Design and synthesis of oxaprozin-1,3,4-oxadiazole hybrids as potential anticancer and antibacterial agents

Article abstract of DOI:10.1002/jhet.3842

In the present study, we report design, synthesis and screening of new novel 5-substituted-2-mercapto-1,3,4-oxadiazole analogues appended to oxaprozin for their in vitro anticancer and antibacterial activity. The synthesised compounds were characterized using various spectroscopic techniques. Furthermore, the structure of 5b (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-5-(ethylthio)-1,3,4-oxadiazole) was unequivocally confirmed by X-ray analysis. Among the series 5c (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-5-(propylthio)-1,3,4-oxadiazole) showed most promising anticancer activity against A549 cancer cell line and all the reported analogues manifested satisfactory safety profiles against human normal cell line HEK293T. The products exhibited good antibacterial activity and among the tested 5j (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-5-([4-fluorobenzyl]thio)-1,3,4-oxadiazole) exhibited most potent.

Full text of DOI:10.1002/jhet.3842

Received: 4 November 2018
DOI: 10.1002/jhet.3842
Revised: 11 September 2019
Accepted: 15 November 2019
A R T I C L E
Design and synthesis of oxaprozin-1,3,4-oxadiazole hybrids
as potential anticancer and antibacterial agents
Parsharamulu Rayam1
| Naveen Polkam1 | Naveen Kuntala1
|
Venkanna Banothu2 | Hasitha Shilpa Anantaraju3 | Yogeeswari Perumal3
Sridhar Balasubramanian4 | Jaya Shree Anireddy1
|
1Centre for Chemical Sciences and
Abstract
Technology, IST, Jawaharlal Nehru
Technological University Hyderabad,
Kukatpally, Hyderabad, Telangana State,
India
2Department of Biotechnology, IST,
Jawaharlal Nehru Technological
University Hyderabad, Kukatpally,
Hyderabad, Telangana State, India
3Department of Pharmacy, Birla Institute
of Technology and Science, Pilani,
Hyderabad, Telangana State, India
4X-ray Crystallography Division,
CSIR-Indian Institute of Chemical
Technology, Hyderabad, Telangana State,
India
In the present study, we report design, synthesis and screening of new novel
5-substituted-2-mercapto-1,3,4-oxadiazole analogues appended to oxaprozin
for their in vitro anticancer and antibacterial activity. The synthesised com-
pounds were characterized using various spectroscopic techniques. Further-
more, the structure of 5b (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-5-(ethylthio)-
1,3,4-oxadiazole) was unequivocally confirmed by X-ray analysis. Among the
series 5c (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-5-(propylthio)-1,3,4-oxadiazole)
showed most promising anticancer activity against A549 cancer cell line and
all the reported analogues manifested satisfactory safety profiles against
human normal cell line HEK293T. The products exhibited good antibacterial
activity and among the tested 5j (2-(2-[4,5-diphenyloxazol-2-yl]ethyl)-
5-([4-fluorobenzyl]thio)-1,3,4-oxadiazole) exhibited most potent.
Correspondence
Jaya Shree Anireddy, Centre for Chemical
Sciences and Technology, IST, Jawaharlal
Nehru Technological University
Hyderabad, Kukatpally, Hyderabad-
500085, India.
Email: jayashreeanireddy@gmail.com
1 | INTRODUCTION
threatening infections in living models thus posing
acquired resistance to many existing drugs, emphasizing
Cancer, the uncontrolled division or abnormal growth of
cells and spread or invade to other parts of the body,
thereby posing severe health consequences. Cancer
remains the second major challenging disorder for the
cause of morbidity and mortality around the world and is
a global challenge in the current scenario.[1] In 2012,
approximately 8.2 million tumor related deaths and 14.1
million new cancer cases are enrolled.[2] Howbeit, the
current chemotherapy methods are insufficient to meet
the expected results and prompted unsatisfactory
results.[3] Another sector of global diseases is ruled by
notorious multidrug-resistant Gram-negative pathogenic
and Gram-positive bacteria which are causing life-
the desperate need for the “miracle agents”[4] to treat
these microorganisms. Therefore, discovery and develop-
ment of new effective drug candidates has attracted
great attention among pharmaceutical and chemistry
researchers in recent years.
1,3,4-oxadiazole constitutes an important privileged het-
erocyclic structure highlighting two nitrogens and an oxy-
gen atom.[5] This pharmacophore has gained greater
attention over years due to exhibition of wide range of bio-
logical profiles[6] owing to the existence of N C O
toxophoric linkage.[3] Development of several techniques
for the facile construction of the scaffold had captured accel-
eration.[7] Over the last decade, approximately 686 patents
J Heterocyclic Chem. 2020;1–12.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
RAYAM ET AL.
have been filed on oxadiazole pharmacophore and its
importance[8] 2,5-Disubstituted oxadiazole subsidiaries have
been encompassed with promising pharmacological profiles
such as anticancer,[9] antitubercular,[10] antimicrobial,[11]
antifungal,[12] antiepileptic,[13] analgesic,[14] antitumour,[15]
carboxylic group as well as considerable damage to renal
system.[25] Albeit several strategies have been proposed,
none of them completely address these symptoms and
thus became the focal point of the present research.
Additionally, it was established from the collected
data, that the oxaprozin and its derivatives as well elicit
anticancer[26] and antibacterial profiles[26a] through a
COX-2 independent mechanism.[27] Bozic et al have
reported that transition metal complexes of oxaprozin
demonstrated for significant antiproliferative activity
against human colon (HCT-116) and breast (MDA-231)
cancer cells.[28] Earlier, Babic et al. have documented on
oxaprozin hydrogels as potential antibacterial agents.[23]
Recently, we have reported etodolac (a non-steroidal
anti-inflammatory drug) based 1,2,3-triazole hybrids
and etodolac-pyridazinones as potential anti-cancer prop-
erties.[26d, e] To the continuation of our current research
programme[29] for the identification of dormant heterocy-
clic compounds, herein, we have designed the synthesis
of hybrid template A (Figure 2) by considering the fol-
lowing points and to ascertain their anticancer and
antibacterial activities. (a) To mask the side effects of
oxaprozin carboxylic motif and convert it to its
1,3,4-oxadiazole bioisoster for safer profiles. (b) Retain
the hydrophobic interactions of oxaprozin.[23] (c) To add
the other vacant position in oxadiazole with thio alkyl
group for the enhancement of lipophilicity and pharma-
cokinetic properties by formation of C S bond which
aids in penetration of the analogues across the cell
wall.[30]
ulcerogenic,[16]
hypoglycaemic,[17]
central
nervous
system,[18] antidepressant,[19] hypnotic[20] and sedative
properties.[21] Few compounds viz., zibotentan, ataluren,
and raltegravir (Figure 1) comprising oxadiazole as core
nucleus are in the late stages of clinical trials substantiat-
ing the valuable privileges of the scaffold. Furthermore,
oxadiazoles are great bioisosters of carbonyl-containing
groups such as amides, esters, hydroxamic and carbamates
which improve hydrogen bond interactions with various
receptors thereby increasing the biological responses to a
remarkable extent.[3] Aside from their latent potential
medicinal applications, they have also captured attention
in material sciences due to electron transporting and hole
blocking abilities.[22]
On the other hand, oxaprozin (Figure 2), one of the
leading non-steroidal anti-inflammatory drug (NSAID) is
a global market drug used for the therapeutic manage-
ment of pain, osteoarthritis, ankylosing spondylitis,
chronic inflammatory disease and bursitis.[23] It acts by
suppressing the prostaglandin (PG) biosynthesis in which
COX-1 and COX-2 are the key intermediates thereby con-
trolling the production of arachidonic acid.[24] However,
its therapeutic usage is restricted due to the development
of side effects such as gastrointestinal injury (GI), peptic
ulceration and aperture due to the presence of corrosive
FIGURE 1 Representative bio-
active compounds featuring
oxadiazole motif
FIGURE 2 Design strategy of hybrid template A based on oxaprozin and 1,3,4-oxadiazole

RAYAM ET AL.
3
2 | RESULTS AND DISCUSSION
structure of compound 5b was verified by a single crys-
tal X-ray diffraction studies (Figure 3).[35]
Among the variety of available methods[31] the synthesis of
previously unreported compounds (5a-p) was accom-
plished by employing conventional (Scheme 1, path a) as
well as microwave irradiation methods (Scheme 1, path b).
Briefly, the compound 3 was prepared in a stepwise man-
ner which involves esterification of the carboxylic group of
Oxaprozin (1) followed by the reaction with hydrazine to
give acid hydrazide 3.[32] Intramolecular cyclization of
3 under strong basic conditions with carbon disulfide
furnished the key intermediate 4 in good yield.[9] Finally,
the reaction of 4 with appropriate electrophilic halides gave
the title compounds 5a-p in excellent yields.[10] Microwave
irradiation (MWI) technique has offered a paradigm shift
in the target synthesis due to its peculiar direct “in core”
heating of the reaction mixture thus diminishing the reac-
tion times.[33] Moreover, it offers myriad advantages such
as acceleration of sluggish transformations, environmen-
tally sustainable conditions, higher yields and strongly pro-
mote the cyclocondensation reaction.[34] Under non-
classical reaction condition (MWI), the reaction rates were
faster than classical heating conditions (Table 1).
2.1 | Biological evaluation
In vitro cytotoxicity of all synthesized compounds was
assessed by employing MTT colorimetric assay[36] against
prostate (PC3) and lung (A549) cancer cell lines
(Table 2). The anticancer drug Doxorubicin was used as a
reference compound in the present study. The results rev-
ealed that the compounds 5a, 5b and 5h showed higher
cytotoxicity against PC3 cells. All of the tested com-
pounds were more potent than the parent scaffold
oxaprozin except 5j for the A549 cells. The compound 5c
exhibited promising cytotoxicity whereas the compounds
5a, 5e and 5h displayed moderate to good cytotoxicity
against A549 cells. From the results, the following SAR
studies can be inferred. Homologation from methyl (5a)
to ethyl (5b) in turns to n-propyl (5c) and then to n-butyl
(5d) decreased the cytotoxicity profile for PC3 cell line
and similar pattern was not observed for A549 cell line
indicating the selectivity of synthesized compounds to a
particular cell line. However, increment in activity was
observed when carbon chain was increased from methyl
to ethyl as in the case of 1-methyl-4-methoxybenzene
(5l) and 1-ethyl-4-methoxybenzene (5p) for both the cell
lines. Introduction of unsaturation (n-propyl (5c) and
allyl (5g)) led to the decrement of cytotoxicity for both
cell lines and further increment of unsaturation that is,
propargylation (5h) led to improvement of activity. For
the analogues phenyl (5i), 4-fluorobenzene (5j),
4-chlorobenzene (5m) and 4-bromobenzene (5n), the
cytotoxicity trend followed the pattern of lesser the -I
effect of the substituent (electronic effect) higher the
activity against A549 cell line. It is well recorded that
cytotoxic medications mitoxantrone and doxorubicin dis-
play cytotoxicity against A549 cells by the generation of
reactive oxygen species, therefore causing apoptosis
through the outflow of caspase-3.[3] Hence, it is trusted
As a representative example, synthesis of 5a was
accomplished in
6 hours by traditional methods
whereas under MW condition, it was achieved in
3 minutes (5a, Table 1). The structures of compounds
5a-p were supported by NMR, IR and HRMS spectral
analysis. On the 1H NMR timescale, disappearance of
NHNH2 proton signals in 3 at δ 4.72 ppm and 4.77 ppm
and appearance of a signal at δ 11.31 ppm (SH) asserted
4. The 13C NMR peaks at δ 162.51 (C-2) ppm and
160.51 (C-5) ppm indicates the formation of
1,3,4-oxadiazole core. Compound 4 IR spectrum dis-
played absorption bands at υmax 1624 cm−1 (C N),
1155 cm−1 ( N N C ) and 1059 cm−1 (C O C) evi-
dent to
C O C
linkage and
N N C
in
oxadiazole frame work. Manifestation of IR signals
between υmax 2771-2924 cm−1 established the formation
of 5a-p due to the presence of S-CH2. Furthermore, the
SCHEME 1 Synthesis of oxaprozin-1,3,4-oxadiazole derivatives

4
RAYAM ET AL.
TABLE 1 Reaction conditions for
the formation of products 5a-p
Microwave
Conventional
Time
(min)
Yield
(%)a
Time
(h)
Yield
(%)a
Compounds R-X
5a
5b
5c
5d
5e
5f
3
4
3
3
3
2
2
94
94
94
93
90
95
94
6
6
6
5
5
5
5
85
83
84
85
82
85
5g
83
5h
5i
3
3
94
93
5
6
85
87
5j
4
3
2
3
92
89
95
94
6
5
5
6
85
85
85
85
5k
5l
5m
5n
5o
3
2
94
95
6
6
83
84
5p
2
87
5
82
aIsolated yield.
also tested on HEK293T human normal cells to study
their toxicity profile and are found to exhibit mediocre
safety profiles.
Additionally, the newly synthesized analogues 5a-p
were screened for their in vitro antibacterial activity
(Table 2) against Gram-positive bacteria (Staphylococcus
aureus) and Gram-negative bacteria (Klebsiella
pneumoniae, Pseudomonas aeruginosa and Escherichia
coli) by Agar well diffusion method.[37,38] As anticipated
and from the bacterial investigations, it is obvious that all
the tested derivatives showed enhanced bacterial inhibi-
tion than the oxaprozin core. Most of them displayed bet-
ter antibacterial activity largely against Gram-negative
strains. Among the tested series 5, compound 5j dis-
played potent antibacterial activity against all the strains
under study except K. pneumoniae and 5c showed good
activity against K. pneumoniae strain. Among the homo-
logues 5a-d, the activity order is compound containing
odd no of carbons that is, methyl (5a), n-propyl (5c)
FIGURE 3 Single-crystal structure of compound 5b
that these new derivatives may act as effective anticancer
agents by the similar mechanism discussed above. The
dose response study revealed that the compound 5c
showed consistent growth of inhibition against A549 cells
(Figure 4). Furthermore, the synthesized derivatives were

RAYAM ET AL.
5
demonstrated higher activity than derivatives with even
no. of carbons ethyl (5b), n-butyl (5d) except for the
K. pneumoniae strain. For the structures allyl (5g), propargyl
(5h) increment of unsaturation led to decrement of activity
for Gram-positive strain and increment for Gram-negative
species except K. pneumoniae. For the series, the halogenated
analogues 4-fluorobenzene (5j), 4-chlorobenzene (5m),
4-bromobenzene (5n), 2-chlorobenzene (5o) demonstrated
higher activity for all the tested strains than non-halogenated
derivatives 1-methyl-4-methoxybenzene (5l), 1-ethyl-4-
methoxybenzene (5p) which might be due to better pharma-
codynamics owing to the presence of halogens. Pharmacoki-
netic and physicochemical properties is due to optimum
lipophilicity, and electronegativity of the halogen bond.[29a]
3 | CONCLUSION
A simple and efficient protocol for the synthesis of novel
hybrids of 1,3,4-oxadiazole appended to oxaprozin, evalu-
ation of cytotoxicity as well as antibacterial activity have
been documented in the present communication. Among
the series, 5c has displayed superior anticancer and
antibacterial activities against A549 and Klebsiella pneu-
monia respectively. 5h has exhibited good anticancer
activity against both cell lines. All these compounds have
elicited mediocre to good safer profiles against HEK293T
normal cell lines.
FIGURE 4 Dose response curve of the most active compound
5c on A549 cell line
TABLE 2 In vitro cytotoxic and antibacterial activities of the designed compounds 5a-p
Cytotoxicity % of inhibition at 25 μM Zone of inhibition (Diameter in mm) at 0.4 mg/50 μL
Compounds
PC3
A549
45.33
31.38
65.04
23.28
45.71
21.23
30.68
45.71
21.19
14.83
23.17
17.86
18.03
20.98
18.68
19.26
17.23
HEK293T
23.63
34.71
33.74
52.63
21.06
20.30
09.30
42.67
29.58
16.45
01.70
−03.40
20.14
23.62
21.18
5.67
S. aureus
14 0.23
12 0.13
15 0.35
13 0.22
15 0.34
13 0.24
17 0.54
15 0.35
17 0.54
18 0.59
17 0.32
15 0.29
16 0.36
16 0.29
17 0.19
15 0.27
12 0.16
—
K. pneumoniae
17 0.38
18 0.55
20 0.89
15 0.35
17 0.42
14 0.32
17 0.47
16 0.45
19 0.98
17 0.63
16 0.48
16 0.58
16 0.42
18 0.67
17 0.31
15 0.35
10 0.12
—
P. aeruginosa
18 0.42
15 0.37
19 0.58
13 0.28
17 0.38
19 0.65
13 0.26
17 0.62
19 0.89
20 0.88
19 0.89
14 0.19
17 0.59
16 0.31
18 0.42
16 0.48
11 0.19
—
E. coli
5a
22.44
21.84
08.04
02.30
12.27
00.93
05.54
32.29
18.10
04.18
11.06
11.64
18.90
18.14
19.45
12.48
17.21
14 0.29
13 0.21
17 0.46
-
5b
5c
5d
5e
16 0.36
17 0.86
14 0.32
15 0.43
20 1.02
22 1.22
21 0.93
15 0.42
18 0.98
19 0.86
19 0.79
17 0.65
12 0.17
—
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
Oxaprozin
Doxorubicin
Ciprofloxacin
20.06
a
b
c
—
—
—
—
—
—
34 0.15
28 0.20
32 0.15
31 0.30
Note: All the values of the average of experiments done in triplicates.
aDoxorubicin IC50 is 2.1 μM.
bDoxorubicin IC50 is 3.3 μM.
cDoxorubicin IC50 is 1.2 μM.

6
RAYAM ET AL.
4 | EXPERIMENTAL
4.1 | General
4.3 | Synthesis of 3-(4,5-diphenyloxazol-
2-yl)propanehydrazide (3)
To the solution of 2 (4.70 g, 15 mmol) in absolute alcohol
(50 mL) was added hydrazine hydrate (1.6 mL, 30 mmol)
and refluxed for about 7 hours. Excess ethanol was dis-
tilled off and the reaction mass was added in to water
and extracted with ethyl acetate (2 × 30 mL). The com-
bined organic layer was dried over sodium sulphate and
evaporated under reduced pressure. The obtained crude
product was purified by recrystallization from ethanol to
furnish 3 as white solid in 95% yield. mp: 108-110ꢀC; Rf:
0.50 (ethyl acetate/hexane, 70/30); IR (KBr, υmax in cm-
1): 3324, 3285 (NH/NH2), 3034 (C H of aromatic), 2921
(C H of aliphatic), 1659 (C O), 1513 (C N), 1437
All the reactions were carried under inert nitrogen atmo-
sphere employing dry solvents. Precoated TLC silica gel
plates (Kieselgel 60 F254, Merck) were used for monitor-
ing reactions. Purification was performed by column
chromatography using silica gel (particle size 100-200
mesh, Merck). Melting points were determined in open
capillary tubes on cintex melting point apparatus and are
uncorrected. A monowave 300 mas 24 (option) (Anton
paar) microwave was used to carry out the microwave
reactions in 30 mL microwave process vials with temper-
ature control by infrared detection temperature sensor.
IR (KBr) spectra were recorded on a Perkin-Elmer
400 FTIR spectrometer (υmax in cm−1) or a Varian 670-IR
FT-IR spectrometer (ATR) in the frequency range of
1
(C C), 1060 (C O C), 762, 692; H NMR (500 MHz,
CDCl3) δ ppm: 7.60 (dd, J = 8.2 Hz, 2H, Ar-H), 7.55 (dd,
J = 8.2 Hz, 2H, Ar-H), 7.31-7.38 (m, 6H, Ar-H), 4.72 (bs,
1H, NH, D2O exchangeable), 4.77 (bs, 2H, NH2, D2O
exchangeable), 3.20 (t, J = 7.4 Hz, 2H, CH2),2.72 (t,
J = 7.4 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ
ppm: 175.36, 162.22, 145.05, 138.69, 135.05, 132.49,
130.07, 129.53, 128.97, 128.64, 29.90, 23.03; ESI-Ms m/z
308.0 (M + H) + .
1
600 to 4000 cm−1. H NMR and 13C NMR spectra were
recorded in CDCl3/DMSO-d6 on a Bruker DRX-300
(300 MHz FT NMR)/Varian Mercury 400 MHz/500 MHz
spectrometer. Proton chemical shifts are presented in δ
ppm regarding TMS. J values are presented in Hz. Mass
spectra were recorded using Jeol SX-102 spectrometer.
High-resolution mass spectra (HRMS) were recorded
with an Agilent Technologies 6510 Q-TOF spectrometer.
4.4 | Synthesis of 5-(2-(4,5-
diphenyloxazol-2-yl)ethyl)-1,3,4-
oxadiazole-2-thiol (4)
4.2 | Synthesis of methyl
3-(4,5-diphenyloxazol-2-yl)propanoate (2)
Mixture of compound 3 (4.23 g, 14 mmol), potassium
hydroxide (0.92 g, 16 mmol) and carbon disulphide
(1.73 mL, 27 mmol) in ethanol (50 mL) was stirred
under reflux for 12 hours, till the evolution of H2S
gas was ceased. The progress of the reaction was
monitored by TLC and excess solvent was distilled.
The residue mass was poured over crushed ice, added
10% hydrochloric acid till the PH of the solution is
5. The precipitated crude product thus obtained was
filtered, washed with water, dried and recrystallized
from ethanol to afford the compound 4 as white solid
in 92% yield. mp: 162-164ꢀC; Rf: 0.42 (ethyl acetate/
hexane, 40/60); IR (KBr, υmax in cm−1): 3078 (C H of
aromatic), 2924 (C H of aliphatic), 2772 (SH), 1624,
To a solution of oxaprozin (5 g, 17 mmol) in methanol
(30 mL), few drops of conc. H2SO4 were added and the
resulting mixture was refluxed for 6 hours. After comple-
tion of the reaction as indicated by TLC, excess methanol
was distilled. The obtained reaction mass was diluted
with 10% sodium bicarbonate solution and extracted with
ethyl acetate and dried over sodium sulphate and
reduced under pressure. The crude product was purified
by recrystallization from ethanol to furnish the title com-
pound 2 as white solid in 95% yield. mp: 56-58ꢀC; Rf: 0.48
(ethyl acetate/hexane, 20/80); IR (KBr, υmax in cm−1):
3049 (C H of aromatic), 2946 (C H of aliphatic), 1734
(C O), 1590 (C N), 1496 (C C), 1060 (C O C),
1
1
763, 700; H NMR (500 MHz, CDCl3) δ ppm: 7.64 (dd,
1510 (C N), 1322, 1155, 1059 (C O C), 766, 799; H
J = 8.2 Hz, 2H, Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H),
7.26-7.38 (m, 6H, Ar-H), 3.74 (s, 3H, OCH3), 3.2 (t,
J = 7.4 Hz, 2H, CH2), 2.92 (t, J = 7.4 Hz, 2H, CH2); 13C
NMR (75 MHz, CDCl3) δ ppm: 173.02, 162.17, 145.07,
138.54, 136.05, 133.49, 132.07, 129.05, 128.97, 128.64,
60.87, 29.90, 23.03; ESI-Ms m/z 308.0 (M + H)+.
NMR (500 MHz, CDCl3) δ ppm: 11.31 (bs, 1H, SH),
7.55-7.63 (m, 4H, Ar-H), 7.26-7.38 (m, 6H, Ar-H),
3.27-3.37 (m, 4H, CH2CH2); 13C NMR (75 MHz,
CDCl3) δ ppm: 162.51, 160.51, 146.05, 135.09, 131.65,
128.74, 128.41, 128.05, 126.50, 24.09, 23.29; ESI-Ms
m/z 350.0 (M + H)+.

RAYAM ET AL.
7
4.5 | General procedure for synthesis of
title compounds (5a-p)
(s, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ ppm: 166.33,
165.41, 160.63, 145.77, 135.21, 133.13, 132.26, 129.93,
129.04, 128.90, 128.77, 128.69, 128.61, 128.18, 127.96,
127.90, 126.51, 25.02, 23.03, 14.55; ESI-Ms m/z 364.0
(M + H)+.
4.5.1 | Conventional synthetic route[10]
To a solution of compound 4 (1.0 eq) in DMF (5 vol),
respected alkyl/substituted benzyl halide (1.2 eq) was
added followed by K2CO3 (1.5 eq) and stirred at ambient
temperature for 5 to 6 hours. The reaction mass was
diluted with water and extracted with EtOAc (2 X
20 mL). Purification of the crude compounds has been
done by using 100-200 silica gel mesh to yield the title
compounds (5a-p) in 83-87% yields.
4.7 | 2-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
5-(ethylthio)-1,3,4-oxadiazole(5b)
White solid; mp: 88ꢀC to 90ꢀC; Rf: 0.40 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3052 (C H of
aromatic), 2927 (C H of aliphatic), 2863 (SCH2), 1576
1
(C N), 1452(C C), 1269, 1111 (C O C), 763, 698; H
NMR (400 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H), 7.40-7.30 (m, 6H,
Ar-H), 3.45-3.35 (m, 4H, CH2), 3.23 (q, J = 7.3 Hz, 2H,
SCH2),1.45 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3) δ ppm: 166.14, 164.63, 160.57, 145.70, 135.16,
132.23, 128.73, 128.62, 128.54, 128.11, 127.84, 126.46,
26.89, 25.01, 22.99, 14.68; HRMS: (ESI m/z) for
C21H20O2N3S calcd: 378.1270, found: 378.1276 (M + H)+;
Crystal Data: C21H19N3O2S (M = 377.45): orthorhombic,
4.5.2 | Microwave one-pot synthetic
route[34a–c]
Alternately, we tested whether the reaction occurs in
diminished time by heating the above reaction mixture at
80ꢀC in DMF (5 vol). Howbeit, the reactants were suc-
cessfully turned in to products within 1 to 2 hours. Next,
we thought of diminishing the reaction time further, by
seeking the help of Microwave where heat transfers to
those components of the reaction mixture, which are sus-
ceptible to microwave polarization, and raises the tem-
perature of sample much faster than conventional
methods.[34d, e] Mixture of compound 3 (1.0 eq), carbon
disulphide (1.2 eq) and potassium hydroxide (1.2 eq) in
ethanol (5 vol) were taken in a closed vessel and irradi-
ated under microwave (MW) at 80ꢀC for 20 minutes.
After completion of the reaction as indicated by TLC,
excess ethanol solvent was distilled off. To the above
reaction mixture, respective alkyl/substituted benzyl
halides (1.2 eq) in DMF (5 vol) were added followed by
K2CO3 (1.2 eq) and it was again irradiated under MW at
100ꢀC for 2-4 minutes. After completion of the reaction,
the reaction crude was diluted with water and extracted
with ethyl acetate (2 × 20 mL). The combined organic
layer was dried over sodium sulphate and evaporated
under reduced pressure. The obtained crude product was
recrystallized from ethanol to furnish 5a-p in 87-95%.
space group Pbca (no. 61),
b = 7.9144(17) Å, c = 46.917(10) Å, V = 3761.3(14) Å3,
Z = 8, T = 294.15 K, μ (MoKα) = 0.193 mm−1
a
=
10.130(2) Å,
,
Dcalc = 1.333 g/mm3, 40 979 reflections measured
(3.472 ≤ 2Θ ≤ 56.668), 4640 unique (Rint = 0.0288) which
were used in all calculations. The final R1 was 0.0622
(I > 2σ[I]) and wR2 was 0.1670 (all data). CCDC 1471510
contains supplementary Crystallographic data for the
structure. [These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html [or from the
Cambridge Crystallographic Data Centre (CCDC),
12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk]].
4.8 | 2-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
5-(propylthio)-1,3,4-oxadiazole (5c)
White solid; mp: 90ꢀC to 92ꢀC; Rf: 0.43 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3058 (C H of
aromatic), 2964 (C H of aliphatic), 2871 (SCH2), 1581
1
4.6 | 2-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
(C N), 1482 (C C), 1152, 1061 (C O C), 765, 695; H
5-(methylthio)-1,3,4-oxadiazole (5a)
NMR (500 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H), 7.40-7.29 (m, 6H,
Ar-H), 3.44-3.33 (m, 4H, CH2), 3.19 (t, J = 7.3 Hz, 2H,
SCH2), 1.81 (m, 2H, SCH2CH2), 1.03 (t, J = 7.3 Hz, 3H,
CH3); 13C NMR (125 MHz, CDCl3) δ ppm: 165.96, 164.65,
160.45, 145.52, 134.99, 132.08, 129.28, 128.75, 128.58,
128.46, 128.38, 127.95, 127.67, 126.29, 34.22, 24.81, 22.81,
22.50, 12.94; ESI-Ms m/z 392.0 (M + H)+.
White solid; mp: 80-82ꢀC; Rf: 0.35 (ethyl acetate/hexane,
30/70); IR (KBr, υmax in cm−1): 3054 (C H of aromatic),
2926 (C H of aliphatic), 2863 (SCH2), 1582 (C N), 1486
(C C), 1168, 1059 (C O C), 763, 695; 1H NMR
(300 MHz, CDCl3) δ ppm: 7.59 (dd, J = 8.2 Hz, 4H, Ar-
H), 7.42-7.31 (m, 6H, Ar-H), 3.50-3.30 (m, 4H, CH2), 2.70

8
RAYAM ET AL.
4.9 | 2-(butylthio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5d)
4.12 | 2-(allylthio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5g)
Light yellow solid; mp: 78ꢀC to 80ꢀC; Rf: 0.48 (ethyl ace-
tate/hexane, 30/70); IR (KBr, υmax in cm−1): 3053 (C H
of aromatic), 2928 (C H of aliphatic), 2861 (SCH2), 1578
Yellow solid; mp: 76ꢀC to 78ꢀC; Rf: 0.43 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3059 (C H of
aromatic), 2926 (C H of aliphatic), 2856 (SCH2), 1583
1
1
(C N), 1478 (C C), 1148, 1058 (C O C), 762, 695; H
(C N), 1481 (C C), 1161, 1059 (C O C), 761, 691; H
NMR (300 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 4H,
Ar-H), 7.41-7.29 (m, 6H, Ar-H), 3.98 (t, J = 7.3 Hz, 2H,
SCH2), 3.50-3.32 (m, 4H, CH2), 2.24-2.12 (m, 4H,
SCH2 CH2 CH2), 1.64 (t, J = 7.3 Hz, 3H, CH3); 13C
NMR (125 MHz, CDCl3) δ ppm: 166.18, 164.95, 160.67,
145.76, 135.22, 132.28, 128.79, 128.68, 128.60, 128.17,
127.90, 126.52, 32.24, 31.23, 25.02, 23.02, 21.72, 13.50;
HRMS: (ESI m/z) for C23H24O2N3S calcd: 406.1583,
found: 406.1585 (M + H)+.
NMR (300 MHz, CDCl3) δ ppm: 7.63 (dd, J = 8.2 Hz, 4H,
Ar-H), 7.44-7.30 (m, 6H, Ar-H), 6.10-5.86 (m, 1H,
CH CH2), 5.35 (d, J = 17.3 Hz, 1H, CH CH2), 5.19 (d,
1H, J = 9.8 Hz, 1H, CH CH2), 3.84 (d, J = 6.7 Hz, 2H,
SCH2), 3.31-3.49 (m, 4H, CH2); 13C NMR (125 MHz,
CDCl3) δ ppm: 166.45, 164.06, 160.61, 145.78, 135.22,
132.27, 131.70, 128.69, 128.61, 128.18, 127.91, 126.53,
119.72, 35.16, 24.98, 23.03; HRMS: (ESI m/z) for
C22H20O2N3S calcd: 390.1270, found: 390.1268 (M + H)+.
4.10 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-(octylthio)-1,3,4-oxadiazole (5e)
4.13 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-(prop-2-yn-1-ylthio)-1,3,4
oxadiazole (5h)
White solid; mp: 85ꢀC to 87ꢀC; Rf: 0.53 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3002 (C H of
aromatic), 2966 (C H of aliphatic), 2924 (SCH2), 1734
(C N), 1689 (C C), 1220, 1093 (C O C); 1H NMR
(300 MHz, CDCl3) δ ppm: 7.63 (dd, J = 8.2 Hz, 4H, Ar-
H), 7.36-7.26 (m, 6H, Ar-H), 3.41-3.37 (m, 4H, CH2), 3.21
(t, J = 7.3 Hz, 2H, SCH2), 1.79-1.75 (m, 2H, SCH2CH2),
1.42-1.27 (m, 13H, CH2[CH2]4CH3); 13C NMR (125 MHz,
CDCl3) δ ppm: 166.16, 164.97, 160.70, 145.77, 135.20,
132.25, 128.77, 128.67, 128.18, 127.91, 126.51, 32.55,
31.76, 29.71, 29.22, 28.98, 28.59, 25.01, 23.02, 22.63, 14.10;
ESI-Ms m/z 462.0 (M + H)+.
White solid; mp: 77ꢀC to 79ꢀC; Rf: 0.40 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3073 (C H of
aromatic), 2923 (C H of aliphatic), 2771 (SCH2), 2274
(C C), 1624 (C N), 1509 (C C), 1154,1058 (C O C),
1
764, 698; H NMR (300 MHz, CDCl3) δ ppm: 7.62 (dd,
J = 8.0 Hz, 4H, Ar-H), 7.42-7.30 (m, 6H, Ar-H), 3.98 (s,
2H, CH2), 3.33-3.51 (m, 4H, CH2 CH2), 2.29 (s, 1H,
C ≡ C H); 13C NMR (125 MHz, CDCl3): δ 166.85,
162.98, 160.54, 145.80, 135.23, 132.25, 128.76, 128.70,
128.62, 128.19, 127.90, 126.53, 76.76, 72.99, 24.95, 23.03,
21.02; HRMS: (ESI m/z) for C22H18O2N3S calcd:
388.1114, found: 388.1114 (M + H)+.
4.11 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-(isobutylthio)-1,3,4-oxadiazole (5f)
4.14 | 2-(benzylthio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5i)
Yellow solid; mp: 80ꢀC to 82ꢀC; Rf: 0.40 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3003 (C H of
aromatic), 2924 (C H of aliphatic), 2784 (SCH2), 1686
Light brown solid; mp: 90ꢀC to 92ꢀC; Rf: 0.46 (ethyl ace-
tate/hexane, 30/70); IR (KBr, υmax in cm−1): 3056 (C H
of aromatic), 2926 (C − H of aliphatic), 2855 (SCH2),
1581 (C N), 1483 (C C), 1152, 1052 (C O C), 766, 699;
1
(C N), 1495 (C C), 1225, 1093 (C O C), 903, 782; H
NMR (300 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 4H,
Ar-H), 7.41-7.28 (m, 6H, Ar-H), 3.47-3.30 (m, 4H, CH2),
3.12 (d, J = 6.7 Hz, 2H, SCH2), 2.13-1.95 (m, 1H, CH),
1.03 (d, J = 6.7 Hz, 6H, CH3); 13C NMR (125 MHz,
CDCl3) δ ppm:165.96164.65, 160.45, 145.52, 134.99,
132.08, 129.28, 128.75, 128.58, 128.46, 128.38, 127.95,
127.67, 126.29, 34.22, 30.72, 24.81, 22.50, 22.81, 12.94;
HRMS: (ESI m/z) for C23H24O2N3S calcd: 406.1573,
found: 406.1576 (M + H)+.
1H NMR (500 MHz, CDCl3)
δ
ppm: 7.62 (dd,
J = 8.8 Hz,2H, Ar-H), 7.56 (dd, J = 8.8 Hz, 2H, Ar-H),
7.41 (dd, J = 8.8 Hz, 2H, Ar-H), 7.39-7.27 (m, 9H, Ar-H),
4.44 (s, 2H, SCH2), 3.44-3.33 (m, 4H, CH2); 13C NMR
(125 MHz, CDCl3): δ 168.68, 167.38, 166.35, 160.57,
145.75, 135.46, 133.34, 132.25, 129.09, 129.12, 128.78,
128.76, 128.66, 128.58, 128.55, 128.52, 128.14, 128.07,

RAYAM ET AL.
9
127.87, 127.84, 126.49, 36.75, 24.90, 23.00; ESI-Ms m/z
440.6 (M + H)+.
Ar-H), 6.61 (d, J = 8.5 Hz, 2H, Ar-H), 5.12 (s, 2H, SCH2),
3.90 (s, 3H, OCH3), 3.45-3.30 (m, 4H, CH2); 13C NMR
(125 MHz, CDCl3) δ ppm: 166.32, 164.39, 160.62, 159.41,
145.77, 135.23, 132.27, 130.40, 128.77, 128.69, 128.62,
128.18, 127.90, 127.37, 126.51, 114.17, 55.30, 36.41, 24.99,
23.02; HRMS: (ESI m/z) for C27H24O3N3S calcd:
470.1532, found: 470.1528 (M + H)+.
4.15 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-([4-fluorobenzyl]thio)-
1,3,4-oxadiazole (5j)
White solid; mp: 78ꢀC to 80ꢀC; Rf: 0.43 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3051 (C H of
aromatic), 2928 (C H of aliphatic), 2864 (SCH2), 1584
(C N), 1481 (C C), 1232, 1154 (Ar-F), 1056, 1019
4.18 | 2-([4-chlorobenzyl]thio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5m)
1
(C O C), 762, 694; H NMR (500 MHz, CDCl3) δ ppm:
7.62 (dd, J = 8.2 Hz, 2H, Ar-H), 7.56 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.43-7.30 (m, 8H, Ar-H), 7.00 (t, J = 8.8 Hz, 2H,
Ar-H), 4.41 (s, 2H, SCH2), 3.46-3.33 (m, 4H, CH2); 13C
NMR (75 MHz, CDCl3): δ 176.94, 175.01, 166.44, 164.02,
163.41, 135.20, 132.21, 131.40, 131.37, 130.86, 130.80,
128.69, 128.58, 128.16, 127.86, 126.55, 126.51, 123.19,
122.94, 122.68, 115.77, 35.96, 24.92, 22.98; HRMS: (ESI
m/z) for C26H21O2N3FS calcd: 458.1333, found: 458.1317
(M+ H)+.
White solid; mp: 84ꢀC to 86ꢀC; Rf: 0.46 (ethyl acetate/hex-
ane, 30/70); IR (KBr, υmax in cm−1): 3063 (C H of aromatic),
2928 (C H of aliphatic), 2838 (SCH2), 1675 (C N), 1485
(C C), 1163, 1093 (C O C), 704, 646; 1H NMR (500 MHz,
CDCl3) δ ppm: 7.61 (dd, J = 8.2 Hz, 2H, Ar-H), 7.57 (dd,
J = 8.2 Hz, 2H, Ar-H), 7.42-7.30 (m, 10H, Ar-H), 4.46 (s, 2H,
SCH2), 3.42-3.34 (m, 4H, CH2); 13C NMR (125 MHz, CDCl3)
δ ppm: 167.25, 163.41, 160.57, 158.55, 146.76, 135.46, 132.55,
131.52, 130.50, 129.56, 129.21, 129.34, 128.55, 128.33, 128.21,
127.07, 127.05, 126.49, 114.54, 34.75, 24.91, 23.07; ESI-Ms
m/z 474.0 (M + H)+, 476.0 (M + 2 + H)+.
4.16 | 2-((4-[benzyloxy] benzyl) thio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5k)
4.19 | 2-([4-bromobenzyl] thio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5n)
White solid; mp: 88ꢀC to 90ꢀC; Rf: 0.43 (ethyl acetate/
hexane, 30/70); IR (KBr, υmax in cm−1): 3047 (C H of
aromatic), 2925 (C H of aliphatic), 2854 (SCH2), 1578
Light brown solid; mp: 90ꢀC to 92ꢀC; Rf: 0.44
(ethylacetate/hexane, 30/70); IR (KBr, υmax in cm−1):
3054 (C H of aromatic), 2927 (C H of aliphatic), 2845
(SCH2), 1585 (C N), 1476 (C C), 1157, 1059 (C O C),
1
(C N), 1484 (C C), 1247, 1029 (C O C), 747, 693; H
NMR (400 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H), 7.48-7.27 (m, 13H,
Ar-H), 6.91 (d, J = 8.5 Hz, 2H, Ar-H), 5.03 (s, 2H, OCH2),
4.40 (s, 2H, SCH2), 3.45-3.30 (m, 4H, CH2); 13C NMR
(100 MHz, CDCl3) δ ppm: 166.23, 164.26, 160.53, 158.54,
145.68, 136.69, 135.15, 132.20, 130.33, 128.70, 128.60,
128.53, 128.08, 127.95, 127.81, 127.59, 127.38, 126.44,
115.01, 69.96, 36.34, 24.91, 22.94; ESI-Ms m/z 546.0
(M + H)+.
1
762, 691; H NMR (500 MHz, CDCl3) δ ppm: 7.60 (dd,
J = 8.2 Hz, 4H, Ar-H), 7.47-7.29 (m, 8H, Ar-H), 6.69 (d,
J = 8.5 Hz, 2H, Ar-H), 4.44 (s, 2H, SCH2), 3.41-3.32 (m,
4H, CH2); 13C NMR (100 MHz, CDCl3) δ ppm: 166.22,
165.26, 161.53, 158.51, 146.68, 137.62, 135.12, 132.21,
130.32, 128.71, 128.65, 128.07, 127.95, 127.84, 127.69,
127.38, 126.44, 125.53, 114.01, 36.35, 23.91, 21.45; ESI-Ms
m/z 518.0 (M + H)+, 520.0 (M + 2 + H)+.
4.17 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-([4-methoxybenzyl]thio)-
1,3,4-oxadiazole (5l)
4.20 | 2-([2-chlorobenzyl]thio)-
5-(2-(4,5-diphenyloxazol-2-yl)ethyl)-
1,3,4-oxadiazole (5o)
Light yellow solid; mp: 90ꢀC to 92ꢀC; Rf: 0.43 (ethyl ace-
tate/hexane, 30/70); IR (KBr, υmax in cm−1): 3043 (C H
of aromatic), 2925 (C H of aliphatic), 2849 (SCH2), 1575
(C N), 1473 (C C), 1146, 1060 (C O C), 764, 686; H
NMR (500 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H), 7.45-7.31 (m, 8H,
White solid; mp: 84ꢀC to 86ꢀC; Rf: 0.42 (ethylacetate/hex-
ane, 30/70); IR (KBr, υmax in cm−1): 3063 (C − H of aro-
matic), 2930 (C − H of aliphatic), 2849 (SCH2), 1703
(C N), 1586 (C C), 1160, 1053 (C O C), 759, 700; H
NMR (500 MHz, CDCl3) δ ppm: 7.64 (dd, J = 8.3 Hz, 2H,
1
1

10
RAYAM ET AL.
Ar-H), 7.54 (dd, J = 8.2 Hz, 2H, Ar-H), 7.41-7.27 (m, 10H,
Ar-H), 5.39 (s, 2H, -SCH2), 3.21 (t, J = 7.4 Hz, 2H, CH2),
2.98 (t, J = 7.4 Hz, 2H, CH2); 13C NMR (125 MHz, CDCl3)
δ ppm:167.38, 166.35, 160.57, 145.75, 135.46, 129.12,
129.09, 128.92, 128.87, 128.78, 128.76, 128.66, 128.58,
128.55, 128.49, 128.14, 128.11, 128.07, 36.75, 24.96, 23.02;
ESI-Ms m/z 474.0 (M + H)+, 476.0 (M + 2 + H)+.
cells and followed by centrifugation to get cell pellet.
Fresh media was added to the pellet to make a cell count
using haemocytometer and plate 100 μL of media with
cells ranging from 5,000 to 6,000 per well in a 96-well
plate. The plate was incubated overnight in CO2 incubator
for the cells to adhere and regain its shape. After 24 hours
cells were treated with the test compounds at 25 μM
diluted using the media to deduce the percentage inhibi-
tion on cancer cells and human normal cells. The cells were
incubated for 48 hours to assay the effect of the test com-
pounds on different cell lines. Zero hour reading was noted
down with untreated cells and also control with 1% DMSO
to subtract further from the 48 hours reading. After 48 hours
incubation, cells were treated by MTT (4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide) dissolved in PBS
(5 mg/mL) and incubated for 3-4 hours at 37ꢀC. The
formazan crystals thus formed were dissolved in 100 μL of
DMSO and the viability was measured at 540 nm on a
multimode reader (Spectra max).
4.21 | 2-(2-(4,5-diphenyloxazol-2-yl)
ethyl)-5-([4-methoxyphenethyl]thio)-
1,3,4-oxadiazole (5p)
Light yellow solid; mp: 90ꢀC to 92ꢀC; Rf: 0.44 (ethyl ace-
tate/hexane, 30/70); IR (KBr, υmax in cm−1): 3055 (C H
of aromatic), 2927 (C H of aliphatic), 2845 (SCH2), 1580
1
(C N), 1447 (C C), 1146, 1024 (C O C), 760, 692; H
NMR (400 MHz, CDCl3) δ ppm: 7.62 (dd, J = 8.2 Hz, 2H,
Ar-H), 7.56 (dd, J = 8.2 Hz, 2H, Ar-H), 7.40-7.29 (m, 6H,
Ar-H), 7.14 (d, J = 8.6 Hz, 2H, Ar-H), 6.84 (d, J = 8.6 Hz,
2H, Ar-H), 3.79 (s, 3H, OCH3), 3.46-3.34 (m, 6H,
CH2 CH2 and overlapping with SCH2 CH2), 3.04 (t,
J = 7.8 Hz, 2H, SCH2 CH2); 13C NMR (125 MHz,
DMSO-d6) δ ppm: 167.08, 163.68, 161.78, 158.41, 145.31,
134.84, 132.31, 131.48, 130.07, 129.39, 129.35, 129.10,
128.75, 128.66, 127.78, 126.79, 114.25, 55.44, 34.52, 33.91,
24.63, 22.59; ESI-Ms m/z 484.0 (M + H)+.
5.2 | In vitro antibacterial activity
A collection of four organisms including Gram-positive
and Gram-negative organisms were used for this study.
Clinical isolates such as Staphylococcus aureus, Klebsiella
pneumoniae, Escherichia coli, Pseudomonas aeruginosa
were obtained from Microbiology laboratory of Osmania
Medical Hospital, Hyderabad. All strains were tested for
purity by standard microbiological methods. The bacte-
rial stock cultures were maintained on Mueller Hinton
Agar (MHA) slants and stored at 4ꢀC. Agar-well diffusion
method[37,38] was employed for evaluation of antibacterial
activity. The bacterial strains were reactivated from stock
cultures by transferring into Mueller Hinton Broth
(MHB) and incubating at 37ꢀC for 18 hours. A final inoc-
ulum containing 106 colony forming units (1 × 106 CFU/
mL) was added aseptically to MHA medium and poured
into sterile Petri dishes. Different test extracts at a con-
centration of 0.4 mg/50 μL were added to wells (8 mm in
diameter) punched on agar surface. Plates were incubated
overnight at 37ꢀC and diameter of inhibition zone (DIZ)
around each well was measured in mm. Experiments
were performed in triplicates. Antibiotics such as Cipro-
floxacin at a concentration of 0.005 mg/mL were used as
positive reference to determine sensitivity of microorgan-
isms tested. DMSO was used as Negative control.
5 | BIOLOGY
5.1 | In vitro anticancer activity
In vitro anticancer activity of the test compounds was stud-
ied using MTT colorimetric assay[36] as per ATCC protocol.
Cell lines that were used for testing in vitro cytotoxicity
included PC-3 derived from human prostate adenocarci-
noma cells (ATCC No. CRL-1435), A549 derived from
human lung carcinoma cells (ATCC No. CCL-185) and
HEK 293 derived from human embryonic kidney cells
(non-cancerous; ATCC No. CRL-1573) which were pro-
cured from American Type Culture Collection, Manassas,
VA, USA. Lung cancer cell line A549 was maintained in
DMEM medium supplemented with 10% new born calf
serum, along with 1% non-essential amino acids, 0.2%
sodium bicarbonate, 1% sodium pyruvate and 1% antibiotic
mixture (10, 000 U penicillin and 10 mg streptomycin per
mL). PC-3, HEK 293 were maintained in RPMI-1640 sup-
plemented with 10% new born calf serum (NBCS), 100 IU/
mL penicillin, 100 mg/mL streptomycin and 2 mM-gluta-
mine. Cell lines were maintained at 37ꢀC in a humidified
5% CO2 incubator (Thermo scientific). Cell lines were
processed by initial trypsinization to detach the adhered
5.3 | X-ray Crystallography
X-ray data for the compounds were collected at room tem-
perature using a Bruker Smart Apex CCD diffractometer

RAYAM ET AL.
11
[13] H. Rajak, T. B. Singh, A. Singh, K. Raghuvanshi, A. K. Sah,
R. Veerasamy, P. C. Sharma, P. R. Singh, M. D. Kharya, Bioorg.
Med. Chem. Lett. 2013, 23, 864.
[14] G. C. Ramaprasad, B. Kalluraya, B. S. Kumar, R. K. Hunnur,
Eur. J. Med. Chem. 2010, 45, 4587.
[15] N. I. Ziedan, F. Stefanelli, S. Fogli, A. D. Westwell, Eur. J. Med.
Chem. 2010, 45, 4523.
[16] B. Shivi, G. Monika, J. Chem. Pharm. Res. 2011, 3, 137.
[17] N. A. Mohamed, Polym. Degrad. Stab. 1994, 44, 33.
[18] P. Singh, P. K. Sharma, J. K. Sharma, A. Upadhyay, N. Kumar,
Org. Med. Chem. Lett. 2012, 2, 8.
[19] O. M. AboulWafa, M. A. El-Metwalli, Arch. Pharm.
(Weinheim) 1992, 325, 603.
[20] S. K. Kashaw, V. Gupta, V. Kashaw, P. Mishra, J. P. Stables,
N. K. Jain, Med. Chem. Res. 2010, 19, 250.
[21] N. R. Gollapalli, N. S. S. Kavuri, P. Kumba, S. Yangalasetty,
R. R. Nadendla, Asian J. Pharm. Anal. Med. Chem. 2015, 3, 20.
[22] S. H. Guang, T. Loon-Seng, Z. Qingdong, N. P. Paras, Chem.
Rev. 2008, 108, 1245.
[23] M. M. Babic, K. M. Antic, J. S. J. Vukovic, B. D. Bozic,
S. B. B. Davidovic, J. M. Filipovic, S. Tomic, J. Mater. Sci. 2015,
50, 906.
with graphite monochromated MoKα radiation
(λ = 0.71073 Å) with ω-scan method.[39] Preliminary lat-
tice parameters and orientation matrices were obtained
from four sets of frames. Integration and scaling of inten-
sity data were accomplished using SAINT program.[39]
The structure was solved by direct methods using
SHELXS97[40] and refinement was carried out by full-
matrix least-squares technique using SHELXL97.[40]
Anisotropic displacement parameters were included for
all non-hydrogen atoms. All H atoms were positioned geo-
metrically and treated as riding on their parent C atoms
[C H = 0.93-0.97 Å and Uiso (H) = 1.5 Ueq (C) for methyl
H or 1.2 Ueq (C) for other H atoms]. The methyl groups
were allowed to rotate but not to tip.
ACKNOWLEDGMENTS
P.R. is sincerely thankful for the financial support from
UGC-New Delhi, India in the form of fellowship. P.R.,
N.P., B.K., and J.S.A. are also thankful to TEQIP-II for
financial support to improve the laboratory facilities.
[24] G. Dannhardt, W. Kiefer, Eur. J. Med. Chem. 2001, 36, 109.
[25] K. Manjunatha, B. Poojary, P. L. Lobo, J. Fernandes,
N. S. Kumari, Eur. J. Med. Chem. 2010, 45, 5225.
ORCID
Parsharamulu Rayam https://orcid.org/0000-0002-
0108-1194
Jaya Shree Anireddy https://orcid.org/0000-0002-4714-
2044
[26] (a) P. Cikla, E. Tatar, I. Kucukguzel, F. Sahin, D. Yurdakul,
A. Basu, R. Krishnan, D. B. Nichols, N. Kaushik-Basu,
S. G. Kucukguzel, S. G. Kucukguzel, Med. Chem. Res. 2013, 22,
5685. (b) J. Deb, J. Majumder, S. Bhattacharyya, S. S. Jana, BMC
Cancer 2014, 14, 567. (c) L. F. Zerbini, M. K. Bhasin, J. F. de
Vasconcellos, J. D. Paccez, X. Gu, A. L. Kung, T. A. Libermann,
Mol. Cancer Ther. 2014, 13, 1929. (d) K. Bhaskar, P. Naveen,
R. Perla, A. Hasithashilpa, Y. Perumal, J. S. Anireddy,
D. G. Sravanthi, N. B. Bathini, RSC Adv. 2017, 7, 23680.
(e) K. Bhaskar, R. Perla, P. Rayam, T. R. Allaka, A. Hasithashilpa,
Y. Perumal, S. Balasubramanian, D. G. Sravanthi, N. B. Bathini,
J. S. Anireddy, ChemistrySelect 2018, 3, 5050.
REFERENCES
[1] S. Detchokul, E. D. Williams, M. W. Parker, A. G. Frauman,
Br. J. Pharmacol. 2014, 171, 5462.
[2] L. A. Torre, F. Bray, R. L. Siegel, J. Ferlay, J. Lortet-Tieulent,
A. Jemal, CA Cancer J. Clin. 2015, 65, 87.
[3] P. Puthiyapurayil, B. Poojary, C. Chikkanna, S. K. Buridipad,
Eur. J. Med. Chem. 2012, 53, 203.
[27] T. S. T. Kamijo, T. K. Y. Nagatomi, Int. J. Urol. 2001, 8, 35.
[28] B. D. Bozic, J. R. Rogan, D. D. Poleti, N. P. Trisovic,
B. D. Bozic, G. S. Uscumlic, Chem. Pharm. Bull. 2012, 60, 865.
[29] (a) N. Polkam, P. Rayam, J. S. Anireddy, S. Yennam,
H. S. Anantaraju, S. Dharmarajan, Y. Perumal, S. S. Kotapalli,
R. Ummanni, S. Balasubramanian, Bioorg. Med. Chem. Lett.
2015, 25, 1398. (b) N. Polkam, V. R. Ramaswamy, P. Rayam,
T. R. Allaka, H. S. Anantaraju, S. Dharmarajan, Y. Perumal,
D. Gandamalla, N. R. Yellu, S. Balasubramanian, Bioorg. Med.
Chem. Lett. 2016, 26, 2562. (c) N. Kuntala, J. Telu, V. Banothu,
S. B. Nallapati, J. S. Anireddy, S. Pal, Med. Chem. Commun.
2015, 6, 1612. (d) T. R. Allaka, N. Polkam, P. Rayam,
J. Sridhara, N. S. Garikapati, R. U. Kotapalli, J. S. Anireddy,
Med. Chem. Res. 2016, 25, 977. (e) J. Mareddy, S. B. Nallapati,
J. Anireddy, Y. P. Devi, L. P. Mangamoori, R. Kapavarapu,
S. Pal, Bioorg. Med. Chem. Lett. 2013, 23, 6721.
[4] M. X. Song, C. J. Zheng, X. Q. Deng, L. P. Sun, Y. Wu,
L. Hong, Y. J. Li, Y. Liu, Z. Y. Wei, M. J. Jin, H. R. Piao, Eur.
J. Med. Chem. 2013, 60, 376.
[5] C. S. De Oliveira, B. F. Lira, J. M. Barbosa-Filho,
J. G. F. Lorenzo, P. F. De Athayde-Filho, Molecules 2012, 17,
10192.
[6] Z. Liu, J. Zhao, X. Huang, Bioorg. Med. Chem. Lett. 2006, 16,
1828.
[7] K. A. Kumar, P. Jayaroopa, G. V. Kumar, Int. J. ChemTech Res.
2012, 4, 1782.
[8] J. Bostrom, A. Hogner, A. Llinas, E. Wellner, A. T. Plowright,
J. Med. Chem. 2012, 55, 1817.
[9] F. Zhang, X. L. Wang, J. Shi, S. F. Wang, Y. Yin, Y. S. Yang,
W. M. Zhang, H. L. Zhu, Bioorg. Med. Chem. 2014, 22, 468.
[10] F. Macaev, Z. Ribkovskaia, S. Pogrebnoi, V. Boldescu,
G. Rusu, N. Shvets, A. Dimoglo, A. Geronikaki, R. Reynolds,
Bioorg. Med. Chem. 2011, 19, 6792.
[30] J. Sindhu, H. Singh, J. M. Khurana, C. Sharma, K. R. Aneja,
Aust. J. Chem. 2013, 66, 710.
[11] K. K. Jha, A. Samad, Y. Kumar, M. Shaharyar, R. L. Khosa,
J. Jain, V. Kumar, P. Singh, Eur. J. Med. Chem. 2010, 45, 4963.
[12] J. N. Sangshetti, A. R. Chabukswar, D. B. Shinde, Bioorg. Med.
Chem. Lett. 2011, 21, 444.
[31] Z. Jakopin, M. S. Dolenc, Curr. Org. Chem. 2008, 12, 850.
[32] J. F. Tang, X. H. Lv, X. L. Wang, J. Sun, Y. B. Zhang,
Y. S. Yang, H. B. Gong, H. L. Zhu, Bioorg. Med. Chem. 2012,
20, 4226.

12
RAYAM ET AL.
[33] R. R. Somani, P. Y. Shirodkar, Der Pharma Chem. 2009, 1, 130.
[34] (a) A. Sharma, P. Appukkuttan, E. Van der Eycken, Chem.
Commun. (Camb.) 2012, 48, 1623. (b) L. S. Breunig,
M. E. Olson, D. A. Harki, Tetrahedron Lett. 2016, 57, 4056.
(c) S. M. Gomha, S. M. Riyadh, Molecules 2011, 16, 8244.
(d) S. Faraji, F. N. Ani, J. Power Sources 2014, 263, 338.
(e) X. Rui, Q. Yan, M. Skylas-Kazacos, T. M. Lim, J. Power
Sources 2014, 258, 19.
[39] B. Sridhar, K. K. Ravi, Acta Crystallogr. 2007, E63, 2887.
[40] B. Christian, G. Hubschle, M. Sheldrick, B. Dittrich, J. Appl.
Crystallogr. 2011, 44, 1281.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[35] CCDC 1471510 contains supplementary Crystallographic data
for the structure 5b. These data can be obtained free of charge
at www.ccdc.cam.ac.uk/conts/retrieving.html.
[36] (a) M. F. Abu Bakar, M. Maryati, A. Rahmat, S. A. Burr,
J. R. Fry, Food Chem. Toxicol. 2010, 48, 1688. (b) D. Gerlier,
N. Thomasset, J. Immunol. Methods 1986, 94, 57.
How to cite this article: Rayam P, Polkam N,
Kuntala N, et al. Design and synthesis of
oxaprozin-1,3,4-oxadiazole hybrids as potential
anticancer and antibacterial agents. J Heterocyclic
Chem. 2020;1–12. https://doi.org/10.1002/jhet.3842
[37] B.
Venkanna,
A.
Uma,
C.
H.
Suvarnalaxmi,
N. Chandrasekharnath, R. S. Prakasham, L. Jayalaxmi, Curr.
Trends Biotechnol. Pharm. 2013, 7, 782.
[38] V. Banothu, C. Neelagiri, U. Adepally, J. Lingam,
K. Bommareddy, Pharm. Biol. 2017, 55, 1155.