Free Radical Scavenging Activity of Coumarin Derivatives . In silico and in vitro Approach

The interest of coumarins as antioxidant agents has attracted much attention in recent years. A quantitative structure-activity relationship (QSAR) study of the DPPH• (2,2-diphenyl-lpicrylhydrazyl) radical scavenging ability of chemical compounds, based on the 0-3D DRAGON molecular descriptors and an artificial neural networks (ANN) technique was developed. The built mathematical model showed a correlation coefficient for the training set (R) = 0.71, an external correlation coefficient (Qext 2 ) = 0.65 and it was used to predict the antioxidant activity of 4-hydroxycoumarin derivatives. Besides, an experimental in vitro assay was developed for the reference compound of this group (4-hydroxycoumarin) and the results obtained confirmed the predictions made by the ANN.


Introduction
SciForum http://sciforum.net/conference/mol2net-1 The development of antioxidant agents has attracted much attention in recent years, because oxidative damage is related to many pathological conditions [1].Several coumarin derivatives have been studied for their biochemical and pharmacological profiles.Some studies suggest that these compounds may significantly affect the function of various mammalian cellular systems.Specifically their antioxidant effect has been explored, because the structural features of this group of compounds suggest that they can probably exhibit this pharmacologic property [2][3][4].The antioxidant capacity can be experimentally measured by several in vitro assays.One of the best-known method is the one based on the capturing of the DPPH• radical [5,6].Chemoinformatics tools have been used in the modeling of the antiradical activity, as well as others biological properties, given their advantages in saving time and resources [7,8].Several statistical and machine learning methods have been widely used in the literature to build models for studying Quantitative Structure Activity Relationships (QSAR).The QSAR studies assess mathematical associations between structural features of the molecules and biological properties.For the last two decades, Artificial Neural Networks (ANN) have increasingly found applicability in QSAR studies, thanks to their ability to map non-linear relations between structural characteristics of chemical compounds and their chemical / biological behavior [9].The objective of this study was to develop an ANN model in order to relate the chemical compounds' scavenging ability of the DPPH• radical with the corresponding structural features, also known as molecular descriptors (MDs).Then, an experiment to predict the antioxidant activity of a group of coumarin derivatives was performed.The coumarin-related compounds used as models in this study; were previously synthesized by the team of Molecular Chemistry at Cnam, Paris.

Modeling:
The mathematical Multilayer Perceptron (MLP) neural model was constructed using DPPH• scavenging capacity of 1329 compounds reported in the literature.This model showed a correlation coefficient (  ) for the training set of 0.71.The predictive ability of the model was assessed using the external validation procedure, yielding a correlation coefficient (   ) of 0.65.Both values are above the limits established for model acceptance [10], and thus indicate the fitness and predictive power of the obtained ANN model.

Prediction:
Recent advances in drug discovery have enabled a dramatic increase in the number of synthetic and naturally occurring molecules that are available for testing using in vitro assays as the scavenging ability of the DPPH• radical.Virtual screening allows for prior assessment of the potential bioactivity of chemical compounds, and thus providing key guidelines in posterior experimental work [11].
In this study, the MLP model obtained was used to predict DPPH• scavenging capacity of coumarin derivatives which were divided into 2 groups, following their structural analogy in function of the posterior analysis of their activity.
The results of the predictions for both groups are shown in Table 1.Group1 (compounds 1-7) and Group2 (compounds 8-15) have significantly different values of pIC50 as it can be noticed.Cyanalogs clearly seems to be less effective in http://sciforum.net/conference/mol2net-1DPPH• radical capturing, because their values of pIC50 are highest (around 4).On the other hand, the pIC50 Wf-analog values are under 3.3.In the case of 4-hydroxycoumarin (number 15) the pIC50 value obtained was of 3.4.These results indicate the superior ability of the compounds Group2 for scavenging the DPPH• radical.To justify the observed trends, a more detailed analysis of the structural characteristics is required.Firstly, the presence of a free hydroxyl group in Wf-analogs may probably favor the higher antioxidant activity of this group.In fact, several research studies have pointed hydroxyl groups as key for antiradical capacity and consequently, antioxidant activity [8,[12][13][14][15].The hydroxyl group is present in the most frequently used reference compounds like: trolox, gallic acid or butylated hydroxytoluene (BHT).

In vitro Assay:
The results obtained with in silico modeling were corroborated by an in vitro study of DPPH• scavenging capacity for the reference compound, 4-hydroxycoumarin.Selecting this compound was specifically based on the prediction results obtained because, according to the model, this molecule has an intermediate pIC50 value.The experimental result of the in vitro assay can provide a comparison criterion for evaluating the neural model and its predictions.The experimental in vitro pIC50 value (2.7) obtained according to the method described below for 4-hydroxycoumarin is to be compared with the prediction value 3.4 given by the model.This result is positive, because the neural model as well as the in vitro assay are in the scale of high activity according to the statistical range established by the built Data and also compared to the value obtained experimentally for BHT (2.10) use as reference.It may thus be suggested that the 4hydroxycoumarin possesses significant radical scavenging ability, and therefore it can be considered as a candidate for antioxidant agent; although more analyses are necessary to ensure that.
The results obtained in the in vitro assay confirmed the predicting power of designed ANN model, and consequently its applicability in the search for new antioxidant compounds.An in vitro study of antioxidant activity of warfarine analogues is currently underway.
. Table 1.Predictions of the pIC50 values.In vitro DPPH• assay: The free radical scavenging activity of the 4-hydroxycoumarin was measured using the stable DPPH• radical, according to Blois´s method [16].Briefly, 0.1 mM solution of DPPH• in methanol was prepared and this solution (1 mL) was added to a sample solution in methanol (3 mL) at different concentrations (150-750 µg/mL).The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm.BHT was use for comparison.The procedure was triplicate to ensure the results.The capability to scavenge the DPPH• radical was expressed as IC50 (concentration of antioxidant that produces 50% of absorbance inhibition).

Conclusions
The scavenging capacity of the DPPH• radical is one of the most extended method to evaluate the in vitro antiradical activity.An MLP neural network was constructed to relate the structure of 1329 molecules and their antiradical activity.The obtained model showed adequate fitness and a good predictive power and was thus used to predict the antioxidant activity of 15 coumarin derivatives.The in silico predictions were further corroborated by an in vitro assay for one of the molecules considered as the reference for this set of compounds, and the obtained IC50 value was similar to the value predicted by the MLP model.