Synthesis and anticonvulsant activity of some novel 7-(benzylamino) -1H-benzo[b][1,4]diazepine-2,4(3H, 5H)-dione derivatives

Article abstract of DOI:10.1007/s00044-017-2089-1

A series of novel 7-(benzylamino)-1H-benzo[b][1,4]diazepine-2,4(3H, 5H)-dione derivatives were synthesized and screened for their anticonvulsant activities by the maximal electroshock (MES) test, subcutaneous pentylenetetrazol (scPTZ) test, and their neurotoxicity was evaluated by the rotarod neurotoxicity test (Tox). The study showed that the compound 8c (7-(4-fluorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]diazepine-2,4(3H,5H)-dione) was the most promising compound, with median effective dose (ED₅₀) of 36.5 mg/kg, and protective index (PI) values of 4.5 in the MES test. In the scPTZ test, compound 8c also exhibited better anticonvulsant activity (ED₅₀ = 68.2 mg/kg), and the ED₅₀ values of compounds 8c were smaller than that of the reference antiepileptic drugs carbamazepine (ED₅₀ CloseSPigtSPi 100 mg/kg), phenytoin (ED₅₀ CloseSPigtSPi 300 mg/kg), and sodium valproate (ED₅₀ = 149 mg/kg). The possible structure activity relationship was discussed.

Full text of DOI:10.1007/s00044-017-2089-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2089-1
ORIGINAL RESEARCH
Synthesis and anticonvulsant activity of some novel 7-
(benzylamino) -1H-benzo[b][1,4]diazepine-2,4(3H, 5H)-dione
derivatives
Yun-Peng Shao1 Rong-Bi Han1,2 Hui-Fan Wu1 Feng-Yu Piao1,2
●
●
●
Received: 9 August 2016 / Accepted: 25 September 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of novel 7-(benzylamino)-1H-benzo[b]
[1,4]diazepine-2,4(3H, 5H)-dione derivatives were synthe-
sized and screened for their anticonvulsant activities by the
maximal electroshock (MES) test, subcutaneous pentyle-
netetrazol (scPTZ) test, and their neurotoxicity was eval-
uated by the rotarod neurotoxicity test (Tox). The study
showed that the compound 8c (7-(4-fluorobenzylamino)-
1,5-dimethyl-1H-benzo[b][1,4]diazepine-2,4(3H,5H)-dione)
was the most promising compound, with median effective
dose (ED50) of 36.5 mg/kg, and protective index (PI) values
of 4.5 in the MES test. In the scPTZ test, compound 8c also
exhibited better anticonvulsant activity (ED50 = 68.2 mg/
kg), and the ED50 values of compounds 8c were smaller
than that of the reference antiepileptic drugs carbamazepine
(ED50 > 100 mg/kg), phenytoin (ED50 > 300 mg/kg), and
sodium valproate (ED50 = 149 mg/kg). The possible struc-
ture activity relationship was discussed.
Introduction
Epilepsy is the most frequent neurologic disorder char-
acterized by excessive temporary neuronal discharge, which
affects up to 1% of the world’s population (Stafstrom 2006).
Although the current drugs provide adequate seizure control
in many patients, it is roughly estimated that up to 28–30%
of patients are poorly treated with the available antiepileptic
drugs (AEDs) (Brodie 2001). Therefore, continued search
for safer and more effective AEDs is urgently necessary. In
recent years, many efforts have been devoted to developing
novel compounds as potential anticonvulsant agents (Eadie
and Vajda 2012; Coleman et al. 2014; Reimers 2014; Kaur
et al. 2016; Mittapalli and Roberts 2014).
Benzazepine derivatives exhibit broad pharmacological
activity. Many members of this family are widely used as
anti-anxiety, anti-seizures, analgesic, antidepressive and
hypnotic or anti-inflammatory agents (Rajarao et al. 2007;
Guerrini et al. 2006; Kumar and Joshi* 2008). Triazole
compounds also have a wide variety of biological activities,
and the introduction of a triazole ring to some active
molecules may significantly improve the biological activity
of the parent molecule due to the superposition of biological
activity (Zhang et al. 2012; Piao* et al. 2011; Piao et al.
2012). The hypothesis was that a triazole ring may have
higher affinity for the receptor and enhance anticonvulsant
activity, and there were some similar design reports
(Dawood et al. 2006; Moreau et al. 1998). In our previous
work, a series of 8-alkoxy-5,6-dihydro-4H-benzo[f][1,2,4]
triazolo [4,3-a]azepine derivative were synthesized, among
these compounds, compound 8-pentyloxy -5,6-dihydro-4H-
benzo[f][1,2,4]triazolo[4,3-a]azepine (compound II) dis-
played the best anticonvulsant activity, having ED50 values of
17.5 mg/kg in the maximal electroshock (MES) test, com-
pound 7-butoxy-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
●
Keywords 1H-benzo[b][1,4]diazepine-2,4(3H,5H)-dione
anticonvulsant activity structure-activity relationship
●
* Feng-Yu Piao
fypiao4989@126.com
1
Department of Chemistry, College of Science, Yanbian
University, 133002 Yanji, Jilin Province, People’s Republic of
China
2
Key Laboratory of Natural Resources of Changbai Mountain &
Functional Molecules (Yanbian University), Ministry of
Education, Yanji 133002 Jilin Province, People’s Republic of
China

Med Chem Res
Scheme 1 The synthesis route
of compunds 5a–I, 8a–I, and 12
(compound I) showed good anticonvulsant activity in the
MES model (ED50 = 52.8 mg/kg) (Wei et al. 2009; Piao*
et al. 2013; Jin et al. 2010).
Pharmacology
The MES test is regarded as the pharmacologic model of
grand mal, and the scPTZ test as the pharmacologic model
of petit mal seizures. Therefore we carried out those two
tests to evaluate the anticonvulsant activity of the synthe-
sized compounds. The anticonvulsant activity and neuro-
toxicity of the synthesized compounds 5a–i and 8a–i were
evaluated by the National Institute of Neurological Dis-
orders and Stroke at the National Institute of Health
(Bethesda, MD, USA) using established procedures (Krall
et al. 1978; Porter et al. 1984).
In preliminary screening (phase I), compounds 5a–i did
not show anticonvulsant activity at a test dose of 100 mg/kg
in both the MES and scPTZ tests, which may be attributed
to these compounds can not pass through the blood-brain
barrier due to their strong molecular polarity.
The results of preliminary (phase I) screening of com-
pounds 8a–i are summarized in Table 1. In both the MES
and scPTZ tests, compounds 8a–i exhibited anticonvulsant
activity at the dose of 100 mg/kg. However, they were
inactive at the dose of 30 mg/kg. As a result of preliminary
screening, compounds 8a–i were subjected to phase II trials
for quantification of their anticonvulsant activity (indicated
by ED50) and neurotoxicity (indicated by TD50) in mice
(Table 2).
Analyzing the activities of the synthesized compounds
the following structure–activity relationships were obtained.
In the anti-MES test, comparison of the halogen substituted
derivatives indicated that different halogen atoms con-
tributed to the anticonvulsant activity in the order of F > Cl
> Br; comparing the derivatives with different F (Cl)-
The amide bond is a key functional group in organic
chemistry. It plays a major role in the elaboration and
composition of biological and chemical systems (Valeur*
and Bradley* 2009; Bode 2006; Cupido et al. 2007). Many
antiepileptic drugs contain an amide bond, for example,
Phenytoin, Diazepam, Oxazepam, Fludiazepam, Pheno-
barbital, Carbamazepine, Oxcarbazepine and Progabide.
Generally, primary and secondary amines can form
hydrogen bonds as either donor or acceptor. The hydro-
phobic phenyl group can increase molecular lipophilicity
and the ability to penetrate the blood–brain barrier.
According to the isostere theory, the numbers of the
outer electrons of secondary amino group and O atom (
) are the same. In this study, an alkoxyl group was
replaced by an alkylamino group to prepare 7-(benzyla-
mino)-1H-benzo[b][1,4]diazepine-2,4(3H, 5H)-dione deri-
vatives, and their anticonvulsant activities were evaluated
by the MES test, the scPTZ test, and their neurotoxicity was
evaluated by the rotarod neurotoxicity test (Tox).
Results and discussion
Chemistry
Target compounds synthesized according to the method
shown in Scheme 1. The structures of all the new com-
1
pounds were confirmed by IR, H NMR, 13C NMR, mass
spectrometry (MS), and elemental analyses.

Med Chem Res
substitution positions on the benzyl ring, their activity order
was p-F > m-F > o-F, p-Cl > m-Cl > o-Cl. The antic-
onvulsant activity of compounds containing substituted
benzyl amine (o-F, m-F, p-F, o-Cl, m-Cl, p-Cl, p-Br) was
stronger than that of the compound with non-substituted
benzyl amine (8i). One electron donor derivative (8h) was
also designed and prepared, and its activity was lower than
that of the compound with non-substituted benzyl amine
(8i). Among these derivatives, the p-F-substituted derivative
8c exhibited the strongest anticonvulsant activity with ED
50 value of 36.5 mg/kg.
The anti-pentylenetetrazole activities of compounds 8a–i
were stronger than that of the marked antiepileptic drug
carbamazepine and Sodium valproate, and 8c was amongst
the most active compound with ED50 of 68.2 mg/kg, which
is much lower than that of carbamazepine (ED50 > 100 mg/kg)
and sodium valproate (149 mg/kg).
Table 1 Phase-I evaluation of anticonvulsant activity in mice (i.p.)
In general, the anticonvulsant activity of an organic
compound might be increased after the introduction of a
halogen atom.
Com. R1
Dosage (mg/kg) MESa
scPTZb
0.5h 4hc 0.5 h 4 hc
Pentylenetetrazole has been reported to produce seizures
by inhibiting γ-aminobutyric acid (GABA) neurotransmis-
sion (Okada et al. 1989; Feigenspan and Bormann 1994).
GABA is the main inhibitory neurotransmitter substance in
the brain, and is widely implicated in epilepsy. Inhibition of
GABAergic neurotransmission has been shown to promote
and facilitate seizures (Loscher 1982), while enhancement
of GABAergic neurotransmission is known to inhibit or
attenuate seizures. The synthesized compounds might
enhance GABAergic neurotransmission.
The anticonvulsant effects of the drugs that showed
antagonism against MES in the MES test models were
considered to be realized through inhibiting the voltage-
gated Na+ channel, i.e., activating the inhibitor for the
Na+ channel (Adam et al. 2005; Helen and Lily 2006).
8a
8b
8c
8d
8e
8f
8g
8h
8i
CH2C6H4(o-F)
100
100
100
100
100
100
100
4/5 0/5 4/5
4/5 1/5 5/5
4/5 0/5 5/5
4/5 0/5 5/5
4/5 1/5 5/5
5/5 0/5 5/5
4/5 0/5 4/5
4/5 0/5 5/5
4/5 0/5 4/5
0/5
0/5
1/5
0/5
0/5
0/5
1/5
0/5
0/5
CH2C6H4(m-F)
CH2C6H4(p-F)
CH2C6H4(o-Cl)
CH2C6H4(m-Cl)
CH2C6H4(p-Cl)
CH2C6H4(p-Br)
CH2C6H4(p-CH3) 100
PhCH2 100
a
Maximal electroshock seizure test (number of animals protected/
number of animals tested)
b
subcutaneous pentylene tetrazole test (number of animals protected/
number of animals tested)
c
compounds are metabolized/excreted at 4 h
Table 2 Phase II quantitative
anticonvulsant data in mice (test
drug administered i.p.)
a
b
Comp.
ED50
MES
TD50
PIc
scPTZ
Rotarod toxicity
MES
scPTZ
8a
51.6 (48.7–57.0)d
48.1(45.5–56.5)
36.5(31.1–41.9)
54.7 (47.4–63.5)
47.2 (42.7–55.4)
43.5 (44.9–54.5)
50.1 (43.1–58.6)
64.7 (59.1–70.1)
56.1 (50.7–68.9)
9.5 (8.1–10.4)
79.0 (70.7–88.7)d
75.2 (70.7–88.7)
68.2 (59.6–77.9)
79.2 (70.7–88.7)
76.1 (67.2–86.1)
73.3 (63.6–84.6)
75.1 (64.1–90.3)
80.8 (71.6–91.2)
76.1 (67.2–86.1)
>300
190.7 (162.5–223.7)d
183.8 (153.8–219.8)
164.3 (150.2–176.0)
164.1 (154.0–169.5)
220.7 (199.0–242.2)
183.9 (164.2–205.9)
210.3 (202.9–226.7)
197.7 (171.4–228.1)
190.7 (162.5–223.8)
65.5 (52.5–72.9)d
71.6 (45.9–135)d
3.7
3.8
4.5
3.0
4.7
4.2
4.2
3.1
3.4
6.9
8.1
3.2
1.6
2.4
8b
2.4
8c
2.4
8d
2.1
8e
2.9
8f
2.5
8g
2.8
8h
2.5
8i
2.5
PHTe
CBZe
PBe
VPAe
a
<0.22
<0.22
5.2
8.8 (5.5–14.1)
>100
21.8 (21.8–25.5)
272 (247–338)
13.2 (5.8–15.9)d
149 (123–177)d
69 (62.8–72.9)d
426 (369–450)d
2.9
ED50-median effective dose required to assure anticonvulsant protection in 50% animals
TD50-median toxic dose eliciting minimal neurological toxicity in 50% animals
b
c
d
e
PI protective index (TD50/ED50
)
95% confidence limits given in parentheses
Date from (Ucar et al. 1998)

Med Chem Res
Table 3 Effects of compound
7g on chemically-induced
seizures in mice
Chemical substances Comp. Doses (mg/kg)
N
Test time (h) Clonic (%) Tonic (%) Death (%)
DMSO
CBZ
8c
–
10 0.5
10 0.5
10 0.5
10 0.5
10 0.5
10 0.5
100
100
20
100
20
0
100
10
3-MP
BIC
50
50
–
80
DMSO
CBZ
8c
100
100
20
100
0
100
60
50
50
0
30
The anticonvulsant effect of our synthesized compounds
might be achieved through inhibiting the voltage-gated Na+
channel.
Compound 8c showed an antagonism toward bicuculline-
induced seizures, which suggested that it exerts antic-
onvulsant activity partially through GABAA-mediated
mechanisms.
Compounds 12 and 5a–i did not show anticonvulsant
activity in two experimental models of epilepsy at a test
dose of 100 mg/kg. The reason for this observation is that
the compound 12 and 5a–i may be difficult to cross the
blood brain barrier due to the strong polarity of molecule.
To further investigate the effects of the anticonvulsant
activity in several different models, compounds 8c was
tested against convulsions induced by chemical substances,
including 3-mercaptopropionic acid and bicuculline. Com-
pound 8c was administered into mice i.p. at a dose of
50 mg/kg, which was higher than their ED50 value and far
below their TD50 value. The reference drug carbamazepine
was also administered i.p. at a dose of 50 mg/kg.
In the 3-mercaptopropionic acid-induced seizure model,
carbamazepine inhibited clonic seizures, tonic seizures and
death at the rate of 0, 80, and 90%, respectively. In com-
parison, compound 8c showed entirely inhibition of the
tonic seizures and partial inhibition of the clonic seizures
and death induced by 3-mercaptopropionic acid. Compound
8c showed inhibition at the rate of 80, 100 and 20%,
respectively (Table 3). In the bicuculline model, carbama-
zepine inhibited clonic seizures, tonic seizures and death
induced by isoniazid at the rate of 0, 100, and 40%,
respectively; and compound 8c did not affect the clonic
seizures and showed partial inhibition of the tonic seizures
and death induced by isoniazid. Compound 8c showed
inhibition at the rate of 80, 100, and 70%, respectively
(Table 3).
Conclusions
A series of novel 7-(benzylamino)-1H-benzo[b][1,4]diaze-
pine-2,4(3H, 5H)-dione derivatives were synthesized and
tested for anticonvulsant activity using MES and scPTZ
screens, and the neurotoxicity was determined applying the
rotorod test. Compounds (8a–i) displayed moderate antic-
onvulsant activity, and compound 8c was amongst the most
active compound in both MES and scPTZ test. The ED50 of
compounds 8b–g, 8i in the anti-MES test were better than
the ED50 of compound I (Fig. 1) and Sodium valproate
(272 mg/kg) but higher than that of phenobarbital, valpro-
ate, carbamazepine, compound II (Fig. 2). In the scPTZ test,
the anticonvulsant activities of compounds 8a–i were much
stronger than that of compound I (inactive at 100 mg/kg)
and better than that of the marked antiepileptic drug car-
bamazepine and Sodium valproate. Compounds 8a–i
showed broad-spectrum anti-epileptic activity.
Experimental section
Chemistry
3-Mercaptopropionic acid is a competitive inhibitor of
GABA synthesis enzyme glutamate decarboxylase (GAD),
and it inhibits the synthesis of GABA resulting in decreased
GABA level in the brain (Löscher and Schmidt 1988).
Compound 8c showed weak antagonism to 3-
mercaptopropionic acid-induced seizures, suggesting that
it might activate GAD or inhibit aminotransferase (GABA-
T) in the brain.
Melting points were determined on X-5 microscope melting
point apparatus, which were uncorrected. Infrared (IR)
spectra were recorded (in KBr) on a Fourier transform
infrared (IRPRESTIGE-21). 1H-NMR spectra were mea-
sured on an AV-300 (Bruker, Switzerland), and all chemical
shifts were given in parts per million relative to tetra-
methylsilane. Mass spectra were measured on a 6400 QQQ
LC/MS (Agilent Technologies, USA). Combustion analyses
(C, H, and N) were performed on a PE-2400 (SHI-
MADZU). Microanalyses of C, N, and H were performed
using a Heraeus CHN Rapid Analyzer. The major chemicals
Bicuculline (BIC) is a competitive antagonist of GABAA
receptor. BIC produces convulsions through its antagonism
of GABAA receptor (Macdonald and Barker 1978).

Med Chem Res
Fig. 1 The structures of
compounds I, II and target
compounds
(methanol: ethyl acetate = 1: 100) to afford compounds
5a–i (Salvatroe et al. 2011; Srivastava et al. 1999).
General procedure for preparation of compound 6
To the stirred mixture of the compound 3 (8mmol) in DMF
(30 ml), sodium hydride (25.6 mmol, 80%) was added
portionwise over 0.5 h whilst maintaining the temperature
between 25–30 °C. Than CH3I (1.6 mL dissolved in 5 mL
DMF) was slowly added to the reaction mixture and stirring
was continued for another 24 h, to which of the water 30 mL
was added and stirring was continued for 24 h between
25–30 °C, then filtered, and the filter cake was washed with
distilled water until the filtrate is neutral to pH paper and
dried under vacuum to yield a pale yellow solid (Weinberg
et al. 2011).
Fig. 2 The anticonvulsant activity of compounds I, II, and targe
compounds
General procedure for preparation of compound 7
were purchased from Alderich Chemical Corporation. All
other chemicals were of analytical grade.
The compound 4 was prepared according to reference
(Shang* et al. 2009).
The compound 6 (3 mmol) and isopropanol (12 ml) were
placed in a single-necked round-bottomed flask, and stirred
at 80 °C 10 min. To the stirred mixture, hydrazine (0.17 mL,
80%) and Pd/C (34.14 mg, 10%) were added and stirring is
continued at 80 °C until the reaction completion was
checked by thin layer silica gel plates, and then filtered with
celite bed, and the filter cake was washed with isopropanol,
the filtrate was concentrated to give a pale yellow solid,
which was dissolved in dichloromethane and washed with
saturated brine. The organic layer was dried over anhydrous
magnesium sulfate, and concentrated to give a white solid,
which was dried under vacuum to afford the pure product.
General procedure for the preparation of compounds 5a–i
The compound 4 (191 mg, 1 mmol), K2CO3 (152 mg, 1.1
mmol), KI (199 mg, 1.2 mmol) and DMF (7 ml) were
placed in a three-necked round-bottomed flask, and stirred
at room temperature 30 min. To the stirred mixture, benzyl
bromide (1.5 mmol dissolved in 3 mL DMF) was slowly
added and stirring is continued at 80 °C. Completion of the
reaction was checked using thin layer silica gel plates. After
removing the solvent under reduced pressure, the residue
was dissolved in 60 mL of saturated brine, extracted with
Ethy1 acetate (60×3), and dried over anhydrous MgSO4.
Evaporation of the solvents gave a crude product, which
was purified by silica gel column chromatography
General procedure for preparation of compounds 8a–i
The compound 7 (1 mmol), K2CO3 (1.5 mmol), KI (1.8
mmol) and DMF (7 ml) were placed in a three-necked
round-bottomed flask, and stirred at room temperature
30 min. To the stirred mixture, benzyl bromide (1.5 mmol

Med Chem Res
dissolved in 3 mL DMF) was slowly added and stirring is
continued at 80 °C until the reaction completion was
checked by thin layer silica gel plates. After removing the
solvent under reduced pressure, the residue was dissolved in
60 mL of saturated brine, extracted with dichloromethane
(60 × 3), and dried over anhydrous MgSO4, and evapora-
tion of the solvents gave a crude product, which was
purified by silica gel column chromatography (Dichlor-
omethane: Methanol = 40: 1) to afford compounds 8a–i.
added and stirring is continued at 80 °C until the reaction
completion was checked by thin layer silica gel plates. After
removing the solvent under reduced pressure, the residue
was dissolved in 60 ml of saturated brine, extracted with
Ethy1 acetate (60 × 3), and dried over anhydrous MgSO4.
Evaporation of the solvents gave a crude product, which
was purified by silica gel column chromatography (acetone:
ethyl acetate = 2: 1) to afford compounds 12.
7-(2-fluorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5a)
General procedure for preparation of compound 9
Acetonitrile (2.0 mL) and triethylamine (0.3 mL) were
placed in a three-necked round-bottomed flask, to which
P2S5 (0.414 g, 1.86 mmol) was added slowly in an ice bath
and stirred until dissolved. Then compound 3 (0.376 g, 1.7
mmol) was added while stirring. The mixture was refluxed
for 3 h in a nitrogen atmosphere. Then filtered, and the filter
cake was washed with dichloromethane and water, which
was dried under vacuum to afford the pure product.
Yield: 53%, white, mp. >250 °C. IR (KBr, ν, cm−1): 3150
(N–H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
1
d6): δ 10.16 (1H, s, CO–NH), 9.95 (1H, s, CO–NH),
7.03–7.48 (4H, m, Ar–H), 6.84 (1H, d, J = 8.4 Hz, Ar–H),
6.33–6.50 (3H, m, Ar–H and NH), 4.26 (2H, d, J = 5.4 Hz,
benzyl CH2), 3.08 (2H, s, CH2). 13C NMR (75 MHz, DMSO-
d6): δ 166.5 (C-4), 166.0 (C-2), 161.0 (d, JC-F = 242.7 Hz, C-
2, phenyl), 146.6 (C-7), 131.5 (C-5a), 130.1 (d, JC-F = 4.4 Hz,
C-6, phenyl), 129.4 (d, JC-F = 7.4 Hz, C-1, phenyl), 127.1 (d,
JC-F = 14.3 Hz, C-4, phenyl), 123.9 (C-8), 120.0 (C-9a),
General procedure for preparation of compound 10
115.8 (d,em>JC-F
= 21.2 Hz, C-3, phenyl), 110.4 (C-9), 104.8
The compound 9 (269 mg, 2 mmol) reacted with methyl
hydrazine carboxylate (310 mg, 5 mmol) in n-butanol 10
mL at 130 °C for 48 h, the solvent was removed under
reduced pressure, and the residue dissolved with ethy1
acetate and washed with water three times. The ethy1
acetate layer was dried over anhydrous MgSO4, filtered, and
concentrated the solvent, and the residue was purified by
recrystallization (ethanol/ N, N-dimethylformamide) to
afford compound 10 (Zhang et al. 2012).
(C-6), 46.5 (C-3), 40.8 (CH2, benzyl). MS (APCI) m/z (%):
299.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C, 64.21; H,
4.71; N, 14.04. Found: C, 64.27; H, 4.76; N, 14.09.
7-(3-fluorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5b)
Yield: 51%, white, mp. >250 °C. IR (KBr, ν, cm−1): 3150
(N–H), 1690 (C=O). 1H NMR (300 MHz, δ, ppm, DMSO-
d6): δ 10.13 (1H, s, CO–NH), 9.92 (1H, s, CO–NH), 7.25–7.50
(4H, m, Ar–H), 6.84 (1H, d, J = 8.4 Hz, Ar–H), 6.38–6.45 (2H,
m, Ar–H and NH), 6.28 (1H, d, J = 2.4 Hz, Ar–H), 4.22 (2H,
d, J = 5.79 Hz, benzyl CH2), 3.08 (2H, s, CH2). 13C NMR
(75 MHz, CDCl3): δ 166.5 (C-4), 166.0 (C-2), 163.0 (d, JC-F
= 241.7 Hz, C-3, phenyl), 146.6 (C-7), 144.0 (d, JC-F = 6.6
Hz, C-1, phenyl), 131.5 (C-5a), 130.9 (d, JC-F = 8.0 Hz, C-5,
phenyl), 123.8 (C-8), 120.0 (C-9a), 114.4 (d, JC-F = 21.3 Hz,
C-4, phenyl), 114.1 (d, JC-F = 20.9 Hz, C-2, phenyl), 110.4
(C-9), 104.8 (C-6), 46.5 (C-3), 45.6 (CH2, benzyl). MS (APCI)
m/z (%): 299.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C,
64.21; H, 4.71; N, 14.04.Found: 64.27; H, 4.76; N, 14.09.
General procedure for preparation of compound 11
The compound 10 (269 mg, 1 mmol), Pd/C (11.38 mg,
10%) and isopropanol (5 ml) were placed in a single-necked
round-bottomed flask, and stirred at 80 °C 10 min. To the
stirred mixture, hydrazine (1 mL, 80%) was added and
stirring is continued at 80 °C for 3 h until the reaction
completion was checked by thin layer silica gel plates, the
solvent was removed under reduced pressure and the resi-
due dissolved with DMF, and then filtered, concentrated the
solvent, the residue was dried under vacuum to afford the
pure product.
7-(4-fluorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5c)
General procedure for preparation of compounds 12
The compound 11 (239 mg, 1 mmol), K2CO3 (152 mg, 1.1
mmol), KI (199 mg, 1.2 mmol) and DMF (7 ml) were
placed in a three-necked round-bottomed flask, and stirred
at room temperature 30 min. To the stirred mixture benzyl
bromide (1.1 mmol dissolved in 3 mL DMF) was slowly
Yield: 51%, white, mp. >250 °C. IR (KBr, ν, cm−1): 3150
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
d6): δ 10.15 (1H, s, CO-NH), 9.94 (1H, s, CO-NH),
7.34–7.43 (2H, m, Ar-H), 7.10–7.19 (2H,m, Ar-H), 6.82
(1H, d, J = 8.67 Hz, Ar-H), 6.36–6.45 (2H, m, Ar-H), 6.30
1

Med Chem Res
(1H, s, NH), 4.20 (2H, d, J = 5.34 Hz, benzyl CH2), 3.07
(2H, s, CH2). 13C NMR (75 MHz, DMSO-d6): δ166.5 (C-
4), 166.0 (C-2), 163.4 (d, JC-F = 240.0 Hz, C-3, phenyl),
146.8 (C-7), 136.8 (C-1, phenyl), 131.5 (C-5a), 129.8 (d,
JC-F = 8.0 Hz, C-2 and C-6, phenyl), 123.8 (C-8), 119.9 (C-
9a), 115.7 (d, JC-F = 21.1 Hz, C-4, phenyl), 110.5 (C-9),
104.8 (C-6), 46.5 (C-3), 45.6 (C-benzyl). MS (APCI) m/z
(%): 299.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C,
64.21; H, 4.71; N, 14.04. Found: 64.27; H, 4.76; N, 14.09.
6.37–6.44 (2H, m, Ar-H), 6.26 (1H, d, J = 2.4 Hz, NH),
4.20 (2H, d, J = 6.0 Hz, benzyl CH2), 3.06 (2H, s, CH2).
13C NMR (75 MHz, DMSO-d6): δ 166.5 (C-4), 166.0 (C-2),
146.7 (C-7), 19.8 (C-1, phenyl), 131.8 (C-4, phenyl), 131.5
(C-5a), 129.7 (C-3, and C-5, phenyl), 128.9 (C-2 and C-6,
phenyl), 123.8 (C-8), 120.0 (C-9a), 110.4 (C-9), 104.7 (C-
6), 46.5 (C-3), 45.6 (CH2, benzyl). MS (APCI) m/z (%):
315.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C, 60.86;
H, 4.47; N, 13.31. Found: C, 60.80; H, 4.43; N, 13.38.
7-(2-chlorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5d)
7-(4-bromobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5g)
Yield: 52%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
Yield: 54%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
1
1
d6): δ 10.13 (1H, s, CO-NH), 9.94 (1H, s, CO-NH),
7.40–7.45 (2H, m, Ar-H), 7.36–7.26 (3H, m, Ar-H), 6.84
(1H, d, J = 8.67 Hz), 6.39–6.46 (1H, m, Ar-H), 6.28 (1H, s,
Bz-NH), 4.29 (2H, d, J = 8.67 Hz, benzyl CH2), 3.08 (2H,
s, CH2). 13C NMR (75 MHz, DMSO-d6): δ 166.5 (C-4),
166.0 (C-2), 146.7 (C-7), 137.4 (C-1, phenyl), 130.0 (C-2,
phenyl), 129.6 (C-5a), 129.9 (C-4, phenyl), 129.4 (C-6,
phenyl), 129.1 (C-3, phenyl), 123.9 (C-5, phenyl), 121.0
(C-8), 120.1 (C-9a), 110.4 (C-9), 104.5 (C-6), 45.7 (C-3),
43.0 (CH2, benzyl). MS (APCI) m/z (%): 315.1 (M+, 100);
Anal. Calcd. for C12H13N3O2: C, 60.86; H, 4.47; N, 13.31.
Found: C, 60.80; H, 4.43; N, 13.38.
d6): δ 10.13 (1H, s, CO-NH), 9.92 (1H, s, CO-NH),
7.32–7.38 (4H,m, Ar-H), 6.79 (1H, d, J = 8.7 Hz, Ar-H),
6.37–6.44 (2H, m, Ar-H), 6.26 (1H, d, J = 2.4 Hz, Bz-NH),
4.20 (2H, d, J = 6 Hz, benzyl CH2), 3.06 (2H, s, CH2). 13C
NMR (75 MHz, DMSO-d6): δ166.5 (C-4), 166.0 (C-2),
146.7 (C-7), 140.6 (C-1, phenyl), 131.5 (C-5a), 128.9 (C-3
and C-5, phenyl), 128.0 (C-2 and C-6, phenyl), 127.3 (C-4,
phenyl), 123.8 (C-8), 120.9 (C-9a), 110.5 (C-9), 104.7 (C-
6), 46.5 (C-3), 45.6 (CH2, benzyl). MS (APCI) m/z (%):
359.0 (M+, 100); Anal. Calcd. for C12H13N3O2: C, 53.35;
H, 3.62; N, 11.67. Found: C, 53.30; H, 3.58; N, 11.60.
7-(4-methylbenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5h)
7-(3-chlorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5e)
Yield: 52%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
1
Yield: 52%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
1
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
d6): δ 10.14 (1H, s, CO-NH), 9.92 (1H, s, CO-NH), 7.23
(2H, d, J = 7.98 Hz, Ar-H), 7.12 (2H, d, J = 7.8 Hz, Ar-H),
6.80 (1H, d, J = 8.7 Hz, Ar-H), 6.25–6.43 (3H, m, Ar-H and
NH), 4.16 (2H, s, benzyl CH2), 3.07 (2H, s, >CH2). 13C
NMR (75 MHz, DMSO-d6): δ166.5 (C-4), 166.0 (C-2),
146.7 (C-7), 140.6 (C-1, phenyl), 131.5 (C-5a), 129.0 (C-3,
and C-5, phenyl), 127.9 (C-2 and C-6, phenyl), 127.3 (C-4,
phenyl), 123.8 (C-8), 120.9 (C-9a), 110.5 (C-9), 104.7 (C-
6), 47.3 (C-3), 45.6 (CH2, benzyl). 21.3 (CH3). MS (APCI)
m/z (%): 295.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C,
69.14; H, 5.80; N, 14.23. Found: C, 69.10; H, 5.81; N,
14.29.
d6): δ 10.15 (1H, s, CO-NH), 9.96 (1H, s, CO-NH),
7.26–7.48 (4H, m, Ar-H), 6.84 (1H, d, J = 8.67 Hz,),
6.39–6.46 (2H, m, Ar-H), 6.28 (1H, s, NH), 4.29 (2H, d, J
= 5.7 Hz, benzyl CH2), 3.08 (2H, s, CH2). 13C NMR (75
MHz, DMSO-d6): δ166.5 (C-4), 166.0 (C-2), 146.7 (C-7),
137.4 (C-1, phenyl), 133.0 (C-2, phenyl), 131.6 (C-5a),
129.9 (C-4, phenyl), 129.4 (C-3, phenyl), 129.1 (C-6,
phenyl), 127.9 (C-5, phenyl), 124.0 (C-8), 120.1 (C-9a),
110.4 (C-9), 104.7 (C-6), 46.5 (C-3), 45.6 (CH2, benzyl).
MS (APCI) m/z (%): 315.1 (M+, 100); Anal. Calcd. for
C12H13N3O2: C, 60.86; H, 4.47; N, 13.31. Found: C, 60.80;
H, 4.43; N, 13.38.
7-(benzylamino)-1H-benzo[b][1,4]diazepine-2,4(3H,5H)-
dione (5i)
7-(4-chlorobenzylamino)-1H-benzo[b][1,4]diazepine-2,4
(3H,5H)-dione (5f)
Yield: 50%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
1
Yield: 51%, white, mp. >220 °C. IR (KBr, ν, cm−1): 3150
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
1
(N-H), 1690 (C=O). H NMR (300 MHz, δ, ppm, DMSO-
d6): δ 10.15 (1H, s, CO-NH), 9.93 (1H, s, CO-NH),
7.17–7.50 (5H, m, Ar-H), 6.81 (1H, d, J = 8.64 Hz, Ar-H),
6.36–6.45 (2H, m, Ar-H), 6.31 (1H, s, Bz-NH), 4.21 (2H, d,
d6): 10.12 (1H, s, CO-NH), 9.92 (1H, s, CO-NH),
7.32–7.38 (4H,m, Ar-H), 6.79 (1H, d, J = 8.7 Hz, Ar-H),

Med Chem Res
J = 5.7 Hz, benzyl CH2), 3.07 (1H, s, CH2). 13C NMR (75
MHz, DMSO-d6): δ 166.5 (C-4), 166.0 (C-2), 146.9 (C-7),
137.4 (C-1, phenyl), 131.5 (C-5a), 129.0 (C-3 and C-5,
phenyl), 127.9 (C-2 and C-6, phenyl), 127.3 (C-4, phenyl),
123.8 (C-8), 119.8 (C-9a), 110.4 (C-9), 104.5 (C-6), 46.5
(C-3), 45.6 (CH2, benzyl). MS (APCI) m/z (%): 281.1 (M+,
100); Anal. Calcd. for C12H13N3O2: C, 68.31; H, 5.37; N,
14.94. Found: C, 68.38; H, 5.32; N, 14.90.
(C-6), 44.7 (C-3), 42.1 (CH2, benzyl), 35.6 (CH3), 35.4
(CH3). MS (APCI) m/z (%): 327.1 (M+, 100); Anal. Calcd.
for C12H13N3O2: C, 66.04; H, 5.54; N, 12.84. Found: C,
65.99; H, 5.58; N, 12.90.
7-(3-fluorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8b)
Yield: 55%, white, mp. 66.1–68.6 °C. IR (KBr, ν, cm−1):
3375(N-H), 1676(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.29–7.38 (1H, m, Ar-H), 6.97–7.18 (1H, m,
Ar-H), 6.54 (1H, dd, J = 2.4, 9.0 Hz, Ar-H), 6.39 (1H, d, J
= 2.4 Hz, Ar-H), 4.47 (1H, s, NH), 4.37 (2H, s, benzyl
CH2), 3.32 (6H, s, -CH3), 3.28 (2H, s, -CH2-). 13C NMR
(75 MHz, CDCl3): δ 166.2 (C-4), 166.0 (C-2), 163.4 (d,
JC-F = 244.8 Hz, C-3, phenyl), 146.3 (C-7), 141.6 (d, JC-F
= 6.7 Hz, C-1, phenyl), 137.5 (C-5a), 130.6 (d, JC-F = 8.3
Hz, C-5, phenyl), 127.2 (C-9a), 123.9 (C-6, phenyl), 122.9
(C-8), 114.7 (d, JC-F = 21.2 Hz, C-4, phenyl), 114.2 (d, JC-F
= 21.8 Hz, CH2, benzyl), 44.7 (C-3), 35.6 (CH3), 35.5
(CH3). MS (APCI) m/z (%):327.1 (M+, 100); Anal. Calcd.
for C12H13N3O2: C, 66.04; H, 5.54; N, 12.84. Found: C,
65.99; H, 5.58; N, 12.90.
7- nitro -1, 5-dimethyl-1H-benzo[b][1,4]diazepine-2,4(3H,
5H)-dione (6)
Yield: 92%, pale yellow, 75.5–76.9 °C. IR (KBr, ν, cm−1):
1
1691 (C=O). H NMR (300 MHz, δ, ppm, CDCl3): δ 8.23
(1H, d, J = 9.0 Hz, Ar-H), 8.18 (1H, dd, J = 2.7, 9.0 Hz,
Ar-H), 7.49 (1H, d, J = 2.7 Hz, Ar-H), 3.47–362 (8H, m,
CH2 and CH3). 13C NMR (75 MHz, CDCl3): δ 165.0 (C-4),
164.9 (C-2), 145.0 (C-7), 141.2 (C-5a), 136.6 (C-9a), 123.3
(C-8), 121.2 (C-9), 118.5 (C-6), 44.1 (C-3), 35.9 (CH3),
35.9 (CH3). MS (APCI) m/z (%): 249.1 (M+, 100); Anal.
Calcd. for C12H13N3O2: C, 53.01; H, 4.45; N, 16.86.
Found: C, 52.95; H, 4.40; N, 16.80.
7-amino-1, 5-dimethyl-1H-benzo[b][1, 4]diazepine-2,4(3H,
5H)-dione (7)
7-(4-fluorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8c)
Yield: 97%, white, mp. 62.5–65.3 °C. IR (KBr, ν, cm−1):
1
3344 (N-H), 1696 (C=O). H NMR (300 MHz, δ, ppm,
Yield: 54%, white, mp. 62.5–64.6 °C. IR (KBr, ν, cm−1):
3387(N-H), 1676(C=O).. 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.31–7.39 (2H, m, Ar-H), 7.01–7.13 (3H, m,
Ar-H), 6.55 (1H, dd, J = 2.4, 9.0 Hz, Ar-H), 6.41 (1H, d, J
= 2.4 Hz, Ar-H), 4.37 (1H, s, NH), 4.33 (2H, s, benzyl
CH2), 3.33 (6H, s, -CH3), 3.30 (2H, s, -CH2-). 13C NMR
(75 MHz, CDCl3): δ 166.2 (C-4), 166.0 (C-2), 162.4 (d,
JC-F = 244.4 Hz, C-4, phenyl), 146.4 (C-7), 137.5 (C-5a),
134.4 (C-1, phenyl), 129.2 (d, JC-F = 8.0 Hz, C-2 and C-6,
phenyl), 127.1 (C-9a), 123.9 (C-8), 115.9 (d, JC-F = 21.3 Hz,
C-3 and C-5, phenyl), 111.7 (C-9), 105.3 (C-6), 47.7 (CH2,
benzyl), 44.7 (C-3), 35.6 (CH3), 35.5 (CH3). MS (APCI) m/
z (%):327.1 (M, 100); Anal. Calcd. for C12H13N3O2: C,
66.04; H, 5.54; N, 12.84. Found: C, 65.99; H, 5.58; N,
12.90.
CDCl3): δ 7.07 (1H, d, J = 8.7 Hz, Ar-H), 6.61 (1H, dd, J
= 2.7, 8.7 Hz, Ar-H), 6.55 (1H, d, J = 2.7 Hz, Ar-H), 3.92
(2H, s, NH2), 3.33–3.42 (8H, m, CH2 and CH3). 13C NMR
(75 MHz, CDCl3): δ 166.0 (C-4), 155.7 (C-2), 145.0 (C-7),
137.1 (C-5a), 127.6 (C-9a), 123.8 (C-8), 113.4 (C-9), 107.5
(C-6), 44.5 (C-3), 35.4 (CH3), 35.3 (CH3). MS (APCI) m/z
(%): 219.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C,
60.26; H, 5.98; N, 19.17. Found: C, 60.20; H, 5.94; N,
19.11.
7-(2-fluorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8a)
Yield: 50%, white, mp. 61.1–63.3 °C. IR (KBr, ν, cm−1):
3416(N-H), 1676(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.25–7.42 (2H, m, Ar-H), 7.08–7.17 (3H, m,
Ar-H), 7.07 (1H, d, J = 8.7 Hz, Ar-H), 6.57 (1H, dd, J =
2.7, 8.7 Hz, Ar-H), 6.45 (1H, d, J = 2.7 Hz, NH), 4.42 (3H,
s, NH and benzyl CH2), 3.30 (8H, s, CH2 and CH3). 13C
NMR (75 MHz, CDCl3): δ 166.2 (C-4), 166.0 (C-2, phe-
nyl), 161.1 (d, JC-F = 240.0 Hz, C-2, phenyl), 146.3 (C-7),
137.5 (C-5a), 129.7 (d, JC-F = 4.2 Hz, C-6, phenyl), 129.5
(d, JC-F = 8.2 Hz, C-1, phenyl), 127.2(C-9a), 125.6 (d, JC-F
= 14.3 Hz, C-4, phenyl), 124.6 (C-5, phenyl), 123.9 (C-8),
115.8 (d, JC-F = 21.2 Hz, C-3, phenyl), 111.7 (C-9), 105.3
7-(2-chlorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8d)
Yield: 51%, white, mp. 59.1–61.4 °C. IR (KBr, ν, cm−1):
3348(N-H), 1678(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.37–7.45 (2H, m, Ar-H), 7.23–7.30 (2H, m,
Ar-H), 7.07 (1H, d, J = 9.0Hz, Ar-H), 6.55 (1H, dd, J = 2.4,
9.0 Hz, Ar-H), 6.41 (1H, d, J = 2.4 Hz, Ar-H), 4.55 (1H, s,
NH), 4.47 (2H, s, benzyl CH2), 3.33 (6H, s, -CH3), 3.31
(2H, s, -CH2--).13C NMR (75 MHz, CDCl3): δ 166.3 (C-4),

Med Chem Res
166.0 (C-2), 146.3 (C-7), 137.5 (C-5a) 135.9 (C-1, phenyl),
133.6 (C-2, phenyl), 130.0 (C-3, phenyl), 129.3 (C-4,
phenyl), 127.3 (C-9a), 127.1(C-5, phenyl), 123.8 (C-8),
111.7 (C-9), 105.4 (C-6), 47.9 (CH2, benzyl), 44.7 (C-3),
35.6 (CH3), 35.5 (CH3). MS (APCI) m/z (%): 343.1 (M,
100); Anal. Calcd. for C12H13N3O2: C, 62.88; H, 5.28; N,
12.22. Found: C, 62.80; H, 5.24; N, 12.28.
-CH3), 3.31 (2H, s, -CH2-). 13C NMR (75 MHz,CDCl3): δ
166.2 (C-4), 166.0 (C-2), 146.3 (C-7), 137.8 (C-1, phenyl),
137.4 (C-5a), 132.1 (C-3 and C-5, phenyl), 129.2 (C-2 and
C-6, phenyl), 127.2 (C-9a), 124.0 (C-8), 121.5 (C-4, phe-
nyl), 111.7 (C-9), 105.4 (C-6), 47.8 (CH2, benzyl), 44.7 (C-
3), 35.6 (CH3), 35.5 (CH3). MS (APCI) m/z (%): 387.1
(M+, 100); Anal. Calcd. for C12H13N3O2: C, 55.68; H,
4.67; N, 10.28. Found: C, 55.60; H, 4.62; N, 10.20.
7-(3-chlorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8e)
7-(4-methylbenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione(8h)
Yield: 53%, white, mp. 73.3–75.7 °C. IR (KBr, ν, cm−1):
3358(N-H), 1678(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.37 (1H, s, Ar-H), 7.23–7.35 (3H, m, Ar-H),
7.08 (1H, d, J = 8.7 Hz, Ar-H), 6.54 (1H, dd, J = 2.7, 8.7
Hz, Ar-H), 6.40 (1H, d, J = 2.7 Hz, Ar-H), 4.47 (1H, s,
NH), 4.36 (2H, s, benzyl CH2), 3.33 (6H, s, –CH3), 3.30
(2H, s, -CH2-). 13C NMR (75 MHz, CDCl3): δ 166.3 (C-4),
166.0 (C-2), 146.3 (C-7), 141.0 (C-1, phenyl), 137.5 (C-5a)
130.0 (C-2, phenyl), 127.9 (C-3, C-6, phenyl), 127.5 (C-4,
phenyl), 127.2 (C-9a), 125.5(C-5, phenyl), 123.9 (C-8),
111.7 (C-9), 105.3 (C-6), 46.0 (CH2, benzyl), 44.7 (C-3),
35.6 (CH3), 35.5 (CH3). MS (APCI) m/z (%): 343.1 (M+,
100); Anal. Calcd. for C12H13N3O2: C, 62.88; H, 5.28; N,
12.22. Found: C, 62.80; H, 5.24; N, 12.28.
Yield: 48%, white, mp. 74.1–76.5 °C. IR (KBr, ν, cm−1):
3358(N-H), 1678(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.26 (2H, d, J = 7.8 Hz, Ar-H), 7.17 (2H, d,
J = 7.8 Hz, Ar-H), 7.08 (1H, d, J = 9.0 Hz, Ar-H), 6.56
(1H, dd, J = 2.7, 9.0 Hz, Ar-H), 6.42 (1H, d, J = 2.7 Hz,
Ar-H), 4.37 (1H, s, NH), 4.30 (2H, s, benzyl CH2),
3.29–3.40 (8H, m, CH2 and CH3). 13C NMR (75 MHz,
CDCl3): δ 166.3 (C-4), 166.0 (C-2), 146.7 (C-7), 137.5 (C-
1, phenyl), 137.4 (C-5a), 135.6 (C-4, phenyl), 129.7 (C-3
and C-5, phenyl), 127.6 (C-2 and C-6, phenyl), 126.9 (C-
9a), 115.7 (C-8), 111.7 (C-9), 105.2 (C-6), 48.2 (C-benzyl,
CH2), 44.7 (C-3), 35.6 (CH3), 35.5 (CH3), 21.3 (C-phenyl,
CH3). MS (APCI) m/z (%): 323.9 (M+, 100); Anal. Calcd.
for C12H13N3O2: C, 70.57; H, 6.55; N, 12.99. Found: C,
70.50; H, 5.60; N, 12.92.
7-(4-chlorobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8f)
7-(benzylamino)-1,5-dimethyl-1H-benzo[b][1,4]diazepine-
2,4(3H,5H)-dione (8i)
Yield: 52%, white, mp. 59.9–62.2 °C. IR (KBr, ν, cm−1):
3354(N-H), 1676(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.28–7.36 (4H, m, Ar-H), 7.07 (1H, d, J =
8.7 Hz, Ar-H), 6.54 (1H, dd, J = 2.7, 8.7 Hz, Ar-H), 6.40
(1H, d, J = 2.7 Hz, Ar-H), 4.49 (1H, s, NH), 4.34 (2H, s,
benzyl CH2), 3.32 (6H, s, -CH3), 3.30 (2H, s, -CH2-). 13C
NMR (75 MHz, CDCl3): δ 166.2 (C-4), 166.0 (C-2), 146.3
(C-7), 137.4 (C-1, phenyl), 137.3 (C-5a), 133.4 (C-4, phe-
nyl), 129.2 (C-2 and C-6, phenyl), 128.8 (C-3 and C5,
phenyl), 127.1 (C-9a), 124.0 (C-8), 111.7 (C-9), 105.4 (C-
6), 47.8 (C-benzyl), 44.7 (C-3), 35.6 (CH3), 35.5 (CH3). MS
(APCI) m/z (%): 343.1 (M+, 100); Anal. Calcd. for
C12H13N3O2: C, 62.88; H, 5.28; N, 12.22. Found: C, 62.80;
H, 5.24; N, 12.28.
Yield: 49%, white, mp. 77.4–79.8.0 °C. IR (KBr, ν, cm-1):
3363(N-H), 1678(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.26–7.40 (5H, m, Ar-H), 7.06 (1H, d, J =
8.7 Hz, Ar-H), 6.56 (1H, dd, J = 2.4, 8.7 Hz, Ar-H), 641
(1H, d, J = 2.4 Hz, Ar-H), 4.35 (3H, s, NH and benzyl
CH2), 3.32 (6H, s, N-CH3), 3.29 (2H, s, -CH2-). 13C NMR
(75 MHz, CDCl3): δ 166.2 (C-4), 166.0 (C-2), 146.6 (C-7),
138.7 (C-1, phenyl), 137.4 (C-5a), 129.1 (C-3 and C-5,
phenyl), 127.8 (C-4, phenyl), 127.6 (C-2 and C-6, phenyl),
127.0 (C-9a), 115.7 (C-8),111.7 (C-9), 105.2 (C-6), 48.5
(C-benzyl, CH2), 44.7 (C-3), 35.6 (CH3), 35.5 (CH3). MS
(APCI) m/z (%): 309.1 (M+, 100); Anal. Calcd. for
C12H13N3O2: C, 69.88; H, 6.19; N, 13.58. Found: C, 69.80;
H, 6.15; N, 13.51.
7-(4-bromobenzylamino)-1,5-dimethyl-1H-benzo[b][1,4]
diazepine-2,4(3H,5H)-dione (8g)
7-nitro-1H-benzo[b][1, 4]diazepine-2, 4(3H, 5H)-dithione
(9)
Yield: 59%, white, mp. 73.8–75.9 °C. IR (KBr, ν, cm−1):
3350(N-H), 1678(C=O). 1H NMR (300 MHz, δ, ppm,
DMSO-d6): δ 7.49 (2H, d, J = 8.4Hz, Ar-H), 7.26 (2H, d, J
= 8.4Hz, Ar-H), 7.08 (1H, d, J = 8.7Hz, Ar-H), 6.54 (1H,
dd, J = 2.4, 8.7 Hz, Ar-H), 6.40 (1H, d, J = 2.4 Hz, Ar-H),
4.51 (1H, s, NH), 4.33 (2H, s, benzyl CH2), 3.33 (6H, s,
Yield: 85%. mp. >220 °C. IR (KBr, ν, cm−1): 3150(N-H),
1
1690(C=O). H NMR (300 MHz, δ, ppm, DMSO-d6): δ
12.84 (1H, s, CS-NH), 12.69 (1H, s, CS-NH), 8.09–8.22
(2H, m, Ar-H), 7.50 (1H, d, J = 8.9 Hz, Ar-H), 4.18 (2H, s,

Med Chem Res
CH2). 13C NMR (75 MHz, DMSO-d6): δ 196.0 (C-4), 195.5
(C-2), 144.5 (C-7), 137.3 (C-9a), 132.5 (C-5a), 124.2 (C-9),
121.5 (C-6), 118.7 (C-8), 62.0 (C-3). MS (APCI) m/z (%):
253.0 (M+, 100); Anal. Calcd. for C12H13N3O2: C, 42.68;
H, 2.79; N, 16.59. Found: C, 42.60; H,2.76; N, 16.51.
Pharmacology
Anticonvulsant effects in the MES and scPTZ test
The MES test, sc-PTZ test, and rotarod test were carried out
by the Antiepileptic Drug Development Program, Epilepsy
Branch, National Institutes of Health, Bethesda, MD, USA.
(Krall et al. 1978; Porter et al. 1984). All compounds were
tested for anticonvulsant activity with Swiss mice in the
18–22 g weight range purchased from the Laboratory of
Animal Research, College of Pharmacy, Yanbian Uni-
versity. The tested compounds were dissolved in DMSO. In
phase I screening (Table 1) each compound was adminis-
tered at three dose levels (30, 100, and 300 mg/ kg ip) with
anticonvulsant activity and neurotoxicity assessed at 30 min
and 4h intervals after administration. Anticonvulsant effi-
cacy was measured in the MES test and the sc-PTZ test. In
the MES test, seizures were elicited with a 60 Hz alternating
current of 50 mA intensity in mice. The current was applied
via corneal electrodes for 0.2 s. Abolition of the hind-leg
tonic-extensor component of the seizure indicated protec-
tion against the spread of MES-induced seizures. The sc-
PTZ test involved subcutaneous injection of a convulsant
dose of pentylenetetrazol (95mg/kg in mice). Elevation of
the pentylenetrazol-induced seizure threshold was indicated
by the absence of clonic spasms for at least 5s duration over
a 30 min period following administration of the test com-
pound. Anticonvulsant drug-induced neurologic deficit was
detected in mice by using the rotarod ataxia test. Antic-
onvulsant activity was expressed in terms of the median
effective dose (ED50), and neurotoxicity was expressed as
the median toxic dose (TD50). For determination of the
ED50 and TD50 values, groups of 10 mice were given a
range of intraperitoneal doses of the tested compounds until
at least three points were established in the range of
10–90% seizure protection or minimal observed neuro-
toxicity. From the plots of these data, the respective ED50,
TD50 values and 95% confidence intervals were calculated
by means of Trimmed Spearman-Karber method (Okada
et al. 1989). Simultaneously clinical application of anti-
epileptic drugs phenytoin, carbamazepine, phenobarbital
and valproate as a control was carried out. The results are
shown in Table 2.
2-nitro-9H-bis[l,2,4]triazolo[4,3-a: 3′,4′-d][I,5]
benzodiazepine (10)
Yield: 98%. mp. >220 °C. IR (KBr, ν, cm-1): 3128(N-H),
1
1690(C=O). H NMR (300 MHz, δ, ppm, DMSO-d6): δ
9.24 (1H, s, triazole CH), 9.20(1H, s, triazole CH), 8.78
(1H, s, Ar-H), 8.52 (1H, d, J = 8.94 Hz, Ar-H), 8.14 (1H, d,
J = 8.94 Hz, Ar-H), 4.59 (2H,s, CH2). 13C NMR (75 MHz,
DMSO-d6): δ 150.2 (C-8a), 150.1(C-9a), 147.4(C-2), 145.2
(C-9), 145.0 (C-12), 131.6 (C-4a), 127.3 (C-13a), 127.2(C-
4), 124.7(C-3), 121.4 (C-1), 22.6 (C-9). MS (APCI) m/z
(%): 269.1 (M, 100); Anal. Calcd. for C12H13N3O2: C,
49.07; H, 2.62; N, 36.42. Found: C, 49.00; H, 2.60; N,
36.48.
2-amine-9H-bis[l,2,4]triazolo[4,3-a: 3′,4′-d][I,5]
benzodiazepine (11)
Yield: 98%. mp. >220 °C. IR (KBr, ν, cm−1): 3128(N-H),
1
1690(C=O). H NMR (300 MHz, δ, ppm, DMSO-d6): δ
9.24 (1H, s, triazole CH), 8.92 (1H, s, triazole CH), 7.51
(1H, s, d, J = 8.6 Hz, Ar-H), 6.88 (1H, s, Ar-H), 6.81 (1H,
d, J = 8.6 Hz, Ar-H), 5.90 (2H, s, NH2), 4.59 (2H, s, CH2).
13C NMR (75 MHz, DMSO-d6): δ 150.2 (C-8a), 150.1(C-
9a), 147.4 (C-2), 145.2(C-6), 127.2 (C-12), 126.3 (C-13a),
126.2 (C-4), 114.9(C-3), 114.7(C-4a), 121.4 (C-1), 22.6 (C-
9). MS (APCI) m/z (%): 239.1 (M+, 100); Anal. Calcd. for
C12H13N3O2: C, 55.22; H, 3.79; N, 40.98. Found: C, 55.29;
H, 3.75; N, 40.92.
2-(4-chlorobenzylamino)-9H-bis[l,2,4]triazolo[4,3-a: 3′,4′-
d][I,5]benzodiazepine (12)
mp. >220 °C. IR (KBr, ν, cm−1): 3128(N-H), 1690(C=O).
1H NMR (300 MHz, δ, ppm, DMSO-d6): δ 9.24 (1H, s,
triazole CH), 9.20(1H, s, triazole CH), 7.40–7.10 (5H, m,
Ar–H), 6.61–6.50 (2H,m, Ar–H), 4.35 (3H, s, Bz-NH and
benzyl CH2), 3.81 (2H,s, –CH2–). 13C NMR (75 MHz,
DMSO-d6): δ 160.2 (C-8a), 160.1(C-9a), 147.4(C-2), 145.2
(C-6), 145.0 (C-12), 138.0 (C-1, phenyl), 132.3 (C-4, phe-
nyl), 130.2(C-4), 129.6(C-12b), 129.2(C-2 and 6, phenyl),
128.4(C-3 and 5, phenyl), 118.2(C-4a), 113.5 (C-3), 95.8
(C-1), 48.1(C-benzyl), 22.6 (C-9). MS (APCI) m/z (%):
363.1 (M+, 100); Anal. Calcd. for C12H13N3O2: C, 59.43;
H, 3.88; N, 26.95. Found: C, 59.50; H, 3.85; N, 26.90.
Neurotoxicity screening (NT)
The NT of the compounds was measured in mice by the
rotarod test. The mice were trained to stay on an accel-
erating rotarod of diameter 3.2 cm that rotated at 10 rpm.
Trained animals were given i.p. injection of the test com-
pounds. Neurotoxicity was indicated by the inability of the
animal to maintain equilibrium on the rod for at least 1 min

Med Chem Res
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Drug Discov Devel 10:768
Dawood KM, Abdel-Gawad H, Rageb EA, Ellithey M, Mohamed HA
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3-MP induced seizures test
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cages and observed for 0.5 h. The number of clonic and
tonic seizures as well as the number of deaths was noted
(Arnoldi et al. 1990). The results are shown in Table 3.
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Bicuculline-induced seizures test
At 30 min after the administration of compounds, the ani-
mals (10 mice in one group) were given a subcutaneous
dose of 2.7 mg/kg for Bicuculline (within 15–45 min after
preparation due to instability). Individual mice were then
placed in isolation cages and observed for at least 30 min for
the presence or absence of clonic seizures, and tonic sei-
zures, and lethality was also recorded (Bernasconi et al.
1988). The results are shown in Table 3.
Acknowledgements This work was supported by the National
Natural Science Foundation of China (No. 21162031).
Rajarao SJ, Platt B, Sukoff SJ, Lin Q, Bender CN, Nieuwenhuijsen
BW, Ring RH, Schechte LE, Rosenzweig-Lipson S, Beyer CE
(2007) Neuropeptides 41:307–320
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
Reimers A (2014) Seizure 23:585–591
interests.
Salvatroe RN, Yoon CH, Jung KW (2011) Tetrahedron 57:7785–7811
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Srivastava SK, Singh PM, Bhaduri AP (1999) Synth Commun
29:2085–2091
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