Synthesis and antimycobacterial properties of N-substituted 6-amino-5-cyanopyrazine-2-carboxamides

Article abstract of DOI:10.1016/j.bmc.2010.12.054

A series of fifteen new compounds related to pyrazinamide (PZA) were synthesized, characterized with analytical data and screened for antimycobacterial, antifungal and antibacterial activity. The series consists of 6-chloro-5-cyanopyrazine-2-carboxamide a

Full text of DOI:10.1016/j.bmc.2010.12.054

Bioorganic & Medicinal Chemistry 19 (2011) 1471–1476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimycobacterial properties of N-substituted
6-amino-5-cyanopyrazine-2-carboxamides
Jan Zitko a, , Martin Dolezal a, Michaela Svobodova b, Marcela Vejsova a, Jiri Kunes a,
⇑
Radim Kucera a, Petr Jilek a
a Faculty of Pharmacy, Charles University, Heyrovskeho 1203, Hradec Kralove 500 05, Czech Republic
b Department of Microbiology, Regional Hospital, Kyjevska 44, Pardubice 532 03, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fifteen new compounds related to pyrazinamide (PZA) were synthesized, characterized with
analytical data and screened for antimycobacterial, antifungal and antibacterial activity. The series
consists of 6-chloro-5-cyanopyrazine-2-carboxamide and N-substituted 6-amino-5-cyanopyrazine-2-
carboxamides, derived from the previous by nucleophilic substitution with various non-aromatic amines
(alkylamines, cycloalkylamines, heterocyclic amines). Some of the compounds exerted antimycobacterial
Received 14 September 2010
Revised 27 December 2010
Accepted 28 December 2010
Available online 1 January 2011
activity against Mycobacterium tuberculosis equal to pyrazinamide (12.5–25 lg/mL). More importantly,
Keywords:
6-chloro-5-cyanopyrazine-2-carboxamide and 5-cyano-6-(heptylamino)pyrazine-2-carboxamide were
active against Mycobacterium kansasii and Mycobacterium avium, which are unsusceptible to PZA. Basic
structure–activity relationships are presented. Only weak antifungal and no antibacterial activity was
detected.
Antitubercular agents
Antifungal activity
Antibacterial activity
Pyrazinamide analogues
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
leading to disruption of membrane transport and energetics.4 The
POA enters the cell by passive diffusion strongly dependent on
According to WHO report1 estimates, there were 9.27 million
new cases and 13.7 million prevalent cases of tuberculosis (TB)
in 2007. Although the absolute number of incident cases of TB is
increasing due to population growth, the relative numbers peaked
at 142 cases per 100,000 population in 2004 and have been
decreasing by now. However, the rate of this decline is very slow,
less than 1% per year. TB remains a serious epidemiological prob-
lem especially in underdeveloped countries. Co-infection with
HIV and rising resistance to currently used anti-TB drugs are
alarming facts. 0.5 million new cases of TB in 2007 were multi-
drug-resistant tuberculosis (resistance to, at least, isoniazid and
rifampicin, MDR-TB). More than half of this MDR-TB cases
appeared in patients previously not treated for TB.
Pyrazinamide (PZA) is one of the most important anti-TB drugs.
Along with rifampicin, it’s the only used active substance to posses
so called sterilizing activity—the ability to kill the dormant non-
growing tubercle bacilli of low metabolism activity.2 The killing
of these persisters is a crucial factor in shortening the therapy
course and avoiding relapses. PZA is a prodrug which is activated
by means of deamination catalyzed by pyrazinamidase to form
the active pyrazinoic acid (POA). It’s mode of action is proposed
by Zhang.3 POA accumulates inside the mycobacterial cell, thus
pH (greater in acidic conditions). The accumulation also increases
in non-growing bacilli, because the POA efflux mechanism is an en-
ergy consuming process.
Some PZA analogues and derivatives (especially 5-chloropyraz-
inamide,5,6 esters of pyrazinoic acid and esters of 5-chloropyrazi-
noic acid6) were also shown to inhibit the FAS I (Fatty Acid
Synthase I) pathway, impairing the building of normal mycobacte-
rial cell wall.
TB remains a serious problem of today. There is a call for devel-
oping new antituberculars to broaden the palette of used sub-
stances, overcome the rising resistance of mycobacteria, shorten
the therapy course, limit the adverse effects and reduce relapses.
The previously mentioned interesting and unique properties of
pyrazinamide and/or its derivatives (sterilizing activity, multiple
mechanisms of action) motivate further research in this field. Re-
cently, promising results were published by Chung et al.7 (ami-
nomethylene pyrazinamide analogues) and Ancizu et al.8
(quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives). Other
ongoing studies are concerned with the pharmacokinetics, bio-
availability and dosage regimen of PZA as reported in the review
by Mitchison et al.9
This study is focused on non-aromatically N-substituted
6-amino-5-cyanopyrazine-2-carboxamides. Dolezal et al.10 pre-
pared N-substituted 3-amino-5-cyanopyrazine-2-carboxamides,
position isomers of the compounds involved in his study, with no
antimycobacterial activity.
⇑
Corresponding author. Tel.: +420 495067272; fax: +420 495067167.
E-mail address: jan.zitko@faf.cuni.cz (J. Zitko).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.054

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J. Zitko et al. / Bioorg. Med. Chem. 19 (2011) 1471–1476
Scheme 1. Synthesis of final compounds and the side reaction.
of reaction concerning pyrazine carbonitrile group was previously
2. Results and discussion
2.1. Chemistry
described by Foks14,15 and used by Shirai.16 In our study, this
reaction appeared mainly with primary amines. The amidine by-
products were beyond the interest of this study, therefore only
two of them (16 and 17) were completely characterized to confirm
the presumed structure. The by-products had almost identical Rf
values with corresponding intended products (silica, hexane/ethyl
acetate) and therefore were not discovered earlier than after NMR
analysis of what seemed to be a pure compound at TLC. The mod-
ification of mobile phase by addition of 1% (v/v) of acetic acid
increased the delta Rf sufficiently (>0.2). To limit the formation of
by-products, it was decided to replace the excess of amine by
indifferent organic base (triethylamine).
3-Chloropyrazine-2-carbonitrile was purchased from ChemPur
(Karlsruhe, Germany). Its identity was confirmed by NMR spectra
and melting point available in literature.11,12 3-Chloropyrazine-2-
carbonitrile underwent homolytic amidation12 performed by form-
amide and ammonium persulfate (see Scheme 1, a) to form the de-
sired 6-chloro-5-cyanopyrazine-2-carboxamide (1). The position of
the carboxamide group was confirmed indirectly by chemical ap-
proach. Compound 1 was dehydrated (d) by phosphorus oxychlo-
ride to yield dicarbonitrile compound identical with 3-
chloropyrazine-2,5-dicarbonitrile described by Palek et al.13
Final compounds 2–15 were prepared by nucleophilic substitu-
tion (Scheme 1, b) of chlorine in 6-chloro-5-cyanopyrazine-2-car-
boxamide (1) with various non-aromatic amines, both primary
and secondary. The reaction proceeded very quickly even at labo-
ratory temperature. The first approach used 2.5-fold excess of
amine to bind the formed HCl. However, the excess of amine led
to the formation of undesired amidine by-product by means of
nucleophilic addition of amine to carbonitrile group (c). This type
2.2. Antimycobacterial activity
The starting compound itself, 6-chloro-5-cyanopyrazine-2-car-
boxamide (1), showed good activity against Mycobacterium tuber-
culosis fully comparable with PZA (see Table 1). More
importantly, it showed the same activity against tested MOTTs
(Mycobacteria Other Than Tuberculosis), which are completely
unsusceptible to PZA. Methylamino moiety in the place of chlo-
rine led to the lost of activity. Along with the increasing length
of the alkyl chain (and lipophilicity), the activity appeared again
to culminate in compounds with hexylamino (7) and heptylami-
no substitution (8). This activity was slightly better than the
activity of starting compound and comparable to PZA (against
M. tuberculosis). Compound 8 exerted mild activity even against
MOTTs. Octylamino derivative (9) was inactive again, although
we have to question whether this wasn’t particularly because
of its worse solubility in the culture medium. Therefore we
might propose that there is a lipophilicity optimum in this series
(see Fig. 1). Compounds with cycloalkylamino substitution (10–
12) and secondary alkylamino substitution (13–15) were
inactive.
This study has discovered an interesting antimycobacterial
activity of 6-chloro-5-cyanopyrazine-2-carboxamide (1) and few
6-alkylamino-5-cyanopyrazine-2-carboxamides (7, 8) derived
from the previous by nucleophilic substitution. Dolezal et al.10 pre-
viously prepared a series of 3-alkylamino-5-cyanopyrazine-2-car-
boxamides (i.e., position isomers of the discussed compounds)
which possessed no antimycobacterial activity. This fact suggests
that the activity of the compounds presented in this article might
be structurally specific and connected with specific substitution
of the pyrazine ring. The presence of chlorine bound to pyrazine
Table 1
Antimycobacterial activity as MIC [lg/mL] in comparison with PZA
Compd
M. tbc H37Rva
M. kansasiib
M. aviumc
M. aviumd
C log P
1
25
25
25
25
À0.606
À0.348
0.181
0.710
2
3
4
5
6
7
8
9
10
11
12
13
14
15
PZA
200
100
50
>200
>200
>200
>200
>200
>200
100
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
100
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
12.5
12.5
>200
>200
>200
>200
>200
>200
>200
12.5–25
1.239
1.768
2.297
2.826
3.355
1.124
1.683
2.242
0.761
À0.445
À1.006
À0.676
a
b
c
CNCTC My 331/88.
CNCTC My 235/80.
CNCTC My 80/72.
CNCTC My 152/73.
d

J. Zitko et al. / Bioorg. Med. Chem. 19 (2011) 1471–1476
1473
Figure 1. Relationship between lipophilicity (C log P) and antitubercular activity against M. tbc expressed as a function f (1/MIC).
ring in compound 1 led to antimycobacterial activity against MOT-
3. Conclusion
Ts, which are known to lack pyrazinamidase needed to convert
inactive PZA to pyrazinoic acid. Therefore we might speculate that
compound 1 could have different mode of action than PZA. This is
in accordance with previously observed fact that 5-chloropyrazin-
amide is active against MOTTs and this activity is probably based
on FAS I pathway inhibition.5,6 Results for compounds 2–9 suggest
that the activity is (not surprisingly) lipophilicity dependant,
highest activity possessed by lipophilic compounds with hexylami-
no and heptylamino substitution. Octylamino derivative (9) might
have failed due to low solubility in the testing medium. On the
other hand, lipophilicity is obviously not the sole determinant,
for example, inactive compound 12 (cycloheptylamino substitu-
tion) has C log P (calculated) similar to the most active 7. Of course
steric effects and the overall flexibility of alkyl/cycloalkyl chain
take part in this case.
ifteen new compounds were prepared, characterized with ana-
lytical data and screened for antimycobacterial, antifungal and
antibacterial activity. No significant antifungal activity was de-
tected and none of the tested compounds possessed any antibacte-
rial activity against tested species. Compounds 1, 7 and 8 exerted
antimycobacterial activity equal to PZA concerning M. tuberculosis.
From alkylamino substituted compounds, the highest activity was
found for hexylamino and heptylamino substitution. Obviously,
lipophilicity is an important factor for antimycobacterial activity.
The most promising fact about this series is the discovery of activ-
ity against MOTTs (Mycobacterium kansasii, Mycobacterium avium)
in compounds 1 and 8. Especially MIC = 25 lg/mL for 1 against
all tested mycobacteria species is worth pointing out. Having in
mind the fact that M. kansasii and M. avium lack the pyrazinami-
dase needed for PZA activation, we might speculate that these
compounds might have another mode of action different from
PZA/POA itself.
2.3. Antifungal activity
Only very weak antifungal activity was found as summarized in
Table 2 for compound 1 and 6. This activity was negligible in com-
parison with standard fluconazole and nystatin. The rest of tested
compounds were inactive even at highest concentrations achieved
Concerning our future plans, the cytotoxicity in VERO cells will
be determined for the most active substances. Inspired by the rel-
atively promising activity of some of the prepared compounds we
decided to prepare a similar series of N-substituted 3-aminopyr-
azine-2,5-dicarbonitriles to determine whether carboxamide moi-
ety is necessary for the activity. This series is about to be finished
and results will be published soon. We’d also like to examine the
activity of compounds derived from 1 by substitution with lipo-
philic substituents, especially benzylamines. If we succeed to push
the activity to a higher level, we will arrange for in vivo antimyco-
bacterial testing.
in the testing, which were 500
lmol/L for 2, 3, 4, 5, 7, 12, 13, 14, 15;
250 mol/L for 10; and 125 mol/L for 8, 9, 11.
l
l
2.4. Antibacterial activity
None of the tested compounds exerted any activity against
tested bacterial species.
4. Experimental
Table 2
Antifungal activity MIC
standards fluconazole and nystatin
[lmol/L] of selected compounds in comparison with
4.1. Chemicals and instrumentation
Compd Hoursa CAb
CT
CK
CG
TB
AF
AC
TM
All chemicals (unless stated otherwise) were purchased from
Sigma–Aldrich (Schnelldorf, Germany). The reaction process and
the purity of final compounds were checked using Merck Silica
60 F254 TLC plates (Merck, Darmstadt, Germany). Flash chromatog-
raphy of the final compounds was run on automated chromato-
graph CombiFlash Rf (Teledyne Isco, Lincoln, NE, USA) using
columns filled with Kieselgel 60, 0.040–0.063 mm (Merck, Darms-
tadt, Germany); gradient elution (hexane/ethyl acetate, 1% (v/v) of
acetic acid), detection wavelength 260 nm. NMR analysis was
performed on Varian Mercury VX-BB 300 (Varian, Palo Alto, CA,
1
24
48
24
48
24
48
24
48
125
125
250
>500 >500 >500 >500 >500 250
>500 >500 >500 >500 >500 250
>250 62.5 250
62.5
62.5
>250
6
>250 250
125
>250 >250 250
>250 >250 >250 >250 >250
62.5 250
250 500
FLU
NYS
0.24
0.24
0.98
>500 125
>500 250
>500 >500 7.81
>500 >500 125
1.95 1.95 1.95 1.95 1.95 15.62 3.9
3.9 1.95 1.95 3.9 31.25 7.81
1.95 3.9
a
72 and 120 h for TM.
See Section 4.4 for the code explanation.
b

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J. Zitko et al. / Bioorg. Med. Chem. 19 (2011) 1471–1476
USA) at 300 MHz for 1H and 75 MHz for 13C. The chemical shifts as
d values in ppm are indirectly referenced to tetramethylsilane
(TMS). IR spectra were recorded in KBr blocks on Nicolet Impact
400 (Nicolet, Madison, WI, USA). The mass spectra were recorded
in 50% ACN using LCQ Advantage Max ion-trap mass spectrometer
(Thermo Finnigan, San Jose, CA, USA) with APCI ionization. Elemen-
tary analysis was performed on CE Instruments EA-1110 CHN ana-
lyser (CE Instruments, Wigan, United Kingdom). Melting points
were determined on Stuart SMP30 melting point apparatus (Bibby
Scientific Limited, Staffordshire, UK) and are uncorrected.
4.2.4. 5-Cyano-6-(propylamino)pyrazine-2-carboxamide (4)
Prepared according to the general procedure. Yellow solid.
Yield: 82%. Mp 175–176 °C. IR (cmÀ1): 3463, 3350, 2960, 2876,
2227, 1674, 1573, 1536, 1512, 1231, 1206, 888, 799, 690. 1H
NMR (300 MHz, DMSO) d: 8.31 (1H, s, H3), 8.06 (1H, br s, NH2),
7.88 (1H, br s, NH2), 7.78 (1H, t, J = 6.3 Hz, NH), 3.45 (2H, q,
J = 6.3 Hz, NCH2), 1.64–1.47 (2H, m, CH2), 0.88 (3H, t, J = 6.3 Hz,
CH3). 13C NMR (75 MHz, DMSO) d: 164.8, 154.3, 145.6, 130.6,
115.7, 115.0, 42.1, 22.0, 11.5. MS (APCI, Pos.): m/z 206 (M+H)+.
Anal. Calcd for C9H11N5O (205.22): C, 52.67; H, 5.40; N, 34.13.
Found: C, 52.60; H, 5.39; N, 34.09.
4.2. Chemistry
4.2.5. 6-(Butylamino)-5-cyanopyrazine-2-carboxamide (5)
Prepared according to the general procedure. Yellow solid.
Yield: 78%. Mp 157–159 °C. IR (cmÀ1): 3467, 3348, 3185, 2957,
2871, 2229, 1689, 1579, 1537, 1511, 1234, 1206, 893, 800, 692.
1H NMR (300 MHz, DMSO) d: 8.30 (1H, s, H3), 8.05 (1H, br s,
NH2), 7.90 (1H, br s, NH2), 7.79 (1H, t, J = 6.5 Hz, NH), 3.48 (2H, q,
J = 6.5 Hz, NCH2), 1.59–1.45 (2H, m, CH2), 1.40–1.23 (2H, m, CH2),
0.88 (3H, t, J = 6.5 Hz, CH3). 13C NMR (75 MHz, DMSO) d: 164.8,
154.3, 145.7, 130.5, 115.8, 115.1, 40.2, 31.0, 19.7, 14.0. MS (APCI,
Pos.): m/z 220 (M+H)+. Anal. Calcd for C10H13N5O (219.24): C,
54.78; H, 5.98; N, 31.94. Found: C, 54.71; H, 6.04; N, 31.88.
4.2.1. 6-Chloro-5-cyanopyrazine-2-carboxamide (1)
7.5 g of 3-chloropyrazine-2-carbonitrile (0.054 mol) was dis-
solved in 50 mL (1.26 mol) of formamide. The mixture was stirred
at 90 °C and 13 g (0.057 mol) of (NH4)2S2O8 was added portion
wise in 1.5 h period. The mixture was kept at 90 °C for another
hour and then cooled to rt. Approx. 70 mL of water was poured into
the reaction flask, stirred, and the mixture was let to stand over-
night. Undissolved precipitate (product) was separated by filtra-
tion. The filtrate was extracted by chloroform for 12 h to gain
another portion of product. Combined portions were purified by
flash chromatography (hexane/ethyl acetate, silica). White solid.
Yield: 41%. Mp 245–247 °C. IR (cmÀ1): 3452, 3288, 3057, 1697,
1681, 1317, 1171, 1090, 963, 905, 799, 711. 1H NMR (300 MHz,
DMSO) d 9.25–9.22 (1H, m, H3), 8.47 (1H, br s, NH2), 8.16 (1H, br
s, NH2). 13C NMR (75 MHz, DMSO) d 163.0, 149.4, 146.8, 142.5,
131.7, 114.7. MS (APCI, Pos.): not ionized. Anal. Calcd for
C6H3ClN4O (182.57): C, 39.47; H, 1.66; Cl, 19.42; N, 30.69. Found:
C, 39.44; H, 1.72; Cl, 19.39; N, 30.68.
4.2.6. 5-Cyano-6-(pentylamino)pyrazine-2-carboxamide (6)
Prepared according to the general procedure. Yellow solid.
Yield: 88%. Mp 172–174 °C. IR (cmÀ1): 3463, 3345, 3193, 2954,
2939, 2863, 2230, 1687, 1577, 1533, 1507, 1225, 1195, 927, 803,
706. 1H NMR (300 MHz, DMSO) d: 8.30 (1H, s, H3), 8.05 (1H, br s,
NH2), 7.90 (1H, br s, NH2), 7.78 (1H, t, J = 6.1 Hz, NH), 3.47 (2H, q,
J = 6.1 Hz, NCH2), 1.62–1.46 (2H, m, CH2), 1.37–1.19 (4H, m, CH2),
0.85 (3H, t, J = 6.1 Hz, CH3). 13C NMR (75 MHz, DMSO) d: 164.8,
154.3, 145.7, 130.5, 115.7, 115.1, 40.3, 28.7, 28.5, 22.1, 14.1. MS
(APCI, Pos.): m/z 234 (M+H)+. Anal. Calcd for C11H15N5O (233.27):
C, 56.64; H, 6.48; N, 30.02. Found: C, 56.68; H, 6.55; N, 29.98.
4.2.2. 5-Cyano-6-(methylamino)pyrazine-2-carboxamide (2)
Prepared according to the general procedure (see Section
4.2.18) with following changes applied. 5 mL of 30% (m/m) aque-
ous solution of methylamine was placed to a flask slightly heated
to approx. 40 °C. Gaseous methylamine was produced by discon-
tinuous adding of 10% (m/m, aq solution) KOH. The gas was dried
by waterless K2CO3 and let to bubble through the reaction mixture.
The completion of the reaction was checked by TLC chromatogra-
phy. Yellow solid. Yield: 55%. Mp 274–275 °C. IR (cmÀ1): 3493,
3371, 3153, 2924, 2854, 2228, 1692, 1586, 1536, 1512, 1233,
1200, 898, 805, 707. 1H NMR (300 MHz, DMSO) d 8.31 (1H, s,
H3), 8.13 (1H, br s, NH2), 7.90 (1H, br s, NH2), 7.82–7.72 (1H, m,
NH), 2.94 (3H, d, J = 4.7 Hz, NCH3). 13C NMR (75 MHz, DMSO) d
164.8, 154.7, 145.7, 130.5, 115.7, 115.4, 27.9. MS (APCI, Pos.): m/z
178 (M+H)+. Anal. Calcd for C7H7N5O (177.16): C, 47.46; H, 3.98;
N, 39.53. Found: C, 47.40; H, 4.02; N, 39.48.
4.2.7. 5-Cyano-6-(hexylamino)pyrazine-2-carboxamide (7)
Prepared according to the general procedure. Yellow solid.
Yield: 84%. Mp 145–147 °C. IR (cmÀ1): 3449, 3345, 3130, 2961,
2926, 2859, 2228, 1693, 1581, 1535, 1514, 1237, 1199, 899, 803,
708. 1H NMR (300 MHz, DMSO) d 8.30 (1H, s, H3), 8.04 (1H, br s,
NH2), 7.90 (1H, br s, NH2), 7.77 (1H, t, J = 6.2 Hz, NH), 3.47 (2H, q,
J = 6.2 Hz, NCH2), 1.62–1.45 (2H, m, CH2), 1.38–1.17 (6H, m, CH2),
0.84 (3H, t, J = 6.2 Hz, CH3). 13C NMR (75 MHz, DMSO) d 164.8,
154.3, 145.6, 130.5, 115.7, 115.0, 40.2, 31.2, 28.7, 26.2, 22.3, 14.1.
MS (APCI, Pos.): m/z 248 (M+H)+. Anal. Calcd for C12H17N5O
(247.30): C, 58.28; H, 6.93; N, 28.32. Found: C, 58.22; H, 7.00; N,
27.34.
4.2.8. 5-Cyano-6-(heptylamino)pyrazine-2-carboxamide (8)
Prepared according to the general procedure. Yellow solid.
Yield: 72%. Mp 135–136 °C. IR (cmÀ1): 3449, 3349, 3136, 2959,
2925, 2855, 2228, 1694, 1583, 1536, 1512, 1232, 1202, 896, 803,
708. 1H NMR (300 MHz, CDCl3) d 8.66 (1H, s, H3), 7.42 (1H, br s,
NH2), 6.27 (1H, br s, NH2), 5.57 (1H, t, J = 6.3 Hz, NH), 3.51 (2H, q,
J = 6.3 Hz, NCH2), 1.75–1.61 (2H, m, CH2), 1.48–1.22 (8H, m, CH2),
0.88 (3H, t, J = 6.3 Hz, CH3). 13C NMR (75 MHz, CDCl3) d 164.8,
154.0, 143.8, 132.2, 117.2, 114.8, 41.4, 31.7, 29.0, 28.9, 26.9, 22.5,
14.0. MS (APCI, Pos.): m/z 262 (M+H)+. Anal. Calcd for C13H19N5O
(261.32): C, 59.75; H, 7.33; N, 26.80. Found: C, 59.85; H, 7.42; N,
26.69.
4.2.3. 5-Cyano-6-(ethylamino)pyrazine-2-carboxamide (3)
Prepared according to the general procedure with following
changes applied. 5 mL of liquid ethylamine (Bp 16.6 °C) was
placed into flask cooled with ice. The flask was let to warm up
slowly, the vapors of ethylamine were dried by waterless
K2CO3 and let to bubble through the reaction mixture. The com-
pletion of the reaction was checked by TLC chromatography. Yel-
low solid. Yield: 59%. Mp 192–193 °C. IR (cmÀ1): 3484, 3358,
3289, 2925, 2223, 1687, 1587, 1534, 1508, 1233, 1208, 909,
815, 689. 1H NMR (300 MHz, DMSO) d 8.31 (1H, s, H3), 8.10
(1H, br s, NH2), 7.89 (1H, br s, NH2), 7.80 (1H, t, J = 5.1 Hz,
NH), 3.59–3.44 (2H, m, NCH2), 1.13 (3H, t, J = 7.1 Hz, CH3). 13C
NMR (75 MHz, DMSO)
d 164.8, 154.1, 145.7, 130.6, 115.7,
4.2.9. 5-Cyano-6-(oktylamino)pyrazine-2-carboxamide (9)
Prepared according to the general procedure. Yellow solid.
Yield: 73%. Mp 135–136 °C. IR (cmÀ1): 3442, 3341, 3131, 2913,
2851, 2231, 1697, 1578, 1536, 1504, 1232, 1202, 895, 803, 710.
115.2, 35.5, 14.5. MS (APCI, Pos.): m/z 192 (M+H)+. Anal. Calcd
for C8H9N5O (191.19): C, 50.26; H, 4.74; N, 36.63. Found: C,
50.30; H, 4.79; N, 36.58.

J. Zitko et al. / Bioorg. Med. Chem. 19 (2011) 1471–1476
1475
1H NMR (300 MHz, CDCl3) d 8.65 (1H, s, H3), 7.42 (1H, br s, NH2),
6.28 (1H, br s, NH2), 5.60 (1H, t, J = 6.3 Hz, NH), 3.50 (2H, q,
J = 6.3 Hz, NCH2), 1.75–1.56 (2H, m, CH2), 1.46–1.18 (10H, m,
CH2), 0.87 (3H, t, J = 6.3 Hz, CH3). 13C NMR (75 MHz, CDCl3) d
164.8, 154.0, 143.8, 132.1, 117.2, 114.8, 41.4, 31.7, 29.2, 29.1,
29.0, 26.9, 22.6, 14.0. MS (APCI, Pos.): m/z 276 (M+H)+. Anal. Calcd
for C14H21N5O (275.35): C, 61.07; H, 7.69; N, 25.43. Found: C,
61.11; H, 7.75; N, 25.39.
132.7, 117.4, 115.5, 54.4, 46.7, 45.8. MS (APCI, Pos.): m/z 247
(M+H)+. Anal. Calcd for C11H14N6O (246.27): C, 53.65; H, 5.73; N,
34.13. Found: C, 53.57; H, 5.80; N, 34.09.
4.2.15. 5-Cyano-6-morpholinopyrazine-2-carboxamide (15)
Prepared according to the general procedure. Yellow solid.
Yield: 76%. Mp 194–195 °C. IR (cmÀ1): 3430, 3188, 2871, 2220,
1697, 1547, 1533, 1433, 1271, 1198, 1121, 988, 885, 797, 711. 1H
NMR (300 MHz, DMSO) d 8.51 (1H, s, H3), 8.27 (1H, br s, NH2),
7.93 (1H, br s, NH2), 3.89–3.82 (4H, m, OCH2), 3.75–3.68 (4H, m,
NCH2). 13C NMR (75 MHz, DMSO) d 164.5, 154.5, 144.6, 132.8,
117.4, 115.6, 66.0, 46.9. MS (APCI, Pos.): m/z 234 (M+H)+. Anal.
Calcd for C10H11N5O2 (233.23): C, 51.50; H, 4.75; N, 30.03. Found:
C, 51.52; H, 4.80; N, 30.07.
4.2.10. 5-Cyano-6-(cyclopentylamino)pyrazine-2-carboxamide
(10)
Prepared according to the general procedure. Yellow solid.
Yield: 62%. Mp 195–196 °C. IR (cmÀ1): 3449, 3337, 3138, 2956,
2924, 2856, 2231, 1693, 1577, 1533, 1505, 1231, 1203, 895, 803,
708. 1H NMR (300 MHz, DMSO) d 8.31 (1H, s, H3), 8.08 (1H, br s,
NH2), 7.90 (1H, br s, NH2), 7.63 (1H, d, J = 4.7 Hz, NH), 4.65–4.49
(1H, m, NCH), 2.05–1.88 (2H, m, CH2), 1.76–1.62 (2H, m, CH2),
1.62–1.44 (4H, m, CH2). 13C NMR (75 MHz, DMSO) d 164.8, 153.9,
145.6, 130.6, 115.8, 115.1, 52.1, 31.8, 23.9. MS (APCI, Pos.): m/z
232 (M+H)+. Anal. Calcd for C11H13N5O (231.25): C, 57.13; H,
5.67; N, 30.28. Found: C, 57.16; H, 5.75; N, 30.32.
4.2.16. 6-(Ethylamino)-5-(N-ethylcarbamimidoyl)pyrazine-2-
carboxamide (16)
By-product derived from (3). Yellow solid. Yield: 15% (related to
starting compd). Mp 150–151 °C. IR (cmÀ1): 3443, 3330, 3173,
2965, 2931, 2866, 1682, 1638, 1579, 1533, 1269, 1194, 1142,
866, 795, 731. 1H NMR (300 MHz, DMSO) d 10.79 (1H, br s, NH),
8.20 (1H, s, H3), 7.92 (1H, br s, NH2), 7.70 (1H, br s, NH2), 6.70
(2H, br s, NH), 3.49 (2H, q, J = 7.1 Hz, NCH2), 3.18 (2H, q,
J = 7.1 Hz, NCH2), 1.22 (3H, t, J = 7.1 Hz, CH3), 1.18 (3H, t,
J = 7.1 Hz, CH3). 13C NMR (75 MHz, DMSO) d 165.8, 155.2, 152.3,
143.2, 130.4, 126.6, 42.0, 34.8, 16.2, 14.7. MS (APCI, Pos.): m/z
237 (M+H)+. Anal. Calcd for C10H16N6O (236.27): Elemental Analy-
sis: C, 50.83; H, 6.83; N, 35.57. Found: C, 50.91; H, 6.74; N, 35.51.
4.2.11. 5-Cyano-6-(cyclohexylamino)pyrazine-2-carboxamide
(11)
Prepared according to the general procedure. Yellow solid.
Yield: 67%. Mp 191–192 °C. IR (cmÀ1): 3446, 3334, 3128, 2922,
2856, 2232, 1690, 1581, 1537, 1508, 1240, 1203, 894, 810, 710.
1H NMR (300 MHz, DMSO) d 8.30 (1H, s, H3), 8.09 (1H, br s,
NH2), 7.90 (1H, br s, NH2), 7.44 (1H, d, J = 7.6 Hz, NH), 4.31–4.06
(1H, m, NCH), 1.86–1.54 (5H, m, CH2), 1.47–1.26 (4H, m, CH2),
1.21–1.02 (1H, m, CH2). 13C NMR (75 MHz, DMSO) d 164.8, 153.4,
145.7, 130.6, 115.8, 114.9, 49.1, 32.0, 25.4, 24.9. MS (APCI, Pos.):
m/z 246 (M+H)+. Anal. Calcd for C12H15N5O (245.28): C, 58.76; H,
6.16; N, 28.55. Found: C, 58.84; H, 6.30; N, 28.63.
4.2.17. 6-(Octylamino)-5-(N-octylcarbamimidoyl)pyrazine-2-
carboxamide (17)
By-product derived from (9). Yellow solid. Yield: 22% (related to
starting compd). Mp 110–111 °C. IR (cmÀ1): 3443, 3391, 3208,
2957, 2919, 2851, 1676, 1649, 1624, 1583, 1568, 1533, 1518,
907, 798, 721. 1H NMR (300 MHz, CDCl3) d 10.53 (1H, br s, NH),
8.41 (1H, s, H3), 7.61 (1H, br s, NH2), 5.73 (1H, br s, NH2), 5.63
(2H, br s, NH), 3.51–3.36 (2H, m, NCH2), 3.26–3.06 (2H, m,
NCH2), 1.85–1.55 (6H, m, CH2), 1.55–1.16 (22H, m, CH2), 0.93–
0.84 (6H, m, CH3). 13C NMR (75 MHz, CDCl3) d 166.6, 154.5,
152.8, 141.8, 131.2, 127.3, 47.9, 40.6, 31.8, 31.1, 29.6, 29.4, 29.3,
29.1 28.7, 27.8, 27.4, 22.6, 14.1. MS (APCI, Pos.): m/z 405 (M+H)+.
Anal. Calcd for C22H40N6O (404.59): Elemental Analysis: C, 65.31;
H, 9.96; N, 20.77. Found: C, 65.20; H, 9.98; N, 20.65.
4.2.12. 5-Cyano-6-(cycloheptylamino)pyrazine-2-carboxamide
(12)
Prepared according to the general procedure. Yellow solid.
Yield: 70%. Mp 193–195 °C. IR (cmÀ1): 3447, 3335, 3126, 2924,
2907, 2850, 2232, 1698, 1578, 1536, 1508, 1234, 1206, 895, 806,
711. 1H NMR (300 MHz, DMSO) d 8.30 (1H, s, H3), 8.03 (1H, br s,
NH2), 7.91 (1H, br s, NH2), 7.51 (1H, d, J = 8.0 Hz, NH), 4.43–4.27
(1H, m, NCH), 1.90–1.36 (12H, m, CH2). 13C NMR (75 MHz, DMSO)
d 164.8, 153.2, 145.6, 130.5, 115.8, 115.1, 51.3, 33.8, 28.0, 23.9. MS
(APCI, Pos.): m/z 260 (M+H)+. Anal. Calcd for C13H17N5O (259.31): C,
60.21; H, 6.61; N, 27.01. Found: C, 60.13; H, 6.65; N, 27.02.
4.2.18. General coupling procedure
100 mg (0.548 mmol) of 6-chloro-5-cyanopyrazine-2-carbox-
amide (1) was dissolved in dry THF together with 67 mg
(0.657 mmol, 1.2 eq.) triethylamine. One equivalent of correspond-
ing amine was diluted with approx. 2 mL of THF and added drop-
wise to the reaction mixture during approx. 15 min while
stirring. The mixture turned yellow and was stirred for next
15 min at rt. The completion of the reaction was checked by TLC
chromatography. The mixture was filtered and the product was ab-
sorbed on silica by solvent evaporation. The product was purified
by flash chromatography (hexane/ethyl acetate gradient elution
with 1% (v/v) of acetic acid, silica).
4.2.13. 5-Cyano-6-(diethylamino)pyrazine-2-carboxamide (13)
Prepared according to the general procedure. Yellow solid.
Yield: 76%. Mp 120–122 °C. IR (cmÀ1): 3449, 3417, 3150, 2917,
2979, 2935, 2219, 1691, 1557, 1527, 1504, 1229, 1205, 1166,
892, 803, 713. 1H NMR (300 MHz, DMSO) d 8.38 (1H, s, H3), 8.38
(1H, br s, NH2), 7.92 (1H, br s, NH2), 3.75 (4H, q, J = 6.9 Hz,
NCH2), 1.20 (6H, t, J = 6.9 Hz, CH3). 13C NMR (75 MHz, DMSO) d
164.7, 152.5, 145.0, 131.0, 118.0, 112.2, 43.6, 13.3. MS (APCI,
Pos.): m/z 220 (M+H)+. Anal. Calcd for C10H13N5O (219.24): C,
54.78; H, 5.98; N, 31.94. Found: C, 54.70; H, 6.12; N, 31.85.
4.3. Evaluation of in vitro antimycobacterial activity
4.2.14. 5-Cyano-6-(4-methylpiperazin-1-yl)pyrazine-2-
carboxamide (14)
Microdilution panel method. The antimycobacterial assay was
provided by Regional Hospital in Pardubice (Czech Republic). Five
strains were used: M. tuberculosis H37Rv CNCTC My 331/88,
M. kansasii CNCTC My 235/80, M. avium CNCTC My 80/72 and
M. avium CNCTC My 152/73 (Czech National Collection of Type
Cultures, National Institute of Public Health, Prague, Czech
Republic). Tested compounds were dissolved in DMSO, diluted
Prepared according to the general procedure. Yellow solid.
Yield: 76%. Mp 188–189 °C. IR (cmÀ1): 3373, 2932, 2220, 1672,
1551, 1447, 1428, 1226, 1198, 897, 790, 715. 1H NMR (300 MHz,
DMSO) d 8.48 (1H, s, H3), 8.23 (1H, br s, NH2), 7.94 (1H, br s,
NH2), 3.83 (4H, t, J = 4.9 Hz, NCH2), 2.43 (4H, t, J = 4.9 Hz, NCH2),
2.21 (3H, s, CH3). 13C NMR (75 MHz, DMSO) d 164.5, 154.6, 144.7,

1476
J. Zitko et al. / Bioorg. Med. Chem. 19 (2011) 1471–1476
with Šula’s semisynthetic medium (Trios, Prague, Czech Republic)
and placed into microdilution panel. Tested species were added
in the form of suspension in isotonic saline solution. Compounds
were tested at final concentrations of 200, 100, 50, 25, 12.5, 6.25
ton Dickinson, Detroit, MI), buffered to pH 7.0. Controls consisted
of medium and DMSO alone. The final concentration of DMSO in
the test medium did not exceed 1% (v/v) of the total solution com-
position. The minimum inhibitory concentration (MIC), defined as
95% inhibition of bacterial growth as compared to control, was
determined after 24 and 48 h of static incubation at 35 °C.
and 3.125 lg/mL. The final concentration of DMSO did not exceed
1% (v/v). It was confirmed that at this concentration DMSO itself
did not affect the growth of mycobacteria. PZA was used as a stan-
dard. The cultures were grown in Šula’s semisynthetic medium at
pH 5.5 and 37 °C. The antimycobacterial activity was determined
visually after 14 days (6 days for M. kansasii) of incubation as
4.6. Lipophilicity calculations
C log P values (the logarithm of n-octanol/water partition co-
efficient P based on established chemical interactions) were calcu-
lated using CS ChemOffice Ultra ver. 11.0 (CambridgeSoft,
Cambridge, MA, USA).
MIC [lg/mL], that is, the lowest used concentration of tested sub-
stance which inhibited the growth of mycobacteria.
4.4. Evaluation of in vitro antifungal activity
Acknowledgments
In vitro antifungal screening was carried out using microdilu-
tion broth method.17 Compounds were dissolved in DMSO and di-
This study was supported by the Grant Agency of Charles Uni-
versity in Prague (B-CH/120509) and by the Ministry of Education
of the Czech Republic (MSM0021620822 and SVV-2010-261-001).
luted in
a twofold manner with RPMI 1640 medium with
glutamine. The final concentration of DMSO in the test medium
did not exceed 2.5% (v/v) of the total solution composition. Static
incubation was performed in RPMI 1640 medium with glutamine
buffered to pH 7.0 (3-morpholinopropane-1-sulfonic acid), in dark
and humid, at 35 °C for 24 and 48 h, 72 and 140 h for Trichophyton
mentagrophytes, respectively. Drug-free controls were included.
Fluconazole (FLU) and nystatin (NYS) were used as standards.
Tested species: Candida albicans ATCC 44859 (CA), Candida tropical-
is 156 (CT), Candida krusei E28 (CK), Candida glabrata 20/I (CG), Tri-
chosporon beigelii 1188 (TB), Aspergillus fumigatus 231 (AF), Absidia
corymbifera 272 (AC), Trichophyton mentagrophytes 445 (TM).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.054. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
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