Molecular docking, synthesis and biological screening of mefenamic acid derivatives as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejphar.2017.02.051

Drug induced gastrointestinal ulceration, renal side effects and hepatotoxicity are the main causes of numerous Non-Steroidal Anti-inflammatory Drugs (NSAIDs). Cyclooxygenase-2 (COX-2) inhibitors discovered to decrease the gastrointestinal issues, but unfortunately, most of them are associated with major cardiovascular adverse effects. Along these lines, various new strategies and frameworks were developed wherein basic alterations of the present medications were accounted for. The aim of the study was to prepare derivatives of mefenamic acid to evaluate anti-inflammatory activity with fewer adverse reactions. In this study, molecular docking investigations of outlined derivatives were done utilizing Protein Data Bank (PDB ID-4PH9). Synthesis of heterocyclic compounds was carried out utilizing Dicyclohexylcarbodiimide/4-Dimethylaminopyridine (DCC/DMAP) coupling. Acute toxicity prediction was performed using free online GUSAR (General Unrestricted Structure-Activity Relationships) software. The study indicated most of the compounds under safe category. In-vitro pharmacological assessment of heterocyclic compounds was done for COX-1 and COX-2 enzymes for the determination of selectivity. In vivo pharmacological screening for anti-inflammatory activity and ED₅₀ value were determined utilizing carrageenan induced rat paw edema. Gastro intestinal safety study was carried out on selected compounds and found to be devoid of any gastric ulcer toxicity. Most of the compounds indicated high scores as compared to standard during molecular modelling, analysis and displayed interactions with active amino acids of a COX-2 enzyme. The pharmacological screening uncovered that compound substituted with p-bromophenyl indicated maximum potency.

Full text of DOI:10.1016/j.ejphar.2017.02.051

European Journal of Pharmacology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Molecular docking, synthesis and biological screening of mefenamic acid
derivatives as anti-inflammatory agents
Jignasa K. Savjania,⁎, Suja Mulamkattilb, Bhavesh Variyaa,c, Snehal Patela
a
Institute of Pharmacy, Nirma University, S.G.Highway, Ahmedabad, Gujarat 382481, India
Beiersdorf. Nivea India, plot no: SM-9/1, Sanand-2, Industrial Estate, Ahmedabad, Gujarat 382110, India
Zydus Research Centre, Sarkhej-Bavla N.H. No. 8A, Moraiya, Ahmedabad, Gujarat 382210, India
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Drug induced gastrointestinal ulceration, renal side effects and hepatotoxicity are the main causes of numerous
Non-Steroidal Anti-inflammatory Drugs (NSAIDs). Cyclooxygenase-2 (COX-2) inhibitors discovered to decrease
the gastrointestinal issues, but unfortunately, most of them are associated with major cardiovascular adverse
effects. Along these lines, various new strategies and frameworks were developed wherein basic alterations of
the present medications were accounted for. The aim of the study was to prepare derivatives of mefenamic acid
to evaluate anti-inflammatory activity with fewer adverse reactions. In this study, molecular docking
investigations of outlined derivatives were done utilizing Protein Data Bank (PDB ID-4PH9). Synthesis of
heterocyclic compounds was carried out utilizing Dicyclohexylcarbodiimide/4-Dimethylaminopyridine (DCC/
DMAP) coupling. Acute toxicity prediction was performed using free online GUSAR (General Unrestricted
Structure-Activity Relationships) software. The study indicated most of the compounds under safe category. In-
vitro pharmacological assessment of heterocyclic compounds was done for COX-1 and COX-2 enzymes for the
determination of selectivity. In vivo pharmacological screening for anti-inflammatory activity and ED50 value
were determined utilizing carrageenan induced rat paw edema. Gastro intestinal safety study was carried out on
selected compounds and found to be devoid of any gastric ulcer toxicity. Most of the compounds indicated high
scores as compared to standard during molecular modelling, analysis and displayed interactions with active
amino acids of a COX-2 enzyme. The pharmacological screening uncovered that compound substituted with p-
bromophenyl indicated maximum potency.
Acute toxicity study
Anti-inflammatory agents
COX-2 inhibitors
Gastro intestinal safety study
GUSAR
Molecular modelling
1. Introduction
(Penning et al., 1997), rofecoxib (Vioxx®), (Prasit et al., 1999)
valdecoxib (Kalgutkar et al., 2000) and etoricoxib (Riendeau et al.,
Anti-inflammatory drugs are used to treat pain and inflammation
associated with musculoskeletal muscle and joints. Inflammation is a
result of an increase in the level of prostanoids which are also
responsible for the protection of the gastric mucosal membrane
(Kalgutkar et al., 2002). Cyclooxygenase (COX) enzyme is involved in
the rate-limiting step for the synthesis of different prostaglandins and
thromboxanes from arachidonic acid (Marnett et al., 1999). The COX-1
enzyme is responsible for the cytoprotection and the COX-2 enzyme is
inducible and responsible for the biosynthesis of Prostaglandins in
inflammatory tissues (Dogné et al., 2006; Kalgutkar et al., 2002;
Marnett et al., 1999). Unfortunately, anti-inflammatory agents dis-
covered till now suffer from major side-effects. Classical NSAIDs are
associated with adverse effects like gastric ulceration, hepatotoxicity,
anemia etc. In order to overcome these side effects various selective
COX-2 inhibitors were discovered. As a result, celecoxib (Celebrex®)
2001) were marketed as selective COX-2 inhibitors for the treatment of
inflammation (Bansal et al., 2014). However, in the year 2004, Merck
& Co., voluntarily withdrawn rofecoxib from the market due to severe
cardiovascular adverse events. The adverse cardiovascular toxicity
related to selective COX-2 inhibitors is due to the increased level of
TXA2 in platelet (Patrono and Baigent, 2014). Kalgutkar et al. synthe-
sized various derivatives of classical NSAIDs (Aljadhey et al., 2010).
They have synthesized various amide and ester derivatives of meclo-
fenamic acid and indomethacin, which showed selectivity for the COX-
2 enzyme. There are several reports which revealed that derivative
preparation of well-known NSAIDs showed better results (Kalgutkar
et al., 2000; Talley et al., 2000; Woods et al., 2001). Moreover, the
gastric irritation associated with NSAIDs and COX-2 inhibitors is free
of the route of the administration totally, mitigating any rationalization
that removing the carboxylic acid moiety would bring about less gastro-
⁎
Corresponding author.
E-mail address: Jignasasavjani@gmail.com (J.K. Savjani).
http://dx.doi.org/10.1016/j.ejphar.2017.02.051
Received 8 February 2017; Received in revised form 27 February 2017; Accepted 28 February 2017
0014-2999/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Savjani, J.K., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.02.051

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
intestinal irritation due to some sort of local effect. The points of
interest offered by the utilization of the particular COX-2 inhibitors
urged to evaluate the pharmacological movement of novel heterocyclic
compounds that could be potential anti-inflammatory agents. Hence,
preparation of amide derivatives of NSAIDs can be utilized as the
classical approach for the development of more efficacious anti-
inflammatory agents, which is our real objective.
ethyl acetate: hexane (0.5: 4.5) as the solvent system. After completion
of the reaction the pale yellow product was extracted using DCM after
repeated washing with brine and sodium bicarbonate.
2.3.2. Synthesis of 2-(2,3-dimethylphenylamino)-N-p-tolylbenzamide
3b
Same as compound 3a; in place of aniline, p-toluidine was used
(0.002 mol) and the reaction was carried out for 15 h afforded a yellow
colour product of 3b.
2. Materials and methods
2.1. Molecular docking studies
2.3.3. Synthesis of 2-(2,3-dimethylphenylamino)-N-(4-fluorophenyl)
benzamide 3c
Docking was used to predict both ligand orientation and binding
affinity with the COX-2 enzyme. The response of designed molecules at
the specific active site of the crystallographic structure of the protein
was determined. SYBYL-X 1.2 software was used to build all the
compounds and energy minimization was carried out using a conjugate
gradient algorithm with a gradient convergence value of 0.01 kcal/
Mol Å. Partial atomic charges were calculated using the Gasteiger
Huckel method. To examine the binding affinities of the designed
compounds with the COX-2 enzyme, docking of these compounds
using GOLD 5.2 was performed. PDB ID- 4PH9 with a resolution of the
1.8 Å cocrystal structure was downloaded from RCSB (Research
Collaboratory for Structural Bioinformatics) protein data bank for
docking study. The result of docking analysis was obtained in terms
of GOLD score and was compared with the standard drug (Ganga
Reddy et al., 2016).
Same as compound 3a; in place of aniline, p-fluoroaniline was used
(0.002 mol) and the reaction was carried out for 12 h afforded a yellow
colour product of 3c.
2.3.4. Synthesis of 2-(2,3-dimethylphenylamino)-N-(2-chlorophenyl)
benzamide 3d
Same as compound 3a; in place of aniline, o-chloroaniline was used
(0.002 mol) and the reaction was carried out for 15 h afforded a dark
yellow colour product of 3d.
2.3.5. Synthesis of 2-(2,3-dimethylphenylamino)-N-(4-bromophenyl)
benzamide 3e
Same as compound 3a; in place of aniline, p-bromoaniline was
used (0.002 mol) and the reaction was carried out for 15 h afforded a
yellow colour product of 3e.
2.2. LD50 prediction using GUSAR software
2.3.6. Synthesis of 2-(2,3-dimethylphenylamino)-N-(2-(piperazin-1-
yl)ethyl)benzamide 3f
In silico acute toxicity prediction was performed using GUSAR
(General Unrestricted Structure-Activity Relationships) software.
GUSAR is a free online software, which include information about
the acute toxicity of around 10,000 chemical structures. The software
predicts activity of compounds based on QSAR (Quantitative Structure
Activity Relationship) models. The data represented as the LD50 values
(log10 (mmol/kg) of the compounds for different routes of adminis-
tration like oral, subcutaneous, intravenous and Intraperitoneal. The
toxicity class of the given compound was reported, according to the
OECD (Organization for Economic Co-operation and Development)
classification project of chemical substance (Korobko, 2016).
Same as compound 3a; in place of aniline, 2-aminoethyl piperazine
was used (0.002 mol) and the reaction was carried out for 15 h afforded
a yellow colour product of 3f.
2.4. Synthesis of benzimidazole and benzthiazole derivatives (4a-4b)
2.4.1. Synthesis of 2-(1H-benzo[d]imidazole-2-yl)-N-(2,3-dimethyl-
phenyl)benzenamine 4a
To 0.1 mol of 3g in a 100 ml round bottom flask, 0.1 mol of glacial
acetic acid and Con. HCl was added. The mixture was refluxed at 80 °C
for 2.5 h. After the completion of the reaction, crushed ice was added to
the reaction mixture with continuous stirring. The reaction mixture
was neutralized with NaHCO3 until precipitates were observed. The
precipitates were filtered under the vacuum and washed with cold
water. The product of 4a, was purified using column chromatography
(5% ethyl acetate: hexane).
2.3. Synthesis of amide derivatives of mefenamic acid (3a-h)
Synthesis of amide derivatives was carried out using borosilicate
glass wares. REMI rota mantle and magnetic stirrers were used for
heating, refluxing as well as stirring of the reaction mixtures. Solvent
recovery was done using Rotary Vacuum evaporator (Buchi type).
Precoated Silica Gel TLC plates (MERCK) were used for monitoring the
progress of the reaction. UV chamber was used for the determination of
progress of a reaction. Melting point was measured using the paraffin
bath or by melting point apparatus (VEEGO corporation). IR spectra
were recorded on JASCO FTIR by KBr dispersion method. Mass
Spectra were recorded on BRUKER using ESI as an ion source. 1H
NMR and 13C NMR spectra were recorded on BRUKER 400 MHz
instrument using TMS as an internal standard.
2.4.2. Synthesis of 2-(1H-benzo[d]thiazol-2-yl)-N-(2,3-dimethyl-
phenyl)benzenamine 4b
Same as compound 4a; in place of 3g, 3h (0.002 mol) and the
reaction was carried out for 17 h afforded a pale yellow colour product
of 4b.
2.5. Pharmacological Screening
2.5.1. In vitro COX-1 and COX-2 enzymatic assay
2.3.1. Synthesis of 2-(2,3-dimethylphenylamino)-N-phenylbenzamide
3a
In vitro COX-I and COX-2 inhibition assay was performed for
standard and synthesized compounds. Evaluation of COX inhibitory
activity was carried out using a COX colorimetric inhibitor screening
assay kit (Cayman Chemicals, USA) (Jang, 1997).
The assay kit includes both ovine COX-1 and human recombinant
COX-2 enzymes in order to screen isozyme-specific inhibitors. The
assay measures the peroxidase activity by monitoring the appearance of
oxidized TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) at
590 nm.
To a mixture of 0.002 mol of 2-(2,3-dimethylphenylamino)benzoic
acid and 0.0022 mol of aniline in DCM (dichloromethane) and DMAP
(dimethyl amino pyridine) was added with continuous stirring. After
30 min of stirring at 0 °C, the cooled solution of DCC (N,N`-dicyclo-
hexylcarbodiimide) (1.1 equiv) was added in to the above reaction
mixture and allowed to stir at room temperature under the N2
environment for 15 h. The reaction was monitored using TLC using
2

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
2.5.2. Study of effect of heterocyclic derivatives on the animal model
of carrageenan-induced paw edema
Table 1
Docking results of mefenamic acid analogs.
All experiments and protocols described in the present study were
approved by the Institutional Animal Ethics Committee (IAEC) of the
Institute of Pharmacy, Nirma University, Ahmedabad as per guidelines
of the committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), the Ministry of Social Justice
and Empowerment, the Government of India. Protocol numbers are
IP/PCOL/MPH/17/006 and IP/PCEM/FAC/20/028. Healthy Wistar
rats (250–300 g) were procured from the Torrent Research Centre,
Gandhinagar, Ahmedabad. Animals were housed in groups of 6
animals in the animal house of Nirma University, Ahmedabad under
controlled conditions of temperature 23 2 °C, relative humidity, 55
5%, and photo-schedule (12 h light, and 12 h dark). Animals had free
access to food and purified water ad libitum. Animals were acclimatized
for one week before starting the experiment and randomized into
different groups.
Compounds
GOLD score
Interaction
Mefenamic acid
47.17
60.51
57.92
45.77
58.51
57.7
54.35
58.66
57.43
TYR 386
TYR386, ASN383, THR213
HIS389
3a
3b
3c
3d
3e
3f
TYR386
TYR386, HIS208
TYR386, ASN383
TYR386
HIS208, THR207, TYR 386
TYR386, HIS208
4a
4b
the mefenamic acid derivatives, phenyl, 2-chlorophenyl, 4-tolyl and 4-
bromophenyl analogs demonstrated higher scores than standard except
3c as shown in the Table 1.
The animals were divided in to five groups- normal control group,
disease control group, animals treated with mefenamic acid (12.8 mg/
kg), animal treated with test compounds 3e (12.85 mg/kg) and 3f
(12.85 mg/kg). 0.1 ml of 1% w⁄ v carrageenan solution was adminis-
tered into the plantar side of the right hind paw of the rats (Chigayo
et al., 2014). In the plethysmometer, mercury displacement method
was used for the determination of the volume of the injected paw. The
paw volume was measured 5hr after the injection of carrageenan. The
difference between the left paw volume and the right paw volume gave
the actual edema volumes. Paw volumes are physical indicators of
inflammation in early as well as the chronic phase of the disease. Using
the following formula the % inhibition of paw volume was calculated.
3.2. Computer aided acute toxicity prediction
The free online GUSAR software has been employed for the
prediction of acute toxicity of compounds under investigation. The
values obtained for different routes of the administration of mefenamic
acid derivatives as shown in Table 2. The LD50 values by Intravenous
and oral administration were found to be between 55 to 130 and 866–
2735 respectively.
3.3. Physical characterization of mefenamic acid derivatives
The synthetic protocol for the target molecules involves a single
step. Mefenamic acid forms amide bond via DCC/DMAP coupling
using different primary amines (Scheme 1). Structure elucidation of
synthesized compounds was done by using IR, Mass, NMR (both 1H
NMR and 13C NMR) spectroscopy (Bali et al., 2012; Vasantha et al.,
2015).
Increase in Paw Volume = VD − VO
Where, VD=Paw Volume at 5 h, VO=Paw Volume at 0 min
% Inhibition in Paw Volume = VD − VO/VO × 100
2.5.3. Determination of ED50 value of the selected compound on an
animal model of carrageenan-induced paw edema
3.3.1. Characterization
of
2-(2,3-dimethylphenylamino)-N-
ED50 value for the selected compound was calculated to determine
the dose required to cause a therapeutic effect in 50% of a population.
In another set of experiment animals were randomized into eight
groups- normal control group, disease control group, mefenamic acid
(6.4 mg/kg, 12.8 mg/kg, 25.6 mg/kg), test compounds treated with 3e
(6.4 mg/kg, 12.8 mg/kg, 25.6 mg/kg). Using the same method paw
edema was induced and calculated. ED50 values were estimated by
back-transforming a linear regression model for percent inhibition as a
function of log (dose).
phenylbenzamide 3a
75% yield, m.p. 205–210 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
8.98 (s, 1 H) 7.78 (s, 1 H) 7.48 (t, J=7.56 Hz, 3 H) 7.29 (t, J=8 Hz, 2 H)
7.16 (t, J=7.96 Hz, 1 H) 7.06 (t, J=8.28 Hz, 2 H) 6.98 (t, J=7.76 Hz,
1 H) 6.88 (d, J=7.32 Hz, 1 H) 6.82 (d, J=8.4 Hz, 1 H) 2.24 (s, 3 H) 2.12
(s, 3 H) 13C NMR (400 MHz, CDCl3) δ PPM: 166.92, 146.63, 138.17,
137.08, 136.69, 131.72, 130.28, 128.09 (2 C), 126.30, 124.97, 124.79,
123.61, 120.43, 119.60 (2 C), 115.82 (2 C), 114.09, 19.63, 12.94. ESI-
MS: m/z=316 [M]+.
2.5.4. The effect of heterocyclic derivative on the peptic ulcer
GI side-effects were evaluated after single-dose administration of
drugs. After one hour of administration of selected compounds, various
parameters were evaluated for gastrointestinal toxicity. All animals
were killed and stomachs were removed, opened along the greater
curvature and rinsed with saline to remove gastric contents and blood
clots. Each stomach was examined grossly and the degree of ulceration
was graded as 0, 0.5, 1, 2, 3, 4, 5 and 6 for no lesions or normal
stomach, hyperemia (red coloration), hemorrhagic spots. Grade 1–5
describes small ulcers, many small ulcers, many small and large ulcers
and stomach full of ulcers with perforations respectively.
3.3.2. Characterization
tolylbenzamide 3b
of
2-(2,3-dimethylphenylamino)-N-p-
81% yield, m.p. 200–205 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
9.04 (s, 1 H) 7.78 (s, 1 H) 7.54 (d, J=7.88 Hz, 1 H) 7.45 (d, J=8.36 Hz,
2 H) 7.22 (d, J=7.12 Hz, 1 H) 7.14 (m, J=7.88 Hz, 3 H) 7.05 (t,
J=7.76 Hz, 1 H) 6.95 (d, J=7.36 Hz, 1 H) 6.89 (d, J=7.84 Hz, 1 H)
6.75(t, J=7 Hz, 1 H) 2.34 (s, 3 H) 2.31 (s, 3 H) 2.19 (s, 3 H) 13C NMR
(400 MHz, CDCl3) δ PPM: 167.91, 147.60, 139.28, 138.11, 135.12,
134.40, 132.65, 131.29, 129.64 (2 C), 127.31, 125.95, 125.82, 121.40,
120.79 (2 C), 117.02, 116.84, 115.10, 20.95, 20.68, 13.99. ESI-MS: m/
z=330 [M]+.
3. Results
3.3.3. Characterization
of
2-(2,3-dimethylphenylamino)-N-(4-
fluorophenyl)benzamide 3c
3.1. Docking studies
48% yield, m.p. 190–195 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
9.18 (s, 1 H) 7.87 (d, J=7.88 Hz, 2 H) 7.15 (d, J=9.68 Hz, 2 H) 7.01 (t,
J=7.68 Hz, 2 H) 6.93 (d, J=7.12 Hz, 2 H) 6.67 (d, J=8.48 Hz, 2 H) 6.56
(t, J=7.44 Hz, 1 H) 5.22 (s, 1 H) 2.255 (s, 3 H) 2.107 (s, 3 H) 13C NMR
(400 MHz, CDCl3) δ PPM: 169.17, 149.44, 138.73, 138.24, 134.19,
To inspect the binding affinities of compounds with the COX-2
enzyme, docking of these compounds utilizing both SYBYL X1.2 and
GOLD 5.2 (PDB ID-4PH9 for mefenamic acid) was performed. Among
3

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
Table 2
Acute toxicity prediction using GUSAR online software.
1
Compound code
R
/X
LD
(mg/kg)
50
Intraperitoneal route
Intravenous route
Oral route
3a
3b
3c
3d
3e
3f
C6H5
4-MeC6H4
4-F-C6H4
2-Cl-C6H4
4-Br-C6H4
N-(2-piperazin−1-yl)ethyl
X=NH
2171 out of AD
2895 out of AD
2206 (Non-toxic)
1852 (Non-toxic)
2289 out of AD
369.6 (Class IV)
277.9 out of AD
757.1 out of AD
130.3 (Class IV)
119.5 (Class IV)
115.6 (Class IV)
77.550 (Class IV)
157.6 (Class IV)
55.330 (Class IV)
72.480 (Class IV)
127.2 (Class IV)
2032 (Class V)
2735 (Class V)
2028 (Class V)
1395 (Class IV)
1931(Class IV)
866.9 (Class IV)
1451 (Class IV)
1724 (Class IV)
4a
4b
X=SH
Out of AD: Compound is out of applicability domain of models
132.46, 131.49 (2 C), 126.76 (2 C), 125.94 (2 C), 123.06 (2 C), 116.07
141.17, 140.67, 138.51, 132.23, 129.85, 126.55, 124.34, 121.04,
120.42, 119.53, 43.76, 47.12, 49.83, 51.66, 34.35, 37.78, 20.78,
14.13. ESI-MS: m/z=352.47 [M]+.
(2 C), 113.69 (2 C), 110.77, 20.68, 14.03. ESI-MS: m/z=334.8 [M]+.
In the second step ring closure was carried out using Con. HCl and
glacial acetic acid as shown in ring closure mechanism is involved as
shown in the Scheme 2.
3.3.4. Characterization
chlorophenyl)benzamide 3d
of
2-(2,3-dimethylphenylamino)-N-(2-
80% yield, m.p. 200–202 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
9.18 (s, 1 H) 7.87 (dd, J=6.52 Hz, 2 H) 7.17 (m, 3 H) 7.15 (m,
J=7.08 Hz, 2 H) 7.08 (d, J=7.6 Hz, 1 H) 7.01 (d, J=7.8 Hz, 2 H) 6.93
(d, J=7.28 Hz, 1 H) 5.22 (s, 1 H) 2.257 (s, 3 H) 2.108 (s, 3 H) 13C NMR
3.4. Physical Characterization of benzimidazole and benzthiazole
derivatives (4a-4b)
(400 MHz, CDCl3)
δ
PPM: 168.11, 148.38, 137.67, 137.18,
133.13(2 C), 131.41, 130.43 (2 C), 125.70 (2 C), 124.88 (2 C), 122.00
(2 C), 115.01 (2 C), 112.63 (2 C), 109.71, 19.62, 12.97. ESI-MS: m/
z=350.1 [M]+.
3.4.1. Characterization of 2-(1H-benzo[d]imidazole-2-yl)-N-(2,3-
dimethylphenyl)benzenamine 4a
68.3% yield, m.p. 190–195 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
10.59 (s, 1 H) 7.827 (m, J=8.28 Hz, 2 H) 7.706 (dd, J=7.92 Hz, 1 H)
7.34 (t, J=7.0 Hz, 1 H) 7.25 (t, J=7.04 Hz, 1 H) 7.20 (d, J=7.84 Hz,
1 H) 7.15 (t, J=7.2 Hz, 1 H) 7.05 (t, J=7.48 Hz, 1 H) 6.93 (m,
J=7.12 Hz, 2 H) 6.76 (t, J=8.28 Hz, 1 H) 5.2 (s 1 H) 2.24 (s, 3 H)
2.09 (s, 3 H) 13C NMR (400 MHz, CDCl3) δ PPM: 169.24, 153.42,
14p5.42, 139.45, 138.23, 134.19, 131.61(2 C), 130.66, 127.70, 126.78
(2 C), 124.68 (2 C), 123.68, 119.82, 118.69, 116.61 (2 C), 115.78,
20.76, 14.24. ESI-MS: m/z=313 [M]+.
3.3.5. Characterization
of
2-(2,3-dimethylphenylamino)-N-(4-
bromophenyl)benzamide 3e
38% yield, m.p. 210–215 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
8.948 (s, 1 H) 6.626 (d, J=7.2 Hz, 1 H) 6.808 (d, J=8.36 Hz, 1 H) 7.22
(d, J=7.12 Hz, 1 H) 7.14 (m, J=7.88 Hz, 3 H) 7.05 (t, J=7.76 Hz, 1 H)
6.95 (d, J=7.36 Hz, 2 H) 6.89 (d, J=7.84 Hz, 1 H) 6.75(t, J=7 Hz, 1 H)
5.18 (s, 1 H)2.34 (s, 3 H) 2.31 (s, 3 H) 13C NMR (400 MHz, CDCl3) δ
PPM: 167.91, 147.60, 139.28, 138.11, 135.12, 134.40, 132.65, 131.29,
129.64 (2 C), 127.31, 125.95, 125.82, 121.40, 120.79 (2 C), 117.02,
116.84, 20.68, 13.99. ESI-MS: m/z=395.3 [M]+.
3.4.2. Characterization
of
2-(1H-benzo[d]thiazol-2-yl)-N-(2,3-
dimethylphenyl)benzenamine 4b
3.3.6. Characterization
(piperazin-1-yl)ethyl)benzamide 3f
of
2-(2,3-dimethylphenylamino)-N-(2-
42.1% yield, m.p. 150–155 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
10.59 (s, 1 H) 7.84 (m, J=7.5 Hz, 2 H) 7.71 (dd, J=7.8 Hz, 1 H) 7.2 (t,
J=7.3 Hz, 1 H) 7.17 (t, J=7.7 Hz, 1 H) 7.08 (d, J=7.3 Hz, 1 H) 6.97 (t,
J=7.6 Hz, 1 H) 6.83 (t, J=7.2 Hz, 1 H) 6.80 (m, J=7.4 Hz, 2 H) 6.54 (t,
J=7.6 Hz, 1 H) 2.25 (s, 3 H) 2.09 (s, 3 H) 13C NMR (400 MHz, CDCl3)
δ PPM: 169.31, 153.40, 146.54, 138.40, 137.25, 134.19, 131.68(2 C),
130.81, 127.45, 126.27 (2 C), 125.47 (2 C), 123.68, 119.81, 118.63,
115.84 (2 C), 115.95, 20.75, 14.15. ESI-MS: m/z=330 [M]+.
73.7% yield, m.p. 150–152 °C, 1H NMR (400 MHz, CDCl3) δ PPM:
9.08 (s, 1 H) 7.71 (d, J=7.2 Hz 1 H) 7.30 (d, J=7.62 Hz, 1 H) 6.89 (m,
J=7.9 Hz, 2 H) 6.41 (d, J=7.12 Hz, 1 H) 6.30 (d, J=7.24 Hz, 2 H) 5.05
(s, 1 H) 4.12(m, J=12.3 Hz, 2 H) 3.89 (m, J=12.8 Hz, 2 H) 3.5 (t,
J=11.8 Hz, 4 H) 3.40 (t, J=12.0 Hz, 4 H) 2.89 (s, 1 H) 2.3 (s, 3 H) 2.22
(s, 3 H) 13C NMR (400 MHz, CDCl3) δ PPM: 169.15, 143.12, 142.56,
Scheme 1. Reagents and conditions: (i) DCC, DMAP, DCM, 15 h stirring.
4

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
NH2
X
(i)
H
NH
N
NH
COOH
O
X
3g:
3h:
X=NH2
X=SH
2g:
2h:
X=NH
X=SH
1
2
(ii)
NH
N
X
4a:
4b:
X=NH
X=S
Scheme 2. Reagents and conditions: (i) DCC, DMAP, DCM, 15 h stirring; (ii) Conc. HCl, Glacial acetic acid, reflux at 80 °C for 2.5 h with stirring.
3.5. Biological screening
3.5.1. In-vitro pharmacological evaluation of heterocyclic derivatives
using colorimetric assay kit
The in-vitro screening results showed that celecoxib exhibited very
weak inhibitory activity against COX- 1 enzyme (4.76%) when com-
pared with mefenamic acid (42.86%). The synthesized compounds 3e
and 3 f exhibited COX-1 inhibitory activity 42.86% and 33.33%
respectively. On the other hand, celecoxib exhibited the superior
inhibitory profile against the COX-2 enzyme (100%) when compared
with mefenamic acid (75.71%) and other investigated compounds 3e
(80.05%) and 3 f (68.78%). However, 3 f has more selectivity towards
COX-2 enzyme and was more than that of mefenamic acid. The COX-2
to COX-1 selectivity ratio of compound 3 f, was found to be 2.063,
while mefenamic acid produced an approximate selectivity ratio of
1.767 (Table 3).
Fig. 1. Anti-inflammatory activity of heterocyclic compounds. aP < 0.05 represents the
significance level when compared with normal control group, bP < 0.01 compared with
disease control group, Each group consists of 6 animals. Values are expressed as Mean
S.E.M, normal control, disease control, disease treated with mefenamic acid(12.85 mg/
kg), 3e-disease treated with 3e (12.85 mg/kg), 3f-disease treated with 3f (12.85 mg/kg).
selected compounds produced a higher inhibition of edema formation
in the carrageenan treated rats as shown in Fig. 1. Their anti-
inflammatory activities were comparable to or higher than that of
mefenamic acid. Compound 3e showed significant anti-inflammatory
activity, was subjected to ED50 determination for further comparison
between different standard drugs. Compound 3e dose-dependently
reduced carrageenan-induced paw edema compared with vehicle-
treated rats, showing an ED50 value of 17.09 mg kg. Mefenamic acid
also reduced paw edema (An ED50 value of 20.17 mg kg) (Table 4).
3.5.2. In-vivo pharmacological evaluation
From the in-vitro evaluation, compounds which showing better
results were subjected for in-vivo pharmacological evaluation. During
the late phase of edema formation, the volume of the carrageenan
injected paw increased rapidly and progressively to reach a maximum
of 58% after 5 h as compared to the normal control group. All the
Table 3
% inhibition and IC50 values of heterocyclic derivatives.
Compound Code % inhibition at 10 µM
Selectivity COX-II IC
50
COX-I
COX-II
3a
3b
3c
3d
3e
3f
4a
4b
38.095
33.333
23.810
19.048
42.857
33.333
38.095
52.381
4.762
13.53
0.355
0.609
1.231
1.243
1.868
2.063
0.651
0.904
21.071
1.269
1.767
1.026
NP
NP
NP
NP
2.131
NP
NP
NP
NP
Table 4
Determination of ED50 Value.
20.294
29.314
23.677
80.05
68.775
24.804
47.353
100.34
78.571
75.714
29.313
S.No
Groups
Dose in mg
Difference in volume
% inhibition
1
2
3
4
5
6
7
Disease Control
Mefenamic acid
Mefenamic acid
Mefenamic acid
Treated with 3e
Treated with 3e
Treated with 3e
–
1.3
100
6.4
0.3
0.55
0.4
1.075
0.875
0.275
15.5
28.1
65.2
17.31
32.7
78.85
12.8
25.6
6.4
12.8
25.6
Celecoxib
Indomethacin
Mefenamic acid
Aspirin
61.905
42.857
28.571
NP
5.342
NP
5

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
3.5.3. Effect of heterocyclic derivative on peptic ulcer
No lesions were found in the rat treated with heterocyclic deriva-
tives at 12.8 mg/kg dose. Similarly, an oral administration of mefe-
namic acid at 12.8 mg/kg dose did not showed incidence and size of
ulcers in treated rats.
COX-1 and COX-2 evaluation for inhibition activities using a COX
colorimetric screening assay method consisting of ovine COX-1 and
human recombinant COX-2 enzymes. The IC50 values of potent
compounds against COX-1 and COX-2 (μM) were calculated from the
concentration inhibition response curve. The outcomes demonstrated
that all the selected compounds were very weak inhibitors of COX-1
contrasted with mefenamic acid except from 4b. All the compounds
were found to be strong inhibitors of COX-2, however, their selectivity
for the COX-2 protein was not as much as that of celecoxib.
Compounds 3e and 3 f had the highest selectivity ratio among all
the investigated compounds. In spite of the fact that the selected
compounds were less potent inhibitors of COX-2 contrasted with
celecoxib.
There has been considerable exertion in the pharmaceutical in-
dustry to recognize a COX-2 selective inhibitor with mitigating proper-
ties, yet without the unfavorable effects related with conventional
NSAIDs (Bansal et al., 2014). In the present study, the heterocyclic
derivatives were subjected to COX-1 and COX-2 evaluation of inhibi-
tion activities using a COX Colorimetric Screening assay kit consisting
of ovine COX-1 and human recombinant COX-2 enzymes. The IC50
values of potent compounds against COX-1 and COX-2 (μM) were
calculated from the concentration inhibition response curve.
Carrageenan-induced rat paw edema is used as a working model of
inflammation in search for new anti-inflammatory drug (Vinegar et al.,
1969). Both the selected compounds produced a higher inhibition of
edema formation in the carrageenan- induced rat paw edema. Their
anti-inflammatory activities were comparable to or higher than that of
mefenamic acid. The results of carrageenan-induced rat paw edema
assay suggest that the vast majority of the recently examined com-
pounds hindered intense periods of inflammation. It can be expected
that they may interact with known mediators of inflammation, for
example, cytokines (Süleyman and Büyükokuroğlu, 2001).
In addition, carrageenan is known to actuate macrophages and
polymorphonuclear cells. These activated cells secrete COX-2 enzyme,
the protein which is responsible for the synthesis of cytokines involved
in inflammation (Li et al., 2005). In this manner, inhibition of COX-2
can add to the mechanism of action underlying the anti-inflammatory
effect of compounds under test. Subsequently authors suggested that
investigated compounds have suppressed the acute stage of inflamma-
tion by hindering the synthesis of PGs by COX-2 inhibition. Various
reviews have proposed that NSAID may assume an essential part in the
etiology of perforated peptic ulcers. Non-steroidal calming drugs
inhibit the synthesis of prostaglandins which have cytoprotective
impacts in the upper gastrointestinal tract, and in addition suppressing
gastric acid secretion. In the present study, the stomach was analyzed
from the animal and scoring was carried out. Mefenamic acid indicated
slight ulcers while none of the newly synthesized compounds demon-
strated any ulcerogenic impact on the gastric mucosa of treated rats.
It has been accounted for that, rather than being exclusively
because of inhibition of gastric protection managed by COX-1, stomach
ulceration brought on by NSAIDs may likewise because of topical
toxicity as an outcome of their physical and chemical characteristics
(Smale and Bjarnason, 2003). Their acidic attributes disrupt the
defensive mucus layer and uncover the underlying cells to the acidic
pH of the stomach. Moreover, acidic NSAIDs may accumulate because
of ion trapping inside intestinal enterocytes achieving concentration
that leads to uncoupling of mitochondrial oxidative phosphorylation
(Krause et al., 2003).
4. Discussion
Cyclooxygenase is a key enzyme responsible for metabolism of
arachidonic acid into several prostaglandins. Prostaglandins formed via
COX-1 activity control renal perfusion, promote platelet aggregation
and provide gastro protection by regulating mucous secretion.
Prostaglandins formed via COX-2 activity mediate pain, inflammation,
fever and inhibit platelet aggregation. COX-2 exclusively expressed in
an inducible way in the sites of inflammation while COX-1 is the source
of the same prostaglandins in the gastric epithelium, where they act as
cytoprotective. Research efforts have focused, on the discovery of
selective COX-2 inhibitors. It was initially believed that this class of
drugs would have reduced gastro-intestinal toxicity compared to
classical NSAIDs (FitzGerald, 2003). Although inhibition of both
COX-1 and COX-2 is generally well tolerated, it is associated with a
wide spectrum of potential clinical toxicities. Many adverse events are
attributed to inhibition of the constitutively expressed COX-1 enzyme,
and some of these appear to be significantly reduced through the use of
COX-2-specific inhibitors (Roth, 1988). However, confusion still
surrounds the role of COX-2 selective inhibitors because of an
increased risk of myocardial infarction and other thrombotic events
(Dogné et al., 2004). The points of interest offered by the utilization of
particular COX-2 inhibitors urged to evaluate the pharmacological
action of novel heterocyclic subordinates that could be potential COX-2
inhibitors and to principally examine their expected side effects. The
introduction of new COX-2 inhibitors comparable in efficacy to
celecoxib, the model medication, with lower adverse reactions would
be a milestone and a point of reference to the improvement of
mitigating treatment, which is our main objective.
Due to the adverse effects connected with COX-2 inhibitors,
researchers have been endeavoring to change the structure of com-
monly used anti-inflammatory agents, especially concentrating on the
acidic group. Empowered by the above perceptions numerous deriva-
tives of mefenamic acid were designed. Diverse amines and in addition,
acids were utilized for the synthesis. Compounds were in anticipation
that these would be more active, potent and of having less toxic effects
than existing drugs.
Molecular docking investigation uncovered that the vast majority of
the compounds displayed the same hydrogen bonding interactions as
exhibited with standard with amino acids like TYR 386, ASN 383, THR
213 etc. An obtained docking score revealed that the designed
compounds showed more selectivity for the COX-2 enzyme.
In silico acute toxicity prediction was performed using GUSAR
software. Most of the compounds were found to be out of the
applicability domain of the models during the acute toxicity study
when considered intraperitoneal route except compounds 3c, 3d and
3 f. An In silico investigation of GUSAR software revealed that
compounds 3d–f and 4a-b are slightly toxic (class 4), whereas
compounds 3a–c are practically nontoxic (class 5).
The synthetic protocol for the target molecules involves a single
step. Mefenamic Acid forms amide bond via DCC/DMAP coupling
using different primary amines. The ring closure reaction involves the
same mechanism as in amide coupling. In the second step ring closure
was carried out using Con. HCl and glacial acetic acid. It was observed
that the yield of the compound with bromo substitution at the 4th
position of the phenyl ring found to be lower as compared to chloro
substitution. It was also observed that the yield of the compound with
methyl substitution at the 4th position of the phenyl ring was found to
be higher as compared to other derivatives.
5. Conclusion
Due to the importance and the emerging use of COX-2 inhibitors,
several researchers have been investigating and trying to solve the
issues of toxicity related to the NSAIDs. The amide derivatives of
mefenamic acid were synthesized and tested for anti-inflammatory
activity. According to docking studies by both Sybyl and GOLD suite,
In the present study, the heterocyclic derivatives were subjected to
6

J.K. Savjani et al.
European Journal of Pharmacology xxx (xxxx) xxx–xxx
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Acknowledgments
The authors thank GUJCOST (No. GUJCOST/MRP/14–15/814)
for providing the financial assistant for this project.
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