Design and synthesis of butenolide-based amide derivatives as anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-015-1404-y

Butenolide-based eighteen new amide derivatives (1-18) have been synthesized and evaluated for anti-inflammatory activity. The compounds 9, 17 and 4 exhibited significant in vivo inhibition of 84.69, 76.52 and 76.22 % inflammation, respectively, after 5 h without causing any damage to stomach and liver in comparison with the standard drug indomethacin which showed 79.04 % inhibition. The compounds showing potent anti-inflammatory activity were further evaluated for ex vivo TNF-α suppression. Compounds 9, 17 and 4 significantly suppressed TNF-α concentration to 74.83, 71.74 and 67.11 % as compared to indomethacin which exhibited a suppression of 69.01 %. Compounds 9 and 17 were also found to suppress the expression of COX-2 and NF-κB in the paw tissue. Moreover, compound 9 showed significant analgesic activity (57.03 %) which was comparable to indomethacin (61.03 %).

Full text of DOI:10.1007/s00044-015-1404-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2015) 24:3775–3784
DOI 10.1007/s00044-015-1404-y
ORIGINAL RESEARCH
Design and synthesis of butenolide-based amide derivatives
as anti-inflammatory agents
Yakub Ali1 Mohammad Sarwar Alam1 Hinna Hamid1 Asif Husain2
•
•
•
•
Abhijeet Dhulap3 Sameena Bano1 Chetna Kharbanda1 Syed Nazreen1
Saqlain Haider1
•
•
•
•
Received: 3 September 2014 / Accepted: 28 June 2015 / Published online: 28 July 2015
Ó Springer Science+Business Media New York 2015
Abstract Butenolide-based eighteen new amide deriva-
tives (1–18) have been synthesized and evaluated for anti-
inflammatory activity. The compounds 9, 17 and 4 exhib-
ited significant in vivo inhibition of 84.69, 76.52 and
76.22 % inflammation, respectively, after 5 h without
causing any damage to stomach and liver in comparison
with the standard drug indomethacin which showed
79.04 % inhibition. The compounds showing potent anti-
inflammatory activity were further evaluated for ex vivo
TNF-a suppression. Compounds 9, 17 and 4 significantly
suppressed TNF-a concentration to 74.83, 71.74 and
67.11 % as compared to indomethacin which exhibited a
suppression of 69.01 %. Compounds 9 and 17 were also
found to suppress the expression of COX-2 and NF-jB in
the paw tissue. Moreover, compound 9 showed significant
analgesic activity (57.03 %) which was comparable to
indomethacin (61.03 %).
Keywords Amide derivatives Á Anti-inflammatory Á
TNF-a Á Analgesic Á Ulcerogenic Á Hepatotoxicity Á
Immunohistochemistry
Introduction
A
number of nonsteroidal anti-inflammatory drugs
(NSAIDs) are available in the market and used for the
treatment of inflammatory diseases such as hay fever,
atherosclerosis and rheumatoid arthritis (Husain et al.,
2010). Most of these drugs function through suppressing
the activity of cyclooxygenase (COX) enzyme. However,
almost all of them present a number of unwanted, often
serious, side effects such as gastrointestinal disorder,
ulceration and kidney damage (Sostres et al., 2013).
Therefore, the development of new drugs with potent anti-
inflammatory activity and reduced side effects is still a
great challenge. In the present study, we report the syn-
thesis of molecules which show potent anti-inflammatory
activity with lesser or no ulceration effects.
Butenolidesorfuranonesareimportant classofcompounds
containingunsaturatedc-lactonering. Theyarefoundinmany
naturally occurring molecules such as digitoxigenin
goniobutenolides A & B and lignans (Mukku et al., 2000).
Butenolides have been found to show important biological
activities such as antibacterial, antimicrobial (Husain et al.,
2010) anti-inflammatory, analgesic(Alametal.,2009; Husain
et al., 2011), antitumor, antiviral (Ali et al., 2014) and anti-
cancer activities (Ali et al., 2003). c-Lactone ring present in
the butenolide derivatives is significantly reactive and can be
utilized for the synthesis of amide derivatives. In the present
study, a series of eighteen new amide derivatives based on
butenolide moiety have been synthesized and all the synthe-
sized compounds have been evaluated for their anti-
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1404-y) contains supplementary
material, which is available to authorized users.
& Mohammad Sarwar Alam
msalam5555@gmail.com;
msalam@jamiahamdard.ac.in
1
Department of Chemistry, Faculty of Science, Jamia
Hamdard (Hamdard University), New Delhi 110 062, India
2
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi 110 062, India
3
CSIR Unit for Research and Development of Information
Products, Pune 411038, India
123

3776
Med Chem Res (2015) 24:3775–3784
inflammatory potential. In order to explore the mechanistic
aspects of the activity, the synthesized molecules were sub-
jected to in silico molecular docking studies with respect to
TNF-a target. The compounds showing significant in vivo
anti-inflammatory activity and good binding affinity for TNF-
a in in silico studies were further evaluated for their effect on
TNF-a level (ex vivo). The active compounds were also
assessed for their effect on protein expression of other
proinflammatory mediators, namely NF-jB and COX-2, in
carrageenan-induced paw tissue immunohistochemically.
Furthermore, active compounds were also tested for hepato-
toxicity, LPO and gastric ulceration, as the impact of the
NSAIDs on the stomach is manifested due to the inhibition of
PGE2, which is normally protective as a result of inhibition of
COX-1, resulting in ulceration over time (Vonkeman and Van
de Laar, 2010).
hours. On completion of reaction, excess benzene was
distilled off under vacuum pressure and a solid mass so
obtained was washed with petroleum ether and dried. The
compound obtained was crystallized from ethanol.
Biological evaluations
Anti-inflammatory activity
The anti-inflammatory activity was carried out bythe reported
method (Winter et al., 1962). Animals were divided into
twenty groups of five animals each. Acute paw edema was
induced by sub-plantar injection of 0.1 % freshly prepared
carrageenan suspension in normal saline into the right hind
paw of each rat after 1 h of administrating the test samples.
The negative control was administered with 0.5 % car-
boxymethylcellulose solution. The animals of positive control
group were orally administered with standard indomethacin
(20 mg/kg/b.w.). The test groups were administered orally at
dose of 20 mg/kg b.w. Right hind paw volume was measured
at 3 and 5 h after carrageenan injection with the help of digital
plethysmometer. The percent anti-inflammatory activity was
calculated according to the formula given below.
Experimental protocols
Materials and methods
Preparation of 3-(4-substituted-benzoyl) propionic acid
%Anti-inflammatory activity ¼ ½VC À Vt=VCŠ  100
where Vt represents the mean increase in paw volume in
rats treated with test sample and VC represents the mean
increase in paw volume in control group of rats.
Succinic anhydride (10 g, 10 mmol) was treated with dif-
ferent aryl derivatives (17 g, 10 mmol) in the presence of
anhydrous aluminum chloride (15 g, 11.25 mmol). The
reaction mixture was stirred for 6 h. The desired product
was purified by dissolving in 5 % sodium hydroxide
solution and filtered. Dilute hydrochloric acid was added to
mother liquor to precipitate the product. The solid mass so
obtained was filtered, washed with cold water, dried and
crystallized from acetone to give 8.6 g of the desired
compound as a colorless solid.
Molecular docking studies on TNF-a
Crystallized structure of 2AZ5 was chosen from Protein Data
Bank and used as a target for molecular docking studies.
Undesired water molecules were removed. The protein was
then optimized and minimized to give low-energy target
protein. The grid was generated by selecting indomethacin as
the reference ligand. Finally, the validated grid was used for
docking with new unknown ligands to predict their docking
score. Two-dimensional chemical structures were converted
into 3-D structures, which were further subjected to OPLS-
2005 force field to generate single low-energy 3-D structure
for each input structure. During this step, chiralities were
maintained. Docking was carried using Glide software. It
was carried using extra precision and write XP descriptor
information. This generates favorable ligand poses which
were subjected to evaluation and minimization of grid
approximation. Scoring was then carried on energy-mini-
mized poses to generate Glide score.
General procedure for the synthesis of 2-arylidene-4-(4-
substituted-phenyl)-but-3-en-4-olides
Solution of 3-(4-substituted-benzoyl) propionic acid (0.71 g,
3 mmol) and 5-(4-chloro-phenyl)-furan-2-carbardehyde
(3 mmol) in acetic anhydride (15 ml) with a pinch of sodium
acetate was refluxed for 4 h under anhydrous conditions.
After completion of the reaction, the contents were poured on
crushed ice in small portions while stirring. A solid mass
separated out, which was filtered, washed with water and
crystallized from a mixture of methanol and chloroform.
General procedure for the synthesis of N-substituted-
benzyl-2-[5-(4-chloro-phenyl)-furan-2-ylmethylene]-4-
oxo-4-substituted-phenyl-butyramide
TNF-a assay
TNF-a assay was carried out by the previously reported
method (Ali et al., 2015). The right hind paws of animals
Butenolides (3 mmol) and different substituted benzy-
lamines (4 mmol) were refluxed in dry benzene for two
123

Med Chem Res (2015) 24:3775–3784
3777
used for anti-inflammatory activity were cut at the level of
the calcaneus bone after 5 h of dosing. Paws were washed
in saline and gently centrifuged at 4000 rpm for 30 min in
order to recover oedemantous fluid. The fluid recovered
was then filtered through Millipore cutoff filter
(10,000 mol. wt.) to remove traces of blood cells if any
(Millipore, Bedford, MA, USA). The levels of TNF-a was
determined using a commercially available ELISA kit (e-
Bioscience, San Diego, CA.) according to the manufac-
turer’s instruction.
thiobarbituric acid reagent were added to 1 ml suspension
taken from the supernatant. The tubes were kept in a
shaking water bath for 30 min at 80 °C. After 30 min,
tubes were kept in ice-cold water for 10 min and cen-
trifuged at 3000 rpm for 15 min. The absorbance of
supernatant was read at 540 nm at room temperature
against the blank.
Hepatotoxicity study
For this study, the dosing pattern was kept the same as that
for ulcerogenic studies. The experimental rats were killed
after 5 h of the administration of the test samples and
standard drug. Their liver specimens were removed and
stored in 10 % formalin solution (Kharbanda et al., 2014).
Immunohistochemistry
Sections of paw tissues (5 lm thick) embedded in paraffin
were deparaffinized in xylene followed by dehydration in
graded ethanol and finally rehydrated in running tap water.
Sections were boiled in 10 mM citrate buffer (pH 6.0) for
5–7 min and incubated with hydrogen peroxide for 15 min
and then rinsed with 1X PBST (0.05 % Tween-20).
Blocking solution was applied for 10 min, and then, sec-
tions were incubated with diluted (1:100) primary anti-
bodies, purified rabbit polyclonal anti-NF-jB antibody
(BioLegend) and rabbit polyclonal anti-COX-2 antibody
(Bio Vision), overnight at 4 °C in humid chamber. Further
processing was done according to the instructions of Ultra
Vision plus Detection System Anti-Polyvalent, HRP/DAB
staining kit (Thermo scientific system). The peroxidase
complex was visualized with 3,30-diaminobenzidine
(DAB). Lastly, the slides were counterstained with hema-
toxylin, cleaned in xylene, and dehydrated with ethanol,
and then, DPX mounting microscopic (BX 51 Olympus)
analysis was done at 40 X magnification.
Analgesic activity
The writhing test in swiss albino mice was carried out
using the previously reported method (Koster et al., 1959).
Swiss albino mice (35–40 g) of either sex were divided
into different groups of five animals, and dosing was done
as in case of anti-inflammatory activity. After 30 min of
test sample administration, 0.6 % acetic acid solution was
given to mice, intraperitoneally. The number of muscular
contractions was counted over a period of 10 min after
acetic acid injection. The data represent the total number of
writhes observed during 10 min and are expressed as
writhing numbers.
Results and discussion
Chemistry
Ulcerogenic activity
The compounds showing good anti-inflammatory activity
were further evaluated for their ulcerogenic activity by
reported method (Vogel and Vogel, 1997). Oral adminis-
tration of test samples and standard drug (indomethacin)
was given at a dose of 60 mg/kg b.w. which is thrice the
dose used for anti-inflammatory activity. Control rats
received only vehicle, i.e., the suspension of 1 % methyl
cellulose. Animals were killed 5 h after sample adminis-
tration, and their stomach were extracted carefully.
2-Arylidene-4-(4-substituted-phenyl)-but-3-en-4-olides were
synthesized by reacting 3-(4-fluoro-benzoyl)-propionic acid
with 5-(4-chloro-phenyl)-furan-2-carbardehyde in the pres-
ence of sodium acetate in acetic anhydride. N-substituted-
benzyl-2-[5-(4-chloro-phenyl)-furan-2-ylmethylene]-4-oxo-
4-substituted-phenyl-butyramide (1–18) were prepared by
reacting butenolides with different benzylamines in dry
benzene. The structural confirmation of target compounds
(1–18) was done by 1H NMR, IR and mass spectral data as
well as by elemental analysis (Scheme 1). 1H NMR spectra
showed two singlets for one proton each at d 6.5–6.8 and d
7.4–7.9 ppm for NH and the olefinic hydrogen of the
arylidene substituent, respectively. A singlet for two
protons at d 4.89–4.94 ppm was assigned to the benzyl
protons. Other singlet for two protons appearing at d
4.89–4.94 ppm corresponded to CH2 to proton adjacent to
olefinic bond.
Lipid peroxidation activity
The reported method was followed for evaluating LPO
activity (Ohkawa et al., 1979). The gastric mucosa scraped
with two glass slides was weighed (100 mg) and homog-
enized in 1.8 ml of 1.15 % ice-cold KCl solution. In total,
0.5 ml of 30 % trichloroacetic acid and 0.5 ml of 0.8 %
123

3778
Med Chem Res (2015) 24:3775–3784
Anti-inflammatory activity
I
R
R
OH
O
O
All the synthesized compounds were evaluated for their anti-
inflammatory activity by carrageenan-induced rat paw edema
model. The results of anti-inflammatory activity are shown in
Table 1. Among all the synthesized compounds, the com-
pound 9 showed maximum anti-inflammatory activity with
% inhibition of 76.44 % at 3 h and 84.69 % at 5 h which was
better than the standard drug indomethacin (72.34 % at 3 h
and 79.04 % at 5 h). Other compounds 17, 4, 15, 1 and 14
also showed comparably significant anti-inflammatory
activities at 5 h of inflammation (76.52, 76.22, 73.69, 70.43
and 70.13 %, respectively) than the standard drug.
II
O
O
Cl
O
R
IV
In silico docking studies
O
The synthesized compounds were docked for in silico study
against TNF-a target. Molecular docking studies provided
insights into molecular binding modes of molecules inside
the large pocket of TNF alpha receptor. Crystallized structure
of 2AZ5 protein was chosen as a target for molecular docking
studies with the specific ligand indomethacin which inhibits
it. In order to analyze the binding pattern and energies of new
R
O
O HN
Cl
R1
Scheme 1 (I) Anhydrous AlCl3, succinic anhydride; (II) 5-(4-chloro-
phenyl)-furan-2-carbardehyde, Ac2O, sodium acetate, reflux; (III) dry
benzene, aromatic benzylamines
Table 1 Anti-inflammatory activity of synthesized compounds
Compounds
R
R2
Change in paw volume (ml)
mean SEM
% Inhibition
3 h
3 h
5 h
5 h
Control
–
–
2.534 0.016
1.52 0.014**
1.66 0.020**
1.73 0.023**
1.79 0.021*
1.55 0.021**
1.67 0.022**
1.77 0.018*
1.69 0.016**
1.72 0.022**
1.52 0.014**
1.83 0.025*
1.76 0.019*
1.72 0.021*
1.78 0.026*
1.62 0.020**
1.62 0.022**
1.79 0.023*
1.532 0.019**
1.69 0.013**
2.564 0.018
1.42 0.014**
1.63 0.016**
1.70 0.018**
1.77 0.018*
1.53 0.012**
1.62 0.018**
1.82 0.014*
1.63 0.010**
1.66 0.020**
1.42 0.014**
1.80 0.021*
1.72 0.014*
1.68 0.021*
1.82 0.033*
1.61 0.019**
1.58 0.032**
1.79 0.019**
1.51 0.011**
1.71 0.028**
–
–
Indomethacin
–
–
72.34
67.43
60.94
55.65
73.70
64.89
56.07
63.52
60.48
76.44
53.19
57.14
61.09
57.00
68.23
70.60
56.68
75.98
63.98
79.04
70.43
63.95
57.50
76.22
69.24
53.54
66.56
66.41
84.69
56.90
60.62
64.93
55.42
70.13
73.69
67.57
76.52
63.29
1
p-H
H
2
p-H
o-Cl
p-F
H
3
p-H
4
p-F
5
p-F
o-Cl
p-F
H
6
p-F
7
p-Cl
p-Cl
p-Cl
p-Br
p-Br
p-Br
p-OCH3
p-OCH3
p-OCH3
p-CH3
p-CH3
p-CH3
8
o-Cl
p-F
H
9
10
11
12
13
14
15
16
17
18
o-Cl
p-F
H
o-Cl
p-F
H
o-Cl
p-F
Data are analyzed by one-way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean SEM from five observations where * p \ 0.05;
** p \ 0.01
123

Med Chem Res (2015) 24:3775–3784
3779
Fig. 1 Docking images of active compound 9 and standard indomethacin, respectively
molecules, the compounds were docked individually against
the generated grid. The synthesized compounds showed good
binding energies ranging from 48.28 to 49.63 kcal/mol.
Among all the synthesized molecules, the most promising
molecules were 9, 17, 4, 15, 14 and 1 with the glide score
-8.19, -8.15, -7.88, -7.84, -7.53 and -7.03, respec-
tively, where the glide score of reference ligand (in-
domethacin) was -5.02 only. Compound 9 was found to
form hydrogen bond with LUC:121 residue and p–p stacking
with TYR-B:59 of the target protein (Fig. 1a).Whereas ref-
erence ligand indomethacin was found to be aligned perfectly
with the hydrophobic pocket of the TNF-a protein (Fig. 1b),
the glide score and binding energies of all the synthesized
compounds are shown in Table 2.
was found that compounds 9, 17 and 4 suppressed the TNF-a
concentrationto74.83,71.74and67.11 % incomparisonwith
the standard indomethacin which inhibited TNF-a concen-
tration to 69.01 %. Other compounds showed moderate
inhibition of TNF-a concentration. From the results, it is clear
that compounds 9, 17 and 4 showing significant in vivo anti-
inflammatory activity also significantly decreased the level of
TNF-a in carrageenan-induced animals.
Immunohistochemistry
During the process of inflammation, several proinflammatory
mediators such as TNF-a, COX-2, NF-jB and iNOS are
activated. Hence, the most active compounds 9 and 17 were
also evaluated for protein expressions of COX-2 and NF-jB
using immunohistochemistry. The results are shown in
Fig. 3a, b, respectively. It was found that COX-2 and NF-jB
expression in the animal paw tissue was increased in car-
rageenan-treatedanimals(groupII) ascomparedtothecontrol
group (I). The treatment of animals with active compounds 9
and 17 showed significant reduction in the number of cells
Ex vivo TNF-a assay
In order to confirm the mode of action, the active compounds
were screened for their effect on TNF-a expression. The
results of TNF-a level are shown in Fig. 2. Carrageenan sig-
nificantly increased TNF-a concentration in the animals. It
123

3780
Med Chem Res (2015) 24:3775–3784
Table 2 Docking results of synthesized compounds
Legends
Glide score
Glide energy
M.W.
QPlog Po/w
QPlogS
PSA
Indomethacin
-5.02
-7.03
-7.38
-6.55
-7.88
-7.12
-6.32
-8.15
-7.58
-8.19
-6.11
-7.11
-7.04
-6.77
-7.53
-7.84
-6.46
-8.15
-7.66
-33.09
-46.98
-46.76
-44.84
-47.43
-47.43
-44.61
-48.00
-48.40
-49.90
-42.28
-42.74
-48.11
-45.69
-51.43
-49.63
-45.21
-47.87
-46.61
357.00
4.28
-5.31
84.32
1
455.94
6.093
6.425
6.23
-6.203
-6.787
-6.284
-6.659
-6.453
-6.272
-6.754
-7.105
-6.878
-6.787
-7.593
-7.403
-6.42
63.57
2
490.385
473.93
64.325
60.459
63.56
3
4
473.93
6.296
6.443
6.249
6.411
6.74
5
508.375
491.921
490.385
524.83
61.103
60.701
63.29
6
7
8
62.08
9
508.375
534.836
569.281
552.826
485.966
520.411
503.956
469.959
504.412
487.957
6.624
6.524
7.002
6.778
6.129
6.447
6.300
6.309
6.706
6.441
60.423
60.538
64.085
62.193
71.863
69.585
71.954
60.485
62.311
60.463
10
11
12
13
14
15
16
17
18
-6.699
-6.649
-6.509
-7.238
-6.593
resulting in ulceration over time. Compounds 9, 17, 4 and
15 did not cause any epithelium tissue damage or gastric
ulceration (Fig. 4), whereas standard drug indomethacin
caused significant epithelium tissue damage and showed
traces of gastric ulceration in stomach of the animals.
Lipid peroxidation assay
Compounds 9, 17, 4 and 15 were also evaluated by lipid
peroxidation assay. The results of LPO assay are shown in
Fig. 5. LPO is measured as nmol of MDA per 100 mg of
gastric mucosa tissue. The level of lipid peroxidation in the
carrageenan-induced animals was found significantly
increased; however, the level of LPO was found to decrease
significantly in the presence of the standard drug indo-
methacin and test compounds 9, 17, 4 and 15. The active
compounds 9 and 17 caused better reduction in lipid per-
oxidation as compared to the standard drug indomethacin.
Fig. 2 Effect of synthesized compounds on carrageenan-induced
TNF-a cytokine level in the paw tissue. Data are analyzed by one-way
ANOVA followed by Dunnett’s ‘t’ test and expressed as mean
SEM from five observations where *p \ 0.05, **p \ 0.01
showing expression of these proteins as compared to the
standard drug indomethacin.
Hepatotoxicity studies
Ulcerogenic activity
The results of liver histopathology studies of compounds 9,
17, 4 and 15 are shown in Fig. 6. It was observed that the
compounds 9, 17, 4 and 15 did not cause any damage to the
liver as compared to the standard drug indomethacin which
caused significant inflammation to the portal and centri-
zonal vein and sinusoidal dilation.
Compounds showing significant anti-inflammatory activity
as well as ex vivo TNF-a suppression were further checked
for ulcerogenic effects, as NSAIDs affect the stomach due
to the inhibition on PGE2 as a result of reduction in COX-1
123

Med Chem Res (2015) 24:3775–3784
3781
Fig. 3 a Paw tissue expressions of COX-2 activation. Representative
photomicrographs magnification (940) group I (only control), group
II (only carrageenan), group III (carrageenan ? standard drug
Representative photomicrograph magnification (940) group I (only
control), group II (only carrageenan), group III (carrageenan ? stan-
dard drug indomethacin), group IV (carrageenan ? 9) group
(carrageenan ? 17)
indomethacin), group IV (carrageenan ? 9) group
rageenan ? 17). b Paw tissue expressions of NF-kB activation.
V
(car-
123

3782
Med Chem Res (2015) 24:3775–3784
Fig. 4 Histopathology of rat stomach in Albino Wistar rats. Low-power (910) and high-power photomicrographs (940) of stomach wall of the
animal groups administered with compounds 9, 17, 4 and 15 standard drug indomethacin and control group, respectively
analgesic activity are shown in Table 3. Among these
compounds, compound 9 showed maximum analgesic
activity (57.03 %) which was comparable with the standard
drug indomethacin (61.13 %). The compounds 14, 15, 17
and
4 also showed considerable analgesic activity
(51.17–55.07 %).
Structure activity relationship
From the biological studies, following structure activity rela-
tionship (SAR) can be drawn for anti-inflammatory activity
Fig. 5 Lipid peroxidation assay of active compounds
Cl
O
Analgesic activity
O
HN
Those compounds which showed significant anti-inflam-
matory activity were also tested for their analgesic activity
by acetic acid-induced writhing test. The results of
O
R1
R
123

Med Chem Res (2015) 24:3775–3784
3783
Fig. 6 Histopathology of rat liver in Albino Wistar rats. Low-power (910) and high-power photomicrographs (940) of stomach wall of the
animal groups administered with compounds 9, 17, 4 and 15 standard drug indomethacin and control group, respectively
•
•
•
For compounds having R = H, F and R1 = H, o-Cl and
p-F, the in vivo anti-inflammatory activity was found to
increase in the order: p-F \ o-Cl \ H.
Above order was reversed, i.e., p-F [ o-Cl [ H for the
compounds having R = Cl, Br, OCH3 and R1 = H,
o-Cl, p-F.
Table 3 Antinociceptive activity of active compounds
Group
Number of writhes in 10 min
% Protection
Control
102.4 1.60
39.8 1.28**
60.0 1.37*
53.6 1.72**
50.0 1.41**
58.0 2.12*
56.5 1.16*
44.0 1.37**
47.6 1.77**
47.8 2.15**
46.0 1.41**
–
Indomethacin
61.13
41.40
47.65
51.17
43.35
44.72
57.03
53.51
53.32
55.07
1
For compounds having R = CH3 and R1 = H, o-Cl,
p-F, the anti-inflammatory activity increases in the
order of o-Cl [ H [ p-F.
5
4
7
8
9
14
15
17
Conclusion
We have presented a facile route for the formation of
butenolide-based amide derivatives. Out of the eighteen
synthesized compounds, compounds 9, 17 and 4 were
found to have anti-inflammatory and analgesic properties
Data are analyzed by one-way ANOVA followed by Dunnett’s ‘t’ test
and expressed as mean
SEM from five observations where
* p \ 0.05; ** p \ 0.01
123

3784
Med Chem Res (2015) 24:3775–3784
Ali Y, Alam MS, Hamid H, Husain A, Kharbanda C, Syed N, Haider
S, Bano S (2015) Attenuation of inflammatory mediators,
oxidative stress and toxic risk evaluation of Aporosa lindleyana
Bail bark extract. J Ethnopharmacol 155:1513–1521
Husain S, Alam MM, Shaharyar M, Sukhbir L (2010) Antimicrobial
activities of some synthetic butenolides and their pyrrolone
derivatives. J Enzyme Inhib Med Chem 25:54–61
Husain S, Alam MM, Drabu S, Nitin K, Alam M, Ahmad A (2011)
Synthesis and biological evaluation of some new pyridazinone
derivative. J Enzyme Inhib Med Chem 26:742–748
comparable to the standard drug indomethacin. Com-
pounds 9 and 17 significantly reduced the level of TNF-a,
COX-2 and NF-jB as compared to the standard drug
indomethacin without causing any damage to stomach and
liver. Therefore, compounds 9, 17 and 4 might be consid-
ered as promising candidates for development of new and
safer anti-inflammatory agents.
Acknowledgments The authors thank Vice Chancellor, Dr. G. N.
Qazi for providing necessary facilities to the Department of Chem-
istry. Yakub Ali acknowledges Hamdard National Fellowship (HNF)
for providing financial assistance.
Kharbanda C, Alam MS, Hamid H, Bano S, Haider S, Syed N, Ali Y,
Javed K (2014) Trapa natans L. root extract suppresses
hyperglycemic and hepatotoxic effects in STZ-induced diabetic
rat model. J Ethnopharmacol 151:931–936
Koster R, Anderson M, Beer EJ (1959) Acetic acid for anti-
nociceptive screening. Fed Proceed 18:412–416
Conflict of interest The authors report no conflict of interest.
Mukku JK, Speiting M, Loatsch H, Helmke
E
(2000) New
Nat Prod
butenolides from two marine streptomycetes.
63:1570–1572
J
References
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal Biochem
95:351–358
Sostres C, Gargallol Carla J, Lanas A (2013) Nonsteroidal anti-
inflammatory drugs and upper and lower gastrointestinal
mucosal damage. Arthritis Res Ther 15(Suppl 3):S3
Vogel HG, Vogel HW (1997) Drug discovery and evaluation-
pharmacological assays. Springer, Berlin, p 1231
Vonkeman HE, Van de Laar MA (2010) Nonsteroidal anti-inflam-
matory drugs: adverse effects and their prevention. Semin
Arthritis Rheum 39:294–312
Winter CA, Risley EA, Nuss GW (1962) Carrageenan induced edema
in hind paw of rat assay for anti-inflammatory drugs. Proc Soc
Exp Biol 111:544–547
Alam MM, Hasan SM, Husain A, Anwer T (2009) Synthesis and
pharmacological evaluation of 2(3H)-furanones and 2(3H)-
pyrrolones, combining analgesic and anti-inflammatory proper-
ties with reduced gastrointestinal toxicity and lipid peroxidation.
Eur J Med Chem 44:2636–2642
Ali AM, Shahram H, Jamshid C, Ali H, Farshid L, Fen-Tair WL, Tai
S, Kak-Shan Y, Chi-Feng LJ, Moti K, Ramasamy X, Cuihua P,
Manijeh H (2003) Design, synthesis, and anticancer activity of
phosphonic acid diphosphate derivative of adenine-containing
butenolide and its water-soluble derivatives of paclitaxel with
high antitumor activity. Bioorg Med Chem 11:4303–4313
Ali Y, Alam MS, Hinna H, Husain A (2014) 2(3H)-Pyrrolone–A
biologically active scaffold (a review). Orient J Chem 30:1–16
123