Novel 1,3,4-oxadiazole/oxime hybrids: Synthesis, docking studies and investigation of anti-inflammatory, ulcerogenic liability and analgesic activities

Article abstract of DOI:10.1016/j.bioorg.2016.09.005

A novel group of 1,3,4-oxadaiazoles, a group known for their anti-inflammatory activity, is hybridized with nitric oxide (NO) releasing group, oxime, for its gastro-protective action and potential synergistic effect. The synthesized hybrids were evaluated for their anti-inflammatory, analgesic, antioxidant and ulcerogenic activities. Most of the tested compounds showed excellent anti-inflammatory activity with compound 8e being more active than indomethacin. They also showed moderate analgesic activity but no antioxidant one. The ability of the synthesized compounds to inhibit COX-1 and COX-2 is studied and the prepared compounds were able to inhibit both COXs non-selectively with IC₅₀s of 0.75–70.50?μM. Docking studies revealed the mode of interaction of the tested compounds into the empty pocket of the isozymes. All of the synthesized compounds interact with COXs active site with energy scores comparable to that of ibuprofen. All compounds showed a safer profile on the stomach tissue integrity compared to conventional NSAIDs. The designed strategy was applied to ibuprofen to introduce ibuprofen/oxadiazole/NO hybrid. The synthesized ibuprofen hybrid is a promising alternative to ibuprofen having similar anti-inflammatory activity but with safer GIT profile.

Full text of DOI:10.1016/j.bioorg.2016.09.005

Accepted Manuscript
Novel 1,3,4-Oxadiazole/Oxime Hybrids: Synthesis, Docking Studies and In-
vestigation of Anti-inflammatory, Ulcerogenic liability and Analgesic activities
Heba S. Abd-Ellah, Mohamed Abdel-Aziz, Mai E. Shoman, Eman A.M. Beshr,
Tamer S. Kaoud, Al-Shaimaa F.F. Ahmed
PII:
S0045-2068(16)30136-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.09.005
YBIOO 1951
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 July 2016
13 September 2016
16 September 2016
Please cite this article as: H.S. Abd-Ellah, M. Abdel-Aziz, M.E. Shoman, E.A.M. Beshr, T.S. Kaoud, A.F.F. Ahmed,
Novel 1,3,4-Oxadiazole/Oxime Hybrids: Synthesis, Docking Studies and Investigation of Anti-inflammatory,
Ulcerogenic liability and Analgesic activities, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.
2016.09.005
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Novel 1,3,4-Oxadiazole/Oxime Hybrids: Synthesis, Docking Studies
and Investigation of Anti-inflammatory, Ulcerogenic liability and
Analgesic activities
Heba S. Abd-Ellaha, Mohamed Abdel-Aziza, Mai E. Shomana,*, Eman A.M. Beshra,
Tamer S. Kaouda,b, Al-Shaimaa F. F. Ahmedc
a Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
b Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712,
USA
c Department of Pharmacology and Toxicology Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
Abstract
A novel group of 1,3,4-oxadaiazoles, a group known for their anti-inflammatory
activity, is hybridized with nitric oxide (NO) releasing group, oxime, for its gastro-
protective action and potential synergistic effect. The synthesized hybrids were
evaluated for their anti-inflammatory, analgesic, antioxidant and ulcerogenic activities.
Most of the tested compounds showed excellent anti-inflammatory activity with
compound 8e being more active than indomethacin. They also showed moderate
analgesic activity but no antioxidant one. The ability of the synthesized compounds to
inhibit COX-1 and COX-2 is studied and the prepared compounds were able to inhibit
both COXs non-selectively with IC50.s 0f 0.75-70.50 M. Docking studies revealed
the mode of interaction of the tested compounds into the empty pocket of the
isozymes. All of the synthesized compounds interact with COXs active site with
energy scores comparable to that of ibuprofen. All compounds showed a safer profile
on the stomach tissue integrity compared to conventional NSAIDs. The designed
strategy was applied to ibuprofen to introduce ibuprofen/ oxadiazole/ NO hybrid. The
synthesized ibuprofen hybrid is a promising alternative to ibuprofen having similar
anti-inflammatory activity but with safer GIT profile.
Key words: 1,3,4-Oxadiazole, Nitric Oxide, Anti-inflammatory, Analgesic, NSAIDs,
COX inhibitors, ibuprofen.
*Address correspondence to:
Dr. Mai E. Shoman
Department of Medicinal chemistry
Faculty of Pharmacy, Minia University, Egypt
Email: Shoman_mai@yahoo.com, mai_shoman@mu.edu.eg
Phone: +20 11116757931; Fax: +20 862369075
1. Introduction
Non–steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs
for treating inflammation, pain, and fever through inhibition of cyclooxygenases
1

(COXs) [1,2]. COXs are the key enzymes responsible for prostaglandin H2
biosynthesis which is the precursor for the inflammatory mediators; prostaglandins,
thromboxanes, and prostacyclins [2,3]. COXs are present in two major isoforms
COX-1 and COX-2 [3,4]. COX-1 is constitutively synthesized, present in most tissues
and plays an important role in physiological homeostasis, while COX-2 is synthesized
in response to inflammatory stimuli and present in the site of inflammation [5,6]. In
long-term use, NSAIDs are frequently associated with systemic and local
gastrointestinal (GI) side effects [7,8]. The systemic side effects are due to non-
selective inhibition of COXs, while the free carboxylic functional group is responsible
for the local GI tract irritation [9].
Different strategies are adopted to obtain an anti-inflammatory agent devoid of these
side effects. Replacing the free carboxylic functional group with different heterocyclic
bioisosters such as 1,3,4 oxadiazole [10], 1,2,4-triazole [11,12], and 1,3,4 thiadiazole
[12] decreases the gastric upset and enhances the anti-inflammatory activity. Also,
selective COX-2 inhibitors such as celecoxib [13], rofecoxib [14], etoricoxib [15], and
valdecoxib [16] preserve the cytoprotective prostaglandins that contribute to
physiological homeostasis and decrease GI and renal toxicities [17]. Unfortunately,
rofecoxib and valdecoxib recently showed serious cardiovascular adverse effects and
have been withdrawn from the market [18]. Another promising strategy was applied
through designing NSAIDs-hybrids that release nitric oxide (NO) as a gastro-
protective mediator that decreases the GI tract damage induced by the parent drug
[19]. The NO-NSAIDs hybrids have comparable anti-inflammatory and analgesic
activity with more safety profile [20,21]. The mount of nitric oxide released control
either it shows pro-inflammatory or anti-inflammatory effects. In low concentrations,
NO produced by endothelial nitric oxide synthase (eNOS) has anti-inflammatory
activity due to inhibition of the adhesion and migration of inflammatory cells [22]. In
contrast, overproduction of NO by inducible nitric oxide synthase (iNOS) leads to
leukocyte infiltration in inflamed tissue and increases vascular permeability [23].
Additional beneficial effects of NO are to keep the integrity of gastric mucosa through
increasing mucosal blood flow and enhancing the resistance of the mucosal cell to
ulceration [24,25]. NO can also provide the same protective functions of
prostaglandins [26]. It increases the mucus and bicarbonate secretion that serve as
mucosal defence against injury and helps in ulcer healing [27]. NO is considered an
important signalling molecule in cardiovascular system. It prevents platelet
aggregation, adhesion of leukocyte to endothelial cells and also preserves vascular
physiology [28,29].
Based on aforementioned information, herein, we report the design and synthesis of
novel 1,3,4-oxadiazole/oxime hybrids with the oxadiazole ring offering an important
pharmacophore that has a promising anti-inflammatory [30,31] and analgesic [32]
activities. It also acts as a bioisoster for the free carboxylic group in conventional
NSAIDs. Hybridization with oxime group; a NO donating group is also designed for
potential synergistic and gastro-protective effect and also to minimize any potential
cardiovascular side effects. Additionally, incorporating the designed 1,3,4-
oxadiazole/oxime hybrid into ibuprofen is also applied to examine the potential
2

synergistic anti-inflammatory effect and minimize ulcerative profile of the parent
ibuprofen.
2. Results and discussion.
2.1. Chemistry
The designed 2-(5-Phenyl-1,3,4-oxadiazol-2-yl)thio)-N-(4-acetylphenyl)acetamides
7a-e were prepared starting with the esterification of benzoic acid derivatives using
usual Fischer esterification [33] followed by hydrazide formation 5a-e. The hydrazide
formed is cyclized using carbon disulfide and potassium hydroxide to yield 1,3,4-
oxadiazoles 6a-e (Scheme 1). The ketonic intermediates 7a-e are formed through
coupling of 1,3,4-oxadiazole derivatives with N-(4-acetylphenyl)-2-bromoacetamide 2.
Hybrids of NO releasing compounds 8a-e are obtained via conversion of the ketone
group into an oxime one through a condensation reaction of compounds 7a-e with
hydroxylamine hydrochloride (Scheme 1). Formation of the designed oximes 8a-e
were confirmed through the appearance of a singlet signal of OH group in the offset
1
region (11.09-11.11 ppm) in H NMR and also through shift of ketonic C=O group
from (196.79-197.01) to C=N at (152.57-162.76) ppm in 13C NMR.
A similar procedure was employed to synthesize an ibuprofen/oxadiazole/oxime
hybrid 14 (Scheme 2). Ibuprofen/oxadiazole hybrid was synthesized through the
cyclization of the corresponding hydrazide 11 using the same previously described
procedure. Reaction of oxadiazole with N-(4-acetylphenyl)-2-bromoacetamide 2 gave
the corresponding ketone 13, which is further reacted with NH2OH.HCl to yield the
oxime 14. Formation of the oxime was also confirmed through the appearance of a
1
singlet signal of OH group in the offset region (11.09 ppm) in H NMR and also
through shift of the ketonic C=O from (196.95) to C=N at (152.47) ppm in 13C NMR.
Scheme 1: Synthesis of substituted N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-((5-
3

phenyl-1,3,4-oxadiazol-2-yl)thio) acetamides 8a-e.
Scheme 2: Synthesis of N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-((5-(1-(4-isobutyl
phenyl)ethyl)-1,3,4-oxadiazol-2-yl)thio)acetamide 14.
2.2. Nitric oxide release
The amount of nitric oxide released from the synthesized compounds 7a, 8a-e and 14
was measured using Griess colorimetric method [34] after incubation in either
phosphate buffer (pH 7.4) or HCl (0.1 N, pH 1) for 1 h. The amount of NO was
measured relative to NO released from standard NaNO2 solution, calculated as
percentage of NO released (mol/mol) and listed in Table 1. The results revealed the
ability of the tested oximes 8a-e and 14 to release small amounts of NO at phosphate
buffer of pH 7.4 after 1 h (0.37-5.46 mol/mol, Table 1), while the parent ketone 7a
did not show any amount of NO at the specified conditions confirming that oxime
group is the only source for NO in the designed structures.
On the other hand, there is no release of NO at pH 1 and this may support the fact that
NO-donating moieties (oximes) are weakly hydrolysed in the gastric lumen and
confirms that the suggested gastro-protective action of NO is mediated systemically
[11,35].
2.3. Biological evaluation
2.3.1. Anti-inflammatory activity
Anti-inflammatory activity of the synthesized compounds 7a-e, 8a-e, -13, and 14 was
tested using carrageenan-induced rat paw oedema method described by Winter et al
[36]. Compounds were administered via the intraperitoneal route in equimolar doses
to (0.05 mol, 17mg/Kg) of the standard drug (indomethacin), 30 min before
carrageenan injection at the right hind paw of adult albino male rats. Mean changes in
4

the paw oedema thickness were recorded every hour for 4 hours after carrageenan
injection. The anti-inflammatory activity was calculated as the percentage of
reduction in oedema thickness induced by carrageenan and was determined using the
following formula:
(VR- VL)control – (VR-VL) treated
% of oedema inhibition =
------------------------------------------- x100
(VR-VL) control
Where VR represents the mean right hind paw thickness and VL represents the mean
left hind paw thickness. (VR- VL)control represents the mean increase in paw thickness
in the control groups of rats. (VR- VL)treated represents the mean increase in paw
thickness in rats treated with the tested compounds.
Results are expressed as % mean ± standard error of mean (SEM) and listed in Table
1. Results revealed that most of the synthesized compounds have significant anti-
inflammatory activity compared to that of indomethacin and most of them reach their
maximum activity at 3-4 h interval. In series I, ketones 7a-e, compound 7a had
excellent anti-inflammatory activity with % inhibition of 100.00 while compounds 7b,
7c and 7e showed comparable activity to indomethacin with % inhibition of 86.67,
83.33 and 83.33, respectively. Compound 7d had good anti-inflammatory activity
(70 % reduction in inflammation, Table 1). Thus compounds 7a elicited promising
anti-inflammatory activity of 120 % potency relative to indomethacin at 4 h. In series
I, unsubstituted phenyl ring had the highest anti-inflammatory activity among the
tested derivatives. Addition of electron withdrawing group decreased the anti-
inflammatory activity from 100 % to 86 %. Addition of electron donating group also
decreased the anti-inflammatory activity from 100 % to 78 %.
In series II, oximes 8a-e, compounds 8e and 8c have excellent anti-inflammatory
activity with % inhibition of 96.67 and 96.66, respectively while compounds 8a and
8d have comparable anti-inflammatory activity to indomethacin with % inhibition of
87.50 and 83.33, respectively. Compounds 8b exhibited moderate anti-inflammatory
activity with 64.17 % reduction in inflammation (Table 1). Compounds 8a, 8c and 8e
elicited promising anti-inflammatory activity of 105 %, 115 %, and 116 % potency
relative to indomethacin at 4 h. In series II, addition of electron withdrawing group
decreased the anti-inflammatory activity from 87.50 % to 64.17 % while the presence
of monomethoxy and trimethoxy groups increased the anti-inflammatory activity
from 87.50 % to 96.66 % and 96.67 %, respectively. Addition of dimethoxy group
decreases the anti-inflammatory activity from 87.50 % to 83.33 %.
The results obtained from measuring the oedema thickness are confirmed by weighing
granuloma formed in right hind paw at 4 h. Percentage of inhibition in granuloma
weight relative to control was calculated and listed in Table 1. Compounds 7a, 7b, 8a,
8c and 8e have the highest anti-inflammatory activity of % inhibition in granuloma
weight of 81.33, 72.14, 77.57, 82.49, and 84.17 relative to 66.96 % of indomethacin.
5

Addition of NO releasing group to ketones 7a-e did not uniformly increase the
observed anti-inflammatory activity of the tested ketones suggesting a minor role for
NO in such activity. The observed activity was hypothetically explained by the ability
of these compounds to inhibit COXs. This hypothesis was tested using in vitro
inhibition assays and docking of the tested compounds on both COX-1 and COX-2.
Inhibition assays and docking studies are discussed later in sections 2.3.5 and 2.4.
Transforming the free carboxylic group of ibuprofen into 1,3,4-oxadiazole derivative
13 caused a slight increase in the anti-inflammatory activity for the first 3 h (from
75 % into 100 % in the second hour and from 77 to 86 at the third one). The anti-
inflammatory activity of compound 13 decreased over the fourth hour. Addition of
oxime moiety into compound 14 decreased the activity into 70 % at the third hour. So
compound 13 is considered an alternative to ibuprofen but probably with a shorter
duration of action.
Histological examination of the granuloma of the right hind paw was done for rats
sacrificed at 4h post evaluation of anti-inflammatory activity of oxime 8e,
indomethacin, and control to confirm the elicited anti-inflammatory activity for the
tested compounds. Carrageenan untreated granuloma showed non-intact tissues with
signs of neutrophil infiltration and tissue injury (Fig. 1D). Tissues of rats treated with
indomethacin were more intact with fewer sites of oedema and infiltration of
inflammatory cells (Fig. 1C). Sections of Compound 8e treated rats showed minimal
injury and appeared as healthy as normal tissue sections (Fig. 1A and B). The use of
NO donating oxime provided healthier tissues than those treated with indomethacin
and this might be attributed to the release of NO and its protective effect on the tissues
examined. Compound 8e had an excellent anti-inflammatory profile with safer effect
on tissues.
6

Table 1. Percentage of NO released from compounds 7a, 8a-e, and 14 using Griess colorimetric method and % of oedema inhibition exhibited
by compounds 7a-e, 8a-e, 13, 14, ibuprofen, and indomethacin using carrageenan induced paw oedema method.
Potency a
Granuloma weight
Compound NO release
Oedema inhibition
(% mean ± standard error, n=5)
(% mol/mol)
No.
inhibition
(%)
4 h
0
(% mean ± standard error)
1 h
0
2 h
0
3 h
0
4 h
0
4 h
Control
0
0
0
66.66±0.07**** 86.66±8.16** 93.33±6.66*** 100.00±5.27*** 120.00
60.00±4.08*** 93.33±4.08*** 86.67±8.16** 86.67±6.23*** 104.01
45.83±4.16** 62.50±4.16** 79.19±7.97** 83.33±0.05*** 100.00
53.33±4.81**** 66.67±8.27*** 60.00±7.28*** 70.00±5.21**** 84.00
81.33±±1.40
72.14±1.87
68.93±1.44
61.89±1.90
69.95±2.84
77.57±1.74
60.65±0.35
82.49±0.88
64.74±3.91
84.17±1.32
63.12±1.93
55.06±4.91
76.74±2.08
66.96±1.21
7a
7b
7c
7d
7e
8a
8b
8c
-----
-----
-----
-----
2.88
4.73
5.46
5.38
4.24
-----
0.37
------
------
66.67±0.06**** 75.00±4.81** 79.17±7.97** 83.33±9.62*
58.33±4.81** 66.67±0.08**** 91.67±4.81** 87.50±7.97**
54.17±7.97* 66.67±6.80** 66.67±0.05**** 64.17±2.50***
100.00
105.00
77.00
66.66±7.45** 70.00±8.16** 86.66±6.23*** 96.66±6.23*** 115.99
45.83±7.97* 58.33±8.33* 75.00±4.81** 83.33±6.80** 100.00
60.00±6.66** 76.67±4.08*** 100±0.05**** 96.67±6.23*** 116.00
8d
8e
13
63.33±3.33*** 100±0.10**** 88.89±4.08*** 76.67±4.08***
33.33±7.45 75.00±6.66** 70.00±6.23** 60.00±8.49*
92.01
72.00
14
Ibuprofenb
Indomethacinc
60.00±8.49* 75.00±6.66*** 77.78±6.23*** 77.78±6.23*** 104.01
54.33±5.46*** 66.67±7.27*** 86.66±7.27*** 83.33±5.14**** 100.00
a Potency was expressed as % of edema inhibition of the tested compounds relative to % of edema inhibition of indomethacin at 4 h.
b
Ibuprofen dose = 10 mg/Kg, c Indomethacin dose = 17 mg/Kg
*Significantly different from control group at p= 0.05, ** significantly different from control group at p= 0.01, *** significantly different from control group at p= 0.001, and ****
significantly different from control group at p= 0.0001.
7

A
B
C
D
Fig. 1. Microscopic features (hemoxylene stain) of carrageenan induced oedema
formed in the right hind paw of A) rats treated with compound 8e (10x); B)
rats treated with compound 8e (20x); C) rats treated with indomethacin; D)
untreated control rats. White arrows indicate sites of tissue injury.
2.3.2. Analgesic activity
Analgesic activity of compounds 7a-e, 8a-e, 13, 14, indomethacin, and paracetamol
was evaluated using tail flick method [37]. Tested compounds were administered
intraperitoneally in a dose similar to that used in anti-inflammatory screening (0.05
mol, equimolar to 17 mg/Kg indomethacin). Paracetamol was used as a reference
drug and administered in two doses (0.05 mol, 0.50 mol). Results were expressed as a
percentage of increase in the latency time of the treated group relative to untreated
control group and listed in Table 2. Paracetamol administered in equimolar dose to
the tested compounds (0.05 mol, 7.2 mg/Kg) did not show any significant analgesic
activity (19.99 %). The test was repeated using a dose reported in similar studies (0.50
mol, 72 mg/Kg) [37], and the analgesic activity was found to be (123.90 %). Most of
drug treated groups have shown significant increase in tail flick latency time
8

compared to control. Compound 8b had excellent analgesic activity with 196.30 %
increase in tail flick latency time compared to 123.90 % of paracetamol. Compounds
7b, 7a, 8a, and 8e exhibited good analgesic activity with mean percentage of 130.60
%, 118.30 % and 102.50 %, 97.63 %, respectively. The rest of the compounds show
weak analgesic activity (36.93-80.34 %, Table 2). Analysing data revealed that both
unsubstituted and p-Cl substitution is favoured for analgesic activity. The role of NO
in such activity is not confirmed, as NO donating oximes did not introduce a uniform
increase in the measured activities.
The good analgesic activity of the tested compounds along with their ability to reduce
inflammation probably suggests that COX inhibition is their mode of action (see
sections 2.3.5 and 2.4).
Since the tail flick method is measuring central analgesic activity [38], the ability of
the synthesized compounds to penetrate blood brain barrier (BBB) might affect their
action. Lipophilicity and molecular size of drugs are important factors affecting the
penetration of drugs into central nervous system (CNS) [39]. Compounds with
moderate lipophilicity are better to penetrate BBB since very lipophilic compounds
tend to be highly protein bound and bind to lipid membranes[40]. Also, the entry of a
compound into the cerebrospinal fluid (CSF) is proportional to the square root of the
molecular mass, so the penetration of large hydrophilic compounds into the CSF is
low [41]. Results mentioned earlier suggested that analgesic activity is favoured with
p-Cl and unsubstituted derivatives. This might be partially explained by the higher
lipophilicity and smaller molecular size of such derivatives. Compound 8e, with best
anti-inflammatory activity, was expected to have a higher analgesic activity. The
decrease in lipophilicity (log p 3.17, Table 2) and large molecular mass (Table 2) of
such compound might hinder its passage into the CSF and decrease its central
analgesic activity. Compounds 13 and 14 with the highest value of log p (5.76 and
6.06, respectively) have low analgesic activity that may also be explained by their
large size and inability to diffuse into the BBB. While compound 8b, with low anti-
inflammatory activity, has moderate lipophilicity (log p = 4.16, Table 2), its smaller
size might contribute to a higher diffusion rate through the bilayer membrane and
hence a better analgesic activity.
2.3.3. Measurement of lipid peroxidation level
Lipid peroxidation was used as a measure for anti-oxidant activity. It was determined
in serum via measuring thiobarbituric acid reactive species (TBARS; sometimes
referred to as malondialdehyde, MDA) as a marker of oxidative stress [42] according
to the method described by Buege and Aust [43].
Lipid peroxidation products including MDA, 4-hydroxynonenal, and others react with
thiobarbituric acid (TBA) to form a pink colored adduct which is measured
colourmetrically at max 532nm. This method was used to evaluate the antioxidant
activity of the synthesized compounds 7a-e, 8a-e as well as indomethacin. Level of
9

TBARS of serum of rats sacrificed 4 h post drug administration. Results were
calculated as the concentration of TBARS in nmol/mL (Table 2). They showed that
samples obtained from animals treated with compound 7a had lower amount of
TBARS indicating a slight reduction in lipid peroxidation process and a slight
antioxidant activity (18 %, Table 2). The rest of compounds caused a slight increase
in the oxidative stress, (Table 2). NSAIDs are reported to increase the serum level of
reactive oxygen species (ROS) [44], therefore the observed increase in oxidative
stress might support the assumption that the tested compounds reveal their anti-
inflammatory and analgesic activity via COX inhibition.
2.3.4. Ulcerogenic liability
The synthesized compounds 7a-e, 8a-e, 13, 14, indomethacin, and ibuprofen were
assessed for their ulcerogenic liability. Results were obtained from the post mortem
studies of rats sacrificed 4 h after anti-inflammatory evaluation. All synthesized
compounds exhibited excellent safe profile compared to that of indomethacin (UI
=35). These finding is supported with the histological examination of the gastric
mucosa of compound 8e (Fig. 2A), which shows normal histology of rat gastric wall
while indomethacin increased vascular congestion, infiltration of inflammatory cells
and sloughing in mucosal layer as signs of mucosal ulcer. The high safety profile is
probably due to bioisosteric replacement of carboxylic function group of conventional
NSAIDs with 1,3,4-oxadiazole ring and additional protective effects from NO
released from compounds 8a-e. Compound 13 showed an intact stomach lining
compared to ibuprofen (UI= 20), so it is considered a safer alternative to ibuprofen
with comparable activity. However, adding a nitric oxide donating group in to
compound 14 enhanced safety profile on stomach lining with slightly weaker anti-
inflammatory
action.
10

Table 2. Analgesic activity, Log p , square root of molecular mass, level of TBARS, and ulcer index of compounds7a-e, 8a-e, 13, 14,
indomethacin, and paracetamol.
Thiobarbituric acid
Ulcer index
)mean ± standard
error)
Analgesic activitya
(% mean ± standard error,
n=5)
reactive speciesc
(concentration of TBARS in
nmol/mL, n=5)
Square root of
molecular mass
Log Pb
Compound No.
4.52±0.02
3.68±0.05
5.64±0.02
4.84±0.01
5.26±0.05
4.74±0.02
6.09±0.05
5.86±0.03
5.17±0.02
5.46±0.01
5.19±0.02
4.97±0.05
6.08±0.04
6.06±0.02
Nd
9±0.15
3±0.02
3±0.05
4±0.18
2±0.10
5±0.05
1±0.04
2±0.10
Zero±0.01
zero±0.10
zero±0.05
3± 0.15
zero±0.07
35± 0.35
Nd
Control
0
118.30±2.98
130.60±6.02
68.51±7.65
41.89±4.85
68.41±3.31
102.50±6.80
196.30±8.84
80.34±6.65
36.93±2.84
97.63±8.53
65.47±4.58
2.37±3.33
-----
3.16
3.72
3.03
2.90
2.78
3.55
4.16
3.42
3.29
3.17
5.76
6.06
3.75
0.55
0.55
------
18.79
19.67
19.57
20.32
21.05
19.19
20.05
19.95
20.69
21.40
20.91
21.26
18.90
12.29
12.29
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
13
14
Indomethacind
Paracetamole
Paracetamolf
143.70±9.35
19.99±4.42
123.90±9.45
Nd
Nd
a Analgesic activity using tail flick method and calculated as % of increase in latency time compared to control.
b
Log p calculated by Crippen's fragmentation, ChemBioDraw Ultra 14.00.
c Lipid peroxidation level measured as the concentration of TBARS and expressed in nmol/mL
11

e
d Indomethacin dose = 17 mg/Kg; paracetamol dose 7.2 mg/Kg ; fparacetamol dose 72 mg/Kg
Nd not determined.
12

A
A
B
B
Fig. 2. Histological examination of the stomach lining for rats treated with A)
compound 8e (4x); B) Indomethacin (4x).
2.3.5. In vitro COX inhibition assay.
Compounds 7a-b, 8c, 8e and 13; compounds with the highest anti-inflammatory
activity; were selected for examining their ability to inhibit both COXs. Their effect is
studied on both ovine COX-1 (using SC-560 as a reference) [45] and on human
recombinant COX-2 (using Dup-697 as a reference) [46] using florescent inhibitor
screening assay kit (Cayman Chemical, Ann Arbor, MI, USA). The results were
recorded, dose response curves were built (Fig. 3) and the concentration required to
produce 50% inhibition of either COX-1 or 2 (IC50) is calculated.
All the examined compounds exhibited a low IC50 values on both COXs supporting
that the described activity occurred though COX inhibition. They exhibited low
inhibition to COX-1 compared to indomethacin ( IC50 of 0.1 M) [47] while they
were potent inhibitors against COX-2 compared to indomethacin (IC50 of 24.60 M)
[48]. No significant difference was observed in IC50s measured on both COX-1 and 2
indicating no selectivity on either isozymes. The presence of an electron withdrawing
group (p-cl) in compound 7b increased the measured IC50 to 10 M. Also the
presence of the long alkyl group from the ibuprofen structure in compound 13
dramatically decreased the ability of the compound to inhibit the enzyme. The
presence of small electron donating methoxy group in compound 8c achieved the best
COX-1 enzyme inhibition. Introduction of a trimethoxy function group (compound
8e) resulted in the best COX-2 enzyme inhibition and that may attributed to the bulky
nature of trimethoxy group which may make it a better fit on the allosteric pocket of
COX-2 active site ( see docking at section 2.4).
Moreover, both functional groups used (ketones and oximes) showed good inhibition
of both COXs suggesting a minimal role of NO in the measured activities.
13

Table 3. In vitro COX-1 and COX-2 enzyme inhibition for compounds 7a, b, 8c, 8e
and 13 compared to SC-560 and Dup-697.
Compound
COX-1
IC50 (M)a
COX-2
IC50 (M)a
SIb
(mean ± SEM, n=3)
(mean ± SEM, n=3)
1.2±0.15
10.3±1.4
0.98±0.25
2.12±0.28
70.3±9.1
7.1±1.4
8.9±1.0
3.3±0.21
0.75±0.10
70.5±10.9
----
0.17
1.15
0.3
2.8
1
7a
7b
8c
8e
13
SC-560
0.066±0.008
---
DuP-697
----
0.07±0.01
-
a Values in the table represent IC50 (μM) and % inhibition at saturation
b In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
14

A
B
C
D
E
Fig. 3. The effect of compounds 7a (A), 7b (B), 8c (C), 8e (D) and 13 (E) on the
activity of ovine COX-1 () and human recombinant COX-2 ().The results
are expressed as percent of control in the absence of inhibitor (100 %
maximum activity).
15

2.4. Docking studies
Docking of the synthesized compounds 7a-e, 8a-e, 13, 14, and ibuprofen was done on
both COX-1 and COX-2 enzymes to suggest the possible binding modes between the
compounds and the enzymes active site. Docking experiments were done using MOE
2014 software. Target compounds were constructed into the builder interface of MOE
program, the energy was minimized until a RMSD (root mean square deviations)
gradient of 0.01 Kcal/mol and RMS (Root Mean Square) distance of 0.1 Å with
MMFF94X (Merck molecular force field 94x) force-field and the partial charges were
automatically calculated. X-ray crystallographic structure of the ligand-enzyme
complex were downloaded from protein data bank (www.rcsb.org); COX-1 enzyme
(pdb: 1eqg) and COX-2 enzyme (pdb: 1cx2).
Enzymes were prepared for docking studies by deleting the ligand, adding hydrogen
atoms, checking the atom connection and type with automatic correction. Then the
potential of the receptor was fixed and docking of the designed compounds is done
into the 3D structure of the catalytic site of COX-1 and COX-2 enzymes. The
obtained poses were studied and the poses showed best ligand-enzyme interactions
were selected and stored for energy calculations.
Table 4 lists the docking energy scores for the synthesized compounds and ibuprofen
(the specific substrate for COX-1), and detailed interactions formed from the tested
compounds with the amino acid residues in the empty pocket of both isozymes. All of
the synthesized compounds gave energy scores from 10.01 to 14.52 Kcal/mol relative
to that of ibuprofen (9.58 and 5.67, Table 4) supporting that COX inhibition is a
reasonable mechanism explaining the analgesic and anti-inflammatory activities
observed with those compounds. All the synthesized compounds have higher energy
scores at COX-2 active site (10.55-12.59 Kcal/mol) compared to that of ibuprofen
(5.67 Kcal/mol).
It is obvious from the results that there is no significant difference was observed in
energy scores obtained for interaction with COX-1 and COX-2 suggesting no
selectivity on either isozymes. The same result was shown before in in vitro COX
inhibition assays. (section 2.3.5)
There is no significant difference between energy scores of all the tested compounds
and ibuprofen, so energy scores could not be used in explaining differences observed
in activities. Detailed study of the binding interactions might offer an explanation for
such differences. Table. 4, shows the number and modes of interactions formed from
the tested compounds with the amino acid residues in the empty pocket of both
isozymes.
Compound 8e with the highest anti-inflammatory activity, showed the best affinity to
COX-2 receptor (energy score -12 Kcal/mol, Table 4, IC50 = 0.75 M, Table 3)
compared that of ibuprofen and the other examined structures. Compound 8e forms
the highest amount of interaction with the active site of COX-2. It forms two
hydrogen bonds with Glu524, two hydrogen bonds with both Arg120 and Tyr385, and
one hydrophobic interaction with Arg513, Fig. 4B, 5B.
16

It also shows the highest amount of interaction with the COX-1 enzyme active site,
Table. 4. It forms 3 hydrogen bonds with Arg120, Tyr355 and Arg83, Fig. 4A, 5A.
The strong binding stabilize the complex formed between compound 8e and the
enzymes and those two amino acids Arg 120 and Tyr 355 are reported to play an
important role in the binding of drugs into COX-1 active site [49],[50].
In series I, compounds 7a-e, compound 7a and 7b (with high anti-inflammatory
activity) have the highest amount of interactions with the empty cavities of COX-1
and COX-2, Table 4, supplementary Information. Compounds 7c, 7d, and 7e show
lower amount of interaction with COXs active site in accordance with anti-
inflammatory data of this group. In series II, compounds 8a-e, compounds 8e and 8c
(with high anti-inflammatory activity) have the highest number of interactions of
COXs active site, Table 4 and supplementary information. Compounds 8a and 8d
showed lower number of interactions Table 4 and supplementary information.
Compound 13 has higher amount of interaction relative to the corresponding oxime
14. Compound 14, having low anti-inflammatory activity, forms only one hydrogen
bond with Arg 120 in the pocket of COX-1, Fig. 6A, 7A and one with Arg 513 in the
pocket of COX-2, Fig. 6B, 7B.
17

Table 4: Types of interactions and Energy scores for the complexes formed from compounds 7a-e, 8a-e, 13 and 14 and the active sites of
both COX-1 and COX-2.
Types of interactions
Energy scores
Compound
COX-1
COX-2
COX-1
COX-2
Ibuprofen
- Two hydrogen bonding with Arg120 (3.60 and
3.83ºA).
- Two hydrogen bonding with Arg120 (2.84 and -9.58
3.30ºA).
-5.67
-One hydrogen bonding with Tyr 355 (2.76 ºA)
-One hydrogen bonding with Arg83 (3.17 ºA)
- One hydrogen bonding with Arg120 (2.76 ºA).
-One hydrogen bonding with Arg 120 (2.90ºA)
-One hydrogen bonding with Glu 524(2.94 ºA)
- One hydrophobic interaction with Arg 513
(4.51ºA).
-One hydrogen bonding with Tyr355 (2.81ºA)
-One hydrogen bonding with Tyr385 (3.07 ºA)
- One hydrophobic interaction with Ala527
(3.85ºA).
-11.50
-11.13
-10.90
7a
-One hydrogen bonding with Arg83 (3.17 ºA)
- One hydrogen bonding with Arg120 (2.71ºA).
-11.37
7b
-One hydrogen bonding with Arg83 (3.11ºA)
- One hydrogen bonding with Arg120 (2.73ºA).
-One hydrogen bonding with Arg 513 (3.43ºA)
-One hydrogen bonding with Glu 524(3.48 ºA)
-11.79
-11.24
-11.82
-10.92
7c
7d
7e
- One hydrogen bonding with Arg120 (2.89ºA).
-One hydrogen bonding with Arg120 (2.68 ºA)
- One hydrophobic interaction with Ala527
(4.05ºA).
-One hydrogen with Arg 513 (3.21 ºA).
-One hydrogen with Lys83 (2.94 ºA).
-11.68
-11.54
- One hydrogen bonding with Arg120 (2.57ºA).
-One hydrophobic interaction with Tyr355 (3.52
ºA).
18

-One hydrogen bonding with Arg 120 (2.78 ºA)
- One hydrogen bonding with Arg83 (3.14 ºA).
-One hydrogen bonding with Met 522 (2.54 ºA). -One hydrogen bonding with Tyr 385 (3.08 ºA)
-One hydrogen bonding with Ser 530 (3.29 ºA).
-One hydrogen bonding with His 90 (3.06 ºA).
-11.38
-11.49
-12.59
-10.55
8a
8b
-One hydrophobic interaction with Ala 527 (4.42
ºA).
-One hydrogen bonding with Met522 (2.72 ºA)
-One hydrogen bonding with Arg120 (3.46 ºA).
- One hydrogen bonding with Arg83 (2.77 ºA).
-One hydrogen bonding with Glu524 (2.93 ºA) .
-One hydrogen with Arg120 (2.97 ºA).
-One hydrogen with Tyr385 (3.19 ºA).
-One hydrophobic interaction with Arg513 (4.52
ºA).
-11.76
-10.75
8c
-One hydrogen bonding with Arg120 (2.79 ºA)
- One hydrogen bonding with Arg83 (2.75ºA).
-One hydrogen bonding with Tyr355 (3.20 ºA)
-One hydrogen bonding with Arg120 (3.13 ºA).
- One hydrogen bonding with Arg83 (3.68 ºA)
-One hydrogen bonding with Arg120 (2.81 ºA)
- One hydrogen bonding with Lys83 (2.92ºA).
- Two hydrogen bonding with Glu524 (2.91 and
3.11 ºA) .
-One hydrogen with Arg120 (3.03 ºA).
-One hydrogen with Tyr385 (3.03 ºA).
-One hydrophobic interaction with Arg513 (4.23
ºA).
-12.41
-14.52
-11.02
-12.56
8d
8e
-Two hydrogen bonding with Arg120 (2.74 and
2.74 ºA).
-One hydrogen bonding with Glu524 (2.50 ºA) .
-One hydrogen with Arg513 (3.00 ºA).
-One hydrogen with Lys83 (3.30 ºA).
-10.80
-11.02
-11.81
13
14
-One hydrogen bonding with Arg120 (2.65ºA).
-One hydrophobic interaction with Arg513 (4.4 ºA) -10.01
19

A)
B)
Fig. 4. 2D drawings of the compound 8e interacted with both active sites of A) COX-
1 and B) COX-2 showing interactions with different amino acid residues found in the
active site.
20

A)
B)
Fig. 5. 3D drawings of the compound 8e interacted with both active sites of A) COX-
1 and B) COX-2 showing interactions with different amino acid residues found in the
active site.
21

A)
B)
Fig. 6. 2D drawings of the compound 14 interacted with both active sites of A) COX-
1 and B) COX-2 showing interactions with different amino acid residues found in the
active site.
22

A)
B)
Fig. 7. 3D drawings of the compound 14 interacted with both active sites of A) COX-
1 and B) COX-2 showing interactions with different amino acid residues found in the
active site.
23

3. Experimental
3.1. Chemistry
Chemicals and solvents used were of analytical grade. Progress of the reactions was
monitored by thin layer chromatography with ethyl acetate/methylene chloride (1:1)
as the mobile phase on pre–coated Merck silica gel 60 F254 aluminum sheets.
Melting points were determined on Stuart electro-thermal melting point apparatus and
were uncorrected. IR spectra were recorded on Nicolet iS5 (ATR) FT-IR spectrometer
1
at Minia University. H NMR spectra were recorded on Bruker Avance III 400 MHz
and 13C spectra were recorded on Bruker AG, Switzerland, 100 MHz . High
resolution mass spectra were collected via Thermo Scientific Q Exactive™
Orbitrap mass spectrometer.
3.1.1. Synthesis of N-(4-acetylphenyl)-2-bromoacetamide 2 [51].
3.1.2. Substituted methyl benzoate 4a-e [52,33].
3.1.3. Substituted benzohydrazides 5a-e [53,33].
3.1.4. Substituted 1,3,4-oxadiazole-2(3H)-thione 6a-e.
3.1.4.1. Substituted 1,3,4-oxadiazole-2(3H)-thione 6a-c [54].
3.1.4.2. 5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 6d.
A mixture of 5d (11.90 g, 0.05 mol), potassium hydroxide (3 g, 0.05 mol), carbon
disulfide (0.17 mol, 10 mL) in absolute ethanol (50 mL) was heated under reflux
with stirring for 12 h. The solvent was evaporated under vaccum. The residue was
dissolved in water and acidified with hydrochloric acid (10 %). The formed
precipitate was filtered, washed with water, dried, and recrystallized from absolute
ethanol to give yellowish white solid Yellowish white solid (7.10 g, 60 % yield); mp
237-238 °C; IR (KBr, cm-1): 3246 (NH), 1621 (C=N); 1H NMR (DMSO-d6) 8.00 (s,
1H, NH), 7.42 (d, 1H, J = 8.4 Hz, Ar-H),s, 1H, Ar-H) 7.10 (d, 1H, J = 8.4 Hz,
Ar-H), 3.80 (s, 6H, 2-OCH3); 13C NMR (DMSO-d6) 177.33, 160.73,152.27,
149.29, 130.47, 119.88, 114.74, 112.12, 55.80, 55.70.
3.1.4.3. 5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 6e [55, 56].
3.1.5. General procedure for the synthesis of compounds 7a-e.
To a stirred mixture of the appropriate oxadiazole 6a-e (1.00 mmol) and compound 2
(0.26 g, 1.00 mmol) in acetonitrile (30 mL), triethylamine (0.12 g, 1.20 mmol) was
added. The mixture was stirred at room temperature and the immediately formed
precipitate was filtered off, washed with methanol, and recrystallized from absolute
ethanol.
3.1.5.1. 2-(5-Phenyl-1,3,4-oxadiazol-2-ylthio)-N-(4-acetylphenyl)acetamide7a [57].
White solid (0.14 g, 56% yield); mp 200-201°C; reported 184-188°C.
3.1.5.2. 2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-acetylphenyl)
acetamide 7b [58].
Yellowish white solid (0.20 g, 54% yield); mp 218-219°C; reported 211-212 °C.
3.1.5.3. 2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-acetylphenyl)
acetamide 7c.
Yellowish white solid (0.13 g, 65 % yield); mp 206-207°C; IR (KBr, cm-1): 3258
(NH), 1685 (COCH3), 1666 (CONH); 1H NMR (DMSO-d6) 10.79 ( s, 1H, NH),
(d, 2H, J = 8.7 Hz, Ar-H), 7.89 (d, 2H, J = 8.9 Hz, Ar-H), 7.73 (d, 2H, J = 8.7
Hz, Ar-H), 7.12 (d, 2H, J = 8.9 Hz, Ar-H), 4.37 (s, 2H, CH2), 3.85 (s, 3H, OCH3),
24

2.54 (s, 3H, CH3); 13C NMR (DMSO-d6) 197.01, 166.11, 165.63, 162.93, 162.55,
143.38, 132.57, 130.07, 128.72, 118.94, 118.76, 115.36, 56.01, 37.35, 26.93; HRMS:
m/z calculated for C19H16N3O4S [M-1]-: 382.08670, found: 382.08722.
3.1.5.4. 2-(5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-acetylphenyl)
acetamide 7d.
Yellow solid (0.23 g, 56 % yield); mp 208-209°C; IR (KBr, cm-1): 3311 (NH), 1686
(COCH3), 1664 (CONH); 1H NMR (DMSO-d6)  11.01 (s, 1H, NH),d, 2H, J =
8.6 Hz, Ar-H), 7.76 (d, 2H, J = 8.6 Hz, Ar-H), 7.52 (dd, 1H, J = 8.5 and 1.5 Hz, Ar-
H), 7.41 (d, 1H, J = 1.5 Hz, Ar-H), (d, 1H, J = 8.5 Hz, Ar-H), 4.41 (s, 2H, CH2),
13
3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 2.52 (s, 3H, CH3); C NMR (DMSO-d6) 
196.79, 165.90, 165.43, 162.69, 152.04, 149.24, 143.17, 132.18, 129.67, 120.10,
118.57, 115.32, 112.10, 109.12, 55.75, 55.68, 36.86, 26.42; HRMS: m/z calculated
for C20H18N3O5S [M-1]- : 412.09726, found: 412.09787.
3.1.5.5. 2-(5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-(4-acetyl
phenyl)acetamide 7e.
White solid (0.24 g, 55 % yield); mp 187-188°C; IR (KBr, cm-1): 3311 (NH), 1681
(COCH3), 1658 (CONH); 1H NMR (DMSO-d6) 10.88 (s, 1H, NH), 7.95 (d, 2H, J
= 8.7 Hz, Ar-H), 7.74 (d, 2H, J = 8.7 Hz, Ar-H), 7.21 (s, 2H, Ar-H), 4.40 (s, 2H,
CH2), 3.83 (s, 6H, 2OCH3), 3.74 (s, 3H, OCH3), 2.54 (s, 3H, CH3); 13C NMR
(DMSO-d6) : 197.00, 166.08, 165.60, 163.60, 153.93, 143.42, 141.05, 132.56,
130.04, 118.92, 118.59, 104.27, 60.70, 56.62, 37.37, 26.92; HRMS: m/z calculated
for C21H20N3O6S [M-1]-: 442.10783, found: 442.10840.
3.1.6.General procedure for the synthesis of compounds 8a-e.
A mixture of the appropriate ketone derivatives 7a-e (1.00 mmol), hydroxylamine
hydrochloride (0.35 g, 5.00 mmol) and anhydrous sodium acetate (0.08 g, 1.00 mmol)
in absolute ethanol (30 mL) was heated under reflux for 10-30 min. and then left to
cool to room temperature. The separated solid was filtered off, washed with dil.
ammonia solution (10 %) and then with distilled water, dried, and recrystallized from
aqueous ethanol affording the corresponding oximes 8a-e.
3.1.6.1. N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-((5-phenyl-1,3,4-oxadiazol-2-
yl)thio)acetamide 8a.
Yellowish white solid (0.15 g, 42 % yield); mp 210-211°C; IR (KBr, cm-1): 3253 (OH,
1
NH), 1659 (CONH); H NMR (DMSO-d6)  11.09 (s, 1H, OH), 10.54 (s, 1H, NH),
- (m, 2H, Ar-H), 7.71-7.46 (m, 7H, Ar-H), 4.35 (s, 2H, CH2), 2.12 (s, 3H,
CH3); 13C NMR (DMSO-d6)  165.40, 165.25, 163.60, 152.57, 139.15, 132.43,
132.23, 129.6, 126.54, 126.30, 123.11, 119.01, 36.85, 11.34; HRMS: m/z calculated
for C18H15N4O3S [M-1]-: 367.08703, found: 367.08734.
3.1.6.2. N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-((5-(4-chlorophenyl)-1,3,4-
oxadiazol-2-yl)thio)acetamide 8b.
Yellowish white solid (0.26 g, 62 % yield); mp 231-232°C; IR (KBr, cm-1): 3381 (OH,
1
NH), 1681 (CONH), 1636 (C=N). H NMR (DMSO-d6)  11.11 (s, 1H, OH), 10.56
(s, 1H, NH), d, 2H, J = 8.0 Hz, Ar-H), 7.65-7.61 (m, 6H, Ar-H), 4.37 (s, 2H,
CH2), 2.13 (s, 3H, CH3); 13C NMR (DMSO-d6) 165.43, 164.89, 164.16, 152.82,
139.42, 137.23, 132.72, 130.08, 128.65, 126.61, 122.33, 119.32, 37.32, 11.84;
HRMS: m/z calculated for C18H14ClN4O3S [M-1]-: 401.04806, found: 401.04883.
3.1.6.3. N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-((5-(4-methoxyphenyl)-1,3,4-
oxadiazol-2-yl)thio)acetamide 8c.
Yellowish white solid (0.20 g, 52 % yield); mp 220-221°C; IR (KBr, cm-1): 3286 (OH,
1
NH), 1664 (CONH), 1612 (C=N); H NMR (DMSO-d6)  11.11 (s, 1H, OH), 10.54
25

(s, 1H, NH),( d, 2H, J = 8.8 Hz, Ar-H), 7.67-7.59 (m, 4H, Ar-H), 7.12 (d, 2H, J
= 8.8 Hz, Ar-H), 4.35 (s, 2H, CH2), 3.86 (s, 3H, OCH3), 2.14 (s, 3H, CH3);13C NMR
(DMSO-d6) 165.33, 165.26, 162.70, 162.26, 152.53, 139.14, 132.39, 128.38,
126.27, 118.97, 115.42, 114.98, 55.55, 36.84, 11.32; HRMS: m/z calculated for
C19H17N4O4S [M-1]-: 397.09760, found: 397.09796.
3.1.6.4. N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-((5-(3,4-dimethoxyphenyl)-
1,3,4-oxadiazol-2-yl)thio)acetamide 8d.
Yellow solid (0.20 g, 47 % yield); mp 198-199°C; IR (KBr, cm-1): 3307 (OH, NH),
1655 (CONH); 1H NMR (DMSO-d6) 11.10 (s, 1H, OH), 10.86 ( s, 1H, NH),  (d,
2H, J = 8.9 Hz, Ar-H), 7.61 (d, 2H, J = 8.9 Hz, Ar-H), 7.53 (dd, 1H, J = 8.4 Hz and
1.5 Hz, Ar-H), 7.43 (d, 1H, J = 1.5 Hz, Ar-H), (d, 1H, J = 8.5 Hz, Ar-H), 4.38
13
(s, 2H, CH2), 3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 2.12 (s, 3H, CH3); C NMR
(DMSO-d6) : 165.94, 165.42, 165.43, 162.76, 152.52, 149.25, 139.29, 132.31,
126.20, 120.11, 118.95, 115.34, 112.12, 109.14, 55.75, 55.69, 36.78, 11.32; HRMS:
m/z calculated for C20H19N4O5S [M-1]-: 427.10816, found: 427.10867
3.1.6.5.
N-(4-(1-(Hydroxyimino)ethyl)phenyl)-2-((5-(3,4,5-trimethoxyphenyl)-
1,3,4-oxadiazol-2-yl)thio)acetamide 8e.
White solid (0.17 g, 46 % yield); mp 227-228°C; IR (KBr, cm-1): 3289 (OH, NH),
1662 (CONH); 1H NMR (DMSO-d6)  11.11 (s, 1H, OH),10.59 (s,1H,NH), 7.62 (s,
4H, Ar-H), s, 2H, Ar-H), 4.37 (s, 2H, CH2), 3.83 (s, 6H, 2OCH3), 3.74 (s, 3H,
13
OCH3), 2.13 (s, 3H, CH3); C NMR (DMSO-d6)  165.59, 165.49, 163.66, 153.93,
152.82, 141.05, 139.46, 132.72, 126.60, 119.30, 118.61, 104.27, 60.71, 56.61, 37.34,
11.84; HRMS: m/z calculated for C21H21N4O4S [M-1]-: 457.11818, found: 457.11954.
3.1.7. Methyl 2-(4-isobutylphenyl)propanoate 10 [59].
3.1.8. 2-(4-Isobutylphenyl)propanehydrazide 11 [60].
3.1.9. 5-(1-(4-Isobutylphenyl)ethyl)-1,3,4-oxadiazole-2(3H)-thione 12 [60].
3.1.10. Synthesis of N-(4-acetylphenyl)-2-((5-(1-(4-isobutylphenyl)ethyl)-1,3,4-
oxadiazol-2-yl)thio)acetamide 13.
Triethylamine (0.12 g, 1.20 mmol) was added to oxadiazole 12 (0.44 g, 1.00 mmol)
and compound 2 (0.26 g, 1.00 mmol) in acetonitrile (30 mL). The mixture was stirred;
a precipitate is immediately formed, filtered off, washed, and recrystallized from
absolute ethanol. to give a yellow solid of 13 (0.20 g, 47 % yield); mp 71-73°C; IR
1
(KBr, cm-1): 3460 (NH), 1675 (COCH3, CONH); H NMR (DMSO-d6),: 10.76 ( s,
1H, NH), 7.95 (d, 2H, J = 8.0 Hz, Ar-H), 7.71 (d, 2H, J = 8.0 Hz, Ar-H), 7.16 (d, 2H,
J = 7.6 Hz, Ar-H), 7.06 (d, 2H, J = 7.6 Hz, Ar-H) 4.41 (q, 1H, J = 7.0 Hz, CH-CH3),
4.30 (s, 2H, CH2-S), 3.06 (d, 2H, J = 7.2 Hz, CH2-Ar), 2.50 (s, 3H, CH3CO), 2.37 (d,
3H, J = 7.0 Hz, CH3-CH), 1.79-1.73 (m, 1H, CH(CH3)2), 0.83 (d, 6H, J = 6.4 Hz,
(CH3)2CH); 13C NMR (DMSO-d6)  196.95, 170.36, 165.88, 163.48, 143.42,
140.66,138.07, 132.50, 129.42, 129.34, 127.45, 118.90, 40.62, 37.18, 36.49, 29.98,
27.32, 22.62, 19.77; HRMS: m/z calculated for C24H26N3O3S [M-1]-: 436.17004,
found: 436.16946.
3.1.11. Synthesis of N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-((5-(1-(4-isobutyl-
phenyl)ethyl)-1,3,4-oxadiazol-2-yl)thio)acetamide 14.
A mixture of compound 13 (0.44 g, 1.00 mmol), hydroxylamine hydrochloride (0.35
g, 5.00 mmol) and anhydrous sodium acetate (0.08 g, 1.00 mmol) in absolute ethanol
(30mL) was heated under reflux for 10-30 min. and then left to cool. The separated
solid was filtered off, washed with dil. ammonia solution (10%) and distilled water,
26

dried, and recrystallized from aqueous ethanol to give a white precipitate in (0.80 g,
1
60 % yield); mp 158-160 °C; IR (KBr, cm-1): 3383 (OH, NH), 1662 (CONH); H
NMR (DMSO-d6), 11.09 (s, 1H, OH), 10.51 (s, 1H, NH),-m, 4H, Ar-H),
(d, 2H, J = 8.7 Hz, Ar-H), 7.07 (d, 2H, J = 8.7 Hz, Ar-H), q, J = 7.2
Hz,CH-CH3), 4.26 (s, 2H, CH2-S), 2.38 (d, 2H, J = 6.8 Hz, CH2-Ar), 2.13 (s, 3H,
CH3-C=NOH), 1.79-1.75 (m, 1H, CH), 1.59 (d, 3H, J = 7.2 Hz, CH3-CH), 0.83 (d,
6H, J = 6.4 Hz, (CH3)2CH); 13C NMR (DMSO-d6)  170.14, 165.22, 163.35, 152.47,
140.52, 139.41,, 132.85, 129.57, 127.26, 126.36, 119.13, 44.55, 36.87, 36.34,
29.85, 22.62, 19.85, 12.10; HRMS: m/z calculated for C24H27N4O3S [M-1]-:
451.18093, found: 451.18066.
3.2. Nitric oxide release
The amount of nitric oxide released from the tested compounds of different forms:
starting ketone 7a, NO-donating oximes 8a-e, and 14 was measured using Griess
colorimetric method [34]. A solution of the appropriate compound (20 µL) in
dimethylsulfoxide (DMSO) was added to 2 mL of 1:1 v/v mixture of 50 mM
phosphate buffer (pH 7.4) with MeOH, containing 5×10-4 M of L-cysteine. The final
concentration of drug was 10-4 M. After 1 h at 37 ºC, 1 mL of the reaction mixture
was treated with 250 µL of Griess reagent [sulfanilamide (2 g), N-
naphthylethylenediamine dihydrochloride (0.2 g), 85% phosphoric acid (10 mL) in
distilled water (final volume: 100 mL)]. After 10 min at room temperature, the
absorbance was measured at max 546 nm. Sodium nitrite standard solutions (10-80
nmol/mL) were used to construct the calibration curve. The same procedure was
repeated using different solutions of the test compounds under the same conditions
using 0.1 N HCl of pH 1 instead of phosphate buffer of pH 7.4. The results were
expressed as amount of NO released relative to a theoretical maximum release of 1
mol NO/mol of test compound.
3.3. Biological evaluation
3.3.1. Screening of anti-inflammatory activity
Anti-inflammatory activity of the different compounds was tested using the
carrageenan-induced paw oedema method described by winter et al, [36]. Briefly,
male albino rats were randomly assigned to different groups (5 rats each). Rats
received either control (vehicle) or equimolar doses of the test compounds by an
intraperitoneal injection. Thirty minutes later, the rats were challenged by a
subcutaneous injection of 0.05 mL of 1% solution of carrageenan into the plantar side
of the right hind paw while the left paw was used as a control. The size of the paw
oedema was recorded every hour for 4 h to determine the decrease in oedema
thickness. At the end of the experiment, rats were sacrificed and the weights of the
right and left paws were measured. Carrageenan-injected and control paws were also
processed for Histopathological examination. The difference between the average
values of weights of the left paws and the corresponding right ones were calculated
and compared to standard drugs.
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3.3.2. Screening of analgesic activity
Analgesic activity of the different compounds was tested using the standard tail flick
method [37]. Briefly, male albino rats were randomly assigned to different groups.
Tail flick analgesimeter (Analgesy-meter LE7106, Panlab, Harvard apparatus) was
used to determine the reaction time for each rat before and after the administration of
the test compound. Radiant heat was applied to the lower third of the rat tail and the
time in which the rat tries to escape was recorded as the reaction time. Reaction times
were recorded thirty minutes after intraperitoneal administration of equimolar doses
of the test compounds. The difference between the average values of reaction time for
each group and the corresponding pretest values were calculated and compared to
standard drugs.
3.3.3. Measurement of lipid peroxidation level
Antioxidant activity was evaluated through determination of thiobarbituric acid
reactive species (TBARS; sometimes referred to as malondialdehyde, MDA).
Aliquots of 0.5 mL of serum of sacrificed albino male rats were added to standard
centrifuge tubes. Then distilled water (0.5 mL) was added to each test tube. A mixture
of thiobarbituric acid/trichloroacetic acid/HCl (15% TBA, 0.2 N HCl and 0.37 %
TCA, 2 mL) was added to the centrifuge tube, then tubes were covered with
aluminium foil and incubated in a boiling water bath for 15 minutes, allowed to cool,
and centrifuged for 15 minutes at 5000 rpm. The supernatant was filtered and the
intensity of the pink colored adduct was measured in the supernatants at max = 532
nm. Concentration of TBARS was calculated from standard calibration curve and
expressed as nmol/ml.
3.3.4. Ulcerogenic liability
The synthesized compounds 7a–e, 8a–e, 13, 14, indomethacin, and ibuprofen were
evaluated for their ulcerogenic liability according to the reported procedure [61].
Ulcerogenic potential was evaluated 4 h post intraperitoneal administration of the
compounds under investigation. The stomachs were removed, collected, opened along
the greater curvature, washed with distilled water and cleaned gently by dipping in
saline. Examination of mucosal layer was done using magnifying lens to detect
macroscopically visible lesions. The number of lesions if any was counted and
recorded. Ulcers were classified into levels, level I, in which ulcer area is less than 1
mm2, level II, in which ulcer area in the range of 1-3 mm2, and level III, in which
ulcer area is more than 3 mm2, and this rated according their areas in mm2. The
following parameters were calculated according to the following formula:
Ulcer index (UI) = 1×(number of ulcers level I) + 2 (number of ulcers level II) + 3
(number of ulcers level III).
3.3.5. In vitro COX inhibition assay
COX fluorescent inhibitor screening assay kit (catalog number 700100, Cayman
chemical, Ann Arbour, MI, USA) has been employed to investigate the isozymes
28

specifity of some of the synthesized compounds 7a, 7b, 8c, 8e, and 13 following the
procedure suggested by manufacturer. Briefly, in 96- well plate, either ovine COX-1
or human re-combinant COX-2 has been incubated with different concentrations of
each tested compound in presence of the assay buffer (100mM Tris-HCl, pH 8.0),
heme and the fluorometric substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) for
20 min at room temperature. The reaction started after addition of arachidonic acid
solution for 2 min at room temperature. Fluorescence of resorufin that is produced by
the reaction of between PGG2 and the fluorometric substrate were analysed. Dup-697
(selective COX-2 inhibitor) and SC-560 (selective COX-1 inhibitor) were used as
reference compounds. The measured fluorescence intensity is proportional to the
amount of PGG2 present in each well during the reaction. IC50 (µM) that corresponds
to the concentration of the inhibitor that causes 50 % inhibition of COX-1 or COX-2
activity was calculated from the dose response curve of inhibition (triplicate
determinations). Dose-response curves of data conforming to inhibition were fitted to:
Where: V0 is the observed rate; I is the concentration of inhibitor I; V' is the observe
rate in the absence of the inhibitor; V∞ is the observed rate constant at saturating the
inhibitor, I; IC50 is the concentration that leads to half the maximal change in V0.
3.3.6. Statistical analysis
The results of all the four methods namely, anti-inflammatory activity, ulcerogenic
liability, analgesic activity, and antioxidant activity were expressed as mean ± SEM.
Results were statistically analysed by using ANOVA (two-way classification
analysis). A probability value of less than 0.05 (P < 0.05) was considered to be
statistically significant.
3.4. Docking studies.
Docking of the synthesized compounds 7a-e, 8a-e, 13, 14 and ibuprofen was done on
both COX-1 and COX-2 enzymes to suggest the possible mechanism of their action
and obtain the possible binding modes between the compounds and the enzymes
active site. Docking experiments were done using MOE 2014 software. Target
compounds were constructed into the builder interface of MOE program, the energy
was minimized until a RMSD (root mean square deviations) gradient of 0.01
Kcal/mol and RMS (Root Mean Square) distance of 0.1 Å with MMFF94X (Merck
molecular force field 94x) force-field and the partial charges were automatically
calculated. X-ray crystallographic structure of the ligand-enzyme complex was
29