SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL IODOPHTHALAZINEDIONE DERIVATIVES AS ANTICONVULSANT AGENTS

In view of their expected anticonvulsant activity, some new derivatives of phthalazine (I, II, IIIa-f, IV, V, VIa-f and VIIa-h) were designed and synthesized by condensation of Iodophthalic anhydride with hydrazine hydrate to produce iodophthalazindione I which heating with Alc. KOH to give compound II. The compounds IIIa-f resulted from condensation of compound II with different alkylcholoroacetates. Compound IIb reacted with ammonia, hydrazinehydrate and different alkyl amines resulted compounds IV, V (hydrazide) and VI respectively. Compound V (hydrazide) react with different aromatic aldehydes to produce compounds VIIa-h. the final compounds were structurally elucidated basis of IR , 1 HNMR, Ms Carbon, hydrogen and nitrogen analysis. The final compounds were evaluated for anticonvulsant activity using pentylenetetrazole (ptz) to induce convulsion and phenobarbital as reference standard. The compounds I, II, IV, V, VIa, VIb, VIc, VId, VIf, VIIa, VIIb, VIIc and VIIg were active than Phenobarbital as standard reference the remain compounds less active than Phenobarbital. To under stand the molecular significant of these results we compared the binding modes of these compounds. Acknalewlgement: Many thanks for Dr. Ahmed Mansour assist. Prof. of pharmacology and Dr. Farg Sherbiny lecturer of organic chemistry


INTRODUCTION
Phthalazine derivatives received much attention in recent years a wing to both their biological significance and pharmaceutical applications (Napoletano, et al., 2001;Napoletano, et al., 2001;Napoletano, et al., 2000;Curia, et al., 2002;Imamura, et al., 2003).Many phthalazine derivatives have been reported as anticonvulsants (Siva, et al., 2002;Ayyad, 2008;El-Helby, et al., 2001;El-Helby, et al., 2002) Some phthalazines were reported to possess anticonvulsant activity against minimal electrical sock (MES) and exhibited significant decrease in elevated motor activity (Siva, et al., 2002).On the other hand, it has been reported that a lot of compounds containing arylidene moiety possess good anticonvulsant activity and have a big role in MAO inhibitory action (Eid, et al., 1991).On the light of these findings, we decided to design and synthesize novel derivatives of iodophthalazine containing arylidene moiety and other derivatives (ester, amide) to evaluate their anticonvulsant activity and to compare the difference in the pharmacological effects.As a result, we found that the synthesized compounds (I, II, III a-f , IV, V, VI a-f and VII a-h ) have appreciable anticonvulsant activity.
. The following scheme illustrate our experimental work.

RESULTS AND DISCUSSION
The synthesized compounds I, II , III a_f , IV , V , VI a-f and VII a_f were confirmed by melting point spectral data and C , H , and N analysis .Also anticonvulsant activities of the synthesized compounds (I -VII a_f ) were determined using the pentylenetetrazole (ptz).Seizure threshold test (Swinyard, et al., 1982) preliminary screening results showed that all compounds (I -VII a_f ) exhibit anticonvulsant activity the compounds are divided into two groups all of them are evaluated at three different dose levels the first at 25, 50 and the second at 50, 100 and 200 mg using Phenobarbital sodium at three different dose levels (50, 100 and 200 mg) as positive control.The test compounds were injected intrapretoneal ( ip ) followed by subcutaneous ( sc ) ( ptz ) at 70 mg / kg aminimal dose that produce threshold seizures in approximately 100% of control animals ( the compounds I , II , III a -d , VI d -f and VII f -h are tested at three different dose levels ( 25 , 50 , and 100 mg ) and revealed anticonvulsant activity equal to or more than Phenobarbital as positive control , while the compounds III e , III f , IV , V , VI a , VI b , VI c , VII d , VII e tested at three different dose levels ( 50 , 100 and 200mg ) and revealed anticonvulsant activity less than Phenobarbital.In general, there is no one of all tested compounds less than 80% active as Phenobarbital.The activity of tested compounds due to Phormocophoric group in phthalazine nucleolus which have CO -N -N -CO which different in there positions in quinoxaline and qiunazoline N -CO -CO -N and CO -N -CO-N, respectively which have anticonvulsant activity (Ossman et al., 1986;Aziza, et al., 1996).Some of the ester groups have activity less than Phenobarbital due to the ester functional group which undergo esterase enzyme but the other groups have amide which is difficult to breakage by amidase enzyme comparing with esterase enzyme.
All tested compounds showed 33.3% protection at the dose (25mg, 50mg), 66,6% protection at the dose( 50mg ,100mg) and 100% protection at the dose (100mg, 200mg) .This kind of protection is an indication for the potential anticonvulsant activity of the tested compounds particularly against absence seizures.Anticonvulsant activity (While, et al., 1995) could be associated with some undesirable effects such as sedation.N.B.GABA is the major inhibitory transmitter in the mammalian CNS, which controls the excitability of many central pathways.The GABA is the receptor complex mediates the flux of chloride ions across nerve membranes and may be related to the anticonvulsant action of GABAmimetic drugs (Reddy, et al., 1997;Wood, et al., 1980) the principal mode of action of this transmitter occurs by modulation of the GABAchloride ion channel complex as attempt explore the possible GABA-mimetic activity of the active compounds.

Anticonvulsant activity
The animals were under taken with approval from ethics committee approval (23PD/3/12/18R) of Al-Azhar University, Cairo, Egypt.All the trials were carried out according to the respective internationally valid guidelines.Anticonvulsant activity of our compounds was evaluated according to the method reported by Soaje-Echaque and-Lim (1962) using Swiss albino adult male mice, weighing 20-59 gm.They were obtained from an animal Facility (Animal house.Department of pharmacology and toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.Mice were housed is stainless steel wire floored cages without any stressful stimuli.Animals were kept under well-ventilated conditions at room temperature (25-30 o C).They were fed on an adequate standard laboratory chow (El-Nasr Co., Abou-Zabal, Egypt) and allowed to acclimatize with free access to food and water for 24 hrs period before testing except during the short time they were removed from the cages for testing.Albino mice were randomly arranged in groups each comprising 12 animals.Phenobarbital sodium (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA) was used as a reference drug for comparison.Pentylenetetrazole (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA) was used to induce convulsion in the experimental animals.All synthesized phthalazine were tested for evolution of their anticonvulsant activities.The test compounds were suspend in normal saline with the aid of Tween 80 (Medical union Pharmaceuticals Co., Ismailia, Egypt)test compounds were intraperitoneally (i.p) injected in dose ranging from 25-200 mg/kg animal weight using the same dosing volume of (0.2 ml per 25 gm.Pentylenetetrazole was dissolved in normal saline in 2% concentration and was given (sc) in dose 50-200 mg/kg (70 mg/kg as a minimal dose that produce threshold seizure in approximately 100% of control animals ) using the same dosing volume.All drugs were freshly prepared to the desired concentration just before use.The percentage protection per each dose and the dose which makes protection for 50% of animals (ED 50 ) was calculated using INSTAT2 program (ICS Philadelphia, PA, USA) presented in table (4) the activity of test compounds in comparison to Phenobarbital or relative potency of the test compounds to Phenobarbital was calculated and used for comparison among compounds under test as shown in table (4).

Experimental Section
All melting points were measured on a griffin melting point apparatus (Griffin, Valdosta, GA, USA) and are corrected.The infrared spectra were recorded asKBr discs on a nicolet IR 200 (thermo fisher scientific, Barrington, USA) at the pharmaceutical analytical unit, Faculty of Pharmacy, Al-Azhar University, Cairo Egypt.The 1 HNMR spectra were ran using TMS as an internal standard (Aldrich Chem.CO., Milwaukee, Wi, USA) on varian Mercury VXr-300 NMR (Varian Palo., Alto, CA, USA) at the Micro Analytical Center, Faculty of Sciences Cairo, University Giza, Egypt.
Mass spectra were performed on varian MAT 311-A (70eV) (Varian, San Fernando, GA, USA) at the Micro analytical center of Cairo University Giza, Egypt, Elemental analyses (C,H,N) were performed on a Perkin-Elmer 2400.
Analyzer (Perkin-Elmer, Nor walk, C.T., USA) at the Micro analytical unit of Cairo University, Giza, Egypt.All compounds were within ± 0.4% of the theoretical.All chemicals used for synthesis were purchased from Sigma-Aldrich Chemical Co.Milwaukee, WI, USA.

Anticonvulsant activity
The animal were under taken with approval from ethics committee approval #23PD/3/12/18R) of Al-Azhar University, Cairo, Egypt.All the trials were carried out according to the respective internationally valid guidelines.Anticonvulsant activity of our compounds was evaluated according to the method reported by Soaje-Echaque and-Lim (11)  using Swiss albino adult male mice, weighing gm.They were obtained from an animal Facility (Animal house.Department of pharmacology and toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.Mice were housed is stainless steel wire floored cages without any stressful stimuli.Animals were kept under well-ventilated conditions at room temperature (25-30 o C).They were fed on an adequate standard laboratory chow (El-Nasr Co., Abou-Zabal, Egypt) and allowed to acclimatize with free access to food and water for 24 hrs period before testing except during the short time they were removed from the cages for testing.Albino mice were randomly arranged in groups each comprising 12 animals.Phenobarbital sodium (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA) was used as a reference drug for comparison.Pentylenetetrazole (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA) was used to induce convulsion in the experimental animals.All synthesized phthalazine were tested for evolution of their anticonvulsant activities.The test compounds were suspend in normal saline with the aid of Tween 80 (Medical union Pharmaceuticals Co., Ismailia, Egypt)test compounds were intraperitoneally (i.p) injected in dose ranging from 25-200 mg/kg animal weight using the same dosing volume of (0.2 ml per 25 gm.Pentylenetetrazole was dissolved in normal saline in 2% concentration and was given (i.p) in dose 50-200 mg/kg using the same dosing volume.All drugs, were freshly prepared to the desired concentration Just before use.The percentage protection per each dose and the dose which makes protection for 50% of animals (ED 50 ) was calculated using INSTAT2 program (ICS Philadlphia, PA, USA).
m a ti c a ld e h y d e g li c a l a c e ti c a c id

Figure 1 .
Figure 1.Compounds II , IV , V , VI a , VI b , VI e VI f , VII b , VII c , VII f , and VII g showed anticonvulsant activity more than Phenobarbital.While compounds I , VI d , and VII a revealed activity equal to Phenobarbital.The rest compounds are anticonvulsant activity less than Phenobarbital but not less than 80% of its activity.

Table 2 .
Physical data