Synthesis of novel 7-benzylamino-2H-1,4-benzoxazin-3(4H)-ones as anticonvulsant agents

Article abstract of DOI:10.1016/j.ejmech.2007.08.006

A series of 7-benzylamino-2H-1,4-benzoxazin-3(4H)-ones were synthesized using 2-amino-5-nitrophenol as a starting material. Their anticonvulsant activities were evaluated by the maximal electroshock test (MES test) and their neurotoxicity was evaluated by the rotarod neurotoxicity test (Tox.). The MES test showed that 7-(4-fluorobenzylamino)-2H-1,4-benzoxazin-3(4H)-one 4b was the most potent with ED₅₀ value of 31.7 mg/kg and protective index (PI = TD₅₀/ED₅₀) value of 7.2. To explain the possible mechanism of anticonvulsant activity, the compound 4b was tested in sc-PTZ test, isoniazid test and strychnine test.

Full text of DOI:10.1016/j.ejmech.2007.08.006

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European Journal of Medicinal Chemistry 43 (2008) 1216e1221
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 7-benzylamino-2H-1,4-benzoxazin-3(4H )-ones
as anticonvulsant agents
Zhong-Tai Piao a,b, Li-Ping Guan b, Li-Ming Zhao b, Hu-Ri Piao b, Zhe-Shan Quan a,b,
*
a Key Laboratory of Organism Functional Factors of the Changbai Mountain, Ministry of Education, Yanbian University,
Yanji, Jilin Province 133002, China
b College of Pharmacy, Yanbian University, Jilin Province 133000, China
Received 19 May 2007; received in revised form 18 July 2007; accepted 9 August 2007
Available online 11 September 2007
Abstract
A series of 7-benzylamino-2H-1,4-benzoxazin-3(4H )-ones were synthesized using 2-amino-5-nitrophenol as a starting material. Their anti-
convulsant activities were evaluated by the maximal electroshock test (MES test) and their neurotoxicity was evaluated by the rotarod neuro-
toxicity test (Tox.). The MES test showed that 7-(4-fluorobenzylamino)-2H-1,4-benzoxazin-3(4H )-one 4b was the most potent with ED50 value
of 31.7 mg/kg and protective index (PI ¼ TD50/ED50) value of 7.2. To explain the possible mechanism of anticonvulsant activity, the compound
4b was tested in sc-PTZ test, isoniazid test and strychnine test.
Ó 2007 Published by Elsevier Masson SAS.
Keywords: 1,4-Benzoxazin-3(4H )-one; Anticonvulsant; MES; sc-PTZ; Isoniazid; Strychnine
1. Introduction
hypothesis was that the introduction of a substituted benzyla-
mino into the 7th position of 2H-benzo[b][1,4]oxazin-3(4H )-
one would increase the lipophilic property of the compounds
and increase their permeability to the bloodebrain barrier
thus probably enhances their anticonvulsant activity. The new
compounds were evaluated as anticonvulsant agents in experi-
mental epilepsy models, i.e., maximal electroshock test (MES),
the most active compound (4b) was tested in pentylenetetrazole
(sc-PTZ) test, isoniazid test and strychnine test, and the possi-
ble mechanism of action was conjectured.
1,4-Benzoxazin-3(4H )-one derivatives have shown various
biological activities, such as anti-inflammatory [1], antiulcer
[2], antiplatelet [3], antihypertensive [4], and antifungal [5],
antimicrobial [6], neuropeptide Y (NPY) Y5 receptor antago-
nists [7], serotonin reuptake inhibitors [8], prostacyclin receptor
agonists, etc. [9]. The synthesis and anticonvulsant activity of
compounds 4e7 in this paper, however, has not been reported.
In our search for new compounds with anticonvulsant activ-
ity, 2H-1,4-benzoxazin-3(4H )-one (compound I) showed
a slight positive anticonvulsant activity with an effective dose
of 300 mg/kg in the anti-MES test. In order to obtain com-
pounds with better anticonvulsant activity, we synthesized 7-
benzylamino-2H-1,4-benzoxazin-3(4H )-ones (4ae4n) using
2H-1,4-benzoxazin-3(4H )-one (I) as the lead compound. The
2. Result and discussion
2.1. Chemistry
Target compounds 4ae4n were synthesized according
to Scheme 1. 7-Nitro-2H-1,4-benzoxazin-3(4H )-one (com-
pound 2) was prepared using 2-amino-5-nitrophenol
(compound 1) as starting material, which reacted further
with 2-chloroacetyl chloride, reacting time was 4 h with yield
of 75% [15]. Based on the reference, TEBA was added as
* Corresponding author. College of Pharmacy, Yanbian University, No. 121,
JuZi Street, Yanji City, Jilin Province 133000, China. Tel.: þ86 433 2660606;
fax: þ86 433 2660568.
E-mail address: zsquan@ybu.edu.cn (Z.-S. Quan).
0223-5234/$ - see front matter Ó 2007 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2007.08.006

Z.-T. Piao et al. / European Journal of Medicinal Chemistry 43 (2008) 1216e1221
1217
H
H
N
N
O
O2N
O
O
OH
O2N
O
H2N
R
R
e
c
a
b
N
H
N
H
O
O
N
NH2
O
N
H
O
CH3
2
1
4a-4n
3
6
f
d
CH3
N
H
N
O
R
O
R
O
N
H
O
N
O
5
CH3
7
a, ClCH2COCl b, Fe/HCl c, RCHO/NaBH4 d, ArCOCl e, (CH3)2SO4/Acetone f, (CH3)2SO4/Toluene
Scheme 1. The synthesis route of target compounds.
activator and refluxing for 2 h, the yield was increased to 87%
which was consonant with the reported method [16].
7-Nitro-2H-1,4-benzoxazin-3(4H )-one (compound 2) was
reduced by iron powder in hydrochloride condition to product
7-amino-2H-1,4-benzoxazin-3(4H )-one (compound 3). Com-
pounds 4ae4n were obtained through the condensation reac-
tion of compound 3 with suitable benzaldehyde in benzene
and further reduced by sodium borohydride in methanol at
room temperature in moderate yields.
Compound 5 was synthesized by 7-amino-2H-1,4-benzoxa-
zin-3(4H )-one (compound 3) and benzoyl chloride. Synthesis
of compounds 6 and 7 was achieved by reacting compound 4b
with dimethyl sulfate [17]. These, however, were reacted in
different temperature. Compounds 6 and 7 were obtained at
35e40 ꢀC and 110e115 ꢀC, respectively.
ComparedthederivativeswithdifferentF-substitutedposition
on the benzyl ring, their activity order was p-F > o-F > m-F.
p-F substituted derivative 4b (7-(4-fluorobenzylamino)-2H-
benzo[b][1,4]oxazin-3(4H )-one) was the strongest in all tested
compounds with ED50 value of 31.7 mg/kg, and exhibited the
lowest neurotoxicity with TD50 value of 228.2 mg/kg, a higher
protective index (PI ¼ TD50/ED50, 7.2) was gained than refer-
ence drug Phenytoin Sodium. And activity order of the atom Cl
substituted derivatives was consistent with atom F substituted
derivatives, it was p-Cl > o-Cl > , m-1Cl, 2,4-Cl2.
Five electron-donor derivatives were also designed and pre-
pared, containing p-CH3, p-OH, p-OCH3, p-OC2H5, 3,4-
OCH2Oe. The pharmacology test revealed that their activities
were lower than halogen substituted derivatives and the activ-
ity order was 3,4-OCH2Oe > p-OC2H5 > p-CH3 > p-OH,
p-OCH3.
7-Benzoylamino derivative 5 showed distinctly lower anti-
convulsant activity than 7-benzylamino derivative 4a with
ED50 value higher than 150 mg/kg. This may be due to the li-
pophilic property that was decreased for the existence of car-
bon group, decreased their permeability to the bloodebrain
barrier thus probably weakened their anticonvulsant activity.
Compound 4b was methylized to products 4-methyl deriv-
atives 6 and 7, which were tested as potent anticonvulsant
agents. But the anticonvulsant activity decreased obviously
after this change. 4-Methyl blocked the combination of amide
and receptor caused a decreased activity. The result proved
that the amide structure in benzoxazin-3(4H )-one may be
the active center combined with the receptor, which was essen-
tial group for the anticonvulsant.
Furthermore, the most active compound 4b was tested
against the convulsant induced by the chemical substances,
containing PTZ, Isoniazid and Strychnine. Compound 4b
was administered i.p. at dose of 100 mg/kg, which was be-
tween the ED50 and TD50 value. Reference drug Carbamaze-
pine was administered i.p. at dose of 50 mg/kg.
2.2. Pharmacology
The results of pharmacology test of all synthesized com-
pound and reference drugs were shown in Table 1. Most of
compounds showed remarkable anticonvulsant activity. Com-
pound I exhibited anti-MES effect only under the high dose
of 300 mg/kg, whereas compounds 4ae4e, 4g, 4i, 4j, 4m
and 4n showed anti-MES effect at the dose of <150 mg/kg.
We reason that the increase in anticonvulsant activity might
be due to their easier passage across biological membranes af-
ter the introduction of the alkylamino at the 7th position of
compound I.
Analyzing the activities of synthesized compounds 4ae4n,
the following SAR was gained.
Generally, the anticonvulsant activity of organic compound
may be increased remarkably after the introduction of a halo-
gen atom. So, some halogen substituted derivatives were de-
signed and synthesized in this paper. Comparison of the
halogen substituted derivatives indicated that different halogen
atoms contributed to the anticonvulsant activity in the order of
F > Cl > Br; the introduction of atom F on the benzyl ring
gained stronger activity.
As shown in Table 2, compound 4b completely inhibited
the clonic seizures induced by sc-PTZ, but the reference
drug did not show any inhibition activity. The results in the

1218
Z.-T. Piao et al. / European Journal of Medicinal Chemistry 43 (2008) 1216e1221
Table 1
Quantitative anticonvulsant data in mice (test drug administered i.p.)
R`
N
O
N
O
R
O
N
H
O
R``
4a-4n, 5-7
I
MES,a ED50 (mg/kg)
PIc
b
TD50 (mg/kg)
Compound
R
R0
R00
I
4a
d
eCH2C6H5
d
d
300
>300
182.5 (114.53e290.81)
d
2.9
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eCH3
eCH3
63.4 (39.78e101.01)
31.7 (19.89e50.50)
76.1 (47.74e121.22)
54.8 (34.37e87.28)
65.7 (41.23e104.69)
>150
4b
4c
eCH2C6H4( p-F)
eCH2C6H4(m-F)
eCH2C6H4(o-F)
eCH2C6H4( p-Cl)
eCH2C6H4(m-Cl)
eCH2C6H4(o-Cl)
eCH2C6H3(2,4-Cl2)
eCH2C6H4( p-Br)
eCH2C6H4( p-CH3)
eCH2C6H4( p-OH)
eCH2C6H4( p-OCH3)
eCH2C6H4( p-OC2H5)
eCH2C6H4(3,4-OCH2Oe)
eCOC6H5
228.2 (143.21e363.63)
228.2 (143.21e363.63)
7.2
3.0
4d
4e
182.6(114.25e290.11)
>300
dd
>300
dd
3.3
>4.6
d
>3.3
d
4f
4g
91.3 (57.29e145.47)
>150
4h
4i
131.5 (82.49e209.46)
131.5 (82.49e209.46)
>150
>300
>300
>2.3
>2.3
d
4j
4k
dd
4l
4m
>150
dd
>300
d
3.3
91.3 (57.29e145.47)
63.4 (39.79e101.03)
>150
4n
5
>300
dd
dd
>300
>4.7
d
6
eCH2C6H4( p-F)
eCH2C6H4( p-F)
>150
d
7
Phenytoin
Carbamazepine
eCH3
91.3 (57.29e145.47)
9.5 (8.1e10.4)
9.2 (6.9e11.7)
>3.3
6.9
65.5 (52.5e72.9)
74.4 (59.1e87.5)
8.1
a
Maximal electroshock test.
b
c
d
Minimal meurotoxicity was determined by the rotarod test after the tested compounds were administrated 30 min.
PI ¼ TD50/ED50
Minimal meurotoxicity was not tested.
.
Table 3 demonstrated that reference drug Carbamazepine in-
hibited the clonic seizures, tonic seizures and death induced
by Isoniazid at the rate of 10%, 10% and 0%, respectively.
But compound 4b completely controlled the clonic seizures,
tonic seizures and death induced by Isoniazid.
might have effects on GABA-ergic neurotransmission and gly-
cine system.
3. Experimental section
As PTZ and Isoniazid have been shown to interact with the
GABA neurotransmitter and the GABA receptor complex
[18e20], antagonism of PTZ and Isoniazid-induced seizures
suggests that the compound 4b might have effects on
GABA-ergic neurotransmission.
On the other hand, in the strychnine-induced seizure model
(Table 4), the control mice were administered vehicle only and
all animals died caused by strychnine. Compound 4b inhibited
the clonic seizures, tonic seizures and death by 40%, 30% and
10%, respectively, and this was consistent with reference drug
Carbamazepine. It is known that strychnine directly antago-
nize the inhibitory spinal reflexes of glycine [21]. Compound
4b therefore, might cause seizure suppression by acting on
glycine inhibitory mechanisms.
3.1. Chemistry
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) on a FT-
IR1730. 1H NMR spectra were measured on a AV-300
(Bruker, Switzerland), and all chemical shifts were given in
parts per million relative to tetramethysilane. Mass spectra
were measured on an HP1100LC (Agilent Technologies,
USA). Elemental analyses were performed on a 204Q CHN
(PerkineElmer, USA). Microanalyses of C, N, and H were
performed using a Heraeus CHN Rapid Analyzer. The major
chemicals were purchased from Alderich Chemical Corpora-
tion. All other chemicals were of analytical grade.
Table 2
Effect of compound 4b on PTZ-induced convulsant in mice
In conclusion, in the present study, through the introduction
of substituted benzylamino to the 7th position of 2H-1,4-ben-
zoxazin-3(4H )-one, we found that 7-(4-fluorobenzylamino)-
2H-1,4-benzoxazin-3(4H )-one (4b) possessed the most potent
anticonvulsant acitivity with ED50 value of 31.7 mg/kg and PI
value of 7.2. Compound 4b showed antagonism of PTZ and
Isoniazid-induced seizures suggests that the compound 4b
Compound
Doses
(mg/kg) (h)
Test time Clonic
Tonic
seizures (%) seizures (%) (%)
Lethality
DMSO
Carbamazepine 50
4b 100
0.5
100
100
0
20
20
0
10
0
0.5
0.5
0

Z.-T. Piao et al. / European Journal of Medicinal Chemistry 43 (2008) 1216e1221
1219
Table 3
Effect of compound 4b on Isoniazid-induced convulsant in mice
one portion at a time. When NaBH4 addition was over
(about 2e4 h), the solvent was removed under reduced pres-
sure. The residue was dissolved in dichloromethane (30 ml),
washed with water (30 ml ꢁ 3) and dried over anhydrous
MgSO4. Evaporation of the solvents gave a crude product,
which was purified by silica gel column chromatography
with ethyl acetateepetroleum ether (1:1 or 2:1) to a solid.
Compound
Doses
(mg/kg) (h)
Test time Clonic
Tonic
seizures (%) seizures (%) (%)
Lethality
DMSO
Carbamazepine 50
4b 100
0.5
100
10
0
100
10
0
100
0
0.5
0.5
0
3.2.1. 7-(Benzylamino)-2H-1,4-benzoxazin-3(4H )-one (4a)
M.p. 138e140 ꢀC; yield 94%. 1H NMR (DMSO, 300 MHz)
d 4.18 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.10e7.37
3.1.1. 7-Nitro-2H-1,4-benzoxazin-3(4H )-one (2)
Compound (10 g, 0.065 mol), sodium bicarbonate
1
(21.8 g, 0.26 mol) and TEBA (14.8 g, 0.065 mol) were placed
into a round-bottomed flask containing 200 ml of acetonitrile,
and the mixture was put in an iceesalt bath to keep the tem-
perature 0e5 ꢀC, then, 2-chloroacetyl chloride (9.5 g,
0.084 mol) was added with moderate speed. 2-Chloroacetyl
chloride (9.5 g, 0.084 mol) was added with moderate speed
and reacted for 40 min. Replaced the iceesalt bath with oil-
bath, heated to reflux for 2 h, then remove the solvent under
reduced pressure. The resultant product was purified by re-
crystallization with ethanolewater (1:1). M.p. 219e222 ꢀC,
yield ¼ 87%. 1H NMR (DMSO, 300 MHz) d 4.48 (s, 2H,
eCOCH2), 7.69 (d, J ¼ 7.3 Hz, 1H, C8eH), 7.75 (d,
J ¼ 9.0 Hz, 1H, C5eH), 8.30 (d, J ¼ 9.0 Hz, 1H, C6eH),
9.85 (s, 1H, COeNH).
(m, 8H, AreH), 10.26 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3408 (NH), 1703 (C]O). MS m/z 255 (M þ 1). Anal. Calcd
for C15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C,
71.07; H, 5.36; N, 10.78.
3.2.2. 7-(4-Fluorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4b)
M.p. 170e172 ꢀC; yield 92%. 1H NMR (DMSO, 300 MHz)
d 4.23 (s, 2H, AreCH2), 4.44 (s, 2H, eCOCH2), 6.24e7.37
(m, 7H, AreH), 10.26 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3421 (NH), 1689 (C]O). MS m/z 273 (M þ 1). Anal. Calcd
for C15H13FN2O2: C, 66.17; H, 4.81; N, 10.29. Found: C,
66.31; H, 5.12; N, 10.37.
3.2.3. 7-(3-Fluorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4c)
3.1.2. 7-Amino-2H-1,4-benzoxazin-3(4H )-one (3)
Compound 2 (8.84 g, 0.046 mol) and iron powder (7.7 g,
0.138 mol) were placed into a three-necked round-bottomed
flask containing 200 ml ethanol (95%). The mixture was
stirred and heated with a blade stirrer and oil-bath until the
temperature attains 80e85 ꢀC. Then 4 ml of hydrochloric
acid (1.2 M) was added and the mixture was stirred at 80e
85 ꢀC for 4 h. The mixture was filtered immediately with
a hot filter under reduced pressure. Adjusting the filtrate’s
pH value to 7e8 using saturated NaHCO3 resulted in precip-
M.p. 134e136 ꢀC; yield 51%. 1H NMR (DMSO, 300 MHz)
d 4.27 (s, 2H, AreCH2), 4.45 (s, 2H, eCOCH2), 6.23e7.31
(m, 7H, AreH), 10.25 (s, 1H, eCONH). IR (KBr) cmꢂ1
3421 (NH), 1689 (C]O). MS m/z 273 (M þ 1). Anal. Calcd
for C15H13FN2O2: C, 66.17; H, 4.81; N, 10.29. Found: C,
66.40; H, 4.59; N, 10.39.
:
3.2.4. 7-(2-Fluorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4d)
1
itation of solid 3 (5.6 g). M.p. 212e215 ꢀC, yield ¼ 75%. H
M.p. 136e138 ꢀC; yield 38%. 1H NMR (DMSO, 300 MHz)
d 4.16e4.24 (m, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.0e
NMR (DMSO, 300 MHz) d 4.41 (s, 2H, eCOCH2), 4.88
(s, 2H, eNH2), 6.17e6.57 (m, 3H, AreH), 10.27 (s, 1H,
eCONH).
7.3 (m, 7H, AreH), 10.27 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3421 (NH), 1689 (C]O). MS m/z 273 (M þ 1). Anal. Calcd
for C15H13FN2O2: C, 66.17; H, 4.81; N, 10.29. Found: C,
66.41; H, 4.92; N, 10.34.
3.2. General procedure for the preparation of
7-benzylamino-2H-1,4-benzoxazin-3(4H )-ones (4ae4n)
3.2.5. 7-(4-Chlorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4e)
Compound 3 (2 g, 0.012 mol) was placed into a round-
bottomed flask containing 120 ml methanol. Benzaldehydes
(0.015 mol) diluted by methanol was dropwise added into
the mixture. After stirring for 1e4 h, NaBH4 (1.76 g,
0.038 mol) was divided into multiple portions and added
M.p. 138e140 ꢀC; yield 44%. 1H NMR (DMSO, 300 MHz)
d 4.19 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.14e7.93
(m, 7H, AreH), 10.27 (s, 1H, eCONH). IR (KBr) cmꢂ1
3415 (NH), 1678 (C]O). MS m/z 290 (M þ 1). Anal. Calcd
for C15H13ClN2O2: C, 62.40; H, 4.54; N, 9.70. Found: C,
62.46; H, 4.21; N, 9.86.
:
Table 4
Effect of compound 4b on Strychnine-induced convulsant in mice
Compound
Doses
(mg/kg) (h)
Test time Clonic
Tonic
seizures (%) seizures (%) (%)
Lethality
3.2.6. 7-(3-Chlorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4f)
M.p. 172e174 ꢀC; yield 39%. 1H NMR (DMSO, 300 MHz)
d 4.20 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.16e7.37 (m,
7H, AreH), 10.28 (s, 1H, eCONH). IR (KBr) cmꢂ1: 3415
DMSO
Carbamazepine 50
4b 100
0.5
100
40
100
30
100
0
0.5
0.5
40
30
10

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Z.-T. Piao et al. / European Journal of Medicinal Chemistry 43 (2008) 1216e1221
(NH), 1678 (C]O). MS 290 m/z (M þ 1). Anal. Calcd for
C15H13ClN2O2: C, 62.40; H, 4.54; N, 9.70. Found: C, 62.06;
H, 4.56; N, 9.86.
3.2.13. 7-(4-Ethoxybenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4m)
M.p. 138e141 ꢀC; yield 50%. 1H NMR (DMSO, 300 MHz)
d 1.32 (t, 3H, J ¼ 7.85 Hz, eCH2CH3), 3.97 (2H, J ¼ 7.0,
eOCH2), 4.09 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2),
5.97e7.24 (m, 7H, AreH), 10.26 (s, 1H, eCONH). IR
(KBr) cmꢂ1: 3371 (NH), 1685 (C]O). MS m/z 299
(M þ 1). Anal. Calcd for C17H18N2O3: C, 68.44; H, 6.08; N,
9.39. Found: C, 68.38; H, 5.95; N, 9.66.
3.2.7. 7-(2-Chlorobenzylamino)-2H-1,4-benzoxazin-
3(4H )-one (4g)
M.p. 160e163 ꢀC; yield 33%. 1H NMR (DMSO, 300 MHz)
d 4.19 (s, 2H, AreCH2), 4.41 (s, 2H, eCOCH2), 6.14e7.56
(m, 7H, AreH), 10.28 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3415 (NH), 1678 (C]O). MS m/z 290 (M þ 1). Anal. Calcd
for C15H13ClN2O2: C, 62.40; H, 4.54; N, 9.70. Found: C,
62.38; H, 4.36; N, 9.52.
3.2.14. 7-(3,4-Methylenediooxybenzylamino)-
2H-1,4-benzoxazin-3(4H )-one (4n)
M.p. 170e173 ꢀC; yield 60%. 1H NMR (DMSO, 300 MHz)
d 4.09 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 5.95 (s, 2H,
eOCH2O), 6.04e6.91 (m, 7H, AreH), 10.26 (s, 1H,
eCONH). IR (KBr) cmꢂ1: 3404 (NH), 1701 (C]O). MS
m/z 299 (M þ 1). Anal. Calcd for C16H14N2O4: C, 64.42; H,
4.73; N, 9.39. Found: C, 64.39; H, 4.79; N, 9.69.
3.2.8. 7-(2,4-Dichlorobenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4h)
M.p. 165e169 ꢀC; yield 55%. 1H NMR (DMSO, 300 MHz)
d 4.23 (s, 2H, AreCH2), 4.41 (s, 2H, eCOCH2), 6.12e7.78
(m, 6H, AreH), 10.30 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3415 (NH), 1678 (C]O). MS m/z 323 (M þ 1); Anal. Calcd
for C15H112Cl2N2O2: C, 55.75; H, 3.74; N, 8.67. Found: C,
55.49; H, 3.46; N, 8.52.
3.2.15. 7-Benzoylamino-2H-1,4-benzoxazin-3(4H )-one (5)
The benzoyl chloride (1.1 g, 0.0078 mol) was added to
a round-bottomed flask containing toluene (30 ml) which dis-
solved compound 3 (1 g,0.006 mol) and sodium bicarbonate
(1 g, 0.012 mol). The stirred reaction mixture was refluxed
for 6 h. After removing the solvent under reduced pressure,
the residue was dissolved in dichloromethane (30 ml), washed
with water (30 ml ꢁ 3) and dried over anhydrous MgSO4.
Evaporation of the solvents gave a crude product, which was
purified by silica gel column chromatography with ethyl acet-
ateepetroleum ether (1:1) to a white solid. M.p. 217e219 ꢀC;
yield ¼ 11%. 1H NMR (DMSO, 300 MHz) d 4.56 (s, 2H,
eCOCH2), 6.83e7.93 (m, 8H, AreH), 10.25 (s, 1H,
eCONH), 10.67 (s, 1H, ArCONH). IR (KBr) cmꢂ1: 3361
(NH), 1656 (C]O). MS m/z 269 (M þ 1). Anal. Calcd for
C15H12N2O3: C, 67.16; H, 4.51; N, 10.44; O, 17.89.
3.2.9. 7-(4-Bromobenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4i)
M.p. 143e146 ꢀC; yield 56%. 1H NMR (DMSO, 300 MHz)
d 4.18 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.14e7.50
(m, 7H, AreH), 10.27 (s, 1H, eCONH). IR (KBr) cmꢂ1
:
3415 (NH), 1678 (C]O). MS m/z 334 (M þ 1). Anal. Calcd
for C15H13BrN2O2: C, 54.07; H, 3.93; N, 8.41. Found: C,
54.26; H, 4.19; N, 8.31.
3.2.10. 7-(4-Methylbenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4j)
M.p. 170e173 ꢀC; yield 55%. 1H NMR (DMSO, 300 MHz)
d 2.25 (s, 3H, eCH3), 4.14 (s, 2H, AreCH2), 4.39 (s, 2H,
eCOCH2), 6.05e7.21 (m, 7H, AreH), 10.26 (s, 1H,
eCONH). IR (KBr) cmꢂ1: 3404 (NH), 1703 (C]O). MS
m/z 269 (M þ 1). Anal. Calcd for C16H16N2O2: C, 71.62; H,
6.01; N, 10.44. Found: C, 71.76; H, 5.86; N, 10.65.
3.2.16. 7-(4-Fluorobenzylamino)-4-methyl-
2H-1,4-benzoxazin-3(4H )-one (6)
Compound 4b (0.5 g, 0.0018 mol) and dimethyl sulfate
(0.91 g, 0.0072 mol) were placed into a round-bottomed flask
containing 30 ml of toluene, potassium hydroxide powder was
added (0.4 g, 0.0072 mol) while the mixture was stirred. The
mixture was stirred at 35e40 ꢀC for 16 min. Then the solvent
was removed under reduced pressure on a rotary evaporator
below 40 ꢀC. The resulting solid residue was partitioned be-
tween water and dichloromethane, and the aqueous layer
was extracted with dichloromethane (30 ml ꢁ 3). The com-
bined layers of dichloromethane were dried over anhydrous
MgSO4. Evaporation of the solvents gave a crude product,
which was purified by silica gel column chromatography
with ethyl acetateepetroleum ether (2:3) to a light yellow
3.2.11. 7-(4-Hydroxybenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4k)
M.p. 192e196 ꢀC; yield 54%. 1H NMR (DMSO, 300 MHz)
d 4.05 (s, 2H, AreCH2), 4.40 (s, 2H, eCOCH2), 6.14e7.13
(m, 7H, AreH), 9.22 (s, 1H, AreOH), 10.26 (s, 1H,
eCONH). IR (KBr) cmꢂ1: 3415 (NH), 1668 (C]O). MS
m/z 271 (M þ 1). Anal. Calcd for C15H14N2O3: C, 66.66; H,
5.22; N, 10.36. Found: C, 66.93; H, 5.32; N, 10.06.
3.2.12. 7-(4-Methoxybenzylamino)-2H-1,4-
benzoxazin-3(4H )-one (4l)
M.p. 179e181 ꢀC; yield 58%. 1H NMR (DMSO, 300 MHz)
d 3.71 (s, 3H, eOCH3), 4.11 (s, 2H, AreCH2), 4.40 (s, 2H,
eCOCH2), 6.01e7.25 (m, 7H, AreH), 10.26 (s, 1H,
eCONH). IR (KBr) cmꢂ1: 3371 (NH), 1685 (C]O). MS
m/z 285 (M þ 1). Anal. Calcd for C16H16N2O3: C, 67.59; H,
5.67; N, 9.85. Found: C, 67.71; H, 5.42; N, 10.12.
liquid; yield 70%. H NMR (DMSO, 300 MHz) d 3.32 (s,
3H, eCONCH3), 4.28 (s, 2H, AreCH2), 4.57 (s, 2H,
1
eCOCH2), 6.35e7.36 (m, 7H, AreH). IR (KBr) cmꢂ1
:
3439 (NH), 1691.57 (C]O). MS m/z 287 (M þ 1). Anal.
Calcd for C16H15FN2O2: C, 67.12; H, 5.28; N, 9.78. Found:
C, 66.86; H, 9.64; N, 10.01.

Z.-T. Piao et al. / European Journal of Medicinal Chemistry 43 (2008) 1216e1221
1221
3.2.17. 7-((4-Fluorobenzyl)(methyl)amino)-4-methyl-
2H-1,4-benzoxazin-3(4H )-one (7)
treated with 4b. The positive control group received Carbama-
zepine, 50 mg/kg.
Compound 4b (0.6 g, 0.0022 mol) and dimethyl sulfate
(1.1 g, 0.0088 mol) were placed into a round-bottomed flask
containing 30 ml of toluene, potassium hydroxide powder
was added (0.49 g, 0.0088 mol) while the mixture was stirred.
The mixture was stirred at 110e115 ꢀC for 40 min, and then
removing the solvent under reduced pressure on a rotary evap-
orator. The resulting solid residue was partitioned between
water and dichloromethane, and the aqueous layer was ex-
tracted with dichloromethane (30 ml ꢁ 3). The combined
layers of dichloromethane were dried over anhydrous
MgSO4. Evaporation of the solvents gave a crude product,
which was purified by silica gel column chromatography
with ethyl acetateepetroleum ether (2:3) to a light yellow
3.4.3. Strychnine-induced seizures test [12]
At 30 min after the administration of the compounds, the
animals received a s.c. injection of a solution of strychnine
chlorhydrate in saline (1.2 mg/kg). The mice were placed in
individual cages and observed for 30 min. The time of onset
of the seizure, the number of clonic seizures, tonic seizures,
and the lethality were recorded.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (no. 30460151).
1
liquid; yield 76.2%. H NMR (DMSO, 300 MHz) d 3.02 (s,
3H, eCONCH3), 3.32 (s, 3H, eCH2NCH3), 4.51 (s, 2H,
AreCH2), 4.57 (s, 2H, eCOCH2), 6.40e7.36 (m, 7H,
AreH). IR (KBr) cmꢂ1: 1687 (C]O). MS m/z 301 (M þ 1).
Anal. Calcd for C17H17FN2O2: C, 67.99; H, 5.71; N, 9.33.
Found: C, 67.89; H, 5.89; N, 9.28.
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