Synthesis and Biological Activity Evaluation of Novel 5-Methyl-7-phenyl-3 H -thiazolo[4,5- b ]pyridin-2-ones

: A series of 5-methyl-7-phenyl-3 H -thiazolo[4,5- b ]pyridin-2-ones has been designed, synthesized, and characterized by spectral data. Target compounds were screened for their antimicrobial activity against some pathogenic bacteria and fungi, and most of them showed moderate activity, especially compound 3g , which displayed the potent inhibitory effect against Pseudomonas aeruginosa and Escherichia coli with MIC value of 0.21 µ M. The active thiazolopyridine derivatives 3c , 3f , and 3g were screened for their cytotoxicity effects on HaCat, Balb/c 3T3 cells using MTT assay, which revealed promising results. In silico assessment for compounds 3c , 3f , and 3g also revealed suitable drug-like parameters and ADME properties. The binding interactions of the most active compound 3g were performed through molecular docking against MurD and DNA gyrase, with binding energies and an inhibitory constant compared to the reference drug ciproﬂoxacin. The tested thiazolo[4,5- b ]pyridines constitute an exciting background for the further development of new synthetic antimicrobial agents.

Given our interest in searching and studying new low molecular weight compounds among fused pyridines, herein, we report the synthesis, structure elucidation, and in vitro antimicrobial, cytotoxic activity evaluation and docking studies of some novel 5-methyl-7phenyl-3H-thiazolo [4,5-b]pyridin-2-ones. This manuscript is intended to draw attention to the chemistry and pharmacology of the thiazolopyridines and its further examination as potent drug candidates.

General Information
All materials were purchased from commercial sources and used without purification. Melting points were measured in open capillary tubes and were uncorrected. The elemental analyses were performed using the Thermo Scientific FlashSmart Elemental Analyzer. 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 . Chemical shifts (δ) are given in ppm units relative to tetramethylsilane as reference (0.00). LC-MS was performed using a system with an Agilent 1100 Series HPLC equipped with diode-array detector and Agilent LC\MSD SL mass-selective detector using chemical ionization at atmospheric pressure (APCI). The reaction mixture was monitored by thin-layer chromatography (TLC) using Silufol-254 plates. Starting 4-amino-5H-thiazol-2-one and benzylideneacetones were prepared according to reported methods [36,37].

Synthesis and Characterization of Compounds
General Procedure for the Synthesis of 5-Methyl-7-Phenyl-3H-Thiazolo[4,5-b]Pyridin-2-Ones 3a-g A mixture of benzylideneacetone (1.0 mmol) and 4-amino-5H-thiazol-2-one (1.0 mmol) was refluxed for 3 h in glacial acetic acid (10 mL) (monitored by TLC). After completion, the reaction mixture was cooled and left overnight at room temperature. The solid precipitates were filtered off, washed with methanol (5-10 mL), and recrystallized from a mixture of DMF: acetic acid (1:2) or glacial acetic acid. The resulting powders were filtered and washed with acetic acid, water, methanol, and diethyl ether, successively. The final products were dried at room temperature until constant weight.

Antimicrobial Activity
The synthesized compounds were tested in vitro for their antibacterial and antifungal activities using the agar diffusion and serial dilutions resazurin-based microdilution assays (RBMA) [38,39]. For this purpose, 100 µL (1 mg/mL) of the tested compound was placed in an agar well with a diameter of 5.5 mm. The diameter of the growth retardation was measured using a micrometer with an error of 0.1 mm. Dimethyl sulfoxide (DMSO), vancomycin (discs), ciprofloxacin (discs), and clotrimazole (discs) were used as a control. Pure DMSO was used as a solvent due to the poor solubility of the test compounds in dilute DMSO. In addition, Mueller-Hinton agar and Saburo agar (for fungi) were used, and Petri dishes were incubated at 37 • C for 24 h for bacteria and at 25 • C for 24-48 h for fungi. The RBMA method involved the introduction of a 96-well plate 50 µL of nutrient medium (Mueller-Hinton broth or glucose broth), 50 mL of a suspension of the microorganism (McFarland 2.0), and 100 µL of tested compounds, with the addition of 15 µL of 0.02% resazurin in each well. Sixteen reference and clinical microbial and fungal strains were used (Table 1), previously identified by the MALDI TOF system (Bruker, Bremen, Germany), and 16S rRNA gene sequences. All clinical strains were multidrug-resistant or extensively drug-resistant with different antibiotic resistance patterns. Clinical strains were isolated from a patient with healthcare-associated infections from regional hospitals. All testing was repeated in triplicate.

MTT Assay
The cytotoxic effect of new thiazolopyridine derivatives on pseudonormal cell lines was evaluated using MTT assay. The 4 × 10 4 cells/mL human epidermal keratinocytes (HaCat cell line) or normal murine fibroblasts (BALB/c 3T3 cell line) were seeded in 96-well plates in 100 µL DMEM medium, supplemented with 10% FBS and incubated for 24 h to attach at 37C in CO 2 incubator. The HaCaT human skin keratinocyte cell line and normal murine fibroblasts (BALB/c 3T3) were kindly gifted by the R.E. Kavetsky Institute of experimental pathology, oncology and radiobiology NAS of Ukraine (Kyiv, Ukraine). The next day, they were treated with studied compounds at concentrations 10, 25, 50, 150, and 250 µM. After 72 h compound exposure, 20 µL MTT reagent was added, incubated for 4 h, and measured according to the manufacturer's recommendations (Merck KGaA, Burlington, MA, USA).

Molecular Docking
Molecular docking is the most widespread method for the calculation and prediction of protein-ligand interactions. Before docking procedures, we analyzed available literature data for choosing the most logical and appropriate biological targets. There are literally reported 4-thiazolidinones derivatives, which demonstrate antibacterial activities through MurD ligase inhibition [40]. Moreover, the well-known DNA gyrase inhibitors fluoroquinolones are based on the core, which are quite similar to 3H-thiazolo [4,5-b]pyridin-2-one. Additionally, structurally related 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-based derivatives were identified as DNA gyrase inhibitors [41]. That is why MurD ligase (PBD code 2Y67) [42] and DNA gyrase (PBD code 2XCT) [43] were chosen for docking in silico simulations with the aim of receiving the necessary data for a suggestion about the possible mechanism of antibacterial activity for our lead compound 3g.
The X-ray crystal structures of target proteins were downloaded from the Protein Data Bank (RCSB PDB: Homepage. Available online: https://www.rcsb.org/ (accessed on 20 August 2021)). The AutoDock Tools were used for preparing the enzymes for docking, which includes removing all bound ligands and water molecules, adding polar hydrogen atoms, and defining all rotatable bonds [44]. Moreover, Kollman charges were computed and spread over all atoms in residues. The chemical structure of 3g was made using Biovia Draw and energy minimization was carried out by Hyperchem 7.5 using an AM1 quantum technique until the RMS gradient was less than 0.01 kcal /(mol Å). The threedimensional grid boxes were created with the size 50 × 50 × 50 xyz points, the spacing between grid points was 0.375 Å, and the grid maps representing the intact ligand in the actual docking target site were made by Auto Grid Tool. For more accurate results, we changed Lamarckian Genetic Algorithm (LGA) parameters from the default setting to enhanced, including 100 runs, 300 populations, and 2,50,0000 energy evaluations, a mutation rate of 0.02, and a crossover rate of 0.80 [45]. The interactions were estimated in terms of binding energy (kcal/mol) and inhibition constant (µM) along with the number of hydrogen bonds formed with the surrounding amino acid residues. The validations of selected docking parameters were performed by redocking of the initial ligands from the used enzyme structures. The accuracies of the redocking experiments were assessed by calculating the root-mean-square deviations (RMSD) and cutting off the results with RMSD > 2. Visualization and interpretation of obtained data were performed by Discovery Studio Visualizer of Dassault Systemes Biovia ® (Vélizy-Villacoublay, Yvelines, France).

Synthesis
In this article, we performed the condensation reaction of 4-amino-5H-thiazol-2-one with benzylideneacetone, leading to 5-methyl-7-phenyl-3H-thiazolo[4,5-b]pyridin-2-ones using conventional thermal heating conditions (Scheme 1). The starting benzylideneacetones 2a-g were synthesized according to the known literature procedures by reaction of aromatic aldehydes and acetone in an aqueous solution of potassium hydroxide and were subsequently used directly without additional purification. Analytical and spectral data ( 1 H and 13 C NMR) confirmed the structure of the synthesized compounds (copies of the corresponding spectra are presented in the Supplementary Material). The 1 H NMR spectra of the fused pyridine analogs 3a-g exhibit singlets for the CH group of the pyridine ring at~7 ppm and broad peaks due to the NH-functionalities around 12 ppm. In the 13 C NMR spectra of the synthesized compounds, the signals observed at δ 160.6-162.0 are assigned to the carbonyl group (C=O). Carbon signals of the methyl group were observed at δ 23.7-23.8 ppm.
using conventional thermal heating conditions (Scheme 1). The starting benzylideneacetones 2a-g were synthesized according to the known literature procedures by reaction of aromatic aldehydes and acetone in an aqueous solution of potassium hydroxide and were subsequently used directly without additional purification. Analytical and spectral data ( 1 H and 13 C NMR) confirmed the structure of the synthesized compounds (copies of the corresponding spectra are presented in the Supplementary Material). The 1 H NMR spectra of the fused pyridine analogs 3a-g exhibit singlets for the CH group of the pyridine ring at ~ 7 ppm and broad peaks due to the NH-functionalities around 12 ppm. In the 13 C NMR spectra of the synthesized compounds, the signals observed at δ 160.6-162.0 are assigned to the carbonyl group (C=O). Carbon signals of the methyl group were observed at δ 23.7-23.8 ppm.

Biological Activity
Antimicrobial resistance (AMR) is a global healthcare problem, and there is a close interrelationship between the progressing of AMR in the world and the increase in mortality from infectious diseases, mainly through healthcare-associated infections (HAIs) [46]. According to a WHO prognosis, mortality from AMR pathogens in the world by 2050 will take first place in the overall structure of causes of death and will exceed the mortality of cancer. According to the WHO recommendation, discovering new synthetic molecules with antimicrobial action is one of the leading short-term approaches to control AMR pathogens. 4-Thiazolidinone-based derivatives are one of the promising classes of compounds with a polypharmacological profile, which contain several reaction centres in the basic heterocycle and are relatively easily adapted for chemical modification. That is why this class of chemical compounds is useful in the search for a "drug-like molecule" with antimicrobial action, effective against the variable mechanisms of AMR, including HAIs pathogens.
Thus, according to the screening of the antimicrobial activity of 5-methyl-7-phenyl-3H-thiazolo [4,5-b]pyridin-2-ones, the best activity was shown by the compound 3g, which showed antifungal activity against fungi of the genus Candida, selective action against Gram-positive microorganism Micrococcus luteus, and selective action on few Gram-negative microorganisms (Table 1 and Figure 3). In Figure 3, it is shown that compounds 3a (light blue colour), 3c (grey colour), 3f (green colour), and 3g (dark clue colour) have a bigger growth inhibition zone than solvent control (brown colour).

Biological Activity
Antimicrobial resistance (AMR) is a global healthcare problem, and there is a close interrelationship between the progressing of AMR in the world and the increase in mortality from infectious diseases, mainly through healthcare-associated infections (HAIs) [46]. According to a WHO prognosis, mortality from AMR pathogens in the world by 2050 will take first place in the overall structure of causes of death and will exceed the mortality of cancer. According to the WHO recommendation, discovering new synthetic molecules with antimicrobial action is one of the leading short-term approaches to control AMR pathogens. 4-Thiazolidinone-based derivatives are one of the promising classes of compounds with a polypharmacological profile, which contain several reaction centres in the basic heterocycle and are relatively easily adapted for chemical modification. That is why this class of chemical compounds is useful in the search for a "drug-like molecule" with antimicrobial action, effective against the variable mechanisms of AMR, including HAIs pathogens.
Thus, according to the screening of the antimicrobial activity of 5-methyl-7-phenyl-3H-thiazolo[4,5-b]pyridin-2-ones, the best activity was shown by the compound 3g, which showed antifungal activity against fungi of the genus Candida, selective action against Grampositive microorganism Micrococcus luteus, and selective action on few Gram-negative microorganisms (Table 1 and Figure 3). In Figure 3, it is shown that compounds 3a (light blue colour), 3c (grey colour), 3f (green colour), and 3g (dark clue colour) have a bigger growth inhibition zone than solvent control (brown colour).
Compounds 3f and 3d exhibited the highest antifungal activity, both for fungi of the genus Candida and fungus of the genus Saccharomyces. These compounds have also been active against some Gram-negative microorganisms, particularly against clinical strains of Citrobacter freundii and Achromobacter xylosoxidans.
been active against some Gram-negative microorganisms, particularly against clinical strains of Citrobacter freundii and Achromobacter xylosoxidans.
Four compounds (3a, 3c, 3f, 3g) showed significant antimicrobial activity against both clinical and reference Gram-negative microorganisms. The tested compounds did not show significant antimicrobial activity against Gram-positive microorganisms, except for the compound 3g, active against Micrococcus luteus. Four lead-compounds (Table 2) were tested for the minimum inhibitory concentration (MIC). The best activity showed compound 3g against Gram-negative microorganisms (0.21 μM) and reference strain Candida albicans (0.83 μM). To know how normal human cells react to synthesized lead-compounds 3c, 3g, and 3f, we tested their toxicity on two human pseudonormal cell lines: human epidermal keratinocytes (HaCat) and normal murine fibroblasts (BALB/c 3T3) (Figure 4). In general, the HaCat cell line is more sensitive than the BALB/c 3T3 cell line to the action of our discovered compounds, but any reaches its IC50 value after 72h of compound exposure. The compound 3f shows the lowest toxicity effect on the pseudonormal cells. The tested compounds did not show significant antimicrobial activity against Grampositive microorganisms, except for the compound 3g, active against Micrococcus luteus. Four lead-compounds (Table 2) were tested for the minimum inhibitory concentration (MIC). The best activity showed compound 3g against Gram-negative microorganisms (0.21 µM) and reference strain Candida albicans (0.83 µM). To know how normal human cells react to synthesized lead-compounds 3c, 3g, and 3f, we tested their toxicity on two human pseudonormal cell lines: human epidermal keratinocytes (HaCat) and normal murine fibroblasts (BALB/c 3T3) (Figure 4). In general, the HaCat cell line is more sensitive than the BALB/c 3T3 cell line to the action of our discovered compounds, but any reaches its IC 50 value after 72h of compound exposure. The compound 3f shows the lowest toxicity effect on the pseudonormal cells.
According to obtained results, the tested compound 3g possesses a low ability to damage pseudonormal cells, had high antifungal activity, and revealed a promising area for further research.  According to obtained results, the tested compound 3g possesses a low ability to damage pseudonormal cells, had high antifungal activity, and revealed a promising area for further research.

Molecular and Pharmacokinetic Properties
The physical properties and the ADMET parameters of active compounds 3c, 3f, and 3g were calculated using the SwisAdme online server of the Swiss Institute of Bioinformatics [47]. As suggested by these data, all compounds are suited to Lipinski's rule of five and may exhibit high gastrointestinal absorption and cross the blood-brain barrier (except 3g). The predicted lipophilicity of synthesized compounds given by log Po/w revealed good permeability and oral absorption through the cell membrane. Our tested compounds did not become p-glycoprotein substrates, suggesting that they could not be

Molecular and Pharmacokinetic Properties
The physical properties and the ADMET parameters of active compounds 3c, 3f, and 3g were calculated using the SwisAdme online server of the Swiss Institute of Bioinformatics [47]. As suggested by these data, all compounds are suited to Lipinski's rule of five and may exhibit high gastrointestinal absorption and cross the blood-brain barrier (except 3g). The predicted lipophilicity of synthesized compounds given by log Po/w revealed good permeability and oral absorption through the cell membrane. Our tested compounds did not become p-glycoprotein substrates, suggesting that they could not be associated with the excretion of the drug. The negative skin permeability of the tested compounds may exhibit low skin permeation across the cell membrane. All the predictive data allow for the considering of compounds 3f, 3g, and 3c as prospective drug-like candidates in further in-depth studies (Table 3).

Molecular Docking Studies
Docking studies have revealed that the lead-compound 3g demonstrated good binding energy towards selected protein targets compared to the reference inhibitor N21 and ciprofloxacin (Table 4). Molecule bonds to the active site of the protein by three hydrogen bonds with the LYS319 (1.92 Å), SER415 (1.87 Å), and ARG425 (3.09 Å). Summary binding energy increases owing to additional Pi-Pi T-shaped, Alkyl, and Pi-Alkyl hydrophobic interaction with the number of lipophilic amino acids ALA414, LEU416, PHE422, ILE 139. Additionally, LYS115, GLU157, and ASN138 form weak carbon-hydrogen bonds with two methoxy groups of 3g. However, the length of the molecule is quite small, and there is an absence of interaction with THR36, which is quite necessary for efficient MurD inhibition ( Figure 5).
The docking results for compound 3g suggest that these compounds form key interactions with important residues at the binding site DNA gyrase ( Figure 6). The compound makes a strong connection inside the active site of DNA gyrase owing to three hydrogen bonds with the SER1084 (1.81 Å), ASP437 (2.01Å), and GLY459 (2.15 Å). Additionally, the position of the molecule is stabilized by Pi-Pi-staked interaction between phenyl, 3H-thiazolo [4,5-b]pyridine cores and nucleotides DG9 and DT8. Moreover, the 3-methoxy group and the thiazole motif form Pi-Alkyl connections with ARG458 and DG9. Ciprofloxacin also forms a hydrogen bond with SER1084 and Pi-alkyl interaction with ARG458, and interactions with the amino acids, as mentioned above, are crucial for the expression of its antibacterial activity. Binding energies and inhibitory constant are equal to ciprofloxacin results, which suggest the good antibacterial potential of compound 3g. The docking results for compound 3g suggest that these compounds form key interactions with important residues at the binding site DNA gyrase ( Figure 6). The compound makes a strong connection inside the active site of DNA gyrase owing to three hydrogen bonds with the SER1084 (1.81 Å), ASP437 (2.01Å), and GLY459 (2.15 Å). Additionally, the position of the molecule is stabilized by Pi-Pi-staked interaction between phenyl, 3H-thiazolo [4,5-b]pyridine cores and nucleotides DG9 and DT8. Moreover, the 3-methoxy group and the thiazole motif form Pi-Alkyl connections with ARG458 and DG9. Ciprofloxacin also forms a hydrogen bond with SER1084 and Pi-alkyl interaction with ARG458, and interactions with the amino acids, as mentioned above, are crucial for the expression of its antibacterial activity. Binding energies and inhibitory constant are equal to ciprofloxacin results, which suggest the good antibacterial potential of compound 3g.   The docking results for compound 3g suggest that these compounds form key interactions with important residues at the binding site DNA gyrase ( Figure 6). The compound makes a strong connection inside the active site of DNA gyrase owing to three hydrogen bonds with the SER1084 (1.81 Å), ASP437 (2.01Å), and GLY459 (2.15 Å). Additionally, the position of the molecule is stabilized by Pi-Pi-staked interaction between phenyl, 3H-thiazolo [4,5-b]pyridine cores and nucleotides DG9 and DT8. Moreover, the 3-methoxy group and the thiazole motif form Pi-Alkyl connections with ARG458 and DG9. Ciprofloxacin also forms a hydrogen bond with SER1084 and Pi-alkyl interaction with ARG458, and interactions with the amino acids, as mentioned above, are crucial for the expression of its antibacterial activity. Binding energies and inhibitory constant are equal to ciprofloxacin results, which suggest the good antibacterial potential of compound 3g.

Conclusions
Novel 5-methyl-7-phenyl-3H-thiazolo [4,5-b]pyridin-2-ones have been synthesized and characterized by spectral and elementary analyses. The synthesized compounds were assessed for their antimicrobial properties against clinical and reference strains and cytotoxicity capacities against HaCat and BALB/c 3T3 cell lines. The antimicrobial screening led to identifying the active compound 3g with the highest activity against Pseudomonas aeruginosa and Escherichia coli (MIC = 0.21 µM). Molecular and pharmacokinetic parameters of the promising active compounds showed satisfactory bioavailability and drug-likeness properties. Computational docking studies of compound 3g displaying the potent inhibitory effect suggest their good fitting in the active sites of MurD and DNA gyrase via different types of interactions.