Synthesis and Evaluation of Anticonvulsant Activity of Some Schiff Bases of 7-Amino-1,3-dihydro-2H-1,4-benzodiazepin-2-one

Article abstract of DOI:10.1002/cbdv.202000342

A variety of 1,3-dihydro-2H-1,4-benzodiazepin-2-one azomethines and 1,3-dihydro-2H-1,4-benzodiazepin-2-one benzamide were prepared, characterized and evaluated for the anticonvulsant activity in the rat using picrotoxin-induced seizure model. The prepared 1,3-dihydro-2H-1,4-benzodiazepin-2-one azomethine derivatives emerged potentially anticonvulsant molecular scaffolds exemplified by compounds, 7-{(E)-[(4-nitrophenyl)methylidene]amino}-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, 7-[(E)-{[4-(dimethylamino)phenyl]methylidene}amino]-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, 7-{(E)-[(4-bromo-2,6-difluorophenyl)methylidene]amino}-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one and 7-[(E)-{[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]methylidene}amino]-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one. All these four compounds have shown substantial decrease in the wet dog shake numbers and grade of convulsions with respect to the standard drug diazepam. The most active compound, 7-[(E)-{[4-(dimethylamino)phenyl]methylidene}amino]-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one, exhibited 74 % protection against convulsion which was higher than the standard drug diazepam. Furthermore, to identify the binding mode of the interaction amongst the target analogs and binding site of the benzodiazepine receptor, molecular docking study and molecular dynamic simulation were carried out. Additionally, in silico pharmacokinetic and toxicity predictions of target compounds were carried out using AdmetSAR tool. Results of ADMET studies suggest that the pharmacokinetic parameters of all the target compounds were within the acceptable range to become a potential drug candidate as antiepileptic agents.

Full text of DOI:10.1002/cbdv.202000342

Title: Synthesis and Evaluation of Anticonvulsant Activity of Schiff Base
of 1,4-Benzodiazepine Amine
Authors: Manoj Bhanudas Gawande, Pankaj R. Nilkanth, Sujit Ghorai,
Arulmozhi S, Kiran Dhawale, sharad Shelke, and Tansir
Ahamad
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000342
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10.1002/cbdv.202000342
Chemistry & Biodiversity
Chem. Biodiversity
Synthesis and Evaluation of Anticonvulsant Activity of Some Schiff Base of
1,4-Benzodiazepine Amine
Pankaj R. Nilkanth,a Sujit K. Ghorai,b Arulmozhi Sathiyanarayanan,c Kiran Dhawale,c Tansir Ahamad,d
Manoj B. Gawande,*,e,f Sharad N. Shelke*,a
aDepartment of Chemistry, S.S.G.M. College, Kopargaon, Dist-Ahmednagar, Maharashtra, 423601, India. E-mail: snshelke@yahoo.co.in
bSyngenta Biosciences Pvt. Ltd., Santa Monica Works, Corlim, Ilhas, Goa, 403110, India.
cDepartment of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth, Erandwane, Pune, Maharashtra, 411038, India.
dDepartment of Chemistry, College of Science, King Saud University, Riyadh-11451, Saudi Arabia.
eRegional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, Šlechtitelů 27, Olomouc, Czech Republic, 78371.
fInstitute of Chemical Technology Mumbai-Marathwada Campus, Jalna, Maharashtra 431203, India.
Email: manoj.gawande@upol.cz
Abstract: A variety of 1,4-benzodiazepine azomethines (3a-n) and 1,4-benzodiazepine benzamide (4) were prepared, characterized and evaluated for the
anticonvulsant activity in the rat using picrotoxin-induced seizure model. The prepared 1,4-benzodiazepine azomethine derivative emerged potentially
anticonvulsant molecular scaffolds exemplified by compounds, 7-(4-nitrophenyl)methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3c), 7-
[4-(dimethylamino)phenyl]methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3e), 7-[(4-bromo-2,6-difluoro-phenyl)methyleneamino]-5-
phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3l) and 7-[3-(4-fluorophenyl)-1-phenyl-pyrazol-4-yl]methyleneamino-5-phenyl-1,3-dihydro-1,4-
benzodiazepin-2-one (3n). All these four compounds have shown substantial decrease in the wet dog shake numbers and grade of convulsions with
respect to the standard drug diazepam. Most active compound, 7-[4-(dimethylamino)phenyl]methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-
2-one (3e) exhibits 74 % protection against convulsion which was higher than standard drug diazepam. Furthermore, to identify the binding mode of the
interaction amongst the target analogs and binding site of the benzodiazepine receptor, molecular docking study and molecular dynamic simulation were
carried out. Additionally, in silico pharmacokinetic and toxicity predictions of target compounds were carried out using AdmetSAR tool. Results of ADMET
studies suggest that the pharmacokinetic parameters of all the target compounds were within the acceptable range to become a potential drug candidate
as antiepileptic agents.
Keywords: Epilepsy; 1,4-benzodiazepines; Azomethine; Anticonvulsant; Molecular docking.
Introduction
Epilepsy is a disorder of central nervous system in which excessive and rapid neuronal discharge in the brain results in loss of consciousness and
convulsions. Approximately 60 million world populations are suffering from this critical ailment, making it one of the most common neurological diseases.
[1] There are many antiepileptic drugs (AEDs) available for the cure epilepsy. Depending upon the mechanism of the actions, currently available marketed
AEDs classified into three major categories, which includes inhibition of sodium channel function (e.g. phenytoin, carbamazepine), [2] inhibition of calcium
channel function (e.g. ethosuximide) [3] and enhancement of GABA action (e.g. benzodiazepines, barbiturates). [4, 5] Other mechanism includes inhibition
of glutamate release, the block of glutamate receptor and the combination of the above actions, often coupled with an additional mechanism (e.g.
valproic acid, gabapentin). [6] Despite, almost 30 % patient’s compliance is not observed to currently available antiepileptic treatment, while in some
critical cases multidrug therapy is required. This leads to significant side effects, like toxicity of neurons, depression, central nervous system (CNS) related
disorders, cosmetic (gingival hyperplasia) as well as severe hepatotoxicity and megaloblastic anaemia. [7, 8] Therefore, there is an important necessity to
develop novel antiepileptic agent with a high degree of selectivity and specificity.
In recent years, the discovery of the novel antiepileptic agents primarily focused on the reduction of seizures in symptomatic fashion. Literature survey
suggests that pharmacophore-based ligand drug design is an excellent approach for the development of novel antiepileptic agents. [9] It relies on the
knowledge of old and new anticonvulsant active drugs that interact to the biological target of interest. The main aim of this approach is the structural
modification of known AEDs, with the acuity of designing molecules with a more efficacious and comparatively less or no critical side effect compare to
available AEDs. [10] Here, we have selected benzodiazepine as active pharmacophore (Figure 1) for the development of novel AEDs. Benzodiazepines are
one of the privileged class of nitrogen containing heteroaromatic compound in the area of pharmaceuticals and mainly acting on the central nervous
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system. Among the benzodiazepines, 1,4-benzodiazepine derivatives are the most abundant and have a well time-honoured position in antiepileptic
therapy. [11, 12] In addition to their established anticonvulsant activity, [13] 1,4-benzodiazepine also demonstrates many other important biological activities

such as, anti-anxiety, [14] hypnotic, [15] NNRTI of HIV-1 reverse transcriptase, [16 18] anti-arrhythmic agents, [19, 20] antiproliferative, [21] anti-platelet anti-ulcer,
[22] vasopressin antagonist activity [23] as well as anti-inflammatory agents. [24] The early structure-activity relationship (SAR) study [25, 26] indicated that the
potency of benzodiazepine is mainly determined by substitution at 7th position, whereas any substitutions at positions 6, 8 and 9 may decrease the
activity. SAR study also promotes the presence of carbonyl function at 2nd position, phenyl ring at 5th position, the presence of double bond between 4th
and 5th position in diazepine ring B and no alkyl substitution or a less bulky alkyl group at N-1, as shown in Figure 1. On the other hand, compounds

containing azomethine group in the structure are known as Schiff bases represents a vital class of medicinally important compounds. [27 29] Thus, a strong
hypothesis was proposed that combination of 1,4-benzodiazepine as pharmacophore along with azomethine at the C7-position would also display potent
anticonvulsant effects.
Thus, we have decided to react 7-amino nitrazepam (2) with substituted aryl aldehydes 8a-n to form a library of Schiff bases (Figure 2) and evaluate their
anticonvulsant activity. Though some Schiff bases of 7-amino nitrazepam (2) known in the literature, [30] those were never evaluated for anticonvulsant
activity.
Figure 1 Molecular hybridization of 1,4-benzodiazepine and azomethine having drug potency.
In this research work, we have reported rational design, synthesis and anticonvulsant activity of some 1,4-benzodiazepine azomethine analogue 3a-n and
a benzamide derivative of 1,4-benzodiazepine 4. Further, molecular docking of the synthesized compounds was carried out on the GABAA receptor to
identify the binding mode of the interactions. Moreover, in-silico ADMET and the molecular property predictions of all the derivatives were reported.
Based on the information gathered from pharmacological activity and molecular docking, SAR was summarized and potential lead molecules were
identified.
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Figure 2 Design strategies for the synthesis of azomethine derivative of 1,4-benzodiazepine as anticonvulsant agents.
Results and Discussion
Chemistry
Target compounds 3a-n and 4 were synthesized according to scheme 1. Synthesis of the key building block 7-amino nitrazepam (2) was achieved in three
steps starting from commercial 2-amino-5-nitro-benzophenone (5). Acylation of 2-amino-5-nitro-benzophenone (5) with chloroacetyl chloride 6 in
toluene at 110 oC afforded 2-chloroacetamido-5-nitrobenzophenone (7). [31] Amide 7 in the presence of hexamethylenetetramine (HMTM) and NH4OAc in
absolute ethanol at 78 oC gave nitrazepam 1. [32] Reduction of nitrazepam 1 with SnCl2.H2O in EtOH ensued in the formation of 7-amino nitrazepam (2). [33]
Azomethine derivative of 1,4-benzodiazepine 3a-n were synthesized by the reaction of 7-amino nitrazepam (2) and appropriate aldehydes 8a-n in ethanol
at 78 oC with catalytic amount of glacial acetic acid. Amide 4 was obtained by the coupling of 7-amino nitrazepam (2) and 4-fluoro benzoyl chloride (9) in
the presence of triethyl amine in CH2Cl2. The structures of the target compounds were characterized by spectral methods. The physicochemical data of
synthesized analogues are depicted in Table 1.
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Scheme 1 Reagents and conditions: (i) Toluene, 110 oC, 2 h, 98 %; (ii) HMTM, NH4OAc, EtOH, 78 oC, 6 h, 71 %; (iii) SnCl2.2H2O, EtOH, ultrasonicated, 24 oC, 2 h, 90 %; (iv) EtOH,
cat. Glacial CH3COOH, 78 oC, 1-6 h; (v) Et3N, CH2Cl2, 24 oC, 2 h, 65 %.
Table 1 Physicochemical data to assess Lipinski’s rule of five and BBB permeability of the synthesized compounds.
Compound
M.W.a
Log Pb
BBB
HBDd
HBAe
Permiabilityc
3a
3b
3c
3d
3e
3f
339.14
357.13
384.12
397.14
382.18
435.04
435.04
451.01
425.12
4.05
4.21
2.93
3.87
4.33
5.04
5.04
5.44
5.13
0.9931
0.9952
0.9982
0.9580
0.9641
0.9914
0.9914
0.9903
0.9911
1
1
1
1
1
1
1
1
1
3
4
4
5
4
4
4
3
7
3g
3h
3i
4
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3j
402.11
393.11
3.06
4.52
0.9750
0.9926
1
1
5
6
3k
3l
3m
3n
4
453.03
389.15
499.18
373.12
251.10
5.19
5.05
6.31
3.15
1.38
0.9914
0.9931
0.9945
0.9920
0.9844
1
1
1
2
2
5
3
6
4
3
2
1
281.08
1.56
0.9802
3
6
a Molecular weight of the compound, b partition coefficient, c Blood brain barrier permeability, d Hydrogen bond
donor, e Hydrogen bond acceptor
Pharmacology
All the synthesized 1,4-bezodiazepine (BZD) derivatives were screened for the anticonvulsant property in PTZ induced seizure model in rats. This test was
used to identify compounds that decrease the seizure threshold. Here, time of onset of convulsions, time of onset of tonic clonic convulsions and
convulsion grades were recorded in rat for all the target compounds.
Anticonvulsant activities of synthesized compounds (3a-n, 4 and 2)
Effect of BZD derivatives in Time of onset of Convulsions
There was a significant increase (p<0.001) in time of onset of convulsions of compound 3b to 3n and 4 treated groups similar to standard drug diazepam
as compared to the convulsive control group. However, there was no change in the onset of convulsions in 3a and 2 treated groups. In the current study
(Figure 3(a)), it was detected that compounds 3c, 3e, 3l and 3n display a significant enhancement of time of onset of convulsions as compared to
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convulsive control.
Figure 3 a) Effect of compounds 3a-3n, 4, 2 and standard drug diazepam on time of onset of convulsions in PTZ induced seizure model in rat. b) Effect of compounds 3a-3n, 4, 2
and standard drug diazepam on time of onset of tonic clonic convulsions in PTZ induced seizure model in rat. c) Effect of compounds 3a-3n, 4, 2 and standard drug diazepam on
number of wet dog shake in PTZ induced seizure model in rat. d) Effect of compounds 3a-3n, 4, 2 and standard drug diazepam on grade of convulsions in PTZ induced seizure
model in rat. (Values are represented as mean ± SEM, n = 8, One-way ANOVA followed by Dunnet’s ‘t’ test, $$p<0.01, $$$p<0.001 when compared to Normal control, *p<0.05,
**p<0.01, ***p<0.001 when compared to Convulsive control).
Effect of BZD derivatives in Time of onset of Tonic-Clonic convulsions
There was a significant increase (p<0.001) in time of onset of tonic-clonic convulsions in the compounds 3a to 3n, 4 and 2 treated group similar to
standard drug diazepam as compared to the, convulsive control group. Further, (Figure 3 (b)), it was observed that derivatives 3c, 3e, 3l, 3n show a
significant increase in the time of onset of tonic-clonic convulsions as compared to the convulsive control group.
Effect of BZD derivatives in wet dog shake
There was a significant decrease (p< 0.01) in wet dog shake numbers in the compounds 3c, 3e, 3h, 3l, and 3n treated groups similar to standard drug
diazepam treated group as compared to the convulsive control group. However, no change was noticed in the wet dog shakes in other BZD derivative
treated groups (Figure 3 (c)). Further, it was noted that there are compounds 3e, 3h and 3l shown superior activity as compared to standard drug
diazepam.
Effect of BZD derivatives in Grade of Convulsions
There was a significant decrease (p<0.01) in the grade of convulsions in the compound 3c, 3e, 3l and 3n treated group similar to standard drug diazepam
treated the group as compared to the convulsive control group. However, there was no change in the grade of convulsions in other BZD derivative treated
groups (Figure 3(d)). From the entire set of compounds, 3e shows the most potent activity as compared to standard drug diazepam. In the current
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research work, it is evident that BZD derivatives specifically 3c, 3e, 3l and 3n were able to ameliorate the epileptic seizures induced by PTZ in laboratory
animals.
Here, in PTZ-induced acute clonic convulsion model, most active compound 3c, 3e, 3l and 3n display maximum protection against convulsion in the range
of 52 % to 74 %. (Figure 4) While, Diazepam show 61.90 % protection against convulsion. The results of anti-convulsion activity strongly revealed
compound 3e show better activity compare to diazepam with 74.40 % protection against convulsion.
Figure 4 Anticonvulsant activity of the tested compounds using PTZ model.
Acute oral toxicity study
Notably, no significant change in respiratory, reflexes, ocular signs, muscle tone, skin, cardiovascular and gastrointestinal parameters was observed in 3a-
3n, 4, 2 treated groups with a comparison to a normal control group of animals administered with the vehicle. However, there was a considerable
reduction in motor activity in 3a-3n, 4, 2 treated groups when compared to a normal control group of animals administered with the vehicle. The animals
treated with compounds 3c, 3e, 3h, 3l and 3n at the doses of 50 mg/kg body weight has been found to have lost the righting reflex which indicated that
treatment with 3c, 3e, 3h, 3l and 3n at 50 mg/kg caused a hypnotic effect in the rat.
In Silico molecular docking study
Molecular docking study was accomplished for the identification of the binding mode of interaction and to confirm whether the anticonvulsant activity of
compounds is mediated via the BZD receptor. Using the gradient optimization algorithm and an empirical scoring function the molecular docking of most
active analogue 3e and standard drug Diazepam in the active site of GABAA receptor were carried out by AutoDockVina tool. Molecular docking score of
diazepam and most active compound 3e were found to be -8.904 and -8.265 respectively. Notably, a standard drug diazepam interacts via hydrogen
bonding with Ser204 amino acid in A chain of GABAA receptor. While, core ring of diazepam interacts with amino acids namely Thr206, Ala79, Leu140,
Tyr159 and Thr142 by hydrophobic interactions. In Figure 5, the most important residues in the binding mode of diazepam are Thr206, Tyr 209, His101,
Tyr159, and Phe77 are presented. Further, π-π interactions were also observed with Tyr159 and Tyr209 amino acids respectively. Most active compound
3e was witnessed to bind at the active site of the GABAA receptor with similar binding mode as observed in the diazepam. In compound 3e, hydrogen
bonding interaction was noticed with His101 While, hydrophobic interaction was detected with amino acids namely Leu, Ser, Asp, Thr, Val, Tyr. Further,
π- π interactions were noted with Phe77, Tyr209 and Tyr159 amino acids as similar to that of standard drug diazepam. Moreover, the results of the
interaction of compound 3e at the active site of the GABAA receptor strongly revealed that it’s binding pattern and mode of interaction resembles the
standard drug diazepam. 2D interaction of diazepam and compound 3e is represented in Figure 5 respectively.
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Figure 5 a) 2D interaction of diazepam with active site of GABAA receptor. b) 2D interaction of most active compound 3e with active site of GABAA receptor.
Molecular dynamics simulations
Molecular dynamics study was carried out to check the stability of the most active compound 3e and diazepam on GABAA receptor. For the analysis of
trajectories of molecular dynamics simulation RMSD and radius of gyration calculated and explored. RMSD value of compound 3e and diazepam are
depicted in Figure 6. The RMSD trajectories clearly demonstrated that complex with compound 3e and diazepam were equilibrated in the range of RMSD
value 0.25 to 0.5 nm. The results of RMSD value indicate that ligand was not fluctuating after binding to the active site of receptor. Further, binding
pattern of most active compound 3e and diazepam was almost similar. Further, the rigidity of protein-ligand complexes can be explained using the radius
of gyration (Rg) parameter obtained from MD simulation trajectories. The Rg values were recorded from the trajectories and plotted against time and
depicted in Figure 7. Rg trajectories in Figure 7 suggest that all system was little bit fluctuating during the simulation process in the case of compound 3e
as well as diazepam.
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Figure 6 a) RMSD vs. time of protein-ligand complexes for compound 3e b) RMSD vs. time of protein-ligand complexes for diazepam
Figure 7 a) Radius of gyration vs. time of protein-ligand complexes for compound 3e b) Radius of gyration vs. time of protein-ligand complexes for diazepam.
ADMET properties prediction of the target analogues
Drug solubility is one of the important parameter which plays an essential role in the pharmacokinetics and pharmacodynamics of the drug. It plays a role
from the administration of drug, absorption and also during the activity on targeted part or metabolism and excretion from the human body. ADMET
prediction of target analogs was accomplished by the use of online software AdmetSAR. Different permeability parameters such as Blood Brain Barrier
(BBB) penetration, Caco-2 cell permeability, Human Intestinal Absorption (HIA), renal organic cation transport and AMES toxicity test were predicted. P-
glycoprotein is one of the membrane efflux transporters having an important role in the determining the absorption, distribution, metabolism, excretion,
and toxicological behaviours of some drugs and molecules in development and also plays a major role in the multidrug resistance (MDR) phenomenon. P-
gp substrates recognition at the early stages of the drug discovery process is very important. Cytochrome enzymes (CYPs) are involved in the metabolism
and biosynthesis of endogenous molecules. So, the study of metabolism-related parameter provides hints to overcome any sign of toxicity. Results
suggest that all the synthesized analogs were able to absorb from human intestine and there were no signs of toxicity or mutagenicity in the compounds.
Results of ADMET study is depicted in Table 2. (Supporting data)
SAR summarization based on molecular docking and pharmacological study
The biological study delivered preliminary insight regarding SAR. Results of pharmacological activity suggested that the presence of azomethine
functionality with suitably substituted aromatic ring plays a significant role in the biological activity. All the synthesized compounds contain same 1,4-
benzodiazepine amine moiety which, did not show anticonvulsant activity. Whereas, 1,4-benzodiazepine amine attached to aromatic moiety having
electron withdrawing or bulky groups at 7th position via azomethine demonstrated significant anticonvulsant activity. In the compounds 3c, 3e, 3l and 3n
where electron withdrawing groups such as Br or NO2 and bulky group like dimethyl amine present at the para- position of phenyl ring or substituted
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pyrazole lead to a substantial decrease in the grade of convulsion (Figure 8). In case of unsubstituted phenyl ring or ortho- and meta- position substituted
aromatic ring present in the compounds 3a, 3f, 3g, 3i, 3k revealed moderate to poor biological activity.
Figure 8 Structures of most active compounds.
Conclusions
The primary aim of the present research work was to develop novel 1,4-benzodiazepine derivatives as anticonvulsant agents. The preliminary
anticonvulsant evaluation was performed in rats using classical Picrotoxin induced epileptic model. From the entire dataset, compounds 3c, 3e, 3l and 3n
displayed significantly better anticonvulsant activity in comparison to standard drug diazepam. Further, there was also a substantial decrease in grade of
convulsions in 3c, 3e, 3l and 3n treated groups when compared to the convulsive control group. Additionally, molecular docking study was undertaken to
retrieve information regarding the mode of interaction at the active site. Further, in silico ADMET results and in- vivo acute oral toxicity study also
indicated that the designed azomethine derivatives have very good pharmacokinetic profile to become a potential drug candidate. Based on these results,
we have identified novel analogs as potential leads. Our future endeavor is the optimization of the lead molecules to find potent and safe anticonvulsant
agents.
Experimental Section
Chemistry
All the solvents and reagents were purchased commercially and were used without further purification. SRS-OptiMelt digital melting point apparatus was
employed to determine the melting point of all synthesized compounds and were uncorrected. 1H and 13C NMR spectra were recorded on a Bruker Avance
II-400 spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shift values are given in ppm (δ) relative to TMS and coupling
constants J are given in Hz. Agilent QTOF 6520 mass spectrometer operating at an ionization potential of 70 eV were used to perform the HRMS analysis.
IR spectra were recorded on Shimadzu DRS Prestige 21. Partition coefficient (log P) values were calculated using the CS ChemDraw Ultra version 12.01,
by CambridgeSoft.Com.
Procedure for the synthesis of intermediates (7, 1 and 2)
Synthesis of 2-chloroacetamido-5-nitrobenzophenone (7)
To a stirred solution of 2-amino-5-nitro-benzophenone (5) (2.42 g, 10 mmol) in toluene (24 mL) under nitrogen, was added chloroacetyl chloride 6 (10
mmol), heated to reflux and allowed to stir at the same temperature for 2 h. The reaction mixture was then cooled to room temperature and
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concentrated in vacuum. The crude was purified by crystallization from ethanol (24 mL) at room temperature to give 2-chloroacetamido-5-
nitrobenzophenone (7) as white crystalline solid (3.11 g), Yield: 98 %, M.p. 175–176 oC.
Synthesis of 7-nitro-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (1)
To a stirred solution of 2-chloroacetamido-5-nitrobenzophenone (7) (3.5 g, 11 mmol) in ethanol (50 mL) were added hexamethylenetetramine (HMTM; 3.4
g, 24 mmol) and ammonium acetate (1.86 g, 24 mmol). The reaction mixture was then heated to reflux and permitted to stir at the same temperature for
6 h. The volatiles were evaporated, water (35 mL) was added and the resulting suspension was stirred at 60 °C for 30 min. The suspension was cooled to
15 °C, filtered and dried under vacuum. To the crude product was added toluene (10 mL) and heated at 70 °C for 30 min. The obtained suspension was
cooled to 10 °C, filtered, and the solid was washed with cold toluene (2× 3 mL) to afford 7-nitro-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (1) as
yellow solid (2.2 g), Yield: 71 %, M.p. 224–225 oC.
Synthesis of 7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (2)
A mixture of 7-nitro-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (1) (2.81 g, 10 mmol) and tin chloride dihydrate (10.4 g, 55 mmol) in ethanol (30 mL)
was ultrasonicated at room temperature for 2 h. The reaction mixture was diluted with water (120 mL), made alkaline with aqueous ammonia and
extracted with dichloromethane (3 × 100 mL). The combined organic layer was dried over sodium sulphate and concentrated under reduced pressure to
give 7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (2) as a yellow solid (2.2 g).
7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (2)
Yellow solid, Yield: 90 %. M.p. 244  245 oC. IR (KBr, cm-1): 3329  3337 (N-H), 2849  3204 (C-H), 1676 (C=O), 1570  1603 (C=N), 1500 (C=C), 1340 (C-N). 1H
NMR (400 MHz, DMSO-d6) δ: 4.05 (br. s, H2C(3)); 5.18 (br. s, H2N, D2O exchangeable); 6.40 (d, J = 2.20 Hz, HC(6)); 6.78 (dd, J = 8.56, 2.32 Hz, HC(8)); 6.93
(d, J = 8.56 Hz, HC(9)); 7.38  7.57 (m, HC(2', 3', 4', 5' and 6')); 10.05 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.0 (C(3)); 113.5
(C(6)); 118.1 (C(8)); 122.1 (C(9)); 127.3 (C(11)); 128.1 (C(3' and 5')); 129.2 (C(10)); 129.2 (C(2' and 6')); 129.9 (C(4')); 139.5 (C(1')); 144.0 (C(7)); 169.7 (C(5));
170.0 (C(2)). HR-MS: 252.1138 ([M + H]+, C15H14N3O+; calc. 252.1131).
General procedure for the synthesis of compounds (3a–n)
A mixture of the appropriate aromatic aldehydes (8a–n) (1.01 mmol) and 7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (2) (1.0 mmol) in
ethanol (7.0 mL) and few drops of glacial acetic acid was refluxed for 2-4 h, until 7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one is fully consumed.
The reaction mixture was cooled to room temperature and the precipitates formed were collected by suction filtration. The crude product was washed
thoroughly with ethanol (2 × 3 mL), to afford fine powder solid products (3a–n) in good yield.
7-(benzylideneamino)-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3a)
Off-white solid, Yield: 80 %. M.p. 123  124 oC. IR (KBr, cm-1): 2831  3061 (C-H), 1662  1680 (C=O), 1568  1628 (C=N), 1489 (C=C). 1H NMR (400 MHz,
DMSO-d6) δ: 4.16 (br. s, H2C(3)); 7.08 (d, J = 2.01 Hz, HC(6)); 7.31 (d, J = 8.78 Hz, HC(9)); 7.40  7.59 (m, HC(2', 3', 4', 5', 6', 8, 15, 16 and 17)); 7.87 (d, J =
6.56 Hz, HC(14 and 18)); 8.59 (s, HC(12)); 10.61 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.1 (C(3)); 122.0 (C(9)); 122.9 (C(6));
124.5 (C(8)); 126.9 (C(11)); 128.3 (C(15 and 17)); 128.7 (C(3' and 5')); 128.8 (C(2' and 6')); 129.2 (C(14 and 18)); 130.2 (C(16)); 131.6 (C(4')); 135.8 (C(10)); 137.9
(C(13)); 139.0 (C(1')); 145.5 (C(7)); 161.1 (C(12)); 169.4 (C(5)); 170.2 (C(2)). HR-MS: 340.1458 ([M + H]+, C22H18N3O+; calc. 340.1444).
7-(4-fluorophenyl)methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3b)
Yellow solid, Yield: 85 %. M.p. 221  222 oC. IR (KBr, cm-1): 1682 (C=O), 1587  1626 (C=N), 1487 (C=C), 1014 (C-F). 1H NMR (400 MHz, DMSO-d6) δ: 4.17 (br.
s, H2C(3)); 7.08 (d, J = 2.20 Hz, HC(6)); 7.26  7.58 (m, HC(2', 3', 4', 5', 6', 8, 9, 15 and 17)); 7.93 (dd, J = 8.56, 5.84 Hz, HC(14 and 18)); 8.59 (s, HC(12));
10.61 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.0 (C(3)); 115.7 (C(15)); 116.0 (C(17)); 122.0 (C(9)); 122.8 (C(6)); 124.6 (C(8)); 126.9
(C(11)); 128.3 (C(3' and 5')); 129.2 (C(2' and 6')); 130.2 (C(4')); 130.9 (C(14)); 131.0 (C(18)); 132.5 (C(13)); 137.9 (C(10)); 138.9 (C(1')); 145.4 (C(7)); 159.7 (C(12));
164.0 (d, 1J(C,F)= 247.84 Hz, C(16)); 169.3 (C(5)); 170.2 (C(2)). HR-MS: 358.1365 ([M + H]+, C22H17FN3O+; calc. 358.1350).
7-(4-nitrophenyl)methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3c)
Yellow solid, Yield: 85 %. M.p. 281  282 oC. IR (KBr, cm-1): 2955  3186 (C-H), 1670 (C=O), 1599  1628 (C=N), 1489  1495 (C=C), 1342 and 1520 (NO2). 1H
NMR (400 MHz, DMSO-d6) δ: 4.17 (br. s, H2C(3)); 7.19 (d, J = 2.08 Hz, HC(6)); 7.33 (d, J = 8.68 Hz, HC(9)); 7.40  7.55 (m, HC(2', 3', 4', 5' and 6')); 7.65 (dd,
J = 8.56, 2.20 Hz, HC(8)); 8.12 (d, J = 8.68 Hz, HC(14 and 18)); 8.31 (d, J = 8.68 Hz, HC(15 and 17)); 8.78 (s, HC(12)); 10.66 (br. s, HN, D2O
exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.1 (C(3)); 122.1 (C(9)); 123.5 (C(6)); 123.9 (C(15 and 17)); 124.7 (C(8)); 126.9 (C(11)); 128.3 (C(3' and 5'));
129.2 (C(2' and 6')); 129.6 (C(14 and 18)); 130.3 (C(4')); 138.6 (C(10)); 138.9 (C(1')); 141.3 (C(13)); 144.6 (C(7)); 148.8 (C(16)); 159.1 (C(12)); 169.3 (C(5)); 170.2
+
(C(2)). HR-MS: 385.1306 ([M + H]+, C22H17N4O3 ; calc. 385.1295).
Methyl- 4-(2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-7-yl)iminomethylbenzoate (3d)
Yellow solid, Yield: 76 %. M.p. 209  210 oC. IR (KBr, cm-1): 1724 (ester C=O), 1676 (amide C=O), 1605  1618 (C=N), 1491 (C=C), 1109 and 1275 (C-O). 1H
NMR (400 MHz, DMSO-d6) δ: 3.87 (s, OCH3); 4.17 (br. s, H2C(3)); 7.15 (br. s, HC(6)); 7.31 (d, J = 8.56 Hz, HC(9)); 7.39  7.56 (m, HC(2', 3', 4', 5' and 6'));
7.62 (d, J = 7.96 Hz, HC(8)); 7.95 – 8.15 (m, HC(14, 15, 17 and 18)); 8.70 (s, HC(12)); 10.63 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6)
11
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10.1002/cbdv.202000342
Chemistry & Biodiversity
Chem. Biodiversity
δ: 52.3 (OCH3); 57.1 (C(3)); 122.1 (C(9)); 123.3 (C(6)); 124.6 (C(8)); 127.0 (C(11)); 128.3 (C(3' and 5')); 128.8 (C(2' and 6')); 129.3 (C(14 and 18)); 129.5 (C(15 and
17)); 130.3 (C(4')); 131.7 (C(16)); 138.3 (C(10)); 138.9 (C(1')); 139.8 (C(13)); 144.0 (C(7)); 160.0 (C(12)); 165.8 (C=O, ester); 169.3 (C(5)); 170.2 (C(2)). HR-MS:
+
398.1512 ([M + H]+, C24H20N3O3 ; calc. 398.1499).
7-[4-(dimethylamino)phenyl]methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3e)
Yellow solid, Yield: 70 %. M.p. 248  249 oC. IR (KBr, cm-1): 1686 (amide C=O), 1585  1610 (C=N), 1481 (C=C), 1362 (C-N, aromatic). 1H NMR (400 MHz,
DMSO-d6) δ: 2.98 (s, N(CH3)2); 4.15 (br. s, H2C(3)); 6.73 (d, J = 8.80 Hz, HC(15 and 17)); 6.97 (d, J = 2.20 Hz, HC(6)); 7.26 (d, J = 8.68 Hz, HC(9)); 7.40 
7.56 (m, HC(2', 3', 4', 5', 6' and 8)); 7.67 (d, J = 8.80 Hz, HC(14 and 18)); 8.36 (s, HC(12)); 10.53 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz,
DMSO-d6) δ: 39.6 (N(CH3)2); 57.0 (C(3)); 111.3 (C(15 and 17)); 121.9 (C(9)); 122.4 (C(6)); 123.5 (C(13)); 124.4 (C(8)); 126.9 (C(11)); 128.2 (C(3' and 5')); 129.2
(C(2' and 6')); 130.1 (C(4')); 130.3 (C(14 and 18)); 136.9 (C(10)); 139.0 (C(1')); 146.5 (C(7)); 152.4 (C(16)); 160.2 (C(12)); 169.4 (C(5)); 170.1 (C(2)). HR-MS:
383.1878 ([M + H]+, C24H23N4O+; calc. 383.1866).
7-[(3-bromo-2-fluoro-phenyl)methyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3f)
Yellow solid, Yield: 72 %. M.p. 222  223 oC. IR (KBr, cm-1): 1684 (C=O), 1610 (C=N), 1491 (C=C), 1016 (C-F). 1H NMR (400 MHz, CDCl3) δ: 4.31 (br. s, H2C(3));
6.98  7.06 (m, HC(17)); 7.10 (d, J = 1.24 Hz, HC(6)); 7.14  7.27 (m, HC(8)); 7.30  7.42 (m, HC(3', 4', 5' and 9)); 7.46  7.61 (m, HC(2', 6' and 16)); 7.98 (t,
J = 6.72 Hz, HC(18)); 8.58 (s, HC(12)); 9.57 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, CDCl3) δ: 56.7 (C(3)); 109.6 (C(15)); 122.2 (C(9)); 123.5
(C(17)); 124.7 (C(6)); 124.9 (C(13)); 125.4 (C(8)); 126.9 (C(4')); 128.0 (C(11)); 128.3 (C(3' and 5')); 129.7 (C(2' and 6')); 130.4 (C(18)); 136.4 (C(16)); 137.4 (C(10));
139.1 (C(1')); 146.6 (C(7)); 153.2 (C(12)); 159.0 (d, 1J(C,F)= 252.22 Hz, C(14)); 170.7 (C(5)); 172.0 (C(2)). HR-MS: 436.0457 ([M + H]+, C22H16BrFN3O+; calc.
436.0455).
7-[(4-bromo-2-fluoro-phenyl)methyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3g)
Off-white solid, Yield: 70 %. M.p. 221  222 oC. IR (KBr, cm-1): 2843  3178 (C-H), 1678 (C=O), 1599  1608 (C=N), 1492 (C=C), 1012 (C-F). 1H NMR (400 MHz,
DMSO-d6) δ: 4.16 (br. s, H2C(3)); 7.14 (d, J = 2.32 Hz, HC(6)); 7.30 (d, J = 8.68 Hz, HC(9)); 7.41  7.54 (m, HC(2’, 3', 4', 5', 6’ and 17)); 7.62 (dd, J = 8.62, 2.38
Hz, HC(8)); 7.71 (dd, J = 10.21, 1.65 Hz, HC(15)); 7.97 (t, J = 8.13 Hz, HC(18)); 8.68 (s, HC(12)); 10.63 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz,
DMSO-d6) δ: 57.0 (C(3)); 119.5 (C(15)); 122.1 (C(9)); 122.7 (C(13)); 123.5 (C(6)); 124.3 (C(8)); 125.5 (C(16)); 126.9 (C(11)); 128.2 (C(17)); 128.2 (C(3' and 5'));
129.2 (C(4')); 129.2 (C(2' and 6')); 130.2 (C(18)); 138.4 (C(10)); 138.9 (C(1')); 145.0 (C(7)); 152.8 (C(12)); 161.6 (d, 1J(C,F)= 255.86 Hz, C(14)); 169.3 (C(5)); 170.2
(C(2)). HR-MS: 436.0456 ([M + H]+, C22H16BrFN3O+; calc. 436.0455).
7-[(4-bromo-2-chloro-phenyl)methyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3h)
Yellow solid, Yield: 75 %. M.p. 219  220 oC. IR (KBr, cm-1): 2885  3180 (C-H), 1697 (C=O), 1606 – 1620 (C=N), 1487 (C=C), 694 (C-Cl), 586 (C-Br). 1H NMR
(400 MHz, DMSO-d6) δ: 4.17 (br. s, H2C(3)); 7.11 (d, J = 2.08 Hz, HC(6)); 7.31 (d, J = 8.56 Hz, HC(9)); 7.39  7.54 (m, HC(2’, 3', 4', 5' and 6’ )); 7.56  7.67 (m,
HC(8 and 15)); 7.84 (d, J = 1.59 Hz, HC(17)); 8.00 (d, J = 8.56 Hz, HC(14)); 8.75 (s, HC(12)); 10.64 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz,
DMSO-d6) δ: 57.1 (C(3)); 122.1 (C(9)); 123.6 (C(6)); 124.3 (C(8)); 125.4 (C(11)); 126.9 (C(16)); 128.2 (C(3' and 5')); 129.2 (C(2' and 6')); 129.7 (C(4')); 130.2
(C(15)); 130.8 (C(14)); 131.8 (C(10)); 132.2 (C(17)); 135.8 (C(13)); 138.5 (C(1')); 138.9 (C(18)); 144.9 (C(7)); 155.7 (C(12)); 169.2 (C(5)); 170.2 (C(2)). HR-MS:
452.0155 ([M + H]+, C22H16BrClN3O+; calc. 452.0160).
7-[2-fluoro-4-(trifluoromethyl)phenyl]methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3i)
Pale yellow solid, Yield: 77 %. M.p. 228  229 oC. IR (KBr, cm-1): 2895  3196 (C-H), 1680 (C=O), 1612  1620 (C=N), 1492 (C=C), 1132 (C-F). 1H NMR (400
MHz, DMSO-d6) δ: 4.17 (br. s, H2C(3)); 7.18 (d, J = 1.36 Hz, HC(6)); 7.32 (d, J = 8.56 Hz, HC(9)); 7.39  7.55 (m, HC(2’, 3', 4', 5' and 6’ )); 7.64  7.68 (m,
HC(8 and 15)); 7.81 (d, J = 10.40 Hz, HC(17)); 8.23 (t, J = 7.52 Hz, HC(18)); 8.78 (s, HC(12)); 10.66 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz,
DMSO-d6) δ: 57.0 (C(3)); 113.9 (C(15)); 121.5 (C(17)); 121.7 (CF3); 122.1 (C(9)); 123.8 (C(6)); 124.5 (C(8)); 126.9 (C(11)); 127.0 (C(13)); 128.3 (C(3' and 5')); 129.1
(C(18)); 129.2 (C(2' and 6')); 130.2 (C(4')); 132.8 (C(16)); 138.7 (C(10)); 138.9 (C(1')); 144.7 (C(7)); 152.6 (C(12)); 161.5 (d, 1J(C,F)= 253.67 Hz, C(14)); 169.3
(C(5)); 170.2 (C(2)). HR-MS: 426.1239 ([M + H]+, C23H16F4N3O+; calc. 426.1224).
7-[(4-fluoro-3-nitro-phenyl)methyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3j)
Pale yellow solid, Yield: 80 %. M.p. 258  259 oC. IR (KBr, cm-1): 3063  3288 (C-H), 1691 (C=O), 1612 (C=N), 1487 (C=C), 1342 and 1531 (NO2), 1012 (C-F). 1H
NMR (400 MHz, DMSO-d6) δ: 4.18 (br. s, H2C(3)); 7.17 (d, J = 2.32 Hz, HC(6)); 7.33 (d, J = 8.68 Hz, HC(9)); 7.40  7.55 (m, HC(2’, 3', 4', 5' and 6’ )); 7.62 (dd,
J = 8.68, 2.32 Hz, HC(8)); 7.71 (dd, J = 11.13, 8.80 Hz, HC(17)); 8.29 (ddd, J = 8.65, 4.31, 2.08 Hz, HC(18)); 8.61 (dd, J = 7.46, 1.96 Hz, HC(14)); 8.73 (s,
HC(12)); 10.65 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.0 (C(3)); 119.2 (C(17)); 122.1 (C(9)); 123.1 (C(6)); 124.8 (C(8)); 126.0
(C(14)); 126.9 (C(11)); 128.3 (C(3' and 5')); 129.2 (C(2' and 6')); 130.3 (C(4')); 133.00 (C(13)); 135.6 (C(18)); 137.2 (C(15)); 138.4 (C(10)); 138.9 (C(1')); 144.5 (C(7));
+
156.0 (d, 1J(C,F) = 265.34 Hz, C(16)); 158.0 (C(12)); 169.3 (C(5)); 170.2 (C(2)). HR-MS: 403.1216 ([M + H]+, C22H16FN4O3 ; calc. 403.1201).
5-phenyl-7-[(2,4,6-trifluorophenyl)methyleneamino]-1,3-dihydro-1,4-benzodiazepin-2-one (3k)
Pale yellow solid, Yield: 75 %. M.p. 241  242 oC. IR (KBr, cm-1): 3034  3196 (C-H), 1687 (C=O), 1587  1620 (C=N), 1483 (C=C), 1043 (C-F). 1H NMR (400
MHz, DMSO-d6) δ: 4.16 (br. s, H2C(3)); 7.06 (d, J = 2.32 Hz, HC(6)); 7.26  7.57 (m, HC(2’, 3', 4', 5', 6’, 8, 9, 15 and 17)); 8.53 (s, HC(12)); 10.62 (br. s, HN,
12
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10.1002/cbdv.202000342
Chemistry & Biodiversity
Chem. Biodiversity
D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.0 (C(3)); 101.6 (t,2J(C,F)= 26.78 Hz, C(15 and 17)); 110.4 (t,2J(C,F)= 12.84 Hz, C(13)); 122.1 (C(9));
122.8 (C(8)); 124.3 (C(6)); 126.9 (C(11)); 128.2 (C(3' and 5')); 129.2 (C(2' and 6')); 130.2 (C(4')); 138.3 (C(10)); 139.0 (C(1')); 145.7 (C(7)); 150.9 (C(12)); 161.8 (dd,
1J(C,F)= 256.59 Hz, 3J(C,F)= 15.67 Hz, C(14 and 18)); 163.4 (dt, 1J(C,F)= 250.76 Hz, 3J(C,F)= 16.77 Hz, C(16)); 169.3 (C(5)); 170.2 (C(2)). HR-MS: 394.1176 ([M +
H]+, C22H15F3N3O+; calc. 394.1162).
7-[(4-bromo-2,6-difluoro-phenyl)methyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3l)
Pale yellow solid, Yield: 70 %. M.p. 228  229 oC. IR (KBr, cm-1): 2970  3049 (C-H), 1680 (C=O), 1612 (C=N), 1494 (C=C), 1045 (C-F). 1H NMR (400 MHz,
DMSO-d6) δ: 4.17 (br. s, H2C(3)); 7.08 (d, J = 2.20 Hz, HC(6)); 7.31 (d, J = 8.56 Hz, HC(9)); 7.40  7.62 (m, HC(2’, 3', 4', 5', 6’, 8, 15 and 17)); 8.54 (s,
HC(12)); 10.63 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.0 (C(3)); 112.7 (C(13)); 116.2 (C(15)); 116.4 (C(17)); 122.1 (C(9)); 122.9
(C(8)); 124.3 (C(6)); 124.7 (C(16)); 126.9 (C(11)); 128.2 (C(3' and 5')); 129.2 (C(2' and 6')); 130.2 (C(4')); 138.5 (C(10)); 138.9 (C(1')); 145.6 (C(7)); 151.0 (C(12));
161.0 (d, 1J(C,F)= 266.79 Hz, C(14 and 18)); 169.3 (C(5)); 170.2 (C(2)). HR-MS: 454.0358 ([M + H]+, C22H15BrF2N3O+; calc. 454.0361).
7-[1-naphthylmethyleneamino]-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3m)
Yellow solid, Yield: 72 %. M.p. 251  252 oC. IR (KBr, cm-1): 2947  3053 (C-H), 1689 (C=O), 1614 (C=N), 1489 (C=C). 1H NMR (400 MHz, DMSO-d6) δ: 4.19 (br.
s, H2C(3)); 7.20 (d, J = 1.84 Hz, HC(6)); 7.35 (d, J = 8.68 Hz, HC(9)); 7.40  7.73 (m, HC(2’, 3', 4', 5', 6’, 2’’, 3’’, 8 and 15)); 8.01 (d, J = 7.95 Hz, HC(1’’)); 8.09
(d, J = 8.07 Hz, HC(4’’)); 8.13 (d, J = 7.21 Hz, HC(16)); 9.15 (d, J = 8.32 Hz, HC(14)); 9.20 (s, HC(12)); 10.63 (br. s, HN, D2O exchangeable). 13C NMR (100
MHz, DMSO-d6) δ: 57.1 (C(3)); 122.0 (C(4'')); 123.1 (C(9)); 124.4 (C(3'')); 124.6 (C(6)); 125.4 (C(8)); 126.3 (C(15)); 127.0 (C(11)); 127.5 (C(1'')); 128.3 (C(3' and
5')); 128.7 (C(2'')); 129.3 (C(2' and 6')); 130.2 (C(14)); 130.4 (C(4')); 130.7 (C(18)); 130.8 (C(17)); 132.1 (C(16)); 133.4 (C(10)); 137.9 (C(13)); 139.0 (C(1')); 146.0
(C(7)); 160.9 (C(12)); 169.4 (C(5)); 170.2 (C(2)). HR-MS: 390.1612 ([M + H]+, C26H20N3O+; calc. 390.1601).
7-[3-(4-fluorophenyl)-1-phenyl-pyrazol-4-yl]methyleneamino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (3n)
Pale yellow solid, Yield: 88 %. M.p. 267  268 oC. IR (KBr, cm-1): 1685 (C=O), 1593  1622 (C=N), 1496 (C=C), 1012 (C-F). 1H NMR (400 MHz, DMSO-d6) δ:
4.16 (br. s, H2C(3)); 7.03 (d, J = 2.08 Hz, HC(6)); 7.26  7.54 (m, HC(2’, 3', 4', 5', 6’, 2’’, 6’’, 3’’’, 4’’’, 5’’’, 8 and 9)); 7.84 (dd, J = 8.44, 5.62 Hz, HC(3’’ and 5’’));
7.98 (d, J = 7.95 Hz, HC(2’’’ and 6’’’)); 8.48 (s, HC(14)); 9.11 (s, HC(12)); 10.58 (br. s, HN, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6) δ: 57.1
(C(3)); 115.4 (C(3'')); 115.6 (C(5'')); 118.8 (C(2''' and 6''')); 119.6 (C(13)); 122.0 (C(9)); 122.9 (C(6)); 124.2 (C(8)); 126.9 (C(11)); 127.2 (C(4''')); 128.2 (C(3' and 5'));
128.4 (C(1'')); 129.2 (C(2' and 6')); 129.6 (C(3''' and 5''')); 129.7 (C(14)); 130.1 (C(4')); 130.7 (C(2'')); 130.8 (C(6'')); 137.5 (C(10)); 138.8 (C(1')); 139.0 (C(1'''));
146.0 (C(7)); 151.6 (C(15)); 152.8 (C(12)); 162.4 (d, 1J(C,F)= 244.20 Hz, C(4'')); 169.4 (C(5)); 170.1 (C(2)). HR-MS: 500.1894 ([M + H]+, C31H23FN5O+; calc.
500.1881).
Synthesis of 4-fluoro-N-(2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-7-yl)benzamide (4)
To a stirred solution of 7-amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (2) (1.2 mmol) and triethyl amine (2.4 mmol) in dichloromethane (3.0 mL)
under nitrogen 4-fluorobenzoyl chloride (9) (1.32 mmol) was added at 24 oC and stirred for 2 h. The reaction mixture was quenched with water and the
precipitate formed was collected by filtration. The crude solid was washed thoroughly with dichloromethane (2 × 2 mL) and water (2 × 2 mL) and dried to
obtain 4-fluoro-N-(2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-7-yl)benzamide (4) as off-white solid.
4-fluoro-N-(2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-7-yl)benzamide (4)
Off-white solid, Yield: 65 %. M.p. 285  286 oC. IR (KBr, cm1): 2839  3063 (C-H), 1683 and 1647 (C=O), 1608 (C=N), 1500 (C=C), 1012  1028 (C-F). 1H NMR
(400 MHz, DMSO-d6) δ: 4.14 (br. s, H2C(3)); 7.24 (d, J = 9.03 Hz, HC(9)); 7.33 (t, J = 8.22 Hz, HC(15 and 19)); 7.42  7.56 (m, HC(2’, 3', 4', 5' and 6’ )); 7.71 (d,
J = 2.26 Hz, HC(6)); 7.95  8.06 (m, HC(8, 16 and 18)); 10.35 (br. s, HN(12), D2O exchangeable); 10.49 (br. s, HN(1), D2O exchangeable); 13C NMR (100
MHz, DMSO-d6) δ: 57.0 (C(3)); 115.2 (C(16)); 115.4 (C(18)); 121.3 (C(9)); 121.4 (C(6)); 123.7 (C(8)); 126.4 (C(11)); 128.3 (C(3' and 5')); 129.3 (C(2' and 6')); 130.2
(C(15)); 130.3 (C(19)); 130.4 (C(4')); 130.9 (C(14)); 133.9 (C(10)); 135.5 (C(1')); 139.0 (C(7)); 164.1 (d, 1J(C,F)= 247.90 Hz, C(17)); 164.4 (C(13)); 169.3 (C(5)); 170.2
(C(2)). HR-MS: 374.1307 ([M + H]+, C22H17FN3O2+; calc. 374.1299).
PHARMACOLOGY
In vivo anticonvulsant activity of target compounds
Adult male Wistar rats (180-200g) were purchased from National Institute of Biosciences, Pune and housed for one week at the institute animal house
separately in groups of six animals per cage at standard laboratory conditions. Animals had free access to food and water ad libitum. The research
protocol was approved by the Institutional Animal Ethics Committee and performed in accordance with the guidelines of Committee for Control and
Supervision of Experimentation on Animals (CPCSEA), Government of India on animal experimentation (CPCSEA approval no -
CPCSEA/PCP/PCL03/2018). The rats were divided into 19 groups of 8 animals each. Group 1 served as normal control and was treated with vehicle 2 % w/v
of Tween 80 orally. Group 2 served as Picrotoxin convulsive control and was treated with vehicle 2 % w/v of Tween 80 and Picrotoxin was administered at
3 mg/kg i.p. Group 3 served as a standards group and received Diazepam 1 mg/kg orally. Group 4 to 19 served as 3a-3n, 4 and 2 (2.5 mg/kg). The animals
were administered orally with respective treatment 24 hours before the experimentation and at 1 hour before the experimentation. After which, the
Picrotoxin was injected at a dose of 3 mg/kg, i.p. to all the animals except normal control group. The time of onset of convulsions, their severity was
13
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10.1002/cbdv.202000342
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scored; a number of wet dog shakes and time of appearance of tonic response as well as the death of animals, if any were noted. The following were the
guidelines used to grade the severity of convulsions: O- No convulsive behavior, 1-head or body twitching, 2- clonic forelimb convulsion, 3 –rearing, 4-
falling back, 5- when 3 or 4 last for more than 5 min, 6- tonus, 7 –convulsion for more than 10 min. The data were analyzed by One-way ANOVA followed

36]
by Dunnet’s ‘t’ test using GraphPad Prism v. 5 demos. The values are expressed as mean ± SEM, n = 8. p<0.05 was considered significant. [34
Acute oral toxicity study
Adult female Wistar rats (180-200g) were used to carry out an acute toxicity study. As per OECD (Organization for economic co-operation and
development) guidelines 423, acute oral toxicity study was carried out for compounds 3a to 3n, 4 and 2. The animals were alienated into 17 groups of 3
animals each as vehicle control and compounds 3a-3n, 4, 2 groups. Overnight fasted healthy female Wistar Albino rats (n = 3) were administered orally
compound 3a-3n, 4, 2 (in 2 % w/v Tween 80) at a dose of 5 mg/kg body weight. After dosing, the animals were observed individually at least once during
the first 30 min, and thereafter periodically during the first 24 h, with special attention given during the first 4 h, and subsequently daily thereafter, for a
total of 14 days. Here, different signs of toxicity such as changes in skin and fur, eyes, mucous membrane, respiratory, CNS activity, and the behavior
pattern were observed. Main Attention was focused to observe tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. As per OECD
guidelines, acute toxicity test was repeated with doses of 50 mg/kg body weight.
In silico molecular docking
Ligand Preparation
The structures of all the derivatives were converted into 3D before analysis. Merck Molecular Force Field (MMFF) with distance-dependent dielectric
function and energy gradient of 0.001 kcal/mol with iteration limit to 10,000 was utilized to perform energy optimization of all the molecules. [37] On the
basis of energy minimization is that the drug binds to effectors/receptors in the most stable form, i.e., the minimum energy form. The optimized
compounds were subjected to conformational analysis and energy minimization using Monte Carlo conformational search. Low energy conformers of all
the structures were generated and which was utilized further for analysis. The conformational analysis includes moving the atoms of a molecule in such a
way that the total energy of the system is reduced based on empirical representation of the interaction energy of the atoms of a molecule. MMFF method
was used to minimize each atom in the designed ligands. [38]
Protein preparation and molecular docking
Ernst et al. have reported the homology model of the diazepam-bound GABAA receptor which was retrieved from the supplementary material of their
published paper. [39] All the water molecules were removed from the crystal structure of GABAA receptor. AutoDockVina tool was used for the protein
synthesis and Grid generation. Polar hydrogens were added into the structure and Gasteiger charges were computed and applied accordingly. Missing
residues in the proteins were also added at the time of preparation. Molecular docking study of Diazepam and most active compound 3e were executed
with AutoDockVina tool. A docking grid box was built with 40, 40 and 40 points in x = 43.640, y = 43.866 and z = 9.3290 directions. Using the gradient
optimization algorithm and an empirical scoring function the molecular docking was conducted to generate the best binding affinity or fitness of protein-
ligand binding poses between compounds as GABAA receptor. The best binding conformations of ligands were selected and analyzed using
AutoDockVina Tool as well as in Discovery Studio 4.0.
Molecular dynamics simulation
Molecular dynamics simulation was carried out to understand the dynamic behavior of protein-ligand complex. Here, best docked complexes of
compound 3e and diazepam with GABAA receptor were used for MD simulation study using Gromacs 5.1.1. [40, 41] 7.0 pH was used to fix the protonation
states to their normal states. The CHARMM27 all atoms force field was used for the simulation. To setup the system protein was surrounded by a cubic
water box of SPC3 water molecules and it was extended 10 Å from the protein. The topology of the small molecules was generated using the SwissParam.
As a part of system setup, the periodic boundary condition (PBC) was applied in all around the molecule. Suitable numbers of Na+ and Cl- charges were
used to neutralize the system. Each system was minimized using the steepest descent algorithm for 10,000 steps. For each docked complex 1 ns of time
span MD simulation was performed at constant pressure (1 atm) and temperature (300 K). At each 1 ps the iterations were documented to analysis of MD
simulations. The root mean-square deviation (RMSD) and radius of gyration were used to analyze the performance of each system. [42]
Prediction of ADMET properties for the designed derivatives
In the process of drug discovery, during the lead identification and optimization ADMET (absorption, distribution, metabolism, excretion, and toxicity)
prediction plays an important role. ADMET properties were predicted using the online tool AdmetSAR. [43] It is free software available online which was
accessed on 10th September 2018. It provides all the data related to ADMET properties.
Supplementary Material
Supporting information for this article given separately.
Acknowledgements
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10.1002/cbdv.202000342
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PN would like to thank Dr. Bhanu Manjunath, Director, Syngenta Research & Technology Centre, Goa, Dr. Sitaram Pal, HOD, Process Research, Syngenta
Research & Technology Centre, Goa and the management of the Syngenta Research & Technology Centre, Goa, for their continuous encouragement and
support. We are grateful to the Principal Dr. S. R. Thopate and Dr. A. B. Nikumbh (HOD), S.S.G.M College, Kopargaon, Ahmednagar (MH) for providing
research facilities and constant encouragement. We are also thankful to Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth
(Deemed to be University), Pune (MH) for providing necessary facility to carry out pharmacological study. The author (Tansir Ahamad) thanks to
Researchers Supporting Project number (RSP-2020/06), King Saud University, Riyadh, Saudi Arabia. The author gratefully acknowledges the support by
the Operational Program Research, Development and Education - European Regional Development Fund (project no.
CZ.02.1.01/0.0/0.0/16_019/0000754) of the Ministry of Education, Youth and Sports of the Czech Republic.
Author Contribution Statement
PN performed all experiments, purified all compounds, analyzed the data and summarized the results. AS and KD tested all compounds for their
anticonvulsant activity. TA helped in the compiling the data of the manuscript. SG, MG and SNS conceived and designed this research and wrote the
manuscript. All authors have contributed to the final version and approved the final manuscript.
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