Insights into the antioxidant activity of phenolic compounds: Synthesis and electrochemical study of new series of hydroxycoumarins

“Abstract.” Phenolic compounds are bioactive substances widely distributed in the vegetable kingdom. They act as natural antioxidants, and their presence contributes to the color, flavor and aroma of food. This group of micronutrients is composed of one or more aromatic benzene rings with one or more hydroxyl groups and their redox properties are related with their chemical structure characteristics. The knowledge of their redox potentials may help the food industry, because when phenolic compounds are oxidized they could affect the quality of the wines, beers, grape juices, etc. Coumarins are a large family of compounds, of natural and synthetic origin, that show important biological activities. Therefore, they occupy an important place in the study of natural products and synthetic organic chemistry. Recent studies pay special attention to their antioxidative, anticarcinogenic and enzymatic inhibition properties. Their preparation, and the versatility of the synthetic methodology, allowed us obtaining a wide family of compounds with substituent in different positions in the molecule. The election of these derivatives has considered the later pharmacological evaluation. The


Introduction
In recent years, phytochemical compounds have been showing a great interest due to their presence in bioactive dairy products.Fruits, vegetables, oil and wine have been studied as health protectors because of the antioxidant potential of their phenolic compounds.These compounds are known to be the most common secondary metabolites in the vegetable kingdom. 1,2,3he fusion of a pyrone with a benzene ring gives rise to a class of heterocyclic compounds known as benzopyrones or coumarins. 4Coumarins are a wide group of compounds present in remarkable amounts in the nature. 5Representatives of this group occur in the vegetable kingdom, either in free or combined state. 6Due to their structural variability, they are an elite class of compounds which occupy an important role in synthetic organic chemistry. 7oumarins have been attracting considerable interest due to their numerous biological activities, usually associated to low toxicity, depending on their substitution pattern. 8There are many possible permutations offered by substitution and conjugation, and this readily explains why so many synthetic analogues featuring coumarin structural motif are investigated due to their wide range of biological properties. 9In the literature, coumarins have been described as anticancer, 10,11 antioxidant, 12,13 antimicrobial, 14 antiviral, 15 vasorelaxant, 16 anti-inflammatory 17 and enzymatic inhibitors. 18,19,20,21ndeed, some coumarins are now commercially available as medicines.

Chemistry
The coumarin derivatives 1-10 15,22,23,24 were efficiently synthesized according to the protocol outlined in Scheme 1.The general conditions and the compounds characterization were described in the experimental section.

Results and discussion
All the described 3-arylcoumarins (compounds 1-10) were efficiently synthesized, characterized and evaluated for their antioxidant functionality.Relevant aspects concerning the electrochemistry of this new series of synthesised coumarins can be obtained.Considering that the electrochemical behaviour of these compounds depends on their structural features, useful information on their antioxidant functionality can be drawn.For this purpose cyclic (CV), differential pulse (DPV), at different pH values, and ORAC-FL experiments for some of the hydroxyl derivatives were performed.
Voltammetric study of compound 6 at pH 7.0, figure 1, shows on the first scan an oxidation peak at E p1 = + 0.650 V corresponding with an irreversible reaction.A value of E p -E p/2 = + 0.050 V indicates that one electron is involved in the first oxidation process.A reduction peak, on the inverse sense, at E p2c = + 0.044 V could also be seen, corresponding to the reduction of the oxidation products formed during oxidation of peak P 1 .The reversibility of peak 2 was confirmed in the second scan where a new oxidation peak appears at lower potentials, E p2a = + 0.076 V.This reversible oxidation process corresponding with peak P 2 occurs with transference of two electrons.When two hydroxyls groups are present in the molecule, as compound 7, the differential pulse voltammogram should show two oxidation peaks due to both oxidizable groups.Figure 2A presents the first and second scan of compound 7.The first one shows an oxidation peak with a W 1/2 ~200 mV suggesting that could be two oxidation peaks.This is confirmed on figure 2B, on the second scan, when the baseline is subtracted.First scan shows also another oxidation peak at lower potentials, called P 2 at E p2 = + 0.167 V.This potential value can be compared with that one for a catecol group.On the second scan an oxidation product peak, P 3 , appears at E p3 = + 0.047 V.As was seen before though CV, P 3 corresponds to the oxidation product formed after P 1 oxidation.The strong adsorption of the oxidation products, which blocked the electrode surface, makes possible to observe more easily on the second scan both oxidation peaks, P 1 and P 1' .Oxidation processes due to P 1 and P 1' occur with one electron transference in each case.A value of W 1/2 P3 = + 0.057 V indicates that one electron is involved on the oxidation process.The value of W 1/2 obtained for P 2 was + 0.054 V indicating that two electron are involved in the oxidation process.This would confirm that P 2 is an oxidation product of P 1 corresponding with a catecol group.

A B
The peak P 1 potential of each compound is displaced to more negative values with increasing pH.A slope of proximately + 0.059 V suggests that the oxidation processes involve the same number of electrons and protons.P 1 and P 1' occurs by transference of one electron and one proton whereas, P 2 and P 3 , both oxidation products, occur by transference of two electrons and two protons.
Oxidation product peaks of each compound involved two electrons and two protons.Oxidation products showed a very low oxidation potential.This behaviour, which is related to their molecular structure, clearly shows their good antioxidant properties.Also, ORAC-FL indexes were calculated by fluorescence measurements, comparing with the reference compound Trolox.The values obtained in ORAC-FL experiment are showed in table 1. Positions of the hydroxyl groups were important factors in the antioxidant activities against peroxyl and hydroxyl radicals of the coumarins derivatives.

Conclusions
We synthesized a new series of compounds using an efficient and versatile synthetic route.The experimental results demonstrated that using electrochemical methods as CV and DPV, it can be clarified the mechanism of electron transfer of a new series of 3arylcoumarins.Compound 7 is the best antioxidant candidate since it has the lowest oxidative potential and also the highest ORAC-FL value of this new series.

Table 1 .
ORAC-FL results of some of the described compounds