Synthesis and evaluation of antioxidant and antibacterial activities of new substituted bis(1,3,4-oxadiazoles), 3,5-bis(substituted) pyrazoles and isoxazoles

Article abstract of DOI:10.1016/j.bmcl.2011.04.142

Two series of five membered heterocyclic bis(1,3,4-oxadiazole) derivatives 2(a-h) and 3,5-bis(substituted)pyrazoles, isoxazoles 3(a,b,d-i), 4(a-c) were synthesized via oxidative cyclization of some diaroylhydrazones using chloramine-T and cyclocondensatio

Full text of DOI:10.1016/j.bmcl.2011.04.142

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3536–3540
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
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Synthesis and evaluation of antioxidant and antibacterial activities of new
substituted bis(1,3,4-oxadiazoles), 3,5-bis(substituted) pyrazoles and isoxazoles
Ebraheem Abdu Musad a, Riyaz Mohamed b, Bahjat Ali Saeed c, Bannikuppe S. Vishwanath b,
K. M. Lokanatha Rai a,
⇑
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
b Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
c Department of Chemistry, College of Education, University of Basrah, Basrah, Iraq
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of five membered heterocyclic bis(1,3,4-oxadiazole) derivatives 2(a–h) and 3,5-bis(substi-
tuted)pyrazoles, isoxazoles 3(a,b,d–i), 4(a–c) were synthesized via oxidative cyclization of some diar-
oylhydrazones using chloramine-T and cyclocondensation reaction with hydrazine hydrate and
hydroxylamine hydrochloride, respectively. The newly synthesized compounds were screened for antiox-
idant and anti-microbial activities. Compounds 2(b), 3(b), and 4(a) showed higher antioxidant activity at
Received 18 March 2011
Revised 27 April 2011
Accepted 29 April 2011
Available online 6 May 2011
10 lg/ml while compounds 2(a), 3(a), 3(f), and 4(a) exhibited better anti-microbial activity at 100 lg/ml
Keywords:
Methylene-linked bis(1,3,4-oxadiazole)
derivatives
3,5-Bis(substituted) pyrazoles
Isoxazoles
compared with standard vitamin C and ciprofloxacin, respectively. Structures of newly synthesized com-
pounds were confirmed by elemental analysis and spectral IR, 1H NMR, and 13C NMR data.
Ó 2011 Elsevier Ltd. All rights reserved.
Antioxidant
Antibacterial
The wide occurrence of the heterocycles in bioactive natural
products, pharmaceuticals, and agrochemicals1,2 has made them
as important synthetic targets. 1,3,4-Oxadiazoles, pyrazoles, and
isoxazoles represent a class of heterocyclic compounds of great
importance in biological chemistry. For instance, compounds pos-
sessing 1,3,4-oxadiazole, pyrazole, and isoxazole moiety show anti-
cancer3–6 activity. Substituted 1,3,4-oxadiazoles, pyrazoles and
isoxazoles have revealed antibacterial,7–9 antioxidant,8–11 insecti-
cidal properties, etc.12–14 Literature studies reveal that 2,5-disubsti-
tuted-1,3,4-oxadiazoles have been synthesized either by
microwave irradiation of hydrazide and carboxylic acid mixture15
or by thermal/acid catalyzed cyclization of 1,2-diacylhydrazines.16
2,5-Disubstituted-1,3,4-oxadiazoles have also been synthesized
by oxidative cyclization of semicarbazone/hydrazone using chlor-
amines-T as an oxidant.17 Cyclocondensation reactions are useful
tools for constructing pyrazoles and isoxazoles18,19 cyclocondensa-
tion of hydrazine hydrate and hydroxylamine hydrochloride to b-
diketones is of synthetic interest, since the products 3,5-disubsti-
tuted pyrazoles, 3,5-disubstituted isoxazoles, and their derivatives
obtained are the versatile intermediates for the synthesis of bifunc-
tional compounds. The literature survey leading to synthesis of
methylene-linked bis(1,3,4-oxadiazol) and 3,5-bis(substituted)
pyrazole, 3,5-bis(substituted)isoxazole indicate the lack of refer-
ence available. In our laboratory Rai et al. extensively used chloram-
ines-T for the generation 2,5-disubstituted-1,3,4-oxadiazoles from
acylhydrazones.20 On the other hand, we have recently reported
the synthesis of 3,5-bis(substituted)pyrazoles and 3,5-bis(substi-
tuted)isoxazoles via cyclocondensation reaction of hydrazine
hydrate, hydroxylamine hydrochloride with some diaroylhydraz-
ones under selevothermal conditions.21 With this background, it
is considered worthwhile to prepare bis(heterocycle) bearing
1,3,4-oxadiazole moieties furthermore 3,5-bis(substituted) pyra-
zoles and isoxazoles starting from simple diaroylhydrazones and
screen them for antioxidant and antibacterial activities. The present
report deals with the synthesis and biological evaluation of hitherto
unknown methylene-linked bis (heterocycle) bearing 1,3,4-oxadi-
azole moieties.
The desired starting material malonodihydrazide was prepared
by condensation of diethylmalonate with hydrazine hydrate in a
molar ratio of 1:2, under refluxing ethanol. The resultant was
subjected to other condensation with corresponding aromatic
aldehydes by employing a well known method available in the
literature22 afforded diaroylhydrazones 1(a–i) (Scheme 1).
The oxidation of diaroylhydrazones 1(a–i) with chloramines-T
in a molar ratio of 1:2 to generate nitrile imines followed by intra-
molecular cyclization with adjacent carbonyl groups yielded the
⇑
Corresponding author. Tel.: +91 9972165994; fax: +91 821 2421263.
E-mail address: alhashmi.ibrahim@yahoo.com (K.M. Lokanatha Rai).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.142

E. A. Musad et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3536–3540
3537
O
O
O
O
refluxe
NH2NH2.H2O
H2N
NH2
OEt
EtO
ethanol/1.5h
N
H
N
H
2 mole
1 mole
R1
R2
R3
R3
R2
O
O
O
refluxe
H2N
NH2
R2
R1
R1
N
H
N
H
O
O
ethanol/0.5h
H
N
N
R3
2 mole
N
H
N
1 mole
H
1 (a-i)
1a: R = H , R = N(Me) , R =H
1
2
2
3
1b: R = H , R = OH , R = H
1
2
3
1c: R = H , R = H , R = OH
1
2
3
1d; R = H , R = Cl , R = H
1
2
3
1e; R = R = R = H
1
2
3
1f ; R = H , R = OMe , R = H
1
2
3
1g; R = H , R = R = OMe
1
2
3
1h; R = R = R = OMe
1
2
3
1i ; R = H , R = NO , R = H
1
2
2
3
1
3
1
Scheme 1. Synthesis of (N0 E,N0 E)-N0 ,N03-dibenzylidenemalonohydrazide derivatives.
methylene-linked bis(1,3,4-oxadiazole) derivatives 2(a–h) which
were identified by NMR spectroscope (Scheme 2).
oles 2(a–h) showed the absence of NH frequency in the region
3100–3200 cmꢀ1 and showed a new peak at 1581–1678 cmꢀ1
due to C@N frequency that confirms the formation of the products.
The 13C NMR and elemental data analysis further confirm the
structures of the desired products.
The diaroylhydrazones were 1(a–b,d–i) further treated with
hydrazine hydrate, meanwhile the diaroylhydrazones 1(a,d,h)
were treated with hydroxylamine hydrochloride to get the desired
products 3(a,b,d–i) and 4(a–c) (Scheme 3). The probable mecha-
nism of formation of isoxazoles 4(a–c) involves the attack of nitro-
gen atom of hydroxylamine hydrochloride on the carbonyl carbon
that follows the oxime pathway to yield the intermediate which
undergoes intramolecular cyclization 4(a–c).
The 2,2-diphenyl-1-picryl-hydrazyl (DPPHÅ) radical scavenging
activity (RSA) evaluation is a standard assay in antioxidant activity
studies and offers a rapid technique for screening the RSA of spe-
cific compounds or extracts.24–27 The interaction of synthesized
bis(1,3,4-oxadiazole) series 2(a–h) with stable DPPH free radical
indicates their free radical scavenging ability. Majority of the
tested compounds in these series showed low to moderate interac-
1H NMR spectra of compounds 2(a–h) showed singlets in the
region of d 2.86–3.35 ppm that correspond to –CH2– group at-
tached to the oxadiazole rings. Aromatic protons and other substit-
uents are at the expected region.23
tion with the DPPH radical at 10 lg/mL concentration. Maximum
DPPH RSA was observed in compound 2(e) (p <0.05), which does
not have a substituent on the phenyl ring (Table 1). The presence
of either electron-donating or electron-withdrawing groups on
the phenyl ring at positions 3,4,5 might not favor the activity. Com-
pounds 2(a–d) which contain –N(CH3)2 or –OH or Cl groups on the
phenyl ring at position 4 showed low to moderate radical scaveng-
ing activity but lower than compound 2(e), whereas the com-
pounds 2(f–h) that contain methoxy groups on the phenyl ring
at positions 3,4,5, did not show any activity. On the other hand,
the 3,5-bis(substituted) pyrazoles and isoxazoles series 3(a,b,d–
According to the 1H NMR spectra of compounds 3(a,b,d–i) and
4(a–c) also showed singlets in the region of d 7.27–8.60 ppm that
correspond to methine protons of pyrazole and isoxazole rings,
other singlets appear in the d 7.56–8.79 ppm region that corre-
spond to the methane proton adjacent to phenyl rings. Further-
more the 1H NMR of pyrazoles 3(a,b,d–i) showed broad signals
in the region of d 9.00–12.28 ppm that correspond to amino proton
of hydrazide and pyrazole rings. Aromatic protons and other sub-
stituents are at the expected region. The IR spectrum of oxadiaz-
R3
R2
R2
R3
R3
R2
O
N
N
R1
N
N
R1
R1
O
O
O
Chloramine-T/2mole
ethanol/refluxe 4h
N
N
N
H
N
H
R1
1 mole
1 (a-h)
R3
R2
2 (a-h)
Scheme 2. Synthesis of bis(1,3,4-oxadiazole) derivatives.

3538
E. A. Musad et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3536–3540
R1
R1
R2
R2
NH
N
R3
N
N
R2
N
H
O
N
H
H 2
H 2
R2
R3
R3
R2
N
H 2
N
e/ethanol 3h
3 (a,b,d-i)
R1
R1
reflux
reflux
O
O
e/ethanol 3h
NH
2
N
N
R1
R1
OH.HCl
N
H
N
H
R2
R2
O
N
R3
N
1 (a,b,d,e,f,g,h,i) for pyrazoles
1 (a,d,h) for isoxazoles
N
R2
N
H
N
H
4 (a-c)
Scheme 3. Synthesis of 3,5-bis(substituted)pyrazoles and isoxazoles.
Table 1
Antioxidant activity of synthesized novel series of bis(1,3,4-oxadiazole) derivatives (2a–h) and 3,5-bis(substituted) pyrazoles (3a,b,d–i) and isoxazoles derivatives (4a–c)
Compound
R
DPPH radical scavenging activity (%) at 10
l
g/ml
Anti-lipid peroxidation (%) at 40
lg/ml
Me
N
2a
28 3.0
46.3 1.1
Me
OH
2b
2c
36 2.0
37 4.0
53.4 0.92
48.6 1.8
HO
Cl
2d
2e
2f
40 2.0
41.3 2.5
NA
16.2 1.4
33.4 1.8
40.5 1.1
OMe
OMe
OMe
OMe
OMe
2g
2h
NA
ND
28.5 3.89
44.3 1.25
OMe
Me
N
3a
3b
59.2 2.1
83 1.8
ND
Me
OH
58.6 2.1

E. A. Musad et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3536–3540
3539
Table 1 (continued)
Compound
R
DPPH radical scavenging activity (%) at 10
40 1.3
lg/ml
Anti-lipid peroxidation (%) at 40
ND
lg/ml
Cl
3d
3e
3f
34.2
3
44 1.08
46.3 1.4
OMe
OMe
OMe
44.3 2.1
47.1 1.8
OMe
OMe
3g
3h
ND
ND
52.4
2
OMe
NO2
3i
74.2
90.6
4
2
NA
Me
N
4a
4b
4c
54.5 0.85
Me
Cl
37.4 6.0
54 4.0
NA
OMe
OMe
OMe
48.4 1.21
Vitamin C
Vitamin E
—
—
93.2 0.8
ND
ND
70 1.8
Antioxidant and anti-lipid peroxidation activities were expressed in percentage compared with standard vitamin C and E, respectively. NA = not active. ND = not determined.
The data represent mean value (SEM) of three duplicates.
i), 4(a–c) showed scavenging of DPPH radical at varying degrees
when compared with the standard antioxidant vitamin C. The
results are represented in Table 1. The maximum antioxidant activ-
ity was observed in compounds in the following order
4(a) > 3(b) > 3(i), which is comparable to that of standard vitamin
C at a similar concentration. The other eight compounds exhibited
good to moderate RSA in the order 3(a) > 4(c) > 3(h) >
3(g) > 3(f) > 3(d) > 4(b) > 3(e) (Table 1). The presence of either
electron-donating or electron-withdrawing groups on the phenyl
ring mostly favor the activity particularly with a strong electron-
donating group such as –N(CH3)2 or a strong electron-withdrawing
group such as NO2. Antioxidant activity of these compounds is re-
lated with their electron or hydrogen radical donating ability to
DPPH radical, so that they become stable diamagnetic molecules.
This might be the reason for the higher antioxidant activity of
the second series of compounds 3(a,b,d–i) and 4(a–c).
tion at 40 lg/mL concentration with a varying degree when com-
pared with standard biological antioxidant vitamin E (Table 1).
Compound 2(b), (p < 0.05) which has –OH group on the phenyl ring
at position 4 showed maximum inhibition. However, inhibition
activity was found to be lower than that of the reference com-
pound vitamin E. Compound 2(d), which has chlorine as the elec-
tron-withdrawing substituent on phenyl ring at position 4,
exhibited the lowest inhibition. In general, it appears that the pres-
ence of electron-donating groups on the phenyl ring favors the
activity. This might be the reason for the enhancement of activity
of the other compounds, which showed moderate activities in
the order 2(c) > 2(a) > 2(h) > 2(f). However, compound 2(e) was
found to show lower activity when compared with compound
2(c), 2(a), 2(h), and 2(f), since it does not have a substituent on
the phenyl ring. Further, synthesized pyrazole 3(a,b,d–i) and isox-
azoles 4(a–c) series showed that majority of the compounds had
moderate inhibitive action, which follows the order
3(b) > 4(a) > 4(c) > 3(g) > 3(e) (Table 1). In these series, the pres-
ence of the electron-donating group on the phenyl ring at position
4 such as –N(CH3)2, –OH and –OMe at positions 3,4,5 enhanced the
activity when compared to the activity of compound 3(e). The
Evaluation of anti-lipid peroxidation property of newly synthe-
sized compounds was performed by the formation of thiobarbitu-
ric acid reactive species (TBARS) using egg yolk homogenate as
lipid-rich media. The result showed that all newly synthesized
compounds inhibited the ferric chloride-induced lipid peroxida-

3540
E. A. Musad et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3536–3540
Table 2
2(a), 3(a), 3(f), and 4(a) showed maximum antibacterial activity
in comparison with their corresponding standard drug. Further de-
tailed studies are required to understand the mechanism of action
of these compounds.
Antibacterial activity of synthesized novel series of pyrazoles and isoxazoles
derivatives (2a–h, 3a,b,d–i and 4a–c)
Compound
S. aureus
B. subtilis
E. coli
P. aeruginosa
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3d
3e
13 0.56
12 1.24
10 0.86
9
1
5
1.65
1.27
0.85
7
9
4
7
9
0.25
1.32
0.52
1.32
1.65
2
1.5
13 1.16
0.98
10 0.73
1.16
11 1.07
0.58
10 1.24
1
8
0.97
1.35
Acknowledgments
6
NA
5
6
NA
13 1.24
7
9
NA
8
18 0.53
5
9
Author expresses his sincere gratitude to the Council of Scien-
tific and Industrial research, India for financial assistance and Uni-
versity of Mysore, Mysore, for providing the laboratory facilities to
carry out this work.
0.93
0.89
8
1.07
1.29
10 0.89
1.32
10 1.31
NA
7
3
20 0.72
NA
9
1.52
1.06
7
6
9
1.07
0.89
1.29
13 1.32
Supplementary data
4
5
1.16
1.07
4
4
1.03
1.03
1.37
1.65
3f
3g
3h
3i
4a
4b
4c
28 1.73
10 1.37
17 1.07
20 0.83
1.16
12 1.05
1.12
15 1.81
0.73
18 0.58
13 1.23
NA
11 1.09
10 1.24
19 0.73
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.142.
8
6
9
8
0.87
1.47
0.67
9
1.65
7
References and notes
20 0.72
14 0.96
13 0.54
29 0.85
12 0.89
8
8
1.14
6
9
0.76
0.87
10 1.09
18 1.25
11 0.67
26 1.31
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determined. The data represent mean value (SEM).
presence of a strong electron-withdrawing group like nitro group
on the phenyl ring at position 4 did not favor the activity. This
might be the reason for the inactivity of compound 3(i).
The synthesized compounds were evaluated for in vitro anti-
microbial activity against various bacterial strains using the agar
disc diffusion method. The results are presented in Table 2. The re-
sult showed that compounds 2(a), 3(a), 3(f), and 4(a) exhibited
maximum antibacterial activity against all the tested microorgan-
isms at a concentration of 100 lg/mL similar to that of the stan-
dard antibiotic ciprofloxacin. The activity is considerably affected
by substituents present at the para position of phenyl ring. It has
been observed that the presence of a strong electron-donating
group specially –N(CH3)2 group enhances the electron conjugation
throughout the structure. The conjugation of electrons through the
compound 3(f) is faster than the compounds 2(a), 3(a), and 4(a).
This might be the reason of the highly active of compound 3(f) than
other substituents at same position. Also the results in Table 2 re-
vealed that the absence of substituents on the phenyl ring and the
presence of strong electron-withdrawing group such as NO2 and Cl
groups also did not favor the activity.
Bis(1,3,4-oxadiazole)derivatives 2(a–h) and 3,5-bis(substi-
tuted)pyrazoles or isoxazoles 3(a,b,d–i) and 4(a–c) were synthe-
sized and their in vitro antibacterial and antioxidant activities
have been evaluated. Compounds 2(b), 3(b), and 4(a) having hy-
droxyl and N,N dimethyl moiety at para position demonstrated po-
tent antioxidant and anti-lipid peroxidation while compounds
27. Experimental details and characterization data for new compounds are
provided in the Supplementary data.