A new class of ibuprofen derivatives with reduced gastrotoxicity

Article abstract of DOI:10.1021/jm0108799

A new series of nonsteroidal antiinflammatory drugs (NSAIDs) obtained by linking ibuprofen to selected furoxan moieties and to related furazans were synthesized and tested for their antiinflammatory, antiaggregatory, and ulcerogenic properties. All the de

Full text of DOI:10.1021/jm0108799

J . Med. Chem. 2001, 44, 3463-3468
3463
A New Cla ss of Ibu p r ofen Der iva tives w ith Red u ced Ga str otoxicity
Marco L. Lolli,† Clara Cena,† Claudio Medana,† Loretta Lazzarato,† Giuseppina Morini,‡ Gabriella Coruzzi,*,‡
Stefano Manarini,§ Roberta Fruttero,† and Alberto Gasco*,†
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy,
and Istituto di Farmacologia, Universita` degli Studi di Parma, via Volturno 39, 43100 Parma, Italy,
and Istituto di Ricerche Farmacologiche Mario Negri, Laboratorio di Interazione tra Cellule Ematiche e Vascolari,
Dipartimento di Medicina e Farmacologia Vascolare, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (CH), Italy
Received March 15, 2001
A new series of nonsteroidal antiinflammatory drugs (NSAIDs) obtained by linking ibuprofen
to selected furoxan moieties and to related furazans were synthesized and tested for their
antiinflammatory, antiaggregatory, and ulcerogenic properties. All the derivatives are endowed
with antiinflammatory activity comparable to that of ibuprofen, but, unlike this drug, they
display reduced acute gastrotoxicity. The masking of the ibuprofen-free carboxylic group seems
to be principally at the basis of this reduced topical irritant action. The two furoxan derivatives
8 and 9 also trigger potent antiaggregatory effects, principally as a consequence of their NO-
donor ability.
In tr od u ction
described as gastrointestinal-sparing prodrugs.7 The
behavior of this class of drugs is very interesting, but
quite complex and not completely understood.
Nonsteroidal antiinflammatory drugs (NSAIDs) are
an important class of compounds that display a number
of effects as a consequence of their ability to block
cyclooxygenase (COX) involved in the first step of the
arachidonic acid cascade.1 COX exists in two isoforms
named COX-1 and COX-2, respectively. The first is
constitutively expressed in the stomach, the kidneys,
and platelets and is considered important in mucosal
protection and platelet function. COX-2 is inducible and
plays a major role in prostaglandin biosynthesis in
inflammatory cells.2 Classical NSAIDs, such as ibupro-
fen 3, inhibit both the isoforms. The major limitation
to long term use of NSAIDs in therapy is the increased
risk of gastrointestinal ulceration and complications
from ulceration, such as bleeding and perforation.1
There is strong evidence that inhibition of COX-1 rather
than inhibition of COX-2 underlies this gastrointestinal
toxicity.3
Nitric oxide (NO) is an endogenous mediator that has
important functions in mammalian tissues.4 It is syn-
thesized by the enzyme nitric oxide synthase (NOS),
which also exists in constitutive and inducible forms,
both present in the stomach. Here NO is able to protect
gastric mucosa by a number of mechanisms including
promotion of mucus secretion and increasing mucosal
blood flow.3
There is interesting evidence that furoxans (1,2,5-
oxadiazole 2-oxides, general structure 2 in Chart 1) are
able to generate nitric oxide NO (family name), in the
presence of thiol cofactors.8,9 By introducing appropriate
substituents to the ring, it is possible to modulate the
rate and the amount of NO production.10 The overall
reaction mechanism appears to be very complex and
direct NO• release or intermediate formation of nitroxyl
anion (NO-) or both could be involved. Both the redox
forms are able to activate soluble guanylate cyclase
(sGC).11 The profile of furoxans as vasodilators is similar
to that of nitrates, but they, differently from the latter,
could lack significant tolerance development.12,13 All this
renders furoxan derivatives flexible tools in designing
NO-donor “hybrid” drugs, potentially useful for the
treatment of cardiovascular diseases. In previous works,
we discussed the use of furoxan substructures to prepare
“hybrids” with either R1-antagonist,14 or â1-antagonist,15
as well as Ca2+ blocker16 and NO-dependent vasodilat-
ing properties. We also obtained interesting compounds
in which the combination of furoxan moieties with a H2-
antagonist pharmacophoric group resulted in in vivo
gastroprotective inhibitors of acid secretion.17
As a further development of our research in this field,
we have now designed NSAIDs (derivatives 8, 9) in
which ibuprofen 3 is joined by an ester linkage to two
furoxan moieties selected for their ability to release NO.
In this paper, we report on the synthesis, antiinflam-
matory, and antiaggregatory properties as well as
gastric effects of these compounds. Related furazan
derivatives 8a and 9a were taken as controls since they
are unable to release NO. Ibuprofen and its γ-nitrox-
ypropyl and propyl esters 11 and 12, respectively, are
also considered as references (Chart 2).
On this basis, molecules endowed with mixed cy-
clooxygenase inhibiting and NO-releasing properties
were recently designed.5,6 These compounds (general
structure 1 in Chart 1) were obtained by conjugating
traditional NSAIDs to NO-donor nitroxy groups. Very
recently nitrosothiol esters of diclofenac were also
* To whom correspondence should be addressed: Dipartimento di
Scienza e Tecnologia del Farmaco, Via P. Giuria, 9, I-10125 Torino,
Italy. Telephone: 0039 011 6707670. Fax: 0039 011 6707972. E-mail:
gasco@pharm.unito.it.
† Universita` degli Studi di Torino.
Ch em istr y. Syntheses of the final compounds con-
sidered for biological investigation are outlined in
Scheme 1. The starting materials were the propanol
‡ Universita` degli Studi di Parma.
§ Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario
Negri Sud.
10.1021/jm0108799 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/12/2001

3464 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Lolli et al.
Ch a r t 1
Ta ble 1. NO Release Data and Ibuprofen Detected in Human
Plasma and in Acidic Medium (pH ) 1) after Hydrolysis of
Esters 8, 8a , 9, 9a , 11, and 12
Ch a r t 2
% ibuprofena
-
% NO2
human plasma
6 h
pH ) 1
2 h
compound
(mol/mol)a,b
8
8a
9
9a
11
12
34.7 ( 1.8
9.5 ( 0.2
3.2 ( 0.1
46.8 ( 1.8
26.0 ( 1.0
35.6 ( 1.2
8.9 ( 1.0
27.1 ( 3.4
22.9 ( 1.5
12.9 ( 0.9
13.0 ( 1.1
6.5 ( 0.5
6.1 ( 0.5
22.3 ( 2.8
11.7 ( 1.0
derivatives 5 and 5a , obtained by action of 1,3-propan-
diol on 3,4-bis(benzenesulfonyl)furoxan 4 and on related
furazan 4a , in the presence of sodium hydroxide. Nu-
cleophilic displacement, by action of thiophenol in
MeOH/THF solution (potassium salt), of the remaining
benzenesulfonyl group of these compounds afforded
intermediates 6 and 6a , respectively. Final structures
8, 9 and 8a , 9a were synthesized by coupling ibuprofen
chloride 7 with the appropriate intermediates, in me-
thylene chloride solution in the presence of pyridine.
Structure 11 was obtained following the standard
reaction sequence reported at the bottom of Scheme 1.
All the structures were confirmed by 1H and 13C NMR
spectroscopy. For the intermediate 5, we proposed a
3-benzenesulfonyl structure18 on the basis of the reac-
tivity of 4 with ethanol in the presence of sodium
hydroxide.19 13C NMR resonances of the endocyclic C-3
(110.35) and C-4 (158.79) carbons supported this as-
signment.20 Structures 6 and 8 are consequently fixed.
All values are mean ( SEM.10
reaction.
Determined by Griess
a
b
other competitive reactions can occur in addition to
oxidation,8 for our purposes nitrite is a convenient index
of NO production trend. The results, expressed as
percent NO2 (mol/mol), are summarized in Table 1.
These same products were unable to afford nitrite under
-
the same conditions in the acidic medium (pH ) 1).
The amount of ibuprofen produced by each ibuprofen
ester after 6 h of incubation in human plasma at 37 °C
and over 2 h in acidic medium (pH ) 1) was detected
by HPLC. The results are reported in Table 1.
P h a r m a cologica l Resu lts a n d Discu ssion
To evaluate the thiol-induced NO-generation, the
appropriate NO-donor compound was dissolved in pH
7.4 buffered water-methanol mixture and incubated at
37 °C for 1 h, in the presence of 1:5 molar excess of
cysteine. The use of a cosolvent was necessary because
of the low water solubility of the compounds. Nitrite,
which is an important air oxidation product of nitric
oxide, was determined by Griess reaction.10 Nitrite
detection may indicate the previous presence of NO.
While it is true that in our experimental conditions
An tiin fla m m a tor y Activity. All the compounds
were tested for their antiinflammatory activity in the
carrageenan-induced rat foot paw edema assay. The
injection of carrageenan into the paw pad produced a
marked increase in paw volume within 1 h of its
administration and reached its maximal effect after 4-6
h. Ibuprofen 3, administered by intragastric route at 120
mg/kg just prior to the carrageenan injection, signifi-
cantly reduced (48.5%) paw edema at 4 h (Table 2).
Sch em e 1

Ibuprofen Derivatives with Reduced Gastrotoxicity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3465
Ta ble 2. Antiinflammatory Activity of Tested Compounds on
Carragenan-Induced Rat Paw Edema and Gastrotoxicity
Studies
Ta ble 3. Antiaggregatory Activity of the Tested Compounds on
Arachidonic Acid Induced PRP Aggregation
antiaggregatory activity
antiinflammatory
b
pIC50
activitya
gastrotoxicityb
% inhibition
compound
(100 µM)a,b
+ HbO2 40 µM
% inhibition of paw
volume increasec
lesion index
(mm)c
c
c
c
c
ibuprofen 3
8
4.94 ( 0.005
6.21 ( 0.01
4.07 ( 0.001
5.57 ( 0.03
dose
dose regimen,e
6 h
8a
compound regimen
4 h
6 h
11.7 ( 6.6
9
d
5.30 ( 0.07
d
d
9a
11
12
aspirin
13.9 ( 3.8
19.6 ( 7.4
25.7 ( 3.5
ibuprofen 3
c
d
48.5 ( 8.8f 30.3 ( 8.9
37.4 ( 8.9f 41.5 ( 6.3f
52.8 ( 6.1f 63.3 ( 6.6h
40.7 ( 14.5f 36.2 ( 11.5f
48.3 ( 10.1f 54.4 ( 9.5h
41.8 ( 10.0f 40.0 ( 11.2f
37.6 ( 6.9f 49.6 ( 7.7f
e
4.24 ( 0.13
d
d
5.0 ( 2.2
8
8a
9
9a
11
12
0g
c
5.7 ( 1.7g
0g
d
4.89 ( 0.26
b
e
a
Maximal concentration tested. All values are mean ( SEM
(n ) 5). c For these compounds, a complete concentration-response
curve could be performed; therefore, pIC50s are reported in the
1.3 ( 0.9g
0.7 ( 0.7g
3.8 ( 0.9g
e
e
d
d
next column. Aggregation did not reach 50% of control effect:
a
pIC50s could not be calculated.
Determined in conscious rat in the carrageenan rat paw model.
b
Determined in conscious rat after i.g. administration of test
compounds. c All values are mean ( SEM. Dose equimolar to 40
mg/kg of ibuprofen. e Dose equimolar to 120 mg/kg of ibuprofen.
Compounds were administered to 6 rats/groups. f P < 0.05 vs
d
induction, and the acute toxicity should be principally
influenced by the former component. The reduced toxic-
ity of 12 could be partly due to the masking of the free
carboxylic group in 3, by ester prodrug formation, with
the consequent reduction of the topical irritating action.
Also, all the other esters of the series were definitely
less irritating than ibuprofen, when studied in the same
conditions (Table 2). In fact, the ibuprofen production
by all the compounds during their lumen transit is likely
to be low as a consequence of their quite good stability
in acidic medium (Table 1).
Another possible reason for the low ulcerogenicity of
the compounds could be a consequence of their inhibi-
tory effects on COX-2 rather than COX-1. A recent
report shows that this is the case of a series of ester
and amide derivatives of indomethacin.24 Preliminary
results obtained using a human whole blood test25 show
that our compounds display a certain degree of COX-2
selective inhibition. However, the interpretation of this
behavior is complex because, owing to different incuba-
tion times required in the COX inhibition experiments
(1 h for COX-1, 24 h for COX-2), enzyme inhibition is
the result of a combined action of the prodrugs as well
as ibuprofen derived from them, and the relative
importance of these two components is different in the
two tests. To overcome this problem, we are extending
the study by employing ovine isolated COX-1 and COX-2
enzymes.
Finally, it is known that nitric oxide is able to trigger
gastroprotection by a number of mechanisms and NO-
donors are capable of reducing gastrotoxicity in some
experimental models.26 Thus, this third component
should be taken into account to explain the behavior of
the NO-donor esters 8, 9, and 11. However, the inability
of NO-releasing ibuprofen derivatives to produce nitrite
in the acidic medium suggests that NO release is
unlikely to occur in the gastric lumen.
P la telet Aggr ega tion . Antiaggregatory effects of the
compounds were studied on arachidonic acid (AA)
induced platelet aggregation of human platelet rich
plasma (PRP); in the case of NO-donor derivatives 8, 9,
11, the experiments were repeated in the presence of
oxyhaemoglobin (HbO2), a known scavenger of NO. The
results are shown in Table 3, where the antiaggregatory
activity of aspirin is also reported for comparison. Data
are expressed as % of inhibition of aggregation at
maximal concentration tested or as pIC50 ( SEM, when
g
vehicle (ANOVA and Dunnett’s test). P < 0.01 vs ibuprofen-
h
treated group (ANOVA and Newman-Keuls test). P < 0.01 vs
vehicle (ANOVA and Dunnett’s test).
However, its inhibitory effect did not reach significance
after 6 h. Ibuprofen propyl ester 12, at 4 h, induced a
significant reduction (37.6%) of the edema volume, this
effect being actually increased after 6 h (49.6%). All the
other products of the series displayed significant anti-
inflammatory activity, with a comparable efficacy (Table
2). Compounds differed in terms of the dose needed to
achieve a significant antiinflammatory effect. In fact,
benzenesulfonyl derivatives 8 and 8a elicited their
action at the lower dose tested, and their effect was
evident at 4 and 6 h. Phenylthiofuroxan 9, phenylthio-
furazan 9a , and the nitroxy ester 11 were effective at
the higher dose tested, and again the effect was present
at 4 and 6 h. The antiinflammatory activity of all the
compounds of the series, could partly reside in their
behavior as prodrugs of 3. Indeed, as shown in Table 1,
after a 6 h incubation in human plasma, the compounds
afforded a significant amount of ibuprofen (Table 1),
although to different degrees. In any case, other factors
could be involved; for example, the products themselves
could directly inhibit COX.
Acu te Ga str ic Mu cosa l Da m a ge. All the com-
pounds were assessed for their ulcerogenic properties
in conscious rats. The extent of gastric lesions were
measured 4 and 6 h after the intragastric administra-
tion of the compounds, and the results were expressed
as “lesion index”. Ibuprofen 3, administered at 120 mg/
kg, time-dependently induced macroscopically visible
lesions, characterized by mucosal necrosis and haem-
orrhage, lesion index being 15.4 and 25.7 at 4 and 6 h,
respectively (Table 2). By contrast, propyl ester 12 was
significantly less ulcerogenic than the parent compound
at 6 h (lesion index ) 3.8, Table 2).
There are two main components of gastric damaging
properties of NSAIDs, an irritating effect dependent
upon direct contact with the mucosa, related to the
presence of acidic groups in the molecules, and a
systemic effect attributable to the inhibition of prostag-
landin synthesis.21-23 Both components contribute to the
ulcerogenic activity of ibuprofen 3.21 Each of these
mechanisms has a different time course for the injury

3466 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Lolli et al.
(0.75 g, 6.6 mmol) dissolved in dry MeOH (10 mL), was added
under nitrogen over 2 h to a stirred solution of 5 (2.0 g, 6.6
mmol) in dry THF (30 mL) kept at -15 °C. The reaction
mixture was stirred for an additional hour at -15 °C. The
solution was allowed to reach room temperature and then was
concentrated under vacuum without heating. The residue was
treated with EtOAc, and the resulting mixture was washed
twice with water. The organic layer dried and evaporated
under vacuum at room temperature gave a residue that was
purified by flash chromatography (petroleum ether (40-60)/
EtOAc 7.5/2.5 v/v). The pure title compound was obtained as
a pale yellow oil, yield 45%. 1H NMR (CDCl3) δ 1.59 (s, 1H,
-CH2OH); 2.04 (qi, 2H,-CH2CH2OH); 3.73 (t, 2H, -CH2CH2OH);
it could be calculated by nonlinear regression analysis.
All the derivatives were able to inhibit the aggregation
induced by AA in PRP in a concentration-dependent
manner. Furazan and propyl derivatives 8a , 9a , and 12
were less potent than ibuprofen. By contrast, the NO-
donor 8 was more potent than ibuprofen, while com-
pounds 9 and 11 were approximately equipotent with
the parent drug. The potency of these three NO-donors
parallels their ability to produce nitrite and, in com-
parison with reference compounds, it follows the se-
quence 8 > 9 > aspirin ) ibuprofen . 11. The
antiaggregatory activity of the NO-donors 8, 9, and 11
was reversed by oxyhaemoglobin, indicating an involve-
ment of nitric oxide.
4.52 (t, 2H, FuroxanOCH2CH2-); 7.3-7.5 (m, 5H, C6H5S-). 13
C
NMR (CDCl3) δ 31.25 (-CH2CH2OH); 58.68 (-CH2CH2OH);
67.53 (FuroxanOCH2CH2-); 104.79 (Furoxan C(3)); 128.63,
129.02, 129.50, 131.81 (C6H5S-); 163.4 (Furoxan C(4)). MS (EI)
m/z 268 (M)+. Anal. (drying conditions: room temperature; 15
days; pressure < 1 mmHg) for C11H12N2O4S C, H, N.
Con clu sion s
The introduction of furoxan and furazan moieties into
the molecule of ibuprofen does not alter the antiinflam-
matory activity. The products described in the present
investigation exhibit reduced gastric ulcerogenicity
when compared with ibuprofen. A number of heteroge-
neous mechanisms could underlie the loss of ulcerogenic
properties. Since both the NO donor and the non-NO-
donor derivatives are devoid of these effects, it is
assumable that the prodrug formation is primarily at
the basis of reduced topical irritant action, by masking
the free carboxylic group of ibuprofen.
Ibuprofen derivatives also display antiaggregatory
activity to a different degree. It is evident that the
furoxan analogues, which are able to release NO, are
endowed with an antiaggregatory activity superior to
ibuprofen and aspirin.
3-(4-(P h en ylth io)fu r a za n -3-yloxy)p r op a n ol (6a ). The
title compound was obtained as pale yellow oil in the same
manner as analogue 6. In this case, the addition of potassium
benzenethiolate solution was performed at room temperature
and the resulting mixture was heated at 40 °C for 48 h to
1
complete the reaction. Yield 68%. H NMR (CDCl3) δ 1.50 (s,
1H, -CH2OH); 1.98 (qi, 2H, -CH2CH2OH); 3.66 (t, 2H, -CH2CH2-
OH); 4.45 (t, 2H, FurazanOCH2CH2-); 7.4-7.6 (m, 5H, C6H5S-
). 13C NMR (CDCl3) δ 31.36 (-CH2CH2OH); 58.67 (-CH2CH2-
OH); 69.66 (-FurazanOCH2CH2-); 127.86, 129.13, 129.38,
132.67 (C6H5S-); 144.06 (Furazan C(4)); 163.39 (Furazan C(3)).
MS (EI) m/z 252 (M)+. Anal. (drying conditions: room tem-
perature; 15 days; pressure < 1 mmHg) for C11H12N2O3S ‚ 0.2
H2O C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Ester s 8,
8a , 9, 9a , 10. Dry pyridine (0.55 mL, 6.8 mmol) and a solution
of the appropriate propanol derivative (3.4 mmol) in dry
CH2Cl2 (7.0 mL) were added dropwise to a solution of 2-(4-
isobutylphenyl)propionyl chloride 7 (0.78 g, 3.4 mmol) in dry
CH2Cl2 (5.0 mL). The mixture was stirred for 2 h, then diluted
with CH2Cl2 (20 mL). The resulting solution was washed in
sequence with 0.5 M HCl (4 × 10 mL), 1 M NaOH (2 × 10
mL), and saturated aqueous NaCl and then dried. Solvent
removal under reduced pressure at room temperature afforded
a crude oily residue that was purified by flash chromatography.
The expected pure compound was obtained as an oily pale
yellow material. Chromatographic eluents and yields of the
products were as follows.
In conclusion, some of the products described herein
might be potential clinical candidates as safer antiin-
flammatory and antiaggregatory drugs.
Exp er im en ta l Section
Ch em istr y. Melting points were measured with a capillary
apparatus (Bu¨chi 530) and are uncorrected. All the compounds
1
were routinely checked by IR (Shimadzu FT-IR 8101 M), H,
and 13C NMR (Bruker AC-200; the following abbreviations
were used to indicate the peak multiplicity: s ) singlet; d )
doublet; t ) triplet; qt ) quartet; qi ) quintet; n ) nine lines;
m ) multiplet) and mass spectrometry (Finnigan-Mat TSQ-
700). Silica gel (Merck Kieselgel 60) 70-230 mesh ASTM was
employed for column chromatography. HPLC analyses were
performed by using a diode array UV detector (Shimadzu
LC10A); the figures in brackets are standard errors (( SEM).
Anhydrous magnesium sulfate was used as drying agent of
the organic phases. Analysis (C, H, N) of the new compounds
was performed by REDOX (Monza), and the results are within
( 0.4% of the theoretical. Structures 4,19 4a ,15 5,18 7,27 and
1228 were synthesized according to literature methods.
3-(4-(Ben zen esu lfon yl)fu r a za n -3-yloxy)p r op a n ol (5a ).
The title compound was obtained in the same manner as
analogue 5 (lit.18). Yield 58%, mp 67 °C from isopropyl ether.
1H NMR (CDCl3) δ 1.61 (t, 1H, -CH2OH); 2.10 (qi, 2H, -CH2-
CH2OH); 3.82 (qt, 2H, -CH2CH2OH); 4.55 (t, 2H, FurazanOCH2-
CH2-); 7.6-8.1 (m, 5H, C6H5SO2-). 13C NMR (CDCl3) δ 31.22
(-CH2CH2OH); 58.81 (-CH2CH2OH); 71.12 (FurazanOCH2-
CH2-); 128.60, 129.53, 135.37, 137.88 (C6H5SO2-); 148.67
(Furazan C(4)); 161.18 (Furazan C(3)). MS (CI) m/z 285
(M+1)+. Anal. (drying conditions: 40 °C; 18 h, pressure < 1
mmHg) for C11H12N2O5S C, H, N.
3-(3-(Ben zen esu lfon yl)fu r oxa n -4-yloxy)p r op yl 2-(4-
isobu tylp h en yl)p r op ion a te (8): eluent (petroleum ether
(40-60)/EtOAc 8/2 v/v); yield 55%. 1H NMR (CDCl3) δ 0.87
(d, 6H, (CH3)2CH-); 1.50 (d, 3H, Ar(CH3)CH-); 1.82 (n, 1H,
(CH3)2CHCH2-); 2.15 (qi, 2H, -COOCH2CH2-); 2.42 (d, 2H,
(CH3)2CHCH2-); 3.71 (qt, 1H, Ar(CH3)CH-); 4.26 (m, 2H,
-COOCH2CH2-); 4.33 (t, 2H, -CH2CH2OFuroxan); 7.0-7.2 (m,
4H, (CH3)2CHCH2C6H4-); 7.6-8.1 (m, 5H, C6H5SO2-). 13C NMR
(CDCl3) δ 18.16 (Ar(CH3)CH-); 22.19 ((CH3)2CHCH2-); 27.63
(-COOCH2CH2-); 29.99 ((CH3)2CHCH2-); 44.81, 44.90 (Ar(CH3)-
CH-/(CH3)2CHCH2-); 60.07 (COOCH2CH2); 67.52 (-CH2CH2-
OFuroxan); 110.30 (Furoxan C(3)); 126.88, 129.21, 137.39,
140.55 (Phenyl); 128.37, 129.56, 135.47, 137.91 (C6H5SO2-);
158.56 (Furoxan C(4)); 174.39 (-COOCH2CH2-). MS (EI) m/z
488 (M)+. Anal. (drying conditions: 40 °C; 15 days; pressure
< 1 mmHg) for C24H28N2O7S C, H, N.
3-(4-(Ben zen esu lfon yl)fu r a za n -3-yloxy)p r op yl 2-(4-
isobu tylp h en yl)p r op ion a te (8a ): eluent (petroleum ether
(40-60)/EtOAc 8.5/1.5 v/v); yield 66%. 1H NMR (CDCl3) δ 0.88
(d, 6H, (CH3)2CH-); 1.49 (d, 3H, Ar(CH3)CH-); 1.86 (n, 1H,
(CH3)2CHCH2-); 2.11 (qi, 2H, -COOCH2CH2-); 2.43 (d, 2H,
(CH3)2CHCH2-); 3.69 (qt, 1H, Ar(CH3)CH-); 4.17 (t, 2H,
-COOCH2CH2-); 4.29 (t, 2H, -CH2CH2OFurazan); 7.0-7.2 (m,
4H, (CH3)2CHCH2C6H4-); 7.6-8.1 (m, 5H, C6H5SO2-). 13C NMR
(CDCl3) δ 18.17 (Ar(CH3)CH-); 22.24 ((CH3)2CHCH2-); 27.73
(-COOCH2CH2-); 30.04 ((CH3)2CHCH2-); 44.86 (Ar(CH3)CH-)/
((CH3)2CHCH2-); 60.07 (-COOCH2CH2-); 69.96 (-CH2CH2-
3-(3-(P h en ylth io)fu r oxa n -4-yloxy)p r op a n ol (6). A solu-
tion of potassium benzenethiolate (20 mL), prepared by mixing
under nitrogen and stirring benzenethiol (0.70 mL, 6.6 mmol)
dissolved in dry MeOH (10 mL) and potassium tert-butoxide

Ibuprofen Derivatives with Reduced Gastrotoxicity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3467
OFurazan); 126.87, 129.21, 137.40, 140.58 (Phenyl); 128.76,
129.57, 135.34, 137.72 (C6H5SO2-); 149.74 (Furazan C(4));
160.92 (Furazan C(3)); 174.37 (COOCH2CH2). MS (EI) m/z 472
(M)+. Anal. (drying conditions: 40 °C; 15 days; pressure <1
mmHg) for C24H28N2O6S C, H, N.
(M)+. Anal. (drying conditions: 40 °C; 3 days; pressure < 1
mmHg) for C16H23NO5 C, H, N.
Hyd r olysis in Hu m a n P la sm a . A solution of each com-
pound (0.0438 µmol) in acetonitrile (5 µL) was added to human
plasma (495 µL) at 37 °C. After 6 h of incubation, the reaction
mixture was diluted with acetonitrile containing 0.1% trifluo-
roacetic acid (1:1.5 v/v). Serum was separated by centrifugation
(9600g, 15 min) and assayed by HPLC for ibuprofen produc-
tion, using a Merck Purospher RP-18 column (250 × 4 mm; 5
µm particles) at 40 °C, eluting (1 mL/min) with water/
acetonitrile/TFA 25/75/0.1 (v/v/v).
Hyd r olysis in Acid ic Med iu m . A solution of each com-
pound (0.25 mM) in acetonitrile (560 µM) was added to a HCl
solution (pH 1, 1440 µL) at 37 °C. After 2 h of incubation,
ibuprofen and corresponding ester were detected by HPLC,
according to the previously described method.
3-(3-(P h en ylt h io)fu r oxa n -4-yloxy)p r op yl 2-(4-Isob u -
t ylp h en yl)p r op ion a t e (9): eluent (petroleum ether (40-
1
60)/EtOAc 9.5/0.5 v/v); yield 64%. H NMR (CDCl3) δ 0.89 (d,
6H, (CH3)2CH-); 1.49 (d, 3H, Ar(CH3)CH-); 1.84 (n, 1H,
(CH3)2CHCH2-); 2.05 (qi, 2H, -COOCH2CH2-); 2.44 (d, 2H,
(CH3)2CHCH2-); 3.69 (qt, 1H, Ar(CH3)CH-); 4.11 (t, 2H,
-COOCH2CH2-); 4.29 (t, 2H, -CH2CH2OFuroxan); 7.0-7.2 (m,
4H, (CH3)2CHCH2C6H4-); 7.3-7.4 (m, 5H, C6H5S-). 13C NMR
(CDCl3) δ 16.17 (Ar(CH3)CH-); 22.21 ((CH3)2CHCH2-); 27.65
(-COOCH2CH2-); 30.04 ((CH3)2CHCH2-); 44.84, 44.92 ((CH3)2-
CHCH2-/Ar(CH3)CH-)); 60.14 (-COOCH2CH2-); 66.61 (-CH2CH2-
OFuroxan); 104.75 (Furoxan C(3)); 126.91, 129.52, 137.39,
140.59 (Phenyl); 128.48, 129.04, 129.23, 131.92 (C6H5S-);
163.75 (Furoxan C(4)); 174.37 (-COOCH2CH2-). MS (EI) m/z
456 (M)+. Anal. (drying conditions: room temperature; 15 days;
pressure < 1 mmHg) for C24H28N2O5S C, H, N; Calculated C
63.14; H 6.18; N 6.14; Found: C 63.70; H 6.34; N 6.06.
Detection of Nitr ite. A solution of the appropriate com-
pound (20 µL) in dimethyl sulfoxide (DMSO) was added to 2
mL of 1:1 v/v mixture either of 50 mM phosphate buffer (pH
7.4) or of a HCl solution (pH 1) with MeOH, containing 5 ×
10-4 M L-cysteine. The final concentration of drug was 10-4
M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated
with 250 µL of Griess reagent [sulfanilamide (4 g), N-
naphthylethylenediamine dihydrochloride (0.2 g), 85% phos-
phoric acid (10 mL) in distilled water (final volume: 100 mL)].
After 10 min at room temperature, the absorbance was
measured at 540 nm (Shimatzu UV-2501PC spectrophotom-
eter). Sodium nitrite standard solutions (10-80 nmol/mL) were
used for the calibration curve. The yields in nitrite expressed
3-(4-(P h en ylt h io)fu r a za n -3-yloxy)p r op yl 2-(4-isob u -
t ylp h en yl)p r op ion a t e (9a ): eluent (petroleum ether (40-
1
60)/EtOAc 9.5/0.5 v/v); yield 74%. H NMR (CDCl3) δ 0.89 (d,
6H, (CH3)2CH-); 1.49 (d, 3H, Ar(CH3)CH-); 1.85 (n, 1H,
(CH3)2CHCH2-); 1.98 (qi, 2H, -COOCH2CH2-); 2.45 (d, 2H,
(CH3)2CHCH2-); 3.69 (qt, 1H, Ar(CH3)CH-); 4.03 (t, 2H,
-COOCH2CH2-); 4.24 (t, 2H, -CH2CH2OFurazan); 7.1-7.2 (m,
4H, (CH3)2CHCH2C6H4-); 7.3-7.5 (m, 5H, C6H5S-). 13C NMR
(CDCl3) δ 18.13 (Ar(CH3)CH-); 22.21 ((CH3)2CHCH2-); 27.76
(-COOCH2CH2-); 30.00 ((CH3)2CHCH2-); 44.85, 44.92 (Ar(CH3)-
CH-/(CH3)2CHCH2-)); 60.21 (-COOCH2CH2-); 66.78 (-CH2CH2-
OFurazan); 126.92, 129.37, 137.40, 140.56 (Phenyl); 127.72,
129.20, 129.20, 132.91 (C6H5S-); 144.02 (Furazan C(4)); 163.30
(Furazan C(3)); 174.33 (-COOCH2CH2-). MS (EI) m/z 440 (M)+.
Anal. (drying conditions: 40 °C; 48 h; pressure < 1 mmHg)
-
as NO2 % (mol/mol) ( SEM. No production of nitrite was
observed in the absence of L-cysteine.
P h a r m a cology. (a ) An t iin fla m m a t or y Act ivit y a n d
Ga str otoxicity. All the compounds were initially dissolved
in DMSO and then diluted in 1% carboxymethylcellulose. The
final concentration of DMSO was 5%. Each agent was prepared
immediately before use and administered intragastrically in
a volume of 10 mL/kg.
(1) Ca r r a geen a n -In d u ced P a w Ed em a . Male Wistar
strain rats (Harlan, Italy), weighing 180-200 g, were deprived
of food but not of water for 24 h before the experiment. The
edema was induced by injecting 0.1 mL of 1% carrageenan
suspended in 1% carboxymethylcellulose, into the plantar
surface of the right hind foot of each rat. Foot volume was
measured 4 h after carrageenan injection; foot volume increase
was recorded with respect to the volume measured before
carrageenan. The edema reduction in treated animals was
expressed as percentage inhibition of the edema observed in
the corresponding control group, considered as 100. Measure-
ments were conducted using a water plethysmometer (Basile,
Comerio, Italy). Groups of rats (n ) 6) were given ibuprofen
40 and 120 mg/kg or equimolar doses of the other compounds.
The higher dose was tested only when the lower one was
inactive. All compounds were administered immediately before
a carrageenan injection. Control rats received the vehicle only.
The results obtained are presented as mean ( SEM. Statistical
analysis was performed with ANOVA followed by Dunnett’s
test.
(2) Ga str ic Mu cosa l Da m a ge. Male Wistar strain rats
(Harlan, Italy), weighing 180-200 g, were deprived of food but
not of water for 24 h before the experiment. Groups of rats (n
) 6) were given ibuprofen 120 mg/kg, or equimolar doses of
the other compounds. The rats were sacrified 6 h after the
administration of the compounds. The stomachs were removed,
opened along the lesser curvature, and examined under a
stereomicroscope for the presence of macroscopically visible
lesions. Each individual haemorrhagic lesion was measured
along its greatest length (<1 mm ) rating of 1; 1-2 mm )
rating of 2; > 2 mm ) rating according to their length in mm).
The overall total was designated as the “lesion index”. The
results obtained are presented as mean ( SEM. Statistical
analysis was performed with ANOVA followed by Newman-
Keuls test.
C
24H28N2O4S C, H, N.
3-Br om op r op yl 2-(4-Isob u t ylp h en yl)p r op ion a t e (10):
eluent (petroleum ether (40-60)/EtOAc 9.5/0.5 v/v); yield
1
67%. H NMR (CDCl3) δ 0.90 (d, 6H, (CH3)2CH-); 1.50 (d, 3H,
Ar(CH3)CH-); 1.85 (n, 1H, (CH3)2CHCH2-); 2.11 (qi, 2H,
-COOCH2CH2-); 2.45 (d, 2H, (CH3)2CHCH2-); 3.28 (t, 2H,
-CH2CH2CH2Br); 3.70 (qt, 1H, Ar(CH3)CH-); 4.21 (m, 2H,
-COOCH2CH2-); 7.1-7.3 (m, 4H, (CH3)2CHCH2C6H4-). 13C
NMR (CDCl3) δ 18.15 (Ar(CH3)CH-); 22.22 ((CH3)2CH-); 30.41
(-COOCH2CH2-); 31.47 ((CH3)2CHCH2-); 32.59 (-CH2CH2CH2-
Br); 44.87, 44.96 ((CH3)2CHCH2-/Ar(CH3)CH-); 62.08 (-CH2CH2-
CH2Br); 126.94, 129.22, 137.52, 140.49 (Phenyl); 174.40
(-COOCH2-). MS (EI) m/z 326/328 (M)+. Anal. (drying condi-
tions: 40 °C; 3 days; pressure < 1 mmHg) for C16H23BrO2 C,
H.
3-Nitr oxyp r op yl 2-(4-Isobu tylp h en yl)p r op ion a te (11).
A solution of compound 10 (0.88 g, 2.7 mmol) in a small
amount of dry CH3CN was added at room temperature to a
stirred solution of AgNO3 (1.83 g, 10.7 mmol) in dry CH3CN
(10 mL). Stirring was continued over 24 h and then for 4 h at
40 °C. The reaction mixture was filtered, and the collected solid
was washed with CH3CN. The combined organic phases were
dried and treated with CH2Cl2 (20 mL). The solution was
washed with water, dried, and evaporated under vacuum. The
residue was purified by flash chromatography [petroleum ether
(40-60)/isopropyl ether 9.5/0.5 v/v] to give the expected
product as yellow oil (lit.29). Yield 49%. 1H NMR (CDCl3) δ 0.92
(d, 6H, (CH3)2CH-); 1.51 (d, 3H, Ar(CH3)CH-); 1.84 (n, 1H,
(CH3)2CHCH2-); 1.98 (qi, 2H, -COOCH2CH2-); 2.47 (d, 2H,
(CH3)2CHCH2-); 3.72 (qt, 1H, Ar(CH3)CH-); 4.19 (m, 2H,
-COOCH2CH2-); 4.36 (t, 2H, -CH2CH2ONO2); 7.1-7.3 (m, 4H,
(CH3)2CHCH2C6H4-). 13C NMR (CDCl3) δ 17.98 (Ar(CH3)-
CH-); 22.13 ((CH3)2CH-); 26.07 (-COOCH2CH2-); 29.97 ((CH3)2-
CHCH2-); 44.79, 44.84 ((CH3)2CHCH2-/Ar(CH3)CH-); 60.15
(-CH2CH2CH2ONO2); 69.44 (-CH2CH2ONO2); 126.85, 129.19,
137.36, 140.49 (Phenyl); 174.22 (-COOCH2-). MS (EI) m/z 309
(b) P la telet Aggr ega tion Assa y. Venous blood was ob-
tained from consenting healthy human subjects who had not

3468 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Lolli et al.
(13) Hecker, M.; Vorhoff, W.; Bora, A. T.; Mordvintcev, P. I.; Busse,
R.; Characterization of Furoxans as a New Class of Tolerance-
Resistant Nitrovasodilators. Naunyn-Schmiedebergs Arch. Phar-
macol. 1995, 351, 426-432.
(14) Fruttero, R.; Boschi, D.; Di Stilo, A.; Gasco, A. The Furoxan
System as a Useful Tool for Balancing “Hybrids” with Mixed
R1-Antagonist and NO-like Vasodilator Activities. J . Med. Chem.
1995, 38, 4944-4949.
(15) Boschi, D.; Di Stilo, A.; Cena, C.; Lolli, M.; Fruttero, R.; Gasco,
A. Studies on Agents with Mixed NO-Dependent Vasodilating
and â-Blocking Activities. Pharm. Res. 1997, 14, 1750-1758.
(16) Di Stilo, A.; Visentin, S.; Cena, C.; Gasco, A. M.; Ermondi, G.;
Gasco, A. New 1,4-Dihydropyridines Conjugated to Furoxanyl
Moieties, Endowed with Both Nitric Oxide-like and Calcium
Channel Antagonist Vasodilator Activities. J . Med. Chem. 1998,
41, 5393-5401.
(17) Bertinaria, M.; Sorba, G.; Medana, C.; Cena, C.; Adami, M.;
Morini, G.; Pozzoli, C.; Coruzzi, G.; Gasco, A. Synthesis and
Pharmacological Characterization of New H2-Antagonists Con-
taining NO-Donor moieties, endowed with Mixed Antisecretory
and Gastroprotective Activities. Hel. Chim. Acta 2000, 83, 287-
299.
taken any drug for at least two weeks. Volunteers were
informed that blood samples were obtained for research
purposes and that their privacy would be protected. PRP was
prepared by centrifugation of citrated blood at 200g for 20 min.
Aliquots (300 µL) of PRP were added into an aggregometer
cuvette (Elvi) and aggregation was recorded as increased light
transmission under continuous stirring (1000 rpm) at 37 °C
for 5 min after addition of the stimulus. AA was used as
platelet activator at a concentration producing 85-95% of the
maximal aggregation (0.5-1 mM). The inhibitory activity of
the compounds was tested by adding their solution or the
solvent (DMSO) to PRP 10 min before addition of the stimulus
at 37 °C. Haemoglobin was added to the platelets 12 min before
addition of the platelet activator at 40 µM final concentration.
The maximal amount (0.5%) of DMSO added to PRP or to the
platelet suspension did not affect platelet function. The anti-
aggregating activity of tested compounds was evaluated as %
of inhibition of platelet aggregation in respect with the control
samples. When inhibition at maximal concentration exceeded
50%, pIC50 values were calculated by nonlinear regression
analysis.
(18) Cena, C.; Visentin, S.; Di Stilo, A.; Boschi, D.; Fruttero, R.; Gasco,
A. Studies on Agents with Mixed NO-Dependent and Calcium
Channel Antagonistic Vasodilating Activities. Pharm. Res. 2001,
18, 157-165.
Ack n ow led gm en t. This work was partially sup-
(19) Sorba, G.; Ermondi, G.; Fruttero, R.; Galli, U.; Gasco, A.
Unsymmetrically Substituted Furoxans. XVI. Reaction of Ben-
zenesulfonyl Substituted Furoxans with Ethanol and Ethaneth-
iol in Basic Medium. J . Heterocyclic Chem. 1996, 33, 327-334.
(20) Fruttero, R.; Sorba, G.; Ermondi, G.; Lolli, M.; Gasco, A.
Unsymmetrically Substituted Furoxans. XVII. Structural In-
vestigations in Benzenesulfonylfuroxan Derivatives and Related
Compounds. Il Farmaco 1997, 52, 405-410.
(21) Cioli, V.; Putzolu, S.; Rossi, V.; Scorza Barcellona, P.; Corradino,
C. The Role of Direct Tissue Contact in the Production of
Gastrointestinal Ulcers by Antiinflammatory Drugs in Rats.
Toxicol. Appl. Pharmacol. 1979, 50, 283-289.
(22) J ones, G. In Design of Prodrugs; Bundgaard, H., Ed.; Elsevier:
Amsterdam, 1985.
(23) Shanbhag, V. R.; Crider, A. M.; Gokhale, R.; Harpalani, A.; Dick,
R. M. Ester and Amide Prodrugs of Ibuprofen and Naproxen:
Synthesis, Antiinflammatory Activity and Gastrointestinal Tox-
icity. J . Pharm. Sci. 1992, 81, 149-154.
(24) Kalgutkar, A. S.; Marnett, A. B.; Crews, B. C.; Remmel, R. P.;
Marnett, L. J . Ester and Amide Derivatives of the Non Steroidal
Antiinflammatory Drug, Indometacin, as Selective Cyclooxyge-
nase-2 Inhibitors. J . Med. Chem. 2000, 43, 2860-2870.
(25) Patrignani, P.; Panara, M. R.; Greco, A.; Fusco, O.; Natoli, C.;
Iacobelli, S.; Cipollone, F.; Ganci, A.; Creminon, C.; Maclouf, J .;
Patrono, C. Biochemical and Pharmacological Characterization
of the Cyclooxygenase Activity of Human Blood Prostaglandin
Endoperoxide Synthases. J . Pharmacol. Exp. Ther. 1994, 271,
1705-1712.
(26) MacNaughton, W. K.; Cirino, G.; Wallace, J . L. Endothelium-
derived Relaxing Factor (Nitric Oxide) has Protective Actions
in the Stomach. Life Sci. 1998, 45, 1869-1876.
(27) Larsen, R. D.; Corley, E. G.; Davis, P.; Reider, P. J .; Grabowski,
E. J . J . R-Hydroxy Esters as Chiral Reagents: Asymmetric
Synthesis of 2-Arylpropionic Acids. J . Am. Chem. Soc. 1989, 111,
7650-7651.
ported by a MURST grant.
Refer en ces
(1) Insel, P. A. Analgesic-antipyretic and antiinflammatory agents
and drugs employed in the treatment of gout. In The Pharma-
cological Basis of Therapeutics, 9th ed.; Hardman, J . G.; Limbird,
L. E.; Molinoff, P. B.; Ruddon, R. W.; Gilman, A. G., Eds.
McGraw-Hill: New York, pp 1996, 617-657.
(2) Griswold, D. E.; Adams, L. Constitutive Cyclooxygenase (COX-
1) and Inducible Cyclooxygenase (COX-2): Rationale for Selec-
tive Inhibition and Progress to Date. Med. Res. Rev. 1996, 16,
181-206.
(3) Wallace, J . L.; Granger, D. N. The Cellular and Molecular Basis
of Gastric Mucosal Defence. FASEB 1996, 10, 731-740.
(4) Kerwin, J . F., J r.; Lancaster, J . R., J r.; Feldman, P. L. Nitric
Oxide: A New Paradigm for Second Messenger. J . Med. Chem.
1995, 38, 4343-4362.
(5) Wallace, J . L.; Cirino, G. The Development of Gastrointestinal-
Sparing Non Steroidal Antiinflammatory Drugs. Trends Phar-
macol. Sci. 1994, 15, 405-406.
(6) Del Soldato, P.; Sorrentino, R.; Pinto, A. NO-Aspirins: a Class
of New Antiinflammatory and Antithrombotic Agents. Trends
Pharmacol. Sci. 1999, 20, 319-323.
(7) Bandarage, U. K.; Chen L.; Fang X.; Garvey, D. S.; Gilarin, A.;
J anero, D. R.; Gordon Letts, L.; Mercer, G. J .; Saha, J . K.;
Schroeder, J . D.; Shumway M. J .; William Tam S. Nitrosothiol
Esters of Diclofenac: Synthesis and Pharmacological Charac-
terization as Gastrointestinal-Sparing Prodrugs. J . Med. Chem.
2000, 43, 4005-4016.
(8) Feelisch, M. In Methods in Nitric Oxide Research; Feelisch, M.,
Stamler, J . S., Eds.; J ohn Wiley: Chichester, 1996.
(9) Scho¨nafinger, K. Heterocyclic NO Prodrugs. Il Farmaco 1999,
54, 316-320.
(10) Sorba, G.; Medana, C.; Fruttero, R.; Cena, C.; Di Stilo, A.; Galli,
U.; Gasco, A. Water Soluble Furoxan Derivatives as NO Pro-
drugs. J . Med. Chem. 1997, 40, 2288; 463-469, and references
therein.
(11) Fukuto, J . M.; Chiang, K.; Hszieh, R.; Wong, P.; Chaudhuri, G.
The Pharmacological Activity of Nitroxyl: A Potent Vasodilator
with Activity Similar to Nitric Oxide and/or Endothelium-
Derived Relaxing Factor. J . Pharmacol. Exp. Ther. 1992, 263,
546-551.
(12) Bohn, H.; Brendel, J .; Martorana, P. A.; Scho¨nafinger, K.
Cardiovascular Actions of Furoxan CAS 1609, a Novel Nitric
Oxide Donor. Br. J . Pharmacol. 1995, 114, 1605-1612.
(28) Hisamitsu Pharmaceutical Co., Inc. J P 57, 206, 640 [82, 206,
640], 1982, Appl. 82/11, 769, 1982. Chem Abstr. 1983, 98,
125656y.
(29) Xiong, C.; Changgeng, Q. Nonsteroidal antiinflammatory agents
capable of releasing nitric oxide, their preparing method and
use. CN 1, 144, 092, 1997, Appl. 95, 109, 791, 1995. Chem. Abstr.
1998, 128, 226234c.
J M0108799