5-Ene-rhodanine-3-carboxylic acids as potential antimicrobial and antiparasitic agents

© 2020 A. P. Kryshchyshyn-Dylevych; 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


Introduction
4-Thiazolidinone derivatives have been known as a source of drug-like molecules with the studied hypoglycemic, anticancer, anti-inflammatory, antituberculosis and antimicrobial ef-fects [1][2][3]. The inhibition activity against protozoa such as Trypanosoma ssp. [4][5][6] or Plasmodium falciparum [7] had been also investigated for thiazole/thiazolidinone-and re-A. P. Kryshchyshyn-Dylevych Bioorganic Chemistry ISSN 1993-6842 (on- lated heterocycles-based compounds. 2,4-Thiazolidinedione, rhodanine (2-thioxo-4-thiazolidinone), 2-alkyl(aryl)-substituted, and 2-R-amino(imino)-substituted 4-thiazolidinone subtypes provide the major part of antimicrobial, antidiabetic, anti-inflammatory and anticancer lead-compounds and drug candidates [1,8,9]. 5-Ene-thiazolidinones including the 4-thiazolidinone-3-carboxylic acids derivatives [10,11] are of special interest taking into account their pharmacological profiles, the feasibility of structure optimization as well as the toxicity profile [12,13]. Despite falling out of favor with medicinal chemists, 5-ene-4-thiazolidinones should not be treated as panassay interference compounds (PAINS) only [14] and still hold a promise for providing active drug-like molecules [15]. One more direction of the 5-ene-4-thiazolidinones study is the search for novel antibacterial agents [16], great part of which comprise rhodanine-3-carboxylic acids. On the one hand, antibacterial drugs have been saving millions of lives since the discovery of penicillin, but on the other hand, their extensive usage had pushed the anti bio-tic resistance mechanisms in bacteria. Surviving of species with such mechanisms challenges therapeutic options of the infectious diseases treatment [17], which needs new classes of antibiotics based on the chemi cal structures different from those used today. The development of novel safe and efficacious antibacterials remains the topical task of medicinal chemistry and health care system worldwide. Reviewing the latest li tera ture data, it should be mentioned that a class of 5-ene-2-thioxo-4-thiazolidininone-3-carboxylic acids is characterized as a source of agents with excellent antimicrobial activity including MDR strains. For example, 3-α-carboxyethyl-5-benzylidenerhodanine derivatives showed moderate to good MIC values against MRSA pathogen panel [18]; a series of 5-(2-hydroxybenzylidene)-rhodanines possessed the MIC values in 32-256 μg/ mL range against S. aureus, E. faecalis, and H. influenza and were experimentally characterized as novel inhibitors of bacterial DNA gyrase [19]; para-N,N-benzylidenediphenylamine substituted rhodanine-3-alkanecarboxylic acids were active against Gram   5-Ene-rhodanine-3-carboxylic acids as potential antimicrobial and antiparasitic agents (+) pathogens with MIC = 1.95 μg/mL [20]. Rhodanine-3-carboxylic acids with an arylhydrazone fragment displayed the excellent activity against MDR methicillin-resistant and quinolone-resistant S. aureus with MIC of 2-4 μg mL −1 [21]. With a broad exposure to antibiotics and immunosuppression, the incidence of opportunistic fungal pathogens such as Candida albicans has increased [22,23]. The compounds revealing antifungal activity are also presented among 2-thioxo-4-thiazolidinones. A derivative of 2-(rhodanine-3-yl)-3-phenylpropanoic acid showed the micromolar ran ges of MIC towards the isolates of Gram (+) and Gram (-) bacteria including the vancomycin resistant strains as well as Candida albicans and was not toxic to mouse murine macrophages and human keratinocytes [24]. Taking into account the literature data on antibacterial, antifungal and antiparasitic properties of thiazolidinone derivatives, the feasibility of their synthesis and further chemical optimization, the aim of presented research was the design of potentially active antimicrobials on the base of rhodanine-3-carboxylic acids.

Chemistry
All chemicals were of the analytical grade and commercially available. All reagents and solvents were used without further purification and drying. NMR spectra were determined with Varian Mercury 400 (400 MHz) spectrometer, in DMSO-d 6 using tetramethylsilane as an internal standard. Elemental analyses (C, H, N) were performed at the Perkin-Elmer 2400 CHN analyzer and the results were with-in ± 0.4 % of the theoretical values. Mass spectra were obtained using electrospray ionization (ESI) techniques on an Agilent 1100 Series LCMS. The purity of the compounds was checked by thin-layer chromatography performed with Merck Silica Gel 60 F254 aluminum sheets.

Synthesis of ethyl 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoate (Ic).
The 3-(4-oxo-2-thioxothiazolidin-3-yl)propanoic acid (0.1 mole) was refluxed in the ethanol medium (200 mL) with adding catalytic amounts of concentrated sulfuric acid for 5 hours. After cooling the reaction mixture to the room temperature, it was neutralized with potassium carbonate to pH 7.0 and the formed precipitate (potassium sulfate) was filtered off. The obtained filtrate was evaporated under vacuo to yield the product as viscous yellow liquid that was used in further reactions without additional purification.
In the case of IIb and IIc synthesis, a mixture of 4-oxo-2-thioxothiazolidine-3-propanoic acid or its ethyl ester (0.1 mol), ethyl ortho-formate (0.12 mol) and 40-50 ml of acetic anhydride was refluxed for 1.5 hour. The reaction mixture was poured into water and the product was extracted with ethylacetate. The ethylacetate layer was evaporated under vacuo and the obtained solid product was recrystallized at first from the mixture of acetic acid/ water with the second recrystallization from the mixture of toluene/hexane for IIb (method A) or from ethanol only for IIc. Purification of the mixture of products after the first crystallization (acetic acid/water) by column chromatography was performed in the system of solvents: aceton/hexane/NH 4 OH = 50:50:1.

Antibacterial and antifungal activity screening
The synthesized compounds were screened for their in vitro antibacterial and antifungal activities using the agar diffusion method and the broth microdilution method (Resazurin Reduction-Based Assay) [27]. A total of 12 microorganisms were tested.  [28] and isolated from patients with health-care-associated infections.
The tested compounds (at the concentration of 1mM) were inoculated into a 5.5±0.5 mm diameter well (50 µL per well) containing suspension of the culture of microorganisms (McFarland 2.0) on the agar plate (meat peptone agar or Saburo agar for fungi). DMSO was used as a control. A compound with the diameter of growth inhibition more than 10 mm was considered as a hit-compound [27].

Antitrypanosomal activity screening
Bloodstream forms of Trypanosoma brucei brucei strain 90-13 and Trypanosoma brucei gambiense Feo strain were cultured in HMI9 medium supplemented with 10 % FCS at 37 °C in an atmosphere of 5 % CO 2 [29]. In all experiments, log-phase cell cultures were harvested by centrifugation at 3000×g and immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells [30]. Drug stock solutions were prepared in DMSO. Trypanosoma brucei bloodstream forms (10 5 cells/mL) were cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 mL. After the 72 h incubation, resazurin solution was added in each well at the final concentration of 45 mM and fluorescence was measured at 530 nm and 590 nm absorbance after further 4 h incubation. The percentage of inhibition of the parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that in the absence of drug. DMSO was used as a control. The concentration inhibiting 50 % of parasite growth (IC 50 ) was given as the mean +/-the standard deviation of three independent experiments.

Antibacterial and antifungal activity
Five of the synthesized compounds III were tested towards a series of Gram (+) and Gram (-) bacteria and four yeasts strains (Table 1) Table 2) that corresponds to the concept of polypharmacological approach [33]. Noteworthy, such small molecules as the studied rhodanine analogs hold a privileged position for polypharmacology and may be successfully utilized in the fragment-based drug discovery too [34,35].  The selectivity indexes calculated as a ratio of cytotoxic concentration against normal fibroblasts CC 50 to the antitrypanosomal IC 50 values, indicate very good perspective for the study of rhodanine-3-carboxylic acids as potential antitrypanosomals with low toxicity parameters.
The established dual antitrypanosomal and antifungal actions are consistent with the recently developed new effective oral monotherapy of HAT with antifungal drug Fexinidazole [5]. propanoate IIIf. The pyridine-rhodanine-3-carboxylic hybrids showed good ratios of Trypanosoma ssp. inhibition and low cytotoxicity towards human fibroblasts. Such dual action of the rhodanine-3-carboxylic acids derivatives is an important issue that may be used in the polypharmacological approach as well as the fragment-based drug discovery.