Antiinflammatory, gastrosparing, and antiplatelet properties of new NO-donor esters of aspirin

Article abstract of DOI:10.1021/jm020969t

A new series of NSAIDs in which aspirin is joined by an ester linkage to furoxan moieties, with different ability to release NO, were synthesized and tested for NO-releasing, antiinflammatory, antiaggregatory, and ulcerogenic properties. Related furazan d

Full text of DOI:10.1021/jm020969t

J . Med. Chem. 2003, 46, 747-754
747
An tiin fla m m a tor y, Ga str osp a r in g, a n d An tip la telet P r op er ties of New NO-Don or
Ester s of Asp ir in
Clara Cena,† Marco L. Lolli,† Loretta Lazzarato,† Elena Guaita,‡ Giuseppina Morini,‡ Gabriella Coruzzi,‡
Stuart P. McElroy,§ Ian L. Megson,§ Roberta Fruttero,† and Alberto Gasco*,†
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy,
Dipartimento di Anatomia Umana, Farmacologia e Scienze Medico-Forensi, Universita` degli Studi di Parma,
via Volturno 39, 43100 Parma, Italy, and Centre for Cardiovascular Science, Biomedical Sciences, University of Edinburgh,
Edinburgh EH8 9XD (UK)
Received J une 18, 2002
A new series of NSAIDs in which aspirin is joined by an ester linkage to furoxan moieties,
with different ability to release NO, were synthesized and tested for NO-releasing, antiin-
flammatory, antiaggregatory, and ulcerogenic properties. Related furazan derivatives, aspirin,
its propyl ester, and its γ-nitrooxypropyl ester were taken as references. All the products
described present an antiinflammatory trend, maximized in derivatives 12, 16, and 17, they
are devoid of acute gastrotoxicity, principally due to their ester nature, and show an antiplatelet
activity primarily determined by their ability to release NO. They do not behave as aspirin
prodrugs in human serum.
Ch a r t 1
In tr od u ction
Nonsteroidal antiinflammatory drugs (NSAIDs) are
widely used to treat the effects of inflammation through
inhibition of cyclooxygenase enzymes (COX).1 The major
limitation to long term therapeutic use of these products
is their gastrotoxicity; in 1997, 16500 patients with
rheumatoid arthritis or osteoarthritis died from the
gastrointestinal toxic effects of NSAIDs in the US.2 Two
approaches have emerged over recent years to improve
the safety profile of these drugs. First, selective inhibi-
tors of the inducible isoform of cyclooxygenase, COX-2,
which plays a major role in prostaglandin biosynthesis
in inflammatory cells, have been described.3 Celecoxib
1 and Rofecoxib 2 (see compounds in Chart 1) are typical
examples that were recently marketed. Second, (NO)-
releasing NSAIDs are being investigated.4 (In this paper
we use NO as a family name, embracing all the NO-
redox species. When necessary we specify which redox
form we refer to.) The fundamental rational of this
approach is that NO is able to protect the gastric mucosa
by a number of mechanisms, including promotion of
mucous secretion, increased mucosal blood flow, and
decreased adherence of neutrophils to the gastric vas-
cular endothelium.5 NO-NSAIDs are still in develop-
ment and have yet to reach the market. The prototype
of NSAIDs is aspirin 3. This drug is commonly used to
alleviate the symptoms of inflammation and for the
long-term prophylaxis against myocardial infarction and
stroke, on account of its antithrombotic properties.1
Aspirin displays gastrotoxicity as a consequence of
topical injury, due to its acidic nature, and of systemic
effects largely related to its ability to inhibit both COX
isoforms (COX-1, COX-2).6 Among NO-releasing NSAIDs,
NO-aspirins have received particular attention7,8 and
NCX-4016 4 has been shown to have a number of
potential benefits over the parent compound.9 The
behavior of this class of drugs is complex and not fully
understood.
As a development of our previous work on NO-
releasing drugs,10 we have now designed new NSAIDs
(Scheme 1, derivatives 9, 12, 14, 16, 17) in which aspirin
is joined by an ester linkage to furoxan moieties,
selected on the basis of their different NO-releasing
properties. In this paper, we report the synthesis and
antiinflammatory and antiaggregatory properties of
these products, as well as their gastric ulcerogenic
effects. Related furazan derivatives (Scheme 1, deriva-
tives 9a , 12a , 14a , 16a , 17a ) were used as NO-deficient
references. Aspirin 3, its propyl ester 5, as well as its
γ-nitrooxypropyl ester 6 (Scheme 1), were also consid-
ered as references.
Ch em istr y
All the products considered for biological investigation
were prepared by standard methods as outlined in
Scheme 1. Furoxanyloxypropyl esters of aspirin 9, 12
and furazanyloxypropyl analogues 9a , 12a were syn-
thesized by treatment of the appropriate alcohol (7, 7a ,
* To whom correspondence should be addressed. Telephone 0039
011 6707670, fax 0039 011 6707972, e-mail alberto.gasco@unito.it.
† Universita` degli Studi di Torino.
‡ Universita` degli Studi di Parma.
§ University of Edinburgh.
10.1021/jm020969t CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/01/2003

748 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Cena et al.
Sch em e 1
11, 11a ) with acetylsalicylic acid chloride (8) in toluene
solution, in the presence of pyridine. Furoxanylmethyl
esters 14, 16 were prepared by reaction of 3 with the
corresponding (bromomethyl)furoxans 13, 15 in aceto-
nitrile as solvent and in the presence of triethylamine
as base. The related furazans 14a , 16a were obtained
from 14, 16, respectively, by reduction with trimethyl
phosphite. Cyano derivatives 17, 17a were obtained by
trifluoroacetic anhydride dehydration of parent amides
(16, 16a ) in THF solution, in the presence of pyridine.
Finally, nitrooxy derivative 6 was synthesized by action
of AgNO3 on 3-bromopropyl ester of aspirin (18) using
acetonitrile as solvent.
NO-Relea se a n d Hyd r olysis. NO (family name)
release was evaluated by detection of nitrite (Griess
reaction).11 The use of a cosolvent was necessary because
of the low water solubility of the compounds. Therefore,
NO-donor compounds were dissolved in pH 7.4 buffered
water/methanol mixture and incubated at 37 °C for 1
h, in the presence of 1:5 molar excess of L-cysteine.
These same products were unable to generate nitrite
Ta ble 1. NO Release Data
% NO2- (mol/mol)a,b
compound
L-Cys 5 × 10-4
M
6
9
12
14
16
17
0.18 ( 0.05
37.1 ( 0.3
0.35 ( 0.08
0
3.8 ( 0.1
32.3 ( 0.8
a
b
All values are mean ( SEM. Determined by Griess reaction,
after incubation for 1 h at 37 °C in pH 7.4 buffered water/methanol
mixture, in the presence of 1:5 molar excess of L-cysteine.
under the same conditions but at pH ) 1. The results,
expressed as % mol/mol of NO2-, are presented in Table
1. Nitrite detection is only a crude measure of NO
release. NO2- production is, in fact, strongly dependent
on the medium, the concentration, and the nature of
the thiol employed, and it is accompanied by other
reactions between furoxan system and thiol cofactor.
Nitrite detection does not give information about the
single NO-redox forms involved in the release and on

New NO-Donor Esters of Aspirin
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 749
F igu r e 1. Antiinflammatory effects of tested compounds on carrageenan-induced paw edema in rat. The compounds were
administered by intragastric route at a dose equimolar with aspirin 3, 120 mg/kg, at the same time as carrageenan, and their
effects were evaluated 3 h later. Results are expressed as % of inhibition of paw volume increase. Edema volume in vehicle-
treated group was taken as 100. *P < 0.05; **P < 0.01 vs vehicle (ANOVA and Newman-Keuls test). Values are mean ( SEM
(n ) 6-8 per group).
Ta ble 2. Amount of Unaltered Compound Detected in Human
increase in paw volume within 1 h. Aspirin 3, admin-
Serum after 1 min and 1 h Hydrolysis
istrated by intragastric route at a dose of 120 mg/kg at
the same time as carrageenan, significantly reduced
% unaltered compounda,b % unaltered compounda,b
compound
1 min
1 h
(55.17% ( 13.88) paw edema at 3 h. Analysis of Figure
1 shows a trend toward antiinflammatory action for all
of the compounds tested, at a dose equimolar with 120
mg/kg of aspirin. In particular, furoxan derivatives 12,
16, 17 displayed significant antiinflammatory proper-
ties, comparable to that of aspirin. This activity cannot
reside in their behavior as prodrugs of 3. Indeed, all the
products were completely or largely transformed within
1 min, and negligible amounts of derivatives 9, 9a , 12,
and 12a were observed after 1 h when incubated in
human serum (Table 2), but formation of aspirin among
metabolites was never observed. Only salicylic acid and
salicylate were qualitatively detected in different ratios
for each compound. This behavior is in keeping with the
knowledge that the elimination of the negative charge
of the aspirin molecule (pKa ) 3.5) by esterification
renders the acetyl group extremely susceptible to en-
zymatic cleavage.14
aspirin (3)c
5
6
9
9a
12
12a
14
14a
16
16a
17
17a
95.0 ( 3.9
43.1 ( 1.9
0
0
0
0
43.7 ( 3.0
9.3 ( 2.2
47.6 ( 5.6
8.1 ( 1.4
38.8 ( 5.8
3.8 ( 2.1
39.3 ( 5.9
3.9 ( 2.0
2.5 ( 1.2
0
0
0
0
0
0
0
0
0
0
0
a
b
All values are mean ( SEM. Detected by HPLC after
incubation in serum at 37 °C. c Half-time values evaluated for
aspirin in these hydrolysis conditions were in keeping with data
reported in ref 25.
-
the eventual formation of NO2 directly from furoxan
system. Moreover, under the adopted conditions, the
different NO-redox forms eventually involved in the
process can undergo a variety of reactions in addition
to nitrite production.12 Since the NO produced by
endothelial cells is generally assumed to be nitric oxide
(NO•), we also determined the ability of the products to
afford this redox form, when dissolved in plasma. These
measurements were performed in fresh human plasma
for compounds 9 and 17 by an electrochemical method,
using a Clark type electrode.13 NO signals obtained with
derivatives 9 and 17 were integrated and the area under
the curve reported in Figure 3.
HPLC detection was used to determine the amount
of unaltered compound remaining after incubation in
human serum for 1 min and 1 h (Table 2). Over this
period, no production of aspirin was observed. All the
products were largely unaltered after 1 h incubation in
mixtures of acetonitrile and HCl.
Acu te Ga str ic Mu cosa l Da m a ge. All the products
were assessed for their ulcerogenic properties in con-
scious rats. The development of gastric lesions was
assessed 3 h after the intragastric administration of the
compounds and lesions were quantified by determina-
tion of the “lesion index”, on the basis of their greatest
length in millimeters. Aspirin 3, administrated at 120
mg/kg, produced macroscopically detectable gastric
damage, characterized by mucosal necrosis and haem-
orrhage (lesion index ) 22.0 ( 1.0; Figure 2). Acute
gastric toxicity of aspirin is reported to be largely
dependent on the presence of the carboxylic group.15 As
a consequence, ester derivatives of aspirin could be
presumed to produce markedly less gastric damage than
the parent compound. In agreement with our expecta-
tions, the present results provide evidence that all the
ester derivatives, when administered at doses equimolar
with aspirin, were almost devoid of ulcerogenic proper-
ties (Figure 2). On account of the fact that compounds
devoid of NO exhibited impressive reduced acute toxic-
ity, the ability to release NO appears to be of little
relevance to this effect. NSAIDs are largely reported to
damage gastric and intestinal mucosa by both local and
systemic action, each of these mechanisms having a
different time course for the induction of the injury.16
While tested compounds appear to be almost devoid of
acute ulcerogenic toxicity, their effects following chronic
administration remain to be determined.
Since NO-releasing aspirins do not produce nitrite at
low pH and they are scarcely hydrolyzed in acidic
conditions, it is unlikely that NO release occurs in the
gastric lumen. It is more likely to happen following
systemic absorption.
P h a r m a cologica l Resu lts a n d Discu ssion
An tiin fla m m a tor y Activity. All the compounds
were tested for their antiinflammatory activity in the
carrageenan rat paw edema assay. The injection of
carrageenan into the hind paw produced a marked

750 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Cena et al.
F igu r e 2. Gastric ulcerogenic effects of tested compounds in rat. The compounds were administered by intragastric route at a
dose equimolar with aspirin 3, 120 mg/kg, and the stomachs were examined 3 h later. Gastric lesions were quantified by
determination of the lesion index, on the basis of their greatest length in millimeters. They all produced significantly less gastric
damage than aspirin 3 (P < 0.01; ANOVA and Newman-Keuls test). Values are mean ( SEM (n ) 6-8 per group).
Ta ble 3. Antiaggregatory Activity of the Tested Compounds on ADPa -Induced PRP Aggregation
+ HbO2 40 µM
+ODQ 100 µM
% inhibition
% inhibition
% inhibition
compound
pIC50 (µM) ( SEM
( SEM (50 µM)b
pIC50 (µM) ( SEM
5.17 ( 0.03
( SEM (50 µM)b
pIC50 (µM) ( SEM
4.86 ( 0.10
( SEM (50 µM)b
5
c
18.1 ( 9.0
18.3 ( 9.2
d
6
c
9
5.95 ( 0.10
d
d
9a
12
12a
14
14a
16
16a
17
17a
c
3.3 ( 4.2
18.9 ( 2.9
16.9 ( 5.6
17.8 ( 9.5
23.2 ( 12.5
d
7.6 ( 3.7
d
14.8 ( 7.0
c
c
c
c
4.52 ( 0.13
c
7.21 ( 0.09
c
c
28.6 ( 7.3
c
22.6 ( 10.9
6.46 ( 0.07
d
5.37 ( 0.10
d
d
a
b
Aspirin 3 was not taken as a reference compound since it displays negligible effects on ADP-induced aggregation. Maximal
concentration tested. c Inhibition did not reach 50% of control effect: pIC50s could not be calculated. For these compounds a complete
d
concentration-response curve could be performed, therefore pIC50s are reported in the previous column.
Ta ble 4. Antiaggregatory Activity of the Tested Compounds on Collagen-Induced PRP Aggregation
+ HbO2 40 µM
+ODQ 100 µM
% inhibition
% inhibition
pIC50 (µM)
( SEM
% inhibition
compound
pIC50 (µM) ( SEM
( SEM (50 µM)a
pIC50 (µM) ( SEM
( SEM (50 µM)a
( SEM (50 µM)a
aspirin (3)
5
6
9
9a
12
12a
14
14a
16
16a
17
17a
4.68 ( 0.11
c
b
14.4 ( 7.0
12.3 ( 5.5
c
14.8 ( 10.1
1.9 ( 2.5
0.7 ( 6.9
18.6 ( 17.5
1.5 ( 1.7
c
b
5.54 ( 0.20
b
b
b
b
b
4.20 ( 0.02
b
7.22 ( 0.10
b
4.84 ( 0.14
c
5.08 ( 0.14
c
b
14.4 ( 6.0
b
18.5 ( 7.5
16.7 ( 10.1
c
27.9 ( 17.8
5.97 ( 0.05
c
5.21 ( 0.17
c
a
b
Maximal concentration tested. Inhibition did not reach 50% of control effect: pIC50s could not be calculated. c For these compounds
a complete concentration-response curve could be performed, therefore pIC50s are reported in the previous column.
P la telet Aggr ega tion . Antiaggregatory effects of the
compounds were studied either on ADP or collagen-
induced platelet aggregation of human platelet rich
plasma (PRP). ADP induces aggregation by a pathway
independent of the arachidonic acid cascade, while
collagen is arachidonic acid-dependent. In the case of
NO-donor furoxan derivatives, the experiments were
repeated in the presence of either the NO-scavenger
oxyhemoglobin (HbO2), or the selective inhibitor of
soluble guanylate cyclase (sGC) ODQ. The results are
shown in Tables 3 and 4. Where possible, results are
expressed as pIC50 ( SEM, as determined by nonlinear
regression analysis. In experiments where pIC50 estima-
tion was precluded because inhibition of maximal
response did not reach 50%, we report % inhibition at
maximal concentration tested (50 µM). Inspection of
Tables 3 and 4 indicates that the antiaggregatory
potency of furoxan derivatives is strongly dependent on
their ability to produce NO. In this respect, the studied
NO-donors can be classified in three groups: good NO-
donors (derivatives 9, 17), intermediate NO-donors
(derivative 16) and poor NO-donors (derivatives 6, 12,
14). In effect, the most potent antiaggregatory com-
pounds of the series are the benzenesulfonyl and the
cyano substituted furoxans 9 and 17 respectively. These
compounds generated correspondingly high levels of
nitrite, while the cyano derivative 17 released the most
NO in plasma (Figure 3) and proved to be the most

New NO-Donor Esters of Aspirin
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 751
presence of ODQ when collagen was used as an agonist.
This activity was very similar to that detected in ADP-
induced aggregation. This residual activity could again
be due to heme independent activation of sGC and/or
to inhibition of aggregation consequent to deactivation
of collagen receptors, e.g. by nitration of tyrosine
residues of the protein.18 The parent furazan was
practically inactive at the maximal concentration tested
(50 µM), irrespective of the agonist used. The behavior
of 9 paralleled that of 17 but, in keeping with its minor
ability to release NO in plasma, was less active. Furox-
ans 12 and 14, which are very poor NO-donors, display
very poor antiaggregatory properties, similar to the
analogue furazans 12a , 14a . This is also the behavior
of the nitrooxy derivative 6 and of the related nitrooxy-
deficient compound 5. The furoxan amide 16, which has
intermediate capacity to produce NO, has an intermedi-
ate potency as an antiaggregatory agent. As expected,
the related furazan was inactive.
F igu r e 3. NO generation from derivatives 9 and 17 in fresh
human plasma at 37 °C, as assessed by electrochemical
determination.13 *P < 0.05 (unpaired t-test, n ) 5).
Con clu sion s
Several key points emerge from the results of this
study: first, the products described in the present work
present an antiinflammatory trend, maximized in de-
rivatives 12, 16, and 17; second, the lack of acute
gastrotoxicity is principally due to the ester nature of
the compounds; third, their antiplatelet action is pri-
marily determined by their ability to release NO; finally,
they do not behave as aspirin prodrugs in human serum.
Exp er im en ta l Section
Ch em istr y. Melting points were measured with a capillary
apparatus (Bu¨chi 530) and are uncorrected. All the compounds
1
were routinely checked by IR (Shimadzu FT-IR 8101 M), H
and 13C NMR (Bruker AC-200; the following abbreviations
were used to indicate the peak multiplicity: s ) singlet; d )
doublet; t ) triplet; qt ) quartet; qi ) quintet; si ) sixtet; n )
nine lines; m ) multiplet), and mass spectrometry (Finnigan-
Mat TSQ-700). Flash column chromatography was performed
on silica gel (Merck Kiesekgel 60, 230-400 mesh ASTM) using
the indicated eluents. Anhydrous magnesium sulfate was used
as the drying agent of the organic extracts. Elemental analysis
of the new compounds was performed by REDOX (Monza), and
the results are within (0.4% of the theoretical values. HPLC
analyses were performed by using a diode array UV detector
(Shimadzu LC10A); the figures in brackets are standard errors
((SEM). Structures 7,10 7a ,10 8,19 10,20 10a ,20 13,21 1521 were
synthesized according to literature methods. Inert atmosphere
indicates that the reaction was performed in flame- or oven-
dried glassware under a positive pressure of dry nitrogen or
argon.
F igu r e 4. Antiaggregatory activity of derivative 17 compared
to aspirin 3 in (a) ADP (8 µM) and (b) collagen (2.5 mg/mL)-
induced platelet aggregation in human platelet rich plasma
(PRP). The role of NO and soluble guanylate cyclase in the
inhibitory effect of 17 was investigated using the NO scaven-
ger, oxyhaemoglobin (10 µM), and the soluble guanylate
cyclase inhibitor, ODQ (100 µM), respectively.
3-(3-P h en ylfu r oxa n -4-yloxy)p r op a n ol (11). NaOH (50%
w/w; 1.16 g; 14.48 mmol) was added over a period of 40 min to
a stirred solution of 4-nitro-3-phenylfuroxan 10 (3.00 g; 14.48
mmol) and 1,3-propandiol (11.4 mL; 14.50 mmol) in THF (50
mL). The resulting mixture was stirred at room temperature
for 12 h and then an excess of 50% w/w NaOH (400 mg; 5.00
mmol) was added to complete the reaction. The mixture was
concentrated under reduced pressure and then poured into
ice-water (40 mL). The resulting mixture was extracted with
CH2Cl2 (20 + 10 + 10 mL), the organic layers were dried and
concentrated under reduced pressure. The crude product was
purified by flash chromatography (eluent: petroleum ether
(40-60)/EtOAc from 8/2 v/v to 6/4 v/v). The pure title com-
pound was obtained as a white crystalline solid. Yield 27%.
powerful antiaggregatory agent. Its potency with respect
to collagen-induced aggregation was about 100-fold
greater than that of aspirin (Figure 4). In addition,
derivative 17 showed potent COX-1-independent inhibi-
tion of ADP-induced aggregation. In both tests, the
inhibition was concentration-dependent and the con-
centration-effect curve is right shifted in the presence
of either HbO2 or ODQ. When ADP was used as an
agonist, the product still displayed poor antiaggregatory
properties in the presence of ODQ, suggesting that it
might inhibit aggregation by a cGMP independent
mechanism, e.g., nitrosation of cysteine residues of ADP
receptors17 and/or activation of sGC by a heme-inde-
pendent mechanism, e.g., through the sulfydryl sites of
enzyme.18 The product also showed poor activity in the
1
Mp 57 °C from diisopropyl ether. H NMR (CDCl3) δ 1.74 (s,
1H, CH2OH); 2.18 (qi, 2H, CH2CH2OH); 3.89 (m, 2H, CH2CH2-
OH); 4.67 (t, 2H, furoxan-OCH2CH2); 7.48-8.14 (m, 5H,
aromatic protons). 13C NMR (CDCl3) δ 31.4 (CH2CH2OH); 58.8

752 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Cena et al.
(CH2CH2OH); 67.7 (furoxan-OCH2CH2); 107.5 (furoxan C(3));
122.3 (C6H5 C(1)); 126.0/128.7 (C6H5 C(2)/C(3)); 130.4 (C6H5
C(4)); 162.2 (furoxan C(4)). MS (EI) m/z 236 (M)+. Anal. (drying
conditions: 40 °C, 13 h, pressure < 1 mmHg) for C11H12N2O4
C, H, N.
3-(4-P h en ylfu r a za n -3-yloxy)p r op a n ol (11a ). The title
compound was obtained from 3-nitro-4-phenylfurazan 10a in
the same manner as analogue 11. Pale yellow oil. Yield 77%.
1H NMR (CDCl3) δ 1.67 (broad, 1H, CH2OH); 2.17 (qi, 2H, CH2-
CH2OH); 3.88 (t, 2H, CH2CH2OH); 4.63 (t, 2H, furazan-OCH2-
CH2-); 7.45-7.99 (m, 5H, aromatic protons). 13C NMR (CDCl3)
δ 31.6 (CH2CH2OH); 58.8 (CH2CH2OH); 69.7 (furazan-OCH2-
CH2); 125.0 (C6H5 C(1)); 127.2/128.8 (C6H5 C(2)/C(3)); 130.5
(C6H5 C(4)); 145.0 (furazan C(3)); 163.5 (furazan C(4)). MS (EI)
m/z 220 (M)+. Anal. (drying conditions: 42 °C, 13 h, pressure
< 1 mmHg) for C11H12N2O3 C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Ester s
9, 9a , 12, a n d 12a . A solution of 8 (2.20 g; 11 mmol) in dry
toluene (25 mL) and a solution of dry pyridine (900 µL; 11
mmol) in dry toluene (25 mL) were added, over a period of 40
min, to a stirred solution, kept under inert atmosphere at room
temperature, of the appropriate propanol derivative (com-
pounds 7, 7a , 11, 11a ; 9.99 mmol). The mixture was then
stirred at room temperature. When necessary an excess of the
two reagents was added to complete the reaction. The mixture
was washed in sequence with water (20 mL), 1 M HCl (2 × 20
mL), and saturated solution of Na2CO3 (2 × 20 mL), dried,
and concentrated under reduced pressure. The crude material
was purified by flash chromatography to give the pure
compound as white solid.
3-(3-(Ben zen esu lfon yl)fu r oxa n -4-yloxy)p r op yl 2-Ace-
toxyben zoa te (9). The mixture was stirred for 12 h, an excess
(30%) of the two reagents was added, and then the mixture
was stirred for 24 h. Flash chromatography eluent: petroleum
ether (40-60)/EtOAc 8/2 v/v. Yield 77%. White crystalline
solid. Mp 94 °C from diisopropyl ether. 1H NMR (CDCl3) δ 2.35/
2.35 (s, 3H, OCOCH3)/(qi, 2H, COOCH2CH2CH2-); 4.49 (t, 2H,
COOCH2CH2CH2); 4.58 (t, 2H, COOCH2CH2CH2); 7.11-8.09
(m, 9H, aromatic protons). 13C NMR (CDCl3) δ 20.9 (COOCH2-
CH2CH2); 27.9 (OCOCH3); 60.8 (COOCH2CH2CH2); 67.8
(COOCH2CH2CH2); 110.3 (furoxan C(3)); 122.6 (C6H4 C(1));
123.8 (C6H4 C(3)); 126.0 (C6H4 C(5)); 126.4 (SO2C6H5 C(3));
129.6 (SO2C6H5 C(2)); 131.5 (C6H4 C(6)); 134.0 (C6H4 C(4));
135.6 (SO2C6H5 C(4)); 137.9 (SO2C6H5 C(1)); 150.7 (C6H4 C(2));
158.7 (furoxan C(4)); 164.0 (COOCH2CH2CH2); 169.6 (OCOCH3).
MS (CI) m/z 463 (M + 1)+. Anal. (drying conditions: rt, 12 h,
pressure < 1 mmHg) for C20H18N2O9S C, H, N.
C(1)); 123.7 (C6H4 C(3)); 125.9 (C6H4 C(5)); 126.0 (C6H5 C(3));
126.4 (C6H5 C(2)); 130.3 (C6H4 C(6)); 131.4 (C6H4 C(4)); 134.0
(C6H5 C(4)); 150.6 (C6H4 C(2)); 162.0 (furoxan C(4)); 164.0
(COOCH2CH2CH2); 169.5 (OCOCH3). MS (EI) m/z 398 (M)+.
Anal. (drying conditions: rt, 24 h, pressure < 1 mmHg) for
C
20H18N2O7 C, H, N.
3-(4-P h en ylfu r a za n -3-yloxy)p r op yl 2-Acetoxyben zoa te
(12a ). The mixture was stirred for 3 h. Flash chromatography
eluent: petroleum ether (40-60)/EtOAc 85/15 v/v. Yield 83%.
1
White crystalline solid. Mp 54 °C from diisopropyl ether. H
NMR (CDCl3) δ 2.33 (s, 3H, OCOCH3); 2.41 (qi, 2H, COOCH2-
CH2CH2); 4.51/4.63 (t, 2H, COOCH2CH2CH2)/(t, 2H, COOCH2-
CH2CH2); 7.08-8.02 (m, 9H, aromatic protons). 13C NMR
(CDCl3) δ 20.8 (COOCH2CH2CH2); 28.2 (OCOCH3); 61.2
(COOCH2CH2CH2); 69.3 (COOCH2CH2CH2); 122.8 (C6H4 C(1));
123.7 (C6H4 C(3)); 124.9 (C6H5 C(1)); 125.9 (C6H4 C(5)); 127.2
(C6H5 C(3)); 128.8 (C6H5 C(2)); 130.5 (C6H4 C(6)); 131.4 (C6H4
C(4)); 133.9 (C6H5 C(4)); 145.0 (furazan C(4)); 150.6 (C6H4 C(2));
163.3 (furazan C(3)); 164.1 (COOCH2CH2CH2); 169.4 (OCOCH3).
MS (CI) m/z 383 (M + 1)+. Anal. (drying conditions: rt, 13 h,
pressure <1 mmHg) for C20H18N2O6 C, H, N.
(3-Meth ylfu r oxa n -4-yl)m eth yl 2-Acetoxyben zoa te (14).
A solution of 4-bromomethyl-3-methylfuroxan 13 (1.00 g; 5.18
mmol) in dry CH3CN (25 mL) was added dropwise to a stirred
solution, under inert atmosphere at room temperature, of 3
(2.54 g; 13.50 mmol) and triethylamine (1.88 mL; 13.50 mmol)
in dry CH3CN (50 mL). The mixture was stirred for 24 h and
then concentrated under reduced pressure. The crude solid
material was dissolved with Et2O (40 mL), and the resulting
solution was washed with water (20 mL) and a saturated
solution of NaHCO3 (20 mL), dried, and concentrated under
reduced pressure. The crude material was purified by flash
chromatography (eluent: petroleum ether (40-60)/AcOEt 8:2
v/v) to give the title compound as a pale yellow oil. Yield 81%.
1H NMR (CDCl3) δ 2.23/2.29 (s, 3H, OCOCH3)/(s, 3H, furoxan-
CH3); 5.36 (s, 2H, COOCH2); 7.12-8.04 (m, 4H, aromatic
protons). 13C NMR (CDCl3) δ 7.6 (furoxan-CH3); 20.8 (OCOCH3);
56.5 (COOCH2); 112.1 (furoxan C(3)); 121.7 (C6H4 C(1)); 123.9
(C6H4 C(3)); 126.1 (C6H4 C(5)); 131.5 (C6H4 C(6)); 134.7 (C6H4
C(4)); 150.8 (C6H4 C(2)); 153.3 (furoxan C(4)); 163.3 (COOCH2);
169.4 (OCOCH3). MS (EI) m/z 292 (M)+. Anal. (drying condi-
tions: 42 °C, 13 h, pressure < 1 mmHg) for C13H12N2O6 C, H,
N.
(3-Ca r ba m oylfu r oxa n -4-yl)m eth yl 2-Acetoxyben zoa te
(16). The title compound was obtained in the same manner
as 14 using 4-bromomethyl-3-carbamoylfuroxan 15. Reaction
time: 48 h. CH2Cl2 was used in the workup of the reaction
mixture. Flash chromatography eluent: petroleum ether (40-
60)/EtOAc 7:3 v/v. White crystalline solid. Yield 66%. Mp 139
°C from water. 1H NMR (CDCl3) δ 2.35 (s, 3H, OCOCH3); 5.65
(s, 2H, COOCH2); 6.30 (broad, 1H, CONH2); 7.12-8.09 (m, 4H,
aromatic protons); 7.50 (broad, 1H, CONH2). 13C NMR (CDCl3)
δ 20.9 (OCOCH3); 57.1 (COOCH2); 110.3 (furoxan C(3)); 122.0
(C6H4 C(1)); 123.9 (C6H4 C(3)); 126.0 (C6H4 C(5)); 131.8 (C6H4
C(6)); 134.4 (C6H4 C(4)); 150.8 (C6H4 C(2)); 154.0/155.6 (furoxan-
CONH2)/(furoxan C(4)); 169.5 (OCOCH3); 163.2 (COOCH2). MS
(CI) m/z 322 (M + 1).+ Anal. (drying conditions: rt, 24 h,
pressure < 1 mmHg) for C13H11N3O7 C, H, N.
3-(4-(Ben zen esu lfon yl)fu r a za n -3-yloxy)p r op yl 2-Ace-
toxyben zoa te (9a ). The mixture was stirred for 12 h. Flash
chromatography eluent: petroleum ether (40-60)/EtOAc from
8/2 to 75/25 v/v. Yield 84%. White crystalline solid. Mp 56 °C
from diisopropyl ether. 1H NMR (CDCl3) δ 2.30 (qi, 2H,
COOCH2CH2CH2); 2.36, (s, 3H, OCOCH3); 4.41/4.53 (t, 2H,
COOCH2CH2CH2)/(t, 2H COOCH2CH2CH2); 7.11-8.12 (m, 9H,
aromatic protons). 13C NMR (CDCl3) δ 20.9 (COOCH2CH2CH2);
28.0 (OCOCH3); 60.7 (COOCH2CH2CH2); 70.2 (COOCH2-
CH2CH2); 122.7 (C6H4 C(1)); 123.8 (C6H4 C(3)); 126.0 (C6H4
C(5)); 128.8 (SO2C6H5 C(3)); 129.6 (SO2C6H5 C(2)); 131.5 (C6H4
C(6)); 134.5 (C6H4 C(4)); 135.4 (SO2C6H5 C(4)); 137.79 (SO2C6H5
C(1)); 148.7 (furazan C(4)); 150.7 (C6H4 C(2)); 161.1 (furazan
C(3)); 164.0 (COOCH2CH2CH2); 169.5 (OCOCH3). MS (CI) m/z
447 (M + 1)+. Anal. (drying conditions: rt, 12 h, pressure < 1
mmHg) for C20H18N2O8S C, H, N.
(4-Meth ylfu r azan -3-yl)m eth yl 2-Acetoxyben zoate (14a).
A solution of 14 (3.84 g; 13.14 mmol) in trimethyl phosphite
(50 mL) was refluxed for 24 h and then cooled at room
temperature. Trimethyl phosphite was distilled off at atmo-
spheric pressure by using a Claisen distillation head. The
residue was dissolved in CH2Cl2 (50 mL), and the resulting
solution was washed with H2O (2 × 50 mL), dried, and
concentrated under reduced pressure. The crude material was
purified by flash chromatography (eluent: petroleum ether
(40-60)/EtOAc 8:2 v/v). The title compound was obtained as
3-(3-P h en ylfu r oxa n -4-yloxy)p r op yl 2-Acetoxyben zoa te
(12). The mixture was stirred for 3 h, an excess (20%) of the
two reagents was added, and then the mixture was stirred for
2 h. Flash chromatography eluent: petroleum ether (40-60)/
EtOAc from 8/2 to 7/3 v/v. Yield 77%. White crystalline solid.
1
1
Mp 84 °C from diisopropyl ether. H NMR (CDCl3) δ 2.34 (s,
a pale yellow oil. Yield 75%. H NMR (CDCl3) δ 2.26/2.45 (s,
3H, OCOCH3); 2.39 (qi, 2H, COOCH2CH2CH2); 4.52/4.67 (t,
2H, COOCH2CH2CH2)/(t, 2H, COOCH2CH2CH2); 7.08-8.10 (m,
9H, aromatic protons). 13C NMR (CDCl3) δ 20.8 (COOCH2CH2-
CH2); 28.1 (OCOCH3); 61.1 (COOCH2CH2CH2); 67.4 (COOCH2-
CH2CH2); 107.4 (furoxan C(3)); 122.2/122.7 (C6H5 C(1))/(C6H4
3H, OCOCH3)/(s, 3H, furazan-CH3); 5.47 (s, 2H, COOCH2);
7.11-8.10 (m, 4H, aromatic protons). 13C NMR (CDCl3) δ 8.1
(furazan-CH3); 20.7 (OCOCH3); 54.7 (COOCH2); 121.8 (C6H4
C(1)); 123.9 (C6H4 C(3)); 126.1 (C6H4 C(5)); 131.7 (C6H4 C(6));
134.6 (C6H4 C(4)); 150.1 (C6H4 C(2)); 150.1/150.8 (furazan C(3))/

New NO-Donor Esters of Aspirin
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 753
(furazan C(4)); 163.5 (COOCH2); 169.4 (OCOCH3). MS (CI) m/z
277 (M + 1)+. Anal. (drying conditions: 40 °C for 8 h then rt
for 48 h, pressure < 1 mmHg) for C13H12N2O5 C, H, N.
in CH3CN (50 mL). The resulting clear solution was heated
at 40 °C for 60 h. The mixture was filtered, and the filtrate
was concentrated under reduced pressure. The residue was
dissolved in CH2Cl2 (50 mL), and the resulting solution washed
with H2O (2 × 50 mL), dried, and concentrated under reduced
pressure. The crude material was purified by flash chroma-
tography (eluent: petroleum ether (40-60)/EtOAc 8/2 v/v) to
give the title compound as a pale yellow oil (lit.23). Yield 80%.
1H NMR (CDCl3) δ 2.36 (s, 3H, OCOCH3); 2.19 (qi, 2H,
COOCH2CH2); 4.40 (t, 2H, COOCH2CH2); 4.61 (t, 2H, CH2-
CH2CH2ONO2); 7.10-8.03 (m, 4H, aromatic protons). 13C NMR
(CDCl3) δ 20.8 (COOCH2CH2); 26.3 (OCOCH3); 60.8 (COOCH2);
69.6 (CH2CH2CH2ONO2); 122.7 (C6H4 C(1)); 123.7 (C6H4 C(3));
125.9 (C6H4 C(5)); 131.4 (C6H4 C(4)); 134.0 (C6H4 C(6)); 150.6
(C6H4 C(2)); 164.0 (COOCH2); 169.5 (OCOCH3). MS (CI) m/z
284 (M + 1)+. Anal. (drying conditions: 40 °C, 48 h, pressure
< 1 mmHg) for C12H13NO7 C, H, N.
P r op yl 2-Acetoxyben zoa te (5). The title compound was
obtained from 1-propanol in the same manner as 18. Reaction
time 96 h. Flash chromatography eluent: petroleum ether
(40-60)/diisopropyl ether 9/1 v/v. Colorless oil (lit.24). Yield
57%. 1H NMR (CDCl3) δ 1.02 (t, 3H, CH2CH2CH3); 1.77 (si,
2H; COOCH2CH2); 2.36 (s, 3H, OCOCH3); 4.24 (t, 2H, COOCH2-
CH2); 7.08-8.06 (m, 4H, aromatic protons). 13C NMR (CDCl3)
δ 10.3 (CH2CH2CH3); 20.8/21.8 (OCOCH3)/(COOCH2CH2); 66.5
(COOCH2CH2); 123.3 (C6H4 (C1)); 123.6 (C6H4 (C3)); 125.6
(C6H4 (C5)); 131.6 (C6H4 (C4)); 133.6 (C6H4 (C6)); 150.5 (C6H4
(C2)); 164.4 (COOCH2); 169.5 (OCOCH3). MS (CI) m/z 223 (M
+ 1).+ Anal. for C12H14O4 C, H.
Detection of Nitr ite. A solution of the appropriate com-
pound (20 µL) in dimethyl sulfoxide (DMSO) was added to 2
mL of 1/1 v/v mixture of 50 mM phosphate buffer (pH 7.4) with
MeOH, containing 5 × 10-4 M L-cysteine. The final concentra-
tion of the compound was 10-4 M. After 1 h at 37 °C, 1 mL of
the reaction mixture was treated with 250 µL of Griess reagent
[sulfanilamide (4 g), N-naphthylethylenediamine dihydrochlo-
ride (0.2 g), 85% phosphoric acid (10 mL) in distilled water
(final volume: 100 mL)]. After 10 min at room temperature,
the absorbance was measured at 540 nm (Shimatzu UV-
2501PC spectrophotometer). Sodium nitrite standard solutions
(10-80 nmol/mL) were used for the calibration curve. The
yields in nitrite were expressed as NO2- % (mol/mol) ( SEM.
No production of nitrite was observed in the absence of
L-cysteine.11
Detection of NO• in Hu m a n P la sm a . Citrated venous
blood was obtained from healthy volunteers who had not taken
any drug for at least two weeks. Platelet poor plasma (PPP)
was prepared by centrifugation at 1000g for 10 min. Aliquots
(2.0 mL) of PPP were equilibrated at 37 °C for 10 min. Radical
NO was detected with a Clark-type electrode (ISO-NO, World
Precision Instruments, Stevenage, UK), calibrated using di-
ethylamine diazeniumdiolate (DEA/NO; 100 nM-10 mM) in
phosphate-buffered saline (pH 4.0; DEA/NO generates 2 mol
equiv of NO spontaneously at pH < 5.0). The area under the
curve (AUC) was integrated for each DEA/NO concentration,
generating a linear calibration curve. NO generated from our
novel compounds in plasma was evaluated by integrating the
area under the curve, and the results were expressed as NO
concentration (nM).
(4-Ca r ba m oylfu r a za n -3-yl)m eth yl 2-Acetoxyben zoa te
(16a ). The title compound was obtained from 16 in the same
manner as 14a . Reaction time: 13 h. White crystalline solid.
Yield 64%. Mp 106-107° C from diisopropyl ether. 1H NMR
(CDCl3) δ 2.33 (s, 3H, OCOCH3); 5.73 (s, 2H, COOCH2); 6.22
(broad, 1H, CONH2); 6.78 (broad, 1H, CONH2); 7.11-8.09 (m,
4H, aromatic protons). 13C NMR (CDCl3) δ 20.9 (OCOCH3);
55.7 (COOCH2); 122.1 (C6H4 C(1)); 123.8 (C6H4 C(3)); 126.0
(C6H4 C(5)); 131.9 (C6H4 C(6)); 134.3 (C6H4 C(4)); 147.2
(furazan C(4)); 150.8 (C6H4 C(2)); 151.4 (furazan C(3)); 158.1
(furazan-CONH2); 163.3 (COOCH2); 169.6 (OCOCH3). MS (CI)
m/z 306 (M + 1)+. Anal. (drying conditions: rt, 24 h, pressure
< 1 mmHg) for C13H11N3O6 C, H, N.
(3-Cya n ofu r oxa n -4-yl)m eth yl 2-Acetoxyben zoa te (17).
Trifluoroacetic anhydride (1.73 mL; 7.04 mmol) was added,
over a period of 90 min, to a ice-cold stirred solution, kept
under inert atmosphere, of 16 (1.2 g; 3.74 mmol) and dry
pyridine (600 µL; 7.48 mmol) in dry THF (40 mL). The mixture
was slowly warmed at room temperature and then concen-
trated under reduced pressure. Water (20 mL) and Et2O (40
mL) were added to the residue, and, after separation, the
organic layer was washed with 0.5 M HCl (2 × 20 mL), dried,
and concentrated under reduced pressure. The crude material
was purified by flash chromatography (eluent: petroleum
ether (40-60)/EtOAc 85:15 v/v) obtaining the title compound
as a sticky oil that becomes a white solid during drying. Yield
1
78%. Mp 57 °C (trituration with diisopropyl ether). H NMR
(CDCl3) δ 2.37 (s, 3H, OCOCH3); 5.44 (s, 2H, COOCH2); 7.14-
8.10 (m, 4H, aromatic protons). 13C NMR (CDCl3) δ 20.8
(OCOCH3); 55.95 (COOCH2); 96.1 (furoxan C(3)); 104.7 (furoxan-
CN); 120.9 (C6H4 C(1)); 124.0 (C6H4 C(3)); 126.1 (C6H4 C(5));
131.6 (C6H4 C(6)); 135.0 (C6H4 C(4)); 151.1 (C6H4 C(2)); 152.2
(furoxan C(4)); 162.8 (COOCH2); 169.4 (OCOCH3). MS (CI) m/z
304 (M + 1)+. Anal. (drying conditions: 42 °C, 15 h, pressure
< 1 mmHg) for C13H9N3O6 C, H, N.
(4-Cya n ofu r a za n -3-yl)m eth yl 2-Acetoxyben zoa te (17a ).
The title compound was obtained from 16a in the same
manner as 17. White crystalline solid. Yield 80%. Mp 52-53
°C. 1H NMR (CDCl3) δ 2.34 (s, 3H, OCOCH3); 5.60 (s, 2H,
COOCH2); 7.13-8.13 (m, 4H, aromatic protons). 13C NMR
(CDCl3) δ 20.6 (OCOCH3); 54.2 (COOCH2); 106.6 (furazan-CN);
121.0 (C6H4 C(1)); 123.9 (C6H4 C(3)); 126.1 (C6H4 C(5)); 131.7
(C6H4 C(6)); 132.2 (furazan C(4)); 135.0 (C6H4 C(4)); 151.1
(C6H4 C(2)); 152.0 (furazan C(3)); 162.9 (COOCH2); 169.5
(OCOCH3). MS (EI) m/z 287 (M)+. Anal. (drying conditions:
40 °C, 48 h, pressure < 1 mmHg) for C13H9N3O5 0.25 H2O C,
H, N.
3-Br om op r op yl 2-Acetoxyben zoa te (18). A solution of 8
(1.00 g; 5.03 mmol) in dry toluene (25 mL) and a solution of
dry pyridine (410 µL; 5.03 mmol) in dry toluene (25 mL) were
added, over a period of 2 h, to a stirred solution, kept under
inert atmosphere at room temperature, of 3-bromopropanol
(1.40 g; 910 µL; 10.06 mmol) in dry toluene (20 mL). The
mixture was stirred at room temperature for 48 h and then
washed with water (2 × 40 mL), 1 M HCl (2 × 40 mL), and
saturated solution of NaHCO3 (2 × 40 mL), dried, and
concentrated under reduced pressure. The crude material was
purified by flash chromatography (eluent: petroleum ether
(40-60)/EtOAc 95/5 v/v) to give the title compound as a
colorless oil (lit.22). Yield 66%. 1H NMR (CDCl3) δ 2.33 (qi, 2H,
COOCH2CH2); 2.40 (s, 3H, OCOCH3); 3.53 (t, 2H, CH2CH2CH2-
Br); 4.44 (t, 2H, COOCH2CH2); 7.10-8.04 (m, 4H, aromatic
protons). 13C NMR (CDCl3) δ 20.9 (COOCH2CH2); 29.2
(OCOCH3); 31.6 (CH2CH2CH2Br); 62.64 (COOCH2CH2); 122.9
(C6H4 C(1)); 123.7 (C6H4 C(3)); 125.9 (C6H4 C(5)); 131.5 (C6H4
C(4)); 133.9 (C6H4 C(6)); 150.6 (C6H4 C(2)); 164.1 (COOCH2);
169.6 (OCOCH3). MS (CI) m/z 301-303 (M + 1)+. Anal. (drying
conditions: 40 °C, 48 h, pressure < 1 mmHg) for C12H13BrO4
C, H.
Hyd r olysis in Hu m a n Ser u m . A solution of each com-
pound (1.75 × 10-2 M) in acetonitrile (5 µL) was added to
human serum (Sigma-Aldrich) (495 µL) preheated at 37 °C.
After appropriate time of incubation the reaction mixture was
diluted with acetonitrile containing 0.1% trifluoroacetic acid
(1:1.5 v/v) in order to deproteinize the serum. Supernatant was
separated by centrifugation (9600g, 10 min) and analyzed by
HPLC. Chromatographic detection of the unaltered compounds
was accomplished using a Merck Purospher RP-18 column (250
× 4 mm; 5 µm particles size) termostatised at 40 °C, eluting
with a flow-rate of 1 mL/min. Mobile phase system was
consisting of linear gradient of 0.1% acqueous TFA (el. A) and
acetonitrile containing 0.1% TFA (el. B): a gradient profile of
25% B to 80% B in 15 min was used. The column effluent was
monitored at 224 nm; quantitation of the compounds was done
3-Nitr ooxyp r op yl 2-Acetoxyben zoa te (6). AgNO3 (11.28
g; 66.4 mmol) was added to a solution of 18 (2.50 g; 8.30 mmol)

754 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Cena et al.
by measurement of the peak areas in relation to those of
standards chromatographed under the same conditions. The
absence of aspirin among the hydrolysis products was con-
firmed according to the methods reported in ref 25.
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In h ibition of P la telet Aggr ega tion in Vitr o. Venous
blood was obtained from healthy volunteers who had not taken
any drug for at least two weeks. Volunteers were informed
that blood samples were obtained for research purposes and
that their privacy would be protected. Platelet rich plasma
(PRP) was prepared by centrifugation of citrated blood at 200g
for 20 min. Aliquots (300 µL) of PRP were added into
aggregometer (Elvi) cuvettes and aggregation was recorded as
increased light transmission under continuous stirring (1000
rpm) at 37 °C for 5 min after addition of the stimulus. ADP
(10 µM) or collagen (2.5 mg/mL) were used as platelet activa-
tors in PRP. The inhibitory activity of the compounds was
tested by addition of drug to PRP 10 min before addition of
the stimulus (ADP or collagen). Drug vehicle (<0.5% DMSO)
added to PRP or to the platelet suspension served as a control
and did not affect platelet function. The role of NO and sGC
in the inhibitory effect of 17 was investigated using the NO
scavenger, oxyhaemoglobin (10 µM), and the sGC inhibitor,
ODQ (100 µM), respectively.
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The antiaggregatory activity of tested compounds was
evaluated as % inhibition of platelet aggregation compared to
control samples. When inhibition of aggregation at maximal
inhibitor concentration exceeded 50%, pIC50 values were
calculated by nonlinear regression analysis.
Ack n ow led gm en t. This work was partially sup-
ported by a MURST grant.
J M020969T