Novel pyrazoline amidoxime and their 1,2,4-oxadiazole analogues: Synthesis and pharmacological screening

Article abstract of DOI:10.1016/j.bmcl.2013.06.042

A novel series of pyrazoline amidoxime (2a-d) and pyrazoly-1,2,4-oxadiazole (3a-p) and (4) of pharmacological significance have been synthesised. Structures of newly synthesised compounds were characterized by spectral studies. New compounds were screened

Full text of DOI:10.1016/j.bmcl.2013.06.042

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel pyrazoline amidoxime and their 1,2,4-oxadiazole analogues:
Synthesis and pharmacological screening
a
Srikantamurthy Ningaiah a, Umesha K. Bhadraiah a, , Shubakara Keshavamurthy ,
⇑
Chethan Javarasetty b
a Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 570 005, India
b Department of Biotechnology, Manasagangotri, University of Mysore, Mysore 570 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of pyrazoline amidoxime (2a–d) and pyrazoly-1,2,4-oxadiazole (3a–p) and (4) of pharma-
cological significance have been synthesised. Structures of newly synthesised compounds were charac-
terized by spectral studies. New compounds were screened for their in vitro antioxidant, antimicrobial
and antiinflammatory activities. Among the synthesized compounds, compound 2a, 3l and 3o were found
to be active antimicrobial agents in addition to having potent antioxidant activity, while the compound 3f
showed promising antiinflammatory activity in comparison with standard drug.
Received 4 April 2013
Revised 6 June 2013
Accepted 13 June 2013
Available online xxxx
Keywords:
Bis-heterocycles
Amidoxime
Ó 2013 Elsevier Ltd. All rights reserved.
EDCÁHCl
Antioxidant
Antimicrobial
Antiinflammatory
Although pyrazoles are less common in nature, they became
ubiquitous motif in numerous pharmaceutically active com-
pounds. This is due to their wide spectrum of biological activity
and easy preparation. In 1884 Knorr discovered the antipyretic ac-
tion of a pyrazole derivative in man and he named the compound
antipyrine. This stimulated interest in pyrazole chemistry. Pyrazole
and its synthetic analogues have been found to exhibit antidia-
betic,1 antidepressant,2 anticonvulsant,3 antimicrobial,4 analgesic,5
and antitumour6 activity and also serves as human acyl-CoA: cho-
lesterol acyltransferase inhibitors.7 In fact, Celecoxib, a pyrazole
derivative is now widely used in the market as antiinflammatory
drug.8 The 1,2,4-oxadiazole, known as an ester isostere, is present
in various biologically active compounds, such as benzodiazepine
receptor ligands, muscarinic receptor agonists and 5-HT3 receptor
antagonists.9 1,2,4-oxadiazole derivatives possess human tryptase
inhibitory activity,10 antitrypanosomal activity,11 b-amyloid imag-
ing agents in Alzheimer’s disease,12 genotoxic activity,13 peptide
inhibitory activity,14 antihyperglycemic activity,15 potential Com-
bretastatin A-4 (CA-4) analogs16 and oxadiazole mannich bases
show antimyco-bacterial activity.17
Oxygen Species). Balanced oxidants (ROS) generation and detoxifi-
cation in a normal cellular metabolism is important to keep the
mammalian cells in a healthy condition. When a cell fails to detox-
ify the excessive ROS generated as a result of damaging species or
low level of antioxidants they enter into a state of oxidative stress
and is damaged.18 High level of ROS can cause damage to cell struc-
ture, nucleic acids, membrane lipids and proteins.19 They also
damage purine and pyrimidine bases of DNA molecule, thus lead-
ing to mutation.20 Oxidative stress on a cell due to high concentra-
tion of ROS can lead to a variety of disorders including cancer,
neurodegenerative disorder, atherosclerosis and aging.21 Many
studies have suggested that antioxidants or other compounds that
can neutralize free radicals may be of pivotal interest in the pre-
vention of vascular diseases and some forms of cancer.22
The incidence of microbial infections has increased dramatically
in recent years.23 The wide spread use of antimicrobial drugs has
resulted in the development of resistance to these drugs by patho-
genic microorganisms and consequently, this has led to serious
health hazards.24 Though there are a number of antimicrobial
drugs such as ampicillin, posaconazole etc., the preparation of
these drugs involve multi steps and expensive chemicals. Thus, in-
tense efforts in antimicrobial drug discovery are still needed to de-
velop more promising, economical, and effective drugs for use in
the clinical arena.25
Antioxidants play a vital role in the defense mechanism against
oxidative damage induced by free radicals and ROS (Reactive
⇑
Corresponding author. Tel.: +91 9480477620; fax: +91 8212419292.
E-mail addresses: kbu68umesha@rediffmail.com, orgchemsri@gmail.com (U.K.
Amidoxime is sometimes considered as bioisostere for carbox-
ylic group, and there are some successful examples of drug
Bhadraiah).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.042
Please cite this article in press as: Ningaiah, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.042

2
S. Ningaiah et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
pyrazole incorporated 1,2,4-triazoles and benzoxazoles,29 pyrazole
Cl
integrated with 1,3,4-oxadiazole,30 1-arylmethyl-3-aryl-1H-pyra-
zole linked with 1,2,4-oxadiazole were synthesized and observed
in the enhancement of pharmacological effect.31 Series of 1,2,4-
oxadiazoles were prepared using amidoxime by acylation with
acid chlorides, anhydrides or by reaction of amidoxime with car-
bonyl-containing compounds (ester, acids, aldehydes), or via inter-
action of amidoxime with acid amides, nitriles in presence of ZnCl2,
or in the course of cycloaddition of N-oxides to nitriles.32–38
Prompted by these observations, we hereby report the synthesis
of pyrazolyl-amidoximes and bis (heterocycle) bearing pyrazole
and 1,2,4-oxadiazoles starting from inexpensive reagents in a sim-
ple procedure and evaluate them for in vitro antioxidant, antimi-
crobial and antiinflammatory activities.
The target compounds were prepared as shown in Scheme 1.
(see Supplementary Informationfor detailed experimental proce-
dure) The key intermediates 4,5-dihydro-1,3-diphenyl-1H-pyra-
zole-5-carbonitrile (1a–d) were synthesized utilizing a reported
method.39 The formation of pyrazolyl-1,2,4-oxadiazoles (3a–p)
can be explained based on the plausible mechanism illustrated in
(Scheme 2). Carboxyl group activation with EDCÁHCl proceeds sim-
ilarly to other carbodiimide couplings. An O-acylisourea intermedi-
ate is formed by the reaction of a carboxylic acid and the EDCÁHCl.
The O-acylisourea is a highly reactive species that readily reacts
with amidoxime (2a–d) to obtain N-acyl-intermediate (5). EDCÁHCl
is transformed to the corresponding water soluble urea (6) during
coupling reactions. Finally pyrazolyl-1,2,4-oxadiazoles (3a–p) was
achieved by cyclodehydration of intermediate (5).
O
N
OH
N OH
NH2
N
NH2
S
(A)
(B)
Figure 1. (A) Cardiotonic (B) abtiarthritic.
Ar1
Ar1
N
H
Ar1
(i)
(ii)
NH2
OH
N
N
CN
N
NH
N
N
(1a-d)
Ar2
(2a-d)
N
Ar1
Ar1
N
N
(v)
Ar2
(iii), (iv)
N
N
N
N
O
N
O
As reported in the literature the regioselectivity of the nitrile
group in compound 1a was further confirmed by the single crystal
X-ray studies40 of 2a wherein amidoxime group is attached to the
5th carbon of the pyrazoline ring. The ORTEP diagram is as shown
in Figure 2.
(3a-p)
(4)
Scheme 1. Reagents and conditions: (i) acrylonitrile, chloramine-T, EtOH, reflux
3 h; (ii) NH2OHÁHCl, Na2CO3, aq. EtOH, reflux 5–6 h; (iii) Ar2-COOH, EDCÁHCl, CH2Cl,
0–30 °C 1–2 h, then rt for 6 h; (iv) heating 110 °C, 12 h; (v) Br2, CH2Cl2, reflux 1 h.
The structures assigned to the compounds were substantiated
by their analytical and other spectral data. The structure of the
compound (2a) was confirmed by single crystal X-ray studies.
The IR spectra of all the synthesized compounds showed character-
istic signals at 1640–1595 cmÀ1 for (C@N), 1570–1460 cmÀ1 for
(C@C), 1260–1285 cmÀ1 for (C–N), and 1240–1230 cmÀ1 for
(C–O). The absence of NH2 stretch at 3248 cmÀ1 and appearance
of C@N at 1620 cmÀ1, also absence of NH2 and OH peak at 5.49
and 9.3 ppm, respectively, in 1H NMR confirms the formation of
oxadiazole 3a.
candidates containing amidoxime moiety (Fig. 1).26,27 A number of
amidoximes have already been used or are currently being used in
clinical trials as prodrugs for amidines, due to their ability to re-
lease NO.28 Nevertheless, the main application of this building
blocks subtype in drug design is related to the construction of
1,2,4-oxadiazole ring.
Pyrazoline/pyrazole incorporated with other biologically active
heterocycles like oxazole, oxadiazole, triazole, thiazole, thiadiazole
etc., was designed in an attempt to obtain synergistic chemother-
apeutic activity with higher selectivity and less toxicity. Recently,
In the 1H NMR spectra of compound 3a, Ha, Hb and Hc protons
of the pyrazoline ring were seen as a doublet of doublet. Among
Scheme 2. Proposed mechanism for pyrazoly-1,2,4-oxadiazole formation.
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S. Ningaiah et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Figure 2. Perspective diagram of the molecule (2a) with 50% probability displacement ellipsoids.
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. The maximum anti-
oxidant activity of compounds was observed in the following order
3o > 3h P 3p > 3f. Particularly, the compound 3o showed more
promising DPPH RSA as compared to that of standard ascorbic acid.
This could be due the presence of electron donating three-OCH3 and
one-OH group on benzene rings. The compounds 3c, 3g and 3k have
shown poor activity even if they process –OCH3 group at ortho posi-
tion. This indicates that the ortho-substituted benzene ring is less
interactive with DPPH radical may be due to immensity of methoxy
group and hence poor activity. It is interesting to notice that the
compound 2d with only one-OH group on benzene ring shows good
interaction with DPPH radical and showed propitious DPPH RSA.
The presence of free –NH2 and –OH group in the amidoximes
(2a–d) might add on to the electron donating capacity to DPPH rad-
ical thus increasing in activity. This is further supported by the fact
that the compounds other than 3o, 3h, 3p, and 3f, showed less
activity than compounds (2a–d), while, 3f has shown more potent
activity by reducing power determination. This may be due to
two halogen atoms on benzene rings. All other compounds showed
low to moderate reducing power activity. An intriguing lipid perox-
d=3.63-3.66 ppm
d=3.10-3.20 ppm
dd, J=17.5, 9.0 Hz
dd, J=21.0, 12.5 Hz
Ha
N
Hb
N
O
N
N Hc
d=4.59-4.63 ppm
dd, J=12.5, 5.5 Hz
Figure 3. Proton chemical shifts and couplings of 3a.
Ha, Hb and Hc protons, Hc is the most deshielded one due to its
close proximity to nitrogen and oxadiazole moiety and it appeared
as a doublet of doublets. The methylene protons of the pyrazoline
ring (Ha and Hb), exhibited a typical ABX spin system with Hc as a
doublet of doublets. (Fig. 3). In addition, the single crystal studies
of 2a confirmed that the connectivities of these protons to the car-
bons were also in accordance with the assigned structure.
The mass spectrum of all the compounds showed molecular ion
peak at M+1 corresponding to its molecular formula, which con-
firmed its chemical structure. The IR, NMR, mass spectra and ele-
mental analysis supported the structure of various synthesized
bis-heterocycles (3a–p).
idation inhibitory activity was observed for compound 2a at 10 lg/
mL concentration. This may be due to the ability of amidoximes to
donate NO under mild oxidants.44 NO can serve as a chain-termi-
nating antioxidant and reduces oxidative injury to mammalian cells
by attenuation of metal/peroxide oxidative chemistry, as well as li-
pid peroxidation.45–47 It has also been proposed that, at higher level
NO can enhance ROS toxicity due to its rapid reaction with superox-
All the synthesized compounds 2–4 were evaluated for their
in vitro antioxidant activity by various methods such as DPPH rad-
ical scavenging assay,41 reducing power determination42 and lipid
ide (OÀ) and CO2 to form highly reactive oxidants peroxynitrite
peroxidation inhibitory activity43 at three (10, 50, 100
lg/ml) dif-
2
(ONOOÀ) and nitrosoperoxocarbonate anion (ONOOCO2À) respec-
tively. Thus NO can act as pro-oxidant at higher concentrations.48
Prompted by these observations, we found out the effective concen-
tration (EC) of compound 2a required for lipid peroxidation inhibi-
tory activity. From the Table 2, it is clear that compound 2a showed
(ꢀthreefold) enhanced lipid peroxidation inhibitory activity than
ferent concentrations. The results of in vitro antioxidant activity
are represented in Table 1. The investigation of antioxidant screen-
ing revealed that some of the tested compounds showed moderate
to good antioxidant activity. The interaction of pyrazolyl-1,2,4-oxa-
diazole (3a–p) with stable DPPH free radical indicates their free
radical scavenging ability. Of all the compounds in these series,
3f, 3h, 3o and 3p showed good interaction with the DPPH radical.
The antioxidant activity of these compounds is related with their
vitamin-E at 0.1
lg/mL concentration. The remaining compounds
showed moderately good activity.
Please cite this article in press as: Ningaiah, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.042

Table 1
Antioxidant activity of synthesized novel series of pyrazole–amideoxime (2a–d) and pyrazoly-1,2,4-oxadiazole (3a–p)
Compounds
Ar1
Ar2
% DPPH radical scavenging assay
% Reducing power determination
% Lipid peroxidation inhibitory activity
10
g/ml)
50
100
10
g/ml)
50
100
10
g/ml)
50
100
(
l
(
l
(l
2a
2b
—
—
16 0.022
29 0.013
28 0.091
39 0.032
51 0.099
69 0.086
08 0.091
11 0.083
12 0.082
28 0.016
56 0.088
68 0.023
32 0.012
18 0.012
15 0.019
12 0.080
14 0.026
14 0.063
Cl
2c
—
—
18 0.092
33 0.013
55 0.066
10 0.019
22 0.027
62 0.082
10 0.021
16 0.011
14 0.023
O
2d
3a
3b
24 0.016
18 0.076
23 0.022
41 0.086
28 0.036
42 0.012
76 0.055
39 0.032
67 0.079
12 0.036
06 0.019
12 0.086
34 0.011
16 0.016
22 0.092
79 0.045
58 0.061
65 0.012
12 0.012
21 0.063
34 0.053
18 0.020
40 0.042
48 0.238
20 0.016
57 0.022
73 0.761
HO
Cl
3c
14 0.012
26 0.191
34 0.082
56 0.111
44 0.016
66 0.093
10 0.016
12 0.019
22 0.082
29 0.016
54 0.082
68 0.061
11 0.086
14 0.056
31 0.130
52 0.026
60 0.052
71 0.048
O
O
3d
O
3e
3f
23 0.226
29 0.10
24 0.016
40 0.183
53 0.099
30 0.117
68 0.082
82 0.093
64 0.025
13 0.086
16 0.016
12 0.019
26 0.092
32 0.082
23 0.016
62 0.012
86 0.082
68 0.061
31 0.010
34 0.044
21 0.088
44 0.058
58 0.080
38 0.097
71 0.276
83 0.045
67 0.128
Cl
Cl
Cl
Cl
Cl
3g
O
O
3h
30 0.226
59 0.168
86 0.298
14 0.086
29 0.092
75 0.012
30 0.095
52 0.058
73 0.446
O

3i
19 0.078
22 0.181
20 0.036
36 0.289
52 0.008
41 0.049
51 0.247
72 0.410
53 0.056
11 0.016
12 0.019
11 0.086
20 0.082
23 0.016
24 0.092
64 0.082
58 0.061
65 0.012
26 0.456
34 0.014
21 0.051
40 0.025
48 0.133
33 0.121
62 0.085
70 0.011
57 0.206
O
O
O
O
3j
Cl
3k
O
O
3l
29 0.151
30 0.191
31 0.055
48 0.096
51 0.080
53 0.041
67 0.075
70 0.021
76 0.086
13 0.016
14 0.019
18 0.086
23 0.082
26 0.016
24 0.092
74 0.082
76 0.061
75 0.012
34 0.013
40 0.885
32 0.113
49 0.018
61 0.025
45 0.044
76 0.024
80 0.066
73 0.029
O
3m
3n
HO
HO
Cl
O
O
O
O
O
HO
HO
3o
34 0.065
63 0.034
91 0.091
16 0.016
26 0.082
76 0.082
42 0.091
62 0.032
87 0.078
3p
4
31 0.035
18 0.076
47 0.155
28 0.036
86 0.229
39 0.032
12 0.011
10 0.086
30 0.016
14 0.092
76 0.066
65 0.012
34 0.013
26 0.296
58 0.038
40 0.114
78 0.016
67 0.178
HO
Ascorbic acid
Vitamin-E
—
—
—
—
32 0.018
ND
57 0.083
ND
94 0.012
ND
14 0.183
ND
32 0.112
ND
82 0.163
ND
ND
47 0.203
ND
62 0.188
ND
92 0.110
Values are expressed as mean standard deviation (n = 3).

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S. Ningaiah et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 2
Effective concentration (EC) of 2a for lipid peroxidation inhibitory activity
Compound
Conc. in
0.01
lg/ml
0.05
0.1
0.25
0.5
0.75
1.0
2.5
5.0
2a
0.7 0.203
1.2 0.203
1.3 0.203
2.0 0.203
8.4 0.203
3.4 0.203
8.0 0.203
4.1 0.203
8.2 0.203
6.0 0.203
8.5 0.203
7.7 0.203
9.5 0.203
9.3 0.203
14.7 0.203
17.0 0.203
21.0 0.203
26.0 0.203
Vitamine-E
Table 3
‘Antimicrobial activity of pyrazole–amideoxime (2a–d) and pyrazolyl-1,2,4-oxadiazole (3a–p) based on the measurement of the zone of the inhibition grow’
Compounds
Antibacterial activity
Gram positive
B. cereus
Antifungal activity
Gram negative
S. aureus
50 g/
ml SD ml SD
E. coli
K. pneumonia
50 g/ 100
ml SD ml SD
S. flexneri
50 g/
ml SD ml SD
A. flavus
A. niger
50 g/
ml SD ml SD
50
lg/
100
l
g/
l
100
l
g/
50
l
g/
100
l
g/
l
l
g/
l
100
l
g/
50
l
g/
100
l
g/
l
100 lg/
ml SD ml SD
ml SD ml SD
ml SD ml SD
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
02 0.16 03 0.10 03 0.23 06 0.11 02 0.13 07 0.10 02 0.09 08 0.23 03 0.22 10 0.10 04 0.19 04 0.22 02 0.20 03 0.16
04 0.18 08 0.15 03 0.50 05 0.10 03 0.08 10 0.08 08 0.11 10 0.25 08 0.13 12 0.11 06 0.14 10 0.50 08 0.09 08 0.12
04 0.12 16 0.13 02 0.18 12 0.02 03 0.11 08 0.11 06 0.21 09 0.24 06 0.15 10 0.15 05 0.11 10 0.10 10 0.12 12 0.22
03 0.10 03 0.06 02 0.24 08 0.53 10 0.05 16 0.07 08 0.11 12 0.11 12 0.08 20 0.14 05 0.06 12 0.42 10 0.22 14 0.52
04 0.13 08 0.27 02 0.13 08 0.16 03 0.15 08 0.11 04 0.22 06 0.08 04 0.13 08 0.12 09 0.05 12 0.55 10 0.35 14 0.11
06 0.11 10 0.11 08 0.16 10 0.17 04 0.17 18 0.23 05 0.10 08 0.13 06 0.12 10 0.22 12 0.08 14 0.48 11 0.28 20 0.18
02 0.09 08 0.25 02 0.17 06 0.24 04 0.25 06 0.22 03 0.12 06 0.19 03 0.12 08 0.11 10 0.18 12 0.69 08 0.44 14 0.20
08 0.12 14 0.21 08 0.13 12 0.16 08 0.23 12 0.09 08 0.52 14 0.18 07 0.20 10 0.18 10 0.14 14 0.19 12 0.46 18 0.14
05 0.05 08 0.11 03 0.22 11 0.17 04 0.17 10 0.11 06 0.15 08 0.11 06 0.11 08 0.16 12 0.16 18 0.11 09 0.08 18 0.08
06 0.09 12 0.16 06 0.04 12 0.19 08 0.15 10 0.18 06 0.16 10 0.12 08 0.19 12 0.12 15 0.12 25 0.19 20 0.15 26 0.10
04 0.11 08 0.13 05 0.42 10 0.10 04 0.11 08 0.14 06 0.10 08 0.21 06 0.23 08 0.20 09 0.11 10 0.20 10 0.22 20 0.11
08 0.12 14 0.19 06 0.36 12 0.20 08 0.30 12 0.13 09 0.11 11 0.26 10 0.22 16 0.19 14 0.15 21 0.18 14 0.13 27 0.16
03 0.18 12 0.17 02 0.22 12 0.11 06 0.18 08 0.08 06 0.12 08 0.22 07 0.09 08 0.14 07 0.13 10 0.16 10 0.41 18 0.20
04 0.15 13 0.22 05 0.19 12 0.14 08 0.22 10 0.19 07 0.25 10 0.18 08 0.11 08 0.18 12 0.11 18 0.10 12 0.36 20 0.12
04 0.06 08 0.21 04 0.35 08 0.10 06 0.20 08 0.11 07 0.33 08 0.11 06 0.12 10 0.21 10 0.14 12 0.08 12 0.21 22 0.23
12 0.08 16 0.14 10 0.22 18 0.16 08 0.15 16 0.17 08 0.40 18 0.16 10 0.20 24 0.14 16 0.13 20 0.16 16 0.11 30 0.16
08 0.13 10 0.06 06 0.45 10 0.14 08 0.11 14 0.12 10 0.17 12 0.21 08 0.32 18 0.23 10 0.21 18 0.11 12 0.41 18 0.31
10 0.11 16 0.33 08 0.04 18 0.20 07 0.18 12 0.14 08 0.19 12 0.18 10 0.22 18 0.19 14 0.52 20 0.52 18 0.60 26 0.23
12 0.02 18 0.07 10 0.11 18 0.06 10 0.31 16 0.04 12 0.02 18 0.23 14 0.08 22 0.07 14 0.11 26 0.41 18 0.33 30 0.10
06 0.06 10 0.13 08 0.15 12 0.19 12 0.28 20 0.11 10 0.16 16 0.17 14 0.14 22 0.11 12 0.19 20 0.25 14 0.14 22 0.21
05 0.11 08 0.22 03 0.41 06 0.13 04 0.52 06 0.21 03 0.12 06 0.08 03 0.18 09 0.23 10 0.15 10 0.41 09 0.23 18 0.08
3m
3n
3o
3p
4
Chloramphenicol 10 0.07 18 0.11 09 0.11 16 0.14 10 0.04 18 0.06 12 0.18 20 0.09 10 0.18 24 0.21
—
—
—
—
Fluconazole 16 0.18 28 0.08 20 0.36 30 0.11
—
—
—
—
—
—
—
—
—
—
aZone of inhibition (Mean six replicate standard deviation).
The synthesized compounds were evaluated for in vitro antimi-
crobial activity against Bacillus cereus (MTCC 8372), Staphylococcus
aureus (MTCC 96), (gram-positive bacteria), Escherichia coli (MTCC
724), Klebsiella pneumonia (MTCC 3384), Shigella flexneri (MTCC
1457), (gram-negative bacteria) and two fungi Aspergillus flavus
(MTCC 873), Aspergillus niger (MTCC 281) by disc diffusion
method.49 The results are summarized in Tables 3 and 4. The re-
sults revealed that, compounds 3l and 3o showed excellent antimi-
crobial activity against all the tested strains of microbes, while
compounds 2d, 3d, 3h and 3p exhibited good to potent antimicro-
bial activity. Of the five tested bacterial strains, gram-positive bac-
teria were inhibited mostly by compounds 2c, 3d, 3i, 3j and 3k at a
The newly synthesized compounds 2–4 were tested for in vitro
antiinflammatory activity.50–52 Compared to the standard, they
have shown acceptable antiinflammatory activity. In vitro antiin-
flammatory activity of compounds is summarized in Table 5. The
results revealed that the compounds, 3f, 3m, 3p and 2d exhibited
moderate antiinflammatory activities. Among all the tested com-
pounds 3f was found to be more potent. The compounds 2a, 3e,
3o, 2b and 3n, showed good activity, while others showed weak
to moderate activities.
In summary, a series of novel pyrazoline amidoximes (2a–d)
and their 1,2,4-oxadiazole analogues (3a–p) and 4 was prepared
with moderate to good yields. The entire series of compounds were
characterized by IR, NMR and mass spectral data. All the synthe-
sized compounds 2–4 have been investigated for their in vitro anti-
oxidant, antimicrobial and antiinflammatory activity. Among the
synthesized compounds, compound 2a showed superior antilipid
peroxidation activity, 3o showed promising DPPH radical scaveng-
ing activity, while the compounds 3l and 3o showed excellent anti-
microbial activity, compound 3f showed potent antiinflammatory
activities in comparison with standard drug. Accordingly, these no-
vel classes of pyrazoline amidoximes and their 1,2,4-oxadiazole
analogues presented in our laboratory emerged as a valuable lead
series that might be useful as antioxidant, antibacterial, antifungal
and antiinflammatory agents and hence promising candidates for
concentration of 100 lg/mL was due to the presence of electron
donating –OCH3 group. While the gram-negative bacteria were
inhibited by compounds 2d, 3n, 3m and 3p this may be due to
the presence of –OH group. This was further confirmed by the fact
that the compounds containing both –OH and –OCH3 groups were
active against both types of bacteria. The activity is considerably
affected by substituents present at the para position of phenyl ring.
The compounds containing –OCH3 group on ortho position (3c, 3g,
3k,) were less active against both bacterial and fungal strains.
While the compounds containing electron withdrawing –Cl group
were less active against bacterial strains but they possess good
antifungal activity.
Please cite this article in press as: Ningaiah, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.042

S. Ningaiah et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
7
Table 4
Antimicrobial activity (MIC) of compounds pyrazole–amideoxime (2a–d) and pyrazoly-1,2,4-oxadiazole (3a–p)
Compounds
Antibacterial activity
Gram positive
B. cereus
Antifungal activity
Gram negative
S. aureus
g/ml
E. coli
K. pneumonia
g/ml
S. flexneri
g/ml
A. flavus
A. niger
g/ml
lg/ml
l
l
g/ml
l
l
lg/ml
l
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
50
30
35
40
30
25
55
20
30
25
35
20
45
40
35
10
20
15
10
25
35
10
—
40
40
50
50
50
20
55
20
50
20
30
25
55
30
35
15
25
20
15
20
55
10
—
50
40
55
15
50
30
35
20
45
20
35
20
25
25
25
20
20
25
15
10
40
10
—
40
20
25
20
30
30
55
20
25
25
25
15
25
25
25
20
15
25
10
15
50
10
—
40
20
25
10
20
25
50
25
25
20
25
15
25
25
25
15
20
15
10
10
55
10
—
30
25
30
35
15
10
15
10
10
10
15
10
25
10
15
10
15
10
10
10
15
—
50
20
15
15
15
10
20
10
15
5
15
10
10
10
10
10
10
5
3m
3n
3o
3p
5
10
20
—
4
Chloramphenicol
Fluconazole
5
5
a(Mean six replicate standard deviation).
Supplementary data
Table 5
Supplementary data (experimental details, IR, NMR, MS and ele-
mentary analysis) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.042.
In vitro antiinflammatory activity of compounds (2a–d) and (3a–p)
Compounds
Mean absorbance SD
% Inhibition of denaturation
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
0.2456 0.018
0.3210 0.007
0.2498 0.020
0.3501 0.026
0.2489 0.023
0.3102 0.010
0.2501 0.011
0.2566 0.052
0.3228 0.010
0.3600 0.041
0.3165 0.022
0.3098 0.026
0.2488 0.010
0.3178 0.020
0.2566 0.031
0.2622 0.019
0.3523 0.008
0.3198 0.018
0.3215 0.026
0.3365 0.022
0.2764 0.010
0.3721 0.013
61.70
59.06
23.78
63.48
23.33
53.71
23.93
27.15
59.96
78.39
56.83
53.51
23.29
57.48
27.15
29.93
74.58
58.47
59.31
69.74
36.96
84.39
—
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