Cyclic enaminone as new chemotype for selective cyclooxygenase-2 inhibitory, anti-inflammatory, and analgesic activities

Article abstract of DOI:10.1016/j.ejmech.2019.111601

The cyclic enaminone moiety has been identified as a new scaffold for selective inhibition of cyclooxygenase-2 with anti-inflammatory and analgesic activities. The designed cyclic enaminones have been synthesized conveniently through the development of a new catalyst-free methodology and evaluated for cyclooxygenase (COX-1 and COX-2) inhibitory activities. Three compounds 7d, 8, and 9 predominantly inhibited COX-2 with selectivity index of 74.09, 19.45 and 108.68, respectively, and were assessed for in vivo anti-inflammatory activity in carrageenan induced rat paw edema assay. The anti-inflammatory activity of 7d was comparable to that of celecoxib at a dose of 12.5 mg/kg. However, the compounds 8 and 9 were more/equally effective as anti-inflammatory agent compared to celecoxib at the doses of 12.5 mg/kg and 25 mg/kg and also exhibited anti-inflammatory activity comparable to that of diclofenac. The therapeutic potential of the most active compound 9 was further assessed by performing in vivo thermal and mechanical hyperalgesia tests using various models that revealed its analgesic activity. The in vivo non-ulcerogenicity of 9 revealed the gastrointestinal safety as compared to the non-selective COX inhibitor indomethacin. The in vitro antioxidant activity and in vivo experiments on heart rate and blood pressure provided the cardiovascular safety profile of 9. The molecular docking studies rationalize the COX-2 selectivity of the newly found anti-inflammatory compounds 7d, 8, and 9.

Full text of DOI:10.1016/j.ejmech.2019.111601

Journal Pre-proof
Cyclic enaminone as new chemotype for selective cyclooxygenase-2 inhibitory, anti-
inflammatory, and analgesic activities
Raj Kumar, Nirjhar Saha, Priyank Purohit, Sanjeev K. Garg, Kapileswar Seth, Vachan
S. Meena, Sachin Dubey, Khyati Dave, Rohit Goyal, Shyam S. Sharma, Uttam C.
Banerjee, Asit K. Chakraborti
PII:
S0223-5234(19)30735-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.111601
EJMECH 111601
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 June 2019
Revised Date: 5 July 2019
Accepted Date: 6 August 2019
Please cite this article as: R. Kumar, N. Saha, P. Purohit, S.K. Garg, K. Seth, V.S. Meena, S. Dubey, K.
Dave, R. Goyal, S.S. Sharma, U.C. Banerjee, A.K. Chakraborti, Cyclic enaminone as new chemotype for
selective cyclooxygenase-2 inhibitory, anti-inflammatory, and analgesic activities, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111601.
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Graphical Abstract
Cyclic enaminone as new chemotype for selective
cyclooxygenase-2 inhibitory, anti-inflammatory,
and analgesic activities
†
†
†
†
†
Raj Kumar , Nirjhar Saha , Priyank Purohit , Sanjeev K. Garg , Kapileswar Seth , Vachan
§
§
‡
$
‡
S. Meena , Sachin Dubey , Khyati Dave , Rohit Goyal, Shyam S. Sharma , Uttam C.
Banerjee and Asit K. Chakraborti *
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
§
†
†
Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
§
Department of Pharmaceutical Technology (Biotechnology), National Institute of
Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar 160 062,
Punjab, India.
‡
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Sector 67, S. A. S. Nagar 160 062, Punjab, India.
$
Indo-Soviet Friendship (ISF) College of Pharmacy, Moga, 142 001, Punjab, India (present
address: School of Pharmaceutical Sciences, Shoolini University, Solan, HP, 173212,
India).
*
Corresponding Author: Tel: 91-(0)-172 229 2027; Fax: 91-(0)-172-2214692. E-mail:
akchakraborti@niper.ac.in; akchakraborti@rediffmail.com.
The cyclic enaminone have been identified as new chemotypes for selective
cyclooxygenase-2 inhibitory, anti-inflammatory, and analgesic activities with
gastrointestinal tract (GIT) and cardiovascular (CVS) safety.

Cyclic enaminone as new chemotype for selective
cyclooxygenase-2 inhibitory, anti-inflammatory, and
analgesic activities
†
,£
†
†
†
†
Raj Kumar , Nirjhar Saha , Priyank Purohit , Sanjeev K. Garg , Kapileswar Seth , Vachan S.
§
§
‡
$
‡
§
Meena , Sachin Dubey , Khyati Dave , Rohit Goyal, Shyam S. Sharma , Uttam C. Banerjee
†
*
and Asit K. Chakraborti
†
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
§
Department of Pharmaceutical Technology (Biotechnology), National Institute of
Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar 160 062, Punjab,
India.
‡
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Sector 67, S. A. S. Nagar 160 062, Punjab, India.
$
Indo-Soviet Friendship (ISF) College of Pharmacy, Moga, 142 001, Punjab, India (present
address: School of Pharmaceutical Sciences, Shoolini University, Solan, HP, 173212, India).
£
Present Address: Department of Pharmaceutical Sciences and Natural Products, Central
University of Punjab, Mansa Road, Bathinda-151001, India.
*
Corresponding Author: Tel: 91-(0)-172 229 2027; Fax: 91-(0)-172-2214692. E-mail:
akchakraborti@niper.ac.in; akchakraborti@rediffmail.com.
1

Abstract
The cyclic enaminone moiety has been identified as a new scaffold for selective inhibition of
cyclooxygenase-2 with anti-inflammatory and analgesic activities. The designed cyclic
enaminones have been synthesized conveniently through the development of a new catalyst-free
methodology and evaluated for cyclooxygenase (COX-1 and COX-2) inhibitory activities. Three
compounds 7d, 8, and 9 predominantly inhibited COX-2 with selectivity index of 74.09, 19.45
and 108.68, respectively, and were assessed for in vivo anti-inflammatory activity in carrageenan
induced rat paw edema assay. The anti-inflammatory activity of 7d was comparable to that of
celecoxib at a dose of 12.5 mg/kg. However, the compounds 8 and 9 were more/equally effective
as anti-inflammatory agent compared to celecoxib at the doses of 12.5 mg/kg and 25 mg/kg and
also exhibited anti-inflammatory activity comparable to that of diclofenac. The therapeutic
potential of the most active compound 9 was further assessed by performing in vivo thermal and
mechanical hyperalgesia tests using various models that revealed its analgesic activity. The in
vivo non-ulcerogenicity of 9 revealed the gastrointestinal safety as compared to the non-selective
COX inhibitor indomethacin. The in vitro antioxidant activity and in vivo experiments on heart
rate and blood pressure provided the cardiovascular safety profile of 9. The molecular docking
studies rationalize the COX-2 selectivity of the newly found anti-inflammatory compounds 7d,
8
, and 9.
Keywords: Cyclic enaminone, catalyst free novel methodology, new anti-inflammatory
chemotypes, analgesic activity, gastrointestinal and cardiovascular safety evaluation.
2

1
. Introduction
Inflammation is the body’s natural way of handling infection and tissue damage.[1, 2] Various
diseases such as asthma,[3] osteo- and rheumatoid arthritis,[4] psoriasis,[5] nephropathy,
cancer,[6] etc. involve uncontrolled inflammatory cascades during their progression.[7]
Inflammation ultimately leads to pain which is the most prevalent symptom needing medical
attention and more than 60% people in the US in the year 2014 had been diagnosed with at least
one inflammatory chronic condition.[8-10] The use of non-narcotic (acetaminophen and aspirin)
usually referred as non-steroidal anti-inflammatory drugs (NSAIDs) and narcotic (opioids)
analgesic agents remain to be the mainstays of pain management. Recent approaches for
therapeutic intervention of pain uses anti-inflammatory-analgesic agents that are selective
cyclooxygenase 2 (COX-2) inhibitors with reduced gastrointestinal side effects such as celecoxib
(1)[11] and rofecoxib (2)[12] (Figure 1) introduced in the late 1990s and are generally referred as
COXIBs.
Insert Figure 1 here.
However, allergic problems associated with 1[13] due to the presence of the sulfonamide
moiety and the recent withdrawal of 2 and valdecoxib (3) due to adverse cardiovascular side
effects[14] raise concern over the safe use of the currently approved drugs that are associated
with poor tolerability, unfavorable side effects, long-term safety, abuse, and inconvenience of
use.[15] These press the need to find new chemotype as efficacious and safe COX-2 selective
anti-inflammatory agents. The findings on the over expression of COX-2 in breast, head and
neck, lung, and skin cancer cells project COX-2 as the drug target for cancer treatment.[16] The
role of COX-2 has recently been explored in the pathogenesis of a number of diseases other than
3

lammation related pain such as schizophrenia,[17] depression,[18] epilepsy,[19, 20]
Parkinsonism,[20] ischemic brain injury,[21] diabetic peripheral neuropathy[22] and
proliferation of mycobacterium tuberculosis.[23] These additional therapeutic benefits of
selective COX-2 inhibitors further put the emphasis on finding new COX-2 selective
chemotypes.
1
.1 Design
In general, the selective COX-2 inhibitors belong to a class of diarylheterocycles in which two
aryl moieties are attached to a central heterocyclic ring[24] and the search for novel structural
scaffold[25] for COX inhibition remains an active area of research. We took cognizance of the
fact that the central ring of 2 represents α,β-unsaturated carbonyl system[26] and the findings on
the COX inhibitory activities of acyclic α,β-unsaturated carbonyl compounds.[27-29]
This encouraged us to envisage the cyclic enaminone (I) as a new template for selective COX-
2
inhibition (Figure 2) as the enone moiety maintained the essential interactions with active site
of the COX enzyme when docked into COX-2 pocket and may be devoid of the side effects
related to the sulfonamide/sulfonate group. The common interactions for both rofecoxib and
chalcones such as H-bond formation with Ser530, pi-pi stacking interactions with Arg513,
Phe518 and Tyr355, and hydrophobic interactions with Leu531 were considered for the design of
the cyclic enaminones as COX-2 inhibitors. Moreover, cyclic enaminones (particularly E121)
have been reported with anti-inflammatory properties in the recent past for the treatment of
asthma[30] and inflammatory bowel disease (IBD)[31] by moderating pro-inflammatory
signalling pathways involving multiple cytokines such as TNF-α, IL-10, Th1 and Th2,
neutrophil apoptosis, chemotaxis and superoxide release.[32, 33] However, to the best of our
4

knowledge cyclic enaminones have never been explored for their anti-inflammatory effects via
COX-2 inhibition.
Further, as the newly designed enaminone scaffold I does not have the structural resemblance
with the 1,2-diarylheterocyclic framework compounds belonging to this class may not be
associated with the cardiovascular side effects. As a matter of fact, lumiracoxib (4) (Figure 1),
the most selective COX-2 inhibitor that does not belong to the 1,2-diarylheterocyclic class, does
not exhibit any cardiotoxicity and its discontinuation for clinical use is due to other reason (i.e.,
hepatotoxicity).
Insert Figure 2 here.
We wish to report herein cyclic enaminones as new chemotype for cyclooxygenase inhibitory,
anti-inflammatory, and analgesic activities of along with the safety pharmacological profile of
the most potent compounds.
2
. Results and discussion
2
.1 Chemistry
The synthesis of cyclic enaminones involves the condensation of an amine with cyclic 1,3-
diones (Scheme 1) and there has been continuous efforts towards the development of synthetic
methodologies.[34]
Insert Scheme 1 here.
However, the generation of new hit/lead requires a convenient and high yielding synthetic
method for timely supply of the designed molecules for biological evaluation.[35] Further, the
necessity to enrich the medicinal chemists’ tool box[36] is increasingly being felt important that
preaches the need to further improve the existing methodologies addressing the key green
chemistry issues.[37] These prompted us to develop an efficient and catalyst-free methodology
for a convenient synthesis of cyclic β-enaminones.
5

While designing a new methodology for an easy and convenient preparation of cyclic β-
enaminones we looked into the various possible pathways of formation of the synthetic target
during the condensation of an amine with a cyclic 1,3-dione (Scheme 2). The reaction may
proceed via two distinct pathways: (i) nucleophilic attack by the amine on one of the carbonyl
groups of the cyclic β-diketone to form the imine IIIa which may tautomerize to the cyclic β-
enaminone (Path a);[38] (ii) conjugate addition of the amine on the enol form of the cyclic β-
diketone, followed by intramolecular proton transfer (via Ib) from the ammonium nitrogen to
2
the OH group (leaving group activation to form IIb/IIIb) and concomitant removal of H O to
[
39]
form the desired cyclic β-enaminone (Path b).
Insert Scheme 2 here.
The necessity in performing the reactions under heating in most of the reported methodologies
could be to facilitate the elimination of water in either of these two pathways. Anticipating that
some catalytic assistance might drive the reaction under milder condition particularly through the
‘path a’ involving the intermediacy of the imine IIIa (Scheme 2) we performed the reaction
under the catalytic influence of Mg(ClO [40, 41] and ZrOCl ·8H O[42] as they are known to
4
)
2
2
2
promote imine formation at room temperature. However, these resulted in the formation of
complex product mixture, perhaps as the result of bis-imine formation. Thus, we thought of a
catalyst-free reaction condition. Since the reaction (Scheme 2) proceeds through ionic
intermediates/mechanism the choice of a suitable solvent with appropriate polarity would
facilitate the enaminone formation. However, whether the reaction would proceed through ‘path
a’ or ‘path b’ would be dependent on to what extent the starting cyclic β-diketone exists in the
keto or enol form. Generally in non-polar solvent the keto form exists exclusively or
predominantly[43] whereas polar solvents (particularly protic polar) direct the equilibrium
6

towards the enol from.[44] Moreover, apart from the popularly considered dielectric constant (ε),
the other less frequently considered physico-chemical parameters such as the acceptor number
(AN) and donor number (DN) also play crucial role in promoting organic reactions.[45,46]
Although a polar solvent is expected to provide a conducive environment through the ease of
formation of the charged intermediates (Ia, Ib, IIb/IIIb) in driving the reaction towards the
product, it should not have the ability to form hydrogen bond (H-B) with the intermediates Ia,
Ib, IIb/IIIb. The H-B formation of the oxyanion site in Ia with the solvent (H-B donor: protic
polar solvent) would retard the intramolecular proton transfer to convert Ia to IIa and be
detrimental to the product formation. In ‘path b’ the H-B formation of IIb with the solvent would
shield the oxyanionic charge in IIb (and not permit the formation of IIIb) and hence be
inhibitory for effective removal of water molecule in the final stage. On the other hand, a solvent
with H-B acceptor ability/property (aprotic polar) would not permit conversion of Ia to IIa (path
a) or Ib to IIb (path b) through the intramolecular proton transfer and hence would be
detrimental for the formation of the desired product. We hypothesized that an effective solvent
should be the one with appropriate polarity along with a balanced/optimal value of AN and DN.
Therefore, the most desirable properties should be high ε but low AN and DN values. However,
the most pronounced effect is expected to be due to DN values and solvents with lower DN
values would be preferred as the higher DN value will establish a more effective molecular level
interaction between the solvent and Ia and Ib through H-B formation with the ammonium
hydrogen and would slow down the intramolecular proton transfer to convert Ia to IIb and Ib to
IIb. As each step of the reaction (path a or b) involves an ionic mechanism during the progress
of the reaction it would require a polar solvent which, however, should not be protic polar i.e.,
7

devoid of H-B donor and acceptor properties so that the solvent participation/intervention with
Ia and Ib – IIIb can be minimal to drive the reaction towards the product.
To find the best reaction condition, cyclohexane-1,3-dione 5a was treated with aniline 6a in
various nonpolar, weakly polar, polar and protic polar solvents (Table 1). The cyclic β-
enaminone 3-phenylaminocyclohex-2-enone (7a) was formed with 85 and 96% conversion
(HPLC) at rt after 6 h in DCM and DCE, respectively. Next best results were obtained with
PhMe and MeNO exhibiting 70% conversion. The better performance of DCM and DCE as the
2
reaction medium compared to the other solvents is due to the negligible DN values of DCM (1.0)
and DCE (0, reference solvent). The inferiority of DCM to DCE could be due to its lower ε and
higher AN values compared to those of DCE (DCM: ε = 8.93, AN = 20.4; DCE: ε = 10.38, AN =
1
6.7).[47] The lesser conversion to 7a observed in CHCl compared to that in DCM/DCE could
3
be due to its low polarity (ε = 4.89) apart from its higher AN (23.1) (better H-B donor) and DN
4.0) value. [48] The negligible AN and DN values of PhMe make it an effective reaction
medium (70% conversion to 7a) although it has lower ε (2.38) value (due to which it is less
effective than DCM/DCE). The significantly lower DN value (2.7) of MeNO makes it the most
(
2
effective amongst the aprotic polar solvents. The use of various alcohols (protic polar solvents)
led to poor conversion (34-55%) to 7a perhaps due to the H-B formation involving these solvents
and the oxyanionic species Ia, Ib, IIb/IIIb that retards the elimination of water to form the final
condensation product. This might be the reason for the requirement of heating the reaction
mixture in the reported procedures. Since water is known to exhibit ‘ambiphilic dual activation’
effect in promoting various organic reactions[49-51] we performed the reaction in water in the
absence of any catalyst/addendum but no significant amount of product formation was
observed.[52] The reaction did not proceed under neat conditions also. However a recent study
8

reported the formation of product under neat condition but only at high temperature i.e.120
ºC.[53] The ineffectiveness of the various protic polar solvents could be attributed due to the
higher AN (27.5 - 54.8) and DN (19 - 21.1) values despite of the fact that these solvents are more
polar (higher ε values: 12.47 - 78.36) compared to DCE. For the panel of aprotic polar solvents
used, the lesser conversion (40-61%) observed with MeCN, DMF, and DMSO may be attributed
to their higher DN values (14.1-29.8) compared to that of MeNO (2.7), the most effective
2
solvent (70% conversion) in this category, although they are more polar (ε values are 35.94,
3
2
6.71, and 46.45, respectively) than MeNO (ε = 35.87).
Insert Table 1 here.
After having the optimized reactions conditions in hand, the reaction scope was further
explored for the synthesis of desired cyclic β-enaminones (7a-7u) in high yields (Table 2) by
treating various cycloalkane-1,3-diones (5) with different aryl, aryl alkyl and alkyl amines (6) in
DCE at rt.
Insert Table 2 here.
The rate of reaction for cyclic β-enaminone formation was governed by nucleophilicity of the
amines. The reactions with aliphatic amines required lesser time (0.5 - 2 h) compared to those
with of aromatic amines (2.5 - 12 h). The formation of compound 7s took longer time due to the
electron deficient nature of the amine group in 2-aminobenzothiazole (6s).
1
In the H NMR spectral data, the aliphatic protons (methylene protons) of five or six-
membered cyclic enones appeared between 1-3 ppm as either triplet or multiplet due to the
coupling with adjacent methylene protons. The olefenic proton and the aromatic amine proton
appeared between 5-6 ppm as sharp intense singlet and between 5-8 ppm as broad singlet
respectively. The olefenic proton appeared as sharp singlet as there are no adjacent proton to this
9

olefenic proton. The peak of proton attached to nitrogen of enaminone has always been observed
as broad singlet due to intermediate rate of proton exchange. Aromatic protons of aryl group
appeared between 6-8.5 ppm. Methoxy proton of 7c appeared as singlet at 3.8 ppm and benzylic
proton of 7o appeared at 4.20 ppm.
1
3
In the C NMR spectral data, the carbonyl carbon has been appeared as very weak intense
1
3
peak due to the long relaxation time of carbonyl carbon. In C NMR, the carbon of carbonyl
group of enone has largest chemical shift (190-220 ppm) indicating it as most deshielded carbon
2
due to sp hybridization and attachment of electronegative oxygen to it. The α-carbon of enone
system appeared at relatively upfield position (80-110 ppm) with respect to the β-carbon (150-
1
70 ppm) of enone as the α-carbon is more shielded than the β-carbon due to resonance.
Aromatic carbons appeared between 110-170 ppm and the aliphatic carbons appeared between
0-60 ppm depending upon the electron-withdrawing group attached to the carbon. Quaternary
1
carbons appeared as weak intense peak than the peak of primary carbons. In the IR spectral data,
frequency of the carbonyl group appeared at relatively lower frequency region than the usual
ketones as there is the partial double bond character of the carbonyl group due to the presence of
resonance in the enaminone system.
2
2
.2 Biological Evaluation
.2.1 In vitro studies: determination of % COX inhibition and IC50
In vitro COX-1 and COX-2 inhibitory activities (µM) of the synthesized compounds were
determined using COX inhibitor screening assay kit (ovine COX-1 and human recombinant
COX-2) (Catalog number 560131, Cayman chemical company) with 96-well plates (Table 3).
Insert Table 3 here.
Initially, twenty-one compounds (7a-7u) were evaluated for COX-1 and COX-2 inhibitory
activities at 10 µM concentration (Table 3). Out of these, seventeen compounds (7a-e, 7i-p, and
10

7
s-7u) showed preference for COX-2 inhibition and 7d was found to be the most effective
(enzyme inhibitory potency: COX-1 = 12.89%; COX-2 = 88.63%). The compound 7g showed
almost equal potency for COX-1 (46.47%) and COX-2 (47.12%) inhibition. Three compounds
exhibited selectivity towards inhibition of COX-1 (7f; 30.98%, 7h; 65.54% and 7m; 43.49%)
than COX-2 (7f; 24.22%, 7h; 41.89% and 7m; 22.25%). The COX-2 selectivity as well as
inhibitory potency were found to be influenced by various factors such as the ring size of the
enone moiety, the presence of substitution on the enone ring system, and more significantly on
the nature of the amine and the substituent present on the amine (in case of aromatic amines).
Decrease of the size of the enaminone moiety from six membered to five membered (compare 7p
with 7u, 7e with 7m, and 7d with 7n) led to decrease in inhibitory potency and selectivity. The
substitution in the alicyclic ring also resulted in decrease of enzyme inhibitory potency and
COX-2/COX-1 selectivity (compare 7d with 7l, 7e with 7k, and 7p with 7t). Preference for
COX-2 inhibition was observed with enaminones derived from aromatic/heteroaromatic amines
and cyclic amines than those prepared from aryl/heteroaryl alkyl amines. The enzyme inhibitory
activity was also influenced by the electronic effect. The enaminones derived from aromatic
amines having methyl/methoxy/thiomethyl groups (7b, 7c, and 7i) and halogen (7d, 7e, 7j-l, and
7
n) substitutions were found to possess better COX-2 inhibition than COX-1 (except for 7m).
On the other hand, those bearing electron withdrawing substituent (7f–h) exhibited decreased
selectivity.
2
.2.2 Optimization of Lead Structure
Since 7d was found to be the most potent and selective COX-2 inhibitor out of the initially
synthesized twenty-one cyclic enaminones, we planned to optimize the lead structure through
structural manipulation. However, as any structural variation in putting substitution on the enone
moiety or the decrease in its ring size as well as structural variation on the amine moiety resulted
11

in decrease in COX-2 inhibitory potency, the NH group appeared to be the only handle for
further structural modification of 7d. The N-tert-butyloxycarbonylation of 7d was carried out by
treating it with (BOC) O (1.1 equiv) under the catalytic influence of DMAP (10 mol%) in DCM
2
at rt for 1 h to afford 8 (Scheme 3). The N-acetylation of 7d was performed using Ac O (1.1
2
equiv) in the presence of DMAP (10 mol%) in DCM under reflux for 6 h to afford the N-
acetylated product 9 (Scheme 3).
Insert Scheme 3 here.
The compounds 8 and 9 were subjected to COX-1 and COX-2 inhibition at 10 µM
concentration (Table 3). Compared to the parent compound 7d, the N-BOC derivative 8 showed
a decrease in COX-2 inhibition (88.63% to 73.46%) while COX-1 inhibition (12.89% to
3
0.50%) was increased indicating that N-BOC protection of the lead structure has detrimental
effect on COX-2 inhibitory potency as well as selectivity. However, the N-acetylated compound
9
emerged as the most potent and selective COX-2 inhibitor (COX-2: 96.94%; COX-1: 10.80%).
As 7d, 8, and 9 were most effective amongst the cyclic β-enaminones with respect to
preference for COX-2 inhibition, we determined the IC50 value for COX-1 and COX-2 inhibition
for these compounds so as to determine the COX-2 selectivity index (SI) that could be the
indicative of their potency as compared to the known COX-2 selective inhibitor celecoxib (1)
(
Table 4). The compound 7d exhibited very good COX-2 inhibitory potency (COX-2 IC50
.513 µΜ) and selectivity (COX-2 SI = 74.09). Compared to the parent compound 7d, the N-
BOC derivative 8 showed a decrease in COX-2 potency (COX-2 IC = 0.915 µΜ) as well as
=
0
5
0
selectivity (COX-2 SI = 19.45). However, the N-acetylated compound 9 emerged as the most
potent (COX-2 IC50 = 0.367 µΜ) and selective COX-2 inhibitor (COX-2 SI = 108.68) and more
effective than celecoxib (Table 4) for selective COX-2 inhibition.
12

Insert Table 4 here.
2
2
.2.3 In vivo studies
.2.3.1 Carrageenan-induced rat paw edema
The in vivo anti-inflammatory activity for the three newly found selective COX-2 inhibitors
7
d, 8, and 9 was evaluated by carrageenan-induced acute rat paw edema in Wistar albino rats[54]
at two doses (Table 5). Acute administration of carrageenan caused significant (p<0.05) paw
edema as compared to normal rat paw volume.[55] Administration of synthesized compounds
7
d, 8 and 9 in two doses 12.5 and 25 mg/kg showed marked (p<0.05) efficacy in preventing paw
edema as determined by the decrease of the rat paw volume in comparison to respective
carrageenan control and the effects were maximum at 3 h. Among these three compounds,
compound 9 exhibited the highest anti-inflammatory activity comparable to the standard drug
celecoxib (1).
Intraplantar
injection
of carrageenan led
to
time-dependent
development
of
peripheral inflammation, which resulted in a significant increase in the levels of tumor necrosis
factor α (TNF-α) and interleukin 1 (IL-1) β, nitric oxide (NO) and prostaglandin E (PGE ) and
2
2
also iNOS and COX-2 protein
Insert Table 5 here.
2
.2.3.2 Indomethacin induced gastric ulceration
For the GIT safety assessment, oral indomethacin at a dose of 20 mg/kg (4 mg/mL in 0.1% aq
Tween 80) was used to observe the lesions and ulcer. Based on their intensity, the ulcers were
given scores as follow:
0
= no ulcer, 0.5 = Red color, 1 = Red spot, 1.5 = Haemmoragic streaks, 2 = Deep ulcer, 3 =
Perforation.
13

Insert Figure 3 here.
Ulcer score of Normal control group: There was no evidence of presence of ulcers in the
normal control animals and thus the score given to this group was zero.
Ulcer score of Indomethacin treated group: There was presence of ulcers in the
indomethacin (20 mg/kg) treated group in the form of haemorrhagic streaks and thus the score
was 1.5.
Ulcer score of Compound 9 treated group: There was no evidence of presence of ulcers in
the intestine of the rats treated with 9 (50 mg/kg) and thus the score given to this group was zero.
In GI safety studies, 9 (30 mg/kg) did not produce any lesions and ulcers although similar dose
of indomethacin induced ulcer. The increase of the dose of 9 to 50 mg/kg also did not produce
ulcer suggesting its potential therapeutic benefit for treatment against inflammatory disorder for
ulcer patients.
2
.2.3.3 Determination of Analgesic activity
During inflammatory stimulus, COX-2 isoform is induced leading to the induction of various
inflammatory mediators like bradykinin and histamine which trigger release of IL-6, PGαα,
TNF-α etc. All of these all mediators are responsible for pain. Hence, inhibition of COX-2 is
associated with the analgesic effect for pain relief.[56] Thus, the analgesic action of 7d, 8, and 9
was evaluated in different pain modals.
2
.2.3.3.1 Hot plate studies
A potent analgesic activity is also the marker for the marked inhibition of COX-1 and COX-2
cascades responsible for nociception. The potency of all the three newly found selective COX-2
14

inhibitors 7d, 8 and 9 was evaluated by in vivo hot plate test (Eddy’s anaelgesiometer method)
57] using Wistar albino rats[54] at two dose levels (Table 6). Before treatment, the animals of
[
the control group showed normal pain response against thermal heating. The treatment with 9, 8,
and 7d at the 12.5 and 25 mg/kg doses showed marked (p<0.05) efficacy in reducing the pain
perception as evidenced by increased reaction time (sec) in comparison to the control animals at
respective time (min) and the effects were maximum at 120 min. Among these three compounds,
9
showed pronounced analgesic effects comparable to that of the standard drug celecoxib at 120
min.
Insert Table 6 here.
.2.3.3.2 Carrageenan-induced inflammatory pain studies
A) Thermal hyperalgesia (Hargreaves Plantar Test)[58]: In thermal hyperalgesia test,
2
(
compared to carrageenan induced inflammatory pain, when 9 was administered at a dose 30
mg/kg, it showed significant reduction in pain parameters at 3 h although the overall effect was
not a complete reversal. Better reduction of pain parameters was observed by an increase of the
dose of 9 to 50 mg/kg. At oral doses of 30 and 50 mg/kg 9 showed significant increase in the
thermal paw withdrawal latency as compared to the carrageenan treated animals with a better
efficacy at the dose of 50 mg/kg comparable to that of celecoxib (Table 7).
Insert Table 7 here.
(B) Mechanical hyperalgesia: In mechanical hyperalgesia test, significant increase in the paw
withdrawal latency was observed after 3 h of administration of 9 at the doses of 50 and 30
mg/kg, compared to that observed with carrageenan treated animals in both of the mechanical
tests i.e., Vonfrey test [59](Table 8) and Randall selittotest [60](Table 9) with better efficacy at
15

the higher dose (i.e. 50 mg/kg). Although 9 was less effective in comparison to celecoxib at the
dose of 30 mg/kg a much-improved effect was obtained at 50 mg/kg in both the tests.
Insert Table 8 here.
Insert Table 9 here.
2
.2.3.4 Antioxidant activity (DPPH scavenging assay) [61]
The antioxidant activity of therapeutic agents exerts beneficiary effect on cardiac system
because oxidative stress is closely related to the cardiovascular disease.[62, 63] The DPPH is a
stable free radical that can accept an electron or hydrogen radical to form a stable diamagnetic
molecule. DPPH radical reacts with various electron donating molecules (reducing agents or
antioxidants) and results the formation of colorless 2,2'-diphenyl-1-picryl hydrazine. Reduction
of the DPPH radicals can be estimated quantitatively by measuring the decrease in absorbance at
5
17 nm. The anti-oxidant activity of 7d, 8, 9 was evaluated and expressed as % scavenging and
presented by IC value (Table 10).
5
0
Insert Table 10 here.
The findings of anti-oxidant assay using DPPH method revealed that 8 and 9 have significant
anti-oxidant activity with IC values of 42.61 and 47.97, respectively. However, 7d showed less
5
0
anti-oxidant profile with IC value of 71.24. Ascorbic acid was used as the standard and showed
5
0
IC 23.41. The data in Table 10 suggest that these compounds may exert beneficiary effects on
5
0
cardiac system which is the important issue of COX-2 selective compounds.
2
.2.3.5 Cardiovascular system (CVS) safety pharmacology core battery studies [61]
The reason of cardiovascular risks of COX-2 inhibitors is attributed to many factors. It may not
only be due to the drug class but also is associated with the drug structure related problems and
16

patient genetics. The dose of the drug also has influence on the cardiac toxicity. Although
generally the cardiac toxicity is manifested after long term use, [64] a randomized trial on
rofecoxib revealed that it increases systolic blood pressure (SBP) in 24 h compared to that with
celecoxib. Therefore initial studies on the vital function of the heart may reveal the acute cardiac
toxicity [65] and a prior reflection of acute cardiac toxicity may be assessed on the basis of heart
rate and blood pressure. Thus, it was planned to measure the effect on heart rate and BP during
the treatment with 9.
Effect of 9 (50 mg/kg) on heart rate and BP
The heart rate and BP were measured in rats under the administration of 9 at the maximum
dose of 50 mg/kg. There was no significant difference in SBP, DBP and the heart rate after
treatment with 9 as compared to normal control animals. The results (expressed as Mean ±
S.E.M with n= 4) (Figure 4) reflect the safety profile of 9 although it may not necessarily
recommend to avoid the investigation after long term use.
Insert Figure 4 here.
2
.2.4 In silico studies: Molecular docking
To understand the rationale of COX selectivity and its correlation with the structure of the
most potent COX-2 selective inhibitors molecular docking studies were performed. The docking
of 7a, 7d, 8, 9 were performed individually on the COX-1 (3KK6.pdb) [66] and COX-2
(6COX.pdb) [67] active sites using the GOLD.[68-70]
Rationalisation of COX-2 selectivity of 7a, 7d, 8, and 9 through computational studies and
correlation with the coxibs (celecoxib and lumiracoxib)
For the in silico docking studies with the COX-2 enzyme, celecoxib was used as the gold
standard. Celecoxib showed similar docking pose and interactions as SC-558 inside the COX-2
active site. Celecoxib maintained four major interactions with Gln192, His90, Ser353 and
17

Leu352 in the COX-2 active site. The hydrogen atom attached to the N atom of the sulfonamide
…
moiety of celecoxib forms hydrogen bond with the carbonyl oxygen of Gln192 (N-H O=C-
…
…
Gln192, 2.5 Å), Leu352 (N-H O=C-Leu352, 1.9 Å) and Ser353 ((N-H O=C-Ser353, 2.8 Å).
The oxygen atom of the sulfonamide moiety of celecoxib (1) forms hydrogen bond with the
…
imidazole NH of His90 (S=O H-N-His90, 2.8 Å).[71]
Lumiracoxib (4) was docked inside the COX-2 active site, by replacing celecoxib from the
active site of COX-2. It holds two major interactions with Tyr 385 and Ser 530 same as those
observed with celecoxib. The carbonyl oxygen of lumiracoxib forms two strong hydrogen bonds
with Tyr 385 (C=O….. H-O- Tyr 385, 2.62 Å) and Ser 530 (C=O …. H-O–Ser 530, 2.5 Å).[72]
The compounds 7a, 7d, 8, and 9 were docked individually. No significant interaction with the
residue of active site of COX-2 was observed with 7a (See supplementary data; Table S1).
However, 7d showed three strong hydrogen bonding interaction (Figure 5) through the carbonyl
(C = O) oxygen atom of the cyclohexenone to the Val 523 (N-H….O=C-Val 523, 2.9 Å), Ser
5
30 (C=O….H–O-Ser 530, 3.0 Å) and Tyr 385 (C=O…H-O-Tyr 385, 3.1 Å). The docking score
was 43.94.
Insert Figure 5 here.
The enaminone 8 showed strong hydrogen bonding interaction similar to that of 7d, through
the oxygen atom of cyclohexanone (Figure 6) to the hydrogen atom of hydroxyl group of the
Ser530 and Tyr 385 with 2.85 and 2.73 Å distance, respectively. These interactions were similar
to those observed with lumiracoxib. However, the docking score (58.71) of 8 is better than that
of lumiracoxib (4).
Insert Figure 6 here.
18

The enaminone 9 showed similar kind of interaction (Figure 7) as observed with 8, 7d and
lumiracoxib. The carbonyl group of the cyclohexanone moiety established interaction with Ser
5
30 (C=O….H –O-Ser 530 2.88 Å) and Tyr 385 (C=O…H-O-Tyr 385. 2.28 Å).
Insert Figure 7 here.
Overall 7d, 8, and 9 have similarity with lumiracoxib (4) with respect to the various
interactions in the active site of COX-2 (through Ser 530 and Tyr 385). As a matter of fact, 9
shows better docking score then lumiracoxib (See supplementary data; Table S1). In view of the
similarity of 7d, 8, and 9 with lumiracoxib in interacting with the active site of COX-2 it may be
anticipated that the therapeutic potential of 7d, 8, and 9 would be similar to that of lumiracoxib,
which is less cardiotoxic than other COX-2 inhibitors (See supplementary data; Table S1). All
the compounds 7d, 8, and 9 were docked into the COX-1 active site and observed to interact
with a smaller number of active site residues (See supplementary data; Table S2) as compared to
those of COX-2 providing a rationale their COX-2 selectivity.
Comparison of the docking pose of 7d, 8, and 9 with that of the marketed COX-2 selective
drugs celecoxib and lumiracoxib (See supplementary data; Figure S1) revealed that 7d, 8, and 9
have ‘V shape’ docking pose similar to that of celecoxib and lumiracoxib.
3
. Conclusions
The present study describes cyclic enaminone moiety as a novel scaffold for COX inhibitory
activity along with a new synthetic methodology for easy accessibility of cyclic enaminones with
distinct advantages over the reported methodologies e.g., room temperature and catalyst-free
reaction conditions without the requirement of special apparatus. The cyclic enaminones 7d, 8
and 9 were found to be selective COX-2 inhibitors with the selectivity index of 74.09, 19.45 and
1
08.68, respectively, and exhibited significant in vivo anti-inflammatory activity in carrageenan
19

induced rat paw oedema assay at different (12.5 mg/kg and 25 mg/kg) doses. The anti-
inflammatory potency of 7d was comparable to that of celecoxib at a dose of 12.5 mg/kg and 8
and 9 were more/equally effective compared to celecoxib at the doses of 12.5 mg/kg and 25
mg/kg. Among the three compounds, 9 showed pronounced analgesic effects comparable to the
standard drugs. In thermal and mechanical hyperalgesia tests, 9 showed significant analgesic
effect at a dose of 50 mg/kg. The in vivo GIT safety studies revealed 9 was found to be
nonulcerogenic even in the higher dose of 50 mg/kg in comparison to the fact that the non-
selective COX inhibitor indomethacin (20 mg/kg) showed the presence of ulcers. The
antioxidant activity of 7d, 8, and 9 makes these compounds beneficial for vital heart functions
which was confirmed by safety pharmacology study of the most potent compound 9 as did not
show any significant difference in SBP (systolic blood pressure), DBP (diastolic blood pressure),
and heart rate at 50 mg/kg dose compared to normal control animals. Molecular docking studies
rationalize the COX-2 selectivity of the newly found anti-inflammatory 7d, 8, and 9 and their
similarity with lumiracoxib with respect to interaction in the COX-2 active site suggest less
propensity for adverse cardiovascular effect.
4
. Experimental Section
4
.1 General Chemistry
The glasswares used were thoroughly washed and dried in an oven and the experiments
were carried out with required precautions. All chemicals were purchased from Aldrich, Alfa-
aesar, Lancaster and Fluka Chemicals and used as received. Chemicals and all solvents were
commercially avialable and used without further purification. Melting points were measured with
Gupta scientific, India melting point apparatus. The IR spectra were recorded either on KBr
1
pellets (for solids) or neat (for liquids) on a Nicolet Impact 410 FTIR spectrometer. The H and
20

1
3
C NMR spectra were recorded on a Bruker Avance 300 or 400 MHz NMR spectrometer in the
indicated solvent using TMS as internal standard. Chemical shifts (δ) are given in ppm and J
1
3
values are given in Hz. The C NMR spectra were fully decoupled. Splitting patterns were
designated as s, singlet; brs, broad singlet; d, doublet; dd, doublet of doublet; t, triplet; dt, doublet
of triplet and m, multiplet. The mass spectrometry of the samples was carried out under
atmospheric pressure chemical ionization (APCI) and electron ionization (EI) mode on
Thermofisher LTQ-XL and Thermofisher Scientific GCMS mass spectrometers respectively.
Open column chromatography, thin layer chromatography (TLC) was performed on Silica gel
®
[
CDH/Avra silica gel 60-120 mesh, F254 and Merck silica gel respectively]. Evaporation of
solvents was performed at reduced pressure, using a Bǘchi rotary evaporator. The products were
purified either by trituration with EtOAc:Hexane or by column chromatography on silica-gel
(#60–120; eluent EtOAc:Hexane). Elemental analysis of the purified compounds was performed
using Thermofisher gas chromatography based elemental analyzer. The purity of all the tested
compounds was assessed by reverse phase analytical HPLC on Shimadzu and found to be >96%.
The HPLC analyses were performed on an instrument equipped with a photodiode array detector
and by using a C-18-DB column (4.6 mm × 25 cm). Water-acetonitrile was used as eluent at a
gradient of 60-80% over 30 minutes with detection at 305 nm and a flow rate of 1 mL/min.
Synthetic procedure for preparation of 3-Phenylaminocyclohex-2-enone (7a) [73]: To a
solution of cyclohexan-1,3-dione (0.28 g, 2.5 mmol) in DCE (5 mL), aniline (0.255 g, 2.75
mmol, 1.1 equiv) was added at rt. After the completion of the reaction (TLC, 6 h), solvent was
evaporated under reduced pressure. The residue obtained was triturated using ethyl acetate and
hexane mixture to afford the pure product 7a (0.397 g, 85%) as white solid. mp: 175-177 °C. IR
-1 1
(
KBr): 3262, 2943, 1601, 1590, 1574, 1525, 1243, 1182, 704 cm . H NMR (400 MHz, CDCl ):
3
21

δ 1.98-2.05 (m, 2H, CH
2 2 2
), 2.34 (t, J = 6.6 Hz, 2H, CH ), 2.50 (t, J = 6.6 Hz, 2H, CH ), 5.56 (s,
1
3
1
H, C=C-H), 6.70 (brs, 1H, NH), 7.13-7.17 (m, 3H, Ar-H), 7.29-7.33 (m, 2H, Ar-H). C NMR
(
100 MHz, CDCl ): δ 21.85, 29.75, 36.49, 99.76, 123.93, 125.55, 129.33, 138.09, 162.29,
3
+
1
98.34. MS (APCI) m/z 188.28 (M+H) . The remaining reactions were carried out following this
general procedure. The physical data (mp, IR, NMR, MS) of the known compounds were in
complete agreement with those reported in the literature (See supplementary data). The
following compounds were unknown.
4
1
1
.1.1 3-(4-Acetylphenylamino)cyclohex-2-enone (7h): Yield: 0.458 g, 80% (yellow solid), mp:
20-122 °C. IR (KBr): 3269, 3181, 3095, 2947, 1677, 1574, 1531, 1411, 1358, 1250, 1179,
-
1 1
139, 959 cm . H NMR (400 MHz, CDCl
), 2.55 (t, J = 6.2 Hz, 2H, CH ), 2.57 (s, 3H, COCH
.0, 1.8 Hz, 2H, Ar-H), 7.33 (brs, 1H, NH), 7.90 (dd, J = 7.0, 1.8 Hz, 2H, Ar-H). C NMR (100
3
): δ 2.01-2.07 (m, 2H, CH
2
), 2.38 (t, J = 6.2 Hz, 2H,
CH
2
2
3
), 5.79 (s, 1H, C=C-H), 7.22 (dd, J =
1
3
7
MHz, CDCl ): δ 21.71, 26.49, 29.82, 36.50, 101.77, 121.67, 129.86, 132.98, 143.14, 160.75,
3
+
1
96.89, 198.92. MS (EI) m/z 229 (M ). Anal. calcd. for C H NO :C, 73.34; H, 6.59; N, 6.11;
1
4
15
2
Found C, 73.22; H, 6.66; N, 6.15.
4
.1.2 3-(4-Methylsulfanylphenylamino)cyclohex-2-enone (7i): Yield: 0.523 g, 90% (off-white
solid), mp: 120-122 °C. IR (KBr): 3246, 2941, 2919, 1599, 1563, 1505, 1397, 1246, 1180, 1135,
-
1 1
9
2
55 cm . H NMR (400 MHz, CDCl ): δ 1.98-2.05 (m, 2H, CH ), 2.34 (t, J = 6.2 Hz, 2H, CH ),
3 2 2
.47-2.50 (m, 5H, CH , SMe), 5.50 (s, 1H, C=C-H), 6.52 (brs, 1H, NH), 7.07 (d, J = 8.5 Hz, 2H,
2
1
3
Ar-H), 7.21 (d, J = 8.5 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl
3
): δ 16.27, 21.84, 29.75,
+
3
6.47, 100.06, 124.56, 127.74, 135.28, 135.69, 161.97, 198.17. MS (EI) m/z 233 (M ). Anal.
calcd. for C H NOS:C, 66.92; H, 6.48; N, 6.00; S, 13.74; Found C, 67.11; H, 6.64; N, 6.11; S,
1
3
15
1
3.97.
22

4
.1.3 3-(5-Chloro-2-fluorophenylamino)cyclohex-2-enone (7j): Yield: 0.507 g, 85% (yellow
-1
solid), mp: 125-127 °C. IR (KBr): 3221, 3032, 2948, 1598, 1530, 1495, 1255, 1181, 1143 cm .
1
H NMR (400 MHz, CDCl ): δ 2.01-2.08 (m, 2H, CH ), 2.37 (t, J = 6.2 Hz, 2H, CH ), 2.53 (t, J
3
2
2
=
6.2 Hz, 2H, CH ), 5.52 (s, 1H, C=C-H), 6.49 (brs, 1H, NH), 7.02-7.11 (m, 2H, Ar-H), 7.33 (dd,
2
1
3
J = 6.8, 2.4 Hz, 1H, Ar-H). C NMR (100 MHz, CDCl ): δ 21.70, 29.55, 36.43, 101.33, 117.08,
3
1
17.30, 125.18, 126.07, 126.15, 127.29, 127.42, 129.46, 129.50, 152.74, 155.19, 161.01, 198.63.
+
MS (EI) m/z 239 (M ).Anal. calcd. for C12
H11ClFNO:C, 60.14; H, 4.63; N, 5.84; Found C,
6
0.11; H, 4.75; N, 5.92.
.1.4 3-(4-Fluorophenylamino)-5,5-dimethylcyclohex-2-enone (7k): Yield: 0.552 g, 95%
4
(
yellow solid), mp: 184-186 °C. IR (KBr): 3247, 3054, 1610, 1583, 1533, 1507, 1225, 1147, 825
-1 1
cm . H NMR (400 MHz, CD OD): δ 1.13 (s, 9H, CH ), 2.23 (s, 2H, CH ), 2.47 (s, 2H, CH ),
3
3
2
2
1
3
5
.36 (s, 1H, C=C-H), 7.14-7.18 (m, 2H, Ar-H), 7.23-7.26 (m, 2H, Ar-H). C NMR (100 MHz,
CD OD): δ 28.38, 33.73, 43.50, 50.65, 97.06, 117.02, 117.25, 127.59, 127.67, 135.86, 160.78,
3
+
1
6
63.20, 166.64, 200.52. MS (APCI) m/z 234.3 (MH ). Anal. calcd. for C H FNO:C, 72.08; H,
1
4
16
.91; N, 6.00; Found C, 72.21; H, 6.98; N, 6.12.
4
.1.5 3-(4-Fluorophenylamino)cyclopent-2-enone (7m): Yield: 0.377 g, 79% (yellow solid),
-1 1
mp > 200 °C (dec.). IR (KBr): 3257, 3012, 1637, 1508, 1312, 1188, 762 cm . H NMR (400
MHz, CD OD): δ 2.45-2.48 (m, 2H, CH ), 2.83-2.85 (m, 2H, CH ), 5.42 (s, 1H, C=C-H), 7.13-
.17 (m, 2H, Ar-H), 7.25-7.28 (m, 2H, Ar-H). C NMR (100 MHz, CD OD): δ 29.54, 34.18,
3
2
2
1
3
7
1
3
01.19, 116.98, 117.21, 124.87, 137.37, 160.20, 162.62, 177.84, 209.20. MS (APCI) m/z 192.3
+
(
MH ).Anal. calcd. for C H FNO:C, 69.10; H, 5.27; N, 7.33; Found C, 69.33; H, 5.31; N, 7.40.
1
1
10
4
.1.6 3-(Piperidin-1-ylamino)cyclohex-2-enone (7r): Yield: 0.441 g, 91% (yellow solid), mp:
-1
1
68-170 °C. IR (KBr): 3427, 3208, 3055, 2937, 1676, 1570, 1536, 1361, 1229, 1185, 706 cm .
23

1
H NMR (300 MHz, CDCl
3 2 2
): δ 1.42 (m, 2H, CH ), 1.61-1.68 (m, 5H, CH ), 1.92-2.08 (m, 4H,
1
3
2
CH ), 2.24-2.34 (m, 5H, CH ), 5.26 (s, 1H, C=C-H), 5.67 (s, 1H, NH). C NMR (100 MHz,
2
2
CDCl ): δ 21.95, 23.25, 25.58, 27.29, 36.79, 56.40, 97.56, 162.30, 197.83. MS (APCI) m/z 195.1
3
+
(
MH ). Anal. calcd. for C H N O:C, 68.01; H, 9.34; N, 14.42; Found C, 67.94; H, 9.48; N,
11
18
2
1
4.37.
4
.1.7 3-(Benzothiazol-2-ylamino)cyclohex-2-enone (7s): Yield: 0.457 g, 75% (Pale yellow
-1 1
semi-solid), IR (KBr): 3270, 3031, 1610, 1567, 1505, 1240, 1190, 752 cm . H NMR (300 MHz,
CDCl + CD OD): δ 2.05-2.10 (m, 2H, CH ), 2.40-2.44 (m, 2H, CH ), 2.65-2.69 (m, 2H, CH ),
3
3
2
2
2
6
2
1
4
.93 (s, 1H, C=C-H), 7.24-7.29 (m, 2H, Ar-H & NH), 7.38-7.43 (m, 1H, Ar-H), 7.70-7.76 (m,
+
H, Ar-H). MS (APCI) m/z 245.1 (MH ). Anal. calcd. for C13
1.47; S, 13.12; Found C, 64.07; H, 5.15; N, 11.57; S, 13.24.
12 2
H N OS:C, 63.91; H, 4.95; N,
.1.8 3-[(Furan-2-ylmethyl)amino]cyclopent-2-enone (7u): Yield: 0.362 g, 82% (pale brown
-1 1
solid), mp: 143-145 °C. IR (KBr): 3175, 2924, 2831, 1660, 1570, 1308, 1197cm . H NMR (400
MHz, CDCl ): δ 2.35 (s, 2H, CH ), 2.60 (s, 2H, CH ), 4.30 (s, 2H, NHCH ), 5.10 (s, 1H, C=C-
3
2
2
2
1
3
H), 6.25 (s, 1H, Het-H), 6.32 (s, 1H, Het-H), 6.69 (brs, 1H, NH),7.35 (s, 1H, Het-H). C NMR
(
100 MHz, CDCl ): δ 28.03, 33.70, 41.86, 99.40, 108.14, 110.47, 142.46, 150.09, 177.06,
3
+
2
05.08. MS (APCI) m/z 178.1 (MH ). Anal. calcd. for C10
2
H11NO :C, 67.78; H, 6.26; N, 7.90;
Found C, 67.54; H, 6.44; N, 8.03.
Synthesis of (4-Chlorophenyl)-(3-oxocyclohex-1-enyl)carbamic acid tert-butyl ester (8):
To a solution of 7d (0.221 g, 1 mmol, 1 equiv) in DCM (5 mL), (BOC) O (0.191 g, 1.1 mmol,
2
1
.1 equiv) in presence of DMAP (10 mol%) was added at rt. After the completion of the reaction
(TLC, 1 h), the reaction mixture was adsorbed on silica gel (#60-120), purified through column
chromatography (EtOAc:Hexane) to afford the product 8 (0.256 g, 80%) as white solid. mp: 113-
24

-1 1
1
15 °C. IR (KBr):2922, 2853, 1727, 1642, 1586, 1459, 1375, 1298, 1244, 1155 cm . H NMR
t
(
400 MHz, CDCl ): δ 1.40 (s, 9H, Bu), 1.98-2.06 (m, 2H, CH ), 2.36 (t, J = 6.0 Hz, 2H, CH ),
3
2
2
2
.79 (t, J = 6.0 Hz, 2H, CH ), 5.40 (s, 1H, C=C-H), 7.05 (dd, J = 6.4, 2.0 Hz, 2H, Ar-H), 7.36
2
1
3
(
dd, J = 6.4, 2.0 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl ): δ 22.86, 27.94, 29.55, 36.67,
3
+
8
3.05, 117.26, 129.31, 133.60, 139.14, 152.00, 162.36, 199.11. MS (EI) m/z 321 (M ).Anal.
calcd. for C H ClNO : C, 63.45; H, 6.26; N, 4.35; Found: C, 63.57; H, 6.41; N, 4.50.
1
7
20
3
4
.1.9 N-(4-Chlorophenyl)-N-(3-oxo-cyclohex-1-enyl)acetamide (9): To a solution of 7d (0.221
g, 1 mmol, 1 equiv) in DCM (5 mL), Ac O (0.112 g, 1.1 mmol, 1.1 equiv) in presence of DMAP
2
(10 mol%) was added at refluxing condition. After the completion of the reaction (TLC, 6 h), the
reaction mixture was adsorbed on silica gel (#60-120), purified through column chromatography
(EtOAc:Hexane) to afford 9 (0.197 g, 75%) as off-white semi-solid. IR (KBr): 2923, 2854, 1658,
-1 1
1
594, 1491, 1240 cm . H NMR (300 MHz, CDCl ): δ 1.97-2.04 (m, 5H, CH & CH ), 2.38 (t, J
3 2 3
=
6.0 Hz, 2H, CH ), 2.82 (t, J = 6.0 Hz, 2H, CH ), 5.38 (s, 1H, C=C-H), 7.13 (d, J = 8.3 Hz, 2H,
2
2
1
3
Ar-H), 7.45 (d, J = 8.3 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl ): δ 22.83, 25.25, 29.55,
3
3
2
5
6.86, 119.48, 129.78, 130.90, 135.05, 139.26, 162.76, 170.48, 199.46. MS (APCI) m/z
+
64.00(MH ). Anal. calcd. for C H ClNO : C, 63.76; H, 5.35; N, 5.31; Found: C, 63.93; H,
1
4
14
2
.44; N, 5.23.
4
.2. Biological Evaluation
Animal studies were carried out after approval of Institutional Animal Ethics Committee
(
IAEC). Male SD rats (weighing around 180-250 g) were used for study. The animals were
ο
housed in room maintained at approximately 23±1 C temperature and humidity of 55±5% with
1
2 h light/dark cycle. Free access to food and water was allowed. The animals were acclimatized
for at least 24 h before the initiation of experiment and observed for any sign of disease. Body
25

weights were determined on a weekly basis. The animals were maintained under proper
condition till the termination of experiment.
4
.2.1 Cyclooxygenase Inhibition Studies
In vitro COX-1 and COX-2 inhibitory activities (µM) were determined using COX inhibitor
screening assay kit (ovine COX-1 and human recombinant COX-2) with 96-well plates as per the
manufacturer’s protocol through direct measurement of PGF2α produced by SnCl reduction of
2
2
COX-derived PGH (Catalog number 560131, Cayman chemical company). The samples for
determination of initial activity of COX-1, COX-2 and inhibitor screening were prepared by
adding 950 µL reaction buffer, 10 µL heme, 10 µL of COX-1/COX-2 enzyme and 20 µL of
DMSO (inhibitor dissolved in DMSO in case of inhibitor screening) in separate test tubes. The
COX-1/COX-2 background samples were prepared by adding 970 µL reaction buffer, 10 µL
heme and 10 µL of inactivated COX-1/COX-2 enzyme (inactivated by incubating in boiling
water for 3 min) in the test tube. Reactions were initiated by adding 10 µL of arachidonic acid
(AA) to each test tube and incubated at 37 °C for 2 min. The enzyme catalysis was quenched by
adding 50 µL of 1 M HCl. PGH thus formed was reduced to PGF2α by adding 100 µL of SnCl
2
2
solution to each test tube. The quantification of prostaglandin formed in each well was done
using specific prostaglandin antiserum and reading the 96-well plate at 405 nm using ELISA
plate reader. The results of this assay have been represented in terms of the percent inhibition of
COX-1 and COX-2 enzymes at 10 µM of inhibitor/standard concentration. The IC values of the
5
0
selected test compounds and standard drug were calculated from concentration-inhibition
response curve.
26

4
.2.2 Carrageenan-Induced Paw Edema Method
The acute anti-inflammatory effect was evaluated by the carrageenan-induced paw edema
assay in Wistar albino rats following the reported protocol [54] in compliance with the relevant
laws on approval with IAEC (Institutional animal ethics committee). The rats were divided into
the following groups: carrageenan control, test compounds, celecoxib and diclofenac as
standards, each comprising six animals. Acute edema was induced by subplantar administration
of 0.1 mL of carrageenan (1%). The test compounds were suspended in 1% Tween-80
suspension and administrated intraperitoneally. Paw volume was measured prior to injection of
carrageenan (0 h) and then at an interval of 1 h up to 3 h using a plethysmograph. The results are
reported as paw volume expressed in mL. The change in paw volume was measured using the
formula:
%
reduction of inflammation = [1 - (V
Where V is the change in paw volume in the test compound treated group, and V
in paw volume in the control group.
t c
/V )×100]
t
c
the change
4
.2.3 Hot plate method
The analgesic activity was carried out using Eddy’s hot plate test. Wistar albino rats of either
sex (180-200 g) were used and divided into different groups: control receiving saline, test
compounds (7d, 8, 9, celecoxib, and diclofenac). The test compounds were suspended in 1%
Tween-80 suspension and administrated in two dose levels: 12.5 and 25 mg/kg intra peritoneal.
o
The temperature of hot plate was set at 50±5 C. The latency of pain responses (reaction time) in
the form of paw licking and jumping responses were noted against thermal stimuli. The cutoff
time was fixed at 15 sec. to avoid paw damage. The experimental procedures used was in
confirmation with the guidelines of CPCSEA and duly approved from IAEC.
27

4
.2.4 Carrageenan-induced inflammatory pain
All the pain parameters were measured prior to carrageenan injection. Paw edema was induced
by subplantar injection of 0.1 ml 1% of carrageenan (suspended in saline) into the left hind paw.
Positive control was dissolved in 1% Tween-80 and was administered orally 1 h before
carrageenan administration. The pain parameters were measured prior to the injection of
carrageenan (0 h) and then at predetermined intervals of 1 h up to 5 h. For each animal, analgesic
activity was measured by inflammatory hyperalgesia model of Hargreaves and mechanical
hyperalgesia.
4
.2.5 Behavioral Parameters
A) Hargreaves Planter Test (Thermal Sensitivity): The Hargreaves Planter (Ugo Basile
7370) used the mild radiant heat to measure thermal nociception in cutaneous hyperalgesia.
(
3
Cutoff time was 15 sec. The time for foot withdrawal response characterizes the pain response
measurement.[58]
(B) von Frey Test (Mechanical Sensitivity): Electro-vonfrey anaesthesiometer (IITC, USA)
was used to assess the mechanical hyperalgesia. The animals were kept on a perforated platform
and von Frey hairs were applied underside of their paw. The pressure (g) at which rats withdraw
their paw was recorded. Three consecutive readings were taken at an interval of 5 min.[59]
(C) Randall-Selitto Test (Paw pressure test): This method uses the Randall-Sellito apparatus
(ALMEMO 2390-5) measured the pain by observing the reaction to gradually increasing
pressure on an inflamed paw. Pain is present if the animal starts to exhibit the fight or struggle
response.[60]
28