Synthesis and Antibacterial Activity of Analogs of 5-Arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiazoli-din-4-one

In an effort to develop new antimicrobial agents, 3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiazolidin-4-one (4) was synthesized by reaction of thiocarbonylbisthioglycolic acid with ethyl (4-methyl-2-oxo-2H-chromen-7-yloxy)aceto- hydrazide (3), which was prepared in turn from 7-hydroxy-4-methylcoumarin (1). The condensation of compound 4 with different aromatic aldehydes afforded a series of 5-(arylidene)-3-(4-methylcoumarin-7-yloxyacetyl-amino)-2-thioxo-1,3-thiozolidin-4-one analogs 5a–h. The structures of these synthetic compounds were elucidated on the basis of IR, 1H-NMR and 13C-NMR spectral data and ESI-MS spectrometric analysis. Compounds 5a–h were examined for their antibacterial activity against several strains of Gram-positive and Gram-negative bacteria.


Introduction
Bacterial disease control, including the food safety issue, has continuously attracted researchers' attention from various fields. The use of preservatives and pathogen antagonists had been reported as a means of protecting the microbiological safety of fresh and processed food products [1][2][3][4]. Although some antagonists exhibited significant inhibition of bacterial growth, they were too toxic to be utilized long term. In the present study, we hoped to explore new lead compounds with natural skeletons which could be modified for further investigation as antimicrobial agents applied to food preservation. 7-Hydroxy-4-methylcoumarin derivatives with heterocyclic moieties possess diverse biological properties such as antibacterial [5][6][7], antifungal [8,9], anticancer [10], enzyme-inhibitory [11], and antioxidant activities [7,9]. On the other hand, thiazolidin-4-ones are important compounds due to their broad range of biological activities including anticancer [12][13][14], virus-inhibitory [15], HIV-inhibitory [16], and enzyme-inhibitory activities [14]. These observations prompted our interest in synthesizing some new 7-hydroxy-4-methylcoumarin derivatives bearing 2-thioxo-1,3-thiozolidin-4-one substituents and evaluate their antibacterial potential.
In the IR spectrum of compound 4, in addition to the lactone and amide carbonyl group absorption at 1709 cm −1 , the stretching band in a high frequency region (1771 cm −1 ) indicated the presence of C=O bonds in a thiazolidine ring. A new signal with intensity of 2H appearing at 4.47 ppm in the 1 H-NMR spectrum was attributed to the methylene group of the thiazolidine ring. Another signal also with intensity of 2H appearing in the downfield region at 4.96 ppm was attributed to the oxymethylene protons (OCH 2 ). In terms of unexpected results, the signals of the methylene protons in 4 were split instead of being a singlet. The splitting of these signals could be explained by a non-first order splitting effect.
Comparing the 13 C-NMR spectra of 3 [7] and 4, three more signals appeared in 4, two of which were at around 160 ppm corresponding to the signals of the carbon atoms in the thioxo group and carbonyl group, whereas the last one was at 33.4 ppm corresponding to the signal of the saturated carbon atom of the thiazolidine ring. The spectral data of 4 as well as the agreement of the predicted mass with molecular mass determined by HR-MS confirmed that a 4-oxo-2-thioxothiazolidine ring was formed. Scheme 1. Synthetic route for the preparation of compounds 5a-h. HO OH  In the IR spectrum of compounds 5a-h, there were shifts of the absorption of the carbonyl group at 1771 cm −1 to lower frequencies (1721-1755 cm −1 ), in agreement with the formation of a conjugated system between the carbonyl group and the benzylidene moiety. Comparison of the 1 H-NMR spectra of 5a-h with the 1 H-NMR of 4 showed not only the disappearance of the methylene proton's signal at 4.47 ppm, but also appearance of additional aromatic proton signals at 6.84-8.36 ppm and methylidene proton signals at 7.82-8.86 ppm. The signal of the oxymethylene protons (OCH 2 ) in compounds 5a-h appeared at 5.00-5.04 ppm. The signals of these protons, like the signals of the methylene protons OCH 2 in compound 4, were also split by a non-first order splitting effect. Therefore, they did not appear as singlets.

Determination of the in Vitro Antimicrobial Activity
Compounds 5a-h were examined for antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria) at concentrations of 0.1% and 0.2% according to the reported method with minor modifications [18]. As shown in Table 1, most of the compounds 5a-g at 0.1% exhibited low antimicrobial activity, with antimicrobial inhibition zone diameters of less than 15 mm. However, at the concentration of 0.2%, most of these compounds showed average activity (the antimicrobial diameters were 15 mm to 20 mm) against certain bacteria including Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria), Bacillus subtilis and Staphylococcus aureus (Gram-positive bacteria). In addition, the synthetic compounds 3 and 4 did not show any significant inhibition of bacterial growth in our preliminary screening and therefore the data were not included. The minimum inhibitory concentration (MIC) value is a measure to define the antibacterial activity of a compound and is defined as the lowest concentration of drug that inhibits visible growth. Compounds 5a-h were subjected to examination of their MIC values according to the reported method [19] and the data are shown in Table 2. The two-fold microdilution broth method was used and all of the tested samples demonstrated inhibitory effects in a concentration-dependent manner. However, only 5c, 5g and 5h exhibited any significant inhibition against S. aureus with MIC values of 50 μg/mL.
a MIC > 50 μg/mL and not determined.

General Procedures
All starting materials were purchased from Merck (Darmstadt, Germany) and used without purification. Melting points were measured in open capillary tubes on a Gallenkamp melting point apparatus. The structures of all compounds were confirmed by IR, NMR and HR-MS spectra. IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer using KBr pellets. The 1 H-NMR spectra were recorded on a Bruker Avance spectrometer at 500 MHz using DMSO-d 6 as solvent, while the 13 C-NMR, HSQC, HMBC spectra were recorded at 125 MHz. The data are given in parts per million (ppm) and are referenced to an internal standard of tetramethylsilane (TMS, δ 0.00 ppm). The spin-spin coupling constants (J) are given in Hz. Peak multiplicities are reported as s (singlet), d (doublet), dd (double-doublet), t (triplet), q (quartet), and m (multiplet). The MS spectra were recorded on a Bruker microTOF-Q 10187 spectrometer or on a Varian FT-ICR-MS 910 spectrometer.

Determination of the in Vitro Antimicrobial Activity
The compounds 5a-h at concentrations of 0.1% and 0.2% were examined for antimicrobial activity against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25923) (Gram-negative bacteria), Bacillus subtilis (ATCC 11774) and Staphylococcus aureus (ATCC 11632) (Gram-positive bacteria) according to the reported method with minor modifications [18]. A mixture of meat extract (5.0 g), peptone (5.0 g), NaCl (5.0 g), agar (20.0 g) and distilled water (1000 mL) was stirred to dissolve the ingredients and then sterilized in an autoclave to give the MPA environment for growing the bacteria. The mixture was poured to Petri dishes. The Petri dishes then were put in a sterile cabinet for 24 h. After infusion of the particular bacteria into the MPA environment in the Petri dishes, a hole was drilled in the center of the dish. DMSO solution (0.1 mL) of the particular chemical at a concentration of 0.1% or 0.2% was dripped into the hole. The samples were placed in a refrigerator for 4-8 h, and then incubated at room temperature for 24 h. The inhibiting zone was measured by the (D − d) value expressed in millimeters (mm), where D was the diameter of inhibited zone and d was the diameter of the hole. The evaluation was based on the following criteria: D − d ≥ 25 mm: very strong antibacterial activity; D − d ≥ 20 mm: strong antibacterial activity; D − d ≥ 15 mm: medium antibacterial activity; D − d ≤ 15 mm: weak antibacterial activity. Each experiment was performed in triplicate.

Minimum Inhibitory Concentration (MIC) Determination
The amount of growth in the wells containing test samples was compared with the amount of growth in the control wells when determining the growth end points. When a single skipped well occurred, the highest MIC was read. Each experiment was performed in triplicate. Streptomycin and tetracyclin were used as positive controls for Gram-positive bacteria and Gram-negative bacteria, respectively.

Conclusions
Eight new 5-arylidene-3-(4-methylcoumarin-7-yloxyacetylamino)-2-thioxo-1,3-thiozolidin-4-one analogs 5a-h were successfully synthesized. The structures of these compounds were determined by IR, 1 H-NMR, 13 C-NMR and HR-ESI-MS spectral data. Most of the compounds 5a-h exhibited significant activity against Bacillus subtilis and Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa at a concentration of 0.2%. Further structural modification could be performed to improve the bioactivity and may prove useful in developing new therapeutic anti-microbial agents.