Synthesis and Anticonvulsant Activity of Various Mannich and Schiff Bases of 1,5-Benzodiazepines

Benzodiazepines have a various behavioral effects in addition to their anxiolytic action. There is every reason to believe that the BZ/GABA receptor complex is involved in these effects, since GABAmimetic manipulations modify the effect of BZ in tests of convulsive activity, motor function, and appetitive behavior. 1,5-Benzodiazepines are biologically important molecules and are extensively used clinically as analgesic, hypnotic, sedative, and antidepressive agents. Hence, 1,5-Benzodiazepines were synthesized by condensation of o-phenylenediamine and ketones, for example, cyclohexanone and acetone in presence of sulfated zirconia (catalyst). Mannich bases were synthesized with acetophenone, p-nitroacetophenone, p-chloroacetophenone, and formaldehyde. Schiff bases were synthesized using Mannich base of 1,5-benzodiazepines with p-chloroaniline and p-chlorophenylsemicarbazide in the presence of glacial acetic acid. All the synthesized compounds were characterized by 1H NMR and IR spectral analyses. All the synthesized derivatives were evaluated at the dose of 30 mg/kg b.w for anticonvulsant activity by isoniazid induced convulsion model, and the compounds NBZD-3 and NBZD-8 were found to be the most active among all compounds. Among all the synthesized derivatives, compounds NBZD-13 and NBZD-17 were found to be the most active among all compounds using thiosemicarbazide induced model. Although NBZD-8, NBZD-10, and NBZD-18 are the compounds which had shown good anticonvulsant activity and have an advantage over that, they were not sedative.


Introduction
A benzodiazepine is a psychoactive drug whose core chemical structure is the fusion of a benzene ring and a diazepine ring. The first benzodiazepine, chlordiazepoxide (Librium), discovered accidentally by Leo Sternbach in 1955 and made available in 1960 by Hoffmann La Roche, which has also marketed diazepam (Valium) since 1963 [1]. 1,5-Benzodiazepines constitute an important class of psychopharmaca [2], in particular as tranquilizers and also as potent Virucides and nonnucleoside inhibitors of HIV-1 reverse transcriptase [3].
Benzodiazepine has a traditional place in antiepileptic therapy. The clinical use of BZDs can be divided into two categories. First, in the acute treatment of seizures as drugs of choice in status epilepticus and also in some cases of febrile seizures. Second, the BZDs are utilized in long-term therapy of certain seizures types primarily in the pediatrics' population [4].
There are some differences between the effects of 1,5and 1,4-benzodiazepines. A greater therapeutic potential and lower incidence of side effects were described for 1,5-BZDs when compared to 1,4-BZDs. 1,5-BZD is used as adjuvant therapy in resistant cases of epilepsies [5]. BZDs exhibit potent anticonvulsant actions in a wide variety of animal seizures models. They are particularly effective against seizures induced by electroshock [6], and various chemoconvulsants, in kindled seizures and in absence seizures [7].
Although, the first benzodiazepine was introduced as a drug nearly 35 years ago, the research in this area is still very active and is directed towards the synthesis of compounds with enhanced pharmacological activity [13]. The chemical structure of the benzodiazepines seems at first sight to be unique among the various types of central depressant drugs [14]. 1,5-Benzodiazepines derivatives show a large number of pharmacological properties such as they acted as sedatives [15], Cerebrovasodialators [16], neuroleptics [17], antispasmodic [18], anticonvulsant [19], tranquilizing agents [20], antibacterial [21], and psoriasis [22] and used for the treatment of small pox [23].

Experimental Procedure
Starting material and reagents were procured from commercial chemical suppliers. All the chemicals and solvents used were of laboratory grade. Melting points were determined in open capillary tubes and are uncorrected. IR spectra (KBr, cm −1 ) were recorded on Perkin Elmer Spectrometer, 1H NMR-(δ, ppm) spectra were recorded on a Brucker 300 MHz NMR spectrometer using TMS as an internal standard. The purity of compounds and progress of the reaction were checked by TLC using silica gel-G as adsorbent.

Procedure for Preparation of Fused
Scheme 6: Synthesis of various Mannich base derivatives of 1,5-benzodiazepines.

Cl
Cl

Chemical Induced
Model. Ten mice of either sex with a weight of 22-25 g were treated with the test compounds (30 mg/kg b.w) or the standard (e.g., diazepam 10 mg/kg b.w) by i.p. administration. Controls received the vehicle only. 30 min after i.p. treatment, the animals were injected with a subcutaneous dose of (300 mg/kg, s.c) isoniazid, thiosemicarbazide (20 mg/kg, s.c). The occurrence of clonic seizures, tonic seizures, and death or recovery were recorded after 0.5 hr, 1 hr, 2 hr, and 4 hr, respectively, for isoniazid induced convulsion (Table 3) and also Thiosemicarbazide induced convulsion (Table 4).

Neurotoxicity Screen.
Minimal motor impairment was measured in mice by the rotarod test. The mice were trained to stay on an accelerating rotarod that rotates at 20 revolutions per minute. The rod diameter was 3.2 cm. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of the three trials. The dose at which the animals were unable to grasp the rotarod was determined (Tables 3 and 4). DOSE: Test drug: 30 mg/Kg b.w i.p.

Sedative-Hypnotic
Activity. This test was performed with the test substances in a dose of 30 mg/kg by phenobarbitone induced narcosis in rats. The compounds in PEG (polyethylene glycol) were administered i.p to a group of six rats. After 30 min, rats were then placed on their back and loss of righting reflex was taken as onset of sleep. The time taken by the rats to awake was noted. A control was also performed after pretreatment with test substances vehicle (PEG) and injected phenobarbitone (Table 5). Table 1. Table 2.

Anticonvulsant Activity Using Chemical Induced Method.
See Tables 3 and 4.

Conclusion
All the synthesized derivatives were evaluated at the dose of 30 mg/kg b.w for anticonvulsant activity by isoniazid induced convulsion model and the compounds NBZD-3 and NBZD-8 were found to be most active among all compounds. Among all the synthesized derivatives, compounds NBZD-13, and NBZD-17 were found to be most active among all compounds using thiosemicarbazide induced model. Activity of the drugs interfering with motor coordination was checked by the rotarod test. None of the synthesized compounds were found to be neurotoxic at a dose of 30 mg/kg b.w among all the tested compounds. The compounds NBZD-1, NBZD-3, NBZD-4, NBZD-7, NBZD-11, NBZD-12, NBZD-14, NBZD-15, NBZD-17, NBZD-20, NBZD-21 were found to cause sedation. Although NBZD-8, NBZD-10, and NBZD-18 are the compounds which had shown good anticonvulsant activity and have an advantage over that, they were not sedative. Sulfated zirconia is one of the important agent that has attracted much attention recently because of its superacidity, nontoxicity, and low cost. Sulfated zirconia catalyzes many reactions under very mild condition in vapor as well as liquid phase. (1) In the basic structure of benzodiazepine, early SAR studies indicated that the seven-membered imino ring B was essential for its affinity towards the BZbinding site.

Structure Activity Relationship
(2) 4-5 Carbimino double bond has also been shown to substantially contribute to the binding affinity of compound. Saturation leads to complete less of activity. It acts as a two-electron donor site.
(3) The primary chemical moieties of the compounds which contribute to high receptor binding affinity are restricted to positions 7, 2, 1. (4) Molar refractivity is the most important parameter at position 1, suggesting that the molecular size of the substituent needs to be restricted at position 1 for effective ligand binding. Compounds NBZD-1 and NBZD-8 have less substituent as compared to other and hence more active.
(5) Compounds NBZD-6, NBZD-10, and NBZD-11 were found to be moderately active anticonvulsant action, hence this shows that chloro-substituted derivatives are rather good anticonvulsant agent as compared to nitro-substituted derivatives.
(6) Among these synthesized compounds which have methyl groups at the 2nd and 4th position, NBZD-13, and NBZD-18 were found to be most active.
Hence it shows that good activity compounds are preferred with less substitution the at 1st position.