Synthesis and Characterization of New Thiazolidinones Containing Coumarin Moieties and Their Antibacterial and Antioxidant Activities

New coumarin derivatives, namely (2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-phenylthiazolidin-3-yl)acetamide, N-(2-(3-methoxyphenyl)-4-oxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide, 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-(2,3,4trimethoxyphenyl)thiazolidin-3-yl)acetamide and N-(2-(4-bromophenyl)-4-oxothiazolidin-3-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide) were synthesized starting from 4-methyl-7-hydroxycoumarin. The structures of the obtained compounds were confirmed by analytical IR and NMR spectra to elucidate the different positions of protons and carbons and as well as theoretical studies (DFT/B3LYP). The new compounds were screened for antibacterial activity. Most of them are more active against E. coli S. aureus and B. subtilis than standard references.

Def2-SVP EMSL Basis Set Exchange was employed for each atom [21]. Values of selected geometrical parameters are listed in Table 1 and optimized geometry for 5a is depicted in Figure 1.   Some conclusions can be extracted from the results obtained. The calculated values for length and angles are similar for compounds 5a-d. This fact suggests that the R group has no influence on interatomic distances and angles. Additionally the dihedral angles 1-2-3-4, 2-3-4-5 and 3-4-5-1 indicate that the thiazolidinone ring in the compounds is not in a plane and that fact is not dependent on the R group. The plane constituted by the thia-ring is perpendicular to that of the coumarin group (for exemple the angle 2-5-6′-7′ = 113.0 in 5a). It is important to point out that the 5c thia-ring displays a different orientation that those for the other calculated compounds. Important information on the chemical groups present in compounds 5a-d can be obtained from their calculated vibrational spectra. As example, intensities and their harmonic wave numbers calculated with the method DFT/B3LYP and the corresponding experimental values of the compound 5c are grouped in Table 2. The values of the theoretical IR frequencies are in agreement with the experimental ones. The largest shifting is less than 10% that could be due to the approximations used in the DFT/B3LYP method; consequently the proposed structures were supported by theoretical calculations.
The NBO analysis allows the determination of the atomic natural charges. Table 3 reports the natural charges of the acid protons in compound 5c calculated at DFT/B3LYP level of theory. We find that proton H 12 carried by the nitrogen was the most acidic.

Antioxidant Activities
The imbalance between reactive oxygen species (ROS) and antioxidant defence mechanisms leads to oxidative modification in cellular membrane or intracellular molecules. Phytochemicals like phenolics, commonly found in plants, could constitute strong natural antioxidants and could play an important role in health care system.
The effect of the different synthetic compounds on DPPH radical scavenging was compared to those of Trolox, using as positive control, and appreciated by the determination of the IC 50 values. The results are shown in Figure 2 and listed in Table 4.  As shown in Figure 2, DPPH test revealed that increase in compounds concentration resulted in increase in free radical-scavenging activity in a dose dependent manner. Based on the IC 50 values, the most active compound is 5c.

ABTS Radical Cation Decolourization Assay
As shown for DPPH scavenging, the obtained results indicate the higher capacity of 5a-d to quench ABTS as compared to the synthetic antioxidant Trolox (Figure 3 and Table 5).

Antibacterial Activity
The antibacterial activity of the tested compounds against a series of bacteria and fungi is shown in Table 6. All the compounds 5 displayed maximum activity against P. vulgaris. The compound 5b is highly active against E. coli. The compounds 5b and 5d also showed very good activity against B. megaterium, while compounds 5a and 5c showed good activity against S. aureus.
Our results also showed that the purified compounds had great potential for antibacterial activity against a panel of microorganisms. There is evidence in the literature that Gram-positive bacteria are more sensitive to plant extracts and essential oil than Gram-negative bacteria [22]. On the basis of inhibition zone diameters values P. vulgaris was more sensitive to the purified compounds than the other Gram positive bacteria. The observed differences in the inhibition zones within pathogenic bacteria could be probably due to cell membrane permeability or other genetic factors.

General
All reagents were obtained from commercial sources. Solvents were either commercially obtained as analytical grade or freshly distilled prior to use. Analytical thin layer chromatography was carried out on precoated silica gel 60F254 plates using either UV absorption or iodine staining for visualization. Column chromatography was carried out using 100-200 mesh silica gel. 1 H-NMR and 13 C{ 1 H}-NMR spectra were recorded on a Bruker DRX 300 MHz instrument (300/75 MHz); chemical shift values are reported relative to tetramethylsilane as internal standard. The IR spectra were recorded on a Thermo Mattson IR300 and Nicolet 510 PET spectrometers. Elemental microanalysis was carried out using a model 5500-Carlo Erba C.H.N.S.O elemental analyzer instrument.

DPPH Test
The DPPH test aims to measure the capacity of the compounds for scavenging the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH . ) by donation of hydrogen atom or an electron [23] If the compounds have the capacity to scavenge the DPPH free radical, the initial blue/purple solution will change to a yellow colour due to the formation of diphenylpicrylhydrazine. Scavenging capacity was read spectrophotometrically by monitoring the decrease of the absorbance at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity.
The percent DPPH scavenging effect was calculated using the following equation: where A control is the absorbance of the control reaction were the sample is replaced by 500 µL ethanol. Tests were carried out in triplicate.

ABTS Radical Cation Decolourization Assay
The potential of N-(2-aryl-4-oxo-thiazolidin-3-yl)-2-(4-(2-aryl-4-oxo-thiazolidin-3-ylcarbamoyl)methyl)-2-oxo-2H-chromen-7-yloxy)-acetamides 5a-d to scavenge free radicals was also assessed by checking their ability to quench ABTS . depicted by the concentration-dependent decolourization of ABTS . . ABTS radical-scavenging activity of 5a-d was determined according to Güven et al. [24]. The ABTS . cation radical was 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 was diluted with ethanol to get an absorbance of 0.700 ± 0.020 at 734 nm. In a final volume of 1 mL, the reaction mixture comprised 950 µL of ABTS . solution and 50 µL of 5a-d at various concentrations. The reaction mixture was homogenized and its absorbance was recorded at 734 nm. Ethanol blanks were run in each assay, and all measurements were done after at least 6 min. Similarly, the reaction mixture of standard group was obtained by mixing 950 µL of ABTS . solution and 50 µL of Trolox. As for the antiradical activity, ABTS scavenging ability was expressed as IC 50 (µg/mL). The inhibition percentage of ABTS radical was calculated using the following formula: ABTS scavenging effect % = {[(A 0 − A 1 )]/A 0 } × 100% where A 0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. All samples were analyzed in triplicate.

Antibacterial Activity
Antimicrobial activity of the purified compounds was evaluated by means of agar-well diffusion assay according to Güven et al. [25] with some modifications. The nutrient agar broth prepared by the usual method, was inoculated aseptically with 0.5 mL of 24 h old subculture of S. aureus ATCC 2353, B. megaterium, P. vulgaris, and E. coli ATCC1225 in separate conical flasks at 40-50 °C and mixed well by gentle shaking. About 25 mL of the contents of the flask were poured and evenly spread in petridish (90 mm in diameter) and allowed to set for two h. The cups (10 mm in diameter) were formed by the help of borer in agar medium and filled with 0.04 mL (40 μg/mL) solution of sample in DMF. The plates were incubated at 37 °C for 24 h and the control was also maintained with 0.04 mL of DMF in similar manner and the zones of inhibition of the bacterial growth were measured in millimetre

Conclusions
A series of new coumarin-based thiazolidinone compounds were successfully synthesized, characterized and tested for their antibacterial and antioxidant activities. Most of the synthesized compounds are more active against E. coli and S. aureus than standard references. Some of the compounds were found equipotent to ampicillin, but less active than other used standard drugs