In this work, the SERS behaviour and electronic properties of N-(thiazol-2-yl)-3-(2-trifluoromethyl)benzamido)pyrazine-2-carboxamide (TFB) were investigated using experimental vibrational spectroscopy supported by DFT calculations. The optimized structure, IR, Raman, and SERS spectra were analyzed to understand the molecular vibrations, adsorption behaviour, and charge-transfer interactions of TFB on silver nanoparticles. Two possible adsorption configurations of TFB with an Ag6 cluster were modelled to identify the most favourable binding orientation. The calculated adsorption energies and thermodynamic parameters confirmed that TFB interacts spontaneously with the silver cluster, with the configuration involving the carbonyl, phenyl, and thiazole regions showing slightly stronger stabilization. Frontier molecular orbital analysis revealed a remarkable decrease in the HOMO-LUMO energy gap after adsorption, indicating enhanced charge-transfer ability and chemical reactivity. Molecular electrostatic potential, ELF, LOL, NCI, and RDG analyses further confirmed that the carbonyl oxygen, thiazole sulfur, and heterocyclic nitrogen atoms are the main active sites responsible for interaction with the silver surface. The concentration-dependent SERS spectra showed clear enhancement and small frequency shifts in carbonyl, C=N, C-N, NH, and ring vibrational modes, supporting adsorption through heteroatom-assisted coordination and metal-molecule charge transfer. The HOMO-LUMO energy gap was reduced from 3.84 to 1.37 (SE1) and 1.12 eV (SE2) due to the adsorption of Ag6 clusters, which also introduced new electronic states close to the Fermi level. Strong charge-transfer interactions between the ligand and Ag6 cluster were encouraged by these modifications, which caused noticeable bathochromic shifts in the absorption maxima from 366 nm to 1324 and 1873 nm. The adsorption structures showed much greater Raman activity than the isolated TFB molecule, and the improved electronic delocalization and metal–ligand orbital hybridization lead to a large SERS enhancement. The combined experimental and theoretical analyses offer a detailed understanding of the adsorption mechanism and electronic interactions of TFB on silver nanostructures. The observed enhancement in spectroscopic response highlights the crucial role of molecule–metal coupling and charge-transfer processes, demonstrating the suitability of TFB as a model system for SERS investigations. Lastly, adsorption energy is also calculated using MD simulations on Ag(111) slab. These results provide valuable insights for the development of sensitive SERS platforms aimed at the detection of biologically active heterocyclic carboxamide compounds.

In this work, the structural, vibrational, electronic, and adsorption properties of N-(5-chloropyrazin-2-yl)-4-ethylbenzamide (CEB) were investigated through a combined experimental and theoretical approach. FT-IR, Raman, and concentration-dependent SERS spectra were analyzed with the support of density functional theory calculations. The optimized geometry, vibrational assignments, frontier molecular orbitals, molecular electrostatic potential, density of states, and UV-Vis transitions were examined to understand the intrinsic electronic nature of CEB and its interaction with silver clusters. Two adsorption models, involving Ag6 interaction near the carbonyl and NH sites, were explored. The calculated adsorption and thermodynamic parameters revealed that both complexes are stable, with the NH-bound configuration showing stronger interaction and greater spontaneity. The reduced HOMO-LUMO energy gaps of the adsorbed systems indicated enhanced charge-transfer behavior and increased chemical reactivity relative to the isolated molecule. Concentration-dependent SERS analysis showed that the 10-3 M system exhibits the strongest enhancement, with clear shifts and intensity modulation in ring and functional-group vibrations due to the molecule-surface interactions. Non-covalent interaction and RDG analyses confirmed that the stabilization of the complexes arises mainly from van der Waals interactions together with localized attractive contacts. MD simulations in water, methanol, and DMSO further demonstrated solvent-dependent stability, with DMSO providing the most compact and stable environment. Overall, the study provides a detailed molecular-level understanding of the adsorption behavior, spectral enhancement, and reactivity of CEB on silver surfaces, highlighting its relevance for SERS-based sensing and spectroscopic characterization.

Tuberculosis (TB) remains a significant global health challenge due to the rapid emergence of drug resistance. Despite substantial progress in anti-TB drug development, effective treatment options are limited. In this study, we report the synthesis and biological evaluation of pyrazinamide (PZA) derivatives with 5-alkyl and 5-alkanamido modifications, designed to enhance antimycobacterial activity by increasing lipophilicity and improving penetration of the lipid-rich mycobacterial cell wall. A positive correlation between the length of the 5-alkyl chain and antimycobacterial activity was observed, with maximal potency achieved with the heptyl substituent (4: 5-heptylpyrazine-2-carboxamide, MIC_M. tuberculosis H37Rv = 3.13 μg/mL). In series C with phenyl substitution on the C-2 carboxamide, different simple substituents were tolerated on the benzene ring (both electron-donating and electron-withdrawing, both lipophilic and hydrophilic), and the length of the alkyl chain was the main determinant of the antimycobacterial activity. Compound 23 (5-hexyl-N-(3-trifluoromethylphenyl)-pyrazine-2-carboxamide) exerted MIC = 3.13 μg/mL and selectivity index (SI, compared to HepG2 cells) >25. Notably, the tested compounds exhibited significant activity against multidrug-resistant (MDR) Mycobacterium tuberculosis strains while maintaining favorable selectivity profiles and low cytotoxicity. In contrast, 5-alkanamido derivatives (series B and D) were devoid of antimycobacterial activity. Mechanistic investigations revealed that unlike PZA, the 5-alkyl pyrazinamide derivatives are not hydrolyzed by mycobacterial pyrazinamidase (PncA), indicating a distinct mode of action. While molecular modeling initially suggested enoyl-ACP reductase (InhA) as a potential target of series C, subsequent experimental validation disproved this hypothesis; thus, the precise mechanism of action remains to be elucidated.

This study investigates the vibrational, electronic, and adsorption properties of N-(5-Chloropyrazin-2-yl)-2-hydroxybenzamide (CHB) using surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT). Concentration-dependent SERS spectra (10-3-10-6 M) reveal strong interactions between CHB and silver colloids. DFT calculations at the B3LYP level, including an Ag6 cluster model, support experimental findings, showing good agreement in vibrational features and adsorption-induced spectral shifts. The carbonyl (C=O) and amine (N-H) groups are identified as primary binding sites, contributing to charge transfer and enhancement mechanisms. Electronic analyses (HOMO-LUMO, DOS, and UV-Vis) indicate reduced energy gaps and increased delocalization upon adsorption. MEP, ELF, LOL, and RDG analyses highlight stronger interaction at the carbonyl site, while thermodynamic results suggest energetically favorable NH adsorption. Overall, adsorption is exothermic and feasible, providing insights into molecule-metal interactions and aiding the design of efficient SERS-based sensing systems.

A series of pyrazinamide-derived 1,2,3-triazoles featuring systematic chlorination of the pyrazine ring and diverse aryl substituents was synthesized and evaluated for antimycobacterial activity. Biological activity screening revealed broad-spectrum antimycobacterial activity and good selectivity toward mycobacteria over other pathogens, with 11 of the prepared compounds showing activity against Mycobacterium tuberculosis (Mtb) H37Ra and/or Mtb H37Rv (MIC ≤62.5 μg/mL). Structure-activity relationship analysis showed that 5-Cl substitution on the pyrazine ring was associated with improved antimycobacterial activity, with the best MIC values observed for compound 7 against Mtb H37Ra (MIC = 1.98 μg/mL) and compound 37 against Mtb H37Rv (MIC = 1.56 μg/mL). The tested compounds retained activity against drug-resistant Mtb isolates and partially against naturally resistant Mycobacterium abscessus, while showing low in vitro cytotoxicity in the HepG2 cell line and favorable selectivity indices. During advanced cytotoxicity testing, both compounds 7 and 37 displayed substantially lower hemolytic activity than bedaquiline, indicating a favorable erythrocyte safety profile within the tested concentration ranges. In vivo toxicity testing on Galleria mellonella showed low acute toxicity for both compounds. Mechanistic studies on compounds 7, 27, 31, and 37 revealed reduced biosynthesis of fatty acids and derived lipids, a phenotype consistent with interference with the Fatty Acid Synthase I (FAS I) system.

The Takeda G protein-coupled receptor 5 (TGR5), also known as GPBAR1 (G protein-coupled bile acid receptor), is a membrane-type bile acid receptor that regulates blood glucose levels and energy expenditure. These essential functions make TGR5 a promising target for the treatment of type 2 diabetes and metabolic disorders. Currently, most research on developing TGR5 agonists focuses on modifying the structure of bile acids, which are the endogenous ligands of TGR5. However, TGR5 agonists with nonsteroidal structures have not been widely explored. This study aimed at discovering new TGR5 agonists using bile acid derivatives as a basis for a computational approach. We applied a combination of pharmacophore-based, molecular docking, and molecular dynamic (MD) simulation to identify potential compounds as new TGR5 agonists. Through pharmacophore screening and molecular docking, we identified 41 candidate compounds. From these, five candidates were selected based on criteria including pharmacophore features, a docking score of less than 9.2 kcal/mol, and similarity in essential interaction patterns with a reference ligand. Biological assays of the five hits confirmed that Hit-3 activates TGR5 similarly to the bile acid control. This was supported by MD simulation results, which indicated that a hydrogen bond interaction with Tyr240 is involved in TGR5 activation. Hit-3 (CSC089939231) represents a new nonsteroidal lead that can be further optimized to design potent TGR5 agonists.

Tuberculosis (TB) is recognized as the second leading cause of death globally from a single infectious agent, following SARS-CoV-2 pneumonia. Aminoacyl-tRNA synthetases (aaRS) are essential enzymes responsible for attaching amino acids to their cognate tRNAs. These enzymes represent a promising set of targets for selective drug design due to the divergence between prokaryotic and eukaryotic aaRS. Recently, a new class of 3-aminopyrazine- 2-carboxamide derivatives has been identified as potent inhibitors of prolyl-tRNA synthetase (ProRS) from Mycobacterium tuberculosis (Mtb). These compounds exhibit significant antimycobacterial activity against Multi Drug Resistant strains of Mtb and demonstrate cytotoxicity against HepG2 human hepatocellular carcinoma cells. In this study, we present the crystal structures of MtbProRS in complex with five distinct 3-aminopyrazine-2-carboxamide derivatives. Structural analysis reveals that these inhibitors compete with ATP for the binding site of MtbProRS. Importantly, a critical hydrogen bond between the Glu144 of MtbProRS and the amino group of the carboxamide is identified, providing insights into the selective inhibition of MtbProRS over human ProRS.

Quinazolinone derivatives have emerged as promising scaffolds in antimicrobial drug discovery. This work focuses on the design, synthesis, and evaluation of novel quinazolinone-based compounds and predicts their potential to interact with mycobacterial penicillin-binding proteins (PBPs). Relying on established structure-activity relationships of antibacterial quinazolinones, a total of 53 compounds belonging to three different structural types are synthesized and biologically evaluated for antimycobacterial, antibacterial, and antifungal activities. Biological evaluations reveal selective efficacy against Mycobacterium tuberculosis with minimum inhibitory concentrations (MICs) as low as 6.25 mu g mL-1 for some derivatives, and this activity is preserved against drug-resistant strains. Molecular docking studies suggest a potential allosteric binding site in mycobacterial PBP 1A (PonA1, UniProt ID: P71707), and subsequential molecular dynamics confirm stable binding with key stabilizing interaction between the carbonyl oxygen of the quinazolinone and either ARG399 or ASP474. These findings suggest quinazolinone derivatives as viable candidates for further development as non-beta-lactam PBP inhibitors, addressing the urgent need for new antitubercular therapies.

Infectious diseases, including bacterial, fungal, and viral, have once again gained urgency in the drug development pipeline after the recent COVID-19 pandemic. Tuberculosis (TB) is an old infectious disease for which eradication has not yet been successful. Novel agents are required to have potential activity against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB. In this study, we present a series of 2-phenyl-N-(pyridin-2-yl)acetamides in an attempt to investigate their possible antimycobacterial activity, cytotoxicity on the HepG2 liver cancer cell line, and-as complementary testing-their antibacterial and antifungal properties against a panel of clinically important pathogens. This screening resulted in one compound with promising antimycobacterial activity-compound 12, MICMtb H37Ra = 15.625 μg/mL (56.26 μM). Compounds 17, 24, and 26 were further screened for their antiproliferative activity against human epithelial kidney cancer cell line A498, human prostate cancer cell line PC-3, and human glioblastoma cell line U-87MG, where they were found to possess interesting activity worth further exploration in the future.

This comprehensive study provides insight into the antibacterial action of a recently published 2-chloro-N-(oxazol-2-yl)isonicotinamide (AB15), intending to assess its potential as a candidate adjuvant molecule to support existing antibacterial drugs. Within the determination of the antibacterial effect, a promising activity against a member of the ESKAPE group with reduced treatment options, biofilm producer, Acinetobacter baumannii, was recognized (MIC of AB15 ranged from 15.63 to 62.5 µM). In addition, AB15 exhibited bactericidal activity and non/low-toxicity in vitro (IC50 > 1000 µM using HK-2 cells) and in vivo (LD50 > 500 mg/kg of body weight of the Galleria mellonella larvae, for both intra-hemocoel and per oral administration routes). Checkerboard assay revealed additive and synergistic interactions of AB15 and last-resort antibiotic drug, colistin (CST). Moreover, attention was also given to a frequently overlooked antibiofilm activity — the ability to suppress bacterial dissemination from microbial biofilms, and parameter MBDC (minimum biofilm dissemination concentration) was introduced. The study of the antibiofilm activity of AB15 and CST, both acting individually, or in AB15 + CST combination, revealed that AB15 has significant potential to suppress bacterial dissemination from biofilm formed by a clinical isolate Acinetobacter baumannii and that it contributes to this effect when combined with CST. Finally, AB15 + CST combination demonstrated significantly greater biocompatibility towards human erythrocytes than CST acting individually at an equivalent antibiofilm-effective concentration. The role of AB15 as a promising adjuvant molecule to CST is also supported by its distinct mechanism of action, which reduces the risk of antimicrobial resistance emergence. To conclude, AB15 exhibits several essential attributes that support its designation as a promising antibiotic adjuvant.

Quinazolinone derivatives have emerged as promising scaffolds in antimicrobial drug discovery. This work focuses on the design, synthesis, and evaluation of novel quinazolinone‐based compounds and predicts their potential to interact with mycobacterial penicillin‐binding proteins (PBPs). Relying on established structure‐activity relationships of antibacterial quinazolinones, a total of 53 compounds belonging to three different structural types were synthesized and biologically evaluated for antimycobacterial, antibacterial, and antifungal activities. Biological evaluations revealed selective efficacy against Mycobacterium tuberculosis with minimum inhibitory concentrations (MICs) as low as 6.25 µg/mL for some derivatives, and this activity was preserved against drug‐resistant strains. Molecular docking studies suggested a potential allosteric binding site in mycobacterial Penicillin‐binding protein 1A (PonA1, UniProt ID: P71707), and subsequential molecular dynamics confirmed stable binding with key stabilizing interaction between the carbonyl oxygen of the quinazolinone and either ARG399 or ASP474. These findings suggest quinazolinone derivatives as viable candidates for further development as non‐β‐lactam PBP inhibitors, addressing the urgent need for new antitubercular therapies.

A series of 33 (E)-N'-benzylidenepyrazine-2-carbohydrazides and their derivatives were synthesized and tested for biological activity. Benzylidene derivatives with 2-OH substitution on the phenyl ring (18: R = 2-OH, 21: R = 2,3-diOH, and 22: R = 2,4-diOH) exhibit various biological activities. Compounds 18 and 21 demonstrate antimycobacterial activity against Mycobacterium tuberculosis H37Ra, M. tuberculosis H37Rv, and M. aurum, with minimum inhibitory concentration values ranging from 15.625 to 62.5 μg mL-1. Compounds 18, 21, and 22 show mild cytotoxicity on several human cell lines (IC50 ranging from 70.2 to 500 μM). Crystallographic studies confirm the (E)-configuration of compound 18 and a nearly planar molecular conformation. Due to their structural similarity with salicylaldehyde isonicotinoyl hydrazone (SIH), a known iron chelator, selected compounds were tested for iron-chelating properties, revealing comparable or superior activity. Mechanistic assays targeting enoyl-[acyl carrier protein] reductase (InhA), isocitrate lyase (ICL), and lipid/mycolic acid biosynthesis show no significant inhibition, suggesting a nonspecific mechanism potentially linked to iron chelation. A correlation is observed between chelating activity and cytotoxicity, while antimycobacterial activity appears to involve additional mechanisms. Pharmacokinetic studies with compound 18 reveal no specific plasma metabolites, and no significant metabolites are detected after incubation with human liver microsomes.

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).

Background: The development of novel antimicrobial drugs is an essential part of combatting the uprising of antimicrobial resistance. Proper hit-to-lead development is crucially needed. Methods & results: We present a hit-expansion study of N-pyrazinyl- and N-pyridyl-hydroxybenzamides with a comprehensive determination of structure-activity relationships. The antimicrobial screening revealed high selectivity to staphylococci along with antimycobacterial activity with the best value of 6.25 μg/ml against Mycobacterium tuberculosis H37Rv. We proved an inhibition of proteosynthesis and a membrane depolarization of methicillin-resistant Staphylococcus aureus. Conclusion: Our results are a good starting point for further development of new antimicrobial compounds, where the next step would be tuning the potential between relatively nonspecific membrane depolarization effect and specific inhibition of proteosynthesis.

Tuberculosis is the number one killer of infectious diseases caused by a single microbe, namely Mycobacterium tuberculosis (Mtb). The success rate of curing this infection is decreasing due to emerging antimicrobial resistance. Therefore, novel treatments are urgently needed. As an attempt to develop new antituberculars effective against both drugs-sensitive and drug-resistant Mtb, we report the synthesis of a novel series inspired by combining fragments from the first-line agents isoniazid and pyrazinamide (series I) and isoniazid with the second-line agent 4-aminosalicylic acid (series II). We identified compound 10c from series II with selective, potent in vitro antimycobacterial activity against both drug-sensitive and drug-resistant Mtb H37Rv strains with no in vitro or in vivo cytotoxicity. In the murine model of tuberculosis, compound 10c caused a statistically significant decrease in colony-forming units (CFU) in spleen. Despite having a 4-aminosalicylic acid fragment in its structure, biochemical studies showed that compound 10c does not directly affect the folate pathway but rather methionine metabolism. In silico simulations indicated the possibility of binding to mycobacterial methioninetRNA synthetase. Metabolic study in human liver microsomes revealed that compound 10c does not have any known toxic metabolites and has a half-life of 630 min, overcoming the main drawbacks of isoniazid (toxic metabolites) and 4-aminosalicylic acid (short half-life).

Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).

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