Clean Procedure and DFT Study for the Synthesis of 2-Amino-3-ethoxycarbonyl-4-( aryl )-4 H-pyrano-[ 3 , 2-c ]-chromene-5-ones Derivatives : A Novel Class of Potential Antimicrobial and Antioxidant Agents

2-Amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]-chromene-5-ones 5 is synthesized by the two-component reaction of 4hydroxycouamrin with ethyl-2-cyano-3-(aryl) acrylate 3.e structures of the obtained compounds are con�rmed by analytical IR, H, and C-NMR spectra to elucidate the different positions of protons and carbons and as well as theoretic studies (DFT/B3LYP). All the newly synthesized compounds are screened for their antibacterial. Furthermore, these compounds showed antioxidant activities of different extents with respect to individual compounds as well as to the antioxidant methods. e compounds 5a–e were found to be the most active antioxidant in the series then Trolox, which makes the investigated complexes a promising new class of antibacterial activity compounds.

Several natural or synthetic coumarins with various hydroxyl and other substituents are found to inhibit lipid peroxydation and to scavenge hydroxyl radicals and superoxide anions [17].4H-chromenes with amino and cyano groups are also the synthons of some special natural products.
In continuation of the search for such potent molecules and as a part of our ongoing research, we plan to synthesize the hybrid structure having the lipohophilic functionality on the pyran ring.e current work highlights the synthesis as well as the theoritical studies of the important coumarin scaffold.e title compound is synthesized by the reaction of 4hydroxycoumarin and (E) ethyl-2-cyano-3-(aryl) acrylate 3 in the presence of piperidine as a base catalyst and at re�ux of DMC.Condensation of 1 with arylaldehydes in the presence of piperidine at room temperature gives the corresponding (E) ethyl-2-cyano-3-(aryl) acrylate 3, Scheme 1 TLC is used to monitor the progress of the reaction; the structures of products are characterized by 1 HNMR along with 13 CNMR; and FT-IR spectral techniques, and they are in agreement with the structure of (E) ethyl-2cyano-3-(aryl) acrylate 3. e IR spectrum of compound 3a shows a band in the region of 1664.62 cm −1 which is the characteristic for C=O.It shows strong bands at 2372 cm − e FT-IR spectra of compounds 5e shows absorption bands at 1582-1586 cm −1 corresponded with C=O and C-O linkage and 3705 cm −1 is observed due to the -NH group.e 1 H NMR spectra of 5e displays a signal at  6.76 ppm that is ascribable to the NH 2 protons.A characteristic singlet proton signal at  2.49 ppm is assigned to the CHAr proton.In addition, the aromatic protons (both coumarinic and aryl) are observed between  6.91 and  7.92 ppm (see Section 4), and the expected triplet for methylic protons is observed at  1.26 ppm.e singlet at  3.03 ppm is assigned to the three protons of the methoxy group.
Indeed the  To further con�rm the structure of the product 5 a systematic theoretical treatment of these compounds is performed by using the DFT/B3LYP approach implemented in the Gaussian 09 series of programs [18].e B3LYP hybrid functional has been used in describing potential energy surfaces (PES).e geometries of compounds 5 are fully optimized using analytic gradients.e harmonic vibrational frequencies of the stationary points of the PES have been calculated at the same level of theory in order to identify the local minima as well as to estimate the corresponding zero-point vibrational energy (ZPE) [19,20].
For each atom no pseudopotential is used.A Def2-SVP EMSL basis set exchange is employed for each atom [21].Optimized geometry for 5c is depicted in Figure 1.Values of selected geometrical parameters are listed in Table 1.
e value of the dihedral angle (3-3 ′ -5 ′ -6 ′ = 96.8○ ) shows that the aryl group is orthogonal to the plan of the tricyclic structure 5. e bond length C 4 ′ -5 ′ in compound 5c is more elongated than its counterparts in 5b-d, this is caused by the steric hindrance of the two methyl groups in positions 2 and 5. Another important information concerning the nature of the groups present in the structure 5 can be obtained by calculating the vibrational frequencies of these  compounds.Values and intensities of the harmonic wave numbers calculated with the DFT/B3LYP method, and the corresponding experimental values of the compound 5c are presented in Table 2. e values of the theoretical IR frequencies are in agreement with the experimental values with a shi to larger frequency values less than 10%.is shi is due to the approximations used in the DFT/B3LYP method.
e NBO analysis allows the determination of the atomic natural charges reports the natural charges of the acid protons in compound 5c calculated at DFT/B3LYP level of theory.We �nd that the protons Ha and Ha ′ carried by the nitrogen atom are the most acidic Table 3. DPPH is a free radical and accepts one electron or one hydrogen radical to become a stable diamagnetic molecule [22].e reduction capability of the DPPH radical is determined by the decrease in absorbance induced by 2amino-3-ethoxycarbonyl-4-(aryl)-4Hpyrano-[3,2-c]-chromene-5-ones 5. Brie�y, 1.5 mL ethanolic solution of the synthesized compounds (0.2 mM) is added to 1.5 mL (0.2 mM) solution of DPPH radical in ethanol (�nal concentration of DPPH and synthesized compounds is 0.1 mM).e mixture is shaken vigorously and le standing for 30 min.Aer this, the absorbance at 534 nm is determined, and the percentage of scavenging activity is calculated using the following formula:

Antibacterial and Antioxidant Studies
where  b : absorbance of 0.1 mM ethanolic solution of DPPH at 534 nm,  s : absorbance of 0.1 mM ethanolic solution of test compound at 534 nm, and  m : absorbance of ethanolic mixture of the drug and DPPH at 534 nm.
Trolox was used as reference compound.All tests and analyses are done on triplicate and average on three samples.e results are illustrated in Scheme 4.
Among the compounds from the 2-amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]-chromene-5-ones 5 series, 5b showed moderate antioxidant activity.e activity exhibited by the compound 5e is the highest.In addition, the experimental data shows that compound 5a scavenges free radicals better than Trolox.us, we can conclude that substituents on the aryl group do not in�uence signi�cantly the antioxidant activity.
One parameter that has been introduced recently for the interpretation of the results from the DPPH method is the effective concentration or EC 50 value (otherwise called the IC 50 value), which is de�ned as the concentration of substrate that causes 50% loss of the DPPH activity (color) and corresponds to the endpoint of the titration.In all cases, any residual (yellow) color from the reduced form or any nonspeci�c absorbance from the sample should be considered in de�ning the �endpoint� of the titration, that is, the 50% point.Additionally, this IC 50 parameter has also the drawback of that the higher the antioxidant activity, 4   4.
From inspection of Table 4, it is evident that compounds 5e are more active than Trolox.
ABTS radical-scavenging activity of 2-amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]chromene-5-ones 5 is determined according to Re et al. [23].e ABTS +• .e cation radical is produced by the reaction between 5 mL of 14 mM ABTS solution and 5 mL of 4.9 mM potassium persulfate (K 2 S 2 O 8 ) solution, stored in the dark at room temperature for 16 h.Before use, this solution is diluted with ethanol to get an absorbance of 0.700 ± 0.020 at 734 nm.�n a �nal volume of 1 mL, the reaction mixture comprised 950 L of ABTS ± solution and 50 L of the 2-amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]xchromene-5-ones 5 at various concentrations.e reaction mixture is homogenized and its absorbance is recorded at 734 nm.Ethanol blanks were run in each assay, and all measurements were done aer at least 6 min.Similarly, the as EC 50 (⟨mu⟩ g/mL).e inhibition percentage of ABTS radical was calculated using the following formula: where  0 is the absorbance of the control at 30 min, and  1 is the absorbance of the Sample at 30 min.All samples are analyzed in triplicate.
e variation in the percentage of inhibition (PI) is almost constant starting from a value of the concentration equal to 1.34 mM.In addition, the synthesized products of the type 5 have antioxidant activities better than Trolox.Indeed, the antioxidant capacity seems to be attenuated when the concentration increases in the medium.is can be explained by the existence of the peroxide sites which are susceptible for oxidizing when the concentration increases.We have just shown that the synthesized 2-amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]-chromene-5-ones 5 has a good antioxidant activity under weak concentration, but it proves to be necessary to determine the reaction time necessary to highlight the antioxidant effect to be able to use these derivatives in pharmacy.e EC 50 values exhibited by 2-amino-3-ethoxycarbonyl-4-(aryl)-4H-pyrano-[3,2-c]-chromene-5-ones 5 are summarized in Table 5. e 2-amino-3-ethoxycarbonyl-4-(aryl)-4Hpyrano-[3,2-c]-chromene-5-ones 5 are shown to be efficient antioxidants, showing higher free radical scavenging activity than Trolox.
ese compounds have a remarkable oxidizing capacity which explains their susceptibility to �x free radicals DPPH and ABTS •+ .[23,24] using Mueller-Hinton agar medium was employed to study the preliminary antibacterial activity of 3a-e against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa.Preparation of base-layer medium, agar medium, and peptone water is done as per the standard procedure.Each test compound (10 mg/mL) is prepared by dissolving 50 mg in 5 mL of dimethylformamide, and is used for testing same cup-plate method using PDA medium is employed to study the preliminary antifungal activity of 3a-e against Candida albicans and A. niger.e PDA medium was purchased from HImedia Laboratories Ltd, Mumbai, India.Preparation of nutrient broth, sub-culture, base-layer medium and PDA medium was done as per the standard procedure.For each test compound, a 50 g/cup was used for testing.e cups of 9 mm diameter were made by scooping out medium with a sterilized cork borer in a petri dish which was streaked with organisms.e solutions of each compound were added separately in the cups, and petri dishes were subsequently inoculated.Ampicillin and Griesofulvin (6 g/cup and 25 g/cup, resp.) were used as standard reference drugs and dimethylformamide (DMF) used as control which did not show any inhibition.Zone of inhibition produced by each compound was measured in mm, and the results are presented in Table 6.

Antibacterial Activity. e cup-plate method
All the tested compounds have shown antibacterial activity to some extent.Among the tested compounds 3b and 3d, show very good activity against the tested organisms.
Compounds 3a and 3e are moderate antibacterial activity.All the compounds synthesized possess electron-releasing groups, on both the aromatic rings.erefore from the results, it is evident that compounds having electronreleasing groups like methyl, hydroxyl, and methoxy may be responsible for antibacterial activity.