Antimicrobial activity of some 5-aminomethylene-2-thioxo- 4-thiazolidinones

S. М. Holota, G. О. Derkach, V. V. Zasidko © 2019 S. М. Holota et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC: 616-093 + 547.789


Introduction
The antimicrobial drug discovery is an actual and important area in modern bioorganic and medicinal chemistry [1,2]. The emergence of microbial cells resistance to known antimicrobial drugs is the main problem and simultaneously the main motive force for deep research in this field [3][4][5]. Despite the success in this process, the current antibiotic detection model does not deliver new agents at a rate sufficient to combat the current level of antibiotic resistance. A number of biological, pharmacological, chemical and sometimes philosophical approaches are proposed for the solution of this problem: a) deep understanding of the complex mechanisms of existing antibiotics action; b) hybridization and activity synergism of some substances with antimicrobial effect; c) search for narrow spectrum antiobiotics; d) screening of potential antimicrobial agents among the new classes of chemical compounds, etc [6][7][8]. The 4-thiazolidinone derivatives represent considerable interest for de novo design of antibacterial agents. Potential antibacterial ligands such as selective and multiinhibitors of Mur B, C, D, E, F; penicillin -binding proteins inhibitors (РВРs); inhibitors of β-lactamase А and С; inhibitors of peptide deformylase; inhibitors of mannosyl transferase 1 (PMT1) were identified among these heterocycles [9][10][11][12][13]. Also, in some structure -activity relationship studies (SAR analysis) it had been shown that incorporation of aromatic amino acids into organic compounds significantly improves the potency and selectivity of antibacterial activity [14]. As part of our research in the field of biologically active heterocycles [15][16][17][18], herein we report the antimicrobial properties of new 5-enamine-4-thiazolidones with L-βphenyl-α-alanine fragment in molecules [19]. Noteworthy, we have previously established an interesting antitumor and antitrypanosomal activity of this class of compounds. Moreover, the activity type significantly depends on the structure features of enamine fragment in C5 position of 2-thioxo-4-thiazolidinone core. Taking into account the above facts, it is promising to study other types of activity of the compounds as a realization of the polypharmacological strategy in the design of potential drug-like molecules among 4-thiazolidinones [20,21].

Materials and Methods
Chemistry. Synthetic procedure and physicalchemical properties of compounds 1-13 have been described earlier [19].
Antimicrobial activity The antimicrobial activity of the synthesized compounds was determined using a method of diffusion into agar. Nutrient agar (0,5 % peptone, 0.3 % beef extract, 1.5 % agar, 0.5 % sodium chloride, distilled water, pH ~ 6.8) was used as a nutrient medium. The test-cultures suspensions (in concentration 1×10 7 CFU/ml), standardized previously by the optical standard of turbidity, were uniformly sown in Petri dishes with the nutrient agar. Aliquots (20 μL) of 0.1 % of the test compounds (concentration 1000 μg/ml) in EtOH/DMSO/water (2:1:1) were placed into wells (diameter of 4.0±0.1 mm) in agar in Petri dishes with test microbes. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone of microbial growth. The plates were incubated for 24 h at 37 •C. The inhibition zone appeared after 24 h and was measured in mm around the well in each plate. Digital images of culture growth on dishes [were] obtained and processed with a computer program UTHSCSA ImageTool 2.0 (The University of Texas Health Science Center in San Antonio, © 1995-1996) for calculation of growth inhibition zone diameters. The experiments were performed in triplicate, and standard deviation was calculated. The experiments were carried out on microorganism strains, which were isolated in the laboratory of the microbiology research of the Department of Microbiology, Virology and Immunology of the Ivano-Frankivsk National Medical University from ambulatory patients. The following isolated clinical strains of conditionally pathogenic bacterial strains were used: methicillin-sensitive Staphylococcus aureus (MSSA); methicillin-resistant Staphylococcus aureus (MRSA); methicillin-resistant Staphylococcus haemolyticus (MRSH); (extended spectrum β-lactamase (ESβL) producing Gram-negative bacteria Escherichia coli; Klebsiella pneumoniae; Pseudomonas aeruginosa; yeasts Candida albicans; Candida tropicalis. Test-cultures were identified using chemical micro-tests "STAPHYtest 16" and "ENTEROtest 24" (Lachema, Czech Republic). Fungi cultures were identified on the basis of 40 biochemical tests using the VITEK 2 system with the VITEK 2 YST ID card (bioMerieux, France). Antimicrobial drug sensitivity patterns of used microbail strains are presented in Table 1. The sensitivity of strains to antibiotics was determined by disco-diffusion method and serial dilutions in agar. The minimum inhibitory concentrations (MICs) of the compounds were determined using the microdilution susceptibility method [22]. Microorganism suspensions were inoculated to the corresponding wells. Plates were incubated at 36 •C for 18 h for bacteria and fungi, respectively. The presence of the microorganism growth in the bouillon (bouillon turbidity) suggested that concentration of the compound was insufficient to suppress its viability. The first lowest concentration of the tested compounds (from a series of dilutions), where the bacterial growth was not visually determined was considered to be the minimum inhibitory concentration (MIC). The estimation of interaction with amoxicillin and co-amoxiclav (amoxicillin/clavulanic acid) for synthesized compounds has been performed on the growing medium with subbacteriostatic concentration of oxacillin (1/4-1/16 MIC) relative to resistant strains [23]. The following isolated clinical strains of conditionally pathogenic bacterial strains with resistance to β-lactam antibiotics were used: ESβL (β-lactamase of the extended action spectrum)producing Klebsiella pneumonie; methicillinresistant Staphylococcus haemolyticus (MRSH) with atypical penicillin-binding protein PBP2* and β-lactamase activities. The production of the atypical penicillin-binding protein PBP2* was determined in the latex agglutination reaction (Slidex® MRSA Detection, bioMerieux, France). The results have been processed by variation statistics methods.

Results and Discussion
The screening of the data reveal that almost all tested compounds demonstrated a moderate antibacterial effect against both Gram-positive and Gram-negative strains (Fig. 1, Tables 2, 3).
The best levels of zone inhibition and MIC values were observed against the MRSA. Six compounds showed a satisfactory activity against the MRSA and derivatives 7 and 11 were the most active with MIC 3.12 µg/mL and 12.5 µg/mL respectively. The compounds 2, 4, 9, 12 were characterized by a slightly lower inhibitory activity and their MIC for    The derivatives 4, 9, 11 and 13 were estimated in the interaction with amoxicillin and co-amoxiclav (amoxicillin/clavulanic acid) against multidrug resistant clinical isolates of ESβL + K. pneumonie and MRSH (Tables 4,5).
According to the preliminary interaction screening results, the derivative 13 displays promising synergistic activity with amoxicillin against ESβL + K. pneumonie strain. The similar results were obtained for derivatives 4 and 9 with amoxicillin against MRSH. However, any positive activity changes in the combinations of tested compounds with co-amoxiclav were not observed against both ESβL + K. pneumonie and MRSH.
The SAR analysis showed that the antibacterial effect of compounds 1-13 depends on the structure features of the enamine fragment. The compound 1 with unsubstituted NH 2group displayed the equivalent activity level to compound 2 with phenyl group. However, the activity level of these compounds was not satisfactory. Introduction of halogen atoms (F, Cl) into position 4 of benzene ring improved the activity (compounds 3, 4), but additional NO 2 -or MeO-groups and change [in the] halogen position (compounds 5, 6) provoke a decrease in the activity. The derivative 9 with 4-ethylsulfanylthiosulfonylphenyl substituent was the most active and demonstrated a good effect against all tested microorganisms with MIC 25 µg/mL. The change of 4-EtS-group in 9 to 4-NH 2 -group (compound 7) provides an increasing selectivity against MRSA (MIC 3.12 µg/mL) but generally decreasing activity. The similar pattern of activity decreasing was observed for the transformation of the sulfonylgroup into position 2 of benzene ring and the additional introduction of CH 3 -group and morpholine cycle (compound 8). Also, compound 10 with isosteric COOEt-group showed a lower activity than 9. The derivative with unsubstituted thiazole rings (11) displayed good level of activity against E. coli (MIC 3.12 µg/ mL) and MRSA (MIC 25 µg/mL), but introduction of 2,4-dichlorobenzyl-fragment into thiazole ring (compound 12) provides a lower activity. The presence of 4H-[1.2.4]-triazol-3ylamine-subtituent (13) was good, especially for antifungal activity. So, from the SAR viewpoint it was not established clear dependence of the influence of electron-donating or electron-withdrawing groups on the activity realization.

Conclusions
The antimicrobial screening of 13 new 2-thioxo-4-thiazolidinones against Grampositive, Gram-negative microorganisms and Candida fungi was performed and the results are described in this paper. It was found that some derivatives have potential antimicrobial activity against S. aureus methicillin-resistant (MRSA) strain, Ps. aeruginosa, C. albicans and are attractive as a novel template for the design of new synthetic antibacterial/antifungal agents. Some derivatives displayed promising synergistic activity with amoxicillin against multiresistant strain of clinical isolates of ESβL + K. pneumonie and MRSH and can be used for the development of new combined antimicrobial chemotherapeutic agents.