Rationally synthesized coumarin based pyrazolines ameliorate carrageenan induced inflammation through COX-2/pro-inflammatory cytokine inhibition

Article abstract of DOI:10.1039/c8md00457a

In the present work, coumarin based pyrazolines (7a-g) have been synthesized and investigated for their in vitro and in vivo anti-inflammatory potential. Amongst the synthesized compounds, compounds 7a, 7d and 7f exhibited significant in vitro anti-inflammatory activity as compared to the standard etoricoxib. Keeping this in mind, in vivo investigations were carried out via carrageenan induced inflammation and acetic acid induced writhing models in male Wistar rats and compound 7a was found to possess appreciable anti-inflammatory and analgesic potential. The mode of action of compound 7a was also investigated by using substance P as the biomarker, which shows promising results. Further, the selectivity of the most active compound 7a against the cyclooxygenase enzyme was supported by molecular docking studies which reveal that compound 7a has greater binding affinity towards COX-2 over COX-1 and 5-LOX enzymes. In silico ADME analysis of compound 7a confirms the drug-like characteristics and the in vivo acute toxicity study showed the safety of the compound even up to a 2000 mg kg^?1 dose. Thus, compound 7a was identified as an effective anti-inflammatory agent, and can be explored for further analgesic/anti-inflammatory drug design and development.

Full text of DOI:10.1039/c8md00457a

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Rationally Synthesized Coumarin Based Pyrazolines ameliorates carrageenan induced
inflammation through COX-2/Pro-inflammatory cytokine inhibition
Priyanka Chandel,a Anoop Kumar,b Nishu Singla,a Anshul Kumar,a Gagandeep Singh,c*
Rupinder Kaur Gilla*
a Deparment of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga-142001, Punjab, India.
bDepartment of Pharmacology, ISF College of Pharmacy, Moga-142001, Punjab, India.
cDepartment of Chemistry, Indian Institute of Technology-Ropar-140001, Punjab, India.
*Corresponding author: G. Singh, E-mail: gagandeep.singh@iitrpr.ac.in;
R.K. Gill, E-mail: rupinder.pharmacy@gmail.com;
Tel.: +91 1636 324200; fax: +91 1636 239515.
Abstract
In the present work, coumarin based pyrazolines (7a-g) have been synthesized and investigated
for their in-vitro and in-vivo anti-inflammatory potential. Amongst the synthesized compounds,
compound 7a, 7d and 7f exhibited significant in vitro anti-inflammatory activity as compared to
the standard etoricoxib. Keeping this in mind, the in-vivo investigations were carried out via
carrageenan induced inflammation and acetic acid induced writhing models in male Wistar rats
and the compound 7a was found to possess appreciable anti-inflammatory and analgesic
potential. The mode of action of compound 7a was also investigated by using substance P as
biomarker, which shows promising results. Further, the selectivity of most active compound 7a
against cyclooxygenase enzyme was supported via the molecular docking studies which reveals
that compound 7a has more binding affinity towards COX-2 over the COX-1 and 5-LOX
enzymes. In-silico ADME analysis of compound 7a confer the drug like characteristics and in
vivo acute toxicity study showed the safety of the compound even up to 2000 mg kg-1 dose.
Thus, compound 7a was identified as an effective anti-inflammatory agent, and can be explored
for further analgesic/anti-inflammatory drug design and development.
Keywords: Coumarin-pyrazolines; COX-2 selective; Anti-inflammatory activity; Cytokine
inhibition; Molecular docking analysis.
1

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1. Introduction
Inflammation is a body’s defense response against injury, infectious agents and autoimmune
reactions.1 However, the outcome of chronic inflammation involves the tissue destruction and
fibrosis, which may lead to the rheumatoid arthritis,2 cancer,3,4 neurodegenerative disorder5 and
cardiovascular diseases.6,7 Several markers played an important role in inflammation including
cytokine receptors, nuclear factor kappa-B (NF-κB), nitric oxide synthase (NOS), tumor necrosis
factor alpha (TNF-α), interferons, chemokines and pro-inflammatory enzymes COX-2 and LOX
(lipoxygenase).8-10 Among them, cyclooxygenase and lipoxygenase are the real culprits, and
thus, are the primary targets of the anti-inflammatory agents.11 Cyclooxygenases causes the
inflammation via arachidonic acid metabolism by catalyzing the formation of prostaglandins H2,
a precursor for the biosynthesis of prostacyclins, prostaglandins, and thromoboxane that virtually
affects diverse biological processes such as regulation of immune function, and maintenance of
renal blood flow, reproductive biology, and gastrointestinal integrity.12 Studies have
demonstrated that COX exists in multiple isoforms, each with its own physiological expression
and function.13 Two main isoforms are COX-1, designated as ‘housekeeping’ enzyme present in
almost all cells and tissues, and regulates homeostasis and blood clotting, whereas, COX-2 is an
inducible enzyme expressed in cells that mediates inflammation such as synoviocytes,
macrophages, and monocytes leading to the synthesis of the prostanoids involved in acute and
chronic inflammatory conditions. In this context, non-steroidal anti-inflammatory drugs
(NSAIDs) are well recognized drugs for the treatment of inflammatory diseases which exerts
their therapeutic effects by preventing the metabolism of arachidonic acid via inhibition of COX
enzymes.14 However, the conventional NSAIDs, including non-selective COX-1/2 inhibitors
such as aspirin, ibuprofen, indomethacin and diclofenac are associated with gastric side effects,
whereas, selective COX-2 inhibitors (COXIBs) such as celecoxib 1 prevent these side effect of
non-selective NSAIDs.15-18 The severe side effects of clinically used NSAIDS include
gastrointestinal lesions,19 cardiovascular diseases20,21 and renal injury,22 which necessitated the
development of new chemical entities having higher efficacy and low/no side effects.
Coumarins have attracted intense interest due to their wide range of applications in
pharmacological chemistry such as anticancer,23 anti HIV,24 antimicrobial,25 anticoagulant,
antioxidant,25,26 antiulcer,27 dyslipidemic,28 antitumor,29 and anti-inflammatory.30 Literature
2

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reports revealed variedly substituted coumarins, pyrazoles, isoxazoles/isoxazolidines and
oxadiazoles etc. as potential anti-inflammatory and analgesic agents via inhibition of
cycloxygenase/pro-inflammatory cytokines (Figure 1).31-38
R
COOCH3
COOCH3
COOCH3
COOCH3
N
N
O
O
N
O
N
NH
O
N
O
NH
N
O
O
O
O
O
R = CF3, Cl, F
III
J. Heterocyclic Chem.,
IV
I
II
J. Heterocyclic Chem., Bioorg. Med. Chem. Lett.,
J. Enzyme Inhib. Med. Chem.,
2001
, 38, 717-722
2001
2014
2011
, 26, 767-776
, 38, 717-722
, 24, 4638-4642
O
Cl
N
R
R1
N
NH
O
HN
NH
O
N
N
N
H
O
O
N
N
R2
N
O
N
H
N
O
O
N
H
R1 = H, R2 = H
R1 = Br, R2 = H
N
N
N
O
O
O
O
O
IX
J. Med. Chem.,
2018
VII
Eur. J. Med. Chem., Eur. J. Med. Chem.,
2017 2018
VIII
VI
V
Eur. J. Med. Chem.,
2014
ACS Omega,
2018
, 61, 7929-7941.
, 127, 210-222
, 153, 56-64
, 77, 185-192
, 3, 5825-5845
Figure 1: Variedly substituted molecules as potential anti-inflammatory/analgesic agents.
It is well established that a common structural feature of selective COX-2 inhibitors includes the
presence of two vicinal aryl rings or 1,3-aryl groups attached with central five or six-membered
heterocyclic or carbocyclic motif. On the other hand, various marketed COX-2 inhibitors
comprises sulfone moiety which was found to be responsible for their prooxidant activity. This
prooxidant effect could be related with the adverse effects observed with rofecoxib and
etoricoxib due to which these drugs have not been approved by FDA for the U.S. market.39a On
the basis of these considerations, the present work describes the rational design and synthesis of
coumarin-pyrazoline in which coumarin nucleus was envisaged as a probable replacement for
one of the aryl ring in selective COX-2 inhibitor (celecoxib 1), and 3-acetyl pyrazoline as central
five membered carbocyclic ring with the underlying anticipation that the designed compounds
would have a selective COX-2 inhibitory effect with low/no side-effects. Further, the ortho- two
aryl groups present in marketed selective COX-2 inhibitors were replaced with meta- two aryl
moieties in the newly synthesized compounds (7a-g) resulting in more bulky compounds, that
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could maximize the interaction with the hydrophobic residues within COX-2 active site and
enhance COX-2 selectivity (Figure 2).
Earlier coumarin-pyrazolines have been reported for their anti-tumor activity,39b however, to the
best of our knowledge, their anti-inflammatory activity has not been reported yet, therefore,
keeping in mind their pharmacological potential and their structural resemblance with selective
COX-2 inhibitors such as celecoxib, herein, we have re-synthesized coumarin-pyrazoline
derivatives 7a and 7e along with some novel derivatives 7b, 7c, 7d, 7f and 7g and evaluated
them for detailed anti-inflammatory and analgesic activity.
Figure 2: Marketed COX-2 selective inhibitors and design considerations for targeted coumarin-
pyrazolines (7a-g).
2. Results and Discussion
2.1. Chemistry
The synthetic methodology employed for the synthesis of target compounds is illustrated in
Scheme 1. The reaction of salicylaldehyde 2 with ethyl acetoacetate 3 in the presence of
piperidine at RT yielded the 3-acetyl coumarin 4.40 Literature reports both acid-base catalyzed
methods for the preparation of coumarin-chalcones derivatives, for instance, in the presence of
piperidine (at 80oC),41, 42 10% NaOH43 and 40% NaOH.44 Herein, we have reported the synthesis
of coumarin-chalcones by the reaction of 3-acetyl coumarin 4 with substituted benzaldehydes 5
using few drops of conc. H2SO4 in glacial acetic acid in excellent yields. Subsequently, chalcone
4

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6 was cyclized in the presence of hydrazine hydrate in glacial acetic acid to furnish the desired
coumarin-pyrazolines 7a-g in high yields (Table 1).45
The synthesized compounds were purified by column chromatography over silica gel and
1
13
1
characterized spectroscopically (IR, H NMR, C NMR and HRMS). For instance, in the H-
NMR spectrum of compound 7b, the appearance of a double doublet (dd) of one proton at δ 5.50
ppm, was ascribed to chiral C-5 proton of pyrazoline, whereas, the methylene hydrogens of
pyrazoline ring were appeared as two double doublets at 3.88 (1H) and 3.42 (1H) ppm. Two
singlets at δ 3.81 ppm and δ 3.77 ppm were ascribed to OCH3 protons, whereas, a singlet at δ 2.43
13
ppm correspond to CH3 protons. The C NMR resonance of acetyl carbonyl was appeared at
169.15 and others corroborated well with the structure of compound 7b. The peak corresponding to
the mass of 7b with m/z 423.1569 (calcd m/z 423.1556 [M+H]+) was observed in the high
resolution mass spectra (HRMS), which in agreement with the molecular formula for assigned
structure.
R
O
O
O
CHO
O
O
Piperidine
RT
5
H
+
O
R
conc.H2SO4, CH3COOH,
RT, 18-24 h
O
OH
4
2
6
3
O
N
O
N
R
N2H4.H2O
O
O
O
O
7a-g
CH3COOH,
80oC, 8 h
R = H, 4-OCH3, 2-OCH3,
3,4,5-OCH3, 4-Cl, 4-NO2, 4-OH
Scheme 1
7a-g
.
. Synthesis of Coumarin-pyrazolines
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Table 1: Reaction time and (%) yields for compound 7a-g
Compound
R
4-OCH3
3,4,5-tri-OCH3
4-Cl
4-NO2
H
Time (h)
(%) yield
82.1
7a
7b
7c
7d
7e
7f
8
8
8
8
8
8
8
85.5
78.1
73.5
66.1
65.0
75.9
4-OH
2-OCH3
7g
2.2. Pharmacological Evaluation
2.2.1. In vitro anti-inflammatory activity:
All the prepared compounds (7a-g) were evaluated32 for their potential to inhibit COX-1 and
COX-2 using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals Inc.,
Ann Arbor, MI, USA) and results obtained are presented in Table 2. The results of inhibition
assay reveal that all the prepared compounds exhibits COX-2 selective inhibition as compare to
COX-1. In particular, compound 7a selectively inhibit COX-2 (IC50 = 0.41µM) over COX-1
which is much comparable to Etoricoxib (IC50 0.23µM). In addition, compound 7d and 7f were
also shows potent inhibitory activity against COX-2 with IC50 = 1.79 and 2.39µM, respectively.
Table 2: In vitro COX-1 and COX-2 inhibitory activity of coumarin-pyrazolines 7a-g
Compound Code
COX inhibition (IC50, µM)
COX-1 COX-2
22.4 0.41
7a
7b
7c
7d
7e
7f
7g
>30
ND
16.2
ND
20
4.35
ND
1.79
ND
2.39
4.37
0.23
>30
>30
Etoricoxib
ND-Not determined
Inhibition of albumin denaturation
Denaturation of proteins is a well-documented cause of inflammation.46, 47 Thus, the synthesized
compounds were investigated for their protective ability against albumin denaturation that could
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indicate their anti-inflammatory capability. The results shown in Table S1 and Figure 3a
indicate that synthesized compounds were effective in protecting heat induced albumin
denaturation in a concentration dependent manner. Particularly, compound 7a has shown more
significant protection against denaturation. At 100µg/ml, compound 7a has shown 70%
protection as compared to standard (72%).
Membrane stabilization assays
The human red blood cell (HRBC) membrane stabilization has been used as an approach to
examine the in-vitro anti-inflammatory activity because the erythrocyte membrane is analogous
to the lysosomal membrane and its stabilization implies that the compounds may well stabilize
the lysosomal membranes.48,49 Stabilization of lysosomal is important in limiting the
inflammatory response by preventing the release of lysosomal constituents of activated
neutrophils, such as bacterial enzymes and proteases, which causes further tissue inflammation
and damage upon extracellular release. Therefore, to check the effect of synthesized compounds
on lysosomal membrane, membrane stabilization assays (heat and hypotonicity induced
hemolysis) have been performed.
Heat induced hemolysis:
The synthesized coumarin-pyrazolines (7a-g) inhibited the heat induced hemolysis in
concentration dependent manner as shown in Table S2 and Figure 3b. Among all the
synthesized compounds, compound 7a has shown more significant protection against the
damaging effect of the heat compared to the control. At 10, 25, 50 and 100 µg/ml, the compound
7a has exhibited protection against heat induced hemolysis by 60, 63, 67 and 69%, respectively
as compared to standard Etoricoxib 65, 73, 78 and 76%, respectively.
Hypo-tonicity induced hemolysis:
The results showed that synthesized coumarin-pyrazolines at concentration range (10-100
μg/ml) protect the erythrocyte membrane against lysis induced by hypotonic solution (Table S3
and Figure 3c). Among all the synthesized compounds, compound 7a has shown more
significant protection against the damaging effect of the hypotonic solution compared to control.
At 10, 25, 50 and 100 µg/ml, the compound 7a has exhibited protection against hypotonicity
induced hemolysis by 62, 63, 67 and 68%, respectively as compared to standard Etoricoxib 52,
62, 66 and 69%, respectively.
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Figure 3: In vitro anti-inflammatory activity a) inhibition of albumin denaturation b) inhibition of heat
induced hemolysis c) inhibition of hypotonicity induced hemolysis. Data is expressed as mean
± S.D. Significantly different ap< 0.05 as compared to Etoricoxib, bp< 0.05 as compared to 7a,
cp<0.05 as compared to 7b using ANOVA.
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2.2.2. In vivo anti-inflammatory activity
The synthesized coumarin-pyrazolines were further tested for in vivo anti-inflammatory activity
using carrageenan paw edema method.38 Male Wistar rats (4–6 weeks old), weight between 150
to 200 g were selected for the in vivo study. Animals were divided into following five groups:
Group I was treated with vehicle and served as control and group II was treated with carrageenan
(0.05ml of 1%). In other groups (Group III-X), the compounds were administered at doses of 25
and 50 mg kg−1. After 30 min of the treatment with Etoricoxib or the test compounds, acute
inflammation was induced by subplantar injection of 0.05 mL of freshly prepared 1% suspension
of carrageenan. The volume displacement was measured by using plethysmography and recorded
after 3h, 6 h and finally at 24h. The displacement volume was significantly increased in
carrageenan treated group of animals as compared to control group. Among the synthesized
compounds, compounds 7a, 7d and 7f showed decrease in displacement volume as compared to
carrageenan group in time dependent manner. There was no significant reduction in
displacement volume when doses were increased from 25 to 50 mg kg-1. Among all tested
compounds, at 0.5, 3, 6 & 24 hrs, compound 7a has shown more significant reduction in
displacement volume as that of standard drug (Figure 4). Particularly, compound 7a at 24 h
reduced the displacement volume remarkably by 67.39% which is similar to standard Etoricoxib,
whereas, compounds 7d and 7f reduced the displacement volume by 52.17 and 55.43%,
respectively as compared to carrageenan.
Figure 4: In-vivo anti-inflammatory activity of synthesized compounds (7a-g). Data is expressed
as mean ± S.D. ap<0.001vs control, bp<0.001 vs carrageenan.
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2.2.3. Effect on cytokines level
The immune cells modulate the inflammatory responses by release of pro-inflammatory
cytokines and contribute to the inflammatory and autoimmune disorders. Thus, the cytokines
level were estimated by using rat TNF-α, IL-6, and IL-1β immunoassay kit (KRISHGEN
Biosystem, Ashley Ct, Whittier, CA).38 The tested compounds 7a, 7d and 7f have shown
significant decrease in the level of TNF- α, IL-6 and IL-1β which was increased by carrageenan.
However, compound 7a has shown more significant (p<0.001) reduction in the level of TNF-α,
IL-6, and IL-1β as compared to compound 7d and 7f (Figure 5).
Figure 5: Effect of i.p. administration of compounds 7a, 7d and 7f on the serum levels of
#
*
**
cytokines. Data is expressed as mean ± S.D. p<0.001 vs control, p<0.05, p<0.01,
***p<0.001 vs carrageenan.
2.2.4. In vivo analgesic activity
The analgesic activity of most potent compound 7a was assessed by the acetic acid induced
abdominal constriction (writhing) test.38 Animals were divided into three groups of six animals
each. Group 1 animals were administered with vehicle and served as vehicle control. Group II
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were administered compound 7a and Group III animals were administered Etoricoxib. One hour
after vehicle and drug administration, each animal was injected with 3% (v/v) acetic acid, 300
mg/kg i.p. The number of stretches or writhes (arching of the back, elongation of the body and
extension of the forelimbs) were counted cumulatively over a period of 20 min. The compound
7a has been observed to significantly reduce the abnormal writhings by 70.5% as compared to
control, whereas, Etoricoxib produced 57.6% inhibition of writhing (Figure 6).
Figure 6: Effect of Compound 7a on acetic acid induced writhing. All the values are expressed
as mean ± S.E.M. a p < 0.05 Vs control, b p < 0.05 Vs Etoricoxib.
.
2.2.5. Studies Supporting the Probable Mode of Action of the Compounds
It is well documented that the substance P (SP) is an important neuropeptide involved in
neurogenic inflammation and it exerts various pro-inflammatory actions on immune-cells,
including macrophages. Prostaglandin E2 (PGE2), a potent lipid mediator produced in nearly
every cell type where it is synthesized from arachidonic acid via the actions of cyclooxygenase
enzymes (COX-1 and COX-2).50 COX-1 is constitutively expressed and important for normal
regulation of vascular activity and gastrointestinal function, whereas, COX-2 is an inducible
enzyme stimulated by numerous mitogens, cytokines, oxidants, and microbial products.51 In
particular, COX-2 is primarily responsible for the synthesis of PGE2, and elevates inflammation
by increasing vasodilation, vascular permeability, and edema in numerous respiratory diseases,
such as acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis, and lung
cancer. Some studies suggest that COX-2 and PGE2 might be associated with Substance P-
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related inflammatory responses.52-54 Thus, to study the potential target of this compound,
substance P (COX and LOX pathway stimulator) was administered to the animals 30 min before
the administration of most potent compound 7a and then the acetic acid was injected after 30
min.55 Pretreatment of rat with substance P results in the reversal of analgesic effect of
compound 7a as shown in Figure 7. Further, the compound 7a possesses poor inhibition of 5-
LOX (IC50 of 12μM) as compared to COX-2 (IC50 of 0.41μM) which clearly indicated that
COX-2 may be the probable target of 7a. These results suggest the compound 7a has analgesic
effect in acetic acid induced algesia due to inhibition of cyclooxygenase-2 pathway.
Figure 7: Effect of substance P pretreatment on analgesic effect of compound 7a. All the values
are expressed as mean ± S.E.M. a p < 0.05 Vs control, b p < 0.05 Vs compound 7a.
2.2.6. Acute toxicity studies
Acute toxicity studies on most active compound were carried out on either sex rats as stated in
OECD guidelines.61 Four groups of animals were included in this study with three animals per
group. The first group of animals was served as control and the second, third, and fourth groups
were treated with tested compound 7a at varied doses upto 2000 mg kg−1, respectively, after 4 h
of fasting. All the animal groups were continuously observed for the first 4 h and periodically for
24 h. After 14 days, histological studies were performed via H and E staining by sacrificing one
animal each from the control and the highest dose (2000 mg kg−1). The animals treated with
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under investigation compound 7a at the dose of 50 mg/kg displayed no behavioral changes,
whereas, the animals administered with the dose of 300 and 2000 mg/kg produces some
irritation, itching, sneezing occasionally after 1-4 h of treatment. However, it is to be mention
here that these symptoms diminish within the next 6 h and no significant changes were observed
in the post-mortem analysis of myocardium, liver or kidney tissues of the animals administered
with the highest dose of compound 7a (2000 mg kg-1) as compared to the control group (Figure
8).
Figure 8: Photomicrographs of hematoxylin-eosin stained sections of control myocardium (A),
kidney (B), and liver (C) and compound 7a (2000 mg kg−1)-treated myocardium (D),
kidney (E), liver (F). The pictures were taken with light microscope at ×200
magnification
2.2.7. Molecular docking study
Molecular docking study was performed using Gold score 5.1 software to predict and compare
the selectivity of coumarin-pyrazoline derivatives against COX-2 (PDB 3LN1),56,57 COX-1
(PDB 3KK6)56,58 and 5-LOX (PDB 3V99) enzymes.56,59 The affinities of synthesized compounds
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were evaluated in terms of Gold score and it was observed that the synthesized compounds 7a-g
showed good binding interaction with the active site of COX-2 enzyme as compared to COX-1
and 5 LOX, which were also taken in consideration to evaluate the selectivity (Table S4).
Further, molecular docking results revealed that the most potent compound 7a was fitted well to
the active site of COX-2 with Gold score = 88.25, as compare to celecoxib (Gold Score = 96.42;
Table S4). The carbonyl of coumarin is involved in hydrogen bonding with Tyr341 and actetyl
group of pyrazoline interacts with Ser516. The aromatic ring of coumarin also showed π-H
hydrophobic interaction with Val 509 (Figure 9A). This may explain the selectivity of the
synthesized coumarin pyrazolines against COX-2 enzyme. The complete overlay of standard
celecoxib was observed with compound 7a as shown in Figure 9B. The docking results suggest
that compound 7a may potentially and selectively target the COX-2 enzyme. In silico ADME
study on synthesized compounds 7a-g was performed using the Swiss ADME predictor to
predict the absorption, distribution, metabolism and elimination (ADME) parameters (Table S5).
It was observed that all the coumarin pyrazolines 7a-g, showed the ADME properties in the
acceptable range, with no violation according to the Lipinski’s rule of five.60 Therefore, these
compounds can be further utilized to be developed as drug candidates.
Figure 9: A) Docking of compound 7a in the active site of COX-2; B) Overlay representation of
celecoxib with compound 7a.
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3. Conclusion
Coumarin based pyrazolines 7a-g were synthesized and evaluated for in-vitro and in-vivo anti-
inflammatory activity. From the in-vitro and in-vivo experiments, compound 7a was identified as
the highly active anti-inflammatory agent comparable to the standard Etoricoxib. Compound 7a
significantly suppress the cytokines level and showed remarkable analgesic and anti-
inflammatory activity with minimum toxicity risk. Testing of this compound on Wistar rat
showed reversal of algesia and inflammation with comparable efficacy to the standard drug
Etoricoxib by targeting specifically COX-2. Further, molecular docking studies supported that
COX-2 is the possible selective target for mode of action of compound 7a, which was also
evident by in vivo mechanistic studies using substance P as biomarker. The obtained results
suggest that compound 7a would be used as potential lead for the development of efficient anti-
inflammatory agents.
Acknowledgements
Authors are thankful to Mr. Praveen Garg, Chairman, ISF College of Pharmacy, Moga, Punjab,
for providing necessary infrastructure and his continuous support and encouragement.
Conflict of interest
The authors declare that they have no conflict of interests.
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GRAPHICAL ABSTRACT
Leave for space
Rationally Synthesized Coumarin Based Pyrazolines
ameliorates carrageenan induced inflammation through
COX-2/Pro-inflammatory cytokine inhibition
Priyanka Chandel,a Anoop Kumar,b Nishu Singla,a Anshul Kumar,a
Gagandeep Singh,c* Rupinder Kaur Gill a*
a
Deparment of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga-142001, Punjab, India.
bDepartment of Pharmacology, ISF College of Pharmacy, Moga-142001, Punjab, India.
cDepartment of Chemistry, Indian Institute of Technology- Ropar-140001, Punjab, India.