Pyrazine-based compounds are of great importance in medicinal chemistry. Due to their heteroaromatic nature, they uniquely combine properties of heteroatoms (polar interactions) with the properties of aromatic moieties (nonpolar interactions). This review summarizes results of a systematic analysis of RCSB PDB database focused on important binding interactions of pyrazine-based ligands cocrystallized in protein targets. The most frequent interaction of pyrazine was hydrogen bond to pyrazine nitrogen atom as an acceptor, followed by weak hydrogen bond with pyrazine hydrogen as donor. We also identified intramolecular hydrogen bonds within pyrazine ligands, π-interactions, coordination to metal ions, and few halogen bonds in chloropyrazines. In many cases the binding mode of the pyrazine fragment was complex, involving a combination of several interactions. We conclude that pyrazine as a molecular fragment should not be perceived as a simple aromatic isostere but rather as a readily interacting moiety of drug-like molecules with high potential for interactions to proteins.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, 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.

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.Molecules 2020, 25, 138 2 of 20 several compounds containing a pyrazine core directly connected to a sulfonamide moiety-like in our case-are already documented in the literature with a wide range of pharmacological applications [9]. Examples (Figure 2) include zibotentan, an endothelin receptor antagonist with antitumor activity for prostate cancer [10]; halogenated N-(pyrazinyl)benzenesulfonamides, which are clathrin-coated pit (CCP) chemokine receptor antagonists used in treating chemokine mediated diseases (such as asthma) [11]; and sulfamethoxypyrazine (sulfalene), which is an antibacterial sulfonamide agent [12]. As for antitubercular activity of sulfonamides, a fixed combination of sulfamethoxazole [4-amino-N-(5-methylisoxazol-3-yl)benzenesulfonamide]-trimethoprim [5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine], also known as co-trimoxazole, has in vitro and in vivo antimycobacterial activity against drug-resistant strains of Mycobacterium tuberculosis (Mtb), and it is used for such cases [13,14]. Sulfamethoxazole is a known inhibitor of bacterial dihydropteroate synthase (DHPS), while trimethoprim is an inhibitor of dihydrofolate reductase (DHFR) [15,16]. Molecules 2020, 25, x 2 of 20 Figure 1. Design rationale: general structure of (a) pyrazinecarboxamides = amides; (b) N-pyrazinylbenzamides = retro-amides; and (c) title compounds = sulfonamides.

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Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), each year causing millions of deaths. In this article, we present the synthesis and biological evaluations of new potential antimycobacterial compounds containing a fragment of the first-line antitubercular drug pyrazinamide (PZA), coupled with methyl or ethyl esters of selected amino acids. The antimicrobial activity was evaluated on a variety of (myco)bacterial strains, including Mtb H37Ra, M. smegmatis, M. aurum, Staphylococcus aureus, Pseudomonas aeruginosa, and fungal strains, including Candida albicans and Aspergillus flavus. Emphasis was placed on the comparison of enantiomer activities. None of the synthesized compounds showed any significant activity against fungal strains, and their antibacterial activities were also low, the best minimum inhibitory concentration (MIC) value was 31.25 µM. However, several compounds presented high activity against Mtb. Overall, higher activity was seen in derivatives containing l-amino acids. Similarly, the activity seems tied to the more lipophilic compounds. The most active derivative contained phenylglycine moiety (PC-d/l-Pgl-Me, MIC < 1.95 µg/mL). All active compounds possessed low cytotoxicity and good selectivity towards Mtb. To the best of our knowledge, this is the first study comparing the activities of the d- and l-amino acid derivatives of pyrazinamide as potential antimycobacterial compounds.

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 MICH37Ra = 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.

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, MICMtb = 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 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.

The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins are comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which is followed by uptake into the intoxicated cell and activation of the host’s adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Ac〈3Gal or Neu5Gc〈3Gal sequence, e.g. the gangliosides GM3, GD1a and Neu5Ac〈3-/Neu5Gc〈3-neolactotetraosylceramide and Neu5Ac〈3-/Neu5Gc〈3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.