Diclofenac 1,3,4-oxadiazole derivatives; biology-oriented drug synthesis (BIODS) in search of better non-steroidal, non-acid antiinflammatory agents

Article abstract of DOI:10.2174/1573406414666180321141555

Background: Inflammation is defined as the response of immune system cells to damaged or injured tissues. The major symptoms of inflammation include increased blood flow, cellular influx, edema, elevated cellular metabolism, reactive oxygen species (ROS) nitric oxide (NO) and vasodilation. This normally protective mechanism against harmful agents when this normal mechanism becomes dysregulated that can cause serious illnesses including ulcerative colitis, Crohn’s disease, rheumatoid arthritis, osteoarthritis, sepsis, and chronic pulmonary inflammation. Method: In this study synthetic transformations on diclofenac were carried out in search of better non-steroidal antiinflammatory drugs (NSAIDs), non-acidic, antiinflammatory agents. For this purpose diclofenac derivatives (2-20) were synthesized from diclofenac (1). All derivatives (2-20) and parent diclofenac (1) were evaluated for their antiinflammatory effect using different parameters including suppression of intracellular reactive oxygen species (ROS), produced by whole blood phagocytes, produced by neutrophils, and inhibition of nitric oxide (NO) production from J774.2 macrophages. The most active compound also evaluated for cytotoxicity activity. Results: Diclofenac (1) inhibited the ROS with an IC of 3.9 ± 2.8, 1.2 ± 0.0 μg/mL respectively 50 and inhibited NO with an IC₅₀ of 30.01 ± 0.01 μg/mL. Among its derivatives 4, 5, 11, 16, and 20, showed better antiinflammatory potential. The compound 5 was found to be the most potent inhibitor of intracellular ROS as well as NO with IC₅₀ values of 1.9 ± 0.9, 1.7 ± 0.4 μg/mL respectively and 7.13 ± 1.0 μg/mL, respectively, and showed good inhibitory activity than parent diclofenac. The most active compounds were tested for their toxic effect on NIH-3T3 cells where all compounds were found to be non-toxic compared to the standard cytotoxic drug cyclohexamide. Conclusion: Five derivatives were found to be active. Compound 5 was found to be the most potent inhibitor of ROS and NO compared to parent diclofenac 1 and standard drugs ibuprofen and L-NMMA, respectively. The most active compounds 1, 4, 5, 11 and 20 were found to be non-toxic on NIH-3T3 cells. Compound 4, 5, and 20 also showed good antiinflammatory potential, compound 11 and 16 showed moderate and low level of inhibition, respectively.

Full text of DOI:10.2174/1573406414666180321141555

674
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Medicinal Chemistry, 2018, 14, 674-687
RESEARCH ARTICLE
ISSN: 1573-4064
eISSN: 1875-6638
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Factor:
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Diclofenac 1,3,4-Oxadiazole Derivatives; Biology-Oriented Drug Synthesis
(BIODS) in Search of Better Non-Steroidal, Non-Acid Antiinflammatory
Agents
Shazia Shaha,c, Arshiaa, Nida Siraj Kazmia, Almas Jabeena, Aisha Faheemb, Nida Dastagirb, Tariq
Ahmedb, Khalid Mohammed Khana,*, Shakil Ahmedd, Abeer Razaa and Shahnaz Perveene
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Ka-
b
rachi, Karachi-75270, Pakistan; Dr. Panjwani Center for Molecular Medicine and Drug Research, International Cen-
c
ter for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan; Department of Chemistry,
d
Federal Urdu University of Art, Science and Technology, Pakistan; Industrial Analytical Center at H. E. J. Research
Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-
e
75270, Pakistan; PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: Background: Inflammation is defined as the response of immune system cells to dam-
aged or injured tissues. The major symptoms of inflammation include increased blood flow, cellu-
lar influx, edema, elevated cellular metabolism, reactive oxygen species (ROS) nitric oxide (NO)
and vasodilation. This normally protective mechanism against harmful agents when this normal
mechanism becomes dysregulated that can cause serious illnesses including ulcerative colitis,
Crohn’s disease, rheumatoid arthritis, osteoarthritis, sepsis, and chronic pulmonary inflammation.
Method: In this study synthetic transformations on diclofenac were carried out in search of better
non-steroidal antiinflammatory drugs (NSAIDs), non-acidic, antiinflammatory agents. For this
purpose diclofenac derivatives (2-20) were synthesized from diclofenac (1). All derivatives (2-20)
and parent diclofenac (1) were evaluated for their antiinflammatory effect using different parame-
ters including suppression of intracellular reactive oxygen species (ROS), produced by whole
blood phagocytes, produced by neutrophils, and inhibition of nitric oxide (NO) production from
J774.2 macrophages. The most active compound also evaluated for cytotoxicity activity.
A R T I C L E H I S T O R Y
Received: July 31, 2017
Revised: February 14, 2018
Accepted: March 08, 2018
Results: Diclofenac (1) inhibited the ROS with an IC50 of 3.9 ± 2.8, 1.2 ± 0.0 μg/mL respectively
and inhibited NO with an IC50 of 30.01 ± 0.01 μg/mL. Among its derivatives 4, 5, 11, 16, and 20,
showed better antiinflammatory potential. The compound 5 was found to be the most potent in-
hibitor of intracellular ROS as well as NO with IC50 values of 1.9 ± 0.9, 1.7 ± 0.4 μg/mL respecti-
vely and 7.13 ± 1.0 μg/mL, respectively, and showed good inhibitory activity than parent di-
clofenac. The most active compounds were tested for their toxic effect on NIH-3T3 cells where all
compounds were found to be non-toxic compared to the standard cytotoxic drug cyclohexamide.
DOI:
10.2174/1573406414666180321141555
Conclusion: Five derivatives were found to be active. Compound 5 was found to be the most po-
tent inhibitor of ROS and NO compared to parent diclofenac 1 and standard drugs ibuprofen and
L-NMMA, respectively. The most active compounds 1, 4, 5, 11 and 20 were found to be non-toxic
on NIH-3T3 cells. Compound 4, 5, and 20 also showed good antiinflammatory potential, com-
pound 11 and 16 showed moderate and low level of inhibition, respectively.
Keywords: Synthesis, diclofenac, 1,3,4-oxadiazole, antiinflammatory parameters, ROS suppression, and NO inhibition.
1. INTRODUCTION
tion of leucocytes such as neutrophils and macrophages from
blood to damaged cells, irritants and pathogens etc for their
repair and removal [1]. During inflammation, inflammatory
mediators including reactive oxygen and nitrogen species,
leukotrienes, prostaglandins, cytokines etc are released that
increases the vascular permeability and leucocytes migration
to the site of inflammation [2]. The major symptoms of in-
flammation include increased blood flow, cellular influx,
edema, elevated cellular metabolism reactive oxygen species
Inflammation is defined as the response of immune sys-
tem cells to damaged or injured tissues that involve migra-
*Address correspondence to this author at the H. E. J. Research Institute of
Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan; Tel: 0092-21-34824910;
Fax: 0092-21-34819018-9; E-mails: khalid.khan@iccs.edu;
drkhalidhej@gmail.com
1875-6638/18 $58.00+.00
© 2018 Bentham Science Publishers

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 675
Cl
O
OH
O
N
Cl
O
NH
Cl
OH
O
OH
O
O
Aspirin
Diclofenac
Indometacin
O
OH
F
F
H
N
OH
F
O
Flufenamic acid
Ibuprofen
Fig. (1). Reported antiinflammatory drug having acidic moiety.
marketed antiinflammatory drug (diclofenac) were carried
out and evaluate their antiinflammatory potentials. As 1,3,4-
oxadiazole also possess good antiinflammatory activity [23,
24]. To the best of our knowledge only compounds 1-4 were
previously reported while remaining analogs are new.
(ROS) nitric oxide (NO) and vasodilation [3]. This normally
protective mechanism against harmful agents when this
normal mechanism becomes dysregulated that can cause
serious illnesses including ulcerative colitis, Crohn’s disease,
rheumatoid arthritis, osteoarthritis, sepsis, and chronic pul-
monary inflammation [4].
2. MATERIALS AND METHOD
The most favored class of drug in use for controlling
various pathological conditions is the non-steroidal anti-
inflammatory drugs (NSAIDs) [5]. The mode of action of
NSAIDs involves the repression of prostaglandin biosynthe-
sis from arachidonic acid by inhibiting the enzyme cy-
clooxygenases (COXs) [6]. Among the most admired
NSAIDs is diclofenac sodium. This drug is accepted in more
than 120 countries across the world, and is ranked 30th
among the top 200 drugs [7].
Methanol, hydrazine hydrate, carbon disulfide, phenacyl
halide, aryl halides, and potassium hydroxide were pur-
chased from Sigma-Aldrich, USA and used as received
without purification. Diclofenac was donated by a local
pharmaceutical company. TLC plates were pre-coated silica
gel aluminium plates (Kieselgel 60, 254, E. Merck). Visuali-
zation of TLC chromatograms was carried out under UV
light at wavelengths of 254 and 365 nm. Melting points of all
the compounds were determined on Stuart® SMP10 melting
point apparatus and are uncorrected. EI-MS has recorded on
Chemical name of diclofenac is 2-(2, 6-dichloranilino)
phenylacetic acid. Diclofenac has potential biological activi-
ties including antibacterial [8], antitumour [9], anticancer
[10, 11], however, the major use of diclofenac is as an
antiinflammatory drug [11, 12]. The suppressive effect of
diclofenac on ROS produced from rat peritoneal neutrophils
was also reported previously [13]. Unfortunately, all
NSAIDs drugs such as diclofenac, indomethacin, aspirin,
flufenamic acid, phenylbutazone, and ibuprofen have gastro-
intestinal tract side effects which is mainly due to the contact
of open carboxylic moiety (pKa = 3.5-5.5) in these drugs to
gastrointestinal tract [14, 15] (Fig. 1). Therefore, our re-
search group continuously engaged to combat this evil dis-
ease in an efficient manner by synthesizing different func-
tionally oriented molecules and evaluate their antiinflam-
matory activities in past [16-21].
1
a Finnigan MAT-311A (Germany) mass spectrometer. H-
NMR experiments were carried out on Avance Bruker AM
300, 400, and 500 MHz machines. 13C-NMR experiments
were carried on Avance Bruker AM 300, 400 and 500 MHz
instruments at 75, 100, and 125 MHz, respectively. Infrared
(IR) spectra were recorded on JASCO IR-A-302 spectropho-
tometer.
3. EXPERIMENTAL
3.1. Antiinflammatory Assays
3.1.1. Oxidative Burst Inhibition by Chemiluminescence
Technique
Oxidative burst studies using luminol enhanced chemi-
luminescence technique were performed as described by
Mesaik et al. with some modifications [25]. The assay was
performed on whole blood and neutrophils isolated from
blood of healthy human volunteers using luminol as a probe
BIODS science consists of some modification of previ-
ously known drug to reduce its side effect, increase activity
potential etc. In the current study, we used the BIODS ap-
proach [22], syntheses of 1,3,4-oxadiazole derivatives of

676 Medicinal Chemistry, 2018, Vol. 14, No. 7
Shah et al.
and zymosan as an activator. Briefly 25 ꢀL of diluted whole
blood in HBSS++ or 25 ꢀL of isolated neutrophils (1 x 106
cells/mL) were incubated with 25 ꢀL of different concentra-
tions of compounds (1, 10, 25 and 100 ꢀg/mL) each in tripli-
cate in white half area 96 well plates (Costar, NY, USA).
The plates were incubated at 37 °C for 15 min in the thermo-
stat chamber of a luminometer (Labsystems, Helsinki, Fin-
land). 25 ꢀL of (7 x 10-5 M) luminol (Research Organics,
Cleveland, OH, USA) and, 25 ꢀL serum opsonized zymosan
(SOZ) 2 mg/mL (Fluka, Buchs, Switzerland) was then added
into the wells. The plates were then read in luminometer for
50 min and results were recorded as total integral readings as
relative light units (RLU) [25].
3.1.4.2. Diclofenac Methyl Ester (2)
Diclofenac acid (5 g) was dissolved in methanol (80 mL)
with stirring and sulfuric acid (3 mL) was added drop wise
with continuous stirring and the mixture was heated under
reflux for 15 min. After completion of the reaction (TLC
analysis), it was cooled to room temperature and poured onto
crushed ice (250 g) to obtain crystalline solid (2) which was
filtered, washed with water followed by 5% sodium hydro-
gen carbonate solution, finally with water and dried in air.
3.1.4.3. Diclofenac Hydrazide (3)
Diclofenac ester (2 g) was dissolved in 20 mL methanol then
hydrazine hydrate (10 mL) was added while keeping in ice
bath and stirred for 48 h. Progress of the reaction was moni-
tored through TLC. A solid was formed when poured on to
ice, filtered and dried in air.
3.1.2. Nitric Oxide Assay
The mouse macrophage cell line J774.2 (European Col-
lection of Cell Cultures, UK) was cultured in 75 mL flasks
IWAKI (Asahi Techno Glass, Japan) in DMEM Sigma-
Aldrich (Steinheim, Germany) that contained 10% fetal bo-
vine serum GIBCO (NY US) supplemented with streptomy-
cin/penicillin 1%. Flasks were kept at 37 °C in atmosphere
of humidified air containing 5% CO2, cells were seeded in
96-well plate (1 x 106 cells/mL), and were stimulated by 30
ꢀg/mL E. coli lipopolysaccharide (LPS) (DIFCO Laborato-
ries Michigon, USA) and test compounds were added at
three different concentrations (1, 10, 100 ꢀg/mL). The plates
were incubated at 37 °C in 5% CO2 and humidified air for 48
hours. The cell culture supernatants were collected. nitrite
accumulation in J774.2 cell culture supernatant was meas-
ured using the Griess method described previously [26].
3.1.4.4. Diclofenac Oxadiazole-2-thione (4)
To a solution of diclofenac hydrazide (1.0 g in 50 mL of
ethanol), potassium hydroxide (0.5 g) and carbon disulfide
(12 mL) were added drop wise at room temperature and the
reaction was allowed to reflux for 24 h. After completion of
reaction (TLC analysis), a potassium salt of the product was
obtained which was acidified with HCL (conc.) with stirring
condition to obtain a solid product which was filtered,
washed with distilled water, and dry at room temperature.
3.1.4.5. Derivatives of Diclofenac Oxadiazole Thione (5-20)
Diclofenac oxadiazole thione (0.352 g), ethanol (15 mL),
triethyl amine (0.1 mL) and phenacyl halide and other hal-
ides (1 mmol) were added in a round-bottomed flask and
refluxed at 100 °C for 5 h. After completion of the reaction,
mixture was placed in refrigerator to obtain precipitates. The
solid was filtered, washed with water and dried in vacuum.
3.1.3. MTT Cytotoxicity Assay
Cytotoxicity of compounds on NIH-3T3 fibroblast cells
(ATCC, Manassas, USA) was evaluated by using the stan-
dard MTT colorimetric assay. Briefly, 100 ꢀL of 6 ꢁ 104
cells/mL in DMEM supplemented with 10% FBS were
plated into 96-wells flat bottom plate and incubated over-
night at 37°C in 5% CO2. Three different concentrations of
test compound (1, 10 and 100 ꢂg/mL) were added to the
plate in triplicates and incubated for 48 h. 50 ꢂL of 0.5
mg/mL MTT was added to each well and plates were then
further incubated for 4 h. MTT was aspirated and 100 ꢂL of
DMSO was then added to each well. The extent of MTT
reduction to formazan within cells was calculated by measur-
ing the absorbance at 540 nm, using spectrophotometer
(Spectra Max plus, Molecular Devices, CA, USA). The cyto-
toxic activity was recorded as concentration causing 50%
growth inhibition (IC50) for 3T3 cells [27].
4. RESULT AND DISCUSSION
4.1. Chemistry
Diclofenac derivative was synthesized by different steps
under refluxing conditions. The diclofenac (1) in the pres-
ence of methanol and sulphuric acid was converted into
methyl ester 2 and then this ester converted hydrazide 3 with
hydrazine hydrate in methanol. Resulting hydrazide 3 was
transformed to oxadiazole derivative 4 in carbon disulfide
under basic conditions. Final products 5-20 were obtained by
reaction of oxadiazole derivative with different phenacyl
halides and aryl halides in the presence of ethanol and
triethyl amine (Scheme 1). Chemical structures of all of
these derivatives were established by H-NMR, 13C-NMR,
1
3.1.4. General Procedure
3.1.4.1. Diclofenac (1)
EI-MS, and IR spectroscopy.
4.2. Antiinflammatory Activity of Diclofenac (1) and its
Derivatives
Diclofenac sodium (5 g) was dissolved in water with stir-
ring followed by the addition of 2 mL of 2 N HCl drop wise
to obtain the precipitate of diclofenac acid then addition of
ice water for the termination of reaction. A solid was formed,
filtered, washed with cold water and dry at room tempera-
ture. The structure was identified by using spectroscopic
techniques including, 1H-, 13C-NMR, EI-MS, ESI-MS,
HRMS, and IR.
Diclofenac 1 and all derivatives (Fig. 2) were initially
tested to check their inhibitory potential on intracellular reac-
tive oxygen species (ROS) produced from human whole
blood phagocytes at single concentration of 25 ꢀg/mL. The
compounds showing inhibitory potential were then tested on
two different parameters of inflammation. The active com-

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 677
5
Cl
6
5
Cl
5
4
6
Cl
6
CH3OH
4
CS2, KOH
NH2-NH2.H2O
O Me
4
3
1
3
1
3
1
Conc. H2SO4
OH
NH
12
2
NH
12
2
NH
12
13
2
Cl
14
13
14
Cl
13
7
Cl
14
11
10
NHNH2
7
11
10
7
11
10
8
O
8
O
8
(91)
O
9
9
(2)
(3)
5
5
Cl
6
Cl
6
1
4
4
3
1
3
R-X
NH
12
2
NH
12
N
N
2
N
N
15
13
R
13
Cl
Cl
SH
7
S
7
14
O
14
15
O
8
10
8
10
9
9
(4)
Scheme 1. Syntheses of oxadiazole derivatives of dicleofenac.
(5-20)
Fig. (2). Structure-activity relationship of diclofenac and oxadiazole 4 and compound 5.
pounds were further evaluated for their inhibitory effect on
the production of ROS from isolated neutrophils and on ni-
tric oxide (NO) generated from lipopolysaccharide (LPS)
stimulated J774.2 macrophages (Table 1).
into ester 2 and hydrazide 3 the activity was found to be di-
minished. Conversion of hydrazide 3 into its oxadiazole de-
rivative 4 it regains the inhibitory activity on ROS f 3.1.4.1.
rom whole blood and neutrophils with IC50 values of 5.7
1.5 and 6.7 0.7 ꢀg/mL, respectively. However potent inhi-
bition of NO IC50 9.34 0.8 ꢀg/mL was observed as compa-
red to parent 1 as well as standard L-NMMA. Conversion of
4 into 5 gave a most potent inhibitior of ROS as well as NO
of this series having an IC50 of value 1.9 0.9 and 1.7 0.4
ꢀg/mL on whole blood and neutrophils, respectively, and
7.13 1.0 ꢀg/mL on nitric oxide (NO) (Fig. 3).
In this study diclofenac 1 inhibited the ROS with an IC50
value 3.9 2.8 and 1.2 0.0 ꢀg/mL from human whole blo-
od cells and isolated neutrophils, respectively. It also mode-
rately inhibited NO with an IC50 value of 30.1 0.0 ꢀg/mL
compared to the standard L-NMMA IC50 24.2 0.8 ꢀg/mL.
However, when the acid group of diclofenac was converted

678 Medicinal Chemistry, 2018, Vol. 14, No. 7
Shah et al.
Fig. (3). Structure-activity relationship of phenacyl analogs of substituted oxadiazole dicleofenac 5, 11, and 16.
The oxadiazole derivative of diclofenac 4 reacts with dif-
ferents phenacyl halide to afford compounds 5-17. Phenacyl
derivative of 1,3,4-oxadiazole diclofenac 5 was found to be
most active compound which showed superior antiinflam-
matory activity than parent diclofenac 1 as well as standard
such as ibuprofen which is used for ROS inhibition having
an IC50 values of 11.2 1.9 and 2.5 0.6 ꢀg/mL for whole
blood and isolated neutrophils, respectively, and from L-
NMMA which was used as standard for nitric oxide (NO)
inhibition IC50 = 24.2 0.8 ꢀg/mL. Compound 11, a substi-
tuted phenacyl derivative of oxadiazole having a bromo resi-
due at position 3 showed moderate level of inhibition having
an IC50 value of 21.7 11.0, 5.0 0.9 and 14.8 2.0 ꢀg/mL
for whole blood, neutrophils ROS, and nitric oxide inhibiti-
on, respectively. While compound 10 having a bromo group
at postion 4 was found to be inactive. Hydroxy substituted
phenacyl derivative 16 showed low level of inhibition on
whole blood ROS and neutrophil ROS IC50 = 31.2 5.2, 9.0
1.0 ꢀg/mL respectively. Where as moderate inhibition of
nitric oxide IC50 = 26.2 3.5 ꢀg/mL was observed (Fig. 4).
The cytotoxicites of most active derivatives 1, 4, 5, 11 and
20 were tested on NIH-3T3 cells where all compounds were
found to be non-toxic as compared to the cyclohexamide
which was used as a standard cytotoxic drug (Table 2).
Chloro, nitro, methoxy and phenyl substituted phenacyl
oxadiazoles 7, 8, 9, 12-15, and 17, respectively were found
to be inactive. 1,3,4-Oxadiazole diclofenac 4 was also reac-
ted with varyingly substituted aryl halide and afforded
dcompounds 18, 19, and 20. Among them only compound 20
having a 2-bromo benzyl substituent showed good activity
with IC50 values of 4.5 1.7, 3.4 0.5, and 21.9 1.15
ꢀg/mL for whole blood, neutrophil ROS and NO inhibition,
respectively. While chloro substituted derivatives 18 and 19
were found to be inactive (Figs. 5, 6, 7).
5. SPECTROSCOPIC DATA OF SYNTHETIC COM-
POUNDS
5.1. 2-(2-(2,6-Dichlorophenylamino)phenyl)acetic Acid
(1)
M.p. 152-154°C (lit. 153-154°C [28]); Rf: 0.31 (ethyl a-
cetate/hexanes, 2:8); 1H-NMR (400 MHz, DMSO-d6): ꢁ 12.7
(s, 1H, OH), 7.52 (d, 2H, J(3,4),(5,4) = 7.8 Hz, H-3, H-5),

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 679
Fig. (4). Structure-activity relationship of benzyl analogs of oxadiazole dicleofenac 20.
Fig. (5). 1,3,4-Oxadiazole derivative of dicleofenac.
Table 1. Anti-inflammatory activity of diclofenac (1) and its derivatives (2-20).
ROS Inhibition
ROS Inhibition
NO Inhibition
IC50 SD
(ꢀg/mL)
Compound
Structure
Whole Blood
Neutrophils
IC50 SD (ꢀg/mL)
IC50 SD (ꢀg/mL)
1
3.9 2.8
1.2 0.0
30.0 0.0
2
NAb
NDc
NDc
Table 1. contd…

680 Medicinal Chemistry, 2018, Vol. 14, No. 7
Shah et al.
ROS Inhibition
Whole Blood
IC50 SD (ꢀg/mL)
ROS Inhibition
Neutrophils
IC50 SD (ꢀg/mL)
NO Inhibition
Compound
Structure
IC50 SD
(ꢀg/mL)
3
NAb
NDc
NDc
4
5
5.7 1.5
6.7 0.7
9.34 0.8
7.13 1.0
1.9 0.9
1.7 0.4
6
7
8
NAb
NAb
NAb
NDc
NDc
NDc
NDc
NDc
NDc
9
NAb
NAb
NDc
NDc
NDc
NDc
10
11
21.7 11.0
4.9 0.9
14.8 2.0
12
NAb
NDc
NDc
Table 1. contd…

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 681
ROS Inhibition
Whole Blood
ROS Inhibition
NO Inhibition
IC50 SD
Compound
13
Structure
Neutrophils
IC50 SD (ꢀg/mL)
IC50 SD (ꢀg/mL)
(ꢀg/mL)
NAb
NDc
NDc
14
15
16
NAb
NAb
NDc
NDc
NDc
NDc
31.2 5.2
9.0 1.0
26.2 3.5
17
18
19
20
NAb
NAb
NDc
NDc
NDc
NDc
NAb
NDc
NDc
4.5 1.7
3.4 0.5
21.9 1.1
SDa means Standard deviation; NAb = Not Active; NDc = Not determined; Standard: Ibuprofen, (whole blood) IC50 = 11.2 1.9 ꢀg/mL; (neutrophil) IC50 = 2.5
0.6 ꢀg/mL; NG monomethyl L-arginine acetate (L-NMMA) nitric oxide IC50= 24.2 0.8.
Table 2. Cytotoxicity of active compounds on NIH-3T3 cells. The IC50 values were calculated using three doses (1, 10, 100 ꢀg/mL)
of each compound. Values are expressed as mean SD of experiments done in triplicate. Results were compared with
cyclohexamide as a standard cytotoxic drug.
Compounds
IC50 SDa (ꢀg/mL)
1
4
29.8 0.4
32.0 0.6
Table 2. contd…

682 Medicinal Chemistry, 2018, Vol. 14, No. 7
Shah et al.
Compounds
IC50 SDa (ꢀg/mL)
5
76.7 6.0
>100
11
20
>100
Cyclohexamide
0.13 0.02
SDa means Standard deviation.
ꢅꢃꢀ8ꢀ
ꢅꢂꢀ8ꢀ
ꢅꢁꢀ8ꢀ
ꢅꢀꢀ8ꢀ
ꢄꢀ8ꢀ
ꢃꢀ8ꢀ
ꢂꢀ8ꢀ
ꢁꢀ8ꢀ
ꢀ8ꢀ
ꢅꢀꢀꢉj$LIꢋ
ꢅꢀꢉj$LIꢋ
ꢅꢉj$LIꢋ
ꢅ
ꢂ
ꢅꢅ
ꢅꢃ
ꢁꢀ
ꢆQJꢇ`QC
ꢆQI]Q%JRꢈ
Fig. (6). The graph represents the inhibitory effect of compounds on oxidative burst. Compounds were tested at three different concentrations
(1, 10 and 100 ꢀg/mL). Results are presented in relative light units (RLU) and oxidative burst activity of whole blood phagocytes using lumi-
nol as a probe. Each vertical bar represents a mean of triplicate. Results were compared to the control. Where Control is zymosan activated
whole blood phagocytes with no drug.
50 mg/mL 5 mg/mL 0.5 mg/mL
70
60
50
40
30
20
10
0
1
4
5
11
16
20
Control
Blank
Compounds
Fig. (7). The graph represents the inhibitory effect of compounds on Nitric oxide (NO) production from mouse macrophage J774.2 cell line.
Compounds were tested at three different concentrations (0.5, 5 and 50 ꢀg/mL). Each vertical bar represents a mean of triplicate. Results were
compared to the control. Where Control = Cells activated by lipopolysaccharide (LPS) with no drug and Blank = Cells without LPS.
7.23 (s, 1H, NH), 7.20 (m, 2H, H-10, H-11), 7.07 (t, 1H,
(4/3,5) = 8.0 Hz, H-4), 6.86 (t, 1H, J(9/10,11) = 8.0 Hz, H-9),
(C-O); Anal. Calcd for C14H11Cl2NO2: C, 56.78; H, 3.74; Cl,
23.94; N, 4.73; O, 10.80; Found: C, 56.77; H, 3.73; N, 4.72.
J
6.28 (d, 1H, J (8,9) = 8.0 Hz, H-8), 3.68 (s, 2H, CH2); EI-MS:
m/z (rel. abund. %), 298 [M+2]+ (29.4.7), 296 [M]+ (18.5),
278 (4.7), 242 (40.0), 214 (100.0), 179 (26.3), 151 (12.5),
109 (6.9); HREI-MS: m/z Calcd for C14H11Cl2NO2 [M]+
296.1486, Found 296.1485; IR (KBr, cm-1): 3347 (O-H),
3362 (N–H), 1670 (C=O), 1569 (C=C), 1259 (C-N), 1180
5.2. Methyl 2-(2-(2,6-dichlorophenylamino)phenyl)ace-
tate (2)
Yield: 96 %; m.p. 98-100°C (lit. 97-99°C [28]); Rf: 0.36
1
(ethyl acetate/hexanes, 2:8); H-NMR (400 MHz, DMSO-

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 683
d6): ꢀ 7.53 (d, 2H, J(3,4),(5,4) = 8.0 Hz, H-3, H-5), 7.22 (m, 2H,
H-9, H-10), 7.06 (d, 1H, J (11,10) = 6.0 Hz, H-11), 7.03 (t, 1H,
NH), 7.06 (t, 1H, J(4/3,5) = 7.5 Hz, H-4) 6.83 (t, 1H, J(9/8,10) =
7.5 Hz, H-9), 6.19 (d, 1H, J(8,9) = 8.0 Hz, H-8), 5.05 (s, 2H,
16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z: 470.0 (M+);
HR-ESI-MS m/z Calcd for C23H17Cl2N3O2S [M]+ 470.3710,
found 470.3711; IR (KBr, cm-1): 3214 (N-H), 1586 (C=N),
1509 (C=C), 1489 (C=S), 1170 (C-O); Anal. m/z Calcd
C23H17Cl2N3O2S: C, 58.73; H, 3.64; Cl, 15.07; N, 8.93; O,
6.80; S, 6.82 Found: C, 58.72; H, 3.63; N, 8.92.
J
(4/3,5) = 7.6 Hz, H-4), 6.25 (d, 1H, J (8,9) = 8.0 Hz, H-8), 3.77
(s, 2H, CH2), 3.64 (s, 3H, OCH3); EI-MS: m/z (rel. abund.
%), 311 [M+2]+ (24.7), 309 [M]+ (36.7), 277 (9.6), 242
(39.2), 214 (100.0), 179 (10.2), 151 (7.6); HREI-MS: m/z
calcd for C15H13Cl2NO2 [M]+ 309.0323, Found 309.0322; IR
(KBr, cm-1): 3382 (N-H), 1660 (C=O), 1579 (C=N), 1569
(C=C), 1259 (C-N), 1170 (C-O); Anal. Calcd for
C15H13Cl2NO2: C, 58.08; H, 4.22; Cl, 22.86; N, 4.52; O,
10.32; Found: C, 58.07; H, 4.21; N, 4.51.
5.6. 3-(3,4-Dichlorobenzyl)-5-(2-(2,6-dichlorophenylami-
no)benzyl)-1,3,4-oxadiazole-2(3H)-thione (6)
Yield: 91%; m.p 120-122°C; Rf: 0.51 (ethyl aceta-
5.3. 2-(2-(2,6-Dichlorophenylamino)phenyl)acetohydra-
zide (3)
1
te/hexane, 2:8) H-NMR (400 MHz, DMSO-d6): ꢀ 7.68 (d,
1H, J(18, 21) = 1.2 Hz, H-18), 7.52 (s, 1H, NH), 7.50 (d, 2H,
(3,4),(5,4) = 8.0 Hz, H-3, H-5), 7.36 (dd, 1H, J(22,18) = 1.2 Hz
(22,21) = 8.4 Hz, H-22) 7.23 (t, 1H, J(4/3,5) = 8.0 Hz, H-4), 7.16
Yield: 97%; m.p. 132-134°C C (lit. 134-136°C [29]); Rf:
J
J
1
0.35 (ethyl acetate/hexanes, 2:8); H-NMR (400 MHz, DM-
SO-d6): ꢀ 9.46 (s, 1H, NH), 9.51 (s, 1H, NH), 7.51 (d, 2H,
(d, 1H, J(11, 10) = 7.6 Hz, H-11), 7.09 (m, 2H, H-8, H-10),
6.85 (t, 1H, J(9/8,10) = 7.6 Hz, H-9), 6.19 (d, 1H, J(21,22) = 8.1
Hz, H-21), 4.44 (s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2); 13C-
NMR: (100 MHz, DMSO-d6): ꢀ 166.5 (C=S), 162.4 (C-14),
142.9 (C-2, C-6), 138.1 (C-17), 136.8 (C-19), 131.5 (C-20),
130.9 (C-18), 130.8 (C-21), 130.6 (C-22), 130.5 (C-3, C-5),
130.2 (C-7), 129.2 (C-1), 129.1 (C-11), 128.2 (C-9), 126.4
(C-10), 122.2 (C-8), 120.4 (C-12), 115.5 (C-4), 34.2 (13-
CH2), 27.3 (16-CH2); ESI-MS m/z: 511.0 (M+); HR-ESI-MS
m/z Calcd for C22H15Cl4N3OS [M]+ 515.2510, Found
511.2511; IR (KBr, cm-1): 3211 (N-H), 1545 (C=N), 1543
(C=C), 1430 (C=S), 1187 (C-O); Anal. m/z Calcd
C22H15Cl4N3OS: C, 51.68; H, 2.96; Cl, 27.74; N, 8.22; O,
3.13; S, 6.27, Found: C, 51.67; H, 2.95; N, 8.21.
J
(3,4),(5,4) = 8.4 Hz, H-3, H-5), 7.16 (m, 2H, H-10, H-11), 7.04
(t, 1H, J(4/3,5) = 7.6 Hz, H-4), 6.85 (t, 1H, J(9/10,11) = 7.6 Hz, H-
9), 6.29 (d, 1H, J (8,9) = 8.0 Hz, H-8), 4.30 (s, 2H, CH2), 3.50
(s, 2H, NH2); 13C-NMR: (100 MHz, DMSO-d6): ꢀ , 170.7
C
(C=O), 142.9 (C-1), 137.1 (C-7), 130.2 (C-3, C-5),), 129.2
(C-3, C-5), 127.1 (C-8), 125.3 (C-9), 124.8 (C-10), 120.6 (C-
10), 116.0 (C-4), 37.6 (CH2); EI-MS: m/z (rel. abund. %),
311 [M+2] + (2.4), 309 [M]+ (3.8), 278 (58.2), 242 (17.2),
214 (100.0), 179 (13.0), 151 (6.9); HREI-MS: m/z Calcd for
C14H13Cl2N3O [M]+ 309.0436, Found 309.0435; IR (KBr,
cm-1): 3387 (N-H), 1670 (C=O), 1599 (C=N), 1560 (C=C),
1299 (C-N); Anal. Calcd for C14H13Cl2N3O: C, 54.21; H,
4.22; Cl, 22.86; N, 13.55; O, 5.16; Found: C, 54.20; H, 4.21;
N, 13.54.
5.7. 1-(4-Chlorophenyl)-2-(5-(2-(2,6-dichlorophenylami-
no)benzyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)ethanone
5.4. 5-(2-(2,6-Dichlorophenylamino)benzyl)-1,3,4-oxadia-
zole-2-thiol (4)
(7)
Yield: 97%; m.p. 160-162°C (lit. 162-164°C [30]); Rf:
Yield: 91%; m.p 118-120°C; Rf: 0.50 (ethyl aceta-
1
1
0.38 (ethyl acetate/hexanes, 2:8); H-NMR (400 MHz, DM-
te/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): ꢀ 8.03 (d,
SO-d6): ꢀ 14.28 (s, 1H, SH), 7.52 (d, 2H, J(3,4),(5,4) = 8.4 Hz,
H-3, H-5), 7.23 (m, 2H, H-10, H-11), 7.12 (s, 1H, NH), 7.09
(t, 1H, J(4/3,5) = 6.9 Hz, H-4), 6.86 (t, 1H, J(9/10,11) = 7.2 Hz, H-
9), 6.19 (d, 1H, J (8,9) = 8.0 Hz, H-8), 4.24 (s, 2H, CH2); 13C-
NMR: (100 MHz, DMSO-d6): ꢀ 177.8 (C-14), 162.5 (C-13),
143.1 (C-7), 136.9 (C-1), 131.8 (C-1), 131.0 (C-2, C-6),
129.1 (C-3, C-5), 128.3 (C-4), 126.4 (C-8), 121.3 (C-12),
120.3 (C-9), 115. (C-10), 27.6 (CH2); EI-MS: m/z (rel. a-
bund. %), 353 [M+2]+ (24.3), 351 [M]+ (32.6), 291 (63.6),
278 (54.6), 256 (19.6), 214 (100.0), 131 (35.4); HREI-MS:
m/z Calcd for C15H11Cl2N3OS [M]+ 352.2383, Found
352.2382; IR (KBr, cm-1): 3367 (N-H), 2979 (S-H), 1579
(C=N), 1500 (C=C), 1294 (C-N), 1286 (C=S); Anal. Calcd
for C15H11Cl2N3OS: C, 51.15; H, 3.15; Cl, 20.13; N, 11.93;
O, 4.54; S, 9.10; Found: C, 51.14; H, 3.14; N, 11.92.
2H, J(20,19),(22,23) = 8.4 Hz, H-20, H-22), 7.64 (d, 2H,
J
(19,20),(23,22) = 8.4 Hz, H-19, H-23), 7.53 (d, 2H, J(3,4),(5,4) = 8.1
Hz, H-3, H-5), 7.24 (t, 1H, J(10/9,11) = 7.5 Hz, H-10), 7.16 (d,
1H, J (11,10) = 7.5 Hz, H-11), 7.12 (s, 1H, NH), 7.08 (t, 1H,
J
(4/3,5) = 6.9 Hz, H-4), 6.84 (t, 1H, J(9/8,10) = 6.0 Hz, H-9), 6.21
(d, 1H, J (8,9) = 8.1 Hz, H-8), 5.03 (s, 2H, 16-CH2), 4.35 (s,
2H, 13-CH2); ESI-MS m/z: 504.0 (M+); HR-ESI-MS m/z
Calcd for C23H16Cl3N3O2S [M]+ 504.8160, Found 504.8161;
IR (KBr, cm-1): 3221 (N–H), 1678 (C=O), 1562 (C=N), 1528
(C=C),1423 (C=S), 1026 (C–O); Anal. m/z Calcd for for for
C23H16Cl3N3O2S: C, 54.72; H, 3.19; Cl, 21.07; N, 8.32; O,
6.34; S, 6.35 Found: C, 54.71; H, 3.18; N, 8.33.
5.8. 1-(3,4-Dichlorophenyl)-2-(5-(2-(2,6-dichlorophenyl-
amino)benzyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)ethano-
ne (8)
5.5. 2-(5-(2-(2,6-Dichlorophenylamino)benzyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)-1-phenylethanone (5)
Yield: 94%; m.p 178-180°C; Rf: 0.52 (ethyl aceta-
te/hexanes, 2:8)1H-NMR (400 MHz, DMSO-d6): ꢀ 8.22 (d,
1H, J(19,22) = 1.6 Hz, H-19), 7.96 (dd, 1H, J(22,19) = 2.0 Hz
Yield: 95%; m.p 128-130°C; Rf: 0.55 (ethyl aceta-
1
te/hexanes, 2:8); H-NMR (500 MHz, DMSO-d6): ꢀ 8.01 (d,
J(22,23) = 8.4 Hz, H-22), 7.84 (d, 1H, J(23,22) = 8.4 Hz, H-23),
2H, J(3,4),(5,4) = 7.5 Hz, H-3, H-5), 7.70 (t, 1H, J(10/9,11) = 7.5
Hz, H-10) 7.57 (t, 2H, J(19/18,20), (21/20,22) = 7.5 Hz, H-19, H-
21), 7.52 (d, 2H, J(18,19),(22,21) = 7.5 Hz, H-18, H-22) 7.23 (t,
1H, J(20/19,21) = 8.0 Hz, H-20) 7.15 (m, 1H, H-11), 7.14 (s, 1H,
7.52 (d, 2H, J(3,4),(5,4) = 8.0 Hz, H-3, H-5), 7.23 (t, 1H, J(10/9,11)
= 8.0 Hz, H-10), 7.16 (d, 1H, J(11,10) = 7.6 Hz, H-11), 7.11 (s,
1H, NH), 7.07 (t, 1H, J(4/3,5) = 8.0 Hz, H-4), 6.83 (t, 1H,
J
(9/8,10) = 7.6 Hz, H-9), 6.19 (d, 1H, J (8,9) = 8.0 Hz, H-8), 5.03

684 Medicinal Chemistry, 2018, Vol. 14, No. 7
Shah et al.
(s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z: 539.0
(M+); HR-ESI-MS m/z Calcd for C23H15Cl4N3O2S [M]+
539.2611, Found 539.2610; IR (KBr, cm-1): 3265 (N-H),
1629 (C=O), 1532 (C=N), 1576 (C=C), 1425 (C=S), 1070
(C-O); Anal. m/z Calcd for for for C23H15Cl4N3O2S: C,
51.23; H, 2.80; Cl, 26.30; N, 7.79; O, 5.93; S, 5.95 Found: C,
51.23; H, 2.82; N, 7.78.
1545 (C=N), 1540 (C=C), 1463 (C=S), 1165 (C-O); Anal.
m/z Calcd C24H18BrCl2N3O2S: C, 51.17; H, 3.22; Br, 14.19;
Cl, 12.59; N, 7.46; O, 5.68; S, 5.69 Found: C, 51.16; H, 3.21;
N, 7.45.
5.12. 2-(5-(2-(2,6-Dichlorophenylamino)benzyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)-1-(3-nitrophenyl)ethanone (12)
Yield: 92%; m.p 123-125°C; Rf: 0.50 (ethyl aceta-
5.9. 3-(4-Chlorobenzyl)-5-(2-(2,6-dichlorophenylamino)
1
te/hexanes, 2:8); H-NMR (400 MHz, DMSO-d6): ꢀ 8.70(s,
benzyl)-1,3,4-oxadiazole-2(3H)-thione (9)
1H, H-23), 8.52 (d, 1H, J (21,20) = 8.4 Hz, H-21), 8.44 (d, 1H,
(19,20) = 8.0 Hz, H-19), 7.88 (t, 1H, J(20/19,21) = 8.0 Hz, H-20),
Yield: 90%; m.p 110-112°C; Rf: 0.51 (ethyl aceta-
J
1
te/hexanes, 2:8); H-NMR (500 MHz, DMSO-d6): ꢀ 7.23 (d,
7.52 (d, 2H, J(3,4),(5,4) = 7.5 Hz, H-3, H-5), 7.23 t, 1H, J(10/9,11)
= 8.0 Hz, H-10), 7.16 (d, 1H, J (11,10) = 7.6 Hz, H-11 ), 7.12
(s, 1H, NH), 7.07 (t, 1H, J(4/3,5) = 8.0 Hz, H-4), 6.83 (t, 1H,
2H, J(3,4),(5,4) = 8.0 Hz, H-3, H-5), 7.19 (d, 2H, J(18,19),(21,22)
=
8.0 Hz, H-18, H-21) 7.37 (t, 1H, J(20/21,22) = 7.5 Hz, H-20),
7.23 (m, 2H, H-10, H-11) 7.12 (s, 1H, NH), 7.08 (t, 1H,
J
(9/8,10) = 7.6 Hz, H-9), 6.19 (d, 1H, J (8,9) = 8.0 Hz, H-8) 5.14
J
(4/3,5) = 8.0 Hz, H-4) 6.86 (t, 1H, J(9/8,10) = 7.5 Hz, H-9), 6.21
(s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z: 515.0
(M+); HR-ESI-MS m/z Calcd for C23H16Cl2N4O4S [M]+
515.3685, Found 515.3684; IR (KBr, cm-1): 3215 (N–H),
1692 (C=O), 1572 (C=N), 1579 (C=C), 1423 (C=S), 1041
(C-O); Anal. m/z Calcd for for C23H16Cl2N4O4S: C, 53.60; H,
3.13; Cl, 13.76; N, 10.87; O, 12.42; S, 6.22, Found: C,
53.62; H, 3.12; Cl, N, 10.88.
(d, 1H, J(8,9) = 7.5 Hz, H-8), 4.42 (s, 2H, 16-CH2), 4.35 (s,
2H, 13-CH2); 13C-NMR: (75 MHz, DMSO-d6): ꢀ , 166.4
C
(C=S), 142.9 (C-14), 136.8 (C-2, C-6), 135.8 (C-20), 132.2
(C-17), 131.5 (C-7), 130.7 (C-12), 130.5 (C-19, C-21), 129.1
(C-18, C-22), 128.4 (C-3, C-5), 128.1 (C-9), 126.4 (C-8),
122.2 (C-9), 120.4 (C-10), 34.8 (13-CH2), 27.2 (16-CH2);
ESI-MS m/z: 476.0 (M+); HR-ESI-MS m/z Calcd for
C22H16Cl3N3OS [M]+ 476.8059, Found 476.8058; IR (KBr,
cm-1): 3210 (N-H), 1546 (C=N), 1548 (C=C), 1436 (C=S),
1187 (C–O); Anal. m/z Calcd C22H16BrCl2N3OS: C, 55.42;
H, 3.38; Cl, 22.31; N, 8.81; O, 3.36; S, 6.72 Found: C, 55.41;
H, 3.36; N, 8.80.
5.13. 5-(2-(2,6-Dichlorophenylamino)benzyl)-3-(2-(4-ni-
trophenyl)-2-thioxoethyl)-1,3,4-oxadiazol-2(3H)-one (13)
Yield: 90%; m.p 135-137°C; Rf: 0.49 (ethyl aceta-
1
te/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): ꢀ 8.37 (d,
2H, J(20,19),(22,23) = 9.0 Hz, H-20, H-22), 8.24 (d, 2H,
5.10. 1-(4-Bromophenyl)-2-(5-(2-(2,6-dichlorophenylami-
no)benzyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)ethanone
(10)
J
(19,20),(23,22) = 8.7 Hz, H-19, H-23) 7.52 (d, 2H, J(3,4),(5,4) = 8.0
Hz, H-3, H-5) 7.24 (m, 2H, H-10, H-11), 7.11 (s, 1H, NH),
7.08 (t, 1H, J(4/3,5) = 6.9 Hz, H-4), 6.84 (t, 1H, J(9/8,10) = 7.5
Hz, H-9), 6.20 (d, 1H, J(8, 9) = 8.1 Hz, H-8), 5.10 (s, 2H, 16-
CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z: 515.0 (M+); HR-
ESI-MS m/z Calcd for C23H16Cl2N4O4S [M]+ 515.3685,
Found 515.3684; IR (KBr, cm-1): 3215 (N-H), 1689 (C=O),
1557 (C=N), 1545 (C=C),1434 (C=S), 1180 (C–O); Anal.
m/z Calcd for C23H16Cl2N4O4S: C, 53.60; H, 3.13; Cl, 13.76;
N, 10.87; O, 12.42; S, 6.22, Found: C, 53.62; H, 3.11; N,
10.86.
Yield: 92%; m.p 148-150°C; Rf: 0.53 (ethyl aceta-
1
te/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): ꢀ 7.95 (d,
2H, J(23,22),(19,20) = 8.4 Hz, H-23, H-19), 7.78 (d, 2H,
J(20,19),(22,23) = 8.4 Hz, H-20, H-22), 7.53 (d, 2H, J(3,4),(5,4) = 8.1
Hz, H-3, H-5), 7.24 (t, 1H, J(10/9,11) = 8.2 Hz, H-10), 7.16 (m,
2H, H-11, NH), 7.08 (t, 1H, J(4/3,5) = 7.5 Hz, H-4), 6.84 (t,
1H, J(9/8,10) = 7.2 Hz, H-9), 6.20 (d, 1H, J (8,9) = 8.0 Hz, H-8),
5.02 (s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z:
549.0 (M+); HR-ESI-MS m/z Calcd for C23H16BrCl2N3O2S
[M]+ 549.2670, Found 549.2671; IR (KBr, cm-1): 3285 (N-
H), 1689 (C=O), 1552 (C=N), 1568 (C=C), 1445 (C=S),
1090 (C-O); Anal. m/z Calcd for C23H16BrCl2N3O2S: C,
50.29; H, 2.94; Br, 14.55; Cl, 12.91; N, 7.65; O, 5.83; S,
5.84 Found: C, 50.28; H, 2.92; N, 7.62.
5.14. 2-(5-(2-(2,6-Dichlorophenylamino)benzyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)-1-(4-methoxyphenyl)ethanone
(14)
Yield: 91%; m.p 130-132°C; Rf: 0.51 (ethyl aceta-
1
te/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): ꢀ 8.00 (d,
5.11. 1-(3-Bromophenyl)-3-(5-(2-(2,6-dichlorophenylami-
no)benzyl)-2-thioxo-1,3,4-oxadiazol-3(2H)-yl)propan-1-
one (11)
2H, J(19,20),(23,22) = 8.8 Hz, H-19, H-23), 7.53 (d, 2H,
J
(20,21),(22,23) = 8.0 Hz, H-20, H-23), 7.23 (t, 1H, J(9/8,10) = 8.0
Hz, J(10/9,11) = 8.0 Hz, H-9, H-10), 7.16 (d, 2H, J(3,4),(5,4) = 8.4
Hz, H-3, H-5), 7.07 (d, 1H, J (11,10) = 7.5 Hz, H-11), 7.04 (s,
1H, NH), 6.84 (t, 1H, J(4/3,5) = 6.9 Hz, H-4), 6.20 (d, 1H, J(8,9)
= 8.0 Hz, H-8), 4.99 (s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2);
Yield: 94%; m.p 134-136°C; Rf: 0.52 (ethyl aceta-
1
te/hexanes, 2:8); H-NMR (300 MHz, DMSO-d6): ꢀ 8.15 (s,
1H, H-23), 8.01 (d, 1H, J(21,20) = 9.8 Hz, H-21), 7.90 (d, 1H,
13C-NMR: (75 MHz, DMSO-d6): ꢀ , 190.6 (C=O), 166.2
C
J(19,20) = 9.8 Hz, H-19), 7.55 (m, 3H, H-3, H-5, H-10), 7.24
(C=S), 163.6 (C-14), 162.9 (C-2, C-6), 142.8 (C-1), 136.9
(C-19, C-23), 131.5 (C-20, C-22), 130.7 (C-3, C-5), 130.4
(C-7), 129.1 (C-18), 128.1 (C-21), 127.8 (C-12), 126.3 (C-
4), 122.2 (C-8), 120.4 (C-9), 115.5 (C-10), 114.0 (C-4), 55.6
(OCH3), 40.9 (13-CH2), 27.1 (C-16); ESI-MS m/z: 499.0
(M+); HR-ESI-MS m/z Calcd for C24H19Cl2N3O3S [M]+
(t, 1H, J(20/19,21) = 8.1 Hz, H-20), 7.16 (m, 1H, H-10), 7.12 (s,
1H, NH), 7.08 (t, 1H, J(4/3,5) = 7.5 Hz, H-4) 6.84(t, 1H, J(9/8,10)
= 7.5 Hz, H-9), 6.21 (d, 1H, J(8,9) = 7.8 Hz, H-8), 5.04 (s, 2H,
16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z: 476.0 (M+);
HR-ESI-MS m/z Calcd for C22H16Cl3N3OS [M]+ 476.8059,
Found 476.8058; IR (KBr, cm-1): 3212 (N-H), 1650 (C=O),

Diclofenac 1,3,4-Oxadiazole Derivatives
Medicinal Chemistry, 2018, Vol. 14, No. 7 685
499.0524, Found 499.0522; IR (KBr, cm-1): 3221 (N-H),
1678 (C=O), 1562 (C=N), 1528 (C=C),1423 (C=S), 1026
(C-O); Anal. m/z Calcd for for for C24H19Cl2N3O3S; C,
57.61; H, 3.83; Cl, 14.17; N, 8.40; O, 9.59; S, 6.41 Found: C,
57.60; H, 3.82; N, 8.41.
C29H21Cl2N3O2S: C, 63.74; H, 3.87; Cl, 12.98; N, 7.69; O,
5.86; S, 5.87 Found: C, 63.73; H, 3.86; N, 7.68.
5.18. 3-(2,4-Dichlorobenzyl)-5-(2-(2,6-dichlorophenylami-
no)benzyl)-1,3,4-oxadiazole-2(3H)-thione (18)
Yield: 92%; m.p 128-130°C; Rf: 0.50 (ethyl aceta-
1
5.15. 2-(5-(2-(2,5-Dichlorophenylamino)benzyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)-1-(3-methoxyphenyl)ethan-1-
one (15)
te/hexanes, 2:8); H-NMR (500 MHz, DMSO-d6): ꢀ 7.23 (d,
2H, J(3,4),(5,4) = 8.0 Hz, H-3, H-5), 7.19 (d, 2H, J(18,19),(21,22)
=
8.0 Hz, H-18, H-21) 7.37 (t, 1H, J(20/21,22) = 7.5 Hz, H-20),
7.23 (m, 2H, H-10, H-11) 7.12 (s, 1H, NH), 7.08 (t, 1H,
(4/3,5) = 8.0 Hz, H-4) 6.86 (t, 1H, J(9/8,10) = 7.5 Hz, H-9), 6.21
Yield: 91 %; m.p 132-134°C; Rf: 0.48 (ethyl aceta-
1
J
te/hexanes, 2:8); H-NMR (500 MHz, DMSO-d6): ꢀ 7.61 (d,
(d, 1H, J(8,9) = 7.5 Hz, H-8), 4.42 (s, 2H, 16-CH2), 4.35 (s,
2H, 13-CH2); 13C-NMR: (75 MHz, DMSO-d6): ꢀ 166.4
(C=S), 142.9 (C-14), 136.8 (C-2, C-6), 135.8 (C-20), 132.2
(C-17), 131.5 (C-7), 130.7 (C-12), 130.5 (C-19, C-21), 129.1
(C-18, C-22), 128.4 (C-3, C-5), 128.1 (C-9), 126.4 (C-8),
122.2(C-9), 120.4 (C-10), 34.8 (13-CH2), 27.2 (16-CH2);
ESI-MS m/z: 511.0 (M+); HR-ESI-MS m/z Calcd for
C22H15Cl4N3OS [M]+ 511.251, Found 511.250; IR (KBr, cm-
1): 3215 (N-H), 1546 (C=N), 1549 (C=C), 1431 (C=S), 1185
(C-O); Anal. m/z Calcd C22H15Cl4N3OS: C, 51.68; H, 2.96;
Cl, 27.74; N, 8.22; O, 3.13; S, 6.27 2 Found: C, 51.66; H,
2.94; N, 8.20.
1H, J(10,11) = 7.5 Hz, H-10), 7.52 (d, 2H, J(3,4),(5,4) = 8.0 Hz, H-
3, H-5), 7.48 (s, 1H, NH), 7.47 (d, 1H, J (19,20) = 8.0 Hz, H-
19), 7.27 (m, 2H, H-20, H-23), 7.16 (d, 2H, J (11,10) = J (21,20)
=
10.5 Hz, H-11, H-21) 7.06 (t, 1H, J(4/3,5) = 7.5 Hz, H-4), 6.83
(t, 1H, J(9/8,10) = 7.5 Hz, H-9), 6.20 (d, 1H, J (8,9) = 8.0 Hz, H-
8) 5.03 (s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2), 3.81 (s, 3H,
OCH3); ESI-MS m/z: 500.0 (M+); HR-ESI-MS m/z Calcd for
C24H19Cl2N3O3S [M]+ 500.3970, Found 500.3971; IR (KBr,
cm-1): 3315 (N-H), 1691 (C=O), 1579 (C=N), 1576
(C=C),1443 (C=S), 1049 (C-O); Anal. m/z Calcd for
C24H19Cl2N3O3S: C, 57.61; H, 3.83; Cl, 14.17; N, 8.40; O,
9.59; S, 6.41, Found: C, 57.62; H, 3.82; Cl, 14.15; N, 8.41.
5.19. 2,6-Dichloro-N-(2-((5-(2,6-dichlorobenzylthio)-1,3,4-
5.16. 2-(5-(2-(2,6-Dichlorophenylamino)benzyl)-2-thioxo-
1,3,4-oxadiazol-3(2H)-yl)-1-(2-hydroxyphenyl)ethanone
(16)
oxadiazol-2-yl)methyl)phenyl)aniline (19)
Yield: 96%; m.p 132-134°C; Rf: 0.50 (ethyl aceta-
1
te/hexanes, 2:8) H-NMR (500 MHz, DMSO-d6): ꢀ 7.36 (d,
Yield: 94%; m.p 164-166°C; Rf: 0.47 (ethyl aceta-
te/hexanes, 2:8); 1H-NMR (300 MHz, DMSO-d6): ꢀ 11.13 (s,
1H, OH), 7.81 (dd, 1H, J(23,22) = 2.4 Hz J(23,21) = 6.3 Hz, H-
23), 7.53 (d, 3H, J(3,4),(5,4), (21,20) = 8.1 Hz, H-3, H-5, H-21),
7.18 (m, 2H, H-10, H-11), 7.05 (d, 1H, J (22,23) = 7.2 Hz, H-
22), 7.01 (s, 1H, NH), 6.97 (t, 1H, J(4/3,5) = 7.8 Hz, H-4), 6.85
(t, 1H, J(9/8,10) = 8.1 Hz, H-9), 6.19 (d, 1H, J (8,9) = 7.5 Hz, H-
8), 4.97 (s, 2H, 16-CH2), 4.35 (s, 2H, 13-CH2); ESI-MS m/z:
486.0 (M+); HR-ESI-MS m/z Calcd for C23H17Cl2N3O3S
[M]+ 486.3704, Found 486.3703; IR (KBr, cm-1): 3215 (N-
H), 1692 (C=O), 1572 (C=N), 1579 (C=C),1423 (C=S), 1041
(C–O); Anal. m/z Calcd for for C23H17Cl2N3O3S : C, 56.80;
H, 3.52; Cl, 14.58; N, 8.64; O, 9.87; S, 6.59 Found: C, 56.82;
H, 3.51; N, 8.63.
2H, J(3,4),(5,4) = 7.5 Hz, H-3, H-5), 7.34 (d, 2H, J(19,20),(21,20)
=
8.0 Hz, H-19, H-21) 7.37 (t, 1H, J(20/21,22) = 7.5 Hz, H-20),
7.23 (m, 2H, H-10, H-11), 7.12 (s, 1H, NH), 7.08 (t, 1H,
J(4/3,5) = 8.0 Hz, H-4) 6.86 (t, 1H, J(9/8,10) = 7.5 Hz, H-9), 6.21
(d, 1H, J(22, 21) = 8.0 Hz, H-22), 4.62 (s, 2H, 16-CH2), 4.39 (s,
2H, 13-CH2); ESI-MS m/z: 511.9 (M+); HR-ESI-MS m/z
Calcd for C22H15Cl4N3OS [M]+ 511.2510, Found 511.2511;
IR (KBr, cm-1): 3217 (N-H), 1540 (C=N), 1544 (C=C),1430
(C=S), 1183 (C-O); Anal. m/z Calcd C22H16BrCl2N3OS: C,
51.68; H, 2.96; Cl, 27.74; N, 8.22; O, 3.13; S, 6.27, Found:
C, 51.67; H, 2.97; N, 8.21.
5.20.
N-(2-((5-(2-Bromobenzylthio)-1,3,4-oxadiazol-2-
yl)methyl)phenyl)-2,6-dichloroaniline (20)
Yield: 93%; m.p 124-126°C; Rf: 0.52 (ethyl aceta-
5.17. 1-(Biphenyl-4-yl)-2-(5-(2-(phenylamino)benzyl)-2-
thioxo-1,3,4-oxadiazol-3(2H)-yl)ethanone (17)
1
te/hexanes, 2:8) H-NMR (400 MHz, DMSO-d6): ꢀ 7.63 (d,
1H, J(19, 20) = 8.4 Hz, H-19), 7.52 (d, 2H, J(3,4),(5,4) = 8.0 Hz,
H-3, H-5) 7.47 (dd, 1H, J(8,11) = 1.3 Hz, J(8,9) = 6.0 Hz, H-8),
7.27 (m, 4H, H-10, H-11, H-20, H-21) 7.13 (s, 1H, NH),
Yield: 90%; m.p 160-162°C; Rf: 0.56 (ethyl aceta-
1
te/hexanes, 2:8): H-NMR (400 MHz, DMSO-d6): ꢀ 8.08 (d,
2H, J(19,20),(23,22) = 8.4 Hz, H-19, H-23), 7.87 (d, 2H,
7.50), 7.09 (t, 1H, J(4/3,5) = 8.0 Hz, H-4) 6.86 (t, 1H, J(9/8,10)
=
J
(20,19),(22,23) = 8.0 Hz, H-20, H-22), 7.77 (d, 2H, J(25,26),(29,28) =
8.0 Hz, H-9), 6.21 (d, 1H, J(22, 21) = 8.1 Hz, H-22), 4.51 (s,
2H, 16-CH2), 4.37 (s, 2H, 13-CH2); 13C-NMR: (100.0 MHz,
DMSO-d6): ꢀ 166.6 (C=S), 162.2 (C-14), 142.9 (C-2, C-6),
136.8 (C-17), 135.3 (C-18), 132.8 (C-7), 131.5 (C-12), 131.4
(C-21), 130.6 (C-22), 130.0 (C-3, C-5), 129.1 (C-19), 128.2
(C-20), 127.9 (C-11), 126.4 (C-8), 123.9 (C-9), 122.1 (C-
10), 120.4 (C-1), 115.5 (C-4), 36.6 (13-CH2), 27.2 (16-CH2);
ESI-MS m/z: 519.0 (M+); HR-ESI-MS m/z Calcd for
C22H16BrCl2N3OS [M]+ 521.2569, Found 521.2568; IR (K-
Br, cm-1): 3219 (N-H), 1542 (C=N), 1540 (C=C),1438
(C=S), 1181 (C-O); Anal. m/z Calcd C22H16BrCl2N3OS: C,
7.6 Hz, H-25, H-29), 7.35( d, 4H, J(20,19),(22,23) = 8.0 Hz, H-20,
H-22), 7.53 (m, 4H, H-3, H-5, H-23, H-28), 7.45 (t, 1H,
J(10/9,11) = 8.0 Hz, H-10), 7.23 (t, 1H, J(27/26,28) = 8.0 Hz, H-
27), 7.17 (m, 2H, H-11, NH), 7.07 (t, 1H, J(4/3,5)= 8.0 Hz, H-
4), 6.84 (t, 1H, J(9/8,10) = 8.0 Hz, H-9), 6.20 (d, 1H, J (8,9) = 8.0
Hz, H-8), 5.08 (s, 2H, 16-CH2), 4.36 (s, 2H, 13-CH2); ESI-
MS m/z: 546.0 (M+); HR-ESI-MS m/z Calcd for
C29H21Cl2N3O2S [M]+ 546.4669, Found 546.4668; IR (KBr,
cm-1): 3281 (N-H), 1688 (C=O), 1562 (C=N), 1598
(C=C),1423 (C=S), 1086 (C-O); Anal. m/z Calcd for for for

686 Medicinal Chemistry, 2018, Vol. 14, No. 7
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CONCLUSION
In this study, nineteen analogs of diclofenac were synthe-
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of diclofenac 1 and its derivatives were performed. Five de-
rivatives were found to be active. Compound 5 was found to
be the most potent inhibitor of ROS and NO compared to
parent diclofenac 1 and standard drugs ibuprofen and L-
NMMA, respectively. The most active compounds 1, 4, 5, 11
and 20 were found to be non-toxic on NIH-3T3 cells. Com-
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of inhibition, respectively. These compounds might have the
potential to develop a novel non-steroidal (NSAIDs), non-
acidic antiinflammatory agent. Therefore, optimization of
these molecules, in vivo and detailed toxic studies are our
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ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are the
basis of this research.
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CONSENT FOR PUBLICATION
Not applicable.
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Mesaik, M.A.; Khan, K.M.; Rahat, S.; Zia-Ullah.; Choudhary, M.I.;
Murad, S.; Siddiqui, R.A. Immunomodulatory properties of syn-
thetic imidazolone derivatives. Lett. Drug Des. Discov., 2005, 2,
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Khan, K.M.; Ziaullah.; Lodhi, M.A.; Jalil, S.; Choudhary, M.I.;
Atta-ur-Rahman. Synthesis and anti-inflammatory activity of some
selected aminothiophene analogs. J. Enz. Inhib. Med. Chem., 2006,
21, 139-143.
Choudhary, M.I.; Jalil, S.; Nawaz, S.A.; Khan, K.M.; Tareen, R.B.
Antiinflammatory and lipoxygenase inhibitory compounds from
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Khan, K.M.; Ambreen, N.; Mughal, U.R.; Jalil, S.; Perveen, S.
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Azizuddin.; Mesaik, M.A.; Choudhary, M.I.; Khan, K.M.; Tareen,
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lated from vitex agnus-castus. J. Chem. Soc., Pak., 2012, 34, 971-
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Ullah, S.; Halimi, S.M.A.; Fakhri, M.I.; Khan, K.M.; Khan, I.;
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El Ashry el, SH.; El Tamany el, S.H.; Abd El Fattah Mel, D.; Aly,
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Asif, M.; Choudhary, M.I. In silico and in vitro immunomodulatory
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
This work was supported by the Higher Education Com-
mission (HEC) Pakistan (Project No. 20-1910), under the
National Research Program for Universities.
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