CHARACTERIZATION AND PHARMACOLOGICAL EVALUATION OF NOVEL SUBSTITUTED ISATIN DERIVATIVES

51 | P a g e TO SYNTHESIZE, CHARACTERIZATION AND PHARMACOLOGICAL EVALUATION OF NOVEL SUBSTITUTED ISATIN DERIVATIVES D B Joshi Department of Pharmaceutical Chemistry, Shrinathji Institute of Pharmacy, Nathdwara (Raj) Article Info: Received 10 January 2020; Accepted 06 February 2020 DOI: https://doi.org/10.32553/jbpr.v9i1.719 Corresponding author: D B Joshi Conflict of interest statement: No conflict of interest ABSTRACT: Series of novel Schiff bases of Isatin the equimolar amines and 5-Dicrboxymethyl (R=COO2Me) substituted isatins (1 mmol of each) were added to 96% w/w ethanol (20 mL) containing 8 drops of glacial acetic acid. The mixture was heated under reflux for 5 h and then cooled to room temperature. The resulting solid was collected by filtration, washed with cold ethanol and dried in open air. The derivatives thus prepared had sufficient analytical purity. anticonvulsant activity performed by method as Animals were weighed and numbered. Mice were divided into 7 groups of six animals each. Group 1 served as control which was treated with vehicle (2% v/v Tween 80), group 2 was treated with standard drug phenytoin (25 mg/kg, i.p.) and groups 3– 7 were treated with newly synthesized oxadiazole derivatives (25 mg/kg, i. p.). One hour after injection, the animals were subjected to electro shock through ear electrodes of 80 mA for 0.2 sec by electroconvulsiometer AND ANTI-inflammatory activity measured by Weigh the animals and number them. Mark the animals with picric acid for individual animal identification. Divide rats into 5 groups of 6 rats each. Note the initial paw volume of each rat by dipping just beyond tibio-tarsal junction by mercury displacement method. The pharmacological screening of the synthesized compounds showed anti convulsant activity ranging from 56.2 % to 76.3 % inhibition of epileptic seizures in mice, where as the standard drug Phenytoin showed 83.95 % inhibition of epileptic seizures in mice. The compound iiih4 from each group was found to be nearly potent to Phenytoin which is used as standard drug. Antiinflammatory activity ranging from 31.09 to 63.11 % inhibition of rat paw edema volume after 3 hours, whereas the standard drug Indomethacin showed 62.06 % inhibition of rat paw edema volume after 4 hours. The compound iiih3 was found to be nearly more potent then indomethacin which is used as standard drug


INTRODUCTION
Isatin (1H-indole-2,3-dione, Figure 1) was first obtained by Erdman and Laurent in 1841 as a product from the oxidation of indigo by nitric and chromic acids.

Figure 1: 1H-indole-2,3-dione
The synthetic versatility of isatin has led to the extensive use of this compound in organic synthesis. Three reviews have been published regarding the chemistry of this compound: the first by Sumpter, in 1954 [1], a second by Popp in 1975 [2], and the third on the utility of isatin as a precursor for the synthesis of other heterocyclic compounds [3].
In nature, isatin is found in plants of the genus Isatis [4], in Calanthe discolor LINDL.

Instrumentation:
Spectroscopic data were recorded with following instrument; Fourier Transform Infra-Red spectra (FTIR) were recorded on Shimadzu FTIR-8400S spectrophotometer using potassium bromide pellets and sodium chloride cell. Nuclear Magnetic Resonance spectra (1H-NMR) were recorded on JEOL-300 MHz spectrophotometer in CDCl3 using TMS as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm). Mass spectra were recorded on HEWLETT 180017, PACKARD GCD System mass spectrophotometer using electron ionization detector. Anticonvulsant activity checked by Electroconvulsometer (LABTECH).

General procedure for the synthesis of Schiff bases:
Equimolar amines and 5-Dicrboxymethyl (R=COO2Me) substituted isatins (1 mmol of each) were added to 96% w/w ethanol (20 mL) containing 8 drops of glacial acetic acid. The mixture was heated under reflux for 5 h and then cooled to room temperature. The resulting solid was collected by filtration, washed with cold ethanol and dried in open air. The derivatives thus prepared had sufficient analytical purity. [24][25][26][27][28][29]

ANTI-CONVULSANT ACTIVITY:
All antiepileptic drugs (AEDs) are rigorously study in animals, particularly rodents, before they are given to patients. Understanding how drugs are screened in animals is useful to the clinician, since the screening process is valuable in predicting the type of seizure in which the drug would be efficacious, as well as determining the mechanism of the drug's anti-seizure action. Indeed, the dramatic discovery of the antiseizure properties of phenytoin was identified by Merritt and Putnam in 1938 using the electroshock-induced seizure model. [30][31]

Animals:
Animals Albino mice, weighing 18-30 g, were used for experiments. The animals were kept in colony cages (6 mice each), maintained on a standard pellet diet with water, and left for 2 days for acclimatization before the experimental session. The food was withdrawn on the day before the experiment, but free access to water was 56 | P a g e allowed. All experiments were carried out according to the suggested ethical guidelines for the care of laboratory animals. [32]

Selection of experimental animals:
Healthy Albino wistar male rats weighing between 18-30 g were used for the evaluation of anti-convulsant activity.

Laboratory conditions:
The rats were housed comfortably in a group of six in a single clean plastic cage with a metal frame lid on its top. Environmental room should be 22°C (± 3°C) relative humidity was kept at least 30 % and preferably not exceed 70 % other than during room cleaning the aim was maintained between 50-60%. Lighting was artificial, the sequence being 12 hours light and 12 hours.

Food and water:
All animals were allowed free access to water and standard palletized laboratory animal diet.

Bedding:
In the present study animals were provided with clean paddy husk bedding. Bedding was changed every alternate day to maintain proper hygienic conditions.
The anticonvulsant activity was carried out by maximal electrical shock induced convulsion method in albino mice.
Animals to be use:

Test compounds
:-suspension of compounds were prepared and administered intra peritoneal similar to that of standard drug. Apparatus: -Syringes (1 ml, 2 ml), sample tubes.

Experimental design and procedure:
Animals were weighed and numbered. Mice were divided into 7 groups of six animals each. Group 1 served as control which was treated with vehicle (2% v/v Tween 80), group 2 was treated with standard drug phenytoin (25 mg/kg, i.p.) and groups 3-7 were treated with newly synthesized oxadiazole derivatives (25 mg/kg, i. p.). One hour after injection, the animals were subjected to electro shock through ear electrodes of 80 mA for 0.2 sec by electroconvulsiometer and the duration of time for extensor response was noted and the activity was expressed in terms of % protection. [33] All the results are expressed as mean ± SEM. The % inhibition of epileptic seizures was calculated by using the formula, Percent (%) protection = VC -VT/VC X 100, Where, VT-Mean time in test group, VC-Mean time in control group.

Animals
For the biological evaluation Albino wistar rats, (200-300 g), were used. The animals were kept in colony cages (6 rats each), maintained on a standard pellet diet with water, and left for 2 days for acclimatization before the experimental session. They kept on fast for 16 hour before the experiment, but free access to water. Experiments were carried out according to the ethical guidelines for the care of laboratory animals.

Selection of experimental animals:
Healthy Albino wistar male rats weighing between 200-300 g. were used for the evaluation of anti-inflammatory activity. The animals were obtained from Zydus research centre, Ahmedabad.

Laboratory conditions:
The rats were housed comfortably in a group of six in a single clean plastic cage with a metal frame lid on its top. Environmental room should be 22°C (± 3°C) relative humidity was at least 30 % and preferably not exceed 70 % other than during room cleaning the aim was to maintain between 50-60%. Lighting was to be artificial, the sequence being 12 hours light and 12 hours.

Anti-inflammatory activity:
The anti-inflammatory activity of synthesized indole derivatives were carried out using carrageenan induced rat hind paw edema method.
Inflammation inducing agent:-carrageenan solution (1%w/v) in saline solution was prepared and injected (0.1ml) in sub planter region to induce paw edema. Chemicals:-Tween-80 Standard drug:-Indomethacin (3 mg/kg) aqueous suspension was prepared using solution of tween-80 as a suspending agent.

Experimental design and procedure:
Weigh the animals and number them. Mark the animals with picric acid for individual animal identification. Divide rats into 5 groups of 6 rats each. Note the initial paw volume of each rat by dipping just beyond tibiotarsal junction by mercury displacement method, so that every time the paw is dipped in the mercury column up to the fixed mark to ensure constant paw volume. The animals were deprived of food overnight (allowed free access to water) and synthetic compounds were administered once before 30 minutes the injection of carrageenan. Dose volume not exceeding 0.5ml/100gm intra peritoneal was administered.
Group I:-The solvent control received normal saline.
Group III:-Received indole derivative-4.7 at a dose of 3 mg/kg suspended in 1%w/v tween-80 Group IV:-Received indole derivative-4.8 at a dose of 3 mg/kg suspended in 1%w/v. tween-80 Group V:-Received indole derivative-4.9 at a dose of 3 mg/kg suspended in 1%w/v tween-80.
After 30 minutes of test compound administration, 0.1ml of 1%w/v of carrageenan in normal saline was injected in to the sub planter region of the left hind paw of rat. Immediately after the carrageenan injection, the volume of its displacement was measured using plethysmometer. [37][38][39]. The reading was recorded at 0, ½, 1, 2, 3 hrs.
The % inhibition of edema was calculated at the end of 3 hrs by using the formula. [30] Percent (%) inhibition = 1 -Vt/Vc X 100, Where Vt: -edema volume in test group, Vc: -edema volume in control group Results were expressed as mean ± standard deviation.

RESULT AND DISCUSSION:
6.1 Screening of Anti-Convulsant Activity:

CONCLUSION:
The pharmacological screening of the synthesized compounds showed anti convulsant activity ranging from 56.2 % to 76.3 % inhibition of epileptic seizures in mice, where as the standard drug Phenytoin showed 83.95 % inhibition of epileptic seizures in mice. The compound iiih4 from each group was found to be nearly potent to Phenytoin which is used as standard drug. Structure resembles in above compound have significant to standard drugs were structure having substitute isatin ring with penta-cyclic ring with amine group give better anti-convulsant activity.
The pharmacological screening of the synthesized compounds showed anti-inflammatory activity ranging from 31.09 to 63.11 % inhibition of rat paw edema volume after 3 hours, whereas the standard drug Indomethacin showed 62.06 % inhibition of rat paw edema volume after 4 hours. The compound iiih3 was found to be nearly more potent then indomethacin which is used as standard drug. Except then above mention compound has shown less activity then indomethacin. Iiih3 compound was possess 5-methoxy (-OCH 3 ) with 4-hydroxy benzahydrazide responsible for anti-inflammatory activity.