New hybrids based on benzimidazole and diazepine moieties: design, synthesis, characterization, molecular docking studies and their in vitro interactions with benzodiazepine receptors

Abstract Benzodiazepines are one of the most widely prescribed pharmacologic agents in the world. They are employed for numerous indications, including anxiety, insomnia, muscle relaxation, relief from spasticity caused by central nervous system pathology and epilepsy. In this work, we have synthesized some new hybrids based on benzimidazole and diazepine scaffolds from the reaction of suitable benzimidazole derivatives with glycine. NMR spectra, IR and mass as well as elemental analyses approved the structure of the title compounds. In vitro interactions of the title compounds were also examined on recombinant benzodiazepine receptors (αxβ2/3γ2, x = 1–3, 5) expressed in HEK293 cells. The results indicated that the title compounds exhibited suitable affinity for α1β2 γ2 subtype (K i = 16–29 nM). To achieve deeper insight into their interactions with benzodiazepine receptors, molecular dynamics simulation was employed. According to the results obtained from the molecular dynamics simulation, Pro85, Leu103, Pro101, Gln102, Ile79, Ser80, Pro17, Leu82 and Val84 interact with the most potent ligand by hydrophobic interactions and Asp86 and Leu87 interact with the ligand by hydrogen bond interactions. Communicated by Ramaswamy H. Sarma


Introduction
Benzodiazepines are a basic class of anxiolytics or tranquilizers compounds for treating conditions of general anxiety, and their synthesis and properties as such will be considered separately (Alexander & Joshi, 2020;Griessner et al., 2021;Kowark et al., 2022;van Beek et al., 2020).The mechanism of action of benzodiazepines is related to their interaction with specific benzodiazepine receptors, considering that as a result of binding, the affinity of inhibitory Gamma-Aminobutyric Acid (GABA) neurotransmitters to their respective receptors is increased, which strengthens the inhibitory action of the GABA (Costa, 2019;Faye et al., 2020;Cicala & Hartley, 1967;Kim et al., 2020;Vossen et al., 2020).The examined drugs flurezepam, temazepam and triazolam evidently raise the inhibitory effect of GABA on the The central nervous system (CNS).GABAA receptor pentamers are composed of subunits from at least five different families: a (1-6), b (1-4), c (1-3), q(1-3), d, e, p and h, among which the a, b and c subunits are not only necessary for the modulation of benzodiazepines but are also the basis of GABAergic neuronal plasticity.This receptor heterogeneity is actually considered promising target sites for the development of new selective therapeutic tools in CNS disorders (Sperk et al., 2020).Benzodiazepines can enhance the activity of the inhibitory GABA-ergenic system of the brain (Ben-Ari, 2021).Benzodiazepines can bind with specific moieties on the chloride ionophore and they do not react directly with the GABA receptors (Salzman, 2020).The increase in the flow of chloride ions through GABA-activated ion channels is the results of the benzodiazepin-receptor interaction and allosteric changes in the GABA receptors, which in turn increases the inhibitory activity of GABA (Solomon et al., 2019).
On the other hand, optimization of the benzimidazole scaffold as an important bioactive heterocyclic compound has resulted in many drugs like albendazole, mebendazole, thiabendazole as anti-helmintics; omeprazole, lansoprazole, pantoprazole as proton pump inhibitors; astemizole as antihistaminic; enviradine as antiviral; candesarten cilexitil and telmisartan as antihypertensives (Kabi et al., 2022;Madawali et al., 2019;Mulugeta & Samuel, 2022;Pathare & Bansode, 2021;Tahlan et al., 2019).Some studies on the anxiolytic activity of the compounds containing privileged scaffolds of benzodiazepine and benzimidazole in their structure have also been reported (Keenan et al., 1998;Maltsev et al., 2021).Moreover, potential anxiolytic agents, pyrido(1,2-a]benzimidazoles have been introduced as a new structural class of ligands for the benzodiazepine binding site on GABA-A receptors (Maryanoff et al., 1995).Molecular docking analysis of a2-containing GABAA receptors with benzimidazoles derivatives has been recently investigated by Bouayyadi et al. (2020).
Within this context and in continuation of our research work on the synthesis of new bioactive heterocyclic compounds (Faramarzi et al., 2020;Karimi et al., 2019;Mohammadi et al., 2020;Ramezani et al., 2018;Rastegarnia et al., 2020;Sobhani et al., 2019), in this work, benzimidazoles and benzodiazepines were combined and some new selective ligands 6,8-dihydroimidazo[4 0 ,5 0 :3,4]benzo [1,2-e][1, 4]diazepines for Bz/GABAA subtypes have been obtained.The results of their in vitro interactions with benzodiazepine receptors indicate that the new compounds showed high affinity for the subtype containing the R1 subunit.Molecular dynamics simulation was also used to gain a deeper insight into the interactions of the title compounds with benzodiazepine receptors.

Equipment and materials
The FTIR spectra were verified on potassium bromide pellets using a Tensor 27 spectrometer and only noteworthy absorptions were listed.The 13 C NMR (75 MHz) and 1 H NMR (300 MHz) spectra were obtained on a Bruker Avance DRX-300 spectrometer.Chemical shifts are described in ppm downfield from TMS as internal standard; the coupling constant is given as J value in Hz.The mass spectrum was recorded on a Varian Mat, CH-7 at 70 eV and ESI mass spectrum was measured using a Waters Micromass ZQ spectrometer.Elemental analysis was accomplished on a Thermo Finnigan Flash EA microanalyzer.Melting points were measured on an Electrothermal type-9100 melting-point apparatus.

Binding studies
GABA A /Bz receptor subtypes were achieved from NEN Life Sciences Products.All other chemicals were of reagent grade and were obtained from commercial suppliers.The binding assays to transfected cells membranes were accomplished as previously described (Besnard et al., 1997).In brief, the cell lines membranes were incubated in a volume of 500 lL which contained (axb2/3c2, x ¼ 1-3, 5) GABA A /Bz receptor subtypes at a concentration of 1-2 nM and the test compounds in the range 10 À 9 -10 À 5 M. Nonspecific binding was assigned by 10 À 5 M diazepam.Assays were incubated to equilibrium for 1 h at 4 � C. The potencies of the new synthesized compounds to inhibit (axb2/3c2, x ¼ 1-3, 5) GABA A /Bz receptor subtypes binding in the presence and absence of GABA were analyzed.The differences obtained were uttered as the GABA ratio, namely the ratios of the K i values obtained in the absence of GABA over the K i values obtained in the presence of GABA.

Simulation details
All molecular dynamics simulations were performed with Gromacs package version 5.1.2and CHARMM27 all atom force fields.Because the force field parameters for studied compounds are not present in the default version of the Gromacs software.The structure of the compounds 6a-d was optimized by using of B3LYP density functional method with the basis function of 6-31 G � .To control the optimization, frequency calculations were performed and virtual frequencies were not observed.All ab initio calculations were done by GAMESS software package (Schmidt et al., 1993).
The SwissParam web server (Zoete et al., 2011) was used to determine the force field parameters of the compounds 6a-d.Energy minimization was done using the steepest descent method (Luenberger & Ye, 1984) to eliminate the primary kinetic energy in each of the simulation boxes and to eliminate inappropriate contacts between the atoms.Each simulation box achieved a two-stage equilibrium in NVT and NPT ensemble.At this stage, the time of equilibration was considered 5 ns with time step 2 fs.Finally, molecular dynamics was performed by solving the second Newton equation for 100 ns with the 2 fs time step.The PME algorithm was used to calculate electrostatic interactions (Essmann et al., 1995).LINCS algorithm (Hess et al., 1997) was employed to fix the chemical bonds between the atoms of the protein and SETTLE algorithm (Miyamoto & Kollman, 1992) in the case of solvent molecules.To fix a constant temperature and pressure during the simulations, systems components were coupled with V-rescale and Nose-Hoover thermostat (Bussi et al., 2007) respectively, in each of the equilibration steps and molecular dynamics simulations.

Synthesis and structure of the new compounds 6a-d
The demand to discover new drugs based on diazepine scaffold such as Anxiolytics with fewer side effects, more optimized biocompatibility and convenient synthesis by commercially available and cheaper molecules prompted us to synthesize new compounds on this basis.
The spectral and analytical data were used to confirm the structure of the imidazobenzodiazepines 6a-d.For example, in the 1 H NMR spectrum of compound 6a, protons of methylene group appears at d 3.21 ppm and protons of methyl groups are observed at d 3.67 and 4.17 ppm.Also, there are four doublet signals (d ¼ 7.39, 7.89, 8.11 and 8.56 ppm) and a singlet signal (d ¼ 8.17 ppm) assignable to seven protons of aromatic rings.The 13 C NMR spectrum of compound 6a shows a signal at 169.2 ppm assigned to carbon for carbonyl group (C ¼ O).In addition, the 13 C NMR spectrum of compound 6a showed 12

In vitro binding studies
It is well-known that the R1-containing receptors play an important role in sedation and hypnosis/sleep control and therefore R1-selective ligands can represent valid aids in the treatment of insomnia.New diazepines 6a-d were screened for their binding interaction on recombinant rats (axb2/3c2, x ¼ 1-3, 5) GABA A /Bz receptor subtypes.As can be seen from Table 1, compounds 6a-d reveal a complete selectivity for the R1 subtype in comparison with diazepam.
Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces between the two molecules (Talhout et al., 2003).As can be deduced from Table 1, when methylene or methoxy groups are inserted in the structure of the title compounds (6c and 6d),  binding affinity increases compared to compounds 6a and 6b.Compound 6d with R 1 ¼ Me and R 2 ¼ OMe groups exhibits the best affinity for the R1 subtype (K i ¼ 16 ± 4 nM) compared to other title compounds.On the other hand, compound 6a with R 1 ¼ Me and R 2 ¼ Cl groups shows the lowest affinity for the R1 subtype (K i ¼ 29 ± 4 nM).
A condition necessary for the expression of anxiolytic activity of benzodiazepines is the presence of an electronegative group on C7 of the benzodiazepine system.The presence of a phenyl group on C5 of the system also increases the pharmacological activity of these compounds.As depicted in Scheme 1, there are aryl and N electronegative groups in the compounds 6a-d in similar positions compared to benzodiazepines which confirm their suitable selectivity for the R1 subtype.

Molecular docking studies
In this section, two types of calculations have been performed; first, molecular dynamics simulation and then docking calculation.Since the protein has several segments and considering all the segments in docking complicates the calculation, molecular dynamics simulation calculation was used to select the appropriate segment.Then, by sampling, the molecular dynamics simulation of the selected segment was used for docking calculations.
Molecular dynamics simulation was used to get a deeper insight into the interactions of the title compounds 6a-d with benzodiazepine receptors (a1).In Figure 1, the optimized structures of the compounds 6a-d have been shown.
The GABA A receptor with pdb code 1UW6 in the protein data bank was placed in the center of the simulation box (Jansen et al., 2008).This strain is the X-ray structure of the acetylcholine-binding protein with 2.2 Å resolution.The structure of this protein has been used in many cases as a model of GABA type A receptor in docking calculations of various compounds (Jansen et al., 2008).In the single cell of this strain, there are four different parts as shown in Figure 2.
As depicted in Figure 3, each part of the strain has five chains, which are called alpha-1, beta-1, alpha-2, beta-2 and gamma, respectively.The results have shown that GABA has two connection areas in the contact surface of alpha-1 with beta-1 and alpha-2 with beta-2.Also, the binding site of benzodiazepines has been reported in the area of the contact surface of gamma and alpha-1 (Zhu et al., 2018).
To select the appropriate segment from among the four segments shown in Figure 2, the molecular dynamics simulation of each segment was performed using Gromac software and CHARMM27 force field.To remove the initial kinetic  energy of the system and remove inappropriate contacts between atoms, the gradient descent algorithm was used to optimize the designed systems.Then, during two stages with a simulation time of 1 ns and a time step of 2 fs, they reached equilibrium in two sets of NVT and NPT of each of the designed systems, respectively.In the final stage, molecular dynamics simulation was performed for 50 ns with a time step of 2 fs.V-resclae and Nose-Hoover algorithms were used to control the temperature and pressure of the system components, respectively.The chemical bonding of non-solvent components was determined by LINCS algorithm and the chemical bonding of solvent molecules by SETTLE  algorithm.The root-mean-square-deviation (RMSD) graph of the alpha carbon of each subunit of each segment was calculated using the following equation, and the results are shown in Figure 4.
According to Figure 4, it can be seen that the stability of chains is more preserved in segment 2. Therefore, this segment was chosen for docking.To sample the simulation done, the free energy analysis method was used.Figure 5 shows the calculated Gibbs free energy for RMSD and R g vectors.
The structure corresponding to the maximum free energy was sampled from the simulation.This sampled structure was used for docking calculations.For example, 3D schematic representations of the interactions for compound 6a and diazepam in binding site of type A GABA protein have been shown Figures 6 and 7. Docking results of compound 6a-d have also been presented in Table 2 and compared to docking results of Diazepam and benzodiazepine.3D schematic representations of the interactions for compound 6b-d can be found in Supporting Information (Figures S1-S3).
The residues that form the binding site of the studied compounds and are common with the residues of the diazepam binding site are highlighted.From the docking results of the protein and different ligands in Table 2, it was found that the most interaction between the protein and the ligands is as follows: 6d > 6b > 6c > 6a.
As mentionedin binding studies, the compound 6d showed the best affinity for the R1 subtype (K i ¼ 16 ± 4 nM) and compound 6a with R 1 ¼ Me and R 2 ¼ Cl groups  revealed the lowest affinity for the R1 subtype (K i ¼ 29 ± 4 nM).Therefore, the results of the simulation show a good agreement with the experimental observations.In Table 2, the length of hydrogen bonds in angstroms is also presented.Imberty et al. (1991) have stated the relationship between hydrogen bond length and hydrogen bond strength as follows: Therefore, all hydrogen bonds calculated for the studied compounds fall within the range of strong interactions.Also, the strength of hydrogen bond for ligand 6d is higher than others.
By comparing the sequence of GABA with the list of sequences that participated in the interaction between ligands and the GABA respirator (Table 2), it can be seen that most of the desired sequences are in the same binding sites reported in Figure 3.
The electrophilicity index is known as a measure of toxicity of a compound and can be calculated from quantum calculations.The following equation was used to calculate this parameter.
In this equation, I, A and g are ionization potential, electron affinity and global hardness respectively.The obtained values are reported in Table 2.The greater the electrophilicity index value, the greater the toxicity of the drug.According to the table, it can be seen that the toxicity of substance 6d is lower than the others.

Conclusion
Four new imidazobenzodiazepines ligands were obtained from the reaction of appropriate derivatives of benzimidazole with glycine in HOAC in good yields.Their spectral and analytical data confirmed the structures of the title compounds.The binding interactions of the new compounds were also studied on recombinant benzodiazepine receptors.The results of binding interactions exhibited that the title compounds have a complete selectivity for the R1 subtype in comparison with diazepam (K i ¼ 16-29 nM).To explore the binding site of GABA protein with title ligands, molecular dynamics simulation was utilized.After the optimization of the ligands, the appropriate segment was selected by Gromax software and Gromos force field.Then, 3D schematic representations of the interactions for compounds 6a-d in binding site of type A GABA protein were obtained.Docking results of compound 6a-d were also compared to docking results of diazepam.The results showed that the most interaction between the protein and the ligands is in compound 6d with R 1 ¼ Me and R 2 ¼ OMe in which the results of the docking reveal a good agreement with the experimental observations.Also, an investigation on the effects of the new title compounds in rodents is in progress and will soon be published elsewhere.
carbon signals in the 114.3-157.3ppm region due to the carbon residue in the aromatic rings.The IR spectrum of compound 6a in KBr displays an absorption band at 1691 cm À 1 assignable to the C ¼ O group.Furthermore, the results of the mass spectroscopy (m/z 338 [M þ ) and elemental analyses strongly support the structure of compound 6a.

Figure 1 .
Figure 1.The Optimized structures of the compounds 6a-d.

Figure 2 .
Figure 2. Different segments of the 1UW6 single cell.

Figure 3 .
Figure 3. Subunits along with their positions in 1UW6.

Figure 4 .
Figure 4.The RMSD graph of the alpha carbon of each subunit of each segment; Black A, Red B, Green C, Blue D, Yellow E.

Figure 5 .
Figure 5.The calculated Gibbs free energy for RMSD and R g vectors.

Figure 6 .
Figure 6.3D schematic representations of the interactions for compound 6a in binding site of type A GABA protein.

Figure 7 .
Figure 7. 3D schematic representations of the interactions for Diazepam in binding site of type A GABA protein.
a K i values characterize the means ± SEM derived from three independent experiments, conducted in triplicate.b Diazepam was applied for comparison purposes in this set of assays.

Table 2 .
Comparison of the interaction status of different ligands with type A GABA protein.