2. World Health Organization. Global Tuberculosis Control:
Surveillance, Planning, Financing. WHO Report 2002.
WHO/CDS/TB/2002.295.
3. World Health Organization. Global Tuberculosis Report 2012.
WHO/HTM/TB/2012.6.
4. Goletti, D.; Weissman, D.; Jackson, R.W.; Graham, N.M.;
Vlahov, D.; Klein, R.S.; Munsiff, S.S.; Lʼ Ortona, L.; Cauda, R.;
Fauci, A.S. J. Immunol. 1996, 157, 1271.
For all tested compounds and strains, only weak antibacterial
activity was found for compound 2g (Staphylococcus epidermidis
H 6966/08; MIC = 62.5 µM), for further details see
Supplementary data. The rest of tested compounds were inactive
even in highest concentrations used in the testing, which were
500 µM.
Concerning the mechanism of action, PZA acts mainly as a
prodrug, which is enzymatically hydrolysed via mycobacterial
pyrazinamidase (PncA, EC 3.5.1.19) to form pyrazinoic acid
(POA).34 On the other hand, some PZA derivatives proved to be
active in non-hydrolysed carboxamide form; e.g. 5-Cl-PZA as a
FAS I inhibitor.6,8,9,11,13 5-Cl-PZA possessed in vitro activity
against mycobacterial strains with low pyrazinamidase activity as
well.7 To elucidate, whether the most active alkylamino
derivatives of PZA could underwent hydrolysis by PncA or
rather function in non-hydrolysed form, a docking study was
performed.
5. Asai, M. Yakugaku Zasshi, 1961, 81, 1475.
6. Cynamon, M.H.; Speirs, R.J.; Welch, J.T. Antimicrob. Agents
Chemother. 1998, 42, 462.
7. Ahmad, Z.; Tyagi, S.; Minkowski, A.; Almeida, D.; Nuermberger,
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Grosset, J.H. Indian J Med Res. 2012, 136, 808.
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2167.
10. Zimhony, O.; Vilcheze, C.; Arai, M.; Welch, J.T.; Jacobs, W.R.
Antimicrob. Agents Ch. 2007, 51, 752.
11. Ngo, S.; Zimhony, O.; Chung, W.J.; Sayahi, H.; Jacobs, W.R.;
Welch, J.T. Antimicrob. Agents Ch. 2007, 51, 2430.
12. Brennan, P.J. Tuberculosis 2003, 83, 91.
Crystallographic structure of PncA was recently described in
detail.35 The substrate binding cavity in PncA is a narrow crevice
(approximately 10 × 7 Å) containing catalytic triad made of
residues Lys96, Asp8 and Cys138. On the opposite site of the
crevice, three histidine residues (His51, His57 and His71) and
Asp49 hold the Fe ion on. According to the orientation of PZA
needed for its catalytic conversion as described in literature35, the
substituents on C5 and C6 of the pyrazine nucleus would
protrude towards the end of the cavity formed by the Trp68. The
distance between the PZA ring and the Trp68 plane is
approximately 4–5 Å (see Supplementary data), which is little
space to accommodate any larger substituent. Therefore, we
expected that the 5-alkylamino and 6-alkylaminopyrazine-2-
carboxamides with longer alkyl chains would have to be
displaced from the position needed for the catalytic
transformation. This was confirmed by docking study described
in Supplementary data. To conclude, neither 5-alkylamino nor
6-alkylamino derivatives of PZA will be converted to
corresponding carboxylic acids by the PncA.
13. Sayahi, H.; Pugliese, K.M.; Zimhony, O.; Jacobs, W.R. Jr.;
Shekhtman, A.; Welch, J.T. Chem. Biodivers. 2012, 9, 2582.
14. Zitko, J.; Doležal, M.; Svobodová, M.; Vejsová, M.; Kuneš, J.;
Kučera, R.; Jílek, P. Bioorg. Med. Chem. 2011, 19, 1471.
15. Zitko, J.; Jampílek, J.; Dobrovolný, L.; Svobodová, M.; Kuneš, J.;
Doležal, M. Bioorg. Med. Chem. Lett. 2012, 22, 1598.
16. Freundlich, J.S.; Wang, F.; Vilcheze, C.; Gulten, G.; Langley, R.;
Schiehser, G.A.; Jacobus, D.R.; Jacobs, W.R.; Sacchettini, J.C.
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17. Luckner, S.R.; Liu, N.; am Ende, C.W.; Tonge, P.J.; Kisker, C. J
Biol Chem. 2010, 285, 14330.
18. Hiltunen, J.K.; Schonauer, M.S.; Autio, K.J.; Mittelmeier, T.M.;
Kastaniotis, A.J.; Dieckmann, C.L. J. Biol. Chem. 2009, 284,
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19. Bernardi, L.; Palamidessi, G.; Leone, A.; Larini, G. Gazz. Chim.
Ital. 1961, 91, 1431.
20. Matulenko, M.A.; Lee, C.H.; Jiang, M.; Frey, R.R.; Cowart, M.D.;
Bayburt, E.K.; DiDomenico, S. Bioorg. Med. Chem. 2005, 13,
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21. Clayden, J. Organic Chemistry. Oxford University Press: Oxford,
2008.
22. Abe, H.; Shigeta, Y.; Uchimaru, F.; Okada, S.; Ozasayma, E.
Methyl 6-methoxypyrazine-2- carboxylate. JP Patent 44012898,
1969; Chem. Abstr. 1969, 71, 112979y
23. Montedison S.p.A . Process for the preparation of 2-
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24. Palamidessi, G.; Vigevani, A.; Zarini, F. J. Heterocycl.
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25. Ellingson, R.C.; Henry, R.L. J. Am. Chem. Soc., 1949, 71, 2798.
26. Abe, H.; Shigeta, Y.; Uchimaru, F.; Okada, S.; Ozasayma, E.
Substituted pyrazinecarboxamide derivatives. JP Patent 46037596,
1971
27. Foks, H.; Pancechowska, D.; Sawlewicz, J. Buraczewska, M.;
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V.K.; Ferguson, R.M.; Gilman, R.H. J. Clin. Microbiol. 1998, 36,
362.
New series of 5-alkylamino and 6-alkylaminopyrazine-2-
carboxamides were synthesized, characterized by analytical data
and screened for antimycobacterial activity. 6-Octylamino-
pyrazine-2-carboxamide (2i) showed the highest activity against
M. tuberculosis H37Rv (MIC = 1.56 µg/mL i.e. 6 µM), broadest
spectrum of activity as well as highest selectivity index for
M. tuberculosis H37Rv (SI = 25.8) within all compounds. Based
on the results, we presume 6-octylaminopyrazine-2-carboxamide
(2i, 100-fold more active than 6-Cl-PZA) could serve as model
structure for further modifications. Presented study also
confirmed the previously reported positive influence of long
alkylamino chains on antimycobacterial activity.
29. Zhang, Y.; Scorpio, A.; Nikaido, H.; Sun, Z. J. Bacteriol. 1999,
181, 2044.
Acknowledgments
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31. Kratky, M.; Vinsova, J.; Volkova, M.; Buchta, V.; Trejtnar, F.;
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This work was financially supported by GAUK B-CH/710312, IGA NT
13346 (2012) and SVV-2013-267-001. The publication is co-financed by the
European Social Fund and the state budget of the Czech Republic. Project no.
CZ.1.07/2.3.00/20.0235, the title of the project: TEAB.
Supplementary data
Supplementary data associated with this article can be found, in online
version:
35. Petrella, S.; Gelus-Ziental, N.; Maudry, A.; Laurans, C.;
Boudjelloul, R.; Sougakoff, W. PLoS One 2011, 6, e15785.
References and notes
1. World Health Organization. Global Tuberculosis Report 2013.
WHO/HTM/TB/2013.11.