Synthesis and pharmacological characterization of a novel nitric oxide-releasing diclofenac derivative containing a benzofuroxan moiety

Article abstract of DOI:10.1016/j.ejmech.2010.02.034

1-Oxy-benzo[1,2,5]oxadiazol-5-ylmethyl [2-(2,6-dichloro-phenylamino)-phenyl]-acetate, a new diclofenac derivative bearing a benzofuroxan heterocyclic moiety in its structure, was prepared by the reaction of sodium diclofenac and 5-bromomethyl-benzo[1,2,5]oxadiazole 1-oxide. Pharmacological characterization of this modified diclofenac maintained the anti-inflammatory activity similar to its parent compound assayed in vitro and in vivo. The ulcerogenic properties of native diclofenac were not observed with this modified compound, despite the inhibition of prostaglandin E₂ gastric content. The better gastric tolerability seems to be related to nitric oxide release ability.

Full text of DOI:10.1016/j.ejmech.2010.02.034

European Journal of Medicinal Chemistry 45 (2010) 2489e2493
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological characterization of a novel nitric oxide-releasing
diclofenac derivative containing a benzofuroxan moiety
*
Paulo Sérgio de Carvalho, Marta Maróstica, Alessandra Gambero , José Pedrazzoli Jr.
Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Av. São Francisco de Assis 218, 12916-900 Bragança Paulista, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
1-Oxy-benzo[1,2,5]oxadiazol-5-ylmethyl [2-(2,6-dichloro-phenylamino)-phenyl]-acetate, a new diclofe-
nac derivative bearing a benzofuroxan heterocyclic moiety in its structure, was prepared by the reaction
of sodium diclofenac and 5-bromomethyl-benzo[1,2,5]oxadiazole 1-oxide. Pharmacological character-
ization of this modified diclofenac maintained the anti-inflammatory activity similar to its parent
compound assayed in vitro and in vivo. The ulcerogenic properties of native diclofenac were not observed
with this modified compound, despite the inhibition of prostaglandin E2 gastric content. The better
gastric tolerability seems to be related to nitric oxide release ability.
Received 29 October 2009
Received in revised form
12 February 2010
Accepted 15 February 2010
Available online 20 February 2010
Keywords:
Furoxans
Ó 2010 Elsevier Masson SAS. All rights reserved.
Non-steroidal anti-inflammatory drugs
Gastric tolerability
Prostaglandin E2
Cyclooxygenases
1. Introduction
gastric functions such as mucosal blood flow and, consequently, the
mucus production. Nitric oxide is also able to interact with cas-
The beneficial effects of non-steroidal anti-inflammatory drugs
(NSAIDs) are always balanced by their side effects; the major
undesirable effect being related to the gastrointestinal system. In
recent years, a number of novel approaches have been taken to
develop gastrointestinal-sparing NSAIDs. One of these was the
establishment of selective cyclooxygenase (COX)-2 inhibitors in the
therapeutic routine. The ability to inhibit the COX-2 isoform
provides these compounds with anti-inflammatory properties that
are comparable to standard NSAIDs in many situations. In contrast
to the standard NSAIDs, these compounds present a reduced
capacity to induce gastric ulceration since they lack the ability to
inhibit prostaglandin derived-COX-1, which is physiologically
expressed in the stomach [1]. Nevertheless, selective COX-2 drugs
interfere strongly with ulcer healing, since COX-2 is induced in the
stomach during injury and adaptive cytoprotection mechanisms
[2]. Another important and recent finding is the increased inci-
dence of thrombotic and cardiovascular events when COX-2
selective drugs are prescribed long term [3]. A second approach is
the addition of a nitric oxide (NO)-releasing moiety to classical
NSAIDs, producing the NO-NSAIDs. The NO released by these
NSAIDs exerts beneficial effects on the mucosa by modulating
pases, resulting in a decreased synthesis of Th-1 derived cytokines,
such as IL-1
, IL-18 and IFN- [4]. The advantage of this approach is
the maintenance of the original activity of classical NSAIDs that do
not modify the dynamics of prostanoid synthesis combined with
reduced gastric side effects [5].
Sodium diclofenac (1) (Fig. 1), a NSAID widely used in chronic
treatments, has a very reactive carboxylate group that can be used
to incorporate new substituents to its structure. Taking into
consideration the beneficial effects of the nitric oxide described
above, it would be of interest to synthesize and investigate the
pharmacological properties of a diclofenac derivative containing
a heterocyclic ring system, such as benzofuroxan, that is able to
release NO (2).
In this study, we describe the synthesis and pharmacological
characterization of a novel modified diclofenac, which has a ben-
zofuroxan heterocyclic moiety on its structure joined to the parent
drug by an ester linkage.
b
g
2. Results and discussion
2.1. Chemistry
Because of their ability to release nitric oxide molecules under
physiological conditions, much attention has been recently paid to
furoxans and benzofuroxans [6e9]. These heterocyclics can be
* Corresponding author. Tel.: þ55 11 2454 8982; fax: þ55 11 2454 1825.
E-mail address: alessandragambero@saofrancisco.edu.br (A. Gambero).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.034

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P.S. de Carvalho, et al. / European Journal of Medicinal Chemistry 45 (2010) 2489e2493
multiplets were also observed in the spectra of benzofuroxan 4
7.00e7.60) and bromide 5 ( 7.10e7.70). A tautomeric equilib-
O Na
Cl
(d
d
rium between the two benzofuroxan isomeric structures
(Scheme 2), that is very fast at room temperature, is responsible for
the coalescence of the individual signals into a very broad
multiplet. The occurrence of this phenomenon has been previously
observed in many benzofuroxan derivatives [15]. In the 13C NMR
spectrum of 6, the signals related to the benzofuroxan aromatic
carbons were not observed, while in the spectra of 4 and 5, the
O
NH
O
N
Cl
O
N
1
2
aromatic carbons originated two broad multiplets (4:
d 114.0 and
Fig. 1. Chemical structures of sodium diclofenac (1) and benzofuroxan heterocyclic
system (2).
135.0; 5: 115.0 and 131.0) because of the very fast tautomeric
d
equilibrium. The mass spectrum of 6 showed a peak at m/z 277,
corresponding to the radical-ion formed by loss of a hydrox-
ymethyl-benzofuroxan molecule from the molecular-ion; the base
peak was viewed at m/z 214, presumably originated from subse-
quent losses of chlorine atom and carbon monoxide (Scheme 3).
prepared by various synthetic routes, including oxidation of
1,2-dioximes [10], thermal decomposition of 2-nitro-arylazides
[11,12] and oxidation of 2-nitroanilines with sodium hypochlorite
[12,13] or iodobenzene diacetate [14].
With the aim of preparing a new derivative of diclofenac that
contains a heterocyclic moiety with NO-releasing properties, we
hypothesized that a nucleophilic substitution reaction could be
used to link a benzofuroxan moiety to the reactive carboxylate
group of diclofenac. As shown in Scheme 1, we first prepared the
benzofuroxan 4 from 4-methyl-2-nitro-aniline (3) through an
oxidation promoted by sodium hypochlorite. This reaction pro-
ceeded smoothly and furnished the desired heterocycle with a 79%
yield after recrystallization. The conversion of 4 to the bromide 5
was carried out in refluxing carbon tetrachloride by the action of
N-bromosuccinimide in the presence of a catalytic amount of
benzoyl peroxide. After purification by flash chromatography, 5 was
obtained with an 85% yield. We were also able to isolate a small
amount (7% yield) of the corresponding dibromo derivative, as
indicated by GCMS analysis.
2.2. Pharmacology
The compound 6 was compared to the diclofenac in vitro for
COX-1 and COX-2 inhibitory effect using the human whole blood
assay. The anti-inflammatory activities of the two drugs were then
studied, in vivo, using the rat paw edema test, and gastric tolera-
bility to the drugs was accessed by measuring gastric prosta-
glandin, following oral administration to rats.
2.2.1. Anti-inflammatory activity
The novel modified diclofenac (compound 6) was prepared as
described above and tested in vitro and in vivo to evaluate its
pharmacological activity. As shown in Fig. 2, incubation of hepa-
rinized whole blood with 1 and 100
mM of diclofenac and
With the bromide 5 in hands, we had a suitable substrate to
prepare the desired diclofenac derivative. The nucleophilic substi-
tution was performed by mixing sodium diclofenac and 5 in stoi-
chiometric amounts using DMF as solvent. The reaction was
completed after 2 h at room temperature, and the modified diclo-
fenac 6 was isolated in 89% yield after purification by flash
chromatography.
The ester 6 was chemically characterized by its IR, NMR and
mass spectra. The IR spectrum showed bands at 3266 cmꢀ1 (NeH
stretch), 1716 cmꢀ1 (C]O stretch), 1256 and 1151 cmꢀ1 (CeO
stretches). An interesting feature observed in its 1H NMR spectrum
is the fact that all the three hydrogens attached to the benzofuroxan
compound 6, before LPS challenge, resulted in a significant reduc-
tion in PGE2 production. When the TXB2 production was assessed in
serum samples, clot formation was obtained from whole blood
previously incubated with the test drug. At a concentration of 1 and
100
mM, the original diclofenac promoted a significant inhibition,
while the compound 6 was only effective at 100
mM, suggesting that
compound 6 was more selective for COX-2, in vitro (Fig. 3).
The use of a classical experimental model of inflammation
demonstrated that the subplantar injection of carrageenan caused
a dose-dependent increase in rat hind paw oedema, compared to
that observed in the saline-injected contralateral paw (data not
shown). When rats received 100 mg kgꢀ1 of diclofenac or
system are viewed as a broad multiplet between
d
7.20e7.60. Broad
compound
6
intraperitoneally, both treatments evoked
H3C
N
N
O
N
H3C
NO2
NH2
Br
O
N
a
b
O
O
3
5
4
ONa
Cl
O
N
O
N
O
O
c
NH
NH
O
Cl
Cl
Cl
6
1
Scheme 1. Reagents and conditions: a. 12% NaClO, EtOH/H2O, 4 ꢁC, 40 min (79%); b. NBS (1.2 equivalent), (PhCO)2O2 (0.1 equivalent), CCl4, refl., 5 h (85%); c. Bromide 5
(1.0 equivalent), DMF, 25 ꢁC, 2 h (89%).

P.S. de Carvalho, et al. / European Journal of Medicinal Chemistry 45 (2010) 2489e2493
2491
R
O
N
N
R
N O
N O
R
O
N
O
O
N
Tautomer I
Tautomer II
Scheme 2. Rearrangement occurring in benzofuroxan systems.
a pronounced decreased in paw volume at 2, 3 and 4 h after the
carrageenan injection (Fig. 4). The AUC for diclofenac was
2.01 ꢂ 0.31, while for compound 6 the AUC was 2.00 ꢂ 0.45, indi-
cating that there were no differences between the two compounds.
The AUC obtained for the control was 4.01 ꢂ 0.42, demonstrating
that the structural modification maintained the original pharma-
cological activity in vivo.
4. Experimental protocols
4.1. Chemistry
All reagents were obtained from commercial sources and used
without further purification. Carbon tetrachloride and N,N-dime-
thylformamide were dried according to literature procedures [18].
Column chromatography (flash chromatography) [19] was per-
formed on silica gel 60 (Aldrich, 230e400 mesh). Thin layer chro-
matography was carried out on TLC aluminium sheets (silica gel 60
Merck). Melting points were determined on a FishereJohns appa-
ratus and are uncorrected. IR spectra were recorded on an ABB
FTLA2000 spectrophotometer. NMR spectra were obtained on
a GEMINI 300 BB instrument at 300 MHz for 1H and 75.5 MHz for
13C spectra. Low resolution mass spectra were determined on
a Shimadzu GCMS-QP5050A apparatus at 70 eV. High resolution
mass spectra were obtained on a VG-Autospec spectrometer at
70 eV.
2.2.2. Acute ulcerogenicity and PGE2 gastric content
The usual side effects of these compounds were evaluated by
their oral administration. These procedures, applied with native
diclofenac (30 mg Kgꢀ1) caused damage in the gastric mucosa that
consisted of hemorrhagic lesion that was significantly different
from that of the control animals that received only vehicle. In
contrast, the administration of an equimolar dose of compound 6
provoked lesions in the rat stomach that were much less extensive
than those induced by the original compound (Fig. 5), confirming
the previous literature reports regarding the ability of NO-releasing
NSAIDs to demonstrate anti-inflammatory properties, but not
ulcerogenic side effects [4,5,16]. The PGE2 content in the gastric
mucosa was reduced in both the original diclofenac and compound
6 groups, presenting different values from the control group,
showing that the gastric sparing is due to NO release rather than
the maintenance of the local production of PGE2 (Fig. 6). Finally, in
order to evaluate the involvement of nitric oxide in the reduction of
ulcerogenicity of compound 6, its ability to release NO was ensured
by measuring nitric oxide-releasing properties in phosphate buffer,
4.1.1. 5-Methyl-benzo[1,2,5]oxadiazole 1-oxide (4)
4-Methyl-2-nitro-aniline (1.52 g, 10.0 mmol) was added to
a stirred solution of sodium hydroxide (0.44 g, 11.0 mmol) in 96%
ethanol (15 mL). The resulting red solution was cooled to 4 ꢁC and
a sodium hypochlorite solution (12%, 20 mL) was added dropwise.
The reaction mixture was stirred at 4 ꢁC for 40 min and the
precipitate was filtered under reduced pressure and washed with
cold water (20 mL). The crude material was purified by recrystal-
lization from 70% ethanol to furnish 4 (1.18 g, 79%) as small yellow
needles: mp 94e95 ꢁC (lit. [20] mp 97 ꢁC); IR (KBr) 1619,1595,1528,
pH 7.4, in the presence of L-cysteine, relative to nitric oxide released
from standard sodium nitrite solution. Compound 6 (10ꢀ4 M)
produces in vitro 372 ꢂ 11 nM of nitrite. NOR-3, a potent NO-
donnor, at same experimental condition produces 2706 ꢂ 412 nM
[17]. Vehicle and native diclofenac were not able to release any
nitrite at same experimental condition.
1490, 793, 570 cmꢀ1; 1H NMR (CDCl3)
m, 3 H); 13C NMR (CDCl3)
22.1, 114.0 (br m), 135.0 (br m); MS m/z
d 2.41 (s, 3 H), 7.00e7.60 (br
d
(%) 150 (M, 87), 134 (38), 105 (27), 89 (69), 77 (59), 65 (100).
4.1.2. 5-Bromomethyl-benzo[1,2,5]oxadiazole 1-oxide (5)
N-Bromosuccinimide (1.34 g, 7.52 mmol) and benzoyl peroxide
(151 mg, 0.62 mmol) were added to a stirred solution of 4 (939 mg,
6.25 mmol) in dry carbon tetrachloride (60 mL). The reaction
mixture was heated under reflux (77 ꢁC) for 5 h and then cooled to
room temperature. The precipitate (succinimide) was filtered and
washed with carbon tetrachloride (10 mL). The solvent was
removed under reduced pressure to produce a brown oil, which
was submitted to flash chromatography (10% ethyl acetate:
hexanes) to yield 5 (1.22 g, 85%) as an amorphous yellow solid: TLC
3. Conclusions
The present data demonstrates that the new diclofenac,
obtained from the native compound by adding a benzofuroxan
moiety on its structure, possesses the expected anti-inflammatory
activity, without the deleterious gastrointestinal effects which is
normally present in typical NSAIDs. This approach could really be
a good alternative to gastro sparing anti-inflammatory non-
steroidal drugs.
loss of
HOCH2
N
O
N
O
N
O
Cl
- Cl , - CO
C21H15Cl2N3O4
m/z 443 (M, 1%)
N
H
Cl
Cl
m/z 214 (100%)
m/z 277 (11%)
Scheme 3. MS fragments of 6.

2492
P.S. de Carvalho, et al. / European Journal of Medicinal Chemistry 45 (2010) 2489e2493
2.0
50
40
30
20
10
0
1.5
1.0
*
*
*
*
***
1
***
100
***
1
***
100
0.5
0.0
Basal Vehicle
0.01
0.01
*
DC ( M)
compound 6 ( M)
*
0
1
2
3
4
5
LPS (10 g/mL)
Time (h)
Fig. 2. Prostaglandin E2 concentrations in plasma samples from blood aliquots stim-
ulated with LPS (10
diclofenac (0.01e100
m
g/mL) previously incubated with different concentrations of
Fig. 4. Effect of intraperitoneally administration of diclofenac (C; 100 mg kgꢀ1) and
mM) or compound 6 (0.01e100
mM). Data are mean ꢂ S.E.M of 3
compound 6 ( ; equivalent molar dose) in volume of rat paw oedema induced by
;
experiments. p < 0.01 when compared with blood aliquot previously incubated with
DMSO (0.1%).
intraplantar injection of carrageenan. Data are mean ꢂ S.E.M of 6 animals. *p < 0.05
when compared with group treated with DMSO (,; 0.1%).
Rf: 0.25 (10% ethyl acetate:hexanes); mp 73e74 ꢁC (lit. [21] mp
(benzofuroxan aromatic carbons were not observed due to very fast
tautomeric equilibrium); MS m/z (%) 443 (M, 1), 429 (13), 427 (18),
277 (11), 242 (26), 214 (100); HRMS m/z calc for C21H15Cl2N3O4
443.0440, found 442.9870, m/z calc for C13H9ClNþ 214.0423, found
213.9983.
75 ꢁC); IR (KBr) 1620, 1594, 1538, 1015, 655, 574 cmꢀ1
;
1H NMR
(CDCl3) d d 31.6,
4.44 (s, 2H), 7.10e7.70 (br m, 3H); 13C NMR (CDCl3)
115.0 (br m), 131.0 (br m); MS m/z (%) 230 (12), 228 (M, 12), 149
(100), 133 (60), 89 (76), 76 (30).
4.1.3. 1-Oxy-benzo[1,2,5]oxadiazol-5-ylmethyl [2-(2,6-dichloro-
phenylamino)-phenyl]-acetate (6)
4.2. Pharmacology
Bromide 5 (180 mg, 0.79 mmol) was added to a solution of
sodium diclofenac (250 mg, 0.79 mmol) in dry N,N-dime-
thylformamide (7 mL). The reaction mixture was stirred at room
temperature (25 ꢁC) for 2 h. After this time, water (20 mL) was
added and the mixture was extracted with dichloromethane (three
portions of 15 mL). After the combination of the organic layers, the
resulting solution was washed with saturated sodium chloride
(10 mL) and dried over anhydrous sodium sulfate. The solvent was
evaporated to give a brown oil which was submitted to flash
chromatography (20% ethyl acetate:hexanes) to yield 6 (312 mg,
89%) as a crystalline yellow solid: TLC Rf: 0.23 (20% ethyl acetate:
hexanes); mp 108e109 ꢁC; IR (KBr) 3266, 1716, 1627, 1601, 1256,
4.2.1. Human whole blood assay (WBA)
Whole blood was collected by venupuncture from healthy
subjects who had not taken any NSAIDs during the 2 weeks
preceding the study. For COX-1 assays, blood was immediately
aliquoted in 1 mL volumes into individual glass tubes containing
test agents or vehicle (0.1% vol/vol dimethyl sulfoxide; DMSO) and
allowed to clot at 37 ꢁC for 60 min. Serum was separated by
centrifugation (10 min at 2000 rpm) and kept at ꢀ20 ꢁC until assay
for TXB2.
For COX-2 assays, 1 mL whole blood samples containing heparin
(IU/mL) were incubated both in the absence and in the presence of
1151 cmꢀ1 1H NMR (CDCl3)
; d 3.91 (s, 2H), 5.17 (s, 2H), 6.55
LPS (10
mg/mL) plus test agents or vehicle. Incubation was then
(d, J ¼ 8.0 Hz, 1H), 6.65 (s, 1H), 6.94e7.01 (m, 2H), 7.15 (td,
continued for a further 18 h, after which the tubes were centrifuged
(10 min at 2000 rpm) and the plasma was removed and kept at
ꢀ20 ꢁC until assay for PGE2 [22].
J1 ¼8.0 Hz, J2 ¼ 1.5 Hz,1H), 7.24 (d, J ¼ 8.0 Hz,1H), 7.32 (d, J ¼ 8.0 Hz,
2H), 7.20e7.60 (br m, 3H); 13C NMR (CDCl3)
d 38.4, 65.2,118.5,122.3,
123.7, 124.2, 128.3, 128.9, 129.4, 130.9, 137.6, 142.5, 171.6
Concentrations of TXB2 and PGE2 in samples were determined
using ELISA kits (GE Healthcare, UK).
8
7
6
5
4
20
**
15
10
5
3
*
2
1
**
**
0
vehicle 0.01
1
100 0.01
1
100
0
DC ( M)
compound 6 ( M)
vehicle
DC
compound 6
Fig. 3. Thromboxane B2 concentrations in serum samples from blood clotted aliquots
previously incubated with different concentrations of diclofenac (0.01e100 M) or
compound 6 (0.01e100
M). Data are mean ꢂ S.E.M of 3 experiments. *p < 0.05 and
**p < 0.01 when compared with blood aliquot previously incubated with DMSO (0.1%).
Fig. 5. Mucosal ulcerogenic effects of orally administered diclofenac (50 mg kgꢀ1) and
compound 6 (equivalent molar dose). Data are mean ꢂ S.E.M of 5 animals. *p < 0.05
when compared with group treated with DMSO (0.1%). The lesion index was described
in Methods.
m
m

P.S. de Carvalho, et al. / European Journal of Medicinal Chemistry 45 (2010) 2489e2493
2493
15000 g at 4 ꢁC. The supernatant of each sample was used for
determination of PGE2 by EIA using a PGE2 kit.
17500
15000
12500
10000
7500
5000
2500
0
4.2.5. Assay of in vitro NO release
Solutions of test agents (compound 6, native diclofenac and
NOR-3) were prepared in DMSO and 10
mL was added to 1 mL of
50 mM phosphate buffer (pH 7.4) in presence of
L
-cysteiꢀn4e (5 mM),
for 1 h, at 37 ꢁC. The final drug concentration was 10 M. Each
sample was used for determination of nitrite content using
a Nitrate/Nitrite Colorimetric Assay kit.
**
**
DC
4.2.6. Statistical analysis
All data are expressed as the mean ꢂ S.E.M. Comparisons among
groups were performed using one-way analysis of variance fol-
lowed by the Student's t test or Dunnett multiple comparisons test,
when convenient. An associated probability (p value) of less than
5% was considered significant.
vehicle
compound 6
Fig. 6. Effects of orally administered diclofenac (50 mg kgꢀ1
) and Compound 6
(equivalent molar dose) on gastric mucosal prostaglandin E2 contents in rat stomachs.
Data are mean ꢂ S.E.M of 5 animals. **p < 0.01 when compared with group treated
with DMSO (0.1%).
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Male Wistar rats (180e200 g) free of specific pathogens were
obtained from CEMIB (State University of Campinas, Campinas, SP,
Brazil). The experiments were performed in accordance with the
principles outlined by the Brazilian College for Animal Experi-
mentation (COBEA).
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4.2.3. Carrageenan-induced paw edema model
Initially, the animals received the anti-inflammatory
compounds or vehicle intraperitoneally (i.p.). Thirty minutes later,
the animals were anaesthetized with halothane (inhaled). A sub-
plantar injection of carrageenan (1 mg/paw) in 0.1 mL of saline was
made into one paw of the rat. The contralateral paw received only
0.1 mL of saline. The paw volume was assessed immediately before
the carrageenan injection and was considered as the basal
measurement, and thereafter at 0.5, 1, 2, 3 and 4 h, using a hydro-
plethysmometer (model 7150, Ugo Basile). The results are
expressed as the increase in paw volume (mL) calculated by sub-
tracting the basal volume. The area under the time-course curve
(AUC0e2h) was also calculated using the trapezoidal rule and the
results expressed as total edema volume (mL h) by comparison
with the vehicle rats [23].
4.2.4. Evaluation of gastric ulcerogenic activity and mucosal
prostaglandin E2 content
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