Synthesis and biological evaluation of N-alkyl-3-(alkylamino)-pyrazine-2-carboxamides

Article abstract of DOI:10.3390/molecules20058687

A series of N-alkyl-3-(alkylamino)pyrazine-2-carboxamides and their N-alkyl-3-chloropyrazine-2-carboxamide precursors were prepared. All compounds were characterized by analytical methods and tested for antimicrobial and antiviral activity. The antimycobacterial MIC values against Mycobacterium tuberculosis H37Rv of the most effective compounds, 3-(hexylamino)-, 3-(heptylamino)- and 3-(octylamino)-N-methylpyrazine-2-carboxamides 14-16, was 25 μg/mL. The compounds inhibited photosystem 2 photosynthetic electron transport (PET) in spinach chloroplasts. This activity was strongly connected with the lipophilicity of the compounds. For effective PET inhibition longer alkyl chains in the 3-(alkylamino) substituent in the N-alkyl-3-(alkylamino)pyrazine-2-carboxamide molecule were more favourable than two shorter alkyl chains.

Full text of DOI:10.3390/molecules20058687

Molecules 2015, 20, 8687-8711; doi:10.3390/molecules20058687
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of N-Alkyl-3-(alkylamino)-
pyrazine-2-carboxamides
1
,
1
1
1
1
Lucia Semelkova *, Klara Konecna , Pavla Paterova , Vladimir Kubicek , Jiri Kunes ,
1
1
2
3
4
Lucie Novakova , Jan Marek , Lieve Naesens , Matus Pesko , Katarina Kralova ,
1
1,
Martin Dolezal and Jan Zitko *
1
Faculty of Pharmacy, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005,
Czech Republic; E-Mails: konecna@faf.cuni.cz (K.K.); pavla.paterova@fnhk.cz (P.P.);
kubicek@faf.cuni.cz (V.K.); kunes@faf.cuni.cz (J.K.); novakoval@faf.cuni.cz (L.N.);
marej1aa@faf.cuni.cz (J.M.); dolezalm@faf.cuni.cz (M.D.)
2
Laboratory of Virology and Chemotherapy, Rega Institute KU Leuven, Minderbroedersstraat 10,
Leuven B-3000, Belgium; E-Mail: lieve.naesens@rega.kuleuven.be
3
Department of Environmental Ecology, Faculty of Natural Sciences, Comenius University,
Mlynska Dolina CH-2, Bratislava 84215, Slovakia; E-Mail: matus.pesko@gmail.com
4
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska Dolina CH-2,
Bratislava 84215, Slovakia; E-Mail: kralova@fns.uniba.sk
*
Authors to whom correspondence should be addressed; E-Mails: semelkol@faf.cuni.cz (L.S.);
jan.zitko@faf.cuni.cz (J.Z.); Tel.: +420-495-067-275 (L.S.); +420-495-067-272 (J.Z.);
Fax: +420-495-518-002 (L.S. & J.Z.).
Academic Editor: Derek J. McPhee
Received: 20 April 2015 / Accepted: 8 May 2015 / Published: 14 May 2015
Abstract: A series of N-alkyl-3-(alkylamino)pyrazine-2-carboxamides and their N-alkyl-3-
chloropyrazine-2-carboxamide precursors were prepared. All compounds were
characterized by analytical methods and tested for antimicrobial and antiviral activity. The
antimycobacterial MIC values against Mycobacterium tuberculosis H37Rv of the most
effective compounds, 3-(hexylamino)-, 3-(heptylamino)- and 3-(octylamino)-N-methyl-
pyrazine-2-carboxamides 14‒16, was 25 μg/mL. The compounds inhibited photosystem 2
photosynthetic electron transport (PET) in spinach chloroplasts. This activity was strongly
connected with the lipophilicity of the compounds. For effective PET inhibition longer alkyl
chains in the 3-(alkylamino) substituent in the N-alkyl-3-(alkylamino)pyrazine-2-
carboxamide molecule were more favourable than two shorter alkyl chains.

Molecules 2015, 20
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Keywords: pyrazinamide; pyrazine; alkylation; aminodehalogenation; antimycobacterial
activity; inhibition of photosynthetic electron transport; structure-activity relationships
1
. Introduction
Pyrazines are symmetrical heterocyclic aromatic organic compounds. Pyrazine is a weaker base than
pyridine, pyridazine and pyrimidine. Pyrazine derivatives occur in many natural sources and can be
synthesized chemically or biologically. In animals and plants, they are considered to be alert signal
molecules, functioning as deterrents or attractants depending on the circumstances without having
harmful or beneficial effect themselves. Pyrazines contribute to many aromas and flavours of heated
foods such as beef products, toasted barley, cocoa, coffee, peanuts, popcorn, potato chips, rye crisp bread
and roasted filberts, as well as in fresh foods like tomatoes, peas, green bell peppers, asparagus, kohlrabi
and dairy products. The pyrazine moiety is an important part of many clinically used drugs, including
anticancer, diuretic [1], antidiabetic, antithrombotic, antidepressants or anti-infective (antituberculotics,
bactericides and fungicides) agents, and offers many possibilities in drug development [2–4].
Tuberculosis (TB) remains a serious global problem and the need for new drugs is still actual [5–7].
One of the most important drugs for TB treatment is pyrazinamide (PZA), which belongs to the
first-line drugs for TB treatment and has been used extensively since 1980s. PZA has an excellent
sterilizing effect on semi-dormant tubercle bacilli and, when used in combination with rifampicin, helps
in shortening the duration of treatment [8]. One of the mechanisms of action is competitive inhibition of
NADPH binding to Mycobacterium tuberculosis Fatty Acid Synthase I. This enzyme is devoted to the
synthesis of common fatty acids and of specific mycolic acids, which constitute the major lipid
component of the envelope and form the external mycomembrane. 5-Chloropyrazine-2-carboxamide
analogues act as well as PZA [9,10]. 5-(Alkylamino)pyrazine-2-carboxamides and
6
-(alkylamino)pyrazine-2-carboxamides prepared previously by Servusová et al. [11], showed
an interesting in vitro whole cell antimycobacterial activity. Based on these results we decided to prepare
a series of 3-(alkylamino)pyrazine-2-carboxamides, to evaluate the importance of positional isomerism
in these derivatives.
Several herbicides acting as photosynthesis inhibitors (acylanilides, thioacylanilides, phenylcarbamates,
ureas, etc.) have (thio)carbamoyl group, -NHCO- or -NHCS-, in their molecules [12–14]. Some
commercially available herbicides act by reversible binding to photosystem 2 (PS2), a membrane-protein
complex in the thylakoid membranes which catalyses the oxidation of water and the reduction of
plastoquinone [15] and thereby inhibit photosynthetic electron transport (PET). Experimental studies
have established that many PS 2 herbicides bind non-covalently to a 32 kDa protein in the PS2 complex
and inhibit electron transfer between primary electron acceptor—quinone Q
A
and the secondary electron
acceptor—quinone Q on the reducing side of PS2 [16]. Numerous PS2 herbicides contain hydrophobic
B
components as well as a flat polar component. The function of the hydrophobic components is to increase
the lipid solubility of the entire herbicide molecule and to fit the hydrophobic surface of the herbicide
binding site and it is assumed that the flat polar component binds electrostatically at a highly polar
protein site [17]. Using EPR spectroscopy it was shown that tyrosine radicals Tyr
Z
and Tyr which are
D

Molecules 2015, 20
situated in D and D
8689
1
2
proteins on the donor side of PS2 interacted with some organic compounds, e.g.,
substituted benzanilides [18] or substituted 2,6-disubstituted pyridine-4-thiocarboxamides [19] or
anilides of pyrazine-2-carboxylic acids [14,20] and due to this interaction interruption of the
photosynthetic electron transport occurred.
This study is focused on preparation of alkyl substituted derivatives of PZA. More specifically, it
deals with the length of the alkylamino chain in position 3 and its influence on biological effects in
comparison with previously evaluated 5- and 6-alkylamino isomers. Antimycobacterial activity of all
prepared compounds was determined and compounds were evaluated also for inhibition of photosynthetic
electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The structure-activity relationships
between the chemical structure and in vitro biological activities of evaluated compounds are discussed.
2
. Results and Discussion
2
.1. Chemistry
We prepared a series of pyrazinamide alkylamino derivatives according to the general procedures
shown in Scheme 1. 3-Chloro-N-methylpyrazine-2-carboxamide (1) was previously prepared by
Allen et al. [21], 3-chloro-N-propylpyrazine-2-carboxamide (3) by Zhu et al. [22] and N-methyl-3-
(
methylamino)pyrazine-2-carboxamide (9) by Albert et al. [23], but they were not tested
for any biological activity. The other 27 compounds are novel. Precursors 1–8 were synthesized
from commercially available 3-chloropyrazine-2-carbonitrile by hydrolysis (Scheme 1, step a) to the
corresponding acid, conversion to the acyl chloride (step b) and its aminolysis (step c) by the
corresponding amine. Final products 9–30 were prepared in yields ranging from 41% to 98% by
aminodehalogenation (step d) in a microwave reactor with a focused field.
O
N
N
CN
Cl
N
N
COOH
Cl
N
N
Cl
a
b
Cl
7
5%
c
O
O
N
N
R1
N
N
R1
d
N
N
H
H
NH
R2
Cl
9
- 30
1 - 8
46%-64%
4
1%-98%
2 2
Reagents and Conditions: (a) NaOH, H O, reflux 7–8 h, HCl; (b) SOCl , DMF, toluene, 95 °C, 1 h; (c) alkylamine,
TEA, acetone, RT, overnight; (d) alkylamine, pyridine, methanol, MW: 150 °C, 30 min, 100 W.
Scheme 1. Synthesis of final compounds 1–30.
1
13
All compounds were characterized by H-, C-NMR and IR spectroscopy, solid compounds by
melting points and elemental analysis, and liquid compounds by HRMS.

Molecules 2015, 20
.2. Lipophilicity
Lipophilicity is one of the most important drug characteristics. It plays a significant role in penetration
8690
2
through biological membranes.
Table 1. Structure of prepared compounds, calculated (log P) and experimentally measured
(log k) lipophilicity parameters, biological activity against M. tuberculosis H37Rv expressed
as minimal inhibitory concentration (MIC) and PET-inhibiting activity expressed by
IC50 value.
MIC M. tuberculosis
No.
Structure
R1
R2
log P
log k
IC50 [mmol/L]
[
µg/mL]
>100
>100
>100
>100
>100
100
1
2
3
4
5
6
7
8
9
CH
3
-
−0.17 −0.747
ND
C
2
C
3
C
4
H
5
7
9
-
0.16
0.65
1.07
1.49
1.90
2.32
2.74
−0.591
−0.414
−0.209
0.012
ND
H
H
-
ND
-
4.014
1.390
1.067
0.256
0.055
5.646
ND
C
C
C
C
5
H
11
13
15
17
-
-
6
7
8
H
H
H
0.239
-
0.470
100
-
0.702
50
CH
3
−0.86 −0.279
−0.52 −0.052
>100
>100
>100
100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
2
C
3
C
4
H
5
7
9
H
H
−0.04
0.38
0.80
1.21
1.63
2.05
0.166
0.401
0.638
0.876
1.118
1.359
ND
0.523
0.544
0.125
0.065
0.064
ND
CH
3
C
C
C
C
5
H
11
13
15
17
3
50
6
7
8
H
H
H
25
25
25
CH
−0.52 −0.063
>100
>100
>100
>100
50
C
2
C
3
C
4
H
5
7
9
−0.19
0.30
0.72
1.13
1.55
1.97
2.39
0.79
1.62
2.46
3.29
4.12
4.96
2.2
0.163
0.382
0.617
0.855
1.094
1.337
1.579
0.600
1.077
1.557
2.041
2.503
2.981
ND
H
H
2.047
1.090
0.301
0.078
0.048
0.056
1.303
0.202
0.105
0.272
1.123
0.752
0.002
2
C H
5
C
C
C
C
5
H
11
13
15
17
6
7
8
H
H
H
50
50
50
C
3
H
H
7
9
C
3
4
H
H
7
>100
50
C
4
C
9
C
C
C
C
5
H
11
13
15
17
C
C
C
C
5
H
H
H
H
11
13
15
17
>100
>100
>100
>100
6
7
8
H
H
H
6
7
8
DCMU
INH
−0.64
0.1−0.39
Experimentally determined values of lipophilicity log k and calculated values of log P of prepared
compounds are summarized in Table 1 together with corresponding IC50 values related to PET inhibition

Molecules 2015, 20
8691
in spinach chloroplasts and MIC values related to antimycobacterial activity against M. tuberculosis.
According to their structure, the prepared compounds could be divided into four groups: N-alkyl-3-
chloropyrazine-2-carboxamides (group A; 1–8), 3-(alkylamino)-N-methylpyrazine-2-carboxamides
(
group B; 9–16), 3-(alkylamino)-N-ethylpyrazine-2-carboxamides (group C; 17–24) and N-alkyl-3-
alkylamino)pyrazine-2-carboxamides (group D; 25–30). At comparable log P values the experimentally
(
determined log k values of N-alkyl-3-chloropyrazine-2-carboxamides (group A) were lower than the
log k values estimated for compounds of groups B, C and D (Figure 1). The dependences of log k vs.
log P were linear for eight compounds of group A (1) as well as for 22 compounds belonging to groups
B, C and D (2) and the corresponding correlations provided excellent results of statistical analysis:
logꢀꢁ ꢂ ꢃ0.700ꢄꢅ 0.021ꢆ ꢇ 0.499ꢄꢅ 0.013ꢆ log ꢈ
r ꢂ 0.998; ꢀn ꢂ 8; ꢀs ꢂ 0.036; ꢀF ꢂ 1400.4
(1)
logꢀꢁ ꢂ 0.213ꢄꢅ 0.007ꢆ ꢇ 0.557ꢄꢅ 0.004ꢆ log ꢈ
r ꢂ 0.9995; ꢀn ꢂ 22; ꢀs ꢂ 0.026; ꢀF ꢂ 22116.7
(2)
Figure 1. Plot of experimentally measured log k parameter on calculated log P.
2
.3. Biological Activity
.3.1. Antimycobacterial Activity
Compounds 9–30 were tested for in vitro whole cell activity against M. tuberculosis H37Rv,
2
M. kansasii and M. avium. Precursors 1–8 were tested against M. tuberculosis H37Rv only. The most
active substances against M. tuberculosis were compounds 14–16, i.e., compounds with long alkyl
chains (hexyl, heptyl and octyl) in the molecule. However, their activity expressed as minimal inhibitory
concentration (MIC) was only 25 μg/mL (corresponding to 105.8, 99.9 and 94.6 μmol/L, respectively),
while the activity of standard INH was 0.1–0.39 μg/mL (i.e., 0.7–2.9 μmol/L).
The antimycobacterial activity of compounds was closely connected with lipophilicity of the
compounds (Table 1, Figure 2). From the series of N-alkyl-3-chloropyrazine-2-carboxamides (group A)
the MIC values could be estimated only for three compounds and the results indicate that a strong
increase in antimycobacterial activity was observed up to log k = 0.702. The dependence of log (1/MIC)

Molecules 2015, 20
8692
on log k for 3-(alkylamino)-N-methylpyrazine-2-carboxamides (group B) was bilinear and strong
2
activity increase was observed for log k from 0.401 (12, R = C
4
H
9
) to 0.876 (14, C H13), while with
6
further lipophilicity increase up to log k = 1.359 the inhibitory activity expressed in molar concentrations
slightly increased (14–16).
Figure 2. Dependence of antimycobacterial activity of tested compounds against
M. tuberculosis H37Rv on log k.
Considerably lower antimycobacterial activity against M. tuberculosis of 3-(alkylamino)-N-
ethylpyrazine-2-carboxamides (group C) in comparison with 3-(alkylamino)-N-methylpyrazine-2-
carboxamides (group B) with comparable lipophilicity indicates that substitution of –CONHCH
substituent in position 2 of the pyrazine ring with –CONHC accompanied with the reduction of alkyl
chain length by one CH group in 3-alkylamino substituent led to significant activity decrease (Table 1).
For example, compound 14 (group B, R = CH
aliphatic alkyl chains, lipophilicity of log k = 0.876 and MIC = 25 μg/mL. Compound 21 (group C,
3
2 5
H
2
1
2
3
, R = C H13) has a total number of seven carbons in
6
1
2
R = C
2
H
5
, R = C H11) has an equal number of carbons in aliphatic alkyl chains, similar lipophilicity of
5
log k = 0.855, but MIC = 50 μg/mL, i.e., approx. half of the activity of compound 14. Consequently,
from the aspect of antimycobacterial activity at comparable lipophilicity, longer alkyl chain in
3
-(alkylamino) substituent is favourable.
Only one compound from group D containing two butyl groups in its molecule (26, R = R = C
1
2
4
H )
9
was found to be active against M. tuberculosis H37Rv and its activity was comparable with compound 22
from group C with similar lipophilicity (Figure 2).
In comparison to derivatives with alkylamino chain in position 5 or 6 previously prepared
by Servusová et al., i.e., 5-octylaminopyrazine-2-carboxamide (MIC = 6.26 μg/mL) and 6-octylamino-
pyrazine-2-carboxamide (MIC = 1.56 μg/mL) [11], the compounds presented in this paper were
generally of lower activity. This decrease in activity can be attributed to the change of the alkylamino
substituent to position 3 on the pyrazine core. On the other hand, compounds 14–16 showed better
activity than compounds previously prepared by Jandourek et al., i.e., 3-hexylamino-, 3-heptyamino-
and 3-octylaminopyrazine-2-carboxamide (MIC = 50 μg/mL) [24]. This indicates that formal
N-methylation of the carboxamide moiety slightly increases the activity. The activity of tested

Molecules 2015, 20
8693
compounds (group B, C, D) against M. kansasii was in general low (MIC >100 μg/mL) and only for
compounds 21–24 MIC = 50 μg/mL was estimated. There was no activity against M. avium.
2
.3.2. Antibacterial and Antifungal Activity
This evaluation was performed in order to obtain results for antifungal and antibacterial activity
against eight fungal strains and eight bacterial strains of clinical significance. The most effective
1
compound against Trichophyton mentagrophytes was 8 (R = C
8
H17) with MIC = 62.5 µmol/L, the
results of other active compounds are shown in Table 2.
Table 2. Antifungal activity of studied compounds against Trichophyton mentagrophytes.
Compound No.
MIC [µmol/L]
6
7
8
250
125
62.5
250
250
125
250
125
125
1
1
1
2
2
2
3
4
5
1
2
3
The dependence of antifungal activity of tested compounds against T. mentagrophytes is shown in
Figure 3. Although the MIC values were estimated only for two compounds of group A, it is evident
that antifungal activity of these compounds was significantly higher than the activity of compounds from
group B and C with comparable lipophilicity.
Figure 3. Dependence of antifungal activity of tested compounds against T. mentagrophytes on log k.
This indicates that the presence of chloro substituent in position 3 of pyrazine ring significantly
contributed to antifungal activity. For compounds of group B and D the antifungal activity against
T. mentagrophytes increased approximately linearly with increasing compound lipophilicity up to

Molecules 2015, 20
8694
log k ≈ 1.334 and with further increase of lipophilicity significant loss of activity was observed. This
indicates that for antifungal activity of tested compounds the total lipophilicity of substituents in position
2
and 3 of pyrazine ring is determinant. Compounds 6–8 were active against Aspergillus fumigatus and
Trichosporon asahii (MIC = 125 µmol/L). Compound 16 showed low activity against Staphylococcus
aureus (MIC = 250 µmol/L) and compounds 15, 19, 23 and 24 against Staphylococcus epidermidis
(
MIC = 250 µmol/L) in antibacterial assays.
2.3.3. Antiviral Activity
All substances were tested for their activity against diverse DNA and RNA viruses. The virus panel
included pathogens of medical importance such as herpesviruses, HIV and influenza virus. None of the
prepared compounds displayed any antiviral activity.
2.3.4. Cytotoxicity Assay
In vitro cytotoxicity assays on HeLa and Vero cells were performed using standard assays. The results
were expressed as minimum compound concentration (MCC) that causes a microscopically detectable
alteration of normal cell morphology. No cytotoxicity was detected up to the highest tested concentration
of 100 µmol/L, except for 30 (MCC = 100 µmol/L, Vero cells). The highest tested concentration for 28
in Vero cells assay was 4 µmol/L due to solubility issues.
2
.3.5. Evaluation of Photosynthetic Electron Transport (PET) Inhibiting Activity
The PET-inhibiting activity was expressed by negative logarithm of IC50 value (compound concentration
in mol/L causing 50% inhibition of PET). The IC50 values of studied pyrazine-2-carboxamides are shown
in Table 1. The PET-inhibiting activity of N-alkyl-3-chloropyrazine-2-carboxamides (group A)
expressed as log (1/IC50) increased linearly (r = 0.977, n = 5) with increasing lipophilicity of the
1
1
compounds from log k = −0.209 (4, R = C
4
H
9
) to log k = 0.702 (8, R = C
8
H
17) (Figure 4, black circles).
1
1
The less lipophilic compounds of group A 1 (R = CH
3
, log k = −0.747), 2 (R = C
2
H , log k = −0.591)
5
1
and 3 (R = C
3
H , log k = −0.414) were inactive. Linear dependence of PET-inhibiting activity on the
7
lipophilicity expressed as log k (r = 0.9778, n = 6) was observed also for 3-(alkylamino)-N-
methylpyrazine-2-carboxamide (group B, 9–16) (Figure 4, red circles), however N-methyl-3-
2
(
pentylamino)pyrazine-2-carboxamide (13
,
R = C
5
H11) showed lower activity as expected. On the other
hand, for N-alkyl-3-(alkylamino)pyrazine-2-carboxamides (group D, 25–30) bilinear dependence of log
1
2
(
1/IC50) on log k was estimated with optimum log k = 1.557 (27, R = R = C
5
H
11) (Figure 4, blue
13, log k = 2.041),
15, log k = 2.503) and 30 (R = R = C H17, log k = 2.981) showed strong linear
1
2
triangles). The activity of more lipophilic compounds of this series 28 (R = R = C H
6
1
2
1
2
2
9 (R = R = C
7
H
8
decrease with increasing compound lipophilicity. In the series of 3-(alkylamino)-N-ethylpyrazine-2-
carboxamides (group C, 17–24) the PET-inhibiting activity increased linearly with increasing
2
2
lipophilicity of the compounds between log k = 0.382 (19, R = C
3
H
7
) and log k = 1.337 (23, R = C
7
H
15
)
2
and with further prolongation of the alkyl chain to R = C
8
H
17 (24, log k = 1.579) slightly decreased
(
Figure 4, green triangles). The less lipophilic compounds of this series, 3-(methylamino)-N-

Molecules 2015, 20
8695
2
ethylpyrazine-2-carboxamide (17, R = CH
3
, log k = −0.063) and 3-(ethylamino)-N-ethypyrazine-2-
2
carboxamide (18, R = C
2
H , log k = 0.163) were not active.
5
Figure 4. Dependence of PET-inhibiting activity on lipophilicity of studied compounds
expressed as log k.
The comparison of PET-inhibiting activity N-alkyl-3-chloropyrazine-2-carboxamides of (group A;
–8) with that of N-alkyl-3-(alkylamino)pyrazine-2-carboxamides (group D; 25–30) showed that IC50
1
values of N-propyl-3-(propylamino)pyrazine-2-carboxamide (25, log k = 0.600, MIC = 1.303 mmol/L)
and N-butyl-3-(butylamino)pyrazine-2-carboxamide (26, log k = 1.077, MIC = 0.202 mmol/L) were 23.7
times and 3.67 times higher than that of N-octyl-3-chloropyrazine-2-carboxamide (8, log k = 0.702,
MIC = 0.055 mmol/L). Consequently, it is evident that for effective PET inhibition substituent with
longer alkyl chain in the molecule is more favourable than substituents with two shorter alkyl chains.
N-pentyl-3-(pentylamino)pyrazine-2-carboxamide (27, MIC = 0.105 mmol/L, log k = 1.557) was the
most effective compound of group D.
From comparison of 3-(alkylamino)-N-methylpyrazine-2-carboxamid (group B; 9–16) and
3-(alkylamino)-N-ethylpyrazine-2-carboxamide (group C; 17–24) at comparable compound lipophilicity
up to log k ≈ 1.117 compounds of group B showed higher PET-inhibiting activity than compounds of
group C, however, in the compound lipophilicity range from log k = 1.117 to log k =1.579 the differences
between activities of the compounds belonging to both groups were low. While the IC50 value of
3
-(propylamino)-N-ethylpyrazine-2-carboxamide (19, IC50 = 2.047 mmol/L, log k = 0.382) was 3.9-fold
higher than that of 3-(butylamino)-N-methylpyrazine-2-carboxamide (12, IC50 = 0.523 mmol/L,
log k = 0.401), the IC50 value of 3-(pentylamino)-N-ethylpyrazine-2-carboxamid (21, IC50 = 0.301 mmol/L,
log k = 0.855) was only 2.4-fold higher than that of 3-(hexylamino)-N-methylpyrazine-2-carboxamide
(
14, IC50 = 0.125, log k = 0.876).
The loss of a biological activity observed for amphiphilic compounds upon elongation of their
hydrophobic (hydrocarbon) part is called ‘cut-off’ effect [25,26]. The hydrophobic parts of such
compounds interact with lipidic parts of biological (including thylakoid) membranes. It could be noted
that water solubility of compounds with longer alkyl chain is limited and too large values of compound
partition coefficient did not enable the penetration of such molecules through hydrophilic (aqueous)

Molecules 2015, 20
8696
regions of biological membranes. Thus, the final concentration of long-chain compounds in the
membrane will be lower than that of compounds with shorter alkyl chain, and this phenomenon is
connected with loss of biological activity. According to the free volume theory the extent of membrane
disturbance due to incorporation of compound containing alkyl chain depends on the size of free volume
created under its alkyl chain which can be then filled up with chains of neighbouring lipids as well as on
the partition coefficient of the compounds [25–28]. Therefore the most effective disturbance of the
membrane and thus the highest inhibitory activity is shown by compounds with middle alkyl chain length
ensuring not only sufficiently high free volume under alkyl chain but also high concentration of the
compound in the membrane due to suitable value of compound partition coefficient.
Moreover compounds with CONH group(s) in their molecules can interact with amino acid
constituents in proteins resulting in the loss of their function. It should be stressed that the CONH group
is characteristic of many herbicides [12,29] and it could also to a certain extent contribute to
their inhibitory effects. For example, the determined IC50 value related to PET inhibition for
N,N'-bis(2-dimethylaminoethyl)ethanediamide (which does not contain a long alkyl chain) was found to
be 4.0 mmol/L, while the corresponding IC50 value estimated for 3,8-diaza-4,7-dioxodecane-1,10-
diylbis(nonyldimethyl)ammonium bromide was 1.74 mmol/L [30]. This indicates that CONH groups in
spacer of this surfactant molecule participated on the resulting inhibitory effects. Because studied
pyrazine-2-carboxamide derivatives do not contain in their structure longer alkyl chain than octyl, by
analogy with above mentioned surfactants it could be assumed that CONH group also participates in
PET inhibition, as it was shown previously by fluorescence experiments [24,31].
Strong dependence of the PET-inhibiting activity on the alkyl chain length of the alkoxy substituent
was estimated previously for esters of 2-, 3- and 4-alkoxy substituted phenylcarbamic acids
(
alkyl = methyl – decyl). For these compounds the dependence of log (1/IC50) vs. alkyl chain length
showed a typical quasi-parabolic course with maximum effect at 6–8 carbon atoms in the alkyl chain of
piperidinoethylesters [13], 7–9 carbon atoms in the alkyl chain of dimethylaminoethylesters [32] and
8–9 carbon atoms in the alkyl chain of piperidinopropyl esters of alkoxyphenylcarbamic acids [33,34].
Because the tested pyrazine-2-carboxamides inhibited Hill reaction, similarly to previously studied
pyrazine derivatives [14,20], they can be considered as photosystem 2 (PS2) inhibitors. The PS2
inhibitors can act on the donor and/or the acceptor side of PS2. Interaction of N-phenylpyrazine-2-
•
carboxamides with the D intermediate which is situated at 161st position in D
2
protein occurring on the
donor side of PS2 was confirmed previously by EPR spectroscopy. Due to this interaction the
photosynthetic electron transport from the oxygen evolving complex to the reaction centre of PS2 was
impaired and consequently, the electron transport between PS2 and PS1 was inhibited.
2
,5-Diphenylcarbazide (DPC) is an artificial electron donor acting in Z/D intermediate on the donor
side of PS2 [35]. After addition of DPC to chloroplasts which activity had been inhibited by studied
compounds to 75%, the PET was gradually restored with increasing DPC concentration. However, for
complete restoration of PET, approximately five-fold higher DPC concentration was necessary compared
to the applied concentration of inhibitor. This indicates that the studied compounds could damage PET
also in the section between P680 and secondary quinone acceptor Q
binding of compounds with herbicidal activity, e.g., atrazine or metribuzine, was found to be altered by
DPC presumably because of overlapping binding domain in the Q pocket, however DPC on the Q site
affected plastoquinone reduction only at relatively high concentrations [36,37]. For PS2 hebicides such
B
on the acceptor side of PS2. The
B
B

Molecules 2015, 20
8697
are DCMU or atrazin also a second binding site situated on the donor side of PS2 near Z/D intermediates
and the high-affinity Mn-binding sites was described by several researchers [38–40]. Based on these
finding as well as on above mentioned results related to interaction of N-phenylpyrazine-2-carboxamides
•
with the D intermediate on the donor side of PS2, we assume similar site of action also for the studied
pyrazinamide alkylamino derivatives.
The three amino acids with aromatic ring side chains—phenylalanine, tyrosine and especially
tryptophan—are sensitive to the local electrostatic environment in proteins and they will undergo
fluorescence wavelength and/or intensity changes upon whatever functional process a protein
performs [41]. Interaction of substituted pyrazine-2-carboxamides with residues of aromatic amino acids
(AAA), mainly tryptophan and tyrosine occurring in photosynthetic proteins situated predominantly in PS2,
was documented by the quenching of AAA fluorescence at 334 nm (Figure 5A). Figure 5A presents
fluorescence emission spectra of AAA of untreated spinach chloroplasts and of chloroplasts treated with
1
2
increasing concentrations of compound 16 (R = CH
3
, R = C H17). As shown in this Figure, the
8
quenching of the fluorescence of aromatic amino acids at 334 nm increased with increasing
concentration of pyrazine derivative In Figure 5B linear dependences of fluorescence intensity of AAA
1
2
(
expressed as % of control) on concentration of compounds 27 (R = R = C H13) and 16 are shown. The
6
extent of fluorescence decline with increasing compound concentration expressed by slope of
this dependence correlated with IC50 values determined using Hill reaction (16: IC50 = 64.0 μmol/L,
slope = −0.668, r = 0.989 and 27: IC50= 105.0 μmol/L, slope = −0.211, r = 0.992). These results indicate
that interaction of studied pyrazine derivatives with aromatic amino acids residues occurring in
photosynthetic proteins contributed to PET inhibition.
Figure 5. (A) Fluorescence emission spectra of aromatic amino acids in suspension of
spinach chloroplasts without and with compound 16 (c = 0, 10, 25, 49, 74 and 123 μmol/L;
the curves from top to bottom); (B) Dependence of intensity of aromatic amino acids
fluorescence emission band (expressed as % of control) at 340 nm in suspension of spinach
chloroplasts on the concentration of studied compounds: 27 and 16. Excitation wave
length λ = 275 nm; chlorophyll concentration 10 mg/L.

Molecules 2015, 20
8698
The quenching of the fluorescence of aromatic amino acids in the presence of 5-bromo- and
3
,5-dibromo-2-hydroxy-N-phenylbenzamides [42] and ring-substituted 2-hydroxynaphthalene-1-
carboxanilides [31] was observed previously.
3. Experimental Section
3.1. General Information
All chemicals were of reagent or higher grade of purity and were purchased from Sigma-Aldrich
(
Steinheim, Germany), unless stated otherwise. The progress of the reaction was checked by Thin Layer
®
Chromatography (TLC) (Alugram Sil G/UV254, Machery-Nagel, Postfach, Germany) with UV
detection using wavelength 254 nm. Microwave assisted reactions were performed in a CEM Discover
microwave reactor with a focused field (CEM Corporation, Matthews, NC, USA) connected to
an Explorer 24 autosampler (CEM Corporation) and this equipment was running under CEM’s
SynergyTM software for setting and monitoring the conditions of reactions. The temperature of the
reaction mixture was monitored by internal infrared sensor. All obtained products were purified by
®
preparative flash chromatograph CombiFlash Rf (Teledyne Isco Inc., Lincoln, NE, USA). The type of
elution was gradient, using the mixture of hexane (LachNer, Neratovice, Czech Republic) and ethyl
acetate (Penta, Prague, Czech Republic) as mobile phase. Silica gel (0.040–0.063 nm, Merck, Darmstadt,
Germany) was used as the stationary phase. NMR spectra were recorded on Varian Mercury-VxBB 300
1
13
with frequencies 299.95 MHz for H and 75.43 MHz for C or Varian VNMR S500 (499.87 MHz for
1
13
H and 125.71 MHz for C) spectrometers (Varian Corporation, Palo Alto, CA, USA). Chemical shifts
were reported in ppm (δ) and were referred indirectly to tetramethylsilane via signal of solvent (2.49 for
1
13
H and 39.7 for C in DMSO-d
6
). Infrared spectra were recorded with spectrometer FT-IR Nicolet 6700
(
Thermo Scientific, Waltham, MA, USA) using attenuated total reflectance (ATR) methodology.
Elemental analyses were measured with EA 1110 CHNS Analyzer (Fisons Instruments S. p. A., Carlo
Erba, Milano, Italy). UHPLC system Acquity UPLC I-class (Waters, Millford, MA, USA) coupled to
high resolution mass spectrometer (HRMS) Synapt G2Si (Waters, Manchester, UK) based on Q-TOF
were used for HRMS spectra measurement. Chromatography was performed using Acquity UPLC BEH
C18 (2.1 × 100 mm, 1.7 um) column using gradient elution with acetonitrile and 0.1% formic acid at
flow-rate 0.4 mL/min. Electrospray ionization was operated in positive mode. The ESI spectra were
recorded in the range 50–1200 m/z using leucine-enkefaline as a lock mass reference and sodium formate
for mass calibration. Melting points were assessed by SMP3 Stuart Scientific (Bibby Sterling Ltd.,
Staffordshire, UK) in open capillary and are uncorrected. Lipophilicity parameter log P was calculated
by software CS ChemBioDraw Ultra 13.0 (CambridgeSoft, Cambridge, MA, USA).
3.2. Synthesis
The starting compound 3-chloropyrazine-2-carbonitrile (Fluorochem Ltd., Hadfield, Derbyshire,
UK) was hydrolysed to 3-chloropyrazine-2-carboxylic acid (3-Cl-POA) in basic aqueous solution.
-Chloropyrazine-2-carbonitrile (35.8 mmol) was added into 10% solution of sodium hydroxide
125 mmol, 3.5 equiv). The reaction mixture was stirred and heated under reflux in an oil bath for
approximately 7–8 h. The progress of reaction was monitored by TLC in system butanol/acetic acid/water
3
(

Molecules 2015, 20
8699
(
4:1:5). The reaction mixture was acidified by 10% solution of hydrochloric acid to pH 3. Newly-emerged
crystals were suctioned.
-Cl-POA (1.0 g, 6.3 mmol) was dispersed in dry toluene (approx. 50 mL) with thionyl chloride
1.4 mL, 18.9 mmol, 3 equiv.) and a catalytic amount (1–2 drops) of N,N-dimethylformamide (DMF).
The reaction mixture in round bottomed flask was stirred and heated in an oil bath under a condenser at
5 °C for approximately 1 h. Solvents were evaporated in vacuo and the residue was azeotroped with
3
(
9
dry toluene (3 × 20 mL) to remove the unreacted SOCl
which was used without further purification.
2
to yield crude acyl chloride [43] as brown solid,
The whole amount of 3-chloropyrazine-2-carbonyl chloride prepared in the previous step was
dissolved in dry acetone. An appropriate alkylamine (18.9 mmol, 3 equiv.) along with triethylamine
(
0.64 g, 6.3 mmol, 1 equiv.) were added to the reaction mixture and stirred at laboratory temperature
overnight [43]. The progress of reaction was checked by TLC in system hexane/ethyl acetate
1:1 or 2:1). The reaction mixture was adsorbed to silica by removing the solvents in vacuo and the
product was purified by flash chromatography using gradient elution with ethyl acetate in hexane.
Precursors 1–8 were recrystallized from EtOH/H O if needed.
(
2
Aminodehalogenation of N-alkyl-3-chloropyrazine-2-carboxamides 1–8 was completed using a
microwave reactor with focused field. Precursors 1–8 (1 mmol) were dissolved in methanol (3 mL),
appropriate alkylamine (3 mmol, 3 equiv.) and pyridine (80 mg, 1 mmol, 1 equiv.) as a base were added.
Conditions used for synthesis were 150 °C, 1 h, 100 W. The progress of reaction was monitored by TLC
in system hexane/ethyl acetate (1:1). The reaction mixture was adsorbed to silica by removing the
solvents in vacuo and the product was purified by flash chromatography using gradient elution with
ethyl acetate in hexane. Methylamine was used as 33% (m/m) aqueous solution.
3
.3. Analytical Data
3
-Chloro-N-methylpyrazine-2-carboxamide (1) [21]. Pale yellow solid. Yield 63.8%; m. p.
−1
1
1
04.8–106.7 °C; IR (ATR-Ge, cm ): 3303 (N-H, CONH), 1662 (C=O, CONH); H-NMR (300 MHz,
) δ 8.51 (d, J = 2.3 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H), 7.57 (s, 1H), 3.02 (d, J = 5.1 Hz, 3H);
13C-NMR (75 MHz, CDCl
) δ 162.62, 148.14, 145.56, 143.28, 140.52, 26.39; Elemental analysis: calc.
for C ClN O (MW 171.58): 42.00% C, 3.52% H, 24.49% N; found 42.17% C, 3.87% H, 24.37% N.
CDCl
3
3
6
H
6
3
3
-Chloro-N-ethylpyrazine-2-carboxamide (2). Pale yellow solid. Yield 50.8%; m. p. 110.5–112.2 °C;
−1
1
IR (ATR-Ge, cm ): 3285 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, CDCl
J = 2.2 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H), 7.54 (s, 1H), 3.55–3.45 (m, 2H), 1.26 (t, J = 7.3 Hz, 3H);
13C-NMR (75 MHz, CDCl
δ 161.86, 148.19, 145.51, 143.36, 140.49, 34.63, 14.62; Elemental analysis:
calc. for C ClN O (MW 185.61): 45.30% C, 4.34% H, 22.64% N; found 45.59% C, 4.71% H, 23.12% N.
3
) δ 8.51 (d,
3
7 8
H
3
3
-Chloro-N-propylpyrazine-2-carboxamide (3) [22]. Pale brown solid. Yield 45.0%; m. p. 93.3–95.0 °C;
−1
1
IR (ATR-Ge, cm ): 3281 (N-H, CONH), 1656 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.76
(
(
t, 1H), 8.68 (d, J = 2.5 Hz, 1H), 8.62 (d, J = 2.5 Hz, 1H), 3.27–3.18 (m, 2H), 1.59–1.46 (m, 2H), 0.90
1
3
t, J = 7.4 Hz, 3H); C-NMR (75 MHz, DMSO-d
2.34, 11.54; Elemental analysis: calc. for C
found 48.25% C, 5.54% H, 21.36% N.
6
) δ 163.74, 148.67, 145.29, 145.12, 142.62, 40.76,
O (MW 199.64): 48.13% C, 5.05% H, 21.05% N;
2
8
H10ClN
3

Molecules 2015, 20
8700
N-Butyl-3-chloropyrazine-2-carboxamide (4). Pale brown solid. Yield 45.8%; m. p. 78.9–79.4 °C; IR
−1
1
(
ATR-Ge, cm ): 3276 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.75 (s,
1
1
1
5
H), 8.68 (d, J = 2.5 Hz, 1H), 8.62 (d, J = 2.5 Hz, 1H), 3.30–3.22 (m, 2H), 1.55–1.44 (m, 2H),
1
3
.41–1.28 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H); C-NMR (75 MHz, DMSO-d
6
) δ 163.69, 148.67, 145.28,
O (MW 213.67):
45.11, 142.62, 38.64, 31.08, 19.68, 13.81; Elemental analysis: calc. for C
9
H
12ClN
3
0.59% C, 5.66% H, 19.67% N; found 50.32% C, 6.12% H, 19.74% N.
3
-Chloro-N-pentylpyrazine-2-carboxamide (5). Pale yellow solid. Yield 43.9%; m. p. 80.6–82.2 °C; IR
−1
1
(ATR-Ge, cm ): 3275 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
)
δ 8.75 (s, 1H), 8.68 (d, J = 2.5 Hz, 1H), 8.65–8.59 (m, 1H), 3.29–3.20 (m, 2H), 1.56–1.43 (m, 2H),
1
3
1
1
5
.39–1.17 (m, 4H), 0.88 (t, 3H); C-NMR (75 MHz, DMSO-d ) δ 163.68, 148.67, 145.29, 145.11,
6
42.64, 38.93, 28.70, 28.66, 21.98, 14.13; Elemental analysis: calc. for C10
2.75% C, 6.20% H, 18.46% N; found 52.31% C, 6.64% H, 18.02% N.
H
14ClN O (MW 227.69):
3
3
-Chloro-N-hexylpyrazine-2-carboxamide (6). Pale brown solid. Yield 46.9%; m. p. 77.3–78.1 °C; IR
−1
1
(
ATR-Ge, cm ): 3277 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.74 (t,
1
1
1
2
H), 8.68 (d, J = 2.5 Hz, 1H), 8.61 (d, J = 2.5 Hz, 1H), 3.29–3.20 (m, 2H), 1.56–1.44 (m, 2H),
1
3
.37–1.20 (m, 6H), 0.87 (t, 3H); C-NMR (75 MHz, DMSO-d ) δ 163.68, 148.68, 145.28, 145.12,
6
42.63, 38.96, 31.10, 28.94, 26.16, 22.25, 14.09; Elemental analysis: calc. for C11
H
16ClN O (MW
3
41.72): 54.66% C, 6.67% H, 17.38% N; found 54.30% C, 6.89% H, 16.96% N.
3
-Chloro-N-heptylpyrazine-2-carboxamide (7). Pale brown solid. Yield 40.3%; m. p. 82.1–83.0 °C; IR
−1
1
(ATR-Ge, cm ): 3275 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.51 (d,
J = 2.3 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H), 7.54 (s, 1H), 3.49–3.40 (m, 2H), 1.68–1.57 (m, 2H),
1
3
1
3
2
.38–1.23 (m, 8H), 0.86 (t, 3H); C-NMR (75 MHz, CDCl ) δ 161.93, 148.23, 145.48, 143.44, 140.49,
3
9.78, 31.68, 29.41, 28.90, 26.89, 22.55, 14.03; Elemental analysis: calc. for C12
55.75): 56.36% C, 7.09% H, 16.43% N; found 56.41% C, 7.37% H, 16.07% N.
H
18ClN O (MW
3
3
-Chloro-N-octylpyrazine-2-carboxamide (8). Pale yellow solid. Yield 47.1%; m. p. 85.4–86.6 °C; IR
−1
1
(
ATR-Ge, cm ): 3277 (N-H, CONH), 1660 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.74 (t,
1
1
1
H), 8.68 (d, J = 2.4 Hz, 1H), 8.61 (d, J = 2.6 Hz, 1H), 3.29–3.20 (m, 2H), 1.55–1.44 (m, 2H),
13
.35–1.20 (m, 10H), 0.84 (t, 3H); C-NMR (75 MHz, DMSO-d
6
) δ 163.67, 148.67, 145.28, 145.11,
42.63, 38.95, 31.42, 28.98, 28.85, 28.82, 26.49, 22.28, 14.15; Elemental analysis: calc. for C13
H
20ClN O
3
(
MW 269.77): 57.88% C, 7.47% H, 15.58% N; found 57.97% C, 7.25% H, 15.67% N.
N-Methyl-3-(methylamino)pyrazine-2-carboxamide (9) [23]. Pale yellow solid. Yield 66.9%; m. p.
−
1
7
5.1–76.1 °C (in lit. 73 °C [23]); IR (ATR-Ge, cm ): 3363, 3275 (N-H, CONH, NH), 1660 (C=O,
1
CONH; H-NMR (300 MHz, CDCl
3
) δ 8.58 (s, 1H), 8.16 (d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.61 (d,
1
3
J = 2.5 Hz, 1H), 3.01 (d, J = 4.9 Hz, 3H), 2.95 (d, J = 5.2 Hz, 3H); C-NMR (75 MHz, CDCl
3
) δ 167.05,
1
5
55.20, 146.29, 128.90, 126.88, 27.23, 25.77; Elemental analysis: calc. for C
7
H
10
N
4
O (MW 166.18):
0.59% C, 6.07% H, 33.71% N; found 50.23% C, 6.11% H, 33.85% N.

Molecules 2015, 20
8701
3
-(Ethylamino)-N-methylpyrazine-2-carboxamide (10). Yellow solid. Yield 83.1%; m. p. 60.9–62.8 °C;
−1
1
IR (ATR-Ge, cm ): 3376, 3306 (N-H, CONH, NH), 1641 (C=O, CONH); H-NMR (500 MHz, CDCl
3
)
δ 8.63 (s, 1H), 8.14 (d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.60 (d, J = 2.3 Hz, 1H), 3.51–3.45 (m, 2H), 2.95
1
3
(
d, J = 5.1 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H); C-NMR (126 MHz, CDCl
3
) δ 167.12, 154.49, 146.21,
1
6
28.89, 126.52, 35.24, 25.72, 14.59; Elemental analysis: calc. for C
8
H
12
N
4
O (MW 180.21): 53.32% C,
.71% H, 31.09% N; found 52.89% C, 6.80% H, 30.73% N.
N-Methyl-3-(propylamino)pyrazine-2-carboxamide (11). Yellow solid. Yield 84.4%; m. p. 32.1–33.9 °C;
−1
1
IR (ATR-Ge, cm ): 3367, 3314 (N-H, CONH, NH), 1645 (C=O, CONH); H-NMR (500 MHz, CDCl
3
)
δ 8.74 (s, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.89 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 3.45–3.40 (m, 2H), 2.95
1
3
(
d, J = 5.2 Hz, 3H), 1.70–1.62 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H); C-NMR (126 MHz, CDCl
54.43, 145.76, 128.81, 126.78, 42.38, 25.76, 22.47, 11.58; Elemental analysis: calc. for C
MW 194.24): 55.65% C, 7.27% H, 28.85% N; found 55.66% C, 7.30% H, 28.48% N.
3
) δ 167.05,
1
9
H
14
N O
4
(
-
1
3
-(Butylamino)-N-methylpyrazine-2-carboxamide (12). Brown liquid. Yield 40.9%; IR (ATR-Ge, cm ):
1
3367, 3314 (N-H, CONH, NH), 1654 (C=O, CONH); H-NMR (500 MHz, CDCl
3
) δ 8.69 (s, 1H), 8.13
(
d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.42 (m, 2H), 2.95 (d, J = 5.1 Hz, 3H),
1
3
1
1
.66–1.58 (m, 2H), 1.47–1.39 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H); C-NMR (126 MHz, CDCl
3
) δ 167.10,
+
54.51, 145.95, 128.80, 126.67, 40.27, 31.33, 25.75, 20.23, 13.79; HRMS [M+H] : calc. for C10
H
16
4
N O
(
MW 208.27): 209.1397; found 209.1403.
−1
N-Methyl-3-(pentylamino)pyrazine-2-carboxamide (13). Yellow liquid. Yield 88.9%; IR (ATR-Ge, cm ):
1
3
395, 3320 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (500 MHz, CDCl
3
) δ 8.73 (s, 1H), 8.12
(
d, J = 2.4 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.44 (q, J = 7.1, 5.5 Hz, 2H), 2.94 (d,
J = 5.1 Hz, 3H), 1.67–1.60 (m, 2H), 1.40–1.31 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H); 13C-NMR
(
126 MHz, CDCl
3
) δ 167.02, 154.33, 145.68, 128.76, 126.80, 40.63, 29.20, 28.92, 25.75, 22.38, 13.92;
O (MW 222.29): 223.1553; found 223.1562.
+
HRMS [M+H] : calc. for C11
H
18
N
4
3
-(Hexylamino)-N-methylpyrazine-2-carboxamide (14). Yellow liquid. Yield 88.5%; IR (ATR-Ge,
−1
1
cm ): 3407, 3310 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (500 MHz, CDCl
H), 8.13 (d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.42 (m, 2H), 2.95 (d,
J = 5.1 Hz, 3H), 1.67–1.60 (m, 2H), 1.43–1.36 (m, 2H), 1.33–1.26 (m, 4H), 0.90–0.84 (m, 3H);
3
) δ 8.71 (s,
1
13C-NMR (126 MHz, CDCl
) δ 167.06, 154.42, 145.83, 128.78, 126.74, 40.65, 31.53, 29.22, 26.76,
3
+
25.76, 22.52, 13.99; HRMS [M+H] : calc. for C12
H
20
4
N O (MW 236.32): 237.1710; found 237.1720.
−
1
3
-(Heptylamino)-N-methylpyrazine-2-carboxamide (15). Yellow liquid. Yield 81.3%; IR (ATR-Ge, cm ):
1
3406, 3310 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.66 (s, 1H), 8.13
(
d, J = 2.4 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.39 (m, 2H), 2.95 (d, J = 5.1 Hz, 3H),
1
3
1
.69–1.58 (m, 2H), 1.43–1.23 (m, 8H), 0.91–0.82 (m, 3H); C-NMR (75 MHz, CDCl
54.71, 146.32, 128.84, 126.48, 40.53, 31.73, 29.31, 29.04, 27.08, 25.77, 22.60, 14.05; HRMS [M+H]+:
O (MW 250.35): 251.1866; found 251.1875.
3
) δ 167.19,
1
calc. for C13
H
22
N
4

Molecules 2015, 20
8702
−1
N-Methyl-3-(octylamino)pyrazine-2-carboxamide (16). Yellow liquid. Yield 90.3%; IR (ATR-Ge, cm ):
1
3
408, 3319 (N-H, CONH, NH), 1656 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.66 (s, 1H), 8.13
(
d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.58 (d, J = 2.4 Hz, 1H), 3.47–3.39 (m, 2H), 2.95 (d, J = 5.2 Hz, 3H),
13
1
1
.68–1.57 (m, 2H), 1.44–1.20 (m, 10H), 0.90–0.82 (m, 3H); C-NMR (75 MHz, CDCl ) δ 167.18,
3
54.70, 146.33, 128.82, 126.45, 40.52, 31.79, 29.32, 29.29, 29.17, 27.11, 25.75, 22.61, 14.05; HRMS
+
[M+H] : calc. for C14
H
24
N O (MW 264.37): 265.2023; found 265.2030.
4
−1
N-Ethyl-3-(methylamino)pyrazine-2-carboxamide (17). Yellow liquid. Yield 73.8%; IR (ATR-Ge, cm ):
1
3
408, 3326 (N-H, CONH, NH), 1651 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.61 (s, 1H), 8.16
(
d, J = 2.4 Hz, 1H), 7.87 (s, 1H), 7.61 (d, J = 2.5 Hz, 1H), 3.47–3.36 (m, 2H), 3.01 (d, J = 4.9 Hz, 3H),
1
3
1
2
.23 (t, J = 7.3 Hz, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.29, 155.22, 146.11, 128.84, 126.96, 33.97,
+
7.25, 14.75; HRMS [M+H] : calc. for C
8
H
12
4
N O (MW 180.21): 181.1084; found 181.1092.
−1
N-Ethyl-3-(ethylamino)pyrazine-2-carboxamide (18). Yellow liquid. Yield 89.0%; IR (ATR-Ge, cm ):
1
3
1
3
1
1
395, 3321 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.79 (t, J = 6.1 Hz,
H), 8.70 (t, J = 5.6 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 3.45–3.35 (m, 2H),
1
3
.33–3.20 (m, 2H), 1.19–1.05 (m, 6H); C-NMR (75 MHz, DMSO-d
6
) δ 165.99, 154.21, 146.37,
O (MW 194.24):
+
29.44, 126.65, 34.80, 33.61, 14.92, 14.73; HRMS [M+H] : calc. for C
9
H
14
N
4
95.1240; found 195.1246.
−1
N-Ethyl-3-(propylamino)pyrazine-2-carboxamide (19). Brown liquid. Yield 95.3%; IR (ATR-Ge, cm ):
1
3
395, 3309 (N-H, CONH, NH), 1652 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.72 (s, 1H), 8.13
(
(
d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.60 (d, J = 2.3 Hz, 1H), 3.48–3.37 (m, 4H), 1.73–1.59 (m, 2H), 1.23
1
3
t, J = 7.3 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.42, 154.77, 146.19,
O (MW 208.27):
+
1
28.80, 126.53, 42.27, 33.96, 22.50, 14.78, 11.63; HRMS [M+H] : calc. for C10
H
16
N
4
2
09.1397; found 209.1402.
−
1
3
-(Butylamino)-N-ethylpyrazine-2-carboxamide (20). Yellow liquid. Yield 97.5%; IR (ATR-Ge, cm ):
1
3396, 3314 (N-H, CONH, NH), 1651 (C=O, CONH); H-NMR (500 MHz, CDCl
3
) δ 8.77 (s, 1H), 8.12
(
d, J = 2.5 Hz, 1H), 7.88 (s, 1H), 7.60 (d, J = 2.5 Hz, 1H), 3.48–3.39 (m, 4H), 1.66–1.59 (m, 2H),
13
1
.47–1.38 (m, 2H), 1.23 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H); C-NMR (126 MHz, CDCl
3
) δ
+
1
66.25, 154.36, 145.52, 128.70, 126.90, 40.37, 33.97, 31.30, 20.22, 14.74, 13.78; HRMS [M+H] : calc.
for C11
H
18
4
N O (MW 222.29): 223.1553; found 223.1563.
−1
N-Ethyl-3-(pentylamino)pyrazine-2-carboxamide (21). Yellow liquid. Yield 93.7%; IR (ATR-Ge, cm ):
1
3
395, 3309 (N-H, CONH, NH), 1652 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.68 (s, 1H), 8.13
(
d, J = 2.4 Hz, 1H), 7.89 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.36 (m, 4H), 1.69–1.58 (m, 2H), 1.41–1.31
1
3
(m, 4H), 1.23 (t, J = 7.2 Hz, 3H), 0.90 (t, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.42, 154.77, 146.27,
+
1
28.76, 126.46, 40.48, 33.94, 29.24, 28.97, 22.41, 14.76, 13.94 HRMS [M+H] : calc. for C12
H
20
4
N O
(
MW 236.32): 237.1710; found 237.1720.
−1
N-Ethyl-3-(hexylamino)pyrazine-2-carboxamide (22). Yellow liquid. Yield 71.2%; IR (ATR-Ge, cm ):
1
3
396, 3315 (N-H, CONH, NH), 1654 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.67 (s, 1H), 8.13

Molecules 2015, 20
8703
(
d, J = 2.4 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.37 (m, 4H), 1.68–1.57 (m, 2H),
13
1
1
.45–1.27 (m, 6H), 1.23 (t, J = 7.3 Hz, 3H), 0.88 (t, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.44, 154.79,
+
46.28, 128.77, 126.48, 40.53, 33.95, 31.55, 29.26, 26.80, 22.54, 14.78, 14.02 HRMS [M+H] : calc. for
C
13
H
22
N
4
O (MW 250.35): 251.1866; found 251.1876.
N-Ethyl-3-(heptylamino)pyrazine-2-carboxamide (23). Yellow liquid. Yield 82.4%; IR (ATR-Ge,
−1
1
cm ): 3396, 3310 (N-H, CONH, NH), 1653 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.67 (s,
1
2
1
H), 8.13 (d, J = 2.4 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.3 Hz, 1H), 3.47–3.37 (m, 4H), 1.69–1.57 (m,
1
3
H), 1.42–1.20 (m, 11H), 0.85 (t, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.44, 154.79, 146.28, 128.77,
+
26.48, 40.52, 33.95, 31.73, 29.30, 29.03, 27.08, 22.59, 14.78, 14.04; HRMS [M+H] : calc. for
C
14
H
24
4
N O (MW 264.37): 265.2023; found 265.2032.
−1
N-Methyl-3-(octylamino)pyrazine-2-carboxamide (24). Yellow liquid. Yield 70.4%; IR (ATR-Ge, cm ):
1
3
395, 3309 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.67 (s, 1H), 8.13
(
d, J = 2.4 Hz, 1H), 7.88 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.47–3.37 (m, 4H), 1.68–1.58 (m, 2H), 1.42–1.20
13
(
m, 13H), 0.85 (t, 3H); C-NMR (75 MHz, CDCl
3
) δ 166.44, 154.79, 146.29, 128.77, 126.48, 40.53,
+
3
3.96, 31.80, 29.32, 29.29, 29.17, 27.12, 22.61, 14.78, 14.05; HRMS [M+H] : calc. for C15
H
26
4
N O (MW
2
78.40): 279.2179; found 279.2190.
−1
N-Propyl-3-(propylamino)pyrazine-2-carboxamide (25). Yellow liquid. Yield 97.6%; IR (ATR-Ge, cm ):
1
3
395, 3315 (N-H, CONH, NH), 1657 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.71 (s, 1H), 8.13
(
d, J = 2.3 Hz, 1H), 7.95 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 3.45–3.30 (m, 4H), 1.76–1.55 (m, 4H), 1.03–0.94
1
3
(
m, 6H); C-NMR (75 MHz, CDCl
3
) δ 166.56, 154.83, 146.27, 128.82, 126.50, 42.26, 40.81, 22.85,
+
2
2.50, 11.64, 11.45; HRMS [M+H] : calc. for C11
H
18
4
N O (MW 222.29): 223.1553; found 223.1563.
−1
N-Butyl-3-(butylamino)pyrazine-2-carboxamide (26). Yellow liquid. Yield 92.9%; IR (ATR-Ge, cm ):
1
3
398, 3312 (N-H, CONH, NH), 1657 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.68 (s, 1H), 8.13
(
d, J = 2.4 Hz, 1H), 7.92 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H), 3.48–3.34 (m, 4H), 1.68–1.52 (m, 4H),
1
3
1
.49–1.33 (m, 4H), 0.97–0.89 (m, 6H); C-NMR (75 MHz, CDCl
3
) δ 166.51, 154.80, 146.27, 128.78,
+
1
26.51, 40.20, 38.83, 31.63, 31.36, 20.27, 20.13, 13.83, 13.73; HRMS [M+H] : calc. for C13
H
22
4
N O
(
MW 250.35): 251.1866; found 251.1874.
−1
N-Pentyl-3-(pentylamino)pyrazine-2-carboxamide (27). Yellow liquid. Yield 93.8%; IR (ATR-Ge, cm ):
1
3
398, 3312 (N-H, CONH, NH), 1657 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.68 (s, 1H), 8.13
(
d, J = 2.4 Hz, 1H), 7.92 (s, 1H), 7.60 (d, J = 2.3 Hz, 1H), 3.47–3.33 (m, 4H), 1.69–1.54 (m, 4H), 1.42–1.29
1
3
(
m, 8H), 0.93–0.86 (m, 6H); C-NMR (75 MHz, CDCl
3
) δ 166.49, 154.79, 146.26, 128.78, 126.52,
+
4
2
0.52, 39.12, 29.27, 29.11, 28.99, 22.43, 22.35, 13.96; HRMS [M+H] : calc. for C15
H
26
4
N O (MW
78.40): 279.2179; found 279.2190.
−1
N-Hexyl-3-(hexylamino)pyrazine-2-carboxamide (28). Yellow liquid. Yield 89.0%; IR (ATR-Ge, cm ):
1
3
2
2
394, 3313 (N-H, CONH, NH), 1655 (C=O, CONH); H-NMR (300 MHz, DMSO-d
6
) δ 8.80–8.71 (m,
H), 8.20 (d, J = 2.4 Hz, 1H), 7.71 (d, J = 2.5 Hz, 1H), 3.36 (q, J = 6.5 Hz, 2H), 3.21 (q, J = 6.8 Hz,
1
3
H), 1.57–1.42 (m, 4H), 1.34–1.18 (m, 12H), 0.86–0.79 (m, 6H); C-NMR (75 MHz, DMSO-d ) δ
6

Molecules 2015, 20
8704
1
66.12, 154.37, 146.33, 129.35, 126.60, 39.94, 38.72, 31.19, 29.22, 28.93, 26.41, 26.29, 22.24, 14.05;
+
HRMS [M+H] : calc. for C17
H
30
4
N O (MW 306.45): 307.2492; found 307.2498.
−1
N-Heptyl-3-(heptylamino)pyrazine-2-carboxamide (29). Yellow liquid. Yield 67.7%; IR (ATR-Ge, cm ):
1
3
1
1
1
2
398, 3307 (N-H, CONH, NH), 1658 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.70 (t, J = 5.0 Hz,
H), 8.14 (d, J = 2.4 Hz, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.60 (d, J = 2.5 Hz, 1H), 3.48–3.33 (m, 4H),
13
.69–1.54 (m, 4H), 1.43–1.21 (m, 16H), 0.91–0.83 (m, 6H); C-NMR (75 MHz, CDCl ) δ 166.46,
3
54.74, 146.16, 128.77, 126.59, 40.58, 39.17, 31.74, 31.71, 29.60, 29.30, 29.04, 28.95, 27.10, 26.95,
+
2.60, 22.56, 14.06, 14.03; HRMS [M+H] : calc. for C19
H
34
N O (MW 334.51): 335.2805; found 335.2809.
4
−1
N-Octyl-3-(octylamino)pyrazine-2-carboxamide (30). Yellow liquid. Yield 94.1%; IR (ATR-Ge, cm ):
1
3
394, 3310 (N-H, CONH, NH), 1658 (C=O, CONH); H-NMR (300 MHz, CDCl
3
) δ 8.69 (t,
J = 5.3 Hz, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.60 (d, J = 2.5 Hz, 1H), 3.48–3.33
1
3
(
m, 4H), 1.70–1.54 (m, 4H), 1.41–1.23 (m, 20H), 0.90–0.83 (m, 6H); C-NMR (75 MHz, CDCl
3
) δ
1
2
66.50, 154.80, 146.29, 128.78, 126.54, 40.55, 39.16, 31.80, 31.76, 29.59, 29.33, 29.30, 29.24,
+
9.16, 27.13, 26.98, 22.60, 14.07; HRMS [M+H] : calc. for C21
H
38
4
N O (MW 362.56): 363.3118;
found 363.3123.
3
.4. Determination of Lipophilicity by HPLC (Log k)
Instrumentation: Agilent Technologies 1200 SL liquid chromatograph with Diode-array Detector SL
G1315C (Agilent Technologies Inc., Colorado Springs, CO, USA); pre-column ZORBAX XDB-C18
μm, 4 × 4 mm, Part No. 7995118-504 (Agilent Technologies Inc.) and column ZORBAX Eclipse
5
XDB-C18 5 μm, 4.6 × 250 mm, Part No. 7995118-585 (Agilent Technologies Inc.). The separation
process was controlled by Agilent ChemStation, version B.04.02 extended by spectral module (Agilent
Technologies Inc.). Mobile phase consisted of MeOH (HPLC grade, 70%) and H
Grade, 30%). Conditions: Flow rate 1.0 mL/min, sample injection volume 20 μL, column temperature
0 °C, detection wavelength 210 nm, monitor wavelength 270 nm. Retention times (tR) were measured
2
O (HPLC-Milli-Q
3
in minutes. The dead time of the system (tD) was determined as the retention time of KI methanol
solution. Capacity factors k for individual compounds were calculated according to the formula
k = (tR − tD)/tD. Log k, calculated from the capacity factor k, is used as the lipophilicity index converted
to log scale.
3
.5. Biological Assays
3
.5.1. Evaluation of in Vitro Antimycobacterial Activity
Microdilution panel method. Tested strains M. tuberculosis H37Rv CNCTC My 331/88, M. kansasii
Hauduroy CNCTC My 235/80 and M. avium ssp. avium Chester CNCTC My 80/72 were obtained from
Czech National Collection of Type Cultures (CNCTC), National Institute of Public Health, Prague,
Czech Republic. Culturing medium was Middlebrook 7H9 broth (Sigma-Aldrich) with the addition of
glycerol (Sigma-Aldrich) and OADC supplement (Himedia, Mumbai, India). Isoniazid was used as
standard. Tested compounds were dissolved and diluted in DMSO and mixed with growth media to final
concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μg/mL. The final concentration of DMSO did

Molecules 2015, 20
8705
not exceed 1% (v/v) and did not affect the growth of mycobacteria. The cultures were grown in
Middlebrook 7H9 medium at 37 °C in humid dark atmosphere. The antimycobacterial activity was
determined using Alamar Blue colouring after 14 days of incubation as MIC (μg/mL). This evaluation
was done in cooperation with Department of Clinical Microbiology, University Hospital in Hradec
Králové, Czech Republic.
3
.5.2. Evaluation of in Vitro Antibacterial Activity
Microdilution broth method [44]. Antibacterial evaluation was performed against bacterial strains
from Czech Collection of Microorganisms (Brno, Czech Republic) (Staphylococcus aureus CCM
516/08, Escherichia coli CCM 4517, Pseudomonas aeruginosa CCM 1961) or clinical isolates from
4
Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové,
Charles University in Prague, Czech Republic (Staphylococcus aureus H 5996/08-methicilin resistant
(MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08, Klebsiella pneumoniae D
1
1750/08, Klebsiella pneumoniae J 14368/08-ESBL positive). All strains were subcultured on Mueller-
Hinton agar (MHA) (Difco/Becton Dickinson, Detroit, MI, USA) at 35 °C and maintained on the same
medium at 4 °C. The compounds were dissolved in DMSO, and the antibacterial activity was determined
in Mueller-Hinton liquid broth (Difco/Becton Dickinson), 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 composition. 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. The standards were neomycin, bacitracin, penicillin G, ciprofloxacin and
phenoxymethylpenicilin.
3
.5.3. Evaluation of in Vitro Antifungal Activity
Antifungal evaluation was performed using microdilution broth method [45] against 8 fungal strains
(
Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii
188, Aspergillus fumigates 231, Absidia corymbifera 272 and Trichophyton mentagrophytes 445).
1
Compounds were dissolved in DMSO and diluted in a twofold manner with RPMI 1640 medium
with glutamine buffered to pH 7.0 (3-morpholinopropane-1-sulfonic acid). The final concentration
of DMSO in the tested medium did not exceed 2.5% (v/v) of the total solution composition. Static
incubation was performed in the dark and humid, at 35 °C for 24 and 48 h (respectively 72 and 120 h
for Trichophyton mentagrophytes). The MIC was defined as 80% inhibition of control (50% IC for
filament fungi). Drug-free controls were included. The standards were amphotericin B, voriconazole,
nystatin and fluconazole.
3.5.4. Antiviral Evaluation
Antiviral activity in cell culture assessed by cytopathic effect (CPE) reduction assays with a broad
panel of viruses [46–48]. The following viruses were examined on human embryonic lung fibroblast
−
cells: herpes simplex virus type 1 (HSV-1); a thymidine kinase-deficient (TK ) HSV-1 KOS strain
resistant to acyclovir; herpes simplex virus type 2 (HSV-2); vaccinia virus; human adenovirus type 2;

Molecules 2015, 20
8706
and vesicular stomatitis virus (VSV). The viruses examined on human cervix carcinoma HeLa cells
were: VSV; Coxsackie B4 virus; and respiratory syncytial virus (RSV). African Green Monkey Vero
cells were used to determine the antiviral effect on para-influenza-3 virus; reovirus-1; Sindbis virus;
Coxsackie B4 virus and Punta Toro virus. Human influenza A/H1N1, A/H3N2 and B viruses were
assessed on Madin-Darby canine kidney (MDCK) cells. Activity against human immunofeficiency virus
(
HIV) type 1 and type 2 was studied in human MT-4 lymphoblast cells. To perform the tests, the virus
was added to semiconfluent cell cultures in 96-well plates and simultaneously serial dilutions of the test
compounds were added. The plates were incubated until clear CPE was reached (typically 3–6 days).
Microscopic scoring was then performed to determine the antiviral activity [expressed as 50% effective
concentration (EC50)]. In the case of HIV-1, HIV-2 and influenza virus, virus-induced CPE was
determined by the colorimetric formazan-based MTS cell viability assay.
3.5.5. Study of Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and
Kralova [49]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Electron Scientific Instruments, Madison, WI,
USA), using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to
Kralova et al. [50], and the rate of photosynthetic electron transport (PET) was monitored as
a photoreduction of DCPIP. The measurements were carried out in phosphate buffer (0.02 mol/L,
pH 7.2) containing sucrose (0.4 mol/L), MgCl
2
(0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll
2
content was 30 mg/L in these experiments and the samples were irradiated (~100 W/m with 10 cm
distance) with a halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples
(
suspension temperature 22 °C). The studied compounds were dissolved in DMSO due to their limited
water solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity
in spinach chloroplasts. The inhibitory efficiency of the studied compounds was expressed by IC50
values, i.e., by molar concentration of the compounds causing 50% PET inhibition relative to the
untreated control. The comparable IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-
®
dimethylurea (DCMU, Diuron ) was about 1.9 μmol/L.
3
.5.6. Study of Fluorescence of Aromatic Amino Acids in Spinach Chloroplasts
The fluorescence emission spectra of aromatic amino acids (AAA) in spinach chloroplasts
were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation
wavelength λex = 275 nm for monitoring AAA fluorescence, excitation slit 20 nm and emission
slit 10 nm. The samples were kept in the dark for 2 min before measuring. The phosphate buffer used
for dilution of the chloroplast suspension was the same as described above. Due to low aqueous solubility
the compounds were added to chloroplast suspension in DMSO solution. The DMSO concentration in
all samples was the same as in the control (10%). The chlorophyll concentration in chloroplast
suspension was 10 mg/L.

Molecules 2015, 20
. Conclusions
We prepared thirty alkylamino pyrazinamide derivatives. We focused on modification of carboxamide
8707
4
moiety (formal N-alkylation) and introduction of an alkylamino substituent to position 3 of the pyrazine
ring. According to the antimycobacterial assay results, the most favourable substitutions were N-methyl
for the carboxamide moiety and long (C
compounds against M. tuberculosis H37Rv were 14–16 (MIC = 25 μg/mL) with log k = 0.876–1.359
log P = 1.21–2.05). The other compounds showed lower or no activity. Compound 8 was the most
6
-C ) alkylamino substituents in position 3. The most active
8
(
effective compound against Trichophyton mentagrophytes (MIC 62.5 μmol/L) in antifungal assays.
None of tested compounds produce any significant antibacterial or antiviral activity. Prepared compounds
were not toxic on HeLa and Vero cells.
The studied compounds inhibited photosynthetic electron transport (PET) in spinach chloroplasts.
The PET-inhibiting activity was strongly connected with the lipophilicity of the compounds. The
PET-inhibiting activity of N-alkyl-3-chloropyrazine-2-carboxamides, 3-(alkylamino)-N-methyl-
pyrazine-2-carboxamides as well as 3-(alkylamino)-N-methylpyrazine-2-carboxamides increased
linearly with increasing lipophilicity of the compounds. On the other hand, the dependence of
PET-inhibiting activity of 3-(alkylamino)-N-methylpyrazine-2-carboxamides on lipophilicity showed
1
2
bilinear course, the optimum being compound 27 (R = R = C H11). For effective PET inhibition longer
5
alkyl chain in 3-(alkylamino) substituent in the molecule was more favourable than two shorter alkyl
chains in N-alkyl-3-(alkylamino)pyrazine-2-carboxamides. The interaction of studied compounds with
aromatic amino acids situated mainly in photosystem 2 was confirmed by fluorescence experiments.
Acknowledgments
This work was co-financed by the European Social Fund and the state budget of the Czech Republic.
Project TEAB no. CZ.1.07/2.3.00/20.0235. This study is also supported by the Grant Agency of Charles
University, project B-CH/1594214, SVV 260 183 and by the Project APVV-0061-11. The publication
is co-financed by the European Social Fund and the state budget of the Czech Republic: Post-doctoral
Project no. CZ.1.07/2.3.00/30.0061.
Author Contributions
L.S. was responsible for the preparation, identification and purity analysis of the compounds, and
manuscript preparation. K.Kon. ensured biological testing on microbial and fungal strains and P.P. on
mycobacterial strains. V.K. measured lipophilicity of compounds (log k). J.K. measured NMR spectra.
L.Nov. performed HRMS analysis. L.Nae. ensured antiviral assays and cytotoxicity testing. K.Kra. and
M.P. ensured measuring and interpretation of PET-inhibiting activity. J.M., M.D. and J.Z. participated
on the design of new compounds, management of the study and manuscript. All authors have read and
approved the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
8708
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Sample Availability: Samples of the compounds are available from the authors.
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