One-pot synthesis of potential antioxidant agents , 3-carboxylate coumarin derivatives

A simple and efficient route to synthesize 3-carboxylate coumarin derivatives through three-component one-pot reaction in a single step has been recommended. This method provides a direct and rapid access to get 3-carboxylate coumarin derivatives. The structures of these synthetic products were identified and the antioxidant activities were tested by inhibiting DPPH· and ·OH radicals capacities. The data suggested that 2H-1-penzopyran-3-carboxylicacid, 7,8-dihydroxy-2-oxo-, ethyl ester showed excellent activity in reducing both DPPH· and ·OH radicals in concentration-dependent manners with IC50 value of 83.1 μg/ml in DPPH· radicals and less than 0.12 mg/ml in the ·OH scavenging activity.

Coumarin-3-carboxylate is one of the important coumarin derivatives, and an important intermediate that could be used in the synthesis of coumarins (Song et al., 2003).Coumarin-3-acyl derivatives were tested in vitro for their distinct human monoamine oxidase A and B (hMAO-A and hMAO-B) inhibitory activity, especially the 3-ethyl ester coumarin ring.They are considered as highly potent and selective hMAO-B inhibitors with IC 50 values in the nanomolar range (Secci et al., 2011).7,8dihydroxy-coumarin and TGF-β1 have a synergistic effect on strongly induced, rat adipose-derived mesenchymal stem cells (ADMSCs) that are differentiated from the cartilage (Liu et al., 2013).
A one-pot reaction, one simple and rapid method, consists of a combination of three components that are used to generate new products in a single step.This resource-effective method is also used in the emerged multicomponent reactions (MCRs) (Karami et al., 2012) without isolating the intermediate.The economical use of material in this reaction along with post-processing is associated with a less tedious process of recycling and regenerating the catalyst.Compared with multistep reactions, it would be a convenient and greener way to get series coumarins.

EXPERIMENTAL General
With the exception of piperidine (technically pure), all reagents used in this study were analytically pure.The products were synthesized using DF-101D solar collector heating thermostat magnetic stirrer.The melting points were measured using melting point apparatus.The synthesized compounds were purified by recrystallization and analyzed by thin-layer chromatography (TLC), melting point determination (X-6), and HPLC (5 μm, 250 × 4.60 mm Gemini, C 18 preparation column, Phenomenex).Infrared spectra was recorded in KBr and determined on a Perkin Elmer fourier transform infrared (FT-IR) spectrometer. 1H and 13 C NMR spectra were recorded on a Bruker 500 MHz Nuclear Magnetic Resonance Spectrometer.

Synthesis procedure
2 H-1-benzopyran-3-carboxylic acids, 7-hydroxy-2-oxo-, ethyl ester.2,4-Dihydroxybenzaldehyde (5.522 g, 0.040 mol), diethyl malonate (6.8 ml, 0.045 mol) with ethyl alcohol (25 ml), piperidine (0.5 ml), were added to a round-bottom flask(250 ml) that was equipped with a magnetic stirrer and spherical condenser.A small amount of glacial acetic acid was added to this flask to dissolve these chemicals.This mixture was heated to about 85°C for 3 h.The completion of this reaction was monitored by thin layer chromatography (TLC) using EtOAc: petroleum (3:1) as eluent.After completion of this reaction, the mixture was transferred to a beaker containing 35 ml of water.Then, the beaker was cooled and leached.The filter cake was washed thrice using 50% ethyl alcohol.

The antioxidant activity test
Assay for the DPPH• radical-scavenging activity: a series of sample solutions was prepared in dimethyl sulfoxide (0.5 ml).The concentrations of these solutions ranged between 10 to 200 μg ml -1 .3.0 ml of DPPH• solution in 95% alcohol was added to each of these solutions.The reaction mixtures were protected from light and incubated at room temperature for 30 min.The absorption was read at 517 nm and the mean value was measured for three duplicated readings.The ascorbic acid was used as a positive control.The scavenging activity was determined from the following equation (Lin et al., 2008;Tyagi et al., 2005;Tantry et al., 2012).
Assay for the scavenging effect on hydroxyl radicals: The scavenging effect was evaluated using the hydroxyl radical system that was generated by the Fenton reaction (Heo et al., 2005) with a minor modification.Briefly, samples were dissolved in dimethyl sulfoxide at 0 (control), 0.04, 0.08, 0.16, 0.24, 0.40 and 0.80 mg/ml.The reaction mixture consisted of the following reagents: 2 ml of salicylic acid and absolute ethanol solution (9 mM), 2 ml of FeSO 4 (9 mM), 2 ml of H 2 O 2 (9 mM), and 2 ml samples of varying concentrations.The absorbance of this mixture was measured at 510 nm after incubating it at 37°C for 30 min.The hydroxyl radicalscavenging rate was calculated with the following equation (Sun et al., 2010).
Hydroxyl radical-scavenging rate (%) = 100 × [1 -(A s -A w ) / A c ] A s : absorbance of the mixture solution containing 2 ml sample; A w : absorbance of the mixture solution in which 2 ml water was replaced with 2 ml H 2 O 2 ; A c : absorbance of the mixture solution in which 2 mL water was replaced with either 2 ml sample or vitamin C.

RESULTS AND DISCUSSION
Herein we gave detailed description of the synthesized compounds.In accordance with the references described in the procedure, the melting points of compounds differed by about 5 to 6°C.This could be attributed to the difference in the usage of the dissolved solvent and the measurement condition effects.The products were found to be highly purified as the melting point of every compound altered by only 1 to 2°C.The crystals of all compounds were yellow in appearance (Valizadeh and Azimi, 2011), except for the crystals of the compound 6hydroxycoumarin-3-carboxylic acid ethyl ester.The crystals of this compound appeared yellowish green in color.The high performance liquid chromatography (HPLC) data is summarized and illustrated through Figure 1.The mean peak area of the samples was over 97%, and this indicated the high purity of products.While performing the experiments, the sample size was set in accordance with the concentration of every sample.In this case, a good, symmetrical, mean peak shape was obtained, which benefited from effective separation, high purity products, and medium sample size.
The reported data was identical with the standard spectra data (Lin et al., 2008;Horváth et al., 2005;Gong and Ding, 2006).Figure 2 displays the infrared (IR) spectra of 7,8-hydroxycoumarin-3-carboxylic acid ethyl ester.On the other hand, Table 1 illustrates those of other coumarins.They could also be recognized by 1 H NMR and 13 C NMR analysis (Table 2).Figure 3 displayed the partial nuclear magnetic resonance (NMR) spectra of compounds.

Conclusion
In summary, we applied a simple way to get a series of ethyl coumarin-3-carboxylate containing hydroxyl group using the one-pot and multicomponent synthesis methods.These synthesized compounds have been elaborately elucidated, with the confirmation of their organic structures.This illustrates that workup reaction condition could be used in the synthesis of these products.Among the synthesized coumarins,7,8dihydroxyl coumarin displayed excellent activities in reducing both DPPH• and •OH radicals.7,8-dihydroxyl coumarin was found to be more active than ascorbic acid under the concentration of 0.12 mg/ml.7-hydroxyl coumarin and 8-hydroxyl coumarin were found to be more active than ascorbic acid within some limits