Synthesis Of Schiff Bases Of Indole-3-Carboxaldehyde Derivatives Using Biginelli Reaction And Their Antioxidant Evaluation

Biginelli reaction, which involves the interaction of ethyl acetoacetate, thiourea, and an aromatic aldehyde. Biginelli products (3,4-dihydropyrimidin-2(1H)-ones) are interesting starting materials due to their significant therapeutic  and pharmacological properties[1]. Additionally the dihydropyrimidinones found to exhibit a wide range of biological activities such as antiviral,anticancer, antibacterial, anti-inflammatory, antimalarial, antitubercular, antidiabetic, anti-epileptic, etc[2]. Pyrimidine is one of the most important nucleus in medicinal chemistry. The synthesis of novel derivatives of pyrimidines remains as main focus in drug discovery. The synthesis of newer generation of pyrimidines would help for further development of better medicinal agents[3]. Indole nucleus also possess a wide range of therapeutic activity. Indole derivatives have found to exhibit potent antifungal, analgesic , anti-inflammatory and  antiamoebic activity. Based on preliminary findings indole-3-carboxaldehyde has potent antioxidant activity[4]. The formation of free radical is assosciated to the normal metabolism of aerobic cell[5]. The inherited oxygen consumption of cell leads to the production of series of oxygen free radicals and this interact with the lipid molecules generates new radicals such as superoxides, hydroxides and lipid peroxides which interact with biological system in a cytotoxic manner[6]. The uncontrolled production of free radicals is responsible for several pathological processes such as tumours(prostate and colon cancers) and coronary heart disease[7]. Even though  lot of study regarding the biological activity of indole-3-carboxaldehyde has been carried out ,the antioxidant activity of indole-3-carboxaldehyde linked with dihydropyrimidinones has not been done.

MATERIALS AND METHODS

All the solvents and reagents used were of laboratory grade. Melting points were determined by an open capillary melting point apparatus and were uncorrected. Thin layer chromatography (TLC) was used to determine the purity of compound synthesized respectively. TLC Merck precoated plates were identified by iodine vapour. Solvent system used for developing the chromatogram was ethylacetate : toluene (9:1). The infrared spectrum was taken on SHIMADZU IR Spirit.

EXPERIMENTAL WORK

Synthesis of biginelli compound

The intial steps involve the reaction between thiourea ,ethylacetoacetae and various substituted aromatic aldehydes.to a round bottom flask 0.15 mole of thiourea ,0.1 mole of ethylacetoacetate and 0.1 mole of substituted aromatic aldehyde dissolved in 20ml ethanol and add 2-3 drops of HCl and reflux for 2 hours.The reaction mixture was then poured into 100 ml ice cold water with stirring and left overnight at room temperature. Then this mixture is filtered and dried.  Recrystallisation done using ethanol. Recrystallised product is used in the next step. Similar procedure was followed for various substituted aromatic aldehydes. The purity of the compounds was determined by thin layer chromatography.

Synthesis of carbohydrazido derivative

A mixture of 0.1 mole of biginelli compound(1st step product) and 0.1 mole of hydrazine hydrate were dissolved in 20 ml of ethanol. To this add 4 drops of conc. sulphuric acid and refluxed for 3 -4 hours. The reaction mixture was  evaporated to obtain a residue. The residue is then recrystallised from ethanol. The purity of the compounds was determined by thin layer chromatography.

Synthesis of schiff bases of Indole-3-carboxaldehyde derivatives

A mixture of 0.01 mole of hydrazido product(2ndstep product) and 0.01 mole of indole-3-carboxaldehyde dissolved in ethanol along with 5 ml of glacial acetic acid were refluxed for 4-5 hours. The reaction mixture was then poured into ice cold water in a beaker, filtered and dried. The precipitate was then recrystallised from ethanol. The purity of the compounds was determined using thin layer chromatography.

SCHEME OF THE WORK

STEP1

STEP2

STEP3

Physiochemical properties of the synthesized compounds

DP1 :-

6-methyl-4-(2-nitrophenyl)-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-1H-indole 

IRspectra KBr(cm-1):  1516.91(NO2 stretching), 2922.92(Ar-H stretching), 3221.87(NH stretching), 1696.28(C=O stretching), 2937.06(CH stretching)

DP2 :-

4-(4-fluorophenyl)-6-methyl-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazidoe-1H-indole

IRspectra KBr(cm-1): 3119.78 (Ar-H stretching), 3414.35 (NH stretching), 2973.7(CH  stretching), 1648.36(C=O stretching)

DP3 :-

4-(4-chlorophenyl)-6-methyl-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-1-H-indole

IRspectra KBr(cm-1): 799.44(C-Cl stretching), 3234.40(Ar-H stretching), 2923.88(NH stretching), 1703.03 (C=O stretching), 2973.3(CH stretching)

DP4 :-

6-methyl-4-(4-methylphenyl)-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-1H-indole

IRspectra KBr(cm-1): 3115.44 (Ar-H stretching), 3536.33 (NH stretching), 1518.75(NO2 stretching), 1648.36(C=O stretching), 2931.75(CH stretching), 2926.93(CH stretching)

DP5 :-

6-methyl-4-(4-nitrophenyl)-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-1H-indole

IRspectra KBr(cm-1): 2925.35 (Ar-H stretching), 3223.52 (NH stretching), 1518.75(NO2 stretching), 1698.55(C=O stretching), 2937.06(CH stretching)

DP6 :-

4-(2-chlorophenyl)-6-methyl-2-sulfanylidene-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide-1H-indole

IRspectra KBr(cm-1): 3235.50(Ar-H stretching), 2925.75(NH stretching), 1705.55 (C=O stretching), 797.30(C-Cl stretching), 2916.32(CH stretching)

ANTIOXIDANT ACTIVITY

DPPH Radical scavenging activity

The newly synthesized compounds were screened for their antioxidant activity by DPPH free radical scavenging activity.  The compounds under study were dissolved in distilled ethanol (50 ml) to prepare 1000 ?M stock solution. Solutions of different concentrations (10 ?M, 50?M,100 ?M, 200 ?M and 500 ?M) were prepared by serial dilutions and the antioxidant activity was studied. Compound of different concentrations were prepared in distilled ethanol, 1ml of each compound solutions having different concentrations (10 ?M, 50 ?M, 100 ?M, 200 ?M and 500 ?M) were taken in different test tubes; 4ml of a 0.1Mm ethanol solution of DPPH was added and shaken vigorously. The tubes were then incubated in the dark room at RT for 20 min. A DPPH blank was prepared without compound, and ethanol was used for the baseline correction. Changes (decrease) in the absorbance at 517 nm were measured using a UV-visible spectrophotometer and the remaining DPPH was calculated. The percent decrease in the absorbance was recorded for each concentration, and percent quenching of DPPH was calculated on the basis of the observed decreased in absorbance of the radical. The percentage of inhibition was calculated using the formula:

Percentage Inhibition (%) = [(Ao-A1) / Ao X 100]

Where Ao is the absorbance of the control (blank, without compound)  and A1 is the absorbance of the compound. The percentage inhibition of internal standard ascorbic acid was also measured and IC50 values was calculated and compared with that of the newly synthesized compounds.

Antioxidant activity of synthesized compounds

           
       

DISCUSSION