Design, synthesis and evaluation of nitric oxide releasing derivatives of 3-n-butylphthalide as antiplatelet and antithrombotic agents

Article abstract of DOI:10.1039/c1ob05478c

Novel nitric oxide (NO) releasing derivatives (7a-7l) of 3-n-butylphthalide (NBP) were designed and synthesized. Compound 7e inhibited the adenosine diphosphate (ADP), thrombin (TH) and arachidonic acid (AA)-induced in vitro platelet aggregation, superior to NBP and aspirin, released moderate levels of NO, and improved aqueous solubility relative to NBP. Furthermore, 7e exhibited greater antithrombotic activity than NBP and aspirin in rats, and protected against collagen and adrenaline-induced thrombosis in mice. Therefore, NO-releasing NBP derivatives possessed potent antiplatelet aggregation and antithrombotic activity. Our findings may aid in the design of new therapeutic agents for the treatment of thrombosis-related ischemic stroke.

Full text of DOI:10.1039/c1ob05478c

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PAPER
Design, synthesis and evaluation of nitric oxide releasing derivatives of
3-n-butylphthalide as antiplatelet and antithrombotic agents†
Xuliang Wang,a Yang Li,a Qian Zhao,b Zhenli Min,a Chao Zhang,b Yisheng Lai,a Hui Ji,*b Sixun Penga and
Yihua Zhang*a
Received 26th March 2011, Accepted 12th April 2011
DOI: 10.1039/c1ob05478c
Novel nitric oxide (NO) releasing derivatives (7a–7l) of 3-n-butylphthalide (NBP) were designed and
synthesized. Compound 7e inhibited the adenosine diphosphate (ADP), thrombin (TH) and
arachidonic acid (AA)-induced in vitro platelet aggregation, superior to NBP and aspirin, released
moderate levels of NO, and improved aqueous solubility relative to NBP. Furthermore, 7e exhibited
greater antithrombotic activity than NBP and aspirin in rats, and protected against collagen and
adrenaline-induced thrombosis in mice. Therefore, NO-releasing NBP derivatives possessed potent
antiplatelet aggregation and antithrombotic activity. Our findings may aid in the design of new
therapeutic agents for the treatment of thrombosis-related ischemic stroke.
Nitric oxide (NO) is a critical regulator of many physiolog-
Introduction
ical and pathological processes.7 The endothelium-derived NO
Thromboembolic diseases, including myocardial infarction, acute
participates in the regulation of blood pressure, vascular tone,
atherosclerosis, pulmonary embolism and ischemic stroke, are
platelet function, neurotransmission and cerebral circulation in
the leading causes of morbidity and mortality worldwide.1 The
formation of a thrombus is usually initiated by blood vessel injury,
which triggers platelet aggregation and adhesion of platelets to
the vessel wall.2 When acute thrombosis occurs in intracerebral
arteries, the formed thrombus and embolus can block the cerebral
blood flow (CBF), leading to the development of brain infarction
and ischemic stroke.3 Apparently, thrombosis is a key process in the
pathogenesis of acute ischemic stroke. Hence, development of new
drugs with antiplatelet aggregation and antithrombotic activities
will be of great significance in treatment of ischemic stroke.
The racemic 3-n-butylphthalide (NBP) was approved by the
State Food and Drug Administration (SFDA) of China as a new
drug mainly for the treatment of ischemic stroke in 2002.4 Previous
studies have demonstrated that NBP can inhibit platelet aggrega-
tion and thrombosis, improve microcirculation, and reduce the
brain infarct volume, providing clinical benefit to patients with
ischemic stroke.5 Unfortunately, NBP has poor aqueous solubility,
limiting its clinical application,4 Notably, the treatment of NBP
with another antiplatelet drug improves the therapeutic efficacy.6
Therefore, new derivatives of NBP may be more promising for the
treatment of ischemic stroke.
the cardiovascular and cerebrovascular systems.8 In particular,
NO can activate the soluble guanylyl cyclase (sGC) and increase
in cGMP, which acts as the second messenger to induce vasodi-
latation and inhibit platelet aggregation.9 Indeed, increasing NO
production either from endothelial NOS (eNOS) or NO-releasing
drugs (NO-donors) mimicking eNOS-derived NO has already
been used as a therapeutic approach for thrombotic complications,
such as myocardial infarction and ischemic stroke.10 Furthermore,
augmentation of NO production can improve CBF and promote
angiogenesis, benefiting patients with cerebral ischemia.11
Accordingly, we hypothesized that new NO-releasing derivatives
of NBP could display synergistic effects on platelet aggrega-
tion and thrombosis. Therefore, a series of novel NO-releasing
derivatives of NBP (NO-NBP) were designed and synthesized,
and their in vitro antiplatelet activities, NO-releasing ability,
aqueous solubility and in vivo antithrombotic potency were
evaluated.
Results and discussion
Strategy for the design of NO-NBP
aCenter of Drug Discovery, China Pharmaceutical University, Nanjing,
P. R. China, 210009. E-mail: zyhtgd@sohu.com; Fax: +86-25-83271015;
Tel: +86-25-83271015
Our previous studies and those of others have demonstrated that
conjugation of an NO-donor moiety with a “native” molecule
enhances therapeutic effect and/or reduces adverse effect of the
native molecule.12 In this study, we designed a novel class of
compounds by connecting the NO-donors (nitrates) to 2-(1-
hydroxypentyl) benzoic acid 1 through different linkers. Com-
pound 1, a ring-opening derivative of NBP, could be converted
bDepartment of Pharmacology, China Pharmaceutical University, Nanjing,
P. R. China, 210009. E-mail: huijicpu@163.com; Fax: +86-25-86021369;
Tel: +86-25-86021369
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of 6a–6f, 7a–7l and 8, HPLC conditions chromatograms of 7a–7l.
See DOI: 10.1039/c1ob05478c
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Scheme 1 Strategy for the design of NO-NBP.
to NBP very quickly in vivo.13 The linkers included hydroxy-
substituted cinnamic acid, like ferulic acid and p-hydroxyl cin-
namic acid, which has both antioxidant and antiplatelet aggre-
gation activity.14 In addition, amines, such as morpholine and
diethylamine, were introduced via a substituted acetate linkage to
the side chain of 1, respectively, to improve aqueous solubility of
these derivatives. We expected that the ester bonds of NO-NBP
would be cleaved in vivo by esterases to release 1, which would
subsequently undergo ring closure to generate NBP, and cinnamic
acid derivatives as well as NO (Scheme 1). These bioactive
compounds would synergistically inhibit platelet aggregation and
thrombosis.
compounds (e.g. 7d, 7e and 7k) at 1.0 mM displayed more potent
inhibition on platelet aggregation in vitro and 7e inhibited platelet
aggregation by 88.5% (Table 1).
Given that 7e is composed of three moieties, NBP precurosor
8, butyl ferulate (FAE), and organic nitrate 9, we further charac-
terized the inhibitory activity of each moiety on the ADP-induced
platelet aggregation in vitro. We found that 8, 9 and FAE at
a concentration of 200 mM inhibited the ADP-induced platelet
aggregation (36.3%, 32.5% and 16.0%, respectively) although each
moiety was less potent than 7e (51.1%, Fig. 1), suggesting that
these three components in 7e may synergistically inhibit the ADP-
induced platelet aggregation in vitro.
Chemistry
The synthesis of NO-NBP 7a–7l is illustrated in Scheme 2.
The bromo-substituted esters 4a–4f were prepared as described
previously,15 and converted to the corresponding nitrates 5a–
5f using AgNO3 in CH3CN. NBP was generated by reacting
commercially available 2-formylbenzoic acid 2 with Grignard
reagent n-BuMgBr in 85% yield. Subsequently, the NBP was
subjected to saponification and sequential acidification to pH 3–4
using dilute HCl solution at -10–0 ◦C to form 1 in 98% yield.
Treatment of 1 with chloroacetyl chloride yielded the acylated
compound 3, which was esterified with 5a–5f in the presence
of 1,3-dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) to offer esters 6a–6f. Because 1 was found
to be very easily lactonized to NBP at room temperature, it
is necessary to handle 1 below 0 ◦C during the acidification
and in the preparation of 3. The esters 6a–6f were condensed
with diethylamine or morpholine, and treated with anhydrous
ethereal HCl to generate the corresponding hydrochlorides 7a–
7l in 65–84% yields based on the two-step process. In addition,
3 was condensed with diethylamine to offer compound 8, a NBP
precursor. 1,4-Butanediol was treated with HNO3 and Ac2O in
EtOAc to give its mononitrate 9.16 All of new compounds were
Fig. 1 Inhibition of 7e, 8, 9 and FAE on the ADP-induced platelet
aggregation in vitro. Rabbit platelet suspensions were preincubated with
testing compound (200 mM) at 37 ◦C for 5 min and exposed to 10 mM
of ADP, followed by continually monitoring. Rabbit platelet suspensions
that had been treated with vehicle and exposed to ADP were used as
positive controls. Data are expressed as mean SD of each group (n = 4)
for two separate experiments. * P < 0.05, **P < 0.01 vs. 7e, determined
by Student’s t test.
Next, we determined whether the NO-releasing ability of 7e
could contribute to its inhibitory activity on the ADP-induced
platelet aggregation in rabbit PRP. Notably, the inhibitory effect
of 7e was enhanced upon treatment of 1 mM glutathione (GSH),
which is known to promote the NO release from nitrates,
suggesting that NO contributed to the inhibitory effect of 7e
on the ADP-induced platelet aggregation in PRP (Fig. 2). In
contrast, treatment with 10 mM of haemoglobin (Hb), a known
NO quencher,18 significantly reduced the inhibition of 7e on the
ADP-induced platelet aggregation. Our data indicate that the
nitrate moiety in 7e is important for its strong inhibition of the
further purified and characterized by IR, ESI-MS, 1H NMR, 13
C
NMR, and HRMS, and individual compounds with a purity of
>95% were used for subsequent experiments.
In vitro antiplatelet effect of NO-NBP
The effects of individual compounds on the adenosine diphos-
phate (ADP)-induced in vitro platelet aggregation in rabbit platelet
rich plasma (PRP) were preliminarily evaluated using Born’s
turbidimetric method.17 In comparison with the reference drugs
NBP and aspirin (ASP), a well-known antiplatelet agent, several
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Scheme 2 Synthetic route of compounds 7a–7l, 8 and 9. Reagents and conditions: (a) AgNO3, CH3CN, 60 ◦C, 2 h; (b) (i) n-BuMgBr, Et2O, -5 to 0 ◦C,
5 h; (ii) 1 M HCl, RT, 0.5 h; (c) (i) NaOH, CH3OH–H2O, reflux, 0.5 h; (ii) 1 M HCl, -10 to 0 ◦C; (d) ClCH2COCl, Et3N, DMAP, CH2Cl2, -10 ◦C; (e)
DCC, DMAP, CH2Cl2, RT, 5 h; (f) diethylamine or morpholine, Et3N, DMF, RT, 8 h; (g) 1 M ethereal HCl, 0 ◦C, 1 h; (h) diethylamine, Et3N, CH2Cl2,
RT, 12 h; (i) HNO3, Ac2O, EtOAc, 0 ◦C, 12 h.
ADP-induced platelet aggregation. Given that NO can activate
sGC and result in an increase in the levels of cGMP, inhibiting
platelet aggregation, we further tested whether inhibition of sGC
could modulate the inhibitory effect of 7e on the ADP-induced
platelet aggregation. We found that treatment with 25 mM of 1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a well known
inhibitor of sGC, significantly mitigated the inhibitory effect of
7e on the ADP-induced platelet aggregation. These data further
supported that NO produced by 7e contributed to its potent
antiplatelet aggregation activity through the sGC pathway.
Further analysis revealed that 7e inhibited the ADP, thrombin
(TH), and arachidonic acid (AA)-induced platelet aggregation in
PRP in a concentration-dependent manner and the inhibitory
potency of 7e on the platelet aggregation induced by these
aggregators ranked in the order AA > ADP > TH, which was
similar to that of NBP (Table 2, Fig. 3). In comparison with
NBP and ASP, 7e displayed much higher inhibition on the ADP-
and TH-induced platelet aggregation, but comparable inhibition
on the AA-induced platelet activation (Table 3), indicating that
different activation mechanisms of the three aggregators inducing
platelet aggregation are involved and affect the inhibitory activities
of 7e, which remain to be investigated.
Correlation of NO produced by NO-NBP with their antiplatelet
aggregation activities
To examine the potential relationship between NO produced by
NO-NBP and their antiplatelet aggregation activities, the levels of
NO produced by 7e, 7k, 7d, 7c and 7i were determined by Griess
assay (Fig. 4).19 We found that the levels of NO produced by 7e,
7k, 7d, 7c and 7i were 0.3, 0.28, 0.2, 0.05, 0.04 mg ml-1, respectively.
Further analysis revealed that the levels of NO produced by
these NO-NBP were correlated positively with the potencies
of these compounds in inhibiting platelet aggregation in vitro
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Table 1 The effect of compounds on the ADP (10 mM)-induced platelets
aggregation in vitroa
Compd. (1.0 mM)
R1
R2
n
% inhibition
Aspirin
NBP
7a
—
—
OMe
—
—
—
—
3
73.7
51.4
23.4
7b
7c
7d
7e
7f
7g
7h
7i
OMe
OMe
OMe
OMe
OMe
H
4
5
3
4
5
3
4
5
3
4
5
29.4
18.5
60.6
88.5
30.3
21.4
35.7
19.8
31.2
70.5
19.3
Fig. 2 The NO formed by 7e contributes to the inhibition of 7e on the
ADP-induced platelet aggregation in vitro. Rabbit platelet rich plasma
(PRP) were treated with 7e (200 mM) in presence or absence of 1 mM
of GSH, 10 mM of haemoglobin (Hb) or 25 mM of ODQ, respectively
and exposed to ADP. The platelet aggregation was determined. Data
are expressed as mean SD of each group (n = 4) from two separate
experiments. * P < 0.05, **P < 0.01 vs. 7e alone, determined by Student’s
t test.
Table 2 The IC50 values of 7e, aspirin, and NBP inhibiting rabbit platelet
aggregation in vitro
IC50 (mM)
Compd.
ADP (10 mM)
TH (0.5 U/mL)
AA (1.0 mM)
Aspirin
NBP
7e
0.82
> 1.0a
0.24
> 1.0b
> 1.0c
0.41
0.04
0.10
0.16
H
a A 51.3% inhibition of platelet aggregation was observed at maximal
concentration of 1.0 mM. b A 48.4% inhibition of platelet aggregation was
observed at maximal concentration of 1.0 mM. c A 31.3% inhibition of
platelet aggregation was observed at maximal concentration of 1.0 mM.
H
7j
H
in aqueous solubility of NO-NBP could be associated with
their enhanced bioactivities. We determined the solubility of
individual compounds as described previously.20 As shown in
Table 3, the saturated concentrations of compounds 7e, 7d and
7k with strong antiplatelet aggregation activity were 0.98, 0.62
and 0.81 mM, respectively, which were higher than that of control
NBP (0.53 mM). In contrast, the saturated concentrations of
compounds 7c and 7i with poor antiplatelet aggregation activity
were 0.36 mM and 0.39 mM, which were less than that of
NBP. Further analysis indicated that the saturated concentrations
of these compounds were correlated significantly with their
antiplatelet aggregation activities (R2 = 0.96, P < 0.01, determined
7k
7l
H
H
a Rabbit platelet suspensions were preincubated with 1.0 mM of each
compound tested at 37 ◦C for 5 min followed by the addition of ADP
(10 mM). Data are expressed as mean % of inhibition from two separate
experiments.
(R2 = 0.98, P < 0.01, determined by logistic regression analysis).
These, together with observation that treatment with the NO
quencher, Hb, mitigated the inhibitory effect of 7e on the ADP-
induced platelet aggregation, clearly indicate that the moderate
levels of NO produced by these NO-NBP are crucial for their
antiplatelet aggregation activities.
Table 3 The solubility of NBP and selected NO-NBPa
Compd.
Solubility (mM)
Compd.
Solubility (mM)
NBP
7c
7d
0.53
0.36
0.62
7e
7k
7i
0.98
0.81
0.39
Aqueous solubility of NO-NBP
a Data are expressed as mean concentration of individual compounds
saturating in 5 ml saline at 25 ◦C.
Since poor solubility of NBP affects its clinical application and
therapeutic efficacy, we further examined whether an increase
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Fig. 3 Compound 7e inhibits the ADP-, TH- and AA-induced platelet aggregation in a dose-dependent manner. PRP were treated in duplicate with
vehicle alone or with the indicated doses of 7e and exposed to 10 mM of ADP (a), 0.5 U mL-1 of TH (b), and 1.0 mM of AA (c), respectively. The platelet
aggregation was determined. The PRP treated with vehicle and individual aggregators were used as positive controls and data are expressed as mean %
of different doses of 7e relative to the positive controls from two separate experiments. Intra-group variation was less than 7%. Treatment with 1 mM of
7e did not cause detectable platelet aggregation (data not shown).
Fig. 5 Effect of ASP, NBP, 7e and 7k on the thrombus formation in rats.
Male SD rats were randomized and treated orally with vehicle control,
ASP, NBP, 7e, and 7k for consecutive 5 days and the formation of thrombus
Fig. 4 Variable levels of NO were produced by selected NO-NBP
in vitro. The levels of NO produced by selected NO-NBP were determined
by Griess assay. Individual compounds were incubated for the indicated
periods and reacted with Griess reagent at 30 ◦C for 10 min, followed by
measuring at 540 nm. The levels of NO produced by individual compounds
were calculated, according to the standards of different concentrations of
nitrate. Data are expressed as means of individual compounds tested at
each time point and intra-group variations were less than 10%.
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 < 0.01 vs. the control group,
determined by Student’s t test.
7e treated rats were also slightly less than that in the NBP-treated
rats (29.97 7.01 mg) and ASP (30.73 6.08 mg), indicating that
7e has greater antithrombotic activity in vivo.
by logistic regression analysis). These data clearly demonstrate
that NO-NBP have better solubility, which contribute to their
high antiplatelet aggregation activity. The improved solubility of
these compounds may enhance their abilities to target the lesion
areas and promote clinical applications.
Inhibitory effect of 7e on the thrombosis in mice
In parallel, we finally evaluated the antithrombotic effect of 7e
on a mouse model of thromboembolism. Male Swiss mice were
randomized and administered orally with vehicle (0.1% CMC-
Na) alone, equimolar doses of 7e, ASP, and NBP, respectively. Two
hours after the treatment, the rats were injected intravenously with
collagen and adrenaline for inducing thromboembolism and the
development of hemiplegia and death in the rats were monitored
within 15 min. Apparently, treatment with 7e significantly reduced
the onset of hemiplegia and death in mice and its protective effect
was similar to that of NBP and Asp in this model (Table 4).
Antithrombotic activities of 7e and 7k in rats
Next, we investigated the effects of compounds 7e and 7k on
the formation of thrombus in a rat extra-corporeal circulation
of arteriovenous (A-V) cannula model. Male SD rats were
randomized and treated orally with 0.1% CMC-Na (negative
control), ASP, NBP, 7e and 7k at equimolar doses (1.58 ¥ 10-4
mol kg-1) for 5 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. 5, the thrombus
weights in the rats receiving 7e (27.95 7.32 mg) and 7k (35.27
4.85 mg) were significantly less than that of the negative control
group (53.37 7.35 mg, P < 0.01), and the thrombus weights in the
Discussion and conclusions
Analysis of structure and activity relationship (SAR) revealed
that 7a–7l with different linkers displayed variable antiplatelet
aggregation activities. The varied length of carbon chain
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Table 4 The effect of 7e on the thromboembolism-induced death and hemiplegia in mice
Group
Dose (mg kg-1)
Number of mice
Number of death in 5 min and hemiplegia in 15 min
Protection rate (%)
Control
Aspirin
NBP
—
100
100
342
17
15
17
18
11
3
5
35.3
80.0a
70.6a
72.2a
7e
5
a P < 0.05 vs. the control group.
connecting to ferulic acid or p-hydroxyl cinnamic acid in those
compounds appeared to affect their antiplatelet aggregation
activities. Evidentially, 7b, 7e, 7h and 7k with a four-carbon linker
displayed the strongest antiplatelet aggregation activity in the
same subgroup. It is possible that varied linkers have different
abilities to modulate the structure, stability, metabolism and
penetrability of these compounds, which affect the NO production
and NBP releasing, leading to various bioactivities of NO-NBP.
Furthermore, the amino component substituted in the side-chain
of NO-NBP was important for their bioactivities. The compounds
(7d–7f and 7j–7l) bearing a diethylamino side-chain exhibited
stronger inhibitory effects than the compounds (7a–7c and 7g–7i)
bearing a morpholino side-chain. Indeed, 7e containing a four-
carbon linker and a diethylamino side-chain produced the highest
levels of NO and displayed the best aqueous solubility and the
strongest biological activities both in vitro and in vivo among the
compounds tested. However, the precise mechanisms underlying
the SAR of NO-NBP remain to be further investigated.
In summary, we designed and synthesized a series of NO-
releasing derivatives of NBP and found that some NO-NBP had a
strong inhibitory effect on platelet aggregation in vitro, particularly
for 7e, which was the most potent in inhibiting the ADP-, TH-
and AA-induced platelet aggregations, superior to NBP and ASP.
Furthermore, several NO-NBP produced moderate levels of NO
in vitro and exhibited better solubility, which were correlated with
their antiplatelet aggregation activities. Notably, our preliminary
pharmacokinetic study showed that 7e could generate the parent
molecule NBP under physiological conditions, which is consistent
with our working hypothesis.21 Interestingly, all the three structural
moieties (NBP precursor 8, nitrate 9 and FAE) had moderate
antiplatelet aggregation activity, and treatment with NO quencher
or sGC inhibitor mitigated the inhibitory effect of 7e on the ADP-
induced platelet aggregation. Furthermore, 7e exhibited greater
antithrombotic activity in rats, and protected against the collagen
and adrenaline induced thrombosis in mice. These data suggest
that these structural moieties may act synergistically inhibiting
platelet aggregation and thrombosis. Therefore, our findings may
aid in the design of new therapeutic reagents for the treatment of
thrombosis-related ischemic stroke and other vascular diseases.
(TLC) on silica gel 60 F254 plates (250 mm; Qingdao Ocean Chem-
ical 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 characterized by
HPLC analysis (Shimadzu LC-2010A HPLC system) and high
resolution mass spectrometry (Agilent technologies LC/MSD
TOF). Target compounds 7a–7l with a purity of >95% were used
for subsequent experiments (see the ESI†).
( )-3-Butyl-1(3H)-isobenzofuranone (NBP)22
To a solution of magnesium (0.48 g, 20.0 mmol) in anhydrous
Et2O (5 mL) was added 1-bromobutane (2.74 g, 20.0 mmol) under
nitrogen atmosphere until the Grignard reaction had started. After
complete addition, the reaction mixture was refluxed for 1 h and
allowed to cool to RT. A solution of 2 (1.50 g, 10.0 mmol) in
anhydrous Et2O (30 mL) was added dropwise to the Grignard
solution at -5 ◦C. After completion by addition, the reaction
mixture was stirred at RT for 5 h. The reaction was quenched by
addition of a saturated aqueous solution of NH4Cl (10 mL). The
mixture was acidified with 1 M HCl (15 mL) to pH 2 and stirred for
1 h at RT. The solution was extracted with Et2O (50 mL ¥ 3), dried
over anhydrous Na2SO4, and evaporated in vacuo. The resulting
residue was purified by column chromatography (petroleum ether–
EtOAc 15 : 1 v/v) to afford NBP as a light yellow oil (1.61 g, 85%).
ESI-MS: m/z 213 [M + Na]+. 1H NMR (CDCl3, 300 Hz, d): 0.93(t,
3H, CH3, J = 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).
( )-2-(1-Hydroxypentyl)benzoic acid (1)
To a solution of NBP (1.24 g, 6.52 mmol) in MeOH–H2O (10 mL,
1 : 1 v/v) was added NaOH (0.28 g, 7.18 mmol). The reaction
mixture was refluxed for 0.5 h. The solvent was removed under
reduced pressure and dissolved in water (10 mL), followed by
acidification with 1 M HCl to pH 3–4 at –5-0 ◦C. The mixture was
extracted with cold Et2O (5 mL ¥ 3) and quickly used for the next
step without any purification.
( )-2-[1-(2-Chloroacetoxy)pentyl]benzoic acid (3)
Experimental section
To a solution of compound 1 (1.35 g, 6.52 mmol) and Et3N
(2.72 mL, 19.6 mmol) in CH2Cl2 (100 mL) was added 2-
chloroacetyl chloride (2.72 mL, 19.6 mmol) 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 separated and dried with anhydrous Na2SO4.
After removal of solvent, the residue was recrystallized from
General
Bromo-substituted esters 4a–4f,15 and NO donor moiety 9
were prepared as described previously.16 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
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n-hexane to afford 3 as white crystals (1.11 g, 60%, over two steps).
Mp 67–68 ◦C. ESI-MS: m/z 283 [M - H]-. IR (KBr): 1412, 1691,
1734, 2958, 3450 cm-1. 1H NMR (CDCl3, 300 Hz, d): 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, d): 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.
(E)-4-(Nitrooxy)butyl 3-(4-hydroxyphenyl)acrylate (5e)
The title compound was obtained as a pale yellow solid (84%),
◦
mp 73–74 C. ESI-MS: m/z 282 [M + H]+, 304 [M + Na]+. H
NMR (CDCl3, 300 Hz, d): 1.86–1.88 (m, 4H, 2¥CH2), 4.26 (t, 2H,
CH2ONO2, J = 5.5 Hz), 4.53 (t, 2H, OCH2, J = 6.0 Hz), 6.26 (d,
1H, CH , J = 16.1 Hz), 6.82–6.87 (m, 2H, ArH), 7.42–7.46 (m,
2H, ArH), 7.61 (d, 1H, CH , J = 16.0 Hz).
1
(E)-5-(Nitrooxy)pentyl 3-(4-hydroxyphenyl)acrylate (5f)
General procedure for the synthesis of nitrate 5a–5f23
The tit◦le compound was obtained as a pale yellow solid (87%), mp
75–76 C. ESI-MS: m/z 296 [M + H]+, 318 [M + Na]+. 1H NMR
(CDCl3, 300 Hz, d): 1.50–1.56 (m, 2H, CH2), 1.72–1.85 (m, 4H,
2¥CH2), 4.23 (t, 2H, CH2ONO2, J = 6.3 Hz), 4.56 (t, 2H, OCH2,
J = 6.5 Hz), 6.25 (d, 1H, CH , J = 16.0 Hz), 6.82–6.87 (m, 2H,
ArH), 7.42–7.47 (m, 2H, ArH), 7.62 (d, 1H, CH , J = 15.9 Hz).
To a solution of corresponding bromo-substituted esters 4a–4f
(5.0 mmol) in CH3CN (15 mL), was added AgNO3 (7.5 mmol)
at RT. The suspension was heated at 70◦ C for 2 h while being
protected from light. The mixture was filtered and the filtrate
concentrated. The residue was dissolved in CH2Cl2 (50 mL) and
washed with water, brine, in turn, and dried with anhydrous
Na2SO4, and finally concentrated to give the crude title compound
(78–85%).
General procedure for the synthesis of compounds 6a–6f
To a solution of acylated compound 3 (2.1 mmol) and the
corresponding nitrates 5a–5f (2.0 mmol) in anhydrous CH2Cl2
(25 mL) was added DMAP (0.1 mmol) and the solution was left
stirring at 0 ◦C for 10 min. DCC (2.1 mmol) was then added
and the solution was left stirring at RT for 6 h. The solution
was then filtered, and the filtrate washed successively with 1 M
HCl, a saturated aqueous solution of NaHCO3, water, and brine.
The solution was then dried with anhydrous Na2SO4, filtered, and
solvent removed under reduced pressure. The resulting residue
was purified by flash chromatography (petroleum ether–EtOAc
5 : 1 v/v) to give the title compounds (70–83%).
(E)-3-(Nitrooxy)propyl 3-(4-hydroxy-3-methoxyphenyl) acrylate
(5a)
The tit◦le compound was obtained as a pale yellow solid (78%), mp
60–61 C. ESI-MS: m/z 298 [M + H]+, 320 [M + Na]+. 1H NMR
(CDCl3, 300 Hz, d): 2.12–2.20 (m, 2H, CH2), 3.89 (s, 3H, OCH3),
4.33 (t, 2H, CH2ONO2, J = 6.0 Hz), 4.61 (t, 2H, OCH2, J = 6.3
Hz), 6.27 (d, 1H, CH =, J = 15.9 Hz), 6.91–6.93 (m, 1H, ArH),
7.03–7.09 (m, 2H, ArH), 7.59 (d, 2H, CH =, J = 16.0 Hz).
(E)-4-(Nitrooxy)butyl 3-(4-hydroxy-3-methoxyphenyl) acrylate
(5b)
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {2-methoxy-4-[2-
(3-nitrooxypropoxycarbonyl)-vinyl]}phenyl ester (6a)
The title compound was obtained as a pale yellow solid (80%),
The title compound was obtained as a colourless waxy solid (75%),
mp 63–65 ◦C. ESI-MS: m/z 581 [M + NH4]+, 586 [M + Na]+. 1H
NMR (CDCl3, 300 Hz, d): 0.85 (t, 3H, CH3, J = 6.9 Hz), 1.26–
1.46 (m, 4H, 2¥CH2), 1.86–1.95 (m, 2H, CH2), 2.12–2.20 (m, 2H,
CH2), 3.89 (s, 3H, OCH3), 4.09 (m, 2H, COCH2Cl), 4.33 (t, 2H,
CH2ONO2, J = 6.0 Hz), 4.62 (t, 2H, OCH2, J = 6.0 Hz), 6.40 (d,
1H, CH , J = 15.9 Hz), 6.67–6.71 (m, 1H, CH), 7.19–7.26 (m,
3H, ArH), 7.42 (m, 1H, ArH), 7.60 (m, 2H, ArH) 7.68 (d, 1H,
CH , J = 16.0 Hz), 8.15 (d, 1H, ArH, J = 7.8 Hz). 13C NMR
(CDCl3, 300 Hz, d): 166.5, 166.4, 164.5, 151.6, 144.8, 143.5, 141.7,
133.3, 133.1, 131.0, 127.6, 127.2, 126.2, 123.5, 121.4, 117.7, 111.4,
74.9, 69.9, 60.5, 55.9, 41.0, 36.5, 27.9, 26.5, 22.4, 13.9. HRMS for
C27H30NO10ClNa ([M + Na]+) calcd: 586.1456, Found: 586.1466.
◦
mp 63–64 C. ESI-MS: m/z 312 [M + H]+, 334 [M + Na]+. H
NMR (CDCl3, 300 Hz, d): 1.81–1.89 (m, 4H, 2¥CH2), 3.93 (s, 3H,
OCH3), 4.24 (t, 2H, CH2ONO2, J = 5.5 Hz), 4.52 (t, 2H, OCH2,
J = 6.0 Hz), 6.27 (d, 1H, CH , J = 16.1 Hz), 6.91–6.93 (m, 1H,
ArH), 7.03–7.09 (m, 2H, ArH), 7.61 (d, 1H, CH , J = 16.0 Hz).
1
(E)-5-(Nitrooxy)pentyl 3-(4-hydroxy-3-methoxyphenyl) acrylate
(5c)
The tit◦le compound was obtained as a pale yellow solid (83%), mp
67–68 C. ESI-MS: m/z 326 [M + H]+, 348 [M + Na]+. 1H NMR
(CDCl3, 300 Hz, d): 1.50–1.56 (m, 2H, CH2), 1.72–1.85 (m, 4H,
2¥CH2), 3.92 (s, 3H, OCH3), 4.23 (t, 2H, CH2ONO2, J = 6.3 Hz),
4.48 (t, 2H, OCH2, J = 6.5 Hz), 6.25 (d, 1H, CH , J = 16.0 Hz),
6.91–6.93 (m, 1H, ArH), 7.03–7.09 (m, 2H, ArH), 7.62 (d, 1H,
CH , J = 15.9 Hz).
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {2-methoxy-4-[2-
(4-nitrooxybutoxycarbonyl)-vinyl]}phenyl ester (6b)
The title compound was obtained as a colourless waxy solid (74%),
mp 62–64 ◦C. ESI-MS: m/z 595 [M + NH4]+, 600 [M + Na]+. 1H
NMR (CDCl3, 300 Hz, d): 0.85 (t, 3H, CH3, J = 7.1 Hz), 1.24–1.47
(m, 4H, 2¥CH2), 1.86–2.04 (m, 6H, 3¥CH2), 3.89 (s, 3H, OCH3),
4.10 (m, 2H, COCH2Cl), 4.26 (t, 2H, CH2ONO2, J = 6.0 Hz), 4.52
(t, 2H, OCH2, J = 6.0 Hz), 6.41 (d, 1H, CH , J = 15.9 Hz), 6.67–
6.71 (m, 1H, CH), 7.20–7.26 (m, 3H, ArH), 7.42 (m, 1H, ArH),
7.61 (m, 2H, ArH) 7.68 (d, 1H, CH , J = 16.0 Hz), 8.15 (d, 1H,
ArH, J = 7.8 Hz). 13C NMR (CDCl3, 300 Hz, d): 166.7, 166.4,
(E)-3-(Nitrooxy)propyl 3-(4-hydroxyphenyl)acrylate (5d)
The title compound was obtained as a pale yellow solid (80%),
◦
mp 68–69 C. ESI-MS: m/z 268 [M + H]+, 290 [M + Na]+. H
NMR (CDCl3, 300 Hz, d): 2.12–2.19 (m, 2H, CH2), 4.33 (t, 2H,
CH2ONO2, J = 6.1 Hz), 4.61 (t, 2H, OCH2, J = 6.8 Hz), 6.28 (d,
1H, CH , J = 16.0 Hz), 6.82–6.87 (m, 2H, ArH), 7.41–7.46 (m,
2H, ArH), 7.61 (d, 2H, CH , J = 16.0 Hz).
1
5676 | Org. Biomol. Chem., 2011, 9, 5670–5681
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164.5, 151.6, 144.4, 143.5, 141.6, 133.4, 133.1, 131.0, 127.6, 127.3,
126.2, 123.5, 121.3, 118.0, 111.4, 74.9, 63.6, 55.9, 41.0, 36.5, 27.9,
25.1, 23.7, 22.4, 13.9. HRMS for C28H32NO10ClNa ([M + Na]+)
calcd: 600.1612, Found: 600.1624.
300 Hz, d): 0.86 (t, 3H, CH3, J = 6.9 Hz), 1.30–1.45 (m, 4H,
2¥CH2), 1.52–1.59 (m, 2H, CH2), 1.73–2.03 (m, 6H, 3¥CH2), 4.09
(m, 2H, COCH2Cl), 4.23 (t, 2H, CH2ONO2, J = 6.3 Hz), 4.48 (t,
2H, OCH2, J = 6.3 Hz), 6.43 (d, 1H, CH , J = 16.0 Hz), 6.68
(m, 1H, CH), 7.26–7.30 (m, 2H, ArH), 7.43 (m, 1H, ArH), 7.60–
7.63 (m, 4H, ArH), 7.69 (d, 1H, CH , J = 16.0 Hz), 8.14 (d, 1H,
ArH, J = 7.8 Hz). 13C NMR (CDCl3, 300 Hz, d): 166.9, 166.6,
164.9, 152.2, 143.8, 143.7, 133.4, 132.3, 130.8, 129.4, 129.4, 127.7,
127.2, 126.4, 122.4, 122.4, 118.0, 74.8, 73.0, 64.1, 41.0, 36.3, 28.3,
27.9, 26.5, 23.7, 22.5, 22.4, 13.9. HRMS for C28H32NO9ClNa ([M
+ Na]+) calcd: 584.1663, Found: 584.1664.
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {2-methoxy-4-[2-
(5-nitrooxypentoxycarbonyl)-vinyl]}phenyl ester (6c)
The title compound was obtained as a colourless waxy solid (70%),
mp 60–61 ◦C. ESI-MS: m/z 609 [M + NH4]+, 614 [M + Na]+. 1H
NMR (CDCl3, 300 Hz, d): 0.85 (t, 3H, CH3, J = 7.2 Hz), 1.29–
1.44 (m, 4H, 2¥CH2), 1.52–1.60 (m, 2H, CH2), 1.75–1.93 (m, 6H,
3¥CH2), 3.89 (s, 3H, OCH3), 4.09 (m, 2H, COCH2Cl), 4.24 (t, 2H,
CH2ONO2, J = 6.4 Hz), 4.48 (t, 2H, OCH2, J = 6.4 Hz), 6.42 (d,
1H, CH , J = 15.9 Hz), 6.68 (m, 1H, CH), 7.19–7.26 (m, 3H,
General procedure for the synthesis of compounds 7a–7l
To a solution of esters 6a–6f (0.45 mmol) and Et3N (1 mL) in
DMF (5 mL) was added the appropriate amine (morpholine
or diethylamine, 1.35 mmol) and the solution was left stirring
at RT for 8 h. The solution was then filtered, and the filtrate
was reconstituted in EtOAc (20 mL) and water (20 mL). The
organic phase was washed with water and brine. The solution
was then dried with anhydrous Na2SO4, filtered, and solvent
removed under reduced pressure. The amino derivatives were
purified by flash chromatography (petroleum ether–EtOAc 3 : 1
v/v), to which EtOAc (0.5 mL) and 1 M ethereal HCl (0.5 mL)
was added dropwise at 0 ◦C. The solution was left stirring at room
temperature for 1 h and filtered to give the title compounds (65–
84%).
ArH), 7.42 (m, 1H, ArH), 7.60 (m, 2H, ArH), 7.67 (d, 1H, CH
,
J = 16.0 Hz), 8.15 (d, 1H, ArH, J = 7.8 Hz). 13C NMR (CDCl3, 300
Hz, d): 166.8, 166.4, 164.5, 151.5, 144.2, 143.5, 141.5, 133.5, 133.1,
131.0, 127.6, 127.3, 126.2, 123.4, 121.3, 118.2, 111.3, 74.9, 73.0,
64.1, 55.9, 41.0, 36.5, 28.3, 27.9, 26.5, 22.5, 22.4, 13.9. HRMS for
C29H34NO10ClNa ([M + Na]+) calcd: 614.1769, Found: 614.1780.
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {4-[2-(3-nitrooxy-
propoxycarbonyl)-vinyl]}phenyl ester (6d)
The title compound was obtained as a colourless waxy solid (79%),
mp 61–62 ◦C. ESI-MS: m/z 551 [M + NH4]+. 1H NMR (CDCl3,
300 Hz, d): 0.87 (t, 3H, CH3, J = 6.9 Hz), 1.26–1.50 (m, 4H,
2¥CH2), 1.87–1.95 (m, 2H, CH2), 2.12–2.20 (m, 2H, CH2), 4.08
(m, 2H, COCH2Cl), 4.33 (t, 2H, CH2ONO2, J = 6.0 Hz), 4.61 (t,
2H, OCH2, J = 6.0 Hz), 6.42 (d, 1H, CH , J = 16.0 Hz), 6.68
(m, 1H, CH), 7.28–7.31 (m, 2H, ArH), 7.36 (m, 1H, ArH), 7.40–
7.63 (m, 4H, ArH), 7.72 (d, 1H, CH , J = 16.0 Hz), 8.15 (d, 1H,
ArH, J = 8.1 Hz). 13C NMR (CDCl3, 300 Hz, d): 166.6, 166.5,
164.9, 152.3, 144.3, 143.7, 133.4, 132.2, 130.8, 129.4, 129.4, 127.7,
127.2, 126.4, 122.5, 122.5, 117.7, 74.8, 69.9, 60.5, 41.0, 36.3, 27.9,
26.5, 22.4, 13.9. HRMS for C26H28NO9ClNa ([M + Na]+) calcd:
556.1350, Found: 556.1358.
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {2-methoxy-
4-[2-(3-nitrooxypropoxycarbonyl)-vinyl]}phenyl ester
hydrochloride (7a)
The title compound◦was obtained as a white solid (75%, for two
steps), mp 103–105 C. ESI-MS: m/z 615 [M + H]+. IR (KBr):
759, 1035, 1641, 1739, 2955, 3069 cm-1. 1H NMR (CDCl3, 300 Hz,
d): 0.85 (t, 3H, CH3, J = 6.3 Hz), 1.31 (m, 4H, 2¥CH2), 1. 92 (m,
2H, CH2), 2.17 (m, 2H, CH2), 3.36 (m, 4H, 2¥NCH2), 3.90 (s, 3H,
OCH3), 3.94 (m, 2H, COCH2N), 4.04 (m, 4H, 2¥OCH2), 4.34 (t,
2H, CH2ONO2, J = 6.0 Hz), 4.62 (t, 2H, COOCH2, J = 6.3 Hz),
6.42 (d, 1H, CH , J = 16.0 Hz), 6.75 (m, 1H, CH), 7.17–7.27
(m, 3H, ArH), 7.43–7.67 (m, 3H, ArH), 7.70 (d, 1H, CH , J =
15.9 Hz), 8.18 (d, 1H, ArH, J = 7.8 Hz), 13.78 (br s, 1H, NH+).
13C NMR (CDCl3, 300 Hz, d): 166.5, 164.5, 164.3, 151.5, 144.6,
142.6, 141.5, 133.5, 133.4, 131.1, 128.0, 127.2, 126.3, 123.4, 121.4,
117.8, 111.4, 75.5, 70.0, 64.1, 64.1, 60.6, 56.0, 55.6, 51.1, 51.1, 36.2,
27.8, 26.5, 22.3, 13.9. HRMS for C31H39N2O11 ([M + H]+) calcd:
615.2554, found: 615.2568.
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {4-[2-(4-nitrooxy-
butoxycarbonyl)-vinyl]}phenyl ester (6e)
The title compound was obtained as a colourless waxy solid (83%),
mp 60–62 ◦C. ESI-MS: m/z 565 [M + NH4]+. 1H NMR (CDCl3,
300 Hz, d): 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, d): 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.
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {2-methoxy-
4-[2-(4-nitrooxybutoxycarbonyl)-vinyl]}phenyl ester hydrochloride
(7b)
The title compound was obtained as a white solid (77%, for two
steps), mp 102–104 ◦C. ESI-MS: m/z 629 [M + H]+. IR (KBr): 760,
1
1038, 1632, 1741, 2872, 2958, 3064 cm-1. H NMR (CDCl3, 300
Hz, d): 0.85 (t, 3H, CH3, J = 6.3 Hz), 1.31 (m, 4H, 2¥CH2), 1. 88 (m,
6H, 3¥CH2), 3.45 (m, 4H, 2¥NCH2), 3.90 (s, 3H, OCH3), 4.01 (m,
4H, 2¥OCH2), 4.15 (m, 2H, COCH2N), 4.26 (t, 2H, CH2ONO2,
J = 6.0 Hz), 4.53 (t, 2H, COOCH2, J = 6.0 Hz), 6.42 (d, 1H,
CH , J = 15.9 Hz), 6.77 (m, 1H, CH), 7.17–7.28 (m, 3H, ArH),
( )-(E)-2-(1-Chloroacetoxypentyl)benzoic acid {4-[2-(5-nitrooxy-
pentoxycarbonyl)-vinyl]}phenyl ester (6f)
The title compound was obtained as a colourless waxy solid (81%),
mp 57–59 ◦C. ESI-MS: m/z 579 [M + NH4]+. 1H NMR (CDCl3,
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7.43–7.65 (m, 3H, ArH), 7.67 (d, 1H, CH , J = 15.9 Hz), 8.19 (d,
1H, ArH, J = 7.8 Hz), 13.50 (br s, 1H, NH+). 13C NMR (CDCl3,
300 Hz, d): 166.6, 164.5, 163.8, 151.5, 144.3, 142.4, 141.4, 133.5,
133.4, 131.1, 128.1, 127.2, 126.4, 123.4, 121.4, 118.2, 111.4, 75.7,
72.6, 63.8, 63.8, 63.6, 56.0, 55.1, 50.8, 50.8, 36.2, 27.8, 25.1, 23.7,
22.3, 13.9. HRMS for C32H41N2O11 ([M + H]+) calcd: 629.2710,
found: 629.2723.
Hz), 8.21 (d, 1H, ArH, J = 7.8 Hz), 12.73 (br s, 1H, NH+). 13C
NMR (CDCl3, 300 Hz, d): 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-(Diethylaminoacetoxy)pentyl]benzoic acid {2-metho-
xy-4-[2-(5-nitrooxypentoxycarbonyl)-vinyl]}phenyl ester
hydrochloride (7f)
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {2-methoxy-
4-[2-(5-nitrooxypentoxycarbonyl)-vinyl]}phenyl ester (7c)
The title compound◦was obtained as a white solid (70%, for two
The title compound◦was obtained as a white solid (80%, for two
steps), mp 106–107 C. ESI-MS: m/z 629 [M + H]+. IR (KBr):
758, 1253, 1633, 1733, 2872, 2953, 3073 cm-1. 1H NMR (CDCl3,
300 Hz, d): 0.86 (t, 3H, CH3, J = 6.7 Hz), 1.37 (m, 7H, 2¥CH2
and CH3), 1.48 (t, 3H, CH3, J = 7.2 Hz), 1.56 (m, 2H, CH2),
1.73–1.86 (m, 4H, 2¥CH2), 1. 93 (m, 2H, CH2), 3.22–3.43 (m, 4H,
2¥NCH2), 3.89 (s, 3H, OCH3), 4.00 (m, 2H, COCH2N), 4.24 (t,
2H, CH2ONO2, J = 6.3 Hz), 4.49 (t, 2H, COOCH2, J = 6.3 Hz),
6.42 (d, 1H, CH , J = 15.9 Hz), 6.77 (m, 1H, CH), 7.20 (m,
3H, ArH), 7.40–7.65 (m, 3H, ArH), 7.70 (d, 1H, J = 16.0 Hz),
8.20 (d, 1H, ArH, J = 7.5 Hz), 12.79 (br s, 1H, NH+). 13C NMR
(CDCl3, 300 Hz, d): 166.7, 164.5, 164.4, 151.5, 144.0, 142.6, 141.3,
133.6, 133.4, 131.2, 128.1, 127.2, 126.1, 123.3, 121.3, 118.4, 111.4,
75.3, 73.0, 64.1, 55.9, 48.6, 48.5, 48.2, 36.1, 28.2, 27.9, 26.5, 22.4,
22.3, 13.9, 10.1, 10.0. HRMS for C33H45N2O10 ([M + H]+) calcd:
629.3074, found: 629.3086.
steps), mp 102–103 C. ESI-MS: m/z 643 [M + H]+. IR (KBr):
1
759, 1032, 1636, 1742, 2951, 3068 cm-1. H NMR (CDCl3, 300
Hz, d): 0.85 (t, 3H, CH3, J = 6.6 Hz), 1.31 (m, 4H, 2¥CH2), 1.55
(m, 2H, CH2), 1.73–1.85 (m, 4H, 2¥CH2), 1.92 (m, 2H, CH2), 3.43
(m, 4H, 2¥NCH2), 3.90 (s, 3H, OCH3), 4.03 (m, 6H, 2¥OCH2
and COCH2N), 4.23 (t, 2H, CH2ONO2, J = 6.3 Hz), 4.53 (t, 2H,
COOCH2, J = 6.3 Hz), 6.43 (d, 1H, CH , J = 15.9 Hz), 6.76
(m, 1H, CH), 7.18–7.20 (m, 3H, ArH), 7.43–7.65 (m, 3H, ArH),
7.67 (d, 1H, CH , J = 15.9 Hz), 8.18 (d, 1H, ArH, J = 8.1 Hz),
13.40 (br s, 1H, NH+). 13C NMR (CDCl3, 300 Hz, d): 166.8, 164.5,
163.9, 151.5, 144.1, 142.4, 141.3, 133.6, 133.4, 131.1, 128.1, 127.2,
126.4, 123.3, 121.3, 118.4, 111.4, 75.7, 73.0, 64.1, 63.8, 63.8, 56.0,
55.1, 50.9, 50.9, 36.2, 28.2, 27.8, 26.5, 22.3, 22.3, 13.9. HRMS for
C33H43N2O11 ([M + H]+) calcd: 643.2867, found: 643.2881.
( )-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {2-metho-
xy-4-[2-(3-nitrooxypropoxycarbonyl)-vinyl]}phenyl ester
hydrochloride (7d)
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {4-[2-(3-
nitrooxypropoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7g)
The title compound◦was obtained as a white solid (78%, for two
steps), mp 109–110 C. ESI-MS: m/z 601 [M + H]+. IR (KBr):
759, 1252, 1631, 1731, 2953, 3078 cm-1. 1H NMR (CDCl3, 300 Hz,
d): 0.86 (t, 3H, CH3, J = 6.9 Hz), 1.36 (m, 7H, 2¥CH2 and CH3),
1.48 (t, 3H, CH3, J = 7.2 Hz), 1.92 (m, 2H, CH2), 2.17 (m, 2H,
CH2), 3.21–3.42 (m, 4H, 2¥NCH2), 3.89 (s, 3H, OCH3), 3.99 (m,
2H, COCH2N), 4.32 (t, 2H, CH2ONO2, J = 6.0 Hz), 4.62 (t, 2H,
COOCH2, J = 6.3 Hz), 6.42 (d, 1H, CH , J = 16.0 Hz), 6.77 (m,
1H, CH), 7.18 (m, 3H, ArH), 7.47–7.65 (m, 3H, ArH), 7.69 (d,
1H, CH , J = 15.9 Hz), 8.21 (d, 1H, ArH, J = 7.8 Hz), 12.78 (br
s, 1H, NH+). 13C NMR (CDCl3, 300 Hz, d): 166.4, 164.5, 164.4,
151.5, 144.6, 142.6, 141.4, 133.5, 133.4, 131.3, 128.1, 127.1, 126.1,
123.4, 121.4, 117.9, 111.4, 75.3, 70.0, 60.6, 56.0, 48.6, 48.5, 48.2,
36.1, 27.9, 26.5, 22.3, 13.9, 10.1, 10.0. HRMS for C31H41N2O10 ([M
+ H]+) calcd: 601.2761, found: 601.2773.
The title compound◦was obtained as a white solid (70%, for two
steps), mp 100-102 C. ESI-MS: m/z 585 [M + H]+. IR (KBr):
856, 1166, 1631, 1739, 2859, 2951, 3069 cm-1. 1H NMR (CDCl3,
300 Hz, d): 0.86 (t, 3H, CH3, J = 6.9 Hz), 1.33 (m, 4H, 2¥CH2),
1.91 (m, 2H, CH2), 2.17 (m, 2H, CH2), 3.44 (m, 4H, 2¥NCH2),
3.59–4.02(m, 4H, 2¥OCH2), 4.22 (m, 2H, COCH2N), 4.34 (t, 2H,
CH2ONO2, J = 6.0 Hz), 4.62 (t, 2H, COOCH2, J = 5.7 Hz), 6.43
(d, 1H, CH , J = 15.9 Hz), 6.78 (m, 1H, CH), 7.27–7.30 (m, 2H,
ArH), 7.47 (m, 1H, ArH), 7.60–7.69 (m, 4H, ArH), 7.73 (d, 1H,
CH =, J = 16.0 Hz), 8.18 (d, 1H, ArH, J = 8.1 Hz), 13.54 (br s, 1H,
NH+). 13C NMR (CDCl3, 300 Hz, d): 166.5, 164.8, 163.9, 152.2,
144.2, 142.8, 133.7, 132.3, 131.0, 129.4, 129.4, 128.2, 127.0, 126.5,
122.4, 122.4, 117.9, 75.5, 70.0, 63.8, 63.8, 60.5, 55.0, 50.7, 50.7,
36.1, 27.8, 26.5, 22.3, 13.9. HRMS for C30H37N2O10 ([M + H]+)
calcd: 585.2448, found: 585.2460.
( )-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {2-metho-
xy-4-[2-(4-nitrooxybutoxycarbonyl)-vinyl]}phenyl ester
hydrochloride (7e)
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {4-[2-(4-
nitrooxybutoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7h)
The title compound was obtained as a white solid (65%, for two
The title compound was obtained as a white solid (84%, for two
steps), mp 106–108 ◦C. ESI-MS: m/z 615 [M + H]+. IR (KBr): 759,
1254, 1630, 1747, 2861, 2956, 3444 cm-1. 1H NMR (CDCl3, 300 Hz,
d): 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
steps), mp 101–103 ◦C. ESI-MS: m/z 599 [M + H]+. IR (KBr): 856,
1
1166, 1631, 1739, 2859, 2951, 3068 cm-1. H NMR (CDCl3, 300
Hz, d): 0.86 (t, 3H, CH3, J = 6.3 Hz), 1.34 (m, 4H, 2¥CH2), 1.90
(m, 6H, 3¥CH2), 3.42 (m, 4H, 2¥NCH2), 4.01(m, 2H, COCH2N),
4.07 (m, 4H, 2¥OCH2), 4.26 (t, 2H, CH2ONO2, J = 6.1 Hz), 4.53
(t, 2H, COOCH2, J = 6.0 Hz), 6.43 (d, 1H, CH , J = 16.0 Hz),
6.77 (m, 1H, CH), 7.27–7.30 (m, 2H, ArH), 7.47 (m, 1H, ArH),
7.62–7.68 (m, 4H, ArH), 7.73 (d, 1H, CH , J = 16.0 Hz), 8.18 (d,
1H, ArH, J = 8.1 Hz), 13.50 (br s, 1H, NH+). 13C NMR (CDCl3,
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300 Hz, d): 166.7, 164.8, 164.1, 152.1, 143.8, 142.8, 133.7, 132.4,
131.0, 129.4, 129.4, 128.1, 127.0, 126.6, 122.4, 122.4, 118.2, 75.4,
72.6, 63.9, 63.9, 63.6, 55.3, 51.0, 51.0, 36.1, 27.8, 25.1, 23.7, 22.3,
13.9. HRMS for C31H39N2O10 ([M + H]+) calcd: 599.2605, found:
599.2616.
122.3, 122.3, 118.2, 75.2, 72.6, 63.6, 48.6, 48.5, 48.3, 36.0, 27.9,
25.1, 23.7, 22.3, 13.9, 10.1, 10.0. HRMS for C31H41N2O9 ([M +
H]+) calcd: 585.2812, found: 585.2823.
( )-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {4-[2-(5-
nitrooxypentoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7l)
( )-(E)-2-[1-(Morpholinoacetoxy)pentyl]benzoic acid {4-[2-(5-
nitrooxypentoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7i)
The title compound◦was obtained as a white solid (81%, for two
steps), mp 104–106 C. ESI-MS: m/z 599 [M + H]+. IR (KBr):
885, 1158, 1635, 1738, 2857, 2961, 3070 cm-1. 1H NMR (CDCl3,
300 Hz, d): 0.87 (t, 3H, CH3, J = 6.6 Hz), 1.36 (m, 7H, 2¥CH2 and
CH3), 1.48 (t, 3H, CH3, J = 6.9 Hz), 1.57 (m, 2H, CH2), 1.73–1.86
(m, 4H, 2¥CH2), 1.92 (m, 2H, CH2), 3.30–3.49 (m, 4H, 2¥NCH2),
4.00 (m, 2H, COCH2N), 4.24 (t, 2H, CH2ONO2, J = 6.4 Hz), 4.49
(t, 2H, COOCH2, J = 6.3 Hz), 6.43 (d, 1H, CH , J = 15.9 Hz),
6.78 (m, 1H, CH), 7.19–7.29 (m, 2H, ArH), 7.48 (m, 1H, ArH),
7.57–7.67 (m, 4H, ArH), 7.71 (d, 1H, CH , J = 15.9 Hz), 8.20 (d,
1H, ArH, J = 7.8 Hz), 12.75 (br s, 1H, NH+). 13C NMR (CDCl3,
300 Hz, d): 166.8, 164.7, 164.5, 152.0, 143.6, 142.9, 133.7, 132.5,
131.1, 129.4, 129.4, 128.2, 127.0, 126.3, 122.3, 122.3, 118.4, 75.2,
73.0, 64.1, 48.6, 48.5, 48.3, 36.0, 28.2, 27.9, 26.5, 22.4, 22.3, 13.9,
10.1, 10.0. HRMS for C32H43N2O9 ([M + H]+) calcd: 599.2969,
found: 599.2980.
The title compound◦was obtained as a white solid (75%, for two
steps), mp 100–101 C. ESI-MS: m/z 613 [M + H]+. IR (KBr):
857, 1162, 1638, 1739, 28569, 2954, 3071 cm-1. 1H NMR (CDCl3,
300 Hz, d): 0.86 (t, 3H, CH3, J = 6.8 Hz), 1.33 (m, 4H, 2¥CH2),
1.50–1.60 (m, 2H, CH2), 1.73–1.83 (m, 4H, 2¥CH2), 1.91 (m, 2H,
CH2), 3.44 (m, 4H, 2¥NCH2), 4.05(m, 4H, 2¥OCH2), 4.11 (m,
2H, COCH2N), 4.23 (t, 2H, CH2ONO2, J = 6.3 Hz), 4.49 (t, 2H,
COOCH2, J = 6.5 Hz), 6.43 (d, 1H, CH , J = 16.0 Hz), 6.78 (m,
1H, CH), 7.26–7.29 (m, 2H, ArH), 7.47 (m, 1H, ArH), 7.61–7.67
(m, 4H, ArH), 7.70 (d, 1H, CH , J = 16.0 Hz), 8.17 (d, 1H, ArH,
J = 8.1 Hz), 13.50 (br s, 1H, NH+). 13C NMR (CDCl3, 300 Hz,
d): 166.8, 164.8, 163.9, 152.1, 143.6, 142.8, 133.7, 132.4, 131.0,
129.4, 129.4, 128.1, 127.0, 126.5, 122.3, 122.3, 118.4, 75.5, 73.0,
64.1, 63.8, 63.8, 55.1, 50.9, 50.9, 36.1, 28.2, 28.0, 27.8, 22.3, 22.3,
13.9. HRMS for C32H41N2O10 ([M + H]+) calcd: 613.2761, found:
613.2772.
( )-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid (8)
To a solution of acylated compound 3 (0.71 g, 2.5 mmol) and
Et3N (1 mL) in CH2Cl2 (15 mL) was added diethylamine (0.55,
7.5 mmol) and the solution was left stirring at room temperature
for 12 h. The solvent was removed under reduced pressure and
purified by flash chromatography (MeOH–EtOAc 1 : 10 v/v) to
give the 8 as a colourless waxy solid (0.36 g, 45%). Mp 69–70 ◦C.
ESI-MS: m/z 322 [M + H]+. 1H NMR (CDCl3, 500 Hz, d): 0.83 (t,
3H, CH3, J = 7.5 Hz), 1.14–1.77 (m, 6H, 2¥CH3, J = 7.0 Hz), 1.29–
1.37 (m, 4H, 2¥CH2), 1.87–1.88 (m, 2H, CH2), 2.91–2.96 (q, 4H,
2¥CH2, J = 7.0 Hz), 3.62–3.76 (m, 2H, COCH2N), 6.69 (m, 1H,
CH), 7.21–7.26 (m, 1H, ArH), 7.34–7.43 (m, 2H, ArH), 7.80 (d,
1H, ArH, J = 7.5 Hz), 10.67 (br s, 1H, COOH). 13C NMR (CDCl3,
500 Hz, d): 172.2, 168.1, 140.8, 133.1, 130.3, 130.0, 127.1, 125.7,
74.7, 51.8, 47.0, 47.0, 36.3, 27.8, 22.3, 13.8, 10.5, 10.5. HRMS for
C18H28NO4 ([M + H]+) calcd: 322.2018, found: 322.2022.
( )-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {4-[2-(3-
nitrooxypropoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7j)
The title compound◦was obtained as a white solid (83%, for two
steps), mp 107–110 C. ESI-MS: m/z 571 [M + H]+. IR (KBr):
1
885, 1171, 1635, 1739, 2858, 2955, 3069 cm-1. H NMR (CDCl3,
300 Hz, d): 0.87 (t, 3H, CH3, J = 6.9 Hz), 1.36 (m, 7H, 2¥CH2 and
CH3), 1.48 (t, 3H, CH3, J = 7.2 Hz), 1.92 (m, 2H, CH2), 2.17 (m,
2H, CH2), 3.13–3.49 (m, 4H, 2¥NCH2), 4.00 (m, 2H, COCH2N),
4.34 (t, 2H, CH2ONO2, J = 6.0 Hz), 4.62 (t, 2H, COOCH2, J =
6.3 Hz), 6.43 (d, 1H, CH , J = 16.0 Hz), 6.78 (m, 1H, CH), 7.27–
7.30 (m, 2H, ArH), 7.48 (m, 1H, ArH), 7.57–7.69 (m, 4H, ArH),
7.73 (d, 1H, CH , J = 15.9 Hz), 8.20 (d, 1H, ArH, J = 7.5 Hz),
12.75 (br s, 1H, NH+). 13C NMR (CDCl3, 300 Hz, d): 166.5, 164.7,
164.5, 152.1, 144.1, 142.9, 133.7, 132.3, 131.1, 129.4, 129.4, 128.2,
127.0, 126.3, 122.3, 122.3, 117.9, 75.2, 70.0, 60.6, 48.6, 48.5, 48.3,
36.0, 27.9, 26.5, 22.3, 13.9, 10.1, 10.0. HRMS for C30H39N2O9 ([M
+ H]+) calcd: 571.2656, found: 571.2668.
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 cen-
trifuging 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
aggregation was measured by Born’s turbidimetric method using
a Platelet-Aggregometer (LG-PABER-I Platelet-Aggregometer,
Beijing). Briefly, PRP (240 ml) was pre-treated in duplicate with
vehicle, different concentrations of individual compounds or the
reference drugs for 5 min and exposed to 10 mM◦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 monitored
longitudinally by optical density. Platelet aggregation was induced
by ADP (final concentration 10 mM), TH (final concentration
( )-(E)-2-[1-(Diethylaminoacetoxy)pentyl]benzoic acid {4-[2-(4-
nitrooxybutoxycarbonyl)-vinyl]}phenyl ester hydrochloride (7k)
The title compound◦was obtained as a white solid (85%, for two
steps), mp 106–108 C. ESI-MS: m/z 585 [M + H]+. IR (KBr):
1
885, 1165, 1630, 1738, 2858, 2957, 3064 cm-1. H NMR (CDCl3,
300 Hz, d): 0.87 (t, 3H, CH3, J = 6.9 Hz), 1.36 (m, 7H, 2¥CH2 and
CH3), 1.48 (t, 3H, CH3, J = 7.2 Hz), 1.86–1.93 (m, 6H, 3¥CH2),
3.22–3.50 (m, 4H, 2¥NCH2), 4.01 (m, 2H, COCH2N), 4.27 (t, 2H,
CH2ONO2, J = 5.7 Hz), 4.53 (t, 2H, COOCH2, J = 5.7 Hz), 6.43
(d, 1H, CH , J = 15.9 Hz), 6.78 (m, 1H, CH), 7.27–7.30 (m, 2H,
ArH), 7.49 (m, 1H, ArH), 7.58–7.68 (m, 4H, ArH), 7.72 (d, 1H,
CH , J = 15.9 Hz), 8.20 (d, 1H, ArH, J = 7.5 Hz), 12.70 (br s, 1H,
NH+). 13C NMR (CDCl3, 300 Hz, d): 166.6, 164.7, 164.5, 152.1,
143.8, 142.9, 133.7, 132.4, 131.1, 129.4, 129.4, 128.2, 127.0, 126.3,
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0.5 U mL-1) or AA (final concentration 1.0 mM). 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 individual compounds was evaluated as
percent inhibition of platelet aggregation compared to positive
controls that had been pre-treated with vehicle alone and exposed
with the inducer samples. The IC50 values of most compounds
were determined by nonlinear regression analysis and the percent
inhibition at the maximal concentration tested (1.0 mM) was
calculated.
was determined in a UV755B spectrophotometer, eventually dilut-
ing the solution (with MeOH) 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. The saturated solution was filtered with a Millipore
0.45-mm filter to remove solid compound 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-1), A = absorbance
of standard solution, A¢ = absorbance of saturated solution, and
C¢ = concentration of saturated solution (mg mL-1).
Antithrombotic activity assay in rats
The antithrombotic activities of individual compounds were tested
in male SD rats, as previously described.24 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 5 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
collected and immediately weighed. The wet weight of a thrombus
was calculated by subtracting the weight of the 5 cm dry cotton
from the total weight and the antiplatelet aggregation activities of
individual compounds were expressed as percent inhibition related
to untreated controls.
Acknowledgements
We thank Prof. Jide Tian, University of California Los Angeles,
Los Angeles, USA, for his help with the manuscript. This study
was supported by a grant from the Major National Science and
Technology Program of China for Innovative Drug during the
Eleventh Five-Year Plan Period (No. 2009ZX09103-095).
Notes and references
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