Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen derivatives as anti-inflammatory agents

Article abstract of DOI:10.1007/s00706-015-1557-x

A series of furoxan-based nitric oxide releasing ibuprofen derivatives were synthesized and tested for their anti-inflammatory, analgesic, ulcerogenic, lipid peroxidation, and hepatotoxic properties. The compounds exhibited more protection than ibuprofen with regard to gastric toxicity. Among the tested compounds 4-[2-[2-(4-isobutylphenyl)propanamido]ethoxycarbonyl]-3-methylfuroxan and 4-[2-[2-(4-isobutylphenyl)propanoyl]hydrazinecarbonyl]-3-phenylfuroxan emerged as most active anti-inflammatory agents with reduced gastrotoxicity. The results showed that incorporation of NO donating group caused a moderate increase in anti-inflammatory activity with a marked decrease in gastric ulcerations compared to their parent drug ibuprofen. A molecular docking study of all the compounds was also performed to provide the binding modes of COX-1 enzyme. Among all the titled compounds, 4-[2-[2-(4-isobutylphenyl)propanamido]ethoxycarbonyl]-3-methylfuroxan was found to be most potent and have high docking score showing favorable orientation within the COX-1 binding site.

Full text of DOI:10.1007/s00706-015-1557-x

Monatsh Chem
DOI 10.1007/s00706-015-1557-x
ORIGINAL PAPER
Synthesis, characterization, and biological evaluation of furoxan
coupled ibuprofen derivatives as anti-inflammatory agents
Mohd Amir1 Mohd Wasim Akhter1 Ozair Alam1
•
•
Received: 4 May 2015 / Accepted: 9 August 2015
Ó Springer-Verlag Wien 2015
Abstract A series of furoxan-based nitric oxide releasing
ibuprofen derivatives were synthesized and tested for their
anti-inflammatory, analgesic, ulcerogenic, lipid peroxidation,
and hepatotoxic properties. The compounds exhibited more
protection than ibuprofen with regard to gastric toxicity.
Among the tested compounds 4-[2-[2-(4-isobutylphenyl)
propanamido]ethoxycarbonyl]-3-methylfuroxan and 4-[2-
[2-(4-isobutylphenyl)propanoyl]hydrazinecarbonyl]-3-phenyl-
furoxan emerged as most active anti-inflammatory agents
with reduced gastrotoxicity. The results showed that
incorporation of NO donating group caused a moderate
increase in anti-inflammatory activity with a marked
decrease in gastric ulcerations compared to their parent drug
ibuprofen. A molecular docking study of all the compounds
was also performed to provide the binding modes of COX-1
enzyme. Among all the titledcompounds, 4-[2-[2-(4-
isobutylphenyl)propanamido]ethoxycarbonyl]-3-methyl-
furoxan was found to be most potent and have high docking
score showing favorable orientation within the COX-1
binding site.
Graphical abstract
Keywords Furoxan Á Ibuprofen Á Nitric oxide donors Á
Anti-inflammatory Á Gastric toxicity Á Hepatotoxic
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of pain, fever, and inflam-
mation [1]. The worldwide NSAIDs market for both
occasional and chronic users has been conservatively
estimated at over 60 million people, and certain NSAIDs
(aspirin, naproxen, ibuprofen) are among the most popular
over-the-counter medications [2, 3]. Chronic NSAIDs
therapy effectively reduces the symptoms of many painful
arthritic syndromes, but invites adverse gastrointestinal
(GI) complications ranging from stomach irritation to life
threatening GI ulceration and bleeding [4, 5]. At the tissue
level, the most common clinical manifestation of NSAIDs-
related GI damage is a combination of gastroduodenal
erosions and ulcerations often called NSAIDs-induced
gastropathy [6], affecting at least 25 % of chronic NSAIDs
patients. NSAIDs exert their anti-inflammatory effect
mainly through inhibition of cyclooxygenase (COXs), key
enzymes in prostaglandin (PG) biosynthesis from arachi-
donic acid [7, 8]. There are at least two mammalian COX
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-015-1557-x) contains supplementary
material, which is available to authorized users.
& Mohd Amir
mamir_s2003@yahoo.co.in
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Hamdard University, New Delhi 110 062, India
123

M. Amir et al.
isoforms, COX-1 and COX-2 [9, 10]. COX-1 is the con-
stitutive isoform of COX, which performs a housekeeping
function to synthesize prostaglandins, which regulate nor-
mal cell activity. Thus, the nonselective inhibition of both
cyclooxygenase isoenzymes (COX-1 and COX-2) by tra-
ditional ulcerogenic NSAIDs clearly illustrated the clinical
need for a new generation of non ulcerogenic selective
COX-2 inhibitors [11, 12]. This was a turning point which
stimulated pharmaceutical companies to seek selective
COX-2 inhibitors that would be free from unwanted gas-
trointestinal toxicity. This effort led to the discovery and
development of a number of selective COX-2 inhibitors,
viz., celecoxib, rofecoxib, valdecoxib, parecoxib, and
etoricoxib [13]. But careful prospective examination of
coxibs has revealed unexpected cardiovascular adverse
effects [14]. Therefore, selective COX-2 inhibitors may not
be the proper strategy to overcome the gastric toxicity of
conventional NSAIDs due to its adverse cardiovascular
effects. One of the most important strategies used to
overcome NSAIDs side effects is to design nitric oxide
(NO) donating NSAIDs (NO-NSAIDs) which are capable
of generating gastroprotective agent NO [15, 16]. Hybrid
molecules comprised of NSAID and nitric oxide (NO)
donor moieties are devoid of adverse cardiovascular effects
of selective COX-2 inhibitors, and also decreases GI tox-
icity observed on long-term use of traditional NSAIDs
[17]. It has been reported that an increased generation of
endothelial NO or release of NO from a nitric oxide donor
drugs produces beneficial effects such as reduction of blood
pressure and prevention of atherosclerosis [18]. At
nanomolar concentrations, NO reversibly activates soluble
guanylate cyclase (sGC) by 400 folds, catalyzing the
conversion of guanosine triphosphate to cyclic guanosine
monophosphate (cGMP) [19]. Elevation of cGMP relaxes
smooth muscles in blood vessels, inhibits platelet aggre-
gation and blocks the adhesion of white cells to blood
vessels walls [20]. Besides these cardiovascular effects,
NO is also recognized as a critical mediator of gastroin-
testinal mucosal defense. NO is able to protect gastric
mucosa by a number of mechanisms including promotion
of mucus secretion, increased mucosal blood flow resulting
in enhanced mucosal resistance to ulceration. NO also
prevents adherence of neutrophils to the gastric vascular
endothelium [21]. NO also increases the ability of mucous
cells to undergo healing and repair of the existing ulcers
[22].
property of furoxan moiety [24]. Thiol induced release of
NO from furoxan moiety involves attack by a thiolate
anion at C-3 and/or C-4 of the furoxan ring followed by
ring opening and subsequent release of NO [25]. Encour-
aged by these observations and in continuation of our
ongoing research program [26–29] to discover new and
useful agents for treatment of anti-inflammatory disease,
we report herein the synthesis and pharmacological pro-
file of furoxan derivatives of ibuprofen as potential NO
donor drugs (Fig. 1).
Results and discussion
Chemistry
Substituted furoxans were synthesized from crotonalde-
hyde and cinnamaldehyde [30–34] (Fig. 2). Compound 8
was synthesized by treating 2-(4-isobutylphenyl)propanoyl
chloride (7) with ethylenediamine to form an intermediate
N-(2-aminoethyl)-2-(4-isobutylphenyl)propanamide which
on further treatment with 4-(chlorocarbonyl)-3-methyl-
furoxan (6a) resulted in the formation of 4-[2-[2-(4-
isobutylphenyl)propanamido]ethylcarbamoyl]-3-methylfur-
oxan (9). The compound 7 was treated with ethylene glycol
to form an intermediate 2-hydroxyethyl 2-(4-isobutylphenyl)-
propionate (10) which on treatment with 6a resulted in the
formation of 4-[2-[2-(4-isobutylphenyl)propanoyloxy]etho-
xycarbonyl]-3-methylfuroxan (11). The ethyl ester of
ibuprofen (12) on treatment with 2-aminoethanol afforded
Fig. 1 General structure of hybrid models
It has been reported that furoxans (1,2,5-oxadiazole-2-
oxides) are biologically active compounds that are capable
of releasing nitric oxide in the presence of thiol cofactors
[23]. Compared to other NO donating agents, furoxans
possess favorable pharmacological properties, since they
frequently release NO slowly resulting in a longer duration
of action. The absence of tolerance is also an important
Fig. 2 Different substituted furoxans
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
the intermediate N-(2-hydroxyethyl)-2-(4-isobutylphenyl)-
propanamide (13). The final compounds 4-[2-[2-(4-
isobutylphenyl)propanamido]ethoxycarbonyl]-3-substituted
furoxan (14a, 14b) were prepared by treating intermediate
13 with 6a and 6b in the presence of anhydrous dichlor-
omethane and triethyl amine. The compounds 4-[[2-(4-
isobutylphenyl)propanoyloxy]methyl]-3-substituted-furoxan
(15a, 15b) were prepared by reacting intermediate 7 with
4-(hydroxymethyl)-3-substituted furoxan. Synthesis of these
compounds is outlined in Scheme 1.
anhydrous dichloromethane. The compound 4-[5-[1-(4-
isobutylphenyl)ethyl]-1,3,4-oxadiazol-2-yl]-3-methyl-
furoxan (18) was synthesized by refluxing hydrazide with
4-carboxy-3-methylfuroxan in phosphorus oxychloride.
The intermediate compound 5-[1-(4-isobutylphenyl)ethyl]-
1,3,4-thiadiazol-2-amine (19) was synthesized by refluxing
the thiosemicarbazide and 2-(4-isobutylphenyl)propanoic
acid with an excess of phosphorus oxychloride [31]. The
final compound 4-[[5-[1-(4-isobutylphenyl)ethyl]-1,3,4-
thiadiazol-2-ylimino]methyl]-3-methylfuroxan (20) was
synthesized by refluxing intermediate 19 and 4-formyl-
3-methylfuroxan for 8 h. The compound 4-[5-[1-(4-iso-
butylphenyl)ethyl]-1,3,4-thiadiazol-2-ylcarbamoyl]-3-
methylfuroxan (21) was prepared by treating 19 with
4-(chlorocarbonyl)-3-methylfuroxan. Synthesis of these
compounds is outlined in Scheme 2. All the compounds
The compounds 4-[[2-[2-(4-isobutylphenyl)propanoyl]-
hydrazono]methyl]-3-substituted-furoxan (16a, 16b) were
prepared by reacting 2-(4-isobutylphenyl)propionic acid
hydrazide [30] with intermediate 6a and 6b in the presence
of concentrated sulfuric acid. The compounds 4-[2-[2-(4-
isobutylphenyl)propanoyl]hydrazinecarbonyl]-3-substi-
tuted-furoxan (17a, 17b) were synthesized by treating the
hydrazide with intermediate 6a, 6b in presence of
1
showed characteristic peaks at appropriate d values in H
NMR and 13C NMR spectral data.
Scheme 1
123

M. Amir et al.
Scheme 2
Nitric oxide release
nitrite solution and calculated as amount of NO released
(mol/mol) [32] and listed in Table 1.
The NO releasing properties of the synthesized furoxan
derivatives of ibuprofen 9, 11, 14a, 14b, 15a, 15b, 16a,
16b, 17a, 17b, 18, 20, and 21 were assessed in vitro in both
phosphate buffer of pH 7.4 and 0.1 N HCl of pH 1 by using
Griess reagent. The reaction was carried out in the presence
of L-cysteine as a source of the SH group. It was found that
a reduced thiol group from endogenous L-cysteine could
mediate the release of NO from furoxan derivatives. The
amount of NO released from the tested compounds was
measured relative to NO released from standard sodium
The results of measurement of NO release revealed that
the tested compounds could only release NO at pH 7.4.
Linker groups which attach furoxan with ibuprofen had
significant effect on NO releasing capacity of compounds.
The compound 14a having an amide–ester linkage and a
methyl group in the furoxan ring and compound 17b
having an amide–amide linkage and a phenyl group in the
furoxan ring showed maximum nitric oxide releasing
capacity. The compound 21 having amide linkage and a
thiadiazole ring showed minimum nitric oxide releasing
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
Table 1 The amount of NO released from tested compounds in phosphate buffer (pH = 7.4)
Compound
Amount of NO released/mol mol-1 SEM
Compound
Amount of NO released/mol mol-1 SEM
9
0.48 0.036
0.48 0.108
0.64 0.042
0.59 0.030
0.58 0.083
0.50 0.177
0.41 0.061
16b
17a
17b
18
0.39 0.034
0.45 0.060
0.63 0.050
0.39 0.041
0.42 0.033
0.27 0.047
11
14a
14b
15a
15b
16a
20
21
capacity. Other compound of the series having an amide-
Schiff base, 1,3,4-oxadiazole, 1,3,4-thiadiazole/Schiff base
spacers showed moderate nitric oxide releasing capacity.
On the other hand the tested compounds, were not able to
release NO at pH 1, which suggest that NO donating fur-
oxan derivatives of ibuprofen are weakly hydrolysed in the
gastric lumen and this confirmed that the suggested gastro
protective action of NO is mediated systemically [32].
activity at an equimolar oral dose related to 70 mg/kg
ibuprofen. Compounds 11, 14a, 14b, 15a, 15b, 16a, 16b,
17a, and 17b showed analgesic activity ranging from 48.86
to 75.87 % whereas standard drug ibuprofen showed
73.45 % inhibition. It was noted that compound 14a having
amide–ester linkage with a methyl group at furoxan ring
showing highest anti-inflammatory activity also exhibited
highest analgesic activity (75.87 %), whereas in compound
14b when methyl group was replaced by a phenyl group
there was slight decrease in analgesic activity (70.95 %).
The compound 17b having an amide–amide linkage and a
phenyl group at furoxan ring also showed good analgesic
activity (72.12 %). Compound 16b having an amide–schiff
base linkage showed poor analgesic activity (48.86 %).
The remaining compound showed moderate to good anal-
gesic activity (Table 2).
Anti-inflammatory activity
All the synthesized compounds were screened for their anti-
inflammatory activity by carrageenan induced rat paw edema
method of Winter et al. The results revealed that compounds
14a, 14b, and 17b exhibited excellent anti-inflammatory
activity (86.10, 83.39, and 82.03 %, respectively), at an
equimolar oral dose relative to 70 mg/kg ibuprofen which
showed 74.40 % inhibition after 4 h. Compounds 11, 15a,
and 15b showed anti-inflammatory activity comparable to
standard drug ibuprofen i.e. 74.07, 73.82, and 71.35 %,
respectively. Compounds 16a, 16b, and 17a also showed
good anti-inflammatory activity, whereas compounds 9, 18,
20, and 21 showed lesser degree of activity. It was interesting
to note that although compound 17a and 17b have similar
amide–amide linkage but there is difference in their anti-
inflammatory activity. The high activity of compound 17b
having a phenyl group could be a consequence of their
inhibitory effects on COX-2 along with the COX-1 enzyme.
The poor activity of compound 18 (58.81 %) and 20
(58.13 %) may be due to the absence of carbonyl group
which is required for binding with COX-1 enzyme. Thus it
was concluded that furoxan derivatives of ibuprofen having
amide-ester linkage (14a, 14b) or ester linkage (15a, 15b) or
amide–amide linkage (17b) showed very good activity.
These results indicated that the incorporation of NO donating
furoxan moieties to ibuprofen has not only retained but
showed enhanced anti-inflammatory activity (Table 1).
Ulcerogenic activity
The compounds 11, 14a, 14b, 15a, 15b, 17a, and 17b
which showed significant anti-inflammatory and good
analgesic activity were further tested for their acute
ulcerogenicity. The compounds were tested at an equimolar
oral dose relative to 210 mg/kg ibuprofen. The maximum
reduction in ulcerogenic activity (0.166 0.10) was found
in compound 14a having an ester amide linkage and a
methyl group at the furoxan ring. Compound 17b having an
amide–amide linkage and a phenyl group at furoxan ring
also showed reduced ulcerogenic potential (0.250 0.11).
Compounds 15a and 11 having an ester and ester–ester
linkage, respectively, showed ulcerogenicity equal to
standard drug ibuprofen (0.666 0.10) which may be due
to in vivo hydrolysis of ester into carboxylic group
resulting in topical irritating action. Rest of the tested
compounds also showed better GI safety profile as com-
pared to ibuprofen (Table 3). Thus, the results indicated
that nitric oxide released from the ibuprofen–furoxan
derivatives have a significant effect on decreased gastric
toxicity (Fig. 4). The decreased severity index may be
explained by the release of NO that increased mucosal
blood flow resulting in enhanced mucosal resistance to
ulceration and/or an enhanced ability of the NO donating
Analgesic activity
The compounds that showed anti-inflammatory activity
higher than 65 % were further tested for their analgesic
123

M. Amir et al.
Table 2 Anti-inflammatory activity of furoxan derivatives using carrageenan-induced paw edema in rats
Compound
Anti-inflammatory activity % inhibition SEM#
Compound
Anti-inflammatory activity % inhibition SEM#
After 3 h
After 4 h
After 3 h
After 4 h
9
61.48 0.86
68.59 0.98
81.48 0.79
79.83 0.61
70.91 0.82
70.66 0.66
71.90 0.66
64.74 0.93a
71.35 1.35c
86.10 0.67a
83.39 0.86a
74.07 0.97
73.82 0.70
74.40 0.61
16a
16b
17a
17b
18
65.12 1.10
63.30 0.88
64.96 0.84
77.52 0.91
55.21 0.83
55.04 0.87
58.84 0.98
67.80 1.13b
66.27 0.88a
67.63 0.61a
82.03 0.90a
58.81 0.82a
58.13 1.16a
61.52 0.89a
11
14a
14b
15a
15b
Ibuprofen
#
20
21
Relative to standard and data were analyzed by Student’s t test for n = 6
a
b
d
p \ 0.0001
p \ 0.001
p \ 0.5
Table 3 Analgesic, ulcerogenic and lipid peroxidation activities of selected compounds
Compound
Analgesic activity
Ulcerogenic activity
(severity index SEM)
nmol MDA content SEM/
100 mg tissue
Pre-treatment/normal
0 h (s)
Post-treatment/after
4 h (s)
% Inhibition
11
1.72 0.01
1.69 0.00
1.51 0.01
1.77 0.01
1.64 0.00
1.60 0.01
1.58 0.01
1.52 0.01
1.47 0.00
–
2.86 0.02
2.98 0.01
2.59 0.02
2.91 0.02
2.76 0.03
2.46 0.01
2.34 0.01
2.56 0.01
2.54 0.01
–
67.62 1.52c
75.87 0.99d
70.95 0.75c
63.80 0.96a
68.71 0.86c
53.24 1.29a
48.86 1.17a
68.27 1.57c
72.12 0.61d
–
0.666 0.10
0.166 0.10c
0.333 0.10c
0.666 0.10
0.583 0.08
–
6.40 0.09b
3.67 0.03a
4.87 0.05a
5.07 0.05a
5.71 0.11a
–
14a
14b
15a
15b
16a
16b
–
–
17a
0.500 0.00d
0.250 0.11c
0.000 0.00
0.666 0.10
5.29 0.09a
3.70 0.07a
3.41 0.08
6.95 0.06
17b
Control
Ibuprofen
#
1.60 0.01
2.78 0.01
73.45 0.76
Relative to standard and data were analyzed by Student’s t test for n = 6
a
b
c
d
p \ 0.0001
p \ 0.001
p \ 0.05
p \ 0.5
furoxan derivatives to cross the gastric mucosal layer prior
to the subsequent release of NO.
also showed reduction in lipid peroxidation. The compound
14a having amide–ester linkage and compound 17b having
amide–amide linkage showed maximum reduction
3.67 0.03 and 3.70 0.07, respectively, whereas com-
pound 11 having ester–ester linkage showed minimum
reduction 6.40 0.09 nmol MDA/100 mg tissue. Thus,
these studies showed that hybrid molecules have inhibited
the induction of gastric mucosal lesion, and the results
further suggested that their protective effect might be
related to the inhibition of lipid peroxidation in the gastric
mucosa and due to the release of nitric oxide from furoxan
derivatives of ibuprofen (Table 3).
Lipid peroxidation activity
All the compounds screened for ulcerogenic activity were
also analyzed for their lipid peroxidation. The lipid per-
oxidation was measured as nanomoles of malondialdehyde
(MDA/100 mg) of gastric mucosa tissue. Ibuprofen
exhibited high lipid peroxidation 6.95 0.06 whereas
control group showed 3.41 0.081. It was found that all
the furoxan derivatives showing less ulcerogenic activity
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
Hepatotoxic and histopathological studies
orientation within the COX-1 binding site. The binding
mode of compound 14a is exactly same as the co-crystal
ligand (IBPA: 701) which was represented in 2D ligand
interaction diagram (Supplementary Material). The docked
pose of the compound 14a showed the hydrogen bond
The compounds 14a and 17b, furoxan derivatives of
ibuprofen showing potent anti-inflammatory and analgesic
activities with reduced ulcerogenicity and lipid peroxida-
tion, were further studied for their hepatotoxic effect. Both
compounds were studied for their effect on biochemical
parameters (serum enzyme, total protein, and total albu-
min). Liver histopathological testing of these compounds
was also carried out. As shown in Table 4, activities of
liver enzyme SGOT, SGPT, alkaline phosphatase, total
protein and total albumin where less than the standard drug
ibuprofen. The histopathological studies of the liver sam-
ples do not show any significant pathological changes in
comparison to standard drug ibuprofen (Fig. 3). No hepa-
tocyte necrosis or degeneration was seen in any of the
samples.
˚
interaction with amino acid ARG 120 (O….HN, 2.04 A)
˚
and TYR 355 (O….HO, 2.13 A). The ARG 83 (O….HN,
˚
1.57 A) makes an additional hydrogen bond with the
oxygen atom of side chain of oxadiazole ring (Fig. 4).
Interestingly 14a also forms a p–p interaction between
oxadiazole ring and same residue of ARG 120 (oxadia-
˚
zole…C = 0. 3.97 A) whereas co-crystal ligand IBPA:
701 forms three hydrogen bonds with ARG 120 (O…HN,
-
˚
˚
˚
1.72 A; O …HN, 1.70 A) and TYR 355 (O…HO, 1.63 A)
(Fig. 4). The isobutyl group also occupied hydrophobic
side-pocket of COX-1 surrounded by the residues LEU
352, MET 522, LEU 384, and PRO 86 diagram (Supple-
mentary Material).
In silico ADME/molecular docking
Further docked pose of compounds 17b and 18 against
COX-1 were also represented in Fig. 4. The oxygen atom
of carbonyl group of compound 17b formed strong
Oral bioavailability is considered to play an important role
for the development of bioactive molecules as therapeutic
agents. Most of the compounds have more than 80 %
human oral absorption. From all these pharmacokinetic
parameters (Table 5), it is concluded that none of the
compounds violated Lipinski’s and Jorgensen’s parame-
ters, making them potentially promising drug candidates
for the treatment of inflammation.
hydrogen bonds with ARG 120 and ARG 83 approximately
-
˚
˚
at distances of 1.86 A (O…HN) and 1.90 A (O …HO),
respectively, as shown in Fig. 4. Furthermore, two p–p
stacking and one p-cation with ARG 120 (phenyl…=C;
oxadiazole…=C and N?….=C) were also observed at
˚
distances of 4.49, 3.85, and 4.21 A, respectively (Supple-
mentary Material). It was observed that the carbonyl group
in the molecule is important for hydrogen bond interaction
with amino acids of COX-1, but there are compounds
without having carbonyl group also formed hydrogen bond
such as compound 18 and its docked pose was shown in
Fig. 4. The compound 18 showed the hydrogen bond
All the compounds are well fitted in the active sites of
COX-I. The dG binding energy of compounds were cal-
culated by Prime MM-GBSA, Maestro 10.1 and found in
the range of 170.0 to -383.15 kJ/mol for COX-I. The
docking scores and binding energy related with inter-
molecular interactions of the titled compounds and co-
crystals ligand with the active site of COX-1 is summarized
in Table 6.
˚
interaction with amino acid ARG 120 (N….HN, 1.72 A)
˚
and TYR 355 (O….HO, 1.82 A). Interestingly compound
also formed a p-cation interaction between oxadiazole ring
and same residue of ARG 120 (oxadiazole
Among all the titled compounds studied for COX-1,
compound 14a was found to be most potent and have high
docking score. The compound 14a also assumes favourable
?
˚
N …C = 4.40 A) and p–p stacking with TYR 355 (phe-
˚
nyl…C = 5.31 A). The superimposed structure of
Table 4 Effect of compounds 14a and 17b on serum enzymes, total proteins, and total albumin
Compound
SGOT/units cm-3#
SGPT/units cm-3#
Alkaline phosphatase#
Total protein g/100 cm3#
Total albumin g/100 cm3#
Control
Ibuprofen
14a
149.76 0.81
155.91 1.49
143.99 1.36a
146.03 1.43a
31.54 1.46
44.84 1.17
33.53 1.20a
25.99 0.64a
32.50 0.45
35.06 0.47
27.17 0.51a
23.14 0.32a
1.69 0.012
1.88 0.011
1.82 0.021c
1.69 0.031b
1.59 0.017
1.70 0.014
1.68 0.018d
1.57 0.020b
17b
#
Relative to standard and data were analyzed by Student’s t test for n = 6
a
b
c
d
p \ 0.0001
p \ 0.001
p \ 0.05
p \ 0.5
123

M. Amir et al.
Fig. 3 a Control: section of
liver showing normal
arrangement of hepatocytes in
the centrizonal area. CV
Centrizonal area (9400).
b Ibuprofen: section of liver
showing evident centrizonal
sinusoidal dilatation (9400).
c Compound 14a: section of
liver showing mild centrizonal
sinusoidal dilatation (9400).
d Compound 17b: section of
liver showing mild centrizonal
sinusoidal dilatation (9400)
compounds 14a and 17b with co-crystal ligand IBPA: 107
were also shown in Fig. 5. The binding orientations of title
superimposed structures are same as co-crystal IBPA: 107.
promoted mucus secretion and increased mucosal blood
flow resulting in enhance mucosal resistance to ulceration.
Thus, the use of hybrid molecules containing NO releasing
furoxan moieties looks as promising approach to improve
the safety profile of NSAIDs. Furthermore, molecular
docking study was performed to provide the binding pat-
terns of the compound 14a, 17b, and 18 into the binding
sites of COX-1 (PDB code: 1EQG) enzymes. The study
showed that 14a has favorable orientation within the COX-
1 enzyme binding site and have a high docking score. In
view of these studies, the compound 14a could be a subject
of further investigations for searching potential new anti-
inflammatory molecules.
Conclusions
Thirteen furoxan derivatives were synthesized by attaching
furoxan moieties into the molecule of ibuprofen by
replacing its carboxylic acid with various linker groups i.e.,
amide–amide, amide-ester, ester–ester, ester, amide–Schiff
base, 1,3,4-oxadiazole/1,3,4-thiadiazole, and amide/Schiff
base linkages. All the synthesized compounds were eval-
uated for their anti-inflammatory and nitric oxide releasing
properties. Compounds 11, 14a, 14b, 15a, 15b, 17a, and
17b showing high anti-inflammatory and analgesic activity
were also tested for their ulcerogenic potential and lipid
peroxidation. It was noted that compounds 14a and 17b
showed maximum anti-inflammatory and analgesic activ-
ity. These compounds exhibited reduced gastric
ulcerogenicity when compared with ibuprofen. It is
assumable that masking the free carboxylic group of
ibuprofen may have reduced its topical irritant action.
Furthermore, pronounced gastroprotective activity of these
compounds might attribute to the release of NO that
Experimental
All chemicals for synthesis were supplied by Merck
(Darmstadt, Germany) and S.D. Fine Chemicals (Delhi,
India). The melting points of newly synthesized com-
pounds were determined in one-end open capillary tubes on
a Hicon melting-point apparatus. IR spectra (KBr) were
1
recorded on a Jasco FT-IR spectrometer. H NMR spectra
were measured on Bruker 300 MHz and Bruker Avance
400 MHz instruments. 13C NMR spectra were measured on
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
Table 5 ADME parameters of all the synthesized compounds
Comp. MWa
Dipole SASAb
PSAc
Donord Accpte LogPo/wf logSg
PCacoh
Human
oral
%
oral
Human Rule
of
Rule
of
threej
HB
HB
absorption absorption fivei
Range \500
1–12.5 300–1000 7–200 B5
B10 \5
-6.5 to
0.5
[500
great
1 Low
2 Med
3 High
[80 %
high
B1
B1
\25 poor
\25 %
poor
9
374.439 9.608 741.805 128.259 2
376.408 6.624 624.96 114.897 0
8
2.073
-4.831
-3.093
-5.018
-6.718
-4.034
-5.731
-5.375
-5.53
116.284
509.364
123.165
115.286
674.298
794.885
214.052
243.494
103.781
135.748
310.573
199.751
94.749
3
3
3
1
3
3
3
3
3
1
3
3
3
76.053
91.328
78.319
85.842
96.057
100
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
1
0
0
0
11
7
2.721
2.384
3.756
3.156
4.561
3.15
14a
14b
15a
15b
16a
16b
17a
17b
18
375.424 8.264 741.407 125.559 1
437.494 8.141 842.857 125.188 1
7.5
7.5
5
318.372 6.332 602.183 88.594
380.443 6.076 691.731 87.201
0
0
5
330.386 11.429 673.476 105.965 1
392.457 11.647 682.197 104.373 1
346.385 7.045 681.058 131.725 0.5
408.456 6.828 770.283 129.863 0.5
5.5
5.5
6.5
6.5
5.5
6
87.1
4.114
2.612
4.023
2.89
93.748
78.327
88.67
-5.217
-6.83
328.37 6.178 641.547 92.835
371.456 6.555 708.566 96.908
0
0
-4.771
-5.495
-5.513
88.473
88.121
77.68
20
3.416
2.623
21
387.456 5.325 711.183 122.081 1
7.5
a
Molecular weight of the molecule
b
c
d
e
f
˚
Total solvent accessible surface area in square angstroms using a probe with a 1.4 A radius
Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms
Estimated number of hydrogen bonds that would be donated by the compound to water molecules in an aqueous solution
Estimated number of hydrogen bonds that would be accepted by the compound from water molecules in an aqueous solution
Predicted octanol/water partition coefficient
Predicted aqueous solubility, log S. S in mol dm-3 is the concentration of the solute in a saturated solution that is in equilibrium with the
crystalline solid
g
h
Predicted apparent Caco-2 cell permeability in nm/s. Caco-2 cells are a model for the gut-blood barrier
i
Lipinski’s violations
j
Jorgensen’s violations
4-[2-[2-(4-Isobutylphenyl)propanamido]ethylcarbamoyl]-
Bruker Avance-400 instrument (100 MHz) with complete
proton decoupling. Chemical shifts are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS).
Elemental analyses (C, H, and N) were undertaken with a
CHNS Vario EL III (Elementar Analysen Systeme GmbH,
Germany) and the results are within 0.4 % of theoretical
values. The reactions were monitored by silica gel-GF
coated aluminum plates and visualized by iodine vapors
and ultraviolet light as visualizing agents. Mass (MS)
spectral data were recorded on a Jeol SR-102 (FAB)
spectrometer. Removal of solvents was carried out at
reduced pressure using a rotary evaporator. Compounds 1
[33], 2 [34], 3 [35], 4 [36], 5a, 5b [37] and 6a, 6b [37] were
synthesized according to methods reported in literature.
Intermediate compounds 8, 10, and 13 were synthesized
according to literature method by slightly modifying
reaction conditions [38].
3-methylfuroxan (9, C19H26N4O4)
Ethylene diamine (0.017 mol) and 50 cm3 glacial acetic
acid were slowly mixed together resulting in the formation
of a white solid which dissolved gradually at room
temperature to form
a
colorless solution. 2-(4-
Isobutylphenyl)propionyl chloride (7, 0.01 mol) was then
added drop wise to this solution with continuous stirring at
room temperature. The mixture was stirred for 2 h, then
diluted with water and the pH was adjusted to 12 with
NaOH solution. The solution was filtered to remove some
white precipitate and the filtrate was extracted with
CH2Cl2. The organic layer was separated and washed with
saturated NaCl solution and water. It was dried and
distilled under reduced pressure to afford the intermediate
N-(2-aminoethyl)-2-(4-isobutylphenyl)propanamide (8)
which was used immediately for the next step of the
123

M. Amir et al.
reaction. A solution of 4-(chlorocarbonyl)-3-methylfuroxan
(6a, 0.01 mol) in 5 cm3 anhydrous THF in the presence of
0.5 cm3 triethylamine was added drop wise at room
temperature to a stirred solution of intermediate 8 in
20 cm3 CH2Cl2. The stirring was continued for 6 h and
reaction mixture was then poured into ice cold water. The
organic layer was separated, washed with water, dried and
distilled under reduced pressure. The product thus obtained
was purified by silica gel column chromatography using
EtOAc/hexane as eluent to afford pure compound 9. Yield
25 %; m.p.: 98–100 °C; IR (KBr): vꢀ = 3299, 3272 (NH),
1
1676, 1656 (C=O), 1593 (C=N) cm-1; H NMR (CDCl3):
d = 0.86 (d, 6H, J = 6.3 Hz, (CH3)2CH), 1.45 (d, 3H,
J = 7.2 Hz, CH3-CH), 1.86 (m, 1H, (CH3)2CH-CH2), 2.35
(s, 3H, CH3), 2.41 (d, 2H, J = 7.2 Hz, (CH3)2CH-CH2),
3.26 (t, 2H, J = 5.1 Hz, CH2), 3.47 (t, 2H, J = 5.1 Hz,
CH2), 3.56 (q, 1H, J = 7.2 Hz, CH3CH), 5.83 (bs, 1H,
CH3-CH-CONH), 7.05–7.22 (m, 4H, ArH), 7.64 (bs, 1H,
CONHfuroxan) ppm; 13C NMR (CDCl3): d = 176.12,
157.82, 150.63, 141.01, 138.11, 129.71, 127.27, 112.36,
46.68 (CH), 44.96 (CH2), 40.34 (CH2), 39.17 (CH2), 30.18
(CH), 22.41 ((CH3)2), 18.41 (CH3), 8.48 (CH3) ppm; MS:
m/z = 375 (M??1).
Table 6 Docking scores and binding energy of compounds at the
active sites of COX-1
Compound
COX-I (PDB ID: 1EQG)
XP Docking scores
dG Binding energy
(kJ/mol)
9
-9.864
-10.683
-11.634
-11.055
-8.700
-10.336
-7.012
-8.316
-9.364
-8.891
-9.467
-8.510
-8.579
-9.868
-74.537
-79.256
-76.938
-76.692
-78.105
-91.515
-79.904
-68.081
-40.601
-52.857
-79.256
-66.343
-71.044
-103.143
11
14a
14b
15a
15b
16a
16b
17a
17b
18
4-[2-[2-(4-Isobutylphenyl)propanoyloxy]ethoxycarbonyl]-
3-methylfuroxan (11, C19H24N2O6)
Ethylene glycol (0.015 mol) and 20 cm3 dry CH2Cl2 were
slowly mixed together at room temperature. 2-(4-
Isobutylphenyl)propionyl chloride (7, 0.01 mol) in 5 cm3
dry CH2Cl2 was added drop wise to this mixture with
continuous stirring in ice cold condition. The stirring was
continued for 5 h and progress of the reaction was
monitored by TLC using 30 % ethyl acetate-hexane
solvent system. After completion, it was washed twice
20
21
IBPA:701
Fig. 4 Docked pose of compounds (i) co-crystal IBPA: 701, (ii) 14a, (iii) 17b, and (iv) 18 represented as tube in the binding site of COX-I
showing hydrogen bond interaction (yellow dash lines) with ARG 120, TYR 355 and ARG 83. The amino acids are represented as thin tube
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
Fig. 5 Superimpose docked 3D image and binding orientation of compound (i) 14a (green color) with co-crystal ligand (IBPA: 701, turquoise
color) (ii) 17b (orange color) with co-crystal ligand (IBPA:701, turquoise color) in COX-I site
with water, dried, and distilled under reduced pressure to
afford the intermediate 2-hydroxyethyl 2-(4-
petroleum ether. A solution of 6a (0.01 mol) in 5 cm3
anhydrous THF was added drop wise to a stirred solution of
intermediate 13 in 30 cm3 dry CH2Cl2 in the presence of
triethylamine (0.01 mol). The stirring was continued for
5 h, after which the reaction mixture was poured into ice
cold water. The organic layer was separated, washed with
water, dried and distilled under reduced pressure. The
residue thus obtained was purified by silica gel column
chromatography using EtOAc/hexane as eluent to afford
pure compound 14a. Yield 32 %; m.p.: 94–96 °C; IR
(KBr): vꢀ = 3308 (NH), 1755 (C=Oester), 1655 (C=Oamide),
isobutylphenyl)propionate (10) which was used immedi-
ately for next step of the reaction. A solution of 6a
(0.01 mol) in 5 cm3 anhydrous THF in the presence of
0.5 cm3 triethylamine was added drop wise at room
temperature to a stirred solution intermediate 10 in
30 cm3 dry CH2Cl2. The stirring was continued for 6 h
and the reaction mixture was poured into ice cold water.
The organic layer was separated, washed twice with water,
dried, and distilled under reduced pressure. The pale
yellow residue thus obtained was purified by silica gel
column chromatography using EtOAc/hexane as eluent to
afford pure compound 11 as a pale yellow semisolid
material. Yield 30 %; IR (KBr): vꢀ = 1755, 1748 (C=O),
1
1604 (C=N) cm-1; H NMR (CDCl3): d = 0.87 (d, 6H,
J = 6.6 Hz, (CH3)2CH), 1.48 (d, 3H, J = 7.2 Hz, CH3-
CH), 1.82 (m, 1H, (CH3)2CH-CH2), 2.32 (s, 3H, CH3), 2.43
(d 2H, J = 7.2 Hz (CH3)2CH-CH2), 3.52 (t, 2H,
J = 5.1 Hz, CH2), 3.61 (q, 1H, J = 7.2 Hz, CH3CH),
4.44 (t, 2H, J = 5.1 Hz, CH2), 5.69 (bs, 1H, CH3-CH-
CONH), 7.06–7.20 (m, 4H, ArH) ppm; 13C NMR (CDCl3):
d = 174.96, 157.90, 148.82, 140.97, 138.09, 129.68,
127.32, 112.22, 65.30 (OCH2), 46.67 (NHCH2), 44.80
(CH), 38.28 (CH2), 30.18 (CH), 22.35 ((CH3)2), 18.56
(CH3), 8.51 (CH3) ppm; MS: m/z = 376 (M??1).
1
1610 (C=N) cm-1; H NMR (CDCl3): d = 0.88 (d, 6H,
J = 6.6 Hz, (CH3)2CH), 1.47 (d, 3H, J = 7.2 Hz, CH3-
CH), 1.83 (m, 1H, (CH3)2CH-CH2), 2.25 (s, 3H, CH3), 2.42
(d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 3.73 (t, 2H,
J = 5.4 Hz, CH2), 4.41 (t, 2H, J = 5.4 Hz, CH2), 4.54
(q, 1H, J = 7.2 Hz, CH3CH), 7.02–7.21 (m, 4H, ArH)
ppm; 13C NMR (CDCl3): d = 175.10, 163.22, 152.63,
140.11, 137.21, 129.73, 126.21, 110.30, 56.62 (CH2), 54.96
(CH2), 41.54 (CH), 40.36 (CH2), 29.98 (CH), 22.38
((CH3)2), 17.31 (CH3), 8.70 (CH3) ppm; MS: m/z = 378
(M??1).
4-[2-[2-(4-Isobutylphenyl)propanamido)ethoxy]carbonyl]-
3-phenylfuroxan (14b, C24H27N3O5)
It was prepared by following the procedure of compound
14a by taking the starting material 4-(chlorocarbonyl)-3-
methyl furoxan (6b). The residue thus obtained was
purified by silica gel column chromatography using
EtOAc/hexane as eluent to afford pure compound 14b.
Yield 28 %; m.p.: 56 °C; IR (KBr): vꢀ = 3318 (NH), 1758
4-[2-[2-(4-Isobutylphenyl)propanamido]ethoxycarbonyl]-
3-methylfuroxan (14a, C19H25N3O5)
Ethyl 2-(4-isobutylphenyl)propionate (12) was added to
ethanolamine (0.02 mol) in 30 cm3 CH2Cl2 with stirring.
The mixture was refluxed for 8 h, cooled at room
temperature and the solvent was removed by distillation
at reduced pressure. Pale brown oil was obtained which
became a sticky solid on cooling in the refrigerator.
Subsequent grinding of the sticky solid with acetone and
diethyl ether gave a white solid which was the intermediate
N-(2-hydroxyethyl)-2-(4-isobutylphenyl)propanamide
(13). The compound thus obtained was recrystallized with
1
(C=Oester), 1650 (C=Oamide), 1601 (C=N) cm-1; H NMR
(CDCl3): d = 0.85 (d, 6H, J = 6.6 Hz, (CH3)2CH), 1.50
(d, 3H, J = 7.2 Hz, CH3CH), 1.79 (m, 1H, (CH3)2CH-
CH2), 2.40 (d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 3.52–3.61
(3H, q of CH3CH and t of CH2), 4.34 (t, 2H, J = 5.4 Hz,
CH2), 5.72 (bs, 1H, CH3-CH-CONH), 7.03–7.15 (m, 4H,
ArH), 7.40–7.60 (m, 5H, ArH) ppm; 13C NMR (CDCl3):
d = 174.89, 169.57, 140.81, 138.20, 133.17, 130.10,
123

M. Amir et al.
129.72, 129.65, 129.63, 128.40, 127.39, 63.36 (OCH2),
46.70 (NCH2), 45.11 (CH), 39.36 (CH2), 30.14 (CH), 22.38
((CH3)2), 18.23 (CH3) ppm; MS: m/z = 438 (M??1).
Progress of the reaction was monitored by TLC using a
mixture of ethyl acetate and n-hexane (2:3). After
completion, reaction mixture was poured into ice cold
water. The crude residue thus obtained was purified by
silica gel column chromatography using EtOAc/hexane as
eluent to afford pure compound 16a as a solid product.
Yield 52 %; m.p.: 70–72 °C; IR (KBr): vꢀ = 3201 (NH),
4-[[2-(4-Isobutylphenyl)propanoyloxy]methyl]-3-methyl-
furoxan (15a, C17H22N2O4)
4-(Hydroxymethyl)-3-methylfuroxan (0.01 mol) was dis-
solved in 20 cm3 dry toluene and 0.1 cm3 triethylamine
was added to it. To this solution, 2-(4-isobutylphenyl)pro-
pionyl chloride (7) in 5 cm3 dry toluene was added drop
wise with continuous stirring. The reaction mixture was
further stirred for 4 h and then poured into ice cold water.
The organic layer was separated, washed with water, dried,
and distilled under reduced pressure. The crude residue,
thus, obtained was purified by silica gel column chro-
matography using EtOAc/hexane as eluent to afford pure
compound 15a as a pale yellow oily material. Yield 52 %;
1682 (C=O), 1594 (C=N) cm-1 1H NMR (CDCl3):
;
d = 0.81 (d, 6H, J = 6.6 Hz, (CH3)2CH), 1.46 (d, 3H,
J = 7.2 Hz, (CH3)2CH-CH2), 3.64 (q, 1H, J = 7.2 Hz,
CH3CH), 6.99–7.12 (m, 4H, ArH), 8.22 (s, 1H, N=CH),
10.53 (bs, 1H, CONH) ppm; 13C NMR (CDCl3):
d = 175.59, 140.54, 140.10, 139.62, 134.09, 130.23,
127.02, 111.34, 44.37 (CH), 43.58 (CH2), 31.28 (CH),
22.76 ((CH3)2), 18.12 (CH3), 8.35 (CH3) ppm; MS:
m/z = 331 (M??1).
1
IR (KBr): vꢀ = 1743 (C=O), 1596 (C=N) cm-1; H NMR
4-[[2-[2-(4-Isobutylphenyl)propanoyl]hydrazono]methyl]-
3-phenylfuroxan (16b, C22H24N4O3)
(CDCl3): d = 0.81 (d, 6H, J = 6.3 Hz, (CH3)2CH), 1.42
(d, 3H, J = 7.2 Hz, CH3-CH), 1.76 (m, 1H, (CH3)2CH-
CH2), 2.10 (s, 3H, CH3), 2.35 (d, 2H, J = 7.2 Hz, (CH3)2-
CH-CH2), 3.64 (q, 1H, J = 7.2 Hz, CH3CH), 4.51 (s, 2H,
OCH2), 7.01–7.52 (m, 4H, ArH) ppm; 13C NMR (CDCl3):
d = 172.96, 149.52, 142.17, 139.18, 133.57, 128.61,
126.43, 54.47 (OCH2), 40.80 (CH), 38.20 (CH2), 29.06
(CH), 21.39 ((CH3)2), 15.47 (CH3), 8.37 (CH3) ppm; MS:
m/z = 319 (M??1).
It was prepared by following the procedure of compound
16a by taking the starting material 4-formyl-3-phenyl-
furoxan. The crude residue thus obtained was purified by
silica gel column chromatography using EtOAc/hexane as
eluent to afford pure compound 16b as a pale yellow oily
material. Yield 38 %; IR (KBr): vꢀ = 3213 (NH), 1692
(C=O), 1583 (C=N) cm-1; 1H NMR (CDCl3): d = 0.90 (d,
6H, J = 6.3 Hz, (CH3)2CH), 1.51 (d, 3H, J = 7.2 Hz,
CH3-CH), 1.85 (m, 1H, (CH3)2CH-CH2), 2.45 (d, 2H,
J = 7.2 Hz, (CH3)2CH-CH2), 3.71 (q, 1H, J = 7.2 Hz,
CH3CH), 7.12–761 (m, 9H, ArH), 8.01 (s, 1H, N=CH),
10.01 (bs, 1H, CONH) ppm; 13C NMR (CDCl3):
d = 179.91, 140.86, 137.07, 133.81, 131.95, 131.01,
130.67, 129.45, 128.97, 127.62, 127.08, 127.38, 45.05
(CH), 44.08 (CH2), 30.20 (CH), 22.41 ((CH3)2), 18.18
(CH3) ppm; MS: m/z = 393 (M??1).
4-[[2-(4-Isobutylphenyl)propanoyloxy]methyl]-3-phenyl-
furoxan (15b, C22H24N2O4)
It was prepared by following the procedure of compound
15a by taking the starting material 4-(chlorocarbonyl)-3-
phenyl furoxan (6b). The crude residue thus obtained was
purified by silica gel column chromatography using
EtOAc/hexane as eluent to afford pure compound 15b as
a pale yellow solid product. Yield 43 %; m.p.: 48 °C; IR
4-[2-[2-(4-Isobutylphenyl)propanoyl]hydrazinecarbonyl]-
3-methylfuroxan (17a, C17H22N4O4)
(KBr): vꢀ = 1750 (C=O), 1601 (C=N) cm-1 1H NMR
;
(CDCl3): d = 0.80 (d, 6H, J = 6.6 Hz, (CH3)2CH), 1.41
(d, 3H, J = 7.2 Hz, CH3-CH), 1.75 (m, 1H, (CH3)2CH-
CH2), 2.34 (d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 3.63 (q,
1H, J = 7.2 Hz, CH3CH), 4.49 (s, 2H, OCH2), 6.99–7.49
(m, 9H, ArH) ppm; 13C NMR (CDCl3): d = 173.78, 140.
87, 137.02, 136.74, 131.94, 131.24, 129.58, 129.42,
129.29, 127.62, 127.30, 127.16, 54.51 (OCH2), 45.05
(CH), 32.69 (CH2), 30.20 (CH), 22.41 ((CH3)2), 18.14
(CH3) ppm; MS: m/z = 381 (M??1).
2-(4-Isobutylphenyl)propionic acid hydrazide (0.01 mol))
was dissolved in 20 cm3 dry toluene and triethylamine
(0.01 mol) was added to it. Intermediate compound 6a in
5 cm3 dry toluene was added to this solution drop wise
with continuous stirring. The reaction mixture was further
stirred for 3 h at room temperature and then was poured
into ice cold water. The organic layer was separated,
washed with water, dried, and distilled under reduced
pressure. The residue, thus, obtained was purified by silica
gel column chromatography using EtOAc/hexane as eluent
to afford pure compound 17a. Yield: 64 %; m.p.: 86–
88 °C; IR (KBr): vꢀ = 3237, 3231 (NH), 1703, 1680 (C=O),
4-[[2-[2-(4-Isobutylphenyl)propanoyl]hydrazono]methyl]-
3-methylfuroxan (16a, C17H22N4O3)
2-(4-Isobutylphenyl)propionic acid hydrazide (0.01 mol)
and 4-formyl-3-methylfuroxan (0.01 mol) was dissolved in
30 cm3 ethanol. The reaction mixture was heated until
clear solution was obtained and few drops of conc. sulfuric
acid were added to it. The solution was refluxed for 6-8 h.
1
1612 (C=N) cm-1; H NMR (CDCl3): d = 0.91 (d, 6H,
J = 6.6 Hz, (CH3)2CH), 1.56 (d, 3H, J = 7.2 Hz, CH3-
CH), 1.81 (m, 1H, (CH3)2CH-CH2), 2.35 (s, 3H, CH3), 2.45
(d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 3.67 (q, 1H,
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
J = 7.2 Hz, CH3CH), 7.09–7.26 (m, 4H, ArH), 7.85 (bs,
1H, CONH), 8.71 (bs, 1H, CONH) ppm; 13C NMR
(CDCl3): d = 173.52, 162.41, 147.45, 141.23, 133.37,
129.93, 128.30, 111.23, 41.53 (CH), 39.76 (CH2), 29.26
(CH), 21.27 ((CH3)2), 16.75 (CH3), 8.21 (CH3) ppm; MS:
m/z = 347 (M??1).
obtained. Few drops of conc. sulfuric acid were added to
the solution and it was refluxed for 8 h. The reaction
mixture was cooled at room temperature and poured into
crushed ice. An oily compound was obtained which was
separated, washed with water and dried. It was purified by
silica gel column chromatography using EtOAc/hexane as
eluent to afford pure compound 20 as an oily light yellow
material. Yield 46 %; IR (KBr): vꢀ = 1612 (C=N), 789 (C-
4-[2-[2-(4-Isobutylphenyl)propanoyl]hydrazinecarbonyl]-
3-phenylfuroxan (17b, C22H24N4O4)
S-C) cm-1
;
1H NMR (CDCl3): d = 0.88 (d, 6H,
It was prepared by following the procedure of compound
17a by taking the starting material 4-(chlorocarbonyl)-3-
phenylfuroxan (6b). The residue thus obtained was purified
by silica gel column chromatography using EtOAc/hexane
as eluent to afford pure compound 17b. Yield 64 %; m.p.:
93–95 °C; IR (KBr): vꢀ = 3232, 3231 (NH), 1687, 1682
(C=O), 1597 (C=N) cm-1; 1H NMR (CDCl3): d = 0.88 (d,
6H, J = 6.6 Hz, (CH3)2CH), 1.51 (d, 3H, J = 7.2 Hz,
CH3-CH), 1.83 (m, 1H, (CH3)2CH-CH2), 2.43 (d, 2H,
J = 7.2 Hz, (CH3)2CH-CH2), 3.75 (q, 1H, J = 7.2 Hz,
CH3CH), 7.10–8.10 (m, 9H, ArH), 9.16 (bs, 1H, CONH),
9.41 (bs, 1H, CONH) ppm; 13C NMR (CDCl3):
d = 174.78, 164.78, 149.38, 142.33, 138.37, 133.25,
131.23, 129.32, 129.04, 128.82, 127.96, 127.23, 45.25
(CH), 44.53 (CH2), 29.62 (CH), 21.41 ((CH3)2), 16.18
(CH3) ppm; MS: m/z = 409 (M??1).
J = 6.6 Hz, (CH3)2CH), 1.49 (d, 3H, J = 7.2 Hz, CH3-
CH), 1.81 (m, 1H, (CH3)2CH-CH2), 2.42 (s, 3H, CH3), 2.44
(d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 4.51 (q, 1H,
J = 7.2 Hz, CH3CH), 7.07–7.26 (m, 5H, 4 ArH and
N=CH) ppm; 13C NMR (CDCl3): d = 170.22, 167.34,
154.56, 147.42, 139.25, 137.65, 128.78, 127.31, 111.43,
45.23 (CH), 38.54 (CH2), 29.37 (CH), 22.56 ((CH3)2),
20.34 (CH3), 8.78 (CH3) ppm; MS: m/z = 372 (M??1).
4-[5-[1-(4-Isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-yl]car-
bamoyl]-3-methylfuroxan (21, C18H21N5O3S)
Intermediate compound 19 (0.01 mol) was dissolved in
25 cm3 dry toluene and triethylamine (0.01 mol) was
added to it. Compound 6a in 5 cm3 dry toluene was added
to the solution drop wise with continuous stirring. The
reaction mixture was further stirred for 3 h and progress of
the reaction was monitored by TLC using TEF (5:4:1)
solvent system. After completion, it was poured into ice
cold water. The oily compound thus obtained was
separated, washed with water, and dried. It was purified
by silica gel column chromatography using EtOAc/hexane
as eluent to afford pure compound 21 as an oily light
yellow material. Yield 45 %; IR (KBr): vꢀ = 3022 (NH),
4-[5-[1-(4-Isobutylphenyl)ethyl]-1,3,4-oxadiazol-2-yl]-3-
methylfuroxan (18, C17H20N4O3)
2-(4-Isobutylphenyl)propionic acid hydrazide (0.01 mol)
was dissolved in 10 cm3 phosphorus oxychloride and to it
was added 4-carboxy-3-methylfuroxan (5a, 0.01 mol). The
reaction mixture was refluxed for 5 h, cooled to room
temperature, and poured on to crushed ice. The solution
was neutralized with sodium bicarbonate (10 %), an oily
compound was obtained which was separated, washed with
water and dried. It was purified by silica gel column
chromatography using EtOAc/hexane as eluent to afford
pure compound 18 as an oily pale yellow material. Yield
1
1701 (C=O), 1600 (C=N), 794 (C-S-C) cm-1; H NMR
(CDCl3): d = 0.88 (d, 6H, J = Hz, (CH3)2CH), 1.56 (d,
3H, J = 7.2 Hz, CH3-CH), 1.80 (m, 1H, (CH3)2CH-CH2),
2.38 (s, 3H, CH3), 2.45 (d, 2H, J = 7.2 Hz, (CH3)2CH-
CH2), 4.50 (q, 1H, J = 7.2 Hz, CH3CH), 7.00–7.37 (m,
4H, ArH), 12.54 (bs, 1H, CONH) ppm; 13C NMR (CDCl3):
d = 172.12, 168.13, 158.91, 147.82, 139.12, 137.23,
128.57, 126.31, 111.21, 44.83 (CH), 38.65 (CH2), 29.32
(CH), 22.05 ((CH3)2), 20.11 (CH3), 8.01 (CH3) ppm; MS:
m/z = 388 (M??1).
38 %; IR (KBr): vꢀ = 1595 (C=N) cm-1
;
1H NMR
(CDCl3): d = 0.80 (d, 6H, J = 6.6 Hz, (CH3)2CH), 1.54
(d, 3H, J = 7.2 Hz, CH3-CH), 1.82 (m, 1H, (CH3)2CH-
CH2), 2.36 (d, 2H, J = 7.2 Hz, (CH3)2CH-CH2), 2.44 (s,
3H, CH3), 4.37 (q, 1H, J = 7.2 Hz, CH3CH), 7.04–7.18
(m, 4H, ArH) ppm; 13C NMR (CDCl3): d = 170.64,
155.31, 144.71, 141.53, 136.37, 129.81, 127.05, 111.17,
44.99 (CH), 37.09 (CH2), 30.19 (CH), 22.36 ((CH3)2),
19.40 (CH3), 8.77 (CH3) ppm; MS: m/z = 329 (M??1).
Measurement of nitric oxide release
To a solution of furoxan derivatives of ibuprofen (20 mm3)
in dimethyl sulfoxide (DMSO) was added to 2 cm3 of 1:1
(v/v) mixture of 50 mM phosphate buffer (pH 7.4) with
MeOH, containing 5 9 10-4 M L-cysteine. The final con-
centration of drug was 10-4 M. After 1 h at 37 °C, 1 cm3
of the reaction mixture was treated with 250 mm3 of Griess
reagent [4 g sulfanilamide, 0.2 g naphthylene diamine
dihydrochloride, 10 cm3 85 % phosphoric acid, in distilled
4-[5-[1-(4-Isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-ylim-
ino]methyl]-3-methylfuroxan (20, C18H21N5O2S)
An equimolar amount of 5-[1-(4-isobutylphenyl)ethyl]-
1,3,4-thiadiazol-2-amine (19, 0.01 mol) and 4-formyl-3-
methylfuroxan (1) was dissolved in 25 cm3 ethanol. The
reaction mixture was heated until clear solution was
123

M. Amir et al.
water (final volume 100 cm3)]. After 10 min at room
temperature the absorbance was measured at 540 nm.
Sodium nitrite standard solutions (10–80 nmol/cm3) were
used to construct the calibration curve. The same procedure
was repeated using different solutions of the test com-
pounds under the same conditions using 0.1 N HCl of pH 1
instead of phosphate buffer of pH 7.4. The results were
expressed as the percentage of NO released relative to a
theoretical maximum release of 1 mol NO/mol of test
compounds [39].
% anti-inflammatory activity = 100 Â ð1 À Vt=VcÞ
where Vt and Vc are the volume edema in test compounds
and control groups, respectively.
Analgesic activity
Analgesic activity was evaluated by tail immersion method
[41] using Swiss albino mice (25–30 g) of either sex
selected by random sampling technique. The standard drug,
ibuprofen and test compounds were administered orally
(70 mg/kg body weight) as a suspension using 0.5 % w/v
carboxymethyl cellulose as a vehicle. The lower 5 cm
portion of the tail was gently immersed into thermostati-
cally controlled water at 55 0.5 °C. The time in seconds
for tail withdrawal from the water was taken as the reaction
time with a cut of time of immersion, set at 10 s for both
control as well as treated groups of animals. The reaction
time was measured before and after 4 h interval of the
administration of test compounds and standard drugs.
Pharmacology
The synthesized compounds were evaluated for their anti-
inflammatory, analgesic, ulcerogenic and lipid peroxida-
tion, hepatotoxic and histopathological properties. The
Wister rats and albino mice used in the present study were
housed and kept in accordance with the Hamdard Univer-
sity Animal Care Unit, which applies the guidelines and
rules laid down by the Committee for the Purpose of
Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Social Justice and Empowerment,
Government of India. All the test compounds and standard
drug were administered in the form of solution (0.5 % w/v
carboxymethyl cellulose as a vehicle) by an oral route.
Each group consisted of six animals. All the animals were
procured from the CPCSEA and maintained in colony
cages at 25 2 °C, relative humidity of 45–55 %, under a
12 h light and dark cycle and were fed a standard animal
feed. All the animals were climatised for a week before
use. The anti-inflammatory activity of the test compounds
were compared with the control. The analgesic, ulcero-
genic, and lipid peroxidation activities were compared with
the standard drug ibuprofen. Data were analyzed by stu-
dent’s t test for n = 6.
Acute ulcerogenicity
Acute ulcerogenesis test was performed according to Cioli
et al. [42] using Wistar rats (180–200 g) of either sex. The
animals were divided into various groups each group
consisting of six rats. All the rats were fasted for 24 h with
free access to water. The control groups of animals were
administered, 0.5 % CMC solution intraperitoneally. One
group was administered with standard drug ibuprofen
orally in a dose of 210 mg/kg once a daily for 3 days. The
remaining group of animals was administered with test
compounds through the same route. The animals were
immediately fed and kept for 17 h after dose administra-
tion. After 17 h they were killed and dissected for the
estimation of ulcerogenic activity. The stomach was dis-
sected out and washed with running water and opened
along the greater curvature and carefully observed with
magnifying glass. For each stomach the mucosal damage
was assessed according to the following scoring system:
0.5: redness, 1.0: spot ulcers, 1.5: hemorrhagic streak, 2.0:
ulcers [3 but B5, 3.0: ulcers [5. The mean score of each
treated group minus the mean score of control group was
regarded as severity index of gastric mucosal damage.
Anti-inflammatory activity
Anti-inflammatory activity was carried out by the car-
rageenan induced paw edema test in Wistar albino rats by
Winter et al. method [40]. The standard drug, ibuprofen
and test compounds were given orally (70 mg/kg body
weight) as a suspension using 0.5 % w/v carboxymethyl
cellulose as a vehicle. One hour later foot paw edema was
induced by injecting 0.1 cm3 of 1 % carrageenan subcu-
taneously into the planter portion of the right hind paw of
each rat. Initial paw volume was measured immediately by
mercury plethysmometer. The paw volume was again
measured after the time interval of 3 and 4 h. The per-
centage inhibition of inflammation was calculated for the
standard drug and other test compounds and comparison
was made. The percentage inhibition of inflammation was
calculated according to the formula
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined
according to the method of Ohkawa et al. [43]. After the
evaluation of stomach for ulcers the gastric mucosa of
glandular portion was scrapped, weighed (100 mg) and
homogenized in pestle and mortar and homogenate was
prepared in 1.8 cm3 of ice cold 1.15 % KCl solution. The
123

Synthesis, characterization, and biological evaluation of furoxan coupled ibuprofen…
homogenate was supplemented with 0.2 cm3 of 8.1 %
sodium dodecyl sulfate (SDS), 1.5 cm3 of acetate buffer,
and 1.5 cm3 of 0.8 % thiobarbituric acid (TBA). The
mixture was incubated at 95 °C for 60 min on boiling
water bath then extracted with a mixture of n-butanol :
pyridine (15:1, v/v; 5 cm3) by shaking vigorously for
1 min and kept in ice for 2 min. Organic layer of reaction
mixture were centrifuged at 3000 rpm for 10 min and
absorbance was measured at 532 nm on UV spectropho-
tometer. The results were expressed as nmol MDA/100 mg
tissue.
the receptors were prepared by using LigPrep and Protein
preparation wizard.
To predict good anti-inflammatory activity on a basis of
structure, a molecular docking studies of all the compounds
were carried out by taking X-ray crystal structure data to
establish the interaction of the synthetic compounds with
COX-1 (PDB code: 1EQG) Molecular docking studies
mainly involve selection and preparation of appropriate
protein, grid generation, ligand preparation followed by
docking and its analysis. The docking score, binding free
energy, and hydrogen bonds and p–p interaction formed
with the surrounding amino acids are used to predict their
binding affinities and proper alignment of these compounds
at the active site of the COX-1 enzyme.
Hepatotoxic studies
The study was carried out on Wistar albino rats of either
sex weighing 150–200 g. The animals were divided into
three groups of six rats each. Group I was kept as control
and received only vehicle (0.5 % w/v solution of CMC in
water), while group II and III received compound 14a and
17b, respectively, in 0.5 % w/v solution of CMC in water
for 15 days. After the treatment (15 days) blood was
obtained from all the groups of rats by puncturing the retro-
orbital plexus. Blood samples were allowed to clot for
45 min at room temperature and serum was separated by
centrifugation at 2500 rpm for 15 min and analyzed for
various biochemical parameters. Assessment of liver
function such as serum glutamate oxaloacetate transami-
nase (SGOT) and serum glutamate pyruvate transaminase
(SGPT) were estimated by a reported method [44]. The
alkaline phosphatase, total protein and total albumin were
measured according to reported procedures [45, 46].
Histopathological studies were also carried out by reported
method [47]. The rats were killed under light ether anes-
thesia after 24 h of the last dosage; the liver was removed
and washed with normal saline, and stored in formalin
solution. Sections of 5–6 microns thickness were cut,
stained with haematoxylin and eosin, and then studied
under an electron microscope.
Acknowledgments The authors are grateful to University Grants
Commission (UGC) New Delhi for financial support F. No. 36-1-08/
2008(SR). Authors are also thankful Central Instrumentation Facility
(CIF) Faculty of Pharmacy for spectral analysis of compounds and to
Dr. Ambrish Kumar Tiwari, In-charge animal house, Jamia Hamdard,
for providing animals. The authors are also thankful to Dr.
A. Mukharjee, MD, Department of Pathology, All India Institute of
Medical Sciences (AIIMS), New Delhi, for carrying out histopatho-
logical studies.
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