Novel hybrids of optically active ring-opened 3-n-butylphthalide derivative and isosorbide as potential anti-ischemic stroke agents

Article abstract of DOI:10.1021/jm4001693

In search of novel anti-ischemic stroke agents with higher potency than a known drug 3-n-butylphthalide (NBP), a series of hybrids ((S)- and (R)-5a-f) from optically active ring-opened NBP derivative and isosorbide were synthesized for evaluating their anti-ischemic stroke activity. Compound (S)-5e displayed the strongest activity in inhibiting the adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation in vitro, with 10.0- and 8.4-fold more effectiveness than (S)-NBP, respectively. Furthermore, (S)-5e was stable in artificial gastrointestinal fluids and could penetrate the blood-brain barrier (BBB) with an appreciate lipid/water partition coefficient relative to (S)-NBP. More importantly, oral treatment with (S)-5e protected from acute thrombosis and inhibited the ischemia/reperfusion-related brain injury in animals. Our findings suggest that (S)-5e may be promising for further evaluation for the intervention of ischemic stroke.

Full text of DOI:10.1021/jm4001693

Article
pubs.acs.org/jmc
Novel Hybrids of Optically Active Ring-Opened 3‑n‑Butylphthalide
Derivative and Isosorbide as Potential Anti-Ischemic Stroke Agents
Xiaoli Wang,†,‡,# Linna Wang,†,§,# Tingting Li,†,§,# Zhangjian Huang,†,‡ Yisheng Lai,†,‡ Hui Ji,*,†,§
Xiaolong Wan,∥ Jinyi Xu,*,†,‡ Jide Tian,⊥ and Yihua Zhang*,†,‡
‡
§
†State Key Laboratory of Natural Medicines, Center of Drug Discovery, Department of Pharmacology, China Pharmaceutical
University, Nanjing 210009, P. R. China
∥Department of Chemistry, Shanghai University, Shanghai 200444, P. R. China
⊥Department of Molecular and Medical Pharmacology, University of CaliforniaLos Angeles, Los Angeles, 90095, United States
S
* Supporting Information
ABSTRACT: In search of novel anti-ischemic stroke agents with
higher potency than a known drug 3-n-butylphthalide (NBP), a series of
hybrids ((S)- and (R)-5a−f) from optically active ring-opened NBP
derivative and isosorbide were synthesized for evaluating their anti-
ischemic stroke activity. Compound (S)-5e displayed the strongest
activity in inhibiting the adenosine diphosphate (ADP) and arachidonic
acid (AA)-induced platelet aggregation in vitro, with 10.0- and 8.4-fold
more effectiveness than (S)-NBP, respectively. Furthermore, (S)-5e was
stable in artificial gastrointestinal fluids and could penetrate the blood−
brain barrier (BBB) with an appreciate lipid/water partition coefficient
relative to (S)-NBP. More importantly, oral treatment with (S)-5e
protected from acute thrombosis and inhibited the ischemia/
reperfusion-related brain injury in animals. Our findings suggest that
(S)-5e may be promising for further evaluation for the intervention of ischemic stroke.
moderate and is often administered together with other
antiplatelet drug(s).16 A previous study suggests that the low
efficacy of NBP is attributed to its poor aqueous solubility.17
However, the efficacy of 2-(1-hydroxypentyl)-benzoate
(HPBA), a ring-opened derivative of NBP with an improved
aqueous solubility, and other HPBA derivatives is still not
satisfactory.18,19 Therefore, new strategies are needed for the
development of drugs for ischemic stroke.
Isosorbide (IS) is a diuretic drug and has been used for the
treatment of hydrocephalus and glaucoma. Meanwhile IS is also
an excellent scaffold and has been used for generating many of
its derivatives with potent vasodilation and anti-inflammatory
activity.20−22 Accordingly, we hypothesize that new hybrids of
optically active ring-opened NBP derivative and IS may have
potent activity against ischemic stroke.
In this study, a series of novel hybrids of optically active ring-
opened NBP derivative and IS were synthesized by inducing
various substituted amines to improve the aqueous solubility,
e.g., diethylamine, N-methyl piperazine, and morpholine to the
side chain of HPBA via a substituted acetate linkage,
respectively. We found that the promising compound, (S)-5e,
displayed potent antiplatelet aggregation, antithrombotic
activity, and anti-ischemic stroke activity in vitro and in vivo.
INTRODUCTION
■
Ischemic stroke is a leading cause of human morbidity and
mortality worldwide.1 Currently, stroke has approximately
caused the loss of 5 million people each year, and the mortality
rate of stroke is increasing.2 Previous studies have suggested
that the pathogenesis of ischemic stroke is attributed to the
interaction of multiple factors, including genetic high risk,
thrombosis, and chronic inflammatory diseases such as
hypertension, diabetes, and others.3−5 During the process of
ischemic stroke, the platelet aggregation-related thrombosis
limits sufficient blood flow in the special region of the brain and
leads to ischemic inflammation and brain damage.6,7 Although
many drugs are available for the intervention of ischemic stroke,
the efficacy of these drugs is not satisfactory. Hence, the
development of new drugs for the effective and safe treatment
of ischemic stroke will be of great significance.
The 3-n-butylphthalide (NBP) is an anti-ischemic stroke
drug that was approved by the State Food and Drug
Administration (SFDA) of China in 2002. NBP can inhibit
platelet aggregation and thrombosis-associated oxidative stress,
improve microcirculation, and mitigate ischemic injury in the
brain.8−10 NBP is a racemic molecule containing a chiral center.
A pair of enantiomers, (S)- and (R)-NBP, have been
synthesized. Pharmacologically, these two enantiomers have
similar potency to NBP, but (S)-NBP is slightly better in a few
cases.11−15 However, the efficacy of NBP or (S)-NBP is
Received: February 1, 2013
Published: March 19, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Compounds (S)- and (R)-5a−f
a
Reagents and conditions: (a) (i) 2 M NaOH, CH3OH−H2O, 50 °C, 0.5 h; 5% HCl, −10−0 °C; (ii) (S)-α-methylbenzylamine, Et2O, −20 to −10
°C; acetone, recrystallization repeated 2 times; (b) (i) NaOH, CH3OH−H2O, 50 °C, 0.5 h; (ii) 5% HCl, −10−0 °C; (c) CH3COCl, Et3N, DMAP,
CH2Cl2, −10 °C, 5 h; (d) IS, DCC, DMAP, CH2Cl2, room temp, 8−12 h; (e) ClCH2COCl, Et3N, DMAP, CH2Cl2, −10 °C, 5 h; (f) corresponding
amines, K2CO3, acetone, room temp, 6−10 h. (g) (i) 2 M NaOH, CH3OH−H2O, 50 °C, 0.5 h; 5% HCl, −10−0 °C; (ii) (R)-α-methylbenzylamine,
Et2O, −20 to −10 °C; acetone, recrystallization repeated 2 times.
a
Scheme 2. Synthesis of Compounds 6−14
a
Reagents and conditions: (a) N-methyl piperazine, K2CO3, acetone, reflux, 8 h; (b) 6, DCC, DMAP, CH2Cl2, room temp, 10 h; (c) H2, Pd/C,
EtOH, room temp, 4 h; (d) Faming HNO3, AcOH, Ac2O, 0 °C to room temp, 2.5 h.
acidification to form the ring-opened acid (HPBA), which was
reacted with (S)-α-methyl-benzylamine to yield the diaster-
eoisomeric salt, followed by alkali treatment and lactonization
under acid conditions to give (S)-NBP (ee >99%). Similarly,
RESULTS AND DISCUSSION
■
Chemistry. The target compounds (S)- and (R)-5a−f were
synthesized from chemical resolution of racemic NBP (Scheme
1).23 NBP was subjected to saponification and sequential
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Table 1. The IC50 Values of (S)- and (R)-5a−f against Platelet Aggregation in Vitro
IC50 (mM)
IC50 (mM)
compd
ADP (10 μM)
AA (1 mM)
compd
ADP (10 μM)
AA (1 mM)
control
IS
(R)-5b
(S)-5c
(R)-5c
(S)-5d
(R)-5d
(S)-5e
(R)-5e
(S)-5f
(R)-5f
0.68 0.11
0.80 0.13
1.00 0.16
0.45 0.05
0.94 0.17
0.07 0.01
0.36 0.07
0.49 0.09
0.75 0.12
0.56 0.07
0.64 0.05
0.91 0.12
0.32 0.06
0.80 0.09
0.05 0.01
0.27 0.04
0.36 0.06
0.63 0.10
1.35 0.12
0.70 0.10
1.15 0.22
0.36 0.04
1.68 0.31
0.42 0.05
0.68 0.08
(S)-NBP
(R)-NBP
b
Ticlid
c
ASP
0.14 0.01
0.21 0.03
0.57 0.09
0.20 0.01
(S)-5a
(R)-5a
(S)-5b
0.33 0.05
0.71 0.06
0.31 0.04
a
b
IC50 values (a dose achieved 50% inhibition of platelet aggregation) are expressed as mean SD. Ticlid is an inhibitor of ADP-induced platelet
c
aggregation. ASP is an inhibitor of AA-induced platelet aggregation.
(R)-NBP (ee >99%) was obtained with (R)-α-methyl-benzyl-
amine. Subsequently, (S)-NBP underwent alkali hydrolysis and
acidification to form acid (S)-1 that was treated with acetyl
chloride or chloroacetyl chloride to give the acylated
compounds (S)-2 or (S)-3, which was esterified with IS in
the presence of 1,3-dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP) to offer ester (S)-5a or (S)-4.
Compound (S)-4 was condensated with corresponding amines
to generate the target compounds (S)-5b−f. Similarly, (R)-5a−
f were synthesized from (R)-NBP. The synthesis of 6−10 was
illustrated in Scheme 2. Compound 6 was obtained by
condensation of (S)-2 with N-methyl piperazine. Treatment
of 6 with 7 or 8 led to ester 9 or 10. Debenzylation of 9 or 10
with Pd/C in hydrogen atmosphere gave hydroxy compound
11 or 12, which was then treated with fuming HNO3, AcOH,
and Ac2O to provide mononitrate 13 or 14. Compounds 7 and
8 were prepared by benzylation of IS, as described previously.24
All of the new compounds were purified by column
of IS (1.68 mM), (S)-NBP (0.42 mM), and ASP (0.14 mM),
respectively.
Analysis of individual moieties (IS, 6, 11, and 12) of (S)-5e
as well as mononitrate 13 and 14 revealed that they at 1 mM
had similar levels of inhibitory activity (6.94−24.10%), which
were significantly lower than that of (S)-5e (50.10%) in
inhibiting the ADP-induced platelet aggregation in vitro (P <
0.001, Figure 1A). Interestingly, compounds 11−14 had
comparable levels of inhibitory activity, suggesting that the
nitrate moiety may not be necessary and can be replaced by
HPBA.
1
chromatography and characterized by IR, ESI-MS, H NMR,
13C NMR, and HRMS. The individual compounds with a
chemical purity of >95% and optical purity (ee) of >99%
(determined by chiral HPLC analysis) were used for
subsequent experiments.
Pharmacology: Antiplatelet Aggregation Effect In
Vitro. The inhibitory effects of target compounds on platelet
aggregation in vitro were determined using Born’s turbidimetric
method.25 Ticlopidine hydrochloride (Ticlid) and aspirin
(ASP) are inhibitors of adenosine diphosphate (ADP)- and
arachidonic acid (AA)-induced platelet aggregation, respec-
tively26,27 and were used as positive controls. The IS, (S)-, and
(R)-NBP were used as control compounds. At least five
different concentrations of individual compounds were tested,
respectively, and data were calculated and expressed as the IC50
values.
As shown in Table 1, (S)-5a−f displayed near 2-fold higher
inhibitory activity than that of (R)-5a−f in ADP- and AA-
induced platelet aggregation, indicating (S)-isomers were more
potent than (R)-isomers in inhibition of both ADP- and AA-
induced platelet aggregation, which was consistent with (S)-
and (R)-NBP. In addition, the (S)-isomers, except for (S)-5c,
were more potent than (S)-NBP. Notably, (S)-5e was the most
potent, significantly superior to IS, (S)-NBP, Ticlid, and ASP,
and its IC50 value (0.07 mM) on ADP-induced platelet
aggregation was 19.3-, 10.0-, and 5.1-fold less than that of IS
(1.35 mM), (S)-NBP (0.70 mM), and Ticlid (0.36 mM),
respectively, and the IC50 value (0.05 mM) on AA-induced
platelet aggregation was 33.6-, 8.4-, and 2.8-fold less than that
Figure 1. (A) Inhibitory effects of (S)-5e, IS, 6, and 11−14 (0.1 mM
each) on ADP-induced platelet aggregation in vitro. Data are
expressed as mean
SD of each group (n = 6) and analyzed by
one-way analysis of variance (ANOVA) followed by post hoc Tukey
test. ***P < 0.001 vs the (S)-5e group. (A) Inhibitory effects of (S)-5e
and combinations of its moieties on ADP-induced platelet aggregation
in vitro. Data are expressed as mean SD of each group (n = 6) and
analyzed by ANOVA followed by post hoc Tukey test. ***P < 0.001
vs the (S)-5e group.
Next, we investigated whether administration of (S)-5e alone
is better than combination administration of its corresponding
moieties in inhibition of ADP-induced platelet aggregation in
vitro. The results indicated that the inhibitory activity of (S)-5e
(0.1 mM) was significantly more potent than that of (S)-NBP
(0.2 mM) together with IS (0.1 mM), 6 (0.2 mM) with IS (0.1
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a
Table 2. The Concentrations of (S)-5e in the Artificial Gastric and Intestinal Juices
concentrations (μM)
gastrointestinal fluid
0 h
1 h
2 h
4 h
8 h
artificial gastric juice
4.92 0.21
4.97 0.14
4.89 0.29
4.86 0.11
5.00 0.11
5.04 0.19
4.79 0.20
4.89 0.10
4.96 0.28
4.92 0.17
artificial intestinal juice
a
Data are expressed as mean SD of the concentrations of (S)-5e at each time point of each group (n = 6) and analyzed by ANOVA and post hoc
Tukey test.
mM), 6 (0.1 mM) with 11 (0.1 mM), or 6 (0.1 mM) with 12
(0.1 mM) in inhibiting the ADP-induced platelet aggregation in
vitro (P < 0.001, Figure 1B).
In parallel, we investigated the antithrombotic activity of (S)-
5e in a rat arterio-venous (A-V) shunt model.30 Male Sprague−
Dawley (SD) rats were randomized and treated with 80 or 340
mg/kg (which was equal mass or equal molar to that of (S)-
NBP) of (S)-5e, 80 mg/kg of (S)-NBP, or vehicle by gavage
daily for seven days. Two hours after the last treatment, all
animals were subjected to the arteriovenous shunt using a 14
cm polyethylene catheter containing a 6 cm silk thread for 15
min. We found that the wet and dry thrombus weights in the
(S)-5e-treated rats, similar to other treatment groups of rats,
were significantly less than that in the vehicle controls (P <
0.001, Figure 3). Interestingly, the wet thrombus weights in the
(S)-5e-treated (340 mg/kg) rats were significantly less than
that in the (S)-NBP-treated rats (P < 0.01), indicating that (S)-
5e had stronger antithrombotic activity in this model. These
two independent lines of evidence demonstrate that (S)-5e has
potent antithrombotic activity in vivo.
Lipid/Water Partition Coefficient and Aqueous Sol-
ubility of (S)-5e. The BBB protects the brain from other
toxicants and infections, and it is well-known that whether a
drug could pass the BBB depends on an appropriate lipid/water
partition coefficient. We calculated the log D values of (S)-5e
and (S)-NBP using ACD/Laboratories Professional Software31
and tested their aqueous solubility, as described previously.32
Interestingly, the log D value of (S)-5e (log D3.6 1.87) was less
than that of (S)-NBP (log D7.0 3.19), whereas the aqueous
solubility of (S)-5e (11.2 mg/mL) was greater than that of (S)-
NBP (0.53 mg/mL), indicating that (S)-5e had an appropriate
lipid/water partition coefficient33 and a better aqueous
solubility relative to (S)-NBP.
Stability of (S)-5e in Artificial Gastrointestinal Fluids.
Theoretically, an ideal anti-ischemic stroke drug should be
rapidly absorbed into blood circulation and enter the target
tissues following oral administration. Alternatively, the drug has
to be tolerant to gastrointestinal fluids for later absorption.
Thus, we tested the stability of (S)-5e in artificial gastric juice
(pH 1.5) and artificial intestinal juice (pH 6.8) by high-
performance liquid chromatography (HPLC).28 We found that
incubation of (S)-5e in these types of fluids at 37 °C for 8 h did
not significantly alter the contents of (S)-5e (Table 2),
suggesting that (S)-5e was stable in these extreme conditions.
Antithrombotic Activities of (S)-5e In Vivo. We next
evaluated the antithrombotic effects of (S)-5e in vivo using two
models of thrombosis in which platelet activation is critical
factor.
The first one was acute vascular occlusion initiated by
intravascular platelet induced aggregation through infusion of a
mixture of collagen and adrenaline into the jugular vein.29 Male
ICR mice were randomized and treated orally with vehicle,
ASP, (S)-NBP, or (S)-5e for seven days. Two hours after the
last administration, individual mice were injected intravenously
with a mixture of collagen (0.21 mg/kg) and adrenaline (44.5
μg/kg) to induce acute systemic vascular thromboembolism.
We observed that treatment with (S)-5e at equal mass (160
mg/kg) or molar (680 mg/kg) to (S)-NBP had similar
protection to that of (S)-NBP and ASP (Figure 2). The
protective effect of (S)-5e at 680 mg/kg was slightly higher
than that of others and significantly greater than that of vehicle
alone (P < 0.05, Log-rank test). Hence, treatment with (S)-5e
efficiently inhibited acute thrombosis in mice.
The BBB Penetration of (S)-5e. To test the BBB
penetration of (S)-5e, we determined the concentration of
(S)-5e in mouse brain and plasma at different time points (0.5,
1.0, 2.0, 4.0, and 8.0 h) following oral administration (680 mg/
kg) and calculated the ratios of the concentrations of (S)-5e in
the brain to plasma (C brain/C plasma).34 As shown in Table 3 and
Figure 4A, the concentrations of (S)-5e in the brain, similar to
that in plasma, rapidly increased and gradually declined. The
mean ratios of the concentrations of (S)-5e in the brain to
plasma gradually decreased (Figure 4B). These results suggest
that (S)-5e may be rapidly absorbed and penetrate to the BBB
in mice.
Treatment with (S)-5e Inhibits Ischemia/Reperfusion
(I/R)-Related Brain Injury in Rats. The occlusion of middle
cerebral artery (MCAO) is the most common reason for
inducing I/R-related brain injury in the clinic. To gain insight
into the therapeutic potential of (S)-5e, we examined the
impact of treatment with (S)-5e on I/R-related brain injury in a
rat I/R model. Male SD rats were randomly treated orally with
0.5% sodium carboxyl methyl cellulose (CMC-Na) (sham
group and vehicle group), (S)-NBP (80 mg/kg), and (S)-5e
(80 mg/kg and 340 mg/kg, which were equal mass and equal
molar to (S)-NBP, respectively) for seven days. Two hours
after the last treatment, the rats were subjected to MCAO for
Figure 2. The survival of mice. Male ICR mice were treated with the
indicated doses of each drug or vehicle for seven days. Two hours after
the last treatment, individual mice were injected intravenously with a
mixture of collagen and adrenaline for inducing acute thrombosis. The
survival of individual mice was monitored up to 15 min post injection.
Data are expressed as mean % of the surviving mice in individual
groups (n = 15). *P < 0.05, **P < 0.01 vs the vehicle group.
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Figure 3. Effects of (S)-5e, (S)-NBP, and ASP on thrombus wet weights (A) and dry weights (B) in rats. SD rats were treated with the indicated
doses of each drug by gavage daily for seven days. Two hours after the last treatment, the rats were subjected to the A-V shunt using a silk thread for
15 min. The wet and dry weights were measured. Data are expressed as mean SD of each group (n = 12) and analyzed by ANOVA and post hoc
Tukey test. ***P < 0.001 vs the sham group, ##P < 0.01 vs the (S)-NBP group.
a
Table 3. The Concentrations of (S)-5e in the Brain and Plasma
concentrations (ng/mL)
distribution
0.5 h
1.0 h
2.0 h
4.0 h
8.0 h
brain
408.3 32.7
320.2 63.5
260.2 31.7
241.0 63.5
173.7 26.5
186.2 25.8
54.3 14.4
16.7 8.54
69.7 14.1
plasma
126.5 28.9
a
Data are expressed as mean SD of the concentrations of (S)-5e in the brain and plasma at each time point after a single oral dose of 680 mg/kg in
mice (n = 6).
Figure 4. (A) Concentrations of (S)-5e in brain and plasma after a single oral dose of 680 mg/kg in mice. (B) The mean brain-to-plasma ratio of
(S)-5e after a single oral dose of 680 mg/kg in mice. Data are presented as mean SD (n = 6).
two hours, followed by reperfusion. The neurological function
of individual rats was evaluated using Longa’s method35 24 h
after reperfusion (Figure 5). As expected, there was no obvious
neuronal abnormality detected in the sham-operated group
while the neurological deficit scores in the vehicle-treated I/R
rats significantly increased (P < 0.001). In contrast, the
neurological deficit scores in the (S)-5e-treated I/R rats, but
not in the (S)-NBP-treated group, were significantly reduced, as
compared with the vehicle-treated controls (P < 0.01).
Similarly, analysis of the infarct size by the 2,3,5-
triphenyltetrazolium chloride (TTC) assay indicated that in
comparison with that in the vehicle-treated controls, the infarct
sizes in the brains of the (S)-NBP or (S)-5e-treated I/R rats
were significantly reduced, particularly in those with higher
dose of (S)-5e (P < 0.001) (Figure 6A). In addition, treatment
with (S)-5e (80 and 340 mg/kg) and (S)-NBP (80 mg/kg)
significantly reduced the water contents in rat brain (Figure
6B).
Figure 5. Effects of (S)-5e and (S)-NBP on neurological deficit score
in rats subjected to I/R (n = 12). Data are assessed 24 h after
reperfusion and analyzed by Kruskal−Wallis test, followed by the
Mann−Whitney U test. ***P < 0.001 vs the sham group, #P < 0.05 vs
the vehicle group.
Histologically, to examine the protective effects of (S)-5e
against cortical neuronal damage, experiments on hematoxylin
and eosin (H&E) staining were performed. While necrotic
neurons, neuronal perikarya shrinkage, perivascular inflamma-
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Figure 6. (A) Effects of (S)-5e and (S)-NBP on infarction after cerebral I/R in rats. (a) Infarcted brain areas were visualized using TTC staining.
Representative examples from each treatment group were shown. (b) Quantitative analysis of the infracted brain regions. The ratios of infarcts area
to whole brain areas were calculated. Data are expressed as mean SD (n = 6) and analyzed by ANOVA followed by post hoc Tukey test. ***P <
0.001 vs the vehicle group, **P < 0.01 vs the vehicle group. (B) Effects of (S)-5e and (S)-NBP on brain edema after cerebral I/R in rats. Data are
expressed as mean SD (n = 6) and analyzed by ANOVA followed by post hoc Tukey test. **P < 0.01 vs the sham group, #P < 0.05 vs the vehicle
group. (C) Protective effects of (S)-5e and (S)-NBP on neuronal injury in the ischemic cerebral cortex of rats (n = 6). Cerebral sections were stained
with hematoxylin and eosin then were examined under light microscope. Representative images from individual groups (n = 6) are shown
(magnification, × 200).
tory infiltration, and vacuolization were observed in the brain
tissues of the vehicle-treated rats, there were a few necrotic
neurons, less inflammatory infiltrates and abnormal changes in
the brains of the (S)-5e or (S)-NBP-treated rats, particularly in
those with higher dose of (S)-5e. These data indicated that
treatment with (S)-5e significantly inhibited the I/R-related
injury in the brains of rats.
and (R)-5a−f displayed different antiplatelet aggregation
activity. In general, (S)-isomers exhibited much stronger
inhibitory effects than (R)-isomers in both ADP- and AA-
induced platelet aggregation. Additionally, various substituted
amines in the side chain of target compounds exerted variable
effects. Significantly, the (S)-5e bearing an N-methyl piperazino
moiety in side chain exhibited the strongest inhibitory activity
among all compounds. Interestingly, individual moieties of (S)-
5e inhibited the ADP-induced platelet aggregation but their
inhibitory activity were much less than (S)-5e. Moreover,
treatment with (S)-5e alone had better antiplatelet aggregation
CONCLUSIONS
■
Analysis of structure and antiplatelet aggregation activity
relationship (SAR) revealed that the target compounds (S)-
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compound (R)-2 was obtained as a white crystal (1.63 g, 65%, over
two steps); mp 65−66 °C; [α]2D7 +37.8° (c 1.00 CHCl3). IR (KBr),
ESI-MS, 1H NMR, and 13C NMR spectral data were identical to those
of (S)-2.
activity than either of the four different combinations from its
corresponding moieties. These results suggest that individual
moieties of (S)-5e may act synergistically.
In summary, a series of novel hybrids ((S)- and (R)-5a−f) of
optically active ring-opened NBP derivative and IS were
synthesized, and compound (S)-5e displayed the most potent
activity in inhibiting the ADP- and AA-induced platelet
aggregation in vitro, superior to IS, (S)-NBP, ASP, and Ticlid.
Furthermore, (S)-5e was stable in artificial gastrointestinal
fluids and rapidly absorbed and distributed into the brain.
Moreover, (S)-5e exhibited higher antithrombotic activity than
(S)-NBP and ASP in vivo. Most importantly, treatment with
(S)-5e inhibited the I/R-related neurobehavioral dysfunction,
the infarct brain size, the brain-water contents, and the brain
damage in rats. Therefore, (S)-5e may be valuable for the
intervention of ischemic stroke. We are interested in further
investigation of the mechanisms underlying the action of (S)-5e
in inhibiting thrombosis and ischemic stroke.
(S)-(−)-2-[1-(2-Chloroacetoxy)pentyl]benzoic Acid ((S)-3).
Compound (S)-3 was synthesized in the same manner as compound
(S)-2. Starting from compound (S)-1 (2.08 g, 10.00 mmol) and 2-
chloroacetyl chloride (4.16 mL, 30.00 mmol), compound (S)-3 was
obtained as a white crystal (1.85 g, 65%, over two steps); mp 67−68
°C; [α]2D7 −40.8° (c 1.00 CHCl3). MS (ESI): m/z 283 [M − H]−. IR
1
(cm−1, KBr): νmax 1412, 1691, 1734, 2958, 3450. H NMR (300 Hz,
CDCl3): 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), 4.11−4.32 (m, 2H, COCH2Cl),
6.71−6.78 (m, 1H, OCHCH2), 7.36−7.42 (m, 1H, ArH), 7.56−7.62
(m, 2H, ArH), 8.08 (d, 1H, ArH, J = 8.1 Hz), 10.89 (brs, 1H, COOH).
13C NMR (75 Hz, CDCl3): 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.
(R)-(+)-2-[1-(2-Chloroacetoxy)pentyl]benzoic Acid ((R)-3).
Compound (R)-3 was synthesized in the same manner as compound
(S)-3. Starting from compound (R)-NBP (1.90 g, 10.00 mmol),
compound (R)-3 was obtained as a white crystal (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 identical to those
of (S)-3.
EXPERIMENTAL SECTION
■
Chemistry. Melting points are uncorrected and were measured in
open capillary tubes using a Gallenkamp melting point apparatus. H
1
2,5-Bis{2-[(S)-(+)-1-(2-Chloroacetoxy)]pentyl}benzoate-
1,4:3,6-dianhydro-D-glucitol ((S)-4). To a stirred solution of (S)-3
(1.85 g, 6.5 mmol) and isosorbide (0.48 g, 3.25 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 (1.47 g, 7.2 mmol) was then
added, and the solution was left stirring at room temperature for 6 h.
The solution was then filtered, and the filtrate was 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 (PE/EtOAc = 5/1,
v/v) to obtain the compound (S)-4 as a light-yellow oil (2.03 g, 92%);
[α]2D7 +48.0° (c 0.06 CHCl3). MS (ESI): m/z 696.3 [M + NH4]+. IR
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 (ESI).
Analytical and preparative TLC were 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). The
target compounds (S)- and (R)-5a−f with a chemical purity of >95%
and optical purity (ee) of >99% were used for subsequent
experiments(see the ESI†).
1
(cm−1, KBr): νmax 756, 1275, 1462, 1723, 2957. H NMR (300 Hz,
All animal experiments were performed in accordance with the
Animal (Scientific Procedures) Act 2009 (P. R. China), and the
experimental protocols were approved by the Animal Research
Protection Committee of China Pharmaceutical University, Nanjing,
China.
(S)-(−)-2-(1-Acetoxypentyl)benzoic Acid ((S)-2). To a stirred
solution of (S)-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 −10−0 °C. The mixture was
extracted with cold Et2O (10 mL × 3) to get (S)-1 and quickly used
for the next step without any purification.
A solution of acetyl chloride (2.12 mL, 30.00 mmol) in dry CH2Cl2
(10 mL) was added dropwise to a mixture of (S)-1 (2.08 g, 10.00
mmol), Et3N (4.17 mL, 30.00 mmol), and DMAP (1.00 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 room temperature. The organic layer was washed
with water, dried, filtered, and evaporated to dryness. The residue was
recrystallized from n-hexane to obtain (S)-2 as a white crystal (1.70 g,
68%, over two steps); mp 65−66 °C; [α]2D7 −39.5° (c 1.00 CHCl3).
MS (ESI): m/z 249.1 [M − H]−. IR (cm−1, KBr): νmax 1412, 1691,
1734, 2958, 3450. 1H NMR (300 Hz, CDCl3): δ 0.93 (t, 3H, CH3, J =
8.5 Hz), 1.37−1.42 (m, 4H, 2 × CH2), 1.88−1.91 (m, 2H, CH2),
2.13−2.33 (m, 3H, COCH3), 6.61−6.72 (m, 1H, OCHCH2), 7.37−
7.40 (m, 1H, ArH), 7.56−7.62 (m, 2H, ArH), 8.05 (d, 1H, ArH, J =
8.1 Hz), 10.98 (brs, 1H, COOH). 13C NMR (75 Hz, CDCl3): 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.
CDCl3): δ 0.89 (t, 6H, 2 × CH3, J = 6.6 Hz), 1.00−1.46 (m, 8H, 4 ×
CH2), 1.70−1.89 (m, 4H, 2 × CH2), 3.91−4.40 (m, 8H, 2 × OCH2, 2
× CH2Cl,), 4.71 (t, 1H, OCH, J = 4.5 Hz), 5.07 (t, 1H, OCH, J = 4.8
Hz), 5.33−5.50 (m, 2H, 2 × COOCH), 6.59−6.67 (m, 2H, 2 ×
OCHCH2), 7.26−7.94 (m, 8H, ArH). 13C NMR (75 MHz, CDCl3): δ
170.34, 166.54, 162.02, 161.94, 146.04, 144.91, 134.15, 134.07, 133.97,
133.89, 131.33, 131.23, 127.85, 127.59, 126.41, 126.38, 86.13, 86.01,
81.20, 81.05, 74.85, 74.83, 72.89, 72.87, 40.97, 36.43, 36.29, 29.68,
28.09, 27.94, 22.42, 22.35, 13.94, 13.92. HRMS (ESI): m/z calcd for
C34H40Cl2O10 [M + H]+ 679.1999; found 679.2097.
2,5-Bis{2-[(R)-(+)-1-(2-chloroacetoxy)]pentyl}benzoate-
1,4:3,6-dianhydro-D-glucitol ((R)-4). Compound (R)-4 was synthe-
sized in the same manner as compound (S)-4. Starting from
compound (R)-3 (1.79 g, 6.30 mmol), compound (R)-4 was obtained
as a light-yellow oil (1.92 g, 90%); [α]2D7 +23.7° (c 0.06 CHCl3). IR
1
(KBr), ESI-MS, H NMR, 13C NMR, and HRMS spectral data were
identical to those of (S)-4.
2,5-Bis{2-[(S)-(+)-2-(1-acetoxypentyl)]}benzoate-1,4:3,6-dia-
nhydro-D-glucitol ((S)-5a). To a stirred solution of (S)-2 (1.70 g, 6.8
mmol) and isosorbide (0.50 g, 3.4 mmol) in anhydrous CH2Cl2 (60
mL) was added DMAP (0.3 mmol), and the solution was left stirring
at 0 °C for 10 min. DCC (1.68 g, 8.2 mmol) was then added, and the
solution was left stirring at room temperature for 6 h. The solution was
then filtered, and the filtrate was 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 (PE/EtOAc = 5/1, v/v) to obtain
the compound (S)-5a as a light yellow oil (1.95 g, 94%); [α]2D7 +36.7°
(c 0.06 CHCl3); ee = 99.7%. MS (ESI): m/z 628.4 [M + NH4]+. IR
1
(cm−1, KBr): νmax 763, 1247, 1461, 1728, 2958. H NMR (300 Hz,
(R)-(+)-2-[1-(2-Chloroacetoxy)pentyl]benzoic Acid ((R)-2).
Compound (R)-2 was synthesized in the same manner as compound
(S)-2. Starting from compound (R)-1 (2.08 g, 10.00 mmol),
CDCl3): δ 0.89 (t, 6H, 2 × CH3, J = 6.8 Hz), 1.10−1.55 (m, 8H, 4 ×
CH2), 1.70−1.91 (m, 4H, 2 × CH2), 2.06 (s, 6H, 2 × CH3), 3.91−4.16
(m, 4H, 2 × OCH2), 4.72 (t, 1H, OCH, J = 4.4 Hz), 5.07 (t, 1H,
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Article
OCH, J = 4.4 Hz), 5.39−5.50 (m, 2H, 2 × COOCH), 6.46−6.58 (m,
2H, 2 × OCHCH2), 7.30−7.95 (m, 8H, ArH). 13C NMR (75 MHz,
CDCl3): δ 170.15, 170.01, 165.39, 165.20, 142.20, 142.09, 134.15,
134.01, 132.62, 132.47, 130.50, 130.41, 127.21, 127.06, 126.39, 126.25.
HRMS (ESI): m/z calcd for C34H42O10 [M + H]+ 611.2778; found
611.2796.
2,5-Bis{2-[(R)-(+)-2-(1-acetoxypentyl)]}benzoate-1,4:3,6-dia-
nhydro-D-glucitol ((R)-5a). Compound (R)-5a was synthesized in
the same manner as compound (S)-5a. Starting from compound (R)-2
(1.63 g, 6.52 mmol), compound (R)-5a was obtained as a light-yellow
oil (1.79 g, 90%); [α]2D7 +20.0° (c 0.06 CHCl3); ee = 99.8%. HRMS
(ESI): m/z calcd for C34H42O10 [M + H]+ 611.2778; found 611.2804.
IR (KBr), ESI-MS, 1H NMR, and13C NMR spectral data were identical
to those of (S)-5a.
ee = 99.8%. MS (ESI): m/z 781.1 [M + H]+. IR (cm−1, KBr): νmax 763,
1258, 1462, 1723, 2956. 1H NMR (300 MHz, CDCl3): δ 0.89 (t, 6H, 2
× CH3, J = 6.7 Hz), 1.26−1.37 (m, 8H, 4 × CH2), 1.81−1.86 (m, 4H,
2 × CH2), 2.55 (t, 8H, 4 × NCH2CH2O, J = 4.5 Hz), 3.15−3.30 (m,
4H, 2 × NCH2COO), 3.71 (t, 8H, 4 × NCH2CH2O, J = 4.4 Hz),
3.99−4.15 (m, 4H, 2 × OCH2), 4.72 (t, 1H, OCH, J = 4.5 Hz), 5.04
(t, 1H, OCH, J = 5.0 Hz), 5.39−5.50 (m, 2H, 2 × COOCH), 6.54−
6.64 (m, 2H, 2 × OCHCH2), 7.29−7.95 (m, 8H, ArH). 13C NMR (75
MHz, CDCl3): δ 169.44, 169.41, 165.99, 165.79, 143.63, 143.48,
132.57, 132.47, 130.38, 130.28, 127.99, 127.88, 127.33, 127.21, 126.34,
126.19, 86.12, 81.05, 78.75, 74.63, 73.35, 73.18, 73.01, 70.67, 66.76,
59.70, 59.67, 53.26, 36.44, 36.38, 28.01, 27.96, 22.44, 22.42, 13.97,
13.94. HRMS (ESI): m/z calcd for C42H56N2O12 [M + H]+ 781.3833;
found 781.3987.
General Method for the Preparation of Compounds (S)- and
(R)-5b−f. To a solution of (S)-4 or (R)-4 (0.2 mmol) and K2CO3 (55
mg, 0.4 mmol) in acetone (10 mL) was added the corresponding
amine, and the solution was left stirring at room temperature for 6−10
h. The solution was then filtered, and the filtrate was reconstituted in
EtOAc (20 mL) and solvent removed under reduced pressure. The
amino derivatives were purified by flash chromatography (CH2Cl2/
MeOH = 100/1−30/1, v/v), to give the title compounds (55−70%).
2,5-Bis{2-[(S)-(+)-1-(2-(diethylamino)acetoxy)]pentyl}-
benzoate-1,4:3,6-dianhydro-D-glucitol ((S)-5b). The title com-
pound was obtained as a light yellow oil, 66% yield; [α]2D7 +33.2° (c
0.05 CHCl3); ee = 99.0%. MS (ESI): m/z 753.3 [M + H]+. IR (cm−1,
KBr): νmax 763, 1258, 1461, 1725, 2958. 1H NMR (300 MHz, CDCl3):
δ 0.89 (t, 6H, 2 × CH3, J = 6.3 Hz), 1.01−1.62 (m, 12H, 4 × NCH3),
1.36−1.41 (m, 8H, 4 × CH2), 1.81−1.88 (m, 4H, 2 × CH2), 2.59−
2.67 (m, 8H, 4 × NCH2), 3.19−3.37 (m, 4H, 2 × NCH2COO), 3.99−
4.16 (m, 4H, 2 × OCH2), 4.73 (t, 1H, OCH, J = 4.5 Hz), 5.04 (t, 1H,
OCH, J = 5.0 Hz), 5.39−5.50 (m, 2H, 2 × COOCH), 6.53−6.62 (m,
2H, 2 × OCHCH2), 7.27−7.95 (m, 8H, ArH). 13C NMR (75 MHz,
CDCl3): δ 170.76, 169.95, 166.00, 165.81, 143.89, 143.76, 132.50,
132.38, 130.34, 130.24, 127.88, 127.76, 127.33, 127.21, 126.39, 126.25,
86.13, 81.06, 78.74, 74.60, 73.36, 72.79, 72.61, 70.68, 54.10, 47.57,
36.51, 36.46, 28.03, 27.99, 22.45, 22.42, 13.96, 13.93, 12.35. HRMS
(ESI): m/z calcd for C42H60N2O10 [M + H]+ 753.4248; found
753.4320.
2,5-Bis{2-[(R)-(+)-1-(2-morpholinoacetoxy)]pentyl}benzoate-
1,4:3,6-dianhydro-D-glucitol ((R)-5d). The title compound was
obtained as a light-yellow oil, 64% yield; [α]2D7 +14.6° (c 0.07 CHCl3);
ee = 99.5%. HRMS (ESI): m/z calcd for C42H56N2O12 [M + H]+
781.3833; found 781.3998. IR (KBr), ESI-MS, 1H NMR, and 13C
NMR spectral data were identical to those of (S)-5d.
2,5-Bis{2-[(S)-(+)-1-(2-(4-methylpiperazin-1-yl)acetoxy)]-
pentyl}benzoate-1,4:3,6-dianhydro-D-glucitol ((S)-5e). The title
compound was obtained as a light-yellow oil, 72% yield; [α]2D7 +20.4°
(c 0.05 CHCl3); ee = 99.4%. MS (ESI): m/z 807.4 [M + H]+. IR
1
(cm−1, KBr): νmax 750, 1260, 1462, 1723, 2958. H NMR (300 MHz,
CDCl3): δ 0.89 (t, 6H, 2 × CH3, J = 6.3 Hz), 1.26−1.35 (m, 8H, 4 ×
CH2), 1.84−1.86 (m, 4H, 2 × CH2), 2.53 (s, 6H, 2 × NCH3), 2.79−
2.82 (m, 16H, 8 × NCH2), 3.19−3.37 (m, 4H, 2 × NCH2COO),
3.99−4.08 (m, 4H, 2 × OCH2), 4.73 (t, 1H, OCH, J = 4.5 Hz), 5.05
(t, 1H, OCH, J = 5.0 Hz), 5.39−5.49 (m, 2H, 2 × COOCH), 6.53−
6.63 (m, 2H, 2 × OCHCH2), 7.30−7.95 (m, 8H, ArH). 13C NMR (75
MHz, CDCl3): δ 179.96, 169.60, 166.02, 165.91, 143.72, 143.63,
132.54, 132.43, 130.34, 130.25, 128.02, 127.90, 127.26, 127.20, 126.37,
126.23, 86.13, 81.05, 78.75, 74.61, 73.35, 73.10, 72.92, 70.68, 59.47,
54.84, 52.96, 45.94, 36.45, 28.01, 22.45, 13.93. HRMS (ESI): m/z
calcd for C44H62N4O10 [M + H]+ 807.4466; found 807.4550.
2,5-Bis{2-[(R)-(+)-1-(2-(4-methylpiperazin-1-yl)acetoxy)]-
pentyl}benzoate-1,4:3,6-dianhydro-D-glucitol ((R)-5e). The title
compound was obtained as a light-yellow oil, 70% yield; [α]2D7 +8.6° (c
0.05 CHCl3); ee = 99.6%. HRMS (ESI): m/z calcd for C44H62N4O10
2,5-Bis{2-[(R)-(+)-1-(2-(Diethylamino)acetoxy)]pentyl}-
benzoate-1,4:3,6-dianhydro-D-glucitol ((R)-5b). The title com-
pound was obtained as a light-yellow oil, 60% yield; [α]2D7 +19.2° (c
0.05 CHCl3); ee = 99.3%. HRMS (ESI): m/z calcd for C42H60N2O10
1
[M + H]+ 807.4466; found 807.4576. IR (KBr), ESI-MS, H NMR,
and 13C NMR spectral data were identical to those of (S)-5e.
2,5-Bis{2-[(S)-(+)-1-(2-(4-(2-hydroxyethyl)piperazin-1-yl)-
acetoxy)]pentyl}benzoae-1,4:3,6 dianhydro-D-glucitol ((S)-5f).
The title compound was obtained as a light yellow oil, 67% yield. [α]D27
+12.4° (c 0.05 CHCl3). ee = 99.5%. MS (ESI): m/z 867.4 [M + H]+.
1
[M + H]+ 753.4248; found 753.4334. IR (KBr), ESI-MS, H NMR,
and 13C NMR spectral data were identical to those of (S)-5b.
2,5-Bis{2-[(S)-(+)-1-(2-(pyrrolidin-1-yl)acetoxy)]pentyl}-
benzoate-1,4:3,6-dianhydro-D-glucitol ((S)-5c). The title com-
pound was obtained as a light yellow oil, 58% yield; [α]2D7 +26.7° (c
0.06 CHCl3); ee = 99.9%. MS (ESI): m/z 749.4 [M + H]+. IR (cm−1,
KBr): νmax 750, 1260, 1462, 1723, 2958. 1H NMR (300 MHz, CDCl3):
δ 0.89 (t, 6H, 2 × CH3, J = 6.6 Hz), 1.35−1.37 (m, 8H, 4 × CH2),
1.79−1.86 (m, 8H, 4 × NCH2CH2), 1.95−1.97 (m, 4H, 2 × CH2),
2.61 (s, 8H, 4 × NCH2), 3.34−3.50 (m, 4H, 2 × NCH2COO), 4.11−
4.32 (m, 4H, 2 × OCH2), 4.72 (t, 1H, OCH, J = 4.5 Hz), 5.04 (t, 1H,
OCH, J = 4.9 Hz), 5.35−5.50 (m, 2H, 2 × COOCH), 6.51−6.63 (m,
2H, 2 × OCHCH2), 7.27−7.95 (m, 8H, ArH). 13C NMR (75 MHz,
CDCl3): δ 170.12, 170.12, 165.83, 165.83, 143.77, 143.63, 132.54,
132.43, 130.34, 130.27, 127.22, 127.20, 126.39, 126.30, 126.23, 126.15,
86.13, 81.06, 76.61, 75.57, 74.61, 73.36, 72.83, 70.68, 56.80, 53.76,
36.44, 29.67, 27.98, 23.80, 22.47, 13.94. HRMS (ESI): m/z calcd for
C42H56N2O10 [M + H]+ 749.3935; found 749.4020.
1
IR (cm−1, KBr): νmax 763, 1247, 1460, 1723, 2958. H NMR (300
MHz, CDCl3): δ 0.89 (t, 6H, 2 × CH3, J = 6.3 Hz), 1.26−1.35 (m, 8H,
4 × CH2), 1.83−1.85 (m, 4H, 2 × CH2), 2.48−2.56 (m, 20H, 8 ×
NCH2, 2 × NCH2CH2OH), 3.16−3.31 (m, 4H, 2 × NCH2COO),
3.48−3.65 (m, 4H, 2 × NCH2CH2OH), 3.89−4.15 (m, 4H, 2 ×
OCH2), 4.46−4.50 (m, 1H, OH), 4.72−4.75 (m, 1H, OH), 4.98 (t,
1H, OCH, J = 4.5 Hz), 5.04 (t, 1H, OCH, J = 5.0 Hz), 5.37−5.49 (m,
2H, 2 × COOCH), 6.54−6.61 (m, 2H, 2 × OCHCH2), 7.30−7.95 (m,
8H, ArH). 13C NMR (75 MHz, CDCl3) δ 179.96, 169.60, 166.02,
165.91, 143.72, 143.63, 132.54, 132.43, 130.34, 130.25, 128.02, 127.90,
127.26, 127.20, 126.37, 126.23, 86.13, 81.05, 78.75, 74.61, 73.35,
73.10, 72.92, 70.68, 59.55, 59.34, 54.84, 52.96, 45.94, 36.45, 28.01,
22.45, 13.93. HRMS (ESI): m/z calcd for C46H66N4O12 [M + H]+
867.4677; found 867.5745.
2,5-Bis((R)-(+)-1-(2-(4-(2-hydroxyethyl)piperazin-1-yl)-
acetoxy)]pentyl}benzoae-1,4:3,6-dianhydro-D-glucitol ((R)-5f).
The title compound was obtained as a light-yellow oil, 67% yield;
[α]2D7 +4.6° (c 0.05 CHCl3); ee = 99.3%. HRMS (ESI): m/z calcd for
C46H66N4O12 [M + H]+ 867.4677; found 867.5763. IR (KBr), ESI-MS,
1H NMR, and 13C NMR spectral data were identical to those of (S)-5f.
2,5-Bis{2-[(R)-(+)-1-(2-(pyrrolidin-1-yl)acetoxy)]pentyl}-
benzoate-1,4:3,6-dianhydro-D-glucitol ((R)-5c). The title com-
pound was obtained as a light yellow oil, 58% yield; [α]2D7 +17.4° (c
0.10 CHCl3); ee = 99.8%. HRMS (ESI): m/z calcd for C42H56N2O10
1
[M + H]+ 749.3935; found 749.4036. IR (KBr), ESI-MS, H NMR,
and 13C NMR spectral data were identical to those of (S)-5c.
2,5-Bis{2-[(S)-(+)-1-(2-morpholinoacetoxy)]pentyl}benzoate-
1,4:3,6-dianhydro-D-glucitol ((S)-5d). The title compound was
obtained as a light-yellow oil, 67% yield; [α]2D7 +28.8° (c 0.05 CHCl3);
(S)-2-{1-[2-(4-Methylpiperazin-1-yl)acetoxy]pentyl}benzoic
Acid (6). To a solution of compound (S)-3 (0.57 g, 2.0 mmol) and
K2CO3 (0.76 g, 4.0 mmol) in acetone (20 mL) was added N-methyl
piperazine (0.66 mL, 6.0 mmol), and the solution was left stirring at
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room temperature for 8 h. The solvent was removed under reduced
pressure and purified by flash chromatography (CH2Cl2/MeOH = 10/
1, v/v) to give the 6 as a light-yellow oil (0.34 g, 48%); [α]2D7 −36.3° (c
1.00 CHCl3). MS (ESI): m/z 349.2 [M + H]+. IR (cm−1, KBr): νmax
2-Hydroxy-5-{2-[(S)-(+)-1-(2-(4-methylpiperazin-1-yl)-
acetoxy)]pentyl}benzoate-1,4:3,6-dianhydro-D-glucitol (12).
Compound 12 was synthesized in the same manner as compound
11. Starting from compound 10 (0.51g, 0.90 mmol), compound 12
was obtained as a light-yellow oil (0.39 g, 91%); [α]2D7 +36.2° (c 2.00
CHCl3). HRMS (ESI): m/z calcd for C25H36N2O7 [M + H]+
477.2523; found 477.2621. IR (KBr), ESI-MS, 1H NMR, and 13C
NMR spectral data were identical to those of compound 11.
1
1383, 1584, 1764, 2360, 2932. H NMR (300 Hz, CDCl3): δ 0.96 (t,
3H, CH3, J = 8.2 Hz), 1.33−1.44 (m, 4H, 2 × CH2), 1.86−1.90 (m,
2H, CH2), 2.23−2.34 (m, 3H, NCH3), 2.46 (s, 8H, 4 × NH2), 3.32 (s,
2H, NH2COO), 6.65−6.79 (m, 1H, OCHCH2), 7.37−7.40 (m, 1H,
ArH), 7.56−7.62 (m, 2H, ArH), 8.05 (d, 1H, ArH, J = 8.1 Hz), 11.02
(brs, 1H, COOH). 13C NMR (75 Hz, CDCl3): δ 172.0, 169.4, 142.6,
133.6, 129.8, 128.5, 126.7, 125.7, 73.8, 56.8, 55.2, 52.5, 43.0, 35.7, 26.8,
22.5, 14.0. HRMS (ESI): m/z calcd for C19H28N2O4 [M + H]+
349.2049; found 349.2130.
2-{2-[(S)-(+)-1-(2-(4-Methylpiperazin-1-yl)acetoxy)]pentyl}-
benzoate-5-nitrooxy-1,4:3,6-dianhydro-D-glucitol (13). A solu-
tion of compound 11 (0.40 g, 0.84 mmol) was added, dropwise, to an
ice-cooled solution of fuming nitric acid (90%, 2 mL) in AcOH/Ac2O
(10 mL, 1:1, v/v). The reaction was stirred in an ice-bath for 0.5 h and
then at room temperature for 2 h. The reaction solution was poured to
the ice water and extracted with CH2Cl2 (20 mL × 3). The combined
organic phases were dried, filtered, and evaporated to dryness. The
residue was purified by column chromatography (CH2Cl2/MeOH =
50/1, v/v) to obtain the compound 13 as a light-yellow oil (0.20 g,
46%); [α]2D7 +22.4° (c 2.00 CHCl3). MS (ESI): m/z 522 [M + H]+. IR
(cm−1, KBr): νmax 1281, 1384, 1637, 1717, 2360, 2908, 3447. 1H NMR
(300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.3 Hz), 1.33−1.45 (m, 4H,
2 × CH2), 1.70−1.85 (m, 2H, CH2), 2.28−2.35 (m, 3H, NCH3),
2.54−2.79 (m, 8H, 4 × NCH2), 3.35−3.50 (m, 2H, COCH2N), 3.72−
4.03 (m, 4H, 2 × OCH2), 4.05 (d, 1H, ONO2CH, J = 5.1 Hz), 5.17 (t,
1H, OCH, J = 5.1 Hz), 5.40 (t, 1H, OCH, J = 5.0 Hz), 5.48 (d, 1H,
COOCH, J = 5.2 Hz), 6.54−6.65 (m, 1H, OCHCH2), 7.39−7.41 (m,
1H, ArH), 7.54−7.66 (m, 2H, ArH), 7.95 (d, 1H, ArH, J = 8.4 Hz).
13C NMR (75 Hz, CDCl3): δ 169.6, 166.1, 143.5, 132.5, 130.4, 128.0,
2-{2-[(S)-(+)-1-(2-(4-Methylpiperazin-1-yl)acetoxy)]pentyl}-
benzoate-5-benzyloxy-1,4:3,6-dianhydro-D-glucitol (9). To a
stirred solution of compound 7 (0.24 g, 1.00 mmol) and compound
6 (0.34 g, 0.98 mmol) in anhydrous CH2Cl2 (60 mL) was added
DMAP (0.1 mmol), and the solution was left stirring at 0 °C for 10
min. DCC (0.25 g, 1.2 mmol) was then added, and the solution was
left stirring at room temperature for 8 h. The solution was then
filtered, and the filtrate was 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 (PE/EtOAc = 5/1, v/v) to obtain
the compound 9 as a light-yellow oil (0.50 g, 90%); [α]2D7 +6.0° (c 2.00
CHCl3). MS (ESI): m/z 567.4 [M + H]+. IR (cm−1, KBr): νmax 1124,
1
1259, 1455, 1659, 1722, 2925, 3360. H NMR (300 Hz, CDCl3): δ
0.89 (t, 3H, CH3, J = 7.0 Hz), 1.33−1.42 (m, 4H, 2 × CH2), 1.82−
1.93 (m, 2H, CH2), 2.28 (s, 3H, NCH3), 2.47−2.57 (m, 8H, 4 ×
NCH2), 3.32−3.40 (m, 2H, OCOCH2), 3.96−4.01 (m, 4H, 2 ×
OCH2), 4.36 (d, 1H, COH, J = 4.9 Hz), 4.39 (t, 1H, OCH, J = 4.9
Hz), 4.59 (s, 2H, OCH2Ar), 4.96 (t, 1H, OCH, J = 5.0 Hz), 5.36 (d,
1H, OCH, J = 5.7 Hz), 6.56−6.65 (m, 1H, OCHCH2), 7.27−7.35 (m,
6H, ArH), 7.49−7.51 (m, 2H, ArH), 7.90 (d, 1H, ArH, J = 8.0 Hz).
13C NMR (75 Hz, CDCl3): δ 169.6, 166.0, 143.7, 137.6, 132.4, 132.0,
130.4, 130.2, 128.5, 127.9, 127.7, 127.2, 126.2, 126.1, 86.4, 83.3, 80.7,
74.6, 73.2, 73.1, 71.4, 70.3, 59.4, 54.8, 54.8, 52.9, 52.9, 46.0, 36.6, 28.1,
22.7,14.1. HRMS (ESI): m/z calcd for C32H42N2O7 [M + H]+
567.2992; found 567.2302.
127.3, 126.2, 88.6, 88.2, 81.6, 80.5, 80.5, 73.4, 70.2, 59.2, 54.7, 54.7,
52.7, 52.7, 45.8, 36.4, 29.7, 28.0, 22.4. HRMS (ESI): m/z calcd for
C25H35N3O9 [M + H]+ 522.2373; found 522.2406.
2-Nitrooxy-5-{2-[(S)-(+)-1-(2-(4-methylpiperazin-1-yl)-
acetoxy)]pentyl}benzoate-1,4:3,6-dianhydro-D-glucitol (14).
Compound 14 was synthesized in the same manner as compound
13. Starting from compound 12 (0.39 g, 0.82 mmol), compound 14
was obtained as a light-yellow oil (0.18 g, 43%); [α]2D7 +35.1° (c 2.00
CHCl3). HRMS (ESI): m/z calcd for C25H35N3O9 [M + H]+
522.2373; found 522.2418. IR (KBr). ESI-MS, 1H NMR, and 13C
NMR spectral data were identical to those of compound 13.
Antiplatelet Aggregation Effect In Vitro. Blood samples were
withdrawn from the carotid artery of individual rabbits and mixed with
3.8% sodium citrate solution (9/1, v/v), followed by centrifuging at
500 rpm for 10 min at room temperature. After the collection of the
resulting platelet-rich plasma (PRP), the residue was centrifuged at
3000 rpm for another 10 min at room temperature to obtain platelet-
poor plasma (PPP). The PRP was adjusted with PPP in order to
obtain platelet counts of 400−450 × 109 Pl/L. The effects of individual
compounds on the platelet aggregation were determined by Born’s
turbidimetric method using a four-channel aggregometer (LG-PABER-
I Platelet-Aggregometer, Beijing, China) within 3 h after blood
collection. Briefly, PRP (240 μL) was preincubated with vehicle
DMSO, positive controls of Ticlid or ASP, or different concentrations
(0.1, 0.2, 0.4, 0.8, and 1.6 mM) of individual compounds (30 μL) for 5
min at 37 °C, followed by the addition of 10 μM of adenosine 5′-
diphosphate sodium salt (ADP, Sigma-Aldrich, USA) or 0.33 mM of
arachidonic acid (AA, Cayman Chemical, USA), respectively, to
induce the platelet aggregation. The maximum aggregation rate
(MAR) was recorded within 5 min at 37 °C. The inhibition rate of the
tested compounds on platelet aggregation was calculated with the
following formula: Inhibition rate (%) = (100% − MAR of tested
compound/MAR of vehicle). The IC50 value of each compound was
calculated accordingly.
2-Benzyloxy-5-{2-[(S)-(+)-1-(2-(4-methylpiperazin-1-yl)-
acetoxy)]pentyl}benzoate-1,4:3,6-dianhydro-D-glucitol (10).
Compound 10 was synthesized in the same manner as compound 9.
Starting from compound 8 (0.24 g, 1.00 mmol) and compound 6
(0.34 g, 0.98 mmol), compound 10 was obtained as a light-yellow oil
(0.51 g, 92%). [α]2D7 +14.7° (c 2.00 CHCl3). HRMS (ESI): m/z calcd
for C32H42N2O7 [M + H]+ 567.2992; found 567.2320. IR (KBr), ESI-
1
MS, H NMR, and 13C NMR spectral data were identical to those of
compound 9.
2-{2-[(S)-(+)-1-(2-(4-Methylpiperazin-1-yl)acetoxy)]pentyl}-
benzoate-2-hydroxy-1,4:3,6-dianhydro-D-glucitol (11). A mix-
ture of compound 9 (0.50 g, 0.88 mmol) and 10% Pd/C in methanol
(10 mL) was allowed to stir under an atmosphere of H2 (45 psi) at
room temperature for 4 h. The reaction mixture was filtered and
evaporated to dryness. The residue was purified by flash chromatog-
raphy (CH2Cl2/MeOH = 30/1, v/v) to obtain the compound 11 as a
light-yellow oil (0.40 g, 95%); [α]2D7 +14.3° (c 2.00 CHCl3). MS (ESI):
m/z 477.3 [M + H]+. IR (cm−1, KBr): νmax 170, 1260, 1456, 1721,
1
2955. H NMR (300 Hz, CDCl3): δ 0.89 (t, 3H, CH3, J = 6.8 Hz),
1.34−1.45 (m, 4H, 2 × CH2), 1.75−1.85 (m, 2H, CH2), 2.27 (s, 3H,
NCH3), 2.32−2.57 (m, 8H, 4 × NCH2), 3.31−3.48 (m, 2H,
OCOCH2), 3.72−4.03 (m, 4H, 2 × OCH2), 4.05 (t, 1H, OHCH, J
= 5.0 Hz), 4.43 (d, 1H, C−OH, J = 4.4 Hz), 4.63 (t, 1H, OCH, J = 4.9
Hz), 5.00 (t, 1H, OCH, J = 5.0 Hz), 5.38 (d, 1H, OCH, J = 5.2 Hz),
6.58−6.70 (m, 1H, OCHCH2), 7.30−7.35 (m, 1H, ArH), 7.50−7.54
(m, 2H, ArH), 7.90 (d, 1H, ArH, J = 8.2 Hz). 13C NMR (75 Hz,
CDCl3): δ 169.6, 166.1, 143.5, 132.5, 130.4, 128.0, 127.3, 126.2, 88.6,
88.2, 81.6, 80.5, 80.5, 73.4, 70.2, 59.2, 54.7, 54.7, 52.7, 52.7, 45.8, 36.4,
28.0, 22.4, 14.0. HRMS (ESI): m/z calcd for C25H36N2O7 [M + H]+
477.2523; found 477.2605.
Stability of (S)-5e in the Gastrointestinal Fluids. Artificial
gastric juice (500 mL) was prepared by adding 5.0 g of pepsin to water
and adjusting the pH to 1.5 with HCl. Then 500 mL of artificial
intestinal juice was prepared by adding 3.4 g of potassium dihydrogen
phosphate and 5.0 g of trypsin to water and then adjusting the pH to
6.8 with sodium hydroxide. (S)-5e (20 μL of 50 μM) was mixed in
triplicate with 180 μL of artificial gastric juice or intestinal juice and
incubated at 37 °C for 0, 1, 2, 4, and 8 h. The mixture was exposed to
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triple volumes of acetonitrile to terminate the reaction. The mixture
was vortexed for 2 min and centrifuged at 12000 rpm for 5 min. The
supernatant samples (20 μL each) were subjected to HPLC analysis
using Shimadzu 20A series (Shimadzu, Kyoto, Japan) on Sapphire C18
analytical column (250 mm × 4.6 mm, i.d., 5 μm, Sepax Technologies,
InC., Delaware, USA). The mobile phase comprised of methanol−
water (90/10, v/v) at a flow rate of 1.0 mL/min with the detection
wavelength at 260 nm.
Thromboembolism in Mice. Male Swiss mice (30−35 g, from
ICR animal colony) were purchased from B&K Universal Group,
Shanghai, China. Animals were housed in specific pathogen free facility
in a 12 h light/dark cycle with free access to food and water. The mice
were subjected to inducing acute systemic vascular thromboembolism
by infusion of a mixture of collagen and adrenaline.29 Briefly, the mice
were randomized and treated with vehicle dimethyl sulfoxide
(DSMO), ASP (160 mg/kg), (S)-NBP (160 mg/kg), or (S)-5e (160
or 680 mg/kg) by gavage daily for seven days. Two hours after the last
administration, the mice were injected intravenously with a mixture of
0.21 mg/kg of collagen and 44.5 μg/kg of adrenaline. The survival of
individual mice was monitored for 15 min.
Antithrombotic Activity Assay in Rats. Male Sprague−Dawley
(SD) rats (250−280 g) were randomized and divided into five groups
(n = 12 per group): (1) sham (0.5% CMC-Na), (2) vehicle (A-V +
0.5% CMC-Na), (3) A-V + (S)-NBP (80 mg/kg), (4) A-V + (S)-5e
(340 mg/kg), (5) A-V + (S)-5e (80 mg/kg). The rats were treated
with the indicated compound with the indicated dose by gavage daily
for seven days. Two hours after the last treatment, the rats were
anesthetized by intraperitoneally injecting with chloral hydrate (300
mg/kg) and subjected to the arteriovenous shunt procedure, as
described previously with modifications.30 The left jugular vein and the
right carotid artery of individual rats were inserted with a saline-filled
polyethylene catheter (14 cm in the length, containing a 6 cm silk
thread (4#)) for maintaining for 15 min, allowing the formation of a
thrombus along the silk thread. The shunt was subsequently removed,
and the silk thread with thrombus were collected and weighed
immediately. Subsequently, the silk thread was dried at room
temperature for 24 h to determine the dry mass. Both the wet and
dry weights of a thrombus were further subtracted by the weight of the
dry 6 cm silk thread.
Aqueous Solubility Assays. Individual compounds at ca. 1 mg
were dissolved in 10 mL of methanol and the maximum UV
absorption of each compound was determined in a UV755B
spectrophotometer, eventually diluting the solution (with CH3OH)
as necessary. A saturated solution of each compound was then
prepared by stirring magnetically a small volume of normal saline in
the presence of excess compound for 5 h at 25 °C. The saturated
solution was filtered with a 0.45 mm filter (Millipore) and measured
by UV-spectrometry at the wavelength determined. Total solubility
was determined by the relationship: C′ = A′CA−1, where C = the
concentration of standard solution (mg/mL), A = absorbance of
standard solution, A′ = absorbance of saturated solution, and C′ =
concentration of saturated solution (mg/mL).
coupled with a Security Guard C18 guars column (4 mm × 3.0 mm,
Phenomenex, Torrance, CA, USA) using gradient elution with water
containing 0.1% formic acid as solvent A and methanol as solvent B.
The gradient elution conditions were: B was kept 20% for 2 min,
increased to 80% at 2.5 min and kept for 3.5 min, and returned to 20%
at 6.5 min and kept for 1.5 min. The flow rate was 0.3 mL/min and the
injection volume was 10 μL. The column temperature was maintained
at 37 °C. The mass spectrometer equipped with an electrospray
ionization source was operated with a gas (N2) flow of 1.5 mL/min
and detector voltage of 1.6 kV. The heat block temperature was 200
°C, and the curved desolvation line (CDL) temperature was
maintained at 250 °C. LC-ESI-MS was performed in positive
selected-ion monitoring mode. (S)-5e was detected at m/z 807.7.
The Cerebral Ischemia/Reperfusion (I/R) Model in Rats. Male
SD rats (280−320 g) were randomly divided into five groups (n = 12
per group): (1) sham (0.5% CMC-Na), (2) vehicle (I/R + 0.5%
CMC-Na), (3) I/R + (S)-NBP (80 mg/kg), (4) I/R + (S)-5e (340
mg/kg), (5) I/R + (S)-5e (80 mg/kg). The rats were treated with the
indicated compound at the indicated dose by gavage daily for seven
days. Two hours after the last treatment, the rats were anesthetized
with chloral hydrate (300 mg/kg, ip) and subjected to a middle
cerebral artery occlusion (MCAO), as described previously with some
modification. Briefly, the right common carotid artery (CCA), internal
carotid artery (ICA), and external carotid artery (ECA) were exposed.
A 40 monofilament nylon suture (Beijing Sunbio Biotech, Beijing,
China) was carefully inserted from the ECA stump into the ICA and
was gently advanced to occlude the origin of the right middle cerebral
artery (MCA) until a light resistance was felt (18−20 mm from CCA
bifurcation). Two hours later, the suture was withdrawn to restore
blood flow (reperfusion). The sham group of rats received a sham
operation by inserting the filament into the ICA and immediately
withdrawing it. The core body temperature was monitored and
maintained at 37
throughout surgical procedures until the rats completely recovered
0.5 °C with a heating pad and warm light
from anesthesia.
Neurological Deficit Scores. Neurological deficits of individual
rats were evaluated at 24 h post reperfusion by the Longa’s method in
a blinded manner.35 Tests were always performed between 10 and 11
a.m. to exclude the possible interference of circadian rhythm-related
behavioral changes.
Measurement of Infarct Size. At 24 h post reperfusion, some rats
(n = 6) from each group were sacrificed and their brains were rapidly
removed onto ice and coronally sectioned (2 mm). The sections were
immediately incubated in 2% TTC for 30 min at 37 °C and fixed in
10% formalin overnight. The infarct areas (white) in individual
sections were measured and analyzed using Image-Pro Plus (Version
6.0). The infarct size was expressed as the percentage size in the total
area of whole brain.
Measurement of Brain-Water Content. At 24 h post
reperfusion, some rats (n = 6) form each group were sacrificed and
their brains were rapidly removed onto ice, followed by isolating the
ischemic hemispheres. The tissue samples were immediately weighed
to obtain the wet weight. The brains were then dried at 100 °C for 24
h and determined for its dry weight. The percentage of brain water
content was calculated as (wet weight − dry weight)/wet weight
×100%.
Histopathological Assessment. At 24 h post reperfusion, some
rats (n = 6) form each group were anesthetized with chloral hydrate
and perfused transcardially with saline and 4% paraformaldehyde,
followed by embedding in paraffin. The paraffin-embedded tissue
sections (5 μM) were stained with hematoxylin and eosin (H&E) and
examined under a light microscope.
The BBB Penetration of (S)-5e In Vivo Assay. The Male ICR
mice (30−35 g) were treated with a single dose (680 mg/kg) of (S)-5e
by gavage. The mice were sacrificed at 0.5, 1.0, 2.0, 4.0, and 8.0 h post
treatment. Their blood samples were collected, and their brain tissues
were dissected, washed with cold saline, and weighed. Their blood
plasma samples were prepared by centrifuging and stored on ice. The
individual brains were homogenized in 3 volumes of saline. Individual
plasma and homogenized brain samples (50 μL each) were mixed with
triple volumes of acetonitrile to precipitate proteins and centrifuged at
12000 rpm for 5 min. The supernatants (10 μL each) were used for
HPLC analysis.
HPLC analysis of (S)-5e in the prepared plasma and brain lysates
was performed on a LC-MS-2020 liquid chromatograph mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with a SIL-20A
autosampler, two LC-20AD pumps, a CTO-20A column oven, and an
electrospray-ionization (ESI) interface using Shimadzu LC-MS
Solution (version 5.42). Separation was performed on a shim-pack
VP-ODS column (5 μm, 2.1 mm × 150 mm, Shimadzu, Kyoto, Japan)
ASSOCIATED CONTENT
■
S
* Supporting Information
Tables of compound purity measured by HPLC and compound
optical purity measured by chiral HPLC. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*For Y.Z.: phone, +86-25-83271015; fax, +86-25-83271015; E-
mail, zyhtgd@163.com For H.J.: phone, +86-25-86021369; fax,
+86-25-86635503; E-mail, huijicpu@163.com. For J.X.: phone,
+86-25-83271445; fax, +86-25-83302827; E-mail, jinyixu@
china.com.
■
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was 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).
ABBREVIATIONS USED
■
ADP, adenosine diphosphate; AA, arachidonic acid; NBP, 3-n-
butylphthalide; ASP, aspirin; BBB, blood−brain barrier; SFDA,
State Food and Drug Administration; HPBA, 2-(1-hydrox-
ypentyl) benzoic acid; IS, isosorbide; DCC, 1,3-dicyclohex-
ylcarbodiimide; DMAP, 4-dimethylaminopyridine; ANOVA,
one-way analysis of variance; A-V, arterio-venous; SD,
Sprague−Dawley; I/R, ischemia/reperfusion; PRP, platelet
rich plasma; PPP, platelet-poor plasma; MAR, maximum
aggregation rate; SAR, structure−activity relationship; HPLC,
high-performance liquid chromatography; ESI, electrospray-
ionization; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenylte-
trazolium bromide; MCAO, middle cerebral artery occlusion;
CMC-Na, sodium carboxyl methyl cellulose; TTC, 2,3,5-
triphenyltetrazolium chloride; H&E, hematoxylin and eosin;
MDA, malondialdehyde; DSMO, dimethyl sulfoxide; CDL,
curved desolvation line; CCA, common carotid artery; ICA,
internal carotid artery; ECA, external carotid artery; MCA,
middle cerebral artery
(20) Gilmer, J. F.; Moriarty, L. M.; Lally, M. N.; Clancy, J. M.
Isosorbide-Based Aspirin Prodrugs II. Hydrolysis Kinetics of
Isosorbide Diaspirinate. Eur. J. Pharm. Sci. 2002, 16, 297−304.
(21) Moriarty, L. M.; Lally, M. N.; Carolan, C. G.; Jones, M.; Clancy,
J. M.; Gilmer, J. F. Discovery of a “True” Aspirin Prodrug. J. Med.
Chem. 2008, 51, 7991−7999.
(22) Jones, M.; Inkielewicz, I.; Medina, C.; Santos-Martinez, M. J.;
Radomski, A.; Radomski, M. W.; Lally, M. N.; Moriarty, L. M.;
Gaynor, J.; Carolan, C. G.; Khan, D.; O’Byrne, P.; Harmon, S.;
Holland, V.; Clancy, J. M.; Gilmer, J. F. Isosorbide-Based Aspirin
Prodrugs: Integration of Nitric Oxide Releasing Groups. J. Med. Chem.
2009, 52, 6588−6598.
(23) Yang, J. H.; Zhang, Y. X.; Feng, Y. P. Synthesis of Optically
Active 3-n-Butylphthalide. CN Patent ZL99109673.8, 1999.
(24) Abenhaïm, D.; Loupy, A.; Munnier, L.; Tamion, R.; Marsais, F.;
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