We prepared a series of substituted N-(pyrazin-2-yl)benzenesulfonamides as an attempt to investigate the effect of different linkers connecting pyrazine to benzene cores on antimicrobial activity when compared to our previous compounds of amide or retro-amide linker type. Only two compounds, 4-amino-N-(pyrazin-2-yl)benzenesulfonamide (MIC = 6.25 μg/mL, 25 μM) and 4-amino-N-(6-chloropyrazin-2-yl)benzenesulfonamide (MIC = 6.25 μg/mL, 22 μM) exerted good antitubercular activity against M. tuberculosis H37Rv. However, they were excluded from the comparison as they—unlike the other compounds—possessed the pharmacophore for the inhibition of folate pathway, which was proven by docking studies. We performed target fishing, where we identified matrix metalloproteinase-8 as a promising target for our title compounds that is worth future exploration.

Antimicrobial drug resistance is currently one of the most critical health issues. Pathogens resistant to last-resort antibiotics are increasing, and very few effective antibacterial agents have been introduced in recent years. The promising drug candidates are often discontinued in the primary stages of the drug discovery pipeline due to their unspecific reactivity (PAINS), toxicity, insufficient stability, or low water solubility. In this work, we investigated a series of substituted N-oxazolyl- and N-thiazolylcarboxamides of various pyridinecarboxylic acids. Final compounds were tested against several microbial species. In general, oxazole-containing compounds showed high activity against mycobacteria, especially Mycobacterium tuberculosis (best MIC_H37Ra = 3.13 µg/mL), including the multidrug-resistant strains. Promising activities against various bacterial and fungal strains were also observed. None of the compounds was significantly cytotoxic against the HepG2 cell line. Experimental measurement of lipophilicity parameter log k’_w and water solubility (log S) confirmed significantly (typically two orders in logarithmic scale) increased hydrophilicity/water solubility of oxazole derivatives in comparison with their thiazole isosteres. Mycobacterial β-ketoacyl-acyl carrier protein synthase III (FabH) was suggested as a probable target by molecular docking and molecular dynamics simulations.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNA^Pro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC₅₀ values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MIC_Mtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MIC_Mtb = 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.