Synthesis and evaluation of substituted 5-(3-chloro-2-oxo-4-phenylazetidin- 1-ylamino)pyrimidine-2,4,6(1H,3H,5H)-triones against pentylenetetrazole-induced convulsant in mice

Article abstract of DOI:10.1007/s00044-011-9760-8

This study was designed to synthesis of substituted 5-(3-chloro-2-oxo-4- phenylazetidin-1-ylamino)pyrimidine-2,4,6(1H,3H,5H)-triones followed by evaluation against pentylenetetrazole-(PTZ) induced convulsant in mice. The titled compoundswere confirmed by

Full text of DOI:10.1007/s00044-011-9760-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2300–2306
DOI 10.1007/s00044-011-9760-8
ORIGINAL RESEARCH
Synthesis and evaluation of substituted 5-(3-chloro-2-oxo-4-
phenylazetidin-1-ylamino)pyrimidine-2,4,6(1H,3H,5H)-triones
against pentylenetetrazole-induced convulsant in mice
•
Karan Deep Singh Puri Shailja Sood
•
Arunachalam Muthuraman
Received: 4 October 2010 / Accepted: 19 July 2011 / Published online: 29 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract This study was designed to synthesis of substi-
tuted 5-(3-chloro-2-oxo-4-phenylazetidin-1-ylamino)pyrimi-
dine-2,4,6(1H,3H,5H)-triones followed by evaluation against
pentylenetetrazole-(PTZ) induced convulsant in mice. The
titled compounds were confirmed by IR and 1H-NMR spectral
techniques. Pre-treatment of compound 4c showed significant
anticonvulsant activity at 40 mg/kg which was comparable to
that of PTZ and sodium valproate pre-treated groups. The
results show the importance of barbituric acid derivative (i.e.,
compound 4c (R = p-OH, m-OCH3) for this anti-convulsant
activity. It may be due to its anti-oxidative and neuroprotec-
tive potential. Therefore, compound 4c emerged as the most
active molecules in the management of convulsive disorder.
children and young people among the developed and
developing countries. Moreover, prevalence rates are 3.6 to
4.2 per 1000 children (Blom et al., 1978; Skinner et al.,
2010). The incidence of epilepsy is higher in men than in
women and appears to be higher in African-Americans
than in Caucasians (Haerer et al., 1986). In fact, epilepsy is
most frequent neurological disorder after cerebrovascular
disease and dementia in elder people (Kramer, 2001).
Cardinal feature of epilepsy syndromes are the predispo-
sition of recurrent unprovoked seizures and convulsions.
Seizures, in turn, are sudden brief attacks of altered con-
sciousness; motor, sensory, cognitive, psychic, or auto-
nomic disturbances; or inappropriate behavior caused by
abnormal excessive or synchronous neuronal activity in the
brain, whereas, convulsions are violent and involuntary
contractions of voluntary muscles (Tuchman et al., 2010;
Kossoff and Andermann, 2010). Epilepsy is associated
with high prevalence of reproductive disorders and leads to
infertility (Kim et al., 2010). Electrical discharges alter the
secretion of pituitary hormones and results in the repro-
ductive dysfunction (Kim et al., 2010). Therefore, emerg-
ing therapies are needed for these disorders.
Keywords Anti-convulsant Á Barbituric acid Á
Mus musculas Á Pentylenetetrazole
Introduction
Epilepsy is one of the most common neurodegenerative
disorders and is affecting at least 50 million people
worldwide. Epilepsy is major neurological problem in
Barbiturate therapy for acute brain insults achieves a
better clinical outcome than the standard treatment and
number of studies reporting disappointing results for both
traumatic brain injury (DeDeyne, 2010; Molina et al.,
2009) and acute cerebral ischemia (Chang et al., 2008).
Moreover, a high incidence of adverse effects have been
reported in association with barbiturate therapy including
cardio-respiratory depression, impaired white cell function,
hypokalemia, stroke, severe cutaneous reactions, hepatic,
and renal injury (Shaik and Mehvar, 2010; Mamishi et al.,
2009; English and Davis, 2010; Hashizume et al., 2002).
Barbiturates were considered as drug of choice in only
intensive care unit (Thuong et al., 2008). On the other
K. D. S. Puri Á S. Sood Á A. Muthuraman
Rayat Institute of Pharmacy, Nawanshahr District,
Near Ropar, Railmajra, Punjab 144533, India
A. Muthuraman (&)
Rayat Institute of Pharmacy, District Shaheed Bhagat Singh
Nagar, Railmajra, Punjab 144533, India
e-mail: arunachalammu@gmail.com
Present Address:
A. Muthuraman
Department of Pharmaceutical Sciences & Drug Research,
Punjabi University, Patiala, Punjab 147002, India
123

Med Chem Res (2012) 21:2300–2306
2301
hand, long-time continuous administration of barbiturates
is associated with an increase of the incidence of side
effects including immunosuppression (Stover and Stocker,
1998). However, barbiturates exert pleiotropic effects in
vivo. The neuroprotective effect of barbiturates was ini-
tially attributed to their ability to reduce cerebral metabo-
lism (Wakamatsu et al., 2009). Paradoxically, barbiturate
therapy reduces CBF, CMRO2 and increases cerebral
vascular resistance in patients with severe head injury (Neil
and Dale, 2009). Acute and chronic intracranial hyperten-
sion is also significantly reduced with a concomitant ele-
vation in cerebral perfusion pressure (Cordato et al., 2003).
Several studies, since 1970 suggested that barbiturate
therapy has a major impact on the prognosis of patients
with intractable intracranial hypertension and other neu-
rological disorders (An et al., 2010; Ghahremanzadeh
et al., 2010). Further, the currently available, other anti-
convulsants drugs (AEDs) are also effective in reducing the
severity and number of seizures in less than 70% of
patients. Moreover, their usage is associated with unde-
sirable side effects ranging from cosmetic (gingival
hyperplasia) to life threatening diseases such as hepato-
toxicity and megaloblastic anemia (Greenwood, 2000;
Llompart-Pou et al., 2007). Therefore, new molecules with
safer and more effective anti-epileptic drugs are needed for
the management of epileptic disorder. This study focused
on the synthesis and evaluation of pharmacologically
active substituted 5-(3-chloro-2-oxo-4-phenylazetidin-1-
ylamino)pyrimidine-2,4,6(1H,3H,5H)-triones in the man-
agement of epileptic disease.
experimental protocol. Each animal was used only once.
The experimental protocol was duly approved by Institu-
tional Animal Ethics Committee (IAEC) and care for the
animals was taken as per the guidelines of Committee for
the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Ministry of Environment and Forest,
Government of India (Reg No: 874/ac/05/CPCSEA).
Synthesis of substituted 5-(3-chloro-2-oxo-4-
phenylazetidin-1-ylamino)pyrimidine-
2,4,6(1H,3H,5H)-triones
Synthesis of 5-bromopyrimidine-2,4,6(1H,3H,5H)-trione
(1)
A suspension of barbituric acid (12.8 g, 0.1 mol) in excess
of glacial acetic acid was prepared and to this bromine
(10.29 ml, 0.2 mol) was added dropwise. After complete
addition of bromine, the reaction mixture was stirred for
10 h and poured into ice-cold water and then left overnight
at room temperature and the completion of reaction was
monitored by TLC. The resultant precipitates were filtered,
thoroughly washed with distilled water, dried, and crys-
tallized from methanol to yield of compound 1 (47.65%),
mp 196°C, IR (KBr): 648 (C–Br), 1338 (C–N), 1715
1
(C=0), 3300 cm-1 (N–H). H-NMR (DMSO-d6): 11.27 (s,
2H, 2NHCO), 5.5 (s, 1H, CH–Br).
Synthesis of 5-hydrazinylpyrimidine-2,4,6(1H,3H,5H)-
trione (2)
The mixture of compounds 1 (20.6 g, 0.1 mol) and
hydrazine hydrate (9.8 ml, 0.2 mol) in methanol was
refluxed for 8 h and the completion of reaction was mon-
itored by TLC. The solvent was removed under reduced
pressure and crushed ice was added to it. The solid
obtained was filtered, washed with distilled water, and
crystallized from methanol to give compound 2 (58.25%),
mp 210°C, IR (KBr): 1292 (N–N), 1349 (C–N), 1720
(C=0), 3221 (NH–NH2), 3286 cm-1 (N–H), 1H-NMR
(DMSO-d6): 11.23 (s, 2H, 2NHCO), 5.3 (d, 1H, CH–NH–
NH2), 4.7 (s, 3H, NH–NH2).
Materials and methods
Drugs and chemicals
Chemicals such as barbituric acid, acetic acid, hydrazine
hydrate, benzaldehyde, anisaldehyde, vanillin, chloroacetyl
chloride, and triethylamine were obtained from SD Fine
Chemicals, Loba Chemicals, Nice Chemicals and Merck
Chemicals. Standard reference drugs sodium valproate
(VPA) and pentylenetetrazole (PTZ) were provided as gift
sample from Ranbaxy laboratory, Mumbai. All the
reagents used in this study were of analytical grade.
Synthesis of 5-(2-benzylidenehydrazinyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (3a)
Animals
To a solution of (2) (15.8 g, 0.1 mol) in methanol, anis-
aldehyde and few drops of acetic acid were added. The
resultant solution was refluxed for 8 h and poured into ice-
cold water. The completion of reaction was monitored by
TLC. The precipitates thus obtained were filtered, washed
with distilled water, dried, and crystallized from methanol
to yield compound 3a.
The mice weighing 15–25 g of either sex were obtained
from National Institute of Pharmaceutical Education and
Research (NIPER), Mohali. The animals were housed under
standard laboratory conditions maintained under a natural
light and dark cycle, and had free access to food and water.
A 12 h light–dark cycle was maintained throughout the
123

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Med Chem Res (2012) 21:2300–2306
Acute toxicity studies
3a. (51.23%), mp 230°C, IR (KBr): 1270 (N–N), 1340
(C–N), 1610 (C–C), 1670 (C–N), 1713 (C=0), 3100 (C–H),
1
3290 (N–H) cm-1, H-NMR (DMSO-d6): 11.22 (s, 2H,
Acute toxicity studies were performed based on Organi-
zation of Economical Cooperation and Development
(OECD) guidelines for the synthesized compound. Syn-
thesized compounds (4a, 4b, and 4c) were administrated
(100, 200, and 1000 mg/kg, i.p.) in three different groups,
each groups comprised of three mice. Acute toxicity signs
and mortality were observed. The LD50 value was deter-
mined by calculating the geometric means of the lowest
dose that caused death and the highest dose for which the
animals survived as described method of Lorke (1983).
2NHCO), 8.60 (s, 1H, =CH–Ar), 8.25–7.85 (m, 5H, Ar–H),
5.2 (d, 1H, CH–NH), 4.5 (s, 1H, CH–NH).
3b. (58.82%), mp 185°C, IR (KBr): 1050 (C–O–C),
1290 (N–N), 1335 (C–N), 1600 (C–C), 1670 (C–N), 1713
1
(C=0), 3100 (C–H), 3280 (N–H) cm-1, H-NMR (DMSO-
d6): 11.24 (s, 2H, 2NHCO), 8.59 (s, 1H, =CH–Ar),
8.20–7.80 (m, 4H, Ar–H), 5.4 (d, 1H, CH–NH), 4.4 (s, 1H,
CH–NH), 3.40 (s, 3H, OCH3).
3c. (63.25%), mp 246°C, IR (KBr): 1060 (C–O–C),
1290 (N–N), 1300 (C–N), 1600 (C–C), 1700 (C=0), 3150
(C–H), 3240 (N–H), 3420 (O–H) cm-1, 1H-NMR (DMSO-
d6): 12.22 (s, 1H, OH), 11.25 (s, 2H, 2NHCO), 8.58 (s, 1H,
=CH–Ar), 8.20–7.80 (m, 3H, Ar–H), 5.5 (d, 1H, CH–NH),
4.2 (s, 1H, CH–NH), 3.45 (s, 3H, OCH3).
Induction of kindled seizure by administration of PTZ
PTZ-induced seizure test was performed according to the
method of Olivera et al. (2004). Mice (15–25 g) were
subjected for the administration of PTZ (60 mg/kg, s.c.) for
the development of seizures. Animals were observed for a
period of 30 min post-PTZ administration. The parameters
noted were mean onset of time of convulsions, duration of
convulsions, and % protection. Sodium valproate (80 mg/
kg, i.p.) was used as a standard drug and was injected
30 min before PTZ administration. Further, all the newly
synthesized barbituric acid derivatives (4a, 4b, and 4c)
were tested for their anticonvulsant activity.
Synthesis of 5-(3-chloro-2-oxo-4-phenylazetidin-1-
ylamino)pyrimidine-2,4,6(1H,3H,5H)-trione (4a)
The mixture of compounds 3a (2.46 g, 0.01 mol) in
dimethylformamide (DMF) and chloroacetyl chloride
(1.12 ml, 0.01 mol) with catalytic amount of triethylamine
was placed in round bottom flask and was refluxed at water
bath for 10 h. The completion of reaction was confirmed by
TLC. The resulting reaction mixture was then poured into
ice-cold water and filtered. The product was crystallized
from methanol to give compound 4a (52.84%), mp 260°C,
m/z (322) IR (KBr): 780 (C–Cl), 1280 (N–N), 1342 (C–N),
Experimental protocol for anticonvulsant studies
in mice
1600 (C–C), 1715 (C=O), 3105 (C–H), 3275 (N–H) cm-1
.
1H-NMR (DMSO-d6): 11.20 (s, 2H, 2NHCO), 8.30–7.80
(m, 5H, Ar–H), 5.90 (s, 1H, CH–Ar), 5.3 (d, 1H, CH–NH),
4.5(s, 1H, CH–NH), 4.1 (d, 1H, CH–Cl). C13H11ClN4O4
(Combound 4a): Calcd.: C, 48.38; H, 3.44; Cl, 10.99; N,
17.36; O, 19.83; Found: C, 48.71; H, 3.64; Cl, 10.65; N,
17.58; O, 19.63. 4b (54.34%), mp 247°C, m/z (352)IR
(KBr): 782 (C–Cl), 1340 (N–N), 1610 (C–C), 1715 (C=O),
Nine groups, each comprising of six mice weighing about
15–25 g, were employed in this study.
Group I (Normal control group): Mice were subjected to
administration of 1 ml of saline (0.9% w/v, i.p.) on the
day of experiment.
Group II (Negative control group): Mice were subjected
to administration of pentylenetetrazole (PTZ, 60 mg/kg,
s.c.) for the development of seizures on the day of
experiment.
3110 (C–H), 3280 (N–H) cm-1 1H-NMR (DMSO-d6):
,
11.20 (s, 2H, 2NHCO), 8.25–7.80 (m, 4H, Ar–H), 5.8 (s,
1H, CH–Ar), 5.4 (d, 1H, CH–NH), 4.5(s, 1H, CH–NH), 4.2
(d, 1H, CH–Cl), 3.42 (s, 3H, OCH3). C14H13ClN4O5
(Compound 4b): Calcd.: C, 47.67; H, 3.71; Cl, 10.05; N,
15.88; O, 22.68; Found: C, 47.83; H, 3.66; Cl, 10.41; N,
15.61; O, 22.93. 4c (62.06%), mp 291°C, m/z (368) IR
(KBr): 780 (C–Cl), 1305 (N–N), 1600 (C–C), 1715 (C=O),
Group III (Positive control group): Mice were subjected
to administration of sodium valproate (80 mg/kg, i.p.)
before 30 min of PTZ administration on the day of
experiment.
Group IV and V (Compound 4a): Mice were subjected to
pretreatment with compound 4a (10 and 40 mg/kg, i.p.)
before 30 min of PTZ administration on the day of
experiment.
1
3150 (C–H), 3245 (N–H) cm-1, 3420 (O–H) cm-1. H-
NMR (DMSO-d6): 12.25 (s, 1H, OH), 11.22 (s, 2H,
2NHCO), 8.15–7.65 (m, 3H, Ar–H), 5.8 (s, 1H, CH–Ar),
5.3 (d, 1H, CH–NH), 4.5(s, 1H, CH–NH), 4.2 (d, 1H, CH–
Cl), 3.39 (s, 3H, OCH3). C14H13ClN4O6 (Compound 4c):
Calcd.: C, 45.60; H, 3.55; Cl, 9.61; N, 15.19; O, 26.03;
Found: C, 45.72; H, 3.39; Cl, 9.49; N, 15.41; O, 26.31.
Group VI and VII (Compound 4b): Mice were subjected
to pretreatment with compound 4b (10 and 40 mg/kg,
i.p.) before 30 min of PTZ administration on the day of
experiment.
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Med Chem Res (2012) 21:2300–2306
2303
Group VIII and IX (Compound 4c): Mice were subjected
to pretreatment with compound 4c (10 and 40 mg/kg,
i.p.) before 30 min of PTZ administration on the day of
experiment.
Statistical analysis
All the results were expressed as standard error of means
( SEM). The data were statistically analyzed by one-way
analysis of variance (ANOVA) followed by Tukey’s mul-
tiple range tests by using Sigma stat Version-2.0 Software.
The P-value \0.05 was considered to be statistically
significant.
Biochemical evaluation
Following the behavioral testing, the animals were decap-
itated under ether anesthesia, and the brains were quickly
removed, cleaned with ice-cold saline, and stored at
-80°C. Brain tissue samples were thawed and homoge-
nized with 10 times (w/v) ice-cold 0.1 M phosphate buffer
(pH 7.4) using a glass homogenizer. Aliquots of homoge-
nates from rat brain were used to determine the lipid per-
oxidation, reduced glutathione, nitric oxide, and carbonyl
protein.
Results and discussions
Synthesized compound
Melting points of synthesized compounds were measured
using Buchi melting point apparatus and are uncorrected.
The 1H-NMR spectra were recorded on a Bruker AC-300F,
300 MHz instrument using DMSO-d6 as solvent, and tet-
ramethyl silane (TMS) as internal reference standard. Thin
layer chromatography was performed on silica gel thin
layer chromatography plates using ethyl acetate–hexane
(7:3 v/v) as the mobile phase.
Estimation of thiobarbituric reactive substances (TBARS)
Estimation of TBARS level as an index of lipid peroxi-
dation product (MDA; malondialdehyde) was determined
by thiobarbituric acid (TBA) reaction as described method
of Ohkawa et al. (1979). The absorbance was determined
spectrophotometrically at 543 nm.
5-hydrazinylpyrimidine-2,4,6(1H,3H,5H)-trione (2)
5-bromopyrimidine-2,4,6(1H,3H,5H)-trione (1) was pre-
pared by carrying out bromination of barbituric acid in the
presence of acetic acid which was followed by reaction
with hydrazine hydrate to obtain compound 5-hydrazinyl-
Estimation of tissue reduced glutathione
Estimation of reduced glutathione (GSH) level as an index
of oxidative stress marker was determined in the tissue
homogenate as described method of Ellman (1959). The
absorbance was determined spectrophotometrically at
412 nm.
1
pyrimidine-2,4,6(1H,3H,5H)-trione (2) (Fig. 1). H-NMR
spectrum exhibited two singlet at d 11.27 (s, 2H, NHCO)
and d 5.5 ppm (s, 1H, CH–Br) for the compound 1. IR band
for C–Br stretching was observed at 648 cm-1. The com-
pound 2 exhibited singlet at d 4.7 (s, 3H, CH–NH–NH2).
IR band at 1292 (N–N) cm-1 further supported the
structure.
Estimation of nitrite level
Estimation of nitrite (NO2-) and nitrate (NO3-) were
estimated by Griess reaction as an index of NO production
as described method of Cortas and Wakid (1990). The
absorbance was determined spectrophotometrically at
543 nm.
Substituted 5-(2-benzylidenehydrazinyl)pyrimidine-
2,4,6(1H,3H,5H)-triones (3a–c)
Reaction of compound 2 with different aromatic aldehydes
in the presence of methanol afforded compounds 3a, 3b,
and 3c (Fig. 1). The structure of these compounds was
Estimation of carbonyl protein content
1
elucidated by spectral analysis. H-NMR spectrum exhib-
Estimation of carbonyl protein content was determined by
the method described of Yan et al. (1995). The absorbance
was determined spectrophotometrically at 370 nm.
ited singlet at d 8.60 (1H, =CH–Ar) and multiplet at d
8.25–7.85 (5H, Ar–H) for the compound 3a. A peak at
1670 (C–N) cm-1 appeared in IR spectrum. Similarly,
anisaldehyde derivative 3b spectrum showed singlet at d
3.40 (3H, OCH3), and also a peak at 1050 (C–O–C) cm-1
in IR spectrum. For compound 3c, spectrum exhibited two
singlets at d 12.22 (1H, OH) at d 3.45 (3H, OCH3). IR
spectrum band was also observed at 3420 (O–H) cm-1
(Fig. 1).
Estimation of total protein content
Estimation of total protein concentration was estimated
according to the method of Lowry et al. (1951). The absor-
bance was determined spectrophotometrically at 750 nm.
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2304
Med Chem Res (2012) 21:2300–2306
Fig. 1 The synthesis of 5-(3-
chloro-2-oxo-4-phenylazetidin-
1-ylamino)pyrimidine-
2,4,6(1H,3H,5H)-triones from
5-bromopyrimidine-
2,4,6(1H,3H,5H)-trione from
barbituric acid by bromination,
hydrazination, attachment of
aromatic ring, and cyclization
reaction. Compound (1)
5-bromopyrimidine-
2,4,6(1H,3H,5H)-trione; (2)
5-hydrazinylpyrimidine-
O
O
O
O
O
HN
HN
HN
HN
NH2NH2.H2O
Br2/CH3COOH
HN
HN
O
O
Br
O
NHNH2
O
(1)
(2)
ArCHO
/CH3COOH
2,4,6(1H,3H,5H)-1trione; (3a)
R = H; (3b) R = p-OCH3; (3c)
R = p-OH, m-OCH3; (4a)
R = H; (4b) R = p-OCH3; (4c)
R = p-OH, m-OCH3
R
O
ClCH2COCl
/(CH3CH2)3
HN
HN
R
O
O
CHNH N CH
HN
CH
O
CHNH N
Cl
O
HN
O
O
(3a-3c)
(4a-4c)
Substituted 5-(3-chloro-2-oxo-4-phenylazetidin-1-
ylamino)pyrimidine-2,4,6(1H,3H,5H)-triones (4a–c)
or reactive species scavengers in a dose-dependent manner.
The results obtained from sodium valproate showed sig-
nificant protection against PTZ-induced convulsions as
depicted in Table 1. Pretreatment of compound 4a, 4b, and
4c attenuated the convulsions induced by PTZ in a dose-
dependent manner. Further, it was noted that all com-
pounds show significant anticonvulsant activity at high
dose level which is comparable with standard reference
drug, i.e., sodium valproate. Among all the newly synthe-
sized barbituric acid derivatives, compound 4c showed
83% protection at higher dose level whereas, at lower dose
showed 66% protection which was comparable to that of
standard drug sodium valproate. It was found that the
presence of hydroxyl and methoxy group in the molecular
framework has shown significant anticonvulsant activity.
Some studies reported that PTZ-induced convulsions are
attenuated by phenyl-butyl-nitrone, tocopherol, glutathi-
one, melatonin, high doses of ascorbate, and sodium val-
proate (Oliveira et al., 2004; Bashkatova et al., 2004).
Compound 3a, 3b, and 3c undergoes cycloaddition with
chloroacetylchloride in the presence of triethyl amine to
yield compound 4a, 4b, and 4c (Fig. 1). These compounds
1
have been characterized by IR and H NMR studies. The
formation of compound 4a was evidenced by appearance
of signal at d 4.1 due to (d, 1H, CH–Cl) in azetidinone ring
1
and IR band at 780 (C–Cl) cm-1. The H NMR spectra of
4b showed the azetidinone ring proton d 4.2 (d, 1H, CH–
Cl). The later compound showed IR bands at 1775 (C=O)
cm-1. Similarly, azetidinone derivative 4c exhibited dou-
blet at doublet d 4.2 (d, 1H, CH–Cl). A peak at (C=O)
cm-1 appeared in IR spectrum.
Acute toxicity studies
Acute toxicity studies were performed on the basis of
OECD guidelines, for the synthesized compound (4a, 4b
and 4c) as described method of Lorke (1983). No toxicity
or death was observed for synthesized compounds on
administration of a dose of 100, 200, and 1000 mg/kg. At
1000 mg/kg dose, there were slight behavioral changes and
70% mortality was found. Based on this observation, a
dose of 20 and 40 mg/kg of synthesized compounds were
selected for the evaluation of their anti-convulsant
activities.
Effect of compound 4a, 4b, and 4c in PTZ-induced
biochemical changes
Literature survey revealed that PTZ-induced convulsion
and brain bio & neurochemical changes can be prevented
or attenuated by various antioxidants or reactive species
scavenging molecule (Aldarmaa et al., 2010). Table 2
shows the changes of PTZ-induced brain biochemical
changes (i.e., TBARS, GSH, nitrite level, and carbonyl
protein content) in mice. PTZ is potentially induced bio-
chemical abnormality in mice brain. Pretreatment of
compound 4a, 4b, and 4c attenuated the alteration bio-
marker changes in a dose-dependent manner. Further, it
was noted that compound 4c shows significant biochemical
Effect of compound 4a, 4b, and 4c in PTZ-induced
convulsant
PTZ is a well-known GABAA antagonist. PTZ-induced
convulsions can be prevented or attenuated by antioxidants
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Med Chem Res (2012) 21:2300–2306
2305
Table 1 Effect of compounds 4a, 4b, and 4c on PTZ-induced convulsion in mice
Groups
Dose
Mean onset of time (s)
Duration of seizure (s)
% Protection
Naive mice
(0.9% w/v, NaCl)
60 mg/kg
80 mg/kg
20 mg/kg
40 mg/kg
20 mg/kg
40 mg/kg
20 mg/kg
40 mg/kg
258.61 6.42
261.34 6.42
300.23 2.61a
263.13 4.34b
267.48 6.24a
270.48 1.29b
278.44 2.16a
282.91 3.98b
290.58 4.34acd
a
4.38 2.1
4.56 3.1
1.1 0.4a
4.21 2.6b
2.73 2.5a
3.75 1.7b
2.31 2.6a
1.98 1.7b
1.60 0.6acd
b
0
0
PTZ
PTZ ? Sodium valproate
PTZ ? Compound 4a
PTZ ? Compound 4a
PTZ ? Compound 4b
PTZ ? Compound 4b
PTZ ? Compound 4c
PTZ ? Compound 4c
100
16
16
33
66
66
83
Data were expressed as standard error of mean (SEM, n = 6), P \ 0.05 versus PTZ control group, P \ 0.05 versus sodium valproate
pretreated group,
c,d
P \ 0.05 versus compound 4a and 4b (20 mg/kg) pretreated group, respectively
Table 2 Effect of compounds 4a, 4b, and 4c on PTZ-induced biomarker changes in mice
Groups
Dose
TBARS (nmol/mg
of protein)
GSH (lmol/mg
of protein)
Nitrite level (lmol/mg
of protein)
Carbonyl protein
(nmol/mg of protein)
Naive mice
0.9% w/v, NaCl
60 mg/kg
80 mg/kg
20 mg/kg
40 mg/kg
20 mg/kg
40 mg/kg
20 mg/kg
40 mg/kg
26.34 0.34
52.26 0.11
28.13 0.54a
48.36 0.28b
44.04 0.16b
47.11 0.09b
42.91 0.16b
35.19 0.13ac
30.68 0.26acd
a
0.093 0.004
0.011 0.002
0.089 0.003a
0.031 0.006b
0.036 0.002b
0.043 0.007b
0.047 0.001b
0.081 0.002ac
0.084 0.005acd
0.55 0.03
0.46 0.01
0.54 0.04a
0.46 0.02b
0.48 0.01b
0.47 0.09b
0.49 0.02b
0.53 0.02ac
0.54 0.01acd
b
18.14 0.24
31.56 0.58
19.06 0.39a
30.74 0.61b
29.61 0.42b
27.53 0.71b
25.94 0.64
21.68 0.32ac
20.48 0.53acd
PTZ
PTZ ? Sodium valproate
PTZ ? Compound 4a
PTZ ? Compound 4a
PTZ ? Compound 4b
PTZ ? Compound 4b
PTZ ? Compound 4c
PTZ ? Compound 4c
Data were expressed as standard error of mean (SEM, n = 6), P \ 0.05 versus PTZ control group, P \ 0.05 versus sodium valproate
pretreated group,
c,d
P \ 0.05 versus compound 4a and 4b (20 mg/kg) pretreated group, respectively
alteration at high dose level which is comparable with
standard reference drug, i.e., sodium valproate. Recent
report evidenced that accumulation of PTZ-induced mito-
chondria derived reactive oxygen and nitrogen species has
reported in the deleterious effects on the brain tissue leads
to convulsant action (Aldarmaa et al., 2010). We examined
the anticonvulsant effects of barbiturate derivatives on
PTZ-induced seizures in mice and the possible mechanisms
of protection against oxidative damage of brain tissue. Our
study indicated that PTZ causes the rise in oxidative stress
marker such as TBARS, nitrite, and carbonyl protein levels
and decreases the reduced glutathione level in brain
homogenates. Similar results were observed in other lab-
oratories (Aldarmaa et al., 2010; Uma Devi et al., 2006).
The variety barbiturate derivatives exhibit a different of
biological activities, such as central nervous depression,
sedative-hypnosis, anti-depression, muscle relaxant, local
anesthesia, anti-bacterial, anti-fungal, and anti-convulsant
activities (Uciechowska et al., 2008; Yan et al., 2009; Kidwai
et al., 2005). In our research, 5-(3-chloro-2-(4-hydroxy-3-
methoxyphenyl)-4-oxoazetidin-1-ylamino)pyrimidine-2,4,6
(1H,3H,5H)-trione, i.e., compound 4c as the lead compound
showed a positive anticonvulsant activity with an effective
dose of 40 mg/kg in the PTZ-induced convulsant model.
Conclusion
To conclude, we can state that the substituted 5-(3-chloro-
2-oxo-4-phenylazetidin-1-ylamino)pyrimidine-2,4,6(1H,3H,
5H)-triones may be served as the potential candidate for the
management of epileptic disorders due to its potential of
antioxidant property. Moreover, the elaborative studies are
necessary to explore their molecular mechanism of its
anticonvulsant activity.
Acknowledgment Thanks to Dr. A.C. Rana and all faculty mem-
bers of Rayat Institute of Pharmacy for their encouragement and
support. We are also grateful to Rayat & Bahra Educational and
Research Trust for their unconditional help to carry out this project.
Conflict of interest There was no conflict of interest in this study.
123

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Med Chem Res (2012) 21:2300–2306
barbiturate coma on adrenal response in patients with traumatic
brain injury. J Endocrinol Invest 30:393–398
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