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.

Department of Clinical Microbiology Faculty 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

See more in PubMed

World Health Organization Global Tuberculosis Report 2018. WHO/CDC/TB/2018.20. [(accessed on 20 February 2019)]; Available online: http://www.who.int/tb/publications/global_report/en/

World Health Organisation . The End TB Strategy. Global Strategy and Targets for Tuberculosis Prevention, Care and Control after 2015. WHO; Geneva, Switzerland: 2016. [(accessed on 20 February 2019)]. Available online: http://www.who.int/tb/ post2015_ TBstrategy.pdf?ua=1.

Singh P., Mishra A.K., Malonia S.K., Chauhan D.S., Sharma V.D., Venkatesan K., Katoch V.M. The Paradox of Pyrazinamide: An Update on the Molecular Mechanisms of Pyrazinamide Resistance in Mycobacteria. J. Commun. Dis. 2006;38:288–298. PubMed

Tripathi R.P., Tewari N., Dwivedi N., Tiwari V.K. Fighting tuberculosis: An old disease with new challenges. Med. Res. Rev. 2005;25:93–131. doi: 10.1002/med.20017. PubMed DOI

Zhang Y., Mitchison D. The curious characteristics of pyrazinamide: A review. Int. J. Tuberc. Lung Dis. 2003;7:6–21. PubMed

Zimhony O., Cox J.S., Welch J.T., Vilcheze C., Jacobs W.R., Jr. Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nat. Med. 2000;6:1043–1047. doi: 10.1038/79558. PubMed DOI

Zimhony O., Vilcheze C., Arai M., Welch J.T., Jacobs W.R., Jr. Pyrazinoic acid and its n-propyl ester inhibit fatty acid synthase type I in replicating tubercle bacilli. Antimicrob. Agents Chemother. 2007;51:752–754. doi: 10.1128/AAC.01369-06. PubMed DOI PMC

Shi W.L., Chen J.Z., Feng J., Cui P., Zhang S., Weng X.H., Zhang W., Zhang Y. Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis. Emerg. Microbes Infect. 2014;3:e58. doi: 10.1038/emi.2014.61. PubMed DOI PMC

Kim H., Shibayama K., Rimbara E., Mori S. Biochemical Characterization of Quinolinic Acid Phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and Inhibition of Its Activity by Pyrazinamide. PLoS ONE. 2014;9:e100062. doi: 10.1371/journal.pone.0100062. PubMed DOI PMC

Shi W., Zhang X., Jiang X., Yuan H., Lee J.S., Barry C.E., 3rd, Wang H., Zhang W., Zhang Y. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 2011;333:1630–1632. doi: 10.1126/science.1208813. PubMed DOI PMC

Yang J., Liu Y., Bi J., Cai Q., Liao X., Li W., Guo C., Zhang Q., Lin T., Zhao Y., et al. Structural basis for targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide. Mol. Microbiol. 2015;95:791–803. doi: 10.1111/mmi.12892. PubMed DOI

Dillon N.A., Peterson N.D., Feaga H.A., Keiler K.C., Baughn A.D. Anti-tubercular Activity of Pyrazinamide is Independent of trans-Translation and RpsA. Sci. Rep. 2017;7:6135. doi: 10.1038/s41598-017-06415-5. PubMed DOI PMC

Simoes M.F., Valente E., Gomez M.J.R., Anes E., Constantino L. Lipophilic pyrazinoic acid amide and ester prodrugs Stability, activation and activity against M. tuberculosis. Eur. J. Pharm. Sci. 2009;37:257–263. doi: 10.1016/j.ejps.2009.02.012. PubMed DOI

Semelkova L., Konecna K., Paterova P., Kubicek V., Kunes J., Novakova L., Marek J., Naesens L., Pesko M., Kralova K., et al. Synthesis and Biological Evaluation of N-Alkyl-3-(alkylamino)-pyrazine-2-carboxamides. Molecules. 2015;20:8687–8711. doi: 10.3390/molecules20058687. PubMed DOI PMC

Semelkova L., Jandourek O., Konecna K., Paterova P., Navratilova L., Trejtnar F., Kubíček V., Kuneš J., Doležal M., Zitko J. 3-Substituted N-Benzylpyrazine-2-carboxamide Derivatives: Synthesis, Antimycobacterial and Antibacterial Evaluation. Molecules. 2017;22:495. doi: 10.3390/molecules22030495. PubMed DOI PMC

Zitko J., Franco F., Paterova P. Synthesis and anti-infective evaluation of 5-amino-N-phenylpyrazine-2-carboxamides. Ceska Slov. Farm. 2015;64:19–24. PubMed

Kajino M., Morimoto S., Inaba A., Nagaya H. Preparation and Formulation of Quinazoline Derivatives as Allergy Inhibitors. 9914203. WO. 1999 Mar 25;

Luo H., Shi J., Lu L., Wu F., Zhou M., Hou X., Zhang W., Ding Z., Li R. Molecular dynamics-based self-organizing molecular field analysis on 3-amino-6-arylpyrazines as the ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase inhibitors. Med. Chem. Res. 2014;23:747–758. doi: 10.1007/s00044-013-0665-6. DOI

Bouz G., Juhas M., Niklova P., Jandourek O., Paterova P., Janousek J., Tůmová L., Kovalíková Z., Kastner P., Doležal M., et al. Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-infectives and Abiotic Elicitors. Molecules. 2017;22:1797. doi: 10.3390/molecules22101797. PubMed DOI PMC

Eric V., Bradley M. Amide Bond Formation: Beyond the Myth of Coupling Reagents. Chem. Soc. Rev. 2009;38:606–631. PubMed

Shalaeva M., Caron G., Abramov Y.A., O’Connell T.N., Plummer M.S., Yalamanchi G., Farley K.A., Goetz G.H., Philippe L., Shapiro M.J. Integrating Intramolecular Hydrogen Bonding (IMHB) Considerations in Drug Discovery Using Delta logP As a Tool. J. Med. Chem. 2013;56:4870–4879. doi: 10.1021/jm301850m. PubMed DOI

Hubbard T.A., Brown A.J., Bell I.A.W., Cockroft S.L. The Limit of Intramolecular H-Bonding. JACS. 2016;138:15114–15117. doi: 10.1021/jacs.6b09130. PubMed DOI

Nagy P.I. Competing Intramolecular vs. Intermolecular Hydrogen Bonds in Solution. Int. J. Mol. Sci. 2014;15:19562–19633. doi: 10.3390/ijms151119562. PubMed DOI PMC

Hsiao C.H., Tsai T.F., Hsueh P.R. Characteristics of skin and soft tissue infection caused by non-tuberculous mycobacteria in Taiwan. Int. J. Tuberc. Lung Dis. 2011;15:811–817. doi: 10.5588/ijtld.10.0481. PubMed DOI

Gupta A., Bhakta S. An integrated surrogate model for screening of drugs against Mycobacterium tuberculosis. J. Antimicrob. Chemother. 2012;67:1380–1391. doi: 10.1093/jac/dks056. PubMed DOI

Servusova B., Paterova P., Mandikova J., Kubicek V., Kucera R., Kunes J., Doležal M., Zitko J. Alkylamino derivatives of pyrazinamide: Synthesis and antimycobacterial evaluation. Bioorg. Med. Chem. Lett. 2014;24:450–453. doi: 10.1016/j.bmcl.2013.12.054. PubMed DOI

Servusova-Vanaskova B., Jandourek O., Paterova P., Kordulakova J., Plevakova M., Kubicek V., Kucera R., Garaj V., Naesens L., Kunes J., et al. Alkylamino derivatives of N-benzylpyrazine-2-carboxamide: Synthesis and antimycobacterial evaluation. MedChemComm. 2015;6:1311–1317. doi: 10.1039/C5MD00178A. DOI

Zitko J., Servusova B., Janoutova A., Paterova P., Mandikova J., Garaj V., Vejsová M., Marek J., Doležal 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

Servusova-Vanaskova B., Paterova P., Garaj V., Mandikova J., Kunes J., Naesens L., Jilek P., Dolezal M., Zitko J. Synthesis and Antimicrobial Evaluation of 6-Alkylamino-N-phenylpyrazine-2-carboxamides. Chem. Biol. Drug Des. 2015 doi: 10.1111/cbdd.12536. in press. PubMed DOI

El Bouazzi O., Hammi S., Bourkadi J.E., Tebaa A., Tanani D.S., Soulaymani-Bencheikh R., Badrane N., Bengueddour R. First line anti-tuberculosis induced hepatotoxicity: Incidence and risk factors. Pan Afr. Med. J. 2016;25:167. doi: 10.11604/pamj.2016.25.167.10060. PubMed DOI PMC

Tostmann A., Boeree M.J., Peters W.H.M., Roelofs H.M.J., Aarnoutse R.E., van der Ven A.J.A.M., Dekhuijzen P.N. Isoniazid and its toxic metabolite hydrazine induce in vitro pyrazinamide toxicity. Int. J. Antimicrob. Agents. 2008;31:577–580. doi: 10.1016/j.ijantimicag.2008.01.022. PubMed DOI

Ellingson R.C., Henry R.L., McDonald F.G. Pyrazine Chemistry. I. Derivatives of 3-Aminopyrazinoic Acid. JACS. 1945;67:1711–1713. doi: 10.1021/ja01226a028. DOI

Dermer O.C., King J. N-BENZYLAMIDES AS DERIVATIVES FOR IDENTIFYING THE ACYL GROUP IN ESTERS1,2. JOC. 1943;8:168–173. doi: 10.1021/jo01190a008. DOI

Clark J., Neath G., Smith C. Heterocyclic Studies. 7. Action of Methoxyamine and Methylhydrazines on 4-Hydroxypteridine and Its Methyl Derivatives. J. Chem. Soc. C-Org. 1969:1297–1301. doi: 10.1039/j39690001297. DOI

EUCAST DISCUSSION DOCUMENT E.Dis 5.1 Determination of Minimum Inhibitory Concentrations (MICs) of Antibacterial Agents by Broth Dilution. Clin. Microbiol. Infect. 2003;9:1–7. PubMed

EUCAST DEFINITIVE DOCUMENT E.DEF 7.3.1 . Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal Agents for Yeasts. EUCAST; Växjö, Sweden: 2017. pp. 1–21.

EUCAST DEFINITIVE DOCUMENT E.DEF 9.3.1 . Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal Agents for Conidia Forming Moulds. EUCAST; Växjö, Sweden: 2017. pp. 1–23.