Synthesis of indoline-thiazolidinone hybrids with antibacterial and antifungal activities

© 2020 Y. Konechnyi 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 ISSN 0233-7657 Biopolymers and Cell. 2020. Vol. 36. N 5. P 381–391 doi: http://dx.doi.org/10.7124/bc.000A3A


Introduction
In the era of rapidly growing number of patients suffering from various infectious diseases, the searching for novel compounds with antibiotic activity is one of the global challenges in overcoming antimicrobial resistance [1]. In this context, prominent interest belongs to heterocyclic compounds, which are one of the most valuable sources of novel chemical entities with diverse biological activity due to their unique ability to mimic the structure of peptides and to bind reversibly to diverse biotargets [2]. Among the design strategies in drug discovery, considerable interest has been paid to thiazole-based heterocycles [3][4][5]. Thiazole/thiazolidinone derivatives constitute an important class of therapeutic agents in medicinal chemistry including antitrypanosomal [6], antiviral [7,8], anticancer [9][10][11], antioxidant [12,13], anti-inflammatory [14][15][16] activities and also display a pivotal role as antimicrobial and antifungal agents [17,18]. Thus, a thiazole ring is present in several drugs, such as penicillin, monobactam, sulfathiazole, thiabendazole and nizatidine, making this heterocyclic fragment ideal for construction more potent and safer drug candidates, especially in the therapy of infectious diseases (Fig. 1). The antimicrobial activity evaluation is also actual and promising for thiazole-based compounds. Thus, among thiazolidinones, especially rhodanines have been identified several lactamases [19], Sortase A (SrtA) [20], Peptide deformylase [21], Protein mannosyl transferase 1 [22], UDP-galactopyranose mutase (UGM) [22], UDP-N-acetylmuramate/L-alanine ligase (MurC) [23] and Dolichol phosphate mannose [24] synthase inhibitors.
It was envisaged, that the combination of thiazolidinone with other pharmacophores, especially indole fragment, would generate molecular templates with new pharmacological profile and lower toxicity [25]. Thus, among indole-based derivatives a set of potent plant hormones [26], essential amino acids [27], neurotransmitters [27], as well as a large number of commercial drugs and biologically ac- tive molecules have been identified [28][29][30].
In continuation of this theme, we designed and synthesized new non condensed heterocyclic compounds containing 4-oxo-2-thioxothiazolidine (rhodanine), and pharmacologically attractive indole moieties (Fig. 2). The evaluation of their antimicrobial and antifungal activity in vitro against several reference and clinical strains was carried out.

Chemistry
The starting rhodanine-3-propanoic/ethanesulfonic acids 1,2 were obtained according to the known synthetic methods [17,31]. Melting points were measured in open capillary tubes and were uncorrected. The elemental analyses were performed using the Perkin-Elmer 2400 CHN analyzer. The analyses indicated by the symbols of the elements or functions were within ±0.4 % of the theoretical values. The 1 H and 13 C NMR spectra were recorded on Varian Gemini ( 1 H at 400 and 13 C at 100 MHz) instrument in DMSO-d 6 using tetramethylsilane as an internal standard.
Chemical shifts are reported in ppm units with use of δ scale. Mass spectra were obtained using electrospray (ES) ionization techniques on an Agilent 1100 Series LCMS.

Antimicrobial and antifungal activities in vitro (agar diffusion method)
The antimicrobial activity of the synthesized compounds was studied by the method of diffusion into agar. Aliquots (50 μL) of 0.1 % tested compound solution in the mixture of EtOH : DMSO : water (2:1:1) were placed into wells in agar in Petri dishes with test microbes. The antimicrobial activity was evaluated by measuring the diameter of the zone of inhibition of microbial growth. The plates were incubated for 24 h at 37 ˚C. The inhibition zone that appeared after 24 h around the well in each plate was measured in mm. Experiments were in triplicates and standard deviation was calculated. All compounds were tested against six Gram-negative (reference strains Pseudomonas aeruginosa (ATCC 27853 (F-51) [33,34]. In vitro antifungal activity was determined by using Sabouraud Agar plates. All results were compared with those of the DMSO.
The MICs of the compound assays were carried out using the microdilution susceptibility method. The microorganism suspensions were inoculated to the corresponding wells. The plates were incubated at 36 ºC for 18 h for bacteria and fungi, respectively. The minimum inhibitory concentrations of the compounds were recorded as the lowest concentration of each compound in the tubes with no turbidity (i.e. no growth) of inoculated bacteria/fungi.

Acute toxicity
The experiments were conducted on white male mice weighing 23-25 g. Compounds were dissolved in saline solution (0.9 % NaCl) with l-2 drops of Polysorbate 80 (Tween-80 ® ). After dissolution they were administered to mice by the oral route. The LD 50 was evalu-ated for 4 or 5 different doses each on 6 animals and calculated by the Litchfield-Wilcoxon method [35,36].

Chemistry
The general methods for synthesis of target indoline substituted rhodanines are depicted in Scheme 1. The starting rhodanine-3-propa noic/ ethanesulfonic acids were synthesized per the procedure reported previously [17,31]. The target 5-indolylmethylene-3-substituted rhodanines were obtained by the Knoevenagel reaction in the medium of acetic acid in the presence of sodium acetate. Additionally, 5-indolylmethylenerhodanine-3-propanoic acid 3 was transformed into corresponding ester 4 via heating with alcohols in the presence of an acid as a catalyst. The structures of synthesized compounds were elucidated by the spectral data ( 1 H and 13 C NMR). The 1 H NMR spectra of compounds (3-6) CH 2 protons of the alkyl fragment in position N-3 appear as a triplet at δ 2.63-2.77 and 4.24-4.30 ppm, respectively. The chemical shift of the methylene group (=CH) of synthesized 5-indolidenederivatives is insignificantly displaced in the weak magnetic field, δ 7.93-8.09 ppm, and clearly indicated that only Z-isomers were obtained in the Knoevenagel reaction of rhodanines with indole-carbaldehydes. The signals of NH group of indole fragment appeared as single-proton singlets displaced in the weak magnetic field at δ 11.46-12.41 ppm. In the 13 C NMR spectra the characteristic signals of (thio)carbonyl carbons at δ ~166. .1 ppm and the signals of methylene group (130.3-135.6 ppm) are observed.

Biological activity
The synthesized compounds were screened for their in vitro antibacterial and antifungal activities using the agar diffusion method [32]. A total of 15 microorganisms which consisted of eleven bacteria, three yeasts and one mold fungi were tested. Clinical and rarely found clinical strains were multidrug resistant [37] and isolated from patient with health-careassociated infections. All the synthesized compounds exhibited varying degree of inhibitory effect on the growth of different tested strains at a dose of 50 μg per well (Table 1 and Fig.  3). The synthesized compounds showed different mean zone of inhibition in the range of 00-19.4 mm against tested microorganisms. DMSO was used as control. Compounds 3 and 4 show good antifungal activity against Candida albicans, Candida dubliniensis, Aspergillus niger and did not possess significant antibacterial activity. Compound 5 showed the highest activity against Escherichia coli, Bacillus licheniformis, Staphylococcus aureus, Staphylococcus lugnuniensis. Compound 6 displayed moderate activity against Raoultella terrigena. No significant antifungal activity was observed for compounds 5 and 6.
The minimum inhibitory concentrations (MIC) for the most active compounds 3-6 against several microorganisms were calculated using the broth microdilution method (Resazurin Reduction-Based Assay) [33,34] ( Table 2). The tested compounds exhibited the inhibitory activity against clinical strains Escherichia coli, Staphylococcus lentus and Candida albicans with MIC 00-50 µg/mL. Two compounds showed a moderate activity (dilution 1:4) against Candida albicans inhibitory activity with MIC 50 μg/mL against Candida albicans. Compound 6 was inactive against tested microorganisms. Interestingly, the tested compounds at dilution 1:1 possessed a slight inhibitory activity with MIC 50.0 µg/mL.
The SAR analysis showed that the antibacterial effect of compounds 3-6 depends on the substituents at C-5 and N-3 positions of 2-thioxo-1,3-thiazolidin-4-one (rhodanine) core. The 5-enerhodanine-3-propanoic acid 3 with indol-3-ylmethylene fragment in the mol- ecule displayed the equivalent antimicrobial activity to its ester 4. The derivative 5 with ethanesulfonic acids substituent of rhodanine core at N-3 and indol-3-ylmethylene fragment at C-5 was the most active and demonstrated a good effect against all tested bacteria with   For the synthesized compounds, the acute toxicity in mice was studied and their medium lethal doses (LD 50 value) were determined. The stock solutions of the compounds used in this study were prepared immediately before usage and increasing amounts of substances (100-1000 mg/kg) were injected intraperitoneally. The LD 50 values were calculated according to Litchfield and Wilcoxon. The synthesized compounds showed low acute toxicity in mice with the LD 50 values within the range of 300-350 mg/kg (Table 3). Table 3. Acute toxicity of the target compounds. Compound LD 50 , mg/kg 3 960.0 ± 64. 5 4 1005.0 ± 67.0 5 1050.6 ± 67. 5 6 900.0 ± 57.0

Conclusions
The new 5-indolylmethylenerhodanine-3-propanoic/ethanesulfonic acids derivatives 3-6 were synthesized via the Knoevenagel reaction from appropriate rhodanine-3-propanoic/ethanesulfonic acids and indolecarbaldehydes. The antimicrobial screening of synthesized compounds against reference and clinical strains bacteria and fungi was performed. It was found that some derivatives have potential antimicrobial activity against Escherichia coli, Staphylococcus lentus and Candida albicans and are attractive as a novel template for the design of new synthetic antibacterial/antifungal agents.