Application of Green Chemistry Principle in Synthesis of Phenytoin and Its Biogical Evaluation as Anticonvulsant Agents

Phenytoin (5,5’-dipenylimidazolidine-2,4-dione) is the prime example of anticonvulsant agent. According to reported procedure, it is synthesized by condensation of benzil and urea in presence of base (30% w/v NaOH) using ethanol as solvent which itself acts as CNS stimulant. Removal of solvent after synthesis is most difficult and non-assured process. In case of phenytoin transformation in polymorphism plays an important role when solvent other than water is used. About 30% extra cost is calculated if solvent other than water is used. Therefore by application of green chemistry principle phenytoin was synthesized by condensation of benzil and urea in presence of base (30 % NaOH) and water as green solvent. This compound was characterized on the basis of its spectral (IR, H NMR) data and evaluated for anticonvulsant activity using MES induced and PTZ induced seizure models in Swiss albino mice. Significant anticonvulsant activity was found by using 25 mg/kg and 50 mg/kg of phenytoin compared with standard phenytoin at 25 mg/kg dose.


Introduction
Green chemistry is the design of chemical products and process that eliminates the use and generation of hazardous substances 1 .Using green solvent, like water, synthesis of biologically active moiety with high percentage yield as well as purity is the one of objective of green chemistry.Purity of few drugs like CNS acting required high profile of purity and safety for pertaining biological activity.Synthesis of CNS acting moiety with at par purity could be achieved by omitting interfering solvents.Epilepsy is a common neurological condition, affecting 0.5 to 1% of the population worldwide (45-10 million people) 2 .Epilepsy may be defined as paroxysmal, self-sustaining and self-limiting cerebral dysarrhythmia characterized by an abnormal and excessive electroencephalogram (EEG) discharge and a loss of consciousness.Major types of epilepsies are Generalized seizure and Partial seizure 3 .Phenytoin (5, 5-diphenylimidazolidine-2,4dione) is the first anticonvulsant agent often cited as a prime example of anticonvulsant acting as a sodium channel blocker 4,5 .Generally, according to reported procedure, it is synthesized by condensation of benzil and urea in presence of base (30% w/v NaOH) using ethanol as solvent which itself acts as CNS stimulant 6 .Removal of solvents after synthesis is most difficult and non-assured process.In case of Phenytoin transformation in polymorphism plays an important role when solvent other than water is used.About 30% extra cost is calculated if solvent other than water is used.Therefore in present work, Phenytoin was synthesized by condensation of benzil and urea in presence of base (30% NaOH) and water, a green solvent and anticonvulsant activity was performed by MES and PTZ induced seizure models.

Experimental
All the chemicals used for synthesis were of LR (Laboratory Reagent) grade.TLC (Thin Layer Chromatography) was performed on microscopic glass slides (2 x 7.5 cm) coated with silica gel-G, using chloroform: ethyl acetate (7:3) as a solvent systems and the spots were visualized by exposure to iodine vapours.The IR spectrum of synthesized compound was recorded on Shimadzu 8400-S FT-IR Spectrophotometer using potassium bromide.The 1 H NMR was recorded in DMSO-D6 using NMR Varian-Mercury 300MHz spectrometer and chemical shifts are given in parts per million, downfield from tetramethylsilane (TMS) as an internal standard from University of Pune.

General procedure
5.3 g (0.025 mol) of benzil, 3 g (0.05 mol) of urea, 15 mL of 30% w/v sodium hydroxide solution and 40 mL of water was placed in a 250 mL round bottom flask and refluxed for 2 hours.After cooling to room temperature, the reaction mixture was poured into 100 mL of water with stirring.It was allowed to stand for 15 minutes and filtered under suction to remove the insoluble by-product.The filtrate thus obtained was cooled and acidified by using concentrate hydrochloric acid.The precipitates obtained were separated by filtration.The crude product obtained was washed with cold water.IR and 1 H NMR spectra of synthetised phenytoin are represented by Figure 1 and Figure 2 respectively.The physical and spectral data matched with that of standard given in Table 1, 2 and 3 respectively.

Anticonvulsant activity
The Anticonvulsant activity of the compound was measured by MES (Maximal electroshock) induced seizure test and PTZ (Pentylenetetrazole) induced seizure test.
MES induced seizure test 7,8 The Swiss albino mice of either sex weighing 25-30 g were used for the test.They were divided into four groups each containing 6 mice.0.1% gum acacia solution was used as a vehicle for preparation of suspension of Phenytoin.Control group was injected with normal saline solution or gum acacia solution 10 mL/kg (p.o.).Phenytoin 20 mg/kg (i.p.) was used as a standard.Each group test 1 and test 2 was treated orally with 20 mg/kg and 40 mg/kg (p.o.) of Phenytoin respectively.They all received current of 48mA for 2s duration through electroconvulsiometer using ear electrodes after 60 min of oral administration of test formulation.The animals were observed closely for 2 minutes.The duration of tonic flexion and latency of onset of clonus was recorded.A complete abolition of hind limb tonic extension was considered as 100% protection.The ability to prevent this feature was considered as an indication of anticonvulsant activity.Data of MES induced seizure test of synthesized phenytoin is given in Table 4 and represented with the Figure 3.  Low dose-25 mg/kg, High dose-50 mg/kg body wt. of Swiss albino mice; Series 1 -tonic flexion, series 2-tonic extension, series 3-clonic phase PTZ induced seizure test 9 ) Swiss albino mice of either sex with a body weigh between 18 & 12 g, were used the test compound or the reference drug was injected subcutaneous or Intraperitoneal or given orally two group of 10 mice.Another group of 10 mice serve as control 15 min after oral administration subcutaneously injection 30 min after intraperitoneal injection or 60 min, after oral administration, 60 mg per kg PTZ was injected subcutaneously.Each animal was placed in to an individual plastic cage, for observation lasting in seizures and tonic clonic convulsion was recovered.At least 80% of the animals in the control group were show to convulsion.Data of PTZ induced seizure test is given in Table 4 and represented with the Figure 4.

Conclusion
Phenytoin is synthesized by application of principle of green chemistry with high purity profile as well as having safety by omitting the use of ethanol.Water a green solvent is used instead of ethanol in synthesis of phenytoin .There is reduction in time and ultimately cost as compare to conventional procedure of synthesis of phenytoin.Thus we conclude that the synthesized compound have potential in the green chemistry.

Figure 3 .
Figure 3. MES induced test on mice of synthetic compound Phenytoin after oral administration (Where As Control group -Saline water, Std.Drug -Phenytoin 25 mg/kg, Test compound phenytoin,.

Table 1 .
Physical data of phenytoin

Table 2 .
IR Spectral data of phenytoin

Table 4 .
Data of Anticonvulsant activity of Synthetic Compound Phenytoin on MESinduced seizures in mice.

Table 4 .
Data of anticonvulsant activity by PTZ-induced seizures in mice (Values are expressed as mean + S.E.M. n=6 ** p < 0.01 * p < 0.05 compared with vehicle control ANOVA followed by Dunnet's t-test)

Figure 4 .
PTZ induced test on mice of Synthetic compound Phenytoin after oral administration '-dphenylimidazoldine-2,4-dione The compound has shown significant anticonvulsant activity with increase latency of onset of phases in Time (s) as compared to standard in MES induced seizure test.PTZ induced seizure test have shown 61.23% and 69.52% inhibition of onset of death.