Substituted N-benzylpyrazine-2-carboxamides: Synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules171113183

A series of twelve amides was synthesized via aminolysis of substituted pyrazinecarboxylic acid chlorides with substituted benzylamines. Compounds were characterized with analytical data and assayed in vitro for their antimycobacterial, antifungal, antibacterial and photosynthesis-inhibiting activity. 5-tert-Butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) has shown the highest antimycobacterial activity against Mycobacterium tuberculosis (MIC = 6.25 μg/mL), as well as against other mycobacterial strains. The highest antifungal activity against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for 5-chloro-N-(3- trifluoromethylbenzyl)- pyrazine-2-carboxamide (2, MIC = 15.62 μmol/L). None of the studied compounds exhibited any activity against the tested bacterial strains. Except for 5-tert-butyl-6-chloro-N-benzylpyrazine-2-carboxamide (9, IC₅₀ = 7.4 μmol/L) and 5-tert-butyl-6-chloro-N-(4-chlorobenzyl) pyrazine-2-carboxamide (11, IC₅₀ = 13.4 μmol/L), only moderate or weak photosynthesis-inhibiting activity in spinach chloroplasts (Spinacia oleracea L.) was detected.

Full text of DOI:10.3390/molecules171113183

Molecules 2012, 17, 13183-13198; doi:10.3390/molecules171113183
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Substituted N-Benzylpyrazine-2-carboxamides:
†
Synthesis and Biological Evaluation
Barbora Servusová 1,*, Drahomíra Eibinová 1, Martin Doležal 1, Vladimír Kubíček 2,
Pavla Paterová 3, Matúš Peško 4 and Katarína Kráľová 5
1
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy,
Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
E-Mails: eibid5aa@faf.cuni.cz (D.E.); dolezalm@faf.cuni.cz (M.D.)
2
Department of Biophysics and Physical Chemistry, Faculty of Pharmacy, Charles University in Prague,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; E-Mail: kubicek@faf.cuni.cz
3
Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05 Hradec Králové,
Czech Republic; E-Mail: pavla.paterova@fnhk.cz
4
Department of Ecosozology and Physiotactics, Faculty of Natural Sciences, Comenius University,
Mlynská Dolina Ch-2, 84215 Bratislava, Slovakia; E-Mail: matus.pesko@gmail.com
5
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská Dolina CH-2,
842 15 Bratislava, Slovakia; E-Mail: kralova@fns.uniba.sk
†
Preliminary results related to the topic of this article were presented at The Fifteenth International
Electronic Conference on Synthetic Organic Chemistry (ECSOC-15, https://docs.google.com/
viewer?url=http://www.sciforum.net/presentation/599/pdf&chrome=true), 1–30 November 2011.
* Author to whom correspondence should be addressed; E-Mail: barbora.servusova@faf.cuni.cz;
Tel.: +420-495-067-272; Fax: +420-495-512-423.
Received: 22 October 2012; in revised form: 31 October 2012 / Accepted: 2 November 2012 /
Published: 6 November 2012
Abstract: A series of twelve amides was synthesized via aminolysis of substituted
pyrazinecarboxylic acid chlorides with substituted benzylamines. Compounds were
characterized with analytical data and assayed in vitro for their antimycobacterial,
antifungal, antibacterial and photosynthesis-inhibiting activity. 5-tert-Butyl-6-chloro-N-(4-
methoxybenzyl)pyrazine-2-carboxamide (12) has shown the highest antimycobacterial
activity against Mycobacterium tuberculosis (MIC = 6.25 µg/mL), as well as against other
mycobacterial strains. The highest antifungal activity against Trichophyton mentagrophytes,
the most susceptible fungal strain tested, was found for 5-chloro-N-(3-trifluoromethylbenzyl)-

Molecules 2012, 17
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pyrazine-2-carboxamide (2, MIC = 15.62 µmol/L). None of the studied compounds
exhibited any activity against the tested bacterial strains. Except for 5-tert-butyl-6-chloro-
N-benzylpyrazine-2-carboxamide (9, IC50 = 7.4 µmol/L) and 5-tert-butyl-6-chloro-N-(4-
chlorobenzyl)pyrazine-2-carboxamide (11, IC50 = 13.4 µmol/L), only moderate or weak
photosynthesis-inhibiting activity in spinach chloroplasts (Spinacia oleracea L.) was detected.
Keywords: pyrazinamide analogues; lipophilicity determination; in vitro antimycobacterial;
antifungal and photosynthesis inhibitory activity
1. Introduction
Tuberculosis (TB) is considered to be one of the most frequent and widespread nowadays infectious
diseases especially in developing countries. In 2010, there were about 8.8 million new cases of TB and
1.4 million deaths (including deaths from TB among HIV-positive people) [1]. Anti-TB drug
resistance is a major public health problem that threatens progress made in TB care and control
worldwide. Particularly dangerous forms of TB are multidrug-resistant TB (MDR-TB) and extensively
drug-resistant TB (XDR-TB) [1]. Furthermore, TB and HIV synergistically influence each other’s
progress and lead to the increased need of new antituberculars [2].
Pyrazinamide (PZA), a nicotinamide analogue, is one of the most important first-line drugs used in
TB-therapy [3]. Along with rifampicin, PZA has sterilizing activity (the ability to kill the semi-dormant
mycobacteria) which is a crucial factor in shortening the duration of therapy [4]. PZA, as a prodrug
that requires bacterial enzymes to generate the biologically active molecule, enters mycobacterial cell
via passive diffusion and it is activated by pyrazinamidase/nicotinamidase (EC 3.5.1.19) to form
pyrazinoic acid (POA) [5]. Pyrazinamidase/nicotinamidase is encoded by the pncA gene and mutation
of this gene is primarily responsible for resistance to PZA [6]. POA’s intracellular accumulation
lowers pH in mycobacterial cell, thus leading to inhibition of membrane transport and depletion of
energy [7]. Otherwise, the demonstration that PZA and POA inhibit Mycobacterium tuberculosis fatty
acid synthase-I (FAS-I) in whole-cell and cell-free assays suggests that the disruption might be a
consequence of the inhibition of membrane synthesis [8–10]. Reversible binding of both PZA and
POA to M. tuberculosis FAS-I has been definitively confirmed by Saturation Transfer Difference
NMR spectroscopy (STD-NMR), a NMR technique used to characterize ligand–protein interactions [11].
Boshoff et al. [12] reported that FAS-I is not the target of PZA. However, FAS-I has been proposed
and confirmed as a target of pyrazinamide derivatives, e.g., 5-chloropyrazinamide [8,9]. Finally,
another specific target for POA, ribosomal protein S1 (RpsA), has been identified in recent study [13].
RpsA is a vital protein involved in protein translation and the ribosome-sparing process of
trans-translation and its role in M. tuberculosis is multifaceted.
Several pyrazine derivatives were found to possess herbicidal activity summarized in review
paper [14]. Many pyrazinamide derivatives inhibited photosynthetic electron transport (PET) in plant
chloroplasts [14–17] and they were found to act as photosystem (PS) 2 inhibitors. Using EPR
spectroscopy it was found that 5-tert-butyl-N-(3-hydroxy-4-chlorophenyl)-pyrazine-2-carboxamide
and 5-tert-butyl-6–chloro-N-(3-fluorophenyl)-pyrazine-2-carboxamide interacted with the D

Molecules 2012, 17
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intermediate, i.e., with the tyrosine radical which is situated at 161th position on D2 protein occurring
on the donor side of PS 2 [18]. Due to this interaction, PET from the oxygen evolving complex to
the reaction centre of PS 2 is impaired. However, an experiment with artificial electron donor
1,5-diphenylcarbazide (DPC) with known site of action in Z/D intermediate confirmed that also some
members of the PET chain between Z/D intermediate and plastoquninone were partially damaged in
the light by these carboxamides. Chlorophyll a fluorescence quenching due to treatment of chloroplast
suspension with above mentioned pyrazinamides indicated their interaction with pigment-protein
complexes in PS 2 [18].
In this study, we focused on binuclear pyrazinamide analogues containing the -CONH-CH2- bridge,
namely on N-benzylpyrazine-2-carboxamides. Earlier studies have shown some interesting
anti-mycobacterial activity in a series of substituted N-phenylpyrazine-2-carboxamides [16,19] and
become a pattern for substitution of aromatic ring in benzylamines. The aim of this work was to find the
structure-activity relationships (SAR) in the series of substituted N-benzylpyrazine-2-carboxamides,
i.e., to study the influence of incorporated methylene moiety in the connecting bridge and to continue
in the study of the substituent variability influence on the biological activity.
2. Results and Discussion
2.1. Chemistry
Synthesis of 5-chloropyrazine-2-carboxylic acid chloride [20] from 5-hydroxypyrazine-2-carboxylic
acid, as well as synthesis of final compounds 1–12, is shown in Scheme 1. Condensation of chlorides
of 5-chloropyrazine-2-carboxylic [20], 6-chloropyrazine-2-carboxylic [21] and 5-tert-butyl-6-
chloropyrazine-2-carboxylic [22] acids with unsubstituted or ring-substituted benzylamines yielded a
series of twelve amides. Reactions proceeded under mild conditions, yields of products ranged within
59–91%, and analytical data were fully consistent with the proposed structures. Specific substituents
R1, R2 and R3 of individual compounds 1–12 are listed in Table 1.
Scheme 1. Synthesis and structure of the 5-chloropyrazine-2-carbonyl chloride and final
products 1–12.
Reagents: (a) SOCl2, DMF, toluene; (b) TEA, acetone.

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Table 1. Comparison of the calculated lipophilicity (log P, Clog P) with the determined
log k values of the studied compounds 1–12. IC50 values related to PET inhibition
in spinach chloroplasts in comparison with the standard 3-(3,4-dichlorophenyl)-1,1-
dimethylurea (DCMU).
Compounds
R1
H
R2
R3
H
log P
1.56
2.48
2.12
1.43
1.56
2.48
2.12
1.43
3.69
4.61
4.25
3.56
–
Clog P
2.3852
3.2682
3.0982
2.3042
2.3852
3.2682
3.0982
2.3042
4.2112
5.0942
4.9242
4.1302
–
log k
0.1862
0.4384
0.4150
0.1655
0.2002
0.4507
0.4335
0.1838
1.1215
1.3638
1.3511
0.8071
–
IC50 [µmol/L]
1623.0
345.8
604.0
ND
1
Cl
2
H
Cl
3-CF3
4-Cl
4-OCH3
H
3
H
Cl
4
H
Cl
5
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
–
H
H
ND
6
3-CF3
4-Cl
4-OCH3
H
1207.0
ND
7
H
8
9
H
ND
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
–
7.4
10
3-CF3
4-Cl
4-OCH3
–
36.3
11
13.4
12
121.6
1.9
DCMU
ND not determined due to their low solubility in the testing medium.
2.2. Lipophilicity
Lipophilicity, one of the most important physicochemical properties of the compound, which seems
to be a key factor related to the cell transmembrane transport and other biological processes, can
either be determined experimentally or predicted by means of commercially available programmes.
In this work Log P/Clog P values of the compounds 1–12 were calculated using the program CS
ChemBioDraw Ultra version 12.0 (CambridgeSoft, Cambridge, MA, USA) and also measured by
means of the RP-HPLC determination of capacity factors k with subsequent calculation of log k. The
results are shown in Table 1 and illustrated in Figure 1.
The lowest lipophilicity was shown by 5-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (4),
while 5-tert-butyl-6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (10) was the most
lipophilic compound of this series. Based on log k values, lipophilicity increased for substituents in
pyrazine part in the following order: 5-chloropyrazine < 6-chloropyrazine < 5-tert-butyl-6-chloropyrazine.
In the case of substituents in the benzyl part of the molecule lipophilicity increased this way:
4-OCH3 < H < 4-Cl < 4-CF3. The dependence of the calculated Clog P values on the measured log k
parameters showed an approximate linearity and the corresponding correlation can be expressed by the
following regression equation:
(1)
Clog P = 2.170 (±0.131) log k + 2.086 (±0.096)
r = 0.9650 s = 0.197 F = 276.1 n = 12
The differences between experimentally determined log k and calculated Clog P values were
observed for 5-chloro and 6-chloro group in pyrazine part. This may be caused by the used calculating
program for Clog P, that does not distinguish difference between substituent’s lipophilicity in position

Molecules 2012, 17
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C(5) and C(6) in pyrazine part of molecule. Consequently, it can be assumed that log k values specify
lipophilicity within this series of compounds more precisely than calculated Clog P values.
Figure 1. Plot of calculated Clog P (CS ChemBioDraw Ultra version 12.0) on
experimentally measured log k parameter. Linear regression parameters.
2.3. Biological Activity
2.3.1. In Vitro Antimycobacterial Evaluation
All synthesized compounds were assayed in vitro towards Mycobacterium tuberculosis and several
Mycobacteria Other Than Tuberculosis (MOTTs) (see Table 2). The most active derivatives against
M. tuberculosis were compounds 8, 10 and 12, whose minimal inhibition concentrations (MICs) were
6.25 µg/mL. These activities were fully comparable with PZA. More importantly, 5-tert-butyl-6-
chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) showed significant activity against tested
MOTTs, which are unsusceptible to PZA. The vast majority of compounds exhibited only modest
antimycobacterial activity expressed as MIC in µg/mL, or with respect to the molecular weight of final
products in µmol/L (see Table 2).
The obtained results provide some insights into the SAR in this miniseries. With respect to the
benzyl part, the most suitable substituents are 4-methoxy and 3-trifluoromethyl groups, especially with
disubstitution in the pyrazine moiety. The most significant substitutions in the pyrazine part are
chlorine in the C(6) and tert-butyl in the C(5) position.
There is no clear dependence between lipophilicity (log k, see Table 1) and antimycobacterial
activities in this series, however the most lipophilic compound 5-tert-butyl-6-chloro-N-(3-
trifluoromethylbenzyl)pyrazine-2-carboxamide (10, log k = 1.3638) displayed one of the highest
activity against M. tuberculosis. On the other hand, 6-chloro-N-(4-methoxybenzyl)pyrazine-2-

Molecules 2012, 17
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carboxamide (8) with the lowest lipophilicity (log k = 0.1838) showed the same activity against
M. tuberculosis as compound 10 (MIC = 6.25 µg/mL).
Table 2. Antimycobacterial and antifungal activity of presented compounds in comparison
with standards: pyrazinamide (PZA), isoniazid (INH) and fluconazole (FLU).
MIC [µg/mL]
TM e MIC
Compounds
M. tuberculosis
H37Rv a
25 (100)
25 (79)
M. avium b
M. avium c
M. kansasii d
[µmol/L]
1
2
100
25
100
100
100
50
100
50
125/125
15.62/15.62
125/125
250/250
250/500
62.5/125
125/125
500/500
250/500
250/250
250/500
250/500
3
12.5 (44)
25 (90)
100
100
100
25
100
100
50
4
5
12.5 (50)
12.5 (39)
12.5 (44)
6.25 (22)
25 (82)
100
100
12.5
50
6
100
100
100
100
25
7
100
100
25
8
9
50
10
11
12
6.25 (16)
12.5 (36)
6.25 (18)
6.25–12.5
(50–101)
1.56 (11)
–
25
25
50
50
50
12.5
6.25
3.125
PZA
>100
>100
>100
–
INH
FLU
12.5–25
–
12.5
–
12.5
–
–
1.95/3.91
a CNCTC My 331/88; b CNCTC My 80/72; c CNCTC My 152/73; d CNCTC My 235/80; e TM Trichophyton
mentagrophytes 445, evaluated after 72 h/120 h.
2.3.2. In Vitro Antifungal Evaluation
The evaluation of in vitro antifungal activity of the studied compounds was performed against
eight fungal strains. Except for 5-chloro- (2) and 6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-
carboxamides (6), only weak antifungal activity was found. In comparison with fluconazole (the standard,
MIC = 1.95 µmol/L after 72 h) compounds 2 (MIC = 15.62 µmol/L) and 6 (MIC = 62.5 µmol/L) exhibited
moderate in vitro antifungal activity against Trichophyton mentagrophytes (TM), the most susceptible
fungal strain evaluated. For the results see Table 2.
2.3.3. In Vitro Antibacterial Evaluation
All prepared compounds were tested for their in vitro antibacterial activity [23,24] against eight
bacterial strains, namely against: Staphylococcus aureus CCM 4516/08, Staphylococcus aureus
H 5996/08–methicilin resistant, Staphylococcus epidermidis H 6966/08, Pseudomonas aeruginosa
CCM 1961, Escherichia coli CCM 4517. Enterococcus sp. J 14365/08, Klebsiella pneumoniae
D 11750/08 and Klebsiella pneumoniae J 14368/08–ESBL positive. None of the synthesized
compounds exhibited any activity against the tested strains.

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2.3.4. Inhibition of Photosynthetic Electron Transport (PET)
All studied compounds were evaluated for their photosynthetic electron transport (PET) inhibition
in spinach chloroplasts, which was reflected in the inhibition of oxygen evolution rate. The
photosynthesis-inhibiting activity of the compounds has been expressed as IC50 values. Compounds 4,
5, 7 and 8 were not tested due to their low solubility in tested medium. The IC50 values varied in the
range from 7.4 to 1,623.0 µmol/L, see Table 1. The activity of the majority of compounds was
moderate or relatively low when compared with the standard 3-(3,4-dichlorophenyl)-1,1-dimethylurea
(DCMU, IC50 = 1.9 µmol/L).
Figure 2. Linear dependence between lipophilicity (expresed as logarithm of retention
factor, log k) and photosynthesis-inhibiting activity log (1/IC50) [mol/L]. of synthesized
compounds 1–12.
The most efficient inhibitors of this series were compounds 9 (IC50 = 7.4 µmol/L) and 11
(IC50 = 13.4 µmol/L). In general, photosynthesis-inhibiting activity of the studied compounds is
dependent on their lipophilicity (compare compounds 9–12, Table 1). PET inhibition of compounds
1–12 has increased linearly with increasing lipophilicity expressed as log P or log k (see Figure 2) and
the corresponding correlations can be expressed by the following regression equations:
(2)
log (1/IC50) = 1.626 (±0.534) + 0.717 (±0.164) log P
r = 0.8722 s = 0.472 F = 19.08 n = 8
log (1/IC50) = 2.475 (±0.266) +1.785 (±0.303) log k
r = 0.92358 s= 0.370 F = 34.82 n = 8
(3)
The results of statistical analysis were improved if log k instead log P was used. This finding is in
agreement with above mentioned assumption that log k values specify lipophilicity within this series of

Molecules 2012, 17
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compounds more precisely than calculated log P values. Log k values of the most active inhibitors
were about 1.2.
The effects of the studied compounds on the photosynthetic apparatus of spinach chloroplasts were
investigated by studying chlorophyll a (Chla) fluorescence. Fluorescence emission spectra of Chla in
spinach chloroplasts treated with compound 2 are shown in Figure 3A. The decreased intensity of the
emission band at 686 nm belonging to the pigment-protein complexes in photosystem 2 [25] suggested
PS 2 as the site of action of the studied inhibitors. The extent of perturbation of chlorophyll a-protein
complexes in the thylakoid membrane was reflected as decreased fluorescence of the pigment
(see Figure 3B). Similar decrease of Chla fluorescence in plant chloroplasts was also observed
previously after treatment with substituted benzanilides [26] and salicyanilides [27].
Figure 3. (A) Fluorescence emission spectra of chlorophyll a in untreated spinach
chloroplasts in the presence of compound 2: 0, 0.11, 0.21 and 0.42 mmol/L (curves from
top to bottom; ex = 436 nm). (B) Dependence of fluorescence intensity of chlorophyll a on
concentration of compounds 2 (squares), 11 (triangles) and 6 (circles).
100
100
A
B
80
60
40
20
0
90
80
70
6
2
11
600
650
700
750
0.0
0.2
0.4
0.6
0.8
1.0
 [nm]
c [mmol/L]
Interaction of the studied compounds with aromatic amino acids, which are present in the proteins
of spinach chloroplasts situated in PS 2, was documented by the quenching of their fluorescence at
334 nm. Figure 4 presents fluorescence emission spectra of aromatic amino acids of untreated spinach
chloroplasts and of chloroplasts treated with increasing concentrations of compound 6 (see Figure 4A)
as well as dependence of fluorescence intensity of chlorophyll a on concentration of compounds 2
(squares), 11 (triangles) and 6 (circles) (see Figure 4B). Binding of these compounds to aromatic
amino acids occurring in photosynthetic proteins contribute to PET inhibition.
By the addition of DPC, an artificial electron donor acting in Z/D intermediate on the donor side
of PS 2, to chloroplasts treated with the studied compounds in which PET was inhibited at about
80-90%, PET was restored only to 77-88%. This indicates that the site of PET inhibition is situated not
only on the donor side of PS 2 in the section between the primary electron donor of PS 2 (H2O) and
Z/D intermediate but also in the photosynthetic transport chain from P 680 to plastoquinone
QB occurring on the acceptor side of PS 2. Similar sites of action were determined previously for
5-tert-butyl-N-(3-hydroxy-4-chlorophenyl)-pyrazine-2-carboxamide and 5-tert-butyl-6–chloro-N-(3-
fluorophenyl)-pyrazine-2-carboxamide [18].

Molecules 2012, 17
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Figure 4. (A) Fluorescence emission spectra of aromatic amino acids in untreated spinach
chloroplasts in presence of compound 6: 0, 0.011, 0.026, 0.037, 0.053 and 0.11 mmol/L
(curves from top to bottom; ex = 275 nm). (B) Dependence of fluorescence intensity of
aromatic amino acids on concentration of compounds 2 (squares), 11 (triangles) and 6 (circles).
1500
100
A
B
1000
500
0
80
60
40
2
11
6
300
350
400
450
500
0.00 0.02 0.04 0.06 0.08 0.10 0.12
c [mmol/L]
 [nm]
3. Experimental
3.1. General
All organic solvents used for the synthesis were of analytical grade. All chemicals were purchased
from Sigma-Aldrich (Schnelldorf, Germany). The reactions were monitored using Merck Silica 60 F254
TLC plates (Merck, Darmstadt, Germany). Compounds were purified using an automated
chromatograph CombiFlash Rf (Teledyne Isco, Lincoln, NE, USA) using columns filled with Kieselgel
60, 0.040–0.063 mm (Merck, Darmstadt, Germany); gradient elution (hexane/ethyl-acetate), detection
wavelength 260 nm, monitor wavelength 280 nm. NMR analysis was performed on Varian Mercury
1
VX-BB 300 (Varian, Palo Alto, CA, USA) at 300 MHz for H and 75 MHz for 13C. Chemical
shifts were recorded as δ values in parts per million (ppm) and were indirectly referenced to
tetramethylsilane (TMS). IR spectra were recorded in KBr blocks on Nicolet Impact 400 (Nicolet,
Madison, WI, USA). Elementary analysis was performed on CE Instruments EA-1110 CHN analyser
(CE Instruments, Wigan, UK). Melting points were determined on Stuart SMP30 melting point
apparatus (Bibby Scientific Limited, Staffordshire, UK) and are uncorrected.
3.2. Synthesis of N-Benzylpyrazine-2-carboxamides
To a solution of 6-chloropyrazine-2-carboxylic [21] or 5-tert-butyl-6-chloropyrazine-2-carboxylic [22]
acid (0.5 mmol) in dry toluene (20 mL) was added 1.5 equivalent of thionyl chloride (0.75 mmol). The
reaction mixture was heated to reflux for about 1 h. Then, the excess of thionyl chloride was removed
by repeated evaporation with dry toluene in vacuo. The crude acyl chloride was dissolved in dry
acetone (20 mL) and added dropwise to a stirred solution of the corresponding benzylamine
(0.5 mmol) with triethylamine (0.5 mmol) in dry acetone (10 mL). Then, the reaction mixture was
stirred at room temperature for about 1 h. The reaction was monitored using TLC with hexane/ethyl
acetate 2:1 mixture as eluent. After this time, the solution was evaporated till dryness with silica gel
and purified using a flash column chromatography (40 g column, gradient elution hexane/ethyl-acetate).

Molecules 2012, 17
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In the case of 5-chloropyrazine-2-carbonyl chloride [20] synthesis, 5-hydroxypyrazine-2-carboxylic
acid (Sigma-Aldrich) was used as starting material. During the reaction with thionyl chloride, the
formation of acyl chloride occurs simultaneously with the nucleophilic substitution of the hydroxyl
group for chlorine. Dimethylformamide (DMF) was added to the reaction mixture as catalyst [28].
3.3. Data of Prepared Target Compounds
N-Benzyl-5-chloropyrazine-2-carboxamide (1). White crystalline compound. Yield: 81%; m.p.
101.9–103.1 °C; 1H-NMR (CDCl3) δ 9.21 (s, 1H, H3), 8.49 (s, 1H, H6), 7.98 (bs, 1H, NH), 7.40–7.26
13
(m, 5H, H2′, H3′, H4′, H5′, H6′), 4.67 (d, 2H, J = 6.0 Hz, NCH2); C-NMR (CDCl3) δ 162.0, 152.1,
144.0, 142.6, 142.4, 137.5, 128.8, 127.8, 127.7, 43.5; IR (cm−1) 3384 (N-H), 1660 (C=O); Anal. Calcd.
For C12H10ClN3O (247.68): 58.19% C, 4.07% H, 16.97% N; Found: 58.38% C, 4.23% H, 16.89% N.
5-Chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (2). White crystalline compound. Yield:
1
65%; m.p. 91.3–92.7 °C; H-NMR (CDCl3) δ 9.20 (s, 1H, H3), 8.51 (s, 1H, H6), 8.09 (bs, 1H, NH),
13
7.65–7.40 (m, 4H, H2′, H4′, H5′, H6′), 4.72 (d, 2H, J = 6.3 Hz, NCH2); C-NMR (CDCl3) δ 162.2,
152.3, 144.0, 142.5, 142.3, 138.6, 131.2, 131.1 (q, J = 32.6 Hz), 129.3, 124.6 (q, J = 3.8 Hz), 124.4 (q,
J = 3.8 Hz), 123.6 (q, J = 272.9 Hz), 43.0; IR (cm−1) 3385 (N-H), 1665 (C=O); Anal. Calcd. For
C13H9ClF3N3O (315.68): 49.46% C, 2.87% H, 13.31% N; Found: 49.51% C, 2.96% H, 13.48% N.
5-Chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (3). White crystalline compound. Yield: 79%;
m.p. 89.9–91.3 °C; 1H-NMR (CDCl3) δ 9.20 (s, 1H, H3), 8.50 (s, 1H, H6), 7.98 (bs, 1H, NH), 7.35–7.25
(m, 4H, H2′, H3′, H5′, H6′), 4.63 (d, 2H, J = 6.2 Hz, NCH2); 13C-NMR (CDCl3) δ 162.1, 152.2, 144.0,
142.5, 142.4, 136.1, 133.6, 129.2, 128.9, 42.9; IR (cm−1) 3327 (N-H), 1672 (C=O); Anal. Calcd. For
C12H9Cl2N3O (282.13): 51.09 % C, 3.22% H, 14.89% N; Found: 50.96% C, 3.14% H, 14.97% N.
5-Chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (4). White crystalline compound. Yield: 80%;
m.p. 123.2–124.0 °C; 1H-NMR (CDCl3) δ 9.19 (s, 1H, H3), 8.48 (s, 1H, H6), 7.90 (bs, 1H, NH), 7.30–7.25
(m, 2H, H2′, H6′), 6.99–6.62 (m, 2H, H3′, H5′), 4.59 (d, 2H, J = 5.9 Hz, NCH2), 3.80 (s, 3H, CH3);
13C-NMR (CDCl3) δ 161.9, 159.2, 152.0, 144.0, 142.6, 142.4, 129.6, 129.3, 114.2, 55.3, 43.1; IR
(cm−1) 3388 (N-H), 1659 (C=O); Anal. Calcd. For C13H12ClN3O2 (277.71): 56.22% C, 4.36% H,
15.13% N; Found: 56.26% C, 4.34% H, 15.01% N.
N-Benzyl-6-chloropyrazine-2-carboxamide (5). White crystalline compound. Yield: 87%; m.p.
58.3–59.7 °C; 1H-NMR (DMSO-d6) δ 9.48 (t, 1H, J = 6.3 Hz, NH), 9.19 (s, 1H, H3), 8.81 (s, 1H, H5),
13
7.34–7.20 (m, 5H, H2′, H3′, H4′, H5′, H6′), 4.50 (d, 2H, J = 6.3 Hz, NCH2); C-NMR (DMSO-d6) δ
163.1, 148.1, 143.8, 143.6, 143.1, 139.4, 128.4, 127.5, 127.0, 42.5; IR (cm−1) 3371 (N-H), 1670
(C=O); Anal. Calcd. For C12H10ClN3O (247.68): 58.19% C, 4.07% H, 16.97% N; Found: 58.33% C,
4.12% H, 17.08% N.
6-Chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (6). White crystalline compound. Yield:
1
63%; m.p. 68.1–69.8 °C; H-NMR (CDCl3) δ 9.33 (s, 1H, H3), 8.77 (s, 1H, H5), 8.03 (bs, 1H, NH),
13
7.64–7.42 (m, 4H, H2′, H4′, H5′, H6′), 4.73 (d, 2H, J = 6.3 Hz, NCH2); C-NMR (CDCl3) δ 161.8,
147.6, 147.5, 143.6, 142.1, 138.5, 131.2 (q, J = 32.4 Hz), 130.9, 129.3, 124.6 (q, J = 3.9 Hz), 124.5 (q,

Molecules 2012, 17
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J = 4.0 Hz), 124.0 (q, J = 272.9 Hz), 43.1; IR (cm−1) 3324 (N-H), 1672 (C=O); Anal. Calcd. For
C13H9ClF3N3O (315.68): 49.46% C, 2.87% H, 13.31% N; Found: 49.57% C, 2.71% H, 13.56% N.
6-Chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (7). White crystalline compound. Yield: 91%;
m.p. 93.2–94.5 °C; 1H-NMR (CDCl3) δ 9.32 (s, 1H, H3), 8.76 (s, 1H, H5), 7.97 (bs, 1H, NH), 7.37–7.31
(m, 2H, H2′, H6′), 7.28–7.20 (m, 2H, H3′, H5′), 4.63 (d, 2H, J = 6.1 Hz, NCH2); 13C-NMR (CDCl3) δ
161.7, 147.5, 147.4, 143.7, 142.0, 135.9, 133.6, 129.2, 128.9, 42.9; IR (cm−1) 3387 (N-H), 1667
(C=O); Anal. Calcd. For C12H9Cl2N3O (282.13): 51.09% C, 3.22% H, 14.89% N; Found: 51.23% C,
3.37% H, 14.76% N.
6-Chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (8). White crystalline compound. Yield: 89%;
m.p. 71.4–72.6 °C; 1H-NMR (CDCl3) δ 9.32 (s, 1H, H3), 8.73 (s, 1H, H5), 7.88 (bs, 1H, NH), 7.31–7.25
(m, 2H, H2′, H6′), 6.91–6.85 (m, 2H, H3′, H5′), 4.59 (d, 2H, J = 6.0 Hz, NCH2), 3.80 (s, 3H, CH3);
13C-NMR (CDCl3) δ 161.5, 159.2, 147.5, 147.2, 144.0, 142.0, 129.5, 129.3, 114.2, 55.3, 43.1; IR
(cm−1) 3370 (N-H), 1662 (C=O); Anal. Calcd. For C13H12ClN3O2 (277.71): 56.22% C, 4.36% H,
15.13% N; Found: 56.34% C, 4.38% H, 15.03% N.
N-Benzyl-5-tert-butyl-6-chloropyrazine-2-carboxamide (9). White crystalline compound. Yield: 61%;
1
m.p. 69.7–71.3 °C; H-NMR (DMSO-d6) δ 9.35 (t, 1H, J = 6.4 Hz, NH), 9.04 (s. 1H, H3), 7.33–7.20
(m, 5H, H2′, H3′, H4′, H5′, H6′), 4.48 (d, 2H, J = 6.4 Hz, NCH2), 1.48 (s, 9H, CH3); 13C-NMR
(DMSO-d6) δ 162.8, 162.0, 145.5, 142.4, 140.1, 139.3, 128.4, 127.6, 127.0, 42.6, 38.6, 28.2; IR (cm−1)
3399 (N-H), 1669 (C=O); Anal. Calcd. For C16H18ClN3O (303.79): 63.26% C, 5.97% H, 13.83% N;
Found: 63.39% C, 6.13% H, 13.67% N.
5-tert-Butyl-6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (10). White crystalline
1
compound. Yield: 59%; m.p. 74.4–75.3 °C; H-NMR (CDCl3) δ 9.21 (s, 1H, H3), 7.98 (bs, 1H, NH),
7.60 (s, 1H, H2′), 7.58–7.45 (m, 3H, H4′, H5′, H6′), 4.72 (d, 2H, J = 6.4 Hz, NCH2), 1.53 (s, 9H, CH3);
13C-NMR (CDCl3) δ 164.5, 162.3, 145.9, 140.8, 140.2, 138.8, 131.2, 131.1 (q, J = 32.3 Hz), 129.3,
124.5 (q, J = 3.9 Hz), 124.4 (q, J = 3.9 Hz), 123.9 (q, J = 272.9 Hz), 42.9, 38.9, 28.3; IR (cm−1) 3373
(N-H), 1676 (C=O); Anal. Calcd. For C17H17ClF3N3O (371.78): 54.92% C, 4.61% H, 11.30% N;
Found: 54.86% C, 4.53% H, 11.35% N.
5-tert-Butyl-6-chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (11). White crystalline compound.
1
Yield: 82%; m.p. 67.5–68.7 °C; H-NMR (CDCl3) δ 9.19 (s, 1H, H3), 7.91 (bs, 1H, NH), 7.39–7.19
13
(m, 4H, H2′, H3′, H5′, H6′), 4.62 (d, 2H, J = 6.3 Hz, NCH2), 1.52 (s, 9H, CH3); C-NMR (CDCl3) δ
164.4, 162.1, 140.9, 140.1, 136.2, 133.5, 131.5,129.2, 128.9, 42.7, 38.9, 28.3; IR (cm−1) 3376 (N-H),
1659 (C=O); Anal. Calcd. For C16H17Cl2N3O (338.23): 56.82% C, 5.07% H, 12.42% N; Found:
56.76% C, 5.18% H, 12.25% N.
5-tert-Butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12). White crystalline compound.
1
Yield: 84%; m.p. 71.3–72.7 °C; H-NMR (CHCl3) δ 9.19 (s, 1H, H3), 7.84 (bs, 1H, NH), 7.31–7.25
(m, 2H, H2′, H6′), 6.91–6.85 (m, 2H, H3′, H5′), 4.59 (2H, d, J = 6.0 Hz), 3.80 (3H, s), 1.52 (9H, s); 13C
NMR (CDCl3) δ 164.1, 161.9, 159.1, 141.1, 140.2, 140.1, 129.7, 129.3, 114.1, 55.3, 42.9, 38.8, 28.2;
IR (cm−1) 3324 (N-H), 1673 (C=O); Anal. Calcd. For C17H20ClN3O2 (333.81): 61.17% C, 6.04% H,
15.13% N; Found: 61.23% C, 6.17% H, 15.01% N.

Molecules 2012, 17
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3.4. HPLC Lipophilicity Determination (Capacity Factor k/ Calculated Log k)
An Agilent Technologies 1200 SL liquid chromatography system equipped with a Diode-Array
Detector SL G1315C, chromatographic pre-column ZORBAX XDB-C18 5 µm, 4 × 4 mm, Part No.
7995118-504 and column ZORBAX Eclipse XDB-C18 5 µm, 4.6 × 250 mm, Part No. 7995118-585
(Agilent Technologies Inc., Colorado Springs, CO, USA) were used. The separation process was
controlled by Agilent ChemStation, version B.04.02 extended by spectral module (Agilent
Technologies Inc.). A solution of MeOH (HPLC grade, 70%) with H2O (HPLC-Milli-Q Grade, 30%)
was used as mobile phase. The total flow of the column was 1.0 mL/min, injection 20 µL, column
temperature 30 °C. 210 nm as detection wavelength and 270 nm as monitor wavelength were chosen.
The KI methanol solution was used for the dead time (TD) determination. Retention times (TR) of
synthesized compounds were measured in minutes. The capacity factors k were calculated using
Microsoft Excel according to formula k = (TR − TD)/TD, where TR is the retention time of the solute
and TD denotes the dead time obtained via an unretained analyte. Log k, calculated from the capacity
factor k, is used as the lipophilicity index converted to log P scale.
3.5. Lipophilicity Calculations
Log P (the logarithm of the partition coefficient for n-octanol/water) and Clog P (the logarithm of
n-octanol/water partition coefficient P based on established chemical interactions) were calculated
using the program CS ChemBioDraw Ultra version 12.0 (CambridgeSoft, Cambridge, MA, USA).
3.6. Biological Methods
3.6.1. Evaluation of In Vitro Antimycobacterial Activity
Microdilution panel method. Antimycobacterial evaluation was shielded by Department of Clinical
Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University in
Prague, Czech Republic. Four mycobacterial strains were used: M. tuberculosis H37Rv CNCTC My
331/88, M. avium CNCTC My 80/72, M. avium CNCTC My 152/73 and M. kansasii CNCTC My
235/80 (Czech National Collection of Type Cultures, National Institute of Public Health, Prague,
Czech Republic). Tested compounds were dissolved in DMSO (to final concentrations 100, 50, 25,
12.5, 6.25, 3.125 and 1.563 µg/mL), diluted 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. The final concentration of DMSO did not exceed 1% (v/v), this concentration
of DMSO did not affect the growth of mycobacteria. The cultures were grown in Šula’s semisynthetic
medium at pH 6.0 and 37 °C. The antimycobacterial activity was determined visually after 14 days
(6 days for M. kansasii) of incubation as minimally inhibition concentration (MIC, µg/mL), i.e., the
lowest concentration of tested substance which inhibited the growth of mycobacteria.
3.6.2. Evaluation of In Vitro Antifungal Activity
The Department of Medical and Biological Sciences at the Faculty of Pharmacy in Hradec Králové,
Charles University in Prague, Czech Republic, performed the antifungal susceptibility assays, which

Molecules 2012, 17
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was carried out using microdilution broth method [29,30]. 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. Drug-free controls were included. Fluconazole
was used as standard. The MICs were determined after 24 and 48 h, respectively after 72 and 120 h for
Trichophyton mentagrophytes (TM), of static incubation in dark at 35 °C. Tested species: Candida
albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188,
Aspergillus fumigates 231, Absidia corymbifera 272 and Trichophyton mentagrophytes 445 (TM).
3.6.3. Study of the Inhibition of Oxygen Evolution rate in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovičová and
Kráľová [31]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific, Madison, WI, USA) using an
artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to Kráľová et al. [33]
and the rate of photosynthetic electron transport (PET) was monitored as a photo-reduction of DCPIP.
The measurements were carried out in a phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose
(0.4 mol/L), MgCl2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in
these experiments and the samples were irradiated (~100 W/m2) from a 10 cm distance with 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 (PET). The inhibitory efficiency of the studied compounds was expressed as the
IC50 values, i.e., molar concentration of the compounds causing 50% decrease in the oxygen
evolution relative to the untreated control. The comparable IC50 value for a selective herbicide
3-(3,4-dichlorophenyl)-1,1-dimethylurea (Diurone®, DCMU) was about 1.9 µmol/L [33].
3.6.4. Study of Fluorescence of Chlorophyll a and Aromatic Amino Acids in Spinach Chloroplasts
The fluorescence emission spectra of chlorophyll a (Chla) and aromatic amino acids in spinach
chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using
excitation wavelength ex = 436 nm for monitoring fluorescence of Chla and ex = 275 nm for
monitoring fluorescence of aromatic amino acids, excitation slit 20 nm and emission slit 10 nm. The
samples were kept in the dark 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 a 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.
4. Conclusions
A series of twelve binuclear pyrazinamide analogues containing -CONH-CH2- bridges was
synthesized by the condensation of substituted pyrazine-2-carboxylic acid chlorides with the
corresponding benzylamines. The final products were characterized by analytical data and evaluated

Molecules 2012, 17
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for their in vitro antimycobacterial, antifungal, antibacterial and photosynthesis-inhibiting activity.
Lipophilicity of the compounds was determined using RP-HPLC method and calculated using
predicting program CS ChemBioDraw Ultra version 12.0. The obtained values were compared with
each other and graphically expressed as dependence of calculated Clog P on log k. Compounds 8, 10
and 12 had in vitro antimycobacterial activity against M. tuberculosis comparable with PZA
(MIC = 6.25 µg/mL). More importantly, 5-tert-butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-
carboxamide (12) was active against tested MOTTs (M. kansasii and M. avium), which are
unsusceptible to PZA. No clear dependence between lipophilicity and antimycobacterial activity has
been found in this series. The highest antifungal activity (MIC = 15.6 µmol/L) against susceptible
strain (Trichophyton mentagrophytes) was observed for 5-chloro-N-(3-trifluoromethylbenzyl)pyrazine-
2-carboxamide (2), other compounds showed only weak or none in vitro antifungal activity. None of
the studied compounds exhibited any significant activity against tested bacterial strains. 5-tert-butyl-6-
chloro-N-benzylpyrazine-2-carboxamide (9, IC50 = 7.4 µmol/L). The most active compounds in
inhibition of PET in spinach chloroplasts (Spinacia oleracea L.) were 5-tert-butyl-6-chloro-N-
benzylpyrazine-2-carboxamide (9, IC50 = 7.4 µmol/L) and 5-tert-butyl-6-chloro-N-(4-chlorobenzyl)-
pyrazine-2-carboxamide (11, IC50 = 13.4 µmol/L). Based on the obtained results it can be assumed,
that in this series of pyrazinamide derivatives the antimycobacterial and antifungal activities did not
depend directly on compound’s lipophilicity. On the other hand, PET-inhibiting activity increased
linearly with compound’s lipophilicity.
Acknowledgements
This work was financially supported by GAUK B-CH/710312, IGA NT 13346 (2012) and
SVV-2012-265-001 as well as by the Slovak Grant Agency VEGA (grant No. 1/0612/11) and by
Sanofi-Aventis Pharma Slovakia. The publication is, a result of the project implementation: Support of
establishment, development, and mobility of quality research teams at the Charles University, project
number CZ.1.07/2.3.00/30.0022, supported by The Education for Competitiveness Operational
Programme (ECOP) and co-financed by the European Social Fund and the state budget of the Czech
Republic. Authors wish to thank Ida Dufková for performing in vitro antifungal and antibacterial
screening, Jiří Kuneš for recording of NMR spectra and Jan Zitko for language corrections.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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