Water-soluble nitric-oxide-releasing acetylsalicylic acid (ASA) prodrugs

Article abstract of DOI:10.1002/cmdc.201300105

A series of water-soluble (benzoyloxy)methyl esters of acetylsalicylic acid (ASA), commonly known as aspirin, are described. The new derivatives each have alkyl chains containing a nitric oxide (NO)-releasing nitrooxy group and a solubilizing moiety bonded to the benzoyl ring. The compounds were synthesized and evaluated as ASA prodrugs. After conversion to the appropriate salt, most of the derivatives are solid at room temperature and all possess good water solubility. They are quite stable in acid solution (pH1) and less stable at physiological pH. In human serum, these compounds are immediately metabolized by esterases, producing a mixture of ASA, salicylic acid (SA), and of the related NO-donor benzoic acids, along with other minor products. Due to ASA release, the prodrugs are capable of inhibiting collagen-induced platelet aggregation of human platelet-rich plasma. Simple NO-donor benzoic acids 3-hydroxy-4-(3-nitrooxypropoxy)benzoic acid (28) and 3-(morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoic acid (48) were also studied as representative models of the whole class of benzoic acids formed following metabolism of the prodrugs in serum. These simplified derivatives did not trigger antiaggregatory activity when tested at 300μM. Only 28 displays quite potent NO-dependent vasodilatatory action. Further in vivo evaluation of two selected prodrugs, {[2-(acetyloxy)benzoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-4-[3-(nitrooxy)propoxy]benzoate{dot operator}HCl (38) and {[2-(acetyloxy)benzoyl]oxy}methyl 3-(morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoate oxalate (49), revealed that their anti-inflammatory activities are similar to that of ASA when tested in the carrageenan-induced paw edema assay in rats. The gastrotoxicity of the two prodrugs was also determined to be lower than that of ASA in a lesion model in rats. Taken together, these results indicated that these NO-donor ASA prodrugs warrant further investigation for clinical application.

Full text of DOI:10.1002/cmdc.201300105

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A new look for aspirin: Acetylsalicylic
acid (ASA) derivatives bearing NO-re-
leasing nitrooxy group and a solubilizing
moiety at the benzoyl ring were de-
signed as ASA prodrugs. When evaluat-
ed, all the products displayed good an-
tiaggregatory properties, and two deriv-
atives (shown) also exhibited improved
in vivo anti-inflammatory properties and
decreased gastrotoxicity when com-
pared with ASA.
B. Rolando, L. Lazzarato, M. Donnola,
E. Marini, S. Joseph, G. Morini, C. Pozzoli,
R. Fruttero,* A. Gasco
&& – &&
Water-Soluble Nitric-Oxide-Releasing
Acetylsalicylic Acid (ASA) Prodrugs
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DOI: 10.1002/cmdc.201300105
Water-Soluble Nitric-Oxide-Releasing Acetylsalicylic Acid
(ASA) Prodrugs
Barbara Rolando,[a] Loretta Lazzarato,[a] Monica Donnola,[a] Elisabetta Marini,[a] Sony Joseph,[a]
Giuseppina Morini,[b] Cristina Pozzoli,[b] Roberta Fruttero,*[a] and Alberto Gasco[a]
A series of water-soluble (benzoyloxy)methyl esters of acetylsa-
licylic acid (ASA), commonly known as aspirin, are described.
The new derivatives each have alkyl chains containing a nitric
oxide (NO)-releasing nitrooxy group and a solubilizing moiety
bonded to the benzoyl ring. The compounds were synthesized
and evaluated as ASA prodrugs. After conversion to the appro-
priate salt, most of the derivatives are solid at room tempera-
ture and all possess good water solubility. They are quite
stable in acid solution (pH 1) and less stable at physiological
pH. In human serum, these compounds are immediately me-
tabolized by esterases, producing a mixture of ASA, salicylic
acid (SA), and of the related NO-donor benzoic acids, along
with other minor products. Due to ASA release, the prodrugs
are capable of inhibiting collagen-induced platelet aggregation
of human platelet-rich plasma. Simple NO-donor benzoic acids
3-hydroxy-4-(3-nitrooxypropoxy)benzoic acid (28) and 3-(mor-
pholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoic acid (48)
were also studied as representative models of the whole class
of benzoic acids formed following metabolism of the prodrugs
in serum. These simplified derivatives did not trigger antiag-
gregatory activity when tested at 300 mm. Only 28 displays
quite potent NO-dependent vasodilatatory action. Further in
vivo evaluation of two selected prodrugs, {[2-(acetyloxy)ben-
zoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-4-[3-(nitrooxy)pro-
poxy]benzoate·HCl (38) and {[2-(acetyloxy)benzoyl]oxy}methyl
3-(morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoate oxa-
late (49), revealed that their anti-inflammatory activities are
similar to that of ASA when tested in the carrageenan-induced
paw edema assay in rats. The gastrotoxicity of the two pro-
drugs was also determined to be lower than that of ASA in
a lesion model in rats. Taken together, these results indicated
that these NO-donor ASA prodrugs warrant further investiga-
tion for clinical application.
Introduction
Nitric oxide (NO)-donor acetylsalicylic acid (ASA) derivatives are
a class of products that has received particular attention in
recent years.[1,2] The pharmacological rationale for developing
these agents is that NO displays gastrosparing properties and
consequently could be expected to decrease the well-known
gastrotoxicity of ASA, without negatively effecting its antith-
rombotic and anti-inflammatory properties.[3–5] NO-donor ASA
(NO-ASA) derivatives have the general structure 1, in which
the carboxylic group of ASA is linked through an enzymatically
labile ester bridge to a substructure containing a NO-donor
moiety, typically belonging to the nitrate, furoxan, or NONOate
class. Examples of such hybrid agents have been reported in
the literature.[6–8] The pharmacological action of these substan-
ces is complex, and there is no clear evidence to date that
they are true prodrugs of ASA, namely that they are capable of
releasing ASA in plasma or other tissues. The absence of a neg-
ative charge, which conversely is present on ASA at physiologi-
cal pH (ASA, pKa =3.5), renders their acetyloxy group highly
susceptible to enzymatic cleavage.[9] These products should
thus be considered prodrugs of salicylic acid (SA) rather than
of ASA.
[a] Dr. B. Rolando, Dr. L. Lazzarato, Dr. M. Donnola, Dr. E. Marini, Dr. S. Joseph,
Prof. R. Fruttero, Prof. A. Gasco
We recently showed that (nitrooxyacyloxy)methyl esters of
ASA and related carbonates are true ASA prodrugs (2 and
3).[10,11] In these structures, the deacetylation rate constant is
lower than the hydrolytic rate constant of the carbonyloxyme-
thyloxycarbonyl moiety, and consequently, the products are
able to release ASA in amounts that depend on the structure
of the NO-donor acyl moiety. One problem with these double
Dipartimento di Scienza e Tecnologia del Farmaco
Universitꢀ degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino (Italy)
E-mail: roberta.fruttero@unito.it
[b] Prof. Dr. G. Morini, Dr. C. Pozzoli
Dipartimento di Neuroscienze
Universitꢀ degli Studi di Parma, Via Volturno 39, 40100 Parma (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300105.
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esters is their poor water solubility; many are hydrophobic in
nature. This study describes a new series of (nitrooxyacyloxy)-
methyl esters of ASA, which are true ASA prodrugs and that
possess good water solubility and are solid at room tempera-
ture. In these products, the NO-donor nitrooxyacyloxy moiety
comprises the benzoyloxy scaffold, bearing a nitrooxyalkyloxy
chain at the para position, and a solubilizing substructure at
the meta position. In most of the derivatives (38–44), the solu-
bilizing substructure is a metabolically labile aminoacyloxy
group (I); however, in the case of compound 49, the morpholi-
nomethyl solubilizing substructure is directly joined to the aro-
matic ring. The synthesis, physical properties (solubility, stabili-
ty in different media, release of ASA), and the in vitro platelet
antiaggregatory profile of these new prodrugs are discussed.
The in vivo gastrotoxicity and anti-inflammatory properties are
also reported for two selected compounds (38 and 49).
moval of the BOC protection under acidic conditions (HCl in di-
oxane) yielded desired compounds 38–43 (Scheme 3). For the
preparation of dinitrooxy-substituted prodrug 44, 3,4-dihydrox-
ybenzaldehyde (4) was alkylated with allylbromide to give un-
Results and Discussion
Chemistry
The products bearing aminoacy-
loxy groups were obtained as
described in Schemes 1–3. Com-
mercially available 3,4-dihydroxy-
benzaldehyde (4) was bromoal-
kylated with 1,3-dibromopro-
pane in acetonitrile to give 5,
which in turn was transformed
by action of silver nitrate into
the related nitrooxy analogue
(7). This intermediate was react-
ed under inert atmosphere with
the appropriate tert-butoxycar-
bonyl (BOC)-protected amino
acid in dichloromethane in the
presence of 1-ethyl-3-(3-dime-
thylaminopropyl)carbodiimide
hydrochloride (EDC·HCl) and 4-
(dimethylamino)pyridine (DMAP)
to afford aminoacyloxy-substitut-
ed aldehydes 9, 11–14. Corre-
sponding acids 15, 17–20 were
obtained by oxidation with po-
tassium permanganate in ace-
tone. Acid 16 and its intermedi-
ates 10 and 8 were obtained in
the same manner as its short-
chain derivative 15, the only dif-
ference being that the starting
material was chloro-substituted
compound 6 (Scheme 1). Cou-
pling of acid derivatives 15–20
with ASA chloromethyl ester 29,
Scheme 1. Reagents and conditions: a) Br(CH2)3Br or Br(CH2)4Cl, KHCO3, CH3CN, 708C, 4–14 h; b) AgNO3, CH3CN,
708C, 14 h; c) EDC·HCl, DMAP, N-BOC-amino acid, CH2Cl2, RT, 3 h; d) KMnO4, acetone, 08C!RT, 1 h; e) BrCH2CH=
CH2, KHCO3, CH3CN, 708C, 4 h; f) EDC·HCl, DMAP, HOOC(CH2)2NHBOC, CH2Cl2, RT, 3 h; g) AgNO3, I2, CH3CN, 708C,
in the presence of cesium car-
bonate, gave oxymethyloxy
esters 30–35. Subsequent re- 16 h.
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saturated aldehyde 21. The latter was treated with BOC-pro-
tected b-alanine to give 22, following the general conditions
described above for the preparation of the BOC-protected ami-
noacyloxy substituted aldehydes. This intermediate was trans-
formed by action of iodine and silver nitrate into dinitrooxyest-
er 23, which in turn was oxidized with potassium permanga-
nate to the corresponding acid (24). Final compound 44 was
obtained from 24 through intermediate 36 using the proce-
dures described above for the preparation of the aminoacy-
loxy-substituted prodrugs from the related acids. Simple NO-
donor benzoic acid derivative 28 was prepared as shown in
Scheme 2. In particular, ester derivative 27 was obtained from
the methyl ester of 3,4-dihydroxybenzoic acid 25, in the same
manner as its aldehyde analogue 7. Intermediate 27 was then
hydrolyzed to the corresponding
Scheme 2. Reagents and conditions: a) Br(CH2)3Br, KHCO3, CH3CN, 708C, 5 h;
b) AgNO3, CH3CN, 708C, 14 h; c) NaOH, THF/H2O (1:1), 408C, 4 h.
carboxylic acid (28) by using
a solution of sodium hydroxide
in tetrahydrofuran/water with
heating to 408C.
Target compound 49 contain-
ing
a morpholino moiety as
a solubilizing group was synthe-
sized as shown in Scheme 4.
Ester 45 was treated with 1,3-di-
bromopropane in acetonitrile to
give bromo compound 46.
Treatment of this product with
a solution of silver nitrate in ace-
tonitrile transformed it into the
nitrooxy analogue (47), which
was subsequently hydrolyzed to
corresponding acid 48. The acid
was finally coupled with 29 to
give the desired product 49.
All of the final products were
successfully transformed into the
hydrochloride salts, with the sole
exception of morpholino deriva-
tive 49, which was characterized
as an oxalate. Most of these salts
are solid at room temperature
with well-defined melting points
(Table 1). Two products were oils
at room temperature: com-
pounds 39 and 41, both of
which are long-chain derivatives
of 38, the former at the aminoa-
cyloxy chain, and the latter at
the nitrooxyalkyloxy moiety.
Compound 44, derived from 38
by the introduction of a second
nitrooxy group at the a-position,
is a foam at room temperature.
Scheme 3. Reagents and conditions: a) Cs2CO3, DMF, RT, 24 h; b) 1,4-dioxane HCl, CH2Cl2, RT, 3 h.
Scheme 4. Reagents and conditions: a) Br(CH2)3Br, CH3CN, K2CO3, 708C, 6 h; b) AgNO3, CH3CN, 708C, 14 h; c) 1n
NaOH, MeOH, RT, 4 h; d) 29, Cs2CO3, DMF, RT, 24 h.
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Table 1. Physicochemical characterization, stability in buffer solutions, and stability in human serum of test compounds.
Compd
mp[a] [8C]
Solubility[b]
[gLꢀ1
Stability in buffer
Stability and ASA released in human serum
Platelet aggregation
[h]
]
pH 1.0
pH 7.4
t1/2[d] [min]
t1/2 [min]
[%] max[g]
AUC0–10 min
IC50[i] [mm]
% unchanged[c]
ASA[10]
38
39
40
41
42
43
44
–
3.0
56
52
110
32
86
70
63
2.0
90
88
98
70
90
70
90
80
98
–
90
57
86
12
10
10
15
20
98[e]
–
63
<1
<1
<1
<1
<1
<1
<1
2.2[f]
–
–
55
50
57
50
55
56
50
30
–
954
54 (49–60)
23 (20–28)
47 (41–54)
40 (34–48)
139 (126–154)
107 (97–119)
35 (31–40)
93 (71–123)
77 (70–84)
0.87ꢁ0.44[j]
7.4ꢁ5.0[j]
94.5–95.5
oil
60.5–62.5
oil
65.5–69.0
87.5–88.5
foam
135–139 dec.
528.0
336.2
381.6
217.3
232.0
397.5
251.8
189.2
–
49
28
48
–
–
–
–
–
–
–
–
–
[a] Melting point (mp). [b] Water solubility; data are the mean (n=3); standard error of the mean (SEM) <1. [c] Percent unchanged after 3 h; data are the
mean (n=3); SEM <1%. [d] n=3; SEM <0.5. [e] Value is the percent unchanged after 3 h at pH 7.4. [f] n=3; SEM=0.2. [g] Maximal amount (%) of ASA re-
leased—data are the percent of the initial concentration of the compound; n=3; SEM <1.5%. [h] Area under the curve (AUC) values at 10 min. [i] IC50
values derived from 3–5 experiments; 95% confidence levels are given in parentheses. [j] Due to the low activity of the compound, the IC50 value could
not be calculated; value given is inhibition (%) ꢁSEM at 300 mm.
Physicochemical evaluation
all products are similar and fall within the range 50–57%.
Table 1 also gives The area under the curve (AUC) values for
ASA release measured after a ten minute incubation period do
not entirely parallel the corresponding maximal ASA release
values (Table 1), which suggests that the substitution pattern
at the benzoyl ring exerts some influence on the hydrolysis of
the carbonyloxymethyloxycarbonyl moieties.
Water solubility
The water solubility of all compounds described here are given
in Table 1. All products display good water solubility, in most
cases higher than that of ASA (3 gLꢀ1, The Merck Index 14th
Edition). For the solid hydrochloride salts, a general trend was
observed—higher solubility was associated with a lower melt-
ing point.
The products are more stable at physiological pH (phos-
phate buffer) than in serum; after an incubation period of
three hours, the sole metabolite detected was III. The hydroly-
sis strictly followed first-order kinetics, and the observed
pseudo-first-order constants (kobs) were calculated from the
slopes of linear plots of the logarithm of the remaining ester
against time. The corresponding half-lives (Table 1) were ob-
tained from Equation (1).
Hydrolysis studies
Most of the prodrugs described here (38–44) contain three
substructures susceptible to hydrolysis: the acetyloxy group,
the aminoacyloxy group, and the carbonyloxymethyloxycar-
bonyl moiety. The possible routes whereby these products can
be hydrolyzed are shown in Scheme 5. Hydrolysis was assessed
by high-performance liquid chromatography (HPLC) in pH 1
and pH 7.4 buffer solutions, as well as in human serum.
t1=2 ¼ 0:693=kobs
ð1Þ
The most stable prodrugs were found to be compounds 39
(86 min), 38 (57 min), and 44 (20 min), bearing a b-aminopro-
pionyloxy chain at the 3-position of the benzoyl ring. The
other compounds exhibited t1/2 values in the range 10–15 mi-
nutes. In pH 1 buffer solution, all products remained 70–90%
unchanged after three hours, and only hydrolysis product III
had formed at this point (Table 1).
In human serum, hydrolysis is catalyzed by carboxylesteras-
es—ubiquitous enzymes that display broad substrate specifici-
ty; the same ester can often be hydrolyzed by more than one
enzyme.[12] In human serum, the fate of all compounds was
very similar; the observed half-lives were less than one minute
(Table 1). The metabolites were monitored for six hours. After
ten minutes, only ASA, SA, IV, and traces of II and III were pres-
ent. This indicates that the aminoacyloxy and carbonyloxyme-
thyloxycarbonyl moieties hydrolyze simultaneously and much
more rapidly than the related acetyloxy functionalities. After
six hours, only SA and IV, accompanied by trace amounts of
ASA, were present. Figure 1 shows the concentration over time
of the metabolites of compound 38, as an example of the per-
formance of the class as a whole. The relationship shown is an
interesting result and shows that these products are true ASA
prodrugs. Maximal ASA production is reached for all com-
pounds after around two to five minutes (Table 1). Values for
Unlike the products discussed above, prodrug 49 bears
a water-solubilizing morpholinomethyl group directly linked to
the benzoyl scaffold, rather than through a vulnerable ester
bridge. Consequently, only the acetyloxy and carbonyloxyme-
thyloxycarbonyl moieties undergo hydrolysis, with a conse-
quent decrease in the number of possible metabolites
(Scheme 5). Compound 49 is more stable in serum than the
other prodrugs discussed above, due to the lack of the very
vulnerable aminoacyloxy function. Like the other prodrugs, its
deacetylation rate constant is lower than the hydrolytic rate
constant of the carbonyloxymethyloxycarbonyl moiety, which
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Pharmacological studies
Platelet antiaggregatory activity
The antiaggregatory activity of
the new NO-ASA derivatives was
assessed through a collagen-in-
duced platelet aggregation of
human platelet-rich plasma (PRP)
assay, using ASA as a reference
agent. The inhibitory effect of
a
compound was tested by
adding it to PRP ten minutes
before addition of the stimulus.
All products displayed a concen-
tration-dependent
inhibitory
effect. Their antiaggregatory po-
tencies, expressed as IC50 values,
are given in Table 1. As previous-
ly mentioned, compounds IV
and 48 are the metabolites
formed rapidly under the action
of serum esterases from the pro-
drugs under study. The antiag-
gregatory action of 28 and 48,
chosen as prototypes of these
acid metabolites, was also evalu-
ated. No antiaggregatory activity
was observed when the prod-
ucts were tested at a concentra-
tion of 300 mm, meaning that
NO does not play a significant
role in the antiaggregatory
effect of these prodrugs and
that this activity is largely due to
their capacity to produce ASA.
The relationship between AUC0–
and IC50 values for the
10 min
entire series of prodrugs studied
is depicted in Figure 3.
Vasodilator activity
Scheme 5. Possible hydrolytic routes of prodrugs bearing a) aminoacyloxy and b) morpholino solubilizing sub-
structures.
All the prodrugs can be expect-
ed to display vasodilator proper-
ties in vivo, due to the rapid for-
enables ASA to be present among the metabolites. The hydrol-
ysis strictly follows first-order kinetics. Its half-life, calculated
from the observed pseudo-first-order rate constant, is 2.2 mi-
nutes. Figure 2 shows the concentrations of the metabolites
over time for this product. The maximum concentration of ASA
is reached after six minutes, and the amount of ASA released
(% max and AUC values) is lower than the values measured for
all other prodrugs (Table 1). After six hours, only 48, SA, and
traces of ASA were present. The compound is very stable both
at pH 1 and at physiological pH.
mation of the relative NO-donor benzoic acid metabolites (IV
and 48) under the action of plasma esterases. The vasodilator
activity of two selected acids, 28 and 48, chosen as prototypes
of these metabolites, was evaluated on endothelium-denuded
rat aorta strips, precontracted with phenylephrine.
Compound 28, which contains the meta-hydroxybenzoic
acid moiety that is common to all the aminoacyloxy-substitut-
ed prodrugs, was found to relax the contracted tissue in a con-
centration-dependent manner (EC50 =11 ꢁ1 mm). When the ex-
periments were repeated in the presence of 1 mm 1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a known inhibi-
tor of soluble guanylate cyclase (sGC), the potency of 28 de-
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Figure 3. Correlation between ASA released during the first 10 min of incu-
bation in human serum (AUC0–10) and antiaggregatory activity (IC50).
trations of up to 100 mm. While compound 48 contains the
same para-nitrooxy alkyl chain as 28, the water solubilizing
group is covalently attached to the benzoic acid scaffold. The
lipophilic–hydrophilic balance of the two compounds differs
(28, logD7.4 =ꢀ0.80; 48, logD7.4 =ꢀ1.30) as do their steric and
electronic properties. At physiological pH, compound 28 exists
Figure 1. a) Concentration over time of prodrug 38 and its metabolites (28
and 37) in human serum during an incubation period of 3 h. b) A detail look
at the concentration of compounds during the first 10 min of incubation.
Values are mean of n=3 experiments; SEM<1.
principally as an anionic species (anion form >99%; pKa(COOH)
=
4.41) while 48 exists as a mixture of the anion and zwitterion
species (anion form=54%, zwitterion form=46%; pKa(COOH)
3.90, pKa(morpholine) =7.33).
=
As was observed in our assay, these diverse physicochemical
profiles give the two compounds different capacities both to
reach and to interact with the putative target enzyme involved
in NO production (i.e., sGC), thus affording different vasodilator
behavior. However, despite these observations, compound 48
could still release NO in a more complex biological environ-
ment; experiments incubating this compound in liver homoge-
nate pointed to a time-dependent production of nitrite and ni-
trate (NOx), the oxidized metabolites of NO derived from bio-
transformation of organic nitrates (data not shown).
Anti-inflammatory activity
NO-donor ASA prodrugs 38 and 49, and ASA as a reference
drug, were tested for their anti-inflammatory activity using
a carrageenan-induced paw edema test in conscious rats. The
intraplantar injection of carrageenan produced an immediate
increase in paw volume, with the inflammatory response peak-
ing four to five hours after carrageenan injection. The intragas-
tric administration of ASA (120 mgkgꢀ1) just prior to carrageen-
an injection led to a significant decrease in edema (57.4ꢁ
6.8%; p<0.01 versus vehicle-treated controls) assessed three
hours after ASA administration. Both prodrugs 38 and 49 ad-
ministered at doses equimolar to that used for ASA (i.e.,
120 mgkgꢀ1) caused a significant decrease in paw edema
(54.9ꢁ6.6% and 51.5ꢁ4.3%, respectively; p>0.01 versus ve-
hicle-treated controls). The extent of decrease achieved follow-
ing the administration of the two prodrugs was comparable to
that observed with ASA (Figure 4a).
Figure 2. a) Concentration over time of prodrug 49 and its metabolites in
human serum during an incubation period of 3 h. b) A detail look at the
concentration of compounds during the first 10 min of incubation. Values
are mean of n=3 experiments; SEM<1.
creased (EC50 >100 mm). This observation is in keeping with
the proposed mechanism of vasodilation through NO-induced
activation of sGC.
Conversely, upon evaluation in the same assay, compound
48 was found to be inactive as a vasodilator, even at concen-
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type of the acid metabolites derived from the aminoacyloxy-
substituted prodrugs, is endowed with NO-dependent vasodi-
lator activity, unlike compound 48, in which the solubilizing
group remains covalently attached to the benzoic acid scaf-
fold. The related prodrugs 38 and 49 display in vivo anti-in-
flammatory properties and decrease gastrotoxicity in rats.
Based on their pharmacological and physicochemical pro-
files, the compounds described here represent an improved
class of NO-donor ASA prodrugs more suitable for clinical ap-
plication than related less soluble prodrugs previously de-
scribed.
Figure 4. a) Anti-inflammatory effects of ASA and NO-donor ASA prodrugs
38 and 49 on carrageenan-induced paw edema in conscious rats. Results
are expressed as the percent inhibition of edema versus the vehicle-treated
Experimental Section
group (set to 100). p <0.01 versus vehicle (ANOVA, followed by Dunnett’s
**
Synthesis
test). Values are the mean ꢁSEM (n=6 rats per group). b) Gastric ulcerogen-
ic effects of ASA and NO-donor ASA prodrugs 38 and 49 in conscious rats.
Gastric lesions were measured along the greatest length, and the cumulative
length (mm) was designated as the lesion index for each stomach. All com-
General: Flash column chromatography was performed on silica
gel (Merck kiesel gel 60, 230–400 mesh ASTM). PE refers to petrole-
um ether 40–60. The progress of the reactions was monitored by
thin layer chromatography (TLC) on 5 cmꢁ20 cm plates, with
a layer thickness of 0.25 mm. Anhydrous Na2SO4 was used as the
drying agent of organic phases during work-up. Organic solvents
were removed in vacuo using a water bath at 308C.
pounds tested produced significantly less gastric damage than ASA.
**
p <0.01 (ANOVA and the Newman–Keuls test). Values are means ꢁSEM
(n=6 rats per group).
Acute gastric mucosal damage
1H and 13C NMR spectra were recorded on a Bruker Avance 300 at
300 and at 75 MHz, respectively, using SiMe4 as an internal stan-
dard. The following abbreviations are used to indicate peak multi-
plicity: s=singlet, d=doublet, t=triplet, m=multiplet, br s=
broad signal. Low-resolution mass spectra were recorded using
a Finnigan-Mat TSQ-700 mass spectrometer using chemical ioniza-
tion (CI) with isobutane; ESI mass spectra were recorded using
a Waters Micromass Quattro micro API. Melting points (mp) were
determined using a capillary apparatus (Buchi 540) and are uncor-
rected. Elemental analyses (C, H, N) were performed by Redox Ana-
lyticals (Monza, Italy), and results for all compounds tested were
within 0.4% of the theoretical values. Compounds 29[13] and 45[14]
were synthesized as described in the literature.
NO-donor ASA prodrugs 38 and 49, and ASA as a reference
drug, were administered intragastrically to conscious rats, and
after three hours, the lesion index of each rat stomach was as-
sessed. At a dose of 120 mgkgꢀ1, ASA treatment resulted in
the development of macroscopically detectable lesions located
in the gastric mucosa, characterized by necrosis and hemor-
rhage (LI=55.3ꢁ10.4 mm). In contrast, when administered at
a dose equimolar to 120 mgkgꢀ1 ASA, neither prodrug caused
macroscopically detectable damaging effects on the gastric
mucosa (Figure 4b).
4-(3-Bromopropoxy)-3-hydroxybenzaldehyde (5): A solution of 4
(5.00 g, 36.22 mmol), Br(CH2)3Br (7.35 mL, 72.44 mmol) and KHCO3
(4.35 g, 43.46 mmol) in CH3CN (100 mL) was heated at reflux for
4 h. The mixture was then poured into H2O (50 mL) and extracted
with EtOAc (50 mL); the organic layer was washed with saturated
aq NaHCO3 (2ꢁ50 mL) and extracted with 2n NaOH (2ꢁ50 mL).
The aqueous extracts were acidified with 6m HCl and then extract-
ed with EtOAc (3ꢁ50 mL). The combined organic layers were
dried, filtered, and concentrated in vacuo to give 5 as a pale
yellow semisolid (4.4 g, 47%): 1H NMR (300 MHz, CDCl3): d=2.43
(qi, 2H, OCH2CH2), 3.60 (t, 2H, CH2Br), 4.32 (t, 2H, OCH2), 5.88 (s,
1H, OH), 7.01 (d, 1H, C6H3), 7.42 (m, 1H, C6H3), 7.44–7.46 (m, 1H,
C6H3), 9.85 ppm (s, 1H, CHO); 13C NMR (75 MHz, CDCl3): d=29.3,
31.8, 66.9, 111.2, 114.4, 124.6, 130.8, 146.2, 150.9, 191.1 ppm; MS
(CI): m/z 259/261 [M+H]+.
Conclusions
We have developed a new class of (benzoyloxy)methyl esters
of acetylsalicylic acid (ASA), commonly known as aspirin. These
new derivatives have both alkyl chains containing NO-releasing
nitrooxy groups and aminoacyloxy solubilizing moieties ap-
pended to the benzoyl ring. When converted to the corre-
sponding salts, most of the derivatives were solid, with well-
defined melting points, and the entire class display good
water solubility and acid stability.
The compounds are rapidly metabolized under physiological
conditions. In human serum, under the action of esterases, the
aminoacyloxy and the carbonyloxymethyloxycarbonyl moieties
simultaneously hydrolyze, much more rapidly than the related
acetyloxy functionalities. The resulting mixture of metabolites
includes significant amounts of ASA, and consequently these
compounds can be considered true NO-donor ASA prodrugs.
All of the compounds display good antiaggregatory proper-
ties as evaluated in a collagen-induced platelet aggregation
assay using human platelet-rich plasma. This activity is roughly
related to the amount of ASA released in the first ten minutes
of incubation. The benzoic acid derivative containing a meta-
hydroxybenzoic acid moiety (28), which was studied as a proto-
4-(4-Chlorobutoxy)-3-hydroxybenzaldehyde (6): A solution of 4
(1.00 g, 7.24 mmol), 1-bromo-4-chlorobutane (2.50 mL, 21.7 mmol)
and KHCO3 (870 mg, 8.69 mmol) in CH3CN (20 mL) was heated at
reflux for 14 h. The mixture was then poured into H2O (30 mL) and
extracted with EtOAc (30 mL); the organic layer was washed with
saturated aq NaHCO3 (30 mL) and extracted with 2n NaOH (3ꢁ
30 mL). The aqueous extracts were acidified with 6m HCl and then
extracted with EtOAc (3ꢁ30 mL). The combined organic layers
were dried, filtered, and concentrated in vacuo to give 6 as
a white solid (1.0 g, 60%): mp: 58–598C (PE/toluene, 1:1 v/v);
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1H NMR (300 MHz, CDCl3): d=1.95–2.12 (m, 4H, OCH2CH2CH2), 3.65
(t, 2H, CH2Cl), 4.20 (t, 2H, OCH2CH2), 5.93 (s, 1H, OH), 6.97 (d, 1H,
C6H3), 7.41–7.46 (m, 2H, C6H3), 9.84 ppm (s, 1H, CHO); 13C NMR
(75 MHz, CDCl3): d=26.9, 29.6, 45.0, 68.9, 111.4, 114.8, 125.0, 131.2,
146.7, 151.6, 191.6 ppm; MS (CI): m/z 229/231 [M+H]+.
Chromatography conditions and characterization data for these
products are given in the Supporting Information.
General procedure for the preparation of N-BOC-protected de-
rivatives 9–14, 22: A stirred solution of BOC-protected amino acid
(9.38 mmol) in dry CH2Cl2 (60 mL) under an inert atmosphere was
treated at RT with EDC·HCl (3.96 g, 20.66 mmol) and DMAP (0.12 g,
0.92 mmol). After 30 min, the appropriate phenol derivative
(1.70 g, 7.05 mmol) was added. The reaction reached completion
after 3 h. The reaction mixture was washed with H2O (50 mL),
dried, filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography. Chromatography conditions and
characterization data for these products are given in the Support-
ing Information.
4-(Allyloxy)-3-hydroxybenzaldehyde (21): A solution of 4 (2.00 g,
14.48 mmol), allyl bromide (1.20 mL, 14.48 mmol) and KHCO3
(1.74 g, 17.37 mmol) in CH3CN (20 mL) was heated at reflux for 4 h.
The mixture was then poured into H2O (50 mL) and extracted with
EtOAc (50 mL); the organic layer was washed with saturated aq
NaHCO3 (2ꢁ50 mL) and extracted with 2n NaOH (2ꢁ50 mL). The
aqueous extracts were acidified with 6m HCl and then extracted
with EtOAc (3ꢁ50 mL). The combined organic layers were dried, fil-
tered, and concentrated in vacuo to give 21 as a pale yellow solid
(1.0 g, 40%): mp: 57–608C (iPr2O); 1H NMR (300 MHz, CDCl3): d=
4.69 (d, 2H, OCH2), 5.34–5.46 (m, AMX-like system, 2H, CH=CH2),
5.99–6.13 (m, AMX-like system, 2H, CH=CH2 +OH), 6.96 (d, 1H,
C6H3), 7.38–7.46 (m, 2H, C6H3), 9.83 ppm (s, 1H, CHO); 13C NMR
(75 MHz, CDCl3): d=69.9, 111.5, 114.4, 119.2, 124.4, 130.6, 131.8,
146.3, 150.9, 191.2 ppm; MS (CI): m/z 179 [M+H]+.
2-[2,3-Bis(nitrooxy)propyl]-5-formylphenyl 3-[(tert-butoxycarbo-
nyl)amino]propanoate (23): I2 (0.70 g, 2.78 mmol) was added por-
tionwise to a stirred solution of 22 (0.97 g, 2.78 mmol) and AgNO3
(0.47 g, 2.78 mmol) in CH3CN (10 mL) at ꢀ158C. At the end of the
addition, stirring was continued for 1 h. Additional AgNO3 (1.18 g,
6.95 mmol) was then added, and the mixture was heated to 708C
for 16 h. After cooling, the mixture was filtered through Celite, and
the filtrate was concentrated in vacuo. The residue was dissolved
in H2O (40 mL) and extracted with EtOAc (3ꢁ20 mL). The com-
bined organic layers were washed with brine (20 mL), dried, fil-
tered, and concentrated in vacuo. The crude product was purified
by flash chromatography (PE/EtOAc, 80:20 v/v) to give 23 as
a yellow oil (0.41 g, 31%): 1H NMR (300 MHz, CDCl3): d=1.46 (s,
9H, tBu), 2.84 (t, 2H, CH2CH2NH), 3.51–3.56 (m, 2H, CH2CH2NH),
4.38 (d, 2H, OCH2CH), 4.69–4.90 (m, AMX-like system, 2H,
CHCH2ONO2), 5.03 (svbr, 1H, NH), 5.62–5.66 (m, AMX-like system,
1H, CHCH2ONO2), 7.08 (d, 1H, C6H3), 7.64–7.65 (m, 1H, C6H3), 7.76–
7.81 (m, 1H, C6H3), 9.90 ppm (s, 1H, CHO); 13C NMR (75 MHz,
CDCl3): d=14.2, 27.9, 28.4, 34.5, 36.0, 60.5, 65.8, 68.4, 70.0, 113.0,
123.9, 130.0, 131.3, 140.2, 153.9, 155.9, 179.9, 189.8 ppm; MS (CI):
m/z 473 [M+H]+.
Methyl 4-(3-bromopropoxy)-3-hydroxybenzoate (26): A solution
of methyl 3,4-dihydroxybenzoate (2.10 g, 12.5 mmol), Br(CH2)3Br
(3.80 mL, 37.5 mmol) and KHCO3 (1.50 g, 14.53 mmol) in CH3CN
(20 mL) was heated at reflux for 5 h. The mixture was then poured
into H2O (50 mL) and extracted with EtOAc (2ꢁ30 mL); the com-
bined organic layers were washed with brine (10 mL), dried, fil-
tered, and concentrated in vacuo. The crude product was purified
by flash chromatography (PE/EtOAc, 9:1!7:3 v/v) to give 31 as
a white solid (0.91 g, 25%): mp: 106.5–107.58C (iPr2O); 1H NMR
(300 MHz, CDCl3): d=2.39 (qi, 2H, OCH2CH2), 3.58 (t, 2H, CH2Br),
3.88 (s, 3H, OCH3), 4.26 (t, 2H, OCH2CH2-), 5.75 (br s, 1H, OH), 6.90
(d, 1H, C6H3), 7.57–7.61 ppm (m, 2H, C6H3); 13C NMR (75 MHz,
CDCl3): d=29.4, 31.8, 50.0, 66.7, 110.8, 115.9, 122.8, 132.1, 145.3,
149.4, 166.8 ppm; MS (CI): m/z 289/291 [M+H]+.
Methyl 4-(3-bromopropoxy)-3-(morpholin-4-ylmethyl)benzoate
(46): A solution of 27 (2.00 g, 7.90 mmol) in CH3CN (12 mL) was
treated with K2CO3 (1.66 g, 12.0 mmol) and 1,3-Br(CH2)3Br (3.96 mL,
39.0 mmol), and the mixture was heated at reflux for 6 h. The reac-
tion mixture was then cooled, poured into H2O (50 mL), and ex-
tracted with EtOAc (3ꢁ50 mL). The combined organic layers were
washed with H2O (25 mL) and brine (25 mL), then dried, filtered,
and concentrated in vacuo. The crude product was purified by
flash chromatography (PE/EtOAc, 60:40 v/v) to give 46 as a color-
less oil (1.12 g, 38%): 1H NMR (300 MHz, CDCl3): d=2.36 (qi, 2H,
OCH2CH2), 2.48 (t, 4H, CH2NCH2), 3.53 (s, 2H, ArCH2N), 3.64–3.73 (m,
6H, CH2OCH2, CH2Br), 3.89 (s, 3H, OCH3), 4.19 (t, 2H, OCH2), 6.90 (d,
1H, C6H3), 7.94 (d, 1H, C6H3), 8.01 ppm (s, 1H, C6H3); 13C NMR
(75 MHz, CDCl3): d=29.8, 32.2, 51.9, 53.7, 56.7, 65.5, 67.0, 110.8,
122.4, 126.1, 130.6, 132.3, 160.6, 166.9 ppm; MS (CI): m/z 372/374
[M+H]+.
General procedure for the preparation of carboxylic acids 15–
20, 24: KMnO4 (0.52 g, 3.27 mmol) was added to a stirred solution
of the appropriate aldehyde (2.18 mmol) in acetone (50 mL) at
08C. The reaction was allowed to reach RT; it was complete after
1 h (TLC, CH2Cl2/MeOH, 95:5 v/v). Oxalic acid was added, and the
mixture was filtered. The filtrate was diluted with CH2Cl2 (100 mL),
and the organic layer was washed with H2O (2ꢁ50 mL), then dried,
filtered, and concentrated in vacuo to give the desired product.
Characterization data for these products are given in the Support-
ing Information.
3-Hydroxy-4-(3-nitrooxypropoxy)benzoic acid (28): A solution of
27 (0.56 g, 2.06 mmol) in THF (10 mL) and H2O (10 mL) was treated
with NaOH pellets (165 mg, 4.13 mmol). The mixture was heated
to 408C for 4 h. The residue was treated with EtOAc (10 mL) and
H2O (10 mL). After separation, the aqueous layer was acidified with
6n HCl and extracted with EtOAc (2ꢁ20 mL). The combined or-
ganic layers were dried, filtered, and concentrated in vacuo to give
28 as a yellow solid (0.31 g, 59%): mp: 166.5–169.58C (toluene);
1H NMR (300 MHz, [D6]DMSO): d=2.14 (qi, 2H, OCH2CH2), 4.12 (t,
2H, OCH2CH2), 4.74 (t, 2H, CH2ONO2), 7.00 (d, 1H, C6H3), 7.35–7.42
(m, 2H, C6H3), 9.27 (br s, 1H, OH), 12.58 ppm (br s, 1H, COOH);
13C NMR (75 MHz, [D6]DMSO): d=26.0, 64.4, 70.8, 112.4, 116.1,
121.4, 123.4, 146.2, 150.4, 167.0 ppm; MS (CI): m/z 258 [M+H]+.
General procedure for the preparation of nitrooxy derivatives 7,
8, 27, 47: A solution of the appropriate bromo/chloro derivative
(1.93 mmol) and AgNO3 (0.82 g, 4.82 mmol) in CH3CN (15 mL) was
stirred at 708C for 14 h. Brine was then added to precipitate excess
AgNO3, and the mixture was filtered through Celite. The filtrate
was concentrated in vacuo, and the residue was partitioned be-
tween EtOAc (50 mL) and H2O (50 mL). After separation, the aque-
ous layer was extracted with EtOAc (2ꢁ10 mL). The combined or-
ganic layers were dried, filtered, and concentrated in vacuo. The
crude product was purified, if necessary, by flash chromatography.
3-(Morpholin-4-ylmethyl)-4-[3-(nitrooxy)propoxy]benzoic
acid
(48): A solution of 29 (0.56 mmol) in MeOH (5 mL) and 1n NaOH
(2 mL) was stirred at RT for 4 h. The solvent was then removed in
vacuo, the residue was redissolved in H2O (10 mL), the pH was ad-
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justed to 7, and the product extracted in continuous cycle for 18 h
with EtOAc (200 mL). The organic layer was dried, filtered, and con-
centrated in vacuo to give 48 as colorless oil (0.15 g, 67%): H NMR
(300 MHz, CDCl3): d=2.28 (qi, 2H, OCH2CH2), 2.79 (m, 4H,
CH2NCH2), 3.77 (m, 4H, CH2OCH2), 3.85 (s, 2H, NCH2Ar), 4.21 (t, 2H,
OCH2), 4.74 (m, 2H, CH2ONO2), 7.07 (d, 1H, C6H3), 7.98 (d, 1H, C6H3),
8.04 ppm (s, 1H, C6H3); 13C NMR (75 MHz, CDCl3): d=28.0, 54.2,
57.1, 66.1, 66.9, 71.9, 112.2, 123.7, 126.4, 133.0, 134.7, 161.8,
171.0 ppm; MS (CI): m/z 341 [M+H]+.
3.13 (m, 2H, CH2CH2NH2), 4.21 (t, 2H, OCH2CH2), 4,63 (t, 2H,
CH2ONO2), 6.14 (s, 2H, OCH2O), 7.26–7.34 (m, 2H, C6H3 +C6H4), 7.44
(t, 1H, C6H4), 7.71–7.77 (m, 1H, C6H4), 7.82–7.83 (m, 1H, C6H3), 7.93–
8.00 (m, 2H, C6H3 +C6H4), 8.22 ppm (br s, 2H, NH2); 13C NMR
(75 MHz, CDCl3): d=21.0, 26.6, 31.0, 35.5, 65.0, 70.1, 80.0, 112.4,
121.7, 122.0, 124.0, 124.8, 126.2, 129.9, 132.3, 134.7, 139.1, 151.0,
154.4, 163.1, 164.1, 169.0, 169.7 ppm; MS (ESI+): m/z 521 [M+H]+;
Anal. calcd for C23H24N2O12·HCl: C 49.60, H 4.52, N 5.03, found: C
49.24, H 4.50, N 5.04.
1
General procedure for the preparation of ASA esters 30–37, 49:
A solution of 29 (0.58 g, 2.52 mmol) in DMF (8 mL) was treated
with the appropriate carboxylic acid derivative (2.52 mmol) and
Cs2CO3 (0.41 g, 1.26 mmol). The mixture was stirred at RT for 24 h,
then poured into H2O (30 mL) and extracted with EtOAc (3ꢁ5 mL).
The combined organic layers were dried, filtered, and concentrated
in vacuo. The crude product was purified by flash chromatography.
Chromatography conditions and characterization data for these
products are given in the Supporting Information.
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-
4-[3-(nitrooxy)butoxy]benzoate·HCl (39): The crude product was
purified by flash chromatography (H2O/CH3CN/HCl, 60:40:0.1 v/v/v)
to give 39 as a colorless oil (0.57 g, 50%): 1H NMR (300 MHz,
[D6]DMSO+CDCl3): d=1.78–1.94 (m, 4H, OCH2CH2CH2), 2.28 (s, 3H,
CH3CO), 3.09 (t, 2H, CH2CH2NH), 3.21 (t, 2H, CH2CH2NH), 4.13 (t, 2H,
OCH2CH2), 4.58 (t, 2H, CH2ONO2), 6.14 (s, 2H, OCH2O), 7.17–7.21 (m,
2H, C6H3 +C6H4), 7.42 (t, 1H, C6H4), 7.81 (t, 1H, C6H4), 7.79–7.80 (m,
1H, C6H3), 7.94–8.03 (m, 2H, C6H3 +C6H4), 8.34 ppm (br s, 2H, NH2);
13C NMR (75 MHz, [D6]DMSO+CDCl3): d=21.4, 23.8, 25.5, 31.9,
35.4, 68.7, 73.8, 80.6, 113.7, 121.4, 122.7, 124.7, 125.0, 126.9, 130.3,
132.3, 135.5, 139.6, 151.2, 155.4, 163.5, 164.3, 169.0, 169.7 ppm; MS
(ESI+): m/z 535 [M+H]+; Anal. calcd for C24H26N2O12·HCl·2.5H2O: C
46.80, H 5.24, N 4.55, found: C 46.74, H 4.71, N 4.38.
{[2-(Acetyloxy)benzoyl]oxy}methyl 3-hydroxy-4-(3-nitrooxypro-
poxy)benzoate (37): Eluent: PE/EtOAc (90:10!70:30 v/v); white
solid (0.42 g, 37 %); mp: 84.5–85.58C; 1H NMR (300 MHz, CDCl3):
d=2.26 (qi, 2H, -OCH2CH2-), 2.35 (s, 3H, CH3CO-), 4.22 (t, 2H,
OCH2CH2-), 4.65 (t, 2H, -CH2ONO2), 5.62 (s, 1H, OH), 6.16 (s, 2H,
-OCH2O-), 6.85 (d, 1H, C6H3), 7.12 (d, 1H, C6H4), 7.32 (t, 1H, C6H4),
7.56–7.67 (m, 3H, C6H3 +C6H4), 8.07–8.10 ppm (m, 1H, C6H4);
13C NMR (75 MHz, CDCl3): d=21.0, 26.9, 65.1, 69.6, 79.8, 110.8,
116.5, 122.1, 122.5, 123.5, 124.0, 126.1, 132.3, 134.6, 145.4, 149.8,
151.0, 163.1, 164.7, 169.8 ppm; MS (CI): m/z 450 [M+H]+; Anal.
calcd for C20H19NO11: C 53.46, H 4.26, N 3.12, found: C 53.08, H 4.42,
N 3.10.
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(2-amino)acetoxy]-4-[3-(ni-
trooxy)propoxy]benzoate·HCl (40): The crude product was tritu-
rated with dry Et2O to give 40 as a white solid (0.43 g, 40%): mp:
1
60.5–62.58C; H NMR (300 MHz, CDCl3): d=2.16 (m, 2H, OCH2CH2),
2.31 (s, 3H, CH3CO), 3.99 (m, 2H, CH2NH2 +OCH2CH2), 4.20 (t, 2H,
CH2NH2 +OCH2CH2), 4.53 (t, 2H, CH2ONO2), 6.16 (s, 2H, OCH2O),
6.83 (d, 1H, C6H3), 7.08 (d, 1H, C6H4), 7.27 (t, 1H, C6H4), 7.55 (t, 1H,
C6H4), 7.71 (s, 1H, C6H3), 7.86 (d, 1H, C6H3), 8.03 (d, 1H, C6H4),
8.69 ppm (br s, 3H, NH3+); 13C NMR (75 MHz, CDCl3): d=20.9, 26.5,
40.4, 65.2, 70.5, 79.9, 112.5, 121.5, 122.0, 124.0, 124.3, 126.2, 130.2,
132.2, 134.7, 138.5, 151.0, 154.2, 158.4, 163.0, 163.9, 165.7,
169.7 ppm; MS (ESI+): m/z 507 [M+H]+; Anal. calcd for
C22H20N2O12·HCl·0.5H2O: C 47.88, H 4.38, N 5.08, found: C 47.82, H
4.35, N 5.37.
{[2-(Acetyloxy)benzoyl]oxy}methyl 3-(morpholin-4-ylmethyl)-4-
[3-(nitrooxy)propoxy]benzoate·H2C2O4 (49): The crude product
was purified by flash chromatography (PE/EtOAc 60:40 v/v) to give
{[2-(acetyloxy)benzoyl]oxy}methyl 4-(morpholin-4-ylmethyl)-3-[3-(ni-
trooxy)propoxy]benzoate (0.38 g) as a colorless oil. To a solution of
{[2-(acetyloxy)benzoyl]oxy}methyl 4-(morpholin-4-ylmethyl)-3-[3-(ni-
trooxy)propoxy]benzoate in EtOAc (3 mL) was added a solution of
H2C2O4 (0.06 g, 1 equiv) in EtOAc (2 mL), and the title compound
(0.25 g, 20%) was obtained by filtration as a white solid; mp: 135–
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(4-amino)butanoyl]oxy-4-
[3-(nitrooxy)propoxy]benzoate·HCl (41): The crude product was
purified by flash chromatography (H2O/CH3CN/HCl, 70:30:0.1 v/v/v)
to give 41 as a colorless oil (0.48 g, 42%): 1H NMR (300 MHz,
CDCl3): d=2.14–2.18 (m, 4H, OCH2CH2 +CH2CH2NH2), 2.32 (s, 3H,
CH3CO), 2.74 (t, 2H, COCH2CH2), 3.10–3.20 (m, 2H, CH2CH2NH2),
4.07 (t, 2H, OCH2CH2), 4.56 (t, 2H, CH2ONO2), 6.12 (s, 2H, OCH2O),
6.90 (d, 1H, C6H3), 7.09 (d, 1H, C6H4), 7.29 (t, 1H, C6H4), 7.56 (t, 1H,
C6H4), 7.75–7.76 (m, 1H, C6H3), 7.90–7.94 (m, 1H, C6H3), 8.02–8.06
(m, 1H, C6H4), 8.30 ppm (br s, 3H, NH3+); 13C NMR (75 MHz, CDCl3):
d=21.0, 22.6, 26.7, 30.6, 39.2, 65.0, 70.0, 79.9, 112.4, 121.7, 122.0,
124.0, 124.8, 126.2, 129.8, 132.2, 134.7, 139.4, 151.0, 154.5, 163.1,
164.1. 169.7, 170.4 ppm; MS (ESI+): m/z 535 [M+H]+; Anal. calcd
for C24H26N2O12·HCl: C 44.58, H 3.81, N 4.00, found: C 44.24, H 3.86,
N 3.81.
1
1398C (dec.); H NMR (300 MHz, [D6]DMSO): d=2.17–2.25 (m, 5H,
-OCH2CH2-+-COCH3), 2.71 (m, 4H, —CH2NCH2-), 3.64 (m, 4H,
-CH2OCH2-), 3.83 (s, 2H, -NCH2Ar), 4.20 (t, 2H, -OCH2-), 4.74 (t, 2H,
-CH2ONO2), 6.14 (s, 2H, -OCH2O-), 7.19 (d, 1H, C6H4), 7.27 (d, 1H,
C6H3), 7.42 (t, 1H, C6H4), 7.73 (t, 1H, C6H3), 7.96–8.05 (m, 3H, C6H3 +
C6H4), 8.06 ppm (s, 1H, C6H4); 13C NMR (75 MHz, [D6]DMSO): d=
20.9, 26.3, 52.7, 54.9, 65.2, 65.3, 71.3, 80.5, 112.2, 120.5, 122.2,
124.5, 126.7, 131.8, 132.1, 133.2, 135.4, 150.5, 161.6, 162.6, 163.1,
164.3, 169.3 ppm; MS (ESI+): m/z 533 [M+H]+; Anal. calcd for
C25H17N2O11·H2C2O4: C 52.09, H 4.86, N 4.50, found: C 52.04, H 4.82,
N 4.48.
General procedure for the preparation of amino acid derivatives
38–44: A solution of the appropriate BOC-protected intermediate
(2.00 mmol) in dry dioxane HCl 2.36m (12.6 mL, 29.0 mmol) was
stirred under inert atmosphere for 3 h. The mixture was concen-
trated in vacuo. Purification details and characterization data for
these products are given below.
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(2-amino)propanoyl]oxy-4-
[3-(nitrooxy)propoxy]benzoate·HCl (42): The crude product was
purified by flash chromatography (H2O/CH3CN/HCl, 80:20:0.1!
60:40:0.1 v/v/v) to give 42 as a white solid (0.5 g, 44%): mp: 65.5–
69.08C; 1H NMR (300 MHz, CDCl3): d=1.78 (d, 3H, CHCH3), 2.09–
2.11 (m, 2H, OCH2CH2), 2.32 (s, 3H, CH3CO), 4.03 (m, 2H, OCH2CH2),
4.40 (m, 1H, CHCH3), 4.53 (t, 2H, CH2ONO2), 6.12 (s, 2H, OCH2O),
6.84 (d, 1H, C6H3), 7.10 (d, 1H, C6H4), 7.29 (t, 1H, C6H4), 7.65 (t, 1H,
C6H4), 7.75 (s, 1H, C6H3), 7.86 (d, 1H, C6H3), 8.02 (d, 1H, C6H4),
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-
4-[3-(nitrooxy)propoxy]benzoate·HCl (38): The crude product was
triturated with dry Et2O to give 38 as a white solid (0.99 g, 89%):
mp: 94.5–95.58C; 1H NMR (300 MHz, [D6]DMSO): d=2.16 (qi, 2H,
OCH2CH2), 2.25 (s, 3H, CH3CO), 3.01–3.06 (m, 2H, CH2CH2NH2), 3.11–
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8.84 ppm (br s, 3H, NH3+); 13C NMR (75 MHz, CDCl3): d=16.1, 21.0,
26.6, 65.1, 70.1, 79.9, 112.4, 121.6, 122.0, 124.0, 124.4, 126.2, 130.3,
132.2, 134.7, 138.5, 151.0, 154.2, 163.0, 163.9, 167.8, 169.7 ppm; MS
(ESI+): m/z 521 [M+H]+; Anal. calcd for C23H24N2O12·HCl·0.5H2O: C
48.82, H 4.63, N 4.95, found: C 48.47, H 4.44, N 5.00.
and quantitated the remaining test compound and the hydrolysis
products (ASA, SA, nitrooxy-substituted carboxylic acid).
HPLC analyses were performed with a HP1100 chromatograph
system (Agilent Technologies, Palo Alto, USA) equipped with a qua-
ternary pump (model G1311A), a membrane degasser (G1379A),
and a diode-array detector (DAD; model G1315B) integrated into
the HP1100 system. Data were analyzed using the HP ChemStation
system (Agilent Technologies). The analytical column was a Nucleo-
sil 100-5 C18 Nautilus (250ꢁ4.6 mm, 5 mm particle size) (Macherey–
Nagel). The samples were analyzed using a gradient method, em-
ploying a mobile phase consisting of CH3CN/H2O with 0.1% TFA:
40:60 (v/v) over the first 2 min, increasing to 60:40 at 11 min, re-
maining at 60:40 until 16 min, and then returning to 40:60 at
20 min. The flow-rate was 1.2 mLminꢀ1. The injection volume was
20 mL (Rheodyne, Cotati, CA). The column effluent was monitored
at 226 nm (for all prodrugs and the majority of metabolites) and at
240 nm (for salicylic acid) referenced against a 600 nm wavelength.
Quantitation was done using calibration curves for compounds
and the metabolites, chromatographed under the same conditions;
the linearity of the calibration curves was determined in a concen-
tration range of 1–200 mm (r2 >0.99).
{[2-(Acetyloxy)benzoyl]oxy}methyl3-[2-amino-3-hydroxybutoxy]-
4-(3-nitrooxypropoxy)benzoate·HCl (43): The crude product was
triturated with dry Et2O to give 43 as a white solid (0.82 g, 70%):
mp: 87.5–88.58C; 1H NMR (300 MHz, CDCl3): d=1.40 (d, 3H,
CHCH3), 2.08 (m, 2H, OCH2CH2), 2.24 (s, 3H, CH3CO), 3.95 (t, 2H,
OCH2CH2), 4.26 (m, 1H, CHCH3), 4.35 (m, 1H, OH), 4.44 (t, 2H,
CH2ONO2), 6.01 (s, 2H, OCH2O), 6.90 (d, 1H, C6H3), 7.00 (d, 1H,
C6H4), 7.22 (t, 1H, C6H4), 7.47 (t, 1H, C6H4), 7.73 (s, 1H, C6H3), 7.82
(d, 1H, C6H3), 7.95 (d, 1H, C6H4), 8.83 ppm (br s, 3H, NH3+);
13C NMR (75 MHz, CDCl3): d=13.2, 19.5, 19.9, 25.5, 64.1, 65.2, 69.1,
79.0, 120.5, 120.9, 122.9, 125.2, 129.3, 131.1, 133.7, 137.4, 150.0,
153.2, 162.0, 162.2, 162.9, 165.1, 168.7 ppm; MS (ESI+): m/z 551
[M+H]+; Anal. calcd for C24H26N2O13·HCl·H2O: C 48.94, H 4.96, N
4.75, found: C 48.96, H 4.70, N 4.79.
{[2-(Acetyloxy)benzoyl]oxy}methyl-3-[(3-[aminopropanoyl)oxy]-
4-[2,3-bis(nitrooxy)propoxy]benzoate·HCl (44): The crude product
was triturated with dry Et2O to give 44 as a colorless foam (0.97 g,
1
79%): H NMR (300 MHz, [D6]DMSO): d=2.26 (s, 3H, CH3CO), 3.02–
Biological evaluation
3.13 (m, 4H, CH2CH2NH2), 4.46–4.62 (m, AMX-like system, 2H,
OCH2CH), 4.85–5.06 (m, AMX-like system, 2H, CHCH2ONO2), 5.82–
5.87 (m, 1H, CHCH2ONO2), 6.15 (s, 2H, OCH2O), 7.28 (d, 1H, C6H3),
7.36–7.47 (m, 2H, C6H3 +C6H4), 7.71–7.74 (m, 1H, C6H4), 7.76–7.77
(m, 1H, C6H3), 7.95–8.00 (m, 2H, C6H3 +C6H4), 8.33 ppm (br s, 2H,
NH2); 13C NMR (75 MHz, [D6]DMSO): d=20.6, 31.0, 34.3, 66.2, 69.7,
77.4, 80.3, 113.8, 121.5, 121.9, 124.2, 124.5, 126.3, 129.6, 131.5,
135.1, 138.8, 150.1, 153.7, 163.4, 166.4, 168.1, 169.0 ppm; MS (ESI+):
m/z 582 [M+H]+. Due to the high hygroscopicity and instability of
the product at RT, elemental analysis was not possible. The purity
was determined by HPLC analysis to be >99%.
Ethical statement: Both the concept and protocols for studies in-
volving animals were approved by the Italian Ministry of Health,
and as few animals as possible were used in each experiment.
Where human tissue was used, volunteers, who were treated ac-
cording to the Helsinki protocol for biomedical experimentation,
gave their informed consent for the use of samples for research
purposes.
Animals: Male Wistar rats were obtained from Harlan Laboratories,
San Pietro al Natisone, Italy and individually housed in hanging
stainless-steel cages with grid floors at constant room temperature
(25ꢁ18C) and humidity (60ꢁ5%), with an artificial 12:12 h light/
dark cycle.
Evaluation of the physicochemical properties
Solubility: The solubility of test compounds in water was deter-
mined at 25ꢁ18C. Excess amounts of test compound was added
to water in test tubes, and the mixtures were sonicated for 10 min
and then stirred for a further 30 min. This time was chosen to mini-
mize hydrolysis during the test. After filtration, an aliquot of the fil-
trate was diluted with an appropriate amount of water, and ana-
lyzed by HPLC for quantitation of remaining intact compound and
any degradation products.
Inhibition of human platelet aggregation in vitro: Venous blood
samples were obtained from healthy volunteers who had not
taken any drug for at least two weeks. Platelet-rich plasma (PRP)
was prepared by centrifugation of citrated blood at 200 g for
20 min. Aliquots (500 mL) of PRP were placed in aggregometer
(Chrono-log 4902D) cuvettes, and aggregation was recorded as in-
creased light transmission under continuous stirring (1000 rpm) at
378C for 10 min after addition of the stimulus. Collagen at a sub-
maximal concentration (0.8–1.5 mgmLꢀ1) was used as platelet acti-
vator in PRP. The compounds under study were preincubated with
PRP for 10 min before addition of the stimulus (collagen). Drug ve-
hicle alone (0.5% DMSO) added to PRP did not affect platelet func-
tion in control samples. The antiaggregatory activity of the tested
compounds is expressed as percent inhibition of platelet aggrega-
tion compared with vehicle control samples. For most of the active
compounds, IC50 values could be calculated by nonlinear regres-
sion analysis; when this was not possible, percent inhibition at
maximal concentration tested (300 mm) is reported. The number of
experiments was 3–5 for IC50 values, and 3 for determination of
percent inhibition.
Stability: For evaluation in buffers, a solution of test compound
(10 mm) in CH3CN/H2O (50:50, v/v) was added to 0.1m HCl (pH 1),
50 mm phosphate buffer (pH 7.4) preheated to 378C. The final
compound concentration was 100 mm. The resulting solution was
maintained at 37ꢁ0.58C and, at appropriate time intervals, a 20 mL
aliquot of reaction solution was analyzed by RP-HPLC, as described
below. For evaluation in human serum, a solution of test com-
pound (10 mm) in CH3CN/H2O (50:50, v/v) was added to human
serum (male AB plasma, Sigma) preheated to 378C. The final com-
pound concentration was 200 mm. The resulting solution was incu-
bated at 37ꢁ0.58C and, at appropriate time intervals, 300 mL of re-
action mixture were withdrawn and added to 300 mL of CH3CN
containing 0.1% trifluoroacetic acid (TFA), in order to deproteinize
the serum. The sample was sonicated, vortexed, and then centri-
fuged for 10 min at 2150 g. The clear supernatant was filtered
through 0.45 mm PTFE filters (Alltech) and analyzed by RP-HPLC, as
described below. The reverse-phase HPLC procedure separated
Vasodilator activity: Thoracic aortas were isolated from male Wistar
rats weighing 180–200 g. The endothelium was removed, and the
vessels were helically cut: 4–6 strips were obtained from each
aorta. The tissue was mounted under 1.0 g tension in organ baths
containing 30 mL of Krebs bicarbonate buffer, with the following
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composition: NaCl (111.2 mm), KCl (5.0 mm), CaCl2 (2.5 mm), MgSO4
(1.2 mm), KH2PO4 (1.0 mm), NaHCO3 (12.0 mm), glucose (11.1 mm),
maintained at 378C and gassed with 95% O2/5% CO2 (pH 7.4). The
aortic strips were allowed to equilibrate for 120 min and then con-
tracted with l-phenylephrine (1 mm). When the response to the ag-
onist reached a plateau, cumulative concentrations of the vasodi-
lating agent were added. Results are expressed as the EC50 value;
data are the mean ꢁSEM of three experiments. The effects of ODQ
(1 mm) on relaxation were evaluated in a separate series of experi-
ments, in which the compound was added to the organ bath
5 min before contraction. Responses were recorded by an isomet-
ric transducer connected to the MacLab System PowerLab. Addi-
tion of drug vehicle (1% DMSO) had no appreciable effect on con-
traction.
the mean ꢁSEM. Statistical analysis was performed with ANOVA,
followed by the Newman–Keuls test.
Acknowledgements
This work was supported by a grant from the Italian Ministry for
University and Research (MIUR), Studi e Ricerche Finalizzate 40%
Roma (PRIN2009, 2009K4NFZ4_002).
Keywords: anti-inflammatory agents · acetylsalicylic acid ·
nitric oxide · prodrugs · water solubility
Anti-inflammatory activity: Acute edema was induced in conscious
male Wistar rats weighing 180–200 g by intraplantar injection of
1% carrageenan (0.1 mL suspended in 1% carboxymethylcellulose
(CMC)) into the right hind paw. Immediately after carrageenan in-
jection, compound or vehicle (1% CMC) was administered intragas-
trically to different groups of rats in a volume of 10 mLkgꢀ1. ASA
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was administered as a reference drug at the dose of 120 mgkgꢀ1
,
and test compounds were administered at a dose equimolar to
120 mgkgꢀ1 ASA. Groups of six animals were used. Paw volume
was measured with a water plethysmometer (Basile, Comerio, Italy)
immediately before carrageenan injection and 3 h afterwards. The
edema volume decrease in treated animals was expressed as per-
cent inhibition of the edema observed in vehicle-treated animals,
taken as 100. Data are the mean ꢁSEM. Statistical analysis was per-
formed with ANOVA, followed by Dunnett’s test.
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Gastrotoxicity: Male Wistar rats weighing 180–200 g were deprived
of food but not of water 24 h before the experiments. By the intra-
gastric route, groups of rats (n=6) were given ASA 120 mgkgꢀ1 or
equimolar doses of test compound, using 1% CMC as the vehicle.
Rats were killed 3 h after administration of the compound. Immedi-
ately after death, the stomach was removed, opened along the
lesser curvature, and examined to assess mucosal lesions. The
stomach was laid on a flat surface under a stereomicroscope. The
glandular mucosa was examined, and each individual hemorrhagic
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. Data are
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Received: March 5, 2013
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