Synthesis, anti-inflammatory activities and docking studies of amide derivatives of meclofenamic acid

Article abstract of DOI:10.1007/s11696-016-0102-7

NSAIDs constitute a heterogeneous class of pharmacological agents widely prescribed for the treatment of inflammation, pain and edema, as well as osteoarthritis, rheumatoid arthritis and musculoskeletal disorders. This class of drugs has proved efficacious on account of their analgesic, anti-pyretic and anti-inflammatory activities, but gastrointestinal toxicity exists as the biggest problem associated with their chronic use. Many attempts have been made to structurally modify conventional NSAIDs as selective COX-2 inhibitors based on the old and still prevalent common belief that selective inhibition of COX-2 would provide safer NSAIDs. The present work thus focused on the synthesis of amide derivatives of one of the conventional non-selective NSAID, meclofenamic acid utilizing the one pot procedure involving a selective agent, bis (2-oxo-3-oxazolidinyl) phosphonic chloride. The synthesized compounds were tested for their in vivo inflammatory activity using carrageenan rat paw edema assay, and were subsequently docked on COX-2 PDB code 4COX to have better insights into their mechanism of action. The amide derivative with N-4-methoxybenzyl moiety (TSN4) proved to have anti-inflammatory potential (72.8%) better than meclofenamic acid (56.75%). This compound also docked with the highest dock score among the synthesized compounds and was found to have both hydrogen bonding with Arg120 and Tyr355 and hydrophobic interactions with Val349, Leu352, Ser353, Tyr385, Trp387, Met522, Val523, Ala527 and Ser530. N-4-methoxybenzyl amide derivative (TSN4) followed by benzyl amide derivative (TSN1) of meclofenamic acid were identified as potential anti-inflammatory compounds in both in vivo and in silico studies.

Full text of DOI:10.1007/s11696-016-0102-7

Chem. Pap.
DOI 10.1007/s11696-016-0102-7
ORIGINAL PAPER
Synthesis, anti-inflammatory activities and docking studies
of amide derivatives of meclofenamic acid
Tamanna Narsinghani1 Rajesh Sharma1
•
Received: 30 May 2016 / Accepted: 2 September 2016
Ó Institute of Chemistry, Slovak Academy of Sciences 2016
Abstract NSAIDs constitute a heterogeneous class of
pharmacological agents widely prescribed for the treatment
of inflammation, pain and edema, as well as osteoarthritis,
rheumatoid arthritis and musculoskeletal disorders. This
class of drugs has proved efficacious on account of their
analgesic, anti-pyretic and anti-inflammatory activities, but
gastrointestinal toxicity exists as the biggest problem
associated with their chronic use. Many attempts have been
made to structurally modify conventional NSAIDs as
selective COX-2 inhibitors based on the old and still
prevalent common belief that selective inhibition of COX-
2 would provide safer NSAIDs. The present work thus
focused on the synthesis of amide derivatives of one of the
conventional non-selective NSAID, meclofenamic acid
utilizing the one pot procedure involving a selective agent,
bis (2-oxo-3-oxazolidinyl) phosphonic chloride. The syn-
thesized compounds were tested for their in vivo inflam-
matory activity using carrageenan rat paw edema assay,
and were subsequently docked on COX-2 PDB code 4COX
to have better insights into their mechanism of action. The
amide derivative with N-4-methoxybenzyl moiety (TSN4)
proved to have anti-inflammatory potential (72.8%) better
than meclofenamic acid (56.75%). This compound also
docked with the highest dock score among the synthesized
compounds and was found to have both hydrogen bonding
with Arg120 and Tyr355 and hydrophobic interactions with
Val349, Leu352, Ser353, Tyr385, Trp387, Met522,
Val523, Ala527 and Ser530. N-4-methoxybenzyl amide
derivative (TSN4) followed by benzyl amide derivative
(TSN1) of meclofenamic acid were identified as potential
anti-inflammatory compounds in both in vivo and in silico
studies.
Keywords NSAIDs Á COX Á Meclofenamic acid Á Anti-
inflammatory activity Á Docking
Introduction
The rheumatic disorders are characteristic with high-fre-
quency. Every seventh inhabitant on the Earth has some
rheumatic disorders, in every third family there is a
member with a rheumatic problem. More than 200 rheu-
matic diseases are known. About 300 million people in the
world are only those suffering from arthritis and osteo-
porosis (Stoilov 2008). With over 30 million people taking
daily a non-steroidal anti-inflammatory drug (NSAID),
NSAIDs constitute one of the most used classes of drugs in
modern medicine and are the mainstay in the treatment of
inflammation, pain and edema, as well as osteoarthritis,
rheumatoid arthritis and musculoskeletal disorders.
NSAIDs exert their analgesic, anti-inflammatory and anti-
pyretic effects through inhibition of cyclo-oxygenase
(COX) enzyme, which is involved in the synthesis of
prostaglandins responsible for fever, pain, sensitization and
inflammation. Two isoforms of COX have been identified,
COX-1 and COX-2. COX-1 is constitutively expressed in
most tissues and is believed to generate prostaglandins
(PGs) for normal physiological functions, while COX-2 is
characterized by rapid induction through a variety of
stimuli, including mitogens, hormones, cytokines, and
growth factors (Narsinghani and Sharma 2014). The huge
number of drugs in this class is placed in two major cate-
gories as—the non-selective COX inhibitors and selective
& Rajesh Sharma
rbsm73@yahoo.co.in
1
School of Pharmacy, Devi Ahilya Vishwavidyalaya,
Takshashila Campus, Khandwa Road, Indore, MP, India
123

Chem. Pap.
COX inhibitors (Reddy and Roy 2013). NSAIDs inhibit
both isoforms of COX; but with variations in affinities and
selectivities. The non-selective inhibitors are the older,
conventional, non-selective NSAIDs that inhibits both
isoforms; COX-1 and COX-2 are preferential towards
COX-1. There are no well-established structural reasons
for preferential COX-1 inhibition in conventional NSAIDs.
However, it is an established fact that inhibitors with a
preference for an isoform are generally also ‘‘tight-bin-
ders’’ of that isoform. This tight-binding is time-dependent
and involve two or three steps. In view of this concept of
tight-binding, the conventional NSAIDs are sub-classified
as (1) irreversible inhibitor, such as the time-dependent
inhibitor Aspirin (2) slow reversible (tight-binding) inhi-
bitors also termed as Class I inhibitors which include COX-
1 selective inhibitors like indomethacin and flurbiprofen,
which are competitive inhibitors with slow tight-binding
kinetic profiles (3) time-independent Class II compounds,
such as non-selective, competitive and reversible NSAID
like ibuprofen (4) mixed-type class, Class III which are
neither competitive, reversible nor classic time-dependent
inhibitors but instead are classified as slow reversible
inhibitors, e.g. meloxicam (Khan et al. 2015).
of enhancing the potency while reducing the unwanted GI
side-effects at the same time. The present work thus deals
with the modification of carboxylate moiety of meclofe-
namic acid into its corresponding amide derivatives, eval-
uating their anti-inflammatory potential and performing
docking of these synthesized derivatives on COX-2
enzyme.
Experimental
Physical measurements
Melting point of all the compounds was determined by
open capillary melting point apparatus and is uncorrected.
Thin layer chromatography was performed using prepared
precoated silica gel-G TLC plates. The IR spectra were
recorded on FTIR, Shimadzu-8400S at School of Phar-
macy, DAVV, Indore (MP). Mass spectra of compounds
were obtained on JEOL-AccuTOF JMS-T100LC Mass
Spectrometer having a DART (Direct Analysis in Real
Time) source at Sophisticated Analytical Instrumentation
1
Facility (SAIF), CDRI, Lucknow. H-NMR and 13C-NMR
However, ulceration of the upper gastrointestinal (GI)
tract is a well-known adverse effect of NSAIDs, which is
related to inhibiting PG synthesis in tissues where PGs are
responsible for physiological homeostasis and synthesis of
these gastroprotective PGs is affected by inhibition of
constitutive isoform of COX, i.e. COX-1. Selective inhi-
bition of COX-2 is desirable to avoid gastric side-effects
generally associated with inhibition of COX-1, which is
responsible for gastric mucosal protection.
spectra of compounds were obtained on Bruker DRX-300
(300 MHz FT NMR) at SAIF, CDRI, Lucknow. Elemental
analysis was also carried out using Elementar Vario EL III
Carlo Erba 1108 at SAIF, CDRI, Lucknow.
Synthesis of compounds
All the chemicals were of synthetic grade and were pur-
chased from Aldrich and alfa-aesar. As depicted in
Scheme 1, a reaction mixture containing meclofenamic
acid (3.64 mmol) and BOP-Cl [bis(2-oxo-3-oxazolidinyl)
phosphonic chloride)] (3.64 mmol), in 10 ml of anhydrous
dichloromethane was treated with anhydrous triethylamine
(7.28 mmol) and allowed to stir at room temperature for
5 min. The mixture was then treated with the appropriate
amine (4 mmol) and stirred overnight at room temperature.
Following dilution with dichloromethane (30 mL), the
organic solution was washed with water (2 9 25 mL),
Many attempts have been made to convert older, con-
ventional, non-selective NSAIDs into their corresponding
ester or amide derivatives to confer COX-2 selectivity
(Narsinghani and Sharma 2014). Indomethacin (Kalgutkar
et al. 2000a, b, 2005); ketoprofen (Rajic et al. 2010; Zarghi
and Ghodsi 2010); flurbiprofen (Halen et al. 2006);
naproxen (Sadek et al. 2013), piroxicam (de Miranda et al.
2012); and lonazolac (Ismail et al. 2009) have been suc-
cessfully converted into selective COX-2 inhibitors.
However, the methodology utilized in NSAID modification
does not follow a general scheme. In principle, the strategy
consisted of introducing larger substituents to fit into the
active site volume of COX-2 (Narsinghani and Sharma
2014).
CH3
CH3
3'
Cl
Cl
4'
5'
2'
1'
BOP-Cl, Et3N
RNH2
Sir James Black, winner of the 1988 Nobel Prize in
Physiology or Medicine, said that ‘the most fruitful basis
for the discovery of a new drug is to start with an old drug’
(Raju 2000). Keeping in mind this statement from the
Noble Laureate, the conventional non-selective NSAID
meclofenamic acid was converted into its corresponding
amide derivatives (Kalgutkar et al. 2002) with the objective
NH
O
NH
6'
Cl
1
Cl
COOH
6
5
NHR
2
3
4
Scheme 1 Synthesis of title compounds. BOP-Cl bis(2-oxo-3-oxa-
zolidinyl) phosphonic chloride), Et3N Triethylamine, RNH2 Amine
123

Chem. Pap.
dried (MgSO4), filtered, and the solvent concentrated in
vacuo. The crude amides were chromatographed [ethyl
acetate: n-hexane (10:90)] to get the pure compounds.
129.32 (C-3), 131.33 (C-5), 132.67 (C-60), 134.2 (C-30),
137.3 (C-20), 137.87 (C-10), 146.42 (C-1), 171.42 (CONH);
MS (DART-MS) m/z: 377.13 (M ? H)?; Mol. formula:
S
TSN6: R =
CH2
TSN1: R = -CH2C6H5
TSN2: R = -C6H11
TSN7: R = -CH2C6H4(4-CF3)
TSN8: R = -CH2C6H3(3,4-diCl)
TSN9: R = -CH2-C6H11
CH3
TSN3: R = -CH2(CH2)5CH3
TSN4: R = -CH2C6H4(4-OCH3)
TSN5: R = -CH2C6H4(4-Cl)
TSN10: R =
CH
CH2
OC6H5
N-Benzyl-2-(2,6-dichloro-3-methyl-phenylamino)-
benzamide (TSN1)
C20H22Cl2N2O; Exact mass: 376.11; Elemental analysis for
C20H22Cl2N2O (MW = 377.32) in wt% calc. C = 63.67,
H = 5.88, N = 7.42, O = 4.24 and found to be
C = 63.29, H = 5.95, N = 6.82, O = 4.76.
Yield: 65%; Rf = 0.4; m.p. 105–110 °C; IR (KBr) m
(cm-1): 1645 [C=O(amide)], 1455, 1607 (C=C aromatic),
3308 (N–H); 1H-NMR (300 MHz, CD3OD) d: 2.40 [s, 3H,
CH3], 4.59 [s, 2H, CH2], 6.28–6.31 [d, 1H, ArH],
6.77–6.82 [t, 1H, ArH], 7.16–7.40 [m, 8H, ArH], 7.61–7.63
[dd, 1H, ArH]; 13C-NMR (300 MHz, CD3OD) d: 20.49 (30-
CH3), 43.28 (CH2-benzyl), 115.7 (C-6), 119.5 (C-2), 119.7
(C-4), 126.74 (C-50), 127.54 (C-40), 128.85 (C-200 and 600),
129.09 (C-400), 129.11 (C-300 and 500), 129.26 (C-3), 131.38
(C-5), 131.89 (C-60), 133.14 (C-30), 134.35 (C-20), 137.2
(C-10), 138.1 (C-1), 146.79 (C-100), 172.14 (CONH); MS
(DART-MS) m/z: 385.1 (M ? H)?; Mol. formula: C21-
H18Cl2N2O; Exact mass: 384.08; Elemental analysis for
C21H18Cl2N2O (MW = 385.29) in wt% calc. C = 65.46,
H = 4.71, N = 7.27, O = 4.15 and found to be
C = 64.58, H = 4.50, N = 6.92, O = 4.52.
(2,6-Dichloro-3-methyl-phenylamino)-N-heptyl-
benzamide (TSN3)
Yield: 70%; Rf = 0.48; m.p. 64–70 °C; IR (KBr) m (cm-1):
1650 [C=O(amide)], 1450, 1610 (C=C aromatic), 3330 (N–
H); 1H-NMR (300 MHz, CD3OD) d: 0.86–0.91 [t, 3H,
CH3(C-700)], 1.31–1.38 [m, 9H, CH2 (alkyl side
chain)],1.59–1.66 [m, 2H, CH2 (alkyl side chain)], 2.39 [s,
3H, CH3], 3.35–3.39 [t, 2H, CH2 (C-100)], 6.27–6.29 [d, 1H,
ArH], 6.76–6.81 [m, 1H, ArH], 7.13–7.21 [m, 2H, ArH],
7.32–7.35 [d, 1H, ArH], 7.54–7.57 [dd, 1H, ArH]; 13C-
NMR (300 MHz, CD3OD) d: 14.58 (C-700), 20.78 (30-CH3),
23.81 (C-600), 28.23 (C-300), 30.31 (C-400), 30.69 (C-200),
33.14 (C-500), 40.87 (C-100), 115.53 (C-6), 119.33 (C-2),
119.97 (C-4), 127.6 (C-50), 129.06 (C-40), 129.18 (C-3),
131.27 (C-5), 132.78 (C-60), 134.17 (C-30), 137.25 (C-20),
137.93 (C-10), 146.26 (C-1), 171.95 (CONH); MS (DART-
MS) m/z: 393.26 (M ? H)?; Mol. formula: C21H26Cl2N2-
O; Exact mass: 392.14; Elemental analysis for C21H26Cl2-
N2O (MW = 393.35) in wt% calc. C = 64.12, H = 6.66,
N = 7.12, O = 4.07 and found to be C = 64.83,
H = 7.52, N = 6.99, O = 4.23.
N-Cyclohexyl-2-(2,6-dichloro-3-methyl-
phenylamino)-benzamide (TSN2)
Yield: 68%; Rf = 0.46; m.p. 120–125 °C; IR (KBr) m
(cm-1): 1658 [C=O(amide)], 1452, 1602 (C=C aromatic),
1
3320 (N–H); H-NMR (300 MHz, CD3OD) d: 1.22–1.49
[m, 6H, H (C-300,C-400,C-500)], 1.67–2.00 [m, 5H, H (C-100,
C-200,C-600)], 2.4 [s, 3H, CH3], 6.27–6.29 [d, 1H, ArH],
6.77–6.82 [t, 1H, ArH], 7.15-7.21 [m, 2H, ArH], 7.34–7.36
[d, 1H, ArH], 7.54–7.57 [dd, 1H, ArH]; 13C-NMR
(300 MHz, CD3OD) d: 20.52 (30-CH3), 23.1 (C-300 and 500),
27.5 (C-400), 34.2 (C-200 and 600), 48.1 (C-100), 115.62 (C-6),
119.37(C-2), 119.76 (C-4), 127.68 (C-50), 129.01 (C-40),
(2,6-Dichloro-3-methyl-phenylamino)-N-(4-
methoxybenzyl)-benzamide (TSN4)
Yield: 72%; Rf = 0.35; m.p. 100–110 °C; IR (KBr) m
(cm-1): 1646 [C=O(amide)], 1485, 1610 (C=C aromatic),
123

Chem. Pap.
1
3315 (N–H); H-NMR (300 MHz, CD3OD) d: 2.4 [s, 3H,
126.45 (C-400), 127.48 (C-50), 129.05 (C-40), 129.36 (C-3),
131.45 (C-5), 132.78 (C-60), 134.32 (C-30), 137.15 (C-20),
137.54 (C-10), 141.28 (C-1), 146.56 (C-100), 171.16
(CONH); MS (DART-MS) m/z: 391.09 (M ? H)?; Mol.
formula: C19H16Cl2N2OS; Exact mass: 390.04; Elemental
analysis for C19H16Cl2N2OS (MW = 391.31) in wt% calc.
C = 58.32, H = 4.08, N = 7.16, O = 4.09, S = 8.19 and
found to be C = 58.14, H = 4.94, N = 6.54, O = 4.25,
S = 5.86.
CH3], 3.78 [s, 3H, OCH3], 4.52–4.58 [d, 2H, CH2],
6.27–6.30 [d, 1H, ArH], 6.7-6.81[t, 1H, ArH], 6.88–6.91
[d, 2H, ArH], 7.17–7.23 [m, 2H, ArH], 7.30–7.37 [m, 3H,
ArH], 7.58–7.61 [dd, 1H, ArH]; 13C-NMR (300 MHz,
CD3OD) d: 20.65 (30-CH3), 44.82 (CH2-benzyl), 54.21
(OCH3), 114.24 (C-300 and 500), 115.21 (C-6), 119.12 (C-2),
119.67 (C-4), 126.65 (C-50), 129.07 (C-40), 129.23 (C-200
and 600), 129.32 (C-3), 131.23 (C-5), 131.88 (C-60), 133.10
(C-30), 134.25 (C-20), 137.12 (C-10), 138.21 (C-100), 146.34
(C-1), 158.15 (C-400), 172.02 (CONH); MS (DART-MS)
m/z: 415.15 (M ? H)?; Mol. formula: C22H20Cl2N2O2;
Exact mass: 414.09; Elemental analysis for C22H20Cl2N2-
O2 (MW = 415.31) in wt% calc. C = 63.62, H = 5.16,
N = 6.75, O = 7.7 and found to be C = 63.37, H = 5.61,
N = 6.19, O = 7.85.
2-(2,6-Dichloro-3-methyl-phenylamino)-N-4-
trifluoromethyl-benzyl)-benzamide (TSN7)
Yield: 77%; Rf = 0.4; m.p. 130–144 °C; IR (KBr) m
(cm-1): 1635 [C=O(amide)], 1495, 1607 (C=C aromatic),
1
3315 (N–H); H-NMR (300 MHz, CD3OD) d: 2.4 [s, 3H,
CH3], 4.67 [s, 2H, CH2], 6.28–6.31 [d, 1H, ArH] 6.79–6.83
[t, 1H, ArH], 7.17–7.24 [m, 4H, ArH], 7.34–7.37 [d,1H,
ArH], 7.56–7.67[m, 4H, ArH]; 13C-NMR (300 MHz,
CD3OD) d: 20.75 (30-CH3), 43.86 (CH2-benzyl), 115.55
(C-6), 119 (C-2), 119.3 (C-4), 124.42 (CF3), 125.08 (C-300
and 500), 126.54 (C-50), 127.58 (C-200 and 600), 129.06 (C-40),
129.19 (C-4000), 129.31 (C-3), 131.59 (C-5), 133.23 (C-60),
134.49 (C-3 ), 137.16 (C-20), 138.01 (C-10), 145.19 (C-1),
146.79 (C-100), 172.05 (CONH); MS (DART-MS) m/z:
453.14 (M ? H)?; Mol. formula: C22H17Cl2F3N2O; Exact
mass: 452.07; Elemental analysis for C22H17Cl2F3N2O
(MW = 453.28) in wt% calc. C = 58.29, H = 3.78,
N = 6.18, O = 3.53 and found to be C = 57.46,
H = 4.75, N = 6.01, O = 3.67.
N-(4-chlorobenzyl)-(2,6-dichloro-3-methyl-
phenylamino)-benzamide (TSN5)
Yield: 67%; Rf = 0.4; m.p. 98–112 °C; IR (KBr) m (cm-1):
1652 [C=O(amide)],1465, 1605 (C=C aromatic), 3325 (N–
1
H); H-NMR (300 MHz, CD3OD) d: 2.40 [s, 3H, CH3],
4.56 [s, 2H, CH2], 6.28–6.30 [d, 1H, ArH], 6.77–6.82 [t,
1H, ArH], 7.17-7.23 [m, 2H, ArH], 7.32–7.39 [m,5H,
ArH], 7.61–7.63 [dd, 1H, ArH]; 13C-NMR (300 MHz,
CD3OD) d: 20.47 (30-CH3), 44.12 (CH2-benzyl), 115.12
(C-6), 119.02 (C-2), 119.34 (C-4), 125.14 (C-50), 126.22
(C-40), 129.1 (C-300 and 500), 129.23 (C-200 and 600), 129.42
(C-3), 131.62 (C-5), 131.75 (C-60), 133.23 (C-400), 134.65
(C-30), 136.51 (C-20), 137.12 (C-10), 138.15 (C-100), 146.57
(C-1), 171.23 (CONH); MS (DART-MS) m/z: 419.16
(M ? H)?; Mol. formula: C21H17Cl3N2O; Exact mass:
N-(3,4-dichlorobenzyl)-2-(2,6-dichloro-3-methyl-
phenylamino)-benzamide (TSN8)
418.04;
Elemental
analysis
for
C21H17Cl3N2O
(MW = 419.73) in wt% calc. C = 60.09, H = 4.85,
N = 6.67, O = 3.81 and found to be C = 59.26,
H = 5.08, N = 6.12, O = 3.92.
Yield: 70%; Rf = 0.4; m.p. 144–148 °C; IR (KBr) m
(cm-1): 1640 [C=O(amide)], 1469, 1613 (C=C aromatic),
1
3331 (N–H); H-NMR (300 MHz, CD3OD) d: 2.4 [s, 3H,
CH3], 4.55–4.57 [s, 2H, CH2], 6.28–6.31 [d, 1H, ArH],
6.78–6.83 [t,1H, ArH], 7.17–7.24 [m, 2H, ArH], 7.31–7.37
[m, 2H,ArH], 7.47–7.54 [m, 2H, ArH], 7.62–7.65 [dd, 1H,
ArH]; C-NMR (300 MHz, CD3OD) d: 20.23 (30-CH3),
2-(2,6-dichloro-3-methyl-phenylamino)-N-thiophen-
2-yl-methyl-benzamide (TSN6)
13
Yield: 75%; Rf = 0.33; m.p. 128–132 °C; IR (KBr) m
(cm-1): 1648 [C=O(amide)], 3083 (C-H str. in thiophene),
1470, 1600 (C=C aromatic), 3310 (N–H); 1H-NMR
(300 MHz, CD3OD) d: 2.4 [s, 3H, CH3], 4.74 [s, 2H, CH2],
6.27–6.30 [d, 1H, ArH], 6.76–6.81 [t, 1H, ArH], 6.94–6.97
[m, 1H, ArH], 7.06-7.07 [m, 1H, ArH], 7.17–7.23 [m, 2H,
ArH], 7.28–7.30 [dd, 1H, ArH], 7.34–7.37 [d, 1H, ArH],
7.56–7.59 [dd, 1H, ArH]; 13C-NMR (300 MHz, CD3OD)
d: 20.34 (30-CH3), 42.62 (CH2-thiophenyl), 115.12 (C-6),
119.12 (C-2), 119.55 (C-4), 123.23 (C-300), 125.72 (C-500),
44.65 (CH2-benzyl), 115.09 (C-6), 119.12 (C-2), 119.54
(C-4), 126.36 (C-50), 127.52 (C-40), 128.15 (C-600), 128.89
(C-200), 129.06 (C-3), 129.18 (C-500), 130.65 (C-400), 131.45
(C-5), 133.48 (C-300), 134.78 (C-60), 135.42 (C-30), 136.78
(C-20), 137.28 (C-10), 138.36 (C-1), 146.68 (C-100), 171.18
(CONH); MS (DART-MS) m/z: 453.07 (M ? H)?; Mol.
formula: C21H16Cl4N2O; Exact mass: 452; Elemental
analysis for C21H16Cl4N2O (MW = 454.18) in wt% calc.
C = 55.53, H = 3.55, N = 6.17, O = 3.52 and found to
be C = 55.8, H = 3.90, N = 6.01, O = 3.8.
123

Chem. Pap.
N-Cyclohexylmethyl-2-(2,6-dichloro-3-methyl-
phenylamino)-benzamide (TSN9)
Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Social Justice and Empowerment,
Government of India.
Yield: 75%; Rf = 0.5; m.p. 134–142 °C; IR (KBr) m
(cm-1): 1655 [C=O(amide)], 1450, 1604 (C=C aromatic),
3318 (N–H); 1H-NMR (300 MHz, CD3OD) d: 1.155–1.366
[m, 6H, H(C-300, C-400, C-500)], 1.595–1.855 [m, 5H, H(C-
100,C-200,C-600)], 2.4 [s, 3H, CH3], 3.21-3.23 [s, 2H, CH2],
6.27–6.29 [d, 1H, ArH], 6.77–6.82 [t, 1H, ArH], 7.16–7.21
[m, 3H, ArH], 7.34–7.36 [d, 1H, ArH], 7.54–7.57 [d, 1H,
Male or female Sprague–Dawley rats with a body
weight between 100 and 150 g were used. To insure uni-
form hydration, the rats received 5 ml of water by stomach
tube (controls) or the test drug dissolved or suspended in
the same volume. Thirty minutes later, the rats were
challenged by a subcutaneous injection of 0.05 ml of 1%
solution of carrageenan into the plantar side of the left hind
paw. The paw volume was measured plethysmographically
(LE7500 Digital Plethysmometer, Panlab Harvard Appa-
ratus) immediately after injection, again 1, 2, 3 h after
challenge and compared with the initial paw volume of
each rat for determining the edema volume (Winter et al.
1962). The difference between the initial and subsequent
paw volume reading gave the actual edema volume. The
percent inhibition of inflammation/protection was calcu-
lated using the formula:
13
ArH]; C-NMR (300 MHz, CD3OD) d: 20.22 (30-CH3),
23.7 (C-300 and 500), 26.8 (C-400), 34.25 (C-200 and 600), 42.75
(C-100), 47.91 (CH2-cyclohexyl), 115.23 (C-6), 119.29 (C-
2), 119.64 (C-4), 127.85 (C-50), 129.12 (C-40), 129.35 (C-
3), 131.09 (C-5), 132.45 (C-60), 134.21 (C-30), 137.44 (C-
20), 137.97 (C-10), 146.12 (C-1), 171.45 (CONH); MS
(DART-MS) m/z: 391.24 (M ? H)?; Mol. formula: C21-
H24Cl2N2O; Exact mass: 390.13; Elemental analysis for
C21H24Cl2N2O (MW = 391.33) in wt% calc. C = 64.45,
H = 6.18, N = 7.16, O = 4.09 and found to be C = 64.4,
H = 8.05, N = 6.96, O = 4.3.
ðVt À V0Þ
À ðVt À V0ÞtreatedÂ100
coðntVrotl À V0Þ
where Vt represents paw volume at given time ‘t’; V0: paw
%inhibition ¼
control
(–)-2-(2,6-dichloro-3-methyl-phenylamino)-N-(2-
phenoxy-propyl)-benzamide (TSN10)
volume before giving any treatment.
Statistical analysis
Yield: 70%; Rf = 0.43; m.p. 110–118 °C; IR (KBr) m
(cm-1): 1660 [C=O(amide)], 1469, 1604 (C=C aromatic),
1
Anti-inflammatory activity was expressed as mean paw
volume standard error of the mean (SEM) and
mean SEM percentage inhibition of edema in treated
animals in comparison with the control group. The statis-
tical significance of differences between control and treated
groups was determined by ANOVA, followed by Dunnett’s
test. A value of p \ 0.05 was considered to be statistically
significant.
3316 (N–H); H-NMR (300 MHz, CD3OD) d:1.26-1.36 [t,
3H, CH3 of propyl], 2.4 [s, 3H, CH3], 3.58–3.61 [s, 2H,
CH2 of propyl], 4.67–4.73 [m, 1H, O–CH of propyl],
6.26–6.28 [d, 1H, ArH], 6.73–6.78 [m, 1H, ArH],
6.86–6.91 [t,1H, ArH], 6.97–7.00 [d, 2H, ArH], 7.16–7.26
[m, 4H, ArH], 7.34–7.36 [d, 1H, ArH], 7.46–7.49 (dd, 1H,
13
ArH]; C-NMR (300 MHz, CD3OD) d: 20.46 (30-CH3),
21.12 (CH3 of propyl), 47.92 (CH2 of propyl), 74.92 (O–
CH of propyl), 114. 6 (C-200 and 600), 115.21 (C-6), 119.42
(C-2), 119.65 (C-4), 121.5 (C-400), 127.57 (C-50), 129.08
(C-40), 129.17 (C-3), 129.33 (C-300 and 500), 131.24 (C-5),
133.23 (C-60), 134.46 (C-30), 137.18 (C-20), 138.49 (C-10),
146.62 (C-1), 158.12 (C-100), 171.12 (CONH); MS (DART-
MS) m/z: 429.16 (M ? H)?; Mol. formula: C23H22Cl2N2-
O2; Exact mass: 428.11; Elemental analysis for C23H22-
Cl2N2O2 (MW = 429.34) in wt% calc. C = 63.62,
H = 4.85, N = 6.75, O = 7.7 and found to be C = 64.21,
H = 5.85, N = 6.15, O = 7.89.
Docking of synthesized compounds on COX-2
enzyme
Docking studies were performed using Surflex Dock
module of SYBYL X 2.1.1 (Tripos Inc, St. Louis, MO,
USA). The title compounds were docked on COX-2 PDB
ID 4COX (COX-2 in complex with indomethacin); reso-
˚
lution 2.9 A. This COX-2 enzyme is a dimer and it is
reported that COX-2 is functionally active as a monomer,
and thus docking was carried out on chain A of the
enzyme. The protomol was generated by extracting the
ligands, removing all water molecules and other co-factors.
In addition to this, polar hydrogen atoms and
AMBER7FF99 charges were added. Generation of proto-
mol was carried out by keeping the threshold and bloat
values as 0.5 and 1, respectively. The synthesized com-
pounds and the standard NSAIDs tested for in vivo anti-
Anti-inflammatory activity
Carrageenan rat paw edema assay
The anti-inflammatory activity was performed using car-
rageenan rat paw edema assay as per the guidelines and
rules laid down by the Committee for the Purpose of
123

Chem. Pap.
inflammatory activities in present study were subjected to
docking on this validated protomol generated from COX-2
PDB 4COX. Finally, the docked ligands were ranked on
the basis of various scoring functions, primarily single
consensus score (C-Score) reporting the output of total
score (Raghavendra et al. 2012).
addition to these spectral data, elemental analysis was also
in good agreement with the reported values.
Anti-inflammatory activity
All the synthesized compounds were evaluated for their
anti-inflammatory activity by carrageenan rat paw edema
assay at the dose of 14 mg/kg along with the standard
drugs meclofenamic acid and indomethacin (Fig. 1). Two
derivatives, TSN4 and TSN1 were found to display high
potency in protection of carrageenan induced inflammation
at 1, 2 and 3 h. of inflammation (p \ 0.001). Compound
TSN4 was the most active compound of the series with
percentage inhibition of 72.8% as compared to meclofe-
namic acid (56.75% inhibition) and comparable to indo-
methacin (75.15%). It was followed by TSN1 (70.35%),
TSN3 (66.55%) and TSN10 (63.75%). TSN7 (54.28%)
showed comparable percentage inhibition of edema to
meclofenamate (56.75%), while TSN8 (15.56%) was
observed to be least active among the series of synthesized
compounds.
Results and discussion
Chemistry
The synthesis of the title compounds was carried out by
converting the carboxylate moiety of meclofenamic acid to
amide functionality utilizing the one pot procedure
involving
a
selective agent, bis(2-oxo-3-oxazo-
lidinyl)phosphonic chloride (BOP-Cl) serving as the car-
boxylic acid activator (Diago-Meseguer and Palomo-Coll
1980). The desired derivatives were prepared by treating
the mixture of acid and BOP-Cl in anhydrous dichlor-
omethane with appropriate amine in the presence of BOP-
Cl and triethylamine. The reaction was monitored by per-
forming TLC using ethyl acetate: n-hexane as the mobile
phase (2:8). This one pot procedure afforded the desired
compounds in 60–75% yield. The pure compounds
obtained after column chromatography were characterized
Analysis of structure–activity relationship of synthe-
sized compounds revealed that the electron-releasing group
substituted on the para position of benzyl group attached to
amide nitrogen of TSN4 resulted in most significant anti-
inflammatory activity (p \ 0.001) when compared to the
control group. The second most promising among the
synthesized compounds was TSN1 with N-benzyl group
linked to the amide functionality (p \ 0.001). Substitution
of electron withdrawing groups resulted in decreased anti-
inflammatory activity when compared to TSN4 and TSN1,
as evident from comparable percentage inhibition of TSN8
with 4-trifluoromethyl group with meclofenamate and loss
of activity in TSN5 and TSN8 with 4-chloro and 3,4-
dichloro groups. In addition to this, substitution of alkyl
1
by FTIR, H-NMR, 13C-NMR and mass spectral analysis.
The C=O str. in amides was observed between
1635–1660 cm-1 and N–H str. was seen between 3308 and
3331 cm-1. 1H-NMR spectra depicted methyl signal at 2.4
d and aromatic protons between 6.27 and 7.67. In 13C-
NMR spectra, methyl signal was seen at 20 d while the
carbonyl carbon was observed at 171 d. Mass spectra of all
the synthesized compounds depict the presence of
[M ? H]? along with isotopic peaks for chlorine. In
Fig. 1 Acute anti-inflammatory
effect of tested amide
derivatives (TSN1-TSN10) on
carrageenan induced rat paw
edema, in comparison with
standard. Data shown are means
(n = 6) SEM, *p \ 0.05,
TSN1
TSN2
TSN3
TSN4
TSN5
TSN6
TSN7
TSN8
TSN9
80
ns
60
40
20
0
**p \ 0.01, all unmarked
***p \ 0.001, ns: p [ 0.05
when compared with control
**
ns
**
**
*
TSN10
Meclofenamic acid
Indomethacin
ns
*
*
1
2
3
Time (in hrs.)
123

Chem. Pap.
chain in the form of TSN3 (heptyl amide) also resulted in
potent anti-inflammatory activity when compared to
meclofenamic acid.
group in between cyclohexyl and amide nitrogen (TSN9),
there was a fall in activity in comparison to TSN2.
Results of biological evaluation highlighted the signifi-
cant anti-inflammatory activity of compounds TSN4, TSN1
followed by TSN3 and TSN10 predominantly at second
and third hour of inflammation (Fig. 1), i.e. in the second
phase of carrageenan induced inflammation, which is
sensitive to drugs like indomethacin (Vinegar et al. 1969).
Furthermore, on substituting 2-phenoxy propyl group to
amide moiety (TSN10), significant reduction in edema was
observed with respect to control group, but the reduction
was lower than TSN4, TSN1 and TSN3. Heterocyclic ring
in the form of thiophene moiety (TSN6) was also tried and
this compound displayed moderate anti-inflammatory
activity (44.4%) in comparison to meclofenamate and
indomethacin. When saturated cyclic ring (cyclohexyl) was
attached directly to amide nitrogen, percentage inhibition
of inflammation decreased and upon inserting a methylene
Molecular docking
To gain insights into the anti-inflammatory activities of
synthesized compounds and correlate the in vivo results to
the structure of title compounds, molecular docking was
carried out on COX-2 PDB ID 4COX. The docking
methodology adopted was validated by redocking the
extracted ligand (indomethacin) and overlapping the
docked and co-crystallized ligands. The two ligands over-
lapped in the same position (Fig. 2).
The newly synthesized compounds (TSN1-TSN10)
docked and occupied the same binding site as that of
celecoxib, indomethacin and meclofenamic acid (Fig. 3).
Figure 3 depicts the overlapped view of ball and stick
models of TSN1, TSN4 and standards.
Indomethacin was shown to have hydrogen bond inter-
actions between its oxygen of benzoyl moiety and hydroxyl
˚
oxygen of Ser530 (2.02 A), and second, oxygen atom of
˚
acidic group and hydrogen atom of Arg120 (2 A). Cele-
coxib displayed two hydrogen bond interactions, viz. one
between oxygen of sulfonamide group and hydrogen atom
˚
of Ser530 (2.73 A) and the other between fluorine atom of
Fig. 2 Comparison of redocking results of ligand (indomethacin) to
X-ray crystallographic mode of binding (model coloured green
represent docked ligand while model coloured orange is experimen-
tally verified binding pose) (colour figure online)
trifluoromethyl group and hydrogen atom of Arg120 (2.45
˚
A). The nitrogen atom of amide group of TSN4 formed
Fig. 3 Overlap of ball and stick
models of docked compounds:
TSN1 (red–orange colour),
TSN4 (magenta colour),
Celecoxib (cyan colour),
Indomethacin (green colour)
and meclofenamic acid (blue
colour) displaying hydrogen
bond interactions (yellow
coloured dotted lines) with
amino acids in the active site of
COX-2, PDB Code 4COX
(COX-2 co-crystallized with
indomethacin) (colour
figure online)
123

Chem. Pap.
Fig. 4 Binding map of top
scoring compound (TSN4) on
COX-2 (4COX); yellow
coloured dotted lines indicate
hydrogen bonding interactions
along with the distances, black
dotted lines indicate
V349
L352
hydrophobic interactions
(colour figure online)
S353
H
N
OCH3
C
O
Cl
N
H3C
H
W387
Y385
Cl
2.44 Å
1.85 Å
Y355
M522
R120
V523
A527
S530
hydrogen bond (Fig. 4) with hydroxyl oxygen of Tyr355
˚
(2.44 A) while the carbonyl oxygen of amide group of this
While analysing the hydrophobic interactions of docked
compounds (Fig. 5), it was seen that amino acids, viz.
Val349, Leu352, Ser353, Tyr385, Trp387, Met522, Val523
and Ala527 were enclosing TSN4, TSN1, celecoxib,
indomethacin and meclofenamic acid.
compound was involved in hydrogen bonding with
˚
hydrogen of Arg120 (1.85 A). Meclofenamic acid dis-
played three hydrogen bond interactions with Met522 (1.85
˚
A), Val523 (2.61 A) and Ala527 (2.5 A).
˚
˚
The amide moiety and the N-4-methoxybenzyl and N-
benzyl groups of TSN4 (Figs. 4, 6a) and TSN1 (Fig. 6b),
respectively, are seen in close vicinity to Val349, Leu352
and Ser353 similar to indomethacin. Indomethacin is
known to place its indoly-2-methyl group at alpha position
that inserts itself in lipophilic pocket above Val349 to form
a strong inhibitor-enzyme complex (Kurumbail et al. 1996;
Prusakiewicz et al. 2004) as depicted in Fig. 6c. A variety
of COX inhibitors are known to have hydrophobic inter-
actions with these amino acids (Fig. 6d). Interactions of
title compounds with these residues are in good agreement
with biological activity.
Research findings have reported that interaction between
Arg120 and the carboxylate moiety of arachidonic acid is the
fundamental requirement for COX reaction to happen
(Bhattacharyya et al. 1996; Malkowski et al. 2000). The most
active compound (TSN4) is also displaying hydrogen bond
interaction with Arg120. Another important residue known
to bind carboxylate of arachidonate through phenolic
hydroxyl is Tyr355, which plays a structural role in COX
inhibition by various NSAIDs (Rowlinson et al. 2003). Our
most potent compound (TSN4) is also involved in hydrogen
bonding with phenolic hydroxyl of Tyr355 (Fig. 4).
123

Chem. Pap.
Fig. 5 Hydrophobic amino
acids (yellow coloured space
fill) enclosing ball and stick
models of docked compounds:
TSN1 (red–orange colour),
TSN4 (magenta colour),
Celecoxib (cyan colour),
Indomethacin (green colour)
and meclofenamic acid (blue
colour) displaying hydrogen
bond interactions with amino
acids in the active site of COX-
2, PDB Code 4COX (COX-2
co-crystallized with
indomethacin) (colour
figure online)
Fig. 6 Binding poses of ball
and stick models of a TSN1,
b TSN4, c indomethacin and
d meclofenamic acid complexed
with COX-2 enzyme showing
hydrogen bonding (yellow
dotted lines) and hydrophobic
interactions (colour
figure online)
123

Chem. Pap.
Chem Score
Table 1 Surflex-Dock GeomX score of synthesized compounds and standards
Compd
C-Score
Crash Score
Polar Score
G Score
PMF Score
D Score
Indomethacina
TSN4
8.33
6.43
5.86
5.35
5.28
5.09
4.93
4.33
4.31
4.31
4.29
4.23
2.69
-1.61
-3.49
-4.22
-0.69
-3.66
-3.54
-3.74
-4.55
-5.98
-1.69
-4.58
-6.24
-6.31
1.08
1.07
0.00
0.00
0.00
0.04
1.16
1.22
0.05
0.97
2.59
0.61
1.05
-240
-292
-303
-189
-327
-301
-265
-292
-320
-197
-263
-333
-319
-67.7
-63.6
-77.9
-60.0
-45.7
-87.6
-99.4
-57.2
-40.2
-53.7
-62.1
-56.8
-57.9
-172
-171
-161
-461
-172
-179
-156
-170
-170
-114
-147
-186
-193
-37.2
-45.5
-40.9
-33.4
-42.3
-48.6
-41.4
-45.5
-42.1
-35.0
-39.8
-46.9
-50.3
TSN1
Celecoxibb
TSN3
TSN10
TSN7
TSN6
TSN2
Meclofenamic acid
TSN9
TSN5
TSN8
a
Extracted co-crystallized ligand (4COX)
Extracted co-crystallized ligand (3LN1)
b
Table 2 Summary of results of docking analysis and in vivo anti-inflammatory activity
Compd.
Dock
%
Interacting amino
˚
HB:HA
Residues involved in hydrophobic interactions
Score inhibition acids (HBD A)
(3 h)
TSN1
TSN2
5.86
4.31
70.35
27.08
–
–
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527, Ser530
˚
Tyr355 (2.32 A)
Tyr355 (OH):O::Lig(H):NH
Val349, Leu352, Ser353, Tyr385, Trp387,
Met522, Val523, Ala527
(anthranilic acid)
–
TSN3
TSN4
5.28
6.43
66.55
72.80
–
Val349, Leu352, Ser353, Tyr355, Leu384,
Trp387, Met522, Val523, Ala527, Ser530
˚
Arg120 (1.85 A)
Arg120(NH2):H::Lig(O):CONH Val349, Leu352, Ser353, Tyr385, Trp387,
Met522, Val523, Ala527, Ser530
Tyr355 (OH):O::Lig(H):NH
(anthranilic acid)
˚
Tyr355 (2.44 A)
TSN5
TSN6
TSN7
4.23
4.33
4.93
18.91
44.4
–
–
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527
˚
Arg120 (1.9 A)
Arg120
(NH2):H::Lig(O):CONH
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527, Ser530
˚
Arg120 (1.82A)
54.28
Arg120
(NH2):H::Lig(O):CONH
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527
˚
Tyr355 (2.43 A)
Tyr355 (OH):O::Lig(H):NH
(anthranilic acid)
˚
Ser530 (2.32 A)
Ser530(CH2OH):H::Lig(F):CF3
˚
Arg120 (1.94A)
TSN8
TSN9
2.69
4.29
15.56
25.02
Arg120
(NH2):H::Lig(O):CONH
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Val523, Ala527
Arg120 (1.82 and Arg120 (NH2):HH12 and
HH22::Lig(O):CONH
Val349, Leu352, Ser353, Trp387,
Met522,Val523, Ala527
˚
2.31 A)
˚
Tyr355 (2.53 A)
Tyr355 (OH):O::Lig(H):NH
(anthranilic acid)
˚
Arg120 (3 A)
TSN10
5.09
63.75
Arg120 (NH2):H::Lig(O):2-
phenoxy-propyl
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527, Ser530
123

Chem. Pap.
Table 2 continued
Compd.
Dock
%
Interacting amino
˚
HB:HA
Residues involved in hydrophobic interactions
Score inhibition acids (HBD A)
(3 h)
˚
Met522 (1.85 A)
Meclofenamic 4.31
acid
56.75
Met522 (COOH)
O::Lig(H):COOH
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Ser530
˚
Val523 (2.61 A)
Val523
(COOH):O::Lig(H):COOH
˚
Ala527 (2.5 A)
Ala527 (NH2) H::Lig(O):O
attached to acidic proton
Indomethacina 8.33
75.15
Arg120
(NH2):H::Lig(O):COOH
Val349, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Met522, Val523, Ala527
˚
Arg120 (2 A)
˚
Ser530 (2.02 A)
Ser530
(CH2OH):H::Lig(O):C=O
(benzoyl)
a
Extracted co-crystallized ligand (4COX) used as reference structure to validate the docking method
Table 1 summarizes the results obtained from docking
in the form of various scores. TSN6, TSN2, TSN9, TSN5
and TSN8 were having crash scores (the degree of inap-
propriate penetration into the protein) [-4.5 kcal/mol,
suggesting inappropriate penetration into the binding site
of COX-2 enzyme. Compounds TSN3, TSN10 and TSN7
were having better C-Scores as is evident from their good
anti-inflammatory activities comparable to meclofenamate.
G-score values indicating hydrogen bonding, complex
(ligand–protein), and internal (ligand–ligand) energies
were better for TSN1 and TSN4 than celecoxib, indo-
methacin, meclofenamic acid and other synthesized com-
pounds. TSN1 was preferred over TSN4 on the basis of
Helmholtz free energies of interactions for protein–ligand
atom pairs (Potential of Mean Force, PMF Scores). In
addition to this, TSN7 and TSN10 also displayed signifi-
cant PMF Scores. D-Score values indicating charge and
van der Waals interactions between the protein and the
ligand were better for celecoxib but TSN4 and TSN1 were
having better D-Scores over meclofenamic acid. Chem-
score points for hydrogen bonding, lipophilic contact,
rotational entropy, along with an intercept term (Chem
Score) places TSN4 above TSN1 and the standard
NSAIDs. Polar Score represents the contribution of
hydrogen bonding to the total score. The tabulated scoring
functions are in favour of indomethacin, followed by TSN4
and TSN1, celecoxib, TSN3, TSN10, TSN7, and finally
meclofenamic acid.
Conclusions
To summarize and conclude the present findings,
reported herein is the synthesis of amide derivatives of
meclofenamic acid, their in vivo biological evaluation
and subsequent docking on COX-2 enzyme. The in vivo
studies of screened compounds revealed the promising
anti-inflammatory activity of compound TSN4 with N-
(4-methoxybenzyl) group followed by TSN1 with N-
benzyl group. The results obtained from docking analy-
sis are in good correlation with anti-inflammatory
activity of title compounds. Further insights into the
structures of docked complexes revealed hydrogen bond
and hydrophobic interactions with the crucial residues
(Arg120, Val329, Leu352, Ser353, Tyr355, Tyr385,
Trp387, Val523, Ala527 and Ser530), reported for COX-
2 inhibition. Both the in silico and in vivo studies have
placed compound TSN4 on the top followed by TSN1.
These two compounds thus can be further explored to
improve their anti-inflammatory activity and safety
profile.
Acknowledgements The authors are thankful to UGC and AICTE
for the financial assistance and to the Head, SAIF, CDRI, Lucknow
for providing mass, 1H, 13C NMR spectral data and elemental analysis
data.
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