Anticonvulsant Potential of Certain New (2E)-2-[1-Aryl-3-(1H-imidazol-1-yl)propylidene]-N-(aryl/H)hydrazinecarboxamides

Anticonvulsant potential and neurotoxicity of certain new imidazole-containing arylsemicarbazones 6a–p are reported. The test compounds 6a–p exhibited anticonvulsant activity mainly in the scPTZ screen. Compound 6p emerged as the most active surrogate displaying 100% protection at a dose level of 636 μmol/kg in the scPTZ screen without any neurotoxicity. The assigned (E)-configuration of the title compounds 6a–p was confirmed via single crystal X-ray structure of compound 6g.


Introduction
Epilepsy is one of the most common neurological disorders affecting nearly 50 million of mankind with the majority of cases being in developing countries [1]. Despite the availability of many new antiepileptic drugs (AEDs) and the remarkable strides in this research field, estimates suggest that about 20-30% of patients have seizures that are resistant to the available antiepileptic medications [2]. The currently approved antiepileptics have dose-related adverse effects such as ataxia, hepatotoxicity, gingival hyperplasia, and megaloblastic anaemia [3][4][5]. Therefore, the development of newer, more effective, and more selective antiepileptic agents with lesser side effects remains a major focus of antiepileptic drug research.
An examination of the literature revealed that aralkylimidazoles, namely, nafimidone (I) and danzimol (II) (Figure 1), exhibited anticonvulsant activity. Compounds I and II are structurally unrelated to the currently available anticonvulsants and they are independently discovered. Additionally, they showed anticonvulsant profile similar to that of phenytoin or carbamazepine while they are more potent than barbiturates and valproic acid [6][7][8][9].
Arylsemicarbazones featuring the general structural skeleton III (Figure 1) have been recognized as a promising pharmacophore for anticonvulsants and a sizable number of arylsemicarbazone derivatives showed anticonvulsant activity [10,11].
In a search for new anticonvulsant chemical entities, we report herein the synthesis and characterization of certain new imidazole-containing arylsemicarbazones 6ap as hybrid structures incorporating both imidazole and arylsemicarbazone moieties in order to gain insight into their anticonvulsant potential (phase I) as well as their neurotoxicity. The importance of the presence of a primary amide group for the anticonvulsant activity of the target compounds 6a-p was also explored. Additionally, the configuration of the title compounds 6a-p around the imine double bond was examined via single crystal X-ray crystallography of compound 6g.

Experimental
All melting points were determined using Electrothermal Capillary melting point apparatus and are uncorrected.  Subsequently, Mannich bases (100 mmol) were dissolved in water (100 mL) and imidazole (200 mmol) was added. The reaction mixture was heated to reflux for five hours, cooled and the precipitated solids were collected by filtration to give ketones 5a-c which were pure enough to be used in the next step.

General Procedure for the Synthesis of the Target Com-
pounds 6a-f, 6h-k, and 6m-p. A solution containing the appropriate arylsemicarbazide 3a-f (10 mmol), appropriate ketone 5a-c (10 mmol), and few drops of glacial acetic acid in ethanol (15 mL) was stirred at room temperature for 18 hrs. The reaction mixture was rotovapped and the residue was The Scientific World Journal 3 crystallized from ethanol to give the title compounds 6a-f, 6h-k, and 6m-p.

Materials and Methods
(1) Materials. Adult male Swiss albino mice weighing 18-25 g were used in our investigations and were purchased from Animals House Colony of the National Research Centre, Cairo, Egypt. Animals were housed under standardized conditions (room temperature 23 + 2 ∘ C; relative humidity 55 + 5%; 12 hrs-light/dark cycle) and had free access to tap water and standard mice chow throughout the whole experimental period. All animal procedures were performed in accordance with the Ethics Committee of the National Research Center and in accordance with the recommendations for the proper care and use of laboratory animals "Canadian Council on Animal Care Guidelines, 1984. " After seven days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups consisting of 6 mice each. All the test compounds were suspended in 7% tween-80 saline solutions.  Aqueous solution of scPTZ (85 mg/kg) was administered in a loose fold of skin on the back of the mice necks half an hour after intraperitoneal (i.p.) injection of the test compounds 6a-p and the animals were observed during the following 0.5 hr for the occurrence of seizures. A threshold convulsion was defined as one episode of clonic convulsions which persisted for at least a 5-second period. Absence of even a single 5-second episode of clonic spasms during the period of observation is taken as the end point in this test [19].
(b) Maximal Electroshock Seizure (MES) Screen. Electroconvulsions were turned out half an hour after i.p. injection of the test compounds 6a-p, by a current (fixed current intensity of 25 mA, 0.2 s stimulus duration) delivered via earclip electrodes by a Rodent Shocker Generator (constant-current stimulator Type 221, Hugo Sachs Elektronik, Freiburg, Germany). The criterion for the occurrence of seizure activity was the tonic hind limb extension (i.e., the hind limbs of animals outstretched 180 ∘ to the plane of the body axis) [20].
(c) Neurotoxicity. In this test, the animals were trained to maintain equilibrium on a rotating 1-inch-diameter knurled plastic rod at a speed of 6 rev/min for at least 1 min in each of the three trials using rotarod device (UGO Basile, 47600, Varese, Italy). Only animals that fulfill this criterion were included into the experiment. The selected trained animals were classified into groups; six mice were used in the control group as well as in the experimental groups. The animals in the experimental groups were given the test compounds 6a-p in 7% aqueous suspension of Tween 80 via i.p. route; meanwhile, the control group received the vehicle. Thirty minutes later, the mice were placed again on the rotating rod and the neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min [21].

Results and Discussion
3.1. Chemistry. Synthetic strategy to synthesize arylsemicarbazides 3a-f was previously reported [12]. Thus, the appropriate aniline derivative 1a-f was allowed to react with ethyl chloroformate to give carbamates 2a-f. Compounds 2a-f were refluxed with hydrazine hydrate to yield arylsemicarbazides 3a-f (Scheme 1).
The target compounds 6a-p were synthesized as portrayed in Scheme 2. The appropriate acetophenone derivative 4a-c was transformed into the penultimate ketones 5a-c in a two-step reaction sequence as previously reported [13]. Ketones 5a-c were allowed to react with the appropriate arylsemicarbazide 3a-f and/or semicarbazide hydrochloride in ethanol at room temperature to give the respective title compounds 6a-p in moderate yields.

Crystal Data for Compound 6g.
Single crystal X-ray crystallography is a doubtless decisive analytical tool which can confirm the configuration of the imine double bond in the target compounds 6a-p. Fortunately, we have succeeded to get single crystals of compound 6g which were suitable for X-ray crystallography as a representative example for the synthesized target compounds 6a-p. Accordingly, the assigned (E)-configuration of compound 6g was established via its single crystal X-ray structure (Figure 2      The chemical structures of the title compounds 5a-p were confirmed via IR, 1 H NMR, 13 C NMR, and mass spectral data.

Anticonvulsant Activity and SAR.
The preliminary anticonvulsant activity (phase I screening) of the target compounds 6a-p was determined according to the phase I tests of the Antiepileptic Drug Development (ADD) program. ADD program was developed by National Institute of Neurological Disorders and Stroke (NINIDS) and it includes the subcutaneous pentylenetetrazole (scPTZ) screen and the maximal electroshock seizure (MES) screen [22]. scPTZ and MES screens are considered as the "gold standard" seizure model screens where they are used to identify compounds that elevate seizure threshold and to indicate the ability of the test compounds to prevent seizure spread, respectively. Additionally, acute toxicity from antiepileptic drugs in rodents is almost invariably manifested by neurological deficits. These include sedation, altered motor activity, ataxia, and impaired righting reflexes. These effects of antiepileptic drugs are often summarized by the term "neurotoxicity. " Minimal neurological deficit, such as impaired motor function, can be detected by standardized test, that is, by the rotarod test [21]. The obtained data expressed as % protection for anticonvulsant activity of the test compounds 6a-p as well as their neurotoxicity are presented in Table 1.
Nafimidone (I) and danzimol (II) are aralkylimidazole derivatives exhibiting anticonvulsant activity. On the other hand, it has been reported that a sizable number of arylsemicarbazones show anticonvulsant activity in both scPTZ and MES screens [10,11]. Additionally, arylsemicarbazones interact at a putative binding site in vivo which was designated as a hydrogen bonding area and an aryl binding site in order to exert their anticonvulsant activity. Therefore, it is likely that the pharmacophoric descriptors in arylsemicarbazones are thought to be a hydrogen bonding semicarbazone group and a lipophilic aryl ring which align at their complementary areas on the macromolecular complex in vivo [23]. The presence of imidazole moiety and hydrogen bonding area in the title compounds 6a-p could improve their binding with the complementary receptor binding area in vivo. Accordingly, compound 6g was synthesized as a hybrid structure containing both imidazole and arylsemicarbazone moieties to be screened as a new chemical entity with anticonvulsant potential. Compound 6g displayed only 50% protection in the scPTZ screen at dose level of 972 mol/kg and was devoid of any activity in the MES screen.
Substitution of the aromatic ring in 6g with a substituent endowed with a negative inductive effect like bromosubstituent gave compound 6l. Compound 6l showed better anticonvulsant activity than that of 6g in both scPTZ (100% protection at a dose level of 818 mol/kg) and MES screens (16% protection at a dose level of 744 mol/kg). Additionally, the terminal hydrogen of the amide moiety of 6g and 6l has been replaced with aryl moiety containing substituents endowed with different electronic and steric properties in order to investigate the influence of the primary amide group on the anticonvulsant activity of this type of imidazole-containing arylsemicarbazones. Accordingly, compounds 6a-f, 6i-k, and 6m-p were synthesized and screened for their anticonvulsant potential. In compounds 6a-f, compound 6f, bearing 2,4-dichloro substituents, is the most active candidate having 100% protection in the scPTZ screen at a dose level of 684 mol/kg without any neurotoxicity, whereas in compounds 6i-k, compound 6i, containing 2-methyl substituent, is the most active congener displaying 100% protection in the scPTZ screen and 16% protection in MES screen at a dose level of 645 and 586 mol/kg, respectively.
In compounds 6m-p, compound 6p, bearing 2,4dichloro substituents, emerged as the most active drug-like candidate among all the synthesized compounds with 100% protection in the scPTZ screen at a dose level of 636 mol/kg without any neurotoxicity.
In summary, the synthesized compounds 6a-p elevate seizure threshold according to their anticonvulsant activity in the scPTZ animal model screen. Additionally, the presence of primary amide group in this type of imidazole-containing arylsemicarbazones is not essential for their anticonvulsant activity in the scPTZ screen.

Conclusion
Synthesis, characterization, and anticonvulsant activity (phase I) of new imidazole-containing arylsemicarbazones 6a-p have been successfully achieved. Compounds 6a-p exhibited anticonvulsant activity principally in the scPTZ screen with compound 6p which emerged as the most active congener at a dose level of 636 mol/kg without any neurotoxicity. Moreover, the evoked anticonvulsant activity of the title compounds 6a-p in the scPTZ screen does not necessitate the presence of a primary amidic moiety in their structure. The assigned (E)-configuration of the title compounds 6a-p was confirmed via single crystal X-ray structure of compound 6g.

Additional Materials
Crystallographic data have been deposited with the Cambridge Crystallographic Data Center (supplementary publication number CCDC-945587). Copies of the data may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, UK.