(Nitrooxyacyloxy)methyl esters of aspirin as novel nitric oxide releasing aspirins

Article abstract of DOI:10.1021/jm900587h

A series of (nitrooxyacyloxy)methyl esters of aspirin were synthesized and evaluated as new NO-donor aspirins. Different amounts of aspirin were released in serum from these products according to the nature of nitrooxyacyloxy moiety present. In the aromat

Full text of DOI:10.1021/jm900587h

5058 J. Med. Chem. 2009, 52, 5058–5068
DOI: 10.1021/jm900587h
(Nitrooxyacyloxy)methyl Esters of Aspirin as Novel Nitric Oxide Releasing Aspirins
Loretta Lazzarato,† Monica Donnola,† Barbara Rolando,† Konstantin Chegaev,† Elisabetta Marini,† Clara Cena,†
Antonella Di Stilo,† Roberta Fruttero,† Stefano Biondi,‡ Ennio Ongini,‡ and Alberto Gasco*,†
†Dipartimento di Scienza e Tecnologia del Farmaco, Universitaꢀ degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy, and
‡Nicox Research Institute, Via Ariosto 21, 20091, Bresso (Milano), Italy
Received May 6, 2009
A series of (nitrooxyacyloxy)methyl esters of aspirin were synthesized and evaluated as new NO-donor
aspirins. Different amounts of aspirin were released in serum from these products according to the
nature of nitrooxyacyloxy moiety present. In the aromatic series, there is a rather good linear correlation
between the amount of aspirin released and the potencies of the products in inhibiting platelet
aggregation induced by collagen. Both the native compounds and the related nitrooxy-substituted acid
metabolites were able to relax rat aorta strips precontracted with phenylephrine, in keeping with a NO-
induced activation of the sGC as a mechanism that underlies the vasodilator effect. The products here
described are new improved examples of NO-donor aspirins containing nitrooxy groups. They could
represent an alternative to the use of aspirin in a variety of clinical applications.
Introduction
a number of mechanisms.10 In addition, it is able to maintain
micro- and macrovascular homeostasis11-13 as well as to trigger
anti-inflammatory and analgesic effects.14 Therefore, this “gas-
eous solution” to the old problem of aspirin gastrotoxicity
received great attention. A number of NO-donor moieties have
been joined through an ester linkage to the carboxylic group
of the drug. They include substructures containing nitrooxy
functions,15,16 furoxan,17 and N-diazeniumdiolate moieties
(Chart 1).18-20 The former two classes of products are rapidly
metabolized in serum, plasma, and a number of cell fractions
with little or no formation of aspirin,16,21-23 while for the latter
no specific study of aspirin release has been reported thus far. In
this paper, we describe a new class of (nitrooxyacyloxy)methyl
esters of this drug, showing that a number of them are stable in
acid and in physiological pH solution but are able to release
relevant amount of aspirin when incubated in human serum.
This double ester moiety was chosen in view of its rapid
hydrolysis in serum.24 Antiaggregatory and NO-dependent
vasodilator properties of all these new products are discussed
as well.
Nonsteroidal anti-inflammatory drugs (NSAIDsa) are the
most commonly used agents for the treatment of pain and
inflammation. Over 30 million people are treated with these
products every day.1 The prototype of NSAIDs is aspirin, which,
in addition to anti-inflammatory and analgesic properties, dis-
plays antithrombotic effects and, consequently, protects against
ischemic vascular disorders, including myocardial and cerebral
infarction. It also exerts some beneficial effects against colorectal
cancer.2-5 The activities of aspirin are largely due to its ability to
inhibit irreversibly COX enzymes, preferentially the COX-1
isoform with respect to COX-2.6,7 The most important limit in
using this drug is its strong gastrotoxicity due to systemic and
local irritant effects.6,7 The former are thought to be dependent
on the inhibition of COX-1 isoform, which is largely expressed in
the gastric epithelial cells, and the latter are closely associated
with the presence in the molecule of a free carboxylic group. A
strategy followed to reduce the local gastric drawback was the
masking of this function through pro-drug formation. Indeed, a
number of these compounds display reduced gastrotoxicity.8
However, the problem with this approach is the high enzymatic
liability of the o-acetyloxy group in these products. This is due to
the loss of the negative charge that is present in aspirin at
physiologic conditions following dissociation of its carboxylic
function (pKa = 3.5).9 Consequently a large part of these
substances are not true pro-drugs because they are rapidly
metabolized to salicylic acid in human serum without any
formation of relevant amounts of aspirin. Another strategy to
decrease the gastrotoxicity of aspirin is the combination of
aspirin with nitric oxide (NO)-donor moieties. Indeed, it is
known that NO is able to display gastrosparing actions through
Results and Discussion
Chemistry. The development of this new class of NO-
donor aspirins required the availability of a number of
nitrooxy substituted carboxylic acids. Products 1-4, 14,
19, and 26 were obtained following procedures already
described in literature, while acids 11-13, 18, 21-23, and
25 were synthesized according to the pathways reported in
Scheme 1. The compounds 11-13 were obtained starting
from the 3-bromopropoxy substituted benzaldehydes 5-
7, which after treating with AgNO3 in acetonitrile solution
afforded the corresponding 3-nitrooxypropoxy analogues
8-10. These latter intermediates treated with KMnO4 in
acetone solution gave the expected corresponding acids.
p-Nitrooxypropyl substituted benzoic acid 18 was pre-
pared starting from allylphenyl substituted dioxolane 15,
*To whom correspondence should be addressed. Phone: þ39-011-
670-7670. Fax: þ39-011-670-7286. E-mail: alberto.gasco@unito.it.
a Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs;
PRP, platelet rich plasma; NO, nitric oxide; sGC, soluble guanylate
cyclase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.
r
pubs.acs.org/jmc
Published on Web 08/04/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5059
Chart 1. Examples of NO-donors Aspirin
Scheme 1a
a Reagents and conditions: (a) AgNO3, CH3CN, 70 °C; (b) KMnO4, acetone; (c) (Sia)2BH, dry THF then NaOH, H2O2; (d) H2O/MeOH, 4 M HCl,
60 °C; (e) Ph3P, NBS, CH3CN; (f) mCPBA, CH2Cl2, -78 °C; (g) 85%H2O2, CF3COOH; (h) I2, AgNO3, CH3CN rt then AgNO3, CH3CN reflux.
which was transformed into alcohol 15a by action of
disiamylborane ((Sia)2BH) and then of H2O2 in the pre-
sence of NaOH. Compound 15a, after partial purifica-
tion, was immediately hydrolyzed with 4 M HCl to the
aldehyde 16. This intermediate was subjected to the action
of N-bromosuccinimide (NBS) in acetonitrile solution in
the presence of triphenylphosphine (Ph3P) to give 16a that
was immediately treated with AgNO3, giving rise to the
aldehyde 17. The related acid 18 was obtained following
the same procedure used to prepare 11-13. The com-
pound 21 was synthesized from the corresponding bromo
derivative 20 by action of AgNO3 in acetonitrile solution.
Acids 22 and 23 were prepared from 21 for m-chloro-
perbenzoic acid and H2O2 oxidation, respectively. Acid 25
was prepared by action of I2 and AgNO3 in acetonitrile
solution on 4-allylthiobenzoic acid (24).
(27) in DMF solution (Scheme 2). The remaining products
were obtained according to the pathways outlined in
Scheme 3. Oxidation of 38 with m-chloroperbenzoic acid
and oxone provided sulfoxide and sulfone models 41 and
42, respectively. The action of acetaldehyde on acetylsali-
cylic acid chloride 43 produced the adduct 44 that was
coupled with acid 21 in DMF solution in the presence of
Cs2CO3 to produce the final compound 45. Nitration of
intermediate 40 with 65% HNO3 in the presence of acetic
anhydride gave rise to the pyridine derivative 46. Finally,
product 32 afforded 47 through HCl hydrolysis. This latter
was used as a reference to follow the hydrolysis of 32 in
human serum.
Hydrolysis Studies. The possible hydrolytic routes of
(nitrooxyacyloxy) aspirin esters are reported in Scheme 4.
For compounds to release a meaningful amount of aspir-
in, the rate constant of the deacetylation k2 must be slower
than the hydrolytic constant k1. The stabilities of the
target products were assessed by high-performance liquid
chromatography (HPLC) in buffered solutions at pH 1.0
Most of the NO-aspirins described in the present work,
namely products 28-39, and the intermediate 40 were
prepared by action of the cesium salt of appropriate acid
prepared in situ on chloromethyl 2-(acetyloxy)benzoate

5060 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Lazzarato et al.
Scheme 2a
a Reagents and conditions: (a) Cs2CO3, DMF.
and 7.4 as well as in human serum. The results are
reported in Table 1. After 3 h of incubation in acid
solution, both the aliphatic and the aromatic NO-donor
aspirins resulted unchanged for about 98% with the only
exception of 46 containing the electron poor pyridine
ring. This product was transformed slightly more exten-
sively (unchanged 84%). A similar behavior was observed
at physiological pH, but in this case, in addition to
46 (unchanged for 60%) also 41 and 42, containing the
electron-withdrawing SO or SO2 groups on the benzene
ring, displayed less stability compared to the other terms
of the series (unchanged for 90% and for 70%, respec-
tively). Quite different results were obtained in serum in
which the hydrolysis of a variety of esters is catalyzed by
carboxylesterases. These enzymes are ubiquitous and dis-
play a broad substrate specificity. Frequently the same
ester can be hydrolyzed by more than one of these en-
zymes.31 It was assumed that (acyloxy)alkyl esters are
hydrolyzed to the corresponding hydroxyalkyl esters,
which immediately and spontaneously decompose to
the related carboxylic acids and aldehydes.24 According
to Scheme 4, aspirin, salicylates, nitrooxy-substituted
carboxylic acids, and salicylic acid were detected during
the hydrolysis. The time course of the metabolites de-
tected over 10 min and 2 h in the case of products 32 and 36
is reported in Figure 1 and Figure 2 as examples. For all
the products, the final metabolites (6 h) were salicylic acid
and nitrooxy-substituted carboxylic acids. The hydrolysis
strictly followed first-order kinetics. The observed pseu-
do-first-order rate constants (kobs) were calculated from
the slopes of linear plots of the logarithm of the remaining
ester against time: the corresponding half-lives were
obtained from eq 1.
t1=2 ¼ 0:693=kobs
ð1Þ
In Table 1, the maximal amounts of aspirin detected for each
product, expressed as % of the initial ester concentration, are
also collected. Analyses of the data show that all the com-
pounds are very quickly hydrolyzed in serum. A number of
products display t1/2 < 1 min, and this figure does not exceed
5.4 min in the others. The peak of aspirin released in the
aliphatic nitrooxyacyl series (28-31) ranges from 11% to
20%. The products of the aromatic series are definitively
better aspirin releasing compounds. In the set of mononi-
trooxy-alkyloxy substituted models, the release follows the
series 33 > 34 . 32. The very low release of aspirin from the
o-derivative is probably due to the negative effect of steric
hindrance on hydrolysis exerted by the substituent group in
proximity of the ester function. The dinitrooxy substituted
compound 35 appears to be more susceptible to the enzy-
matic cleavage of acetyloxy group than the mononitrooxy
structurally related 34. All the other compounds we consid-
ered bear a p-substituent at the phenyl ring of benzoic acid.
This is due to their easier synthetic availability, but the good
results obtained with 33 indicates that the m-substitution
could also be worthy of investigation. The products 36 and
38, bearing mononitrooxypropyl- and mononitrooxypro-
pylthio- chains, respectively, are among the best aspirin
releasing products of the series. Moving to the dinitrooxy
analogues 37 and 39, this capacity decreases. The change in
the oxidation level of the sulfur chain of 38 affords the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5061
Scheme 3a
a Reagents and conditions: (a) mCPBA, CH2Cl2, -78 °C; (b) oxone, H2O/MeOH; (c) CH3CHO, ZnCl2, CH2Cl2; (d) 21, Cs2CO3, DMF;
(e) (CH3CO)2O, 65% HNO3, CH2Cl2; (f) 6 M HCl, 1,4-dioxane, 70 °C.
taking aspirin as a reference. The inhibitory activity of the
compounds was tested by addition of product to PRP 10 min
before addition of the stimulus. All the products displayed a
concentration dependent inhibitory effect. Their antiaggre-
gatory potency, expressed as IC50, are reported in Table 1. By
contrast, no antiaggregatory activity was observed when the
related nitrooxy substituted carboxylic acids, which are
rapidly formed from the parent drugs under the action of
plasma esterases, were tested at 300 μM concentration. This
finding is consistent with the known inability of platelets to
efficiently effect NO-release from organic nitrates.32 As
expected, the products of the aromatic series are definitively
more active than the products of aliphatic series. The low
activity of these latter compounds prevented us from deter-
mining their IC50 values. The areas under the aspirin release
curves (AUC) of all the products, measured after 10 min of
incubation, are reported in Table 1. In the aromatic series
there is a rather good linear correlation (r2=0.8732) between
these values and the corresponding IC50 values (Figure 3),
despite the different medium in which they were measured,
serum and plasma, respectively (eq 2, the standard error of
regression coefficients is given within parentheses).
Scheme 4. Possible
(Acyloxy)alkyl Esters to Salicylic Acid
Hydrolytic
Routes
of
Aspirin
sulfoxide 41 and the sulfone 42, which are very good aspirin
releasing compounds. As expected, the introduction of a
methyl group on the methylene moiety of the acyloxymethyl
substructure of 38 to give 45 weakens the production of
aspirin, reasonably for steric reason. Finally, the good
behavior of the pyridine derivative 46 indicates that other
interesting NO-donor aspirins could be designed from the
use of other π-deficient heterocycle carboxylic acids.
IC50 ¼ -0:27ð ( 0:03ÞAUC þ 174ð ( 12Þ
n ¼ 11, r2 ¼ 0:8732, F ¼ 61:98
ð2Þ
Platelet Antiaggregatory Activity. Antiaggregatory effects
of the new NO-aspirins were studied on collagen induced
platelet aggregation of human platelet rich plasma (PRP),
Vasodilator Activities. The vasodilator activity of the new
NO-donor aspirins was evaluated on endothelium denuded

5062 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Lazzarato et al.
Table 1. Stability of the Compounds 28-39, 41, 42, 45, and 46 in Buffered Solutions (Percentage of Unchanged Compound after 3 h); Stability in
Human Serum (Half-Life, Percent of Maximal Amounts of Aspirin Released and AUC Values at 10 min) and Antiaggregatory Activities
stability in buffered solutions % unchanged at 3 ha
stability in human serum
platelet aggregation
% max of aspirin AUC at
releasedc
IC50 (μM)
(CL 95%)
% inhibition (
compd
pH 1.0
pH 7.4
t
1/2 (min)b
10 min
SEM at 300 μMd
aspirin
28
29
90
99
99
99
99
99
99
99
99
98
99
99
98
98
98
98
84
90
99
99
100
99
98
98
99
99
100
99
99
98
90
70
99
60
63
54 (49-60)
d
<1
<1
<1
<1
<1
1.4
3.4
5.4
1.9
3.4
2.4
4.6
<1
<1
4.4
<1
10.1
10.6
17.6
9.8
93.6
84.6
17 ( 3
8.6 ( 5.8
29 ( 3
d
d
d
30
31
153.7
86.6
43.6
36 ( 14
32
33
5.0
163 (132-198)
100 (81-122)
138 (105-181)
140 (119-163)
41 (35-49)
97 (82-115)
45 (36-56)
129 (116-145)
72 (66-78)
48.3
39.8
22.4
65.2
34.2
61.7
43.0
70.7
59.2
29.4
58.0
387.6
236.7
105.2
521.6
220.1
403.3
192.2
301.0
515.8
144.6
542.8
34
35
36
37
38
39
41
42
38 (34-44)
d
45
46
27 ( 4
20 (15-25)
a SEM e 1%. b SEM e 0.2. c SEM e 2.5%. d Due to the low activity of the compound, IC50 could not be calculated: in this case the percent of
inhibition is reported at 300 μM.
Figure 2. Time course of the metabolites of product 36 at 10 min
and at 2 h incubation time in human serum: values are mean ( SEM
(SEM e 5; number of determinations=3).
Figure 1. Time course of the metabolites of product 32 at 10 min
and at 2 h incubation time in human serum: values are mean ( SEM
(SEM e 5; number of determinations=3).
greater hydrophilicity, which limits the penetration into the
vascular smooth muscle cell. When the experiments were
repeated in the presence of 1 μM ODQ (1H-[1,2,4]oxa-
diazolo[4,3-a]quinoxalin-1-one), a decrease in the potencies
was observed, in keeping with a NO-induced activation of
the sGC as a mechanism which underlies the vasodilator
effect.
rat aorta strips precontracted with phenylephrine. The vaso-
dilator action of the nitrooxy-substituted acids, intermedi-
ates in the synthesis of the target compounds and which are,
together with salicylic acid, the final metabolites of the
hydrolysis of these products, were assessed as well. As a
nitrooxy-substituted acid for compound 46, we used
6-(nitrooxymethyl)pyridine-2-carboxylic acid (48). All the
products were able to relax the contracted tissue in a
concentration-dependent manner. Their potencies, ex-
pressed as EC50, are collected in Table 2. The nitrooxy-
substituted acids are always less potent than the parent
compounds. The case of 36 and of the related acid 18 is
reported in Figure 4 as an example. This is likely due to their
Conclusions
We were able to develop a class of (nitrooxyacyloxy)methyl
esters of aspirins which behave as true NO-donor aspirins.
The aspirin release, evaluated in serum, follows a pseudo-first-
order kinetic. The amount of aspirin released is strongly

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5063
Figure 4. Concentration response curves for vasodilator activity of
the compound 36 and of its corresponding nitrooxy-substituted acid
18 in the absence and in the presence of ODQ.
Figure 3. Linear correlation between AUC values of aspirin re-
leased in human serum from the indicated compounds and the
corresponding antiaggregatory IC50 values.
3
3
-OCH2-, JHH =6.0 Hz), 4.71 (t, 2H, -CH2ONO2, JHH
6.0 Hz), 6.98 (d, 1H, C6H4), 7.06 (t, 1H, C6H4), 7.53-7.59 (m,
1H, C6H4), 7.82-7.85 (m, 1H, C6H4), 10.48 (s, 1H, -CHO). 13
=
dependent on the nature of acyloxy moiety. The best results
were obtained using aromatic nitrooxyacyloyl moieties. In the
case of the benzoyl derivatives, the most active compounds
are those bearing NO-donor chains at p- or at m-position. All
the products display in vitro vasodilator activities included the
nitrooxy-substituted acids formed following metabolism. The
products here described are new improved examples of NO-
donor aspirins containing nitrooxy groups. They could repre-
sentanimproved alternative tothe use of aspirin ina varietyof
clinical applications. Studies are in progress aimed at examin-
ing their oral and dermal delivery characteristics.
C
NMR (CDCl3) δ 27.0, 64.2, 69.6, 112.3, 121.2, 124.9, 128.8,
136.0, 160.6, 189.3. MS (CI) m/z 226 (M þ 1)þ.
3-(3-Formylphenoxy)propyl Nitrate (9). Yield 90%. 1H NMR
(CDCl3) δ 2.25 (qi, 2H, -CH2CH2ONO2), 4.14 (t, 2H, -OCH2,
3JHH=6.0 Hz), 4.69 (t, 2H, -CH2ONO2, 3JHH=6.3 Hz), 7.13-
7.21 (m, 1H, C6H4), 7.38-7.50 (m, 3H, C6H4), 9.97 (s, 1H,
-CHO). 13C NMR (CDCl3) δ 26.9, 63.9, 69.8, 112.6, 121.8,
124.0, 130.2, 137.8, 159.0, 192.0. MS (CI) m/z 226 (M þ 1)þ.
3-(4-Formylphenoxy)propyl Nitrate (10). Yield 93%. 1H
NMR (CDCl3) δ 2.26 (qi, 2H, -CH2CH2ONO2), 4.16 (t, 2H,
3
3
-OCH2-, JHH =6.0 Hz), 4.69 (t, 2H, -CH2ONO2, JHH
=
Experimental Section
6.3 Hz), 7.00 (d, 2H, C6H4), 7.84 (d, 2H, C6H4), 9.89 (s, 1H,
-CHO). 13C NMR (CDCl3) δ 26.9, 64.0, 69.7, 115.2, 130.3,
132.0, 163.4, 190.8. MS (CI) m/z 226 (M þ 1)þ.
Synthesis. 1H and 13C NMR spectra were recorded on a
Bruker Avance 300 at 300 and 75 MHz, respectively, using
SiMe4 as the internal standard, and the following abbreviations
were used to indicate the peak multiplicity: s = singlet, d =
doublet, t=triplet, qt=quartet, qi=quintet, m=multiplet, br s=
broad signal. Low resolution mass spectra were recorded with a
Finnigan-Mat TSQ-700. Melting points were determined with a
capillary apparatus (Buchi 540). Flash column chromatography
was performed on silica gel (Merck Kieselgel 60, 230-400 mesh
ASTM); PE stands for 40-60 petroleum ether. The progress of
the reactions was followed by thin layer chromatography (TLC)
on 5 cm ꢀ 20 cm plates with a layer thickness of 0.25 mm.
Anhydrous magnesium sulfate was used as the drying agent for
the organic phases. Organic solvents were removed under
vacuum at 30 °C. Preparative HPLC was performed on a
Lichrospher C18 column (250 mm ꢀ 25 mm, 10 μm) (Merck
Darmstadt, Germany) with a Varian ProStar mod-210 with
Varian UV detector mod-325. Elemental analyses (C, H, N)
were performed by REDOX (Monza), and the results are within
(0.4% of the theoretical values. Compounds 1,25 2,26 3,27 4,28
5,33 6,33 7,34 14,29 19,29 20,35 24,36 26,30 27,37 43,38 and 4830 were
synthesized according to literature. Compound 15 was synthe-
sized with standard method39 starting from 4-allylbenzalde-
hyde.29
3-(4-Formylphenyl)propyl Nitrate (17). A solution of NaBH4
(11.3 g, 0.30 mol) in dry THF (250 mL) was slowly added
to amylene (115 mL, 1.1 mol) stirred at 0 °C. Then BF3 Et2O
3
(27.5 mL, 0.22 mol) was added in 30 min to the mixture
maintained at 0 °C. After 5.5 h, a solution of 15 (4.20 g,
22.1 mmol) in dry THF (100 mL) was slowly added and the
stirring was continued for 24 h. Then to the mixture, cooled at
0 °C, H2O (140 mL), NaOH 3 M (140 mL), and H2O2 30%
(210 mL) were added and the resulting mixture was heated at
40 °C for 1.5 h. After separation, the organic layer was washed
with H2O (100 mL), dried, filtered, and concentrated under
reduced pressure. The crude product so obtained was purified by
flash chromatography (PE/EtOAc 8/2 to 6/4 v/v) to give
3-[4-(1,3-dioxolan-2-yl)phenyl]propan-1-ol (15a) as a colorless
oil. HCl (4 M, 20 mL) was added to a stirred solution of 15a (4.50g,
21.61 mmol) in MeOH/H2O 1/1 (90 mL). After 2 h, the reaction
was completed; the mixture was concentrated under reduced
pressure and extracted twice with CH2Cl2 (50 mL). The com-
bined organic layers were washed with brine (20 mL), dried,
filtered, and concentrated under reduced pressure to give 16 as a
pale-yellow oil. Yield 97%.
NBS (4.49 g, 25.21 mmol) was added portionwise to a
solution of 16 (3.45 g, 21.01 mmol) and Ph3P (6.06 g,
23.11 mmol) in dry CH3CN (50 mL), stirred at 0 °C. After
30 min, the reaction was completed, the mixture was diluted
with CH2Cl2 (50 mL), washed twice with H2O (50 mL), dried,
filtered, and concentrated under reduced pressure. AgNO3
(7.14 g; 42.02 mmol) was added to a solution of the crude
in CH3CN (50 mL), and the mixture was heated at 70 °C for
1 h. Then brine was added to precipitate the excess of
AgNO3, the mixture was filtered through celite, and the
residue was diluted with CH2Cl2 (50 mL) and washed with
H2O (50 mL). The organic layer was dried, filtered,
and concentrated under reduced pressure. The crude pro-
duct was purified by flash chromatography (PE/EtOAc
80/20 v/v) to give the title compound as a pale-yellow oil.
General Procedure for the Preparation of 8, 9, 10. A solution of
the appropriate bromo derivative (2.20 g, 9.05 mmol) and
AgNO3 (3.10 g, 18.10 mmol) in CH3CN (25 mL) was stirred
at 70 °C for 1 h. Then brine was added to precipitate the excess of
AgNO3, the mixture was filtered through celite and concen-
trated under reduced pressure. The residue was treated with
CH2Cl2 (50 mL) and H2O (50 mL). After separation, the
aqueous layer was extracted twice with CH2Cl2 (50 mL). The
combined organic layers were dried, filtered, and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (PE/EtOAc 90/10 v/v) to give the compound
as a pale-yellow oil.
3-(2-Formylphenoxy)propyl Nitrate (8). Yield 70%. 1H NMR
(CDCl3) δ 2.31 (qi, 2H, -CH2CH2ONO2), 4.20 (t, 2H,

5064 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Lazzarato et al.
Table 2. Vasodilator Activities of the Products 1-4, 11-14, 18, 19, 21-23, 25, 28-39, 41, 42, 45, 46, and 48
a The maximal concentration tested (100 μM) cannot reach the 50% of the effect. b The maximal concentration tested (30 μM due to insolubility limits)
cannot reach the 50% of the effect.
Yield 76%. 1H NMR (CDCl3) δ 2.04-2.14 (m, 2H,
65.5, 69.5, 112.8, 118.3, 121.9, 133.6, 135.0, 157.8, 167.7. MS
(CI) m/z 242 (M þ 1)þ.
3
-CH2CH2ONO2), 2.83 (t, 2H, -CH2-, JHH = 7.5 Hz),
3
4.47 (t, 2H, -CH2ONO2, JHH = 6.6 Hz), 7.36 (d, 2H,
3-[3-(Nitrooxy)propoxy]benzoic Acid (12). Melting point:
110-111 °C (from iPr2O); white solid; yield 75%. 1H NMR
(DMSO-d6) δ 2.16 (qi, 2H, -CH2CH2ONO2), 4.12 (t, 2H,
-OCH2-), 4.70 (t, 2H, -CH2ONO2), 7.19-7.21 (m, 1H,
C6H4), 7.83 (d, 2H, C6H4), 9.99 (s, 1H, -CHO). 13C NMR
(CDCl3) δ 28.3, 32.3, 72.3, 129.4, 130.5, 135.3, 147.8, 192.2.
MS (CI) m/z 210 (M þ 1)þ.
General Procedure for the Preparation of 11, 12, 13, 18.
KMnO4 (2.00 g, 12.58 mmol) was added to a solution of the
appropriate aldehyde (8.39 mmol) in acetone (25 mL) and stirred
at 0 °C. The reaction was allowed to reach rt, and it was completed
after 1 h. Oxalic acid was added until the color of the mixture
became green, and then the mixture was filtered and the filtrate was
diluted with CH2Cl2 (50 mL). The organic layer was washed with
H2O (50 mL) and then dried, filtered, and concentrated under
reduced pressure.
C6H4), 7.40-7.71 (m, 3H, C6H4), 13.0 (br s, 1H, -COOH). 13
C
NMR (DMSO-d6) δ 26.6, 64.6, 71.3, 114.9, 119.7, 122.2, 130.1,
132.6, 158.7, 167.5. MS (CI) m/z 242 (M þ 1)þ.
4-[3-(Nitrooxy)propoxy]benzoic Acid (13). Melting point:
1
137-138 °C (from iPr2O/iPrOH); white solid; yield 81%. H
NMR (DMSO-d6) δ 2.20 (qi, 2H, -CH2CH2ONO2), 4.18 (t, 2H,
-OCH2-, 3JHH=6.0 Hz), 4.73 (t, 2H, -CH2ONO2, 3JHH=6.3
Hz), 7.05 (d, 2H, C6H4), 7.93 (d, 2H, C6H4), 12.7 (s, 1H,
-COOH). 13C NMR (DMSO-d6) δ 26.5, 64.7, 71.3, 114.6,
123.5, 131.8, 162.3, 167.4. MS (CI) m/z 242 (M þ 1)þ.
2-[3-(Nitrooxy)propoxy]benzoic Acid (11). The crude product
so obtained was purified by flash chromatography (PE/EtOAc/
HCOOH 90/10/0.1 v/v/v) to give the title compound as a
colorless oil; yield 47%. 1H NMR (CDCl3) δ 2.34 (qi, 2H,
4-[3-(Nitrooxy)propyl]benzoic Acid (18). Melting point:
125.5-126.5 °C (from iPr2O); white solid; yield 89%. 1H
NMR (CDCl3) δ 2.09 (qi, 2H, -CH2CH2ONO2), 2.82 (t, 2H,
3
-CH2CH2ONO2), 4.30 (t, 2H, -OCH2-, JHH = 6.0 Hz),
C6H4CH2-, 3JHH=7.5 Hz), 4.46 (t, 2H, -CH2ONO2, 3JHH
=
3
4.73 (t, 2H, -CH2ONO2, JHH=6.0 Hz), 7.03 (d, 1H, C6H4),
6.3 Hz), 7.30 (d, 2H, C6H4), 8.06 (d, 2H, C6H4), 11.7 (br s, 1H,
-COOH). 13C NMR (CDCl3) δ 28.0, 31.9, 72.0, 127.6, 128.6,
130.7, 146.7, 171.9. MS (CI) m/z 226 (M þ 1)þ.
7.11 (t, 1H, C6H4), 7.53-7.59 (m, 1H, C6H4), 8.09-8.12 (m, 1H,
C6H4), 11.0 (br s, 1H, -COOH). 13C NMR (CDCl3) δ 26.9,

Article
4-[3-(Nitrooxy)propylthio]benzoic Acid (21). A solution of 20
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5065
General Procedure for the Preparation of 28-40. To a solution
of the appropriate carboxylic acid (1.00 mmol) in DMF (5 mL)
were added Cs2CO3 (0.50 mmol) and after 10 min 27
(1.00 mmol). The mixture was stirred for 24 h and then poured
in H2O (10 mL) and extracted with Et2O (3 ꢀ 10 mL). The
combined organic layers were washed with a saturated solution
of NaHCO3 (10 mL), dried, filtered, and concentrated under
reduced pressure. The crude product so obtained was purified by
flash chromatography. Chromatographic eluents and yields of
the products were as follow.
(2.70 g, 10.0 mmol) and AgNO3 (3.40 g, 20.0 mmol) in CH3CN
(50 mL) was stirred at 70 °C for 5 h. Then brine was added to
precipitate the excess of AgNO3, and the mixture was filtered
through celite and concentrated under reduced pressure. The
residue was treated with CH2Cl2 (50 mL) and H2O (50 mL).
After separation, the aqueous layer was extracted twice with
CH2Cl2 (50 mL). The combined organic layers were dried,
filtered, and concentrated under reduced pressure. The crude
product was purified by flash chromatography (PE/EtOAc/
HCOOH 80/20/0.1 v/v/v) to give the title compound as a white
solid. Yield 80%; mp 103.5-105.5 °C (from iPr2O/iPrOH). 1H
NMR (CD3OD) δ 2.07 (qi, 2H, -CH2CH2ONO2), 3.13 (t, 2H,
{[6-(Nitrooxy)hexanoyl]oxy}methyl 2-(Acetyloxy)benzoate
(28). Eluent (PE/EtOAc 9/1 v/v); colorless oil; yield 70%. H
1
NMR (CDCl3) δ 1.41-1.49 (m, 2H), 1.64-1.78 (m, 4H)
(-CH2CH2CH2CH2ONO2), 2.36 (s, 3H, CH3COO-), 2.41
(t, 2H, -OCOCH2-, 3JHH=7.2 Hz), 4.41 (t, 2H, -CH2ONO2,
3JHH=6.6 Hz), 5.95 (s, 2H, -OCH2O-), 7.12 (d, 1H, C6H4),
7.33 (t, 1H, C6H4), 7.61 (t, 1H, C6H4), 8.07 (d, 1H, C6H4).
13C NMR (CDCl3) δ 21.2, 24.3, 25.3, 26.7, 33.8, 73.1, 79.4,
122.2, 124.3, 126.4, 132.4, 135.0, 151.4, 163.2, 169.9, 172.2. MS
(CI) m/z 370 (M þ 1)þ.
3
3
-SCH2-, JHH = 7.2 Hz), 4.60 (t, 2H, -CH2ONO2, JHH
=
6.3 Hz), 7.38 (d, 2H, C6H4), 7.93 (d, 2H, C6H4). 13C NMR
(CD3OD) δ 27.4, 28.9, 72.8, 127.9, 128.8, 131.3, 144.4, 169.4.
MS (CI) m/z 258 (M þ 1)þ.
4-[3-(Nitrooxy)propyl-1-sulfinyl]benzoic Acid (22). A solution
of 70% mCPBA (0.25 g, 1.0 mmol) in dry CH2Cl2 (10 mL) was
slowly added to a solution of 21 (0.26 g, 1.0 mmol) in dry CH2Cl2
(15 mL), stirred at -78 °C. At the end of the addition, the
reaction was completed. The mixture was poured in 10%
Na2SO3 (50 mL), the layers separated, and the aqueous
layer extracted twice with Et2O (50 mL). The organic layers
were dried, filtered, and concentrated under reduced pressure.
The crude product so obtained was purified by flash chroma-
tography (PE/EtOAc/HCOOH 80/20/0.1 v/v/v) to give the title
compound as a white solid. Yield 55%; mp 154.5-155.0 °C. 1H
NMR (DMSO-d6) δ 1.70-1.90 (m, 1H, -CHaHbCH2ONO2),
2.00-2.14 (m, 1H, -CHaHbCH2ONO2), 2.86-2.95 (m, 1H,
SOCHaHb-), 3.15-3.35 (m, 1H, SOCHaHb-), 4.58 (t, 2H,
{[5,6-Bis(nitrooxy)hexanoyl]oxy}methyl 2-(Acetyloxy)benzo-
ate (29). Eluent (PE/EtOAc 8/2 v/v); colorless oil; yield 36%.
1H NMR (CDCl3) δ 1.74-1.83 (m, 4H, -CH2CH2CH-),
2.36 (s, 3H, CH3COO-), 2.45-2.48 (m, 2H, -OCOCH2-),
4.39-4.46 (m, 1H, -CHaHbONO2), 4.68-4.74 (m, 1H,
-CHaHbONO2), 5.25-5.28 (m, 1H, -CHONO2-), 5.95
(s, 2H, -OCH2O-), 7.12 (d, 1H, C6H4), 7.34 (t, 1H, C6H4),
7.61 (t, 1H, C6H4), 8.08 (d, 1H, C6H4). 13C NMR (CDCl3) δ
20.0, 21.0, 28.4, 33.0, 71.0, 78.7, 79.2, 121.8, 124.0, 126.2, 132.2,
134.9, 151.1, 163.0, 169.6, 171.3. MS (CI) m/z 431 (M þ 1)þ.
{[7-(Nitrooxy)heptanoyl]oxy}methyl 2-(Acetyloxy)benzoate
(30). Eluent (PE/EtOAc 8/2 v/v); colorless oil; yield 52%.
1H NMR (CDCl3) δ 1.34-1.43 (m, 4H), 1.61-1.71 (m, 4H)
(-CH2CH2CH2CH2CH2ONO2), 2.36-2.42 (m, 5H, CH3-
3
-CH2ONO2, JHH = 6.3 Hz), 7.80 (d, 2H, C6H4), 8.12 (d,
2H, C6H4), 13.32 (br s, 1H, COOH). 13C NMR (DMSO-d6)
δ 19.7, 51.7, 73.0, 125.1, 131.1, 133.7, 149.4, 167.4. MS (CI) m/z
274 (M þ 1)þ.
3
COO- þ -OCOCH2-), 4.41 (t, 2H, -CH2ONO2, JHH =6.6
Hz), 5.94 (s, 2H, -OCH2O-), 7.12 (d, 1H, C6H4), 7.33 (t, 1H,
C6H4), 7.61 (t, 1H, C6H4), 8.07 (d, 1H, C6H4). 13C NMR (CDCl3)
δ 21.0, 24.3, 25.3, 26.5, 28.4, 33.7, 73.2, 79.2, 122.0, 124.1, 126.2,
132.2, 134.7, 151.2, 163.0, 169.7, 171.1. MS (CI) m/z 384 (M þ 1)þ.
{[6,7-Bis(nitrooxy)heptanoyl]oxy}methyl 2-(Acetyloxy)benzo-
ate (31). The crude product was purified partially by flash
chromatography (PE/EtOAc 8/2 v/v) and then by RP18 pre-
parative HPLC (flow 39 mL/min, λ 226 nm, CH3CN/H2O 65/35
v/v, injection 2 mL, solution 100 mg/mL); colorless oil; yield
4-[3-(Nitrooxy)propyl-1-sulfonyl]benzoic Acid (23). A solu-
tion of 85% H2O2 (0.31 g, 7.76 mmol) in CF3COOH (2 mL)
was slowly added to a suspension of 21 (0.50 g, 1.94 mmol)
in CF3COOH (5 mL) stirred at 0 °C. The reaction was allowed
to reach rt, and it was completed after 1.5 h. The mixture was
poured into ice/water and the white solid so obtained
was filtered. The crude product was purified by flash chro-
matography (CH2Cl2/EtOAc/HCOOH 95/5/0.1 v/v/v) to give
the title compound as a white solid. Yield 74%; mp 172-173.5
1
1
30%. H NMR (CDCl3) δ 1.45-1.51 (m, 2H), 1.65-1.76 (m,
°C. H NMR (DMSO-d6) δ 1.97 (qi, 2H, -CH2CH2ONO2),
3
4H) (-CH2CH2CH2CH-), 2.35 (s, 3H, CH3COO-), 2.42 (t,
3.51 (t, 2H, SO2CH2CH2-, JHH = 7.5 Hz), 4.56 (t, 2H,
3
3
2H, -OCOCH2-, JHH = 6.9 Hz), 4.38-4.45 (m, 1H,
-CH2ONO2, JHH = 6.3 Hz), 8.04 (d, 2H, C6H4), 8.19 (d,
2H, C6H4), 13.6 (br s, 1H, COOH). 13C NMR (DMSO-d6)
δ 20.1, 50.9, 71.2, 128.1, 130.1, 135.5, 142.0, 166.0. MS (CI)
m/z 290 (M þ 1)þ.
-CHaHbONO2), 4.67-4.72 (m, 1H, -CHaHbONO2), 5.15-
5.28 (m, 1H, -CHONO2-), 5.95 (s, 2H, -OCH2O-), 7.12
(d, 1H, C6H4), 7.34 (t, 1H, C6H4), 7.60 (t, 1H, C6H4), 8.08 (d, 1H,
C6H4). 13C NMR (CDCl3) δ 20.9, 24.0, 24.2, 29.0, 33.4, 71.1,
78.9, 79.2, 121.9, 124.1, 126.2, 132.2, 134.8, 151.1, 163.0, 169.7,
171.8. MS (CI) m/z 445 (M þ 1)þ.
4-{[2,3-Bis(nitrooxy)propyl]thio}benzoic Acid (25). Iodine
(8.2 g, 32.38 mmol) was added portionwise to a stirred solution
of 24 (6.30 g, 32.38 mmol) and AgNO3 (5.50 g, 32.38 mmol) in
CH3CN (100 mL) kept at -15 °C. At the end of the addition, the
stirring was continued for 1 h. Then AgNO3 (11.0 g,
64.76 mmol) was added, and the mixture was heated at 70 °C
for 16 h. After cooling, the mixture was filtered through celite.
The filtrate was concentrated under reduced pressure, dissolved
in water (50 mL), and extracted with CH2Cl2 (3 ꢀ 100 mL).
The combined organic layers were washed with brine (50 mL),
dried, filtered, and concentrated under reduced pressure. The
crude product was purified by flash chromatography (PE/
EtOAc/HCOOH 80/20/0.1 v/v/v) to give the title compound
as white solid. Yield 60%; mp 132-133 °C (toluene). 1H NMR
(DMSO-d6) δ 3.50-3.63 (m, 2H, -SCH2-), 4.77-4.86 (m,
1H, -CHaHbONO2), 5.00-5.05 (m, 1H, -CHaHbONO2),
5.52-5.58 (m, 1H, -CH(ONO2)-), 7.53 (d, 2H, C6H4), 7.87
(d, 2H, C6H4), 13.00 (br s, 1H, -COOH). 13C NMR (DMSO-d6)
δ 30.5, 71.3, 78.6, 127.8, 128.7, 130.5, 141.1, 167.2. MS (CI) m/z
319 (M þ 1)þ.
({2-[3-(Nitrooxy)propoxy]benzoyl}oxy)methyl 2-(Acetyloxy)-
benzoate (32). Eluent (PE/EtOAc 90/10 v/v); colorless oil; yield
73%. 1H NMR (CDCl3) δ 2.21 (qi, 2H, -CH2CH2ONO2), 2.36
(s, 3H, CH3COO-), 4.12 (t, 2H, -OCH2CH2-, 3JHH=5.7 Hz),
3
4.72 (t, 2H, -CH2ONO2, JHH = 6.3 Hz), 6.16 (s, 2H,
-OCH2O-), 6.94 (d, 1H, C6H4), 7.01 (t, 1H, C6H4), 7.12
(d, 1H, C6H4), 7.33 (t, 1H, C6H4), 7.50 (t, 1H, C6H4), 7.60 (t,
1H, C6H4),7.91 (d, 1H, C6H4), 8.11 (d, 1H, C6H4). 13C NMR
(CDCl3) 20.9, 26.9, 64.3, 69.9, 79.5, 113.1, 118.6, 120.7, 122.0,
124.0, 126.2, 132.3, 132.6, 134.6, 134.7, 151.2, 158.7, 163.1,
164.4, 169.7. MS (CI) m/z 434 (M þ 1)þ.
({3-[3-(Nitrooxy)propoxy]benzoyl}oxy)methyl 2-(Acetyloxy)-
benzoate (33). Eluent (PE/EtOAc 90/10 v/v); colorless oil; yield
65%. 1H NMR (CDCl3) δ 2.23 (qi, 2H, -CH2CH2ONO2), 2.36
(s, 3H, CH3COO-), 4.12 (t, 2H, -OCH2CH2-, 3JHH=6.0 Hz),
3
4.67 (t, 2H, -CH2ONO2, JHH = 6.3 Hz), 6.19 (s, 2H,
-OCH2O-), 7.12 (d, 2H, C6H4), 7.30-7.40 (m, 2H, C6H4),

5066 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Lazzarato et al.
7.58-7.73 (m, 2H, C6H4), 7.71 (d, 1H, C6H4), 8.09 (d, 1H,
C6H4). 13C NMR (CDCl3) 21.0, 26.9, 63.8, 69.8, 79.9, 114.9,
120.7, 122.0, 123.0, 124.0, 126.2, 129.7, 130.2, 132.3, 134.7,
151.1, 158.5, 163.0, 165.0, 169.7. MS (CI) m/z 434 (M þ 1)þ.
({4-[3-(Nitrooxy)propoxy]benzoyl}oxy)methyl 2-(Acetyloxy)-
benzoate (34). Eluent (PE/EtOAc 8/2 v/v); colorless oil; yield
47%. 1H NMR (CDCl3) δ 2.23 (qi, 2H, -CH2CH2ONO2), 2.35
(s, 3H, CH3COO-), 4.12 (t, 2H, -OCH2CH2-, 3JHH=5.7 Hz),
4.66 (t, 2H, -CH2ONO2,), 6.17 (s, 2H, -OCH2O-), 6.91 (d,
2H, C6H4), 7.11 (d, 1H, C6H4), 7.32 (t, 1H, C6H4), 7.59 (t, 1H,
C6H4), 8.03-8.10 (m, 3H, C6H4). 13C NMR (CDCl3) δ 21.0,
26.8, 63.8, 69.6, 79.8, 114.2, 121.6, 122.1, 124.0, 126.1, 132.3,
132.3, 134.6, 151.1, 162.8, 163.2, 164.8, 169.7. MS (CI) m/z 434
(M þ 1)þ.
CH3COO-), 3.55 (br s, 1H, -CH2OH), 4.75 (s, 2H, -CH2OH),
6.14 (s, 2H, -OCH2O-), 7.02 (d, 1H, C6H4), 7.22 (t, 1H,
C6H4), 7.48-7.52 (m, 2H, C6H4 þ C5H3N), 7.74 (t, 1H,
C6H4), 7.90-8.00 (m, 2H, C5H3N). 13C NMR (CDCl3) δ 19.9,
63.5, 79.4, 120.8, 123.0, 123.4, 123.8, 125.2, 131.2, 133.8,
136.9, 144.6, 150.1, 160.0, 161.9, 162.6, 168.8. MS (CI) m/z
346 (M þ 1)þ.
[(4-{[3-(Nitrooxy)propyl]sulfinyl}benzoyl)oxy]methyl 2-(Acet-
yloxy)benzoate (41). A solution of 70% mCPBA (0.25 g,
1.0 mmol) in dry CH2Cl2 (7 mL) was slowly added to a solution
of 38 (0.45 g, 1.0 mmol) in dry CH2Cl2 (7 mL) and stirred at
-78 °C. At the end of the addition, the reaction was completed.
The mixture was poured in 10% Na2SO3 (50 mL), the layers
separated, and the acqueous layer was extracted twice with Et2O
(50 mL). The organic layers were dried, filtered, and concen-
trated under reduced pressure. The crude product so obtained
was purified by flash chromatography (PE/EtOAc 6/4 v/v) to
give the title compound as a colorless oil. Yield 73%. 1H NMR
(CDCl3) δ 1.97-2.05 (m, 1H, -SOCH2CHaHb-), 2.23-2.33
(m, 1H, -SOCH2CHaHb-), 2.37 (s, 3H, CH3COO-), 2.76-
2.85 (m, 1H, -SOCHaHb-), 2.98-3.07 (m, 1H, -SOCHaHb-),
4.52-4.58 (m, 2H, -CH2ONO2), 6.21 (s, 2H, -OCH2O-), 7.13
(d, 1H, C6H4), 7.33 (t, 1H, C6H4), 7.61 (t, 1H, C6H4), 7.70 (d, 2H,
C6H4), 8.10 (d, 1H, C6H4), 8.26 (d, 2H, C6H4). 13C NMR
(CDCl3) δ 19.7, 21.0, 52.3, 71.1, 79.9, 121.8, 124.1, 124.1,
126.2, 131.0, 131.6, 132.2, 134.9, 149.1, 151.2, 162.9, 164.2,
169.7. MS (CI) m/z 466 (M þ 1)þ.
({4-[2,3-Bis(nitrooxy)propoxy]benzoyl}oxy)methyl 2-(Acetyl-
oxy)benzoate (35). Eluent (PE/EtOAc 8/2 v/v); colorless oil;
yield 37%. 1H NMR (CDCl3) δ 2.36 (s, 3H, CH3COO-),
3
4.31 (d, 2H, -OCH2CH- JHH=5.4 Hz), 4.75-4.81 (m, 1H,
-CHaHbONO2), 4.90-4.96 (m, 1H, -CHaHbONO2), 5.61-
5.64 (m, 1H, -CHONO2-), 6.17 (s, 2H, -OCH2O-), 6.94
(d, 2H, C6H4), 7.11 (d, 1H, C6H4), 7.32 (t, 1H, C6H4), 7.59 (t,
1H, C6H4), 8.05-8.09 (m, 3H, C6H4). 13C NMR (CDCl3) δ 20.9,
64.7, 68.6, 76.4, 79.8, 114.2, 122.0, 122.7, 124.0, 126.1, 132.2, 132.4,
134.7, 151.0, 161.6, 163.1, 164.6, 169.7. MS (CI) m/z 495 (M þ 1)þ.
({4-[3-(Nitrooxy)propyl]benzoyl}oxy)methyl 2-(Acetyloxy)-
benzoate (36). Eluent (PE/EtOAc 9/1 v/v); colorless oil; yield
52%. 1H NMR (CDCl3) δ 2.07 (qi, 2H, -CH2CH2ONO2),
2.36 (s, 3H, CH3COO-), 2.80 (t, 2H, -CH2CH2CH2-,
[(4-{[3-(Nitrooxy)propyl]sulfonyl}benzoyl)oxy]methyl 2-(Ace-
tyloxy)benzoate (42). Oxone (0.40 g, 0.55 mmol) was added to a
stirred solution of 38 (0.10 g, 0.22 mmol) in MeOH (3 mL) and
H2O (1 mL). Two hours later, the reaction was completed, and
the mixture was diluted with H2O (10 mL) and extracted with
CH2Cl2 (3ꢀ10 mL). The combined organic layers were dried,
filtered, and concentrated under reduced pressure. The crude
product so obtained was purified by flash chromatography
(PE/EtOAc 8/2 v/v) to give the title compound as a color-
less oil. Yield 88%. 1H NMR (CDCl3) δ 2.20 (qi, 2H,
-CH2CH2ONO2), 2.36 (s, 3H, CH3COO-), 3.21 (t, 2H,
3
3JHH = 7.2 Hz), 4.44 (t, 2H, -CH2ONO2, JHH = 6.3 Hz),
6.19 (s, 2H, -OCH2O-), 7.12 (d, 1H, C6H4), 7.26-7.35 (m,
3H, C6H4), 7.59 (t, 1H, C6H4), 8.02-8.11 (m, 3H, C6H4).
13C NMR (CDCl3) δ 21.0, 28.0, 31.8, 72.0, 79.7, 122.0, 124.0,
126.1, 127.1, 128.6, 130.5, 132.3, 134.7, 146.6, 151.1, 163.1,
165.0, 169.7. MS (CI) m/z 418 (M þ 1)þ.
({4-[2,3-Bis(nitrooxy)propyl]benzoyl}oxy)methyl 2-(Acetyl-
oxy)benzoate (37). Eluent (PE/EtOAc 8/2 v/v); colorless oil;
yield 60%. 1H NMR (CDCl3) δ 2.35 (s, 3H, CH3COO-),
3.03-3.18 (m, 2H, -CH2CH-), 4.40-4.46 (m, 1H, -CHa-
HbONO2), 4.70-4.75 (m, 1H, -CHaHbONO2), 5.42-5.50
(m, 1H, -CHONO2-), 6.19 (s, 2H, -OCH2O-), 7.12 (d, 1H,
C6H4), 7.26-7.35 (m, 3H, C6H4), 7.59 (t, 1H, C6H4), 8.06-8.10
(m, 3H, C6H4). 13C NMR (CDCl3) δ 21.0, 35.6, 60.4, 70.1, 78.7,
124.5, 126.4, 128.2, 129.9, 131.3, 132.2, 135.6, 141.4, 151.9,
159.6, 161.6, 169.5, 171.2. MS (CI) m/z 479 (M þ 1)þ.
3
-SO2CH2CH2-, JHH = 7.2 Hz), 4.56 (t, 2H, -CH2ONO2,
3JHH=6.3 Hz), 6.22 (s, 2H, -OCH2O-), 7.13 (d, 1H, C6H4),
7.34 (t, 1H, C6H4), 7.61 (t, 1H, C6H4), 8.01 (d, 2H, C6H4), 8.10
(d, 1H, C6H4), 8.29 (d, 2H, C6H4). 13C NMR (CDCl3) δ 20.6,
21.0, 52.3, 70.2, 80.1, 121.7, 124.1, 126.2, 128.3, 131.1, 132.2,
134.0, 135.0, 143.1, 151.2, 162.9, 163.6, 169.7. MS (CI) m/z 482
(M þ 1)þ.
[(4-{[3-(Nitrooxy)propyl]thio}benzoyl)oxy]methyl 2-(Acetyl-
oxy)benzoate (38). Eluent (PE/EtOAc 9/1 v/v); colorless oil
that became solid on standing; yield 70%. 1H NMR (CDCl3)
δ 2.10 (qi, 2H, -CH2CH2ONO2), 2.35 (s, 3H, CH3COO-),
1-Chloroethyl 2-(Acetyloxy)benzoate (44). To a solution of 43
(10.0 g, 50.35 mmol) in dry CH2Cl2 (100 mL) stirred under N2 at
room temperature dry ZnCl2 (0.14 g) was added. After 15 min,
the reaction mixture was cooled at -15 °C and a solution of
CH3CHO (2.80 mL, 50.35 mmol) in dry CH2Cl2 (30 mL) was
slowly added. The reaction mixture was allowed to reach room
temperature and was stirred for 48 h. Then it was washed with
H2O (100 mL) and a saturated solution of NaHCO3 (100 mL),
dried, filtered, and concentrated under reduced pressure. The
crude product was purified by flash chromatography (PE/
EtOAc 90/10 v/v) to give the title compound as a colorless oil
that became solid on standing. Yield 64%; mp 42.5-45.5 °C. 1H
NMR (CDCl3) δ 1.89 (d, 3H, CH3CH-, 3JHH=6.0 Hz), 2.36 (s,
3H, CH3COO-), 6.73 (q, 1H, CH3CH-, 3JHH=6.0 Hz), 7.13
(d, 1H, C6H4), 7.33 (t, 1H, C6H4), 7.59 (t, 1H, C6H4), 8.03 (d, 1H,
C6H4). 13C NMR (CDCl3) δ 21.0, 25.3, 81.1, 122.0, 124.0, 126.1,
131.9, 134.7, 151.0, 162.0, 169.5. MS (CI) m/z 243/245 (M þ 1)þ.
1-[(4-{[3-(Nitrooxy)propyl]thio}benzoyl)oxy]ethyl 2-(Acetyloxy)-
benzoate (45). To a solution of 21 (0.50 g, 2.06 mmol) in DMF
(5 mL) Cs2CO3 (0.34 g, 1.03 mmol) was added and after 10 min
44 (0.50 g, 2.06 mmol). The mixture was stirred for 4 days and
then poured in H2O (30 mL) and extracted with Et2O (3 ꢀ
20 mL). The combined organic layers were washed with a
saturated solution of NaHCO3 (30 mL), dried, filtered, and
concentrated under reduced pressure. The crude product was
3
3.09 (t, 2H, -SCH2CH2-, JHH = 7.9 Hz), 4.57 (t, 2H,
-CH2ONO2, 3JHH=6.3 Hz), 6.18 (s, 2H, -OCH2O-), 7.12
(d, 1H, C6H4), 7.30-7.34 (m, 3H, C6H4), 7.59 (t, 1H, C6H4),
7.99 (d, 2H, C6H4), 8.08 (d, 1H, C6H4). 13C NMR (CDCl3)
δ 21.0, 26.1, 28.1, 71.0, 79.8, 122.0, 124.0, 125.9, 126.1, 126.9,
130.6, 132.3, 134.7, 143.7, 151.1, 163.1, 164.8, 169.7. MS (CI)
m/z 450 (M þ 1)þ.
[(4-{[2,3-Bis(nitrooxy)propyl]thio}benzoyl)oxy]methyl
2-(Acetyloxy)benzoate (39). Eluent (PE/EtOAc 8/2 v/v); col-
orless oil; yield 36%. 1H NMR (CDCl3) δ 2.36 (s, 3H,
CH3COO-), 3.20-3.28 (m, 1H, -SCHaHb-), 3.35-3.42
(m, 1H, -SCHaHb-), 4.63-4.69 (m, 1H, -CHaHbONO2),
4.86-4.91 (m, 1H, -CHaHbONO2), 5.28-5.36 (m, 1H,
-CHONO2-), 6.19 (s, 2H, -OCH2O-), 7.12 (d, 1H, C6-
H4), 7.33 (t, 1H, C6H4), 7.42 (d, 2H, C6H4), 7.60 (t, 1H, C6H4),
8.03-8.11 (m, 3H, C6H4). 13C NMR (CDCl3) δ 21.0, 31.4,
69.3, 77.2, 79.9, 122.0, 124.0, 126.2, 127.4, 128.3, 131.0,
132.3, 134.7, 141.1, 151.1, 163.0, 164.5, 169.7. MS (CI) m/z
511 (M þ 1)þ.
{[2-(Acetyloxy)benzoyl]oxy}methyl 6-(Hydroxymethyl)pyrid-
ine-2-carboxylate (40). Eluent (CH2Cl2/EtOAc 9/1 v/v); pale-
yellow oil; yield 17%. 1H NMR (CDCl3) δ 2.26 (s, 3H,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5067
purified by flash chromatography (PE/EtOAc 9/1 v/v) to give
the title compound as a colorless oil. Yield 25%. 1H NMR
(CDCl3) δ 1.72 (d, 3H, CH3CH-, 3JHH=5.4 Hz), 2.10 (qi, 2H,
-CH2CH2ONO2), 2.30 (s, 3H, CH3COO-), 3.10 (t, 2H,
Alto, CA) equipped with a quaternary pump (model G1311A), a
membrane degasser (G1379A), and a diode-array detector
(DAD) (model G1315B) integrated in the HP1100 system. Data
analysis was done using a HP ChemStation system (Agilent
Technologies). The injection volume was 20 μL (Rheodyne,
Cotati, CA). The analytical column was a Nucleosil 100-5C18
Nautilus (250 mm ꢀ 4.6 mm, 5 μm particle size) (Macherey-
Nagel) eluting with a flow rate of 1.2 mL/min. The samples were
analyzed using a gradient method employing a mobile phase
consisting of acetonitrile/water with 0.1% trifluoroacetic acid
55/45 over the first 4 min, grading to 70/30 to 6 min, keeping 70/
30 until 15 min and then back to 55/45 to 20 min. The column
effluent was monitored at 226 nm (for compounds, aspirin, and
nitrooxy-substituted carboxylic acids) and at 240 nm (for sali-
cylic acid and salicylates) referenced against a 600 nm wave-
length. Quantitation was done by comparison of peak areas with
standards chromatographed under the same conditions.
Inhibition of Platelet Aggregation in Vitro. Venous blood
samples were obtained from healthy volunteers who had not
taken any drug for at least two weeks. Volunteers, who were
treated according to Helsinki protocol for biomedical experi-
mentation, gave their informed consent to the use of blood
samples for research purposes. Platelet-rich plasma (PRP) was
prepared by centrifugation of citrated blood at 210g for 20 min.
Aliquots (500 μL) of PRP were added into aggregometer
(Chrono-log 4902D) cuvettes, and aggregation was recorded
as increased light transmission under continuous stirring
(1000 rpm) at 37 °C for 10 min after addition of the stimulus.
Collagen at submaximal concentration (0.8-1.5 μg/mL) was
used as a platelet activator in PRP. Compounds under study
were preincubated with PRP 10 min before addition of the
stimulus (collagen). Vehicle alone (0.5% DMSO) added to PRP
did not affect platelet function in control samples. At least five
experiments for each compound were performed.
The antiaggregatory activity of tested compounds is evalu-
ated as % inhibition of platelet aggregation compared to control
samples. For most active compounds, IC50 values could be
calculated by nonlinear regression analysis, otherwise % inhibi-
tion at maximal concentration tested (300 μM) is reported.
Vasodilator Activities. Thoracic aortas were isolated from
male Wistar rats weighing 180-200 g. As few animals as
possible were used. The purposes and the protocols of our
studies have been approved by the Ministero della Salute,
Rome, Italy. The endothelium was removed, the vessels were
cut helically, and three strips were obtained from each aorta.
The tissues were mounted under 1.0 g tension in organ baths
containing 30 mL of Krebs-bicarbonate buffer with the follow-
ing composition (mM): NaCl 111.2, KCl 5.0, CaCl2 2.5, MgSO4
1.2, KH2PO4 1.0, NaHCO3 12.0, glucose 11.1, maintained at
37 °C and gassed with 95% O2 - 5% CO2 (pH=7.4). The aortic
strips were allowed to equilibrate for 1.5 h and then contracted
with 1 μM L-phenylephrine. When the response to the agonist
reached a plateau, cumulative concentrations of the vasodilat-
ing agent were added. Results are expressed as EC50 ( SEM
(μM). The effects of 1 μM ODQ on relaxation were evaluated in
a separate series of experiments in which it was added 5 min
before the contraction. With this protocol, the inhibitor is
preincubated for at least 30 min before the addition of the
vasodilator compound. Responses were recorded by an iso-
metric transducer connected to the MacLab System PowerLab.
Addition of the drug vehicle, DMSO, had no appreciable effect
on contraction level. At least five experiments for each com-
pound were performed.
3
3
-SCH2-, JHH = 6.9 Hz), 4.58 (t, 2H, -CH2ONO2, JHH
6.0 Hz), 7.11 (d, 1H, C6H4), 7.27-7.34 (m, 4H, C6H4
=
þ
CH3CH-), 7.58 (t, 1H, C6H4), 7.97 (d, 2H, C6H4), 8.04 (d,
1H, C6H4). 13C NMR (CDCl3) δ 19.8, 21.0, 26.1, 28.2, 71.0,
89.6, 122.5, 123.9, 126.1, 126.4, 127.0, 130.5, 132.0, 134.4, 143.3,
150.9, 162.4, 164.0, 169.6. MS (CI) m/z 463 (M þ 1)þ.
{[2-(Acetyloxy)benzoyl]oxy}methyl 6-[(Nitrooxy)methyl]pyri-
dine-2-carboxylate (46). A solution of 40 (0.10 g, 0.29 mmol) in
(CH3CO)2O (0.30 mL) was slowly added to a mixture of 65%
HNO3 (0.10 mL) and (CH3CO)2O (0.20 mL) and stirred at 0 °C.
Then the reaction mixture was allowed to reach room tempera-
ture, and the stirring was continued for 2 h. The mixture was
poured into H2O (10 mL) and extracted with CH2Cl2 (5ꢀ5 mL).
The organic layers were dried, filtered, and concentrated under
reduced pressure. The crude product so obtained was purified by
flash chromatography (CH2Cl2/EtOAc 95/5 v/v) to give the title
compound as a colorless oil that became solid on standing;
mp 73-78 °C; yield 50%. 1H NMR (CDCl3)
δ 2.37
(s, 3H, CH3COO-), 5.67 (s, 2H, -CH2ONO2), 6.26 (s, 2H,
-OCH2O-), 7.13 (d, 1H, C6H4), 7.33 (t, 1H, C6H4), 7.58-7.64
(m, 2H, C5H3N), 7.93 (t, 1H, C6H4), 8.09-8.17 (m, 2H, C6H4 þ
C5H3N). 13C NMR (CDCl3) δ 21.0, 73.9, 80.5, 121.8, 124.0,
125.4, 125.6, 126.2, 132.3, 134.8, 138.4, 147.0, 151.2, 153.8,
162.8, 163.2, 169.7. MS (CI) m/z 391 (M þ 1)þ.
({2-[3-(Nitrooxy)propoxy]benzoyl}oxy)methyl 2-(Hydroxy-
benzoate (47). 6 M HCl (4 mL) was added to a solution of 32
(0.20 g, 0.46 mmol) in 1,4-dioxane (4 mL) and the mixture was
heated at 70 °C for 6 h. Then it was poured in H2O (20 mL) and
extracted with CH2Cl2 (3 ꢀ 30 mL). The combined organic
layers were dried, filtered, and concentrated under reduced
pressure. The crude product was purified by flash chroma-
tography (PE/EtOAc 90/10 v/v) to give the title compound as
1
a colorless oil; yield 78%. H NMR (CDCl3) δ 2.22 (qi, 2H,
-CH2CH2ONO2), 4.13 (t, 2H, -OCH2CH2-, 3JHH=5.7 Hz),
3
4.72 (t, 2H, -CH2ONO2, JHH = 6.0 Hz), 6.23 (s, 2H,
-OCH2O-), 6.87-7.04 (m, 4H, C6H4), 7.46-7.54 (m, 2H,
C6H4), 7.89-7.93 (m, 2H, C6H4), 10.46 (s, 1H, OH). 13C
NMR (CDCl3) δ 26.9, 64.2, 69.8, 79.5, 111.4, 113.1, 117.7,
118.3, 119.5, 120.7, 130.3, 132.6, 134.8, 136.6, 158.8, 162.0,
164.2, 169.0. MS (CI) m/z 392 (M þ 1)þ.
Evaluation of Stability in Buffered Solutions and in Human
Serum. Hydrolysis in Acidic Medium (pH 1.0) and in Phosphate
Buffer (pH 7.4). A 2 mL aliquot of 0.5 mM solution of each
compound in acetonitrile was diluted to 10 mL using HCl to
reach pH 1.0 or phosphate buffer 50 mM to obtain pH 7.4. The
resulting solution was maintained at 37 ( 0.5 °C and at appro-
priate time intervals a 20 μL aliquot of reaction solution was
analyzed by RP-HPLC. All experiments were performed in
triplicate.
Hydrolysis in Human Serum. A solution of each compound
(10 mM) in acetonitrile was added to human serum (sterile-
filtered from human male AB plasma, Sigma-Aldrich) pre-
heated at 37 °C; the final concentration of the compound was
250 μM. The resulting solution was incubated at 37 ( 0.5 °C and
at appropriate time intervals 300 μL of the reaction mixture was
withdrawn and added to 450 μL of acetonitrile containing 0.1%
trifluoroacetic acid in order to deproteinize the serum. The
sample was sonicated, vortexed, and then centrifuged for 10
min at 2150g, and the clear supernatant was filtered by 0.45 μm
PTFE filters (Alltech) and analyzed by RP-HPLC. All experi-
ments were performed at least in triplicate.
The reverse-phase HPLC procedure allowed separation and
quantitation of the remaining compound and of the products of
hydrolysis (aspirin, salicylic acid, salicylate, and nitrooxy-sub-
stituted carboxylic acid). HPLC analyses were performed with a
HP 1100 chromatograph system (Agilent Technologies, Palo
Acknowledgment. This work was supported by a MIUR
grant (COFIN 2005).
Supporting Information Available: Elemental analyses. This
material is available free of charge via the Internet at http://
pubs.acs.org.

5068 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Lazzarato et al.
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