Synthesis and evaluation of hexahydropyrrolo[3,4-d]isoxazole-4,6-diones as anti-stress agents

Article abstract of DOI:10.1016/j.ejmech.2011.11.037

A series of 2,3-diphenyl-5-(naphthalen-1-yl)-4H-2,3,3a,5,6,6a- hexahydropyrrolo[3,4-d]isoxazole-4,6-dione derivatives were synthesized via 1,3-dipolar cycloaddition of azomethine N-oxides with N-(α-naphthyl) maleimide. The pyrrolo-isoxazole derivatives were assigned cis- and trans- configurations (3-A and 3-B) with respect to proton C₃-H on azomethinic carbon on the basis of their ¹H NMR. The reaction proceeds through cis- endo addition rule indicating the predominance of cis isomer. The cis- and trans- isomers of a prototype compound 3a i.e., compound 3a-A and compound 3a-B were evaluated for anti-stress activity in immobilization-induced acute stress. Compound 3a-A (5 and 10 mg/kg) and compound 3a-B (10 mg/kg) attenuated immobilization stress-induced behavioral alterations in Swiss albino mice suggesting that pyrrolo-isoxazole may serve as lead molecule for the development of anti-stress agents.

Full text of DOI:10.1016/j.ejmech.2011.11.037

European Journal of Medicinal Chemistry 48 (2012) 81e91
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of hexahydropyrrolo[3,4-d]isoxazole-4,6-diones
as anti-stress agents
*
Rahul Badru, Preet Anand, Baldev Singh
Department of Chemistry, Punjabi University, Patiala 147002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 2,3-diphenyl-5-(naphthalen-1-yl)-4H-2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6-
Received 9 September 2011
Received in revised form
22 November 2011
Accepted 23 November 2011
Available online 1 December 2011
dione derivatives were synthesized via 1,3-dipolar cycloaddition of azomethine N-oxides with N-(
a
-
naphthyl)maleimide. The pyrrolo-isoxazole derivatives were assigned cis- and trans- configurations (3-A
and 3-B) with respect to proton C3-H on azomethinic carbon on the basis of their 1H NMR. The reaction
proceeds through cis- endo addition rule indicating the predominance of cis isomer. The cis- and trans-
isomers of a prototype compound 3a i.e., compound 3a-A and compound 3a-B were evaluated for anti-
stress activity in immobilization-induced acute stress. Compound 3a-A (5 and 10 mg/kg) and compound
3a-B (10 mg/kg) attenuated immobilization stress-induced behavioral alterations in Swiss albino mice
suggesting that pyrrolo-isoxazole may serve as lead molecule for the development of anti-stress agents.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Cycloaddition
Pyrrolo-isoxazoles
Stereoisomers
Acute immobilization stress
Behavioral alterations
1. Introduction
prominent role of tyrosine hydroxylase enzyme has been postu-
lated in stress models as the studies have shown its increased
Stress has been described as a sum total of all the reactions of
the body that disturb the normal physiological equilibrium and
result in a state of threatened homeostasis. Exposure to stress
stimuli induces various changes in the body including alteration in
behavior, autonomic function, hyper-activation of hypothalamus-
pituitary adrenal (HPA) axis and thus, secretion of hormones
including adrenocorticotropin hormone (ACTH) and corticosterone
[1]. Stress-induced hyper-activation of HPA axis leads to various
diseases such as hypertension [2], immunosuppression [3], repro-
ductive dysfunction [4], mental depression, schizophrenia, amnesia
and neurodegeneration [5]. Currently, the effective drugs are not
available in modern medicine to counter stress and associated
behavioral changes and therefore, the identification of new effec-
tive anti-stress agents is required for effective stress management.
Previous studies revealed that isoxazoline moieties possess
varied biological activities like anti cancer [6], anti-tubercular [7],
tyrosine phosphatase inhibitory activity [8], antifungal [9], anti-
influenza virus [10], glycoprotein IIb/IIIa receptor antagonists [11],
expression in different brain and adrenal tissue following stressful
conditions [16]. Furthermore, the drugs showing tyrosine hydrox-
ylase inhibitory activity has been reported to exhibit potent anti-
stress effects in different models [17]. The studies have shown
that pyrrolo-isoxazoles possess the tyrosine hydroxylase inhibitory
activity [18]. An increased activity of tyrosine hydroxylase leads to
increase in norepinephrine release which is responsible for stress
related behavioral alterations. It has been shown that pyrrolo-
isoxazole carboxylic acid derivatives decrease catecholamine and
5-HT levels in brain [19]. Furthermore, structurally related iso-
xazoline derivatives have also been reported to exhibit anti-stress
effects in an acute model of immobilization stress in rats [20].
Based on these it is hypothesized that pyrrolo-isoxazole derivatives
may be potentially exploited for anti-stress activity. Therefore, the
present study was designed to synthesize series of new pyrrolo-
isoxazole derivatives and evaluate one of the prototype chemical
moieties (cis- and trans- isomers) for anti-stress activity in
immobilization model.
analgesic and anti-inflammatory [12],
b-adrenergic receptor
antagonist properties [13], anti-HIV [14], anti-convulsant [15]. The
2. Chemistry
With viable reaction conditions and under inert nitrogen
atmosphere, 1,3-dipolar cycloaddition reaction between several
different substituted nitrones [21] and N-(
a-naphthyl)maleimide
* Corresponding author. Tel.: þ91 9988302741 (mobile).
E-mail address: drbaldev.chem@gmail.com (B. Singh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.037

82
R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
the range 1781e1787 cmꢀ1 due to imide carbonyl groups. In the 1H
NMR spectrum of trans diastereomer C3-H and C6a-H were ob-
tained as doublets at 5.5e5.6 and 5.0e5.1 with J z 7.88e8.04 Hz
and 8.92e9.12 Hz respectively on coupling with proton C3a-H,
while proton C3a-H was obtained at 4.3 as double doublet with
J z 8.24e8.88 Hz and 8.12e8.28 Hz on coupling with protons C3-H
and C6a-H respectively (3-B). However in the 1H NMR spectra of cis
diastereomer C3-H and C6a-H were obtained as doublets at
Table 1
Synthesis
xazole-4,6(5H,6aH)-dione derivatives.
of
dihydro-5-(naphthalen-1-yl)-2,3-diphenyl-2H-pyrrolo[3,4-d]iso-
d
O
d
O
N
N
O
d
5.3e5.4 and 5.0 with J z 7.88e7.96 Hz and 8.40e8.48 Hz
respectively on coupling with proton C3a-H, while proton signal for
C3a-H was obtained at 4.3 as double doublet with
X
d
J z 8.28e8.92 Hz and 8.10e8.18 Hz on coupling with protons C3-H
and C6a-H respectively (3-A). In all the reactions studied the cis and
trans isomers were obtained nearly in 3:2 ratio indicating the
predominance of endo-type transition state over the exo-type [24].
Compound - 3
Compound
X
% Yield
A:B
Melting point in ꢁC
A
B
3. Pharmacology
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
H
74
82
88
78
84
92
88
86
82
88
78
84
85
80
78
72
53:47
62:38
60:40
64:36
62:38
60:40
63:37
64:36
58:42
53:47
58:42
62:38
58:42
64:36
58:42
60:40
205e206
220e222
222e223
215e217
189e191
192e193
222e223
232e233
197e199
210e212
202e204
208e210
198e199
206e207
215e216
238e240
198e199
205e206
202e204
206e207
180e182
185e186
210e212
205e206
192e193
197e199
188e190
186e187
192e194
184e185
202e203
220e222
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-OH
3-OH
4-OH
2-OCH3
3-OCH3
4-OCH3
2-NO2
3-NO2
4-NO2
One of the pyrrolo-isoxazole derivatives i.e., compound 3a was
considered as a prototype and its two isomers (cis- and trans-) in
three different doses 2 mg/kg, 5 mg/kg and 10 mg/kg were evalu-
ated for potential anti-stress activity in Swiss albino mice. Diaz-
epam (2 mg/kg) was employed as a standard anti-stress agent. Mice
were subjected to acute immobilization stress and resulting
behavioral alterations were evaluated using actophotometer, social
interaction and open field tests.
4. Results and discussion
4.1. Biological activity
4.1.1. Effect of compound 3a-A and compound 3a-B on head dips
and rearings in the hole-board test in immobilization subjected mice
The head dips are considered as an index of curiosity or explo-
ration; while the frequency of rearing reflects the exploration of
novel surroundings. In immobilization subjected mice, the
frequency of head dips and rearing was decreased significantly as
compared to the normal control group. Treatment with the
compound 3a-A (5 and 10 mg/kg i.p.) and diazepam (2 mg/kg i.p.)
significantly attenuated immobilization stress-induced decrease in
the frequency of head dips and rearing. However, treatment of the
compound 3a-A at lower concentrations (2 mg/kg i.p.) did not
modulate stress-induced changes in the frequency of head dips and
rearing in a significant manner.
Treatment with the compound 3a-B (10 mg/kg i.p.) attenuated
stress-induced decrease in the frequency of head dips and rearing.
However, the comparative effect of trans- isomer on restoration of
head dips and rearing was significantly less as compared to corre-
sponding cis- isomer at the same dose level of 10 mg/kg. Treatment
with compound 3a-B (2 and 5 mg/kg i.p.) did not modulate
immobilization stress-induced decrease in the frequency of head
dips and rearing (Figs. 1 and 2). Furthermore, per se treatment with
compound 3a-A (10 mg/kg i.p.) and compound 3a-B (10 mg/kg i.p.)
did not modulate the frequency of head dips and rearing in the
normal mice.
has been examined and found to be independent of the nature of
substituents as is evident from their relative yields (Table 1) with
electron-rich (entry 3b, 3c, 3d, 3h, 3i, 3j, 3k, 3l and 3m), electron-
neutral (entry 3a), electron-poor (entries 3e, 3f, 3g, 3n, 3o and 3p),
o-substituted (entries 3b, 3e, 3h, 3k and 3n), m-substituted (entries
3c, 3f, 3i, 3l and 3o) and p-substituted (entries 3d, 3g, 3j, 3m and
3p) benzaldehyde substrates.
The starting compounds, azomethine N-oxides 1 (Scheme 1)
and N-(a-naphthyl) maleimide 2 (Scheme-2) were synthesized by
following similar procedure as reported in literature [22,23]. The
compound 1 reacted with compound 2 in sodium dried toluene
under refluxing conditions to afford a mixture of diastereoisomers,
which were separated and were characterized as cis and trans 4H-
2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6edione deriva-
tives 3aep (Scheme 3).
A 1,3-dipolar cycloaddition reaction between azomethine N-
oxide 1 and maleimide 2 seems to occur from the less hindered face
of the double bond of malemide ring to afford a mixture of
stereoisomers 3-A and 3-B [24] as indicated by TLC and 1H NMR
analysis, which were separated by flash column chromatography.
The two diastereomers were isolated in approximately 3:2 ratio.
In their I.R. spectra these derivatives exhibit a strong absorption
band (nmax) in the range 1703e1725 cmꢀ1 and a shoulder band in
Scheme 1. Synthesis of azomethine N-oxides.

R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
83
Scheme 2. Synthesis of N-(a-naphthyl)maleimide.
Scheme 3. Cycloaddition of azomethine N-oxide and N-(a-naphthyl)maleimide leading to the synthesis of diastereoisomers.
4.1.2. Effect of compound 3a-A and compound 3a-B on line crossing
and rearing in open field test in immobilization subjected mice
The line crossings are taken as an indicative of motor activity
and the frequency of rearing reflects the exploration of novel
surroundings. Immobilization stress for 120 min decreased the
number of line crossings and frequency of rearing as compared to
the normal control group. Treatment with the compound 3a-A (5
and 10 mg/kg) and diazepam (2 mg/kg i.p.) attenuated immobili-
zation stress-induced decrease in the number of line crossings and
frequency of rearing in a significant manner.
18
Head Dips in Hole Board test
16
14
12
10
8
b
b
b
b
a
6
4
2
0
Normal
Stress
Compound Compound Compound Compound Compound Compound Diazepam
3a-A (2 3a-A (5 3a-A (10 3a-B (2 3a-B (5 3a-B (10 (2mg/kg)
mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in in stress
stress stress stress stress stress stress
Fig. 1. Assessment of the exploratory behavior of animals in terms of number of head dips in 10 min time interval in the hole board test for investigating the anti-stress effects of
different doses of compounds 3a-A and 3a-B. Results are represented as mean ꢂ S.E.M. With n ¼ 10 in each group.a p < 0.05 as compared to the normal control;b p < 0.05 as
compared to the stress control.

84
R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
Rearing in Hole Board Test
16
14
12
10
8
b
b
b
b
a
6
4
2
0
Normal
Stress
Compound Compound Compound Compound Compound Compound Diazepam
3a-A (2 3a-A (5 3a-A (10 3a-B (2 3a-B (5 3a-B (10 (2mg/kg)
mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in in stress
stress stress stress stress stress stress
Fig. 2. Assessment of the exploratory behavior of animals in terms of frequency of rearing in 10 min time interval in the hole board test for investigating the anti-stress effects of
different doses of compounds 3a-A and 3a-B. Results are represented as mean ꢂ S.E.M. With n ¼ 10 in each group.a p < 0.05 as compared to the normal control;b p < 0.05 as
compared to the stress control.
Treatment with the compound 3a-B (10 mg/kg i.p.) attenuated
stress-induced decrease in the number of line crossings and
frequency of rearing. However, the restorative effects of trans-
isomer on line crossings and frequency of rearing was significantly
less as compared to corresponding cis- isomer at the same dose
level of 10 mg/kg. Treatment with compound 3a-B (2 and 5 mg/kg
i.p.) failed to attenuate immobilization stress-induced decrease
in the number of line crossings and frequency of rearing (Figs. 3
and 4).
the partner). Treatment with the compound 3a-A (5 and 10 mg/kg)
and diazepam (2 mg/kg i.p.) significantly attenuated immobiliza-
tion stress-induced non-social behavior. Treatment with the
compound 3a-B (10 mg/kg i.p.) attenuated stress-induced increase
in non-social behavior. However, the comparative effect of trans-
isomer on restoration of social behavior was significantly less as
compared to corresponding cis- isomer at the same dose level of
10 mg/kg. Treatment with compound 3a-B (2 and 5 mg/kg i.p.)
failed to attenuate immobilization stress-induced increase in non-
social behavior (Table 2).
4.1.3. Effect of compound 3a-A and compound 3a-B on following
and avoidance in the social interaction test in immobilization
subjected mice
In immobilization subjected mice, the non-social behavior
(avoiding the partner) was more predominant as compared to the
normal control group, which exhibited social behavior (following
4.1.4. Effect of compound 3a-A and compound 3a-B on the
locomotor activity in actophotometer test in immobilization stress
subjected mice
Single exposure of immobilization stress of 120 min led to
significant decrease in the locomotor activity in actophotometer
Line Crossings in Open Field Test
140
120
100
80
b
b
b
b
a
60
40
20
0
Normal
Stress
Compound Compound Compound Compound Compound Compound Diazepam
3a-A (2 3a-A (5 3a-A (10 3a-B (2
3a-B (5 3a-B (10 (2mg/kg)
mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in in stress
stress stress stress stress
stress stress
Fig. 3. Assessment of spontaneous, exploratory and ambulatory activity in terms of the line crossings in 10 min time interval in the open field test for investigating the anti-stress
effects of different doses of compounds 3a-A and 3a-B. Results are represented as mean ꢂ S.E.M. With n ¼ 10 in each group.a p < 0.05 as compared to the normal control;b p < 0.05
as compared to the stress control.

R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
85
Rearings in Open Field Test
45
40
35
30
25
20
15
10
5
b
b
b
b
a
0
Normal
Stress
Compound Compound Compound Compound Compound Compound Diazepam
3a-A (2 3a-A (5 3a-A (10 3a-B (2 3a-B (5 3a-B (10 (2mg/kg) in
mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in stress
stress stress stress stress stress stress
Fig. 4. Assessment of spontaneous, exploratory and ambulatory activity in terms of the frequency of rearing in 10 min time interval in the open field test for investigating the anti-
stress effect of different doses of compounds 3a-A and 3a-B. Results are represented as mean ꢂ S.E.M. With n ¼ 10 in each group.a p < 0.05 as compared to the normal control;b
p < 0.05 as compared to the stress control.
test. Administration of compound 3a-A (5 and 10 mg/kg i.p.) and
diazepam (2 mg/kg i.p.) attenuated immobilization stress-induced
decrease in decrease in the locomotor activity in a significant
manner. Treatment with the compound 3a-B (10 mg/kg i.p.)
attenuated stress-induced decrease in locomotor activity. However,
the comparative effect of trans- isomer on restoration of locomotor
activity was significantly less as compared to corresponding cis-
isomer at the same dose level of 10 mg/kg. Treatment with
compound 3a-B (2 and 5 mg/kg i.p.) failed to attenuate immobili-
zation stress-induced decrease in locomotor activity (Fig. 5).
In the present investigation, immobilization-induced acute
stress resulted in significant behavioral alterations including
decrease in locomotor activity (decrease in frequency of counts
in the actophotometer test); spontaneous and orientation-
investigating activities (decrease in head dips and rearings in the
hole board test along with decrease in total line crossings and
rearings in the open-field test) and alteration of social behavior
(decrease in time of following and an increase in time of
avoidance in the social-interaction test). The earlier studies from
our institute have shown that stress produces behavioral alter-
ations including decrease in locomotor activity, exploratory
behavior and social behavior [25].
In the present study, administration of compound 3a-A (5 mg/
kg and 10 mg/kg) 30 min prior to immobilization stress attenuated
immobilization-induced different behavioral alterations in
a significant manner. However, its trans- isomer compound 3a-B
attenuated stress-induced behavioral alterations only at higher
dose with relatively lesser efficacy at comparable dose level sug-
gesting the prominent anti-stress activity of cis- isomer as
compared to its corresponding tran- isomer. Diazepam, employed
as a standard anti-stress agent, also attenuated immobilization-
stress induced behavioral alterations. Compound 3a-A and
compound 3a-B (cis- and trans- isomers, respectively) were
selected as a representative molecules of synthesized pyrrolo-
isoxazole derivatives to explore whether pyrrolo-isoxazole mole-
cule serve as lead with potential anti-stress activity. These
compounds contain unsubstituted C-phenyl ring while all others
are the derivatives of these compounds with different substitutions
with electron withdrawing and electron donating at C-phenyl ring.
Immobilization-induced stress has been the most frequently
employed model of stress because stressor is of sufficient intensity
to alter most of the stress responsive biological systems. Immobi-
lization is a complex stressor and includes physical as well as
psychological dimensions. The struggling and muscular exertion of
the mice during the process of immobilization represents a physical
dimension; while limited range of movement along with exposure
in an open area represents the psychological dimensions [26].
Furthermore, immobilization stress produces behavioral alter-
ations in a reversible manner and animal tend to recover from the
stress after a definite time interval depending upon the duration of
immobilization stressor. This is important as far as the timing of
administration of drug is concerned and the effect of the drug will
vary with the change in the time of administration of the drug. The
detailed pharmacokinetic parameters of compound 3a-A including
its t1/2 are not known, however, the data in hand suggests that drug
remain active in the body for sufficient period of time (at least
Table 2
Assessment of social and non-social behavior in the 10 min social interaction test by
noting time of following and avoiding the partner for evaluating the anti-stress
effect of different doses of compounds 3a-A and 3a-B. Results are represented as
mean ꢂ S.E.M. With n ¼ 10 in each group.
Experimental groups
Social interaction test
Following (s)
Avoidance (s)
Normal
Stress
498.5 ꢂ 27.5
40.6 ꢂ 12.9a
58.6 ꢂ 9.2
101.5 ꢂ 27.5
559.4 ꢂ 12.9a
541.4 ꢂ 9.2
Compound 3a-A (2 mg/kg) in stress
Compound 3a-A (5 mg/kg) in stress
Compound 3a-A (10 mg/kg) in stress
Compound 3a-B (2 mg/kg) in stress
Compound 3a-B (5 mg/kg) in stress
Compound 3a-B (10 mg/kg) in stress
Diazepam (2 mg/kg) in stress
145.0 ꢂ 10.9b
275.4 ꢂ 85.2b
49.5 ꢂ 6.9
455.0 ꢂ 10.9b
324.6 ꢂ 85.2b
550.5 ꢂ 6.9
58.4 ꢂ 8.3
541.6 ꢂ 8.3
179.7 ꢂ 12.2
295.6 ꢂ 13.5
420.3 ꢂ 8.3b
304.4 ꢂ 13.2b
a
p < 0.05 as compared to the normal control.
p < 0.05 as compared to the stress control.
b

86
R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
Locomotor Activity in Actophotometer Test
200
180
160
140
120
100
80
b
b
b
b
a
60
40
20
0
Normal
Stress
Compound Compound Compound Compound Compound Compound Diazepam
3a-A (2 3a-A (5 3a-A (10 3a-B (2 3a-B (5 3a-B (10 (2mg/kg) in
mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in mg/kg) in stress
stress stress stress stress stress stress
Fig. 5. Assessment of the locomotor activity in terms of counts in 5 min time interval in the actophotometer test for investigating the anti-stress effects of different doses of
compounds 3a-A and 3a-B. Results are represented as mean ꢂ S.E.M. With n ¼ 10 in each group.a p < 0.05 as compared to the normal control;b p < 0.05 as compared to the stress
control.
3e4 h) including the time of stressor and subsequent behavioral
evaluation.
6.1.3. General procedure for synthesis of N-(
a
-naphthyl)maleimide
-naphthyl)amine
Maleic anhydride (4.9 g, 50 mmol) and N-(
a
(7.15 g, 50 mmol) were condensed in toluene (50 mL) to get the
maleamic acid. Further the maleamic acid (4.82 g, 20 mmol) was
cyclised to maleimide in acetic anhydride (10 mL, 100 mmol) along
with catalytic amount of anhydrous sodium acetate (0.82 g,
10 mmol) by refluxing the mixture for 1.5 h on water bath and then
poured in ice cold water. Maleimide precipitated out in this manner
was characterized by melting point, TLC and IR spectroscopy.
5. Conclusion
A series of novel pyrrolo-isoxazole derivatives were synthesized
in good to excellent yields. The compound 3a-A (cis- isomer)
exhibited significant anti-stress activity in immobilization-induced
acute stress suggesting that pyrrolo-isoxazole may serve as
potential lead for development of effective anti-stress agents.
6.1.4. General procedure for cycloadditions
An oven-dried flask was cooled under a stream of nitrogen and
charged with azomethine N-oxide 1 (0.985 g, 5 mmol), N-(a-
6. Experimental
naphthyl)maleimide 2 (1.115 g, 5 mmol) and sodium dried toluene
(25 mL). The flask was equipped with a reflux condenser and the
mixture was refluxed for 3.5e4 h until the substrates were
consumed as judged by TLC. On completion the reaction mixture
was concentrated in vacuo and the precipitated compound was
filtered. The crude product consists of a mixture of cis and trans
isomers which was subjected to flash column chromatography over
silica gel (230e400 mesh) using hexane: ethyl acetate mixture as
eluent.
6.1. Chemistry
6.1.1. General
All reactions were carried out under a nitrogen atmosphere in
oven-dried glassware. Melting points reported are uncorrected.
IR spectra were recorded on a Perkin Elmer RX I FTIR Spectro-
photometer using KBr pellets. 1H NMR and 13C NMR spectra were
recorded on Bruker spectrometer at 400 MHz and 300 MHz,
respectively, with TMS as internal standard. Mass spectra were
recorded on Thermo Scientific, LTQ-XL LCMS. Elemental analysis
was carried out using Elementar Vario MICRO cube CHN
analyzer.
6.1.4.1. cis-Dihydro-5-(naphthalen-1-yl)-2,3-diphenyl-2H-pyrrolo
[3,4-d]isoxazole-4,6(5H,6aH)-dione (3a-A). Compound obtained as
white solid (0.82 g, 53%); mp: 205e206 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
¼ 4.3 (dd,1H, J ¼ 8.12 and 8.76 Hz); 5.0 (d,1H, J ¼ 8.48 Hz);
5.4 (d, 1H, J ¼ 7.88 Hz); 6.7e7.9 (m, 17H); 13C NMR (300 MHz, [D6]
6.1.2. General procedure for synthesis of azomethine N-oxides
Nitrobenzene (4.16 mL, 41 mmol) and ammonium chloride
(2.5 g, 46 mmol) were put into 100 mL water taken in a 500 mL
beaker and mixture was stirred with mechanical stirrer. 5.9 g
(83 mmol) zinc dust was added in portions to the stirring mixture
so that the temperature of mixture does not rise above 60e70 ꢁC.
After complete addition of zinc dust, the reduction completed after
temperature of mixture began to fall down. Filtered the mixture
and to the filtrate was added stoichiometric amount of aromatic
aldehyde and stirred the mixture till solid precipitated out. The
synthesis of product was confirmed by TLC and further by recording
the melting points of the nitrones obtained.
DMSO):
d
¼ 54.9, 70.3, 77.2, 118.7, 119.9, 122.8, 124.4, 125.1, 125.7,
126.2, 126.4, 126.6, 126.7, 127.5, 128.0, 128.5, 128.6, 128.8, 129.0,
129.4, 133.5, 134.5, 135.1, 146.7, 147.1, 171.9, 173.8; IR (KBr pellets):
1723, 1786 cmꢀ1(C]O); MS: m/z: 420 [M]þ, Anal. Calcd for
C27H20N2O3: C, 77.14; H, 4.76; N, 6.67, Found: C, 77.28; H, 4.74; N,
6.68.
6.1.4.2. trans-Dihydro-5-(naphthalen-1-yl)-2,3-diphenyl-2H-pyrrolo
[3,4-d]isoxazole-4,6(5H,6aH)-dione (3a-B). Compound obtained as
white solid (0.73 g, 47%); mp: 198e199 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.28 and 8.88 Hz); 5.0 (d, 1H,
J ¼ 8.96 Hz); 5.6 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m, 17H); 13C NMR

R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
87
(300 MHz, [D6]DMSO):
d
¼ 54.9, 70.5, 77.4, 118.7, 120.1, 122.0, 122.8,
C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found: C, 77.63; H, 5.01; N,
6.52.
124.3, 125.1, 125.8, 126.6, 126.8, 127.5, 127.8, 128.0, 128.3, 128.5,
128.6, 128.8, 129.4, 133.5, 134.5, 135.1, 146.9, 147.5, 172.1, 174.2; IR
(KBr pellets): 1724, 1786 cmꢀ1(C]O); MS: m/z: 420 [M]þ, Anal.
Calcd for C27H20N2O3: C, 77.14; H, 4.76; N, 6.67, Found: C, 77.31; H,
4.74; N, 6.68.
6.1.4.8. trans-3-(4-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3d-B). Compound ob-
tained as white solid (0.61 g, 36%); mp: 206e207 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.14 and 8.48 Hz);
6.1.4.3. cis-3-(2-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3b-A). Compound ob-
tained as white solid (1.10 g, 62%); mp: 220e222 ꢁC; 1H NMR
5.0 (d, 1H, J ¼ 8.92 Hz); 5.6 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m, 16H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 17.7, 55.7, 65.4, 76.5, 112.3, 120.2,
121.3, 123.0, 123.9, 124.4, 124.7, 125.1, 125.7, 126.2, 126.4, 126.8,
127.2, 127.5, 129.2, 132.3, 133.5,135.7,136.9, 145.3,146.0,147.1,172.2,
173.3; IR (KBr pellets): 1724, 1781 cmꢀ1(C]O); MS: m/z: 434 [M]þ,
Anal. Calcd for C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found: C,
77.67; H, 4.99; N, 6.59.
(400 MHz, CDCl3):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.14 and 8.28 Hz);
5.0 (d, 1H, J ¼ 8.48 Hz); 5.4 (d, 1H, J ¼ 7.88 Hz); 6.7e7.9 (m, 16H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 17.7, 55.1, 65.4, 76.4, 113.7, 119.8,
122.9, 123.6, 124.2, 124.4, 125.5, 126.4, 126.6, 126.9, 127.3, 127.8,
128.3, 129.4, 131.8, 133.0, 133.3, 134.9, 135.2, 142.8, 143.1, 143.7,
172.2, 173.3; IR (KBr pellets): 1725, 1786 cmꢀ1(C]O); MS: m/z: 434
[M]þ, Anal. Calcd for C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found:
C, 77.05; H, 5.16; N, 6.46.
6.1.4.9. cis-3-(2-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-
2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3e-A). Compound ob-
tained as white solid (1.18 g, 62%); mp: 189e191 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.16 and 8.48 Hz); 5.0 (d, 1H,
6.1.4.4. trans-3-(2-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3b-B). Compound ob-
tained as white solid (0.67 g, 38%); mp: 205e206 ꢁC; 1H NMR
J ¼ 8.40 Hz); 5.3 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 56.8, 68.2, 77.2, 116.8, 118.0, 120.6, 122.3,
122.9, 123.5, 124.1, 125.2, 125.5, 126.2, 126.4, 126.6, 127.0, 127.3, 127.6,
128.2, 128.8, 129.5, 131.4, 133.5, 145.9, 146.3, 172.7, 173.8; IR (KBr
pellets): 1724, 1784 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ,
Anal. Calcd for C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C,
70.99; H, 4.22; N, 6.02.
(400 MHz, CDCl3):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.16 and 8.24 Hz);
5.0 (d,1H, J ¼ 8.92 Hz); 5.5 (d,1H, J ¼ 8.04 Hz); 6.7e7.9 (m, 16H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 17.7, 56.0, 65.4, 76.5, 113.9, 120.1,
121.3,123.6,124.2,124.7,125.1,125.3,125.8,126.1,126.4,127.2,127.6,
128.1, 129.1, 131.9, 133.1, 134.7, 135.8, 143.5, 144.9, 145.3, 173.1, 173.3;
IR (KBr pellets): 1725, 1786 cmꢀ1(C]O); MS: m/z: 434 [M]þ, Anal.
Calcd for C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found: C, 77.10; H,
5.09; N, 6.46.
6.1.4.10. trans-3-(2-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3e-B). Comp-
ound obtained as white solid (0.72 g, 38%); mp: 180e182 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.12 and 8.38 Hz); 5.0 (d, 1H,
6.1.4.5. cis-3-(3-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3c-A). Compound ob-
tained as white solid (1.14 g, 60%); mp: 222e223 ꢁC; 1H NMR
J ¼ 9.12 Hz); 5.5 (d, 1H, J ¼ 7.88 Hz); 6.7e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 57.0, 68.7, 77.2, 116.9, 118.2, 120.8, 122.5,
123.1, 123.7, 124.1, 125.1, 125.5, 126.0, 126.2, 126.4, 126.8, 127.0, 127.4,
128.2, 128.9, 129.7, 133.6, 135.4, 146.7, 148.1, 173.4, 173.9; IR (KBr
pellets): 1724, 1784 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ,
Anal. Calcd for C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C, 71.04;
H, 4.23; N, 6.04.
(400 MHz, CDCl3):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.12 and 8.48 Hz);
5.0 (d, 1H, J ¼ 8.40 Hz); 5.4 (d, 1H, J ¼ 7.88 Hz); 6.7e7.9 (m, 16H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 17.7, 55.1, 65.3, 76.5, 112.5, 120.8,
123.2, 123.7, 124.4, 124.6, 125.2, 126.1, 126.4, 126.6, 127.5, 127.8,
128.0, 129.1, 132.0, 133.2, 133.3, 135.7, 135.9, 146.8, 147.9, 148.1, 173.1,
173.4; IR (KBr pellets): 1725, 1783 cmꢀ1(C]O); MS: m/z: 434 [M]þ,
Anal. Calcd for C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found: C,
77.74; H, 5.22; N, 6.25.
6.1.4.11. cis-3-(3-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3f-A). Comp-
ound obtained as white solid (1.25 g, 60%); mp: 192e193 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.12 and 8.92 Hz); 5.0 (d, 1H,
6.1.4.6. trans-3-(3-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3c-B). Compound ob-
tained as white solid (0.76 g, 40%); mp: 202e204 ꢁC; 1H NMR
J ¼ 8.40 Hz); 5.3 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 55.1, 67.4, 77.0,117.3,118.7,120.5,122.4,123.1,
123.6, 124.3, 124.7, 125.6, 125.7, 126.5, 127.3, 128.2, 128.5, 128.8, 129.0,
129.3, 129.7, 133.5, 134.7, 146.1, 147.9, 172.9, 174.1; IR (KBr pellets): 1714,
1783 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ, Anal. Calcd for
C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C, 71.16; H, 4.13; N, 6.19.
(400 MHz, CDCl3):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.16 and 8.80 Hz);
5.0 (d, 1H, J ¼ 8.92 Hz); 5.6 (d, 1H, J ¼ 7.96 Hz); 6.7e7.9 (m, 16H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 17.7, 56.0, 65.4, 76.8, 112.3, 120.0,
121.2, 123.6, 124.2, 124.5, 124.9, 125.7, 126.1, 126.2, 126.5, 127.0,
127.7,127.9,129.0,132.0,133.3,135.7,135.9,146.8,147.9,148.0,172.2,
173.3; IR (KBr pellets): 1725, 1782 cmꢀ1(C]O); MS: m/z: 434 [M]þ,
Anal. Calcd for C28H22N2O4: C, 77.42; H, 5.07; N, 6.45, Found: C,
77.69; H, 5.21; N, 6.28.
6.1.4.12. trans-3-(3-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3f-B). Comp-
ound obtained as white solid (0.83 g, 40%); mp: 185e186 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.12 and 8.88 Hz); 5.0 (d, 1H,
J ¼ 9.00 Hz); 5.5 (d, 1H, J ¼ 8.00 Hz); 6.8e7.9 (m, 16H); 13C NMR
6.1.4.7. cis-3-(4-tolyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-2H-
pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3d-A). Compound ob-
tained as white solid (1.08 g, 64%); mp: 215e217 ꢁC; 1H NMR
(300 MHz, [D6]DMSO):
d
¼ 55.8, 68.0, 77.2, 117.5, 118.9, 120.7, 122.6,
123.2, 123.6, 124.4, 124.7, 125.3, 125.7, 126.0, 127.1, 127.7, 128.3, 128.6,
128.9, 129.1, 129.4, 133.4, 134.8, 146.3, 148.1, 173.4, 174.2; IR (KBr
pellets): 1714, 1786 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ,
Anal. Calcd for C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C,
71.62; H, 4.21; N, 6.18.
(400 MHz, CDCl3):
8.80 Hz); 5.0 (d, 1H, J ¼ 8.48 Hz); 5.4 (d, 1H, J ¼ 7.96 Hz); 6.7e7.9
(m, 16H); 13C NMR (300 MHz, [D6]DMSO):
d
¼ 2.2 (S, 3H); 4.3 (dd, 1H, J ¼ 8.10 and
d
¼ 17.7, 56.0, 65.3,
76.5, 112.3, 120.1, 122.2, 123.1, 123.5, 124.3, 125.2, 125.5, 125.9,
126.3, 127.1, 127.3, 127.6, 128.1, 131.8, 132.9, 133.3, 134.3, 135.0,
146.0, 147.7, 148.1, 173.1, 173.3; IR (KBr pellets): 1724,
1781 cmꢀ1(C]O); MS: m/z: 434 [M]þ, Anal. Calcd for
6.1.4.13. cis-3-(4-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3g-A). Comp-
ound obtained as white solid (1.26 g, 63%); mp: 222e223 ꢁC; 1H NMR

88
R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.12 and 8.40 Hz); 5.0 (d, 1H,
C
27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.59; H, 4.63;
J ¼ 8.48 Hz); 5.3 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 13C NMR
N, 6.47.
(300 MHz, [D6]DMSO):
d
¼ 54.9, 70.3, 77.2, 118.1, 119.3, 122.3, 123.8,
124.5, 125.3, 125.7, 126.6, 127.5, 127.7, 128.0, 128.3, 128.6, 128.8, 129.1,
129.3, 129.6, 130.8, 133.5, 135.4, 146.3, 147.1, 172.9, 174.1; IR (KBr
pellets): 1719, 1784 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ,
Anal. Calcd for C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C,
71.09; H, 4.08; N, 6.29.
6.1.4.19. cis-3-(4-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3j-A). Comp-
ound obtained as white solid (1.02 g, 53%); mp: 210e212 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.16 and 8.48 Hz); 5.1 (d, 1H,
J ¼ 8.44 Hz); 5.3 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 9.9 (s, 1H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 54.9, 70.3, 77.2, 119.3, 122.1, 122.6,
6.1.4.14. trans-3-(4-chlorophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3g-B). Comp-
ound obtained as white solid (0.74 g, 37%); mp: 210e212 ꢁC; 1H NMR
124.3, 125.0, 125.6, 126.2, 127.4, 127.6, 127.9, 128.1, 128.3, 128.5, 128.7,
128.9, 129.3, 132.9, 134.2, 134.9, 145.7, 146.8, 147.4, 172.1, 173.3; IR
(KBr pellets): 1706,1785 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal. Calcd
for C27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.06; H, 4.59; N,
6.39.
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.16 and 8.80 Hz); 5.0 (d, 1H,
J ¼ 9.12 Hz); 5.5 (d, 1H, J ¼ 8.04 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 55.3, 68.7, 77.2, 118.7, 120.3, 121.1, 123.6,
124.2, 124.5, 125.5, 126.2, 127.3, 127.7, 128.1, 128.3, 128.5, 128.7, 129.1,
129.4, 129.7, 130.5, 132.9, 135.1, 145.7, 146.8, 173.4, 174.2; IR (KBr
pellets): 1720, 1784 cmꢀ1(C]O); MS: m/z: 454 [M]þ, 456 [Mþ2]þ,
Anal. Calcd for C27H19N2O3Cl: C, 71.36; H, 4.18; N, 6.17, Found: C, 71.13;
H, 4.09; N, 6.31.
6.1.4.20. trans-3-(4-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3j-B). Comp-
ound obtained as white solid (0.90 g, 47%); mp: 197e199 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.12 and 8.42 Hz); 5.0 (d, 1H,
J ¼ 8.96 Hz); 5.6 (d, 1H, J ¼ 7.96 Hz); 6.8e7.9 (m, 16H); 9.9 (s, 1H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 55.0, 70.5, 77.2, 118.9, 122.0, 122.6,
6.1.4.15. cis-3-(2-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3h-A). Comp-
ound obtained as white solid (1.20 g, 64%); mp: 232e233 ꢁC; 1H NMR
124.4, 125.1, 125.7, 126.4, 126.6, 126.9, 127.1, 127.5, 127.7, 128.0, 128.3,
128.5, 128.7, 129.0, 129.4, 133.5,135.1, 146.2, 147.6, 171.9, 174.0; IR (KBr
pellets): 1703, 1786 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal. Calcd for
C27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.11; H, 4.62; N, 6.38.
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.12 and 8.76 Hz); 5.1 (d, 1H,
J ¼ 8.40 Hz); 5.3 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m, 16H); 9.9 (s, 1H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 54.9, 70.5, 77.0, 118.7, 122.0, 122.4,
6.1.4.21. cis-3-(2-methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3k-A). Comp-
ound obtained as white solid (1.02 g, 58%); mp: 202e204 ꢁC; 1H
NMR (400 MHz, CDCl3):
and 8.76 Hz); 5.0 (d, 1H, J ¼ 8.40 Hz); 5.3 (d, 1H, J ¼ 7.96 Hz); 6.8e7.9
(m, 16H); 13C NMR (300 MHz, [D6]DMSO):
118.8, 120.0, 122.1, 122.8, 124.3, 125.1, 125.8, 126.3, 126.7, 127.5, 127.7,
128.0, 128.3, 128.6, 128.9, 129.1, 129.4, 133.5, 134.5, 135.1, 146.9, 147.4,
171.9, 173.8; IR (KBr pellets): 1705, 1781 cmꢀ1(C]O); MS: m/z: 450
[M]þ, Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C,
74.71; H, 4.86; N, 6.24.
124.1, 124.8, 125.3, 125.6, 126.3, 127.4, 127.6, 127.8, 128.0, 128.3, 128.5,
128.7, 128.9,129.4,133.5,134.2,134.7, 146.3, 147.7, 171.7, 173.9; IR (KBr
pellets): 1703, 1782 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal. Calcd for
C27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.53; H, 4.57;
N, 6.35.
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.14
d
¼ 54.8, 65.3, 70.5, 77.3,
6.1.4.16. trans-3-(2-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3h-B). Comp-
ound obtained as white solid (0.67 g, 36%); mp: 205e206 ꢁC; 1H
NMR (400 MHz, CDCl3):
1H, J ¼ 8.96 Hz); 5.5 (d, 1H, J ¼ 7.96 Hz); 6.7e7.9 (m, 16H); 9.9 (s, 1H);
13C NMR (300 MHz, [D6]DMSO):
123.3, 124.4, 125.1, 125.6, 126.3, 127.0, 127.4, 127.6, 127.9, 128.1, 128.4,
128.6, 128.7, 129.0, 133.5, 134.2, 134.9, 136.1, 146.8, 148.1, 171.9, 174.1;
IR (KBr pellets): 1703, 1782 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal.
Calcd for C27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.49; H,
4.56; N, 6.32.
d
¼ 4.3 (dd, 1H, J ¼ 8.14 and 8.68 Hz); 5.1 (d,
d
¼ 54.9, 70.6, 77.2, 119.8, 122.8,
6.1.4.22. trans-3-(2-methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3k-B). Comp-
ound obtained as white solid (0.74 g, 42%); mp: 188e190 ꢁC; 1H NMR
(400 MHz, CDCl3):
8.42 Hz); 5.1 (d, 1H, J ¼ 9.00 Hz); 5.5 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m,
16H); 13C NMR (300 MHz, [D6]DMSO):
119.9, 122.0, 122.8, 124.4, 125.9, 126.2, 126.6, 127.5, 127.8, 128.0, 128.2,
128.5, 128.7, 128.9, 129.1, 133.4, 133.7, 134.5, 135.5, 146.2, 147.0, 172.1,
174.2; IR (KBr pellets): 1705, 1783 cmꢀ1(C]O); MS: m/z: 450 [M]þ,
Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C,
74.76; H, 4.88; N, 6.24.
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.20 and
d
¼ 54.9, 65.4, 70.3, 77.4,118.7,
6.1.4.17. cis-3-(3-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3i-A). Comp-
ound obtained as white solid (1.04 g, 58%); mp: 197e199 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.16 and 8.76 Hz); 5.1 (d, 1H,
J ¼ 8.48 Hz); 5.3 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m, 16H); 9.9 (s, 1H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 54.9, 70.5, 77.0, 118.7, 121.9, 124.3,
6.1.4.23. cis-3-(3-methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3l-A). Comp-
ound obtained as white solid (1.17 g, 62%); mp: 208e210 ꢁC; 1H NMR
(400 MHz, CDCl3):
8.48 Hz); 5.0 (d, 1H, J ¼ 8.48 Hz); 5.3 (d, 1H, J ¼ 7.92 Hz); 6.7e7.9 (m,
16H); 13C NMR (300 MHz, [D6]DMSO):
119.9, 122.1, 122.9, 124.3, 124.6, 125.1, 126.0, 127.2, 127.5, 127.9, 128.1,
128.4, 128.7, 129.0, 129.3, 132.9, 133.2, 133.9, 145.7, 146.0, 146.4, 172.1,
173.3; IR (KBr pellets): 1703, 1782 cmꢀ1(C]O); MS: m/z: 450 [M]þ,
Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C,
74.89; H, 4.91; N, 6.17.
125.7, 125.9, 126.2, 126.6, 126.8, 127.4, 127.7, 128.0, 128.2, 128.4, 128.6,
128.7, 128.9, 129.1, 133.2, 134.9, 146.3, 146.5, 147.1, 171.7, 173.9; IR (KBr
pellets): 1706, 1784 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal. Calcd for
C27H20N2O4: C, 74.31; H, 4.59; N, 6.42, Found: C, 74.55; H, 4.63; N, 6.44.
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.18 and
d
¼ 54.9, 65.4, 70.5, 77.2,119.7,
6.1.4.18. trans-3-(3-hydroxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3i-B). Comp-
ound obtained as white solid (0.75 g, 42%); mp: 192e193 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.3 (dd, 1H, J ¼ 8.14 and 8.78 Hz); 5.1 (d, 1H,
J ¼ 8.92 Hz); 5.5 (d,1H, J ¼ 8.04 Hz); 6.8e7.9 (m, 16H); 9.9 (s, 1H); 13C
NMR (300 MHz, [D6]DMSO):
d
¼ 54.9, 70.6, 77.2, 119.9, 122.8, 125.1,
125.5, 125.9, 126.3, 126.5, 126.7, 127.0, 127.5, 127.8, 128.2, 128.5, 128.6,
128.8, 129.1, 129.4, 133.4, 134.5, 135.1, 146.9, 147.4, 171.9, 174.1; IR (KBr
pellets): 1704, 1784 cmꢀ1(C]O); MS: m/z: 436 [M]þ, Anal. Calcd for
6.1.4.24. trans-3-(3-Methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3l-B). Comp-
ound obtained as white solid (0.72 g, 38%); mp: 186e187 ꢁC; 1H NMR

R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
89
(400 MHz, CDCl3):
8.48 Hz); 5.1 (d, 1H, J ¼ 8.96 Hz); 5.5 (d, 1H, J ¼ 7.96 Hz); 6.7e7.9 (m,
16H); 13C NMR (300 MHz, [D6]DMSO):
120.0, 122.1, 123.2, 123.5, 124.6, 124.9, 125.2, 125.6, 126.1, 126.5, 127.0,
127.4, 127.7, 128.0, 129.1, 132.0, 133.2, 133.5, 135.7, 146.8, 147.9, 173.1,
173.4; IR (KBr pellets): 1703, 1783 cmꢀ1(C]O); MS: m/z: 450 [M]þ,
Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C,
74.87; H, 4.90; N, 6.17.
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.20 and
pellets): 1723, 1781 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.93; H, 4.43; N,
8.97.
d
¼ 55.1, 65.4, 70.3, 77.3, 119.7,
6.1.4.30. trans-3-(3-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3o-B). Comp-
ound obtained as white solid (0.76 g, 42%); mp: 202e203 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.20 and 8.62 Hz); 5.0 (d, 1H,
J ¼ 9.12 Hz); 5.6 (d, 1H, J ¼ 7.92 Hz); 6.8e7.9 (m, 16H); 13C NMR
6.1.4.25. cis-3-(4-Methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3m-A). Comp-
ound obtained as white solid (1.11 g, 58%); mp: 198e199 ꢁC; 1H NMR
(300 MHz, [D6]DMSO):
d
¼ 56.9, 69.1, 77.3, 112.5, 120.0, 121.1, 123.5,
124.2, 124.4, 124.6, 125.1, 126.1, 126.4, 126.6, 127.5, 127.8, 128.0, 132.2,
133.4, 133.7, 134.9, 136.1, 146.8, 147.1, 147.7, 173.4, 174.2; IR (KBr
pellets): 1724, 1783 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.91; H, 4.39;
N, 8.98.
(400 MHz, CDCl3):
8.76 Hz); 5.0 (d, 1H, J ¼ 8.48 Hz); 5.3 (d, 1H, J ¼ 7.88 Hz); 6.7e7.9 (m,
16H); 13C NMR (300 MHz, [D6]DMSO):
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.16 and
d
¼ 54.9, 65.3, 70.5, 77.2, 118.7,
119.9, 122.0, 122.6, 124.3, 125.0, 125.6, 126.2, 127.4, 127.7, 128.0, 128.1,
128.3, 128.5, 128.7, 128.9, 133.5, 134.2, 134.9, 146.3, 146.5, 147.1, 171.7,
173.9; IR (KBr pellets): 1703, 1783 cmꢀ1(C]O); MS: m/z: 450 [M]þ,
Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C, 74.81;
H, 4.84; N, 6.07.
6.1.4.31. cis-3-(4-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-
2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3p-A). Compound ob-
tained as white solid (1.00 g, 60%); mp: 238e240 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.14 and 8.40 Hz); 5.0 (d, 1H,
J ¼ 8.48 Hz); 5.4 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 13C NMR
6.1.4.26. trans-3-(4-methoxyphenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3m-B). Comp-
ound obtained as white solid (0.80 g, 42%); mp: 192e194 ꢁC; 1H NMR
(300 MHz, [D6]DMSO):
d
¼ 56.8, 68.2, 77.2, 113.9, 114.7, 121.4, 122.3,
122.5, 122.7, 123.3, 124.9, 125.1, 125.7, 126.1, 126.6, 127.0, 127.7, 127.9,
128.3, 129.0, 129.3, 129.5, 133.4, 146.0, 146.9, 172.8, 173.9; IR (KBr
pellets): 1725, 1787 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.83; H, 4.05; N, 9.11.
(400 MHz, CDCl3):
8.52 Hz); 5.1 (d, 1H, J ¼ 9.00 Hz); 5.5 (d, 1H, J ¼ 7.92 Hz); 6.8e7.9 (m,
16H); 13C NMR (300 MHz, [D6]DMSO):
d
¼ 3.9 (S, 3H, OCH3); 4.2 (dd, 1H, J ¼ 8.18 and
d
¼ 54.9, 65.4, 70.3, 77.2, 118.7,
119.8, 122.1, 122.6, 124.4, 125.1, 125.6, 125.9, 126.3, 126.5, 127.3, 127.6,
128.1, 128.3, 128.6, 129.0, 132.5, 132.8, 133.1, 133.5, 134.2, 135.0, 171.9,
174.0; IR (KBr pellets): 1704, 1783 cmꢀ1(C]O); MS: m/z: 450 [M]þ,
Anal. Calcd for C28H22N2O4: C, 74.67; H, 4.89; N, 6.22, Found: C, 74.81;
H, 4.84; N, 6.08.
6.1.4.32. trans-3-(4-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3p-B). Comp-
ound obtained as white solid (0.67 g, 40%); mp: 220e222 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.18 and 8.42 Hz); 5.0 (d, 1H,
J ¼ 9.12 Hz); 5.6 (d, 1H, J ¼ 7.96 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 56.9, 68.7, 77.3, 114.1, 114.8, 121.5, 122.4,
6.1.4.27. cis-3-(2-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-
2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3n-A). Compound ob-
tained as white solid (1.19 g, 64%); mp: 206e207 ꢁC; 1H NMR
122.7, 123.3, 123.8, 124.1, 124.6, 125.1, 125.8, 126.3, 126.6, 127.0, 127.9,
128.3, 128.7, 129.4, 133.5, 143.5, 145.1, 146.7, 173.3, 174.1; IR (KBr
pellets): 1724, 1787 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.87; H, 4.10; N, 9.11.
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.14 and 8.88 Hz); 5.0 (d, 1H,
J ¼ 8.40 Hz); 5.4 (d, 1H, J ¼ 7.92 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 56.5, 68.3, 76.9, 112.7, 113.5, 120.4, 121.3,
6.2. Biological evaluation
121.6, 121.9, 122.2, 122.6, 123.3, 124.0, 124.9, 125.7, 126.2, 126.7, 127.5,
127.7, 128.1, 129.4, 133.5, 145.4, 146.0, 148.3, 172.9, 174.0; IR (KBr
pellets): 1725, 1786 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.34; H, 4.11;
N, 9.24.
6.2.1. Animals
Swiss albino male mice, weighing 20e25 g, were employed in
the present study. Animals were fed on standard laboratory diet
and water ad libitum. The experimental protocol was approved by
the Institutional Animal Ethics Committee and care of the animals
was carried out as per the guidelines of the Committee for the
Purpose of Control and Supervision of Experimental Animals
(CPCSEA), Ministry of Environment and Forests, Government of
India (Reg.No.-107/1999/CPCSEA).
6.1.4.28. trans-3-(2-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-
phenyl-2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3n-B). Comp-
ound obtained as white solid (0.67 g, 36%); mp: 184e185 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.14 and 8.76 Hz); 5.0 (d, 1H,
J ¼ 8.96 Hz); 5.6 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 56.7, 68.3, 77.0, 112.9, 113.7, 121.3, 122.5,
6.2.2. Induction of acute immobilization stress
122.7, 123.3, 123.6, 124.1, 124.5, 124.9, 125.1, 125.6, 126.0, 127.3, 127.5,
127.7, 128.7, 129.9, 133.5, 145.9, 146.1, 147.3, 173.4, 174.1; IR (KBr
pellets): 1725, 1787 cmꢀ1(C]O); MS: m/z: 465 [M]þ, Anal. Calcd for
C27H19N3O5: C, 69.68; H, 4.09; N, 9.03, Found: C, 69.36; H, 4.13; N,
9.27.
Acute stress was produced as per the method described by Kvet-
nanskyand Mikulai [27]. Micewere immobilized in the proneposition
with all four limbs fixed to the immobilization board with adhesive
tape. The head was also fixed with a metal loop over the neck area,
with a consequent limitation of head motion. The mice were kept
immobilized in this position for 2 h for inducing acute stress.
6.1.4.29. cis-3-(3-Nitrophenyl)-dihydro-5-(naphthalen-1-yl)-2-phenyl-
2H-pyrrolo[3,4-d]isoxazole-4,6(5H,6aH)-dione (3o-A). Compound ob-
tained as white solid (1.05 g, 58%); mp: 215e216 ꢁC; 1H NMR
6.2.3. Behavioral measurements
All the animals were acclimatized for 5 min on behavioral test
equipments for three days before initiating the actual experimental
protocol. It is necessary to acclimatize the animals to the test
apparatus in the behavioral studies to avoid potentially confound-
ing effects induced by novelty of the testing apparatus that in-turn
reduces the variation in the experimental data. After exposure of
(400 MHz, CDCl3):
d
¼ 4.2 (dd, 1H, J ¼ 8.18 and 8.74 Hz); 5.0 (d, 1H,
J ¼ 8.48 Hz); 5.4 (d, 1H, J ¼ 7.88 Hz); 6.8e7.9 (m, 16H); 13C NMR
(300 MHz, [D6]DMSO):
d
¼ 56.9, 69.7, 77.2, 112.3, 120.5, 120.8, 121.1,
121.6, 121.9, 123.2, 123.5, 123.9, 124.3, 124.6, 125.2, 126.1, 126.5, 127.1,
127.7, 127.9, 129.0, 132.1, 135.3, 146.8, 148.0, 172.9, 174.1; IR (KBr

90
R. Badru et al. / European Journal of Medicinal Chemistry 48 (2012) 81e91
immobilization stress, the battery of behavioral tests were per-
formed in animals with the sequence of actophotometer, the hole
board, the open field, and the social interaction with a time gap of
5 min between the successive behavioral tests. All behavioral test
equipments were cleaned after each test.
6.2.4.2. Group II: stress control. The mice were subjected to single
episode of immobilization stress and the different behavioral tests
were performed as described in group I.
6.2.4.3. Group III, IV, V: compound 3a-A (2, 5 and 10 mg/kg) in stress
control. The synthesized compound 3a-A was administered in
different doses (2, 5 and 10 mg/kg) 60 min before subjecting the
animals to stress and the different behavioral tests were performed
as described in group I.
6.2.3.1. Hole board test. The hole board test was employed to assess
the exploratory behavior of animals. The hole board consisted of
a wooden box measuring 68 cm ꢃ 68 cm. The walls were 40 cm
high, and the box was raised 28 cm above the ground on a stand.
Four holes (4 cm in diameter) were cut into the floor of the appa-
ratus: each hole was 28 cm from a corner of the box along the
diagonal from the corner to the centre. The floor of the box was
marked out into four outer areas and one central area using black
masking tape. The central area was delineated by four lines of tape
each 20 cm from one of the walls, while the four outer areas were
marked out by diagonal lines of tape running from the corners of
the floor to the corners of the central square. The four holes were
thus located at the corners of the central area. The animals were
assessed for 10 min during which the number of rearing (repre-
senting exploration in the novel surroundings) and number of head
dips were recorded [28].
6.2.4.4. Group VI, VII, VIII: compound 3a-B (2, 5 and 10 mg/kg) in
stress control. The synthesized compound 3a-B was administered
in different doses (2, 5 and 10 mg/kg) 60 min before subjecting the
animals to stress and the different behavioral tests were performed
as described in group I.
6.2.4.5. Group IX: diazepam (2 mg/kg i.p.) in stress con-
trol. Diazepam was employed as standard drug and was adminis-
tered(2mg/kg)60minbefore subjectingtheanimals to stress and the
different behavioral tests were performed as described in group I.
6.2.4.6. Group X: compound 3a-A (10 mg/kg) per se. The synthe-
sized compound 3a-A (10 mg/kg) was administered in normal
animals and the different behavioral tests were performed as
described in group I.
6.2.3.2. Open field test. The open field test has been considered as
a non-conditioned anxiety test based on the creation of a conflict
between the exploratory drive of the rat and its innate fear to
exposure to an open area. The open field test has been employed to
assess the spontaneous activity, general exploration and ambula-
tion of the rodents. The open field consisted of a wooden box
90 cm ꢃ 90 cm ꢃ 38 cm positioned in a dimly lighted room. The
walls were painted black, while the floor was painted white and
was divided by 1 cm wide black lines into 25 squares of
17 cm ꢃ 17 cm (16 peripheral squares and 9 central squares). The
mice were placed in the centre of the open field for 10 min and
during this period; the number of line crossings and rearings were
noted [29].
6.2.4.7. Group XI: compound 3a-B (10 mg/kg) per se. The synthe-
sized compound 3a-B (10 mg/kg) was administered in normal
animals and the different behavioral tests were performed as
described in group I.
6.2.4.8. Group XII: DMSO (vehicle) in stress control. The vehicle for
compound 3a-A and compound 3a-B was administered 60 min
before subjecting the animals to stress and the different behavioral
tests were performed as described in group I.
6.2.5. Statistical analysis
6.2.3.3. Social interaction test. The social interaction test has been
widely used as an anxiety related paradigm. The social interaction
test was carried out in the same box in which open field test was
performed. During the 10 min test, the duration of social behaviors
was noted in terms of following the partner which also included
sniffing the partner, contact interaction (physical contact with
mutual responses and orientation toward the other), climbing over
or burrowing under it, and walking around it. A mouse is consid-
ered to be socially interacting when in close proximity to, and
facing the test partner. The remaining time interval was considered
as avoidance of partner and non-social behavior which included
actively turning away, keeping the approaching interaction partner
at distance with forepaws in an upright posture, freezing when
approached by the conspecific animal, self-grooming and remain-
ing alone [25].
The results were expressed as mean ꢂ standard error of means
(S.E.M.). The results were analyzed using one-way ANOVA followed
by post-hoc analysis using Tukey’s Multiple Comparison test for
comparison between different groups. The p < 0.05 was considered
to be statistically significant.
Acknowledgement
The authors acknowledge the financial support from UGC New
Delhi and are grateful to Department of Chemistry, Punjabi
University, Patiala, India for providing technical facilities and
financial assistance for the work.
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