Novel 4-Thiazolidinone Derivatives as Anti-Infective Agents: Synthesis, Characterization, and Antimicrobial Evaluation

A series of new 4-thiazolidinone derivatives was synthesized, characterized by spectral techniques, and screened for antimicrobial activity. All the compounds were evaluated against five Gram-positive bacteria, two Gram-negative bacteria, and two fungi, at concentrations of 50, 100, 200, 400, 800, and 1600 µg/mL, respectively. Minimum inhibitory concentrations of all the compounds were also determined and were found to be in the range of 100–400 µg/mL. All the compounds showed moderate-to-good antimicrobial activity. Compounds 4a [2-(4-fluoro-phenyl)-3-(4-methyl-5,6,7,8-tetrahydro-quinazolin-2-yl)-thiazolidin-4-one] and 4e [3-(4,6-dimethyl-pyrimidin-2-yl)-2-(2-methoxy-phenyl)-thiazolidin-4-one] were the most potent compounds of the series, exhibiting marked antimicrobial activity against Pseudomonas fluorescens, Staphylococcus aureus, and the fungal strains. Thus, on the basis of results obtained, it may be concluded that synthesized compounds exhibit a broad spectrum of antimicrobial activity.


General Procedure for the Synthesis of Compounds
(3a-3c). Equimolar solution of dicarbonyl compounds and guanidine in ethanol was refluxed at 78 ∘ C for 8 hr. The reaction mixture was then concentrated to dryness under reduced pressure and the residue was partitioned in ethyl acetate. The organic layer was successively washed with water and then finally with brine. The organic layer was dried over sodium sulphate and the solvent was removed under reduced pressure to get the products (3a-3c) [49]. The progress of the reaction was monitored by TLC, using 5% methanol in chloroform.

General Procedure for the Synthesis of Compounds (4a-4f).
A solution of 3a-3c (10 mmol) and various substituted aldehydes (20 mmol) was stirred in THF, under ice cold conditions for 5 min, followed by the addition of mercaptoacetic acid (30 mmol). After 5 min, DCC (12 mmol) was added to the reaction mixture at 0 ∘ C and the reaction mixture stirred for an additional 5 hr at room temp. DCU was removed by filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was extracted with ethyl acetate. The organic layer was successively washed with 5% aqueous citric acid, water, and 5% aqueous sodium hydrogen carbonate and then finally with brine. The organic layer was dried over sodium sulphate and the solvent was removed under reduced pressure to get the products (4a-4f) [60]. The progress of the reaction was monitored by TLC, using the solvent system methanol : chloroform (2 : 98).

Preparation of the Samples and Standard Solution.
The compounds (4a-4f) were dissolved in 10% DMSO at the concentrations of 50, 100, 200, 400, 800, and 1600 g/mL, respectively. Norfloxacin and fluconazole, used as the standard drugs for antibacterial and antifungal studies, respectively, were also dissolved in 10% DMSO at the concentrations of 10 g/mL.

Method.
Antimicrobial activity of the synthesized compounds was evaluated by cup-plate method. Nutrient broth suspension of test microorganism (10 mL) was added to 100 mL of sterile molten nutrient agar growth media (cooled to 45 ∘ C), mixed well, and poured on to sterile petri plates. The agar was allowed to solidify and was then punched to make six wells/cups, using a 6 mm sterile cork borer (separate borer for each organism), to ensure proper distribution of wells in the periphery and one well in the centre. Agar plugs were removed and 50 L solution of test samples (each compound in six concentrations) was poured into the corresponding marked well using micropipette. Triplicate plates of each organism were prepared. The plates were left at room temperature for 2 h to allow diffusion of samples and then incubated face upward, at corresponding temperature of each organism, for 48 h [61]. The diameters of zone of inhibition were measured to the nearest millimeter (the cup size also included) and are presented in Table 2.

Determination of Minimum Inhibitory Concentration (MIC).
A series of glass tubes, containing different concentrations of the synthesized compounds (in 10% DMSO), with nutrient broth was inoculated with the required quantity of the inoculums to obtain a suspension of microorganisms which contained 10 5 colony forming units per milliliter. One growth control tube was prepared without the addition of the compounds or the microorganisms. The tubes were incubated at 37 ∘ C for 24 h. The turbidity produced in each       tube was recorded on a UV-visible spectrometer [62,63]. The observed MICs ( g/mL) are presented in Table 3.

Results and Discussion
4-Thiazolidinones were synthesized in two steps. In the first step, 2-aminopyrimidine derivatives were synthesized by the reaction of 1,3-dicarbonyl compounds with guanidine. Finally, the compounds (4a-4f) were synthesized by reaction of the compounds of step 1 with substituted aromatic aldehydes and mercaptoacetic acids, using DCC as intramolecular cyclizing agent. Characteristic peaks were observed for N-H stretching, C=O stretching, and C-N stretching. The IR spectra of the 4-thiazolidinone derivatives exhibited C=O lactam amide stretching vibration in the range of 1637-1728 cm −1 .
[M] + /[M + 1] + peaks were observed for the synthesized compounds. 1 H-NMR spectra of the compounds indicated the presence of two diastereotopic protons at C-5 position and one single proton at C-2 position; doublets were obtained in the region of 3.07-3.47 ppm. A doublet integrated for one proton appeared at the value of 2.37-2.74 ppm. This can be attributed to the C-2 proton of the 4-thiazolidinone ring.
The antimicrobial activity was observed at 50, 100, 200, 400, 800, and 1600 g/mL, respectively (Table 2). Minimum inhibitory concentrations of the synthesized compounds were also determined, in nutrient broth by tube dilution method. MICs were in the range of 100-500 g/mL, which were recorded as the optical density, at 530 nm.
Thus, the study provides a lead for the syntheses and evaluation of more 4-thiazolidinone derivatives for antimicrobial activity, as the same could lead to the discovery of some promising agents.