Searching for new NO-donor aspirin-like molecules: A new class of nitrooxy-acyl derivatives of salicylic acid

Article abstract of DOI:10.1021/jm701104f

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

Full text of DOI:10.1021/jm701104f

1894
J. Med. Chem. 2008, 51, 1894–1903
Searching for New NO-Donor Aspirin-like Molecules: A New Class of Nitrooxy-acyl Derivatives
of Salicylic Acid†
Loretta Lazzarato,‡ Monica Donnola,‡ Barbara Rolando,‡ Elisabetta Marini,‡ Clara Cena,‡ Gabriella Coruzzi,§ Elena Guaita,§
Giuseppina Morini,§ Roberta Fruttero,‡ Alberto Gasco,*,‡ Stefano Biondi,| and Ennio Ongini|
Dipartimento di Scienza e Tecnologia del Farmaco, UniVersità degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy, Dipartimento di
Anatomia Umana, Farmacologia e Scienze Medico-Forensi, Sezione di Farmacologia, UniVersità degli Studi di Parma, Via Volturno 39,
40100 Parma, Italy, and Nicox Research Institute, Via Ariosto 21, 20091, Bresso (Milano), Italy
ReceiVed September 5, 2007
A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing
nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable
in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent
upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin
when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed
reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to
inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were
capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner.
Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer
alternatives to aspirin in different clinical settings.
Introduction
(-ONO2). Prototypes of such structures 2 (NCX4040), 3
(NCX4016), and 4 (NCX4060) are reported in Chart 1.
Subsequently, a second family of NO-donor aspirins 5 (Chart
1) was developed,9 which, despite a close similarity with the
first-generation molecules, contains furoxan derivatives as NO-
donor groups. Unlike the previous NO-donor aspirins that seem
to require enzymatic metabolism for NO release,10–12 these
products proved to release NO under the action of thiols.13
Intracellular release induced by glutathione is potentiated by
ascorbic acid.14 Recently, an additional class of NO-donor
NSAIDs, including aspirins 6 (Chart 1), has been proposed.
They have moieties, containing an ONN(O)N substructure,
linked to the carboxy group through a methylene bridge.15 In
serum, these compounds are metabolized to a mixture of
products, including N-diazeniumdiolate anions, which are able
to release NO spontaneously.
It is becoming more and more evident that NO-donor aspirins
display a number of effects exceeding their original intended
use. They display a variety of actions, including anti-inflam-
matory and analgesic effects, antiplatelet and vasodilator proper-
ties, and beneficial effects in the treatment of restenosis and
myocardial ischemia. In addition, a number of them proved to
be more potent than aspirin in inhibiting the proliferation of
cancer cell lines, including prostate cancer cells.16–19 Thus, 3,
the prototype of NO-donor aspirins, was found to enhance the
preventive and therapeutic effectiveness of the antitumor
immunity elicited by cancer vaccination.20 Finally, a possible
use of NO aspirins in the treatment of type-2 diabetes has been
claimed.18 The mechanism of action of NO aspirins is still under
investigation, also taking into account that the simple esters of
aspirin and, in particular, 3 are often not true aspirin prodrugs.
Some of them are rapidly metabolized, with little or no
formation of aspirin, when incubated in serum, plasma, and rat
liver subcellular fractions.9,12 This behavior is in keeping with
the knowledge that the loss of the negative charge by the aspirin
molecule, because of the esterification of the COOH group (pKa
) 3.5), makes the acetoxy moiety extremely susceptible to
enzymatic cleavage.21 Interestingly, it was recently found that
Aspirin (1) (Chart 1) is a well-established drug, belonging
to the class of nonsteroidal anti-inflammatory drugs (NSAIDs).
These products are widely used because of their anti-inflam-
matory, analgesic, antipyretic, and platelet anti-aggregatory
properties. The pharmacological basis of these beneficial effects
are related, at least in part, to their ability to inhibit two
cyclooxygenase isoenzymes, COX-1 and COX-2, which are
involved in the production of prostanoids from arachidonic
acid.1,2 Aspirin is unique among NSAIDs because it modifies
both isoforms covalently by acetylating a serine residue (Ser530
)
positioned in the arachidonic acid-binding channel of the
enzyme.3 It is considered a nonselective COX inhibitor, although
it displays a somewhat higher inhibitor potency in COX-1
assays. The major drawback of the drug is a significant
gastrotoxicity that is responsible for gastric ulceration, exacerba-
tion of peptic ulcer symptoms, gastrointestinal hemorrhage,
erosive gastritis, and in some cases, death.1,2,4 Nitric oxide (NO)-
releasing aspirins are an interesting class of products that were
originally designed to reduce gastrotoxicity of the parent
aspirin.5,6 They are hybrid drugs that combine aspirin properties
with gastroprotection exerted by NO, which they are able to
release. NO-induced gastroprotection occurs through a number
of mechanisms, including an increase of gastric microcirculation
and mucous and bicarbonate secretion, as well as the inhibition
of neutrophil adhesion to vascular endothelium of gastric
microcirculation.7,8 These products were originally obtained by
joining, through a simple ester bridge, the carboxylic group of
aspirin with moieties containing NO-donor nitrooxy groups
†
A portion of this work was presented as an invited lecture at the 41st
International Union of Pure and Applied Chemistry (IUPAC) World
Chemistry Congress, August 5–11, 2007, Turin, Italy.
* To whom correspondence should be addressed. Telephone: +39-011-
670-7670. Fax: +39-011-670-7286. E-mail: alberto.gasco@unito.it.
‡
Università degli Studi di Torino.
Università degli Studi di Parma.
Nicox Research Institute.
§
|
10.1021/jm701104f CCC: $40.75
2008 American Chemical Society
Published on Web 02/23/2008

Nitrooxy-acyl DeriVatiVes of Salicylic Acid
Chart 1. Examples of NO-Donor Aspirin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1895
Chart 2. NO-Donor “Aspirin-like” Compounds
gave 25 that yielded the target model 33 after subsequent
oxidation with KMnO4. The remaining mononitrooxy derivative
28 was prepared by direct coupling of 27 with salicylic acid in
dry tetrahydrofuran (THF), in the presence of dry pyridine. The
preparation of the dinitrooxy-substituted final products 51–55
is reported in Scheme 2. Two of them required the dinitrooxy-
substituted alkanoic acids 35 and 36 as starting materials. The
former is a product known in the literature,24 while the latter
was prepared by action of I2 and AgNO3 in acetonitrile solution
on hept-6-enoic acid (34). These products were transformed into
the corresponding acyl chlorides 37 and 38 and then coupled
with salicylaldehyde to give 48 and 49. Oxidation of these
intermediates afforded the final target compounds 53 and 54.
All of these reactions were carried out under conditions similar
to those described for the preparation of the mononitrooxy-
substituted product 33 from 23. To prepare 51, 52, and 55, the
unsaturated acids 39–41 were used as starting materials. Acids
39 and 41 were transformed in the corresponding active
intermediates through the reaction with N,N′-dicyclohexylcar-
bodiimide (DCC); these intermediates were then coupled to
salicylaldehyde in dry CH2Cl2 solution in the presence of 4-N,N′-
dimethylaminopyridine (DMAP) to give 43 and 45. To prepare
aldehyde 44, the best results were obtained transforming 40 into
the corresponding acyl chloride 42 and then coupling 42 with
salicylaldehyde, under the conditions already described. The
unsaturated aldehydes 43–45 were transformed into the corre-
sponding dinitrooxy derivatives 46, 47, and 50 by action of I2
and AgNO3 in CH3CN solution. The aldehydes 47 and 50
afforded the desired final products by usual oxidation with
KMnO4, while aldehyde 46 was oxidated using mild conditions
(NaClO2 and H2O2).
neither aspirin nor NO contributes to the antitumor effect of 2,
4, which, in contrast, is due to the quinone methides formed
after carboxylic ester hydrolysis.22,23 As a development of our
work in this area, we designed a new class of NO-donor
“aspirin-like” compounds (Chart 2). These products are formally
derived from aspirin by substituting acyl groups containing
nitrooxy NO-donor moieties for the acetyl group. They are not
prodrugs of aspirin but true aspirin/NO-donor hybrids. All of
the NO-donor aspirins known thus far were designed by merging
NO-donor moieties at the carboxylic site of aspirin. In this paper,
we describe the synthesis, stability in different buffer solutions
and human serum of these products, as well as preliminary
pharmacological characterization including in vivo anti-inflam-
matory, gastrosparing properties, in vitro anti-aggregatory
activity, and cGMP-dependent vasodilation.
Chemistry
Mononitrooxy-substituted compounds were synthesized ac-
cording to the pathways reported in Scheme 1. The preparation
of the products 29–32 was carried out starting from the bromo-
substituted alkanoic acids 7–10. These starting materials were
transformed into the corresponding acyl chlorides 11–14 by
SOCl2 in dry CH2Cl2, in the presence of a few drops of dry
N,N-dimethylformamide (DMF). The acyl chlorides were con-
jugated to salicylaldehyde in dry CH2Cl2, in the presence of
dry pyridine to give 15–18. The action of AgNO3 on these
intermediates in refluxing acetonitrile afforded the corresponding
mononitrooxy-substituted aldehydes 19–22 that gave rise to the
final compounds 29–31, under the action of KMnO4. For the
final compound 32, mild oxidative reagents (NaClO2 and H2O2)
were used, because better yields were obtained. The preparation
of the target products 28 and 33 was carried out starting from
the nitrooxy-substituted alkanoic acids 23 and 26. These acids
were transformed into the corresponding acyl chlorides 24 and
27 by the action of SOCl2. Coupling of 24 with salicylaldehyde
under the same conditions used to prepare the aldehydes 15–18
Results and Discussion
Stability in Aqueous Buffer Solutions and Human
Serum. The stability of all of the final products was studied by
high-performance liquid chromatography (HPLC) in aqueous
buffer solutions of pH 1 and 7.4 as well as in human serum
(Table 1). Salicylic acid (56) and the nitrooxyalkanoic acids
were the only transformation products. In an acidic medium,
the stability, after 3 h of incubation, ranged from 85 to 97%.
At pH 7.4, most of the products showed a stability >90%, with
the only exception of products 28 and 51, for which only 55
and 20% remained unchanged over the same time period. A
different situation occurred when the compounds were incubated

1896 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Lazzarato et al.
Scheme 1a
a Reagents and conditions: (i) SOCl2, dry CH2Cl2, dry DMF, (ii) salicylaldehyde, dry CH2Cl2, dry Py, (iii) AgNO3, CH3CN, 70 °C, (iv) KMnO4, acetone
for n ) 1–4 and R ) H and CH3; 80% NaClO2, 30% H2O2, CH3CN for n ) 5 and R ) H, (v) SOCl2, dry THF, dry DMF, and (vi) salicylic acid, dry THF,
dry Py.
in serum, in which they can undergo enzymatic metabolism.
All of them were hydrolyzed, following a first-order kinetic
mechanism. In Figure 1, the hydrolysis in serum of 31 is
reported as an example. The observed pseudo-first-order rate
constants (kobs) for the hydrolysis were calculated from the
slopes of linear plots of the logarithm of the remaining ester
against time; the corresponding half-lives (t1/2) were obtained
from eq 1 (Table 1).
anti-inflammatory effects comparable to those caused by aspirin,
belong to both the class of mononitrooxy derivatives (com-
pounds 30, 31, and 33) and that of dinitrooxy derivatives
(compounds 52–54).
As aforementioned, the anti-inflammatory effects of NSAIDs
can be partly explained by the inhibition of prostanoid produc-
tion following the inhibition of COX enzymes. More recently,
it was found that the irreversible acetylation of COX-2 isoform
by aspirin may contribute to its anti-inflammatory activity,
through the synthesis of aspirin-triggered lipoxins (ATLs).27
Because the drug is rapidly deacetylated under the action of
esterases present in blood, intestinal mucosa, and tissues, such
as liver and kidney,28,29 it has been proposed that salicylic acid
may also contribute to its anti-inflammatory action.30 It is known
that rodents (rats and guinea pigs) tend to metabolize ester-
containing drugs, including aspirin, much faster than humans.25,28
Furthermore, NO may display anti-inflammatory actions of its
own when produced in the appropriate amounts.31–33 In
particular, both NO and salicylate are able to inhibit NF-κB, a
transcription factor involved in the synthesis of inflammatory
cytokines, cytokine receptors, and adhesion molecules in a
variety of cells.30,34–37 Actually, 3 was shown to inhibit this
factor and suppress the processing of IL-1ꢀ and IL-18, two
inflammatory cytokines, by inhibiting caspase-1 activity.17,38
Studies dedicated to this question are necessary to clarify
whether the mechanisms underlying the in vivo anti-inflamma-
tory properties of this new class of NSAIDs involve combina-
tions of COX effects of the native products, the metabolite
salicylic acid, and possibly NO release.
t1/2 ) 0.693/kobs
(1)
Analysis of these parameters shows that they are influenced
by the structure of the ester chain. In the mononitrooxy series,
some products are less stable than aspirin, while others as stable
as or more stable than it. The stability increases with the length
of the chain, but when the number of carbons become >6
(compound 32), it decreases. The presence of two methyl groups
at the R position of the ester function (compound 33) induces
strong stabilization. A similar situation occurs in the dinitrooxy
series, but all of these compounds are definitively more stable
than the related monosubstituted analogues. This picture might
be justified by a different ability of the models to interact with
the esterase enzyme and/or bind with plasma proteins, following
their different lipophilicity and stereoelectronic properties.25,26
Antiinflammatory Activity. All of the products, including
aspirin (1) and salicylic acid (56) as references, were tested on
carrageenan-induced paw edema in conscious rats. The injection
of carrageenan into rat hind paw produced an immediate paw
swelling, which reached a peak at 4–5 h. Aspirin, administered
by intragastric route at 120 mg/kg, just prior to carrageenan
injection, significantly reduced (48.1 ( 2.9%) paw edema at
3 h, when compared to vehicle-treated animals (Figure 2). Also,
a number of the new NO-donor products were able to reduce
paw edema in a significant or highly significant manner, when
administered intragastrically at a dose equimolar to aspirin (120
mg/kg; Figure 2). The most active compounds, which induce
Acute Gastric Mucosal Damage. All of the compounds,
including aspirin (1) and salicylic acid (56) as references, were
assessed for their ulcerogenic properties in conscious rats. The
development of gastric lesions was assessed 3 h after intragastric
administration of the compounds, and lesions were quantified
by determining the “lesion index” on the basis of their greatest

Nitrooxy-acyl DeriVatiVes of Salicylic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1897
Scheme 2a
a Reagents and conditions: (i) I2, AgNO3, CH3CN room temperature and then AgNO3, CH3CN reflux, (ii) SOCl2, dry CH2Cl2, dry DMF, (iii) salicylaldehyde,
dry CH2Cl2, dry Py, (iv) DCC, dry CH2Cl2 and then salicylaldehyde, dry CH2Cl2, DMAP, and (v) KMnO4, acetone for n ) 1–3 and R ) H and CH3; 80%
NaClO2, 30% H2O2, CH3CN for n ) 0 and R ) H.
Table 1. Stability in Aqueous Buffers and Human Serum, Anti-aggregatory, and Vasodilator Activity of the Products 28–33 and 51–55
stability
aqueous buffers
percent unchanged at 3 h (%)
anti-aggregatory activity
vasodilator activity
human serum
t1/2 (h) ( SD
IC50 (CL 95%) (µM)
[+50 µM ODQ]
percent inhibition (
SEM at 300 µMa
compound
pH 1.0
pH 7.4
EC50 (µM) ( SEM
1
28
>90
>90
>90
1.06 ( 0.08
0.26 ( 0.01
54 (49–60)
162 (129–204)
[186 (172–200)]
30 (24–37)
[24 (17–34)]
97 (85–110)
[91 (76–110)]
a
inactive
a
126 (98–160)
[119 (105–134)]
a
55
37 ( 2b
23 ( 6b
21 ( 2b
29
30
>97
>97
>95
>95
0.47 ( 0.01
0.76 ( 0.01
31
32
33
51
>97
>97
>97
>85
>95
>95
>95
20
2.03 ( 0.11
1.19 ( 0.08
9.03 ( 0.22
0.28 ( 0.01
27 ( 10
26 ( 6
14 ( 1b
8.1 ( 1.4b
6.2 ( 0.9b
9.2 ( 0.9b
52
53
54
55
>85
>85
>85
>97
>95
>95
>95
>95
4.09 ( 0.08
4.87 ( 0.07
6.25 ( 0.17
15.2 ( 0.40
15 ( 8
6.7 ( 5.8
8.2 ( 1.2b
5.8 ( 0.7b
3.1 ( 0.7b
5.3 ( 0.6b
a
inactive
inactive
a Because of the low activity of the compound, IC50 could not be calculated. In this case, the percent of inhibition is reported at 300 µM. b In the presence
of 1 µM ODQ, EC50 values were >100 µM.
length in millimeters (Figure 3). Aspirin displays strong
gastrotoxicity despite its ability to generate ATLs, which are
endowed with gastroprotective effects as well.39 In the present
study, aspirin, administered at 120 mg/kg, produced macro-
scopically detectable gastric damage, characterized by mucosal
necrosis and hemorrhage (lesion index ) 53.5 ( 5.2%). All of
the NO-donor products described here displayed greatly reduced
gastrotoxicity when administered at a dose equimolar to aspirin.
There are two effects that contribute to gastrotoxicity of
conventional acidic NSAIDs: the systemic and local irritant

1898 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Lazzarato et al.
a role of NO release in the reduced gastrotoxicity, it must be
remembered that these products are metabolized to salicylic acid,
which is unable to induce gastric mucosal damage (see Figure
3 and ref 30).
Platelet Anti-aggregatory Activity. Anti-aggregatory effects
of the compounds were studied on collagen-induced platelet
aggregation of human platelet-rich plasma (PRP). It is known
that collagen-induced aggregation occurs through a pathway
dependent upon the arachidonic acid cascade.43 The results
expressed as IC50 or, when IC50 could not be calculated, as a
percentage of inhibition at the maximal concentration tested are
reported in Table 1. Analysis of the data show that the most
active compounds were 29 > 30 > 28, belonging to the
mononitrooxy series and the dinitrooxy-substituted model 51.
To determine whether NO-mediated stimulation of soluble
guanylate cyclase (sGC) was involved in their anti-aggregatory
action, these products were also tested in parallel in the presence
of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a well-
known inhibitor of sGC. The ineffectiveness of ODQ on the
anti-aggregatory activities of compounds 28–30 and 51 excludes
any involvement of NO-mediated stimulation of sGC in keeping
with the known inability of platelets to effect NO release from
organic nitrates.14,44 This strengthens the possibility that platelet
COX-1 inhibition is the main underlying mechanism.
Figure 1. Time courses for compound 31 and salicylic acid 56 in
human serum at 37 °C. Values are mean ( SEM (SEM e 2; number
of determinations g 4).
Vasodilator Activity. It is known that organic nitrates display
vasodilator activities. The generally accepted mechanism of this
action involves their conversion in vascular smooth-muscle cells
into NO with consequent activation of the sGC. In turn, sGC
increases the intracellular levels of cyclic guanosine-3,5-
monophosphate (cGMP), with consequent activation of cGMP-
dependent protein kinase (cGK-I) and vasodilation.45 To
highlight the NO-releasing activity of the compounds described
in the present work, their vasodilatory effects were evaluated
on denuded rat aorta strips precontracted with phenylephrine.
All of the products were capable of relaxing the contracted tissue
in a concentration-dependent manner, in keeping with the
presence in the structures of NO-donor nitrooxy functions. The
vasodilator potencies, expressed as EC50, are shown in Table
1. In the mononitrooxy series, the potency increases with the
length of the linear lateral chain. The most active term was the
branched product 33, which bears two methyl groups at the R
carbon to ester function. Similar behavior is shown by the
dinitrooxy-substituted compounds, but in this series, the branched
compound 55 is less active than 54, containing the longest linear
lateral chain, and as active as 53, which is homologous,
immediately inferior to 54. With the length of the chain being
equal, the dinitrooxy-substituted compounds are always more
active than the corresponding mononitroxy analogues, in keeping
with the statement that the number of nitrate groups determines
reactivity and potency of organic nitrates.46 When the vasodilator
experiments were repeated in the presence of 1 µM ODQ, a
decrease in the potencies was observed, in keeping with NO-
induced activation of the sGS.
Figure 2. Anti-inflammatory effects of aspirin (1), salicylic acid (56)
and NO-donor aspirin-like molecules (28–33 and 51–55) on carrag-
eenan-induced paw edema in conscious rats. The aspirin-like com-
pounds were administered by intragastric route at a dose equimolar to
aspirin, 120 mg/kg, at the same time as carrageenan, and their effects
were evaluated 3 h later. Results are expressed as a percentage of
inhibition of edema observed in the vehicle-treated group. This edema
was considered arbitrarily as 100. (/) p < 0.05. (//) p < 0.01 versus
vehicle (ANOVA, followed by Dunnett test). Values are mean ( SEM
(n ) 8–10 rats per group).
Figure 3. Gastric ulcerogenic effects of aspirin (1), salicylic acid (56),
and NO-donor aspirin-like molecules (28–33 and 51–55) in conscious
rats. The aspirin-like compounds were administered by intragastric route
at a dose equimolar to aspirin, 120 mg/kg, and the stomachs were
examined 3 h later. Gastric lesions were measured along the greatest
length, and the cumulative length in millimeters was designated as the
“lesion index” for each stomach. All of the compounds tested produced
significantly less gastric damage than aspirin (//) p < 0.01; (ANOVA
and Newman-Keuls test). Values are mean ( SEM (n ) 8–10 rats
per group).
Conclusions
effects.40,41 The former is dependent upon COX inhibition, in
particular, the COX-1 isoform in gastric epithelial cells. The
latter is the result of a number of events, including perturbation
of physicochemical properties of phospholipids and “ion trap-
ping” of the drugs into surface epithelial cells. These two events
are tightly linked to the pKa and the lipophilicity of acid
NSAIDs.42 From this point of view, the new products developed
here show acid properties (pKa ) 3.85–3.69) close to that of
aspirin and greater lipophilicity (log P ) 1.60–3.22; log Paspirin
) 1.14) (unpublished data). While it is plausible to hypothesize
We have been able to successfully prepare a new class of
“aspirin-like” products obtained through a novel approach,
which implies the merging of nitrooxy-acyl moieties at the
phenol site of salicylic acid. Generally speaking, these com-
pounds are stable in acidic media and at physiological pH, but
they are hydrolyzed when incubated in serum. This new class
of products displays anti-inflammatory activity in the carrag-
eenan-induced paw edema test, with several compounds being
as potent as aspirin; nevertheless, they show reduced or no

Nitrooxy-acyl DeriVatiVes of Salicylic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1899
-CHO). 13C NMR (CDCl3) δ: 23.8, 27.6, 32.4, 33.5, 34.0, 123.5,
126.4, 128.1, 131.0, 135.3, 151.5, 171.7, 188.8. MS (CI) m/z: 299/
301 (M + 1)+.
gastrotoxicity when compared to this lead. Several components
may contribute to their in vivo behavior, including salicylic acid
formation, NO release, and variable linkers. Furthermore, some
of them are able to block platelet aggregation induced by
collagen in human plasma-rich platelets. All of the products
trigger cGMP-dependent vasodilator actions when tested on
precontracted rat aorta strips, according to the presence in their
structures of nitrooxy NO-donor moieties. Additional pharma-
cological and biochemical studies, mainly addressing COX-1/
COX-2 inhibition, are necessary to highlight action mechanisms
of these products and understand whether selected members of
this new class of NSAIDs might represent possible safer
alternatives to aspirin in different clinical settings.
2-Formylphenyl 7-Bromoheptanoate (18). Eluent [95:5 PE/
1
EtOAc (v/v)]. Pale yellow oil. Yield 62%. H NMR (CDCl3) δ:
1.47–1.55 (m, 4H), 1.78–1.96 (m, 4H, -CH2CH2CH2CH2CH2Br),
3
2.70 (t, 2H, -OCOCH2-, JHH ) 7.3 Hz), 3.45 (t, 2H, -CH2Br,
3JHH ) 6.7 Hz), 7.19 (d, 1H, C6H4), 7.42 (t, 1H, C6H4), 7.65 (t,
1H, C6H4), 7.90 (d, 1H, C6H4), 10.12 (s, 1H, -CHO). 13C NMR
(CDCl3) δ: 24.4, 27.8, 28.2, 32.5, 33.8, 33.9, 123.5, 126.4, 128.1,
131.2, 135.3, 151.6, 171.9, 188.7. MS (CI) m/z: 313/315 (M +
1)+.
2-Formylphenyl 2,2-Dimethyl-3-(nitrooxy)propanoate (25). In
this case, the formation of acyl chloride and the next synthetic step
are very slow, and 1 week of stirring was required. Eluent [90:10
1
PE/EtOAc (v/v)]. Pale yellow oil. Yield 45%. H NMR (CDCl3)
Experimental Section
δ: 1.51 (s, 6H, -CH3), 4.69 (s, 2H, -CH2ONO2), 7.15 (d, 1H,
C6H4), 7.43 (t, 1H, C6H4), 7.64 (t, 1H, C6H4), 7.88 (d, 1H, C6H4),
10.07 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 22.4, 42.7, 77.5, 123.3,
126.8, 128.2, 131.8, 135.3, 151.0, 172.8, 188.5. MS (CI) m/z: 268
(M + 1)+.
2-Formylphenyl Pent-4-enoate (44). Eluent [95:5 PE/EtOAc
(v/v)]. Pale yellow oil, immediately used in the next synthetic step.
Yield 65%.
2-Formylphenyl 5,6-Bis(nitrooxy)hexanoate (48). Eluent [90:
10 f 80:20 PE/EtOAc (v/v)]. Pale yellow oil. Yield 56%. 1H NMR
(CDCl3) δ: 1.89–1.98 (m, 4H, -CH2CH2CH2CH-), 2.74–2.78 (m,
2H, -OCOCH2-), 4.49–4.55 (dd, 1H, AMX-like system, -CHaH-
bONO2), 4.73–4.83 (dd, 1H, AMX-like system, -CHaHbONO2),
5.36–5.39 (m, 1H, AMX-like system, -CHONO2), 7.17 (d, 1H,
C6H4), 7.44 (t, 1H, C6H4), 7.66 (t, 1H, C6H4), 7.86 (d, 1H, C6H4),
10.02 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 19.9, 28.5, 33.0, 71.1,
78.8, 123.5, 126.7, 128.0, 133.0, 135.4, 150.6, 171.1, 189.3. MS
(CI) m/z: 343 (M + 1)+.
Synthesis. 1H and 13C nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker Avance 300 at 300 and 75 MHz,
respectively, using SiMe4 as the internal standard. Low-resolution
mass spectra were recorded with a Finnigan-Mat TSQ-700. Melting
points were determined with a capillary apparatus (Büchi 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 × 20 cm plates, with a layer
thickness of 0.2 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 × 25 mm, 10 µm) (Merck
Darmstadt, Germany) with a Varian ProStar mod-210 with Varian
UV detector mod-325. Elemental analyses (C, H, and N) were
performed by REDOX (Monza), and the results are within ( 0.4%
of the theoretical values. Compounds 8,47 10,48 23,49 26,50 35,24
and 4151 were synthesized according to the literature.
2-Formylphenyl Hept-6-enoate (49). Eluent [85:15 PE/EtOAc
(v/v)]. Pale yellow oil. Yield 58%. 1H NMR (CDCl3) δ: 1.55–1.67
(m, 2H), 1.80–1.90 (m, 4H, -CH2CH2CH2CH2CH-), 2.71 (t, 2H,
General Procedure for the Preparation of 15, 16, 17, 18,
25, 44, 48, and 49. SOCl2 (6.55 mmol) and a few drops of dry
DMF were added to a solution of the appropriate carboxilic acid
7, 8, 9, 10, 23, 35, 36, and 40 (5.46 mmol) in dry CH2Cl2 (15 mL),
stirred under N2 at room temperature. The stirring was continued
for 2 h at room temperature. The solution of the acyl chloride thus
obtained was slowly added to a stirred solution of salicylaldehyde
(4.37 mmol) and dry Py (8.19 mmol) in dry CH2Cl2 (10 mL) kept
under N2 at 0 °C. The reaction was allowed to reach room
temperature and then stirred for 2.5 h. Then, the mixture was washed
with 2 M HCl (3 × 10 mL). The combined organic layers were
dried, filtered, and concentrated under reduced pressure. The crude
product thus obtained was purified by flash chromatography.
Chromatographic eluents and yields of the products were as follows.
2-Formylphenyl 4-Bromobutanoate (15). Eluent [90:10 PE/
3
-OCOCH2-, JHH ) 7.2 Hz), 4.50 (dd, 1H, AMX-like system,
-CHaHbONO2), 4.78 (dd, 1H, AMX-like system, -CHaHbONO2),
5.29–5.37 (m, 1H, AMX-like system, -CHONO2), 7.17 (d, 1H,
C6H4), 7.43 (t, 1H, C6H4), 7.65 (t, 1H, C6H4), 7.87 (d, 1H, C6H4),
10.1 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 24.0, 24.3, 29.0, 33.4,
71.2, 79.0, 119.9, 123.5, 128.1, 132.2, 135.4, 151.0, 171.4, 189.0.
MS (CI) m/z: 357 (M + 1)+.
General Procedure for the Preparation of 19, 20, 21, and
22. A solution of the appropriate bromo derivative 15, 16, 17, and
18 (18.4 mmol) and AgNO3 (46.0 mmol) in CH3CN (150 mL) was
stirred at 70 °C for 7 h. 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 thus obtained was purified by
flash chromatography. Chromatographic eluents and yields of the
products were as follows.
1
EtOAc (v/v)]. Pale yellow oil. Yield 80%. H NMR (CDCl3) δ:
3
2.34–2.36 (m, 2H, -CH2CH2Br), 2.89 (t, 2H, -OCOCH2-, JHH
3
) 7.1 Hz), 3.57 (t, 2H, -CH2Br, JHH ) 6.4 Hz), 7.18 (d, 1H,
C6H4), 7.43 (t, 1H, C6H4), 7.66 (t, 1H, C6H4), 7.88 (d, 1H, C6H4),
10.1 (s br, 1H, CHO). 13C NMR (CDCl3) δ: 27.4, 32.3, 32.5, 123.5,
126.5, 128.0, 132.0, 134.8, 151.0, 171.0, 188.9. MS (CI) m/z: 271/
273 (M + 1)+.
2-Formylphenyl 4-(Nitrooxy)butanoate (19). Eluent [90:10 PE/
1
EtOAc (v/v)]. Pale yellow oil. Yield 84%. H NMR (CDCl3) δ:
2-Formylphenyl 5-Bromopentanoate (16). Eluent [90:10 PE/
2.17–2.27 (m, 2H, -CH2CH2ONO2), 2.82 (t, 2H, -OCOCH2-,
3JHH ) 7.1 Hz), 4.61 (t, 2H, -CH2ONO2, 3JHH ) 6.2 Hz), 7.18 (d,
1H, C6H4), 7.44 (t, 1H, C6H4), 7.65 (t, 1H, C6H4), 7.87 (d, 1H,
C6H4), 10.0 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 22.1, 30.1, 71.8,
123.5, 126.7, 127.9, 132.7, 134.9, 150.6, 170.8, 189.1. MS (CI)
m/z: 254 (M + 1)+.
1
EtOAc (v/v)]. Pale yellow oil. Yield 57%. H NMR (CDCl3) δ:
1.91–2.05 (m, 4H, -CH2CH2CH2Br), 2.70 (t, 2H, -OCOCH2-,
3
3JHH ) 6.8 Hz), 3.48 (t, 2H, -CH2Br, JHH ) 6.3 Hz), 7.18 (d,
1H, C6H4), 7.40 (t, 1H, C6H4), 7.64 (t, 1H, C6H4), 7.88 (d, 1H,
C6H4), 10.1 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 23.2, 31.9, 32.9,
33.1, 123.5, 126.5, 128.2, 131.7, 135.3, 151.3, 171.4, 188.8. MS
(CI) m/z: 285/287 (M + 1)+.
2-Formylphenyl 5-(Nitrooxy)pentanoate (20). Eluent [90:10
1
PE/EtOAc (v/v)]. Pale yellow oil. Yield 64%. H NMR (CDCl3)
2-Formylphenyl 6-Bromohexanoate (17). Eluent [90:10 PE/
δ: 1.88–1.95 (m, 4H, -CH2CH2CH2ONO2), 2.75 (m, 2H,
-OCOCH2-), 4.55 (m, 2H, -CH2ONO2), 7.19 (d, 1H, C6H4), 7.43
(t, 1H, C6H4), 7.66 (t, 1H, C6H4), 7.89 (d, 1H, C6H4), 10.1 (s, 1H,
-CHO). 13C NMR (CDCl3) δ: 20.9, 26.2, 33.3, 72.7, 123.5, 126.5,
128.0, 132.2, 135.4, 151.0, 171.2, 188.9. MS (CI) m/z: 268 (M +
1)+.
1
EtOAc (v/v)]. Pale yellow oil. Yield 68%. H NMR (CDCl3) δ:
1.57–1.65 (m, 2H), 1.78–1.83 (m, 2H), 1.89–1.97 (m, 2H,
-CH2CH2CH2CH2Br), 2.70 (t, 2H, -OCOCH2-, 3JHH ) 7.5 Hz),
3.44 (t, 2H, -CH2Br, 3JHH ) 6.7 Hz), 7.19 (d, 1H, C6H4), 7.39 (t,
1H, C6H4), 7.64 (t, 1H, C6H4), 7.88 (d, 1H, C6H4), 10.10 (s, 1H,

1900 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
2-Formylphenyl 6-(Nitrooxy)hexanoate (21). Eluent [90:10 PE/
Lazzarato et al.
AMX-like system, -CHaHbONO2), 4.95–5.00 (dd, 1H, AMX-like
system, -CHaHbONO2), 5.81–5.84 (m, 1H, AMX-like system,
-CHONO2), 7.18 (d, 1H, C6H4), 7.49 (t, 1H, C6H4), 7.67 (t, 1H,
C6H4), 7.86 (d, 1H, C6H4), 9.96 (s, 1H, -CHO). 13C NMR (CDCl3)
δ: 34.0, 70.3, 74.9, 123.4, 127.2, 127.6, 134.3, 135.6, 149.4, 167.1,
189.6. MS (CI) m/z: 315 (M + 1)+.
2-Formylphenyl 4,5-Bis(nitrooxy)pentanoate (47). Eluent [90:
10 PE/EtOAc (v/v)]. Pale yellow oil. Yield 53%. 1H NMR (CDCl3)
δ: 2.10–2.27 (m, 2H, -CH2CH2CH-), 2.87 (t, 2H, -OCOCH2-,
3JHH ) 6.9 Hz), 4.57 (dd, 1H, AMX-like system, -CHaHbONO2),
4.86 (dd, 1H, AMX-like system, -CHaHbONO2), 5.52 (m, 1H,
AMX-like system, -CHONO2), 7.17 (d, 1H, C6H4), 7.46 (t, 1H,
C6H4), 7.64 (t, 1H, C6H4), 7.85 (d, 1H, C6H4), 9.98 (s, 1H, -CHO).
13C NMR (CDCl3) δ: 24.2, 29.2, 71.1, 78.0, 123.5, 126.8, 127.8,
133.7, 135.5, 150.1, 170.6, 189.5. MS (CI) m/z: 329 (M + 1)+.
2-(2,2-Dimethyl)-formylphenyl 5,6-Bis(nitrooxy)hexanoate
(50). Eluent [90:10 PE/EtOAc (v/v)]. Pale yellow oil. Yield 55%.
1H NMR (CDCl3) δ: 1.37 (s, 6H, -CH3), 1.82–1.95 (m, 4H,
-CH2CH2CH-), 4.48–4.54 (dd, 1H, AMX-like system, -CHaH-
bONO2), 4.77–4.82 (dd, 1H, AMX-like system, -CHaHbONO2),
5.30–5.37 (m, 1H, AMX-like system, -CHONO2), 7.11 (d, 1H,
C6H4), 7.41 (t, 1H, C6H4), 7.65 (t, 1H, C6H4), 7.87 (d, 1H, C6H4),
10.0 (s, 1H, -CHO). 13C NMR (CDCl3) δ: 24.6, 25.1, 35.2, 42.4,
71.2, 79.6, 123.4, 126.6, 128.3, 132.2, 135.3, 151.1, 175.4, 188.8.
MS (CI) m/z: 371 (M + 1)+.
2-{[3-(Nitrooxy)propanoyl]oxy)}benzoic Acid (28). SOCl2
(2.43 mL, 33.3 mmol) and a few drops of dry DMF were added to
a solution of 3-(nitrooxy)propionic acid (3.0 g, 22.2 mmol) in dry
THF (20 mL), stirred under N2 at room temperature; the stirring
was continued for 3 h at room temperature. The solution of the
acyl chloride thus obtained was slowly added to a stirred solution
of salycilic acid (3.07 g, 22.2 mmol) and dry Py (2.7 mL, 33.3
mmol) in dry THF (40 mL) kept under N2 at 0 °C. The mixture
was allowed to reach room temperature, and the stirring was
continued overnight. The mixture was diluted with Et2O (90 mL)
and washed twice with 2 M HCl (60 mL). The organic layer was
dried, filtered, and concentrated under reduced pressure. The crude
product was partially purified by flash chromatography [97:3
CH2Cl2/MeOH (v/v)]. The crude solid thus obtained was crystallized
by toluene. Yield 46%. mp 86–88 °C (from toluene). 1H NMR
(CDCl3) δ: 3.09 (t, 2H, -OCOCH2-, 3JHH ) 6.4 Hz), 4.87 (t, 2H,
-CH2ONO2, 3JHH ) 6.4 Hz), 7.16 (d, 1H, C6H4), 7.39 (t, 1H, C6H4),
7.65 (t, 1H, C6H4), 8.16 (d, 1H, C6H4), 10.0 (s vvbr, 1H, -COOH).
13C NMR (CDCl3) δ: 32.2, 67.6, 121.8, 123.9, 126.6, 132.7, 135.2,
150.8, 168.3, 169.8. MS (CI) m/z: 256 (M + 1)+.
1
EtOAc (v/v)]. Pale yellow oil. Yield 80%. H NMR (CDCl3) δ:
1.52–1.62 (m, 2H), 1.77–1.89 (m, 4H, -CH2CH2-
3
CH2CH2ONO2), 2.68 (t, 2H, -OCOCH2-, JHH ) 7.4 Hz), 4.49
3
(t, 2H, -CH2ONO2, JHH ) 6.5 Hz), 7.17 (d, 1H, C6H4), 7.41 (t,
1H, C6H4), 7.64 (t, 1H, C6H4), 7.88 (d, 1H, C6H4), 10.10 (s, 1H,
-CHO). 13C NMR (CDCl3) δ: 24.1, 25.1, 26.5, 33.7, 73.0, 123.5,
126.5, 128.1, 131.7, 135.3, 151.3, 171.6, 188.9. MS (CI) m/z: 282
(M + 1)+.
2-Formylphenyl 7-(Nitrooxy)heptanoate (22). Eluent [90:10
1
PE/EtOAc (v/v)]. Pale yellow oil. Yield 79%. H NMR (CDCl3)
δ: 1.47–1.52 (m, 4H), 1.75–1.83 (m, 4H, -CH2CH2-
3
CH2CH2CH2ONO2), 2.69 (t, 2H, -OCOCH2-, JHH ) 7.4 Hz),
4.47 (t, 2H, -CH2ONO2, 3JHH ) 6.6 Hz), 7.17 (d, 1H, C6H4), 7.41
(t, 1H, C6H4), 7.64 (t, 1H, C6H4), 7.88 (d, 1H, C6H4), 10.10 (s, 1H,
-CHO). 13C NMR (CDCl3) δ: 24.4, 25.4, 26.6, 28.4, 33.9, 73.2,
123.5, 126.4, 128.1, 131.4, 135.4, 151.5, 171.8, 188.9. MS (CI)
m/z: 296 (M + 1)+.
2-Formylphenyl But-3-enoate (43). To a solution of vinyl acetic
acid (3.0 mL, 35.3 mmol) in dry CH2Cl2 (50 mL), stirred under
inert atmosphere, DCC (2.18 g, 31.7 mmol) was added. After 1 h,
salicylaldehyde (3.0 mL, 28.2 mmol) and DMAP (0.43 g, 3.53
mmol) were added. The reaction was completed after 2 h. The
mixture was filtered, and the filtrate was washed with H2O (20 mL)
and brine (20 mL). The organic layer was dried, filtered, and
concentrated under reduced pressure. The crude product was purified
by flash chromatography [95:5 PE/EtOAc (v/v)] to give the title
1
compound as a yellow oil. Yield 67%. H NMR (CDCl3) δ: 3.46
(d, 2H, -CH2CHdCH2), 5.28–5.36 (m, 2H, -CH2CHdCH2),
6.02–6.11 (m, 1H, -CH2CHdCH2), 7.20 (d, 1H, C6H4), 7.41 (t,
1H, C6H4), 7.64 (t, 1H, C6H4), 7.89 (d, 1H, C6H4), 10.11 (s, 1H,
-CHO). 13C NMR (CDCl3) δ: 38.9, 119.7, 123.4, 126.5, 128.0,
129.2, 131.2, 135.3, 151.5 169.8, 188.7. MS (CI) m/z: 191 (M +
1)+.
2-Formylphenyl (2,2-Dimethyl)-hex-5-enoate (45). The title
compound was prepared in the same manner as 43. Eluent [98:2
PE/EtOAc (v/v)]. Yellow oil. Yield 47%. 1H NMR (CDCl3) δ: 1.40
(s, 6H, -CH3) 1.80–1.86 (m, 2H, -CH2CH2CH-), 2.12–2.20 (m,
2H, -CH2CH2CH-), 5.10 (dd, 1H, AMX-like system,
-CHdCHaHb), 4.86 (dd, 1H, AMX-like system, -CHdCHaHb),
5.85 (m, 1H, AMX-like system, -CHdCH2), 7.14 (d, 1H, C6H4),
7.38 (t, 1H, C6H4), 7.63 (t, 1H, C6H4), 7.92 (d, 1H, C6H4), 10.15
(s, 1H, -CHO). 13C NMR (CDCl3) δ: 24.8, 29.4, 39.6, 42.8, 115.0,
123.3, 126.2, 128.3, 129.9, 135.3, 138.0, 152.5, 175.9, 188.3. MS
(CI) m/z: 247 (M + 1)+.
General Procedure for the Preparation of 36, 46, 47, and
50. Iodine (9.79 mmol) was added portion-wise to a stirred solution
of the appropriate unsatured compounds 34, 43, 44, and 45 (9.79
mmol) and AgNO3 (1.66 g, 9.79 mmol) in CH3CN (100 mL) kept
at -15 °C. At the end of the addition, the stirring was continued
for 1 h. Then, AgNO3 (19.6 mmol) was added, and the mixture
was heated at 70 °C until the disappearance of the starting material,
as checked by TLC. After cooling, the mixture was filtered through
Celite. The filtrate was concentrated under reduced pressure,
dissolved in water (50 mL), and extracted with EtOAc (4 × 50
mL). The combined organic layers were dried, filtered, and
concentrated under reduced pressure. The crude product thus
obtained, when necessary, was purified by flash chromatography.
Chromatographic eluents and yields of the products were as follows.
6,7-Dinitrooxyheptanoic Acid (36). Yellow oil. Yield 88%. 1H
NMR (CDCl3) δ: 1.50–1.55 (m, 2H), 1.67–1.80 (m, 4H,
-CH2CH2CH2CH2OCO-), 2.41 (t, 2H, -OCOCH2-, 3JHH ) 7.1
Hz), 4.48 (dd, 1H, AMX-like system, -CHaHbONO2), 4.76 (dd,
1H, AMX-like system, -CHaHbONO2), 5.26–5.33 (m, 1H, AMX-
like system, -CHONO2), 9.66 (s br, 1H, -COOH). 13C NMR
(CDCl3) δ: 24.0, 24.3, 27.4, 33.5, 71.2, 78.6, 179.5. MS (CI) m/z:
253 (M + 1)+.
General Procedure for the Preparation of 29, 30, 31, 33,
52, 53, 54, and 55. KMnO4 (4.38 mmol) was added to a stirred
solution of the appropriate aldehyde 19, 20, 21, 25, 47, 48, 49 and
50 (2.92 mmol) in acetone (20 mL) kept at 0 °C. The reaction was
allowed to reach room temperature and was completed after 3 h.
Oxalic acid was added. The mixture was filtered, and the filtrate
was diluted with CH2Cl2 (20 mL). The organic layer was washed
with H2O (20 mL) and then dried, filtered, and concentrated under
reduced pressure. The crude product was purified by crystallization.
2-{[4-(Nitrooxy)butanoyl]oxy}benzoic Acid (29). mp 70.5–
71.5 °C [from 50:50 PE/toluene (v/v)]. White solid. Yield 52%.
1H NMR (DMSO-d6) δ: 2.05 (qi, 2H, -CH2CH2ONO2), 2.71 (t,
2H, -OCOCH2-, 3JHH ) 6.0 Hz), 4.63 (t, 2H, -CH2ONO2, 3JHH
) 6.0 Hz), 7.21 (d, 1H, C6H4), 7.39 (t, 1H, C6H4), 7.65 (t, 1H,
C6H4), 7.94 (d, 1H, C6H4), 13.13 (s, 1H, -COOH). 13C NMR
(DMSO-d6) δ: 21.4, 29.7, 72.6, 123.7, 123.8, 126.1, 131.3, 133.8,
150.0, 165.5, 170.9. MS (CI) m/z: 270 (M + 1)+.
2-{[5-(Nitrooxy)pentanoyl]oxy}benzoic Acid (30). mp 48.5–50.5
°C [from 70:30 PE/toluene (v/v)]. White solid. Yield 56%. 1H NMR
(CDCl3) δ: 1.89 (m, 4H, -CH2CH2CH2ONO2), 2.66 (m, 2H,
-OCOCH2-), 4.47 (m, 2H, -CH2ONO2), 7.12 (d, 1H, C6H4), 7.37
(t, 1H, C6H4), 7.63 (t, 1H, C6H4), 8.12 (d, 1H, C6H4), 12.1 (s br,
1H, -COOH). 13C NMR (CDCl3) δ: 20.9, 26.3, 33.5, 73.0, 122.2,
124.1, 126.4, 132.7, 135.2, 151.3, 170.5, 171.7. MS (CI) m/z: 284
(M + 1)+.
2-Formylphenyl 3,4-Bis(nitrooxy)butanoate (46). Eluent [90:
10 PE/EtOAc (v/v)]. The orange solid was treated with iPr2O to
1
obtain the title compound as a pale yellow solid. Yield 15%. H
NMR (CDCl3) δ: 3.18 (d, 2H, -OCOCH2-), 4.71–4.77 (dd, 1H,

Nitrooxy-acyl DeriVatiVes of Salicylic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1901
2-{[6-(Nitrooxy)hexanoyl]oxy}benzoic Acid (31). mp 68.0–70.0
°C [from 75:25 PE/toluene (v/v)]. White solid. Yield 82%. 1H NMR
(DMSO-d6) δ: 1.41–1.77 (m, 6H, -CH2CH2CH2CH2ONO2), 2.60
v)]. 1H NMR (CDCl3) δ: 1.36–1.54 (m, 4H), 1.74–1.81 (m, 4H,
3
-CH2CH2CH2CH2CH2ONO2) 2.64 (t, 2H, -OCOCH2-, JHH
3
) 7.3 Hz), 4.44 (t, 2H, -CH2ONO2, JHH ) 6.6 Hz), 7.12 (d,
3
(t, 2H, -OCOCH2-, JHH ) 7.3 Hz), 4.55 (t, 2H, -CH2ONO2,
1H, C6H4), 7.36 (t, 1H, C6H4), 7.63 (t, 1H, C6H4), 8.10 (d, 1H,
C6H4), 11.53 (s, 1H, -COOH). 13C NMR (CDCl3) δ: 24.2, 25.4,
26.6, 28.6, 33.9, 73.3, 122.3, 124.0, 126.2, 132.4, 135.0, 151.2,
170.3, 172.1. MS (CI) m/z: 312 (M + 1)+.
3JHH ) 6.0 Hz), 7.20 (d, 1H, C6H4), 7.39 (t, 1H, C6H4), 7.65 (t,
1H, C6H4), 7.94 (d, 1H, C6H4), 13.10 (s, 1H, -COOH). 13C NMR
(DMSO-d6) δ: 23.5, 24.4, 25.7, 33.1, 73.6, 123.7, 124.1, 126.0,
131.3, 133.7, 150.0, 165.6, 171.5. MS (CI) m/z: 298 (M + 1)+.
2-{[2,2-Dimethyl-3-(nitrooxy)propanoyl]oxy}benzoic Acid
(33). mp 95.0–96 °C [from 75:25 PE/toluene (v/v)]. White solid.
Yield 61%. 1H NMR (CDCl3) δ: 1.47 (s, 6H, -CH3), 4.67 (s, 2H,
-CH2-), 7.10 (d, 1H, C6H4), 7.37 (t, 1H, C6H4), 7.64 (t, 1H, C6H4),
8.13 (d, 1H, C6H4), 12.1 (s vvbr, 1H, -COOH). 13C NMR (CDCl3)
δ: 22.3, 42.5, 77.5, 122.2, 123.8, 126.5, 132.6, 135.1, 150.9, 170.2,
172.8. MS (CI) m/z: 284 (M + 1)+.
2-{[3,4-Bis(nitrooxy)butanoyl]oxy}benzoic Acid (51). The title
compound was prepared in the same manner as 32. mp 128–
1
129 °C (from toluene). Yield 68%. H NMR (CDCl3) δ: 3.15 (d,
2H, -OCOCH2-), 4.69–4.76 (dd, 1H, AMX-like system, -CHaH-
bONO2), 4.92–4.98 (dd, 1H, AMX-like system, -CHaHbONO2),
5.77–5.84 (m, 1H, AMX-like system, -CHONO2), 7.16 (d, 1H,
C6H4), 7.41 (t, 1H, C6H4), 7.67 (t, 1H, C6H4), 8.15 (d, 1H, C6H4),
10.90 (s vvbr, 1H, -COOH). 13C NMR (CDCl3) δ: 34.0, 70.2,
74.8, 121.5, 123.7, 126.9, 132.8, 135.3, 138.4, 150.5, 167.4. MS
(CI) m/z: 331 (M + 1)+.
2-{[4,5-Bis(nitrooxy)pentanoyl]oxy}benzoic Acid (52). mp
92.5–93.0 °C [from 45:55 PE/toluene (v/v)]. White solid. Yield
89%. 1H NMR (CDCl3) δ: 2.13–2.25 (m, 2H, -CH2CH2CH-),
Evaluation of Stability in Aqueous Buffer Solutions and
Human Serum. Hydrolysis in Acidic Medium (pH 1) and
Phosphate Buffer (pH 7.4). A solution of each compound (10 mM)
in acetonitrile was added to 0.1 M HCl or phosphate buffer at pH
7.4 (50 mM) preheated at 37 °C. The final concentration of the
compound was 250 µM. The resulting solution was maintained at
37 ( 0.5 °C, and at appropriate time intervals, a 20 µL aliquot of
reaction solution was analyzed by RP-HPLC.
3
2.83 (t, 2H, -OCOCH2-, JHH ) 6.0 Hz), 4.54 (dd, 1H, AMX-
like system, -CHaHbONO2), 4.84 (dd, 1H, AMX-like system,
-CHaHbONO2), 5.50 (m, 1H, AMX-like system, -CHONO2), 7.13
(d, 1H, C6H4), 7.40 (t, 1H, C6H4), 7.66 (t, 1H, C6H4), 8.14 (d, 1H,
C6H4), 11.0 (s vvbr, 1H, COOH). 13C NMR (CDCl3) δ: 24.6, 29.8,
71.4, 78.2, 122.0, 124.2, 126.9, 133.0, 135.6, 151.3, 169.9, 171.2.
MS (CI) m/z: 345 (M + 1)+.
2-{[5,6-Bis(nitrooxy)hexanoyl]oxy}benzoic Acid (53). mp
101.5–102.5 °C [from 50:50 PE/toluene (v/v)]. White solid. Yield
72%. 1H NMR (DMSO-d6) δ: 1.73–1.86 (m, 4H, -CH2CH2CH-),
Hydrolysis in Human Serum. A solution of each compound
(10 mM) in acetonitrile was added to human serum (Sigma)
preheated 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, 500 µL of the reaction mixture was
withdrawn and added to 750 µL of acetonitrile containing 0.1%
trifluoroacetic acid to deproteinize the serum. The sample was
sonicated, vortexed, and then centrifuged for 10 min at 2150g. The
clear supernatant was filtered by 0.45 µm PTFE filters (Alltech)
and analyzed by RP-HPLC.
3
2.64 (t, 2H, -OCOCH2-, JHH ) 6.0 Hz), 4.73 (dd, 1H, AMX-
like system, -CHaHbONO2), 4.96 (dd, 1H, AMX-like system,
-CHaHbONO2), 5.46 (m, 1H, AMX-like system, -CHONO2), 7.19
(d, 1H, C6H4), 7.39 (t, 1H, C6H4), 7.64 (t, 1H, C6H4), 7.93 (d, 1H,
C6H4), 13.3 (s br, 1H, -COOH). 13C NMR (DMSO-d6) δ: 19.5,
27.5, 32.8, 71.7, 80.0, 123.7, 123.9, 126.0, 131.3, 133.7, 150.0,
165.5, 171.2. MS (CI) m/z: 359 (M + 1)+.
2-{[6,7-Bis(nitrooxy)heptanoyl]oxy}benzoic Acid (54). The
crude product was purified by preparative HPLC (Lichrospher 250-
25 C18, 60:40:0.1 CH3CN/H2O/TFA, flow of 39 mL/min, λ of 224
nm, injection of 2 mL, solution at 100 mg/mL) to give the title
compound as a white solid. Yield 89%. mp 92.5–93.0 °C [from
The reverse-phase HPLC procedure allowed for the separation
and quantitation of the remaining compound and salicylic acid.
HPLC analyses were performed with a HP 1100 chromatograph
system (Agilent Technologies, Palo 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 performed using a HP
ChemStation system (Agilent Technologies). The analytical column
was a Nucleosil 100-5C18 Nautilus (250 × 4.6 mm, 5 µm particle
size) (Macherey-Nagel). The mobile phase consisted of acetonitrile/
water (55:45) with 0.1% trifluoroacetic acid, and the flow rate was
1.2 mL/min. The injection volume was 20 µL (Rheodyne, Cotati,
CA). The column effluent was monitored at 226 nm (for com-
pounds) and 240 nm (for salicylic acid) referenced against a 600
nm wavelength. Quantitation was performed by a comparison of
peak areas with standards chromatographed under the same
conditions.
Anti-inflammatory Activity. Male Wistar rats, weighing 180–200
g (Harlan, S. Pietro al Natisone, Italy) were individually housed in
hanging stainless-steel cages with grid floors, at constant room
temperature (25 ( 1 °C) and humidity (60 ( 5%), with an artificial
12:12 h light/dark cycle. Edema was induced in conscious rats by
intraplantar injection into the right hindpaw of 0.1 mL of 1%
carrageenan, suspended in 1% carboxymethylcellulose (CMC).
Immediately after carrageenan injection, compounds or vehicle (1%
CMC) were administered intragastrically to different groups of rats
in a volume of 10 mL/kg. Salicylic acid and NO aspirin derivatives
were administered at a dose equimolar to 120 mg/kg aspirin. Groups
of 6–8 animals were used. The paw volume was measured with a
water plethysmometer (Basile, Comerio, Italy) immediately before
carrageenan injection and 3 h afterward. The edema reduction in
treated animals was expressed as percent inhibition of the edema
observed in vehicle-treated animals, considered as 100. The results
obtained are presented as mean ( standard error of the mean (SEM).
Statistical analysis was performed with analysis of variation
(ANOVA) followed by the Dunnett test.
1
45:55 PE/toluene (v/v)]. H NMR (CDCl3) δ: 1.51–1.65 (m, 2H),
1.74–1.86 (m, 4H, -CH2CH2CH2CH-), 2.67 (t, 2H, -OCOCH2-,
3JHH ) 6.0 Hz), 4.47 (dd, 1H, AMX-like system, -CHaHbONO2),
4.74 (dd, 1H, AMX-like system, -CHaHbONO2), 5.30 (m, 1H,
AMX like system, -CHONO2), 7.13 (d, 1H, C6H4), 7.38 (t, 1H,
C6H4), 7.65 (t, 1H, C6H4), 8.11 (d, 1H, C6H4), 8.49 (s br, 1H,
-COOH). 13C NMR (CDCl3) δ: 23.9, 24.3, 29.0, 33.6, 71.1, 78.9,
121.8, 124.0, 126.4, 132.5, 135.3, 151.1, 170.0, 172.2. MS (CI)
m/z: 373 (M + 1)+.
2-{[2,2-Dimethyl-5,6-bis(nitrooxy)hexanoyl]oxy}benzoic Acid
(55). mp 72.0–73.0 °C [from 70:30 PE/toluene (v/v)]. White solid.
Yield 49%. 1H NMR (CDCl3) δ: 1.40 (s, 6H, -CH3), 1.78–1.93
(m, 4H, -CH2CH2CH-), 4.45–4.52 (dd, 1H, AMX-like system,
-CHaHbONO2), 4.73–4.79 (dd, 1H, AMX-like system, -CHaH-
bONO2), 5.27–5.34 (m, 1H, AMX-like system, -CHONO2), 7.07
(d, 1H, C6H4), 7.37 (t, 1H, C6H4), 7.64 (t, 1H, C6H4), 8.11 (d,
1H, C6H4), 11.75 (s vvbr, 1H, -COOH). 13C NMR (CDCl3) δ:
24.6, 25.0, 25.6, 35.2, 42.1, 71.2, 79.4, 122.3, 123.7, 126.3,
132.4, 135.0, 151.2, 170.2, 175.4. MS (CI) m/z: 387 (M + 1)+.
2-{[7-(Nitrooxy)heptanoyl]oxy}benzoic Acid (32). To a solu-
tion of 22 (2.6 g, 8.80 mmol) in CH3CN (20 mL) kept at 0 °C
were added a solution of KH2PO4 (0.80 g) in H2O (10 mL) and
30% H2O2 (1.1 mL, 9.68 mmol) and dropwise a solution of 80%
NaClO2 (1.40 g, 12.3 mmol) in H2O (12 mL). After 2 h, the
reaction was completed. Na2SO3 was added to destroy the excess
of H2O2. After acidification with 6 M HCl, the mixture was
diluted with H2O (100 mL) and extracted twice with CH2Cl2
(100 mL). The organic layer was dried, filtered, and concentrated
under reduced pressure. The crude product was crystallized from
70:30 PE/toluene (v/v) to give the title compound as a white
solid. Yield 71%. mp 47.0–49.0 °C [from 70:30 PE/toluene (v/

1902 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Lazzarato et al.
Gastrotoxicity. Male Wistar rats, weighing 180–200 g (Harlan,
S. Pietro al Natisone, Italy) were individually housed in hanging
stainless-steel cages with grid floors, at constant room temperature
(25 ( 1 °C) and humidity (60 ( 5%), with an artificial 12:12 h
light/dark cycle. They were deprived of food but not water 24 h
before the experiments. Groups of rats (n ) 8–10) were given 120
mg/kg aspirin by intragastric route or equimolar doses of the
compounds under study (1% vehicle CMC). Rats were sacrificed
3 h after the administration of the compounds. Immediately after
the sacrifice, the stomachs were removed, opened along the lesser
curvature, and examined for the assessment of mucosal lesions.
The stomachs were laid on a flat surface under a stereomicroscope.
The glandular mucosa was examined, and each individual hemor-
rhagic lesion was measured along its greatest length (<1 mm, rating
) 1; 1–2 mm, rating ) 2; >2 mm, rating according to their greatest
length). The lengths of the lesions were summed to give an overall
total, designated as the lesion index, for each stomach. The results
obtained are presented as mean ( SEM. Statistical analysis was
performed with ANOVA followed by the Newman-Keuls test.
Inhibition of Human Platelet Aggregation in Vitro. Venous
blood samples were obtained from healthy volunteers who had not
taken any drugs for at least 2 weeks. Volunteers, who were treated
according to the Helsinki protocol for biomedical experimentation,
gave their informed consent to the use of blood samples for research
purposes. PRP was prepared by centrifugation of citrated blood at
200 g 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 the addition of the stimulus.
Collagen at submaximal concentrations (0.8–1.5 µg/mL) was used
as the platelet activator in PRP. Compounds under study were
preincubated with PRP 10 min before the addition of the stimulus
(collagen). Vehicle alone (0.5% DMSO) added to PRP did not affect
platelet function in control samples. The role of NO and sGC in
the inhibitory effect was investigated using the sGC inhibitor, ODQ
(50 µM). At least five experiments for each compound were
performed.
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