Substituted 5-aroylpyrazine-2-carboxylic acid derivatives: Synthesis and biological activity

Article abstract of DOI:10.1016/S0014-827X(03)00163-0

Homolytic aroylation of pyrazine nucleus with various substituted aromatic carbaldehydes afforded a series of 5-aroylpyrazine-2-carboxylic acid derivatives. The synthetic approach, analytical and spectroscopic data of all compounds synthesized, their preliminary in vitro evaluation of antituberculotic and antifungal activities, cytotoxicity data and subsequent SAR studies are presented. Among all derivatives prepared, only 5-(4-chlorobenzoyl)-pyrazine-2-carbothioamide (3d) showed promising activity (90% inhibition) against Mycobacterium tuberculosis. The highest antifungal effect (MIC<1.95 μmol ml^-1) against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for 5-benzoylpyrazine-2-carbothioamide (3a). Thioamides exhibited higher in vitro antimicrobial activity than the corresponding amides.

Full text of DOI:10.1016/S0014-827X(03)00163-0

Il Farmaco 58 (2003) 1105ꢁ1111
/
www.elsevier.com/locate/farmac
Substituted 5-aroylpyrazine-2-carboxylic acid derivatives: synthesis
and biological activity
M. Dolezal a,*, J. Jamp´ılek a, Z. Osicka a, J. Kunesˇ b, V. Buchta c, P. V´ıchova´ d
a Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kra´love´, Charles University in Prague, Heyrovske´ho
1203, 50005 Hradec Kra´love´, Czech Republic
b Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kra´love´, Charles University in Prague, Heyrovske´ho 1203, 50005
Hradec Kra´love´, Czech Republic
c Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kra´love´, Charles University in Prague, Heyrovske´ho 1203, 50005
Hradec Kra´love´, Czech Republic
d Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hradec Kra´love´, Charles University in Prague, Heyrovske´ho 1203, 50005
Hradec Kra´love´, Czech Republic
Received 3 February 2003; accepted 24 May 2003
Abstract
Homolytic aroylation of pyrazine nucleus with various substituted aromatic carbaldehydes afforded a series of 5-aroylpyrazine-2-
carboxylic acid derivatives. The synthetic approach, analytical and spectroscopic data of all compounds synthesized, their
preliminary in vitro evaluation of antituberculotic and antifungal activities, cytotoxicity data and subsequent SAR studies are
presented. Among all derivatives prepared, only 5-(4-chlorobenzoyl)-pyrazine-2-carbothioamide (3d) showed promising activity
(90% inhibition) against Mycobacterium tuberculosis. The highest antifungal effect (MICB
1.95 mmol mlꢀ1) against Trichophyton
mentagrophytes, the most susceptible fungal strain tested, was found for 5-benzoylpyrazine-2-carbothioamide (3a). Thioamides
/
exhibited higher in vitro antimicrobial activity than the corresponding amides.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Aroylpyrazines; Homolytic aroylation; In vitro antimycobacterial activity; In vitro antifungal activity; MTT assay; Structureꢁ
/activity
relationships
1. Introduction
niazid and rifampicin). The current recommended
strategy is facing two problems: multidrug resistance
and HIV/AIDS pandemic [1,2]. Additionally, in patients
with impaired cellular immunity (HIV-syndrom), myco-
bacterial and fungal (Aspergillus, Histoplasma, etc.)
infections predominate and may coexist [3].
There are two basic approaches to get a new drug for
TB: (i) synthesis of analogues, modifications or deriva-
tives of existing compounds for shortening and impro-
ving TB treatment; and (ii) searching novel structures,
that the TB organism ‘has never seen before’, for the
treatment of multidrug-resistant TB [4].
Pyrazinamide (PZA), an analogue of nicotinamide, is
a unique first-line TB drug, which is involved in short-
ening the TB therapy. PZA is a prodrug that requires
activation or conversion at acid pH condition to its
active form, pyrazinoic acid (POA), by the pyrazinami-
dase/nicotinamidase enzyme encoded by pncA gene of
One-third of the world’s population is infected with
tuberculosis (TB), therefore, TB today still represents
one of the major problems for public health worldwide.
After a long period in which it seemed to be declining,
an unexpected return has been recorded for this disease.
Each year there are 8.7 million new cases of TB and
estimated 1.7 million deaths. If current control efforts
are not massively expanded, TB will kill more than 40
million people over the next 25 years. The existing
recommended TB treatment is based on 2 months of
four drugs (isoniazid, rifampicin, pyrazinamide, and
ethambutol) followed by 4 months of two drugs (iso-
* Corresponding author.
E-mail address: dolezalm@faf.cuni.cz (M. Dolezal).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00163-0

1106
M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ1111
/
susceptible Mycobacterium tuberculosis. The target of
PZA/POA appears to be the membrane, i.e. fatty acid
synthase-I [5]. One of the effective methods that can lead
to new drug discoveries is the bioisosteric replacement of
a functional group.
To this mixture 80% tert-BuO2H (13.5 g, 120 mmol) and
a solution of FeSO4×7H2O (33.4 g, 120 mmol) in water
/
were added simultaneously. The temperature must not
exceed 10 8C. The resulting mixture was stirred for
additional 2 h, during which the temperature was
allowed to rise to 20 8C. The reaction product (yellow
crystals) was filtered and crystallized from mixture
EtOH/H2O.
This paper is the continuation of our interest in
pyrazine-2-carboxylic acid derivatives, previous studies
[6ꢁ11] showed that alkylation, amidation or substitu-
/
tion of the pyrazine ring with chlorine increases
antituberculotic activity in series of functional deriva-
tives pyrazine-2-carboxylic acid. Pyrazine nucleus rea-
dily undergoes radical aroylation reactions using the
appropriate benzaldehyde with tert-butyl hydroperox-
ide/Fe2ꢂ as the initiator [12]. Aim of this work is: (i) the
radical aroylation of pyrazine nucleus study; (ii) in vitro
screening of antituberculotic, antifungal activity, and
cytotoxic properties of newly prepared aroylpyrazines;
(iii) the comparison of their lipophilicity parameters;
and finally (iv) the SAR study of functional modifica-
tions in 5-aroylpyrazine-2-carboxylic acid series, i.e.
carboxamides, carbonitriles, carbothioamides, and
methyl esters of pyrazine-2-carboxylic acid.
2.2.2. 5-Benzoylpyrazine-2-carboxamide (1a)
Yield 40%, m.p. 205ꢁ
for C12H9N3O2 (226.2): 68.43% C, 3.99%; H, 18.49% N;
found: 68.29% C, 4.15% H, 18.41% N, log Pꢃ1.859
/
206 8C (205 8C [13]). Anal. Calc.
/
/
0.43. IR spectrum: 3432 (NH amide), 1672 (CO amide),
1598 (CO aroyl), 1580 (Ph), 1413, 1274, 1178 (pyrazine).
1H NMR spectrum: d 9.25d 1H Jꢃ
/
1.32 (H3), 9.16ꢁ
7.94m 2H (H2?, H6?), 7.76ꢁ7.67m
7.52m 2H (H3?, H5?). 13C NMR
/
9.13m 1H (H6), 8.01ꢁ
/
/
1H (H-4?), 7.61ꢁ
/
spectrum: d 192.4, 164.8, 151.5, 146.6, 144.1, 142.4,
135.4, 134.2, 131.0, 128.9.
2.2.3. 5-(2-Hydroxybenzoyl)-pyrazine-2-carboxamide
(1b)
Yield 24%, m.p. 245ꢁ
C12H9N3O3 (243.2): 59.26% C, 3.73% H, 17.28% N;
found: 59.28% C, 3.70% H, 17.11% N, log Pꢃ2.149
/
246 8C. Anal. Calc. for
2. Experimental
/
/
2.1. Instrumentation
0.47. IR spectrum: 3427 (NH amide), 3233 (OH), 1673
(CO amide), 1625 (CO aroyl), 1601 (Ph), 1437, 1247,
1180 (pyrazine). 1H NMR spectrum: d 10.44s 1H (OH),
TLC was performed on Silufol UV 254 plates
toluene
(Kavalier, Votice) using a mixture of acetoneꢁ
/
9.19d 1H Jꢃ
1H (NH2), 7.99bs 1H (NH2), 7.65ꢁ
7.53ꢁ7.44m 1H (H4?), 6.99ꢁ
6.89m 2H (H3?, H5?). 13C
/
1.50 (H3), 9.03d 1H Jꢃ/1.49 (H6), 8.42bs
(1:1) or acetoneꢁEtOAc (1:1). Silica gel plates were
/
/
7.58m 1H (H6?),
visualized in addition to the solution of 2,4-dinitrophe-
nylhydrazine in NaOH (detection of carbonyl group).
Melting points were determined on a Kofler block, and
are uncorrected. Elemental analyses were obtained using
an EA 1110 CHNS-O CE apparatus (Fisons Instru-
ments S.p.A., Milan). The IR spectra were recorded on a
Nicolet Impact 400 spectrometer in KBr pellets. The 1H
and 13C NMR spectra were measured for DMSO (or
CDCl3) solutions with a Varian Mercury-Vx BB 300
spectrometer operating at 300 MHz (1H NMR spectra)
and 75 MHz (13C NMR spectra). Chemical shifts were
recorded as d values in parts per million (ppm), and
were indirectly referenced to tetramethylsilane via the
/
/
NMR spectrum: d 195.0, 164.8, 158.8, 152.7, 146.4,
142.6, 135.5, 131.7, 123.5, 119.6, 117.2.
2.2.4. 5-(4-Hydroxybenzoyl)-pyrazine-2-carboxamide
(1c)
Yield 11%, m.p. 235ꢁ
C12H9N3O3 (243.2): 55.17% C, 4.24% H, 16.09% N;
found: 55.48% C, 4.34% H, 16.10% N, log Pꢃ1.549
/
236 8C. Anal. Calc. for
/
/
0.45. IR spectrum: 3420 (NH amide), 3231 (OH), 1701
(CO amide), 1634 (CO aroyl), 1600 (Ph), 1448, 1281,
1171 (pyrazine). 1H NMR spectrum: d 10.55s 1H (OH),
9.23d 1H Jꢃ
1H (NH2), 7.99bs 1H (NH2), 7.96ꢁ
6.94ꢁ
6.87m 2H (H3?, H5?). 13C NMR spectrum: d
/
1.35 (H-3), 9.06d 1H Jꢃ/1.35 (H6), 8.43bs
solvent signal (7.26 for H and 77.0 for 13C). Multi-
/
7.88m 2H (H2?, H6?),
1
plicities are given together with the coupling constants
(in Hz). Log P values were computed using a program
ACD/log P ver. 1.0 (Advanced Chemistry Development
Inc., Toronto).
/
190.2, 164.9, 163.2, 152.6, 146.2, 143.8, 142.2, 134.0,
126.7, 115.6.
2.2.5. 5-(4-Chlorobenzoyl)-pyrazine-2-carboxamide
(1d)
2.2. 5-Aroylpyrazine-2-carboxamides (1aꢁ
/
1d)
Yield 16%, m.p. 225ꢁ
C12H8ClN3O2 (261.7): 55.08% C, 3.08% H, 16.06% N;
found: 55.46% C, 3.12% H, 15.99% N, log Pꢃ2.579
0.45. IR spectrum: 3425 (NH amide), 1702 (CO amide),
1674 (CO aroyl), 1589 (Ph), 1477, 1400, 1274, 1177
/
226 8C. Anal. Calc. for
2.2.1. General procedure
A stirred mixture of the carbaldehyde (120 mmol),
and pyrazine-2-carboxamide (40 mmol) in 50% H2SO4
/
/
(80 ml) and 99% AcOH (80 ml) was cooled to ꢀ5 8C.
/

M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ
/1111
1107
1
(pyrazine). H NMR spectrum: d 10.73bs 2H (NH2),
9.33d 1H Jꢃ1.51 (H3), 9.20d 1H Jꢃ1.51 (H6), 7.94ꢁ
7.86m 2H (H2?, H6?), 6.94ꢁ
6.86m 2H (H3?, H5?). 13C
2.3.5. 5-(4-Chlorobenzoyl)-pyrazine-2-carbonitrile (2d)
Yield 16%, m.p. 225ꢁ226 8C. Anal. Calc. for
C12H8ClN3O2 (261.7): 55.08% C, 3.08% H, 16.06% N;
found: 55.46% C, 3.12% H, 15.99% N, log Pꢃ2.589
0.44. IR spectrum: 2225 (CN), 1674 (CO aroyl), 1589
(Ph), 1477, 1400, 1274, 1177 (pyrazine). 1H NMR
/
/
/
/
/
NMR spectrum: d 189.3, 163.6, 152.6, 147.3, 145.7,
133.9, 130.9, 126.1, 116.1, 115.7.
/
/
spectrum (CDCl3): d 9.53ꢁ
/
9.50m 1H (H3), 9.22ꢁ
/
2.3. 5-Aroylpyrazine-2-carbonitriles (2aꢁ2d)
/
9.21m 1H (H-6), 8.04ꢁ
/
7.97m 2H (H2?, H6?), 7.68ꢁ
/
7.59m 2H (H3?, H5?). 13C NMR spectrum (CDCl3):d
191.9, 155.5, 147.2, 143.6, 139.2, 135.1, 129.1, 128.0,
117.2.
2.3.1. General procedure
A mixture of compound 1 (10 mmol) and POCl3 (110
mmol) is stirred at 110 8C for 1 h. Then the mixture
cooled at 10 8C and poured on ice. Temperature must
not exceed 50 8C. The aqueous solution was extracted
with CHCl3. The solvent was dried over MgSO4 and
evaporated in vacuo. The reaction product (red crystals)
was filtered and crystallized from mixture EtOH/H2O.
2.4. 5-Aroylpyrazine-2-carbothioamides (3aꢁ
/
3d)
2.4.1. General procedure
A compound 2 (4 mmol) was dissolved in MeOH (50
ml) and (NH4)2S (4 ml, 20% wt.% solution in water) was
added. A reaction mixture was kept at 5 8C for 24 h.
Then the mixture was filtered, MeOH evaporated and
the product (yellow crystals) crystallized from EtOH.
2.3.2. 5-Benzoylpyrazine-2-carbonitrile (2a)
Yield 91%, m.p. 93ꢁ
(208.2): 68.89% C, 3.37% H, 20.09% N; found: 68.63%
C, 3.28% H, 19.65% N, log Pꢃ1.8690.43. IR spec-
trum: 2220 (CN), 1670 (CO aroyl), 1597 (Ph), 1447,
1
1275, 1156 (pyrazine). H NMR spectrum (CDCl3): d
/
94 8C. Anal. Calc. for C12H7N3O
2.4.2. 5-Benzoylpyrazine-2-carbothioamide (3a)
/
/
Yield 81%, m.p. 119ꢁ120 8C. Anal. Calc. for
/
C12H9N3OS (242.2): 59.24% C, 3.73% H, 17.27% N,
13.18% S; found: 59.56% C, 3.77% H, 17.22% N, 12.93%
9.36ꢁ
/
9.34m 1H (H3), 9.30ꢁ
/
9.28m 1H (H6), 8.01ꢁ
/
7.95m
S, log Pꢃ
/
2.0490.49. IR spectrum: 3443 (NH amide),
/
2H (H2?, H6?), 7.78ꢁ
/
7.69m 1H (H4?), 7.62ꢁ
/
7.54m 2H
(H3?, H5?). 13C NMR spectrum (CDCl3): d 191.5, 151.4,
147.4, 145.9, 134.8, 134.4, 131.3, 130.9, 128.8, 116.1.
1660 (CO aroyl), 1598 (Ph), 1447, 1273, 1173 (pyrazine).
1H NMR spectrum: d 10.50bs 1H (NH2), 10.19bs 1H
(NH2), 9.60d 1H Jꢃ
8.01ꢁ7.95m 2H (H2?, H6?), 7.74ꢁ
7.60ꢁ
7.52m 2H (H3?, H5?). 13C NMR spectrum: d
/
1.51 (H3), 9.09d 1H Jꢃ/1.51 (H-6),
/
/
7.67m 1H (H-4?),
/
2.3.3. 5-(2-Hydroxybenzoyl)-pyrazine-2-carbonitrile
(2b)
Yield 92%, m.p. 132ꢁ
C12H7N3O2 (225.2): 64.00% C, 3.13% H, 18.66% N;
found: 64.33% C, 3.09% H, 18.27% N, log Pꢃ2.159
0.47. IR spectrum: 3210 (OH), 2210 (CN), 1626 (CO
aroyl), 1605 (Ph), 1447, 1248, 1149 (pyrazine). 1H NMR
spectrum: d 10.49s 1H (OH), 9.30d 1H Jꢃ
192.4, 192.3, 150.6, 147.9, 144.1, 143.1, 135.5, 134.2,
131.0, 128.8.
/
133 8C. Anal. Calc. for
2.4.3. 5-(2-Hydroxybenzoyl)-pyrazine-2-carbothioamide
(3b)
Yield 85%, m.p. 171ꢁ173 8C. Anal. Calc. for
C12H9N3O2S (259.2): 55.59% C, 3.50% H, 16.21% N,
12.36% S; found: 56.85% C, 3.62% H, 15.9% N, 12.23%
/
/
/
/
1.37 (H3),
1.65
9.18d 1H Jꢃ1.37 (H6), 7.62dd 1H Jꢃ7.69 Jꢃ
/
/
/
S, log Pꢃ
/
2.3390.52. IR spectrum: 3440 (NH amide),
/
(H6?), 7.56ꢁ
/
7.47m 1H (H4?), 7.01ꢁ6.91m 2H (H3?, H5?).
/
1636 (CO aroyl), 1601 (Ph), 1437, 1247, 1180 (pyrazine).
1H NMR spectrum: d 10.50bs 1H (NH2), 10.39bs 1H
13C NMR spectrum: d 193.7, 158.7, 152.8, 147.7, 144.5,
135.7, 131.4, 130.9, 123.0, 119.5, 117.2, 116.1.
(OH), 10.19bs 1H (NH2), 9.60d 1H Jꢃ
1H Jꢃ1.51 (H6), 8.01ꢁ7.95m 1H (H-6?), 7.74ꢁ
1H (H4?), 7.60ꢁ
7.52m 2H (H3?, H5?). 13C NMR
/1.51 (H3), 9.09d
/
/
/7.67m
2.3.4. 5-(4-Hydroxybenzoyl)-pyrazine-2-carbonitrile
(2c)
Yield 90%, m.p. 163ꢁ
C12H7N3O2 (225.2): 64.00% C, 3.13% H, 18.66% N;
found: 64.36% C, 2.95% H, 18.37% N, log Pꢃ1.549
/
spectrum: d 195.0, 192.5, 158.7, 151.7, 147.9, 144.3,
141.6, 135.5, 131.7, 123.5, 119.6, 117.2.
/
164 8C. Anal. Calc. for
/
/
2.4.4. 5-(4-Hydroxybenzoyl)-pyrazine-2-carbothioamide
0.44. IR spectrum: 3397 (OH), 2244 (CN), 1640 (CO
aroyl), 1586 (Ph), 1437, 1280, 1149 (pyrazine). 1H NMR
(3c)
Yield 88%, m.p. 199ꢁ200 8C. Anal. Calc. for
/
spectrum: d 10.53s 1H (OH), 9.33d 1H Jꢃ
9.20d 1H Jꢃ1.51 (H6), 7.94ꢁ7.86m 2H (H2?, H6?),
6.94ꢁ
6.86m 2H (H3?, H5?). 13C NMR spectrum: d
189.3, 163.6, 152.6, 147.3, 145.7, 133.9, 130.9, 126.1,
116.1, 115.7.
/
1.51 (H3),
C12H9N3O2S (259.2): 51.98% C, 4.00% H, 15.15% N,
11.56% S; found: 52.04% C, 3.97% H, 15.19% N, 11.44%
/
/
/
S, log Pꢃ
1647 (CO aroyl), 1601 (Ph), 1438, 1278, 1148 (pyrazine).
1H NMR spectrum: d 10.50bs 1H (OH), 9.93d 1H Jꢃ
/
1.739
/
0.50. IR spectrum: 3432 (NH amide),
/

1108
M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ1111
/
1.47 (H3), 9.17d 1H Jꢃ
/
1.47 (H6), 8.53bs 1H (NH2),
H2SO4 (0.5 ml) was added and the mixture was refluxed
for 2 h under the Soxhlet-extractor, where dry Potasite
(70 g) was located. Then the mixture was cooled,
neutralized with KHCO3 and filtered. MeOH was
evaporated and the product (white crystals) was crystal-
lized from EtOH.
8.06ꢁ
/
7.97m 2H, (H2?, H6?), 7.21ꢁ7.18m 2H (H3?, H5?).
/
13C NMR spectrum: d 193.1, 191.9, 164.1, 162.6, 151.7,
144.8, 142.5, 129.2, 116.3.
2.4.5. 5-(4-Chlorobenzoyl)-pyrazine-2-carbothioamide
(3d)
Yield 83%, m.p. 219ꢁ
/
220 8C. Anal. Calc. for
C12H8ClN3OS (277.7): 51.90% C, 2.90% H, 15.13% N,
2.6.2. Methyl-5-benzoylpyrazine-2-carboxylate (5a)
Yield 60%, m.p. 101ꢁ
C13H10N2O3 (242.2): 64.46% C, 4.16% H, 11.56% N;
found: 64.77% C, 4.04% H, 11.46% N, log Pꢃ1.909
/
103 8C. Anal. Calc. for
11.55% S; found: 51.82% C, 2.86% H, 15.03% N, 11.39%
S, log Pꢃ
1647 (CO aroyl), 1601 (Ph), 1438, 1278, 1148 (pyrazine).
1H NMR spectrum (CDCl3): d 9.91d 1H Jꢃ
1.51 (H3),
9.22bs 1H (NH2), 9.15d 1H Jꢃ1.51 (H6), 8.17ꢁ8.09m
/
2.769
/
0.50. IR spectrum: 3439 (NH amide),
/
/
0.41. IR spectrum: 1700 (CO acid), 1670 (CO aroyl),
1600 (Ph), 1437, 1280, 1149 (pyrazine). 1H NMR
spectrum (CDCl3): d 9.22s 1H (H3), 9.10s 1H (H6),
/
/
/
2H (H2?, H6?), 7.75bs 1H (NH2), 7.55ꢁ
/
7.47m 2H (H3?,
7.58ꢁ
/
7.47m 2H (H2?, H6?), 7.39ꢁ7.22m 3H (H3?, H4?,
/
H5?). 13C NMR spectrum (CDCl3): d 192.3, 190.1,
150.5, 145.0, 144.5, 142.9, 140.6, 133.5, 132.5, 128.8.
H5?), 4.01s 3H (CH3). 13C NMR spectrum (CDCl3): d
192.0, 164.2, 159.0, 145.4, 145.3, 144.2, 134.0, 130.9,
128.5, 126.9, 53.1.
2.5. 5-Aroylpyrazine-2-carboxylic acids (4aꢁ
/
4b)
2.6.3. Methyl-5-(2-hydroxybenzoyl)-pyrazine-2-
carboxylate (5b)
2.5.1. General procedure
A compound 1 (40 mmol) was refluxed in 10%
solution of NaOH for 10 h, then the solution was
filtered with charcoal and the solution was neutralized
by 10% HCl. White crystalline product was obtained.
Yield 83%, m.p. 126ꢁ
C13H10N2O4 (258.2): 60.47% C, 3.90% H, 10.85% N;
found: 60.25% C, 3.60% H, 10.75% N, log Pꢃ2.199
0.44. IR spectrum: 3398 (OH), 1730 (CO acid), 1640
1
(CO aroyl), 1595 (Ph), 1437, 1280, 1149 (pyrazine). H
/
127 8C. Anal. Calc. for
/
/
2.5.2. 5-Benzoylpyrazine-2-carboxylic acid (4a)
NMR spectrum (CDCl3): d 11.85s 1H (OH), 9.41ꢁ
7.38m 1H (H3), 9.24ꢁ9.22m 1H (H6), 8.04dd 1H Jꢃ
8.24, Jꢃ 1.38 (H6?), 7.62ꢁ7.53m 1H (H4?), 7.11ꢁ7.07m
1H (H3?), 6.97ꢁ
6.89m 1H (H5?), 4.10s 3H (CH3). 13C
/
Yield 94%, m.p. 148ꢁ
C12H8N2O3 (228.2): 63.16% C, 3.53% H, 12.28% N;
found: 63.26% C, 3.75% H, 12.07% N, log Pꢃ2.459
/
150 8C. Anal. Calc. for
/
/
/
/
/
/
/
/
0.43. IR spectrum: 1692 (CO acid), 1645 (CO aroyl),
1589 (Ph), 1437, 1280, 1149 (pyrazine), (in literature
1
1692 and 1658 [14]). H NMR spectrum: d 9.27s 1H
NMR spectrum (CDCl3): d 194.9 164.2, 163.7, 152.4,
145.1, 144.1, 144.0, 137.8, 133.7, 119.2, 118.7, 118.2,
53.5.
(H3), 9.24s 1H (H6), 8.03ꢁ
/
7.95m 2H H-2? (H6?), 7.77ꢁ
/
7.68m 1H (H4?), 7.63ꢁ
/
7.52m 2H (H3?, H5?). 13C NMR
spectrum: d 192.2, 164.8, 151.4, 145.4, 145.0, 144.1,
135.3, 134.1, 130.9, 128.7.
2.7. Antimycobacterial assay
Antimycobacterial evaluation was carried out in
Tuberculosis Antimicrobial Acquisition and Coordina-
ting Facility (TAACF), Southern Research Institute,
Birmingham, AL, USA, which is a part of the National
Institutes of Health (NIH). Primary screening of all
compounds was conducted at 6.25 mg mlꢀ1 against M.
tuberculosis H37Rv in BACTEC 12B medium using the
BACTEC 460 radiometric system [15]. Compounds
showing at least 90% inhibition at 6.25 mg mlꢀ1 in this
primary screening were retested at lower concentrations
against M. tuberculosis H37Rv to determine the mini-
mum inhibitory concentration (MIC) in a broth micro-
dilution Alamar Blue assay (MABA). The MIC was
defined as the lowest concentration causing a decrease in
fluorescence of 99% relative to controls.
2.5.3. 5-(2-Hydroxybenzoyl)-pyrazine-2-carboxylic acid
(4b)
Yield 91%, m.p. 141ꢁ
C12H8N2O4 (244.2): 59.02% C, 3.30% H, 11.47% N;
found: 59.12% C, 3.39% H, 11.32% N, log Pꢃ2.479
/
142 8C. Anal. Calc. for
/
/
0.46. IR spectrum: 3395 (OH), 1700 (CO acid), 1620
1
(CO aroyl), 1589 (Ph), 1447, 1280, 1149 (pyrazine). H
NMR spectrum: d 10.46bs 1H (OH), 9.22d 1H Jꢃ
/
1.38
(H3), 9.12d 1H Jꢃ1.37 (H6), 7.62dd 1H Jꢃ7.69 Jꢃ
/
/
/
1.38 (H6?), 7.54ꢁ
/
7.46m 1H (H4?), 7.00ꢁ6.91m 2H (H3?,
/
H5?). 13C NMR spectrum: d 194.8, 164.9, 158.7, 152.6,
145.2, 144.4, 143.5, 135.4, 131.4, 123.4, 119.4, 117.2.
2.6. Methyl-5-aroylpyrazine-2-carboxylates (5aꢁ
/
5b)
2.8. In vitro antifungal susceptibility testing
2.6.1. General procedure
An anhydrous compound 4 (20 mmol) was dissolved
in anhydrous MeOH (100 ml). To the solution 98%
The broth microdilution test [16,17] was used for the
assessment of in vitro antifungal activity of the synthe-

M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ
/1111
1109
Scheme 1.
sized compounds and ketoconazole (standard) against
Candida albicans ATCC 44859, Candida tropicalis 156,
Candida krusei E28, Candida glabrata 20/I, Trichos-
poron beigelii 1188, Trichophyton mentagrophytes 445,
Aspergillus fumigatus 231, and Absidia corymbifera 272.
The procedure was performed with twofold dilutions of
the compounds in RPMI 1640 buffered to pH 7.0 with
0.165 mol of 3-morpholino-propane-1-sulfonic acid. The
final concentrations of the compounds ranged from
1000 to 0.975 mmol lꢀ1. Drug-free controls were
included. The MICs were determined after 24 h and 48
h of static incubation at 35 8C. With T. mentagrophytes,
the final MICs were read after 72 and 120 h of
incubation.
2.9. Cytotoxicity assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide) test was first described by Mosmann
[18] and improved in subsequent years by several other
investigators [19]. Cytotoxicity measurements were
based on the growth inhibition of Caco-2 cells (human
intestinal epithelial cells). An assay was performed after
24 h exposure to 50 ml mlꢀ1 of 5% DMSO solutions of
the tested agents. After incubation, 20 ml of MTT
solution (5 mg mlꢀ1) were added to each well. The
mitochondria of viable cells reduce the pale yellow MTT
to a violet formazan: the more viable cells are present in
well, the more MTT will be reduced to formazan. When

1110
M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ1111
/
incubation for 90 min was stopped, 100 ml DMSO were
added to each well. When formazan crystals had been
dissolved, the optical densities at 550 nm wavelength of
the samples were read on a Bio-rad Microplate reader
(Bio-rad Laboratories, Austria). The results of in vitro
them were prepared newly. Primary in vitro antimyco-
bacterial screening was provided for all 16 compounds,
in vitro antifungal screening was performed for 11
compounds, cytotoxic assay was performed for eight
derivatives.
cytotoxic activity were expressed as an EC50ꢀthe dose
/
In vitro antimycobacterial activity tended to be
proportional to molecular weight and lipophilicity of
these compounds. Therefore, in carbothioamides and
methyl esters studied some considerable in vitro biolo-
gical activity was determined in the functional deriva-
tives carboxylic acid series. Both the highest activity
(90% inhibition) against M. tuberculosis and the highest
of compound (in mmol lꢀ1) that inhibits proliferation
rate of Caco-2 cells by 50% as to the control untreated
cells.
3. Results and discussion
lipophilicity (log Pꢃ2.76) of all compounds studied was
/
found for 5-(4-chlorobenzoyl)-pyrazine-2-carbothioa-
mide (3d). This compound was retested (MABA) to
determine the actual minimal inhibitory concentration
Pyrazine-2-carboxamide was used as the starting
material for radical aroylation using substituted benzal-
dehydes. Radical aroylation is a homolytic chain oxida-
tion in liquid phase catalysis by means of salts of
transition metals (redox homogeneous catalysis). The
principle of this method is described [20] and according
to various different modification of radical aroylation,
some substituted pyrazin-2-carboxylic acid derivatives
were prepared [13,14,21].
(MICꢀ
12.5 mg mlꢀ1). Only other two compounds (3b,
/
5b) exhibited more than 50% inhibition, both of them
with hydroxylic group in ortho position of benzene
moiety of the structure (Table 1).
The evaluation of in vitro antifungal activity of the
synthesized compounds was performed against eight
fungal strains. The results showed no activity against
majority of fungal strains tested. Only compound 1d and
Prepared
(method according to Heinisch) [14] were transformed
to appropriate carbonitriles 2aꢁd and carbothioamides
3aꢁd. Corresponding carboxylic acids 4a, 4b and methyl
5-aroylpyrazine-2-carboxamides
1aꢁ/d
especially carbothioamides 3aꢁ
in vitro antifungal activity against T. mentagrophytes
(MICꢃ1.95ꢁ
62.5 mmol mlꢀ1). Other carbothioamide,
5-(4-chlorobenzoyl)-pyrazine-2-carbothioamide (3d)
/c exhibited a promising
/
/
/
/
esters 5a, 5b were prepared from 1a, 1b (Scheme 1). Our
study describes the synthesis and properties of 16
compounds; two of them were characterized previously
by Sato [13] (1a) and Heinisch [14] (4a), 14 derivatives of
precipitated in the RPMI medium. 5-Benzoylpyrazine-
2-carbothioamide (3a) had comparable activity against
Table 1
Antituberculotic activity, antifungal activity, cytotoxicity, selectivity index (SI), and lipophilicity (calculated log P) of compounds 1aꢁ
/
4d in
comparison with standards
Comp.
Mycobacterium tuberculosis H37Rv % inhibi-
tion at 6.25 (mg mlꢀ1
Trichophyton mentagrophytes 445 MIC Caco-2 cells EC50
d
SI
log P
)
(mmol mlꢀ1
)
(mmol lꢀ1
)
1a
1b
8
250/ꢀ
/
250
0.67
12.50
2.7
1.859
2.149
1.549
2.579
1.869
119.6 2.159
28.9 1.549
2.589
5153.9 2.049
1913.0 2.339
1.739
2.769
2.459
2.479
1.909
2.199
0.379
4.019
/
0.43
0.47
0.45
0.45
0.43
0.47
0.44
0.44
0.49
0.52
0.50
0.50
0.43
0.46
0.41
0.44
0.35
0.66
2
250/ꢀ
/
250
50.0
/
b
1c
1d
0
ꢀ/
500/ꢀ
/500
/
b
a
16
10
5
ꢀ/
62.5/ꢀ
/62.5
/
2a
2b
500/500
125/250
250/500
250/500
/
14.95
/
2c
3
7.22
a
/
2d
3a
12
39
60
21
90
15
4
/
B/
1.95/B
/1.95
10.05
7.48
/
3b
3.91/7.81
/
a
3c
3d
3.91/7.81
b
/
a
/
4a
4b
ꢀ
/
500/ꢀ
500/ꢀ
500/ꢀ
250/ꢀ
/
500
500
500
250
18.96
4.72
37.9
9.4
/
ꢀ
/
/
/
a
5a
5b
48
53
ꢀ
/
/
/
a
ꢀ/
/
/
c
Pyrazinamide
Ketoconazole
/
0.98/1.95
/
a
Not tested.
Precipitated in the testing medium.
b
c
MICꢃ
/
12.5 mg mlꢀ1, data from Refs. [6ꢁ
After 72/120 h.
/
8].
d

M. Dolezal et al. / Il Farmaco 58 (2003) 1105ꢁ
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/
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/
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ˇ
[6] M. Dolezal, J. Hartl, A. Lycˇka, V. Buchta, Z. Odlerova´, Synthesis
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cells with the MTT reagent for approximately 2ꢁ4 h, a
/
detergent solution is added to lyse the cells and
solubilize the coloured crystals. The samples are read
using a microplate reader at a wavelength of 550 nm.
The intensity of colour produced is directly proportional
to the number of viable cells. Therefore, the measure-
ment of optical densities by MTT assay of some tested
compounds was problematic due to interference of
coloured samples (yellow or red) with formazan absor-
bance maximum at 550 nm. Lower solubility, resp.
precipitation in the testing medium of compounds
studied was other difficult trouble. The selectivity index
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/
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/
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Farmaco 57 (2002) 71ꢁ78.
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/
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/
[13] N. Sato, H. Kadota, Studies on pyrazine 23. Homolytic acylation
(1a) possessed the highest cytotoxicity (IC50ꢃ
mmol lꢀ1, SIꢃ
2.7). The in vitro antifungal most
effective compound 5-benzoylpyrazine-2-carbothioa-
mide (3a) exerted EC50ꢃ 5000.
10.05 mmol lꢀ1, SIꢀ
There is no general result about the structureꢁcytotoxi-
/
0.67
of 2-amino-3-cyanopyrazine and the related-compounds with
/
alpha-keto acids*/a synthesis of 5-acyl-3-aminopyrazinecar-
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/
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/
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/
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This study was supported by the Ministry of Educa-
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and MSM 111600002). Antimycobacterial data were
provided by the Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF) through a research
and development contract with the U.S. National
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