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.

Department of Clinical Microbiology University Hospital Sokolská 581 500 05 Hradec Králové Czech Republic

Faculty of Pharmacy in Hradec Králové Charles University Heyrovského 1203 500 05 Hradec Králové Czech Republic

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Tacconelli E., Carrara E., Savoldi A., Harbarth S., Mendelson M., Monnet D.L., Pulcini C., Kahlmeter G., Kluytmans J., Carmeli Y., et al. Discovery, research, and development of new antibiotics: The WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 2018;18:318–327. doi: 10.1016/S1473-3099(17)30753-3. PubMed DOI

Glaziou P., Floyd K., Raviglione M.C. Global Epidemiology of Tuberculosis. Semin. Respir. Crit. Care Med. 2018;39:271–285. doi: 10.1101/cshperspect.a017798. PubMed DOI

World Health Organization . WHO Global Tuberculosis Report 2019. World Health Organization; Geneva, Switzerland: 2019.

Gagneux S. Ecology and evolution of Mycobacterium tuberculosis. Nat. Rev. Microbiol. 2018;16:202–213. doi: 10.1038/nrmicro.2018.8. PubMed DOI

Hutchison J.M., Zhang Y., Waller S. Nontuberculous Mycobacteria Infection: Source and Treatment. Curr. Pulmonol. Rep. 2019;8:151–159. doi: 10.1007/s13665-019-00237-8. DOI

Jeon D. Infection Source and Epidemiology of Nontuberculous Mycobacterial Lung Disease. Tuberc. Respir. Dis. 2019;82:94–101. doi: 10.4046/trd.2018.0026. PubMed DOI PMC

Darwish A.A.E., El Mahalawy I., El Dahdouh S.S.A., Elkholy R.M., El Beshbishy M.I.A. Nontuberculous mycobacterium as a hidden cause of noncystic fibrosis bronchiectasis. Egypt. J. Chest Dis. Tuberc. 2020;69:46–50.

Nath H., Ryoo S. First- and Second-Line Drugs and Drug Resistance. In: Mahboub B., editor. Tuberculosis-Current Issues in Diagnosis and Management. InTech; Rijeka, Croatia: 2013. DOI

Saltini C. Chemotherapy and diagnosis of tuberculosis. Respir. Med. 2006;100:2085–2097. doi: 10.1016/j.rmed.2006.09.015. PubMed DOI

Cynamon M.H., Speirs R.J., Welch J.T. In vitro antimycobacterial activity of 5-chloropyrazinamide. Antimicrob. Agents Chemother. 1998;42:462–463. PubMed PMC

Dolezal M., Zitko J., Jampilek J. Pyrazinecarboxylic Acid Derivatives with Antimycobacterial Activity. In: Cardona P.-J., editor. Understanding Tuberculosis-New Approaches to Fighting Against Drug Resistance. InTech; Rijeka, Croatia: 2012. DOI

Dolezal M., Kesetovic D., Zitko J. Antimycobacterial evaluation of pyrazinoic acid reversible derivatives. Curr. Pharm. Des. 2011;17:3506–3514. doi: 10.2174/138161211798194477. PubMed DOI

Zitko J., Servusova B., Paterova P., Mandikova J., Kubicek V., Kucera R., Hrabcova V., Kunes J., Soukup O., Dolezal M. Synthesis, antimycobacterial activity and in vitro cytotoxicity of 5-chloro-N-phenylpyrazine-2-carboxamides. Molecules. 2013;18:14807–14825. doi: 10.3390/molecules181214807. PubMed DOI PMC

Zitko J., Servusova B., Janoutova A., Paterova P., Mandikova J., Garaj V., Vejsova M., Marek J., Dolezal M. Synthesis and antimycobacterial evaluation of 5-alkylamino-N-phenylpyrazine-2-carboxamides. Bioorg. Med. Chem. 2015;23:174–183. doi: 10.1016/j.bmc.2014.11.014. PubMed DOI

Jena L., Kashikar S., Kumar S., Harinath B.C. Comparative proteomic analysis of Mycobacterium tuberculosis strain H37Rv versus H37Ra. Int. J. Mycobacteriol. 2013;2:220–226. doi: 10.1016/j.ijmyco.2013.10.004. PubMed DOI

Heinrichs M., May R., Heider F., Reimers T.B., Sy S., Peloquin C., Derendorf H. Mycobacterium tuberculosis Strains H37ra and H37rv have equivalent minimum inhibitory concentrations to most antituberculosis drugs. Int. J. Mycobacteriol. 2018;7:156–161. doi: 10.4103/ijmy.ijmy_33_18. PubMed DOI

Sood S., Yadav A., Shrivastava R. Mycobacterium aurum is Unable to Survive Mycobacterium tuberculosis Latency Associated Stress Conditions: Implications as Non-suitable Model Organism. Indian J. Microbiol. 2016;56:198–204. doi: 10.1007/s12088-016-0564-x. PubMed DOI PMC

Servusova B., Vobickova J., Paterova P., Kubicek V., Kunes J., Dolezal M., Zitko J. Synthesis and antimycobacterial evaluation of N-substituted 5-chloropyrazine-2-carboxamides. Bioorg. Med. Chem. Lett. 2013;23:3589–3591. doi: 10.1016/j.bmcl.2013.04.021. PubMed DOI

Joseph T., Varghese H.T., Panicker C.Y., Viswanathan K., Dolezal M., Van Alsenoy C. Spectroscopic (FT-IR, FT-Raman), first order hyperpolarizability, NBO analysis, HOMO and LUMO analysis of N-[(4-(trifluoromethyl)phenyl]pyrazine-2-carboxamide by density functional methods. Arab. J. Chem. 2017;10:S2281–S2294. doi: 10.1016/j.arabjc.2013.08.004. DOI

Holzer W., Eller G.A., Datterl B., Habicht D. Derivatives of pyrazinecarboxylic acid: 1H, 13C and 15N NMR spectroscopic investigations. Magn. Reson. Chem. 2009;47:617–624. doi: 10.1002/mrc.2437. PubMed DOI

Franzblau S.G., Witzig R.S., McLaughlin J.C., Torres P., Madico G., Hernandez A., Degnan M.T., Cook M.B., Quenzer V.K., Ferguson R.M., et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J. Clin. Microbiol. 1998;36:362–366. doi: 10.1128/JCM.36.2.362-366.1998. PubMed DOI PMC

European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin. Microbiol. Infect. 2003;9:1–7. doi: 10.1046/j.1469-0691.2003.00790.x. PubMed DOI

Arendrup M.C., Meletiadis J., Mouton J.W., Lagrou K., Hamal P., Guinea J., Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST) Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal agents for Yeasts. [(accessed on 5 March 2020)];2017 EUCAST Definitive Document E.Def 7.3.1. Available online: http://www.eucast.org/astoffungi/methodsinantifungalsusceptibilitytesting/susceptibility_testing_of_yeasts/

Arendrup M.C., Meletiadis J., Mouton J.W., Lagrou K., Hamal P., Guinea J., Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST) Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal agents for Conidia Forming Moulds. [(accessed on 5 March 2020)];2017 EUCAST Definitive Document E.Def 9.3.1. Available online: http://www.eucast.org/astoffungi/methodsinantifungalsusceptibilitytesting/susceptibility_testing_of_moulds/

Design, Synthesis and Antimicrobial Evaluation of New N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)(hetero)aryl-2-carboxamides as Potential Inhibitors of Mycobacterial Leucyl-tRNA Synthetase