Imidazo[4′,5′:3,4]Benzo[1,2-e][1,4]Diazepins as New Heterocyclic Systems: Synthesis, Characterization and Their in Vitro Interactions with Benzodiazepine Receptors

Abstract In this work, some new heterocyclic systems 10-(aryl)-3-alkyl-6-methyl-6,8-dihydroimidazo[4′,5′:3,4]benzo[1,2-e][1, 4]diazepin-7(3H)-ones were obtained from the reaction of o-N-methylamino-ketone benzimidazoles with glycine in good yields. The structures of the new compounds were confirmed by elemental analyses, IR, mass, and NMR spectra. Also, their interactions were studied in vitro on recombinant benzodiazepine receptors (αxβ2/3γ2, x = 1–3, 5) expressed in HEK293 cells. The results revealed that the new compounds displayed suitable affinity for α1β2 γ2 subtype (K i = 19–31 nM).


Introduction
Benzodiazepines have emerged as extremely effective drugs for treating neuropathic conditions. They are used in premedication before operational interventions in order to achieve ataraxia in the patient, as an adjuvant supplementary drug in treating epilepsy, tetanus, and other pathological conditions accompanied by skeletal muscle hypertonicity. [1][2][3] The anxiolytic effect of benzodiazepines is specific and unique, and it differs from sedative and hypnotic drugs of other classes. [4][5][6] Chlordiazepoxide was the first representative of this large group of compounds and it was synthesized in the 1930s and introduced into medical practice at the end of the 1950s. 7 Research data indicate that the mechanism of action of benzodiazepines lies in the stimulation of the benzodiazepin-GABA receptor complex. 8,9 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. 10 Benzodiazepines can enhance activity of the inhibitory GABA-ergenic system of the brain. 11 Benzodiazepines can bind with specific moieties on the chloride ionophore and they do not react directly with the GABA receptors. 12 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. 13 Furthermore, benzimidazole scaffolds have established as noteworthy heterocyclic compounds because of their existence in a wide range of bioactive compounds such as anti-parasitic, 14 anti-convulsant, 15 analgesic, 16 antiulcer, 17,18 anti-hypertensive, 19 antiviral, 20 anti-cancer, 21 anti-fungal, 22 anti-inflammatory and anti-coagulants agents 23,24 as well as proton pump inhibitors. 25 Optimization of the benzimidazole substituents 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. Also, benzimidazoles play a significant role in determining the function of a number of biologically important metal complexes. 26 In continuation of our research work on the synthesis of new bioactive heterocyclic compounds 27-32 based on the aforementioned literature data, in this work, 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 synthesized by the reaction of benzimidazole derivatives with appropriate reagents. The results of their in vitro interactions with benzodiazepine receptors indicate that the new compounds exhibit high affinity for the subtype containing the R1 subunit.

Equipment and materials
The FT-IR 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; 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.
All solvents were dehydrated according to standard procedures. Compounds 1a-e 33 and 3a-e, 34 4a-e and 5a-e 35 were obtained according to the published methods. Other reagents were commercially available.

General procedure for the synthesis of compounds 6a-e
A mixture of compounds 5a-e (17 mmol), glycine (17 mmol), water (50 mL), and triethylamine (15 mL) was stirred at rt for 6 h. Then, the mixture was treated with glacial acetic acid (100 mL) and heated to reflux for 6 h. After the mixture cooled, as much acetic acid was evaporated under reduced pressure, and crude product was purified by crystallization from ethanol to give new compounds 6a-e. 10-

Binding studies
GABA A /Bz receptor subtypes were obtained 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 carried out as previously described. 36 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 À9 -10 À5 M. Nonspecific binding was defined by 10 À5 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 compared. The differences obtained were expressed 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.

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-e were screening for their binding interaction on recombinant rat (axb2/3c2, x ¼ 1-3, 5) GABA A /Bz receptor subtypes. As can be seen from Table 1, compounds 6a-e exhibit a complete selectivity for the R1 subtype in comparison with diazepam. Binding affinity decreases with increased branching at the first carbon but increases with increasing the length of a linear alkyl substituent, suggesting that steric hindrance and hydrophobic interactions play dominant roles in binding. 37 In these compounds (6a-e), binding affinity increases upon adding a methylene group to the derivatives of diazepines 6a-e. Compound 6d with R ¼ Bu group shows the best affinity for the R1 subtype (K i ¼ 19 ± 4 nM) compared to other the title compounds. On the other hand, compound 6e with R ¼ iso-bu group displays the lowest affinity for the R1 subtype (K i ¼ 31 ± 4 nM). It seems that the steric effects of the isobutyl group have a significant result on binding interaction.
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. On the other hand, as depicted in Scheme 1, there are an aryl and N electronegative groups in compounds 6a-e in similar positions compared to benzodiazepines which confirms their suitable selectivity for the R1 subtype.
The structures of the new compounds were established by spectral and analytical data. In the 1 H NMR spectrum of these compounds, two protons of methylene group were appeared at d 3.20-3.25 ppm. The binding interactions of the new compounds were also studied on recombinant rat (axb2/3c2, x ¼ 1-3, 5) GABA A /Bz receptor subtypes. The results revealed that the title compounds have a complete selectivity for the R1 subtype in comparison with diazepam (K i ¼ 19-31 nM) and the binding affinity increases upon adding a methylene group to the derivatives of new diazepines.
Also, investigation on the effects of the new benzodiazepines in rodents is in progress and will soon be published elsewhere.

Disclosure statement
No potential conflict of interest was reported by the author(s).