Synthesis and antimycobacterial evaluation of substituted pyrazinecarboxamides

Article abstract of DOI:10.1016/j.ejmech.2007.07.013

Unsubstituted, halogenated and/or alkylated pyrazine-2-carboxylic acid amides connected via -CONH- bridge with substituted anilines were synthesized using currently known synthetic pathways. The synthetic approach, analytical, spectroscopic, lipophilicity and biological data of 20 newly synthesized compounds are presented. Structure-activity relationships among the chemical structures, the antimycobacterial, antifungal, photosynthesis inhibiting and antialgal activity of the evaluated substituted N-phenylpyrazine-2-carboxamides are discussed. 5-tert-Butyl-6-chloro-N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (19) has shown the highest activity against Mycobacterium tuberculosis H₃₇Rv (MIC = 3.13 μg/mL). The highest antifungal effect against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (14, MIC = 62.5 μmol/mL). The highest reduction of chlorophyll content in Chlorella vulgaris was found for pyrazine-2-carboxylic acid (3-trifluoromethylphenyl)amide (9, IC₅₀ = 12.1 μmol/L).

Full text of DOI:10.1016/j.ejmech.2007.07.013

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1105e1113
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antimycobacterial evaluation of substituted
pyrazinecarboxamides
a
a
a
a
Martin Dolezal a, , Pavlina Cmedlova , Lukas Palek , Jarmila Vinsova , Jiri Kunes ,
*
Vladimir Buchta b, Josef Jampilek c, Katarina Kralova d
a Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b Department of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University in Prague, Sokolska 581,
500 05 Hradec Kralove, Czech Republic
c Zentiva a.s., U Kabelovny 130, 102 37 Prague 10, Czech Republic
d Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska Dolina, 842 15 Bratislava, Slovak Republic
Received 15 February 2007; received in revised form 24 May 2007; accepted 12 July 2007
Available online 2 August 2007
Abstract
Unsubstituted, halogenated and/or alkylated pyrazine-2-carboxylic acid amides connected via eCONHe bridge with substituted anilines
were synthesized using currently known synthetic pathways. The synthetic approach, analytical, spectroscopic, lipophilicity and biological
data of 20 newly synthesized compounds are presented. Structureeactivity relationships among the chemical structures, the antimycobacterial,
antifungal, photosynthesis inhibiting and antialgal activity of the evaluated substituted N-phenylpyrazine-2-carboxamides are discussed. 5-tert-
Butyl-6-chloro-N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (19) has shown the highest activity against Mycobacterium tuberculosis
H37Rv (MIC ¼ 3.13 mg/mL). The highest antifungal effect against Trichophyton mentagrophytes, the most susceptible fungal strain tested,
was found for N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (14, MIC ¼ 62.5 mmol/mL). The highest reduction of chlorophyll content
in Chlorella vulgaris was found for pyrazine-2-carboxylic acid (3-trifluoromethylphenyl)amide (9, IC50 ¼ 12.1 mmol/L).
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrazinecarboxamides; Antimycobacterial; Antifungal; Photosynthesis inhibiting; Antialgal activity; Lipophilicity determination; Structureeactivity
relationships; In vitro
1. Introduction
original compounds made more accessible by the design of
new, or the modernisation of, synthetic transformations. The
evaluation of pyrazinoic acid, the pharmacologically active
principle of first line anti-tuberculosis drug pyrazinamide, is
intensively focused on. The minimal structure of pyrazine
ring with an acyl moiety is sufficient for fatty acid synthase
type I (FAS I) and antimycobacterial activity of some pyrazi-
necarboxylic acid derivatives [2].
In connection with our research focused on antimycobac-
terial active pyrazinecarboxylic acid derivatives we are
interested in binuclear analogues with the eCONHe bridge
[3e5]. The amide function, as considered in this work, is
based on the bivalent moiety eCONHe, which can form
centrosymmetric dimer pairs with the peptidic carboxamido
group of some peptides, needed for binding to the receptor
Tuberculosis is still a major global disease infecting one
third of the world’s population and killing almost two million
people each year. Multidrug-resistant strains of Mycobacte-
rium tuberculosis in conjunction with the spread of HIV infec-
tion, often lead to the treatment failure. Thus, much effort is
being made to develop novel anti-tuberculosis drugs which
are more safe and efficacious [1]. There are two basic strate-
gies for antimycobacterial drug development. The first is the
extraordinary molecular diversity. The second results from
* Corresponding author. Tel.: þ420495067389; fax: þ420495067167.
E-mail address: dolezalm@faf.cuni.cz (M. Dolezal).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.013

1106
M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
site, possibly by forming a hydrogen bond. Various com-
pounds possessing eCONHe group have been found to in-
hibit photosynthetic electron transport [6e9]. Previous
studies [4,5,10] showed that alkylation, amidation or aroyla-
tion of the pyrazine ring or substitution of pyrazine with
chlorine can increase the antituberculotic and/or antifungal
activity in series of functional derivatives of pyrazinecarbox-
ylic acid [11]. Some of these derivatives influenced produc-
tion of flavonolignans in an in vitro culture [12].
This study is continuation of our previous articles dealing
with the N-heterocyclic derivatives as potential drugs [3e
5,10e14] and is concerned with the synthesis of a series of am-
ides prepared from pyrazinecarboxylic acids and various ring-
substituted anilines. The aim of this work is to find the struc-
tureeactivity relationship in the mentioned series, i.e. to con-
tinue the study of the substituent variability influence on the
antimycobacterial, antifungal, photosynthesis inhibiting or
antialgal activities, and to determine the importance of in-
creased hydro/lipophilic properties for biological effect of the
newly prepared substituted pyrazinecarboxamides.
Experimentally determined lipophilicity values (log K values)
were lower than the corresponding calculated log P values.
The linear dependence of log K vs. log P is shown in Fig. 1.
The statistical parameters of this correlation (r ¼ 0.969;
P < 0.0001; n ¼ 20) confirmed accordance between experi-
mental log K values with the corresponding calculated log P
values. The lowest lipophilicity was shown by N-(4-fluoro-
phenyl)pyrazine-2-carboxamide (2), whereas 5-tert-butyl-6-
chloro-N-(2-trifluoromethylphenyl)pyrazine-2-carboxamide (18)
possessed the highest lipophilicity.
Based on the log K data, the lipophilicity of the substituents
in the aromatic part of the molecule increased in the following
order: 4-F < 2-Cl-5-OH < 4-CH3 < 3-CF3 < 2-CF3. This is in
accordance with the values of corresponding p-parameters
characterizing hydrophobicity of the substituents, namely 0.15
for F, 0.76 for 2-Cl, ꢀ0.50 for 5-OH, 0.60 for 4-CH3 and 1.1
for 3-CF3 [17]. For 2-CF3 the value of the p-parameter is
not published, from the comparison of pꢀ values of 2-CF3
(1.34) and 3-CF3 (1.49) the lower lipophilicity of 2-CF3 is
evident.
The differences between the experimental (log K ) and the
calculated lipophilicity parameters (log P) were observed for
3-CF3 and 2-CF3 substituents. This could be connected with
the fact that the calculating program for log P does not distin-
guish between different lipophilicities of substituents in ortho,
meta and para positions. Anilides substituted by the 2-CF3
moiety showed higher hydrophobicity than 3-CF3 substituted
compounds, probably due to0 the intramolecular interactions
of the substituent in the C(2) position of the anilide part of
the molecule with the carboxamide (eCONHe) bridge in
the individual compounds.
2. Result and discussion
2.1. Chemistry
The synthesis of the amides is shown in Scheme 1. Con-
densation of chlorides of pyrazine-2-carboxylic acid [15],
6-chloropyrazine-2-carboxylic acid [16], 5-tert-butylpyra-
zine-2-carboxylic acid [4] or 6-chloro-5-tert-butylpyrazine-2-
carboxylic [4] acid with ring-substituted anilines yielded a
series of the substituted N-phenylpyrazine-2-carboxamides
1e20.
It can be assumed that capacity factor K/calculated log K
values specify lipophilicity within the individual series of
compounds.
2.2. Lipophilicity
Hydrophobicity parameters of anilides 1e20 were calcu-
lated (log P values) and measured by means of RP-HPLC
determination of capacity factor K and subsequently log K
was evaluated. The values of calculated lipophilicity (log P)
of compounds ranged from 1.60 to 5.30. The increasing
lipophilicity of the substituents on the pyrazine ring (H < 6-
Cl < 5-tert-butyl < 6-Cl, 5-tert-butyl) affects the resulting
lipophilicity of the compounds within individual series.
log K values evaluated from the capacity factor K specify
lipophilicity within these individual series of compounds.
2.3. Biological activity
2.3.1. In vitro antimycobacterial activities
All compounds prepared were evaluated on their in vitro
antimycobacterial susceptibility. Some interesting results
were obtained, as shown in Tables 1 and 2. The highest activ-
ity against M. tuberculosis H37Rv was found for N-(3-trifluor-
omethylphenyl)pyrazine-2-carboxamide (4, 99% inhibition)
and for 6-chloro-5-tert-butyl-N-(3-trifluoromethylphenyl)pyra-
zine-2-carboxamide (19, 95% inhibition). Other derivatives
NH2
O
R2
R2
X
N
N
COOH
X
N
N
COCl
X
N
N
SOCl2
N
H
R1
R1
R1
1-20
R1 = H, tert-butyl
R2 = 4-CH3, 3-CF3, 2-CF3, 4-F, 2-Cl-5-OH
X = H, Cl
Scheme 1. Synthesis of the discussed substituted pyrazine-2-carboxamides 1e20.

M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
1107
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
>90% inhibition in this primary screen (i.e. MIC >6.25 mg/
mL) were further evaluated to determine their actual minimum
inhibitory concentration (MIC) in the MABA. Simultaneously,
the compounds were tested for their cytotoxicity, i.e. the deter-
mination of their 50% inhibitory concentrations (IC50) in
VERO cells. At the conclusion of secondary screening, the
MIC and IC50 values are formed into a ratio termed selectivity
index (SI). The SI is defined as the ratio of the measured IC50,
in VERO cells, to the MIC. Compound 19 showed a good
MIC, but poor selectivity for mycobacteria (SI ¼ >3.2). Fi-
nally, N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (4)
with an MIC ꢁ 6.25 mg/mL and an SI level of 10 or greater
can be considered as an active compound at the second level
of antimycobacterial evaluation (see Table 2) [18,19].
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
In general, it can be concluded that with the exception of
compounds 1, 2 and 19 the increase of compound lipophilicity
resulted in decreased antimycobacterial activity. On the other
hand it is evident that the biological activity does not depend
exclusively on the compound lipophilicity but it is probably
affected also by electron accepting or withdrawing power of
the substituents on the benzene ring. A large positive Hammett
constant (s-value) implies high electron-withdrawing power
by inductive and/or resonance effect, relative to H; a large neg-
ative s-value implies high electron-releasing power relative to
H (the corresponding s-values are 0.062 for 4-F, 0.430 for
log P
Fig. 1. Dependence of experimentally determined log K values on calculated
log P values of compounds 1e20.
showing lower activities were N-p-tolylpyrazine-2-carbo-
xamide (5, 86%), 6-chloro-N-(3-trifluoromethylphenyl)
pyrazine-2-carboxamide (9, 77%), and 6-chloro-N-p-tolylpyr-
azine-2-carboxamide (10, 71%) (see Table 1).
In the tuberculosis antimicrobial acquisition and coordinat-
ing facility (TAACF) program compounds 4 and 19 effecting
Table 1
Antimycobaterial evaluation (% of inhibition), antifungal susceptibility (MIC), OER inhibition in spinach chloroplasts (IC50), and reduction of chlorophyll content
in C. vulgaris (IC50) of compounds 1e20 in comparison with the standards: pyrazinamide (PZA), fluconazole (FLU) and diurone (DCMU)
Comp.
Structure
X
Antimycobacterial
evaluation
Antifungal
susceptibility
(MICb, mmol/mL)
OER inhibition
(IC50, mmol/L)
Chlorophyll reduction
R1
R2
IC50
(mmol/L)
% of Control
ꢂS.D.d
(% inhibition
at 6.25 mg/mL)
1
H
H
2-Cl-5-OH
4-F
0
0
>250/>250
>500/>500
>250/>250
>500/>500
>500/>500
>250/>250
500/>500
500/>500
125/125
722
480
376
130
1475
624
384
557
229
e
e
97.1 ꢂ 10.6
89.9 ꢂ 5.7
91.7 ꢂ 4.9
91.5 ꢂ 2.7
e
2
H
H
3
H
H
2-CF3
3-CF3
4-CH3
2-Cl-5-OH
4-F
31
99
86
15
44
26
77
71
0
e
4
H
H
e
70.9
e
5
H
H
6
Cl
Cl
Cl
Cl
Cl
H
H
103.0 ꢂ 8.3
e
7
H
32.3
e
12.1
37.4
e
8
H
2-CF3
3-CF3
4-CH3
2-Cl-5-OH
4-F
87.8 ꢂ 4.3
e
9
H
10
11
12
13
14
15
16
17
18
19
20
PZA
FLU
DCMU
a
H
250/250
1524
c
e
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
(CH3)3C
e
>125/>125
500/>500
250/250
122 ꢂ 9.8
94.5 ꢂ 4.6
e
H
25
7
524
55
e
32.9
e
H
2-CF3
3-CF3
4-CH3
2-Cl-5-OH
4-F
H
23
61
0
62.5/62.5
125/125
283
164
625
103
205
173
73
85.2 ꢂ 5.1
91.8 ꢂ 2.2
98.0 ꢂ 6.7
101.6 ꢂ 9.0
94.4 ꢂ 2.0
90.1 ꢂ 2.0
92.9 ꢂ 2.4
e
H
e
e
Cl
Cl
Cl
Cl
Cl
e
e
e
>125/>125
>500/>500
250/250
15
6
e
2-CF3
3-CF3
4-CH3
e
e
95
0
250/250
>500/>500
e
e
e
a
e
e
e
e
e
e
1.95/3.91
e
e
e
7.3
e
e
e
1.9
e
MIC ¼ 12.5 mg/mL [10].
b
c
d
Against T. mentagrophytes after 72 h/120 h.
Not tested due to their low solubility in DMSO.
IC50 was determined only for five compounds, for the other 15 compounds an average of Chl content decrease in the concentration range 0.83e100 mmol/L was
determined.

1108
M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
Table 2
determined for five compounds (5, 7, 9, 10, and 13) and the
highest effect was found for 6-chloro-N-(3-trifluoromethylphe-
nyl)pyrazine-2-carboxamide (9, IC50 ¼ 12.1 mmol/L).
Primary antimycobaterial evaluation (% of inhibition), actual minimum inhib-
itory concentration (MIC), cytotoxicity (IC50) and selectivity index (SI) of
compounds 4 and 19
The inhibitory activity of compounds 3, 6 and 5 increased
linearly with increasing lipophilicity of the compound
(log P ¼ 1.60, 2.72 and 3.61, respectively). However, further
lipophilicity increases (compound 7, log P ¼ 4.02) led to re-
duced activity. The inhibition of chlorophyll content by other
investigated compounds (1, 2, 4, and 8e12) was low in the
whole studied concentration range (0.83e100 mmol/L), and
for the majority of these compounds did not exceed 10%
(i.e. 90% of the control). The results are shown in Table 1.
Comp. Structure
% Inhibition at MIC IC50
6.25 mg/mL (mg/mL) (mg/mL)
SI
X
R1
R2
3-CF3 99
Cl (CH3)3C 3-CF3 95
4
19
H
H
6.25
3.13
>62.5
>10
>10
>3.2
3-CF3, and ꢀ0.170 for 4-CH3) [20]. However, Hammett con-
stants reflecting electron accepting or withdrawing power of
ortho substituents are not used because steric effects can inter-
fere with the purely electronic effects. Relatively low lipophi-
licity is typical also for some drugs with antimycobacterial
activity, e.g. ethionamide (log P ¼ 1.22 ꢂ 0.29), isoniazide
(log P ¼ ꢀ0.89 ꢂ 0.24), and pyrazinamide (log P ¼ ꢀ0.37
ꢂ 0.35).
3. Conclusion
Thehighest activity against M. tuberculosis H37Rv was found
for N-(3-trifluoromethylphenyl)pyrazine-2-carboxamide (4,
MIC ¼ 6.25 mg/mL) and for 6-chloro-5-tert-butyl-N-(3-trifluor-
omethylphenyl)pyrazine-2-carboxamide (19, MIC ¼ 3.13 mg/
mL). In the TAACF program compounds effecting <90% inhi-
bition in this primary screen (MIC > 6.25 mg/mL) are not gener-
ally evaluated further. On other side, inactive compounds may
still have significant inhibitory activity, and this data should
not be ignored; analogues, derivatives, and alterations in physi-
cal properties may confer drastic changes in biological effects.
Therefore, synthesis and evaluation of other pyrazinecarboxylic
acid derivatives are necessary to broaden the structureeactivity
data.
The more lipophilic substituents (CF3 and CH3) in positions
3 and 4 on the benzene part of molecule possess some positive
influence on biological activity of the compounds studied.
Based on the obtained results it could be assumed that the bi-
ological activity of the studied pyrazinecarboxamides did not
depend exclusively on the compound lipophilicity but it was
also affected by electron accepting or withdrawing power of
the substituents on the benzene ring. In general it can be con-
cluded that the most effective antimycobacterial compounds
possess lower lipophilicity than the most active inhibitors of
photosynthetic electron transport.
2.3.2. In vitro antifungal activities
The evaluation of in vitro antifungal activity of the synthe-
sized compounds was performed against eight fungal strains.
The results showed no interesting activity against the majority
of fungal strains tested. Only compound 5-tert-butyl-N-(3-tri-
fluoromethylphenyl)pyrazine-2-carboxamide (14) exhibited
some moderate in vitro antifungal activity against Trichophy-
ton mentagrophytes, the most susceptible fungal strain evalu-
ated (MIC ¼ 62.5 mmol/mL). This activity is the only modest
one in comparison with fluconazole, the standard (MIC ¼
3.91 mmol/mL after 120 h, see Table 1). The negative results
of antifungal screening do not allow us to draw detailed con-
clusions on any structureeactivity relationships.
2.3.3. In vitro herbicidal activities
The majority of all the compounds studied inhibited
photosynthetic electron transport in spinach chloroplasts (see
Table 2). Based on the obtained results it could be concluded
that the activity of investigated compounds related to inhibi-
tion of oxygen evolution rate (OER) depends on lipophilicity
and also on the electron accepting or withdrawing power of
the substituents. However, the values of their inhibitory activ-
ities were rather low. At higher applied concentrations the
solubility of some compounds in the suspensions of spinach
chloroplasts was limited. The most effective inhibitor was
compound 13 (5-tert-butyl-N-(2-trifluoromethylphenyl)pyra-
zine-2-carboxamide, IC50 ¼ 55 mmol/L). Among the three
most active compounds 13, 20, and 17 the optimal values of
lipophilicity ranged from log P ¼ 4.02e4.41. On the other
hand, for the group of compounds 16, 19, and 18 with the
highest lipophilicity the OER-inhibiting activity showed a
decrease with increasing compound lipophilicity. The log P
values of efficient inhibitors of photosynthetic electron trans-
port with CONH group in their molecule are 2.78 ꢂ 0.38 (diur-
one) and 3.15 ꢂ 0.57 (linurone), corresponding with literature
[21]. Some of the compounds under study reduced the chloro-
phyll content in Chlorella vulgaris. The IC50 values related to
reduction of chlorophyll content in C. vulgaris could only be
4. Experimental
4.1. Chemistry
4.1.1. Instrumentation and chemicals
All organic solvents used for the synthesis were of analyt-
ical grade. The solvents were dried and freshly distilled under
argon atmosphere. The reactions were monitored and the
purity of the products was checked by TLC (Silufol UV
254, Kavalier Votice, Czech Republic) using developing sol-
vents petroleum ether/EtOAc (9:1). The plates were visualized
using UV light (254 nm). Melting points (uncorrected) were
determined on Boetius PHMK 05 (VEB Kombinat Nagema,
Radebeul, Germany). Elemental analyses were performed on
an automatic microanalyser CHNS-O CE instrument (FISONS
EA 1110, Milano, Italy). UV spectra (l, nm) were determined
on a Waters Photodiode Array Detector 2996 (Waters Corp.,

M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
1109
Milford, MA, U.S.A.) in ca. 9 ꢃ 10ꢀ4 mol methanolic solution
and log 3 (the logarithm of molar absorption coefficient 3) was
calculated for the absolute maximum lmax of individual target
compounds. Infrared spectra were recorded in Nicolet Impact
1.58 ꢂ 0.46. TLC: RF ¼ 0.78. UV (nm) lmax/log 3: 221.7/
3.48. IR (KBr) cmꢀ1: 3440 (NeH), 1665 (C]O), 1595 (phe-
nyl), 1523 (NeH), 1409, 1318, 1027 (pyrazine). 1H NMR
(300 MHz, DMSO-d6) d: 10.84 (1H, br s, NH), 9.29 (1H, d,
J ¼ 1.7 Hz, H3), 8.92 (1H, d, J ¼ 2.5 Hz, H6), 8.80 (1H, dd,
J ¼ 2.5 Hz, J ¼ 1.4 Hz, H5), 7.98e7.87 (2H, m, H20, H60),
and 7.26e7.15 (2H, m, H30, H50). 13C NMR (75 MHz,
DMSO-d6) d: 161.9, 158.8 (d, J ¼ 240.8 Hz), 148.0, 145.2,
144.3, 143.4, 134.8 (d, J ¼ 2.6 Hz), 122.7 (d, J ¼ 7.8 Hz),
and 115.5 (d, J ¼ 22.0 Hz).
1
400 spectrometer in KBr pellets. H and 13C NMR spectra
were recorded on
a
Varian Mercury-Vx BB 300
(299.95 MHz for 1H and 75.43 MHz for 13C) and Varian
(Palo Alto CA, U.S.A.) in CDCl3 or DMSO-d6 solutions at
ambient temperature. The chemical shifts d are given in ppm
related to tetramethylsilane (TMS) as internal standard. The
coupling constants (J ) are reported in Hz.
The purity of the final compounds was checked by HPLC,
see Section 2.2. The detection wavelength 210 nm was chosen.
Peaks in the chromatogram of the solvent (blank) were de-
ducted from peaks in the chromatogram of the sample solu-
tion. The purity of the individual compounds was
determined from area peaks in the chromatogram of the sam-
ple solution.
4.1.3.3. N-(2-Trifluoromethylphenyl)pyrazine-2-carboxamide
(3). Yield 71%. Anal. Calcd for C12H8F3N3O (267.2):
53.94% C, 3.02% H, 15.73% N. Found: 54.29% C, 3.05%
H, 15.53% N. Mp 123 ꢄC. HPLC purity 99.66%. log K:
0.6911. log P: 2.33 ꢂ 0.44. TLC: RF ¼ 0.77. UV (nm) lmax
/
log 3: 221.7/3.29. IR (KBr) cmꢀ1: 3370 (NeH), 1704
(C]O), 1593 (phenyl), 1457 (NeH), 1319, 1298, and 1112
1
(pyrazine). H NMR (300 MHz, CDCl3) d: 10.26 (1H, br s,
4.1.2. General procedure for pyrazinecarboxamide
synthesis
NH), 9.52 (1H, d, J ¼ 1.5 Hz, H3), 8.84 (1H, d, J ¼ 2.5 Hz,
H6), 8.64 (1H, dd, J ¼ 2.5 Hz, J ¼ 1.5 Hz, H5), 8.54 (1H, d,
J ¼ 7.4 Hz, H30), 7.71e7.59 (2H, m, H50, H60), and 7.33e
7.24 (1H, m, H40). 13C NMR (75 MHz, CDCl3) d: 161.0,
147.8, 144.7, 144.0, 142.7, 134.9, 133.0, 126.2 (q,
J ¼ 5.4 Hz), 124.6, 124.0 (q, J ¼ 272.9 Hz), 123.2, and 120.0
(q, J ¼ 29.7 Hz).
A mixture of acid, i.e. pyrazine-2-carboxylic [15], 6-chlor-
opyrazine-2-carboxylic [16], 5-tert-butylpyrazine-2-carbox-
ylic [3] or 5-tert-butyl-6-chloropyrazine-2-carboxylic [3]
acids (50.0 mmol) and thionyl chloride (5.5 mL, 75.0 mmol)
in dry toluene (20 mL) was refluxed for about 1 h. The excess
of thionyl chloride was removed by repeated evaporation with
dry toluene in vacuo. The crude acyl chloride dissolved in dry
acetone (50 mL) was added drop wise to a stirred solution of
the corresponding substituted amine (50.0 mmol) in 50 mL of
dry pyridine kept at room temperature. After the addition was
complete, stirring was continued for another 30 min. The reac-
tion mixture was then poured into 100 mL of cold water and
the crude amide was collected and recrystallized from aqueous
ethanol.
4.1.3.4. N-(3-Trifluoromethylphenyl)pyrazine-2-carboxamide
(4). Yield 61%. Anal. Calcd for C12H8F3N3O (267.2):
53.94% C, 3.02% H, 15.73% N. Found: 53.94% C, 2.93%
H, 15.62% N. Mp 109 ꢄC. HPLC purity 98.81%. log K:
0.5585. log P: 2.49 ꢂ 0.44. TLC: RF ¼ 0.69. UV (nm) lmax
/
log 3: 221.7/3.44. IR (KBr) cmꢀ1: 3327 (NeH), 1680
(C]O), 1593 (phenyl), 1450 (NeH), 1337, and 1126 (pyra-
1
zine). H NMR (300 MHz, CDCl3) d: 9.80 (1H, br s, NH),
9.52 (1H, d, J ¼ 1.4 Hz, H3), 8.84 (1H, d, J ¼ 2.5 Hz, H6),
8.61 (1H, dd, J ¼ 2.5 Hz, J ¼ 1.4 Hz, H5), 8.08e8.05 (1H,
m, H20), 8.00e7.94 (1H, m, H40), 7.52 (1H, t, J ¼ 8.0 Hz,
H50), and 7.46e7.40 (1H, m, H60). 13C NMR (75 MHz,
CDCl3) d: 160.9, 147.9, 144.7, 143.8, 142.4, 137.7, 131.6 (q,
J ¼ 32.7 Hz), 129.7, 123.8 (q, J ¼ 272.6 Hz), 122.8, 121.3
(q, J ¼ 4.0 Hz), and 116.5 (q, J ¼ 4.1 Hz).
4.1.3. Data of prepared target compounds
4.1.3.1.
N-(2-Chloro-5-hydroxyphenyl)pyrazine-2-carboxa-
mide (1). Yield 47%. Anal. Calcd for C11H8ClN3O2 (249.5):
52.92% C, 3.23% H, 16.83% N. Found: 49.31% C, 3.85%
H, 16.72% N. Mp 223e224 ꢄC. HPLC purity 99.29%. log K:
0.4527. log P: 1.64 ꢂ 0.41. TLC: RF ¼ 0.78. UV (nm) lmax
/
log 3: 232.3/3.38. IR (KBr) cmꢀ1: 3348 (NeH), 1671
(C]O), 1593 (phenyl), 1529 (NeH), 1402, 1321, 1023 (pyr-
4.1.3.5. N-p-tolylpyrazine-2-carboxamide (5). Yield 44%.
Anal. Calcd for C12H11N3O (213.2): 67.59% C, 5.20% H,
19.71% N. Found: 67.34% C, 5.57% H, 19.55% N. Mp
148 ꢄC. HPLC purity 99.56%. log K: 0.4595. log P:
1.60 ꢂ 0.40. TLC: RF ¼ 0.70. UV (nm) lmax/log 3: 218.2/
3.47. IR (KBr) cmꢀ1: 3348 (NeH), 1671 (C]O), 1593
1
azine). H NMR (300 MHz, DMSO-d6) d: 10.27 (br s, 1H,
NH), 9.93 (br s, 1H, OH), 9.32 (d, 1H, J ¼ 1.40 Hz, H3),
8.97 (d, 1H, J ¼ 2.50 Hz, H6), 8.83e8.81 (m, 1H, H5), 7.87
(d, 1H, J ¼ 2.70 Hz, H60), 7.32 (d, 1H, J ¼ 8.80 Hz, H30),
6.61 (dd, 1H, J ¼ 8.80 Hz, J ¼ 2.70 Hz, H40). 13C NMR
(75 MHz, DMSO-d6) d: 160.9, 157.1, 148.7, 144.0, 143.9,
143.6, 134.5, 130.0, 113.5, 113.2, 109.3.
1
(phenyl), 1523 (NeH), 1402, 1321, and 1023 (pyrazine). H
NMR (300 MHz, CDCl3) d: 9.61 (1H, br s, NH), 9.51 (1H,
d, J ¼ 1.5 Hz, H3), 8.79 (1H, d, J ¼ 2.5 Hz, H6), 8.58 (1H,
dd, J ¼ 2.5 Hz, J ¼ 1.5 Hz, H5), 7.68e7.60 (2H, m, AA0,
BB0, H20, H60), 7.24e7.16 (2H, m, AA0, BB0, H30, H50), and
2.35 (3H, s, CH3). 13C NMR (75 MHz, CDCl3) d: 160.4,
147.4, 144.6, 144.5, 142.3, 134.6, 134.5, 129.6, 119.7,
and 20.9.
4.1.3.2. N-(4-Fluorophenyl)pyrazine-2-carboxamide (2). Yield
83%. Anal. Calcd for C11H8FN3O (217.2): 60.83% C, 3.71%
H, 19.35% N. Found: 60.40% C, 4.15% H, 19.45% N. Mp
154e155 ꢄC. HPLC purity 99.21%. log K: 0.4416. log P:

1110
M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
4.1.3.6.
6-Chloro-N-(2-chloro-5-hydroxyphenyl)pyrazine-2-
4.1.3.10. 6-Chloro-N-p-tolylpyrazine-2-carboxamide (10). Yield
59%. Anal. Calcd for C12H10ClN3O (247.7): 58.19% C, 4.07%
H, 16.97% N. Found: 58.11% C, 4.45% H, 16. 32% N. Mp
134 ꢄC. HPLC purity 99.39%. log K: 0.7627. log P:
2.72 ꢂ 0.41. TLC: RF ¼ 0.81. UV (nm) lmax/log 3: 222.7/
3.37. IR (KBr) cmꢀ1: 3370 (NeH), 1694 (C]O), 1592
carboxamide (6). Yield 58%. Anal. Calcd for C11H7Cl2N3O2
(284.1): 46.51% C, 2.48% H, 14.79% N. Found: 46.16% C,
3.42% H, 14.705% N. Mp 255 ꢄC. HPLC purity 98.72%.
log K: 0.7801. log P: 2.76 ꢂ 0.43. TLC: RF ¼ 0.72. UV (nm)
lmax/log 3: 233.5/3.37. IR (KBr) cmꢀ1: 3365 (NeH), 1700
(C]O), 1593 (phenyl), 1528 (NeH), 1350, 1290, 1108 (pyr-
1
(phenyl), 1531 (NeH), 1320, 1168, and 1144 (pyrazine). H
1
azine). H NMR (300 MHz, DMSO) d: 10.10 (1H, br s, NH),
NMR (300 MHz, CDCl3) d: 9.39 (1H, d, J ¼ 0.5 Hz, H3),
9.34 (1H, br s, NH), 8.79 (1H, s, H5), 7.67e7.59 (2H, m,
AA0, BB0, H20, H60), 7.24e7.17 (2H, m, AA0, BB0, H30,
H50), and 2.35 (3H, s, CH3). 13C NMR (75 MHz, CDCl3) d:
159.2, 147.4, 144.1, 142.2, 134.9, 134.3, 129.7, 119.9, and
20.9.
9.93 (1H, br s, OH), 9.26 (1H, s, H3), 9.11 (1H, s, H5), 7.71
(1H, d, J ¼ 2.8 Hz, H60), 7.33 (1H, d, J ¼ 8.7 Hz, H30), and
6.64 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.8 Hz, H40). 13C NMR
(75 MHz, DMSO) d: 159.9, 157.1, 148.3, 147.0, 143.9,
142.2, 134.4, 130.0, 114.4, 113.7, and 110.2.
4.1.3.7. 6-Chloro-N-(4-fluorophenyl)pyrazine-2-carboxamide
(7). Yield 59%. Anal. Calcd for C11H7ClFN3O (251.7):
52.50% C, 2.80% H, 16.70% N. Found: 52.40% C, 3.205%
H, 17.07% N. Mp 131e132 ꢄC. HPLC purity 98.03%. log K:
4.1.3.11. 5-tert-Butyl-N-(2-chlor-5-hydroxyphenyl)pyrazine-2-
carboxamide (11). Yield 35%. Anal. Calcd for C15H16ClN3O2
(305.8): 58.92% C, 5.27% H, 13.74% N. Found: 58.39% C,
6.335% H, 13.83% N. Mp 239e240 ꢄC. HPLC purity
99.34%. log K: 1.0654. log P: 3.33 ꢂ 0.42. TLC: RF ¼ 0.87.
UV (nm) lmax/log 3: 225.2/3.29. IR (KBr) cmꢀ1: 3376 (Ne
H), 2992, 2960, 2926, 2915 (tert-butyl), 1690 (C]O), 1595
(phenyl), 1528 (NeH), 1320, 1250, 11320 (pyrazine). 1H
NMR (300 MHz, DMSO) d: 10.24 (1H, br s, NH), 9.93 (1H,
br s, OH), 9.23 (1H, d, J ¼ 1.6 Hz, H3), 8.93 (1H, d,
J ¼ 1.6 Hz, H6), 7.92 (1H, d, J ¼ 2.9 Hz, H60), 7.32 (1H, d,
J ¼ 8.8 Hz, H30), 6.60 (1H, dd, J ¼ 8.8 Hz, J ¼ 2.9 Hz, H40),
and 1.39 (9H, s, CH3). 13C NMR (75 MHz, DMSO) d:
167.8, 160.9, 157.2, 142.3, 141.2, 140.5, 134.6, 129.9,
113.1, 113.0, 108.9, 37.1, and 29.6.
0.7557. log P: 2.70 ꢂ 0.48. TLC: RF ¼ 0,82. UV (nm) lmax
/
log 3: 229.9/3.49. IR (KBr) cmꢀ1: 3375 (NeH), 1710
(C]O), 1594 (phenyl), 1533 (NeH), 1351, 1302, 1119 (pyr-
1
azine). H NMR (300 MHz, DMSO) d: 10.74 (1H, br s, NH),
9.22 (1H, d, J ¼ 0.6 Hz, H3), 9.05 (1H, s, H5), 7.93e7.83 (2H,
m, H20, H60), and 7.27e7.17 (2H, m, H30, H50). 13C NMR
(75 MHz, DMSO) d: 160.8, 159.0 (d, J ¼ 241.0 Hz), 147.7,
147.1, 145.3, 142.6, 134.5 (d, J ¼ 2.6 Hz), 123.0 (d,
J ¼ 8.1 Hz), and 115.5 (d, J ¼ 22.3 Hz).
4.1.3.8. 6-Chloro-N-(2-trifluoromethylphenyl)pyrazine-2-car-
boxamide (8). Yield 47%. Anal. Calcd for C12H7ClF3N3O
(301.7): 47.78% C, 2.34% H, 13.93% N. Found: 47.44% C,
2.31% H, 13.65% N. Mp 148e150 ꢄC. HPLC purity
99.69%. log K: 0.8602. log P: 3.45 ꢂ 0.46. TLC: RF ¼ 0.84.
UV (nm) lmax/log 3: 220.5/3.26. IR (KBr) cmꢀ1: 3384 (NeH),
1707 (C]O), 1594 (phenyl), 1543 (NeH), 1322, 1301, and
4.1.3.12. 5-tert-Butyl-N-(4-fluorophenyl)pyrazine-2-carboxa-
mide (12). Yield 47%. Anal. Calcd for C11H16FN3O (273.3):
65.92% C, 5.90% H, 15.37% N. Found: 64.92% C, 6.405%
H, 15.525% N. Mp 178 ꢄC. HPLC purity 99.58%. log K:
0.9928. log P: 3.27 ꢂ 0.47. TLC: RF ¼ 0.84. UV (nm) lmax
/
1
1109 (pyrazine). H NMR (300 MHz, CDCl3) d: 9.98 (1H, br
log 3: 222.9/3.34. IR (KBr) cmꢀ1: 3361 (NeH), 2984, 2961,
2920, 2906 (tert-butyl), 1680 (C]O), 1591 (phenyl), 1522
s, NH), 9.39 (1H, d, J ¼ 0.6 Hz, H3), 8.84 (1H, d, J ¼ 0.6 Hz,
H5), 8.46 (1H, d, J ¼ 8.2 Hz, H30), 7.73e7.59 (2H, m, H50,
H60), and 7.35e7.27 (1H, m, H40). 13C NMR (75 MHz,
CDCl3) d: 160.6, 159.8, 147.9, 147.7, 143.5, 142.1, 133.0,
126.3 (q, J ¼ 5.2 Hz), 125.0, 123.9 (q, J ¼ 272.9 Hz), 123.5
and 120.4 (q, J ¼ 32.7 Hz).
1
(NeH), 1318, 1150 (pyrazine). H NMR (300 MHz, DMSO)
d: 10.45 (1H, br s, NH), 9.19 (1H, d, J ¼ 1.5 Hz, H3), 8.84
(1H, d, J ¼ 1.5 Hz, H6), 7.96e7.86 (2H, m, H20, H60),
7.25e7.15 (2H, m, H30, H50), and 1.39 (9H, s, CH3). 13C
NMR (75 MHz, DMSO) d: 166.9, 162.0, 158.7 (d,
J ¼ 240.8 Hz), 142.6, 142.6, 139.9, 134.9 (d, J ¼ 2.6 Hz),
122.6 (d, J ¼ 8.0 Hz), 115.5 (d, J ¼ 22.3 Hz), 37.0,
and 29.6.
4.1.3.9. 6-Chloro-N-(3-trifluoromethylphenyl)pyrazine-2-car-
boxamide (9). Yield 62%. Anal. Calcd for C12H7ClF3N3O
(301.7): 47.78% C, 2.34% H, 13.93% N. Found: 48.09% C,
2.72% H, 13.97% N. Mp 109 ꢄC. HPLC purity 98.71%. log K:
4.1.3.13. 5-tert-Butyl-N-(2-trifluoromethylphenyl)pyrazine-2-
carboxamide (13). Yield 47%. Anal. Calcd for C16H16
F3N3O (323.3): 59.44% C, 4.99% H, 13.00% N. Found:
59.47% C, 5.10% H, 13.05% N. Mp 82.5 ꢄC. HPLC purity
99.89%. log K: 1.2622. log P: 4.02 ꢂ 0.46. TLC: RF ¼ 0.88.
UV (nm) lmax/log 3: 224.0/3.27. IR (KBr) cmꢀ1: 3356 (Ne
H), 2961, 2907, 2869 (tert-butyl), 1698 (C]O), 1593 (phe-
0.8362. log P: 3.61 ꢂ 0.47. TLC: RF ¼ 0.80. UV (nm) lmax
/
log 3: 219.8/3.35. UV (nm) lmax/log 3: 232.3/3.38. IR (KBr)
cmꢀ1: 3351 (NeH), 1680 (C]O), 1603 (phenyl), 1545 (Ne
1
H), 1330, 1169, and 1131 (pyrazine). H NMR (300 MHz,
CDCl3) d: 9.52 (1H, br s, NH), 9.40 (1H, s, H3), 8.84 (1H, s,
H5), 8.07 (1H, br s, H20), 7.99e7.94 (1H, m, H40), 7.58e7.49
(1H, m, H50), and 7.49e7.42 (1H, m, H60). 13C NMR
(75 MHz, CDCl3) d: 159.6, 147.9, 147.5, 143.4, 142.3, 137.4,
131.6 (q, J ¼ 32.7 Hz), 129.8, 123.7 (q, J ¼ 272.6 Hz), 123.0,
121.7 (q, J ¼ 3.7 Hz), and 116.7 (q, J ¼ 4.0 Hz).
1
nyl), 1539 (NH), 1322, 1171, and 1118 (pyrazine). H NMR
(300 MHz, CDCl3) d: 10.27 (1H, br s, NH), 9.40 (1H, d,
J ¼ 1.4 Hz, H3), 8.70 (1H, d, J ¼ 1.4 Hz, H6), 8.57 (1H, d,
J ¼ 8.2 Hz, H30), 7.70e7.58 (2H, m, H50, H60), 7.31e7.22

M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
1111
(1H, m, H40), and 1.45 (9H, s, CH3). 13C NMR (75 MHz,
CDCl3) d: 168.1, 161.5, 143.1, 141.0, 139.4, 135.1, 133.0,
126.2 (q, J ¼ 5.2 Hz), 124.3, 124.1 (q, J ¼ 272.8 Hz), 123.1,
119.8 (q, J ¼ 29.7 Hz), 37.1, and 29.7.
UV (nm) lmax/log 3: 228.7/3.33. IR (KBr) cmꢀ1: 3370
(NeH), 2978, 2956, 2929 (tert-butyl), 1700 (C]O), 1594
(phenyl), 1532 (NeH), 1327, 1262, 1148 (pyrazine). 1H
NMR (300 MHz, DMSO) d: 10.62 (1H, br s, NH), 9.12 (1H,
s, H3), 7.91e7.81 (2H, m, H20, H60), 7.26e7.16 (2H, m,
H30, H50), and 1.50 (9H, s, CH3). 13C NMR (75 MHz,
DMSO) d: 163.0, 160.9, 158.9 (d, J ¼ 241.4 Hz), 145.4,
142.8, 140.6, 134.6 (d, J ¼ 2.6 Hz), 122.9 (d, J ¼ 8.0 Hz),
115.5 (d, J ¼ 22.3 Hz), 38.7, and 28.2.
4.1.3.14. 5-tert-Butyl-N-(3-trifluoromethylphenyl)pyrazine-2-
carboxamide (14). Yield 38%. Anal. Calcd for C16H16F3N3O
(323.3): 59.44% C, 4.99% H, 13.00% N. Found: 59.83% C,
5.09% H, 13.17% N. Mp 84.5 ꢄC. HPLC purity 99.81%.
log K: 1.1338. log P: 4.18 ꢂ 0.46. TLC: RF ¼ 0.86. UV (nm)
lmax/log 3: 217.0/3.35. IR (KBr) cmꢀ1: 3350 (NeH), 2974,
2938, 2911, 2874 (tert-butyl), 1684 (C]O), 1604 (phenyl),
1543 (NeH), 1339, and 1133 (pyrazine). 1H NMR
(300 MHz, CDCl3) d: 9.78 (1H, br s, NH), 9.40 (1H, d,
J ¼ 1.7 Hz, H3), 8.63 (1H, d, J ¼ 1.7 Hz, H6), 8.08 (1H, br
s, H20), 7.98e7.92 (1H, m, H40), 7.51 (1H, t, J ¼ 8.0 Hz,
H50), 7.44e7.39 (1H, m, H60), and 1.45 (9H, s CH3). 13C
NMR (75 MHz, CDCl3) d: 168.2, 161.4, 143.1, 140.9, 139.1,
138.0, 131.6 (q, J ¼ 32.4 Hz), 129.7, 123.8 (q,
J ¼ 272.6 Hz), 122.7, 121.1 (q, J ¼ 4.0 Hz), 116.4 (q,
J ¼ 4.0 Hz), 37.1, and 29.7.
4.1.3.18. 5-tert-Butyl-6-chloro-N-(2-trifluoromethylphenyl)pyr-
azine-2-carboxamide (18). Yield 26%. Anal. Calcd for
C16H15ClF3N3O (357.8): 53.72% C, 4.23% H, 11.75% N.
Found: 54.06% C, 3.89% H, 11.82% N. Mp 81 ꢄC. HPLC pu-
rity 99.75%. log K: 1.6560. log P: 5.14 ꢂ 0.48. TLC:
RF ¼ 0.89. UV (nm) lmax/log 3: 229.9/3.26. IR (KBr) cmꢀ1
:
3366 (NeH), 2980, 2961, 2936, 2909, 2874 (tert-butyl, tri-
fluoromethyl), 1715 (C]O), 1592 (phenyl), 1532 (NeH),
1323, 1290, and 1120 (pyrazine). 1H NMR (300 MHz,
CDCl3) d: 9.99 (1H, br s, NH), 9.26 (1H, s, H3), 8.49 (1H,
d, J ¼ 8.2 Hz, H30), 7.72e7.57 (2H, m, H50, H60), 7.33e7.24
(1H, m, H40), and 1.56 (9H, s, CH3). 13C NMR (75 MHz,
CDCl3) d: 165.0, 160.2, 146.0, 140.6, 140.2, 134.7, 133.0,
126.3 (q, J ¼ 5.2 Hz), 124.7, 124.0 (q, J ¼ 272.9 Hz), 123.3,
120.2 (q, J ¼ 30.0 Hz), 39.1, and 28.2.
4.1.3.15. 5-tert-Butyl-N-p-tolylpyrazine-2-carboxamide (15).
Yield 83%. Anal. Calcd for C16H19N3O (269.4): 71.35% C,
7.11% H, 15.60% N. Found: 71.58% C, 7.38% H, 15.62%
N. Mp 143 ꢄC. HPLC purity 99.08%. log K: 1.0222. log P:
3.28 ꢂ 0.40. TLC: RF ¼ 0.86. UV (nm) lmax/log 3: 222.9/
3.38. IR (KBr) cmꢀ1: 3351 (NeH), 2977, 2957, 2922, 2906,
2872 (tert-butyl, methyl), 1676 (C]O), 1592 (phenyl), 1520
(NH), 1314, and 1147 (pyrazine). 1H NMR (300 MHz,
CDCl3) d: 9.60 (1H, br s, NH), 9.39 (1H, d, J ¼ 1.4 Hz, H3),
8.61 (1H, d, J ¼ 1.4 Hz, H6), 7.68e7.61 (2H, m, AA0, BB0,
H20, H60), 7.23e7.15 (2H, m, AA0, BB0, H30, H50), 2.35
(3H, s, CH3), and 1.44 (9H, s, CH3). 13C NMR (75 MHz,
CDCl3) d: 167.6, 160.9, 142.9, 141.5, 138.9, 134.9, 134.2,
129.6, 119.7, 37.0, 29.7, and 20.9.
4.1.3.19. 5-tert-Butyl-6-chloro-N-(3-trifluoromethylphenyl)pyr-
azine-2-carboxamide (19). Yield 21%. Anal. Calcd for
C16H15ClF3N3O (357.8): 53.72% C, 4.23% H, 11.75% N.
Found: 53.49% C, 4.60% H, 12.03% N. Mp 71.8 ꢄC. HPLC
purity 98.37%. log K: 1.5271. log P: 5.30 ꢂ 0.48. TLC:
RF ¼ 0.84. UV (nm) lmax/log 3: 232.3/3.28. IR (KBr) cmꢀ1
:
3375 (NeH), 2985, 2960, 2940, 2910, 2870 (tert-butyl, tri-
fluoromethyl), 1720 (C]O), 1603 (phenyl), 1530 (NeH),
1325, 1250, and 1158 (pyrazine). 1H NMR (300 MHz,
CDCl3) d: 9.99 (1H, br s, NH), 9.04 (1H, s, H3), 8.63 (1H,
d, J ¼ 1.7 Hz, H6), 8.08 (1H, br s, H20), 7.98e7.92 (1H, m,
H40), 7.51 (1H, t, J ¼ 8.0 Hz, H50), 7.44e7.39 (1H, m, H60),
1.34 (9H, s and CH3). 13C NMR (75 MHz, CDCl3) d: 168.2,
161.4, 144.9, 140.9, 139.1, 138.5, 131.6 (q, J ¼ 32.4 Hz),
129.7, 123.8 (q, J ¼ 272.6 Hz), 122.7, 120.9 (q, J ¼ 4.0 Hz),
119.4 (q, J ¼ 4.0 Hz), 37.1, 31.0, and 29.7.
4.1.3.16. 5-tert-Butyl-6-chloro-N-(2-chloro-5-hydroxyphenyl)-
pyrazine-2-carboxamide (16). Yield 69%. Anal. Calcd for
C15H15Cl2N3O2 (340.2): 52.96 % C, 4.44% H, 12.35% N.
Found: 52.525% C, 5.765% H, 12.12% N. Mp 253 ꢄC.
HPLC purity 99.29%. log K: 1.4335. log P: 4.45 ꢂ 0.44.
TLC: RF ¼ 0.92. UV (nm) lmax/log 3: 227.6/3.28. IR (KBr)
cmꢀ1: 3386 (NeH), 2990, 2960, 2939 (tert-butyl), 1705
(C]O), 1591 (phenyl), 1529 (NeH), 1340, 1260, 1049 (pyr-
4.1.3.20. 5-tert-Butyl-6-chloro-N-p-tolylpyrazine-2-carboxa-
mide (20). Yield 31%. Anal. Calcd for C16H18ClN3O
(303.8): 63.26% C, 5.97% H, 13.83% N. Found: 63.17% C,
5.87% H, 13.69% N. Mp 162 ꢄC. HPLC purity 99.18%.
log K: 1.3205. log P: 4.41 ꢂ 0.42. TLC: RF ¼ 0.83. UV (nm)
lmax/log 3: 227.9/3.39. IR (KBr) cmꢀ1: 3376 (NeH), 2978,
2956, 2929, 2867 (tert-butyl, methyl), 1701 (C]O), 1596
1
azine). H NMR (300 MHz, DMSO) d: 10.03 (1H, br s, NH),
9.93 (1H, br s, OH), 9.16 (1H, s, H3), 7.76 (1H, d, J ¼ 2.8 Hz,
H60), 7.33 (1H, d, J ¼ 8.7 Hz, H30), 6.63 (1H, dd, J ¼ 8.7 Hz,
J ¼ 2.8 Hz, H40), and 1.50 (9H, s, CH3). 13C NMR (75 MHz,
DMSO) d: 163.8, 159.9, 157.1, 145.3, 141.4, 140.3, 134.4,
130.0, 114.0, 113.5, 109.8, 38.8, and 28.2.
1
(phenyl), 1538 (NeH), 1317, 1260, and 1148 (pyrazine). H
NMR (300 MHz, CDCl3) d: 9.31 (1H, br s, NH), 9.26 (1H,
s, H3), 7.67e7.60 (2H, m, AA0, BB0, H20, H60), 7.23e7.16
(2H, m, AA0, BB0, H30, H50), 2.35 (3H, s, CH3), and 1.55
(9H, s, CH3). 13C NMR (75 MHz, CDCl3) d: 164.4,
159.6, 141.2, 140.2, 134.6, 134.5, 129.6, 119.9, 38.9, 28.3,
and 20.9.
4.1.3.17. 5-tert-Butyl-6-chloro-N-(4-fluorophenyl)pyrazine-2-
carboxamide (17). Yield 61%. Anal. Calcd for C15H15ClFN3O
(307.8): 58.54% C, 4.91% H, 13.65% N. Found: 58.265% C,
5.40% H, 13.98% N. Mp 171e172 ꢄC. HPLC purity
99.53%. log K: 1.3488. log P: 4.39 ꢂ 0.49. TLC: RF ¼ 0.93.

1112
M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
4.2. Lipophilicity HPLC determination
(capacity factor K/calculated log K )
the Promega CellTiter 96 Non-radioactive Cell Proliferation
Assay. At the conclusion of secondary screening, the MIC
and IC50 values are formed into a ratio termed selectivity in-
dex (SI). At an SI level of 10 or greater, a compound is con-
sidered active at the second level [18,19]. The results are
presented in Tables 1 and 2.
The HPLC separation module Waters Alliance 2695 XE
and Waters Photodiode Array Detector 2996 (Waters Corp.,
Milford, MA, U.S.A.) were used. The chromatographic
column SymmetryÒ C18 5 mm, 4.6 ꢃ 250 mm, Part No.
WAT054275 (Waters Corp., Milford, MA, U.S.A.) was used.
The HPLC separation process was monitored by Millen-
nium32Ò Chromatography Manager Software, Waters 2004
(Waters Corp., Milford, MA, U.S.A.). The mixture of MeOH
p.a. (70.0%) and H2O-HPLCeMili-Q Grade (30.0%) was
used as a mobile phase. The total flow of the column was
1.0 mL/min, injection 30 mL, column temperature 30 ꢄC and
sample temperature 10 ꢄC. The detection wavelength 210 nm
was chosen. The KI methanolic solution was used for the
dead time (TD) determination. Retention times (TR) were mea-
sured in minutes.
The capacity factors K were calculated using the Millen-
nium32Ò Chromatography Manager Software according to
the formula K ¼ (TRꢀTD)/TD, where TR is the retention time
of the solute, whereas TD denotes the dead time obtained via
an unretained analyte. log K, calculated from the capacity fac-
tor K, is used as the lipophilicity index converted to log P
scale. The log K values of the individual compounds are sum-
marized in Table 1 and Section 4.1.3.
4.4.2. In vitro antifungal susceptibility testing
The broth microdilution test [10,22] was used for the as-
sessment of in vitro antifungal activity of the synthesized com-
pounds against Candida albicans ATCC 44859 (CA), Candida
tropicalis 156 (CT), Candida krusei E28 (CK), Candida glab-
rata 20/I (CG), Trichosporon asahii 1188 (TB), Aspergillus
fumigatus 231 (AF), Absidia corymbifera 272 (AC), and
T. mentagrophytes 445 (TM). Fluconazole was used as a refer-
ence drug. The procedure was performed with twofold dilution
of the compounds in RPMI 1640 medium (Sevapharma,
Prague, Czech Republic) buffered to pH 7.0 with 0.165 mol
of 3-morpholinopropane-1-sulfonic acid. The final concentra-
tions of the compounds ranged from 0.975 mmol/L to
500 mmol/L. Drug-free controls were included. The minimal
inhibitory concentrations (MICs) were determined after 24 h
and 48 h of static incubation at 35 ꢄC. With T. mentagro-
phytes, the final MICs were determined after 72 h and 120 h
of incubation. The results of all compounds in vitro tested
against T. mentagrophytes, the most susceptible fungal strain,
are summarized in Table 1.
4.3. Lipophilicity calculations
4.4.3. Herbicidal activities
log P, i.e. the logarithm of the partition coefficient P for
n-octanol/water, was calculated using the commercially avail-
able program ACD/log P version 1.0 (Advanced Chemistry
Development Inc., Toronto, Canada). The results are summa-
rized in Table 1 and Section 4.1.3.
4.4.3.1. Study of inhibition of oxygen evolution rate in spinach
chloroplasts. Chloroplasts were prepared by the procedure of
Walker from spinach (Spinacia oleracea L.) [23]. The inhibi-
tion of photosynthetic electron transport (PET) in spinach
chloroplasts was determined spectrophotometrically (Kontron
Uvikon 800, Kontron, Muenchen, Germany) using an artificial
electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) ac-
cording to Kralova et al. [24] and the rate of photosynthetic
electron transport 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),
MgCl2 (0.005 mol/L) and NaCl (0.015 mol/L). The chloro-
phyll content was 30 mg/L in these experiments and the sam-
ples were irradiated (w100 W/m2) from a 10-cm distance with
a halogen lamp (250 W) using a 4-cm water filter to prevent
warming of the samples (suspension temperature 22 ꢄC).
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 (concentration)
of the studied compounds has been expressed by IC50 values,
i.e. by molar concentration of the compounds causing 50%
decrease in the oxygen evolution relative to the untreated con-
trol. The comparable IC50 value for a selective herbicide
3-(3,4-dichlorophenyl)-1,1-dimethylurea (diurone, DCMU)
was about 1.9 mmol/L [25]. The results are summarized in
Table 1.
4.4. Biological methods
4.4.1. Antimycobacterial assay
Antimycobacterial evaluation was carried out in the tuber-
culosis antimicrobial acquisition and coordinating facility
(TAACF), Southern Research Institute, Birmingham, AL,
U.S.A., which is a part of the National Institutes of Health
(NIH). Primary screening of all compounds was conducted
at 6.25 mg/mL against M. tuberculosis H37Rv (ATCC27294)
in BACTEC 12B medium using both BACTEC 460 radiomet-
ric system and the Microplate Almar Blue Assay (MABA).
Compounds demonstrating at least 90% inhibition in the pri-
mary screen were tested at lower concentration against M. tu-
berculosis H37Rv to determine the MIC testing by MABA.
The MIC is defined as the lowest concentration effecting a re-
duction in fluorescence of 90% relative to controls. Concurrent
with the determination of MICs, the compounds were tested
for their cytotoxicity (determination of 50% inhibitory con-
centrations, IC50) in VERO cells at concentrations less than
or equal to 62.5 mg/ml or 10 times the MIC for M. tuberculosis
H37Rv. After 72 h exposure, viability is assessed on the basis
of cellular conversion of MTT into a formazan product using

M. Dolezal et al. / European Journal of Medicinal Chemistry 43 (2008) 1105e1113
1113
[2] O. Zimhony, C. Vilcheze, M. Arai, J.T. Welch, W.R. Jacobs Jr., Antimi-
crob. Agents Chemother. 51 (2007) 752.
4.4.3.2. Reduction of chlorophyll content in the green algae
C. vulgaris Beij. The green algae C. vulgaris Beij. were culti-
vated statically at room temperature according to Kralova
et al. [26] (photoperiod 16 h light/8 h dark; photosynthetic ac-
tive radiation 80 mmol/m2 s, pH 7.2). The effect of the com-
pounds on algal chlorophyll (Chl) content was determined
after seven-day cultivation in the presence of the tested com-
pounds. The Chl content in the algal suspension was deter-
mined spectrophotometrically (Kontron Uvikon 800,
Kontron, Munchen, Germany) after extraction into methanol
according to Wellburn [27]. The Chl content in the suspen-
sions at the beginning of the cultivation was 0.01 mg/L. The
applied compound concentrations were as follows:
0.83 mmol/L, 4.2 mmol/L, 8.3 mmol/L, 25 mmol/L, 50 mmol/L,
75 mmol/L, and 100 mmol/L. Because of the low solubility
of the studied compounds in water, these were dissolved in
DMSO. DMSO concentration in the algal suspensions did
not exceed 0.25% and the control samples contained the
same DMSO amount as the suspensions treated with the tested
compounds. The antialgal activity of the compounds was ex-
pressed as IC50 (the concentration of the inhibitor causing
a 50% decrease in content of chlorophyll as compared with
the control sample) or as percentage of the control determined
for the studied concentration range (0.83e100 mmol/L) with
the corresponding standard deviation (S.D.). The comparable
IC50 value for a selective herbicide DCMU was about
7.3 mmol/L [23]. The results are summarized in Table 1.
[3] K. Dlabal, M. Dolezal, M. Machacek, Collect. Czech. Chem. Commun.
58 (1993) 452.
[4] M. Dolezal, J. Hartl, M. Miletin, M. Machacek, K. Kralova, Chem. Pap.
53 (1999) 126.
[5] M. Dolezal, L. Palek, J. Vinsova, V. Buchta, J. Jampilek, K. Kralova,
Molecules 11 (2006) 242.
[6] N.E. Good, Plant. Physiol. 36 (1961) 788.
[7] K. Kralova, F. Sersen, J. Cizmarik, Chem. Pap. 46 (1992) 266.
[8] K. Kralova, F. Sersen, M. Miletin, J. Hartl, Chem. Pap. 52 (1998) 52.
[9] L. Kubicova, M. Sustr, K. Kralova, V. Chobot, J. Vytlacilova, L. Jahodar,
P. Vuorela, M. Machacek, J. Kaustova, Molecules 8 (2003) 756.
[10] M. Dolezal, J. Jampilek, Z. Osicka, J. Kunes, V. Buchta, P. Vichova,
Farmaco 58 (2003) 1105.
[11] M. Dolezal, J. Hartl, M. Miletin, Folia Pharm. Univ. Carol. 25 (2000) 15.
[12] L. Tumova, K. Gallova, J. Rimakova, M. Dolezal, J. Tuma, Acta Physiol.
Plant. 27 (2005) 357.
´
[13] R. Musiol, J. Jampılek, V. Buchta, L. Silva, H. Niedbala, B. Podeszwa,
A. Palka, K. Majerz-Maniecka, B. Oleksyn, J. Polanski, Bioorg. Med.
Chem. 14 (2006) 3592.
[14] J. Vinsova, K. Cermakova, A. Tomeckova, M. Ceckova, V. Buchta,
J. Jampilek, P. Cermak, J. Kunes, M. Dolezal, F. Staud, Bioorg. Med.
Chem. 14 (2006) 5850.
[15] H. Foks, J. Sawlewicz, Acta Polon. Pharm. 21 (1964) 429.
[16] Y. Abe, Y. Shigeta, F. Uchimaru, S. Okada, E. Ozasayama, Japan Patent
69 12,898, 1969. Chem. Abstr. 71 (1969) 112979y.
´
´
´
´
[17] P.M. Lopez-Roldan, J. Lopez de Alda, D. Barcelo, Anal. Bioanal. Chem.
378 (2004) 599.
[18] http://www.taacf.org/about-TAACF.htm (13 February 2007).
[19] L. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004.
[20] F.E. Norrington, R.M. Hyde, G.G. Williams, R. Wooton, J. Med. Chem.
18 (1975) 604.
Acknowledgments
[21] C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in
Chemistry and Biology, John Wiley & Sons, New York, 1979, pp.
1e379.
This study was supported by the Ministry of Education of
the Czech Republic (MSM0021620822), and by the Slovak
Scientific Grant Agency VEGA (No. 1/0089/03). Antimyco-
bacterial data were provided by the Tuberculosis Antimicro-
bial Acquisition and Coordinating Facility (TAACF) through
a research and development contract with the U.S. National
Institute of Allergy and Infectious Diseases.
[22] National Committee for Clinical Laboratory Standards, Reference
Method for Broth Dilution Antifungal Susceptibility Testing of Yeast:
Approved Standard, NCCLS Document, M27-A, NCCLS, Villanova,
PA, U.S.A, 1997.
[23] D.A. Walker, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in
Enzymology Part C, Vol. 69, Academic Press, New York, 1980, pp.
94e104.
[24] K. Kralova, F. Sersen, E. Sidoova, Chem. Pap. 46 (1992) 348.
[25] C. Fedke, Biochemistry and Physiology of Herbicide Action, Springer
Verlag, BerlineHeidelbergeNew York, 1982.
References
[26] K. Kralova, F. Sersen, M. Melnik, J. Trace Microprobe Tech. 16 (1998)
491.
[27] A.R. Wellburn, J. Plant. Physiol. 144 (1994) 307.
[1] P. Nunn, B. Williams, K. Floyd, Ch. Dye, G. Elzinga, M. Raviglione,
Nat. Rev. Immunol. 5 (2005) 819.