Searching for new NO-donor aspirin-like molecules: Furoxanylacyl derivatives of salicylic acid and related furazans

Article abstract of DOI:10.1016/j.bmc.2011.08.018

A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furazans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid (pH 1) and physiological solutions (pH 7.4), and

Full text of DOI:10.1016/j.bmc.2011.08.018

Bioorganic & Medicinal Chemistry 19 (2011) 5852–5860
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Searching for new NO-donor aspirin-like molecules: Furoxanylacyl
derivatives of salicylic acid and related furazans
Loretta Lazzarato a, Clara Cena a, Barbara Rolando a, Elisabetta Marini a, Marco Lucio Lolli a,
Stefano Guglielmo a, Elena Guaita b, Giuseppina Morini b, Gabriella Coruzzi b, Roberta Fruttero a,
Alberto Gasco a
⇑
,
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
b Istituto di Farmacologia, Università degli Studi di Parma, Via Volturno 39, 40100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furaz-
ans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid
(pH 1) and physiological solutions (pH 7.4), and in human serum. No compound exhibited COX-inhibitory
Received 13 June 2011
Revised 27 July 2011
Accepted 9 August 2011
Available online 16 August 2011
activity against COX-1 and COX-2 isoforms, when tested up to 100 lM, respectively, on isolated platelets
and on monocytes. Phenylsulfonyl- and cyano-substituted furoxans inhibited platelet aggregation
induced by collagen in human platelet-rich plasma, through a cGMP dependent mechanism. Furoxan
derivatives displayed cGMP-dependent vasodilator activities, tested on rat aorta strips precontracted
with phenylephrine. All products showed anti-inflammatory activity similar to that of ASA, tested on rats
by the carrageenan-induced paw edema assay. Unlike ASA, all products showed markedly reduced
gastrotoxicity in a rat lesion model.
Keywords:
NO-aspirins
NO-NSAIDs
Multitarget drugs
Anti-inflammatory activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
for the antithrombotic effect of ASA. By contrast, inhibition of the
COX-2 enzyme, prevalently an inducible isoform expressed by
The major limitation on the prolonged use of aspirin (ASA) 1
(Fig. 1) is its gastrotoxicity, responsible for gastric ulceration, exac-
erbation of peptic ulcer symptoms, gastrointestinal hemorrhage,
erosive gastritis, delay in ulcer healing and, in some cases,
death.1–3
inflammatory stimuli in many tissues, is largely responsible for
the drug’s anti-inflammatory, analgesic, and antiproliferative ac-
tions.9 ASA’s gastrotoxicity involves local and systemic mecha-
nisms.1,10,11 Local mechanisms include interaction with
phospholipids, weakening of the hydrophobic surface barrier in
membranes, and the diffusion and subsequent entrapment of the
drug into the mucosal cell, with consequent trapping of hydrogen
ions, or both. These events principally depend on the product’s pKa
and lipophilicity. The systemic mechanisms are mainly related to
the drug’s ability to inhibit the COX-1 enzyme present in gastric
epithelial cells, and consequently to block the production of pros-
taglandins, known to be crucial in defending the gastric mucosa. In
view of the gastrosparing and anti-inflammatory actions exerted
by nitric oxide (NO),12,13 one of the strategies proposed to over-
come the problem of ASA’s gastrotoxicity is that of linking the drug
with an appropriate NO-donor moiety. Most such products are ob-
tained by linking the NO-donor moieties to the carboxylic site of
aspirin through an ester linkage (Fig. 1A).14–17 Recently, we pro-
posed a new class of NO-donor aspirin-like molecules obtained
by substituting the acetyl group of aspirin with acyl groups, con-
taining nitrooxy NO-donor moieties (Fig. 1B).18 In order to explore
the influence exerted by the nature of the NO-donor moiety on the
pharmacological profile of this new type of products, we here de-
scribe the synthesis and pharmacological activity of a new class
This is a prominent problem, in view of the widespread use of
ASA to treat headache, rheumatic pain and inflammation, as well
as for its effective antithrombotic activity.1,4,5 A number of new
therapeutic perspectives are emerging for this enigmatic and
intriguing drug, including its ability to reduce the risk of colorectal
adenoma or cancer, and that of the recurrence of colorectal ade-
noma in high risk patients.6–8 The beneficial pharmacological ac-
tions of ASA are predominantly dependent on its ability to
inhibit both isoforms of the COX-enzyme, with a preference for
the COX-1 isoform, with consequent inhibition of prostanoid pro-
duction.9 Unlike the other nonsteroidal anti-inflammatory drugs
(NSAIDs), ASA irreversibly inhibits both COX-1 and COX-2, by
forming a covalent bond with the serine residue (Ser530) positioned
in the arachidonic acid-binding channel of the enzymes. COX-1 is
prevalently a constitutive enzyme, present in many tissues and
cells, including platelets, and it is thought to be largely responsible
⇑
Corresponding author.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.018

L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
5853
4, 4a, 5 and 5a, were obtained by oxidation of the corresponding
alcohols 2, 2a, 3 and 3a with Jones reagent in acetone solution. To
prepare carbamoyl- and cyano-substituted compounds 8, 8a, 10
and 10a, 4-bromomethylfuroxan-3-carboxamide (6) was treated
with methyl thioglycolate in acetonitrile solution in the presence
of Et3N to give the ester 7. Ester 7, containing the furoxan ring,
was reduced with trimethyl phosphite (P(OCH3)3) to give the result-
ing furazan ester 7a. Dehydration of 7 and 7a with trifluoroacetic
anhydride in THF, in the presence of pyridine (Py), afforded 9 and
9a. Hydrolysis of 7, 7a, 9 and 9a with 6 M hydrochloric acid in diox-
ane yielded the expected acids. The preparation of the final products
13, 13a, 14, 14a, 17, 17a, 18 and 18a is depicted in Scheme 2. The
appropriate acids were transformed into the corresponding chlo-
rides 11, 11a, 12, 12a, 15, 15a, 16 and 16a. These products were
immediately coupled with salicylic acid in THF or CH2Cl2 solution
in the presence of pyridine to give the target compounds.
2.2. Stability in aqueous buffered solutions and human serum
The stability of all end products was studied in aqueous solu-
tions at pH 1 and 7.4, as well as in human serum. The progress
of the transformation was evaluated by high-performance liquid
chromatography (HPLC). The results are shown in Table 1. All prod-
ucts remained largely unchanged after 3 h of incubation in acidic
medium. At physiological pH, both furoxans and furazan analogues
were transformed into salicylic acid, accompanied prevalently by
the native heterocyclic acids. The transformation strictly followed
first-order kinetics. The observed pseudo-first-order rate constants
(kobs) and the half-lives (t1/2, Table 1) were determined by fitting
the remaining ester against time with one-phase exponential de-
cay equation (Graph Pad, Prism software vers. 5).
Figure 1. (a) Structure of aspirin and general structures of NO-donor aspirin co-
drugs; (b) general structure of NO-donor salicylic acid co-drugs.
of compounds, obtained by substituting acyl groups containing
NO-donor furoxan moieties for acetyl group of ASA (compounds
13, 14, 17 and 18, Fig. 2). Furoxans are NO-donors able to release
NO through a mechanism different from that occurring for the
organic esters of nitric acid. It is generally accepted that they can
produce NO under the action of thiol cofactors,19 while nitrooxy
derivatives require principally enzymatic metabolism.20 This new
class of products might be expected to display a pharmacological
profile different from that observed for the nitrooxy analogues,21
in the light of both the different structure and the different NO-re-
lease mechanism of the corresponding NO-donor moieties. In this
study, the related furazan derivatives 13a, 14a, 17a and 18a
(Fig. 2), devoid of NO-releasing (des-NO) properties, are also con-
sidered, for comparison purposes.
Analysis of Table 1 shows that the products containing a sulfur
atom
a-positioned to the ester function are less stable than the
others, and that the stability of pairs of related furazan and furoxan
esters is similar. Different results were obtained working in human
serum, where the compounds can interact with serum esterases
and other serum proteins, in particular with albumin. After 2 h,
stoichiometric amounts of salicylic acid were recovered from car-
bamoyl- and cyano-substituted compounds containing sulfur, 17,
17a, 18 and 18a. Conversely, the native acid components were only
partly recovered because of their instability in this medium. In the
case of the remaining products, the formation of salicylic acid was
not stoichiometric, but was accompanied by a number of other
metabolites. In all cases, the disappearance of the initial products
followed a pseudo-first-order kinetics, with half-lives markedly
lower than those measured at physiological pH, and falling within
a narrower range.
2. Results and discussion
2.1. Chemistry
2.3. COX inhibitory activity
The synthesis of the end products required the preliminary
preparation of the carboxylic acid 4, 4a, 5, 5a, 8, 8a, 10 and 10a
(Scheme 1). Phenyl- and phenylsulfonyl-substituted compounds
Compounds were assayed for COX-1 and COX-2 inhibition.
COX-1 inhibitory activity was evaluated on isolated human
Figure 2. Structure of the new compounds.

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L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
Scheme 1. Reagents and conditions: (i) Jones reagent, acetone, 0 °C; (ii) HSCH2COOCH3, Et3N, CH3CN; (iii) (CF3CO)2O, Py, THF, 0 °C; (iv) 6 M HCl, dioxane, 70 °C; (v) P(OCH3)3,
reflux.
platelets. In this assay, washed platelets were treated with differ-
ent concentrations of the products for 30 min, before stimulation
of thromboxane B2 (TXB2) production with the calcium ionophore
A23187. After a fixed time, the cells were lysed and the TXB2 pro-
duced was quantitated by EIA.
COX-2 inhibitory activity was evaluated on isolated human
monocytes, in which COX-2 protein expression had been induced
by overnight lipopolysaccharide (LPS) treatment. After addition
of arachidonic acid, PGE2 production was assessed by EIA. Neither
the NO-donor furoxan derivatives nor the des-NO furazan ana-
logues were able to inhibit either COX isoform, when tested up
let-rich plasma. The results, expressed as IC50, or as percentage of
inhibition at the highest concentration tested when IC50 could
not be calculated, are reported in Table 2. The data show that the
furazan derivatives display either no activity or very weak activity.
The only exception is the amide compound 17a, which displays
low antiaggregatory action. Compounds in the furoxan series
display very weak antiaggregatory action, while the phenylsulfo-
nyl- and the cyano-substituted members 14 and 18 behave as
potent antiaggregatory agents. The potency of these two furoxan
derivatives decreased if the tests were carried out in the presence
of ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one),
a well-
to 100
l
M. This is probably due to the steric bulk of the heterocy-
known inhibitor of soluble guanylate cyclase (sGC), an enzyme that
mediates the antiaggregatory effects of NO. This is thus in keeping
with the involvement of NO in the antiaggregatory effects of furox-
ans. The NO-donor aspirin obtained by linking the 3-cyanofuroxan-
4-yl-methyl substructure present in 18 to the carboxylic group of
the lead, through an ester bridge, possessed potent antiaggregatory
activity. This activity was shown to be due to abundant NO-release
in the platelets, following the synergistic effect of high intracellular
concentrations of glutathione (GSH) and ascorbate in these kind of
cells.22 The furoxan amide compound 17 acts as a rather good anti-
aggregatory agent; however, its antiplatelet activity was found not
clic acyl moieties, which prevent binding of the products to the ac-
tive cleft of the enzyme, and then its acylation. Conversely a
number of nitrooxyacyl derivatives of salicylic acid, previously de-
scribed by our group, were shown to be potent irreversible inhib-
itors of both enzymes.21
2.4. Platelet anti-aggregatory activity
Both the furoxan and the furazan series were studied for their
action on collagen-induced platelet aggregation of human plate-

L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
5855
Scheme 2. Reagents and conditions: (i) SOCl2, dry THF; (ii) Py, salicylic acid, dry THF; (iii) SOCl2, dry CH2Cl2; (iv) Py, salicylic acid, dry CH2Cl2.
the contracted tissue in a concentration-dependent manner. The
vasodilator potencies, expressed as EC50, are shown in Table 2.
Their order of potency was 14 > 18 > 13 P 17. When the experi-
Table 1
Stability in aqueous buffered solutions and human serum of aspirin (1) and of
compounds 13, 13a, 14, 14a, 17, 17a, 18 and 18a incubated at 37 °C
ments were repeated in the presence of 1 lM ODQ, the potencies
Compound
Stability
Buffered solutions
decreased, in keeping with NO-induced activation of the sGC being
the mechanism underlying this effect. The most potent products,
namely 14 and 18, also displayed the most potent NO-dependent
antiaggregatory activity. Two properties of this new class of vaso-
dilators that differ from those of their nitrooxy analogues are of
interest: their greater potency and the probable lack of tolerance
development. This latter aspect was studied in vivo for the simple
4-hydroxymethylfuroxan-3-carboxyamide (CAS 1609),23 whose
substructure is present in 17, and for the simple 4-ethoxy-3-phen-
ylsulfonylfuroxan (CHF 2363),24 whose substructure is present in
14. Both these two products were found to be devoid of tolerance
development.
Human serum t1/2
(min)c
pH 1% unchanged at
3 ha
pH 7.4 t1/2
(min)
b
1
13
13a
14
14a
17
17a
18
18a
90
98
98
96
97
97
97
96
96
–
63
35
40
30
35
27
27
13
14
288
318
360
370
45
50
33
38
a
b
c
SEM 6 1%.
%Unchanged at 3 h = 90; SEM 6 1%.
SEM 6 0.2.
2.6. Anti-inflammatory activity
All products in the furoxan and furazan series were tested on
carrageenan-induced paw edema in conscious rats, together with
aspirin (ASA) and salicylic acid. Injection of carrageenan into the
rat’s hind paw produced immediate swelling, which peaked at 4–
5 h. Aspirin, administered by the intragastric route at 120 mg/kg
just prior to carrageenan injection, significantly reduced
(43.0 3.3%) paw edema at 3 h, compared to vehicle-treated ani-
mals (Table 2). Salicylic acid acted similarly, reducing paw edema
by 37%. The furoxan products significantly inhibited paw edema,
from 37% to 55%, when administered intragastrically at doses equi-
molar with aspirin 120 mg/kg (Table 2). The most efficacious inhib-
itors were the phenylsulfonyl- and cyano-substituted furoxans 14
and 18, for which inhibition reached 50% and 55%, respectively.
Either the NO released by the products,25 and/or the salicylic acid
deriving from their metabolic transformation, could be involved
in this activity.26 The finding that the des-NO furazan analogues
also displayed potent anti-inflammatory action (about 40%
inhibition) appears to minimize the role played by NO in the ob-
served effects, suggesting conversely that salicylic acid plays a par-
amount role. Indeed, both salicylic acid and the present series of
to be influenced by the presence of ODQ, suggesting that NO-
dependent sGC activation plays little or no role in this action. As
in the case of the related furazancarboxyamide 17a, an antiaggre-
gatory mechanism that is neither COX- nor NO-dependent must be
involved. Indeed, preliminary results obtained with these two
products indicate that they behave as antagonists at the thrombox-
ane receptor (IC50 (CL 95%)
lM: 38 (37–38) and 39 (37–42) for
compound 17 and 17a, respectively). The above picture indicates
that the potential value of this new series of furoxanylacyl deriva-
tives of salicylic acid as anti-thrombotic agents is limited, since
none of these compounds act as irreversible inhibitors of the
COX-enzymes, unlike aspirin and some its nitrooxyacyl
analogues.21
2.5. Vasodilator activity
The vasodilatory effects of the furoxan derivatives were evalu-
ated on endothelium-denuded rat aorta strips, pre-contracted with
phenylephrine. All the furoxan products were capable of relaxing

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L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
Table 2
Antiaggregatory, vasodilator, anti-inflammatory and ulcerogenic activity of ASA, salicylic acid and compounds 13, 13a, 14, 14a, 17, 17a, 18 and 18a
Compound
Antiaggregatory activity
Vasodilator activity
Anti-inflammatory activity
Ulcerogenic
activity
IC50 (CL 95%)
54 (49–60)
l
M [+50
l
M ODQ]
%Inhibition SEM at
300
EC50
ODQ]
(
l
M) SEM [+1
lM
%Inhibition of paw volume
increasec
Lesion index
(mm)d
l
M
ASA (1)
Salicylic acid
13
13a
14
///
43.0 3.3⁄⁄
36.8 4.2⁄⁄
37.6 4.8⁄⁄
38.6 5.4
50.0 3.8
1.1 0.6⁄⁄⁄
0.3 0.2⁄⁄⁄
1.2 0.5⁄⁄⁄
0.4 0.4⁄⁄⁄
a
a
17.0 0.2
12
11 2b
///
0.014 0.003
[0.97 0.03]
///
1
7.8 (5.5–11)
49.6 9.6⁄⁄
[152 (121–190)]
a
14a
17
2.6 2.6
38.8 10.1⁄⁄
39.4 4.8⁄⁄
0.3 0.2⁄⁄⁄
0.0 0.0⁄⁄⁄
66 (57–76)
23
3
[59 (46–74)]
144 (110–190)
7.3 (5.1–10)
b
17a
18
39.6 9.4⁄⁄
54.9 8.1⁄⁄
0.3 0.2⁄⁄⁄
1.2 1.2⁄⁄⁄
0.040 0.010
[2.4 0.4]
///
[88 (80–97)]
a
18a
36
2
38.9 3.8⁄⁄
0.2 0.2⁄⁄⁄
a
b
c
Due to the low activity of the compound, IC50 could not be calculated. In this case the percentage inhibition is reported at 300
In the presence of 1 M ODQ , EC50 values were >100 M.
Determined in the carrageenan–induced paw edema model at 3 h in rats. Compounds were administered by intragastric route at doses equimolar with ASA 120 mg/kg.
lM.
l
l
Edema volume in vehicle–treated group was taken as 100%; * P < 0.05; ** P < 0.01 versus vehicle (Dunnett’s test).
d
Determined in rats after intragastric administration of the compounds at doses equimolar with ASA 120 mg/kg. Gastric lesions were determined 3 h later; *** P < 0.001 vs.
ASA (Newman–Keuls test).
compounds are ineffective as inhibitors of prostanoid synthesis un-
der in vitro conditions, while being as effective as aspirin in inhib-
iting the rat paw edema. As for the mechanism involved, it has
been proposed that the inhibition of COX-2 enzyme induction
(via inhibition of NFkB signaling) may represent the major mode
of action of salicylates in vivo.26 Conversely, in some of the previ-
ously-described nitrooxy analogues, COX-acylation might also con-
tribute to their in vivo anti-inflammatory activity.
from those of the nitrooxy analogues previously described, due
to both the different structure and the different NO-release mode
of the respective NO-donor moieties. In particular in this new ser-
ies of compounds, the antiaggregatory activity is dependent on the
thiol-induced NO-release by furoxan moieties in platelets and the
products are unable to inhibit COX-enzyme. The comparison with
the corresponding des-NO furazans indicates that salicylic acid
formed following hydrolysis under the action of serum esterases,
is the main responsible of the anti-inflammatory activity of the ser-
ies. The designed products are an emblematic example of how the
appropriate selection of the NO-releasing group is a critical aspect
in designing NO-hybrid compounds.
2.7. Acute gastric mucosal damage
All compounds, including aspirin and salicylic acid as reference
standards, were assessed for their ulcerogenic properties in con-
scious 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 length in millimeters (Table 2). In the present study
aspirin, administered at 120 mg/kg, produced macroscopically-
detectable gastric damage, characterized by mucosal necrosis and
hemorrhage (lesion index = 50 3.8). By contrast salicylic acid,
tested under the same conditions, showed much lower gastrotox-
icity, in keeping with findings that it lacks this side effect in
experimental animals.26 All the NO-donor products, as well as their
des-NO furazan analogues, also displayed greatly reduced gastro-
toxicity when administered at doses equimolar with aspirin. In
the light of the fact that the products are not COX-inhibitors, and
of the finding that the des-NO furazan analogues are devoid of gas-
trotoxicity (Table 2), a possible explanation for this finding is that
they behave in vivo essentially as pro-drugs of salicylic acid. By
contrast, in the COX-inhibitor nitrooxyacyl analogues, a role of
NO in the reduced gastrotoxicity must be considered.
4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Bruker Avance 300
at 300 and 75 MHz, respectively, using TMS as internal standard.
Low resolution mass spectra were recorded with a Finnigan-Mat
TSQ-700. Melting points were determined with a capillary appara-
tus (Büchi 540). Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230–400 mesh ASTM); PE stands
for 40–60 petroleum ether. Progress of the reactions was moni-
tored by thin layer chromatography (TLC) on 5 Â 20 cm plates with
a layer thickness of 0.25 mm. Anhydrous magnesium sulfate was
used as drying agent for the organic phases. Organic solvents were
removed under vacuum at 30 °C. Preparative HPLC was performed
on a LichrospherÒ C18 column (250 Â 25 mm, 10
lm) (Merck
Darmstadt, Germany) with a Varian ProStar mod-210 with Varian
UV detector mod-325. Elemental analyses (C, H, N) were per-
formed and the results are within 0.3% of the theoretical values,
for compound 17a the results are within 0.5% of the theoretical
values. Compounds 2,22 2a,22 3,27 3a,28 529 and 6,30 were synthe-
sized as indicated in the literature. CAS1609 was a gift from Sano-
fi-Aventis Deutschland GmbH.
3. Conclusions
A new series of salicylic acid derivatives, containing NO-donor
furoxan moieties linked to the hydroxyl group through an ester
bridge, were designed together with the corresponding des-NO
furazan analogues. The furoxan derivatives display antiaggregato-
ry, vasodilator and anti-inflammatory properties, and show very
limited gastrotoxicity when tested in conscious rats. The mecha-
nisms responsible for these properties are somewhat different
4.1.1. General procedure for the preparation of 4, 4a and 5a
A solution of Jones reagent 2.5 M (19 mL, 46.62 mmol) was
added to a stirred solution of the appropriate alcohol (18.65 mmol)
in acetone (150 mL), cooled at 0 °C. The mixture was allowed to

L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
5857
reach rt and stirred for 4 h. iPrOH (10 mL) was added and the mix-
ture was concentrated under reduced pressure. The residue was
dissolved in EtOAc (150 mL) and extracted with a saturated solu-
tion of NaHCO3 (3 Â 20 mL). The aqueous layers were acidified
with 6 M HCl and extracted twice with EtOAc (50 mL). The com-
bined organic layers were dried and concentrated under reduced
pressure to give the desired compound.
(10 mL) and twice with 0.5 M HCl (10 mL). The organic layer was
dried and concentrated under reduced pressure. The crude product
was purified by flash chromatography (PE/EtOAc 90:10 v/v) to give
the desired compound.
4.1.4.1. Methyl 2-[(3-cyanofuroxan-4-yl)methyl]thioacetic acid
(9). 89% yield; colorless oil. 1H NMR (300 MHz, CDCl3, TMS):
d = 3.30 (s, 2H, –CH2COOCH3), 3.74 (s, 3H, CH3), 3.93 ppm (s, 2H,
–CH2S–); 13C NMR (75 MHz, CDCl3, TMS): d = 26.1, 32.9, 52.9,
96.6, 105.0, 154.4, 169.6 ppm; MS m/z 230 (M+H)+.
4.1.1.1. 3-(3-Phenylfuroxan-4-yloxy)propanoic acid (4). 59%
yield; white solid; mp = 136.5-137 °C (from toluene). 1H NMR
(300 MHz, DMSO-d6, TMS): d = 3.01 (t, J = 6.0 Hz, 2H, –CH2COOH),
4.78 (t, J = 6.0 Hz, 2H, –OCH2–), 7.41–7.50 (m, 3H, C6H5), 8.05–
8.07 (m, 2H, C6H5), 11.41 ppm (br s, 1H, COOH); 13C NMR
(75 MHz, DMSO-d6, TMS): d = 33.3, 66.7, 107.3, 121.9, 125.7,
128.8, 129.3, 161.9, 171.6 ppm; MS m/z 251 (M+H)+.
4.1.4.2. Methyl 2-[(4-cyanofurazan-3-yl)methyl]thioacetic acid
(9a). 84% yield; colorless oil. 1H NMR (300 MHz, CDCl3, TMS):
d = 3.24 (s, 2H, –CH2COOCH3), 3.74 (s, 3H, CH3), 4.10 ppm (s, 2H,
–CH2S–); 13C NMR (75 MHz, CDCl3, TMS): d = 23.3, 32.8, 52.8,
106.9, 132.7, 153.9, 169.6 ppm; MS m/z 214 (M+H)+.
4.1.1.2. 3-(4-Phenylfurazan-3-yloxy)propanoic acid (4a). 71%
yield; white solid; mp = 105.5–108 °C (from toluene). 1H NMR
(300 MHz, DMSO-d6, TMS): d = 3.00 (t, J = 6.0 Hz, 2H, –CH2COOH),
4.74 (t, J = 6.0 Hz, 2H, –OCH2–), 7.43–7.47 (m, 3H, C6H5), 7.92–
7.96 (m, 2H, C6H5), 11.41 ppm (br s, 1H, COOH); 13C NMR
(75 MHz, DMSO-d6, TMS): d = 34.4, 68.0, 125.4, 127.9, 129.6,
131.3, 145.6, 163.7, 177.0 ppm; MS m/z 235 (M+H)+.
4.1.5. General procedure for the preparation of 8, 8a, 10 and 10a
To a solution of the appropriate methyl ester (6.3 mmol) in
dioxane (30 mL), 6 M HCl (30 mL) was added and the mixture
heated at 70 °C for 4 h. After concentration under reduced pres-
sure, the residue was dissolved in EtOAc (30 mL) and the solution
washed with H2O (50 mL), and extracted twice with a saturated
solution of NaHCO3 (30 mL). The aqueous layers were acidified
and extracted twice with EtOAc (30 mL). The organic layers were
washed with brine (50 mL), dried and concentrated under reduced
pressure to give the desired product.
4.1.1.3. 3-[(4-Phenylsulfonyl)furazan-3-yloxy]propanoic acid
(5a). 68% yield; mp = 99.5–103.5 °C (from toluene). 1H NMR
(300 MHz, DMSO-d6, TMS): d = 2.80 (t, J = 5.8 Hz, 2H, –CH2COOH),
4.55 (t, J = 5.8 Hz, 2H, –OCH2–), 7.76 (t, 2H, C6H5), 7.92 (t, 1H,
C6H5), 8.07 (d, 2H, C6H5), 12.62 ppm (br s, 1H, COOH); 13C NMR
(75 MHz, DMSO-d6, TMS): d = 33.3, 69.8, 128.5, 130.1, 136.1,
136.7, 148.6, 160.7, 171.3 ppm; MS m/z 299 (M+H)+.
4.1.5.1. 2-[(3-Aminocarbonylfuroxan-4-yl)methyl]thioacetic acid
(8). 83% yield; white solid; mp = 110–112 °C (from toluene). 1H
NMR (300 MHz, DMSO-d6, TMS): d = 3.36 (s, 2H, –CH2COOH),
4.05 (s, 2H, –CH2S–), 7.79 (br s, 1H, CONH2), 8.45 (br s, 1H, CONH2),
12.69 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6, TMS):
d = 26.3, 33.3, 110.3, 155.8, 156.6, 171.0 ppm; MS m/z 234 (M+H)+.
4.1.2. Methyl 2-[(3-aminocarbonylfuroxan-4-yl)methyl]thioace
tic acid (7)
To
a
solution of 4-bromomethylfuroxan-3-carboxyamide
(3.1 g, 14 mmol) in CH3CN (100 mL) methyl thioglycolate
(1.3 mL, 14 mmol) and Et3N (1.95 mL, 14 mmol) were added.
After 1 h the mixture was diluted with CH2Cl2 (100 mL), washed
with H2O (100 mL), 1 M NaOH (50 mL), brine (50 mL), dried and
concentrated under reduced pressure to give the title compound
as a white solid, 72% yield; mp = 92.5–93.5 °C (from iPr2O). 1H
NMR (300 MHz, CDCl3, TMS): d = 3.33 (s, 2H, –CH2COOCH3),
3.75 (s, 3H, -CH3) 4.16 (s, 2H, –CH2S–), 6.27 (br s, 1H, CONH2),
7.58 ppm (br s, 1H, CONH2); 13C NMR (75 MHz, CDCl3, TMS):
d = 26.7, 33.1, 52.7, 110.1, 156.1, 156.2, 170.3 ppm; MS m/z 248
(M+H)+.
4.1.5.2. 2-[(4-Aminocarbonylfurazan-3-yl)methyl]thioacetic acid
(8a). 67% yield; white solid; mp = 151–157 °C (toluene). 1H NMR
(300 MHz, DMSO-d6, TMS): d = 3.29 (s, 2H, –CH2COOH), 4.14 (s,
2H, –CH2S–), 8.18 (br s, 1H, CONH2), 8.54 (br s, 1H, CONH2),
12.75 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6, TMS):
d = 23.3, 33.4, 148.3, 153.8, 158.4, 170.9 ppm; MS m/z 218 (M+H)+.
4.1.5.3. 2-[(3-Cyanofuroxan-4-yl)methyl]thioacetic acid (10). 97%
yield; white solid; mp = 87–88 °C (from toluene). 1H NMR (300 MHz,
DMSO-d6, TMS): d = 3.35 (s, 2H, –CH2COOH), 4.05 (s, 2H, –CH2S–),
12.64 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6, TMS):
d = 25.9, 33.3, 97.8, 105.7, 155.4, 170.7 ppm; MS m/z 216 (M+H)+.
4.1.3. Methyl 2-[(4-aminocarbonylfurazan-3-yl)methyl]thioace
tic acid (7a)
4.1.5.4. 2-[(4-Cyanofurazan-3-yl)methyl]thioacetic acid (10a). 92%
yield; white solid; mp = 34–37 °C (from toluene). H NMR (300 MHz,
CDCl3, TMS): d = 3.32 (s, 2H, –CH2COOH), 4.16 (s, 2H, –CH2S–),
10.87 ppm (br s, 1H, COOH); 13C NMR (75 MHz, CDCl3, TMS):
d = 23.6, 33.1, 107.2, 133.0, 154.1, 175.7 ppm; MS m/z 200 (M+H)+.
1
A solution of 7 (3.1 g, 12.54 mmol) in P(OMe)3 (20 mL) was
heated under reflux for 17 h. The mixture was then poured into
iced 6 M HCl (200 mL) and the white solid thus obtained was fil-
tered. The crude product was purified by flash chromatography
(PE/EtOAc 70:30 v/v) to give the title compound as a white solid,
90% yield; mp = 83–84 °C (from iPr2O). 1H NMR (300 MHz, CDCl3,
TMS): d = 3.29 (s, 2H, –CH2COOCH3), 3.73 (s, 3H, CH3), 4.24 (s,
2H, –CH2S–), 6.29 (br s, 1H, CONH2), 6.83 ppm (br s, 1H, CONH2);
13C NMR (75 MHz, CDCl3, TMS): d = 23.8, 33.1, 52.6, 146.9, 152.9,
158.7, 170.3 ppm; MS m/z 232 (M+H)+.
4.1.6. General procedure for the preparation of 13, 13a, 14 and
14a
SOCl2 (334 lL, 4.58 mmol) and a few drops of dry DMF were
added to a solution of the appropriate carboxylic acid (3.82 mmol)
in dry THF (20 mL), stirred under N2 at rt. Stirring was continued
for 7 h at rt. The solution of the acyl chloride thus obtained was
4.1.4. General procedure for the preparation of 9 and 9a
slowly added to
3.82 mmol) and dry Py (463 l
kept under N2 at 0 °C. The mixture was allowed to reach rt and
then stirred overnight. The mixture was diluted with Et2O
(50 mL) and washed twice with 2 M HCl (50 mL). The combined
organic layers were dried and concentrated under reduced
a
stirred solution of salicylic acid (0.53 g,
L, 5.73 mmol) in dry THF (20 mL)
To
1.21 mmol) in dry THF (10 mL) stirred under N2 at 0 °C, dry pyri-
dine (200 L, 2.42 mmol) and, dropwise, (CF3CO)2O (320 L,
a
solution of the appropriate amide derivative (0.3 g,
l
l
2.3 mmol) were added. After 20 min the reaction is complete; the
mixture was diluted with Et2O (15 mL), washed with H2O

5858
L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
pressure. The crude product was purified by preparative HPLC
(Lichrospher 250–25 C18, CH3CN/H2O/TFA 50:50:0.1, flow 39 mL/
min, k224 nm, injection 4 mL, solution 51 mg/mL) to give the de-
sired compound.
(s, 2H, –CH2S–), 7.21 (d, 1H, C6H4), 7.41 (t, 1H, C6H4), 7.67 (t, 1H,
C6H4), 7.82 (br s, 1H, CONH2), 7.95 (d, 1H, C6H4), 8.48 ppm (br s,
1H, CONH2); 13C NMR (75 MHz, DMSO-d6, TMS): d = 26.2, 33.1,
110.0, 119.1, 123.4, 126.3, 131.4, 133.9, 149.8, 155.6, 156.3,
165.4, 168.4 ppm; MS m/z 354 (M+H)+. Anal. Calcd for
4.1.6.1. 2-({3-[(3-Phenylfuroxan-4-yl)oxy]propanoyl}oxy)benzo
ic acid (13). 61% yield; white solid; mp = 152–154 °C (from tolu-
ene). 1H NMR (300 MHz, DMSO-d6, TMS): d = 3.27 (t, J = 6.0 Hz, 2H,
–CH2COO–), 4.80 (t, J = 6.0 Hz, 2H, –CH2O–), 7.14–8.07 (m, 9H, C6H4
+ C6H5), 13.16 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6,
TMS): d = 33.9, 66.5, 107.9, 122.3, 124.1, 124.2, 126.6, 126.7, 129.3,
131.1, 131.9, 134.3, 150.3, 162.4, 165.8, 169.4 ppm; MS m/z 371
(M+H)+. Anal. Calcd for C18H14N2O7: C, 58.38; H, 3.81; N, 7.56.
Found: C, 58.55; H, 3.91; N, 7.34.
C13H11N3O7S: C, 44.19; H, 3.14; N, 11.89. Found: C, 43.80; H,
3.18; N, 12.00.
4.1.7.2. 2-{[(4-Aminocarbonylfurazan-3-yl)methyl]thioacetoxy}
benzoic acid (17a). Purified by flash chromatography (eluent
CH2Cl2/MeOH/HCOOH 98:2:0.1 v/v/v) to give the title compound
in 69% yield as white solid; mp = 154–157 °C (from toluene). 1H
NMR (300 MHz, DMSO-d6, TMS): d = 3.70 (s, 2H, –CH2COO–), 4.25
(s, 2H, –CH2S–), 7.21 (d, 1H, C6H4), 7.39 (t, 1H, C6H4), 7.68 (t, 1H,
C6H4), 7.95 (d, 1H, C6H4), 8.20 (br s, 1H, CONH2), 8.57 (br s, 1H,
CONH2), 13.20 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-
d6, TMS): d = 23.4, 33.3, 123.7, 126.3, 131.4, 133.9, 148.1, 149.8,
153.2, 158.2, 165.4, 168.3, 170.7 ppm; MS m/z 338 (M+H)+. Anal.
Calcd for C13H11N3O6S 0.5 H2O: C, 45.09; H, 3.49; N, 12.13. Found:
C, 45.27; H, 3.36; N, 11.65.
4.1.6.2. 2-({3-[(4-Phenylfurazan-3-yl)oxy]propanoyl}oxy)benzo
ic acid (13a). 37% yield; white solid; mp = 141–146 °C (from tol-
uene). 1H NMR (300 MHz, DMSO-d6, TMS): d = 3.25 (t, J = 6.0 Hz,
2H, –CH2COO–), 4.78 (t, J = 6.0 Hz, 2H, –CH2O–), 7.18–7.98 (m,
9H, C6H4 + C6H5), 13.16 ppm (br s, 1H, COOH); 13C NMR (75 MHz,
DMSO-d6, TMS): d = 33.8, 68.2, 123.9, 124.0, 124.4, 126.5, 127.4,
129.4, 131.3, 131.7, 134.1, 145.4, 150.1, 163.3, 165.6, 169.2 ppm;
MS m/z 355 (M+H)+. Anal. Calcd for C18H14N2O6: C, 61.02; H,
3.98; N, 7.91. Found: C, 61.13; H, 4.02; N, 7.74.
4.1.7.3. 2-{[(3-Cyanofuroxan-4-yl)methyl]thioacetoxy}benzoic
acid (18). Purified by reverse phase flash chromatography (eluent
H2O/CH3CN/TFA 60:40:0.1 v/v/v) to give the title compound in 38%
yield as white solid; mp = 121.5–124.5 °C (from toluene). 1H NMR
(300 MHz, DMSO-d6, TMS): d = 3.82 (s, 2H, –CH2COO–), 4.20 (s, 2H,
–CH2S–), 7.19 (d, 1H, C6H4), 7.42 (t, 1H, C6H4), 7.67 (t, 1H, C6H4),
7.96 (d, 1H, C6H4), 13.18 ppm (br s, 1H, COOH); 13C NMR
(75 MHz, DMSO-d6, TMS): d = 25.4, 32.8, 98.7, 106.2, 123.4, 123.7,
126.4, 131.4, 133.9, 149.8, 155.8, 165.3, 168.2 ppm; MS m/z 336
(M+H)+. Anal. Calc. for C13H9N3O6S: C, 46.57; H, 2.70; N, 12.53.
Found: C, 46.27; H, 2.76; N, 12.27.
4.1.6.3. 2-[(3-{[(3-Phenylsulfonyl)furoxan-4-yl]oxy}propanoyl)
oxy]benzoic acid (14). 32% yield; white solid; mp = 169.5–
170 °C (from toluene). 1H NMR (300 MHz, DMSO-d6, TMS):
d = 3.16 (t, J = 6.0 Hz, 2H, –CH2COO–), 4.71 (t, J = 6.0 Hz, 2H, –
CH2O–), 7.21 (d, 1H, C6H4), 7.43 (t, 1H, C6H4), 7.63–7.70 (m, 3H,
C6H4 + C6H5), 7.86 (t, 1H, C6H4), 7.98 (d, 1H, C6H4), 8.08 (d, 2H,
C6H5), 13.16 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6,
TMS): d = 33.3, 66.6, 110.4, 123.6, 123.7, 126.3, 128.2, 129.8,
131.5, 133.9, 136.0, 137.1, 149.8, 158.6, 165.4, 168.6 ppm; MS m/
z 435 (M+H)+. Anal. Calcd for C18H14N2O9S: C, 49.77; H, 3.25; N,
6.45. Found: C, 49.67; H, 3.28; N, 6.53.
4.1.7.4. 2-{[(4-Cyanofuroxan-3-yl)methyl]thioacetoxy}benzoic
acid (18a). Purified by preparative HPLC (Lichrospher 250–25
C18, CH3CN/H2O/TFA 45:55:0.1, flow 39 mL/min, k 224 nm, injec-
tion 3 mL, solution 51 mg/mL) to give the title compound in 46%
yield as a white solid; mp = 114–115.5 °C (from toluene). 1H
NMR (300 MHz, DMSO-d6, TMS): d = 3.76 (s, 2H, –CH2COO–), 4.35
(s, 2H, –CH2S–), 7.16 (d, 1H, C6H4), 7.41 (t, 1H, C6H4), 7.68 (t, 1H,
C6H4), 7.95 (d, 1H, C6H4), 13.19 ppm (br s, 1H, COOH); 13C NMR
(75 MHz, DMSO-d6, TMS): d = 23.1, 33.3, 107.7, 123.3, 123.6,
126.4, 131.4, 133.7, 133.9, 149.8, 155.1, 165.3, 168.3 ppm; MS m/
z 320 (M+H)+. Anal. Calcd for C13H9N3O5S 1.5 H2O: C, 45.09; H,
3.49; N, 12.13. Found: C, 45.18; H, 3.17; N, 12.47.
4.1.6.4. 2-[(3-{[(4-Phenylsulfonyl)furazan-3-yl]oxy}propanoyl)
oxy]benzoic acid (14a). 38% yield; white solid; mp = 139.5–
140 °C (from toluene). 1H NMR (300 MHz, DMSO-d6, TMS):
d = 3.16 (t, J = 5.8 Hz, 2H, –CH2COO–), 4.71 (t, J = 5.8 Hz, 2H, –
CH2O–), 7.21 (d, 1H, C6H4), 7.43 (t, 1H, C6H4), 7.63–7.70 (m, 3H,
C6H4 + C6H5), 7.86 (t, 1H, C6H5), 7.98 (d, 1H, C6H4), 8.08 (d, 2H,
C6H5),13.16 ppm (br s, 1H, COOH); 13C NMR (75 MHz, DMSO-d6,
TMS): d = 33.4, 69.0, 123.6, 123.7, 126.3, 128.6, 130.0, 131.4,
133.9, 136.0, 136.6, 148.6, 149.8, 160.7, 165.3, 168.6 ppm; MS m/
z 419 (M+H)+. Anal. Calcd for C18H14N2O8S 0.25 H2O: C, 51.12; H,
3.46; N, 6.62. Found: C, 51.00; H, 3.30; N, 6.54.
4.2. Stability studies
4.2.1. Evaluation of stability in aqueous buffered solutions
A solution of each compound (10 mM) in acetonitrile was added
to HCl 0.1 M or to phosphate buffer pH 7.4 (50 mM) preheated at
4.1.7. General procedure for 17, 17a, 18 and 18a
SOCl2 (190 lL, 2.57 mmol) and dry DMF (1 mL) were added to a
solution of the appropriate carboxylic acid (2.15 mmol) in dry
CH2Cl2 (10 mL), stirred under N2 at rt. Stirring was continued for
1 h at rt. The solution of the acyl chloride thus obtained was slowly
added to a stirred solution of salicylic acid (0.24 g, 1.72 mmol) and
37 °C; the final concentration of the compound was 100
resulting solution was maintained at 37 0.5 °C and, at appropri-
ate time intervals, a 20 L aliquot of reaction solution was ana-
lM. The
l
lyzed by RP-HPLC, as described below.
dry Py (260 lL, 3.22 mmol) in dry CH2Cl2 (20 mL) kept under N2 at
0 °C. The mixture was allowed to reach rt and then stirred over-
night. The mixture was washed twice with 2 M HCl (20 mL). The
combined organic layers were dried and concentrated under re-
duced pressure. The crude product was purified as follows.
4.2.2. Stability in human serum
A solution of each compound (10 mM) in acetonitrile was added
to human serum (Sigma) preheated to 37 °C; the final concentra-
tion of the compound was 200
incubated at 37 0.5 °C and, at appropriate time intervals, 300
of reaction mixture were withdrawn and added to 300 L of aceto-
lM. The resulting solution was
lL
4.1.7.1.
2-{[(3-Aminocarbonylfuroxan-4-yl)methyl]thioacet-
l
oxy}benzoic acid (17). Purified by flash chromatography (eluent
PE/EtOAc/HCOOH 70:30:0.1 v/v/v) to give the title compound in
69% yield as white solid; mp = 143.5–144.5 °C (from toluene). 1H
NMR (300 MHz, DMSO-d6, TMS): d = 3.74 (s, 2H, –CH2COO–), 4.15
nitrile containing 0.1% trifluoroacetic acid, 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 lm PTFE filters (Alltech) and analyzed by RP-HPLC.

L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
5859
The reverse-phase HPLC procedure separated and quantitated
the remaining compound and salicylic acid. HPLC analyses were
performed with a HP 1100 chromatograph system (Agilent Tech-
nologies, Palo Alto, CA, USA) equipped with a quaternary pump
(model G1311A), a membrane degasser (G1379A), and a diode-ar-
ray detector (DAD) (model G1315B) integrated in the HP1100 sys-
tem. Data analysis was done by the HP ChemStation system
(Agilent Technologies). The analytical column was a Nucleosil
who were treated according to Helsinki protocol for biomedical
experimentation, gave their informed consent to the use of blood
samples for research purposes. Platelet rich plasma (PRP) was pre-
pared by centrifugation of citrated blood at 200g for 20 min. Ali-
quots (500 lL) 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 minutes after addition of the stimulus. Collagen at submaximal
100-5C18 Nautilus (250 Â 4.6 mm, 5
l
m particle size) (Mache-
concentration (0.8–1.5 l
g mLÀ1) was used as platelet activator in
rey-Nagel). The mobile phase consisting of acetonitrile/water
PRP. Compounds under study were preincubated with PRP
10 min before addition of the stimulus (collagen). Vehicle alone
(0.5% DMSO) added to PRP did not affect platelet function in con-
trol samples. The role of NO and sGC in the inhibitory effect was
(55:45) with 0.1% trifluoroacetic acid and the flow-rate was
1.2 mL/min. The injection volume was 20 lL (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 done using calibration
curves of compounds and relative metabolites chromatographed
under the same conditions; the linearity of the calibration curves
investigated using the sGC inhibitor, ODQ (50
lM). At least five
experiments for each compound were performed.
To study the antagonism at the thromboxane receptor human
blood was anticoagulated with citrate solution and treated with
1 mM acetylsalicylic acid; PRP was prepared as described above.
PRP samples were incubated with compounds or vehicle (0.5%
was determined in a concentration range of 1–200 l
M (r2 >0.99).
4.3. Anti-inflammatory activity
DMSO) and challenged with the agonist U-46619 (0.5–2.5 lM).
Incubation and registration times were the same as described pre-
viously. At least four experiments for each compound were
performed.
The antiaggregatory activity of tested compounds is evaluated
as% inhibition of platelet aggregation compared to control samples.
For most active compounds IC50 values could be calculated by non-
linear regression analysis, otherwise% inhibition at maximal con-
Male Wistar rats, weighing 180–200 g (Harlan, S. Pietro al Nati-
sone, 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 (CMC, 1%) were administered
centration tested (300
lM) is reported.
intragastrically to different groups of rats in
10 mL kgÀ1
Salicylic acid and NO-aspirin derivatives were
administered at a dose equimolar with aspirin 120 mg kgÀ1
Groups of 6–8 animals were used. Paw volume was measured with
a water plethysmometer (Basile, Comerio, Italy) immediately
before carrageenan injection and 3 h afterwards. The edema reduc-
tion in treated animals was expressed as percentage inhibition of
the edema observed in vehicle-treated animals, considered as
100. The results obtained are presented as mean SEM. Statistical
analysis was performed with ANOVA followed by Dunnett test.
a volume of
4.6. Vasodilator activity
.
.
Thoracic aortas were isolated from male Wistar rats weighing
180–200 g. As few animals as possible were used. The purposes
and the protocols of our studies have been approved by Ministero
della Salute, Rome, Italy. The endothelium was removed and the
vessels were helically cut: three strips were obtained from each
aorta. The tissues were mounted under 1.0 g tension in organ baths
containing 30 mL of Krebs-bicarbonate buffer with the following
composition (mM): NaCl 111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2,
KH2PO4 1.0, NaHCO3 12.0, glucose 11.1, maintained at 37 °C and
gassed with 95% O2-5% CO2 (pH = 7.4). The aortic strips were al-
4.4. Gastrotoxicity
lowed to equilibrate for 120 min and then contracted with 1
-phenylephrine. When the response to the agonist reached a pla-
teau, cumulative concentrations of the vasodilating agent were
added. Results are expressed as EC50 SE ( M). The effects of
M ODQ on relaxation were evaluated in separate series of
experiments in which it was added to the organ bath 5 min before
the contraction. Responses were recorded by an isometric trans-
ducer connected to the MacLab System PowerLab. Addition of the
drug vehicle (DMSO) had no appreciable effect on contraction
level.
lM
Male Wistar rats, weighing 180–200 g (Harlan, S. Pietro al Nati-
sone, 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 of water 24 h before the exper-
iments. Groups of rats (n = 8–10) were given aspirin 120 mg kgÀ1
by intragastric route or equimolar doses of the compounds under
study (vehicle CMC 1%). Rats were sacrificed 3 h after the adminis-
tration 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 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, desig-
nated as the lesion index, for each stomach. The results obtained
are presented as mean SEM. Statistical analysis was performed
with ANOVA followed by Newman–Keuls test.
L
l
1
l
4.7. COX-1 inhibition
In order to evaluate COX-1 inhibitory activity, washed platelets
from individual donors were prepared as described elsewhere.18 Ali-
quots of platelets (200 l
L at 108 cells mLÀ1) were incubated with
inhibitors at different concentrations (diluted from 400-fold con-
centrated solutions in DMSO) for 30 min at 37 °C, before addition
of calcium ionophore (A23187, 2
lM). After 10 min at 37 °C, the
reactions were terminated with 100
lL of methanol. TXB2 in the
supernatant was determined by enzyme immunoassay (Cayman
Chemical). Percent inhibition of TXB2 production in compound-trea-
ted samples was calculated by comparison with untreated control
samples. Background (no ionophore) TXB2 production was sub-
tracted from each value obtained in the presence of ionophore.
4.5. 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. Volunteers,

5860
L. Lazzarato et al. / Bioorg. Med. Chem. 19 (2011) 5852–5860
11. Lichtenberger, L. M.; Zhou, Y.; Dial, E. J.; Raphael, R. M. J. Pharm. Pharmacol.
4.8. COX-2 inhibition
2006, 58, 1421.
12. Wallace, J. L.; Granger, D. N. FASEB J. 1996, 10, 731.
13. Wallace, J. L.; Chin, B. C. Drug Today 1997, 33, 371.
In order to evaluate COX-2 inhibitory activity, human mono-
cytes were isolated from buffy coats obtained from the SIMT (Ban-
ca del sangue, AOU San Giovanni Battista Hospital, Turin, Italy).
Mononuclear cells were isolated as described elsewhere.18 The
14. Bandarage, U. K.; Janero, D. R. Mini-Rev. Med. Chem. 2001, 1, 57.
15. Lazzarato, L.; Donnola, M.; Rolando, B.; Chegaev, K.; Marini, E.; Cena, C.; Di
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monocytes (106 cells mLÀ1) were incubated with LPS (10 g mLÀ1
l )
for 18 h at 37 °C in a 5% CO2 atmosphere. Aliquots of LPS-stimu-
lated monocytes were centrifuged (300g, 8 min) and resuspended
in fresh FCS-free RPMI-1640 medium. Each compound (diluted
from 200-fold concentrated solutions in DMSO) was added at dif-
ferent concentrations to the monocyte suspensions; the aliquots
of monocytes were then incubated at 37 °C in a 5% CO2 atmosphere
for 30 min, prior to stimulation with arachidonic acid (100 lM) for
15 min. Cell suspensions were centrifuged and cell-free superna-
tants were stored at À20 °C until eicosanoid determination was
completed. PGE2 in the supernatant was determined by EIA (Cay-
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