INTRODUCTION:

Epilepsy is not a singular disease entity but a variety of disorders reflecting underlying brain dysfunction that may result from many different causes.¹Therefore, there is continuing demand for new anticonvulsant agents. So, there is an urgent requirement for the discovery and development of some novel anticonvulsant agents with more selective activity and lower toxicity for the effective treatment of epilepsy. Several five-member aromatic systems having three heteroatom at symmetrical positions such as thiadiazoles have been studied extensively owing to their interesting pharmacological activities.

The word epilepsy usually describes a group of common chronic neurological disorders characterized by recurrent unprovoked seizures due to synchronous neuronal activity in the brain. Several new drugs have appeared on the market, the development of novel agents, particularly compounds effective against complex partial seizures remains a major focus of antiepileptic drug research.² A review on new structural entities having anticonvulsant activity has recently appeared.³ The anticonvulsants are a diverse group of pharmaceuticals used in the treatment of epilepticseizures. The goal of an anticonvulsant is to suppress the rapid and excessive firing of neurons that start a seizure.

The structural diversity and biological importance of nitrogen containing heterocycles have made them attractive targets for synthesis over many years. They are found in various natural products and have been identified as products of chemical and biological importance. Pyrimidine is considered to be a resonance hybrid of the charged and uncharged cannonical structures; its resonance energy has been found to be less than benzene or pyridine. The pyrimidine moiety is a versatile lead molecule in pharmaceutical development and has a wide range of biological activities. In the past few years, the therapeutic interest of pyrimidine derivatives in pharmaceutical and medicinal field has been given a great attention to the medicinal chemist. Pyrimidine ring is fused to various heterocycles, that represent an important class of heterocyclic compounds having wide range of applications.^4,5 The existing methods for the preparation of triazolopyrimidines are based on heterocyclic hydrazones.Pyrimidines and their derivatives are considered to be important for drugs and agricultural chemicals. A large number of pyrimidine derivatives are reported to exhibit antimycobacterial,⁶ antitumor,⁷ antiviral,⁸ anticancer,⁹ anti-inflammatory¹⁰ and antimicrobial¹¹ activities. We have reported the synthesis and biological activity of 1-(5-bromo-2-chloropyrimidin-4-yl)hydrazine derivatives.¹²In the present study, a series of new pyrimidine analogues, 3(a-f) have been synthesized and their anticonvulsant activity were determined.

MATERIALS AND METHODS:

All solvents and reagents were purchased from Sigma Aldrich Chemicals Pvt Ltd. Melting range was determined by GLNR SELEC apparatus. The new compounds were analyzed with FT-IR spectrophotometer (Agilent FT-IR ATR Cary 630) in the range of 7000-350 cm^-1. ¹H NMR spectra were recorded on Bruker DRX -500 spectrometer at 400 MHz using d₆-DMSO as solvent and TMS as an internal standard. The mass spectra of the samples were recorded using the instrument LC-MSD-Trap-XCT.

Synthesis of 1-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (2)

A mixture of 5-bromo-2,4-dichloropyrimidine (1) (0.01 mol) in methanol was taken and cooled to 0–5 °C in an ice bath. Trietheylamine (0.01 mol) was added to the cold reaction mixture and then hydrazine hydrate (0.012 mol) was added slowly at 5-10 °C. The reaction mass was allowed to stir at room temperature for 1h. The solid thus obtained was filtered, washed with chilled water and dried to afford compound 2 (Yellow solid). Yield- 83 %. ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.05 (s, 1H, NH), 7.80 (s, 1H, py-H), 4.30 (s, 2H, NH₂).

General procedures for the synthesis aryl-(5-bromo-2-chloro-pyrimidine-4-yl)hydrazone 3(a-f)

Compound 2 was dissolved in ethanol and aryl aldehyde was added to it. The content were refluxed on a water bath for 1 h and allowed to stand at room temperature. The crystalline solid thus obtained, was filtered, washed with ethanol and dried to afford compound 3(a-f).

1-(4-Chlorobenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3a)

The product obtained from (2) (0.01 mol) and 4-chlorobenzaldehyde (0.02 mol). Brown solid, Yield: 84 %, M.p.: 155-157 °C. FT-IR (KBr, cm⁻¹): 3435 (NH), 2950 (C-H), 1610 (C=N), 1375 (C-N), 715 (C-Cl), 520 (C-Br). ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.38 (s, 1H, NH), 8.51 (s, 1H, Py-H), 8.48 (s, 1H, CH), 7.91 (d, 1H, J = 8.20 Hz, Ar-H), 7.87 (d, 1H, J = 8.10 Hz, Ar-H), 7.72 (s, 1H, Ar-H). MS (ESI) m/z: 381.56.

1-(2-Fluorobenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3b)

The product obtained from (2) (0.01 mol) and 2-fluorobenzaldehyde (0.012 mol). Yellow solid, Yield: 88 %, M.p.: 174-176 °C.FT-IR (KBr, cm⁻¹): 3490 (NH), 2935 (C-H), 1613 (C=N), 1370 (C-N), 1090 (C-F), 725 (C-Cl). ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.74 (s, 1H, NH), 8.29 (s, 1H, Py-H), 8.26 (s, 1H, CH), 7.20 (d, 1H, J = 7.42 Hz, Ar-H), 7.03 (d, 1H, J = 7.15 Hz, Ar-H). MS (ESI) m/z: 373.1.

1-(4-Methoxybenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3c)

The product obtained from (2) (0.01 mol) and 4-methoxyenzaldehyde (0.012 mol). White solid, Yield: 85 %, M.p.: 151-153 °C. FT-IR (KBr, cm⁻¹): 3447 (NH), 2930 (C-H), 1612 (C=N), 1375 (C-N), 720 (C-Cl). ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.21 (s, 1H, NH), 8.52 (s, 1H, Py-H), 8.47 (s, 1H, CH), 7.40 (t, 1H, J = 7.84 Hz, Ar-H), 7.31 (d, 1H, J = 7.64 Hz, Ar-H), 7.25 (s, 1H, Ar-H), 7.06 (d, 1H, J = 7.61 Hz, Ar-H), 3.81 (s, 3H, OCH₃). MS (ESI) m/z: 342.7.

1-(3-Methoxybenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3d)

The product obtained from (2) (0.01 mol) and 3-methoxybenzaldehyde (0.012 mol). White solid, Yield: 92 %, M.p.: 141-143 °C. FT-IR (KBr, cm⁻¹): 3349 (NH), 2928 (C-H), 1618 (C=N), 1375 (C-N), 725 (C-Cl).¹H NMR (DMSO-d₆, 400 MHz) δ: 11.31 (s, 1H, NH), 8.74 (s, 1H, Py-H), 8.49 (s, 1H, CH), 7.51-7.50 (m, 2H, Ar-H), 7.20 (t, 1H, J = 6.60 Hz, Ar-H). MS (ESI) m/z: 350.0.

1-(2-Chlorobenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3e)

The product obtained from (2) (0.01 mol) and 2-chlorobenzaldehyde ( 0.012 mol). White solid, Yield: 92 %, M.p.: 143-145 °C. FT-IR (KBr, cm⁻¹): 3345 (NH), 2930 (C-H), 1615 (C=N), 1380 (C-N), 725 (C-Cl).¹H NMR (DMSO-d₆, 400 MHz) δ: 11.31 (s, 1H, NH), 8.74 (s, 1H, Py-H), 8.49 (s, 1H, CH), 7.51-7.50 (m, 2H, Ar-H), 7.20 (t, 1H, J = 6.60 Hz, Ar-H). MS (ESI) m/z: 350.0.

1-(2-Methoxybenzylidene)-2-(5-bromo-2-chloropyrimidin-4-yl)hydrazine (3f)

The product obtained from (2) (0.01 mol) and 2-methoxybenzaldehyde (0.02 mol). White solid, Yield: 85 %, M.p.: 155-157 °C. FT-IR (KBr, cm⁻¹): 3440 (NH), 2945 (C-H), 1614 (C=N), 1372 (C-N), 715 (C-Cl).¹H NMR (DMSO-d₆, 400 MHz) δ: 11.38 (s, 1H, NH), 8.51 (s, 1H, Py-H), 8.48 (s, 1H, CH), 7.91 (d, 1H, J = 8.20 Hz, Ar-H), 7.87 (d, 1H, J = 8.10 Hz, Ar-H), 7.72 (s, 1H, Ar-H). MS (ESI) m/z: 381.56.

Anticonvulsant activity

Animals:

Male wistar rats procured from National Institute of Nutrition, Hyderabad (190-220 g) were used in the present study. The animals were kept in individual cages for one week to acclimatize for the laboratory conditions. They were allowed to free access of water and food. All the experimental procedures were carried out in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The study was reviewed and approved by the Institutional Animal Ethics Committee, G Pulla Reddy College of Pharmacy, Hyderabad, India.

Maximal Electroshock Seizure Model (MES):

Maximal electroshock seizure model was used in the present study to evaluate the anticonvulsant activity of the compounds on male wistar rats. Seizures were induced in rats by delivering electro shock of 150 mA for 0.2 s by means of a convulsiometer through a pair of ear clip electrodes. The test compounds (100 mg/kg) were administered by oral route in the form of solution (The compounds were dissolved in 1% sodium carboxymethyl cellulose), 30 minutes before the maximal electroshock seizure test. The animals were observed closely for 2 minutes. The percentage of inhibition of seizure relative to control was recorded and calculated.¹⁵Phenytoin (100 mg/kg) was used as a standard drug.

Neurotoxicity screening:

The minimal motor impairment was measured in mice by the rotorod test. Acute neurological toxicity in mice was evaluated by rotorod test.¹⁵The mice were trained to stay on the accelerating rotorod that rotates at 10 revolutions per minute. The rod diameter was 3.2 cm. Trained animals were administered with the test compounds at dose of 100 mg/kg. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one minute in each of the three trails. Phenytoin was used as a standard drug.

RESULTS:

Spectral studies

Hydrazinopyrimidine (1) was synthesized according to the reported procedure.¹³ The reaction of 5-bromo-2,4-dichloropyrimidine with hydrazine hydrate in methanol to afford the corresponding hydrazino-pyrimidine (2). Hydrazino-pyrimidine, 2 which was reacted with substituted benzaldehyde to afford aryl-(5-bromo-2-chloropyrimidine-4-yl)hydrazone 3(a-f). Newpyrimidine derivatives,3(a-f) were synthesized according to Scheme 1. The chemical structures of all the synthesized compounds are tabulated in Table 1. Readily available starting materials and simple synthesizing procedures make this method is very attractive and convenient for the synthesis of various pyrimidines.

Anticonvulsant activity

Antiepileptic drug research has for several decades focused on identifying new potential drugs based on their anticonvulsant activity against single acute seizures induced by various stimulators, usually in mice and rats. All established antiepileptic drugs have anticonvulsant activity in at least MES model.¹⁴In the present study, the anticonvulsant activity of the synthesized compounds was evaluated by MES model at the dose of 100 mg/kg and the results are summarized in Table 2.

Table 2: Effect of compounds in the maximal electroshock seizure test.

Treatment	E/F	% Protection
3a	5.49	42.61
3b	2.07	68.15
3c	6.25	41.32
3d	6.25	41.34
3e	6.53	44.76
3f	6.25	41.35
Standard	1.96	75.85
Control (Vehicle)	8.20	-
E/F = Extension/Flexion [Decrease in ratio of extension phase (in seconds)/flexion phase (in seconds]. % Protection = (control

DISCUSSION:

Formation of products was confirmed by recording their ¹H NMR, FT-IR and mass spectra. The absence of N-H absorption bands in the IR spectra conﬁrmed that the synthesized compounds. The appearance of a medium to strong absorption band from 1610 to 1618 cm^-1 is due to the stretching vibration of C=N bond formation in the synthesized compounds. The ¹H NMR data of 3(a-f) showed, singlet in the region of δ, 8.74-8.29 (pyrimidine ring) and 8.49-8.26 (CH group), respectively. The mass spectra of all the synthesized compounds showed molecular ion peaks, which are in agreement with their molecular formula. The mass spectra of all the synthesized compounds showed molecular ion peaks, which are in agreement with their molecular formula.

Compound 3b was shown good protective effect on MES induced seizure, and the effect was nearer to that of standard (phenytoin). Similarly, compounds 3e and 3a showed moderate protective effects and a significant difference in protectiveness were observed when compared to standard group. All the compounds were examined for their neurotoxicity using rotorod method given in the dose of 100 mg/kg. None of the compounds showed neurotoxicity (Table 3). The compound 3bshowed 68.15% protection in comparison to phenytoin which completely inhibited the convulsions produced by electro-convulsometer, which having electron withdrawing groups showed excellent anticonvulsant activity.

Table 3: Neurotoxicity screening of the compounds.

Compound	Neurotoxicity Screen
Compound	0.5 h	1h	2h	4h
3a	0/4	0/4	0/4	0/4
3b	0/4	0/4	0/4	0/4
3c	0/4	0/4	0/4	0/4
3d	0/4	0/4	0/4	0/4
3f	0/4	0/4	0/4	0/4
Standard	0/4	0/4	0/4	0/4
The data in the table represent ratio between the numbers of the animals that exhibited neurotoxicity against the number of tested animals.

ACKNOWLEDGMENT:

One of the authors (L. Mallesha) is grateful to the UGC-SWRO, Bengaluru for financial support Minor Research Project, Order No. 2177-MRP/15-16/KAMY013/UGC-SWRO.The Authors sincerely thank JSS Mahavidyapeeta, JSS College of Arts, Commerce and Science and JSS Research Foundation, Mysuru, for providing research facilities to carry out this work.

CONFLICT OF INTEREST:

The authors report no conflict of interest.

REFERENCES:

1. Loscher W. New visions in the pharmacology of anticonvulsion. European Journal of Pharmaceutical Sciences. 1998; 342: 1–13.

2. Goel KK. A review on second generation antiepileptic drugs. Indian Journal of Pharmaceutical Education and Research. 2012; 2: 20-27.

3. Dalkara S, Karakurt A. Recent progress in anticonvulsant drug research: strategies for anticonvulsant drug development and applications of antiepileptic drugs for non-epileptic central nervous system disorders. Current Topics in Medicinal Chemistry. 2012; 12: 1033-1071.

4. Om P, Rajesh K, Ravi K, Prikshit T, Kuhad RC. Organoiodine(III)mediated synthesis of 3,9-diaryl- and 3,9-difuryl-bis-1,2,4-triazolo[4,3-a][4,3-c]pyrimidines as antibacterial agents. European Journal of Medicinal Chemistry. 2007; 42: 868-872.

5. Guetzoyan, Lucie J, Spooner, Robert A, Lord, Mike J, Roberts, Lynne M, Clarkson, Guy J. Simple oxidation of pyrimidinylhydrazones to triazolopyrimidines and their inhibition of shiga toxin trafficking. European Journal of Medicinal Chemistry. 2010; 45: 275-283.

6. Kumar A, Sinha S, Chauhan PM. Synthesis of novel antimycobacterial combinatorial libraries of structurally diverse substituted pyrimidines by three-component solid phase reactions. Bioorganic and Medicinal Chemistry Letters. 2012; 12: 667-669.

7. Baraldi PG, Pavani MG, Nunez M, Brigidi P, Vitali B, Gambari R, Romagnoli R. Antimicrobial and antitumor activity of N-heteroimine-1,2,3-dithiazoles and their transformation in triazolo- imidazo- and pyrazolopyrimidines. Bioorganic and Medicinal Chemistry Letters. 2002;10: 449-456.

8. Nasr MN, Gineinah MM. Pyrido [2,3-d]pyrimidines and pyrimido [5′,4′:5,6]pyrido[2,3-d]pyrimidines as new antiviral agents: synthesis and biological activity.Archives of Pharmacal Research. 2002; 335: 289-295.

9. Sondhi SM, Johar M, Rajvanshi S, Dastidar SG, Shukla R, Raghubir R, Lown JW. Anticancer, anti-inflammatory and analgesic activity evaluation of heterocyclic compounds synthesized by the reaction of 4-isothiocyanato-4-methylpentan-2-one with substituted o-phenylenediamines, o-diaminopyridine and (un)substituted o-diamino pyrimidines. Australian Journal of Chemistry. 2001; 54: 69-74.

10. Gangjee A, Vidwans A, Elzein E, McGuire JJ, Queener SF, Kisliuk RL. Synthesis, antifolate and antitumor activities of classical and nonclassical 2-amino-4-oxo-5- substituted-pyrrolo[2,3-d]pyrimidines. Journal of Medicinal Chemistry. 2001; 44: 1993-2003.

11. Kumar N, Singh G, Yadav AK. Synthesis of some new pyrido[2,3-d]pyrimidines and their ribofuranosides as possible antimicrobial agents. Heteroatom Chemistry. 2001; 12: 52-56.

12. Fathalla OA, Zeid IF, Haiba ME, Soliman AM, Abd-Elmoez SI, El-Serwy WS. Synthesis, antibacterial and anticancer evaluation of some pyrimidine derivatives. World Journal of Chemistry. 2009; 4: 127-132.

13. Castel-Branco MM, Alves GL, Figueiredo IV, Falcao AC, Caramona MM.The maximal electroshock seizure model in the preclinical assessment of potential new antiepileptic drugs.Methods and Findings in Experimental and Clinical Pharmacology. 2009; 31: 101-106.

14. Mohana KN, Prasanna kumar BN, Mallesha L. Synthesis and biological activity of some pyrimidine derivatives. Drug invention today. 2013; 5: 216-222.

15. Hossein H,Marjan NA.Anticonvulsant, sedative and muscle relaxant effects of carbenoxolone in mice. BMC Pharmacology. 2003; 3: 1-6.

Received on 20.11.2017 Modified on 22.12.2017

Asian J. Research Chem. 2018; 11(2):282-286.

DOI:10.5958/0974-4150.2018.00053.6