Synthesis and anticonvulsant activity of clubbed thiazolidinone – barbituric acid and thiazolidinone – triazole derivatives

A new series of clubbed thiazolidinone–barbituric acid and thiazolidinone–triazole derivatives was synthesized to study the effect of a hydrophobic unit, hydrogen bonding domain and electron-donor group on the compounds’ anticonvulsant activity. The structures of the synthesized compounds were confirmed by their spectroscopic data and elemental analysis. All compounds were evaluated for their anticonvulsant activity in two animal models of seizures, viz. maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ). The compounds were also evaluated for neurotoxicity. Compounds 4g, 4i, 5g and 5i exhibited excellent anticonvulsant activity in both animal models of seizure.


Introduction
Epilepsy is a neurological disorder characterized by unprovoked seizures, and affects at least 50 million people worldwide.There is a continuing demand for new anticonvulsant agents as it has not been possible to control every kind of seizure with the currently available antiepileptic drugs.About one third of patients do not respond well to current multiple drugs therapy. 1,25][6] Phenobarbital 7a and mephobarbital 7b are well-known barbituric acid derivatives which are used for the treatment of epilepsy.These drugs are very effective in controlling the seizures but they suffer from major side effects such as sedation, and hypnosis.Substituted heterocyclic/substituted aryl systematic variation at the 5position of the barbituric [8][9][10] or thiobarbituric [11][12][13] acids nucleus remarkably increases the antiepileptic activity.Furthermore, thiazolidinone derivatives [14][15][16][17] are also well known for their pronounced anticonvulsant activity.9][20] In continuation of our program on the pharmacological evaluation of clubbed triazoles [21][22][23][24][25][26] we discuss herein the synthesis and anticonvulsant activity of clubbed thiazolidinones, with an in vivo efficacy approach 27 to furnish drugs which are more potent and at the same time better tolerated than existing drugs. 28We have selected three easy screening models, the MES seizure model, the PTZ seizure model, and the rotarod procedure to get a first hint for efficacy and safety.
ARKAT USA, Inc. Particularly, the compounds 4g and 4i have been found to be most potent in the series while from the rest of the compounds, 4l, 4m, 4o, 4q and 4s have shown better activity compared to the other tested compounds The next-stage compounds, i.e., 5f-5j characterized by the incorporation of an electrondonor hydroxyl group containing a 4-hydroxybenzylidenylimino group at the 2-position, while having the small hydrogen moiety at N-4 of the triazole ring at the 3rd position of the thiazolidinone.While evaluating the anticonvulsant activity, it was observed that compounds 5a-5e exhibited a less-to moderate-degree of anticonvulsant activity, which suggested that we should go for different substitutions.The next step of modification provided us with the most potent group of compounds, i.e., 5f-5j.All these compounds showed potent anticonvulsant activity, however compounds 5f, 5g and 5i, showed better response as anticonvulsant drugs, than the other substituted derivatives.Further, the next substitutions of this series could afford us 5l, 5o, and 5s, which showed moderate anticonvulsant activity, while the rest were inactive.
The structural requirement for maintaining anticonvulsant activity was the presence of a hydroxyl -OH function at the 4-position of the phenyl ring, as seen with compounds 4f-4j and 5f-5j.This requirement was further evidenced by compounds 4k-4y and 5k-5y where the -OH function was replaced by a -Cl, CH 3 or -NO 2 moiety.The complete loss of activity due to the disappearance of this function could be explained in terms of interaction at the binding site by the pharmacophoric models which were previously proposed [30][31][32][33] (Scheme 2).In these models, it has been reported that the existence of a hydrophobic unit (Ar), an electron donor group (D) and hydrogen-bonding domain (HBD) was essential for anticonvulsant activity, as evidenced by the active drugs, such as carbamazepine or phenytoin, fulfilling these demands.As shown in Scheme 2, the replacement of the hydroxyl group responsible for hydrogen bonding in compounds 4f-4j and 5f-5j also resulted in the lack of a HBD leading to abolishment of the activity seen with compounds 4k-4y and 5k-5y.From the present study, four compounds (4g, 4i, 5g and 5i) have emerged as the lead compounds.Further structural modifications of these molecules might lead to the discovery of more potent anticonvulsant agents with lower neurotoxicity.1][32][33] The essential structural requirements are indicated by dotted rectangles (Ar, hydrophobic unit; D, electron donor group; HBD, hydrogen-bonding domain).

Conclusions
The present study revealed that some of the thiazolidinone-barbituric acids and thiazolidinonetriazoles possessed a broad spectrum of anticonvulsant activity with less or no neurotoxicity.Ten compounds exhibited protection in the seizure models, viz., MES, and scPTZ, and 4g, 4i, 5g and 5i have emerged as the most active compounds in these models with no neurotoxicity.We conclude that further structural modifications of these molecules might lead to the discovery of more potent anticonvulsant agents with still lower neurotoxicity.

Experimental Section
General Procedures.The melting points were recorded on an Electrothermal apparatus and are uncorrected.The structures of synthesized compounds were assigned based on their analytical and spectroscopic properties. 1 H-NMR spectra were recorded on a Bruker Avance 300 MHz instrument, using DMSO-d 6 as solvent and TMS as internal standard; the chemical shifts (δ) are reported in ppm and coupling constants (J) are given in Hz.Signal multiplicities are represented by s (singlet), d (doublet), t (triplet), ds (double singlet), dd (double doublet), m (multiplet) and bs (broad singlet).Mass spectra were recorded on a Finnigan LCQ mass spectrometer.Elemental analyses were performed on a Heraeus CHN-Rapid Analyzer.Analytical figures were within ±/0.4% of the theoretical values.The purity of the compounds was checked on Merck precoated silica gel 60 F-254.
Chloroacetyl chloride (1 mmol) was added with vigorous stirring, followed by addition of triethylamine (1 mL) and potassium carbonate (1 mmol).The reaction mixture was then refluxed for 4-5 h.At the end of the 5th hour, the benzene was distilled off.The residue was washed with 5 mL of water.An oily product was obtained which was dissolved in a minimum quantity of acetone to obtain a precipitate which was filtered off, dried and crystallized.

Pharmacology
For the anticonvulsant studies, male albino mice (CF-1 strain, 18-25 g) and male albino rats (Sprague-Dawley, 100-150 g) were used as experimental animals.The animals were allowed to ARKAT USA, Inc.
adapt to the laboratory environment for one week before the experiments started.All experiments with drug injection were then carried out within one week to minimize the effect of increasing age on drug susceptibility.Each animal was used for only one experiment.Experimental animals were kept in groups of six in plastic cages at controlled temperature (22 ± 2°C) and humidity (about 55 ± 15 %) with a 12-h light cycle beginning at 6 a.m.They received standard laboratory rodent chow and tap water ad libitum.
Anticonvulsant screening scPTZ test 35,36 Pentylenetetrazole (PTZ, Sigma Chemicals, USA) was used as convulsant.PTZ was dissolved in normal saline.The mice were divided into groups of six each, keeping the group weights as equal as possible.All the synthesized compounds 4a-y and 5a-y were suspended in 5% aqueous gum acacia to give a concentration of 1% (w/v).The test compounds were injected i.p. into each group.The control animals were injected with vehicle only.Thirty minutes later, for the administration of either vehicle or test compounds, the animals were injected with pentylenetetrazole (90 mg/kg, s.c.).This dose of pentylenetetrazole was shown to produce convulsions in all untreated mice and these animals exhibited 100% mortality during 24 h.The mice that survived after 24 h were considered to be protected.The number of protected animals in each group was recorded and the anticonvulsant activity of compounds 4a-y and 5a-y were represented as the percent protection.MES test 37,38 Mice were divided into different groups with six mice in each group.Suspensions of the compounds/standard in 0.5% Tween-80 in saline were administered intraperitoneally (i.p.) at three dose levels (30, 100, 300 mg kg -1 ).The untreated group was administered the same volume of the vehicle.A drop of 0.9% saline was instilled in each eye prior to the application of electrodes (Centroniks Electroconvulsiometer).The mice were subjected to electrical shock delivered through the corneal electrodes for 0.2 s (40 mA, 50 Hz, AC).Failure to extend the hind limbs to an angle with the trunk greater than 90° is defined as protection.The seizure pattern was recorded at 0.5-and 4 hours after administration of the dose (Tables 5 and 6). 39he Rotarod test has been performed to detect the motor deficit in mice.Animals were divided into the groups (each of six) and trained to stay on an accelerating rotarod that rotates at 10 revolutions per minute.The rod diameter was 3.2 cm.Trained animals (able to stay on the rotarod for at least two consecutive periods of 90 s) were given an i.p. injection of the test compounds in the doses of 30, 100 and 300 mg/kg.Neurological deficit 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 animal fell off the rod, was determined, and the data are presented in Tables 5 and 6.

Table 1 .
Physical and analytical data of compounds

Table 2 .
Physical and analytical data of compounds 5a-5y