A hit expansion of 3-benzamidopyrazine-2-carboxamide: Toward inhibitors of prolyl-tRNA synthetase with antimycobacterial activity.

This study presents an exploration of the chemical space around derivatives of 3-benzamidopyrazine-2-carboxamides, previously identified as potent antimycobacterial compounds with predicted binding to mycobacterial prolyl-transfer RNA synthetase. New urea derivatives (Series-1) were generally inactive, probably due to their preference for cis-trans conformation (confirmed by density functional theory calculations and experimentally by nuclear overhauser effect spectroscopy NMR). Series-2 (3-benzamidopyrazine-2-carboxamides with disubstituted benzene ring) demonstrated that substituents larger than fluorine are not tolerated in the ortho position of the benzene ring. This series brought two new compounds (21: R = 2-F, 4-Cl and 22: R = 2-F, 4-Br) with in vitro activity against Mycobacterium tuberculosis H37Rv as well as multidrug-resistant clinical isolates, with minimum inhibitory concentration ranging from 6.25 to 25 µg/mL. The lactone-type derivatives 4H-pyrazino[2,3-d][1,3]oxazin-4-ones (Series-3) were inactive, but solvent stability studies of compound 29 indicated that they might be developed to usable lactone prodrugs of inhibitors of mycobacterial aspartate decarboxylase (PanD).

Design, synthesis and biological evaluation of substituted 3-amino-N-(thiazol-2-yl)pyrazine-2-carboxamides as inhibitors of mycobacterial methionine aminopeptidase 1.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug resistance. New compounds with new mechanisms of action are therefore needed. We present the design, synthesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target - mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines. Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv). Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted benzamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is discussed.

Inhibitors of aminoacyl-tRNA synthetases as antimycobacterial compounds: An up-to-date review.

Aminoacyl-tRNA synthetases (aaRSs) are crucial for the correct assembly of amino acids to cognate tRNA to maintain the fidelity of proteosynthesis. AaRSs have become a hot target in antimicrobial research. Three aaRS inhibitors are already in clinical practice; antibacterial mupirocin inhibits the synthetic site of isoleucyl-tRNA synthetase, antifungal tavaborole inhibits the editing site of leucyl-tRNA synthetase, and antiprotozoal halofuginone inhibits proline-tRNA synthetase. According to the World Health Organization, tuberculosis globally remains the leading cause of death from a single infectious agent. The rising incidence of multidrug-resistant tuberculosis is alarming and urges the search for new antimycobacterial compounds, preferably with yet unexploited mechanism of action. In this literature review, we have covered the up-to-date state in the field of inhibitors of mycobacterial aaRSs. The most studied aaRS in mycobacteria is LeuRS with at least four structural types of inhibitors, followed by TyrRS and AspRS. Inhibitors of MetRS, LysRS, and PheRS were addressed in a single significant study each. In many cases, the enzyme inhibition activity translated into micromolar or submicromolar inhibition of growth of mycobacteria. The most promising aaRS inhibitor as an antimycobacterial compound is GSK656 (compound 8), the only aaRS inhibitor in clinical trials (Phase IIa) for systemic use against tuberculosis. GSK656 is orally available and shares the oxaborole tRNA-trapping mechanism of action with antifungal tavaborole.

Towards Novel 3-Aminopyrazinamide-Based Prolyl-tRNA Synthetase Inhibitors: In Silico Modelling, Thermal Shift Assay and Structural Studies.

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.

5-Alkylamino-N-phenylpyrazine-2-carboxamides: Design, Preparation, and Antimycobacterial Evaluation.

According to the World Health Organization, tuberculosis is still in the top ten causes of death from a single infectious agent, killing more than 1.7 million people worldwide each year. The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is an imperative to develop new compounds with potential antimycobacterial activity. As a part of our continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain (propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c, MIC = 3.91 µg/mL or 13.02 µM, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78 µg/mL or 2.39 µM, SI > 20). In a complementary screening, we evaluated the in vitro activity against bacterial and fungal strains of clinical importance. We observed no antibacterial activity and sporadic antifungal activity against the Candida genus.

N-Pyrazinoyl Substituted Amino Acids as Potential Antimycobacterial Agents-The Synthesis and Biological Evaluation of Enantiomers.

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 ʟ-amino acids. Similarly, the activity seems tied to the more lipophilic compounds. The most active derivative contained phenylglycine moiety (PC-ᴅ/ʟ-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 ᴅ- and ʟ-amino acid derivatives of pyrazinamide as potential antimycobacterial compounds.

Derivatives of 3-Aminopyrazine-2-carboxamides: Synthesis, Antimicrobial Evaluation, and in Vitro Cytotoxicity.

We report the design, synthesis, and in vitro antimicrobial activity of a series of N-substituted 3-aminopyrazine-2-carboxamides with free amino groups in position 3 on the pyrazine ring. Based on various substituents on the carboxamidic moiety, the series is subdivided into benzyl, alkyl, and phenyl derivatives. The three-dimensional structures of the title compounds were predicted using energy minimization and low mode molecular dynamics under AMBER10:EHT forcefield. Compounds were evaluated for antimycobacterial, antibacterial, and antifungal activities in vitro. The most active compound against Mycobacterium tuberculosis H37Rv (Mtb) was 3-amino-N-(2,4-dimethoxyphenyl)pyrazine-2-carboxamide (17, MIC = 12.5 µg/mL, 46 µM). Antimycobacterial activity against Mtb and M. kansasii along with antibacterial activity increased among the alkyl derivatives with increasing the length of carbon side chain. Antibacterial activity was observed for phenyl and alkyl derivatives, but not for benzyl derivatives. Antifungal activity was observed in all structural subtypes, mainly against Trichophyton interdigitale and Candida albicans. The four most active compounds (compounds 10, 16, 17, 20) were evaluated for their in vitro cytotoxicity in HepG2 cancer cell line; only compound 20 was found to exert some level of cytotoxicity. Compounds belonging to the current series were compared to previously published, structurally related compounds in terms of antimicrobial activity to draw structure activity relationships conclusions.

Substituted N-(Pyrazin-2-yl)benzenesulfonamides; Synthesis, Anti-Infective Evaluation, Cytotoxicity, and In Silico Studies.

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.

Design, Synthesis and Evaluation of N-pyrazinylbenzamides as Potential Antimycobacterial Agents.

Three series of N-(pyrazin-2-yl)benzamides were designed as retro-amide analogues of previously published N-phenylpyrazine-2-carboxamides with in vitro antimycobacterial activity. The synthesized retro-amides were evaluated for in vitro growth inhibiting activity against Mycobacterium tuberculosis H37Rv (Mtb), three non-tuberculous mycobacterial strains (M. avium, M. kansasii, M. smegmatis) and selected bacterial and fungal strains of clinical importance. Regarding activity against Mtb, most N-pyrazinylbenzamides (retro-amides) possessed lower or no activity compared to the corresponding N-phenylpyrazine-2-carboxamides with the same substitution pattern. However, the active retro-amides tended to have lower HepG2 cytotoxicity and better selectivity. Derivatives with 5-chloro substitution on the pyrazine ring were generally more active compared to their 6-cloro positional isomers or non-chlorinated analogues. The best antimycobacterial activity against Mtb was found in N-(5-chloropyrazin-2-yl)benzamides with short alkyl (2h: R² = Me; 2i: R² = Et) in position 4 of the benzene ring (MIC = 6.25 and 3.13 µg/mL, respectively, with SI > 10). N-(5-Chloropyrazin-2-ylbenzamides with hydroxy substitution (2b: R² = 2-OH; 2d: R² = 4-OH) on the benzene ring or their acetylated synthetic precursors possessed the broadest spectrum of activity, being active in all three groups of mycobacterial, bacterial and fungal strains. The substantial differences in in silico calculated properties (hydrogen-bond pattern analysis, molecular electrostatic potential, HOMO and LUMO) can justify the differences in biological activities between N-pyrazinylbenzamides and N-phenylpyrazine-2-carboxamides.

Synthesis of Novel Pyrazinamide Derivatives Based on 3-Chloropyrazine-2-carboxamide and Their Antimicrobial Evaluation.

Aminodehalogenation of 3-chloropyrazine-2-carboxamide with variously substituted benzylamines yielded a series of fifteen 3-benzylaminopyrazine-2-carboxamides. Four compounds possessed in vitro whole cell activity against Mycobacterium tuberculosis H37Rv that was at least equivalent to that of the standard pyrazinamide. MIC values ranged from 6 to 42 µM. The best MIC (6 µM) was displayed by 3-[(4-methylbenzyl)amino]pyrazine-2-carboxamide (8) that also showed low cytotoxicity in the HepG2 cell line (IC50 ≥ 250 µM). Only moderate activity against Enterococcus faecalis and Staphylococcus aureus was observed. No activity was detected against any of tested fungal strains. Molecular docking with mycobacterial enoyl-ACP reductase (InhA) was performed to investigate the possible target of the prepared compounds. Active compounds shared common binding interactions of known InhAinhibitors. Antimycobacterial activity of the title compounds was compared to the previously published benzylamino-substituted pyrazines with differing substitution on the pyrazine core (carbonitrile moiety). The title series possessed comparable activity and lower cytotoxicity than molecules containing a carbonitrile group on the pyrazine ring.