Solvent-free , Environmentally Benign Syntheses of Some Imines and Antioxidant Activity

Environmentally benign, economically feasible, and solvent-free syntheses of series of imines by the condensation of a substituted hydroxynaphthyl ketone with several substituted iodoanilines under grinding approach are described. Imines were further tested for antioxidant activity; most of them show moderate activity.


INTRODUCTION
In recent years, the use of hazardous and toxic solvents in chemical laboratories, chemical industry has been considered a very serious problem for the health, safety of workers, and environmental pollution.For these purposes, the emerging area of green chemistry plays an important role into the development of synthetic strategies [1,2].Grinding technique, which is reacting under solvent-free conditions, is one of the green synthetic routes that has gained popularity towards designing the structure of new molecules [3][4][5][6].Grinding technique has been increasingly used in organic synthesis as compared to traditional methods [7][8][9], because these reactions are not only of interest from an economical point of view, but also in many cases they offer considerable advantages in terms of yield, selectivity, and simplicity of reaction procedure with high atom efficiency.Many well known reactions have been reported under solvent-free environments using the grinding technique [10][11][12][13][14][15][16][17].

MATERIAL AND METHODS
Melting points were determined in open capillary tubes and are uncorrected.FT-IR spectra were recorded in KBr pellets on a Perkin-Elmer [8201] spectrometer. 1H NMR spectra were recorded on a Gemini 300-MHz instrument in DMSO-d6 as the solvent and TMS was used as an internal standard.The mass spectra were recorded on SHIMADZU (GCMS-QP 1000 EX) GC-EI-MS spectrometer.Elemental analyses were performed on a Perkin-Elmer 240 CHN elemental analyser.The reactions were carried out in open glass mortar and pestle.Purification of the compound was indicated using TLC (ethyl acetate / hexane (0.25 mL: 0.25 mL, v/v) as the mobile phase).

General procedure for the synthesis of imines
A mixture of the hydroxyketone 1 (0.01 mol)

Full Paper
Orbital: Electron.J. Chem.7 (3): 208-214, 2015 209 and substituted anilines 2 (0.01 mol) were ground with a pestle in an open mortar at room temperature for 2-3 minutes.To this reaction mixture sulfuric acid (0.002 mmol) was added and grinding continued for 4-7 minutes.On completion of reaction as monitored by TLC, the light greenish-coloured solid was separated out.The obtained solid was diluted with cold water and isolated by simple Buchner filtration and recrystallized from ethanol to give pure imines 3a-x as light yellow crystals.
Scheme 1. Synthesis of imines under solvent-free conditions using grinding technique 3a-x

Antioxidant activity
The following antioxidant methods were used to evaluate the antioxidant properties of our test compounds.

1) DPPH• Scavenging Activity
) is a stable free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule (Scheme 2).Due to its singlet electron, the methanolic solution of DPPH shows a strong absorption band at 517 nm.DPPH radical reacts with various electron-donating molecules (reducing agents or antioxidants).When electrons become paired off, bleaching of the DPPH solution is what we observe as a result.This results in the formation of the colourless 2,2'-diphenyl-1picrylhydrazine.Reduction of the DPPH radicals can be estimated quantitatively by measuring the decrease in absorbance at 517 nm.Procedure: Equal volumes of 100 μM DPPH in methanol were added to different concentrations of test compounds (0 -200 μM/mL) in methanol, mixed well and kept in dark for 20 min.The absorbance at 517 nm was measured using the spectrophotometer UV-1650 (Shimadzu) [28].Plotting the percentage DPPH• scavenging against concentration gave the standard curve and the percentage scavenging was calculated from the following equation: IC50 was obtained from a plot between concentration of test compounds and percent scavenging.Ascorbic acid was used as standard for comparison.

2) Nitric Oxide Scavenging Activity
Nitric oxide (NO) will be generated by sodium nitroprusside in the solution.In the presence of an antioxidant or nitric oxide scavenger the amount of NO generated will be less.The excess NO will be estimated by Griess reagent, which is the mixture of sulphanilic acid and naphthylethylenediamine dihydrochloride.The nitric oxide will give pinkcoloured complex estimated at 540 nm.
Procedure: To a reaction mixture (6 mL) containing sodium nitroprusside (10 mM, 4 mL), phosphate buffer saline (PBS, 1.0 mL) and 1.0 mL of different concentration of test compounds/standard were incubated at 25 0 C for 150 min.After incubation, 0.5 mL of the reaction mixture containing nitrate was removed and 1.0 mL of sulphanilic acid was added, mixed well, and allowed to stand for 5 min for completion of diazotisation.Then 1.0 mL of naphthylethylenediamine dihydrochloride was added, mixed, and allowed to stand for 30 min in dark at room temperature.The absorbance of these solutions was measured at 540 nm against the corresponding blank solution without sodium nitroprusside [29].The % scavenging and IC50 values were determined as explained in the DPPH assay.The standard rutin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[α-Lrhamnopyranosyl-(1→6)-β-D-glucopyranosyloxy]-4H-chromen-4-one) were used for comparison antioxidant activity of synthesized compounds.

RESULTS AND DISCUSSION
The present studies describe the reactions carried out using grindstone technique, simply by mixing corresponding substituted hydroxyketones 1 and substituted anilines 2. The mixture was ground together in mortar with pestle at room temperature for 2-3 minutes, and then a catalytic amount of sulfuric acid was added to this grinded reaction mixture.The grinding was continued for 4-7 minutes and the progress of reaction was monitored on thin layer chromatography (TLC).The completion of reaction was indicated by wetting with the formation of yellow-coloured reaction mixture.The solid obtained was easily separated by using cold water and simple Buchner filtration; final purification was achieved by crystallization from ethanol to give pure samples of Schiff bases 3a-x (Scheme 1).The reasons to use grinding technique [30] in the synthesis of title compounds, which make the reaction procedure simple, short reaction time, increase the purity of the resulting products and enhance the quantitative yield.Another advantage of the method is that it is too consistent with green chemistry approach because it does not require heating or microwave irradiation.It occurs at room temperature and is completely free from organic solvents during both the reaction and separation of the product; except for the recrystallization of product.Therefore, the method avoids the use of organic solvents during the reaction, leading to easy isolation of product.
In order to optimize the capability and efficiency of the present method, we carried out the reaction above by conventional method using ethanol as reaction solvent (Table 1).We found that solidstate reaction occurs more efficiently and more selectively than does the solution-based reaction.This happens because the molecules in the crystal are arranged tightly and regularly.Thus, in grindstone technique, the reaction occurs efficiently in terms of clean reaction conditions, operationally being simple, and short reaction time giving quantitative yields of product and environmental-and eco-friendliness.
The results of antioxidant activity expressed as IC50 value with two different antioxidant agents are shown in Table 2.The compound 3m and 3n, tested using the DPPH scavenging method, showed IC50 value at 70.13, 70.28 µM, when compared with that of the standard ascorbic acid at 69.08 µM, respectively.However, the compounds 3d and 3k did not show significant activities.Further, the antioxidant studies carried out using NO scavenging method, the only compound 3m and 3w showed IC50 values at 92.04 and 91.25 µM in comparison with standard.The compound 3f and 3j did not show antioxidant activity.

CONCLUSION
In conclusion, we reported short a library of imines 3a-x by the condensation of substituted hydroxyketones with substituted anilines under solvent-free conditions using the grinding method.The method is efficient, convenient in terms of simple reaction procedure, short reaction time, increasing the purity of resulting products, and enhances the quantitative yield.Further preliminary in vitro antioxidant activity of newly synthesized compounds reveals that 3m, 3n and 3w shows significant activity in comparison with standard antioxidant ascorbic acid and rutin.The remaining compound shows comparative antioxidant activity.Compound 3d and 3k did not show significant activity (NSA) in comparison with ascorbic acid.Similarly compound 3f, 3j, and 3l were also did not possess antioxidant activity in comparison with rutin.Thus, the present study is useful in the field of medicinal chemistry.

Table 1 .
Comparison of grinding technique reaction with conventional method.

Table 2 .
Antioxidant scavenging of newly synthesized imines 3a-x at different concentration.