Anticonvulsant and toxicity evaluation of newer 4H-benzo[1,4]oxazin-3-ones: The effect of two hydrogen bonding domains

Article abstract of DOI:10.1002/ardp.201000098

A series of (Z)-2-(substituted aryl)-N-(3-oxo-4-(substituted carbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl) hydrazine carboxamides (6a-r) was synthesized using 2-amino-5-nitrophenol as a starting material. All the synthesized compounds possessed two hydrogen-bonding domains and their effect on the activity was studied thereof. The anticonvulsant activity was assessed by the maximal electroshock test (MES), subcutaneous pentylenetetrazole test (scPTZ) and intraperitoneal thiosemicarbazide test (ipTSC). Compounds (6b, 6h, 6i, and 6p) were found to be the most potent of the series as they showed 83-100% protection in the MES test. They also displayed considerable activity in the chemically induced seizure tests. Most of the tested compounds were devoid of the neurotoxic and hepatotoxic effects. A series of (Z)-2-(substituted aryl)-N-(3-oxo-4-(substituted carbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin- 7-yl) hydrazine carboxamides (6a-r) was synthesized. Four compounds were found to be very promising as far as efficacy and safety is concerned. They may act as lead molecules for future investigations. Copyright

Full text of DOI:10.1002/ardp.201000098

Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
657
Full Paper
Anticonvulsant and Toxicity Evaluation of Newer 4H-
Benzo[1,4]oxazin-3-ones: The Effect of Two Hydrogen
Bonding Domains
1
1
1
1
Nadeem Siddiqui , Ruhi Ali , M. Faiz Arshad , Waquar Ahsan , Sharique Ahmed , and
2
1
M. Shamsher Alam
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi, India
Department of Biochemistry, Faculty of Medicine, 7th October University, Misurata, Libya
2
A series of (Z)-2-(substituted aryl)-N-(3-oxo-4-(substituted carbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-
7-yl) hydrazine carboxamides (6a–r) was synthesized using 2-amino-5-nitrophenol as a starting material.
All the synthesized compounds possessed two hydrogen-bonding domains and their effect on the activity
was studied thereof. The anticonvulsant activity was assessed by the maximal electroshock test (MES),
subcutaneous pentylenetetrazole test (scPTZ) and intraperitoneal thiosemicarbazide test (ipTSC).
Compounds (6b, 6h, 6i, and 6p) were found to be the most potent of the series as they showed 83–
100% protection in the MES test. They also displayed considerable activity in the chemically induced
seizure tests. Most of the tested compounds were devoid of the neurotoxic and hepatotoxic effects.
Keywords: Anticonvulsant activity / MES / Neurotoxicity / scPTZ / ipTSC
Received: April 1, 2010; Revised: June 14, 2010; Accepted: June 23, 2010
DOI 10.1002/ardp.201000098
Introduction
derivatives of benzoxazine have displayed several other
activities such as anti-inflammatory [4], antiulcer [5], antipla-
telet [6], antihypertensives [7], and antifungal [8], antimicro-
bial [9], neuropeptide Y (NPY) Y5 receptor antagonists [10],
serotonin reuptake inhibitors [11], prostacyclin receptor ago-
nists [12], etc. Recently some benzoxazine derivatives are
tested for their antimicrobial activity [13] and inhibitory
activity on human leukocyte elastase [14].
Epilepsy is the second most common disorder after stroke [1].
Despite the development of several anticonvulsants, the treat-
ments of epilepsy remain still inadequate. Studies indicate
that a significant group of patients (20–30%) are resistant to
the currently used therapeutic agents [2]. Adverse effects of
established antiepileptic drugs such as ataxia, hepatotoxicity,
gingival hyperplasia and megaloblastic anemia are a major
area of concern for the scientists worldwide.
To determine the effect of two hydrogen-bonding domains
(HBDs) on the activity [15], we designed the compounds in a
way to keep two HBDs, to which, were attached two hydro-
phobic aryl (A) rings. The two hydrogen-bonding domains
were present in semicarbazone (HBD1) and carbothioamides
(HBD2) moiety. The aryl ring (A1) attached to the HBD1 was
substituted with electron withdrawing groups and the aryl
ring (A2) that was attached to the HBD2 was substituted with
electron releasing groups. The prototype structure of the
synthesized compounds showing all the essential pharmaco-
phoric elements is depicted as Fig. 1.
The 2H-1,4-benzoxazin-3-(4H)-ones have been studied inten-
sively as important heterocyclic system for building natural
and designed synthetic compounds. Increasing evidence
suggests that benzoxazinone derivatives possess broad spec-
trum of biological activities and are less investigated for
anticonvulsant activity. While searching for compounds with
potential antiepileptic activity we directed our attention to
the benzoxazinone derivatives possessing varied pharmaco-
logical properties. Apart from anticonvulsant activity [3],
Results and Discussion
Correspondence: Nadeem Siddiqui, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062,
India.
E-mail: nadeems_03@yahoo.co.in
Fax: þ91 11 26059688 x 5307
Synthesis
The target compounds were synthesized according to the
Scheme 1. The starting material 2-amino-5-nitrophenol was
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

6
58
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
yl) urea (3). The reaction of compound (3) with hydrazine
hydrate yielded N-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl) hydrazinecarboxamide (4) which was then condensed
with different aryl aldehydes to yield corresponding (Z)-2-
A2
(
substituted aryl)-N-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-
-yl) hydrazinecarboxamides (5a–f), The titled compounds
6a–r) were obtained on further condensation of compounds
S
NH
HBD2
D
7
(
N
O
O
(5a–f) with different aryl isothiocyanates.
O
A1
N
Pharmacology
All the pharmacological tests were carried out with the
permission from Institutional Animal Ethics committee
N
H
N
H
HBD1
(
IAEC), form no. 527. Animals were obtained from Central
HBD1 = Hydrogen bonding domain -1; HBD2 = Hydrogen bonding domain -2
A1 = Hydrophobic aryl ring -1; A2 = Hydrophobic aryl ring -2
D = Electron donor moiety
Animal House Facility, Hamdard University, New Delhi. The
investigations were conducted on male albino mice (25–30 g)
at a dose of 30 mg/kg i.p.
Figure 1. Prototype structure of the final compounds showing pre-
sence of two hydrogen bonding domains and two hydrophobic aryl
rings
Anticonvulsant activity
cyclized to 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (1) on
treatment with chloroacetylchloride, which was reduced
by iron powder in hydrochloric acid to afford 7-amino-2H-
benzo[b][1,4]oxazin-3(4H)-one (2). This, on further treatment
with sodium cyanate in presence of glacial acetic acid was
converted into 1-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
The compounds were screened for their anticonvulsant
activity by maximal electroshock seizure (MES) test subcu-
taneous pentylenetetrazole-induced seizure (scPTZ) test and
intraperitoneal thiosemicarbazide-induced seizure (TSC)
tests and the possible neurotoxicity was assessed by the
rotarod method. The results of anticonvulsant activity
NO2
OH
NO2
O
H N
2
O
(
i)
(ii)
(iii)
N
H
O
N
H
O
NH2
1
2
H N
2
NH
O
NH
NH
O
(
iv)
H N
2
(v)
O
O
N
H
O
N
H
O
3
4
NH
NH
O
NH
NH
O
N
C H R
N
6
4
(vi) C H R
N
6
4
O
O
N
H
O
O
(
5a-f)
(
6a-r)
HN
C H R'
S
6
4
R = H, 2-Cl, 4-Cl, 4-OH, 2-NO
R′ = H, CH , OCH
Reagents and Conditions: (i) Chloroacetylchloride, reflux (ii) Fe/HCl, reflux (iii) sodium cyanate, glacial AcOH
iv) hydrazine hydrate, ethanol, reflux (v) aryl aldehydes, ethanol, reflux (vi) phenyl isothiocyanates, abs. ethanol
2
, 3-NO
2
3
3
(
Scheme 1. Synthetic route to the titled compounds (6a–r).
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Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
Newer 4H-Benzo[1,4]oxazin-3-ones as anticonvulsants
659
Table 1. Physicochemical data of synthesized compounds (6a–r)
a
b
Cmpd.
R
R’
Molecular formula
m. wt.)
M.p.
(8C)
R
f
value
% yield
Elemental analysis % calcd. (found)
(
C
H
N
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
H
H
H
H
CH
3
C
C
C
23
24
24
H
H
H
19
21
21
N
N
N
5
5
5
O
O
O
3
3
4
S (445.49)
S (459.52)
S (475.52)
190
182
200
180
187
192
180
187
192
201
200
204
189
182
187
189
182
187
0.6
0.5
0.4
0.5
0.6
0.6
0.5
0.4
0.5
0.6
0.5
0.5
0.4
0.3
0.6
0.3
0.5
0.4
70
64
78
59
73
75
78
70
73
75
79
83
81
79
78
74
75
71
61.60 (61.54)
62.34 (62.24)
60.09 (60.11)
57.11 (57.17)
57.90 (57.84)
55.73 (55.71)
57.16 (57.22)
57.96 (57.88)
55.74 (55.69)
59.14 (59.18)
60.13 (60.15)
59.92 (59.86)
55.81 (55.84)
56.90 (56.94)
56.56 (56.61)
55.82 (55.78)
56.91 (56.86)
56.92 (56.87)
3.91 (3.88)
3.82 (3.86)
4.11 (4.19)
3.20 (3.26)
3.68 (3.73)
3.20 (3.26)
3.28 (3.35)
3.60 (3.64)
3.22 (3.16)
3.81 (3.84)
3.91 (4.01)
4.03 (4.11)
3.50 (3.46)
3.69 (3.66)
3.47 (3.51)
3.56 (3.62)
3.64 (3.67)
3.43 (3.49)
15.31 (15.40)
14.71 (14.79)
14.11 (14.18)
14.00 (14.13)
13.72 (13.81)
13.20 (13.14)
14.01 (13.96)
13.70 (13.74)
13.21 (13.26)
14.54 (14.58)
14.66 (14.59)
14.82 (14.85)
16.91 (16.88)
16.10 (16.14)
16.32 (16.36)
16.91 (16.95)
16.12 (16.08)
16.30 (16.34)
OCH
H
CH
OCH
H
CH
OCH
H
CH
OCH
H
CH
OCH
H
CH
OCH
3
2-Cl
2-Cl
2-Cl
4-Cl
4-Cl
4-Cl
4-OH
4-OH
4-OH
2-NO
2-NO
2-NO
3-NO
3-NO
3-NO
C
23
H
H
H
H
H
H
18ClN
20ClN
20ClN
18ClN
20ClN
20ClN
5
O
5
O
5
O
5
O
5
O
5
O
3
3
4
3
3
4
S (479.94)
S (493.97)
S (509.96)
S (479.94)
S (493.97)
S (509.96)
3
C
C
C
C
C
24
24
23
24
24
3
3
3
C
23
H
H
H
H
H
H
H
H
H
19
N
21
N
21
N
18
N
20
N
20
N
18
N
20
N
20
N
5
5
5
6
6
6
6
6
6
O
O
O
O
O
O
O
O
O
4
S (461.49)
4
S (475.52)
5
S (491.52)
5
S (490.49)
5
S (504.52)
6
S (520.52)
5
S (490.49)
5
S (504.52)
6
S (520.52)
3
C
C
C
C
C
C
C
C
24
24
23
24
24
23
24
24
3
2
2
2
2
2
2
3
3
3
3
a
Solvent of crystallization: ethanol.
Solvent system: benzene/acetone (8:2), toluene/ethylacetate/formic acid (5:4:1).
b
Table 2. Anticonvulsant activity and neurotoxicity screening of compounds (6a–r)
a
Anticonvulsant activity (Percentage protection)
Compound
Neurotoxicity screen
MES
scPTZ
TSC
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0;h
ꢀ
ꢀ
ꢀꢀ
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
66
83
66
33
50
33
66
83
83
50
33
16
50
16
16
83
66
66
66
83
X
X
X
X
–
33
X
X
X
–
–
33
X
þ
ꢁ
þ
X
X
X
ꢁ
þ
þ
X
X
X
X
X
X
þ
þ
þ
þ
þ
þ
ꢁ
þ
X
X
X
þ
þ
þ
X
X
X
X
X
X
þ
þ
þ
þ
þ
ꢀ
ꢀ
ꢀ
16
33
X
X
X
33
50
66
X
X
X
–
66
66
X
X
X
X
X
X
50
50
X
X
X
16
16
50
X
X
X
X
X
X
33
66
66
ꢀꢀ
83
50
50
50
ꢀ
ꢀ
ꢀ
ꢀꢀ
83
ꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
100
100
50
33
33
66
83
X
X
X
X
X
X
83
50
66
X
X
X
X
50
33
33
X
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀ
83
66
83
66
50
50
33
—
ꢀ
ꢀꢀ
ꢀ
ꢀ
33
50
—
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
Phenytoin
Phenobarbital
100
100
100
100
—
100
—
83
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀ
ꢀꢀꢀ
100
100
a
Number of animals tested (n ¼ 6); dose ¼ 30 mg/kg i.p.
ꢀ
ꢀꢀ
ꢀꢀꢀ
P < 0.05, P < 0.01, P < 0.001; Solvent used: propylene glycol (0.1 mL, i.p.)
The (þ) sign indicates 50% or more passed the neurotoxicity screening whereas, the dash (-) indicates 50% or more failed the
neurotoxicity screening; (X) denotes not tested; (—) denotes absence of activity at the administered dose
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N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
Table 3. Enzyme estimation of the most active compounds
Compound
Alkaline phosphatase (Mean ꢂ SEM)
SGOT level (Mean ꢂ SEM)
SGPT level (Mean ꢂ SEM)
Control
17.95 ꢂ 0.702
18.97 ꢂ 0.812
138.57 ꢂ 1.852
139.37 ꢂ 1.725
133.89 ꢂ 2.005
136.37 ꢂ 0.574
138.21 ꢂ 1.131
27.20 ꢂ 1.553
26.99 ꢂ 1.653
23.94 ꢂ 1.186
25.54 ꢂ 0.823
26.27 ꢂ 1.528
6
6
6
6
b
h
i
ꢀꢀ
22.79 ꢂ 0.847
20.43 ꢂ 0.621
18.97 ꢂ 0.822
p
Number of animals tested (n ¼ 6).
Significantly different from control P < 0.01
Mean ꢂ SEM values were calculated using ANOVA followed by Dunnett’s multiple comparison test.
ꢀ
ꢀ
together with the neurotoxicity data are presented in Table 2.
can also be concluded that these compounds do not cause any
mineral (Mg) or Vitamin (Vit C) deficiency.
In the MES test, compounds (6a, 6b, 6c, 6g, 6h, 6i, 6p, 6q, and
6
r) exhibited a higher degree of anticonvulsant activity by
showing protection from seizures in 66–100% animals.
Compounds (6h and 6i) were found to display 100% protec-
tion after 4.0 h in the MES screen (p < 0.001). The most
potent compounds against MES test were selected for
further investigations against seizures induced by penty-
lenetetrazole and thiosemicarbazide to prove their diverse
anticonvulsant activity and speculate about their possible
mechanism of action. In scPTZ and ipTSC tests, compounds
Conclusion
All the synthesized compounds possessed the pharmaco-
phoric elements necessary for the anticonvulsant activity.
The result from the present study suggests that the benzox-
azinone derivatives might have inhibited or attenuated PTZ-
induced seizures in mice by enhancement of GABAergic
neurotransmission. The antagonism of thiosemicarbazide-
induced seizures suggests that the compounds might have
inhibited aminotransferase in the brain. In the neurotoxicity
screen all the tested compounds except (6b) successfully
passed the test without any sign of neurological deficit.
Four compounds were found to be very promising as far as
efficacy and safety is concerned. They may act as lead mol-
ecules for future investigations.
(
6b, 6c, 6g, 6h, 6i, 6p, 6q, and 6r) showed 16–66% protection
that indicated mild to moderate activity in both the
models. Pentylenetetrazole have been reported to produce
seizures by inhibiting g-amino butyric acid (GABA)
neurotransmission, which is the main inhibitory neuro-
transmitter in the brain.
The structure–activity relationship study revealed that the
substitutions at the phenyl ring (A1) attached to the semi-
carbazone moiety (HBD1) played a major role in determining
the activity of the compounds as the unsubstituted deriva-
tives and the chloro- and nitro-substitution at the para- and
meta-position respectively were the most active of the series.
Substitutions by other functional groups at other positions
led to decrease in the activity. In particular, the p-chloro
substituted compounds were the most potent and showed
activity in all the screens. The other phenyl ring (A2) that was
attached to the carbothioamide moiety (HBD2) did not have
any marked effect on the activity.
Experimental
Chemistry
All chemicals used for the experimental work were supplied by E.
Merck India Ltd., Central Drug House, s.d fine chemicals, and
Qualigens. Thin layer chromatography was performed on pre-
coated silica gel plates using silica gel G (160–120 mesh, Merck).
Iodine vapors and UV lamps were used for visualization of TLC
spots. Melting points were determined in open glass capillary
1
and are uncorrected. H-NMR spectra were obtained with Bruker
4
00 MHz instrument in DMSO-d
6 3
/CDCl using tetramethylsilane
[
(CH Si] as internal standard. Chemicals shifts are recorded in
3 4
)
Liver Enzyme Estimation
Liver Enzyme estimation of the most active compounds was
carried out and the results are shown in Table 3. Compounds
parts per million (ppm). The infrared spectra were recorded in
KBr on Bio-Rad FTIR spectrometer.
(6b, 6h, 6i, and 6p) were selected for the study and found that
7-Nitro-2H-benzo[b][1,4]oxazin-3(4H)-one 1
all the values were comparable to the control and the changes
seen were not significant except in case of compound (6h) that
showed an elevated alkaline phosphatase value with p < 0.01.
Hence it can be concluded that the compounds do not have
any adverse affect on the liver or bone tissues. Since the
compounds (6b, 6i, and 6p) do not lower the ALP values it
2-Amino-5-nitro phenol (10 g, 0.06 mol) and NaHCO
0
2
to keep the temperature at 0–58C. To this mixture, 2-chloroace-
tylchloride (9.5 g, 0.08 mol) was added with moderate speed,
reacted for 40 min and refluxed on an oil bath for 2 h.
Solvent was removed under reduced pressure to obtain the
3
(21.8 g,
.26 mol) were placed into a round bottom flask containing
00 mL acetonitrile. The mixture was put in an ice-salt bath
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Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
Newer 4H-Benzo[1,4]oxazin-3-ones as anticonvulsants
661
resultant product 1 which was purified by recrystallization with
ethanol/H O (1:1).
2
(
-
Z)-2-Benzylidene-N-3-oxo-4-(4-tolylcarbamothioyl)
3,4-dihydro-2H-benzo[b][1,4] oxazin-7-yl)-
hydrazinecarboxamide 6b
FT-IR (KBr) (cm ): 3489 (NH), 2392 (CH str.), 1695 (C–O), 1339
7
-Amino-2H-benzo[b][1,4]oxazin-3(4H)-one 2
A mixture of compound 1 (8.84 g, 0.46 mol), iron powder (7.7 g,
.13 mol), and 200 mL ethanol (95%) was stirred and heated on an oil
ꢁ1
1
(
4
7
CH ¼ N), 1213 (C–S); H NMR (DMSO-d
–
6
) d (ppm): 2.35 (s, 3H,CH
), 6.41–7.09 (m, 3H, Ar-H), 7.12–7.21 (m, 3H, Ar-H),
.04–7.49 (m, 2H, Ar-H), 7.53–7.64 (m, 2H, Ar-H), 8.04–8.13 (m, 2H,
3
),
0
.68 (s, 2H, CH
2
bath until the temperature attains 80–858C. 4 mL HCl (1.2 M) were
added and refluxed for 4 h at 80–858C. After completion of reaction,
the mixture was filtered immediately while hot under reduced
Ar-H), 8.69 (s, 1H, –CH), 9.64 (s, 1H, NH-Ar), 10.21 (s, 1H, –N-NH).
–
–
pressure and the filtrate was neutralized with NaHCO
the pH was adjusted to 7–8, resultant in precipitation of solid 2.
3
until
(Z)-2-Benzylidene-N-(4-(4-methoxyphenylcarbamothioyl)-
3-oxo-3,4-dihydro-2H-benzo[b][1,4] oxazin-7-yl)-
hydrazinecarboxamide 6c
1
-(3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl) urea 3
FT-IR (KBr) (cmꢁ1): 3509 (NH), 2931 (CH str), 1695 (C–O), 1339
To a solution of sodium cyanate in a minimal quantity of water,
glacial acetic acid (5 mL) was added. This solution was heated with
compound 2 (5.6 g, 0.01 mol) in ethanol until the content of
the mixture became turbid and the volume remained half of
the original volume. The content was added to ice-cool water.
The solid 3 obtained was filtered off and dried.
CH–N), 1213 (C–S); 1H-NMR (DMSO-d
(
6
) d (ppm): 3.83 (s, 3H,
), 6.40–6.86 (m, 3H, Ar-H), 7.12–7.74 (m,
H, Ar-H), 8.01–8.07 (m, 2H, Ar-H), 8.71 (s, 1H, –CH), 9.66 (s, 1H,
NH-Ar), 10.18 (s, 1H, –N-NH).
3 2
OCH ), 4.60 (s, 2H, CH
7
(
(
Z)-2-(2-Chlorobenzylidene)-N-(3-oxo-4-
phenylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]
N-(3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)
hydrazinecarboxamide 4
To the warm hydrazine hydrate solution of compound 3 in
ethanol, conc. NaOH was added and refluxed for 6 h. Reaction
mixture was poured into crushed ice and the solid compound 4
obtained was filtered off and dried.
oxazin-7-yl)hydrazinecarboxamide 6d
FT-IR (KBr) (cm ): 3511 (NH), 2926 (CH str.), 1692 (C –– O), 1339 (CH ––
N), 1218 (C –– S); H-NMR (DMSO-d
7
1
ꢁ1
1
6
) d (ppm): 4.67 (s, 2H, CH
.36 (m, 7H, Ar-H), 7.60 (s, 1H, Ar-H), 7.78–8.10 (m, 3H, Ar-H), 9.29 (s,
H, –– CH), 9.68 (s, 1H, NH-Ar), 10.25 (s, 1H, –– N-NH).
2
), 7.10–
(
Z)-2-(2-Chlorobenzylidene)-N-(3-oxo-4-(4-
tolylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4] oxazin-
-yl)hydrazinecarboxamide 6e
General procedure for synthesis of (Z)-2-substituted aryl-
N-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)
hydrazinecarboxamides 5a–f
The solution of compound 4a in glacial acetic acid (5 mL) and
ethanol (10 mL) was heated to boiling and refluxed with benzal-
dehyde (0.12 mol) for 5 h. The refluxed solution was cooled to room
temperature and kept overnight. The solid 5a was collected out,
washed with methanol, dried and recrystallized from methanol to
get the pure compound. All other compounds of the series 5b–f
were also prepared by using respective aromatic aldehydes by the
above specified procedure with slight variation in reaction time.
7
ꢁ
1
FT-IR (KBr) (cm ): 3512 (NH), 2850 (CH str.), 1699 (C –– O), 1342 (CH ––
N), 1226 (C –– S); H-NMR (DMSO-d
7
1
1
6
) d (ppm): 2.41 (s, 3H, CH
.30 (m,7H, Ar-H), 7.58 (s, 1H, Ar-H), 7.77–8.12 (m, 3H, Ar-H), 4.27 (s,
H, –– CH), 9.65 (s, IH, NH, -Ar), 10.21 (s, 1H, –– N-NH).
3
), 7.10–
(Z)-2-(2-Chlorobenzylidene-N-(4-(4-
methoxyphenylcarbamothioyl-(3-oxo-3,4-dihydro-2H-
benzo[b][1,4]oxazin-7-yl)hydrazinecarboxamide 6f
ꢁ
1
FT-IR (KBr) (cm ): 3523 (NH), 2922 (CH str.), 1689 (C –– O), 1341 (CH –– N),
1
1
082 (C –– S); H-NMR (DMSO-d
m, 8H, Ar-H), 7.50 (s, 1H, Ar-H), 7.78–8.12 (m, 3H, Ar-H), 9.21 (s, 1H, ––
CH), 9.68 (s, 1H, NH-Ar), 10.24 (s, 1H, –– N-NH).
6 3
) d (ppm): 3.92 (s, 3H, OCH ), 7.19–7.28
General procedure for synthesis of (Z)-2-substituted aryl-
N-(3-oxo-4-(substituted carbamothioyl)-3,4-dihydro-2H-
(
benzo[b][1,4]oxazin-7-yl) hydrazine carboxamides 6a–r
To the boiling solution of compound 5a (0.01 mol) in anhydrous
ethanol (20 mL), substituted phenyl isothiocyanate (0.01 mol)
was added drop wise and the reaction mixture was refluxed
for 6 h. The solid obtained after subsequent concentration
and cooling was filtered, washed with ethanol, dried and recrys-
tallized from ethanol to give the pure product 6a. All other
compounds 6b–r were also prepared by using different substi-
tuted phenyl isothiocyanates (0.01 mol).
(Z)-2-(4-Chlorobenzylidene)-N-(3-oxo-4-
(phenylcarbamothioyl)-3,4-dihydro-2H-benzo[b] [1,4]
oxazin-7-yl) hydrazinecarboxamide 6g
FT-IR (KBr) (cm ): 3505 (NH), 2885 (CH str.), 1695 (C–O), 1341
ꢁ
1
–
1
(CH–N), 1220 (C–S); H-NMR (DMSO-d ) d (ppm): 4.66 (s, 2H, CH ),
–
7.11–7.34 (m, 8H, Ar-H), 7.58 (s, 1H, Ar-H), 7.70–8.15 (m, 3H, Ar-H),
–
6
2
9.27 (s, 1H, –CH), 9.68 (s, 1H, NH-Ar), 10.25 (s, 1H, –N-NH).
–
–
(
-
Z)-2-Benzylidene-N-(3-oxo-4-(phenylcarbamothioyl)
3,4-dihydro-2H-benzo[b][1,4] oxazin-7-yl)-
(Z)-2-(4-Chlorobenzylidene)-N-(3-oxo-4-(4-
tolylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
hydrazinecarboxamide 6a
FT-IR (KBr) (cmꢁ1): 3499 (NH), 2025 (CH str.), 1688 (C–O), 1037
yl)hydrazinecarboxamide 6h
FT-IR (KBr) (cm ): 3508 (NH), 2931 (CH str.), 1695 (C–O), 1339 (CH–
ꢁ1
1
–
–
1
(
C–S); H-NMR (DMSO-d
6
) d (ppm): 4.66 (s, 2H, CH
2
), 6.44–7.12 (m,
N), 1213 (C–S); H-NMR (DMSO-d ) d (ppm): 2.43 (s, 3H, CH ), 7.10–
–
6
3
3
H, Ar-H), 7.18–7.95 (m, 10H, Ar-H), 8.74 (s, 1H, –CH), 9.62 (s, 1H,
NH-Ar), 10.16 (s, 1H, –N-NH).
7.36 (m, 8H, Ar-H), 7.58 (s, 1H, Ar-H), 7.75–8.10 (m, 3H, Ar-H), 9.29 (s,
1H, –CH), 9.68 (s, 1H, NH-Ar), 10.24 (s, 1H, –N-NH).
–
–
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
62
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
1
7
H, Ar-H), 7.09–7.27 (m, 3H, Ar-H), 7.61–7.89 (m, 4H, Ar-H), 7.89–
.99 (m, 2H, Ar-H), 8.10–8.16 (m, 2H, Ar-H), 9.08 (s, 1H, –CH), 9.75
(Z)-2-(4-Chlorobenzylidene-N-(4-(4-
methoxyphenylcarbamothioyl)-3-oxo-3,4-dihydro-2H-
(
s, 1H, NH-Ar), 10.27 (s, 1H, –N-NH).
benzo[b][1,4]oxazin-7-yl) hydrazinecarboxamide 6i
FT-IR (KBr) (cm ): 3508 (NH), 2931 (CH str.), 1695 (C –– O), 1339 (CH ––
ꢁ1
1
(Z)-2-(3-Nitrobenzylidene)-N-(3-oxo-4-
N), 1213 (C –– S); H-NMR (DMSO-d
7
1
) d (ppm): 3.81 (s, 3H, OCH
.31 (m, 8H, Ar-H), 7.63 (s, 1H, Ar-H), 7.79–8.09 (m, 3H, Ar-H), 9.27 (s,
H, –– CH), 9.67 (s, 1H, NH-Ar), 10.25 (s, 1H, –– N-NH).
3
), 7.11–
6
(phenylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-
7-yl) hydrazinecarboxamide 6p
FT-IR (KBr) (cm ): 3505 (NH), 2921 (CH str.), 1695 (C–O), 1510
ꢁ1
1
(
(
Z)-2-(4-Hydroxybenzylidene)-N-(3-oxo-4-
phenylcarbamothioyl)-3,4-dihydro-2H-
(CH–N), 1213 (C–S); H-NMR (DMSO-d
.38–6.56 (m, 1H, Ar-H), 7.17–7.24 (m, 3H, Ar-H), 7.61–7.83 (m, 4H,
Ar-H), 7.93–7.95 (m, 2H, Ar-H), 8.10–8.16 (m, 2H, Ar-H), 9.08 (s, 1H,
CH), 9.40 (s, 1H, NH-Ar), 10.20 (s, 1H, –N-NH).
6 2
) d (ppm): 4.73 (s, 2H, CH ),
6
benzo[b][1,4]oxazin-7-yl) hydrazinecarboxamide 6j
FT-IR (KBr) (cm ): 3511 (NH), 2931 (CH str.), 1650 (C–O),
–
–
ꢁ1
–
1
1
2
350(CH–N), 1200 (C–S); H-NMR (DMSO-d
H, CH ), 6.44 (s, 1H, OH), 7.11–7.25 (m, 8H, Ar-H), 7.65 (s, 1H,
6
) d (ppm): 4.31 (s,
(Z)-2-(3-Nitrobenzylidene)-N-(3-oxo-4-(4-
tolylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl) hydrazinecarboxamide 6q
FT-IR (KBr) (cm ): 3500 (NH), 2931 (CH str.), 1655 (C–O), 1550
– –
(CH–N), 1200 (C–S); H-NMR (DMSO-d ) d (ppm): 2.49 (s, 3H, CH ),
6 3
6.76–6.92 (m, 1H, Ar-H), 7.13–7.34 (m, 3H, Ar-H), 7.62–7.83 (m, 4H,
2
Ar-H), 7.72 (m, 1H, Ar-H), 8.02–8.12 (m, 2H, Ar-H), 8.68 (s, 1H, –
CH), 9.62 (s, 1H, NH-Ar), 10.16 (s, 1H, –N-NH).
ꢁ
1
1
(Z)-2-(4-Hydroxybenzylidene)-N-(3-oxo-4-(4-
tolylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
Ar-H), 7.92–7.97 (m, 2H, Ar-H), 8.34–8.47 (m, 2H, Ar-H), 9.08 (s, 1H,
–CH), 9.69 (s, 1H, NH-Ar), 10.23 (s, 1H, –N-NH)
yl) hydrazinecarboxamide 6k
FT-IR (KBr) (cm ): 3500 (NH), 2990 (CH str.), 1660 (C–O), 1330
ꢁ
1
–
–
1
(
CH–N), 1250 (C–S); H-NMR (DMSO-d
6
1
6
) d (ppm): 2.31 (s, 3H, CH
.44 (s, 1H, OH), 7.09–7.21 (m, 8H, Ar-H), 7.59 (s, 1H, Ar-H), 7.70 (m,
H, Ar-H), 8.02–8.12 (m, 2H, Ar-H), 8.68 (s, 1H, –CH), 9.62 (s, 1H,
2
),
(Z)-2-(3-Nitrobenzylidene)-N-(4-(4-
methoxyphenylcarbamothioyl)-3-oxo-3,4-dihydro-2H-
NH-Ar), 10.16 (s, 1H, –N-NH).
benzo[b][1,4]oxazin-7-yl) hydrazinecarboxamide 6r
FT-IR (KBr) (cm ): 3550 (NH), 1695 (C –– O), 1570 (CH –– N), 1213
ꢁ1
(Z)-2-(4-Hydroxybenzylidene)-N-(4-(4-
methoxyphenylcarbamothioyl)-3-oxo-3,4-dihydro-2H-
1
(
(
C –– S); H-NMR (DMSO-d
m, 1H, Ar-H), 7.23–7.43 (m, 3H, Ar-H), 7.67–7.84 (m, 4H, Ar-H),
6 3
) d (ppm): 3.74 (s, 3H, OCH ), 6.55–6.70
benzo[b][1,4]oxazin-7-yl) hydrazinecarboxamide 6l
FT-IR (KBr) (cm ): 3500 (NH), 2990 (CH str.), 1670 (C–O), 1350
7.89–7.99 (m, 2H, Ar-H), 8.14–8.36 (m, 2H, Ar-H), 9.08 (s, 1H, ––
ꢁ
1
CH), 9.63 (s, 1H, NH-Ar), 10.46 (s, 1H, –
–
N-NH).
1
(
CH–N), 1220 (C–S); H-NMR (DMSO-d
OCH ), 6.45 (s, 1H, OH), 7.01–7.25 (m, 8H, Ar, H), 7.65 (s, 1H,
6
) d (ppm): 3.79 (s, 3H,
3
Pharmacology
Ar-H), 7.72 (m, 1H, Ar-H), 8.02–8.14 (m, 2H, Ar-H), 8.60 (s, 1H, –
CH), 9.62 (s, 1H, NH-Ar), 10.16 (s, 1H, –N-NH).
The pharmacological testing of all the final compounds were
performed according to the standard protocol given by epilepsy
branch of the National Institute of Neurological Disorders and
Stroke (NINDS) following the protocol adopted by the
Antiepileptic Drug Development (ADD) program.
(Z)-2-(2-Nitrobenzylidene)-N-(3-oxo-4-
(phenylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-
The investigations were conducted on albino mice of either
sex (25–30 g) at a dose of 30 mg/kg i.p. The albino mice were
kept under standard conditions at an ambient temperature of
7
-yl) hydrazinecarboxamide 6m
ꢁ
1
FT-IR (KBr) (cm ): 3505 (NH), 2921 (CH str.), 1655 (C–O), 1510
1
(
CH–N), 1200 (C–S); H-NMR (DMSO-d
.58–6.62 (m, 1H, Ar-H), 7.10–7.25 (m, 3H, Ar-H), 7.63–7.81 (m, 4H,
Ar-H), 7.93–7.95 (m, 2H, Ar-H), 8.10–8.16 (m, 2H, Ar-H), 9.08 (s, 1H,
CH), 9.69 (s, 1H, NH-Ar), 10.21 (s, 1H, –N-NH).
6 2
) d (ppm): 4.72 (s, 2H, CH ),
2
5 ꢂ 28C and allowed free access to food and water except at
6
the time they were brought out of the cage. All the experimen-
tal protocols were carried out with the permission from
Institutional Animal Ethics committee (IAEC), form no. 527.
Animals were obtained from Central Animal House Facility,
Hamdard University, New Delhi. Registration no. and date of
registration is 173/CPCSEA, 28 Jan. 2000.
–
–
–
(Z)-2-(2-Nitrobenzylidene)-N-(3-oxo-4-(4-
tolylcarbamothioyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-
yl) hydrazinecarboxamide 6n
FT-IR (KBr) (cm ): 3500 (NH), 2931 (CH str.), 1655 (C–O), 1550
ꢁ1
Anticonvulsant activity
1
(
CH–N), 1200 (C–S); H-NMR (DMSO-d
.59–6.62 (m, 1H, Ar-H), 7.11–7.24 (m, 3H, Ar-H), 7.63–7.81 (m, 4H,
Ar-H), 7.94–7.97 (m, 2H, Ar-H), 8.10–8.17 (m, 2H, Ar-H), 9.08 (s, 1H,
CH), 9.69 (s, 1H, NH-Ar), 10.25 (s, 1H, –N-NH).
6 3
) d (ppm): 2.33 (s, 3H, CH ),
6
Maximal electroshock seizure (MES) test
The compounds were screened for their anticonvulsant activity
by electroshock seizure method [16]. Seizures were elicited with a
–
–
–
6
0 Hz alternating current of 50 mA intensity in mice. The cur-
(Z)-2-(2-Nitrobenzylidene)-N-(4-(4-
methoxyphenylcarbamothioyl)-3-oxo-3,4-dihydro-2H-
rent was applied via corneal electrodes for 0.2 s. Protection
against the spread of MES induced seizures was defined as the
abolition of the hind limb and tonic maximal extension com-
ponent of the seizure. At 30 min after the administration of the
compounds, the activities were evaluated in MES test.
benzo[b][1,4]oxazin-7-yl) hydrazinecarboxamide 6o
FT-IR (KBr) (cm ): 3550 (NH), 1695 (C–O), 1339 (CH–N), 1213 (C–
ꢁ1
1
S); H-NMR (DMSO-d
6 3
) d (ppm): 3.85 (s, 3H, OCH ), 6.59–6.60 (m,
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 10, 657–663
Newer 4H-Benzo[1,4]oxazin-3-ones as anticonvulsants
663
Subcutaneous Pentylenetetrazole-induced seizure (scPTZ) References
test
At 30 min after the administration of the compounds, the sub-
cutaneous dose of pentylenetetrazole (85 mg/kg) at which 100%
of the animals showed convulsive reaction was determined by a
dose-percent effect curve. This dose was then administered to
animals 30 min after different treatments, and the presence and
absence of clonic spasm were recorded in the animals and the
anticonvulsant activity of the compounds was represented as
percentage protection [17].
[1] R. S. Fischer, W. Van Emde Boas, W. Blume, C. Elger,
P. Genton, P. Lee, Eplepsia 2005, 46, 470–472.
[2] W. J. Currey, D. L. Kulling, Am. Fam. Phys. 1998, 57, 513–
520.
[
3] P. Zhang, E. A. Terefenko, A. Fensome, Z. Zhang, Y. Zhu,
J. Cohen, R. Winneker, J. Wrobel, J. Yardley, Bioorg. Med.
Chem. Lett. 2002, 12, 787–790.
[
[
[
[
4] S. Wahidulla, J. Bhattachargee, J. Ind. Inst. Sci. 2001, 4, 485–
4
90.
5] Y. Kastura, S. Nishino, H. Takasugi, Chem. Pharm. Bull. 1991,
1, 2937–2943.
Thiosemicarbazide-induced seizure (ip TSC) test
1
At 30 min after the administration of the compounds, the intra-
peritoneal dose of thiosemicarbazide (50 mg/kg) at which 100%
of the animals showed convulsive reaction was determined by a
dose-percent effect curve. This dose then administered to
animals 1h after different treatments, latency as well as duration
of convulsions was recorded during the 2.5 h following thiose-
micarbazide administration. The doses were calculated which
prevented 50% of the treated animals from tonic convulsions
6] A. R. Katritzky, L. M. Pacureanu, Slavov, D. A. Dobchev,
M. Karelson, Bioorg. Med. Chem. 2006, 15, 7490–7500.
7] N. Kajino, Y. Shibouta, K. Nishikawa, K. Meguro, Chem.
Pharm. Bull. 1991, 11, 5593–5598.
[8] R. Fringuelli, D. Pietrella, F. Schiaffella, A. Guarraci, S.
Perito, F. Bistoni, A. Vecchiarilli, Bioorg. Med. Chem. 2002,
10, 1681–1686.
[18].
[9] S. Alper-Hayta, E. Aki-Sener, B. Tekiner-Gulbas, I. Yildiz,
I. Yalcin, N. Alanlar, Eur. J. Med. Chem. 2006, 41, 1398–1404.
[10] S. Deswal, N. Roy, Eur. J. Med. Chem. 2006, 41, 552–557.
Neurotoxicity test
This test was performed using the rotorod method [19]. At
[11] S. H. Zhao, J. Berger, R. D. Clark, S. G. Sethofer, N. E. Krauss,
J. M. Brothers, R. S. Martin, D. L. Misner-Schwab, L. Alexandrova,
Bioorg. Med. Chem. Lett. 2007, 17, 3504–3507.
30 min after the administration of the compounds, the animals
were tested on a knurled plastic rod of 1 inch diameter rotating
at 6 rpm for 1 min. Neurotoxicity was indicated by the inability
of an animal to maintain equilibrium in each of three trials.
[
[
[
12] M. Ohno, Y. Tanaka, M. Miyamoto, T. Takeda, K. Hoshi,
M. Yamada, Bioorg. Med. Chem. Lett. 2006, 14, 2005–2021.
13] B. P. Mathew, A. Kumar, S. Sharma, P. Shukla, M. Nath, Eur. J.
Med. Chem. 2010, 45, 1502–1507.
14] A. Arcadi, C. Asti, L. Brandolini, G. Caseilli, F. Marinelli,
V. Ruggieri, Bioorg. Med. Chem. Lett. 2009, 9, 1291–1294.
Liver Enzyme Estimation
To find out the toxic effects, if any, of the synthesized compounds
on liver, the biochemical parameters such as serum glutamate
oxaloacetate transaminase (SGOT) and serum glutamate pyru-
vate transaminase (SGPT) were estimated by the Reitman and
Frankel’s method [20]. Alkaline phosphatase (ALP) was measured
by using King’s method [21].
[15] S. N. Pandeya, A. S. Raja, J. P. Stables, J. Pharm. Sci. 2002, 57,
266–271.
[16] R. J. Krall, J. K. Penry, B. G. White, H. J. Kupferberg, E. A.
Swinyard, Epilepsia 1978, 19, 409–428.
[17] G. N. Kelekci, S. Yabanoglu, E. Kupeli, U. Salgin, A. A. Bilgin,
Bioorg. Med. Chem. 2007, 15, 5775–5786.
[
[
18] W. Loscher, D. Schimdt, Epilepsy Res. 1988, 2, 145–181.
Authors are thankful to the department of Pharmaceutical Chemistry,
Faculty of Pharmacy Hamdard University, for providing necessary
facilities to carry out the research work.
19] X. Y. Sun, Y. J. Jin, F. N. Li, G. Li, K. Y. Chai, Z. S. Quan, Arch.
Pharm. Res. 2006, 29, 1080–1085.
[
[
20] S. Reitman, S. Frankel, Am. J. Clin. Pathol. 1957, 28, 56–63.
21] E. J. King, A. R. Armstrong, Can. Med. Assoc. J. 1934, 31, 376–
381.
The authors have declared no conflict of interest.
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com