Anticonvulsant Profiles of Certain New 6-Aryl-9-substituted-6,9-diazaspiro-[4.5]decane-8,10-diones and 1-Aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones

Synthesis and anticonvulsant potential of certain new 6-aryl-9-substituted-6,9-diazaspiro[4.5]decane-8,10-diones (6a–l) and 1-aryl-4-substituted-1,4-diazaspiro[5.5]undecane-3,5-diones (6m–x) are reported. The intermediates 1-[(aryl)(cyanomethyl)amino]cycloalkanecarboxamides (3a–f) were prepared via adopting Strecker synthesis on the proper cycloalkanone followed by partial hydrolysis of the obtained nitrile functionality and subsequent N-cyanomethylation. Compounds 3a–f were subjected to complete nitrile hydrolysis to give the respective carboxylic acid derivatives 4a–f which were cyclized under mild conditions to give the spiro compounds 5a–f. Ultimately, compounds 5a–f were alkylated or aralkylated to give the target compounds 6a–i and 6m–u. On the other hand, compounds 6j–l and 6v–x were synthesized from the intermediates 5a–f through alkylation, dehydration and finally tetrazole ring formation. Anticonvulsant screening of the target compounds 6a–x revealed that compound 6g showed an ED50 of 0.0043 mmol/kg in the scPTZ screen, being about 14 and 214 fold more potent than the reference drugs, Phenobarbital (ED50 = 0.06 mmol/kg) and Ethosuximide (ED50 = 0.92 mmol/kg), respectively. Compound 6e exhibited an ED50 of 0.019 mmol/kg, being about 1.8 fold more potent than that of the reference drug, Diphenylhydantoin (ED50 = 0.034 mmol/kg) in the MES screen. Interestingly, all the test compounds 6a–x did not show any minimal motor impairment at the maximum administered dose in the neurotoxicity screen.


Introduction
Epilepsy is a group of neurological disorders characterized by excessive abnormal bioelectrical functions of the brain leading to recurrent unprovoked seizures [1,2]. It affects about 1% of the global population with the majority of cases being in the developing countries [3]. Estimates suggest that approximately 20%-30% of patients are not adequately controlled by the available antiepileptic medications [4,5]. Furthermore, the clinically used antiepileptics display serious side effects such as ataxia, hepatotoxicity, gingival hyperplasia and megaloblastic anaemia [6][7][8]. Therefore, there is a substantial need for novel, more effective and more selective antiepileptic agents with lesser side effects.
Diketopiperazines (DKPs) are the smallest cyclic peptides known, commonly biosynthesized from amino acids by a large variety of organisms [9]. They are privileged structures for the discovery of new lead compounds. They display attractive chemical characteristics, such as resistance to proteolysis, mimicking of peptidic pharmacophoric groups, conformational rigidity and donor as well as acceptor groups for hydrogen bonding which might influence interactions with biological targets [10].
Incorporation of lipophilic moieties in the scaffold of new bioactive chemical entities could improve their anticonvulsant potential. Accordingly, cyclohexane and/or cyclopentane moieties were embedded in the skeleton of the new 2,6-DKP derivatives 6a-x aiming to enhance their anticonvulsant activity. On the other hand, the tetrazole moiety is a bioisostere of carboxylic acid functionality and it is an integrated part in the construction of certain anticonvulsants [14,15]. Therefore, compounds 6j-l and 6v-x, bearing a tetrazole moiety, were synthesized and screened for their anticonvulsant potential.

Chemistry
Syntheses of the target compounds 6a-x and their intermediates are depicted in Schemes 1-3. Thus, cyclopentanone and/or cyclohexanone were allowed to react with the appropriate commercially available aniline derivative and potassium cyanide in glacial acetic acid under Strecker synthesis conditions to give the respective nitrile derivatives 1a-f. The nitrile group of compounds 1a-f was subjected to hydrolysis under acidic conditions using sulfuric acid at ambient temperature to yield the amide derivatives 2a-f. Subsequently, cyanomethylation of the secondary amine moiety of compounds 2a-f was successfully achieved using potassium cyanide, paraformaldehyde and formaldehyde to furnish the corresponding compounds 3a-f (Scheme 1).

Scheme 1. Synthesis of compounds
The target compounds 6a-i and 6m-u as well as their intermediates 4a-f and 5a-f were obtained as portrayed in Scheme 2. Thus, the nitrile moiety in compounds 3a-f was hydrolysed via reflux in sodium hydroxide solution to yield the corresponding carboxylic acid derivatives 4a-f. Cyclization of the latter compounds 4a-f was successfully realized using ethylenediamine in 4 N HCl solution to give the respective spiro compounds 5a-f according to our previously developed procedure [16]. The imide functionality of compounds 5a-f was alkylated under phase transfer catalysis conditions using the appropriate alkyl/aralkyl halide to give the target compounds 6a-i and 6m-u. Scheme 2. Synthesis of compounds 4a-f, 5a-f, 6a-i and 6m-u. (CH 2  The synthesis of the intermediates 7a-f and 8a-f as well as the target compounds 6j-l and 6v-x were successfully achieved as illustrated in Scheme 3. Synthesis of compounds 6j-l and 6v-x was commenced with the reaction of compounds 5a-f with chloroacetamide to give the corresponding compounds 7a-f. Dehydration of compounds 7a-f using trifluoroacetic anhydride furnished the respective penultimate cyanomethyl derivatives 8a-f. Elaboration of the cyano group of compounds 8a-f to the tetrazolyl moiety was acquired using sodium azide in the presence of aluminium chloride to yield the desired compounds 6j-l and 6v-x. Scheme 3. Synthesis of compounds 7a-f, 8a-f, 6j-l and 6v-x. Reagents and conditions: (i) Acetone, K2CO3, tetrabutylammonium bromide, reflux 7 h; (ii) Triflouroacetic anhydride, THF, cooling, 0-5 °C, 2 h, ammonium bicarbonate; (iii) NaN3, AlCl3, cooling then reflux 24 h.

Anticonvulsant Activity
The test compounds 6a-x were subjected to preliminary anticonvulsant evaluation (Phase I screening) according to the protocol given by the Epilepsy Section of the National Institute of Neurological Disorders and Stroke (NINIDS) using the standard procedure adopted by the Antiepileptic Drug Development (ADD) program [17]. Those include the 'gold standard' screens, namely subcutaneous Pentylenetetrazole (scPTZ) screen and the maximal electroshock seizure (MES) screen. The former screen identifies compounds that elevate seizure threshold while the latter one measures the ability of the test compound to prevent seizure spread. Compounds exhibited 100% protection against induced seizures, were subjected to median effective dose (ED50) estimation and minimal motor impairment (neurotoxicity) evaluation.
It has been indicated that PTZ-induced seizures can be prevented by drugs that reduce T-type Ca 2+ currents such as Ethosuximide and also by drugs that enhance gamma amino butyric acid type A (GABAA) receptor-mediated inhibitory neurotransmission such as Phenobarbital [18].
The results of the initial anticonvulsant screening of the test compounds 6a-x are given in Table 1. The evaluation indicated that, all the compounds were effective in scPTZ screen while most of them were effective in MES screen. scPTZ screen showed that, compound 6g (R 1 = 4-OCH3 and R 2 = -CH2COOCH3) was the most potent congener in the cyclopentane series 6a-l, displaying 100% protection against PTZ-induced seizure at dose level of 0.0086 mmol/kg as compared with Phenobarbital (0.13 mmol/kg) and Ethosuximide (1.06 mmol) which were used as reference standards.
The different congeners of the cyclohexane series 6m-x showed a decrease in the anticonvulsant potential in the following decreasing order: Concerning the MES test, the dose which exerted 100% anticonvulsant protection in the scPTZ screening has been selected. In this screening test, all of the compounds showed protection in half or more of the tested mice after 0.5 h post administration except compounds 6i, 6j, 6k, 6m, 6u and 6w. On the other hand, compounds 6l, 6p, 6r, 6s and 6t were devoid from anticonvulsant activity. Meanwhile, 6e (R 1 = 4-CH3, R 2 = -CH2Ph) exhibited 100% protection at dose level of 0.032 mmol/kg being more potent than the reference drug, Diphenylhydantoin, which exerted the same protection at a dose level of 0.16 mmol/kg. It is worthwhile to mention that, compound 6e displayed 100% protection against both scPTZ and MES-induced seizures in mice.
Compounds showed 100% protection in scPTZ and/or MES screens, were subjected to median effective dose (ED50) estimation as well as to minimal motor impairment (neurotoxicity) evaluation. Table 2 summarizes ED50 of the selected test compounds along with their neurotoxicity evaluation.
Compound 6g gave an ED50 of 0.0043 mmol/kg ≡ 1.5 mg/kg in the scPTZ screen being about 14 and 214 fold more potent than the reference drugs, Phenobarbital (ED50 = 0.06 mmol/kg ≡ 13.2 mg/kg) and Ethosuximide (ED50 = 0.92 mmol/kg ≡ 130 mg/kg), respectively. In the MES screen, only compound 6e showed 100% protection against induced seizures with ED50 of 0.019 mmol/kg ≡ 7.0 mg/kg being about 1.8 fold more potent than that of the reference drug, Diphenylhydantoin (ED50 = 0.034 mmol/kg ≡ 9.5 mg/kg [19]). Interestingly, all the test compounds did not show any minimal motor impairment at the maximum administered dose in the neurotoxicity screen.

Chemistry
All melting points were determined using Electrothermal Capillary melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded as thin film (for oils) in NaCl discs or as KBr pellets (for solids) with JASCO FT/IR-6100 spectrometer and values are represented in cm −1 . 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were carried out on Jeol ECA 500 MHz spectrometer using TMS as internal standard and chemical shift values were recorded in ppm on δ scale. The 1 H-NMR data were represented as follows: chemical shifts, multiplicity (s. singlet, d. doublet, t. triplet, m. multiplet, br. broad), number of protons, and type of protons. The 13 C-NMR data were represented as chemical shifts and type of carbons. Mass spectral data were obtained with electron impact (EI) ionization technique at 70 eV from a Finnigan Mat SSQ-7000 Spectrometer. Elemental analyses were carried out in Microanalytical Units at National Research Centre and Cairo University. Silica gel TLC (thin layer chromatography) cards from Merck (silica gel precoated aluminum cards with fluorescent indicator at 254 nm) were used for thin layer chromatography. Visualization was performed by illumination with UV light source (254 nm). Column chromatography was carried out on silica gel 60 (0.063-0.200 mm) obtained from Merck.

General Procedure for the Synthesis of 1-(Arylamino)cycloalkanecarbonitriles (1a-f)
A solution of potassium cyanide (9.75 g, 0.15 mol) in water (25 mL) was added drop-wise to a solution of cycloalkanone (0.15 mol) and the appropriate aniline derivative (0.15 mol) in glacial acetic acid (75 mL). The reaction mixture was stirred mechanically at room temperature for 24 h. The precipitated product was filtered off, washed with water, dried and recrystallized from petroleum ether (40-60 °C) to afford 1a-f. The spectral data of compounds 1a-f were consistent with the published ones (cited below).
3.1.2. General Procedure for the Synthesis of 1-(Arylamino)cycloakanecarboxamides (2a-f) The appropriate nitrile derivative 1a-f (0.125 mol) was dissolved in cold concentrated sulfuric acid (20 mL). After remaining at room temperature for 48 h, the reaction mixture was poured over crushed ice and rendered alkaline with 25% ammonium hydroxide solution. The precipitated amide was filtered off, washed with water, dried and recrystallized from ethanol to give 2a-f. The spectral data of compounds 2a-f were consistent with the published ones (cited below).
3.1.3. General Procedure for the Synthesis of 1-[(Aryl)(cyanomethyl)amino]cycloalkanecarboxamides (3a-f) Paraformaldehyde (1.52 g, 0.05 mol) was added to a solution of the appropriate 1-(arylamino)cycloakanecarboxamides (2a-f) (0.05 mol) in glacial acetic acid (30 mL). A solution of potassium cyanide (3.9 g, 0.06 mol) was added drop-wise to the stirred and cooled (15 °C) reaction mixture. The temperature was raised gradually to 45 °C over 30 min and was maintained at 50-60 °C for 3 h. After cooling to 35 °C, a 37% formaldehyde solution (5 mL) was added and the reaction mixture was stirred at room temperature for 18 h. Water (30 mL) was added, the reaction mixture was cooled and neutralized with 10% sodium carbonate solution. The precipitated product was extracted with CH2Cl2 (3 × 50 mL), washed with water (2 × 30 mL), dried (Na2SO4) and evaporated under vacuum to give the anticipated compounds 3a-f. The crude 3a-f were pure enough to be used in the following step without any further purification. The spectral data of compounds 3a-f were consistent with the published ones (cited below).
1-[(Cyanomethyl)(4-methoxyphenyl)amino]cyclopentanecarboxamide (3c) [16] A mixture of the appropriate cyanomethyl derivative 3a-f (0.01 mol) and NaOH (0.48 g, 0.012 mol) in 50% aqueous ethanol (25 mL) was stirred under reflux for 18 h, utill complete evolution of ammonia was ceased. The ethanol was removed by evaporation under vacuum. The residue was extracted with ethyl acetate (2 × 15 mL) and the aqueous layer was acidified with 2 N HCl. The acidic layer was extracted with ethyl acetate (3 × 15 mL), dried (Na2SO4) and evaporated under reduced pressure to yield compounds 4a-f. The crude 4a-f were pure enough to be used in the following step without any further purification. The spectral data of compounds 4a-f were consistent with the published ones (cited below).
Neurotoxicity [34]: This test is designed to detect minimal neurological deficit. 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 three trials using a rotarod device (UGO Basile, 47600, Varese, Italy). Only animals that fulfill this criterion were included in the experiment. The selected trained animals were classified into control and experimental groups. The animals in the experimental groups were given the reference drug or one of the test compounds via i.p. route at doses which exerted 100% protection in the PTZ test; 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.

Determination of the ED50
Anticonvulsant activity of the test compounds was expressed in term of median effective dose (ED50) that is, the dose of drug required to produce the required biological response in 50% of animals. For determination of the ED50, groups of 8 mice were given a range of i.p. doses of the test compound until at least three points were established in the range of 15%-84% seizure protection. From the plot of these data, the respective ED50 value and the confidence limits were calculated [18].