Synthesis and Antimicrobial Activity Evaluation of Novel 4-Thiazolidinones Containing a Pyrone Moiety

Abstract A series of thiosemicarbazones 3a–d and 4-thiazolidinones 5a–d, 7a–d, and 9a–h were synthesized and evaluated for their in vitro antimicrobial activity. Condensation of 3-acetyl-4-hydroxy-6-methyl-2H pyran-2-one1 (dehydroacetic acid) with thiosemicarbazide 2a–d in ethanol at room temperature yielded the thiosemicarbazones 3a–d. These compounds were exploited to synthesize the 4-thiazolidinones 5a–d via their reactions with ethyl 2-bromo propionate4. Derivatives 7a–d were prepared by reaction of the thiosemicarbazones 3a–d with phenyl bromoacetate 6. The 4-thiazolidinones 9a–h were obtained by treatment of compound 3a or 3c with maleimide derivatives 8a–d in refluxing ethanol, under sulfuric acid catalysis. All compounds were screened in vitro for their antibacterial and antifungal activities against five human pathogens microorganisms: Escherichia coli ATCC 25992, Pseudomonas aeruginosa ATCC 27852, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 25923, and Candida albicans. GRAPHICAL ABSTRACT


INTRODUCTION
In recent years, 4-thiazolidinones and their derivatives have become among the most extensively investigated compounds. They constitute an important group of heterocyclic compounds, displaying a broad spectrum of biological activity. [1][2][3] They have found uses, for example, as antimicrobial [4][5][6] and anti-inflammatory drugs [7][8][9] and anti-HIV derivatives. [10][11][12] The thiazolidinone ring system belongs to the privileged structure in modern medicinal chemistry, particularly in discovery of new antitumor and anti-angiogenic agents.
A literature survey revealed that many different protocols have been developed in a way that allows the synthesis of 4-thiazolidinone skeletons, and several reactants or heterocyclic compounds have been employed for the preparation of 4-thiazolidinone moiety. [13,14] The main synthesis routes to 4-thiazolidinones involve three components (an aldehyde, an amine or thiosemicarbazide, and mercaptoacetic acid or halogenoester), either in one-or two-step processes. [15][16][17][18][19][20] We report in this article the syntheses of a new class of structurally novel 4-thiazolidinone derivatives, incorporating two known bioactive heterocyclic nuclei such as pyrone and 4-thiazolidinone, known to be active towards (G þ and G -) and yeast. Pyrones and their fused derivatives have attracted a great deal of interest because of their wide range of biological activities. [21][22][23] The incorporation of another heterocyclic moiety in pyrone either in the form of a substituent or as a fused component often leads to incredible diverse biological activity.
In this study, new 4-thiazolidinones 5a-d, 7a-d, and 9a-h were synthesized starting from commercially dehydroacetic acid, which possesses three active sites in positions 2, 3, and 4 corresponding to ester, acetyl, and hydroxyl groups respectively for nucleophilic substitution by amine. It has been reported that the carbonyl of acetyl group in position 3 in dehydroacetic acid proved to be the more reactive site toward coupling reaction with nucleophilic reagents. [24] In this context, we prepared thiosemicarbazones 3, via treatment of dehydroacetic acid with thiosemicarbazides 2, to investigate its reactivity toward some electrophilic reagents. All synthesized compounds were tested for their in vitro antimicrobial properties against Gram-positive, Gram-negative, and antifungal bacteria.

Chemistry
In continuation of our ongoing work aiming at the development of efficient methods for the synthesis of heterocyclic compounds, [25] we carried out an efficient preparation of 4-thiazolidinones derivatives 5a-d and 7a-d (Scheme 1). We also report a new synthetic route to the formation of 4-thiazolidinone compounds 9a-h, from reaction of the precursors 3a or 3c and maleimide derivatives 8a-h in refluxing ethanol.
In our study, we initially isolated the thiosemicarbazones intermediates 3a-d in quantitative yields (81-91%) after stirring the thiosemicarbazides 2a-d with dehydroacetic acid 1 in ethanol at room temperature. Spectral 1 H NMR data of thiosemicarbazones 3a-d indicated that these compounds exist as a tautomeric equilibrium between the enol (I) and the ketone (II) forms in an average ratio of 1:2, represented in Scheme 2.
For example, concerning 3a, the chemical shift of the singlet corresponding to methyl protons of the pyrone unit was observed at d: 2.12 ppm in form (I) and at d: 2.16 ppm in form (II). The methylene proton (CH) appeared as a singlet at d: 5.93 ppm (I) and at d: 6 ppm in form (II). Methyl imine protons resonated at d: 2.4 ppm in form (I) and at d: 2.03 ppm in form (II). The 13 C NMR spectrum exhibited a signal at 184 ppm assigned to the C = S bond. The infrared (IR) spectra showed bands around 1150-1156 cm À1 and 1575 cm À1 assigned to C = S and C = N bending vibrations, from the thiosemicarbazone fragment.
Subsequently, 4-thiazolidinone compounds 5a-d were synthesized in good yields as described in Table 1, by reaction of the intermediates 3a-d, ethyl 2-bromo propionate 4, and 3 equiv of sodium acetate anhydrous in the presence of two drops of concentrated sulfuric acid or 0.12 mol% of keggin heteropolyacid [26,27] (H 3 PW 12 O 4 , n H 2 O) in refluxing acetonitrile (Scheme 1).
As shown in Table 1, compounds 5a-d were formed efficiently when we used H 3 PW 12 O 4 , n H 2 O as catalyst comparatively with sulfuric acid use. The reaction times were shortened from 2 h to 30 min for derivatives 5a-b, 1 h to 15 min for compound 5c, and 30 to 5 min for 5d. However, the reaction yields are slightly lower compared with sulfuric acid use.  To improve our studies, we applied the same methodology to synthesize 4thiazolidinones 7a-d from compounds 3a-d and phenyl bromoacetate 6. The products 7a-d were isolated in excellent yields as shown in Table 1.
The appearance of the double signal at 1.53 (J ¼ 7 Hz) ppm due to -CH 3 and the quadruplet signal at 4.43 ppm (J ¼ 7 Hz, J ¼ 14 Hz) attributed to -CH in 1 H NMR confirms the formation of 4-thiazolidinones 5a-d. The IR spectra of 4-thiazolidinone showed absorption bands at 1711-1718 and 1313-1357 cm À1 corresponding to the C=O and the N=C-S groups of thiazolidinone ring. Moreover, in the 13 C NMR spectrum, signals at 17.4, 43, and 178 ppm were assigned to CH 3 , CH, and C=O (thiazolidinone) carbons.
The signal of methylene (CH 2 ) which appeared at 4.5 ppm in 1 H NMR and at 34 ppm in 13 C NMR corroborates the structures 7a-d.
The proposed mechanism of formation of 4-thiazolidinones 5 or 7 is illustrated in Scheme 3.

NOVEL 4-THIAZOLIDINONES
The formation of 5 or 7 was assumed to proceed through a nucleophilic attack of the sulfur atom of the thiosemicarbazones 3 onto the bromoester, followed by the intramolecular cyclization with elimination of ethanol and regeneration of sulfuric acid.
We also described a simple new strategy of the preparation of thiazolidinones 9. The reaction of 1 equiv of the key intermediates 3a or 3c with the appropriate maleimide 8a-h in the presence of two drops of concentrated sulfuric acid in refluxing ethanol for 3 to 4 h afforded structures 9a-h in moderate yield (46-52%). The solid was obtained after purification by column chromatography (eluent: ethyle acetate) or by recrystallization in acetonitrile as represented in Scheme 4.
The formation of compounds 9a-h results from initial attack of the sulfur atom of thiosemicarbazone group on the double bond of maleimide followed an intermediate A (not isolated). The ring opening of maleimide is obtained by nucleophic attack of nitrogen azote NR on the functional carbonyl, affording thiazolidinone structures 9a-h (Scheme 5).
For example, in IR spectra, the formation of compound 9a was confirmed through the presence of absorption bands around 1341 and 1704 cm À1 attributed to N = C-S-C = N and C = O bending vibrations. Consequently, the absence of the signal at 184 ppm in 13 C NMR spectra, attributed to C=S, was also a parameter considered for the confirmation of the cyclization reaction. The 1 H NMR spectra

Antimicrobial Evaluation
Antibacterial activity of all compounds was determined by the well diffusion method [28] and the diameter of inhibition zone was measured as indicated by Nataraja et al. [29] and Vinod et al. [30] Antimicrobial activity of the synthesized products 3a-d, 5a-d, 7a-d, and 9a-h was evaluated against five microorganisms, known to cause some infections in humans: Escherichia coli ATCC 25992, Pseudomonas aeruginosa ATCC 27852, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 25923, and Candida albicans. The results obtained (Table 2) revealed that compounds 3a, 3b, 7c, and 9a-h (except 9f) showed bactericidal activity toward Gram-positive bacteria Staphylococcus aureus ATCC43300. Compounds 7a-d (except 7b) demonstrated the greatest inhibition activity against Staphylococcus aureus ATCC 25923. All the tested compounds, 3b-d, 5a-d, 7c, 7d, and 9a-e (except 9d) are distinguished by a strong inhibitory activity on the growth of the Gram-negative bacteria Pseudomonas aeruginosa ATCC 27852. Compounds 3a-d (except 3c) and 7d are the most active against Candida albicans. Very low inhibitory activity on the growth of Escherichia coli ATCC 25992 is observed for compounds 3a, 5a, 7c, and 9a, c. As shown in Table 2, some compounds did not show any activity toward Gram-positive bacteria, Gram-negative bacteria, and Candida albicans.  Many synthetic compounds feature activity against microorganisms. The inhibitory activity increases in the compounds having substitution. [31,32] However, it is reported that these compounds show a variety of pharmacological properties such as antibacterial, antifungal, and biological activities. [33,34] The results obtained in this study indicate that the attachment of hydrogen, methyl, or amid groups at the 5-position of 4-thiazolidinone moiety gained the different activities of all expected compounds 3.

SUPPORTING INFORMATION
Full experimental detail, 1 H and 13 C NMR spectra, and results of antimicrobial activity for this article can be accessed on the publisher's website.