Recyclable CuO nanoparticles-catalyzed synthesis of novel-2,5-disubstituted 1,3,4-oxadiazoles as antiproliferative, antibacterial, and antifungal agents

Article abstract of DOI:10.1007/s00044-014-1025-x

A series of new 2,5-disubstituted 1,3,4-oxadiazoles have been conveniently synthesized through an oxidative C-O coupling by direct C-H bond activation of N-aroyl-N-arylidinehydrazines using a catalytic quantity of CuO nanoparticles. Twenty compounds have been synthesized in good to excellent yields (75-90 %). All the synthesized compounds were evaluated for their in vitro antiproliferative, antibacterial, and antifungal activity. Compounds 8d and 10d are more promising antiproliferative agents with IC₅₀ value of 3.66 and 3.89 μM in MCF-7 cell line, and compounds 8a and 10a were showed more potent antifungal activity than standard drug.

Full text of DOI:10.1007/s00044-014-1025-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1025-x
ORIGINAL RESEARCH
Recyclable CuO nanoparticles-catalyzed synthesis of novel-2,5-
disubstituted 1,3,4-oxadiazoles as antiproliferative, antibacterial,
and antifungal agents
•
M. S. R. Murty Raju Penthala Sudheer Kumar Buddana
•
•
•
R. S. Prakasham Pompi Das Sowjanya Polepalli
•
•
•
N. Jain Sreedhar Bojja
Received: 13 February 2014 / Accepted: 25 April 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of new 2,5-disubstituted 1,3,4-oxadi-
azoles have been conveniently synthesized through an
oxidative C–O coupling by direct C–H bond activation of
N-aroyl-N-arylidinehydrazines using a catalytic quantity of
CuO nanoparticles. Twenty compounds have been syn-
thesized in good to excellent yields (75–90 %). All the
synthesized compounds were evaluated for their in vitro
antiproliferative, antibacterial, and antifungal activity.
Compounds 8d and 10d are more promising antiprolifer-
ative agents with IC50 value of 3.66 and 3.89 lM in MCF-7
cell line, and compounds 8a and 10a were showed more
potent antifungal activity than standard drug.
Introduction
Among five-membered aromatic heterocycles, 1,3,4-oxa-
diazoles are of particular interest in different areas of
medicinal and pesticide chemistry (Dogan et al., 2002; Shi
et al., 2001). Within drug discovery and development, a
number of compounds containing an oxadiazole moiety are
in late stage clinical trials, including Zibotentan as an
anticancer agent (James and Growcott, 2009), Raltegravir
as an antiretroviral drug for the treatment of HIV infection
(Summa et al., 2008), and ABT-751 as tubulin polymeri-
zation inhibitor (Ouyang et al., 2006). It is clear that
oxadiazoles are having a large impact on multiple drug
discovery programs across a variety of disease areas,
including anticancer, antibacterial, antifungal, antiviral,
analgesic, anti-inflammatory, antihypertensive, anticon-
Keywords 1,3,4-Oxadiazoles Á CuO nanoparticles Á
Antiproliferative Á Antibacterial Á Antifungal activity
¨
vulsant, and anti-diabetic properties (Bostrom et al., 2012;
Oliveira et al., 2012).
Natural products represent a significant source of inspi-
ration for the design of structural analogs with improved
pharmacological profiles. As we know, salicylic acid was
identified in willow bark extracts as an active anti-inflam-
matory compound. To our knowledge, the natural products
salicylihalamides A and B are potent inhibitors of the vac-
uolar ATPase (V-ATPase) and are anticancer agents (Su-
gimoto et al., 2009). In particular, oxadiazoles derived from
salicylic acid were established as hypnotic drug Fenadiazole
(1), anticancer (2) (Aboraia et al., 2006), antiproliferative,
EGFR inhibitory (3) (Liu et al., 2012), PI3K inhibitory,
potential immunosuppressive (4) (Zhang et al., 2012), anti-
convulsant activities (5) (Zarghi et al., 2005a), and oxadi-
azoles derived from thiosalicylic acid act as benzodiazepine
receptor agonists (6) (Zarghi et al., 2005b) (Fig. 1).
Due to the biological significance of oxadiazoles that are
derived from salicylic acid and thiosalicylic acid, we have
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1025-x) contains supplementary
material, which is available to authorized users.
M. S. R. Murty (&) Á R. Penthala
Medicinal Chemistry and Pharmacology Division, Discovery
Laboratory, Indian Institute of Chemical Technology, Uppal
Road, Hyderabad 500007, India
e-mail: msrmurty@ymail.com; msrmurty@iict.res.in
S. K. Buddana Á R. S. Prakasham
Bioengineering and Environmental Center, Indian Institute of
Chemical Technology, Uppal Road, Hyderabad 500007, India
P. Das Á S. Polepalli Á N. Jain
Centre for Chemical Biology, Indian Institute of Chemical
Technology, Uppal Road, Hyderabad 500007, India
S. Bojja
Inorganic and Physical Chemistry Division, Indian Institute of
Chemical Technology, Uppal Road, Hyderabad 500007, India
123

Med Chem Res
Fig. 1 Representative structure of biological active oxadiazoles derived from salicylic and thiosalicylic acid
developed a new protocol for the synthesis of 2,5-disub-
stituted 1,3,4-oxadiazoles from salicylic and thiosalicylic
acid with an expectation to find more potent antiprolifer-
ative, antibacterial, and antifungal agents.
groups as well as intra-molecular nucleophiles, are ideal,
and atom-economical processes can be used for the con-
struction of heterocyclic architectures (Stokes and Driver,
2011). A few reports have appeared in the literature on Cu
catalyzed, C(sp3)–H, and C(sp2)–H bond activation and its
further application to C–O bond formation (Kumar et al.,
2011; Saberi and Heydari, 2013; Zhao et al., 2012; Zhang,
2010). Therefore, it is of utmost importance to develop
economically and environmentally more viable procedure
for the synthesis of 2,5-disubstituted 1,3,4-oxadiazoles
from N-aroyl-N-arylidinehydrazines utilizing recyclable
CuO nanoparticles as catalyst.
Various reagents have been reported for the synthesis of
2,5-disubstituted 1,3,4-oxadiazoles such as sodium chlorate
platinum (Singh et al., 2013), hypervalent iodine’s (Prakash
et al., 2010), ceric ammonium nitrate (Dabiri et al., 2006),
chloramine-T (Jedlovska and Lesko, 1994), lead dioxide in
acetic acid (Milcent and Barbier, 1983), bromine/sodium
acetate (Werber et al., 1977), potassium permanganate
(Rostamizadeh and Housaini, 2004), mercuric oxide/iodine
(Flidallah et al., 2002), trichlorophosphate (Lee et al.,
2001), phosphorus oxychloride (Al-Talib et al., 1990),
sulphuric acid (Short and Long, 1969), Cu(OTf)2 (Guin
et al., 2011), and CuI (Kawano et al., 2009).
Nano CuO is a heterogeneous catalyst and has become
an attractive both from economic and industrial points. The
CuO nano-catalyzed reactions provide the advantages of
high atom efficiency, simplified isolation of product easy
recovery, and recyclability of the catalyst in several
organic transformations (Babu and Ramasamy, 2011; Rout
et al., 2012; Kantam et al., 2008).
Yet, the disadvantages associated with these methods
are usage of expensive reagents, hazardous materials, multi
step processes, long reaction times, and formation of
undesired side products. In case of Cu(OTf)2 catalyzed
imine C–H functionalization, and C–H activation/arylation
of 2-substituted 1,3,4-oxadiazole with CuI seems some
advantages, but these catalytic systems i.e., Cu(OTf)2 and
CuI are not recyclable.
Inspired by the recent advantages of Cu-catalyzed C–H
functionalization processes, we hypothesized that inex-
pensive CuO nano-catalyst should also be capable of pro-
moting C–O cyclization reactions of N-aroyl-N-
arylidinehydrazine derivatives. Below, we describe a new
process for the preparation of 2,5-disubstituted 1,3,4-oxa-
diazoles through aerobic imine C(sp2)–H functionalization
starting with N-aroyl-N-arylidinehydrazines. The method-
ology relies on a simple reaction system and inexpensive
CuO nano as a catalyst.
The C–H functionalization strategy has received sub-
stantial attention because of its economic, sustainable, and
environmentally benign alternative for C–C and C–het-
eroatom bond formation. In the case of intra-molecular
C–H hetero functionalization, heteroatoms act as directing
123

Med Chem Res
Scheme 1 Synthesis of N-aryl-N-arylidinehydrazine derivatives from salicylate or methyl thiosalicylate. Reagents and conditions: (i) allyl (or)
benzyl chloride, KOH, MeOH, 6 h reflux; (ii) NH2NH2.H2O, MeOH, 5 h reflux; (iii) R2-CHO, AcOH, EtOH, 2 h reflux
Scheme 2 CuO nanoparticle catalyzed synthesis of 2,5-disubstituted 1,3,4-oxadiazoles 8–11(a–e). Reaction conditions: N-aryl-N-arylidinehydr-
azines 4–7(a–e) (1.0 mmol), CuO nanoparticles (0.1 mmol), Cs2CO3 (1.0 mmol), in DMSO, 9–10 h at 80 °C
Results and discussion
products 8–11(a–e) were formed in good yields (75–90 %).
The structures of the products 8–11(a–e) (Scheme 2,
Table 1) were confirmed by NMR and HRMS spectral
data.
Chemistry
Considering the biological significance of 1,3,4-oxadiaz-
oles in particular derived from salicylic acid and thiosali-
cylic acid, we have developed a straightforward and
versatile protocol with CuO nano for the synthesis of a
novel series 2,5-disubstituted 1,3,4-oxadiazoles. CuO
nano-catalyst successfully performs the C–H functionali-
zation followed by an intra-molecular C–O cross-coupling
to afford cyclized products from N-aroyl-N-arylidinehydr-
azine derivatives.
The occurrence of the above reaction is based on both
the catalyst and base. In the absence of either catalyst or
base, the reaction did not yield any product even after long
reaction times, which suggests that a metal/base combi-
nation is required for the reaction to occur.
Optimization of reaction conditions
N-aroyl-N-arylidinehydrazine derivatives were synthe-
sized according to Scheme 1. Reaction of methyl salicylate
or methyl thiosalicylate 1 with appropriate allyl or benzyl
chloride methanolic KOH solution afforded corresponding
products 2(a–d). Acid hydrazides 3(a–d) were readily
prepared by treatment of 2(a–d) with hydrazine hydrate in
methanol (Zarghi et al. 2005b). Acid hydrazides 3(a–
d) were refluxed with aryl aldehydes in the presence of
acetic acid afforded the pure N-aroyl-N-arylidinehydra-
zines 4–7(a–e) (Okimoto and Chiba, 1990; Prakash et al.,
2010).
Initially, the reaction conditions such as solvent, base,
amount of base, amount of catalyst, and temperature were
optimized. The data are listed in Table 2.
From the data given in Table 2, it is concluded that
among the five different solvents such as acetonitrile
(CH3CN), N,N-dimethylformamide (DMF), dimethyl sulf-
oxide (DMSO), ethanol (EtOH), and water (H2O), the
solvent dimethyl sulfoxide is an efficient one (Table 2,
entries 1–5). Similarly among the various carbonate salts
like Na2CO3, K2CO3 and Cs2CO3, Cs2CO3 is found to be a
better one (Table 2, entries 3, 6 and 7). A 10 mol% amount
of CuO nanoparticles is found to be the optimum amount of
catalyst (Table 2, entries 3, 10, 11 and 12). Excess or less
than 10 mol% CuO decreases the yield of product. It is
noted that temperature has significant effect on the effi-
ciency of present catalytic system. The product 11a was
obtained in 90 % yield after stirring at 80 °C in DMSO.
In a typical procedure, N-aroyl-N-arylidinehydrazines
4–7(a–e) (1.0 mmol) was carried out with Cs2CO3
(1.0 mmol) and commercially available CuO nano
(\50 nm particle size) (10 mol %) in DMSO by stirring at
80 °C for 9–10 h, under an air atmosphere. After com-
pletion of the reaction (monitored by TLC), expected
123

Med Chem Res
Table 1 Synthesis of 2,5-disubstituted 1,3,4-oxadiazoles 8–11(a–e)b,c
Entry
Compound
Time (h)
9.5
Yield (%)a
83
mp (°C)
8a
98–100
N
N
O
N
S
O
8b
8c
10
10
81
75
127–129
76–78
N
N
O
O
S
F
N
N
O
F
S
F
F
8d
8e
10
10
77
79
82–84
N
N
F
O
F
F
S
O
102–104
N
N
O
O
S
N
N
9a
9b
9
88
82
176–178
188–190
O
N
S
O
N
9.5
N
O
O
S
F
N
9c
N
S
9
81
118–120
O
F
123

Med Chem Res
Table 1 continued
Entry
Compound
Time (h)
10
Yield (%)a
84
mp (°C)
9d
138–140
F
F
N
N
F
O
F
F
S
O
N
9e
9.5
86
85
80
79
78
77
90
83
157–159
103–105
144–146
79–81
N
O
O
S
10a
10b
10c
l0d
l0e
N
N
9.5
10
10
10
10
9
O
N
O
N
O
N
O
O
O
N
F
N
O
F
O
N
F
F
117–119
109–111
101–103
177–179
N
F
O
F
F
O
N
O
N
O
O
O
N
N
11a
11b
O
N
O
N
O
N
9.5
O
O
O
123

Med Chem Res
Table 1 continued
Entry
Compound
Time (h)
Yield (%)a
85
mp (°C)
F
11c
130–132
N
9
N
O
F
O
F
F
N
11d
11e
N
O
N
88
134–136
10
F
O
F
F
O
N
84
143–145
9.5
O
O
O
a
Isolated yield
b
Reaction conditions: N-aroyl-N-arylidinehydrazines 4–7(a–e) (1.0 mmol), CuO nano (10 mol%), Cs2CO3 (1.0 mmol), in DMSO
c
Structures of synthesized compounds was confirmed by NMR and HRMS spectral data
Lower or higher temperature makes the reaction slower
form an active five coordinated intermediate (B) (Guin
et al., 2011; Rout et al., 2012) under basic condition, which
on oxidative addition may give intermediate (C) (Zhang,
2010; Guin et al., 2011). The intermediate (C) undergoes
an intra-molecular C–O bond formation via reductive
elimination to afford the product and regenerating the
catalyst under oxygen atmosphere.
(Table 2, entries 13, 14, 15 and 16). When the reaction was
operated in nitrogen, the cyclizations were considerably
less efficient, suggesting that O2 played a vital role in the
catalytic cycle.
With these results in hand, we sought to examine the
scope and generality of the method. N-aroyl-N-aryl-
idinehydrazines derived from arylaldehydes possessing di-
OMe as electron-donating group gave substituted 1,3,4-
oxaziazoles 8e, 9e, 10e, and 11e in good yields. Similarly
substrates possessing F as electron-withdrawing group
underwent C–H functionalization to give 1,3,4-oxaziazoles
8c, 8d, 9c, 9d, 10c, 10d, 11c, and 11d in better yields.
N-aroyl-N-arylidinehydrazines substrates originating from
heterocyclic aldehyde like pyridine gave excellent yields of
the products 8a, 9a, 10a, and 11a. Also it was observed that
electron-withdrawing substituent on the arylaldehydes
improved the yields as in the case of 10c, 10d, 11c, and
11d probably through an increment of the acidity of the
imine proton thereby facilitating the metalation step.
On the basis of observation, a typical mechanism pro-
posed for CuO nanoparticles-catalyzed oxidative C–O
coupling by direct C–H bond activation of N-aroyl-N-
arylidinehydrazines is given in Scheme 3. CuO nanopar-
ticles (A) bearing high reactive morphologies expectedly
In the next step, the recycling ability of the catalyst was
studied, since it is very important for industrial applications.
After completionofthe first reaction, the reaction mixture was
centrifuged to separate the catalyst and washed several times
with ethyl acetate and oven dried at 80 °C overnight. A new
second reaction was then performed with fresh solvent and
reactant under identical conditions. Using this approach, our
catalyst could be reused for three times with almost consistent
activity (Table 3). After three cycles, the fresh and used cat-
alysts were well characterized by powder XRD, SEM, and
TEM. The crystallinity of CuO nanoparticles (NPs) was
confirmed by powder X-ray diffraction (Fig. 2), and the
observed diffraction peak positions and intensities are in good
agreement with the reported values (JCPDS file No. 05-661)
and could be indexed with lattice planes. These NPs retained
˚
itsmonoclinic structure with latticeparameters:a = 4.688 A,
˚
˚
˚
b = 3.422 A, c = 5.131 A, b = 99.506, and V = 82.31 A.
SEM and TEM techniques were employed to study the
123

Med Chem Res
Table 2 Screening of reaction conditions for compound 11a
S.NO
Solvents
Base
Amount of base (mmol)
Amount of catalyst (mol%)
Temp (°C)
Yield (%)a
1
CH3CN
DMF
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Na2CO3
K2CO3
1
1
1
1
1
1
1
2
3
1
1
1
1
1
1
1
10
10
10
10
10
10
10
10
10
0
80
80
80
80
80
80
80
80
80
80
80
80
40
60
100
120
45
2
82
3
DMSO
EtOH
90
4
30
5
H2O
Trace
6
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
75
7
80
8
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
39
9
26
10
11
12
13
14
15
16
No reaction
5
50
48
23
40
32
18
15
10
10
10
10
All the reactions were performed with 1.0 mmol of 2-(benzyloxy)-N’-(pyridin-3-ylmethylene)benzohydrazide
a
Isolated yield
Table 3 Recycling of the catalysta
No of cycle
Yield (%)b
recovery (%)
1
2
3
a
90
87
85
96
94
90
Reactions were monitored by TLC
Isolated yield
b
morphologies and particle size of the fresh and the recovered
catalysts after three cycles as represented in Figs. 3 and 4.
Selected Area Electron Diffraction (SAED) patterns shown as
inset in Fig. 4 are also in consonance with the power XRD
patterns reconfirming that the crystalline nature of the CuO
nanoparticles is intactevenafter threecyclesand has excellent
reusability and chemical stability.
Scheme 3 Proposed mechanism for CuO nanoparticles-catalyzed
oxidative C–O coupling by direct C–H bond activation of N-aryl-N-
arylidinehydrazines
123

Med Chem Res
Pharmacology
the human cell lines, some bacterial and fungal strains in
comparison with some standard drugs in a trial to get more
potent and less toxic agents.
The aim of the present study was to illustrate the effect of
newly synthesized 2,5-disubstituted 1,3,4-oxadiazoles on
Antiproliferative activity
The prepared oxadiazoles were evaluated to determine their
antiproliferative activities against a panel of four different
human tumor cells from cervix(HeLa), breast(MCF-7),
lung(A549), and neuroblastoma(IMR32) (HeLa, MCF7,
A549, and IMR32 were obtained from American Type
culture collection) using the sulforhodamine B (SRB) assay
method (Reddy et al., 2011). Combretastatin A-4 was used
as standard drug. The IC50 values are listed in Table 4.
From the screening results in Table 4, it was observed
that compounds 8–11(a–e) exhibited moderate to good
antiproliferative activity. Two of the most active com-
pounds are 8d and 10d, with IC50 values against the four
tested human cancer cell lines ranging from 3.66 to
7.03 lM and 3.89 to 4.86 lM, respectively. Compound
10d was more cytotoxic than CA4 against all tested four
human cancer cell lines, while 8d was less active than CA4
only against HeLa cells. We have also observed that
compound 9d was more potent against MCF-7 with IC50
Fig. 2 Powder XRD patterns a fresh and b after third cycle of CuO
nanoparticles
Fig. 3 Scanning Electron
Microscope (SEM) images
a fresh and b after third cycle of
CuO nanoparticles
Fig. 4 Transmission Electron Microscope (TEM) images a fresh and b after third cycle of CuO nanoparticles
123

Med Chem Res
Table 4 Cytotoxic effects of compounds 8–11(a–e) on HeLa, MCF7,
A549, and IMR32 human cancer Cellsa
Table 5 In vitro Antibacterial activities of oxadiazoles 8–11(a–
e) using well-plate method
Entry IC50 (lM)b
Entry
Diameter of growth of inhibition zone (mm)a
HeLa
MCF-7
A549
IMR32
E. coli
B. subtilis
M. luteus
K. pnemoniae
8a
12.69 1.5
21.16 2.5
19.09 2.1
7.03 3.2
11.66 1.9
15.09 2.7
27.3 2.8
3.66 – 2.7
33.64 1.9
13.32 1.0
12.72 1.2
6.85 – 2.0
10.68 1.3 [100 2.5
15.04 4.3 [100 6.2
19.61 1.0
29.15 1.6
42.23 9.5
4.93 – 3.9
8a
24
18
21
17
23
17
0
20
15
14
13
22
16
12
0
25
17
18
17
25
19
17
15
16
20
14
14
17
15
15
15
15
16
17
16
33
23
18
16
13
23
20
13
11
0
8b
8b
8c
8c
8d
8d
8e
32.6 2.3 [100 2.5
8e
9a
27.96 4.4
17.6 1.6
30.46 9.2
11.48 2.4
25.95 2.7
13.78 1.3
13.4 8.6
9.55 2.0
4.46 – 3.0
14.02 2.7
9.55 2.3
18.32 2.3
12.97 1.1
5.44 – 2.7
18.08 1.2
28.35 1.6
36.76 1.2
4.51 – 2.5
18.7 1.7
12.92 1.7
9.83 5.8
6.82 2.6
3.89 – 3.6
10.02 1.4
8.73 3.6
8.44 2.0
8.14 9.6
21.81 4.0
6.91 2.0
4.63 0.15
9a
9b
16.36 1.4
20.03 8.3
18.77 1.4
23.03 4.9
24.16 9.8
36.2 3.7
13.58 2.1
4.86 – 2.1
29.3 5.2
17.04 1.2
24.96 1.2
23.02 9.3
6.14 – 2.2
42.58 1.4
80.8 4.8
9.22 3.5
55.14 1.5
10.53 6.5
10.2 6.4
8.74 6.5
4.28 – 3.6
11.2 3.3
11.95 2.1
13.21 2.1
19.46 1.0
9.08 2.0
15.13 4.2
5.72 0.13
9b
9c
9c
0
9d
9d
0
0
9e
9e
14
0
17
0
18
0
10a
10b
10c
l0d
l0e
11a
11b
11c
11d
11e
CA4c
10a
10b
15
16
15
20
20
18
14
16
14
36
20
12
12
18
14
15
13
13
13
33
22
14
14
17
17
14
13
16
13
35
10c
l0d
l0e
11a
11b
11c
11d
8.75 1.8
17.39 1.3
11e
5.36 0.12
6.45 0.19
Streptomycin
a
The compounds which show good activity are in bold
Values, including diameter of the well, means of three replicates
a
Cell lines were treated with different concentration of compounds
for 48 h as mentioned in materials and methods section. Cell viability
was measured employing SRB assay
enzymes. It has been evident from that fluorine-substituted
heterocycles have got significant place in modern medici-
nal chemistry (Pushpan et al., 2012).
b
IC50 values (in lM) are indicated as mean SD (standard devi-
ation) of three independent experiments
c
Combretastatin A-4 (CA4) was used as reference drug
value 4.51 lM, and compound 11d was active against
A549, HeLa with IC50 value 6.14, 5.44 lM, respectively.
In case of 11(a–e) series, all the compounds showed sig-
nificant cytotoxicity; compounds 11a and 11e are showed
IC50 value ranging from 6.91 to 9.55 lM against MCF7
and HeLa cell lines, while the compounds 11b and 11c are
showed IC50 value 8.44 and 8.14 lM only on MCF7 cell
line. All other remaining compounds 8(a–c), 8e, 9(a–c), 9e,
10(a–c), 10c, and 10e showed moderate activity. Among
the synthesized compounds, the compounds having per-
fluorophenyl (8d, 9d, 10d, 11d) substitution at 5-position
to 1,3,4-oxadiazole ring have shown significant effect
against all the cell lines screened. The increased activity is
attributed to the presence of fluorine atoms (highly elec-
tronegative) in the molecules, which increases the lipo-
philicity and affects the partitioning of molecules into
membranes and facilitates hydrophobic interactions of the
molecules with specific binding sites on either receptors or
Antibacterial activity
All the synthesized compounds 8–11(a–e) were also eval-
uated for their in vitro antibacterial activity against two
gram-positive bacteria namely, Bacillus subtilis (MTCC
736), Micrococcus luteus (MTCC 106), two gram-negative
bacteria namely, Escherichia coli (MTCC 40) and Kleb-
siella pneumoniae (MTCC 3384) by using broth dilution
method (Prakash et al., 2011). Streptomycin was used as a
control drug for antibacterial activity. The results of the
antibacterial studies are presented in Tables 5 and 6. In
general, the antibacterial activity profile differed with type
of bacterial strain and nature of compounds. For example,
compounds 8a and 8e displayed moderate inhibitory
activity on both gram-positive and gram-negative bacteria.
In case of gram-positive bacteria, 8b, 9a, 9e, 10e, and 11b
compounds, while in the case of gram-negative bacteria,
8b, 8c, 9a, 10b, 10e, 11a, and 11d compounds showed
123

Med Chem Res
Table 6 Minimum inhibitory concentrations (MIC) (in lg/mL) of
compounds 8–11(a–e) using broth dilution method
Table 7 In vitro antifungal activities of oxadiazoles 8–11(a–e) using
well-plate method
Entry
MlC (lg/mL)a
8aEntry
Diameter of growth of inhibition Zone (mm)a
E. coli
B. subtilis
M. luteus
K. pnemoniae
A. niger
A. terreus
A. fumigatus
8a
37.5
50
37.5
100
100
100
37.5
100
100
na
37.5
75
37.5
37.5
75
8a
40
15
0
30
12
0
30
20
21
17
0
8b
8b
8c
50
75
8c
8d
75
75
100
37.5
50
8d
15
0
13
0
8e
37.5
75
37.5
50
8e
9a
9a
0
0
0
9b
na
75
100
150
na
9b
0
0
0
9c
na
150
150
50
9c
0
0
0
9d
na
na
9d
0
0
0
9e
100
na
75
50
9e
0
0
0
10a
na
150
100
100
150
150
75
na
10a
35
15
14
15
20
20
18
14
16
14
20
35
12
14
12
18
14
15
13
13
13
22
30
14
16
15
15
15
15
16
17
16
25
10b
150
150
150
75
75
37.5
100
100
75
10b
10c
150
150
100
150
75
10c
l0d
10d
l0e
10e
11a
75
75
11a
l1b
75
75
150
150
100
150
10
11b
11c
150
100
100
10
150
150
150
12
100
100
150
12
11c
l1d
11d
l1e
11e
Streptomycin
Cycloheximide
a
a
Mean of three replicates, na not active
Values, including diameter of the well, means of three replicates
antibacterial activity ranging from 15 to 20 mm zone, and
the compounds 8a, 8e, 9a, 10b, 10e, 11a exhibited efficient
zone of inhibition (Table 5). The compounds 8d, 10c, 10d,
11c, 11e were possessed moderate efficiency in inhibiting
the growth of all tested strains. Further E.coli growth was
not inhibited by 9b, 9c, 9d, and 10a. Among all the newly
synthesized compounds 8a, 8e showed broad spectrum
antibacterial activity (for all tested strains MIC 37.5 lg/
mL), and compounds 8b, 10b showed antibacterial activity
against Klebsiella pneumoniae (MIC 37.5 lg/mL)
(Table 6). Critical evaluation of the above data suggested
that each of the synthesized compounds is specific in nature
especially in regulating the microbial growth.
showed maximum zone of inhibition. Among these 8a and
10a, compounds showed superior antifungal activity as
compared with standard drug cycloheximide (Table 7).
This manifested that an allyloxy and allylthio group-con-
taining oxadiazole derivatives (8a and 10a) exhibited
excellent antifungal activity rather than benzyloxy and
benzylthio group containing oxadiazole derivatives (9a and
11a), while the compounds 10b and 10c showed promising
activity. Compounds 8e, 9a–e did not exhibit antifungal
activity. The compound 8c depicted more efficient zone of
inhibition against A. fumigatus and is not effective against
A. niger and A. terreus. From the data, the compound 8a
revealed 40 mm of inhibitory zone against A. niger, while
standard antifungal drug cycloheximide showed only
20 mm of inhibitory zone for the same fungal strains.
Similar trend has been noticed with compound 10a
(Table 7). The evaluation data indicated that the presence
of pyridyl group on the 5-position of oxadiazole moiety
and the allyl substituent attached to phenolic or thiophen-
olic functionality are more effective for antifungal activity.
To the best our knowledge, such type of work has not been
reported so far.
Antifungal activity
The in vitro antifungal activity of synthesized compounds
8–11(a–e) was determined against three fungal strains
belonging to Aspergillus genus by using well-plate method
(Prakash et al., 2011; Zovko et al., 2012). The results are
summarized in Table 7. It is evident that among all the
compounds 8a, 8b, 8d, 10a, 10d, 10e, 11a, 11b, and 11d
123

Med Chem Res
2-(2-(allylthio)phenyl)-5-(benzo[d][1,3]dioxol-5-yl)-
Experimental protocols
General
1,3,4-oxadiazole (8b)
1
Light gray solid; Yield: 81 %; mp 127–129 °C; H NMR
(CDCl3, 500 MHz): d 8.0 (d, J = 8.0 Hz, 1H, Ar–H), 7.70
(d, J = 8.0 Hz, 1H, Ar–H), 7.60 (d, J = 2.0 Hz, 1H, Ar–
H), 7.50–7.43 (m, 2H, Ar–H), 7.33–7.28 (m, 1H, Ar–H),
6.97–6.92 (m, 1H, Ar–H), 6.07 (s, 2H, O–CH2–O),
5.96–5.86 (m, 1H, =CH), 5.27 (dd, J = 17.0, 1.0 Hz, 1H,
=CH2), 5.15 (dd, J = 10.0, 1.0 Hz, 1H, =CH2), 3.67 (d,
J = 6.0 Hz, 2H, S–CH2); 13C NMR (CDCl3, 75 MHz): d
164.1, 163.3, 150.5, 148.1, 137.8, 132.5 (=CH), 131.0,
129.8, 128.1, 125.1, 122.0, 118.5 (=CH2), 108.7, 106.9,
Analytical thin-layer chromatography (TLC) was carried out
using silica gel 60 F254 pre-coated plates. Visualization was
accomplished with UV lamp or I2 stain. All products were
characterized bytheir NMR and HRMSspectra. The 1H NMR
(300 and 500 MHz) spectra were recorded on Bruker Avance
spectrometer, and 13C NMR (75 MHz) spectra were recorded
on Bruker Avance spectrometers using TMS as an internal
standard; Chemical shifts were reported in parts per million
(ppm, d) downfield from tetra methyl silane. ESI and HRMS
were recorded on ‘‘High Resolution QSTAR XL hybrid MS/
MS system, Applied bio systems’’ under Electron Spray
Ionization conditions preparing sample solutions in methanol.
Powder X-ray diffraction (XRD) analyses were performed
using a Make Bruker, Model: D8-Advance, Detector: Lynx-
Eye. Scanning Electron Microscopy (SEM) analyses were
performed using a Make: Hitachi S- 3000 N scanning elec-
tron microscope. Transmission Electron Microscope (TEM)
analyses were performed using Philips TECNAI F12 FEI
transmission electron microscope (TEM). Melting points
were recorded on Buchi R-535 apparatus and are uncorrected.
106.7, 101.7 (O–CH2–O), 36.2 (S–CH2): HRMS
339.0792 [M?H]? Calcd for C18H14N2O3S : 339.0797.
:
2-(2-(allylthio)phenyl)-5-(2,6-difluorophenyl)-1,3,4-
oxadiazole (8c)
Light red solid; Yield: 75 %; mp 76–78 °C; 1H NMR
(CDCl3, 300 MHz): d 8.00 (dd, J = 7.5, 3.7 Hz, 1H, Ar–
H), 7.57–7.46 (m, 2H, Ar–H), 7.41–7.29 (m, 1H, Ar–H),
7.17–7.07 (m, 3H, Ar–H), 6.00–5.84 (m, 1H, =CH), 5.28
(dd, J = 16.6, 1.5, Hz, 1H, =CH2), 5.16 (dd, J = 9.8,
1.5 Hz, 1H, =CH2), 3.68 (d, J = 6.7 Hz, 2H, S–CH2); 13C
NMR (CDCl3, 75 MHz): 162.4, 158.9 (d, J = 4.3 Hz,
ArC–F), 133.4, 132.5 (=CH), 131.8, 131.4, 130.5, 130.2,
128.9, 128.3, 126.0, 125.2, 118.5 (=CH2), 112.4, 112.2,
36.3 (S–CH2): HRMS : 331.0705 [M?H]? Calcd for
C17H12F2N2OS : 331.0711.
A typical procedure for synthesis of 2,5 disubstituted
1,3,4-oxadiazoles 8–11(a–e)
In a typical experiment, the reaction of N-aroyl-N-aryl-
idinehydrazines 4–7(a–e) (1.0 mmol) was carried out with
Cs2CO3 (1.0 mmol), CuO nanoparticles (10 mol%) in DMSO
by stirring at 80 °C for 9–10 h. After completion of the reac-
tion (as monitored by thin-layer chromatography (TLC)), the
reaction mixture was centrifuged to separate the catalyst and
washed several times with ethyl acetate. The filtrate was con-
centrated, and the residue was purified by column chroma-
tography on silica gel (hexane/ethyl acetate, 80/20) to afford
pure product 2,5-disubstituted 1,3,4-oxadiazoles (75–90 %).
2-(2-(allylthio)phenyl)-5-(perfluorophenyl)-1,3,4-
oxadiazol (8d)
1
Light yellow solid; Yield: 77 %; mp 82–84 °C; H NMR
(CDCl3, 300 MHz): d 8.03–7.95 (m, 1H, Ar–H), 7.58–7.46
(m, 2H, Ar–H), 7.42–7.28 (m, 1H, Ar–H), 5.99–5.83 (m,
1H, = CH), 5.28 (dd, J = 16.6, 1.5, Hz, 1H, =CH2), 5.17
(dd, J = 9.0, 1.5 Hz, 1H, =CH2), 3.69 (d, J = 6.7 Hz, 2H,
S–CH2); 13C NMR (CDCl3, 75 MHz): 160.0, 139.8, 138.6
(d, J = 4.4 Hz, ArC–F), 132.4 (=CH), 131.8, 130.3, 128.6,
125.4, 122.0, 118.6 (=CH2), 36.4 (S–CH2): HRMS :
385.0424 [M?H]? Calcd for C17H9F5N2OS: 385.0428.
2-(2-(allylthio)phenyl)-5-(pyridine-3-yl)-1,3,4-
oxadiazole (8a)
1
Light yellow solid; Yield: 83 %; mp 98–100 °C; H NMR
(CDCl3, 300 MHz): d 9.37 (s, 1H, Ar–H), 8.80 (d,
J = 3.7 Hz, 1H, Ar–H), 8.46 (m, 1H, Ar–H), 8.04 (d,
J = 7.7 Hz, 1H, Ar–H), 7.54–7.47 (m, 3H, Ar–H), 7.38–7.30
(m, 1H, Ar–H), 5.99–5.84 (m, 1H, =CH), 5.29 (dd, J = 16.9,
1.5 Hz, 1H, =CH2), 5.17 (dd, J = 10.0, 1.5 Hz, 1H, =CH2),
3.69 (d, J = 6.6, 2H, S–CH2); 13C NMR (CDCl3, 75 MHz):
d 164.2, 162.3, 152.3, 147.9, 138.1, 134.2 (6 ArC), 132.4
(=CH), 131.5, 130.2, 128.5, 125.8, 125.4, 123.8, 122.5 (7
ArC), 118.7 (=CH2), 36.4 (S–CH2): HRMS : 296.0845
[M?H]? Calcd for C16H13N3OS : 296.0852.
2-(2-(allylthio)phenyl)-5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazole (8e)
1
Light gray solid; Yield: 79 %; mp 102–104 °C; H NMR
(CDCl3, 300 MHz): d 8.00 (d, J = 7.5 Hz, 1H, Ar–H),
7.75–7.65 (m, 2H, Ar–H), 7.47 (t, J = 2.2 Hz, 2H, Ar–H),
7.36–7.28 (m, 1H, Ar–H), 6.99 (dd, J = 8.3, 2.2 Hz, 1H,
Ar–H), 6.00–5.81 (m, 1H, =CH), 5.27 (dd, J = 18.1,
1.5 Hz, 1H, =CH2), 5.15 (dd, J = 11.3, 1.5 Hz, 1H, =CH2),
123

Med Chem Res
4.0 (s, 3H, O–CH3), 3.97 (s, 3H, O–CH3), 3.67 (dd,
J = 6.7, 1.5 Hz, 2H, S–CH2); 13C NMR (CDCl3,
75 MHz): 164.1, 151.7, 148.9, 137.6, 132.3 (=CH), 130.9,
129.6, 127.9, 124.9, 120.2, 118.3 (=CH2), 116.1, 110.8,
109.1, 55.8 (O–CH3), 55.7 (O–CH3), 36.0 (S–CH2): HRMS
: 355.1103 [M?H]? Calcd for C19H18N2O3S : 355.1110.
(s, 2H S–CH2–Ph); 13C NMR (CDCl3, 75 MHz): 164.7,
154.3, 146.9 (d, J = 6.0 Hz, ArC–F), 143.3 (d,
J = 3.8 Hz, ArC–F), 139.7 (d, J = 6.6 Hz, ArC–F), 138.9,
135.8, 131.8, 130.1, 128.8, 128.4, 127.3, 125.4, 121.5
(ArC), 38.2 (S–CH2–Ph): HRMS : 435.0576 [M?H]?
Calcd for C21H11F5N2OS : 435.0585.
2-(2-(benzylthio)phenyl)-5-(pyridine-3-yl)-1,3,4-
2-(2-(benzylthio)phenyl)-5-(3,4-dimethoxyphenyl)-
oxadiazole (9a)
1,3,4-oxadiazole (9e)
Light yellow solid; Yield: 88 %; mp 176–178 °C; 1H NMR
(CDCl3, 300 MHz): d 9.35 (dd, J = 2.0, 0.7 Hz, 1H, Ar–
H), 8.79 (dd, J = 4.9, 1.7 Hz, 1H, Ar–H), 8.46–8.41 (m,
1H, Ar–H), 8.02 (dd, J = 7.3, 1.1 Hz, 1H, Ar–H),
7.53–7.42 (m, 5H, Ar–H), 7.39–7.28 (m, 4H, Ar–H), 4.24
(s, 2H, S–CH2–Ph); 13C NMR (CDCl3, 75 MHz): 164.3,
162.3, 152.3, 147.9, 138.5, 136.0, 134.2, 131.6, 130.1,
128.8, 128.7, 128.5, 127.4, 125.5, 123.8, 122.5, 120.4
(ArC), 38.4 (S–CH2–Ph) : HRMS : 346.1001 [M?H]?
Calcd for C20H15N3OS : 346.1008.
White solid; Yield: 86 %; mp 157–159 °C; 1H NMR
(CDCl3, 300 MHz): d 8.02 (d, J = 7.3 Hz, 1H, Ar–H),
7.71 (dd, J = 8.3, 1.8 Hz, 1H, Ar–H), 7.64 (d, J = 1.7 Hz,
1H, Ar–H), 7.50–7.27 (m, 8H, Ar–H), 6.98 (d, J = 8.4 Hz,
1H, Ar–H), 4.23 (s, 2H, S–CH2–Ph), 3.96 (s, 3H, O–CH3),
3.93 (s, 3H, O–CH3); 13C NMR (CDCl3, 75 MHz): 151.9,
149.2, 138.2, 136.1, 131.2, 130.0, 128.9, 128.5, 127.3,
125.4, 122.9, 120.5, 116.4, 111.0, 109.5 (ArC), 56.0(O–
CH3), 38.4(S–CH2–Ph): HRMS : 405.1260 [M?H]? Calcd
for C23H20N2O3S : 405.1267.
2-(benzo[d][1,3]dioxol-5-yl)-5-(2-(benzylthio)phenyl)-
2-(2-allyloxy)phenyl)-5-(pyridine-3-yl)-1,3,4-
1,3,4-oxadiazole (9b)
oxadiazole (10a)
1
Light Yellow solid; Yield: 82 %; mp 188–190 °C; 1H
NMR (CDCl3, 300 MHz): d 7.64 (d, J = 8.1 Hz, 2H, Ar–
H), 7.57–7.45 (m, 4H, Ar–H), 7.40–7.23 (m, 4H, Ar–H),
6.95 (d, J = 8.1 Hz, 2H, Ar–H), 6.09 (s, 2H, O–CH2–O),
4.24 (s, 2H, S–CH2–Ph); 13C NMR (CDCl3, 75 MHz):
164.1, 163.3, 150.2, 148.1, 137.8, 132.5, 131.0, 129.8,
128.1, 125.1, 122.0, 121.7, 108.7, 107.5, 106.9 (ArC),
101.7 (O–CH2–O), 36.2 (S–CH2–Ph) : HRMS : 389.0946
[M?H]? Calcd for C22H16N2O3S : 389.0954.
Yellow solid; Yield : 85 %; mp 103–105 °C; H NMR
(CDCl3, 300 MHz): d 9.35 (d, J = 1.5 Hz, 1H, Ar–H),
8.79 (dd, J = 4.5, 1.5 Hz, 1H, Ar–H), 8.47–8.42 (m, 1H,
Ar–H), 8.09 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H), 7.57–7.46
(m, 2H, Ar–H), 7.17–7.05 (m, 2H, Ar–H), 6.20–6.07 (m,
1H, =CH), 5.58 (dd, J = 17.3, 1.5 Hz, 1H, =CH2), 5.36
(dd, J = 10.5, 1.5 Hz, 1H, = CH2), 4.73 (d, J = 5.2 Hz,
2H, O–CH2); 13C NMR (CDCl3, 75 MHz): 164.0, 162.2,
156.7, 152.0, 147.7, 133.9 (6 ArC), 133.2 (=CH), 132.3,
130.5, 123.7, 120.9 (4 ArC), 117.8 (=CH2), 113.1 (ArC),
69.3 (O–CH2): HRMS : 280.1079 [M?H]? Calcd for
C16H13N3O2 : 280.1080.
2-(2-(benzylthio)phenyl)-5-(2,6-difluorophenyl)-1,3,4-
oxadiazole (9c)
White solid; Yield: 81 %; mp 118–120 °C; 1H NMR
(CDCl3, 300 MHz): d 7.99 (d, J = 7.5 Hz, 1H, Ar–H),
7.62–7.22 (m, 9H, Ar–H), 7.19–7.06 (m, 2H, Ar–H), 4.23
(s, 2H, S–CH2–Ph); 13C NMR (CDCl3, 75 MHz): 164.2,
162.3 (d, J = 4.3 Hz, ArC–F), 158.8, 156.4, 138.5, 136.0,
133.3, 131.4, 131.2, 130.0, 128.8, 128.4, 127.2, 125.3,
122.2, 112.4, 112.1 (ArC), 38.1 (S–CH2–Ph): HRMS :
381.0863 [M?H]? Calcd for C21H14F2N2OS : 381.0867.
2-(2-allyloxy)phenyl)-5-(benzo[d][1,3]dioxol-5-yl)-
1,3,4-oxadiazole (10b)
Light yellow solid; Yield : 80 %; mp 144–146 °C; 1H
NMR (CDCl3, 300 MHz): d 8.04 (dd, J = 7.7, 1.7 Hz, 1H,
Ar–H), 7.68 (dd, J = 8.1, 1.5 Hz, 1H, Ar–H), 7.58 (d,
J = 1.5 Hz, 1H, Ar–H), 7.54–7.45 (m, 1H, Ar–H),
7.14–7.02 (m, 2H, Ar–H), 6.93 (d, J = 8.3 Hz, 1H, Ar–H),
6.19–6.08 (m, 1H, =CH), 6.06 (s, 2H, O–CH2–O), 5.59 (dd,
J = 17.1, 1.1 Hz, 1H, =CH2), 5.34 (dd, J = 10.5, 1.1 Hz,
1H, =CH2), 4.71 (d, J = 4.9 Hz, 2H, O–CH2); 13C NMR
(CDCl3, 75 MHz): 164.1, 163.0, 156.6, 150.3, 148.1, 132.7
(=CH), 132.4, 130.4, 121.8, 120.9, 117.6 (=CH2), 113.1,
108.7, 106.9, 101.7 (O–CH2–O), 69.3 (O–CH2): HRMS :
323.1022 [M?H]? Calcd for C18H14N2O4 : 323.1026.
2-(2-(benzylthio)phenyl)-5-(perfluorophenyl)-1,3,4-
oxadiazole (9d)
Light yellow solid; Yield: 84 %; mp 138–140 °C; 1H NMR
(CDCl3, 300 MHz): d 7.98 (d, J = 7.5 Hz, 1H, Ar–H),
7.49–7.42 (m, 2H, Ar–H), 7.39–7.21 (m, 6H, Ar–H), 4.23
123

Med Chem Res
CH2–Ph); 13C NMR (CDCl3, 75 MHz): 164.6, 161.1,
157.7, 152.7, 147.9, 134.1, 133.3, 130.6, 128.6, 128.1,
127.4, 126.5, 123.8, 121.1, 120.0, 117.7, 113.2 (ArC), 70.7
(O–CH2–Ph): HRMS : 330.1234 [M?H]? Calcd for
C20H15N3O2 : 330.1237.
2-(2-(allyloxy)phenyl)-5-(2,6-difluorophenyl)-1,3,4-
oxadiazole (10c)
1
Light yellow solid; Yield : 79 %; mp 79–81 °C; H NMR
(CDCl3, 300 MHz): d 8.07 (dd, J = 7.5, 2.2 Hz, 1H, Ar–H),
7.62–7.47 (m, 2H, Ar–H), 7.17–7.03 (m, 4H, Ar–H),
6.18–6.03 (m, 1H, =CH), 5.57 (dd, J = 17.3, 1.5 Hz, 1H,
=CH2), 5.31 (dd, J = 10.5, 1.5, 1H, =CH2), 4.72 (d,
J = 4.5 Hz, 2H, O–CH2); 13C NMR (CDCl3, 75 MHz):
164.1, 162.3 (d, J = 4.9 Hz, ArC–F), 158.9 (d, J = 4.9 Hz,
ArC–F), 156.8, 133.1, 132.3 (=CH), 130.6, 120.8, 120.4,
117.6 (=CH2), 113.3, 112.4, 112.1, 69.5 (O–CH2): HRMS :
315.0937 [M?H]? Calcd for C17H12F2N2O2 : 315.0939.
2-(benzo[d][1,3]dioxol-5-yl)-5-(benzyloxy)phenyl)-
1,3,4-oxadiazole (11b)
Light yellow solid; Yield : 83 %; mp 177–179 °C; 1H
NMR (CDCl3, 300 MHz): d 8.13 (dd, J = 8.3, 1.5 Hz, 1H,
Ar–H), 7.66 (dd, J = 8.3, 1.5 Hz, 1H, Ar–H), 7.59–7.53
(m, 3H, Ar–H), 7.49–7.36 (m, 3H, Ar–H), 7.18–7.11 (m,
2H, Ar–H), 6.94 (d, J = 8.3 Hz, 2H, Ar–H), 6.07 (d,
J = 3.7 Hz, 2H, O–CH2–O), 5.23 (s, 2H, O–CH2–Ph);
13CNMR (CDCl3, 75 MHz) : 163.9, 156.5, 150.4, 148.2,
136.2, 132.9, 130.5, 128.5, 128.0, 127.4, 121.9, 121.8,
121.0, 113.0, 108.7, 108.6, 106.8 (ArC), 101.7 (O–CH2–
O), 70.5 (O–CH2–Ph) : HRMS : 395.1001 [M?Na]? Calcd
for C22H16N2O4 : 395.1002.
2-(2-(allyloxy)phenyl)-5-(perfluorophenyl)-1,3,4-
oxadiazole (10d)
1
Light gray solid; Yield : 78 %; mp 117–119 °C; H NMR
(CDCl3 300 MHz): d 8.06 (dd, J = 7.7, 1.5 Hz, 1H, Ar–H),
7.57–7.50 (m, 1H, Ar–H), 7.15–7.04 (m, 2H, Ar–H), 6.17–6.03
(m, 1H, =CH), 5.54 (dd, J = 17.1, 1.3 Hz, 1H, =CH2), 5.33
(dd, J = 10.5, 1.3 Hz, 1H, =CH2), 4.72 (d, J = 5.0 Hz, 2H, O–
CH2); 13C NMR (CDCl3, 75 MHz): 164.7, 157.0, 146.9 (d,
J = 12.6 Hz, ArC–F), 133.6, 132.3(=CH), 130.7, 121.0,
117.9(=CH2), 113.3, 112.4, 69.6(O–CH2): HRMS : 369.0653
[M?H]? Calcd for C17H19F5N2O2 : 369.0657.
2-(2-(benzyloxy)phenyl)-5-(2,6-difluorophenyl)-1,3,4-
oxadiazole (11c)
White solid; Yield: 85 %; mp 130–132 °C; 1H NMR
(CDCl3, 300 MHz): d 7.59–7.52 (m, 12H, Ar–H), 5.28 (s,
2H, O–CH2–Ph); 13C NMR (CDCl3, 75 MHz): 161.8 (d,
J = 5.4 Hz, ArC–F), 159.7 (d, J = 4.5 Hz, ArC–F), 157.5,
133.8, 133.7, 133.6, 130.8, 128.4, 127.7, 126.9, 121.1,
112.5, 112.3, 102.8 (ArC), 70.5 (O–CH2–Ph): HRMS :
365.1093 [M?H]? Calcd for C21H14F2N2O2 : 365.1096.
2-(2-(allyloxy)phenyl)-5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazole (10e)
Light yellow solid; Yield : 77 %; mp 109–111 °C; 1H
NMR (CDCl3, 300 MHz): d 8.06 (dd, J = 7.4, 2.1 Hz, 1H,
Ar–H), 7.70 (dd, J = 8.4, 2.1 Hz, 1H, Ar–H), 7.66 (d,
J = 2.1 Hz, 1H, Ar–H), 7.52–7.46 (m, 1H, Ar–H),
7.13–7.03 (m, 2H, Ar–H), 6.98 (dd, J = 8.4, 1.0 Hz, 1H,
Ar–H), 6.17–6.08 (m, 1H, =CH), 5.60 (dd, J = 16.9,
2.1 Hz, 1H, =CH2), 5.32 (dd, J = 10.6, 2.1 Hz, 1H, =CH2),
4.71 (d, J = 4.2 Hz, 2H, O–CH2), 3.98 (s, 3H, O–CH3),
3.96 (s, 3H, O–CH3); 13C NMR (CDCl3, 75 MHz): 164.3,
163.0, 156.5, 151.6, 149.1, 132.7, 132.4 (=CH), 130.4,
120.8, 120.2, 117.5 (=CH2), 113.0, 110.9, 109.2, 69.2 (O–
CH2), 55.9 (O–CH3): HRMS : 339.1335 [M?H]? Calcd
for C19H18N2O4 : 339.1339.
2-(2-(benzyoxy)phenyl)-5-(perfluorophenyl)-1,3,4-
oxadiazole (11d)
Light yellow solid; Yield : 88 %; mp 134–136 °C; 1H
NMR (CDCl3, 300 MHz): d 8.36 (dd, J = 7.9, 1.8 Hz, 1H,
Ar–H), 7.57–7.53 (m, 2H, Ar–H), 7.51–7.48 (m, 3H, Ar–
H), 7.43 (m, 1H, Ar–H), 7.18 (t, J = 7.9 Hz, 1H, Ar–H),
7.14 (d, J = 8.2 Hz, 1H, Ar–H), 5.23 (s, 2H, O–CH2–Ph);
13C NMR (CDCl3, 75 MHz): 161.6, 156.6, 134.9, 134.4 (d,
J = 6.5 Hz, ArC–F), 133.9, 133.0, 129.4, 129.1, 128.5,
122.1, 112.5 (ArC), 70.5(O–CH2–Ph): HRMS : 419.0804
[M?H]? Calcd for C21H11F5N2O2 : 419.0818.
2-(2-(benzyloxy)phenyl)-5-(pyridine-3-yl)-1,3,4-
oxadiazole (11a)
2-(2-(benzyloxy)phenyl)-5-(3,4-dimethoxyphenyl)-
1
Light gray solid; Yield: 90 %; mp 101–103 °C; H NMR
1,3,4-oxadiazole (11e)
(CDCl3, 300 MHz): d 9.37 (s, 1H, Ar–H), 9.22 (s, 1H, Ar–
H), 8.83 (d, J = 4.1 Hz, 1H, Ar–H), 8.75 (d, J = 4.1 Hz,
1H, Ar–H), 8.48–8.41 (m, 1H, Ar–H), 8.22–8.13 (m, 1H,
Ar–H), 7.88 (dd, J = 7.7, 1.3 Hz, 1H, Ar–H), 7.61–7.35
(m, 4H, Ar–H), 7.21–7.03 (m, 2H, Ar–H), 5.29 (s, 2H, O–
Light orange solid; Yield: 84 %; mp 143–145 °C; 1H NMR
(CDCl3, 300 MHz): d 8.11 (dd, J = 7.5, 1.5 Hz, 1H, Ar–
H), 7.61 (d, J = 1.7 Hz, 1H, Ar–H), 7.58–7.47 (m, 4H, Ar–
H), 7.43–7.30 (m, 2H, Ar–H), 7.20–7.08 (m, 2H, Ar–H),
123

Med Chem Res
7.06–6.95 (m, 1H, Ar–H), 6.92–6.86 (m, 1H, Ar–H), 5.25
(s, 2H, O–CH2–Ph), 3.96 (s, 3H, O–CH3), 3.88 (s, 3H, O–
CH3); 13C NMR (CDCl3, 75 MHz): 132.9, 130.6, 128.5,
127.9, 127.1, 121.9, 121.1, 120.3, 113.2, 110.9, 109.3,
108.8, 106.9, 101.8 (ArC), 70.4(O–CH2–Ph), 56.0(O–
grown in potato dextrose broth (PDB) for 24 and 48 h,
respectively. The inoculated flasks were incubated at
150 rpm at 37 °C for bacterial strains and at 30 °C for
fungal strains for active cultures.
The synthesized compounds were dissolved in DMSO to
get a final concentration of one mg/ml. In order to ensure
that the solvent had no effect on bacterial and fungal
growth, a control test was also performed containing
inoculated broth supplemented with only DMSO at the
same dilution used in the experiments.
CH3), 55.9(O–CH3): HRMS
C23H20N2O4: 389.1495.
: 389.1499 Calcd for
In vitro cytotoxic activity studies
The cell lines used in this study (HeLa, MCF-7, A549, and
IMR32) were purchased from the American Type Culture
Collection (ATCC, United States). All these cell lines were
grown in Dulbecco’s modified Eagle’s medium (containing
10 % fetal bovine serum (FBS) in a humidified atmosphere
of 5 % CO2 at 37 °C). Cells were trypsinized when sub-
confluent from T25 flasks/60-mm dishes and seeded in
96-well plates. The synthesized oxadiazoles were evaluated
for their in vitro cytotoxicity in four different human cancer
cell lines. A protocol of 48 h continuous drug exposure was
used, and a SRB cell proliferation assay (Reddy et al.,
2011) was used to estimate cell viability or growth. The
cell lines were grown in the media containing 10 % fetal
bovine serum and were seeded into 96-well microtiter
plates in 100 lL aliquots at plating densities depending on
the doubling time of individual cell lines. The microtiter
plates were incubated at 37 °C, 5 % CO2, 95 % air, and
100 % relative humidity for 24 h prior to addition of
experimental drugs and were incubated for 48 h with dif-
ferent doses (1, 10, 100 lM) of prepared derivatives. After
48 h incubation at 37 °C, cell monolayers were fixed by the
addition of 100 lL of 10 % (wt/vol) cold trichloroacetic
acid and incubated at 4 °C for 1 h. The supernatant was
discarded, and the cells were then stained with 0.057 %
SRB dissolved in 1 % acetic acid for 30 min at room
temperature. Unbound SRB was washed away with four
washes of 1 % acetic acid and the bound SRB stain, rep-
resenting surviving cells, was dissolved in 50 lL of Tris
base (10 mM). The optical density was determined at
510 nm using a microplate reader (Enspire, Perkin Elmer,
USA).
Zone of inhibition plate tests
Well-plate method (Prakash et al., 2011; Zovko et al.,
2012) was used for both antibacterial and antifungal
activities for measuring the zone of inhibition. All twenty
compounds 8–11(a–e) were used for antimicrobial activ-
ity studies. Sterile NB and PDB plates were prepared, and
the 30 lL of above test organism fermentation broth was
poured on the separate plates and uniformly spread using
sterile spreader. Wells were made with sterile cork borer,
and each well was loaded with exactly 50 lL of sample.
Control (only sterile broth without inoculation) and
standard (drug i.e., streptomycin in case of bacterial
strains and cycloheximide in case of fungal strains) also
placed in separate wells in the similar concentration as
that of synthesized compounds i.e., 50 lg per well. The
plates were first incubated for 20–30 min at 4 °C to allow
the compounds to diffuse into the agar and then subse-
quently incubated for 24 h at 37 °C for bacteria and 48 h
at 30 °C for fungi. After incubation, the zone of growth
inhibition was measured using calibrated scale and
expressed in mm. Experiment was performed triplicate to
minimize the deviations, and average values were
reported.
Minimum inhibitory concentration (MIC) (in lg/mL)
The selected bacterial strains viz., Escherichia coli (MTCC
40), Bacillus subtilis (MTCC 736), Klebsiella pneumonia
(MTCC 3384), and Micrococcus luteus (MTCC 106) were
used for this study using broth dilution method (Prakash
et al., 2011). Exponentially growing cultures of above
strains in one ml volume (OD equal to the turbidity of a
Mac Farland 0.5 standard tube) were inoculated in one ml
NB for bacterial strains. These tubes were supplemented
with final compound at a concentration range from 9.37 to
300 lg/mL broth. The tubes were incubated for 12–16 h at
37 °C, and the turbidity of each tube was measured with
respect to control. MIC values (the lowest concentration of
compound at which growth is completely inhibited) were
determined based on the turbidity data.
Microorganisms and growth conditions
Antimicrobial activity for all synthesized compounds was
evaluated using four different bacterial strains viz., Esch-
erichia coli (MTCC 40), Bacillus subtilis (MTCC 736),
Klebsiella pneumonia (MTCC 3384), and Micrococcus
luteus (MTCC 106) and fungal Aspergillus niger (MTCC
281), Aspergillus terreus (MTCC 1782), and Aspergillus
fumigatus (MTCC 343) strains. All bacterial strains were
grown in nutrient broth (NB), while fungal strains were
123

Med Chem Res
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Conclusion
In conclusion, we have developed an efficient synthetic
strategy toward the synthesis of unsymmetrical 2,5-disub-
stituted 1,3,4-oxadiazoles via oxidative C–O coupling by
direct C–H bond activation of N-aroyl-N-arylidinehydra-
zines mediated by CuO nano. The catalyst CuO nanopar-
ticle is air-stable, recyclable, and used for three cycles with
almost consistent activity. All the synthesized compounds
were subjected to in vitro antiproliferative, antibacterial,
and antifungal activity. In case of cytotoxic studies, the
results revealed that the compounds 8d and 10d are the
most promising cytotoxic agents with IC50 value of 3.66
and 3.89 lM in MCF-7. In case of antimicrobial studies,
compounds 8a, 8e showed broad spectrum antibacterial
activity (for all tested strains MIC 37.5 lg/mL), and the
compounds 8a and 10a displayed potent activity and
exerted antifungal activity better than standard drug. These
results are therefore conclusive in showing that 1,3,4-
oxadiazoles derived from salicylic acid and thiosalicylic
acid bearing perfluorophenyl group at 5-position to oxa-
diazole act as antiproliferative and pyridyl groups at
5-position to oxadiazole act as antimicrobial agents.
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Acknowledgments The authors are thankful to the Director, Indian
Institute ofChemicalTechnology, Hyderabad for theencouragement, RP
thankfultoCSIR, New Delhi, India forthe award ofresearch fellowships.
We thank CSIR for financial support under the 12th Five Year plan
projects ‘‘Affordable Cancer Therapeutics (ACT)’’ (CSC 0301) and
‘‘Small Molecules in Lead Exploration (SMiLE)’’ (CSC0111).
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