Research Article - (2014) Volume 5, Issue 4
A series of new benzamides were synthesized. The chemicals structures were confirmed by elemental analyses 1H NMR and 13C studies. The antioxidant activity of the synthesized compounds was evaluated by square wave voltammetry. A new approach, for antioxidant capacity determination was proposed. It is based on the using of the xanthine-xanthine oxidase system coupled with H2O2 electrochemical sensor.
Keywords: Voltammetry, Antioxidant capacity, Xanthine, Xantine oxidase
Reactive oxygen species (ROS) including superoxide anion (O2-.) hydrogen peroxide, and hydroxyl radical (OH.), are generated naturally in vivo during metabolic processes and keeps in a balance level in normal living organisms . However, when a body is subject to the environmental or behavioral stressors (pollution, sunlight exposure, cigarette smoking, excessive alcohol consumption, etc,), excess ROS are generated . If the excess ROS cannot be scavenged in time, they would attack and induce DNA, proteins and lipids damage, and impede normal cell functions . Therefore, overproduction of ROS is associated with numerous diseases like cancer and Alzheimer’s disease, as well as aging. In living systems, the deleterious effects of ROS can be neutralized by the endogenous and exogenous antioxidant systems . Antioxidants are synthetic or natural substances that prevent or delay the oxidative damage by scavenging the free radicals. Fruits and vegetables are good sources of high amounts of known antioxidants.
The aims of this study are to synthesize derivatives of benzamides, from the arylamines and salicylic acid under the action of thionyl chloride, and tested as antioxidants.
The antioxidant capacity was evaluated, by coupling an amperometric sensor for H2O2 detection, obtained by modification of paste carbon graphite electrode with copper, with xanthine oxidase (XOD) immobilized at silice–xanthine (XA) enzymatic system, as generator of O2-. radicals. The advantages of this strategy consist to [5-16]:
- It works at low applied potential, allowing a significant decrease of the risk of electrochemical interferences;
- The antioxidant capacity evaluation, requiring the monitoring of H2O2 concentration in presence of antioxidant sample as well as in its absence, will be a global estimation of the free radicals (O2-.) and nonradical reactive species, (H2O2) interactions with the investigated ontioxidant (AOX) (Reaction 1).
Synthesis of some benzamides derivatives
Access to these amides requires the preparation of the acid chloride from salicylic acid by the action of thionyl chloride followed by nucleophilic attack of arylamines (Scheme 1). The different molecules (A, B and C) shown in Scheme 2, are obtained and purified by chromatography on silica gel and characterized by 1H and 13C NMR spectroscopy.
Experimental section: Melting points were taking for samples in capillary tubes with an electro-thermal apparatus and are uncorrected. 1H NMR and 13C NMR were recorded on a Bruker Avance DPX250 spectrometre (300 MHz 1H, 75 MHz 13C) using trimethylsilane as the internal standard, chemical shifts were reported in parts per million (ppm, δ units). Coupling constants were reported in units of hertz (Hz). Flash chromatography was performed on silica gel 60 (40–63 mesh). Thin layer chromatography (TLC) was carried out on Merck silica gel 60F254 precoated plates. Visualization was made with ultraviolet light. All organic solvents were distilled immediately prior to use.
General procedure for the synthesis of compounds A, B and C: Salicylic acid (2 g, 0.013 mol) was dissolved in thionyl chloride (16.07 g, 0.13 mol). The mixture was stirred at reflux for 2 hours. After evaporation of the SOCl2, the residue obtained is dissolved in CH2Cl2 (20 mL) and phenylhydrazine (1.75 g, 0.016 mol) and triethylamine (1.36 g, 0.013 mol) were added. The reaction mixture is stirred at room temperature for 5 hours. After hydrolysis with a solution of NaOH (1N), the mixture was extracted with CH2Cl2. The organic phases are combined, dried over MgSO4, filtered and evaporated under reduced pressure. The crude product thus obtained was purified by silica gel chromatography (eluent: ethyl acetate/hexane: 4/6) leading to the desired compound in good yield.
2-hydroxy-N-(4-hydroxyphenyl)benzamide A: This compound was obtained as a white solid (70%). Mp: 155-157°C. 1H NMR (300 MHz, DMSO) d 11.75 (s, 1H, NH), 10.45 (s, 1H, OH), 10.27 (s, 1H, OH), 7.78 (d, J=8,7Hz, 2H, ArH), 7.30 (d, J=8,7Hz, 2H, ArH), 7.10- 6.95 (m, 2H, ArH), 6.85 (t, J=16,8Hz, 1H, ArH), 7.50 (t, J=13,6Hz, 1H, ArH); 13C NMR (75 MHz, DMSO): d 167.8, 159.4, 154.1, 137.2, 131.8, 123.1, 121.5, 120.1, 118.1, 118.0.
N-(4-cyanophenyl)-2-hydroxybenzamide B: This compound was obtained as a (marron Claire) solid (73%). Mp: 164-166°C. 1H NMR (300 MHz, DMSO) d 11.41 (s, 1H, NH), 10.70 (s, 1H, OH), 6.9-79 (m, 8H, ArH); 13C NMR (75 MHz, DMSO): d 164.8, 159.4, 140.2, 133.6, 132.4, 128.9, 122.3, 121.5, 119.9, 116.0, 115.8, 108.2.
N-(4-chlorophenyl)-2-hydroxybenzamide C: This compound was obtained as a white solid (67%). Mp: 185-187°C. 1H NMR (300 MHz, DMSO) d 11.58 (s, 1H, NH), 10.13 (s, 1H, OH), 6.9-8.2 (m, 8H, ArH); 13C NMR (75 MHz, DMSO): d 164.8, 159.4, 134.0, 133.6, 129.9, 129.1, 128.9, 123.0, 121.5, 119.9, 116.0.
Electrochemical experiments were performed using a voltalab potentiostat (model PGSTAT 100, Eco Chemie B. V., Utrecht, The Netherlands) driven by the general purpose electrochemical systems data processing software (voltalab master 4 software).
All the electrochemical experiments were performed in a standard one-compartment three-electrode cell. The reference electrode was SCE and the counter electrode was platinum. All electrode potentials were referred to this reference electrode. The working electrode was copper modified carbon paste electrode (Cu-CPE).
Reagents and solutions
All chemicals were of the highest quality. Graphite powder (spectroscopic grade RWB, Ringsdorff-Werke GmbH, Bonn-Bad Godesberg, Germany) was obtained from Aldrich and was used without further purification. CuSO4 was obtained from Merck chemicals. Deionised water was used to prepare all solution.
Preparation of the electrochemical sensor
The carbon paste unmodified was prepared by adding paraffin oil to carbon powder and thoroughly hand–mixing in a mortar and pestle. The resulting paste was packed into the electrode and the surface was smoothed. The electrochemical sensor was developed by depositing the copper at fixed potential (0.1 V for 1 hour) onto the carbon paste electrode surface.
The device constructed for the measurement of the antioxidant capacity is given in Figure 1. The free radical was generated in column following the reaction 1 and the graph plot, giving reduction H2O2 current density versus [H2O2], was carried out. In the second time, the investigated antioxidant associated to xanthine solution were pouring in column and response behaviour was recorded.
Electrochemical behaviour of the studied molecules
The cyclic voltammograms (CVs) recorded at Cu-CPE in the supporting electrolyte containing or not molecule are presented in Figure 2. In presence of molecule A in medium, we note, higher current densities of the anodic side and the occurrence of a reduction peak in the cathodic scanning.
The square wave (SQW) voltammograms obtained, respectively, in buffer solution (curve a) and buffer solution containing molecule A (curve b) are shown in Figure 3. In the presence of the molecule A in the buffer solution causes the appearance of two redox peaks in the SQW voltammogram. The first one around -1.0 V and the second at 0.4V versus SCE. Peaks can be attributed to redox responses of molecule A.
The same behavior was observed in the presence of the molecule B in the electrolytic solution. The two redox peaks are shifted by about 0.5 V (Figure 4).
A compared square wave voltammograms corresponding to studied molecules shows that only the molecules A and B gives rise to two welldefined redox peaks (Figure 5).
Antioxidant capacity evaluation
Molecule A: The anti-oxidant properties of the studied molecules were evaluated, investigating square wave voltammetry, by comparing the reduction of H2O2 in the presence and absence of the considered molecule.
We can see from Figure 6 that, the progressive addition of the solution containing molecule A, leads to the decrease of the H2O2 reduction current density (in absolute value). The complete inhibition of the reduction reaction of hydrogen peroxide requires 100 μL/100 mL(buffered solution) (Figure 7).
The inhibition of the oxidative capacity of molecule A by H2O2 was investigated in Figure 8. The hydrogen peroxide formed in the silica column is added, to a phosphate buffer solution containing the molecule A, in the electrochemical cell. We note that the antioxidant molecule A power down depending on the amount of H2O2 paid.
The same experiments were conducted for molecules B and C. The same behavior is observed (Figures 9 and 10).
In this work, the antioxidant capacities of three synthesized molecules were investigated based on an electrochemical sensor using copper modified carbon paste electrode as work electrode. The study of SQW voltammograms showed that this sensor had good properties in detection of H2O2 in electrochemical cell. H2O2 is generated in silica column based on xanthine/xanthine oxidase system. The experiment procedure exhibited good stability and reproducibility.
Antioxidant capacity (AOC) of the studied molecules varies in the following sense (Figure 11):
- For concentrations ≤ 60 μL/100mL
AOC (molecule B) ≥ AOC (molecule C) ≥ AOC (molecule A)
- For concentrations ≥ 60 μL/100mL
AOC (molecule C) ≥ AOC (molecule A) ≥ AOC (molecule B)