Synthesis and anti-oxidant activity of coumarinyl chalcones

The ability to inhibit oxidative stress has been established as the prime mechanism in treatment of several disease conditions. In view of this, two new series of coumarin–chalcone hybrid molecules (5a–o and 6a–o) were synthesized using various aromatic aldehydes. The structures of the compounds were confirmed using IR, 1HNMR and mass spectral analyses. The compounds were evaluated for their antioxidant potential against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals in scavenging assays. Compounds 5o and 5k exhibited significant antioxidant potential as compared to the standard drug (ascorbic acid). It can be concluded that the coumarin–chalcone treatment have the potential to be optimized further to generate scaffolds capable to treat many pathological conditions.


Background
Coumarins (2H-1-benzopyran-2-one) (I) contribute to more than 1300 secondary metabolites obtained from plants, bacteria, and fungi and therefore represent the largest class of phenolic substances found in plants [1]. The widespread availability of coumarins in nature has been instrumental for the wide spectrum biological activities exhibited by the natural coumarins. Several synthetic derivatives of coumarins have been explored for activities including antibacterial [2], antifungal [3], anticancer [4], anti-HIV [5], anti-inflammatory [6] etc. Al-Majedy et al. have reported a detailed review on the antioxidant action of synthetic coumarin derivatives [7].
Chalcones (II) are molecules containing a 1,3-diphenylprop-2-en-1-one obtained from the flavonoid class of natural products. Several chalcone compounds have been isolated from plant sources and aplenty synthetic derivatives have been produced in laboratories by substituting on the benzene rings. Most of the biological actions exhibited by chalcones as owed to their antioxidant potential [8].
Reports have been made where linking two distinct moieties together using various functional groups or spacers has resulted in synergizing the action of the resulting molecules [9][10][11][12][13][14][15][16][17]. In persuasion to the reports, we envisaged to fuse coumarin and chalcone nucleuses to form novel conjugates and evaluate the antioxidant potential of the conjugates.

Chemistry
The synthesis of the coumarin-chalcone hybrid molecules was accomplished according the reaction depicted in Scheme 1. The steps of the present scheme were adapted with modifications form the scheme reported by Tandel et al. [18] and Srikrishna et al. [19].

4-Morpholino-2-oxo-2H-chromene-3-carbaldehyde (3)
A solution of morpholine (21.75 g, 20 mmol) in 10 ml of dichloromethane was gradually added under constant stirring to an ice-cooled mixture of 2 (2.09 g, 10 mmol) in 25 mL of dichloromethane. After stirring for 30 min at 0-5 °C, the mixture was washed with three 10 mL portions of water to remove any unreacted morpholine and its salt. The organic phase was dried over MgSO 4 and the solvent was evaporated under reduced pressure. The dry residual flakes were recrystallized from 1,4-dioxane to obtain pure 3 [20].

General method of synthesis of 3-((E)-3-(4-((Z)-benzylideneamino)phenyl)-3-oxoprop-1-enyl)-4-morpholino-2H-chromen-2-one (5a-o)
In a round bottom flask compound 4 (0.0329 mol) was dissolved in methanol (20 mL). Separately, substituted benzaldehyde (0.0329 mol) was dissolved in methanol (20 mL) in a beaker. The solution of substituted benzaldehyde was added drop by drop in to the solution of 4 with continuous stirring. On completion of addition, the mixture was allowed to reflux for 4 h. On completion of reaction, the reaction mixture was poured in to an evaporating dish and the excess of solvent was removed under reduced pressure. The solid obtained crystallized using methanol [22].

In-vitro antioxidant activity
The in vitro antioxidant activity of the synthesized compounds 5a-o and 6a-o was determined by two different methods using ascorbic acid as the standard.

DPPH method
The free radical scavenging activity of the synthesized molecules was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH [23]. The test samples (10-100 μL) were prepared in DMSO and were mixed with 1.0 mL of DPPH solution and filled up with methanol to a final volume of 4 mL.
Absorbance of the resulting solution was measured at 517 nm in a visible spectrophotometer. Ascorbic acid was used as the reference compound. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. Radical scavenging activity was expressed as the inhibition percentage of free radical by the sample and was calculated using the following formula: where Ao is the absorbance of the control (blank, without sample) and At is the absorbance in the presence of the test samples. All tests were performed in triplicate and the results were expressed as mean values ± standard deviations.

Hydroxyl radical scavenging method
The test samples (10-100 μL) were prepared in DMSO and 1 mL of iron EDTA solution, 0.5 mL of EDTA solution, 1 mL of DMSO and 0.5 mL of ascorbic acid were added to it. The mixture was incubated in a boiling water bath at 80 to 90 °C for 15 min. After incubation, 1 mL of ice cold TCA and 3 mL of Nash reagent were added and the reaction mixture was incubated at room temperature for 15 min. The absorbance was read at 412 nm. The % hydroxyl radical scavenging activity is calculated by the following formula where, HRSA is the Hydroxyl Radical Scavenging Activity, Abs control is the absorbance of control and Abs sample is the absorbance of the test solution. Table 1 presents the physical data and chemical structures of all the synthesized compounds.

Antioxidant action
The antioxidant activity displayed by the synthesized compounds against DPPH and hydroxyl radicals is presented in Tables 2 and 3.      1 with Vilsmeier-Haack reagent leading to formylation of the electron rich ring [24]. Compound 2 undergoes nucleophilic aromatic substitution with morpholine to yield the compound 3 which on condensation with amino acetophenone under the conditions of Claisen-Schmidt reaction yielded the chalcone conjugates 4. The coumarin-chalcone conjugates were further condensed at reflux conditions with aromatic aldehydes and methyl amine to obtain compounds 5a-o and 6a-o. The optimization of the reaction for completion and purity was performed throughout using TLC. The structures of the synthesized molecules were characterized by 1 H NMR, IR and mass spectral studies. The 1 H NMR spectra of compound 5a-o exhibited peaks in the region of 3.1-3.9 corresponding to -N(CH 2 ) 2 and -O(CH 2 ) 2 ; 6.7-7.8 due to aromatic protons, the peaks due to α, β-unsaturation of chalcones; 8.2 owing to the imine protons. Additionally, the signals due to the hydroxyl and methoxy protons were also found in the corresponding compounds. In the 1 H NMR spectra of compound 6a-o additional peak at 3.3-3.5 was obtained due the methyl protons of methylamine. The mass