Antimicrobial and cytotoxic activities of thiazolo[4,5- b ]pyridine derivatives

Aim. The screening of antimicrobial and cytotoxic activities of thiazolo[4,5- b ]pyridine derivatives was accomplished. Methods. The antibacterial and antifungal activities of synthesized thiazolopyridines were evaluated in vitro with the agar diffusion and broth microdilution methods using clinical and reference strains of Gram-positive, Gram-negative bacteria and yeasts. The structure-antibacterial/antifungal activity relationships of the screened compounds were established. The target compounds were screened for their cytotoxicity effects on HaCaT and HEK293 cells using MTT assay. Results. The highest antimicrobial activity was observed for compound V 2-oxo-7-thiophen-2-yl-2,3-dihydrothiazolo[4,5- b ]pyridine-5-carboxylic acid with minimal inhibitory concentration (MIC) 12.5 μg/mL against Candida albicans . At the same time, the synthesized compounds were explored in the interaction with amoxicillin against multidrug resistant clinical isolates of ESβL + Klebsiella pneumonie and Staphylococcus haemolyticus (MRSH). The best synergistic activity with amoxicillin was exhibited by compound VI. HaCaT human keratinocytes and HEK293 human embryonic kidney cells demonstrated resistance to the thiazolopyridine derivatives treatment and did not reach the IC 50 value up to 100 µM. Conclusions. The tested thiazolopyridines constitute an interesting background for further development of new chemotherapeutic agents.


Introduction
A wide range of infectious diseases caused by different pathogens is a main focus of the searching for new highly active and low-toxic antimicrobials in modern drug discovery. A special issue in this contest is occupied by heterocyclic compounds, due to their unique ability to mimic the structure of prokaryotic cell metabolites and to bind reversibly to diverse biotargets [1,2]. Thus, considerable interest among antimicrobial drug-design strategies has been paid to thiazole derivatives and their structure-related analogues [3].

Antimicrobial activity
The antimicrobial activity of the synthesized thiazolopyridines was estimated with the agar diffusion method [29]. 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 for in vitro antibacterial activity. In vitro antifungal activity was determined by using Sabouraud Agar plates. 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. Aliquot part (20 μL) of 0.1 % tested thiazolopyridine derivatives (concentration 1000 μg/ml) in ethanol/dimethyl sulfoxide/ water (2:1:1) was placed into wells (diameter of 4.0±0.1 mm) in agar in Petri plates with test microorganisms. Antibacterial and antifungal activities were estimated by measuring the diameter of inhibition zone of microbial growth. The plates were incubated for 24 h at 37 °C for bacteria and for 24 h at 25 °C for fungi. The inhibition zone appeared after 24 h was measured in mm around the well in each plate. The digital images of culture growth on dishes were obtained and processed with a computer program UTHSCSA ImageTool 2.0 (UT Health San Antonio, © 1995-1996) for calculation of diameters of the growth inhibition zone. Each experiment was performed by three independent researchers. The results were expressed as the means ± S.D. The experiments were carried out on the microorganism strains, which were isolated from the ambulatory patients. The following isolated clinical strains of conditionally pathogenic bacteria were used: methicillin-sensitive Staphylococcus aureus (MSSA); methicillin-resistant Staphylococcus aureus (MRSA); methicillinresistant Staphylococcus haemolyticus (MRSH) (extended spectrum β-lactamase (ESβL) pro-ducing); Gram-negative bacteria Escherichia coli; yeasts Candida albicans. All clinical strains were multidrug resistant (MDR) [30] and Candida albicans were resistant to fluconazole and clotrimazole. All compounds were also tested against the reference strains of Staphylococcus aureus (ATCC 25923 (F-49)), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), Klebsiella pneumoniae (ATCC 700603) and Candida albicans (ATCC 885-653) from the culture museum. Testcultures were identified using chemical microtests "STAPHYtest 16" and "ENTEROtest 24" (Erba 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 (bioMérieux Corporate, France).
The sensitivity of strains to antibiotics was determined by disc-diffusion method and serial dilutions in agar. The MICs of the com- pounds were determined using the microdilution methods for antimicrobial susceptibility [31,32]. Microorganism suspensions were inoculated to the corresponding wells and incubated at 36 °C for 18 h for bacteria and at 25 °C for 24 h for fungi. The presence of the microorganism growth in the bouillon (bouillon turbidity) suggested that the 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 the minimum inhibitory concentration (MIC). The estimation of synergy with amoxicillin for synthesized compounds has been performed by comparison of amoxicillin MICs in the presence of compounds in subinhibitory concentrations [33].
The following bacterial strains with the resistance to β-lactam antibiotics were used: EsβL (extended spectrum β-lactamase)-producing Klebsiella pneumonie ATCC 700603; methicillin-resistant Staphylococcus haemolyticus (MRSH) with double mechanisms of β-lactam resistance included both atypical penicillinbinding protein PBP2* and β-lactamase activities. The production of the atypical penicillin-binding protein PBP2* was proved by the latex agglutination reaction (Slidex® MRSA Detection, bioMérieux Corporate, France). The results have been refined by variation statistics methods.

Antiproliferative activity
The

Results and Discussion
Antimicrobial activity of the synthesized thiazolopyridines in vitro was evaluated with the agar diffusion method.   (Tables 3-5). According to the preliminary results of interaction screening, the thiazolopyridine derivative with a styrene fragment in the molecule VI displays the promising synergistic activity with amoxicillin against ESβL + K. pneumonie and MRSH strains. Interestingly, in numerous literature reports, the styrene fragment combined with various heterocyclic systems exhibits a significant synergistic effect with beta-lactam antibiotics [34]. Especially it can be observed for the quinoline/quinaxoline-styrene hybrid molecules structurally related to thiazolopyridines [34,35].
The SAR analysis showed that the antibacterial effect of compounds I-VII did not depend on the substituents at C5 and C7 of thiazolopyridine core. However, the thia zolo [4,5-b] pyridine-5-carboxylic acid V with thienyl substituent was the most active and demonstrated a good effect against Candida albicans with MIC 12.5 µg/mL. Compound V possessed also a slight activity on renal cancer A498 cell line (GP = 73.76 %) as described previously [28].
The antimicrobial activity of thiophene-based derivatives has already been observed in the previous systematic studies, especially for condensed benzothiophene [36], thieno [2,3-d] pyrimidine [37] and thieno[3,2-c]pyrazole derivatives [38]. Additionally, the experimental study revealed that the presence of an amide fragment in thiazolopyridine core of the compound VII is also favorable for antimicrobial potency. The data concerning a critical impact of electron withdrawing groups in amide fragment are also presented in our previous paper about thiazolopyridine-5-carboxylic acid amides as possible anticancer agents [27]. On the other hand, the compounds with phenyl, 4-chlorophenyl, 4-methoxyphenyl, styryl and carboxylic substituents at C5 and C7 on thiazolo [4,5-b]pyridine core were insufficient to show enhanced activity and didn't correlate with other types of activity typical for these compounds [27,28] .
Next, we used the MTT assay to investigate cytotoxicity of the thiazolopyridine derivatives towards the pseudo-normal cell line (HaCaT human keratinocytes and HEK293 human em-  (Figure 3).

Conclusions
The various subtypes of thiazolopyridines I-VII were evaluated for in vitro antibacterial activity against Staphylococcus aureus, # in brackets -zones of partial inhibition of the bacterial growth (bacteriostatic effect).

Funding
The research was funded by the Ukrainian-Chinese program and Ministry of Education and Science of Ukraine, the project M/92-2019, and the National Research Foundation of Ukraine, the project 2020.02/0035.  Fig. 3. Cytotoxic action of thiazolopyridine derivatives I-VII towards HaCaT human keratinocytes and HEK293 human embryonic kidney cells. The effect was measured by the MTT assay after 72 h of cells exposure. Data are presented as the mean ± SD. * -P < 0.05; ** -P < 0.01; *** -P < 0.001 compared with control (non-treated) cells.