation with
brain injury, we examined whether preconditioning of the enan-
tiomers were associated with an improvement of ischemic CBF.
As shown in Fig. 2, MCAO caused more than 70% reduction in
the regional cerebral blood flow (rCBF) compared with the pre-
occlusion baseline values and this almost completely reversed
by reperfusion in all groups. Compared with vehicle and NBP
groups, the two enantiomers and ZJM-289 markedly increased
rCBF during MCAO, and there was no significant difference
between (S)-ZJM-289, (R)-ZJM-289 and ZJM-289 groups.
The neuroprotective effects of (S)- and (R)-ZJM-289 were also
evaluated by examining neurological deficit, infarct volume
and brain water content after three days of reperfusion. NBP,
ZJM-289, (S)- and (R)-ZJM-289 were administered just after the
onset of reperfusion and continued for three days in this study.
Transient two hours of focal cerebral ischemia followed by reper-
fusion for three days induced brain infarct formation and neuro-
logical deficits. The two enantiomers and ZJM-289 appeared to
be equally effective in reducing neurological deficits (Fig. 3a)
and infarct volume (Fig. 3b) while there was a significant differ-
ence between (S)-ZJM-289 and NBP group (P < 0.05). As
shown in Fig. 3c, I/R resulted in apparent brain edema (83.04
1.26%, P < 0.05) compared with sham-operated group (78.82
0.76%), which was also significantly improved in (S)-ZJM-289
(79.28 0.95%), (R)-ZJM-289 (80.63 1.08%) and ZJM-289
2.5. Vascular modulation as another evidence of the
neuroprotective effect of the enantiomers of ZJM-289
At three days after reperfusion, NO production in plasma was
evaluated indirectly by measuring nitrite/nitrate (NOx), and
levels of ET-1, TXB2, 6-keto-PGF1α, cGMP and cAMP in
plasma were quantified by radioimmunoassay or enzyme-linked
immunosorbent assay. In the present study, levels of NOx were
decreased significantly in the vehicle group compared to the
sham group (Fig. 4a), which was in agreement with an earlier
report where the plasma NOx level was reduced in acute stroke.15
Like ZJM-289, its enantiomers distinctly increased plasma NOx
concentration compared to vehicle and NBP groups. As a power-
ful biological marker of developing brain edema,16 the plasma
level of ET-1 was two-folder higher in the vehicle group (82.49
20.66) as compared with sham group (39.99 18.48). Treat-
ment with (S)- and (R)-ZJM-289, significantly reduced plasma
ET-1 level to 43.05 15.06, 47.91 11.14 and 47.34 6.96
pmol L−1, respectively (Fig. 4b), similar to ZJM-289. By con-
trast, NBP had no obvious effect on plasma NOx and ET-1
levels. Furthermore, the ratio of TXB2 : 6-keto-PGF1α was
increased significantly after focal cerebra I/R, and cAMP was
decreased to a very low level, which might contribute to I/R
injury. As shown in Fig. 4c and 4d, the two enantiomers and
ZJM-289 could restore the dynamic balance between TXB2 and
6-keto-PGF1α and elevate plasma cAMP level to the baseline
level. NBP decreased the ratio of TXB2 : 6-keto-PGF1α and
failed to improve the plasma cAMP level. Meanwhile, the enan-
tiomers-, ZJM-289- and NBP-treated rats elevated the plasma
cGMP level relative to the sham-operated rats, but without sig-
nificant difference in these groups.
(80.29
1.13%) groups. Compared with NBP group, (S)-
ZJM-289 showed more intense decline in the brain water
content.
To observe the effects of (S)- and (R)-ZJM-289 on cortical
neuronal damage, HE staining was performed. As shown in
Fig. 3d, in the sham group, the brain tissues were kept intact
and no inflammatory cells infiltrated, cortical neurons kept
eumorphism and arranged well, the cytoplasm was abundant
and the nuclei were centered with clear staining. In contrast,
in the vehicle group, cortical neurons were degenerated
and necrotic, and their arrangement was disordered, and
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Fig. 3 Neuroprotective effects of (S)- and (R)-ZJM-289 following focal cerebral ischemia and reperfusion. Male SD rats received 2 h MCAO fol-
lowed by 3 days reperfusion and treated orally with 0.1% CMC-Na, NBP, ZJM-289, (S)- and (R)-ZJM-289 just after the onset of reperfusion and con-
tinued for 3 days. Sham-operated rats were subjected to the same surgery, except MCAO, and treated orally with 0.1% CMC-Na. Data are expressed as
#
mean SD (n = 10). P < 0.05 vs. sham group, *P < 0.05 vs. vehicle group, △P < 0.05 vs. NBP group, determined with one-way ANOVA followed
by Tukey’s post hoc test. (a) Neurologic deficits were assessed at 3 days after reperfusion according to Longa’s method. (b) Effects of (S)- and
(R)-ZJM-289 on the brain infarct size. Animals were killed at 3 days after reperfusion and infarcted brain regions were visualized using TTC staining.
(c) Effects of (S)- and (R)-ZJM-289 on the brain water content. After 3 days of reperfusion, the brain samples were weighed immediately after
dissection (wet weight) and then dried at 105 °C for 24 h. (d) Cerebral sections were stained with hematoxylin and eosin and examined under light
microscope at 3 days after reperfusion.
2.6. The enantiomers of ZJM-289 protected rat brain capillary
endothelial cells (BCECs) against ischemia-like injury
injury, and treated with ZJM-289, (S)- and (R)-ZJM-289 for two
hours before and during OGD/R. BCECs mortality was assessed
by MTT and LDH assay (by measuring lactate dehydrogenase
activity in the medium), respectively. For detection of cell
apoptosis, cells were labeled with FITC-conjugated Annexin V
(Annexin V-FITC) and propidium iodide (PI). The fluorescence
was examined using a flow cytometry.
BCECs are a primary target of ischemic brain insults and
cerebral endothelial dysfunctions may contribute to postischemic
secondary brain injury.17 In this study, we investigated the pro-
tective effects of (S)- and (R)-ZJM-289 in rat BCECs. The
BCECs in cultures were exposed to oxygen–glucose deprivation
followed by reoxygenation (OGD/R), a model of ischemia-like
As shown in Fig. 5a, the viability of the BCECs exposed
to OGD/R was reduced to 52.6
5.6% compared with the
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Fig. 4 Effects of (S)- and (R)-ZJM-289 on NOx concentration (a), ET-1 level (b), the ratio of TXB2 : 6-keto-PGF1α (c) and cAMP : cGMP level (d)
in plasma. Male SD rats received 2 h MCAO followed by 3 days reperfusion and treated orally with 0.1% CMC-Na, NBP, ZJM-289, (S)- and (R)-
ZJM-289 just after the onset of reperfusion and continued for 3 days. Sham-operated rats were subjected to the same surgery, except MCAO, and
treated orally with 0.1% CMC-Na. Data are expressed as mean SD (n = 10). #P < 0.05 vs. sham group, *P < 0.05 vs. vehicle group, △P < 0.05 vs.
NBP group, determined with one-way ANOVA followed by Tukey’s post hoc test.
control. Pretreatment with (S)- and (R)-ZJM-289, as well as
ZJM-289, significantly attenuated OGD/R-induced cell death
and the cell viability was significantly increased to 81.2 3.7,
70.4 5.5 and 74.0 4.1%, respectively. The cytoprotective
effects of (S)- and (R)-ZJM-289 were also confirmed by LDH
assay (Fig. 5b). Negative annexin V-FITC and PI labeling
(Fig. 5c) showed a near absence of apoptosis and necrosis in
control BCECs. The cultures subjected to OGD/R contained
apoptotic (37.03 2.07%) and necrotic (12.45 1.49%) cells
significantly (P < 0.05) more than the control cultures.
The enantiomers equally and significantly decreased apoptotic
and necrotic cells as ZJM-289, and the protective effect of
(S)-ZJM-289 was stronger than that of NBP.
We have previously reported ZJM-289 as a potent antiplatelet
aggregation and antithrombotic agent. Since it is a racemic com-
pound, we further synthesized the enantiomers of the ZJM-289,
and simultaneously evaluated their antiplatelet, antithrombotic,
and neuroprotective effects in the present study.
Compared with NBP, the two enantiomers and ZJM-289
displayed much stronger inhibitory activity on the ADP- and
TH-induced platelet aggregation, but comparable effect on the
AA-induced platelet activation. Additionally, the two enantio-
mers and ZJM-289 appeared to be almost equally effective in
suppressing thrombus formation in the rat arteriovenous-shunt
thrombosis model and in the mouse pulmonary embolization
model, while (R)-ZJM-289 was somewhat less active.
Previous studies concluded that the ratio of TXA2 : PGI2
modulated vasomotion and thrombosis, which might contribute
to the pathogenic consequences of ischemic stroke.18 Therefore
we examined the contents of TXB2 and 6-keto-PGF1α, the stable
metabolites of TXA2 and PGI2. Our results showed that the
enantiomers of ZJM-289 significantly decreased the ratio of
TXA2 : PGI2 in rats after I/R, which was in agreement with their
3. Discussion and conclusions
It is well-known that platelet activation contributes significantly
to ischemic microvascular occlusion and causes secondary injury
after the initial ischemic insult. Thus, inhibition of platelet aggre-
gation in the cerebral microvessels is of great importance.
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Fig. 5 (S)- and (R)-ZJM-289 protects primary cultured brain capillary endothelial cells (BCECs) against OGD/R-induced cytotoxicity. Primary cul-
tures of rat brain capillary endothelial cells were prepared from 2-week-old rats and maintained in DMEM/F12 supplemented with 20% fetal bovine
serum, heparin (100 U mL−1) and basic fibroblast growth factor (1.5 ng mL−1). BCECs were subjected to 2 h OGD followed by 24 h reoxygenation
(OGD/R) and treated with 1 μM NBP, ZJM-289, (S)- and (R)-ZJM-289 2 h before and during OGD/R, respectively. (a) Effects of (S)- and (R)-
ZJM-289 on the viability of OGD/R injured BCECs, determined using the MTT reduction assay (n = 6). (b) Cell death was assessed by testing LDH
activity in the supernatant culture medium (n = 6). (c) Representative dot plots for quantification of mode of death (n = 3). The mean fluorescence
from endothelial rich populations using FITC-conjugated Annexin V and propidium iodide (PI) were measured by flow cytometry. Data are expressed
#
as mean SD. P < 0.05 vs. control group, *P < 0.05 vs. OGD/R group, △P < 0.05 vs. NBP group, determined with one-way ANOVA followed by
Tukey’s post hoc test.
inhibitory effects on AA-induced platelet aggregation and antith-
rombotic effects. It appears likely that the enantiomers of
ZJM-289 exerted neuroprotective effects against I/R injury
benefiting from their antiplatelet and antithrombotic activities.
Another important and favorable effect imparted by the enan-
tiomers of ZJM-289 on ischemic stroke is associated with their
releasing moderate levels of NO. Compared with NBP, the two
enantiomers intensely increased the CBF in the ischemic region,
probably due to the NO released by these compounds. In
addition to its direct vasodilative effect, NO released by the two
enantiomers may contribute to suppressing the overproduction of
ET-1, a vasoconstrictor involved in severe ischemic brain injury,
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(−)-2-(1-Hydroxypentyl)benzoate (3). To a stirred solution of
compound 2 (2.08 g, 10.00 mmol) in cold Et2O was added
(S)-FEA (1.21 g, 10.00 mmol, ee > 98%) dropwise such that the
internal reaction temperature did not exceed −10 °C. The reac-
tion mixture was allowed to stand for 5–10 min at −20 to
−10 °C and then 6–10 h at RT and filtered to obtain the crude
product as a white solid. The solid was recrystallized repeated
2 times from acetone to obtain compound 3 as white needle-like
crystals (1.58 g, 48% yield for the two steps). mp 123–124 °C,
[α]2D5 −8.94° (c 1.12, CH3OH). IR (KBr): 1392, 1517, 1566,
subsequently enhancing CBF. By contrast, NBP had no such
effects on levels of plasma NO and ET-1, leading to less aug-
mentation of CBF. Furthermore, consistent with our previous
observation that ZJM-289 stimulated NO–sGC–cGMP pathway,
the enantiomers indeed elevated plasma cGMP level in the
present experiments. As the second messenger, cGMP mediates
various physiological responses such as inhibition of platelet
adhesion, neurotransmission and many other processes.19 There-
fore, it seems reasonable to speculate that the neuroprotection
of the two enantiomers, as well as their antiplatelet and
antithrombotic actions, are partly mediated through cGMP
pathways.
Furthermore, we found that pretreatment with the two enantio-
mers and ZJM-289 could protect BCECs against hypoxic-
ischemic injury, which may be partially mediated by NO
released from them. As a result, the two enantiomers and
ZJM-289 could modulate endothelial function by maintaining
NO level in ischemic cerebral vasculature, which contributes not
only to their inhibition of platelet adhesion and aggregation, and
decreased thrombus formation, but also to their neuroprotection.
In conclusion, (S)-ZJM-289, (R)-ZJM-289 and ZJM-289 have
comparable inhibitory effects on platelet aggregation and throm-
bus formation in vitro and in vivo. Additionally, the two enantio-
mers and ZJM-289 appear to be equally effective in a rat model
of focal cerebral I/R. And (S)-ZJM-289 is somewhat better than
(R)-ZJM-289 in a few respects, which might be owing to their
slight discrepancy in metabolism and pharmacokinetics proper-
ties, and the precise mechanisms remain to be investigated. The
present findings may provide new insight into the development
of novel antiischemic stroke agents like ZJM-289.
l
1641, 3411 cm−1. H NMR ((CD3)2CO, 300 Hz, δ): 0.80–1.00
(m, 3H, CH3), l.22–1.50 (m, 4H, CH2CH2), 1.64–1.88 (m, 5H,
CH2, CH3), 3.62 (s, 1H, OH), 4.56 (dd, 1H, CH–NH2, J = 7.2
Hz), 4.94 (dd, 1H, CH–OH, J = 3.4 Hz, 7.2 Hz), 7.14–7.66
(m, 8H, ArH), 7.70–7.84 (dd, 1H, ArH, J = 1.8 Hz, 7.2 Hz).
(+)-2-(1-Hydroxypentyl)benzoate (4). Compound 4 was syn-
thesized in the same manner as compound 3, starting from
compound 2 (2.08 g, 10.00 mmol) and (R)-FEA (1.21 g,
10.00 mmol, ee > 98%), compound 4 was obtained as white
needle-like crystals (1.51 g, 46% yield for the two steps). mp
1
123–124 °C, [α]2D5 +8.89° (c 1.05, CH3OH). IR (KBr) and H
NMR spectral data were identical to those of compound 3.
(−)-3-Butyl-1(3H)-isobenzofuranone ((S)-NBP). To a stirred
solution of compound 3 (1.58 g, 4.83 mmol) in H2O (20 mL)
was added 2 M NaOH and stirred for 0.5 h at RT. The mixture
was extracted with Et2O (10 mL × 3). The water layer was
acidified with 3 M HCl to pH 2 and stirred for 0.5 h at RT. The
solution was then extracted with Et2O (10 mL × 3). The com-
bined organic layers were dried, filtered and evaporated to
dryness to afford (S)-NBP as a light yellow oil (0.87 g, 94.8%).
[α]2D7 −66.6° (c 1.00 CHCl3). ee = 98.1%. ESI-MS: m/z 213
1
[M + Na]+. H NMR (CDCl3, 300 Hz, δ): 0.93 (t, 3H, CH3, J =
4. Experimental section
3.1 Hz), 1.37–1.45 (m, 4H, 2 × CH2), 1.86–1.89 (m, 2H, CH2),
5.48–5.49 (m, 1H, CH), 7.37–7.39 (m, 1H, ArH), 7.56–7.5 (m,
2H, ArH), 8.05 (d, 1H, ArH, J = 6.5 Hz).
4.1 Chemistry
General. NO donor moieties 6 were prepared as described
previously.14 Melting points were determined using a capillary
apparatus (RDCSYI). All of the synthesized compounds were
purified by column chromatography (CC) on silica gel 60
(200–300 mesh) or thin layer chromatography (TLC) on silica
gel 60 F254 plates (250 mm; Qingdao Ocean Chemical
Company, China). Subsequently, they were routinely analyzed
by IR (Shimadzu FTIR-8400S), 1H NMR and 13C NMR (Bruker
ACF-300Q, 300 MHz), and MS (Hewlett-Packard 1100 LC/
MSD spectrometer). The purity of test compounds was charac-
terized by HPLC analysis (Shimadzu LC-2010A HPLC system)
and high resolution mass spectrometry (Agilent technologies
LC/MSD TOF). Target compounds (S)-ZJM-289 and (R)-
ZJM-289 with a purity of >97% were used for subsequent exper-
iments (see the ESI†).
(+)-3-Butyl-1(3H)-isobenzofuranone ((R)-NBP). Compound
(R)-NBP was synthesized in the same manner as compound
(S)-NBP, starting from compound 4 (1.51 g, 4.59 mmol), com-
pound (R)-NBP was obtained as a light yellow oil (0.84 g, 96%).
[α]2D7 +68.4° (c 1.00 CHCl3). ee = 97.4%. IR (KBr), ESI-MS
and 1H NMR spectral data were identical to those of (S)-NBP.
(−)-2-(1-Hydroxypentyl)benzoic acid ((S)-2). Compound (S)-2
was synthesized in the same manner as compound 2. Starting
from compound (S)-NBP (1.90 g, 10.00 mmol), compound (S)-2
(2.08 g, 10.00 mmol) in cold Et2O was used for the next step
without any purification.
(−)-2-[1-(2-Chloroacetoxy)pentyl]benzoic acid ((S)-5). A solu-
tion of 2-chloroacetyl chloride (4.16 mL, 30.00 mmol) in dry
CH2Cl2 (10 mL) was added dropwise to a mixture of (S)-2
(2.08 g, 10.00 mmol), Et3N (4.17 mL, 30.00 mmol) and DMAP
(1.0 mmol) in CH2Cl2 (150 mL) at −10 °C and the solution was
left stirring at −10 °C for 5 h. The mixture was acidified with
1 M HCl to pH 2 and stirred for 1 h at RT. The organic layer was
washed with water, dried, filtered, and evaporated to dryness.
The residue was recrystallized from n-hexane to obtain (S)-5
as white crystals (1.85 g, 65%, over two steps). mp 67–68 °C.
( )-2-(1-Hydroxypentyl)benzoic acid (2). To a stirred solution
of NBP (1.90 g, 10.00 mmol) in CH3OH–H2O (20 mL, 1 : 1 v/v)
was added NaOH (0.80 g, 20.00 mmol). The reaction mixture
was heated at 50 °C for 0.5 h. The solvent was removed under
reduced pressure and dissolved in water (20 mL), followed by
acidification with 5% HCl to pH 3–4 at −10 to 0 °C. The
mixture was extracted with cold Et2O (10 mL × 3) and quickly
used for the next step without any purification.
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[α]2D7 −40.8° (c 1.00 CHCl3). ESI-MS: m/z 283 [M − H]−. IR
brine. The solution was then dried, filtered, and evaporated
to dryness. The residue was purified by flash chromatography
(petroleum ether–EtOAc 3 : 1 v/v), to which EtOAc (2.0 mL)
and 1 M ethereal HCl (2.0 mL) was added dropwise at 0 °C. The
solution was left stirring at RT for 1 h and filtered. The residue
was purified the title compound as a white solid (1.01 g, 82%,
for two steps). [α]2D7 +7.5° (c 1.10 CHCl3). ee = 99.5%. mp
106–108 °C. ESI-MS: m/z 615 [M + H]+. IR (KBr): 759, 1254,
1
(KBr): 1412, 1691, 1734, 2958, 3450 cm−1. H NMR (CDCl3,
300 Hz, δ): 0.93 (t, 3H, CH3, J = 4.2 Hz), 1.37–1.42 (m, 4H,
2 × CH2), 1.88–1.91 (m, 2H, CH2), 4.11 (m, 2H, COCH2Cl),
6.78 (m, 1H, CH), 7.36–7.42 (m, 1H, ArH), 7.56–7.62 (m, 2H,
ArH), 8.08 (d, ArH, J = 8.1 Hz), 10.89 (brs, 1H, COOH). 13C
NMR (CDCl3, 300 Hz, δ): 172.0, 166.5, 140.8, 133.1, 130.3,
130.0, 127.1, 125.7, 74.8, 41.0, 36.3, 27.8, 22.4, 13.8.
1
1630, 1747, 2861, 2956, 3444 cm−1. H NMR (CDCl3, 300 Hz,
(+)-2-[1-(2-Chloroacetoxy)pentyl]benzoic acid ((R)-5). Com-
pound (R)-5 was synthesized in the same manner as compound
(S)-5. Starting from compound (R)-NBP (1.90 g, 10.00 mmol),
compound (R)-5 was obtained as a white crystals (1.79 g, 63%,
over two steps). mp 67–68 °C. [α]2D7 +39.6° (c 1.00 CHCl3). IR
(KBr), ESI-MS, 1H NMR and 13C NMR spectral data were iden-
tical to those of (S)-5.
δ): 0.86 (t, 3H, CH3, J = 6.6 Hz), 1.37 (m, 7H, 2 × CH2 and
CH3), 1.48 (t, 3H, CH3, J = 7.2 Hz), 1.78 (m, 6H, 2 × CH3),
3.26–3.41 (m, 4H, 2 × NCH2), 3.89 (s, 3H, OCH3), 4.00 (m,
2H, COCH2N), 4.27 (t, 2H, CH2ONO2, J = 5.4 Hz), 4.53 (t, 2H,
COOCH2, J = 5.7 Hz), 6.42 (d, 1H, CH, J = 15.9 Hz), 6.76 (m,
1H, CH), 7.19 (m, 3H, ArH), 7.44–7.65 (m, 3H, ArH), 7.68 (d,
1H, CH, J = 16.0 Hz), 8.21 (d, 1H, ArH, J = 7.8 Hz), 12.73
(brs, 1H, NH+). 13C NMR (CDCl3, 300 Hz, δ): 166.4, 164.5,
164.4, 151.5, 144.2, 142.6, 141.4, 133.5, 133.4, 131.2, 128.1,
127.1, 126.1, 123.3, 121.3, 118.2, 111.4, 75.3, 72.7, 63.6, 55.9,
48.6, 48.5, 48.2, 36.1, 27.9, 25.1, 23.7, 22.3, 13.9, 10.1, 10.0.
HRMS for C32H43N2O10 ([M + H]+) calcd: 615.2918, found:
615.2931.
(+)-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {4-[2-(4-nitro-
oxybutoxycarbonyl)vinyl]}phenyl ester ((S)-7). To a stirred solu-
tion of acylated compound (S)-5 (1.42 g, 5.0 mmol) and
compound 6 (1.56 g, 5.0 mmol) in anhydrous CH2Cl2 (60 mL)
was added DMAP (0.2 mmol) and the solution was left stirring
at 0 °C for 10 min. DCC (1.13 g, 5.5 mmol) was then added and
the solution was left stirring at RT for 6 h. The solution was then
filtered, and the filtrate washed sequentially with 1 M HCl, a
saturated aqueous solution of NaHCO3, water, and brine. The
solution was then dried, filtered, and evaporated to dryness. The
residue was purified by flash chromatography (petroleum ether–
EtOAc 5 : 1 v/v) to obtain compound (S)-7 as a colourless waxy
solid (2.27 g, 83%), mp 60–62 °C. [α]2D7 +6.6° (c 1.00 CHCl3).
(−)-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {2-
methoxy-4-[2-(4-nitrooxybutoxycarbonyl)vinyl]}phenyl
ester
hydrochloride ((R)-ZJM-289). Compound (R)-ZJM-289 was
synthesized in the same manner as compound (S)-ZJM-289.
Starting from compound (R)-7 (1.09 g, 2.00 mmol), compound
(R)-ZJM-289 was obtained as a white solid (1.03 g, 84%, for
two steps). mp 106–108 °C. [α]2D5 −7.1° (c 1.12 CHCl3). ee =
98.6%. IR (KBr), ESI-MS, 1H NMR, 13C NMR and HRMS
spectral data were identical to those of (S)-ZJM-289.
1
ESI-MS: m/z 595 [M + NH4]+. H NMR (CDCl3, 300 Hz, δ):
0.86 (t, 3H, CH3, J = 6.9 Hz), 1.26–1.52 (m, 4H, 2 × CH2),
1.85–1.94 (m, 6H, 4 × CH2), 4.00 (m, 2H, COCH2Cl), 4.26
(t, 2H, CH2ONO2, J = 5.8 Hz), 4.53 (t, 2H, OCH2, J = 6.0 Hz),
6.42 (d, 1H, CH, J = 15.9 Hz), 6.68 (m, 1H, CH), 7.26–7.30 (m,
2H, ArH), 7.43 (m, 1H, ArH), 7.55–7.63 (m, 4H, ArH), 7.70 (d,
1H, CH, J = 16.0 Hz), 8.15 (d, 1H, ArH, J = 8.0 Hz). 13C NMR
(CDCl3, 300 Hz, δ): 166.7, 166.6, 164.9, 152.3, 144.0, 143.6,
133.3, 132.3, 130.8, 129.4, 129.4, 127.7, 127.3, 126.4, 122.4,
122.4, 118.0, 74.8, 72.6, 63.6, 41.0, 36.3, 27.9, 25.1, 23.7, 22.4,
13.9. HRMS for C27H30NO9ClNa ([M + Na]+) calcd: 570.1507,
Found: 570.1516.
4.2 Platelet aggregation in vitro
Blood samples were withdrawn from rabbit carotid artery and
mixed with 3.8% trisodium citrate (9 : 1 v/v), followed by centri-
fuging at 500 rpm for 10 min. The supernatants were collected
and used as platelet rich plasma (PRP). Additional samples were
centrifuged at 3000 rpm for 10 min and the supernatants were
collected as platelet poor plasma (PPP). The effect of individual
compounds on the ADP-, TH- and AA-induced platelet aggrega-
tion was measured by Born’s turbidimetric method using a
Platelet-Aggregometer (LG-PABER-I Platelet-Aggregometer,
Beijing). Briefly, PRP (240 μL) was pre-treated in duplicate with
vehicle, different concentrations of NBP, ZJM-289, (S)- and
(R)-ZJM-289 for 5 min and exposed to 10 μM of ADP, 0.5 U
mL−1 of TH, or 1.0 mM of AA incubated at 37 °C for 5 min,
respectively. The formation of platelet aggregation was moni-
tored longitudinally by optical density. Compounds under study
or vehicle alone were added to the PRP samples 5 min before
addition of the aggregating agent. The antiplatelet aggregatory
activity of NBP, ZJM-289, (S)- and (R)-ZJM-289 was evaluated
as percent inhibition of platelet aggregation compared to positive
controls that had been pre-treated with vehicle alone and
exposed to the inducer samples. The IC50 values of these com-
pounds were determined by nonlinear regression analysis or the
percent inhibition at the maximal concentration tested (1.0 mM)
was calculated.
(−)-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {4-[2-(4-nitro-
oxybutoxycarbonyl)vinyl]}phenyl ester ((R)-7). Compound (R)-7
was synthesized in the same manner as compound (S)-7.
Starting from compound (R)-5 (1.42 g, 5.00 mmol), compound
(R)-7 was obtained as a colourless waxy solid (2.16 g, 80%).
1
mp 60–62 °C. [α]2D7 −10.6° (c 1.10 CHCl3). ESI-MS, H NMR,
13C NMR and HRMS spectral data were identical to those
of (S)-7.
(+)-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {2-
methoxy-4-[2-(4-nitrooxybutoxycarbonyl)vinyl]}phenyl
ester
hydrochloride ((S)-ZJM-289). To a stirred solution of (S)-7
(1.09 g, 2.00 mmol) and Et3N (1.1 mL, 8.00 mmol) in DMF
(10 mL) was added diethylamine (0.6 mL, 6.00 mmol) and the
solution was left stirring at RT for 8 h. The solution was then
filtered, and the filtrate was reconstituted in EtOAc (30 mL) and
water (30 mL). The organic layer was washed with water and
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ZJM-289 (172 mg kg−1, i.g.) group, (6) I/R + (R)-ZJM-289
(172 mg kg−1, i.g.) group. The drugs were administered daily,
starting at the onset of reperfusion and continued for 3 days.
4.3 Antithrombotic activity assay in rats
The antithrombotic activities of NBP, ZJM-289, (S)- and (R)-
ZJM-289 were tested in male SD rats, as previously described.20
Briefly, after anesthesia, rats were subjected to surgical exposure
of their neck areas and inserted with an 8 cm polyethylene tube
connecting the left jugular vein and right carotid artery. The
saline-filled shunt was assembled by connecting two cannulae
with a 6 cm slightly curved Tygon tubing containing a 6 cm
long cotton thread. The rats were maintained on extracorporeal
circulation for 15 min, during which a thrombus formed and
adhered to the cotton thread. The shunt was then removed and
the thread with the associated thrombus was withdrawn and
immediately weighed. The wet weight of thrombus was deter-
mined by subtracting the weight of thread from the total wet
weight. Antithrombotic efficacy was indicated by decreases in
the weight of thrombus formed in 15 min of exposure to flowing
blood.
4.5.2 Neurologic scoring. Neurologic scores were evaluated
by a blinded observer at 3 days after reperfusion, according to
Longa’s method.21
4.5.3 Measurement of infarct size. At 3 days after reperfu-
sion, rats were decapitated and the brains were rapidly removed
onto ice and sliced into six 2 mm-thick coronal sections. Sec-
tions were stained with 2% 2,3,5-triphenyltetrazolium chloride
for 30 min at 37 °C followed by overnight immersion in 10%
formalin PBS. The infarcted tissue remained unstained (white),
whereas normal tissue was stained red. The infarct zone was
demarcated and analyzed by image processing software. The
infarcted area was numerically integrated across each section and
was expressed as a percentage of the total area of whole brain.
4.5.4 Brain-water content. The brain samples were weighed
immediately after dissection (wet weight) and then dried at
105 °C for 24 h. The percent of water content was calculated
with the formula (wet weight − dry weight)/wet weight × 100%.
4.4 Effect of compounds on the thrombosis in mice
Male Swiss mice (30–35 g, from ICR animal colony) were
randomized and treated orally with vehicle alone or compounds
at indicated concentrations for two hours, respectively. Sub-
sequently, the mice were injected intravenously with a mixture of
1.0 mg mL−1 of collagen and 44.5 μg mL−1 of adrenaline, and
observed for thrombosis-related death for 5 min and hemiplegia
for 15 min. The antithrombotic effects of NBP, ZJM-289,
(S)- and (R)-ZJM-289 were calculated as percent protection rela-
tive to controls injected with vehicle alone. The experimental
protocols of animal studies were approved by the Animal
Research Care Committee of China Pharmaceutic University.
4.5.5 Histopathology. The ipsilateral hemisphere of ischemic
brain samples was harvested and post-fixed in formalin, and cut
coronally at 2 mm intervals from the frontal pole. Paraffin-
embedded coronal slices sectioned at 5 μm, and stained with
hematoxylin–eosin. Histopathological evaluation was performed
by a pathologist blinded to the treatment group.
4.5.6 Measurement of regional cerebral blood flow (rCBF).
rCBF during I/R was monitored continuously by laser-Doppler
flowmetry (BIOPAC MP 150, CA, USA) after treatment with
NBP, ZJM-289, (S)- and (R)-ZJM-289. At the ischemic hemi-
sphere, a 2 × 2 mm cranial window was drilled through the skull
2.0 mm lateral to the midline and 3.0 mm caudal to bregma. The
laser-Doppler probe was mounted on a micromanipulator and
placed over the brain surface. The analog output from the probe
was fed into an analog-to-digital converter and displayed on a
chart recorder. rCBF was expressed as absolute laser-Doppler
units.
4.5 Neuroprotection against focal cerebral ischemia/
reperfusion (I/R)
4.5.1 Ischemic/reperfusion (I/R) model and drug adminis-
tration. Male Sprague–Dawley rats (250–300 g) were purchased
from B&K Universal Group Limited, Shanghai, China. Animals
were kept under standard laboratory conditions and maintained
in a 12 h light–12 h dark cycle with free access to food and
water. Animals care and treatment were conducted in accordance
with the institutional guidelines. Rats were anesthetized with
chloral hydrate (300 mg kg−1, i.p.) and subjected to middle cere-
bral artery occlusion (MCAO) as described previously with
slight modification.21 Briefly, the right common carotid artery,
internal carotid artery (ICA), and external carotid artery (ECA)
were exposed. A 4-0 monofilament nylon suture (Beijing Sunbio
Biotech Co., Ltd., Beijing, China) with a rounded tip was
inserted into the ICA through the ECA stump and gently
advanced to occlude the MCA. After 2 h of MCAO, the suture
was withdrawn to restore blood flow (reperfusion). Sham-oper-
ated rats were manipulated in the same way, but the MCA was
not occluded. Core body temperatures were monitored with a
rectal probe and maintained at 37 °C 0.5 °C during the whole
procedure.
4.5.7 Measurement of NO, ET-1, TXB2, 6-keto-PGF1α
,
cGMP and cAMP. Carotid artery blood samples were collected
in tubes containing EDTA and centrifuged at 3000 rpm for
10 min, at 4 °C. NO production was evaluated indirectly by
measuring nitrite/nitrate (NOx), the stable metabolites of NO.
NOx content in plasma was measured using colorimetric total
NO assay kit (JianCheng Bioengineering Institute, Nanjing,
China) as described previously.22 Plasma cGMP and cAMP
levels were determined using a commercial enzyme immuno-
assay (R&D Systems, Minneapolis, MN, USA).
Two milliliters of carotid artery blood were collected in tubes
containing 40 μL aprotinin and 30 μL EDTA. These samples
were immediately centrifuged at 3000 rpm for 10 min. Collected
plasma was stored at −80 °C until batched assays were per-
formed. Plasma ET-1 levels were evaluated with ET radio-
immunoassay kit (Beijing Purevalley Biotechnology Co., Ltd.,
Beijing, China) according to the manufacturer’s instructions.
Rats were divided randomly into 6 groups: (1) sham group,
(2) vehicle group (I/R), (3) I/R + NBP (50 mg kg−1, i.g.) group,
(4) I/R + ZJM-289 (172 mg kg−1, i.g.) group, (5) I/R + (S)-
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Three milliliters of carotid artery blood were mixed with
200 μL indomethacin-EDTA·2Na and immediately centrifuged at
3500 rpm for 15 min. Collected plasma was stored at −80 °C
until assayed. The levels of TXB2 and 6-keto-PGF1α were
measured by radioimmunoassay according to the manufacturer’s
instructions (Beijing Purevalley Biotechnology Co., Ltd,
Beijing, China).
(BD FACSCanto, NJ, USA), which discriminated intact cells
(FITC−/PI−), apoptotic cells (FITC+/PI−) and necrotic cells
(FITC+/PI+).
Acknowledgements
This study was financially supported by grants from the
Major National Science and Technology Program of China for
Innovative Drug during the Eleventh Five-Year Plan Period
(no. 2009ZX09103-095) and the Project Program of State
Key Laboratory of Natural Medicines, China Pharmaceutical
University (no. ZJ11176).
4.6 The enantiomers of ZJM-289 protected rat BCECs against
OGD/R injury
4.6.1 Rat BCECs culture. Primary cultures of BCECs were
prepared from 2-week-old Sprague-Dawley rats as described pre-
viously with some modifications.23 Briefly, the isolated cerebral
gray matter was digested by trypsin (0.05%) at 37 °C for 30 min,
then filtered through 145 μm nylon mesh. The filtrate was
filtered through 75 μm nylon mesh and the matter was collected
on the nylon mesh. Then the matter was digested by collagenase
type II (0.1%) at 37 °C for 30 min. Cells were collected by
centrifugation at 1500 rpm for 5 min and resuspended in
DMEM–F12 (1 : 1) with 20% (v/v) fetal bovine serum, 100 U
mL−1 penicillin, 100 U mL−1 streptomycin, 100 U L−1 heparin
and 1.5 ng mL−1 basic fibroblast growth factor. Cells were
seeded in gelatin coated 96- or 6-well plates and cultured in a
humidified incubator in air with 5% CO2 (Thermo Scientific
3110, OH, USA).
Notes and references
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2 G. Stoll, C. Kleinschnitz and B. Nieswandt, Blood, 2008, 112, 3555.
3 N. Mackman, Nature, 2008, 451, 914.
4 B. Chen, Q. Cheng, K. Yang and P. D. Lyden, Stroke, 2010, 41, 2348.
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4.6.2 Oxygen glucose deprivation and reoxygenation (OGD/R)
treatment. BCECs were rinsed twice with phosphate buffered
saline (PBS), and OGD was induced with Earle’s solution
without glucose (pH 7.4, 143 mM NaCl, 5.4 mM KCl, 1.0 mM
MgSO4, 1.0 mM NaH2PO4, 1.8 mM CaCl2, and 2.4 mM
HEPES). The cultures were introduced into an anaerobic
chamber flushed with 5% CO2 and 95% N2 for 15 min. The
chamber was tightly sealed and placed in an incubator at 37 °C
for 2 h. Controls were incubated with Earle’s solution containing
5.6 mM glucose and maintained in an incubator with 5% CO2
atmosphere at 37 °C. After the deprivation period, cultures were
returned back to the normal culture medium under normoxic
conditions for 24 h, corresponding to the reoxygenation period.
NBP, ZJM-289, (S)- and (R)-ZJM-289 were applied to BCECs
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fluorescence was then examined using a flow cytometry
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