In vitro antibacterial and antifungal activity of salicylanilide pyrazine-2-carboxylates

Article abstract of DOI:10.2174/157340612801216346

The development of new antimicrobial agents for the treatment of infectious diseases remains challenging due to the increasing impact of antibiotic resistance. Since salicylanilides and esters of pyrazine-2-carboxylic acid have been described as potential antimicrobials, we have designed and synthesized a series of 2-(phenylcarbamoyl)phenyl pyrazine-2-carboxylates. These were evaluated in vitro for the activity against fungi and Gram-positive and Gram-negative bacteria. All derivatives showed significant antibacterial activity against Gram-positive strains (MIC ≥ 0.98 μmol/L) including methicillin-resistant Staphylococcus aureus. The most active molecule was 5-chloro-2-(3-chlorophenylcarbamoyl)phenyl pyrazine-2-carboxylate. With one exception these esters were at least partly active against fungi tested strains, in particular against mould strains (MIC ≥ 1.95 μmol/L). The most active antifungal agent overall proved to be 2-(4-bromophenylcarbamoyl)-4-chlorophenyl pyrazine-2-carboxylate.

Full text of DOI:10.2174/157340612801216346

732
Medicinal Chemistry, 2012, 8, 732-741
In Vitro Antibacterial and Antifungal Activity of Salicylanilide Pyrazine-2-
carboxylates
Martin Krátkꢀa, Jarmila Vinꢁováa,* and Vladimír Buchtab,c
aDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05
Hradec Králové, the Czech Republic
bDepartment of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University, Sokolská 581,
500 12 Hradec Králové, the Czech Republic
cDepartment of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05
Hradec Králové, the Czech Republic
Abstract: The development of new antimicrobial agents for the treatment of infectious diseases remains challenging due
to the increasing impact of antibiotic resistance. Since salicylanilides and esters of pyrazine-2-carboxylic acid have been
described as potential antimicrobials, we have designed and synthesized a series of 2-(phenylcarbamoyl)phenyl pyrazine-
2-carboxylates. These were evaluated in vitro for the activity against fungi and Gram-positive and Gram-negative bacte-
ria. All derivatives showed significant antibacterial activity against Gram-positive strains (MIC ꢂ 0.98 ꢃmol/L) including
methicillin-resistant Staphylococcus aureus. The most active molecule was 5-chloro-2-(3-chlorophenylcarbamoyl)phenyl
pyrazine-2-carboxylate. With one exception these esters were at least partly active against fungi tested strains, in particu-
lar against mould strains (MIC ꢂ 1.95 ꢃmol/L). The most active antifungal agent overall proved to be 2-(4-
bromophenylcarbamoyl)-4-chlorophenyl pyrazine-2-carboxylate.
Keywords: Antibacterial activity, antifungal activity, in vitro activity, pyrazine-2-carboxylic acid ester, salicylanilide, salicy-
lanilide ester.
1. INTRODUCTION
Pathogenic fungi can also develop resistance to various
drugs [5].
The ubiquitous presence of antibiotic resistance threatens
to end the “golden age” of antibiotic therapy and therefore
affects everyone [1]. Today major problems arise from the
spread of nosocomial antibiotic-resistant bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA), ex-
tended-spectrum ß-lactamases (ESBL)-producing Es-
cherichia coli or Klebsiella spp., multiresistant Pseudomonas
and Acinetobacter sp. as well as Clostridium difficile [2].
The difficulty in treating MRSA is often discussed. The evo-
lution of nosocomial antibiotic-resistant bacteria has required
the development of new agents and approaches to prevent
and overcome drug-resistance. The inappropriate antibiotic
treatment supports MRSA selection, overgrowth and in-
creased virulence [3]. MRSA is growing in prevalence and is
now endemic in many healthcare facilities and communities
[4].
A wide range of substituted salicylanilides (2-hydroxy-N-
phenylbenzamides; Fig. 1) have exhibited significant in vitro
antibacterial activity, especially against Gram-positive bacte-
ria species in low concentrations [6-13]. Salicylanilide esters
with a different organic acid as well as structures containing
an O-acylated salicylanilide substructure have been reported;
these have been shown to have good antibacterial and anti-
fungal properties [14].
O
N
H
OH
1
Fig. (1). Salicylanilide core (2-hydroxy-N-phenylbenzamide).
The situation in the fungal kingdom is a bit different.
Over the past decades there have been a growing number of
immunocompromised patients (e.g. HIV-infected patients)
who have developed opportunistic mycoses caused by an
expanding spectrum of fungal pathogens, including those
with problematic susceptibility to current antifungal drugs.
Salicylanilides possess multiple mechanisms of action
which were briefly summarized in our previous review [14].
It was described that they interfere with the structure and
functions of biomembranes as well as with the uptake and
incorporation of some biogenic compounds. Salicylanilides
also exhibit uncoupling activity and inhibit vitally important
two-component regulatory systems necessary for the regula-
tion of bacterial cell homeostasis.
*Address correspondence to this author at the Heyrovského 1203, 500 05
Hradec Králové, the Czech Republic; Tel: +420 495067343; Fax: +420
495067166, E-mail: vinsova@faf.cuni.cz
Pyrazinamide (Fig. 2), clinically used as the first line oral
antituberculosis agent, has been greatly investigated [15]. Its
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

In vitro Antibacterial and Antifungal Activity
Medicinal Chemistry, 2012, Vol. 8, No. 4 733
O
All synthesized compounds were characterized by ele-
mentary analysis, melting point, IR and NMR spectroscopy.
Elemental analyses (C, H, N) were performed on the auto-
matic microanalyser CHNS-O CE (FISONS EA 1110, Italy).
Melting points were determined on the melting point appara-
tus B-540 (Büchi) using open capillaries; the melting points
are uncorrected. Infrared spectra (KBr pellets) were recorded
on a Nicolet 6700 FT-IR spectrometer in the range of 400 –
4000 cm-1. The NMR spectra were measured in dimethyl
sulfoxide (DMSO)-d6 solutions at ambient temperature in a
N
NH2
2
N
Fig. (2). Pyrazinamide.
O
N
OH
N
1
3
Varian Mercury-VxBB 300 spectrometer (300 MHz for H
and 75.5 MHz for 13C; Varian Comp. Palo Alto, USA). The
chemical shifts ꢀ are given in ppm and referenced against
tetramethylsilane as an internal standard.
Fig. (3). Pyrazinoic (pyrazine-2-carboxylic) acid.
active form, in which it is converted by nicotinami-
dase/pyrazinamidase in vivo in mycobacterial cells, has been
demonstrated to be pyrazine-2-carboxylic (pyrazinoic) acid
(Fig. 3) [15, 16]. Pyrazine-2-carboxylic acid exhibited sig-
nificant activity against Mycobacterium tuberculosis [17] as
well as pyrazinoates (for example [17-19]).
The LogP values represented as the logarithm of the par-
tition coefficient for octan-1-ol/water were calculated using
the software ChemOffice Ultra 11.0 (CambridgeSoft, USA).
2.2. Pharmacology
Esters of pyrazine-2-carboxylic acid expressed a certain
antibacterial activity against Gram-positive strains, but no
activity against E. coli [20]. Some pyrazine-2-carboxylates
have been patented as potential antibacterial drugs [21, 22].
The technique of combining this acid with another antimi-
crobial active compound to form mutual molecules was ap-
plied in the case of penem derivatives inhibiting methicillin-
resistant Staphylococcus aureus [23], macrolides [24, 25],
rifamycines [26], dihydroisoxazole and oxazolidinone [27,
28], in general with a hopeful antimicrobial activity. In one
assay, substituted ethyl pyrazine-2-carboxylate exhibited a
mild inhibition of Candida albicans and Trichophyton men-
tagrophytes growth (MIC 62.5 – 250 ꢀmol/L [18]); other
pyrazine-2-carboxylic esters were inactive against T. menta-
grophytes [29].
Both the antibacterial and antifungal activity of the tested
compounds were determined at the Department of Medical
and Biological Sciences at the Faculty of Pharmacy, Charles
University, Hradec Králové, using the microdilution broth
method modified according to standard M07-A07 [33]. The
minimum inhibitory concentrations (MIC) were assayed as
an 80% (IC80) or higher reduction of growth in comparison
to the control.
2.2.1. Antibacterial Evaluation
In vitro antibacterial activity was assayed against Gram-
positive and Gram-negative strains: Staphylococcus aureus
CCM 4516/08, methicillin-resistant Staphylococcus aureus
H 5996/08 (MRSA), Staphylococcus epidermidis H 6966/08,
Enterococcus sp. J 14365/08; Escherichia coli CCM4517,
Klebsiella pneumoniae D 11750/08, ESBL-positive Kleb-
siella pneumoniae J 14368/08, and Pseudomonas aeruginosa
CCM 1961.
Based on these findings, we designed, synthesized and
evaluated a series of eighteen salicylanilide esters of
pyrazine-2-carboxylic acid.
2. MATERIAL AND METHODS
2.1. Chemistry
The microdilution broth method was used featuring a
Mueller-Hinton broth (HiMedia Laboratories, India) adjusted
to pH 7.4 (±0.2). The investigated compounds were dis-
solved in DMSO to final concentrations ranging from 500 to
0.49 μmol/L. Penicillin G (benzylpenicillin) and zinc salt of
bacitracin were used as comparative drugs. Bacterial inocu-
lum using sterile water was prepared to match the 0.5
McFarland scale (1.5 x 108 CFU/mL). The minimum inhibi-
tory concentrations (MIC) were assayed as an 80% (IC80) or
higher reduction of growth in comparison to the control. The
determination of results was performed visually and spectro-
photometrically (at 540 nm). The MIC values were deter-
As is briefly indicated in the Scheme 1, the esters were
prepared by the methodology previously described using
N,N´-dicyclohexylcarbodiimide [30-32].
All used reagents and solvents were purchased from
commercial sources (Sigma-Aldrich). Commercial grade
reagents were used without further purification. Reactions
were monitored using thin layer chromatography plates
coated with 0.2 mm silica gel 60 F254 (Merck) visualized by
UV irradiation (254 nm).
O
R2
O
R2
O
N
R1
H
N
N
N
+
R1
H
HO
O
OH
N
N
O
Scheme 1. Esterification of salicylanilides by pyrazine-2-carboxylic acid (R1 of esters = 4-Cl, 5-Cl, 4-Br; R2 = 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-
Br, 3-F, 4-F, 3-CF3, 4-CF3).

734 Medicinal Chemistry, 2012, Vol. 8, No. 4
Krátkꢀ et al.
Table 1. Overview of the Prepared Esters 1 and their Calculated logP.
O
R2
N
R1
H
O
N
N
O
R1
R2
logP
R1
R2
logP
1a
1b
1c
1d
1e
1f
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
3-Cl
3-Cl
3.19
3.19
3.19
3.19
3.75
3.75
3.46
3.46
3.46
1j
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Cl
5-Cl
4-Br
4-Cl
4-Br
3-F
3.46
2.79
2.79
2.79
2.79
3.55
3.55
3.82
3.55
1k
1l
4-Cl
3-F
4-Cl
1m
1n
1o
1p
1q
1r
4-F
3,4-diCl
3,4-diCl
3-Br
4-F
4-CF3
4-CF3
4-CF3
3-CF3
1g
1h
1i
3-Br
4-Br
mined after 24 and 48 h of incubation in the dark at 35 °C
(±0.1) in a humid atmosphere.
employed methodology using N,N´-dicyclohexylcarbodi-
imide as a condensation agent is applicable and useful in the
case when a carboxy group in the organic acid being used is
directly bonded to the aromatic ring, not only to the aliphatic
part of the molecule as in previous reports [30-32]. The
yields of esters ranged from 26 to 91%. An overview of the
compounds can be found in Table 1. Calculated logP values
range from 3.19 to 3.82.
2.2.2. Antifungal Evaluation
The inhibitory activity of pyrazine-2-carboxylates was
determined in vitro against four yeast strains (Candida albi-
cans ATCC 44859, Candida tropicalis 156, Candida krusei
E28, Candida glabrata 20/I), and four moulds
–
Trichosporon asahii 1188, Aspergillus fumigatus 231, Ab-
sidia corymbifera 272, and Trichophyton mentagrophytes
445.
3.1.1. 4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl
pyrazine-2-carboxylate (1a)
White solid; yield 57%; mp 161.5-163 °C. IR (KBr pel-
let): 3388, 3080, 1759 (CO ester), 1672, 1594, 1545, 1482,
1406, 1281, 1257, 1191, 1077, 1038, 1016, 856, 810, 770,
The microdilution broth method was used according to
the CLSI M27-A3 and M38 A2 guidelines for yeasts and
moulds [34, 35] in RPMI 1640 with glutamine (KlinLab, the
Czech Republic) buffered to pH 7.0 with 0.165 M of 3-
morpholino-propane-1-sulphonic acid (Sigma-Aldrich, Ger-
many). DMSO served as a diluent for all compounds. In the
yeast, the final size of the inoculum was 5 x 103 ± 0.2
CFU/mL. In the moulds – Aspergillus, Trichophyton and
Absidia corymbifera – the spores were harvested after the
cultivation of a given fungal strain grown on Sabouraud agar
from 3 to 10 days. The final size of the inoculum was 0.5 –
5 x 104 CFU/mL. The sizes of the fungal inocula were
checked using a Bürker’s chamber. Fluconazole and ampho-
tericin B were used as reference drugs. The other conditions
were the same as for the antibacterial assay; the only differ-
ence being that for T. mentagrophytes the final MIC were
determined after 72 and 120 h of incubation. MIC were de-
termined twice and in duplicate.
1
672. H NMR (300 MHz, DMSO): ꢀ 10.72 (1H, bs, NH),
9.35 (1H, d, J=1.3 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz, H5´´),
8.87 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.89 (1H, d, J=2.5
Hz, H3), 7.78 (1H, d, J=2.6 Hz, H2´), 7.75 (1H, d, J=2.5 Hz,
H5), 7.57 (1H, d, J=8.5 Hz, H6), 7.50 (1H, d, J=8.2 Hz,
H6´), 7.32 (1H, t, J=8.2 Hz, H5´), 7.12 (1H, dd, J=2.0 Hz,
J=8.0 Hz, H4´). 13C NMR (75 MHz, DMSO): ꢀ 162.8, 161.9,
149.0, 146.9, 146.4, 145.4, 142.2, 140.2, 133.1, 132.0, 130.8,
130.6, 130.6, 129.4, 125.6, 123.9, 119.6, 118.5. Anal. Calcd.
for C18H11Cl2N3O3 (388.20): C, 55.64; H, 2.86; N, 10.82.
Found: C, 55.71; H, 2.52; N, 10.66.
3.1.2. 5-Chloro-2-(3-chlorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1b)
White solid; yield 53%; mp 166-167.5 °C. IR (KBr pel-
let): 3398, 3072, 3040, 1763 (CO ester), 1671, 1592, 1539,
1483, 1405, 1279, 1262, 1185, 1085, 1040, 1015, 847, 833,
1
3. RESULTS
774, 706, 672. H NMR (300 MHz, DMSO): ꢀ 10.68 (1H,
bs, NH), 9.36 (1H, d, J=1.3 Hz, H3´´), 8.96 (1H, d, J=2.4
Hz, H5´´), 8.88 (1H, dd, J=1.5 Hz, J=2.3 Hz, H6´´), 7.85
(1H, d, J=8.4 Hz, H3), 7.76-7.73 (2H, m, H2´, H6), 7.61
(1H, dd, J=2.0 Hz, J=8.4 Hz, H4), 7.50 (1H, d, J=8.7 Hz,
3.1. Chemistry
Eighteen new salicylanilide esters with pyrazine-2-
carboxylic acid were prepared; we confirmed the fact that the

In vitro Antibacterial and Antifungal Activity
Medicinal Chemistry, 2012, Vol. 8, No. 4 735
H6´), 7.31 (1H, t, J=8.1 Hz, H5´), 7.12 (1H, dd, J=1.6 Hz,
J=8.0 Hz, H4´). 13C NMR (75 MHz, DMSO): ꢀ 163.3, 161.8,
149.0, 148.8, 146.4, 145.4, 142.2, 140.3, 136.0, 133.1, 131.2,
130.6, 128.0, 126.9, 123.9, 123.8, 119.5, 118.5. Anal. Calcd.
for C18H11Cl2N3O3 (388.20): C, 55.69; H, 2.86; N, 10.82.
Found: C, 55.90; H, 2.84; N, 10.94.
Hz, H5´´), 8.88 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.93
(1H, s, H2´), 7.85 (1H, d, J=8.4 Hz, H3), 7.75 (1H, d, J=2.0
Hz, H6), 7.62 (1H, dd, J=2.1 Hz, J=8.4 Hz, H4), 7.56-7.54
(2H, m, H5´, H6´). 13C NMR (75 MHz, DMSO): ꢀ 163.4,
161.8, 149.0, 148.8, 146.4, 145.4, 142.1, 139.0, 136.2, 131.2,
131.1, 130.9, 127.8, 126.9, 125.6, 123.9, 121.3, 120.1. Anal.
Calcd. for C18H10Cl3N3O3 (422.65): C, 51.15; H, 2.38; N,
9.94. Found: C, 50.92; H, 2.47; N, 9.87.
3.1.3. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1c)
3.1.7. 2-(3-Bromophenylcarbamoyl)-4-chlorophenyl pyra-
zine-2-carboxylate (1g)
White solid; yield 53%; mp 182-184 °C. IR (KBr pellet):
3405, 3324, 3082, 2927, 2850, 1746 (CO ester), 1676, 1596,
1539, 1491, 1403, 1286, 1242, 1190, 1090, 1042, 1018, 830,
White solid; yield 49%; mp 165-166 °C. IR (KBr pellet):
3360, 3076, 1756 (CO ester), 1661, 1587, 1526, 1473, 1406,
1274, 1255, 1190, 1087, 1040, 1019, 861, 787, 764, 680.
1H NMR (300 MHz, DMSO): ꢀ 10.69 (1H, bs, NH), 9.35
(1H, d, J=1.3 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz, H5´´), 8.88
(1H, dd, J=1.6 Hz, J=2.4 Hz, H6´´), 7.90-7.87 (2H, m, H2´,
H3), 7.76 (1H, dd, J=2.6 Hz, J=8.7 Hz, H5), 7.57 (1H, d,
J=8.7 Hz, H6), 7.55 (1H, d, J=6.3 Hz, H6´), 7.27-7.24 (2H,
m, H4´, H5´). 13C NMR (75 MHz, DMSO): ꢀ 162.8, 161.9,
149.0, 146.9, 146.4, 145.4, 142.2, 140.4, 132.0, 130.9, 130.8,
130.6, 129.4, 126.8, 125.6, 122.4, 121.6, 118.9. Anal. Calcd.
for C18H11BrClN3O3 (432.66): C, 49.97; H, 2.56; N, 9.71.
Found: C, 50.21; H, 2.66; N, 9.90.
1
801, 769, 712, 656. H NMR (300 MHz, DMSO): ꢀ 10.67
(1H, bs, NH), 9.34 (1H, d, J=1.4 Hz, H3´´), 8.95 (1H, d,
J=2.4 Hz, H5´´), 8.87 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´),
7.88 (1H, d, J=2.5 Hz, H3), 7.75 (1H, dd, J=2.6 Hz, J=8.7
Hz, H5), 7.64 (1H, d, J=8.9 Hz, H6), 7.56 (2H, d, J=8.7 Hz,
H2´, H6´), 7.34 (2H, d, J=8.9 Hz, H3´, H5´). 13C NMR (75
MHz, DMSO): ꢀ 162.6, 161.9, 149.0, 146.9, 146.4, 145.4,
142.2, 137.8, 132.0, 130.8, 130.7, 129.3, 128.8, 127.8, 125.6,
121.7. Anal. Calcd. for C18H11Cl2N3O3 (388.20): C, 55.69;
H, 2.86; N, 10.82. Found: C, 55.50; H, 2.96; N, 10.73.
3.1.4. 5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1d)
3.1.8. 2-(3-Bromophenylcarbamoyl)-5-chlorophenyl pyra-
zine-2-carboxylate (1h)
White solid; yield 43%; mp 178-179 °C. IR (KBr pellet):
3310, 3064, 2928, 2850, 1757 (CO ester), 1655, 1587, 1521,
1489, 1400, 1273, 1242, 1194, 1083, 1043, 1020, 828, 765,
White solid; yield 47%; mp 169-171 °C. IR (KBr pellet):
3398, 3320, 3039, 2928, 1763 (CO ester), 1671, 1587, 1541,
1479, 1403, 1278, 1262, 1185, 1084, 1040, 1015, 848, 673.
1H NMR (300 MHz, DMSO): ꢀ 10.66 (1H, bs, NH), 9.36
(1H, d, J=1.1 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz, H5´´), 8.88
(1H, dd, J=1.3 Hz, J=2.3 Hz, H6´´), 7.87 (1H, d, J=8.2 Hz,
H3), 7.83 (1H, s, H2´), 7.74 (1H, d, J=1.9 Hz, H6), 7.61 (1H,
dd, J=2.0 Hz, J=8.4 Hz, H4), 7.54 (1H, d, J=7.1 Hz, H6´),
7.26-7.23 (2H, m, H4´, H5´). 13C NMR (75 MHz, DMSO): ꢀ
163.3, 161.8, 149.0, 148.8, 146.4, 145.4, 142.2, 140.4, 136.0,
131.2, 130.9, 128.0, 126.9, 126.7, 123.9, 122.4, 121.6, 118.9.
Anal. Calcd. for C18H11BrClN3O3 (432.66): C, 49.97; H,
2.56; N, 9.71. Found: C, 49.76; H, 2.51; N, 9.83.
1
698, 652. H NMR (300 MHz, DMSO): ꢀ 10.63 (1H, bs,
NH), 9.35 (1H, d, J=1.4 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz,
H5´´), 8.88 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.84 (1H, d,
J=8.3 Hz, H3), 7.73 (1H, d, J=2.0 Hz, H6), 7.65-7.58 (3H,
m, H4, H2´, H6´), 7.34 (2H, d, J=8.9 Hz, H3´, H5´). 13C
NMR (75 MHz, DMSO): ꢀ 163.1, 161.8, 149.0, 148.8,
146.4, 145.4, 142.2, 137.8, 135.9, 131.2, 128.8, 128.1, 127.7,
126.8, 123.9, 121.6. Anal. Calcd. for C18H11Cl2N3O3
(388.20): C, 55.69; H, 2.86; N, 10.82. Found: C, 55.89; H,
2.65; N, 10.67.
3.1.5.
4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl
pyrazine-2-carboxylate (1e)
White solid; yield 43%; mp 196.5-198.5 °C. IR (KBr pel-
let): 3379, 3085, 1755 (CO ester), 1673, 1590, 1531, 1477,
1407, 1375, 1282, 1261, 1193, 1098, 1080, 1040, 1016, 859,
3.1.9. 2-(4-Bromophenylcarbamoyl)-4-chlorophenyl pyra-
zine-2-carboxylate (1i)
1
White solid; yield 71%; mp 188.5-190.5 °C. IR (KBr pel-
let): 3406, 3082, 1746 (CO ester), 1675, 1595, 1536, 1488,
1400, 1286, 1242, 1189, 1100, 1041, 1018, 804, 770, 704.
1H NMR (300 MHz, DMSO): ꢀ 10.67 (1H, bs, NH), 9.34
(1H, d, J=1.4 Hz, H3´´), 8.95 (1H, d, J=2.4 Hz, H5´´), 8.87
(1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.88 (1H, d, J=2.6 Hz,
H3), 7.75 (1H, dd, J=2.6 Hz, J=8.7 Hz, H5), 7.60-7.54 (3H,
m, H6, H2´, H6´), 7.47 (2H, d, J=8.9 Hz, H3´, H5´). 13C
NMR (75 MHz, DMSO): ꢀ 162.6, 161.9, 149.0, 146.9,
146.4, 145.4, 142.2, 138.2, 131.9, 131.7, 130.8, 130.7, 129.3,
125.6, 122.0, 115.9. Anal. Calcd. for C18H11BrClN3O3
(432.66): C, 49.97; H, 2.56; N, 9.71. Found: C, 50.09; H,
2.80; N, 10.01.
807, 767, 677. H NMR (300 MHz, DMSO): ꢀ 10.81 (1H,
bs, NH), 9.35 (1H, d, J=1.5 Hz, H3´´), 8.96 (1H, d, J=2.4
Hz, H5´´), 8.87 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.92
(1H, t, J=1.1 Hz, H2´), 7.90 (1H, d, J=2.6 Hz, H3), 7.77 (1H,
dd, J=2.6 Hz, J=8.7 Hz, H5), 7.57 (1H, d, J=8.7 Hz, H6),
7.56-7.54 (2H, m, H5´, H6´). 13C NMR (75 MHz, DMSO): ꢀ
162.9, 161.9, 149.0, 146.9, 146.4, 145.4, 142.2, 138.9, 132.2,
131.1, 130.9, 130.8, 130.4, 129.4, 125.7, 125.6, 121.3, 120.2.
Anal. Calcd. for C18H10Cl3N3O3 (422.65): C, 51.15; H, 2.38;
N, 9.94. Found: C, 51.46; H, 2.30; N, 9.74.
3.1.6.
5-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl
pyrazine-2-carboxylate (1f)
White solid; yield 43%; mp 199.5-201 °C. IR (KBr pel-
let): 3402, 3091, 1744 (CO ester), 1677, 1588, 1533, 1475,
1397, 1375, 1285, 1262, 1183, 1097, 1040, 1018, 914, 854,
823, 767, 633. H NMR (300 MHz, DMSO): ꢀ 10.77 (1H,
bs, NH), 9.36 (1H, d, J=1.4 Hz, H3´´), 8.96 (1H, d, J=2.4
3.1.10. 2-(4-Bromophenylcarbamoyl)-5-chlorophenyl pyra-
zine-2-carboxylate (1j)
1
White solid; yield 50%; mp 180-182.5 °C. IR (KBr pel-
let): 3312, 3065, 2930, 1756 (CO ester), 1655, 1584, 1521,

736 Medicinal Chemistry, 2012, Vol. 8, No. 4
Krátkꢀ et al.
125.6, 122.0 (J=7.9 Hz), 115.5 (J=22.3 Hz). Anal. Calcd. for
C18H11ClFN3O3 (371.75): C, 58.16; H, 2.98; N, 11.30.
Found: C, 58.00; H, 3.15; N, 11.17.
1486, 1398, 1272, 1241, 1194, 1083, 1043, 1020, 868, 826,
764, 696, 653. H NMR (300 MHz, DMSO): ꢀ 10.63 (1H,
1
bs, NH), 9.35 (1H, d, J=1.4 Hz, H3´´), 8.96 (1H, d, J=2.4
Hz, H5´´), 8.88 (1H, dd, J=1.5 Hz, J=2.3 Hz, H6´´), 7.84
(1H, d, J=8.4 Hz, H3), 7.73 (1H, d, J=2.0 Hz, H6), 7.62-7.55
3.1.14. 5-Chloro-2-(4-fluorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1n)
(3H, m, H4, H2´, H6´), 7.46 (2H, d, J=8.9 Hz, H3´, H5´). 13
C
White solid; yield 71%; mp 160-162 °C. IR (KBr pellet):
3386, 3053, 1757 (CO ester), 1668, 1601, 1548, 1508, 1407,
1277, 1192, 1155, 1085, 1041, 1016, 828, 768, 692.
1H NMR (300 MHz, DMSO): ꢀ 10.55 (1H, bs, NH), 9.35
(1H, d, J=1.4 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz, H5´´), 8.88
(1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.84 (1H, d, J=8.3 Hz,
H3), 7.73 (1H, d, J=2.0 Hz, H6), 7.63-7.57 (3H, m, H4, H2´,
H6´), 7.15-7.08 (2H, m, H3´, H5´). 13C NMR (75 MHz,
DMSO): ꢀ 165.4 (J=242.8 Hz), 162.9, 161.8, 149.0, 148.8,
146.4, 145.4, 142.2, 135.8 (J=2.0 Hz), 131.1, 128.2, 126.8,
123.8, 121.9 (J=7.9 Hz), 119.4, 115.5 (J=22.3 Hz). Anal.
Calcd. for C18H11ClFN3O3 (371.75): C, 58.16; H, 2.98; N,
11.30. Found: C, 58.39; H, 3.09; N, 11.09.
NMR (75 MHz, DMSO): ꢀ 163.1, 161.8, 149.0, 148.8,
146.4, 145.4, 142.2, 138.3, 135.9, 131.7, 131.2, 128.1, 126.8,
123.9, 122.0, 115.8. Anal. Calcd. for C18H11BrClN3O3
(432.66): C, 49.97; H, 2.56; N, 9.71. Found: C, 49.74; H,
2.67; N, 9.89.
3.1.11. 4-Chloro-2-(3-fluorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1k)
White solid; yield 61%; mp 155.5-157.5 °C. IR (KBr pel-
let): 3387, 3316, 3081, 2929, 1753 (CO ester), 1668, 1614,
1541, 1491, 1474, 1446, 1287, 1195, 1094, 1043, 1019, 768,
1
674. H NMR (300 MHz, DMSO): ꢀ 10.74 (1H, bs, NH),
9.35 (1H, d, J=1.3 Hz, H3´´), 8.95 (1H, d, J=2.4 Hz, H5´´),
8.87 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.88 (1H, d, J=2.5
Hz, H3), 7.76 (1H, dd, J=2.6 Hz, J=8.7 Hz, H5), 7.57 (1H, d,
J=8.7 Hz, H6), 7.53 (1H, dt, J=1.9 Hz, J=11.5 Hz, H6´),
7.39-7.28 (2H, m, H2´, H5´), 6.93-6.86 (1H, m, H4´). 13C
NMR (75 MHz, DMSO): ꢀ 163.7 (J=241.6 Hz), 162.8,
161.9, 149.0, 146.9, 146.4, 145.4, 142.2, 140.5 (J=11.0 Hz),
132.0, 130.8, 130.7, 130.6 (J=9.4 Hz), 129.3, 125.6, 115.9
(J=2.6 Hz), 110.7 (J=21.1 Hz), 106.8 (J=26.2 Hz). Anal.
Calcd. for C18H11ClFN3O3 (371.75): C, 58.16; H, 2.98; N,
11.30. Found: C, 58.38; H, 3.22; N, 11.42.
3.1.15. 4-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)
phenyl pyrazine-2-carboxylate (1o)
White solid; yield 55%; mp 183.5-185 °C. IR (KBr pel-
let): 3401, 3080, 2928, 1748 (CO ester), 1655, 1600, 1541,
1474, 1409, 1331, 1288, 1242, 1191, 1159, 1085, 1066,
1
1041, 1015, 842, 770, 698. H NMR (300 MHz, DMSO): ꢀ
10.89 (1H, bs, NH), 9.34 (1H, d, J=1.4 Hz, H3´´), 8.95 (1H,
d, J=2.4 Hz, H5´´), 8.87 (1H, dd, J=1.5 Hz, J=2.3 Hz, H6´´),
7.92 (1H, d, J=2.6 Hz, H3), 7.82 (2H, d, J=8.6 Hz, H2´,
H6´), 7.77 (1H, dd, J=2.6 Hz, J=8.7 Hz, H5), 7.66 (1H, d,
J=8.7 Hz, H6), 7.58 (2H, d, J=8.7 Hz, H3´, H5´). 13C NMR
(75 MHz, DMSO): ꢀ 163.1, 161.9, 149.0, 146.9, 146.4,
145.4, 142.4, 142.2, 132.1, 130.8, 130.5, 129.4, 126.2, 125.6,
124.3, 123.9, 120.0. Anal. Calcd. for C19H11ClF3N3O3
(421.76): C, 54.11; H, 2.63; N, 9.96. Found: C, 54.35; H,
2.90; N, 9.66.
3.1.12. 5-Chloro-2-(3-fluorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1l)
White solid; yield 61%; mp 147.5-148.5 °C. IR (KBr pel-
let): 3389, 3106, 2929, 1751 (CO ester), 1670, 1598, 1541,
1442, 1396, 1276, 1196, 1122, 1090, 1042, 1018, 966, 837,
1
768, 674. H NMR (300 MHz, DMSO): ꢀ 10.70 (1H, bs,
3.1.16. 5-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)
phenyl pyrazine-2-carboxylate (1p)
NH), 9.36 (1H, d, J=1.3 Hz, H3´´), 8.96 (1H, d, J=2.4 Hz,
H5´´), 8.88 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 7.84 (1H, d,
J=8.3 Hz, H3), 7.74 (1H, d, J=1.9 Hz, H6), 7.61 (1H, dd,
J=2.0 Hz, J=8.3 Hz, H4), 7.53 (1H, dt, J=1.8 Hz, J=11.5 Hz,
H6´), 7.38-7.27 (2H, m, H2´, H5´), 6.93-6.86 (1H, m, H4´).
13C NMR (75 MHz, DMSO): ꢀ 163.8 (J=241.5 Hz), 163.3,
161.8, 149.0, 148.8, 146.4, 145.4, 142.2, 140.6 (J=11.0 Hz),
136.0, 131.2, 130.6 (J=9.4 Hz), 128.1, 126.8, 123.9, 115.8
(J=2.5 Hz), 110.6 (J=21.1 Hz), 106.8 (J=26.3 Hz). Anal.
Calcd. for C18H11ClFN3O3 (371.75): C, 58.16; H, 2.98; N,
11.30. Found: C, 58.02; H, 3.13; N, 11.48.
White solid; yield 56%; mp 159-161 °C. IR (KBr pellet):
3356, 3068, 1749 (CO ester), 1657, 1593, 1537, 1476, 1410,
1319, 1280, 1250, 1185, 1161, 1087, 1062, 1044, 1019, 843,
1
764, 697. H NMR (300 MHz, DMSO): ꢀ 10.91 (1H, bs,
NH), 9.34 (1H, d, J=1.4 Hz, H3´´), 8.95 (1H, d, J=2.4 Hz,
H5´´), 8.87 (1H, dd, J=1.4 Hz, J=2.4 Hz, H6´´), 7.85 (1H, d,
J=8.4 Hz, H3), 7.82 (2H, d, J=8.5 Hz, H2´, H6´), 7.73 (1H,
d, J=2.0 Hz, H6), 7.62 (1H, dd, J=2.1 Hz, J=8.8 Hz, H4),
7.45 (2H, d, J=6.9 Hz, H3´, H5´). 13C NMR (75 MHz, DM-
SO): ꢀ 163.2, 161.8, 153.0, 149.0, 148.8, 146.4, 145.4,
142.2, 139.0, 135.0, 131.6, 130.1, 128.9, 128.4, 124.8, 124.0,
118.3. Anal. Calcd. for C19H11ClF3N3O3 (421.76): C, 54.11;
H, 2.63; N, 9.96. Found: C, 54.41; H, 2.96; N, 9.85.
3.1.13. 4-Chloro-2-(4-fluorophenylcarbamoyl)phenyl pyra-
zine-2-carboxylate (1m)
White solid; yield 91%; mp 151.5-153.5 °C. IR (KBr pel-
let): 3404, 3326, 3080, 2927, 2849, 1746 (CO ester), 1674,
1624, 1548, 1508, 1473, 1407, 1287, 1251, 1215, 1190,
1157, 1097, 1041, 1019, 831, 768, 652. 1H NMR (300 MHz,
DMSO): ꢀ 10.59 (1H, bs, NH), 9.34 (1H, d, J=1.4 Hz, H3´´),
8.95 (1H, d, J=2.4 Hz, H5´´), 8.87 (1H, dd, J=1.5 Hz, J=2.3
Hz, H6´´), 7.88 (1H, d, J=2.6 Hz, H3), 7.75 (1H, dd, J=2.6
Hz, J=8.7 Hz, H5), 7.63-7.54 (3H, m, H6, H2´, H6´), 7.16-
7.09 (2H, m, H3´, H5´). 13C NMR (75 MHz, DMSO): ꢀ
165.4 (J=248.6 Hz), 162.5, 161.9, 149.0, 146.9, 146.4,
145.4, 142.3, 135.2 (J=2.6 Hz), 131.8, 130.8, 130.7, 129.3,
3.1.17. 4-Bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)
phenyl pyrazine-2-carboxylate (1q)
White solid; yield 54%; mp 165-167 °C. IR (KBr pellet):
3403, 3082, 1746 (CO ester), 1678, 1600, 1541, 1469, 1408,
1331, 1288, 1254, 1187, 1164, 1086, 1066, 1041, 1017, 841,
1
770, 687. H NMR (300 MHz, DMSO): ꢀ 10.89 (1H, bs,
NH), 9.34 (1H, d, J=1.4 Hz, H3´´), 8.95 (1H, d, J=2.4 Hz,
H5´´), 8.87 (1H, dd, J=1.5 Hz, J=2.4 Hz, H6´´), 8.03 (1H, d,
J=2.5 Hz, H3), 7.90 (1H, dd, J=2.5 Hz, J=8.7 Hz, H5), 7.82

In vitro Antibacterial and Antifungal Activity
Medicinal Chemistry, 2012, Vol. 8, No. 4 737
Table 2. In vitro Antibacterial Activity of the Esters
MIC/IC80 [μmol/L]
SA
MRSA
SE
EF
EC
PA
24 h
3.9
48 h
7.81
1.95
3.9
24 h
3.9
48 h
31.25
3.9
24 h
3.9
48 h
62.5
24 h
31.25
3.9
48 h
31.25
7.81
24 h
>125
>125
62.5
48 h
>125
>125
62.5
24 h
>125
7.81
48 h
>125
7.81
1a
1b
1c
0.98
3.9
1.95
3.9
1.95
3.9
1.95
7.81
15.62
7.81
7.81
15.62
7.81
7.81
7.81
3.9
7.81
7.81
15.62
15.62
>125
125
7.81
15.62
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>500
>500
1d
1e
7.81
7.81
7.81
7.81
7.81
7.81
7.81
1.95
3.9
7.81
7.81
7.81
7.81
7.81
7.81
7.81
3.9
7.81
7.81
7.81
7.81
7.81
7.81
7.81
3.9
7.81
7.81
7.81
7.81
7.81
7.81
7.81
7.81
7.81
>125
7.81
7.81
7.81
7.81
3.9
15.62
7.81
15.62
7.81
>125
>125
>125
>125
>125
>125
>125
>125
125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>500
>500
1f
7.81
7.81
1g
15.62
15.62
15.62
15.62
15.62
15.62
>125
15.62
7.81
31.25
15.62
7.81
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
15.62
62.5
1h
1i
1j
15.62
31.25
15.62
>125
15.62
7.81
1k
1l
7.81
7.81
7.81
7.81
7.81
7.81
3.9
7.81
7.81
3.9
15.62
15.62
15.62
7.81
7.81
7.81
3.9
1m
1n
1o
7.81
3.9
>125
125
7.81
7.81
7.81
3.9
7.81
7.81
7.81
3.9
>125
>125
>125
>125
125
>125
>125
>125
>125
125
1p
1q
1r
7.81
7.81
7.81
7.81
7.81
7.81
PNC
BAC
0.98
7.81
0.98
15.62
62.5
15.62
125
250
250
7.81
15.62
15.62
31.25
15.62
>500
>500
SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; EF: Enterococcus sp. J
14365/08; EC: E. coli CCM4517; PA: Pseudomonas aeruginosa CCM 1961; PNC: penicillin G; BAC: bacitracin. Two lowest MIC values for each strain (except E. coli) are bolded.
(2H, d, J=8.5 Hz, H2´, H6´), 7.66 (1H, d, J=8.6 Hz, H6),
7.51 (2H, d, J=8.7 Hz, H3´, H5´). 13C NMR (75 MHz, DM-
SO): ꢀ 163.0, 161.9, 149.0, 147.4, 146.4, 145.4, 142.4,
142.2, 135.1, 132.2, 130.9, 126.2, 125.9, 124.3, 123.9, 120.0,
118.9. Anal. Calcd. for C19H11BrF3N3O3 (466.21): C, 48.95;
H, 2.38; N, 9.01. Found: C, 49.22; H, 2.69; N, 9.24.
NMR (75 MHz, DMSO): ꢀ 163.0, 161.9, 149.0, 146.9,
146.4, 145.4, 142.2, 139.6, 132.1, 130.8, 130.5, 130.2, 129.4,
128.6, 126.0, 125.6, 123.7, 122.9, 119.2. Anal. Calcd. for
C19H11ClF3N3O3 (421.76): C, 54.11; H, 2.63; N, 9.96. Found:
C, 54.29; H, 2.97; N, 10.12.
3.2. Antibacterial Evaluation
3.1.18. 4-Chloro-2-(3-(trifluoromethyl)phenylcarbamoyl)
phenyl pyrazine-2-carboxylate (1r)
The results of the in vitro antibacterial activity of the syn-
thesized esters (1) are presented in Table 2. In this assay, in
the concentration of 250 ꢁmol/L pyrazine-2-carboxylic acid
was completely inactive (data not shown).
White solid; yield 26%; mp 168-170 °C. IR (KBr pellet):
3322, 3065, 2927, 2850, 1712 (CO ester), 1665, 1625, 1568,
1489, 1393, 1310, 1272, 1226, 1154, 1123, 1088, 1051,
1
1018, 890, 786, 700. H NMR (300 MHz, DMSO): ꢀ 10.85
All of the compounds exhibited activity against both S.
aureus strains and except 1m inhibited the growth of S. epi-
dermidis and Enterococcus as well. The methicillin-sensitive
S. aureus strain was susceptible in the range of 0.98 to 7.81
ꢁmol/L. The most efficacious structure was found ester 1b
(0.98/1.95 ꢁmol/L after 24 h and 48 h, respectively); the
(1H, bs, NH), 9.35 (1H, d, J=1.3 Hz, H3´´), 8.95 (1H, d,
J=2.4 Hz, H5´´), 8.86 (1H, dd, J=1.4 Hz, J=2.4 Hz, H6´´),
7.99 (1H, s, H2´), 7.92 (1H, d, J=2.5 Hz, H3), 7.77 (1H, dd,
J=2.5 Hz, J=8.7 Hz, H5), 7.58 (1H, d, J=8.8 Hz, H6), 7.52
(1H, d, J=8.1 Hz, H6´), 7.46-7.40 (2H, m, H4´, H5´). 13C

738 Medicinal Chemistry, 2012, Vol. 8, No. 4
Krátkꢀ et al.
Table 3. In vitro Antifungal Activity of Prepared Esters
MIC/IC80 [μmol/L]
CG TA
CA
CT
CK
AF
AC
TM
24 h
>125
>125
>125
7.81
48 h
24 h
48 h
24 h
>125
>125
>125
7.81
48 h
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
0.18
24 h
>125
>125
>125
7.81
48 h
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
31.25
>125
>125
>125
>125
0.09
24 h
>125
>125
>125
7.81
>125
7.81
>125
>125
7.81
7.81
>125
125
48 h
>125
>125
>125
62.5
>125
7.81
>125
>125
7.81
7.81
>125
>125
>125
15.62
>125
7.81
125
24 h
>125
>125
>125
7.81
48 h
>125
>125
>125
>125
>125
>125
>125
>125
7.81
24 h
>125
>125
>125
7.81
>125
3.9
48 h
>125
>125
>125
7.81
72 h
7.81
7.81
7.81
7.81
120 h
7.81
7.81
7.81
7.81
1a
1b
1c
>125 >125 >125
>125 >125 >125
>125 >125 >125
1d
1e
>125
125
>125
>125
>125
>125
>125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125 >125 >125
>125
>125
>125
>125
7.81
>125
>125
>125
>125
7.81
>125
7.81
>125
15.62
62.5
>125
15.62
7.81
31.25
3.9
>125
15.62
15.62
62.5
3.9
1f
1g
>125
>125
3.9
7.81
7.81
3.9
1h
1i
125
15.62
7.81
7.81
1j
7.81
15.62
>125
>125
>125
15.62
>125
125
7.81
7.81
3.9
7.81
3.9
7.81
3.9
1k
1l
>125
125
>125
125
>125
31.25
>125
15.62
7.81
>125
>125
>125
15.62
62.5
>125
31.25
>125
15.62
3.9
>125
>125
>125
15.62
125
3.9
3.9
3.9
1m
1n
1o
>125
31.25
>125
62.5
>125
7.81
>125
15.62
7.81
7.81
62.5
>125
1.08
4.00
3.9
3.9
3.9
3.9
>125
7.81
3.9
3.9
1p
1q
1r
7.81
>125
>125
>125
0.23
3.9
7.81
3.9
3.9
>125
>125
0.03
>125
>125
0.14
>125
>125
0.03
7.81
3.9
15.62
7.81
15.62
1.95
1.08
17.0
15.62
3.9
>125
1.8
>125
0.18
7.81
1.08
AMB
FLU
0.06
2.00
0.09
3.00
0.11
5.00
2.16
1.08
26.0
1.00
>50.0
>50.0
22.0
>50.0
9.00
>50.0 >50.0
>50.0 >50.0
CA: Candida albicans ATCC 44859; CT: Candida tropicalis 156; CK: Candida krusei E28; CG: Candida glabrata 20/I; TB: Trichosporon asahii 1188; AF: Aspergillus fumigatus
231; AC: Absidia corymbifera 272; TM: Trichophyton mentagrophytes 445; AMB: amphotericin B; FLU: fluconazole. The best MIC values for each strain are bolded.
MRSA strain was affected too, with MIC being for almost
all derivatives equal or only two-fold higher than for the
non-resistant strain (1.95 – 31.25 ꢀmol/L). 1b expressed the
lowest MIC values of 1.95/3.9 ꢀmol/L. Also the same com-
pound best suppressed the growth of S. epidermidis (MIC =
1.95/1.95 ꢀmol/L, generally 1.95 – 62.5 ꢀmol/L) and En-
terococcus (MIC of 3.9/7.81 ꢀmol/L). Enterococcus dis-
played the lowest sensitivity out of four tested Gram-positive
strains.
Gram-negative bacteria showed a higher level of in vitro
resistance. E. coli was affected by 1c with MIC of 62.5
ꢀmol/L and 1l, 1n (125 ꢀmol/L). No compound inhibited the
growth of ESBL-positive K. pneumoniae in the concentra-
tion of 125 ꢀmol/L or lower, and only 1n affected K. pneu-
moniae at 125 ꢀmol/L. Moreover, P. aeruginosa was suscep-
tible in this assay only to 1b and 1c, but with relatively low
MIC (7.81 ꢀmol/L).
3.3. Antifungal Evaluation
These derivatives expressed lower MIC than benzylpeni-
cillin for MRSA, S. epidermidis; an average comparable (but
ranging) expression of MIC was found for Enterococcus
after 24 hours of incubation (Table 2). When compared with
bacitracin, the activity of salicylanilide pyrazine-2-
carboxylates was better (or in some cases comparable) for
MRSA, S. aureus and S. epidermidis. The situation for en-
terococci is more complex and results are not clear.
The prepared esters (1) were investigated in vitro for
their antifungal activity against four yeast and four mould
strains. Free pyrazine-2-carboxylic acid was quite inactive in
this assay, with MIC values of 500 ꢀmol/L and higher (re-
sults not shown). Table 3 summarizes these results.
In summary, besides 1e, which was in our test conditions
inactive (MIC > 125 ꢀmol/L), all esters expressed a clear
significant antifungal activity. Candida spp. were less sus-

In vitro Antibacterial and Antifungal Activity
Medicinal Chemistry, 2012, Vol. 8, No. 4 739
ceptible than moulds – the lowest MIC were 7.81 and 1.95
ꢀmol/L, respectively. The most susceptible to all derivatives
except for 1e was the T. mentagrophytes strain (MIC ranged
between 1.95 and 62.5 ꢀmol/L with 1r being a favourable
derivative); the least susceptible was C. tropicalis, which
was resistant – only 1d expressed some activity (MIC 125
ꢀmol/L). With one exception (1n at C. glabrata) MIC for all
Candida spp. after 48 hours exceeded 125 ꢀmol/L. Three
partly susceptible Candida strains (C. albicans, C. krusei and
C. tropicalis) were inhibited by 1d, 1i, 1j, 1n and 1p, with
the greatest inhibition found in 1d (7.81 ꢀmol/L).
ence on the position of chlorine on the salicylic ring – in
some cases 5-chloro derivatives (1a vs. 1b, 1e and 1f, 1g and
1h, 1m and 1n) are more active, in others 4-chloro deriva-
tives (1c vs. 1d, 1i and 1j, 1k and 1l). 1o and 1p have the
same activities. In the pair of 1o and 1q, 4-Cl and 4-Br are
comparably efficacious. When we evaluated the substituents
on the anilide moiety, there were not many clear connections
among position isomers. Comparing different substituents on
the same positions we can conclude in general that 4´-Br is
slightly superior to 3´-Br and 3´-F to 4´-F. 3´-Cl reduces
more MIC than 3´-Br and 3´-F, 4´-Cl than 4´-Br and 4´-F,
3´-CF3 than 4´-CF3. 4´-Cl is gently more efficacious than 4´-
CF3; on the other hand 4´-CF3 is preferable to 4´-Br and 4´-
F. It also seems that the anilide 3-position brings better re-
sults. The antibacterial activity of more lipophilic salicy-
lanilide esters with 3,4-dichloroaniline moiety (1e, 1f) is not
better than those derived from either 3- or 4-chloroaniline
(1a – 1d).
Half of the esters expressed the significant growth inhibi-
tion of the A. fumigatus strain (1d, 1f, 1i, 1j, 1l, 1n, 1o, 1p,
and 1q), and twelve derivatives (1d, 1f, 1g, 1h, 1i, 1j, 1l, 1n,
1o, 1p, 1q, and 1r) inhibited A. corymbifera with MIC 3.9
ꢀmol/L and higher for both strains. Nine esters (1d, 1f, 1i,
1j, 1l, 1n, 1o, 1p, 1q) were able to stop the growth of T. asa-
hii (MIC ꢁ 7.81 ꢀmol/L).
Lipophilicity is considered only one factor influencing
antimicrobial activity, and in this series a direct relationship
was not found to be present. Good activities were expressed
by a derivative with a high calculated lipophilicity (1r;
3.55), but also by 1k with logP 2.79 (one of the most hydro-
philic esters). 1e, 1f, 1o, 1p, or 1q, the most lipophilic com-
pounds (logP 3.55 – 3.82), exhibited uniform results, but not
the most excellent antibacterial acting. Derivatives with 3- or
4-chloroaniline parts (3.19) were more efficient than both 3-
/4-bromoaniline (3.46) and 3-/4-fluoroaniline (2.79) moie-
ties.
No compound was more effective than the standard an-
timycotic drug fluconazole against Candida albicans. When
esters showed activity against C. krusei, C. glabrata, T. asa-
hii, A. fumigatus and A. corymbifera, they were more active
than fluconazole. The susceptibility of T. mentagrophytes
was dependent on the time of incubation – after 72 hours in
vitro fluconazole showed a stronger effect than four com-
pounds (1f, 1g, 1h, 1q, 1e), whereas 1a, 1b, 1c, 1d, and 1g
were comparable and nine esters favorable; after 120 hours
only 1e showed a higher MIC level than fluconazole.
4. DISCUSSION
Our results concerning salicylanilide pyrazine-2-
carboxylates support earlier findings that salicylanilide esters
with simple organic acids exhibited a good activity against
bacteria, especially Gram-positive cocci [37, 39]. When
compared with N-acetyl-L-phenylalanine salicylanilide es-
ters [30], the derivatives described above stopped the growth
of Gram-positive bacteria more uniformly, whereas these
amino acid derivatives expressed non-constant activity with
MIC ꢁ 0.98 μmol/L. Tetrachlorosalicylanilide (3,5-dichloro-
N-(3,4-dichlorophenyl)-2-hydroxybenzamide), a well-known
member of the salicylanilide group, inhibits various Gram-
positive strains at the concentrations of 0.25 – 0.5 μg/mL [9].
4.1. Antibacterial Activity
All eighteen newly synthesized esters exhibited in vitro
activity against all four Gram-positive cocci, including
MRSA and Enterococcus. The exception was the compound
1m, which was active only against S. aureus. The MIC val-
ues of the esters were only negligibly influenced by the
length of incubation, which is comparable with antibiotic
reference drugs (penicillin, bacitracin). This suggests the
bactericidal rather than bacteriostatic nature of the com-
pounds. The potential of salicylanilide esters with pyrazine-
2-carboxylic acid to inhibit in vitro growth especially of
MRSA and enterococci in relatively low concentrations may
be very promising. This is supported by the fact that these
derivatives are more or similarly efficient in comparison
with the commonly used and well-established antibiotic sub-
stances benzylpenicillin and bacitracin, which is used spe-
cifically for the treatment of topical Gram-positive infections
[36]. The expressed inhibitory action of 1b and 1c against P.
aeruginosa is also significant because it is the first described
low-concentration activity of salicylanilide esters against P.
aeruginosa [7, 30, 37]. This bacterial pathogen represents a
serious threat in hospitals, causing severe nosocomial infec-
tions which are complicated by the bacteria’s resistance to
many antibiotics [38]. Therefore, the in vitro activity of these
two salicylanilide esters may be considered to be of vital
importance.
4.2. Antifungal Activity
All esters except 1e exhibited a significant antifungal ef-
fect on the filamentous strains tested, particularly on T. men-
tagrophytes. In most cases, the antifungal activity is en-
hanced by the presence of chlorine on the position 4 of sali-
cylic ring (1c and 1d, 1e and 1f, 1k and 1l, 1m and 1n, 1o
and 1p); in the counterpart 1o and 1q 4-Cl is more favour-
able than 4-Br. Optimal substituents for the anilide ring rep-
resent 4-Br, 4-CF3 and in one case 3-CF3. The 3,4-
disubstitution by chlorines provides no benefit as compared
with 4-chloro derivatives. 3´-F derivatives expressed a sig-
nificantly better activity than 3´-Cl ones.
Lipophilicity plays a similar role as is the case with anti-
bacterial activity; it is undoubtedly important, but is only one
factor of many in total inhibitory activity, one whose precise
role has not been clearly defined.
Focusing on the structure-activity relationship for the in
vitro inhibition of Gram-positive pathogens, we can postu-
late that the relationship is in this series not a distinct influ-

740 Medicinal Chemistry, 2012, Vol. 8, No. 4
Krátkꢀ et al.
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Received: March 09, 2011
Revised: November 21, 2011
Accepted: November 25, 2011