Novel fused 1,2,3-triazolo-benzodiazepine derivatives as potent anticonvulsant agents: design, synthesis, in vivo, and in silico evaluations

Article abstract of DOI:10.1007/s11030-019-09940-9

Abstract: A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a–o has been synthesized and evaluated in vivo for their anticonvulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. The synth

Full text of DOI:10.1007/s11030-019-09940-9

Molecular Diversity
https://doi.org/10.1007/s11030-019-09940-9
ORIGINAL ARTICLE
Novel fused 1,2,3‑triazolo‑benzodiazepine derivatives as potent
anticonvulsant agents: design, synthesis, in vivo, and in silico
evaluations
1
2
3
4
1
Arefeh Shaꢀe · Maryam Mohammadi‑Khanaposhtani · Mehdi Asadi · Nastaran Rahimi · Parviz Rashidi Ranjbar ·
1
5
5
6
4,7
Jahan B. Ghasemi · Bagher Larijani · Mohammad Mahdavi · Hamed Shafaroodi · Ahmad Reza Dehpour
Received: 29 January 2019 / Accepted: 13 March 2019
©
Springer Nature Switzerland AG 2019
Abstract
A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a–o has been synthesized and evaluated in vivo for their anti-
convulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. The
synthetic approach started with diazotizing 2-aminobenzoic acids 1 to produce 2-azidobenzoic acids 2. Next, reaction of
the latter compounds with propargylamine 3, benzaldehyde 4, and isocyanides 5 led to the formation of the title compounds
6
a–o, in good yields. All the synthesized compounds exhibited high anticonvulsant activity in the PTZ test, comparable
to or better than the standard drug diazepam. Among the tested compounds, N-(tert-butyl)-2-(9-chloro-6-oxo-4H-[1,2,3]
triazolo[1,5-a][1,4]benzodiazepin-5(6H)-yl)-2-(3-bromophenyl)acetamide 6h was the most potent compound in this assay.
Moreover, compounds 6i and 6k showed excellent activity in MES test. Loss of the anticonvulsant eꢀect of compound 6h in
the presence of ꢁumazenil in the PTZ test and appropriate interaction of this compound in the active site of benzodiazepine
(BZD)-binding site of GABA receptor conꢂrm involvement of BZD receptors in the anticonvulsant activity of compound 6h.
A
Graphical abstract
A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a–o have been synthesized and evaluated in vivo for their anti-
convulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. All the
synthesized compounds exhibited high anticonvulsant activity, comparable to or better than the standard drug diazepam in
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-09940-9) contains
supplementary material, which is available to authorized users.
4
*
Mohammad Mahdavi
Experimental Medicine Research Center, Tehran University
of Medical Sciences, Tehran, Iran
Momahdavi@sina.tums.ac.ir
5
*
Ahmad Reza Dehpour
Endocrinology and Metabolism Research Center,
Endocrinology and Metabolism Clinical Sciences Institute,
Tehran University of Medical Sciences, Tehran, Iran
dehpour@sina.tums.ac.ir
1
2
School of Chemistry, College of Science, University
of Tehran, Tehran, Iran
6
7
Department of Pharmacology, Tehran University of Medical
Sciences, Tehran, Iran
Cellular and Molecular Biology Research Center, Health
Research Institute, Babol University of Medical Sciences,
Babol, Iran
Department of Pharmacology, School of Medicine, Tehran
University of Medical Sciences, Tehran, Iran
3
Department of Medicinal Chemistry, Faculty of Pharmacy
and Pharmaceutical Sciences Research Center, Tehran
University of Medical Sciences, Tehran, Iran
Vol.:(0
1
123456789)
3

Molecular Diversity
the PTZ test and compounds 6i and 6k showed excellent activity in MES test. Flumazenil test and in silico docking study
conꢂrm involvement of benzodiazepine receptors in the anticonvulsant activity of these compounds.
Keywords Anticonvulsant · Seizure · 1,2,3-Triazolo-benzodiazepine · Docking study
Introduction
are two eꢀective derivatives of BZDs with 1,2,4-triazole-
benzodiazepine scaꢀold [6]. Biagia et al. in 1996 reported
that derivatives of 1,2,3-triazole fused to benzodiazepine
acted as potent agonists for BZD receptor (Fig. 1, A) [7].
Fused 1,2,3-triazolo-benzodiazepine derivatives such as
compounds B and C were also introduced that acted as
antitumor and serine protease inhibitor agents, respectively
(Fig. 1) [8, 9]. Due to the importance of BZD derivatives,
diverse routes have been reported for the synthesis of these
compounds [10–13].
After cerebrovascular disease and dementia, epilepsy is the
third most frequent neurological disorder aꢀecting approx-
imately 50 million people worldwide [1]. This disease is
caused by the abnormal discharge of cerebral neurons and is
associated with the periodic and unpredictable occurrence of
seizures. Several antiepileptic drugs such as phenobarbital,
sodium valproate, carbamazepine, phenytoin, and diazepam
are available but they fail to control seizures in about 30%
of epileptic patients [2]. In addition, some of these drugs
display several severe side eꢀects [3]. Thus, there is continu-
ing demand to discover and develop new eꢀective and safe
anticonvulsant agents.
In this work, taking into account the anticonvulsant struc-
tures D and E, a novel series of fused 1,2,3-triazolo-benzo-
diazepine derivatives 6a–o were designed using a hybrid
approach to achieve new scaꢀolds as potent anticonvulsant
drugs (Fig. 1) [14]. Figure 2 shows the pharmacophore
model of designed compounds for anticonvulsant activ-
ity [15]. Fused 1,2,3-triazolo-benzodiazepines 6a–o were
tested for their in vivo anticonvulsant activity. Furthermore,
to evaluate the mechanism action of these compounds as
BZD receptor agonists, ꢁumazenil test and in silico molecu-
lar docking studies were also performed.
Benzodiazepines (BZDs) such as diazepam (Valium),
lorazepam (Ativan), clonazepam (Klonopin) and alprazolam
(
Xanax) are a popular class of drugs that are used to treat
anxiety, insomnia, agitation, and seizures [4]. BZDs enhance
the eꢀect of the neurotransmitter GABA through agonist
binding to a speciꢂc domain of GABA receptor known as
A
BZD pocket [5]. With minor changes in structures of BZDs,
agents with the mentioned therapeutic eꢀects and or diꢀer-
ent biologically active compounds with novel applications
can be achieved. For example, alprazolam and estazolam
1
3

Molecular Diversity
Fig. 1 Chemical structures of biological active compounds with 1,2,3-triazolo-benzodiazepine scaꢀold A, B, and C, anticonvulsant compounds
D and B, and designed compounds 6a–o as new anticonvulsant agents
Chemistry
Scheme 1 shows the synthetic route for the synthesis of
fused triazolo-benzodiazepines. In the ꢂrst step, 2-azido-
benzoic acids 2 were obtained from azidation of 2-amin-
obenzoic acids 1 in the presence of NaNO , HCl, and NaN .
2
3
Next, 2-Azidobenzoic acids 2 reacted with propargylamine
3
, benzaldehyde 4, and isocyanide 5 in methanol at reꢁux to
give desired triazolo-benzodiazepines 6a–o in good yields
65–79%).
(
Fig. 2 Pharmacophore model of designed compounds 6a–o for anti-
convulsant activity. A hydrophobic domain, B electron donor moiety,
C H-bonding site, D distal hydrophobic domain
Scheme 1 Synthesis of 1,2,3-triazolo-benzodiazepine derivatives 6a–o
1
3

Molecular Diversity
Pharmacology
derivative 6b, as in compound 6d, decreased anticon-
vulsant activity. Moreover, 3 or 4-bromo derivatives 6e
and 6f showed activity similar to 4-chloro derivative 6c.
Introduction of 9-chloro substituent on benzodiazepine
moiety improve anticonvulsant potency in tert-butyl
derivatives. In this regard, the comparison of percent-
age of clonic seizure threshold of 9-chloro derivatives
6g and 6h with their analogs 6a and 6e revealed that the
chlorine atom had an important role in the anticonvulsant
activities obtained.
Anticonvulsant activity
Anticonvulsant activity against PTZ‑induced seizures
Percentage of clonic seizure threshold in the PTZ test of
target compounds revealed that all of these compounds had
excellent anticonvulsant activity, comparable or more than
the standard drug diazepam (Table 1). Among 1,2,3-tria-
zolo-benzodiazepines 6a–o, compounds 6g, 6h, 6n, and 6o
were the most potent compounds with activity more than
diazepam.
In the cyclohexyl series, the 9-unsubstituented deriva-
tives 6i–m showed approximately the same anticonvul-
sant activity (Table 1). In this series, similar to tert-butyl
series, the most potent compounds were derivatives with
9-chloro substituent on benzodiazepine moiety (6n and
6o). A comparison of anticonvulsant activity of tert-butyl
derivatives with their corresponding cyclohexyl analogs
revealed that cyclohexyl analogs except 6i and 6l were as
active as their tert-butyl analogs. In the case of compounds
6i and 6l, the anticonvulsant activity of these cyclohexyl
derivatives was more than their tert-butyl analogs 6a and
6d, respectively.
Synthesized derivatives 6a–o, structurally, can be divided
to two series: tert-butyl derivatives 6a–h and cyclohexyl
derivatives 6i–o.
Compound 6a with 4-fluoro substituent on pendant
phenyl group is the weakest derivative among synthe-
sized compounds. The presence of a chlorine atom on
pendant phenyl ring, especially in 2-position, led to a
significant increase in activity as observed in compounds
6
b and 6c. On the other hand, adding a second chlorine
atom to 3-position of pendant phenyl group in 2-chloro
Table 1 Anticonvulsant
activities of compounds 6a–o in
PTZ test
R1
R2
R3
Dose (mg/kg)
a
Clonic seizure
threshold (%)
Compound
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
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
Cl
4-F
2-Cl
4-Cl
2,3-Dichloro
3-Br
4-Br
4-F
3-Br
4-F
2-Cl
4-Cl
2,3-Dichloro
3-Br
4-F
Tert-butyl
Tert-butyl
Tert-butyl
Tert-butyl
Tert-butyl
Tert-butyl
Tert-butyl
Tert-butyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
0.145
0.148
0.148
0.161
0.165
0.165
0.155
0.175
0.152
0.158
0.158
0.170
0.175
0.165
44.32
51.68
49.37
45.27
48.19
48.11
54.60*
56.94**
48.23
51.68*
50.43
49.15
48.64
54.60
6
o
Cl
–
4-Br
–
Cyclohexyl
–
0.185
0.1
55.02**
53.87
Diazepam
*
P<0.05; **P<0.01 compared to vehicle group
a
Synthesized compounds and standard drug diazepam were used in an equimolar dose
1
3

Molecular Diversity
Table 2 Anticonvulsant activities of compounds 6a–o in MES test
In vivo neurotoxicity
Compound
Dose (mg/kg)a
Tonic seizure
protection (%)
Compounds 6h and 6k as most potent compounds, respec-
tively, in PTZ and maximal electroshock (MES) tests were
screened for their neurotoxicity in mice by using rotarod
test [18]. Rotarod test is used widely to evaluate neurotox-
icity of new anticonvulsant agents in mice. As shown in
Table 3, these compounds showed neurological deꢂcits less
than diazepam.
Vehicle
–
12.5
75*
75*
62.5*
75*
25
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
0.725
0.74
0.74
0.805
0.805
0.825
0.775
0.875
0.76
0.79
0.79
0.85
0.875
0.825
0.925
0.5
25
Study on mechanism of action
62.5*
62.5*
87.5**
50
100***
25
75*
75*
75*
87.5**
To study the mechanism of action, eꢀect of ꢁumazenil as
an antagonist of BZD receptor on anticonvulsant activity
of compound 6h was evaluated. In this assay, ꢁumazenil
antagonized anticonvulsant activity of the most potent com-
pound 6h in the PTZ test. This ꢂnding conꢂrms that title
compounds can act as agonists for BZD receptors.
Docking study
Diazepam
a
Synthesized compounds and standard drug diazepam were used in
an equimolar dose
P<0.05; **P<0.01; ***P<0.001 compared to vehicle group
To study the interaction mode of the title compounds in the
BZD-binding pocket of GABA receptor (α1β2 γ2), a dock-
A
*
ing study was performed using Auto Dock Tools (version
1
.5.6) [19]. The superposed structure of diazepam and the
most potent compound 6h in the binding pocket is shown in
Fig. 3. The detailed binding mode of diazepam showed that
benzodiazepine moiety interacted with α1 Tyr159 (π–π) and
α1 Tyr 209 (π–π), and α1 Thr206 (hydrogen bond) (Fig. 4a).
Pendant phenyl group of this compound formed a π–π and
a π–anion interactions with γ2 Phe77 and α1 His101. Fur-
thermore, a weak hydrophobic interaction between chlorine
atom and α1 Val211 was also observed.
Anticonvulsant activity against MES‑induced
seizures
The target compounds 6a–o were also screened for their
anticonvulsant activities against maximal electroshock
(
MES)-induced seizure in mice [17]. Among the synthe-
sized compounds, cyclohexyl derivative 6k showed 100%
protection against MES-induced seizure while diazepam
The most active compound 6h established π interactions
with γ2Glu189 and α1 His101 through benzodiazepine and
(
(
standard drug) showed 87.5% protection in this assay
Table 2). Moreover, compound 6i showed protection
equal with diazepam. Other synthesized compounds exhib-
ited less protective percentage of diazepam in the MES-
induced seizure assay.
Table 3 Neurotoxicity evaluation of promising compounds 6h and 6k
Compound
Dose (mg/kg)a Rotarod test
No. of animals fall/
Muscle
no. of animals tested incoordina-
tion (%)
6
h
k
0.875
0.79
0.5
2/4
3/4
4/4
50
75
100
6
Diazepam
a
Synthesized compounds and diazepam were used in an equimolar
dose
Fig. 3 Diazepam (cyan) and most potent compound 6h (pink) super-
imposed in the BZD-binding pocket of GABA receptor (α1β2 γ2)
A
1
3

Molecular Diversity
Fig. 4 a The binding modes of diazepam and b compound 6h in the BZD-binding pocket of GABA receptor (α1β2 γ2)
A
Fig. 5 The binding mode of
compound 6e in the BZD-bind-
ing pocket of GABA receptor
A
(
α1β2 γ2)
1
,2,3-triazole moieties (Fig. 4b). A hydrogen bond between
residues—γ2Glu189, α1 His101, α1 Tyr159, α1 Tyr 209,
α1 Phe99, and γ2 Val190—while compound 6e displayed
interactions with four residues—γ2Glu189, α1 His101,
α1 Tyr159, and α1 Phe99—in the BZD-binding pocket. It
appears that the diꢀerence in the anticonvulsant activity of
these two compounds can be reasonably explained by two
additional interactions of compound 6h with BZD-binding
pocket (α1 Tyr 209 and γ2 Val190) in comparison with com-
pound 6e.
carbonyl unit attached to N-tert-butyl moiety and α1 Tyr159
was formed. Several π interactions were also observed
between pendant phenyl ring of compound 6h and BZD-
binding pocket residues α1 His101 and α1 Tyr 209. 3-Bromo
of pendant phenyl group and 9-chloro of benzodiazepine
moiety of this compound, interacted with α1 Phe99 and γ2
Val190, respectively.
The comparison of interaction modes of the most potent
compound 6h (Fig. 4b) with it 9-unsubstituented analog
6
e (Fig. 5) showed that compound 6h interacted with six
1
3

Molecular Diversity
Conclusion
General procedure for the synthesis of N‑(alkyl)‑2‑(9‑c
hloro‑6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzodiaz‑
epin‑5(6H)‑yl)‑2‑(substituted phenyl)acetamide (6a–o) 2-azi-
dobenzoic acids 2 (1 mmol), propargylamine 3 (1 mmol),
substituted benzaldehydes 4 (1 mmol), and isocyanides 5
(1 mmol) were reꢁuxed in MeOH (10 mL) for 24 h. Then,
the reaction mixture was poured into crushed ice, and the
precipitated products were ꢂltered and dried at 60 °C to
obtain pure compounds 6a–o (64–79%).
In conclusion, we presented design, synthesis, in vivo anti-
convulsant activity of a novel series of 1,2,3-triazolo-ben-
zodiazepine derivatives 6a–n. All synthesized compounds
6
a–o showed high anticonvulsant activity in the PTZ test,
comparable to or more than diazepam as the standard drug.
Among synthesized compounds, the 9-chloro-benzodiaz-
epin derivatives 6g, 6h, 6n, and 6o displayed more potent
activity than diazepam in the PTZ test. Among the synthe-
sized compounds, compounds 6k and 6i were most potent
compounds in MES test. The promising compounds 6h
and 6k showed neurological deꢂcits less than diazepam in
the rotarod test. The experimental investigation of action
of mechanism compound 6h by ꢁumazenil and docking
study of this compound showed this compound can act as
BZD receptor agonist.
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(4‑ꢀuorophenyl)acetamide (6a) White
−
1
solid; yield: 79%, mp 232–235 °C. IR (KBr) (νmax/cm ):
1
3302, 3061, 2965, 1673, 1611, 1546. H NMR (CDCl3,
500 MHz): δ = 1.33 (9H, s), 4.43 (1H, d, J = 15 Hz,
CHdiastrotopic), 4.67 (1H, d, J=15 Hz, CHdiastrotopic), 6.27 (1H,
s, CHChiral), 6.80 (1H, s, CHtriazole), 7.04 (2H, t, J=8.5 Hz),
7
.19–7.32 (2H, m), 7.54 (1H, td, J=7.7, 1.2 Hz), 7.67 (1H,
td, J = 7.8, 1.6 Hz), 7.94 (1H, d, J = 8.1 Hz), 8.08 (1H,
1
3
Experimental
dd, J = 7.9, 1.5 Hz), 8.13 (1H, s) ppm; C NMR (CDCl3,
25 MHz): δ = 28.58, 36.53, 28.58, 36.53, 52.11, 59.98,
116.00, 116.17, 122.57, 126.51, 128.84, 130.59, 130.86,
1
Chemistry
1
32.74, 133.15, 135.28, 138.05, 161.86, 163.84, 166.87,
Melting points of target compounds 6a–o were measured
167.22 ppm. Anal. Calcd for C22H22FN5O2 (407.44): C,
64.85; H, 5.44; N, 17.19. Found: C, 64.71; H, 5.63; N, 17.25.
by a Koꢁer hot stage apparatus and are uncorrected. A
1
Bruker FT-500 spectrometer was used to record H and
1
3
C NMR spectra, using CDCl3 as a solvent and TMS
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
as an internal standard. The IR spectra were obtained by
a Nicolet Magna FTIR 550 spectrometer (in KBr disks).
High-resolution mass spectra were determined with an
Agilent Technology (HP) mass spectrometer operating at
an ionization potential of 70 eV. All reagents and solvents
used in this study were purchased from Aldrich or Merck
Company without the requirement of any puriꢂcation.
diazepin‑5(6H)‑yl)‑2‑(2‑chlorophenyl)acetamide (6b) White
−
1
solid; yield: 78%, mp 273–275 °C. IR (KBr) (νmax/cm ):
1
3305, 3066, 2961, 1675, 1615, 1549. H NMR (CDCl3,
500 MHz): δ = 1.36 (9H, s), 4.42 (1H, d, J = 15 Hz,
CHdiastrotopic), 4.64 (1H, d, J = 15 Hz, CHdiastrotopic), 6.40
(1H, s, CHChiral), 6.91 (1H, s, CHtriazole), 7.30–7.34 (1H, m),
7.38–7.44 (2H, m), 7.55 (1H, t, J=7 Hz), 7.63–7.71 (2H,
m), 7.95(1H, d, J=8 Hz), 8.13 (1H, d, J=8 Hz), 8.24 (1H, s)
1
3
ppm; C NMR (CDCl , 125 MHz): δ=28.58, 36.89, 52.10,
3
General procedure for the synthesis of 2‑azidobenzoic acids 2
59.15, 122.59, 126.41, 127.13, 128.73, 130.32, 130.43,
1
30.56, 132.27, 132.91, 132.97, 133.04, 134.83, 166.09,
A suspension of 2-aminobenzoic acids 1 (21.78 mmol)
166.34 ppm. Anal. Calcd for C22H22ClN5O2 (423.9): C,
62.34; H, 5.23; N, 16.52. Found: C, 62.51; H, 5.48; N, 16.39.
in water (15 mL) and concentrated hydrochloric acid
(
5.55 mL) was cooled to − 5 °C. Then, a solution of
sodium nitrite (22.8 mmol) dissolved in water (4.5 mL)
was prepared and added dropwise to the suspension and
the resulting mixture was stirred for 30 min at − 5 °C.
The reaction mixture (without diazonium salt isolation)
was poured into a solution of sodium azide (24.5 mmol)
in water (4.5 mL) and ice (20 g), and a pale yellow pre-
cipitate formed immediately. The reaction mixture was set
aside overnight. Next, the precipitate was isolated by ꢂltra-
tion, washed with water and dried under reduced pressure
to provide compounds 2 (yield 93%).
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(4‑chlorophenyl)acetamide (6c) White
−
1
solid; yield: 76%, mp 242–243 °C. IR (KBr) (νmax/cm ):
1
3301, 3065, 2959, 1678, 1613, 1544. H NMR (CDCl3,
500 MHz): δ = 1.35 (9H, s), 4.46 (1H, d, J = 15 Hz,
CHdiastrotopic), 4.82 (1H, d, J = 15 Hz, CHdiastrotopic), 6.30
(1H, s, CHChiral), 6.77 (1H, s, CHtriazole), 7.17–7.30 (2H,
m), 7.35 (2H, d, J=8.0 Hz), 7.56 (1H, t, J=7.7 Hz), 7.69
(1H, t, J = 7.5, Hz), 7.96(1H, d, J = 8.1 Hz), 8.09 (1H, d,
13
J=7.9 Hz), 8.24 (1H, s) ppm; C NMR (CDCl3, 125 MHz):
δ = 28.57, 29.65, 36.59, 47.32, 62.12, 122.59, 126.44,
1
28.86, 129.24, 130.30, 132.72, 133.20, 135.01, 139.49,
1
3

Molecular Diversity
1
65.78, 166.92 ppm. Anal. Calcd for C H ClN O (423.9):
N‑(tert‑butyl)‑2‑(9‑chloro‑6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a]
[1,4]benzodiazepin‑5(6H)‑yl)‑2‑(4‑fluorophenyl)acetamide
(6g) White solid; yield: 67%, mp 298–300 °C. IR (KBr)
2
2
22
5
2
C, 62.34; H, 5.23; N, 16.52. Found: C, 62.47; H, 5.11; N,
1
6.65.
−
1
1
(
ν
/cm ): 3279, 3085, 1681, 2968, 1647, 1562. H NMR
max
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]ben‑
zodiazepin‑5(6H)‑yl)‑2‑(2,3‑dichlorophenyl)acetamide
(CDCl , 500 MHz): δ=1.35 (9H, s), 4.46 (1H, d, J=15 Hz,
3
CH
), 4.68 (1H, d, J=15 Hz, CH
), 6.25 (1H,
diastrotopic
diastrotopic
), 7.06 (2H, t, J=8.5 Hz),
triazole
(
(
(
6d) White solid; yield: 69%, mp 270–272 °C. IR (KBr)
s, CH
), 6.83 (1H, s, CH
Chiral
−
1
1
ν
/cm ): 3302, 3064, 2959, 1676, 1614, 1547. H NMR
7.22–7.31 (2H, m), 7.51 (1H, dd, J=8.5, 2.1 Hz), 7.99 (1H,
max
CDCl , 500 MHz): δ=1.34 (9H, s), 4.42 (1H, d, J=15 Hz,
d, J=2.1 Hz), 8.04 (1H, d, J=8.5 Hz), 8.20 (1H, s) ppm.
3
1
3
CH
), 4.78 (1H, d, J=15 Hz, CH
), 6.45 (1H,
C NMR (CDCl , 125 MHz): δ = 28.60, 36.44, 52.18,
3
diastrotopic
diastrotopic
), 7.37 (1H, t, J=7.9 Hz),
triazole
s, CH
), 6.97 (1H, s, CH
60.43, 116.07, 116.24, 119.12 122.57, 124.71, 129.08,
133.49, 134.20, 135.08, 139.36, 161.19, 163.16, 166.07,
166.57 ppm. Anal. Calcd for C H ClFN O (441.89): C,
Chiral
7
.49–7.63 (3H, m), 7.69 (1H, td, J=7.8, 1.6 Hz), 7.96 (1H,
d, J=7.5 Hz), 8.11 (1H, d, J=7.5 Hz), 8.20 (1H, s) ppm;
2
2
21
5
2
1
3
C NMR (CDCl , 125 MHz): δ = 28.54, 36.87, 49.20,
59.80; H, 4.79; N, 15.85. Found: C, 59.69; H, 4.57; N, 16.01.
3
5
1
1
5
9.26, 118.56, 119.07, 121.74, 123.13, 126.40, 128.41,
28.79, 131.28, 132.74, 132.80, 133.18, 136.99, 166.84,
67.59 ppm. Anal. Calcd for C H Cl N O (458.34): C,
N‑(tert‑butyl)‑2‑(9‑chloro‑6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a]
[1,4]benzodiazepin‑5(6H)‑yl)‑2‑(3‑bromophenyl)acetamide
(6h) White solid; yield: 66%, mp 209–211 °C. IR (KBr)
2
2
21
2
5
2
7.65; H, 4.62; N, 15.28. Found: C, 57.46; H, 4.39; N, 15.09.
−
1
1
(
ν
/cm ): 3278, 3083, 1682, 2969, 1645, 1564. H NMR
max
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzodi‑
azepin‑5(6H)‑yl)‑2‑(3‑bromophenyl)acetamide (6e) White solid;
(CDCl , 500 MHz): δ=1.37 (9H, s), 4.45 (1H, d, J=15 Hz,
3
CH
), 4.73 (1H, d, J=15 Hz, CH
), 6.25 (1H,
diastrotopic
diastrotopic
−1
yield: 68%, mp 207–209 °C. IR (KBr) (ν /cm ): 3300, 3064,
s, CH
), 6.90 (1H, s, CH
), 7.16–7.27 (2H, m), 7.46
triazole
max
Chiral
−1 1
2
967, 1674, 1612, 1544 cm . H NMR (CDCl , 500 MHz):
(1H, s), 7.51–7.56 (2H, m), 8.01 (1H, d, J=2.1 Hz), 8.07
3
1
3
δ=1.37 (9H, s), 4.27 (1H, d, J=15 Hz, CH
), 4.78 (1H,
(1H, d, J = 8.5 Hz), 8.20 (1H, s)ppm; C NMR (CDCl ,
diastrotopic
3
d, J=15 Hz, CH
), 6.30 (1H, s), 6.89 (1H, s), 7.15–
125 MHz): δ=28.59, 36.60, 52.26, 60.30, 118.96, 122.60,
123.13, 124.61, 127.50, 129.09, 130.60, 131.82, 132.22,
133.52, 134.20, 136.84, 139.43, 166.16, 166.84 ppm. Anal.
Calcd for C H BrClN O (502.79): C, 52.55; H, 4.21; N,
diastrotopic
7
.27 (2H, m), 7.48 (1H, s), 7.54 (1H, d, J=8 Hz), 7.58 (1H,
t, J=7.7 Hz), 7.71(1H, t, J=7.5 Hz), 7.99 (1H, d, J=8.0 Hz),
1
3
8
1
1
1
.05 (1H, s), 8.12 (1H, d, J=8.0 Hz). C NMR (CDCl ,
3
22 21
5
2
25 MHz): δ=28.59, 36.68, 48.22, 60.06, 118.78, 120.13,
22.61, 123.07, 126.40, 128.86, 130.56, 132.11, 132.74,
32.80, 133.21, 137.03, 166.97, 167.90. EIMS, m/z (%): 470
13.93. Found: C, 52.61; H, 4.35; N, 14.07.
N‑(cyclohexyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(4‑ꢀuorophenyl)acetamide (6i) White
+
81
+ 79
(M
Br, 9), 468 (M Br, 9), 368 (100), 339 (54), 261 (20),
−
1
1
85 (28), 171 (62), 155 (67), 130 (72), 115 (39), 57 (82), 41
solid; yield: 76%, mp 249–251 °C. IR (KBr) (ν /cm ):
max
1
(
25). Anal. Calcd for C H BrN O (468.35): C, 56.42; H,
3235, 3073, 2934, 2858, 1637, 1569. H NMR (CDCl ,
22
22
5
2
3
4
.73; N, 14.95. Found: C, 56.27; H, 4.91; N, 15.12.
500 MHz): δ = 1.00–1.99 (10H, m), 3.80 (1H, m), 4.36
(
1H, d, J = 15 Hz, CH
), 4.82 (1H, d, J = 15 Hz,
diastrotopic
N‑(tert‑butyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(4‑bromophenyl)acetamide (6f) White
CH
), 6.41 (1H, s, CH
), 6.76 (1H, s, CH
),
Chiral
diastrotopic
Chiral
6.87 (1H, d, J=8.0 Hz), 7.05 (2H, t, J=8.3 Hz), 7.16–7.34
−
1
solid; yield: 77%, mp 208–210 °C. IR (KBr) (ν /cm ):
(2H, m), 7.54 (1H, t, J = 7.7 Hz), 7.69(1H, t, J = 7.7 Hz),
max
1
13
3
5
275, 3089, 1683, 2966, 1644, 1564. H NMR (CDCl ,
7.97(1H, d, J= 8.1 Hz), 8.05(1H, d, J = 7.9 Hz) ppm;
C
3
00 MHz): δ = 1.37 (9H, s), 4.14 (1H, d, J = 15 Hz,
NMR (CDCl , 125 MHz): δ = 24.64, 24.66, 25.33, 32.61,
3
CH
), 4.73 (1H, d, J = 15 Hz, CH
), 6.26
diastrotopic
32.77, 36.57, 48.71, 59.65, 115.91, 116.08, 122.57, 126.44,
diastrotopic
(
1H, s, CH
), 6.75 (1H, s, CH
), 7.09–7.25 (2H,
triazole
128.77, 130.56, 132.61, 132.76, 133.14, 135.06, 161.83,
Chiral
+
m), 7.49–7.55 (2H, m), 7.58 (1H, t, J=7.7 Hz), 7.72(1H, t,
163.77, 166.92, 167.27 ppm. EIMS, m/z (%):434 (M , 11),
J=7.7 Hz), 7.98 (1H, d, J=8.1 Hz), 8.12 (1H, d, J=7.9 Hz),
334 (5), 308 (100), 279 (71), 235 (17), 206 (21), 155 (32),
130 (41), 109 (50), 83 (19), 55 (24), 41 (10). Anal. Calcd for
C H FN O (433.48): C, 66.50; H, 5.58; N, 16.16. Found:
1
3
8
3
1
1
.18 (1H, s) ppm; C NMR (CDCl , 125 MHz): δ=28.59,
3
6.61, 52.18, 60.16, 116.91, 118.02, 122.61, 126.41,
2
4
24
5
2
28.90, 130.62, 132.25, 132.75, 133.24, 139.39, 161.32,
C, 66.31; H, 5.62; N, 16.26.
+
81
+
66.93 ppm. EIMS, m/z (%): 470 (M
Br, 7), 468 (M
7
9
Br, 7), 368 (100), 339 (51), 260 (11), 186 (22), 171 (69),
N‑(cyclohexyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(2‑chlorophenyl)acetamide (6j) White
1
55 (41), 130 (53), 115 (27), 89 (18), 57 (53), 41 (19). Anal.
−
1
Calcd for C H BrN O (468.35): C, 56.42; H, 4.73; N,
solid; yield: 75%, mp 240–242 °C. IR (KBr) (ν /cm ):
2
2
22
5
2
max
1
1
4.95. Found: C, 56.22; H, 4.87; N, 15.06.
3233, 3075, 2937, 2861, 1636, 1565. H NMR (CDCl ,
3
1
3

Molecular Diversity
5
4
00 MHz): δ = 1.00–2.02 (10H, m), 3.83 (1H, m), 4.32–
.75 (2H, m, CH2 ), 6.45 (1H, s, CH ), 6.83
CH
), 6.43 (1H, s, CH
), 6.78(1H, s, CH
),
triazole
diastrotopic
Chiral
7.10–7.24 (3H, m),7.45 (1H, s), 7.50 (1H, d, J = 7.7 Hz),
diastrotopic
Chiral
(
1H, s, CH
), 7.03 (1H, d, J = 8 Hz), 7.29–7.36 (1H,
7.53(1H, t, J = 7.7 Hz), 7.69(1H, t, J = 7.7 Hz), 7.96 (1H,
triazole
1
3
m), 7.37–7.44 (2H, m), 7.56 (1H, td, J=7.7, 1.2 Hz), 7.64
d, J = 8.1 Hz), 8.02 (1H, d, J = 7.9 Hz) ppm; C NMR
(
1H, dd, J = 7.5, 2.6 Hz), 7.70(1H, td, J = 7.8, 1.6 Hz),
(CDCl , 125 MHz): δ=24.65, 25.34, 32.60, 32.75, 36.74,
3
1
3
7
.96(1H, d, J = 8 Hz), 8.12(1H, d, J = 7.5 Hz) ppm;
C
48.75, 59.76, 119.95, 122.61, 123.03, 126.34, 127.47,
128.80, 130.52, 131.67, 132.06, 132.65, 132.80, 133.22,
136.99, 162.29, 167.04 ppm. Anal. Calcd for C H BrN O
NMR (CDCl , 125 MHz): δ = 24.62, 24.67, 25.36, 32.63,
3
3
1
1
6
2.70, 48.05, 59.79, 119.55, 121.06, 122.61, 126.43, 127.13,
28.77, 130.41, 130.56, 132.08, 132.91, 133.07, 165.81,
66.33 ppm. Anal. Calcd for C H ClN O (449.93): C,
24
24
5
2
(494.38): C, 58.31; H, 4.89; N, 14.17. Found: C, 58.52; H,
4.78; N, 14.05.
2
4
24
5
2
4.07; H, 5.38; N, 15.57. Found: C, 63.91; H, 5.22; N, 15.73.
N‑(cyclohexyl)‑2‑(9‑chloro‑6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a]
[1,4]benzodiazepin‑5(6H)‑yl)‑2‑(4‑fluorophenyl)acetamide
(6n) White solid; yield: 69%, mp 239–241 °C. IR (KBr)
N‑(cyclohexyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(4‑chlorophenyl)acetamide (6k) White
−
1
−1
1
solid; yield: 79%, mp 248–250 °C. IR (KBr) (ν /cm ):
(ν /cm ): 3234, 3085, 2931, 2865, 1633, 1567. H NMR
max
max
1
3
5
234, 3086, 2930, 2857, 1638, 1569. H NMR (CDCl ,
(CDCl , 500 MHz): δ=1.02–1.99 (10H, m), 3.68–3.92 (1H,
3
3
00 MHz): δ = 1.01–2.02 (10H, m), 3.82 (1H, m), 4.42
m), 4.32–4.70 (2H, m, CH
), 6.27 (1H, s, CH
),
Chiral
2
diastrotopic
(
1H, d, J = 15 Hz, CH
), 4.88 (1H, d, J = 15 Hz,
6.86 (1H, s, CH
), 7.06 (2H, t, J =8.4 Hz), 7.20–7.25
triazole
diastrotopic
CH
), 6.36 (1H, s, CH
), 6.83 (1H, s, CH ),
triazole
(2H, m), 7.52 (1H, dd, J=8.8, 2.0 Hz), 7.97–8.02 (1H, m),
diastrotopic
Chiral
1
3
7
.11 (1H, d, J=7.9 Hz), 7.19–7.30 (2H, m), 7.32–7.43 (2H,
8.06 (1H, d, J=8.5 Hz), 8.23 (1H, d, J=2.5 Hz) ppm; C
m), 7.57 (1H, t, J=7.7 Hz), 7.71(1H, t, J=7.7 Hz), 7.99(1H,
NMR (CDCl , 125 MHz): δ = 24.67, 25.34, 32.69, 32.84,
3
1
3
d, J=8 Hz), 8.10(1H, d, J=8 Hz) ppm; C NMR (CDCl ,
36.56, 48.83, 62.20, 116.05, 116.22, 122.59, 124.66, 129.08,
133.51, 134.08, 134.19, 135.05, 139.40, 160.33, 162.34,
166.10, 167.14 ppm. Anal. Calcd for C H ClFN O
3
1
5
1
25 MHz): δ = 24.66, 25.33, 32.63, 32.81, 36.64, 48.76,
9.73, 119.06, 122.62, 126.37, 128.84, 129.23, 130.27,
32.66, 132.78, 133.23, 134.98, 164.64, 166.98 ppm. EIMS,
2
4
23
5
2
(467.92): C, 61.60; H, 4.95; N, 14.97. Found: C, 61.56; H,
+
37
+ 35
m/z (%): 452 (M Cl, 7), 450 (M Cl, 25), 350 (9), 324
4.81; N, 15.06.
(
(
100), 295 (67), 155 (43), 125 (59), 98 (12), 83 (18), 55
21), 41 (16). Anal. Calcd for C24H24ClN5O2 (449.93): C,
N‑(cyclohexyl)‑2‑(9‑chloro‑6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a]
[1,4]benzodiazepin‑5(6H)‑yl)‑2‑(4‑bromophenyl)acetamide
6
4.07; H, 5.38; N, 15.57. Found: C, 64.26; H, 5.45; N, 15.73.
(
6o) White solid; yield: 65%, mp 277–279 °C. IR (KBr)
max
−
1
1
N‑(cyclohexyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzodi‑
azepin‑5(6H)‑yl)‑2‑(2,3‑dichlorophenyl)acetamide (6l) White
(ν /cm ): 3233, 3081, 2932, 2866, 1637, 1564. H NMR
(CDCl , 500 MHz): δ=1.00–2.03 (10H, m), 3.81 (1H, m),
3
−
1
solid; yield: 70%, mp 241–243 °C. IR (KBr) (ν /cm ):
4.25 (1H, d, J=15 Hz, CH
), 4.90 (1H, d, J=15 Hz,
), 6.76 (1H, d, J=3 Hz),
max
diastrotopic
1
3
5
233, 3084, 2931, 2859, 1635, 1566. H NMR (CDCl ,
CH
), 6.29 (1H, s, CH
diastrotopic Chiral
3
00 MHz): δ = 1.01–1.94 (10H, m), 3.80 (1H, m), 4.46
6.94 (1H, s, CH
), 7.17 (2H, d, J=8.1 Hz), 7.51–7.54
triazole
1
3
(
1H, d, J = 15 Hz, CH
), 4.62 (1H, d, J = 15 Hz,
(3H, m), 8.03–8.06 (2H, m) ppm; C NMR (CDCl ,
diastrotopic
3
CH
CH
), 6.29 (1H, d, J = 3 Hz, CH
), 6.81(1H, s,
Chiral
125 MHz): δ = 24.98, 25.33, 32.68, 32.86, 50.29, 61.08,
122.84, 126.37, 128.62, 128.96, 129.99, 132.32, 132.66,
133.23, 135.08, 139.49, 162.12, 166.69 ppm. Anal. Calcd
for C H BrClN O (528.83): C, 54.51; H, 4.38; N, 13.24.
diastrotopic
), 7.15 (1H, d, J=7.9 Hz) 7.35(1H, t, J=7.9 Hz),
triazole
7
.50–7.61 (3H, m), 7.70 (1H, t, J= 7.5 Hz), 7.97 (1H, d,
1
3
J=8.1 Hz), 8.08(1H, d, J=7.9 Hz) ppm; C NMR (CDCl ,
3
24 23
5
2
1
4
1
1
25 MHz): δ = 24.60, 24.65, 25.33, 32.53, 32.64, 36.87,
8.89, 59.46, 118.34, 122.68, 126.27, 127.50, 128.50,
28.79, 131.18, 132.77, 132.88, 133.18, 134.45, 134.69,
37.28, 166.47, 167.39 ppm. Anal. Calcd for C H Cl N O
Found: C, 54.69; H, 4.55; N, 13.12.
Anticonvulsant activity
24
23
2
5
2
(
484.38): C, 59.51; H, 4.79; N, 14.46. Found: C, 59.33; H,
Animals and drugs Male mice (Pasteur Institute of Iran)
weighing 24–30 g were used as experimental animals. The
animals were housed in a temperature-controlled room
(22± 1 °C) on a 12-h light/dark cycle with free access to
water and food for a 24-h period before testing, except dur-
ing the experiment. Mice were assigned to experimental
groups randomly, and each animal was used only once for
the experiments. Diazepam (Sigma) was used as a reference
drug, and pentylenetetrazole (PTZ, Sigma) was applied to
4
.48; N, 14.57.
N‑(cyclohexyl)‑2‑(6‑oxo‑4H‑[1,2,3]triazolo[1,5‑a][1,4]benzo‑
diazepin‑5(6H)‑yl)‑2‑(3‑bromophenyl)acetamide (6m) White
−
1
solid; yield: 67%, mp 211–213 °C. IR (KBr) (ν /cm ):
max
1
3
5
231, 3084, 2933, 2862, 1632, 1563. H NMR (CDCl ,
3
00 MHz): δ = 0.92–2.03 (10H, m), 3.79 (1H, m), 4.47
(
1H, d, J = 15 Hz, CH
), 4.78 (1H, d, J = 15 Hz,
diastrotopic
1
3

Molecular Diversity
induce convulsions in mice. PTZ was dissolved in physi-
ological saline solution, and diazepam and synthesized
compounds 6a–o were dispersed in carboxymethyl cellulose
Auto dock Tools (version 1.5.6), using previously described
method [19].
Acknowledgements This work was supported by Grants from the
National Institute for Medical Research Development (NIMAD) (Grant
Number: 971149).
(
CMC, 0.5%) [16].
All procedures were carried out in accordance with the
institutional guidelines for animal care and use that are in
compliance with the National Institutes of Health guide for
the care and use of Laboratory animals (NIH Publications
No. 8023, revised 1978). Ethical approval ID from ethics
committee for involving animals in this work is IR.NIMAD.
REC.1397.111.
Compliance with ethical standards
Conflict of interest The authors have declared no conꢁict of interest.
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