Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant, analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibition activity

Article abstract of DOI:10.1080/14756366.2020.1746783

A new series of N′-(substituted phenyl)-2-(1-(4-(methylsulfinyl) benzylidene)?5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide derivatives (1–25) were prepared in good yields in an efficient manner. All the compounds were fully characterised by the elemental analysis and spectral data. Synthesised compounds were evaluated for antioxidant activity by DPPH method. Compounds 7 (R = 3-methoxyphenyl), 3 (R = 4-dimethylaminophenyl) and 23 (R = 2,4,5-trimethoxy phenyl) substitutions were found to be having highly potent antioxidant activity. Compound 3, with para dimethylaminophenyl substitution was found to be having highest antioxidant activity. It was further evaluated in vivo for various analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibitory activity in different animal models. Lead compound 3 was found to be significant anti-inflammatory and analgesic agent. It was also evaluated for ulcerogenic activity and demonstrated significant ulcerogenic reduction activity in ethanol and indomethacin model. The LD₅₀ of compound 3 was found to be 131 mg/kg. The animals treated with compound 3 prior to cisplatin treatment resulted in a significant reduction in COX-2 protein expression when compared to cisplatin-treated group. Sulindac derivative with para dimethylaminophenyl substitution was found to be the most potent antioxidant, anti-inflammatory and analgesic agent as well as with significant gastric sparing activity as compared to standard drug sulindac. Compound 3 significantly downregulated liver tissue COX‐2 gene expression.

Full text of DOI:10.1080/14756366.2020.1746783

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel sulindac derivatives: synthesis,
characterisation, evaluation of antioxidant,
analgesic, anti-inflammatory, ulcerogenic and
COX-2 inhibition activity
Mashooq A. Bhat, Mohamed A. Al-Omar, Nawaf A. Alsaif, Abdulrahman
A. Almehizia, Ahmed M. Naglah, Suhail Razak, Azmat Ali Khan & Naeem
Mahmood Ashraf
To cite this article: Mashooq A. Bhat, Mohamed A. Al-Omar, Nawaf A. Alsaif, Abdulrahman A.
Almehizia, Ahmed M. Naglah, Suhail Razak, Azmat Ali Khan & Naeem Mahmood Ashraf (2020)
Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant, analgesic, anti-
inflammatory, ulcerogenic and COX-2 inhibition activity, Journal of Enzyme Inhibition and Medicinal
Chemistry, 35:1, 921-934, DOI: 10.1080/14756366.2020.1746783
To link to this article: https://doi.org/10.1080/14756366.2020.1746783
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 02 Apr 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 921–934
https://doi.org/10.1080/14756366.2020.1746783
RESEARCH PAPER
Novel sulindac derivatives: synthesis, characterisation, evaluation of antioxidant,
analgesic, anti-inflammatory, ulcerogenic and COX-2 inhibition activity
Mashooq A. Bhata , Mohamed A. Al-Omara, Nawaf A. Alsaifa, Abdulrahman A. Almehiziaa, Ahmed M. Naglahb,c
Suhail Razakd, Azmat Ali Khana and Naeem Mahmood Ashrafe
,
b
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Pharmaceutical
c
Chemistry, Drug Exploration and Development Chair (DEDC), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Peptide
d
Chemistry Department, Chemical Industries Research Division, National Research, Centre, Dokki, Egypt; Department of Community Health
e
Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia; Department of Biochemistry and Biotechnology, The
University of Gujrat, Punjab, Pakistan
ABSTRACT
ARTICLE HISTORY
A new series of N0-(substituted phenyl)-2-(1-(4-(methylsulfinyl) benzylidene)ꢀ5-fluoro-2-methyl-1H-inden-3-
yl) acetohydrazide derivatives (1 – 25) were prepared in good yields in an efficient manner. All the com-
pounds were fully characterised by the elemental analysis and spectral data. Synthesised compounds
were evaluated for antioxidant activity by DPPH method. Compounds 7 (R ¼ 3-methoxyphenyl), 3 (R ¼ 4-
dimethylaminophenyl) and 23 (R ¼ 2,4,5-trimethoxy phenyl) substitutions were found to be having highly
potent antioxidant activity. Compound 3, with para dimethylaminophenyl substitution was found to be
having highest antioxidant activity. It was further evaluated in vivo for various analgesic, anti-inflammatory,
ulcerogenic and COX-2 inhibitory activity in different animal models. Lead compound 3 was found to be
significant anti-inflammatory and analgesic agent. It was also evaluated for ulcerogenic activity and dem-
onstrated significant ulcerogenic reduction activity in ethanol and indomethacin model. The LD50 of com-
pound 3 was found to be 131mg/kg. The animals treated with compound 3 prior to cisplatin treatment
resulted in a significant reduction in COX-2 protein expression when compared to cisplatin-treated group.
Sulindac derivative with para dimethylaminophenyl substitution was found to be the most potent antioxi-
dant, anti-inflammatory and analgesic agent as well as with significant gastric sparing activity as compared
to standard drug sulindac. Compound 3 significantly downregulated liver tissue COX-2 gene expression.
Received 26 December 2019
Revised 6 March 2020
Accepted 16 March 2020
KEYWORDS
Sulindac hydrazide;
hydrazones; antioxidant;
analgesic; anti-
inflammatory; COX-
2 activity
Introduction
Sulindac is an indene derivative NSAID, known to induce ulcer-
ation. New sulindac derivatives are reported as anti-inflammatory9,
anticancer10–12, COX-1 inhibitors13 and have shown PPAR c activ-
ity14. Syntheses of novel derivatives of NSAIDs have improved
their safety profile which resulted in an increased anti-inflamma-
Cyclooxygenase (COX) enzyme catalyses the conversion of arachi-
donic acid to prostaglandin H2 (PGH2), which is converted to
many prostanoids by specific isomerase enzymes because it is an
unstable intermediate. Non-beneficial effects of prostaglandins
include pain and fever associated with inflammation; beneficial
effects include gastro-intestinal protection and platelet function.
The COX-1 and COX-2 are the two isoforms, which are regulated
differently. The cyto-protection in the gastrointestinal (GI) tract is
provided by COX-1 and COX-2 mediates inflammation1.
Non-steroidal anti-inflammatory drugs (NSAIDs) are used to
treat pain and inflammation. Side effects include gastrointestinal
toxicity such as gastro-duodenal perforations, ulcers and bleeding,
ascribed to the inhibition of cyclooxygenase-1 (COX-1). Thus,
selective inhibitors of cyclooxygenase-2 (COX-2) were synthesised
in an attempt to decrease these side effects2–6. Physicians would
prescribe gastro-protective agents with a conventional NSAID,
prior to the introduction of the COX-2 selective inhibitors.
Selectively inhibition of COX-2 enzyme would result in the same
anti-inflammatory benefits as that of non-selective NSAIDs provide
but with less incidences of gastrointestinal side effects7,8. Some
COX-2 inhibitors have also been found to have cardiovascular
side effects.
tory activity with reduced ulcerogenicity15,16
.
It would be desirable to provide an indene derivative having
the anti-inflammatory and analgesic properties of a COX-2 inhibi-
tor NSAID, but which also provides gastric sparing activity. The
aim of this study was to prepare novel sulindac acetohydrazide
derivatives and evaluate their potential antioxidant, analgesic,
anti-inflammatory, ulcerogenic and COX-2 inhibition activity.
Experimental
Chemistry
Materials and methods
Solvents were procured from Merck. Thin layer chromatography
(TLC), was performed on Silica gel 60 F254 coated plates (Merck) to
check the purity of compounds. For performing FT-IR, Perkin
Elmer FT-IR spectrophotometer was used. Melting points were
determined by Gallenkamp melting point apparatus. 1H and 13C
NMR were recorded in Bruker NMR 500/700 MHz and 125/176 MHz
CONTACT Mashooq A. Bhat
mabhat@ksu.edu.sa
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

922
M. A. BHAT ET AL.
spectrophotometer. The samples were run in DMSO-d6 with tetra N’-(4-Chlorobenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-5-flu-
methyl silane (TMS) as an internal standard. Molecular masses of
compounds were determined in GC mass spectroscopy. The CHN
Elementar (Analysensysteme GmbH, Germany) was used for elem-
oro-2-methyl-1H-inden-3-yl)acetohydrazide (2)
Yield: 85%; m. p.: 228–230 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2955
(CH str.), 1664 (C¼O, Str.), 1599 (C¼N str.); 1H NMR (500 MHz,
ental analysis of the compounds. Cisplatin was purchased from DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 4.00 (2H, s,
CH2), 6.72 (1H, s, ¼CH), 7.09–7.78 (11H, m, Ar-H), 8.00 (1H, s,
Sigma-Aldrich, USA. Antibodies against COX-2 and b-actin were
N¼CH), 11.56 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz,
purchased from Abcam (Cat Log No: ab15191).
DMSO–d6): d ¼ 10.9, 29.9, 43.5, 124.3, 128.9, 129.3, 129.7, 130.4,
134.7, 139.0, 146.7, 171.5; MS: m/z ¼ 493.40 [M þ 1]þ; Analysis: for
C27H22ClFN2O2S, calcd. C 65.78, H 4.50, N 5.68, S 6.50%; found C
Synthesis of methyl-5-fluoro-1-f[4-(methane sulfinyl) phenyl]
65.55, H 4.49, N 6.48, S 6.52%.
methylideneg-2-methyl-1H-inden-3-yl] acetate
The sulindac ester was prepared according to the reported
procedure17.
N0-(4-Dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (3)
Yield: 50%; m. p.: 158–160 ꢁC; IR (KBr) cmꢀ1: 3165 (NH str.), 3025
(CH str.), 1655 (C¼O, Str.), 1599 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.82 (3H, s, S¼O CH3), 2.96 (6H, s,
2 ꢂ NCH3), 3.54 (1H, s, CH2), 3.96 (1H, s, CH2), 6.73 (1H, s, ¼CH),
2-[5-Fluoro-1-f[4-(methanesulfinyl)phenyl]methylideneg-2-methyl-
1H-inden-3-yl]acetohydrazide
The methyl ester of sulindac (0.01 mol) and hydrazine hydrate
(99%) (0.2 mol) were refluxed in methanol (50 ml) for 30 h. The 7.12–7.78 (11H, m, Ar-H), 7.92 (1H, s, N¼CH), 11.19 (1H, s, CONH,
D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6): d ¼ 11.0, 29.9, 43.5,
mixture was concentrated, cooled and poured in crushed ice in
112.2, 124.3, 128.5, 128.8, 129.6, 129.9, 130.4, 144.7, 146.6, 170.8;
MS: m/z ¼ 501.77 [M]þ; Analysis: for C29H28FN3O2S, calcd. C 69.44,
H 5.63, N 8.38, S 6.39%; found C 69.17, H 5.62, N 8.35, S 6.41%.
small portions while stirring, and kept for 3–4 h at room tempera-
ture. The solid separated out was filtered, dried and crystallised
from ethanol. The product was carefully checked by thin layer
chromatography.
Colour: yellow; Yield: 70%; m.p.: 120–122 ꢁC; UV kmax
1
N0-(3-Hydroxybenzylidene)-2-1-(4-(methylsulfinyl)benzylidene)-5-
fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (4)
(Methanol) ¼ 327 nm; H NMR (500 MHz, DMSO–d6): d ¼ 2.20 (3H,
s, CH3), 2.82 (3H, s, SOCH3), 3.38 (2H, s, CH2), 4.28 (2H, bs, NH2,
D2O exchg.), 6.71 (1H, t, J ¼ 9.5 Hz, ¼CH), 7.15–7.80 (7H, m, Ar-H),
9.30 (1H, bs, CONH, D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6):
d ¼ 10.88, 31.39, 43.59, 106.74, 106.93, 110.69, 110.87, 123.49,
123.56, 124.39, 129.71, 129.93, 130.40, 133.86, 138.29, 139.07,
140.95, 140.95, 146.69, 147.62, 147.69, 162.63, 163.96, 168.69; MS:
m/z ¼ 370.44 [M]þ; Analysis: for C20H19FN2O2S, calcd. C 64.85, H
5.17, N 7.56, S 8.66%; found C 64.65, H 5.15, N 7.54 S 8.88%.
Yield: 60%; m. p.: 240–242 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3080
(CH str.), 1664 (C¼O, Str.), 1601 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.81 (3H, s, CH3S¼O), 3.99 (1H, s,
CH2), 6.71 (1H, s, ¼CH), 7.10–7.78 (11H, m, Ar-H), 7.97 (1H, s,
N¼CH), 9.65 (1H, s, OH, D2O exchg.), 11.44 (1H, s, CONH, D2O
exchg.); 13C NMR (125.76 MHz, DMSO–d6): d ¼ 10.9, 29.7, 43.5,
110.7, 113.2, 118.7, 124.3, 129.9, 130.4, 135.8, 140.9, 144.0, 158.1,
171.4; MS: m/z ¼ 474.94 [M]þ; Analysis: for C27H23FN2O3S, calcd. C
68.34, H 4.89, N 5.90, S 6.76%; found C 68.07, H 4.90, N 5.88,
S 6.78%.
General procedure for the synthesis of N’-(substituted benzyli-
dene)-2–(1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-
inden-3-yl) acetohydrazide (1–25)
A solution of sulindac acetohydrazide (1.0 mmol) in ethanol
(50 ml) containing appropriate substituted benzaldehydes
(1.1 mmol) and a catalytic amount of glacial acetic acid was
heated under reflux for 3 h. The reaction mixture was added to
the ice cold water in a beaker. The product was precipitated, fil-
tered by vacuum filtration and washed several times with cold
water. The solid was recrystallized from ethanol.
N0-(4-Hydroxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-5-
fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (5)
Yield: 50%; m. p.: 210–212 ꢁC; IR (KBr) cmꢀ1: 3165 (NH str.), 3031
(CH str.), 1652 (C¼O, Str.), 1597 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.18 (1H, s,
CH2), 3.97 (1H, s, CH2), 6.72 (1H, s, ¼CH), 6.82–7.78 (11H, m, Ar-H),
7.95 (1H, s, N¼CH), 9.91 (1H, s, ꢀOH), 11.28 (1H, s, CONH, D2O
exchg.); 13C NMR (125.76 MHz, DMSO–d6): d ¼ 11.0, 29.9, 43.5, 49.0,
106.8, 110.8, 116.1, 124.4, 129.0, 130.4, 144.1, 146.6, 147.4, 147.8,
147.9, 159.6, 159.8, 162.2, 163.6, 165.4, 171.1; MS: m/z ¼ 474.75
[M]þ; Analysis: for C27H23FN2O3S, calcd. C 68.34, H 4.89, N 5.90, S
6.76%; found C 68.12, H 4.90, N 5.91, S 6.75%
2-1-(4-(Methylsulfinyl)benzylidene)-5-fluoro-2-methyl-1H-inden-3-
yl)-N0-benzylideneacetohydrazide (1)
Yield: 65%; m. p.: 138–140 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2980
(CH str.), 1662 (C¼O, Str.), 1591 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.23 (3H, s, CH3), 2.82 (3H, s, S¼O CH3), 3.59 (1H, s,
CH2), 4.00 (1H, s, CH2), 6.71 (1H, s, ¼CH), 7.11–7.78 (12H, m, Ar-H),
8.28 (1H, s, N¼CH), 11.50 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 29.9, 43.5, 124.3, 127.3, 127.5,
129.0, 129.2, 129.7, 129.9, 130.0, 130.3, 130.4, 134.6, 138.4, 139.0,
140.9, 143.8, 146.6, 171.5; MS: m/z ¼ 459.39 [M þ 1]þ; Analysis: for
C27H23FN2O2S, calcd. C 70.72, H 5.06, N 6.11, S 6.99%; found C
70.99, H 5.05, N 6.14, S 6.97%.
N0-(2-Methoxybenzylidene)-2-(1-(4-(methylsulfinyl) benzylidene)-5-
fluoro-2-methyl-1H-inden-3-yl) acetohydrazide (6)
Yield: 55%; m. p.: 173–176 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3007
(CH str.), 1653 (C¼O, Str.), 1564 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.18 (3H, s, CH3), 2.80 (3H, s, S¼OCH3), 3.81 (3H, s,
OCH3), 3.96 (2H, s, CH2), 6.69 (1H, s, ¼CH), 7.0–7.77 (11H, m, Ar-H),
8.36 (1H, s, N¼CH), 11.45 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.6, 14.7, 31.0, 49.0, 56.2, 112.4,
121.2, 127.0, 133.5, 157.0, 159.2, 171.0; MS: m/z ¼ 489.45 [M þ 1]þ;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
923
Analysis: for C28H25FN2O3S, calcd. C 68.83, H 5.16, N 5.73, S 6.56%; N0-(2,3-Dihydroxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-
found C 68.56, H 5.15, N 5.75, S 5.13%.
5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (11)
Yield: 52%; m. p.: 220–222 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3060
(CH str.), 1671 (C¼O, Str.), 1596 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.24 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.62 (1H, s,
CH2), 3.97 (1H, s, CH2), 6.74 (1H, s, ¼CH), 6.84–7.91 (10H, m, Ar-H),
8.42 (1H, s, N¼CH), 9.11 (1H, s, OH, D2O exchg.), 9.21 (1H, s, OH,
D2O exchg.), 11.32 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 31.7, 43.5, 110.7, 111.0, 116.9,
117.1, 117.8, 119.1, 119.6, 120.3, 121.2, 124.4, 130.4, 133.1, 133.8,
138.4, 138.9, 139.0, 140.8, 146.0, 146.6, 146.7, 148.3, 165.6, 171.0;
MS: m/z ¼ 490.70 [M]þ; Analysis: for C27H23FN2O4S, calcd. C 66.11,
H 4.73, N 5.71, S 6.54%; found C 66.33, H 5.72, N 5.68, S 6.53%.
N0-(3-Methoxybenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-
fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (7)
Yield: 65%; m. p.: 130–132 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2960
(CH str.), 1664 (C¼O, Str.), 1588 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.82 (3H, s, CH3S¼O), 3.79 (3H, s,
OCH3), 4.01 (2H, s, CH2), 6.70 (1H, s, ¼CH), 6.99–7.78 (11H, m, Ar-
H), 8.03 (1H, s, N¼CH), 11.52 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 11.0, 29.9, 43.5, 55.5, 93.0, 110.8,
116.3, 120.0, 123.5, 124.3, 129.9, 130.4, 133.7, 136.0, 138.4, 138.7,
139.0, 140.9, 143.7, 146.6, 147.1, 160.0, 171.5; MS: m/z ¼ 488.85
[M]þ; Analysis: for C28H25FN2O3S, calcd. C 68.83, H 5.16, N 5.73, S
6.56%; found C 69.10, H 5.17, N 5.75, S 6.54%.
N0-(2,5-Dihydroxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-
5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (12)
Yield: 55%; m. p.: 180–182 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2915
(CH str.), 1656 (C¼O, Str.), 1570 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.81 (3H, s, S¼OCH3), 4.02 (2H, s,
CH2), 6.72 (1H, s, ¼CH), 7.18–7.96 (10H, m, Ar-H), 8.38 (1H, s,
N¼CH), 8.98 (1H, s, OH, D2O exchg.), 10.22 (1H, s, OH, D2O exchg.),
11.78 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6):
d ¼ 11.0, 29.6, 31.8, 36.2, 106.7, 110.8, 114.0, 117.4, 119.4, 124.4,
129.9, 130.4, 139.0, 140.9, 146.6, 150.3, 162.8, 165.6, 171.0; MS: m/
z ¼ 490.55 [M]þ; Analysis: for C27H23FN2O4S, calcd. C 66.11, H 4.73,
N 5.71, S 6.54%; found C 66.30, H 4.74, N 5.69, S 6.55%.
N0-(2-Nitrobenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-5-flu-
oro-2-methyl-1H-inden-3-yl)acetohydrazide (8)
Yield: 70%; m. p.: 148–150 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3001
(CH str.), 1665 (C¼O, Str.), 1570 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.23 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.62 (1H, s,
CH2), 4.00 (1H, s, CH2), 6.71 (1H, s, ¼CH), 7.06–8.08 (11H, m, Ar-H),
8.42 (1H, s, N¼CH), 11.79 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 29.9, 43.5, 124.4, 125.0, 128.7,
129.7, 129.8, 130.4, 130.9, 133.9, 138.5, 139.0, 139.2, 140.8, 142.6,
147.7, 148.4, 171.8; MS: m/z ¼ 503.22 [M]þ; Analysis: for
C27H22FN3O4S, calcd. C 64.40, H 4.40, N 8.34, S 6.37%; found C
64.60, H 4.41, N 8.36, S 6.39%.
N0-(2,3-Dimethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-
5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (13)
Yield: 62%; m. p.: 203–205 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3014
(CH str.), 1654 (C¼O, Str.), 1565 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.21 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.84 (1H, s,
CH2), 3.78 (6H, s, 2 ꢂ OCH3), 4.00 (1H, s, CH2), 6.71 (1H, s, ¼CH),
7.11–7.78 (10H, m, Ar-H), 8.35 (1H, s, N¼CH), 11.47 (1H, s, CONH,
D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6): d ¼ 10.9, 29.9, 32.0,
43.5, 56.2, 61.6, 106.7, 106.9, 110.7, 114.4, 117.3, 124.3, 130.4,
138.4, 139.0, 139.5, 140.9, 142.5, 146.6, 148.3, 153.1, 162.2, 163.6,
165.7, 171.4; MS: m/z ¼ 518.84 [M]þ; Analysis: for C29H27FN2O4S,
calcd. C 67.16, H 5.25, N 5.40, S 6.18%; found C 67.3.1, H 5.24, N
5.38, S 6.19%.
N0-(3-Nitrobenzylidene)-2-1-(4-(methylsulfinyl)benzylidene)-5-flu-
oro-2-methyl-1H-inden-3-yl)acetohydrazide (9)
Yield: 70%; m. p.: 165–167 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3002
(CH str.), 1654 (C¼O, Str.), 1570 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.21 (3H, s, CH3), 2.82 (3H, s, S¼O CH3), 3.63 (1H, s,
CH2), 4.03 (1H, s, CH2), 6.70 (1H, s, ¼CH), 7.08–8.24 (11H, m, Ar-H),
8.52 (1H, s, N¼CH), 11.73 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 30.0, 43.5, 121.4, 124.3, 124.5,
129.7, 129.8, 130.4, 130.8, 133.4, 136.4, 138.4, 139.0, 140.8, 141.6,
146.6, 148.6, 171.8; MS: m/z ¼ 504.41 [M þ 1]þ; Analysis: for
C27H23FN2O2S, calcd. C 70.72, H 5.06, N 6.11, S 6.99%; found C
71.00, H 5.07, N 6.14, S 6.97%.
N0-(2,4-Dimethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-
5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (14)
Yield: 60%; m. p.: 185–187 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3011
(CH str.), 1654 (C¼O, Str.), 1600 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.21 (3H, s, CH3), 2.84 (3H, s, S¼OCH3), 3.83 (6H, s,
2 ꢂ CH3), 3.84 (2H, s, CH2), 6.63 (1H, s, ¼CH), 7.13–7.74 (10H, m,
Ar-H), 8.32 (1H, s, N¼CH), 11.32 (1H, s, CONH, D2O exchg.); 13C
NMR (125.76 MHz, DMSO–d6): d ¼ 10.9, 29.9, 32.0, 43.5, 55.8, 56.1,
98.5, 106.7, 110.7, 115.3, 123.4, 124.3, 127.0, 129.5, 130.3, 133.4,
138.2, 139.0, 140.8, 142.6, 147.5, 159.4, 162.2, 163.6, 165.4, 171.1;
MS: m/z ¼ 518.91 [M]þ; Analysis: for C29H27FN2O4S, calcd. C 67.16,
H 5.25, N 5.40, S 6.18%; found C 67.33, H 5.26, N 5.42, S 6.17%.
N0-(4-Nitrobenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-5-flu-
oro-2-methyl-1H-inden-3-yl)acetohydrazide (10)
Yield: 80%; m. p.: 220–222 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2962
(CH str.), 1666 (C¼O, Str.), 1591 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.23 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.64 (1H, s,
CH2), 4.00 (1H, s, CH2), 6.71 (1H, s, ¼CH), 7.08–7.97 (11H, m, Ar-H),
8.38 (1H, s, N¼CH), 11.80 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 29.8, 32.0, 43.5, 124.3, 124.4, N0-(3,4-Dimethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzylidene)-
128.2, 128.4, 129.8, 130.4, 139.0, 140.8, 141.4, 146.6, 148.1, 166.3,
171.9; MS: m/z ¼ 504.28 [M þ 1]þ; Analysis: for C27H22FN3O4S, calcd.
C 64.40, H 4.40, N 8.34, S 6.37%; found C 64.62, H 4.39, N 8.36,
S 6.38%.
5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (15)
Yield: 65%; m. p.: 230–232 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3002
(CH str.), 1656 (C¼O, Str.), 1571 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.25 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.58 (1H, s,

924
M. A. BHAT ET AL.
CH2), 3.81 (6H, s, 2 ꢂ OCH3), 4.00 (1H, s, CH2), 6.72 (1H, s, ¼CH), 11.36 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6):
7.01–7.79 (10H, m, Ar-H), 7.80 (1H, s, N¼CH), 11.38 (1H, s, CONH, d ¼ 10.9, 11.0, 15.1, 30.0, 43.5, 55.7, 64.1, 106.8, 108.7, 110.7, 120.6,
D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6): d ¼ 10.9, 30.0, 32.0, 121.7, 124.4, 127.1, 129.9, 130.4, 133.7, 138.3, 138.7, 139.0, 140.9,
43.5, 55.8, 56.0, 108.7, 111.9, 121.8, 122.2, 124.4, 127.3, 129.8, 144.0, 146.6, 147.4, 147.8, 149.5, 150.3, 162.2, 163.6, 165.6, 171.2;
130.4, 138.3, 139.4, 138.3, 139.0, 140.8, 144.0, 146.6, 149.4, 150.9, MS: m/z ¼ 535.19 [M þ 2]þ; Analysis: for C30H29FN2O4S, calcd. C
171.2; MS: m/z ¼ 518.58 [M]þ; Analysis: for C29H27FN2O4S, calcd. C 67.65, H 5.49, N 5.26, S 6.02%; found C 67.42, H 5.50, N 5.24,
67.16, H 5.25, N 5.40, S 6.18%; found C 67.32, H 5.26, N 5.38, S 6.01%.
S 6.17%.
N0-(3,5-Dimethoxy-4-hydroxybenzylidene)-2-(1-(4-(methylsulfinyl)-
N0-(2-Hydroxy-3-methoxymethoxybenzylidene)-2-(1-(4-(methylsulfi-
nyl)benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydra-
benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (20)
Yield: 65%; m. p.: 218–220 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3010
(CH str.), 1654 (C¼O, Str.), 1577 (C¼N str.); 1H NMR (500 MHz,
zide (16)
Yield: 55%; m. p.: 210–212 ꢁC; IR (KBr) cmꢀ1: 3180 (NH str.), 3051
DMSO–d6): d ¼ 2.23 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.84 (6H, s,
(CH str.), 1693 (C¼O, Str.), 1599 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.24 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.61 (1H, s,
CH2), 3.80 (3H, s, OCH3), 4.00 (1H, s, CH2), 6.82 (1H, s, ¼CH),
7.01–7.79 (10 H, m, Ar-H), 8.48 (1H, s, N¼CH), 10.78 (1H, s, OH,
D2O exchg.), 11.85 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.9, 29.9, 31.7, 43.5, 56.2, 106.7,
113.2, 114.1, 118.0, 119.3, 119.4, 119.6, 121.3, 130.4, 138.3, 138.8,
138.9, 139.0, 140.8, 141.0, 147.4, 162.2, 162.3, 163.7, 165.6, 171.2;
MS: m/z ¼ 504.34 [M]þ; Analysis: for C28H25FN2O4S, calcd. C 66.65,
H 4.99, N 5.55, S 6.35%; found C 66.85, H 4.50, N 5.53, S 6.34%.
2 ꢂ OCH3), 3.98 (2H, s, CH2), 6.74 (1H, s, ¼CH), 7.17–7.80 (9H, m,
Ar-H), 8.33 (1H, s, N¼CH), 11.31 (1H, s, CONH, D2O exchg.); 13C
NMR (125.76 MHz, DMSO–d6): d ¼ 10.9, 30.1, 43.5, 56.2, 56.9, 98.2,
106.8, 107.9, 108.3, 110.7, 113.6, 124.3, 130.4, 138.3, 139.0, 140.8,
142.7, 143.6, 146.6, 152.2, 153.6, 165.3, 171.1; MS: m/z ¼ 534.59
[M]þ; Analysis: for C29H27FN2O5S, calcd. C 65.15, H 5.09, N 5.24, S
6.00%; found C 65.30, H 5.10, N 5.22, S 6.01%.
N0-(2,3,4-Trihydroxybenzylidene)-2-(1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (21)
Yield: 50%; m. p.: 195–197 ꢁC; IR (KBr) cmꢀ1: 3180 (NH str.), 3051
(CH str.), 1668 (C¼O, Str.), 1598 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.24 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.59 (1H, s,
CH2), 6.38 (1H, s, ¼CH), 6.71–7.81 (10 H, m, Ar-H), 8.30 (1H, s,
N¼CH), 9.24 (1H, s, OH, D2O exchg.), 11.29 (1H, s, OH, D2O exchg.),
11.73 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz, DMSO–d6):
d ¼ 10.9, 29.7, 31.7, 43.5, 108.0, 111.1, 121.5, 124.4, 130.4, 133.1,
138.8, 139.0, 140.8, 146.7, 147.8, 149.2, 149.5, 162.3, 163.7, 165.2,
170.5; MS: m/z ¼ 504.94 [M-1]þ; Analysis: for C27H23FN2O5S, calcd.
C 64.02, H 4.58, N 5.53, S 6.33%; found C 64.19, H 4.57, N
5.55, S6.31%.
N0-(3-Hydroxy-4-methoxybenzylidene)-2-(1-(4-(methylsulfinyl)ben-
zylidene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (17)
Yield: 58%; m. p.: 208–210 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3023
(CH str.), 1657 (C¼O, Str.), 1569 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.22 (3H, s, CH3), 2.83 (3H, s, S¼OCH3), 3.56 (1H, s,
CH2), 3.82 (3H, s, OCH3), 3.99 (1H, s, CH2), 6.72 (1H, s, ¼CH),
6.98–7.80 (10H, m, Ar-H), 7.92 (1H, s, N¼CH), 9.24 (1H, s, OH, D2O
exchg.), 11.32 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz,
DMSO–d6): d ¼ 10.9, 29.7, 32.0, 43.5, 56.0, 106.7, 112.6, 120.3,
120.7, 127.4, 130.4, 138.4, 138.6, 139.0, 140.8, 144.1, 147.2, 150.2,
162.2, 163.6, 165.5, 171.1; MS: m/z ¼ 504.94 [M]þ; Analysis: for
C28H25FN2O4S, calcd. C 66.65, H 4.99, N 5.55, S 6.35%; found C
66.45, H 4.98, N 5.56, S 6.36%.
N0-(2,3,4-Timethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (22)
Yield: 70%; m. p.: 185–187 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2987
(CH str.), 1651 (C¼O, Str.), 1589 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.21 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.82 (9H, s,
3 ꢂ OCH3), 3.97 (2H, s, CH2), 6.73 (1H, s, ¼CH), 7.18–7.79 (9H, m,
Ar-H), 8.24 (1H, s, N¼CH), 11.35 (1H, s, CONH, D2O exchg.);13C
NMR (125.76 MHz, DMSO–d6): d ¼ 11.0, 30.0, 32.1, 43.6, 56.5, 61.0,
62.3, 106.9, 109.2, 110.9, 121.0, 124.5, 130.0, 130.5, 138.4, 139.1,
139.8, 141.0, 142.1, 142.6, 146.7, 153.0, 155.5, 162.3, 163.7, 165.6,
171.3; MS: m/z ¼ 548.03 [M]þ; Analysis: for C30H29FN2O5S, calcd. C
65.68, H 5.33, N 5.11, S 5.84%; found C 65.45, H 5.34, N 5.13,
S 5.85%.
N0-(3-Ethoxy-4-hydroxybenzylidene)-2-(1-(4-(methylsulfinyl) benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (18)
Yield: 60%; m. p.: 193–195 ꢁC; IR (KBr) cmꢀ1: 3255 (NH str.), 2940
(CH str.), 1661 (C¼O, Str.), 1592 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 1.37 (3H, s, CH3), 2.21 (3H, s, CH3), 2.83 (3H, s,
S¼OCH3), 3.43 (2H, q, OCH2), 4.04 (2H, s, CH2), 6.83 (1H, s, ¼CH),
7.17–7.81 (10H, m, Ar-H), 8.16 (1H, s, N¼CH), 9.49 (1H, s, OH, D2O
exchg.), 11.31 (1H, s, CONH, D2O exchg.); 13C NMR (125.76 MHz,
DMSO–d6): d ¼ 11.0, 15.2, 30.1, 43.6, 54.3, 110.9, 116.0, 121.9,
123.6, 124.4, 126.0, 129.8, 130.0, 133.8, 138.3, 138.7, 139.1, 140.9,
144.4, 146.5, 147.6, 149.4, 162.3, 163.7, 165.6, 171.2; MS: m/
z ¼ 520.49 [M þ 2]þ; Analysis: for C29H27FN2O4S, C 67.16, H 5.25, N
5.40, S 6.18%; found C 67.35, H 5.24, N 5.42, S 6.19%.
N0-(2,4,5-Trimethoxybenzylidene)-2-1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (23)
Yield: 60%; m. p.: 233–235 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2914
(CH str.), 1658 (C¼O, Str.), 1598 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.16 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.63 (9H, s,
N0-(3-Methoxy-4-ethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (19)
Yield: 65%; m. p.: 158–160 ꢁC; IR (KBr) cmꢀ1: 3227 (NH str.), 3100 3 ꢂ OCH3), 3.70 (2H, s, CH2), 6.73 (1H, s, ¼CH), 7.01–7.79 (9H, m,
(CH str.), 1658 (C¼O, Str.), 1603 (C¼N str.); 1H NMR (500 MHz, Ar-H), 8.80 (1H, s, N¼CH), 11.11 (1H, s, CONH, D2O exchg.); 13C
DMSO–d6): d ¼ 1.32 (3H, s, CH3), 2.22 (3H, s, CH3), 2.80 (3H, s, NMR (125.76 MHz, DMSO–d6): d ¼ 10.70, 31.0, 43.5, 52.3, 106.4,
S¼OCH3), 3.76 (3H, s, OCH3), 3.98 (2H, q, OCH2), 4.04 (1H, s, CH2), 110.9, 124.4, 130.4, 138.8, 140.6, 147.2, 160.3, 162.3, 162.6, 163.6,
6.71 (1H, s, ¼CH), 6.96–7.95 (10H, m, Ar-H), 8.17 (1H, s, N¼CH), 171.0; MS: m/z ¼ 544.67 [M-4]þ; Analysis: for C30H29FN2O5S, calcd.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
925
C 65.68, H 5.33, N 5.11, S 5.84%; found C 65.45, H 5.32, N 5.09, 24 h. Water was made available. Control temperature was taken
S 5.85%.
24 h after the yeast injection to determine the pyretic response to
yeast. Temperature taken 1 h prior to drug administration in the
fevered animals, served as a pre drug control. Drugs were given
24 h after the yeast injection and temperature were recorded at
60, 90 and 120 min. after the administration.
N0-(2,4,6-Timethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (24)
Yield: 68%; m. p.: 193–195 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 3001
(CH str.), 1652 (C¼O, Str.), 1584 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.19 (3H, s, CH3), 2.79 (3H, s, S¼OCH3), 3.78 (9H, s,
3 ꢂ OCH3), 3.85 (2H, s, CH2), 6.68 (1H, s, ¼CH), 7.18–7.72 (9H, m, Ar-
H), 8.22 (1H, s, N¼CH), 11.11 (1H, s, CONH, D2O exchg.); 13C NMR
(125.76 MHz, DMSO–d6): d ¼ 10.86, 29.38, 32.0, 43.5, 55.8, 56.3, 91.5,
104.1, 106.8, 110.8, 123.5, 124.4, 129.6, 130.4, 138.6, 139.1, 141.0,
142.9, 146.5, 147.9, 160.3, 162.3, 162.7, 163.7, 171.1; MS: m/
z ¼ 547.97 [M-1]þ; Analysis: for C30H29FN2O5S, calcd. C 65.68, H 5.33,
N 5.11, S 5.84%; found C 65.88, H 5.34, N 5.13, S 5.85%.
Analgesic study by tail flick method
Acute nociception was assessed using a tail flick apparatus (Tail
Flick model DS 20 Sorrel Apelex, France) following the method20.
Briefly, each animal was placed in a restrainer, 2 min. before treat-
ment, and baseline reaction time was measured by focussing an
intensity controlled beam of light on the distal one-third portion
of the animals’ tail. The suspension was orally administered imme-
diately after this step and 25 min. later, the post drug reaction
time was measured. A 10-s cut-off time was used in order to pre-
vent tissue damage.
N0-(3,4,5-Trimethoxybenzylidene)-2-(1-(4-(methylsulfinyl)benzyli-
dene)-5-fluoro-2-methyl-1H-inden-3-yl)acetohydrazide (25)
Yield: 62%; m. p.: 165–167 ꢁC; IR (KBr) cmꢀ1: 3150 (NH str.), 2917
(CH str.), 1652 (C¼O, Str.), 1598 (C¼N str.); 1H NMR (500 MHz,
DMSO–d6): d ¼ 2.23 (3H, s, CH3), 2.82 (3H, s, S¼OCH3), 3.81 (9H, s,
3 ꢂ OCH3), 4.01 (1H, s, CH2), 6.72 (1H, s, ¼CH), 7.05–7.79 (9H, m,
Ar-H), 7.98 (1H, s, N¼CH), 11.50 (1H, s, CONH, D2O exchg.); 13C
NMR (125.76 MHz, DMSO–d6): d ¼ 11.2, 30.2, 43.6, 56.4, 60.7, 104.6,
107.0, 110.9, 123.6, 124.5, 130.0, 130.5, 133.8, 138.8, 139.1, 139.5,
140.9, 143.9, 146.7, 147.3, 147.7, 153.7, 162.4, 163.7, 171.6; MS: m/
z ¼ 547.64 [M-1]þ; Analysis: for C30H29FN2O5S, calcd. C 65.68, H
5.33, N 5.11, S 5.84%; found C 65.43, H 5.32, N 5.13, S 5.82%.
Analgesic activity by hot plate method
The hot plate method used as described by Turner21. The animals
were dropped gently on a hot plate maintained at 55 5.5 ꢁC. The
reaction time was taken as the interval extending from the instant
the animal reached the hot plate until the moment the animal
licked its forefeet or jumped off. The reaction time was measured
10 min before the oral administration of the drug and
þ60 þ 90 þ 120 min after treatments.
Analgesic activity by writhing test
Writhing was induced in mice by intraperitoneal administration of
0.1 ml of 1% acetic acid. The number of writhing movements was
counted for 20 min. The writhing test was performed after the
administration of the vehicle or drug.
DPPH radical scavenging assay
The antioxidant activity was measured based on the scavenging
activity of the stable DPPH free radical. The antioxidant activity
was determined by following the method18. The compounds in
concentration of (100 mg/mL) were added to 3 ml of 0.004% DPPH
solution. Methanol was replaced in the control sample.
Absorbance was determined at 520 nm after 30 min. Butylated
hydroxytoluene (BHT) was used a reference drug. The percent
inhibition was calculated by the following equation: A0 ꢀ At/Ao ꢂ
100, where At ¼ absorbance of compound, A0 ¼ absorbance
of control.
Carrageenan-induced paw edoema in rats
Pedal inflammation in albino rats of either sex was produced
according to the reported method22. An injection wad made of
0.05 ml of 1% carrageenan sodium salt (BDH) into right hind foot of
each rat under the plantar aponeurosis. The test group of rats was
treated orally with drugs 1 h before carrageenan injection. At the
same time, control group was given 5 ml/kg of normal saline and
the reference group was given 100 mg/kg of an aqueous solution
of sulindac. The measurements of foot volume were done by the
displacement technique using a plethysmometer (Apelex, France)
immediately after and þ2 and þ3 h after the injection of carra-
geenan. The inhibitory activity was calculated to the following for-
mula 100 (1 ꢀ a ꢀ x/b ꢀ y); where “b” is the mean paw volume of
control rats after carrageenan injection and “y” before the injection;
whereas” x” is the mean paw volume of treated rats before injec-
tion and “a” is the mean paw volume after carrageenan injection.
Pharmacological activities
Thirty-five Adult wistar male rats weighing 240–260 g, 12–14
weeks’ old and mice were obtained from animal house of
Department of the Community Health Sciences, College of
Applied Medical Sciences, King Saud University, Riyadh, KSA.
Animals were given favourable conditions (temperature 25 ꢁC, 12/
12 h light and dark cycle, and humidity 60 10% and pathogen-
free environment). The rats were fed a dietary formulation of pro-
tein (18.1%), fat (7.1%), carbohydrate (59.3%) and fibre (15.5%)
with food and water being provided ad libitum. The study proto-
col was approved by Ethical Committee of College of Applied
Medical Sciences, King Saud University, Saudi Arabia. (Ethics
Number: CAMS 22 – 39/40).
Ulcer study of drugs using 80% ethanol
The ethanol induced ulcer model was used to study gastro-pro-
tective activity of compound 3. The rats were grouped into five
groups (n ¼ 6). Groups I and II received saline solution and served
as negative-control and ulcer-control, respectively. Group III
received compound 3 (150 mg/kg) orally and served as the experi-
Anti-pyretic study
Hyperthermia was induced in mice by (s.c.) injection of (20 ml/kg) mental drug group. Animals in groups IV received the Sulindac
of a 20% aqueous suspension of brewer’s yeast in the back below (100 mg/kg body weight). After 1 h, all of the groups, except
of nape of the neck19. The animals were fasted for the duration of Group I, received (20 ml/kg) of 80%. The animals were sacrificed

926
M. A. BHAT ET AL.
1 h later under anaesthesia and their stomachs were quickly
removed for further studies23.
ꢃ
ꢃ
Group 3: Cisplatin group, received a single dose of cisplatin
(12 mg/kg, i.p) on the second day of experiment.
Group 4: Cisplatin – compound 3 group, received compound 3
(20 mg/kg i.p) for 7 days and a single dose of Cisplatin (12 mg/
kg) on the second day of experiment, 1 h after the dose of
compound 3.
Group 5: Cisplatin – compound 3 group, received compound 3
(40 mg/kg i.p) for 7 days and a single dose of Cisplatin (12 mg/
kg) on the second day of experiment, 1 h after the dose of
compound 8.
Gastric lesions induced by indomethacin
Suspension of Indomethacin in 1.0% carboxymethylcellulose
(CMC) in water (6 mg/mL) at a dose of (30 mg/kg) body weight
was administered orally. Control rats were treated with vehicle.
Compound 3 was given half an hour prior to Indomethacin
administration at a dose of 150 mg/kg24.
ꢃ
The doses of compound 3 and cisplatin were selected after
performing the pilot experiment.
Determination of malondialdehyde (MDA)
The MDA was measured according to the method by Utley and
others25. The tissue was removed and each tissue was homoge-
nised in 0.15 M KCl to give a 10% W/v homogenate. Aliquots of
homogenate (1 ml) were incubated at 37 ꢁC for 3 h in a metabolic
shaker. Then 1 ml of 10% aqueos trichloroacetic acid was added
and mixed. This was then centrifuged at 4000 rpm for 10 min.
Total of 1 ml of the supernatant was removed and mixed with
1 ml of 0.67% thiobarbituric acid in water and placed in a boiling
water bath for 10 min. The mixture was cooled and diluted with
1 ml of distilled water. The absorbance of the solution was then
read at 535 nm. The content of MDA (nmol/g) was then calculated,
by the reference to a standard curve of MDA solution.
Sample collection and preparation
On the last day of experiment, all animals were terminated and
anaesthesia was made by injecting ketamine/xylazine mixture (75/
2.5 mg/kg,
respectively)
via
the
intraperitoneal
route.
Anaesthetised rats were secured in a supine position and organ
samples were taken from the liver. The liver tissues were quickly
harvested. The tissues were treated with liquid nitrogen and were
used for RNA extraction and immunoblotting.
Extraction of protein and Western blot analysis
The previously developed procedures with slight modifications
were used to perform SDS-PAGE and western blot investigations.
The protein concentration was estimated by Bradford assay. For
western blotting 8–12% polyacrylamide gels were used to resolve
40 lM of protein, transferred on to a nitrocellulose membrane,
probed with appropriate monoclonal primary antibodies, and
detected by super signal west Pico, Dura or Femto
Estimation of non-protein sulfhydryls (NP-SH)
Hepatic non-protein sulfhydryls (NP-SH) was measured according
to the reported method by Sedlak and Lindsay26. The tissue was
homogenised in ice cold 0.02 mmol/L ethylenediaminetetraacetic
acid (EDTA). The Aliquotes of 5 ml of the homogenates were
mixed in 15 ml test tube with 4 ml of distilled water and 1 ml of
50% trichloacetic acid (TCA). The tube was shaken intermittently
for 10 min. and centrifuged 3000 rpm for 10 min. Total of 2 ml
supernatant was mixed with 4 ml of 0.4 mmol/L tris buffer (pH
8.9). Total of 0.1 ml of 5,5-dithiobis (2-nitrobenzoic acid) (BTNB)
was added and the sample was shaken. The absorbance was
measured within 5 min of addition of DTNB at 412 nm against
reagent blank.
Chemiluminescence
Reagent
(Thermo
Scientific,
USA).
Quantification of protein bands was done through measuring
band density using Image J software. The densities of the bands
(normalised to actin) relative to that of the untreated control (des-
ignated as 1.00) were presented as mean SEM of three separate
experiments.
Gene expression analysis
Determination of LD50
Total RNA from frozen liver was extracted by using kit, according
to the manufacturer’s instructions (Promega, CatLog No: Z3101).
The cDNA synthesis was performed using the Applied
BiosystemsTM High-Capacity cDNA Reverse Transcription Kit. The
reaction mixture was prepared containing 10 mL FastStart
Universal SYBR Green Master (Roche, Germany), 6 mM reverse pri-
mers, and 10 mg cDNA, with RNAase free water added to a total
volume of 20 mL. The amplification and real-time analysis were
done for 40 cycles with following factors; 95 ꢁC (10 min.) in order
to activate of FastStart Taq DNA polymerase; 60 ꢁC (1 min.) for
amplification and real-time analysis. The gene expression levels
were determined using 2-DDCT. Primer sequences used are
shown below:
The LD50 (lethal dose 50%) was calculated for compound 3 by
Karber method27. For determination of LD50, an observation was
made for 24 h and symptoms of toxicity and rate of mortality
were noted. Expired animals were counted at the end of the study
P
period for the calculation of LD50. LD50 ¼ LD100
ꢀ
ꢂ (a ꢂ b)/n,
where n is the total number of animals in a group, a is the differ-
ence between two successive doses of administered extract/sub-
stance,
b is the average number of dead animals in two
successive doses, and LD100 is the lethal dose causing 100% death
of all test animals.
COX-2 mRNA expression in cisplatin-induced hepatotoxicity in rats
The animals were divided into five groups and each group with
seven rats.
Primers
Candidate gene primer
ꢃ
ꢃ
Group 1: Control (Normal) group, received a single dose (i.p) of
isotonic saline on the second day of experiment.
Group 2: DMSO group, received a single dose (i.p) of 2%
DMSO on the second day of experiment.
ꢃ
ꢃ
COX-2 F: 50-CACTCATGAGCAGTCCCCTC-30
R: 50-ACCCTGGTCGGTTTGATGTT-30

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
927
H3C
O
O
O
F
HO
F
CH3
CH3
CH OH, H SO ,
Δ
3
2
4
H3C
H3C
(II)
S
S
O
O
(I)
NH NH .H O, CH OH,
Δ
2
2
2
3
F
H2N
NH
O
O
F
NH
N
C2H5OH
CH3
Ar
ArCHO/CH3COOH
CH3
H3C
S
H3C
O
S
(1-25)
O
(III)
Compound
Ar
Compound
Ar
1
2
3
4
5
6
7
8
9
10
11
12
13
Phenyl
14
15
16
17
18
19
20
21
22
23
24
25
2,4-Dimethoxyphenyl
3,4-Dimethoxyphenyl
2-Hydroxy-3-methoxy
4-Chlorophenyl
4-Dimethylaminophenyl
3-Hydroxyphenyl
4-Hydroxyphenyl
2-Methoxyphenyl
3-Methoxyphenyl
2-Nitrophenyl
3-Nitrophenyl
4-Nitrophenyl
2,3-Dihydroxyphenyl
2,5-Dihydroxyphenyl
2,3-Dimethoxyphenyl
3-Hydroxy-4-methoxyphenyl
4-Hydroxy-3-ethoxyphenyl
3-Methoxy-4-ethoxyphenyl
4-Hydroxy-3,5-dimethoxyphenyl
2,3,4-Trihydroxyphenyl
2,3,4-Trimethoxyphenyl
2,4,5-Trimethoxyphenyl
2,4,6-Trimethoxyphenyl
3,4,5-Trimethoxyphenyl
Scheme 1. Synthetic route of compounds (1–25).
Molecular docking of compounds against COX-2 protein
Results and discussion
Three-dimensional structure of the Cox-2 gene was developed
using homology modelling. Modeller 9.17 was employed to pre-
Chemistry
dict the structure using templates (5F1A, 5IKQ, 5F19) down- Scheme 1 illustrates the synthesis of the acetohydrazide deriva-
loaded from PDB. All of the models showed more than 90% tives (1 – 25). The compound acetohydrazide (III) was synthesised
identity with our protein. The predicted structure was further by refluxing methyl ester of sulindac and hydrazine hydrate (99%)
refined by energy minimisation. Finally, the structure was vali- in the presence of absolute ethanol. Sulindac methyl ester (II) was
dated using the Ramachandran plot. Furthermore, three-dimen- synthesised from sulindac by refluxing in methanol with concen-
sional structures of all synthetic compounds and Sulindac were trated sulphuric acid according to the reported procedure.
constructed using Chem 3 D Pro 12.0 version. The protein-ligand
The acetohydrazide (III) was used as a starting material for the
docking analysis was performed using online PatchDock server. synthesis of various substituted sulindac hydrazone derivatives
Three-dimensional structures of protein and ligands were used (1 – 25). The acetohydrazide (III) was reacted substituted benzal-
as input. PatchDock server rated the all possible docking confir- dehydes in ethanol and glacial acetic acid as a catalyst. The aceto-
mations using minimum ACE (Atomic contact energies). Finally, hydrazide was characterised by the appearance of singlet peak for
the docking confirmations were visualised using Pymol the –NH2 protons at d 3.38 ppm and broad singlet for the CONH
and LigPlus.
proton at d 9.30 ppm. The disappearance of NH2 protons at d

928
M. A. BHAT ET AL.
Table 1. Antioxidant activity of compounds (1–25) by DPPH method.
Compound
Scavenging effect % (mean SD)a
1
2
3
4
5
6
7
8
67.40 9.25
39.86 10.80
85.10 6.80
42.20 13.20
45.06 22.57
39.73 15.65
71.03 5.31
18.53 4.86
49.36 5.57
19.26 5.32
33.10 8.41
38.10 10.92
62.93 6.72
23.07 6.95
40.60 14.40
27.66 2.12
51.40 16.66
35.50 9.05
37.90 10.35
29.66 16.93
30.06 3.30
63.56 0.47
78.97 9.36
30.80 2.50
42.33 18.38
90.6 3.83
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
BHT
Figure 1. Comparison of DPPH scavenging activity of compounds (1–25) and
BHT. All values are means of three replicates SD.
3.38 ppm confirmed the structures of sulindac hydrazones.
Carbon-13 NMR confirmed all the carbon atoms of the synthesised
compounds (1–25). Mass spectroscopy confirmed the molecular
weights of compounds. All the compounds were characterised by
their molecular ion peaks. The three methyl protons of the indene
moiety appeared in the range of d 2.18–2.23 ppm. The three pro-
tons of –SOCH3 appear at d 2.81–2.83 ppm. The aromatic protons
appeared in the range of d 6.71–8.24 ppm. Protons of the N¼CH
appeared as a singlet in the range of d 7.92–8.52 ppm and the
proton of CONH appeared as broad singlet at d 9.30–11.80 ppm.
Compound number 3 having highest Scavenging effect represented in bold.
aAll values are means of three replicates SD.
(63.33% inhibition) was observed after 120 min for the compound
3 as compared to reference compound, sulindac.
Antioxidant activity
The synthesised compounds (1–25) were evaluated for their anti-
oxidant activity by DPPH method (Figure 1). All the compounds
exhibit antioxidant activity from (18.53 4.86) to (85.10 6.80) as
compared to standard drug BHT (90.6 3.83) (Table 1).
Hot plate method
Hot plate method was used for testing the analgesic activity. After
30 min, the % inhibition of test compound 3 was 15% as com-
pared to reference drug sulindac with 41.30%.The testing com-
pound 3 expressed highly significant activity of 67.50% inhibition
compared to reference drug sulindac with 86.95 inhibition after
60 min (Table 3).
Structure–activity relationship (SAR)
Compound 3 (R ¼ para dimethylaminophenyl) was found to be
highly potent antioxidant (85.10 6.80). Compounds
1
Acetic acid –induced writhing
(R ¼ phenyl), 7 (R ¼ 3-methoxyphenyl), 23 (R ¼ 2,4,6-trimethoxy-
phenyl), 13 (R ¼ 2,3,dimethoxyphenyl), 22 (R ¼ 2,3,4-trimethoxy-
phenyl) and 17 (R ¼ 3-hydroxy, 4-methoxy) were found to be
significantly active as antioxidants as compared to BHT.
Compounds containing hydroxyl phenyl substitutions were found
to be week antioxidants. Compounds 8 (R ¼ 2-nitrophenyl) and 10
(R ¼ 4-nitrophenyl) were found to be least active as antioxidants.
Electron donating groups like methoxy, were found to have posi-
tive effect while as electron withdrawing groups like nitro, was
found to have negative effect on antioxidant activity. Compound
3 having para dimethylaminophenyl group was found to be the
most potent anti-oxidant compound.
Acetic acid induced writhing was used for testing analgesic activ-
ity. The compound 3 expressed significant analgesic activity of
58.56% inhibition compared to reference drug sulindac with
74.1% inhibition (Table 4).
Yeast-induced hyperthermia
Yeast-induced hyperthermia was used for testing analgesic activity
in mice. The compound
activity of after 120 min as compared to reference drug sulindac
(Table 5).
3 expressed significant analgesic
Anti-inflammatory activity
Analgesic activity
Based on the in vitro antioxidant activity, compound 3 was
selected for in vivo anti-inflammatory activity by carrageenan
induced paw edoema method. The anti-inflammatory activity of
tested compound 3 after 3 and 5 h ranges from 50.52 to 50.54%,
respectively compared to reference drug sulindac, which showed
65.18% after 3 h and 65.02% after 5 h (Table 6). Because of hydra-
zide substitution of para dimethylaminophenyl group, compound
Tail flick method
Tail flick method was used for testing the analgesic activity of
compound 3. After 30 min, the % inhibition of test compound 3
was 3.33% as compared to reference drug sulindac with 13.79%.
The testing compound 3 expressed significant activity of 46.66%
inhibition compared to reference drug sulindac with 82.75 inhib-
ition after 60 min (Table 2). Highly significant analgesic activity 3 presented significant anti-inflammatory activity.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
929
Table 2. Analgesic effect of drugs by Tail flick method in mice.
Reaction time (s) post drug
Treatment (n ¼ 6)
Dose (mg/kg)
Reaction time (s) pre drug
30 m
% Inhibition
60 m
% Inhibition
120 m
% Inhibition
ꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
Compound 3
150
100
5.00 0.36
4.83 0.40
4.83 0.30
5.50 0.34
3.33
13.79
7.33 0.42
8.83 0.79
46.66
82.75
7.16 0.30
11.00 0.57
63.33
127.58
ꢄꢄꢄ
Sulindac
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄ ꢄꢄꢄ
p < 0.01. p < 0.001.
Table 3. Analgesic effect of drugs by Hot Plate method in mice.
Reaction Time (seconds) post drug
Treatment (n ¼ 6) Dose (mg/kg) Reaction Time (seconds) pre drug
30 m
% Inhibition
60 m
% Inhibition
120 m
% Inhibition
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
Compound 3
150
100
6.66 0.33
7.66 0.33
7.66 0.42
10.83 0.47
15
41.30
9.83 0.30
11.83 0.47
47.50
54.34
11.16 0.30
14.33 0.42
67.50
86.95
ꢄꢄꢄ
Sulindac
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄꢄ
p < 0.001.
Table 4. Analgesic effect of drugs by acetic acid-induced writhing in mice.
Treatments (n ¼ 6)
Dose (mg/kg)
Number of writhing in 20 min
% Inhibition
ꢄꢄꢄ
Compound 3
Sulindac
150
100
0.1 ml of 20%
14.66 0.95
8.83 0.70
56.86
74.01
—
ꢄꢄꢄ
Control (acetic acid)
34.00 1.23
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄꢄ
p < 0.001.
Table 5. Effect of compound 3 and sulindac on yeast-induced hyperthermia in mice.
Rectal temperature
after yeast
administration
Rectal temperature ꢁC post drug administration
Normal rectal
temperature
Treatment (n ¼ 6)
Dose (mg/kg)
20 mL/kg of 20%
30 m
60 m
120 m
ꢄꢄꢄ
ꢄ
ꢄꢄꢄ
ꢄꢄꢄ
Compound 3
150
100
35.16 0.10
35.21 0.11
38.38 0.19
38.21 0.31
38.15 0.10
37.46 0.14
37.83 0.15
37.16 0.09
37.33 0.12
36.23 0.08
ꢄꢄꢄ
ꢄꢄ
Sulindac
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄ
ꢄꢄ ꢄꢄꢄ
p < 0.05, p < 0.01, p < 0.001.
Table 6. Anti-inflammatory activity of drugs by carrageenan-induced paw edoema method in albino rats.
Increase paw volume after 3 h
Before Carrageenan
Increase paw volume after 5 h
Group (n ¼ 6)
Only carrageenan
Compound 3
Sulindac
Dose (mg/kg)
Mean SE
Mean SE
Net
% Inhibition
Mean SE
Net
% Inhibition
ꢄꢄꢄ
ꢄꢄꢄ
1.04 0.02
1.02 0.02
1.03 0.03
1.68 0.01
1.34 0.02
1.26 0.03
0.63 0.01
0.31 0.03
0.22 0.01
1.65 0.01
1.33 0.01
1.25 0.03
0.61 0.02
0.30 0.02
1.25 0.03
Compound 3
50.54
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
150
100
50.52
65.18
ꢄꢄ
65.02
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄ ꢄꢄꢄ
p < 0.01, p < 0.001.
Table 7. Ulcer study of drugs using 80% Ethanol.
Treatments
Dose mg/kg
Ulcer Index
% Inhibition
80% Ethanol only
1 mL/200 g Rat
7.33 0.33
6.83 0.40
4.33 0.4
1.33 0.49
—
80% Ethanol only þ Sulindac
80% Ethanol only þ Compound 3
Sulindac only
100
150
100
150
6.81
40.90
ꢄꢄꢄ
Compound 3 only
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄꢄ
p < 0.001.
The significant anti-inflammatory activity of compound 3
was observed
compared to reference drug sulindac with 6.83 0.40 (6.81%
inhibition) (Table 7).
Ulcerogenic activity
The compound 3 was further evaluated for ulcerogenic and lipid
peroxidation activity. Equimolar concentration of compound 3 and
reference drug sulindac was administered as oral doses to the
examined animals. Compound 3 demonstrated highly significant
Ulcer study with indomethacin
Compound 3 was further evaluated for its ulcerogenicity as
compared to indomethacin. Maximum ulcerogenic activity
ulcerogenic reduction activity 4.33 0.40 (40.90% inhibition) as was observed in indomethacin (37.00 1.59) as ulcer index.

930
M. A. BHAT ET AL.
Table 8. Ulcer study of drugs compared with Indomethacin
Treatments
Dose mg/kg
Ulcer Index
% Inhibition
40.09
Indomethacin
Sulindac only
Compound 3 only
30
100
150
37.00 1.59
32.00 3.86
ꢄꢄꢄ
22.16 1.10
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄꢄ
p < 0.001.
Table 9. Anti-oxidant activity, MDA, NP-SH and total protein of drugs in stomach tissue of rat.
Treatments
Dose mg/dL
MDA (nmol/g)
1.03 0.02
NP-SH (nmol/g)
8.29 0.53
Total protein (g/L)
113.37 2.94
Normal saline
80% Ethanol only
80% Ethanol only þ Sulindac
80% Ethanol only þ Compd. 3
Sulindac only
1 mL
1 mL
1 mL þ 100
1 mL þ 150
100
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
6.53 0.56
3.04 0.39
47.90 2.89
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
ꢄꢄꢄ
2.28 0.11
1.59 0.06
1.31 0.04
1.06 0.02
5.08 0.28
93.81 3.05
100.99 2.42
105.78 1.43
114.57 1.89
ꢄꢄꢄ
6.62 0.26
ꢄꢄꢄ
7.00 0.032
ꢄꢄꢄ
Compound 3 only
150
7.52 0.31
All values represent mean SEM. ANOVA, followed by Dunnett’s multiple comparison test.
ꢄꢄꢄ
p < 0.001.
Table 10. LD50 determination of compound 3.
Sulindac (100 mg/kg) also produced the ulcer with ulcer index
(32.00 3.86) whereas compound 3 demonstrated a significant
ulcerogenic reduction activity (22.16 1.10) with 40.09% inhibition
(Table 8).
Compound 3 with para dimethylaminophenyl substitution
was found to be the most potent anti-inflammatory and
analgesic derivative as well as a significant gastric sparing
activity.
ꢄ
Compound 3 Group Dose mg/kg
D. F (a)
Dead M.M (b) Pro. (a b)
1
2
3
4
5
6
100
200
400
800
1600
0
0
100
200
400
800
400
1
3
5
9
0.5
2
4
100
800
3200
2800
6900
1310
2000
7
Exp. Dose
131 mg/kg
MDA, NP-SH, total protein content in gastric tissue
It has been known that the reduction of malondialdehyde (MDA)
content in gastric tissue is consistent with the reduction of ulcero-
genic activity. Compound 3 has shown a maximum reduction
in the lipid peroxidation and gastric ulceration. The MDA content
in compound 3 group was found to be (1.06 0.02 nmol/g)
as compared to the 80% ethanol group (6.53 0.56 nmol/g)
(Table 9).
Toxicity of compound 3
Karber method was used to determine the LD50 of compound 3.
The LD50 of compound 3 was found to be 131 mg/kg (Table 10).
Effect of compound 3 on COX-2 mRNA expression in cisplatin
induced hepatotoxicity in rats
The expression of COX-2 is increased by pro-inflammatory medi-
taors28. Furthermore, the previous studies have revealed the
increased COX-2 mRNA expression in cisplatin-induced hepatotoc-
ity29. Hence we characterised the effect of compound 3 adminis-
tration on cisplatin-induced hepatotoxicity by measuring COX-2
mRNA levels in cisplatin-treated rats, untreated control rats,
DMSO-treated rats, cisplatin–compound 3 treated (20 and 40 mg/
kg) rats. Cisplatin-induced hepatotoxicity was categorised by a sig-
nificant increase in liver tissue gene expression of COX-2
(P < 0.001), when compared to normal values. Co-treatment of cis-
platin-treated rats with compound 3 significantly downregulated
liver tissue gene expression of COX-2 (P < 0.001), as compared to
the cisplatin values. Furthermore, co-treatment of cisplatin-treated
rats with 40 mg/kg dose of compound 3 significantly normalised
liver COX-2 gene expression (Figure 2).
Figure 2. Quantitative real-time PCR analysis for COX-2 mRNA expression in hep-
atocytes of treated and untreated rats with actin as housekeeping gene. Data
analysed by one-way analysis of variance using graph pad prism 8. Asterisks
þþþþ
ꢄꢄꢄꢄ
indicate p < 0.0001 significance from control and vehicle group,
indi-
##
cates p < 0.0001 significance from cisplatin (CP 12 mg/kg) treated group and
indicate p < 0.01 significance difference between CPþ T (20mg/kg) and CP þ T
(40mg/kg) treatment groups. T stands for compound 3 treatment.
Compound 3 administration induced reticence of COX-2 protein
expression in liver tissue
We further examined the anti-inflammatory effect of compound 3
in liver tissues in cisplatin administrated rats by measuring protein

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
931
expression of inflammatory mediators. In this regards, a substantial rats. The animals treated with compound 3 prior to cisplatin treat-
increase in COX-2 protein level was observed in the liver tissues iso- ment resulted in a significant reduction in COX-2 protein expression
lated from cisplatin-administrated rats against untreated, control when compared to cisplatin-treated group (Figure 3).
Figure 3. Effect of compound 3 treatment in regulating COX-2 protein expression in liver tissues of rats inoculated with cisplatin (12mg/kg dose). Immunoblot analysis
of COX-2 and b-actin in rat hepatocytes (n ¼ 7). CP: cisplatin, T: compound 3 treatments.
Table 11. Docking energies of compounds (1–25) and sulindac.
Compound
Docking energies
Compound
Docking energies
1
2
3
4
5
6
7
8
ꢀ81.19
ꢀ257.54
ꢀ440.29
ꢀ417.10
ꢀ413.27
ꢀ96.75
14
15
16
17
18
19
20
21
22
23
24
25
Sulindac
ꢀ247.54
ꢀ298.94
ꢀ252.92
ꢀ266.51
ꢀ227.18
ꢀ220.75
ꢀ150.57
ꢀ220.38
ꢀ457.75
ꢀ421.83
ꢀ466.48
ꢀ77.10
ꢀ392.71
ꢀ317.14
ꢀ388.61
ꢀ345.55
ꢀ242.36
ꢀ428.94
ꢀ275.16
9
10
11
12
13
ꢀ325.99
Figure 4. The orientation of docked compounds with Cox-2. The Sulindac is shown in blue colour while the compound 3 is shown in green colour. (A) The orientation
of docked molecules is shown in sticks format. (B) The orientation of docked molecules is shown in spheres format.

932
M. A. BHAT ET AL.
Figure 5. The orientation of Cox-2 residues making interactions with ligands (green) in 3 D confirmation. (A) The binding of drug Sulindac with Cox-2 protein. The
amino acids residues are making hydrophobic interaction with drug. (B) The binding of compound 3 with Cox-2 protein. The amino acids residues are making two
hydrogen bonds and hydrophobic interaction with the compound. (C) The Cys-32 and Tyr-116 making hydrogen bonds with compound 3.
The binding affinities of all the synthesised compound (1 – 25)
and Sulindac with Cox-2 were predicted using geometry-based
molecular docking. The binding affinities of the compounds were
demonstrated by PatchDock server with ACE values (Table 11).
The lower ACE value is considered to be associated with better
ligand affinity with the enzyme30. Compound 3 showed lower ACE
values (ꢀ440.29 kJ/mol) as compared to the standard drug
Sulindac (ꢀ325.99 kJ/mol). This data suggests a better binding
affinity of the compound 3 with Cox-2 protein as compared to
the Sulindac (Figure 4). The visualisation of the docked complex
revealed two hydrogen bonds by compound 3 with Cys-32 and
Tyr-116, along with many hydrophobic interactions (Figure 5).
While Sulindac showed only the hydrophobic interaction with the
protein in the docked complex (Figure 6). It can be speculated
that the formation of hydrogen bonds by compound 3 is respon-
sible for the better affinity of the compound as compared
to Sulindac.
Conclusion
In conclusion, novel sulindac hydrazide derivatives (1–25) were
synthesised in good yields and characterised by spectral data
and elemental analysis. Chemical modification to the sulindac
acetohydrazide scaffold resulted in twenty-five derivatives with
significant antioxidant activity. Compound 3 containing para
dimethylaminophenyl group was found to be having highly
potent antioxidant activity. It was evaluated for in vivo anti-
inflammatory and various analgesic and ulcerogenic activity dif-
ferent animal models and was found to be significant anti-
inflammatory and analgesic agent. It demonstrated significant
ulcerogenic reduction activity in ethanol and indomethacin
model. The LD50 of compound 3 was found to be 131 mg/kg.
Compound 3 with para dimethylaminophenyl substitution was
found to be the most potent anti-inflammatory and analgesic
derivative as well as
a significant gastric sparing agent.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
933
Figure 6. 2D map of interactions of Cox-2 protein with Sulindac (A) and compound 3 (B).
Compound
3 significantly downregulated liver tissue gene
ORCID
expression of COX-2. The animals treated with compound 3 prior
to cisplatin treatment resulted in a significant reduction in COX-2
protein expression when compared to cisplatin-treated group.
Mashooq A. Bhat
http://orcid.org/0000-0001-8426-4692
Data Availability
Samples of the compounds (1–25) in pure form are available
from authors.
Author contributions
Investigation, Ahmed M. Naglah, Suhail Razak; Methodology,
Abdulrahman A. Almehizia; Administration, Mohamed A. Al-Omar;
Resources; software, Nawaf A. Alsaif; Supervisor, Mashooq A. Bhat;
Manuscript writing and editing, Azmat Ali Khan; Molecular dock-
ing, Naeem Mahmood Ashraf.
References
1. Robert A, Nezamis JE, Lancaster C, et al. Mild irritants pre-
vent gastric necrosis through “adaptive cytoprotection medi-
ated by prostaglandins”. Am J Physiol 1983;245:G113–21.
2. Khatri CK, Indalkar KS, Patil CR, et al. Novel 2-phenyl-4,5,6,7-
tetrahydro[b]benzothiophene analogues as selective COX-2
inhibitors: Design, synthesis, anti-inflammatory evaluation,
and molecular docking studies. Bioorg Med Chem Lett 2017;
27:1721–6.
Disclosure statement
3. Abdelgawad MA, Bakr RB, Azouz AA. Novel pyrimidine-pyri-
dine hybrids: synthesis, cyclooxygenase inhibition, anti-
inflammatory activity and ulcerogenic liability. Bioorg Chem
2018;77:339–48.
No potential conflict of interest was reported by the author(s).
Funding
4. Banerjee AG, Das N, Shengule SA, et al. Synthesis, character-
ization, evaluation and molecular dynamics studies of 5, 6-
diphenyl-1,2,4-triazin-3(2H)-one derivatives bearing 5-
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for fund-
ing this research group no. (RG 1435–006).

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M. A. BHAT ET AL.
substituted 1,3,4-oxadiazole as potential anti-inflammatory 17. Furness B, Hannaford AH, Smith PWG, Tatchell AR, Vogels
and analgesic agents. Eur J Med Chem 2015;101:81–95.
Text book of practical organic chemistry. 5th ed. London:
Addison Wesely Longman, Inc.; 1998:1077–78.
5. Chandrashekharappa S, Venugopala KN, Tratrat C, et al.
Efficient synthesis and characterization of novel indolizines:
exploration of in vitro COX-2 inhibitory activity and molecu-
lar modelling studies. New J Chem 2018;42:4893–901.
6. Banerjee AG, Das N, Shengule SA, et al. Design, synthesis,
evaluation and molecular modelling studies of some novel
5,6-diphenyl-1,2,4-triazin-3(2H)-ones bearing five-member
heterocyclic moieties as potential COX-2 inhibitors: a hybrid
pharmacophore approach. Bioorg Chem 2016;69:102–20.
7. Bhat MA, Ahmed AF, Wen ZH, et al. Synthesis, anti-inflam-
matory and neuroprotective activity of pyrazole and pyra-
zolo[3,4-d]pyridazine bearing 3,4,5- trimethoxyphenyl. Med
Chem Res 2017;26:1557–66.
8. Bhat MA, Al-Omar MA, Ansari MA, et al. Design and synthe-
sis of N-arylphthalimides as inhibitors of glucocorticoid-
induced TNF receptor-related protein, proinflammatory
mediators, and cytokines in carrageenan-induced lung
inflammation. J Med Chem 2015;58:8850–67.
9. Romeiro NC, Leite RD, Lima LM, et al. Synthesis, pharmaco-
logical evaluation and docking studies of new sulindac ana-
logues. Eur J Med Chem 2009;44:1959–71.
18. Braca A, De Tommasi N, Di Bari L, et al. Antioxidant princi-
ples from Bauhinia tarapotensis. J Nat Prod 2001;64:892–5.
19. Loux JJ, De Palma PD, Yankell SL. Antipyretic testing of
aspirin in rats. Toxicol Appl Pharmacol 1972;22:672––5.
20. D’Amour FE, Smith DL. A method for determining loss of
pain sensation. J Pharmacol Exp Ther 1941;72:74–9.
21. Turner RA. Analgesic. In: Turner RA, ed. Screening methods
in pharmacology. London: Academic Press; 1965.
22. Winter CA, Risley EA, Nuss GW. Carrageenin-induced edema
in hind paw of the rat as an assay for antiiflammatory drugs.
Proc Soc Exp Biol Med 1962;111:544–7.
23. Yu C, Mei XT, Zheng YP, Xu DH. Gastroprotective effect of
taurine zinc solid dispersions against absolute ethanol-
induced gastric lesions is mediated by enhancement of anti-
oxidant activity and endogenous PGE2 production and
attenuation of NO production. Eur J Pharmacol 2014;740:
329–36.
24. Bhargava KP, Gupta MB, Tangri KK. Mechanism of ulcero-
genic activity of indomethacin and oxyphenbutazone. Eur J
Pharmacol 1973;22:191–5.
10. Fogli S, Banti I, Stefanelli F, et al. Therapeutic potential of
sulindac hydroxamic acid against human pancreatic and 25. Utley HG, Bernheim F, Hochstein P. Effect of sulfhydryl
colonic cancer cells. Eur J Med Chem 2010;45:5100–7.
11. Mathew B, Hobrath JV, Connelly MC, et al. Diverse amide
analogs of sulindac for cancer treatment and prevention.
Bioorg Med Chem Lett 2017;27:4614–21.
12. Mathew B, Snowden TS, Connelly MC, et al. A small diversity
library of a-methyl amide analogs of sulindac for probing
anticancer structure-activity relationships. Bioorg Med Chem
Lett 2018;28:2136–42.
13. Liedtke AJ, Crews BC, Daniel CM, et al. Cyclooxygenase-1-
selective inhibitors based on the (E)-2’-des-methyl-sulindac
sulfide scaffold. J Med Chem 2012;55:2287–300.
14. Felts AS, Siegel BS, Young SM, et al. Sulindac derivatives
that activate the peroxisome proliferator-activated receptor
gamma but lack cyclooxygenase inhibition. J Med Chem
2008;51:4911–9.
reagents on peroxidation in microsomes. Arch Biochem
Biophy 1967;118:29–32.
26. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and
nonprotein sulfhydryl groups in tissue with Ellman’s reagent.
Anal Biochem 1968;25:192–205.
27. Karber G. Beitrag zur kollektiven Behandlung pharmakolo-
gischer Reihenversuche. Arch Exptl Pathol Pharmakol 1931;
162:480–3.
28. Kim HJ, Kim KW, Yu BP, et al. The effect of age on cyclooxy-
genase-2 gene expression: NF-kappaB activation and
IkappaBalpha degradation. Free Radic Biol Med 2000;28:
683–92.
29. Kim SH, Hong KO, Chung WY, et al. Abrogation of cisplatin-
induced hepatotoxicity in mice by xanthorrhizol is related to
its effect on the regulation of gene transcription. Toxicol
Appl Pharmacol 2004;196:346–55.
30. Ashraf NM, Krishnagopal A, Hussain A, et al. Engineering of
serine protease for improved thermostability and catalytic
activity using rational design. Int J Biol Macromol 2019;126:
229–37.
15. Amir M, Kumar S. Anti-inflammatory and gastro sparing
activity of some new indomethacin derivatives. Arch Pharm
2005;338:24–31.
16. Bhat MA, Al-Omar MA, Raish M, et al. Indole derivatives as
cyclooxygenase inhibitors: synthesis, biological evaluation
and docking studies. Molecules 2018;23:1250.