Design, synthesis and biological evaluation of piperic acid triazolyl derivatives as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2015.01.001

Nineteen novel piperine based triazoles have been synthesized using click chemistry approach and were tested for in vivo anti-inflammatory activity. The most active compounds were evaluated for in vitro TNF-α expression. Compounds 3g and 3f were found to

Full text of DOI:10.1016/j.ejmech.2015.01.001

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Piperic Acid Triazolyl Derivatives as
Potent Anti-inflammatory Agents
Yakub Ali , Mohammad Sarwar Alam , Prof., Hinna Hamid , Asif Husain , Sameena
Bano , Abhijeet Dhulap , Chetna Kharbanda , Syed Nazreen , Saqlain Haider
PII:
S0223-5234(15)00002-1
10.1016/j.ejmech.2015.01.001
EJMECH 7617
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2014
Revised Date: 6 December 2014
Accepted Date: 1 January 2015
Please cite this article as: Y. Ali, M.S. Alam, H. Hamid, A. Husain, S. Bano, A. Dhulap, C. Kharbanda,
S. Nazreen, S. Haider, Design, Synthesis and Biological Evaluation of Piperic Acid Triazolyl Derivatives
as Potent Anti-inflammatory Agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.01.001.
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Graphical abstract:

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Piperic Acid Triazolyl Derivatives as
Potent Anti-inflammatory Agents
Yakub Alia, Mohammad Sarwar Alama*, Hinna Hamida, Asif Husainb, Sameena Banoa,
Abhijeet Dhulapc, Chetna Kharbandaa, Syed Nazreena, Saqlain Haidera.
aDepartment of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New
b
Delhi-110 062, India. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia
c
Hamdard (Hamdard University), New Delhi-110 062, India. CSIR Unit for Research and
Development of Information Products, Pune -411038.
*Correspondence to Prof. Mohammad Sarwar Alam, Email ID msalam@jamiahamdard.ac.in,
msalam5555@gmail.com Phone: +91 11 26059688 (5555), Fax: +91 11 26059663
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Abstract
Nineteen novel piperine based triazoles have been synthesized using click chemistry approach
and were tested for in vivo anti-inflammatory activity. The most active compounds were
evaluated for in vitro TNF-α expression. Compounds 3g and 3f were found to show
significant in vivo inhibition of inflammation, 80.40 % and 76.71%, respectively after 5h in
comparison to piperine (54.72%) and the standard drug indomethacin (77.02%) without
causing any damage to the stomach. Compounds 3g and 3f suppressed TNF-α level by
73.73% and 70.64%, respectively and protein expression of COX-2, NF-κB and TNF-α more
than indomethacin. Moreover, the compound 3g was found to show significant analgesic
activity of 54.09% which was comparable with the indomethacin (57.43%).
Keywords:
Piperine, Click chemistry, TNF-α, Cytotoxicity, Immunohistochemistry,
Molecular docking.
Abbreviations: Tumour necrosis factor (TNF-α), Cyclooxygenase (COX), Lipid peroxidation
(LPO), Non steroidal anti-inflammatory drug (NASIDs)
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1. Introduction
Piperine is a major alkaloid constituent of piper species, including Piper nigrum Linn
and Piper longum Linn. It is commonly used in various traditional systems of medicines [1].
Piperine has been reported to exhibit various types of pharmacological activities such as anti-
inflammatory [2], anti-oxidant [3], anti-tumor [4], anti-asthmatic [5], hepato-protective [6],
anti-thyroid [7], and anti-depressant [8]. Most of non steroidal anti-inflammatory drugs
(NSAIDs) used for the treatment of inflammation show adverse effects such as gastric ulcer
[9], kidney damage [10] and hepatotoxicity [11]. Therefore, the development of NSAIDs with
reduced side effects is still underway all over the world. Plant-derived drugs used in the
traditional systems of medicine for the treatment of pain, hay fever and inflammatory ailments
have received considerable attention as they are cheap and have no or little side effects [12].
1,2,3-triazoles have been reported to possess a wide range of pharmacological properties
including anti-inflammatory, analgesic, anti-microbial, anti-convulsant, anti-neoplastic, anti-
malarial, and anti-viral [13-20]. In view of the biological importance of piperine and 1,2,3-
triazoles as anti-inflammatory agents we have synthesized piperine based 1,2,3-triazoles as
potent anti-inflammatory agents with reduced side effects. We have conjugated these two
moieties under one construct using click chemistry approach. The synthesized molecules were
evaluated for their in vivo anti-inflammatory activity. In silico molecular docking study has
been done to study the binding interactions of the synthesized compounds with TNF-α protein.
The compounds showing significant anti-inflammatory activity and good binding affinity with
TNF-α target were further evaluated for their effect on in vitro TNF-α level. Furthermore
immunohistochemical studies have also been done to explore the effect of the active
compounds on expression of COX-2, NF-Κb and TNF-α protein. The synthesized compounds
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have also been screened for their anti-nociceptive potential. Lipid peroxidation and
ulcerogenic risk evaluation studies have also been carried out.
2. Results and discussion
2.1.
Chemistry
For the synthesis of piperine based 1,2,3-triazole library, propargylated ester of piperic
acid was prepared by reacting piperic acid with propargyl bromide in the presence of cesium
carbonate in dry THF. 1,3-dipolar cycloaddition reaction between the propargylated ester and the
aromatic azides resulted in the synthesis of the final compounds. The structural confirmation of
target compounds (3a-s) was done by 1H NMR (Brucker Avance II 300 NMR Spectrometer) 13C
NMR (Brucker Avance II 400 NMR Spectrometer) and mass spectral (LCQ Fleet) data as well
as elemental analysis (Elementar GMBH) (Scheme-1). In the 1H NMR spectra, the formation of
triazole derivatives was confirmed by the presence of proton of triazole ring as a singlet in the
range of δ 7.99 to 8.54 ppm along with methylene protons appearing at δ 5.35 to 5.87 ppm. The
double doublet of H-3 appeared in the range of δ 7.47 to7.49 ppm and the singlet of H-4
appeared in the range of δ 5.91 to 6.05 ppm (fig A).The remaining two protons H-1and H-2
appeared in aromatic region as expected. ESI-MS of all the compounds showed [M]+ and
[M+H]+ peaks with reasonable intensity.
2.2.
Anti-inflammatory activity
All the synthesized compounds have been tested for their in vivo anti-inflammatory
activity by carrageenan induced rat paw edema model. The results (table 1) indicated that
compounds 3e-g & 3o-q possess significant anti-inflammatory activity. Amongst these
compounds, the compound 3g showed better activity (76.12 % at 3h and 80.40% inhibition at
5h) as compared to the standard drug indomethacin (72.05% at 3h and 77.02 at 5h).
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The compounds 3e, 3f, 3o, 3p, and 3q also showed significant anti-inflammatory activity
(70.81%, 76.71%, 71.21%, 72.97%, and 70.27%) respectively which is comparable to
standard drug indomethacin at 5 h of inflammation (77.02%) but higher than that of piperine
which showed 54.72% inhibition at 5 h.
2.3.
In silico docking studies
It has been reported in earlier studies that piperine can significantly lower the level of
TNF-α, which is one among the important pro-inflammatory cytokines. It is expected that the
anti-inflammatory effect of piperine might attribute to the inhibition of TNF-α.[21] Therefore,
the synthesized piperine derivatives were docked against TNF-α to analyze their binding
pattern and energies inside the receptor pocket. Crystallized structure of 2AZ5 was chosen
from protein data bank and used as a target for molecular docking studies with the specific
ligand indomethacin which inhibits it [22]. The synthesized derivatives were docked
individually against the generated grid and were found to show good binding energies ranging
from -34.24 to -42.41 kcal/ mol. Amongst all the synthesized molecules, the most promising
molecules were 3g, 3a, 3f, 3o, 3h, 3e, 3p, and 3q which exhibited a glide score of -5.81, -
5.80, -5.78, -5.67, -5.61, -5.59,-5.32 and -5.13 respectively whereas the glide score of
compounds piperine and indomethacin were found to be -4.42 and -5.02 respectively. Alike
indomethacin, the most active compound (3g) were found to align perfectly with the
hydrophobic pocket of the TNF-α protein where as piperine was found to form hydrogen
bonding with LYS-98 residue. The glide score, binding energies of all the synthesized
compounds are shown in table 2.
2.4. TNF-α assay
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Compounds showing significant in vivo anti-inflammatory activity were further
evaluated for in vitro TNF–α level in LPS induced RAW 264.7 cell lines. The results are
shown in fig 2. It was found that compound 3g and 3f showed better suppression of 73.73%
& 70.64% in TNF-α concentration as compared to the standard drug indomethacin as well as
piperine which decrease TNF-α level by 67.99% and 49.67% respectively. The compounds
3p (63.58%) and 3o (60.71%) also showed significant decrease in TNF-α concentration.
From the above results, it is clear that compounds 3g and 3f showing significant in vivo anti-
inflammatory activity and glide scores also significantly decreased the level of TNF-α in LPS
induced RAW 264.7 cell line.
2.5.
Cytotoxicity profile
Cytotoxicity effect of active compounds in RAW 264.7 macrophages was carried out
by MTT assay. The results of cytotoxicity assay are shown in Fig 3. The results clearly
indicates that the cell viability of compounds 3f, 3g, 3o and 3p was more than 90% i.e. these
compounds did not cause any abnormal cell death as compared to standard drug indomethacin
which has cell viability of 55% only.
2.6. Antinociceptive activity
Compounds showing significant anti-inflammatory activity were also further tested for
their in vivo analgesic activity by writhing test method. The results of analgesic activity are
shown in table 3. Amongst all the active compounds, compound 3g showed significant
analgesic activity (54.09%) in comparison to standard drug indomethacin (57.43%)
Compounds 3f, 3o and 3p also showed good analgesic activity.
2.7.
Ulcerogenic studies
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The compounds 3f, 3g, 3o and 3p showing most potent in vivo anti-inflammatory
activity were further evaluated for ulcerogenic risk. They were not found to cause any damage
to the stomach of the animal, whereas standard group (indomethacin) treated animals caused
mucosal and epithelium tissue damage. Piperine treated animals also did not show any
damage to the stomach of animals (fig4)
2.8. Lipid peroxidation assay
The result of LPO is shown in fig 5 and it is measured as nmol of MDA per 100 mg of
gastric mucosa tissue. It has been observed that carrageenan increased the level of LPO in
animals. However the level of LPO was found to decrease significantly in the presence of the
standard drug and the test compounds 3f, 3g, 3o and 3p.
2.9.
Immunohistochemistry
The modulation of cellular signalling network involved in induction and activity of pro-
inflammatory cytokines like TNF-α, COX-2 and NF-κB has been considered a paradigm for
preventing inflammation [21]. Therefore in the present study the potential of the active
compounds in suppressing these cytokines was studied. NF-κB plays an important role in
maintaining immune response by regulating the production of cytokines responsible for
inflammation [22]. NF-κB stimulates the expression of enzymes such as nitric oxide synthase
(iNOS) which is involved in the pathogenesis of inflammatory process. Therefore, the activation
of NF-κB acts as a key mediator in controlling inflammatory responses. Cyclooxygenase enzyme
mediates the formation of several biological precursors [23]. Three isoenzyme of COX mainly
are COX-1, COX-2 and COX-3. COX-2 is inducible and gets activated at the site of
inflammation. The effects of active compounds 3f and 3g on paw tissue expression of COX-2
and NF-κB are shown in fig 7a and 7b. Group II i.e. only carrageenan treated group showed
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more intense brown color which clearly indicated increase in the number of cells with COX-2
and NF-κB expression as compared to that of normal control group (I) which showed no
expression of COX-2, NF-κB and TNF-α. The treatment of animals with indomethacin and
active compound 3f and 3g reduced the number of cells showing expression of these proteins.
However compounds 3f and 3g showed more reduction in the expression of these proteins as
compared to standard drug.
3. Structure activity relationship
The structure activity relationship of the synthesized compounds has been analyzed as
follows.
Ring A
O
O
N
3
N
N
4
1
2
R
O
O
Fig A
•
•
Presence of electron withdrawing groups on the aryl moiety decreased the in vivo anti-
inflammatory activity as compared to the presence of electron donating groups.
Better in vivo anti-inflammatory activity was observed for compounds having halogen at
ortho position of the aromatic ring A as compared to halogen attached at para position.
Increasing order of activity found was F< Cl<Br.
•
Compound with methoxy group at ortho position of aromatic ring A (3m) showed better
activity (in vivo) as compared to compound with methoxy at para position (3l ).
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•
•
Compound with methyl group at para position showed less activity (3i) as compared to
when methyl group is at ortho position (3j).
Compound (3q) with di-subsitituted aromatic ring (methyl group) showed better activity as
compared to when mono substituted methyl group is present (3i, 3j).
Position of chlorine groups at aromatic ring (3o and 3p) did not affect the activity.
Ethoxy group substituted compound (3n) showed less activity as compared to compound
having ethyl substituent (3k).
•
•
4. Conclusion
It may be concluded that the compound 3g showed better anti-inflammatory and
significant analgesic activity in vivo as compared to piperine and the standard drug
indomethacin. The anti-inflammatory activity of the compound 3g could be attributed to its
inhibition of TNF-α in addition to the reduction in protein expression of COX-2, NF-Κb and
TNFα. Furthermore, no ulcerogenic and cytotoxic effect was observed for compound 3g.
Therefore, the compound3g may be considered as a promising candidate for development of new
and safer anti-inflammatory agents.
5. Experimental protolcol
5.1.
Isolation of piperine
The coarsely powdered fruits of Piper nigrum (3kg) were extracted three times with
chloroform by cold maceration for 24 h. The extract was filtered and concentrated to dryness at
60oC on a water bath. The residue was dissolved in minimum quantity of chloroform and
addition of diethyl ether to this solution resulted in immediate separation of crude piperine. The
needle-shaped crystals of compound were obtained by repeated crystallization with diethyl ether.
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5.2.
Preparation of piperic acid (1)
Piperine (2g) was dissolved in 200 ml of methanol containing 20% KOH in a 500 ml
reaction flask. The reaction mixture was refluxed with stirring for 48 h. After completion of
hydrolysis and removal of methanol under reduced pressure, a yellow colored oily solid was
obtained which was then dissolved in water (100 mL) and acidified with HCl yielding yellowish
precipitate of piperic acid. Crystallization with methanol gave yellow needle shaped crystals.
5.3.
Synthesis of compound (2)
To a solution of (1) in THF, cesium carbonate and propargyl bromide were added and the
reaction mixture was refluxed with stirring for 12h. After completion of the reaction, the reaction
mixture was poured into crushed ice and extracted with ethyl acetate. The ethyl acetate layer was
then dried over sodium sulfate and concentrated to obtain the crude product. The crude product
(2) was then crystallized from methanol.
5.4.
General procedure for the synthesis of compounds (3a-s)
To a solution of compound (2) (100 mg, 0.03 mol) in t-BuOH: H2O (2:1, 2 ml), sodium
ascorbate (1.2 mg, 0.06 mol) and CuSO4 (1.2 mg, 0.045 mol) were added at room temperature.
To this solution, aromatic azide (0.06 mol) was added and the reaction mixture was stirred for 24
h. After completion of the reaction, the reaction mixture was poured into crushed ice and
extracted with ethyl acetate. The ethyl acetate layer was then dried over sodium sulfate, filtered
and concentrated. The final product was then crystallized with methanol.
5.5.1. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(phenyl)-1H-[1,2,3]trizol-4-ylmethyl
ester (3a): White crystals; yield: 64%; M.p: 145-146οC; Rf : 0.51; FT-IR (cm-1 KBr); 1661, 1594,
1509, 1403. 1H-NMR (300MHz, CDCl3, δppm): 5.40 (s, 2H, CH2), 5.97 (s, 2H, CH2), 6.00 (s, 1H,
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CH=CH-CH=CH), 6.80-7.21 (m,7H, Ar-H), 7.48 (dd,1H, CH=CH-CH=CH, J=10.8, J=14.4),
7.59-7.70 (m, 3H, Ar-H,), 8.01 (s, 1H, triazole ring proton). 13C-NMR (120MHz, CDCl3): 57.75
(OCH2), 101.68 (CH2 piperine), 117.90-149.18 (Ar-C), 166.66 (C=O). ESI-MS m/z: 376 [M+H]+
Anal. Calcd. for C21H17N3O4: C, 67.19; H, 4.56; N, 11.19; found C, 67.45; H, 4.65; N, 11.34.
5.4.2. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-fluoro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3b): White crystals; yield: 71%; M.p: 121-122οC; Rf : 0.53; FT-IR (cm-1 KBr);
1
1668, 1599, 1520, 1421; H-NMR (300 MHz, CDCl3, δppm): 5.41 (s, 2H, CH2), 5.95 (s, 2H,
CH2), 6.00 (s, 1H, CH=CH-CH=CH), 6.66-7.00 (m, 5H, Ar-H), 7.23 (d, 2H, Ar-H, J= 8.4), 7.47
(dd, 1H, CH=CH-CH=CH, J=11.1, J=15), 7.73 (dd, 2H, Ar-H, J= 4.5, J=8.4), 8.07 (s, 1H,
triazole ring proton). 13C-NMR (120MHz, CDCl3): 57.40 (OCH2), 101.44 (CH2 piperine),
116.66-148.73 (Ar-C), 166.96 (C=O). ESI-MS m/z: 416 [M+Na] +. Anal. Calcd. for C21H16FNO4:
C, 64.12; H, 4.10; N, 10.68; found C, 64.38; H, 4.39; N, 10.84.
5.4.3. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-chloro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3c): Brown crystals; yield: 65%; M.p: 176-177οC; Rf : 0.51; FT-IR (cm-1 KBr);
1
1658, 1590, 1515, 1414. H-NMR (300 MHz, CDCl3, δppm): 5.32 (s, 2H, CH2), 5.86 (s, 2H,
CH2), 5.91 (s, 1H, CH=CH-CH=CH), 6.58-6.92 (m, 5H, Ar-H), 7.34-7.45 (m, 3H, Ar-H), 7.62
13
(d, 2H, Ar-H, J=8.4), 7.99 (s, 1H, triazole ring proton). C-NMR (120MHz, CDCl3): 57.40
(OCH2), 102.34 (CH2 piperine), 118.16-148.73 (Ar-C), 166.66 (C=O). ESI-MS m/z: 410
[M+H]+. Anal. Calcd. for C21H16N3O4 : C, 61.54; H, 3.94; N, 10.25; found C,61.10; H, 3.26; N,
10.91.
5.4.4. 5-Benz [1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-bromo-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3d): Brown crystals; yield: 67%; M.p: 188-191οC; Rf : 0.51; FT-IR (cm-1 KBr);
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1668, 1585, 1520, 1410. 1H-NMR (300 MHz, CDCl3, δppm): 5.43 (s,2H, CH2), 5.99 (s, 2H, CH2),
6.05 (s, 1H, CH=CH-CH=CH), 6.71-7.10 (m, 5H, Ar-H), 7.49 (dd, 1H, CH=CH-CH=CH, J=
10.4, J= 14.9), 7.67 (broad, s, 4H, Ar-H), 8.00 (s, 1H, triazole ring proton). 13C-NMR (120MHz,
CDCl3): 57.89 (OCH2), 102.49 (CH2 piperine), 120.93-149.64(Ar-C), 166.10 (C=O). ESI-MS
m/z: 456 [M+2]+ Anal. Calcd. for C21H16BrF3NO4 : C, 55.52; H, 3.35; N, 9.25; found C,55.81; H,
3.51; N, 9.53.
5.4.5. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(2-fluoro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3e): Light yellow crystals; yield: 62%; M.p: 134-135οC; Rf : 0.52; FT-IR (cm-1
1
KBr); 1670, 1590, 1525, 1418. H-NMR (300 MHz, CDCl3, δppm): 5.43 (s, 2H, CH2), 5.96 (s,
2H, CH2), 6.00 (s, 1H, CH=CH-CH=CH), 6.67-7.01(m, 5H, Ar-H), 7.32-7.52 (m, 4H, Ar-H),
13
7.97 (dd, 1H, Ar-H, J=7.8 J= 13.8), 8.19 (s, 1H, triazole ring proton). C-NMR (120MHz,
CDCl3): 57.48 (OCH2), 101.56 (CH2 piperine), 114.67-145.53 (Ar-C), 165.19 (C=O). ESI-MS
m/z: 416 [M+Na]+. Anal. Calcd. for C21H16FNO4 : C, 64.12; H, 4.10; N, 10.68; found C,64.98;
H, 4.61; N, 10.56.
5.4.6. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(2-chloro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3f): White powder; yield: 60%; M.p: 142-143οC; Rf : 0.50; FT-IR (cm-1 KBr);
1660, 1580, 1520, 1410. 1H-NMR (300 MHz, CDCl3, δppm): 5.44 (s, 2H, CH2), 5.89 (s, 2H, CH2),
6.00 (s, 1H, CH=CH-CH=CH), 6.72-7.00 (m, 5H, Ar-H), 7.37 (dd, IH, Ar-H, J=7.8, J=18), 7.47-
13
7.82 (m, 4H, Ar-H), 8.11 (s, 1H, triazole ring proton). C-NMR (120MHz, CDCl3): 57.36
(OCH2), 101.44 (CH2 piperine), 119.33-148.70 (Ar-C), 166.95 (C=O). ESI-MS m/z: 410
[M+H]+. Anal. Calcd. for C21H16N3O4 : C, 61.54; H, 3.94; N, 10.25; found C,61.13; H, 3.34; N,
10.67.
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5.4.7. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(2-bromo-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3g): Brown crystals; yield: 67%; M.p: 180-181οC; Rf : 0.50; FT-IR (cm-1 KBr);
1665, 1598, 1515, 1425. 1H-NMR (300MHz, CDCl3, δppm): 5.41(s, 2H, CH2), 5.95 (s, 2H, CH2),
6.00 (s, 1H, CH=CH-CH=CH), 6.66-7.00 (m, 5H, Ar-H), 7.47 (dd, 1H, CH=CH-CH=CH, J=
10.5, J= 14.7), 7.66 ( broad, s, 4H, Ar-H), 8.00 (s, 1H, triazole ring proton). 13C-NMR (120MHz,
CDCl3): 57.35 (OCH2), 101.44 (CH2 piperine), 119.33-148.74(Ar-C), 166.95 (C=O). ESI-MS
m/z: 456 [M+2]+. Anal. Calcd. for C21H16BrF3NO4 : C, 55.52; H, 3.35; N, 9.25; found C,55.81;
H, 3.51; N, 9.53.
5.4.8. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(3-chloro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3h): White powder; yield: 63%; M.p: 161-162οC; Rf : 0.50; FT-IR ( cm-1 KBr);
1675, 1610, 1530, 1405. 1H-NMR (300MHz, CDCl3, δppm): 5.33 (s, 2H, CH2), 5.87 (s, 2H, CH2),
5.91 (s, 1H, CH=CH-CH=CH), 6.57-6.91 (m, 7H, Ar-H), 7.38 (dd, 1H, Ar-H, J=7.8, J=18), 7.58
(d, 1H,Ar-H, J=7.2), 7.73(s, 1H, Ar-H), 8.11 (s, 1H, triazole ring proton). 13C-NMR (120MHz,
CDCl3): 58.91 (OCH2), 102.18 (CH2 piperine), 120.60-150.23 (Ar-C), 166.59 (C=O). ESI-MS
m/z: 410[M+H]+. Anal. Calcd. for C21H16N3O4 : C, 61.54; H, 3.94; N, 10.25; found C,61.45; H,
3.44; N, 10.80.
5.4.9. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-methyl-phenyl)-1H-[1,2,3] trizol-4-
ylmethyl ester (3i): White crystal; yield: 60%; M.p: 151-152οC; Rf : 0.55; FT-IR ( cm-1 KBr);
1689, 1620, 1513, 1409. 1H-NMR (300MHz, CDCl3, δppm): 2.36 (s, 3H, CH3), 5.45 (s, 2H, CH2),
5.93 (s, 2H, CH2), 6.03 (s, 1H, CH=CH-CH=CH), 6.63-6.98 (m, 5H, Ar-H), 7.32-7.52 (m, 4H,
Ar-H), 7.97 (dd, 1H, Ar-H, J=15.6 J= 18), 8.19 (s, 1H, triazole ring proton). 13C-NMR (120MHz,
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CDCl3): 22.12 (CH3), 58.91 (OCH2), 102.59 (CH2 piperine) 121.68-149.53 (Ar-C), 166.59
(C=O). ESI-MS m/z: 390 [M+H]+. Anal. Calcd. for C22H19N3O4: C, 67.86; H 4.92; N, 10.79;
found C,68.05; H, 4.67; N, 10.57.
5.4.10. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(2-methyl-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3j): White powder; yield: 58%; M.p: 146-147οC; Rf : 0.55; FT-IR ( cm-1 KBr);
1
1666, 1618, 1518, 1420. H-NMR (300MHz, CDCl3): δ 2.39 (s, 3H, CH3), 5.47 (s, 2H, CH2),
5.95 (s, 2H, CH2), 6.01 (s, 1H, CH=CH-CH=CH), 6.70-6.96 (m, 5H, Ar-H), 7.35-7.56 (m, 4H,
Ar-H), 7.97 (dd, 1H, Ar-H, J=7.8, J=13.8), 8.16 (s, 1H, triazole ring proton). 13C-NMR
(120MHz, CDCl3) 22.89 (CH3), 57.81 (OCH2), 101.19 (CH2 piperine), 118.18-147.23 (Ar-C),
166.18 (C=O). ESI-MS m/z: 390 [M+H]+. Anal. Calcd. for C22H19N3O4 : C, 67.86; H, 4.92; N,
10.79; found C,67.45; H, 4.10; N, 10.50.
5.4.11. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-ethyl-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3k): White powder; yield: 61%; M.p: 155-156οC; Rf : 0.54; FT-IR (cm-1 KBr);
1680, 1582, 1510, 1412. 1H-NMR (300MHz, CDCl3, δppm): 1.46 (t, 3H, CH3 J= 6.0), 1.76 (q, 2H,
OCH2 J=6.8), 5.42 (s, 2H, CH2), 5.99, (s, 2H, CH2), 6.01 (s, 1H, CH=CH-CH=CH), 6.75-7.15
(m, 7H, Ar-H), 7.48 (dd, 1H, CH=CH-CH=CH, J= 9.9, J=14.4), 7.60-7.88 (m, 2H, Ar-H,), 8.04
13
(s, 1H, triazole ring proton). C-NMR (120MHz, CDCl3): 18.18 (CH3), 30.12, (CH2), 58.99
(OCH2), 101.90 (CH2 piperine) 119.13-150.24 (Ar-C), 166.18 (C=O). ESI-MS m/z: 404 [M+H]+.
Anal. Calcd. for C23H21N3O4 : C, 68.47; H, 5.25; N, 10.42; found C,68.21; H, 5.45; N, 10.71.
5.4.12. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-methoxy-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3l): White powder; yield: 63%; M.p: 130-131οC; Rf : 0.53; FT-IR ( cm-1 KBr);
1
1680, 1582, 1510, 1418. H-NMR (300MHz, CDCl3, δppm): 3.88 (s, 3H, OCH3), 5.41(s, 2H,
14

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CH2), 5.95 (s, 2H, CH2), 6.00 (s, 1H, CH=CH-CH=CH), 6.66-7.05 (m, 7H, Ar-H), 7.47( dd, 1H,
CH=CH-CH=CH, J=10.8, J=15.3), 7.64 (d, 2H, Ar-H, J= 9.0), 8.01 (s,1H, triazole ring proton).
13C-NMR (120MHz, CDCl3): 55.69 (OCH3), 57.50 (OCH2), 101.43 (CH2 piperine), 114.29-
148.32 (Ar-C), 166.97 (C=O), ESI-MS m/z: 406 [M+H]+. Anal. Calcd. for C22H19N3O5 : C,
64.12; H, 4.10; N, 10.68; found C,64.98; H, 4.61; N, 10.56.
5.4.13. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(2-methoxy-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3m): White cystral; yield%: 59; M.p: 141-142οC; Rf : 0.53; FT-IR ( cm-1 KBr);
1
1668, 1570, 1529, 1429. H-NMR (300MHz, CDCl3, δppm): 3.86 (s, 3H, OCH3), 5.43(s, 2H,
CH2), 5.98 (s, 2H, CH2), 6.03 (s, 1H, CH=CH-CH=CH), 6.70-7.10 (m, 9H, Ar-H), 7.47 (dd,
13
1H,CH=CH-CH=CH , J=10.5, J=15.3), 8.03 ( s,1H, triazole ring proton). C-NMR (120MHz,
CDCl3): 55.18 (OCH3), 57.90 (OCH2), 102.43 (CH2 piperine) 115.19-149.42 (Ar-C), 166.11
(C=O). ESI-MS m/z: 406 [M+H]+. Anal. Calcd. for C22H19N3O5 : C, 64.12; H, 4.10; N, 10.68;
found C,65.08; H, 4.89; N, 10.91.
5.4.14. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-ethoxy-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3n): Light brown; yield: 65%; M.p: 162-163οC; Rf : 0.54; FT-IR ( cm-1 KBr);
1670, 1575, 1520, 1408. 1H-NMR (300MHz, CDCl3, δppm): 1.46 (t, 3H, CH3 J= 6.9), 4.08 (q, 2H,
OCH2 J=6.9), 5.40 (s, 2H, CH2), 5.95, (s, 2H, CH2), 6.00 (s, 1H, CH=CH-CH=CH), 6.75-7.03 (
m, 7H, Ar-H), 7.47 (dd, 1H, CH=CH-CH=CH , J= 10.5, J=15), 7.63 ( d, 2H, Ar-H, J= 8.4), 8.01
13
(s, 1H, triazole ring proton ). C-NMR (120MHz, CDCl3): 20.38 (OCH2CH3), 57.90 (OCH2),
64.12, (OCH2CH3), 101.90 (CH2 piperine) 119.13-150.24 (Ar-C), 166.18 (C=O). ESI-MS m/z:
442 [M+Na]+. Anal. Calcd. for C23H21N3O5 : C, 65.86; H, 5.05; N, 10.02; found C,65.10; H,
5.26; N, 10.35.
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5.4.15. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic
acid
1-(2,4-di-chloro-phenyl)-1H-
[1,2,3]trizol-4-ylmethyl ester (3o): White powder; yield: 67%; M.p: 168-169οC; Rf : 0.50; FT-IR
( cm-1 KBr); 1677, 1570, 1505, 1412. 1H-NMR (300 MHz, CDCl3, δppm): 5.34 (s, 2H, CH2), 5.87
(s, 2H, CH2), 5.91 (s, 1H, CH=CH-CH=CH), 6.58-6.92 (m, 5H, Ar-H), 7.35-7.53 (m, 4H, Ar-H),
8.01 (s, 1H, triazole ring proton). 13C-NMR (120MHz, CDCl3): 57.90 (OCH2), 102.10 (CH2
piperine) 120.43-151.24 (Ar-C), 166.98 (C=O). ESI-MS m/z: 466 [M+Na]+. Anal. for C21H15
Cl2N3O4: C, 56.77; H, 3.40; N, 9.46; found C, 56.89; H, 3.77; N, 9.98.
5.4.16. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic
acid
1-(3,4-di-chloro-phenyl)-1H-
[1,2,3]trizol-4-ylmethyl ester (3p): White powder; yield: 66%; M.p: 171-172οC; Rf : 0.50; FT-IR
1
( cm-1 KBr); 1671, 1577, 1525, 1418. H-NMR (300MHz, CDCl3, δppm): 5.41 (s, 2H, CH2), 5.95
(s, 2H, CH2), 6.00 (s, 1H, CH=CH-CH=CH), 6.72-7.01(m, 5H, Ar-H), 7.63 (s, 2H, Ar-H), 7.47
13
(dd, 1H, Ar-H, J=10.8 J=15), 7.92 (s 1H, Ar-H), 8.09 (s,1H, triazole ring proton). C-NMR
(120MHz, CDCl3): 57.80 (OCH2 piperine), 101.18 (CH2 piperine) 121.33-151.84 (Ar-C), 166.16
(C=O). ESI-MS m/z: 466 [M+Na]+. Anal. Calcd. for C21H15 Cl2N3O4 : C, 56.77; H, 3.40; N, 9.46;
found C,56.98; H, 3.56; N, 10.01.
5.4.17. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic
acid
1-(3,4-di-methyl-phenyl)-1H-
[1,2,3]trizol-4-ylmethyl ester (3q): White powder; yield: 62%; M.p: 164-166οC; Rf : 0.52; FT-IR
1
( cm-1 KBr); 1679, 1575, 1535, 1419. H-NMR (300MHz, CDCl3, δppm): 2.38 (s, 6H, CH3), 5.48
(s, 2H, CH2), 5.97 (s, 2H, CH2), 6.04 (s, 1H, CH=CH-CH=CH), 6.60-7.15(m, 7H, Ar-H),
13
7.31(m, 1H, Ar-H), 7.59 (dd, 1H, Ar-H, J=10.8, J=15.3), 8.15 (s, 1H, triazole ring proton). C-
NMR (120MHz, CDCl3): 22.19 (CH3), 58.45 (OCH2), 101.40 (CH2 piperine) 120.56-148.23 (Ar-
C), 166.68 (C=O). ESI-MS m/z: 404 [M+H]+. Anal. Calcd. for C23H21N3O4 : C, 68.47; H, 5.25;
N, 10.42; found C,68.89; H, 5.39; N, 10.47
16

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5.4.18. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(4-nitro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3r): White powder; yield: 56%; M.p: 180-181οC; Rf : 0.50; FT-IR (cm-1 KBr);
1887, 1587, 1515, 1423. 1H-NMR (300MHz, CDCl3, δppm): 5.43 (s, 2H, CH2), 5.95 (s, 2H, CH2),
6.01 (s, 1H, CH=CH-CH=CH), 6.72-7.01(m, 7H, Ar-H), 7.55 (dd, 1H, Ar-H, J=10.8 J=15), 8.01(
d 1H, Ar-H, J=9.0), 8.22 (s, 1H, triazole ring proton), 8.44 (d, 1H, Ar-H, J=9). 13C-NMR
(120MHz, CDCl3): 56.65 (OCH2), 101.20 (CH2 piperine) 118.97-148.28 (Ar-C), 165.67 (C=O).
ESI-MS m/z: 421 [M+H]+. Anal. Calcd. for C21H16 Cl2N4O6 : C, 60.00; H, 3.84; N, 13.33; found
C,60.27; H, 3.56; N, 13.78.
5.4.19. 5-Benzo[1,3]dioxol-5-yl-penta-2,4-dienoic acid 1-(3-nitro-phenyl)-1H-[1,2,3]trizol-4-
ylmethyl ester (3s): White powder; yield: 52%; M.p: 185-186οC; Rf : 0.50; FT-IR (cm-1 KBr);
1687, 1589, 1514, 1419. 1H-NMR (300 MHz, CDCl3, δppm): 5.35 (s, 2H, CH2), 5.87 (s, 2H, CH2),
5.91 (s, 1H, CH=CH-CH=CH), 6.63-6.92 (m, 5H, Ar-H), 7.40 (dd, 1H, Ar-H, J=10.8 J= 15),
13
8.11-8.26 (m, 4H, ArH), 8.54 (s, 1H, triazole ring proton). C-NMR (120 MHz, CDCl3): 56.35
(OCH2), 102.70 (CH2 piperine), 119.87-149.78 (Ar-C), 165.34 (C=O). ESI-MS m/z: 421
[M+H]+. Anal. Calcd. for C21H16 N4O6 : C, 60.00; H, 3.84; N, 13.33; found C,60.34; H, 3.95; N,
13.39.
6. Pharmacology
6.1.
Anti-inflammatory activity
The anti-inflammatory activity was carried out by the reported method [26]. Animals
were divided into twenty one groups of five animals each. One group kept as a positive control
group was orally administered with standard indomethacin (20mg/kg) and other group kept as a
negative control group and administered with 0.5% carboxymethyl cellulose solution. The
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remaining groups were test groups and administered orally with synthesized compounds at dose
of 20mg/kg b.w. A freshly prepared solution of carrageenan (1.0% in sterile 0.9% NaCl solution)
in a volume of 0.1 ml was injected subcutaneously into the subplantar region of the right hind
paw after 1 h of administration of the test sample. Right hind paw volume was measured at 3 h
and 5 h after carrageenan injection with the help of digital plethysmometer. The percent anti-
inflammatory activity was calculated according to the formula given below.
% Anti-inflammatory Activity = [VC -Vt /VC] x 100
Where, Vt represents the mean increase in paw volume in rats treated with test samples and
VC represents the mean increase in paw volume of rats in control group.
6.2.
Molecular Docking studies on TNF-α
Crystallized structure of 2AZ5 was chosen from Protein Data Bank and used as target for
molecular docking studies. 2AZ5 structure was imported in Schrodinger using Protein
Preparation Wizard. Missing hydrogen and were added using prime interface. Undesired water
molecules were removed. The protein was then optimized and minimized to give low energy and
structurally correct target protein. As the target protein had already the site for reference ligand,
the grid was generated by selecting the ligand as the reference ligand. Finally the grid was
validated and was used for further docking with new unknown ligands to predict their docking
score. Chemical structures were drawn in maestro and geometrically refined by LigPrep module.
In this module 2-D structures were converted into 3-D structures, which were further subjected
to OPLS-2005 force field to generate single low energy 3-D structure for each input structure.
During this step chiralities were maintained. Docking was carried using Schrodinger Glide
software with Extra precision and XP descriptor information. This generates favourable ligand
poses which are further screened through filters to examine spatial fit of the ligand in the active.
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Ligand poses which pass through initial screening are subjected to evaluation and minimization
of grid approximation. Scoring is then carried on energy minimized poses to generate Glide
score.
6.3.
TNF-α Assay
RAW 264.7 cells were cultured in RPMI-1640 medium supplemented with 10% FBS,
penicillin (100 units/mL), and streptomycin sulfate (100m g/mL) in a humidified atmosphere of
5% CO2. The cells were harvested with trypsin-EDTA and diluted to a suspension in fresh
medium. The cells were then seeded in 96-well plates with 2.1 × 105 cells/well, and allowed to
adhere for 1 h. Then the medium was induced with 100 µg/ml LPS (lipopolysaccharide), test
samples (20µM), and incubated for 24 h. The supernatant (50 µL) was then transferred into a 96-
well ELISA plate and TNF-α level were quantified by ELISA kits according to the
manufacturer’s instructions.
6.4.
MTT assay for Cell Viability
RAW 264.7 cells (2 × 105) were cultured in 96-well plate containing DMEM
supplemented with 10% FBS. The cells were stimulated with 20µM test compounds in the
presence of 100 µg/ml LPS for 24 h. Then, the cells were washed twice with DPBS and
incubated for 2h at 37oC with 100 µl of 0.5 mg/ml MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-
diphenyltetrazolium bromide). The medium was discarded and 100 µl dimethyl sulfoxide (DESI-
MSO) was added to it. The absorbance was recorded at 570nm using a microplate reader after 30
min incubation.
6.5.
Antinociceptive activity
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The analgesic activity was carried out by writhing test method using the previously
reported method [27]. Swiss albino mice (35–40 g) of either sex were divided into seven groups
with each group containing five animals. Group I was taken as control and received CMC
suspension only, group II received standard drug indomethacin and rest of the groups were orally
administered with synthesized compounds at a dose of 20mg/kg. After 30 min of test samples
administration, 0.1% acetic acid solution was given to mice intraperitoneally. The number of
muscular contractions was counted over a period of 10 min after acetic acid injection. The data
represents the total number of writhes observed during 10 min and is expressed as writhing
numbers.
6.6.
Ulcerogenic activity
The ulcerogenic study was carried out by earlier reported method [28]. Those compounds
which showed significant anti-inflammatory activity were further evaluated for their ulcerogenic
effects. Control group rats were orally given a suspension of 1% carboxymethylcellulose only.
The standard group and test samples were orally administered at a dose of 60mg respectively
which is three times of the dose used for anti-inflammatory activity. Animals were sacrificed
after 5 h of dosing of standard group and test samples.
6.7.
Lipid peroxidation assay
Lipid peroxidation assay was carried out by reported method [29]. The gastric mucosa
was scraped with two glass slides and weighed (100 mg) and homogenized in 1.8 mL of 1.15%
ice cold KCl solution. One milliliter of suspension medium was taken from the supernatant, 0.5
mL of 30% trichloroacetic acid followed by 0.5 mL of 0.8% thiobarbituric acid reagent were
added to it. The tubes were covered with aluminum foil and kept in a shaking water bath for 30
min at 80°C. After 30 min, tubes were taken out and kept in ice cold water for 10 min. These
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were then centrifuged at 3000 rpm for 15 min. The absorbance of supernatant was read at 540
nm at room temperature.
6.8.
Immunohistochemistry
The paw tissues were fixed in formalin and embedded in paraffin. Sections of 5 µm
thickness were cut onto poly-lysine coated glass slides. Sections were deparafinized three times
(5 min) in xylene followed by dehydration in graded ethanol and finally rehydrated in running
tap water. For antigen retrieval, sections were boiled in 10mM citrate buffer (pH 6.0) for 5-7
min. Sections were incubated with hydrogen peroxide for 15 min to minimize non-specific
staining and then rinsed three times (5 min each) with 1X PBST (0.05% Tween-20). Blocking
solution was applied for 10 min and then sections were incubated with diluted (1:100) primary
anti-bodies, purified rabbit polyclonal anti-NF-κB antibody (BioLegend), rabbit polyclonal anti-
COX-2 antibody (Bio Vision) and polyclonal anti-TNF-α antibody (Bio Vision) overnight at 4oC
in humid chamber. Further processing was done according to the instructions of Ultra Vision
plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use) staining kit (Thermo
scientific system). The peroxidase complex was visualized with 3,3-diaminobenzidine (DAB).
Lastly the slides were counterstained with haematoxylin, cleaned in xylene, dehydrated with
ethanol and after DPX mounting microscopic (BX 51 Olympus) analysis was done at 40X
magnification.[30]
Supplementary data
The supplementary data has been provided with this article
Acknowledgements
The authors wish to express their thanks to Dr G N Qazi, Vice Chancellor, Jamia
Hamdard for providing necessary research facilities. The authors are thankful to Dr. Javed Iqbal,
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Director, Institute of Life Sciences (ILS), Hyderabad, for providing necessary in vitro facilities
and Dr. Parimal Misra for his help in performing in vitro studies. One of the authors, YA is also
thankful to Hamdard National Foundation for providing financial assistance.
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8
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Scheme I:
O
O
O
O
O
OH
N
I
O
1
II
O
O
O
O
2
III
O
O
N
N
N
3a-3s
O
O
Reagents and Conditions: (I) Alcohol, KOH, reflux; (II) CeCO3, dry THF, reflux; (III)
CH3COONa, NaN3, Ar-N3.
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Table1: Anti-inflammatory activity of synthesized compounds.
Compounds
R
Change in paw volume(ml)
mean±(SEM)
% inhibition
3h
5h
3h
5h
1.68±0.029
1.70±0.020
Control
-
-
-
0.608±0.032** 0.54±0.023**
72.05
77.02
(Indomethacin) -
0.676±0.022*
1.12±0.028*
0.87±0.017*
0.83±0.017*
0.89±0.023*
0.68±0.014**
0.58±0.017**
0.56±0.020**
0.71±0.016**
1.01±0.025*
0.91±0.017*
0.83±0.010*
0.88±0.016*
0.76±0.009**
1.10±0.023*
0.64±0.023**
0.632±0.023*
0.68±0.020**
1.39±0.020*
1.44±0.021*
0.670±0.013*
1.11±0.014**
0.85±0.025*
0.81±0.022*
0.87±0.017*
0.65±0.023**
0.56±0.022**
0.51±0.025**
0.69±0.015**
0.98±0.029*
0.88±0.029*
0.80±0.011*
0.86±0.020*
0.72±0.022**
1.14±0.034
53.69
36.98
55.47
57.53
53.56
68.19
74.52
76.12
64.72
46.22
51.50
57.53
53.97
61.64
32.87
69.31
69.58
67.94
19.58
15.61
54.72
38.51
57.02
59.72
55.53
70.81
76.71
80.40
67.81
49.09
54.59
60.00
55.94
65.00
36.48
71.21
72.97
70.27
22.07
15.40
Piperine
3a
-
H
3b
3c
4-F
4-Cl
3d
3e
4-Br
2-F
3f
2-Cl
3g
3h
3i
2-Br
3-Cl
4-CH3
O-CH3
4-C2H5
4-OCH3
2-OCH3
4-OC2H5
2,4-di-Cl
3,4-di-Cl
3,4-di-CH3
4-NO2
3-NO2
3j
3k
3l
3m
3n
3o
3p
3q
3r
0.622±0.04**
0.588±0.08**
0.66±0.016**
1.37±0.013
3s
1.46±0.011*
Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from five observations where * p < 0.05,
**p < 0.01

ACCEPTED MANUSCRIPT
Table2: Docking results of synthesized compounds.
Ligands
Glide score Glide
energy
M.W
QPlog Po/w
QPlogS
PSA
3a
-5.80
-3.82
-4.73
-3.58
-5.59
-5.78
-5.81
-5.61
-4.28
-4.64
-5.09
-3.18
-5.15
-3.30
-5.67
-5.32
-5.13
-4.09
-2.81
-5.02
-4.42
-40.12
-38.22
-37.15
-40.32
-42.36
-42.15
-41.80
-43.98
-34.69
-38.93
-37.30
-33.97
-36.96
-39.91
-43.63
-41.25
-39.76
-37.59
-38.75
-33.09
-24.68
375.38
393.37
409.82
454.27
393.37
409.82
454.27
409.82
389.41
389.41
403.43
405.40
405.40
419.43
444.27
444.27
403.43
420.38
420.38
357.00
285.00
4.17
4.44
4.71
4.78
4.43
4.70
4.78
4.70
4.52
4.49
5.14
4.23
4.27
4.27
3.44
3.11
4.78
3.41
3.44
4.28
3.25
-5.31
-5.68
-6.07
-6.19
-5.57
-5.93
-6.04
-6.06
-5.91
-5.92
-6.71
-5.46
-5.46
-5.28
-5.33
-4.35
-6.37
-5.35
-5.33
-5.31
-3.44
87.20
86.31
86.31
86.31
85.81
78.51
85.34
86.31
86.31
87.19
86.32
95.49
92.33
94.06
85.39
80.32
87.19
132.11
131.19
84.32
47.98
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
Indomethacin
Piperine

ACCEPTED MANUSCRIPT
Table 3: Analgesic activity of active compounds
Group
Number of writhes in 10 min
% Protection
Control
Indomethacin
3e
92.8 ± 1.49
39.4 ± 1.16**
45.8± 1.85*
-
57.43
48.70
3f
45.8± 1.85**
42.6 ± 1.63**
51.0 ± 1.87*
50.64
54.09
45.04
3g
3o
3p
3q
46.4±1.69**
51.2±1.28*
50.00
44.93
Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from five observations where * p < 0.05, **p
< 0.01

ACCEPTED MANUSCRIPT
Fig captions
Fig1: Docking images of active compounds (3g, 3f), standard drug indomethacin and piperine
respectively.
Fig 2: Effect of synthesized compounds on LPS induced TNF-α cytokine level in the RAW
264.7 cell line.
Fig 3: Cytotoxicity assay of active compounds.
Fig4: Histopathology of rat stomach in Albino Wistar rats, photomicrographs (20X) of stomach
wall of the animal groups administered with compound 3g, 3f, 3p, 3o standard drug
indomethacin and control group respectively.
Fig5: Lipid peroxidation assay of active compounds.
Fig6.a: Paw tissue expressions of COX-2 activation. Representative photomicrographs
magnification (40X) Group I (only control), Group II (only carrageenan), Group III (carrageenan
+ standard drug Indomethacin), Group IV (carrageenan +3g) GroupV (carrageenan+ 3f).
Fig6.b: Paw tissue expressions of NFkB activation. Representative photomicrographs
magnification (40X) Group I (only control), Group II (only carrageenan), Group III (carrageenan
+ standard drug Indomethacin), Group IV (carrageenan +3g) Group (carrageenan+ 3f).
Fig6.c: Paw tissue expressions of TNF-α activation. Representative photomicrographs
magnification (40X) Group I (only control), Group II (only carrageenan), Group III (carrageenan
+ standard drug Indomethacin), Group IV (carrageenan +3g) Group (carrageenan+ 3f).