Synthesis and amelioration of inflammatory paw edema by novel benzophenone appended oxadiazole derivatives by exhibiting cyclooxygenase-2 antagonist activity

Article abstract of DOI:10.1016/j.biopha.2018.04.167

Ten new 2(4-hydroxy-3-benzoyl) benzamide-5-phenyl-1,3,4-oxadiazole derivatives (10a–j) were synthesized by coupling 3-benzoyl-4-hydroxybenzoic acid (5) with 2-amino-5-phenyl-1,3,4-oxadiazoles (9a–j). The structures of these compounds were confirmed by IR, ¹H, ¹³C NMR, and mass spectra, and also by elemental analyses. The anti-inflammatory activity of the compounds 10a–j were investigated by screening them against human red blood cells (HRBC) in-vitro. The results reveal that among this series, compound 10j with hydroxy substituent, particularly at the ortho position of the phenyl ring attached to the 5th carbon atom of the oxadiazole ring possess significant membrane stabilizing activity in comparison with the control. Further, in-vivo chick chorioallantoic membrane (CAM) and rat corneal anti-angiogenesis assays were performed to assess the effect of compound 10j on endothelial cell migration. This confirmed that compound 10j inhibits the proliferation of endothelial cells. Anti-inflammatory studies detected the amelioration of carrageen induced rat hind paw edema. Further in-vivo and in-silico approaches revealed the inhibition of inflammatory marker enzyme cyclooxygenase-2 (Cox-2) and myleoperoxidase (MPO). The study reports that the compound 10j effectively act against the inflammatory mediated anti-angiogenic disorders which could be translated into a new drug in future.

Full text of DOI:10.1016/j.biopha.2018.04.167

Biomedicine&Pharmacotherapy103(2018)1446–1455
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Synthesis and amelioration of inflammatory paw edema by novel
benzophenone appended oxadiazole derivatives by exhibiting
cyclooxygenase-2 antagonist activity
Naveen Puttaswamya,b, Vikas H. Malojiaoc, Yasser Hussein Eissa Mohammeda,
Ankith Sherapurac, B.T. Prabhakarc, Shaukath Ara Khanuma,⁎
a
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore, 570005 Karnataka, India
Department of Chemistry, JSS College of Arts, Commerce & Science, Ooty Road, Mysore, 570025, India
Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College (Autonomous), Kuvempu University,
b
c
Shivammogga, 577203, Karnataka, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ten new 2(4-hydroxy-3-benzoyl) benzamide-5-phenyl-1,3,4-oxadiazole derivatives (10a–j) were synthesized by
coupling 3-benzoyl-4-hydroxybenzoic acid (5) with 2-amino-5-phenyl-1,3,4-oxadiazoles (9a–j). The structures
of these compounds were confirmed by IR, 1H, 13C NMR, and mass spectra, and also by elemental analyses. The
anti-inflammatory activity of the compounds 10a–j were investigated by screening them against human red
blood cells (HRBC) in-vitro. The results reveal that among this series, compound 10j with hydroxy substituent,
particularly at the ortho position of the phenyl ring attached to the 5th carbon atom of the oxadiazole ring
possess significant membrane stabilizing activity in comparison with the control. Further, in-vivo chick chor-
ioallantoic membrane (CAM) and rat corneal anti-angiogenesis assays were performed to assess the effect of
compound 10j on endothelial cell migration. This confirmed that compound 10j inhibits the proliferation of
endothelial cells. Anti-inflammatory studies detected the amelioration of carrageen induced rat hind paw edema.
Further in-vivo and in-silico approaches revealed the inhibition of inflammatory marker enzyme cyclooxygenase-
2 (Cox-2) and myleoperoxidase (MPO). The study reports that the compound 10j effectively act against the
inflammatory mediated anti-angiogenic disorders which could be translated into a new drug in future.
Angiogenesis
Benzophenones
Inflammatory
Oxadiazoles
1. Introduction
include the activation of downstream molecules such as prostaglandins
(PGs) which act as local intermediaries of inflammation which is
Inflammation is a part of the multifaceted biological response of
vascularised tissues to damaging stimuli. It is well thought-out as a
homeostatic response intended to destroy or deactivate invading pa-
thogens, eliminate squander and debris, that leads to re-establishment
of normal function, either through a resolution or restore mechanism
[1]. Angiogenesis is considered as a vital constituent of inflammation
and its resolution and both are inter reliant events [2]. Some pattern of
inflammation, notably chronic inflammation, can encourage vessel
augmentation. New vessels may add to a tissue’s altered inflammatory
reaction [2]. Angiogenesis and inflammation, however, remain distinct
cellular events that can come about independently. Many chief tran-
scription factors, such as nuclear factor kappa B, signal transducers and
activators of transcription, the vital mediators of inflammatory sig-
naling. Mutually, these transcription factors converge and regulates
inflammatory pathways [3]. Molecular events related to these processes
regulated by cyclooxygenases (COXs). The COX-1 isoform is expressed
constitutively whereas COX-2 expression is encouraged during specific
pathophysiological conditions or in response to inflammatory stimuli
[3]. Another prime hallmark enzyme in the inflammatory response is
myeloperoxidase (MPO) mainly secreted by activated neutrophils,
portrayed to exhibit powerful pro-oxidative and proinflammatory
properties and likely to facilitate the angiogenic gene regulation [4].
Blockage of chronic inflammation may be likely to constrain angio-
genesis where the stimulus for vascular growth is consequent from in-
flammatory cells [5]. Some anti-inflammatory agents may also have
anti-angiogenic role that is independent of its effects on inflammation,
which add up the fact that agents that have been designed to specifi-
cally inhibit angiogenesis may also inhibit chronic inflammation [6].
Benzophenone based analogue is known to exhibit a broad range of
biological activity. Primarily, it acts as target specific anti-angiogeneic
⁎
Corresponding author.
E-mail address: shaukathara@yahoo.co.in (S.A. Khanum).
https://doi.org/10.1016/j.biopha.2018.04.167
Received 7 March 2018; Received in revised form 21 April 2018; Accepted 23 April 2018
0753-3322/©2018ElsevierMassonSAS.Allrightsreserved.

N. Puttaswamy et al.
Biomedicine&Pharmacotherapy103(2018)1446–1455
pharmacophores which is in the processes of drug development pro-
gression [7–10]. On the other hand now a days an increasing interest
towards the oxadiazole ring system has been observed as it is the basis
for the development of many drugs with a diversity of biological ac-
tivities. Substituted 1,3,4-oxadiazoles have revealed varied pharmaco-
logical activities that include anti-bacterial [11], anti-mycobacterial
[12], anti-fungal [13], anti-inflammatory [14], analgesic [15], anti-
convulsant [16] and anti-cancer [17] properties. Recently our research
group [7,9] has demonstrated that compounds containing oxadiazole
moiety induces cytotoxicity and antiproliferative effect on various
cancer cell lines and anti-angiogenic potential. With a focused interest
in developing a new drug which can specifically target inflammatory
angiogenesis disorder, here we hybridized benzophenone with ox-
adiazole moiety which is reported as having anti-angiogenic potency
[9] with an amide linkage. Based on the aforementioned findings and
docking-simulated interaction we have made an emphasis that among
these newly synthesized series, the compound 10j emerged as a phar-
macological active component particularly inhibiting the COX-2 ac-
tivity.
the disappearance of a broadband between 3312–3490 cm−1 for phe-
nolic OH of compound 2 and the appearance of a band at 1725 cm−1
for C]O of OCOPh in the IR spectrum. Further, the disappearance of a
broad singlet at 5.8 ppm of the OH group of compound 2 and increase
in the number of aromatic protons in the NMR spectrum clearly in-
dicates, the formation of compound 3. Similarly the appearance of a
band at 3328–3403 cm−1 for the OH group in IR spectrum and the
presence of a broad singlet at 9.1 ppm for OH group and decrease in the
number of aromatic protons in 1H NMR spectrum indicates the for-
mation of methyl-3-benzoyl-4-hydroxybenzoate (4). Wherein the for-
mation of 3-benzoyl-4-hydroxybenzoic acid (5) was confirmed by the
presence of the band at 3450–3475 cm−1 for OH of COOH in IR spec-
trum and a broad singlet at 12.8 ppm for COOH group and also by the
disappearance of a peak at 2.9 ppm for CH3 protons of compound 4 in
1H NMR spectrum.
Among the series 10a–j, 2(4-Hydroxy-3-benzoyl) benzamide-5-
(phenyl) 1,3,4-oxadiazole (10a) has been taken as a representative
example to discuss spectral characterization. In the IR spectrum of
compound 10a the disappearance of a band at 3450–3475 cm−1 for
COOH of compound 5 and appearance of a band at 3600 cm−1 for NH
group, similarly disappearance of a singlet at 12.8 ppm for COOH
proton of compound 5, appearance of a signal at 8.1 ppm for NeH
proton and increase in the number of aromatic protons in NMR spec-
trum clearly indicate the acid amine coupling and the formation of
compound 10a.
2. Result and discussion
2.1. Chemistry
The 3-benzoyl-4-hydroxybenzoic acid (5) were synthesized as re-
presented in Scheme 1. Primarily, methyl-4-hydroxybenzoate (2) was
obtained by esterification of 4-hydroxybenzoic acid (1) with methanol
in the presence of catalytic amount of sulfuric acid. Then, compound 2
was benzoylated using benzoyl chloride in the presence of di-
chloromethane (DCM) and triethylamine (TEA) as a base to achieve
methyl-4-(benzoyloxy)benzoate (3). Further, methyl-3-benzoyl-4-hy-
droxybenzoate (4) was obtained by Fries rearrangement of compound 3
and compound 5 was achieved by the hydrolysis of compound 4 with
10% sodium hydroxide. Besides, 1-benzylidenesemicarbazide deriva-
tives (8a–j) were synthesized by reaction of semicarbazide hydro-
chloride (6) with substituted benzaldehydes (7a–j) in the presence of
sodium acetate. Further, 2-amino-5-phenyl-1,3,4-oxadiazole deriva-
tives (9a–j) were obtained by oxidative cyclization of corresponding 1-
benzylidenesemicarbazide derivatives using chloramine T (CAT) [18]
as represented in Scheme 2.
Finally the title compounds 2(4-hydroxy-3-benzoyl) benzamide-5-
phenyl1,3,4-oxadiazoles (10a–j) were achieved as outlined in Scheme
3, by acid amine coupling of compound 5, with compounds (9a–j) in
the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo
[4,5-b]pyridinium 3-oxidhexafluorophosphate, N-[(dimethylamino)-
1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanami-
nium hexafluorophosphate N-oxide (HATU) and TEA [19].
2.2. Biology
2.2.1. Compound 10j emerged as a lead molecule
The erythrocyte haemolysis assay is considered as a simple and
rapid tool for screening of drugs for anti-inflammatory activity. Most of
the anti-inflammatory drugs stabilize the plasma membrane of mam-
malian erythrocytes and thereby inhibit the hypotonicity induced
haemolysis. The plasma membrane of mammalian erythrocytes have
been chiefly used as a model to study membrane stabilizing activity of
the compounds 10a–j [20]. In vitro anti-inflammatory activity (HRBC
membrane stabilization) was performed by increasing concentration (0,
50, 100, 150, 200, 250 μg/ml) of the title compounds 10a–j. The result
demonstrated that compound 10j bearing hydroxy substitution, parti-
cularly at the ortho position of the phenyl ring attached to the 5th
carbon atom of the oxadiazole ring possesses significant membrane
stabilizing activity compared to the control group and hence selected as
a lead compound. The inhibitory concentration (IC50) value of com-
pound 10j is 153.43 μg/ml and for the standard indomethacin it is
121.68 μg/ml, (Table 1). Based on the IC50 values, compound 10j was
chosen as a lead compound.
All the synthesized compounds were characterized by IR, NMR and
mass spectral studies. For example, the IR spectrum of methyl-4-hy-
droxybenzoate (2) showed the presence of OeH stretching at
3312–3490 cm−1, a band for C]O stretching of OCOCH3 at 1672 cm−1
and a signal for CeO stretching at 1210 cm−1. Whereas in 1H NMR
spectrum a signal at 3.8 ppm as a singlet for CH3, protons, a multiplet
around 6.8–7.9 ppm for aromatic protons and a broad signal at 5.8 ppm
for hydroxy proton indicates the formation of compound 2. However,
the formation of methyl-4-(benzoyloxy)benzoate (3) was confirmed by
2.2.2. Structure activity relationship (SAR) and selection of 10j as a potent
anti-inflammatory and anti-angiogenesis agent
The present study was carried out to evaluate the biological sig-
nificance of synthesized benzophenone embedded oxadiazole analogues
10a–j. Based on HRBC membrane assay, among the series 10a–j, the
compound 10j has exhibited a potent inflammatory angiogenesis ac-
tivity with IC50 153.43 μg/ml. From this study it is interesting to note
that the presence of hydroxy substitution, particularly at the ortho
position of the phenyl ring attached to the 5th carbon atom of the
Scheme 1. Synthesis of 3-benzoyl-4-hydroxybenzoic acid
(5).
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Scheme 2. Synthesis of 2-amino-5-phenyl-1,3,4-oxadiazoles (9a–j).
Scheme 3. Synthesis of 2(4-Hydroxy-3-benzoyl) benzamide-5-(phenyl) 1,3,4-oxadiazole (10a–j).
oxadiazole ring, exhibited a good anti-inflammatory and angiogenesis
activity. On the other hand compound 10e, which also comprises of the
hydroxy group, but at para position of the phenyl ring attached to the
5th carbon atom of the oxadiazole ring showed IC50 value as 250 μg/ml
and this indicates that it has not shown remarkable activity.
sections revealed that the primary infiltrating cells in the corneal
stroma after the corneal alkali burn were inflammatory cells. The
number of infiltrating cells in the corneal stroma were radically de-
creased in the compound 10j treated group than in the control group. In
the control group, the corneal epithelium was not repaired, the corneal
stroma was thicker and more swollen than those in the compound 10j
treated group (Fig. 1B).
The chicken chorioallantoic membrane (CAM) assay was deployed
to assess the inhibitory effect of compound 10j on neovasularization,
since it is considered as a more reliable model system to evaluate the
angiopreventive and anti-inflammatory role of a test compound 10j in
in-vivo condition because this assay is a sensitive, inexpensive and easily
feasible in-vivo model system to investigate potentially anti-in-
flammatory compounds [21]. In the present study rVEGF165 induced
neovascularisation in in-vivo CAM model was exposed with compound
10j. A clear formation of a vascular zone around the implanted disc
with compound 10 j as shown in the Fig. 1C and D was a clear evidence
of the regression of neovessels in the developing embryos with com-
pound 10j which indicated the inhibition of angiogenesis.
2.2.3. Inhibition of inflammatory corneal neovascularization (CNV) by
compound 10j
The inflammatory corneal angiogenesis assay is one of the most
reliable models to study the effect of drug on inflammatory angiogen-
esis, ever since the alkali burn creates the local inflammation in the
cornea leading to the sprouting of new blood vessels. Three days after
the corneal alkali burn, CNV occurred in both the control and treated
groups (Fig. 1A). According to the results compared to the cornea of the
control group, cornea in the compound 10j treated group showed less
CNV. The amount of CNV was quantified by measuring the area of
neovascularization and it was found that the area of CNV was sig-
nificantly smaller in the group treated with compound 10j compared
with the control group, which indicated an inhibitory effect of com-
pound 10j on CNV.
2.2.4. Reduction of carrageenan induced paw oedema by compound 10j
Carrageenan induced paw oedema is a well established animal
model system to evaluate the anti-inflammatory drugs as it is acute,
nonimmune, well researched and a highly reproducible model. The
subcutaneous injection of carrageenan results in the release of pro-in-
flammatory agents and the inflammatory reaction is typically quanti-
fied by an increase in paw size (oedema).
Injection of mice with carrageenan caused a significant increase in
the paw weight and percentage was measured after 5 h compared to the
control values (Fig. 2A and B). Pretreatment of carrageenan injected
mice with indomethacin 10 mg/kg body weight (b.w) significantly de-
creased paw volume to 40% and it was measured after 5 h compared to
carrageenan injected mice. Whereas compound 10j (25 mg/kg/b.w)
treated animals revealed significantly decreased paw weight to 30%
and it was measured after 5 h (75%) compared to that of carrageenan
injected mice (Fig. 2).
The haematoxylin and eosin (H and E) staining of the eyeball
Table 1
IC50 values of compounds 10a–j calculated based on HRBC membrane stabili-
zation assay. Based on the IC50 values, compounds 10j was chosen as a lead
compound.
Samples
HRBC membrane stabilization IC50 Value (μg/ml)
Control
10a
10b
10c
10d
10e
10f
–
254
273
234
239
> 250
194
10g
10h
10i
211
> 250
213
10j
153.43
2.2.5. MPO activity of compound 10j
Indomethacin
121.68
MPO corresponds to 1–5% of total protein in human neutrophils
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Fig. 1. Angiogenesis modulatory effect of compound 10j reduces the neovascularization. (A) Representative photographs of the rat cornea indicating the inhibition of
angiogenesis in alkali burnt rat cornea and H and E staining of cornea showing improved corneal gesture with compound 10j treatment. (B) Decrease in total vessel
length after compound 10j treatment in rat cornea. (C) In vivo CAM photos exhibits the angiopreventive effect of compound 10j. (D) Quantification of MVD of CAM.
[22,23]. The crux of the matter is that MPO was found at elevated levels
within and near blood vessels in inflammatory conditions [24]. The
neutrophil migration towards carrageenan induced inflammation in
mouse paws was indirectly determined based on MPO activity in the
target tissue. MPO secretion was a result of the host defense me-
chanism. Due to its importance during inflammatory processes and for
being an indicator of the polymorphonuclear (PMN) presence in tissues,
the MPO has been widely used as an inflammatory marker of both acute
and chronic conditions. Positive control indomethacin treatment re-
sulted in the decreased level of MPO activity. Treatment with com-
pound 10j exhibited similar action in carrageenan induced paw oedema
by significantly preventing the increase in MPO activity induced by
carrageenan when compared to the control group (Fig. 2C).
hydrogen bond exhibited in the COX-2-10j complex have been docu-
mented, together with their distances and angles, by taking into ac-
count the interaction energies of the compound 10j with the active site
of the COX-2 residue, a key active binding site of the model were de-
termined and proved. Moreover, data revealed that the compound 10j
formed hydrogen bonds with the amino acid ASP125, and an ionic in-
teraction between carboxylic group of amino acid ALA86 and atom of
oxadiazole ring of the compound 10j which contributed to the most
stable binding of the S1 conformation from ligand to COX-2 with lowest
binding energy at −7.11 kJ/mol (Fig. 3A–C). Further, the in-silico re-
sults were revalidated using in-vivo samples. By the mode of action of
compound 10j the cellular COX-2 activity were measured in homo-
genate of paw tissue of the paw oedema animal experiment. The result
inferred that compound 10j inhibited COX-2 activity by 26%, which is
due to the presence of hydroxy substitution, particularly at the ortho
position of the phenyl ring attached to the 5th carbon atom of the ox-
adiazole ring.
2.2.6. Compound 10j inhibits COX-2 activity
Prostaglandins are lipid autacoids derived from arachidonic acid.
They both maintain homeostatic regulation and also plays vital role in
mediating pathogenic mechanisms, including the inflammatory re-
sponse. The PG H2 synthase (cyclooxygenase or COX) is the crucial
enzyme in the conversion of arachidonic acid to PGs. The PGs are prime
mediators in physiological event, especially during inflammatory re-
sponse [25]. COX-1 is consecutively expressed protein by most cell
types and they are considered to take part in the PGs synthesis during
physiological processes. Whereas COX-2 usually is absent, during
normal physiological event, but is induced by numerous physiologic
stimuli such as inflammation. Targeting the COX-2 expression level
during inflammatory reaction will be a vital strategy. Following the
promising potency of the compound 10j as anti-inflammatory agent,
and COX-2 being the important factor in inflammatory reactions we
first validated the effectiveness of the compound 10j under in-vivo ap-
proaches (Fig. 3E). On the other hand, by in-silico study the autodock
programme was utilized to produce the protein-10j complex, in order to
understand the interaction between COX-2 protein and compound 10j
as shown in the Fig. 3A that ligand 10j is placed in the center of the
active site and it is stabilized by hydrogen bonding interaction. The
3. Conclusion
The present study has focused on the synthesis of a series of ben-
zophenone integrated oxadiazoles (10a–j) with different substituents.
Among the series 10a–j, compound 10j exhibited potential anti-in-
flammatory and anti-angiogenesis activity. The activity of synthesized
compounds revealed that benzophenone appended oxadiazole plays a
major role in enhancing the activity. From the study it is significant to
note that the presence of hydroxy substituent, particularly at the ortho
position of the phenyl ring attached to the 5th carbon atom of the ox-
adiazole ring exhibited a good anti-inflammatory and anti-angiogenesis
activity. Among the series 10a–j, compound 10e has not shown re-
markable activity, though it comprises the hydroxy group, but at para
position of the phenyl ring attached to the 5th carbon atom of the ox-
adiazole ring.
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Fig. 2. Effect of compound 10j on carrageenan-induced paw oedema in mice. (A) Typical representative macroscopic photographs of paw from the normal, car-
rageenan + saline carrageenan + indomethacin, carrageenan + compound 10j. (B) Paw swelling percentage after 48 h carrageenan injection in the different ex-
perimental animal. (C) Decreased MPO activity in compound 10j treated animal.
4. Materials and methods
CH3), 5.8 (s, 1H, OH), 6.8–7.9 (m, 4H, ArH); 13C NMR (100 MHz) δ:
52.21, 115.3, 121.82, 131.32, 160.34, 167.69; LCMS (M+): (153);
Anal.Calcd. for C8H8O3: C, 63.15; H, 5.30; Found: C, 63.11; H, 5.36%.
4.1. Chemistry
Chemicals were purchased from Sigma Aldrich. Thin layer chro-
matography (TLC) was performed on aluminum backed silica plate
which was visualized by UV-light. Melting points were determined on a
Thomas Hoover capillary melting point apparatus with a digital ther-
mometer. The IR spectra was recorded by the potassium bromide pellet
method on FT-IR Shimadzu 8300 spectrophotometer, NMR spectra
were recorded on a Bruker 400 MHz NMR spectrophotometer in DMSO
and chemical shifts were recorded in parts per million down field from
tetramethylsilane. Mass spectra were obtained with a VG70-70H spec-
trometer and elemental analysis results are within 0.4% of the calcu-
lated value.
4.1.2. General procedure for the preparation of methyl-4-(benzoyloxy)
benzoate (3)
The mixture of compound (2, 0.016 mol), benzoyl chloride
(0.016 mol) and TEA was stirred in a freezing mixture for 45 min and
the reaction was monitored using TLC. After completion of the reaction,
the organic layer was washed with sodium hydroxide, (3 × 10 ml)
followed by distilled water (3 × 15 ml) and brine solution (3 × 10 ml).
The organic solvent was dried over anhydrous sodium sulfate and the
solvent was evaporated under reduced pressure to obtain compound (3)
which was recrystallized from ethanol [27].
Yield: 71%; m.p: 98–101 °C; IR (KBr, γ/cm−1): 1214 (CeO of
OCOCH3); 1315 (CeO of OCOPh), 1670 (C]O of OCOCH3), 1725 (C]
O of OCOPh), 1H NMR (400 MHz, DMSO-d6) δ: 3.4 (s, 3H, CH3), 7.1–8.1
(m, 9H, ArH); 13C NMR (100 MHz) δ: 51.11, 121.43, 127.54, 128.32,
130.12, 130.43, 130.56, 133.98, 134.81, 155.34, 165.21; LCMS (M+):
(257); Anal.Calcd. for C15H12O4: C, 70.31; H, 4.72; Found: C, 70.37; H,
4.76%.
4.1.1. General procedure for the preparation of methyl-4-hydroxybenzoate
(2)
To the solution of 4-hydroxybenzoic acid (1, 0.028 mol) in me-
thanol (25 ml), a catalytic amount of sulfuric acid was added and re-
fluxed for 3 h, the reaction was monitored by TLC. After completion of
the reaction, the organic layer was washed with sodium bicarbonate
(3 × 15 ml), followed by distilled water (3 × 15 ml) and saturated so-
dium chloride solution (3 × 10 ml). The organic layer was dried over
anhydrous sodium sulfate and the solvent was evaporated under re-
duced pressure to obtain compound 2. The crude compound 2 was
further purified by recrystallization with ethanol [26].
4.1.3. General procedure for the preparation of methyl-3-benzoyl-4-
hydroxybenzoate (4)
Compound (3, 0.016 mol) and anhydrous aluminum chloride
(0.032 mol) were heated to 120 °C for 20 min. Then cooled to room
temperature and the complex was cleaved using 10% hydrochloric acid
and the product was filtered, washed thoroughly with water
(3 × 15 ml) and recrystallized with ethanol to afford compound 4 as a
Yield: 83%; m.p: 124–126 °C; IR (KBr, γ/cm−1): 1210 (CeO); 1672
(CO), 3312–3490 (OH), 1H NMR (400 MHz, DMSO-d6) δ: 3.8 (s, 3H,
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Fig. 3. Compound 10j interacts with COX-2 by binding with amino acid ASP125 through the hydrogen-bond. (A) 3D structure of the compound 10j in the active site
C-terminal transactivation COX-2 complexes. (B) Ribbon models of COX-2 and the ligand molecule 10j complexes. (C) Hydrogen-bond interaction of the ligand
molecule 10j with COX-2. (D) The 2D interactions analysis of 10j COX-2. (E) COX-2 activity in mice edematic tissue lysate treated with or without compound 10j.
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pale yellow solid [28].
89%, m.p 150–153 °C; IR (KBr, γ/cm−1): 1549 (C]N), 3343–3365
(OH), 3454 (NeH of NH2); 1H NMR (400 MHz, DMSO-d6) δ: 3.39 (s, 2H,
NH2), 7.13–7.65 (m, 4H, ArH), 9.1 (s, 1H, OH); 13C NMR (100 MHz) δ:
116.92, 118.45, 129.91, 157.22, 159.19, 164.32; LCMS (M+): (178);
Anal.Calcd. for C8H7N3O2: C, 54.24; H, 3.98; N, 23.72; Found: C, 54.24;
H, 3.96; N, 23.81%.
Yield: 83%; m.p 124–126 °C; IR (KBr, γ/cm−1): 1232 (CeO of
OCOCH3), 1312 (CeO of OCOPh), 1676 (C]O of COPh), 1669 (C]O of
OCOCH3), 3328–3403 (OH); 1H NMR (400 MHz, DMSO-d6) δ: 2.9 (s,
3H, CH3), 7.1–8.1 (m, 8H, ArH), 9.1(s, 1H, OH); 13C-NMR (100 MHz) δ:
51.23, 115.21, 119.21, 122.65, 128.23, 130.43, 131.23, 132.65,
135.65, 139.21, 165.21, 166.23, 196.45; LCMS (M+): (257); Anal.
Calcd. for C15H12O4: C, 70.31; H, 4.72; Found: C, 70.23; H, 4.76%.
4.1.5.6. 2-Amino-5-(2,4-dimethoxy)phenyl-1,3,4-oxadiazole (9f). Yield:
83%; m.p: 163–166 °C; IR (KBr, γ/cm−1): 1543 (C]N), 3441 (NeH
of NH2); 1H NMR (400 MHz, DMSO-d6) δ: 3.21 (s, 6H, OCH3), 3.31 (s,
2H, NH2), 7.13–7.65 (m, 3H, ArH), 13C NMR (100 MHz) δ: 56.2,
101.23, 106.12, 130.23, 157.32, 161.37, 164.12; LCMS (M+): (222)
Anal. Calcd. forC10H11N3O3: C, 54.29; H, 5.01; N, 19.00; Found: C,
54.28; H, 5.04; N, 19.03%.
4.1.4. General procedure for the preparation of 3-benzoyl-4-
hydroxybenzoic acid (5)
Compound (4, 0.016 mol) was refluxed with 20 ml of 10% sodium
hydroxide in ethanol for 1 h and then acidified. A white solid separates
out which was filtered and recrystallized with ethanol to achieve
compound 5 [29].
Yield: 83%; m.p138–140 °C; IR (KBr, γ/cm−1): 1678 (C]O of
COPh), 1716 (C]O of COOH), 3312–3355 (OH), 3450–3475 (OH of
COOH); 1H NMR (400 MHz, DMSO-d6) δ: 6.8–7.9 (m, 8H, ArH), 11.2 (s,
1H, OH) 12.8 (s, 1H, COOH); 13C NMR (100 MHz) δ: 121.93, 125.43,
126.72, 130.24, 134.73, 137.06, 138.24, 139.43, 142.32, 165.65,
171.71, 201.52; LCMS (M+): (243); Anal.Calcd. for C14H10O4: C, 69.42;
H, 4.16; Found: C, 69.40; H, 4.09%.
4.1.5.7. 2-Amino-5-(4-chloro)phenyl-1,3,4-oxadiazole (9g). Yield: 79%;
m.p: 210–213 °C; IR (KBr, γ/cm−1): 1584 (C]N), 3469 (NeH of NH2);
1H NMR (400 MHz, DMSO-d6) δ: 3.30 (s, 2H, NH2), 7.13–7.65 (m, 4H,
ArH); 13C NMR (100 MHz) δ: 123.43, 127.32, 129.23, 136.23, 156.43,
163.21; LCMS (M+): (196) (198); Anal. Calcd. for C8H6ClN3O: C, 49.12;
H, 3.09; N, 21.48; Found: C, 49.14; H, 3.06; N, 21.48%.
4.1.5.8. 2-Amino-5-(3-bromo)phenyl-1,3,4-oxadiazole (9h). Yield: 83%;
m.p: 146–148 °C; IR (KBr, γ/cm−1): 1568 (C]N), 3476 (NeH of NH2):
1H NMR (400 MHz, DMSO-d6) δ: 3.29 (s, 2H, NH2), 7.13–7.65 (m, 4H,
ArH), 13C NMR (100 MHz) δ: 124.21, 128.32, 129.23, 126.23, 131.21,
133.61, 156.65, 165.21; LCMS (M+): (239); Anal. Calcd. For
C8H6BrN3O: C, 40.03; H, 2.52; N, 17.50; Found: C, 40.04; H, 2.54; N,
17.53%.
4.1.5. General procedure for the preparation of 2-amino-5-phenyl-1,3,4-
oxadiazole (9a)
A mixture of 1-benzylidenesemicarbazide (8a, 1.9 mmol) and CAT
(2 mmol) in ethanol was refluxed with stirring for 3 h. The sodium
chloride formed in the reaction was filtered off and washings were
evaporated in vacuum and the residue was extracted with 10% hy-
drochloric acid (3 × 10 ml) and washed thoroughly with DCM. The
aqueous layer was neutralized with 10% sodium hydroxide to achieve
compound 9a, which was further purified by recrystallisation with
methanol. The remaining compounds 9b–j were synthesized in the si-
milar manner [18].
4.1.5.9. 2-Amino-5-(2-methoxy)phenyl-1,3,4-oxadiazole
(9i). Yield:
70%; m.p: 132–135 °C; IR (KBr, γ/cm−1): 1551 (C]N), 3442 (NeH
of NH2): 1H NMR (400 MHz, DMSO-d6) δ: 3.28 (s, 3H, OCH3), 3.43 (s,
2H, NH2), 7.13–7.65 (m, 4H, ArH), 13C NMR (100 MHz) δ: 57.2,
110.21, 115.23, 120.32, 122.34, 128.32, 156.21, 157.12, 166.32;
LCMS (M+): (191); Anal. Calcd. forC9H9N3O2: C, 56.54; H, 4.74; N,
21.98; Found: C, 56.56; H, 4.76; N, 21.93%.
4.1.5.1. 2-Amino-5-phenyl-1,3,4-oxadiazole (9a). Yield: 81%; m.p:
240–242 °C; IR (KBr, γ/cm−1): 1581 (C]N), 3475 (NeH of NH2); 1H
NMR (400 MHz, DMSO-d6) δ: 3.34 (s, 2H, NH2), 7.13–7.65 (m, 5H,
ArH); 13C NMR (100 MHz) δ: 124.23, 125.02, 127.32, 128.41, 157.11,
164.32, LCMS (M+): (162); Anal. Calcd. for C8H7N3O: C, 59.62; H,
4.38; N, 26.07; Found: C, 59.64; H, 4.36; N, 26.08%.
4.1.5.10. 2-Amino-5-(2-hydroxy)phenyl-1,3,4-oxadiazole
(9j). Yield:
81%, m.p: 123–126 °C; IR (KBr, γ/cm−1): 1530 (C]N), 3329–3345
(OH), 3464 (NeH of NH2); 1H NMR (400 MHz, DMSO-d6) δ: 3.21 (s, 2H,
NH2), 7.13–7.65 (m, 4H, ArH), 9.6 (s, 1H, OH); 13C NMR (100 MHz) δ:
113.21, 117.21, 126.02, 128.05, 130.91, 155.22, 156.29, 163.12; LCMS
(M+):(178); Anal. Calcd. For C8H7N3O2: C, 54.24; H, 3.98; N, 23.72;
Found: C, 54.14; H, 3.95; N, 23.83%.
4.1.5.2. 2-Amino-5-(4-bromo)phenyl-1,3,4-oxadiazole (9b). Yield: 85%;
m.p: 223–226 °C; IR (KBr, γ/cm−1): 1579 (C]N), 3472 (NeH of NH2);
1H NMR (400 MHz, DMSO-d6) δ: 3.31 (s, 2H, NH2), 7.13–7.65 (m, 4H,
ArH), 13C NMR (100 MHz) δ: 123.23, 125.42, 129.32, 132.41, 156.91,
163.32; LCMS (M+): (239) Anal. Calcd. for C8H6BrN3O: C, 40.03; H,
2.52; N, 17.50; Found: C, 40.24; H, 2.52; N, 17.58%.
4.1.6. General procedure for the preparation of 2(4-hydroxy)-3-benzoyl)
benzamide-5-(phenyl) 1,3,4 oxadiazole (10a)
To the stirring solution of compound (5, 1.9 mmol) in 10 ml of
DMSO, TEA was added, followed by the addition of coupling agent
HATU (2.0 mmol). The mixture was stirred for 20 min at room tem-
perature, to this stirring solution compound 9a was added and the
stirring was continued. Further, the reaction was monitored by TLC,
after completion of the reaction, the reaction mass was treated with
ethyl acetate and the organic layer was washed with 10% sodium bi-
carbonate solution (3 × 15 ml) followed by 10% hydrochloric acid
(3 × 15 ml). Finally the mixture was washed with water (3 × 15 ml),
then the organic layer was evaporated under reduced pressure to
achieve title compound 10a. The crude compound was recrystallized
with ethanol and the remaining compounds 10b–j were synthesized in
the similar manner [19].
4.1.5.3. 2-Amino-5-(3-methoxy)phenyl-1, 3, 4-oxadiazole (9c). Yield:
83%; m.p: 181–183 °C; IR (KBr, γ/cm−1): 3432 (NeH of NH2), 1564
(C]N): 1H NMR (400 MHz, DMSO-d6) δ: 3.32 (s, 3H, OCH3), 3.31 (s,
2H, NH2), 7.13–7.65 (m, 4H, ArH), 13C NMR (100 MHz) δ: 164.65,
161.32, 156.54, 130.41, 127.32, 114.32, 119.65, 111.43, 55.23; LCMS
(M+): (191); Anal. Calcd. for C9H9N3O2: C, 56.54; H, 4.74; N, 21.98;
Found: C, 56.52; H, 4.72; N, 21.68%.
4.1.5.4. 2-Amino-5-(2,6-dimethoxy)phenyl)-1,3,4-oxadiazole
(9d). Yield: 74%; m.p: 154–157 °C; IR (KBr, γ/cm−1): 3443 (NeH of
NH2), 1552 (C]N): 1H NMR (400 MHz, DMSO-d6) δ: 3.36 (s, 6H,
OCH3), 3.39 (s, 2H, NH2), 7.13–7.65 (m, 3H, ArH), 13C NMR (100 MHz)
δ: 165.32, 157.42, 153.17, 148.34, 113.45, 115.92, 115.12, 111.43,
57.23; LCMS (M+) : (222); Anal. Calcd. For C10H11N3O3: C, 54.29; H,
5.01; N, 19.00; Found: C, 54.22; H, 5.06; N, 19.08%.
4.1.6.1. 2(4-Hydroxy-3-benzoyl) benzamide-5-(phenyl) 1,3,4-oxadiazole
(10a). Yield: 83%, m.p: 161–163 °C; IR (KBr, γ/cm−1): 1580 (C]N),
1679 (C]O of COPh), 1680 (C]O of CONH), 3310–3385 (OH), 3600
(NH of CONH), 1H NMR (400 MHz, DMSO-d6) δ: 7.1–8.1 (m, 13H, ArH),
4.1.5.5. 2-Amino-5-(4-hydroxy)phenyl-1,3,4-oxadiazole
(9e). Yield:
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Biomedicine&Pharmacotherapy103(2018)1446–1455
NMR (100 MHz) δ: 115.01, 119.16, 124.24,125.23,126.16, 128.54,
129.23, 129.62, 130.01, 132.43, 132.43, 134.64, 139.52, 157.45,
165.25, 164.55, 164.93, 196.73; LCMS (M+): (419); Anal. Calcd. For
8.1 (s, 1H, CONH), 9.5 (s, 1H, OH); 13C NMR (100 MHz) δ: 119.56,
114.65, 126.12, 126.34, 126.76, 128.42, 128.94, 129.12, 129.54,
130.61, 132.64, 133.03, 139.72, 157.65, 163.23, 163.75, 164.22,
196.43; LCMS (M+): (386); Anal. Calcd. for C22H15N3O4: C, 68.57; H,
3.92; N, 10.90; Found: C, 68.54; H, 3.94; N, 10.93%.
C
22H14ClN3O4: C, 62.94; H, 3.36; N, 10.01; Found: C, 62.94; H, 3.06; N,
10.09%.
4.1.6.2. 2(4-Hydroxy-3-benzoyl) benzamide-5-(4-bromophenyl) 1,3,4-
oxadiazole (10b). Yield: 69%; m.p145–148 °C; IR (KBr, γ/cm−1):,
1584 (C]N), 1675 (C]O of COPh), 1683 (C]O of CONH),
3345–3395 (OH), 3599 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 7.1–8.1 (m, 12H, ArH), 8.3 (s, 1H, CONH), 9.7 (s, 1H, OH); 13C NMR
(100 MHz) δ: 116.01, 118.16, 123.52, 125.34, 126.66, 128.34, 129.12,
129.53, 130.31, 131.89, 132.23, 132.64, 138.62, 157.45, 164.25,
164.75, 165.23, 196.53; LCMS (M+): (465) Anal. Calcd. for
4.1.6.8. 2(4-Hydroxy-3-benzoyl) benzamide-5-(3-bromophenyl) 1,3,4-
oxadiazole (10h). Yield: 79%; m.p:165–168 °C; IR (KBr, γ/cm−1):
1583 (C]N), 1674 (C]O of COPh),1686 (C]O of CONH),
3349–3386 (OH), 3587 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 7.1–8.1 (m, 12H, ArH), 8.3 (s, 1H, CONH), 9.7 (s, 1H, OH); 13C NMR
(100 MHz) δ: 115.71, 119.32, 123.21, 126.44, 126.65, 128.23, 128.45,
129.32, 129.43, 130.42, 131.56, 131.78, 132.46, 132.33, 133.54,
139.62, 156.95, 164.44, 164.66, 165.53, 196.43; LCMS (M+): (465);
Anal. Calcd. for C22H14BrN3O4: C, 56.91; H, 3.04; N, 9.05; Found: C,
56.98; H, 3.05; N, 9.06%.
C
22H14BrN3O4: C, 56.91; H, 3.04; N, 9.05; Found: C, 56.94; H, 3.06;
N, 9.09%.
4.1.6.3. 2(4-Hydroxy-3-benzoyl) benzamide-5-(3-methoxyphenyl)1,3,4-
oxadiazole (10c). Yield: 81%; m.p: 135–138 °C; IR (KBr, γ/cm−1):
1582 (C]N), 1675 (C]O of COPh), 1684 (C]O of CONH),
3339–3374 (OH), 3586 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 3.61 (s, 3H, OCH3) 7.1–8.1 (m, 12H, ArH), 8.6 (s, 1H, CONH), 9.4 (s,
1H, OH); 13C NMR (100 MHz) δ: 57.6, 111.23, 114.31, 115.16, 119.12,
119.34, 126.56, 127.43, 128.34, 129.12, 130.21, 130.41, 132.53,
132.72, 138.12, 157.29, 161.34, 164.31, 164.72, 165.43, 196.51;
LCMS (M+): (416); Anal. Calcd. for C23H17N3O5: C, 66.50; H, 4.12;
N, 10.12; Found: C, 66.54; H, 4.16; N, 10.12%.
4.1.6.9. 2(4-Hydroxy-3-benzoyl) benzamide-5-(2-methoxyphenyl) 1,3,4-
oxadiazole (10i). Yield: 91%; m.p:143–146 °C; IR (KBr, γ/cm−1):
1584 (C]N), 1676 (C]O of COPh), 1682 (C]O of CONH),
3345–3376 (OH), 3576 (NH of CONH); 1H NMR (400 MHz, DMSO-
d6): δ 3.54 (s, 3H, OCH3) 7.1–8.1 (m, 12H, ArH), 8.4 (s, 1H, CONH), 9.6
(s, 1H, OH); 13C NMR (100 MHz) δ: 57.6, 110.51, 115.46, 114.82,
119.64, 121.36, 126.23, 128.34, 129.45, 129.12, 130.43, 132.56,
132.52, 135.23, 139.52, 158.75, 157.59, 164.31, 164.54, 165.45,
196.23; LCMS (M+): (416); Anal. Calcd. for C23H17N3O5: C, 66.50; H,
4.12; N, 10.12; Found: C, 66.53; H, 4.12; N, 10.13%.
4.1.6.4. 2(4-Hydroxy-3-benzoyl) benzamide-5-(2,
6 dimethoxyphenyl)
4.1.6.10. 2(4-Hydroxy-3-benzoyl) benzamide-5-(2-hydroxyphenyl) 1,3,4-
oxadiazole (10j). Yield: 78%; m.p:130–133 °C; IR (KBr, γ/cm−1): 1572
(C]N), 1669 (C]O of CONH), 1678 (C]O of COPh), 3335–3365
(OH), 3582 (NH of CONH); 1H NMR (400 MHz, DMSO-d6) δ: 7.1–8.1
(m, 12H, ArH), 8.5 (s, 1H, CONH), 9.4 (s, 1H, OH) 9.8 (s, 1H, OH) 13C
NMR (100 MHz) δ: 115.71, 119.66, 123.64, 126.55, 126.83,
128.54,128.43, 129.52, 130.31, 131.31, 132.23, 132.63, 133.33,
139.42, 157.43, 164.41, 164.82, 165.64, 196.28; LCMS (M+): (402);
Anal. Calcd. for C22H15N3O5: C, 65.83; H, 3.77; N, 10.47; Found: C,
65.79; H, 3.73; N, 10.48%.
1,3,4-oxadiazole (10d). Yield: 78%; m.p: 135–138 °C; IR (KBr, γ/
cm−1): 1588 (C]N), 1677 (C]O of COPh),1685 (C]O of CONH),
3347–3356 (OH), 3580 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 3.21(s, 6H, OCH3) 7.1–8.1 (m, 11H, ArH), 8.3 (s, 1H, CONH), 9.7 (s,
1H, OH); 13C NMR (100 MHz) δ: 56.6, 111.32, 112.23, 116.01, 118.16,
123.52, 126.66, 128.34, 129.53, 129.12, 130.31, 132.23, 132.64,
138.62, 157.45, 164.25, 164.75, 165.23, 196.53; LCMS (M+): (467);
Anal. Calcd. For C24H19N3O6: C, 64.72; H, 4.30; N, 9.43; Found: C,
64.54; H, 4.29; N, 9.42%.
4.1.6.5. 2(4-Hydroxy-3-benzoyl) benzamide-5-(4-hydroxyphenyl) 1,3,4-
oxadiazole (10e). Yield: 87%; m.p: 143–145 °C; IR (KBr, γ/cm−1):
1576 (C]N), 1672 (C]O of COPh), 1679 (C]O of CONH),
3334–3356 (OH), 3586 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 7.1–8.1 (m, 12H, ArH), 8.3 (s, 1H, CONH), 9.2 (s, 1H, OH), 9.7 (s, 1H,
OH); 13C NMR (100 MHz) δ: 115.32, 116.01, 116.23, 118.16, 119.34,
126.76, 128.53, 128.84, 130.61, 132.23, 133.64, 139.02, 157.43,
158.43, 164.82, 164.81, 165.53, 196.26; LCMS (M+): (402); Anal.
Calcd. for C22H15N3O5: C, 65.83; H, 3.77; N, 10.47; Found: C, 65.84; H,
3.79; N, 10.42%.
4.2. Biology
4.2.1. HRBC membrane stabilization assay
The HRBC membrane stabilization has been used as a method to
investigate the anti-inflammatory activity. Series of compounds 10a–j
were used for the in vitro anti-inflammatory activity [30]. In brief, blood
was collected from healthy human volunteer who had not taken any
non-steroidal anti-inflammatory drugs (NSAIDS) for two weeks prior to
the experiment and mixed with equal volume of sterilized alsever’s
solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.55%
sodium chloride). The blood was centrifuged at 3000 revolutions per
minute (rpm) and packed cells were washed with isosaline (0.9% w/v
sodium chloride) and a 10% suspension of blood was made with iso-
saline. Various concentrations of the compounds 10a–j were prepared
(0, 50, 100, 150, 200 and 250 μg/ml) using DMSO and to each con-
centration 1 ml phosphate buffer, 2 ml hyposaline, and 0.5 ml HRBC
suspension were added. Then the mixture was incubated at 37 °C for
30 min and centrifuged at 3000 rpm for 20 min and the hemoglobin
content in the supernatant solution was estimated spectro-
photometrically at 560 nm. Indomethacin (50, 100, 150, 200 and
250 μg/ml) was used as a reference and a control was prepared without
the compounds 10a–j. The hemolysis percentage was calculated by
assuming the hemolysis produced by the control group as 100%. The
percentage of HRBC membrane stabilization or protection was calcu-
lated using the formula, percent protection = 100 − (OD of compound
treated sample/OD of control × 100).
4.1.6.6. 2(4-Hydroxy-3-benzoyl) benzamide-5-(1,
3 dimethoxyphenyl)
1,3,4-oxadiazole (10f). Yield: 81%; m.p: 167–170 °C; IR (KBr, γ/
cm−1): 1587 (C]N), 1671 (C]O of COPh), 1679 (C]O of CONH),
3343–3365 (OH), 3589 (NH of CONH); 1H NMR (400 MHz, DMSO-d6)
δ: 3.21(s, 6H, OCH3) 7.1–8.1 (m, 11H, ArH), 8.3 (s, 1H, CONH), 9.1 (s,
1H, OH) 13C NMR (100 MHz) δ: 56.32, 57.6, 100.23, 102.31, 106.36,
115.12, 119.74, 126.13, 128.44, 129.32, 129.61, 130.71, 132.43,
132.92, 138.42, 157.69, 158.45, 161.67, 164.31, 164.92, 165.73,
196.63; LCMS (M+): (446); Anal. Calcd. for C24H19N3O6: C, 64.72; H,
4.30; N, 9.43; Found: C, 64.74; H, 4.36; N, 9.42%.
4.1.6.7. 2(4-Hydroxy-3-benzoyl) benzamide-5-(4 chlorophenyl) 1,3,4-
oxadiazole (10g). Yield: 85%; m.p: 205–208 °C; IR (KBr, γ/cm−1):
1578 (C]N), 1679 (C]O of COPh), 1686 (C]O of CONH),
3341–3375 (OH), 3576 (NH of CONH); 1H NMR (400 MHz, DMSO-
d6): δ 7.1–8.1 (m, 12H, ArH), 8.8 (s, 1H, CONH), 9.6 (s, 1H, OH); 13C
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4.2.2. In vivo CAM assay
animals from group II were injected with saline (5 ml/kg), group III
animals were injected with positive control (10 mg/kg/b.w), group IV
animals were injected with a test compound 10j (25 mg/kg/b.w) and
animals were left in a respective cage for 2 h. After 2 h animals from the
control, positive control, and test group were injected with 100 μl of 2%
carrageenan at the right paw. The right paw of all the experimental
animals was observed with different time intervals (2 h, 3 h and 5 h)
and volume of the paw was measured using digital plethysmometer.
After 24 h the right hind paw tissue was rinsed in ice-cold normal
saline, and immediately placed in its four volumes of cold normal saline
and homogenized at 4 °C. Then the homogenate was centrifuged at
12,000 rpm for 5 min. Then the supernatant was stored at −80 °C for
the COX-2, activity assays.
The in vivo CAM assay was performed according to the standard
protocol [8]. In brief, fertilized hen’s eggs were procured from Indian
Veterinary Institute Poultry Department, Bengaluru. All the eggs were
incubated at 37 °C in a humidified incubator (Rotex, India). On the 11th
day of incubation, all the eggs were grouped separately with each group
containing minimum six eggs. Group A served as normal with no
treatment, group B served as control group treated with rVEGF165
(1 μg) (Produced in Molecular Biomedicine laboratory, Department of
Biotechnology, Sahyadri Science College, Shivammogga alone) and
group
C served as a test group, treated with compound 10j
(5 μM) + rVEGF165 (1 μg). The windows were opened after 48 h i.e. on
the 13th day of incubation, inspected and quantified for changes in the
MVD and photographed using Sony steady shot DSC-W610 camera.
4.2.3. Animals and ethics
4.2.7. MPO assays
The animal models used for the study include healthy Swiss albino
The tissue MPO activity of compound 10j, using a standard protocol
method with little modification was performed [33]. The tissue from
the carrageenan induced paw oedema experimental mice were used to
assess the MPO activity, after the 4 h carrageenan administration. The
tissue samples from all groups of animals were placed in 0.75 ml of
80 mM phosphate buffered saline, pH 5.4, containing 0.5% hex-
adecyltrimethylammonium bromide (HTAB), and were then homo-
genized 45 s at 0 °C in a homogenizer. The homogenate was decanted
into a microfuge tube and the vessel was washed with a second 0.75 ml
aliquot of HTAB in the buffer. The washing was transferred to a mi-
crofuge tube, and centrifuged at 12,000 rpm at 4 °C for 15 min. About
30 μl resulted supernatant solution were added to 96-well microtitre
plates, then 200 μl of a mixture containing phosphate buffered saline
(100 μl, 80 mM), pH 5.4, phosphate buffered saline (85 μl, 0.22 mM),
pH 5.4, and hydrogen peroxide (15 μl–0.017%) was added to the wells.
The reaction was initiated by the addition of tetramethyl benzidine-
hydrochloride (20 μl, 18.4 mM) in dimethylformamide. Then the plates
were incubated at 37 °C for 3 min and the reaction was stopped by the
addition of sodium acetate (30 μl of 1.46 M) pH 3.0. Finally, the enzyme
activity was determined calorimetrically using a plate reader, at 630 nm
and expressed as MOD/mg tissue.
male mice weighing 25
150 5.0 g. All the animals were grouped separately and housed in
polyacrylic cages and maintained under standard conditions
(25 2 °C). All procedures described were reviewed and approved by
2.0 g and Swiss albino male rat weighing
the National College of Pharmacy Ethical Committee, Shivammogga,
India, in accordance with the CPCSEA guidelines for laboratory animal
facility (NCP/IAEC/CL/101/05/ 2012-13).
4.2.4. Determination of the lethal dose 50 (LD50) of the compounds 10a–j
For the determination of LD50 of the series of compounds 10a–j,
‘staircase’ method [10] was employed. Healthy Swiss albino male mice
weighing 25
5 g were used and were divided into 12 groups of six
animals in each group for all the compounds. The test compounds 10a–j
were dissolved in DMSO and administered i.p., in increasing levels of
100, 200, 300, 400, and 500 mg/kg body weight of the animals. The
animals were then observed continuously for 3 h for general behavioral,
neurological, and autonomic profiles and then every 30 min for the next
3 h and finally the animals were died after 24 h. The maximum non
lethal and the minimum lethal doses were thus determined. One tenth
and one twentieth of this LD50 dose was selected as the therapeutic dose
for the evaluation of in vivo anti-angiogenic activity of the compounds
10a–j.
4.2.8. COX-2 activity assay
4.2.5. Induction of alkali burn-induced corneal neovascularization
Corneal neovascularization was induced by alkali injury, as re-
ported earlier with little modification [31]. The Swiss albino Wistar rats
were examined and found to have no ophthalmic diseases before alkali
burn. Animals were divided into three groups and each group consists
of 6 animals (n = 6). Group A: normal animal without any treatment,
animals from group B were treated as control animals and the group C
animals served as a 10j treated group.
Corneal alkali burn was generated in the right eye of animals from
group B and group C after general anesthesia by a combined in-
traperitoneal injection of katamine (1 ml/kg) and chlorpromazine
(1 ml/kg), A piece of Whatman filter paper (3-mm diameter) soaked in
4 ml sodium hydroxide (1 mol/l) was applied to the center of the cornea
for 40 s. The alkali treated cornea was then irrigated with 60 ml of
normal saline. The area of corneal neovascularization was quantified by
photographic documentation after 48 h of alkali burn. Animals from
group C were used to treat the alkali burnt cornea by compound 10j
(5 μg). Neovascular inhibitory effect of compound 10j was assessed by
photographic documentation after 24 h of compound 10j treatment.
The COX-2 activity was measured in mice edematic tissue lysate
treated with or without compound 10j in accordance with the pre-
viously described methods with slight modifications [34]. Briefly, after
24 h post treatment paws were homogenized in RIPA buffer and protein
concentration was determined. COX enzymatic activity was determined
with 100 mg of protein in 0.2 ml volume of assay buffer (100 mM tris-
hydrochloride pH 7.8, 5 mM tryptophan, and 5 mM reduced glu-
tathione). Samples were incubated at 37 °C for 15 min in the presence of
an excess arachidonic acid (100 mM). The reaction was stopped by
boiling, and samples were centrifuged at 10,000 rpm for 10 min. Con-
centrations of PGs were measured according to the manufacturer’s in-
structions (Cayman Chemical).
4.2.9. In-silico studies
Molecular modeling investigations were carried out using autodock
tools-1.5.6,4 GB RAM, 5000 GB hard disk, and NVIDIA Quodro FX 4500
graphics card which was employed in the docking studies. The com-
pounds used for docking was converted into 3D with ChemBio3D Ultra
14.0. The mouse COX-2 (4rry model) taken as a model for the human
COX-2 enzyme which was obtained from protein data bank (PDB 4rry).
Using tools available in autodock programmer the proteins and ligands
were downloaded and prepared in three-dimensional atomic co-
ordinates for molecular docking. All molecular modeling experiments
were carried out with carton and ribbon models and the Figures
showing protein-ligand interactions were generated using PyMOL.
4.2.6. Carrageenan-induced paw oedema in mice
Male Swiss albino mice were selected for the study of anti-in-
flammatory effect of compound 10j against carrageenan induced paw
oedema [32]. For this study, experimental animals were divided into
four groups, each group consists of six animals (n = 6). Group I animals
are served as normal animals with no treatment. Prior to experiment
1454

N. Puttaswamy et al.
Biomedicine&Pharmacotherapy103(2018)1446–1455
Conflict of interest statement
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The authors hereby state that they have no conflict of interest.
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Naveen P. gratefully acknowledges the financial support of
UGCMRP(S)-0551-13-14/KAMY013/UGC-SWRO. S. A. Khanum greatly
acknowledges the financial support provided by the Vision Group on
Science and Technology, Bangalore, Government of Karnataka, under
the scheme CISEE (VGST/CISEE/2012-13/282), India. B.T. Prabhakar
and Vikas H.M. gratefully acknowledges for the grant support by VGST
(VGST/P-2/CISEE/GRD-231/2013-14). Also the authors are thankful to
the Principals, JSS College of Arts, Commerce and Science, Mysuru
Yuvaraja’s College, Mysuru and Sahyadri Science College
(Autonomous), Kuvempu University, Shivammogga for providing la-
boratory facilities.
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