Microwave-assisted synthesis of 11-substituted-3,3-dimethyl-2,3,4,5,10,11-hexahydrodibenzo[b,e][1,4]diazepin-1-one derivatives catalysed by silica supported fluoroboric acid as potent antioxidant and anxiolytic agents

Article abstract of DOI:10.1007/s00044-019-02447-w

Keeping in view the successive attributes of benzodiazepines on the central nervous system, we have synthesized a novel series of benzodiazepine derivatives as antioxidant and anxiolytic agents. All the compounds were obtained in good yield by facile synt

Full text of DOI:10.1007/s00044-019-02447-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02447-w
ORIGINAL RESEARCH
Microwave-assisted synthesis of 11-substituted-3,3-dimethyl-
2,3,4,5,10,11-hexahydrodibenzo[b,e][1,4]diazepin-1-one derivatives
catalysed by silica supported fluoroboric acid as potent antioxidant
and anxiolytic agents
Kavita Bhagat1 Atamjit Singh1 Suruchi Dhiman1 Jatinder Vir Singh1 Ramandeep Kaur2 Gurinder Kaur1
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Harmandeep Kaur Gulati1 Palwinder Singh3 Raman Kumar1 Rajan Salwan1 Kajal Bhagat1 Harbinder Singh
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Sahil Sharma1 Preet Mohinder Singh Bedi1
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Received: 13 July 2019 / Accepted: 14 September 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Keeping in view the successive attributes of benzodiazepines on the central nervous system, we have synthesized a novel
series of benzodiazepine derivatives as antioxidant and anxiolytic agents. All the compounds were obtained in good yield by
facile synthesis. To check their antioxidant potential, DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay
was performed. Among all the synthetics seven derivatives (SD-3, SD-5, SD-7, SD-14, SD-18, SD-19, and SD-20) exhibited
IC50 values ranging from 76 to 489 nM. These seven compounds were further evaluated to check their binding abilities
towards the benzodiazepine binding site on GABAA receptors. Three best-fit ligands (SD-3, SD-14 and SD-20) were further
evaluated for their anxiolytic effect by using three in vivo mice models i.e. Elevated Plus Maze, Light & Dark box, and
Mirror Chamber model. The study revealed that compounds SD-3 and SD-20 showed the best anxiolytic effect as compared
to standard drug diazepam even at an oral dose of 1.0 mg/kg.
These authors contributed equally: Kavita Bhagat, Atamjit Singh
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02447-w) contains supplementary
material, which is available to authorized users.
* Harbinder Singh
1
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar, Punjab 143005, India
singh.harbinder40@gmail.com
* Sahil Sharma
2
University Institute of Pharmaceutical Sciences, Panjab
ss.gq2009@gmail.com
University, Chandigarh 160014, India
* Preet Mohinder Singh Bedi
3
Department of Chemistry, Guru Nanak Dev University,
Amritsar, Punjab 143005, India
bedi_preet@yahoo.com

Medicinal Chemistry Research
Graphical Abstract
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Keywords Benzodiazepines Antioxidant activity Molecular modelling studies In silico studies In vivo anxiolytic effect
Introduction
physiological condition of high concentration of ROS i.e
free radicals, that cause molecular damage to cellular
structures. These free radicals are associated with the
development of various chronic disease conditions like
cancer, degenerative and neurodegenerative diseases, heart-
related complications, as well as can be involved in aging
process (Ko et al. 2010), mental disorders (Dean et al. 2009;
Salustri et al. 2010), chronic inflammation, arthritis,
decrease the function of immune system (Pande and Akoh
2009), fibrosis, cirrhosis, and atherosclerosis (Roesler et al.
2007). Therefore, the compounds having the antioxidant
potential could be beneficial to treat these disease
conditions.
Benzodiazepines are the elite class of heterocyclic
compounds that exhibited a wide range of pharmacological
activities and are also considered as one of the most
explored classes as ‘privileged scaffolds’ in both drug dis-
covery and medicinal chemistry (Evans et al. 1988; Pas-
quale et al. 2013; Horton et al. 2003). In contrast to this,
controversy still continues with the use of benzodiazepines
due to the high risk of overdose and long-term risk of
dementia (Gage et al. 2015; Gray et al. 2016). Nevertheless,
they are still recommended for both short-term and long-
term treatment of anxiety due to its strong and better effi-
cacy than other anxiolytic agents (Krystal et al. 2015). 1,4-
benzodiazepine (psychoactive drugs) involves the aug-
mentation of the inhibitory activity of the gamma-
aminobutyric acid (GABA) neurotransmitter at the GABA
receptor, further resulting in therapeutic properties like
sedation, anxiolytic, alcohol withdrawal, anticonvulsant,
Anxiety is a human emotion, commonly experienced by
every person in their lives in unique ways such as when the
problem is occurring during work, before going for exams
and on making important and major decisions (https://www.
nimh.nih.gov/health/topics/anxiety-disorders/index.shtml).
According to the World Health Organisation (WHO) report
2015, it has been found that the rate of the global population
that suffered from an anxiety disorder is estimated to be
3.6%, out of which females were found more prone as
compared to males (http://ghdx.healthdata.org/gbd-results-
tool). Patient suffering from anxiety disorder shows avoid-
ance of perceived threats from the environment as well as
from the internal body (Craske et al. 2009). Pharmacologi-
cally, anxiety disorder mainly associated with the dysfunction
of various monoamine neurotransmitters (gamma-aminobu-
tyric acid (GABA), dopamine, 5-Hydroxytryptamine (ser-
otonin), and norepinephrine in the central nervous system)
(Nash and Nutt 2004; Nutt and Malizia 2001). There are
numerous effective psychological (cognitive behavioural
therapy, CBT) and pharmacological treatments. Various
antidepressants, serotonin noradrenaline-reuptake inhibitors,
selective serotonin-reuptake inhibitors, and benzodiazepines
are available for the treatment of anxiety.
There is a well-known relation between the increase in
oxidative stress and anxiety in rodents (Oliveira et al. 2007;
Masood et al. 2008; Salim et al. 2011). It has been reported
that elevation in oxidative stress in mice hypothalamus
leads to anxiety. Oxidative stress is a cellular or

Medicinal Chemistry Research
and muscle relaxation (Wang et al. 2017). They have
acquired a great reputation in medicinal chemistry for their
broad biological and pharmacological activities (Cortes
et al. 2007; Makaron et al. 2013). These are broadly pre-
scribed psychotropics due to their exceptional CNS
depressant activity (Michelini et al. 1996; Ookura et al.
2008). Diazepam and chlordiazepoxide had set a great
example in the class of antianxiety drugs. Further mod-
ifications and refinements in these heterocycles have been
made and the anxiolytic effect of benzodiazepines has been
evaluated. Moreover, their structural significance and the
practicality of these organic compounds as anxiolytics have
achieved a distinct standard impression in the field of
chemistry as a privileged structure. It has also been revealed
that benzodiazepines are generally unstable and thus form
toxic metabolites in the body. In this quest, researchers
around the globe are making continuous efforts for the
development of new benzodiazepine compounds with lesser
side effects (Skopp et al. 1998; Robertson and Drummer
1998; Coassolo and Aubert 1985). In the recent past,
numerous benzodiazepines were approved by Food & Drug
Administration (FDA) with their improved efficacy as well
as with fewer side effects (Manchester et al. 2017) which
include Remimazolam (Wesolowski et al. 2016), 3-
hydroxyphenazepam (Garibova and Voronina 1986), 4-
chlorodiazepam (Paradis et al. 2013), Adinazolam (Ven-
katakrishnan et al. 2005), Clonazepam (McLean and Mac-
donald 1988), Cloniprazepam (Mortele et al. 2018),
Desalkyfurazepam (Manchester et al. 2018), Des-
chloroetizolam (El-Balkhi et al. 2017), Desmethyl-
flunitrazeapm (fonazepam) (Katselou et al. 2017),
Diclazepam (Moosmann et al. 2014), Etizolam (Connor
et al. 2016), Flubromazepam (Sternbach et al. 1962), Flu-
bromazolam (Lukasik et al. 2016), Phenazepam (Maskell
et al. 2014), Pyrazolam (Moosmann et al. 2013), Nifoxipam
(Katselou et al. 2017), Meclonazepam (Menezes et al. 2012)
(Fig. 1). The seven-membered ring in benzodiazepines has
been found to be responsible for their pharmacological
activities and also found to display excellent binding ability
towards the GABA receptors, due to their conformational
enantiomeric property (Constantinos et al. 2010; Zizhao
et al. 2012). Since 1977, benzodiazepines have been used
globally as the most prescribed medications. Therefore,
these derivatives are the compounds of interest among the
researchers around the globe. Inspired from these findings,
we also have synthesized a novel series of benzodiazepine
compounds for the treatment of anxiety. All the synthesized
compounds were preliminarily tested for their antioxidant
potential by using DPPH assay. Furthermore, the binding
ability of the synthesized compounds towards the benzo-
diazepine binding site on the GABAA receptor was also
determined. The molecules that displayed greater binding
potential with the GABAA receptors were further tested
in vivo to check their anxiolytic effect in mice by using
three models, Elevated Plus Maze model (EPM), Mirror
Chamber model and Light and Dark model.
Experimental
Chemistry
Procurement of the reagents was done from Sigma-
Aldrich, CDH, and Loba, India and used without further
purification. Purity of the reagents was evaluated by
1
comparison with authentic samples and by H NMR and
13C NMR. Avance III HD 500 MHz Bruker Biospin NMR
Spectrometer was used to record the NMR spectras. The
spectra were recorded in DMSO-d6 and CDCl3 relative to
TMS (0.00 ppm). In 1H NMR chemical shifts were
reported in δ values using tetramethyl silane as an internal
standard with a number of protons, multiplicities (s-sing-
let, d-doublet) and coupling constants (J) in Hz in the
solvent indicated. Melting points were determined in open
capillaries and were uncorrected.
Procedure for the preparation of silica adsorbed
tetrafluoroboric acid (HBF4-SiO2)
40% aqueous solution of HBF4 (1.65 g) was added to the
suspension of 13.35 g of silica gel (230–400 mesh) in
40 mL of diethyl ether. The mixture was concentrated and
the residue thus obtained was dried under vacuum for 72 h
at 100 °C to afford 0.5 mmolg−1 HBF4–SiO2.
Procedure for the synthesis of benzodiazepine analogues
In a 50 mL conical flask o-phenylenediamine (1 mmol),
dimedone (2 mmol) benzaldehyde (1 mmol) and a catalytic
amount of silicate fluoroboric acid (5 mol %) was added.
The resulting mixture was placed into the microwave
reactor and exposed to microwave radiation in a microwave
synthesizer operating at 150 °C with the maximum micro-
wave power of 400 W. After the completion of the reaction,
the crude mixture was dissolved in methanol and adsorbed
on silica (60–120#). The desired product was purified by
column chromatography with an increasing percentage of
ethyl acetate in hexane as eluting solvent.
Rest of the compounds were synthesized by the above-
mentioned procedure. The characterization data for all the
synthesized compounds are given below.
11-phenyl-3,3-dimethyl-2,3,4,5,10,11-hexahydrodibenzo[b,
e][1,4]diazepin-1-one (SD-1) Yellowish solid; Yield 85%,
mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz, δ): 1.05–1.11
(6H, m, -CH3), 2.16–2.25 (2H, m, -CH2-), 2.46–2.58 (2H,

Medicinal Chemistry Research
Cl
A
N
N
O
N
H
N
N
N
N
Cl
OH
O
N
N
N
N
Br
Br
N
O
Cl
Cl
N
O
CH3
4-chlorodiazepam
Remimazolam
3-hybdroxyphenazepam
Adinazolam
O
H
O
F
H
N
O
N
N
S
N
N
N
O
N
N
N
O
Cl
N
O2N
Cl
Cl
N
Clonazepam
N-Desalkylflurazepam
Deschloroetizolam
Cloniprazepam
H
N
O
N
O
H
N
S
N
N
N
NH
Cl
N
Cl
N
F
N
Cl
Br
Cl
Etizalom
Desmethylflunitrazepam
N
N
N
Diclazepam
O
Flubromazepam
O
N
O
H
N
H
N
H
N
N
N
OH
O
O
N
Br
N
N
N
N
O
N
O
Br
N
Br
Cl
F
F
Cl
N
Nifoxipam
Flubromazolam
Meclonazepam
Phenazepam
Pyrazolam
B
O
N
R
H
N
O
N
Cl
N
H
Targeted compounds
R = 3,4-dichlorophenyl (SD-3)
R = Naphthyl (SD-20)
Diazepam
Anxiolytic agent
Active at 2 mg/Kg
Potent anxiolytic agents than Diazepam
Active at 1mg/Kg
Fig. 1 a New benzodiazepine-based drugs for the treatment of various CNS disorders; b Comparison of anxiolytic effect of targeted compounds
with the existed benzodiazepine derivative (Diazepam)
11-(4-Chlororphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
m, -CH2-), 5.87 (1H, s, -CH-), 6.48 (1H, s, -CH-),
6.66–6.69 (2H, m, ArH), 6.93–6.95 (2H, m, ArH),
7.05–7.09 (2H, m, ArH), 7.18 (2H, m, ArH), 7.48 (1H, s,
dibenzo[b,e][1,4]diazepin-1-one (SD-2) Yellowish solid;
1
Yield 85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz,
ArH). 13C NMR (CDCl3, 125 MHz, δ): 27.7, 28.8, 32.2,
δ): 1.06–1.12 (6H, m, -CH3), 2.15–2.26 (2H, m, -CH2-),
2.43–2.53 (2H, m, -CH2-), 5.85 (1H, s, -CH-), 6.41 (1H, bs,
45.8, 49.7, 57.5, 110.4, 120.3, 121.3, 121.3, 121.4, 126.8,
-NH-), 6.69–6.72 (2H, m, ArH), 6.86 (1H, m, ArH),
127.0, 128.6, 135.1, 136.9, 146.4, 193.7 (C=O). Anal.
6.92–6.94 (2H, m, ArH), 7.13 (1H, m, ArH), 7.21 (1H, s,
Calcd. for C21H21ClN2O: C, 71.48; H, 6.00; Cl, 10.05; N,
ArH), 7.28 (1H, m, ArH), 7.47 (1H, s, ArH). 13C NMR
7.94; O, 4.53; Found: C, 71.41; H, 5.97; Cl, 10.35; N, 7.54.

Medicinal Chemistry Research
(CDCl3, 125 MHz, δ): 27.7, 28.8, 32.2, 45.8, 49.7, 57.5,
110.4, 120.3, 121.3, 121.3, 121.4, 123.7, 125.7, 129.4,
129.5, 130.4, 131.1, 136.9, 146.4, 154.3, 193.7 (C=O).
Anal. Calcd. for C21H21ClN2O: C, 71.48; H, 6.00; Cl,
10.05; N, 7.94; O, 4.53; Found: C, 71.54; H, 5.99; Cl,
10.32; N, 7.56.
δ): 1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H, m, -CH2-),
2.42–2.50 (2H, m, -CH2-), 5.87 (1H, s, -CH-), 6.40 (1H, bs,
-NH-), 6.69–6.73 (2H, m, ArH), 6.85–6.86 (2H, m, ArH),
6.98–7.03 (3H, m, ArH), 7.08 (1H, m, ArH), 7.14 (1H, m,
ArH). 13C NMR (CDCl3, 125 MHz, δ): 27.7, 28.7, 32.2,
45.8, 49.7, 57.5, 110.4, 120.3, 121.3, 121.3, 121.4, 123.7,
125.7, 129.4, 129.5, 130.4, 131.1, 136.9, 146.4, 160.3,
193.7 (C=O). Anal. Calcd. for C21H21FN2O: C, 74.98; H,
6.29; F, 5.65; N, 8.33; O, 4.76; Found: C, 74.87; H, 6.32; F,
5.22; N, 8.68.
11-(3,4-dichlororphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahy-
drodibenzo[b,e][1,4]diazepin-1-one (SD-3) Yellowish solid;
Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz, δ):
1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H, m, -CH2-),
2.42–2.50 (2H, m, -CH2-), 5.91 (1H, s, -CH-), 6.69–6.72 (2H,
m, ArH), 6.76–6.80 (2H, m,. ArH), 6.98–7.01 (2H, m, ArH),
7.12–7.14 (1H, d, J = 7.5 Hz, ArH), 7.28–7.31 (2H, m, ArH).
13C NMR (CDCl3, 125 MHz, δ): 27.7, 28.8, 32.2, 45.8, 49.7,
57.5, 110.4, 120.3, 121.3, 121.3, 121.5, 126.5, 128.2,
128.6, 131.2, 131.4, 133.2, 135.1, 136.9, 146.4, 193.7
(C=O). Anal. Calcd. for C21H20Cl2N2O: C, 65.12; H,
5.20; Cl, 18.31; N, 7.23; O, 4.13; Found: C, 65.04; H, 5.25;
Cl, 17.91; N, 7.41.
11-(4-Nitrophenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydrodi-
benzo[b,e][1,4]diazepin-1-one (SD-7) Yellowish solid;
1
Yield 95%, mp: 194–195 °C, H NMR (CDCl3, 500 MHz,
δ): 1.18–1.28 (6H, m, -CH3), 2.36–2.38 (2H, m, -CH2-),
2.69–2.75 (2H, m, -CH2-), 6.02 (1H, s, -CH-), 6.65–6.78
(2H, m, ArH), 7.11 (1H, m, ArH), 7.45 (1H, m, ArH), 8.05
(2H, d, J = 8.9 Hz, ArH), 8.94 (2H, m, ArH). 13C
NMR (CDCl3, 125 MHz, δ): 32.7, 33.5, 36.9, 49.2, 54.6,
61.4, 114.2, 125.3, 125.50, 125.87, 127.7, 127.8,
128.2, 133.2, 133.3, 136.1, 142.6, 150.8, 157.7, 160.3,
197.7 (C=O). Anal. Calcd. for C21H21N3O3: C, 69.41;
H, 5.82; N, 11.56; O, 13.21; Found: C, 69.48; H, 5.75;
N, 11.75.
11-(4-Bromophenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-4) Yellowish solid;
Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3,500 MHz, δ):
1.07–1.14 (6H, m, -CH3), 2.15–2.22 (2H, m, -CH2-),
2.42–2.50 (2H, m, -CH2-), 5.85 (1H, s, -CH-), 6.42 (1H, bs,
-NH-), 6.68–6.70 (2H, m, ArH), 6.83 (1H, m, ArH),
6.93–6.95 (2H, m, ArH), 7.12 (1H, m, ArH), 7.20 (1H, s,
ArH), 7.28 (1H, m, ArH), 7.45 (1H, bs, ArH). 13C NMR
(CDCl3, 125 MHz, δ): 27.7, 28.7, 32.2, 45.8, 49.7, 57.5,
110.4, 120.3, 121.2, 121.2, 121.4, 123.7, 125.7, 129.4, 129.5,
130.4, 131.1, 136.9, 146.4, 154.3, 193.7 (C=O). Anal. Calcd.
for C21H21BrN2O: C, 63.48; H, 5.33; Br, 20.11; N, 7.05; O,
4.03; Found: C, 63.54; H, 5.21; Br, 20.35; N, 6.69.
11-(4-methylphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-8) Yellowish solid;
1
Yield 85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz,
δ): 1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H, m, -CH2-),
2.42–2.50 (2H, m, -CH2-), 5.86 (1H, s, -CH-), 6.42 (1H, bs,
-NH-), 6.68–6.71 (2H, m, ArH), 6.86–6.88 (2H, m, ArH),
6.96–6.99 (2H, m, ArH), 7.10–7.11 (2H, d, J = 6.5 Hz, ArH),
7.13–7.14 (2H, d, J = 6.5, ArH). 13C NMR (CDCl3,
125 MHz, δ): 24.3, 27.7, 28.7, 32.2, 45.8, 49.7, 57.5, 110.4,
120.3, 121.3, 121.3, 121.4, 123.7, 125.7, 129.4, 129.5, 130.4,
131.1, 136.9, 146.4, 193.7 (C=O). Anal. Calcd. for
C22H24N2O: C, 79.48; H, 7.28; N, 8.43; O, 4.81; Found: C,
79.41; H, 7.34; N, 8.02.
11-(3-Bromophenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-5) Yellowish solid;
1
Yield 85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz,
δ): 1.04–1.11 (6H, m, -CH3), 2.17–2.28 (2H, m, -CH2-),
2.46–2.58 (2H, m, -CH2-), 5.84 (1H, m, -CH-), 6.40 (1H, m,
ArH), 6.69 (1H, m, ArH), 6.83 (1H, m, ArH), 6.94 (1H, m,
ArH), 7.12 (1H, m, ArH), 7.20 (1H, m, ArH), 7.28 (1H, m,
ArH), 7.45 (1H, m, ArH). 13C NMR (CDCl3, 125 MHz, δ):
20.9, 27.7, 28.7, 30.8, 32.2, 45.8, 49.7, 57.5, 110.4, 120.3,
121.2, 121.4, 123.7, 125.7, 129.5, 130.4, 146.4, 154.2,
193.7 (C=O). Anal. Calcd. for C21H21BrN2O: C, 63.48; H,
5.33; Br, 20.11; N, 7.05; O, 4.03; Found: C, 63.41; H, 5.38;
Br, 20.32; N, 6.65.
11-(4-methoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahy-
drodibenzo[b,e][1,4]diazepin-1-one (SD-9) Yellowish solid;
Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz, δ):
1.07–1.11 (6H, m, -CH3), 2.12–2.22 (2H, m, -CH2-),
2.41–2.49 (2H, m, -CH2-), 5.90 (1H, s, -CH-), 6.44 (1H, bs,
-NH-), 6.68–6.72 (2H, m, ArH), 6.84–6.86 (2H, m, ArH),
6.99–7.03 (2H, m, ArH), 7.01–7.02 (2H, d, J = 6.5 Hz,
ArH), 7.14–7.15 (2H, d, J = 6.5, ArH). 13C NMR (CDCl3,
125 MHz, δ): 27.7, 28.7, 32.2, 45.8, 49.7, 57.5, 61.1,
110.4, 120.3, 121.3, 121.3, 121.4, 123.7, 125.7, 129.4, 129.5,
130.4, 131.1, 136.9, 160.7, 193.7 (C=O). Anal. Calcd.
for C22H24N2O2: C, 75.83; H, 6.94; N, 8.04; O, 9.18; Found:
C, 75.89; H, 6.84; N, 7.69.
11-(4-Fluorophenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-6) Yellowish solid;
1
Yield 85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz,

Medicinal Chemistry Research
11-(3,4-dimethoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexa-
hydrodibenzo[b,e][1,4]diazepin-1-one (SD-10) Yellowish
solid; Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3,
500 MHz, δ): 1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H, m,
-CH2-), 2.42–2.50 (2H, m, -CH2-), 3.73 (6H, s, -OCH3),
5.86 (1H, s, -CH-), 6.46 (1H, bs, -NH-), 6.68–6.72 (2H, m,
ArH), 6.85–6.88 (2H, m, ArH), 7.01–7.03 (3H, m, ArH)
7.09 (1H, m, ArH). 13C NMR (CDCl3, 125 MHz, δ): 27.7,
28.7, 32.2, 45.8, 49.7, 57.5, 61.1, 61.5, 110.4, 118.3, 121.3,
121.3, 121.5, 123.7, 125.7, 125.8, 129.4, 133.4, 134.5,
136.9, 152.6, 155.6, 193.7 (C=O). Anal. Calcd. for
C23H26N2O3: C, 72.99; H, 6.92; N, 7.40; O, 12.68; Found:
C, 73.11; H, 6.98; N, 7.70
C22H24N2O2: C, 75.83; H, 6.94; N, 8.04; O, 9.18; Found: C,
75.77; H, 7.05; N, 7.85.
11-(3-methoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexa-
hydrodibenzo[b,e][1,4]diazepin-1-one (SD-14) Yellow-
ish solid; Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3,
500 MHz, δ): 1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H,
m, -CH2-), 2.42–2.50 (2H, m, -CH2-), 3.74 (3H, s,
-OCH3), 5.86 (1H, s, -CH-), 6.68–6.71 (2H, m, ArH),
6.89–6.92 (2H, m, ArH), 7.15–7.19 (3H, m, ArH),
7.31–7.45 (2H, m, ArH), 7.52 (1H, s, ArH). 13C NMR
(CDCl3, 125 MHz, δ): 27.7, 28.7, 32.2, 45.8, 49.7, 57.5,
61.1, 110.4, 120.3, 121.3, 121.3, 121.4, 123.8, 125.7,
125.8, 128.4, 129.5, 135.1, 136.9, 160.7, 193.7 (C=O).
Anal. Calcd. for C22H24N2O2: C, 75.83; H, 6.94; N, 8.04;
O, 9.18; Found: C, 75.95; H, 6.88; N, 7.24.
11-(2,3-dimethoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexa-
hydrodibenzo[b,e][1,4]diazepin-1-one (SD-11) Yellowish
solid; Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3,
500 MHz, δ): 1.08–1.13 (6H, m, -CH3), 2.14–2.24 (2H, m,
-CH2-), 2.44–2.53 (2H, m, -CH2-), 3.74 (6H, s, -OCH3),
5.92 (1H, s, -CH-), 6.47 (1H, bs, -NH-), 6.66–6.70 (2H, m,
ArH), 6.86–6.88 (2H, m, ArH), 7.03–7.06 (2H, m, ArH),
7.09 (1H, m, ArH), 7.50 (1H, s, ArH). 13C NMR (CDCl3,
125 MHz, δ): 27.7, 28.7, 32.2, 45.8, 49.7, 57.5, 61.1, 61.5,
110.4, 118.3, 121.3, 121.3, 121.5, 123.7, 125.7, 125.8,
129.4, 133.4, 134.5, 136.9, 152.6, 155.6, 193.7 (C=O).
Anal. Calcd. for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40; O,
12.68; Found: C, 73.14; H, 6.85; N, 7.74.
11-(N,N-dimethylaminophenyl)-3,3-dimethyl-2,3,4,5,10,11-
hexahydrodibenzo[b,e][1,4]diazepin-1-one (SD-15) Yellow-
1
ish solid; Yield 85%, mp: 174–176 °C, H NMR (CDCl3,
500 MHz, δ): 1.03–1.11 (6H, m, -CH3), 2.11–2.19 (2H, m,
-CH2-), 2.44–2.51 (2H, m, -CH2-), 2.92 (6H, s, -CH3), 5.93
(1H, s, -CH-), 6.46 (1H, bs, -NH-), 6.68–6.72 (2H, m, ArH),
6.84–6.86 (3H, m, ArH), 6.96–6.99 (3H, m, ArH) 7.16–7.18
(2H, d, J = 6.5 Hz, ArH). 13C NMR (CDCl3, 125 MHz, δ):
27.7, 28.7, 32.2, 44.4, 45.8, 49.7, 57.5, 110.4, 120.3, 121.3,
121.3, 121.4, 123.7, 129.4, 129.8, 134.4, 135.1, 136.9, 155.7,
193.7 (C=O). Anal. Calcd. for C23H27N3O: C, 76.42; H, 7.53;
N, 11.62; O, 4.43; Found: C, 76.32; H, 7.59; N, 11.98.
11-(3,4,5-trimethoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-
hexahydrodibenzo[b,e][1,4]diazepin-1-one (SD-12) Yel-
lowish solid; Yield 85%, mp: 174–176 °C, 1H NMR
(CDCl3, 500 MHz, δ): 1.05–1.10 (6H, m, -CH3), 2.13–2.21
(2H, m, -CH2-), 2.42–2.50 (2H, m, -CH2-), 3.74 (6H, s,
-OCH3), 3.82 (3H, s, -OCH3), 5.86 (1H, s, -CH-), 6.64–6.67
(2H, m, ArH), 6.83 (1H, s, ArH), 6.86–6.88 (2H, m, ArH),
6.94–6.97 (2H, m, ArH), 7.50 (1H, s, ArH). 13C NMR
(CDCl3, 125 MHz, δ): 27.7, 28.7, 32.2, 45.8, 49.7, 57.6,
61.1, 61.2, 61.5, 110.4, 118.3, 121.3, 121.3, 121.5, 123.7,
125.7, 125.8, 133.4, 134.5, 136.9, 143.3, 153.6, 160.5,
193.7 (C=O). Anal. Calcd. for C24H28N2O4: C, 70.57; H,
6.91; N, 6.86; O, 15.67; Found: C, 70.45; H, 6.98; N, 7.12.
11-methyl-3,3-dimethyl-2,3,4,5,10,11-hexahydrodibenzo
[b,e][1,4]diazepin-1-one (SD-16) Yellowish solid; Yield
1
85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz, δ):
1.03 (3H, s, -CH3), 1.05–1.10 (6H, m, -CH3), 2.13–2.21
(2H, m, -CH2-), 2.42–2.50 (2H, m, -CH2-), 5.86 (1H, s,
-CH-), 6.47 (1H, s, ArH), 6.68–6.70 (2H, m, ArH),
6.82–6.85 (2H, m, ArH), 7.52 (1H, s, ArH). 13C NMR
(CDCl3, 125 MHz, δ): 20.0, 27.7, 28.8, 32.2, 45.8, 45.9,
57.5, 117.4, 121.3, 123.3, 124.3, 125.4, 131.0, 135.8,
136.0, 193.7 (C=O). Anal. Calcd. for C16H20N2O: C, 74.97;
H, 7.86; N, 10.93; O, 6.24; Found: C, 75.05; H, 7.76;
N, 10.76.
11-(2-methoxyphenyl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-13) Yellowish solid;
Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz, δ):
1.04–1.12 (6H, m, -CH3), 2.11–2.23 (2H, m, -CH2-),
2.41–2.52 (2H, m, -CH2-), 3.76 (3H, s, -OCH3), 5.84 (1H, s,
-CH-), 6.45 (1H, bs, -NH-), 6.68–6.71 (2H, m, ArH),
6.88–6.90 (2H, m, ArH), 7.12–7.17 (2H, m, ArH) 7.31–7.36
(2H, m, ArH), 7.52 (1H, s, ArH). 13C NMR (CDCl3,
125 MHz, δ): 27.7, 28.7, 32.2, 45.7, 49.7, 57.5, 61.1, 110.4,
120.3, 121.3, 121.3, 121.4, 123.7, 125.7, 125.8, 128.4, 129.5,
135.1, 136.9, 160.7, 193.7 (C=O). Anal. Calcd. for
11-(thiophen-2-yl)-3,3-dimethyl-2,3,4,5,10,11-hexahydrodi-
benzo[b,e][1,4]diazepin-1-one (SD-17) Yellowish solid;
Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz,
δ): 1.07–1.13 (6H, m, -CH3), 2.11–2.22 (2H, m, -CH2-),
2.41–2.50 (2H, m, -CH2-), 5.95 (1H, s, -CH-), 6.46 (1H,
s, ArH), 6.67–6.69 (2H, m, ArH), 6.83–6.86 (2H, m,
ArH), 7.13–7.17 (2H, m, ArH), 7.31 (1H, m, ArH), 7.50
(1H, s, ArH). 13C NMR (CDCl3, 125 MHz, δ): 27.7, 28.8,
32.2, 45.8, 49.7, 57.5, 110.4, 120.3, 121.3, 122.3, 123.4,
125.6, 126.8, 126.9, 127.0, 128.6, 136.9, 145.9, 193.7

Medicinal Chemistry Research
(C=O). Anal. Calcd. for C19H20N2OS: C, 70.34; H, 6.21;
N, 8.63; O, 4.93; S, 9.88; Found: C, 70.24; H, 6.28;
N, 8.83.
as a control. The reaction mixture consisted of 1.5 mL of
DPPH solution and 1 mL of test compounds in a test tube.
The reaction mixture was mixed gently at 37 °C for 1 min and
stored in dark conditions for 30 min. The intensity of colour
was measured by using spectrophotometer SYSTRONICS-
Serial no. 2231 at 517 nm. The % inhibition (DPPH
scavenging) was calculated by using the following equation:
11-(3-methylthiophen-2-yl)-3,3-dimethyl-2,3,4,5,10,11-hex-
ahydrodibenzo[b,e][1,4]diazepin-1-one (SD-18) Yellowish
solid; Yield 85%, mp: 174–176 °C, 1H NMR (CDCl3,
500 MHz, δ): 1.08–1.12 (6H, m, -CH3), 2.22–2.28 (2H, m, -
CH2-), 2.30–2.37 (3H, m, -CH3), 2.50–2.62 (2H, m, -CH2-),
4.41 (1H, bs, -NH-), 6.21 (1H, s, -CH-), 6.47 (1H, m, ArH),
6.67 (1H, m, ArH), 6.76–6.83 (4H, m, ArH). 13C NMR
(CDCl3, 125 MHz, δ): 14.1, 27.4, 27.5, 29.0, 29.1, 32.3,
46.2, 49.7, 51.5, 53.1, 113.0, 119.2, 120.0, 120.1, 121.3,
121.6, 123.7, 123.8, 127.0, 129.9, 131.5, 133.0, 136.6,
137.0, 141.3, 153.3. 193.5 (C=O). Anal. Calcd. for
C20H22N2OS: C, 70.97; H, 6.55; N, 8.28; O, 4.73; S, 9.47;
Found: C, 70.99; H, 6.43; N, 7.92.
Absorbance of controlÀAbsorbance of sample
:
% inhibition ¼
 100
Absorbance of control
Concentration of antioxidant substance causing 50%
(IC50) decrease in the concentration of free radicals was also
determined. All experiments were performed in triplicate
and values were then expressed as mean.
Molecular modelling studies with GABAA receptor
Molecular docking was performed by using Schrödinger
2015-14 Software. All the molecules were prepared using
Ligprep tool of Schrödinger, which convert 1D/2D structures
to 3D and also perform optimization. Finally, OPLS 2005
force field was used to minimize prepared ligand molecules.
Before docking, the least energy state of ligand was needed to
be prepared. The homology model of GABAA receptor was
used for molecular docking studies (Wagner et al. 1998). The
protein model was already refined by developers using protein
preparation wizard of Schrödinger known as Prepwiz. For
docking, prepared protein binding site was used for generat-
ing the grids which had a default set of options with van der
Waals radius of 1.0, thereby allowing the rotation of the
receptor thiol and hydroxyl groups in the receptor site. These
grids were then used to calculate the interaction of the indi-
vidually prepared ligand against the receptor using the extra
precision ligand docking in the glide with nitrogen inversion,
flexible ligand sampling, epik state penalties and ligand van
der Waals scaling radii. We considered only poses having low
energy conformations and good H-bond geometries.
11-cinnamyl-3,3-dimethyl-2,3,4,5,10,11-hexahydrodibenzo
[b,e][1,4]diazepin-1-one (SD-19) Yellowish solid; Yield
85%, mp: 174–176 °C, 1H NMR (CDCl3, 500 MHz, δ):
1.05–1.10 (6H, m, -CH3), 2.13–2.21 (2H, m, -CH2-),
2.42–2.50 (2H, m, -CH2-), 5.85 (1H, s, -CH-), 6.49 (1H, s,
ArH), 6.67–6.72 (3H, m, ArH), 6.93–6.98 (3H, m, ArH),
7.18–7.22 (3H, m, ArH), 7.36–7.38 (2H, m, ArH), 7.48 (1H,
s, ArH). 13C NMR (CDCl3, 125 MHz, δ): 27.7, 28.7, 30.9,
32.3, 46.4, 54.6, 111.8, 120.0, 121.1, 121.2, 123.8, 126.3,
127.1, 128.2, 129.9, 130.1, 130.8, 137.1, 137.2, 152.2, 193.5
(C=O). Anal. Calcd. for C23H24N2O: C, 80.20; H, 7.02; N,
8.13; O, 4.64; Found: C, 80.03; H, 7.22; N, 8.45.
11-(naphthalen-1-yl)-3,3-dimethyl-2,3,4,5,10,11-hexahydro-
dibenzo[b,e][1,4]diazepin-1-one (SD-20) Yellowish solid;
1
Yield 85%, mp: 174–176 °C, H NMR (CDCl3, 500 MHz,
δ): 1.17–1.27 (6H, m, -CH3), 2.26–2.38 (2H, m, -CH2-),
2.54–2.74 (2H, m, -CH2-), 5.96 (1H, m, -CH-), 6.76–6.85
(2H, m, ArH), 6.98–7.05 (2H, m, ArH), 7.04–7.08 (2H, m,
ArH), 7.51–7.65 (3H, m, ArH), 7.81–7.83 (2H, m, ArH),
8.37 (1H, d, J = 10 Hz, ArH). 13C NMR (CDCl3, 125 MHz,
δ): 27.7, 28.6, 32.2, 44.5, 49.8, 53.0, 110.9, 120.2, 122.8,
123.2, 125.6, 127.2, 128.7, 129.3, 131.3, 132.0, 133.5,
133.7, 138.0, 139.7, 155.5, 192.4 (C=O). Anal. Calcd. for
C25H24N2O: C, 81.49; H, 6.57; N, 7.60; O, 4.34; Found: C,
81.34; H, 6.65; N, 7.38.
In vivo anti-anxiety activity
Animals
Animals were approved by the respective university com-
mittees, ‘Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA)’ for
animal experiments with a proposal no. 226/CPCSEA-
2016/17. Swiss albino male mice weighing 25–30 g (three-
week-old) were purchased from Central Research Institute,
Kasauli, Himachal Pradesh, India. Animals were divided
into five experimental groups (n = 5). The first group
received vehicle control, second one received 2 mg/kg
diazepam and remaining groups received three doses (0.5,
1.0 and 2.0 mg/kg) of test compounds. Animals were kept
Antioxidant activity (DPPH assay)
Antioxidant potential of all the synthesized compounds was
tested by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
(Tang et al. 2016). DPPH solution (0.004% w/v) and different
concentrations of test compounds were prepared in methanol
solvent. During the assay, methanol alone was taken as blank
while 1.5 mL DPPH solution with 1 mL methanol was taken

Medicinal Chemistry Research
in a plastic cage and acclimatized for 1 week for adapting to
the environmental conditions before study. Animals were
housed on a 12 h/12 h light/dark cycle at a maintained
temperature of 24 2 °C. The standard animal feed and RO
water was provided during the study. All experiments were
carried out by following the CPSCEA guidelines.
Mice were administered orally with either standard or
test compound dissolved in 0.5% carboxymethylcellulose
(CMC) (<0.2 mL/animal). After 30 min of treatment, ani-
mals were tested as described in the following sections.
consisted of one wall made up of mirror and four walls
which were black. The inner surfaces of the five panels of
the smaller box were lined with mirror panels. The smaller
box of size 28 × 28 × 28 cm was fitted inside the larger box
of size 38 × 38 × 29 cm with the open side facing towards
the mirrored panel of the larger box. The smaller box was
placed exactly in the centre of the larger box so that a wall
(4.75 cm) was created around the periphery of the smaller
box. (4.75 cm wide). In one of the corners of the enclo-
sure, mice were placed as far as possible from the mirrored
panel and the mirrored chamber. The time spent and the
number of entries in the mirrored chamber were recorded
during the interval of 5 min. Entry into the chamber was
considered only when all the four paws were placed on the
floor of the mirrored chamber.
Elevated Plus Maze Model
The wooden elevated plus-maze model was used to evaluate
the anxiolytic effect of the synthesized compounds. The plus-
maze consisted of two open arms of size 30 × 10 cm without
any walls and two closed arms of the same size with 5 cm side
walls. The central area of the plus-maze interconnecting the
four arms was of the size 10 × 10 cm. The elevation of the
plus-maze was kept 30 cm from the floor. During the experi-
ment, the animal was placed initially with four legs in the
central arena facing towards one of the closed arms. The time
spent and a number of entries by the mice were recorded in the
open arm during 5 min of the interval. When the mouse had
clearly crossed the central arena with its four legs, it was
considered to be in the open part.
Statistical analysis
All the in vivo experimental results are expressed as means
S.E.M. Statistical comparisons were made using one-way
analysis of variance (ANOVA). Tukey’s test was used to
analyse the significant differences between the vehicle
control and treated mice.
Results and discussion
Light and Dark Box
Chemistry
The box used in this model was made up of glass, sized 25 ×
50 × 20 cm having one-third portion as a dark and two-third
portion as a light compartment. Initially, mice were placed in
the light compartment. A 40-W light bulb was positioned in the
light compartment 10 cm above the centre. The mice could
move freely between both the compartments through the
opening at the centre. The time spent and the entries made by
the animals in the light compartment were recorded for 5 min.
The mouse was considered to be in the light compartment
when its four legs were clearly in the light compartment.
All the synthetics were synthesized via three-component
condensation of o-phenylenediamine, dimedone and var-
iously substituted aldehydes, by using silicated fluor-
oboric acid in a catalytic amount under microwave
Table 1 Percentage yield of benzodiazepine analogue with various
catalysts
Entry
Catalyst (BA-SiO2)
% age yield
1
2
3
4
5
HNO3−SiO2
H2SO4−SiO2
HClO4−SiO2
HBF4−SiO2
SiO2
40
55
45
71
—
Mirror chamber model
The mirror chamber apparatus consisted of two open-top
boxes made up of black Plexiglas. The larger box
Scheme 1 Model reaction for
the synthesis of benzodiazepine
derivatives

Medicinal Chemistry Research
Table 2 Percentage yield with varying mol % of the catalyst and time of exposure to microwave irradiation of some selected benzodiazepines
derivatives
Structure
Yield (%age)
65
Time (min)
Loading (mol%)
10
15
20
69
67
1
S
H
N
10
15
20
72
92
O
5
89
73
82
N
H
10
15
10
20
76
63
79
73
10
15
1
NO2
20
10
75
95
90
69
77
H
N
O
5
15
20
N
H
10
15
20
10
73
63
10
15
20
1
68
73
H3CO
OCH3
10
15
20
75
83
OCH3
H
N
5
O
79
69
77
N
H
10
15
10
20
73
10
15
53
61
58
1
20
10
63
H
N
5
O
76
69
58
68
15
20
N
H
10
15
20
10
63
The bold values point to the most applicable conditions

Medicinal Chemistry Research
Table 3 Docking scores (GABAA receptor) of all the synthesized benzodiazepine derivatives
Compound
Structure
Antioxidant IC50
(nM)
Dock Score
(kcal/mol)
SD-1
>500
>500
ND
H
N
O
N
H
Cl
SD-2
SD-3
SD-4
ND
H
N
O
N
H
Cl
489
-9.025
Cl
H
N
O
N
H
Br
>500
ND
H
N
O
N
H
SD-5
SD-6
233
-8.662
ND
Br
H
N
O
N
H
F
>500
H
N
O
N
H
NO2
SD-7
SD-8
470
-8.249
ND
H
N
O
N
H
CH3
>500
H
N
O
N
H

Medicinal Chemistry Research
OCH3
SD-9
SD-10
SD-11
>500
>500
>500
ND
ND
ND
H
N
O
N
H
OCH3
OCH3
H
N
O
N
H
H3CO
H3CO
H
N
O
N
H
H3CO
SD-12
OCH3
>500
ND
OCH3
H
N
O
N
H
SD-13
SD-14
SD-15
H3CO
>500
138.55
>500
ND
-8.799
ND
H
N
O
N
H
OCH3
H
N
O
N
H
N
H
N
O
N
H
H
N
SD-16
SD-17
O
O
>500
>500
ND
ND
N
H
S
H
N
N
H

Medicinal Chemistry Research
SD-18
SD-19
S
83.35
76
-7.352
-5.953
H
N
O
N
H
H
N
O
N
H
SD-20
81.26
-9.996
H
N
O
N
H
Diazepam
-8.153
Fig. 2 Structure of a α1β3γ2 GABAA receptor with diazepam binding site
irradiation. It has been previously reported that these
types of reactions are very proficient and eco-friendly to
form the desired products.
Initially, for the synthesis of benzodiazepine derivatives,
a model reaction was performed in order to check the cat-
alytic effectiveness of various silica adsorbed bronsted acids
(BA–silica), (Scheme 1 and Table 1).
From the careful examination of Table 1, it was
observed that fluoroboric acid adsorbed on silica was
highly efficient to catalyse this type of reaction. The high
catalytic impact of HBF4–SiO2 could be explained owing
to its weak protic acidic nature which might have by-pass
the side reaction problems. It was also found that, when
the reactions were catalysed by strong bronsted acids like,
perchloric acid, sulfuric acid and nitric acid, the final
product thus formed was not in such a good yield. From
these particular findings, it was concluded that silica
performed a critical role as an activator to estimate its
catalytic power, in the absence of which reaction did not
proceed well.
Furthermore, to improve the %age yield of the final
product, reaction optimization was carried out by

Medicinal Chemistry Research
Fig. 3 a Diazepam (blue) in its binding pocket of GABAA receptor showing π−π interactions (blue lines): b Compound SD-20 (green) docked in
the binding pocket of Diazepam showing various H-bonding (pink dotted lines) and π−π interactions (blue dotted lines) (colour figure online)
Fig. 4 Diazepam (a) and compound SD-20 (b) showing hydrophobic interactions with various binding site residues
using different amounts of silicate fluoroboric acid under
similar conditions, for a 100% conversion of reactants. A
significant improvement was observed in the yield
with 5 mol% of catalyst in 15 min. The amount more than
5 mol% of catalyst did not improve the results (Table 2).
Therefore, 5 mol% HBF4–silica was capable to catalyse
the synthesis of benzodiazepines in good to high yields
ranging from 67 to 87%.
The structures of synthesized compounds were con-
firmed by using 1H NMR, 13C NMR and elemental analysis
and the spectral data were found in accordance with the
assumed structures.
Antioxidant activity (DPPH assay)
It has been reported that during the compounds with anti-
oxidant potential could treat the anxiety disorder. In this
respect, all the synthesized benzodiazepine derivatives were
evaluated to check their antioxidant potential by using 1,1-
Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
assay. Results of DPPH assay (Table 3) revealed that seven
compounds SD-3, SD-5, SD-7, SD-14, SD-18, SD-19 and
SD-20 have significant antioxidant potential with the IC50
values in the nanomolar range. Among all the synthetics,
compound SD-19 exhibited higher antioxidant activity with

Medicinal Chemistry Research
Fig. 5 2D view of the docked
structure of compound SD-20 in
GABAA receptor binding site
Table 4 Physicochemical parameters of compounds SD-3, SD-14 and SD-20
Compound
Molecular weight
No. of H-
bond dono-
rs
No. of H-
bond
acceptors
Molar Refractivity
Log P
No. of
Lipinski
Violation
SD-3
387
348
424
2
2
2
3
4
3
110.36
107.21
127.52
4.78
3.41
5.17
0
0
0
SD-14
SD-20
the IC50 value of 76 nM whereas compounds SD-18 and
SD-20 also possessed significant activity with their IC50
values of 83.35 and 81.26 nM, respectively. The com-
pounds that displayed IC50 values above 500 nM are sum-
marized in the table as >500 nM.
Software and dock scores were calculated (Cottrell et al.
1987). Seven compounds that exhibited significant anti-
oxidant potential were only selected to analyse by docking
studies i.e SD-3, SD-5, SD-7, SD-14, SD-18, SD-19 and
SD-20. Among the seven compounds, five compounds
exhibited the best dock score as compared to diazepam
(Table 3). SD-20 was found to be the best fit with the
diazepam binding site with the most prominent dock score
of −9.996 kcal/mol, which was further selected to discuss
its binding behaviour.
Molecular modelling studies with GABAA receptor
GABAA receptor is a pentameric ligand-gated ion channel
consisting of five main binding domains viz. two α1, two
β3, and one γ2. Among these binding sites benzodiazepines
have been found to bind on the interface of α1 and γ2
domains (Fig. 2). After the binding of benzodiazepines
(Positive Allosteric Modulators) with GABA receptor, the
central chloride pore opens up and causes an influx of
chloride ions which is associated with the anxiolytic effect.
Most commonly used benzodiazepines are the diazepam
and lorazepam as positive allosteric modulators of GABA
(Simonas et al. 2019).
Binding mode of compound SD-20
In order to see the mode of binding of compound SD-20
into the GABAA receptor binding site, diazepam was used
as a reference for validating the docking procedure. Dia-
zepam in its binding pocket showed π−π interactions with
Tyr159 and Tyr209 residues and also exhibited hydro-
phobic interactions with various binding site residues (Fig.
3a). Compound SD-20, when docked in the binding site
grid of GABAA receptor, exhibited well-docked pose with
a docking score of −9.996 kcal/mol. It showed two
To predict the binding affinity of the synthesized ben-
zodiazepine molecules, diazepam-bound GABAA receptor
homology model was used in Schrödinger 2015-14

Medicinal Chemistry Research
Fig. 6 (Left) Dose−effect relationship of benzodiazepines in three
different animal models: (Right) number of positive entries in the open
field. The values were shown as the mean S.E.M. (n = 5). Similar
notations define not significant difference between the groups at p <
0.05 level, and dissimilar notations define the significant difference
between the groups at p < 0.05 level

Medicinal Chemistry Research
H-bonds through its OH group with Ser204 (1.84 Å) and
Thr206 (1.76 Å) and π−π interactions with Phe77 and
Tyr159 (Fig. 3b). It also interacted with Val209, Val211,
Val202, Phe99, Phe77, Ser158, and Ala79 through various
hydrophobic interactions in the same mode as diazepam did
(Fig. 4). The two-dimensional view of SD-20 within the
GABAA receptor is provided in Fig. 5.
14 did not show any significant effect even at the dose level
of 2 mg/kg. A statistically significant difference was found
between the SD-3 and SD-20 compounds which suggest
that the compound SD-20 displayed better anxiolytic effect
than SD-3 and diazepam at the dose level of 1.0 mg/kg.
Similar results were found with the Light & Dark box and
Mirror Chamber model, which are summarized in Fig. 6.
Therefore, in vivo results were found in accordance with the
molecular docking results which also revealed the SD-20
compound as best fit molecule with the GABAA receptor.
In silico study
To determine whether the three potent compounds (SD-3,
SD-14 and SD-20) fit to the Lipinski rule of five or not,
ChemAxon software MarvinSketch was used for the same
purpose. The results stated that the compounds well fol-
lowed all the properties of the rule (Table 4) that indicate:
(a) the molecular weight of the synthesized compounds
found below 500 Da, i.e. within the range of 348–424; (b)
the H-bond donating property of all the compounds lies
within the limits (<5); (c) H-bond acceptor sites were found
<10; (d) molar refractivities were fallen in the range of
107.21–127.52 that lies well in the permitted range of
40–130 and (e) log P of all the synthetics was found in the
range of 3.41–5.17 which suggested that the compounds
were lipophilic in nature and able to cross the blood brain
barrier. These results indicated that all of the synthetics
could be pharmacologically efficient to be used further in
animal models.
Conclusion
In the present study, various benzodiazepine derivatives
were synthesized by using silicated fluoroboric acid in good
to high yield. All the synthetics were preliminarily eval-
uated to check their antioxidant potential. Seven compounds
with significant antioxidant potential were further stream-
lined to check their binding ability towards the diazepam
binding site of GABAA receptor. Three compounds SD-3,
SD-14 and SD-20 that significantly bind with GABAA
receptor were tested in vivo to assess their antianxiety
activity. Among these three compounds, SD-3 and SD-20 at
1.0 mg/kg oral dose displayed better anxiolytic effect as
compared to standard drug diazepam. Thus, the overall
study distinguished that compounds SD-3 and SD-20 could
act as hit lead molecules for the further development of
anxiolytic agents.
In vivo antianxiety activity
Acknowledgements The authors are grateful to University Grants
Commission (RGNF), Council of Scientific & Industrial Research
(CSIR), Department of Science & Technology (WOS-A) and DST-
FIST for providing funds and are also thankful to Guru Nanak Dev
University, Amritsar for providing various facilities to carry out the
research work.
By carefully examining the antioxidant potential as well as
docking simulations of the synthesized compounds, we
have selected three most prominent molecules SD-3, SD-14
and SD-20 to check their anxiolytic effect in the three
in vivo mice models viz. (1) Elevated Plus Maze Model, (2)
Light and Dark box, and (3) Mirror chamber model. The
compounds were found to show no signs of acute toxicity,
even up to 10 mg/kg in mice.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
The three most potent compounds (SD-3, SD-14 and SD-
20) were tested for their anxiolytic activity by using ele-
vated plus maze model and the time spent by mice in the
open arms and the number of entries were observed and the
data were displayed in Fig. 6. The results were compared
with the diazepam (2 mg/kg) which was used as standard
control. The 0.5% carboxymethylcellulose was taken as
vehicle control. A significant anxiolytic effect was observed
with compounds SD-3 and SD-20 at the dose level of
1.0 mg/kg. The total time taken by the animals in the open
arm treated with SD-3 and SD-20 (1.0 mg/kg) was found
almost similar to the time spent by the animals treated with
2 mg/kg of diazepam. Statistically, there was no significant
difference between results of 1 mg/kg dose of SD-3 and SD-
20, and 2 mg/kg dose of diazepam (Fig. 6). Compound SD-
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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